structure-based design of drugs and other bioactive...

Arun K. Ghosh and Sandra Gemma

Structure-based Design of Drugs and Other Bioactive Molecules Tools and Strategies


Structure-based Design of Drugs andOther Bioactive Molecules

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Structure-based Design of Drugs andOther Bioactive Molecules

Tools and Strategies

The Authors

Prof. Dr. Arun K. GhoshPurdue UniversityDepartment of Chemistryand Department of Medicinal Chemistry560 Oval DriveWest Latayette, INUnited States

Prof. Dr. Sandra GemmaUniversità degli Studi SienaDipartimento di Biotecnologie, Chimica eFarmaciavia Aldo Moro 253100 SienaItaly

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Contents

Preface XIII

1 From Traditional Medicine to Modern Drugs: HistoricalPerspective of Structure-Based Drug Design 1

1.1 Introduction 11.2 Drug Discovery During 1928–1980 11.3 The Beginning of Structure-Based Drug Design 61.4 Conclusions 12

References 13

Part One Concepts, Tools, Ligands, and Scaffolds for Structure-BasedDesign of Inhibitors 19

2 Design of Inhibitors of Aspartic Acid Proteases 212.1 Introduction 212.2 Design of Peptidomimetic Inhibitors of Aspartic Acid Proteases 222.3 Design of Statine-Based Inhibitors 242.4 Design of Hydroxyethylene Isostere-Based Inhibitors 292.5 Design of Inhibitors with Hydroxyethylamine Isosteres 352.5.1 Synthesis of Optically Active a-Aminoalkyl Epoxide 372.6 Design of (Hydroxyethyl)urea-Based Inhibitors 402.7 (Hydroxyethyl)sulfonamide-Based Inhibitors 422.8 Design of Heterocyclic/Nonpeptidomimetic Aspartic

Acid Protease Inhibitors 422.8.1 Hydroxycoumarin- and Hydroxypyrone-Based Inhibitors 442.8.2 Design of Substituted Piperidine-Based Inhibitors 462.8.3 Design of Diaminopyrimidine-Based Inhibitors 502.8.4 Design of Acyl Guanidine-Based Inhibitors 512.8.5 Design of Aminopyridine-Based Inhibitors 532.8.6 Design of Aminoimidazole- and Aminohydantoin-Based Inhibitors 532.9 Conclusions 56

References 56

jV

3 Design of Serine Protease Inhibitors 673.1 Introduction 673.2 Catalytic Mechanism of Serine Protease 673.3 Types of Serine Protease Inhibitors 673.4 Halomethyl Ketone-Based Inhibitors 693.5 Diphenyl Phosphonate-Based Inhibitors 703.6 Trifluoromethyl Ketone Based Inhibitors 733.6.1 Synthesis of Trifluoromethyl Ketones 763.7 Peptidyl Boronic Acid-Based Inhibitors 783.7.1 Synthesis of a-Aminoalkyl Boronic Acid Derivatives 833.8 Peptidyl a-Ketoamide- and a-Ketoheterocycle-Based Inhibitors 853.8.1 Synthesis of a-Ketoamide and a-Ketoheterocyclic Templates 903.9 Design of Serine Protease Inhibitors Based Upon Heterocycles 933.9.1 Isocoumarin-Derived Irreversible Inhibitors 943.9.2 b-Lactam-Derived Irreversible Inhibitors 953.10 Reversible/Noncovalent Inhibitors 973.11 Conclusions 104

References 105

4 Design of Proteasome Inhibitors 1134.1 Introduction 1134.2 Catalytic Mechanism of 20S Proteasome 1134.3 Proteasome Inhibitors 1144.3.1 Development of Boronate Proteasome Inhibitors 1154.3.2 Development of b-Lactone Natural Product-Based Proteasome

Inhibitors 1164.3.3 Development of Epoxy Ketone-Derived Inhibitors 1184.3.4 Noncovalent Proteasome Inhibitors 1204.4 Synthesis of b-Lactone Scaffold 1214.5 Synthesis of Epoxy Ketone Scaffold 1234.6 Conclusions 126

References 126

5 Design of Cysteine Protease Inhibitors 1315.1 Introduction 1315.2 Development of Cysteine Protease Inhibitors with Michael

Acceptors 1325.3 Design of Noncovalent Cysteine Protease Inhibitors 1365.4 Conclusions 140

References 140

6 Design of Metalloprotease Inhibitors 1436.1 Introduction 1436.2 Design of Matrix Metalloprotease Inhibitors 144

VIj Contents

6.3 Design of Inhibitors of Tumor Necrosis Factor-a-ConvertingEnzymes 150

6.4 Conclusions 152References 152

7 Structure-Based Design of Protein Kinase Inhibitors 1557.1 Introduction 1557.2 Active Site of Protein Kinases 1557.3 Catalytic Mechanism of Protein Kinases 1567.4 Design Strategy for Protein Kinase Inhibitors 1567.5 Nature of Kinase Inhibitors Based upon Binding 1607.5.1 Type I Kinase Inhibitors and Their Design 1607.5.2 Type II Kinase Inhibitors and Their Design 1647.5.3 Allosteric Kinase Inhibitors and Their Design 1687.5.4 Covalent Kinase Inhibitors and Their Design 1727.6 Conclusions 177

References 177

8 Protein X-Ray Crystallography in Structure-Based Drug Design 1838.1 Introduction 1838.2 Protein Expression and Purification 1848.3 Synchrotron Radiation 1858.4 Structural Biology in Fragment-Based Drug Design 1868.5 Selected Examples of Fragment-Based Studies 1878.6 Conclusions 196

References 197

9 Structure-Based Design Strategies for Targeting G-Protein-CoupledReceptors (GPCRs) 199

9.1 Introduction 1999.2 High-Resolution Structures of GPCRs 2009.3 Virtual Screening Applied to the b2-Adrenergic

Receptor 2019.4 Structure-Based Design of Adenosine A2A Receptor

Antagonists 2049.5 Structure-Guided Design of CCR5 Antagonists 2079.5.1 Development of Maraviroc from HTS Lead Molecules 2079.5.2 Improvement of Antiviral Activity and Reduction of

Cytochrome P450 Activity 2089.5.3 Reduction of hERG Activity and Optimization of Pharmacokinetic

Profile 2099.5.4 Other CCR5 Antagonists 2139.6 Conclusion 213

References 213

Contents jVII

Part Two Structure-Based Design of FDA-Approved Inhibitor Drugs and DrugsUndergoing Clinical Development 217

10 Angiotensin-Converting Enzyme Inhibitors for the Treatmentof Hypertension: Design and Discovery of Captopril 219

10.1 Introduction 21910.2 Design of Captopril: the First Clinically Approved

Angiotensin-Converting Enzyme Inhibitor 22010.3 Structure of Angiotensin-Converting Enzyme 22510.4 Design of ACE Inhibitors Containing a Carboxylate as

Zinc Binding Group 22810.5 ACE Inhibitors Bearing Phosphorus-Based Zinc Binding Groups 23110.5.1 Phosphonamidate-Based Inhibitors 23210.5.2 Phosphonic and Phosphinic Acid Derivatives: the Path to

Fosinopril 23310.6 Conclusions 234

References 235

11 HIV-1 Protease Inhibitors for the Treatment of HIV Infection and AIDS:Design of Saquinavir, Indinavir, and Darunavir 237

11.1 Introduction 23711.2 Structure of HIV Protease and Design of Peptidomimetic Inhibitors

Containing Transition-State Isosteres 23911.3 Saquinavir: the First Clinically Approved HIV-1 Protease Inhibitor 24111.4 Indinavir: an HIV Protease Inhibitor Containing the

Hydroxyethylene Transition-State Isostere 24611.5 Design and Development of Darunavir 25111.6 Design of Cyclic Ether Templates in Drug Discovery 25211.7 Investigation of Cyclic Sulfones as P2 Ligands 25511.8 Design of Bis-tetrahydrofuran and Other Bicyclic P2 Ligands 25711.9 The “Backbone Binding Concept” to Combat Drug Resistance:

Inhibitor Design Strategy Promoting Extensive BackboneHydrogen Bonding from S2 to S2

0 Subsites 25911.10 Design of Darunavir and Other Inhibitors with Clinical

Potential 26311.11 Conclusions 266

References 266

12 Protein Kinase Inhibitor Drugs for Targeted Cancer Therapy: Designand Discovery of Imatinib, Nilotinib, Bafetinib, and Dasatinib 271

12.1 Introduction 27112.2 Evolution of Kinase Inhibitors as Anticancer Agents 27212.3 The Discovery of Imatinib 27412.4 Imatinib: the Structural Basis of Selectivity 27512.5 Pharmacological Profile and Clinical Development 278

VIIIj Contents

12.6 Imatinib Resistance 27912.7 Different Strategies for Combating Drug Resistance 27912.7.1 Nilotinib and Bafetinib: Optimizing Drug–Target

Interactions 27912.7.2 Dasatinib: Binding to the Active Conformation (the First

Example of Dual Abl/Src Inhibitors) 28412.8 Conclusions 289

References 290

13 NS3/4A Serine Protease Inhibitors for the Treatment of HCV:Design and Discovery of Boceprevir and Telaprevir 295

13.1 Introduction 29513.2 NS3/4A Structure 29613.3 Mechanism of Peptide Hydrolysis by NS3/4A Serine Protease 29913.4 Development of Mechanism-Based Inhibitors 30013.5 Strategies for the Development of HCV NS3/4A Protease

Inhibitors 30313.6 Initial Studies toward the Development of Boceprevir 30413.7 Reduction of Peptidic Character 30813.8 Optimization of P2 Interactions 30913.9 Truncation Strategy: the Path to Discovery of Boceprevir 31213.10 The Discovery of Telaprevir 31413.11 Simultaneous P1, P1

0, P2, P3, and P4 Optimization Strategy:the Path to Discovery of Telaprevir 316

13.12 Conclusions 319References 319

14 Proteasome Inhibitors for the Treatment of Relapsed Multiple Myeloma:Design and Discovery of Bortezomib and Carfilzomib 325

14.1 Introduction 32514.2 Discovery of Bortezomib 32614.3 Discovery of Carfilzomib 33014.4 Conclusions 334

References 334

15 Development of Direct Thrombin Inhibitor, Dabigatran Etexilate,as an Anticoagulant Drug 337

15.1 Introduction 33715.2 Coagulation Cascade and Anticoagulant Drugs 33815.3 Anticoagulant Therapies 34015.4 Structure of Thrombin 34215.5 The Discovery of Dabigatran Etexilate 34515.6 Conclusions 353

References 353

Contents jIX

16 Non-Nucleoside HIV Reverse Transcriptase Inhibitors for theTreatment of HIV/AIDS: Design and Developmentof Etravirine and Rilpivirine 355

16.1 Introduction 35516.2 Structure of the HIV Reverse Transcriptase 35716.3 Discovery of Etravirine and Rilpivirine 36016.4 Conclusions 368

References 370

17 Renin Inhibitor for the Treatment of Hypertension: Design andDiscovery of Aliskiren 373

17.1 Introduction 37317.2 Structure of Renin 37317.3 Peptidic Inhibitors with Transition-State Isosteres 37417.4 Peptidomimetic Inhibitors 37617.5 Design of Peptidomimetic Inhibitors 38017.6 Biological Properties of Aliskiren 39317.7 Conclusions 393

References 394

18 Neuraminidase Inhibitors for the Treatment of Influenza: Design andDiscovery of Zanamivir and Oseltamivir 397

18.1 Introduction 39718.2 Discovery of Zanamivir 40118.3 Discovery of Oseltamivir 40318.4 Conclusions 407

References 408

19 Carbonic Anhydrase Inhibitors for the Treatment of Glaucoma:Design and Discovery of Dorzolamide 411

19.1 Introduction 41119.2 Design and Discovery of Dorzolamide 41219.3 Conclusions 418

References 418

20 b-Secretase Inhibitors for the Treatment of Alzheimer’s Disease:Preclinical and Clinical Inhibitors 421

20.1 Introduction 42120.2 b-Secretase and Its X-Ray Structure 42220.3 Development of First Peptidomimetic BACE Inhibitors 42320.4 X-Ray Structure of Inhibitor-Bound BACE1 42520.5 Design and Development of Selective Inhibitors 42720.6 Design of Small-Molecule Inhibitors with Clinical Potential 43120.7 GRL-8234 (18) Rescued Cognitive Decline in AD Mice 43520.8 BACE1 Inhibitors for Clinical Development 436

Xj Contents

20.8.1 Development of Clinical Inhibitor, AZD3839 43620.8.2 Development of Iminopyrimidinone-Based BACE1 Inhibitors 44020.9 Conclusions 443

References 444

Index 449

Contents jXI

Preface

As our knowledge of the structure and function of proteins has expanded, newtechniques employing this knowledge as the basis for drug design and discoveryhave emerged and taken the lead. The impact of structure-based design strategieshas been dramatic and far-reaching, resulting in the discovery and development ofnumerous FDA-approved drugs, many of which are first-in-class medicines. Majoradvancements in molecular biology and technology have led to in-depth structuralknowledge of new disease-relevant target enzymes. Improvements in X-ray crystal-lographic techniques have created an important database and enabled a betterunderstanding of the role of enzyme–ligand interactions. Progress in computeranalysis has also played a vital role in advancing structure-based design capabilitiessince the 1980s. Today, structure-based design has become one of the mostinnovative and dynamic areas of drug design and discovery.Over the years, the Ghosh laboratories have gained extensive experience with

structure-based design. The development of conceptually novel inhibitors againstHIV-1 protease for the treatment of HIV/AIDS has been an important area ofresearch that led to the design and discovery of darunavir, the first FDA-approvedtreatment for drug-resistant HIV/AIDS. Structure-based design of b-secretase 1(BACE1) inhibitors for the treatment of Alzheimer’s disease also started in theGhosh laboratories with the design and synthesis of the first substrate-basedtransition-state inhibitors, determination of the first X-ray crystal structure ofinhibitor-bound BACE1, followed by design and development of potent and selectiveinhibitors with clinical potential. The Ghosh laboratories have also led the design ofcoronavirus 3CLPro and PLpro inhibitors for possible treatment of SARS/Mers andthe design of methyltransferase inhibitors for possible treatment of dengue virusinfection. Our experience in structure-based design in these diverse areas is detailedwithin this book.A significant body of structure-based design work for many approved therapeutic

drugs and preclinical and clinical candidates has been reported by numerousacademic and pharmaceutical scientists. This work has led to the developmentof tools, strategies, and concepts that aid the process of structure-based design. Asubstantial part of this work has been an integral part of the lecture notes of one ofthe authors for teaching fundamentals and concepts of drug discovery and design tostudents at Purdue University. During these research and teaching endeavors, an

jXIII

important need for writing this book was recognized. Although there are manyelegant reports of the structure-based design of therapeutic drugs that span threedecades now, a systematic presentation of the evolution of the field, principles, andapplications had not yet been compiled. The materials of this treatise are organizedwith these objectives in mind. This book covers a critical overview of the history ofstructure-based drug design, an analysis of the underlying principles, and an up-to-date description of the X-ray techniques and methods that led to the structuredetermination of many important biomolecules. The book also highlights thestructure-based design and drug development process covering a broad array ofFDA-approvedmedications. The reader will gain a sense of how a drug interacts withits biological target at the molecular level and how the drug–target interactions canbe optimized in order to increase affinity with desired physicochemical and drug-like properties. Furthermore, the reader will gain knowledge of how other factorssuch as in vivo efficacy and physicochemical and pharmacokinetic parameters needto be optimized in order to convert a lead compound into a clinical drug structure.Chapter 1 provides a historical perspective of drug discovery encompassing

discovery through serendipity and natural product screening to the evolution ofthe field of structure-based design of today’s medicines. Chapters 2–7 outlinegeneral principles for design of enzyme inhibitors covering aspartic acid proteases,serine proteases, cysteine proteases, metalloproteases, threonine proteases, andprotein kinases. These chapters highlight the key protein–ligand interactions andevolution of ligands, scaffolds, and templates to aid molecular design of leadinhibitors and their optimization. These chapters also cover the synthesis of aselection of ligands, templates, and isosteres generally utilized for structure-baseddesign. Chapter 8 reviews recent progress in gaining high-resolution structuralknowledge of biologically relevant proteins and G-protein-coupled receptors(GPCRs), particularly the methods of X-ray crystallography and their applicationin lead discovery. Chapter 9 covers recent developments in the structure-baseddesign of novel ligands for GPCRs, an exciting new dimension for GPCR research.Chapters 10–20 cover an array of recently FDA-approved drugs developed by

utilizing structure-based design strategies. These chapters highlight themechanismof action associated with each drug class, in-depth structural analysis of protein–ligand interactions, structural design, and optimization of ligand binding to proteinstructures. Chapter 10 is devoted to the design of the first ACE inhibitor, captopril,which marks the beginning of structure-based design. Chapters 11–19 cover thedesign and development of HIV-1 protease inhibitors such as saquinavir, indinavir,and darunavir (Chapter 11); kinase inhibitor drugs imatinib, nilotinib, and dasatinib(Chapter 12); NS3/4A serine protease inhibitor drugs boceprevir and telaprevir forthe treatment of HCV (Chapter 13); proteasome inhibitor drugs bortezomib andcarfilzomib for the treatment of relapsed multiple myeloma (Chapter 14); develop-ment of direct thrombin inhibitor dabigatran etexilate (Chapter 15); non-nucleosideHIV reverse-transcriptase inhibitors etravirine and rilpivirine (Chapter 16); devel-opment of renin inhibitor aliskiren (Chapter 17); neuraminidase inhibitors zana-mivir and oseltamivir for the treatment of influenza (Chapter 18); and carbonic

XIVj Preface

anhydrase inhibitor dorzolamide (Chapter 19) for the treatment of glaucoma. Thelast chapter outlines the development of b-secretase inhibitors that are at variousstages of preclinical and clinical development for possible treatment of Alzheimer’sdisease.Overall, this book will greatly enhance the readers’ understanding of structure-

based design and drug discovery, its potential, underlying principles, feasibility, andlimitations. We believe that the book will be an excellent resource for new andpracticing medicinal chemists, biologists, biochemists, and pharmacologists whoare interested in working in the field of molecular design for discovery anddevelopment of human medicine. Structure-based design has a critical role intoday’s drug design and discovery, and it will continue to play a very prominent rolein drug design and medicinal chemistry endeavors throughout the twenty-firstcentury. We hope that the book will be helpful to researchers involved in drugdiscovery and the pursuit of knowledge in structure-based design and related areas.We gratefully acknowledge the National Institutes of Health for financial support

of our research work.We very much enjoyed working with Drs. Frank Weinreich and Lesley Belfit and

the Wiley-VCH editorial team. We sincerely appreciate their help and supportthroughout this project. We would like to thank Dr. Hiroaki Mitsuya, Dr. JordanTang and Dr. Irene Weber for longstanding and productive research collaboration.We would like to express our appreciation and thanks to our research colleaguesfrom Purdue University, Dr. Venkateswararao Kalapala, Dr. Navanth Gavande,Ms. Heather Osswald, Mr. Anindya Sarkar, Ms. Kelsey Cantwell and Mr. AnthonyTomaine for their invaluable help with proofreading and reviewing of this work. Wewish to convey special thanks and appreciation to Dr. Jody Ghosh for her help andsupport and Mrs. JoAnna Hadley for her help with the manuscript preparation andorganization. Finally, we wish to thank our families for their love, support, andinspiration.

Purdue University Arun K. GhoshPurdue University & University of Siena Sandra Gemma

Preface jXV

1From Traditional Medicine to Modern Drugs: HistoricalPerspective of Structure-Based Drug Design

1.1Introduction

The drug design and discovery process of today is a highly interdisciplinaryresearch endeavor [1–3]. Advances in molecular biology, synthetic chemistry, andpharmacology, as well as technological breakthroughs in X-ray crystallography andcomputational methods have brought dramatic changes to medicinal chemistrypractices during the late twentieth century. Drug design efforts based upon thethree-dimensional structure of a target enzyme have become the hallmark of mod-ern molecular design strategies. This structure-based design approach has revolu-tionized the practice of medicinal chemistry and recast the preclinical drugdiscovery process. Many of the FDA-approved drugs have evolved through struc-ture-based design strategies. By 2012, as many as 35 newly approved drugs haveemanated from structure-based design. The post-genomic era holds huge promisefor the advancement of structure-based design of drugs for new therapies. Humangenome sequencing has now revealed that there are an estimated 20,000–25,000protein-coding human genes, and each gene can code for one protein. These pro-teins are responsible for carrying out all the cellular functions in the human body.These proteins can also be involved in disease pathologies, providing uniqueopportunities and challenges for structure-based design of new drugs. It may beappropriate to review briefly how the first half of the twentieth century was shapedand enriched by a number of seminal discoveries and the advent of new technolo-gies, all of which left an important imprint on today’s drug discovery and medici-nal chemistry. A number of previous reviews have provided some insight [4,5].

1.2Drug Discovery During 1928–1980

The history of medicinal chemistry is marked by examples in which the discoveryof novel drugs relied upon serendipity and clinical observations. It is interesting toconsider the role of chance in unexpected and accidental scientific discoveries.This serendipity is not simply luck. Rather, it is a process of finding significance

1

Structure-based Design of Drugs and Other Bioactive Molecules: Tools and Strategies, First Edition.Arun K. Ghosh and Sandra Gemma.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

and value in the lucky coincidence. As Pasteur observed, “Chance favors the pre-pared mind.” Without engaging creative thinking and analysis, accidents do notlead to discoveries. The discovery of penicillin is a famous example of serendipity[6,7]. Alexander Fleming departed for a vacation in the summer of 1928. He left abacterial culture of Staphylococcus aureus on his laboratory bench. When hereturned a month later, he found that the culture was contaminated by a patch ofblue-green mold that caused the lysis of bacteria. Fleming later demonstrated thatthe mold, Penicillium notatum, produced an active ingredient that he called penicil-lin. The discovery of penicillin was particularly fortunate since the penicillin thatlanded on Fleming’s bacterial culture was not ordinary Penicillium! If it were, itwould not have produced penicillin in high enough concentrations to cause thelysis of bacteria.The discovery of penicillin was not mere luck. Much more subsequent investi-

gation was required before it could be used as an antibiotic. More than a decadelater in the 1940s, Howard Florey and Ernest Chain, with their Oxford team,unveiled its therapeutic potential. During this time, fermentation methods weredeveloped that allowed the effective application of penicillins (Figure 1.1) for thetreatment of bacterial infections in humans. Bactericidal penicillin rapidlyreplaced the bacteriostatic sulfonamide drugs used until then for the treatment ofsome bacterial infections.The discovery of bacteriostatic sulfonamides has its own interesting story of ser-

endipity and intuition [8]. The dye industry was advanced and promoted chemicalmanufacturing to develop new dyes. German chemists working with azo dyesobserved that certain dyes could preferentially stick to and stain bacterial colonies.Could this serve as a way to target bacteria? In 1935, the German biochemistGerhard Domagk, assisted by a group of chemists, synthesized and tested hun-dreds of dyes and finally discovered, by a trial-and-error approach, the potent

N

SHN

OO

H

CO2HMe

Me

Penicillin G (1)

N

SHN

OO

OH

CO2HMe

Me

Penicillin V (2)

N

SHN

OO

H

CO2H

Me

Me

Methicillin (4)

N

SHN

OO

H

CO2H

Me

Me

Amoxicillin (3)

NH2

HOMeO

OMe

Semisynthetic penicillin

Figure 1.1 Structures of penicillins G and V and semisynthetic penicillins.

2 1 From Traditional Medicine to Modern Drugs: Historical Perspective of Structure-Based Drug Design

antibacterial activity of Prontosil rubrum (Figure 1.2). Subsequent studies revealedthat the active moiety of the compound was the 4-aminobenzenesulfonamide moi-ety. Introduction of substituents at both the p-aniline and the sulfonamide groupsled to the development of new sulfanilide derivatives with broad-spectrum activity,improved pharmacokinetic properties, and lowered therapeutic side effects. Thesynthesis of new derivatives became less important due to the discovery and intro-duction of penicillin and subsequently discovered antibiotics.Research on sulfonamide derivatives, however, continued. Close observation of

side effects led to the development of new uses and expansion of this class ofcompounds. The clinical observation that sulfa drugs induced hypoglycemia wasfollowed by studies aimed at maximizing this side effect and dissociating it fromthe bacteriostatic activity. This led to the advent of oral hypoglycemia drugs for thetreatment of diabetes. In 1940, Mann and Keilin discovered the inhibitory activityof sulfanilamide against carbonic anhydrase. This key discovery paved the way forthe subsequent development of diuretic sulfonamides [9].The discovery of the antidepressant agent iproniazid is also due to the clinical

observation of a “side effect” [10]. Both isoniazid (Figure 1.3) and its isopropyl-substituted derivative iproniazid were originally developed as tuberculostaticdrugs. However, it was observed that in contrast to isoniazid, patients treated with

N N NH2

H2N

SH2N

OO

Sulfamethoxazole (6)Prontosil (5) H2N

NH

SOO

ONMe

Figure 1.2 Structures of prontosil and its derivative.

NN

O

NH2

Isoniazid (7)

NN

O

N

Iproniazid (8)

HH

H

N

NH2N NH2

N

O+

_

Minoxidil (9)

SN

N

OO

O

N

HNN

N

O

Sildenafil (10, Viagra)

Figure 1.3 Structures of isoniazids, minoxidil, and sildenafil.

1.2 Drug Discovery During 1928–1980 3

iproniazid experienced elevation of mood. Subsequent studies clarified that theantidepressant activities of iproniazid were due to the inhibition of the centrallyactive enzyme monoamine oxidase (MAO). Iproniazid was approved in 1958 forthe treatment of depression. There are also more recent examples of clinicalobservations leading to the discovery of new drugs. Sildenafil or Viagra, a drugused for the treatment of erectile dysfunction, was originally developed for thetreatment of angina [11,12]. Minoxidil [13,14], originally developed as an antihy-pertensive agent, was later approved for the treatment of hair loss.Serendipity also played a role in the discovery of Librium, the first antianxiety

benzodiazepine, but it did not happen by accident [15,16]. In 1954, Dr. Leo Stern-bach was actively involved in the development of new tranquilizers in the NewJersey laboratories of Hoffmann-La Roche. He decided to explore the chemistry ofbenzheptoxdiazines, a class of compounds he had synthesized 20 years ago insearch of new dyes but whose biological activity was unknown. His research groupsynthesized 40 new derivatives and determined that they were six-membered ringcompounds such as 11 and 12 (Figure 1.4) rather than seven-membered ring com-pounds 13 and 14, as was originally thought. Pharmacological testing showedthese compounds were inactive. As their project on tranquilizers was coming toan end, during their laboratory cleanup work, they realized two of their earliercrystalline derivatives had never been submitted for pharmacological evalua-tion. They decided to send them for biological testing. One of the compoundsthat resulted from the reaction of a quinazoline derivative 15 with methyl-amine showed potent sedative and hypnotic effects. This compound was supe-rior to phenobarbital. Subsequent structural work on the compound led to itscharacterization as benzodiazepine derivative chlordiazepoxide (17, Figure 1.5)known as Librium. This resulted from a rearrangement of the original benzo-fused six-membered heterocycle to afford a benzo-fused seven-membered

NO

NCH2Cl

NO

NCH2NR2

4131

2111

N

N CH2Cl

O-+ N

N CH2NR2

O-+X X

X X

Figure 1.4 Structures of benzheptoxdiazines and quinazoline-3-oxides.


heterocycle 17. This discovery led to the subsequent development of a host ofbenzodiazepines, including diazepam (18, Valium).Natural products have long served as a key source for the development of

numerous new drugs. Biological screening of natural products has proven to beextremely useful. The anticancer agent Taxol was discovered in the 1970s as aresult of a project implemented in 1960 by the American National CancerInstitute consisting of the biological screening of extracts arising from various nat-ural sources [17]. One of the extracts showed promising anticancer activityagainst a wide range of tumors in mice. After the initial discovery, the active com-pound was isolated from the Taxus brevifolia and in 1972 its chemical structure(19, Figure 1.6) was fully characterized [18]. Another important anticancer treat-ment resulting from the screening of natural products was camptothecin (20),which was isolated from Camptotheca acuminata [19]. A number of camptothecinderivatives have been approved and are used in cancer chemotherapy.The antimalarial drug artemisinin (21, Figure 1.7) [20] is also the result of a

screening campaign. After a heavy outbreak of malaria in the 1950s, the spread of

6151

N

N CH2Cl

O-+ N

HN CH2Cl

O-+

NHCH3

CH3NH2

Cl Cl

N

NNHCH3

O-+

Chlordiazepoxide (Librium)

17

N

NO

Diazepam (Valium)

18

Cl Cl

CH3

Figure 1.5 Structures of benzodiazepine derivatives, Librium and Valium.

Me

O

ONH

OH

O

PhAcO O

HO OPh

O

H

OH

OO

O

Taxol (19)

NN

O

O

OHO

Camptothecin (20)

Figure 1.6 Structures of Taxol and camptothecin.

1.2 Drug Discovery During 1928–1980 5

drug-resistant malaria strains raised huge treatment concerns. A Chinese nationalproject implemented a campaign aimed at discovering, isolating, and characteriz-ing natural products as potential antimalarial leads. Phytochemist Tu Youyou andhis colleagues found that the extract of the traditional Chinese herbal remedy Arte-misia annua was effective in a mouse model against malaria. Later, the sesqui-terpene lactone artemisinin was characterized as the active ingredient and itssemisynthetic derivatives, such as artemether (22), are used for the treatment ofmultidrug-resistant malaria.During the 1970s, a number of other important natural products were also

introduced as new drugs. Natural products compactin and mevinolin were iso-lated from Penicillium citrinum and Aspergillus terreus, respectively. Both these nat-ural products showed very potent inhibitory activity of HMG-CoA reductase,responsible for biosynthesis of cholesterol in human liver. Mevinolin (lovastatin)and derivatives of mevinolin (Zocor) were introduced for the treatment of athero-sclerosis by lowering cholesterol levels and inhibiting the enzyme HMG-CoAreductase [21,22].

1.3The Beginning of Structure-Based Drug Design

During the late 1970s, rational design evolved into a strategy for the discovery anddevelopment of new drugs. With the knowledge of the three-dimensional struc-ture of drug targets and their site of interaction with a prototype drug molecule or

Artemisinin (21)

O

O

Me

R

HMe

O

OHO

Compactin (R = H, 23)Mevinolin (R = Me, 24)

O

O

Me

HMe

O

OHO

Zocor (25)

Artemether (22)

OMe

H

H

Me

OO H

MeO

MeO

Me

H

H

Me

OO H

MeO

Me Me

O O

Figure 1.7 Structures of artemisinin and mevinolin and their derivatives.


ligand, logical molecular design based upon target–ligand interactions began totake shape. Early during this practice, X-ray structural information was limited.The X-ray structural data of related enzymes were used to model the targetenzyme. Advances in technology and molecular biology greatly enhanced thepromise of structure-based design. During the 1980s, rapid progress in proteinexpression, purification, and protein crystallography provided detailed structuralknowledge of disease-relevant target proteins. The progress of chemical synthesiswas timely as well. New and efficient reagents, protecting groups, catalytic trans-formations, and multistep chemical synthetic strategies provided the power of cre-ative design capabilities for structure-based design. Drug design throughstructure-based approaches rapidly revolutionized the field of medicinal chemistryand changed the approach toward the identification and optimization ofnovel drugs.In structure-based design, the shape and the electronic features for the binding

site of a specific target protein are generated early on. Also, the crystal structuresof protein and ligand complexes are determined to obtain information on inter-molecular interactions within the protein active site. This molecular insight oftenprovides the bioactive conformation of the ligand for molecular design [23,24].Starting from this key information, structure-based design strategies allow the opti-mization of ligand–protein interactions to improve potency, affinity, and selectivity,while at the same time preserving and optimizing selected drug-like properties.An early example of rational design utilizing structural information of an

enzyme–inhibitor interaction can be traced to the discovery of the angiotensin-converting enzyme (ACE) inhibitor, captopril [25]. Although the X-ray structure ofthe actual ACE was unknown at that time, the structure of a similar enzyme, car-boxypeptidase A, had already been determined. Both carboxypeptidase A and ACEhave a number of common features, including the presence of a zinc ion in theprotease active site. Based on this structural knowledge and a number of peptidiclead ACE inhibitors from snake venoms, investigators at Bristol-Myers Squibb(BMS) modeled the active site of ACE and rationally designed captopril, the firstFDA-approved ACE inhibitor for the treatment of hypertension in 1981.The clinical success of ACE inhibitors fueled a great deal of interest in the devel-

opment of inhibitor drugs against renin, an aspartic acid protease [26]. Renin isresponsible for the regulation of blood pressure, and therapeutic inhibition ofrenin was considered a promising strategy for the development of novel therapiesfor the treatment of hypertension. It was presumed that a successful renin inhibi-tor would possess fewer side effects than ACE inhibitors due to the exquisiteselectivity of renin for a single physiological substrate. Key information in thedevelopment of renin inhibitors was the understanding of the substrate cleavagemechanism and the characterization of the endogenous peptide binding site[27–29]. The X-ray structure of renin was not known; however, a model structureof renin was created based upon the X-ray structure of related aspartic acid prote-ases such as Rhizopus chinensis carboxyl proteinase, endothiapepsin, and otheraspartic acid proteases. The X-ray structural studies with peptide inhibitorsalso provided the details of molecular interactions. Based upon this knowledge,

1.3 The Beginning of Structure-Based Drug Design 7

substrate-based inhibitors were developed to mimic the N-terminal portion ofangiotensinogen, in which the scissile peptide bond was modified based on thetransition-state mimetic concept. Subsequent structure-based optimization of theearly renin inhibitors led to the successful modification of inhibitors withimproved drug-like properties, resulting in the discovery of aliskiren in 2007, thefirst FDA-approved renin inhibitor for the treatment of hypertension [30,31].In the late 1980s, the power of structure-based design was unveiled in the con-

text of the structure-based design and synthesis of HIV protease inhibitors for thetreatment of HIV infection and AIDS. The discovery of the key role of HIV prote-ase in the viral life cycle and the documentation that inhibition of the viral HIV-1protease resulted in noninfectious virions brought hope and urgency to the thera-peutic inhibition of HIV protease [32,33]. Knowledge and expertise gained in thedesign of renin inhibitors, and the determination of the X-ray structure of HIV-1protease at the early stages of inhibitor design, led to rapid progress in structure-based design capabilities [34]. Within a decade, hundreds of X-ray structures ofHIV protease, inhibitor-bound HIV-1 protease, and mutant proteases aided in thedesign of conceptually novel inhibitors. In this context, numerous tools and con-cepts have emerged for the design of novel inhibitors and for addressing issues ofdrug resistance [35,36]. The first HIV-1 protease inhibitor, saquinavir, receivedFDA approval in 1996. Structure-based drug discovery efforts expanded rapidly inmany other areas. As can be seen in Table 1.1, structure-based approaches contrib-uted to the approval of 34 new drugs for the treatment of hypertension, HIV/AIDSchemotherapy, various cancers, and other human diseases through 2012 [37–70].Structure-based drug design approaches have been widely utilized in the design

and development of inhibitors of protein kinases for the treatment of a range ofhuman carcinomas [71–73]. Imatinib was the first example of an anticancer drugspecifically directed at inhibiting a drug target Bcr-Abl fusion protein involved inthe pathogenesis of chronic myelogenous leukemia. Detailed structural studies ofimatinib and Abl kinase complexes provided much molecular insight into imati-nib resistance. The lead compound for imatinib was discovered through high-throughput screening (HTS). Lead optimization to improve potency, selectivity,and pharmacokinetic properties led to the discovery of imatinib. The structuralstudies paved the way for development of other kinase inhibitors. Once proteinkinases were recognized as important drug targets for the development of anti-cancer therapies, much effort was directed toward obtaining structural insightinto the binding sites of various protein kinases. X-ray crystallography was centralto the understanding of binding mode of various classes of inhibitors. This molec-ular insight was extensively utilized in the structure-based design of variouskinase inhibitor drugs.The full potential of structure-based design has yet to be realized. Progress in

structure-based design of ligands for G-protein-coupled receptors (GPCRs) hasbeen growing steadily [74–77]. The recent evolution of techniques for X-ray crystal-lography resulted in the determination of novel GPCR structures at a rapid pace.Many high-resolution X-ray structures of ligand-bound GPCRs provided importantunderstanding of the molecular determinants of ligand binding and receptor


Table 1.1 Drugs derived from structure-based design approaches.

Captopril [37]Approved in 1981

ACE inhibitor (antihypertensive)

S SS

O

O

O O

NHH3C

H3C

Dorzolamide [38]Approved in 1995

Carbonic anhydrase inhibitor (antiglaucoma)

OHN

HN

O

H

H

NH

OPh

NH

O

N

OH2N

Saquinavir [39]Approved in 1995

HIV protease inhibitor (anti-HIV/AIDS)

HN

NH

OH OHN N

O

CH3

O

O

SN

Ritonavir [40]Approved in 1996


NN

ONH

N

OHPh

O

HN

Indinavir [41]Approved in 1996


NS S

S

O

O

O O

NHH3C

H3CO

Brinzolamide [42]Approved in 1999

Carbonic anhydrase inhibitor (antiglaucoma)

N

O NH

HN

OH

O

H

H

CH3

HO

SPh

Nelfinavir [43]Approved in 1999


OHN

SO O

HNO

OO

NH2

Amprenavir [44]Approved in 1999


NH2

NH2

N

OO

OH

HS

H3C

Ph

PhS

N

OH

Ph

Lopinavir [45]Approved in 1999


HN

NH

OO

CH3

CH3 OH ON NH

O

OO

OH

HNHN

ONH2

H

HO

HO

OH

NHZanamivir [46]Approved in 1999

Neuraminidase inhibitor (anti-influenza)

Ph

Ph

(continued)


Table 1.1 (Continued)

O

O CH3

NH2

O

HN

OMe

Oseltamivir [47]Approved in 1999

Neuraminidase inhibitor (anti-influenza)

N

N

MeO

ONO

Gefitinib [49]Approved in 2003

EGFR inhibitor (anticancer)

OHN

NH

HN

Atazanavir [50]Approved in 2003


OHN OCH3

O

N

ONH

H3CO

O

ON

SO O

HNO

OO

NH2POHO OH

Fosamprenavir [51]Approved in 2003


N

N

HN

O

O

MeO

Erlotinib [52]Approved in 2004

EGFR inhibitor (anticancer)

Ximelagatran [53]Approved in 2004

Thrombin inhibitor (anticoagulant)

HN

F

Cl

Ph

PhMeO

NHN

OOHN

O

O

H2N

NHO

N

O

HN

HN

OCl

CF3

HN

OMe

Sorafenib [54]Approved in 2005

VEGFR inhibitor (anticancer)

Udenafil [56]Approved in 2005

PDE-5 inhibitor (erectile dysfunction)

N

NH

NNMeO

S

O Me

NH

O O

NMe

HN

SO O

NCF3

O

MeOH

O

Ph

Me Tipranavir [55]Approved in 2005


Me

HNN

N

N Imatinib [48]Approved in 2001

Chronic myelogenous leukemia

HN

O

NN

Me

Me


OHN

SO O

HNO

O

NH2

Darunavir [58]Approved in 2006


HN

O

Vorinostat [59]Approved in 2006

Histone deacetylase inhibitor (anticancer)

Dasatinib [60]Approved in 2006

Tyrosine kinase inhibitor (antileukemia)

Nilotinib [61]Approved in 2006

BCR-ABL kinase inhibitor (antileukemia)

Ph

O

O

HH

O NHOH

S

NN

NN

Me

N N

HOH

O

HN

Me

Cl

NN

NHN

NHO

NN

MeMe F3C O

MeOOMe

NH2

OHNH

O

Aliskiren [62]Approved in 2007

Renin inhibitor (antihypertensive)

Lapatinib [63]Approved in 2007

Tyrosine kinase inhibitor (anticancer)

Rivaroxaban [64]Approved in 2008

Factor Xa inhibitor (anticoagulant)

Dabigatran [65]Approved in 2008

Thrombin inhibitor (anticoagulant)

N

N

HN

OCl

F

O

N

SMe OO

H

NO

HN

O

SCl

O

NO

O

N

NMe

N

O

HNN

NH2

O

OMe

N

CO2Et

Etravirine [66]Approved in 2008

NNRT inhibitor (anti-HIV/AIDS)

N N

NH

H2NBr

O

NC

Me

Me CN

CONH2

NH

OF N

HMe

MeNH

O

N

Sunitinib [57]Approved in 2006

Multikinase inhibitor (anticancer)

(continued)



activation, a critical step for designing agonists or antagonists. Recent studies havebeen aimed at understanding if this structural information can be effectivelyemployed for the structure-based design of novel, potent, and selective GPCR lig-ands. Structure-based design of novel ligands for GPCRs has become an excitingarea of drug development.

1.4Conclusions

Serendipity and natural product screening may continue to have an important rolein drug design, but it is clear that structure-based design strategies are making asignificant impact on the drug discovery process. The success of this approach isalready evident in 34 FDA-approved drugs on the market through 2012. Numer-ous other drugs developed using this approach are undergoing clinical trials. Nodoubt, the success of structure-based design strategies rests heavily on the struc-tural knowledge of disease-relevant target enzymes and their families. The notablesuccess of drug development in the areas of HIV-1 protease, protein kinase, NS3/4A serine protease, and b-secretase greatly empowered the application of thesestrategies in other areas of drug development. With continual advances in technol-ogy and increasing knowledge of disease mechanisms and protein structures,structure-based design strategies will find wide applications in drug discoveryendeavors.


Vemurafenib [68]Approved in 2011

B-Raf kinase inhibitor (anticancer)

N NH

O

F

F

HN

Cl

SO

O

Ponatinib [70]Approved in 2012

Bcr-Abl inhibitor (antileukemia)

Crizotinib [69]Approved in 2011

c-MET and ALK inhibitor (anticancer)

HN

O

Me

N

N

N

F3CNH2N

NNO

Me

Cl

ClF

NH

Pazopanib [67]Approved in 2009

Multikinase inhibitor (anticancer)

N

N NMe

NN

Me

MeNH

MeSO2NH2

N

NMe


In the post-genomic era, many new and important drug targets are emerging,and structure-based design is expected to offer new opportunities for drug devel-opment. The drug discovery efforts in the area of GPCRs have witnessed signifi-cant breakthroughs with the availability of high-resolution structures of drug-relevant GPCRs. Structure-based design efforts have greatly benefited from rapidprogress in lead generation and validation strategies. Fragment-based screening isproviding early structural knowledge of small-molecule leads. Also, virtual screen-ing has witnessed major improvements with the sophistication of computationalinfrastructure, data sets, and analysis tools. Virtual screening is very important astraditional HTS is often expensive and time consuming and selected compoundlibraries may not have enough diversity.Despite the successful trends of structure-based design strategies, it is impor-

tant to note that the lead optimization and drug design process are driven bymedicinal chemistry efforts. It is this ingenuity and innovation of experiencedmedicinal chemists that will fuel the drug discovery of the future. The ever-increasing knowledge of molecular and structural biology will likely reveal newexciting drug targets. However, for innovative molecular design and synthesis, therole of chemical synthesis will be vital for tomorrow’s new treatments. Structure-based design has not yet reached its full potential and these strategies willundoubtedly play a major role in the drug discovery endeavors in the rest of thetwenty-first century.

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53 Gustafsson, D., Nystrom, J.E., Carlsson, S.,Bredberg, U., Eriksson, U., Gyzander, E.,Elg, M., Antonsson, T., Hoffmann, K.J.,Ungell, A.L., Sorensen, H., Nagard, S.,Abrahamsson, A., and Bylund, R. (2001)The direct thrombin inhibitor melagatranand its oral prodrug H 376/95: intestinalabsorption properties, biochemical andpharmacodynamic effects. Thromb. Res.,101, 171–181.

54 Wilhelm, S.M., Carter, C., Tang, L.Y.,Wilkie, D., McNabola, A., Rong, H., Chen,C., Zhang, X.M., Vincent, P., McHugh, M.,Cao, Y.C., Shujath, J., Gawlak, S., Eveleigh,D., Rowley, B., Liu, L., Adnane, L., Lynch,M., Auclair, D., Taylor, I., Gedrich, R.,Voznesensky, A., Riedl, B., Post, L.E.,Bollag, G., and Trail, P.A. (2004) BAY 43-9006 exhibits broad spectrum oral

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55 Turner, S.R., Strohbach, J.W., Tommasi,R.A., Aristoff, P.A., Johnson, P.D.,Skulnick, H.I., Dolak, L.A., Seest, E.P.,Tomich, P.K., Bohanan, M.J., Horng, M.M.,Lynn, J.C., Chong, K.T., Hinshaw, R.R.,Watenpaugh, K.D., Janakiraman, M.N.,and Thaisrivongs, S. (1998) Tipranavir(PNU-140690): a potent, orally bioavailablenonpeptidic HIV protease inhibitor of the5,6-dihydro-4-hydroxy-2-pyronesulfonamide class. J. Med. Chem., 41,3467–3476.

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57 Mendel, D.B., Laird, A.D., Xin, X.H., Louie,S.G., Christensen, J.G., Li, G.M., Schreck,R.E., Abrams, T.J., Ngai, T.J., Lee, L.B.,Murray, L.J., Carver, J., Chan, E., Moss,K.G., Haznedar, J.O., Sukbuntherng, J.,Blake, R.A., Sun, L., Tang, C., Miller, T.,Shirazian, S., McMahon, G., andCherrington, J.M. (2003) In vivo antitumoractivity of SU11248, a novel tyrosine kinaseinhibitor targeting vascular endothelialgrowth factor and platelet-derived growthfactor receptors: determination of apharmacokinetic/pharmacodynamicrelationship. Clin. Cancer Res., 9, 327–337.

58 Ghosh, A.K., Dawson, Z.L., and Mitsuya,H. (2007) Darunavir, a conceptually newHIV-1 protease inhibitor for the treatmentof drug-resistant HIV. Bioorg. Med. Chem.,15, 7576–7580.

59 Kelly, W.K., O’Connor, O.A., Krug, L.M.,Chiao, J.H., Heaney, M., Curley, T.,MacGregore-Cortelli, B., Tong, W., Secrist,J.P., Schwartz, L., Richardson, S., Chu, E.,Olgac, S., Marks, P.A., Scher, H., andRichon, V.M. (2005) Phase I study of anoral histone deacetylase inhibitor,suberoylanilide hydroxamic acid, inpatients with advanced cancer. J. Clin.Oncol., 23, 3923–3931.

60 Lombardo, L.J., Lee, F.Y., Chen, P., Norris,D., Barrish, J.C., Behnia, K., Castaneda, S.,


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61 Weisberg, E., Manley, P.W., Breitenstein,W., Bruggen, J., Cowan-Jacob, S.W., Ray, A.,Huntly, B., Fabbro, D., Fendrich, G., Hall-Meyers, E., Kung, A.L., Mestan, J., Daley,G.Q., Callahan, L., Catley, L., Cavazza, C.,Mohammed, A., Neuberg, D., Wright, R.D.,Gilliland, D.G., and Griffin, J.D. (2005)Characterization of AMN107, a selectiveinhibitor of native and mutant Bcr-Abl.Cancer Cell, 7, 129–141.

62 Rahuel, J., Rasetti, V., Maibaum, J., Rueger,H., Goschke, R., Cohen, N.C., Stutz, S.,Cumin, F., Fuhrer, W., Wood, J.M., andGrutter, M.G. (2000) Structure-based drugdesign: the discovery of novel nonpeptideorally active inhibitors of human renin.Chem. Biol., 7, 493–504.

63 Rusnak, D.W., Lackey, K., Affleck, K.,Wood, E.R., Alligood, K.J., Rhodes, N.,Keith, B.R., Murray, D.M., Knight, W.B.,Mullin, R.J., and Gilmer, T.M. (2001) Theeffects of the novel, reversible epidermalgrowth factor receptor/ErbB-2 tyrosinekinase inhibitor, GW2016, on the growthof human normal and tumor-derived celllines in vitro and in vivo. Mol. Cancer Ther.,1, 85–94.

64 Roehrig, S., Straub, A., Pohlmann, J.,Lampe, T., Pernerstorfer, J., Schlemmer, K.H., Reinemer, P., and Perzborn, E. (2005)Discovery of the novel antithrombotic agent5-chloro-N-({(5S)-2-oxo-3[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide (BAY59-7939): an oral, direct factor Xa inhibitor.J. Med. Chem., 48, 5900–5908.

65 Sorbera, L.A., Bozzo, J., and Castaner,J. (2005) Dabigatran/dabigatran etexilate:prevention of DVT, prevention of ischemic

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66 Das, K., Clark, A.D., Lewi, P.J., Heeres, J.,deJonge, M.R., Koymans, L.M.H., Vinkers,H.M., Daeyaert, F., Ludovici, D.W., Kukla,M.J., De Corte, B., Kavash, R.W., Ho,C.Y., Ye, H., Lichtenstein, M.A., Andries,K., Pauwels, R., deBethune, M.P., Boyer, P.L., Clark, P., Hughes, S.H., Janssen, P.A.J.,and Arnold, E. (2004) Roles ofconformational and positional adaptabilityin structure-based design of TMC125-R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitorsthat are highly potent and effective againstwild-type and drug-resistant HIV-1 variants.J. Med. Chem., 47, 2550–2560.

67 Sleijfer, S., Ray-Coquard, I., Papai, Z., LeCesne, A., Scurr, M., Schoffski, P., Collin,F., Pandite, L., Marreaud, S., De Brauwer,A., vanGlabbeke, M., Verweij, J., and Blay,J.Y. (2009) Pazopanib, a multikinaseangiogenesis inhibitor, in patients withrelapsed or refractory advanced soft tissuesarcoma: a phase II study from theEuropean Organisation for Research andTreatment of Cancer – Soft Tissue andBone Sarcoma Group (EORTC study62043). J. Clin. Oncol., 27, 3126–3132.

68 Tsai, J., Lee, J.T., Wang, W., Zhang, J.,Cho, H., Mamo, S., Bremer, R., Gillette, S.,Kong, J., Haass, N.K., Sproesser, K., Li,L., Smalley, K.S., Fong, D., Zhu, Y.L.,Marimuthu, A., Nguyen, H., Lam, B., Liu,J., Cheung, I., Rice, J., Suzuki, Y., Luu, C.,Settachatgul, C., Shellooe, R., Cantwell, J.,Kim, S.H., Schlessinger, J., Zhang, K.Y.,West, B.L., Powell, B., Habets, G., Zhang,C., Ibrahim, P.N., Hirth, P., Artis, D.R.,Herlyn, M., and Bollag, G. (2008) Discoveryof a selective inhibitor of oncogenic B-Rafkinase with potent antimelanoma activity.Proc. Natl. Acad. Sci. USA, 105, 3041–3046.

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Part OneConcepts, Tools, Ligands, and Scaffolds for Structure-BasedDesign of Inhibitors

19


2Design of Inhibitors of Aspartic Acid Proteases

2.1Introduction

Over the last two decades, structure-based design has transformed the field ofmedicinal chemistry with the discovery and development of many critical first-in-class medicines. Structure-based design has also led to the evolution of numerousconcepts and tools for drug discovery and development in broad areas of medici-nal chemistry. The sophistication of technology and advancement of X-ray crystal-lographic techniques, particularly the availability of high-intensity synchrotronsources for X-rays, have now provided high-resolution X-ray structures of numer-ous protein–ligand complexes [1]. As a result, molecular insight into the specificligand–binding site interactions of early lead structures with the drug target canbe obtained for lead optimization. First-hand three-dimensional views of theseinteractions, along with the analysis of biological results, very often assist medici-nal chemists with charting the next course of action in molecular design and opti-mization of lead structures. The drug discovery process, by and large, requiresmolecular modifications to improve a molecule’s binding affinity and selectivity,or to enhance physicochemical parameters and pharmacokinetic properties.Structure-based design strategies can significantly reduce extensive molecular iter-ations of the traditional medicinal chemistry approach. As a consequence, thestructure-based design approach can greatly reduce cost and speed up the processof identification of drug candidates for clinical development. As shown in Figure2.1, the determination of an X-ray structure of a substrate-based inhibitor OM99-2complexed with b-secretase was achieved in 2000 [2]. This initial protein–ligandX-ray structure provided critical molecular insight and important drug designtemplates which set the stage for the structure-based design of potent, selectiveinhibitors [3]. Prototype small-molecule and exceedingly potent inhibitors such asGRL-8234 (1) and exceptionally selective b-secretase inhibitor GRL-1439 (2)evolved for possible treatment of Alzheimer’s disease [4,5]. Subsequently, manyclasses of inhibitors, including clinical inhibitors that can cross the blood–brainbarrier and reduce brain amyloid-b peptide, emerged from the structure-baseddesign approach [6–9].


21

Structure-based design provides incredible opportunities for innovations andthis aspect is important for intellectual property issues. The knowledge oforganic synthesis is critically important for the structure-based design pro-cess. Successful molecular design also requires judicial choice of which mole-cules to pursue for synthesis, weighing synthetic feasibility out of manypossible choices. Very often, molecular modification is specific for one situa-tion and may not be applied to another medicinal chemistry project. How-ever, the fundamentals of molecular design are widely applicable to addressissues in drug design and development. In this chapter, we highlight variousdesign concepts and practical synthetic feasibility of ligands, scaffolds, andbioisosteres utilized in successful structure-based design of drugs and clinicalagents.

2.2Design of Peptidomimetic Inhibitors of Aspartic Acid Proteases

For the design of aspartic acid protease inhibitors, a nonhydrolyzable dipeptideisostere can be used as the replacement of the scissile bond [10,11]. Suchdipeptide isosteres mimic the tetrahedral transition state of proteolytic cleavage(Figure 2.2). It was first proposed by Pauling that the interaction of enzyme andsubstrate is the strongest at the transition state. Therefore, an inhibitor that mim-ics the transition state would be a potent competitive inhibitor [12]. Over the years,a variety of isosteres have been developed for the design of numerous potent andselective inhibitors of aspartic acid proteases [13]. The basic design involvedreplacement of the P1 � P1

0 peptide bond of substrate analogs with nonhydrolyz-able dipeptide isosteres. The basic core of these dipeptide isosteres include statine

Figure 2.1 Structure-based design of BACE inhibitors GRL-8234 and GRL-1439.

22 2 Design of Inhibitors of Aspartic Acid Proteases

(7, Figure 2.3), hydroxyethylene (8), reduced amide (9), hydroxyethylamine (11),hydroxyethylsulfonamide (12), and phosphinate isosteres (13). Successful incorpo-ration of these structural scaffolds often improves the binding affinity, activity pro-file, metabolic stability, and oral absorption properties [14].The design of inhibitors by inserting these basic cores has emerged as an impor-

tant strategy for drug discovery and development. There are many FDA-approved

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond

HN

H2N

O P1

O P1'

O

HN

NH

P2'

OP2

OH +

HN

O P1'

NH

P1

O O H O O_

OHH

HN

OH P1'

NH

P1

O O H O O

‡

_

OH

Tetrahedraltransition state

AspAspAsp Asp

3 54

Figure 2.2 Catalytic mechanism of peptide hydrolysis by aspartic acid protease.

HN

P1

OH O

7 (Statine)

HN

P1

OH P1'

O

HN

8 (Hydroxyethylene)HN

NH

P1

P1'

O

HN

9 (Reduced amide)

6 (Tetrahedral transition state)

HN

NH

P1

P1'

O

HN

OHHO

HN N

P1

11 (Hydroxyethylamine)

OH

OHN N

S

P1

12 (Hydroxyethylsulfonamide)

OH P1'

OO

HN P

P1

O P1'

O

HN

13 (Phosphinate)

HN

P1

P1'

O

HN

10 (trans-Olefin)

HN

P1

O P1'

O

HN

14 (Ketodifluoroethylene)

FF

OH

Figure 2.3 Basic dipeptide isosteres for the design of aspartic acid protease inhibitors.

2.2 Design of Peptidomimetic Inhibitors of Aspartic Acid Proteases 23

inhibitor drugs that contain dipeptide isosteres. Particularly, hydroxyethylene andhydroxyethylamine isosteres have been extensively employed in the design ofpotent and selective clinical inhibitors of renin for the treatment of hypertension,HIV-1 protease for the treatment of HIV/AIDS, b-secretase for the possible treat-ment of Alzheimer’s disease, and plasmepsins for possible treatment of malaria[15]. These basic core units themselves do not show any significant inhibitoryactivity; however, incorporation of appropriate P2 and P2

0 ligands specific for S2and S20 subsites generally results in potent and selective inhibitors against the tar-get enzyme. Since the dipeptide isostere core mimics the transition state of sub-strate cleavage, the choice of both P1 and P1

0 substituents would be based uponS1 � S1

0 subsite specificity [16,17].

2.3Design of Statine-Based Inhibitors

Statine was formally named by Tang and coworkers in 1976 [18]. Statine (16) is anunusual amino acid, which contains a 4-amino-3-hydroxy-6-methylheptanoic acid.This was first recognized by Umezawa et al. during their discovery of pepstatinfrom the culture filtrates of various species of Actinomyces [19]. Pepstatin (15) is apentapeptide, which contains two units of statine. It was shown to be a potent inhib-itor of renin and other aspartic acid proteases [20]. Tang and coworkers demon-strated that statine is the major structural element for pepstatin’s aspartic acidprotease inhibitory activity [18]. It mimics the catalytic tetrahedral transition state ofpeptide cleavage. Statine is also prevalent in cytotoxic depsipeptides [21,22]. It is onecarbon shorter than typical dipeptide hydroxyethylene isosteres and lacks the P1

0

side chain. The design of competitive aspartic acid protease inhibitors of porcinepepsin was initially carried out by the incorporation of statine in the small peptides17 and 18, as shown in Figure 2.4 [21]. Potent inhibitors of pig renin were alsodesigned by incorporation of statine in the pig renin substrate [23]. Since then, a

NH

HN

NH

O

OH O HN

OH O

OH

OHN

O O

15 (Pepstatin)

H2N

OH O

OH

16 (Statine)

NH

HN

OH O

OH

O

O

K = 1.2 x 10i-4 M

NH

HN

OH

O

OH OOHN

O

K = 5.65 x 10i-6 M

17 18

Figure 2.4 Structures of pepstatin and early statine analogs.


variety of statine derivatives have been utilized in the design of aspartic acid prote-ase inhibitors, including renin, HIV-protease, b-secretase, and cathepsin D [24–26].Since the discovery of pepstatin, statines have been incorporated in the

design of potent renin, plasmepsin II, and cathepsin D inhibitors [24,27,28].The X-ray crystal structure of statine-derived renin inhibitor L-363564 (Boc-His-Pro-Phe-His-Sta-Leu-Phe-NH2) complexed to aspartic acid protease fromfungus Endothia parasitica was determined [29]. As shown in Figure 2.5, thestructure showed that the statine hydroxyl group forms strong hydrogen bondswith the carboxyl groups of catalytic aspartates Asp32 and Asp215 in the activesite. The phenylalanine side chain is not shown in the X-ray crystal structure.This structure provided evidence of the structural role of statines in the designof inhibitors. Statines are unique in the sense that they are not truly dipeptidemimics. However, structural studies have shown that statines occupy P1 to P1

0

even though they lack a P10 side chain [29]. It is likely that other residues,

such as the P20 side chain of the inhibitor, compensate for the lack of a P1

0

side chain in the S10 subsite. Therefore, for the design of statine-derived inhib-

itors, statines are used as the dipeptide mimic. However, replacement of onesubstrate residue has also resulted in potent inhibitors.Many syntheses of statines and their derivatives have been reported [30–40].

Maibaum and Rich reported a practical synthesis of statines from Boc-protected amino acids [34]. As shown in Figure 2.6, N-Boc-protected aminoacids (19) were reacted with carbonyldiimidazole (CDI) and the resulting imi-dazolines were treated with the magnesium enolate (20) of ethyl malonate. Theresulting c-amino-b-ketoester (21) was reduced by a number of reducingagents to provide a diastereomeric mixture of statine derivatives 22 and 23that can be separated. 4-Amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA)(24), a statine derivative containing a cyclohexylmethyl side chain, was synthe-sized using the above protocol [41]. ACHPA (24) was extensively utilized in the

Figure 2.5 X-ray structure of L-363564 bound to an aspartic acid protease from the fungus E.parasitica. PDB code: 2ER9.

2.3 Design of Statine-Based Inhibitors 25

synthesis of renin inhibitors. In fact, the ACHPA incorporation resulted in 50-fold enhancement in potency over other side chains [24,42].A highly diastereoselective synthesis of statine was developed by Woo using

Evans aldol reaction as the key step (Figure 2.7) [43,44]. The synthesis involved analdol condensation of boron enolate of methylthioacetyl oxazolidinone 25 and N-Boc-leucinal to provide aldol product 26 in >99% de. Desulfurization of the aldolproduct with Raney nickel (Ra-Ni) followed by removal of the chiral oxazolidinonewith sodium ethoxide in ethanol provided the statine ethyl ester 27 conveniently.

NO

O

SMe

O nBu2BOTf,

iPr2NEt

N-Boc-leucinal NO

OO

SMe

HN

OH

Boc

1. Ra-Ni

OEt

OHN

OH

Boc

2. NaOEt, EtOH

25 26

27

Figure 2.7 Highly diastereoselective synthesis of statine.

HN

R

O

OHBoc

OMg

O

OEtO

1. CDI, THF

2.

HN

R

O

Boc

O

OEt

NaBH4,

THF-MeOH

HN

R

OH

Boc

O

OEt

HN

R

OH

Boc

O

OEt+

H2N

OH O

OHH2N

OH O

OH

Statine ACHPA

19 20 21

(16) (24)

2223

Figure 2.6 Practical synthesis of statine and its derivatives.


Kwon and Ko reported a general syn-amino alcohol-based, optically active syn-thesis of statine derivatives [45]. Although the synthetic route is lengthy, thismethod provides an access to non-amino acid-derived side chains dependingupon the choice of Grignard reagent, as shown in Figure 2.8. This methodemployed the ring opening of N-Boc-aziridine 30 with a Grignard reagent in thepresence of a CuBr catalyst. Optically active N-Boc-aziridine 30 can be readily pre-pared from O-benzyl-N-Boc-benzoyl-O-TBS-protected 2-amino-1,3,4-butanetriol29. This protected 2-amino-1,3,4-butanetriol can be prepared from optically activediisopropyl tartrate 28 as described previously [46].For the design of inhibitors with non-amino acid-derived side chains, Ghosh et

al. also developed a general synthesis of statine. The synthesis involved a diaster-eoselective synthesis of functionalized tetrahydrofuran derivatives from opticallyactive 4-phenylbutyrolactone 34, followed by a Lewis acid-catalyzed acyloxycarbe-nium ion-mediated ring opening to form styrene derivatives 38 (Figure 2.9) [40].Oxidative cleavage of the styrene derivative 38 afforded access to anti-aldol seg-ment, which was converted to statine derivative 39 using the Curtiusrearrangement as a key step.Over time, a variety of aspartic acid protease inhibitors were designed by incor-

poration of statine as the transition-state mimic. The design principle involvedinsertion of statine at the scissile site. This strategy resulted in substrate-baseddesign of potent inhibitors (40–42) of human renin, HIV-1 protease, and b-secre-tase (Figure 2.10) [47–49].

OMe

OHN

OH

Boc

Boc

HN OTBS

BnO

OBz

HO O-iPr

iPr-O

OH

OO

1. H2, Pd-C

2. TsCl, Pyr

3. NaH, THF

BocN OTBS

OBz

MgCl

CuBr∙SMe2

Boc

HN OTBS

OBz

1. aq. HF

2. TsCl, Pyr3. Cs2CO3, MeOH

Boc

HN

O1. VinylMgBr, CuI

2. O3, NaOH,

MeOH

28 29

3031

32 33

Figure 2.8 Synthesis of statines with non-amino acid-derived side chain.

2.3 Design of Statine-Based Inhibitors 27

O

NH

HN N

HN

O

OH O

NH

Renin inhibitor

Ki = 1.7 nM (human renin)

HN

OH O

NH

O

HN

HIV-1 protease inhibitor

IC50 = 70 nM (enzyme)

Ac-Phe-Pro-Phe-Val

Ph

O

NH2

HN

OH O

NH

O

HN

β-Secretase inhibitor

IC50 = 300 nM (enzyme)

Ac-Val-Met

O

NH

CO2H

O

HN

Ph

O

OH

40

41

42

Figure 2.10 Structures of statine-derived protease inhibitors.

O OPh

Ph

OH R

H

O OPh

R

OPh

HR

LDA, THF

RX, -78 °C

1. DIBAL-H2. Ph3P=CHCO2Et

H

KHMDS

Zn(OTf)2 (5 mol%)

R

OAc

CO2Et

CO2Et

Ac2O, PhMe

1. OsO4, Oxone

2. (PhO)2P(O)N3, then BnOH

OEt

OHN

OAc

Cbz

O2N

CO2EtTHF

Ph

34 35

36

3938

37

R = p-NO2-Bn

Figure 2.9 Synthesis of statines with designed side chains.


2.4Design of Hydroxyethylene Isostere-Based Inhibitors

As mentioned earlier, the X-ray structural studies of statine-derived inhibitor L-363564 revealed that the 3(S)-hydroxyl of statine residue forms tight hydrogenbonds with the catalytic aspartates. It is not clear, however, if the statine hydroxylgroup occupies the same position as the nucleophilic water molecule or the scis-sile bond carboxyl oxygen. Nevertheless, the X-ray structure indicates that the sta-tine hydroxyl group mimics the transition state at the catalytic center [29].Structurally, statine is unique, as it contains two additional main chain carbonatoms rather than a single a-amino acid. Also, it has one less carbon atom than atypical dipeptide. Although statine lacks a P1

0 side chain, the P20 group takes dif-

ferent orientations to partially fill in the S10 subsite. By analogy, replacement of apeptide bond with a hydroxyethylene functionality (CH(OH)��CH2) should mimicthe tetrahedral transition state of the scissile bond cleavage, as shown in Figure2.11. Indeed, renin inhibitors containing hydroxyethylene isosteres were reportedby Szelke et al. [50,51]. In these inhibitors, hydroxyethylene dipeptide isostereswere incorporated at the scissile site. Inhibitor H261 (44) contains a hydroxyethy-lene isostere in the renin substrate sequence 6–13. This resulted in an extremelypotent inhibitor with an IC50 value of 0.7 nM [52].An X-ray structure of H261-bound endothiapepsin was determined by Blundell

et al. at 2.6 A�resolution [52]. As shown in Figure 2.12, the structure shows that the

P1

H2N

O

OH

α-Amino acid

H2N

OH

Statine

O

OH

P1

HN

O

NH

Dipeptide

P1'

O P1

HN

OH

Hydroxyethylenedipeptide isostere

P1'

O

HN

HOHN

NH

IC50 = 0.7 nM (human renin)

Boc-His-Pro-Phe-His OH

O

O N

NH

O

44 (H261, renin inhibitor)

43

3

16

8

Figure 2.11 Structures of statine, dipeptide, hydroxyethylene isosteres, and inhibitor H261.

2.4 Design of Hydroxyethylene Isostere-Based Inhibitors 29

hydroxyl group of hydroxyethylene dipeptide isostere replaces the water moleculethat is present between the catalytic aspartates Asp32 and Asp215. Furthermore,the hydroxyl group appears to be symmetrically disposed and forms strong hydro-gen bonds with the carboxylates of Asp32 and Asp215. The hydroxyl group mim-ics the transition state of peptide bond cleavage by the catalytic aspartates. Incontrast to statine-containing inhibitors, both P1 and P1

0 residues in inhibitorH261 nicely fill in the hydrophobic pockets in the S1 and S10 subsites. Also, boththe P1 NH and carbonyl form strong hydrogen bonds with the Gly220 backbonecarbonyl and Asp77 backbone NH, respectively. The imidazole side chain of P2

His is not shown in the X-ray structure [52].Over the years, a variety of potent aspartic acid protease inhibitors containing

hydroxyethylene isosteres were designed [15]. As a consequence, many synthesesof these structural scaffolds were developed based upon an a-amino acid-derivedP1 ligand [50–60]. Many renin inhibitors were designed based upon Leu–Val iso-steres and other isosteres containing P1 cyclohexylmethyl and P1

0 Val residues. Alarge number of HIV-1 protease inhibitors were designed based upon a preferredcleavage site at Phe–Phe or Leu–Phe. For the design of b-secretase inhibitors,Leu–Ala isosteres were initially designed based upon the kinetics and specificityof Leu at the P1 site and Ala at the P1

0 site.Initial syntheses of hydroxyethylene isosteres were reported by Szelke et al. and

Rich and coworkers in 1983 [50–53]. The synthesis of the Leu–Leu dipeptide iso-stere by Szelke et al. [50,51] involved conversion of N-phthaloylleucine 45 to thecorresponding bromoketone 47, as shown in Figure 2.13. Reduction of the bromo-ketone 47 and subsequent protection of the hydroxyl group provided bromo etherderivative 48. Reaction of the bromo ether 48 with the dianion of 4-methylpentanoic

Figure 2.12 X-ray structure of endothiapepsin complexed with hydroxyethylene isostere-containinginhibitor H261 (blue; PDB code: 1OEX).


acid provided Leu–Leu dipeptide isostere core 49. Removal of phthaloyl group withhydrazine followed by reaction of the resulting amine with Boc2O furnished Boc-protected acid 50 for inhibitor synthesis.A synthesis of Leu–Val dipeptide isostere was developed by Szelke et al. [50,51].

As shown in Figure 2.14, reaction of the protected bromohydrin 51 with di-tert-butyl malonate anion followed by alkylation with isopropyl iodide provided thediester derivative 52. Acid-catalyzed removal of the THP protecting group and sub-sequent reprotection provided the benzyl derivative 53. Treatment of diester withp-TsOH afforded the Leu–Val isostere core 54, which was converted to Boc deriva-tive 55 as described above.Early on, Rich and Holladay developed a stereoselective synthesis of the Leu–Ala

dipeptide isostere [53,54]. As shown in Figure 2.15, chiral bromo ether 57 wassynthesized using Evans alkylation [61] as the key step. This was converted tothe corresponding Grignard reagent. Reaction of this Grignard reagent with Boc-leucinal afforded a 1: 4 mixture of C-4 diastereomers 58 and 59. Protection of thehydroxyl group as an acetate followed by benzyl ether cleavage afforded the alcoholthat was oxidized with KMnO4 in the presence of nBu4N

þI� to afford the Boc-protected Leu–Ala acid 60 for inhibitor synthesis. The synthesis of Leu–Phe iso-steres was accomplished via the same scheme using 3-phenylpropanoyl oxazolidi-none as the starting material [54]. A number of other approaches to the synthesisof hydroxyethylene dipeptide isosteres have been reported [55–63].

N

OO

O

NN

HBr, EtOAc

N

OO

O

Br

1. NaCNBH3

2. 4-Methoxy- dihydropyranN

OO

O

Br

N

O

OH

O

O

1. i-BuOCOCl,

Et3N

2. CH2N2

LiCHCO2Li

i-Bu

N

OO

O O

OH

OMeO

+ isomer

HN

OH

O

OHBoc

OMeO

45 46

47

5049

48

Figure 2.13 Synthesis of hydroxyethylene isostere by Szelke et al.


NO

O

MePh

O 1. LiHMDS,

BrCH2OBn

2. Ca(BH4)2

3. Ph3P, NBS

Br

Me

OBn

1. Mg, Et2O

2. Boc-leucinal

HN

OH Me

OBnBoc

2. H2, Pd-C

1. Ac2O, DMAP

HN

O Me

O

OHBoc

Ac

+

(major isomer)(minor isomer)

3. KMnO4

HN

OH Me

OBnBoc

56 57

58 59

60

Figure 2.15 Synthesis of Leu–Ala hydroxyethylene isostere by Rich and coworkers.

HN

O

O

OHBoc

N

OO

O

Br

THP

1. NaCH(CO2t-Bu)2

2. NaH, i-PrI

N

OO

OCO2t-Bu

CO2t-Bu

THP

1. HCl, EtOH

2. NaH, BnBr

N

OO

OCO2t-Bu

CO2t-Bu

Bn

p-TsOHN

OO

OCO2H

Bn

1. NH2NH2

2. Boc2O

Bn

5152

53

55

54

toluene

Figure 2.14 Synthesis of Leu–Val hydroxyethylene isostere by Szelke et al.


A practical and stereoselective synthesis of a hydroxyethylene dipeptide isostere wasdeveloped by Herold et al. [62] for the design of renin inhibitors. The following syn-thesis allows variation of both C2 and C5 substituents as the synthesis is not basedupon amino acids. As shown in Figure 2.16, the chiral hexanoic acid derivative 63was readily synthesized using Evans alkylation procedure [61,63]. After removal ofthe chiral auxiliary, the resulting acid was converted to dimethylamide 64 via the acidchloride. Iodolactonization using a slightly modified Yoshida’s procedure [64] pro-vided the c-lactone 65 as the major product. Bromolactonization also proceeded withexcellent diastereoselectivity and yields. Displacement of an iodide with an azidemostly resulted in elimination. However, reaction of a bromide with an azideproceeded well to provide the corresponding azide 66. Exposure of azidolactone 66 ton-butylamine at 40 �C provided a butylamide derivative, which was subjected tohydrogenation to provide hydroxyethylene dipeptide isostere 67 containing a cyclo-hexylmethyl group as the P1 group. This methodology can be adapted to the synthe-sis of hydroxyethylene isosteres with varying substituents at the P1 and P1

0 positions.A highly stereoselective synthesis of a hydroxyethylene dipeptide Phe–Phe iso-

stere was developed by Ghosh et al. [65] for the design of HIV-1 protease inhibitors.This synthesis utilized D-mannose (68) as the chiral starting material. The synthesisprovided enantioselective access to a range of C2 and C5 substituents not limited toamino acid-derived side chains. As shown in Figure 2.17, D-mannose was readilyconverted to glycal, as described by Ireland et al. [66,67]. Ferrier-type rearrangementprovided a 1:1 mixture of methyl glycoside 70, which was converted to the

NO

O O

LDA, THF

Br

Cy

NO

O O

1. LiOH, H2O2

O

N

2. (COCl)2

3. Me2NH, Py

Me

MeI2, AcOHO

X

O

(X = I, Br)

NaN3, DMPU

O

N3

O

H2N

OH

O

HN

Bu

61 63

6465

66 67

62

or NBS, AcOH

Figure 2.16 Synthesis of hydroxyethylene isostere by Herold et al.


corresponding epoxide 71. Opening of the epoxide ring with phenylmagnesiumbromide and subsequent conversion of the resulting alcohol provided azide deriva-tive 72. This was converted to the c-lactone 73, an intermediate that can be used forintroduction of a variety of C-2 substituents. Such alkylated lactones were convertedto numerous potent and selective HIV-1 protease inhibitors.A variety of hydroxyethylene dipeptide isosteres were utilized in the design of

potent inhibitors of renin [68], b-secretase [2,6–8], c-secretase [69], HIV-1 protease[70–72], and plasmepsin E [73]. Figure 2.18 shows representative examples ofaspartic acid protease inhibitors with a hydroxyethylene isostere core replacing the

O

OH

OH

HOHO

HO

O

O

O

OH

O

O

O

OMe

MeOH

PPTS

O

O

OMe

N3

Ph

O

OMe

1. PhMgBr, CuI

2. azidation

1. mCPBA,

BF3∙OEt2

2. H2, Pd-C,

Boc2O

3. LiHMDS, BnI

HN

Ph

O

O

Ph

Boc

Me3Al

PhCH2NH2

HN

OH

PhO

HN

Ph

BnBoc

Refs [66,67]

D-Mannose (68) 69 70

717273

74

Figure 2.17 Synthesis of hydroxyethylene isostere by Ghosh et al.

OHN

HN

OPh

OH

O

β-Secretase inhibitor Ki = 1.2 nM (enzyme)

H2N

HN

NH

HN

HN

CO2H

O

O

O

OH Me

O

Me

OHN

CO2H

HN

OC2HOPh

H2NO

Ph

NH

OPh

O

NH2

HIV-1 protease inhibitor (L-682679) IC50 = 0.6 nM (enzyme)

S NH

HN

Ph

O

OCy

OH

O

NH

Renin inhibitor (CGP 38560) IC50 = 2 nM (enzyme)

OHN

NH

HN

OPh

O

OCy

OH

OH

Plasmepsin I inhibitor (SC-5003) IC50 = 500 nM (enzyme)

OO

NHN

75 (OM99-2) 76

77 78

Figure 2.18 Structures of hydroxyethylene isostere-containing potent inhibitors.


scissile amide. A substrate-based design of a b-secretase inhibitor incorporatingLeu–Ala isostere provided inhibitor OM99-2 (75). Subsequently, an inhibitor-bound X-ray structure of OM99-2 complexed with b-secretase provided detailsof the molecular interactions in the active site [2,3]. HIV-1 protease inhibitorL-682679 (76), with a Phe–Phe hydroxyethylene isostere, was one of the early leadinhibitors at Merck Research Laboratories [74]. Renin inhibitor CGP 38560 (77),containing a hydroxyethylene isostere, was developed at Ciba Geigy [75]. Asecreted aspartic protease (SAP) inhibitor was developed from a renin inhibitorlead A-70450. This modified inhibitor was a selective SAP inhibitor [76,77].

2.5Design of Inhibitors with Hydroxyethylamine Isosteres

Besides statine and hydroxyethylene isosteres, an amino alcohol-derived structuralcore, known as the hydroxyethylamine isostere, was designed to replace a peptidebond at the scissile site and mimic the putative transition state. As shownin Figure 2.19, in this design, the peptide bond is typically replaced with aCH(OH)��CH2�� functionality. In essence, an additional atom is added in theP1 � P1

0 linkage. The design concept for the hydroxyethylamine isostere (79 and80) was first introduced during the design of angiotensin-converting enzyme(ACE) inhibitors by Gordon et al. [78]. Incorporation of hydroxyethylamine core inthe penultimate amide bond of N-benzoyl-Phe-Ala-Pro, a known substrate [79] forACE, led to a 1:1 mixture of diastereomers 81. The mixture showed potent ACEinhibitory activity (IC50¼ 35 nM).The design of renin inhibitors incorporating a hydroxyethylamine core was first

reported by Dann et al. [80]. As shown, introduction of a hydroxyethylamine corein the P1 � P1

0 scissile site led to a potent renin inhibitor 82 with an IC50 value of230 nM. Over the years, the hydroxyethylamine core has been utilized extensivelyin the design and synthesis of a variety of aspartic acid protease inhibitors.

P1

HN

O

NH

Dipeptide

P1'

O P1

HN

OH

Hydroxyethylamine dipeptide isosteres

HN P1'

HN

HOHN

PhO

ACE inhibitorIC50 = 35 nM

Me

O P1

HN

OHN

O

P1'

PhO

N

CO2H

HN

HOHN

Renin inhibitorIC50 = 230 nM

Boc-Phe-His

O NH

Ile-His-OMe

3 8079

81 82

Figure 2.19 Structures of hydroxyethylamine dipeptide isosteres and inhibitors.

2.5 Design of Inhibitors with Hydroxyethylamine Isosteres 35

The design and synthesis of a hydroxyethylamine isostere based upon the pref-erence of Phe–Pro cleavage site was reported by Rich et al. [81]. As can be seen inFigure 2.20, inhibitor JG-365 (84) was designed based upon the partial substratesequence Ac-Ser-Leu-Asn-Phe-Pro-Ile-Val-OMe (83). In the substrate-based designof HIV-1 protease inhibitors, a hydroxyethylamine core was incorporated in placeof the scissile site of Phe–Pro. The hydroxyl has a mixture of (R)- and (S)-diastere-omers (84). The mixture showed a Ki of 0.66 nM. The pure (S)-isomer 85 displayeda Ki of 0.23 nM [81].An X-ray structure of JG-365-bound HIV-1 protease was determined with 2.3A

�

resolution [82]. Although the inhibitor contained a mixture of (R)- and (S)-configu-rations at the hydroxyl chiral carbon, the inhibitor–HIV-1 protease complex onlyshowed the tighter binding with (S)-diastereomer. As shown in Figure 2.21, the

HN

NH

HN

O

O

OPh

OH2N

O

N

O NH

Ile-Val-OMe

HN

NH

HN

O

O

OPh

OHH2N

O

N

O NH

Ile-Val-OMe

HIV-1 protease substrate sequence (partial)

Potent HIV-1 protease inhibitor (JG-365)IC50 = 0.66 nM (enzyme)

(R&S)

HN

NH

HN

O

O

OPh

OHH2N

O

N

O NH

Ile-Val-OMeIC50 = 0.23 nM (enzyme)

(R)

OH

NH

Ac

OH

NH

Ac

OH

NH

Ac

(83) (84)

85

Figure 2.20 Design of substrate-based inhibitors containing hydroxyethylamine isostere.

Figure 2.21 X-ray structure of HIV-1 protease complexed with hydroxyethylamine isostere-containinginhibitor JG-365 (green; PDB code: 7HVP).


inhibitor’s transition-state hydroxyl group is positioned between the side chain car-boxylates of catalytic Asp25 and Asp250 and forms strong hydrogen bonds. A tightlybound water molecule, water-301, forms strong hydrogen bonds with the carbonylsof Phe–Asn and Pro–Ile peptide bonds, as well as with the Ile50 and Ile500 NHs ofthe HIV-1 protease. The inhibitor JG-365 (84) appears to make extensive interac-tions similar to the substrate-derived reduced peptide inhibitor MVT-101 [82,83].The synthesis of hydroxyethylamine core of inhibitor JG-365 (84) involved a sim-

ilar synthetic strategy as Gordon et al. [78]. As outlined in Figure 2.22, tetrapeptidechloromethyl ketone 86 was synthesized from phenylalanine chloromethyl ketoneby stepwise coupling. The chloromethyl ketone 86 was reacted with proline tripep-tide 87 to provide the ketomethyleneamine derivative 88. Reduction of the ketonewith NaBH4 provided the hydroxyethylamine core. Catalytic hydrogenationresulted in the removal of the benzyl group, providing inhibitor JG-365 [81].

2.5.1Synthesis of Optically Active a-Aminoalkyl Epoxide

The synthesis of inhibitors containing a hydroxyethylamine scaffold can be readilyachieved by the opening of an appropriately protected aminoalkyl epoxide 89 by anamine 90, as depicted in Figure 2.23. In general, the aminoalkyl epoxide contains

IC50 = 0.66 nM (enzyme)

HN

Ph

OCl H2N

O NH

Ile-Val-OMe

+Tos-

+

NaI, DMFNaHCO3

1. NaBH4

2. H2, Pd-C

N

O NH

Ile-Val-OMe

R & S mixture

HIV-1 protease inhibitor (JG-365)

Ac-Ser(Bn)-Leu-Asn-

H

Ph

OAc-Ser(Bn)Leu-Asn-N

N

O NH

Ile-Val-OMe

H

Ph

OHAc-Ser-Leu-Asn-N

86 87

84

88

Figure 2.22 Synthesis of hydroxyethylamine isostere-containing inhibitor JG-365.


the P1 side chain and the amine component contains the P10 substituent. A variety

of inhibitors have been designed and synthesized using this strategy. As a conse-quence, synthesis of optically active aminoalkyl epoxides has become a subject ofimmense synthetic interest [55,84–90]. A number of methods were reported basedon an a-amino acid as the starting material. Also, several asymmetric syntheses ofthese chiral oxiranes were reported [91–94]. The main advantage of asymmetricsynthesis is that non-amino acid-derived side chains can be incorporated. Typi-cally, the (R)-hydroxy configuration of the hydroxyethylamine isostere provides amore potent inhibitor than the (S)-hydroxy configuration.Luly et al. reported the synthesis of aminoalkyl epoxide based on readily availa-

ble protected amino acid-derived aldehyde [84]. As shown in Figure 2.24, DIBAL-H reduction of ester 92 provided aldehyde 93, which upon Wittig olefination withmethylenetriphenyl phosphorane afforded the protected allylic amines 94. Epoxi-dation of this alkene with mCPBA in dichloromethane provided excellent threo-selective epoxide 95. A Corey–Chaykovsky epoxidation [95] of amino acid-derivedaldehyde 93 with dimethylsulfonium methylide afforded a mixture (1:1) of diaster-eomeric epoxides 96 in a very good yield [95]. These isomers can be separated bycolumn chromatography.A practical method for the synthesis of aminoalkyl epoxides was reported by

Chen et al. [96]. This method utilized the Kowalski homologation reaction as thekey step [97,98]. As shown in Figure 2.25, a-chloroketone 98 was synthesized insitu by reaction of ethyl ester 97 with 4 equiv of a reagent derived from LDA andICH2Cl and an additional equivalent of LDA at �78 �C. The resulting chloroke-tone 98 was reduced with NaBH4 to provide the chlorohydrin with high diastereo-selectivity (typically �98:2 for a variety of starting esters). Treatment of the

Boc

HN

P1

O

+ H2N P1'HN

P1

Boc

OHHN P1'

O O89 90 91

Figure 2.23 General strategy for the synthesis of hydroxyethylamine isostere.

HN

P1

O

ORBoc

HN

P1

O

HBoc

HN

P1

Boc

m-CPBA

HN

P1

Boc

OHN

P1

Boc

O

Mixture (1:1) of diastereomers

Mostly (S)-isomer

Ref. [84]Ref. [95]

92 93 94

9596

Ph3P=CH2

Me2S+CH2-

Figure 2.24 Synthesis of aminoalkyl epoxide from a-amino acids.


chlorohydrin with KOH in ethanol afforded Boc-protected aminoalkyl epoxide 99in high optical purity. The method is suitable for large-scale synthesis [96]. Otheruseful variations of this method have provided optically pure epoxides with vary-ing N-protecting groups [99].Ghosh et al. devised a practical synthesis of azidoalkyl epoxides in the opti-

cally active form using Sharpless asymmetric epoxidation as the key step [91].As shown in Figure 2.26, commercially available butadiene monoxide 100 wasconverted to an allylic alcohol by reaction with PhMgBr in the presence of acatalytic amount of CuCN. Sharpless asymmetric epoxidation [100,101] of thisallylic alcohol with (�)-diethyl D-tartrate provided the epoxide 101 in highenantiomeric purity [98]. Reaction of this epoxide with diisopropoxytitaniumdiazide as described by Sharpless afforded the azidodiol 102 regioselectively[102]. The diol was converted to epoxide 103 with successive reactions with2-acetoxyisobutyryl chloride and sodium methoxide. This epoxide was con-verted to numerous potent HIV-1 protease inhibitors [91]. The method is alsosuitable for large-scale synthesis of a range of azidoalkyl epoxides with a widevariety of P1 side chains [91].

HN

O

OEtBoc

HN

O

Boc

HN

Boc

O

Mostly (S)-isomer

ICH2Cl (4 equiv)

LDA (5 equiv)

Cl

Ph Ph

Ph

1. NaBH4

2. KOH, EtOH

97 98

99

Figure 2.25 Synthesis of aminoalkyl epoxide from a-chloroketone.

N3O 1. PhMgBr,

CuCN

2. t-BuOOH, (-)-DET, Ti(OiPr)4

Ph

OOH

N3

O

OHOH

Ph

Ti(OiPr)4, TMSN3

Ph

1. AcOCMe2

2. NaOMe

COCl

N3 NOH

Ph O NH

H

HSaquinavir isostere

100 101 102

103(104)

Figure 2.26 Asymmetric synthesis of azidoepoxide.


2.6Design of (Hydroxyethyl)urea-Based Inhibitors

In the past years, a variety of hydroxyethylamine scaffolds have been utilizedin the design and synthesis of inhibitors of renin, HIV-1 protease, b-secretase,and plasmepsins [15]. Hydroxyethylamine-derived HIV-1 protease inhibitors,such as saquinavir and nelfinavir, were approved by the FDA as part of ahighly active antiretroviral treatment (HAART) regimen for treatment of HIVinfections and AIDS [103,104]. A number of variations of the hydroxyethyl-amine isostere also evolved over the years [105,106]. These include the designof (hydroxyethyl)urea- and (hydroxyethyl)sulfonamide-derived transition-stateisosteres.An interesting variation of the hydroxyethylamine isostere was first reported

by a number of investigators when they devised a (hydroxyethyl)urea isostere106 and incorporated it in renin inhibitors [107–109]. In this design, a nitro-gen atom in the isostere replaced the P1

0 a-carbon atom in a dipeptide moiety(105), as shown in Figure 2.27. This modification leaves the carbonyl group ofthe urea in the same place as the P1

0 � P20 dipeptide carbonyl group. A

P1

HN

O

NH

Peptide bonds

P1'

O

HN

Hydroxyethylamine isostere

HN

OHN

Renin inhibitor (CP-69799)IC50 = 300 nM (human plasma renin)

Boc-Phe-His

O

P2'

(Hydroxyethyl)urea

P1

HN

OHN

P1'

(Hydroxyethyl)sulfonamide

P1

HN

OHN

P1'

SOO

P1

HN

OHHN P1'

O

HN

H2N

O

NH

Ph

O

OH

P1

P1'

P2'

O

HN

P2'

105

108

isostere

106

107

79

Figure 2.27 Structures of (hydroxyethyl)urea, hydroxyethylamine and (hydroxyethyl)sulfonamideisosteres.


hydroxyethylamine isostere typically incorporates an additional atom in theP1 � P1

0 linkage; however, a (hydroxyethyl)urea isostere resembles closer todipeptide isostere with no additional atom. CP-69799 (107) is a potent renininhibitor that incorporated the (S)-hydroxyethylene moiety at the scissile site[108]. A comparison of X-ray structures of renin inhibitors containing hydroxy-ethylene (inhibitor H261) and (hydroxyethyl)urea (CP-69799) isosteres revealedthat the kinks of the hydroxyethylene moiety are very similar [52,108]. As aresult, both P1

0 substituents were nicely accommodated in the S10 subsite, as

shown in Figure 2.28.The design of HIV-1 protease inhibitors incorporating (hydroxyethyl)urea

was reported by Getman et al. [110]. A variety of P10 substituents were explored

and it turned out that isobutyl and isopentyl side chains provided the bestresults in combination with tert-butyl urea. These (hydroxyethyl)urea isostereswere readily prepared by opening aminoalkyl epoxide 99 with isopentylamine.The resulting secondary amine was reacted with tert-butyl isocyanate to providethe urea isostere 110. The hydroxy stereochemistry was shown to be importantfor potency. The (R)-hydroxyethylene isomer was preferred over the (S)-isomer.As shown in Figure 2.29, inhibitors with P2 asparagine and P3 quinoline-2-car-boxamide in combination with isopentyl substituent as the P1

0 ligand providedHIV-1 protease inhibitor 111 with excellent enzyme inhibitory and antiviralactivity in CEM cell line. The X-ray cocrystal structure of HIV-1 protease withan inhibitor (112) with n-butyl urea and isobutyl side chain showed that theP2

0n-butyl urea side chain filled in the S10 subsite and the P1

0 isobutyl sidechain occupied the S2

0 subsite [110]. The design of c-secretase inhibitors incor-porating (hydroxyethyl)urea isosteres provided potent inhibitors [111]. A repre-sentative example is shown in Figure 2.29.

Figure 2.28 Overlay of X-ray structures of renin inhibitors H261 (magenta, PDB code: 1OEX)and CP-69799 (green, PDB code: 5ER2) complexed with endothiapepsin.

2.6 Design of (Hydroxyethyl)urea-Based Inhibitors 41

2.7(Hydroxyethyl)sulfonamide-Based Inhibitors

Further modification of the urea functionality with sulfonamide derivatives pro-vided (hydroxyethyl)sulfonamide isostere 108, initially described by Vasquez et al.and Tung et al. (Figure 2.30) [112,113]. In this isostere, the P1

0 � P20 amide of a

hydroxyethylamine isostere-containing inhibitor is replaced with a sulfonamidegroup. A variety of (hydroxyethyl)sulfonamides were incorporated in the designand synthesis of potent inhibitors of HIV-1 protease (115–117) for treatment ofHIV/AIDS [114–116].

2.8Design of Heterocyclic/Nonpeptidomimetic Aspartic Acid Protease Inhibitors

As described above, the design and synthesis of aspartic acid protease inhibitorsgenerally involves replacing the scissile amide bond with an isosteric templatethat mimics the putative transition state of peptide hydrolysis. This classicapproach has been the hallmark of structure-based design over the last three

HN

Ph

O

Boc1. H2N

2. t-BuNCO

HN

OHN

Ph

Boc

O

HN

HN

OHN

PhO

HN

O

OH2N

NH

O

N

γ-Secretase inhibitorIC50 = 30 nM

HIV-1 protease inhibitorIC50 = 3 nM

HN

OHN

PhO

HN

O

OH2N

NH

O

N

HIV-1 protease inhibitorIC50 = 126 nM

HN

OHN

PhO

Ph

HN

O

OO

NH

O

OMe

99 110

111

113112

109

Figure 2.29 Potent HIV-1 protease and c-secretase inhibitors containing (hydroxyethyl)ureaisostere.


decades [117,118]. The core structural templates were extensively used in thedesign and evolution of substrate-based design, peptidomimetic design, andnonpeptide design of inhibitors. Many nonpeptide inhibitors contain featuresto mimic the basic transition state; however, such inhibitors contain no pep-tide bonds or basic amino acid-derived side chains or features. Over the years,many interesting structural classes of inhibitors that are devoid of classictransition-state mimetic features have evolved, but these inhibitors incorporatefunctionalities that form tight interactions with the active site catalytic residues[119]. In this context, many new classes of aspartic acid protease inhibitorshave been designed based upon novel heterocyclic scaffolds, which bind to theactive site aspartic acid residues in entirely different ways, but fulfill the func-tion of the transition-state isosteres. The advent of these structural classesmarked the beginning of another exciting era in structure-based design. Asshown in Figure 2.31, some of these new heterocyclic scaffolds and core tem-plates include dihydropyranone (118) [120,121], piperazine (119) [122], amino-pyridine (120) [123], and acyl guanidine (121) [124].

HN

Ph

O

Boc1. H2N

2.

HN

OHN

S

Ph

Boc

HN

OH

PhO

OH2N

NH

O

N

HN

OHN

S

PhO

HIV-1 protease inhibitorKi = 4.5 pMIC50 = 1.8 nM (cell)

HIV-1 protease inhibitorIC50 = 3 nMEC50 = 7 nM (cell)

HIV-1 protease inhibitor

Ki = 1 nM IC50 = 5 nM (cell)

PhSO2Cl OO

NS

OO

(Hydroxyethyl)sulfonamide isostere

OO

HN

OHN

S

PhO

O

OO

OH

OMeHO

OMe

99 114

115

116 117 (GRL-06579)

109

Figure 2.30 Potent HIV-1 inhibitors containing a (hydroxyethyl)sulfonamide isostere.

2.8 Design of Heterocyclic/Nonpeptidomimetic Aspartic Acid Protease Inhibitors 43

2.8.1Hydroxycoumarin- and Hydroxypyrone-Based Inhibitors

During broad screening efforts, warfarin (122) and phenprocoumon (123) were ini-tially identified as weak HIV-1 protease inhibitors with micromolar inhibitory activity[125,126]. Subsequent kinetic studies demonstrated that both of these inhibitors arecompetitive inhibitors of HIV-1 protease [126]. The X-ray cocrystal structures of phen-procoumon and its methoxy derivative with HIV-1 protease revealed an interestingmode of interaction in the active site. As depicted in Figure 2.32, the 4-hydroxyl

O

O

O P1'

P1

OO

O O

H H

N

O O O

OH

HN

NH

HO

+

P1'P1

Dihydropyranone

Acyl guanidine

N

O O O

OH

P1'P1

+ NH

H

Aminopyridine

N

O

OO O

H H

HN P1'P1

+

Piperazine118 119

120 121

AspAsp

Asp AspAsp Asp

AspAsp

Figure 2.31 Binding mode of nonpeptide inhibitors with unique structural motifs in the activesite of aspartic acid proteases.

Figure 2.32 X-ray cocrystal structure of methoxyphenprocoumon with HIV-1 protease (green;PDB code: 3UPJ) and structures of warfarin and phenprocoumons.


group of 123 (R¼OMe) formed hydrogen bonds with the two catalytic aspartates.The lactone oxygen atom formed hydrogen bonds with the backbone amide NHs ofthe flap residues. It appears that various substituents can be incorporated in theselead structures to interact with residues in the S1, S1

0, and S20 subsites. The uniqueactive site interactions and unprecedented structural motif, as well as X-ray structuralinformation, set the stage for structure-based design of more potent derivatives.As shown in Figure 2.33, incorporation of a 3-aminopropionamide side chain to

interact with the S20 subsite improved the activity of compound 124 to low nano-molar potency [127]. The corresponding pyranone derivatives turned out to be verypotent. Further structure-based optimization resulted in a very potent series ofinhibitors [120,121]. Ultimately, one of these inhibitors, tipranavir 126, became anFDA-approved inhibitor [128]. Both hydroxycoumarin and dihydropyranone struc-tural cores are unique templates for the design of nonpeptide HIV-1 proteaseinhibitors. In principle, these structural features can be utilized in the design andsynthesis of other protease inhibitors.The synthesis of 4-hydroxycoumarin core structure is shown in Figure 2.34.

Horner–Emmons olefination of m-nitropropiophenone 127 afforded an E/Z

O

OH

O

O

OH

OPh

NH

S N

CF3

CH O3

OO

Ki = 38 nM

Ki = 8 pM IC50 = 30 nM (cell)

O

OH

O

NH

Ki = 28 nM

O

NH

Boc124 125

126Tipranavir

Figure 2.33 Hydroxycoumarin- and dihydropyranone-derived HIV-1 protease inhibitors.

NO2O NaH, THF

NO2

t-BuO2C

(MeO)2P(O)-CH2CO2t-Bu

NH

Tr

LDA, methyl salicylate

H2, Pt-C

Ph3CCl, iPr2NEt

NH

Tr

O

t-BuO O

OH Ot-Bu

OCF3CO2H

NH2

OH

O O

127 128 129

130131

Figure 2.34 Synthesis of 4-hydroxycoumarin structural motif.


mixture of a,b-unsaturated ester 128 [129]. Hydrogenation over a platinum cata-lyst resulted in saturation of the olefin and reduction of the nitro group to anamine, which was protected as tritylamine 129. Deprotonation of the ester withLDA followed by reaction of the resulting enolate with methyl salicylate providedb-ketoester 130. Subsequent treatment of this b-ketoester with trifluoroacetic acidprovided the 4-hydroxycoumarin derivative 131, which was further functionalizedto improve potency [129].The synthesis of the 4-hydroxypyrone core was carried out from commercially

available 4-hydroxy-6-methyl-2-pyrone 132, as shown in Figure 2.35. The preparationof benzyl alcohol 133 was achieved by nitration of the corresponding cyclopropylketone followed by reduction of the nitro group and ketone functionality by catalytichydrogenation over a platinum catalyst [127]. Condensation of benzyl alcohol deriva-tive 133 with 6-methyl pyrone 132 in the presence of p-TsOH provided the cyclo-propyl pyrone derivative 134. Alkylation of methyl pyrone, first with LDA and ethylbromide and then with benzyl bromide, provided derivative 135 with ethyl and ben-zyl side chains. Removal of the Cbz group provided amine 136, which was furtherelaborated as P3 ligands, providing a variety of potent HIV-1 protease inhibitors [127].As discussed earlier, considerable progress has been achieved in structure-based

design of a significant number of peptidomimetic inhibitors for a host of asparticacid proteases. However, peptidomimetic inhibitors are not without issues, as theyoften experience problems related to their large size, low oral absorption, and solu-bility. Therefore, structure-based design of nonpeptide inhibitors using heterocyclicor heteroatomic templates would be very promising as drug candidates. Further-more, protein–ligand interactions of such templates will provide opportunities forthe design and creation of new basic structural motifs for structure-based design.

2.8.2Design of Substituted Piperidine-Based Inhibitors

In 1996, high-throughput screening (HTS) of Roche compound collections identi-fied a 3,4-disubstituted alkoxyarylpiperidine 137 as a weak renin inhibitor [122].

O

OH

O

NH

CbzHO+NH

Cbz

O O

OH

p-TsOH

CH2Cl2

NH

Cbz

O O

OH

LDA, EtBr,then PhCH2Br

H2, Pd-CNH2

O O

OH

132 133 134

135136

Figure 2.35 Synthesis of the 4-hydroxypyrone structural motif.


This discovery marked the beginning of the evolution of many new classes of non-peptide aspartic acid protease inhibitors [130]. Such structural motifs are likely tobe applicable to other enzyme and receptor systems. The X-ray structural insightinto the binding mode of the initial alkoxyarylpiperidines in the renin active siteprompted subsequent structure-based design of more potent 3,4,5-trisubstitutedderivatives, such as 139 shown in Figure 2.36.The X-ray cocrystal structural studies of all three early inhibitors (137–139, Fig-

ure 2.36) with recombinant human renin showed that the protonated nitrogen ofthe piperidine ring was positioned between the active site aspartates [122]. Asshown in Figure 2.37, the ring nitrogen of 3,4-disubstituted derivative was in prox-imity to form two strong hydrogen bonds with the catalytic aspartates Asp32 and

HN

Cl

O

OMe

HN

Br

O

HN

OMeO

O

O Cl

Human renin inhibitorIC50 = 26 µM

IC50 = 5 µM

Human renin inhibitorIC50 = 2 nM

IC50 = 1 µM

Plasmepsins I and IIinhibitor

137 138

139

Figure 2.36 Structures of a new class of nonpeptide renin inhibitors.

Figure 2.37 X-ray structure of arylpiperidine with renin (PDB code: 4GJ5).


Asp215. The 3-naphthylmethoxy substituent occupied the hydrophobic S1 and S3subsites of renin. These regions were typically occupied by the P1 cyclohexyl andP3 phenyl side chains of peptidomimetic inhibitor CP-69799 (see Figure 2.28).Interestingly, the large 4-phenyl ring along with its side chain caused an induced-fit adaptation in the active site and a major conformational change in the proteinstructure compared with the apoenzyme.This induced-fit adaptation in the binding site was observed in all X-ray struc-

tures of the alkoxyarylpiperidine class of renin inhibitors. This molecular insightinto the induced-fit adaptation suggests that the binding site of aspartic acid prote-ases possesses latent conformational flexibility. Indeed, 3,4,5-trisubstituted piperi-dine derivative 139 also inhibited plasmepsins I and II from Plasmodiumfalciparum with an IC50 value of 1mM [122]. This result indicates that piperidinesand other related heterocycles can be utilized as general structural templates forthe design of other aspartic acid protease inhibitors.An enantioselective synthesis of the piperidine structural motif was developed by

Rich and coworkers [119]. As shown in Figure 2.38, an enol triflate derived from N-Boc-4-piperidone 140 was subjected to Suzuki coupling conditions [131] to form thecoupling product 141. Sharpless asymmetric dihydroxylation [132] provided the diol142 enantioselectively. Removal of the TBS group followed by Raney nickel reduc-tion afforded optically pure trans-3,4-disubstituted hydroxyarylpiperidine 143. Of par-ticular note, Raney nickel reduction proceeded successfully only after removal of theTBS group. Selective protection of the phenol and alkylation of the resulting alcoholprovided access to the basic piperidine structural motif 144 in optically active form.Many different variations of this piperidine class of inhibitors led to potent

inhibitors [133–135]. As shown in Figure 2.39, an arylpiperidine scaffold was opti-mized to provide exceptionally potent inhibitor 145, which was shown to havegood oral bioavailability in dogs [136]. A number of potent compounds containing

N

O

Boc

2. Pd(PPh3)4, LiCl, Na2CO3

B(OH)2OTBS

NBoc

TBSO

AD-mix-α

CH3SO2NH2

NBoc

TBSO

OHOH

1. TBAF2. Ra-Ni, EtOH

NBoc

HO

OH

1. TIPS-Cl, imidazole

2. NaH, 4-Br-PhCH2Br

NBoc

TIPSO

O

1. LDA, Tf2NPh

140

141 142

143144

Br

Figure 2.38 Synthesis of the piperidine structural motif in optically active form.


novel bridged piperidine scaffolds were also designed. The representative com-pound 146 displayed excellent renin inhibitory activity and oral absorption in rats[137]. An X-ray cocrystal structure showed that the N-9 atom was located betweenthe catalytic aspartates and the N-3 atom was solvent exposed. Researchers atNovartis identified a new piperidine scaffold and carried out structure-based opti-mization to potent renin inhibitors as represented in 147 [135].The bridged piperidine scaffold was prepared from 4-bromocrotonate 148 and

benzylamine [137]. As shown in Figure 2.40, N-alkylation of the benzylamineafforded the corresponding tertiary amine 149, which was subjected to double

OEt

O

PhCH2NH2

Na2CO3, EtOH

EtON

PhO

Br

OEtO

N

HN

EtO

O O

OEt

1. MeNH2, EtOH

2. H2, Pd-C

1. NaH, THF2. Boc2O, Et3N3. resolution

N

NMe Boc

OEtO

O

2. ArBr, n-BuLi ZnCl2, Pd(PPh3)4

1. Tf2O

N

NMe Boc

EtO

OOR

148 149150

151152

Figure 2.40 Synthesis of bridged piperidine structural template in optically active form.

HN

O O

OH

OH

O O

IC50 = 0.06 nMF = 27% (dog)

OMe

MeO

HN

N

OO

IC50 = 0.2 nMF = 24% (rat)

F = 18% (rat)

O

HN

HH

Cl

Cl

(RO-65-7219) (ACT-077825)

HN

NH

O S

HN

O

O

O

MeO

IC50 = 3 nM

N-9 interacts with catalytic Asps

N-3 solvent exposed

145 146

147

Figure 2.39 Structures of potent nonpeptidomimetic renin inhibitors.


Michael reaction to provide piperazine 150 as a 2 : 1 cis/trans mixture. The cis-isomer 150 was recrystallized selectively. Dieckmann cyclization and amine pro-tection afforded the 3,9-diazobicyclononanone. Optical resolution with (þ)-tartaricacid afforded b-ketoester 151. Formation of the vinyl triflate followed by Negishicoupling [138] provided the basic bicyclic scaffold 152 for inhibitor preparation.The 3,5-disubstituted piperidine structural scaffold can be readily synthesized

from commercially available Boc-aminonicotinic acid 153 [135]. As shown in Fig-ure 2.41, catalytic hydrogenation followed by Fmoc protection of the resultingamino acid provided Fmoc derivative 154 as a racemic mixture. Selective removalof the Boc group and subsequent reaction of the amine with tosyl chlorideafforded the basic 3,5-disubstituted piperidine scaffold 155 for inhibitor synthesisby coupling with appropriate amines.

2.8.3Design of Diaminopyrimidine-Based Inhibitors

Holsworth et al. identified diaminopyrimidine-derived compounds such as 156 asweak renin inhibitors through HTS efforts (Figure 2.42) [139]. Lead optimizationresulted in difluoro derivative 157 with a sevenfold improvement in potency. AnX-ray structure of this inhibitor bound to renin revealed a unique mode of bindingin the renin active site. Interestingly, the N0-pyrimidine ring and the 2-NH2 groupformed hydrogen bonds with the catalytic aspartates Asp215 and Asp32. Basedupon the comparison of the binding modes of diaminopyrimidine- and piperidine/ketopiperazine-based inhibitors, a substituted tetrahydroquinoline was incorporatedin place of the arylamine group. As can be seen, the structure-based incorporationof a pentanoate side chain in 159 to interact in the S3 subsite resulted in a potentinhibitor. This class of inhibitors does not contain any chiral center.Besides piperidine-based nonpeptidomimetic protease inhibitors, HTS has iden-

tified a number of other interesting chemotypes that bind to the catalytic

N

HO2C NH

O

O1. H2, Pt/Rh-C

2. Fmoc- succinimide

N

HO2C NH

O

O

Fmoc

1. 4 M HCl

2. TsCl, aq. K2CO3

153 154

155

N

HO2C NH

Fmoc

SOO

Figure 2.41 Synthesis of 3,5-disubstituted piperidine structural scaffold.


aspartates in a unique mode. A number of these inhibitors were subsequentlyoptimized utilizing structure-based design strategies. These heterocyclic scaffoldsinclude acyl guanidine, 2-aminoquinoline, aminopyridine, or spiropiperidine imi-nohydantoin structures. Many potent aspartic acid protease inhibitors, particularlyBACE1 inhibitors, were designed incorporating these core structures. The modeof interaction of these inhibitors is distinct from that of the inhibitors with transi-tion-state isosteres. Furthermore, these scaffolds may potentially be optimizedagainst other aspartic acid proteases.

2.8.4Design of Acyl Guanidine-Based Inhibitors

Cole et al. at Wyeth Research identified acyl guanidine-based structure 160(Figure 2.43) as a weak b-secretase inhibitor (IC50¼ 3.7 mM) through HTS [140].An X-ray cocrystal structure of optimized inhibitor 161 with BACE1 revealed aunique mode of interaction. As shown in Figure 2.43, the acyl guanidine function-ality formed four hydrogen bonds with the catalytic aspartates Asp32 and Asp228.Interestingly, the flap region adopted a flap-open conformation to accommodatethe diarylpyrrole scaffold. In essence, the diarylpyrrole occupied the space of

NN

NH2

NH2

HN

Cl

NN

N

NH2

HN

F

Cl

F

12

3

5

H H

O O

Asp215

Asp32

S1 site

S3 site

NN

NH2

NH2

N

O

NN

NH2

NH2

N

O

OMe

IC50 = 27 μM (renin)

IC50 = 650 nM IC50 = 91 nM

IC50 = 4 μM

O

O

H

156 157

158 159

Figure 2.42 Structures of 2,4-diaminopyrimidine-based renin inhibitors.


Tyr71 in peptidomimetic BACE1 inhibitors and the pyrrole ring appeared to forma p-edge stacking with the phenyl ring of Tyr71. Similar flap-open conformationwas observed in enzyme–inhibitor complexes of renin and pepsin. Subsequentstructure-based modification of the guanidine nitrogen to reach out to the S1

0 sub-site with a 3-propanol side chain as well as the incorporation of an adamantyl sidechain for S1 subsite resulted in improved potency (BACE1, IC50¼ 240 nM) [140].Further variation of ligand binding in the S1 and S3 subsites with a sterically lessdemanding phenyl side chain led to many potent inhibitors, as represented incompound 161 (IC50¼ 0.6mM) [141].A general synthesis of the core acyl guanidine is shown in Figure 2.44. Coupling

of the a-methylketone 163 with enolizable ketone 162 according to the procedure of

Figure 2.43 Structures of acyl guanidine-based BACE1 inhibitors and X-ray cocrystal structure ofan inhibitor with BACE1 (green; PDB code: 2ZE1).

O

Br+

O

Et2NH, ZnCl2

t-BuOH

OO

Glycine, p-TSA

N

CO2H

1. CDI, DMF

2. Guanidine∙HCl

Et3N

N

NH

O

NH

H2N

162 163 164

165166

Figure 2.44 Synthesis of 2,5-disubstituted pyrrole-containing acyl guanidine.


Kulinkovich and coworkers [142] provided the 1,4-diketone 164. Condensation of thisdiketone with glycine provided the pyrrole acetic acid 165. The acid was reacted with1,10-carbonyldiimidazole followed by guanidine hydrochloride to provide the unsub-stituted acyl guanidine 166 for further N-substitution and analog preparation [141].

2.8.5Design of Aminopyridine-Based Inhibitors

An application of fragment screening against b-secretase using HTS and X-ray crys-tallography led to the identification of aminoquinoline and aminopyridine motifs,which bind to the catalytic aspartates in an unprecedented manner [143,144]. Thebinding mode of aminoquinoline fragment 167 is shown in Figure 2.45. Aminopyr-idine fragment 168 was shown to bind to BACE with higher affinity. Structure-based modification resulted in inhibitors (170 and 171) with improvement in affin-ity to low nanomolar concentration. The discovery of these structural motifs led tostructure-based design of a range of low nanomolar b-secretase inhibitors [145].The synthesis of the core aminopyridine template can be carried out from com-

mercially available 2,3-diaminopyridine 172, as depicted in Figure 2.46. Reductiveamination with 3-bromobenzaldeyde provided bromobenzylpyridine-2,3-diamine173. Suzuki coupling with 5-methoxypyridyl-3-boronic acid provided b-secretaseinhibitor 174 with micromolar activity [144].

2.8.6Design of Aminoimidazole- and Aminohydantoin-Based Inhibitors

An aminoimidazole structural core was identified as a b-secretase inhibitorthrough HTS [146]. As shown in Figure 2.47, aminoimidazole derivative 175

N

HN

NH2

NN

N

HN

NH2 N

O

N

HN

NH2

HN

IC50 = 2 mM (BACE1)

IC50 = 310 μM IC50 = 24 μM

IC50 = 9100 nM (R = H)

R

IC50 = 690 nM (R = OCH2Ph)

N

N

NH2

Cl

O

N

N

O

OH

IC50 = 40 nM (BACE1)

H

HO

OO O

H

+

Asp32Asp228

167

168 169

171170

Figure 2.45 Structures of aminopyridine-based BACE1 inhibitors.


showed weak b-secretase inhibitory activity (IC50¼ 38 mM). An X-ray structure ofthis compound complexed with BACE1 revealed unique active site binding withthe catalytic aspartates via a network of hydrogen bonds. The X-ray structureshowed that both the S1 and S2 enzyme subsites can be accessed by substitutionon the aromatic rings. Subsequent structure-based ligand binding optimizationresulted in a low nanomolar inhibitor 176 with an aminohydantoin scaffold. Avariety of very potent BACE1 inhibitors incorporating this scaffold have beenreported [147–149].The synthesis of the aminoimidazole core can be readily achieved, as outlined in

Figure 2.48. Benzonitrile or its derivatives were converted to amines such as 178

N

NH2

NH2NaBH(OAc)3 N

HN

NH2

Br3-BrPhCHO

Pd(PtBu3)4,

Na2CO3

N

HN

NH2 N

OMe

172

173

174

N

OMe(HO)2B

Figure 2.46 Synthesis of substituted aminopyridine template.

Figure 2.47 Structures of BACE1 inhibitors and X-ray cocrystal structure of an inhibitor withBACE1 (magenta; PDB code: 3INF).


S

HN S

S

CN1. ArMgBr

2. NaBH4

NH2

1. CSCl2, NaHCO3

2. CS2, tBuOK

Br

Br

H2N-(CH2)3

NH2

N

HN N

S

Br

NH4OH

tBuOOH

N

N N

H2N

Br

N

N N

H2N

cross-

coupling

177 178

179180

181 182

Figure 2.48 Synthesis of substituted aminoimidazole core.

N + I

Br

Pd(PPh3)2Cl2

CuI, Et3NN

Br

KMnO4,

MgSO4

N

O

O

BrH2N

NH

NH

Me

Na2CO3N

N

N

BrO

NH2

Cross-coupling

N

N

N

O

NH2

183 184 185

186187

188

Figure 2.49 Synthesis of substituted aminohydantoin core.


by reaction with Grignard reagents followed by reduction with NaBH4. This aminewas converted to an isothiocyanate, which upon reaction with CS2 in the presenceof KOtBu afforded 1,3-thiazolidine-2,5-dithione 179 in excellent yield. Condensa-tion of this dithione with various diamines followed by reaction with t-BuO2Hand NH4OH provided access to aminoimidazole core 181 for further inhibitoroptimization [146].A general synthesis of substituted aminohydantoins is shown in Figure 2.49.

Sonogashira coupling of ethynylpyridine and 3-bromoiodobenzene provided acety-lene derivative 185 in good yield [150]. Oxidation of acetylene 185 with KMnO4

afforded the diketone 186. Reaction of 186 with 1-methylguanidine provided theaminohydantoin core 187 for further derivative synthesis [151]. Suzuki couplingwith boronic acids in the presence of suitable palladium catalysts provided accessto a variety of substituted derivatives.

2.9Conclusions

Aspartic acid proteases are involved in the pathogenesis of numerous human dis-eases. Consequently, these enzymes have attracted immense attention from pro-tein biochemists, medicinal chemists, and structural biologists alike. Over time, avariety of tools, concepts, and design principles have been developed. The knowl-edge of protein X-ray structures greatly advanced the structure-based design of avariety of potent and selective inhibitors with clinical potential. Hundreds of X-raystructures of enzyme and inhibitor complexes have been determined, leading to adetailed knowledge of these drug targets and the molecular interactions involvingenzyme inhibition. This chapter outlined the evolution of design concepts, appli-cation of structure-based design strategies, and chemical synthetic routes for anumber of key ligands, scaffolds, and bioisosteres widely used for successfuldesign of drugs.

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114 Kim, E.E., Baker, C.T., Dwyer, M.D.,Murcko, M.A., Rao, B.G., Tung, R.D., andNavia, M.A. (1995) Crystal structure of HIV-1 protease in complex with VX-478, a potentand orally bioavailable inhibitor of theenzyme. J. Am. Chem. Soc., 117, 1181–1182.

115 Ghosh, A.K., Sridhar, P.R., Leshchenko,S., Hussain, A.K., Li, J.F., Kovalevsky,A.Y., Walters, D.E., Wedekind, J.E.,Grum-Tokars, V., Das, D., Koh, Y.,Maeda, K., Gatanaga, H., Weber, I.T.,and Mitsuya, H. (2006) Structure-baseddesign of novel HIV-1 proteaseinhibitors to combat drug resistance.J. Med. Chem., 49, 5252–5261.

116 Ghosh, A.K., Anderson, D.D., Weber, I.T.,and Mitsuya, H. (2012) Enhancing proteinbackbone binding a fruitful concept forcombating drug-resistant HIV. Angew.Chem., Int. Ed., 51, 1778–1802.

117 Rich, D.H. (1990) Peptidase inhibitors, inComprehensive Medicinal Chemistry: TheRational Design, Mechanistic Study andTherapeutic Application of ChemicalCompounds (eds C. Hansch, P.G. Sammes,and J.B. Taylor), Pergamon Press,pp. 391–441.

118 Babine, R.E. and Bender, S.L. (1997)Molecular recognition of protein–ligandcomplexes: applications to drug design.Chem. Rev., 97, 1359–1472.

119 Bursavich, M.G., West, C.W., and Rich,D.H. (2001) From peptides to non-peptide peptidomimetics: design andsynthesis of new piperidine inhibitors ofaspartic peptidases. Org. Lett., 3,2317–2320.

120 Prasad, J.V.N.V., Para, K.S., Lunney, E.A.,Ortwine, D.F., Dunbar, J.B., Ferguson, D.,Tummino, P.J., Hupe, D., Tait, B.D.,Domagala, J.M., Humblet, C., Bhat, T.N.,Liu, B.S., Guerin, D.M.A., Baldwin, E.T.,Erickson, J.W., and Sawyer, T.K. (1994)Novel series of achiral, low-molecular-weight,and potent HIV-1 protease inhibitors. J. Am.Chem. Soc., 116, 6989–6990.

121 Hagen, S., Prasad, J.V.N.V., and Tait, B.D.(2000) Nonpeptide inhibitors of HIVprotease. Adv. Med. Chem., 5, 159–195.

122 Oefner, C., Binggeli, A., Breu, V., Bur, D.,Clozel, J.P., D’Arcy, A., Dorn, A., Fischli,W., Gruninger, F., Guller, R., Hirth, G.,Marki, H.P., Mathews, S., Muller, M.,Ridley, R.G., Stadler, H., Vieira, E.,Wilhelm, M., Winkler, F.K., and Wostl, W.(1999) Renin inhibition by substitutedpiperidines: a novel paradigm for theinhibition of monomeric asparticproteinases. Chem. Biol., 6, 127–131.

123 Murray, C.W., Callaghan, O., Chessari, G.,Cleasby, A., Congreve, M., Frederickson,M., Hartshorn, M.J., McMenamin, R.,Patel, S., and Wallis, N. (2007) Applicationof fragment screening by X-raycrystallography to b-secretase. J. Med.Chem., 50, 1116–1123.

124 Cole, D.C. and Bursavich, M.C. (2010)Nonpeptide BACE1 inhibitors: design andsynthesis, in Aspartic Acid Proteases asTherapeutic Targets, vol. 45 (ed. A.K.Ghosh), Wiley-VCH Verlag GmbH,Weinheim, pp. 481–509.

125 Bourinbaiar, A.S., Tan, X., and Nagorny, R.(1993) Effect of the oral anticoagulant,warfarin, on HIV-1 replication and spread.AIDS, 7, 129–130.

126 Thaisrivongs, S., Tomich, P.K.,Watenpaugh, K.D., Chong, K.T., Howe,W.J., Yang, C.P., Strohbach, J.W., Turner,S.R., Mcgrath, J.P., Bohanon, M.J., Lynn,J.C., Mulichak, A.M., Spinelli, P.A.,Hinshaw, R.R., Pagano, P.J., Moon, J.B.,Ruwart, M.J., Wilkinson, K.F., Rush, B.D.,Zipp, G.L., Dalga, R.J., Schwende, F.J.,Howard, G.M., Padbury, G.E., Toth, L.N.,Zhao, Z.Y., Koeplinger, K.A., Kakuk, T.J.,Cole, S.L., Zaya, R.M., Piper, R.C., andJeffrey, P. (1994) Structure-based design ofHIV protease inhibitors: 4-hydroxycoumarins and 4-hydroxy-2-pyrones as nonpeptidic inhibitors. J. Med.Chem., 37, 3200–3204.

127 Thaisrivongs, S., Janakiraman, M.N.,Chong, K.T., Tomich, P.K., Dolak, L.A.,Turner, S.R., Strohbach, J.W., Lynn, J.C.,Horng, M.M., Hinshaw, R.R., andWatenpaugh, K.D. (1996) Structure-baseddesign of novel HIV protease inhibitors:sulfonamide-containing 4-hydroxycoumarins

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128 Thaisrivongs, S., Skulnick, H.I., Turner,S.R., Strohbach, J.W., Tommasi, R.A.,Johnson, P.D., Aristoff, P.A., Judge, T.M.,Gammill, R.B., Morris, J.K., Romines,K.R., Chrusciel, R.A., Hinshaw, R.R.,Chong, K.T., Tarpley, W.G., Poppe, S.M.,Slade, D.E., Lynn, J.C., Horng, M.M.,Tomich, P.K., Seest, E.P., Dolak, L.A.,Howe, W.J., Howard, G.M., Schwende,F.J., Toth, L.N., Padbury, G.E., Wilson,G.J., Shiou, L.H., Zipp, G.L., Wilkinson,K.F., Rush, B.D., Ruwart, M.J.,Koeplinger, K.A., Zhao, Z.Y., Cole, S.,Zaya, R.M., Kakuk, T.J., Janakiraman,M.N., and Watenpaugh, K.D. (1996)Structure-based design of HIV proteaseinhibitors: sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrones as non-peptidic inhibitors. J. Med. Chem., 39,4349–4353.

129 Thaisrivongs, S., Watenpaugh, K.D.,Howe, W.J., Tomich, P.K., Dolak, L.A.,Chong, K.T., Tomich, C.S.C., Tomasselli,A.G., Turner, S.R., Strohbach, J.W.,Mulichak, A.M., Janakiraman, M.N.,Moon, J.B., Lynn, J.C., Horng, M.M.,Hinshaw, R.R., Curry, K.A., and Rothrock,D.J. (1995) Structure-based design ofnovel HIV protease inhibitors:carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent nonpeptidic inhibitors.J. Med. Chem., 38, 3624–3637.

130 Tice, C.M. and Singh, S.B. (2010)Evolution of diverse classes of renininhibitors through the years, in AsparticAcid Proteases as Therapeutic Targets:Methods and Principles in MedicinalChemistry, vol. 45 (eds A.K. Ghosh, R.Mannhold, H. Kubinyi, and G. Folkers),Wiley-VCH Verlag GmbH, Weinheim,pp. 297–324.

131 Miyaura, N. and Suzuki, A. (1995)Palladium-catalyzed cross-couplingreactions of organoboron compounds.Chem. Rev., 95, 2457–2483.

132 Sharpless, K.B., Amberg, W., Bennani,Y.L., Crispino, G.A., Hartung, J., Jeong,K.S., Kwong, H.L., Morikawa, K., Wang,Z.M., Xu, D.Q., and Zhang, X.L. (1992)

The osmium-catalyzed asymmetricdihydroxylation: a new ligand class and aprocess improvement. J. Org. Chem., 57,2768–2771.

133 Webb, R.L., Schiering, N., Sedrani, R., andMaibaum, J. (2010) Direct renin inhibitorsas a new therapy for hypertension. J. Med.Chem., 53, 7490–7520.

134 Powell, N.A., Clay, E.H., Holsworth, D.D.,Bryant, J.W., Ryan, M.J., Jalaie, M., andEdmunds, J.J. (2005) Benzyl etherstructure–activity relationships in a seriesof ketopiperazine-based renin inhibitors.Bioorg. Med. Chem. Lett., 15, 4713–4716.

135 Ostermann, N., Ruedisser, S., Ehrhardt,C., Breitenstein, W., Marzinzik, A., Jacoby,E., Vangrevelinghe, E., Ottl, J., Klumpp,M., Hartwieg, J.C.D., Cumin, F.,Hassiepen, U., Trappe, J., Sedrani, R.,Geisse, S., Gerhartz, B., Richert, P.,Francotte, E., Wagner, T., Kromer, M.,Kosaka, T., Webb, R.L., Rigel, D.F.,Maibaum, J., and Baeschlin, D.K. (2013) Anovel class of oral direct renin inhibitors:highly potent 3,5-disubstituted piperidinesbearing a tricyclic P-3–P-1 pharmacophore.J. Med. Chem., 56, 2196–2206.

136 Marki, H.P., Binggeli, A., Bittner, B.,Bohner-Lang, V., Breu, V., Bur, D.,Coassolo, P., Clozel, J.P., D’Arcy, A.,Doebeli, H., Fischli, W., Funk, C.,Foricher, J., Giller, T., Gruninger, F.,Guenzi, A., Guller, R., Hartung, T., Hirth,G., Jenny, C., Kansy, M., Klinkhammer,U., Lave, T., Lohri, B., Luft, F.C., Mervaala,E.M., Muller, D.N., Muller, M., Montavon,F., Oefner, C., Qiu, C., Reichel, A.,Sanwald-Ducray, P., Scalone, M.,Schleimer, M., Schmid, R., Stadler, H.,Treiber, A., Valdenaire, O., Vieira, E.,Waldmeier, P., Wiegand-Chou, R.,Wilhelm, M., Wostl, W., Zell, M., and Zell,R. (2001) Piperidine renin inhibitors:from leads to drug candidates. Farmaco,56, 21–27.

137 Bezencon, O., Bur, D., Weller, T., Richard-Bildstein, S., Remen, L., Sifferlen, T.,Corminboeuf, O., Grisostomi, C., Boss, C.,Prade, L., Delahaye, S., Treiber, A., Strickner,P., Binkert, C., Hess, P., Steiner, B., andFischli, W. (2009) Design and preparation ofpotent, nonpeptidic, bioavailable renininhibitors. J. Med. Chem., 52, 3689–3702.


138 Negishi, E., Hu, Q., Huang, Z.H., Qian,M.X., and Wang, G.W. (2005) Palladium-catalyzed alkenylation by the Negishicoupling. Aldrichim. Acta, 38, 71–88.

139 Holsworth, D.D., Jalaie, M., Belliotti, T.,Cai, C., Collard, W., Ferreira, S., Powell,N.A., Stier, M., Zhang, E., McConnell, P.,Mochalkin, I., Ryan, M.J., Bryant, J., Li, T.,Kasani, A., Subedi, R., Maiti, S.N., andEdmunds, J.J. (2007) Discovery of 6-ethyl-2,4-diaminopyrimidine-based smallmolecule renin inhibitors. Bioorg. Med.Chem. Lett., 17, 3575–3580.

140 Cole, D.C., Manas, E.S., Stock, J.R.,Condon, J.S., Jennings, L.D., Aulabaugh,A., Chopra, R., Cowling, R., Ellingboe,J.W., Fan, K.Y., Harrison, B.L., Hu, Y.,Jacobsen, S., Jin, G.X., Lin, L., Lovering,F.E., Malamas, M.S., Stahl, M.L., Strand,J., Sukhdeo, M.N., Svenson, K., Turner,M.J., Wagner, E., Wu, J.J., Zhou, P., andBard, J. (2006) Acylguanidines as small-molecule b-secretase inhibitors. J. Med.Chem., 49, 6158–6161.

141 Cole, D.C., Stock, J.R., Chopra, R.,Cowling, R., Ellingboe, J.W., Fan, K.Y.,Harrison, B.L., Hu, Y., Jacobsen, S.,Jennings, L.D., Jin, G., Lohse, P.A.,Malamas, M.S., Manas, E.S., Moore, W.J.,O’Donnell, M.M., Olland, A.M.,Robichaud, A.J., Svenson, K., Wu, J.J.,Wagner, E., and Bard, J. (2008)Acylguanidine inhibitors of b-secretase:optimization of the pyrrole ringsubstituents extending into the S-1 and S-3 substrate binding pockets. Bioorg. Med.Chem. Lett., 18, 1063–1066.

142 Nevar, N.M., Kel’in, A.V., and Kulinkovich,O.G. (2000) One step preparation of1,4-diketones from methyl ketones andalpha-bromomethyl ketones in the presenceof ZnCl2�t-BuOH�Et2NR as a condensationagent. Synthesis, 9, 1259–1262.

143 Murray, C.W., Callaghan, O., Chessari, G.,Cleasby, A., Congreve, M., Frederickson,M., Hartshorn, M.J., McMenamin, R.,Patel, S., and Wallis, N. (2007) Applicationof fragment screening by X-raycrystallography to b-secretase. J. Med.Chem., 50, 1116–1123.

144 Congreve, M., Aharony, D., Albert, J.,Callaghan, O., Campbell, J., Carr, R.A.E.,Chessari, G., Cowan, S., Edwards, P.D.,

Frederickson, M., McMenamin, R.,Murray, C.W., Patel, S., and Wallis, N.(2007) Application of fragment screeningby X-ray crystallography to the discovery ofaminopyridines as inhibitors ofb-secretase. J. Med. Chem., 50, 1124–1132.

145 Malamas, M.S., Barnes, K., Hui, Y.,Johnson, M., Lovering, F., Condon, J.,Fobare, W., Solvibile, W., Turner, J., Hu,Y., Manas, E.S., Fan, K., Olland, A.,Chopra, R., Bard, J., Pangalos, M.N.,Reinhart, P., and Robichaud, A.J. (2010)Novel pyrrolyl 2-aminopyridines as potentand selective human b-secretase (BACE1)inhibitors. Bioorg. Med. Chem. Lett., 20,2068–2073.

146 Malamas, M.S., Erdei, J., Gunawan, I.,Barnes, K., Johnson, M., Hui, Y., Turner,J., Hu, Y., Wagner, E., Fan, K., Olland, A.,Bard, J., and Robichaud, A.J. (2009)Aminoimidazoles as potent and selectivehuman b-secretase (BACE1) inhibitors.J. Med. Chem., 52, 6314–6323.

147 Zhou, P., Li, Y.F., Fan, Y., Wang, Z.,Chopra, R., Olland, A., Hu, Y., Magolda,R.L., Pangalos, M., Reinhart, P.H., Turner,M.J., Bard, J., Malamas, M.S., andRobichaud, A.J. (2010) Pyridinylaminohydantoins as small moleculeBACE1 inhibitors. Bioorg. Med. Chem.Lett., 20, 2326–2329.

148 Malamas, M.S., Erdei, J., Gunawan, I.,Barnes, K., Hui, Y., Johnson, M.,Robichaud, A., Zhou, P., Yan, Y.F.,Solvibile, W., Turner, J., Fan, K.Y., Chopra,R., Bard, J., and Pangalos, M.N. (2011)New pyrazolyl and thienylaminohydantoins as potent BACE1inhibitors: exploring the S2 0 region. Bioorg.Med. Chem. Lett., 21, 5164–5170.

149 Malamas, M.S., Erdei, J., Gunawan, I.,Turner, J., Hu, Y., Wagner, E., Fan, K.,Chopra, R., Olland, A., Bard, J., Jacobsen,S., Magolda, R.L., Pangalos, M., andRobichaud, A.J. (2010) Design andsynthesis of 5,50-disubstitutedaminohydantoins as potent and selectivehuman b-secretase (BACE1) inhibitors.J. Med. Chem., 53, 1146–1158.

150 Sonogashira, K., Tohda, Y., and Hagihara,N. (1975) Convenient synthesis ofacetylenes: catalytic substitutions ofacetylenic hydrogen with bromoalkenes,

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iodoarenes, and bromopyridines.Tetrahedron Lett., 50, 4467–4470.

151 Malamas, M.S., Barnes, K., Johnson, M.,Hui, Y., Zhou, P., Turner, J., Hu, Y.,Wagner, E., Fan, K., Chopra, R., Olland,A., Bard, J., Pangalos, M., Reinhart, P.,

and Robichaud, A.J. (2010) Di-substitutedpyridinyl aminohydantoins as potent andhighly selective human b-secretase(BACE1) inhibitors. Bioorg. Med. Chem.,18, 630–639.


3Design of Serine Protease Inhibitors

3.1Introduction

Serine proteases are a large family of proteolytic enzymes ubiquitous in botheukaryotes and prokaryotes. Serine proteases play a host of critical roles in numer-ous physiological processes including digestion, blood coagulation (clotting),wound healing, inflammation, cell signaling, and other processes [1–3]. In gen-eral, deregulation of key proteolytic events often leads to the pathogenesis ofmany human diseases including stroke, inflammation, Alzheimer’s disease, can-cer, and arthritis. Not surprisingly, the design of selective serine protease inhibitorshas been a subject of great interest in drug development.

3.2Catalytic Mechanism of Serine Protease

The active site of all serine proteases consists of a catalytic triad of Ser195, His57,and Asp102 (chymotrypsin numbering). The active site also possesses an oxyanionbinding site that is made from the backbone of Ser195 and Gly193 [4]. These keyactive residues are conserved in all serine proteases. The X-ray structural studiesshowed that these residues are superimposable in a majority of serine proteases[5,6]. The catalytic mechanism of serine proteases is depicted in Figure 3.1.As shown, the nucleophilic attack by the hydroxyl group of Ser195 at the carbonylcarbon of the scissile bond via general base catalysis by His57 leads to the tetrahe-dral transition state. This intermediate is stabilized by hydrogen bond formationwith backbone NHs of Ser195 and Gly193 in the oxyanion hole. The tetrahedralintermediate ultimately collapses to cleavage products 2 and 3.

3.3Types of Serine Protease Inhibitors

Over the years, a variety of inhibitors of serine protease have been designed anddeveloped [6–8]. The majority of early inhibitors were covalent inhibitors as they

67


contained an electrophilic group, which formed a covalent bond with the serinehydroxyl of the catalytic triad. The electrophilic groups are referred to as serinetraps or warheads, as shown in Figure 3.2. The choice of serine trap is importantfor the reversible or irreversible nature of the covalent bond formed with the cata-lytic triad. The major limitation of covalent inhibitors has been their lack of

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond

HN

H2NO P1

O P1'

O

HN

NH

P2'

OP2

OH +

NH

O P1'HN

P1

‡


1 2 3

NNH H

O H

Ser195

NN

HO

O

His57

Asp102

NH

O P1'HN

P1_

NN

H H

O

Ser195

NN+ H

O

O

His57

Asp102

Oxyanion hole

H

Figure 3.1 Catalytic mechanism of peptide hydrolysis by serine proteases.

Peptidyl boronic acid

HN

NHP2

O P1

OCl

Chloromethyl ketone

HN

NH

PP2

O P1

O

OPh

Aminoalkyldiphenyl phosphonate

OPh

HN

NHP2

O P1

O

CF3

Trifluoromethylketone

BOH

OH

α-Ketoheterocycle 8

β-Lactam derivative 9

P1

O

HN N

P2

O

ON

O

4 5

6 7

Figure 3.2 Basic structural cores for design of serine protease inhibitors.

68 3 Design of Serine Protease Inhibitors

selectivity and specificity against other proteases in the same class or in the sameclan. For potential therapy development, it is essential that inhibitors be particu-larly selective considering that a large number of physiologically important serine,cysteine, and threonine proteases are prevalent in mammalian systems. The lackof inhibitor selectivity often leads to toxicity and side effects.Inhibitor potency is important, but it cannot be the only aim in an inhibitor

design strategy. The inhibitor must be chemically and metabolically stable, andthe choice of serine trap is critically important. Early serine protease inhibitorsevolved following the discovery of the natural product leupeptin containing analdehyde as the serine trap. The design of serine protease inhibitors incorporatingaldehydes followed. However, the issue of chemical and metabolic stability of alde-hydes led to the development of other electrophilic serine traps. The basic designstrategy for covalent serine protease inhibitors involves selecting a good substrateand then attaching a serine trap/warhead such as chloromethyl ketone (4),diphenyl phosphonate esters (5), trifluoromethyl ketone (6), peptidyl boronic acid(7), various a-ketoheterocycles (8), and b-lactam derivatives (9). Based upon thesewarheads, a variety of irreversible and reversible covalent serine protease inhibi-tors were designed. Halomethyl ketones generally form an irreversible covalentbond with the active site serine hydroxyl group, whereas ketocarbonyl and ketohe-terocycles form a reversible covalent bond with the catalytic serine hydroxyl group.Many irreversible covalent serine proteases were also designed based upon hetero-cycles such as isocoumarins, b-lactams, and others [9].The majority of early covalent inhibitors did not exhibit selectivity against cys-

teine proteases, and thus were not suitable for in vivo application and possessedconsiderable toxicity. Medicinal chemistry efforts using structure-based design ledto the development of many important classes of serine protease inhibitors,including FDA-approved NS3 protease inhibitors against hepatitis C virus, throm-bin inhibitors against venous thrombosis, and elastase inhibitors against inflam-mation, pulmonary, and chronic obstructive pulmonary diseases [6–8].

3.4Halomethyl Ketone-Based Inhibitors

A large number of synthetic serine protease inhibitors were synthesized incorpo-rating functionalities that can alkylate the active site serine residue. These func-tionalities include chloromethyl ketone, dimethylsulfonium salts, and others [6].Because of the high reactivity of these halomethyl ketones, achieving high selectiv-ity has been a major limitation. However, a certain degree of selectivity can beachieved by varying the P1 and P2 amino acids since these serve as the recognitionelements by the enzyme. Inhibitor design strategy involved the identification of apeptide segment corresponding to the sequence of a good substrate and attachingalkylating groups such as haloketones. Serine protease inhibitors exhibit highreactivity and low selectivity as they recognize a wide variety of serine and cysteineproteases [9]. Furthermore, they react with SH-containing molecules such as

3.4 Halomethyl Ketone-Based Inhibitors 69

glutathiones and other nonproteolytic enzymes [10]. As a consequence, these typesof inhibitors typically show toxicity and are not suitable for in vivo application.Peptidyl fluoromethyl ketones, on the other hand, are not quite as reactive, andthe glutathione alkylation is considerably slower than chloromethyl ketone-derivedinhibitors [11]. However, inhibitors containing fluoromethyl ketones are generallyselective for cysteine proteases [12].Structural studies of these peptidyl chloromethyl ketone inhibitors provided

important insight into molecular interactions in the enzyme’s subsites. Thisinsight can be useful for designing selective inhibitors. The X-ray crystal struc-ture of inhibitor 10-bound chymotrypsin in Figure 3.3 showed that the activesite Ser195 and His57 form a tetrahedral hemiketal adduct with the P1 car-bonyl carbon of the inhibitor [12]. The phenylalanine side chain nicely filled inthe S1 specificity pocket.

3.5Diphenyl Phosphonate-Based Inhibitors

Diisopropyl phosphorofluoridate (DFP) is a widely used classic inhibitor of serineproteases [13,14]. It inhibits serine proteases by forming a covalent bond betweenthe active site serine hydroxyl group and phosphorus atom of the inhibitor. Theresulting pentavalent phosphorus adduct resembles a tetrahedral intermediate;however, it possesses very few similarities to the peptide substrate. The DFP hasvery reactive functionalities. It reacts with many proteins and lacks selectivity.Both selectivity and reactivity problems associated with DFP were addressed byLamden and Bartlett [15] as they replaced one of the isopropyl groups with anamino acid. The resulting (aminoalkyl)phosphonyl fluoride derivatives shown inFigure 3.4 displayed improved inhibitory activity against a-chymotrypsin and alsoshowed selectivity over cysteine proteases. However, such phosphonofluoridatesare not very stable as they hydrolyze rapidly in aqueous conditions.

Figure 3.3 Structure of inhibitor 10 and X-ray cocrystal structure of this inhibitor with chymo-trypsin (green; PDB code: 1DLK).


Oleksyszyn and Powers developed a new class of (a-aminoalkyl)diphenyl phos-phonate ester-based inhibitors [16,17]. These inhibitors exhibited good biologicalactivity, were hydrolytically quite stable, and showed no reactivity with acetylcho-linesterase. A number of serine protease inhibitors were designed based upon thediphenyl phosphonate ester functionality. The basic design strategy involvedreplacement of the scissile bond with an (a-aminoalkyl)phosphoric acid residue or(a-aminoalkyl)phosphonates.As shown, potent, selective, and irreversible inhibitors of serine proteases, ela-

stases and chymotrypsin, were designed [15,18–20]. The S1 pockets of trypsin andthrombin show preference for basic amino acids. The corresponding peptidylphosphonates containing P1 Lys, Arg, and ornithine side chains showed very goodactivity and selectivity. Powers and coworkers subsequently designed 4-amidino-phenyl structural core to mimic the arginine side chain [20]. Inhibitors containing4-amidinophenylglycine and 4-amidinophenylalanine phosphonate derivativeswere investigated against thrombin and other coagulation serine proteases. Deriv-atives with the 4-amidinoglycine core showed the best inhibitory activity againsthuman thrombin. The Cbz derivative 13 with a single-amino acid side chainshowed very good inhibitory activity against human plasma kallikrein [18,19], andcompound 14 showed good potency and selectivity against human thrombin.These derivatives were shown to be stable and showed no activity againstacetylcholinesterase.As shown in Figure 3.5, the mode of action of this class of inhibitors involves the

activation of phosphorus in the active site and substitution of phenoxide with theactive site Ser195 hydroxyl group [17]. This class of inhibitors is selective over cysteineproteases. Also, inhibitor selectivity can be improved by incorporating suitable P1and P2 amino acids corresponding to subsite specificity of the target serine protease.

PHN

O

OO

O

FP

HN

O

OO

OPh

OPh

Ph

NH

POPh

OOPh

NHH2N

O

OPh NH

POPh

OOPh

NHH2N

ON

O

Ph

NHBoc

Ph

(chymotrypsin)

(human kallikrein)kobsd/I = 11 000 M-1s-1kobsd/I = 18 000 M-1s-1

kobsd/I = 1200 M-1s-1k2/Ki = 180 000 M-1s-1

(chymotrypsin)

(human thrombin)

11 12

13 14

Figure 3.4 Structures of (aminoalkyl)phosphonyl fluoride and (aminoalkyl)phosphonates.

3.5 Diphenyl Phosphonate-Based Inhibitors 71

The X-ray structural studies of 4-amidinophenylglycine derivative 13 bound tobovine trypsin were carried out by Powers and coworkers [20,21]. The structurerevealed the formation of a tetrahedral phosphorus atom that was covalentlybound to the active site Ser195. As shown in Figure 3.6, the covalent bond forma-tion proceeded with the loss of both phenoxy groups. An interesting crystallo-graphic study with inhibitor Cbz-D-Dpa-Pro-Mpg-(OPh)2 bound to humana-thrombin was carried out to examine the inhibitor binding mode [22]. As itturned out, a 7-day-old inhibitor–thrombin complex showed a reversible penta-coordinated phosphorus intermediate. However, a 12-week-old inhibitor–enzyme

Peptidyl

HN P

R

O

OPh

OPh

OH

Ser195

Peptidyl

HN P

R

O

OPh

OPh

O

Ser195

Peptidyl

HN P

R

O

O

O

NH

N

Gly193

Ser195

Oxyanion hole

S1Substrate

binding siteHis57 His57

Active site Active site

Active site

Figure 3.5 Proposed mode of inhibition of serine protease by phosphonate ester.

Figure 3.6 X-ray cocrystal structure of inhibitor 13 (magenta) complexed with trypsin (PDB code:1MAX).


complex revealed that the inhibitor’s phosphorus formed a covalent bond with thehydroxyl group of Ser195 of the enzyme. In this case, phosphorus was tetracoordi-nated and the oxyanion was involved in interaction with the oxyanion hole.Various peptidyl phosphonates were synthesized, as shown in Figure 3.7.

Reaction of 4-cyanobenzaldehyde 15 with triphenyl phosphite and benzyl carba-mate in acetic acid provided a-amidoalkylation product 16 in good yield [18]. Thecyano group was then converted to amidine 17 by treatment with dry hydrochloricacid and then dry ammonia in methanol in good yield. Removal of the Cbz groupby hydrogenolysis provided aminophosphonate ester 18. Coupling of this aminewith Boc-Phe-(D)-Pro-OH 19 afforded the inhibitor 14.

3.6Trifluoromethyl Ketone Based Inhibitors

Trifluoromethyl ketones have been designed as warheads in the development of anew class of serine protease inhibitors. Imperiali and Abeles introduced trifluoro-methyl ketones as specific serine protease inhibitors, particularly for chymotrypsinand elastase [23]. Incorporation of di- and trifluoromethyl ketones in substrateanalogs was shown to result in potent transition-state inhibitors for a number of

O

H

NC

P(OPh)3, AcOH

O

O NH2

NH

P

NC

Cbz

O

OPh

OPh

1. Dry HCl, EtOH2. Dry NH3, MeOH

NH

P

Cbz

O

OPh

OPh

NH

H2N 2N HCl

NH3

P

O

OPh

OPh

NH

H2N

+Cl-

Ph

15 16

1718 MeOH, Pd/C

CDI, DMF

19

14

NH

POPh

OOPh

NHH2N

ON

O

Ph

NHBoc

ON

O

Ph

NHBoc

OH

Figure 3.7 Synthesis of peptidyl phosphonate ester derivative.

3.6 Trifluoromethyl Ketone Based Inhibitors 73

proteolytic enzymes, including acetylcholinesterase. As shown in Figure 3.8,the design strategy of this class of inhibitors involves the replacement of thescissile bond at the cleavage site with a trifluoromethyl ketone functionality.The nature of the peptidyl side chains at P1, P2, P3, and so on would deter-mine the inhibitor’s specificity [23]. The 13C NMR studies revealed that inwater the trifluoromethyl ketone exists mainly as its hydrated form. Presum-ably, the corresponding hydrated peptidyl trifluoroketone can react with theactive site Ser195 to form a stable hemiketal as shown. Trifluoromethylketone-derived slow-binding serine protease inhibitors of human leukocyteelastase were also reported by Stein et al. [24].Imperiali and Abeles [23] showed that trifluoromethyl derivative 20 was more

potent than difluoromethyl derivative 21 and monofluoromethyl derivative 22, asshown in Figure 3.9. The trifluoromethyl ketone exhibited excellent specificity forchymotrypsin. This inhibitor showed very little to no effect on bovine trypsin or

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond

HN

O P1

O

NH

P2

1

F

FF

(Substrate) (Inhibitor)

HN

O P1

NH

P2

F

FF

H2O

OHHOEnz-OHHN

O P1

NH

P2

F

FF

OHO

Enz-OH

Enz

Figure 3.8 Design strategy of trifluoromethyl ketones as serine protease inhibitors and theirmode of inhibition.

MeHN

NHO

OPh

O

Ki = 1.2 μM

F

F

FMe

HN

NHO

OPh

O

Ki = 25 μM

F

H

F

MeHN

NHO

OPh

O

Ki = 200 μM

F

H

H

20 21

22

Figure 3.9 Structures of fluoromethyl ketone-containing chymotrypsin inhibitors.


porcine pancreatic elastase (PPE). This class of inhibitors initially showed slow-binding reversible competitive inhibition [23–25].The enzyme–inhibitor interaction in the enzyme active site was demonstrated

by Abeles and coworkers [25] by X-ray crystallographic studies of cocrystal oftrifluoromethyl ketone 20 bound to chymotrypsin. As shown in Figure 3.10,catalytic Ser195 formed a covalent bond with the trifluoromethyl ketone car-bonyl and provided a tetrahedral hemiketal intermediate. The hemiketal oxygenwas positioned within hydrogen bonding distance to the backbone amide NHsof Gly193 and Ser195 in the oxyanion hole and the P1 benzyl side chain nicelynestled in the S1 pocket [25].Trifluoromethyl ketones are pharmacologically quite stable. As a result, a variety

of serine protease inhibitors based upon trifluoromethyl ketones were designed,synthesized, and evaluated for their clinical potential. Researchers at AstraZenecadesigned numerous peptidyl trifluoromethyl ketone derivatives as potent humanelastase inhibitors [26–29]. As depicted in Figure 3.11, inhibitor 23 containing dia-rylacylsulfonamide functionality was shown to be a very potent elastase inhibitor[30]. However, such inhibitors were not effective when administered orally in lab-oratory animals. Further optimization of features resulted in the development of anumber of orally active inhibitors containing trifluoromethyl ketones. Both inhibi-tors 24 and 25 were shown to be very potent inhibitors with excellent oral bio-availability in laboratory animals [29,31]. Optically pure compound 25 with an(S)-configuration at the P1 isopropyl side chain became a candidate for clinicaldevelopment for the possible treatment of a host of elastase-implicated respiratorydiseases. Besides trifluoromethyl ketones, pentafluoroethyl ketones were alsoshown to be effective serine protease inhibitors. Furthermore, peptidomimeticpyridone-containing inhibitors were designed as effective inhibitors [27,28].

Figure 3.10 X-ray cocrystal structure of inhibitor 20 (carbon chain, green; F3, yellow) complexedwith chymotrypsin (PDB code: 7GCH).


Compound 26 showed excellent potency and in vivo properties. The X-ray crystalstructure of 26 with porcine pancreatic elastase showed catalytic site interactionssimilar to inhibitor 20 (Figure 3.11). The P1 isopropyl chain occupies the S1pocket. The pyrimidinone carbonyl, 5-sulfonamide NH, and p-aminoaryl group atthe 2-position form a number of hydrogen bonds in the active site [27]. The pyr-imidinone scaffold was also utilized in peptidomimetic cysteine protease inhibi-tors. This will be discussed in more detail later.

3.6.1Synthesis of Trifluoromethyl Ketones

Imperiali and Abeles devised a convenient synthesis of a b-amino alcohol thatserves as the basic building block for trifluoromethyl ketone derivatives containinga P1 side chain [32]. The choice of nitroalkane sets the P1 side chain of thedesigned inhibitor. As shown in Figure 3.12, for the synthesis of phenylalanineside chain at P1, (2-nitroethyl)benzene was condensed with trifluoroacetaldehydehydrate to provide b-nitroalcohol 27 in excellent yield. Reduction of the nitrogroup followed by treatment with concentrated HCl provided the amine salt 28 asa racemic mixture of syn/anti diastereomers. Peptide coupling of this amino alco-hol mixture with optically active acid 29 furnished peptide containing diastereo-meric alcohol. Oxidation of the mixture of trifluoromethyl carbinols with KMnO4

afforded inhibitor 20 as a diastereomeric mixture at the amine center [32].A stereoselective synthesis of a trifluoromethyl ketone containing a valine side

chain was reported by Edwards [33]. Addition of (trifluoromethyl)zinc reagents tothe amino acid-derived aldehyde, such as valinal derivatives, provided the trifluor-omethyl carbinols that can be oxidized with Dess–Martin periodinane to providethe trifluoromethyl ketone without epimerization. Veale et al. developed analternative route for optically active (2R,3S)-amino alcohol 32 [31]. As shown in

O

HN

SO O

ClO

NH

O

N

O NH

O

CF3Ki = 0.5 nM

23

O

NH

O

N

O NH

O

CF3Ki = 1.9 nM

24

MeO

O

NH

O

N

O NH

O

CF3

Ki = 13 nM

25

Ki = 15 nM26

N

N

NH

SMe

OO

OO N

HO

CF3

NH2

MeO

Figure 3.11 Structures of trifluoromethyl ketone-containing inhibitors.


Figure 3.13, condensation of nitroisobutane with trifluoroacetaldehyde provided amixture (syn/anti) of b-nitroalcohols 31. Fractional crystallization followed byreduction of the nitro group furnished anti-diastereomer 32 as a racemate. Themother liquors containing mostly syn-isomer can be epimerized to syn/anti mix-ture with K2CO3. Resolution of anti-diastereomer (�)-32 with D-tartaric acidprovided optically active amino alcohol 33 (2R,3S). In an alternative route, racemicanti-diastereomer 32 was converted to an oxazolidinone. Coupling of this oxazoli-dinone with (�)-menthyl chloroformate followed by recrystallization providedoptically active urethane 34. Treatment of 34 with aqueous KOH afforded optically

O2N

PhCF3

HO

H

OH+

K2CO3

50 °C

O2NCF3

Ph

OH

1. H2, Ra-Ni2. HCl

Cl- H3NCF3

Ph

OH+AcNH

CO2H

DCC, THF

HN

CF3

Ph

OH

NH

O

Me

O

KMnO4aq. NaOH

HN

CF3

Ph

O

NH

O

Me

O

20

(Diastereomeric mixture)

27

28

29

30

Figure 3.12 Synthesis of peptidyl trifluoromethyl ketone derivatives.

O2NCF3

OH OH1. Fractional crystallization2. H2, Ra-Ni

H2NCF3

(+)-32

D-tartaric acidEtOH

H2NCF3

OH

S

R

1. Triphosgene2. i-BuLi, (-)-menthyl chloroformate3. Fractional crystallization

_

H2NCF3

OH

S

RON

O

CF3

O

O

S

R

KOH, H2Odioxane

31 33 (2R,3S)

33 (2R,3S)34

O2N

CF3

O

H+

Figure 3.13 Synthesis of optically active b-aminotrifluoromethyl carbinols.


active 33 (2R,3S). Coupling of this amino alcohol with the appropriate acid fol-lowed by oxidation provided access to inhibitors with a trifluoromethyl ketone con-taining P1 isopropyl side chain.

3.7Peptidyl Boronic Acid-Based Inhibitors

Peptidyl boronic acids are of significant interest in structure-based design anddevelopment of serine protease inhibitors. Historically, simple alkyl and arylboronic acids were first shown to inhibit chymotrypsin and subtilisin [34–36]. TheX-ray crystal structures of subtilisin complexes of phenylboronic acid and phenyl-ethane boronic acid revealed that the aryl boronic acids form a tetrahedral adductwith the Ser195 residue, thus mimicking the putative tetrahedral transition statefor peptide cleavage [37,38]. Also, the aromatic group is nestled in the S1 subsite.This molecular insight suggested that boronic acids can be used as warheads withappropriate subsite-specific amino acid residues to design potent and specificinhibitors. Figure 3.14 depicts how boronic acid in the serine protease active sitemimics the transition state. Interestingly, the negative charge resides on boron, asopposed to the carbonyl oxygen of the actual transition state formed during pep-tide cleavage. Since the atomic size of carbon is comparable to that of boron, thetetrahedral adducts created by boronic acid more closely mimic the actual tetrahe-dral transition state located at the carbon center during peptide cleavage.The boronic acid-based inhibitor design strategy involves replacement of the

scissile bond at the cleavage site with a boronic acid functionality, as shown inFigure 3.14. For the design of potent inhibitors, it is important to incorporate notonly a P1 ligand but also additional complementary residues for enzyme

Peptidyl

HN B

P1 OH

Ser195

Peptidyl

HN B

P1 O

Ser195S1Substrate

binding site His57

OH

OH

OH

OH

NH

O P1'HN

P1

‡


NNH H

O H

Ser195

His57

NH

O P1'HN

P1

NN

H H

O

Ser195

Scissile bond

Tetrahedraltransition-state

mimic

O

P2

NH O

HN

P2'

O

ONH

P2

O

HN

P2'

O

Figure 3.14 Proteolysis by serine protease and boronic acid-based inhibitors.


secondary subsites. Incorporation of appropriate P1, P2, and P3 groups that fulfillspecificity requirements of a particular serine protease may result in tight-bindinginhibitors. Indeed, numerous peptidyl boronic acid-based potent and selectiveinhibitors have been designed. A number of review articles on boronic acid-derived serine protease inhibitors cover this material in detail [39–41]. In recentyears, boronic acid-based inhibitors particularly gained added attention since theFDA approval of bortezomib, a potent and selective proteasome inhibitor for treat-ment of multiple myeloma and mantle cell lymphoma [42,43].To improve specificity of boronic acid-based inhibitors, a-aminoalkyl boronic

acids corresponding to a-amino acids were first developed by Matteson et al.[44]. In particular, 1-acetamido-2-phenylethane boronic acid 36 in Figure 3.15was prepared as a transition-state analog for chymotrypsin. The choice of Pheside chain as P1 was logical as chymotrypsin and subtilisin possess a highlyspecific cleavage site that prefers a large hydrophobic amino acid residue in theS1 site. The affinity of chymotrypsin for both (R)- and (S)-acetamido-2-phenyl-ethane boronic acids was determined on the basis of the rates of hydrolysis ofmethyl hippurate. As it turned out, both isomers showed competitive inhibitionof chymotrypsin. The (R)-isomer binds more tightly than the (S)-isomer, whichis consistent with the stereochemical preference for the L-phenylalanine deriva-tive. In fact, chymotrypsin affinity for (R)-acetamido-2-phenylethane boronicacid was shown to be 14 000-fold greater than N-acetyl-L-phenylalanine amide[45]. Subsequently, a variety of peptidyl boronic acid-based potent inhibitors ofchymotrypsin and elastases were developed.In an effort to inhibit serine proteases leukocyte elastase, pancreatic elastase,

cathepsin G, and chymotrypsin, Kettner and Shenvi designed substrate-basedboronic acid-derived inhibitors [46]. In particular, P1 boro-Phe for chymotryp-sin, P1 boro-Ala for pancreatic elastase, and P1 boro-Val for leukocyte elastasewere selected. MeO-Suc-Ala-Ala-Pro was introduced as the P2 to P5 ligands asthis sequence is the best for the above proteases. As shown in Figure 3.16,these peptidyl boronic acid derivatives 38–40 showed very potent inhibitoryactivity against various serine proteases. Furthermore, inhibitory activity nicelycorresponded to the better substrate sequence for each protease. Kinetic stud-ies showed that inhibitors did not show competitive inhibition, but insteadexhibited “slow-binding inhibition.”

BOH

OHHN B

OH

OH

O

HN B

OH

OH

O

2-Phenylethane- boronic acid

Kd = 4 x 10-5 M

(R)-Acetamido- boro-PheKd = 2.1 x 10-6 M Kd = 5.3 x 10-5 M

(S)-Acetamido- boro-Phe

35 36 37

Figure 3.15 Structures of boronic acid-based serine protease inhibitors.

3.7 Peptidyl Boronic Acid-Based Inhibitors 79

Researchers at DuPont-Merck reported a variety of peptidyl boronic acid-basedthrombin inhibitors. Thrombin is a serine protease that catalyzes the conversionof fibrinogen to fibrin in the blood coagulation cascade. Furthermore, thrombin isan activator of platelets and other coagulation factors. As a result, thrombinbecame an attractive therapeutic target for drug development against pulmonaryembolism, thrombosis, and related diseases [47,48]. Thrombin inhibitors weredesigned based upon (D)-Phe-Pro-Arg, a substrate sequence of thrombin [49,50].Inhibitors containing other active warheads provided effective inhibitors with thissubstrate sequence. As shown in Figure 3.17, inhibitor 41 with a-amino Arg-boronic acid and P2 proline showed a Ki of 3.3 nM. Extension to P3 (D)-Phe pro-vided inhibitor 42 (Dup-714) with a Ki of 0.041 nM, an 80-fold contribution from(D)-Phe. Replacement of the N-acetyl group with N-Boc resulted in remarkablypotent inhibitor 43 with another 10-fold potency enhancement. Based upon theX-ray structure of Dup-714-bound thrombin, subsequently Quan et al. incorpo-rated a biaryl derivative in place of the Phe–Pro dipeptide [51]. As shown, thebiphenyl derivative 44 showed excellent potency against thrombin. Further incor-poration of an ortho-methyl group in the lipophilic S2 and S3 specificity pocketsresulted in inhibitor 45 with a fourfold potency enhancement.The X-ray cocrystal structure of 42-bound a-thrombin was determined to gain

additional molecular insight [52]. As shown in Figure 3.18, the boron atom of theinhibitor formed a covalent bond with the active site Ser195. Boron adopts a tetra-hedral geometry and the boronic acid nicely mimics the tetrahedral transitionstate of proteolysis. As expected, the basic groups of arginine are involved inhydrogen bonding interactions with the backbone carboxyl, water molecules, andAsp189 side chains located in the S1 specificity pocket.Peptidomimetic boronic acid-based hepatitis C virus (HCV) NS3/4A protease

inhibitors were designed and synthesized for treatment of chronic HCV infec-tions, which can lead to progressive liver damage, cirrhosis, and liver cancer [53].The NS3/4A serine protease plays a critical role in virus replication and becamean antiviral drug development target [54,55]. The X-ray crystallographic studies of

38 39 40(Boro-Ala)

Chymotrypsin Ki = 9100 nM Ki = 1200 nM Ki = 3.4 nM (0.16 nM final)

Elastase (Pan) Ki = 18 nM (0.32 nM final) Ki = 30 nM Ki = 270 nM

Elastase (Leu) Ki = 79 nM Ki = 15 nM (0.57 nM final) Ki = 350 nM

HN B

OH

OHO

N

O

HNO

NH

O CO2Me

(Boro-Val)

HN B

OH

OHO

N

O

HNO

NH

O CO2Me

(Boro-Phe)

HN B

OH

OHO

N

O

HNO

NH

O CO2Me

Ph

Figure 3.16 Structures of peptidyl boronic acid-derived serine protease inhibitors.


(Boro-Arg)

HN B

OH

OHO

NHN B

OH

OHO

N

O

HN

O

O

HN

H2N NH

41 Ki = 3.3 nM

Ph HN

H2N NH

42 Ki = 0.041 nM(Dup-714)

43 Ki = 0.004 nM

HN B

OH

OHO

N

O

HN

O

O

Ph HN

H2N NH

44 Ki = 1.7 nM

HN B

OH

OH

O

HN

H2N NH

45 Ki = 0.42 nM

HN B

O

OO

HN

H2N NH

Me H

Figure 3.17 Structures of peptidyl and peptidomimetic thrombin inhibitors.

Figure 3.18 X-ray cocrystal structure of inhibitor 42 (Dup-714) (carbon chain, magenta; boron,green) complexed with a-thrombin (PDB code: 1LHC).


NS3/4A protease revealed that the monomeric enzyme contains two domains: a tryp-sin-like fold and a zinc binding site. The substrate specificity of NS3/4A protease isdifferent from the cellular serine proteases. The peptide substrate shows a P1

cysteine residue. A wide range of NS3/4A protease inhibitors have been designed,including recent FDA approval of telaprevir and boceprevir containing a-ketoamidesas the warheads [56,57].A significant effort has also been devoted to the design of acyclic and cyclic

NS3/4A protease inhibitors that incorporate a-aminoalkyl boronic acids or cyclicboronates as warheads at the P1 site. As shown in Figure 3.19, the boronic acidderivative of boceprevir, compound 46, exhibited a Ki of 10 nM. Extension toinclude a P4 substituent provided compound 47 with a 50-fold improvement inpotency [58]. The X-ray structure of 47-bound NS3/4A protease revealed that cata-lytic Ser139 binds to the boron atom and mimics the tetrahedral transition state ofproteolysis. The P1 cyclobutylmethyl nicely nestles in the S1 subsite. Also, the P4

tert-butyl group with a sulfonamide cap occupied the S4 subsite. The sulfona-mide oxygen forms a hydrogen bond with Cys159 in the S4 region. Compounds48 and 49 with a carbamate containing P2 proline core showed very potentinhibitory activity [59]. The corresponding cyclic boronates also provided potent

N

HN

OO

HN

HN

BOH

OH

O

N

HN

OO

HN

HN

BOH

OH

ON

SOO

46 Ki = 10 nM 47 Ki = 0.2 nM

48 IC50 = 2 nM

N

HN

OO

HNO

BOH

OH

O

O

ON

49 IC50 = 23 nM

N

HN

OO

HNO

B

O

O

ON

50 IC50 = 43 nM

F

O

OH

N

HN

OO

HN

O

ON

O

O

BO

OH

Figure 3.19 Structures and activities of NS3/4A serine protease inhibitors.


inhibitors, as exemplified in compound 49 [60]. Similarly, macrocyclic inhibitor50 with an a-amino cyclic boronate showed good inhibitory activity [61]. The X-ray structure revealed the formation of a tetrahedral intermediate at the boroncenter with the catalytic Ser139.

3.7.1Synthesis of a-Aminoalkyl Boronic Acid Derivatives

The synthesis of optically active a-aminoalkyl boronic acid was developed byMatteson et al. [44]. As shown in Figure 3.20, (þ)-pinanediol benzylboronate (51)was homologated with (dichloromethyl)lithium, as described previously by Matte-son and Majumdar [62], to provide chloro-2-phenylethane boronate 52. Withoutpurification, this was treated with lithiumhexamethyldisilazane followed by aceticanhydride and acetic acid to provide optically active (R)-1-acetamido-2-phenylethaneboronate 54. Treatment with BCl3 furnished the boronic acid derivative 36. The(S)-1-acetamido-2-phenylethane boronate was prepared from (�)-pinanediolbenzylboronate following the same sequence of reactions.A modified synthesis of a-alkylboronic acid is shown in Figure 3.21. As

shown, deprotonation of dichloromethane at �100 �C followed by treatmentwith trimethyl borate provided methyl ester 55 [58]. Transesterification with(þ)-pinanediol followed by treatment with the appropriate Grignard reagentafforded chloro derivatives 57 and 58. Treatment of these chlorides with lithium-hexamethyldisilazane followed by treatment with HCl provided optically activeamine salts 59 and 60, which can be utilized for the synthesis of boronic acid-based inhibitors.The synthesis of a-amino cyclic boronates is shown in Figure 3.22. Chloro-

methyl boronate 61 was prepared from chloroiodomethane, as describedpreviously by Sadhu and Matteson [63]. This was converted to PMB ether 62.Homologation of 62 followed by treatment with (dichloromethyl)lithium, as

36

B

Ph

O

O

51

Li CHCl2 BO

O

52Ph

Cl LiN(SiMe3)2 BO

O

53Ph

(Me3Si)2N

Ac2O, AcOH

BO

O

Ph

HN

O

BCl3BOH

OH

Ph

HN

O

54

(R)-Acetamido- boro-Phe

Figure 3.20 Synthesis of (R)-acetamido-boro-Phe.


described by Matteson and Majumdar [62], furnished a-chloroboronate 63.Displacement of the chloride with lithiumhexamethyldisilazane followed by treat-ment with anhydrous HCl provided amine salt 64. Coupling of this amine withthe appropriate peptide or peptidomimetic acid furnished a carboxamide, whichupon treatment with isobutyl boronic acid and HCl provided the inhibitor witha-amino oxaborole as the P1 ligand [60].

1. LiN(SiMe3)2

nBuLiB

Cl

OMe

OMe

55

Cl

56

EtMgBr orCy-BuCH2MgBr

BO

O

R

Cl

R57 R = CH3

H2CCl

Cl

-100 °C to rt(MeO)3B

B

Cl

O

OCl

HO

HO

58 R = Cy-Bu

BO

OHCl·H2N 2. HCl in ether

59 R = CH360 R = Cy-Bu

Figure 3.21 Synthesis of optically active aminoalkyl boronic acid.

1. LiN(SiMe3)2

1. nBuLi, -78 °C

62

1. nBuLi, ICH2ClBO

OCl

63

H2CI

Cl

(iPrO)3B

2. HCl, dioxane

64

HO

HO

2.BO

OCl

BO

OPMBO

61nBuLi,PMB-OH

2. LiCHCl2, ZnCl2

PMBO

BO

OH3N+

HO

Cl-1. Peptide acid

HATU, DIPEA2. iBuB(OH)2,

HCl

OB

HN

O

Peptide

OH

65

Figure 3.22 Synthesis of inhibitor with a-amino oxaborole as the P1 ligand.


3.8Peptidyl a-Ketoamide- and a-Ketoheterocycle-Based Inhibitors

As we discussed earlier, the electron-withdrawing effect of the trifluoromethylgroup in trifluoromethyl ketone-derived serine protease inhibitors activated thecarbonyl group of the peptidyl ketones toward nucleophilic addition of thehydroxyl group in the active site. The electron-withdrawing effect of ester andamide functionalities was also utilized in the design of a-ketoester- or a-ketoamide-derived transition-state inhibitors [64,65]. Powers and coworkers reported thedesign of potent inhibitors of elastase [66,67]. Ocain and Rich [68] designed a-ketoamide-derived inhibitors of aminopeptidases. The use of a-ketoesters is limitedin drug design because of their potential hydrolytic and metabolic instability [69].However, inhibitors incorporating a-ketoamides as warheads have been widelyutilized in the design of a variety of protease inhibitors. A number of quality reviewscover the design strategies, biological evaluation, X-ray structural studies, andclinical development of these inhibitors [70–72].The design strategy involved the replacement of the scissile bond at the cleavage

site with an a-ketoamide functionality. As shown in Figure 3.23, the mechanismof action involves a nucleophilic attack by the Ser195 hydroxyl group of thrombinat the ketoamide carbonyl group to form a covalent adduct, which turned out to bestable and reversible. For the design of potent inhibitors, it is important to incor-porate P10, P2, and P3 groups that fulfill specificity requirements [73].A range of HCV NS3/4A protease inhibitors were designed and synthesized

incorporating a-ketoamide templates at the scissile site. NS3/4A protease plays acritical role in the HCV replication cycle, and there has been an intensive effort inthe design of small-molecule NS3/4A protease inhibitors [74,75]. Han et al. at

Peptidyl

HN

P1

S1Substrate

binding site

O

Ser195

OHN

P1

Tetrahedral transition-state

mimic

ONH

P2

O

HN

Ketoamide warhead

NH

O

N N H

His57

H

OO

Asp102

HO

Peptidyl

HN

P1

S1

Ser195

HN

O

N N H

His57

H

OO

Asp102

OO

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond (Substrate)(Inhibitor)

Enzyme

Figure 3.23 Design strategy of a-ketoamides as a-thrombin serine protease inhibitors and theirbiological mode of action.

3.8 Peptidyl a-Ketoamide- and a-Ketoheterocycle-Based Inhibitors 85

DuPont Pharmaceuticals reported the design of peptidyl ketoamide inhibitorsbased upon hexapeptide lead structure 66, which was shown to inhibit NS3/4Aprotease with an IC50 of 2.5 mM [76,77]. Based upon the structural insight fromthe X-ray structure of NS3/4A protease, it was determined that the S1 specificitypocket is shallow and hydrophobic. Researchers examined the feasibility of ethyland allyl side chains with ketoamide templates as the P1 ligand. As shown in Fig-ure 3.24, truncation of hexapeptide to tetrapeptide with a ketoamide serine trapresulted in inhibitors with improvement in potency over the hexapeptide lead. Fur-ther modification of P1 with a 2,2-difluoroethyl group provided inhibitor 68 thatshowed more than seven-fold improvement in potency [76]. Structure-based designled to a variety of potent acyclic and cyclic inhibitors with ketoamide templates, asexemplified in compounds 69 and 70 [78–81]. The ketoamide functionality is criti-cal to serine protease inhibitory activity, and the mechanism of NS3/4A proteaseinhibition is similar to the one shown in Figure 3.23. Presumably, the ketoamidecarbonyl is attacked by the Ser195 hydroxyl group of NS3/4A protease to form a

HN

OH

SH

O

ON

O

NHO

NH

O HN

ONH

CO2H

O

HN

O

ON

O

NHO

NH

O HN

ONH2

CO2H

HN

O

OHN

O

NHO

NH

ON

HN

FF

N

HN

O

ON

O

HN

Boc

HN

NHO

O

Ph

HN

O

ON

O

HN

Boc

NH2

O

NMe2

O

66 IC50 = 2.5 μM

67 IC50 = 420 nM

68 IC50 = 60 nM

69 IC50 = 3.8 nM

70 IC50 = 30 nM

HO2CHO2C

OO OH

HN

O

Figure 3.24 Structures and activities of ketoamide-based NS3 protease inhibitors.


covalent adduct that is stable and reversible. The covalent tetrahedral intermediateis stabilized by the residues of His57 and Asp81 in the NS3 protease active site.Boceprevir and telaprevir are two NS3 protease inhibitor drugs that evolvedthrough structure-based design strategies and received FDA approval for the treat-ment of HCV infection [56,57]. Further details are provided in a later section.The a-ketoamide functionalities are structural features of bioactive natural prod-

ucts. As shown in Figure 3.25, cyclotheonamide A [82] and poststatin [83] possessthe a-ketoamide functionality and both compounds were shown to inhibit serineproteases. Cyclotheonamide A (71) was isolated from the Japanese marine spongeTheonella sp. It showed inhibitory activity against serine proteases such asa-thrombin and trypsin [84]. However, it is a more potent inhibitor of trypsinthan a-thrombin. Poststatin was isolated from the culture broth of Streptomycesviridochromogenes, and showed inhibitory activity against prolyl endopeptidase.The a-ketoamide functionality is critically important for the bioactivity of thesenatural products as the reduction of the keto carbonyl abolished much of theenzyme inhibitory properties.The X-ray cocrystal structure of cyclotheonamide-bound thrombin showed that

the active site Ser195 hydroxyl group formed a covalent bond with the ketoamidegroup of cyclotheonamide A (Figure 3.26) [84]. This resulted in a tetrahedral inter-mediate mimicking the transition state of peptide cleavage. The inhibitor’s Argside chain occupied the S1 specificity pocket and formed hydrogen bonds withAsp189. The proline moiety filled in the S2 subsite. As can be expected, reductionof the electrophilic carbonyl of the a-ketoamide functionality to the correspondingalcohol abolished the inhibitory activity against a-thrombin. The structural insightinto the binding interactions of cyclotheonamide A in the thrombin active site fur-ther motivated the design and synthesis of cyclotheonamide derivatives and newthrombin inhibitors incorporating ketoamide and structural variants of ketoamidefunctionalities [85].Edwards et al. developed peptidyl a-ketoheterocycles as a new template for

inactivation of elastase [72,86]. The design of a-ketoheterocycles was based uponthe premise that the ketone carbonyl group would be significantly activated by theelectron-withdrawing effect of the heterocyclic ring. The nucleophilic attack of the

72 Poststatin IC50 = 30 nM (prolyl endopeptidase)

HN

O

OHN

O

NH3

HN

OO N

H

CO2-

+

HN

NH

OO

HN

Ph

O

O NH O

NH

NO

OHC

HO

D

71 Cyclotheonamide A Ki = 180 nM for α-thrombin Ki = 23 nM for trypsin

NH

HN NH2

Figure 3.25 Structures and activities of cyclotheonamide A and poststatin.


active site Ser195 hydroxyl group would then result in a tetrahedral intermediatethat can mimic the proteolytic transition state. They synthesized a series of inhibi-tors with a-ketoheterocycles that demonstrated the feasibility of such design con-cept [86–88]. As shown in Figure 3.27, tripeptidyl a-ketobenzoxazole 73 inhibitedhuman neutrophil elastase (HNE) with a Ki of 3 nM. The importance of keto func-tionality was demonstrated as the corresponding inhibitor with an a-hydroxyben-zoxazole 74 showed a 7000-fold reduction in potency. The ketooxazoline-derivedinhibitor 75 displayed very potent activity against HNE. Replacement of Cbz-Valin 73 with N-Ac-Ala provided inhibitor 76 with a Ki of 73 nM. Kinetic analysis and

Figure 3.26 X-ray cocrystal structure of cyclotheonamide 71 (carbon chain, green) complexedwith a-thrombin (PDB code: 1TMB).

HN

O

O

N

O

HN

O

OPh O

NHN

OH

O

N

O

HN

O

OPh O

N

73 IC50 = 3 nM

74 IC50 = 21000 nM

HN

O

O

N

O

HN

O

OPh O

N

75 IC50 = 0.6 nM

HN

O

O

N

O

HN

O

O

N

76 IC50 = 73 nM

Figure 3.27 Structures and activities of peptidyl a-ketoheterocycle-derived inhibitors.


Lineweaver–Burk plots of inhibitor 73 established that the inhibitor was reversibleand competitive in nature. The X-ray cocrystal structure of 76-bound PPE wasdetermined to obtain insight into the ligand–binding site interactions. PPE sharessimilar substrate specificity as HNE and structural studies show topological simi-larities between the two enzymes. The stereoview of the reported structureshowed that the hydroxyl group of Ser195 formed a covalent bond with the ketonecarbonyl of inhibitor, the benzoxazole nitrogen is involved in a hydrogen bondinginteraction with His57, and the P1 Val residue occupied the S1 site [86].The utility of an a-ketoheterocycle-derived template was further demonstrated

in the design of other serine protease inhibitors. Tsutsumi et al. [89,90] prepared aseries of potent prolyl endopeptidase inhibitors incorporating a range of substi-tuted a-ketoheterocycles. As shown in Figure 3.28, inhibitor 77 showed potentinhibitory activity against prolyl endopeptidase. Costanzo et al. [91] designed aseries of peptidyl ketoheterocycles with a Me(D-Phe)–Pro–Arg tripeptide motif asinhibitors of thrombin and trypsin. Inhibitor 78 displayed potent inhibitory activ-ity against thrombin and showed excellent selectivity against plasmin, tissue-typeplasminogen activator, and streptokinase. Its selectivity against trypsin was moder-ate (16-fold). Inhibitor 79, with a 1-amidinyl-3-piperidine side chain as an argininemimic and an a-ketothiazole group, showed very potent activity against a-throm-bin and exhibited more than 500-fold selectivity against trypsin [92,93]. Althoughcompound 79 appears nonpeptidic in nature, it showed poor oral bioavailability indogs. However, it showed effectiveness in rat arterial models upon intravenousadministration.

NO

N

ON O

Ph

S

77 IC50 = 4.0 nM(prolyl endopeptidase)

HN

O

O

N

O

HN

Me

Ph HN

H2N NH

78 (RWJ-50353) Ki = 0.19 nM (thrombin)

S

N

HN

O

O

N

H2N NH

S

NN

NO

O

Ph

79 Ki = 1.2 nM (thrombin)

Cl

Figure 3.28 Structures and activities of inhibitors with a-ketoheterocycles.


An X-ray structure of 78-bound thrombin was reported by Matthews et al. [94].As can be seen in Figure 3.29, the active site Ser195 hydroxyl group formed acovalent bond with the inhibitor’s ketone carbonyl group. The P1

0 benzothiazolenitrogen in the S10 subsite formed a hydrogen bond with the catalytic His57residue. The carbonyl carbon of inhibitor formed a tetrahedral geometry. TheD-Phe–Pro–Arg motif in the structure showed similar inhibitor–thrombin interac-tions as observed for inhibitor 42-bound thrombin, as shown in Figure 3.18.The methyl group is not shown in the crystal structure.

3.8.1Synthesis of a-Ketoamide and a-Ketoheterocyclic Templates

Han et al. developed a synthetic route to aminoalkyl a-ketoamide derivatives [77].This process can provide access to aminoalkyl a-ketoamides conveniently fromprotected a-amino acids, as shown in Figure 3.30. Reduction of Weinreb amide80 provided an aldehyde, which was converted to cyanohydrin 81. Acid-catalyzedhydrolysis of the cyanide followed by Boc protection afforded a-hydroxy acid 82.Acid 82 was coupled with allylamine to provide allylamide 83. Removal of the Bocgroup followed by coupling of the resulting amine with pentapeptide acid affordedthe corresponding amide. Dess–Martin oxidation of the hydroxy amide providedthe a-ketoamide derivative 84 in good yield.Chen and coworkers reported the synthesis of a-ketoamides using the Henry

reaction as the key step [78]. As shown in Figure 3.31, nitrobutane 85 was con-densed with glyoxalic acid to provide the corresponding nitroaldol product. Reduc-tion of the nitro group followed by Boc protection resulted in Boc derivative 86.Nitrobutane as the starting material provided a propyl side chain for the P1 ligand.The a-hydroxy acid 86 can be converted to a variety of a-ketoamides. As shown,hydroxy acid 86 was converted to a-hydroxyamides 87 and 88 with ammonium

Figure 3.29 X-ray cocrystal structure of inhibitor 78 (carbon chain, green) complexed witha-thrombin (PDB code: 1TBZ).


chloride and glycine ester, respectively. Glycine amide 88 was converted to targetketoamide derivatives by coupling with suitable acids followed by modifiedMoffatt oxidation to the desired product 89.An asymmetric synthesis of a-ketoamide derivatives was reported by Han et al.

during their synthesis of NS3 protease inhibitors [77]. As shown in Figure 3.32,Sharpless asymmetric aminohydroxylation of a,b-unsaturated ester 90 afforded78% yield of amino alcohol derivative 91 with 83% ee [95,96]. Recrystallizationimproved optical purity to 95% ee. Reductive cleavage of the Cbz group followed bycoupling of the resulting amine with a pentapeptide acid provided the correspondingcoupling product 92. This was converted to an a-hydroxyallylamide 93. Dess–Martinoxidation provided a-ketoamide derivative 84 and reaction yields were very good.

HN

NOMe

Boc

O

Me

1. LAH, THF

2. Me2C(OH)CN, Et3N, DCM

HN

CNBoc

OH

1. aq. HCl, dioxane

2. Boc2O, aq. Na2CO3

HN

CO2HBoc

OH AllylamineBOP, i Pr2NEt

HN

Boc

OH

O

HN

2. Peptide-OHBOP, iPr2NEt

HN

Peptide

O

O

HN

3. Dess-Martin oxidation

80 81

8283

84

1. Aq. HCl

Figure 3.30 Synthesis of a-ketoamide template from a-amino acid.

O2N1. Glyoxalic acid, Et3N2. H2, Pd-C then Boc2O

HN

CO2HBoc

OH 1. NH4ClEDCI, NMM

2. Aq. HCl

H2NOH

O

NH2

HCl. H2NOH

O

HN

1. EDCI, NMM Gly-OBn2. aq. HCl

O OBn

1. Peptide-OH coupling2. DMSO, DDC, Cl2CHCO2H

HN

Peptide

O

O

HN

OBnO

8687

88 89

85

HCl.

Figure 3.31 Synthesis of a-ketoamide template from nitroalkane.


For synthesis of inhibitors with ketobenzoxazoles, Edwards et al. utilized con-densation of a nitrile with an aminophenol to construct the benzoxazole ring [86].As shown in Figure 3.33, aldehyde 94, which can be prepared by reduction of aWeinreb amide, was converted to cyanohydrin 95. Treatment of 95 with anhydrous

CO2Me(DHQ)2PHAL,

HN

CO2MeCbz

OH

1. H2, Pd-C2. Peptide-OH coupling

HN

CO2MePeptide

OH

2. Allylamine, BOP, iPr2NEt

HN

Peptide

OH

O

HN

HN

Peptide

O

O

HN

Dess-Martin oxidation

90 91

9293

84

K2[OsO2(OH)4],

1. Aq. LiOH

CbzNClNa

Figure 3.32 Asymmetric synthesis of a-ketoamides from a,b-unsaturated ester.

HN

O

O

N

O

HN

O

O

N

HN

O

HCbzMe2C(OH)CN

Et3N, DCM

HN

OH

CNCbz

1. HCl, EtOH2. o-aminophenol

HN

OH

Cbz

O

N

2. Ac-Ala-Pro-OH EDCI, HOBt

HN

OH

O

N

O

HN

O

O

N

DMSO, EDCI,Cl2CHCO2H

94 95

9697

76

1. H2, Pd-C

Figure 3.33 Synthesis of a-ketobenzoxazole-derived HNE inhibitors.


HCl and ethanol afforded the corresponding iminoether hydrochloride, which wascondensed with 2-aminophenol to provide the a-hydroxybenzoxazole 96. Reduc-tive removal of the Cbz group followed by coupling of the resulting amine withpeptide carboxylic acid furnished peptidyl a-hydroxybenzoxazole 97. Oxidation ofthe alcohol using modified Moffatt conditions provided inhibitor 76. This generalprocedure was utilized for the synthesis of a variety of inhibitors with a-ketoben-zoxazoles and other heterocycles.Costanzo et al. developed a practical route to a-ketobenzothiazole-containing

thrombin inhibitors [91]. As depicted in Figure 3.34, reaction of Weinreb amide98 with lithiobenzothiazole 99 resulted in the corresponding ketone, which wasthen reduced to an a-hydroxybenzothiazole. Although an a-ketobenzothiazole wasthe desired product, the ketone functionality was reduced to alcohol to avoid epi-merization during subsequent reactions. The removal of the Boc group followedby coupling with a peptide carboxylic acid afforded the hydroxybenzothiazolederivative. Dess–Martin oxidation followed by treatment with HF and anisole pro-vided inhibitor 78 (RWJ-50353). This method can be adapted to the synthesis ofinhibitors with other a-ketoheterocycles.

3.9Design of Serine Protease Inhibitors Based Upon Heterocycles

A number of specific heterocyclic compounds have been developed as serine pro-tease inhibitors. These heterocyclic inhibitors are typically acylating agents andinhibit enzymes irreversibly. One of the major drawbacks of such inhibitors isthat the acyl enzymes, once formed, can be rapidly deacylated to restore enzyme

HN

NOMe

Boc

O

Me

1.

2. NaBH4

98

99

100NH

NH

NHTs

N

SLi

3. CF3CO2H

H2NOH

NH

NH

NHTs

S

N

2 TFA

Cbz(Me)-D-Phe-Pro-OH, DCC, HOBt

HN

OH

O

N

ON

Cbz

Ph NH

NH

NH

S

N

Ts

HN

O

O

N

O

HN

Me

Ph NH

H2N NH

S

N 1. Dess-Martin2. HF, anisole

Me

10178 (RWJ-50353)

Figure 3.34 Synthesis of a-ketobenzothiazole-derived thrombin inhibitors.

3.9 Design of Serine Protease Inhibitors Based Upon Heterocycles 93

activity. As a result, geometric and electronic effects were manipulated to developconsiderably more stable heterocyclic inhibitors. A review by Powers et al. nicelycovers a host of heterocyclic inhibitors [6]. We will outline only isocoumarin- andb-lactam-based heterocyclic inhibitors.

3.9.1Isocoumarin-Derived Irreversible Inhibitors

Isocoumarin-derived inhibitors were designed and developed by Harper and Pow-ers [97]. Initially, 3,4-dichloroisocoumarin (102, Figure 3.35) was shown to be ageneral serine protease inhibitor [98]. It did not inhibit aspartic acid proteases,but showed some inhibitory activity against cysteine proteases. To improvepotency and selectivity within serine proteases, substituted derivatives were pre-pared. Substitutions at the 3-, 4-, and 7-positions provided access to selectiveinhibitors. Inhibitors with 3-alkoxy-4-chloroisocoumarins are generally very potentacylating agents [99,100]. Inhibitor 103, with 7-amino and 3-methoxy substitution,provided a potent inhibitor against HLE [97]. Inhibitor 104, with a 3-isothioureido-propoxy substituent, resulted in a very potent inhibitor against bovine trypsin [98].The reactivity of 3-substituents agreed with the substrate preference at the P1 siteof various serine proteases. The X-ray structural studies showed that the substitu-ents at the 3-position are involved in interactions in the S1 site [99,100].The proposed mechanism of action of isocoumarin-based inhibitors involves the

opening of the isocoumarin ring in 7-amino-substituted isocoumarins 105 by theactive site serine hydroxyl group (Figure 3.36). The resulting acyl-enzyme interme-diate 106 is converted to a quinone imine methide intermediate 107. This highlyreactive intermediate can react with active site His57 and form an irreversible

O

ClCl

OH

3,4-Dichloroisocoumarin kobsd/I = 8920 M-1s-1 (HLE)

102

O

ClOMe

OHN

ONH

Ph

Ts

103

kobsd/I = 190 000 M-1s-1 (HLE)

O

ClO

OH2N

104

SH2N

NH+

kobsd/I = 410 000 M-1s-1

(bovine trypsin)

12

3456

7

Figure 3.35 Structures and activities of substituted isocoumarin-derived inhibitors.


complex 108, or it may react with solvent nucleophile to provide 109. The X-raystructure of 7-amino-4-chloro-3-methoxyisocoumarin with PPE showed the forma-tion of an acyl-enzyme complex with Ser195 [99]. An acetate from the solvent wasalso shown to displace the chlorine.

3.9.2b-Lactam-Derived Irreversible Inhibitors

b-Lactam antibiotics, which include penicillins, cephalosporins, and related com-pounds, are the most widely used antimicrobial agents. This class of antibioticsexerts their mechanism of action by inhibiting transpeptidase, the key enzymeresponsible for bacterial cell wall biosynthesis [101]. The development of bacterialresistance to b-lactam antibiotics is a major problem. The mechanism of resist-ance involves the opening of the b-lactam ring by a group of serine hydrolasesknown as b-lactamases [102]. Clavulanic acid is an effective b-lactamase inhibitor.Logically, researchers at Merck showed that the benzyl ester of clavulanic acid 110can inhibit serine protease, and elastase (Figure 3.37) [103]. Subsequently, neutralcephalosporin esters were shown to inhibit human leukocyte elastase, porcinepancreatic elastase, and a-chymotrypsin. As a result, b-lactams, including cephalo-sporins, azetidinones, and penams, were modified to provide irreversible serineprotease inhibitors. A considerable effort was devoted to the synthesis of inhibi-tors of human leukocyte elastase based upon the b-lactam template.The proposed inhibitory mechanism of cephalosporin derivative L-659286 (114)

involved the attack by the active site Ser195 at the b-lactam carbonyl to form atetrahedral intermediate 115, as shown in Figure 3.38 [104,105]. The opening ofthe b-lactam ring leads to the formation of a reactive acyl-enzyme intermediate116. Michael addition of the active site His57 to this intermediate could lead toa stable enzyme–inhibitor complex 117. This intermediate can also undergo

O

ClOR

OHN

RCO2RO

Cl

OHN

R

Ser195HO

Ser195

CO2RO

ON

R

Ser195

(acyl enzyme)

(quinone imine methide)

His57

Im (His57)O

CO2R

OHN

R

Ser195

Nu (solvent)O

CO2R

OHN

R

Ser195

105 106

107

108109

Figure 3.36 Mechanism of inhibition of serine proteases by 7-amino-substituted isocoumarins.

3.9 Design of Serine Protease Inhibitors Based Upon Heterocycles 95

acyl-enzyme hydrolysis to regenerate the active enzyme. The X-ray structuralstudies showed support of this mechanism [105].The b-lactam-based design of thrombin inhibitors was reported by Han et al.

[106]. Thrombin is known to preferentially cleave substrates at the scissile site con-taining the basic amino acids arginine or lysine at P1. Based upon this preference,b-lactam derivatives containing an alkyl guanidine at the 3-position of azetidinone

N

O

O

OO

Ph

OH

110

Benzyl clavulanate

NO

RCONH

111

Cephalosporins

S

XCO2R

112

NO

RCONH

CO2R

SR

Penams

113

NO

R

R

Azetidinone

X

Figure 3.37 Structures of various b-lactam derivatives.

NO

MeO SOO

S

O NN

NN

OOH

Me

Ser195

OHNO

MeO SOO

S

O NN

NN

OOH

Me

O

Ser195

_

115114 (L-659 286)

N

MeOS

OO

O N

O

Ser195

OHN

MeOS

OO

O N

O

Ser195

ON

N

His57

His57

hydrolysis

Active enzyme

116117

Figure 3.38 Mechanism of inhibition of serine proteases by b-lactam.


were shown to be potent thrombin inhibitors in a time-dependent manner [106].Compound 118 in Figure 3.39 showed potent thrombin inhibitory activity. Further-more, this inhibitor prevented thrombin-induced clot formation in human plasma.A series of azetidinone derivatives were designed to inhibit cytomegalovirus

protease by Borthwick et al. [107]. The design of inhibitors was based upon thepremise that the substituents at the 3- and 4-positions on the azetidinone ringwould occupy the S1 and S3 sites, respectively. The substituent on the nitrogenwould access the S10 site. A representative compound 119 showed low micromolarinhibitory potency against human cytomegalovirus (HCMV) protease. Azetidinonederivatives were also designed to inhibit prostate-specific antigen. Inhibitor 120showed inhibitory activity in the nanomolar range [108]. Presumably, it exerted itsinhibitory effect by forming an acyl-enzyme complex with the active site Ser195.

3.10Reversible/Noncovalent Inhibitors

The development of serine protease inhibitors by replacing the scissile bond of apeptide substrate with appropriate serine traps yielded potent and orally activeFDA-approved drugs against NS3 protease. A number of serine protease inhibitordrugs also advanced to several stages of clinical development. As described earlier,structure-based design of inhibitors involving serine traps generally leads topotent inhibitors with a degree of reliability. However, issues of covalent or non-covalent, reversible or irreversible inhibition may be critically relevant for particu-lar therapeutic applications. For instance, a slow-binding drug may not beappropriate in the development of thrombin- or factor Xa-based inhibitor drugs.In this case, the design of reversible, fast-acting inhibitors may be preferable.

NO

HN

NO

O

O

HO

O

118 IC50 = 12 nM(thrombin)

119 Ki = 5.7 μM(HCMV protease)

120 IC50 = 226 nM(prostate-specific antigen)

NO

NH

Me

O

NH

NH

H2N

HCl O

O

OH Me O CO2t Bu

OPh

O

O

Ph

Ph

Figure 3.39 Structures and activities of substituted b-lactam-derived inhibitors.

3.10 Reversible/Noncovalent Inhibitors 97

Based upon molecular insight into ligand–binding site interactions and X-raystructural studies, design and development of potent and selective noncovalentserine protease inhibitors have evolved [109]. A number of small-molecule, non-covalent serine protease inhibitor design approaches are highlighted here.Bajusz et al. reported substrate-based design of potent thrombin inhibitor

D-Phe-Pro-Arg-H (121), as shown in Figure 3.40 [110]. The electrophilic aldehydefunctionality served as the serine trap and formed a covalent bond with the serinehydroxyl group in the active site. Bajusz et al. also prepared inhibitor 122, wherethe aldehyde functionality was replaced with hydrogen [111]. Wiley et al. subse-quently showed that agmatine-derived inhibitor 122 retained significant anticoagu-lant activity in vitro and in vivo [112]. Furthermore, it was shown that inhibitor 122maintained improved selectivity against digestive enzyme trypsin and fibrinolyticenzymes plasmin, n-tPA, and urokinase. Agmatine’s binding affinity is about3–4 kcal/mol weaker than the arginine derivative 121, which forms a covalentbond in the active site. Presumably, agmatine exerts its activity through noncova-lent interactions in the thrombin active site. The X-ray crystal structure of inhibitor122 and thrombin complex revealed that agmatine side chain nestled in the S1specificity pocket of thrombin where the guanidinium functionality formed stronghydrogen bonds with Asp189. The D-Phe–Pro dipeptide unit showed very similarinteractions as the corresponding arginine inhibitor 121. To improve potency andselectivity, Wiley et al. explored the replacement of the agmatine with substituted

HN

O

HO

N

OH2N

Ph HN

H2N NH

Kass = 5.4 x108 l/mol (thrombin)Selectivity

Trypsin: 5.1-fold Plasmin: 390-fold

121

HN

O

N

OH2N

Ph HN

H2N NH

Kass = 5.5 x106 l/mol (thrombin)

SelectivityTrypsin: 33-fold

Plasmin: 4600-fold

122

HN

O

N

OH2N

Ph

H2N NH

SelectivityTrypsin: 130-fold

Plasmin: 26 000-fold

123

Kass = 6.8 x108 l/mol (thrombin)

Figure 3.40 Structures, inhibitory activities, and selectivity profiles of inhibitors.


amidinobenzylamine derivatives [113]. It turned out that p-amidinobenzyl-amine derivative 123 restored the inhibitor’s binding affinity to a similar levelas covalent inhibitor 121. Presumably, the improvement in potency is due toimprovement in noncovalent interactions in the S1 specificity pocket. Interest-ingly, p-amidinobenzylamine also improved selectivity against trypsin and fibri-nolytic enzymes.Lyle et al. at Merck investigated the effect of serine trap deletion from a potent

covalent inhibitor developed in their laboratories [114]. Inhibitor 124 (Figure 3.41)with trans-aminocyclohexylglycine ketoamide as the P1 ligand showed excellentpotency against thrombin and also exhibited good selectivity against bovinetrypsin [115]. The compound was designed based upon a D-Phe-Pro-Arg-H motif,where arginine side chain was replaced with an aminocyclohexyl moiety specifi-cally designed to interact with S1 specificity pocket for thrombin over trypsin. Theother important feature in the structure was the presence of the a-ketoamidefunctionality, which formed a covalent bond with the catalytic serine hydroxylgroup. The deletion of the a-ketoamide warhead from inhibitor 124 resulted incompound 125 with a Ki of 5 nM. Surprisingly, inhibitor 125 showed only 55-foldreduction of potency. This inhibitor still retained significant thrombin inhibitoryactivity. Furthermore, inhibitor 125 displayed much improved selectivity againsttrypsin, presumably due to electronic and hydrophobic fit by the aminocyclohexylring in the S1 specificity pocket. The X-ray structural studies revealed that, in theabsence of the covalent bond between Ser195 and ketoamide warhead in 124, thecyclohexyl ring in inhibitor 125 adopted a more favorable conformation in the S1pocket. This allowed better hydrogen bonding and hydrophobic interactions in theS1 site, thus compensating for the loss of binding energy due to the covalent bondwith serine in inhibitor 124. As shown in Figure 3.42, inhibitor 125 was involvedin forming four strong hydrogen bonds in the S1 and S2 pockets. A comparisonwith a 124-bound structure showed that the Glu192 side chain adjusted itself byrotating �90� around its Ca��Cb bond to maximize the hydrophobic interactionwith the cyclohexyl ring in the S1 site. Furthermore, the aminocyclohexyl groupwas “tension-free” and was involved in a stronger electrostatic interaction withAsp189 [114].

124

Ki = 0.09 nM (human thrombin)Ki = 1150 nM (bovine trypsin)

125

Ki = 5 nM (human thrombin) Ki = 11 000 nM (bovine trypsin)

HN

O

N

O

HN

Ph

Me

OHN

OMe

NH2

HN

O

N

O

HN

Ph

Me

NH2

Figure 3.41 Structures, inhibitory activities, and selectivities of inhibitors.


Subsequent structure-based design efforts toward incorporation of P3 lipophilicgroups led to an improvement in potency. As shown in Figure 3.43, inhibitor126, with a D-diphenylalanine P2 ligand, showed thrombin inhibitory activityof 0.1 nM [116].

Figure 3.42 X-ray crystal structure of inhibitor 125 (carbon chain, green) complexed witha-thrombin (PDB code: 1TOM).

126 Ki = 0.1 nM (X = H)127 Ki = 2.5 pM (X = SO2CH2Ph)

HN

O

N

O

HN

Ph

NH2

Ph

X

128 Ki = 2.1 nM (thrombin)Ki = 6100 nM (trypsin)

HN

O

N

N

NH2

MeO

Me

NHSN

O

O

129 Ki = 0.37 nM (thrombin)Ki = 3300 nM (trypsin)

HN

O

N

O

NH2

F

N N

N

Cl

Figure 3.43 Structures, thrombin inhibitory activities, and selectivities of inhibitors.


Consistent with the precedence of a D-diphenylalanine as a good P2 ligand, theaffinity of inhibitor 126 improved 50-fold compared with 125 [117]. Further incor-poration of phenylmethanesulfonamide at P3 resulted in 127 with furtherimprovement of Ki to 2.5 pM. The sulfone oxygens were expected to form hydro-gen bonds with the Gly219 backbone NH. It showed good oral absorption in dogs.Compound 126 showed better in vivo efficacy than compound 127. Further struc-ture-based iteration cycles led to very potent and selective noncovalent inhibitors128 and 129 showing good efficacy and pharmacokinetic properties in laboratoryanimals [118,119].Diederich and coworkers designed structurally rigid, nonpeptide, noncovalent

thrombin inhibitors by using interactive structure-based design strategies. Asshown in Figure 3.44, benzamidine derivative 130 was designed to form hydrogenbonds with Gly216 and Tyr60 as well as to make hydrophobic interactions in theS1 and S2 sites [120]. Compound 130 was prepared in a racemic form, and showeda Ki of 18mM for thrombin. Removal of the methylene group from the benzamidi-nium needle resulted in inhibitors with an improved potency. Racemic compound131 exhibited a Ki value of 90 nM and showed about eightfold selectivity againsttrypsin [121].In an effort to ascertain the binding mode, X-ray cocrystal structural studies

with thrombin were carried out with racemic inhibitor 131. It turned out that the(3aS,4R,8aS,8bR)-enantiomer was found in the active site. A schematic representa-tion of the X-ray structure is shown in Figure 3.44. The benzamidinium sidechain shows hydrogen bonding interactions with Asp189 in the S1 site. One ofthe succinimide carbonyls forms a hydrogen bond with the Gly216 amide NH in

N N

H

H

Me

NH2

NH

O

O

130 Ki = 18 μM (thrombin)

(racemic)

N N

H

H

O

O

131 Ki = 90 nM (thrombin)

NHH2N

O O

(racemic)

N

N

H

H

O

O

NHHN

OO

OH

Tyr60

HN

OGly216

O O

Asp189

S1 site

ON H

Gly219

HON

H

Ser195

D-pocket

P-pocket

Figure 3.44 Structures of polycyclic inhibitors and schematic representation of the X-ray struc-ture of 131-bound thrombin.


the distal pocket (D-pocket). The piperonyl ring oxygen forms a hydrogen bondwith Tyr60 in the proximal pocket (P-pocket) of the enzyme.Based upon these ligand–binding site interactions, Diederich and coworkers

subsequently incorporated an alkyl substituent at C-1 to improve selectivity againsttrypsin. As shown in Figure 3.45, racemic inhibitor 132 with a C-1 isopropylgroup improved Ki of thrombin. Optical resolution provided enantiomeric inhibi-tors. Inhibitor ent-(þ)-132 with (1R,3aS,4R,8aS,8bR)-configuration showed a Ki of7 nM against thrombin and selectivity of 740-fold against trypsin. ent-(�)-132showed significantly reduced Ki against thrombin. The X-ray crystal structure ofent-(þ)-132-bound thrombin showed that the C-1 isopropyl substituent nestlesnicely in the hydrophobic P-pocket and the piperonyl ring oxygen maintainshydrogen bonding interactions with Tyr60.Ganellin and coworkers reported a general approach to designing noncovalent

and reversible cholecystokinin-inactivating serine protease inhibitors for possibletreatment of obesity [122]. The investigators started with dipeptide lead 133(Figure 3.46), with micromolar affinity to endopeptidase, and successively opti-mized P3 and P1 ligands to provide inhibitor 134 with a nanomolar Ki [123]against tripeptidyl peptidase II (TPPII). Further optimization with an indolinederivative resulted in butabindide (135) as a potent and reversible inhibitor.

N N

H

HO

132 Ki = 13 nM (thrombin)

NHH2N

O O

(racemic)

N N

H

HO

Ki = 7 nM (thrombin)

NHH2N

O O

ent-(+)-132

N N

H

HO

Ki = 5600 nM (thrombin)

NHH2N

O O

ent-(-)-132

and

Selectivity: 740-fold (trypsin) Selectivity: 21-fold (trypsin)

Figure 3.45 Structures, activities, and selectivities of inhibitors.


The structure of endopeptidase is unknown. However, the current strategy to non-covalent inhibitor design starting from the first hydrolysis products may be a gen-eral strategy for endopeptidase inhibitor design.Greco et al. at Johnson & Johnson developed nonpeptide reversible inhibitors of

serine protease cathepsin G [124]. This chymotrypsin-like enzyme is stored in theazurophilic granules of neutrophils and released upon degranulation. Cathepsin Ghas been suggested as a target for a variety of inflammatory conditions. As shownin Figure 3.47, the initial high-throughput screening of compound librariesresulted in a weak lead 136, which showed low micromolar activity. Interestingly,this nonpeptide compound exhibited competitive, reversible inhibition kinetics.An X-ray structure of 136-bound cathepsin G was determined. As can be seen, thestructure revealed that the compound was bound in the active site and it pos-sessed an (R)-configuration. The 2-naphthyl ring occupied the S1 site, whereas the

NO

HN

OH2N

Ki = 570 nM (TPPII)

133

NO

HN

OH2N

Ki = 80 nM (TPPII)

134

NO

HN

OH2N

Ki = 7 nM (TPPII)

135

Figure 3.46 Structures and activities of TPPII inhibitors.

Figure 3.47 Structures of cathepsin G inhibitors and X-ray crystal structure of inhibitor 136 withcathepsin G (magenta; PDB code: 1KYN).


1-naphthyl ring nestled in the S2 specificity pocket. The compound formed a num-ber of hydrogen bonds with His57, Gly193, and Lys192 in the active site. Subse-quently, structure-based incorporation of substituents on the naphthyl ringresulted in inhibitor 137 as a competitive, reversible inhibitor with nanomolaraffinity. Compound 137 showed good selectivity against chymotrypsin and otherserine proteases [124,125].A variety of other carboxamide inhibitors were designed so that the substituents

could nestle in the S3 and S4 subsites. As shown in Figure 3.48, inhibitors weresynthesized by deprotonation of diethyl phosphonate 138 followed by reactionwith naphtho[2,3-c]furan-1,3-dione to provide carboxylic acid 139. Coupling of thisacid with appropriate secondary amines provided convenient access to racemicinhibitors 140 [124].

3.11Conclusions

Serine proteases play important roles in numerous biological processes.Deregulation of these enzymes lead to many human diseases. Over the years, avariety of serine protease inhibitor design tools and strategies have been devel-oped. X-ray crystallography has been extensively utilized in studying theseenzymes as well as in structure-based design of a variety of inhibitors. This chap-ter has outlined design strategies that guided the development of various covalentinhibitors bearing electrophilic warheads to inhibit serine protease activity. Also,

PO

EtO OEt

i-BuLi, THF

O

O

Othen

O

PEtO

O

OEt

OHO

1. R1R2NH, DCC2. TMSBr, Py3. 1N HCl

O

PHO

O

OH

ON

138

139

140

R2

R1

Figure 3.48 Synthesis of carboxamide derivatives of b-ketophosphonic acid.


the structure-based design of a range of peptidomimetic scaffolds and the chemi-cal synthesis of a variety of warheads utilized in the development of approved ther-apeutics have been highlighted.

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78 Chen, K.X., Njoroge, F.G., Pichardo, J.,Prongay, A., Butkiewicz, N., Yao, N.,Madison, V., Girijavallabhan, V. (2005)Design, Synthesis, and Biological Activityof m-Tyrosine-Based 16- and 17-Membered Macrocyclic Inhibitors ofHepatitis C Virus NS3 Serine Protease.J. Med. Chem. 48, 6229–6235.

79 Liu, R., Abid, K., Pichardo, J., Pazienza, V.,Ingravallo, P., Kong, R., Agrawal, S.,Bogen, S., Saksena, A., Cheng, K.-C.,Prongay, A., Njoroge, F.G., Baroudy, B.M.,and Negro, F. (2007) In vitro antiviralactivity of SCH446211 (SCH6), a novelinhibitor of the hepatitis C virus NS3serine protease. J. Antimicrob. Chemother.,59, 51–58.

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114 Lyle, T.A., Chen, Z.G., Appleby, S.D.,Freidinger, R.M., Gardell, S.J., Lewis, S.D.,Li, Y., Lyle, E.A., Lynch, J.J., Mulichak, A.M., Ng, A.S., NaylorOlsen, A.M., andSanders, W.M. (1997) Synthesis,evaluation, and crystallographic analysis ofL-371,912: a potent and selective active-sitethrombin inhibitor. Bioorg. Med. Chem.Lett., 7, 67–72.

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116 Tucker, T.J., Lumma, W.C., Mulichak, A.M., Chen, Z.G., NaylorOlsen, A.M., Lewis,S.D., Lucas, R., Freidinger, R.M., and Kuo,L.C. (1997) Design of highly potentnoncovalent thrombin inhibitors thatutilize a novel lipophilic binding pocket inthe thrombin active site. J. Med. Chem.,40, 830–832.

117 Cheng, L.F., Goodwin, C.A., Schully, M.F.,Kakkar, V.V., and Claeson, G. (1992)Synthesis and biological activity ofketomethylene pseudopeptide analogs asthrombin inhibitors. J. Med. Chem., 35,3364–3369.

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119 Staas, D.D., Savage, K.L., Sherman, V.L.,Shimp, H.L., Lyle, T.A., Tran, L.O.,Wiscount, C.M., McMasters, D.R.,Sanderson, P.E.J., Williams, P.D., Lucas,B.J., Krueger, J.A., Lewis, S.D., White, R.B., Yu, S., Wong, B.K., Kochansky, C.J.,Anari, M.R., Yan, Y., and Vacca, J.P.(2006) Discovery of potent,selective 4-fluoroproline-based thrombininhibitors with improved metabolic

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124 Greco, M.N., Hawkins, M.J., Powell, E.T.,Almond, H.R., Corcoran, T.W., deGaravilla, L., Kauffman, J.A., Recacha, R.,Chattopadhyay, D., Andrade-Gordon, P.,and Maryanoff, B.E. (2002) Nonpeptideinhibitors of cathepsin G: optimization ofa novel beta-ketophosphonic acid lead bystructure-based drug design. J. Am. Chem.Soc., 124, 3810–3811.

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4Design of Proteasome Inhibitors

4.1Introduction

The proteasome, a multicatalytic proteolytic complex localized in the nucleusand cytosol, is responsible for the selective degradation of intracellular proteins.This ubiquitin-dependent proteolysis in eukaryotes regulates a variety of normalcellular functions, including signal transduction, cell cycle control, transcriptionregulation, apoptosis, stress regulation, and immune responses [1]. The ubiquitin-proteasome system is involved in degradation of abnormal proteins, includingmisfolded and mutated proteins that might otherwise disrupt cellular homeostasis[2]. As can be expected, defects within this pathway have been implicated in thepathogenesis of diseases, including inflammation, neurodegenerative diseases,and cancers. Selective inhibition of proteasomes emerged as an attractive targetfor drug discovery. The recent approval of bortezomib (Velcade) for the treatmentof relapsed multiple myeloma set the stage for drug development in academic andpharmaceutical laboratories against proteasomes in other human diseases, mostprominently cancers and neurodegenerative disorders [3,4].

4.2Catalytic Mechanism of 20S Proteasome

The proteolytic component of the ubiquitin-proteasome system is the 26S protea-some, which consists of two 19S regulatory particles, responsible for substraterecognition and unfolding, and a core particle termed as 20S proteasome. Theproteolytic activity of the N-terminal threonine hydrolase occurs in a 700 kDacylindrical shaped structure containing 28 protein subunits arranged in fourstacked rings in a a7b7b7a7 manner. The a-rings possess the regulatory assemblyand the two inner b-rings contain the proteolytic active sites. The proteasomeexhibits at least three distinct proteolytic activities, including the chymotrypsin-like, trypsin-like, and post-glutamyl peptide hydrolytic activity [5]. The catalyticmechanism of threonine protease is shown in Figure 4.1. The X-ray crystallo-graphic studies revealed that the hydroxyl group of N-terminal catalytic threonine

113


1 serves as the catalytic nucleophile and attack at the carbonyl carbon of thescissile bond is assisted through a water molecule [6,7]. The resulting tetrahedraloxyanion is stabilized by hydrogen bonding with the Gly47 amide NH. The tetra-hedral intermediate collapses to acyl-enzyme intermediate, which undergoeshydrolysis to amino acid degradation product and regenerates the catalytic Thr1.

4.3Proteasome Inhibitors

Structurally diverse proteasome inhibitors have been reported. A number of recentreviews covered the development of various proteasome inhibitors and applicationsof such inhibitors [8–10]. The majority of inhibitors fall within two large groups:covalent and noncovalent inhibitors. Since proteasome is a threonine protease, inprinciple, inhibitors can be designed by attachment of an electrophilic “warheadgroup” to a small peptide fragment that can bind to the substrate recognition site.The design of common classes of inhibitors with warhead groups such as chloro-methyl ketones, peptide aldehydes, trifluoromethyl ketones, and a-ketocarbonylsfor serine protease inhibitors was described in Chapter 3. A large number of pro-teasome inhibitors were designed based upon these electrophilic warhead groups.The majority of inhibitors are covalent and irreversible inhibitors. However, somecovalent inhibitors with warheads such as ketocarbonyls and boronates form a

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond

HN

H2NO P1

O P1'

O

HN

NH

P2'

OP2

OH +

NH

O P1'HN

P1‡

Tetrahedral transition state

1 2 3

O H

NH

O P1'HN

P1

NO HH

OH

H

O

NO HH

O

H

H

H+

3 Acyl enzyme

OHN

P1 O

NO HH

OH

H

2

Thr1

N H

HN

Gly47

Gly47

Figure 4.1 Catalytic mechanism of 20S proteasome.

114 4 Design of Proteasome Inhibitors

reversible covalent bond. Noncovalent proteasome inhibitors are reversible bynature. The design of early serine and threonine protease inhibitors with electro-philic traps was reviewed by Powers et al. [11]. In this section, we will outline thedesign of boronate inhibitors, natural product inhibitors containing c-lactam-b-lac-tone cores, and noncovalent inhibitors.

4.3.1Development of Boronate Proteasome Inhibitors

Peptidyl aldehyde inhibitors are, in general, not suitable for drug development dueto their high reactivity and issues of stability. Compound 4 in Figure 4.2 showedgood proteasome inhibitory activity. The X-ray crystal structure of inhibitor 4 withproteasome revealed that the aldehyde functionality formed a hemiacetal adductwith the active site threonine hydroxyl group [12,13]. Preliminary optimization ofligands showed that a leucine side chain was preferred at the P1 position and lipo-philic groups can be accommodated in the P2 and P3 positions. Compound 5 wasthe most potent among a number of inhibitors prepared. Adams et al. also investi-gated other warheads such as trifluoromethyl ketone, ketobenzoxazole, and dike-toamide inhibitors. Interestingly, incorporation of boronic acid in compound 6

Cbz

HN

NH

HN

H

O

O

O

4 Ki = 4 nM

Cbz

HN

NH

HN

H

O

O

O

5 Ki = 0.015 nM

Cbz

HN

NH

HN B

O

O

6 Ki = 0.03 nM

OH

OH

NC(CH2)8 NH

HN B

O

O

7 Ki = 8 nM

O

O

HN

NH2

O2NN

NNH

HN B

O

O

8 Ki = 0.62 nM

OH

OH

NBortezomib

Figure 4.2 Structures and activities of proteasome inhibitors.

4.3 Proteasome Inhibitors 115

resulted in more than 100-fold potency enhancement over peptidyl aldehydeinhibitor 4 [14]. A related peptidyl boronic acid 7 has also shown potent protea-some inhibitory activity [15]. Peptidyl boronic acid 6 showed excellent selectivityover thiol protease cathepsin B (Ki¼ 6.1mM, 200,000-fold selectivity). Further opti-mization of inhibitor 6 provided inhibitor 8 with reduced molecular weight andhigh selectivity over common serine proteases. Successful clinical development ofinhibitor 8 culminated in the FDA approval of bortezomib (Velcade). Subsequentinvestigation addressing the side effects of Velcade resulted in potent andselective second-generation boronate inhibitors 9 and 10 (Figure 4.3) withimproved properties [16,17]. Boronates form reversible adducts with protea-some; however, the dissociation rates are much slower than the proteasome–peptidyl aldehyde complex. The off rate of bortezomib is so slow in cell cultureassay that it is essentially considered irreversible inhibition. Further details ofbortezomib development are described later.

4.3.2Development of b-Lactone Natural Product-Based Proteasome Inhibitors

Natural products play a very important role in proteasome research. Lactacystin (11)is one of the first natural products that showed significant proteasome inhibitoryactivity [18,19]. Lactacystin was isolated from a strain of Streptomyces and acted as aprodrug in vivo. It spontaneously transformed into cell-permeable, active clasto-lacta-cystin-b-lactone (Figure 4.4) known as omuralide (12). Omuralide potently inhibitedchymotrypsin-like (CT-L) proteasome activity in low nanomolar concentration (CT-Lproteasome activity, IC50¼ 49nM) [20,21]. The X-ray crystal structure of yeast protea-some and omuralide complex revealed that opening of the b-lactone ring by thenucleophilic hydroxyl group of the catalytic threonine generated ester adduct 13 (Fig-ure 4.4) [22]. The resulting a-hydroxy functionality appears to form a hydrogen bondwith the amine functionality of the threonine and prevented inhibitor–threonine ester(13) hydrolysis. The resulting a-hydroxy functionality appears to form hydrogenbonds with Phe168 and Thr21. The isopropyl side chain of omuralide nicely packedthe S1 specificity pocket. These noncovalent interactions apparently played an impor-tant role in omuralide’s selectivity. The omuralide family of natural products and theirstructural analogs have shown more specific proteasome inhibitory activity than thepeptidyl aldehyde, but less specific than the epoxy ketone-derived natural products.

NNH

HN B

OOH

O

9 IC50 = 3.8 nM

OH

OH NH

HN B

O

O

10 IC50 = 1.6 nM

OH

OH

Figure 4.3 Structures and potencies of second-generation boronate inhibitors.


Furthermore, the b-lactone natural products [23] do not inhibit cysteine and serineproteases, except for cathepsin A and cytosolic tripeptidyl peptidase II.A number of other b-lactone natural products have shown proteasome inhibi-

tory activity. Salinosporamide A (14, Figure 4.5), also known as marizomib, wasisolated from a marine actinomycete Salinospora tropica [24]. It showed potent

Salinosporamide A14

Thr1

NH

OO

O

OH

HClNH

O O

O

OH

O

H3N

O

HCl

+

NH

OO

O

OH

O

N

O

H

HH2

+

15

16

-

IC50 = 2.6 nM

Me Me

Me

Figure 4.5 Structure of salinosporamide A and its mechanism of proteasome inhibition.

NH

OO

O

OH

Lactacystin11

Omuralide12

NH

OO

O

OH

O

NH2 O

HOH

H

Thr1

NH

O O

O

OH

O

H2N

O

H

S1

OHN

O

H

Thr21

O

HN

Phe168Ph

NH

HOO

O

OH

S

CO2H

HN O

13

Acyl-enzymeintermediate

Figure 4.4 Structures of lactacystin and omuralide and schematic representation of the X-raystructure of omuralide-bound 20S proteasome.


activity against rabbit proteasome (CT-L proteasome activity, IC50¼ 2.6 nM). Itexhibited enhanced activity over omuralide (12). The mechanism of action of sali-nosporamide A involves the opening of the b-lactone ring by the nucleophilichydroxyl group of threonine generating an ester adduct 15, which concomitantlyforms the tetrahydrofuran ring 16 after displacement of the chlorine atom, asshown in the figure 4.5. The prolonged proteasome inhibitory activity of salino-sporamide A is possibly due to full protonation of the amino group of the catalyticthreonine and stabilization of the acyl-enzyme adduct by the tetrahydrofuran ring.Salinosporamide A has been undergoing clinical trials for the treatment of multi-ple myeloma and other cancers [25].Another family of b-lactone-containing proteasome inhibitors includes belacto-

sins A (17) and C (18) shown in Figure 4.6. These natural products were isolatedfrom Streptomyces sp. UCK14 [26]. They exhibited comparable activity (CT-L activ-ity of rabbit 20S proteasome, IC50� 210 nM). The mechanism of action is similarto omuralide, where opening of the b-lactone ring and acylation of the catalyticthreonine hydroxyl group resulted in an acyl-enzyme intermediate [27]. Structuralmodification of this class of natural products provided more potent derivatives 19(homobelactosin C, CT-L activity, IC50¼ 48 nM) and the b-lactone derivative 20(IC50¼ 5.7 nM) [28].

4.3.3Development of Epoxy Ketone-Derived Inhibitors

Antimicrobial natural products eponemycin (21) and epoxomicin (22) in Figure4.7 showed potent antitumor activity specific against B16 murine melanoma[29,30]. These natural products showed very specific proteasome inhibitory

17 Belactosin A

HO2CNHHN

O

OO

O

H2N

18 Belactosin C

HO2CNHHN

O

OO

O

H2N

NH

O

OO

HN

20

O

NH

Cbz19 Homobelactosin C

derivative

BnO2CNH

HN

O

OO

O

NH

( )2

Cbz

IC50 = 210 nM IC50 = 210 nM

IC50 = 48 nM IC50 = 5.7 nM

Ph

Figure 4.6 Structures of belactosins A and C and their derivatives.


activity. The X-ray structure of yeast proteasome complexed with epoxomicin (22)revealed that the N-terminal threonine of the proteasome formed an irreversiblemorpholine adduct 23 with the epoxy ketone [31]. Epoxomicin was shown to haveminimum off-target effect. The unusual formation of the morpholine adduct 23 isshown in Figure 4.7. The epoxy ketone was presumably responsible for its extra-ordinary specificity as cysteine and serine proteases lack a-amine functionalityand can not form such an adduct.Subsequent exploration of P2 to P4 residues resulted in derivatives with potent

and improved properties over bortezomib. The tetrapeptide epoxy ketones 24 (YU-101, Figure 4.8) and 25 (PR-171, also known as carfilzomib) were very potent andselective proteasome inhibitors [32,33]. Tetrapeptide 25 was approved by the FDAfor treatment of relapsed and refractory multiple myeloma in 2012 [34]. Furtherdetails of carfilzomib development are described later.

HN

NH

O OH

O

OHO

21 Eponemycin

HN

NH

O

OOO

OHO

HN

O

22 EpoxomicinNO

Thr1

HN

NH

OO

OHO

HN

ONO

NH

OH

HO O

23 (Morpholine adduct)

IC50 = 8-10 nM IC50 = 30-80 nM (CT-L)

Figure 4.7 Structures of eponemycin, epoxomicin, and morpholine adduct.

HN

NH

O

OO

OHN

O PhHN

Ph

24 (YU-101)

HN

NH

O

OO

OHN

O PhHN

Ph

25 Carfilzomib (PR-171)

O

Me

O

NO

IC50 CI)L-TC( Mn 21-5 = 50 = 6 nM (CT-L)

Figure 4.8 Structures of YU-101 and carfilzomib.


4.3.4Noncovalent Proteasome Inhibitors

A number of cyclic and acyclic peptides were identified as noncovalent protea-some inhibitors. Among them, natural product TMC-95A (26), shown in Figure4.9, and its derivatives have shown potent proteasome inhibitory activity [35,36].TMC-95A and related compounds were isolated from the fermentation broth ofApiospora montagnei Sacc. TC 1093. TMC-95A displayed the most potent proteasomeinhibitory activity against CT-L proteasome (IC50¼ 5.4 nM), T-L (IC50¼ 200nM), andPA (IC50¼ 60nM). The X-ray structural analysis of TMC-95A-bound yeast protea-some revealed that the natural product binds tightly to the proteasome active site ina noncovalent manner [37]. The catalytic Thr1 does not form any covalent interactionwith TMC-95A, but it forms hydrogen bonds with the (Z)-propenyl amide NH in theS1 pocket. Its binding blocks the substrate’s access to the catalytic threonine.The noncovalent inhibition resulted in an antiparallel b-sheet formation betweenTMC-95A and the amino acid residues in the S1 and S2 specificity pockets. TMC-95A showed good selectivity against calpain, cathepsin, and trypsin. Extensive struc-tural modifications of TMC-95A resulted in numerous analogs and these have beenreviewed. Other cyclic peptides that exhibited noncovalent proteasome activityinclude argyrin A [38] and scytonemide A [39].A number of acyclic peptide and peptidomimetic derivatives have been shown

to inhibit proteasome activity. As shown in Figure 4.10, ritonavir (27) and a num-ber of modified statine derivatives, as represented in compound 28, potentlyinhibited proteasome activity [40,41] in a reversible manner. Structural modifica-tion of various acyclic peptides resulted in capped peptide inhibitors [42]. A repre-sentative compound 29 exhibited potent proteasome inhibitory activity. The X-raycocrystal structures of 29 and yeast proteasome showed that the inhibitor binds tothe yeast proteasome in a noncovalent manner [43]. The C-terminal cap binds tothe S1 pocket and the amino acid residues bind to the S2 and S3 subsites. The

NH

HO

NH

O NH

O

NH

HOHO

O

HN

O

O

O

CONH2

HN

OH

NH

O

HN

O

N

OH

OH

O N

O

26

HNO

H

OH

O

NH

NH

O

Gly23

Thr1

OHSer20

O

H2N

H

OH

HN

O

Thr21

S1 site

O

O

S3 site

NH

OTMC-95AAla49IC50 = 5.4 nM (CT-L)

Figure 4.9 Structure of TMC-95A and schematic representation of the X-ray structure of 26-bound yeast proteasome (PDB code: 1JD2).


catalytic threonine did not interact with the inhibitor. The inhibitor structures andmolecular insight into the ligand–binding site interactions provided a number ofstructural platforms for the structure-based design of noncovalent proteasomeinhibitors.

4.4Synthesis of b-Lactone Scaffold

Armstrong and Scutt employed a chelation-controlled stereoselective chlorinationfollowed by cyclization strategy for the synthesis of b-lactone scaffold 30 of(þ)-belactosin A [44]. Barlaam et al. previously developed similar strategy for thesynthesis of b-lactone 31 [45]. As outlined in Figure 4.11, succinate derivative 33was prepared by a stereoselective alkylation (dr 93: 7) of oxazolidinone 32 with tert-butyl bromoacetate followed by removal of the oxazolidinone. Treatment of succi-nate derivative 33 with LiHMDS followed by addition of CCl4 to the resulting eno-late 34 provided b-chlorocarboxylic acid 35. Exposure of 35 to aqueous NaHCO3

afforded desired b-lactone 30.During the synthesis of lactacystin and salinosporamide A, Corey and coworkers

prepared b-lactone scaffold from the corresponding b-hydroxy acids [46,47]. Asshown in Figure 4.12, diol 36 was converted to aldehyde 37 using standard protec-tion, deprotection, desulfurization, and oxidation reaction sequence. Reaction ofaldehyde 37 with isopropenylmagnesium bromide in the presence of trimethyl-chlorosilane afforded the desired allyl alcohol derivative 38 as a single isomer.

OHN

Ph

OH

O

Ph

NH

ONH

N

OS

N

HN

Ph

OH

O

O

NH

NH

Cbz

NH

OMe

O

NH

OMe

OH

HNNH

O

N

NH

O

O

HN

OO

NH

Cl

27 Ritonavir

28

(IC50 = 900 nM)

29

(IC50 = 15 nM)

SN

Figure 4.10 Structures of peptidomimetic and peptide-derived noncovalent inhibitors.

4.4 Synthesis of b-Lactone Scaffold 121

N

O

PMB

CO2MeTBSOO

Me

N

O

PMB

CO2MeTBSO

Me

HO

N

O

PMB

CO2HHO

Me

HO

N

O

PMBMe

O

OHO

MgBr

TMSCl

BOP-ClEt3N

37

3839

40

N

OMeS

Me PMB

HO

CO2MeHO

36

NH

OMe

HO

11O

SOH

CO2H

NH LactacystinO

Figure 4.12 Synthesis of lactacystin b-lactone.

HO

O

CO2But

LiHMDSCCl4, -78 oC

OLi

O

O

OButLi

H

H

HO

O

HtBuO2C

H

HCl

O

O

CO2But

NaHCO3

33

30

O N

O O

Ph

32

1. NaHMDS,tert-butyl

bromoacetate

OO

tBuO2C

31

34

2. LiOH-H2O2

35

30

Figure 4.11 Stereoselective synthesis of b-lactone 30.


Hydrogenation and TBS deprotection followed by saponification of compound 38provided the required hydroxycarboxylic acid 39 for lactonization. Treatment ofhydroxycarboxylic acid 39 with BOP-Cl in the presence of Et3N provided the desiredb-lactone 40 in 90% yield, which was eventually transformed into lactacystin 11.Larionov and Meijere have synthesized b-lactone core of belactosins A and C

using a novel domino acylation/b-lactone strategy [48]. As depicted in Figure 4.13,Mukaiyama aldol reaction of (Z)-silyl ketene acetal 41 with ethyl glyoxylate follow-ing a protocol developed by Evans et al. [49] provided compound 43 with highenantioselectivity and diastereoselectivity (99% ee, syn/anti >40: 1). Selectivehydrolysis of ethyl ester 43 was achieved using 10% aqueous HCl to afford therequired substituted malic acid derivative 44 in 74% yield. Coupling of substitutedmalic acid derivative 44 with amine 45 using EDC, HOAt, and TMP providedb-lactone core of belactosin (46) in 71% yield through a domino acylation/b-lactonereaction sequence.

4.5Synthesis of Epoxy Ketone Scaffold

Crews and coworkers prepared epoxy ketone scaffold of epoxomicin by epoxidationof the respective a,b-unsaturated ketone using alkaline H2O2 following a similar pro-cedure utilized for the synthesis of the epoxy ketone scaffold of dihydroeponemycin

SPh

OOH

O

EtO

SPh

OOH

O

HO

CbzHN

HN

NH3

CO2BnO

Cl

CbzHN

HN

NH

CO2BnO

O

O

H

O

EDC, HOAt,TMP

Sn(OTf)2

(10 mol%)

10% aq. HCl

41

43

44

46

45

N

O

N

O

Ph Ph42

SPh

O SiMe3

HCOCO2Et42 (11 mol%)

Figure 4.13 Domino acylation/b-lactone strategy.

4.5 Synthesis of Epoxy Ketone Scaffold 123

[50,51]. As shown in Figure 4.14, enone 48 was prepared by the reaction of propene-2-yl lithium, prepared in situ by the treatment of 2-bromo-1-propene witht-BuLi, with Boc-leucine Weinreb amide 47. Treatment of enone 48 with H2O2 inthe presence of iPr2NEt provided corresponding epoxy ketones 49 and 50 in 1:1.7diastereomeric ratio. Deprotection of Boc group of 50 using trifluoroacetic acid fol-lowed by coupling of the resulting amine with tripeptide carboxylic acid 51 affordedthe TBS-protected epoxomicin 52.Zhou et al., during their work on the development of proteasome inhibitors with

improved oral bioavailability, have prepared epoxy ketone scaffold by stereospecificepoxidation of allyl alcohol using t-BuOOH in the presence of VO(acac)2 followedby oxidation of the resulting epoxy alcohol [52]. As shown in Figure 4.15, reduc-tion of a,b-unsaturated ketone 53 using NaBH4 and CeCl3�7H2O provided thecorresponding allyl alcohols 54 and 55 in 98% yield with a 6:1 diastereomericratio. Treatment of this mixture with t-BuOOH in the presence of VO(acac)2 pro-vided the epoxy alcohols 56 and 57 in a similar diastereomeric ratio, which weresubsequently used in the next step without any further purification. Oxidation ofepoxy alcohol mixture using Dess–Martin periodinane provided the required epox-ide 58 in 27% yield over two steps.Williams and coworkers have developed a method for the synthesis of epoxy

ketone precursor (61) from optically pure allene 59 and utilized it in the synthesisof epoxomicin [53]. As outlined in Figure 4.16, reaction of allene 59 with DMDOfollowed by treatment of the resulting spirodiepoxide 60 with Bu4NN3 afforded

Boc

HN

N

O

OBoc

HN

O

Br

t-BuLi

+

H2O2, PhCN

48

49 50

NH

N

O

O

O

HN

OTBS

OH

O

51

NH

N

O

O

O

HN

OTBS

NH

O

52O

O

1. TFA

2. HATU, HOAt,iPr2NEt

iPr2NEt

47

(ratio 1 : 1.7)

Boc

HN

O

O Boc

HN

O

O

acid 51

Figure 4.14 Synthesis of epoxy ketone by enone epoxidation.


azido ketone 61 in 54% yield along with minor diastereomeric product (dr 3:1,overall yield 73%). Azide group of 61 was converted to its corresponding amine.Coupling of the amine with the corresponding acid provided epoxomicin precur-sor 62 in 86% yield. Compound 62 was treated with TBAF followed by MsCl toobtain the corresponding mesylate, which was subsequently transformed into thedesired epoxy ketone by treatment with K2CO3.

OTBSH

59

DMDOH

60

OO

OTBS

Bu4NN3

N3

OOH

OTBS61

NH

N

O

O

O

HN

OtBu

NH

O

62O

OHOTBS

1. TBAF2. MsCl, iPr2NEt3. K2CO3

NH

N

O

O

O

HN

OtBu

NH

O

63O

O

Figure 4.16 Spirodiepoxide ring-opening strategy to epoxy ketone.

Cbz

HN

O

Cbz

HN

OH

Cbz

HN

OH

+

Cbz

HN

OH

Cbz

HN

OH

O O

VO(acac)2

t-BuOOH

53 54 55

56 57

(ratio = 6:1)

NaBH4

CeCl3.7H2O

+Cbz

HN

O

O

58

Dess-Martin

Figure 4.15 Stereospecific epoxidation of allyl alcohol.

4.5 Synthesis of Epoxy Ketone Scaffold 125

4.6Conclusions

Proteasomes are critically important in regulating many processes, including celldivision, cell death, signal transduction, and immune surveillance. The design ofproteasome inhibitors has become an important area of anticancer therapy devel-opment. As described in this chapter, the design of early proteasome inhibitorsrelied upon the warhead approaches outlined in Chapter 3. The FDA-approvedproteasome inhibitor, bortezomib exploited a novel boronate warhead and providedvalidation of proteasomes as important anticancer targets. This chapter has out-lined the mechanism of action of a variety of b-lactone- and epoxide-containingnatural products and described the design of selective proteasome inhibitors, includ-ing carfilzomib. This chapter has also depicted synthetic schemes of b-lactone andepoxide warheads that can be utilized in the design of next-generation proteasomeinhibitors with clinical potential.

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52 Zhou, H.-J., Aujay, M.A., Bennett, M.K.,Dajee,M., Demo, S.D., Fang, Y., Ho,M.N.,Jiang, J., Kirk, C.J., Laidig, G.J., Lewis, E.R.,Lu, Y., Muchamuel, T., Parlati, F., Ring, E.,Shenk, K.D., Shields, J., Shwonek, P.J.,Stanton, T., Sun, C.M., Sylvain, C., Woo,T.M., and Yang, J. (2009) Design and synthesisof an orally bioavailable and selective peptideepoxyketone proteasome inhibitor (PR-047).J. Med. Chem., 52, 3028–3038.

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References 129

5Design of Cysteine Protease Inhibitors

5.1Introduction

Cysteine proteases, also known as thiol proteases, are proteolytic enzymes respon-sible for the degradation of proteins [1]. These proteases are widely distributed innature, existing in viruses, bacteria, mammals, fungi, and nearly all plants. Theseenzymes are categorized into three distinct classes based upon their sequencehomology [2,3]: the papain family, the caspase family, and the Picornaviridae fam-ily. The papain family of proteases is the most well known and extensively studied[4]. Cysteine proteases have been implicated in the pathogenesis of numerous dis-ease states, including inflammatory, respiratory, cardiovascular, musculoskeletal,CNS, and cancer. Logically, the design and synthesis of cysteine protease inhibi-tors has been the subject of great interest in medicinal chemistry. The develop-ment of selective inhibitors has been a major challenge as cysteine proteasesbelong to the papain superfamily.The active site of cysteine proteases has some similarities to serine proteases. As

shown, the active site of a cysteine protease is comprised of a catalytic triad of Cys,His, and Asn. The proteolytic mechanism involves the formation of a thiolate–imidazolium ion pair, which provides a highly nucleophilic cysteine thiol. Theattack of the thiolate anion at the amide carbonyl of the scissile bond results in atetrahedral transition state (Figure 5.1), which is stabilized by the oxyanion hole[5]. The tetrahedral transition state breaks down to an acyl-enzyme intermediateand the first cleavage product. Subsequent hydrolysis of the acyl-enzyme interme-diate leads to a second cleavage product and free enzyme.Over the years, many cysteine protease inhibitors were designed by attaching

appropriate electrophilic warheads to the recognition sequence of peptide sub-strates. The warheads utilized in cysteine protease inhibitors include substitutedmethyl ketones, aldehydes, epoxides, aziridines, haloketones, Michael acceptors,and others. The basic strategy for the design of these inhibitors is similar to thatfor the design of serine protease inhibitors, which is described previously. Thedesign of drug-like and selective cysteine protease inhibitors has been reported in anumber of reviews [2,5–8]. Structure-based design of cysteine protease inhibitors

131


resulted in a number of classes of selective inhibitors with clinical potential. We willoutline here the design of cysteine protease inhibitors based upon Michael acceptorscaffolds and noncovalent small-molecule reversible cysteine protease inhibitors asthese inhibitors have received considerable attention in drug development.

5.2Development of Cysteine Protease Inhibitors with Michael Acceptors

Peptidyl or peptidomimetic derivatives containing Michael acceptor electrophilesare an important class of cysteine protease inhibitors [5,6]. These inhibitors arespecific irreversible inhibitors for cysteine proteases and a number of inhibitorshave shown clinical potential. A variety of electrophilic warheads have been devel-oped, including vinyl sulfones, a,b-unsaturated carbonyl derivatives, and relatedcompounds. The inhibitor design strategies involve the replacement of the sub-strate’s scissile amide bond carboxyl with an appropriate Michael acceptor group.In this design, the inhibitor generally mimics the substrate binding from P1 to P3and extends to the S1 specificity pocket. As shown in Figure 5.2, the mechanismof inhibition involves Michael addition of the active site thiolate anion on theb-carbon of the Michael acceptor warhead. Subsequent protonation of the a-carb-anion results in stable thioether adduct [9]. The mechanism was supported by theobservation that a stoichiometric amount of chloride ion was released when achlorinated a,b-unsaturated derivative was employed [10].One of the first inhibitors with a Michael acceptor warhead was the fumarate

derivative 4 (Dc-11), shown in Figure 5.3 [11], which inhibited cathepsins B, H,and L irreversibly. Hanzlik and coworkers demonstrated the utility ofa,b-unsaturated carbonyl and vinyl sulfone derivatives 5 and 6 in the design of

HN

NH

O P1

O P1'

O

HN

NH

P2'

OP2

Scissile bond

HN

H2NO P1

O P1'

O

HN

NH

P2'

OP2

OH +

NH

O P1'HN

P1

‡


1 2 3

S H

Cys25

NN

H

His159

NH

O P1'HN

P1 S NHN

His159H

Cys25

HHNNCys25 Gln19

Oxyanion hole

Figure 5.1 Catalytic mechanism of peptide hydrolysis by cysteine proteases.

132 5 Design of Cysteine Protease Inhibitors

cysteine protease inhibitors of papain and dipeptidyl peptidase-I (DPP-I) [12]. Pep-tide and peptidomimetic vinyl sulfone derivatives were designed by Palmer et al.to inhibit cathepsin, calpains, and cruzain [13].Vinyl sulfones as electrophilic warheads provided many potent inhibitors with

marked specificity. This class of inhibitors did not inhibit serine proteases and arestable toward circulating glutathione. Vinyl sulfone derivative 7 in Figure 5.4 wasdesigned as a falcipain-2 inhibitor to block the malaria parasites. This compoundhas shown efficacy when administered orally to mice [14,15]. A related derivative 8

RHN

P1

S H

Cys

NN

H

His

RHN

P1

S

H

Cys

NN

H

His

RHN

P1

S

Cys

R = SO2R1 or CO2R1

Figure 5.2 Inhibition of a cysteine protease by inhibitors with a Michael acceptor group.

HO

ONH

OHN

O

4, (Dc-11)Kapp = 11 M-1S-1 (cathepsin H)

HN

NH

O

CO2MeO

Ph

HN

NH

OS

OPh

5Ki = 26 μM (papain)

H3N+

O O

6, Ki = 180 μM (DPP-I)

Figure 5.3 Structures and activities of a,b-unsaturated carbonyl and vinyl sulfone-containing cys-teine protease inhibitors.

5.2 Development of Cysteine Protease Inhibitors with Michael Acceptors 133

exhibited potent inhibitory activity against cathepsins B and L and cruzain [16].This compound showed efficacy in a mouse model of Chagas disease and wasselected for clinical development [17,18].The first X-ray crystal structure of vinyl sulfone inhibitor 8 with cathepsin V

revealed that the inhibitor made extensive contacts in the active site, as shownin Figure 5.5. The vinyl sulfone functionality extends into the prime side of thebinding cleft and appears to form hydrogen bonds with the Gln19 side chain aswell as with the Trp189 in the active site. This results in good alignment of theb-carbon with the nucleophilic thiolate anion of Cys25. The inhibitor also formsan antiparallel b-sheet with backbone of Gly68 and an additional hydrogen bondwith Asp162. The enzyme’s thiolate anion could conceivably attack the vinyl sul-fone of either diastereotopic face; however, cathepsin V attacks the si-face ofinhibitor [19].A wide variety of human rhinovirus 3C (HRV 3C) protease inhibitors were

designed and synthesized, incorporating a,b-unsaturated carbonyl derivatives aswarheads. This cysteine protease possesses a unique specificity for Gln at P1. Thiswas exploited in the design of inhibitors. Kong et al. reported the first HRV 3Cprotease inhibitors when they prepared a series of peptidic inhibitors incorporat-ing a,b-unsaturated esters [20]. The peptide parts were selected based upon thesubstrate cleavage site. The representative inhibitor 9 showed an IC50 value of130 nM. The presence of P1 glutamine was critical to inhibitory potency.Dragovich et al. devoted a significant effort to the design of specific inhibitors forHRV 3C protease [21,22]. Peptidyl inhibitor 10 showed selective HRV 3C proteaseinhibitory activity. Subsequent optimization of P2 and P3 ligands resulted in

NH

HN S

N

O O

7 2 nM (falcipain)

Ph

O

O

N

NH

HN S

N

O O

8 (K777 or APC3316)IC50 = < 1 nM (cruzain)

IC50 =

Ph

O

PhO

N

Figure 5.4 Structures of vinyl sulfone-derived cysteine protease inhibitors.


potent peptidomimetic inhibitors 11 and 12 [20]. Inhibitor 12, also known asrupintrivir (AG7088), inhibited HRV14 3Cpro with an IC50 of 13 nM, EC50 of100 nM against 48 different HRV serotypes, and EC50 of 23 nM against serotype14 (Figure 5.6). Due to these promising results, it was selected for clinical develop-ment [23,24].

Figure 5.5 X-ray cocrystal structure of inhibitor 8 (carbon chain, green) complexed with cathep-sin V (PDB code: 1FH0).

HN

HN

O

O

O

O

NH

O

O

NOMe

H

HN

NH

HN

OPh

O

O

O

NH2O

ONH

BocHN

NH

HN

O

O

O

O

NH2O

O

S

9 IC50 = 130 nM (enzyme) 10 IC50 = 56 nM (antiviral)

Me

12 (AG7088, Rupintrivir)IC50 = 13 nM (enzyme)EC50 = 23 nM (antiviral)

HN

HN

O

O

O

O

NH2O

O

S

11 EC90 < 250 nM (antiviral)

F F

Figure 5.6 Structures and activities of HRV 3C protease inhibitors.

5.2 Development of Cysteine Protease Inhibitors with Michael Acceptors 135

The outbreak of severe acute respiratory syndrome (SARS) in 2003 and identifi-cation of a novel coronavirus as the etiological agent led to the recognition of cys-teine proteases SARS-CoV 3CLpro and SARS-CoV PLpro (papain-like protease) astargets for drug design [25,26]. Based upon the similarities of the substrate speci-ficity to HRV 3Cpro, AG7088 was suggested as the starting point to develop novelinhibitors [27,28]. Ghosh et al. modified AG7088 at the P1 site and reported potentinhibitors 13 and 14 shown in Figure 5.7 [29,30]. These compounds were shownto block SARS-CoV replication in cell culture assay without toxicity.The X-ray structure of 14-bound SARS-CoV 3CLpro provided critical ligand–

binding site interactions. As shown in Figure 5.8, the structure revealed that theinhibitor is covalently bonded to the enzyme via a carbon–sulfur bond to the activesite Cys145. The P1 lactam carbonyl formed a hydrogen bond with the imidazolering of His172. The P3 carbonyl group forms a hydrogen bond with the NH ofGlu166. This structure was further exploited in the structure-based design ofmore potent SARS-CoV 3CLpro inhibitors such as compound 15 [31].

5.3Design of Noncovalent Cysteine Protease Inhibitors

As we have seen in the design of noncovalent and reversible inhibitors of serineprotease in Section 3.10, substrate-based design of inhibitors without the electro-philic warhead is a useful strategy for lead generation. Human cathepsin K plays acritical role in bone resorption. In an effort to block bone resorption, noncovalent

13, IC50 = 870 μM (3CLpro)

HN

NOMe

O

HN

OPh

O

O

O

NHO

H

14, IC50 = 800 μM (3CLpro)

HN

NOMe

O

HN

O

O

O

O

NHO

H

15, IC50 = 80 μM (3CLpro)

HN

NH

O

HN

O

O

O

O

NHO

H

HO

Boc

Figure 5.7 Structures and activities of SARS-CoV 3CLpro inhibitors.


cathepsin K inhibitor was designed and synthesized. Toward the design of noncova-lent inhibitors, Kim et al. started with peptidyl aldehyde 16 (Figure 5.9) [32].Removal and replacement of the aldehyde with a substituted aniline provided inhib-itor 17 with a nearly 10-fold loss of potency. A 4-piperidinyl aniline 18 restored theactivity similar to the level of the peptidyl aldehyde inhibitor. This inhibitor showedpotent activity against cathepsin L. Detailed kinetic studies indicated that the

Figure 5.8 X-ray crystal structure of inhibitor 14 (carbon chain, magenta) complexed with SARS-CoV 3CLpro (PDB code: 2ALV).

OHN

NH

H

O

PhO

O

IC50 = 20 nM (cathepsin L)16

OHN

NH

HN

O

PhO

IC50 = 1.1 µM (cathepsin K)17

IC50 = 0.23 µM (cathepsin L)

H

OHN

NH

HN

OPh

O

IC50 = 0.01 µM (cathepsin K)18

IC50 = 0.002 µM (cathepsin L)

N

Figure 5.9 Structures and activities of noncovalent and reversible cathepsin K and L inhibitors.

5.3 Design of Noncovalent Cysteine Protease Inhibitors 137

inhibition was competitive. Furthermore, reversibility of the inhibitors was demon-strated by recovery of enzyme activity after dialysis or dilution.Fairlie and coworkers designed noncovalent caspase-1 inhibitors starting

from peptidyl inhibitors with conventional electrophilic warheads [33]. As shown inFigure 5.10, incorporation of a reduced amide (secondary amine) isostere resultedin potent caspase-1 inhibitors. Inhibitors 19 and 20, with benzyl and cyclohexylamines, respectively, showed selective caspase-1 inhibition over other caspases inthe low nanomolar range. Kinetic studies demonstrated a competitive inhibition. Amodel structure of 19 docked in the active site of caspase-1 showed possible hydro-gen bonding between the phenolic OH and Asp288 carbonyl side chain as well asbetween the secondary amine NH and His237. The P2 and P3 amino acid sidechains packed the respective hydrophobic pockets in the S2 and S3 subsites.Edwards and coworkers from Johnson & Johnson reported a new class of non-

peptidic and noncovalent cathepsin S inhibitors in 2007 [34]. Cathepsin S hasbeen suggested for the development of agents against a range of immune disor-ders. In silico theoretical model of cathepsin S was generated using coordinates ofX-ray crystal structure of cathepsin K. Several compounds were screened usingDOCK against the predictive cathepsin K model that resulted in compound 21 asthe lead inhibitor with micromolar potency (Figure 5.11). This compound wasshown to inhibit cathepsins B, F, and K at 20 mM level and cathepsin L with anIC50 of �2–5mM. Subsequent structural optimization resulted in very potent andcompetitive noncovalent inhibitor 22.A number of unprecedented small-molecule noncovalent and reversible inhibitors

of papain-like protease of SARS-CoV were reported. Following the high-throughput

Ki = 47 nM (caspase-1)

19

NH

HN

NH

O

O

O

HN

CO2H

OH Ki = 128 nM (caspase-1)

20

NH

HN

NH

O

O

O

N

CO2H

Me

Figure 5.10 Structures and activities of noncovalent and reversible caspase-1 inhibitors.

IC50 = 1 µM (cathepsin S)

21

N NN

OHN

F

F

NO

Me

Cl

IC50 = 20 nM (cathepsin S)

22

N NN

ON

NO

H2N

ICN

ONH2

Figure 5.11 Structures and activities of noncovalent and reversible cathepsin S inhibitors.


screening (HTS), Ratia et al. identified a weak noncovalent lead compound 23against SARS-CoV PLpro (Figure 5.12) [35]. Ghosh et al. subsequently optimized thestructure and improved the potency of this inhibitor to nanomolar range [36]. Com-pound 24 inhibited SARS-CoV viral replication in Vero E6 cells. The X-ray cocrystalstructure of inhibitor 24 and SARS-CoV PLpro revealed noncovalent interactions inthe active site of SARS-CoV PLpro where inhibitor binds within the S3 and S4 sub-sites and induces a loop closure that shuts down catalysis. As shown in Figure 5.12,inhibitor 24 forms a number of hydrogen bonds with the PLpro enzyme. Theseinclude a hydrogen bond between the amide NH of inhibitor and highly conservedAsp165, a pair of hydrogen bonds between the carbonyl oxygen and backbone NH ofTyr269 and hydroxyl group of Tyr265, and a hydrogen bond between the anilineamine and Gln270 side chain. Inhibitor 24 showed excellent specificity when testedagainst a host of human deubiquitinating enzymes. Structure-based design led toother potent and orally bioavailable SARS-CoV PLpro inhibitors. A second lead, 25shown in Figure 5.13, from the HTS was also optimized to potent inhibitor 26. TheX-ray crystal structure of 26 with PLpro revealed a unique mode of noncovalent

Figure 5.12 Structures of SARS-CoV PLpro inhibitors and X-ray crystal structure of inhibitor 24with PLpro (carbon chain, green; PDB code: 3E9 S).

NHN

IC50 = 59.2 µM IC50 = 320 nM EC50 = 9 µM (Vero E6 cells)

N HN

O

Me

O

OH

O

OMe

25 26 (GRL-0667)

Figure 5.13 Structures and activities of noncovalent and reversible PLpro inhibitors.

5.3 Design of Noncovalent Cysteine Protease Inhibitors 139

interaction in the active site [37]. Interestingly, the key molecular interactions ofinhibitor 26 are quite different from the active site interactions with 24. The enantio-meric preference of compound is extremely important for inhibitory activity. Thesestructural templates can serve as starting points for the structure-based design ofother noncovalent cysteine protease inhibitors.

5.4Conclusions

The design of cysteine protease inhibitors has become an important area in medic-inal chemistry, particularly in the design of inhibitors against human rhinovirus3C protease and SARS/MERS coronaviruses. As highlighted in this chapter, avariety of Michael-acceptor electrophilic groups, including vinyl sulfone anda,b-unsaturated esters, have been utilized in inhibitor design. These inhibitorsshow their inhibitory properties by forming a covalent bond with the active sitecysteine. Although a number of inhibitors of rhinovirus 3C protease showed clini-cal potential, no cysteine protease inhibitor has been approved as yet. This chapterhas also outlined the structure-based design of a variety of peptidomimetic cysteineprotease inhibitors as well as the design and discovery of noncovalent/reversibleinhibitors. These tools and design strategies will be useful for the next generationof inhibitors with clinical potential.

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33 Loser, R., Abbenante, G., Madala, P.K.,Halili, M., Le, G.T., and Fairlie, D.P. (2010)Noncovalent tripeptidyl benzyl- andcyclohexyl-amine inhibitors of the cysteineprotease caspase-1. J. Med. Chem., 53,2651–2655.

34 Thurmond, R.L., Beavers, M.P., Cai, H.,Meduna, S.P., Gustin, D.J., Sun, S.Q.,Almond, H.J., Karlsson, L., and Edwards,J.P. (2004) Nonpeptidic, noncovalentinhibitors of the cysteine proteasecathepsin S. J. Med. Chem., 47, 4799–4801.

35 Ratia, K., Pegan, S., Takayama, J., Sleeman,K., Coughlin, M., Baliji, S., Chaudhuri, R.,Fu, W.T., Prabhakar, B.S., Johnson, M.E.,Baker, S.C., Ghosh, A.K., and Mesecar,A.D. (2008) A noncovalent class of papain-like protease/deubiquitinase inhibitorsblocks SARS virus replication. Proc. Natl.Acad. Sci. USA, 105, 16119–16124.

36 Ghosh, A.K., Takayama, J., Aubin, Y.,Ratia, K., Chaudhuri, R., Baez, Y.,Sleeman, K., Coughlin, M., Nichols, D.B.,Mulhearn, D.C., Prabhakar, B.S., Baker,S.C., Johnson, M.E., and Mesecar, A.D.(2009) Structure-based design, synthesis,and biological evaluation of a series ofnovel and reversible inhibitors for thesevere acute respiratory syndrome-coronavirus papain-like protease. J. Med.Chem., 52, 5228–5240.

37 Ghosh, A.K., Takayama, J., Rao, K.V., Ratia,K., Chaudhuri, R., Mulhearn, D.C., Lee, H.,Nichols, D.B., Baliji, S., Baker, S.C.,Johnson, M.E., and Mesecar, A.D. (2010)Severe acute respiratory syndromecoronavirus papain-like novel proteaseinhibitors: design, synthesis, protein–ligand X-ray structure and biologicalevaluation. J. Med. Chem., 53, 4968–4979.


6Design of Metalloprotease Inhibitors

6.1Introduction

The metalloproteases are one of the most abundant protease classes in the humangenome. The majority of metalloproteases contain zinc ions, but the presence ofcopper, manganese, cobalt, and others is also known [1,2]. The active site of everymetalloprotease possesses a divalent metal that is critical for catalysis. This metalion is coordinated with three ligands in the protein. The coordinating ligands con-sist of histidine, glutamic acid, aspartic acid, lysine, and arginine. A labile watermolecule takes up the fourth coordination site. The catalytic mechanism of metal-loproteases has been investigated rigorously. The X-ray crystallographic studies ofthermolysin and peptide complexes provided intriguing insight into various stagesof the catalysis [3,4]. As shown in Figure 6.1, the catalytic mechanism of metal-loproteases involves substrate binding of the scissile carbonyl to the zinc ion,which facilitates the positioning of the zinc-bound water molecule closer to thecatalytic glutamate side chain. Subsequent nucleophilic attack by the activatedwater molecule and proton transfer result in the formation of a zinc-complexedtetrahedral intermediate. The scissile bond then gets cleaved and after reshufflingof a proton to the N-terminus of the cleaved peptide, fragments are released andthe catalytic enzyme is regenerated.The design and development of angiotensin-converting enzyme (ACE) inhibi-

tor drugs in the 1980s was one of the hallmark achievements in medicinalchemistry. The details of this development are described in Chapter 10. Thedesign of ACE inhibitors is also well covered by Acharya et. al. [5] and Ganteet. al. [6]. ACE inhibitors have been widely used for regulation of hypertension.In addition, these drugs are now prescribed for the treatment of congestiveheart failure [7]. The success of ACE inhibitor drugs generated significant inter-est in drug discovery and development for other metalloprotease disease tar-gets, particularly matrix metalloproteases (MMPs). There has been a significanteffort toward the development of therapeutic inhibitors of MMPs targeting vari-ous cancers, rheumatoid arthritis, and osteoarthritis. The drug design efforts inthis area led to the design of structurally diverse classes of metalloprotease

143


inhibitors. A number of inhibitors have been through clinical trials. The designof inhibitors and limitations of MMP inhibitors have been well covered in sev-eral reviews [8–11]. In next section, we will briefly outline the design aspects ofmatrix metalloprotease inhibitors.

6.2Design of Matrix Metalloprotease Inhibitors

Matrix metalloproteases are involved in tissue remodeling and degradation of theextracellular matrix (ECM), such as collagens, elastins, gelatin, matrix glycoprotein,and proteoglycans. MMPs also participate in the release of growth factor from theECM, which results in altered cell–cell and cell–matrix interactions [12]. MMPs aremediators of nerve and bone growth, endometrial recycling, wound healing, angio-genesis, and apoptosis [13]. Consequently, MMPs are generally synthesized asinactive, latent enzymes, which are converted to an active form by a mediator acti-vator system such as plasminogen activator or the prohormone convertase furin.The activity of MMPs is regulated by a group of endogenous proteins, termed astissue inhibitor of metalloproteases (TI-MPs), which bind to active MMPs. MMPsare involved in disease pathology of a large number of human disorders, includingcancer invasion and metastasis, rheumatoid arthritis, neuroinflammatory diseases,liver cirrhosis, fibrotic lung disease, atherosclerosis, multiple sclerosis, cardio-myopathy, aortic aneurysm, and many other diseases [14,15].Over the past years, numerous MMP inhibitors have been designed for the possible

treatment of cancer, arthritis, and cardiovascular diseases. The basic principles for

HN

NHO P1

O P1'

O

HN

NH P2'

OP2

Scissile bond

HN

H2NO P1

O P1'

O

HN

NH P2'

OP2

OH +1 2 3

HN

O P1'NH

P1

OH

HZn

OO

_2+

O

‡HN

P1'NH

P1

Zn

OO

2+

O OH_

HTetrahedral

transition state

O

H2N

O P1'

NH

P1

Zn

OO

_2+

OOH+

2 3

Glu

Glu

Glu

MMP

MMP MMP

Figure 6.1 Catalytic mechanism of matrix metalloproteases.

144 6 Design of Metalloprotease Inhibitors

the design of MMP inhibitors involve mimicking the substrate with a shortpeptide or peptidomimetic unit and attaching a chelating zinc binding group (ZBG)that can interact with the zinc ion. The majority of MMP inhibitors incorporated thehydroxamate chelating group. Several other non-hydroxamate ligands have also beendeveloped (Figure 6.2) [9,16]. The inhibitor design strategy has mostly focused on sub-strate mimetic segments. There are at least 28 human MMPs currently known, andthese enzymes show significant sequence homology [17]. These MMPs are classifiedinto subfamilies based upon their substrate specificity: collagenases, gelatinases, stro-melysins, and matrilysins. Another subclass is represented by membrane-type MMPs(MT-MMPs) [18,19]. The design of selective MMP inhibitors has been a major chal-lenge. The X-ray cocrystal structures of MMPs bound to various inhibitors facilitatedstructure-based design of inhibitors [20]. The majority of MMPs possess a hydropho-bic S10 subsite, a shallow S20 subsite, and a solvent-exposed S30 subsite. The mainstructural differences between MMP family members are found at the S10 subsite. Asa result, modification of the P10 group is logically chosen to introduce specificity. Thenature of amino acid 218 (MMP-13 numbering) and amino acids in the specificityloop (244–255) that surround the distal part of zinc are responsible for substrate speci-ficity [21,22].Many of the earlier classes of MMP inhibitors are based upon hydroxamic acid

and a small peptide and peptidomimetic backbone [8–11]. Inhibitors batimastat (4)and marimastat (5), shown in Figure 6.3, are representative examples of hydroxa-mic acid-based inhibitors. These inhibitors showed excellent broad-spectruminhibitory activity against MMP subtypes. Both inhibitors have undergone clinicaltrials. These drugs reached different stages of clinical development ranging fromphase I to phase III; however, development was terminated. The outcome of

HOHN R

O

Hydroxamate

O NR

OH

"Reverse"hydroxamate

HOHN

HN

O

N-Hydroxyurea

Hydroxamate ligands:H

R

Non-hydroxamate ligands:

Phosphonic acidor phosphinic acid

HN

NH O

R

Hydrazide

R' HN

NH

O

OO

R

Pyrimidinetrione

PHO

RX

O(X = OH or R')

O

O

RR'

Pyrone

HON

HO

Hydroxypyridinone

NN

SR'

Thiadiazine

O R

R'

HN

R

Figure 6.2 Representative examples of zinc binding groups.

6.2 Design of Matrix Metalloprotease Inhibitors 145

clinical trials with other MMP inhibitors was also disappointing due to inductionof musculoskeletal pain and other toxicities [22]. There have been major concernsabout broad-spectrum activity, the nonspecific metal chelation ability of hydroxa-mic acid group, and the metabolically labile nature of this chelating group. As aresult, there has been more emphasis on the design of more selective and activesite-directed MMP inhibitors with alternative metal binding groups and also thedevelopment of inhibitors without metal chelating groups.Inhibitor 6 in Figure 6.4 was reported by Martin et al. at British Biotech as a

potent and highly selective inhibitor of MMP-1 [23]. However, it has been

MeHNHN

NH

OHO

OS

O

MeHNHN

NH

OHO

O OH

O

S

4 (Batimastat or BB-94)IC50 (nM)

MMP-1 3MMP-2 4MMP-3 20MMP-7 6MMP-9 4

5 (Marimastat or BB-2516)IC50 (nM)

MMP-1 5MMP-2 6MMP-3 200MMP-7 20MMP-9 3

Figure 6.3 Structures and activities of broad-spectrum MMP inhibitors.

N NH

OH

ON

O

NNH

OHO

6

IC50 (nM)

MMP-1 20

7

IC50 (nM)

MMP-1 14 000MMP-2 529MMP-9 2420MMP-14 20 100

SO

O

NS

NH

OHO

MMP-2 3000MMP-3 10 000MMP-8 200MMP-13 200

MMP-3 1

OO

N

OOH

S OO

O8

IC50 (nM)

MMP-1 >50 000MMP-3 4500MMP-7 >50 000MMP-9 200

MMP-2 12

Figure 6.4 Structures and activities of subtype-selective MMP inhibitors.


postulated that the musculoskeletal syndrome observed with broad-spectrumMMP inhibitors was related to inhibition of MMP-1. Therefore, much researcheffort was devoted to optimize inhibitors that target specific MMPs (MMP-2 andMMP-9 for gelatinases; MMP-13 for collagenase 3) and spare others, particularlyMMP-1. Toward targeting a specific metzincin family, Whitlock et al. from Pfizerdesigned a highly selective MMP-3 (stromelysin) inhibitor 7 by optimizing ligandbinding interactions in the S10 subsite [24]. This inhibitor showed excellent selec-tivity over MMP-1, MMP-2, MMP-9, and MMP-14. Rossello et al. designed inhibi-tor 8 with sulfonamide and hydroxamate functionalities as a selective inhibitor ofgelatinase (MMP-2) [25]. This compound exhibited selective MMP-2 inhibitionover MMP-1, MMP-3, MMP-7, and MMP-9.A significant effort was devoted to design selective inhibitors with carboxylic

acids and other zinc binding groups, as shown in Figure 6.5. Cherney et al.from DuPont Merck reported the design of macrocyclic inhibitors that selec-tively inhibited MMP-8 (collagenases) [26]. As represented in Figure 6.5, theX-ray crystal structure of acyclic inhibitor 9-bound MMP-3 revealed that P1

and P20 residues were located in proximity to each other and extended toward

the solvent-exposed area [27]. Also, the carboxylic acid formed hydrogen bondswith the catalytic zinc. Based upon this insight, the investigators designedcycloamide inhibitor 10 by linking P1 and P2

0 with altered P1–P20 group for

selective inhibition of MMP-8.Zhang et al. from Johnson & Johnson investigated arylsulfone-based MMP

inhibitors with heterocyclic zinc binding groups [28,29]. Inhibitor 11 in Figure6.6, with an N-hydroxy-2-pyridinone as the zinc binding group, exhibited goodinhibition of gelatinases (MMP-9) and also displayed excellent selectivity againstMMP-1 and MMP-3. LeDour et al. reported inhibitor 12 with a hydrazide func-tionality as the zinc binding group [30]. This inhibitor displayed excellent IC50 val-ues of MMP-9, comparable to ilomastat (IC50¼ 0.6 nM), and more than 400-foldselectivity over MMP-3 and MMP-14.The design of potent, selective, nonpeptidic, and non-zinc chelating MMP

inhibitors has evolved. Dublanchet et al. of Pfizer reported the identification of

10

IC50 (nM)

MMP-8 17MMP-1 2500MMP-2 8100MMP-3 13 500MMP-9 6600

O

OH

HN

O

MeHN

O

O

OH

ONH

HN

O

MeHN

O

HN

Ph

OMeP1P2'

Ki (MMP-3) 21 nM9

Figure 6.5 Design of selective macrocyclic inhibitor with a carboxylic acid ZBG.


thiophene amide 13 in Figure 6.7 as a weak inhibitor of MMP-12 after high-throughput screening (HTS) of MMP-12’s catalytic domain in the presence of5mM acetohydroxamate [31]. Subsequent structural and modeling studies sug-gested a unique mode of binding without chelation, and the inhibitor bindinginvolved hydrophobic interactions in the S10 pocket through the aromatic ring aswell as through the formation of a number of hydrogen bonds in the S10 subsite.Further optimization of hydrophobic elements as well as the amide bindingregion, led to the design of compound 14 as a MMP-12 inhibitor. Inhibitor 14exhibited a nanomolar IC50 value (in the presence of acetohydroxamate) andshowed excellent selectivity over MMP-2 and moderate selectivity over otherMMPs. The X-ray crystal structure of 14-bound MMP-12 revealed a unique bind-ing mode without involving the catalytic zinc [32]. As can be seen in Figure 6.8,the inhibitor binding is mostly hydrophobic in nature. The biaryl segment nestlesinto the S10 specificity pocket while the thiophene and phenyl rings pack thehydrophobic pocket surrounding Thr215 and Tyr240. The carboxylic acid forms apair of hydrogen bonds with the Tyr240 backbone NH, and through a water mole-cule with the backbone carbonyl group. The molecular insight from the structuralstudies may lead to further optimization of potency and selectivity.Engel et al. from Aventis Pharma reported pyrimidine dicarboxamides as a new

class of non-zinc and nonpeptide MMP-13 inhibitors [33]. Compound 15 in Figure 6.9

11IC50 (nM)

MMP-2 17.2MMP-1 >1000MMP-3 308MMP-9 7.3MMP-13 13.4

NHO

O

S

O

OMeO O

MeHNHN

NH

HN

O

O

O

S O

O

Br

12IC50 (nM)

MMP-9 3MMP-1 30MMP-2 9.8MMP-3 1700MMP-7 475MMP-14 17,000

NH

Figure 6.6 Structures of selective MMP inhibitors with non-hydroxamate ZBGs.

14

IC50 (nM)

MMP-12 14MMP-13 270MMP-2 >10 000MMP-3 390MMP-8 1700MMP-9 980

IC50 (MMP-12) = 13 µMIC50 (MMP-13) = 24 µM

13S

OMe

NH

O

N

O

S

NH

O

OHNO

Figure 6.7 Structures of selective non-hydroxamate and non-ZBG MMP inhibitors.


was initially identified as a very selective MMP-13 inhibitor with no apparent activityagainst other MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, MMP-12, andMMP-16 up to 100mM). The X-ray cocrystal structural studies of inhibitor 15 andMMP-13 complex revealed no involvement of the catalytic zinc, while the inhibitor

Figure 6.8 X-ray cocrystal structure of inhibitor 14 (carbon chain, green; catalytic zinc, magenta)complexed with MMP-12 (PDB code: 1UTZ).

15IC50 (MMP-13) = 6600 nM

N NNH

NH

O O

N N16

N NNH

NH

O OMe

F

Me

F

IC50 (MMP-13) = 8 nM

17

N N

O

O

S O

O

IC50 (MMP-13) = 23 nM18

IC50 (MMP-13) = 6.7 nM

(No detectable inhibition forMMP-1-3, 7-10, 12, 14, 16up to 100 µM)

(No detectable inhibition forMMP-1-3, 7-10, 12, 14, 16up to 100 µM)

(No detectable inhibition for MMP-1-3, 7-9, 12, 14, 17)

(No detectable inhibition for MMP-1-3, 7-9, 12, 14, 17)

N

NN

O

O

HONH

O

OMe

Figure 6.9 Structures and activities of selective MMP-13 inhibitors.


binds in the S10 pocket and extends into S10 side pocket. Further optimization andreplacement of the pyridyl groups resulted in potent inhibitor 16 with an IC50 value of8nM. Li et al. from Pfizer also reported the identification of thiazolopyrimidinedione17 through their HTS efforts [34]. The X-ray structural studies of 17 with MMP-13revealed that the inhibitor occupied the S10 specificity pocket and there was no interac-tion with the catalytic zinc. Modification of ester and N-benzyl side chains as wellas optimization of the scaffold led to compound 18 as a very potent and selectiveMMP-13 inhibitor. This compound displayed positive cartilage protection in rabbitanimal models of osteoarthritis and exhibited favorable ADMETand safety profiles.

6.3Design of Inhibitors of Tumor Necrosis Factor-a-Converting Enzymes

Tumor necrosis factor-a-converting enzyme (TACE) is a zinc metalloprotease thatcleaves membrane-bound protein pro-tumor necrosis factor-a and releases a17 kDa tumor necrosis factor-a (TNF-a). TNF-a is one of the most common proin-flammatory and immunomodulatory cytokines responsible for numerous inflam-matory disorders, including rheumatoid arthritis, multiple sclerosis, and certaincancerous conditions [35]. Antibodies of TNF-a have shown success in patients as

NHMeNH

HN

HOO

OOH

O

5 (Marimastat)

SN

HN

HOO

N

OO

OMe

19 (CGS 27023A)

N

O

H

HN

HOO

N

O

OCF3

CF3

HN

HOO

Activity (nM)

pTACE 2200 (IC50)

20

21

MMP-1 221 MMP-2 108MMP-9 242

N

O

ONH

NHO

O22 (IK682)

pTACE 2 (IC50)

MMP-1 >5000MMP-2 >3000MMP-9 >2000

pTACE 0.56

MMP-1 30000MMP-2 2050MMP-9 10340MMP-13 1417

Ki (nM)

(Ki)

Activity (nM)

(Ki)

Figure 6.10 Structures and activities of selective TACE inhibitors.


antagonists. Therapeutic inhibition of active TNF-a is regarded as a promisingapproach for the treatment of many inflammatory disorders. A number of inhibi-tor design strategies have evolved based upon earlier work on broad-spectrumMMP inhibitors [36,37]. The design of selective inhibitors of TNF-a is critical forthe reduction of side effects and toxicity. TACE has become a specific target forpossible treatment of rheumatoid arthritis.Based upon homology models and structural studies of TACE and MMPs, a bend-

shaped S10 pocket was identified and targeted for selectivity design. Duan et al. atBMS designed TACE inhibitor lead compound 20 (Figure 6.10) based uponsubstrate-based inhibitor 5 (marimastat) and a nonpeptidic inhibitor 19 (CGS 27023A)reported by Novartis scientists [38–40]. Incorporation of various 4-substitutionson the aromatic ring of compound 20 resulted in improvement in TACE potencyand selectivity over MMPs. Compound 21, with a 3,5-disubstituted benzyloxy substitu-ent, exhibited excellent TACE potency and selectivity over other MMP subtypes. Fur-ther optimization of substituents resulted in orally bioavailable inhibitor 22.The specific design of a methylquinoline substituent on inhibitor 22 is responsi-

ble for the TACE selectivity. Duan et al. subsequently designed non-hydroxamateand nonpeptide TACE inhibitors based upon features of inhibitor 22 and a pyrimi-dine-2,4,6-trione-based MMP inhibitor 23, shown in Figure 6.11, reported by

24 (pTACE IC50 = 1.03 µM)

26Activity (nM)

pTACE 81 (IC50)

MMP-1 >4950MMP-2 >3330MMP-9 >2130MMP-13 >5000

pTACE 2 (IC50)

MMP-2 2170MMP-3 >4500MMP-7 >6370MMP-12 1020

N

O

ONH

NHO

O

22 (IK682)

HN

NH

O

O

O O

23

ON

HN

NH

O

O O

ON

HN

NH

NO

O O

25

N

ONHN

NH

O

O O

N

NS

OO

HN

O

(Ki) (Ki)

Activity (nM)

Figure 6.11 Design of selective non-hydroxamate and non-ZBG TACE inhibitors.

6.3 Design of Inhibitors of Tumor Necrosis Factor-a-Converting Enzymes 151

researchers from BMS and Roche [41–43]. The X-ray structure of 23 revealedthat the catalytic zinc binds to the pyrimidine-2,4,6-trione and this moiety isalso involved in hydrogen bonding interactions in the active site. Based uponthis insight, they initially prepared compound 24, which was less active. However,further optimization of substituents resulted in potent and selective non-hydroxamate inhibitors 25 and 26 [41,44].

6.4Conclusions

To date, more than 300 metalloproteases are known. A number of zinc-containingmetalloproteases are involved in the pathogenesis of human diseases. Metalloen-zymes such as angiotensin-converting enzyme (ACE) and carbonic anhydrase havebeen successfully targeted in the development of FDA-approved inhibitor drugs.The design strategies that led to the development of ACE inhibitors and carbonicanhydrase inhibitors are described in detail in later chapters. A number of othermetalloenzymes have been targeted for drug development. This chapter outlinesthe general design strategies leading to the preparation of a variety of metallopro-tease inhibitors. As depicted, the design of an appropriate tether and metal bind-ing group is responsible for tight binding to the enzyme and effective inhibition ofits catalytic function. The tools and strategies discussed will be useful for develop-ing the next generation of inhibitors with clinical potential.

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29 Zhang, Y.M., Fan, X., Chakaravarty, D.,Xiang, B., Scannevin, R.H., Huang, Z., Ma,J., Burke, S.L., Karnachi, P., Rhodes, K.J.,and Jackson, P.F. (2008) 1-Hydroxy-2-pyridinone-based MMP inhibitors:synthesis and biological evaluation for thetreatment of ischemic stroke. Bioorg. Med.Chem. Lett., 18, 409–413.

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30 LeDour, G., Moroy, G., Rouffet, M.,Bourguet, E., Guillaume, D., Decarme, M.,ElMourabit, H., Auge, F., Alix, A.J.P.,Laronze, J.Y., Bellon, G., Hornebeck, W.,and Sapi, J. (2008) Introduction of the 4-(4-bromophenyl) benzenesulfonyl group tohydrazide analogs of ilomastat leads topotent gelatinase B (MMP-9) inhibitorswith improved selectivity. Bioorg. Med.Chem., 16, 8745–8759.

31 Dublanchet, A.C., Ducrot, P., Andrianjara,C., O’Gara, M., Morales, R., Compere, D.,Denis, A., Blais, S., Cluzeau, P., Courte, K.,Hamon, J., Moreau, F., Prunet, M.L., andTertre, A. (2005) Structure-based designand synthesis of novel non-zinc chelatingMMP-12 inhibitors. Bioorg. Med. Chem.Lett., 15, 3787–3790.

32 Morales, R., Perrier, S., Florent, J.M.,Beltra, J., Dufour, S., De Mendez, I.,Manceau, P., Tertre, A., Moreau, F.,Compere, D., Dublanchet, A.C., andO’Gara, M. (2004) Crystal structures ofnovel non-peptidic, non-zinc chelatinginhibitors bound to MMP-12. J. Mol. Biol.,341, 1063–1076.

33 Engel, C.K., Pirard, B., Schimanski, S.,Kirsch, R., Habermann, J., Klingler, O.,Schlotte, V., Weithmann, K.U., and Wendt,K.U. (2005) Structural basis for the highlyselective inhibition of MMP-13. Chem.Biol., 12, 181–189.

34 Li, J.J., Nahra, J., Johnson, A.R., Bunker, A.,O’Brien, P., Yue, W.S., Ofwine, D.F., Man,C.F., Baragi, V., Kilgore, K., Dyer, R.D., andHan, H.K. (2008) Quinazolinones andpyrido[3,4-d]pyrimidin-4-ones as orallyactive and specific matrix metalloproteinase-13 inhibitors for the treatment ofosteoarthritis. J. Med. Chem., 51, 835–841.

35 Bahia, M.S. and Silakari, O. (2010) Tumornecrosis factor alpha converting enzyme:an encouraging target for variousinflammatory disorders. Chem. Biol. DrugDes., 75, 415–443.

36 Newton, R.C. and Decicco, C.P. (1999)Therapeutic potential and strategies forinhibiting tumor necrosis factor-alpha. J.Med. Chem., 42, 2295–2314.

37 Skotnicki, J.S. and Levin, J.I. (2003) TNF-alpha converting enzyme (TACE) as a

therapeutic target. Annu. Rep. Med. Chem.,38, 153–162.

38 Duan, J.J.W., Chen, L.H., Wasserman, Z.R.,Lu, Z.H., Liu, R.Q., Covington, M.B., Qian,M.X., Hardman, K.D., Magolda, R.L.,Newton, R.C., Christ, D.D., Wexler, R.R.,and Decicco, C.P. (2002) Discovery ofgamma-lactam hydroxamic acids asselective inhibitors of tumor necrosis factoralpha converting enzyme: design,synthesis, and structure–activityrelationships. J. Med. Chem., 45, 4954–4957.

39 Nar, H., Werle, K., Bauer, M.M.T.,Dollinger, H., and Jung, B. (2001) Crystalstructure of human macrophage elastase(MMP-12) in complex with a hydroxamicacid inhibitor. J. Mol. Biol., 312, 743–751.

40 Li, Y.C., Zhang, X.L., Melton, R., Ganu, V.,and Gonnella, N.C. (1998) Solutionstructure of the catalytic domain of humanstromelysin-1 complexed to a potent,nonpeptidic inhibitor. Biochemistry, 37,14048–14056.

41 Duan, J.J.W., Lu, Z.H., Wasserman, Z.R.,Liu, R.Q., Covington, M.B., and Decicco,C.P. (2005) Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione derivatives asselective inhibitors of tumor necrosisfactor-alpha converting enzyme. Bioorg.Med. Chem. Lett., 15, 2970–2973.

42 Foley, L.H., Palermo, R., Dunten, P., andWang, P. (2001) Novel 5,5-disubstitutedpyrimidine-2,4,6-trione as selective MMPinhibitors. Bioorg. Med. Chem. Lett., 11,969–972.

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7Structure-Based Design of Protein Kinase Inhibitors

7.1Introduction

Protein kinases are now validated targets for anticancer drug development. As of2012, there were 22 kinase inhibitors that have received FDA approval for treat-ment of cancers. Kinase targets are also being investigated for the treatment ofother disease states, including neuronal disorders, inflammation, and metabolicdiseases. A number of review articles cover these subjects in detail [1–3]. Thereare more than 500 kinases encoded in the human genome, playing importantroles in signal transduction pathways that regulate all aspects of cellular function[4]. Protein kinases catalyze the transfer of the c-phosphate of adenosine 50-tri-phosphate (ATP) to acceptor hydroxyl groups of serine, threonine, or tyrosine resi-dues of a substrate protein. This protein phosphorylation activates target proteinsand regulates diverse, critical, cellular processes, including cell growth, differenti-ation, and apoptosis. The aberrant kinase activity triggers inappropriate signalingor uncontrolled cell growth, leading to a variety of disease pathologies, particularlycancer. Therefore, the design and discovery of small-molecule kinase inhibitorshas become a major research focus in academic and pharmaceutical laboratories.

7.2Active Site of Protein Kinases

The catalytic domains of all kinases follow a similar three-dimensional arrange-ment and share similar catalytic mechanisms [5]. In the catalytic domain, thereare several functional subdomains presenting conserved amino acid residues [6].As can be seen in Figure 7.1, the core structure is formed by a small N-terminallobe (green) and a large C-terminal lobe (blue) connected by a hinge region (gray).The N-terminal lobe is formed mainly of b-strands, whereas the C-terminal lobe isformed by several a-helices. The ATP binding site is located in the cleft betweenthe N- and C-terminal lobes. The hinge region connecting the N- and C-terminaldomains is formed by a stretch of amino acids that interact through hydrogenbond contacts with the adenine ring of ATP. The P-loop (orange) forms an

155


important interaction with the phosphate group of ATP. The activation state of thekinase is regulated by the position of the activation loop (magenta).

7.3Catalytic Mechanism of Protein Kinases

The catalytic mechanism of the phosphorylation reaction is described in Figure 7.2. Asshown, the ATP is complexed with two Mg2þ ions in the active site. The nontransfer-able a- and b-phosphate groups are held in position by interaction with highly con-served residues in the N-terminal lobe of the protein. In particular, Lys72 interacts withoxygens from the a- and b-phosphate groups, whereas Glu91 stabilizes the Lys72 inter-actions with phosphates. Lys168, which is part of the catalytic loop, is localized in the C-terminal lobe and interacts with the c-phosphate group. The hydroxyl group of the pro-tein substrate residue is positioned to perform the nucleophilic attack at the c-phospho-rus. The Asp166 functions as the catalytic base at the site of phosphoryl transfer. Inorder to facilitate the transfer, the negative charge on the nucleotide is stabilized by thetwoMg2þ ions, Lys168, Lys72, and several backbone NH residues [7,8].

7.4Design Strategy for Protein Kinase Inhibitors

Structure-based design has played a very important role in the development of a vari-ety of small-molecule protein kinase inhibitor drugs approved by the FDA. The

Figure 7.1 Crystal structure of the kinase domain of cAMP-dependent kinase in complex withATP. Magnified ATP binding site: ATP, catalytic, and hinge residues are shown as sticks.

156 7 Structure-Based Design of Protein Kinase Inhibitors

majority of protein kinase inhibitor drugs are orally bioavailable and typically admin-istered once or twice a day dosing which is similar to other conventional therapies.Also, kinase drugs limit toxicity on tissues and bone marrow, as well as cardiovascu-lar side effects [9,10]. Protein X-ray structural studies have had a major impact on thesuccess of kinase inhibitor drug development. Since the first X-ray crystal structureof protein kinase A reported in 1991, more than 1000 X-ray structures have beenreported in the Protein Data Bank, with the majority of them being high-resolutionstructures and covering virtually all kinase families. Many inhibitor-bound X-ray crys-tal structures provided molecular insights into the key active site interactions, whichhave been exploited in the optimization of potency and selectivity of inhibitors.As can be seen in Figure 7.1, the two lobes of the catalytic domain are linked by

a “hinge region.” This region contains critical donor and acceptor groups in thebackbone, which anchor ATP binding and facilitate substrate phosphorylation. Apractical approach to kinase inhibition would be to block this ATP binding site.Indeed, the majority of kinase inhibitors contain binding elements that undergoat least one hydrogen bonding interaction with the hinge region. The inhibitoraffinity and specificity would depend upon additional interactions in the riboseand polar phosphate region occupied by ATP or interactions in the hydrophobic

N

HO OH

O

NN

N

NH2

OP

OP

OP

O

O-

O

O-

O

O-

O-

NH3+

Lys168Mg2+

OH2N

Asn171

NH3+ Mg2+

O OO O O O-

Glu91 Asp184 Asp166

HO

Ser

Adenosine

OP

OP

OP

O

O-

O

O-

O

O-

O-

NH3+

Lys168

Mg2+

OH2N

Asn171

NH3+

Lys72

Mg2+

O OO O O O-

Glu91 Asp184 Asp166

HO

Ser

Lys72

Ad

OP

OP

O-P

O

O-

O

O-

O

O--O NH3+

Lys168Mg2+

OH2N

Asn171

NH3+

Lys72

Mg2+

O OO OO OH

Glu91 Asp184Asp166

O SerAd

(Product)

Figure 7.2 Schematic representation of protein kinase catalytic mechanism.

7.4 Design Strategy for Protein Kinase Inhibitors 157

regions not occupied by ATP. The “hinge” binding elements are critical to achiev-ing good potency as their interactions contribute nearly 40–60% of the total bind-ing energy of inhibitor. Thus, the nature of hinge binding heterocycles withdonor/acceptor functionalities is important to the development of novel inhibitorsagainst various target kinases.Bollag et al. from Plexxikon reported the structure-based design and develop-

ment of PLX4032, a potent and selective inhibitor of oncogenic B-Raf kinase[11]. Azaindole scaffold 1 in Figure 7.3 was identified as one of the initial leadcompounds [12]. Subsequent X-ray structure-based optimization ultimatelyresulted in the design and discovery of PLX4032, which received FDA approvalin 2011 for the treatment of B-Raf mutant melanoma. The X-ray crystal struc-ture of PLX4032 bound to B-Raf kinase (V600E) showed a number of keyinteractions, which are critical to inhibitor’s potency and selectivity. As shown,the azaindole template formed two strong hydrogen bonds with the Cys532backbone NH and Gln530 backbone carbonyl in the “hinge” region of the ATPbinding site. In addition, the sulfonamide functionality formed hydrogenbonds with the backbone NHs of Asp594 and Phe595, respectively, in theDFG loop in active conformation, also known as “DFG-in,” where the asparticacid residue in the DFG motif points toward the ATP binding pocket and thephenylalanine residue points away [11].A number of representative hinge binding heterocyclic scaffolds utilized in the

development of FDA-approved drugs are shown in Figure 7.4. Many of these scaf-folds have been utilized in the design of potent inhibitors of a variety of kinasetargets, including vascular endothelial growth factor receptor (VEGFR), KIT,B-Raf, PLK1, epidermal growth factor receptor (EGFR), kinase insert domainreceptor (KDR), AKT-1, and Chk-1 [1–3]. Most small-molecule kinase inhibitorleads are generally identified by high-throughput, virtual, or fragment-based

Figure 7.3 Structures of azaindole lead, vemurafenib, and X-ray crystal structure of inhibitor 2with B-Raf (V600E) (carbon chain, magenta; PDB code: 3OG7).


screening of compound libraries. To improve diversity and lead structure novelty,libraries of compounds with heterocycles and donor/acceptor functionalities havebeen developed. Particularly, researchers are interested in libraries with core frag-ments that have the potential to interact with the hinge region of the kinase bind-ing site. Once lead structures are identified, medicinal chemistry efforts arepursued to optimize leads through structure-based design, synthesis, X-ray crystal-lography, and biological profiling. The determination of the binding mode in thekinase active site is important, as this information is used to prioritize where andhow to explore substituents to improve potency, selectivity, and compound proper-ties. When crystallography fails, the available X-ray structural information andhomology modeling are reliably used to predict the binding mode for compoundoptimization.

N

NHN

HN O

N

N O

O

Me

NO

HN

Me

Gefitinib (7) Imatinib (4)

N NH

Azaindole

N

N

Quinazoline

N

Pyridine

NN

Me

FCl

Pyrrole-oxindole

HN

SN

Amino-thiazole

Sorafenib (5)

NH

NHO

CF3

Cl

O

N

N

N

N

HN Me

OH

N S

ONH Me

Cl

Dasatinib (6)

N NH

Cl OF

F

Vemurafenib (2)

NO

NHMe

NH

OHN

Me

Me

O

HN

F N

N

NH

ONH

Sunitinib (3)

NH

NH

OUrea

HN S OO

Figure 7.4 Representative heterocycles and functionalities in FDA-approved kinase drugs (2–7).The hinge interactions are shown as dotted lines.

7.4 Design Strategy for Protein Kinase Inhibitors 159

7.5Nature of Kinase Inhibitors Based upon Binding

Kinase inhibitors are classified based upon their binding sites [13]. Small-mole-cule inhibitors that target the ATP binding site of the kinase in its active form aretype I kinase inhibitors. Most of the FDA-approved kinase inhibitor drugs are typeI inhibitors. Kinase inhibitors that target the ATP binding pocket and make inter-actions with an adjacent hydrophobic pocket (hydrophobic pocket II) of ATP aretype II inhibitors. Type III or allosteric inhibitors are those that bind to a hydro-phobic pocket remote to ATP and induce conformational changes of the ATPbinding pocket to modulate kinase activity. Type IV inhibitors are covalent inhibi-tors that form covalent bonds with kinase active site residues, often with a nucleo-philic cysteine residue.

7.5.1Type I Kinase Inhibitors and Their Design

Type I inhibitors are ATP competitive as they bind to the active kinase conforma-tion capable of phosphorylation of the substrate. Since these inhibitors bind to theATP site, which is highly conserved among all kinases, the majority of themexhibit broad reactivity among other members of a target kinase family. The lackof selectivity often leads to side effects and toxicity. However, medicinal chemistryoptimization, particularly of inhibitors extending interaction into hydrophobicregions, provides selective inhibitors for drug development.All kinases have a conserved activation loop that regulates kinase activity.

This activation loop is designated by DFG and APE motifs at the start andend of the loop. The activation loop can adopt many different conformations,ranging from a catalytically active form that can phosphorylate the substrateto an inactive conformation that blocks substrate entry and binding. In theactive conformation “DFG-in,” the aspartic acid residue points towardthe ATP binding pocket. The phenylalanine residue points away, as shown inthe X-ray structure of PLX4032 (2)-bound B-Raf kinase (V600E) in Figure 7.3[11,12].Type I inhibitors possess a heterocyclic scaffold that occupies the adenine bind-

ing site and forms up to three hydrogen bonds similar to adenine of ATP in the“hinge” region. Inhibitor optimization involves the incorporation of substituentsthat can extend into either or both adjacent hydrophobic pockets I and II. Asshown in Figure 7.5, dasatinib (6), a Bcr-Abl kinase inhibitor, was FDA approvedin 2006 for the treatment of adult chronic myelogenous leukemia (CML) [14].Dasatinib is an example of a type I inhibitor, and it was designed from thiazolelead 8. The details of design and discovery are provided in Chapter 12. A pharma-cophore model of the design of an ATP competitive type I inhibitor is shown inFigure 7.5 [13].The X-ray crystal structure of dasatinib-bound human Abl kinase in Figure 7.6

shows that dasatinib occupies the ATP binding site, with the aminothiazole


forming two hydrogen bonds in the hinge domain [15]. The chloromethyl phenylring is orthogonal to the thiazole ring and sits in hydrophobic pocket II nearThr315, where it forms a hydrogen bond with the threonine side chain. The piper-azine group makes van der Waals contact in hydrophobic pocket I near the carbox-ylic acid terminus of the hinge domain. The majority of type I inhibitors do notutilize the ribose or triphosphate binding sites.Harris et al. from GSK reported a series of potent VEGFR inhibitors that bind

within the ATP binding site [16]. This investigation ultimately resulted in the dis-covery of pazopanib for the treatment of solid tumors [17]. As shown in Figure 7.7,

Me

MeMe

NH

O

S

NH2N

Me

NN

NN

N

Me

N

SOH

HO

HN

Me

Cl

HN

NH

O

O

O

Thiazole lead Dasatinib8 6

Hydrophobic pocket II

Hinge binder

Hydrophobic pocket I

Figure 7.5 Design of dasatinib and a pharmacophore model of a type I inhibitor.

Figure 7.6 The X-ray cocrystal structure of inhibitor dasatinib with Abl kinase (carbon chain,green; Phe side chain, turquoise; PDB code: 2GQG).

7.5 Nature of Kinase Inhibitors Based upon Binding 161

fluoropyrimidine derivative 9 was identified as the lead inhibitor after screening asample collection. Initial structural modification based upon the model kinasedomain resulted in potent inhibitor 10. The X-ray crystal structure of 10-boundVEGFR-2 showed its binding in the ATP binding site. Further optimization of lig-and binding led to inhibitor 11, which progressed into full clinical developmentand was ultimately FDA approved as pazopanib [17]. The X-ray structural studiesof 11-bound VEGFR-2 showed that the inhibitor binds to the hinge domain as wellas hydrophobic pockets I and II.Protein kinase CK2 is a serine/threonine kinase. It has been implicated in the

pathologies of a variety of human diseases [18,19]. Nakanishi and coworkersreported the design of potent protein kinase CK2 inhibitors [20]. Phenyl thiadia-zole derivative 12, in Figure 7.8, was identified by virtual screening as a lead

HN

Br

N

NF

NH

Br

9

IC50 = 400 nM (VEGFR-2)

HN

OMe

N

N

NH

10

IC50 = 6.3 nM (VEGFR-2)

11 (Pazopanib)

IC50 = 30 nM (VEGFR-2)

NH

N

Me

MeOOMe

HN N

N

N NN

Me

SO2NH2

Me

Me

IC50 = 21 nM (cell)

Me

Figure 7.7 Structures and activities of VEGFR inhibitors and pazopanib.

NO2

NN

S

HN

12

IC50 = 26.8 μM (CK2α)IC50 = 32.2 μM (CK2α )'

OMe

CO2H

N

S

HN

O

OMe 13

IC50 = 32 nM (CK2α)IC50 = 46 nM (CK2α )'

Figure 7.8 Structures and activities of CK2 inhibitors.


compound against protein kinase CK2. The X-ray cocrystal structure of the corre-sponding benzoic acid derivative with CK2a showed the binding mode of thiadia-zole ring in the ATP adenine site. Subsequent X-ray structure-based modificationled to potent and selective inhibitor 13, which presumably binds to the hingebackbone residues, Glu114 carbonyl and Val116 NH, as well as occupies bothhydrophobic pockets I and II.Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays a critical role in

G2/M phase transition and in key regulation of cell mitosis [21,22]. A number ofPLK1 inhibitors are progressing through clinical development [23,24]. Chen andcoworkers from Roche and Nie and coworkers from Takeda reported the identifi-cation of pyrimidodiazepines as a potent class of ATP-competitive PLK1 inhibi-tors. As shown in Figure 7.9, screening identified N-arylpyrimidin-2-amine 14 asthe hinge binding motif for PLK1 inhibitor [25,26]. Subsequent design from thishit resulted in a variety of 2-arylamino pyrimidodiazepinone derivatives as potentPLK1 inhibitors. Compound 15 exhibited good kinase inhibitory activity; however,cell potency was inadequate. Subsequent structure-based design resulted in potentinhibitors 16 and 17. Inhibitor 17, also known as TAK-960, progressed to clinicaldevelopment. As shown in Figure 7.10, the X-ray crystal structure of 17-boundPLK1 revealed that the inhibitor formed a hydrogen bond in the hinge domainand interacted with hydrophobic pockets I and II. The inhibitor amide NH twistedto make interaction with Leu59 backbone carbonyl, stabilizing the P-loop. The pyr-imidodiazepinone ring carbonyl is involved in a water-mediated interaction withAsp194 [26].

N

NNH

Ar

R

R'

N

N N

N

NH

O

F

F

OMe

Me

MeMe

R

N

N N

N

NH

O

F

F

HO2C

OMe

15

IC50 = 18 nM (PLK1)EC50 = 390 nM (H82 cell)

14

16 (RO3280, R = H)17 (TAK-960, R = F)

IC50 = 2 nM (PLK1)EC50 = 3 nM (HT29 cell)

NH

ON

Figure 7.9 Structures and activities of PLK1 inhibitors.


7.5.2Type II Kinase Inhibitors and Their Design

Type II inhibitors bind to the kinase ATP binding site similar to type I inhibitors,but extend further into an allosteric site, which is available only when the enzymeis in the inactive state. The inhibitor induces a conformational change and theenzyme no longer functions. This state is termed the “DFG-out,” where the aspar-tic acid residue points away from the ATP site and the phenylalanine residuemoves toward the ATP site, exposing a hydrophobic pocket in the allosteric regionadjacent to the ATP binding site [27]. These inhibitors typically contain hetero-cyclic or heteroatomic groups that form one or two hydrogen bonds in the hingeregion and also occupy the allosteric site. Since the allosteric site is not conservedand differs from one kinase to another, inhibitors of this class show better selec-tivity and safety profiles [28].There are only a few approved type II inhibitors: imatinib, nilotinib, and sorafenib.

These inhibitors are some of the most successful kinase inhibitor drugs. Imatinibwas the first FDA-approved kinase inhibitor drug. The details of design and discoveryof imatinib and nilotinib are provided in Chapter 12. There has been a major empha-sis on the design and development of type II inhibitors targeting the inactive kinaseconformation [29–31]. Sorafenib (5) in Figure 7.4 is a multikinase inhibitor that tar-gets Raf, VEGF, and PDGF receptor tyrosine kinase and was FDA approved in 2005for the treatment of renal cell carcinoma and hepatocellular carcinoma [32]. It is anexample of a type II inhibitor. The X-ray crystal structure of sorafenib-bound p38a inFigure 7.11 shows that it occupies the ATP binding site in an inactive DFG-out con-formation [33]. The structure revealed that it forms two hydrogen bonds in the hingedomain, additional hydrogen bonds with the conserved Glu71 and Asp168, stabilizedthe DFG-out conformation, and occupied the allosteric pocket [15]. A pharmacophoremodel of the design of type II inhibitors is shown in Figure 7.11 [13].

Figure 7.10 The X-ray crystal structure of inhibitor 17 (TAK-960) with PLK1 (carbon chain, green;fluorine, magenta; PDB code: 4J53).


Wang et al. from Pfizer reported structure-based design of type II inhibitorsas B-Raf kinase inhibitors [34]. Based upon the X-ray structure of type I andtype II inhibitors bound to B-Raf, isoindoline-1,3-diones 18 and 2,3-dihydroph-thalazine-1,4-diones 19 shown in Figure 7.12 were designed. These scaffoldsshowed potency in a micromolar range against B-Raf. Initial modeling showedthat these inhibitor leads bind to B-Raf in the active DFG-in conformation. In an

Figure 7.11 X-ray crystal structure of sorafenib (5)-bound p38a (carbon chain, green; PDB code:3HEG).

HN

O

ONH

NH

OH

OH

NH

NH

OH

OH

IC50 = 0.338 µM (B-Raf)

HNHN

O

O

IC50 = 0.29 µM (B-Raf)

18 19

HN

O

ONH

Cl

HN

NH

Cl

HN

HNHN

O

O20 IC50 = 10 nM (B-Raf) 21 IC50 = 17 nM (B-Raf)

O OCF3 CF3

Figure 7.12 Structures and activities of B-Raf inhibitors.


effort to design type II inhibitors, lipophilic groups were introduced to interact withthe allosteric pocket formed due to the movement of DFG motif. In particular, sub-stituents were introduced to interact with Glu501 and Asp594. These led to potent B-Raf inhibitors 20 and 21 containing isoindoline and dihydrophthalazine scaffolds.Both inhibitors were evaluated against a panel of kinases and showed excellentselectivity.Oguro et al. from Takeda reported pyrrolopyrimidine-based type II VEGFR-2

kinase inhibitors [35]. As shown in Figure 7.13, functionalization of the pyrrolo-pyrimidine scaffold with phenyl urea provided inhibitors 22 and 23 with markeddifference in potency of meta- and para-derivatives. Further incorporation of sub-stituents on the phenyl ring of the urea provided a variety of derivatives. Com-pound 24, with a meta-trifluoromethyl group, improved VEGFR-2 potency as wellas growth inhibitory activity in human umbilical vein endothelial cells (HUVEC).Further investigation of substituents on the phenyl ether ring resulted in verypotent inhibitor 25. Oral treatment of inhibitor 25 in a xenograft mouse showedantitumor effects. This compound also exhibited an interesting time-dependentinhibition of the nonphosphorylated catalytic site of VEGFR-2 [36].The X-ray crystal structure of 25-bound VEGFR-2 revealed that the inhibitor

binds to the VEGFR-2 in its the inactive conformation [35]. As can be seen inFigure 7.14, one of the pyrimidine nitrogens forms a hydrogen bond with theCys919 backbone NH in the hinge region. The urea functionality forms two hydro-gen bonds with the protein in the conserved Glu885 carbonyl and the backboneNH of Asp1046 of the DFG-out motif. The m-trifluorophenyl moiety occupied theallosteric site created by the conformational change of Phe1047 to the DFG-outconformation.

N

NNMe O

H

NH

NH

O

22 IC50 = 1800 nM (VEGFR-2) N

NNMe O

HN

H

23 IC50 = 33 nM (VEGFR-2)

O

HN

N

NNMe O

HN

24 IC50 = 5.3 nM (VEGFR-2)

O

HN

CF3

25 IC50 = 6.2 nM (VEGFR-2)N

NNMe O

HN

O

HN

CF3

F

IC50 = 4.4 nM (HUVEC) IC50 = 22 nM (HUVEC)

Figure 7.13 Structures and activities of VEGFR-2 inhibitors.


Dai et al. from Abbott Laboratories designed a 3-aminoindazole-based receptortyrosine kinase inhibitor that presumably binds to the KDR, also known as VEGFR-2, in the inactive conformation [37]. As depicted in Figure 7.15, starting from thieno-pyrimidine scaffold 26, the investigators designed 3-aminoindazole 27 by removal ofa CH unit from the pyrimidine ring to give the five-membered ring to mimic the

Figure 7.14 The X-ray crystal structure of inhibitor 25 with VEGFR-2 (carbon chain, turquoise;fluorine, magenta; PDB code: 3VHE).

N

N SR

NH2

NHR'

HN

NH

H2N

NHR'

NNH

H2N

NH2

NNH

H2N

HN

26 27

NH

O

Me

28 29 30 (ABT-869)

NNH

H2N

HN NH

O

Me

R

F

KDR (IC50 nM) FLT3 (IC50 nM) c-KIT (IC50 nM)Compound

28 4790 43 736029 3 12 1730 4 5 16

Figure 7.15 Structures and activities of 3-aminoindazole-based VEGFR-2 inhibitors.


adenine binding site of ATP with the kinase hinge region. Aniline derivative 28showed good potency against the fms-like tyrosine kinase (FLT1) subfamily. Incorpo-ration of diaryl urea at the C4-position of the indazole resulted in a variety of potentcompounds. Compound 29 displayed very good potency against KDR, FLT1, and c-KIT. Compound 30 potently inhibited VEGFR and PDGFR kinases and displayedgood cellular activity as well. It was also shown to have potent oral activity in a xeno-graft mouse model. It has subsequently progressed to clinical development.

7.5.3Allosteric Kinase Inhibitors and Their Design

Allosteric kinase inhibitors are also known as type III kinase inhibitors. They bindto the allosteric site, which is a non-ATP binding site, and modulate ATP bindingto the kinase by making a conformational change that makes the kinase inactive.Allosteric inhibitors exhibit the most selectivity since they occupy the allostericbinding site, which is unique to a particular kinase. Numerous allosteric inhibi-tors have progressed to clinical development and are reported in a number ofreviews [2,13,38,39]. In this section, we will review the structure-based design of afew selected allosteric inhibitors.The mitogen-activated protein (MAP) kinase signaling pathways are involved in

controlling various cellular functions [40,41]. The RAG–MEK–ERK signal transduc-tion pathway is critical to cell growth, differentiation, and apoptosis. Overexpres-sion and activation of MEK/ERK have been implicated in several human cancers.A significant effort has been devoted to the design and synthesis of MEK inhibi-tors. As shown in Figure 7.16, potent allosteric MEK inhibitor 31 was evaluated in

HN

F

F

Cl I

O

NH

O

HN

F

F

F I

O

NH

OOH

OH

R

HNF

Cl Br

O

NH

OHO

NN

31 (CI-1040) 32 R = H (PD 0325901)33 R = Br (PD 318088)

34 (AZD6244)

HN

F I

O

NH

OHO N Me

OMe35

Figure 7.16 Structures of various MEK inhibitors.


the clinic [42]. However, this inhibitor suffered from low systemic exposurebecause of solubility and rapid metabolism issues. To address these issues, deriva-tive 32 (PD-0325901) was prepared for evaluation. Both 31 and 32 have shownexcellent potency in cellular assay (IC50¼ 35 and 0.33 nM, respectively). However,compound 32 showed significantly improved solubility (<1mg/ml for 31 and190mg/ml for 32) [42]. To obtain structural interactions in the active site, the X-raycrystal structure of bromo derivative 33 with human MAP kinase1 (MEK1) andMEK2 was determined [43]. The structural analysis revealed that inhibitor 33 andMgATP bind simultaneously and noncompetitively. The structures showed thatboth MEK1 and MEK2 have a unique inhibitor binding pocket close to the MgATPsite. The inhibitor induces a conformational change in the unphosphorylated stateand locks the enzymes into catalytically inactive states. As shown in Figure 7.17,the hydroxamate oxygen and the carbonyl oxygen form hydrogen bonds with theLys97 side chain. One of the fluorine atoms was located within proximity to inter-act with backbone NHs of Val211 and Ser212. The fluoroiodoaniline piece occu-pied the hydrophobic pocket surrounding Phe209, Ile141, Met143, and Val127.A structurally related derivative 34 (AZD6244) has shown good enzyme inhibi-

tory activity (IC50¼ 14.1 nmol/L). This compound is undergoing clinical develop-ment as well [44]. Wallace et al. from Takeda carried out structure-based designand synthesis of pyrrole derivatives as MEK inhibitors [45]. Compound 35 (MEK1,IC50¼ 18 nM; Colo 205 cell, EC50¼ 12 nM) showed excellent enzyme inhibitoryactivity and cellular potency. The X-ray structural studies revealed that compound35 binds in the MEK1 allosteric site similar to inhibitor 33 in Figure 7.17.

Figure 7.17 The X-ray crystal structure of inhibitor 33 (inhibitor carbon chain, magenta; bro-mine, purple; fluorine, turquoise; iodine, brown) and MgATP (carbon chain, green; Mg2þ,orange; phosphorus, yellow) with MEK1 (PDB code: 1S9J).


AKT, also known as protein kinase B, a serine/threonine kinase, plays a criticalrole in the signal transduction pathways of cell proliferation, apoptosis, angiogene-sis, and diabetes. Dysregulation of AKT pathways leads to numerous human can-cers [46]. The development of inhibitors of AKT pathways has been pursued by anumber of laboratories. Both compounds 36 and 37 in Figure 7.18 were designedand developed at Merck [47,48]. Inhibitor 37 (MK-2206) is potent against AKT1–AKT3 and orally bioavailable. Both compounds are allosteric inhibitors. The X-raystudies of 36-bound AKT1 revealed that the compound binds to AKT1 in an allo-steric binding site [49].Tomita et al. from Takeda reported the structure-based design and development

of cell-active and allosteric inhibitors of focal adhesion kinase (FAK) [50]. FAK is anonreceptor protein tyrosine kinase, also known as PTK2. This kinase was shownto play important roles in cell proliferation, resistance, migration, and invasion[51,52]. Overexpression of FAK is related to a variety of human cancers. A numberof type I dual inhibitors have been evaluated clinically [53,54]. Tomita et al. carriedout a systematic search for an ATP noncompetitive inhibitor. In their HTS efforts,in the presence of a high concentration of ATP, they have identified 1,5-dihydro-pyrazole benzothiazine derivative 38 in Figure 7.19 as a low micromolar inhibitor.The X-ray crystal structural studies of 38 with FAK revealed that the inhibitoroccupied the allosteric binding site. Subsequent structure-based optimizationresulted in derivative 39 with an improvement in potency. However, X-ray struc-tural studies revealed that the pyrazole nitrogens interacted with the hinge regionand the inhibitor did not occupy the allosteric site. To disrupt the hinge binding,alkylation of pyrazole nitrogen 1 provided very potent inhibitor 40 with excellentselectivity against other kinases (PyK2, Aurora, MEK1). Alkylation of nitrogen 2resulted in a loss of potency (IC50 >30 mM). Compound 40 showed poor cell activ-ity (31% inhibition of cell FAK). Optimization of the biphenyl ring provided inhib-itor 41 with improvement in cellular activity (IC50¼ 7.1 mM).The X-ray crystal structure of 41 with FAK showed an allosteric binding mode

similar to 38 [50]. X-ray structures of inhibitors 39 and 41 in the FAK binding siteare shown in Figure 7.20. As can be seen, both inhibitors bind to the FAK binding

N

NH N

NN

N NH

O

36 (Inhibitor VIII)

N

NHN

O

N

NH2

37 (MK-2206)

IC50 = 58 nM (AKT1)IC50 = 210 nM (AKT2)IC50 = 2119 nM (AKT3)

IC50 = 5 nmol/L (AKT1)IC50 = 12 nmol/L (AKT2)IC50 = 65 nmol/L (AKT3)

Figure 7.18 Structures and activities of AKT1 inhibitors.


SN

NHN

Me

O

O

38

IC50 = 0.96 µM (FAK, 0.5 µM ATP)

SN

NHN

Me

O

O

39

IC50 = 0.5 µM (FAK, 0.5 µM ATP)

O

N

HO

41

SN

NN

Me

O

O N

40

Me

SN

NN

Me

O

O

Me1

2

H

IC50 = 0.077 µM (FAK, 0.5 µM ATP) IC50 = 0.64 µM (FAK, 0.5 µM ATP)

Figure 7.19 Structures and activities of FAK inhibitors.

Figure 7.20 The overlay of X-ray crystal structures of inhibitors 39 (carbon chain, green; PDBcode: 4I4E) and 41 (carbon chain, magenta; PDB code: 4I4F) in FAK binding site.


site in different locations. Inhibitor 39 clearly binds to the ATP binding site,whereas inhibitor 41 binds to the allosteric site of the FAK binding site. The pyr-azole group of inhibitor 39 interacts with Glu500 and Cys502 in the hinge region.Also, one of the sulfone oxygens forms a hydrogen bond with the terminal aminogroup Lys454. The terminal hydroxyl group of 39 also formed water-mediatedhydrogen bonds in the ATP binding site. The pyrazole methyl group of 41, on theother hand, is oriented toward the hydrophilic space surrounded by Asp604 andHis544. The terminal tert-butyl group nestled in a hydrophobic pocket surroundedby Met475, Leu486, and Met499 (gatekeeper of FAK).

7.5.4Covalent Kinase Inhibitors and Their Design

Covalent kinase inhibitors form covalent and irreversible bonds to the kinase activesite. The inhibitors often react with the nucleophilic cysteine residue in the activesite. The design strategy for covalent inhibitors involves attaching an electrophilicfunctionality to an appropriate scaffold capable of reacting with the electron-richsulfur of the cysteine residue. Ideally, the inhibitor will first bind in a noncovalentmanner and then form a covalent bond with an appropriate electrophilic function-ality located within proximity to cysteine residues in the ATP binding site. Thecovalent inhibitor should react poorly with glutathiones and thiols of other pro-teins, but react preferably with target cysteine selectively upon binding to thekinase binding site. Various warhead functionalities such as epoxides, aziridines,haloketones, and Michael acceptors can be utilized; however, Michael acceptinggroups have been most commonly utilized in the design of covalent inhibitors. Avariety of covalent kinase inhibitors have been designed. This has been covered ina number of recent review articles [55–57].The EGFR has a Cys797 located in a conserved a-helix at the vicinity of the ATP

binding site [58,59]. This has been specifically targeted in the design of covalentkinase inhibitors. As shown in Figure 7.21, EGFR-selective anilinoquinazolineand anilinoquinoline derivatives were converted to covalent inhibitors 42 and 43[60,61]. The X-ray crystal structures of the scaffolds without the Michael acceptorgroups suggested the optimum position where the a,b-unsaturated carbonylgroups can be attached in 42 and 43 [62]. The mechanism of action involvesMichael addition of the cysteine residue of EGFR to form a covalent bond. Thisresults in blocking ATP binding in the active site and inactivation of the kinase.The X-ray crystal structure of inhibitor 43-bound EGFR revealed covalent bondformation with the Cys797 side chain (Figure 7.21). Also, the quinoline nitrogenformed a hydrogen bond with the Met793 backbone NH in the hinge region.Inhibitor 43 is currently in clinical development [63,64].The c-Jun N-terminal kinase (JNK) is a part of MAP kinase signaling pathways

and plays an important role in cellular responses to mitogenic stimuli, environ-mental stresses, and as an apoptotic agent [65]. There are three isoforms of JNK,known as JNK1, JNK2, and JNK3. They are encoded by three independent genes.Both JNK1 and JNK2 show broad tissue expression profiles, whereas JNK3 is


mainly expressed in the central nervous system. JNK signaling is associated withthe pathophysiology of a number of diseases, including cardiovascular, inflamma-tion, cancer, and neurodegeneration [66].Gray and coworkers designed a variety of covalent JNK inhibitors using phenyl-

aminopyrimidine structural scaffold that is inherent to many potent noncovalentinhibitors, including imatinib [67]. Based upon the proximity of the Cys788 andmethylpiperazine, electrophilic acrylamide was incorporated in place of the methyl-piperazine of imatinib. The resulting inhibitor 44, in Figure 7.22, inhibited JNK1–JNK3 in micromolar range. Assuming imatinib would bind to JNK in an alterna-tive conformation, the flag methyl in 44 was removed as this methyl group wasresponsible for selectivity against c-KIT, Abl, and PDGF relative to other kinases.This provided inhibitor 45 with 4–10-fold improvement in potency. Exploration ofa 1,4-diamine and 1,3-benzamide combination resulted in inhibitor 46 with 500-fold improvement in potency against JNK1–JNK3. The X-ray crystal structure of 46with JNK3 revealed that Cys154 forms a covalent bond with the acrylamide and theaminopyrimidine motif forms two hydrogen bonds with the hinge region. Subse-quently, various methylated derivatives were prepared and extensive biochemicaland cellular assays showed their ability to inhibit JNK activity and selectivity.

Figure 7.21 Structures of EGFR inhibitors 42 and 43 and X-ray crystal structure of inhibitor 43with EGFR (carbon chain, green; PDB code: 2JIV).


Inhibitor 46, which is a relatively selective JNK inhibitor in cells, exhibitedenhanced selectivity upon introduction of the flag methyl group in derivative 47.Mitogen- and stress-activated kinase 1 (MSK1) is a nuclear protein kinase

that regulates transcription downstream of extracellular signal-regulated kin-ases and p38 nitrogen-activated protein kinases via the phosphorylation of thecAMP response element binding protein and histone H3. Overexpression ofMSK1 has been implicated in numerous human cancers [68]. Specific inhibi-tors of MSK1 may be of use in cancer chemotherapy as well as in the study ofMSK function in cells. Taunton and coworkers carried out electrophilic frag-ment-based design of reversible MSK1 inhibitors that exhibited high selectivityover MSK/RSK family kinases [69]. MSK1 is closely related to p90 ribosomalprotein S6 kinase (RSK). Both possess the same kinase domains and a struc-turally homologous cysteine in its C-terminal kinase domain (CTD). In an ear-lier study, Taunton and coworkers reported design of reversible covalentinhibitors of RSK2-CTD by targeting noncatalytic cysteine residues using acryl-amide-based derivatives [70]. As shown in Figure 7.23, compound 48 is highly

44 (JNK-IN-1) 45 (JNK-IN-2)

46 (JNK-IN-7) 47 (JNK-IN-8)

JNK1 JNK2 JNK3Compound

44 7780 4230 775045 809 1140 70946 1.54 1.99 0.7547 4.67 18.7 0.98

(IC50 nM)(IC50 nM) (IC50 nM)

N

NHN

N

NH

H

N

NHN

N

Me

HN

O

HN O

NN

NHN

N

H

HN

O

HN O

N

O

NH

ON

N

NHN

N

NH

Me

O

NH

ON

Figure 7.22 Structures and activities of covalent JNK inhibitors.


Figure 7.23 Structures of RSK1-CTD inhibitors 48 and 49 and X-ray crystal structure of inhibitor49 with RSK2-CTD (carbon chain, magenta; PDB code: 4D9U).

Figure 7.24 Structures of RSK2 inhibitors 50–52 and overlay of X-ray crystal structure of inhibi-tors 50 and 52 with RSK2 (inhibitor 50, carbon chain, green; PDB code: 4JG6; inhibitor 52, car-bon chain, magenta; PDB code: 4JG7).


selective for RSK1-CTD and RSK4-CTD. Kinase profiling showed that only 6 of442 kinases exhibited >90% inhibition. The KD for RSK1-CTD was 0.54 nMand its affinity was 80-fold higher than MAP3K1 and more than 400-foldhigher than STK16, R1PK2, RET, MEK5, and PDGFRB [70]. The X-ray struc-ture of the corresponding tert-butyl derivative 49 revealed that Cys436 was con-nected to the b-carbon of the cyanoacrylate. The other cysteine (Cys560) in thevicinity (�7 A

�) was not able to form a covalent bond. The pyrrolopyrimidine

scaffold formed a number of hydrogen bonds with the Thr493 side chain aswell as the Glu494 backbone carbonyl and Met496 backbone NH. Further-more, the p-tolyl group appeared to pack against the gatekeeper side chain ofThr493 and extended into a hydrophobic pocket.Based upon the above studies, Taunton and coworkers designed a series of cya-

noacrylamide-based inhibitors that showed activity against MSK/RSK family kin-ases, but maintained high selectivity over NEK2 and PLK1, even though theypossess a homologous cysteine residue [69]. As shown in Figure 7.24, compounds50–52 inhibited RSK2 at submicromolar concentrations. Interestingly, the X-raycrystallographic studies of inhibitors 50 and 52 bound to RSK2 revealed that both

Figure 7.25 Structures of RSK2 inhibitors 53 and 54 and the X-ray crystal structure of inhibitor54 with T493M RSK2 (carbon chain, green; PDB code: 4JG8).


inhibitors bind differently in the RSK2 active site. As shown in Figure 7.24, anoverlay of both crystal structures showed that one can optimize inhibitor 50 byappending aromatic substituents at the 3-position of the indazole ring. Basedupon this molecular insight, the investigators designed trimethoxyphenyl-substi-tuted derivative 53, which showed a 20-fold improvement in potency for RSK2;however, its selectivity over NEK2 and PLK1 was poor. Incorporation of a bulkyamide substituent in 54 (Figure 7.25) retained potency for RSK2 and significantlyimproved selectivity over NEK2 and PLK1. A crystal structure of 54 bound toT493M RSK2 revealed the binding mode. The indazole moiety packed againstgatekeeper Met493 and the trimethoxyphenyl derivative nicely occupied the hydro-phobic pocket around Ile428, Met496, and Leu546.

7.6Conclusions

Protein kinases are important drug design targets. This chapter has outlined a vari-ety of structure-based design strategies, including the design of a range of hingebinding heterocycles and functionalities, utilized in the development of FDA-approved drugs. In recent years, many different types of kinase inhibitors have beendesigned and synthesized. This chapter has also described lead discovery, structure-based optimization, selectivity design, and structure–activity studies leading toapproved drugs. A large number of inhibitors known to date have resulted from theavailability of many X-ray crystal structures of inhibitor–kinase complexes. The analy-sis of protein–ligand interactions of a number of selected crystal structures that haveaided the engineering of selectivity and designing of inhibitors to combat resistancehas been provided. The design of kinase inhibitors will continue to be an importantarea in medicinal chemistry. Various tools and strategies described will be valuablefor the development of new and more effective inhibitors.

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40 Davis, R.J. (1993) The mitogen-activatedprotein-kinase signal transduction pathway.J. Biol. Chem., 268, 14553–14556.

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45 Wallace, M.B., Adams, M.E., Kanouni, T.,Mol, C.D., Dougan, D.R., Feher, V.A.,O’Connell, S.M., Shi, L.H., Halkowycz, P.,and Dong, Q. (2010) Structure-baseddesign and synthesis of pyrrole derivativesas MEK inhibitors. Bioorg. Med. Chem. Lett.,20, 4156–4158.

46 Graff, J.R. (2002) Emerging targets in theAKT pathway for treatment of androgen-independent prostatic adenocarcinoma.Expert Opin. Ther. Targets, 6, 103–113.

47 Lindsley, C.W., Zhao, Z., Leister, W.H.,Robinson, R.G., Barnett, S.F., Defeo-Jones,D., Jones, R.E., Hartman, G.D., Huff, J.R.,Huber, H.E., and Duggan, M.E. (2005)Allosteric Akt (PKB) inhibitors: discoveryand SAR of isozyme selective inhibitors.Bioorg. Med. Chem. Lett., 15, 761–764.

48 Hirai, H., Sootome, H., Nakatsuru, Y.,Miyama, K., Taguchi, S., Tsujioka, K., Ueno,Y., Hatch, H., Majumder, P.K., Pan, B.S.,and Kotani, H. (2010) MK-2206, anallosteric Akt inhibitor, enhances antitumorefficacy by standard chemotherapeuticagents or molecular targeted drugs in vitroand in vivo. Mol. Cancer Ther., 9, 1956–1967.

49 Wu, W.I., Voegtli, W.C., Sturgis, H.L.,Dizon, F.P., Vigers, G.P.A., andBrandhuber, B.J. (2010) Crystal structure ofhuman AKT1 with an allosteric inhibitor


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50 Tomita, N., Hayashi, Y., Suzuki, S.,Oomori, Y., Aramaki, Y., Matsushita, Y.,Iwatani, M., Iwata, H., Okabe, A., Awazu,Y., Isono, O., Skene, R.J., Hosfield, D.J.,Miki, H., Kawamoto, T., Hori, A., and Baba,A. (2013) Structure-based discovery ofcellular-active allosteric inhibitors of FAK.Bioorg. Med. Chem. Lett., 23, 1779–1785.

51 Schwock, J., Dhani, N., and Hedley, D.W.(2010) Targeting focal adhesion kinasesignaling in tumor growth and metastasis.Expert Opin. Ther. Targets, 14, 77–94.

52 McLean, G.W., Carragher, N.O., Avizienyte,E., Evans, J., Brunton, V.G., and Frame, M.C. (2005) The role of focal-adhesion kinasein cancer: a new therapeutic opportunity.Nat. Rev. Cancer, 5, 505–515.

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55 Liu, Q.S., Sabnis, Y., Zhao, Z., Zhang, T.H.,Buhrlage, S.J., Jones, L.H., and Gray, N.S.(2013) Developing irreversible inhibitors ofthe protein kinase cysteinome. Chem. Biol.,20, 146–159.

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8Protein X-Ray Crystallography in Structure-Based Drug Design

8.1Introduction

Advances in protein X-ray crystallography have made an enormous impact ondrug discovery. X-ray crystallography is an important technique that allows visual-ization and study of protein structures at the atomic level. This can provide impor-tant molecular insight into the protein function and protein–ligand interactions.The determination of the three-dimensional structure of a protein is a complextask that requires several steps. These include protein expression and purification,protein crystallization, acquisition of diffraction data, and structural determinationand refinement of the three-dimensional models. These techniques have wit-nessed tremendous technical advancements over the years. Structural biology hasestablished a powerful role in both target identification and lead optimization.Also, fragment-based crystallography has become a powerful technique for leaddiscovery. The introduction of synchrotron radiation has greatly enhanced thepower of X-ray crystallography to obtain high-resolution macromolecular struc-tures [1,2]. Furthermore, recent technical advancements in protein engineering,as well as in robotic handling and synchrotron beamlines, are opening new excit-ing opportunities.There are around 90,000 structures in the Protein Data Bank (PDB), of which

almost 80,000 were determined by X-ray crystallographic methods and 10,000were determined by NMR spectroscopic methods. X-ray crystallography dominatesthe field of structural biology. Today, the knowledge of three-dimensional struc-tures of target proteins has greatly accelerated drug discoveries through structure-based design approaches. During early efforts of structure-based design, the three-dimensional structural information was limited. Therefore, the X-ray structures ofrelated enzymes were utilized to generate a comparative model for the target pro-tein. Such models were exploited for optimization of drug–target interactions toimprove potency and selectivity of lead structures. The availability of X-ray struc-tures of the protein drug target and determination of crystal structures of protein–ligand complexes provided more detailed protein–ligand interactions. This struc-tural knowledge has accelerated the drug design and development process [3,4].

183


Drugs such as Agenerase, Viracept, and Relenza were among the first few earlydrugs that benefited from the X-ray crystal structures of the target enzymes. Withthe growth and advancements in structural biology, the three-dimensional struc-tural knowledge of disease-relevant targets also expanded rapidly. As shown inChapter 1 X-ray crystal structure-based design contributed to the approval of 34new drugs through 2012. Structure-based design of a number of these marketeddrugs and advanced clinical candidates will be covered in the next section.

8.2Protein Expression and Purification

The first step in protein crystallization is to obtain a sufficient quantity of the tar-get protein with a suitable degree of purity. The protein can be extracted and purif-ied from a biological matrix, or it can be produced through recombinant methodsor heterologous expression systems. The first method is extremely time consum-ing and can be applied only to proteins that are expressed at a high level. For rareproteins, an amount appropriate for crystallographic studies cannot be obtainedfrom natural sources. Overexpression of the protein through recombinant meth-ods is now the method of choice, not only for rare proteins but also for severalapplications since it presents a number of advantages.Recombinant proteins are often expressed in bacteria, especially in Escherichia

coli. Bacteria can be handled quite easily, thus furnishing affordable culture condi-tions. Moreover, several strains of E. coli are now available with useful propertiessuch as lack of specific proteases that reduce the degradation of the foreign pro-tein, the presence of chaperones that aid in the proper folding of the target pro-tein, availability of wholly or partially synthetic genes with optimized codons thatincrease the protein expression levels, and the availability of several expressionvectors that help the production of fusion proteins that can be easily isolated andpurified from the growth media. Furthermore, recombinant methods allow themodification of the protein to increase its solubility, since the formation of aggre-gates or inclusion bodies lowers the overall yield of the protein. Most eukaryoticproteins can be expressed in E. coli. However, in those cases in which this is notpossible, more labor-intensive but effective eukaryotic cell systems, such as isletcells, yeast, and Chinese hamster ovary cells, are available and very effective.Several methodologies exist for the modification of the target protein in order to

aid expression yield, purification, and crystallizability. Engineering of the targetprotein can be accomplished in several ways [5]. There are techniques that helpprotein purification. Several tags have been developed in order to allow proteinpurification through affinity chromatography, most commonly used is hexa-Histags for Ni affinity chromatography. Other common tags are glutathione S-trans-ferase (GST) and maltose binding protein (MBP). The latter two tags are powerfulways of increasing solubility and are most often used for this reason. Techniquesare available to increase protein crystallizability. Crystallizability is sometimes poordue to the presence of highly flexible regions. Protein engineering can be used for

184 8 Protein X-Ray Crystallography in Structure-Based Drug Design

isolating the stable domains of a protein by eliminating N- or C-terminal flexiblefragments, by isolating domains from multidomain proteins, or, more rarely, byremoving internal flexible loops. Protein engineering is much more convenientthan limited proteolysis, which could result in sample heterogeneity [6]. Moreover,the solubility of a properly folded protein is a function of the hydrophobicity of itssurface. Mutation of single surface amino acids (e.g., derived from model or byscanning mutagenesis) can result in an improvement of solubility. Single-pointmutations are often useful for improving protein solubility, thus enhancingcrystallizability. Moreover, mutation of a single surface amino acid can interferewith protein–protein interactions, thus modifying the overall crystallizability ofthe target protein. Crystallography can be improved by removing sites susceptibleto posttranslational modification that are often detrimental to crystallization. Forexample, removal of glycosylation sites by replacing asparagines with other resi-dues (e.g., aspartates or glutamines) can be useful. Removal of flexible glycosidicgroups through deglycosylating enzymes is also possible [7].

8.3Synchrotron Radiation

The introduction of synchrotron radiation and the subsequent advancement in syn-chrotron beamline contributed to advances in X-ray crystallography. In contrast toclassical X-ray tubes, the synchrotron radiation is produced when electrons are accel-erated centripetally by using appropriate bending magnets. Evolution of facilities fromfirst- to second- and third-generation fully exploited insertion devices (e.g., undula-tors) that are able to intensify the beam. X-ray beams produced at third-generationsynchrotron facilities are laser-like and are characterized by a high-brilliance beam.They allow the reduction of sample exposure time from hours to minutes to seconds.This was paralleled by the development of detectors with greatly reduced exposuretimes. In particular, improvement of intensity of the radiation and continuous spec-tral distribution of synchrotron radiation allowed implementing experiments such asmultiwavelength anomalous diffraction (MAD) and time-resolved crystallographybased on Lane diffraction.The application of MAD is an advantageous solution to the phase problem in

X-ray crystallography since systematic incorporation of selenomethionine becamequite routine due to advancement in recombinant DNA technology. Also, advance-ments in the techniques for cryopreservation of the macromolecular crystalsthrough rapid freezing are of paramount importance for fully exploiting synchro-tron radiation. In this way, the cryopreserved samples acquire resistance to radia-tion damage that is a critical issue for large biological assemblage, small crystals,and MAD phasing experiments that can be run on the same crystal sample.These technical advancements paved the way for microcrystallography that can

analyze crystals smaller than 20 mm in size using beam sizes in the range of1–20mm [8]. These extremely small beamlines are now available at third-generationsynchrotron facilities and are able to generate microbeams with high brilliance and

8.3 Synchrotron Radiation 185

stability. Microcrystallography is particularly useful when crystals of reduced sizeare available; however, it can also be used for illuminating a well-diffracting regionof an imperfect sample or single-crystal regions of multiple-crystal samples. One ofthe most important applications of microcrystallography is in the field of G-pro-tein-coupled receptors [9–11]. Microcrystal sample handling and mounting, in par-ticular for the precision and stability of the beam/crystal interception, is alsocritical to microcrystallography. Progressive robotization of several operationalaspects in data acquisition paved the way for high-throughput crystallography andthe important application of X-ray crystallography in fragment-based drug design.

8.4Structural Biology in Fragment-Based Drug Design

Fragment-based drug design is a methodological approach based on screening ofsmall fragments against a protein target in order to obtain starting hits for thesubsequent optimization process to obtain a lead compound. The process of frag-ment-based drug design starts with the so-called crystallographic screening, con-sisting of soaking crystals of biological targets with cocktails of small fragments.Usually, the fragment libraries consist of about 1,000 fragments divided into cock-tails containing 3–12 compounds. The binding of a fragment is afterward directlymonitored by X-ray diffraction of the target–fragment complex. This techniquewas pioneered by Astex Pharmaceuticals with the approach of high-throughput X-ray fragment screening. The second step is the optimization of the bound frag-ment(s) that usually have low affinity for the target to improve potency. The pre-cise knowledge of the binding mode of the fragment inside the biological targetdrives a more efficient process of fragment optimization. X-ray crystallography isalso a privileged technique for the fragment optimization process.Often prescreening methods can be used for identifying appropriate fragments

whose binding mode is subsequently determined by X-ray crystallography of cocrys-tals. Physical methods necessary for the identification of binding fragments requireextreme sensitivity in order to detect the binding of low-affinity compounds. Surfaceplasmon resonance, ligand-observed NMR, high-concentration screening, and iso-thermal titration calorimetry are some of the methods used for prescreening of thefragment library and can be accompanied by computational methods such as virtualscreening [12]. Regardless of which technique has been used for assessing the bind-ing of the small fragment to the protein target, unambiguous definition of the bind-ing mode of the fragment to the protein should be obtained. X-ray crystallography isalso necessary for the subsequent phase of elaboration of the initial fragment to alead compound. NMR is an alternative technique for the definition of the bindingmode of a fragment to the protein target.Fragments usually have a molecular weight of <300Da and form favorable inter-

actions with their target since they have to overcome an entropic cost (rigid bodyentropic barrier) that is independent of the molecular weight [13]. For fragmentlibraries, the so-called rule of three has been elaborated [14] in analogy to the rule of


five that is applied to lead compounds for predicting or defining oral bioavailability.The rule of three states that suitable fragments have a molecular mass lower than300Da, log P< 3, and up to three hydrogen bond donor and acceptor groups. Dueto the smaller size of fragments with respect to conventional compounds includedin high-throughput screening (HTS) libraries, fragment libraries have a smaller size.Several commercial suppliers are now providing fragment libraries complying withthe rule of three and with high chemical diversity. Since small fragments are formedby a low number of atoms, the chemical space that can be explored is wider, withrespect to higher molecular weight compounds [15–18]. Although their affinity forthe target is low, they bind the target with high efficiency [19–21].Several metrics have been elaborated to estimate ligand efficiency, but fundamen-

tally it is a function of the enthalpy of binding with respect to the number of heavyatoms in a molecule [22,23]. Moreover, fragments have a high degree of comple-mentarity with the target and this level is most likely to decrease if the complexityof the ligand increases [20]. In most cases, in subsequent steps of the optimization,the original fragment maintains a binding mode and forms a number of hydrogenbonds that are retained in the optimized lead compound [24]. From a physico-chemical viewpoint, one of the advantages of small fragments complying with the“rule of three” is their low lipophilicity and their ability to form a large number ofpolar interactions with the target. So, during the optimization procedure, attentionneeds to be paid to avoid an increase in the molecular weight and the lipophilicityin order to gain higher affinity. The development of highly lipophilic lead com-pounds can result in several unwanted biological properties such as promiscuity,tendency for aggregation, increased affinity for P-gp efflux pumps, and increasedhERG affinity [12]. All of these properties can be responsible for high attrition ratesin the subsequent steps of the drug development pipeline.

8.5Selected Examples of Fragment-Based Studies

Several examples of lead compounds that have been developed by fragment-baseddiscovery approaches can be found in the literature and are summarized in severalreviews [12,18,25–29]. Here, we outline a selection of recently reported cases. D-Amino acid oxidase is a flavoenzyme that catalyzes the formation of D-serine.Inhibitors of this enzyme could be potentially useful in the treatment of schizo-phrenia. Since the enzyme is centrally located, inhibitors should be endowed withbrain-penetrating properties in order to validate this enzyme as a drug target inCNS diseases. Recently, a fragment-based drug design approach has been usedfor the identification of novel inhibitors [30]. The binding pocket of previouslyknown inhibitors of this enzyme comprise a complex between the flavin ring ofthe enzyme cofactor and Tyr224. This key residue can occupy two different posi-tions: Tyr224-in and Tyr224-out (Figure 8.1a and b, respectively). In the out confor-mation, the aromatic ring of Tyr224 opens a subpocket that is perpendicular to theoriginal binding pocket.

8.5 Selected Examples of Fragment-Based Studies 187

A library of 3,500 fragments was screened to find hits suitable for subsequentelongation in the subpocket. Most of the hit fragments were small aryl carboxylicacids (as those reported in Figure 8.1a) that have an interaction motif in which thearomatic ring forms p–p stacking interaction with the flavin and the Tyr224 rings,whereas the carboxylic acid forms an electrostatic interaction with Arg283. It wasnoted that the formation of a hydrogen bond with Gly313 induced the Tyr224-outconformation. Among the fragments able to induce the Tyr224-out conformation,the 3-hydroxy-2-pyridone hit 1 (Figures 8.2 and 8.3) was chosen for subsequentelongation. This compound was thought to have a better chance of obtaining celland brain permeability as it does not possess acidic groups.

Figure 8.1 X-ray crystal structures of D-amino acid oxidase displaying the Tyr224 residue in (a)Tyr224-in (PDB code: 2DU8) and (b) Tyr224-out (PDB code: 3CUK) conformations.

NH

OH

O

1IC50 = 1150 nM

NH

OH

O

Cl

NH

OH

O NH

OH

O

Me

3IC50 = 3.9 nM

2aIC50 = 47 nM

2bIC50 = 140 nM

H

Figure 8.2 Structures of the fragment hit and optimized inhibitors.


On the basis of the X-ray structure of 1, it was speculated that introducing a flexiblelinker at C-5 of the pyridone ring would guarantee access to the perpendicular sub-pocket opened by the Tyr224-out conformation. Introduction of small substituents atC-5 resulted in significant increase of the enzyme inhibition of the correspondinginhibitors (2a and 2b, Figure 8.2). The inhibitory potency was further increased whenan unsubstituted aromatic ring was elongated in the perpendicular subpocket througha flexible linker appended at C-5 of the pyridone ring. X-ray cocrystal structure of com-pound 3 revealed a binding mode compatible with the expected one (Figure 8.4).Epigenetic regulation of gene expression is a growing field of research that can

open new opportunities for the discovery of novel treatment options in a numberof human diseases, especially in cancer. Several classes of enzymes regulate theposttranslational modification of histones, mainly acetylation and methylation.These modifications in turn affect the expression state of genes. Among the mostimportant epigenetic targets, histone acetyltransferases are responsible for theacetylation of histone tails (writers), whereas histone deacetylases mediate thedeacetylation of lysine residues of histone tails (erasers). Inhibitors of the latterclass of enzymes have been very recently introduced in therapy for the treatmentof cancer (vorinostat). Other important classes of epigenetic targets are proteinscontaining bromodomains that are able to “read” the acetylated histones.In a study aimed at discovering bromodomain-containing protein 4 (BRD4)

inhibitors, a fragment-based approach was used [31]. For the creation of a frag-ment library, the ZINC database was screened with criteria similar to the “rule ofthree.” The filtered compounds were clustered based on a similarity index. The

Figure 8.3 X-ray crystal structure of fragment 1 with D-amino acid oxidase (PDB code: 3W4I).


library finally consisted of 487 potential hit compounds. The library was enrichedwith potential fragment hits through a molecular docking approach, takingadvantage of available structural information of the binding mode of inhibitors toBRD4(I) and monitoring the formation of an interaction with Asn140 in the bind-ing mode of the fragments. Applying these selection criteria, 41 fragments wereused for crystallographic studies. Fragment 4 (Figure 8.5) was chosen for furtheroptimization and displayed a binding mode similar to that of the acetyl moiety oflysine of the natural substrate in the KAc pocket. In particular, the carbonyl groupof 4 forms a hydrogen bond with the side chain nitrogen of Asn140 and a water-mediated hydrogen bond with Tyr97 (Figure 8.6). Another water-mediated contactwith Asn140 is formed by the NH group of 4.Subsequent optimization of the fragment was based on the analysis of its bind-

ing mode compared with the binding mode of known BRD4(I) inhibitors. Theanalysis revealed that binding could be improved by modifications at m- or p-posi-tions of the aromatic ring. Accordingly, compounds 5a–5c (Figure 8.5) bearingsmall substituents at the aromatic ring showed an improvement of binding withrespect to the fragment hit. Moreover, it was foreseen that the introduction of ahydrophobic group projected close to the WPF shelf by a sulfonamide linker, asobserved for other inhibitors, could improve binding.Introduction of arylsulfonamides at both m- and p-positions of the aromatic ring

of 4 resulted in inhibitors with improved potency. Incorporation of sulfonamide atthe m-position resulted in low micromolar inhibitory activity (6 and 7, Figure 8.7).

Figure 8.4 X-ray cocrystal structure of lead compound 3 with D-amino acid oxidase (PDB code:3W4J).


Compounds 6 and 7 were cocrystallized to determine their binding mode to theprotein target and, although very structurally similar, the two compounds dis-played quite different binding modes (Figure 8.8). In particular, whereas the thio-phene ring of 6 was projected toward the WPF shelf, aided by the rotation of thesulfonamide group that was engaged in the formation of a hydrogen bond net-work with the enzyme, the benzyl group of 7 entered the ZA channel. Moreover,it was evident that the sulfonamide group of 7 was not necessary for orientatingthe projection of the aromatic ring as in 6 and that it could be replaced by anisosteric amide moiety. Subsequent design by merging of the two binding motifs

4

HNS

O

F

HNS

O

Cl H

HNS

O

H2N H

5a IC50 = 24 µM

5b IC50 = 42 µM

HNS

O

H2N NH2

5c IC50 = 51 µM

Figure 8.5 Structures of fragments and optimized lead compounds as BRD4 inhibitors.

Figure 8.6 X-ray cocrystal structure of fragment 4 with BRD4(I) (PDB code: 4HXN).


Figure 8.8 X-ray cocrystal structure of optimized fragments 6 and 7 with BRD4(I) (PDB code:4HXR and 4HXS, respectively).

HNS

O

HN

S SO O

6 IC50 = 4.1 µM

HNS

O

HN

SO O

7 IC50 = 4.1 µM

HNS

O

HN

S SO O

8 IC50 = 0.57 µM

HN

HNS

O

HN

S SO O

9 IC50 = 0.23 µM

HN

OO

S

Figure 8.7 Structures of optimized lead compounds as BRD4(I) inhibitors.


resulted in further improved inhibitors such as compound 8 and 9 (Figure 8.7),with submicromolar inhibitory potency.Hsp90 (heat shock protein 90) is part of a large class of molecular chaperones

that aid in correct folding, maturation, and conformational stability of their clientproteins. A number of Hsp90 client proteins are oncoproteins involved in cancerprogression. Although Hsp90 is highly expressed in most cells, it has been dem-onstrated that inhibition of the function of this protein mainly affects cancer cellsover normal cell lines and led to antiproliferative activity. The N-terminal domainof Hsp90 has ATPase activity and presents a binding site for nucleotides. Severalnatural and synthetic Hsp90 inhibitors have been reported over the years. Thedesign approach for the development of new inhibitors was based on fragmentscreening and optimization [32]. The researchers screened a library formed byaround 2,600 fragments against Hsp90 through ligand-observed NMR. Com-pounds showing binding for the target in the assay were further confirmed in com-petition experiments in the presence of low concentrations of ADP (that in the assayconditions had low affinity for Hsp90). The competition experiments were per-formed both in the absence and in the presence of low concentrations of Mg2þ

ions. The role of Mg2þ ions was to increase the affinity of ADP for the nucleotidebinding site. From these NMR experiments, around 100 compounds were pro-gressed to X-ray crystallography studies and 26 cocrystal structures were obtained.In the next steps of the design approach, the affinity of the compounds for

Hsp90 was assessed through isothermal titration calorimetry (ITC). Among thediscovered hit fragments, the phenol derivative 10 (Figure 8.9), although showingpoor ligand efficiency with respect to other fragments, displayed a binding modeto the nucleotide binding site of Hsp90, which paved the way for extensive optimi-zation. Moreover, the optimization procedure could take advantage of the similarbinding mode displayed by the natural compound radicicol (11).The binding mode of 10 to the enzyme is depicted in Figure 8.10. Several con-

served water molecules are displaced by the binding of the fragment. Moreover,the carbonyl group of 10 engages in a direct hydrogen bonding interaction withThr184, whereas the phenolic group forms a water-mediated hydrogen bond withthe enzyme. Finally, the methoxy group suboptimally fills a hydrophobic pocket.Overlapping the structure of this fragment with that of radicicol highlights that

OHOMe

O N

Me

Me

10IC50 = 790 µM

SubstitutionSubstitution

OH

ClHO

O

O

O

Me

OHH

11 (Radicicol)

Figure 8.9 Structures of lead Hsp90 inhibitor and radicicol.


Figure 8.10 X-ray cocrystal structure of fragment 10 with Hsp90 (PDB code: 2XDL).

OHCl

O N

Me

Me

12aIC50 = 1130 µM

OH

O N

Me

Me

12b IC50 = 45 µM

OH

O N

Me

Me

12c IC50 = 7 µM

OH

O N

Me

Me

12d IC50 = 8.6 µM

O N

Me

Me

HO

O N

Me

Me

HO

13a IC50 = 51 µM

13b IC50 = 134 µM

Figure 8.11 Structures of early Hsp90 inhibitors.


the 2-OH of the resorcinol ring of the natural compound is able to engage in ahydrogen bond formation with Asp93. The formation of this interaction is knownto be critical for achieving good affinity.Based on the X-ray crystal structure, the first step was to optimize the

pocket filled by the methoxy group of 10 by introducing different lipophilic groups(Figure 8.11, 12a–12d). Among them, i-Pr (12c) and t-Bu (12d) gave the bestresults in terms of both affinity and ligand efficiency. Guided by the bindingmode of radicicol, the 4-OH was removed and replaced by a 2-OH, coupled withi-Pr or t-Bu at C-3 of the aromatic ring. However, both compounds (13a and 13b)displayed a drop in affinity with respect to the corresponding 4-OH derivatives 12cand 12d, thus proving that the presence of the 4-OH is important for affinity.Subsequently, researchers focused on the tertiary amide by two different

approaches. First, they tried to introduce an amide with polar substituents withthe aim of engaging the neighboring Lys58 through hydrogen bonding interac-tions (compounds 14 and 15, Figure 8.12). Second, displacement of the side chainof Lys58 was attempted through the introduction of bulky tertiary amides (16a,16b, 17a, and 17b, Figure 8.12). In both cases, the design strategy led to com-pounds with improved affinity. The X-ray cocrystal structural studies showed goodagreement with the design approach (Figure 8.13).Finally, the resorcinol segment of radicicol was merged with the optimized com-

pound 16b. The corresponding compound 18 (Figure 8.14) showed subnanomolaraffinity for the nucleotide binding site. Furthermore, structural modifications of 18aimed at ADME optimization finally led to the clinical candidate 19 (AT13387) [33].

OH

O N

15 IC50 = 2.3 µM

OH

O N

14 IC50 = 1.1 µM

16a IC50 = 0.25 µM (X = t-Bu)16b IC50 = 0.068 µM (X = iPr)

OOH

OHX

O

OHX

O N

17a IC50 = 0.4 µM (X = t-Bu)17b IC50 = 0.128 µM (X = iPr)

N

Figure 8.12 Structures of optimized Hsp90 inhibitors.


8.6Conclusions

X-ray crystallography is a powerful tool for analysis of protein structure andprotein–ligand interactions. With major advances in technology and techniques, itis now possible to determine the structures of protein–ligand complexes whereprotein structures and crystallization conditions are known. For determination ofthe structures of protein–ligand complexes, convenient techniques such as ligandsoaking, cocrystallization, or molecular replacement methods are routinely uti-lized. Also, the improvement of computational technologies has facilitated therapid building of protein models. X-ray crystallography not only is useful for targetidentification but also has a critical role in lead identification and optimization.Further technological advancement may facilitate improved systems to allow broadscreening for drug discovery.

OHiPr

O N

HO

18IC50 = 0.54 nM

OHiPr

O N

HO

N N Me

19 (AT13387)Kd = 0.71 nM

Figure 8.14 Chemical structures of lead and clinical candidate AT13387.

Figure 8.13 Binding mode of the optimized fragments 14 (a) and 16a (b) to Hsp90 (PDB code:2XHT and 2XHX, respectively).


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Alzheimer’s disease: a fragment-baseddrug discovery story. Curr. Opin. Chem.Biol., 17, 320–328.

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9Structure-Based Design Strategies for TargetingG-Protein-Coupled Receptors (GPCRs)

9.1Introduction

G-protein-coupled receptors (GPCRs) constitute the largest family of membraneproteins in the human genome. With more than 1000 members, these receptorsplay critical roles in many physiological and pathophysiological processes. GPCRsare characterized by the presence of seven transmembrane helices that span thecell membrane. They are involved in transducing a variety of extracellular signalsacross the membrane to elicit a cellular response. GPCRs are monomeric recep-tors formed by a single peptide chain that crosses the plasma membrane seventimes [1–3]. The amino-terminal site of the protein is located extracellularly,whereas the carboxy-terminal site is located in the intracellular compartment.Each receptor presents seven transmembrane a-helices (I–VII), three intracellular(ICL1–ICL3) and three extracellular (ECL1–ECL3) loops. The binding site forreceptor agonists is located close to the extracellular region of the receptor. TheGPCR signaling system involves receptor binding with specific ligands, hor-mones, glycoproteins, growth factors, neurotransmitters, cytokines, odorants, andphotons. For photons, neurotransmitters, and small-molecule agonists, the bind-ing site is located in the transmembrane region, whereas peptides and cytokinesmainly interact with the extracellular surface of the receptor. The binding site forthe G protein can be found on the intracellular side of the receptor and is mainlyformed by ICL3 and the carboxy-terminal end of the peptide chain.The G protein is a complex of three proteins: a, b, and c. When the receptor is

in its resting state, the a-subunit of the G protein binds to a GDP molecule andthe bc-subunits are associated with the a-subunit. Upon an agonist binding, thereceptor undergoes a conformational change. The activation of the receptor resultsin the release of GDP from the a-subunit and in the binding of a GTP molecule,followed by dissociation of bc-subunits from the a-subunit. The a-subunit boundto GTP is able to activate or inhibit specific enzymes or ion channels collectivelydefined as “effectors.” The a-subunit remains active until GTP is bound. GTP iseventually hydrolyzed by the intrinsic GTPase activity of the subunit to GDP. TheGDP-bound a-subunit is finally able to reassociate with the bc-subunits, thusreturning to its initial inactive state.

199


A series of different G proteins have been characterized that differ in theiramino acid sequence, receptor with which they interact, and effectors that theyare able to activate or inactivate. The effectors can be enzymes that produce theso-called second messengers or ion channels. One of the most important effectorenzymes is adenylate cyclase that converts ATP into the second messenger cAMP.G proteins can be clustered in Gs or Gi if they activate or inhibit adenylate cyclase,respectively. The second messenger cAMP activates the protein kinase A (PKA)that is able to phosphorylate specific enzymes and control their functions. Theactivity of cAMP is terminated by enzymes called phosphodiesterases. Gq proteinsstimulate the enzyme phospholipase C (PLC) that hydrolyzes phosphatidylinositi-des from the plasma membrane producing inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG). IP3 activates a calcium channel located in intracellular vesi-cles allowing calcium ions to efflux from inside the vesicles to the cytoplasm. Theincrease of intracellular calcium ion concentration causes a series of effects on thecell metabolic pathways that are mainly mediated by the activation of enzymessuch as calcium/calmodulin-dependent kinases. Finally, Go proteins stimulate ionchannels.A single agonist binding to a GPCR can produce several molecules of sec-

ondary messengers, and this sequence of events explains how the stimulationof a GPCR by an extracellular stimulus can be transmitted and amplified intra-cellularly. Although GPCRs could be activated or inhibited by interfering with one ofthe several steps that characterize the transduction machinery, in reality theactivity of GPCRs is mainly controlled by the use of agonists or antagonists ofthe endogenous ligand [4]. GPCRs can exist in two different conformations,the active and the inactive states. In the absence of an endogenous ligand, theequilibrium between the two states is shifted toward the inactive state. The receptorshows a small degree of basal activity that is independent of the presence ofagonist, but depends on the portion of the receptor in its active form. As aconsequence, small molecules that have affinity for GPCRs can present differentintrinsic activity. An agonist activates the receptor, has a higher affinity for the activestate of the receptor and shifts the above-mentioned equilibrium toward the activestate. A neutral antagonist has equal affinity for both active and inactive states, andhence it does not alter the equilibrium among them, but it simply occupies thereceptor, hindering its activation by the endogenous ligand. An inverse agonist hashigher affinity for the inactive state of the GPCR and abolishes the constitutiveactivity of the receptor.

9.2High-Resolution Structures of GPCRs

At present, approximately 40% of all currently FDA-approved drugs target GPCRs.The design of a majority of agonists or antagonists of these receptors has mainlyrelied upon classical ligand-based approaches such as virtual screening, pharma-cophore modeling, and quantitative structure–activity relationship (QSAR) studies.

200 9 Structure-Based Design Strategies for Targeting G-Protein-Coupled Receptors (GPCRs)

The determination of X-ray structures of GPCRs is a considerable challenge. In2000, the X-ray crystal structure of rhodopsin was determined. This has facilitatedthe design strategies of GPCR research [5–7]. Models of other GPCRs were thencreated based upon the rhodopsin structure and such strategies have been widelyused in GPCR-based drug discovery. The determination of high-resolution X-raystructures of drug-relevant GPCRs in 2007 marked the beginning of a new era instructure-based design of ligands targeting GPCRs. The X-ray crystal structures ofthe inverse agonist and agonist-bound human b2-adrenergic receptor were deter-mined in 2007 [8–10]. Subsequently, the X-ray structures of other GPCRs, such asturkey b1- and b2-adrenergic receptors [3,11,12], adenosine A2A receptor (bound toboth antagonist and agonist) [13–16], dopamine D3 receptor [17], CXCR4 [18], andCCR5 receptor [19], were subsequently determined. In addition, the crystal struc-tures of the agonist and antagonists bound to rhodopsin, the b1- and b2-adrenergicreceptors, and the adenosine A2A receptors have been solved [3]. The high-resolution X-ray structures of these receptors have been obtained either by proteinengineering with the T4 lysozyme [8,20] or through a thermostabilization strategy[21]. This impressive progress in GPCR X-ray crystallography opened new oppor-tunities for structure-based design of GPCR ligands.The availability of several high-resolution X-ray crystal structures of GPCRs has

led to structure-based ligand discovery efforts as an alternative to classic analog-based drug discovery approaches that have been the hallmark of the field. Further-more, these high-resolution structures have enabled the generation of morereliable homology models of GPCRs for which high-resolution structures havenot yet been resolved [22–24]. The X-ray structure-based design of novel ligandsfor GPCRs is rapidly growing [3]. In this section, we describe some examples ofvirtual screening approaches aimed at finding novel ligands targeting GPCR recep-tors with high-resolution X-ray structures. Interestingly, these screening efforts aregenerating high hit rates and a good proportion of hit compounds have beenvalidated leads [25]. In addition, we discuss current progress in the state of theart of structure optimization and their utilization in structure-based drug design.

9.3Virtual Screening Applied to the b2-Adrenergic Receptor

The X-ray crystal structure of the b2-adrenergic receptor [9,26] documents the clas-sic seven membrane-spanning a-helices and three extracellular and three intra-cellular loops connecting the transmembrane a-helices (Figure 9.1). In thecrystallized construct, the T4 lysozyme replaces the intracellular loop 3. Theb2-adrenergic receptor binding pocket has a deep cleft, mainly occupied by hydro-phobic residues allowing the formation of several van der Waals interactions withreceptor ligands. On the other hand, the polar residues form a strong directionalelectrostatic interaction with ligands in the binding pocket.As shown in Figure 9.2, carazolol, an inverse agonist of the receptor, binds

deep in the pocket forming several hydrophobic and electrostatic interactions.

9.3 Virtual Screening Applied to the b2-Adrenergic Receptor 201

In particular, the NH moiety of the carbazole ring forms a hydrogen bond withSer203. A complex network of close hydrogen bonding can also be observed at thehydroxyalkylamine moiety, involving the protonatable amine and alcoholic OHfrom carazolol and residues Asn312 and Asp113 of the receptor. The carbazolemoiety is also packed between residues Val114, Phe290, and Phe193.Kolb et al. have carried out virtual screening for the discovery of new struc-

tural hits as receptor ligands [27]. The investigators docked a large library oflead-like compounds (the ZINC database) and ranked their docking scoresbased on steric and electrostatic complementarity with the receptor, while alsotaking into account ligand desolvation energies. Selection of the best rankingcompounds also took into account other parameters such as chemical diversity,and commercial availability. Of these, 25 lead-like structures were finallyselected for evaluation and 6 of them displayed binding affinity for the

Figure 9.1 X-ray crystal structure of the carazolol–b2-adrenergic receptor complex (trans-membrane a-helices, blue; intracellular and extracellular loops, violet; T4 lysozyme, cyan; inverseagonist carazolol, magenta spheres; PDB code: 2RH1).


receptor. The structures of carazolol (1) and other interesting novel hits (2–4)are shown in Figure 9.3.Compound 2 displayed a higher affinity for the receptor. This compound

belongs to a cluster defined as “classic compounds” that present pharmacophoricfeatures similar to those of typical known receptor binders. The predicted bindingmode of compound 2 is similar to the binding mode of carazolol, with the

Figure 9.2 Detailed binding interactions of carazolol within the b2-adrenergic receptor bindingsite (PDB code: 2RH1).

HN

O

OH

NH

MeMe

1, carazolol

O

O

OH

NH

MeMeMe

EtO2C

2, Ki = 0.009 µM

N N

S

MeMe

O

Asp113

O

3, Ki = 1.1 µM

+O

O

O

N

OH

Me

4, Ki = 3.2 µM

Figure 9.3 Chemical structures of carazolol 1 and novel b2-adrenergic receptor ligands 2–4.

9.3 Virtual Screening Applied to the b2-Adrenergic Receptor 203

heterocyclic hydrophobic bicyclic system occupying a similar position as the carba-zole ring. Moreover, polar interactions predicted for 2 are similar to thoseobserved for 1 within the binding site. Compound 2 has displayed a very highbinding affinity for the b2-receptor with a Ki in the low nanomolar range.Compound 3 is less active; however, it belongs to a novel chemical scaffold char-

acterized by low similarity to other reported receptor binders. The interesting fea-ture of compound 3 is the positive polarization of the alkyl chains attached to theheterocyclic nitrogen atoms, which allow it to form charge–charge interactionswith Asp113. Compound 3 does not contain an OH group that can otherwiseinteract with Asp113. Compound 4, on the other hand, interacts with Asp113through the charged pyrrolidine moiety of the phenolic group.

9.4Structure-Based Design of Adenosine A2A Receptor Antagonists

Jaakola et al. obtained the X-ray crystal structure of the adenosine A2A receptor at2.6 A

�resolution in complex with the antagonist ZM241385 (5) using the T4 lyso-

zyme fusion strategy already described for the b2-receptor [13]. The X-ray crystalstructure of ZM241385 bound to adenosine A2A receptor is shown in Figure 9.4.The overall organization of the receptor is similar to that found for the rhodop-

sin and the b2-receptors. However, there are marked differences in the organiza-tion of the extracellular loops, particularly of extracellular loop 2. Also, there aredifferences in relative position of the a-helices, reflecting the modifications of thebinding cavity that in this case is accommodating an antagonist.The antagonist binding cavity is different from that predicted by docking studies

performed on homology models of the receptor. As shown in Figure 9.5,ZM241385 binding is collinear with helix VII and interacts with both extracellularloops 2 and 3. The heterocyclic ring of 5 is anchored in the binding site by ap-stacking interaction with Phe168 on one side of the pocket and by hydrophobicinteraction with Ile274 on the other side. Also, the phenolic ring forms hydropho-bic contacts with Met270 and Leu267. Asn253 forms several hydrogen bonds withthe heterocyclic ring and the side chain furan ring of the inhibitor. The aminogroup is also in close contact with Glu169, whereas the phenolic group forms ahydrogen bond with a structured water molecule.Katritch et al. reported virtual screening of the Molsoft ScreenPub nonredun-

dant screening database using an optimized model of the A2A receptor based onthe X-ray crystal structure [28]. This has resulted in the identification of several hitcompounds. Compounds were divided into clusters based on similarity, and com-pounds to be tested were chosen from each cluster based on docking score, ligandefficiency, and other drug-like parameters. Compounds with high similarity toknown A2A receptor ligands were excluded, as the aim of the study was to discovernovel chemotypes. The binding modes of the selected compounds were predictedto form p-stacking interactions with Phe168 and hydrogen bonds with Asn253and Glu169. Several compounds possess structural features allowing them toextend deep in the binding pocket toward other key amino acid residues. Several


Figure 9.4 X-ray crystal structure of the ZM241385–adenosine A2A receptor complex (trans-membrane a-helices, red; intracellular and extracellular loops, blue; T4 lysozyme, white; reverseagonist ZM241385, yellow spheres; PDB code: 3EML).

Figure 9.5 Detailed binding interactions of ZM241385 within the adenosine A2A receptor bind-ing site (PDB code: 3EML).

9.4 Structure-Based Design of Adenosine A2A Receptor Antagonists 205

compounds displayed significant affinity for the receptor subtype. Notably, com-pounds 6 and 7 (Figure 9.6) showed submicromolar Ki values. In general, thecompounds did not show selectivity for the adenosine A1 receptor subtype,whereas none of them showed affinity for the adenosine A3 receptor subtype. Fur-thermore, most of the A2A binders behaved as antagonists in functional assays.Carlsson et al. reported the structure-based discovery of adenosine A2A receptor

ligands [29]. A virtual screening of the ZINC database was carried out. Each mole-cule of the database was scored based on van der Waals and electrostatic comple-mentarity with the binding site and adding a factor considering the liganddesolvation energy. From these screening efforts, 20 compounds were prioritizedfor evaluation in in vitro assays. Among them, seven compounds were shown tobe receptor binders with Ki values between 200 nM and 8.8 mM. Of particularnote, all binders showed preferential binding for the adenosine A2A receptor sub-type with respect to both A1 and A3 subtypes, displaying negligible or lower affin-ity for both these latter receptor subtypes. Compounds 8 and 9 (Figure 9.7) were

N

N

N

N

N

NH2

NH

OH

O

5 (ZM241385)Ki = 0.0006 µM

N

N

N

NH2

NHOMe

Cl

O

Me

6, Ki = 0.032 µM

N

O

NH2

NC

7, Ki = 0.06 µM

Figure 9.6 Structures of adenosine A2A receptor antagonists ZM241385 and other ligands.

N NH

NNH

O

O

Me

Me

8, Ki = 0.2 µM

HN

HN

NH2

HN

N

NMe

Me

F

+

9, Ki = 0.2 µM

Figure 9.7 Structures of adenosine A2A receptor ligands.


the most interesting ligands and displayed low micromolar affinity. In addition,both compounds are antagonists of the adenosine A2A receptor.

9.5Structure-Guided Design of CCR5 Antagonists

Chemokine receptors are a large family of GPCRs that regulate many biologicalprocesses. They control the trafficking and activation of leukocytes and othercell types in a range of inflammatory and noninflammatory conditions via inter-action with secreted chemoattractant cytokines or “chemokines” [30,31]. Todate, approximately 50 human chemokines and 20 receptors have been discov-ered and classified on the basis of their subfamily specificity. The chemokinereceptors play an important role in several pathophysiological processes, includ-ing cancer (CCR1/2/5/7 and CXCR3/4), HIV (CCR5 and CXCR4), asthma(CCR2/3/4/6), transplant (CCR1/2/5 and CXCR3), diabetes and obesity(CCR2), atherosclerosis (CCR2 and CXCR1/2), skin disease (CCR4), andinflammatory bowel disease (CCR9) [31,32]. Maraviroc (Selzentry) became thefirst small-molecule CCR5 antagonist that has been approved by the FDA in2007 for the treatment of HIV/AIDS. Since then, a number of other chemo-kines targeting NCEs (new chemical entities) have progressed into preclinicaland advanced clinical stages [33].The CCR5 is expressed on the cell surface of a wide range of cell types, predom-

inantly on T lymphocytes and monocytes and macrophages in blood, dendriticand Langerhans cells, and primary and secondary lymphoid organs [34]. CCR5has received extensive attention because of its essential and most predominantrole as a coreceptor for the HIV-1 fusion that allows entry into host target immunecells. In the process of HIV-1 invasion, the attachment of CD4þ cells with R5-tropic HIV-1 gp120 produces a conformational change in gp120. CCR5 acts as acoreceptor, binding to the conformationally changed R5-tropic HIV-1 gp120,which triggers the glycoprotein-mediated fusion of the viral envelope with the cellmembrane and viral entry into target lymphocyte T cells [35]. Therefore, the devel-opment of CCR5 antagonists for the treatment of HIV/AIDS has become an inten-sive area of research.

9.5.1Development of Maraviroc from HTS Lead Molecules

In the late 1990s, researchers at Pfizer started high-throughput screening (HTS)of a collection containing nearly half a million compounds using a chemokineradioligand binding assay to identify CCR5 ligands. Several promising hitsemerged based on the inhibition of binding of radiolabeled MIP-1b to humanCCR5 expressed in HEK-293 cells [36–38]. A number of representative hits areshown in Figure 9.8. The imidazopyridine 14 was selected for further optimiza-tion considering its potency, ligand efficiency, and drug-like properties.

9.5 Structure-Guided Design of CCR5 Antagonists 207

9.5.2Improvement of Antiviral Activity and Reduction of Cytochrome P450 Activity

During optimization of the scaffold, molecular modeling suggested that the pyri-dine “N” could coordinate with CYP heme Asp301. Initially, strategies wereadopted to overcome CYP2D6 inhibition as well as to replace the nitrogen with acarbon. The nitrogen replacement was sought because of the fact that “N” coordi-nated strongly with heme group and resulted in reduction of the lipophilicity ofdiphenylmethylene moiety [37].Accordingly, compound 15 with a benzimidazole ring reduced CYP2D6 activity

more than 15-fold and improved CCR5 binding affinity. The replacement of one lipo-philic phenyl ring from the diphenylmethylene moiety with amide moiety furtherreduced CYP2D6 inhibition. More encouragingly, the replacement of the phenylring with an amide moiety exhibited potent antiviral activity. Several aromatic andaliphatic amide moieties were prepared in a parallel manner. Cyclobutyl derivative 16(Figure 9.9) with (S)-configuration demonstrated excellent ligand efficiency, antiviralactivity (IC90¼ 440nM), and much reduced CYP2D6 inhibition. Modeling studies of

N

ON

10 IC50 = 2 µM

SO

O

NNH2

NO2

12 IC50 = 1.87 µM

Cl

N

O

NN

11 IC50 = 1.28 µM

NOH

13 IC50 = 0.27 µM

N

N

N

N

H3C

14 IC50 = 0.6 µM

OO

Figure 9.8 Structures and potencies of initial HTS hits during high-throughput screening.


16-bound CYP2D6, however, suggested that piperidine “N” could coordinate withheme Asp301. Therefore, it was envisioned that introduction of steric bulk aroundpiperidine “N” may reduce the affinity toward Asp301. Toward this goal, investiga-tors identified that the sterically constrained tropane ring (17) was a good replace-ment for the central piperidine ring. Inhibitor 17 showed similar CCR5 bindingaffinity, low nanomolar antiviral activity, and, most importantly, it did not showCYP2D6 inhibition. SAR studies revealed that (S)-phenyl configuration compoundsremain more potent than (R)-phenyl configuration. Interestingly, both the endo-and exo-benzimidazole diastereoisomers showed similar binding and antiviralpotency [39,40].

9.5.3Reduction of hERG Activity and Optimization of Pharmacokinetic Profile

Although compound 17 showed promising results, it demonstrated 99% inhibi-tion (at 1mM) of the potassium channel of the human ether a-go-go-related gene(hERG). The hERG channel activity was expected as the lead compound 14 also

N

N

N

N

H3C

14 IC50 = 0.6 µM

No antiviral activity

N

N

NH3C

15 IC50 = 1.8 nM

No antiviral activity

HN N

N

NH3C

O

HN

O

"N" coordination with CYP heme

"N" is critical for CCR5 activity, but interactswith CYP Asp301

N

H N

NH3C

IC50 = 40 nM (CYP2D6) IC50 = 710 nM (CYP2D6)

16 IC50 = 4 nM

Antiviral IC90 = 440 nM CYP2D6 IC50 = 5 µM

Antiviral IC90 = 3 nM

17 IC50 = 6 nM

No CYP activityhERG IC50 < 10 nM

Figure 9.9 Structures and activities of CCR5 antagonists 14–17.


possesses hERG activity. Subsequently, other CCR5 analogs that were in clinicaland preclinical development were also reported to have hERG activity. The hERGpotassium channel conducts the rapidly activating delayed rectifier potassium cur-rent (IKr), which plays an important role in controlling cardiac rhythm. Inhibitionof the hERG channel causes prolongation of QT/QTc interval, which ultimatelyleads to the ventricular arrhythmia and cardiac failure. The hERG inhibition effecthalted clinical trials of several drugs and also led to the withdrawal of several mar-keted drugs. Consequently, a prime objective for lead optimization was to reducehERG activity and improve physicochemical properties.Molecular docking studies with a hERG ion channel model suggested that the

addition of a polar group at the amide side chain or at the exo-benzimidazole ringcould reduce hERG affinity. Therefore, several compounds were synthesizedincorporating polar functionality at the amide side chain and screened usinghigh-throughput hERG binding assay, which led to the identification of compound18 (Figure 9.10). The polar compound 18 exhibited impressive binding and

HN

O

HN

O

NN

H N

NH3C

O

HN

O

N

H N

NH3C

O

N N NN

H3C

H3C CH3

HN

21 (Maraviroc)IC50 = 0.2 nM

Antiviral IC90 = 2 nMCYP2D6 IC50 > 50 µM

hERG IC50 > 10 µMOral bioavailability (rat) = 23%

O

N N NN

H3C

H3C CH3

F

F

Antiviral IC90 = 1.5 nM

18 IC50 = 0.8 nM

hERG IC50 > 10 µM Poor absorption

Antiviral IC90 = 0.6 nM

19 IC50 = 8 nM

No hERG and CYP activity HLM t1/2 = 77 min

20 IC50 = 7 nM

Antiviral IC90 = 8 nMhERG = 30% inhibition at 300 nM HLM t1/2 = 55 min

Figure 9.10 Structures and activities of CCR5 antagonists 18–20 and maraviroc 21.


antiviral activity and also reduced the affinity for hERG (IC50> 10 mM). However,this compound showed extremely poor intrinsic cell permeability and there wasno absorption in rat pharmacokinetic studies. The corresponding morpholinederivative 19 also exhibited impressive antiviral activity (IC90¼ 0.6 nM) and nohERG activity, but it was susceptible to P450 degradation in isolated human livermicrosomes [36]. Structural modification at the benzimidazole ring led to the exo-and endo-1,3,4-triazoles with cyclobutyl group at amide side chain. The exo-1,3,4-triazole 20 was more favored and exhibited improved pharmacokinetics, butshowed hERG inhibition (30% at 300 nM) [41]. After critical analysis of SAR data,researchers hypothesized that increasing the ring size of the amide chain couldreduce hERG affinity and improve the antiviral activity. Further incorporation of4,40-difluorocyclohexylamide (21) exhibited excellent binding (IC50¼ 0.2 nM) andantiviral activity (IC90¼ 2 nM), with improved metabolic stability (23% oral bio-availability in rats). Of particular interest, this derivative was devoid of cytochromeP450 (IC50> 50 mM) and hERG activity (IC50> 10 mM). Overall, it has displayedimpressive selectivity, pharmacological efficacy, safety, and pharmacokinetic pro-file. Thus, structure-based design ultimately led to the development of maraviroc21 and subsequent approval by FDA in 2007 for the treatment of HIV/AIDS. Ithas also exhibited compatibility with other drugs in combination therapy [37,42].Recently, Tan et al. cocrystallized the CCR5 receptor with maraviroc 21 (Fig-

ure 9.11) [19]. As shown in Figure 9.11, the crystal structure revealed that

Figure 9.11 X-ray crystal structure of maraviroc21 in complex with CCR5. Hydrogen bondinginteractions are shown in dotted lines. Creamcolor surface is shown for residues involved

in hydrophobic interactions with maraviroc(Tyr108, Phe109, Phe112, and Trp248; PDBcode: 4MBS).


maraviroc binds deeper at the bottom of CCR5 pocket. As stated earlier, the cen-tral ring nitrogen of maraviroc is critical for activity. In fact, the tropane ring nitro-gen is protonated and makes a salt bridge interaction with Glu283. Thecarboxamide nitrogen forms a hydrogen bond with Tyr251 hydroxyl group. Thelength of the carbon chain between carboxamide and tropane nitrogen is impor-tant to retain CCR5 activity, which correlates with the spatial locations of Glu283and Tyr251. The adjacent nitrogen of the triazole moiety forms hydrogen bondswith Tyr37 hydroxyl group and a water molecule. One of the fluorines of the cyclo-hexane ring forms two hydrogen bonds with the hydroxyls of Thr195 and Thr259.In addition, the phenyl group occupies a deep pocket and forms hydrophobicinteractions with five aromatic lipophilic residues lined as Tyr108, Phe109,Phe112, Trp248, and Tyr251. The triazole, tropane, and cyclohexane groups alsofit very nicely into small subhydrophobic pockets of CCR5 (hydrophobic pocketsare shown in Figure 9.12 as a solid surface). These high-resolution insights mayspeed up structure-based drug discovery for the identification of novel chemokineligands for the treatment of HIV-1 infection or other diseases.

N NN

H3CN

F

O

ClF

S

22 (PF-232798)

IC50 = 0.1 nMAntiviral IC90 = 2 nMhERG IC50 > 10 µM

Oral bioavailability (dog) = 31% Phase II clinical trials

23 (GSK163929) IC50 = 4.3 nM

Oral bioavailability (rat)(AUC 272 ng. h/mL);

Clinical candidate

HN

O

N

H N

NH3C

N

F

O

MeN

OO

H

Figure 9.12 CCR5 antagonists developed on the basis of maraviroc scaffold 21.


9.5.4Other CCR5 Antagonists

Several other CCR5 antagonists with different scaffolds have been developed.A number of compounds are in preclinical or advanced clinical stages[33,36,43,44]. During the development of maraviroc, researchers at Pfizerobserved that replacement of benzimidazole or imidazopyridine ring with imi-dazopiperidine ring also reduced significant hERG affinity. This led to the dis-covery of compound 22 (PF-232798, Figure 9.12), which is currently in phaseII clinical trials. This compound exhibited similar antiviral activity as mara-viroc and activity against maraviroc-resistant HIV strains. It also displayed afavorable preclinical profile (Figure 9.12) [36,44]. The researchers at GSK alsocarried out SAR studies of their tropane-like CCR5 leads to improve the hERGprofile. Halogen-substituted sulfonamide derivative 23 exhibited favorableproperties and was selected as a preclinical candidate (GSK163929, 23) [45,46].It is interesting to note that a key success factor in both compounds is thereduction of lipophilicity by introducing a polar group at the periphery, whichis possibly responsible for reduced hERG activity.

9.6Conclusion

G-protein-coupled receptors are widespread and perform vital functions in cellularsignaling and control of physiological processes. With the availability of high-resolution X-ray structures, our knowledge and understanding of the structureand function of GPCRs provide an important foundation for creative structure-based drug design. Structure-based design offers a rational approach to modifynegative interactions and optimize drug-like properties. This chapter has outlinedthe use of virtual screening and fragment-based techniques that led to the identifi-cation of novel lead compounds. The design and discovery of maraviroc andinsight into high-resolution X-ray structures of ligand-bound CCR5 have alsobeen highlighted. This insight may further facilitate the structure-based drugdiscovery and identification of novel therapeutics for the treatment of HIV/AIDSand other diseases.

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Part TwoStructure-Based Design of FDA-Approved Inhibitor Drugs andDrugs Undergoing Clinical Development

217


10Angiotensin-Converting Enzyme Inhibitors for the Treatmentof Hypertension: Design and Discovery of Captopril

10.1Introduction

The late twentieth century marked the beginning of a dramatically new era of drugdiscovery and medicinal chemistry. This period witnessed remarkable progress inthe development of powerful strategies and techniques in drug development thatculminated in the approval of numerous breakthrough medicines. The major con-tributing factors for this progress were the advent of many powerful technologiesand major advances in molecular biology and synthetic organic chemistry. Theelucidation of X-ray crystal structures of many drug-relevant enzyme targets, com-puter-based creation of models, and structural analysis brought new opportunitiesfor developing novel therapies. Since then, the evolution of rational drug designand structure-based design strategies has made a tremendous impact on today’sdrug design and discovery research. The early beginning of a rational approach todrug design, taking into account what is known about the structure-based design,may be found in the story of the discovery of captopril, an angiotensin-convertingenzyme (ACE) inhibitor used for the treatment of hypertension.The renin–angiotensin–aldosterone system (RAAS) is involved in the regulation

of blood pressure [1–4]. In response to a drop in blood pressure (Figure 10.1), thekidneys produce aspartic protease renin. Renin exerts its proteolytic activity on theprotein angiotensinogen, which is produced in the liver. Renin cleaves angiotensi-nogen, thus releasing a decapeptide called angiotensin I. Angiotensin I has noeffect on blood pressure and is activated by a second protease called ACE. ACEcleaves a dipeptide from the carboxy-terminus of angiotensin I, producing theoctapeptide angiotensin II. This peptide interacts with its cellular receptors caus-ing vasoconstriction and stimulation of aldosterone secretion, leading to an overallincrease in blood pressure. Aldosterone is a hormone involved in the control ofthe blood volume and sodium and potassium balance of the organism. Itincreases the reabsorption of sodium ions by the kidneys, leading to increasedretention of water and increase in blood volume, which contributes in the raisingof blood pressure. The enzymatic activity of ACE not only activates angiotensin Ito angiotensin II but also proteolytically degrades bradykinin, raising the bloodpressure by a second mechanism [1]. It has also been demonstrated that ACE has

219


a broad substrate specificity and hydrolyzes several physiologically relevant signal-ing peptides.ACE is a zinc-dependent carboxypeptidase that cleaves two amino acids from the

C-terminus of the target peptide. Due to its broad substrate activity, the cleavagesequence is not strictly specific, in contrast to renin that is strictly selective for angio-tensinogen. It was earlier hypothesized, and later confirmed, that blocking the pro-duction of angiotensin II could produce beneficial effects for the control of bloodpressure in hypertensive patients. The blockade of angiotensin II could be achievedby blocking either of the two enzymes, renin or ACE. The discovery of ACE inhibitor,captopril, illustrates an early example of rational drug design that takes into accountthe enzyme structure andmolecular interactions to guide drug development.

10.2Design of Captopril: the First Clinically Approved Angiotensin-ConvertingEnzyme Inhibitor

Two landmark discoveries paved the way for the rational design of the first ACEinhibitor, captopril. First, the mechanism of peptide hydrolysis catalyzed by ACEwas thought to be similar to that of carboxypeptidase A [5,6]. At the time of thesestudies aimed at developing ACE inhibitors, carboxypeptidase was well character-ized and the X-ray crystal structure of carboxypeptidase was known [7,8]. Thestructure of ACE was not known until 2003; however, the knowledge of

Figure 10.1 Schematic representation of the renin–angiotensin–aldosterone system.

220 10 Angiotensin-Converting Enzyme Inhibitors for the Treatment of Hypertension

carboxypeptidase structure guided the discovery of ACE inhibitors [9]. The secondimportant discovery was the vasoactive peptides isolated from the venom ofBothrops jararaca snake. They were characterized and evaluated for their ACEinhibitory properties [10–13]. Some of these peptides displayed antihypertensiveactivity in vivo [14–17]. This discovery further prompted the search for ACE inhibi-tors with drug-like properties.Carboxypeptidase A cleaves an amino acid residue from the C-terminus of the

substrate peptide. The terminal carboxylate group of the substrate interacts with apositively charged amino acid located at the appropriate position of the enzymeactive site, whereas the aromatic ring of a phenylalanine residue fills ahydrophobic pocket [7,8]. The mechanism of hydrolysis catalyzed by the enzyme(Figure 10.2) [18] involves the activation of a water molecule through coordinationwith a catalytic zinc ion. The activated water molecule performs a nucleophilic attackat the carbonyl carbon of the cleavable amide bond, thus forming a tetrahedral inter-mediate. Collapse of the tetrahedral intermediate releases the hydrolysis products,namely, the free C-terminal amino acid and the remaining peptide. The carboxylicacid terminus of the product peptide binds the zinc ion of the catalytic site.In the 1970s, it was discovered that several dicarboxylic acids were able to inhibit

carboxypeptidase A in a reversible manner [19]. One of the most active of theseacids was L-benzylsuccinic acid (Figure 10.2). Based on the mechanism of hydroly-sis catalyzed by this enzyme, it was hypothesized that this compound mimickedthe by-products of the reaction catalyzed by carboxypeptidase A [20]. Indeed, asdepicted in Figure 10.3, one of the carboxylate groups was hypothesized to mimicthe carboxylate of the product amino acid, presumably forming a salt bridge with apositively charged residue. The phenyl group was believed to occupy the samehydrophobic pocket as the phenylalanine residue of the substrate, whereas thesecond carboxylate group was probably bound to the catalytic zinc ion, thusmimicking the C-terminal carboxylate group of the hydrolyzed peptide.It was thought that carboxypeptidase A and ACE were very similar hydrolytic

enzymes, the only difference being that carboxypeptidase A cleaves single aminoacids from the C-terminus of a peptide, whereas ACE cleaves dipeptides [5,6].Accordingly, the by-product analog design approach elaborated for carboxypepti-dase A was adapted to ACE by increasing the length between the terminal carbox-ylic acid moiety and the zinc binding group. To compensate for the increaseddistance of the ligand binding moieties in ACE, an appropriate linker amino acidresidue was inserted. As summarized in Figure 10.3, the linker was assumed toengage in a hydrogen bonding interaction with specific residues of the enzymeactive site. The resulting by-product analogs were derivatives of succinic acid con-taining three pharmacophoric moieties, responsible for affinity for the enzymeand inhibitory activity: (i) a negatively charged carboxylate group (violet); (ii) ahydrogen bond acceptor group (red); and (iii) a Zn binding group (cyan).The first ACE inhibitor prototype, shown in Figure 10.3, contains a proline ter-

minal amino acid residue. This was chosen because the series of peptides isolatedfrom the venom of B. jararaca as ACE inhibitors contained a terminal proline resi-due [10–13]. The structures of these peptides are shown in Figure 10.4 and their

10.2 Design of Captopril: the First Clinically Approved Angiotensin-Converting Enzyme Inhibitor 221

N-terminal sequence contains a pyroglutamic acid and one tryptophan (red, inFigure 10.4). The central region contains two or three proline and one glutamicacid residue (green, in Figure 10.4). The C-terminal sequence shows Ile–Pro–Proidentical for all peptides (blue, in Figure 10.4).The in-depth analysis of these structures led to the rational design of the first

ACE inhibitor prototype. Combining the structural information arising from thestructures of the ACE inhibitor peptides and the active model of carboxypeptidase

Figure 10.2 Mechanism of peptide hydrolysis catalyzed by carboxypeptidase A and rationale ofinhibitory activity of benzylsuccinic acid as a “by-product analog.”


A adapted to ACE, the prototype ACE inhibitor 7 (Figure 10.5) was synthesizedand biologically evaluated. A series of analogs were also prepared and evaluated tostudy the structure–activity relationships for this class of ACE inhibitors. As sum-marized in Figure 10.5, the length of the linker was explored, maintaining a pro-line as the terminal residue.ACE inhibitory activity progressively increased from n¼ 0 (compound 8) to n¼ 3

methylene residues (7, 9, and 10), whereas a drop in activity was observed when afour-methylene linker was introduced in compound 11 [5]. Branching the linkers

Figure 10.3 Rational design of “by-product analogs” as inhibitors of carboxypeptidase A (left)and ACE (right).

10.2 Design of Captopril: the First Clinically Approved Angiotensin-Converting Enzyme Inhibitor 223

N

HN

O

NH

ON

O O

HN

N

O

OH

NH

O HN

ON

O

N

OCO2H

O

HN

O

V-2

NH

N

O O

HN

N

O

NH

O HN

ON

O

N

OCO2H

O

HN

O OHN

H2N

O

V-6-II

NH

N

O O

HN

N

O

NH

O HN

ON

O

N

OCO2H

O

HN

O OHN

H2N

O

V-7

C-terminalIle-Pro-Pro

N

O

NH

ON

O O

HN

N

O

NH

O HN

ON

O

N

OCO2H V-9

NH

N

O O

HN

N

O

NH

O HN

ON

O

N

OCO2H

O

HN

O

V-6-I

NH

NH

H2N NH

H2N NH

NH

H2N NH

N NH

NH

HN NH2

N-terminal sequence Pyr-M-N-Trp

H2N O

H2N O

H2N O

H2N O

HO O

NH

HN

HN

HN

Middle sequence Pro-X-Pro-Y-Pro-Z or Pro-X-Pro-Gln

HN

NH

O

HN

O

O

HN

HNO

Figure 10.4 Structures of ACE inhibitor peptides isolated from the venom of the snakeB. jararaca.


of 8 and 10 led to two series of compounds (Figure 10.6). Derivatives with stereo-chemically defined methyl groups (compounds 12–17) were introduced at variouspositions on the polymethylene linker. Compound 18 with a mixture of diastereo-mers at the methyl center was also investigated. Incorporation of a methyl group atthe proximal position with respect to the amide bond and having (R)-configurationconsistently showed an improvement in activity (compounds 12 and 16 vscompounds 13 and 17, respectively). The effect of different terminal amino acidresidues on activity was also evaluated. Compounds presenting a proline consis-tently showed better activity than those containing different amino acids.The di- and trimethylene derivatives 7 and 10 were further investigated by

replacing the zinc binding carboxylate with stronger metal chelating moieties. Thethiol derivatives 19 and 20 in Figure 10.7 showed much improved activity versustheir carboxylic acid counterparts. The effect on potency was greater for 19, leadingto a submicromolar inhibitor. Finally, combining the thiol group with the branchedlinker of 12 led to captopril (21), which showed nanomolar potency against ACE.

10.3Structure of Angiotensin-Converting Enzyme

It was not until 2003 that the X-ray crystal structures of ACE and ACE–inhibitorcomplexes were determined. The main reasons for such a delay were the complex-ity of the enzyme and the heavy posttranslational modifications (mainly glycosyla-tion) involved in the maturation of the active form of the enzyme. ACE is atransmembrane enzyme anchored to the cell surface through a hydrophobic22-amino acid sequence located at the C-terminus and spanning the cell membrane.

N

O

O

HOO

OH

8 IC50 = 4800 µM

N

O O

OH

O

HO

N

O O

OHO

HON

O O

OH

N

O O

OH

9 IC50 = 2600 µM

7 IC50 = 330 µM 10 IC50 = 70 µM

11 IC50 > 4000 µM

O

HO

HO

O

Figure 10.5 ACE inhibitory activities of compounds with linear chains.

10.3 Structure of Angiotensin-Converting Enzyme 225

Human ACE has two isoforms: a high molecular weight form, expressed insomatic tissues, containing two catalytic sites at the C- and N-terminal domains,and a low molecular weight form, expressed in male germinal cells (testis ACE),identical to the C-terminal domain of somatic ACE [21]. It has been observedthat the C- and N-terminal domains of somatic ACE have different physiochemicaland functional properties, and, interestingly, the two catalytic domains showdifferent substrate specificity. A particularly important substrate specificity aspect ofthe two domains involves the cleavage of angiotensin I versus bradykinin. Although

HS N

O O

OH N

O O

OHHS

HS N

O O

OHMe

19 IC50 = 0.2 µM 20 IC50 = 9.7 µM

21 IC50 = 0.023 µM

Captopril

Figure 10.7 Structures of thiol-based ACE inhibitors.

12 IC50 = 22 µM

N

O O

OHO

HO

Me

N

O O

OHO

HO

Me

N

O O

OHO

HOMe

N

O O

OHO

HOMe

N

O O

OHMe

O

HO

N

O O

OH

O

HO

13 IC50 = 1480 µM

14 IC50 = 610 µM 15 IC50 = 2600 µM

16 IC50 = 4.9 µM 17 IC50 = 950 µM

N

O O

OHMe

O

HO

18 IC50 = 260 µM

Me

Figure 10.6 ACE inhibitory activities of compounds with branched chains.


bradykinin is equally processed by both domains, the C-terminal domainshows specificity for angiotensin I cleavage. This finding has been considered quiteimportant since bradykinin accumulation is known to mediate some common sideeffects of ACE inhibitors (e.g., persistent cough and angioedema). As a consequence,the development of domain-selective inhibitors could lead to optimized antihyper-tensive drugs with fewer side effects. The crystal structures of testis ACE (similarto the C-terminal domain of somatic ACE) and of the N-terminal domain of somaticACE in the apo form or in complex with inhibitors are now available [9,22–26].However, no domain-selective ACE inhibitors have reached the market so far.The structure of testis ACE has an overall ellipsoid shape and is mainly formed

by a-helices. The active site is located 30A�from the enzyme surface in a groove

that runs along the enzyme and divides it into two subdomains (Figure 10.8). Thecatalytic zinc is coordinated by two histidine residues (His383 and His387) and aglutamate residue (Glu411) (Figure 10.8).The X-ray crystal structure of the captopril–ACE complex has been determined [22].

As shown in Figure 10.9, the binding mode revealed a lot of similarities with thatoriginally hypothesized in the design approach. The sulfhydryl group strongly interactswith the zinc ion within the catalytic site. The carboxylate group of captopril anchorsthe inhibitor through a series of interactions with Lys511, Tyr520, and Gln281. Allof these interactions occur with one carboxylate oxygen, whereas the other one inter-acts with water molecules. Finally, two strong hydrogen bonds are formed betweenthe amide carbonyl group of captopril and two histidines (His353 and His513).Captopril showed good oral bioavailability and efficacy in vivo and was rapidly

approved by the FDA for the treatment of hypertension. Further studies on ACEinhibitors were then aimed at removing the free thiol group of captopril that wasresponsible for several side effects. Skin rash and loss of taste reduced patients’

Figure 10.8 Structure of testis ACE in complex with lisinopril. The insert shows the coordinationpattern of catalytic zinc ion (cyan sphere; PDB code: 1O86).

10.3 Structure of Angiotensin-Converting Enzyme 227

compliance to the antihypertensive therapy. Subsequently, two important classesof ACE inhibitors were developed and characterized by the presence of a carboxyl-ate or a phosphorus-containing functionality as the zinc binding groups.

10.4Design of ACE Inhibitors Containing a Carboxylate as Zinc Binding Group

As depicted in Figure 10.10, isosteric replacement of the methylene in the above-mentioned inhibitor 16 with an NH group led to the dipeptide derivative 22 as alead compound suitable for further optimization [27]. In the carboxyalkanoyl aminoacid series described above (Figures 10.5 and 10.6), the presence of a distal methylgroup (compound 18) was detrimental to inhibitory potency. Contrastingly, in thedipeptide series displayed in Figure 10.10, introduction of a racemic methyl group(in order to compensate for the increased hydrophilicity obtained by the introductionof the NH group) at the equivalent position yielded a consistent improvement ininhibitory activity (inhibitor 23). Systematic exploration of substituents at this posi-tion revealed that large groups could be accommodated. One of the best substituentswas a stereochemically defined phenethyl group, leading to the discovery of enalapri-lat (24) that was approved for therapy as the ethyl ester prodrug (enalapril).Enalaprilat forms strong interactions within the active site of the enzyme [22] (Fig-

ure 10.11). The presence of a carboxylate group as the zinc binding moiety, instead ofthe sulfhydryl group of captopril, allows the formation of an additional hydrogenbond with one of the oxygens of the carboxylate and the Tyr523 phenolic hydroxyl

Figure 10.9 X-ray crystal structure of the testis ACE–captopril complex. Amino acid residuesinvolved in binding to captopril are displayed as sticks. The zinc ion is shown as a cyan CPKsphere (PDB code: 1UZF).


group. Similar to the binding mode of captopril, the amide carbonyl group forms twohydrogen bonds with His353 and His513, whereas the terminal carboxylate interactswith Tyr520 and Lys511. An important hydrogen bond is also formed between theNH of the inhibitor and the main chain carbonyl of Ala354. From the crystal struc-ture, the initial hypothesis that the phenethyl group was able to interact with the S1subsite of the enzyme, which is not reached by captopril, has been confirmed. Thephenyl ring indeed forms hydrophobic interactions with the side chain of residuesforming the S1 pocket, namely, Phe512 and Val518 (yellow residues in Figure 10.12).Following the same principle that led to the design of enalaprilat and starting

from 24 (Figure 10.10), systematic exploration of the central amino acid led to thediscovery that a lysine residue was able to greatly increase binding affinity of com-pound 25 (lisinopril), probably by interacting with the S10 subsite of the enzyme[28]. The X-ray crystal structure of the ACE and lisinopril complex (Figure 10.13)[9] revealed a binding mode similar to that described above for enalaprilat. Theonly difference relies on the interaction of the terminal amino group of lysine inthe inhibitor with a glutamate residue (Glu162) in the S10 subpocket. It is worthmentioning that lisinopril is orally bioavailable, whereas the corresponding mono-ethyl ester does not show oral bioavailability.

N

O O

OHHO

O

Me

N

O O

OHNH

HO

O

Me

N

O O

OHNH

HO

O

MeMeN

O O

OHNH

HO

O

Me

Enalaprilat

N

O O

OHNH

HO

O

25 IC50 = 0.0012 µM Lisinopril

NH2

16 IC50 = 4.9 µM 22 IC50 = 2.4 µM

24 IC50 = 0.0012 µM 23 IC50 = 0.09 µM

Figure 10.10 Chemical structures of carboxylate-based ACE inhibitors.

10.4 Design of ACE Inhibitors Containing a Carboxylate as Zinc Binding Group 229

Figure 10.11 X-ray crystal structure of testis ACE and enalaprilat (24) complex showing majorhydrogen bonding interactions (PDB code: 1UZE).

Figure 10.12 X-ray crystal structure of testis ACE and enalaprilat (24) complex showing surfaceareas surrounding the binding of the phenyl ring of the inhibitor within the S1 subpocket (PDBcode: 1UZE).


10.5ACE Inhibitors Bearing Phosphorus-Based Zinc Binding Groups

Based upon the knowledge that phosphoramidon (26, Figure 10.14), a compoundwith a phosphoramidate group, was able to inhibit thermolysin [29], a zinc-con-taining peptidase similar to ACE, phosphorus-containing functionalities as zincbinding groups in place of carboxylates and thiols were investigated. This func-tionality was considered to be particularly suitable for the development of ACEinhibitors due to its tetrahedral geometry, closely mimicking the tetrahedral tran-sition state of peptide hydrolysis catalyzed by ACE. This assumption was furthersupported when the X-ray crystal structure of phosphoramidon-bound thermolysinwas determined. The structure revealed that the phosphoramidate group occupiedthe position of the cleavable peptide bond of the substrates and the phosphoryl

Figure 10.13 X-ray crystal structure of testis ACE and lisinopril complex (PDB code: 1O86).

NH

HN

PO

HO OO

OHO O

OHOH

MeOH

HN

26 Phosphoramidon

N

OPO

O

CO2H

27 Fosinopril

OO

Me

Figure 10.14 Chemical structures of phosphoramidon and fosinopril.

10.5 ACE Inhibitors Bearing Phosphorus-Based Zinc Binding Groups 231

oxygen was placed within binding distance from the catalytic zinc ion. The phos-phorus-containing groups that were investigated were mainly phosphonamidates,derivatives of phosphonic and phosphinic acids, and their corresponding monoest-ers. These studies finally led to the discovery of fosinopril (27), the only representa-tive of the class of phosphorus-containing ACE inhibitors.

10.5.1Phosphonamidate-Based Inhibitors

Based upon the previously developed model for inhibitor binding to ACE, com-pounds 28–30 (Figure 10.15) were designed based on captopril by replacing the thiolgroup with different phosphorus-containing functional groups [30]. Whereas phos-phonate 28 and phosphate 29 did not show great inhibitory potency, the phosphora-midate 30 showed submicromolar potency toward ACE. Researchers prepared estersof these compounds, as the phosphoramidate derivatives bearing a free OH wereunstable. The choice of the bulky benzyl ester was suggested based on previous stud-ies on ACE inhibitors such as enalapril and lisinopril, in which the phenyl group washypothesized to interact at the S1 subsite of ACE. Taking 30 as lead compound, fur-ther investigation was performed at the benzyl ester by replacing it with a phenoxyester (31). Although this modification led to a decrease in inhibitory potency, isos-teric replacement of the oxygen of the phenoxy group with a methylene linker led tocompound 32, which was as potent as phosphoramidate 30. The higher homolog 33

N

O

PMeO

OCO2HOH

28 IC50 = 9 µM

N

OO

PMeO

OCO2HOH

29 IC50 = 8 µM

N

ONH

P

MeO

OCO2H

OH

30 IC50 = 0.04 µM

N

ONH

PMeO

OCO2HOH

31 IC50 = 0.9 µM

32 IC50 = 0.08 µM

N

ONH

PMeO

CO2HOH

33 IC50 = 0.007 µM

N

ONH

PMeO

CO2HOH

N

ONH

PMeO

CO2HOH

34 IC50 = 0.01 µM

Figure 10.15 Chemical structures of phosphate-, phosphonate-, and phosphoramidate-basedACE inhibitors.


was one order of magnitude more potent than 32, whereas the three-methylenederivative 34 showed a reduction of potency.

10.5.2Phosphonic and Phosphinic Acid Derivatives: the Path to Fosinopril

Phosphonic acid derivatives showed moderate inhibitory activity against ACE. Thestructure–activity studies did not overlap those studied for carboxylic acid deriva-tives of captopril [31]. Compound 36 (Figure 10.16), homolog of 7 shown in Fig-ure 10.5, showed a reduced inhibitory activity with respect to the correspondinglower homolog 35. Also, the introduction of the methyl group (as in 37) that was akey modification in the development of captopril did not result in an increased activity.A slight improvement in activity was obtained when one of the acidic hydroxyl

groups of the phosphonic acid was replaced with a methyl group, leading to phos-phinic acid derivative 38 shown in Figure 10.17 [31]. Given the hypothesis that thephenethyl group of lisinopril and enalapril was able to interact with the S1 subsite ofthe enzyme, a phenyl ring appropriately spaced from the phosphinic acid group wasintroduced, leading to compounds 39–41, with the four-methylene linker leading tothe most potent compound. In addition, the linker between the phosphinic groupand the amide bond was investigated, but without substantial improvement of activity.

N

OP

HO

CO2H

35 IC50 = 8.4 µMO

HO

36 IC50 = 48 µM

N

O

P

O

HOCO2HOH

N

OP

HO

CO2H

37 IC50 = 18 µMO

HOMe

Figure 10.16 Chemical structures of phosphonic acid-based inhibitors.

N

OP

CH3

CO2H

38 IC50 = 3.3 µMO

HO

N

OP

CO2H

39 IC50 = 0.88 µMO

HO

N

OP

CO2H

40 IC50 = 0.22 µM

O

HO

N

OP

CO2H

41 IC50 = 0.18 µMO

OH

Me

Figure 10.17 Chemical structures of phosphinic acid-based inhibitors.

10.5 ACE Inhibitors Bearing Phosphorus-Based Zinc Binding Groups 233

Compound 41 was further investigated and subjected to optimization, as itshowed good antihypertensive activity when administered intravenously. In orderto increase the affinity for ACE, some lipophilic proline derivatives were explored(42–46, Figure 10.18). This kind of modification was previously explored with cap-topril derivatives, but improvement in inhibitory potency was more consistent inthe phosphinic acid series [32]. With the exception of the methoxy group (42), thi-omethyl (43), thiophenyl (44), phenyl (45), and cyclohexyl (46) substituents wereall able to greatly improve potency with respect to the unsubstituted inhibitor. Forall substituents taken into consideration, a stereoselective interaction with theenzyme was not observed, since (R)- and (S)-isomers showed similar activities.The only exception was the phenyl derivative 45, whose (R)-isomer was >15 timesmore potent than the (S)-isomer. Fosinoprilat (46) was then developed into thecorresponding prodrug fosinopril (27), suitable for oral administration.

10.6Conclusions

ACE is an enzyme with a broad specificity, and, as a consequence, its activityaffects several physiological processes apart from controlling blood pressure. Someof the processes controlled by ACE activity and by its reaction product angiotensinII are renal function, hematopoiesis, reproduction, and some functions of theimmune system. At the moment, ACE inhibitors are being used primarily for the

N

OPO

OH

CO2H

46 Fosinoprilat

N

OP

CO2H

42 IC50 = 100 nM

O

OH

OMeR

R

N

OP

CO2H

43 IC50 = 29 nM

O

OH

SMeR

N

OP

CO2H

44 IC50 = 17 nM

O

OH

SPhR

R

N

OP

CO2H

45 IC50 = 7 nM

O

OH

PhR

IC50 = 11 nM

S

Figure 10.18 Chemical structures of phosphinate-based inhibitors.


control of hypertension, and also for the treatment of heart failure and nephropa-thies. However, they are also studied for their effects on tumor growth, erectiledysfunctions, and neurodegenerative diseases such as Alzheimer’s disease.Another interesting feature of the ACE enzyme is that somatic ACE is composedof two catalytic domains (N- and C-terminal domains). Several reports suggest thatthe two catalytic domains have different substrate specificities and biological func-tions. Recently, subtle differences in the structure of these two domains have beenhighlighted and the current challenge for structure-based drug design approachesis the development of domain-selective inhibitors. This is a very exciting field thatwill lead to better comprehension of the physiological role of ACE.The details of the design of the first ACE inhibitor, captopril, may illustrate the

beginning of rational structure-based design. The major advancements in technol-ogy and X-ray crystallographic techniques have allowed further improvement anddevelopment of ACE inhibitors with improved properties. These advancementshave also enabled the application of structure-guided drug development to thedevelopment of newer classes of inhibitors. This early application of drug designguided by structural analysis of a target enzyme formed the foundation of struc-ture-based design approach to drug design and discovery.

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19 Byers, L.D. andWolfenden, R. (1972) Apotentreversible inhibitor of carboxypeptidase A.J. Biol. Chem., 247, 606–608.

20 Byers, L.D. and Wolfenden, R. (1973)Binding of the by-product analogbenzylsuccinic acid by carboxypeptidase A.Biochemistry, 12, 2070–2078.

21 Acharya, K.R., Sturrock, E.D., Riordan, J.F.,and Ehlers, M.R. (2003) ACE revisited: anew target for structure-based drug design.Nat. Rev. Drug Discov., 2, 891–902.

22 Natesh, R., Schwager, S.L., Evans, H.R.,Sturrock, E.D., and Acharya, K.R. (2004)Structural details on the binding ofantihypertensive drugs captopril andenalaprilat to human testicular angiotensinI-converting enzyme. Biochemistry, 43,8718–8724.

23 Corradi, H.R., Schwager, S.L., Nchinda, A.T., Sturrock, E.D., and Acharya, K.R. (2006)Crystal structure of the N domain ofhuman somatic angiotensin I-convertingenzyme provides a structural basis fordomain-specific inhibitor design. J. Mol.Biol., 357, 964–974.

24 Akif, M., Schwager, S.L., Anthony, C.S.,Czarny, B., Beau, F., Dive, V., Sturrock, E.D., and Acharya, K.R. (2011) Novelmechanism of inhibition of humanangiotensin-I-converting enzyme (ACE) bya highly specific phosphinic tripeptide.Biochem. J., 436, 53–59.

25 Akif, M., Masuyer, G., Schwager, S.L.,Bhuyan, B.J., Mugesh, G., Isaac, R.E.,Sturrock, E.D., and Acharya, K.R. (2011)Structural characterization of angiotensin I-converting enzyme in complex with aselenium analogue of captopril. FEBS J.,278, 3644–3650.

26 Anthony, C.S., Corradi, H.R., Schwager, S.L., Redelinghuys, P., Georgiadis, D., Dive,V., Acharya, K.R., and Sturrock, E.D. (2010)The N domain of human angiotensin-I-converting enzyme: the role of N-glycosylation and the crystal structure incomplex with an N domain-specificphosphinic inhibitor, RXP407. J. Biol.Chem., 285, 35685–35693.

27 Patchett, A.A., Harris, E., Tristram, E.W.,Wyvratt, M.J., Wu, M.T., Taub, D.,Peterson, E.R., Ikeler, T.J., tenBroeke, J.,Payne, L.G., Ondeyka, D.L., Thorsett, E.D.,Greenlee, W.J., Lohr, N.S., Hoffsommer, R.D., Joshua, H., Ruyle, W.V., Rothrock, J.W.,Aster, S.D., Maycock, A.L., Robinson, F.M.,Hirschmann, R., Sweet, C.S., Ulm, E.H.,Gross, D.M., Vassil, T.C., and Stone, C.A.(1980) A new class of angiotensin-converting enzyme inhibitors. Nature, 288,280–283.

28 Menard, J. and Patchett, A.A. (2001)Angiotensin-converting enzyme inhibitors.Adv. Protein Chem., 56, 13–75.

29 Komiyama, T., Suda, H., Aoyagi, T., Takeuchi,T., and Umezawa, H. (1975) Studies oninhibitory effect of phosphoramidon and itsanalogs on thermolysin. Arch. Biochem.Biophys., 171, 727–731.

30 Thorsett, E.D., Harris, E.E., Peterson, E.R.,Greenlee, W.J., Patchett, A.A., Ulm, E.H.,and Vassil, T.C. (1982) Phosphorus-containing inhibitors of angiotensin-converting enzyme. Proc. Natl. Acad. Sci.USA, 79, 2176–2180.

31 Petrillo, E.W., Jr. and Ondetti, M.A. (1982)Angiotensin-converting enzyme inhibitors:medicinal chemistry and biological actions.Med. Res. Rev., 2, 1–41.

32 Krapcho, J., Turk, C., Cushman, D.W.,Powell, J.R., DeForrest, J.M., Spitzmiller, E.R., Karanewsky, D.S., Duggan, M., andRovnyak, G. (1988) Angiotensin-convertingenzyme inhibitors. Mercaptan, carboxyalkyldipeptide, and phosphinic acid inhibitorsincorporating 4-substituted prolines.J. Med. Chem., 31, 1148–1160.


11HIV-1 Protease Inhibitors for the Treatment of HIV Infection andAIDS: Design of Saquinavir, Indinavir, and Darunavir

11.1Introduction

The discovery of human immunodeficiency virus (HIV) as the causative agent foracquired immunodeficiency syndrome (AIDS) and subsequent investigation of thebiochemical events critical to the HIV replication cycle led to the recognition of anumber of important targets for drug development. During HIV replication, gagand gag–pol gene products are translated into precursor polyproteins that arecleaved by the virally encoded aspartic acid protease. The resulting products, struc-tural proteins and essential viral enzymes, are assembled to form virions that areready to infect new cells. Because of the critical role of HIV-1 protease in the late-stage viral replication cycle, this enzyme became an attractive target for the devel-opment of antiviral therapies in 1986. Early experiments demonstrated thatinactivation of retroviral protease by either site-specific mutagenesis or chemicalinhibition led to the production of immature and noninfective viral particles [1–5].Consequently, the design and development of HIV-1 protease inhibitors becamean intensive area of research across the world. Therapeutic inhibition of the virallyencoded HIV protease became particularly attractive due to prior knowledge ofmechanism-based inhibition of other aspartyl proteases.HIV-1 protease is an aspartic acid protease. The early development of HIV-1

protease inhibitors was based upon the knowledge of mechanism-based inhibitionof renin, another member of the aspartic acid protease family. Within a decade,hundreds of protease structures in complex with or without inhibitors were deter-mined by X-ray crystallography. NMR techniques were also utilized, but to a lesserextent. This structural knowledge greatly facilitated the structure-based design of avariety of peptidomimetic inhibitors and guided the evolution of inhibitors forclinical development [6,7]. Saquinavir was the first protease inhibitor to receiveFDA approval in 1996 for the treatment of AIDS.Saquinavir was designed by incorporating a hydroxyethylamine dipeptide

isostere to mimic the transition state formed during peptide cleavage by HIV-1protease. It is one of the most potent first-generation HIV-1 protease inhibitors.The approval of several other first-generation protease inhibitors (Figure 11.1)

237


quickly followed. Indinavir was designed based upon a lead structure obtainedfrom renin inhibitors and X-ray structural studies of saquinavir. Protease inhibi-tors were introduced into highly active antiretroviral therapy (HAART) withreverse transcriptase inhibitors or other drugs targeting different viral biochemicaltargets. This significantly reduced mortality and morbidity of patients with HIVinfection and AIDS in the United States and other industrialized nations. Themajority of first-generation protease inhibitors contain substantial peptide-like fea-tures. Also, it was found that rapid emergence of drug resistance quickly rendersthese therapies ineffective.The structure-based design approach led to a second generation of nonpeptidyl

protease inhibitors (Figure 11.2) that were designed and developed specifically tocombat drug resistance. These inhibitors are atazanavir, lopinavir, fosamprenavir,tipranavir, and darunavir. These inhibitors were designed to inhibit HIV-1 variantsresistant to the first-generation inhibitors and to reduce side effects to improvepatients’ adherence to treatment. Among the second-generation inhibitors, daru-navir is particularly potent. Darunavir resulted from structure-based design effortsby promoting extensive interactions with the highly conserved active site proteasebackbone atoms. This “backbone binding concept” has emerged as a useful strat-egy for combating drug resistance [8]. Indeed, in clinical studies, darunavir hasshown a high genetic barrier to the development of resistance (discussed laterin detail).

O

O

N

NH

NHN

OH

O

PhOH

ONH

NHN

H2N

H

O HN

H

H

HN

N

PhO

OH

OHN

NH

PhOH

Ph

HN

O

O

N

S

O

N

2 Indinavir1 Saquinavir

NOH

HN

H

H

HN

SO

OH

3 NelfinavirPh

HOMe

4 Ritonavir

O

OHN

OH

Ph

NS

O

H

OO

NH2

5 Amprenavir

SN

Figure 11.1 Structures of first-generation HIV-1 protease inhibitors.

238 11 HIV-1 Protease Inhibitors for the Treatment of HIV Infection and AIDS

11.2Structure of HIV Protease and Design of Peptidomimetic Inhibitors ContainingTransition-State Isosteres

HIV protease is a homodimeric enzyme, and it is different from themonomeric aspartic proteases such as human cathepsin D or renin. Two flaps(yellow in Figure 11.3) cover the active site of the enzyme and are involved inthe regulation of substrate accessibility to the active site, interaction betweensubstrate and inhibitors, and release of the cleavage products. When theenzyme is bound to an inhibitor (e.g., acetyl pepstatin in Figure 11.3), the flapsare closed and contribute to inhibitor binding [9–11]. In the absence of inhibi-tor, the flaps are highly flexible, and X-ray crystal structures revealed that theyare in an open conformation [12,13]. However, the open flaps do not necessarilyrepresent the lowest-energy conformation of the enzyme but are probably theresult of crystal packing. The enzyme is mainly composed of b-strands, and theactive site triplet (Asp25–Thr26–Gly27) is located in the active site loop (yellowin Figure 11.3), which is stabilized by a network of hydrogen bonding interac-tions. The two catalytic aspartates from each monomer closely interact witheach other and are almost coplanar.

HN

NH

PhOH

Ph

NO

O

NH

7 Lopinavir

CH3 NOH O

HN

O

Ph

NOH

NH

O HN

O

OCH3

N

6 Atazanavir

O

OH

O

SHN

NO O

CF3

9 Tipranavir

O

OHN

OH

Ph

NS

O

H

OO

NH2

O

O

10 Darunavir

HH

O

OHN

O

Ph

NS

O

H

OO

NH2

8 Fosamprenavir

P

O-

O-O

O

Figure 11.2 Structures of second-generation HIV-1 protease inhibitors.

11.2 Structure of HIV Protease and Design 239

The peptide bond is hydrolyzed by a water molecule, which is activated bythe two catalytic aspartate residues. The mechanism of hydrolysis involves theformation of a tetrahedral intermediate. As discussed earlier in Chapter 2, avariety of inhibitors were developed using nonhydrolyzable transition-state iso-steres. Most of the peptidomimetic inhibitors contain a transition-state isostereas a noncleavable peptide bond, which shares a similar binding mode with theinhibitor bound in an extended conformation. In Figure 11.4, the potent

Figure 11.3 X-ray crystal structure of the HIV-1 protease and acetyl pepstatin complex (the twoenzyme monomers, blue and green; the flaps and the active site loops, yellow; acetyl pepstatin,pink spheres; PDB code: 5HVP).

Figure 11.4 Binding mode of acetyl pepstatin within the active site of HIV protease (inhibitor,pink sticks; amino acid residues forming the S and S0 subpockets, green and blue, respectively;amino acids of the flaps, yellow; water molecule, red sphere; PDB code: 5HVP).


inhibitor acetyl pepstatin has been taken as a representative structure [10]. Allpeptide bonds of the inhibitor form hydrogen bonds with enzyme residuesbelonging to the two flaps and to the catalytic loops. The hydroxyl group ofthe transition-state mimetic is close to both catalytic aspartates. Another com-mon feature is a buried water molecule that forms hydrogen bonds with theP2 and P1

0 carbonyls of the inhibitor and the backbone NHs of residues Ile50and Ile500 of the flaps. This highly conserved water molecule has been targetedfor the design of several inhibitors. The side chains of the inhibitor (Pn � Pn

0)occupy the corresponding subsites of the enzyme.Cleavage sites on the gag and gag–pol proteins [14] are shown in Figure 11.5.

Substrate specificity studies suggest that the S1 and S10 subpockets of the enzymeprefer hydrophobic groups, whereas the S2 and S20 subpockets can accommodateboth the hydrophobic and polar groups of the inhibitor. S3 and S30 subsites areless well defined with respect to the above-mentioned subsites.

11.3Saquinavir: the First Clinically Approved HIV-1 Protease Inhibitor

For the discovery of saquinavir (1, Figure 11.1), a transition state-based design strat-egy was employed. The design approach involved the replacement of the peptidebond in a specific cleavage sequence with a nonhydrolyzable hydroxyethylamine iso-stere [15]. The hydroxyethylamine transition-state isostere mimicked the Phe–Procleavage site (Figure 11.5). This cleavage sequence, presenting a proline as the P1

0

residue, is highly specific for the viral enzyme, since none of the human asparticproteases can cleave peptide bonds presenting a proline at P1

0. Therefore, it wasspeculated that mimicking a peptide sequence highly specific for the viral proteasewould help to obtain selective inhibitors that would unlikely inhibit human aspartic

Figure 11.5 Cleavage sites localized in the gag and gag–pol polyprotein.

11.3 Saquinavir: the First Clinically Approved HIV-1 Protease Inhibitor 241

proteases. Once the peptide isostere was set, the flanking amino acid residues wereoptimized through exploration of their interactions with the enzyme through exten-sive structure–activity relationship studies.The tripeptide analog 11a (Figure 11.6) was subjected to extensive structure–

activity relationship studies, in order to increase the inhibitory potency andreduce the peptidic character of the corresponding inhibitors. Early on, it wasrecognized that tert-butyl ester of 11a could be replaced by the isosteric tert-butylamide group (11b) without significant loss of potency. Moreover, itwas observed that the (R)-configuration at the carbon bearing the transition-state mimetic hydroxyl group was slightly preferred over the (S)-stereochemistry

13a R = Cbz, IC50 = 13 nM (R/S IC50 ratio > 7.7)

HN

OHN

ONH

OH2N

O

O

11a X = O, IC50 = 140 nM (R/S IC50 ratio = 0.46)

OX

HN

OHN

ONH

OH2N

O

12 IC50 = 14 nM (R/S IC50 ratio = 1)

O O

HN

OHN

ONH

OH2N

R

OO

HN

O

ONH

HN

ONH

OHO

HN

OHN

ONH

OH2N

OO

HN

O

ONH

OHN

ONH

OHO

14 IC50 = 3.4 nM (R/S IC50 ratio = 19)

R

R

S

S

P-side extended compounds

P'-side extended compounds

11b X = NH, IC50 = 210 nM

13b R = Boc, IC50 = 16 nM (R/S IC50 ratio = >50)

Figure 11.6 Structures of protease inhibitors 11–14.


(R/S IC50 ratio¼ 0.46). Interestingly, other groups developing hydroxyethylene-based transition-state isosteres [11] observed a preference for the (S)-stereo-chemistry at the hydroxyl stereocenter. In particular, it was discovered that com-pounds, such as 12, presenting a two-residue extension at the N-terminal side didnot show any preference for hydroxyl stereochemistry [16]. In contrast, the C-ter-minal extended derivatives, such as 13a or 13b [16], showed a marked preferencefor the (S)-stereochemistry at the hydroxyl-bearing center. Similar stereochemicaltrends were observed for compound 14, showing the importance of both amino-and carboxy-terminal amino acid extensions [11,16].A systematic optimization of inhibitor 11 was then carried out (Figure 11.7). An

improvement in the enzyme inhibitory activity was achieved by extending the hydro-phobic benzyloxycarbonyl group by converting it to a naphthylamide (15a). A furthertwofold improvement in enzyme inhibitory potency was gained by converting the

(R/S IC50 ratio = 0.004)

HN

OHN

ONH

OH2N

ON

(R/S IC50 ratio < 0.004)

HN

OHN

ONH

OH2N

O

O NH

R

HN

OHN

ONH

OH2N

ON

O NH

HN

OHN

ONH

OH2N

O

O NH

N

NH

O

H

H

Ro 31-8959 (saquinavir)

ICEnzyme Antiviral

50 (nM) EC50 (nM)

15a 52 130

15b 23 110

16 2 17

1 <0.4 2

Figure 11.7 Structures and activities of HIV protease inhibitors 15, 16, and saquinavir (1).


naphthylamide to a 2-quinolinylamide (15b). Of particular note, the asparagine resi-due could not be replaced by any other residue without a dramatic loss of potency,suggesting that the side chain carboxamide forms strong interactions with theenzyme. Replacement of the five-membered proline ring with a piperidine ringresulted in a 10-fold increase in enzyme inhibitory activity and 6-fold improvementin antiviral potency (inhibitor 16). The enzyme inhibitory and antiviral potency wasfurther enhanced by introducing a decahydroisoquinoline. This inhibitor (Ro 31-8959) exhibited a marked enzyme IC50 value and very potent antiviral activity. Subse-quent clinical development led to the first HIV-1 protease inhibitor, saquinavir (1),which received FDA approval for the treatment of HIV infection and AIDS.The transition-state hydroxyl stereochemistry is critical to enzyme affinity. As it

turned out, increasing the size of the cyclic amine ring in the hydroxyethylamineisostere from pyrrolidine to piperidine to decahydroisoquinoline led to anincreased selectivity for the inhibitor with (R)-stereochemistry. Indeed, inhibitorswith (R)-1 and (R)-17 hydroxyl stereochemistry were 250 and 30 times morepotent than the corresponding (S)-1 and (S)-17 inhibitors, respectively. It wasalso observed that with the decahydroisoquinoline ring in place, extension of theC-terminal side of the inhibitor resulted in a loss of enzymatic affinity, irrespectiveof the transition-state hydroxyl stereochemistry. Both (R)- and (S)-18 were inactiveat a concentration of >100 nM (Figure 11.8).The X-ray crystal structure of saquinavir and HIV-1 protease complex was deter-

mined to obtain insight into the inhibitor binding properties [17]. This structure hasprovided important insight into the ligand and binding site interactions.

17 IC50 = < 2.7 nM R/S IC50 ratio < 0.027

(R)-18 IC50 > 100 nM(S)-18 IC50 > 100 nM

HN

OHN

ONH

OH2N

O

O

NH

O

H

H

HN

OHN

ONH

OH2N

O

O

NHO

H

H

O

HN

O

O

Figure 11.8 Structures and activities of HIV protease inhibitors 17 and 18.


Interestingly, the binding mode of saquinavir was slightly different from the bindingmode previously observed for proline-containing inhibitors such as 13 [11,16]. Asshown in Figure 11.9, the isosteric hydroxyl group is located between the two cata-lytic aspartates Asp25 and Asp250 in the active site. The carbonyl groups of both theP1 and P1

0 residues of saquinavir formed hydrogen bonds with a structural watermolecule, which in turn also formed hydrogen bonds with the backbone NHs ofIle50 and Ile500. Further hydrogen bonds are formed between the quinoline amideand the backbone atoms of Gly48 and Asp29. Furthermore, hydrogen bonding inter-actions of the asparagine carboxamide with the backbone NH and side chain car-boxyl group of Asp30 explain the importance of this residue at P2. The hydrophobicmoieties of saquinavir, namely, the tert-butyl group, the phenyl ring, and the quino-line rings, make extensive van der Waals contact with S2

0, S1, and S3, respec-tively. These interactions can easily be established between the inhibitor andthe enzyme with the (R)-stereochemistry at the hydroxyl center, thus explain-ing the observed stereochemical preference. The extensive network of enzyme–inhibitor interactions is responsible for saquinavir’s excellent potency [17].A comparison of the X-ray crystal structures of protease complexes with inhibi-

tor 14 (containing a proline as the P10 residue) and saquinavir highlights small but

important differences in the conformation of specific enzyme residues involved invarious binding interactions. Figure 11.10 shows the superimposition of the X-ray

Figure 11.9 X-ray crystal structure of the saquinavir (1) and HIV-1 protease complex (inhibitorcarbon chain, green; hydrogen bonding interactions, dotted lines; enzyme residues, yellow sticks;PDB code: 1HXB).


structures of saquinavir and inhibitor 14 in complex with HIV-1 protease and therespective bound conformations of the Val82–Thr79 residues. As can be seen, toaccommodate the bulky decahydroisoquinoline bicyclic ring of saquinavir, the flapPro81 (red) undergoes a conformational change, thus increasing the size of thecorresponding hydrophobic pocket. Furthermore, both the NH of the tert-butyla-minocarbonyl group of saquinavir and the NH of the Ile residue of inhibitor 14occupy different positions. In saquinavir, the tert-butyl group is projected towardthe S2 pocket, which is effectively filled by this bulky group. Further extensions ofthe P3

0 and P40 ligands may be detrimental to effective ligand binding in this sub-

site. This may explain the reason for the loss of significant inhibitory potency ofcompound 18, which contains extended amino acid-derived ligands at P3

0 and P40.

11.4Indinavir: an HIV Protease Inhibitor Containing the Hydroxyethylene Transition-StateIsostere

Merck researchers screened a collection of compounds previously developed asrenin inhibitors and identified the heptapeptide mimetic 19 (Figure 11.11) as apotent inhibitor of HIV protease [18]. Inhibitor 19 contains a hydroxyethylene tran-sition-state isostere. It was established that the depicted configuration of the transi-tion-state hydroxyl group and the P1

0 benzyl ligand was important for interactionwith the enzyme. Despite the potent enzymatic inhibitory activity, the minimuminhibitory concentration (MIC) of 19 required to inhibit the spread of the virus in

Figure 11.10 Superimposition of bound conformation of saquinavir (1, magenta) and inhibitor14 (green) and corresponding positions of the protease residues 79–82 (PDB codes: 1HXB(saquinavir and HIV-1 complex) and 7HVP (14 and HIV-1 complex)).


infected cells was in the high micromolar range, mainly due to the size and theheavy peptidic character of the compound. Reduction of the molecular weight byelimination of one of the prime-side phenylalanine side chains resulted in inhibi-tor 20 with a loss of inhibitory potency. The removal of the second prime-sidephenylalanine provided inhibitor 21, which restored potency similar to lead inhibi-tor 19. Compound 21 not only was more potent than heptapeptide 19, but alsoshowed an improvement of the cell MIC value.Further removal of the P3

0 phenylalanine and incorporation of benzylamideresulted in compound 22. It displayed similar enzyme inhibitory potency as the

HN

HN

NHO

OH

O

O

O

NH2NH

OHN

O

O

HN

HN

NHO

OH

O

O

O

NH2

O

O NH

HN

NH

OH

O

O

O

NH2

O

OHN

HN

NH

OH

O

O

O

OHN

HN

OH

OO

OHN

19 (L-364505)

20

21

22

23

IC50 (enzyme) = 1.0 nM MIC = 50 µM

IC50 (enzyme) = 20 nM



IC50 (enzyme) = 111 nM MIC = >50 µM

P3

P2

P3'

P2' P2'

Figure 11.11 Structures, enzyme inhibitory potencies, and MIC values of initial HIV proteaseinhibitors 19–23.

11.4 Indinavir: an HIV Protease Inhibitor Containing the Hydroxyethylene Transition-State Isostere 247

starting lead compound 19 and a slightly better cellular activity profile. However, itslack of adequate antiviral activity, high molecular weight, and the presence of peptidecharacter made this inhibitor unsuitable for further development. Consequently, fur-ther optimization was carried out to reduce molecular weight and peptidic features.Inhibitor 23 was obtained by removing the P2

0 amino acid from inhibitor 22, andthis exhibited reasonable potency with a molecular weight of 502Da.In an effort to optimize potency, the researchers explored conformationally

constrained benzo-fused cycloalkyl amide in place of the benzylamide in inhibitor23. The results are summarized in Figure 11.12 [19]. In general, incorporation of

HN

OH

OO

OHN

23IC50 (enzyme) = 111 nM MIC = >50 µM

HN

OH

OO

OHN

24IC50 = 19 nM

HN

OH

OO

OHN

26IC50 = 0.3 nM

CIC = 400 nM

HN

OH

OO

OHN

28IC50 = 259 nM

HO

HO

(R) HN

OH

OO

OHN

25IC50 = 21 nM

(S)

(S)

(R)

HN

OH

OO

OHN

27IC50 = 229 nM

HO

(R)

(S)

(SR)

(SR)

HN

OH

OO

OHN

29 (L-689502) IC50 = 0.45 nM; CIC95 = 12 nM

ON

O

OH

Figure 11.12 Structures and enzyme inhibitory potencies of HIV protease inhibitors 24–29.


four- to six-membered benzo-fused derivatives resulted in a five- to sevenfoldimprovement in potency, with respect to inhibitor 23. It appeared that the aminestereochemistry did not have much influence on inhibitory activity. Further intro-duction of a hydroxyl group on the cyclopentane ring of indane was carried out.As can be seen, the cis-derivative (26) exhibited very potent enzyme inhibitoryactivity and improved antiviral activity. The diastereomeric cis-derivative (27)showed dramatic loss of potency compared with dehydroxy derivative 25. Incorpo-ration of a diastereomeric mixture of trans-aminoindanol also resulted in a loss ofpotency for compound 28. Although compound 26 showed good overall potency, itsuffered from low aqueous solubility and poor pharmacokinetic properties. Toaddress this problem, Merck researchers incorporated a solubilizing group at the4-position of the P1

0 phenyl ring. The resulting compound 29 (L-689502) showedvery good enzyme and antiviral activity [20]. This compound also showed oral bio-availability in dogs (5%). However, subsequent safety studies revealed hepatotoxic-ity associated with this inhibitor.Following the development of compounds 26 and 29, researchers envisioned

incorporating a basic amine functionality by merging the decahydroisoquinolinegroup of saquinavir (Ro 31-8959) in place of the P1 Boc-aminophenethyl ligand of26. As shown in Figure 11.13, the corresponding chimeric derivative 30 displayedgood enzyme inhibitory potency. However, this compound showed only moderateantiviral activity [21].Further investigations focused on replacement of the decahydroisoquinoline

moiety of inhibitor 30 led to a series of potent inhibitors. As shown in Figure 11.14,incorporation of a substituted piperazine derivative in inhibitor 30 providedinhibitors with improved enzyme inhibitory and antiviral activity. The benzyl deriva-tive (33) displayed improvement in cellular potency over the Cbz derivative 32. Itappeared that a large hydrophobic group was necessary to maintain potency.

HN

OH

OO

OHN

OH

HN

OHN

PhO

NH

ONH2

ON

H

H

NH

O

HN

OH

O

OHN

H

H

NH

O

30 IC50 = 7.8 nM; CIC95 = 400 nM

1 Saquinavir

26

Chimeric lead

Figure 11.13 Discovery of chimeric inhibitor 30 from saquinavir (1) and 26.

11.4 Indinavir: an HIV Protease Inhibitor Containing the Hydroxyethylene Transition-State Isostere 249

Incorporation of an 8-quinolinylsulfonyl derivative provided compound 34 withimproved potency, but its oral bioavailability was not improved. Incorporation of a 3-pyridylmethyl group resulted in significantly enhanced aqueous solubility and oralbioavailability. This led to the discovery and development of indinavir (2), which wasapproved by the FDA for the treatment of HIV/AIDS.As shown in Figure 11.15, X-ray crystal structure of indinavir and HIV-1 prote-

ase complex was determined [22]. The structure revealed the formation of a seriesof hydrogen bonding and van der Waals interactions in the active site. The

HN

OH

O

OHNHN

NH

O

IC50 (nM) CIC95 (nM)

31 38 3000

HN

OH

O

OHNN

NH

O 32 0.36 100

O

O

HN

OH

O

OHNN

NH

O 33 0.30 50

HN

OH

O

OHNN

NH

O 34 0.013 12.5-50

S

HN

OH

O

OHNN

NH

O 2 0.56 50.4

N

Indinavir

OON

Figure 11.14 Structures and potencies of HIV protease inhibitors 31–34 and indinavir (2).


transition-state hydroxyl group forms tight hydrogen bonds with the catalyticaspartates. The P1 and P1

0 carbonyl groups formed hydrogen bonds with the struc-tural water, which in turn formed hydrogen bonds with the flap backbone NHs ofIle50 and Ile500. The P2

0 hydroxyl group on the indane moiety formed severalhydrogen bonds with the side chains and backbone atoms of Asp29 and Gly27.The P2

0tert-butylamide NH formed a water-mediated hydrogen bond with the sidechain carbonyl of Asp290.

11.5Design and Development of Darunavir

The design and development of first-generation HIV-1 protease inhibitors markedthe beginning of an unprecedented era of structure-based design of aspartic acidprotease inhibitors. Saquinavir (1) was the first protease inhibitor and receivedFDA approval in late 1995. Saquinavir is the most potent among first-generationFDA-approved protease inhibitors. The X-ray crystal structure of saquinavir-boundHIV-1 protease revealed important structural insights into its interactions in theprotease active site. Of specific interest, the P2 asparagine carboxamide and the P3quinoline carboxamide carbonyls of saquinavir were involved in a number ofhydrogen bonding interactions with the Asp29 and Asp30 NHs in the S2 subsite[17]. During structure–activity studies of saquinavir, it was recognized that the P2

Figure 11.15 X-ray crystal structure of indinavir (2) and HIV-1 protease complex (PDB code:1HSH).

11.5 Design and Development of Darunavir 251

asparagine residue was critical to its high affinity [15]. Presumably, the asparagineside chain is involved in important hydrogen bonding interactions in the activesite. Based upon these structural insights, we became interested in designing aconceptually new class of inhibitors that contain no amide/peptide-like features,but can mimic the specific binding interactions of saquinavir. Although saquinaviris very potent, it possesses multiple amide bonds and contains peptide-like fea-tures that may account for its poor pharmacological properties. Also, the first-generation protease inhibitors led to the quick emergence of resistance. The issueof cross-resistance also limits their effectiveness. In addition, poor bioavailability,high toxicity, and other side effects hamper long-term use of many of these earlyprotease inhibitors.

11.6Design of Cyclic Ether Templates in Drug Discovery

In the beginning, one of our inhibitor design objectives was to develop cyclicether-derived ligands and templates. We particularly envisioned that a suitablypositioned ring oxygen could effectively form similar interactions as the carbonyloxygen of P2/P3 amide bond of saquinavir. Our enthusiasm for cyclic ethersevolved due to the prevalence of such structural templates, particularly five- andsix-membered tetrahydrofurans and tetrahydropyrans, respectively, in a wide vari-ety of bioactive natural products, such as ginkgolides, monensin, and azadirachtin(Figure 11.16) [23–25]. These natural products are devoid of any peptidic features,yet they bind to their biosynthetic enzymes, as well as to the respective target

O OO

Me

Me

MeCO2Na

HO

MeH

O

O

HO Me

HOOMe

MeH

HH

Et MeH

O O

OO

Me

O

OHH

HOO

HO

H H

HH

OH

36, Monensin (Antibiotic)

35, Ginkgolide B (PAF antagonist)

OOH

MeO2CAcO

O

O

OCO2Me

OH

H

OOH

O

O

37, Azadirachtin(Insecticide)

Me

Figure 11.16 Structures of bioactive natural products containing cyclic ethers (highlighted inred).


enzyme/receptor with high affinity. Indeed, nature has been optimizing such tem-plates over millions of years of evolution and making them compatible with vari-ous biological microenvironments [26].Conceptually, the design of inhibitors with such cyclic ether scaffolds is quite

intriguing. Although the ether oxygen can accommodate up to two hydrogenbonds, the cyclic carbon backbone of five- and six-membered rings with definedconformation and limited rotational freedom could offer structural complementar-ity to hydrophobic pockets in the enzyme active site. In addition, unlike peptide-based compounds, the cyclic ether templates are expected to be metabolically stableas they are not subject to degradation by peptidases. Based upon these premises,we investigated the potential of cyclic ether-derived ligands and templates in drugdiscovery and structure-based design of HIV-1 protease inhibitors. These structure-based design efforts culminated in the discovery of a range of exceptionally potentinhibitors with unprecedented resistance profiles. One of these inhibitors is daru-navir (10, TMC-114, UIC-94017), which was approved by the FDA for the treat-ment of patients harboring multidrug-resistant HIV-1 variants [27–31].Our structural insight into the saquinavir-bound HIV-1 protease crystal structure

brought a unique opportunity to examine a number of intriguing molecular designprinciples. In particular, we planned to incorporate natural product-derived molec-ular templates into structure-based drug design. We initially speculated that astereochemically defined tetrahydrofuran ring could be designed in place of theasparagine side chain where the tetrahydrofuran ring oxygen could mimicthe binding of the carbonyl oxygen of the asparagine side chain and the ringcycle could effectively fill in the hydrophobic pocket of the S2 subsite. As shown inFigure 11.17, incorporation of (2S,30R)-tetrahydrofuranyl glycine as the P2 ligand inplace of asparagine resulted in inhibitor 38 with significant potency enhancementover saquinavir (Ki¼ 0.23 nM; CIC95¼ 22 nM). The ring stereochemistry andposition of oxygen are important for potency, as inhibitor 39 with (30S)-ringstereochemistry showed a substantial loss of potency. The removal of the ring oxy-gen also effected nearly a 50-fold loss of inhibitory activity over inhibitor 38 [32,33].As shown in Figure 11.17, we presumed that the (2S,30R)-tetrahydrofuran ringoxygen in 38 is optimally positioned to hydrogen bond with the Asp30 backboneNH and the ring cycle is able to fill in the hydrophobic pocket in the S2subsite more effectively than the corresponding (30S)-tetrahydrofuranyl ring ininhibitor 39.Saquinavir has a high molecular size (�670Da). In an effort to reduce molecu-

lar weight, we removed the P3 quinaldic amide ligand and designed the corre-sponding urethane 41 maintaining the (3S)-tetrahydrofuranyl ring to interactwith the Asp30 backbone NH of the HIV-1 protease. As shown in Figure 11.18,urethane 41 exhibited rather potent inhibitory activity, considering that the molec-ular size of compound 41 is 515Da [33,34]. The stereochemical preference for the(3S)-tetrahydrofuranyl ring is apparent as inhibitor 42 with the (3R)-tetrahydrofur-anyl ring is at least fourfold less potent than inhibitor 41. The corresponding tert-butyl urethane derivative 43 did not exhibit noticeable activity. The X-ray structureof inhibitor 41 and HIV-1 protease complex revealed that the tetrahydrofuran ring

11.6 Design of Cyclic Ether Templates in Drug Discovery 253

oxygen was involved in weak hydrogen bonding interactions with the Asp29 andAsp30 NHs in the S2 site [34].We then investigated the potency enhancing effect of the (3S)-tetrahydrofuranyl

urethane in inhibitors containing hydroxyethylene dipeptide isosteres. As can beseen in Figure 11.19, inhibitor 44 with (3S)-THF as the P2 ligand and a chiralaminoindanol as the P2

0 ligand exhibited excellent enzyme inhibitory and antiviralpotency. The stereochemical preference for the (3S)-isomer at the S2 subsite wasmaintained as inhibitor 45 with the (3R)-THF derivative or the correspondingcyclopentanyl urethane 46 showed significantly lower activity compared with(3S)-THF derivative. Subsequently, researchers at Vertex laboratory incorporated(3S)-THF urethane in the hydroxyethylamine sulfonamide isostere in the design

HNN

OPh

OHN

N H

O NH

O

N

H

HON

-O

O

H

H

H

O

O

HNN

O

ON

N H OO

OO-

HH

Asp25

Asp25'

O NH

O

O

N

H

HON

-O

O

HAsp29

HNO

Gly48HN

H

Asp30

N

O-

O

HH

IC50 = 0.054 nM; CIC95 = 8 nM

O NH

N

OHN

H

H

HN

N

PhO

OH

O

H

IC50 = 5.4 nM; CIC95 = 100 nM

O NH

HN

O HN

H

H

HN

N

PhO

OH

IC50 = 2.6 nM

H

38 39

40

Saquinavir

H

Figure 11.17 Introduction of cyclic ethers in saquinavir structure. Cyclic ligands are highlighted in red.


and discovery of a very potent inhibitor VX-478 (5) [35]. Consistent with the X-raystructural studies of inhibitor 41, the X-ray structure of VX-478-bound HIV-1 pro-tease revealed that the (3S)-THF ring oxygen in this inhibitor was in proximity toform hydrogen bonds with the Asp29 (distance 3.4 A

�) and Asp30 (distance 3.5 A

�)

NHs in the S2 site [36]. Clinical development of this inhibitor ultimately led toFDA approval of amprenavir (5). Later, a phosphate prodrug, fosamprenavir (8),was developed, which showed improved pharmacological properties and wasapproved by the FDA for the treatment of HIV/AIDS.

11.7Investigation of Cyclic Sulfones as P2 Ligands

It was evident from the X-ray studies of 41, and subsequently with VX-478, thatthe (3S)-THF ring oxygen formed weak hydrogen bonds in the S2 subsite. In an

HNN

OPh

OHN

O NH

O

N

H

H

H

H

O

IC50 = 0.054 nM CIC95 = 8 nM

NOH

N

OHN

H

H

HN

N

PhO

OH

O

H

IC50 = 5.4 nMCIC95 = 100 nM

H

38 39

HN

OPh

OHN

N H

O NH

H

HO

N

-O

O

H H

O

O

O

IC50 = 132 nMCIC95 = 800 nM

41

HN

OPh

OHN

O NH

H

H

HO

O

IC50 = 694 nM42

(S)-isomer (R)-isomer

HN

OPh

OHN

O NH

H

H

O

IC50 > 1000 nM43

H

Figure 11.18 Structural evolution of inhibitors with 30-tetrahydrofuranyl urethanes.

11.7 Investigation of Cyclic Sulfones as P2 Ligands 255

effort to strengthen hydrogen bonding with urethane-derived cyclic ligands, wethen speculated that the corresponding cyclic sulfone could form an effectivehydrogen bond as one of the oxygens would be closer to the backbone Asp29and Asp30 NHs in the S2 site. The results of this investigation are highlightedin Figure 11.20. As shown, replacement of (3S)-THF with (3S)-sulfolaneprovided inhibitor 47 with improvement of potency over inhibitor 41 [37,38].Inhibitor 48 with a (3R)-sulfolane also improved potency over inhibitor 42 with(3R)-THF as the P2 ligand. However, the (3S)-sulfolane is preferred over the(3R)-sulfolane by the S2 subsite. Further optimization of van der Waals interac-tions in the S2 hydrophobic pocket led to incorporation of various cis-2-alkyl-substituted sulfolanes. Both inhibitors 49 and 50 displayed improved activityover the unsubstituted sulfolanes. Introduction of both (3S)-hydroxysulfolane-and (2R,3R)-hydroxyisopropyl sulfolane-derived ligands in the hydroxyethyl-amine sulfonamide isostere also provided very potent inhibitors 51 and 52,respectively [28,31]. Modeling studies of inhibitors 47 and 51 suggested that thesulfolane oxygen cis to the carbamate moiety is within hydrogen bonding dis-tance to the amide NHs of Asp29 and Asp30, respectively. This may account forthe enhanced potency of (3S)-hydroxysulfolane-derived inhibitors over the (3S)-hydroxy-THF-derived compounds.

HN

OPh

OHO

Ph

O

IC50 <0.03 nMCIC95 = 3 nM

44O

H HN

OHHN

OPh

OHO

Ph

O

IC50 = 0.03 nMCIC95 = 100 nM

45O

H HN

OH

HN

OPh

OHNO

S

Ki = 0.6 nMIC90 = 40 nM

5 Amprenavir (VX-478)

O

H

O O

NH2

HN

OPh

OHO

Ph

O

IC50 = 0.33 nMCIC95 = 400 nM

46

H HN

OH

(3'S)-THF (3'R)-THF

(3'S)-THF

O

OHN

O

PhO

H

8 Fosamprenavir

P

O-

O-O

NS

O O

NH2

Figure 11.19 Application of (3S)-THF urethane in the development of potent inhibitors.


11.8Design of Bis-tetrahydrofuran and Other Bicyclic P2 Ligands

After developing sulfolane-derived ligands, we became interested in modulatingligand–binding site interactions of the (3S)-sulfolane ligand with stereochemicallydefined bicyclic ligands with cyclic ether features. Presumably, a cyclic ether oxy-gen would form a stronger hydrogen bond with subsite backbone NHs. Basedupon possible interactions of the (3S)-sulfolane in the S2 subsite of HIV-1 protease(Figure 11.21), we envisioned several intriguing possibilities to design bicyclic lig-ands with oxygens in place to interact with the backbone of the viral enzyme andeffectively fill in the S2 hydrophobic site. As outlined with red arrows (designmodes A–D), various bicyclic rings can be formed through the cis-sulfolane oxy-gen and adjacent C-2 carbon of the sulfolane ring. The cis-alkyl substitution at theC-2 carbon is quite feasible as the cis-methyl substitution provided inhibitor 49with a sixfold improvement in enzyme inhibitory activity over unsubstitutedinhibitor 47 (Figure 11.20).As can be seen, our design of the (3aS,4S,6aR)-hexahydro-2H-cyclopenta[b]furan-

4-ol-derived ligand (design mode A) with a saquinavir-based hydroxyethylamine

HN

OPh

OHN

O NH

H

H

O

47IC50 = 76 nMCIC95 = 364 nM

SO O

H HN

OPh

OHN

O NH

H

H

O

48IC50 = 140 nMCIC95 = >800 nM

SO O

H

HN

OPh

OHN

O NH

H

H

O

49IC50 = 11.4 nMCIC95 = 200 nM

SO O

H

CH3

HN

OPh

OHN

O NH

H

H

O

50IC50 = 3.5 nMCIC95 = 50 nM

SO O

HN

OPh

OHNO

S

SO O

H

OO

OMe

51Ki = 1.2 nMIC50 = 19 nM

HN

Ph

OHN

SOO

OMe

52Ki = 1.4 nMIC50 = 18 nM

O

SO O

O

(3'S)-sulfolane (3'R)-sulfolane

H

H

Figure 11.20 Development of P2 cyclic sulfone-derived potent inhibitors.

11.8 Design of Bis-tetrahydrofuran and Other Bicyclic P2 Ligands 257

isostere resulted in inhibitor 53 with a significant improvement in potency overinhibitors with the (3S)-THF ligand (41, Figure 11.18) and (3S)-sulfolane (47, Fig-ure 11.20). We then incorporated a second oxygen on the cyclopentane ring, whichled to the design of the (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-ol-derived ligandor simply bis-tetrahydrofuan (bis-THF) ligand (design mode B). Inhibitor 54 withthe bis-THF ligand exhibited very potent enzyme inhibitory and antiviral activity[39]. Our design of an oxaspiro ligand (design mode C) and incorporation of thisligand in the hydroxysulfonamide isostere provided inhibitor 55 with moderateantiviral activity [30,31]. Our design of a (3aS,5R,6aR)-hexahydro-2H-cyclopenta[b]furan-5-ol-derived ligand (design mode D) or simply cyclopentafuranyl (Cp-THF)ligand resulted in inhibitor 56 with excellent activity [40].We have incorporated the corresponding enantiomeric bis-THF ligand in inhibi-

tor 57, which is less potent than inhibitor 54 (Figure 11.22). To ascertain the con-tribution of the bottom oxygen, we prepared the corresponding deoxy ligand andincorporated it in inhibitor 58. However, this inhibitor showed a significant reduc-tion of potency, indicating the importance of both bis-THF oxygens. The X-raystructure of the inhibitor 54 and HIV-1 complex revealed that both oxygens of thebis-THF ligand are within proximity to form hydrogen bonds with the main chain

O

OHN

OH

PhS

H

O

O

O

OHN

OH

Ph

NS

Ki = 3.7 nMID50 = 265 nM

55

H

N H

O

NH

O

D

CA, B

N

O

OHN

OH

Ph

N

H

H

NH

O

IC50 = 17 nM53

H

OH

HO

OHN

OH

Ph

N

H

H

NH

O

IC50 = 1.8 nMCIC95 = 46 nM

54

O

H

OH

H

O

OHN

OH

Ph

H

A B

DC

O

OO

NH2

OH

H

NS

OO

OH

Ki = 4.5 pMIC50 = 1.8 nM

56

Asp30

Asp29

Figure 11.21 Design and development of bis-tetrahydrofuran and other bicyclic high-affinity P2 ligands.


amide NH of Asp29 and Asp30 residues in the S2 subsite [39]. Incorporationofthis bis-THF ligand in the hydroxysulfonamide isostere resulted in very potentinhibitors, and will be described in the following section. We have also investi-gated inhibitors with hexahydrofuropyranyl derivatives in inhibitors 59 and60. Inhibitor 60 showed potency similar to (3R,3aS,6aR)-bis-THF-containinginhibitor 54 [39,41].

11.9The “Backbone Binding Concept” to Combat Drug Resistance: Inhibitor DesignStrategy Promoting Extensive Backbone Hydrogen Bonding from S2 to S20 Subsites

Following our design and discovery of a range of conceptually novel cyclic ether-and cyclic sulfone-derived high-affinity ligands, we became interested in designinginhibitors that would exhibit exceptional potency against wild-type HIV-1 protease,while maintaining potency against a range of mutant proteases. We have exten-sively compared structural variations and essential molecular interactions in the

IC50 = 6.4 nMCIC95 = 200 nM

57O

OHN

OH

Ph

N

H

H

NH

OO

H

OH

H

IC50 = 1.8 nMCIC95 = 46 nM

54O

OHN

OH

Ph

N

H

H

NH

OO

H

OH

H

O

OHN

OH

Ph

N

H

H

NH

OO

H

OH

H

IC50 = 4.2 nMCIC95 = 100 nM

59

O

OHN

OH

Ph

N

H

H

NH

OO

H

OH

H

IC50 = 1.2 nMCIC95 = 50 nM

60

IC50 = 190 nM58

O

OHN

OH

Ph

N

H

H

NH

OO

H

HH

Figure 11.22 Structures and potencies of inhibitors with other bicyclic P2 ligands.

11.9 The “Backbone Binding Concept” to Combat Drug Resistance 259

X-ray structures of inhibitor bound to wild-type and various mutant proteases. Ofparticular interest, the overlays of several drug-resistant HIV-1 protease variantswith 10–14 mutations and with HIV-2 protease, with 40 or so variations, revealedthat the backbone conformation of the active sites distorted minimally comparedwith wild-type HIV-1 protease [42–44]. This is fundamentally significant as prote-ase cannot alter its overall structure around the active site without compromisingits vital catalytic fitness required for viral replication [8,45,46]. Based upon thisobservation, we hypothesized that an inhibitor maximizing interactions in the pro-tease active site and forming a network of robust hydrogen bonding interactionswith the active site protein backbone of the wild-type HIV-1 protease will likelymaintain these interactions with protease mutants as well. In all likelihood, sucha design strategy targeting the protein backbone would slow the development ofdrug-resistant HIV, since mutations that alter the backbone conformation wouldbe likely to reduce catalytic fitness [8]. In essence, we viewed this inhibitor designstrategy as designing a “molecular crab” capable of tightly gripping the proteinbackbone and holding on in the enzyme active site.Based on this backbone binding concept, our molecular design efforts focused

on promoting extensive hydrogen bonding interactions with the protein backboneatoms from the S2 to S2

0 subsites, as represented in Figure 11.23. It is vital thatthe corresponding P2 and P2

0 ligands would form robust hydrogen bonds inboth the S2 and S20 regions simultaneously. Furthermore, we planned to effec-tively fill the hydrophobic pockets in the active site, thus limiting the virus’s abilityto develop drug resistance. The S1 and S10 subsites are mostly formed by hydro-phobic residues; however, the S2 and S20 subsites contain both hydrophobic andhydrophilic residues. Our inhibitor design effort also involved improvement ofpharmacokinetic properties, oral bioavailability, and half-life to reduce the numberof daily doses.Our initial structure-based design of various cyclic and bicyclic ether-derived P2

ligands was developed based upon the X-ray structure of the saquinavir and HIV-1protease complex. However, the X-ray structure revealed that saquinavir does not

Transition-statebinder B

ackbonebinder

Bac

kbon

ebi

nder

N

N

O

O

H

HS2 site

S2' site

S1' site

S1 site

N

N

O

O

H

H

P2 ligand

P2' ligand

P1 ligand

P1' ligand

Figure 11.23 Inhibitor design model to combat drug resistance.


form any hydrogen bonds with backbone atoms in the S20 pocket. For the design

of inhibitors that can form hydrogen bonds with S2 and S20 subsites based upon

our “backbone binding concept,” we needed to modify functionalities in the P20

ligand of the saquinavir isostere so that it could hydrogen bond in the S20 subsite.We explored manipulations of the saquinavir isostere for this purpose. Also, weutilized the (R)-(hydroxyethyl)sulfonamide isostere [35,47] since it is syntheticallyamenable for incorporation of functionalities that can form hydrogen bonds withbackbone atoms in the S20 subsite. We investigated a number of P2 cyclic and bicy-clic ether-derived ligands in the (R)-(hydroxyethyl)sulfonamide isostere in combi-nation with a P2

0 sulfonamide that can form backbone interactions in the S20 site.

As shown in Figure 11.24, incorporation of (3R,3aS,6aR)-bis-THF as the P2 ligandand p-methoxybenzenesulfonamide as the P2

0 ligand to specifically form hydrogenbonds within the S20 subsite of the dimeric enzyme resulted in exceptionallypotent inhibitor 61 (UIC-94003, later renamed TMC-126) [28,48,49]. This inhibitorexhibited a remarkable antiviral IC50 of 1.4 nM in MT cells. Inhibitor 62, with anonpolar methyl group on the sulfonamide ring, also exhibited potent activity(saquinavir showed a Ki of 1.2 nM in this assay). Inhibitor 63 with enantiomeric

O

OHN

OH

Ph

NS

O

H

OO

O

(UIC-94003, TMC-126)Ki = 14 pM

ID50 = 1.4 nM

OH

H

O

OHN

OH

Ph

NS

O

H

OO

O

63Ki = 1.6 nMID50 = 4.1 nM

OH

H

O

OHN

OH

Ph

NS

O

H

OO

Me

62OH

H

61

Ki = 1.2 nMID50 = 3.5 nM

Figure 11.24 Structures and potencies of inhibitors with hydroxyethylamine sulfonamideisosteres and bis-THF P2 ligands.

11.9 The “Backbone Binding Concept” to Combat Drug Resistance 261

(3S,3aR,6aS)-bis-THF and p-methoxybenzenesulfonamide also exhibited potentactivity; however, inhibitor 61 with (3R,3aS,6aR)-bis-THF exhibited the mostpotent activity, indicating a stereochemical preference for the bis-THF stereo-chemistry for potency [28,31].We have determined a high-resolution X-ray structure of the inhibitor 61 and

HIV-1 protease complex [50]. As seen in Figure 11.25, the structure showed thepresence of a network of hydrogen bonds throughout the active site. In particular,both bis-THF oxygens formed strong hydrogen bonds with the backbone NHs ofAsp29 and Asp30 in the S2 pocket. The para-methoxy oxygen on the P2

0 benzene-sulfonamide appeared to form two hydrogen bonds with both Asp290 and Asp300

NHs in the S20 subsite. Also, the aromatic ring fills the hydrophobic pocket at the

S20 site. The transition-state hydroxyl group of 61 formed strong hydrogen bondswith Asp25 and Asp250. The urethane NH of 61 also formed a hydrogen bondwith the Gly27 backbone carbonyl group. In addition, the phenylmethyl P1 sidechain and the isobutyl P1

0 side chain effectively filled in the hydrophobic pocketsof the protease active site.The X-ray structure appeared to fulfill the criteria of our backbone binding

design concept for combating drug resistance [8,48]. Indeed, inhibitor 61 main-tained enzyme inhibitory potency less than 100 pM to several mutant proteasesknown to be resistant to several first-generation inhibitors. Also, it exhibited excel-lent potency against a wide spectrum of drug-resistant HIV-1 variants with IC50

values ranging from 0.3 to 0.5 nM. Of particular note, viral acquisition of resist-ance to inhibitor 61 was substantially delayed. Furthermore, inhibitor 61

Figure 11.25 X-ray structure of inhibitor 61 with wild-type HIV-1 protease. Inhibitor representedas CPK where hydrogen bonds are shown as dotted lines. (PDB code: 3I7E).


maintained impressive potency with IC50 values ranging from 0.5 to 5.5 nM,against multiprotease inhibitor-resistant HIV-1 strains isolated from patients withdrug-resistant HIV-1 [51–53]. It is conceivable that the formation of a network ofhydrogen bonds with protein backbone from the S2 to S20 sites as well as effectivepacking of the S1 and S10 hydrophobic pockets may be responsible for robust activ-ity of inhibitor 61 against a wide spectrum of drug-resistant HIV variants. Thus,the P2 bis-THF ligand and (R)-(hydroxyethyl)sulfonamide isostere provided anintriguing structural framework for developing a conceptually new generation ofinhibitors that can combat drug resistance.

11.10Design of Darunavir and Other Inhibitors with Clinical Potential

We have also designed a variety of inhibitors containing bis-THF as the P2 ligand incombination with (R)-(hydroxyethyl)sulfonamide isosteres with a variety of P2

0 sul-fonamide functionalities that are capable of forming hydrogen bonds with the back-bone atoms in the S20 site. As shown in Figure 11.26, all the inhibitors displayed

O

OHN

OH

Ph

NS

O

H

OO

Ki = 11 pMIC50 = 1.1 nM

OH

HO

OHN

OH

Ph

NS

O

H

OO

65OH

H

64 (GRL-98065)

Ki = 12 pM ID50 = 5.3 nM

O

O

Ki = 16 pM; IC90 = 4.1 nM(Darunavir)

OH

O

OHN

OH

Ph

NS

O

H

OO

66 OH

H

Ki = 43 pM ID50 = 28 nM

NH2

67 (GRL-00811)Ki = 9 pM

ID50 = 38 nM

O

O

OHN

OH

Ph

NS

O

H

OO

OH

HOH

O

OHN

OH

Ph

NS

O

H

OO

NH2

OH

H

10

Figure 11.26 Structure-based design of inhibitors leading to the development of darunavir.

11.10 Design of Darunavir and Other Inhibitors with Clinical Potential 263

superior enzyme affinity. Inhibitors 64 and 65 have also maintained extraordinaryefficacy against a broad range of resistant strains [8,48]. Inhibitor 10 (also known asTMC-114), however, exhibited the best pharmacokinetic properties and drug resist-ance profiles [54–56]. It was selected for clinical development. Clinical developmentof inhibitor 10 (TMC-114, also known as darunavir) was carried out by Tibotec-Virco, Belgium. Based upon clinical efficacy, inhibitor 10 was subsequentlyapproved by the FDA in 2006 for the treatment of patients carrying multidrug-resistant strains who do not respond to the available treatments. Later, in 2008, itreceived full approval to treat all HIV/AIDS patients, including pediatric patients.HIV-1 protease requires homodimerization of two 99-amino acid monomers to

form an active functional protease. The dimerization of protease monomers resultsin the formation of the HIV-1 protease active site. This is a critical event for thematuration and acquisition of proteolytic activity. Therefore, inhibition of proteasedimerization may offer an additional option to block viral replication. We have dis-covered that darunavir potently inhibits the dimerization of protease [57]. Thus, dar-unavir not only inhibits protease dimerization at the nascent stage, but also potentlyinhibits dimeric catalytically active enzyme that escapes inhibition at the nascentstage. This unique dual mechanism of action of darunavir may be responsible forits potent antiviral activity and durability against emergence of drug resistance.The X-ray crystal structures of HIV-1 protease complexed with inhibitors 10, 64,

and 67 were determined [8,58]. These inhibitors formed a network of effectivehydrogen bonding interactions throughout the active site. The X-ray structures ofdarunavir and saquinavir with HIV-1 protease are overlaid in Figure 11.27 [17,59].The binding mode of darunavir shows its complex network of hydrogen bonding

Figure 11.27 X-ray crystal structure of daruna-vir (10) and HIV-1 protease complex overlaidwith X-ray structure of saquinavir (1)-boundHIV-1 protease. Hydrogen bonds are shown as

dotted lines (red for saquinavir and black fordarunavir) (PDB codes: 2IEN (darunavir) and1HXB (saquinavir)).


interactions with the protein backbone from the S2 to S20 subsites. The central

hydroxyl group interacts with both catalytic aspartates, the urethane NH with thebackbone carbonyl of Gly27, and the buried water molecule bound to Ile50 andIle500 with the carbonyl and sulfonamide oxygen atoms of the inhibitor. Both theP2 bis-THF and P2

0 4-aminosulfonamide functionalities formed hydrogen bondswith the backbone NHs located at the S2 (Asp29 and Asp30) and S20 (Asp300) sub-pockets, respectively. Darunavir also nicely fills the hydrophobic pockets through-out the protease active site. The overlay structures also highlight that both oxygensof bis-THF formed strong hydrogen bonds with NHs located at the S2 (Asp29 andAsp30) and mimics the binding of P2 asparagine carbonyl with Asp30 NH and thebinding of P2/P3 carboxamide carbonyl with Asp29 NH.The unique binding mode, pharmacology of bis-THF ligand, and chemistry

and biology of darunavir attracted immense research interest. As shown inFigure 11.28, a number of very potent protease inhibitors have been designedbased upon the bis-THF ligand [30,41]. Brecanavir 68 developed by GSK (BCV/GW640385) showed femtomolar protease inhibitory activity and subnanomolar

O

OHN

OHN

SO

H

OO

O

O

O

O

S

NH

H

68 Brecanavir (GW640385)

O

OHN

OHN

SO

H

OO

O

O

O PO

OEt

HH

OEt

Ki = 15 fM; IC50 = 0.7 nMKi = 8 pM; IC50 = 3.5 nM

O

OHN

OHN

SO

H

OO

O

O

O

O

HH

69 GS-8374

71 Ki = 11 pM; IC50 = 3.1 nM70 Ki = 14 pM; IC50 = 5.4 nM

O

OHN

OHN

SO

H

OO

OH

HS

NNH

N

O

OHN

OHN

SO

H

OO

O

O

O

O

HH

NO

72 (TMC310911)EC50 = 2.2 nM

Figure 11.28 Structures and activities of bis-THF-derived inhibitors for clinical development.

11.10 Design of Darunavir and Other Inhibitors with Clinical Potential 265

antiviral activity [60]. However, brecanavir’s clinical trials were terminated dueto formulation issues. Inhibitors 70 and 71 were reported by our laboratories.Other bis-THF-derived clinical inhibitors GS-8374 (69) and TMC310911 (72)developed by Gilead Science and Tibotec-Virco, respectively, exhibited verypotent antiviral activity against multidrug-resistant HIV-1 variants [61,62].

11.11Conclusions

The introduction of combination antiretroviral therapy with protease inhibitorshas had a huge impact on the long-term management of HIV infection and AIDSin the United States and other developed countries. HIV infection has become atreatable disease. Early on, anti-HIV drug development efforts with HIV proteaseinhibitors were aimed at finding effective inhibitor drugs that could reduce viralloads in HIV patients. Only a few poorly effective and highly toxic therapeuticoptions were available at that time. Subsequently, the advent of protease inhibitorsand their introduction to HAART treatment regimens led to impressive reductionof mortality in HIV/AIDS patients. However, the low genetic barrier to resistancedemonstrated by several anti-HIV drugs, including protease inhibitors, presentedan urgent need for novel drugs and targets. Drug resistance is an evolutionary andfundamental problem. Therefore, combating resistance requires innovative designstrategies that target drug resistance mechanisms. The design and discovery ofdarunavir exploited structure-based design strategies, maximizing drug–enzymeinteractions specifically targeting the backbone of the HIV-1 protease active sitefrom the S2 to S20 subsites. Darunavir exhibited effectiveness against multidrug-resistant HIV-1 variants and showed a high genetic barrier to resistance in clinicaltrials. The design strategy targeting the protein backbone may prove useful fordesigning antiretroviral agents for other disease targets.

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12Protein Kinase Inhibitor Drugs for Targeted Cancer Therapy:Design and Discovery of Imatinib, Nilotinib, Bafetinib,and Dasatinib

12.1Introduction

Protein kinases function in cellular signaling by catalyzing the transfer of thec-phosphate of ATP to an acceptor hydroxyl group belonging to the serine, threo-nine, or tyrosine residues of a substrate protein. The tight regulation of proteinactivity through phosphorylation is necessary to coordinate several important cel-lular processes such as signal transduction, coordination of the cell cycle, and cellgrowth, cell development, and homeostasis. Altered protein kinase activity canlead to deregulation of cellular processes resulting in the development and pro-gression of the cancer [1]. Targeting protein kinases to develop novel kinase inhib-itors has proven to be a useful approach for the development of chemotherapeuticdrugs. The human kinome consists of 418 putative eukaryotic protein kinasegenes and 40 atypical protein kinase genes [2]. Protein kinases can be clustered innine groups, each one consisting of several families and subfamilies. However,the protein kinase domain structure is broadly conserved in all serine/threonineand tyrosine kinases.Tyrosine kinases are one of the most abundantly studied groups of protein kin-

ases. Members of the tyrosine kinase group are involved in the regulation ofnumerous physiological and pathological biochemical pathways. Special attentionhas been devoted to study their role in the pathophysiology of cancer. Tyrosinekinases represent a major portion of all oncoproteins that play a transforming rolein a plethora of cancers. The tyrosine kinase group can be divided into receptortyrosine kinases (RTKs) and nonreceptor tyrosine kinases (NRTKs). RTKs are cellsurface receptors in numerous polypeptide growth factors, cytokines, and hor-mones. NRTKs, on the other hand, are cytoplasmic enzymes that are involved inthe catalytic transfer of a phosphate group from ATP to a tyrosine in proteins.RTKs are localized at the cellular membrane where they respond to extracellular

stimuli. The stimulation of RTK catalytic activity in response to ligand bindinginitiates a downstream intracellular signaling cascade. Some of the most studiedRTKs are the epidermal growth factor receptors (erbB1 and erbB2), the fibroblastgrowth factor receptor 1 (FGFR1), the vascular endothelium growth factorreceptor (VEGFR), the platelet-derived growth factor receptor (PDGFR), and the

271


insulin-like growth factor receptor 1 (IGFR-1). RTKs mediate cell proliferation andsurvival, migration, and metabolism [3]. It is not surprising that deregulation ofRTK function has been implicated in several aspects of tumor progression, includ-ing cell proliferation, survival, angiogenesis, and tumor dissemination [4]. Thestructural organization of RTKs exhibits a multidomain extracellular portion con-veying ligand specificity, a single transmembrane hydrophobic helix, and a cyto-plasmic portion containing the tyrosine kinase domain and regulatory domains.When the extracellular ligand binds the external domain of the RTK, the resultingreceptor dimerization activates the phosphorylase activity of the catalytic domain.The kinase domain recruits several effector proteins and, among them, other kin-ases, such as Src and Abl, that can be, in turn, activated to give rise to the finalcellular response.The Src kinase family is implicated in the regulation of selected cellular signaling

pathways that control cell growth and differentiation, cell migration and adhesion,cell metabolism, and specialized cell signaling networks [5,6]. All of these functionssuggest that Src may be intimately involved in oncogenesis [5,7]. Accordingly, ele-vated Src expression and activation have been associated with several types oftumors, such as breast cancer, non-small cell lung cancer, brain cancer, and manyothers. The Src family consists of nine members: Lyn, Hck, Lck, Blk, Src, Fyn, Yes,Fgr, and Yrk [8]. The Src was the first tyrosine kinase to be discovered [9] and one ofthe most studied among NRTKs. The history of Src discovery can be traced back tothe pioneering work of Peyton Rous who described the oncogenic properties of thevirus bearing his name (Rous sarcoma virus). Later on, studies of v-Src oncogeneand its cellular counterpart c-Src proto-oncogene were followed by the identificationof their protein product Src, the first tyrosine kinase to be characterized [9,10].The Abl kinase family is formed by two members: the Abelson kinase (Abl) and

its paralog Arg [11]. The Abl kinase has been intensively studied since the discov-ery of the so-called Philadelphia chromosome, in which a reciprocal translocationbetween chromosomes 9 and 22 results in a shortened chromosome 22 [12].The translocation causes a head-to-tail fusion of the Bcr and Abl genes formingthe Bcr-Abl oncogene. Splicing at different Bcr breakpoints can form three fusionproteins, differing by the amounts of fused Bcr sequence [13–15]. The three var-iants are associated with distinct types of leukemia: acute lymphocytic leukemia,chronic myelogenous leukemia (CML), and chronic neutrophilic leukemia [15]. TheBcr-Abl fusion protein acts as an oncoprotein because it is constitutively activatedand localized in the cytoplasm. Both Bcr-Abl localization and activation states trig-ger several signaling paths leading to transformation [13]. Some of these pathwaysare normally activated by growth factor receptors such as EGFR and PDGFR.

12.2Evolution of Kinase Inhibitors as Anticancer Agents

The important role of kinases in the control of cell homeostasis resulted in theexploitation of these enzymes as oncologic drug targets. The last 20 years have

272 12 Protein Kinase Inhibitor Drugs for Targeted Cancer Therapy

witnessed a tremendous explosion of novel kinase inhibitors as lead structuresand identification of potential drugs. To date, 25 kinase inhibitors are currentlyFDA approved for the treatment of various forms of malignancies, and severalothers are in advanced clinical trials [16].Several approved anticancer drugs target RTKs. Among them, gefitinib (1) [17],

erlotinib (2) [18], and lapatinib (3) [19] (Figure 12.1) inhibit the erbB family ofkinases (EGFR and erbB2). These kinase inhibitors are used in therapy for thetreatment of non-small cell lung cancer, pancreatic cancer, and breast cancer.Angiogenesis is critical in sustaining growth of a tumor and favors the meta-

static process. Tumors induce angiogenesis through growth factors such as thevascular endothelium growth factor (VEGF), platelet-derived growth factor(PDGF), and fibroblast growth factor (FGF) that interact with the correspondingRTKs. Inhibiting these kinases has proven to be a powerful strategy for the inhibi-tion of angiogenesis and useful for the development of cancer chemotherapies.Sorafenib (4) (initially characterized as a B-Raf inhibitor) [20] and sunitinib (5)[21] are targeting RTKs, approved for the treatment of renal cell carcinoma, hepa-tocellular carcinoma, or gastrointestinal stromal tumor (GIST).A single mutation in the activation loop of the B-Raf kinase results in destabili-

zation of the inactive conformation and formation of a constitutively active

N

N

MeO

ONO HN

F

Cl

Gefitinib (1)

N

N

O

OHN

Erlotinib (2)

N

N

HN Cl

Lapatinib (3)

ONH

O

NH

NH

OO

NNH

OMeCl

F3C

Sorafenib (4)

NH

NH

Me

Me

O

NH

FO

NEt2

Sunitinib (5)

N NH

Cl O

F

F

HN S

Vemurafenib (6)

OO

MeO

MeO

SO2Me

F

Figure 12.1 Structures of FDA-approved protein kinase inhibitors as anticancer drugs.

12.2 Evolution of Kinase Inhibitors as Anticancer Agents 273

enzyme. This mutation is found in several human cancers, especially in melano-mas. Although sorafenib (4) was initially developed as a B-Raf kinase inhibitor, itsclinical efficacy has been mainly related to the inhibition of angiogenesis throughinterference with the VEGFR family of kinase receptors. The first B-Raf kinaseinhibitor vemurafenib (6) (Figure 12.1) was approved by the FDA in 2011 for thetreatment of B-Raf-mutated metastatic melanoma [22]. This drug was discoveredthrough a fragment-based drug discovery approach. Phase I–III clinical trialsrevealed that treatment with vemurafenib results in a very high response rate andimproved survival with low toxicity profile. Since almost 50% of melanoma cellsexpress an activating mutation in B-Raf kinase, a large proportion of affectedpatients will benefit from this newly approved treatment.Imatinib, nilotinib, and dasatinib are inhibitors of the Abl family of kinases and

have been approved for the treatment of hematological malignancies. In particu-lar, both nilotinib and dasatinib have been designed in order to overcome theselection of resistance by imatinib. Nilotinib maximizes drug–target interactionsin order to increase potency against both Bcr-Abl and its mutated forms. Dasati-nib, a dual Abl/Src inhibitor, exploits a different mechanism for overcoming ima-tinib resistance by targeting a different conformation of the enzyme.

12.3The Discovery of Imatinib

During the early 1990s, Baltimore et al. found that the p210Bcr-Abl gene present onthe Philadelphia chromosome is the major reason for the pathophysiology of CMLin most cases. After the discovery of tyrosine kinase-related Bcr-Abl gene in thecells, the subsequent studies started with the identification of kinase inhibitors.After characterization of many compounds, a novel class of phenylaminopyrimi-dine derivatives demonstrating activity against both serine/threonine and tyrosinekinases was identified [23–26]. The evolution of the phenylaminopyrimidine scaf-fold is depicted in Figure 12.2 (7–10). Introduction of a 3-pyridyl moiety at the 30-position of the pyrimidine ring was found to increase cellular activity [26]. Lateron, during optimization, it was discovered that the presence of differently substi-tuted amides at the 3-position of the diaminophenyl moiety increased activityagainst tyrosine kinases (such as Bcr-Abl and PDGFR). A breakthrough discoverywas that a 6-Me group at the diaminophenyl ring, defined as the “flag methyl,” com-pletely abolished inhibition of protein kinase C (PKC) function. At the time of thesestudies, the selective inhibition of Bcr-Abl kinase by compounds presenting the “flagmethyl” was explained, assuming that the presence of the methyl group forced thetwo aromatic rings into a conformation not compatible with the binding to PKC.Further substitution of the phenyl amide moiety was performed to address poor

physicochemical and pharmacokinetic properties of the lead compound. Additionof N-methylpiperazine to the phenyl amide moiety improved both solubility andoral bioavailability, which ultimately led to the development of imatinib (10). Thepiperazine ring, chosen as the solubilizing moiety, was separated from the


aromatic ring by a methylene spacer in order to avoid potential toxicity issues thatwere known to be associated with the presence of aniline moieties [26]. The selec-tivity profile of imatinib is impressive. It is active against Abl, c-KIT, and PDGFRkinases, whereas it does not show activity against a panel of related kinases, asexemplified in Table 12.1. Furthermore, imatinib potently inhibits all of the Abltyrosine kinases such as cellular Abl (c-Abl), viral Abl (v-Abl), Bcr-Abl, and TEL-Abl. The structural basis of imatinib’s selectivity is due to its binding to aninactive conformation of the kinase domain.

12.4Imatinib: the Structural Basis of Selectivity

The X-ray crystal structure of the Abl kinase and imatinib (10) complexrevealed an interesting binding mode of imatinib in the Abl kinase binding site

N

NHN

N

HN

OMe

N

NHN

HN

O

N

NMe

Me

N

NHN

N

HN

O

N

N

NHN

N

The 3-pyridyl moiety was recognized to confer

cellular activity

The arylamide moiety is responsible for increased

activity against TKs

The methyl “flag” was responsible for the

selectivity over PKCs

The piperazine moiety was introduced to increase

water solubility and necessary for improving PK profile

7

PAP scaffold

8

9

10

Imatinib

Figure 12.2 Evolution of the phenylaminopyrimidinyl (PAP) scaffold through rational drugdesign leading to the development of imatinib (10).

12.4 Imatinib: the Structural Basis of Selectivity 275

(Figure 12.3). The structural analysis provided molecular insight into its observedhigh selectivity with respect to other kinases [27,28]. A close-up view of variousbinding interactions is shown in Figure 12.4. The pyridine and pyrimidine ringsof imatinib occupy the region where the adenine ring of ATP normally binds in adeep cleft located between the N- and C-terminal lobes. The adenine ring of ATPforms two hydrogen bonds with the amino acids in the kinase “hinge region,”namely, Met318 and Glu316. In the case of imatinib, the pyridine moiety formsa hydrogen bond with Met318. The amide group of the inhibitor also forms anetwork of hydrogen bonds with Glu286 and Asp381. The hydrogen bonding net-work between the amide group of the inhibitor, amino acids Glu286, Lys271, andAsp381, and two water molecules in the active conformation stabilizes the imati-nib binding.Structural studies have demonstrated that the conformation of the DFG motif

(Asp–Phe–Gly triad) at the start of the A-loop (activation loop), the conformationof the P-loop (phosphate binding or glycine-rich loop), and the position of thegatekeeper residue play an important role in the selectivity profile of imatinib asan Abl, c-KIT, and PDGFR inhibitor. The A-loop controls the catalytic activityof most kinases by switching between different states in a phosphorylation-dependent manner. X-ray structures revealed that the position of the activationloop is not competent for substrate binding and occupies the mouth of the cata-lytic pocket. The position of the activation loop is one of the most importantaspects of the interaction between imatinib and the Abl kinase, since it deter-mines the binding specificity. Asp381 is a part of the conserved DFG motif, whoseconformation strongly differs from the DFG conformation in the active form ofAbl as well as in the closed conformation of Src. The DFG region has the “in” and“out” conformations and inactive Abl has the DFG “out” conformation in whichthe position of the phenylalanine residue is distinct from that in active protein

Table 12.1 Inhibitory potencies of imatinib.

Protein kinasea IC50 (mM)

c-Ablv-AblP210Bcr-Abl

P185Bcr-Abl

TEL-Abl

0.200.038 (0.1� 0.3b)0.250.250.35

EGFR >100c-Src >100PDGFR 0.05PKA >500PKCa >100PKCd >100c-KITb 0.1

a) Data from Refs [23,24].b) Data from Ref. [26].


Figure 12.3 The X-ray crystal structure of the Abl kinase and imatinib complex. Amino acidresidues and ribbons are colored according to kinase subdomains: N-terminal lobe (green),C-terminal lobe (blue), hinge region (gray), and activation loop (magenta) (PDB code: 1IEP).

Figure 12.4 Close-up view of imatinib hydrogen bonds and hydrophobic interactions. Yellowsurfaces are for residues involved in hydrophobic interactions (Tyr253, Leu370, Phe382, Met290,and Ile313). Hydrogen bonds are shown with dotted lines. (PDB code: 1IEP).

12.4 Imatinib: the Structural Basis of Selectivity 277

kinases, as well as in the inactive form of the highly identical Src kinases [27].The movement of the phenylalanine side chain creates a lipophilic channelbeyond Thr315, (the so-called gatekeeper residue) opening an auxiliary bindingsite. Imatinib forms a critical hydrogen bond with Abl Thr315, which isreplaced by a methionine, threonine, or phenylalanine in many other DFG“out” conformation kinases such as Ser/Thr kinases B-Raf, P38 Map kinase,c-KIT, KDR/VEGFR-2, Flt-3, and Irk receptor kinases. However, imatinib doesnot bind to them except c-KIT, which also has a threonine in the gatekeeperposition and shares high homology with Abl in the DFG region. Imatinib’shigh affinity toward PDGFR is presumably because it adopts a conformationsimilar to Abl in the DFG region.Other than hydrogen bonding, there are a number of van der Waals interac-

tions between protein residues (Tyr253, Leu370, Phe382, Met290, and Ile313)and the aromatic rings of imatinib resulting in an exceptional level of surfacecomplementarity. This is aided by an induced fit of the P-loop that folds on theimatinib structure bringing Tyr253 in close contact with the inhibitor. Finally,the piperazinyl moiety increases the solubility of imatinib, but does not alterthe selectivity profile as it lies along a solvent-accessible hydrophobic environ-ment and the distal nitrogen of the piperazine is engaged in hydrogen bondingwith Ile360 and His361.

12.5Pharmacological Profile and Clinical Development

Imatinib has been demonstrated to inhibit active p210Bcr-Abl tyrosine kinase andother Abl fusion proteins such as p185Bcr-Abl and TEL (ETV6)-Abl at the cellularand subcellular levels. The antiproliferative activity of imatinib is directly relatedto inhibition of autophosphorylation of Bcr-Abl. Dose-dependent inhibition oftumor growth has also been demonstrated in mice injected with Bcr-Abl trans-formed cells after i.p. administration of imatinib. Bcr-Abl tyrosine kinase is essen-tial for leukemic cell survival since it exists only in the leukemic cells but not inthe healthy cells; therefore, imatinib works as a target-oriented therapy to combatcancer. Subsequently, imatinib has shown in vivo activity against PDGF-associatedtumors, including glioblastoma, dermatofibrosarcoma protuberans, and myelodys-plastic syndrome. Imatinib has also demonstrated clinical activity against the KIT-associated GIST and in small-cell lung cancer cell lines experimentally. In phase Iclinical trials conducted on patients affected by Philadelphia chromosome-positiveCML, no maximal tolerated dose was identified, whereas complete hematological(blood leukocyte counts returned to normal) and cytogenetic (Philadelphia chro-mosome was no longer found in the patients’ blood cells) responses wereobserved. In subsequent studies, it was found that hematological and cytogeneticresponses were associated with improved survival times. Further clinical trials ledto FDA approval of imatinib for the treatment of CML, GIST, and a number ofother malignancies [26].


12.6Imatinib Resistance

Although hematological and cytogenetic responses with imatinib are positive,these pharmacological responses are usually for shorter time span and mostpatients ultimately develop drug resistance and undergo disease progression. Inaddition, some patients develop resistance to imatinib that is particularly depen-dent on the stage of the disease at which drug treatment is initiated. Resistancecan arise from amplification and overexpression of the Bcr-Abl gene [29–31],activation of pathways of tumor transformation to compensate those inhibited byimatinib (e.g., overexpression of Lyn kinase, a member of the Src family) [32–34],or by point mutations in the kinase domain of the protein [35–43].Mutations responsible for resistance to imatinib usually act through two differ-

ent mechanisms. A direct mechanism is exploited by those mutations within thebinding site that remove favorable interactions with the ligand or create stericclashes due to enlarged side chains (such as Thr315 or Phe317) [36,37]. Thesemutations preserve the ability of the enzyme to bind ATP and catalyze the phos-phorylation reaction. An indirect mechanism of resistance can be defined bythose mutations that exploit the particular binding mode of imatinib. Point modi-fications that destabilize the closed conformation of the P-loop or mutations of theDFG motif are able to decrease the binding affinity of imatinib since the enzymecannot adopt a competent conformation for binding (Glu255, Tyr253, and Gly250)[35,39,42]. For example, Tyr253 forms a water-mediated hydrogen bond withAsn322 side chain, helping to stabilize the folded P-loop conformation andalso increasing surface complementarity of imatinib; however, the mutation atTyr253 removes this interaction and destabilizes the closed conformation of thekinase domain.

12.7Different Strategies for Combating Drug Resistance

12.7.1Nilotinib and Bafetinib: Optimizing Drug–Target Interactions

Nilotinib and bafetinib (the latter is in phase II clinical trials) are two examples ofdrugs discovered through rational, structure-based drug discovery and engineeredto overcome imatinib resistance. From the imatinib-bound Abl kinase X-ray struc-tures [27,28], it was observed that the shape of the phenyl amide group and theorientation of the basic N-methylpiperazine of imatinib were not optimal for fill-ing the hydrophobic pocket. N-Methylpiperazine lies along a partially hydropho-bic, surface-exposed pocket of the Abl kinase, lined by lipophilic residues Val289,Met290, Val299, and Ala380, and is more amenable for further modification. Itwas also known that introduction of the N-methylpiperazine was originallyperformed to increase water solubility and ultimately oral bioavailability, since

12.7 Different Strategies for Combating Drug Resistance 279

it was expected that the piperazine moiety would be extended toward thesolvent-accessible region.In order to find a better fit and alter the piperazinyl moiety within the hydropho-

bic site, Manley et al. [44] initially probed different aliphatic and aromatic hydro-phobic groups linked to the aromatic system by a urea linker. The urea moiety waspredicted to maintain the critical hydrogen bonding interactions with Glu286 andAsp381 via the preferred (E,E)-conformation. Different aliphatic and aromaticgroups were introduced at the urea functionality (Figure 12.5). The IC50 values for

N

NHN

N

HN

HN

O

IC50 = 110 nM

IC50 = 56 nM

IC50 = 52 nM

IC50 = 280 nM

IC50 = > 5000 nM

N

NHN

N

NH

O

N

CF3

N

Me

Nilotinib (16)IC50 < 30 nM

Cl

Me

11N

NHN

N

HN

HN

O

NEt2

Me

12

N

NHN

N

HN

HN

O

NMe2

Me

13N

NHN

N

HN

HN

OMe

14

NMe2

N

NHN

N

HN

HN

O

N

Me

15

Me

Figure 12.5 The urea analogs of imatinib leading to the final discovery of nilotinib (16).


the analogs with isolated Abl revealed that the size and shape of the urea substitu-ent were important for activity. Aromatic rings bearing small substituents at30-position increased the inhibitory activity, whereas the potency of compoundsrapidly decreased when the size or the position of the substituents was altered, asseen in the 3-diethylaminophenyl (12) and 4-dimethylaminophenyl derivatives(14). Inexplicably, the same compounds were unable to maintain their activityagainst Bcr-Abl-catalyzed autophosphorylation in the cellular assay [44].Consequently, the researchers at Novartis Pharmaceuticals rationalized that

based upon the available crystal structures of the imatinib–Abl complex [27,28]and above structural data [44], a more potent, selective, and also effective com-pound against Bcr-Abl imatinib-resistant mutants could be designed by addingalternative binding groups to the N-methylpiperazine moiety, while retaining anamide pharmacophore to preserve hydrogen bonding interactions with Glu286and Asp381 [44]. This rational approach through structure-based design strategiesresulted in the discovery of nilotinib (16) [45,46].Analysis of the X-ray crystal structure of the Abl–nilotinib complex confirmed

the rationale behind the original design [45–47]. The nilotinib (16) binding modeis very similar to that of imatinib (10) with the amide moiety of nilotinib alsofitting similar to imatinib’s phenyl amide in the DFG region (Figure 12.6).

Figure 12.6 Crystal structures of nilotinib (a) and imatinib (b) in complex with the kinasedomain of Abl. (c) An overlap of (a) and (b) highlighting that nilotinib has an increased surfacecontact with the protein residues (PDB codes: 3CS9 and 1IEP, respectively).


The methylimidazole ring and trifluoromethyl group on the phenyl ring are cor-rectly oriented to obtain better topological fit with respect to protein residues inthe hydrophobic pocket, at the same time retaining water solubility and optimalpredicted absorption properties. Nilotinib has 20-fold higher cellular activity forwild-type Bcr-Abl with respect to imatinib, inhibits the majority of Bcr-Abl1mutants, and also maintains a similar selectivity profile [48]. Due to their verysimilar binding modes, it was expected that mutations of Bcr-Abl amino acidswould affect the affinity of both compounds. However, the better fit of the substi-tuted phenyl amide portion results in a lower contribution of the pyridyl and pyr-imidyl rings to the overall binding energy. Consequently, mutations at the hingeregion or at the P-loop decrease the affinity of nilotinib to a lesser degree thanimatinib. Moreover, due to its intrinsic higher potency, it is still able to inhibitmost mutant enzymes at physiologically relevant concentrations. A better thanexpected activity of nilotinib against the M351Tmutant is also observed. AlthoughM351 does not directly contact imatinib or nilotinib, the M351T mutation proba-bly increases the energy required by the enzyme to adopt the imatinib bindingconformation implying an induced fit of the C-lobe. Nilotinib is probably less sen-sitive to this effect since the imidazole moiety has less critical interactions withthe C-lobe. However, T315I mutant is highly sensitive to binding of both nilotiniband imatinib due to lack of hydrogen bonding and introduction of a steric clash.The discovery of bafetinib was also guided by a similar rationale as nilotinib

[49–51]. In order to fill the hydrophobic pocket formed by lipophilic amino acidresidues Ile293, Leu298, and Leu354 around the phenyl ring adjacent to the piper-azinylmethyl moiety of imatinib, the investigators probed a series of small hydro-phobic groups by substituting 3-position of the aromatic amide, while retainingthe piperazinylmethyl moiety (Figure 12.7). Halogen substitutions demonstratedan increase in activity with respect to hydrogen, and the increase in potency wasdirectly correlated with the size of the halogen in the series of F, Cl, and Br. Theideal group was found to be a trifluoromethyl group that was previously utilized inthe development of nilotinib (16). In order to compensate the increased lipophilic-ity caused by the introduction of CF3 group, the investigators replaced the pyri-dine ring of imatinib (10) with a more polar pyrimidine ring. The proximity ofTyr253 to the pyridine ring and its critical role in stabilizing the inactive confor-mation discouraged the introduction of the bulky polar groups at this ring, as theywould likely result in decreased affinity. Furthermore, replacement of the piperazi-nylmethyl moiety with a stereochemically defined 3-dimethylaminopyrrolidine ledto a slightly less potent compound than the corresponding piperazine derivative,but more selective against Bcr-Abl-positive (K562) than Bcr-Abl-negative (U937)cell lines and endowed with activity against Lyn kinase. Lyn is a tyrosinekinase belonging to the Src family whose overexpression has been implied as oneof the mechanisms leading to imatinib resistance [32–34,51,52]. The X-raystructure of the bafetinib–Abl complex confirmed the expected binding mode(Figure 12.8) [50].Bafetinib (18) is a dual inhibitor of Bcr-Abl and Lyn kinases, which exhibited

25–55-fold higher potency in vitro and at least 10-fold higher potency in vivo than


The pyridine ring isclose to bulky Tyr253

Imatinib

Hydrophobic pocketIle293, Leu298, Leu354

Figure 12.7 The X-ray structure of the imatinib–Abl kinase complex showing hydrophobic pocketaround the aromatic ring (Ile293, Leu298, and Leu354) and proximity of Tyr253 side chain to thepyridyl ring (PDB code: 1IEP).


imatinib. Bafetinib has antiproliferative effects against cells bearing wild-type andat least 12–13 mutated Bcr-Abl proteins (including both imatinib- and some dasa-tinib-resistant mutations). However, it does not inhibit phosphorylation of theT315I mutant. Recently, it has also been reported that bafetinib inhibited Bcr-Abl-positive leukemic cell growth in the CNS. Bafetinib has more affinity for Bcr-Ablthan nilotinib, which only targets Bcr-Abl, Lck, and Lyn kinases [53].

12.7.2Dasatinib: Binding to the Active Conformation (the First Example of DualAbl/Src Inhibitors)

Structural data clarifying the mechanism of resistance to imatinib guided thedevelopment of second-generation targeted agents for subsequent clinical investi-gations [54]. Indeed, the structural elucidation of the binding mode of imatinib

Bafetinib

Figure 12.8 X-ray structure of the bafetinib–Abl kinase complex showing the hydrophobic pocketaround phenyl ring filled by the trifluoromethyl substituent (PDB code: 2E2B).


and later of the crystal structure of pyridopyrimidines bound to the active site ofBcr-Abl, suggested that compounds that are capable of binding to the open confor-mation of the Bcr-Abl site would be able to override resistant mutations, thus sta-bilizing the active form of Bcr-Abl or destabilizing the closed conformation.Dasatinib was the second kinase inhibitor approved in 2006 after imatinib. Sinceit poses a less stringent conformational requirement for kinase binding, it is capa-ble of binding the active form of Bcr-Abl that resembles the binding pocket of Srcand related kinases such as Lck (type I inhibitor). The studies that led to the dis-covery of dasatinib [55–58] started with the screening of an internal compoundcollection at Bristol-Myers Squibb (BMS) against Lck kinase, an enzyme belongingto the Src family of tyrosine kinases. After extensive screening, the 2-aminothia-zole hit compound shown in Figure 12.9 was identified as an ATP-competitiveLck inhibitor.The first optimization studies were performed on the 2-aminothiazole moiety at

2-position leading to tert-butyl thiazol-2-ylcarbamates (20) shown in Figure 12.9. Sub-stitution of the benzamide revealed a preference for 2,6-di- or 2,4,6-trisubstitutedaromatic rings with small hydrophobic groups, such as Me or Cl. Exploration of the

HN

OMe

Me

Me

N

S

Me

H2NHN

O

N

S

Me

HN

O

O

R

HN

O

N

S

Me

HN

R = 2,4,6-triMe or, 2-Me, 6-Cl

O

preferred

Cl

Me

IC50 = 35 nM (hLck)

2-aminothiazole hit compound

20

23

19

S

IC50 = 130 nM (hLck)

21

HN

O

N

S

Me

HN

O

Cl

Me

IC50 = 1400 nM (hLck)

(IC50 = 5 μM hLck)

22

HN

O

N

S

H

HN

O

Cl

Me

4

4

Figure 12.9 Hit to lead progression of the 2-aminothiazole scaffold through structure–activityrelationship studies.


2-aminothiazole moiety was accomplished through a parallel synthetic approachleading to the synthesis of several carbamates, ureas, and carboxamides. Amongthem, several potent compounds were identified. As it turned out, this position canaccommodate small aliphatic and aromatic amide moieties, although phenyl and het-erocyclic thienyl moieties were the preferred functionalities. In the tert-butoxycar-bonyl series, removal of the methyl or its modification resulted in a significantdecrease in potency. However, the cyclopropylcarboxamide coupled to the 4-unsubsti-tuted thiazole ring led to a very potent compound 23.Docking studies suggested that the 2-amidothiazole moiety was able to form a

hydrogen bond with Met319, thus mimicking the ATP adenine ring, whereas theamide moiety formed a hydrogen bond with the Thr316 residue (Figure 12.10).This pattern of hydrogen bonding interactions projects the perpendicular2,6-disubstituted aniline rings toward a hydrophobic pocket, nicely filled by thearomatic ring.The conformation required for binding can be easily adopted from compounds

bearing a 4-unsubstituted thiazole ring. However, this conformation could possesa higher energy than the 4-methylthiazole derivative [58]. Consistent with the pro-posed binding mode, benzothiazole 24 shown in Figure 12.10, displayed an

N

O

N

SN

O

H

O

N

H

MeSO

H

OHMe

HN

O

Me

Cl

N

SHN

O

HN

OCl

Me

IC50 = 35 nM (hLck)IC50 = 884 nM (T cell)

IC50 = 9 nM (hLck)IC50 = 4700 nM (T cell)

23

24

Met319

Thr316

Figure 12.10 Proposed kinase inhibitor interactions from docking studies for compound 23 andthe benzothiazole derivative 24.


impressive binding affinity for Lck, although displayed a significant reduction ofcellular activity.A Lck binding site model was constructed by the investigators on the basis of

published crystal structure of the ATP binding site of active Lck and homologymodeling of Hck structure. The binding model outlined the expected hydrogenbonding interactions with the active site residues. However, the cyclopropyl amidecarbonyl occupied in the hydrophobic pocket without any productive hydrogenbonding interactions. The replacement of the carboxamide moiety with a confor-mationally constrained “amide mimetic” pyrimidine led to a significant improve-ment in cellular activity for compound 26 (Figure 12.11). Furthermore, differentpolar side chains at the 200-position of the pyrimidine ring were introduced toimprove the physicochemical and cell permeability properties. After several substi-tutions, 4-hydroxyethylpiperazine-substituted compound 27 turned out to be oneof the most potent Lck inhibitors in the T-cell proliferation assay [57,58].After broad biochemical studies, dasatinib exhibited a potent pan-tyrosine

kinase inhibitor profile against the Src, Bcr-Abl family, and several other kinases(Table 12.2). Dasatinib is 325-fold more potent than imatinib against wild-typeBcr-Abl and as an Src kinase inhibitor, it inhibits all Bcr-Abl kinase domainmutants except T315I mutant. Clinical development of compound 27 ultimatelyled to FDA approval of dasatinib.The X-ray structure of the Abl–dasatinib complex confirmed the predicted bind-

ing mode of the compound to the open conformation of the enzyme [59]. A com-parison of the X-ray structures of Abl, in complex with dasatinib and imatinib,

HN

O

N

SNH

Cl

Me

N

IC50 = 1.2 nM (hLck)IC50 = 140 nM (T cell)

HN

O

N

SNH

Cl

Me

NN

IC50 = 1.0 nM (hLck) IC50 = 80 nM (T cell)

HN

O

N

SNH

Cl

Me

NN

IC50 = 0.4 nM (hLck)IC50 = 3 nM (T cell)

N

Me

Me

Me

NHO

25 26

27 (Dasatinib)

Figure 12.11 Structures and activities of compounds 25–27.


revealed the substantial difference in the binding mode of the two compoundsthat occupy two different sites of the ATP binding cleft (Figure 12.12). A close-up view of the main interactions of dasatinib with the enzyme is displayed inFigure 12.13. Dasatinib forms three hydrogen bonds with Met318 and Thr315 inthe active site of Abl, whereas an ample series of hydrophobic interactions contrib-uted to strengthening the dasatinib binding. Key hydrophobic interactions of dasati-nib are highlighted in Figure 12.13.

Table 12.2 Inhibitory potencies of dasatinib.

Protein kinasea IC50 (nM)

Lck 0.4Src 0.5Fyn 0.2Yes 0.5Bcr-Abl <1Cdk2 >5000Her1 180Her2 710FGFR1 880

a) Data from Ref. [58].

Figure 12.12 Overlap of dasatinib (orange) and imatinib (blue) structures in complex with thekinase domain of Abl. Inhibitors are shown as spheres, and proteins are represented as ribbons(PDB codes: 2GQG (dasatinib) and 1IEP (imatinib)).


12.8Conclusions

Since the FDA approval of imatinib for the treatment of chronic myelogenous leu-kemia, several kinase inhibitors have been developed for the treatment of different

Figure 12.13 X-ray crystal structure of the Ablkinase–dasatinib complex. Subdomains:N-terminal lobe (green), C-terminal lobe (blue),hinge region (gray), activation loop (magenta),and P-loop (orange). (a) Dasatinib in the

kinase domain of Abl. (b) Close-up view show-ing hydrogen bonding and hydrophobic inter-actions; white surfaces for residues involved inhydrophobic interactions (PDB code: 2GQG).

12.8 Conclusions 289

malignancies and several others are currently under clinical evaluation. Structure-based drug design approaches are playing a very important role in optimization ofpotency and selectivity in the kinase inhibitor-based drug discovery process. Ininitial kinase drug discovery projects, lead structures were typically generated byhigh-throughput screening or by using virtual screening. The availability of X-raycrystal structures resulted in the structure-based/fragment-based approaches forlead discovery and lead optimization. Selectivity is a very important issue in kinaseinhibitor design. Toxicity could arise from a lack of selectivity among differentkinases. Future challenges in this field will be to develop tools and strategies toimprove selectivity. Also, the development of allosteric kinase inhibitors, not inter-acting with the ATP binding site, may provide higher specificity and lower toxicityin kinase inhibitor drugs.

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17 Barker, A.J., Gibson, K.H., Grundy, W.,Godfrey, A.A., Barlow, J.J., Healy, M.P.,Woodburn, J.R., Ashton, S.E., Curry, B.J.,Scarlett, L., Henthorn, L., and Richards, L.(2001) Studies leading to the identificationof ZD1839 (IRESSA): an orally active,selective epidermal growth factor receptortyrosine kinase inhibitor targeted to the


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19 Moy, B., Kirkpatrick, P., Kar, S., and Goss,P. (2007) Lapatinib. Nat. Rev. Drug Discov.,6, 431–432.

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21 Sun, L., Tran, N., Tang, F., App, H., Hirth,P., McMahon, G., and Tang, C. (1998)Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class oftyrosine kinase inhibitors that exhibitselectivity toward particular receptortyrosine kinases. J. Med. Chem., 41,2588–2603.

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25 Zimmermann, J., Buchdunger, E., Mett,H., Meyer, T., Lydon, N.B., and Traxler, P.(1996) Phenylamino-pyrimidine (PAP)derivatives: a new class of potent andhighly selective PDGF-receptorautophosphorylation inhibitors.Bioorg. Med. Chem. Lett., 6,1221–1226.

26 Capdeville, R., Buchdunger, E.,Zimmermann, J., and Matter, A. (2002)Glivec (STI571, imatinib), a rationallydeveloped, targeted anticancer drug. Nat.Rev. Drug Discov., 1, 493–502.

27 Schindler, T., Bornmann, W., Pellicena, P.,Miller, W.T., Clarkson, B., and Kuriyan, J.(2000) Structural mechanism for STI-571inhibition of Abelson tyrosine kinase.Science, 289, 1938–1942.

28 Nagar, B., Bornmann, W.G., Pellicena, P.,Schindler, T., Veach, D.R., Miller, W.T.,Clarkson, B., and Kuriyan, J. (2002) Crystalstructures of the kinase domain of c-Abl incomplex with the small molecule inhibitorsPD173955 and imatinib (STI-571). CancerRes., 62, 4236–4243.

29 leCoutre, P., Tassi, E., Varella-Garcia, M.,Barni, R., Mologni, L., Cabrita, G.,Marchesi, E., Supino, R., and Gambacorti-Passerini, C. (2000) Induction of resistanceto the Abelson inhibitor STI571 in humanleukemic cells through gene amplification.Blood, 95, 1758–1766.

30 Mahon, F.X., Deininger, M.W., Schultheis,B., Chabrol, J., Reiffers, J., Goldman, J.M.,and Melo, J.V. (2000) Selection andcharacterization of BCR-ABL positive celllines with differential sensitivity to thetyrosine kinase inhibitor STI571: diversemechanisms of resistance. Blood, 96, 1070–1079.

31 Weisberg, E. and Griffin, J.D. (2000)Mechanism of resistance to the ABLtyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines.Blood, 95, 3498–3505.

32 Donato, N.J., Wu, J.Y., Stapley, J., Gallick,G., Lin, H., Arlinghaus, R., and Talpaz, M.(2003) BCR-ABL independence and LYNkinase overexpression in chronicmyelogenous leukemia cells selected forresistance to STI571. Blood, 101, 690–698.

33 Dai, Y., Rahmani, M., Corey, S.J., Dent, P.,and Grant, S. (2004) A Bcr/Abl-independent, Lyn-dependent form ofimatinib mesylate (STI-571) resistance isassociated with altered expression of Bcl-2.J. Biol. Chem., 279, 34227–34239.

34 Ptasznik, A., Nakata, Y., Kalota, A.,Emerson, S.G., and Gewirtz, A.M. (2004)Short interfering RNA (siRNA) targetingthe Lyn kinase induces apoptosis inprimary, and drug-resistant, BCR-ABL1(þ)leukemia cells. Nat. Med., 10, 1187–1189.

35 Cowan-Jacob, S.W., Fendrich, G.,Floersheimer, A., Furet, P., Liebetanz, J.,Rummel, G., Rheinberger, P., Centeleghe,

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36 Cowan-Jacob, S.W., Guez, V., Fendrich, G.,Griffin, J.D., Fabbro, D., Furet, P.,Liebetanz, J., Mestan, J., and Manley, P.W.(2004) Imatinib (STI571) resistance inchronic myelogenous leukemia: molecularbasis of the underlying mechanisms andpotential strategies for treatment. Mini Rev.Med. Chem., 4, 285–299.

37 Gorre, M.E., Mohammed, M., Ellwood, K.,Hsu, N., Paquette, R., Rao, P.N., andSawyers, C.L. (2001) Clinical resistance toSTI-571 cancer therapy caused by BCR-ABLgene mutation or amplification. Science,293, 876–880.

38 Hochhaus, A., Kreil, S., Corbin, A.S., LaRosee, P., Muller, M.C., Lahaye, T.,Hanfstein, B., Schoch, C., Cross, N.C.,Berger, U., Gschaidmeier, H., Druker, B.J.,and Hehlmann, R. (2002) Molecular andchromosomal mechanisms of resistance toimatinib (STI571) therapy. Leukemia,16, 2190–2196.

39 Roumiantsev, S., Shah, N.P., Gorre, M.E.,Nicoll, J., Brasher, B.B., Sawyers, C.L., andVan Etten, R.A. (2002) Clinical resistance tothe kinase inhibitor STI-571 in chronicmyeloid leukemia by mutation of Tyr-253in the Abl kinase domain P-loop. Proc. Natl.Acad. Sci. USA, 99, 10700–10705.

40 Shah, N.P., Nicoll, J.M., Nagar, B., Gorre,M.E., Paquette, R.L., Kuriyan, J., andSawyers, C.L. (2002) Multiple BCR-ABLkinase domain mutations confer polyclonalresistance to the tyrosine kinase inhibitorimatinib (STI571) in chronic phase andblast crisis chronic myeloid leukemia.Cancer Cell, 2, 117–125.

41 vonBubnoff, N., Schneller, F., Peschel, C.,and Duyster, J. (2002) BCR-ABL genemutations in relation to clinical resistanceof Philadelphia-chromosome-positiveleukaemia to STI571: a prospective study.Lancet, 359, 487–491.

42 Weisberg, E., Manley, P.W., Cowan-Jacob,S.W., Hochhaus, A., and Griffin, J.D.(2007) Second generation inhibitors ofBCR-ABL for the treatment of imatinib-

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43 Corbin, A.S., La Rosee, P., Stoffregen, E.P.,Druker, B.J., and Deininger, M.W. (2003)Several Bcr-Abl kinase domain mutantsassociated with imatinib mesylateresistance remain sensitive to imatinib.Blood, 101, 4611–4614.

44 Manley, P.W., Breitenstein, W., Bruggen, J.,Cowan-Jacob, S.W., Furet, P., Mestan, J.,and Meyer, T. (2004) Urea derivatives ofSTI571 as inhibitors of Bcr-Abl andPDGFR kinases. Bioorg. Med. Chem. Lett.,14, 5793–5797.

45 O’Hare, T., Walters, D.K., Deininger, M.W.,and Druker, B.J. (2005) AMN107: tighteningthe grip of imatinib. Cancer Cell, 7, 117–119.

46 Weisberg, E., Manley, P., Mestan, J.,Cowan-Jacob, S., Ray, A., and Griffin, J.D.(2006) AMN107 (nilotinib): a novel andselective inhibitor of BCR-ABL. Br. J.Cancer, 94, 1765–1769.

47 Manley, P.W., Stiefl, N., Cowan-Jacob, S.W.,Kaufman, S., Mestan, J., Wartmann, M.,Wiesmann, M., Woodman, R., andGallagher, N. (2010) Structuralresemblances and comparisons of therelative pharmacological properties ofimatinib and nilotinib. Bioorg. Med. Chem.,18, 6977–6986.

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49 Asaki, T., Sugiyama, Y., Hamamoto, T.,Higashioka, M., Umehara, M., Naito, H.,and Niwa, T. (2006) Design and synthesisof 3-substituted benzamide derivatives asBcr-Abl kinase inhibitors. Bioorg. Med.Chem. Lett., 16, 1421–1425.

50 Horio, T., Hamasaki, T., Inoue, T.,Wakayama, T., Itou, S., Naito, H., Asaki, T.,Hayase, H., and Niwa, T. (2007) Structuralfactors contributing to the Abl/Lyn dualinhibitory activity of 3-substitutedbenzamide derivatives. Bioorg. Med. Chem.Lett., 17, 2712–2717.


51 Kimura, S., Naito, H., Segawa, H., Kuroda,J., Yuasa, T., Sato, K., Yokota, A., Kamitsuji,Y., Kawata, E., Ashihara, E., Nakaya, Y.,Naruoka, H., Wakayama, T., Nasu, K.,Asaki, T., Niwa, T., Hirabayashi, K., andMaekawa, T. (2005) NS-187, a potent andselective dual Bcr-Abl/Lyn tyrosine kinaseinhibitor, is a novel agent for imatinib-resistant leukemia. Blood, 106,3948–3954.

52 Deguchi, Y., Kimura, S., Ashihara, E.,Niwa, T., Hodohara, K., Fujiyama, Y., andMaekawa, T. (2008) Comparison ofimatinib, dasatinib, nilotinib and INNO-406 in imatinib-resistant cell lines. Leuk.Res., 32, 980–983.

53 Santos, F.P., Kantarjian, H., Cortes, J., andQuintas-Cardama, A. (2010) Bafetinib, adual Bcr-Abl/Lyn tyrosine kinase inhibitorfor the potential treatment of leukemia.Curr. Opin. Invest. Drugs, 11, 1450–1465.

54 Shah, N.P., Tran, C., Lee, F.Y., Chen, P.,Norris, D., and Sawyers, C.L. (2004)Overriding imatinib resistance with a novelABL kinase inhibitor. Science, 305, 399–401.

55 Wityak, J., Das, J., Moquin, R.V., Shen, Z.,Lin, J., Chen, P., Doweyko, A.M., Pitt, S.,Pang, S., Shen, D.R., Fang, Q., deFex, H.F.,Schieven, G.L., Kanner, S.B., and Barrish,J.C. (2003) Discovery and initial SAR of 2-amino-5-carboxamidothiazoles as inhibitorsof the Src-family kinase p56(Lck). Bioorg.Med. Chem. Lett., 13, 4007–4010.

56 Chen, P., Norris, D., Das, J., Spergel, S.H.,Wityak, J., Leith, L., Zhao, R., Chen, B.C.,Pitt, S., Pang, S., Shen, D.R., Zhang, R., DeFex, H.F., Doweyko, A.M., McIntyre, K.W.,Shuster, D.J., Behnia, K., Schieven, G.L.,and Barrish, J.C. (2004) Discovery of novel2-(aminoheteroaryl)-thiazole-5-carboxamides as potent and orally active

Src-family kinase p56(Lck) inhibitors.Bioorg. Med. Chem. Lett., 14, 6061–6066.

57 Lombardo, L.J., Lee, F.Y., Chen, P., Norris,D., Barrish, J.C., Behnia, K., Castaneda, S.,Cornelius, L.A., Das, J., Doweyko, A.M.,Fairchild, C., Hunt, J.T., Inigo, I., Johnston,K., Kamath, A., Kan, D., Klei, H., Marathe,P., Pang, S., Peterson, R., Pitt, S., Schieven,G.L., Schmidt, R.J., Tokarski, J., Wen, M.L.,Wityak, J., and Borzilleri, R.M. (2004)Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Ablkinase inhibitor with potent antitumoractivity in preclinical assays. J. Med. Chem.,47, 6658–6661.

58 Das, J., Chen, P., Norris, D., Padmanabha,R., Lin, J., Moquin, R.V., Shen, Z., Cook, L.S., Doweyko, A.M., Pitt, S., Pang, S., Shen,D.R., Fang, Q., deFex, H.F., McIntyre, K.W., Shuster, D.J., Gillooly, K.M., Behnia, K.,Schieven, G.L., Wityak, J., and Barrish, J.C.(2006) 2-Aminothiazole as a novel kinaseinhibitor template. Structure–activityrelationship studies toward the discovery ofN-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as apotent pan-Src kinase inhibitor. J. Med.Chem., 49, 6819–6832.

59 Tokarski, J.S., Newitt, J.A., Chang, C.Y.,Cheng, J.D., Wittekind, M., Kiefer, S.E.,Kish, K., Lee, F.Y., Borzillerri, R.,Lombardo, L.J., Xie, D., Zhang, Y., andKlei, H.E. (2006) The structure of dasatinib(BMS-354825) bound to activated ABLkinase domain elucidates its inhibitoryactivity against imatinib-resistant ABLmutants. Cancer Res., 66, 5790–5797.

References 293

13NS3/4A Serine Protease Inhibitors for the Treatment of HCV:Design and Discovery of Boceprevir and Telaprevir

13.1Introduction

Hepatitis C virus (HCV), the causative agent of non-A, non-B hepatitis [1], isa positive-stranded RNA virus of the Flaviviridae family. The virus chronicallyinfects more than 170 million people worldwide, causing 8000–10 000 deathseach year in the United States alone [2]. Around 85% of patients infected byHCV develop chronic hepatitis. In 10–20% of these cases, patients developliver cirrhosis over a 20–25-year period and are at increased risk of developinghepatocellular carcinoma [3]. In the mid-1990s, both academic and industriallaboratories put forth extensive efforts to develop HCV protease inhibitors.However, this task met with a number of challenging hurdles: (1) HCV lifecycle was not well established; (2) lack of cell-based assay and validation ofHCV protease inhibition as a mechanism of viral inhibition; and (3) lack ofanimal models for preclinical drug evaluation. Over a decade, the necessarytools were developed for discovery and development of drugs for HCVtherapy.Drugs approved for the treatment of chronic HCV infection are interferon-

alpha (pegylated and conventional) and ribavirin, administered alone or in com-bination. However, these therapies are effective in only about 60% of cases,depending on the genotype of the virus. Also, they require a long duration oftreatment, and can cause serious side effects. Recently, FDA has approveddirect-acting agents for HCV therapy. The initiative for the development ofdirect-acting antiviral therapies emerged due to advances in knowledge of theHCV virus life cycle.Over the years, structure-based design efforts have been directed toward the

development of drugs specifically targeting post-translational processes [4–6]. Thepositive-stranded RNA genome of the HCV virus is formed by a central open read-ing frame (ORF) flanked by 50- and 30-terminal nontranslated regions (NTRs). The50-NTR contains an internal ribosome entry site (IRES) that allows cap-indepen-dent translation of the viral RNA. The ORF encodes for a polyprotein precursor ofaround 3000 amino acids, which is composed of structural and nonstructural (NS)

295


proteins. The structural proteins include the envelope 1 and 2 (E1 and E2) and thecore proteins. The NS proteins include the p7 ion channel, the protease NS2, themultifunctional protease/RNA helicase NS3 along with its protease cofactorNS4A, the NS4B forming the membranous web, the NS5A protein, and the RNA-dependent RNA polymerase NS5B [7,8].The polyprotein precursor is processed by cellular and viral proteases to

release the mature structural and NS proteins. The NS3 enzyme is involved inthe polyprotein maturation together with the NS2 protease. Specifically, NS3cleaves the polyprotein at the following sites: NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B. It has also been demonstrated that the NS3 enzymecleaves critical host proteins involved in natural antiviral defenses [9–11]. Boce-previr (1, Figure 13.1) and telaprevir (2), the only two direct-acting agentsapproved by the FDA for the treatment of HCV, inhibit the protease activity ofthe NS3 enzyme.

13.2NS3/4A Structure

HCV NS3 is a multifunctional enzyme having NTPase, RNA helicase, andprotease activities. The NTPase and helicase activities are localized at the

HN

HN

O

N

O

HN

O

ONH2

O

1 Boceprevir

HN

NH

N

O

O

O

N

N

2 Telaprevir

NHN

O

OHO

O

OHN

O

O

3 BILN 2061 (ciluprevir)

NO HO

O NH

NS

N

MeOHN

Figure 13.1 Structures of FDA-approved HCV NS3 protease inhibitors 1–3.

296 13 NS3/4A Serine Protease Inhibitors for the Treatment of HCV

Figure 13.2 (a) Crystal structure of the NS3holoenzyme complexed with cofactor NS4A(PDB code: 1CU1). Helicase domain: orange;protease domain: green; NS4A cofactor:

magenta. (b) Representation of the NS3/NS4Aprotease domain (PDB code: 1A1R). N-terminallobe: green; C-terminal lobe: blue; structuralZn2þ is represented as a sphere.

13.2 NS3/4A Structure 297

larger C-terminal domain of the protein, whereas the protease domain issmaller and includes N-terminal residues 1–180 (Figure 13.2a) [12]. The crys-tal structure of the holoenzyme shows an autoinhibited form in which theC-terminal residues of the helicase domain occupy the nonprime side ofthe protease domain. The interaction between the two domains does notseem important to the catalytic activity since the two domains can indepen-dently catalyze their enzymatic reactions. In fact, in most studies, evaluationof protease activity and X-ray structural studies of ligand–protease complexesare performed on recombinant proteins comprising the protease domainalone.The first crystal structure of the NS3 protease domain in the presence of

NS4A cofactor was determined in 1996 [13]. The protease has a chymotrypsin-like fold (Figure 13.2b) and is composed of a C-terminal portion encompassingresidues 120–206 (colored in blue) forming six stranded b-barrels and onestructurally conserved a-helix [14]. The N-terminal part (residues 28–119,cyan) is formed by eight b-barrels (one strand is contributed by NS4A cofactor,magenta). The protease domain is fully functional only when the cofactorNS4A is bound to the N-terminal region of the protease via a central hydro-phobic domain spanning residues 21–34 [15–21]. The presence of the cofactorforces the catalytic triad into an enzymatically competent conformation [22,23].The NS4A cofactor is also probably responsible for association with mem-branes and resistance to proteolytic degradation. The active site residues(His83, Asp107, and Ser165) are disposed in the cleft connecting the N- andC-terminal portions. The substrate binding channel is relatively solventexposed and quite shallow.The protease activity of the NS3 enzyme is necessary to process the polyprotein

resulting from RNA translation [24] at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A,and NS5A/NS5B junctions. The first cleavage event is autocatalytic, whereas theothers occur in a trans cleavage site [15,24–29]. The substrate specificity of NS3has been investigated by several groups using different techniques [30–35]. Thecleavage consensus sequence is comprised of 10 amino acid residues and hasbeen determined as the following: (D/E)-X-X-X-X-C+(A/S)-X-X-X, where X is anyamino acid [24]. The S4 � S2

0 subsites are shown in Figure 13.3. Nomenclature ofSchechter and Berger is used in designating the cleavage sites of the substratepeptide.Early kinetic studies on substrate peptides revealed that amino acids on the

prime side give a low contribution to substrate binding compared with aminoacids on the nonprime side [33–35]. However, with the availability of X-raycrystal structures, it was discovered later that subsites at the prime side couldbe exploited for the development of inhibitors spanning the prime to non-prime subsites. The specificity pocket of the enzyme (S1) is able to accommo-date small amino acid residues such as cysteine or threonine since the size ofthe pocket is limited by the presence of a phenylalanine side chain at the bot-tom of the pocket.


13.3Mechanism of Peptide Hydrolysis by NS3/4A Serine Protease

The catalytic mechanism of NS3/4A serine protease is shown in Figure 13.4. Theactive site residues are the Asp81, His57, and Ser139. The histidine residue actsas a base to accept a proton from the serine hydroxyl group. The Asp81 interactswith the His57 NH, allowing the basic nitrogen of imidazole to deprotonate theserine hydroxyl group (Figure 13.4a). The side chain oxygen atom of Ser139 isconsequently able to form a bond with the carbonyl group of the substrate pep-tide. This results in the formation of a tetrahedral intermediate whose negativecharge at the oxygen atom is stabilized by the so-called oxyanion hole formed by

Figure 13.3 (a) Surface representation of HCV protease domain crystal structure (PDB code:2O8M) with subsites colored in orange (S4), pink (S3), green (S2), cyan (S1), purple (S1

0), andbrown (S2 0). (b) P4 �P2 0 sequence of NS3/4A autocatalytic cleavage site.

13.3 Mechanism of Peptide Hydrolysis by NS3/4A Serine Protease 299

the backbone NHs of Ser139 and Gly137 (Figure 13.4b). The tetrahedral inter-mediate subsequently collapses to release the amino-terminal prime side, lead-ing to the formation of a Ser139 acylated intermediate (Figure 13.4c). A watermolecule then hydrolyzes the acylated intermediate, thus completing thereaction cycle (Figure 13.4d).

13.4Development of Mechanism-Based Inhibitors

The presence of a nucleophilic serine in the active site allowed the development ofmechanism-based inhibitors. This strategy of inhibitor design consists of thepreparation of peptide substrates bearing electrophilic “warheads” as active siteserine traps. These electrophilic warheads, such as aldehydes, trifluoroketones,boronic acids, a-ketoacids, or a-ketoamides, are able to form covalent transition-state analogs by reacting with the hydroxyl group of active site serine, resulting incovalent reversible enzyme inhibition. The first series of HCV NS3/4A inhibitorsoften presented peptides bearing electrophilic “warheads” as active site serinetraps [36–46]. Some examples of these early inhibitors (4–7) and the activities ofsome of their “nonelectrophilic” counterparts are shown in Figure 13.5.

Asp81

O-

HN N

His57

O

H O

N

O

H

Ser139

HN

P

O

NH

OGly137

Asp81

O-

HN N

His57

O

HO

N

O

H

Ser139

HO

P

O

NH

OGly137

(a)

(d)

Asp81

OH

N N

His57

O

HO

N

O

H

Ser139

NH

OGly137

(c)

O

H

O

P

Asp81

O- H N N+

His57

O

H

O

N

O

H

Ser139

NH

OGly137(b)

HN

P

O-

Tetrahedralintermediate

NH2

Figure 13.4 Mechanism of peptide hydrolysis reaction catalyzed by NS3/4A serine protease.


The mechanism of formation of the covalent bond between the electrophilic ketoa-cid group and active site serine is depicted in Figure 13.6. Two possible transition-state analogs can be formed based on the geometry of the a-ketoacid when present-ing its carbonyl group to the serine nucleophile. The resulting transition-state analogswithin the active site differ according to the hydrogen bonding pattern that stabilizesthe covalent complex (Figure 13.6).Using mechanism-based inhibitors bearing an a-ketoacid electrophilic “war-

head,” researchers at IRBM (Rome, Italy) obtained the first NMR solution struc-ture of the NS3/4A protease domain bound to inhibitor 6b [47]. This study was

NH

O

F

O

F

OH

OHN

ONH

O

O

HN

OH

O

O

N

ONH

HN

O

O

CO2H

NH

O

CO2H

AcHN

4a IC50 = 17 µM

HN

OH

SH

O

O

N

ONH

HN

O

ONH

O

CO2H

AcHN

5a IC50 = 28 µM

CO2H

HN

O

O

N

ONH

HN

O

ONH

O

CO2H

H2N

5b IC50 = 0.34 µM

CO2H

O

HN

NH

OF

O

F

OH

OHN

ONH

CO2H

O

O

6a IC50 = 1.7 µM IC50 = 0.33 µM6b

NH

OF

O

F

HN

ONH

CO2H

7a, R = CO2H: Ki = 0.01 nM7b, R = H: Ki = 0.5 nM7c, R = OH: Ki = 30 nM

HN

NH

O

PhPhO

CO2H

O

CO2H

AcHN R

HN

CF3

O

O

N

ONH

HN

O

O

CO2H

NH

O

CO2H

AcHN

4b IC50 = 22 µM

Figure 13.5 Peptide inhibitors bearing electrophilic “warheads” (shown in box).

13.4 Development of Mechanism-Based Inhibitors 301

soon followed by the first X-ray structure of the NS3/4A heterodimer complexedwith the same and one structurally related inhibitor 6a [48].Key ligand–protein interactions are shown in Figure 13.7. As can be observed,

the carbonyl group of the a-ketoacid forms a covalent bond with active siteSer139, whereas the rest of the molecule is rather solvent exposed. The oxyanionhole, formed by the backbone NHs of Gly137 and Ser139, stabilizes the carboxyl-ate group of the inhibitor, whereas the oxygen of the a-ketoacid carbonyl forms ahydrogen bond with the imidazole nitrogen of His57. From the X-ray structure, itappears that the observed tetrahedral intermediate results from a si-attack of theserine nucleophile on the carbonyl group (mechanism shown in Figure 13.6). Incontrast, for human serine proteases such as thrombin and trypsin, a re-sideattack of serine on covalent inhibitors is usually observed. Two more hydrogenbond contacts are established by the inhibitor with protein residues, namely, themain chain carbonyl of Arg155 with the NH of the P1 residue and the NH ofAla157 with the P3 residue carbonyl group. The difluoroleucine moiety fills thesmall selectivity pocket S1 (lined by residues Val132, Cys135, Lys136, Gly137,Ser138, Ser139, and Phe154), whereas the side chain of inhibitor leucine interactswith the shallow S2 pocket (lined by residues His57, Asp81, Arg155, and Ala156).In the series of inhibitors developed by IRBM researchers, the use of the difluoro-c-aminobutyric acid as a suitable isostere for the cysteine side chain is quite inter-esting [42,46–49].

Asp81

O-

HN N

His57

O

H O

N

O

H

Ser139

N

OGly137

OO

O-

HAsp81

O-

HN N+

His57

O

HO

O-O O-

O

NH

HN

O

Ser139 Gly137

Attack from the re-side

Asp81

O-

HN N

His57

O

H O

N

O

H

Ser139

N

OGly137

O

O O-

HAsp81

O-

HN N

His57

O

H

O

O

O

O-

H N

OGly137

N

H

OSer139

Attack from the si-side

Figure 13.6 Tetrahedral intermediates resulting from serine attack from the re-side and si-side ofthe a-ketoacid inhibitor.


13.5Strategies for the Development of HCV NS3/4A Protease Inhibitors

A number of key observations guided the initial strategies for the development ofHCV NS3/4A inhibitors. The identification of the long cleavage consensussequence suggested that amino acid residues made only a few specific interac-tions with the binding site of the enzyme. It appeared that preparing high-affinityligands with low molecular weight would be a challenging task. Examination ofthe first crystal structure of the enzyme showed a nearly featureless substratebinding channel. These observations prompted researchers to prepare ligandsthat can enforce the binding through covalent interactions. Electrophilic serinetraps described earlier were explored for the development of covalent inhibitors[36–46].Another key observation for the development of novel inhibitors was that

prime-side cleavage product peptides were competitive inhibitors of theenzyme [38,50]. Researchers consequently focused on optimizing the interac-tions of the inhibitor peptides with the enzyme and also made efforts towardreducing their peptidic character. Researchers at Boehringer Ingelheim utilizedthis approach for the development of HCV NS3/4A protease inhibitors, which

Figure 13.7 Binding mode of covalent inhibitor 6a in the active site of HCV NS3/4A protease.Subsites surfaces colored as follows: cyan (S1), yellow (S2), pink (S3), and orange (S4) (PDBcode: 1DY8).

13.5 Strategies for the Development of HCV NS3/4A Protease Inhibitors 303

finally led to the development of BILN 2061 (3, Figure 13.1). This clinical can-didate demonstrated the ability to reduce HCV loads in human patients, estab-lishing the proof of concept for targeting HCV NS3 protease for anti-HCVtherapy [51].In-depth kinetic studies also played a role in the development of successful

HCV NS3/4A inhibitors. Kinetic studies on substrate peptides suggested thatamino acids in the prime side contributed less compared with amino acids inthe nonprime side [33–35]. However, it was soon discovered that prime-sideresidues could effectively contribute to the binding of inhibitors, thus aidingthe development of high-affinity compounds. Systematic optimization effortsof noncleavable decapeptides spanning the P6 � P4

0 amino acids led to the dis-covery of very potent peptide inhibitors [52,53]. Several research groups pre-pared carboxamide-based inhibitors [54,55]. The introduction of a ketoamidemoiety in place of the cleavable peptide bond of decapeptides also providedpotent, covalent reversible inhibitors [56]. The initial studies that led to thediscovery and development of boceprevir 1 were also based on similar strate-gies [57,58].

13.6Initial Studies toward the Development of Boceprevir

Early on, researchers at Schering-Plough decided to develop NS3/4A mecha-nism-based inhibitors spanning both the prime and nonprime sides of theenzyme substrate binding site [58]. Other research groups have also investi-gated these types of inhibitors [59]. In particular, it was demonstrated that aglycine residue at P1

0 was ideal for binding and that the free carboxylic groupof glycine was able to interact with Lys136 and Arg109, forming salt bridges orhydrogen bonds shown in Figure 13.8. Based upon preliminary data and X-raycrystal structures of hexapeptide-bound inhibitors, researchers at Schering-Plough reasoned that an appropriately placed P2

0 substituent would be able tofurther interact with Arg109 [59]. To test this hypothesis, compounds basedupon a P3 truncated peptide bearing acidic substituents as the P2

0 derivative(such as 8a, Figure 13.8) were prepared [58]. Such compounds could form saltbridges with the positively charged residues Lys136 and Arg109. As it turnedout, dicarboxylic acids such as 8a were not active at concentrations up to100 mM, whereas the corresponding esters partially recovered some inhibitoryactivity. On the other hand, compounds with P2

0 hydrophobic moieties (suchas 8b) were in general more potent. Further exploration of other groupsrevealed that a phenylglycine residue at P2

0 was the best substituent at thisposition, as shown in compound 8c. Combining the phenylglycine at P2

0 withcyclohexylglycine at P3 resulted in further increased potency. Parallel struc-ture–activity relationship studies on the substituent at P2 led to the observationthat leucine gave better results than proline in all inhibitors investigated.


Optimized inhibitor 9 showed a Ki of 66 nM. The X-ray crystal structure of thisinhibitor in complex with NS3/4A was determined. Subsequent structural anal-ysis provided rationale for potency enhancement by the phenylglycine residueat P2 (Figure 13.9).As can be seen in the X-ray structure in Figure 13.9, the side chains of phenyl-

glycine at P20 and norvaline at P1 formed what is termed a “C-clamp” around the

Lys136 side chain [58]. As expected, the crystal structure also revealed the forma-tion of a covalent bond with inhibitor 9 through the electrophilic ketoamide groupwith the active site serine.This X-ray crystal structure also provided insights for further optimization. A

notable feature of the binding mode shown in Figure 13.9 is the close contactmade by the leucine residue at P2 with Arg155. Attempts to amplify the P2–

Arg155 interaction were explored by incorporating larger side chains at P2

[60]. However, as shown in Figure 13.10, it was soon realized that large ringscould not be accommodated at P2, and an inverse correlation was found

HN

O

O

OH

O

P1 H2NLys136

H2N

NH

NH

Arg109

NHN

HN

NH O

OLi

CO2Li

O

O

O

OO

HN 8a, Ki > 100 µM

NHN

HN

NH O

OLiO

O

O

OO

HN 8b, Ki = 15 µM

NHN

HN

NH O

O

O

O

OO

HN 8c, Ki = 2.1 µM

NH2HNHN

HN

NH O

O

O

O

OO

HN 9, Ki = 0.066 µM

NH2

Can form salt bridges or hydrogen bonds with P1' carboxylic acid groups

P2' groups can engage ininteractions with Arg109

and Lys 136

Inhibitor design strategy

Acidic P2'

HydrophobicP2'

P1' P2'

P2'

O

O

O

O

O

O

O

O

Optimization of P2'

(Optimization of P2 and P3)

P1

Figure 13.8 Structures and activities of early inhibitors and discovery of lead inhibitor 9.

13.6 Initial Studies toward the Development of Boceprevir 305

Figure 13.9 X-ray crystal structure of NS3/4A bound to inhibitor 9. The closed surface of theenzyme is colored according to electrostatic potential (PDB code: 2A4G).

HNO

OMe

Me HN

O

HN

O

Me

Me

Me

OO

HN

NHO

NH2

O

10a X = S: Ki = 0.09 µM10b X = O: Ki = 0.30 µM

11a Ki = 0.05 µM

Lys

Arg

HN

O

Me

OO

HN

NHO

OH

O

X

X

HN

O

HN

O

Me

OO

HN

NHO

OH

O

HN

O

HNHN

O

Me

OO

HN

NHO

OH

O

O

OHN

O

O

HN

OHN O

11b Ki = 0.14 µM

O

9 Ki = 0.066 µM

10c X = S: Ki = 0.25 µM10d X = O: Ki = 0.43 µM

HN

O

Me

OO

HN

NHO

OH

O

HN

OHN O

O

X

X

Figure 13.10 Structure-based optimization of inhibitors.


between inhibitory potency and size of the cyclic substituent: five-memberedsaturated rings containing oxygen or sulfur (10a and 10b) were more favorablethan the larger six-membered rings (10c and 10d). The smaller cyclopropylring (11a) slightly improved potency, and the cyclobutane ring (11b) resultedin a lower activity.Subsequently, a cyclopropylalanine residue was also found to be optimal at P1

and the resulting compound 12 (Figure 13.11) displayed a Ki of 15 nM. An overlayof X-ray crystal structures of compounds 12 and 9 in complex with NS3/4A isshown. It is evident that the P1 residue is able to fill in the S1 subsite. Moreover,the cyclopropyl ring at P2 is firmly packed into the enzyme and is in close contactwith Arg155 [60].

Arg155

Lys136

Ki = 0.015 µM

Figure 13.11 Crystal structure of inhibitor 12 in complex with NS3/4A. Protein surface coloredaccording to electrostatic potential (blue: positive; red: negative). Inhibitor 12 (magenta) issuperimposed with inhibitor 9 (cyan) (PDB code: 2A4R (for 12)).

13.6 Initial Studies toward the Development of Boceprevir 307

13.7Reduction of Peptidic Character

Although inhibitors 9 and 12 displayed good enzyme inhibitory potency, they did notexhibit cellular activity in a subgenomic HCV replicon assay. This is possibly due totheir strong peptidic features. The next optimization goal was to reduce their peptidiccharacter [61]. In order to remove or replace peptide bonds without affecting the bind-ing affinity of the inhibitor, the X-ray crystal structure of 9-bound NS3/4A was furtherexamined. As shown in Figure 13.12, compound 9 forms six hydrogen bonds with theenzyme. The P2 carbonyl forms a hydrogen bond with the main chain NH of Ala157,the NH of the P1 residue forms a hydrogen bond with the main chain carbonyl ofArg155, the ketoamide carbonyl is stabilized by contacts with Ser139 and Gly137, andthe P2

0 cap interacts with main chain carbonyl and NH of Thr42. The only residue ofthe inhibitor not engaged in hydrogen bonding interactions was the P2 residue. Con-sequently, inhibitor optimization strategies were focused at the P3–P2 region. Optimi-zation plans are highlighted in Figure 13.12 [61]. Of particular note, compound 12 hasan identical binding mode and hydrogen bonding pattern as compound 9.Initial attempts at replacing P2 residues with different amino acid isosteres were

unsuccessful. As shown in Figure 13.13, compound 13 (Ki¼ 10mM) was the mostpotent among the inhibitors synthesized. The development of azapeptides wasexplored. Isosteric replacement of the a-carbon of 12 with a nitrogen led to the

Figure 13.12 Binding mode of inhibitor 9 in NS3/4A active site. Protein amino acids forminghydrogen bonds with inhibitor: catalytic residues, gray; protein residues, magenta; inhibitor,cyan. Dotted lines represent hydrogen bonds (PDB code: 2A4G).


synthesis of a small set of azapeptides. The most potent azapeptide was compound14, which displayed a loss of potency (Ki¼ 0.23mM). The X-ray crystal structure of theNS3/4A enzyme in complex with 14 revealed that the nitrogen had a planar geometrythat was different from the tetrahedral geometry of the corresponding carbon of 12.Therefore, investigation of this class of compounds was not pursued further.N-Methylation of the peptide bond was then investigated. This led to compound

15 that showed a significant loss of enzyme inhibitory potency. However, cappingthe C-terminal side with a dimethylamino group led to inhibitor 16, which showedcellular potency in the replicon assay. The C-capped inhibitor lacking N-methylsubstituent, however, did not show any activity in the replicon assay [61].

13.8Optimization of P2 Interactions

The discovery that N-methylation of the leucine at P2 was critical not only toachieve enzymatic potency but also for cellular activity led to further investigation

HNO

O

HN

O

HN

O

O HN

ONH

OOH

O

12 Ki = 0.015 µM

not involved in H-bonds

isosteric replacement

OH

NHN

O

O HN

ONH

OOH

O

13 Ki = 10 µM

HNN

HN

O

O HN

ONH

OOH

O

14 Ki = 0.23 µM

azapeptide

NHN

O

O HN

ONH

OOH

O

15 Ki = 0.12 µM

Me

N

O

HN

O

O

HN

O

HN

ONH

ONMe2

O

16 Ki = 0.06 µM IC90 (replicon) = 0.95 µM

Me

replicon activity

O

HN

O

OO

HN

O

OO

HN

O

O

Figure 13.13 Inhibitor optimization strategies at the P3–P2 area.

13.8 Optimization of P2 Interactions 309

of this structural element. This key discovery paved the way for the development ofSCH6 21, as outlined in Figure 13.14 [62]. Compound 17 with 4,4-dimethyl-substi-tuted proline derivative displayed a very potent activity (Ki¼ 36 nM). In order toexploit the proximity of the P2 residue to Arg155 and Ala156, a bulkier tert-butoxygroup at C-4 was introduced. The activity of the resulting inhibitor 18 was

N

O

HN

O

O HN

ONH

ONMe2

O

Me Me

17 Ki = 36 nM

O

HN

N

O

Me

MeMe

18 Ki = 19 nM IC90 = 2.0 µM (replicon)

20 Ki = 16 nM IC90 = 0.35 µM

NHN

O

O HN

ONH

ONMe2

O

Me Me

Ki = 3.8 nM IC90 = 0.10 µM

Possibility of aring from X-ray

NHN

O

O HN

ONH

ONMe2

O

O

HNO

O

N

O

HN

O

O HN

ONH

ONMe2

O

X

Me Me

NHN

O

O HN

ONH

ONMe2

O

Me

Me Me

19a X = O: Ki = 10 nM; IC90 = 1.8 µM19b X = CH2: Ki = 15 nM; IC90 = 0.9 µM

HN O

O

O

HN O

O

O

HN O

O

O

HN O

O

21 (SCH6)

Figure 13.14 Structures and activities of substituted proline-derived inhibitors 17–21.


improved (Ki¼ 19 nM). Both fused tetrahydrofuran and cyclopentane rings wereaccommodated in inhibitors 19a and 19b, respectively. The ring oxygen atom isnot important for activity. All proline derivatives displayed good IC90 in the repli-con assay. Cyclopropyl-fused proline turned out to be the optimal P2 substituent[63]. Compound 20 retained a potent Ki value and showed an improvement inIC90. Combination of the P2-optimized ligand with previously optimized P1 and P3residues provided compound 21 with a Ki of 3.8nM and an IC90 value of 100nM[62]. This compound exhibited selectivity over human neutrophil elastase (HNE).Its antiviral properties were confirmed against the full-length genotype 2a HCVgenome and by other studies. However, its oral bioavailability in rats and monkeyswas too low to proceed for clinical evaluation.The X-ray crystal structure of NS3/4A in complex with inhibitor 21 (SCH6)

was determined [62]. The structure (Figure 13.15) revealed that the gem-dime-thylcyclopropyl moiety of the proline residue engaged in optimal hydrophobicinteractions with Arg155 and Ala156 through its endo-methyl group, whereasthe cyclopropylalanine P1 residue filled the specificity pocket. Inhibitor 21 isinvolved in a number of important hydrogen bonding interactions with thebackbone of Thr42, Arg155, and Ala157, and with the imidazole NH ofHis57. The P2

0 and P1 hydrophobic moieties formed a nice C-clamp aroundLys136.

Figure 13.15 X-ray structure of inhibitor 21 (SCH6) and NS3/4A complex. Catalytic residues:gray; inhibitor: green; hydrogen bond: dotted lines. Surface is colored based on the electrostaticpotential. (PDB Code: 2FM2).

13.8 Optimization of P2 Interactions 311

13.9Truncation Strategy: the Path to Discovery of Boceprevir

The high molecular weight and peptidic character of inhibitor 21 (SCH6) severelyaffected its pharmacokinetic properties. Consequently, the next round of optimizationfocused on the reduction of molecular weight and improvement of cell-based activity,bioavailability, and selectivity over related serine proteases such as HNE. The firstapproach was truncation at the P0 side of 22 (Figure 13.16). Removal of the dimethy-lamide moiety resulted in benzylamide-capped inhibitor 23 with a Ki of 56nM. Trun-cation of both P1

0 and P20 residues to the corresponding primary a-ketoamide 24a

led to only a twofold decrease in potency with respect to 23, but with a significantreduction of molecular weight. Presumably, the NH2 formed hydrogen bonds withresidues in the active site since the corresponding dimethylamine derivative 24b lostits activity. The a-ketoamide derivative 24a displayed good potency in the repliconassay and showed promising results in preliminary pharmacokinetic studies [64].Compound 24a was chosen for further optimization efforts. A systematic explora-

tion of the size, shape, and polarity of the S1 pocket was carried out (Figure 13.17).

HNO

O

N

O

HN

O

O HN

ONH

ONMe2

O

HNO

O

N

O

HN

O

O HN

ONH

O

HNO

O

N

O

HN

O

OOH

O

22 Ki = 10 nM

23 Ki = 56 nM

24a R = H, Ki = 100 nM24b R = Me, Ki = > 13 µM

24c, Ki = 110 nM

HNO

O

N

O

HN

O

ONR2

O

P1' P2'

MeMe

MeMe

MeMe MeMe

Figure 13.16 Truncation strategy leading to a-ketoamide inhibitors 22–24.


25c 400

25i 50

25h 90

25g 400

25f 150

25e 8

25d 25

25b 150

25a 740

Depth of S1

Me

Shape and size of S1

Polarity of S1

O

O

F

F

FCpd P1 Ki (nM)

Cpd P1 Ki (nM) Cpd P1 Ki (nM)

HNO

O

N

O

HN

O

O

NH2

O

Me Me

MeS1

S3 24a Ki = 100 nM

HNO

O

N

O

HN

O

O

NH2

O

26 Ki = 76 nMHNE/HCV = 684Replicon IC90 = 800 nMRat bioavailability (PO) = 28%

MeMe

Improved selectivity over HNE

P3P1

Figure 13.17 Optimization efforts of P1 and P3 leading to inhibitor 26.

13.9 Truncation Strategy: the Path to Discovery of Boceprevir 313

As can be seen, small ring-containing side chains showed promising results. Thecyclobutyl derivative 25e was the most potent. Also, parallel measurement of inhibi-tory activity against HNE revealed that increasing the size of the ring at P1 resultedin an increase of selectivity, the S1 subsite of HNE being smaller. The cyclobutanederivative 25e displayed optimal PK properties with 28% oral bioavailability in rats,whereas the cyclopropyl derivative showed only 3.4% bioavailability. Further replace-ment of cyclobutyl moiety was explored in order to form polar interactions or hydro-gen bond formation in the active site. The corresponding oxetane rings in 25g and25h or a 2,2,2-trifluoroethyl derivative in 25i did not improve potency. The cyclohex-ylglycine residue at P3 was able to confer good potency. However, a tert-butylglycineresidue at P3 became the group of choice since the corresponding compound 26showed selectivity over HNE. Inhibitor 26 was selected for further optimization.Starting from inhibitor 26, optimization of the P3 capping group was investi-

gated. Although modification of the hydrophobic tert-butyl system did not resultin any significant improvement in binding affinity, the isosteric replacement ofthe carbamate oxygen with an NH group resulted in urea derivatives withenhanced inhibitory potency [64]. Urea derivative 1, (Figure 13.18, SCH 503034,boceprevir), containing equal mixture of two diastereomers at P1 showedenhanced potency and selectivity against HNE. The oral bioavailability of bocepre-vir was in the range of 26–34% in rats, mice, and dogs. The X-ray crystal structureof inhibitor 1 bound to NS3/4A protease was determined. As can be seen, thesecond NH group of the urea derivative was able to form an extra hydrogen bondwith the main chain carbonyl of Ala157. This may explain the reason for signifi-cant potency enhancement of urea derivative over the urethane 26.This inhibitor was then selected for clinical development. Boceprevir can be

combined with PEG-IFN-a-2b which results in no modification of its absorptionand/or elimination [6,65–67]. In both phase II and phase III clinical trials, triplecombination therapy (boceprevir/PEG-IFN-a-2b/ribavirin) after a PEG-IFN-a-2b/ribavirin lead-in treatment, resulted in a significantly higher sustained virologicresponse rate with respect to the control groups. Similar sustained virologicresponse rates were observed in both treatment-naïve and treatment-experiencedpatients [6,65]. Boceprevir received FDA approval for the treatment of chronicHCV genotype 1 infection in 2011.

13.10The Discovery of Telaprevir

The development of HCV protease inhibitors started at Vertex Pharmaceuticalsbased upon truncation of a natural NS5A/5B decapeptide substrate 27 (Fig-ure 13.19). During the initial studies, it was observed that nonprime-side residuescontribute more to the affinity than the prime-side residues. Also, it was recog-nized that truncation beyond three or four amino acids is difficult, as substantialhydrophobic contacts are needed to achieve sufficient affinity. As discussed previ-ously, covalent reversible warheads are essential to obtain good potency rather


than inhibitors that solely rely on ionic interactions. Several inhibitors with “ser-ine trap warheads” were optimized either sequentially or in parallel. Initially, alde-hyde warhead surrogates were extensively used in the design of HCV proteaseinhibitors. After several modifications, researchers at Vertex found that hydropho-bic proline-containing hexapeptide aldehyde 28 (Ki¼ 0.89 mM) retained similarHCV protease affinity as decapeptide inhibitor 27 [68]. To reduce the peptidic char-acter, P5 and P6 residues were then replaced with heterocyclic caps. The tetrapep-tide derivative 29 with a pyrazine cap showed an enzymatic Ki of 12 mM. Thisinhibitor was selected for further optimization.

His57

Gln41

Ser139

Gly137

Ala156

Arg155

Ala157

Ki = 76 nM

Ki = 14 nM

Figure 13.18 Optimization of inhibitor structure 26 to boceprevir (1) and X-ray structure ofboceprevir bound to NS3/4A protease (PDB code: 2OC8).

13.10 The Discovery of Telaprevir 315

13.11Simultaneous P1, P10, P2, P3, and P4 Optimization Strategy: the Path to Discoveryof Telaprevir

The structure-based design approach that led to the discovery of telaprevir (2) isoutlined in Figure 13.20. Early optimization efforts were focused on the P2 resi-due. The hydrophobic proline at P2 was important for inhibitor affinity. Subse-quently, attachment of large hydrophobic groups to the proline was explored,particularly ethers, esters, and carbamate-derived substituents. The crystalstructures of lead inhibitors revealed that different hydrophobic orientations ofthe proline substituent have been accommodated in the S2 pocket. It appearedthat amino acids such as Arg181, Asp107, and His83 in the S2 pocket canmake hydrophobic interactions with the P2 substituent of inhibitor. This inves-tigation led to the identification of carbamate-containing tetrahydroisoquinolyl(THIQ) derivative 30, which exhibited similar enzyme inhibitory potency asdecapeptide 27 (Ki¼ 0.89 mM) [68,69].The P1 substituent was then optimized. As the S1 specificity pocket is lined with

amino acids Leu135, Phe154, and Ala157, this pocket only allows incorporation ofrelatively small hydrophobic P1 substituents. The S1 pocket provides excellentselectivity for thrombin, kallikrein, and factor Xa clotting enzymes, since theseenzymes require basic substitution at P1. Although norvaline and trifluoroethyl

NS5A/5B decamer substrate (27)K i = 0.89 µM

Serine attack and cleavage site

NH

HN

NH

HNH2N

O OH

OCO2H

O

O

ONH

HN

NH

O

O

SH

OH

O HN

SCH3

OOH

NH

OOH

O

NH

P4

Hexapeptide aldehyde 28K i = 0.89 µM

Tetrapeptide aldehyde 29K i = 12 µM

P2

S2

HN

O HN

O

HNH O

N

O

O

Ph

O

HO2CNH

ONHAc

OH

HN

O HN

O

HNH O

N

O

O

Ph

O

NN

O

Figure 13.19 Truncation of natural substrate 27 to tetrapeptide aldehyde (29) as HCV inhibitor.


side chains can be accommodated, the former was chosen because of its easiersynthetic accessibility [68]. The initial aldehyde warhead was replaced as it isunstable. A number of warheads such as carboxylic acids, boronic acids, trifluoro-methyl ketones, a-diketones, a-ketoacids, and a-ketoamides were examined. How-ever, replacement with an a-ketoamide provided inhibitor 31 (Figure 13.20) whichshowed up to a 4-fold improvement in binding affinity due to increased interac-tions and a longer half-life [68,70,71].

31K i = 0.22 µMIC50 = 0.31 µM (replicon)

P4

Cap

30K i = 0.89 µM

HN

O HN

O

HNH O

N

O

O

N

O

NN

O

HN

O HN

O

NH O

N

O

O

N

O

NN

O

O

HN

P1'

P1

P2

32K i = 0.15 µMIC50 = 0.45 µM (replicon)

HN

O HN

O

NH O

N

OO

NN

O

HN

HN

O HN

O

NH O

N

OO

NN

O

HN

CO2H

O

Ph

33K i = 40 nM

K i = 44 nMHNE/HCV >500Replicon IC50 = 350 nMCC50 = 86.5 µMRat bioavailability (PO) = 25%

2 (Telaprevir)

HN

O HN

O

NH O

N

OO

NN

O

HN

P3

Figure 13.20 Landmarks in the design and discovery of telaprevir 2.

13.11 Simultaneous P1, P10, P2, P3, and P4 Optimization Strategy 317

Further modifications at the P10, P3, and P4 with 3-alkylated proline led to inhib-

itor 32 with comparable potency to inhibitor 31. However, inhibitor 32 exhibitedsignificantly improved pharmacokinetic profiles [72]. Inhibitors with cyclopropylketoamides at P1

0, tert-butyl at P3, and cyclohexane side chain at P4 exhibited goodenzyme inhibitory and cellular potency. Incorporation of a bicyclic ketone at P2

resulted in inhibitor 33 with an enzyme Ki of 40 nM. Removal of the ketone func-tionality and incorporation of structural features (P3, P4, and P1

0) to inhibitor 32provided inhibitor 2 (telaprevir) as a potent HCV NS3/4A serine protease inhibitor[69,73,74].X-ray crystal structure of the telaprevir and NS3/4A complex was determined

(Figure 13.21) [75]. Telaprevir (2) forms a tight complex with HCV NS3/4A prote-ase by forming a reversible covalent bond between the hydroxyl group of catalyticSer139 and the carbonyl group of the ketoamide warhead of telaprevir. In addi-tion, two hydrogen bonds are formed between NH groups of the amino acidsGly137 and Ser139 with the amide carbonyl group of telaprevir. It also formshydrogen bonds, like boceprevir with the protease backbone, such as the P1 NHwith the carbonyl oxygen of Arg155, P3 carbonyl oxygen with NH of Ala157, andP3 NH with the carbonyl oxygen of Ala157. The carbonyl of pyrazine forms anadditional hydrogen bond with the OH and NH of Ser159.Telaprevir inhibits HCV NS3/4A protease by a reversible covalent inhibition

mechanism. It may risk binding to other serine proteases. However, telaprevirshowed excellent selectivity (>500-fold) against thrombin, chymotrypsin, trypsin,plasmin, and kallikrein [74]. Telaprevir shows similar potency against HCV NS3/4A protease genotypes 1 and 2, but less activity against genotype 3 protease in

Figure 13.21 Binding mode of telaprevir (2) at the binding site of HCV NS3/4A protease (PDBcode: 3SV6).


enzyme assay. It also exhibited safety and efficacy profiles similar to boceprevir. In2011, FDA approved telaprevir as a direct-acting antiviral drug for the treatment ofthe hepatitis C virus infection [76].

13.12Conclusions

Both boceprevir and telaprevir are serine protease inhibitors approved for thetreatment of HCV infections. Prior to the discovery of these drugs, the develop-ment of drug-like inhibitors against serine proteases was considered extremelychallenging. However, structure-based design guided the successful discovery andoptimization of these inhibitor drugs. The therapeutic efficacy of NS3/4A proteaseinhibitors is currently challenged by the development of resistant viral strains.Another major clinical need regarding NS3/4A protease inhibitors is the develop-ment of inhibitors with a broad specificity and effective against various genotypesof the virus. Recently, structure-based drug design approaches have been exploitedfor the development of new drugs which are active against the main resistantmutants as well as inhibitors with a broader specificity for different genotypes.Several inhibitors are currently under clinical evaluation with promising results.

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9 Seth, R.B., Sun, L., Ea, C.K., and Chen, Z.J.(2005) Identification and characterization ofMAVS, a mitochondrial antiviral signalingprotein that activates NF-kappaB and IRF 3.Cell, 122, 669–682.

10 Li, K., Foy, E., Ferreon, J.C., Nakamura, M.,Ferreon, A.C., Ikeda, M., Ray, S.C., Gale,M., Jr., and Lemon, S.M. (2005) Immuneevasion by hepatitis C virus NS3/4Aprotease-mediated cleavage of the Toll-likereceptor 3 adaptor protein TRIF. Proc. Natl.Acad. Sci. USA, 102, 2992–2997.

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14Proteasome Inhibitors for the Treatment of Relapsed MultipleMyeloma: Design and Discovery of Bortezomib and Carfilzomib

14.1Introduction

The ubiquitin-proteasome system is a biochemical pathway that plays a crucialrole in protein turnover in all eukaryotic cells. This system targets proteins thatare misfolded, damaged, or no longer needed by the cell for degradation. Ubiqui-tin is a 76-amino acid tag that is attached to lysine residues of the target protein.Following tagging, the target protein is further polyubiquitinated. Four enzymefamilies play a role in the ubiquitination process: E1, which activates ubiquitin inan ATP-dependent reaction; E2, which serves as a ubiquitin carrier protein; E3,which transfers and conjugates ubiquitin from the E2–ubiquitin complex to thetarget protein; and E4, which assembles the ubiquitin chain. Polyubiquitinatedproteins are then directed to the 26S proteasome that degrades them into smallpeptides [1–4]. Inhibition of the 21S proteasome may serve as a target for cancerchemotherapy due to the anomalous effects in several cell signaling pathwayscaused by an abnormal ubiquitin-proteasome system [5,6]. The introduction ofbortezomib, a first-in-class proteasome inhibitor currently approved for the treat-ment of multiple myeloma or mantle cell lymphoma, validated this concept [7,8].The 26S proteasome is a protein complex formed by the 20S core particle (CP)

and two regulatory domains (19S). In vertebrates, three different 20S CPs exist:the thymoproteasome (tCP), the immunoproteasome (iCP), and the constitutiveproteasome (cCP); the latter is found in almost all tissues. The determination ofthe first crystal structure of a 20S proteasome from Thermoplasma acidophilum (aprokaryote) [9] revealed a cylindrical shape for this enzyme, which is formed by 28subunits (Figure 14.1). The subunits are assembled into four circles: each of thetwo inner circles is formed by seven b-subunits, whereas each of the two externalcircles is formed by seven a-subunits. In eukaryotic CPs, the same arrangementof subunits is found [10], with the main difference being that seven differenta-subunits (a1–a7) and seven different b-subunits (b1–b7) comprise the circles.The catalytic centers are located in the central channel of the cylinder, and ineukaryotes, only subunits b1, b2, and b5 present catalytic activity. Each subunithas an S1 specificity pocket that regulates substrate preference [11,12].

325


14.2Discovery of Bortezomib

The proteasome is an endoprotease that cleaves peptide bonds of the target proteinthrough the nucleophilic OH of the N-terminal threonine residue of each catalyti-cally active b-subunit. As described in Chapter 4, the initial acyl-enzyme intermedi-ate that is formed is subsequently cleaved by a nucleophilic water molecule torelease the C-terminal end of the hydrolyzed peptide [9,10,13]. The X-ray crystalstructure of aldehyde inhibitor N-acetyl-Leu-Leu-norleucinal (1, Figure 14.2) revealedthat the aldehyde group binds covalently with the Thr1 residue, forming a tetrahe-dral hemiacetal. The norleucine side chain fills the S1 specificity pocket and a hydro-gen bond is formed between residue Gly47 and the peptidic moiety of the inhibitor.The studies that led to the discovery of bortezomib started with the known bind-

ing mode of aldehyde inhibitor 2 and were aimed at increasing potency and selec-tivity. Structure–activity relationship studies revealed that leucine was thepreferred residue at P1, whereas potency of the aldehyde inhibitors could beincreased by introducing large hydrophobic groups at both P2 and P3 (inhibitors 3and 4, Figure 14.2). Although they have high potency, both inhibitors 3 and 4 pre-sented drawbacks regarding their possible development as drug candidates. First,their tripeptidic nature hindered intracellular penetration. Additionally, the pres-ence of the aldehyde warhead resulted in stereochemical liability of the adjacentstereogenic center, and selectivity over cysteine and serine proteases presented anissue due to the ability of aldehydes to react with the active site residues of both

Figure 14.1 Front (a) and (b) upper view of the 20S proteasome. a-Subunits are colored inmagenta (upper and lower rings), whereas b-subunits are colored in blue (central rings) (PDBcode: 1RYP).

326 14 Proteasome Inhibitors for the Treatment of Relapsed Multiple Myeloma

these classes of proteases. In subsequent studies, dipeptidyl derivatives wereinvestigated bearing different electrophilic warheads. In particular, both chloro-methyl or trifluoromethyl ketones (5 and 6, respectively, in Figure 14.3) provedineffective, whereas boronic acid 7 led to a potent inhibitor.The improved potency of 7 derives from the ability of boronates to form a tetra-

hedral intermediate by reacting with the hydroxyl group of Thr1. Moreover, thisintermediate is stabilized by the formation of a hydrogen bond with the NH ofthe oxyanion hole formed by residue Gly47 and the amino group of Thr1. Com-pounds such as 7 are also more selective because they lack P3 and P4 residues topromote interactions with other classes of proteases, such as chymotrypsin and

1

HN

NH

HN

H

O

Me

O

OMe

O

2 Ki = 6 nM

HN

NH

HN

H

O

O

OO

O

3 Ki = 0.24 nM

HN

NH

HN

H

O

O

OO

O

4 Ki = 0.015 nM

HN

NH

HN

H

O

O

OO

O

Figure 14.2 Chemical structures of aldehyde proteasome inhibitors 1–4.

5 Ki = 22000 nM

NH

HN

O

O

OCl

6 Ki = 1400 nM

NH

HN

O

O

O

CF3

7 Ki = 0.03 nM

NH

HN B

O

O OH

OH

HN

Cbz

HN

Cbz

HN

Cbz

Figure 14.3 Structures and activities of proteasome inhibitors 5–7 bearing different electrophilicwarheads.

14.2 Discovery of Bortezomib 327

elastase. Further optimization of 7 was accomplished through the synthesis ofinhibitors 8 and 9 (bortezomib), bearing a pyrazine as the N-capping group andlarge hydrophobic residues at P2 (Figure 14.4).The X-ray crystal structure of bortezomib in complex with the yeast 20S protea-

some showed that, at elevated inhibitor concentrations used for cocrystallizationstudies, it binds to all three active sites of the 20S proteasome [14]. Bortezomibhas different inhibitory potencies against the three enzymatically active siteslocated at the b1-, b2-, and b5-subunits (Figure 14.5). The inhibitor has higherpotency against the b5-subunit, which has chymotryptic-like activity, intermediatepotency against the b1-subunit, which has caspase-like activity, and very lowpotency against the b2-subunit, which has tryptic-like activity.The X-ray structure of bortezomib with 20S proteasome was determined

to obtain insight into the specificity and ligand–binding site interactions [14].

8 Ki = 0.18 nM

NH

HN B

O

O OH

OHN

N

9 Ki = 0.62 nM, Bortezomib

NH

HN B

O

O OH

OHN

N

Figure 14.4 Structures of boronate proteasome inhibitors 8 and 9 (bortezomib).

Figure 14.5 Inner ring of the 20S proteasome from yeast. Enzymatically active b-subunits, blue(b1, b2, and b5); enzymatically inactive b-subunits, cyan; bound inhibitors, magenta spheres(PDB code: 2F16).


Figure 14.6 shows critical interactions between the boronate warhead and the cata-lytically active Thr1 in the active site. As can be seen, the boron atom forms acovalent bond with the nucleophilic oxygen of the Thr1 hydroxyl group. TheGly47 backbone NH appears to stabilize the oxyanion hole through hydrogenbonding with the boronate OH group. Moreover, the Thr1 backbone NH forms atight hydrogen bond with the boronate OH group. The peptide skeleton of theinhibitor forms several hydrogen bonds with key residues. From the illustratedbinding mode, the key role of the pyrazine moiety emerges as it forms a hydrogenbond with Thr22.The leucine side chain of the inhibitor fills the S1 specificity pocket lined by resi-

dues Thr20, Thr31, and Ala49 of the b1-subunit (Figure 14.7). Analysis of the struc-ture shows that bortezomib binds by inducing a fit to Met45 of the b5-subunit.A comparison of bortezomib-bound structure with the structure of native unligatedproteasome shows that the inhibitor’s P1 side chain caused a shift of the Met45side chain. This shift resulted in an enlargement of the S1 specificity pocket.Bortezomib was approved by the FDA in 2003 as a first-in-class proteasome

inhibitor. It showed efficacy for the treatment of multiple myeloma, validating therole of proteasomes as drug targets. However, the drug is administered by intra-venous route and has serious side effects, such as thrombocytopenia and neutrope-nia. Moreover, in 30% of cases, it exerts reversible neurodegenerative effects thatare related to off-target activity [15]. Recently, a large proportion of nonresponsive-ness in newly diagnosed patients and a high frequency of relapses have also beenreported [16,17].

Figure 14.6 X-ray crystal structure of bortezomib with 20S proteasome from yeast. The keyhydrogen bonding interactions in b1-subunit are highlighted (PDB code: 2F16).

14.2 Discovery of Bortezomib 329

14.3Discovery of Carfilzomib

A second proteasome inhibitor, carfilzomib, has been approved by the FDA in2012 for the treatment of patients who are affected by multiple myeloma and didnot respond to at least two therapies. The history of the discovery of carfilzomibstarts from an epoxide-containing tetrapeptidic natural compound named epoxo-micin (10, Figure 14.8).

Figure 14.7 X-ray crystal structure of bortezomib with 20S proteasome from yeast. The occu-pancy of bortezomib in the specificity pocket S1 is highlighted (PDB code: 2F16).

NMe

O

Me

NH

OHN

OOH

NH

O

OMeO

10 Epoxomicin Me

HO NH2

O

MeO

Me

NH2

O

OHO

Thr1

Morpholinecovalent adduct

NMe

O

Me

NH

O HN

OOH

NH

O

NHO

Me O

MeOH

HO

Figure 14.8 Structure of epoxomicin and mechanism of inhibition of the proteasome.


Epoxomicin was discovered by Bristol-Myers Squibb in Tokyo for its antitumoractivity against melanoma [18,19]. Later, Crews and coworkers investigated themechanism of action of epoxomicin and discovered that it was linked to the inhi-bition of the proteasome [20,21]. The X-ray crystal structure of the proteasome–epoxomicin complex [22] revealed that the natural compound inhibited theenzyme by forming a covalent bond, resulting in the formation of a morpholinering. This six-membered ring is formed in two steps, as illustrated in Figure 14.8.The first step involves the nucleophilic attack of the Thr1 oxygen on the carbonylgroup, forming a tetrahedral hemiacetal intermediate. In the second step, the pri-mary amino group of Thr1 performs a second nucleophilic attack toward the epox-ide ring, resulting in its opening and the formation of the morpholine ring. Thismechanism of inhibition also explained the exquisite selectivity of this naturalcompound for the proteasome. The mechanism of formation of the morpholinering is only possible for enzymes in which the catalytic threonine residue has afree terminal amino group. Most serine proteases do not have this peculiarfeature.Using epoxomicin as the lead compound, optimization studies were performed

following a classical structure–activity relationship approach in which the variouspositions of the tetrapeptidic natural compound were investigated, leaving intactthe reactive electrophilic a0,b0-epoxyketone of the natural compound [23]. Asshown in Table 14.1, all-leucine tetrapeptide 11 showed good potency in inhibitingthe chymotrypsin-like activity of the proteasome. The corresponding tripeptidederivative 12 was significantly less potent.

Table 14.1 Structure and inhibitory potency of proteasome inhibitors 10–12.

Compound Kobs/[I] (M�1 s�1)

HNMe

ONH

OHN

ONH

O

OMe

O

11

14 000

Me NH

OHN

ONH

O

OMe

O

12

780

NMe

O

Me

NH

OHN

OOH

NH

O

OMeO

10Epoxomicin

37 000

14.3 Discovery of Carfilzomib 331

Investigation of different substituents at P2 and P3 (inhibitors 13–16) revealedthat bulky aromatic groups were tolerated. As can be seen in Table 14.2, bothphenylalanine and naphthylalanine derivatives were investigated. It appeared thatphenylalanine (derivative 13) is preferred at P2. At P3, naphthylalanine (derivative16) turned out to be the preferred residue. Phenylalanine derivative 15 exhibitedsubstantial loss of potency.



HN

NH

O

OMe

O

13

Ac-Leu-Leu

P2

54 000

HN

NH

O

OMe

O

14

Ac-Leu-Leu

29 000

NH

HN

ONH

O

OMe

O

15

Ac-Leu

P3 8500

NH

HN

ONH

O

OMe

O

16

Ac-Leu

31 000


Exploration of side chain functionalities is shown in Table 14.3. Aromatic sub-stituents such as phenylalanine (17) or naphthylalanine (18) were also favorablefor activity when placed at P4. However, the phenethyl side chain provided themost potent inhibitor 19 in this series.Combining the best groups at the P2 and P4 positions led to optimized inhibitor

20, which showed increased potency over the reference natural compound epoxo-micin. However, the major drawback of this compound was its poor water solubil-ity. To improve solubility, a morpholine ring was introduced as the capping group,leading to carfilzomib (21, Figure 14.9). In clinical trials, carfilzomib showed effi-cacy in the treatment of multiple myeloma in heavily pretreated patients [24]. Car-filzomib is administered by intravenous route and is rapidly cleared from plasma(half-life <1 h). It is mainly metabolized by hydrolases to form inactive fragments,whereas the cytochrome P450 system does not seem to be involved in its metabo-lism [24,25]. One of the notable features of carfilzomib, with respect to bortezo-mib, is the lack of peripheral neuropathic side effects. In fact, carfilzomib is moreselective than bortezomib. However, the latter drug also inhibits serine proteaseHtrA2/Omi, which is expressed in neurons [15].



HNMe

ONH

O

NH O

Me

O

17

Leu-Leu

P437 000

HNMe

ONH

O

NH O

Me

O

18

Leu-Leu

29 000

HNMe

ONH

O

NH O

Me

O

19

Leu-Leu

63 000

14.3 Discovery of Carfilzomib 333

14.4Conclusions

Both bortezomib and carfilzomib are protease inhibitors characterized by the pres-ence of functional groups able to react covalently with the active site threonine (aboronic acid and an epoxyketone moiety, respectively). Both these functionalitieswere not previously exploited in medicinal chemistry, mainly due to their highreactivity. Structure-based drug design played a key role in optimizing the activityand drug-like properties of the initial lead compounds. The discovery of carfilzo-mib represents another exciting journey of isolation, characterization, and biologi-cal studies of a natural product to its development into a clinically approved drug.Once the target of the natural compound was discovered, the presence of an epox-yketone moiety was initially seen as a drawback for its prospective clinical applica-tion since it posed several potential liabilities such as high reactivity, poorselectivity, toxicity, and so on. However, in-depth study of its mechanism of inter-action with the enzyme revealed its intriguing selectivity and the mode of action ofthis molecular fragment. The discovery and development of carfilzomib furtherattests to the fact that nature is an irreplaceable source of new lead structures withnovel mechanisms.

References

1 Reinstein, E. and Ciechanover, A. (2006)Narrative review: protein degradation anddiseases: the ubiquitin connection. Ann.Intern. Med., 145, 676–684.

2 Micel, L.N., Tentler, J.J., Smith, P.G.,and Eckhardt, G.S. (2013) Role ofubiquitin ligases and the proteasomein oncogenesis: novel targets for

HN

O

NH

O HN

ONH

O

OMeO

21 Carfilzomib

NO

HNMe

O

NH

O HN

ONH

O

OMeO

20 Kobs/[I] (M-1s-1) = 166000IC50 = 20 nM

(IC50 = 6 nM)

Figure 14.9 Chemical structures of compound 20 and carfilzomib.


anticancer therapies. J. Clin. Oncol., 31,1231–1238.

3 Hershko, A. (2005) The ubiquitin systemfor protein degradation and some of itsroles in the control of the cell divisioncycle. Cell Death Differ., 12,1191–1197.

4 Dammer, E.B., Na, C.H., Xu, P., Seyfried,N.T., Duong, D.M., Cheng, D., Gearing,M., Rees, H., Lah, J.J., Levey, A.I., Rush, J.,and Peng, J. (2011) Polyubiquitin linkageprofiles in three models of proteolyticstress suggest the etiology of Alzheimerdisease. J. Biol. Chem., 286, 10457–10465.

5 Orlowski, R.Z. and Kuhn, D.J. (2008)Proteasome inhibitors in cancer therapy:lessons from the first decade. Clin. CancerRes., 14, 1649–1657.

6 Nencioni, A., Grunebach, F., Patrone, F.,Ballestrero, A., and Brossart, P. (2007)Proteasome inhibitors: antitumoreffects and beyond. Leukemia, 21,30–36.

7 Richardson, P.G., Mitsiades, C.,Schlossman, R., Ghobrial, I., Hideshima,T., Munshi, N., and Anderson, K.C. (2008)Bortezomib in the front-line treatment ofmultiple myeloma. Expert Rev. AnticancerTher., 8, 1053–1072.

8 O’Connor, O.A. and Czuczman, M.S.(2008) Novel approaches for the treatmentof NHL: proteasome inhibition andimmune modulation. Leuk. Lymphoma, 49(Suppl. 1), 59–66.

9 Lowe, J., Stock, D., Jap, B., Zwickl, P.,Baumeister, W., and Huber, R. (1995)Crystal structure of the 20S proteasomefrom the archaeon T. acidophilumat 3.4 A

�resolution. Science, 268,

533–539.10 Groll, M., Ditzel, L., Lowe, J., Stock, D.,

Bochtler, M., Bartunik, H.D., and Huber, R.(1997) Structure of 20S proteasome fromyeast at 2.4A

�resolution. Nature, 386,

463–471.11 Kish-Trier, E. and Hill, C.P. (2013)

Structural biology of the proteasome. Annu.Rev. Biophys., 42, 29–49.

12 Huber, E.M. and Groll, M. (2012)Inhibitors for the immuno- andconstitutive proteasome: current and futuretrends in drug development. Angew. Chem.,Int. Ed., 51, 8708–8720.

13 Seemuller, E., Lupas, A., Stock, D., Lowe, J.,Huber, R., and Baumeister, W. (1995)Proteasome from Thermoplasmaacidophilum: a threonine protease. Science,268, 579–582.

14 Groll, M., Berkers, C.R., Ploegh, H.L.,and Ovaa, H. (2006) Crystal structureof the boronic acid-based proteasomeinhibitor bortezomib in complex withthe yeast 20S proteasome. Structure, 14,451–456.

15 Arastu-Kapur, S., Anderl, J.L., Kraus, M.,Parlati, F., Shenk, K.D., Lee, S.J.,Muchamuel, T., Bennett, M.K., Driessen,C., Ball, A.J., and Kirk, C.J. (2011)Nonproteasomal targets of the proteasomeinhibitors bortezomib and carfilzomib: alink to clinical adverse events. Clin. CancerRes., 17, 2734–2743.

16 Kuhn, D.J., Orlowski, R.Z., and Bjorklund,C.C. (2011) Second generation proteasomeinhibitors: carfilzomib andimmunoproteasome-specific inhibitors(IPSIs). Curr. Cancer Drug Targets, 11,285–295.

17 Kumar, S. and Rajkumar, S.V. (2008)Many facets of bortezomib resistance/susceptibility. Blood, 112,2177–2178.

18 Hanada, M., Sugawara, K., Kaneta, K.,Toda, S., Nishiyama, Y., Tomita, K.,Yamamoto, H., Konishi, M., and Oki, T.(1992) Epoxomicin, a new antitumor agentof microbial origin. J. Antibiot. (Tokyo), 45,1746–1752.

19 Kim, K.B. and Crews, C.M. (2013) Fromepoxomicin to carfilzomib: chemistry,biology, and medical outcomes. Nat. Prod.Rep., 30, 600–604.

20 Sin, N., Kim, K.B., Elofsson, M., Meng, L.,Auth, H., Kwok, B.H., and Crews, C.M.(1999) Total synthesis of the potentproteasome inhibitor epoxomicin: a usefultool for understanding proteasomebiology. Bioorg. Med. Chem. Lett., 9,2283–2288.

21 Meng, L., Mohan, R., Kwok, B.H., Elofsson,M., Sin, N., and Crews, C.M. (1999)Epoxomicin, a potent and selectiveproteasome inhibitor, exhibitsin vivo antiinflammatory activity.Proc. Natl. Acad. Sci. USA, 96,10403–10408.

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22 Groll, M., Kim, K.B., Kairies, N., Huber, R.,and Crews, C.M. (2000) Crystal structure ofepoxomicin: 20S proteasome reveals amolecular basis for selectivity ofa0,b0-epoxyketone proteasome inhibitors.J. Am. Chem. Soc., 122, 1237–1238.

23 Elofsson, M., Splittgerber, U., Myung, J.,Mohan, R., and Crews, C.M. (1999)Towards subunit-specific proteasomeinhibitors: synthesis and evaluation ofpeptide alpha0,beta0-epoxyketones. Chem.Biol., 6, 811–822.

24 McCormack, P.L. (2012) Carfilzomib: inrelapsed, or relapsed and refractory,multiple myeloma. Drugs, 72,2023–2032.

25 Demo, S.D., Kirk, C.J., Aujay, M.A.,Buchholz, T.J., Dajee, M., Ho, M.N., Jiang,J., Laidig, G.J., Lewis, E.R., Parlati, F.,Shenk, K.D., Smyth, M.S., Sun, C.M.,Vallone, M.K., Woo, T.M., Molineaux,C.J., Bennett, M.K. (2007) Antitumor activityof PR-171, a novel irreversible inhibitor of theproteasome. Cancer Res. 67, 6383–6391.


15Development of Direct Thrombin Inhibitor, Dabigatran Etexilate,as an Anticoagulant Drug

15.1Introduction

Warfarin and heparin have long been used as anticoagulants to treat conditions ofvenous thromboembolism, such as deep venous thrombosis (DVT) and pulmo-nary embolism (PE). DVT and PE occur when there is an abnormal formation ofblood clot within the circulatory system. Thrombosis can also manifest as throm-botic or embolic stroke and myocardial infarction. Therefore, it is not surprisingthat anticoagulants have also been heavily used for prevention of stroke in patientswho suffer from atrial fibrillation. Traditional anticoagulants often required closemonitoring due to their narrow therapeutic window and individualized patientresponse. New orally bioavailable anticoagulants are being developed to provideeffective treatment without the disadvantages of traditional agents.A thrombus is a blood clot formed locally in a blood vessel and is composed of

aggregates of fibrin, platelets, and red blood cells. The thrombus can partially orcompletely occlude the blood vessel in which it forms, thus limiting the bloodflow. The consequent hypoperfusion can damage the affected tissue. An embolusis formed when a thrombus is displaced from its original position and flowsthrough the circulatory system to occlude a vessel in a different location.In arterial thrombosis, the formation of a thrombus is most commonly trig-

gered by the rupture of an atherosclerotic plaque, and is mainly composed ofaggregated platelets known as a white clot. In venous thrombosis, the forma-tion of a thrombus is favored by alterations in the composition of the blood orof the vein walls, or by a reduction of blood flow in large veins. The resultingthrombus is called a red clot and is mainly composed of fibrin and red bloodcells [1].It is estimated that venous thromboembolism is the third most disseminate car-

diovascular disease among the US population. Venous thromboembolism isresponsible for hundreds of thousands of deaths each year. Patients undergoingmajor surgeries such as hip or knee replacement, as well as patients sufferingfrom cardiovascular diseases such as atrial fibrillation, have an increased risk ofdeveloping venous thromboembolism. Other risk factors can be genetic such as

337


mutations in enzymes involved in the coagulation process or can be acquired as aresult of obesity or advanced age [1–3].The blood coagulation process is a central event in the formation of both arterial

and venous thrombi, but it is also a critical physiological process necessary formaintaining hemostasis. Hemostasis is a process that occurs as a consequence ofa vessel injury to stop bleeding and avoid loss of blood from the circulatory system.Hemostasis is a delicate equilibrium with clot formation and dissolution. Anyalteration of this balance can lead to either thrombotic diseases when excessiveclotting occurs or hemorrhagic diseases due to defect of platelets or coagulationfactors. When a blood vessel is injured, there is an initial vascular phase wherevasoconstriction occurs, leading to reduced blood flow. Next is the platelet phasewhere specific proteins (collagen and the von Willebrand factor) allow platelets toadhere to an injured vessel wall. Following adhesion, platelets undergo a series ofmodifications, consisting of shape modification, degranulation to release cytokines,recruitment of more platelets (aggregation), and exposure of fibrinogen receptors.Thrombin, an enzyme involved in the clot formation, is also the most potent

platelet activating factor. It cleaves the membrane-bound protease-activated recep-tors 1, 3, and 4. These events finally form a platelet plug that immediately sealsthe damaged vessel. Next is the clotting phase where an enzymatic cascade is acti-vated, ultimately leading to the formation of the fibrin clot. The enzymes involvedin the coagulation cascade are all serine proteases that are normally present in theblood in their inactive forms (zymogens) and are activated through a proteolyticevent. Platelets are also involved in the fibrin clot formation. Finally, there is thefibrinolytic phase. Activation of the fibrinolytic system leads to dissolution of theclot after the damaged vessel has been repaired or when an undesired thrombus isformed.

15.2Coagulation Cascade and Anticoagulant Drugs

The coagulation cascade is schematically shown in Figure 15.1. Historically, it hasbeen divided into an intrinsic pathway and an extrinsic pathway. The extrinsicpathway is activated following an injury event at the vascular endothelium thatexposes collagen and the tissue factors normally contained within healthy endo-thelial cells. The intrinsic pathway is activated after blood stasis or upon contactwith negatively charged surfaces. Both pathways result in a cascade activation ofseveral serine proteases, mediated by coagulation factors, including enzymes,Ca2þ ions, and phospholipids that ultimately lead to the formation of a clot. Thereare 12 coagulation factors involved in the coagulation cascade that are numberedbased on the order of their discovery (FI–FV and FVII–FXIII, Table 15.1). Eachserine protease enzymatic factor is activated by a proteolytic event, in which thesingle polypeptide chain forming the zymogen is cleaved to form two polypeptidechains held together by disulfide bonds. Active coagulation factors are designatedwith an “a” following the coagulation factor Roman numeral (FIa, FIIa, etc.).

338 15 Development of Direct Thrombin Inhibitor, Dabigatran Etexilate, as an Anticoagulant Drug

The two pathways converge in their common target, finally converting factor Xinto its active form (factor Xa). The latter catalyzes the activation of a smallamount of prothrombin (factor II) into the enzymatically active thrombin (factorIIa) [4]. Activation of this small amount of thrombin is sufficient to amplify theenzymatic cascade, since thrombin activates upstream factors involved in its ownactivation (feedback activation of factors XI, V, and VIII). Thrombin activation is acentral event in the coagulation process, since this enzyme catalyzes the cleavage

Figure 15.1 Schematic representation of bloodcoagulation and factors (Roman numerals)involved in the coagulation cascade. The intrin-sic pathway enzymes, green; extrinsic pathwayenzymes, yellow. Both pathways result in

activation of factor X (orange) which convertsprothrombin (factor II) into active thrombin(cyan). Thrombin converts fibrinogen intomonomeric fibrin (red). The cross-linkingreaction is catalyzed by factor XIIIa (violet).

15.2 Coagulation Cascade and Anticoagulant Drugs 339

of fibrinogen into fibrin monomers. Fibrin monomers form a soluble polymerin which the monomers interact with each other through noncovalent interac-tions. Thrombin also activates factor XIII to XIIIa, and the latter enzyme furtherstabilizes the structure of the fibrin polymer by cross-links (insoluble fibrinpolymer). It also activates platelets that are involved in the coagulation process[4–14]. Due to its central role in the coagulation cascade, thrombin activity istightly regulated in its multiple procoagulant and antifibrinolytic activities,endowing it with a paradoxical anticoagulant activity. The regulatory systemsare extremely important since they avoid excessive clotting. Two main enzymesbelonging to the serpin family, namely, antithrombin III and heparin cofactorII, inhibit thrombin activity. The inhibition of thrombin activity by these twoserpins is enhanced by the presence of heparin [15–17]. Additional regulationoccurs through thrombomodulin, which promotes the anticoagulant activity ofthrombin. In the presence of thrombomodulin, thrombin loses the ability tocleave fibrinogen to fibrin and gains high specificity for the cleavage and activa-tion of two regulatory proteins, namely, the latent circulating protein C and thethrombin-activatable fibrinolysis inhibitor [4,6,18]. Once activated by thrombin,the latent circulating protein C deactivates the coagulation cascade by degradingFVa and FVIIIa.

15.3Anticoagulant Therapies

Traditional anticoagulants mainly relied on the use of coumarin derivativesand heparin. The anticoagulant properties of compounds such as coumarinsand heparins have been known since the early 1900s and have been used inclinical practice since the late 1950s for the prevention and treatment ofthrombotic diseases [19–21].

Table 15.1 Coagulation factors.

Coagulation factor number Common name

Factor I (FI) FibrinogenFactor II (FII) ProthrombinFactor III (FIII) Tissue factorFactor IV (FIV) Ca2þ

Factor V (FV) —

Factor VII (FVII) ProconvertinFactor VIII (FVIII) Antihemophilic factor AFactor IX (FIX) Antihemophilic factor BFactor X (FX) Stuart–Prower factorFactor XI (FXI) —

Factor XII (FXII) Hageman factorFactor XIII (FXIII) Fibrin stabilizing factor


Warfarin is the leading coumarin used in clinical practice. This compoundinterferes with the vitamin K-mediated posttranslational modification of somecoagulation factors. This hampers their proper functioning and results in antico-agulant activity. The posttranslational modifications consist of the carboxylation ofspecific glutamate residues to form carboxyglutamate. The latter group is able tocomplex Ca2þ, which is an important cofactor necessary for coagulation. Coagula-tion factors that require vitamin K-mediated maturation are prothrombin and fac-tors VII, IX, and X. It takes several days of oral therapy for warfarin to beginshowing its anticoagulation effects. Moreover, the therapeutic index of warfarin isextremely low and the plasma concentration of the administered drug is erratic,mainly due to drug–drug and food–drug interactions, hepatic function, and thepatient’s general health status and lifestyle. In order to prevent excessive bleedingand to guarantee administration of sufficient amounts of compound to reachantithrombotic efficacy, frequent blood test monitoring of the anticoagulationeffect is required.Unfractionated heparin is a heterogeneous mixture of glycosaminoglycans,

and, combined with some low molecular weight derivatives, it is used in anti-coagulation therapy. It has indirect thrombin inhibitory activity since it poten-tiates the physiological effects of the enzyme antithrombin. Heparin-activatedantithrombin binds to the active site of thrombin and blocks the fibrin bind-ing site. Due to this indirect mechanism of action, heparin activity relies onthe availability of the target protein in the blood. Moreover, heparin adminis-tration requires constant monitoring of anticoagulation levels. Due to its heavynegative charge, it binds to several plasma proteins, blood cellular compo-nents, and platelets. Binding of heparin to platelets, and the resulting interfer-ence with their function, can cause excessive bleeding. Due to its highmolecular weight (3000–30 000 Da), unfractionated heparin can only beadministered by continuous infusion in hospital settings or through sub-cutaneous administration.The numerous drawbacks of the above-mentioned therapies prompted the

search for alternative, safe, and more effective antithrombotics. Due to its centralrole in the coagulation process, thrombin was recognized early as a therapeutictarget to be directly inhibited by orally available small molecules [19]. Historically,the first direct thrombin inhibitors were derived from blood-sucking organisms,such as leeches. Hirudin, for example, is a potent direct thrombin inhibitorderived from the medicinal leech Hirudo medicinalis. This peptide, having amolecular weight of around 7000 Da, binds thrombin and forms aslow-reversible complex. This inhibition kinetics is responsible for its very highinhibitory potency. Unfortunately, the almost irreversible binding of hirudins tothrombin, coupled with the lack of specific antidotes, results in an increased riskof bleeding during the therapy, restricting the therapeutic safety window. More-over, due to the peptidic nature of hirudin and its derivatives, they cannot be orallyadministered.However, studies involving hirudin and its derivatives confirmed that the

strategy of directly inhibiting thrombin was suitable and promising for the

15.3 Anticoagulant Therapies 341

development of orally available, safe anticoagulant therapies. Several groupsstarted working on the development of low molecular weight, direct thrombininhibitors, with the ultimate goal of developing orally bioavailable drugs. A keydiscovery in this field has been the resolution of the X-ray crystal structure ofthrombin [22–24].

15.4Structure of Thrombin

The inactive precursor of thrombin is called prothrombin and is a membrane-bound protein. Prothrombin is activated by proteolytic maturation to form solu-ble a-thrombin. Human a-thrombin is formed by A-chain and B-chain, com-posed of 36- and 259-amino acid residues, respectively. The structure of maturea-thrombin belongs to the chymotrypsin family, and similar to chymotrypsin,the B-chain forms two b-barrel domains (Figure 15.2). In addition to the charac-teristic fold and structural features of this class of enzymes, a-thrombin pres-ents several insertion loops. The B-chain hosts several functional epitopesresponsible for the regulation of pro- and anticoagulant activities of thrombin.The active site residues (Ser195, His57, and Asp102) are located at the junctionbetween the two b-barrels. His57 and Asp102 are contributed by the N-terminalb-barrel, whereas the oxyanion hole residue and catalytic Ser195 are contributedby the C-terminal b-barrel. The peptide binding domain runs perpendicular tothe two domain junctions [22–26].

Figure 15.2 X-ray crystal structure of the binary complex thrombin and hirudin. The inhibitor isnot shown for clarity. Green: B-chain; red: A-chain, yellow: hirudin; blue: 60D insertion loop.Active site residues (His57, Ser195, and Asp102) are shown as sticks. (PDB Code: 3HTC).


This enzyme has a very narrow specificity that is mediated by the presence ofthe above-mentioned insertion loops. Two of the most important insertion loopsare the 60D and 149 insertion loops. The 60D insertion loop has a hairpin shapeprojected to the outside of the protein. It has an important role in recognition ofsusceptible substrates and inhibitors. At the tip of the loop, Trp60D is solventexposed, whereas Tyr60A covers the S2 binding subsite. The 149 insertion loop ishighly flexible. Both loops are responsible for the high specificity of thrombintoward its substrates since they block access of substrates and inhibitors to thesubstrate binding site.The surface of a-thrombin also presents an uneven electrostatic distribution and

is characterized by the presence of two zones, one with a highly positive electro-static field, known as anion binding exosite I and anion binding exosite II, and theother a negative zone (Figure 15.3).The anion binding exosite I is also called the fibrinogen recognition exosite (res-

idues 67–80). It is formed by the loop centered on K70 and is homologous to theCa2þ binding loop of trypsin and chymotrypsin. The K70 has a positive chargethat occupies the same position as Ca2þ in homologous enzymes. This loop isalso called the autolysis loop since it is susceptible to hydrolysis and autohydroly-sis (tryptic and autocatalytic attack). Human b-thrombin is formed upon excisionof peptide Ile68–Arg77A. It is located at the rim of the access site for substratesapproaching the active site and is also involved in the recognition of fibrinogen.The anion binding exosite I binds not only fibrinogen but also thrombomodulin,PAR1 (receptors on the platelet surface responsible for platelet activation), fibrin,and the direct inhibitor hirudin.

Figure 15.3 Surface representation of the above ternary complex. Color by electrostatic potential(red: negative; blue: positive). Anion binding exosites I and II and the substrate binding channelare shown as dotted circles (PDB: 1DWB).

15.4 Structure of Thrombin 343

The anion binding exosite II binds heparin and related glycosaminoglycans andplatelet receptors. The heparin–antithrombin complex also docks in exosite II. Thecleavage sequence of thrombin substrates is highly specific, and the main featuresof P4–P3

0 substrate residues are summarized in Figure 15.4.The high P1 selectivity in trypsin-like enzymes for Arg is mediated by the pres-

ence of Asp189 at the bottom of the S1 subsite in thrombin. However, thrombincan also cleave substrates bearing a Phe as the P1 residue. A unique feature of thissubsite in thrombin, with respect to other enzymes belonging to the same fam-ily, is the presence of a Glu residue (Glu192) at the entrance of the S1 subsite.The S1

0 subsite is able to accommodate small, polar substrate residues since itssize is limited by the pending side chain of Lys60F (located within the 60Dinsertion loop). In general, there is no preference for specific P3 residues sincethe corresponding residue side chain points away from the enzyme surface. TheS4 subsite, or aryl binding site, is defined by residues Leu99, Ile174, andTrp215. The S2 subsite can accommodate substrate residues bearing small andhydrophobic side chains, such as proline or valine, and is lined by residues 60a–60d belonging to the 60D loop.Some thrombin substrates, such as fibrinogen Aa, FXIII, latent circulating pro-

tein C, and heparin cofactor II, have nonoptimal cleavage sequences, but use addi-tional binding sites (exosites I and II) to increase the rate of hydrolysis bythrombin. Consequently, they are efficiently cleaved by thrombin even thoughthey do not possess an optimal cleavage sequence.

Figure 15.4 P4–P3 0 sequence specificity of thrombin cleavage substrates.


15.5The Discovery of Dabigatran Etexilate

Structure-based design of dabigatran started with compound 1 (Figure 15.5),which was crystallized in 1991 in complex with thrombin and hirudin [27]. X-raycrystal structure is shown in Figure 15.6. As shown in Figure 15.7, compound 1forms two hydrogen bonds between the sulfonamide nitrogen and the backbonecarbonyl of Gly216 and the glycine carbonyl with the backbone NH of Gly216.The sulfonamide oxygens do not form any polar interaction with the enzyme

residues since they are solvent exposed. The positively charged amidine groupforms a salt bridge interaction with Asp189 at the bottom of the specificity pocket.The piperidine ring and the naphthyl moiety of the inhibitors fill the hydrophobicpocket formed by four residues of the 60D insertion loop and residues Trp215 andLeu99 (Figure 15.6). The pocket is divided into two parts: the distal (D)-pocket thatembeds the naphthyl ring and the proximal (P)-pocket that is filled by the piperi-dine ring. The name of the pockets is based on their positions relative to catalyticSer195. Surface representation of inhibitor 1 in the thrombin/hirudin active site isshown in Figure 15.7.Studies for the development of dabigatran started from this crystal structure

and structure–activity relationship (SAR) studies on compound 1, which bindsthrombin with an IC50 of 0.2 mM. Besides being a medium-affinity inhibitor ofthe enzyme, inhibitor 1 did not show good oral absorption and had a short

NH O

HN

N

O

NH2

H2N

S

N

N

NH

S

Me

1 IC50 = 0.2 µM

2 IC50 = 1.5 µM

OO

Asp189

OO

Asp189

NH2H2N

OO

OO

Figure 15.5 Structures and activities of lead inhibitors 1 and 2.

15.5 The Discovery of Dabigatran Etexilate 345

half-life after i.v. injection. Researchers at Boehringer Ingelheim started theirinvestigation by replacing the central glycine linker with various glycine cyclicanalogs [28]. Their goal was to identify a suitable heterocyclic scaffold able toreplace the glycine linker and present groups that interact with the specificitypocket and with the D- and P-pockets in the correct orientation. The expectedoverall advantage would be an increase of potency and a strong reduction inthe peptidic character of the resulting molecules. The researchers decided toignore the hydrogen bond formed by the glycine moiety of 1. Their first leadcompound was the N-methylbenzimidazole 2, which showed an IC50 of 1.5 mMand displayed a longer half-life after i.v. injection. However, severe cardiovas-cular effects were also observed after i.v. administration of 2. The X-ray crystalstructure of 2 in Figure 15.8 showed a binding mode similar to that of 1,where the benzamidine moiety was engaged in the salt bridge interaction withAsp189 at the bottom of the specificity pocket. The phenylsulfonamide ringfilled the D-pocket, whereas the methyl substituent of the benzimidazole ringwas embedded within the P-pocket.

Figure 15.6 X-ray crystal structure of the ter-nary complex thrombin/hirudin and inhibitor 1.Green; B-chain; red: A-chain; blue: insertionloops; yellow: hirudin; the catalytic triad

residues, the Asp189 residue at the bottom ofthe specificity pocket (gray), and the inhibitor(magenta) (PDB code: 1DWD).


Figure 15.8 Binding mode of inhibitor 2. Red: D-pocket; cyan: P-pocket; residues forming thehydrophobic pockets and Asp189 are shown as sticks (PDB code: 1KTT).

Figure 15.7 Surface representation of thethrombin/hirudin and inhibitor 1 complex high-lighting the distal (red) and the proximal (blue)hydrophobic pockets. The specificity pocket is

in yellow. Hydrogen bonds are shown as dottedlines. Residues Asp189 and Gly216 are shownas yellow sticks and inhibitor in magenta (PDBcode: 1DWD).


Investigators started a structure–activity relationship study of the various substit-uents on the benzimidazole scaffold guided by the X-ray crystal structure. Due tothe dimension of the P-pocket, an attempt at increasing the potency was made byreplacing the methyl substituent with larger groups such as ethyl and n-propylgroups in compounds 3 and 4, respectively, shown in Figure 15.9. Surprisingly,these modifications did not improve binding potency even though the pocket waslarge enough to accommodate the alkyl chains at the benzimidazole ring. Thisresult was explained by a negative effect on binding related to the loss of entropyin the binding mode. On the other hand, compound 5, lacking the methyl group,was less active than compound 2, suggesting that the hydrophobic interaction ofthe methyl group is extremely important for binding site interactions.Investigation of the second hydrophobic moiety, namely, the aryl ring, was more

successful since replacement of the phenyl ring with larger groups, such as a 2-naphthyl or various quinolines and isoquinolines, led to encouraging results(inhibitors 6–8, Figure 15.10). The shape of the D-pocket was examined throughan isomeric 1-naphthyl derivative, which was 40-fold less active. Pyridine ringswere also tolerated within this pocket.The main problem with these early leads was that when the enzymatic activity was

tested in the presence of plasma, a marked increase in IC50 values was observed.This indicated that the compounds were binding plasma proteins, meaning that thepharmacokinetic parameters needed to be optimized. Taking the X-ray crystal struc-ture as a guide, it was noted that the sulfonamide NH was largely solvent exposed;so polar groups could be incorporated at this position without any risk of compro-mising enzymatic binding. As speculated, introduction of an acetic acid moietyat the sulfonamide nitrogen improved potency of the corresponding compound 9

N

N

NH

S

HNNH2

3 IC50 = 2.4 µM

N

N

NH

S

HNNH2

4 IC50 = 4.0 µM

MeMe

N

HN

NH

S

HNNH25 IC50 = 19 µM

OO

OO

OO

Lead inhibitor 2

Figure 15.9 Structures and activities of inhibitors 3–5.


(Figure 15.11) in the presence of plasma. However, the observed enzyme IC50 valuerequired further improvement. An examination of the X-ray crystal structure sug-gested that modification of the tether connecting the benzimidazole ring at position2 with the benzamidine ring may lead to improved potency. As it appeared, anextension of this tether could be potentially able to present the aromatic ring andthe positively charged amidine moiety to the specificity pocket in a better conforma-tion for interactions in the binding pocket. As speculated, almost one order of mag-nitude enhancement of potency was observed for compounds 10–12 (Figure 15.11),which contained aliphatic, ethereal, and amine linkers, respectively. The amino deriv-ative 12 resulted in the most potent compound in this series.Starting from this compound 12 and exploiting the newly discovered amino

linker, investigators turned their attention to replacing the quinolinesulfonamidesubstituent with an N-arylcarboxamide functionality. It was assumed that thegeometry of the amide linker would orient the phenyl ring within the D-pocketmore efficiently than the sulfonamide linker. This modification resulted in a seriesof potent compounds (Figure 15.12). Compound 13 exhibited similar enzymeinhibitory activity as compound 12; however, compound 13 showed improvementin pharmacokinetic properties. Further investigation of the length of the linkerconnecting the carboxylic group to the amide nitrogen was carried out. The activ-ity of compound 14 with a three-carbon linker was very similar to compound 13.Compound 15 with a two-carbon linker showed substantial enhancement inpotency with respect to compound 13 with a one-carbon linker and compound 14with a three-carbon linker. In an effort to improve the pharmacokinetic propertiesfurther, replacement of the phenyl ring with a 2-pyridyl ring was carried out. The

N

N

NH

S

Me

HNNH2

6 IC50 = 24 µM

N

N

NH

S

Me

HNNH2

7 IC50 = 0.6 µM

N

N

NH

S

Me

HNNH2

8 IC50 = 0.26 µM

N

2

OO

OO

OO



resulting compound 16 (BIBR 953) showed an IC50 value of 9.3 nM and favorableselectivity profiles against related serine proteases shown in Table 15.2. This com-pound showed very potent anticoagulant activity in vivo and was selected for in-depth studies as a candidate for further development (16, dabigatran).Compound 16 was shown to be effective in animal models after i.v. administration,

but lacked oral bioavailability. Lack of oral activity for 16 could be explained by thepresence of two charged groups in the molecule, leading to a zwitterionic form ofthe drug that cannot be absorbed after oral administration since it is too hydrophilic.This issue was addressed on the basis of previous work, where the two charged func-tionalities were masked with metabolically labile groups. The dabigatran prodrug 17(dabigatran etexilate, Figure 15.13) was subsequently selected for clinical investiga-tion. Dabigatran etexilate was approved by the FDA for the treatment of venousthromboembolism and for stroke prevention in patients with atrial fibrillation.The X-ray crystal structure of the ethyl ester of dabigatran bound to thrombin

(18) has been determined (Figure 15.14) [28]. Although inhibitor 18 displayednanomolar affinity for the enzyme, its binding affinity for thrombin mainly relies

N

N

NS

Me

HNNH2

9 IC50 = 0.12 µM

N

CO2H

OO

10 IC50 = 0.032 µM

N

N

ONS

Me

N

CO2H

OO

11 IC50 = 0.058 µM

N

N

NS

Me

N

CO2H

OO

H

N

N

NNS

Me

N

CO2H

OO

12 IC50 = 0.011 µM

NH

NH2

H

NH

NH2

NH2

NHH



on the salt bridge between the benzamidine moiety and Asp189 within the speci-ficity pocket. The central template of inhibitor 18 is packed in the hydrophobicP-pocket and the pyridine ring is nestled in the D-pocket surrounding Leu99 andIle174. A surface representation of the X-ray structure is shown in Figure 15.15.

Table 15.2 Selectivity profile for compound 16 (BIBR 953, dabigatran) against serine proteases.

Human enzymes Ki (nM)

Thrombin 4.5 � 0.2Factor Xa 3760 � 20Trypsin 50.3 � 0.3Plasmin 1695 � 50tPA 45 360 � 10Activated protein C 20 930 � 10

N

N

N

Me

13 IC50 = 0.010 µM

NH

NH2

N

N

N

Me

O

NH

NH2

N

N

NN

Me

NH

NH2

14 IC50 = 0.010 µM

15 IC50 = 0.0054 µM

N

N

NN

Me

O

NH

NH2

16 IC50 = 0.0093 µM

Dabigatran

H

H

H

H

N

O

HO2C

O

HO2C

N

CO2H

N

CO2H



Figure 15.14 X-ray crystal structure of ethyl ester derivative of dabigatran (18) in complex withthrombin. The carbon chain is shown in green and hydrogen bonds are shown in dotted lines(PDB code: 1KTS).

N

N

NN

Me

O

NH2

N

17 Dabigatran etexilate

OO

H

N

N

NN

N

Me

O

NH

NH2

18 Dabigatran ethyl ester

H

N

O O

O O

Figure 15.13 Structures of inhibitors 17 and 18.


As can be seen, inhibitor effectively filled in the D- and P-pockets and is involvedin extensive hydrophobic contacts in the binding site.

15.6Conclusions

Before the discovery of dabigatran, anticoagulant therapy relied only on the injectableheparin and low molecular weight heparins, or on the orally available drug warfarin.However, both drugs have an extremely reduced therapeutic index and need to beadministered in a hospital setting or with close monitoring of the patient’s coagula-tion status. The development of direct thrombin inhibitors fulfilled a great clinicalneed in the field of anticoagulant therapy. Dabigatran etexilate has been approved byFDA in 2010 for the treatment of venous thromboembolism. It is also approved forthe prevention of stroke in patients with atrial fibrillation. Importantly, dabigatranetexilate administration does not require coagulation monitoring or dose adjustment.This drug represents the first therapeutic alternative to coumarins in over 60 years.

References

1 Mackman, N. (2008) Triggers, targets andtreatments for thrombosis. Nature, 451,914–918.

2 Geerts, W.H., Pineo, G.F., Heit, J.A.,Bergqvist, D., Lassen, M.R., Colwell, C.W.,

and Ray, J.G. (2004) Prevention of venousthromboembolism: the Seventh ACCPConference on Antithrombotic andThrombolytic Therapy. Chest, 126,338S–400S.

Figure 15.15 A surface representation of the X-ray structure of inhibitor 18 and thrombin com-plex (PDB code: 1KTS).

References 353

3 Mahan, C.E., Holdsworth, M.T., Welch,S.M., Borrego, M., and Spyropoulos, A.C.(2011) Deep-vein thrombosis: a UnitedStates cost model for a preventable andcostly adverse event. Thromb. Haemost.,106, 405–415.

4 Mann, K.G. (2003) Thrombin formation.Chest, 124, 4S–10S.

5 Davie, E.W. and Kulman, J.D. (2006) Anoverview of the structure and function ofthrombin. Semin. Thromb. Hemost., 32(Suppl. 1), 3–15.

6 Mann, K.G., Butenas, S., and Brummel, K.(2003) The dynamics of thrombinformation. Arterioscler. Thromb. Vasc. Biol.,23, 17–25.

7 Furie, B. and Furie, B.C. (2008)Mechanisms of thrombus formation. N.Engl. J. Med., 359, 938–949.

8 Licari, L.G. and Kovacic, J.P. (2009)Thrombin physiology and pathophysiology.J. Vet. Emerg. Crit. Care (San Antonio), 19,11–22.

9 Di Cera, E. (2003) Thrombin interactions.Chest, 124, 11S–17S.

10 Bajzar, L., Morser, J., and Nesheim, M.(1996) TAFI, or plasmaprocarboxypeptidase B, couples thecoagulation and fibrinolytic cascadesthrough the thrombin–thrombomodulincomplex. J. Biol. Chem., 271, 16603–16608.

11 Griffin, J.H. (1995) Blood coagulation. Thethrombin paradox. Nature, 378, 337–338.

12 Lorand, L., Downey, J., Gotoh, T., Jacobsen,A., and Tokura, S. (1968) Thetranspeptidase system which crosslinksfibrin by gamma-glutamyl–epsilon-lysinebonds. Biochem. Biophys. Res. Commun., 31,222–230.

13 Coughlin, S.R. (2000) Thrombin signalingand protease-activated receptors. Nature,407, 258–264.

14 Brass, L.F. (2003) Thrombin and plateletactivation. Chest, 124, 18S–25S.

15 Gettins, P.G. (2002) Serpin structure,mechanism, and function. Chem. Rev., 102,4751–4804.

16 Olson, S.T. and Chuang, Y.J. (2002) Heparinactivates antithrombin anticoagulantfunction by generating new interaction sites(exosites) for blood clotting proteinases.Trends Cardiovasc. Med., 12, 331–338.

17 Tollefsen, D.M. (2007) Heparin cofactor IImodulates the response to vascular injury.Arterioscler. Thromb. Vasc. Biol., 27, 454–460.

18 Esmon, C.T. (2003) The protein C pathway.Chest, 124, 26S–32S.

19 Markwardt, F. (2002) Historical perspectiveof the development of thrombin inhibitors.Pathophysiol. Haemost. Thromb., 32 (Suppl. 3),15–22.

20 Nutescu, E.A., Shapiro, N.L., Chevalier, A.,and Amin, A.N. (2005) A pharmacologicoverview of current and emerginganticoagulants. Cleve. Clin. J. Med., 72(Suppl. 1), S2–S6.

21 Gurm, H.S. and Bhatt, D.L. (2005) Thrombin,an ideal target for pharmacological inhibition:a review of direct thrombin inhibitors. Am.Heart J., 149, S43–S53.

22 Rydel, T.J., Ravichandran, K.G., Tulinsky, A.,Bode, W., Huber, R., Roitsch, C., and Fenton,J.W., 2nd (1990) The structure of a complexof recombinant hirudin and human alpha-thrombin. Science, 249, 277–280.

23 Bode, W., Turk, D., and Karshikov, A.(1992) The refined 1.9-A

�X-ray crystal

structure of D-Phe-Pro-Argchloromethylketone-inhibited humanalpha-thrombin: structure analysis, overallstructure, electrostatic properties, detailedactive-site geometry, and structure–functionrelationships. Protein Sci., 1, 426–471.

24 Bode, W. (2006) Structure and interactionmodes of thrombin. Blood Cells Mol. Dis.,36, 122–130.

25 Pineda, A.O., Carrell, C.J., Bush, L.A.,Prasad, S., Caccia, S., Chen, Z.W., Mathews,F.S., and Di Cera, E. (2004) Moleculardissection of Naþ binding to thrombin.J. Biol. Chem., 279, 31842–31853.

26 Page, M.J. and Di Cera, E. (2008) Serinepeptidases: classification, structureand function. Cell. Mol. Life Sci., 65,1220–1236.

27 Banner, D.W. and Hadvary, P. (1991)Crystallographic analysis at 3.0-A

�resolution

of the binding to human thrombin of fouractive site-directed inhibitors. J. Biol.Chem., 266, 20085–20093.

28 Hauel, N.H., Nar, H., Priepke, H., Ries, U.,Stassen, J.M., and Wienen, W. (2002)Structure-based design of novel potentnonpeptide thrombin inhibitors. J. Med.Chem., 45, 1757–1766.


16Non-Nucleoside HIV Reverse Transcriptase Inhibitorsfor the Treatment of HIV/AIDS: Design andDevelopment of Etravirine and Rilpivirine

16.1Introduction

The enzyme reverse transcriptase has a central role in the life cycle of the humanimmunodeficiency virus (HIV) [1–4]. Once inside the host cell, the HIV releasesits genomic RNA, which gets converted into double-stranded DNA in order tobe incorporated in the genome of the infected cell. This function is accomplishedby the enzyme reverse transcriptase [1,5].HIV reverse transcriptase is a heterodimeric enzyme formed by p66 and p51

subunits that has DNA polymerase activity as well as RNase H activity [6,7]. TheDNA polymerization can occur using either RNA or DNA as templates, whereasthe RNase H activity requires RNA as a template in order to degrade the RNA:DNA double strands formed by DNA polymerization reaction. This creates asingle-stranded DNA, which is copied again by the polymerase activity of thetranscriptase enzyme to form double-stranded DNA. This latter DNA caneventually be integrated into the host cell genome, assisted by the activity of theviral enzyme integrase.Due to its key role in the life cycle of the virus, HIV reverse transcriptase

has been targeted since the beginning of the fight against HIV infection.The first drugs to be approved in therapy were nucleoside and nucleotidereverse transcriptase inhibitors [NNRTI] [1,8,9]. These drugs (or prodrugs)are structural analogs of nucleosides and are incorporated in the growingDNA chain by the reverse transcriptase. However, due to the lack of an OHgroup at C-30 of the sugar moiety, their incorporation in the growing DNAchain interrupts the growth of the chain. This class of drugs is also definedas “chain terminators.” Later, a second class of reverse transcriptase inhibi-tors, characterized by a completely different mechanism of inhibition,was discovered and named non-nucleoside reverse transcriptase inhibitors[10–13]. These drugs are allosteric inhibitors of the enzyme since they donot bind to the active site of the enzyme, but bind to a lipophilic pocket ofthe enzyme, located close to the active site moiety. Consistent with the allo-steric mechanism of inhibition, the binding of NNRTIs does not prevent the

355


binding of the nucleotide to the active site, but results in the blocking of thepolymerase activity of the enzyme. Non-nucleoside inhibitors are a veryimportant class of antiviral drugs that are essential components of the so-called highly active antiretroviral therapy together with the proteaseinhibitors [12].One of the major issues associated with the use of non-nucleoside reverse

transcriptase inhibitors was the rapid selection of resistant viral strainsafter exposure to these drugs. Therefore, research efforts have focused on thedevelopment of novel NNRTIs capable of overcoming the most common resist-ance mutations [14]. To date, five NNRTIs are used in therapy (Figure 16.1):nevirapine (1), efavirenz (2), delavirdine (3), etravirine (4), and rilpivirine (5).Nevirapine was the first NNRTI approved in 1996 for the treatment of HIV/AIDS [6].

HN

NN N

Me O

1 Nevirapine

NH

OCl

O

F3C

2 Efavirenz

N NNO

NH

HN

SMe

O OHN

3 Delavirdine

N

N

NH2

Br

O NHMe Me

CN CN

4 Etravirine

N

NHN NHMe Me

CN

5 Rilpivirine

CN

Figure 16.1 Chemical structures of non-nucleoside reverse transcriptase inhibitors currentlyapproved in therapy.

356 16 Non-Nucleoside HIV Reverse Transcriptase Inhibitors for the Treatment of HIV/AIDS

16.2Structure of the HIV Reverse Transcriptase

As previously described, the HIV reverse transcriptase is a heterodimer formed bytwo subunits: p66 and p51 [6,15,16]. The p66 subunit is formed by the DNA poly-merase and RNase H domains (Figure 16.2). The p51 subunit has the sameamino acid sequence as the DNA polymerase domain. However, its active site isnot functional and its role seems to provide a scaffold for the correct polymeriza-tion process catalyzed by the p66 subunit. The DNA polymerase domain is in turndivided into four subdomains: fingers, thumb, palm, and connection. The poly-merase active site resides in the palm and the catalytic residues are three aspar-tates (Asp110, Asp185, and Asp186). For the polymerization reaction, the 30-OHof the primer is located close to a highly conserved structure (residues 227–235)that holds the primer in the correct position for nucleotide incorporation in thegrowing chain [6].Non-nucleoside reverse transcriptase inhibitors bind at a site that is 10A

�away

from the polymerase active site. The binding pocket of these inhibitors existsonly after inhibitor binds. The formation of the non-nucleoside reverse transcrip-tase inhibitor binding pocket requires the movement of several b-sheets. As a

Figure 16.2 Overall structure of HIV reversetranscriptase with non-nucleoside reverse tran-scriptase inhibitor, etravirine. The p51 subunit,white; p66 subunit, red (finger subdomain);palm subdomain, cyan; thumb subdomain,

magenta; connection subdomain, blue; RNaseH subdomain, green; active site residues, balland sticks; bound inhibitor, CPK spheres (PDBcode: 1S6P).

16.2 Structure of the HIV Reverse Transcriptase 357

consequence, the primer grip region is shifted and a rotation of the aromatic ringsof Tyr181 and Tyr188 away from the polymerization site occurs [15–20]. Thenucleotide incorporation is thus hindered and the polymerase reaction is blocked.Non-nucleoside reverse transcriptase inhibitors can be divided into first- and

second-generation inhibitors based on their binding mode and resilience toresistance mutations [21]. In particular, first-generation inhibitors share acommon binding conformation that appears “butterfly-like,” as shown inFigure 16.3. Inhibitor (1) structural elements interacting within the bindingpocket can be defined as wing I, wing II, and body [6,21–23]. As shown inFigure 16.4, wing I forms mainly stacking interactions with residues Tyr181,Tyr188, and Trp229. Wing II, while forms mainly hydrophobic contacts withresidues Lys101, Lys103, Val106, Val179, and Tyr318. The body of first-generation non-nucleoside reverse transcriptase inhibitors interacts with themain chain atoms of Tyr188, Val189, and Gly190 as well as with the side chainatoms of Val106 and Val179. Other residues that are contacted by the inhibi-tors are Leu100 and Leu234. The binding pocket is also lined by amino acidresidues that are part of the primer grip region, namely, Phe227, Trp229, andLeu234 [15]. A surface representation of the active site of reverse transcriptaseand the respective wing residues is shown in Figure 16.5.The efficacy of first-generation non-nucleoside reverse transcriptase inhibi-

tors has been severely hampered by several resistance mutations that decrease

Figure 16.3 Butterfly-like binding conformation of non-nucleoside reverse transcriptaseinhibitors: nevirapine (pink; PDB code: 1VRT), a-APA (green; PDB code: 1HPZ), and9-Cl-TIBO (blue; PDB code: 1TVR).


their inhibitory potency. The reduced inhibitory potency observed with some ofthe most common mutations can be explained because the amino acid resi-dues directly involved in inhibitor binding are mutated [22]. Mutations such asL100I, Y181C, or Y188L directly alter the size, shape, and/or chemical environ-ment of the binding pocket. In particular, the loss of hydrophobic interactionswith aromatic residues Tyr181 and Tyr188, replaced by Cys or Leu residues,strongly affects the inhibitor affinity for the binding pocket. The loss ofpotency observed for the L100I or the G190A mutation is probably due to theincreased volume or shape of the side chain of the mutated enzyme, withrespect to the wild-type side chain. On the other hand, the K103N resistancemutation affects the inhibitor potency due to a kinetic mechanism that, bystabilizing the closed, unbound enzyme conformation, limits the accessibilityof the pocket to inhibitors [24,25].One of the notable features of the non-nucleoside reverse transcriptase inhibitor

binding pocket is its plasticity, making it difficult to predict the binding mode of anovel compound, unless the crystal structure of a close analog is known. Even inthat case, sometimes the introduction of a specific substituent on a known scaf-fold results in a binding mode slightly different from those predicted by moleculardocking studies [16,22,26].

Figure 16.4 Non-nucleoside reverse transcrip-tase inhibitor binding pocket. Residues contact-ing wing I and wing II are displayed as bluesticks. The primer grip region is displayed in

red and residues Trp229, Phe227, and Leu234are shown as sticks. Active site residues aredisplayed as ball and sticks. (PDB code: 1S6P).

16.2 Structure of the HIV Reverse Transcriptase 359

16.3Discovery of Etravirine and Rilpivirine

The strategy to circumvent resistance to non-nucleoside reverse transcriptaseinhibitors at Tibotec was to increase the flexibility of target molecules in order toallow them to adapt to amino acid side chain mutations responsible for resistance.Among several classes of reverse transcriptase inhibitors discovered at Tibotec, aseries of imidoyl thioureas, typified by 8 in Figure 16.6, were investigated. Thesecompounds were previously developed as chain-extended derivatives of a class ofa-APA non-nucleoside reverse transcriptase inhibitors (inhibitor 8, Figure 16.6)and were characterized by potent activity in inhibiting replication of wild-type ormutated HIV-1 strains.Despite their extremely good antiviral activity profile, these compounds were

both chemically and metabolically unstable due to an oxidative cyclization of theimidoyl thiourea group to form a five-membered heterocyclic ring shown in com-pound 9. In order to overcome this issue, the synthesis of cyanoguanidine deriva-tive 10 as a thiourea bioisostere was attempted. However, this compound rapidlycyclized to form the corresponding triazine derivative 11. Although compound 9

Figure 16.5 Surface representation of the reverse transcriptase active site. Residues contactingwing I and wing II are displayed as blue sticks (PDB code: 1S6P).


did not exhibit antiviral activity, the corresponding triazine derivative 11 showed avery promising antiviral profile. It showed low nanomolar activity against wild-type HIV-1 and also showed nanomolar activity against several widespread resist-ant strains [27].Starting from compound 11, structure–activity relationship investigations were

conducted at the wing I aromatic ring (structure 12, Figure 16.6), the X linker,and the substituent Y at C-4 of the triazine ring. Studies on the previous series ofimidoyl thiourea derivatives indicated that the substituent of choice at C-6 of thetriazine ring was an NH-tethered p-CN-phenyl group. In this series of com-pounds, the structure–activity relationship studies indicated that the nature of theX linker did not appreciably alter antiviral potency. A brief summary of structure–activity relationship studies is represented in Figure 16.7. As can be seen, disub-stitution at the 2,6-positions of the wing I aromatic ring was important for potencyagainst both wild-type and resistant strains. Moreover, small 4-substituents

ClCl

NH

HN NH

S

CN

OMe

Me

NH

OH2NCl

Cl

ClCl

N

N NH

S

CN

ClCl

NH

HN NH

N

CN

CN

ClCl N NH

CN

N N

NH2

6 8 IC50 (HIV-1wt) = 2.5 nM

9 (inactive)10

11 IC50 (HIV-1wt) = 6.3 nM

X

ClCl N NH

CN

N N

Y

13 5

12 (for SAR)

Figure 16.6 Structures of reverse transcriptase inhibitors 8–12.

16.3 Discovery of Etravirine and Rilpivirine 361

coupled with 2,6-disubstitution were also tolerated. Small alkylamines at the tria-zine C-4 proved to be tolerated, whereas replacement of the 4-amino group by a4-chloro resulted in loss of activity [27].Antiviral activities of compounds 11 and 13–19 against wild-type HIV-1 and

strains containing single-point mutation are shown in Figure 16.8. Some of themost interesting compounds were the hydroxylamine derivative 13 and the4-amino derivatives 14 and 15. However, compound 13 was metabolicallyunstable. The 4-amino group in compounds 14 and 15 is also susceptible to glu-coronation, making their excretion particularly rapid. Consequently, further workon the triazine scaffold was carried out in order to improve metabolic stability.The removal of the 4-amino group with varying tethers resulted in compounds16–19. As it turned out, compounds 16–19 retained potent antiviral activity againstwild-type and mutated HIV-1 strains [27].

NN

N NH

NH

CN

Cl Cl

NN

N NH

NH2

CN

Me Me

Me

N NH

CN

NN

NH2

Cl Cl

NN

NS NH

CN

Me Me

Me

NN

NHN NH

CN

Me Me

CN

11 IC50 = 6.3 nM 13 IC50 = 2.4 nM 14 IC50 = 0.8 nM

HO

HN N NH

CN

NN

NH2

Me Me

15 IC50 = 1.0 nM 16 IC50 = 0.3 nM 17 IC50 = 0.6 nM

Me

HN N NH

CN

NN

Me Me

Me

H

O N NH

CN

NN

Me Me

Me

H

18 IC50 = 2.0 nM 19 IC50 = 1.0 nM

Figure 16.7 Schematic representation of structure–activity relationships for 1,3,5-triazine asreverse transcriptase inhibitors.


As previously demonstrated for first-generation non-nucleoside reverse tran-scriptase inhibitors, the binding mode of the triazine compounds was similar tothat of the imidoyl thiourea 8 and adopted a “horseshoe” rather than a butterfly-like conformation within the binding pocket [16]. As stated earlier, both wing Iand wing II make several hydrophobic contacts with side chains of amino acids,forming two binding pockets (Figure 16.4). The central triazine ring makes con-tacts with the side chains of Val179 and Leu100. Residue Trp229, part of theprimer grip region, is extremely important for the polymerase reaction catalyzedby the enzyme. Indeed, mutations of this amino acid are never observed inmutant HIV-1 reverse transcriptases. Consequently, an effort to maximize interac-tions between the wing I aromatic residue and the tryptophan ring was attempted.For this purpose, starting from the X-ray crystal structure of 20 bound to wild-typereverse transcriptase, a chloroindole analog (21) was designed and synthesized.The X-ray crystal structures of inhibitor-bound reverse transcriptase were deter-mined. As shown in Figures 16.9 and 16.10, the structures revealed that thebound conformation of compound 21 is more extended than expected comparedwith that of compound 20 (Figure 16.9). The chloroindole wing I binds deeper inthe binding pocket, thus forcing the triazine ring deeper in the pocket too. In thisway, the horseshoe binding mode of typical triazine derivatives cannot be main-tained. Also, the interaction with Lys101 is lost for this compound.

Figure 16.8 Antiviral activities of compounds 11 and 13–19 against wild-type HIV-1 strains andstrains containing single-point mutation. Height of the bars indicates IC50 value (nM). Bar colorrefers to the HIV-1 viral strain.


Overall, the triazine compounds showed an optimized antiviral profile and verygood potency against wild-type and single-point mutant viral strains. However,activity against clinically significant double-mutant strains was not satisfactory.Following the discovery of triazine derivatives, three isomeric pyrimidine deriva-tives were synthesized and evaluated for antiviral activity (22–24, Figure 16.11).These regioisomeric compounds contain a pyrimidine central ring and pendantaromatic rings showing the same substitution pattern of the reference triazinederivative 11. As shown, two of the pyrimidine isomers (22 and 23) showed com-parable potency against wild-type HIV-1. However, pyrimidine 24 did not show

Figure 16.9 Binding mode of inhibitor 20 to the allosteric binding pocket of the wild-type HIVreverse transcriptase (PDB code: 1S9E).

Figure 16.10 Binding mode of inhibitor 21 to the allosteric binding pocket of the wild-type HIVreverse transcriptase (PDB code: 1S9G).


appreciable antiviral activity even at higher concentrations. Antiviral activityagainst single-point mutant HIV-1 strains is shown in Figure 16.12. As can beseen, both compounds 23 and 24 did not exhibit measurable antiviral activityagainst viral strains tested up to 30 mM concentration.Previous structure–activity relationships have established that the 4-amino

group was not important for activity, thus, the synthesis of pyrimidine derivativeslacking the 4-amino group was carried out. The removal of the 4-amino groupfrom the above pyrimidine derivatives resulted in a very potent series of com-pounds. As shown in Table 16.1, inhibitors 25–27 exhibited good activity againstwild-type, single-point mutants, and, most importantly, double-mutant strains [28].The X-ray cocrystal structure of the reverse transcriptase and inhibitor 25 com-

plex (Figure 16.13) revealed that the inhibitor binds to the non-nucleoside reversetranscriptase binding pocket adopting a horseshoe-shaped conformation in whichthe wing I and wing II pharmacophores interacted with lipophilic sites within thebinding pocket. Moreover, hydrogen bonding interactions with Lys101 werestrengthened [16].Trp229 is a key residue that is highly conserved in the reverse transcriptase

active site. An effort was then made to improve interactions with Trp229.Another goal was made to design inhibitors that relied less heavily on theTyr188 and Tyr181 interactions, which are mutated frequently. The introduc-tion of a cyanovinyl substituent at wing I of 25 led to the successful design ofinhibitor 5 (rilpivirine, Figure 16.1 and Table 16.1). This inhibitor wasapproved by the FDA for the treatment of HIV infection and AIDS. The X-ray

NN

NH

NH2

CN

Cl ClN NH

CN

N

NH2

Cl Cl

22 IC50 = 1.0 nM 23 IC50 = 10 nM

24 IC50 >200 nM

N

N NH

NH2

CN

Cl Cl

Figure 16.11 Structures and activities of pyrimidine inhibitors 22–24.


crystal structure of rilpivirine in complex with an engineered HIV reverse tran-scriptase enzyme was reported [26]. The X-ray structure of the inhibitor com-plex using a different crystallization approach was also reported. Bothstructures revealed an interaction of the cyanovinyl substituent with Trp229.However, different binding conformations were observed for rilpivirine in thetwo crystal structures and in the rilpivirine/mutant reverse transcriptase com-plex (Figure 16.14).The resilience of both triazine and pyrimidine classes of non-nucleoside

reverse transcriptase inhibitors was explained by taking into accounttheir flexibility. The notable activity of both etravirine and rilpivirine (4 and 5,Figure 16.1) toward several resistant strains is attributed to their ability to flexand accommodate, allowing these classes of inhibitors to adapt to modifica-tions of the shape of the binding pocket. Moreover, the flexibility of these com-pounds allows for different binding modes within the binding pocket. Thisflexibility makes it challenging to obtain high-resolution X-ray crystal struc-tures of inhibitor–enzyme complexes.The novel pyrimidine scaffold was also exploited for the design of etravirine.

In particular, the pyrimidine ring allowed for the introduction of substituents at

Figure 16.12 Antiviral activities of compounds 22–24 against wild-type HIV-1 and strains con-taining single-point mutation. Height of the bars indicates IC50 value (nM). Bar color refers tothe HIV-1 viral strain. n.a.¼not active at concentrations >30 mM.


the 5-position of the pyrimidine ring, thus exploring a new region of the bindingpocket. This was previously inaccessible for the triazine derivatives. Introductionof specific substituents at the C-5 of the pyrimidine ring resulted in an improve-ment of antiviral potency. As shown in Figure 16.15, all inhibitors (28–31 and 4)have shown excellent antiviral activity against wild-type HIV-1.These compounds were then examined against single-point mutant and double-

mutant strains. As can be seen in Table 16.2, although several inhibitors showedgood profiles, compound 4 exhibited the best profiles against single-point mutantand double-mutant virus strains. Clinical development of compound 4 proceeded,and it was subsequently named as etravirine (compound 4).Both etravirine (inhibitor 4) and rilpivirine (inhibitor 5) have been approved by

the FDA as second-generation non-nucleoside reverse transcriptase inhibitors forthe treatment of patients with HIV infection and AIDS. Etravirine shows a

Table 16.1 Antiviral potency of compounds 25–27.

IC50 (nM)

Inhibitor structure HIV-1 L100I K103N Y181C Y188L L100IþK103N

K103NþY181C

1.0 18 4.3 7.5 48 >10 000 44

26

HN N NH

CNCN

N

Me Me

0.4 34 1.9 7.1 7.8 1086 37

1.1 73 2.7 37 19 798 94

Rilpivirine (5) 0.4 0.4 0.3 1.3 2.0 — 1.0


higher genetic barrier to the selection of resistant viral strains with respect to first-generation non-nucleoside reverse transcriptase inhibitors. Most importantly, etra-virine maintains its efficacy against K103N mutants [29]. Rilpivirine has beenapproved for the treatment-naïve patients. In phase III clinical studies, the efficacyof rilpivirine was compared with that of efavirenz. At a dose of 25mg once daily(in association with two nucleoside/nucleotide reverse transcriptase inhibitors),rilpivirine showed to be equipotent to efavirenz (600mg once daily) and showedbetter tolerability [30,31].

16.4Conclusions

Both etravirine and rilpivirine are second-generation non-nucleoside reverse tran-scriptase inhibitors with diarylpyrimidine scaffolds. These inhibitors weredesigned using structure-based design strategies. Both inhibitor drugs bind to anallosteric binding site of reverse transcriptase. This allosteric binding site islocated close to the active site, but it is distinct from the active site. One particu-larly interesting feature of these inhibitors is that both inhibitors are conforma-tionally flexible and can adapt to the reverse transcriptase binding site. Thisfeature enables these inhibitors to make a more robust interaction with theenzyme even in the presence of mutations. Etravirine (Intelence) was approved by

Figure 16.13 X-ray crystal structure of wild-type reverse transcriptase in complex with inhibi-tor 25. (PDB code: 1S6Q).


the FDA in 2008, and rilpivirine (Edurant) in 2011. Both drugs are active againstHIV resistant to nevirapine and efavirenz. Rilpivirine is more potent than etravir-ine presumably because of the interaction of the cyanovinyl substituent with theTrp229 and is effective against etravirine-resistant viral strains. Rilpivirine isadministered with emtricitabine (a nucleoside reverse transcriptase inhibitor) andtenofovir (a nucleotide analog reverse transcriptase inhibitor).

Figure 16.14 X-ray structures of rilpivirine (inhibitor 5) and wild-type HIV reverse transcriptase.Surface representation highlights the interaction of the cyanovinyl group (green sticks) within thechannel lined by Trp229 (PDB code: 2ZD1).

Table 16.2 Antiviral potency of compounds 28–31 and 4 (etravirine).

IC50 (nM)

Compound HIV-1 L100I K103N Y181C Y188L L100IþK103N

K103NþY181C

28 1.4 7.5 2.6 34 4.8 138 3829 1.4 6.6 1.4 22 5.9 49 2530 1.0 16 1.3 9.3 5.7 282 1431 1.9 31 3.3 30 8.4 205 304 1.4 3.3 1.2 7.0 4.6 19 4.3


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3 Frankel, A.D. and Young, J.A. (1998) HIV-1:fifteen proteins and an RNA. Annu. Rev.Biochem., 67, 1–25.

4 Goto, T., Nakai, M., and Ikuta, K. (1998)The life-cycle of human immunodeficiencyvirus type 1. Micron, 29, 123–138.

5 Jonckheere, H., Anne, J., and De Clercq, E.(2000) The HIV-1 reverse transcription (RT)process as target for RT inhibitors. Med.Res. Rev., 20, 129–154.

6 Kohlstaedt, L.A., Wang, J., Friedman, J.M.,Rice, P.A., and Steitz, T.A. (1992)Crystal structure at 3.5 A

�resolution

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7 diMarzo Veronese, F., Copeland, T.D.,DeVico, A.L., Rahman, R., Oroszlan, S.,Gallo, R.C., and Sarngadharan, M.G. (1986)Characterization of highly immunogenic

O N NH

CN

N

Me Me

28 IC50 = 1.4 nM

CN

Cl

O N NH

CN

N

Me Me

29 IC50 = 1.4 nM

CN

Br

O N NH

CN

N

Me Me

30 IC50 = 1.0 nM

CN

H2N

31 IC50 = 1.9 nM

O N NH

CN

N

NH2

Me Me

CN

Br

Etravirine4 IC50 = 1.4 nM

O N NH

CN

N

Me Me

CN

Ac-NH

Figure 16.15 Structures and antiviral activities of wild-type HIV-1 inhibitors 28–31 and 4.


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8 Mitsuya, H., Weinhold, K.J., Furman, P.A.,St Clair, M.H., Lehrman, S.N., Gallo, R.C.,Bolognesi, D., Barry, D.W., and Broder, S.(1985) 30-Azido-30-deoxythymidine (BWA509U): an antiviral agent that inhibits theinfectivity and cytopathic effect of humanT-lymphotropic virus type III/lymphadenopathy-associated virus in vitro.Proc. Natl. Acad. Sci. USA, 82, 7096–7100.

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�resolution shows

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18 Tantillo, C., Ding, J., Jacobo-Molina, A.,Nanni, R.G., Boyer, P.L., Hughes, S.H.,Pauwels, R., Andries, K., Janssen, P.A., andArnold, E. (1994) Locations of anti-AIDSdrug binding sites and resistancemutations in the three-dimensionalstructure of HIV-1 reverse transcriptase.Implications for mechanisms of druginhibition and resistance. J. Mol. Biol., 243,369–387.

19 Hsiou, Y., Ding, J., Das, K., Clark, A.D., Jr.,Hughes, S.H., and Arnold, E. (1996)Structure of unliganded HIV-1 reversetranscriptase at 2.7 A

�resolution:

implications of conformational changes forpolymerization and inhibitionmechanisms. Structure, 4, 853–860.

20 Rodgers, D.W., Gamblin, S.J., Harris, B.A.,Ray, S., Culp, J.S., Hellmig, B., Woolf, D.J.,Debouck, C., and Harrison, S.C. (1995) Thestructure of unliganded reversetranscriptase from the humanimmunodeficiency virus type 1. Proc. Natl.Acad. Sci. USA, 92, 1222–1226.

21 Campiani, G., Ramunno, A., Maga, G.,Nacci, V., Fattorusso, C., Catalanotti, B.,Morelli, E., and Novellino, E. (2002) Non-nucleoside HIV-1 reverse transcriptase (RT)inhibitors: past, present, and futureperspectives. Curr. Pharm. Des., 8, 615–657.

22 Das, K., Lewi, P.J., Hughes, S.H., andArnold, E. (2005) Crystallography and thedesign of anti-AIDS drugs: conformationalflexibility and positional adaptability areimportant in the design of non-nucleosideHIV-1 reverse transcriptase inhibitors. Prog.Biophys. Mol. Biol., 88, 209–231.

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24 Hsiou, Y., Ding, J., Das, K., Clark, A.D., Jr.,Boyer, P.L., Lewi, P., Janssen, P.A., Kleim,J.P., Rosner, M., Hughes, S.H., and Arnold,E. (2001) The Lys103Asn mutation ofHIV-1 RT: a novel mechanism of drugresistance. J. Mol. Biol., 309, 437–445.

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26 Janssen, P.A., Lewi, P.J., Arnold, E.,Daeyaert, F., deJonge, M., Heeres, J.,Koymans, L., Vinkers, M., Guillemont, J.,Pasquier, E., Kukla, M., Ludovici, D.,Andries, K., deBethune, M.P., Pauwels, R.,Das, K., Clark, A.D., Jr., Frenkel, Y.V.,Hughes, S.H., Medaer, B., De Knaep, F.,Bohets, H., De Clerck, F., Lampo, A.,Williams, P., and Stoffels, P. (2005) Insearch of a novel anti-HIV drug:multidisciplinary coordination in thediscovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2-pyrimidinyl]amino]benzonitrile (R278474, rilpivirine).J. Med. Chem., 48, 1901–1909.

27 Ludovici, D.W., Kavash, R.W., Kukla,M.J., Ho, C.Y., Ye, H., De Corte, B.L.,Andries, K., de Bethune, M.P., Azijn, H.,Pauwels, R., Moereels, H.E., Heeres, J.,Koymans, L.M., de Jonge, M.R., Van Aken,K.J., Daeyaert, F.F., Lewi, P.J.,Das, K., Arnold, E., Janssen, P.A. (2001)Evolution of anti-HIV drug candidates.Part 2: Diaryltriazine (DATA) analogues.Bioorg. Med. Chem. Lett. 11, 2229–2234.

28 Ludovici, D.W., De Corte, B.L., Kukla,M.J., Ye, H., Ho, C.Y., Lichtenstein, M.A.,Kavash, R.W., Andries, K., de Bethune,M.P., Azijn, H., Pauwels, R., Lewi, P.J.,Heeres, J., Koymans, L.M., de Jonge,M.R., Van Aken, K.J., Daeyaert, F.F.,Das, K., Arnold, E., Janssen, P.A. (2001)Evolution of anti-HIV drug candidates.Part 3: Diarylpyrimidine (DAPY)analogues. Bioorg. Med. Chem. Lett. 11,2235–2239.

29 Croxtall, J.D. (2012) Etravirine: a review ofits use in the management of treatment-experienced patients with HIV-1 infection.Drugs, 72, 847–869.

30 Imaz, A. and Podzamczer, D. (2012) Therole of rilpivirine in clinical practice:strengths and weaknesses of the newnonnucleoside reverse transcriptaseinhibitor for HIV therapy. AIDS Rev., 14,268–278.

31 Putcharoen, O., Kerr, S.J., andRuxrungtham, K. (2013) An update onclinical utility of rilpivirine in themanagement of HIV infection intreatment-naive patients. HIV AIDS, 5,231–241.


17Renin Inhibitor for the Treatment of Hypertension:Design and Discovery of Aliskiren

17.1Introduction

Renin is an enzyme produced in the juxtaglomerular cells of the kidneys. It initi-ates a cascade leading to the vasopressor effect of the renin–angiotensin–aldoster-one system [1,2]. Its physiological role is to cleave the Leu10–Val11 peptide bondof angiotensinogen, a protein produced in the liver. The hydrolytic activity of reninreleases decapeptide angiotensin I from angiotensinogen, which is converted toangiotensin II by the action of angiotensin-converting enzyme (ACE). AngiotensinII has a vasopressor effect, mainly due to direct vasoconstriction of blood vesselsand stimulation of aldosterone release. The development of ACE inhibitors for thetreatment of hypertensive patients furnished the proof of concept that interruptingthe renin–angiotensin–aldosterone system was an effective therapeutic strategy tocontrol blood pressure. However, ACE has broad substrate specificity and acts onmultiple physiological substrates with different biological activities. Consequently,this class of drugs is characterized by side effects due to inhibition effects on mul-tiple substrates of ACE. Renin is an aspartic protease with extremely high selectiv-ity, since its only known physiological substrate is angiotensinogen [1]. For thisreason, the enzyme represented an attractive target for medicinal chemists todevelop antihypertensive drugs devoid of the classic side effects of angiotensin-converting enzyme inhibitors.

17.2Structure of Renin

The three-dimensional structure of human recombinant renin [3,4] has a fold sim-ilar to the class of monomeric aspartyl proteases and is mainly composed ofb-strands that fold to form two similar domains (Figure 17.1). Each domain con-tributes one catalytic aspartate to the active site. The catalytic site is located at thecenter of the substrate binding cleft that runs along the intersection of the twodomains. Residues 71–81 form the flap of the enzyme that closes the active sitecleft from the N-terminal side.

373


17.3Peptidic Inhibitors with Transition-State Isosteres

The first design approach for the development of renin inhibitors was the synthe-sis of peptidic molecules bearing the cleavage sequence recognized by renin andreplacing the scissile peptide bond with a transition-state analog [5,6]. The mini-mum peptide sequence that can be cleaved by renin is an octapeptide correspond-ing to amino acids 6–13 of the natural substrate angiotensinogen (compound 1 inTable 17.1, the peptide sequence corresponds to equine angiotensinogen) [7].Based on the sequence of this octapeptide and replacing the P0

1 Leu residue withVal, which is the corresponding residue in human angiotensinogen, transition-state analogs at the scissile bond were synthesized and found to be highly potentrenin inhibitors. Examples of such inhibitors are reported in Table 17.1 wherecompound 2 presents a statine transition-state analog [5].This statin moiety is found twice in the sequence of the nonspecific aspartyl

protease inhibitor pepstatin. Pepstatin is a compound isolated from cultures ofStreptomyces, and although the natural compound is a poor inhibitor of renin,introduction of the statine as a nonscissile moiety of the P4 � P0

3 angiotensin-ogen-based peptide led to the nanomolar inhibitor 2. In compound 3 [8], thetransition-state analog is a reduced peptide, in which the carbonyl group of the scis-sile bond is replaced by a methylene group, resulting in a secondary amine.

Figure 17.1 X-ray crystal structure of recombi-nant human renin. Catalytic aspartates are rep-resented as sticks. Amino acids forming theS1–S4 and S1�S03 pockets are represented as

colored spheres. S4: yellow; S3: green; S2: cyan;S1: magenta, S01: orange; S

02: red; S

03: blue. Flap

residues are colored as orange strip (PDBcode: 2REN).

374 17 Renin Inhibitor for the Treatment of Hypertension: Design and Discovery of Aliskiren

The affinity of this moiety for the enzyme is thought to be dependent upon the for-mation of an electrostatic interaction between the protonatable secondary amine andthe active site aspartates. The third example of a transition-state analog is a hydroxy-ethylene isostere, in which the NH group of the peptide bond has been removed and

Table 17.1 Structures and inhibitory activities of pseudopeptide renin inhibitors with varioustransition-state analogs.

Structure (P5–P4–P3–P2–P1#P01–P02–P03–) IC50 (nM)

His–Pro–Phe–His–Leu#Val–Ile–His– (human angiotensinogen residues 6–13)

N

O

HN

N

NH

HN

NH

O

O

OHN

O

HN

N

HN

NHO

O

O

HN

N

H2NOH

1

—

H2NN

O

HN

N

NH

HN

O

O

OHHN

O

HN

N

HN

NHO

O

O

HN

NO

NH

OH

2

17

NH

N

O

HN

N

NH

HN

NH

O

O

HN

O

HN

N

HN

NHO

O

O

HN

N

HN

O

HN OH

O

NH2

3

10

NH

N

O

HN

N

NH

HN

O

O

OHHN

O

HN

N

HN

NHO

O

O

HN

N

OHO

O

4

0.7

17.3 Peptidic Inhibitors with Transition-State Isosteres 375

the resulting carbonyl moiety is reduced to a stereochemically defined hydroxylgroup (inhibitor 4) [9]. This modification led to one of the most potent renininhibitors with subnanomolar inhibitory potency. Small modifications of theoriginal substrate octapeptide sequence in inhibitors 3 and 4 were introducedin order to solve solubility issues related to the original peptide sequences.Examples of such modifications include capping the N-terminal residue by aBoc group (e.g., compound 4) or introducing a lysine at the C-terminal end ofthe peptide as in compound 3.

17.4Peptidomimetic Inhibitors

The high molecular weight of inhibitors, such as compounds 2–4, and theirstrong peptidic character were not suitable for the development of antihyper-tensive drugs. Antihypertensive therapy requires good stability and oral availa-bility. Some peptidic renin inhibitors showed moderate activity in vivo [5,6],furnishing the proof of principle of renin inhibition for the treatment ofhypertension. However, many pharmaceutical laboratories tried to modify thestructure of the above peptides by developing peptidomimetics characterizedby a lower molecular weight and a reduced peptidic character. The researchefforts around this topic were intense and led to the selection of several clini-cal candidates. The structures of some of these inhibitors (5–9) are shown inFigure 17.2 [10–13].Starting from the pseudopeptide inhibitor 10 (Table 17.2), which contains a

hydroxyethylene isostere as the transition-state analog, researchers at Ciba-Geigy(now Novartis) studied the structure–activity relationships for the various groupsspanning the S3 � S03 region of the enzyme. Attempts were made to reduce themolecular size of the inhibitors, while maintaining inhibitory potency [14].Initially, the carbamate group of 10 was replaced by a pivaloyl amide (11a,

Table 17.2). It was discovered that the NH amide group probably did not functionas a hydrogen bond donor group since it could be replaced by an oxygen (11b)without loss of affinity for the enzyme. Substituting the P0

2 and P03 amino acids

with n-butylamide led to an equipotent inhibitor (12 versus 11b). An improvementin activity was obtained by replacing the Leu side chain with a cyclohexyl moiety atP1 (inhibitor 13), a strategy previously has been applied to hydroxyethylene ana-logs [15,16]. The chemically labile ester group of 13 was converted into the isos-teric ketone (14) without loss of potency. Finally, sulfone (15a), sulfoxide (15b),and sulfide (15c) groups were all tolerated in place of the carbonyl group. Theselatter compounds were all tested as racemates, although a stereoselective interac-tion was observed since the pure enantiomers of sulfone 15a displayed a fivefolddifference in potency.Compound 15a was selected for further tests and was demonstrated to inhibit

plasma renin activity in sodium-depleted marmosets after i.v. or oral administra-tion and lowered their blood pressure. However, in vivo studies in laboratory


animals revealed that compound 15a possessed inadequate oral bioavailability andshowed high biliary excretion. It also lacked suitable pharmacokinetic propertiesfor further development. For similar reasons, the clinical evaluation of compounds5–9 was also dropped by other pharmaceutical laboratories.The X-ray crystal structure of inhibitor 15a in complex with human recombinant

renin was solved at 2.4 A�resolution [17]. The inhibitor occupies the substrate

binding cleft in an extended conformation, as shown in Figure 17.3. The hydroxylgroup of the inhibitor mimicking the tetrahedral intermediate is positioned equi-distant from the catalytic Asp32 and Asp215, forming hydrogen bonds with bothcarboxylate groups.Other important hydrogen bonds are engaged between the inhibitor backbone

and residues Ser219, Thr77, and Ser76 on the nonprime side and Gly34 on theprime side. The histidine heterocyclic nitrogen forms a further hydrogen bond

NH

HN

O

O

OH

S

HN

N

OH

IC50 = 1.0 nM

NH

HN

O

O

OH

SO2

S

N

N

OH

MeNMe Me

NH

HN

O

O

OH

HN

N

N

IC50 = 0.5 nM

O

O

S NN

NN

Me

NH

HN

O

O

OH

S

8

Me

OHN

O

Me

N

HN

O

O

OH

S

N

OH

IC50 = 1.4 nM

NH2

N

OMe

N

OO

5IC50 = 1.1 nM

6

7IC50 = 3.3 nM

9

OO

Figure 17.2 Structures of selected peptidomimetic renin inhibitors as preclinical or clinicalcandidates.

17.4 Peptidomimetic Inhibitors 377

Table 17.2 Structures and inhibitory activities of compounds 10–15 [14].

Compound Structure X IC50 (nM)

10NH

HN

O

O

OHHN

HN

N

HN

NHO

O

O

HN

N

NH2O

O

— 15

11a11b N

H

HN

O

O

OHX

HN

N

HN

NHO

O

O

HN

N

NH2

O

X¼NHX¼O

20

20

1213 N

H

HN

O

O

OHO

HN

N

X

HN

OO

MeX¼ iPrX¼Cy

207

14NH

HN

O

O

OH

HN

N

HN

OO

Me — 6

15a15b15c N

H

HN

O

O

OH

X

HN

N

HN

O

Me

X¼SO2

X¼SOX¼S

2

24


with Ser222. The surface representation of inhibitor 15a bound to renin is shownin Figure 17.4. As can be seen, the isopropyl group occupies the S01 pocket,whereas the other two hydrophobic groups (the cyclohexylmethyl and the benzyl)fill in the S1 and S3 pockets, respectively. The n-butyl group does not form exten-sive interactions within the binding site.

Figure 17.3 X-ray crystal structure of 15a and renin complex. Inhibitor’s carbon chain, pink; thecatalytic aspartates, green; all hydrogen bonding interactions are shown as dotted lines (PDBcode: 1RNE).

Figure 17.4 Surface representation of 15a and renin complex. Enzyme residues forming the S3subsite, yellow; residues forming the S1 pocket, red; residues belonging to both sites, red rectan-gle (PDB code: 1RNE).

17.4 Peptidomimetic Inhibitors 379

17.5Design of Peptidomimetic Inhibitors

One striking and important structural feature that was noted from the renin andinhibitor complexes [17,18] was the size and contiguity of the S1 and S3 bindingpockets. These two large, hydrophobic pockets when, taken together, form anextended “superpocket.” Based on this structural information, some inhibitordesign approaches were based on the chemical connection of the two hydrophobicP1 and P3 moieties of peptidic inhibitors forming P1–P3 extended moieties. Thisapproach was aimed at emphasizing hydrophobic enzyme–inhibitor interactions,thus giving access to truncation strategies in order to reduce the size and peptidiccharacter of the previous series of renin inhibitors [19–21]. Some examples ofthese early P1–P3 extended inhibitors are shown in Figure 17.5. Starting frominhibitor 16, the cyclohexyl and the phenyl moieties were directly connectedthrough an appropriate linker. The bond between the benzylic moiety and the

NHN

NH

HN

OH

OH

O

O

O

SMe

O

16

NSO2

HN

NH

HN

OH

OH

O

OO

NHN

NH

HN

OH

OH

O

O

O

SMe

O

17a IC50 = 110 nM

Me NH

HN

OH

OH

O

OS

Me

IC50 = 0.3 nM18

IC50 = 11 nM

NHN

NH

HN

OH

OH

O

O

O

S

Me

O

17b IC50 = 130 nM

X

19a IC50 = 1600 nM

Me NH

HN

OH

OH

O

OS

Me

19b IC50 = 1100 nM

Figure 17.5 Design approach and structures of P1–P3 extended peptidic renin inhibitors.


amino acid a-carbon was then cleaved, leading to inhibitors 17a and 17b. Com-pound 18 was developed following a similar approach. Inhibitors 17a and 17bwere then truncated at the P1 residue leading to moderately active lead com-pounds 19a and 19b, with reduced molecular weight and fewer peptide bondscompared with 17a and 17b.Optimization of compound 15a was undertaken to develop nonpeptidic com-

pounds as renin inhibitors. As depicted in Figure 17.6, inhibitor 15a was taken asthe reference structure and the goal was to remove the P2 and P4 groups togetherwith their peptide linker to provide nonpeptide inhibitors. Inhibitor’s hydrophobicinteractions were planned to be optimized by linking the P1 and P3 groups. It wasassumed that an appropriate linker would be able to correctly orient these twomoieties in respective binding pockets S1 and S3. In order to improve potency, thegoal was also to place specific hydrogen bond donor/acceptor groups at theextended P1–P3 lipophilic moiety that could target the Ser219 residue [22]. More-over, optimization of specific physicochemical properties such as logP, aqueoussolubility, and polar surface area could be achieved by appropriate substitution ofP02 n-butylamino group, which did not form specific contacts within the binding

cavity. Initial investigation of this design approach was performed on compoundswith the general structures shown in Figure 17.7.The inhibitory activities of compounds 20a–20d, belonging to series A, are

reported in Figure 17.8 [23]. The P1 cyclohexylmethyl moiety is connected through

Figure 17.6 Design strategy that led to the discovery of nonpeptidic renin inhibitors.

17.5 Design of Peptidomimetic Inhibitors 381

HN

O

MeOHH2N

R

Me

X

MeX

N

Me MeO

(a) (b)

(c) (d)

P3

P3 P3

P3P1P1

P1P1

Figure 17.7 General structures of early nonpeptidic renin inhibitors developed by researchers atCiba-Geigy.

HN

O

MeOHH2N

R

Me

HO

20aIC50 = 0.3 µM

20bIC50 = 2.9 µM

20cIC50 = 4.3 µM

20dIC50 = 13 µM

21aIC50 = 0.7 µM

21bIC50 = 2.1 µM

R =

21cIC50 = 3 µM

21dIC50 = 10 µM

Figure 17.8 Structures and inhibitory activities of compounds 20a–20d and 21a–21d.


a three-methylene linker to the P3 residue, in this case represented by a tert-butylgroup. A trans-1,3-disubstituted cyclohexyl junction led to submicromolar inhibi-tor 20a, which proved to be the optimal linker, as both the cis-1,3- and trans-1,4-disubstituted derivatives (20b and 20c, respectively) exhibited poor inhibitoryactivity with respect to 20a. Also, replacement of the cyclohexyl ring with a planar1,3-disubstituted aromatic ring led to a drop in potency (20d).Derivatives 21a–21d contain a different P1–P3 extended hydrophobic group, in

which two geminal methyl groups occupy the S1 pocket and are connectedthrough a methylene linker to an a-naphthyl ring that is projected into the S3binding site [23]. The two-methylene linker is the tether of choice (21a versus21b) while a reduction of activity is observed when the a-naphthyl is replacedwith a b-naphthyl group (21a versus 21c). Introduction of a hydroxymethyl moietyat the naphthyl ring (21d) was designed to form a hydrogen bond with Ser219.However, this modification did not improve potency and it is likely that thedesired hydrogen bond did not take place.In order to form hydrogen bonds with Ser219, a series of tetrahydroquinoline-

based P1–P3 scaffolds were designed. As shown in Figure 17.9, when the naphthylring of 21a was replaced by a tetrahydroquinoline ring connected through a 2Clinker to the quaternary carbon, inhibitor 22 showed an IC50 of 50 nM [22]. Theimportance of the tetrahydroquinoline moiety was investigated by synthesizingthe corresponding aniline derivative (23), which showed an order of magnitudedrop in potency. Alternatively, almost two orders of magnitude increase in inhibi-tory potency was achieved by incorporating a stereochemically defined methyl

HN

O

MeOHH2N Me

O

N

CO2Me

HN

O

MeOHH2N Me

O

N

HN

O

MeOHH2N Me

O

HN

22IC50 = 0.05 µM

23IC50 = 0.4 µM

24IC50 = 0.8 nM

Figure 17.9 Structures of tetrahydroquinoline-based inhibitors 22–24.


ester group at the C-3 of the tetrahydroquinoline ring (24). Modeling studies pre-dicted that the carboxylic oxygen of the ester group was in the appropriate positionto form a hydrogen bond with Ser219.To obtain insight into the binding properties of the inhibitor in renin active site,

the X-ray crystal structure of inhibitor 24 and renin complex was determined [24].Analysis of the X-ray structure revealed an unexpected interaction of the carboxy-methyl group with a previously known S3 subpocket (S

sp3 ) [25], located perpendicu-

larly with respect to the binding cleft (Figure 17.10). This subpocket was nottargeted by any previously known renin inhibitor. The Ssp3 is directed toward theinterior of the enzyme and has a depth of 9A

�. Besides the interaction with the S3

subpocket, compound 24 also formed several hydrogen bonds in the active site.The primary amine group formed hydrogen bonds with both catalytic aspartatesand the carbonyl oxygen of Gly217. The transition-state hydroxyl group formed ahydrogen bond with Asp32. The carbonyl group of the P1–P3 ester appeared toform hydrogen bonds with Ser219 backbone NH as well as with the side chain ofthe hydroxymethyl group.A surface representation of the X-ray structure of inhibitor 24-bound renin is

shown in Figure 17.11. The binding cavities of S1, S3, and Ssp3 are marked and therespective ligands appear to fill these binding pockets. In particular, the methylester functionality on the tetrahydroquinoline ring nestles in the S3 subpocket andforms hydrogen bonds with Ser219 in the subpocket.In addition to the tetrahydroquinoline series of inhibitors, another class of

P1–P3 extended inhibitors was investigated. This class of inhibitors is character-ized by the presence of a benzyl group as the linker projecting either a phenyl ora tert-butyl substituent toward the S3 pocket [26]. The structures of the lead

Figure 17.10 X-ray crystal structure of inhibitor 24 and renin complex. Inhibitor carbon chain,yellow; catalytic aspartates, cyan; hydrogen bonding interactions, dotted lines (PDB code: 2V16).


inhibitors 25 and 26 for this series are shown in Figure 17.12. Both inhibitorswere relatively less potent than the tetrahydroquinoline-based inhibitor 24. How-ever, optimization of substituents in the P1–P3 ligand was planned to improvepotency.The synthesis and biological studies of a series of inhibitors containing various

sterically demanding alkyl and aromatic substituents were carried out [26]. Theresults are summarized in Table 17.3. It appeared that substitution of the methylgroup of 26 with an ethyl (27a) or an isopropyl group (27b) led to improvement ofthe binding affinity. The limits of the S1 pocket size were examined by incorporat-ing sterically demanding groups. Inhibitors 27c and 27d exhibited a decrease inaffinity. The optimal substituent was the isopropyl group in inhibitor 27b. A phe-nyl ring in compound 27e showed a significant loss of potency.

Figure 17.11 Surface representation of the X-ray crystal structure of inhibitor 24 and renin com-plex (PDB code: 2V16).

HN

O

MeOHH2N Me

Me

HN

O

MeOHH2N Me

Me

25IC50 = 3 µM

26IC50 = 2 µM

Figure 17.12 Structures and activities of inhibitors 25 and 26.


The next round of optimization of the extended P1–P3 substituent was focusedon the basis of structural information obtained from the tetrahydroquinolineseries of compounds. It was speculated that introduction of specific substituentson the aromatic ring, capable of forming a hydrogen bond with Ser219, could leadto further enhancement of potency. Molecular modeling and docking studies sug-gested that an appropriately tethered hydrogen bond acceptor group, such as anester or an amide functionality at the meta-position of the aromatic ring (ortho-position to the tert-butyl group) of inhibitor 26, could form the expected hydrogenbond with Ser219. As shown in Table 17.4, the corresponding inhibitors 28a–28dshowed a significant improvement in affinity. Incorporation of an ester group onthe aromatic ring using a methoxy linker provided compound 28a with significantenhancement of potency in purified recombinant renin [26,27]. With the excep-tion of the carboxylic acid derivative 28d, all inhibitors displayed single- or double-digit nanomolar potency.In order to overcome potential problems related to biological stability of the

compounds, researchers examined inhibitory activity not only using purifiedrecombinant human renin in buffer but also in the presence of human plasma.As can be seen in Table 17.4, a reduction of inhibitory potency was also observedwhen the compounds were measured in the presence of plasma. Subsequently,determination of IC50 values was carried out in both purified recombinant reninand plasma renin. An improved plasma renin activity was chosen as an importantcriterion for selection of compounds for in vivo studies.The reduction of inhibitory potency in plasma could be related to instability of

the compounds in plasma, or it could be related to inhibitor binding to plasmaproteins. Regardless, this could effectively reduce the amount of inhibitor availa-ble for enzyme inhibition. This issue is related to the overall lipophilicity of thedrug. In an attempt to reduce the lipophilicity of the compounds, the tert-butylmoiety linked to the aromatic ring was replaced by a less hydrophobic methoxy

Table 17.3 Structures and inhibitory activities of compounds 26 and 27a–27e.

HN

O

MeOHH2N Me

R

Compound R IC50 (mM)

26 Me 227a Et 0.827b Me2CH 0.127c Me2CH CH2 427d Me3C 1.527e Ph 39


group [27]. The results are shown in Table 17.5. Compound 29a showed compara-ble potency to inhibitor 28a with a tert-butyl group in both the purified humanenzyme and plasma renin. The data for compounds 29b and 29c were also encour-aging as well, indicating that the lipophilic pocket of S3 could accommodate themethoxy group without dramatic loss of potency.The methoxy series of compounds was then subjected to further optimization

by modifications of the ortho-alkoxy substituent on the aromatic ring. The objec-tive was to find a suitable replacement of the ester or amide side chains in com-pounds 29a–29c. In particular, such substituted functionalities were expected toform hydrogen bonds with Ser219 in the Ssp3 binding cleft of renin. As shown inTable 17.6, a series of compounds were synthesized with linear alkoxy substituentsof varying chain length (four atoms: 30a and 30b; five atoms: 30c, 30d and 30e;

Table 17.4 Structures and inhibitory activities of compounds 28a–28d.

Compound IC50 (nM)

Purified Plasma

HN

O

MeOHH2N

OMeO

O

28a3 210

HN

O

MeOHH2N

OH2N

O

28b20 460

HN

O

MeOHH2N

O

SOO

28c13 160

HN

O

MeOHH2N

OHO

O

28d 120 —


six atoms: 30f; seven atoms: 30h) at this position. Moreover, the position of theoxygen was shifted along the chain (30g). As can be seen, with the exception ofcompounds 30a and 30h, all inhibitors exhibited IC50 values in the single-digitnanomolar range when tested against purified renin. However, in plasma renin, arange of potency reduction was observed. Inhibitor 30d showed the optimumresults with a plasma renin/purified renin IC50 ratio of 1.To obtain molecular insight into the binding properties of inhibitor 30d in the

renin active site, the X-ray crystal structure of 30d-bound renin was determined[24]. As shown in Figure 17.13, the structure revealed that the extended S1–S3lipophilic pocket was largely occupied by the isopropyl and substituted aromaticring of the inhibitor. The methoxy group projected toward the S3 pocket and themethoxypropyloxy substituent nestled inside the Ssp3 , mainly formed by lipophilicamino acids. Interestingly, the expected hydrogen bond between the distalmethoxy oxygen and Ser219 was not formed. Instead, the distal methoxy oxygenformed a hydrogen bond with the backbone NH of Tyr14. The contribution of thishydrogen bond to the overall binding affinity did not appear to be critical, as theinhibitor 30c with an n-pentyl chain exhibited potent activity. On the other side ofthe binding cleft, the P0

2 n-butyl group occupied the S02 pocket and the amide NHformed a hydrogen bond with Gly34, whereas the amide carbonyl formed a

Table 17.5 Structures and inhibitory activities of compounds 29a–29c.

Compound IC50 (nM)

Purified Plasma

29a

HN

O

MeOHH2N

MeO

OMeO

O

4 340

29b

HN

O

MeOHH2N

MeO

OH2N

O

92 —

29c

HN

O

MeOHH2N

MeO

O

SOO

50 220


second hydrogen bond with the backbone NH of Ser76. The transition-statehydroxyl group formed hydrogen bonds with catalytic Asp32. In contrast to theprevious inhibitor 24, the amino group is not within hydrogen bonding distanceof either catalytic aspartate. Because of this shift in the position of the hydroxyland NH2 groups and it appeared that the P0

1 methyl group only suboptimally occu-pies the S01 pocket.

Table 17.6 Structures and inhibitory activities of compounds 30a–30h.

HN

O

MeOHH2N

R

R IC50 (nM) R IC50 (nM)

Purified Plasma Purified Plasma

MeO

O

30a

OMe

11 38

MeO

O

30e

O

Me

4 32

MeO

O

30b

OH

6 36

MeO

O

30f

OMe

2 22

MeO

O

30c

Me

4 70

MeO

30g

O

O

Me

3 20

MeO

O

30d

OMe

1 1

MeO

O

30h

O

O

Me

19 90


Based upon the X-ray structural information, the P01 methyl group of inhibitor

30d was replaced with an isopropyl group. As shown in Figure 17.14, the resultinginhibitor 31 showed a slight reduction of activity in plasma renin. This was possi-bly due to increase in lipophilicity of inhibitor 31 compared with 30d. In order toimprove activity in plasma renin, modification of the P0

2 n-butylamino group wascarried out.

Figure 17.13 X-ray crystal structure of the 30d and renin complex. Inhibitor’s carbon chain, blue;enzyme residues forming hydrogen bonds, yellow; enzyme surface, white (PDB code: 2V10).

30d IC50 = 1 nM (purified renin) IC50 = 1 nM (plasma renin)

HN

O

MeHOH2N

MeO

O

O

Me

31 IC50 = 1 nM (purified renin) IC50 = 4 nM (plasma renin)

HN

O

HOH2N

MeO

O

O

Me

Figure 17.14 Structures and activities of inhibitors 30d and 31.


It was previously observed that modifications at the P02 part of the inhibitor had

less effect on the binding affinity. Thus, the optimization of the physicochemicalproperties of inhibitor was focused by modifying this section of the inhibitor[11,27,28]. Especially, polar, neutral, and acidic groups could be readily accommo-dated at this position, as well as heterocyclic substituents. In particular, it was pre-viously observed that terminal carboxamides were able to maintain their bindingaffinity in purified renin, while improving inhibitory potency in the presence ofplasma. As shown in Table 17.7, various primary and secondary carboxamideslinked to the peptide bond of the inhibitor through carbon chains of differentlengths and varying steric properties were incorporated [29]. The two best per-forming compounds were 32d and its N-methyl derivative 32f, which showed sub-nanomolar potency in both purified renin and plasma renin. Inhibitor 32d(aliskiren) was chosen for further clinical development.

Table 17.7 Structures and inhibitory activities of compounds 32a–32g.

HN

RO

HOH2N

MeO

O

O

Me

R IC50 (nM) R IC50 (nM)

Purified Plasma Purified Plasma

32a

O

NH2 3 10

32e

O

NMe

Me

3 7

32b

O

NH2Me Me

13 100

32fO

HN

MeMe

Me 0.4 0.7

32cO

HN

Me 3 2

32g

NO

3 3

HN

O

HOH2N

MeO

O

O

Me

O

NH2

MeMe

32d (Aliskiren)

IC50¼ 0.6 nM(renin)IC50¼ 0.6 nM(plasma)


The X-ray crystal structure of the aliskiren and renin complex revealed theoptimal fit of the P1–P3 hydrophobic group within the extended S1–S3 pocketand the tight occupancy of the Ssp3 by the methoxypropyloxy chain of the inhib-itor (Figure 17.15) [24,29]. The P0

1 isopropyl group occupied the S01 pocket. Fur-thermore, the structure highlights the shifting of the inhibitor position in thebinding cleft with respect to inhibitor 30d. The transition-state hydroxyl groupis positioned in a more symmetrical manner between the two catalytic aspar-tates. It formed strong hydrogen bonds with catalytic aspartate Asp32. Also,the position of the amino group of the inhibitor shifted in the binding cleftand now located within hydrogen bonding distance of the other catalytic aspar-tate Asp215. The amine group also formed a hydrogen bond with Gly217, asobserved for 30d.A number of other hydrogen bonding contacts were found in both the

prime and nonprime sides of the inhibitor binding cleft. The two phenoxyoxygens formed water-mediated hydrogen bonds with the side chainhydroxyl group of Ser219. The distal oxygen of the alkoxy chain forms ahydrogen bond with the NH backbone of Tyr14, analogous to that observedfor 30d. On the prime side, the terminal carboxy group forms a series ofwater-mediated hydrogen bonds with several amino acid residues, whereasthe P0

1 amide group forms the usual two hydrogen bonds with Gly34 andSer76. A surface representation of the X-ray structure of renin with aliskirenis shown in Figure 17.16. The S1–S3 pocket accommodated the P1–P3 hydro-phobic ligand. The extended methoxypropyloxy chain occupied the shallowSsp3 site. The P0

1 isopropyl group also optimally filled in the S01 hydrophobicpocket.

Figure 17.15 X-ray crystal structure of aliskiren and renin complex. Inhibitor carbon chain, green;hydrogen bond network is shown in dotted lines (PDB code: 2V0Z).


17.6Biological Properties of Aliskiren

Aliskiren demonstrated excellent selectivity over related human aspartic proteasessuch as cathepsin D, cathepsin E, and pepsin. It has 16% oral bioavailability in mar-mosets and 3% oral bioavailability in humans. Once-daily oral doses of aliskirenlowered blood pressure in sodium-depleted marmosets. Since aliskiren presents a100-fold higher potency in inhibiting human renin over rat renin, preclinical evalua-tion of aliskiren was performed on transgenic rats expressing the human renin andangiotensinogen genes. These transgenic rats can develop high blood pressure andend organ damage (e.g., heart and kidneys), so they are particularly useful for evalu-ating end organ protection. Untreated transgenic rats showed high blood pressure,whereas aliskiren proved to normalize blood pressure after 9 weeks. Moreover, thealiskiren-treated group showed 100% survival, compared with 100% mortality of theuntreated group and 26% mortality of a control group treated with low-dose valsar-tan (an angiotensin II receptor antagonist). Several other studies also demonstratedthe efficacy of aliskiren in reducing mortality and end organ damage [2,22,30–32].Aliskiren proved its safety and efficacy after oral administration in several clinical

trials in which it was tested alone or in combination with other antihypertensiveagents. Aliskiren has a long half-life in plasma (>20h), thus maintaining its effect forlong periods of time, and is well tolerated. This feature is particularly important sinceit increases patient compliance with therapy. Aliskiren was approved by the FDA in2007 as a first-in-class inhibitor of renin for the treatment of hypertension [2,30].

17.7Conclusions

Renin is the first enzyme in the renin–angiotensin–aldosterone system that playsan important role in blood pressure control. It was long recognized that selective

Figure 17.16 Surface representation of the X-ray crystal structure of aliskiren-bound renincomplex. Inhibitor carbon chain is shown in green (PDB code: 2V0Z).


inhibition of renin could lead to effective control of hypertension. From 1975–1995, significant early efforts on substrate-based design and then on structure-based design of renin inhibitors led to discovery of many potent renin inhibitors.Further clinical development was hampered due to high molecular weight, poorpharmacokinetic properties, poor metabolic stability, and low efficacy. Aliskiren isthe first renin inhibitor approved in 2007 as an antihypertensive agent. Aliskirenwas developed through extensive molecular modeling and structure-based designefforts. Clinical and preclinical studies indicated that aliskiren was effective inlowering blood pressure in hypertensive patients. Also, aliskiren alone or in com-bination with other agents showed renal protective effects and relief from endorgan damage. Further benefits of renin inhibitors are being investigated.

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16 Boger, J., Payne, L.S., Perlow, D.S., Lohr, N.S., Poe, M., Blaine, E.H., Ulm, E.H.,Schorn, T.W., LaMont, B.I., and Lin, T.Y.(1985) Renin inhibitors. Syntheses ofsubnanomolar, competitive, transition-stateanalogue inhibitors containing a novelanalogue of statine. J. Med. Chem., 28,1779–1790.

17 Rahuel, J., Priestle, J.P., and Grutter, M.G.(1991) The crystal structures ofrecombinant glycosylated human reninalone and in complex with a transition stateanalog inhibitor. J. Struct. Biol., 107,227–236.

18 Dhanaraj, V., Dealwis, C.G., Frazao, C.,Badasso, M., Sibanda, B.L., Tickle, I.J.,Cooper, J.B., Driessen, H.P., Newman, M.,and Aguilar, C. (1992) X-ray analyses ofpeptide–inhibitor complexes define thestructural basis of specificity for humanand mouse renins. Nature, 357, 466–472.

19 Lefker, B.A., Hada, W.A., Wright, A.S.,Martin, W.H., Stock, I.A., Schulte, G.K.,Pandit, J., Danley, D.E., Ammirati, M.J.,and Sneddon, S.F. (1995) Rational design,synthesis, and X-ray structure of renininhibitors with extended P1 sidechains.Bioorg. Med. Chem. Lett., 5, 2623–2626.

20 Plummer, M.S., Shahripour, A.,Kaltenbronn, J.S., Lunney, E.A.,Steinbaugh, B.A., Hamby, J.M., Hamilton,H.W., Sawyer, T.K., Humblet, C., andDoherty, A.M. (1995) Design and synthesisof renin inhibitors: incorporation oftransition-state isostere side chains thatspan from the S1 to the S3 binding pockets

and examination of P3-modified renininhibitors. J. Med. Chem., 38, 2893–2905.

21 Plummer, M., Hamby, J.M., Hingorani, G.,Batley, B.L., and Rapundalo, S.T. (1993)Peptidomimetic inhibitors of reninincorporating topographically modifiedisosteres spanning the P1ð! P3Þ � P0

1sites. Bioorg. Med. Chem. Lett., 3,2119–2124.

22 Cohen, N.C. (2007) Structure-based drugdesign and the discovery of aliskiren(Tekturna): perseverance and creativity toovercome a R&D pipeline challenge. Chem.Biol. Drug Des., 70, 557–565.

23 Rasetti, V., Cohen, N.C., R€ueger, H.,G€oschke, R., Maibaum, J., Cumin, F.,Fuhrer, W., and Wood, J.M. (1996) Bioactivehydroxyethylene dipeptide isosteres withhydrophobic (P3–P1)-moieties. A novelstrategy towards small non-peptide renininhibitors. Bioorg. Med. Chem. Lett., 6,1589–1594.

24 Rahuel, J., Rasetti, V., Maibaum, J., Rueger,H., Goschke, R., Cohen, N.C., Stutz, S.,Cumin, F., Fuhrer, W., Wood, J.M., andGrutter, M.G. (2000) Structure-based drugdesign: the discovery of novel nonpeptideorally active inhibitors of human renin.Chem. Biol., 7, 493–504.

25 Tong, L., Pav, S., Lamarre, D., Pilote, L.,LaPlante, S., Anderson, P.C., and Jung, G.(1995) High resolution crystal structures ofrecombinant human renin in complex withpolyhydroxymonoamide inhibitors. J. Mol.Biol., 250, 211–222.

26 G€oschke, R., Cohen, N.C., Wood, J.M., andMaibaum, J. (1997) Design and synthesis ofnovel 2,7-dialkyl substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamidesas in vitro potent peptidomimetic inhibitorsof human renin. Bioorg. Med. Chem. Lett., 7,2735–2740.

27 Goschke, R., Stutz, S., Rasetti, V., Cohen,N.C., Rahuel, J., Rigollier, P., Baum, H.P.,Forgiarini, P., Schnell, C.R., Wagner, T.,Gruetter, M.G., Fuhrer, W., Schilling, W.,Cumin, F., Wood, J.M., and Maibaum, J.(2007) Novel 2,7-dialkyl-substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamide transition statepeptidomimetics are potent and orallyactive inhibitors of human renin. J. Med.Chem., 50, 4818–4831.

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28 Boyd, S.A., Fung, A.K., Baker, W.R.,Mantei, R.A., Stein, H.H., Cohen, J.,Barlow, J.L., Klinghofer, V., Wessale, J.L.,and Verburg, K.M. (1994) Nonpeptiderenin inhibitors with good intraduodenalbioavailability and efficacy in dog. J. Med.Chem., 37, 2991–3007.

29 Maibaum, J., Stutz, S., Goschke, R.,Rigollier, P., Yamaguchi, Y., Cumin, F.,Rahuel, J., Baum, H.P., Cohen, N.C.,Schnell, C.R., Fuhrer, W., Gruetter, M.G.,Schilling, W., and Wood, J.M. (2007)Structural modification of the P0

2 positionof 2,7-dialkyl-substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: thediscovery of aliskiren, a potent nonpeptidehuman renin inhibitor active after once

daily dosing in marmosets. J. Med. Chem.,50, 4832–4844.

30 Siragy, H.M., Kar, S., and Kirkpatrick, P.(2007) Aliskiren. Nat. Rev. Drug Discov., 6,779.

31 Muller, D.N., Derer, W., and Dechend, R.(2008) Aliskiren: mode of action andpreclinical data. J. Mol. Med. (Berl.), 86,659–662.

32 Ganten, D., Wagner, J., Zeh, K., Bader, M.,Michel, J.B., Paul, M., Zimmermann, F.,Ruf, P., Hilgenfeldt, U., and Ganten, U.(1992) Species specificity of reninkinetics in transgenic rats harboringthe human renin and angiotensinogengenes. Proc. Natl. Acad. Sci. USA, 89,7806–7810.


18Neuraminidase Inhibitors for the Treatment of Influenza:Design and Discovery of Zanamivir and Oseltamivir

18.1Introduction

Influenza viruses have been responsible for severe pandemics in the last century.Three distinct serological types of influenza virus are known as types A, B, and C[1]. The type A and B influenza viruses are the most pathogenic in humans. Thetype A influenza viruses are further classified based upon the antigenic propertiesof hemagglutinin and sialidase (neuraminidase), two membrane glycoproteinsexpressed on the viral surface [2]. Sixteen antigenically distinct hemagglutinins(H1–H16) and nine distinct neuraminidases (N1–N9) have been characterized[3–5]. Several combinations of hemagglutinins and neuraminidases have beendescribed. Viruses are also further distinguished into two phylogenetically differ-ent groups based on the expressed neuraminidase: group 1 viruses express N1,N4, N5, and N8 subtypes, whereas group 2 viruses contain N2, N3, N6, N7, andN9 neuraminidase glycoproteins [5,6]. Even more interesting is that influenzaviruses change over time. These can be small, continual changes (antigenic drift)or sometimes an abrupt shift (antigenic shift) which lead to a novel virus capableof a new pandemic. The 1918, 1957, and 1968 pandemics were caused by virusescontaining H1N1, H2N2, and H3N2, respectively. Recently, viral types H5N1 andH1N1 have threatened the insurgence of a new pandemic [7–10].Historically, the first drugs identified for the treatment of influenza were aman-

tadine and rimantadine. Their mechanism of action involves the inhibition of theion channel M2. However, this ion channel is present only in type A influenzaviruses, so these drugs are not effective for the treatment of type B influenza.Moreover, resistant strains of the influenza A virus rapidly emerged after treat-ment with channel M2 inhibitors. For these reasons, research efforts for the devel-opment of new antiviral drugs, specifically addressing influenza A and B virus-mediated infections, focused on the design of novel inhibitors of hemagglutininand neuraminidase enzymes. Both enzymes recognize the N-acetylneuraminicacid (a sialic acid), which is the terminal carbohydrate unit of glycoconjugatespresent on the cell membrane of the epithelium of the upper respiratory tract.Hemagglutinin binds to N-acetylneuraminic acid (1) and anchors the virus to the

397


cell membrane. Moreover, this interaction promotes the internalization of thevirus through fusion of the virus envelope with the cell membrane of the targetcell [11,12]. The role of neuraminidase is to cleave the glycosidic bond that linksthe N-acetylneuraminic acid to the glycoconjugate, thus assisting the release ofprogeny virions from the infected cell and the movement of the virus along theepithelium of the upper respiratory tract [13,14].To date, only neuraminidase has been successfully targeted for the development

of antiviral agents for the treatment of influenza A and B [15]. The first neuramin-idase inhibitor introduced in therapy was zanamivir (2a, Figure 18.1) [16], devel-oped by GlaxoSmithKline, and was immediately followed by the discovery of thefirst orally available neuraminidase inhibitor oseltamivir (3) [17], developed byGilead. Both inhibitors were discovered through structure-based drug design.This approach benefited from the wealth of structural information available forneuraminidase, in its apo form, in complex with the natural substrate sialic acidor with inhibitors. More recently, peramivir (4) [18] has been approved for hospi-talized patients not responding to first-line therapies, whereas laninamivir(2b) [19] is currently undergoing phase III clinical trials.Neuraminidase is an integral membrane glycoprotein anchored to the viral

membrane through a hydrophobic N-terminal region. It is a tetramer whose crys-tal structure was solved at 2.9 A

�resolution in 1983 and later refined at 2.2 A

�resolu-

tion [20–22]. The X-ray structure of the N9 subtype was later resolved and did notdisplay significant structural differences with respect to the N2 subtype [23]. Eachmonomer has the shape of a propeller, resulting from the arrangement of sixidentical b-sheets, each formed by four b-strands (Figure 18.2). Among the variousinfluenza virus subtypes that have been sequenced, the main clusters of invariant

O CO2HOH

H

OH

OH

OH

OH

HN

N-acetylneuraminic acid (1)

O CO2HH

HN

OR

OH

OH

HN

R = H, zanamivir (2a)R = Me, laninamivir (2b)

NH2

NH

CO2EtH

NH2

O

HN

oseltamivir (3)

Me

Me

OMeOMe

OMe

OH

CO2H

HN

NH

Me

Me

O

H2N NH

Me

peramivir (4)

Figure 18.1 Chemical structures of N-acetylneuraminic acid (1), zanamivir (2a), laninamivir(2b), oseltamivir (3), and peramivir (4).

398 18 Neuraminidase Inhibitors for the Treatment of Influenza

residues are located within the sialic acid binding site. Sialic acid binds to theenzyme, as shown in Figure 18.3 [24]. The carboxylate group of N-acetylneura-minic acid (1) is held in place by a cluster of three conserved arginines. The acety-lamido group forms two hydrogen bonds with the enzyme: one through thenitrogen atom that makes contact with a water molecule, and another between the

Figure 18.2 Structure of neuraminidase (PDB code: 1NN2).

Figure 18.3 Binding mode of N-acetylneuraminic acid (1) (PDB code: 2BAT).

18.1 Introduction 399

carbonyl oxygen and Arg152. The methyl group fits into a hydrophobic pocket cre-ated by Trp178 and Ile222 residues. The terminal hydroxyl groups of the polyalco-holic chain at C-6 of the pyranose ring form a bidentate hydrogen bond with thecarboxylate group of Glu276.The bound conformation of the pyranose ring on 1 is distorted to a boat

conformation (Figure 18.4), with the carboxylic acid in a pseudoequatorialposition. The carboxylic acid of nonbonded sialic acid has an axial orientation[24,25]. Neuraminidase catalyzes the hydrolysis of the glycosidic bond linkingthe terminal N-acetylneuraminic acid to the remaining glycoconjugate attachedat the surface of the host cell membrane. The mechanism of hydrolysis of theglycosidic bonds appears to involve the formation of a sialosyl cation (oxocar-benium ion, Figure 18.4), in which the positive charge on the intermediate isstabilized by a negatively charged environment [15,26,27]. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (5, Figure 18.4), one of the first neuraminidase inhibitorsidentified, was developed through a transition-state analog design approach inwhich the unsaturated pyranose ring was able to mimic the binding conformationof the putative oxocarbenium ion intermediate. The X-ray crystal structure of neur-aminidase complexed with 5 displays a binding mode overlapping that of sialicacid 1 (Figure 18.5) [28].

free conformation of 1 enzyme-bound conformation of 1

oxocarbenium ion (sialosyl cation)

transition-state analog (5)

O CO2HH

OH

OH

OH

OH

HN

OMeKi = 4.0 µM

O

CO2H

OH

HO

OHHO

HO

AcHN

OHHO

HO

OCO2H

OH

O

HOHO

HO

OH

AcHN

CO2H

HO

AcHN

O

HOHO

HO

OH

AcHN

CO2H

Figure 18.4 Schematic representation of free and bound conformation of 1, oxocarbenium ionintermediate, and transition-state analog 5.


18.2Discovery of Zanamivir

The design strategy that led to the discovery of zanamivir was based on the availa-ble structural data regarding the enzyme, in both its apo form and when bound toinhibitor 5 or to sialic acid (1). Initial designs relied upon molecular modelingstudies [16,26,29]. The surface of the enzyme in the sialic acid binding pocket wassampled through the program GRID. This program is used to calculate the energyof interaction between the enzyme surface and probes characterized by differentchemical properties. The binding pocket surface was sampled with polar, basic,acidic, and hydrophobic probes. In particular, three different hot spots on thesialic acid binding site surface were able to establish favorable interactions with apositively charged amino probe (Figure 18.6). One of these hot spots was localizedaround the binding pocket of the 4-OH group of sialic acid. It was speculated thata basic substituent such as an amino group, when placed at the C-4 of the pyra-nose ring and having the same stereochemistry as the 4-OH group, could form ahydrogen bond with Glu119.The synthesis and biological evaluation of the 4-amino derivative 6 [16] sup-

ported the design hypothesis (Figure 18.7). A second hot spot was located beneaththe C-4 of the pyranose ring, opposite to the hydroxyl group. This led to the syn-thesis of the 4-aminoepimeric derivative of 6 (7) that displayed improved activity

Figure 18.5 Bound conformation of 1 in PDB code: 2BAT (left), 5 in PDB code: 1NNB (right),and 1/5 overlap (center).

18.2 Discovery of Zanamivir 401

compared with transition-state analog 5 (Ki¼ 4 mM). However, it showed loweractivity compared to derivative 6 [29]. In an effort to interact with Glu227, replace-ment of the amine group of 6 with a more basic and larger group was planned.Introduction of a guanidine group at C-4 resulted in a very potent inhibitor 2a.This was later developed as zanamivir (2a).

Figure 18.6 Highlight of the environment surrounding the hydroxyl group at C-4 of sialic acid 1bound to neuraminidase (PDB code: 2BAT).

O CO2HH

NH2

OH

OH

OH

HN

OMe

O CO2HH

NH2

OH

OH

OH

HN

OMe

O CO2HH

HN

OH

OH

OH

HN

zanamivir (2a)Ki = 0.03 nM

NH2

NHOMe

7Ki = 300 nM

6Ki = 40 nM

Figure 18.7 Structures and activities of neuraminidase inhibitors 6, 7, and 2a (zanamivir).


The X-ray crystal structure of zanamivir-bound neuraminidase (Figure 18.8)revealed the molecular interactions of zanamivir within the active site [16,30].Interestingly, the pattern of interactions for the guanidino group appeared to bedifferent from what was speculated by docking studies. In particular, the pre-sumed hydrogen bonding interaction of the guanidine moiety with the Glu119did not form since the Glu side chain moved slightly apart from the guanidinegroup. However, salt bridge and hydrogen bonding interactions were observedwith Glu227. The carboxyl group remained within proximity to engage in an elec-trostatic interaction with the arginine residues.These inhibitors were shown to be effective against influenza A and B viruses.

Furthermore, they were shown to be selective in inhibiting viral, but not mammalianor bacterial neuraminidases. The amino acid composition at the C-4 binding pocketis conserved in different strains of virus A and B neuraminidases. The equivalentbinding site in bacterial and mammalian neuraminidases is different, whichaccounts for the selectivity of zanamivir toward viral neuraminidases [31].An intranasal administrative route was developed in mice since this class of com-

pounds was expected to have limited bioavailability. The intranasal administrativeroute turned out to be successful for zanamivir. Inhibitor 6, which lacked selectivity,showed no activity after in vivo administration against murine viral infection models.However, it showed efficacy when administered via intranasal administration [16,29].

18.3Discovery of Oseltamivir

Developing an orally available neuraminidase inhibitor was the main goal ofthe research program that led to the discovery of oseltamivir (3) [17,32].

Figure 18.8 X-ray crystal structure of neuraminidase and zanamivir complex (PDB code: 1NNC).

18.3 Discovery of Oseltamivir 403

Research efforts in this direction started from the available X-ray crystal struc-tures of the enzyme bound to sialic acid, as well as from the analysis of thepyranose ring conformation following enzyme binding in the X-ray crystalstructure of the 1 and neuraminidase complex. The goal was to mimic thealtered bound conformation of sialic acid using a carbocyclic template contain-ing an endocyclic double bond. The choice of the carbocyclic template was dueto the notion that a carbocyclic ring with an endocyclic double bond would bechemically more stable than the unsaturated pyranose ring of 5. Moreover, pre-vious investigations already outlined that the stereochemistry of the substitu-ents on the carbocyclic scaffold was important for activity. Previous attempts atmimicking the pyranose ring with a benzene ring containing appropriate polarfunctionalities were not able to interact with the polar binding pocket of theenzyme and failed to show enzyme inhibition.Regarding the relative position of the endocyclic double bond with respect to

the carboxylic acid substituent, two possible isomeric carbocyclic structureswere initially designed (8 and 9, Figure 18.9) [17]. Structure 8 was a mimic ofthe transition-state oxocarbenium ion intermediate shown in Figure 18.4,whereas structure 9 presented a double bond at the same position as theknown inhibitor and transition-state mimetic 5. Functional groups appendedto the carbocyclic ring were chosen based on available information regardinginteractions of the enzyme with the specific functional groups. In particular,the carboxylic acid was retained as this functionality interacts with the three-asparagine cluster. The 4-amino group with appropriate stereochemistry wasalso preserved as it was previously demonstrated to boost the inhibitorypotency by forming electrostatic and hydrogen bonding interactions within theacidic binding pocket. Furthermore, the acetylamido group was maintained atthe carbocyclic ring with the appropriate stereochemistry as it formed criticalinteractions in the active site. In place of the glycerol-type aliphatic chain of 5,a hydroxyl group was inserted at C-3, to mimic the electronic features of thetransition-state double bond. Two simple hydroxyl derivatives were preparedand tested in order to understand which regioisomer was more suitable forinteracting with the enzyme. Compound 8 was the most active [17], whereascompound 9 did not show inhibitory activity even when tested at higherconcentrations.The lack of oral bioavailability of previous neuraminidase inhibitors was attrib-

uted to the presence of a number of hydrophilic groups, which was hinderingabsorption through cell membranes. It was recognized that the lipophilic and

CO2H

H2N

AcHNHO

CO2H

H2N

AcHNHO

8 IC50 = 6300 nM 9 not active

13

Figure 18.9 Structures and activities of carbocyclic inhibitors 8 and 9.


hydrophilic balance of the newly designed compounds was an important featurethat needed optimization. In order to improve lipophilicity, researchers at Gileaddecided to optimize the glycerol-like aliphatic chain. From the X-ray crystal struc-ture studies, it was evident that the proximal hydroxyl group of the glycerol-typechain of 1 or 5 was not engaged in polar interactions with the enzyme residues.The terminal hydroxyl group appeared to form a bidentate hydrogen bond withGlu276. It was also noted that one of the carbon atoms of the glycerol-type chainwas able to form a hydrophobic interaction with the enzyme. Based upon thismolecular insight, the researchers elected to optimize hydrophobic interactionswith the enzyme by exploring different aliphatic ethers appended at the 3-positionof the cyclohexene skeleton [17].As shown in Figure 18.10, the C-3 hydroxyl group of 8 was converted to

methyl ether 10a, and this resulted in improved potency. Subsequently, start-ing from methyl ether 10a and gradually increasing the length of the aliphaticchain, a steady increase in activity of the resulting compounds was observed(10a–10c). This result suggested that the chain was filling a hydrophobicpocket at that position. The increase in activity was observed with chainlengths up to three carbons, whereas the n-butyl chain (10d) resulted in a

CO2H

NH2

HN

O

Me O

10a IC50 = 3700 nM

MeCO2H

NH2

HN

O

Me O

CO2H

NH2

HN

O

Me O

CO2H

NH2

HN

O

Me O

Me

10b IC50 = 2000 nM 10c IC50 = 180 nM

CO2H

NH2

HN

O

Me O

CO2H

NH2

HN

O

Me O

Me

Me

Me

CO2H

NH2

HN

O

Me O

Me

10f IC50 = 10 nM 10e IC50 = 200 nM 10d IC50 = 300 nM

10g IC50 = 9 nM 10h, IC50 = 1 nM

Me

Me

Me

MeCO2H

NH2

HN

O

Me O

Me

Me

Figure 18.10 Structures and activities of neuraminidase inhibitors 10a–10h.

18.3 Discovery of Oseltamivir 405

decrease in activity (10d versus 10c). Interestingly, branching of the chainresulted in a positive activity trend (10e–10g). Since stereoisomers 10f and 10gexhibited similar inhibitory potency, it was apparent that the side chain stereo-chemistry was not critical and the ethyl chain with either stereochemistry wasinvolved in filling the hydrophobic pocket. Therefore, the corresponding 3-pentylanalog 10h was designed and synthesized. This derivative exhibited nearly 10-foldpotency enhancement over inhibitors 10f and 10g. Compound 10h (active form ofoseltamivir) exhibited good antiviral activity against laboratory strains and clinicalisolates of influenza virus in cell culture assays. Oseltamivir carboxylate exhibitedEC50 and EC90 values in the range from 0.0008 to >35mM and 0.004 to >100mM,respectively.Although compound 10h displayed good antiviral properties, it was found to be

inadequate for oral formulation due to poor absorption profiles. In order toimprove oral bioavailability, it was formulated as a prodrug (oseltamivir, 3, Figure18.1), in which the free carboxylic group was converted to metabolically labileethyl ester. The drug is formulated as a phosphate salt. Upon oral administration,the drug is rapidly absorbed from the gastrointestinal tract in patients. Hepaticesterases presumably convert the ester prodrug into the active agent oseltamivircarboxylate (Figure 18.11). The absolute oral bioavailability was reported to bearound 80% in humans. Also, oseltamivir carboxylate was detectable in plasmawithin 30min of dosing and maximum drug concentration was reached after3–4 h [33].The X-ray crystal structure of the complex inhibitor 10h and neuraminidase was

determined to obtain molecular insight into the ligand–binding site interactions[32,34]. As shown in Figure 18.12, inhibitor 10h makes a number of critical inter-actions in the neuraminidase active site. It appears that the binding of the ali-phatic ether occurred due to movement of the glutamate carboxylic group awayfrom its original position observed in the X-ray structures of the neuraminidasecomplexes with inhibitors 1 and 5. This movement is induced by inhibitor 10hand is assisted by the formation of an electrostatic interaction between Glu276and Arg224. The movement creates a lipophilic pocket that is perfectly filled bythe 3-pentylether moiety at C-3 of 10h. Unfortunately, the movement of this resi-due is the Achilles’ heel of oseltamivir, rendering it vulnerable to resistant viralstrains. Indeed, in resistant viral strains containing an R292 K substitution,

NH2

HN

O

Me O

Me

MeNH2 . H3PO4

HN

O

Me O

Me

Me

O

O

O

OH

Oseltamivir phosphate Oseltamivir carboxylate

Hepatic esterases

Figure 18.11 Prodrug conversion to active drug.


creation of the lipophilic binding pocket was prevented by the formation of a saltbridge between Lys292 and Glu276 [34].

18.4Conclusions

The crystal structures of neuraminidases were determined in the 1980s. The knowl-edge of structural information and interactions seen with natural substrates andinhibitors formed a strong foundation for structure-based drug design. This struc-tural insight played an important role in structure-based drug design of inhibitorsthat led to the discovery of zanamivir and oseltamivir. Clinical trials for zanamivirrevealed poor oral bioavailability and rapid excretion. Consequently, oseltamivir wasdeveloped as an orally available alternative drug. Since their introduction into ther-apy for the treatment of severe influenza, resistant viral strains have been reportedfor both drugs. In particular, transmission of both the 2009 swine flu pandemic andseasonal influenza A viruses resistant to oseltamivir has been reported. Influenzaviruses continue to cause significant morbidity and mortality today. The threat of asevere pandemic particularly involving resistant viral strains looms large. Furtherdrug development efforts directed toward biochemical targets critical to viral replica-tion are important for the development of new and more effective therapies.

Figure 18.12 X-ray crystal structure of neuraminidase and inhibitor 10h complex (PDB code:2QWK). The Glu276 residue in neuraminidase–1 complex is shown as pink sticks.


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17 Kim, C.U., Lew, W., Williams, M.A., Liu,H., Zhang, L., Swaminathan, S.,Bischofberger, N., Chen, M.S., Mendel, D.B., Tai, C.Y., Laver, W.G., and Stevens, R.C.(1997) Influenza neuraminidase inhibitorspossessing a novel hydrophobic interactionin the enzyme active site: design, synthesis,and structural analysis of carbocyclic sialicacid analogues with potent anti-influenzaactivity. J. Am. Chem. Soc., 119, 681–690.

18 Babu, Y.S., Chand, P., Bantia, S., Kotian, P.,Dehghani, A., El-Kattan, Y., Lin, T.H.,Hutchison, T.L., Elliott, A.J., Parker, C.D.,Ananth, S.L., Horn, L.L., Laver, G.W., andMontgomery, J.A. (2000) BCX-1812 (RWJ-270201): discovery of a novel, highly potent,orally active, and selective influenza


neuraminidase inhibitor through structure-based drug design. J. Med. Chem., 43,3482–3486.

19 Yamashita, M., Tomozawa, T., Kakuta, M.,Tokumitsu, A., Nasu, H., and Kubo, S.(2009) CS-8958, a prodrug of the newneuraminidase inhibitor R-125489, showslong-acting anti-influenza virus activity.Antimicrob. Agents Chemother., 53, 186–192.

20 Varghese, J.N., Laver, W.G., and Colman,P.M. (1983) Structure of the influenza virusglycoprotein antigen neuraminidase at2.9A

�resolution. Nature, 303, 35–40.

21 Colman, P.M., Varghese, J.N., and Laver,W.G. (1983) Structure of the catalytic andantigenic sites in influenza virusneuraminidase. Nature, 303, 41–44.

22 Varghese, J.N. and Colman, P.M. (1991)Three-dimensional structure of theneuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A

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vanDonkelaar, A., Laver, W.G., Webster,R.G., and Colman, P.M. (1991) Refinedatomic structures of N9 subtype influenzavirus neuraminidase and escape mutants.J. Mol. Biol., 221, 487–497.

24 Varghese, J.N., McKimm-Breschkin, J.L.,Caldwell, J.B., Kortt, A.A., and Colman,P.M. (1992) The structure of the complexbetween influenza virus neuraminidaseand sialic acid, the viral receptor. Proteins,14, 327–332.

25 Burmeister, W.P., Henrissat, B., Bosso, C.,Cusack, S., and Ruigrok, R.W. (1993)Influenza B virus neuraminidase cansynthesize its own inhibitor. Structure, 1,19–26.

26 Taylor, N.R. and von Itzstein, M. (1994)Molecular modeling studies on ligandbinding to sialidase from influenza virusand the mechanism of catalysis. J. Med.Chem., 37, 616–624.

27 Chong, A.K., Pegg, M.S., Taylor, N.R., andvon Itzstein, M. (1992) Evidence for asialosyl cation transition-state complex in

the reaction of sialidase from influenzavirus. Eur. J. Biochem., 207, 335–343.

28 Bossart-Whitaker, P., Carson, M., Babu,Y.S., Smith, C.D., Laver, W.G., and Air,G.M. (1993) Three-dimensional structureof influenza A N9 neuraminidase and itscomplex with the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. J. Mol.Biol., 232, 1069–1083.

29 von Itzstein, M., Dyason, J.C., Oliver,S.W., White, H.F., Wu, W.Y., Kok, G.B.,and Pegg, M.S. (1996) A study of theactive site of influenza virus sialidase: anapproach to the rational design of novelanti-influenza drugs. J. Med. Chem., 39,388–391.

30 Varghese, J.N., Epa, V.C., and Colman,P.M. (1995) Three-dimensional structure ofthe complex of 4-guanidino-Neu5Ac2enand influenza virus neuraminidase. ProteinSci., 4, 1081–1087.

31 Holzer, C.T., von Itzstein, M., Jin, B., Pegg,M.S., Stewart, W.P., and Wu, W.Y. (1993)Inhibition of sialidases from viral, bacterialand mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminicacid modified at the C-4 position. Glycoconj.J., 10, 40–44.

32 Kim, C.U., Lew, W., Williams, M.A., Wu,H., Zhang, L., Chen, X., Escarpe, P.A.,Mendel, D.B., Laver, W.G., and Stevens,R.C. (1998) Structure–activity relationshipstudies of novel carbocyclic influenzaneuraminidase inhibitors. J. Med. Chem.,41, 2451–2460.

33 Davies, B.E. (2010) Pharmacokinetics ofoseltamivir: an oral antiviral for thetreatment and prophylaxis of influenza indiverse populations. J. Antimicrob.Chemother., 65 (Suppl. 2), 5–10.

34 Varghese, J.N., Smith, P.W., Sollis, S.L.,Blick, T.J., Sahasrabudhe, A., McKimm-Breschkin, J.L., and Colman, P.M. (1998)Drug design against a shifting target: astructural basis for resistance to inhibitorsin a variant of influenza virusneuraminidase. Structure, 6, 735–746.

References 409

19Carbonic Anhydrase Inhibitors for the Treatment of Glaucoma:Design and Discovery of Dorzolamide

19.1Introduction

Glaucoma is an insidious optic neuropathy that can progress to irreversible blind-ness if left untreated. Glaucoma is divided into two main categories: open-angleglaucoma and closed-angle glaucoma. The most common is open-angle glaucoma[1]. Primary open-angle glaucoma is an acquired, chronic, and progressive diseaseof the optic nerve. Several risk factors have been identified. These includeadvanced age, family history, diabetes mellitus, and elevated intraocular pressure.High intraocular pressure leads to the development of glaucomatous optic nervedamage [1–4].Elevated intraocular pressure is determined by an imbalance between the pro-

duction of aqueous humor by the ciliary body of the eye and its outflow from theeye. The aqueous humor is mainly produced in the posterior chamber of the eyeby a metabolically active process involving the enzyme carbonic anhydrase. Theaqueous humor then enters the anterior chamber of the eye through the pupiland exits through the trabecular meshwork. Accumulation of aqueous humor dueto reduced outflow results in increased intraocular pressure, which in turn dam-ages the optic nerve [5,6].Intraocular pressure can be decreased by using several classes of topical or

systemic drugs such as cholinergic inhibitors, prostaglandins, b-adrenergicreceptor blockers, a-adrenergic receptor agonists, or carbonic anhydraseinhibitors [5]. Inhibition of the enzyme carbonic anhydrase decreases theintraocular pressure by reducing the production of HCO3

�, leading toreduced production of aqueous humor. Currently, dorzolamide and brinzola-mide, and two orally administered carbonic anhydrase inhibitors acetazol-amide and methazolamide (Figure 19.1), are used for the treatment ofglaucoma [1,5,6].Carbonic anhydrase is a ubiquitously expressed enzyme that catalyzes the hydra-

tion of carbon dioxide to form the bicarbonate ion (Equation 19.1). In humans, 16different carbonic anhydrase isozymes with different tissue distributions and sub-cellular localizations are known [7]. Several carbonic anhydrase isozymes are

411


targeted for the treatment of diseases such as edema, glaucoma, obesity, andosteoporosis [8,9].

CO2 þH2O Ð HCO3� þHþ ð19:1Þ

The X-ray structure of human carbonic anhydrase was determined in 1972 [10].The structure was revised in 1988 [11]. Since then, its mechanism of catalysis hasbeen studied in depth and is now understood in great detail [8,11–14]. Carbonicanhydrase is a metalloenzyme containing a zinc ion that is necessary for the cata-lytic activity. The zinc ion is located at the bottom of a 15A

�deep active site cleft

(Figure 19.2). The zinc ion is coordinated by three histidine residues (His119,His96, and His94) and by a water molecule, which is deprotonated in the activeform of the enzyme. The active site cleft contains a hydrophobic side, where thelipophilic carbon dioxide molecule binds.The carbonic anhydrase catalytic mechanism is depicted in Figure 19.3. The

hydroxide anion attacks the carbon of CO2, forming the intermediate bicarbonatecoordinated to the zinc ion. A water molecule subsequently displaces thebicarbonate anion. Afterward, a proton is abstracted from the zinc-bound watermolecule by the His64 residue, leading to the original hydroxide anion and replac-ing the enzyme in its active form.

19.2Design and Discovery of Dorzolamide

Dorzolamide (1, Figure 19.1) is a carbonic anhydrase inhibitor for the treatment ofglaucoma [15] that was designed through a structure-based approach. The goal

S SSO2NH2

HN

Me

Me

Dorzolamide, 1

NN

SNH

SO2NH2Me

O

Acetazolamide, 2

O O

Methazolamide, 3

NN

SN SO2NH2O

Me

NS S

SO2NH2

HN

Me

Brinzolamide, 4

O O

O

Me

Figure 19.1 Chemical structures of dorzolamide (1), acetazolamide (2), methazolamide (3), andbrinzolamide (4).

412 19 Carbonic Anhydrase Inhibitors for the Treatment of Glaucoma

was to develop an active carbonic anhydrase inhibitor after topical administrationinto the eye. Carbonic anhydrase inhibitor acetazolamide (2) has been widely usedfor the treatment of glaucoma by systemic administration. Its use is reserved forshort-term reduction of intraocular pressure. The systemic administration of acet-azolamide is associated with unfavorable side effects such as tingling of extrem-ities, metallic taste, fatigue, depression, weight loss, and metabolic acidosis. Theseside effects are possibly due to the widespread distribution of carbonic anhydraseisozymes in the human tissues and their involvement in numerous physiologicalprocesses. Therefore, localized topical administration of a carbonic anhydraseinhibitor was considered an appropriate strategy to avoid systemic side effects.Unfortunately, acetazolamide has poor water solubility and could not be formu-lated for topical administration. At least 2% drug solubility in water is typicallynecessary for appropriate formulation for topical administration into the eyes. As aconsequence, one research objective was to design more hydrophilic carbonicanhydrase inhibitors. However, it is important to maintain a balance of hydrophilic

Figure 19.2 X-ray crystal structure of carbonic anhydrase II. Zinc ion is shown as a yellow sphere(PDB code: 1CA2).

19.2 Design and Discovery of Dorzolamide 413

and lipophilic properties since excessive hydrophilicity would result in poorabsorption of the drug in the target tissue, whereas excessive lipophilicity wouldhinder the preparation of solutions for topical use.Acetazolamide belongs to a class of sulfonamide-type carbonic anhydrase inhibi-

tors whose unsubstituted sulfonamide group binds the zinc ion of the enzyme byreplacing the hydroxide ion–water complexes to the metal ion [8,14]. In order tofind carbonic anhydrase inhibitors suitable for topical administration, researchersat Merck investigated several carbonic anhydrase inhibitors characterized by thepresence of a primary sulfonamide as the zinc complexing group with differentheteroaromatic rings. Among these structures, a series of thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxides showed interesting inhibitory activity and water solu-bility [16,17]. Compounds (S)- and (R)-5 (Figure 19.4) showed low nanomolarcarbonic anhydrase II inhibitory activity. Intriguingly, these derivatives alsodemonstrated stereochemical preference by the carbonic anhydrase active site. Inparticular, it was observed that the (S)-enantiomer was more potent than the(R)-enantiomer. Introduction of basic moieties in place of the hydroxyl group of 5led to several alkylamino derivatives, all showing improved affinity for the enzymecoupled with increased water solubility. Compound (S)-6 showed a 10-foldincrease in inhibitory potency compared with (S)-5. The (R)-isomer [(R)-6] wasaround 100 times less potent than (S)-6 isomer. To understand the structural basisof this stereoselective enzyme–inhibitor interaction, the X-ray crystal structures ofboth (R)- and (S)-6 enantiomers complexed with carbonic anhydrase II were deter-mined [17].The X-ray structure revealed that both enantiomers engaged in similar interac-

tions with the enzyme. The primary sulfonamide displaced the hydroxide ion andcoordinated the zinc ion. The main difference between the two enantiomeric

Zn2+

OH-

His94 His119His96

C

O

O

Zn2+

O

His94 His119His96

C

O

O-H

Zn2+

O

His94 His119His96

H H

+H2O-HCO3

-

N

NH

His64

Zn2+

OH-

His94 His119His96

+CO2

Figure 19.3 Mechanism of CO2 hydration catalyzed by carbonic anhydrase.


inhibitors resided in their bound or bioactive, conformations (Figure 19.5). In par-ticular, the dihedral angle N��S��C��S (colored in red in Figure 19.5) shows a14� twist between the conformations of the two inhibitors. In the low-energy con-formation of both unbound (S)- and (R)-6, this dihedral angle should have a valueof �90� (ab initio calculations). However, the bound conformations of (S)- and(R)-6 present a dihedral angle value of 144� and 158�, respectively. The more thisdihedral angle is distorted with respect to the low energy value, the more the inter-nal energy of the compound negatively affects the binding energy of the inhibitor–

S SSO2NH2

OH

S SSO2NH2

OH

(S)-5, K i = 6.2 nM (R)-5, K i = 16 nM

O O O O

S SSO2NH2

HN

S SSO2NH2

HN

(S)-6, K i = 0.61 nM (R)-6, K i = 71 nM

O O O O

Me Me Me Me

Figure 19.4 Structures and activities of the thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxides5 and 6.

Figure 19.5 Schematic representation of the bioactive conformations of (S)-6 (left) and (R)-6(right) as shown in the X-ray crystal structure of carbonic anhydrase II–inhibitor complexes.


enzyme complex. The observed difference in the dihedral angle between (S)-6 and(R)-6 is in turn dependent on the conformation of the amino substituent at thethiopyran ring. In the enzyme-bound (S)-6, the alkyl amino side chain has a high-energy pseudoaxial orientation that is necessary as it allows the isobutyl group tobe projected toward an appropriate position for optimal interaction with theenzyme. In the (R)-6 enantiomer, the alkylamino group is positioned in a morefavorable pseudoequatorial conformation to allow the isobutyl group to occupy asimilar position as in enzyme-bound (S)-6. However, the isobutyl group shows aless favorable gauche conformation for (R)-6. These conformational differencesbetween the (S)- and (R)-6 isomers account for the different affinities of the twoenantiomers for the enzyme.In order to stabilize the pseudoaxial alkylamino group conformation in (S)-6, a

stereochemically defined methyl group at the 2-position of the thiopyran ring wasintroduced [18]. In order to compensate for the increased lipophilicity resultingfrom the introduction of an extra methyl group, the isobutylamino substituentwas replaced with an ethylamino group. These modifications led to the identifica-tion of dorzolamide (1) [18,19]. The methyl group is able to control the conforma-tional preference of the thiopyran ring by favoring the conformation in which theethylamino group has a pseudoaxial conformation, thus reducing the energeticpenalty associated with the binding of 6 and increasing the binding affinity (1,Ki¼ 0.37 nM; IC50¼ 0.23 nM). The bioactive conformation and the binding modeof dorzolamide within the enzyme are shown in Figure 19.6. The sulfonamidegroup of 1 replaces the hydroxide ion in its interaction with the zinc atom. More-over, it forms two hydrogen bonds with the backbone nitrogen and the side chainhydroxyl group of Thr199. One of the oxygen atoms of the sulfone moiety forms ahydrogen bond with Gln92.

Figure 19.6 X-ray crystal structure of dorzolamide–carbonic anhydrase II complex (PDB code:1CIL).


One of the main differences between inhibitor-bound carbonic anhydrase II andits apo form resides in the movement of the His64 side chain. The effect of theHis64 side chain shift on inhibitor binding has been studied by comparing thebinding affinity and binding mode of dorzolamide with those of structurallyrelated analogs 7 and 8 (Figure 19.7). Compounds 7 and 8 show an amino and amethylamino substituent at the 4-position of the thiopyran ring, respectively [20],and both have a similar affinity for the enzyme, lower than that of the ethylaminoderivative dorzolamide. In Figure 19.8, the dorzolamide-bound carbonic anhy-drase structure (yellow) superimposed with the 7-bound structure (blue) is shown.In the dorzolamide-bound enzyme, the bulk of the ethylamino substituent forcesthe His64 side chain into a different conformation with respect to the positionoccupied by this residue in the 7-bound structure and in the apoenzyme. The dif-ference between the binding affinity of dorzolamide and 7 has been explained byentropic factors. The His64 imidazole ring is bound to two water molecules inboth the apoenzyme and the 7-bound enzyme. When dorzolamide is bound, theHis64 side chain movement allows the release of one of the two ordered water

S SSO2NH2

NH

CH3 CH3

2

S SSO2NH2

NHMe

7, Ki = 1.52 nM 8, Ki = 1.88 nM

O O O O

Figure 19.7 Structures and activities of carbonic anhydrase inhibitors 7 and 8.

Figure 19.8 Superimposition of the X-ray crystal structure of dorzolamide–carbonic anhydrase IIcomplex (yellow; PDB code: 1CIL) and 7–carbonic anhydrase II complex (blue; PDB code: 1CIM).For the sake of clarity, the structure of dorzolamide has been omitted.


molecules into the bulk solvent, thus resulting in an increase in entropy. It hasbeen suggested that one of the main reasons for the unique affinity of dorzola-mide for carbonic anhydrase, with respect to 7 and 8, is the entropy increase dueto the loss of ordered water molecule around His64.

19.3Conclusions

The development of dorzolamide for the treatment of glaucoma highlights anotherearly, successful application of structure-based design approach. Dorzolamide inhib-its human carbonic anhydrase with a Ki value of 0.37nM. The ophthalmic solution ofdorzolamide reduces elevated intraocular pressure following topical administration.Carbonic anhydrase inhibitors are now widely used as diuretics and antiglau-

coma agents. There are five known families of carbonic anhydrases, which aredistributed in almost all cell/tissue compartments of various organisms. Eachfamily of carbonic anhydrase is formed by several isoforms of the enzyme, andthe development of specific modulators of the various isoforms could lead to thedevelopment of novel drugs effective against diseases such as cancer, obesity,osteoporosis, or infectious diseases.

References

1 Lee, D.A. and Higginbotham, E.J. (2005)Glaucoma and its treatment: a review.Am. J. Health Syst. Pharm., 62, 691–699.

2 Sommer, A. (1989) Intraocular pressureand glaucoma. Am. J. Ophthalmol.,107, 186–188.

3 Klein, B.E., Klein, R., and Lee, K.E. (2004)Heritability of risk factors for primaryopen-angle glaucoma: the Beaver DamEye Study. Invest. Ophthalmol. Vis. Sci.,45, 59–62.

4 Leibowitz, H.M., Krueger, D.E., Maunder,L.R., Milton, R.C., Kini, M.M., Kahn, H.A.,Nickerson, R.J., Pool, J., Colton, T.L.,Ganley, J.P., Loewenstein, J.I., and Dawber,T.R. (1980) The Framingham Eye Studymonograph: an ophthalmological andepidemiological study of cataract,glaucoma, diabetic retinopathy, maculardegeneration, and visual acuity in a generalpopulation of 2631 adults, 1973–1975.Surv. Ophthalmol., 24, 335–610.

5 Zhang, K., Zhang, L., and Weinreb, R.N.(2012) Ophthalmic drug discovery: noveltargets and mechanisms for retinal

diseases and glaucoma. Nat. Rev. DrugDiscov., 11, 541–559.

6 Cvetkovic, R.S. and Perry, C.M. (2003)Brinzolamide: a review of its use in themanagement of primary open-angleglaucoma and ocular hypertension. DrugsAging, 20, 919–947.

7 Imtaiyaz Hassan, M., Shajee, B., Waheed,A., Ahmad, F., and Sly, W.S. (2013)Structure, function and applications ofcarbonic anhydrase isozymes. Bioorg. Med.Chem., 21, 1570–1582.

8 Supuran, C.T. (2008) Carbonic anhydrases:novel therapeutic applications for inhibitorsand activators. Nat. Rev. Drug Discov., 7,168–181.

9 Alterio, V., Di Fiore, A., D’Ambrosio, K.,Supuran, C.T., and De Simone, G. (2012)Multiple binding modes of inhibitors tocarbonic anhydrases: how to design specificdrugs targeting 15 different isoforms?Chem. Rev., 112, 4421–4468.

10 Liljas, A., Kannan, K.K., Bergsten, P.C.,Waara, I., Fridborg, K., Strandberg, B.,Carlbom, U., Jarup, L., Lovgren, S., and


Petef, M. (1972) Crystal structure of humancarbonic anhydrase C. Nat. New Biol., 235,131–137.

11 Eriksson, A.E., Jones, T.A., and Liljas, A.(1988) Refined structure of humancarbonic anhydrase II at 2.0 A

�resolution.

Proteins, 4, 274–282.12 Domsic, J.F., Avvaru, B.S., Kim, C.U.,

Gruner, S.M., Agbandje-McKenna, M.,Silverman, D.N., and McKenna, R. (2008)Entrapment of carbon dioxide in the activesite of carbonic anhydrase II. J. Biol. Chem.,283, 30766–30771.

13 Tu, C.K., Silverman, D.N., Forsman, C.,Jonsson, B.H., and Lindskog, S. (1989) Roleof histidine 64 in the catalytic mechanismof human carbonic anhydrase II studiedwith a site-specific mutant. Biochemistry,28, 7913–7918.

14 Supuran, C.T. (2010) Carbonic anhydraseinhibitors. Bioorg. Med. Chem. Lett., 20,3467–3474.

15 Pfeiffer, N. (1997) Dorzolamide:development and clinical application of atopical carbonic anhydrase inhibitor. Surv.Ophthalmol., 42, 137–151.

16 Ponticello, G.S., Freedman, M.B.,Habecker, C.N., Lyle, P.A., Schwam, H.,Varga, S.L., Christy, M.E., Randall, W.C.,

and Baldwin, J.J. (1987) Thienothiopyran-2-sulfonamides: a novel class of water-solublecarbonic anhydrase inhibitors. J. Med.Chem., 30, 591–597.

17 Baldwin, J.J., Ponticello, G.S., Anderson,P.S., Christy, M.E., Murcko, M.A., Randall,W.C., Schwam, H., Sugrue, M.F., Springer,J.P., and Gautheron, P. (1989)Thienothiopyran-2-sulfonamides: noveltopically active carbonic anhydraseinhibitors for the treatment of glaucoma.J. Med. Chem., 32, 2510–2513.

18 Greer, J., Erickson, J.W., Baldwin, J.J., andVarney, M.D. (1994) Application of thethree-dimensional structures of proteintarget molecules in structure-based drugdesign. J. Med. Chem., 37, 1035–1054.

19 Babine, R.E. and Bender, S.L. (1997)Molecular recognition of protein–ligandcomplexes: applications to drug design.Chem. Rev., 97, 1359–1472.

20 Smith, G.M., Alexander, R.S., Christianson,D.W., McKeever, B.M., Ponticello, G.S.,Springer, J.P., Randall, W.C., Baldwin, J.J.,and Habecker, C.N. (1994) Positions of His-64 and a bound water in human carbonicanhydrase II upon binding threestructurally related inhibitors. Protein Sci.,3, 118–125.

References 419

20b-Secretase Inhibitors for the Treatment of Alzheimer’s Disease:Preclinical and Clinical Inhibitors

20.1Introduction

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder of thebrain that leads to neuronal cell death, memory loss, cognitive decline, andbehavioral changes. Pathologically, AD is defined by the buildup of amyloid pla-ques that are sticky clumps of proteins and neurofibrillary tangles composed ofinsoluble twisted fibers made mostly of protein tau. The main component ofamyloid plaques is a 40–42-amino acid peptide termed b-amyloid (Ab). The Abpeptides are formed by proteolytic processing of one or more isoforms of theamyloid precursor protein (APP), a transmembrane sialoglycoprotein encoded bya single gene on chromosome 21. APP is processed through two alternative path-ways. The primary pathway involves cleavage at Lys16 within the Ab region ofthe protein by the a-secretase enzyme. This cleavage generates the soluble APP.Alternatively, APP can be hydrolyzed by the b-secretase or b-site APP cleavingenzyme (BACE1 or memapsin 2) and c-secretase to generate the Ab. This dysre-gulation in APP processing is an early event in the disease, resulting in anincreased production of the amyloidogenic Ab peptides that lead to the pathologi-cal changes observed in the brains of AD patients. These changes include syn-apse damage, activation of inflammatory processes, oxidative stress, and neuronaldeath [1,2]. Recently, it has been suggested that neurotoxicity may be mediated bysoluble oligomeric forms of Ab species [3–5].Due to the central role of Ab in Alzheimer’s disease pathology, both b- and

c-secretases have been suggested as suitable targets for drug development for thetreatment of AD [6,7]. BACE1 is an attractive target for AD drug development for anumber of reasons. First of all, BACE1 is involved in the rate-limiting step of Abproduction. Second, although its physiological role has not been identified, knock-out transgenic mice do not show any major phenotypical abnormalities, suggest-ing that therapeutic inhibition of BACE1 may be feasible without unwanted sideeffects [8,9]. Finally, BACE1 is an aspartic acid protease and development ofother aspartic acid protease inhibitors has been successfully accomplished (seeChapters 11 and 17). The clinical development of BACE1 inhibitors, however, haspresented different sets of challenges. BACE1 inhibitors not only require

421


satisfactory pharmacokinetic properties but must also penetrate the blood–brainbarrier (BBB) and possess a degree of selectivity to avoid toxicity problems relatedto off-target effects. The selectivity issues of HIV protease and renin inhibitors areless complex as HIV protease has a different substrate specificity with respect tomammalian proteases, and renin has a very high specificity for its substrate. Incontrast, structural and biochemical studies have demonstrated that BACE1 exhib-its broad substrate specificity [10,11]. Moreover, the high degree of homology pre-sented by BACE1 and BACE2, a related aspartic acid protease, has complicated thedesign of selective inhibitors.Despite these major obstacles, many academic and industrial laboratories are

intensely pursuing structure-based design of BACE1 inhibitors for the treat-ment of AD. The first clinical BACE1 inhibitor, CTS-21166, developed atCoMentis, was evaluated in humans in clinical trials [12]. There are currentlytwo other b-secretase inhibitors, AZD3839 from AstraZeneca and MK-8931from Merck Research Laboratories, under clinical evaluation and several otherpreclinical candidates have been developed with promising features [13,14].There are many recent reviews that summarize the progress of the inhibitorstructures [10,15–17]. To provide insight into the evolution of structure-basedBACE1 drug development, the evolution of peptidomimetic BACE1 inhibitorsto an advanced inhibitor GRL-8234 will be described. In addition, an outline ofthe discovery and development of pre-clinical and clinical small-moleculeinhibitors will be discussed.

20.2b-Secretase and Its X-Ray Structure

The X-ray structures of b-secretase in the apo form and with inhibitor complexeshave been determined [18–20]. b-Secretase is a membrane-bound aspartic acidprotease formed by an N-terminal domain on the luminal side, a transmembranedomain, and an intracellular cytosolic C-terminal domain. The enzyme has a bilo-bal structure (Figure 20.1) typical of pepsin family enzymes. The two lobes areformed by the N- and C-terminal domains of the protein and the ligand bindingcleft runs between the two lobes. The aspartic acid catalytic residues Asp32 andAsp228 are located in the center of the substrate binding cleft. A flap moiety par-tially covers the substrate. This flap is flexible and its position varies in the boundand unbound enzymes. The open conformation shows a “bottleneck” in the cleftopening formed by residue Thr72 in the N-terminal domain and residues Arg235,Ser328, and Thr329 in the C-terminal domain. The distance between these resi-dues defines the minimum opening of the cleft for substrate access. In such aconformation, a good degree of flexibility of the side chains of both substrate andenzyme is required in order to allow access of the substrate to the active site. Ithas been speculated that the presence of this bottleneck, limiting the access to thesubstrate cleft, could be responsible for specificity toward the protein substrate ofBACE1.

422 20 b-Secretase Inhibitors for the Treatment of Alzheimer’s Disease

20.3Development of First Peptidomimetic BACE Inhibitors

The aspartic protease BACE1 was independently characterized by five researchgroups [11,21–24]. Since the cloning and identification of BACE1 as a possibletarget for AD intervention, Ghosh et al. designed and synthesized the first mecha-nism-based and substrate-derived potent BACE1 inhibitors [25]. These inhibitorswere initially designed by inserting a Leu–Ala dipeptide transition-state isostere atthe scissile site, mimicking the transition state of the hydrolysis of the Leu–Aladipeptide bond. One of the inhibitors displayed potent BACE1 inhibitory activitywith a Ki value of 1.6 nM (1, Figure 20.2). This specific dipeptide isostere waschosen based on biochemical studies addressing the sequence preference of thecleavage site [11,12]. In particular, it was demonstrated that P5�P5

0 decapeptidederived from the b-secretase cleavage site of APP (SEVKM/DAEFR) containingMet as the P1 residue is poorly hydrolyzed by BACE. However, the decapeptidederived from the Swedish mutant APP (SEVNL/DAEFR) is an excellentsubstrate. As a consequence, a Leu residue was chosen at the P1 position.Although optimal P1

0 residues are Ser or Asp, it was known that the Alaside chain was tolerated at this position [10]. Moreover, a Leu–Ala dipeptideisostere would be more lipophilic than Leu–Asp or Leu–Ser mimetics. Since

Figure 20.1 Ribbon representation of the X-ray crystal structure of BACE1 showing the N-terminal domain (red) and the C-terminal domain (cyan). Catalytic aspartates (Asp32 and Asp228)are shown as blue sticks, the flap in blue, and substrate binding cleft as sticks (PDB code: 1SGZ).

20.3 Development of First Peptidomimetic BACE Inhibitors 423

appropriate lipophilicity is a molecular property that needs to be carefully opti-mized for the development of centrally acting inhibitors, the Leu–Ala dipeptideisostere was more promising as a starting point for further development.Moreover, its synthetic accessibility would guarantee ample exploration of thestructure–activity relationships. The incorporation of the Leu–Ala dipeptide iso-stere in the b-secretase cleavage site of Swedish mutant APP led to the identifica-tion of inhibitor 1 (OM99-2) as a nanomolar inhibitor of BACE1. Subsequently,

Figure 20.2 Design approach to the Leu–Ala transition-state isostere of inhibitor 1 (OM99-2)and subsequent design of peptidomimetic inhibitor 2 as lead compound.


the first crystal structure of inhibitor 1 (OM99-2) and b-secretase complex wasdetermined in 2000 [18]. Shortly after, the X-ray crystal structure of the apo formof the b-secretase was determined [19,20]. Based upon the X-ray structure, pseudo-peptide inhibitor 1 was truncated into small-molecule peptidomimetic inhibitorsas represented in structure 2.

20.4X-Ray Structure of Inhibitor-Bound BACE1

An X-ray crystal structure of the inhibitor 1 and BACE1 complex is shown inFigure 20.3. The inhibitor forms a number of hydrogen bonds with the enzymeresidues. As expected, important hydrogen bonding interactions are formedbetween the transition-state hydroxyl group of the inhibitor and the catalyticAsp32 and Asp228. In the crystal structure, several hydrogen bonds can beobserved between the inhibitor and the corresponding residues in the enzymeactive site. Most of these interactions are conserved among eukaryotic asparticacid proteases. Of particular note, an intramolecular hydrogen bond is formedbetween the side chain of the P4 Glu residue and the Asn at P2. The formation ofthis hydrogen bond possibly helps the orientation of Asn residue to form a hydro-gen bond with Arg235. Furthermore, the P4 carboxylate is close to Arg235 andArg304, probably accounting for the 20-fold decrease in potency of inhibitorOM99-1 lacking the P4 residue. The inhibitor skeleton spanning from P3 to P2

0 isin an extended conformation. However, a key hydrogen bond with the carbonylgroup of the P2

0 residue and the hydroxyl group of Tyr198 tilts the backbone of

Figure 20.3 X-ray crystal structure of BACE1 bound to inhibitor 1. Main hydrogen bonding inter-actions are formed between the inhibitor and the enzyme residues (PDB code: 1FKN).

20.4 X-Ray Structure of Inhibitor-Bound BACE1 425

the inhibitor, deviating from the extended conformation and projecting the P30

and P40 side chains toward the surface of the enzyme. The side chains of the

inhibitor are tightly packed in the corresponding subsites. From the X-ray struc-ture, it is also evident that the S1 and S3 subpockets are nicely filled by the corre-sponding hydrophobic residues of the inhibitor [18]. Accordingly, the S1 and S3subsites are mainly formed by hydrophobic amino acids.This X-ray structure also provided important insight into the difference between

the BACE1 binding site and those of related aspartic proteases belonging to thepepsin family. For example, both the subsites S2 and S4 are quite hydrophilic incontrast to other aspartic proteases of the pepsin family. The detailed characteriza-tion of the binding mode of inhibitor 1 and its main interactions with the enzymeprovided important templates for the structure-based design of BACE1 inhibitors.The molecular binding properties guided subsequent optimization of the inhibitorin order to reduce molecular weight and the peptidic character of inhibitors[10,18].Deletion of inhibitor residues that were not crucial for binding was guided

by the information gained from the above-described X-ray crystal structure(Figure 20.3). The formation of the hydrogen bond between the P2

0 carbonylgroup and the hydroxyl group of Tyr198 caused a turn of the inhibitor backboneat this position, resulting in the tilting of residues P3

0�P40 toward the surface of

the enzyme. As a consequence, these two residues did not form crucial interactionwith the enzyme. We deleted these residues from the inhibitor and replaced themby a benzylamide C-capping group. The benzyl ring was placed since we specu-lated that it could fill the hydrophobic S30 enzyme subpocket.We also speculated that the hydrogen bonding interaction between the aspara-

gine side chain and Arg235 could be optimized through the introduction ofunnatural amino acids at this position. Optimization of the P2 ligand by promot-ing hydrogen bonding with other residues could allow the removal of the P4 Gluresidue. This latter residue was thought to assist the carboxamide moiety of Asnin gaining an orientation optimal for establishing a hydrogen bonding interactionwith Arg235. This structure-guided truncation strategy led to the pentapeptidemimetic 2 as a lead compound for structure–activity relationship studies [12,26].From the X-ray structure, it was evident that larger residues could be accommo-

dated at the P20 position. Accordingly, inhibitor 3, bearing a P2

0 Val residue, wassynthesized. As shown in Figure 20.4, inhibitor 3 displayed a marked increase ininhibitory potency compared with inhibitor 2. Starting from compound 3, optimi-zation of P2 Asn residue was attempted. It appeared that a methylsulfone replac-ing the carboxamide moiety of Asn (inhibitor 4) could be accommodated at S2.The corresponding methyl sulfide was 10-fold less potent, indicating that the sul-fone oxygens may be engaged in hydrogen bonding interactions in the S2 subsite.A carbon chain elongation in inhibitor 5 with a P2 methionine-derived sulfone didnot improve potency. Interestingly, a methionine at P2, as in inhibitor 6, resultedin a threefold increase in potency compared with inhibitor 5. Further attempts todecrease the molecular size by removal of Val P3 residue (inhibitor 7) led to adramatic loss of inhibitory potency [10,26].


20.5Design and Development of Selective Inhibitors

From a therapeutic point of view, BACE1 inhibitor selectivity over other humanaspartic acid proteases, particularly over BACE2 and cathepsin D, is important[27–29]. Although the above inhibitors displayed potent inhibitory activity againstBACE1, they were not selective. Inhibitor 6 showed a BACE2 Ki of 1.2 nM. Ourstructure-based design strategy then focused on developing inhibitors that areselective against BACE2 and cathepsin D. The X-ray crystal structure of theBACE1 and inhibitor 1 complex suggested some critical differences between sub-sites of BACE1 and cathepsin D that could be exploited for achieving selectivity.However, the selectivity issues against BACE2 were more difficult to address sinceprevious studies revealed that the substrate specificities of BACE1 and BACE2

Me

O

HN

HN

HN OO

OHN

OH

OH2N

O

ONH

Ph3, Ki = 5.9 nM

Me

O

HN

HN

HN OO

OHN

SOH

O

O

ONH

Ph4, Ki = 9.4 nM

O

Me

O

HN

HN

HN OO

OHN

OH

O

ONH

Ph5, Ki = 8 nM

SO

O

Me

O

HN

HN

HN OO

OHN

OH

O

ONH

Ph6, Ki = 2.5 nM (BACE1)

S

Me

O

HN

HN

HN OO

O

O OH

NH

Ph7, Ki = 5808 nM

S

Ki = 1.2 nM (BACE2)


20.5 Design and Development of Selective Inhibitors 427

were very similar [29]. We compared both BACE2 and BACE1 structures by ahomology modeling to address this problem.Based upon subtle differences in the residues in the S2 and S3 subsites, our

strategy was to replace substituents at P2 and P3 with small heterocycles to formhydrogen bonds with residues [30]. As can be seen in Figure 20.5, the pyrazolederivative 8 showed reasonable activity with respect to inhibitor 6. However, itimproved the selectivity profile over BACE2, and showed moderate selectivity overcathepsin D. Replacement of the C-terminal benzylamide with an isobutylamideand replacement of the P2 methionine with an S-methylcysteine resulted in inhib-itor 9, with improved potency toward BACE1 and selectivity over BACE2. How-ever, the selectivity toward cathepsin D was only threefold. Oxidation of thesulfide atom in 9 to the corresponding sulfone gave inhibitor 10, showed subna-nomolar BACE1 inhibitory potency and very good selectivity over both BACE2 andcathepsin D. Then, replacement of pyrazolylmethyl urethane in 10 with an oxazo-lylmethyl derivative provided very potent BACE1 inhibitor 11 with excellent selec-tivity over BACE2 and cathepsin D [30].

O NH O

HN

OHO Me

O

HN

OHN

i-Bu

ON

Me

MeS

OOMe

14 811 25O N

HO

HN

OHO Me

O

HN

OHN

NN

Me

Me

SMe

Ph

9

O NH

O

HN

OHO Me

O

HN

OHNi-Bu

NN

Me

MeS

Me

10

O NH O

HN

OHO Me

O

HN

OHN

i-Bu

NN

Me

MeS

OOMe

11

0.3 356 131 (>1150) (>425)

0.12 458 304 (>3800) (>2500)

Ki (nM)

BACE1 BACE2 Cathepsin D

4.4 161 15 (>35) (>3)

8

(selectivity for BACE1)

Figure 20.5 Structures and selectivities of inhibitors 8–11.


The X-ray crystal structure of the inhibitor 10 and BACE1 complex was deter-mined [30]. As shown in Figure 20.6, the structure revealed that the pyrazolenitrogen was located within hydrogen bonding distance from Thr232 side chainhydroxyl group and that the pyrazole methyl groups nicely filled the hydrophobicS3 subpocket. Furthermore, the oxygen of the sulfone was within hydrogen bond-ing distance from Arg235. Presumably, these interactions are responsible for theenhanced selectivity of compound 10. Based upon this molecular insight, we thenredesigned the pyrazole heterocyclic ring to a more stable dimethyl oxazole deriva-tive in inhibitor 11, which resulted in further enhancement in potency and selec-tivity over both BACE2 and cathepsin D.Although inhibitor 11 showed good selectivity, however its cellular potency

required improvement. BACE1 activity in Chinese hamster ovary (CHO) cells wasin the low micromolar range. Our inhibitor optimization strategy then aimed atreducing the peptidic character and molecular size and increasing the lipophilic-ity. The feasibility of functionalized isophthalamide derivatives as the P2–P3 lig-ands was investigated [31,32]. As shown in Figure 20.7, an isophthalic derivativebearing an N-methylsulfonamide as the P2 ligand resulted in inhibitor 12 showinggood potency and selectivity [33]. However, cellular activity was still far fromsatisfactory.To improve lipophilicity, the dimethyl oxazole moiety was replaced by a simple

aromatic ring and the resulting inhibitor 13 displayed only moderate BACE1inhibitory activity. The replacement of the benzylamide with an (R)-a-methylben-zylamide resulted in inhibitor 14 [33]. This inhibitor exhibited potent BACE1activity in Chinese hamster ovary (CHO) cells at nanomolar concentration. Thiscompound also displayed a strong stereochemical preference at the a-benzylamide

Figure 20.6 An X-ray crystal structure of inhibitor 10 (cyan) bound to BACE1. Hydrogen bondinginteractions formed by P2 and P3 ligands are shown (PDB code: 2G94).

20.5 Design and Development of Selective Inhibitors 429

position. The corresponding inhibitor with an (S)-a-methylbenzylamide showed amarked reduction in inhibitory potency (Ki¼ 315 nM). Inhibitor 14 was selectedfor in vivo studies in Tg2576 mice. An intraperitoneal administration of inhibitor14 (8mg/kg dose) showed 30% reduction of plasma Ab40 production levels [33].Since Ab production in this mouse model is brain specific and the efflux of Abfrom brain to plasma occurs rapidly, the observed plasma Ab40 reduction is likelydue to an inhibition of Ab40 production in the brain [33].The X-ray crystal structure of the inhibitor 14 and BACE1 complex was deter-

mined (Figure 20.8) [33]. The structure showed that the hydrophobic and polar

Figure 20.7 Development of BACE1 inhibitors with nanomolar cellular potency.


interactions of the inhibitor P10�P3

0 residues are similar to those of inhibitor 11.The striking differences were observed for the interaction of the nonprime-sideresidues. The sulfone moiety engaged in hydrogen bonding interactions withSer325 and Asn233. The position of the a-methylbenzylamide moiety, however,highlighted the flexibility of the binding cleft of BACE1. In fact, the presence ofthis bulky moiety results in a shift of the 10 s loop [20] that reorients with respectto the 10-bound structure (as evidenced in Figure 20.6). The movement of the 10 sloop opens up a hydrophobic site that can be nicely filled by the phenyl ring of theinhibitor.

20.6Design of Small-Molecule Inhibitors with Clinical Potential

As described, the evolution of the above BACE1 inhibitors was guided by struc-ture-based design cycles through rigorous assessment by using in vitro and in vivoassays. Our X-ray crystallographic analysis provided a better understanding of therelationships and limitations of some of the design parameters. Our initial targetfor BACE1 and cellular activity was in the low nanomolar range. The molecularsize should be targeted near 550–600 Da. Our initial structure-based design strate-gies provided lead inhibitors with many desirable drug properties, such as selec-tivity, in vivo Ab inhibition, and blood–brain barrier penetration in the size rangefrom 600 to 650 Da [34].

Figure 20.8 An X-ray structure of the inhibitor14 (green) and BACE1 complex. Hydrogenbonding interactions formed by P2 and P3 resi-dues are shown. Also, movement of 10 s loopin inhibitor 14 (green) with respect to inhibitor

10 (Figure 20.6) is shown. Surface representa-tion involves amino acids forming the S3–S4subpocket when inhibitor 14 is bound (PDBcode: 2P4J).

20.6 Design of Small-Molecule Inhibitors with Clinical Potential 431

We then investigated BACE1 inhibitors incorporating hydroxyethylamine iso-steres in combination with a variety of functionalized isophthalamide derivativesas the P2–P3 ligands. This led to a series of drug-like BACE1 inhibitors 15–18,shown in Figure 20.9 [35]. A number of inhibitors exhibited improved potency,selectivity, and cellular inhibitory activity. Inhibitor 15 bearing the (R)-a-methyl-benzyl isophthalamide as the P2–P3 ligand and a valine isopropyl amide as the P2

0

ligand displayed only moderate potency against BACE1. When the P20 Val amide

was replaced with an indole sulfonamide derivative, the resulting inhibitor 16showed an improved BACE1 inhibitory activity with a cellular IC50 value of200 nM in CHO cell lines. The replacement of the indole sulfonamide derivativewith a P1

0m-trifluoromethylphenyl ligand was carried out to increase lipophilicityof the inhibitor for better cellular activity. Inhibitor 17 displayed potent BACE1activity with excellent cellular IC50 value. Furthermore, it showed improved

HN

HN

OH

PhO

HN

O

MeN

SOO

Ph

Me

HN

HN

OH

PhO

HN

O

MeN

SOO

Ph

Me

HN

HN

OH

PhO

HN

O

MeN

SOO

Ph

Me

N

SO2Me

HN

HN

OH

PhO

HN

O

MeN

SOO

Ph

Me

O

NH

BACE1 Ki (nM) IC50 (nM)

564 ---

15

27 200

16

17 (GRL-8235)

18 (GRL-8234)

0.61 2.1

1.8 1.0

(Ki = 168 nM, CD)

OMe

CF3

(Ki = 137 nM, BACE2)(Ki = 81 nM, CD)



selectivity against cathepsin D (>275-fold) compared with inhibitor 14. We thenincorporated a m-methoxybenzyl ring as the P1

0 ligand that provided inhibitor 18(GRL-8234). This inhibitor showed very potent BACE1 inhibitory and cellularactivity. Inhibitor 18 has a molecular weight of 658 Da. It possesses drug-like fea-tures, and we selected this compound for in vivo studies. Inhibitor 18 inhibited Abproduction in transgenic mice (Tg2576) when administered intraperitoneally at8mg/kg dose. On average, there was 65% reduction of Ab40 production in plasmaafter 3 h. Inhibitor 18 exhibited modest BACE2 selectivity (Ki¼ 137 nM, 75-fold forBACE1) and cathepsin D selectivity (Ki¼ 81 nM, 45-fold for BACE1) [35].The X-ray crystal structure of the inhibitor 18 and BACE1 complex was deter-

mined to obtain insight into the ligand–binding site interactions. Figure 20.10highlights various hydrophobic and hydrogen bonding interactions in the BACE1active site. The sulfonamide group of the inhibitor forms several hydrogen bondsin the S2 subsite. The carbonyl of isophthalamide group is engaged in polar con-tacts with the flap residue Thr72. The a-methylbenzamide function nicely fits thesubpocket, and the other aromatic rings show extensive hydrophobic interactions.We have also designed potent and selective BACE1 inhibitors based upon the

reduced amide isostere [36]. Inhibitors with this scaffold may particularly beintriguing for the development of novel BACE1 inhibitors since the presence of aprotonatable amine, besides increasing water solubility, could help the corre-sponding inhibitors to better cross cell membranes. As shown in Figure 20.11,(R)-a-methylbenzyl isophthalamide P2–P3 ligand was coupled to a reduced peptideisostere mimicking the transition state of the peptide hydrolysis [36]. This designresulted in inhibitor 19, showing reasonable activity both against the enzyme and

Figure 20.10 X-ray crystal structure of the 18 (GRL-8234) and BACE1 complex. The main hydro-gen bonds are shown as dotted lines. Surface representation of the enzyme highlights inhibitormoieties filling the corresponding subpockets of the enzyme (PDB code: 2VKM).

20.6 Design of Small-Molecule Inhibitors with Clinical Potential 433

in cell-based assays. In order to improve the binding affinity, the X-ray crystalstructure of the inhibitor 1 and BACE1 complex guided the introduction of ahydroxyl group at the P1

0 side chain since it was speculated that it could beengaged in the formation of a hydrogen bond with the hydroxyl group of Tyr198.The resulting inhibitor 20 showed potent BACE1 inhibitory (Ki¼ 17 pM) and cell(IC50¼ 1 nM) activity. Furthermore, this inhibitor exhibited >7000-fold selectivityover BACE2 and >250 000-fold selectivity over cathepsin D. The X-ray crystalstructure of the inhibitor 20 and BACE1 complex was determined. The structurerevealed that the P1

0 hydroxyl group was located in proximity to form a hydrogenbond with Tyr198. The stereochemistry of this hydroxyl group is important as theinhibitor 21 containing the epimeric hydroxyl group showed significant reductionin BACE1 inhibitory activity. The corresponding methyl ether derivative of inhibi-tor 20 also showed decreased potency for inhibitor 22. Furthermore, removal ofthe carbonyl group at the prime side resulted in a loss of inhibitory potency [36].

HN

O

HN

O

NMeS

Me

O O

Ph

Me

NH

PhHN

O

Me

19, K i = 27.1 nM (BACE1) IC50 = 9.5 nM (cell)

HN

O

HN

O

NMeS

Me

O O

Ph

Me

NH

PhHN

O

Me

K i = 0.017 nM (BACE1) IC50 = 1 nM (cell)

K i = 120 nM (BACE2); BACE2/BACE1 >7000K i = 4300 nM (CD); CD/BACE1 >250,000

OH

H

H

HN

O

HN

O

NMeS

Me

O O

Ph

Me

NH

PhHN

O

Me OHHN

O

HN

O

NMeS

Me

O O

Ph

Me

NH

PhHN

O

Me OMe

21, K i = 98.8 nM (BACE1) IC50 >1000 nM (cell)

22, K i = 25 nM (BACE1)

20 (GRL-1439)

Figure 20.11 Structures and activities of BACE1 inhibitors 19–22.


20.7GRL-8234 (18) Rescued Cognitive Decline in AD Mice

In the proof-of-concept experiments, in collaborative studies with Jordan Tang andDavid Holtzman, we demonstrated that inhibitor 18 was able to rescue cognitivedecline in transgenic AD mice [37]. Osmotic pumps were implanted in youngTg2576 mice (5.5 months old) to deliver either inhibitor solution (33.4mg/g/day) orcontrol solvent throughout 220 days of the experiment time. It was observed thatplasma Ab40 and Ab42 of the treated group were about 65% lower than the controlsthroughout (Figure 20.12). Cognitive tests with a Morris water maze at 1.5 and 4.6months of treatment showed no difference between the two groups. However, at6.7 months, the cognitive performance of the treated group was clearly superior tothe controls in time latency, annulus crossing index (ACI), and time in quadrant.In other experiments, it was confirmed that cognitive rescue occurred after 5 and7.5 months of treatment with starting ages of 8 and 9.6 months, respectively.Furthermore, we observed a decrease of plaques and amyloid load, but only a

slight change in Ab oligomer patterns in the brains of treated mice as compared

Figure 20.12 Rescue of age-related cognitivedecline in Tg2576 mice after treatment withBACE1 inhibitor 18. (a) Plasma Ab patterns ofcontrol and inhibitor-treated mice. (b) Cogni-tive performance (left panel: latency time;

right panel: annulus cross-index) of control andtreated mice after 6.7 months of treatment.Cognitive performance of the treated mice wasimproved significantly. (The figure is modifiedfrom Figures 3 and 4 of Ref. [37].)

20.7 GRL-8234 (18) Rescued Cognitive Decline in AD Mice 435

with the controls. We also found no accumulation of Ab precursor protein afterseveral months of inhibitor treatment. These results represent the first directexperimental evidence that the treatment of Tg2576 mice with a BACE1 inhibitor(GRL-8234, 18) rescues the age-related cognitive decline. These observations cor-roborate the idea that Ab accumulation plays a major role in the cognitive declineof Tg2576 mice and support the concept of Ab reduction therapy as a treatment ofAD. Of particular note, treatment of 10-month-old Tg2576 mice with inhibitorGRL-8234 failed to show cognitive rescue, which suggests the need for an earlystart on amyloid reduction treatment in human AD [37].In highly collaborative studies with our laboratories at Purdue University

and Jordan Tang’s laboratories at the Oklahoma Medical Research Foundation,we created the first potent BACE1 inhibitor and determined the first X-raystructure of inhibitor-bound BACE1 that provided structural templates fordrug design. Subsequently, we developed strategies for structure-based designof BACE1 inhibitors and developed tools and strategies for selectivity designagainst BACE2 and cathepsin D. We carried out optimization of a number ofinhibitor classes at CoMentis, a biopharmaceutical company founded by JordanTang and Arun Ghosh. One of the b-secretase inhibitors, CTS-21166 (structurehas not yet been disclosed), emerged as the first BACE1 inhibitor for clinicaldevelopment [12].

20.8BACE1 Inhibitors for Clinical Development

For the development of BACE1 inhibitor drugs, drug candidates need to possessthe ability to cross the blood-brain barrier, show selectivity against physiologicallyimportant aspartyl proteases, and exhibit efficacy by inhibiting the formation ofAb40–Ab42 peptides. Inhibitors with low molecular weight and low number ofhydrogen bond donors (preferably �3) would be more likely to cross BBB, therebyreaching the target enzyme to exert therapeutic effect [34]. Toward these objec-tives, fragment-based drug design has gained attention in recent years. Thisapproach can provide opportunity to identify low molecular weight novel struc-tural scaffolds. Potency and selectivity of such small-molecule inhibitors can beimproved by rational structure-based drug design. By using this approach, a num-ber of small-molecule BACE1 inhibitors with ability to cross BBB have been devel-oped and even completed phase I clinical trials. Two drugs have now entered intothe next phase of clinical trials.

20.8.1Development of Clinical Inhibitor, AZD3839

Folmer and coworkers from AstraZeneca identified 6-propylisocytosine scaffold 23(Figure 20.13) as a novel structural class of BACE1 inhibitors, using NMR-basedfragment screening [38]. Compound 23 displayed 28% inhibition at 1mM in the


surface plasmon resonance (SPR) assay [38,39]. Subsequently, screening of an in-house compound collection containing isocytosine core structures led to the identi-fication of dihydroisocytosine 24, which showed 20% inhibition at 500 mM [39].The X-ray crystal structure of compound 24-bound BACE1 showed that it binds ina less stable pseudoaxial bound conformation shown in 24a (Figure 20.13). Thisconformation has 1.4 kcal/mol higher energy than the corresponding pseudoequa-torial conformation. Investigators at AstraZeneca envisioned that incorporation ofa methyl group at the 6-position may increase the relative stability of the pseudoax-ial conformation, which in turn may improve potency. Accordingly, the resultingcompound 25 was synthesized and its potency was evaluated in both SPR- andFRET-based assays. It displayed improved potency (IC50¼ 140mM, SPR). Furtherstructure-guided refinement of inhibitor 25 provided inhibitor 26 with submicro-molar efficacy [39].The X-ray crystal structure of the inhibitor 26 and BACE1 complex was deter-

mined. The structure revealed that the isocytosine core formed hydrogen bondswith catalytic aspartates Asp32 and Asp228, as shown in Figure 20.14. The oxygenof the 30-methoxy group formed a hydrogen bond with the hydroxyl group ofSer229. The inhibitor’s biphenyl group fills the hydrophobic pockets in the S1 andS3 subsites.Subsequent studies were directed toward the development of potent inhibitors

by incorporating this amidine functionality, as it interacts with catalytic aspartates.These efforts resulted in potent inhibitors 27 and 28 (Figure 20.15) with amino-imidazole and aminoisoindole scaffolds, respectively [40]. Inhibitors 27 and 28displayed low nanomolar potency, albeit with low permeability. Inhibitor 28 wasselected for further modifications to improve pharmacokinetic properties. Fluo-rine was introduced ortho to the amidine moiety to shield the amine functionality

HN

NH2N

O

23

HN

NH2N

O

24

HN N

O

H2N

H

24a

HN

NH2N

O

25

Me

IC50 = 140 µM (SPR)IC50 = 190 µM (FRET)

N

NH2N

O

26

Me

IC50 = 380 nM (FRET)IC50 = 590 nM (Cell)

Me

O

Figure 20.13 Structures and activities of initial BACE1 inhibitors 23–26 containing isocytosinescaffold.

20.8 BACE1 Inhibitors for Clinical Development 437

Figure 20.14 X-ray crystal structure of the inhibitor 26 and BACE1 complex. Key residues ofBACE1 (magenta) interacting with 26 (cyan) are shown as sticks and hydrogen bonds are shownby dotted lines (PDB code: 2VA6).

NN

N

NH2

FF

O

O

27

K i = 23 nM (BACE1)Caco-2: 8

N

NH2

N28

NF

K i = 20 nM (BACE1)Caco-2: 1pKa: 8.4Efflux ratio : >35

N

NH2

N

29

N

N

K i = 93 nM (BACE1)Caco-2: 12pKa: 7.2Efflux ratio : 10

F

N

NH2

N

30 (AZD3839)

N

N

Ki = 26 nM (BACE1); Ki = 372 (BACE2)Caco-2: 23pKa: 7.1Efflux ratio : 3.5

F

F

FH

F

F

FH

Figure 20.15 Structures and activities of BACE1 inhibitors 27–30.


of amidine from solvent by formation of an intramolecular hydrogen bond. Thismay improve permeability and also reduce the pKa. Also, 2-fluoropyridine ininhibitor 28 was replaced with pyrimidine. The resulting inhibitor 29 displayedimproved permeability and reduced Pgp efflux ratio, but there was over fourfoldloss in potency. Further incorporation of difluoromethyl at the ortho-position ofthe pyridine ring of compound 29 restored the potency similar to compound 28.The resulting inhibitor 30 (AZD3839) displayed good permeability and low Pgpefflux ratio. Inhibitor 30 exhibited a 14-fold selectivity against BACE2 (memapsin1) and >1000-fold selectivity over cathepsin D.The X-ray crystal structure of the inhibitor 30 and BACE1 complex provided

molecular insight into its high affinity (Figure 20.16). Inhibitor 30 binds toBACE1 in a flap-open conformation that allows pyridine nitrogen to form a hydro-gen bond with Trp76. The amidine moiety forms hydrogen bonds with thecatalytic Asp32 and Asp228, and one of the pyrimidine nitrogens forms a water-mediated hydrogen bond with the carbonyl of Ser229. The fluorine ortho to theamidine moiety is in close contact with the hydroxyl group of Thr231. Theadjacent phenyl group occupies the hydrophobic S1 pocket. One of the fluorinesfrom difluoromethyl group ortho to pyridine is in close contact with the hydroxylgroups of Ser35 and might be responsible for the improvement in potencycompared with inhibitor 29.AZD3839 (30) was selected for clinical development. It showed high efficacy in

reducing Ab40 and also in reducing the formation of sAPPb in SH-SY5Y cellsoverexpressing wild-type APP695. It displayed plasma stability for at least 24 h.Oral administration of AZD3839 in C57BL/6 mice and guinea pig and

Figure 20.16 X-ray crystal structure of 30-bound BACE1. Inhibitor 30 (magenta) interacting withkey residues of BACE1 (green) is shown as sticks and hydrogen bonds are shown by dotted lines(PDB code: 4B05).


intravenous infusion in monkeys reduced the Ab40, Ab42, and sAPPb levels inplasma, brain, and CSF [40]. AstraZeneca recently completed phase I clinical trialsof AZD3839 on healthy volunteers. The results clearly show that AZD3839 effec-tively reduced the formation of Ab40 and Ab42 in a dose-dependent manner with-out any serious adverse effects [13].

20.8.2Development of Iminopyrimidinone-Based BACE1 Inhibitors

Zhu et al. identified a low-affinity small-molecule BACE1 inhibitor, diphenylimi-nohydantoin 31 (Figure 20.17) by NMR-based fragment screening [41]. Malamaset al. also independently identified inhibitor 31 [42]. Inhibitor 31 displayed goodligand efficacy, pharmacokinetic properties, and selectivity against cathepsin D.Cumming et al. of Merck selected inhibitor 31 for further optimization [43]. An X-ray crystal structure of 31-bound BACE1 showed that amidine moiety of inhibitor31 forms hydrogen bonds with catalytic aspartates Asp32 and Asp228, and one ofthe phenyl groups fills the S1 hydrophobic pocket (Figure 20.18). The second phe-nyl group occupies the space near S20 region. The X-ray crystal structure alsorevealed that the S3 pocket of BACE1 is available for further optimization ofinhibitor 31. Investigators speculated that incorporation of lipophilic groupson the P1 phenyl ring (ring A) would improve the affinity by occupying theS3 pocket. Docking experiments also suggested that 3-biaryl rings can occupyS1–S3 pockets [43].For lead identification, various racemic iminohydantoin analogs having 3-biaryl

groups were screened. Inhibitor 32 obtained by incorporation of a 5-chloropyridine-3-yl group displayed nanomolar activity (Figure 20.19). Further optimization of thesecond phenyl ring (ring B) led to the identification of inhibitor 33 with improvedpotency. An X-ray crystal structure of 33-bound BACE1 revealed that the pyridylgroup occupies the S3 pocket. The chlorine substituent projects into the S3 subpocket

HN

NHNMe

O

31

MW : 265.3 LE : 0.37 (ligand efficiency)

Ki : 7.1 µM (BACE1)

N

NN

OO

O

OO

H

H

H

S1 pocket

Asp228

AB

Cathepsin D: 38% inhibition at 50 µM

Asp32

Figure 20.17 Structure and activity of inhibitor 31 and schematic representation of its bindingmode.


and the amidine functionality forms hydrogen bonds with catalytic aspartates(Figure 20.20). The structure also showed that the (R)-enantiomer is preferred over(S)-enantiomer, as this is the only isomer observed in the X-ray cocrystal structure ofracemic 33-bound BACE1.

Figure 20.18 X-ray crystal structure of 31 (yellow)-bound BACE1 (flap has not been shown forclarity) (PDB code 4DJU).

HN

NHNMe

O

32N

Cl

Ki = 90 nM (BACE1)

HN

NHNMe

O

33N

Cl

IC50 = 380 nMcLogP = 2.4

HN

NHNMe

O

33(R)N

Cl HN

NHNMe

O

34N

Ki = 59 nM (BACE1)

IC50 = 150 nMcLogP = 2.4

Ki = 21 nM (BACE1)IC50 = 82 nMcLogP = 2.4

Ki = 5.4 nM (BACE1)



The efficacy of the (R)-enantiomer of 33 was then evaluated. It showed improve-ment in both enzyme inhibitory and cellular potency. Furthermore, it has shownvery good ligand efficiency (LE¼ 0.44) and 350-fold selectivity against cathepsin D.Replacement of chlorine with a propynyl moiety was sought assuming that thepropynyl group can go even deeper into S3 subpocket. This resulted in inhibitor34 that displayed further improvement in BACE1 and cellular activity. It alsoshowed improved selectivity against cathepsin D (7500-fold).Although inhibitor 34 displayed nanomolar BACE1 inhibitory activity, good

selectivity over cathepsin D, and a ligand efficiency of 0.43, the ratio of cell andenzyme inhibitory potency was still very high (>15) and it did not penetrate thebrain well. Researchers then directed their studies to reduce the ratio of cell andenzyme inhibitory activity. Toward this goal, they speculated that replacing five-membered iminohydantoin with six-membered iminopyrimidinone wouldimprove the basicity of inhibitors. This in turn would allow inhibitors to partitioninto acidic intracellular compartments to inhibit the BACE1-mediated APP proc-essing. Docking studies suggested that substituents on the prime site (substituentR in structure 35, Figure 20.21) might need to be smaller than a phenyl to avoidsteric repulsion with the enzyme. Accordingly, various compounds containing theiminopyrimidinone moiety were designed and evaluated. As shown, the cell toenzyme activity ratio for compound 36 was 1.3 (versus 15 for inhibitor 34); how-ever, compound 36 is not very potent. Replacement of the phenyl group with anisosteric thiophene ring provided compound 37 with improved BACE1 inhibitoryand cellular activity of more than sixfold. Subsequently, compound 38 was pre-pared by replacing the 3-cyanophenyl group with 3-propynylpyridine in order togo deeper into the S3 subpocket. Inhibitor 38 exhibited very potent BACE1 andcellular activity. Also, it displayed 130-fold selectivity over cathepsin D and goodligand efficiency (0.46) [44].

Figure 20.20 X-ray crystal structure of 33-bound BACE1 (PDB code 4DJX) (flap has not beenshown for clarity). Hydrogen bonding interactions of inhibitor 33 (cyan) with catalytic aspartates(yellow) are shown by dotted lines.


Besides CTS-21166 and AZD3839, Merck Research Laboratories announcedclinical development, of a small-molecule BACE1 inhibitor MK-8931 (structurehas not been disclosed). This inhibitor has now completed phase I clinical trialsand started phase II/III clinical trials [14].

20.9Conclusions

After BACE1 was discovered, cloned, and its structure determined, there was arapid development of structure-based design tools and strategies to develop effec-tive treatment for Alzheimer’s disease. The clinical development of BACE1 inhibi-tor drugs, however, is faced with many uncertainties and obstacles. The challengesare numerous, including optimization of pharmacokinetic properties, necessityfor a high degree of selectivity, and requirement for effective penetration of theblood–brain barrier. The presence of specialized transporter proteins that effluxexogenous substances from CNS presents unique difficulties. The development ofsmall-molecule nonpeptidic BACE1 inhibitors is a prolific area of researchwith hundreds of publications and patents. The power of structure-based designis significant and researches continue to build on the knowledge of structure,activity, and enzyme function to overcome obstacles. Structure-based design offersa formidable approach to the complexity and challenges of BACE1 inhibitordevelopment.

HN N

NHMe

OAr1R

Ar2

35R should be smaller in size than phenyl

HN N

NHMe

OMe

36

CN

Ki = 350 nM (BACE1)IC50 = 450 nM

HN N

NHMe

OMe

37

IC50 = 68 nM

S

CN

HN N

NHMe

OMe

38

IC50 = 13 nM

S

N

Ki = 57 nM (BACE1) Ki = 7.8 nM (BACE1)



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Index

aAbl kinase complex 8acetazolamide 411–4142-acetoxyisobutyryl chloride 39N-acetylneuraminic acid 398– binding mode 399– bound conformation 400– carboxylate group 399– chemical structures of 398– hemagglutinin binds 397acetyl pepstatin 239–241acquired immunodeficiency syndrome

(AIDS), see also HIV-1 protease inhibitors;non-nucleoside HIV reverse transcriptaseinhibitors

– approvednewdrugs, for treatment 9–11, 237– CCR5 antagonists 207aldosterone 219, 373aliskiren 8, 11, 391–393– antihypertensive agents 393– biological properties 393– x-ray crystal structure of 392, 393allosteric kinase inhibitors 168– AKT1 inhibitors 170– 1,5-dihydropyrazole benzothiazine

derivative 170– FAK inhibitors 170, 171– MEK inhibitor 168– signaling pathways 168– structure-based design 168– x-ray crystal structure 169, 171Alzheimer’s disease (AD) 24, 421, 439aminoalkyl epoxide synthesis– from a-amino acids 38– from a-chloroketone 39(aminoalkyl)phosphonyl fluoride (aminoalkyl)

phosphonates 714-amino-5-cyclohexyl-3-hydroxypentanoic acid

(ACHPA) 25, 26

4-amino-3-hydroxy-6-methylheptanoic acid 24amprenavir 9, 238, 255, 256amyloid precursor protein (APP) 421, 423,

424, 442angiogenesis 144, 170, 272–274angiotensin-converting enzyme– action 373– structures 225angiotensin-converting enzyme (ACE)

inhibitor 7, 35, 143, 219, 221, 223, 226, 231,234, 373

– activity regulation 223, 225– bearing phosphorus-based zinc binding

groups 231, 232– bradykinin accumulation and 227– design of inhibitors containing a carboxylate

as zinc binding group 228–231– prototype 221, 223– rational design of “by-product analogs” as

inhibitors of 223– vasoactive peptides isolated from venom

221– vs. carboxypeptidase A 221anticancer agent, see Taxolanticoagulant drugs, see dabigatran; direct

thrombin inhibitor; etexilateanticoagulant therapies 340–342Artemisia annua 6artemisinin 5, 6aspartic acid protease inhibitors 22, 42– basic dipeptide isosteres for design of 23– binding mode of nonpeptide inhibitors

with 44– heterocyclic/nonpeptidomimetic inhibitors,

design of 42–44, 50–53– peptide hydrolysis, catalytic mechanism

of 23aspartic protease (SAP) inhibitor 35Aspergillus terreus 6

j449


atazanavir 10, 238, 239azapeptides 308, 309

bBACE1 inhibitors 51, 421, 423, see also

b-secretase inhibitors– acyl guanidine-based 52– aminopyridine-based 53– for clinical development 436– design of small-molecule inhibitors with

clinical potential 431–434– dimethyl oxazole moiety 429– GRL-8234 rescued cognitive decline in AD

mice 435, 436– hydrogen bonding interactions 425, 426– iminopyrimidinone-based BACE1

inhibitors, development 440–443– inserting a Leu–Ala dipeptide transition-state

isostere at 423– b-secretase cleavage site of Swedish mutant

APP led to 424– selective inhibitors, design and

development 427–431– selectivities 428– small-molecule BACE1 inhibitor

MK-8931 443– structures and activities 427, 432, 434, 437,

440, 443– treatment of Tg2576mice with 430, 435, 436– x-ray structure 425–431, 433, 438, 439, 443bafetinib 271, 279, 282–284– affinity for Bcr-Abl 284– design strategy for 283– potency 282– x-ray structure of bafetinib–Abl kinase

complex 284b-amyloid (Ab) 421benzheptoxdiazines 4benzodiazepine derivatives 5bicyclic P2 ligands– design of 257–259– structures and potencies, of inhibitors

with 259bioisosteres 22bis-tetrahydrofuran, design of 257–259blood–brain barrier (BBB) 21, 422, 436blood coagulation 67, 80, 338, 339blood pressure 7, 220, 234, 373, 376, 393, 394– regulation 219Boc-aminonicotinic acid 50boceprevir 82, 87, 296, 304–307– bound to NS3/4A protease, x-ray

structure 315

– crystal structure 307– structure-based optimization 306, 307– truncation strategy 312–314boronic acid 53, 56, 68, 78–84, 116, 327,

334bortezomib 79, 113, 115, 116, 119, 325–330– approved by FDA 329– side effects 329– structure–activity relationship studies 326Bothrops jararaca 221B-Raf kinase inhibitors 12, 165, 274brecanavir 265, 266brinzolamide 9, 411, 412

cCamptotheca acuminata 5captopril 7, 9, 219, 220, 225–228, 232–235– approved by FDA for 227– design of 220carazolol 201–203carbocyclic inhibitors 404carbonic anhydrase inhibitors 3, 411, 418– acetazolamide 412, 413– active site 414– brinzolamide 412– carbonic anhydrase II-inhibitor

complexes 415– catalytic mechanism 412, 414– dorzolamide (see dorzolamide)– methazolamide 412– structures and activities 417carboxypeptidase A 7, 220, 221– benzylsuccinic acid as a “by-product

analog” 221, 222– dicarboxylic acids inhibiting 221– mechanism of peptide hydrolysis catalyzed

by 222– rational design of “by-product analogs” as

inhibitors of 223carfilzomib 119, 325, 330, 333, 334– chemical structure 334– clinical trials 333cathepsin inhibitors 25, 79, 103, 117, 137, 138,

393, 427, 428, 433, 439, 440, 442CCR5 antagonists, structure-guided

design 207– development of maraviroc from HTS lead

molecule 207– GSK163929 213– optimization of pharmacokinetic

profile 209–212– PF-232798 213– reduction of hERG activity 209–212

450j Index

chymotrypsin 70, 71, 79, 103, 318, 328,342, 343

coagulation cascade 80, 338–340coagulation factors 80, 338, 340, 341compactin 6constitutive proteasome (cCP) 325Corey–Chaykovsky epoxidation 38covalent kinase inhibitors 172–177– covalent JNK inhibitors 173, 174– EGFR inhibitors 172, 173– mitogen- and stress-activated kinase 1

(MSK1) 174– RSK1-CTD/RSK2-CTD inhibitors 175–177– signaling pathways 172crizotinib 12cyclic ether templates, in drug discovery

252–255– bioactive natural products containing cyclic

ethers 252– cyclic ethers in saquinavir structure 254– structural evolution of inhibitors 255cyclic sulfones as P2 ligands 255–257cyclopentanyl urethane 254cysteine protease inhibitors 131– catalytic mechanism of peptide hydrolysis

by 132– development with Michael acceptors

132–135– HRV 3C protease inhibitors 135– with Michael acceptor group 133– noncovalent, design of inhibitors 136–140– SARS-CoV 3CLpro inhibitors 136– a,b-unsaturated carbonyl and vinyl

sulfone-containing 133– vinyl sulfone-derived 134cytokines 150, 199, 207, 271, 338

ddabigatran 11, 345–353– binding mode 346, 347– development 345– enzymatic activity and IC50 values 348– ethyl ester derivative 352– examination of x-ray crystal structure 349,

350– to increase of potency and strong reduction in

peptidic character 346, 348– piperidine ring and the naphthyl moiety

345– prodrug (dabigatran etexilate), clinical

investigation 352– structure–activity relationship (SAR)

studies 345, 348

– structures and activities 345, 346, 348–350,349, 351

– surface representation, complex with 347,353

darunavir 11, 239– binding mode 265– bis-THF-derived inhibitors for clinical

development 265– design/development 251, 252, 263–266– no amide/peptide-like features 252– pharmacological/bioavailability/toxicity

properties 252– potently inhibit dimerization of protease 264– structure-based design of inhibitors

leading 263– x-ray crystal structure 264dasatinib 11, 160, 274, 279, 284– approval 285– binding mode 287, 288– binding to active conformation 284–289– design 161– docking studies, 2-amidothiazole moiety to

form hydrogen bond with 286– inhibitory potencies of 288– optimization studies 285– potency 286– x-ray cocrystal structure with Abl kinase 161,

289deep venous thrombosis (DVT) 337delavirdine 3562-deoxy-2,3-didehydro-N-acetylneuraminic

acid 400, 401Dess–Martin periodinane 124diazepam 53,9-diazobicyclononanone 50Dieckmann cyclization 502,3-dihydrophthalazine-1,4-diones 165diisopropyl phosphorofluoridate (DFP) 70dipeptide isosteres 22–24, 29, 31, 35, 41, 423,

424, see also Leu-Ala dipeptide isosteredipeptidyl peptidase-I (DPP-I) 133diphenyl phosphonate-based inhibitors

70–73direct thrombin inhibitor 337, 341, 342, 3533,4-disubstituted alkoxyarylpiperidine 46dorzolamide 9, 412–418– chemical structures 412– development 412–418– dorzolamide-bound carbonic anhydrase

structure, 417– effect of His64 side chain shift on 417– glaucoma treatment 412– properties 413, 414

Index j451

– stabilize pseudoaxial alkylamino groupconformation 416

– suitable for topical administration 414– thiopyran ring conformation 416dorzolamide–carbonic anhydrase II complex– x-ray crystal structure of 417drug discovery– during 1928–1980 1–6– cyclic ether templates in 252–255– during the late 1970s 6–12drug resistance– “backbone binding concept” to combat

259–263– HIV-1 variants 253, 260– malaria strains 6– strategies for combating 279drug–target interactions 274dual Abl/Src inhibitor, see dasatinib

eefavirenz 356, 368, 369Endothia parasitica 25– L-363564 bound to aspartic acid protease 25epoxomicin 118, 119, 123–125, 330, 331, 333– mechanism of inhibition of proteasome 330– structure 330epoxy ketone scaffold, synthesis of 123–125– spirodiepoxide ring-opening strategy 125– stereospecific epoxidation of allyl

alcohol 125– synthesis by enone epoxidation 124erlotinib 10, 273Escherichia coli 184etravirine 11, 355–357, 360–370– antiviral potency of compounds 369– approved by FDA 367– efficacy against K103N mutants 368– structure–activity relationship

investigations 361E2–ubiquitin complex 325

ffibrinogen 80, 338–340, 343, 344fms-like tyrosine kinase (FLT1) subfamily 168– aniline derivative, potency against 168fosamprenavir 10, 238, 239, 255, 256fragment-based studies 187– arylsulfonamides 190– BRD4 inhibitors 189– D-amino acid oxidase 187– drug design 186– epigenetic regulation of gene expression

189

– fragments against Hsp90 through ligand-observed NMR 193

– fragments screened to find hits suitablefor 188

– optimization of fragment 190– structures of fragment hit and optimized

inhibitors 188– x-ray crystal structure 189– ZINC database 189

ggefitinib 10, 159, 273glaucoma 411– carbonic anhydrase inhibitors 411–418– dorzolamide 9, 411–418glutathione S-transferase (GST) 184glycoproteins 144, 199, 207, 398glycosaminoglycans 341, 344G protein 199, 200G-protein-coupled receptors (GPCRs) 8, 12,

186, 199– activated/inhibited by 200– conformations 200– high-resolution structures of 200, 201– signaling system 199Grignard reagents 27GRL-8234 21, 22, 422, 432, 433, see alsoBACE1

inhibitors– rescued cognitive decline, in mice 433, 435,

436– x-ray crystal structure 433

hhalomethyl ketone-based inhibitors 69, 70hemorrhagic diseases 338hemostasis 338heparin 337, 340, 341, 353heparin–antithrombin complex 344hepatitis C virus (HCV) 295– protease inhibitors 295, 298– – cleavage sites of substrate peptide 298– – developments, challenging hurdles 295– – FDA-approved HCV NS3 protease

inhibitors 296– – NS3/4A protease inhibitors (see NS3/4A

serine protease)– – structure-based design 295high-throughput screening (HTS) 8, 13, 46– acyl guanidine-based structure 51– aminopyridine-based BACE1 inhibitors 53– chemotypes 50– development of Maraviroc from HTS lead

molecule 207, 208

452j Index

– 1,5-dihydropyrazole benzothiazinederivative 170

– fragment screening against b-secretaseusing 53

– identified diaminopyrimidine-derivedcompounds 50

– libraries 187– noncovalent and reversible PLpro

inhibitors 139– papain-like protease of SARS-CoV 138– of Roche compound collections 46– weak inhibitor of MMP-12/13 148–150hirudin 342, 343HIV-1 protease 237– binding mode of acetyl pepstatin within

active site 240– gag and gag–pol polyprotein, cleavage

sites 241– require homodimerization 264HIV-1 protease inhibitors 8, 41– containing a (hydroxyethyl)sulfonamide

isostere 43– first-generation HIV-1 protease inhibitors,

structures 238– 4-hydroxycoumarin structural motif 45– 4-hydroxypyrone structural motif 46– inhibitors containing (hydroxyethyl)urea 42– second-generationHIV-1 protease inhibitors,

structures 239– structure-based design 238HIV reverse transcriptase 357–360HMG-CoA reductase 6Horner–Emmons olefination, m-

nitropropiophenone 45HTS, see high-throughput screening (HTS)human immunodeficiency virus (HIV), see

HIV protease inhibitors; HIV reversetranscriptase; rilpivirine

human rhinovirus 3C (HRV 3C) proteaseinhibitors 134

hydroxyethylamine isosteres 24– design of inhibitors with 35–37– JG-365 37– OM99-2 35hydroxyethylene 23, 24hydroxyethylene isostere– based inhibitors 29–35– design of inhibitors with 35–37– inhibitor JG-365 36, 37(hydroxyethyl)sulfonamide-based

inhibitors 42(hydroxyethyl)sulfonamide isosteres 40(hydroxyethyl)urea-based inhibitors 40

hypertension, renin inhibitor 373– aliskiren, biological properties 393– pepstatin 374– peptidomimetic inhibitors 376–379– primary/secondary carboxamides– structure of renin 373, 374– transition-state isosteres 374–376

iimatinib 8, 10, 164, 173, 274, 289– affinity toward PDGFR 278– clinical development 278– crystal structures 281– evolution of phenylaminopyrimidine

scaffold 274, 275– “flag methyl,” completely abolished

inhibition 274– imatinib–Abl complex 281– inhibitory potencies 276– movement of phenylalanine side chain

278– pharmacological profile 278– 3-pyridyl moiety, introduction 274– resistance 279– structural basis of selectivity 275–278– substitution of phenyl amide moiety 274– van der Waals interactions 278– x-ray crystal structures 277immunoproteasome (iCP) 325indinavir 9, 237, 238– benzyl derivative 249– cis-derivative 249– conformationally constrained benzo-fused

cycloalkyl amide 248, 249– containing hydroxyethylene transition-state

isostere 246–251– heptapeptide mimetic 246– hydroxyl group on indane moiety 251– incorporation of 8-quinolinylsulfonyl

derivative 250– merging decahydroisoquinoline group

of 249– minimum inhibitory concentration

246, 247– phenylalanine side chain resulted in 247– removal of phenylalanine and incorporation

of benzylamide 247– replacement of the decahydroisoquinoline

moiety 249influenza, treatment 406– oseltamivir (see oseltamivir)– prodrug conversion to active drug 406– type A and B viruses 397

Index j453

– zanamivir-bound neuraminidase (seezanamivir)

isoindoline-1,3-diones 165

kb-ketophosphonic acid– carboxamide derivatives of 104kinase inhibitors, as anticancer agents 272– FDA-approved protein kinase inhibitors

273

lb-lactone scaffold, synthesis of 121–123– domino acylation/b-lactone strategy 123– synthesis of lactacystin b-lactone 122laninamivir 398lapatinib 11, 273L-benzylsuccinic acid 221Leu–Ala dipeptide isostere 31– design approach, use in 30, 423, 424– lipophilic nature 423– substrate-based design of a b-secretase

inhibitor incorporating (see OM99-2inhibitor)

– synthesis 32librium 5ligand–protein interactions 7, 302lipophilicity 423, 429lopinavir 9, 239

mmaltose binding protein (MBP) 184matrix metalloproteases (MMPs) 143– catalytic mechanism 144– inhibitors, design of 144–150– structures/activities of inhibitors– x-ray crystallographic studies 149mechanism-based inhibitors, see NS3/4A

serine proteasemethazolamide 411, 412mevinolin (lovastatin, Zocor) 6Michael reaction 50microcrystallography 185, 186MMPs, see matrix metalloproteases (MMPs)mutation 184, 185– activation loop of B-Raf kinase 273,

274– G190A 359– M351T 282– single-point 362, 363, 366– single surface amino acid 185– Swedish mutant APP 423, 424– Tyr255 279

nnelfinavir 9, 40, 238neuraminidase 397, 398, 400– complexes 404, 406, 407– structure 399, 403, 407– zanamivir-bound 403neuraminidase inhibitors 397, 398– sialic acid, hydroxyl group at C-4 402– structures and activities 399, 402, 405– x-ray crystal structure 403neurotransmitters 199nevirapine 356, 358, 369nilotinib 11, 164, 274, 280– Abl–nilotinib complex 281– binding mode 281– cellular activity 282– crystal structures 281non-nucleoside HIV reverse transcriptase

inhibitors– antiviral activity profile 360, 362, 363, 366– antiviral potency 367, 369– binding mode 364– binding pocket 359– butterfly-like binding conformation 358– etravirine (see etravirine)– interactions with residues 358– loss of potency 359– p51/66 subunit, structures 357– rilpivirine (see rilpivirine)– side chains of amino acids 363– structure 357–360– structure–activity relationship 361– surface representation of 360– triazine derivatives 364– triazine/pyrimidine, resilience of 366– Trp231 residue 363, 365– wild-type HIV-1 inhibitors– x-ray cocrystal structure 365non-nucleoside reverse transcriptase

inhibitors 356– butterfly-like binding conformation 358nonreceptor tyrosine kinases (NRTKs). 271,

272NS3/4A serine protease– binding mode 303– formation of covalent bond,mechanism 301– optimization of P2 interactions 309–311– peptide hydrolysis 299, 300– peptide inhibitors bearing electrophilic

“warheads” 301– peptidic character, reduction 308, 309– strategies for development, protease

inhibitors 303, 304

454j Index

– structures 296, 298– transition-state analogswithin active site 301

oodorants 199OM99-2 inhibitor 35– design approach 424oseltamivir 10, 397, 398, 403–407– acetylamido group, maintained at 404– 4-amino group with appropriate

stereochemistry 404– antiviral activity 406– chemical structures of 398– choice of the carbocyclic template 404– discovery of 403–407– hydrophobic interaction 405– to improve lipophilicity 405– inhibitory activity 404– isomeric carbocyclic structures 404– oral bioavailability 404, 406– pyranose ring conformation 404– vulnerable to resistant viral strains 406, 407– x-ray crystal structure, complex of

inhibitor 406oxocarbenium ion– bound conformation of 400

ppapain 133pazopanib 12, 161, 162PDGFR kinase, see platelet-derived growth

factor receptor (PDGFR) kinasePenicillium citrinum 6Penicillium notatum 2pepstatin 24, 25peptidic character, reduction 308, 309, see also

NS3/4A serine proteasepeptidomimetic BACE inhibitors, see also

BACE1 inhibitors– design 427–431– development 423–425peptidomimetic inhibitors 22, 239, 240,

376–379– aspartic acid proteases 22–24– design 380–393peptidyl a-ketoamide-based inhibitors 85–90– templates synthesis 90–93peptidyl a-ketoheterocycle-based

inhibitors 85–90– templates synthesis 85–90peptidyl boronic acid-based inhibitors 78–83– a-aminoalkyl derivatives 83, 84peptidyl chloromethyl ketone inhibitors 70

peramivir 398Philadelphia chromosome 274phosphinate-based inhibitors 234plasmepsin II 25Plasmodium falciparum 48platelet-derived growth factor receptor

(PDGFR) kinase 167, 271, 272, 274–276,278

platelet receptors 344Polo-like kinase 1 (PLK1) 163– 2-aryl pyrimidodiazepinone derivatives 163– structures and activities 163– x-ray crystal structure 164polyubiquitinated proteins 325ponatinib 12porcine pancreatic elastase (PPE) 75protease inhibitors, see various specific protease

inhibitorsproteasome inhibitors 113–115, 325– aldehyde proteasome inhibitors, chemical

structures 327– boronate proteasome inhibitors (see

bortezomib)– chymotrypsin-like activity of

proteasome 331– development of boronate proteasome

inhibitors 115, 116– development of epoxy ketone-derived

inhibitors 118, 119– – eponemycin 119– – epoxomicin 119– – morpholine adduct 119– – PR-171 119– – YU-101 119– exploration of side chain functionalities 333– improved potency 327– b-lactone natural product-based proteasome

inhibitors 116–118– – belactosins A and C and derivatives 118– – lactacystin 117– – omuralide 117– – salinosporamide A 117– noncovalent proteasome inhibitors 120, 121– phenylalanine and naphthylalanine

derivatives 332– 20S proteasome, catalytic mechanism 113,

114– 26S proteasome protein complex 325, 326protein crystallization 183, 184Protein Data Bank (PDB) 157, 183protein engineering 184, 185protein kinase inhibitors 155– active site of protein kinases 155, 156

Index j455

– catalytic mechanism of protein kinases156

– design strategy for inhibitors 156–159– nature of kinase inhibitors based upon

binding– protein kinase CK2 inhibitors 162protein–ligand complexes 21, 196protein–ligand interactions 46, 183, 196protein–protein interactions 185protein purification 184protein x-ray crystallography 183prothrombin 339–342pulmonary embolism (PE) 337pyrimidodiazepines 163

qquinazoline-3-oxides 4

rRAAS, see renin-angiotensin-aldosterone

system (RAAS)receptor tyrosine kinases (RTKs) 271– effector proteins/kinases, cellular

response 272– stimulation in response to ligand

binding 271– structural organization 272recombinant DNA technology 185recombinant proteins 184renin 373– complex, x-ray crystal structure 390, 392– recombinant human renin 374renin–angiotensin–aldosterone system

(RAAS) 219, 220renin inhibitors 7, 8, 47– alkoxyarylpiperidine class 48– bridged piperidine structural template in

optically active form 49– containing hydroxyethylene isosteres

(see hydroxyethylene isostere)– 3,5-disubstituted piperidine structural

scaffold 50– H261 and CP-69799, x-ray structures 41– incorporating a hydroxyethylamine core 35– new class of nonpeptide 47– nonpeptidomimetic 49– piperidine structural motif in optically active

form 48– structure-based optimization 8reverse transcriptase inhibitors– antiviral activities of compounds 362, 363– antiviral potency of compounds 367– binding mode of triazine compounds 363

– SAR relationship for triazine 362– structures/activities 361Rhizopus chinensis 7rilpivirine 356, 360–369– administered with 369– antiviral potency of compounds 367– approved by FDA 367– phase III clinical studies 368– potent efficiency 369– x-ray structures 369ritonavir 9rivaroxaban 11

ssaquinavir 8, 9, 237– binding mode 244, 245– conformational changes 246– as first clinically approved inhibitor 241–246– hydroxyethylamine transition-state isostere

241– hydroxyethylene-based transition-state

isosteres 243– introduction of cyclic ethers in 254– molecular size 253– systematic optimization 243– tert-butyl group 246– transition-state hydroxyl

stereochemistry 244– tripeptide analog 242– twofold improvement in enzyme inhibitory

potency 243, 244– x-ray crystallographic study 244SARS-CoV 3CLpro/SARS-CoV PLpro

inhibitors 136–139– targets for drug design 136SAR studies, see structure-activity relationship

(SAR) studiesb-secretase inhibitors 421– AZD3839 422, 436, 438, 439– bilobal structure 422, 423– conformation, flexibility of side chains 422– CTS-21166 422, 436, 443– inhibitor GRL-1439 21– x-ray structures 422, 423c-secretase inhibitors 41– inhibitors containing (hydroxyethyl)urea 42serine protease inhibitors 67– basic structural cores for design of 68– catalytic mechanism, of peptide

hydrolysis 67, 68– design of inhibitors, based on

heterocycles 93, 94– types 67–69

456j Index

severe acute respiratory syndrome (SARS) 136– recognition of cysteine proteases 136small-molecule inhibitors 431, 436– design, with clinical potential 431–434sorafenib 10, 159, 164, 273, 274– x-ray crystal structure 165Staphylococcus aureus 2statine– analogs 24– based inhibitors 24– discovery of 25– oxidative cleavage 27– synthesis 26structure–activity relationship (SAR) studies– ACE inhibitors 223– 4-amino group, not important for

activity 365– 2-aminothiazole scaffold 285– benzimidazole 348– dabigatran 345– increasing ring size of amide chain, reduce

hERG affinity 211– non-nucleoside reverse transcriptase

inhibitors 360, 361– (S)-phenyl configuration compounds 209– 1,3,5-triazine reverse transcriptase

inhibitors 362structure-based design approaches– adenosineA2A receptor antagonists 204–207– BACE inhibitors GRL-8234 and GRL-1439

22– drugs derived from 9–12– x-ray crystallography 182, 183substrate-based inhibitors 8sunitinib 11, 159, 273Suzuki coupling 56synchrotron radiation 185, 186

tTaxol 5Taxus brevifolia 5telaprevir 82, 87, 296– binding mode 318– inhibit HCV NS3/4A protease by 318– landmarks in design/discovery 317– optimization strategy 316–319– truncation beyond three/four amino

acids 314– x-ray crystal structure 318Thermoplasma acidophilum 325– 20S proteasome from 325, 326thieno[2,3-b]thiopyran-2-sulfonamide

7,7-dioxides 415

thiopyran ring 416thrombin– activation 339, 340– inhibition 100, 340– structure 342– substrates 344– surface structure 343– x-ray crystal structure of binary complex

342thrombosis 337thymoproteasome (tCP) 325tipranavir 10, 45, 238, 239tosyl chloride 501,3,5-triazine reverse transcriptase

inhibitors 362–364, 367trifluoroacetic acid 46trifluoromethyl ketones 73–76– cocrystal structure of inhibitor 75– design strategy 74– fluoromethyl ketone-containing

inhibitors 74– stereoselective synthesis containing a valine

side chain 76– synthesis of peptidyl trifluoromethyl ketone

derivatives 77, 78truncation– of natural substrate 316– strategy for boceprevir 312–314– strategy leading to a-ketoamide

inhibitors 312trypsin 343tumor necrosis factor-a (TNF-a) 150tumor necrosis factor-a-converting enzymes

(TACE) 150– design of inhibitors of 150–152– non-Zn TACE inhibitors 151– selective non-hydroxamate inhibitors 151– structures and activities, of selective

inhibitors 150type I kinase inhibitors– dasatinib (see dasatinib)– pharmacophore model 161type II kinase inhibitors 164–168– FDA-approved type II inhibitors 164– structure-based design 165– – as B-Raf kinase inhibitors 165type III kinase inhibitors, see allosteric kinase

inhibitorstyrosine kinases 271

uubiquitin-proteasome system 325udenafil 10

Index j457

vvalium 5VEGFR-2 inhibitors 166– 3-aminoindazole-based 167– meta-trifluoromethyl group, for potency 166– pyrrolopyrimidine-based 166– structures and activities 167vemurafenib 12venous thromboembolism 337vinyl sulfones 133virtual screening 13, 186– agonists/antagonists of receptors GPCRs,

relied upon 200, 201– applied to b2-adrenergic receptor 201–204– phenyl thiadiazole derivative 162– ZINC database 206von Willebrand factor 338vorinostat 11, 189

wwarfarin 44, 337Wittig olefination 38

xximelagatran 10x-ray crystal structures– Abl kinase and imatinib complex 277– aliskiren and renin complex. inhibitor carbon

chain 392– arylpiperidine with renin 47– bafetinib–Abl kinase complex, hydrophobic

pocket around phenyl ring 284– binary complex thrombin and hirudin 342– bortezomib with 20S proteasome from

yeast 329, 330– carazolol–b2�adrenergic receptor

complex 202– carbonic anhydrase II 413– cocrystal structure of fragment with Hsp90

194– D-amino acid oxidase displaying Tyr226

residue in 188, 189– darunavir 264

– dorzolamide–carbonic anhydrase IIcomplex 417

– endothiapepsin complexed withhydroxyethylene isostere-containinginhibitor H263 30

– ethyl ester derivative of dabigatran 352– HIV-1 protease 240– methoxyphenprocoumon with HIV-1

protease PDB code 44– neuraminidase and zanamivir complex

403– NS3/4A bound to inhibitor 306, 311– recombinant human renin 374– renin complex 379, 384, 385– renin inhibitors H263 and CP-69799 41– sorafenib-bound p38a 165– structure bound yeast proteasome 120– ternary complex thrombin/hirudin and

inhibitor 346– testis ACE and enalaprilat complex, major

hydrogen bonding interactions 230– testis ACE and lisinopril complex 231– with wild-type HIV-1 protease, inhibitor

represented as CPK 262– ZM241385–adenosine A2A receptor

complex 205

zzanamivir 9– 4-aminoepimeric derivative 401, 402– chemical structures of 398– complex, x-ray crystal structure 403– discovery of 401–403– effective against influenza 403– evaluation of 4-amino derivative and design

hypothesis 401– guanidine group at C-4 402– intranasal administrative route 403– introduction of guanidine group at 402– structures and activities 402– x-ray crystal structure, complex with

neuraminidase 403ZINC database 189, 202, 206

458j Index

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