ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2015

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 197

Design and Synthesis of HepatitisC Virus NS3 Protease Inhibitors

Targeting Different Genotypes and Drug-ResistantVariants

ANNA KARIN BELFRAGE

ISSN 1651-6192ISBN 978-91-554-9166-6urn:nbn:se:uu:diva-243317

Dissertation presented at Uppsala University to be publicly examined in B41 BMC,Husargatan 3, Uppsala, Friday, 27 March 2015 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Pharmacy). The examination will be conducted in Swedish. Facultyexaminer: Ulf Ellervik (Lunds tekniska högskola).

AbstractBelfrage, A. K. 2015. Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors.Targeting Different Genotypes and Drug-Resistant Variants. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Pharmacy 197. 108 pp. Uppsala:Acta Universitatis Upsaliensis. ISBN 978-91-554-9166-6.

Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerousdirect acting antivirals (DAAs) have reached late stages of clinical trials. Today, severalcombination therapies, based on different DAAs, with or without the need of pegylatedinterferon-α injection, are available for chronic HCV infections. The chemical foundation ofthe approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partlyexplain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors.The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 ofthe virus and were less potent against other genotypes.

The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinonebased HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinicaltrials or approved, potentially with a unique resistance profile and with a broad genotypiccoverage. Successive modifications were performed around the pyrazinone core structure toclarify the structure-activity relationship; a P3 urea capping group was found valuable forinhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket.Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essentialfor inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. Invitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety onthe overall drug-properties and biochemical evaluation of the inhibitors against drug resistantenzyme variants showed retained inhibitory potency as compared to the wild-type enzyme.Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, withrespect to improved PK properties, were also performed on an advanced class of HCV NS3protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclicproline urea scaffold with nano-molar cell based activities.

Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yieldingintroductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methodscan be generally applicable in the synthesis of bioactive compounds containing peptidomimeticscaffolds and carboxylic acid bioisosteres.

Keywords: hepatitis C virus, HCV, NS3 protease inhibitors, structure-activity relationship,2(1H)-pyrazinone, quinazoline, resistance, Pd catalysis

Anna Karin Belfrage, Department of Medicinal Chemistry, Organic PharmaceuticalChemistry, Box 574, Uppsala University, SE-75123 Uppsala, Sweden.

© Anna Karin Belfrage 2015

ISSN 1651-6192ISBN 978-91-554-9166-6urn:nbn:se:uu:diva-243317 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-243317)

Till Johan och Hedda ♥

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nilsson, M., Belfrage, A K., Lindström, S., Wähling, H.,

Lindquist, C., Ayesa, S., Kahnberg, P., Pelcman, M., Benkestock, K., Agback, T., Vrang, L., Terelius, Y., Wikström, K., Hamelink, E., Rydergård, C., Edlund, M., Eneroth, A., Ra-boisson, P., Lin, T-I., de Kock, H., Wigerinck, P., Simmen, K., Samuelsson, B., Rosenquist, Å.* Synthesis and SAR of potent inhibitors of the hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents. Bioorg. Med. Chem. Lett., 2010, 20, 4004-4011.

II Gising, J.,# Belfrage, A K.,# Alogheli, H., Ehrenberg, A., Åkerblom, E., Svensson, R., Artursson, P., Karlén, A., Dan-ielson, U. H., Larhed, M., Sandström, A.* Achiral pyrazinone-based inhibitors of the hepatitis C virus NS3 protease and drug-resistant variants with elongated substituents directed toward the S2 pocket. J. Med. Chem., 2014, 57, 1790-1801.

III Belfrage, A K., Abdurakhmanov, E., Åkerblom, E., Oshalim, A., Gising, J., Skogh, A., Danielson, U. H., Sandström, A. Py-razinone based hepatitis C virus NS3 protease inhibitors target-ing genotypes 1a and 3a, and the drug-resistant enzyme variant R155K. Manuscript.

IV Belfrage, A K.,* Gising, J., Svensson, F., Åkerblom, E., Sköld, C., Sandström, A. Efficient and selective palladium-catalysed C3-urea couplings to 3,5-dichloro-2(1H)-pyrazinones. EurJOC., 2015, 978-986.

V Belfrage, A K., Wakchaure, P., Larhed, M., Sandström, A. Pal-ladium-catalyzed carbonylation of aryl iodides with sulfin-amides. Manuscript.

*Corresponding author. # These authors contributed equally.

Reprints were made with permission from the respective publishers.

Contents

1 Introduction .............................................................................................. 11 1.1 Hepatitis C Virus ............................................................................. 11

1.1.1 General Background .............................................................. 11 1.1.2 Course and Outcome of HCV Infection ................................. 12 1.1.3 The Viral Life Cycle .............................................................. 13 1.1.4 The Viral Proteins .................................................................. 15

1.2 Treatment Strategies ....................................................................... 15 1.2.1 Current Treatment .................................................................. 16

1.3 Resistance to Direct Acting Antiviral Drugs .................................. 19 1.4 HCV NS3 Protease .......................................................................... 21 1.5 Discovery and Development of HCV NS3 Protease Inhibitors ...... 24

1.5.1 A Historical View of the Discovery of HCV NS3 Protease Inhibitors ................................................................................ 25

1.6 Model Systems for Analyzing HCV NS3 Protease Inhibitors ........ 28

2 Aims of the Thesis ................................................................................... 32

3 P2 Quinazoline Substituents in HCV NS3 Protease Inhibitors (Paper I) .................................................................................................... 33 3.1 Pharmacokinetic Aspects in Drug Discovery ................................. 33 3.2 Harmonizing Antiviral Potency with PK Properties in the

Development of HCV NS3 Protease Inhibitors (Paper I) ............... 34 3.3 Synthesis of P2 Quinazoline-Containing HCV NS3 Protease

Inhibitors ......................................................................................... 39

4 2(1H)-Pyrazinone-Based HCV NS3 Protease Inhibitors (Papers II and III) ..................................................................................... 45 4.1 Pyrazinones in Medicinal Chemistry .............................................. 45 4.2 From Tripeptides to Pyrazinone-Based HCV NS3 Protease

Inhibitors ......................................................................................... 46 4.3 Development of Achiral Inhibitors (Paper II) ................................. 46 4.4 Exploration of the P1P1’ Substituents (Paper III) ........................... 56 4.5 P3P4 Urea Elongations (Paper III) .................................................. 61 4.6 Evaluation Towards the Genotype 3a of HCV NS3 Protease

(Paper III) ........................................................................................ 64

5 Synthesis of Pyrazinone-Based HCV NS3 Protease Inhibitors (Papers II and III) ..................................................................................... 66 5.1 Formation of the 2(1H)-Pyrazinone Core ....................................... 66 5.2 Introduction of Urea Substituents ................................................... 68 5.3 Synthesis of P1P1’ Building Blocks ............................................... 70 5.4 Amide Coupling of P3 Pyrazinones and P1P1’ Building Blocks ... 73 5.5 Final Decorations and Modifications .............................................. 74

6 Method Development (Papers IV and V) ................................................. 76 6.1 Palladium as a Catalyst in Organic Synthesis ................................. 76 6.2 C-N Urea Arylation ......................................................................... 76 6.3 Palladium Catalyzed Urea Couplings to 3,5-Dichloro-2(1H)-

Pyrazinones (Paper IV) ................................................................... 77 6.4 Palladium Catalyzed Carbonylations .............................................. 84 6.5 Synthesis of Acyl Sulfinamide, a Versatile Functionality

(Paper V) ........................................................................................ 85

7 Concluding Remarks ................................................................................ 92

8 Acknowledgements .................................................................................. 94

9 References ................................................................................................ 97

Abbreviations

ACCA ADME Ala or A Arg or R Asp or D CD CDI Clint CMC CO CYP3A Cys or C DAA DBU DCM DFT DMAP DME DMSO dppe dppp EC50

ER FRET Gln or Q Gly or G

aminocyclopropane carboxylic acid absorption, distribution, metabolism and excretion alanine arginine aspartic acid candidate drug carbonyl diimidazole intrinsic clearance chemistry, manufacturing and control carbon monoxide cytochrome P450 Isoform 3A cysteine direct-acting antiviral agent 1,8-diazabicyclo[5.4.0]undec-7-ene dichloromethane density functional theory 4-dimethylaminopyridine 1,2-dimethoxyethane dimethylsulfoxide 1,2-bis(diphenylphosphino)ethane 1,3-bis(diphenylphosphino)propane inhibitor concentration giving 50% inhibition in a cell-based system endoplasmic reticulum fluorescence resonance energy transfer glutamine glycine

Gt HATU His or H HPLC

genotype N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluoro-phosphate N-oxide histidine high-performance liquid chromato-graphy

HSPG HTA HCV IFN IRES LC-MS log D7.4 Lys or K Met or M MW NMR NS Papp Pd PEG Phe PK pKa RBV RNA SAR Ser or S SOC SRB SVR THF Thr or T TMS Val or V wt Xantphos

heparan sulfate proteoglycan host-targeting antiviral agent hepatitis C virus interferon internal ribosome entry site liquid chromatography-mass spectrometry logarithm of the distribution coefficient at pH 7.4 lysine methionine microwave nuclear magnetic resonance non-structural apparent permeability coefficents palladium pegylated phenyl alanine pharmacokinetics the acid dissociation constant at loga-rithmic scale ribavirin ribonucleic acid structure-activity relationship serine standard of care scavenger receptor class B sustained virological response tetrahydrofurane threonine trimethylsilyl valine wild-type 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene

11

1 Introduction

1.1 Hepatitis C Virus Forty years ago, in 1974, it was found that a previously unknown infectious agent, different from the known hepatitis A and B viruses, caused transfu-sion-associated hepatitis.1 Fifteen years later, in 1989, the viral agent was characterized and named hepatitis C virus (HCV).2 Another twenty-two years later, in 2011, the first direct-acting antiviral agents (DAAs) against HCV entered the market. This was the beginning of a new era in HCV histo-ry.

1.1.1 General Background Each year, 350 000-500 000 people die from HCV-related liver diseases.3 In the western world, HCV is the primary indication for liver transplantations, and the supply of organs remains less than the demand.4 It is estimated that 130-150 million people worldwide are chronically infected, but the majority of people carrying HCV are still undiagnosed.3,5 HCV is unevenly distribut-ed among the different regions of the world, as well as between age and risk groups within countries.6 The highest rates of prevalence are found in North Africa and the Middle East.7 Approximately 15% of the citizens of Egypt are infected; this high figure is the result of a health campaign running from the 1950s to the 1980s which had the objective of reducing the prevalence of schistosomiasis, a parasitic worm infection. The campaign was successful, but HCV was widely spread among the population at the same time, because of insufficient knowledge about transmittance of the virus. In North Ameri-ca, Australia, Japan and Northern and Western Europe, the prevalence is lower, with an upper limit of 2%.6

Unsafe drug injections are nowadays the most common method for transmission of HCV in developed countries, whereas unscreened blood or poor handling of medical equipment in healthcare are still common reasons in resource-poor countries.8 The virus can also be transmitted sexually or from infected mother to her baby, but these causes of infection are less common.3

Six major genotypes (Gts) and several subtypes of HCV are unevenly dis-tributed over the world (Figure 1). Gt 1 is found worldwide, and is the major variant in America, Europe, Australia and Japan. Gt 2 is found in the indus-

12

trialized countries as well as in South America and Asia. Gt 3, a variant that appears to be increasing in prevalence, is common in Europe, North Ameri-ca, Australia and Southern Asia. Gts 4-6 are isolated to specific countries: Gts 4-5 in Africa and the Middle East and Gt 6 in Southeast Asia.6

Figure 1. Global distribution of HCV genotypes.9 Reproduced with the permission of the publisher.

1.1.2 Course and Outcome of HCV Infection HCV causes both acute and chronic hepatitis. One to two weeks after expo-sure to the virus, HCV ribonucleic acid (RNA) can be detected in the serum. Subsequently, the levels of serum alanine aminotransferase elevate, indica-tive of hepatocyte injury. The symptoms are diffuse, (they include flu-like symptoms, nausea and jaundice) but are often absent during the early phases of the infection. Therefore, infected persons seldom seek medical care at this stage.

About 20% of those infected will recover spontaneously but around 80% will develop a chronic infection, often asymptomatic for up to 20-30 years. Progression to cirrhosis is estimated to occur in 15-20% of those with a chronic infection; cirrhosis can ultimately develop into end-stage liver dis-ease or hepatocellular carcinoma.10 Liver transplantation is the only life-saving alternative at this stage.4

13

Figure 2. A schematic representation of the HCV life cycle. Attachment and entry into the hepatocyte is followed by release of (+) RNA into the cytoplasm, transla-tion by the ribosomes on the endoplasmic reticulum, polyprotein processing into ten mature viral proteins, replication and assembly and release of new virus particles.11 Reproduced with the permission of the owner.

1.1.3 The Viral Life Cycle HCV belongs to the virus family Flaviviridae; it is a single-stranded (+) RNA virus.12 The viral RNA genome is protected by a nucleocapsid layer and the envelope glycoproteins E1 and E2. Replication of HCV mostly oc-curs in the hepatocytes, which are present in the liver.10,13 The HCV viral life cycle is illustrated in Figure 2, and described in the text below. Cell binding and entry: The HCV particles that circulate in the serum of an infected person can be bound to host lipoproteins or immunoglobulins or be present as free HCV particles.14 Both the lipoproteins and the envelope gly-coproteins E1 and E2 are crucial for the interaction between HCV and the host cell.15 Several host proteins are involved in the HCV-host interaction and subsequent cell entry, which takes place by an endocytosis process.11

Some of the host cell proteins bind directly to the envelope glycoproteins, while others have indirect functions essential for binding and entry.15 The tetraspanin CD81 binds directly to E2, but also contributes to post-binding processes which assist in the internalization of the virus particle. The scav-enger receptor class B1 (SRB1) is another direct binding partner for E2. Moreover, SRB1 is a receptor for lipoproteins, which could provide addi-

14

tional interactions with the viral particle. It has also been suggested that SRB1 is involved in the entry of HCV into cells. Unlike the other known host factors, the only identified mission for the heparan sulfate proteoglycans (HSPGs) is to mediate HCV attachment to target cells via E1 and E2. The tight-junction protein claudin 1 interacts with CD81 rather than directly with the E1 or E2, but is nevertheless essential for HCV entry. A second tight-junction protein, occludin, is also important in the HCV entry process, but whether it has direct or indirect interactions with the HCV envelope is not yet understood.13,15 Translation and processing: Inside the cell, the RNA genome is released into the cytoplasm, where translation occurs in ribosomes at the endoplasmic reticulum (ER). The translation of the HCV genome encodes a polyprotein of more than 3000 amino acids, which subsequently cleaves into ten mature structural and non-structural (NS) proteins (Figure 3, further described in 1.1.4). The structural proteins and the p7 protein are processed by the ER signal peptidase. The NS proteins (NS2/3/4A/4B/5A/5B), on the other hand, are processed by viral proteases. These proteins are essential for replication, and for subsequent formation of new virus particles. The NS3 protease is the drug target in focus of this thesis and will be described in more detail in sec-tion 1.4.14,16

RNA replication: After the described cleavages into mature proteins, a repli-cation complex based on several viral proteins is formed on the ER mem-brane.13 A template, the complementary negative-sense RNA strand, is first synthesized, and this is followed by replication of the positive-sense RNA.11 Virion assembly and release: The newly formed positive-sense RNA is suc-cessively incorporated into new virus particles. The assembly of new virus particles is not completely understood, but it has been suggested that the viral envelope is built at the ER membrane. Finally, the virus particles are thought to be released through the secretory pathway.11,13

15

Figure 3. Schematic representation of the organization of the HCV polyprotein. The host cell enzymes, NS2 protease and NS3 protease cleave the protein into structural and non-structural proteins. 1.1.4 The Viral Proteins The structural HCV proteins C, E1 and E2 make up the outer structure of the virion (Figure 3). The core protein (C) forms the nucleocapsid, which sur-rounds the genetic material. E1 and E2 are glycosylated proteins which form a non-covalent complex, integrated in the viral envelope. The membrane protein p7 is thought to form ion channels, and could be important in the maturation and release of the viral particles.14

The zinc-dependent metalloprotease NS2, together with the N-terminal of NS3 protease cleave at the NS2/NS3 junction. In complex with the cofactor NS4A, NS3 protease cleaves at four junctions of the polyprotein (i.e. the junctions between NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B).17

The membrane-bound NS4B protein induces the formation of the mem-branous web, which works as a scaffold for the HCV replication complex.11 Although not fully understood, the phosphoprotein NS5A has multiple func-tions that are essential in the HCV replication process, viral assembly and viral release.11,18 The RNA-dependent RNA polymerase NS5B catalyzes the replication of viral RNA.11

1.2 Treatment Strategies The purpose of therapy is to eliminate HCV from the body (i.e. to achieve a sustained virological response, SVR), and thus to avoid HCV-related liver and extra-hepatic diseases (e.g. fibrosis, cirrhosis and death).19 HCV does not incorporate into the host cellular DNA, meaning that an HCV infection can be cured, in contrast to for example an HIV infection.20,21 There is still no preventative vaccine available, although progress has been made in this research field.22

16

All stages of the HCV life cycle could be targets for DAAs. Indeed, most of the viral proteins described above have been evaluated as drug targets, with varying levels of success.23

NS3 protease has been widely investigated in recent decades and these ef-forts have yielded several inhibitors, both the so-called serine-trapping elec-trophilic inhibitors (Figure 4) and product-based inhibitors (Figure 5), which are currently in varying stages of development. NS5A protein inhibitors and NS5B polymerase inhibitors are also now available as anti-HCV drugs (Fig-ure 6). In addition, so-called host-targeting antivirals (HTAs), which focus on the host factors required for viral replication, are being considered.20,23

Before 2011, the standard of care (SOC) for HCV infection consisted of a weekly injection of the immunomodulatory agent interferon (IFN)-α at-tached to polyethylene glycol (PEG) in combination with the broad-spectrum antiviral ribavirin (RBV). This therapy continued for 24 to 48 weeks, with varying success depending on the genotype of the virus.24 For Gt 1 infection, the SVR ranges from 42% to 46%. For Gts 2 and 3, the SVR is somewhat higher, between 76% and 80%.25 Factors like sex, age, body weight and co-infections also influence the outcome of this treatment regimen. In some cases the side-effects, such as influenza-like symptoms, depression and he-matological abnormalities, cause patients to abandon the treatment.24

Figure 4. The electrophilic HCV NS3 protease inhibitors telaprevir26 and bo-ceprevir27.

1.2.1 Current Treatment In 2011, the “first-wave-first-generation” HCV NS3/4A protease inhibitors, telaprevir28 (IncivekTM, Vertex) and boceprevir27 (VictrelisTM, Merck/Schering) were approved for use in combination with PEG-IFN-α and RBV against Gt 1 infection (Figure 4). The launching of these drugs was the beginning of a new era, demonstrating that DAAs for HCV could reach the market. The cure rate increased to 75% with the addition of telaprevir to SOC and to 70% for boceprevir. Also, a factor of significant importance, the treatment was reduced to 24-28 weeks, compared to 48 weeks with the pre-vious SOC.7 Despite these treatments having similar potency, efficacy, side effects and cost, sales for the third quarter of 2011 to the fourth quarter of

17

2012 were $ 2.11 billion for telaprevir and $ 0.114 billion for boceprevir; telaprevir became the fastest drug in history to reach a billion dollars in sales.5

Figure 5. HCV NS3 protease inhibitors in clinical trials or approved. Simeprevir,29 paritaprevir,30 danoprevir,31 vaniprevir,32 grazoprevir,33 asunaprevir,34 faldaprevir,35 vedroprevir,36 sovaprevir37 and ciluprevir.38

This appeared to be a tremendous step forward in the struggle to cure HCV-infected patients. However, these drugs have disadvantages that have imped-ed broad, lasting success. For instance, both drugs need to be taken in high doses and both are associated with side effects, such as anemia and severe

18

rashes, in addition to the adverse effects associated with PEG-IFN-α and RBV.7 In some patient populations the efficacy is low,5 and the risk of re-sistance development has been detected both in vitro and in vivo for these inhibitors.39–41

Figure 6. Approved DAAs used in anti-HCV therapeutic regimens: sofosbuvir (NS5B nucleoside inhibitor),42 ledipasvir43 and ombitasvir44 (NS5A inhibitors) and dasabuvir (NS5B non-nucleoside inhibitor).45

In 2013 the “second-wave-first-generation” NS3 protease inhibitor sime-previr29 (OlysioTM) (Figure 5) and the NS5B polymerase inhibitor sofos-buvir46 (SovaldiTM) (Figure 6) were approved; these drugs have more benefi-cial dosage regimens, fewer side effects and better pharmacokinetic (PK) properties than previous therapies.47,48 Due to this competition from new anti-HCV drugs, the sale of telaprevir was discontinued in 2014, three years after it entered the market as a successful drug.49 In January 2015 it was an-nounced that Merck planned to stop selling boceprevir by the end of 2015.

The biggest weakness in the current treatment regimens is the reliance on PEG-IFN-α and RBV, as required for preventing the emergence of resistant variants.21,23 Recently, the first all-oral combination therapy, HarvoniTM, reached the market. This contains a fixed-dose combination pill of ledipasvir (NS5A inhibitor)43 (Figure 6) and sofosbuvir42 and is the first approved reg-imen that does not require concurrent administration of PEG-IFN-α or RBV. Soon after, a combination therapy containing simeprevir and sofosbuvir, without PEG-IFN-α or RBV, was also approved.

In December 2014, a so-called “3D” (three DAAs) therapy, Viekira PakTM, developed by AbbVie (Abbott/Enanta), was approved. This new treatment regimen consists of a combination pill, ViekiraxTM, containing the antiviral drugs paritaprevir (NS3 protease inhibitor)30 (Figure 5) and om-

19

bitasvir (NS5A inhibitor)44 (Figure 6) plus ritonavir; it is intended to be add-ed to dasabuvir (ExvieraTM; a non-nucleoside NS5B polymerase inhibitor)45 (Figure 6) with or without RBV, for HCV Gt1 infections. The NS3 protease inhibitor in this therapy, paritaprevir, is highly metabolized by the enzyme cytochrome P450 isoform 3A (CYP3A). As a consequence, ritonavir, which is a strong inhibitor of CYP3A, is needed to prolong the half-life and in-crease the plasma concentrations of paritaprevir. This calls for careful plan-ning and monitoring in order to avoid drug-drug interactions, since CYP3A metabolizes many other drugs. Another concern is the relatively short half-life of the polymerase inhibitor dasabuvir, since it needs to be administered twice daily, which complicates the overall dosage regimen.30,44 However, the advantage of this 3D therapy is that PEG-IFN-α is not needed and the side-effects are relatively mild (e.g. fatigue, itching and nausea).30,45

These drugs will most likely be followed by new therapies as several DAAs (e.g. HCV NS3 protease inhibitors, Figure 5) are currently being evaluated, in various combinations, in clinical trials.23 However, at the same time, the recent approvals have forced the withdrawal of several advanced DAAs in late clinical trials because of the changes in the accessability of HCV drugs and a few additional benefits associated with new DAAs.

1.3 Resistance to Direct Acting Antiviral Drugs Because of fast turnover rate and lack of a proofreading function of the NS5B polymerase, HCV rapidly produces mutated variants which results in a wide genetic diversity of different genotypes (Gts 1 to 6) and subtypes (a, b, c…). In an infected individual an array of slightly different virus strains, so-called quasispecies, can be present.50 Under the pressure of selective anti-viral drugs, resistance-associated amino acid substitutions emerge in the drug target, which is a major challenge in anti-HCV drug discovery.51 The striking similarities in structure among advanced HCV NS3 protease inhibi-tors (Figure 5) could partly explain the cross-resistance that has been ob-served during evaluation of this class of DAAs.18

The strategy in the IFN-free treatments that are now available is to com-bine DAAs of different targets and dissimilar resistance profiles to create a high enough resistance barrier to inhibit the development of resistant vari-ants.21,23 Table 1 summarizes the level of the barrier to resistance, the extent of genotype coverage and the extent of antiviral activity of the different clas-ses of DAA.

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Table 1. Barrier to resistance, genotype coverage and antiviral activity18

NS3 protease

inhibitors

NS5A inhibitors

NS5B nucleoside inhibitors

NS5B non-nucleoside

inhibitors Barrier to resistance

Low Low High Low

Genotype coverage

Narrow/Broad Broad Broad Narrow

Antiviral activity

High High Intermediate/

High Low/

Intermediate

NS3/4A protease inhibitors: Both in vitro and in vivo studies have shown selection of drug-resistant variants under the pressure of NS3/4A protease inhibitors. Several inhibitors in this class show overlapping resistance pro-files. Common positions for amino acid substitutions caused by mutations that lead to resistance are R155, A156 and D168. The R155K/T/Q substitu-tion confers resistance to most NS3 protease inhibitors, both approved and in clinical trials (Table 2).18

Table 2. Substitutions at amino acid positions within the NS3/4A protease associat-ed with resistance 18,52

*In vitro data.

NS5A inhibitors: Although the barrier to resistance is low (Table 1), no cross-resistance with DAAs from other classes has been observed. Thus, NS5A inhibitors may be useful in combination therapies. Indeed, as de-scribed above, the NS5A inhibitor ledipasvir was recently approved in fixed combination with sofosbuvir, without the need for IFN-α and ribavirin18 and the NS5A inhibitor ombitasvir was later approved in combination therapy containing three DAAs.41

NS5B polymerase inhibitors: Since NS5B polymerase is essential for HCV replication, it has been a well-studied target for DAAs.18 The DAAs that are

V36A/M T54S/A V55A Q80R/K R155K/T/Q A156S/T/V D168A/V/T/H D170A/

T

Telaprevir Boceprevir Simeprevir

Paritaprevir Danoprevir Vaniprevir

Grazoprevir Asunaprevir Faldaprevir Vedroprevir * * Sovaprevir * Ciluprevir * * *

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active against NS5B polymerase are divided into two classes: the nucleoside and non-nucleoside polymerase inhibitors.

The nucleoside polymerase inhibitors are associated with a high genetic barrier to the development of resistance (Table 1). Although a single substi-tution close to the active site of the enzyme, i.e. S282T, impaired the potency of this class of inhibitors in vitro, this mutation has shown limited relevance clinically. Moreover, they show a broad genotype coverage.18,53

The non-nucleoside inhibitors bind to allosteric sites distant from the ac-tive site of NS5B polymerase. As a consequence, the barrier to resistance is low, since mutations at this site do not affect the function of NS5B polymer-ase (Table 1).53

1.4 HCV NS3 Protease The HCV NS3 protein consists of a protease and a helicase/nucleoside tri-phosphatase.11 The NS3 serine protease located in the N-terminal, makes up one-third of the NS3 protein. In complex with its cofactor NS4A,17 it plays an important role in the HCV life cycle since it is essential for the formation of infectious viral particles.16,54,55 Cleavage of the NS3/4A site occurs intra-molecularly (i.e. cis), while the other cleavages occur intermolecularly (i.e. trans)54 (Figure 3). In general, Schechter-Berger nomenclature is used when describing interactions between a protease and its substrates or inhibitors (Figure 7).56

Figure 7. A schematic outline of protease-substrate interactions. Schechter-Berger nomenclature56 is used to describe protease-substrate interactions. The sidechains (P) are numbered according to their positions relative to the cleavage site (scissile bond). The corresponding subsites of the protease are denoted S.

The amide bond that is cleaved by the protease is called the scissile bond. The amino acid residues on the N-terminal side are denoted P1, P2, P3, etc., starting from the scissile bond, while the C-terminal residues are named P1’, P2’, P3’, etc., going in the other direction from the scissile bond. The corre-sponding subsites of the protease are termed S1, S2, S3 and S1’, S2’, S3’. The P1 residue in the NS3 protease substrates is essential for substrate recognition, showing specificity for small hydrophobic residues, preferably a

22

cystein, which stack to the aromatic side chain of the phenylalanine (Phe154) positioned in the S1 pocket.57,58

In addition to cleaving the HCV polyprotein, the NS3 protease suppresses the host’s immune response by limiting IFN production.59 Thus, inhibition of the NS3 protease both blocks replication of viral particles and restores the innate immune response. As described above, four NS3 protease inhibitors have been approved as agents in anti-HCV therapies (Figures 4 and 5).

Structure of the NS3 protein In 1996, the protease domain of the NS3 protein was structurally solved by X-ray crystallography, which established a classification into a chymotryp-sin-like serine protease stabilized by coordination to a Zn2+ ion. Unlike other chymotrypsin family proteases, the binding site of the NS3 protein is flat and featureless without specific pockets or cavities.60,61

Figure 8. The full-length NS3 protein and the N-terminal part of NS4A (pdb code 1CU1).62 NS3 protein: grey; NS4A: blue; catalytic triad: magenta; NS3 helicase: green; P6-P1 cleavage product from the NS3/4A cleavage site: orange.

In 1999, the crystal structure of the full-length NS3 protein (i.e. the bifunc-tional protease-helicase) was determined (Figure 8). As a result of this it was found that the protease and the helicase domains are covalently linked by a short solvent-exposed strand. The catalytic site is located in the interface between the protease and the helicase (Figure 8). It is considered possible that the bifunctional protease-helicase makes up for the lack of specific cavi-ties in the protease, resulting in strong interactions with the substrate.62

23

Catalytic mechanism The serine protease, HCV NS3, follows the catalytic mechanism outlined in Figure 9. The catalytic triad comprises Ser139, His57 and Asp81. Ser139 and Gly137 compose the oxyanion hole.

O O-

N

N

OHN

O P1'

P1

NH

N

O

H

HN

H

C term.

N term.

Gly 137Ser 139

His 57

Asp 81

H

O O-

N+

N

OHN

O- P1'

P1

NH

N

O

H

HN

C term.

N term.

Gly 137Ser 139

His 57

Asp 81

H

H

O O-

N

NO-

P1

NH

N

O

H

HN

N term.

Gly 137 Ser 139

His 57

Asp 81

H

O

P1'

H2NC term.

OH

H

O O-

N+

NO-

P1

NH

N

O

H

HN

N term.

Gly 137Ser 139

His 57

Asp 81

HOH

H

O O-

N

NO

P1

NH

NH

HN

N term.

Gly 137Ser 139

Asp 81

HOH

OH

a) b)

c) d)

e)

His 57

Figure 9. General mechanism for serine protease substrate cleavage. a) The sub-strate, positioned in the active site, is attacked at the P1 carbonyl by the activated serine. b) Proton transfer from His57 enables the collapse of the tetrahedral inter-mediate, yielding the covalent acyl complex and releasing the C-terminal fragment. c) Water attacks the complex, which produces a second tetrahedral intermediate. d) Protonation of Ser139 by His57 forms (e) the N terminal cleavage product.63

24

1.5 Discovery and Development of HCV NS3 Protease Inhibitors

The fact that HCV NS3 protease inhibitors are available on the market means that these inhibitors have successfully passed the entire process of drug discovery and development. The goal of drug discovery is to develop and identify candidate drugs (CDs) that have properties favorable for success in the subsequent processes of drug development.

In general, the drug discovery process starts with validation of the drug target and identification of a hit compound (screening libraries of synthe-sized compounds or starting from the natural ligand of the target, e.g. the natural substrates of enzymes). Structure-activity relationship (SAR) studies locate crucial moieties important for biological activity. Lead optimization to improve binding to the target and the in vitro and in vivo PK properties will subsequently support the selection of a CD.64,65

Prior to evaluation in humans, animal models are used to establish the dosage regimen and determine potential toxicities in heart, lungs, brain, kid-ney, liver and digestive system. Chemistry, manufacturing and control (CMC) includes establishment of the physicochemical properties of the compound and the synthetic process for generating it on a kilogram to ton scale. The best formulation is also agreed on. If pre-clinical testings and CMC provide information that supports approval by the regulatory authori-ties, the compound will enter clinical trials.64

Clinical trials comprise three or four stages which can take 5-7 years to complete. In Phase I trials, the safety and dosage are determined in 100-200 healthy volunteers. In Phase II trials, a small group of sick patients is ex-posed to the drug to evaluate efficacy and safety. In the following Phase III trials, a large group of patients is included in order to determine efficacy and safety more reliably. In some cases, a Phase IV study is also carried out; this is called post-market surveillance, and is used to detect any unexpected side effects.64 With a verified desired effect and an established acceptable toxicity and safety profile, the drug can be submitted for marketing approval.

From peptides to peptidomimetics As mentioned above, the starting point in the development of a new drug could be the natural ligand of the target; this is a relevant strategy in HCV drug discovery since NS3 protease inhibitors are often derived from the nat-ural peptide substrates of the protease (see section 1.5.1). Nevertheless, due to their often very flexible and unstable structures, peptides have poor oral bioavailability, are rapidly degraded by proteases, permeate cell membranes poorly, are associated with nonselective receptor binding, and their prepara-tion is often time-consuming.66

A proven strategy for circumventing these difficulties is to develop pep-tide-like (peptidomimetic) compounds designed to exert the same biological

25

effects as the natural substrates but with modified properties such as im-proved stability, bioavailability and selectivity. SAR studies, which identify the most important amino acids in the peptide, are often used in this process. The peptide is systematically truncated to find the shortest sequence needed for affinity. Thereafter, crucial properties like stereochemistry, charge and hydrophobicity can be elucidated by exchanging the amino acids in the pep-tide sequence.67 The peptide backbone can be replaced with building blocks that is less prone to protease cleavages, e.g. the replacement of amide bonds with azapeptides, ureas, sulfonamides or extensions of the peptide chain by introducing a methylene linker or a heteroatom.67,68 The side chains, i.e. the individual amino acids that make up the peptide, are often responsible for strong, specific interactions between the protein and the peptide.67 Thus, a peptidomimetic scaffold needs to allow the substituents to remain in the correct 3D-position in order to achieve the highest potency for the com-pound. Conformational restrictions in the structure, e.g. macrocyclizations or introduction of rigid components into the backbone, enable pre-organization of the bioactive conformation, which decreases the entropic penalty upon binding to the active site. This strategy can therefore improve the potency, and also increase the metabolic stability and permeation characteristics of the compound.

The discovery of the highly potent HCV NS3 protease inhibitor ciluprevir (Figure 5, section 1.2.1) is an example of successful drug design which start-ed from the peptide substrate. The process will be summarized in the follow-ing section.

1.5.1 A Historical View of the Discovery of HCV NS3 Protease Inhibitors

Independently of each other, Istituto di Ricerche di Biologica Molecolare (IRBM) in Italy and Boehringer Ingelheim in Canada found that NS3 prote-ase was inhibited by its own cleavage products.58,69 This higher affinity for the cleavage product than for the corresponding substrate is an unusual prop-erty.69 This pioneering discovery initiated the design and evaluation of pep-tide-based inhibitors that have engaged numerous researchers in both aca-demia and the industry (Figure 10).

Initial modifications Initial optimization of the peptides (Figure 10) revealed that a C-terminal carboxylic acid was crucial for potency.57,58 An acetyl capping group on the N-terminal also increased the potency to some extent.57,70 Replacement of the cysteine in P2 with a proline, which rigidified the backbone, gave hexapep-tide B. Furthermore, the P1 substituent influenced on the potency signifi-

26

cantly, in agreement with other peptide-based inhibitors of serine proteases.58,71

Figure 10. Hexapeptides derived from cleavage products from the NS4A/4B (A)69 and NS5A/5B (B)58 cleavage sites.

The P1 cysteine appeared to be important for substrate specificity, which possibly relied on interactions between the sulfhydryl group and the aro-matic side chain of Phe154 positioned in the S1 pocket.57,58 In order to re-place the metabolically unstable cysteine, a norvaline was identified as a promising alternative (C, Figure 11).70

Figure 11. Replacement of the cysteine in B with norvaline yielded inhibitor C.

In other serine protease inhibitors, the placement of electrophilic derivatives such as α-ketoamides, α-ketoesters, α-ketoacids, etc. in the C-terminal has produced potent inhibitors due to efficient serine trapping.72 Early investiga-tions showed that electrophilic functionalities in this series also gave promis-ing inhibitors. However, inhibition by these molecules was only slightly better, and sometimes even worse, than that by the product-based analogs.70 This finding enabled the development of inhibitors with reduced specificity problems since the electrophilic inhibitors, in theory, could react covalently with numerous nucleophiles present in the host.63 The first approved NS3 protease inhibitors contained α-ketoamides (Figure 4, section 1.2.1). Howev-

27

er, the many side effects associated with these inhibitors were partly respon-sible for the subsequent focus on highly potent product-based inhibitors (Figure 5, section 1.2.1).

Key improvements Introduction of aromatic substituents on the P2 proline in combination with further optimization of the P1 side chain, i.e. introduction of 1-aminocyclopropane carboxylic acid (ACCA), allowed N-terminal truncations (D, Figure 12). One of the key improvements was achieved by placing (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid (vinyl ACCA) at P1, which impressively improved the potency of compounds in this series of inhibitors (E, Figure 12).73,74

Figure 12. Lead optimization process leading to the clinical candidate, ciluprevir.

Macrocyclization Nuclear magnetic resonance (NMR) experiments indicated that peptides in this series were extended in a well-defined configuration (P1-P4), and that the P3 and P1 side chains lay in proximity to each other.75 It was hypothe-sized that the entropic penalty upon binding to the protease could be reduced by pre-organizing the bioactive conformation. Hence the P3 and P1 side chains were connected, yielding a macrocyclic inhibitor.76 The clinical CD, ciluprevir was generated by further lead optimization (Figure 12).77,78

28

Ciluprevir was the first of the HCV NS3 protease inhibitors to enter clini-cal trials.78 Proof-of-concept was established as a result of significant reduc-tion in HCV RNA plasma levels in HCV-infected patients who received ciluprevir.77

Unfortunately, further evaluation of this inhibitor was halted in phase II trials because of cardiotoxicity in rhesus monkeys that received high doses of the agent for four weeks.79 However, the research process, from the initial peptides to the highly potent peptidomimetic clinical candidate, was ground-breaking and motivated the development of the HCV NS3 protease inhibi-tors currently available on the market as anti-HCV therapies (Figure 5, sec-tion 1.2.1).

Further development of product-based inhibitors Simeprevir, developed in a research collaboration between Medivir and Janssen, was the first product-based inhibitor to reach the market (Figure 5, section 1.2.1).29 The characteristic moiety in this compound is the unhydro-lyzable proline mimic cyclopentane, which inverts the back-bone amide.80

The product-based inhibitors, which bind non-covalently to the active site of NS3 protease, all contain a carboxylic acid/acyl sulfonamide at P1; this is involved in hydrogen bonding to the oxyanion cavity.70 Explorations previ-ously undertaken by our group revealed that the replacement of the C-terminal carboxylic acid (pKa ~4.5) with a phenyl acyl sulfonamide (pKa ~5) gave more potent inhibitors.81,82 Furthermore, these inhibitors proved to be highly selective for HCV NS3 protease.83 In parallel, Bristol-Myers Squibb submitted a patent application for HCV NS3 protease inhibitors containing an acyl sulfonamide.84 The two approved inhibitors in this class to date (simeprevir and paritaprevir) contain an acyl sulfonamide, as do the majority of the advanced NS3 protease inhibitors in clinical trials, as shown in Figure 5, which demonstrates the importance of this discovery.

1.6 Model Systems for Analyzing HCV NS3 Protease Inhibitors

Improved understanding of the HCV life cycle, via the development of cell culture models and elucidation of the structures of HCV proteins, has been crucial for the discovery of novel targets and the development of DAAs. The continuing evaluation of drugs with regard to, for example, drug resistance, relies on these techniques.13,85 Various model systems are used in parallel to study the specific functions of a potential anti-HCV drug. These are com-plementary to each other, and yield both detailed and general information regarding the evaluated substance.

29

Enzyme inhibition analysis The NS3 protein, in vivo, comprises a protease and a helicase. However, the truncated protein (isolated NS3 protease) is often used for analysis since the full-length NS3 can be difficult to express and purify, resulting in low yields of the final protein.86 Studies have suggested that the NS3 protease alone is sufficient for proteolytic activity,87 while other investigations have proposed an impact of the helicase on the activity of the protease domain.88,89

The inhibitors presented in this thesis were evaluated in a fluorescence resonance energy transfer (FRET) assay (Figure 13), using the full-length NS3 protein together with part of its co-factor NS4A on both the wild-type enzyme and drug-resistant enzyme variants.

Figure 13. Illustration of a protease activity assay utilizing Fluorescence Resonance Energy Transfer (FRET). The fluorescent signal is detectable upon substrate cleav-age. F = Fluorescent donor; Q = Quenching acceptor.

The enzyme´s capacity to hydrolyze a depsipeptide, in the presence of the evaluated inhibitor, was measured. The depsipeptide, which resembles the natural enzyme substrate, contains a fluorescent group (EDANS) near the N-terminal as well as a quenching group (DABCYL) in the C-terminal part. The fluorescence is quenched in the intact substrate, since these groups are in close proximity to each other. However, when the substrate has been hy-drolyzed by the NS3 protease, the distance between the groups changes, and a fluorescent signal is detected. In the presence of an inhibitor, the velocity of the process changes, resulting in an altered signal. The enzyme-catalyzed conversion rate of substrate into product is described by the Michaelis-Menten equation:

V = [ ]+ [ ]

30

The inhibition is measured at six inhibitor concentrations, in triplicate. Intro-ducing a competitive inhibitor modifies the equation:

V = [ ]× (1 + [ ][ ]) + [ ] This allows the inhibition constant, Ki to be estimated.86 Ki is the dissociation constant for the enzyme-inhibitor complex, defined as: Ki = KD = [E][I]/[EI]; E + I EI

The HCV replicon model In the HCV research area, the main challenge for many years was to estab-lish a trustworthy, cell-based HCV replication system. From the first molec-ular cloning of an HCV genome it took another ten years before a reliable cell culture system could be developed.2,90 It was found that human hepato-ma cells can be transfected by replicon RNA by modifying the viral genome, which enabled monitoring the HCV RNA replication.

The HCV subgenomic replicon RNA uses the segment of the genome coding for the non-structural proteins, controlled by the encephalomyocardi-tis virus internal ribosome entry site (IRES), and a gene coding for re-sistance, driven by the HCV IRES. This allows the selection of transfected cells encompassing the replication machinery.90

In contrast to in vitro assays using isolated NS3 protein, the replicon as-say provides information on how an inhibitor performs in a complex cellular system. The cell-based activities are measured in Huh7 replicon cell lines containing a luciferase readout. The concentration of inhibitor needed to decrease the luciferase signal by 50% is determined (EC50).

91 In this thesis, advanced HCV NS3 protease inhibitors (in late lead optimization stage) were evaluated for their inhibitory potencies in a cell-based replicon assay.

Pharmacokinetic (PK) profiling Different approaches are followed in order to evaluate the compounds with respect to their absorption, distribution, metabolism and excretion (ADME) properties. In vivo PK models resemble the real life environment of a drug, which clearly needs to be evaluated at some point in the drug discovery pro-cess. However, for a medicinal chemist, the isolated in vitro assays provide early information on specific properties, such as solubility, permeability characteristics and metabolic stability, which are useful for the iterative drug discovery process in guiding the optimization of the lead compound.65

In this thesis, a few compounds at the stage of lead selection were evalu-ated for in vitro physicochemical and PK properties, i.e. solubility, metabolic stability (Clint) and permeability coefficient (Papp). The most promising com-pounds in a series of advanced HCV NS3 protease inhibitors, in late lead

31

optimization, were also evaluated for their in vivo liver-to-plasma ratios and PK properties in a rat model.

Molecular modeling Molecular modeling could support the design of novel bioactive molecules. The molecular modeling studies in this project used the HCV NS3/4A prote-ase-helicase crystal structure in complex with a macrocyclic protease inhibi-tor (PDB code 4A92).92 The inhibitors were modeled in Maestro (Schrö-dinger 2011) and induced-fit docking was performed by FLO (QXP 200605).93 Four hydrogen bond constraints, which mimic the hydrogen pat-tern found between the bound cleavage product and the protein, were used to restrict the inhibitor within the active site: between the NH of alanine (Ala157) and the carbonyl of P3, between the backbone carbonyl oxygen of arginine (Arg155) and the NH of P1, and between the NH of glycine (Gly137) and Ser139 and the carbonyl oxygen of P1. Ten unique binding modes were generated using 6000 Monte Carlo perturbation cycles (mcldock) for each inhibitor, and the most plausible binding mode was cho-sen by visual inspection.

In this thesis, molecular modeling was carried out for inhibitors in the py-razinone series in order to clarify the SAR as well as to guide the design of inhibitors for further evaluation.

32

2 Aims of the Thesis

Numerous HCV NS3 protease inhibitors have entered the market and clini-cal trials during the time covered by this thesis. However, at the same time, drug-resistant enzyme variants have appeared which can complicate future treatment of HCV infection. The overall aim of this thesis was:

• to design and synthesize a new lead class of HCV NS3 protease inhibitors based on a pyrazinone scaffold, structurally different from the advanced agents in clinical trials and on the market, pos-sessing a unique resistance profile and potentially with a broad genotypic coverage.

The specific objectives were:

• to synthesize and evaluate the PK properties (in vitro and in vivo) of late stage, macrocyclic HCV NS3 protease inhibitors, contain-ing a P2 quinazoline, in order to increase awareness of how struc-tural features can affect permeability and stability and in the long run influence the bioavailability.

• to investigate the structure-activity relationships (SARs) regarding the inhibition of the wild-type NS3 protease, Gt 1a and Gt 3a, and drug-resistant enzyme variants of pyrazinone based inhibitors.

• to evaluate in vitro PK properties of the pyrazinone based inhibi-

tors to identify how various substituents influence solubility, per-meability and metabolic stability.

• to evaluate procedures for synthesizing the designed compounds,

including establishment of generally applicable Pd catalyzed pro-tocols useful in the synthesis of bioactive molecules.

33

3 P2 Quinazoline Substituents in HCV NS3 Protease Inhibitors (Paper I)

3.1 Pharmacokinetic Aspects in Drug Discovery A revealing paper published in 1988 presented the reasons for the failure of drugs in development.94 Alarmingly, 39% of drugs failed due to poor PK properties and bioavailability. Years of invested money and time were lost, and the introduction of new drugs on the market was delayed. This ultimate-ly affected the patients in need of new pharmaceuticals.

Contemporary drug discovery and development has a different approach. At its best, a drug discovery program is a highly iterative process, where properties such as solubility, permeability and metabolic stability are evalu-ated in parallel with optimizations in terms of binding to the target. A less active compound could have advantageous PK properties which enable a better in vivo therapeutic response and, eventually, might offer more conven-ient dosage regimens for the patient. A successful research program needs to consider and attempt to anticipate how the various properties of a drug coop-erate at its final destination inside the human body.65

Lipinski’s well known “rule-of-five” has, since it was presented in 1997,95 guided the choice of compounds that will proceed in the discovery process. While favorable PK properties and solubility can be predicted from the mo-lecular qualities, the emerging area of demanding and novel targets as well as poor outcomes from big pharma have challenged researchers to think “outside the box”, and this can be rewarding. In the HCV research field, for example, the approved HCV NS3 protease anti-HCV drugs violate at least one of the rules, since they have a molecular weight of >700. One could consider the drug-like properties as guidelines but should also bear in mind that the success of a drug depends on how well various properties are bal-anced with each other. Moreover, oral drug space is likely to expand with improved formulation techniques.96

34

3.2 Harmonizing Antiviral Potency with PK Properties in the Development of HCV NS3 Protease Inhibitors (Paper I)

During the development of the now approved HCV NS3 protease inhibitor simeprevir, which contains a quinoline P2 substituent (Figure 14), other P2 heterocycles were also evaluated (e.g. pyrimidines97, I, Figure 14). The ether linkage found in simeprevir, which connects the P2 core and the P2 hetero-cyclic group, was replaced with a carbamate moiety (II, Figure 14) in anoth-er series.98 However, neither the pyrimidine- nor the carbamate-linked P2 aromatic substituents yielded optimal properties for the inhibitors.29

During these explorations, a novel P2 quinazoline substituent was identi-fied (III, Figure 14); this was combined with a cyclopentane core (as in simeprevir) and a proline urea core in further optimizations. The quinazoline substituent was modified with the goal of balancing antiviral potency with the PK properties.

Figure 14. Pyrimidine, phenyl carbamate and quinazoline as P2 substituents.

Initial modifications The initial lead in this series (1, Figure 15), which contained a 2-phenylquinazoline on the cyclopentyl scaffold, showed excellent permeation through Caco-2 cells, moderate stability in human liver microsomes, high potency in the enzyme assay and moderate potency in the cell-based assay. Introduction of a methoxy moiety in position 7 of the quinazoline (2) im-proved the cell-based potency. The metabolic stability, on the other hand, decreased. This property was adjusted by a fluoro-moiety on the para posi-tion of the phenyl substituent in the quinazoline (3).

35

Figure 15. Initial modifications on the quinazoline substituent. Ki (NS3fl1a): inhibi-tion constant. EC50 (NS31b): cell-based activity. The cut-off values for stability in human liver microsomes (HLM), intrinsic clearance (µL/min/mg): Clint< 30: no risk; 30 < Clint < 92: moderate risk; Clint > 92: high risk. The cut-off values for Caco-2 permeability (cm/s): Papp < 2×10-6: low; 2×10-6 < Papp < 20×10-6: moderate; Papp > 20 × 10-6: high.65

Interestingly, the introduction of a thiazolyl substituent reduced the en-

zyme- and the cell-based activity drastically (4 and 5), in contrast to the out-comes found in the quinoline series, where such a moiety improved the po-tency.29

Figure 16. Left: quinoline-based P2 substituent containing a thiazolyl moiety. Right: quinazoline-based P2 substituent containing a thiazolyl moiety, indicating a possible repulsion between one nitrogen on the quinazoline and the nitrogen in the thiazolyl moiety.

36

A likely reason for the lower potency is that repulsion between the hetero atoms in the thiazolyl moiety and the nitrogens in the quinazoline impeded the bioactive co-planar conformation of the thiazolyl substituent, leading to reduced interactions with the enzyme (Figure 16). A similar reason could possibly explain the drastic decrease in both enzyme and cell-based assays for compounds 6 and 7, i.e. that the non-aromatic rings did not adopt a bio-active conformation, leading to reduced potencies.

The main improvements for the initial optimizations were the addition of a methoxy group in position 7 of the quinazoline, which improved the cell-based potency (2), and the introduction of a fluoro moiety on the phenyl group, which increased the metabolic stability (3).

Table 3. SAR and in vitro PK for the P2 cyclopentane series

Cmpd R1 R2 NS3fl 1a Ki (nM)

NS3 1b EC50 (nM)

aClint

(µL/min/mg)

aPapp (cm/s×10-6)

3

0.2 11 32 20

8

0.1 5 <6 13

9

0.4 10 <6 3

10

0.4 20 10 5

11

0.6 8 11 9

12

>1000 >1000 N.d. N.d.

N.d.: not determined; a See Figure 15.

37

SAR and PK properties for the cyclopentane series Rewardingly, an 8-methyl on the quinazoline drastically improved the meta-bolic stability (8-11, Table 3) as well as preserving the potency. However, the Caco-2 permeability decreased, especially for the analogs 9 and 10 which contained a fluoro moiety. This could be explained by lowered solu-bility due to increased lipophilicity of the compounds, which possibly pro-vided less substance in solution.

Importantly, in the absence of an aromatic moiety on the quinazoline (12), the potency radically decreased, which emphasizes the importance of an aromatic moiety for crucial interactions with the S2 pocket of the NS3 en-zyme. Besides good potency and metabolic stability, there was still room for improvement regarding the Caco-2 permeability to these compounds.

SAR and PK properties for the proline urea series Gratifyingly, when switching to the proline urea scaffold (Table 4), en-hanced Caco-2 permeabilities were obtained for compounds 13-15 compared with the corresponding cyclopentane-based inhibitors 8, 9 and 11. The po-tencies and metabolic stabilities were preserved.

The pyridine-containing inhibitors, 16 and 17, on the other hand, were less potent. Compound 16, with a much lower cell-based activity than the other inhibitors in the series, might be repulsed from the nitrogens on the quinazoline with the ortho nitrogen on pyridine. The Caco-2 cells, however, became totally impermeable to the potent pyridine-containing inhibitor 18, which could be explained by the partially protonated pyridine nitrogen, and the possibly zwitterionic character of the compound.

Two compounds containing an expanded macrocyclic ring (15-membered, 19 and 20) were evaluated. Besides potencies and Caco-2 per-meabilities similar to those of the 14-membered derivatives 13 and 14, they exhibited decreased metabolic stability.

In vivo PK evaluation The evaluated inhibitors exhibited excellent potencies and in vitro drug me-tabolism and PK properties, which endorsed an extended in vivo PK evalua-tion on selected compounds. Since promising properties were detected in all three series, i.e. the cyclopentane series and in both the 14- and 15-membered proline urea series, compounds 8, 13, 14 and 20 were chosen for evaluation in vivo (Table 5).

The data presented in Table 5 include the PK properties after intravenous (iv) injection and after oral administration in Sprague-Dawley rats. A high drug concentration in the liver is essential for this type of compound, since the main location for replication of HCV is the hepatocytes. The urea-based compounds 13, 14 and 20 were all well distributed to the liver, with liver-to-plasma ratios of 32-100. Interestingly, for the cyclopentane derivative 8, the level of compound concentration was not detectable.

38

Table 4. SAR and in vitro PK for the P2 proline series

Cmpd R1 R2

nNS3fl 1a

Ki

(nM)

NS3 1b EC50

(nM)

aClint

(µL/min/mg)

aPapp

(cm/s×10-6)

13

1 0.2 11 17 26

14

1 0.3 16 <6 32

15

1 0.6 3 8 33

16

1 0.9 130 N.d. N.d.

17

1 2.8 13 N.d. N.d.

18

1 0.2 7 14 0.4

19

2 0.3 23 26 38

20

2 0.2 16 20 32

N.d.: not determined. a See Figure 15.

The volume of distribution (0.56-2 L/kg) indicated that the compounds were evenly distributed to blood and tissues. The proline compound, 13, was ab-sorbed more slowly (Tmax, 5 h) and had a higher Cmax (3.4 µM) than the cy-clopentane 8 (Tmax, 2 h; Cmax, 0.66 µM), which resulted in greater exposure to the drug (AUC: 15 µM h, approx. 10500 h·ng/mL vs 3.3 µM h, approx.

39

2400 h·ng/mL). Interestingly, the properties of the 14-membered urea 14 were distinctly different from those of the 15-membered urea 20. Compound 20 had a higher clearance rate, and was associated with a lower Cmax and AUC than compound 14. This correlates with the predicted outcomes from the in vitro evaluations; although the in vitro clearance of compound 20 was not high, it was higher than that of the 14-membered derivative (14). How-ever, the oral bioavailabilities (F) were good for the evaluated compounds, especially for compound 13 (73%).

Table 5. Mean plasma levels and PK parameters after single intravenous and oral administrations in male Sprague-Dawley rats.

8 13 14 20

iv (2 mg/kg, n = 2) Cl (L/h/kg) 1 0.69 0.56 1.6 Vdss (L/kg) 0.56 1.1 2 1.2 AUC (µM h) N.d. 8.4 N.d. N.d. Liver/plasma ratio (6 h) N.d.a 38 N.d. 2.5 Oral (10 mg/kg, n = 2) AUC (µM h) 3.3 15 11 1.7 Cmax (µM) 0.66 3.4 2.6 0.39 Tmax (h) 2 5 5 2 t½ (h) 3.8 N.d. N.d. N.d. F (%) 40 73 N.d. 38

Liver/plasma ratio (6 h) N.d.a 32 42 100 Cl, clearance; Vdss, volume of distribution at steady state; AUC, area under the plasma con-centration-time curve; Cmax, maximum plasma concentration; Tmax, time to maximum plasma concentration; t1/2, plasma half-life; F, percentage of dose reaching systemic circulation. N.d.: not determined. Cut-off values: Low: Cl < 0.6 (L/h/kg); Vd < 1 L/kg; AUC < 500h·ng/mL; Tmax < 1 h; T1/2 < 1 h; F < 20%. High: Cl > 2.7 (L/h/kg); Vd > 10 L/kg; AUC > 2000h·ng/mL; Tmax > 3 h; T1/2 > 3 h; F > 50%; a Levels were not detectable.

3.3 Synthesis of P2 Quinazoline-Containing HCV NS3 Protease Inhibitors

The synthesis of the investigated inhibitors is outlined in the following sec-tions. The P2 quinazoline building blocks were separately prepared (Schemes 1 and 2) and then incorporated into the cyclopentane (Scheme 3) and proline (Scheme 4) scaffolds under Mitsunobu conditions.

Synthesis of the quinazolinol building blocks Two approaches were followed to prepare the quinazolinol building blocks, as outlined in Scheme 1 (25a-c and 28) and Scheme 2 (35a-h).

Starting with the carboxylic acid 21 (Scheme 1), the acyl chloride formed in situ was displaced with ammonia, yielding the primary amide 22. Reduc-

40

tion of the nitro moiety gave the corresponding amine 23. Coupling of 23 and 26 with carboxylic acids generated the amide-containing intermediates 24a-c and 27, in which the rings were subsequently closed to form the quinazolinol building blocks 25a-c and 28, by refluxing in ethanol and water in the presence of sodium carbonate.

Scheme 1. Reagents and conditions: (a) oxalyl chloride, DMF cat., DCM, then NH3, 71%. (b) Raney-Ni, EtOH, H2, 3.5 bar, 95%. (c) R2-COOH, HOBt, EDAC, Et3N, DMF, 55-90%. (d) Na2CO3, EtOH/H2O 1:1, reflux, 77-90%.

The 8-methyl-containing quinazolinols were prepared as summarized in Scheme 2. The hydroxyl on 29 was initially methylated, generating the me-thyl ether 30. The nitrile moiety was subsequently hydrolyzed under basic conditions. This approach gave a mixture of the carboxylic acid 31 (30%) and the primary amide 36 (58%). Both could be utilized, but the carboxylic acid needed protection, which was established via esterification, to obtain 32. Thereafter the coupling of carboxylic acids and hydrolysis of the meth-ylester provided the intermediates 34a-c which were treated under elevated temperatures in formamide to obtain the quinazolinol building blocks 35a-c. The intermediate 36 was coupled with acyl chlorides to generate the amide-containing intermediates 34d-g which were subsequently dissolved in EtOH/water and refluxed in the presence of Na2CO3 to produce the quinazo-linols 35d-h.

41

Scheme 2. Reagents and conditions: (a) MeI, K2CO3, DMF. (b) 2 M NaOH, EtOH. (c) R2-COOH, HOBt, EDAC, Et3N, DMF, 50-56%. (d) 1 M LiOH, EtOH, 60°C. (e) formamide, 150°C, 71-89% over two steps. (f) R2-COCl, pyridine, THF. (g) Na2CO3, EtOH/H2O 1:1, reflux, 60-95% over two steps. 35h was formed as a by-product when (g) was performed in DMF rather than in EtOH.

Synthesis of the cyclopentane-based inhibitors The final cyclopentane-based inhibitors were prepared via two cyclopentane scaffolds. The key operation for generating the final compounds 1, 6 and 7 was a nucleophilic aromatic substitution in order to introduce the quinazo-line moiety.99 However, herein the focus will be on the main pathway, name-ly the route using a Mitsunobu reaction for introduction of the quinazoline building blocks (Scheme 3).

42

Scheme 3. Reagents and conditions: (a) quinazolinol (25a-c, 28, 35a,b,d,e,h, Scheme 1 and 2), PPh3, DIAD, THF, 0°C to rt, overnight, (68-99%). (b) 2:1 DCM/TFA, Et3SiH, 0°C to rt. (c) N-methylhex-5-en-amine, HATU, DIPEA, DMF, 0°C to rt,1-3 h, (60-85%) over two steps. (d) Hoveyda-Grubbs catalyst, 1st or 2nd generation, 1,2-dichloroethane, reflux, 4-12 h (30-80%). (e) 2:1:1 THF/MeOH/H2O, aq 1 M LiOH. (f) EDAC, DCM, rt, 3-12 h, then cyclopropylsulfonamide, DBU, rt, 6-12 h, (10-40%). R1 substituents are included in Table 3.

The building block 3780 was coupled with the quinazolinols 25a-c, 28,

and 35a,b,d,e,h (Schemes 1 and 2) under Mitsunobu conditions, which gen-erated 38a-i in good to excellent yields (68-99%). Selective hydrolysis of the

43

tert-butyl ester was accomplished under acidic conditions, using Et3SiH as a carbocation scavenger, which gave the carboxylic acid intermediates 39a-i. Coupling of the N-methylhex-5-enamine using N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) as coupling reagent produced 40a-i in 60-85% yield. The macrocyclic, 14-membered ring was well formed in 30-80% yield employing Hoveyda-Grubbs catalyst (1st or 2nd). Finally, hy-drolysis of the ethyl ester under basic conditions and successive coupling of the cyclopropylsulfonamide, via the formation of oxazolidinone, yielded the final inhibitors 2-5 and 8-12 in moderate yields (10-40%) after preparative high-performance liquid chromatography (HPLC) purification.

Synthesis of the proline urea-based inhibitors The generation of proline urea-based final inhibitors was accomplished as outlined in Scheme 4. The successful introduction of the quinazolinol moiety late in the procedure was a rewarding synthetic strategy; this enabled fast production of target compounds containing various quinazoline substituents.

N-deprotection of 42100 and further treatment with phosgene delivered the acid chloride 44 in situ which thereafter was coupled with N-methylhex-5-en-1-amine or hept-6-enyl-(p-methoxybenzyl)-amine with a satisfying yield of 85-90% over three steps. Successful macrocyclization (74% yield of 46) followed by selective hydrolysis of the 4-nitrobenzyl ester revealed the alco-hol (80% yield of 47) in 55-65% yield over two steps.

The quinazolinol building blocks 35a-c, e-g were introduced to the 14-membered intermediate 47 under Mitsunobu conditions (30-85%). Finally, ester hydrolysis and coupling to the cyclopropylsulfonamide, as described before, provided the final inhibitors 13-18 with 40-85% yield over two steps after preparative HPLC purification.

In parallel, the 15-membered compounds 53a and 53b were prepared with a total yield of 20% and 12%, respectively, from intermediate 44. The p-methoxybenzyl protecting group was removed under acidic conditions, using DCM/TFA, yielding the target compounds 19 and 20 with 62% and 38% yields, after preparative HPLC purification.

44

Scheme 4. Reagents and conditions: (a) 2:1 DCM/TFA, 0°C to rt, 1.5 h. (b) phos-gene in toluene, THF, NaHCO3, rt, 1 h. (c) DCM, NaHCO3, N-methylhex-5-en-1-amine, or hept-6-enyl-(p-methoxybenzyl)-amine, rt, overnight, 85-90% over three steps. (d) Hoveyda-Grubbs catalyst 2nd generation, 1,2-dichloroethane, reflux, 4-12 h. (e) aq 1 M LiOH, 2:1:1 THF/MeOH/H2O, 0°C, 4 h, 55-65% over two steps. (f) quinazolinol (35a-c, e-g, Scheme 2), PPh3, DIAD, THF, 0°C to rt, overnight, 30-85%. (g) 2:1:1 THF/MeOH/H2O, aq 1 M LiOH, 40-50 °C, overnight. (h) EDAC, DCM, rt, 5h, then cyclopropylsulfonamide, DBU, rt, overnight, 45-80%. (i) 2:1 DCM/TFA, rt, 30 min, 38-62%. R1 substituents are included in Table 4.

45

4 2(1H)-Pyrazinone-Based HCV NS3 Protease Inhibitors (Papers II and III)

4.1 Pyrazinones in Medicinal Chemistry Pyrazinone is a versatile scaffold which has been evaluated as a core struc-ture in protease inhibitors of thrombin, tryptase, tissue factor VIIa, caspase-3 and HCV NS3.101–105 This heterocyclic template has also provided the basis for other types of bioactive compounds such as kinase inhibitors intended for anticancer drugs, µ-opioid receptor agonists with analgesic effects and natu-ral product analogs.106–108

The advantages of the use of the pyrazinone structure in the protease in-hibitors discussed above are correlated with its ability to induce β-strand conformations, which is an important aspect of the recognition and subse-quent binding to an active site in the proteases.109,110 The pyrazinone ring contributes to the rigid structure, forming the hydrogen bond interactions normally found between the natural substrate and the protease (Figure 17).

Figure 17. Hydrogen bond interactions from the peptide, i.e. natural substrate (left) and from the corresponding P3 pyrazinone scaffold (right) to the protease. HBA: hydrogen bond acceptor; HBD: hydrogen bond donor.

Another important aspect is the opportunity for decorating the core struc-ture with various functional groups.111,112 This allows efficient and informa-tive SAR studies to be accomplished using straightforward synthetic proce-dures. Various synthetic approaches, e.g. Pd-catalyzed cross-coupling reac-tions, such as C-N arylations and Suzuki couplings, and amide coupling pro-cedures yielding peptidomimetic inhibitors are presented in chapters 5 and 6.

46

4.2 From Tripeptides to Pyrazinone-Based HCV NS3 Protease Inhibitors

As discussed in section 1.5.1, inhibitors of HCV NS3 protease have often been developed from the cleavage product of its natural substrate. With the aim to develop HCV NS3 protease inhibitors, structurally distinct from the inhibitors in clinical trials, our group shifted focus to alternative tripeptidic HCV NS3 protease inhibitors e.g. 54113 in Figure 18, containing a phenyl-glycine P2 substituent instead of the commonly used proline (i.e. inhibitors in Figure 5, section 1.2.1), and similar P3 and P1P1’ substituents to those found in potent proline-based inhibitors. Subsequently, introduction of a P3 pyrazinone rigidified the core structure and yielded 55 which was considered a promising starting point for development of novel peptidomimetic inhibi-tors.114

Figure 18. Replacing a P3 t-Leucin 54 (left) with a P3 pyrazinone 55 (right).

The P1P1’ substituent was then investigated and an aromatic acyl sulfona-mide (56, Table 6) previously found to be useful in other HCV NS3 protease inhibitors, was equipotent to the P1P1’ found in 55.114 The aromatic P1P1’ enabled further substituents around the P1 and P1’ substituents to be intro-duced.

4.3 Development of Achiral Inhibitors (Paper II) Optimization of this class of compounds was initiated after the above modi-fications. Molecular modeling suggested that the proposed P2 aryl could possibly be moved to the R6 position of the pyrazinone while retaining the same or similar interactions with the S2 pocket. Preliminary results support-ed this idea and inspired further optimizations.114

A carbamate in the 3 position had previously been identified as a promis-ing P3 capping group of the pyrazinone (56, Table 6) and better than other

47

attempts to introduce alkyl and aryl amines and primary amides. However, the chemical stability of the tert-butyl carbamate was unpredictably low, which prompted replacement with a more stable derivative.

Introduction of a P3 urea capping group and relocation of the P2 substituent The 3-tert-butyl carbamate was replaced with a tert-butyl urea moiety, which to our surprise increased the potency of the inhibitor by a factor of ten (Table 6, 57 compared with 56) while also increasing the stability. Compound 57 contained an additional hydrogen which could possibly be involved in inter- or intramolecular bonding.

Subsequent relocation of the aromatic P2 group (R2) to the R6 position of the pyrazinone to yield achiral inhibitor 58 was allowed with only a minor drop in inhibitory potency. Combining a phenylglycine with the R6 benzyl did not further improve the inhibitory potency of compound 59.

Table 6. Influence on inhibitory potency by carbamate-to-urea exchange and the relocation of the R2 group to the R6 position on the pyrazinone.

aNS3fl 1a; SD, standard deviation.

48

To compare the acyl sulfonamide in the C-terminal with a serine-trapping electrophilic group, an α-ketoamide (the same as that in boceprevir, Figure 4, section 1.2.1) was evaluated. As presented in Table 7, this produced the in-hibitors 60 and 61 which were less potent than the acyl sulfonamide deriva-tives 57 and 58 in Table 6.

Accordingly, the achiral compound 58 (Table 6) which contained an aro-matic acyl sulfonamide in P1P1’, was selected for further investigations, as presented in Table 8.

Table 7. Electrophilic serine trap inhibitors containing an α-ketoamide in P1.

aNS3fl 1a; SD, standard deviation

Elongation of the R6 substituent Various substituents, e.g. alkyl, elongated phenyl, naphthyl and bromo- and pyridine-substituted aryls, were introduced and evaluated with respect to their inhibitory constants in order to develop a SAR around the R6 position of the pyrazinone (58, 62-81, Table 8).

The potency of 62, which lacks a substituent in R6 was decreased (1.9 µM vs 0.66 µM for 58). The potency was then increased by adding larger, more lipophilic substituents, as found in 63, 64, 68 and 69. A slight decrease in potency was however found for the naphthyl derivatives 66 and 67. Com-pound 65 which contained an oxygen spacer, also appeared to be less effi-cient. In order to study the preferred orientation of a second aromatic sub-stituent, 70-75 were prepared, biochemically evaluated and subsequently used as precursors for compounds 76-81. Slightly improved potencies were obtained for the bromo-containing phenyl and benzyl derivatives, 70-75, compared with the unsubstituted benzyl analog 58. More interestingly, the corresponding pyridyl substituents revealed limitations for the ortho-derivatives 78 and 81 with a significant drop in inhibitory potency.

49

Table 8. Pyrazinone based HCV NS3 protease inhibitors containing elongated R6

substituents and their Ki values.

aNS3fl 1a; SD, standard deviation

50

Molecular modeling of a pyrazinone-based inhibitor A representative compound from the series (77) was modeled into the prote-ase active site (Figure 19). The binding mode showed backbone interactions with Ala157 and Cys159 and possible edge-to-face interactions between the aromatic P1 group and Phe154. Furthermore, the P2 substituent seemed to be directed towards the S2 pocket, which supported our hypothesis for elongat-ed R6 groups.

An internal hydrogen bond between the urea nitrogen on the P3 capping group and the 4-nitrogen in the pyrazinone (Figure 19) possibly stabilizes the bioactive conformation and could partly explain the gain in inhibitory poten-cy observed by the replacement of a P3 carbamate with a urea moiety. Therefore, the urea functionality was preserved and further evaluated in SAR studies (sections 4.4 and 4.5).

Figure 19. Compound 77 modeled into the boundary between the NS3 protease active site (grey surface) and the helicase domain (green) of the 4A92 crystal struc-ture.92 Hydrogen bond interactions are shown in red dashed lines.

Additionally, for the ortho pyridyl compounds 78 and 81, modeling sug-gested poses where the P2 group was directed out from the S2 pocket, in-stead forming hydrophobic interactions with the pyrazinone core. The back-bone is somewhat distorted in these modes which could explain the loss of inhibitory potency for 78 and 81.

51

The substrate envelope theory and amino acid substitutions in the NS3 protease The resistant HCV mutations that have emerged during the development and evaluation of advanced HCV NS3 protease inhibitors have limited the effi-cacy of these compounds. As previously mentioned (section 1.3) the most common amino acid substitutions that lead to resistance occur at positions R155, A156 and D168 (Figure 20).

Figure 20. Left: Drug resistant amino acid substitutions R155K, A156T and D168V. Right: The electrostatic network in the S2 pocket between R123, D168, R155. The hydrogen bond interctions between the P1 residue and the enzyme, as suggested by the full-length crystal structure.62

Schiffer and co-workers have explained the emergence of drug resistance against HCV NS3 protease inhibitors using the so-called substrate envelope theory115 which they previously have utilized in the design of HIV protease inhibitors showing reduced problems with resistance.116,117 Crystal structures of the NS3 protease domain with its N-terminal products of the different cleavage sites (P7-P1) were analyzed and the consensus volume (substrate envelope) inside the NS3 active site was defined.115 Comparisons between the substrate envelope and the crystal structures of the NS3 protease in com-plex with boceprevir, danoprevir and simeprevir (the structures of these in-hibitors can be found in Figures 4 and 5, section 1.2.1) revealed that these drugs protrude outside the substrate envelope at positions associated with drug-resistant mutations, e.g. R155, D168, etc. These mutations do not affect the viral fit, but selectively disrupt important interactions with the inhibi-tors.118 The large aromatic P2 groups found in danoprevir and simeprevir stack with the guanidinium group of R155, which changes the electrostatic

52

network in the S2 pocket between R123, D168 and R155 (Figure 20). The extensive packing of these big P2 groups will hence be altered by mutations at R155 and D168, with a possibly reduced affinity as a consequence. This could explain why mutations at R155, and especially D168, confer resistance to the inhibitors containing extended P2 groups.

Furthermore, it was found that A156 and R155 not only interacted with the P2 groups of simeprevir and danoprevir, but also had apparent interac-tions with the less bulky P2 group found in boceprevir at places outside the substrate envelope. Accordingly, substitutions at A156 and R155 would alter the interactions with the P2 moieties in all three inhibitors. More recent stud-ies on crystal structures of these inhibitors with the drug-resistant enzyme variants R155K, A156T and D168A supported these suggestions.118 Interest-ingly, grazoprevir (Figure 5, section 1.2.1), an inhibitor that has shown a lower level of resistance to R155K than other protease inhibitors, had a dif-ferent binding conformation with fewer interactions from the P2 group with R155 and D168. These findings could help in the design of novel inhibitors of the NS3 protease encompassing a unique resistance profile.

Evaluation of pyrazinone-based inhibitors against drug resistant enzyme variants One of the driving forces for our development of coming generation HCV NS3 protease inhibitors is the possibility to maintain potency against drug-resistant enzyme variants. Consequently, three inhibitors in the pyrazinone series were evaluated against R155K, A156T and D168V. The selected compounds contained one phenyl glycine inhibitor 57 from Table 6 as well as two compounds from the achiral pyrazinone series, containing one alkyl (69) and one aryl (76) R6 substituent (Table 8). Two advanced HCV NS3 protease inhibitors were also included, namely the electrophilic NS3 prote-ase inhibitor telaprevir (Table 4, section 1.2.1) and the first-in-class, product-based inhibitor ciluprevir (Table 5, section 1.2.1). Vitality values,119 which normalize for the different catalytic abilities of the enzyme variants, were calculated and presented along with the Ki values (Table 9). If the vitality value < 1, the enzyme variant is disfavored in the presence of inhibitor com-pared with the wild-type enzyme. If the value > 1 the amino acid substitution provides a less vulnerable enzyme to the evaluated inhibitor compared with the wild-type enzyme.

Both telaprevir and ciluprevir showed a decrease in potency on the R155K substituted enzyme. A crystal structure of telaprevir in complex with the R155K substituted protease has previously been published by Schiffer and co-workers; this demonstrates disrupted interactions by P2 and P4 of the inhibitor with the protease.118 However, for inhibitors in the pyrazinone se-ries this amino acid substitution was well accepted, with vitality values <1. The Ki values displayed a preference for the aliphatic R6 group in 69 com-pared with 57 and 76.

53

Table 9. Inhibition of the protease activity of the wild-type (wt) and the drug re-sistant enzyme variants A156T, D168V and R155K for the full length NS3 protease, and corresponding vitality values (V).

The inhibition potencies for telaprevir and ciluprevir of wt, A156T and D168V have previous-ly been reported in Dahl et al.119

The amino acid substitution A156T results in increased bulk, which influ-

ences interactions with the P2 and P4 substituents of HCV NS3 inhibitors. Both ciluprevir and telaprevir lost potency against this mutated form of the enzyme. The drop in potency was especially distinct for telaprevir, which displayed a Ki value in a micro molar range. A published crystal structure of telaprevir in complex with the A156T substituted protease has later clarified the large drop in potency;118 the A156T substitution resulted in a steric clash with the P2 moiety of telaprevir which led to a changed position of the inhib-itor in the active site and reduced interactions with R155.118 This alteration also resulted in an extended distance between the ketoamide warhead and the

54

catalytic serine, which reduced the capacity for a covalent bond formation. Although more potent than telaprevir against the A156T protease, the phenyl glycine inhibitor 57 showed a slightly larger drop in inhibition potency com-pared with 69 and 76, which could be because of the altered location of the P2 substituent. Again, the cyclohexyl-containing compound 69 showed the most promising Ki value of the three inhibitors 57, 69 and 76; this was close to the Ki in the presence of the wild-type enzyme.

As previously discussed, the D168V mutation primarily affects inhibitors with large P2 substituents. Ciluprevir, which contains an extended quinoline P2 substituent, dramatically lost inhibitory potency against this mutation. However, telaprevir, which lacks an extended P2 substituent, showed im-proved inhibitory potency and a vitality value less than 1, implying that this compound inhibits the D168V mutated form of the enzyme more effectively than it inhibits the wild-type enzyme. The crystal structure of telaprevir with a D168A substituted protease (note: not D168V) showed a tighter interaction with the D168A mutant than with the wild-type, which also resulted in in-creased interactions with R155 and A156.118 The higher potency against D168V could possibly be explained by similar interactions. The Ki values for the pyrazinone-based inhibitors were about three times larger against D168V mutant than against the wild-type. The vitality values indicated only minor influence on the inhibition of this mutated form of the enzyme. Among the pyrazinones, compound 76, which was less flexible with respect to its P2 substituent than 69, seemed to be slightly less potent against the D168V mutation.

In vitro PK properties for pyrazinone-based inhibitors Although the stage of this drug discovery project could be considered to be between hit and lead selection, the awareness of less suitable PK properties in a prospective candidate drug is of importance for further investigations.

Initially, in silico predictions were performed to estimate pKa and log D7.4

(Table 10). Based on the acyl sulfonamide group, these inhibitors possess acidic properties, with small differences in pKa (4.7-5.1). The higher value was seen in 76, which contains the slightly basic moiety pyridyl, which pos-sibly affected the overall pKa value.

The ideal value for the lipophilicity, estimated at pH 7.4, i.e. the log D7.4, should be between 1 and 3 for well balanced solubility and permeability, and minimal metabolism.65 For this set of compounds, the log D7.4 ranged be-tween 3.8 and 5.0, with the lowest values found for 65 and 76, which con-tained heteroatoms in the R6 substituent. Overall, these log D7.4 values indi-cated low solubility, which could negatively affect the level of absorption. One could expect good permeability, which is associated with high lipo-philicity, but at the same time, an increased risk of metabolism due to bind-ing to metabolic enzymes could be expected.120

55

Table 10. In vitro PK properties, in silico pKa values and log D7.4 values.

57 63 65 68 69 76

In silico pKa 4.7 4.7 4.7 4.7 4.8 5.1

logD7.4 4.2 4.4 3.8 4.3 5.0 4.1

In vitro

Solubility (µM) 38 23 22 21 31 53

Papp a-b

(10-6 cm/s)1.1

± 0.40.3

± 0.24.0

± 1.10.04

± 0.020.12

± 0.097.0

± 1.5 Clint (µL/min/mg) 26 ± 2 <13 17 ± 4 33 ± 13 52 ± 3 17 ± 3

t½ (min) 54 ± 3 >100 81 ± 19 42 ± 16 27 ± 2 82 ± 13

Papp = apparent permeability coefficient; a-b = apical to basolateral; Clint = in vitro clearance; t1/2 = in vitro half-life; Predicted risk of oxidative first-pass metabolism in vivo based on in vitro intrinsic clearance (µL/min/mg), cut-off values: Clint< 30: no risk; 30 < Clint < 92: mod-erate risk; Clint > 92: high risk; Caco-2 permeability, apparent permeability coefficent, Papp (cm/s):121 Papp < 0.2 × 10-6 : low; 0.2 × 10-6 < Papp < 1.6 × 10-6 : moderate; Papp > 1.6 × 10-6 : high.

The in vitro solubility, Caco-2 permeability and metabolic stability were determined, as presented in Table 10. The solubilities were overall classified as moderate, indicating that solubility is not a major issue for this class of compounds despite the predictions made by in silico calculations. The pyri-dine containing inhibitor 76 was the most soluble in the series and gives important insights for further optimizations. Interestingly, when evaluating the permeability, the pyridine compound 76 and the ether compound 65, showed the most promising Papp values. In contrast, the alkyl-containing R6 moieties in 68 and 69 were associated with low Caco-2 permeabilities.

The in vitro intrinsic clearance studies, performed in human liver micro-somes, indicated low or moderate risk of high oxidative first-pass metabo-lism in vivo for this class of compounds, in contrast to the predicted in silico calculations. It was found that the compounds containing alkyls in R6 (57, 68 and 69) were less stable than the aromatic derivatives 63, 65 and 76.

It is interesting to observe the big variation in PK properties for the dif-ferent R6 substituents situated on an otherwise unchanged large scaffold. The predicted in silico data supported to some extent the in vitro PK properties, indicating that a lower log D7.4 could relate to balanced PK properties, as exemplified by 65 and 76 compared with 68 and 69.

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4.4 Exploration of the P1P1’ Substituents (Paper III) The SAR investigation around the R6 position improved understanding of the function of the substituents at this site in the pyrazinone. The importance of a urea as a P3 capping group was also noticed. Further, an aromatic P1P1’ block was identified as a better substituent than a classical serine-trapping, α-keto amide (57 and 58, Table 6, compared to 60 and 61, Table 7) or amino acid based carboxylic acid or acyl sulfonamide.

Preceding these investigations it had been found that the classical acyl sulfonamide (as in 55) gave more promising Ki values than the correspond-ing carboxylic acid.114 The acyl sulfonamide is a widely used carboxylic acid bioisostere, found in most advanced HCV NS3 protease inhibitors (Figure 5, Section 1.2.1).

Based on those previous findings, further investigations were focused on the interactions with the protease from P1 and P1’ substituents and the aro-matic acyl sufonamide building block was considered to be a suitable scaf-fold. The template could be modified on both the P1 and P1’ aromatic moie-ties, which is valuable in SAR studies.

Regioisomers The investigation was started by varying the regioisomer in order to study if there were any preferences for specific isomers (Table 11). Initially, we ex-plored the inhibitory potencies against full length NS3, Gt 1a. Surprisingly, going from lead 69, with the acyl sulfonamide in the ortho position to the meta (82) or para (83) positions did not affect the inhibitory potencies much. The electron-withdrawing trifluoromethyl moiety can have two functionali-ties in this type of compound: affecting the acidity of the acyl sulfonamide hydrogen or providing hydrophobic interactions with the protease. However, the trifluoromethyl group was varied between the ortho (84), meta (85) and para (69) positions with similar inhibitory potencies. Even replacement with a methyl group (86) yielded similar Ki values. These modifications indicated that the P1’ moiety did not have any specific interactions with the enzyme. Moreover, the increased acidity of the acyl sulfonamide hydrogen was not beneficial in terms of stronger interactions with the oxyanion cavity, in con-trast to previous findings for other HCV NS3 protease inhibitors.122

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Table 11. Pyrazinone based HCV NS3 protease inhibitors containing various P1P1’ aromatic acyl sulfonamides.

Cmpd P1P1’ NS3fl 1a

Ki ±SD (µM)

NS3fl 1a R155K

Ki ±SD (µM)

NS3fl 3a Ki ±SD (µM)

69 0.3 ± 0.1a 0.13 ± 0.04 N.d.

82 0.36 ± 0.16 N.d. N.d.

83 0.40 ± 0.28 0.23 ± 0.1 1.0 ± 0.38

84 0.27 ± 0.17 N.d. N.d.

85 0.55 ± 0.27 N.d. N.d.

86 0.26 ± 0.11 N.d. N.d.

87 0.40 ± 0.1 N.d. N.d.

58

Cmpd P1P1’ NS3fl 1a

Ki ±SD (µM)

NS3fl 1a R155K

Ki ±SD (µM)

NS3fl 3a Ki ±SD (µM)

88 0.36 ± 0.11 N.d. N.d.

89 0.21 ± 0.08 0.17 ± 0.04 3.44 ± 1.0

90 0.22 ± 0.11 0.14 ± 0.05 0.93 ± 0.26

91 0.30 ± 0.07 N.d. N.d.

92 0.53 ± 0.2 N.d. N.d.

93 94

0.22 ± 0.06 0.20 ± 0.08

0.05 ± 0.01 0.02 ± 0.01

N.d. N.d.

95 0.17 ± 0.1 0.09 ± 0.05 0.80 ± 0.57

96 >10 N.d. N.d.

N.d.: not determined. aThe Ki value was determined on the same occasion as the other inhibi-tors in this series. (Ki = 0.11 µM in a previous measurement, Table 8).

59

Next, we focused on the P1 aryl, and examined an aryl bromine (87) which was subsequently replaced with vinyl (88) and pyrimidyl (89) moie-ties. A fluorine in the same position was also examined (90), as well as a CF3 group with the acyl sulfonamide in the meta position (91). Although there was a big difference in size and electronic properties, the influence on inhibi-tory potencies was small. Of interest, the bulky pyrimidine (89) was well tolerated but showed no additional interactions with the protease, based on similar Ki values for 89 and 69. These modifications to the P1 aryl, which has potentially more acidic acyl sulfonamides, questioned the importance of an acidic functionality. The alkylated acyl sulfonamide 92 which lacks an acidic hydrogen, supported this assumption, showing a maintained inhibitory potency compared with 69.

Truncation The surprisingly flat SAR obtained from the modifications performed on the P1 and P1’ aromatic moieties resulted in further analysis of truncated deriva-tives. Thus, 93, 94 and the carboxylic acid 96, which lacks the entire P1P1’ aromatic building block, were evaluated (Table 11). Remarkably, similar potencies were retained for the P1 carboxylic acid (93) and for the P1 methyl ester derivative (94). However, removal of the P1 aromatic functionality (96) resulted in a considerable loss of inhibitory potency, verifying the need for the P1 moiety in combination with the P2P3 pyrazinone scaffold. A slightly improved inhibitory potency was achieved for the more flexible inhibitor 95.

Reversed acyl sulfonamides One advantage of the aryl acyl sulfonamides, compared with alkyl analogs, is the possibility to easily reverse the acyl sulfonamide functionality, which could alter the opportunity of reaching interactions in the oxyanion cavity. Accordingly, the inhibitors in Table 12 were evaluated. Again, it was found that even the reversed acyl sulfonamides were allowed. No well defined favorable influence from this rearrangement was found, although slightly lower Ki values were obtained for compounds 97-99 than for 67. However, in combination with the better fitting phenethyl P2 group, as previously found, the potencies were similar for the original acyl sulfonamide (63) as for the reversed analog (100).

The results shown in Tables 11 and 12 indicated that the P1 aryl improved potency. However, the P1’ aromatic moiety did not show any additional benefits. The acidic acyl sulfonamide was not specifically needed for good binding to the protease for these pyrazinone based inhibitors, in contrary to most HCV NS3 protease inhibitors known. Further investigations are needed to confirm and shed light on this observation.

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Table 12. Pyrazinone based HCV NS3 protease inhibitors containing reversed P1P1’ aromatic acyl sulfonamides.

Cmpd P1P1’ R6 NS3fl 1a Ki ±SD (µM)

NS3fl 1a R155K

Ki ±SD (µM)

NS3fl 3a Ki ±SD (µM)

67 A

0.81 ± 0.23a 0.15 ± 0.07 1.3 ± 0.4

97 A

0.51 ± 0.2 0.08 ± 0.04 0.78 ± 0.14

98 A

0.38 ± 0.15 0.12 ± 0.08 0.87 ± 0.18

99 A

0.61 ± 0.14 N.d. N.d.

63 B

0.30 ± 0.13b N.d. N.d.

100 B

0.35 ± 0.1 0.10 ± 0.02 N.d.

N.d.: not determined. a,bThe Ki value was determined on the same occasion as the other inhibi-tors in this series. (aKi = 0.29 µM, bKi = 0.12 µM in a previous measurement, Table 8).

Cyclobutyl A high aromatic content could be a problem in terms of the bioavailability of a potential candidate drug.123 A cyclobutyl moiety was therefore evaluated as an alternative to the aromatic P1 substituent. Spirocyclobutyls have previ-ously been evaluated in electrophilic serine-trap HCV NS3 protease inhibi-tors.124 If promising, the inherent opportunity for incorporating substituents

61

into the spirocyclobutyl group, via a 3-hydroxyl moiety, would be useful in forthcoming SAR studies.

Table 13. Pyraonzine based HCV NS3 protease inhibitors containing P1 cyclobutyl acyl sulfonamides.

Cmpd R1 R2 R6 NS3fl 1a

Ki ±SD (µM)NS3fl 1a R155K

Ki ±SD (µM)

101 Cl A B 0.11 ± 0.14 0.11 ± 0.04

102 Cl A A Isomer a: 0.40 ± 0.13 Isomer b: 0.50 ± 0.17

N.d.

103 H H A >10 N.d.

N.d.: not determined. As presented in Table 13, this modification improved the inhibitory po-

tency about three-fold (101 vs 69 found in Table 11, and 102 vs 58 found in Table 14). The compound containing the free hydroxyl group (103) also lacked the chlorine in position 5 of the pyrazinone (synthesis in section 5.3-5.5) which hampered a truly reliable comparison of the compound. The ab-sence of the benzyl and the C5 chlorine decreased the inhibitory potency substantially. Efforts to further evaluate this spirocyclobutyl moiety would be of interest. Possibly, the P1’ aromatic part could be removed, and the substituent on the ring might enable novel interactions with the protease.

4.5 P3P4 Urea Elongations (Paper III) In 2011, Schiering et al. published the crystal structure of a macrocyclic HCV NS3 protease inhibitor in the presence of the full-length NS3 protein, i.e. the protease and the helicase. This showed interactions between the P4 capping group and the helicase,92 which supported suggestions from our group emphasizing the importance of having the full-length protein for eval-uation of inhibitors and resistance profiling.119 Because of our interest in developing inhibitors that are also potent against known drug-resistant en-zyme variants, the hypothesis of designing inhibitors to fit inside the so-called substrate envelope was appealing. When applied to the pyrazinone-based inhibitors, elongation of the inhibitors to span the P4P5 region, in combination with a relatively small P2 group, could be a possible strategy. A

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cyclic imide (found in 107 and 108, Table 14) that previously had been eval-uated in an electrophilic inhibitor of the HCV NS3 protease125 was examined as a P4P5 substituent in a pyrazinone based inhibitor by molecular modeling (Figure 21).

Figure 21. A close derivative to compound 108 modeled into the boundary between the NS3 protease active site (grey surface) and the helicase domain (green) of the 4A92 crystal structure. Hydrogen bond interactions are shown in red dashed lines.

The molecular modeling of a close derivative of compound 108 (i.e. lack-ing a fluorine on the P1 aryl) showed hydrogen bond interactions between His528, positioned in the helicase, and one of the oxygens on the P4 capping 4,4-dimethylpiperidine-2,6-dione. The backbone interactions with Lys136, Ala157 and Cys159 and an internal hydrogen bond between the urea hydro-gen and the pyrazinone were suggested.

These initial indications of possible interactions with the helicase sup-ported the evaluation of a small series of diverse P3 and P4(P5) capping groups (Table 14). 104 and 105 which contained short P3 capping groups and lacked possible internal hydrogen bond interactions with the pyrazinone, gave lower inhibitory potencies than the reference compound containing the tert-butyl urea (58). Gratifyingly, compounds 106 and 107 which contained elongated P4P5 substituents, showed improved inhibitory potencies com-pared to 58. In combination with a better P2 moiety, the potencies were simi-

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lar to the tert-butyl urea (108 vs 69 found in Table 11), but were well ac-cepted.

Table 14. Pyrazinone based HCV NS3 protease inhibitors containing various ureas in the 3-position of the pyrazinone.

Cmpd R1 R2 R6 NS3fl 1a

Ki ±SD (µM)

NS3fl 1a R155K Ki ±SD (µM)

NS3fl 3a Ki ±SD (µM)

58 H A 1.28 ± 0.38 a 0.58 ± 0.1 N.d.

104 H A 2.82 ± 0.42 N.d. N.d.

105 H A 3.8 ± 1.4 N.d. N.d.

106 F A 0.40 ± 0.18 0.14 ± 0.02 1.9 ± 0.6

107 F A 0.27 ± 0.14 0.13 ± 0.03 1.3 ± 0.6

108 F B 0.30 ± 0.16 0.10 ± 0.01 0.61 ± 0.21

N.d.: not determined. aThe Ki value was determined on the same occasion as the other inhibi-tors in this series. (Ki = 0.66 µM in a previous measurement, Table 6).

Evaluation against the R155K enzyme variant Selected inhibitors in Tables 11-14 were evaluated against the most preva-lent drug-resistant enzyme variant R155K, which is resistant to most of the HCV NS3 protease inhibitors under clinical evaluation or available on the market, as discussed in section 4.3.52 Satisfyingly, in general, this series showed more promising Ki values against the R155K mutant than against the

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wild-type. To some extent, this could be explained by the relatively small P2 substituents compared with the extended P2 blocks found in the advanced inhibitors (Figure 5, section 1.2.1). The inhibitors displayed Ki values rang-ing from 0.02 µM to 0.58 µM which could aid in the future design of opti-mized inhibitors directed against the R155K variant of the virus. Notably, the P1’ truncated inhibitors 93 and 94 were the most active compounds in this series, which accentuates the potential of this class of inhibitors.

4.6 Evaluation Towards the Genotype 3a of HCV NS3 Protease (Paper III)

Gt 3a of HCV is currently considered the most difficult genotype to treat.126 Moreover, there are indications that the prevalence of this genotype is in-creasing and that an infection with Gt 3a is connected with a greater risk of severe liver inflammation, faster progression of fibroses and an increased risk of hepatocellular carcinoma.6

The HCV NS3 protease inhibitors available on the market are not effec-tive against Gt 3a of the virus. The so-called second generation NS3 protease inhibitors (e.g. grazoprevir, Figure 5, section 1.2.1) have addressed this weakness; they have promising SVRs against Gt 3a.33,127

As previously discussed for Gt 1b, NS3 protease inhibitors containing large P2 substituents force R155 into an extended conformation which fa-vors the salt bridge between R123, D168 and R155 in the S2 pocket (Figure 20). Gallo and co-workers have used NMR spectroscopy to structurally characterize Gt 3a.128 This study showed that the previously described salt bridge is lacking in Gt 3a, due to a glutamine (Q) in position 168 and a thre-onine (T) in position 123. This partly explains the lower inhibitory potencies for inhibitors with extended P2 substituents. In presence of inhibitors inter-acting with R155 and Q168 (i.e. containing P2 substituents) the otherwise solvated, charged residue (Arg) will have to compensate for desolvation. Moreover, the flexible side chains of R155 and Q168 become more rigid in presence of inhibitors leading to loss of entropy.128 Recently, it was suggest-ed from molecular dynamics simulation that part of the reduced inihibitory potency of HCV NS3 protease inhibitors against Gt 3a was due to reduced catalytic activity of the enzyme. This was explained by a different distance between the amino acids in the catalytic triad, which results in a less effi-cient acid-base support from His57 (Figure 9, section 1.4).129

Pyrazinone-based inhibitors against genotype 3a The increased understanding of the Gt 3a, i.e. the different properties in the S2 pocket compared with the Gt 1a, partly supported the previously dis-cussed strategy for dealing with drug resistance against R155K and D168V,

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i.e. to design inhibitors with smaller P2 substituents and elongations in the P4P5 region.

A few inhibitors in the pyrazinone series (Tables 11, 12 and 14) have so far been selected for evaluation against this enzyme variant. Notably, alt-hough relatively high Ki values, they differed five- to six-fold, ranging from 0.61 µM to 3.44 µM. The highest Ki value was found for the bulky pyrimi-dine containing inhibitor 89 which fitted well in Gt 1a (0.2 µM) but was less suitable for Gt 3a (3.44 µM). In comparison, inhibitor 108, which encom-passed an elongated urea as a P4 capping group, showed a promising inhibi-tory potency also for Gt 3a (0.61 µM; Gt 1a, 0.30 µM).

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5 Synthesis of Pyrazinone-Based HCV NS3 Protease Inhibitors (Papers II and III)

The P3 pyrazinone-based inhibitors, containing P1P1’ substituents, P4(P5) ureas and varied R6 substituents (Figure 22) can be synthesized in a five- to seven-step procedure starting from aldehyde or alcohol, via the formation of α-aminonitriles and subsequent ringclosure (Scheme 5). Displacement of the C3-chlorine with a urea (Scheme 9) followed by introduction of the P1P1’ building blocks via amide coupling (Scheme 13) provide target compounds. Further decoration of the aromatic R6 group can be achieved in a final step using Suzuki-Miyaura couplings (Scheme 14).

In this chapter, synthetic strategies are outlined and discussed. Optimiza-tion of a C-N urea protocol is further evaluated in chapter 6.2.

Figure 22. A schematic overview of the synthetic strategies for the preparation of pyrazinone based HCV NS3 protease inhibitors.

5.1 Formation of the 2(1H)-Pyrazinone Core The 2(1H)-pyrazinone core structures 113-120 were constructed via a proce-dure previously established by our group.130 The aldehydes 110a-h compris-ing the R6 moiety, which in the pyrazinone is located in position 6, reacted with the glycine benzyl ester (109), forming the imin. Addition of trime-thylsilyl cyanide and microwave irradiation yielded the intermediate α-aminonitrile, which was purified by flushing the crude material through a plug of silica. In order to avoid a symmetrical oxamide in the cyclization step, the α-aminonitrile was first enriched with HCl gas with a subsequent cyclization using oxalyl chloride and microwave heating at 145-170 °C for

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10-25 min, or by reflux over-night, to obtain 113-120 in 28-66% yield over two steps.

The desired R6 groups found in 121-127 were not available as aldehydes. Thus, the corresponding alcohols (112a-g) were first oxidized by sulfur tri-oxide-pyridine complex, dimethylsulfoxide (DMSO) and Et3N in DCM at room temperature. For the naphthyl derivatives (111a and b), the corre-sponding carboxylic acids were first reduced to the alcohols (112a and b) followed by oxidation to aldehydes and a subsequent Strecker-type reaction by in situ addition of TMS-cyanide and 109, under reflux for one hour. The ring closure procedure described previously gave 121-127 in 25-43% yield over three steps.

Scheme 5. Reaction conditions: (a) TMSCN, DIPEA, DME, MW, 110-170 °C, 10 min or reflux, 30 min; (b) LiAlH4, THF, -78 °C to rt; (c) SO3-Py, Et3N, DMSO, DCM, rt, 1 h; (d) Dess-Martin periodinane, DCM, rt, 30 min; (e) 109, TMSCN, DIPEA, reflux, (f) HCl gas, Et2O, then oxalyl chloride, DME, MW, 145 °C for 25 min or reflux over-night (25-66% from 109).

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5.2 Introduction of Urea Substituents The urea moieties in position 3 of the pyrazinone were introduced in a palla-dium (Pd)-catalyzed C-N urea arylation protocol, which was evaluated and established for various urea derivatives as described in section 6.2. However, before the protocol was thoroughly investigated, another strategy was tried, as described in the following section.

Introduction of P4P5 urea via isocyanate In the first attempt to incorporate a sophisticated urea moiety, the C3-chlorine on the pyrazinone was replaced with an amino functionality by ad-dition of 25% NH3 in H2O to 127 at 110 °C for 6 h which gave 128 in 55% yield (Scheme 6). The amino group in 128 was weakly nucleophilic and after unsuccessful trials using pyridine as the base, NaH was applied, which ena-bled the amino functionality to react with the isocyanate 129.125,131 However, the 4,4-dimethylpiperidine-2,6-dione moiety on 130 hydrolyzed to some extent in THF and 1,2-dimethoxyethane (DME) at 100 °C for 15-30 min. Additionally, the benzylester present in 128 partly hydrolyzed resulting in a complicated reaction mixture. After several attempts it was, however, possi-ble to obtain 43% of 130.

Scheme 6. Reaction conditions: (a) 25% NH3 in H2O, 110°C, 55%; (b) 60% NaH, DME, rt, 129, MW 100°C, 15 min, 43%.

Formation of urea building blocks Clearly, the above procedure was unreliable and difficult to master. Thus, a protocol was optimized and evaluated to facilitate the introduction of com-plicated urea building blocks to position 3 of the pyrazinone (section 6.2). The ureas 136 and 137 were prepared as outlined in Scheme 7.

First, the phthalimido group of 131132,133 was deprotected with 40% me-thylamine in H2O. Generation of the 3-pyridine sulfonamide 133 was fol-lowed by alkylation of the nitrogen using methyl iodide and cesium car-bonate (134). Along with the isocyanate 135133 in the following step, the corresponding symmetrical urea was formed, which explained the low yield

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(25%). In the literature it was found that symmetrical ureas can be formed under the described conditions in the presence of a tertiary amine (i.e. the pyridine in this reaction).134 In the absence of pyridine (or any tertiary amine) when synthesizing 137, no byproduct was detected. The desired ure-as were finally obtained by addition of 0.5 M ammonia in dioxane (136, quant. and 137, 93% yield).

Scheme 7. Reaction conditions: (a) 40% CH3NH2 in H2O, rt, 88%; (b) 3-pyridine sulfonylchloride, TEA, DCM, 0°C/rt, 66%; (c) Cs2CO3, CH3I, DMF, 0°C/rt, 76%; (d) i. 4 M HCl in dioxane, rt, ii. sat. NaHCO3 aq., DCM, triphosgene, 0°C/rt; (e) 0.5 M NH3 in dioxane, 0°C/rt.

Interestingly, when treating 133 with 4 M HCl in dioxane in order to re-move the Boc group, followed by treatment with triphosgene to obtain 138, the ring-closed compound 139 was formed in high yields (Scheme 8). There-fore, it was not possible to manufacture the urea via this protocol.

Scheme 8. Reaction conditions: (a) 4 M HCl in dioxane, rt, sat. NaHCO3, DCM, triphosgene, 0°C/rt.

Palladium-catalyzed C-N urea arylation The urea/urea derivatives tert-butyl urea, benzohydrazide, 1-(tert-butyl)imidazolidin-2-one, 136 or 137 were installed via chemoselective Pd-catalyzed C-N arylation of the C3-chlorine on pyrazinones 113-127 (Scheme

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9) to yield 140-158 in 34-89%. It was found that the benzyl ester in some entries was partly or fully converted into the methyl ester due to residues of methanol in the DME. Exchange of the solvent to THF in the preparation of 155 conserved the benzyl ester.

As found previously111,112 the chlorine in position 3 was much more reac-tive than the chlorine in position 5, because of low electron density on the C3. More surprisingly, complete C3 selectivity was also noticed when the reaction conditions were applied to the pyrazinones 118-120 and 124-126, which contained R6 aryl bromide moieties. For similar selectivity patterns under Pd-catalyzed cross coupling, see section 6.2 and density functional theory (DFT) calculations.

Scheme 9. Reaction conditions: (a) tert-butylurea, Benzohydrazide, 1-(tert-butyl)imidazolidin-2-one, 136 or 137, Pd(OAc)2, Xantphos, Cs2CO3, DME or THF, MW 100-110 °C, 15-20 min or reflux, 0.5-1 h.

5.3 Synthesis of P1P1’ Building Blocks The P1P1’ building blocks were prepared as described in Schemes 10-12 to furnish an amino functionality successively acting as nucleophile in the am-ide coupling reactions found in Scheme 13.

Aromatic acyl sulfonamides The various P1P1’ building blocks 161-171 and 174 containing different regioisomers and P1 substituents were prepared as outlined in Scheme 10, utilizing a carbonyl diimidazole (CDI)-promoted coupling of the sulfona-mides with the carboxylic acids (160a-e and 172). In general this reaction worked well (47-80%) but in two reactions the yields were surprisingly low (163, 19% and 173, 26%). The lower yield of 163 can probably be explained by steric hindrance from the trifluoro methyl group situated in the ortho po-sition of the aryl sulfonamide.

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The bromo derivative 166 was further modified by displacing the halogen with a vinyl (169) and a pyrimidyl (170) moiety via microwave-assisted Suzuki coupling (66% and 48% yield, respectively). The sulfonamide nitro-gen on 161 was methylated to 171. Finally, in order to uncover the free amine, the Boc group was removed using 4 M HCl in dioxane, which gave the hydrochloride salt of the amines. The nitro group in 173 was reduced to the amine by Pd catalyzed hydrogenation to give 174 in 92% yield.

Scheme 10. Reaction conditions: (a) Boc2O, NaOH, dioxane; (b) CDI, THF, 66-68 °C, 2-CF3-benzene sulfonamide, 3-CF3-benzene sulfonamide, 4-CF3-benzene sul-fonamide or 4-(CH3)-benzene sulfonamide, DBU, rt; (c) 2,4,6-trivinyl cycloborox-ane pyridine complex or pyrimidine boronic acid, Pd(OAc)2, HP(tBu)3BF4, K2CO3, H2O, DME, MW 100 °C, 15 min; (d) Cs2CO3, MeI, DMF, 65 °C; (e) 4 M HCl in dioxane, rt, quant.; (f) Pd/C (10%), H2, EtOAc, 1 atm, rt, 92%.

P1 cyclobutyl-containing building blocks The spirocyclobutyl intermediate 179 (Scheme 11) was obtained via a four-step procedure previously described,135,136 starting from a 2-(chloromethyl)oxirane, and including epoxide opening (176), cyclization to a cyclobutyl (177),135 selective hydrolysis (178) and Curtius rearrangement (179).136 The ethylester was thereafter hydrolyzed, and coupled with the sulfonamide, in a similar manner to that described above, which gave 181 in an excellent yield (89%) with subsequent Boc deprotection. From 178 and onwards the cyclobutyl moiety contained two isomers (cis and trans).

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Scheme 11. Reaction conditions: (a) BnBr, HgCl2, 160 °C, 55%; (b) 60% NaH, diethyl malonate, dioxane, 0 °C to reflux, 53%; (c) NaOH, H2O/EtOH reflux, 54%; (d) DPPA, EtN3 toluene, t-butoxide, reflux, 40%; (e) LiOH, H2O, THF, rt, quant.; (f) CDI, THF, 66 °C, 4-CF3-benzene sulfonamide, DBU, rt; (g) 4 M HCl in dioxane, rt, quant.

Reversed aromatic acyl sulfonamides For the preparation of the reversed acyl sulfonamides 184-186 we again utilized a nitro moiety as a masked amine (182a-c). Coupling with 4-trifluoromethyl benzoyl chloride gave the intermediates 183a-c in moderate yields (45-58%) as depicted in Scheme 12. Reduction of the nitro group produced the building blocks 184-186 in 65-87% yield. It is known from the literature that acyl sulfonamides containing an amino group in the ortho position are precursors in the preparation of benzothiadiazine analogs by exposure to heat under acidic conditions.137,138 It was indeed noticed that during hydrogenation of the ortho derivative 183a, a byproduct with the actual molecular weight of the ring-closed/dehydrated compound was formed. The amount of byproduct seemed to increase during the prolonged reaction time. Since the desired reduction of the nitro moiety proceeded effi-ciently in only two hours, it was possible to suppress the quantity of the by-product (<5%) and the free amine was immediately coupled with the pyrazi-none scaffold as described in Scheme 13.

Scheme 12. Reaction conditions: (a) 4-pyrrolidino pyridine, pyridine, 4-(CF3)-benzoyl chloride, toluene, rt, N2-atm; (b) Pd/C (10%), H2, EtOAc, 1 atm, rt.

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5.4 Amide Coupling of P3 Pyrazinones and P1P1’ Building Blocks

After urea N-arylation (Scheme 9), the methyl/benzyl esters were hydro-lyzed, followed by peptide coupling between the formed carboxylic acid and the building blocks presented in Schemes 10-12 (Scheme 13).

Scheme 13. Reaction conditions: (a) (i) K2CO3, CH3CN/H2O, MW 100-120 °C, 15-20 min (except for 130), (ii) P1P1’ building block, POCl3, pyridine, -15 °C/rt, 4-89% over two steps, or P1P1’ building block, HATU, DIEA, DCM, rt to 45 °C, 11-68% over two steps.

Initially, we used HATU to promote the coupling. However, the reaction appeared slow and low yielding, probably because of the poor nucleophilici-ty of the P1P1’ building block 161, and much of the starting material re-mained intact in the HATU-promoted coupling (58, 22% yield). Hence, we decided to investigate alternative coupling procedures and found that phos-phoryl chloride in pyridine at low temperatures provided the final inhibitors 62-75, 82, 83, 86, 87, 90-92, 95, 97-100 in 14-89% yield. The low yields of compounds 84 (10%), 85 (4%) and 105 (11%) were partly the result of sev-eral purifications on a silica column, followed by preparative HPLC, which resulted in very small amounts of the pure compounds. These yields could therefore possibly be improved. However, compound 104 (8%) which con-tained the hydrazide moiety as a P3 capping group, was partly unstable and present in at least two tautomeres. This complicated both the introduction of the hydrazide moiety (155, Scheme 9) and purification of the final target compound 104.

During the synthesis of compound 88 we needed to reconsider HATU as a coupling reagent since the vinyl moiety was unstable in the presence of phosphoryl chloride. Interestingly, HATU worked very well, in just 2.5 h reaction time (67%). Compounds 89 (54%), 94 (57%), 102 (68%), 106 (52%), 107 (61%) and 108 (71%) were also successfully coupled in the pres-

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ence of HATU. The low yield of 101 (11%) was due to repeated purification attempts, providing small amounts of the pure target compound.

5.5 Final Decorations and Modifications For some of the inhibitors (76-81, 93, 96 and 103) a final conversion was needed to achieve the desired inhibitors, as described in the two following sections.

Suzuki coupling to the R6 arylbromide Decoration of the aryl bromides 70-75 was achieved under microwave-assisted Suzuki-Miyaura coupling conditions, using 3-pyridyl boronic acid (Scheme 14). The yields ranged between 5% and 69%, with poor outcomes for the sterically hindered ortho derivatives 78 (20%) and 81 (5%) and high-er yields for the meta and para analogs (76, 77, 79 and 80, 41-69%). Alt-hough five equivalents of boronic acid were used, no competing coupling with the C5 chlorine was observed.

Scheme 14. Reaction conditions: (a) 3-Pyridyl boronic acid, Pd(PPh3)2Cl2, Na2CO3, H2O, EtOH, DME, MW, 120 °C, 15 min (5-69%).

Removal of protecting groups The inhibitors 93, 96 and 103 were finally synthesized as described in Scheme 15. The methyl ester in 94 and the benzyl ester in 141 were hydro-lyzed in good to excellent yields (75% and 98%, respectively). The benzyl-protecting group positioned on the spirocyclobutyl hydroxyl in compound 102 was removed via Pd-catalyzed hydrogenation under mild conditions (rt, 1 atm). However, the chlorine in position 5 was also removed, and a shorter reaction time did not selectively remove the benzyl group. Although unde-sirable in this case, from a synthetic point of view this observation was inter-esting since the C5 chlorine is often considered to be more or less unreac-tive.

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Scheme 15. Reaction conditions: (a) K2CO3, CH3CN/H2O, MW 100 °C, 15 min. (b) Pd/C (10%), H2, MeOH, 1 atm, rt, 59%.

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6 Method Development (Papers IV and V)

6.1 Palladium as a Catalyst in Organic Synthesis In 1959, the so-called Wacker oxidation process emphasized the d-block transition metal Pd as a valuable catalyst. Nowadays, Pd is one of the most frequently used transition metals in preparative organic synthesis, as a result of its ability to catalyze C-C and C-heteroatom bond formations with high chemo- and regio-selectivity.139 The importance of the Pd(0)-catalyzed cross-coupling reactions was accentuated in 2010, when the Nobel Prize in Chemistry was awarded to Heck, Negishi and Suzuki for their contributions in this research area.140

The usefulness of Pd can be explained by its inherent fundamental proper-ties. The moderately large atomic size provides a stable, but also wide-ranging, reactive metal. Pd is primarily found in oxidation states 0 or +2, with a tetrahedral (d10) or a square planar (d8) geometry and a low barrier for moving between these states (oxidation/reduction). The relatively electro-negative Pd makes the C-Pd bond rather nonpolar which explains the low reactivity towards polar groups, such as ketones, esters, amides and alde-hydes, etc. These properties combined with a low risk for radical or one-electron reactions endorse Pd as a useful catalyst with relatively few un-wanted side reactions.139,141

Indeed, since the beginning of the 1970s when the C-C bond formation was demonstrated under Pd catalysis, this type of reaction has been widely used and further developed by researchers all over the world, both in aca-demia and the industry. The influence of the steric and electronic properties of ligands led to the design and preparation of numerous ligands which sub-sequently enabled cross couplings, to generate bonds like C-N, C-O and C-S, etc.140

6.2 C-N Urea Arylation In 1995, Buchwald and Hartwig, independently of each other, discovered Pd-catalyzed amination of aryl halides.142,143 The scope of this reaction sub-sequently also covered arylation of less reactive nucleophiles, e.g. amides,144 carbamates145 and sulfonamides.146,147 Six years later, in 2001, Beletskaya and co-workers managed to arylate ureas under Pd-catalyzed conditions.148

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This was an important alternative to the conventional synthesis of ureas us-ing toxic isocyanate or phosgene. The catalytic cycle for Pd-catalyzed ami-dation (Figure 23) includes oxidative addition of aryl halide to the Pd0 spe-cies, creating a PdII complex, followed by the introduction of the nucleophile (e.g. a urea moiety). Subsequently, the catalytic cycle is closed by a reduc-tive elimination, forming the desired C-N bond and regenerating the Pd0

species. Mechanistic studies suggest that the reductive elimination is the rate-determining step for the ureidation reaction.149,150 The development and evaluation of various ligands in Pd-catalyzed amidation reactions indicated that the rate of reductive elimination is increased by steric and electron-deficient ligands on Pd, electron-deficient aryl halides and electron-rich nu-cleophiles.148–152

Several modified protocols have been developed for the arylation of vari-ous ureas, including both mono- and bidentate ligands as well as a variety of aryl halides and substrates.149,150,153–155 Recently, a few heteroaryl halides were investigated as components of the catalytic cycle.156,157 However stud-ies on heterocyclic structures that are interesting as templates in medicinal chemistry are still limited.

Figure 23. The proposed catalytic cycle for the palladium-catalyzed amidation (i.e. ureidation).149 Ar = Aryl, X = Leaving group, L = Ligand.

6.3 Palladium Catalyzed Urea Couplings to 3,5-Dichloro-2(1H)-Pyrazinones (Paper IV)

As discussed in a previous section (4.1) the 2(1H)-pyrazinone molecule has found its application in various medicinal chemistry projects. We found that a tert-butyl urea as a P3-capping group improved the inhibitory potency of pyrazinone-based HCV NS3 protease inhibitors (section 4.3.1). However, the introduction of urea to the C3 position of a pyrazinone is not a well-

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established procedure. In a few patent applications, urea moieties have been installed using a pyrazinone amine and an isocyanate158 or an activated car-bamate.159

A literature survey revealed a considerable number of biologically active N-aryl and N-heteroaryl ureas, which are able to improve the biological sta-bility of peptidomimetic inhibitors.160–162 Given our increased knowledge of C-N arylations, and the potential to exploit the pyrazinone scaffold in vari-ous peptidomimetic compounds, we felt encouraged to explore scope and limitations of a C-N urea arylation to the 3,5-dichloro-2(1H)-pyrazinone (Figure 24).

Figure 24. Pd-catalyzed C-N arylation of urea analogs to the 3,5-dichloro-2(1H)-pyrazinone.

Ligand screen The investigation of urea coupling to pyrazinone was initiated with a ligand screen, in order to analyze the impact of employing mono- or bidentate lig-ands to the protocol (Figure 25). 1-Benzyl-3,5-dichloro-6-methyl-2(1H)-pyrazinone (187a) was selected as a model substrate and tert-butylurea was used as nucleophile. Pd(OAc)2 was chosen as the catalyst, Cs2CO3 was used as base and DME as solvent. All reactions were performed in a dedicated microwave reactor (MW), at 100 °C for 20 min (Table 15).

Figure 25. Ligands evaluated in the optimization study.

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Monodentate ligands (188a,b) and the nitrogen-based bidentate ligand (188c) gave poor yields of 189a (6-11%). Along with the formation of prod-uct, a major byproduct was also detected in entries 1-3, which explained the low yield of product in combination with small amounts of recovered start-ing material. Analysis of the byproduct by NMR and liquid chromatography-mass spectrometry (LC-MS) suggested structure 190a (Figure 26), which originated from two coupled pyrazinones. A possible explanation for the formation of 190a is that the azomethine nitrogen of a second 2(1H)-pyrazinone coordinated to Pd, making the actual C-N formation feasible during the reductive elimination step.

Table 15. Optimization study of C-N urea arylation between the 3,5-dichloro-2(1H)-pyrazinone and tert-butyl urea.

Entry Ligand Base 189a (%)a 187a (%)b

1 188a Cs2CO3 11 3 2 188b Cs2CO3 7 6 3 188c Cs2CO3 6 13 4 188d Cs2CO3 73c 0 5 188e Cs2CO3 63d 0 6 188f Cs2CO3 91 0 7e 188f Cs2CO3 92 0 8f 188f Cs2CO3 91 0 9 188f K2CO3 82 0 10 188f K3PO4 86 0 11 188f Et3N 45 43 12 188f - 22 52 13 - Cs2CO3 2 23 14g - Cs2CO3 0 n.d.

Reaction conditions: 187a (0.5 mmol, 1.0 equiv.), tert-butylurea (1.5 mmol, 3.0 equiv.), Pd(OAc)2 (0.025 mmol, 0.05 equiv.), 188a-f (0.04 mmol, 0.08 equiv.) and Cs2CO3 (1.0 mmol, 2.0 equiv.) in 3.0 mL of DME, 100 °C, 20 min. aIsolated yield. bRecovered starting material 187a. c10% isolated yield of 190b (Figure 26). d4% isolated yield of 190b (Figure 26). e80 °C, 20 min. f100 °C, 10 min. gWithout Pd(OAc)2.

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Figure 26. Byproducts 190a and 190b.

Gratifyingly, an improved outcome, with no formation of this byproduct, was achieved by changing to phosphorous-based bidentate ligands (entries 4-6). However, 1,2-bis(diphenylphosphino)ethane (dppe) (188d, entry 4, 73%) and 1,3-bis(diphenylphosphino)propane (dppp) (188e, entry 5, 63%) caused partial cleavage of the urea moiety, yielding some of the amino com-pound 190b (Figure 26); this was not found when employing 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (188f, entry 6). Moreover, absence of ligand (entry 13) or removal of the Pd catalyst (entry 14), resulted in low or no formation of product. These outcomes further em-phasized the importance of the ligand for the reaction to proceed successful-ly. Consequently, Xantphos was identified as the most suitable ligand in this study and was selected for further optimization studies.

Influence of base and reaction conditions Although a satisfying outcome was achieved via the introduction of Xantphos as ligand, we decided to evaluate a few different bases, as well as the reaction time and temperature, in order to determine the robustness of the protocol. It was found that K2CO3 and K3PO4 gave good yields (82% and 86%, respectively) which were close to the yield found for Cs2CO3 (91%). Et3N, on the other hand, hampered the reaction and resulted in incomplete conversion of starting material with a low yield of 189a (45%). Removal of base gave only partial conversion of starting material and low product yield (22%). Thus, the presence and choice of base were essential for an efficient reaction.

The reaction was performed with Xantphos and Cs2CO3 at 80 °C for 20 min as well as at 100 °C for 10 min (entries 7 and 8). The excellent yields indicated that the protocol was reliable with fast conversion also at decreased temperatures. The milder conditions were examined on the somewhat more complex urea derivatives 1-(tert-butyl)imidazolidin-2-one and 1-(pyridine-3-yl)urea (80 °C, 20 min and 100 °C, 10 min, respectively) which resulted in incomplete conversion of 187a. Bearing in mind that the protocol should allow installation of a wide variety of ureas, these control experiments sup-

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ported the choice of conditions found in entry 6 (100 °C, 20 min) for the extended study.

Scope and limitations A broad investigation to cover a wide variety of ureas was carried out next, including aliphatic, aromatic and sterically hindered ureas (Table 16). Over-all, the protocol performed well with most of the ureas. The aliphatic methyl, dimethyl and ethyl ureas were arylated in good to excellent yields (82-94%, entries 2-4) as was the sterically hindered cyclic urea (96% yield, entry 5). The reaction protocol also handled the morpholino urea well (189f, 82% yield).

Table 16. Palladium-catalyzed C3 coupling of various ureas to 3,5-dichloro2(1H)-pyrazinone.

Entry Urea Product Yield (%)a

1 189a 91

2 189b 94

3

189c 90

4 189d 82

5

189e 96

6 189f 82

7

189g 191

45 41

82

8

189h 50

9

189i 67

10

189j 85b

11

189k 84

12 189l 71

13

189m 96c

14

189n 56 67c

15

189o Tracesc

Reaction conditions: The reaction was performed under microwave irradiation in a sealed vial: 187a (0.5 mmol, 1.0 equiv.), urea analog (entries 1-15) (0.75 - 1.5 mmol, 1.5 – 3.0 equiv.), Pd(OAc)2 (0.025 mmol, 0.05 equiv.), Xantphos (0.04 mmol, 0.08 equiv.) and Cs2CO3 (1.0 mmol, 2.0 equiv.) in 3.0 mL of DME, 100 °C, 20 - 25 min. aIsolated yield. b25 min reac-tion time. c1.5 equiv. urea.

When the reaction conditions were applied to the allyl urea (entry 7) the

byproduct 191 (41%, Figure 27) negatively affected the formation of 189g (45%). At least two pathways could possibly give the byproduct 191. Either the more hindered nitrogen of the allylurea was coupled to the pyrazinone followed by hydrolysis of the urea, or the free amine of allylurea hydrolyzed in situ competed as a component in the catalytic cycle. Despite a low yield, these results presented a reasonable approach to the introduction of this chemical handle which can be utilized in further modifications, including macrocyclizations,163 epoxidations164 and aziridinations,164 etc., which are reactions widely performed to prepare bioactive compounds.163,164

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Figure 27. Byproduct 191 formed in entry 7, Table 16.

The hydroxyethylurea, with potentially two nucleophilic positions availa-ble, produced only the desired product (189h). However, during purification on a silica column, the product decomposed and provided a lower amount of 189h (50%) than expected. The biuret (entry 9) provided 189i in 67% yield, despite some di-arylation (<10%) of the biuret.

In general, the protocol handled the aromatic ureas very well (entries 10-12, 71-85% yield). In order to reach full conversion of the basic pyridyl urea (entry 10), a 25 min reaction time was required (189j, 85%). The fairly acid-ic hydroxyl group (entry 11) was well tolerated in the protocol, giving 189k in 84% yield. Gratifyingly, 5H-dibenzo[b,f]azepine-5-carboxamide (entry 12) gave 189l in 71% yield despite the sterically congested urea.

One important investigation within this set of reactions was to examine the selectivity pattern between the 3,5-dichloro-2(1H)-pyrazinone and the chloro-, bromo- and iodophenyl ureas (entries 13-15). The purification of the bromo compound (189n, 56%) was complicated due to the presence of by-products which most likely originated from the excess urea reactant. In an attempt to reduce the amount of byproducts formed, the reaction was per-formed using 1.5 equiv. of 4-bromophenyl urea. This was a fruitful modifi-cation, which indeed reduced the amount of byproducts and facilitated the purification (189n, 67% yield). The same strategy was followed in entries 13 and 15, using the chloro- and iodophenyl ureas, respectively. Interestingly, the outcomes of these two reactions were entirely different. The chloro-phenyl derivative (entry 13) gave an excellent yield (189m, 96%) while the 4-iodophenyl urea (entry 15) only provided traces of product 189o. The po-tentially higher reactivity of the 4-iodophenyl urea compared to the C3 chlo-rine on the 2(1H)-pyrazinone 187a and polymerization of the iodophenyl urea could explain the low amount of product formed. We decided to scruti-nize the selectivity pattern by performing DFT calculations.

DFT calculations have previously been used to investigate the oxidative addition of aryl halides to Pd complexes.165,166 However, the oxidative addi-tion of the 3,5-dichloro-2(1H)-pyrazinone, which has two potentially reac-tive positions for this type of reaction, has not been investigated. Initially, the DFT calculations showed a free energy requirement of 30 kJ mol-1 for oxidative addition to the C3 position, compared with 104 kJ mol-1 for the C5 chlorine. The expected trend in free energy requirement between the various aryl halides was verified, i.e. Cl > Br > I with energies of 107 kJ mol-1, 72 kJ

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mol-1 and 60 kJ mol-1, respectively. Thus, it is suggested that the chlorine in the C3 position is more reactive than the iodophenyl urea with respect to oxidative addition. The initial hypothesis suggesting that polymerization of the iodo phenyl urea could account for the poor formation of product was therefore contradictory to the DFT calculations. For that reason, the reaction was further examined by a few control experiments.

First, we ran a reaction following the conditions in entry 1, Table 16, with the addition of 3 equiv. of 3-iodotoluene. Minor amounts (<10%) of 189a were formed, in contrast to the outcome for entry 6 (91%). This indicated that the presence of an aryl iodide somehow impeded the reaction. It was further noted that 3-iodotoluene did not react with tert-butylurea. Moreover, the byproduct 190a (Figure 26) was observed, implying a suboptimal cata-lytic system for the desired reaction to occur. Subsequently, a control reac-tion was performed in the absence of the pyrazinone 187a. Minor amounts (<10%) of 3-iodotoluene coupled to tert-butylurea, which also supported the idea that aryl iodides prohibited the C-N urea formation. Taken together, these experiments verified the DFT calculations suggesting the C3 chlorine on the pyrazinone was more prone to oxidative addition to Pd than aryl io-dides.

Although limited to urea containing aryl bromides and chlorides, the re-sults presented in entries 13 and 14 allow for selective couplings to the C3 chlorine on the pyrazinone, which permit further elaborations on the in-stalled aryl, e.g. Pd cross-coupling reactions.140

6.4 Palladium Catalyzed Carbonylations The initial efforts to provide amides and esters via amino- and alkoxy-carbonylations of aryl or vinyl halides by the use of a Pd catalyst and carbon monoxide (CO) gas were presented by Heck and co-workers in 1974.167,168 A proposed mechanism of a typical carbonylation reaction is outlined in Figure 28.139 The oxidative addition of aryl halide generates the PdII complex, fol-lowed by insertion of CO. The nucleophile then attacks the electron poor PdII complex, which expels the formed carbonyl compound. Finally, a reductive elimination step follows, which regenerates the Pd0 catalyst.169

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Figure 28. Proposed catalytic cycle for the carbonylation of aryl halides.L = Ligand, Ar = Aryl, X = Leaving group.

An interesting feature regarding the carbonylation chemistry is the possi-bility of delivering a wide variety of compounds by simply changing the nucleophile. This makes it a suitable reaction in the development of com-pound libraries of biologically active analogs. A major drawback, however, is the use of gaseous CO, which requires special equipment that can endure high pressure.170 Moreover, the CO gas is toxic and flammable, which calls for caution when performing these experiments. Gas-free alternatives are therefore highly valuable. As a matter of fact, several crystalline and liquid CO sources are frequently used in small-scale synthetic procedures, where CO is released in situ by an activator or heat and is subsequently incorpo-rated into the catalytic cycle.171–174

Mo(CO)6, a gas-free CO source, has been widely explored by researchers at our department.174 Recently, Skydstrup and co-workers developed a two-chamber reaction system172 which enabled ex situ generation of CO. This system has been further evaluated using Mo(CO)6 as the CO source.175

6.5 Synthesis of Acyl Sulfinamide, a Versatile Functionality (Paper V)

In line with our overarching goal to investigate bioactive molecules and bi-oisosteres that are useful to drug discovery, we identified acyl sulfinamide (I) (Figure 29) as an interesting functionality found in different bioactive molecules.176–178 Acyl sulfinamide has been considered as a carboxylic acid bioisostere due to its slightly acidic proton (ca. pKa 6)179 in, for example, HCV NS3 protease inhibitors.180 Besides its direct application in drug dis-covery, acyl sulfinamide also works as a precursor in the preparation of sul-fonimidamides (II)181–187 and acyl sulfonimidamides (III and IV)188,189 (Fig-ure 29).

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Figure 29. Acyl sulfinamide (I), sulfonimidamide (II), acyl sulfonimidamides (III), (IV).188,189

These acyl sulfinamide derivatives have recently been evaluated in drug discovery-related research. The sulfonimidamide (II) has been recognized as a sulfonamide bioisostere in γ-secretase inhibitors.186,190 PK evaluation re-vealed increased solubility, decreased lipophilicity, and decreased plasma protein binding compared with derivatives containing a sulfonamide.186 Cy-clic sulfonimidamides have also been evaluated as novel carboxylic acid bioisosteres, since they are associated with higher Caco-2 permeability and lower efflux than the carboxylic acid isostere, tetrazole.191 Moreover, the acyl sulfonimidamides (III and IV) have been identified as potential carbox-ylic acid bioisosteres based on their in vitro PK properties.192

Preparation of acyl sulfinamide Acyl sulfinamide has typically been constructed from sulfinamide and car-boxyl acid anhydride, by exposure to n-BuLi or NaH at low temperatures. 179,193 With an interest in further evaluating acyl sulfinamides and derivatives thereof as carboxylic acid bioisosteres, we took the opportunity to facilitate the synthesis of this functionality to make it more accessible.

The capacity of Pd-catalyzed carbonylation is expansive, which is demon-strated by the number of reported functionalities installed by various car-bonylation protocols, e.g. amides, carboxylic acids, esters, ketones and al-kynes.170,194 However, to the best of our knowledge, sulfinamide has not yet been evaluated as a nucleophile in this type of reaction. If a satisfying proto-col could be attained, varied aromatic moieties could be introduced next to the carbonyl functionality, which should be of interest in diverse library syn-theses for SAR studies as well as for adjustment of other properties im-portant to drug design (Figure 30).

Figure 30. A schematic description of a Pd catalyzed carbonylation reaction.

Initially, the reaction conditions previously developed for Mo(CO)6-based carbonylative transformations for the functionalization of sulfonimidamides (III) (Figure 29) were applied.188,189 A two-vial system was used (Table 17) and chamber 1 (C1) was charged with sulfinamide (192a), 4-iodoanisole, Pd(OAc)2 and Et3N in 1,4-dioxane. Thereafter, Mo(CO)6 together with DBU,

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which has been suggested to facilitate the release of CO from Mo(CO)6,174 in

1,4-dioxane were added to chamber 2 (C2). The two vials were capped, and the reaction system was heated to 80 °C for 6 h (entry 1, Table 17). The sub-strate 192a was fully consumed but purification only provided 20% yield of the desired product (193a). It was found that a byproduct, the thioester 194 (Figure 31)195 was generated along with the product, which explained the relatively low yield.

Table 17. Optimization of reaction conditions between sulfinamide and 4-iodoanisol.

Entry Base Temp Time Yield (%)a

1 Et3N 80 6 202b Et3N 80 3 173b Et3N 80 2 124c Et3N 60 1 05c Et3N 70 18 226 K2CO3 70 2 717 K2CO3 80 2 798d K2CO3 80 2 329e K2CO3 80 1 16

Reagents and conditions: C1: sulfinamide (0.354 mmol), 4-iodoanisol (0.885 mmol), base (0.885 mmol), Pd(OAc)2

(0.018 mmol) and 1,4-dioxane (2 mL). C2: Mo(CO)6 (0.885 mmol), DBU (1.563 mmol) and 1,4-dioxane (2 mL). aIsolated yields. b0.1 equiv. Xantphos. c2 equiv. DMAP. dCO (75 psi). eCO (50 psi).

Figure 31. Byproduct (194) formed using Et3N as base in the carbonylation reaction (Table 17).

These disappointing, yet challenging, results needed to be addressed be-fore an acceptable yield could be obtained. We planned to successively vary the reaction time, investigate the impact of ligand, and stimulate the nucleo-philic attack of the sulfinamide on the catalytic system.

We speculated that a prolonged reaction time could support the formation of 194 at the expense of 193a by reducing unreacted starting material to thiol. Therefore, the reaction time was reduced to 3 h and Xantphos, a ligand that potentially enhances the progress of the reaction, was added (entry 2).196 However, a similar yield to that in entry 1 was obtained with no obvious

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advantage from the ligand. Shorter reaction time (entry 3) further lowered the yield of 193a.

Next we considered an approach for investigating whether the introduc-tion of the sulfinamide was the problematic stage. 4-Dimethylaminopyridine (DMAP) has previously been applied for enhancing the carbonylation of poor or sterically hindered nucleophiles by acting as an acylating agent.197 However, in this protocol, no improvement was obtained (entries 4 and 5). A temperature of 60 °C (entry 4) was not sufficient for the reaction to com-mence, since neither product nor byproduct were detected after 1 h of heat-ing. At 70 °C with an extended reaction time (entry 5), it was noted that after about 2-3 h of reaction the amount of 193a did not increase, indicating that the catalytic system at that point was no longer active.

To summarize these initial investigations, it was found that the byproduct 194 was formed in entries 1-3 and 5, regardless of varying reaction condi-tions and the presence of ligand and DMAP. A likely explanation is that the reaction conditions in these entries enabled the reduction of sulfinamide to the corresponding thiol which subsequently competed with 192a in the nu-cleophilic attack on the Pd complex. As mentioned, after 2-3 h of reaction, the formation of product seemed to halt. This could be explained by poison-ing of the Pd catalyst, due to the presence of thiol.198,199

We considered that the choice of base could have affected the outcome of the reaction, so the organic base Et3N in chamber 1 was exchanged to K2CO3 which dramatically increased the formation of product (entry 6). Most im-portantly, the byproduct 194 was not detected and full consumption of 192a after 2 h at 70 °C was realized, with a slightly increased yield at 80 °C (entry 8).

It has previously been confirmed that reduction-sensitive moieties, like the nitro group, are viable under carbonylation conditions, provided that a two-chamber system is used.175 A nitro group could be considered as a masked amine, useful for further modifications, so this equipment widens the application of carbonylations. When employing the sulfinamide as a component in the Pd-catalyzed carbonylation protocol, our results indicated that the two-vial system was crucial for successful formation of product. In a single vial reaction several byproducts were formed, most likely due to the sensitive sulfinamide group. Moreover, under molybdenum-free conditions using CO-gas, a pressurized reactor was employed. Lower yields were ob-tained (entries 8 and 9, 32% and 16%, respectively) which emphasized the benefits of a two-compartment system for this reaction.

Scope and limitations To evaluate the protocol, electron-deficient, -neutral and -rich aryl and het-eroaryl iodides were investigated, using aryl and alkyl sulfinamides follow-ing the conditions in entry 7 (Table 17). For all the presented entries, full

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consumption of the sulfinamide was reached. Yields in the range of 34% to 79% were obtained (Table 18).

Table 18. Scope of aryl iodides in the carbonylative synthesis of acyl sulfinamides.

Entry R-SO-NH2 Ar-I Product

Yield (%)a

1 192a

193a 79

2 ’’ 193b 76

3 ’’ 193c 46

4 ’’

193d 68

5 ’’

193e 58

6 ’’ 193f 65

7 ’’

193g 51

8 ’’

193h 38

9 ’’

193i 66

10 ’’

193j 41

90

11 ’’

193k 38

12 ’’ 193l 71b

13 192b

193m 69

14 ’’

193n 76

15 ’’

193o 62

16 ’’

193p 34

17 192c

193q 49

18 ’’

193r 53

Reagents and conditions: C1: sulfinamide (0.354 mmol), aryl iodide (0.885 mmol), K2CO3 (0.885 mmol), Pd(OAc)2

(0.018 mmol) and 1,4-dioxane (2 mL). C2: Mo(CO)6 (0.885 mmol), DBU (1.563 mmol) and 1,4-dioxane (2 mL), 80 °C, 2 h; aIsolated yields; bReaction time, 3 h.

The aryl iodides with para and meta methoxy substituents (193a, 193b

and 193m) and the electron-neutral aryl iodides (193d-f, 193n, 193o) gave the highest yields.200 For the ortho iodoanisole (entry 3), steric hindrance probably affected the outcome of the reaction, since this has been observed previously.188

Among the aryl iodides containing electron-withdrawing substituents in the para position, the cyano derivative (entry 9) gave a markedly higher yield (193i, 66%) than 193g 51%, 193h 38%, 193p 34% or 193r 53%. The nitro moiety was conserved, with no reduction to the corresponding aniline. The heteroaryl derivatives (entries 10-12) were relatively well achieved. Notably, the trityl-protected imidazole (193k) was stable under the present reaction conditions with no deprotection detected (38%). A comparable high yield was obtained for the possible PdII-coordinating pyridyl derivative

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(193l, 71%). However, the 2-iodopyridyl only provided traces of product (not included in the table). The achievable introduction of heteroaryl iodides broadens the scope of this protocol, providing means for elaboration with PK properties in for example the design of bioactive molecules.

Surprisingly, for the benzene sulfinamide (entries 17 and 18), the differ-ence in yield between the electron-rich 4-iodoanisole and the electron-poor para-nitro aryliodide was small (193q, 49% and 193r, 53%), in contrast to previous entries. It is important to mention that purification in some entries was challenging. The compound adhered to the silica, possibly due to the slightly acidic proton on the acyl sulfinamide nitrogen. For the compounds containing electron-withdrawing aryls, and thus an increased acidic character of the acyl sulfinamide functionality, this phenomenon appeared to be more distinct. This could be a reason for the generally lower isolated yields of compounds containing electron-withdrawing aryl substituents. Unfortunate-ly, when the reaction conditions were applied to the chiral sulfinamides, 192a and 192b, partial racemization seemed to occur as suggested by optical rotation.

Arylbromides did not work under these relatively mild conditions. A few attempts were made to identify a suitable catalytic system by adding a ligand or changing the Pd source (e.g. Xantphos, Palladacycle/Fu salt, Pd(PPh3)4, Pd(dppf)Cl2/DCM) and by raising the temperature to 120 °C. However, so far a protocol that works smoothly also with aryl bromide has not yet been established.

To summarize chapter 6, a chemoselective and robust palladium-catalyzed N-arylation protocol to install various ureas at the 3-position of the 3,5-dichloro-2(1H)-pyrazinone scaffold was developed. Bidentate ligands, Xantphos in particular, were superior to monodentate ligands and in pres-ence of aryl chloride and aryl bromide, the C-3 chlorine was selectively dis-placed. Subsequently, a novel palladium-catalyzed protocol for the car-bonylative synthesis of acyl sulfinamides, was developed. The two-chamber system, using Mo(CO)6 as CO-source, and the choice of base, K2CO3, were crucial for the successful carbonylation of various aryl/heteroaryl iodides with the sensitive sulfinamide functionality.

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7 Concluding Remarks

The work presented in this thesis was based on two drug discovery projects within the HCV area, both aiming to inhibit the drug target, NS3 protease. The criteria differed with respect to the various stages of discovery they rep-resented. In the P2 quinazoline macrocyclic series, the lead structure was optimized for improved PK properties along with sub-nanomolar Ki and nanomolar EC50 values. The pyrazinone series, on the other hand, repre-sents an early stage of drug discovery aiming for new lead compounds, which could be further optimized into coming generation of HCV NS3 pro-tease inhibitors. The main findings are summarized below:

• Improved Caco-2 permeability and promising in vivo PK properties for

inhibitors containing the optimized P2 quinazoline were obtained by re-placing the cyclopentane scaffold with the proline urea analog.

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For the P3 pyrazinone-based inhibitors, the following main results were obtained during the evaluation and optimization of the substituents around the core structure:

• A urea moiety in the C3 position improved both stability and inhibitory potency compared with the carbamate analog. Inhibitors containing P4P5-ureas were prepared and evaluated and indicated allowance for substituents in this area.

• Relocation of the P2 group to the R6 position was well accepted and resulted in achiral inhibitors with improved inhibitory potencies for elongated R6 moieties. Moreover, the R6 substituents influenced the PK, with favorable properties for a pyridyl moiety.

• The resistance profile for this class of inhibitors showed retained inhibi-

tory potencies against known drug-resistant variants of the virus, i.e. R155K, A156T and D168V. Initial evaluation against genotype 3a dis-played promising inhibitory potencies for a set of inhibitors with Ki val-ues 0.6-3.4 µM.

• Based on evaluation of several P1P1’ building blocks, preliminary re-

sults suggested that the acyl sulfonamide did not improve the inhibitory potency. The P1’ aryl did not appear to have any specific interactions with the S1’ pocket, as supported by comparable inhibitory potencies for truncated derivatives. It was found that the P1 aryl in combination with the P3 pyrazinone and a C3 urea were important for sub-micromolar Ki values, suggesting that this could be the new lead structure.

• An efficient Pd-catalyzed C-N urea arylation to the C3 position of the

pyrazinone was developed and successively applied to inhibitors with elongated P4P5 urea substituents. In line with our interest in identifying carboxylic acid bioisosteres, a novel Pd-catalyzed carbonylation pro-tocol for sulfinamides yielding acyl sulfinamides was developed.

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8 Acknowledgements

I have had the privilege to work with many skillful and nice researchers at the De-partment of Medicinal Chemistry. You have made these years so enjoyable and rewarding. Thank you all! I would like to express my sincere gratitude to the follow-ing people: My excellent supervisor, Docent Anja Sandström, for your enthusiasm and confi-dence in the projects and in me. You are a true inspiration both in research and in teaching and you have gently guided me during these years. Docent Eva Åkerblom, your experience and caring have been invaluable when supporting me, regardless of good or bad results. I will miss your engagement in the HCV project and our always fruitful and pleasant discussions over a cup of tea. My assistant supervisor, Prof. Mats Larhed, for sharing your experience and show-ing me what is possible to accomplish with limited time.

Former and present members of the HCV team: Anja, Eva, Anna L, Pernilla, Johan and Krzysztof for sharing ideas and experiences during the group meetings and in the lab. Angelica, Eldar, Sofia and Prof. Helena Danielson, for the biochemical evalua-tion and for discussions regarding assays. Dr. Richard Svensson and Prof. Per Ar-tursson and for providing me with PK data and new insights into the properties of these molecules. Hiba and Prof. Anders K for molecular modeling and for the beau-tiful pictures in this thesis. The Pd group for contributing to my C-N urea arylation-project. Fredrik and Docent Christian for the DFT calculations. Anna S and Anna O, for skillful laboratory work during your master thesis work and SOFOSKO project. Dr. Prasad, for your contribution and positive attitude in my “last but not least”-project. Dr. Sanyaj, for your enthusiasm and chemistry suggestions. Antona Wag-staff, for excellent linguistic revision of this thesis. Linda Å, Drs Patrik, Rebecca, Eva and Anja for constructive criticism and comments on my thesis. Rebecca and Anna L, your open arms when I moved to Uppsala made me feel like home. You shared my “first-grey-hair”-moment and I will never forget our trip to San Diego; we are probably better scientists than surfers! Charlotta W, Linda A and Pernilla, for early on sharing your experience what PhD studies is about. Ulrika for good support and valuable insights. Per and Patrik, for your company in Boston and in New York and for crossing Manhattan carrying heavy back-packs in the dark.

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Gunnar, for making the equipment working with us and not against us. Your support is appreciated and the lab is empty on Wednesdays! My room-mates: Vivek, Suresh and Johanna for a nice atmosphere and for chats about chemistry and life. Karin, for kindly serving me coffee and cake during weekends of thesis-writing. Anna S and Linda Å for your open office door and for inspiring talks about life, society, and matters outside the life of chemistry. Hiba, for lunches sharing our thoughts. Sara and Rebecka, for all fun and nice lunch and coffee breaks! Bobo, for knocking on my closed office door just to make sure I was still alive during the last months of hard work. Gunilla, for taking such good care of OFK and for supportive company during late nights. Prof. Anders H, for spreading your genuine passion for medicinal chemistry. My mentor Dr Jenny Ekegren, for valuable conversations during the first part of my PhD studies. Former colleagues at Medivir who inspired me to take this journey! Some of you have remained good friends. Bertil Samuelsson, Åsa Rosenquist and Xiao-Xiong Zhou for accepting me at Medivir and guiding me in industrial medicinal chemistry but also supporting my inherent wish to complete a PhD. Nils-Gunnar Johansson for introducing me to the challenging world of nucleoside analogs. Per-Ola Johansson for excellent supervision during my master thesis work at Linköping University and for letting me synthesize my first protease inhibitors. Members of the Pharmaceutical PhD Student Council (FDR) 2010-2011, the Doc-toral Board 2010-2011 and the Academic Senate 2011-2012, for inspiring and inter-esting discussions and good work. The Swedish Academy of Pharmaceutical Sciences and Anna-Maria Lundins sti-pendiefond for making it possible to attend international conferences. Thanks to: Stiftelsen Hanna Roos af Hjelmsäters testamentsfond, stiftelsen Hierta af Petersens fond, Farmaceutiska fakultetens jubelfeststipendium, Cronhielms fond, Stiftelsen von Krusenstiernas fond, Pauli OG stiftelse and Belfrageska släktföreningen for recogni-tion and support during these years. Annika, vi delade underbara, roliga, galna och ibland slitsamma år i Stockholm. Alla våra samtal har varit och är ovärderliga! Robert, så fint att du kom in i Annikas liv och att vi har fått lära känna varandra. Ert paradis på Rindö har blivit ett härligt ställa att umgås på. Sofie, tänk att vi till slut hamnade i samma stad igen! Du är en så fin vän som tror på mig och min förmåga när jag själv tvivlar. Jag hoppas vi snart kan spela in vår nästa skiva; den senaste inspelningen har några år på nacken… David, vi tar en lunch-dejt snart för vi befinner ju oss löjligt nära varandra om dagarna! Ylva, jag önskar ofta att Uppsala låg på västkusten. Det var underbart att vara med dig och din fina familj i somras!

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Pia, Ulrika och Lotta med familjer, jag missade julgransplundringen i år, men fram-över kan ni lita på min närvaro! Lotta, det är så härligt att få hänga med dig och din Felix. Vi har följts åt på ett spe-ciellt sätt och har sett hur mirakel kan ske. Än är de inte över! Jan, Jörgen, Per och Anna, förra året blev det två maffiga 50-årskalas, och jag ser fram emot fler stora som små händelser att fira. Alltid fint att vara med er, vänner! Kerstin, vilken lyx att ha dig så nära! Du har en förmåga att förgylla med en bukett tulpaner, eller något litet ”smått och gott” och stöttar på så många sätt i vardagen! Ingvar, jag är så glad att vi hann lära känna varandra och jag hoppas att du hade gillat avhandlingen! Annika, Pelle, Jesper, Emrik, Astor och Mira, jag flyttade solo till Uppsala, och fick inom ett halvår en stor, extra-familj. Jag ser fram emot lite mer umgänge efter en lång ”upptagen” tid. Mina älskade bröder, Erik och Hans, vi får se till att träffas oftare för jag saknar er! Ni är så kloka på många sätt, och jag behöver lite syskonhäng snart! Jennyh, för att du finns. Din kämparanda är inspirerande! Mina fina, goa brorsbarn Emil och Jenni-fer, hoppas ni väljer att plugga i Uppsala om sådär 10-15 år, för jag vill se er oftare! Farmor, det känns lite ledsamt att du inte fick vara med in i mål! Du som inte hade möjlighet till studier som ung tyckte att en doktorsexamen var ett alldeles självklart val för mig! Mamma och Pappa, för att ni alltid har trott på mig och ställt upp när det har be-hövts, inte minst med all flytthjälp mellan olika städer och lägenheter genom åren. Det var fint att få fira en tidig jul med er här i Uppsala när den senaste julledigheten blev väldigt begränsad. Ser fram emot Strömstad i sommar med salta bad och fix-ande med huset. Johan♥! Du är en fantastisk person och en superfin pappa med enorm kapacitet. Du stöttar och tror på mig, och du är den förste jag frågar om råd – vad det än gäller! Hoppas jag kan fixa det där som kallas hemarbete snart för nu är det i jämställdhet-ens namn din tur att tänka på annat! Hedda☼! Du är en väldigt klok ”snart 3-åring” som börjat fundera över vad det är för spännande jag håller på med på jobbet eftersom ”det aldrig är stängt”! Kanske du en dag vill läsa den här boken och finna svaren på egen hand?... Älskar dig!

Anna Karin, 8 February 2015

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