chemical modifications for activity and property ...m044w353q/... · bachelor of science in...

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Chemical Modifications for Activity and Property Optimization: Drug Discovery Efforts for

Neglected Tropical Diseases

By Kelly A. Bachovchin

Bachelor of Science in Molecular Biology, Kean University

Master of Science in Biotechnology, Kean University

A dissertation submitted to

The Faculty of

The College of Science of

Northeastern University

in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

November 26th, 2019

Dissertation directed by

Michael P. Pollastri

Professor of Chemistry and Chemical Biology

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Acknowledgements

First and foremost, I would like to thank Northeastern University and the Department of

Chemistry and Chemical Biology for allowing me to pursue my doctoral studies. I would

particularly like to thank my committee members, Dr. Robert Hanson, Dr. Roman Manetsch, and

Dr. Leila Deravi for all your insight on this project.

I would like to acknowledge the hard work of our collaborators who, without their efforts,

none of this work would have been possible: Dr. Kojo Mensa-Wilmot, Dr. Miguel Navarro, Dr.

Rosario Diaz, Dr. Gloria Ceballos-Perez, Dr. Domingo Rojas-Barros, Dr. Maria Santos Martinez-

Martinez, Dr. Pilar Manzano, and Dr. Richard Sciotti. Thank you to AstraZeneca for providing all

the in vitro ADME data.

Thank you to my undergraduate advisor Professor James Robert Merritt for introducing

me to life in the lab and mentoring me through my master’s thesis. Without you I would have never

decided to pursue a PhD.

To my Pollastri Lab coworker and labmates, thank you to everyone who has given me

advice and helped me day to day along the way. A special thank you to Dr. Seema Bag who showed

me the ropes in my early days and Dr. Baljinder Singh who picked up the job in her place. To Dr.

Dana Klug for sharing your intermediates and all of the countless hours you spent maintaining the

LCMS. To Dr. Melissa Buskes and Dr. Quillon Simpson for editing and helping with the writing

of this dissertation. A very special thank you to Dr. Lori Ferrins for not only all your edits but for

being a pillar of support over the years, I never would have finished without you.

Thank you to all my “PL2” labmates for keeping the lab a pleasant space to be every day,

including Dr. Meaghan Fallano, Dr. Enrico Mongeau, Dr. Andrew Spaulding, Dr. Hitesh Jalani,

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Dr. Naresh Gunaganti, Dr. Alex Muthengi, and all the wonderful undergraduate researchers. I

would like to especially thank Mitch Rivers who always kept the conversation interesting and for

being the best undergrad mentee I could ask for.

A special thank you goes to my support system outside of Northeastern. To my family,

Mom, Dad, Julie, Cory, and Hailey who supported my decisions even when they took me hundreds

of miles away, who tolerated my absence and stood by me throughout the years, and whose love

and support I’ve cherished for so many years. To Eliza Miller who I started day one with at

Northeastern, and who believed in me and helped me believe in myself even from thousands of

miles away. To my salsa family, especially Andres, Hernan, Amy, Limar, Amelia, Maureen, Celia,

and Beatriz, who have become my family away from home, and given me a community to escape

to when times were hard in the lab.

I want to extend my deepest gratitude to my advisor, Professor Michael Pollastri. Thank

you for always having a box of tissues handy. Most importantly, thank you for believing I could

finish and for all of your help, guidance, and understanding along the way.

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Abstract of Dissertation

Neglected tropical diseases (NTDs) affect the lives of billions of people across the globe,

and disproportionately affect the most poverty-stricken populations. Current chemotherapeutics

present many challenges with difficult treatment regimens and toxic side effects prevalent;

resistance has also been observed for many. It has been shown that repurposing chemical matter

for use in NTD drug development is a successful strategy to discover new drugs, accelerating the

drug discovery process, and requiring less monetary investment. To this end, we have employed

target-class and lead repurposing strategies using human kinase inhibitors to identify inhibitors of

four parasitic diseases: human African trypanosomiasis (HAT, caused by Trypanosoma brucei),

Chagas disease (T. cruzi), leishmaniasis (Leishmania donovani), and malaria (Plasmodium

falciparum).

The first project outlined in this dissertation describes the process of repurposing the drug

lapatinib and subsequent optimizations that led to advanced hits for T. brucei inhibition. Through

the application of known medicinal chemistry strategies we have successfully modulated the

aqueous solubility of the series. This has led to new, potent compounds that inhibit multiple

parasites besides T. brucei, including T. cruzi, L. donovani, and P. falciparum, each with their own

structure-activity relationship optimization pathways.

The second project involves the prioritization of a cluster of 2,4-substituted azaindoles

from a high-throughput screen (HTS) of a kinase-targeted inhibitor library against T. brucei. The

hit compound from this cluster, NEU-1200, showed high in vivo clearance rates. Strategic

modifications were made to NEU-1200 to reduce the clearance and these modifications led to

NEU-5123 which showed improved in vivo clearance rates and good exposure in mice brains, an

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important factor for treating stage II of HAT. NEU-5123 was tested in an acute efficacy model for

HAT and showed an extension of life of 8.8 days when dosed at 10 mg/kg/day, however at higher

doses was highly toxic. Toxicity of this series will be addressed in future work.

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Table of Contents

Acknowledgements ......................................................................................................................... 2

Abstract of Dissertation .................................................................................................................. 4

Table of Contents ............................................................................................................................ 6

List of Figures ............................................................................................................................... 10

List of Schemes ............................................................................................................................. 13

List of Tables ................................................................................................................................ 15

List of Abbreviations .................................................................................................................... 18

Chapter 1: Background and Introduction ................................................................................ 21

1.1 Neglected tropical diseases (NTDs)........................................................................................ 21

1.1.1 Human African Trypanosomiasis (HAT) ............................................................................ 23

1.1.2 Other parasitic diseases ........................................................................................................ 24

1.1.2.1 Chagas disease .................................................................................................................. 25

1.1.2.2 Leishmaniasis .................................................................................................................... 26

1.1.2.3 Malaria .............................................................................................................................. 28

1.2 Current treatments for NTDs .................................................................................................. 29

1.2.1 Current treatments for HAT ................................................................................................. 29

1.2.2 Current treatments for Chagas disease ................................................................................. 31

1.2.3 Current treatments for leishmaniasis .................................................................................... 32

1.2.4 Current treatments for malaria ............................................................................................. 33

1.3 NTD drug development .......................................................................................................... 34

1.3.1 Repurposing chemical matter ............................................................................................... 35

1.3.1.1 Drug repurposing .............................................................................................................. 36

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1.3.1.2 Target repurposing ............................................................................................................ 41

1.3.1.3 Target-class repurposing ................................................................................................... 44

1.3.1.4 Lead repurposing ............................................................................................................... 49

1.4 Compound optimization .......................................................................................................... 52

Chapter 2: Property optimization of lapatinib derived analogs for human African

trypanosomiasis (HAT) drug discovery .................................................................................... 55

2.1 Introduction ............................................................................................................................. 55

2.1.1 Repurposing lapatinib for Trypanosoma brucei inhibition .................................................. 55

2.1.2 Identification of NEU-1912 and NEU-1953 as leads for drug development ....................... 57

2.1.3 Methods for improving aqueous solubility .......................................................................... 61

2.1.3.1 Salt formation.................................................................................................................... 62

2.2 Salt screens.............................................................................................................................. 65

2.2.1 Salt formations of NEU-1953 .............................................................................................. 65

2.2.1.1 Evaluation of NEU-1953 salt forms ................................................................................. 66

2.2.2 Salt screen of NEU-1912 ..................................................................................................... 67

2.2.2.1 Synthesis of NEU-1912 .................................................................................................... 67

2.2.2.2 Salt formations of NEU-1912 ........................................................................................... 69

2.2.2.3 Evaluation of NEU-1912 salt forms ................................................................................. 69

2.3 Increasing Fsp3: Design, synthesis, and evaluation of NEU-1912 and NEU-1953 matched

pairs with saturated head groups ................................................................................................... 70

2.3.1 Selection of saturated primary amines as replacements for aromatic head groups of NEU-

1912 and NEU-1953 ..................................................................................................................... 70

2.3.2 Synthesis of saturated head group replacements, NEU-1953 scaffold ................................ 71

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2.3.3 Synthesis of saturated head group replacements, NEU-1912 scaffold ................................ 75

2.3.4 Evaluation of saturated head group replacements, NEU-1912 and NEU-1953 scaffolds and

matched pairs ................................................................................................................................ 76

2.4 Crossover compounds between NEU-4363 and NEU-1912 ................................................... 79

2.4.1 Synthesis of crossovers ........................................................................................................ 80

2.4.2 Evaluation of crossovers ...................................................................................................... 82

2.5 Evaluation of lapatinib analogs against other parasites .......................................................... 84

2.5.1 Evaluation of lapatinib analogs against Trypanosoma cruzi ............................................... 84

2.5.2 Evaluation of lapatinib analogs against Leishmania ............................................................ 85

2.5.3 Evaluation of lapatinib analogs against P. falciparum ........................................................ 86

2.6 Summary ................................................................................................................................. 87

Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT

drug discovery ............................................................................................................................. 91

3.1 Introduction ............................................................................................................................. 91

3.1.1 High-throughput screen of a kinase targeted library against T. brucei in collaboration with

GSK............................................................................................................................................... 91

3.1.2 Organization of HTS hits and identification of NEU-1200 as a potent inhibitor of T. brucei

growth ........................................................................................................................................... 92

3.2.3 In vivo pharmacokinetic (PK) evaluation of NEU-1200...................................................... 96

3.2 Design, synthesis, and evaluation of NEU-1200 first-generation analogs ............................. 97

3.2.1 Rationale of NEU-1200 first-generation analog design ....................................................... 97

3.2.2 Synthesis of NEU-1200 and first-generation analogs .......................................................... 99

3.2.3 Evaluation of NEU-1200 first-generation analogs ............................................................ 107

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3.2.4 Pharmacokinetic analysis of NEU-5123 ............................................................................ 118

3.2.5 In vivo evaluation of NEU-5123 ........................................................................................ 119

3.2.6 Kinase selectivity evaluation of NEU-5123 and NEU-5449 ............................................. 121

3.3 Design, synthesis, and evaluation of NEU-1200 second-generation analogs ....................... 122

3.3.1 Rationale of NEU-1200 second-generation analog design ................................................ 122

3.3.2 Synthesis of NEU-1200 second-generation analogs .......................................................... 123

3.3.3 Evaluation of NEU-1200 second-generation analogs ........................................................ 124

3.4 Evaluation of cluster compounds against other parasites ..................................................... 128

3.5 Summary ............................................................................................................................... 131

3.6 Future Directions .................................................................................................................. 134

References ................................................................................................................................... 135

Chapter 4: Experimental section ............................................................................................. 149

4.1 General methods ................................................................................................................... 149

4.2 Experimental details.............................................................................................................. 150

4.2.1 Experimental procedures for Chapter 2 ............................................................................. 150

4.2.2 Experimental procedures for Chapter 3 ............................................................................. 187

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List of Figures


Figure 1-1. Life cycle of Trypanosoma brucei. ............................................................................ 24

Figure 1-2. Life cycle of Trypansoma cruzi. ................................................................................ 26

Figure 1-3. Life cycle of Leishmania. ........................................................................................... 27

Figure 1-4. Life cycle of Plasmodium falciparum. ....................................................................... 28

Figure 1-5. Current treatments for HAT. ...................................................................................... 30

Figure 1-6. Drugs in clinical trials for HAT. ................................................................................ 31

Figure 1-7. Current treatments for Chagas disease. ...................................................................... 32

Figure 1-8. Current treatments for leishmaniasis. ......................................................................... 33

Figure 1-9. Current treatments for malaria. .................................................................................. 34

Figure 1-10. The drug development pipeline and different entry points for repurposing

campaigns. .................................................................................................................................... 35

Figure 1-11. Categorization cascade of the different types of repurposing strategies. ................. 36

Figure 1-12. FDA-approved drugs identified as active against T. b. rhodesiense. ....................... 38

Figure 1-13. FDA-approved drugs identified as active against T. cruzi and that could serve as

starting points for further optimization to mitigate toxicity.......................................................... 39

Figure 1-14. FDA-approved drugs shown to inhibit TcPAT12. ................................................... 40

Figure 1-15. Antifungal azoles repurposed for Chagas disease and used in combination with

DB766 to enhance killing of L. donovani. .................................................................................... 41

Figure 1-16. Progression of the diaminothiazole hit to GSK3186899/DDD853651. ................... 43

Figure 1- 17. Human PDE inhibitors that entered medicinal chemistry optimization for T. brucei.

....................................................................................................................................................... 44

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Figure 1-18. Hit to lead compounds resulting from target-guided SAR explorations of

TbrPDEB1..................................................................................................................................... 44

Figure 1-19. The HDAC inhibitor, vorinostat, shows promising in vivo efficacy against L.

donovani when absorbed by gold nanoparticles ........................................................................... 45

Figure 1-20. Anticancer drugs shown to moderately reduce parasitemia in the livers of mice

infected with L. donovani ............................................................................................................. 47

Figure 1-21. Structure of lapatinib-derived analog NEU-4438. ................................................... 48

Figure 1-22. Structures of delaminid and its optimized analogs. ................................................. 49

Figure 1-23. Isatinoid compound NEU-4391 identified from a lead repurposing HTS campaign.

....................................................................................................................................................... 50

Figure 1-24. Most active compound (56) from a kinase library screen and optimized compound

(57) for T. brucei growth inhibition. ............................................................................................. 51



Figure 2-1. Optimizations of EGFR inhibitor lapatinib to lead compound NEU-617.................. 56

Figure 2-2. Summary of modifications made to NEU-1045. ....................................................... 58

Figure 2-3. Modifications to NEU-961 resulting in NEU-1953. .................................................. 59

Figure 2-4. Comparison of lipophilicity versus aqueous solubility in first and second generation

lapatinib analogs. .......................................................................................................................... 61

Figure 2-5. Clinical candidate AMG-517 and its analog with improved aqueous solubility. ...... 64

Figure 2-6. Comparison of parent compound NEU-1953 and analog NEU-4363. ...................... 80

Figure 2-7. Summary of modifications made to parent compounds NEU-1912 and NEU-1953. 88

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drug discovery ............................................................................................................................. 91

Figure 3-1. Predicted metabolically labile sites of NEU-1200 ..................................................... 98

Figure 3-2. Summary of analog design plans. .............................................................................. 99

Figure 3-3. Crystal structure of 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b] pyridine. .............. 103

Figure 3-4. Peripheral blood levels of NEU-5123 in mice. ........................................................ 119

Figure 3-5. Efficacy of NEU-5123 in mice after a 10 mg/kg/day dose. ..................................... 120

Figure 3-6. Efficacy of NEU-5123 in mice after a 30 mg/kg/day dose. ..................................... 121

Figure 3-7. Human kinase profiles of NEU-5123 and NEU-5449. ............................................ 122

Figure 3-8. Graph of multiple cell line toxicity values of all synthesized NEU-1200. .............. 128

Figure 3-9. Correlation of T. cruzi potency and L6 host cell toxicity. ....................................... 130

Figure 3-10. SAR/SPR summary for synthesized NEU-1200 analogs. ...................................... 132

Figure 3-11. Compound progressions of the series. ................................................................... 133

Figure 3-12. Summary of future directions for analog design. ................................................... 134

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List of Schemes



Scheme 2-1. Synthesis of 6-bromoquinolone core ....................................................................... 67

Scheme 2-2. Synthetic route for NEU-1912 tail group................................................................. 68

Scheme 2-3. Synthesis of NEU-1912. .......................................................................................... 68

Scheme 2-4. Synthetic route for NEU-1953 tail group................................................................. 71

Scheme 2-5. Saturated head group matched pairs to NEU-1953 template synthesis. .................. 72

Scheme 2-6. Synthetic attempts via SNAr conditions using NEU-1953 7-bromo-4-

chloroquinoline core for intermediate to final compounds. .......................................................... 75

Scheme 2-7. Synthetic route for various head group analogs. ...................................................... 76

Scheme 2-8. Synthesis of 1-methylhomopiperazine tail group .................................................... 81

Scheme 2-9. Synthesis of 6-position substituted matched pairs of NEU-1912 and NEU-4363. .. 81

Scheme 2-10. Synthesis of 7-position substituted matched pair of NEU-1912 and NEU-4363. . 82


drug discovery ............................................................................................................................. 91

Scheme 3-1. Synthesis of intermediates 3.2a-c. ......................................................................... 100

Scheme 3-2. Synthesis of analogs truncated at the 2-position. ................................................... 100

Scheme 3-3. Synthesis of boronate coupling partners 3.7a,b. .................................................... 101

Scheme 3-4. Synthesis of analogs truncated at the 4-position. ................................................... 102

Scheme 3-5. Initial synthesis of analogs with modifications at the 4-position. .......................... 103

Scheme 3-6. Modified synthesis of compounds with 4-position modifications. ........................ 104

Scheme 3-7. Attempted synthesis of analogs with modifications at the 2-position. .................. 105

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Scheme 3-8. First successful synthesis of analogs with modifications at the 2-position. .......... 106

Scheme 3-9. Modified synthesis of analogs with modifications at the 2-position. .................... 107

Scheme 3-10. Synthesis of second-generation NEU-1200 analogs. ........................................... 124

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List of Tables


Table 1-1. The 20 neglected tropical diseases as defined by the World Health Organization ..... 21

Table 1-2. Targeted compound criteria for hit and lead compounds. ........................................... 54



Table 2-1. Comparison of NEU-1045 to NEU-1912 and NEU-961 to NEU-1953. ..................... 58

Table 2-2. Determination of approximate solubility of NEU-1953 in various organic solvents. . 65

Table 2-3. Salts formed of NEU-1953 compared to the free base. ............................................... 66

Table 2-4. Conditions for salt formations of NEU-1912. ............................................................. 69

Table 2-5. Salts formed of NEU-1912 compared to the free base. ............................................... 70

Table 2-6. Synthetic attempts via Buchwald-Hartwig cross coupling using NEU-1953

chlorinated core + tail for final compound synthesis. ................................................................... 73

Table 2-7. Synthetic attempts via SNAr conditions using NEU-1953 chlorinated core + tail for

final compound synthesis. ............................................................................................................. 74

Table 2-8. Saturated head group analogs of NEU-1953, T. brucei activities and physicochemical

properties....................................................................................................................................... 78


properties....................................................................................................................................... 79

Table 2-10. Evaluation of NEU-1912 and NEU-4363 matched pairs. ......................................... 83

Table 2-11. Evaluation of lapatinib analogs against Trypanosoma cruzi and T. cruzi host cells

(C2C12)......................................................................................................................................... 85

Table 2-12. Evaluation of lapatinib analogs against Leishmania and B10R host cells. ............... 86

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Table 2-13. Evaluation of lapatinib analogs against common strains of P. falciparum. .............. 87


drug discovery ............................................................................................................................. 91

Table 3-1. Compound properties for a cluster of 2,4-substituted 1H-pyrrolo[2,3b]pyridines and

selected compound NEU-1200. .................................................................................................... 93

Table 3-2. Multi-parameter optimization (MPO) scoring for predicting CNS exposure. ............ 93

Table 3-3. Subset of cluster 32 compounds from initial HTS typified by a 2,4-substituted

pyrrolo[2,3-b]pyridine core........................................................................................................... 95

Table 3-4. PK evaluation of NEU-1200 following IV administration. ......................................... 97

Table 3-5. PK evaluation of NEU-1200 following IP administration. ......................................... 97

Table 3-6. Activity of truncated analogs of NEU-1200 against T. brucei. ................................. 108

Table 3-7. Activities and clearance rates of 4-position variant first-generation analogs of NEU-

1200............................................................................................................................................. 110


1200............................................................................................................................................. 112

Table 3-9. Other ADME measurements of NEU-1200 and its first-generation analogs. ........... 116

Table 3-10. Toxicity of NEU-1200 and its first-generation analogs against HepG2 and MRC5

cell lines. ..................................................................................................................................... 117

Table 3-11. Individual blood pharmacokinetic parameters of evaluated compounds after

intraperitoneal administration of 10 mg/kg single dose (target dose) to female NMRI mice (n=3).

..................................................................................................................................................... 118

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Table 3-12. Blood and brain levels (ng/mL in blood, ng/g in brain) of NEU-5123, after


LLOQ=50 ng/mL (blood). .......................................................................................................... 119

Table 3-13. Activities and clearance rates of second-generation analogs of NEU-1200. .......... 125

Table 3-14. Toxicity of second-generation NEU-1200 analogs against HepG2 and MRC5 cell

lines. ............................................................................................................................................ 127

Table 3-15. Potencies and toxicities of NEU-1200 analogs against T. cruzi and Leishmania. .. 129

Table 3-16. Potencies of NEU-1200 analogs against various P. falciparum strains. ................. 131

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List of Abbreviations

Ac Acetyl

ACT Artemisinin-combination therapy

ADME Absorption, distribution, metabolism, excretion

Aq Sol Aqueous solubility

ATP Adenosine triphosphate

AUC Area under the curve

BCS Biopharmaceutical classification system

CDC Centers for disease control

CL Cutaneous leishmaniasis

cLogP Calculated partition coefficient

Cmax Maximum concentration

CNS Central nervous system

CYP51 lanosterol 14α-demethylase

DALY Disability-adjusted life year

DCE Dichloroethane

DCM Dichloromethane

DIPEA Diisopropylethylamine

DNA Deoxyribonucleic acid

DNDi Drugs for neglected diseases initiative

EC50 Half maximal effective concentration

EGFR Epidermal growth factor receptor

eq equivalent

FDA Food and drug administration

Fsp3 Fraction of sp3 hybridized carbon atoms

GI Gastrointestinal

GPCR G-protein coupled receptor

GSK GlaxoSmithKline

GSK3β Glycogen synthase kinase-3 beta

HAT Human African trypanosomiasis

HBD Hydrogen bond donor

HDAC Histone deacetylase

hERG human Ether-a-go-go-Related Gene

HLM Clint Human liver microsomal intrinsic clearance rate

HTS Hight-throughput screen

IP Intraperitoneal

IPA Isopropanol

IRK Insulin receptor tyrosine kinase

IV Intravenous

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KOAc Potassium acetate

Lck Lymphocyte-specific protein tyrosine kinase

LCMS Liquid chromatography mass spectrometry

LDA Lithium diisopropylamide

LE Ligand efficiency

LLE Lipophilic ligand efficiency

M2 Muscarinic receptor 2

M3 Muscarinic receptor 3

MPO Multi-parameter optimization

MTD Maximum tolerated dose

mTOR Mammalian target of rapamycin

MW Molecular weight

NaOH Sodium hydroxide

NECT Nifurtimox eflornithine combination therapy

NIH National institutes of health

NMP N-methylpyrrolidone

NTD Neglected tropical disease

PAT12 Polyamine transporter 12

PdCl2(dppf)·CH2Cl2

[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II),

complex with dichloromethane

PDE Phosphodiesterase

PDE4 Phosphodiesterase 4

PDEB1 Phosphodiesterase B1

PDEB2 Phosphodiesterase B2

PDGFR Platelet-derived growth factor receptor

PI3K Phosphoinositide kinase-3

PK Pharmacokinetic

PPB Plasma protein binding

RH Clint Rat hepatocyte intrinsic clearance rate

SAR structure-activity relationship

SNAr Nucleophilic aromatic substitution

SPR Structure-property relationship

Src Proto-oncogene tyrosine-protein kinase

t1/2 Time at half the maximum concentration

tBu tert-butyl

tBuOH tert-butanol

tBuOK Potassium tert-butoxide

tbuXPhos 2-Di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl

TC50 Half maximal toxic concentration

THF Tetrahydrofuran

tmax Time at maximum concentration

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TPSA Topological polar surface area

UV Ultraviolet

VEGFR Vascular endothelial growth factor receptor

VL Visceral leishmaniasis

WHO World health organization

Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene

XPhos 2-Dicyclohexylphosphino-2',4',5'-triisopropylbiphenyl

μwave Microwave

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Chapter 1: Background and Introduction

1.1 Neglected tropical diseases (NTDs)

The World Health Organization (WHO) defines the 20 neglected tropical diseases (NTDs)

as communicable diseases prevalent throughout the tropical regions of the world (Table 1-1).

These diseases affect over 1 billion people in 149 countries, and disproportionately affect

impoverished societies, costing their developing economies billions of dollars.1-2 A more

comprehensive assessment of the burden of these diseases is identified by the number of disability-

adjusted life years (DALYs) lost; DALYs are the sum of years of life lost due to premature death

or disability.3 NTDs are commonly caused by parasitic infections, and while some infections can

result in death, it is the chronic, debilitating nature of most of these diseases that accounted for

26.06 million DALYs in 2010.4

Table 1-1. The 20 neglected tropical diseases as defined by the World Health Organization1

Disease Category of infection

• Chagas disease

• Human African trypanosomiasis

• Leishmaniasis

Protozoan

• Dracunculiasis

• Echinococcosis

• Foodborne trematodiases

• Lymphatic filariasis

• Onchocerciasis

• Schistosomiasis

Helminth

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• Soil-transmitted helminthiases

• Taeniasis/Cysticercosis

• Buruli ulcer

• Leprosy

• Trachoma

• Yaws

Bacterial

• Dengue and Chikungunya

• Rabies

Viral

• Mycetoma, chromoblastomycosis and

other deep mycoses

Fungal

• Scabies and other ectoparasites Ectoparasitic

• Snakebite envenoming Venom

There are treatments available for NTDs, but their adverse side effects, poor dosing

regimens, and accessibility to the populations that need them make them non-ideal. Areas of

infection tend to be remote rural or suburban slums where the average person lives on less than

$2.00 US per day.3 Low to no return on investments have caused the pharmaceutical industry to

shy away from neglected disease discovery programs. New treatments are therefore needed that

are safe, orally bioavailable, and have low costs to produce. A target product profile for each of

these diseases was identified by the WHO and Drugs for Neglected Diseases initiative (DNDi) and

will be discussed later in this chapter. With the help of academic institutions and non-profit

organizations in partnership with industry, progress has been made toward eradicating these

diseases.2

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1.1.1 Human African Trypanosomiasis (HAT)

Human African trypanosomiasis (HAT) is an endemic disease in 36 sub-Saharan African

countries. In 2009, continued efforts to eliminate this disease by the year 2020 brought the number

of reported cases below 10,000 for the first time in 50 years. In 2017, there were 1,446 new cases

reported.5 However, poor healthcare infrastructure, indistinguishable symptoms, and co-infections

of other disease cause the number of cases to be underreported.

The disease is caused by an infection of the protozoan parasite Trypanosoma brucei. There

are two subspecies of T. brucei that are infective to humans. T. b. gambiense is predominantly

found in western sub-Saharan Africa and is the cause for 98% of reported cases, where T. b.

rhodesiense is predominantly found in the eastern region. An infection of T. b. gambiense causes

a chronic form of HAT that can take months to years to progress to stage 2, and T. b. rhodesiense

causes an acute version which can progress to stage 2 within a few weeks.6 Both forms of the

disease are fatal if left untreated.

T. brucei belong to the order Kinetoplastida, characterized by the presence of densely

packed DNA called a kinetoplast which can be found in the mitochondria.7 This order is also

characterized by the presence of a flagella, a cellular structure most commonly studied to

determine in which stage of reproduction a parasite may be.7-8 Trypanosomatids characteristically

have four main developmental stages they progress through during proliferation and transmission

between hosts.

Infection begins when a metacyclic trypomastigote enters the blood stream of a host after

the bite from an infected tsetse fly vector (genus Glossina). The hemolymphatic, first stage of

infection begins here as the parasite proliferates generating feverish responses and flu-like

symptoms (Figure 1-1). Antibodies begin to be made by the hosts immune system that eliminates

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most of the parasites present, however some parasites evade this destruction and survive.9-10 Other

symptoms include headache, weakness, and joint stiffness.6 Eventually, the parasites will invade

the central nervous system (CNS), including the brain, thus starting stage 2 of the disease. The

beginning of stage 2 is dependent on the subspecies of infection. More severe symptoms are

experienced during this stage including sleep pattern disruptions, coma, and eventually death.

Figure 1-1. Life cycle of Trypanosoma brucei.

Source: Public Health Image Library, provided by CDC-DPDx, Alexander J. da Silva and

Melanie Mosier. http://phil.cdc.gov/phil/details.asp?pid=3418

1.1.2 Other parasitic diseases

Though the work in the following chapters focuses on developing treatments for HAT,

compounds synthesized were also screened for their potential use against other diseases using a

parasite hopping approach. These other diseases are also caused by protozoan parasites and include

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Chagas disease, Leishmaniasis, and malaria. Structure-activity optimizations are not performed

specifically for the parasites that cause these diseases, but if cross-screening resulted in interesting

results new potential projects could arise from these data.

1.1.2.1 Chagas disease

Chagas disease is caused by a related kinetoplastid parasite, T. cruzi. There are 65-100

million people are at risk of infection in Mexico, Central, and South America.11 Though the number

of reported cases has dropped from 16-18 million to 6-7 million because of vector control and

blood transfusion programs, there are still 12,000 annual deaths accounted for from this disease.11

There are also about 300,000 documented cases of Chagas disease in the United States.12 Hosts

are most commonly infected after having their blood come into contact with the feces of the

triatomine bug vector. The life cycle of T. cruzi can be seen in Figure 1-2, which coincides with

the two clinically relevant stages of the disease.

First, triatomine bugs become infected with the non-dividing infective trypomastigote form

of the parasite after biting an infected host. The trypomastigotes transform into epimastigotes in

the triatome bug’s midgut. The parasites then develop into metacyclic trypomastigotes which are

excreted through the bug’s feces onto the skin of a human host who will be self-infected after

scratching the skin at the site of the feces, allowing the parasites to enter the blood stream. Thus

begins the first clinical stage of the disease, an acute form where mild symptoms can be

experienced including redness and swelling at the infection site and mild fever.9

Parasites will then go on to invade the human host cells, where they transform into non-

infective amastigotes which multiply by binary fission. Amastigotes develop into trypomastigotes

that can go on to infect other human host cells. Being an intracellular infection is a key factor in

26

the difficulty of killing these parasites; a drug would need to overcome another barrier, the host

cell membrane. It is at this point that patients enter a chronic, asymptomatic form of this disease.

An estimated 70-80% of infected people will live asymptomatically for the rest of their life without

any detectable parasites in their blood stream. The remaining 20-30% of infected people will

experience heart rhythm abnormalities resulting in sudden death, or a dilated heart, esophagus, or

colon resulting in related complications.9

Figure 1-2. Life cycle of Trypansoma cruzi.



1.1.2.2 Leishmaniasis

Leishmaniasis is another disease caused by a kinetoplastid containing trypanosomatid.10

There are 21 of 30 species of the Leishmania parasite that are human infective.13 The human

infective species include the L. donovani complex (L. donovani, L. infantum, L. chagasi), L.

27

mexicana complex (L. mexicana, L. amazonensis, L. venezuelensis), the subgenus Vianna (L. [V.]

brazielensis, L. [V.] guyanensis, L. [V.] panamensis, L. [V.] peruviana), and L. tropica, L. major,

and L. aethiopica.10 This disease can be found in 88 countries throughout the Americas and some

parts of Asia, putting about 350 million people at risk for infection.13

Figure 1-3. Life cycle of Leishmania.



Infections begin after a person is bitten by an infected female phlebotomine sandfly. The

life cycle of Leishmania can be seen in Figure 1-3. The infective promastigote form of the parasite

becomes phagocytized by host cells where they transform into their dividing amastigote form. The

type of disease that results from infection is dependent on the host, and species of parasite. There

are two major forms of the disease cutaneous (CL) and visceral leishmaniasis (VL). Symptoms of

CL include skin lesions that resemble volcanoes, raised edges with craters in the middle, at the site

http://phil.cdc.gov/phil/details.asp?pid=3400

28

of the sandfly bite.14 VL symptoms include fever, weight loss, enlarged spleen, and low blood cell

counts, and eventually leads to death.13

1.1.2.3 Malaria

Malaria is another disease cause by a protozoan parasite and is the leading cause of illness

and death in the poor, tropical regions of the world.14 Though it is prevalent in tropical regions, it

is not deemed neglected by the WHO because of the large effort dedicated to combat this disease.

Malaria control efforts have greatly increased in the last decade, yet in 2016 an estimated 445,000

people died of malaria, the major victims being young children in sub-Saharan Africa.15 Young

children and pregnant women are at the highest risk of infection due to underdeveloped, and

decreased immunity to the disease, respectively.15

Figure 1-4. Life cycle of Plasmodium falciparum.



29

Plasmodium falciparum, the most common species of the parasite, are transmitted to their

human hosts by the Anopheles mosquito.16 The life cycle for P. falciparum can be seen in Figure

1-4.16 Sporozoites are injected into the hosts blood stream causing flu-like symptoms, the other

stage to this disease is the infection of the liver where parasites can remain dormant for years and

can lead to relapse even after the parasites have been cleared from the blood stream. Depending

on the tissues and organs the parasites invade, severe malaria disease accompanied by more severe

symptoms can be observed. Other Plasmodium species include P. vivax, P. ovale, and P. malariae

which are the species that cause recurring malaria and can stay dormant in the liver, the most

predominantly spread being P. vivax.16

1.2 Current treatments for NTDs

There are no vaccines currently for any of the NTDs. These diseases are managed mainly

through vector control and chemotherapeutics.6, 17-20 Current treatments are limited mainly due to

toxic side effects for patients and grueling dosing regimens.

1.2.1 Current treatments for HAT

Treatments for HAT are specific to the stage the disease is in as well as the subspecies

causing the infection. There are two treatments for stage 1 of the disease (shown in Figure 1-5),

pentamidine and suramin, though the disease has low rates of diagnosis in stage 1 due to the

mildness of symptoms and inaccessibility to clinics. Pentamidine is only used to treat T. b.

gambiense infections and suramin is only effective against T. b. rhodesiense infections.

30

Figure 1-5. Current treatments for HAT.

Eflornithine, originally developed as an anticancer treatment, was a repurposed drug for

treating stage 2 T. b. gambiense infections and is far less toxic than the arsenic based drug,

melarsoprol. Eflornithine was originally used as an intravenous (IV) monotherapy,21 but showed

31

toxicity and evidence of resistance.22-23 With the approval of nifurtimox in 2009, NECT became

the first in class treatment for stage 2; the co-administration of nifurtimox with eflornithine reduces

the amount of eflornithine that needs to be injected into the patient for treatment by 50%.23

However, NECT still requires multiple doses making it logistically challenging to administer in

areas of sub-Saharan Africa. Its position as the first line treatment for stage 2 is expected to change

with the introduction of the oral drug fexinidazole (6), which was recently approved for use by the

European Medical Agency.24-25

Fexinidazole is taken orally once daily for 10 days, and, although somewhat less effective

than the first-line NECT (91.2% vs 97.6% effective, respectively),26-27 fexinidazole is an overall

improvement given that it is an oral drug with shorter treatment course. Another orally available

drug, acoziborole (7, Figure 1-6) is in Phase II/III clinicals trials for treatment of HAT.28

Figure 1-6. Drugs in clinical trials for HAT.

1.2.2 Current treatments for Chagas disease

There are currently only 2 drugs for the treatment of Chagas disease, nifurtimox (3) and

benznidazole (8) (Figure 1-7). Both drugs are orally bioavailable but are more effective on

parasites that have not yet invaded cells and thus are the first in line treatments for the acute phase

of the disease. These treatments are also used to treat the chronic phase (once T. cruzi has invaded

host cells) though they are not as effective and a more rigorous dosing regimen is needed.29-30

32

Benznidazole is more readily used as a treatment for Chagas disease because it has fewer side

effects than nifurtimox.31 Unfortunately, there have been signs of resistance from certain strains

of T. cruzi against both of these treatments.32

Figure 1-7. Current treatments for Chagas disease.

1.2.3 Current treatments for leishmaniasis

Multiple factors must be taken into consideration when treating leishmaniasis, making it

particularly difficult to treat. These factors include the type of infection, availability of the drug,

age of the patient, and whether the patient is pregnant or nursing.20, 33 The first in line treatments

for CL (and alternatives for VL) are injectable pentavalent antimonials pentostam (9) and

meglumine antimoniate (10) (Figure 1-8).18, 20 One of the most common side effects of these drugs

is a change in electrocardiograms of about 50% of patients.18, 34 The 3 other treatments for

leishmaniasis are amphotericin B (11), miltefosine (12), and paromomycin (13) that were

repurposed for their current indications (see 1.3.1 Repurposing chemical matter) (Figure 1-8).

Amphotericin B is a treatment that require intravenous dosing and has a high cost per treatment.18

Miltefosine, the first orally available treatment for leishmaniasis displays toxic side effects.18

Lastly, paromomycin (13) is administered by intramuscular injection to treat VL18 or can be

applied topically as a treatment for CL due to is poor absorption across the GI tract.35

33

Figure 1-8. Current treatments for leishmaniasis.

1.2.4 Current treatments for malaria

The current treatments used to treat uncomplicated malaria are combination therapies of

artemisinin (14) and other antimalarial drugs such as amodiaquine (15), lumefantrine (16),

mefloquine (17), and sulfadoxine(18)/pyrimethamine (19) (Figure 1-9).36-37 These artemisinin-

combination therapies (ACTs) are 90% effective in patients.37-38 Infections caused by P. vivax

34

require treatments to be effective in both blood and liver. P. vivax infections are mainly treated by

using ACT plus primaquine (20) to clear the liver stage parasites.37

One of the biggest challenges to treating malaria is the rapidly evolving strains of these

parasites.36 Partial resistance to artemisinins emerged in southeast Asia in the 2000s causing drug

resistant strains of the parasite.39-41 Chloroquine(21)-resistant P. falciparum has also spread to

most disease prevalent areas.41

Figure 1-9. Current treatments for malaria.

1.3 NTD drug development

The most cost and time efficient methods need to be utilized to develop new treatments for

NTDs. Repurposing chemical matter is one way to cut down on both cost and time. Compounds

are also cross-screened against the various aforementioned parasites due to the organisms’

35

biological similarities.42 Cross-screening can provide new starting points for compound

development against different pathogens, and is a form of repurposing.

1.3.1 Repurposing chemical matter

In classic drug development programs the time between bringing concept to production

takes on average 10-12 years and $1.8 billion.43 The length of stages (Figure 1-10) in the drug

discovery process can vary based on the project and challenges encountered. Late stage failures of

discovery programs can occur, and drug attrition rates are reportedly high.44 NTD drug

development is especially daunting because of the low return on investment when a drug goes to

market. These factors lower the incentive for NTD development by for-profit organizations.

Figure 1-10. The drug development pipeline and different entry points for repurposing

campaigns.

One way to enter the development pipeline later and with less money invested, is through

repurposing chemical matter and utilizing prior knowledge of developed drugs and drug targets.

Repurposing has become an established strategy in NTD drug discovery and is discussed below.

There are four different types of repurposing: drug, target, target-class, and lead repurposing.45 As

shown in Figure 1-10 repurposing allows for different entry points along the development process

ultimately abbreviating the timeline. The type of repurposing strategy utilized in a project depends

36

on the source of the chemical matter and the way in which compounds are optimized. The

categorization of repurposing strategies is summarized in Figure 1-11.

Figure 1-11. Categorization cascade of the different types of repurposing strategies.

The application of repurposing can be powerful due to the often, large knowledge base that

is already in the public domain and that can help illuminate a path along which the desired

selectivity and ADME profiles may be achieved. This can be helpful given the resource-limited

nature of NTD drug discovery programs. Specific examples of each of the repurposing strategies

are discussed herein.

1.3.1.1 Drug repurposing

The most direct and streamlined approach to repurposing is drug repurposing, as it

repositions an already approved drug for a new indication. Directly repurposed drugs do not

require any further chemical optimization and can be tested in Phase II clinical trials (Figure 1-

10). The understanding is that a repurposed approved drug has already been evaluated for safety

and bioavailability. Later stage clinical trials are used to assess the drugs ability to be effective for

37

the new indication. Drug repurposing has a proven track record of identifying successful

treatments for NTDs, including leishmaniasis and HAT, and remains an excellent strategy to

identify new therapies.

Some of the current therapies to treat NTDs have resulted from drug repurposing. For

example, originally developed as an anticancer treatment, eflornithine (4) has been repurposed for

treating Stage 2 of gambiense HAT infections. Amphotericin B (11), was isolated from

Streptomyces nodosus in 1955 and was originally used as an antifungal agent.46 Currently it is used

to treat leishmaniasis as described in Section 1.2.3. Miltefosine (12) is the first available oral

treatment for leishmaniasis and was originally developed as an anticancer drug, though it does

display toxic side effects.18 Lastly, another repurposed treatment for leishmaniasis, paromomycin

(13), was originally developed as an oral antibiotic to treat intestinal bacterial infections.18 Given

its poor absorption across the GI tract, it is administered by intramuscular injection to treat VL,18

or it can be applied topically as a treatment for CL.35

Other drug repurposing campaigns have been pursued in the effort to identify new

treatments for NTDs. One such campaign was pursued by Kaiser and colleagues, which assembled

100 approved drugs with target product profiles (TPP) that matched the requirements as outlined

by the DNDi including oral bioavailability, safety, and moderate price. The list of approved drugs

was also chosen based on their registered indications, mechanisms of action and chemical class.

The drugs were then screened against T. brucei, T. cruzi and L. donovani in parallel with current

treatments for the parasites.47 Two antibiotics, nifuroxazide (22) and nitrofurantoin (23) (Figure

1-12), had better EC50 values (EC50 = 0.03, 0.5 µM respectively) against T. b. rhodesiense than

nifurtimox (EC50 = 1.4 µM). The rheumatoid arthritis drug, auranofin (24) (Figure 1-12), was also

more potent against T. b. rhodesiense (EC50 = 0.01 µM), but when tested in a mouse model was

38

found to be inactive. The antifungal azoles were found to be a promising class of inhibitors against

T. cruzi with EC50 values ranging from 0.003 to 0.3 µM with selectivity index values >100 against

L6 rat myoblast cells. The antibacterial, clofazimine (25) (Figure 1-12), emerged as the only active

compound (EC50 = 0.95 µM) against intracellular L. donovani amastigotes with an acceptable 10-

fold selectivity against mouse peritoneal macrophages. Overall, the study identified several

interesting starting points for future repurposing ventures.47

Figure 1-12. FDA-approved drugs identified as active against T. b. rhodesiense.47

Another screen of FDA approved drugs was reviewed by Hernandez et. al. 48 This screen

utilized 1,148 compounds from the Selleckchem library of FDA-approved drugs and the National

Institutes of Health (NIH) Clinical Collection.49 Promising compounds ifenprodil (26), ziprasidone

(27), clemastine (28), clofibrate (29), and azelastine (30) (Figure 1-13) were more active than

nifurtimox when tested in vitro against T. cruzi. Although the compounds were potent and

bioavailable, the free blood concentrations caused toxicity issues normally associated with their

primary human target in the central nervous system. These compounds were also antagonist of the

39

H1 histamine receptor. Thus, although originally intended for direct repurposing, further

optimization of these drugs is required to mitigate toxicity.

Figure 1-13. FDA-approved drugs identified as active against T. cruzi and that could serve as

starting points for further optimization to mitigate toxicity.48-49

Previously, retinoic acid (31) and retinol acetate (32) (Figure 1-14) were shown to inhibit

L. donovani promastigote growth and drastically lower the amount of polyamines produced.50 T.

cruzi is unable to synthesize polyamines de novo making TcPAT12, a high-affinity spermidine

transporter51, essential to the survival of the parasite.52 With TcPAT12 as the intended target for

inhibition, a drug repurposing program was started using retinol acetate as the template for

desirable ligands. A structural similarity screen of 2,924 commercially available drugs was

performed53 and a virtual screen of these drugs vs. TcPAT12 identified potential inhibitors.

Compounds of interest were subsequently screened in vitro against T. cruzi. Compounds

structurally related to retinoic acid and retinol acetate were hypothesized to inhibit TcPAT12 which

40

was confirmed when isotretinoin (33) (an acne treatment; Figure 1-14) was shown to inhibit

TcPAT12 and kill T. cruzi trypomastigotes (EC50 = 130 nM).53

Figure 1-14. FDA-approved drugs shown to inhibit TcPAT12.50, 53

It has been shown that fungicidal compounds and compounds containing imidazoles,

pyrimidines, and triazoles (compounds 34-36, Figure 1-15) that inhibit lanosterol 14α-

demethylase (CYP51) can also inhibit similar biosynthetic pathways in kinetoplastids.54

Repurposing CYP51 antifungals for kinetoplastid diseases has been widely explored.54

Posaconazole (34) (Figure 1-15), one said antifungal, reached clinical trials for chronic Chagas

disease.55 Patients treated with 100 mg or 400 mg twice daily tested negative for T. cruzi DNA

during the treatment period (with the exception of two patients in the 100 mg/kg twice daily group).

Upon follow-up, however 92% and 81% of patients in the 100 mg/kg and 400 mg/kg twice daily

groups, respectively, tested positive for T. cruzi DNA compared to 38% of the benznidazole-

treated group.55 These results suggest that the TcCYP51 pathway is only essential for rapidly

dividing parasite and not for more dormant forms.56 The lack of clinical efficacy with CYP51

inhibitors has led to their deprioritization by the DNDi.56-57

41

Figure 1-15. Antifungal azoles repurposed for Chagas disease and used in combination with

DB766 to enhance killing of L. donovani.54, 58

Although antifungal azoles as monotherapies have been deprioritized, repurposing them in

combination therapies is prominent in leishmaniasis drug discovery, likely due to the current use

of the repurposed antifungal, amphotericin B. Combinations of the antifungal azoles, posaconazole

(34) and ketoconazole (35) with the arylimidamide, DB766 (36) (Figure 1-15), enhanced the

killing of L. donovani in vitro over the respective monotherapies.54, 58 The most promising

combination, which involved posaconazole, resulted in an 81% reduction of liver parasitemia in

L. donovani-infected mice over either the DB766 (40% reduction) or posaconazole (21%

reduction) monotherapies.59 Posaconazole has also been tested against T. b. brucei with an EC50

of 8.5 µM. Treating mice with a combination of posaconazole and eflornithine extended the

survival of infected mice over either monotherapy, and led to a comparable extension of life to

that of NECT.60

1.3.1.2 Target repurposing

The two factors used to categorize a program as target repurposing are the identification of

a validated parasite target and the need to re-optimize a hit compound. The parasite target also has

42

a homologous mammalian species target for which inhibitors have already been discovered. Using

these known inhibitors narrows the screening scope of chemical matter to be evaluated, speeding

hit identification.

A classic target repurposing program utilized chemical matter to target T. brucei glycogen

synthase kinase-3 (TbGSK3), an enzyme important to parasite development as demonstrated by

knockdown experiments.61 The homologous protein in humans is GSK3β and is implicated in

diabetes and neurodegenerative diseases.62 GSK3β has 52% amino acid identity to TbGSK3 and

was used to generate a homology model.63 A library consisting of kinase inhibitors was docked

into the homology model of TbGSK3 and the resulting hits were screened in vitro against the

enzyme. Hits from the in vitro screen were prioritized based on their ligand efficiencies leading to

the identification of diaminothiazole 37 (Figure 1-16) (EC50 = 2 µM).63 Hit-to-lead optimizations

were carried out on 37, however in vitro results revealed that potency against T. brucei no longer

tracked with TbGSK3 binding, suggesting that the series of compounds had a different molecular

target and that further optimization would need to be phenotypically driven.63

In an extension of this work on TbGSK3β,63 the DDU and GSK identified the hit compound

37 as an inhibitor of growth of L. donovani.64-65 New chemical scaffolds were synthesized and

evaluated, identifying pyrazolopyrimidine 38 that offered improved potency and metabolic

stability.65 Further optimization to improve the aqueous solubility resulted in

GSK3186899/DDD853651 (39, Figure 1-16) which, when dosed at 25 mg/kg orally twice a day,

demonstrated comparable activity to miltefosine and reduced parasitemia by 99%.64 This

compound is currently undergoing further preclinical development for VL.64

43

Figure 1-16. Progression of the diaminothiazole hit to GSK3186899/DDD853651.63-65

Another target repurposing program by Bland et al. focused on T. brucei phosphodiesterase

B1 and B2 (TbrPDEB1 and TbrPDEB2, respectively).66 It has been shown by knockdown of both

genes through RNA interference that together they are essential for parasite survival, but not

individually.67-68 Human PDE inhibitors are shown to be active against both parasite enzymes69,

and TbrPDEB1 and TbrPDEB2 have catalytic domains 30-35% similar in amino acid sequence

to human PDEs.70 A library of compounds with chemotypes known to bind human PDE was

screened against TbrPDEB1, and compounds that had EC50 values <20 µM were subsequently

screened against TbrPDEB2. A human PDE4 inhibitor, Piclamilast (40, Figure 1-17), was

moderately active (TbrPDEB1 EC50 = 4.7 μM, TbrPDEB2 EC50 = 11.4 μM). Analogs were

synthesized as part of a structure-based drug discovery effort and had EC50 values in the

micromolar range.66 Cilomilast (41), rolipram (42) and GSK-256066 (43) (Figure 1-17) were

pursued by Amata et al. 71-72 and Ochiana et al.73 Their optimizations efforts revealed the

challenging nature of decoupling parasite and human enzyme potencies and thus the series was

deprioritized.

44

Figure 1- 17. Human PDE inhibitors that entered medicinal chemistry optimization for T.

brucei.66, 71-73

In parallel, another group pursued TbrPDEB1 as a target for HAT and identified an hPDE4

hit series (typified by 44, NPD-001; Figure 1-18) after a high throughput screen (HTS) of 400,000

compounds.74 The chemical matter used for the HTS was not biased and thus this program is not

strictly speaking “target repurposing”. However, the subsequent medicinal chemistry campaigns

to optimize this series are consider as such and resulted in lead compounds 45 and 46.75-78

Figure 1-18. Hit to lead compounds resulting from target-guided SAR explorations of

TbrPDEB1.74-78

1.3.1.3 Target-class repurposing

The differentiating factor between target-class and target repurposing is whether a specific

human target has homology to a specific parasite target. In target-class repurposing a specific target

is unknown, but a family of targets that are similar in humans and parasites is used to bias the

chemical matter used in hit identification screening. Compounds shown to engage this shared

family of targets are considered. For instance, parasites express essential proteins to their survival,

45

such as histone deacetylases and kinases, for which their homologous human counterparts have

been widely studied. Chemical matter for the human target class is then screened in phenotypic

assays, evaluating for parasite death or growth inhibition. The chemical matter that is screened is

late-stage but requires further optimization for desired effects.

As stated above, histone deacetylases (HDACs) are proteins essential for transcription,

gene expression and the regulation of chromatin structure. 18 HDACs have been categorized into

classes based on genetic sequence similarities and cofactor dependence and have been implicated

as viable drug targets in many human diseases, including cancer,79 neurodegenerative,80 metabolic

and immunological diseases.81 Sirtuins, also known as class III HDACs, function in gene

expression, DNA repair and apoptosis. Kinetoplastid sirtuins have been shown to be essential to

parasite survival.81-84 A review by Hailu summarizes numerous HDAC inhibitors that have been

screened against T. brucei, T. cruzi, L. major and L. donovani.81 Vorinostat (47, Figure 1-19), a

human HDAC inhibitor, and some of its derivatives were screened against L. donovani and L.

infantum. All compounds that advanced to in vivo efficacy studies were unsuccessful.

Interestingly, when the compounds were absorbed by gold nanoparticles and retested, good in vivo

efficacy without toxicity was demonstrated.85

Figure 1-19. The HDAC inhibitor, vorinostat, shows promising in vivo efficacy against L.

donovani when absorbed by gold nanoparticles.81, 85

46

As previously described, trypanosomes express essential protein kinases and they account

for approximately 2% of their respective genomes.86-87 A genomic analysis of T. brucei, T. cruzi,

and L. major identified the presence of 176, 190 and 196 orthologous protein kinases,

respectively.86-87 In 2014, 10 FDA approved anti-cancer drugs, including protein kinase inhibitors

sunitinib (48, EC50 = 1.1 µM), sorafenib (49, EC50 = 3.7 µM) and lapatinib (50, EC50 = 2.5 µM)

(Figure 1-20), were evaluated for their activity against L. donovani amastigotes.88 Each showed a

similar bioactivity to those of the current antileishmanial drugs, amphotericin B (11) (EC50 = 0.02

µM) and miltefosine (12) (EC50 = 1.0 µM).88 Evaluation of the kinase inhibitors, depicted in

Figure 1-20, in a mouse model of Leishmania infection showed moderate reduction in the levels

of liver amastigotes (41%, 36% and 30% respectively). This was the first demonstration that

Leishmania survival in vivo could be challenged by protein kinase inhibitors.88 Sorafenib,

originally designed to target vascular endothelial growth factor receptors (VEGFR) and platelet-

derived growth factor receptors (PDGFR), was unique in that it showed activity across several

Leishmania strains. Although sunitinib, sorafenib and lapatinib were moderately effective in vitro

and in vivo, further optimizations are needed to improve their efficacy.

47

Figure 1-20. Anticancer drugs shown to moderately reduce parasitemia in the livers of mice

infected with L. donovani.88

Lapatinib, originally developed as an epidermal growth factor receptor (EGFR) inhibitor

for breast cancer, was also identified as a micromolar inhibitor of T. b. brucei growth.89 Subsequent

optimizations will be discussed in depth in Chapter 2. Structural modifications designed to

improve the solubility and in vitro clearance of the lapatinib-derived analogs led to NEU-4438

(51, Figure 1-21).90 Undetectable levels of parasitemia were observed in 3 out of 4 mice in an in

vivo efficacy study of 51. The compound was administered orally at 100 mg/kg for six days with

an additional 50 mg/kg given on day 6. However, after dosing was stopped parasitemia rebounded

on day 11. Compound 51 was also tested against a panel of 45 human kinases due to its origins as

a kinase inhibitor. Results showed 51 potently inhibited three kinases: insulin receptor tyrosine

kinase (IRK), lymphocyte-specific protein tyrosine kinase (Lck) and proto-oncogene tyrosine-

protein kinase (Src).90 An Ames test was also performed and 51 and its metabolites were negative

for genotoxicity. One major liability comes with its reactivity to human GPCRs and ion channels

(>50% inhibition at 10 µM). These included muscarinic M2 (85% inhibition), muscarinic M3

48

(52% inhibition), nicotinic acetylcholine α1 receptors (69% inhibition), human ether-a-go-go

(hERG, 94% inhibition), and the norepinephrine transporter (71% inhibition).90

Figure 1-21. Structure of lapatinib-derived analog NEU-4438.

A nitroaromatic structural class of compounds are well-known antiparasitic agents.91-92 The

most successful story for repurposing of a nitroaromatic is the first oral monotherapy for both

stages of T. b. gambiense infections, fexinidazole (6).24 Fexinidazole was first reported as being

effective against T. cruzi, trichomonads and Entamoeba histolytica in 1981,93 and T. brucei in

1983.94 A lack of market incentive caused the discontinuation of development of the drug.26 DNDi

rediscovered the drug in a compound mining campaign that used a targeted library of 700

nitroheterocycles in their screen for inhibition of various parasites.91 Fexinidazole has a good

overall safety profile though nitroaromatic compounds are often found to be toxic.

Another repurposed nitroheterocycle, delaminid (52, Figure 1-22), an approved treatment

for multi-drug resistant tuberculosis, was screened against L. donovani promastigotes and

amastigotes, and identified as a nanomolar inhibitor of growth.95 Delaminid showed similar

clinical efficacy to that of miltefosine when dosed at 30 mg/kg once daily for five days. Further in

vivo efficacy studies showed that decreasing the dosing regimen to 1 mg/kg twice daily increased

49

efficacy. However, because of the complicated pharmacokinetic/pharmacodynamic relationships

human dosing prediction are difficult. Other studies have shown that delaminid meets most of the

TPP criteria (except in cost per patient) and could be considered a good candidate for further

progression as an antileishmanial agent.95

Figure 1-22. Structures of delaminid and its optimized analogs.

Based on the potential of delaminid, Thompson et al. screened other anti-tubercular

nitroimidazoles against L. donovani.96-97 Subsequent optimization studies yielded a preclinical

candidate, 53 (Figure 1-22), which decreased parasitemia by 99% in a chronic leishmaniasis

hamster model.97 During the development of 53, Thompson et al. then sought a backup compound

with a similar scaffold, leading to the identification of 54.98

1.3.1.4 Lead repurposing

Lastly, lead repurposing takes gene-family-targeting (like target-class repurposing)

chemical matter to identify potent and selective hit compounds. The difference from target-class

repurposing is that the chemical matter consists of early stage compounds.

50

Our lab used a lead repurposing approach with the kinase family in mind, beginning with

a biased library of known human kinase inhibitors (and will be discussed in depth in Chapter 3).

This library was screened in collaboration with GlaxoSmithKline (GSK) to identify potential

inhibitors of T. brucei. Inhibitors of parasite growth by 50% in a single concentration assay were

progressed to dose response and selectivity assays.99 Prioritized compounds were then clustered

based on structural similarities.

Various medicinal chemistry hit-to-lead optimization programs have commenced since the

publication of the initial screening results and are in different stages of development.100-102 An

isatinoid-containing series from this screen was pursued by Klug et al. with a focus on improving

potency and aqueous solubility while maintaining its good physicochemical properties.100

Combining potency with a favorable absorption, distribution, metabolism, excretion (ADME)

profile proved challenging as revealed by SARs and structure-property relationships (SPRs).

NEU-4391 (55, Figure 1-23), was progressed to a mouse PK study, however, there was low

exposure of the compound in both the blood and brain. Based on these data, the series was not

worth further pursuit.100

Figure 1-23. Isatinoid compound NEU-4391 identified from a lead repurposing HTS campaign.

51

A similar lead repurposing campaign was pursued by DDU. A kinase inhibitor library was

screened against T. b. rhodesiense identifying 121 compounds that inhibited parasite growth by

50% at 5 µM.103 These hits were subsequently screened in a T. b. brucei proliferation assay to

determine EC50 values. The most active compound 1H-Imidazo[4,5-b]pyrazin2(3H)-one (56,

Figure 1-24, EC50 = 80 nM) had good selectivity over the MRC-5 host cell line (EC50 >50 µM).

Swapping the 1H-imidazo[4,5-b]pyrazin2(3H)-one core, which is associated with static parasite

growth, for a 1H-pyrazolo[3,4-b]pyridine core, seen in compound 57 (Figure 1-24), yielded cidal

compounds. Compound 57 was progressed to an in vivo PK study due to its favorable profile for

potency (EC50 = 0.09 µM), solubility (> 250 µM), metabolic stability (3.6 mL*min-1g-1) and

plasma protein binding (89% bound). However, the molecule had low exposure levels (Cmax = 580

ng*mL-1) and was rapidly cleared (t1/2 = 0.7 h). Thus, further work on this compound series is

required.

Figure 1-24. Most active compound (56) from a kinase library screen and optimized compound

(57) for T. brucei growth inhibition.

In summary, numerous repurposing campaigns have been employed in the effort to find

new therapeutics to treat HAT, Chagas disease and leishmaniasis. Each of the four types of

repurposing strategies has key differences, including whether a specific target is known or

whether optimization of a hit compound is required. Whilst direct drug repurposing is the most

52

efficient repurposing strategy, the most recent example, fexinidazole, came from a target-class

repurposing approach. Fexinidazole stands as an excellent example of the value of repurposing

chemical matter for urgently-needed kinetoplastid drugs.

1.4 Compound optimization

After identifying a hit compound from a repurposing campaign (except direct drug

repurposing) compounds need to be further optimized to eventually become drugs. Not only are

they optimized for potency, but they must have optimal ADME properties in order to become

orally dosed drugs. Recently, development programs have been focused on optimizing compound

properties using different tools as discussed below.

In the 1990s, combinatorial chemistry was widely used to synthesize large libraries of

compounds. These combinatorial libraries allowed for a larger chemical space to be explored,

however the space for clinically relevant drugs were in small clusters. Thus, combinatorial libraries

led to compound with poor ADME properties.104 To filter these large libraries, Lipinski analyzed

the effect of various compound properties on solubility and permeability for published clinical

candidates. This analysis led to a set of “rules” that would help predict drug-likeness.104-105 These

rules stated that a given compound was more likely to be soluble and permeable, and therefore

druglike, if it had fewer than 5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular

weight less than 500 g/mol, and a calculated Log P (cLogP) less than 5.105

Veber et. al. performed a similar analysis to Lipinski utilizing the orally bioavailable

compounds at what is now GSK.106 This analysis took into account the number of rotatable bonds,

total number of hydrogen bonding atoms, and polar surface area as good predictors of whether a

compound would be effectively orally bioavailable.106 The criteria stated that a compound with 10

53

or fewer rotatable bonds and a polar surface area less than 140 Å would have a high probability of

being orally bioavailable in rats.106

In developing a drug, high potency is not the be all and end all for compound progression.

Rather good compounds are ones that make favorable interactions with a target. Calculated

properties for the efficiency of the binding for a compound can help assess hit and early lead

compounds when making go/no-go decisions. Ligand efficiency (LE) characterizes the efficiency

of a compound based on the number of heavy atoms it contains (LE = 1.37×pEC50/# of heavy

atoms).107 The LE essentially helps normalize potency for molecular size. An LE value greater

than 0.3 is a good starting point for optimization efforts. Lipophilic ligand efficiency (LLE) is

another of these calculations which determines a compound’s efficiency based on the lipophilicity

of the molecule (LLE = pEC50-cLogP).108-109 The LLE predicts whether a compound is potent

because it is making a favorable interaction with the target of interest, or it is greasy and interacting

with the intended target to allow it’s escape from water.

Structure-property optimizations are equally as important as structure-activity

optimizations. Compounds that do not reach the intended target due to poor ADME, but still make

specific, favorable interactions are irrelevant regardless of high potency. Table 1-2 shows the

desired properties of compounds pursued as part of discovery and development efforts in the

Pollastri lab.

54

Table 1-2. Targeted compound criteria for hit and lead compounds.

Assay Hit criteria Lead criteria

Potency pEC50 T.b. brucei > 7 > 7.5

T.b. brucei LLE ≥ 4

pEC50 T. cruzi > 5.3 > 6.5

T. cruzi LLE ≥ 4

pEC50 Leish spp > 5 > 6

Leish LLE ≥ 4

pEC50 P. fal > 7 > 7.5

P. fal LLE ≥ 4

Toxicity HepG2 TC50

MRC5 TC50

10 × EC50

10 × EC50

10 × EC50

10 × EC50

ADME &

Phys.

Properties

MW

cLogP

LogD

RH Clint

HLM Clint

Aq Sol (μM)

PPB (%)

≤ 360

≤ 3

≤ 2

≤ 27

≤ 47

> 10

≤ 95

≤ 360

≤ 3

≤ 2

≤ 5

≤ 9

> 100

≤ 95

PK Free plasma

concentration >

10xEC50 for given

parasite

Free plasma

concentration >

EC99 for given

parasite

Efficacy T.b. brucei,

T. cruzi,

Leish spp.,

P. fal

Reduction,

control, or

elimination of

parasitemia

following oral or

IP dosing at

levels that

provide sufficient

exposure (above)

Control, or

elimination of

parasitemia

following oral or

IP dosing at

levels that

provide

sufficient

exposure (above) MW = molecular weight (g/mol); RH Clint = rat hepatocyte clearance rate (μL/min/106 cells); HLM Clint = human liver

microsomal clearance rate (μL/min/mg of protein); PPB = plasma protein binding.

55


trypanosomiasis (HAT) drug discovery

2.1 Introduction

2.1.1 Repurposing lapatinib for Trypanosoma brucei inhibition

T. brucei cells are known to express essential protein kinases. Previous studies have shown

that the parasite growth factor, transferrin, is acquired from host cells through receptor-mediated

endocytosis, and the EGFR inhibitor lapatinib, shown in Figure 2-1 blocks this endocytosis from

occurring.110-111 As a part of a target-class repurposing approach to streamline the drug discovery

process, lapatinib was screened against T. brucei brucei Lister 427112 resulting in a potency in the

low micromolar range (pEC50 = 5.84) and 4-fold selectivity over human HepG2 cells (pTC50 =

5.21).112 With lapatinib as the initial hit molecule, optimizations were then made to improve the

overall potency of the molecule and selectivity for parasite inhibition versus host cells. The

summary of these optimizations can be seen in Figure 2-1.

Chemical modifications were first made to the left-hand side of the molecule, otherwise

referred to as the “tail”. A broad diversity of tail groups was explored, leading to the identification

of NEU-369, a compound equipotent to lapatinib (pEC50 = 5.86) with a larger selectivity over

HepG2 cells (TC50 < 4.82). Head group (shown in green, Figure 2-1) modifications were made

exploring various substitutions on the benzyl group. All head group modifications showed similar

potencies to that of lapatinib, including removal of the benzyl group (NEU-555, Figure 2-1, pEC50

= 6.08, pTC50 < 4.82). Another set of tail group substitutions led to the most potent compound,

NEU-617 (pEC50 = 7.38, pTC50 < 4.70).112 NEU-617 was evaluated in a mouse pharmacokinetic

model, after a single oral 40 mg/kg dose of NEU-617, plasma concentrations of the drug exceeded

56

the EC50 by approximately 4-fold for 10 hours.112 This led to the evaluation of NEU-617 in a mouse

efficacy model as a proof of concept for the further exploration of this chemotype.

Figure 2-1. Optimizations of EGFR inhibitor lapatinib to lead compound NEU-617.

57

The efficacy study of NEU-617 was performed in mice that were infected with 104 T. b.

brucei cells.112 A dose of 40 mg/kg/day was administered orally or intraperitoneally 24 hr after

infection with T. b. brucei.112 Parasitemia was undetectable for 3 days in infected mice, though

drug-related toxicity was observed.112

With NEU-617 in hand, an exploration of the core region of the molecule was undertaken

to determine the importance of nitrogen atom positioning as part of a multi-parasite cross screening

campaign.113 Quinazoline core replacements included quinoline, isoquinoline, cinnoline,

phthalazine, 3-cyanoquinoline, and two thienopyrimidine isomers. Switching from the quinazoline

core to the quinoline resulted in NEU-1045 (Figure 2-1). NEU-1045 had improved potency over

lapatinib (pEC50 = 6.43) with 70-fold selectivity over HepG2 cells. The position of attachment for

the tail group was explored as well as various tail groups as seen in NEU-961 (pEC50 = 7.10, pTC50

< 5.39) (Figure 2-1), which was one of the most potent compounds to date. Perhaps not

unexpectedly based on the analogs’ high molecular weights and lipophilicities, the

physicochemical properties were poor, such as aqueous solubility. These results suggested

improving the pharmacokinetic properties of the compound to be the next step in the hit

development of this series.

2.1.2 Identification of NEU-1912 and NEU-1953 as leads for drug

development

The next round of analog design and synthesis focused on improving the “drug-like”

properties104 and lead quality of the compounds. Lead quality is assessed by calculating lipophilic

ligand efficiencies (LLE = pEC50 - cLogP).114 It was hypothesized that truncation of the large,

lipophilic head group and replacement with small polar heterocycles would lead to an

improvement in the overall ADME profile.115 All compounds synthesized had improved calculated

58

physicochemical properties as compared to parent compound NEU-1045.115 A summary of

modifications to NEU-1045 can be seen in Figure 2-2. From this set of compounds NEU-1912

(Figure 2-2) was synthesized and evaluated. Table 2-1 compares NEU-1045 to analog NEU-

1912.

Figure 2-2. Summary of modifications made to NEU-1045.

Table 2-1. Comparison of NEU-1045 to NEU-1912 and NEU-961 to NEU-1953.

Compound

Hit

Targeted

Values

NEU-1045 NEU-1912 NEU-961 NEU-1953

Tbb pEC50 > 7 6.43 7.70 7.10 6.37

Molecular weight

(g/mol) ≤ 360 569.6 447.5 617.1 398.5

cLogP ≤ 3 5.76 2.72 6.43 2.09

logD (pH 7.4) ≤ 2 5.5 3.3 5.0 1.8

Human plasma

protein binding

(PPB) (%)

≤ 95 100 96 99 88

Aqueous

Solubility (μM) > 10 2 1.4 0.3 44

LLE (pEC50 –

cLogP) ≥ 4 0.67 4.98 0.67 4.28

With improved properties as compared to NEU-1045, NEU-1912 was tested in a mouse

pharmacokinetic study. Female BALB/c mice were dosed intraperitoneally at 10 mg/kg (n=18).

NEU-1912 showed sufficient plasma exposure (>120×EC50 for 4 h) but low brain exposure

59

(brain:plasma = 0.1 at 0.25 hr after dosing). The compound also displayed rapid clearance in

human liver microsomes (HLM) at 207.5 μL/min/mg protein and rat hepatocytes (RH) at 31.5

μL/min/106 cells. Although the properties were improved with the identification of NEU-1912,

ADME optimization was still needed.

Figure 2-3. Modifications to NEU-961 resulting in NEU-1953.

Similar modifications to NEU-961 (Figure 2-3) were made to improve physicochemical

properties of the 7-subsituted quinoline series.116-117 Analogs were synthesized with head group

truncations using small, polar, heterocyclic head groups. Tail group modifications were also made

and crossover compounds combining the most promising head and tail groups were synthesized.

A summary of the modifications made to NEU-961 is depicted in Figure 2-3. Compound NEU-

60

1953116-117 (Figure 2-3) was a result of these modifications. Table 2-1 shows a comparison

between NEU-961 and crossover analog NEU-1953. Although NEU-1953 was a sub-micromolar

inhibitor of T. b. brucei proliferation (pEC50 = 6.37), had an improved LLE as compared to NEU-

961, and was sufficiently water soluble (44 μM), the clearance rates for HLM and RH were still

high (180 μL/min/mg protein and 130 μL/min/106 cells respectively).

NEU-1953 was progressed into a mouse efficacy study. After infection with T. b. brucei,

mice were dosed orally at 60 mg/kg for three days and then at 70 mg/kg for the remaining 3 days

of the study since there was no observable toxicity. Infected control mice were infected and dosed

orally with vehicle only. However, there was no significant difference in the levels of parasitemia

in the treated versus control mice.

The next step in the development process would be to further improve the absorption,

distribution, metabolism, and excretion (ADME) properties of the two advanced hit compounds

NEU-1912 and NEU-1953 to improve the pharmacokinetic properties and in vivo efficacy. It was

originally hypothesized that the lapatinib analogs had poor solubility because they were highly

lipophilic. Truncation of the large, lipophilic head group of lapatinib should therefore have

increased the aqueous solubility. However, an analysis comparing the clogP values versus aqueous

solubilities (Figure 2-4) of the lapatinib analogs synthesized up to this point showed that there

was no correlation between low lipophilicity and increased aqueous solubility.

61

Figure 2-4. Comparison of lipophilicity versus aqueous solubility in first and second generation

lapatinib analogs.

Since the lack of solubility was not due to lipophilicity, a new hypothesis was required:

due to the numerous aromatic rings in the analogs, the compounds would most likely have a flat

3-dimensional shape and multiple hydrogen bond donors/acceptors, allowing the compounds to

stack on top of one another. Solvation by breaking up this crystal packing would be energetically

unfavorable and therefore solubility would be low.118 There are various ways to address low

compound solubility, which are outlined below.

2.1.3 Methods for improving aqueous solubility

In the mid-1990s, the Biopharmaceutical Classification System (BCS) was introduced to

classify drugs based on their aqueous solubility and permeability rates.119 There are four classes

of compounds: class I (soluble, permeable), class II (poorly soluble, permeable), class III (soluble,

poorly permeable), and class IV (poorly soluble, poorly permeable). Improving the solubility of

62

compounds in classes II and IV can help improve oral bioavailability.120 Numerous drug

development failures have been due to poor solubility of drug candidates.121

Drug solubility can be broken down into 3 parts. First, intermolecular forces between

compound molecules must be broken. Second, a void must be formed in the solvent produced by

the breaking of solvent-solvent intramolecular forces. Lastly, the solute must enter the void made

in the solvent. With these steps considered there are two determinants of drug solubility, the energy

needed to break intermolecular forces of the solid being dissolved and the solvation energy.118

Poor aqueous solubility of chemical matter is a consistent problem in the drug discovery

process. Almost 40% of marketed drugs and 75% of drugs in the discovery pipeline have low water

solubility.122-123 The trend towards production of insoluble compounds has caused the

pharmaceutical industry to identify different strategies to circumvent their solubility woes.

Medicinal chemistry strategies used to chemically modify compounds to increase the aqueous

solubility include increasing charge and polarity,124-125 reducing planarity,126-127 or reducing

aromatic ring content.128-129 Two of these strategies were employed.

2.1.3.1 Salt formation

Forming salts of compounds is one of the most common strategies to increase their aqueous

solubility.125 Compounds that are ionizable can be paired with suitable counter ions and potentially

raise their dissolution rates and oral absorption.123 It was demonstrated in the 1950s that the free

acid form of weakly acidic compounds had slower dissolution rates than that of their associated

salts.130-131 One example of using a salt to increase aqueous solubility is that of delveridine. The

initial solubility of the free base of delveridine was 143 μg/mL, however, the mesylate salt of

delveridine was 320 mg/mL, a 2000-fold increase.132 Salt forms are mostly identified and selected

63

through a trial and error process, and using them to enhance aqueous solubility has its advantages

and disadvantages.124, 133

The screening of potential counter ions for salt formation can be performed in a high-

throughput manner where the drug is dissolved in different solvents and precipitated from solvent

by the addition of various counter ions.134-135 This makes salt formation a simple way to drastically

increase solubility without making chemical modifications. However, it has been shown that solids

formed after counter ion screening may not necessarily be salt forms, yet just precipitated parent

compound or counter ion forming agent.125 Partial salt forms can be produced under some

conditions resulting in salts co-crystallized with free base forms of a molecule thus affecting

dissolution rates.136 A potentially useful salt form would need to be confirmed by remaking the hit

on a larger scale and performing a thorough analysis by x-ray diffraction and crystallography.

Remaking a salt form could be made complicated by partial salts and co-crystals.

2.1.3.2 Increasing sp3 hybridized carbon content

Molecules with a planar shape have an increased probability of tightly crystal packing

together and crystal packing has been suggested to increase a compound’s melting point.126-127 It

has been shown that increasing the fraction of sp3 hybridized carbons can decrease the melting

points of compounds and improve solubility.126 Lovering et. al. analyzed the molecular weights

fraction of sp3 hybridized carbon atoms (Fsp3 = number of sp3 hybridized carbon atoms/total

number of carbon atoms) for all compounds published after 1980 from all phases of the discovery

process.126 Their results showed that compounds that reached later stages of the development

process showed higher Fsp3 content.126

Ishikawa and Hashimoto analyzed different examples of reducing molecular planarity that

led to the increase in solubility.127 One such example included clinical candidate, AMG-517

64

(Figure 2-5), produced at Amgen which showed poor thermodynamic aqueous solubility (<1

μg/mL in PBS).137 Chemical modifications to the terminal phenyl ring resulted in the compound

AMG-517b which showed a 13-fold improvement in the thermodynamic aqueous solubility (13

μg/mL in PBS).137

Figure 2-5. Clinical candidate AMG-517 and its analog with improved aqueous solubility.

A study performed by Ritchie et. al. analyzed 280 compounds from the discovery pipeline

for the effect that aromatic ring count had on developability and physicochemical properties.128

The analysis showed that as compounds progressed through the pipeline the number of aromatic

rings decreased and the average number of aromatic rings in oral drugs is 1.6. Approximately

31,000 compounds in the GSK database were used in analyzing the relationship between aqueous

solubility and aromatic ring count.128 It was concluded that solubility decreases dramatically with

the increase in the number of aromatic rings, specifically over a total amount of 2 aromatic rings.

In a follow-up study it was shown that replacement of aromatic rings with heteroaliphatic rings

were beneficial for aqueous solubility.129

We have applied both salt screening and Fsp3 modulation strategies to the problem of

solubility as it applies to NEU-1912 and NEU-1953 described in the sections following.

65

2.2 Salt screens

2.2.1 Salt formations of NEU-1953

The first step of the salt screen began with the qualitative analysis of NEU-1953’s

solubility in various organic solvents (scaled up NEU-1953 provided by Dr. Baljinder Singh)

(Table 2-2). A suitable solvent was identified as one that could dissolve the compound eventually

but not too readily. Approximately 2 mg of NEU-1953 were added to 7 different vials. Seven

different solvents were added to the vials in increments of 200 μL until the compound was

completely dissolved. Solutions that reached 2.5 mL in volume were heated and sonicated; such

was the case for isopropanol, tetrahydrofuran, acetonitrile, ethyl acetate, and water. However, for

these solvents, the compound did not completely dissolve. Both methanol and ethanol successfully

dissolved NEU-1953 with less than 1 mL of solvent. Ethanol was ultimately chosen for the salt

screen of NEU-1953 as the larger volume needed would be better for measuring and handling

when performing the salt screen. Less solid compound would be needed to saturate the solution

and thus require fewer equivalents of acid to be added for a salt form to precipitate.

Table 2-2. Determination of approximate solubility of NEU-1953 in various organic solvents.

Vial Solvent Amount of

NEU-1953

Volume of solvent needed to

dissolve NEU-1953

completely (μL)

1 Methanol 2.0 mg 500

2 Ethanol 2.2 mg 800

3 Isopropanol 2.1 mg 2500

4 Tetrahydrofuran 1.9 mg 2500

5 Acetonitrile 2.5 mg > 2500

6 Ethyl Acetate 1.9 mg > 2500

7 Water 2.5 mg > 2500

66

For the salt formations of NEU-1953, 20 mg of compound was suspended in 4 mL of

ethanol and heated until fully dissolved. Various acids (1 equivalent of 1M acid in ethanol) were

then added to the solution of NEU-1953. If a precipitate formed, it was then collected by vacuum

filtration and washed with ethanol.

2.2.1.1 Evaluation of NEU-1953 salt forms

A common strategy used to increase the kinetic aqueous solubility of a series is through

the formation of salts at acidic or basic centers on compounds.21, 22 A range of salts of NEU-1953

were studied (Table 2-3), taking advantage of the basic nitrogen atoms within the compound. The

hydrochloride (2.1) and sulfate (2.2) salt forms remained unchanged in potency and both

thermodynamic and kinetic solubility, while the citrate salt (2.3) showed a modest increase in

solubility, both thermodynamic (25%) and kinetic. The methanesulfonate salt (2.4) demonstrated

a 4-fold increase in the kinetic solubility which could be the reason for the slight increase in

potency.

Table 2-3. Salts formed of NEU-1953 compared to the free base.

Compound

Salt form (1953) Salt

ratio

T.b.b.

pEC50

(µM)

Thermodynamic

solubility (µM)

Kinetic

solubility

(µM)

HLM Clint

(µL/min/mg)

NEU-1953 Free base - 6.4 44 25 180

2.1 Hydrochloride 4:1 6.5 42 25 160

2.2 Sulfate 1:1 6.3 39 25 170

2.3 Citrate 2:1 6.3 61 50 190

2.4 Methanesulfonate 2:1 6.7 16 100 170

67

2.2.2 Salt screen of NEU-1912

2.2.2.1 Synthesis of NEU-1912

The synthesis of 6-bromoquinolone was carried out via the Gould-Jacobs sequence

following previously reported protocols.115 4-Bromoaniline was coupled through conjugate

addition with ethoxymethylenemalonate to provide diester 2.5. Cyclization was achieved in

refluxing diphenyl ether to generate ester 2.6, followed by hydrolysis, and decarboxylation to give

quinolinone 2.8.

Scheme 2-1. Synthesis of 6-bromoquinolone core.115

Reagents and conditions: a) diethyl 2-(ethoxymethylene)malonate, 100 °C, 2.5 hr (90%); b)

diphenyl ether, reflux 2 hr (91%); c) 2.5 M sodium hydroxide, reflux, 3 hr (97%); d) diphenyl

ether, reflux, 3 hr (97%).

The synthesis of 4-(morpholinosulfonyl)phenyl tail group began first with reaction of 4-

bromophenylsulfonyl chloride with morpholine to give bromide 2.9. The boronic ester 2.10 was

generated via a Miyaura coupling with bis(pinacolato)diboron.

68

Scheme 2-2. Synthetic route for NEU-1912 tail group.

Reagents and conditions: a) morpholine, THF, rt, 12 hr (82%); b) B2pin2, KOAc,

PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 85 °C, 3 hr (assumed quantitative).

With core (2.8) and tail (2.10) groups in hand, the two were coupled using Suzuki

conditions to give 4-((morpholinosulfonyl)phenyl)quinolone 2.11. Deoxychlorination in neat

phosphorus oxychloride provided the chloroquinoline 2.12. Finally, 4-aminopyrimidine was

attached to the core using Buchwald-Hartwig cross coupling conditions, resulting in the final

compound NEU-1912 (2.13).

Scheme 2-3. Synthesis of NEU-1912.

Reagents and conditions: a) (4-(morpholinosulfonyl)phenyl)boronic acid pinacol ester, 6-

bromoquinolin-4(1H)-one, TEA, Pd(OAc)2, 85 °C, 12 h (83%); b) Phosphorus oxychloride, 106

°C, 2 h, (70%); c) pyrimidine-4-amine, tBuOK, xantphos, Pd2(dba)3, 101°C, 24 h (92%).

69

2.2.2.2 Salt formations of NEU-1912

The first step of the salt screen began with the scale up synthesis of NEU-1912 as discussed

in section 2.2.1. A solvent screen similar to the one done for NEU-1953 was performed and

isopropanol was determined to be a suitable solvent for the salt formation of NEU-1912.

Experiments (shown in Table 2-4) that resulted in the precipitation of the salts from the solution,

were deemed successful. Salts were then collected by vacuum filtration and washed with

isopropanol.

Table 2-4. Conditions for salt formations of NEU-1912.

Entry

Mass of

NEU-1912

(mg)

Volume of

isopropanol

(mL)

Acid

(1 eq.) Observation

1 2.3 1.6 Sulfuric ppt

2 2.1 1.46 Hydrochloric crystals after

4 days

3 2.1 1.46 Phosphoric ppt

4 2.4 1.67 Methane

sulfonic acid ppt

5 1.9 1.32 p-toluene

sulfonic acid ppt

6 2.3 1.6 Citric acid No ppt

7 2.0 1.39 Acetic Acid No ppt

8 2.2 1.53 Formic Acid No ppt

2.2.2.3 Evaluation of NEU-1912 salt forms

Various salts forms of NEU-1912 were studied as compared to the free base form of the

compound. In this case, only the thermodynamic solubility was tested. Thermodynamic solubilities

70

were virtually unchanged for all of the salt forms. The T. brucei activities of the salt forms also

remained unchanged.

Table 2-5. Salts formed of NEU-1912 compared to the free base.

Compound

Salt form (1912) Salt

ratio

T.b.b.

pEC50

(µM)

Thermodynamic

solubility (µM)

2.13 Free base - 7.6 1.4

2.14 Phosphate 1:1 7.7 2.0

2.15 Sulfate 1:1 7.6 6.0

2.16 p-toluenesulfonate 1:1 7.6 2.0

2.17 Methanesulfonate 1:1 7.6 3.0

2.3 Increasing Fsp3: Design, synthesis, and evaluation of NEU-1912 and NEU-

1953 matched pairs with saturated head groups

2.3.1 Selection of saturated primary amines as replacements for aromatic

head groups of NEU-1912 and NEU-1953

The second strategy pursued to increase the solubilities of NEU-1953 and NEU-1912 was

to increase the sp3 hybridized atom count and thus reduce the number of aromatic rings in the

structure. The region of focus for modification was the “head-group” since previous SAR studies

showed that changes to this region were highly tolerated in terms of potency. A virtual library of

analogs was enumerated by using JChem reactor (ChemAxon Inc.) and a set of aliphatic, cyclic

primary amines that were available in pre-weighed quantities (Frontier Scientific). Using Vortex

(Dotmatics Inc.) the library was shaped on the basis of clogP (<5) and molecular weight (<500

g/mol). Final compounds containing 6 and 5-membered rings with one heteroatom were selected

from the remaining compounds for synthesis.

71

2.3.2 Synthesis of saturated head group replacements, NEU-1953 scaffold

In an attempt to diversify analogs in the final step of the synthesis, building up the

chlorinated core + tail groups (2.21) was the main priority. First the tail group was synthesized

starting with 5-bromo-2-chloropyrimidine via SNAr displacement of the chlorine with 1-

methylpiperazine (Scheme 2-4). The resulting bromide was then borylated using Miyaura

conditions.

Scheme 2-4. Synthetic route for NEU-1953 tail group.

Reagents and conditions: a) N-methylpiperazine, DIPEA, t-butanol, 90 °C, 12h (%); B2pin2,

KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145 °C, microwave, 1 h (assumed quantitative).

The borylated tail group was subsequently coupled with 7-bromoquinolone (synthesized

and provided by Dr. Baljinder Singh) to give quinolone 2.20 (Scheme 2-5). The core + tail scaffold

was then chlorinated by deoxy-chlorination with neat phosphorus oxychloride.

72

Scheme 2-5. Saturated head group matched pairs to NEU-1953 template synthesis.

Reagents and conditions: a) 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester,

Cs2CO3, Pd(PPh3)4, 3:1 dioxane/H2O, 130 °C μwave, 15 min (96%), or PdCl2(dppf)·CH2Cl2, 90

°C, 12 hr (85%); b) Phosphorus oxychloride, 106 °C, 3 hr (71%); c) 1-methylpiperidin-4-amine or

2-(pyrrolidin-1-yl)ethan-1-amine, Pd2(dba)3, XPhos, KOtBu, 90 °C, 18 hr (47%, 59%

respectively) d) various SNAr conditions (no isolated products).

With desired chlorinated core + tail in hand, we attempted to synthesize final compounds

with saturated head groups determined by the virtual library as previously stated. It has been

previously shown that Buchwald-Hartwig aminations utilizing various ligands and catalysts have

been successful for coupling aliphatic primary amines to aromatic heterocycles.138-142 The results

from attempts to apply Buchwald conditions with aliphatic cyclic amines are shown in Table 2-6.

Although the use of these conditions was successful at converting starting material to product,

hydrolysis of the starting material and dehalogenation were common side products. The production

of these side products, coupled with the high polarity of the final products, made purification of

these compounds by both normal and reverse phase difficult.

73

The most successful set of conditions was the use of XPhos as a ligand with heating at 90

°C overnight. These conditions were successful at converting enough starting material to product

for two of the desired amine substitutions. However, these conditions were not successful for other

amines because of the similarity in polarities of the de-halogenated starting material and products

for that of cyclohexylamine and 4-aminotetrohydropyran. The products and side products were not

separable by column chromatography and therefore other routes to final compounds were

explored.

Table 2-6. Synthetic attempts via Buchwald-Hartwig cross coupling using NEU-1953

chlorinated core + tail for final compound synthesis.

Entry Amine Ligand Catalyst Conditions Conversion1

1

Xantphos Pd2(dba)3

tBuOK,

anhydrous 1,4-

dioxane, 130

°C, 1 h

70%*

2

Xantphos Pd2(dba)3

tBuOK, 1,4-

dioxane, 150

°C, 1 h

65%*

3

Xantphos Pd2(dba)3 tBuOK, tbuOH,

90 °C, 12 h 60%*

4

tbuXPhos Pd2(dba)3

tBuOK, 1,4-

dioxane, 90 °C,

12 h

60%*

5

(2.22a)

XPhos Pd2(dba)3

tBuOK, 1,4-

dioxane, 90 °C,

12 h

60%

(47% isolated)

6

(2.22b) XPhos Pd2(dba)3

tBuOK, 1,4-

dioxane, 85 °C,

12 h

80%

(59% isolated)

7

XPhos Pd2(dba)3

tBuOK, 1,4-

dioxane, 85 °C,

12 h

35%*

8

XPhos Pd2(dba)3

tBuOK, 1,4-

dioxane, 90 °C,

12 h

55%*

1 Monitored by LCMS, conversion based on integration of UV peaks at 254 nm. *0% isolated yield.

74

Since the side products from the Buchwald coupling were difficult to separate from the

products, SNAr conditions were also explored on the core + tail scaffold. Various conditions were

used and are shown in Table 2-7. Conventional heating and microwave irradiation, up to 200 °C,

were both used but only trace amounts of product were observed. A variety of solvents were also

tried, but again there was no significant conversion for any of the solvents tried.

Table 2-7. Synthetic attempts via SNAr conditions using NEU-1953 chlorinated core + tail for

final compound synthesis.

Entry Amine Solvent Conditions Conversion1

1

IPA DIPEA, 90 °C, 48 h 0%

2

IPA DIPEA, 90 °C, 48 h 0%

3

tBuOH DIPEA, μwave 150 °C, 2 h trace

4

nBuOH DIPEA, μwave 180 °C, 1 h 0%

5

NMP DIPEA, μwave 200 °C, 1 h trace

1Monitored by LCMS, conversion based on integration of UV peaks at 254 nm.

With many failed attempts at synthesizing the NEU-1953 analogs with a generalizable

synthetic scheme, a new synthetic route was established and shown in Scheme 2-6. Starting with

7-bromoquinolone, the dihalogenated quinoline 2.23 was synthesized via deoxy-halogenation.

SNAr conditions were then used to selectively add various saturated primary amines at the 4-

position of the quinoline. Bromide intermediates 2.24a-2.24f were subjected to Suzuki conditions

using boronic ester 2.19 as the coupling partner to give final compounds 2.25a-f.

75

Scheme 2-6. Synthetic attempts via SNAr conditions using NEU-1953 7-bromo-4-

chloroquinoline core for intermediate to final compounds.

Reagents and conditions: a) POCl3, reflux, 18 hrs (81%); b) saturated primary amine, DIPEA, n-

butanol, 200 °C, microwave, 3 hrs (22-98%); c) B2pin2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145

°C, microwave, 1 hr (assumed quantitative); d) Cs2CO3, Pd(PPh3)4, 3:1 dioxane/H2O, 80 °C, 48

hrs (11-44%).

2.3.3 Synthesis of saturated head group replacements, NEU-1912 scaffold

Synthesis of the NEU-1912 matched pairs (Scheme 2-7) began with 6-bromoquinolone

(2.8) which was coupled to 4-(phenylsulfonyl)morpholine boronic acid (2.10) under Suzuki

conditions. Deoxy-halogenation afforded chlorinated compound 2.12. Subsequent chlorine

displacements with various primary aliphatic amines under SNAr conditions provided the desired

final compounds 2.26a-h.

76

Scheme 2-7. Synthetic route for various head group analogs.

Reagents and conditions: a) 4-(phenylsulfonyl)morpholine-boronic acid pinacol ester,

Dichlorobis(triphenylphosphine)palladium(II), TEA, ethanol/water, 100 °C, microwave, 2 h (%);

b) Phosphorus oxychloride, 106 °C, 2 h (82%); c) saturated primary amine, DIPEA, n-butanol,

200 °C, microwave, 3 h (20-80%).

2.3.4 Evaluation of saturated head group replacements, NEU-1912 and

NEU-1953 scaffolds and matched pairs

Comparing the parent compound NEU-1953 to its saturated head group matched pairs, the

analog bearing the cyclohexylamine, NEU-5144 (Table 2-8), showed a modest improvement in

solubility and potency over NEU-1953, but was less metabolically stable. Tertiary amines (NEU-

4920, NEU-5375, and NEU-4987) all exhibited improved solubility with moderate-to-excellent

anti-trypanosomal potencies. Compound NEU-4987 is 20-fold more potent than NEU-1953 with

a 10-fold improvement in aqueous solubility. However, its high plasma protein binding (100%)

and toxicity against HepG2 cells are liabilities that make it less desirable for progression.

Replacement of the headgroup with the tetrahydropyrans (NEU-5139 and NEU-5326) maintained

activity against T. brucei, when compared to NEU-1953, and improved aqueous solubility 15-fold,

77

but increased toxicity against HepG2 cells. The primary alcohol compound NEU-5376, was the

only matched pair to NEU-1953 that showed a loss in potency (4-fold), though the aqueous

solubility was improved.

Saturated head group compounds with the NEU-1912 scaffold (Table 2-9) were similar to

the NEU-1953 analogs in that their aqueous solubilities were improved; however anti-

trypanosomal potencies were all less than that of the parent compound NEU-1912. The most potent

compound of this series, NEU-5536, is 24-fold less potent than NEU-1912, however this is still in

our target range for favorable anti-typanosome activity. Matched pairs in Table 2-8 and Table 2-

9 can also be compared. The NEU-1953 analogs (Table 2-8) have pEC50 values about a log unit

greater than the NEU-1912 analogs (Table 2-9).

78


properties.

Compound R T.b.b.

pEC50

Aq. Sol.

(µM)

Human

PPB

(%)

HLM Clint

(µL/min/mg

of protein)

HepG2

pTC50 LLE

NEU-1953

6.37 44 87 180 < 4.52 4.3

NEU-5144

(2.25a)

6.54 98 85 300 ntf 2.8

NEU-4920

(2.22a)

7.27 >1000 ndb ndc ntf 5.3

NEU-5375

(2.25b)

6.77 660 ndd ndd 5.60 3.7

NEU-4987

(2.22b) 7.66 590 100 nde 5.52 5.3

NEU-5139

(2.25c)

6.42 760 50 94 4.82 4.5

NEU-5326

(2.25d) 6.44 760 47 170 4.79 4.0

NEU-5376

(2.25e)

5.74 980 63 160 < 5.44 3.0

NEU-6734

(2.25f)

7.56 836 50 240 pending 4.8

SCYX-7158a 6.54

nt = not tested. nd = not determined. aSCYX-7158 (acoziborole Ch 1. cmpd 7) was used as a control. bCompound

lost, likely due to unspecific binding. cAssay failed due to poor curve fitting. dPoor MS response. eLow recovery. fNot tested initially and compounds with a superior profile overall had already been identified.

79


properties.

Compound Head group T.b.b.

pEC50

Aq. Sol.

(µM)

Human

PPB

(%)

HLM Clint

(µL/min/mg

of protein)

HepG2

pTC50 LLE

NEU-1912

7.68 3.7 98 236 < 4.47 4.9

NEU-5533

(2.26a)

5.78 8.1 96 100 4.47 1.9

NEU-5537

(2.26b)

5.43 790 58 23 4.58 3.3

NEU-6735

(2.26c)

5.44 811 69 13 pending 2.2

NEU-5536

(2.26d) 6.30 870 73 28 5.03 3.8

NEU-5825

(2.26e)

6.04 7.0 70 59 4.80 4.0

NEU-5534

(2.26f) 5.60 38 74 70 4.49 3.1

NEU-5535

(2.26g)

5.42 140 72 43 4.49 2.6

NEU-5824

(2.26h)

6.12 200 82 110 4.68 3.3

SCYX-7158a 6.54 aSCYX-7158 (acoziborole Ch 1. cmpd 7) was used as a control.

2.4 Crossover compounds between NEU-4363 and NEU-1912

As part of the optimization of NEU-1953, the tail group region of the molecule was also

explored for increasing the sp3 carbon content.90 For example, the homopiperazine-containing

NEU-4363 (Figure 2-6, Aq. Sol. = 985 μM) displayed improved solubility compared to its parent

80

compound NEU-1953 (Aq. Sol. = 44 μM). With the success of substituting the N-

methylhomopiperazine for N-methylpiperazine, it was hypothesized that the addition of the NEU-

4363 tail group to the NEU-1912 scaffold would also have improved aqueous solubility.

Figure 2-6. Comparison of parent compound NEU-1953 and analog NEU-4363.

2.4.1 Synthesis of crossovers

First the tail group for the crossover compounds was synthesized using a similar scheme

(Scheme 2-8) as those previously mentioned. The chlorine of 5-bromo-2-chloropyrimidine was

displaced using 1-methylhomopiperazine and bromide 2.27 was subjected to Miyaura borylation

conditions to yield boronic acid pinacol ester 2.28. This borylated compound 2.28 was used crude

in subsequent reactions assuming a quantitative yield.

Next, 6-position substituted matched pairs of NEU-1912 and NEU-4363 were synthesized

via Scheme 2-9. Using previously synthesized 6-bromoquinolone 2.8, deoxychlorination in neat

phosphorus oxychloride yielded dihalogenated core 2.29 in 80% yield. Aromatic substitution of

the chloride using sodium hydride and appropriate amine heterocycle resulted in bromide

intermediates 2.30a and 2.30b. Suzuki coupling with previously synthesized boronic acid pinacol

ester 2.28 gave final compounds NEU-5374 (2.31b) and NEU-5377 (2.31a) in moderate yields.

81

Scheme 2-8. Synthesis of 1-methylhomopiperazine tail group.

Reagents and conditions: a) 1-methyl-1,4-diazepane, diisopropylethylamine, THF/IPA (1:1), 80

°C, 18 hr (91%); b) B2pin2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 85 °C, 18 hr (assumed

quantitative).

Scheme 2-9. Synthesis of 6-position substituted matched pairs of NEU-1912 and NEU-4363.

Reagents and conditions: a) POCl3, reflux, 18 hr (80%); b) pyrazin-2-amine or pyrimidin-4-

amine, NaH, DMF, rt, 48 hr (36%, 27% respectively); c) 2.28, Pd(PPh3)4, Cs2CO3, EtOH/H2O

(1:1), 85 °C, 12 hr (50%, 42% respectively).

The 7-position matched pair analog NEU-5030 was synthesized via a different route than

the other matched pairs via Scheme 2-10. First, 7-bromoquinolone was coupled with tail group

2.28 using Suzuki conditions. The quinolone 2.32 was then chlorinated using phosphorus

oxychloride in moderate yield. Similar aromatic substitution conditions as above were used to

82

attach the pyrimidine-4-amine head group, which was successful to provide the final compound

NEU-5030 (2.34) but was low yielding.

Scheme 2-10. Synthesis of 7-position substituted matched pair of NEU-1912 and NEU-4363.

Reagents and conditions: a) 2.28, PdCl2(dppf)·CH2Cl2, Cs2CO3, dioxane, 90 °C, 12 hr (77%); b)

POCl3, reflux, 18 hr (69%); c) pyrimidin-4-amine, NaH, DMF, 90 °C, 12 hr (14%).

2.4.2 Evaluation of crossovers

Previously, the replacement of 1-methylpiperazine (NEU-1953) with 1-

methylhomopiperazine (NEU-4363, Aq. Sol. = 985 μM) was shown to significantly increase the

aqueous solubility of the scaffold. It was hypothesized that the tail-group from NEU-4363 could

be used to increase the aqueous solubility of the NEU-1912 scaffold as well, while maintaining

the higher potency against T. brucei of NEU-1912. Matched pairs of NEU-1912 and NEU-4363

were therefore synthesized varying the substitution pattern and head groups. Using the NEU-4363

head group (pyrazine-2-amine) and substitution of the tail group at the 6-position (the substitution

83

pattern of NEU-1912) (NEU-5377) showed almost 1000 times higher solubility than NEU-1912

and was comparable to NEU-4363. However, there was a 2-log unit loss in potency of NEU-5377

as compared to NEU-1912 and 1-log unit loss in potency as compared to NEU-4363. The clearance

rate of NEU-5377 was lower when substitution was at the 6 versus 7 position.

Varying the head group in the matched analog to NEU-4363, compound NEU-5030,

showed similar in vitro results as NEU-4363. Changing both the head group and substitution

position of NEU-4363 as seen in NEU-5374 was the most detrimental to potency but had similar

solubility and clearance rates to that of NEU-4363. All the matched pairs showed an improvement

in aqueous solubility (as compared to NEU-1912) and reduction in potency as compared to both

parent molecules.

Table 2-10. Evaluation of NEU-1912 and NEU-4363 matched pairs.

Compound Tail

group

Tail

pos. R

T.b.b.

pEC50

(µM)

Aq. Sol.

(µM)

Human

PPB

(%)

HLM Clint

(µL/min/mg

of protein)

HepG2

pTC50 LLE

NEU-1912 A 6 C 7.70 1.4 96 210 < 4.47 5.0

NEU-5374

(2.31b) B 6 C 5.20 907 61 87 < 4.44 2.5

NEU-5030

(2.34) B 7 C 6.26 933 66 74 < 4.44 3.6

NEU-5377

(2.31a) B 6 D 5.49 990 81 48 < 4.44 3.4

NEU-4363* B 7 D 6.79 985 75 77 4.48 4.7 *Synthesized by Dr. Lori Ferrins

84

2.5 Evaluation of lapatinib analogs against other parasites

2.5.1 Evaluation of lapatinib analogs against Trypanosoma cruzi

Compounds synthesized and discussed above were also screened against Trypanosoma

cruzi (Table 2-11), the causative agent of Chagas disease. Many of the above synthesized analogs

had favorable hit activity inactive against T. cruzi (see hit criteria Table 1-2). NEU-5535 (pEC50

= 6.48) showed the most promise for development having the highest potency, being selective over

host cells, and showing a moderate LLE value (3.67). As shown in Table 2-9, NEU-5535 has a

moderate in vitro clearance rate of 43 µL/min/mg of protein, and acceptable levels of plasma

protein binding (72%) and aqueous solubility (140 μM). These criteria would make NEU-5535 a

good molecule to pursue for further optimization to increase T. cruzi potency and selectivity and

reduce clearance rates.

85

Table 2-11. Evaluation of lapatinib analogs against Trypanosoma cruzi and T. cruzi host cells

(C2C12).

Compound T. cruzi

pEC50

C2C12

pTC50 T. cruzi LLE

T. cruzi

selectivity

(pEC50-pTC50)

NEU-5144 < 5.00 < 5.00 < 1.24 0

NEU-5375 < 5.00 5.74 < 1.92 < -0.74

NEU-4987 5.68 5.41 3.30 0.27

NEU-5139 < 5.00 5.02 < 3.08 < -0.02

NEU-5326 5.94 5.42 3.55 0.51

NEU-5376 < 5.00 5.09 < 2.31 < -0.09

NEU-6734 pending pending pending pending

NEU-5537 5.89 5.39 3.78 0.50

NEU-6735 pending pending pending pending

NEU-5536 5.73 5.78 3.22 -0.05

NEU-5825 5.02 5.65 2.97 -0.63

NEU-5534 5.78 < 5.00 3.27 > 0.77

NEU-5535 6.48 < 5.00 3.67 > 1.48

NEU-5824 5.33 5.23 2.45 0.09

NEU-5377 < 5.00 5.01 < 2.85 < -0.0

NEU-5374 < 5.00 < 5.00 < 2.34 0

NEU-5030 < 5.00 < 5.00 < 2.34 0

2.5.2 Evaluation of lapatinib analogs against Leishmania

Most compounds synthesized and discussed above were also screened against Leishmania

major and some against L. donovani (Table 2-12), causative agents of Leishmaniasis. All of the

lapatinib analogs that were tested were shown to be inactive against L. major, L. donovani or both

except NEU-5139 which had a favorable potency for L. donovani hit criteria ( hit criteria found in

Table 1-2). Thought NEU-5139 has favorable potency, selectivity for host cells would need to be

addressed if pursued further.

86

Table 2-12. Evaluation of lapatinib analogs against Leishmania and B10R host cells.

Compound L. donovani

pEC50

L. donovani

LLE

B10R

pTC50

L. donovani

selectivity

(pEC50 – pTC50)

L. major

amastigote

pEC50

NEU-5144 < 5.00 < 1.24 5.64 < -0.638 -

NEU-5375 < 5.00 < 1.92 6.00 < -1.00 5.65

NEU-4987 < 5.00 < 2.61 5.41 < -0.407 4.59

NEU-5139 5.39 3.47 5.44 -0.0428 4.61

NEU-5326 5.06 2.68 5.50 -0.438 4.62

NEU-5376 < 5.00 < 2.31 5.01 < -0.006 -

NEU-6734 pending pending pending pending pending

NEU-5533 - - - - 4.65

NEU-5537 - - - - 4.67

NEU-6735 pending pending pending pending pending

NEU-5536 - - - - 4.67

NEU-5825 < 5.00 < 2.95 5.43 < -0.429 4.66

NEU-5534 - - - - 4.67

NEU-5535 - - - - 4.67

NEU-5824 < 5.00 < 2.13 < 5.00 0 4.68

NEU-5377 < 5.00 < 2.85 5.19 < -0.188 4.61

NEU-5374 < 5.00 < 2.34 5.03 < -0.0302 4.61

NEU-5030 < 5.00 < 2.34 < 5.00 0 < 4.43

2.5.3 Evaluation of lapatinib analogs against P. falciparum

Compounds synthesized and discussed above were also screened against 3 strains of

Plasmodium falciparum, the causative agent of malaria (D6, W2, and C235 strains, Table 2-13).

All compounds tested were active against the three strains of P. falciparum. NEU-1953 saturated

head group matched pairs were more potent than the NEU-1912 saturated head group matched

pairs. Matched pairs of NEU-1912 and NEU-4363 were moderately potent against the 3 strains of

P. falciparum. Compounds with the NEU-1953 scaffold and saturated head groups would be good

starting points for further optimization, focusing on the high clearance rates. However, analogs

with the NEU-1912 scaffold and saturated head groups containing nitrogen atoms (NEU-5537,

87

NEU-6735, and NEU-5536) may be a more favorable starting point for P. falciparum optimization

because of their good potencies, high aqueous solubility, and moderate clearance rates.

Table 2-13. Evaluation of lapatinib analogs against common strains of P. falciparum.

Compound P. falciparum

D6 pEC50

P. falciparum

W2 pEC50

P. falciparum

C235 pEC50

NEU-5375 7.28 7.41 7.17

NEU-4987 7.62 7.60 7.60

NEU-5139 7.60 7.32 7.64

NEU-5326 7.58 7.36 7.57

NEU-5376 7.43 7.10 7.39

NEU-6734 pending pending pending

NEU-5533 7.32 6.83 7.30

NEU-5537 7.37 6.98 7.28

NEU-6735 pending pending pending

NEU-5536 7.52 6.90 7.08

NEU-5825 6.69 6.40 6.53

NEU-5534 6.44 6.16 6.45

NEU-5535 6.39 6.15 6.43

NEU-5824 7.12 6.76 6.97

NEU-5377 6.84 6.49 6.74

NEU-5374 6.61 6.06 6.44

NEU-5030 6.79 6.64 6.84

2.6 Summary

Lapatinib was previously shown to be active against T. brucei, the causative agent of

HAT. Several rounds of optimization led to the advanced hit compounds NEU-1912 and NEU-

1953. Though, these compounds had low solubility that was not driven by lipophilicity. Various

strategies were pursued to increase the aqueous solubility of these lead compounds, two of which

are described above, salt formation and increasing sp3 atom content, and summarized in Figure

2-7.

88

Figure 2-7. Summary of modifications made to parent compounds NEU-1912 and NEU-1953.

Salts of basic compounds NEU-1912 and NEU-1953 were formed by mixing the parent

compounds dissolved in an organic solvent with various acids. Forming salts of NEU-1912 and

NEU-1953 were unsuccessful at increasing the thermodynamic aqueous solubilities.

Thermodynamic solubility is important to increase because compounds are tested in vitro and in

vivo after reaching an equilibrium in solution. However, salt formation has a greater effect on

89

increasing dissolution rates (kinetic solubility). In the future, a more in depth look at pH of salt

formation solutions would need to be performed though should not be a high priority for this class

of compounds.

Replacement of the aromatic head groups with various saturated, cyclic amines was carried

out by two synthetic means: using Buchwald coupling chemistry and SNAr conditions. Buchwald

conditions were screened and resulted in two final compounds, but products were difficult to purify

due to the inability to separate the highly soluble final compounds with the de-halogenated starting

material which was a side product of the reaction. SNAr conditions were found to be more

successful and more easily purified using column chromatography.

A small library of NEU-1912 and NEU-1953 matched pair analogs were synthesized and

were successful at improving the aqueous solubility of these compounds. However, none of the

saturated head group analogs were sufficient for further progression as HAT therapies due to low

potency against T. brucei or unfavorable clearance rates. One of the saturated head group analogs,

NEU-5535 did show moderate potency against T. cruzi and could be a good starting point for

further optimization against this parasite. All of the saturated head group analogs were potent

against 3 strains of P. falciparum, NEU-5537, NEU-6735, and NEU-5536 being the most

promising compounds, with moderate clearance rates.

Matched pair analogs of NEU-1912 and advanced lead compound NEU-4363 were also

synthesized to evaluate the effect of the 1-methylhomopiperazine moiety on solubility. All

matched pairs showed improved aqueous solubility but reduced potency against T. brucei as

compared to their parent analogs.

90

The future of this work would likely consist of synthesizing more analogs with increased

sp3 carbon content focusing on improving potency against T. brucei and in vitro clearance rates.

Other work would utilize the above-mentioned compounds for further development against T.

cruzi and P. falciparum. Development of structure-activity relationships for these parasites would

be the first step, utilizing the improved physicochemical properties. More broadly, this work shows

that increasing the sp3 carbon content is beneficial to improving aqueous solubility. This strategy

could therefore be used to improve the aqueous solubility of other previously synthesized lapatinib

analogs with varying scaffolds.

91


drug discovery

3.1 Introduction

3.1.1 High-throughput screen of a kinase targeted library against T. brucei in

collaboration with GSK

As previously discussed, the pathogenic parasites targeted in the Pollastri lab have similar

enzymes among one another as well as similar enzymes to humans. One such essential target class

is the kinase family of enzymes.86 The overlap of these targets in parasites and humans led to the

idea to use a lead repurposing45 approach to discovering new hit compounds that inhibit T. brucei.

The work was performed in collaboration with GSK and the OpenLab Foundation.

The study began with the selection of a suitable kinase-targeted compound library to test

in the HTS.99 The overall library of 42,444 compounds was composed of three different subsets.

One subset of 2,979 members, was composed of compounds from the GSK screening collection

that had structural similarity with a Tanimoto score greater than 70% to compounds that were

previously reported as inhibitors of PI3K/mTOR.143 The second subset of 367 members, was

composed of compounds from the published kinase inhibitor set from GSK.144 Finally, the last

subset of 39,098 members, were general kinase inhibitors from the GSK screening collection.99

The total compound library was tested in HTS format in a single concentration (4 μM)

whole-cell assay against T. b. brucei Lister 427 strain.143, 145 The growth inhibition of the parasites

was assessed in the log-phase. From the original set, 6,368 compounds showed greater than 50%

growth inhibition. These compounds were retested to confirm their activities resulting in 4,574

compounds that were progressed to dose response studies against T. b. brucei and HepG2 host

92

cells. Compounds that had a pEC50 ≥ 6 and were 100-fold more selective for T. b. brucei inhibition

over HepG2 cells were selected for progression (797 compounds).99

3.1.2 Organization of HTS hits and identification of NEU-1200 as a potent

inhibitor of T. brucei growth

Progressed compounds were grouped into clusters based on structural similarities resulting

in 59 clusters and 53 singleton compounds. These compounds were subjected to cidality and rate

of action assays, and chemical properties were computed to aid in the prioritization of clusters for

advancement.

A cluster of 2,4-subsituted 1H-pyrrolo[2,3b] pyridines, colloquially known as 2,4-

substituted azaindoles (cluster 32), was ultimately chosen for prioritization. Various factors were

considered during prioritization including ligand efficiency (LE),107 lipophilic ligand efficiency

(LLE),146 aqueous solubility, multi-parameter optimization (MPO) score,147 cidality, and speed-

to-kill and are displayed in Table 3-1. A LE > 0.3 is considered a good starting point for chemical

optimization and is the characterization of efficiency based on the heavy-atom count in the

molecule ((1.37*pEC50)/number of heavy atoms).107 LLE (pEC50 – clogP) takes into account

whether favorable potency results are caused by effective lipophilic within the biological target(s)

rather than simply escaping a hydrophilic environment; the targeted LLE value is > 4.109 Lastly,

MPO scores consider multiple chemical properties (shown in Table 3-2) and is thought to be a

good predictor of a compound’s ability to enter the brain.147

93

Table 3-1. Compound properties for a cluster of 2,4-substituted 1H-pyrrolo[2,3b]pyridines and

selected compound NEU-1200.

Property Desired value NEU-1200

value

Cluster average

(23 compounds)

T. b. brucei pEC50 > 7 7.73 6.6

HepG2 pTC50 < 5 4.30 4.2

T. b. gambiense pEC50 > 7 7.93 -

T. b. rhodesiense pEC50 > 7 8.20 -

MW (g/mol) ≤ 360 331 360

clogP ≤ 3 2.81 2.2

logD (7.4) ≤ 2 1.41 2.8

HBD ≤ 0.5 1 -

pKa ≤ 8 8.8 -

TPSA (Å2) 40 < TPSA ≤ 90 49.74 81

MPO score ≥ 5 5.4 3.8

T. b. brucei pEC50

(18h)

≥ 6 6.89 -

T. b. brucei pEC99 ≥ 6 6.57 -

LE ≥ 0.3 0.42 0.32

LLE ≥ 4 4.52 3.2

Human PPB (%) < 90 80 -

Aqueous Solubility

(μM)

> 50 630 >10

Fast Yes Yes 52%

Cidal Yes Yes 13%

Table 3-2. Multi-parameter optimization (MPO) scoring for predicting CNS exposure.

Property Desirable range NEU-1200 value (score)

clogP ≤ 3 2.8 (1)

clogD ≤ 2 1.41 (1)

MW (g/mol) ≤ 360 330 (1)

TPSA (Å2) 40 < TPSA ≤ 90 49.74 (1)

HBD ≤ 0.5 1 (0.833)

pKa ≤ 8 8.8 (0.600)

Score sum 5.4

94

Compound rate-to-kill was determined using an assay that quantified, at increasing

incubation times, living cells (based on ATP content).148 Cell viability was assessed at 6, 12, 18,

and 24 hrs. Compounds tested that showed low cell viability before 18 hours were considered to

be fast acting (pEC50>6 at 18 hr). An additional assay was performed to determine cidality (or

irreversibility), where the compounds were washed out from the cell culture medium after 18 hours

of incubation and cell density data was continued to be collected after washout to see if growth

recovery occurred. A minimum trypanocidal concentration is considered to be the compound

concentration that stops 99% of the growth recovery or pEC99. Compounds were deemed cidal if

their pEC99 > 6 after 72 hrs of incubation.99

The 2,4-substitued azaindole cluster 32 showed good overall properties with compounds

that were both fast-acting and cidal. The cluster has an average MPO score of 3.8, the cluster

average LE is good, and the LLE is modest. Lastly, this cluster had an average aqueous solubility

of 10 μM. Some representative structures from the initial cluster can be seen in Table 3-3. The

best compound from the series was NEU-1200 (Table 3-1) which was progressed to a mouse

pharmacokinetic (PK) study.

95

Table 3-3. Subset of cluster 32 compounds from initial HTS typified by a 2,4-substituted

pyrrolo[2,3-b]pyridine core.

NEU-

#### R1 R2 R3

T.b.b

pEC50 LLE

Aq.

Sol.

(μM)

MPO

score

1200

-H 7.73 4.52 630 5.44

1878

-H 6.93 3.72 >200 4.65

1201

-Cl 7.44 3.71 32 4.34

1705

-H 6.64 1.96 nd 5.22

1700

-H 6.70 4.22 219 4.24

1702

-H 6.77 3.55 286 4.28

1699

-Cl 6.68 4.67 298 4.50

1711

-H 6.53 2.50 273 3.50

1708

-H 7.18 0.94 nd 2.03

96

1202

-H 7.64 4.35 28 3.87

1707

-H 6.13 3.46 24 2.56

1204

-H 6.26 4.05 146 3.54

1205

-H 6.54 5.23 834 4.48

1703

-H 6.04 2.62 >400 4.28

3.2.3 In vivo pharmacokinetic (PK) evaluation of NEU-1200

NEU-1200 had promising properties and was therefore tested in a mouse PK model. The

results obtained for NEU-1200 following intravenous (IV; 1 mg/kg) administration to female

97

NMRI mice (n=3) are shown in Table 3-4. The PK parameters have been obtained from

concentration versus time profiles measured in whole blood. NEU-1200 exhibits moderate to high

clearance, high volume of distribution, and a short half-life.

Table 3-4. PK evaluation of NEU-1200 following IV administration.

Mean Standard Deviation

t1/2 (hr) 1.1 0.4

Cl (mL/min/kg) 101.0 4.8

AUC(0-∞) (hr*ng/mL) 165.2 7.8

Vss (L/kg) 9.0 1.5

The results obtained for NEU-1200 following intraperitoneal (IP; 5 mg/kg, saline, pH 4.8)

administration to female NMRI mice (n=3) are shown in Table 3-5. The PK parameters have been

obtained from concentration versus time profiles measured in whole blood. NEU-1200 exhibits an

average Cmax of 393 ng/ml and an average AUC (0-∞) of 614 hr*ng/mL.

Table 3-5. PK evaluation of NEU-1200 following IP administration.

Mean Standard Deviation

t1/2 (hr) 1.2 0.4

Cmax (ng/mL) 393.0 66.9

AUC(0-∞) (hr*ng/mL) 613.9 50.2

3.2 Design, synthesis, and evaluation of NEU-1200 first-generation analogs

3.2.1 Rationale of NEU-1200 first-generation analog design

Results from the PK study showed that NEU-1200 was highly cleared due to the metabolic

liabilities in the structure. The areas predicted to be most metabolically labile (using MetaSite,

98

predictive metabolism software in use at GSK) are shown in Figure 3-1. Not surprisingly, the most

metabolically labile structure is the methyl on the pyrazole.

Figure 3-1. Predicted metabolically labile sites of NEU-1200. Image generated using MetaSite.

During oxidative metabolism cytochrome P450 enzymes catalyze the oxidation of drugs in

the liver.149 N-demethylation is a well-known metabolic pathway in the breakdown of drugs in the

body.149 Benzylic positions, like the one depicted by a lighter red circle in Figure 3-1, are also

common areas of cytochrome P450 oxidation due to the activation of the carbon by a heteroatom

and sp2 hybridized carbon adjacent to the benzylic position.149 Another oxidative metabolism

product could be the formation of the N-oxide of one or more of the various nitrogen atoms in the

molecule.149

Based on these predictions, compounds were designed to vary the amine pendant at the 2-

position and the pyrazole at the 4-position, in order to block metabolism from occurring at these

sites. Other compounds were synthesized due to the likelihood that they would be metabolites of

the parent compound, and we wished to know whether these metabolites might have biological

activity themselves. Other compounds were synthesized to identify a pharmacophore and to

Dark blue = highest metabolic liability

Dark red = next highest

99

elucidate structure-activity (SAR) and structure-property relationships (SPR). Figure 3-2 shows

the summary for the plan of modifications to be made.

Figure 3-2. Summary of analog design plans.

3.2.2 Synthesis of NEU-1200 and first-generation analogs

The synthesis of boronate coupling partners 3.2a-c for future Suzuki couplings were carried

out via the sequence in Scheme 3-1. Chlorine displacement was carried out using the appropriate

amine. The resulting bromide intermediates 3.1a-c were then borylated using Miyaura borylation

conditions.

100

Scheme 3-1. Synthesis of intermediates 3.2a-c.

Reagents and conditions: a) ethanolamine, triethylamine, anhydrous DCM, -10 °C to rt, 18 hr

(82%); b) secondary amine, THF, rt, 18 hr (82%); c) B2Pin2, PdCl2(dppf)·CH2Cl2, KOAc, dioxane,

80-85 °C, 18 hr (90%).

The first round of analogs synthesized were truncated analogs. The synthesis of analogs

truncated at the 2-postion (Scheme 3-2) started with 4-bromo-7-azaindole or tosyl-protected

bromide 3.3, which were coupled to various boronic acids and boronic acid pinacol esters using

Suzuki conditions in various yields. Tosylated analogs were tested in vitro (Table 3-6) and then

detosylated using 3.0 M aqueous sodium hydroxide at 150 °C in the microwave to give final

compounds 3.5a and 3.5b (Table 3-6).

Scheme 3-2. Synthesis of analogs truncated at the 2-position.

Reagents and conditions: a) various boronic acids/boronic acid pinacol esters, Na2CO3,

PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 130-150 °C μwave, 10-30 min (17-86%).

101

Preparation of benzylamine boronate coupling partners were synthesized according to

Scheme 3-3. Starting with 4-bromobenzyl bromide, the benzyl bromide was substituted with

dimethylamine or diethylamine to give intermediates 3.6a and 3.6b, respectively. The resulting

bromide was then subjected to Miyaura borylation conditions to give the appropriate boronic acid

pinacol ester.

Scheme 3-3. Synthesis of boronate coupling partners 3.7a,b.

Reagents and conditions: a) 4-bromobenzylbromide, dimethylamine or diethylamine, hexanes, 0

°C – rt, 18 hr (92%, 70% respectively); b) B2Pin2, PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 80-85 °C,

18 hr (yield assumed quantitative).

Analogs truncated at the 4-position (Scheme 3-4) were synthesized starting with 7-

azaindole which was protected using tosyl chloride under basic conditions. The 2-postion was

selectively iodinated via deprotonation using LDA and treatment with molecular iodine. Iodinated

compound 3.9 was coupled in good yields to various boronic acids and esters including those

synthesized above according to Scheme 3-3. The resulting tosylated compounds (which were also

tested in vitro, Table 3-6) were subsequently deprotected under basic conditions and are evaluated

in Table 3-6.

102

Scheme 3-4. Synthesis of analogs truncated at the 4-position.

Reagents and conditions: a) 4-methylbenzenesulfonyl chloride, tetrabutylammonium

hydrogensulfate, NaOH, DCM, rt, 12 hr (73%); b) n-butyllithium, diisopropylamine, I2, THF, -78

°C – rt, 12 hr (39%); c) various boronic acids/ pinacol esters, Cs2CO3, PdCl2(dppf)·CH2Cl2, 3:1

dioxane/H2O, 150 °C μwave, 10 min (80%), d) 3M NaOH, dioxane, 150 °C μwave, 15 min.

Compounds modified at the 4-position started with 4-chloro-7-azaindole which was

protected with tosyl chloride as shown in Scheme 3-5. Where previous schemes utilized the

bromo-azaindole (3.3), this scheme utilized the chloride 3.12 to aid in the purification of compound

3.13 (as the corresponding bromide starting material co-eluted with the desired product). The

crystal structure (obtained by Dr. Bo Li at Boston College) of intermediate 3.13 was obtained to

confirm the position of iodination (Figure 3-3). Using compound 3.13 led to an increase in the

regioselectivity of the first Suzuki reaction, occurring first at the 2-postion to give compound 3.14.

With a bromine at the 4-postion, test reactions showed a mixture of products, including reaction

at both the 2- and 4-positions. Chloride 3.14 was subsequently reacted with various boronic acids

and esters to give tosyl protected compounds 3.15a-e. Finally, the tosylated compounds were

deprotected under basic conditions to yield final compounds 3.16a-e which are evaluated in Table

3-7.

103

Scheme 3-5. Initial synthesis of analogs with modifications at the 4-position.

Reagents and conditions: a) 4-methylbenzenesulfonyl chloride, tetrabutylammonium

hydrogensulfate, NaOH, DCM, rt, 12 hr (81%); b) lithium diisopropylamide, I2, THF, -78 °C – rt,

12 hr (88%); c) 3.7a, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 80 °C, 12 hr (76%), d)

various boronic acids/boronic acid pinacol esters, Na2CO3,

tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 90 °C, 48 hr (28-72%), e) 3M NaOH,

dioxane, 150 °C μwave, 15 min (33-63%).

Figure 3-3. Crystal structure of 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b] pyridine. Obtained by

Dr. Bo Li, [Boston College]

104

Other final compounds modified at the 4-position were prepared via the modified synthetic

route shown in Scheme 3-6 which utilized the high-yielding aldehyde intermediate 3.17. Boronic

acids/pinacol esters were coupled at the 4-position in moderate yields to give aldehyde

intermediates 3.18a-f. Reductive amination with dimethylamine hydrochloride salt gave protected

intermediates 3.19a-f. Detosylation under basic conditions afforded the requisite final compounds

in varying yields which are evaluated in Table 3-7.

Scheme 3-6. Modified synthesis of compounds with 4-position modifications.

Reagents and conditions: a) (4-formylphenyl)boronic acid, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1

dioxane/H2O, 85 °C, 12 hr (95%); b) various boronic acids/boronic acid pinacol esters, Na2CO3,

tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 90 °C, 48 hr (39-85%); c)

dimethylamine hydrochloride, triethylamine, NaBH4, DCE, rt, 12 hr (13-84%); d) 3M NaOH,

dioxane, 150 °C μwave, 15 min (10-92%).

The next type of compounds to be synthesized were the 2,4-subsituted analogs that were

modified at the pendant dimethylamine of NEU-1200. The first synthesis attempt (Scheme 3-7)

started with previously-synthesized compound 3.2 and iodination at the 2-position in a similar

fashion as shown in Scheme 3-3. This would have allowed for a handle to perform Suzuki

couplings with various boronic acids/pinacol esters, which would have facilitated parallel

105

synthesis and late stage diversification. Unfortunately, the iodination reaction did not go to

completion and the starting material (3.4a) and product (3.21) were inseparable using

chromatography.

Scheme 3-7. Attempted synthesis of analogs with modifications at the 2-position.

Reagents and conditions: a) n-butyllithium, diisopropylamine, I2, THF, -78 °C – rt, 12 hr; b)

various boronic acids/pinacol esters, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 80 °C, 12

hr; c) 3M NaOH, dioxane, 150 °C μwave, 15 min.

The first successful synthesis for analogs with modifications at the 2-position is shown in

Scheme 3-8. Starting with intermediate 3.13, various boronic acids and pinacol esters were used

as coupling partners in Suzuki reactions, after which the resulting chlorides were subjected to a

second Suzuki reaction to yield protected compounds 3.23a-j. Final compounds were elucidated

after tosyl deprotection under basic conditions and are evaluated in Table 3-8.

106

Scheme 3-8. First successful synthesis of analogs with modifications at the 2-position.

Reagents and conditions: a) various boronic acids/pinacol esters, Na2CO3, PdCl2(dppf)·CH2Cl2,

3:1 dioxane/H2O, 80 °C, 12 hr (29-99%); b) (1-methyl-1H-pyrazol-4-yl)boronic acid pinacol

ester, Na2CO3, tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 80 °C, 12 hr (4-

99%), e) 3M NaOH, dioxane, 150 °C μwave, 15 min (21-97%).

Scheme 3-9 shows the modified synthetic route for the 2-position analogs. The

intermediate 3.23f was utilized in two different ways. In the first route, a reductive amination with

piperazine was performed to yield intermediate 3.25. which could then be deprotected under basic

conditions to yield final compound 3.26a. Alternatively as a means to diversify at the last possible

step in the synthesis, 3.23f could be deprotected first followed by various reductive aminations to

yield final compounds 3.26b-k evaluated in Table 3-8.

107

Scheme 3-9. Modified synthesis of analogs with modifications at the 2-position.

Reagents and conditions: a) various amines, NaBH4, DCE, rt, 12 hr (10-64%); b) 3M NaOH,

dioxane, 150 °C μwave, 15 min (37% or 79%).

3.2.3 Evaluation of NEU-1200 first-generation analogs

Truncated matched pair analogs (Table 3-6) were synthesized using similar substituents as

presented in the initial cluster. Tosylated intermediates of these compounds were also tested for T.

brucei inhibition. NEU-4472 is the 2-postion truncated analog and NEU-4844 is the 4-position

truncated analog of parent compound NEU-1200. Truncation in either position results in loss of

activity against T. brucei. Tosylated intermediate (NEU-4877) to NEU-1200 also shows loss of

activity. These data suggest that substitutions at both positions are needed in order to retain activity

against the parasite.

108

Table 3-6. Activity of truncated analogs of NEU-1200 against T. brucei.

Entry R1 R2 R3 T.b.b

pEC50

NEU-1200

-H

7.73

NEU-4472

3.3

-H -H

4.92

NEU-4813

3.2

-Ts -H

<4.40

NEU-4877

-Ts

5.24

NEU-4844

3.17

-H

-H 5.23

NEU-4845

3.18

-H

-H 4.80

NEU-4812

3.16

-Ts

-H 4.40

NEU-4815

3.5

-H -H

<4.40

NEU-4814

3.4

-Ts -H

4.81

109

NEU-4786

3.6

-H -H

4.75

NEU-4471

3.7

-H -H

<4.88

Table 3-7 shows the modifications made to NEU-1200 at the 4-position of the molecule.

NEU-4928 was synthesized as a potential metabolite of NEU-1200 and had similar potency. The

HLM clearance is higher than that of NEU-1200. Methylated pyrazole analogs NEU-4929 and

NEU-4927 have similar activities to NEU-1200. NEU-4927 had a similar clearance rate in rat

hepatocytes as compared to parent compound. The phenyl methyl sulfone compound NEU-4917

showed a slight reduction in potency, though is still acceptable, and also showed a moderate

reduction in clearance rates against both human liver microsomes and rat hepatocytes.

Sulfonamides NEU-5828 and NEU-5829 showed a log unit decrease in potency against T. brucei

and NEU-5828 had increased clearance rates. Pyridyl compound NEU-5446 was less potent and

had higher clearances rates. Methyl substituted pyridines (NEU-5449 and NEU-5452) were

similar in potency but had similar clearance rates to matched analog NEU-5446. The increased

clearance rates of the 4-position modified analogs that do not contain pyrazoles suggest that the

primary metabolism of those compounds occurs at the dimethylamine pendant area of the

molecule.

110


1200.

Entry R T.b.b

pEC50

HLM Clint

(µL/min/mg

of protein)

RH Clint

(µL/min/106

cells)

Hit

Criteria > 7 ≤ 47 ≤ 27

Lead

Criteria > 7.5 ≤ 9 ≤ 5

NEU-1200

7.73 25 25

NEU-4927

7.96 25 19

NEU-4928

7.85 64 26

NEU-4929

7.52 34 34

NEU-4917

7.17 18 13

NEU-5828

6.31 45 37

111

NEU-5829

6.32 nt nt

NEU-5446

6.70 19 38

NEU-5447

6.75 56 64

NEU-5449

6.83 43 38

NEU-5452

6.27 42 19

nt = not tested

The 2-postion of the azaindole was explored through various modifications keeping the

pyrazole in place for matched pair analysis (Table 3-8). Removal of the methylene carbon (NEU-

5323) drastically increased clearance and resulted in a 10x loss in potency. Extension of the

dimethyl to the diethyl (NEU-4973) resulted in similar potency and clearance rates to NEU-1200.

Tying the ethyl chains into a pyrrolidine ring (NEU-5123) was beneficial to reducing clearance

rates in both human liver microsomes and rat hepatocytes. Methylation of the benzyl position

(NEU-6072) yields a similar microsomal clearance rate and reduced rat hepatocyte clearance rate

as compared to NEU-1200 but did not show extra benefit to clearance reduction as compared to

matched pair NEU-5123. NEU-4995 extends the pyrrolidine ring to a piperidine which slightly

increased the potency but also the human liver microsomal clearance (pEC50=7.92, HLM Clint=30

112

μL/min/mg protein). Piperazine analog NEU-5936 was similar to the piperidine in terms of

clearance but showed 10-fold improvement in potency (pEC50=8.70). Though still active

(pEC50=7.70), methylation of the piperazine (NEU-6076) loses the improvement of potency

afforded by NEU-5936 but improves the rat hepatocyte clearance rate to an acceptable level (6.5

μL/min/106 cells). Replacing the nitrogen of the piperazine with an oxygen to form a morpholine

(NEU-5937) increased the clearance rates well above the acceptable level.


1200.

Entry R T.b.b.

pEC50

HLM Clint

(µL/min/mg

of protein)

RH Clint

(µL/min/106

cells)

Hit

Criteria > 7 ≤ 47 ≤ 27

Lead

Criteria > 7.5 ≤ 9 ≤ 5

NEU-5323

6.85 110 300

NEU-1200

7.73 25 25

NEU-4973

7.76 28 17

NEU-5123

7.71 13 20

NEU-6072

7.77 28 15

113

NEU-4995

7.92 30 19

NEU-5936

8.70 35 21

NEU-6076

7.70 27 6.5

NEU-5937

7.48 75 140

NEU-5938

8.16 18 16

NEU-5124

7.62 3.0 17

NEU-6018

8.26 <3.0 14

NEU-6048

7.24 54 >300

NEU-6025

6.82 74 16

NEU-6044

7.69 13 12

NEU-6049

7.78 5.2 9.4

NEU-6047

7.26 92 >300

NEU-4974

7.30 nda nda

NEU-5448

5.26 ndb ndc

NEU-5306

7.23 27 5.3

114

NEU-6073

7.06 46 8.4

NEU-4918

6.73 77 160

nd = not determined; acompound detected only in first sample; bpoor optimization; cfailed to create

analytical method.

The next set of 2-position modifications explored various secondary amines (Table 3-8).

Removal of one of the methyl groups from NEU-1200 resulted in analog NEU-5938 which

showed almost a half of a log unit increase in potency (pEC50 = 8.16) and an acceptable reduction

in intrinsic clearance rates. The best compound of this series was NEU-6018, which extended the

methyl chain of NEU-5124 to an isopropyl substituent. NEU-6018 had an improved pEC50 of 8.26,

human liver microsome clearance rate (< 3.0 µL/min/mg protein), and rat hepatocyte clearance

rate (14 µL/min/106 cells). Though not as potent as NEU-6018, pyrrolidine ethyl amine NEU-

6044, and a pyrrolidine analog attached directly to the phenyl group (NEU-6049) both had very

good clearance rates which were comparable to NEU-6018 and improved over parent compound

NEU-1200.

Compounds NEU-6047, NEU-4974, and NEU-5448 were synthesized as putative

oxidative metabolites of NEU-1200. Benzyl alcohol NEU-6047, not surprisingly, had increased

clearance rates and a reduction in potency. Aldehyde NEU-4974 and carboxylic acid NEU-5448

were both less potent than the parent compound, especially NEU-5448 by two log units. Since the

aldehyde and carboxylic acid would be the subsequent metabolite after the initial metabolism of

NEU-1200 to alcohol NEU-6047, they were not tested in the clearance assays, and assumed the

clearance rates would be lower than NEU-6047.

115

Other ADME properties, including aqueous solubility, human plasma protein binding, and

logD at pH 7.4, were also assessed for all the first-generation analogs and are shown in Table 3-

9. Promising compounds NEU-5123, NEU-5124, NEU-6018, NEU-6044, and NEU-6049 all had

very high solubilities, were not excessively protein bound, and had low logD values.

Host cell toxicities of all first-generation analogs were assessed in MRC5 human lung cell

line. Most compounds were also tested in HepG2 cells, a human liver cancer cell line. All

compounds tested showed no potent activity in these cell lines with pEC50 values less than 5.50,

shown in Table 3-10. Most first-generation compounds were 100 times more selective for T. b.

brucei inhibition versus MRC5 host cells, with 4 exceptions (NEU-5829, NEU-5452, NEU-5306,

and NEU-5448) which had difference values below 2 log units.

116

Table 3-9. Other ADME measurements of NEU-1200 and its first-generation analogs.

Entry Aq. Sol.

(μM)

Human PPB

(%)

LogD 7.4

Hit Criteria > 10 ≤ 95 ≤ 2

Lead Criteria > 100 ≤ 95 ≤ 2

NEU-1200 630 80 2.7

NEU-4917 8 87 2.9

NEU-4918 3 97 3.5

NEU-4927 1000 85 2.9

NEU-4928 483 85 2.0

NEU-4929 68 86 3.0

NEU-4973 832 89 2.9

NEU-4974 < 1 100 3.7

NEU-4995 39 91 3.4

NEU-5123 1 89 2.6

NEU-5124 841 76 1.7

NEU-5306 2 nda 2.8

NEU-5323 4 99 ndb

NEU-5446 2.9 99 3.9

NEU-5447 348 89 3.3

NEU-5449 647 93 3.7

NEU-5452 68 94 3.6

NEU-5828 < 1 94 3.4

NEU-5936 29 91 3.3

NEU-5937 2 99 3.6

NEU-5938 476 nda 1.4

NEU-6018 902 80 1.9

NEU-6025 750 nda 2.5

NEU-6044 580 nda 2.3

NEU-6047 30 nda 3.5

NEU-6048 3 97 3.5

NEU-6049 641 81 2.0

NEU-6072 733 86 3.0

NEU-6073 5 nda 3.1

NEU-6076 60 87 3.0 nd = not determined; alow recovery; bfailed to create analytical method.

117

Table 3-10. Toxicity of NEU-1200 and its first-generation analogs against HepG2 and MRC5

cell lines.

Entry HepG2

pTC50

MRC5

pTC50

Tbb pEC50 – MRC5 pTC50

NEU-1200 4.26 4.98 2.75

NEU-4927 nd 4.86 3.10

NEU-4928 4.69 5.01 2.84

NEU-4929 nd 4.82 2.70

NEU-4917 4.50 4.97 2.20

NEU-5828 5.16 <4.30 >2.01

NEU-5829 4.72 4.58 1.74

NEU-5446 nd <4.30 >2.40

NEU-5447 4.52 <4.30 >2.45

NEU-5449 4.57 <4.30 >2.53

NEU-5452 nd <4.30 >1.97

NEU-5323 4.61 <4.30 >2.55

NEU-4973 4.50 4.81 2.95

NEU-5123 4.72 4.76 2.95

NEU-4995 5.29 5.19 2.73

NEU-5936 4.57 5.79 2.91

NEU-6076 <4.59 4.92 2.78

NEU-5937 4.57 <4.30 >3.18

NEU-5938 4.50 5.46 2.70

NEU-5124 4.66 4.73 2.89

NEU-6018 4.54 5.88 2.38

NEU-6048 nd <4.78 >2.46

NEU-6025 4.56 4.78 2.04

NEU-6044 4.82 5.05 2.64

NEU-6049 nd 5.25 2.53

NEU-6072 4.84 5.05 2.72

NEU-5306 nd <5.26 >1.97

NEU-5448 nd <4.30 >0.96

NEU-4974 <5.30 <4.30 >3.00

NEU-6047 6.13 4.30 2.96

NEU-4918 nd <4.30 >2.43

NEU-6073 4.52 <4.30 2.76

118

3.2.4 Pharmacokinetic analysis of NEU-5123

Compound NEU-5123 was progressed to a mouse PK study due to its favorable in vitro

properties. First, NEU-5123 was dosed intraperitoneally at 10 mg/kg in 3 female NMRI mice.

Blood samples were taken from each of the mice to determine the Cmax, tmax, AUC, and t1/2 shown

in Table 3-11. The average Cmax value was well over 10×EC50 (7.15 ng/mL) and was reached after

30 minutes post injection. Notably, the half-life of NEU-5123 compared to NEU-1200 was

increased from 1.1 hours to 2 hours, showing that the clearance rate of NEU-5123 was improved

over NEU-1200.

Table 3-11. Individual blood pharmacokinetic parameters of evaluated compounds after


a r< 0.95 and/or only two points to define terminal half life

Measurements were also taken of drug levels in the blood versus the brain in 3 mice (Table

3-12). Measurements were taken at 0.5 hours and 4 hours after an intraperitoneal dose of 10 mg/kg.

After 30 minutes the brain to blood ratio was 0.648 which increased to 1.39 after 4 hours post

injection. Figure 3-3 shows the concentrations of NEU-5123 in the blood and brain of 3 mice.

Results show that exposure levels are well over 10×EC50 (EC50 = 7.61 ng/mL) for the duration of

the study.

Compound Cmax

(ng/mL)

tmax

(h)

AUC0-t

(ng·hr/ml)

AUC

(ng·hr/ml)

t1/2

(hr)

NEU-5123 Mean 520

0.50 1300 1500 2.0

SD 110 190 250 0.78

119

Table 3-12. Blood and brain levels (ng/mL in blood, ng/g in brain) of NEU-5123, after


LLOQ=50 ng/mL (blood).

Sampling

Time (hr) Matrix Mean SD

0.5

Blood 580 62

Brain 380 83

Brain/Blood

Ratio 0.65 0.11

4

Blood 170 2.8

Brain 230 18

Brain/Blood

Ratio 1.4 0.087

Figure 3-4. Peripheral blood levels of NEU-5123 in mice.

3.2.5 In vivo evaluation of NEU-5123

With decreased in vivo clearance rates and good exposure in the blood and brain, NEU-

5123 was progressed to an acute in vivo efficacy study. First a maximum tolerated dose (MTD)

study was performed. Mouse 1 was dosed with 5+15+30 m/kg to total 50 mg/kg/day, and Mouse

0 2 4 6 8 1 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

N E U -5 1 2 3 ; 1 0 m g /k g ip

M e a n B lo o d / B ra in c o n c e n tra t io n s

T im e (h r )

Blo

od

co

nc

en

tra

tio

n (

ng

/mL

) /

Bra

in c

on

ce

ntr

ati

on

(n

g/g

)

B lo o d C o n c e n tra tio n (n g /m L )

B lo o d C o n c e n tra tio n (n g /m L )

B ra in C o n c e n tra t io n (n g /g )

120

2 was dosed with 50+50 mg/kg to total 100 mg/kg/day. After a dose of 50 mg/kg was achieved the

mice showed toxicity and pain signs, and died within 24 hrs.

For the acute efficacy study NEU-5123 was dosed first at 10 mg/kg/day (Figure 3-4) which

showed an average extension of life of 8.8 days as compared to control mice. The dose of NEU-

5123 was then increased to 30 mg/kg/day (Figure 3-5). This higher dose resulted in the death of

all treated mice by day 5, confirming that this compound is highly toxic. This contradicts the in

vitro data which showed that NEU-5123 did not potently inhibit host cell lines and the selectivity

window was greater than 2 log units.

Figure 3-5. Efficacy of NEU-5123 in mice after a 10 mg/kg/day dose.

Efficacy Model (Acute)

Days

0 5 10 15 20 25 30

% S

urv

iva

l

0

20

40

60

80

100

Control (Vehicle) NEU5123 - 10 mg/kg/day

121

Figure 3-6. Efficacy of NEU-5123 in mice after a 30 mg/kg/day dose.

3.2.6 Kinase selectivity evaluation of NEU-5123 and NEU-5449

Since these compounds came from a kinase targeted library, it was hypothesized that they

could have off-target human kinase activity. Compounds NEU-5123 and NEU-5449 were profiled

at 5 μM for their percent inhibition against a panel of 45 human kinases (Figure 3-6). “Potent”

inhibition was defined as > 50%. NEU-5123 potently inhibited 14 kinases while NEU-5449

potently inhibited 32 kinases. This suggest further optimization is needed to tune selectivity versus

human kinases. The mouse kinome contains 510 orthologs for the 518 human kinases.150 It is

possible that the toxicity observed in the efficacy studies could be due to NEU-5123 hitting

multiple essential host kinases.

Efficacy Model (Acute)

Days

0 5 10 15 20 25 30

% S

urv

ival

0

20

40

60

80

100

Control (Vehicle) NEU5123 - 30 mg/kg/day

122

Figure 3-7. Human kinase profiles of NEU-5123 and NEU-5449.

3.3 Design, synthesis, and evaluation of NEU-1200 second-generation analogs

3.3.1 Rationale of NEU-1200 second-generation analog design

The PK study described above showed that NEU-5123 displayed reduced in vivo clearance

rates relative to NEU-1200. NEU-6018, which was synthesized after the testing of NEU-5123,

showed lower in vitro clearance rates suggesting that it would also be successful in vivo. Second-

generation analogs utilized the benzyl isopropylamine pendant at the 2-position while varying the

4-position to see if further improvements in the clearance could be obtained in combination with

the isopropylamine. NEU-6018 also showed improved activity (pEC50=8.26) over NEU-5123

(pEC50=7.71), and second-generation analogs would probe the activity range of new 4-position

123

substituents. Matched pair analogs with 4-position groups that were correlated with low compound

toxicity were synthesized in an attempt to reduce toxicity level of NEU-6018 while maintaining

its high potency. The next goal for this project would be to increase potency and selectivity to

enable lower doses in the animal studies to see a positive effect.

3.3.2 Synthesis of NEU-1200 second-generation analogs

Second-generation analogs were synthesized in a similar fashion as above (Scheme 3-10).

Starting with dihalide 3.13, Suzuki reaction at the 2-position with 4-formylphenylboronic acid

gave aldehyde 3.17 in 95% yield. Reductive amination with isopropylamine gave chloride 3.27a

in 94% yield. These high yielding reactions allowed for scale-up synthesis of intermediate 3.27a

for subsequent late stage Suzuki reactions at the 4-position (3.28a-h). Detosylation under basic

conditions gave final compounds 3.29a-3.29h. Chloride 3.17 was also coupled with two different

aryl substituents using Suzuki conditions to yield aryl aldehydes 3.27b and 3.27c. Intermediate

3.27b was first deprotected to give 3.28i followed by reductive amination with isopropylamine

(3.29i) and boc-deprotection with trifluoracetic acid to yield final compound 3.30. Aldehyde

intermediate 3.27c was coupled with pyrrolidine using reductive amination conditions. Subsequent

detosylation under basic conditions yielded final compound 3.29j. All second-generation

compounds are evaluated in Table 3-13 for their in vitro T. b. brucei activities.

124

Scheme 3-10. Synthesis of second-generation NEU-1200 analogs.

Reagents and conditions: (4-formylphenyl)boronic acid, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1

dioxane/H2O, 85 °C, 12 hr (95%); b) isopropylamine or pyrrolidine, NaBH3CN, 10% acetic

acid/methanol, 50 °C, 18 hr (94%, 93%); c) appropriate boronic acids/pinacol esters, Na2CO3,

tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 85-90 °C, 18-48 hr (42-99%); d) 3M

NaOH, dioxane, 150 °C μwave, 15 min (31-69%), e) TFA, DCM, rt, 18 hr (59%).

3.3.3 Evaluation of NEU-1200 second-generation analogs

A library of second-generation 2,4-substituted azaindole analogs were analyzed for their

T. brucei inhibition and in vitro clearance rates (Table 3-13). In general, almost all analogs

maintained or improved clearance relative to NEU-6018. Substituting the methyl on the pyrazole

with a tetrahydropyran (NEU-6509) or piperidine (NEU-6097) further improved the clearance but

potency was reduced by a log unit. Changing the attachment position of the pyrazole to the 5-

position (NEU-6508) reduced the potency two log units (pEC50=6.77) though the clearance rates

were still acceptable. The unsaturated piperidine analog (NEU-6476) had a similar human liver

microsomal clearance rate (<3 μL/min/mg protein) and a 10-fold improved clearance rate of 1.1

125

μL/min/106 cells but had a 2-log unit reduction in potency as compared to NEU-6018. Pyridine

analogs NEU-6098, NEU-6477, and NEU-6478 had moderate clearance rates and reduced

potencies. NEU-6098, which has the nitrogen atom in the 3-position, has a higher pEC50 (7.82)

than the analog with a nitrogen atom in the 4-position (NEU-6477, pEC50=7.32), suggesting that

the position of the nitrogen atom could be beneficial to making a favorable interaction with a

potential target. Finally, sulfonamide NEU-6111 and methyl sulfone analog NEU-6712 lose

potency as compared to their matched pair parent molecules (NEU-6018 and NEU-5123, Table

3-8). NEU-6475 maintains its potency as compared to its matched pair (NEU-4917, Table 3-7).

Table 3-13. Activities and clearance rates of second-generation analogs of NEU-1200.

Entry R1 R2 T.b.b.

pEC50

HLM Clint

(µL/min/mg

protein)

RH Clint

(µL/min/106

cells)

Hit Criteria > 7 ≤ 47 ≤ 27

Lead

Criteria > 7.5 ≤ 9 ≤ 5

NEU-6018

8.26 <3 14

NEU-6077

6.71 59 10

NEU-6097

7.23 <3 1.8

126

NEU-6509

7.28 6.3 7.2

NEU-6508

6.77 17 10

NEU-6476

6.73 <3 1.1

NEU-6098

7.82 27 42

NEU-6477

7.32 16 13

NEU-6478

6.58 15 7.4

NEU-6111

6.64 6.8 8.0

NEU-6475

7.15 <3 5.8

NEU-6712

6.85 pending pending

127

Toxicities for second-generation analogs can be found in Table 3-14. All compounds tested

were found to be minimally active in HepG2 toxicity assays except for parent compound NEU-

6018 (pTC50 = 4.54). Similar results for seen for MRC5 host cell toxicity. Compounds NEU-6508

and NEU-6509 were not active against MRC5 cells with pTC50 values of 4.93 and 4.68

respectively. However, second-generation compounds were less selective than their first-

generation matched pairs. Most second-generation analogs had <100x selectivity windows.

Table 3-14. Toxicity of second-generation NEU-1200 analogs against HepG2 and MRC5 cell

lines.

Entry HepG2 pTC50 MRC5 pTC50 Tbb pEC50 -

MRC5 pTC50*

NEU-6018 4.54 5.88 2.38

NEU-6077 5.06 5.05 1.66

NEU-6097 5.86 5.80 1.43

NEU-6509 - 4.68 2.60

NEU-6508 - 4.93 1.84

NEU-6476 5.06 4.86 1.87

NEU-6098 5.40 5.26 2.56

NEU-6477 5.42 5.48 1.84

NEU-6478 5.32 5.11 1.47

NEU-6111 5.17 - 1.47*

NEU-6475 5.23 5.18 1.97

NEU-6712 - - - *HepG2 pTC50 value used instead of MRC5 pTC50

We note that the mammalian cell toxicity varies significantly based on cell lines. A plot of

the toxicity values for four different host cell lines for all cluster 32 compounds synthesized can

be seen in Figure 3-7. The two alternative host cell line (L6 and THP-1) are the host cells for T.

cruzi and Leishmaniasis assays respectively (data shown in Table 3-15). This plot shows how

widely toxicity varies for most of the compounds. Further exploration of this series is therefore

needed to better understand structure-toxicity relationships.

128

Figure 3-8. Graph of multiple cell line toxicity values of all synthesized NEU-1200.

3.4 Evaluation of cluster compounds against other parasites

A selection of analogs (selected on the basis of available quantities) was screened against

other protozoan parasites when enough material was available. Table 3-15 shows the potencies of

cluster 32 compounds against Trypanosoma cruzi, Leishmania major and L. donovani.

Compounds that were active against T. cruzi showed low selectivity versus L6 host cells. Anti-

trypanosomal activity correlates with host cell toxicity as shown in Figure 3-8. A similar trend

occurs for potency and toxicity when testing compounds against L. major and THP-1 host cells.

Potent compounds against L. major were toxic against host cells, for example NEU-5447

(pEC50=7.43, THP-1 pTC50=5.66). All cluster 32 compounds tested against L. donovani were

inactive.

4

4.5

5

5.5

6

6.5

7

7.5N

EU-1

20

0N

EU-1

93

6N

EU-4

47

1N

EU-4

47

2N

EU-4

78

6N

EU-4

81

2N

EU-4

81

3N

EU-4

81

4N

EU-4

81

5N

EU-4

84

4N

EU-4

84

5N

EU-4

87

7N

EU-4

91

7N

EU-4

91

8N

EU-4

92

7N

EU-4

92

8N

EU-4

92

9N

EU-4

97

3N

EU-4

97

4N

EU-4

99

5N

EU-5

12

3N

EU-5

12

4N

EU-5

30

6N

EU-5

32

3N

EU-5

44

6N

EU-5

44

7N

EU-5

44

8N

EU-5

44

9N

EU-5

45

2N

EU-5

82

8N

EU-5

82

9N

EU-5

93

6N

EU-5

93

7N

EU-5

93

8N

EU-6

01

8N

EU-6

02

5N

EU-6

04

4N

EU-6

04

7N

EU-6

04

8N

EU-6

04

9N

EU-6

07

2N

EU-6

07

3N

EU-6

07

6N

EU-6

07

7N

EU-6

09

7N

EU-6

09

8N

EU-6

11

1N

EU-6

47

5N

EU-6

47

6N

EU-6

47

7N

EU-6

47

8N

EU-6

50

8N

EU-6

50

9N

EU-6

71

2

pTC

50

Toxicity values (in vitro)

L6 THP-1 HepG2 MRC5

129

Table 3-15. Potencies and toxicities of NEU-1200 analogs against T. cruzi and Leishmania.

Entry T. cruzi

pEC50

L6

pTC50

L. major

pEC50

L. donovani

pEC50

THP-1

pTC50

NEU-1200 - >6.21 - - >6.21

NEU-1936 5.00 4.65 - <5.30 4.35

NEU-4471 4.97 4.30 - <5.30 4.51

NEU-4472 <4.70 - - <5.30 4.49

NEU-4786 <4.70 <4.30 - <5.30 <4.30

NEU-4812 <4.70 5.05 - <5.30 <4.30

NEU-4813 <4.70 4.34 - - <4.30

NEU-4814 <4.70 - - <5.30 <4.30

NEU-4815 <4.70 <4.30 - <5.30 <4.30

NEU-4844 <4.70 <4.30 - <5.30 <4.30

NEU-4845 - >6.21 - <5.30 <4.30

NEU-4877 4.77 - - <5.30 4.51

NEU-4917 - 5.73 - - 4.68

NEU-4918 - 5.81 - - 6.01

NEU-4927 7.22 >6.21 - - >6.21

NEU-4928 7.32 >6.21 7.00 - 7.35

NEU-4929 6.62 >6.21 - - >6.21

NEU-4973 6.93 >6.21 - - >6.21

NEU-4974 5.38 4.91 - <5.30 6.03

NEU-4995 7.06 >6.21 - - >6.21

NEU-5123 6.99 >6.21 7.26 - 6.86

NEU-5124 6.83 >6.21 6.65 <5.30 >6.21

NEU-5306 5.78 >6.21 - <5.30 >6.21

NEU-5323 - >6.21 5.77 - 6.03

NEU-5446 5.87 5.39 - - 5.65

NEU-5447 6.03 5.43 7.43 - 5.66

NEU-5448 <4.70 4.46 - <5.30 4.60

NEU-5449 6.05 5.22 7.34 - 5.55

NEU-5452 5.25 4.96 - - 4.80

NEU-5828 - 5.56 5.20 - -

NEU-5829 - 5.71 4.69 - -

NEU-5936 6.92 >6.21 7.27 - >6.21

NEU-5937 6.29 6.03 6.16 - >6.21

NEU-5938 6.75 >6.21 6.82 - >6.21

NEU-6018 7.15 >6.21 7.19 - >6.21

NEU-6025 5.93 5.67 6.28 <5.30 5.74

NEU-6044 6.20 6.14 6.35 <5.30 -

NEU-6047 6.28 >6.21 6.62 <5.30 -

NEU-6048 6.41 >6.21 6.90 - -

NEU-6049 7.09 >6.21 7.49 - -

NEU-6072 7.12 - - <5.30 -

130

NEU-6073 6.40 - - - -

NEU-6076 6.93 - - - -

NEU-6077 5.56 - - - -

NEU-6097 <4.70 5.37 5.21 - 4.66

NEU-6098 - - 6.95 - >6.21

NEU-6111 - - 4.67 - -

Figure 3-9. Correlation of T. cruzi potency and L6 host cell toxicity.

Solid line = 0 selectivity; dotted line = 10x selectivity. Compounds between the two lines are < 10x.

Synthesized compounds from the cluster 32 series were also tested for their activities

against various strains of P. falciparum. The pEC50 values for D6, W2, and C235 strains were

mostly between 6 and 7 (Table 3-16). Compounds with secondary amines in the 2-position showed

slightly higher potencies than tertiary amines.

131

Table 3-16. Potencies of NEU-1200 analogs against various P. falciparum strains.

Entry P. fal D6

pEC50

P. fal W2

pEC50

P. fal C235

pEC50

NEU-4928 6.60 6.96 6.59

NEU-5123 6.83 6.67 6.59

NEU-5124 6.84 6.82 6.56

NEU-5323 6.18 6.60 5.35

NEU-5447 6.18 6.50 5.99

NEU-5449 5.98 6.22 5.72

NEU-5828 6.64 6.45 6.38

NEU-5829 7.00 7.11 6.87

NEU-5936 6.88 6.73 6.70

NEU-5937 6.31 6.25 6.05

NEU-5938 6.57 6.59 6.53

NEU-6018 7.04 6.74 6.68

NEU-6044 7.19 6.80 6.98

NEU-6047 5.90 5.86 5.70

NEU-6048 5.37 5.20 5.12

NEU-6049 6.86 6.50 6.72

NEU-6072 6.23 6.38 6.08

NEU-6073 6.31 6.39 6.24

NEU-6076 6.39 6.44 6.18

NEU-6077 6.59 6.04 6.27

NEU-6097 6.84 6.89 6.69

NEU-6098 6.50 6.59 6.50

NEU-6111 6.49 6.42 6.29

NEU-6475 6.93 6.76 6.42

NEU-6476 6.49 6.23 6.26

NEU-6477 6.89 6.73 6.58

NEU-6478 6.78 6.72 6.45

NEU-6508 6.40 6.18 6.00

NEU-6509 6.78 6.37 6.14

3.5 Summary

A lead repurposing approach was taken to screen a kinase inhibitor library against T.

brucei, the causative agent of HAT. The HTS resulted in 797 primary hit compounds, from which

a cluster of 2,4-substituted azaindoles were chosen to progress. This cluster, deemed cluster 32,

132

was chosen for its good physicochemical properties, potency and selectivity, fast-acting and cidal

nature, and predictability for brain penetration, essential for stage 2 HAT. NEU-1200 was the best

compound from the initial cluster and pursued further in a PK study. The results of the study

showed high clearance rates, most likely due to metabolism of the methyl groups associated with

the pyrazole and pendant dimethylamine.

Structural modifications were made at the 2- and 4-positions of the azaindole core with the

focus on improving the in vitro clearance rates. An SAR summary can be seen in Figure 3-10.

NEU-5123 was shown to have improved in vitro clearance rates and comparable potency with

NEU-1200 and was thus progressed to a PK study. Results showed that NEU-5123 was not rapidly

cleared and showed good exposure in the blood and brain of the mice. Upon dosing in an acute

efficacy model, NEU-5123 was shown to extend mouse survival by 8.8 days when dosed at 10

mg/kg/day, however it was found to be highly toxic when dosed at 30 mg/kg/day. A human kinase

selectivity panel showed that NEU-5123 potently inhibited 14 human kinases.

Figure 3-10. SAR/SPR summary for synthesized NEU-1200 analogs.

133

Second-generation analogs were then synthesized to test the generalizability of using the

isopropylamine pendant to keep clearance rates low, while varying the 4-position substituent in an

attempt to increase the selectivity window. Second-generation compounds were shown to have

lower selectivity windows than their matched pairs and none were progressed further. This

suggests that the substituent at the 2-position may be more important for regulating the toxicity of

these compounds.

Finally, when cross-screening these compounds against other parasites a trend was seen

for correlation of increasing potency and toxicity, especially in T. cruzi and L6 cellular assays.

Although these compounds were also potent against multiple strains of P. falciparum the high

toxicity makes them unideal for further progression. Further work must be done to understand the

structure-toxicity relationship of the compounds in this series. The overall compound progression

can be seen in Figure 3-11.

Figure 3-11. Compound progressions of the series.

134

3.6 Future Directions

The second-generation of analogs resulted in NEU-6097, which showed reduced toxicity

in L6 and THP-1 cell lines. To determine if there is a correlation between toxicity in these cell

lines and in vivo toxicity, a maximum tolerated dose study should be performed. New analogs

should be synthesized to get a better understanding of structure-toxicity relationships suggested

modifications are summarized in Figure 3-12.

Figure 3-12. Summary of future directions for analog design.

135

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Chapter 4: Experimental section

4.1 General methods

All starting materials were commercially procured and were used without further

purification, unless specified. Reaction solvents were purified by passage through alumina

columns on a purification system manufactured by Innovative Technology (Newburyport, MA).

For microwave reactions a Biotage® Initiator+ or CEM Discover® SP was used and the

absorbance was set in accordance with the recommendations set by the manufacturer. NMR

spectra were obtained on Varian NMR systems, operating at 400 or 500 MHz for 1H acquisitions.

LCMS analysis was performed using a Waters Alliance reverse phase HPLC (columns Waters

SunFire C18 4.6 × 50 mm, 3.5 μm, or Waters SunFire C8 4.6 × 50 mm, 3.5 μm), with single-

wavelength UV−visible detector and LCT Premier time-of-flight mass spectrometer (electrospray

ionization) or Waters Micromass ZQ detector (electrospray ionization), or using Agilent 1100

series LCMS instrument, with the binary system water/acetonitrile containing 0.1 % of formic acid

as eluent. Optical rotations were obtained on a Jasco P-2000. Purification of intermediates and

final compounds was performed using silica gel chromatography on a Biotage® IsoleraTM One

Flash purification system. Where required, final compounds were purified by preparative reverse

phase HPLC (columns Waters Symmetry RP8 30 × 50 mm, 5 μm column, or OBD RP18 30 × 50

mm, 5 μm), with a single wavelength UV−visible detector and Waters Micromass ZQ

(electrospray ionization). All final compounds have purities greater than 95% based upon LC/MS

analysis.

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4.2 Experimental details

4.2.1 Experimental procedures for Chapter 2

General Procedure for Salt Formation of NEU-1953:

The qualitative solubility of 1 was determined in various solvents (methanol, ethanol, isopropanol,

tetrahydrofuran, acetonitrile, ethyl acetate, and water). For each of the solvents, 2.0 mg of 1 as

added to vials. Solvents were added in 200 μL increments until the solid was completely dissolved.

With a solubility of about 2.75 mg/mL, ethanol was chosen as the solvent for the salt screen.

Compound 1 (20 mg, 0.05 mmol) was dissolved in ethanol (4 mL) using sonication and heat. Stock

solutions of various acids were made (1 M in ethanol). Acid solution was added to solution of 1

until a precipitate formed (different amounts needed for each acid). The precipitate was collected

by vacuum filtration and washed with ethanol.

Hydrochloride Salt (2.1).

1H NMR (500 MHz, DMSO-d6) δ ppm 9.04 (s, 2H), 8.91 (s, 1H), 8.81−8.86 (m, 1H), 8.78 (d, J =

5.9 Hz, 1H), 8.38−8.44 (m, 2H), 8.27−8.34 (m, 2H), 8.08−8.13 (m, 1H), 7.52−7.65 (m, 2H), 2.79

(s, 3H).

Sulfate Salt (2.2).

1H NMR (500 MHz, DMSO-d6) δ ppm 10.79−11.01 (m, 1H), 9.07 (s, 2H), 8.86−8.94 (m, 3H),

8.53−8.59 (m, 2H), 8.50 (d, J = 2.9 Hz, 1H), 8.28−8.34 (m, 2H), 3.34−3.40 (m, 2H), 3.17 (s, 3H),

2.88 (s, 3H).

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Citrate Salt (2.3).

1H NMR (500 MHz, DMSO-d6) δ ppm 9.77−9.85 (m, 1H), 8.98 (s, 2H), 8.73 (s, 2H), 8.57 (d, J =

8.8 Hz, 1H), 8.31−8.35 (m, 2H), 8.23−8.26 (m, 1H), 8.17−8.20 (m, 1H), 7.96−8.02 (m, 1H), 4.34

(br. s., 13H), 3.85−3.96 (m, 4H), 3.72−3.83 (m, 7 H), 3.40−3.48 (m, 8 H), 2.53−2.71 (m,14H),

2.42 (s, 4H).

Methanesulfonate Salt (2.4).

1H NMR (500 MHz, DMSO-d6) δ ppm 10.63−10.88 (m, 1H), 9.62−9.94 (m, 1H), 9.07 (s, 2H),

8.77−8.96 (m, 4H), 8.41−8.62 (m, 3H), 8.30 (d, J= 1.5 Hz, 2H), 4.62−4.98 (m, 2H), 2.87 (s, 4H),

2.32 (s, 6 H).

Diethyl 2-(((4-bromophenyl)amino)methylene)malonate. (2.5) 4-Bromoaniline (5.00 g, 29.1

mmol) and diethyl 2-(ethoxymethylene)malonate (5.87 mL, 29.1 mmol) were combined in a vial

and stirred at room temperature until homogenous. The mixture was then heated at 100 °C for 2.5

h. The mixture was cooled to room temperature and the product was triturated with hexanes, giving

a precipitate. The precipitate was then collected by vacuum filtration, washed with hexanes, and

dried under vacuum overnight to afford the title compound as a white powder (9.47 g, 90% yield).

LCMS rt 3.64 min, [M+H]+ 341.9, 343.9 m/z; 1H NMR (500 MHz, CDCl3) ppm 11.00 (d, J=13.7

Hz, 1 H), 8.46 (d, J=13.7 Hz, 1 H), 7.48 (d, J=8.8 Hz, 2 H), 7.02 (d, J=8.8 Hz, 2 H), 4.28 (dq,

J=28.3, 6.8 Hz, 4 H), 1.36 (dt, J=24.9, 7.3 Hz, 6 H).

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Ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate. (2.6) In a 3 neck flask with a stir bar,

diethyl 2-(((4-bromophenyl)amino)methylene)malonate (9.47 g, 27.7 mmol) was added with 6 mL

of diphenyl ether. The mixture was heated to reflux for 2 h with nitrogen bubbling into a side neck.

A precipitate formed and was collected by vacuum filtration, washed with hexanes, and dried under

vacuum overnight to afford the title compound as a tan powder (7.47 g, 91% yield). LCMS rt 2.13

min, [M+H]+ 295.9, 297.9 m/z; *Insufficient solubility to obtain an NMR spectrum.

6-Bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.7) In round bottom flask with a stir bar,

ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate (7.47 g, 25.2 mmol) and 2.5 M sodium

hydroxide (200 mL) were added. The suspension was heated to reflux for 3 h. The solution was

cooled to room temperature and acidified with aqueous hydrochloric acid to pH2 (3M, 25 mL).

The precipitate was collected by filtration, washed with water and dried under a vacuum to afford

the title compound as a light tan solid (6.57 g, 97% yield). LCMS rt 2.36 min, [M+H]+ 267.9,

269.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 14.97 (br. s., 1 H), 13.52 (br. s., 1 H), 8.92 (s,

1 H), 8.31 (d, J=2.5 Hz, 1 H), 8.01 (dd, J=8.8, 2.2 Hz, 1 H), 7.75 (d, J=9.1 Hz, 1 H).

153

6-Bromoquinolin-4(1H)-one (2.8) 6-Bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.57

g, 24.5 mmol) was dissolved in 46 mL of diphenyl ether. The reaction mixture was heated to reflux

for 3 h. The reaction mixture was cooled to room temperature, triturated with hexanes, and the

precipitate formed was collected by vacuum filtration to afford the title compound as a light tan

solid (5.32 g, 97% yield). LCMS rt 1.80 min, [M+H]+ 223.9, 225.9 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 11.92 (br. s., 1 H), 8.16 (d, J=2.4 Hz, 1 H), 7.95 (d, J=6.8 Hz, 1 H), 7.79 (dd,

J=8.8, 2.4 Hz, 1 H), 7.52 (d, J=8.8 Hz, 1 H), 6.08 (d, J=7.3 Hz, 1 H).

4-((4-Bromophenyl)sulfonyl)morpholine (2.9, 3.1b) 4-Bromobenzenesulfonyl chloride (200 mg,

0.78 mmol) was dissolved in THF (2 mL). Morpholine (74 μL) was added and the reaction mixture

was left stirring overnight at room temperature. The reaction mixture was neutralized with

saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times. The combined

organic layers were washed with water and with brine, dried using sodium sulfate, filtered, and

concentrated under reduced pressure to afford the title compound as white solid (196 mg, 82%

yield). LCMS [M+H]+ 307.1 m/z (Br79), 309.1 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm

7.89 (d, J=8.3 Hz, 2 H), 7.67 (d, J=8.8 Hz, 2 H), 3.54 - 3.72 (m, 4 H), 2.79 - 2.95 (m, 4 H).

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4-((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine (2.10, 3.2b) 4-

((4-Bromophenyl)sulfonyl)morpholine (196 mg, 0.640 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-

bi(1,3,2-dioxaborolane) (243 mg, 0.960 mmol), [1,1′-

Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane

(PdCl2(dppf)·CH2Cl2) (26.1 mg, 0.032 mmol), and potassium acetate (188 mg, 1.92 mmol) were

added to a vial that was subsequently sealed and vacuum purged with nitrogen. A degassed solution

of dioxane (4 mL) was added to the vial. The reaction mixture was heated at 85 °C overnight and

monitored by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under

reduced pressure. The resulting residue was used crude in the next reaction. A quantitative yield

was assumed. LCMS [M+H]+ 354.1 m/z. 75% product, 25% boronic acid of product by LC trace.

6-(4-(Morpholinosulfonyl)phenyl)quinolin-4(1H)-one (2.11) 6-Bromoquinolin-4(1H)-one (250

mg, 1.12 mmol), (4-morpholinosulfonyl)phenyl)boronic acid (302 mg, 1.12 mmol), triethylamine

(467 μL, 3.35 mmol), bis(triphenylphosphine)palladium(II) dichloride (10 mg, 0.014 mmol) were

155

dissolved in a 1:1 solution of ethanol/water (10 mL). The reaction mixture was refluxed for 3 h.

The ethanol was removed by rotary evaporation; a precipitate formed and was collected by vacuum

filtration to afford the title compound as an off-white solid (349 mg, 84% yield). LCMS [M+H]+

371.0 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 11.92 (br. s., 1 H), 8.43 (d, J=2.0 Hz, 1 H), 8.08

(dd, J=8.5, 2.2 Hz, 1 H), 8.03 (d, J=8.8 Hz, 2 H), 7.96 (d, J=7.3 Hz, 1 H), 7.83 (d, J=8.8 Hz, 2 H),

7.69 (d, J=8.8 Hz, 1 H), 6.10 (d, J=7.3 Hz, 1 H), 3.61 - 3.69 (m, 4 H), 2.88 - 2.95 (m, 4 H).

4-((4-(4-Chloroquinolin-6-yl)phenyl)sulfonyl)morpholine. (2.12) 6-(4-

(Morpholinosulfonyl)phenyl)quinolin-4(1H)-one (342 mg, 0.942 mmol) was dissolved in neat

phosphorus (V) oxychloride (3 mL) and refluxed for 2.5 h after which the solvent was removed

by vacuum distillation. The crude residue was cooled in an ice water bath, diluted with

dichloromethane and neutralized with saturated sodium bicarbonate solution. The layers were

separated, and the aqueous layer extracted 3 times with dichloromethane. The combined organic

layers were washed with sodium bicarbonate and brine, dried with sodium sulfate, filtered, and

concentrated. The crude product was purified by column chromatography, eluting with a mobile

phase of 2% methanol/dichloromethane to afford the title compound as an off-white solid (249

mg, 69% yield). LCMS [M+H]+ 389.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.90 (d, J=4.4

Hz, 1 H), 8.47 (d, J=2.0 Hz, 1 H), 8.22 - 8.30 (m, 2 H), 8.15 (d, J=8.3 Hz, 2 H), 7.89 (d, J=8.3 Hz,

2 H), 7.85 (d, J=4.9 Hz, 1 H), 3.57 - 3.74 (m, 4 H), 2.82 - 3.07 (m, 4 H).

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6-(4-(Morpholinosulfonyl)phenyl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-1912 (2.13) 4-((4-

(4-Chloroquinolin-6-yl)phenyl)sulfonyl)morpholine (50 mg, 0.128 mmol), pyrimidine-4-amine

(24.5 mg, 0.257 mmol), potassium tert-butoxide (14.4 mg, 0.126 mmol), xantphos (3.0 mg, 0.005

mmol), and tris(dibenzylideneacetone)dipalladium(0) (5.0 mg, 0.005 mmol) were added to a vial

which was vacuum purged with nitrogen, after which 1,4-dioxane (2 mL) was added. The reaction

mixture was heated at 101 °C overnight. The mixture was cooled to room temperature, poured

over water, and extracted with ethyl acetate 3 times. The combined organic layers were washed

with water and brine, dried over sodium sulfate, filtered, and concentrated. The product was

purified by column chromatography, eluting with a mobile phase of 4% methanol/dichloromethane

to afford the title compound as a pale yellow solid (39.4 mg, 68% yield). LCMS [M+H]+ 448.1

m/z; 1H NMR (500 MHz, DMSO-d6) ppm 10.05 (s, 1 H), 8.79 - 8.85 (m, 2 H), 8.76 (d, J=2.0

Hz, 1 H), 8.52 (d, J=5.9 Hz, 1 H), 8.38 (d, J=4.9 Hz, 1 H), 8.14 - 8.21 (m, 3 H), 8.10 - 8.14 (m, 1

H), 7.87 - 7.93 (m, 2 H), 7.30 (dd, J=5.9, 1.0 Hz, 1 H), 3.64 - 3.70 (m, 4 H), 2.89 - 2.97 (m, 4 H).

5-Bromo-2-(4-methylpiperazin-1-yl)pyrimidine. (2.18) 5-Bromo-2-chloropyrimidine (2.00 g, 10.3

mmol), 1-methylpiperazine (4.59 mL, 41.4 mmol), diisopropylethylamine (5.40 mL, 31.0 mmol)

were dissolved in tert-butanol (35 mL). The reaction mixture was heated at 85 °C overnight. Water

157

was added, a precipitate formed and was collected by vacuum filtration to afford the title

compound as a peach colored solid (2.30 g, 86% yield). LCMS [M+H]+ 335.0 m/z (Br79), 337.0

m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.44 (s, 2 H), 3.64 - 3.72 (m, 4 H), 2.30 - 2.37

(m, 4 H), 2.20 (s, 3 H).

2-(4-Methylpiperazin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine. (2.19) 5-

Bromo-2-(4-methylpiperazin-1-yl)pyrimidine (100 mg, 0.389 mmol), 4,4,4',4',5,5,5',5'-

octamethyl-2,2'-bi(1,3,2-dioxaborolane) (296 mg, 1.17 mmol), PdCl2(dppf)·CH2Cl2 (22.2 mg,

0.027 mmol), and potassium acetate (114 mg, 1.17 mmol) were added to a vial that was

subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane (5 mL) was

added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.

The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure.

The resulting residue was used crude in the next reaction. A quantitative yield was assumed. LCMS

[M+H]+ 301.2 m/z. 50% product, 50% boronic acid of product by LC trace.

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7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one. (2.20) 2-(4-methylpiperazin-1-

yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (1.2 eq) was dissolved in a 3:1

dioxane/water (0.13 M) mixture. To this was added 7-bromoquinolin-4(1H)-one (1 eq), potassium

carbonate (3 eq) and PdCl2(dppf)·CH2Cl2 (0.05 eq) and the reaction mixture was degassed before

being transferred to the microwave for 30 min at 130 °C (high absorbance). Reaction monitored

by LCMS analysis and once complete, the reaction mixture was filtered through Celite and the

crude material was purified by column chromatography, eluting with the specified mobile phase.

A precipitate was isolated by vacuum filtration and washed with water to afford the title compound

as a tan solid, (411 mg, 85%). LCMS [M+H]+ 322.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm

8.77 (s, 1 H), 8.08 - 8.15 (m, 1 H), 7.87 - 7.92 (m, 1 H), 7.68 - 7.72 (m, 1 H), 7.57 - 7.63 (m, 1 H),

6.31 (s, 2 H), 5.99 - 6.05 (m, 1 H), 3.76 - 3.85 (m, 4 H), 2.34 - 2.42 (m, 4 H), 2.22 (s, 3 H).

4-chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline. (2.21) 7-(2-(4-methylpiperazin-

1-yl)pyrimidin-5-yl)quinolin-4(1H)-one (103 mg, 0.32 mmol) was suspended in neat phosphorus

oxychloride (excess, 1.0 mL). The reaction mixture was refluxed overnight. Phosphorus

oxychloride was then removed by vacuum distillation. The resulting residue was put in an ice bath,

159

diluted with dichloromethane, and quenched using a saturated aqueous sodium bicarbonate

solution. The aqueous layer was extracted 3 times with dichloromethane. The combined organic

layers were washed with water and a saturated sodium chloride solution, before being dried with

sodium sulfate. The volatiles were removed in vacuo to obtain the title compound as a light tan

solid (77.2 mg, 71%). LCMS [M+H]+ 340.1 m/z (Cl35), 342.2 m/z (Cl37); 1H NMR (500 MHz,

CHLOROFORM-d) ppm 8.80 (d, J=4.9 Hz, 1 H), 8.73 (s, 2 H), 8.31 (d, J=8.8 Hz, 1 H), 8.23 (d,

J=1.5 Hz, 1 H), 7.78 - 7.83 (m, 1 H), 7.48 (d, J=4.9 Hz, 1 H), 3.91 - 3.98 (m, 4 H), 2.51 (t, J=4.9

Hz, 4 H), 2.37 (s, 3 H).

7-(2-(4-Methylpiperazin-1-yl)pyrimidin-5-yl)-N-(1-methylpiperidin-4-yl)quinolin-4-amine, tri-

formate salt. NEU-4920 (2.22a) 4-Chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline

(39) (20 mg, 0.059 mmol), 1-methylpiperidin-4-amine (14.8 µL, 0.12 mmol),

tris(dibenzylideneacetone)dipalladium(0) (2.7 mg, 0.003 mmol), 2-dicyclohexylphosphino-

2′,4′,6′-triisopropylbiphenyl (2.8 mg, 0.006 mmol), and potassium tert-butoxide (19.8 mg, 0.177

mmol) were added to a vial that was filled with nitrogen and evacuated three times. Dry, degassed

dioxane (1 mL) was then added. The reaction mixture was heated at 90 °C overnight. LCMS

analysis revealed no remaining starting material and a mass consistent with the desired product.

The reaction mixture was filtered through Celite and purified by column chromatography eluting

160

with 0-20% (10% ammonium hydroxide/methanol)/dichloromethane. The material was further

purified by prep HPLC (method: 99-95_6p5min) to afford the title compound as a yellow solid

(12 mg, 47%). LCMS [M+H]+ 418.1 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.82 (s,

2 H), 8.52 (d, J=8.8 Hz, 1 H), 8.47 (s, 3 H), 8.41 (d, J=6.8 Hz, 1 H), 8.01 (s, 1 H), 7.93 (d, J=8.8

Hz, 1 H), 6.94 (d, J=7.3 Hz, 1 H), 4.06 (br. s., 5 H), 3.50 (br. s., 2 H), 3.06 (br. s., 2 H), 2.95 (t,

J=5.1 Hz, 4 H), 2.62 (s, 6 H), 2.27 (d, J=12.7 Hz, 2 H), 2.03 - 2.17 (m, 2 H).

7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-4-amine.

NEU-4987 (2.22b) 4-chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline (39) (20 mg,

0.059 mmol), 2-(pyrrolidin-1-yl)ethan-1-amine (14.8 µL, 0.12 mmol),

Tris(dibenzylideneacetone)dipalladium(0) (2.7 mg, 0.003 mmol), 2-Dicyclohexylphosphino-

2′,4′,6′-triisopropylbiphenyl (2.8 mg, 0.006 mmol), and potassium tert-butoxide (19.8 mg, 0.177

mmol) were added to a vial that was filled with nitrogen and evacuated three times. Dry, degassed

dioxane (1.5 mL) was then added. The reaction mixture was heated at 90 °C overnight. LCMS

analysis revealed no remaining starting material and a mass consistent with the desired product.

The reaction mixture was filtered through Celite and purified by column chromatography eluting

with 2-20% (5% ammonium hydroxide/methanol)/dichloromethane to afford the title compound

as a yellow solid (36 mg, 59%). LCMS [M+H]+ 418.3 m/z; 1H NMR (399 MHz, DMSO-d6) ppm

8.87 (s, 2 H), 8.40 (d, J=5.9 Hz, 1 H), 8.25 (d, J=8.8 Hz, 1 H), 8.01 (s, 1 H), 7.75 (d, J=8.8 Hz, 1

161

H), 7.20 (br. s., 1 H), 6.45 (d, J=5.1 Hz, 1 H), 3.81 (m, J=4.4 Hz, 1 H), 3.38 - 3.47 (m, 1 H), 2.73

(t, J=6.6 Hz, 2 H), 2.55 (br. s., 4 H), 2.38 (t, J=4.8 Hz, 4 H), 2.18 - 2.26 (m, 3 H), 1.70 (br. s., 4

H).

6-Bromo-4-chloroquinoline.151-152 (2.23) 7-Bromoquinolin-4(1H)-one (500 mg, 1.12 mmol) was

dissolved in phosphorus oxychloride (3 mL). The reaction mixture was refluxed overnight. Excess

solvent was removed by vacuum distillation. The remaining brown residue was neutralized using

saturated sodium bicarbonate solution. The resulting precipitate was collected by vacuum filtration

and washed with water to afford the title compound as a light tan solid (440 mg, 81% yield). LCMS

[M+H]+ 241.8 m/z (Br79), 243.9 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.79

(#1, d, J=4.9 Hz, 1 H), 8.32 (#5, s, 1 H), 8.12 (#3, d, J=9.3 Hz, 1 H), 7.75 (#4, dt, J=8.9, 1.6 Hz, 1

H), 7.52 (#2, dd, J=4.6, 1.2 Hz, 1 H).

General Procedure A.

Compound 11 was dissolved in n-butanol (0.1 M). Appropriate saturated primary amine (4 equiv)

and diisopropylethylamine (10 equiv) were added. The reaction was heated in the microwave at

200 °C for 3 h. n-Butanol was removed in vacuo, and the reaction mixture was purified by column

chromatography, eluting with specified mobile phase.

162

7-Bromo-N-cyclohexylquinolin-4-amine. (2.24a) Prepared via General Procedure A. The crude

material was purified by column chromatography eluting with 1-20% methanol/dichloromethane

to afford the title compound as an off-white solid (26 mg, 41%). LCMS [M+H]+ 305.1 m/z (Br79),

307.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.49 (d, J=5.9 Hz, 1 H), 8.12

(d, J=1.5 Hz, 1 H), 7.57 (d, J=8.8 Hz, 1 H), 7.46 (dd, J=9.0, 1.7 Hz, 1 H), 6.44 (d, J=5.4 Hz, 1 H),

4.93 (d, J=6.8 Hz, 1 H), 3.43 - 3.56 (m, 1 H), 2.08 - 2.21 (m, 2 H), 1.78 - 1.89 (m, 2 H), 1.66 - 1.76

(m, 1 H), 1.39 - 1.52 (m, 2 H), 1.22 - 1.38 (m, 3 H).

N1-(7-bromoquinolin-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine. (2.24b) Prepared via

General Procedure A. The crude material was purified by column chromatography, eluting with

1-20% methanol/dichloromethane to afford the title compound as an off-white solid (15.8 mg,

22%). LCMS [M+H]+ 348.1 m/z (Br79), 350.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-

d) ppm 8.51 (d, J=4.9 Hz, 1 H), 8.13 (d, J=2.0 Hz, 1 H), 7.56 (d, J=8.8 Hz, 1 H), 7.48 (dd, J=8.8,

2.0 Hz, 1 H), 6.45 (d, J=5.4 Hz, 1 H), 4.83 (d, J=6.8 Hz, 1 H), 3.39 - 3.49 (m, 1 H), 2.33 (s, 6 H),

2.24 - 2.32 (m, 2 H), 2.12 - 2.22 (m, 1 H), 2.04 (d, J=12.7 Hz, 2 H), 1.27 - 1.51 (m, 4 H).

163

7-bromo-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine. (2.24c) Prepared via General

Procedure A. The crude material was purified by column chromatography eluting with 1-20%

methanol/dichloromethane to afford the title compound as a yellow solid (46 mg, 74%). LCMS

[M+H]+ 307.0 m/z (Br79), 309.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.28

(d, J=5.4 Hz, 1 H), 7.95 (d, J=2.0 Hz, 1 H), 7.73 (d, J=9.3 Hz, 1 H), 7.42 (dd, J=8.8, 2.0 Hz, 1 H),

6.39 (d, J=5.9 Hz, 1 H), 3.98 (dt, J=11.5, 3.3 Hz, 2 H), 3.75 (s, 1 H), 3.66 (tt, J=10.7, 4.2 Hz, 1

H), 3.50 (td, J=11.8, 2.2 Hz, 2 H), 1.98 - 2.05 (m, 2 H), 1.56 - 1.67 (m, 2 H).

7-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-amine. (2.24d) Prepared via General

Procedure A. The crude material was purified by column chromatography eluting with 5-15%

methanol/dichloromethane. The resulting residue was washed with hexanes, after which the title

compound was collected by vacuum filtration as a yellow solid (47 mg, 71%). LCMS [M+H]+

321.1 m/z (Br79), 323.0 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.37 (d, J=5.4 Hz, 1

H), 8.22 (d, J=8.8 Hz, 1 H), 7.93 (d, J=2.0 Hz, 1 H), 7.55 (dd, J=8.8, 2.0 Hz, 1 H), 7.39 (t, J=5.4

Hz, 1 H), 6.52 (d, J=5.4 Hz, 1 H), 3.85 (dd, J=11.2, 2.9 Hz, 2 H), 3.24 - 3.30 (m, 2 H), 1.91 - 2.01

(m, 1 H), 1.68 (d, J=12.7 Hz, 2 H), 1.25 (qd, J=12.2, 4.4 Hz, 2 H), 1.07 (s, 2 H).

164

(1R,2R)-2-((7-bromoquinolin-4-yl)amino)cyclohexan-1-ol. (2.24e) Prepared via General

Procedure A, heating temperature was 180 °C instead of 200 °C. The crude material was purified

by column chromatography eluting with 1-8% methanol (+10% ammonium

hydroxide)/dichloromethane, to afford the product compound as an off-white solid (95 mg, 98%).

LCMS [M+H]+ 321.3 m/z (Br79), 323.1 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.58

(d, J=9.3 Hz, 1 H), 8.41 (d, J=6.8 Hz, 2 H), 8.11 (d, J=2.0 Hz, 1 H), 7.76 (dd, J=9.3, 2.0 Hz, 1 H),

6.86 (d, J=6.3 Hz, 1 H), 5.00 (br. s., 1 H), 3.53 - 3.66 (m, 2 H), 2.84 - 3.06 (m, 1 H), 1.86 - 1.97

(m, 2 H), 1.64 - 1.76 (m, 2 H), 1.28 - 1.51 (m, 3 H).

7-bromo-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinolin-4-amine (2.24f) Prepared via General

Procedure A, heating temperature was 180 °C instead of 200 °C. The crude material was purified

by column chromatography eluting with 1-20% methanol/dichloromethane, to afford the product

compound as a light brown solid (196 mg, 71%).

LCMS [M+H]+ 335.0 m/z (Br79), 337.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d)

ppm 8.48 (d, J=5.9 Hz, 1 H), 8.15 (d, J=1.5 Hz, 1 H), 7.70 (d, J=8.8 Hz, 1 H), 7.49 (dd, J=8.8, 2.0

Hz, 1 H), 6.42 (d, J=5.4 Hz, 1 H), 5.44 (br. s., 1 H), 4.00 (dd, J=11.2, 3.9 Hz, 2 H), 3.36 - 3.45 (m,

4 H), 2.30 - 2.75 (m, 1 H), 1.72 - 1.80 (m, 2 H), 1.69 (d, J=12.7 Hz, 2 H), 1.34 - 1.48 (m, 2 H).

165

N-cyclohexyl-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-amine, mono-formate salt.

NEU-5144 (2.25a) The appropriate boronate (1.2 equiv) was dissolved in a 3:1 dioxane/water

(0.13 M) mixture. To this was added 7-bromoquinolin-4(1H)-one (2.24a) (1 equiv), potassium

carbonate (3 equiv), and PdCl2(dppf)·CH2Cl2 (0.05 equiv), and the reaction mixture was degassed

before being transferred to the microwave for 30 min at 130 °C (high absorbance). The reaction

was monitored by LCMS analysis and once complete, the reaction mixture was filtered through

Celite and the crude material was purified by column chromatography, eluting with eluting with

1-20% methanol/dichloromethane. The material was further purified by prep HPLC (method: 95-

70_6p5min). The residue was triturated with dichloromethane and the subsequent precipitate was

isolated by vacuum filtration; the title compound was afforded as tan solid (7.3 mg, 11%). LCMS

[M+H]+ 403.3 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.88 (s, 2 H), 8.45 (d, J=9.3 Hz, 1 H),

8.40 (d, J=5.4 Hz, 1 H), 8.15 (s, 2 H), 8.00 (s, 1 H), 7.81 (d, J=8.8 Hz, 1 H), 7.31 - 7.40 (m, 1 H),

6.60 (d, J=5.4 Hz, 1 H), 3.82 (br. s., 4 H), 2.41 (t, J=4.6 Hz, 4 H), 2.24 (s, 3 H), 2.00 (br. s., 2 H),

1.80 (br. s., 2 H), 1.67 (d, J=11.7 Hz, 1 H), 1.35 - 1.48 (m, 4 H), 1.13 - 1.26 (m, 1 H). 1H NMR

(500 MHz, METHANOL-d4) ppm 8.87 (s, 2 H), 8.55 (d, J=8.8 Hz, 1 H), 8.38 (br. s., 2 H), 8.35

(d, J=6.8 Hz, 1 H), 7.99 (br. s., 1 H), 7.96 (d, J=9.3 Hz, 1 H), 6.90 (d, J=6.8 Hz, 1 H), 4.13 (br. s.,

4 H), 3.78 - 3.90 (m, 1 H), 3.08 (br. s., 4 H), 2.74 (s, 3 H), 2.12 (d, J=9.8 Hz, 2 H), 1.92 (d, J=10.7

Hz, 2 H), 1.78 (d, J=12.7 Hz, 1 H), 1.46 - 1.64 (m, 4 H), 1.32 (d, J=10.7 Hz, 1 H).

166

General Procedure B.

The appropriate boronate (1.2 equiv) was dissolved in a 1:2 water/ethanol (0.13 M) mixture. To

this were added 2.24b-f (1 equiv), cesium carbonate (4 equiv), and

tetrakis(triphenylphosphine)palladium(0) (5 mol %), and the reaction mixture was degassed before

being transferred to the microwave for 10 min at 130°C (high absorbance). The reaction was

monitored by LCMS analysis and once complete, the reaction mixture was filtered through Celite

and the crude material was purified by column chromatography, eluting with the specified mobile

phase.

N1,N1-Dimethyl-N4-(7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-yl)cyclohexane-1,4-

diamine. NEU-5375 (2.25b) Prepared via General Procedure B with some exceptions. The

reaction mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction

was heated for 48 h at 80 °C. The crude material was purified by column chromatography eluting

with 4-10% methanol (10% ammonium hydroxide)/dichloromethane to afford the title compound

as an off-white solid, 28 mg (27%). LCMS [M+H]+ 446.3 m/z; 1H NMR (500 MHz, DMSO-d6)

ppm 8.86 (s, 2 H), 8.37 (d, J=5.4 Hz, 1 H), 8.34 (d, J=8.8 Hz, 1 H), 7.99 (d, J=2.0 Hz, 1 H), 7.71

(dd, J=8.8, 2.0 Hz, 1 H), 6.79 (d, J=7.8 Hz, 1 H), 6.48 (d, J=5.4 Hz, 1 H), 3.78 - 3.83 (m, 4 H),

167

3.41 - 3.50 (m, 1 H), 2.39 (t, J=5.1 Hz, 4 H), 2.23 (s, 3 H), 2.20 (s, 7 H), 2.08 (d, J=10.7 Hz, 2 H),

1.87 (d, J=11.2 Hz, 2 H), 1.39 (m, J=14.2 Hz, 4 H).

7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine.

NEU-5139 (2.25c) Prepared via General Procedure B. The crude material was purified by

column chromatography eluting with 1-20% methanol (+50% ammonium

hydroxide)/dichloromethane. The resulting product was triturated with ether and the precipitate

was collected by vacuum filtration to afford the title compound as a light brown solid (22 mg,

36%). LCMS [M+H]+ 405.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.74 (s, 2 H), 8.36

(d, J=5.9 Hz, 1 H), 8.27 (d, J=8.8 Hz, 1 H), 7.92 (s, 1 H), 7.69 (dd, J=8.8, 1.5 Hz, 1 H), 6.65 (d,

J=5.9 Hz, 1 H), 3.99 - 4.08 (m, 2 H), 3.85 - 3.95 (m, 5 H), 3.60 (m, J=11.2, 11.2 Hz, 2 H), 2.52 (t,

J=5.1 Hz, 4 H), 2.34 (s, 3 H), 2.01 - 2.11 (m, 2 H), 1.68 - 1.82 (m, 2 H).

168

7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-

amine. NEU-5326 (2.25d) Prepared via General Procedure B with some exceptions. The reaction

mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction was heated

for 18 h at 80 °C. The crude material was purified by column chromatography eluting with 2-20%

methanol/dichloromethane. The compound was further purified by column chromatography

eluting with 1-8% methanol (+5% ammonium hydroxide)/dichloromethane to afford the title

compound as a colorless solid, (26 mg, 44%). LCMS [M+H]+ 419.2 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 8.86 (s, 2 H), 8.38 (d, J=5.4 Hz, 1 H), 8.31 (d, J=8.8 Hz, 1 H), 8.00 (d, J=1.5

Hz, 1 H), 7.72 (dd, J=8.8, 2.0 Hz, 1 H), 7.25 (t, J=5.6 Hz, 1 H), 6.46 (d, J=5.9 Hz, 1 H), 3.86 (dd,

J=11.2, 2.9 Hz, 2 H), 3.77 - 3.83 (m, 4 H), 3.28 (m, J=10.3 Hz, 2 H), 3.18 (t, J=6.1 Hz, 2 H), 2.38

(t, J=4.9 Hz, 4 H), 2.22 (s, 3 H), 1.93 - 2.03 (m, 1 H), 1.69 (dd, J=12.5, 1.2 Hz, 2 H), 1.26 (qd,

J=12.2, 3.9 Hz, 2 H).

(1R,2R)-2-((7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-yl)amino)cyclohexan-1-ol.

NEU-5376 (2.25e) Prepared via General Procedure B with some exceptions. The reaction

169

mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction was heated

for 48 h at 80 °C. The crude material was purified by column chromatography eluting with 4-8%

methanol (+10% ammonium hydroxide)/dichloromethane to afford the title compound as a light

brown solid, (46 mg, 38%). [α]D22 + 0.57 ± 0.28 (c 1.1, 1:10 DCM/CH3OH); LCMS [M+H]+ 419.2

m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.87 (s, 2 H), 8.35 (d, J=2.0 Hz, 1 H), 8.34 (s, 1 H),

7.99 (d, J=2.0 Hz, 1 H), 7.72 (dd, J=8.8, 2.0 Hz, 1 H), 6.72 (d, J=7.8 Hz, 1 H), 6.51 (d, J=5.9 Hz,

1 H), 4.74 (br. s., 1 H), 3.92 (br. s., 1 H), 3.74 - 3.85 (m, 4 H), 3.52 - 3.62 (m, 1 H), 2.35 - 2.43 (m,

4 H), 2.23 (s, 3 H), 1.98 (m, J=12.2 Hz, 2 H), 1.68 (m, J=2.0 Hz, 2 H), 1.31 (m, J=2.9 Hz, 4 H).

N-cyclohexyl-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-amine, mono-formate salt.

NEU-6734 (2.25f) Prepared via General Procedure B. The crude material was purified by column

chromatography, eluting with 1-20% methanol/dichloromethane. The residue was triturated with

dichloromethane and the subsequent precipitate was isolated by vacuum filtration, the title

compound was afforded as a grey solid (93 mg, 44%). LCMS [M+H]+ 433.4 m/z; 1H NMR (500

MHz, METHANOL-d4) δ ppm 8.70 (s, 2 H), 8.34 (d, J=5.4 Hz, 1 H), 8.14 (d, J=8.3 Hz, 1 H), 7.90

(d, J=1.5 Hz, 1 H), 7.61 (dd, J=8.8, 1.5 Hz, 1 H), 6.44 (d, J=5.9 Hz, 1 H), 3.83 - 3.97 (m, 6 H),

3.35 - 3.45 (m, 4 H), 2.49 (t, J=4.9 Hz, 4 H), 2.33 (s, 3 H), 1.65 - 1.74 (m, 5 H), 1.27 - 1.39 (m, 2

H).

170

General procedure C.

4-((4-(4-chloroquinolin-6-yl)phenyl)sulfonyl)morpholine (50 mg, 0.13 mmol) was dissolved in 1

mL of n-butanol. The respective amine (4 eq) and diisopropylethylamine (10 eq) were added to

the solution which was subsequently heated overnight at 180 °C in a sealed vial. The solvent was

removed, and the resulting solid was purified by column chromatography using conditions

described below.

N-cyclohexyl-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine NEU-5533 (2.26a) Prepared via

General Procedure C. The reaction was purified by column chromatography using a mobile

phase of 0-100% ethyl acetate/dichloromethane followed by 0-20% methanol/ethyl acetate, to

afford the title compound as a light tan solid (8 mg, 14% yield). LCMS [M+H]+ 452.2 m/z; 1H

NMR (500 MHz, METHANOL-d4) ppm 8.86 (d, J=1.5 Hz, 1 H), 8.36 (d, J=6.8 Hz, 1 H), 8.26

- 8.32 (m, 1 H), 8.08 (d, J=8.8 Hz, 2 H), 7.96 (d, J=8.8 Hz, 1 H), 7.93 (d, J=8.3 Hz, 2 H), 6.95 (d,

J=7.3 Hz, 1 H), 3.70 - 3.74 (m, 4 H), 3.63 (d, J=11.2 Hz, 2 H), 2.98 - 3.04 (m, 4 H), 2.14 (d, J=12.2

Hz, 2 H), 1.91 (d, J=13.2 Hz, 2 H), 1.78 (d, J=13.2 Hz, 1 H), 1.47 - 1.65 (m, 4 H).

171

N-(1-methylpiperidin-4-yl)-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine NEU-5537

(2.26b) Prepared via General Procedure C. The reaction was purified by column chromatography

using a mobile phase of 0-30% methanol/ethyl acetate, to afford the title compound as an orange

solid (12 mg, 20% yield). LCMS [M+H]+ 467.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66

(s, 1 H), 8.40 (d, J=5.4 Hz, 1 H), 8.13 (d, J=8.3 Hz, 2 H), 7.97 - 8.02 (m, 1 H), 7.87 (dd, J=8.5, 6.1

Hz, 3 H), 7.08 (d, J=7.3 Hz, 1 H), 6.57 (d, J=5.4 Hz, 1 H), 3.62 - 3.69 (m, 4 H), 2.93 (d, J=3.9 Hz,

4 H), 2.84 (d, J=11.2 Hz, 2 H), 2.22 (d, J=8.3 Hz, 4 H), 2.06 (t, J=11.2 Hz, 2 H), 1.99 (d, J=11.7

Hz, 2 H), 1.67 (d, J=11.2 Hz, 2 H).

N1,N1-dimethyl-N4-(6-(4-(morpholinosulfonyl)phenyl)quinolin-4-yl)cyclohexane-1,4-diamine

NEU-6735 (2.26c) Prepared via General Procedure C. The reaction was purified by column

chromatography using a mobile phase of 1-20% methanol/dichloromethane, to afford the title

compound as a light brown solid (64 mg, 50% yield). LCMS [M+H]+ 495.2 m/z; 1H NMR (500

MHz, METHANOL-d4) δ ppm 8.52 (d, J=1.5 Hz, 1 H), 8.35 (d, J=5.9 Hz, 1 H), 8.01 (d, J=8.3 Hz,

2 H), 7.96 (dd, J=8.8, 1.5 Hz, 1 H), 7.87 (d, J=8.3 Hz, 1 H), 7.83 (d, J=8.3 Hz, 2 H), 6.61 (d, J=5.9

172

Hz, 1 H), 3.66 - 3.72 (m, 4 H), 3.58 (br. s., 1 H), 2.93 - 3.01 (m, 4 H), 2.58 (br. s., 1 H), 2.45 (s, 6

H), 2.23 (br. s., 2 H), 2.06 (br. s., 2 H), 1.54 (m, J=9.5, 9.5 Hz, 4 H).

6-(4-(morpholinosulfonyl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-4-amine NEU-5536

(2.26d) Prepared via General Procedure C. The reaction was purified by column chromatography

using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-20% methanol/ethyl

acetate, to afford the title compound as a dark yellow solid (35.3 mg, 59% yield). LCMS [M+H]+

467.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.65 (br. s., 1 H), 8.43 (d, J=5.4 Hz, 1 H), 8.13

(d, J=7.8 Hz, 2 H), 8.03 (d, J=8.8 Hz, 1 H), 7.88 (dd, J=11.7, 8.8 Hz, 3 H), 7.53 (br. s., 1 H), 6.54

(d, J=4.9 Hz, 1 H), 3.65 (d, J=4.4 Hz, 4 H), 3.49 (br. s., 2 H), 2.93 (br. s., 4 H), 2.81 (br. s., 2 H),

2.61 (br. s., 3 H), 2.34 (s, 1 H), 1.72 (br. s., 4 H), 1.23 (br. s., 2 H).

6-(4-(morpholinosulfonyl)phenyl)-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine NEU-5825

(2.26e) Prepared via General Procedure C. The reaction was purified by column chromatography

using a mobile phase of 0-10% methanol/ethyl acetate, to afford the title compound as an off white

173

solid (29 mg, 50% yield). LCMS [M+H]+ 454.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66

(s, 1 H), 8.41 (d, J=5.9 Hz, 1 H), 8.13 (d, J=8.8 Hz, 2 H), 8.01 (dd, J=8.5, 1.7 Hz, 1 H), 7.87 (t,

J=8.3 Hz, 3 H), 7.10 (d, J=7.3 Hz, 1 H), 6.65 (d, J=5.9 Hz, 1 H), 3.95 (d, J=9.8 Hz, 2 H), 3.77 -

3.87 (m, 1 H), 3.63 - 3.68 (m, 4 H), 3.50 (t, J=11.5 Hz, 2 H), 2.93 (d, J=4.4 Hz, 4 H), 1.98 (d,

J=14.2 Hz, 2 H), 1.66 (dd, J=11.2, 3.4 Hz, 2 H).

6-(4-(morpholinosulfonyl)phenyl)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-amine NEU-

5534 (2.26f) Prepared via General Procedure C. The reaction was purified by column

chromatography using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-

20% methanol/ethyl acetate, to afford the title compound as a light tan solid (11.7 mg, 28% yield).

LCMS [M+H]+ 468.2 m/z; 1H NMR (500 MHz, METHANOL-d4) δ ppm 8.57 (s, 1 H), 8.38 (d,

J=6.3 Hz, 1 H), 8.02 - 8.09 (m, 3 H), 7.93 (d, J=8.8 Hz, 1 H), 7.89 (d, J=8.8 Hz, 2 H), 6.67 (d,

J=6.3 Hz, 1 H), 3.97 (dd, J=11.7, 3.4 Hz, 2 H), 3.69 - 3.76 (m, 4 H), 3.43 (t, J=11.2 Hz, 2 H), 3.37

(d, J=6.8 Hz, 2 H), 2.98 - 3.04 (m, 4 H), 2.07 - 2.18 (m, 1 H), 1.92 (s, 1 H), 1.79 (d, J=13.2 Hz, 2

H), 1.36 - 1.48 (m, 2 H).

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(1R,2R)-2-((6-(4-(morpholinosulfonyl)phenyl)quinolin-4-yl)amino)cyclohexan-1-ol NEU-5535

(2.26g) Prepared via General Procedure C. The reaction was purified by column chromatography

using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-20% methanol/ethyl

acetate, to afford the title compound as an off white solid (34.5 mg, 57% yield). LCMS [M+H]+

468.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66 (s, 1 H), 8.36 (d, J=5.4 Hz, 1 H), 8.13 (d,

J=8.3 Hz, 2 H), 7.98 (d, J=9.8 Hz, 1 H), 7.82 - 7.89 (m, 3 H), 7.01 (d, J=7.8 Hz, 1 H), 6.59 (d,

J=5.4 Hz, 1 H), 4.78 (br. s., 1 H), 3.66 (t, J=4.4 Hz, 4 H), 3.59 (br. s., 1 H), 2.92 (br. s., 4 H), 1.92

- 2.07 (m, 2 H), 1.64 - 1.77 (m, 2 H), 1.23 - 1.43 (m, 4 H).

6-(4-(morpholinosulfonyl)phenyl)-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinolin-4-amine.

NEU-5824 (2.26h) Prepared via General Procedure C. The reaction was purified by column

chromatography using a mobile phase of 0-20% methanol/ethyl acetate, to afford the title

compound as an off white solid (49.6 mg, 80% yield). LCMS [M+H]+ 482.2 m/z; 1H NMR (500

MHz, DMSO-d6) δ ppm 8.64 (s, 1 H), 8.41 (d, J=5.4 Hz, 1 H), 8.12 (d, J=8.3 Hz, 2 H), 8.01 (dd,

J=8.8, 1.5 Hz, 1 H), 7.83 - 7.91 (m, 3 H), 7.42 (br. s., 1 H), 6.51 (d, J=5.4 Hz, 1 H), 4.10 (d, J=4.9

175

Hz, 1 H), 3.85 (dd, J=11.0, 3.2 Hz, 2 H), 3.66 (t, J=4.4 Hz, 4 H), 3.25 - 3.31 (m, 2 H), 3.17 (d,

J=4.4 Hz, 2 H), 2.93 (br. s., 4 H), 1.61 - 1.72 (m, 4 H), 1.24 (d, J=10.2 Hz, 2 H).

1-(5-bromopyrimidin-2-yl)-4-methyl-1,4-diazepane. (2.27) 5-bromo-2-chloropyrimidine (1.00 g,

5.17 mmol), 1-methyl-1,4-diazepane (771 μL, 6.20 mmol), diisopropylethylamine (1.17 mL, 6.72

mmol) were dissolved in a 1:1 solution of THF/IPA (18 mL). The reaction mixture was heated at

80 °C overnight. A precipitate formed and was collected by vacuum filtration to afford the title

compound as a white solid (1.28 g, 91% yield). LCMS [M+H]+ 271.1 m/z (Br79), 273.0 m/z (Br81);

1H NMR (500 MHz,DMSO-d6) δ ppm 8.50 (s, 2H), 3.95 (br. s., 2H), 3.74 (br. s., 2H), 3.16 (d, J

= 4.9 Hz, 4H), 2.66 (br. s., 3H), 2.13 (br. s., 2H).

1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane. (2.28)

1-(5-bromopyrimidin-2-yl)-4-methyl-1,4-diazepane (250 mg, 0.922 mmol), 4,4,4',4',5,5,5',5'-


176






[M+H]+ 237.1 m/z (mass of boronic acid), 100% by LC trace.

6-bromo-4-chloroquinoline. (2.29) 6-bromoquinolin-4(1H)-one (171 mg, 0.763 mmol) was

dissolved in neat phosphorus oxychloride (3 mL). The reaction mixture was refluxed for 12 h.

Phosphorus oxychloride was removed by vacuum distillation. The remaining brown residue was

cooled to 0 °C and neutralized using saturated sodium bicarbonate solution. A precipitate formed

and was collected by vacuum filtration to afford the title compound as a light tan solid (149 mg,

80% yield). LCMS [M+H]+ 241.9 m/z (Br79), 243.9 m/z (Br81); 1H NMR (500 MHz, DMSO-d6)

ppm 8.89 (d, J=4.4 Hz, 1 H), 8.36 (d, J=1.5 Hz, 1 H), 8.05 - 8.08 (m, 1 H), 8.00 - 8.04 (m, 1 H),

7.84 (d, J=4.9 Hz, 1 H)

177

7-bromo-N-(pyrazin-2-yl)quinolin-4-amine. (2.30a) To a suspension of sodium hydride (40 mg,

0.29 mmol) in dry DMF (1 ml) was added pyrazin-2-amine (68.6 mg, 0.72 mmol) under nitrogen,

upon which the reaction mixture turned bright yellow. To this was added a solution of 6-bromo-4-

chloroquinoline (70 mg, 0.289 mmol) in dry DMF (1 ml), accompanied by a color change from

yellow, to dark orange, to brown. The reaction was stirred under nitrogen at room temperature for

48 h. Upon completion the reaction mixture was quenched with saturated aqueous ammonium

chloride. A precipitate was observed and collected by vacuum filtration and washed with water.

The crude material was purified by column chromatography, eluting with 1-20%

methanol/dichloromethane to afford the title compound as a light-yellow solid (31.2 mg, 36%


9.77 - 9.83 (m, 1 H), 8.78 - 8.82 (m, 1 H), 8.73 - 8.77 (m, 1 H), 8.69 - 8.73 (m, 1 H), 8.39 - 8.44

(m, 1 H), 8.32 - 8.36 (m, 1 H), 8.17 - 8.22 (m, 1 H), 7.91 (s, 2 H).

6-bromo-N-(pyrimidin-4-yl)quinolin-4-amine. (2.30b) To a suspension of sodium hydride (40 mg,

0.29 mmol) in dry DMF (1 ml) was added pyrimidin-4-amine (68.6 mg, 0.72 mmol) under

178

nitrogen, upon which the reaction mixture turned bright yellow. To this was added a solution of 6-

bromo-4-chloroquinoline (70 mg, 0.289 mmol) in dry DMF (1 ml), accompanied by a color change

from yellow, to dark orange, to brown. The reaction was stirred under nitrogen at room temperature

for 48 h. Upon completion the reaction mixture was quenched with saturated aqueous ammonium

chloride. A precipitate was observed and collected by vacuum filtration and washed with water.

The crude material was purified by column chromatography, eluting with 1-20%

methanol/dichloromethane to afford the title compound as a light-yellow solid (23.5 mg, 27%

yield). LCMS [M+H]+ 301.1 m/z (Br79), 303.0 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) δ ppm

9.90 (s, 1 H), 8.81 (m, J=3.4 Hz, 2 H), 8.70 (d, J=2.0 Hz, 1 H), 8.51 (d, J=5.9 Hz, 1 H), 8.43 (d,

J=5.4 Hz, 1 H), 7.92 - 7.97 (m, 1 H), 7.89 (m, J=2.0 Hz, 1 H), 7.29 (dd, J=5.9, 1.0 Hz, 1 H).

7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrazin-2-yl)quinolin-4-amine. NEU-5377

(2.31a) 7-bromo-N-(pyrazin-2-yl)quinolin-4-amine (29 mg, 0.096 mmol) was dissolved in a 1:1

mixture of ethanol/water (1 mL). To this was added 1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane (2 equiv), cesium carbonate (3 equiv), and

tetrakis(triphenylphosphine)palladium(0) (5 mol%), and the reaction mixture was degassed before

heating at 85 °C for 12 h. The reaction mixture was monitored by LCMS analysis and, once

complete, the reaction mixture was filtered through Celite and the crude material was purified by

column chromatography, eluting with a mobile phase of 1-20% methanol/dichloromethane,

179

followed by 3-10% (1% ammonium hydroxide/methanol)/dichloromethane. The title compound

was afforded as a yellow solid (19.8 mg, 50% yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 9.71 (s, 1 H), 8.94 (s, 2 H), 8.72 (d, J=1.0 Hz, 1 H), 8.66 - 8.70 (m, 2 H),

8.34 - 8.37 (m, 2 H), 8.19 (d, J=2.9 Hz, 1 H), 8.06 (d, J=1.5 Hz, 1 H), 7.99 - 8.03 (m, 1 H), 3.88 -

3.93 (m, 2 H), 3.83 (t, J=6.1 Hz, 2 H), 2.62 - 2.67 (m, 2 H), 2.47 - 2.50 (m, 2 H), 2.27 (s, 3 H),

1.91 (s, 2 H).

6-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-

5374 (2.31b) 6-bromo-N-(pyrimidin-4-yl)quinolin-4-amine (21 mg, 0.070 mmol) was dissolved

in a 1:1 mixture of ethanol/water (1 mL). To this was added 1-methyl-4-(5-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane (2 equiv), cesium carbonate (3 equiv), and

tetrakis(triphenylphosphine)palladium(0) (5 mol%), and the reaction mixture was degassed before

heating at 85 °C for 12 h. The reaction mixture was monitored by LCMS analysis and, once

complete, the reaction mixture was filtered through Celite and the crude material was purified by

column chromatography, eluting with a mobile phase of 7-20% methanol/dichloromethane,

followed by 3-10% (1% ammonium hydroxide/methanol)/dichloromethane. The title compound

was afforded as a yellow solid (12 mg, 42% yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 9.84 (s, 1 H), 8.92 (s, 2 H), 8.82 (d, J=1.0 Hz, 1 H), 8.75 (d, J=4.9 Hz, 1 H),

8.61 (d, J=1.5 Hz, 1 H), 8.52 (d, J=5.9 Hz, 1 H), 8.37 (d, J=5.4 Hz, 1 H), 8.06 - 8.10 (m, 1 H), 8.02

180

- 8.06 (m, 1 H), 7.30 (dd, J=5.9, 1.0 Hz, 1 H), 3.88 - 3.93 (m, 2 H), 3.83 (t, J=6.3 Hz, 2 H), 3.17

(d, J=4.4 Hz, 1 H), 2.62 - 2.67 (m, 2 H), 2.27 (s, 3 H), 1.91 (dt, J=11.7, 5.9 Hz, 2 H).

7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one. (2.32) 7-bromoquinolin-

4(1H)-one (155 mg, 0.691 mmol), 1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)pyrimidin-2-yl)-1,4-diazepane (1.3 equiv), cesium carbonate (3 equiv), and PdCl2(dppf)CH2Cl2

(3 mol %) were added to a vial that was vacuum purged with nitrogen. A mixture of degassed 3:1

dioxane/water (6 mL) was added to the vial. The reaction mixture was heated at 90 °C for 12 h.

The reaction mixture was monitored by LCMS analysis and, once complete, the reaction mixture

was filtered through Celite and the crude material was purified by column chromatography, eluting

with a mobile phase of 1-20% methanol/dichloromethane. The title compound was afforded as a

tan solid (178 mg, 77% yield). LCMS [M+H]+ 336.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm

8.75 (s, 2 H), 8.10 (d, J=8.3 Hz, 1 H), 7.88 (d, J=7.8 Hz, 1 H), 7.71 (s, 1 H), 7.58 (dd, J=8.3, 1.5

Hz, 1 H), 6.00 (d, J=7.3 Hz, 1 H), 3.87 - 3.97 (m, 2 H), 3.81 (t, J=6.3 Hz, 2 H), 3.05 (t, J=7.1 Hz,

2 H), 2.37 (br. s., 2 H), 1.93 - 2.00 (m, 1 H), 1.89 (s, 3 H), 1.81 (s, 2 H).

181

4-chloro-7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinoline. (2.33) 7-(2-(4-methyl-1,4-

diazepan-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one (178 mg, 0.530 mmol) was dissolved in neat

phosphorus oxychloride (3 mL). The reaction mixture was refluxed for 12 h. Phosphorus

oxychloride was removed by vacuum distillation. The remaining brown residue was cooled to 0

°C and neutralized using saturated sodium bicarbonate solution. The aqueous suspension was

extracted 3 times with dichloromethane. The combined organic layers were dried using sodium

sulfate, filtered, and concentrated under reduced pressure to afford the title compound as a beige

solid (129 mg, 69% yield). LCMS [M+H]+ 354.1 m/z (Cl35), 356.1 m/z (Cl37); 1H NMR (500 MHz,

DMSO-d6) δ ppm 8.98 (s, 2 H), 8.86 (d, J=4.9 Hz, 1 H), 8.39 (s, 1 H), 8.26 (d, J=8.8 Hz, 1 H),

8.13 (d, J=8.8 Hz, 1 H), 7.75 (d, J=4.9 Hz, 1 H), 3.87 (br. s., 2 H), 3.34 (br. s., 3 H), 3.12 (br. s., 2

H), 3.03 (d, J=17.6 Hz, 2 H), 2.61 (br. s., 2 H), 2.11 (br. s., 2 H).

182

7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-

5030 (2.34) To a suspension of sodium hydride (56.5 mg, 1.03 mmol) in dry DMF (1 ml) was

added pyrimidin-4-amine (80.6 mg, 0.848 mmol) under nitrogen. To this was added a solution 4-

chloro-7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinoline (100 mg, 0.283 mmol) in dry

DMF (1 ml), accompanied by a color change from yellow to dark orange. The reaction was stirred

under nitrogen at 90 °C for 12 h. Upon completion the reaction mixture was quenched with

saturated aqueous ammonium chloride and the resulting solution was lyophilized to dryness. The

crude material was purified by column chromatography, eluting with 0-20%

methanol/dichloromethane, to afford the title compound as a bright-yellow solid (16 mg, 14%

yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 9.92 (s, 1 H), 8.92 (s, 2

H), 8.76 - 8.81 (m, 2 H), 8.44 - 8.51 (m, 2 H), 8.31 (d, J=5.1 Hz, 1 H), 8.24 (s, 1 H), 7.98 (d, J=8.8

Hz, 1 H), 7.29 (d, J=5.9 Hz, 1 H), 4.09 (d, J=5.1 Hz, 1 H), 3.87 - 3.94 (m, 2 H), 3.82 (t, J=6.2 Hz,

2 H), 3.17 (d, J=5.1 Hz, 1 H), 2.61 - 2.68 (m, 2 H), 2.27 (s, 3 H), 1.85 - 1.95 (m, 2 H).

183

Cellular biological assays

The protocols for the biological assays of Leishmania major amastigotes, Plasmodium falciparum

D6, W2, C235, and HepG2 cell toxicity were performed as previously described.153-157 The

compounds 1-11 and 35-37 were evaluated against T. cruzi and host cell toxicity (NIH3T3) using

previously reported protocol;153, 158 and rest all of the compounds were tested against T. cruzi and

host cell toxicity (C2C12) as described here.

T. cruzi in vitro assay. C2C12 (ATCC CRL-1772) and T.cruzi (CaI-72) trypomastigotes were

collected from culture flasks and counted. For each assay, a batch infection was prepared by

mixing cells and parasites in solution (DMEM media supplemented with 10% FBS, 1x

penicillin/streptomycin). Multidrop Combi (Thermo Fisher Scientific) was used to dispense 10ul

of that solution in the assay plates containing the compounds. Plates were incubated at 37C, 5%

CO2 incubators for 72h. After incubation, 5ul/well is aspirated using EL-406 (Biotek) and we

dispensed 5ul of 8% paraformaldehyde (PFA) to fix the samples using the Multidrop Combi. After

at least 30 minutes incubation with PFA solution at room temperature, we aspirated the content

and wash one time with isotonic buffer. Next, we added DAPI solution (5ug/ml) for nucleic acid

staining for at least 30 minutes before reading the plates in ImageXpress MicroXL (Molecular

Devices). The images were analyzed by a custom module algorithm in MetaXpress (Molecular

Devices) to determine the number of host cells and number of parasites per well. The averages of

the parasite/cell ratio from the set of controls determined the 100% efficacy normalized activity

(positive control average) and 0% efficacy normalized activity (negative control average).

184

L. donovani in vitro assay. B10R (kindly provided by Martin Olivier, McGill University) and L.

donovani were collected from culture flasks and counted. For each assay, a batch infection was

prepared by mixing cells and parasites in solution (DMEM media supplemented with 10% FBS,

1x penicillin/streptomycin). Multidrop Combi was used to dispense 10ul of that solution in the

assay plates containing the compounds. Plates are incubated at 37C, 5% CO2 incubators for 72h.

After incubation, 5ul/well is aspirated using EL-406 (Biotek) and we dispensed 5ul of 8%

paraformaldehyde (PFA) to fix the samples using the Multidrop Combi. After at least 30 minutes

incubation with PFA solution at room temperature, we aspirated the content and wash one time

with isotonic buffer. Next, we add DAPI solution (5ug/ml) for DNA staining for at least 30 minutes

before reading the plates in ImageXpress MicroXL (Molecular Devices). The images were

analyzed by a custom module algorithm in MetaXpress (Molecular Devices) to determine the

number of host cells and number of parasites per well. Each plate contained wells with positive

controls (non-infected) and wells with negative controls (infected not-treated). The averages of the

parasite/cell ratio from the set of controls determined the 100% efficacy normalized activity

(positive control average) and 0% efficacy normalized activity (negative control average).

Host cell toxicity in vitro assay. To assess the host cell cytotoxicity for both T. cruzi and L.

donovani screening assays, we first determine the average number of host cells in the negative

control wells that are infected and untreated with any compound. To assess the cell viability from

each well with a tested compound, we divided the number of host cells from that well by the

average number of cells from the negative controls from that same plate. The result informed the

percentage of cell relative to an average without any toxicity. Calculating the average number of

cells from negative controls minus three standard deviations from that average it is always around

185

50% cell toxicity. Being that the case, we considered <50% cell viability as a sign of observed

toxicity caused by the tested compound.

ADME Experiment Protocols

Assays were run by AstraZeneca.

Aqueous pH 7.4 Solubility. Compounds were dried down from 10 mM DMSO solutions using

centrifugal evaporation technique. Phosphate buffer (0.1 M pH 7.4) was added and StirStix were

inserted in the glass vials, with shaking then performed at a constant temperature of 25 °C for 20-

24 h. This step was followed by double centrifugation with a tip wash in between, to ensure that

no residues of the dried compounds interfere. The solutions were diluted before analysis and

quantification using LC/MS/MS was performed.

Log D7.4. Shake-flask octanol-water distribution coefficient was determined at pH 7.4 (Log D7.4).

The aqueous solution used is 10 mM sodium phosphate pH 7.4 buffer. The method has been

validated for Log D7.4 ranging from -2 to 5.0.

Human Plasma Protein Binding (PPB). PPB was determined using equilibrium dialysis (RED

device) to separate free from bound compound. The amount of compound in plasma (10 µM initial

concentration) and in dialysis buffer (pH 7.4 phosphate buffer) was measured by LC-MS/MS after

equilibration at 37 °C in a dialysis chamber to give the fraction unbound (fu); percent bound is

calculated and reported.

186

Human Liver Microsomal Clint. In vitro intrinsic clearance was determined from human liver

microsomes using a standard approach.159 Following incubation and preparation, the samples were

analyzed using LC/MS/MS. Refined data were uploaded to IBIS and are displayed as Clint

(intrinsic clearance) in μL/min/mg.

Rat Hepatocyte Clint. In vitro intrinsic clearance was determined from rat hepatocytes using a

standard approach.159 Following incubation and preparation, the samples were analyzed using

LC/MS/MS. Refined data are uploaded to IBIS and are displayed as Clint (intrinsic clearance)

μL/min/1 million cells.

Calculated LogP and LogD values. Both LogP and LogD predictions are based on a modified

version of the method160 where the predicted partition coefficients are composed of the

molecules’ atomic increments.

187

4.2.2 Experimental procedures for Chapter 3

4-Bromo-N-(2-hydroxyethyl)benzenesulfonamide.161-162 (3.1a) Ethanolamine (23.4 μL, 0.391

mmol) and triethylamine (109 μL, 0.783 mmol) were added to a flask containing anhydrous DCM

(1 mL) at -10°C. 4-Bromobenzenesulfonyl chloride (100 mg, 0.391 mmol) was dissolved in

anhydrous DCM (1 mL) and then added dropwise to the stirring solution of amines. The reaction

mixture was left stirring and allowed to warm to room temperature overnight. A white precipitate

formed and filtered off. The filtrate was collected and concentrated to afford the title compound as

white solid (89.5 mg, 82% yield). LCMS [M+H]+ 279.9 m/z (Br79), 281.9 m/z (Br81).

4-((4-bromophenyl)sulfonyl)morpholine.163 (3.1b) 4-bromobenzenesulfonyl chloride (200 mg,

0.78 mmol) was dissolved in THF (2 mL). Morpholine (74 μL) was added and the reaction mixture

was left stirring overnight at room temperature. The reaction mixture was neutralized with

saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times. The combined

organic layers were washed with water and with brine, dried using sodium sulfate, filtered, and

concentrated under reduced pressure to afford the title compound as white solid (196 mg, 82%

yield). LCMS [M+H]+ 307.1 m/z (Br79), 309.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-

188

d) ppm 7.71 (d, J=8.3 Hz, 2 H), 7.63 (d, J=8.8 Hz, 2 H), 3.76 (t, J=1.0 Hz, 4 H), 3.01 (t, J=1.0

Hz, 4 H).

1-((4-bromophenyl)sulfonyl)-4-methylpiperazine.163 (3.1c) 4-bromobenzenesulfonyl chloride (200

mg, 0.78 mmol) was dissolved in THF (2 mL). 1-methylpiperazine (95 μL) was added and the

reaction mixture was left stirring overnight at room temperature. The reaction mixture was

neutralized with saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times.

The combined organic layers were washed with water and with brine, dried using sodium sulfate,

filtered, and concentrated under reduced pressure to afford the title compound as light yellow solid

(198 mg, 79% yield). LCMS [M+H]+ 320.0 m/z (Br79), 322.0 m/z (Br81); 1H NMR (500 MHz,

CHLOROFORM-d) ppm 7.66 - 7.70 (m, 2 H), 7.59 - 7.64 (m, 2 H), 3.04 (br. s., 4 H), 2.48 (t,

J=4.6 Hz, 4 H), 2.28 (s, 3 H).

189

N-(2-hydroxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide. (3.2a)

4-bromo-N-(2-hydroxyethyl)benzenesulfonamide (441 mg, 1.57 mmol), 4,4,4',4',5,5,5',5'-



subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,

15 mL) was added to the vial. The reaction mixture was heated at 80 °C overnight and monitored

by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under reduced

pressure, and then purified by silica gel column chromatography using a mobile phase of 0-100%

ethyl acetate/dichloromethane to afford the title compound as a light orange solid (464 mg, 90%

yield). LCMS [M+H]+ 328.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 7.84 - 7.89 (m, 2 H),

7.78 - 7.82 (m, 2 H), 7.68 (t, J=5.9 Hz, 1 H), 4.68 (t, J=5.6 Hz, 1 H), 3.31 - 3.36 (m, 2 H), 2.77 (q,

J=6.3 Hz, 2 H), 1.31 (s, 12 H), 1.16 (d, J=4.4 Hz, 2 H).

190

4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine. (3.2b) 4-((4-

bromophenyl)sulfonyl)morpholine (196 mg, 0.640 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-

bi(1,3,2-dioxaborolane) (243 mg, 0.960 mmol), PdCl2(dppf)·CH2Cl2 (26.1 mg, 0.032 mmol), and

potassium acetate (188 mg, 1.92 mmol) were added to a vial that was subsequently sealed and

vacuum purged with nitrogen. A degassed solution of dioxane (4 mL) was added to the vial. The

reaction mixture was heated at 85 °C overnight and monitored by LCMS. The reaction mixture

was filtered through a bed of Celite, concentrated under reduced pressure. The resulting residue

was used crude in the next reaction. A quantitative yield was assumed. LCMS [M+H]+ 354.1 m/z.

75% product, 25% boronic acid of product by LC trace.

1-methyl-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)piperazine. (3.2c) 1-

((4-bromophenyl)sulfonyl)-4-methylpiperazine (195 mg, 0.611 mmol), 4,4,4',4',5,5,5',5'-


191






[M+H]+ 367.2 m/z, 85% pure by LC trace.

4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine.164 (3.3) 4-bromo-1H-pyrrolo[2,3-b]pyridine (300

mg, 1.52 mmol) and 4-methylbenzenesulfonyl chloride (580 mg, 3.05 mmol) were dissolved in

dichloromethane (3 mL). Tetrabutylammonium hydrogensulfate (15.5 mg, 0.045 mmol) and

sodium hydroxide (3M, 1.9 mL) were added to the reaction mixture. The reaction mixture was

stirred at room temperature for 1 hour, monitoring by TLC, after which the reaction mixture was

quenched with saturated ammonium chloride solution. The organic layer was collected, and the

aqueous layer was extracted 3x with dichloromethane. The combined organic layers were washed

with water and then with brine, dried with sodium sulfate, filtered, and concentrated under reduced

pressure. The reaction mixture was then purified by column chromatogr5aphy using a mobile

phase of 0-10% ethyl acetate/hexanes to afford the title compound as a white, powdery solid (241

g, 45% yield). LCMS [M+H]+ 351.0 m/z (Br79), 353.0 m/z (Br81); 1H NMR (500 MHz,

CHLOROFORM-d) ppm 8.23 (d, J=5.4 Hz, 1 H), 8.07 (d, J=8.3 Hz, 2 H), 7.79 (d, J=3.9 Hz, 1

H), 7.36 (d, J=5.4 Hz, 1 H), 7.29 (d, J=8.3 Hz, 2 H), 6.65 (d, J=3.9 Hz, 1 H), 2.38 (s, 3 H).

192

4-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. NEU-4813 (3.4a) 4-Bromo-1-

tosyl-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.285 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan -2-yl)-1H-pyrazole (71.1 mg, 0.342 mmol), sodium carbonate (90.5 mg, 0854

mmol), and PdCl2(dppf)·CH2Cl2 (2.33 mg, 0.002 mmol) were added to a microwave vial that was

vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.8 mL) was added to

the vial. The reaction mixture was microwaved at 150 °C for 10 minutes. The reaction mixture

was monitored by LCMS analysis and, once complete, the reaction mixture was filtered through

Celite and the crude material was purified by column chromatography, eluting with a mobile phase

of 10-100% ethyl acetate/hexanes to afford the title compound as a colorless solid (75 mg, 75%

yield). LCMS [M+H]+ 353.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.37 (d, J=5.4

Hz, 1 H), 8.08 (d, J=8.8 Hz, 2 H), 7.88 (s, 1 H), 7.78 (s, 1 H), 7.76 (d, J=4.4 Hz, 1 H), 7.27 (d,

J=8.3 Hz, 2 H), 7.20 (d, J=4.9 Hz, 1 H), 6.78 (d, J=4.4 Hz, 1 H), 3.99 (s, 3 H), 2.36 (s, 3 H).

193

4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. NEU-4814 (3.4b) 4-bromo-1-

tosyl-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.285 mmol), (4-(methylsulfonyl)phenyl)boronic acid

(68.3 mg, 0.342 mmol), sodium carbonate (90.5 mg, 0.854 mmol), and PdCl2(dppf)·CH2Cl2 (2.33

mg, 0.002 mmol) were added to a microwave vial that was vacuum purged with nitrogen. A

mixture of degassed 3:1 dioxane/water (2.8 mL) was added to the vial. The reaction mixture was

microwaved at 150 °C for 10 minutes. The reaction mixture was monitored by LCMS analysis

and, once complete, the reaction mixture was filtered through Celite and the crude material was

purified by column chromatography, eluting with a mobile phase of 10-60% ethyl acetate/hexanes

to afford the title compound as a colorless solid (104 mg, 86% yield). LCMS [M+H]+ 427.1 m/z;

1H NMR (500 MHz, CHLOROFORM-d) ppm 8.52 (d, J=4.9 Hz, 1 H), 8.11 (d, J=8.3 Hz, 2 H),

8.08 (d, J=8.3 Hz, 2 H), 7.83 (d, J=3.9 Hz, 1 H), 7.78 (d, J=8.3 Hz, 2 H), 7.30 (d, J=8.3 Hz, 2 H),

7.25 (d, J=5.4 Hz, 1 H), 6.71 (d, J=4.4 Hz, 1 H), 3.13 (s, 3 H), 2.38 (s, 3 H).

194

4-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine. NEU-4471 (3.4c) 4-bromo-1H-pyrrolo[2,3-

b]pyridine (50 mg, 0.254 mmol), pyridin-4-ylboronic acid (37.4 mg, 0.304 mmol), sodium

carbonate (80.7 mg, 0.761 mmol), and PdCl2(dppf)·CH2Cl2 (2.1 mg, 0.002 mmol) were added to

a microwave vial that was vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water

(2.5 mL) was added to the vial. The reaction mixture was microwaved at 130 °C for 30 minutes.

The reaction mixture was monitored by LCMS analysis and, once complete, the reaction mixture

was filtered through Celite and the crude material was purified by column chromatography, eluting

with a mobile phase of 0-20% methanol/dichloromethane to afford the title compound as a pale

yellow solid (41 mg, 83% yield). LCMS [M+H]+ 195.9 m/z; 1H NMR (500 MHz, DMSO-d6)

ppm 11.94 (br. s., 1 H), 8.74 (d, J=6.3 Hz, 2 H), 8.34 (d, J=4.9 Hz, 1 H), 7.78 (d, J=5.9 Hz, 2 H),

7.61 - 7.64 (m, 1 H), 7.30 (d, J=4.9 Hz, 1 H), 6.68 (dd, J=3.4, 2.0 Hz, 1 H).

195

N-(2-hydroxyethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide. NEU-4786 (3.4d) 4-

bromo-1H-pyrrolo[2,3-b]pyridine (20 mg, 0.103 mmol), N-(2-hydroxyethyl)-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (42 mg, 0.128 mmol), sodium carbonate

(41 mg, 0.385 mmol), and PdCl2(dppf)·CH2Cl2 (3.1 mg, 0.003 mmol) were added to a microwave

vial that was vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.0 mL)

was added to the vial. The reaction mixture was microwaved at 130 °C for 30 minutes. The reaction

mixture was monitored by LCMS analysis and, once complete, the reaction mixture was filtered

through Celite and the crude material was purified by column chromatography, eluting with a

mobile phase of 40-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to

afford the title compound as a pale yellow solid (7 mg, 17% yield). LCMS [M+H]+ 318.1 m/z; 1H

NMR (500 MHz, DMSO-d6) ppm 11.90 (br. s., 1 H), 8.33 (d, J=4.9 Hz, 1 H), 7.97 - 8.03 (m, 2

H), 7.93 - 7.97 (m, 2 H), 7.74 (t, J=5.9 Hz, 1 H), 7.58 - 7.63 (m, 1 H), 7.27 (d, J=4.9 Hz, 1 H),

6.66 (dd, J=3.4, 1.5 Hz, 1 H), 4.72 (t, J=5.6 Hz, 1 H), 3.41 (m, J=5.4 Hz, 2 H), 2.86 (q, J=6.2 Hz,

2 H).

196

4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine.164 NEU-4472 (3.5a) 4-bromo-1H-

pyrrolo[2,3-b]pyridine (50 mg, 0.254 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (63.3 mg, 0.304 mmol), sodium carbonate (80.7 mg, 0.761

mmol), and PdCl2(dppf)·CH2Cl2 (2.1 mg, 0.002 mmol) were added to a microwave vial that was

vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.5 mL) was added to

the vial. The reaction mixture was microwaved at 130 °C for 30 minutes. The reaction mixture

was monitored by LCMS analysis and, once complete, the reaction mixture was filtered through

Celite and the crude material was purified by column chromatography, eluting with a mobile phase

of 70-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title

compound as a yellow solid (47 mg, 93% yield). LCMS [M+H]+ 198.9 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 11.65 (br. s., 1 H), 8.45 (s, 1 H), 8.15 (d, J=4.9 Hz, 1 H), 8.10 (s, 1 H), 7.43 -

7.55 (m, 1 H), 7.24 (d, J=4.9 Hz, 1 H), 6.78 (dd, J=3.4, 2.0 Hz, 1 H), 3.94 (s, 3 H).

197

4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine. NEU-4815 (3.5b) 4-(4-

(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (30.0 mg, 0.070 mmol) was dissolved

in dioxane (0.5 mL). Sodium hydroxide (2.5M, 0.295 mL) was added and the reaction mixture was

microwaved at 150 °C for 15 minutes. The reaction mixture was then concentrated under reduced

pressure and purified by silica gel column chromatography eluting with a mobile phase 0-100%

ethyl acetate/dichloromethane to afford the title compound as a yellow solid (9 mg, 47%). LCMS

[M+H]+ 273.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 11.93 (br. s., 1 H), 8.34 (d, J=4.9 Hz,

1 H), 8.07 - 8.11 (m, 2 H), 8.02 - 8.07 (m, 2 H), 7.62 (t, J=3.2 Hz, 1 H), 7.28 (d, J=4.9 Hz, 1 H),

6.65 (dd, J=3.2, 1.7 Hz, 1 H), 3.30 (s, 3 H).

1-(4-bromophenyl)-N,N-dimethylmethanamine.165 (3.6a) 4-bromobenzylbromide (500 mg, 2.00

mmol) was dissolved in hexanes (2 mL) and the solution then cooled to 0 °C. Dimethylamine (2M,

4.0 mL) was added dropwise. The reaction mixture was left stirring overnight and allowing it to

warm to room temperature. A white precipitate formed and was filtered off. The filtrate was

concentrated under reduced pressure to afford the title compound as a yellow liquid (394 mg, 92%

198


7.67 (d, J=8.3 Hz, 2 H), 7.49 (d, J=8.3 Hz, 2 H), 2.91 (s, 2 H), 2.67 (s, 6 H).

N-(4-bromobenzyl)-N-ethylethanamine.166 (3.6b) 4-bromobenzylbromide (200 mg, 0.800 mmol)

was dissolved in hexanes (1 mL) and the solution then cooled to 0 °C. Diethylamine (331 μL, 3.20

mmol) was added dropwise. The reaction mixture was left stirring overnight and allowing it to

warm to room temperature. A white precipitate formed and was filtered off. The filtrate was

concentrated under reduced pressure to afford the title compound as a white solid (137 mg, 70%

yield). LCMS [M+H]+ 243.0 m/z (Br79), 245.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-

d) ppm 7.43 (d, J=8.3 Hz, 2 H), 7.22 (d, J=8.3 Hz, 2 H), 3.51 (s, 2 H), 2.51 (q, J=6.8 Hz, 4 H),

1.03 (t, J=7.1 Hz, 6 H).

N,N-dimethyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanamine.167 (3.7a)

1-(4-bromophenyl)-N,N-dimethylmethanamine (361 mg, 1.69 mmol), 4,4,4',4',5,5,5',5'-


0.05 mmol), and potassium acetate (496 mg, 5.06 mmol) were added to a microwave vial that was

subsequently sealed and vacuum purged with nitrogen. Anhydrous dioxane (10 mL) was added to

199

the flask. The reaction mixture was microwaved at 130 °C for 40 minutes and monitored by LCMS.

The reaction mixture was filtered through a bed of Celite and concentrated under reduced pressure

and then purified by silica gel column chromatography using a mobile phase of 1-20% (10%

ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a brown solid

(325 mg, 74% yield). LCMS [M+H]+ 262.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm

7.78 (d, J=7.8 Hz, 2 H), 7.33 (d, J=7.8 Hz, 2 H), 3.46 (s, 2 H), 2.25 (s, 6 H), 1.35 (s, 12 H).

N-ethyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)ethanamine.168 (3.7b) N-(4-

bromobenzyl)-N-ethylethanamine (124 mg, 0.513 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-

bi(1,3,2-dioxaborolane) (194.5 mg, 0.769 mmol), PdCl2(dppf)·CH2Cl2 (12.6 mg, 0.015 mmol),

and potassium acetate (151.2 mg, 1.54 mmol) were added to a vial that was subsequently sealed

and vacuum purged with nitrogen. Anhydrous dioxane (3 mL) was added to the flask. The reaction

mixture was heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered

through a bed of Celite and concentrated under reduced pressure. The product was used crude in

the subsequent reaction, assuming quantitative yield. LCMS [M+H]+ 290.1 m/z.

200

tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxylate.

(3.7c) tert-butyl 2-(4-bromophenyl)pyrrolidine-1-carboxylate (243 mg, 0.74 mmol),

4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (284 mg, 1.12 mmol),

PdCl2(dppf)·CH2Cl2 (30.4 mg, 0.037 mmol), and potassium acetate (219 mg, 2.23 mmol) were

added to a vial that was subsequently sealed and vacuum purged with nitrogen. Anhydrous dioxane

(5 mL) was added to the vial. The reaction mixture was heated at 85 °C overnight and monitored

by LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under

reduced pressure. The product was used crude in the subsequent reaction, assuming quantitative

yield. LCMS [M+H]+ 318.2 m/z (minus t-butyl fragment mass), 90% pure by LC trace.

1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.8) 1H-pyrrolo[2,3-b]pyridine (200 mg, 1.69 mmol) and 4-

methylbenzenesulfonyl chloride (645 mg, 3.39 mmol) were dissolved in dichloromethane (8.6

mL). Tetrabutylammonium hydrogen sulfate (17.2 mg, 0.050 mmol) and sodium hydroxide (3M,

2.1 mL) were added to the reaction mixture. The reaction mixture was stirred at room temperature

for 1 hour, monitoring by TLC, after which the reaction mixture was quenched with saturated

ammonium chloride solution. The organic layer was collected and the aqueous layer was extracted

3x with dichloromethane. The combined organic layers were washed with water and then with

201

brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure. The reaction

mixture was then purified by column chromatography using a mobile phase of 0-70% ethyl

acetate/hexanes to afford the title compound as a white solid (339 mg, 73% yield). LCMS [M+H]+

273.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.43 (dd, J=4.9, 1.5 Hz, 1 H), 8.08 (d,

J=8.3 Hz, 2 H), 7.84 (dd, J=7.8, 1.5 Hz, 1 H), 7.73 (d, J=3.9 Hz, 1 H), 7.27 - 7.29 (m, 2 H), 7.17

(dd, J=7.8, 4.4 Hz, 1 H), 6.59 (d, J=3.9 Hz, 1 H), 1.57 (s, 3 H).

2-Iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.9) To a flame dried flask was added anhydrous THF

(2 mL) and diisopropylamine (178.8 μL, 1.27 mmol). The solution was cooled to 0 °C and n-

butyllithium (505 μL, 1.21 mmol) was added. The solution was then cooled to -78 °C and 1-tosyl-

1H-pyrrolo[2,3-b]pyridine (300 mg, 1.10 mmol) in anhydrous THF (1 mL) was added dropwise.

The reaction mixture was left stirring for 3 h, allowing it to warm to -20 °C after which iodine

(363 mg, 1.43 mmol), dissolved in anhydrous THF (1 mL), was added dropwise. The reaction

mixture was left stirring overnight and allowing it to warm to room temperature. The reaction

mixture was then quenched with saturated aqueous ammonium chloride solution. The organic layer

was collected and the aqueous layer washed 3x with DCM. The combined organic layers were

washed with brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure.

The reaction mixture was then purified by silica gel column chromatography, using a gradient of

0-20% ethyl acetate/hexanes to afford the title compound as tan solid (173 mg, 39% yield). LCMS

[M+H]+ 399.0 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.40 (dd, J=4.9, 1.5 Hz, 1 H),

202

8.10 (d, J=8.3 Hz, 2 H), 7.72 (dd, J=7.8, 2.0 Hz, 1 H), 7.29 (s, 2 H), 7.14 (dd, J=7.8, 4.9 Hz, 1 H),

6.99 (s, 1 H), 2.38 (s, 3 H).

N,N-dimethyl-1-(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. (3.10a) 2-iodo-

1-tosyl-1H-pyrrolo[2,3-b]pyridine (32 mg, 0.08 mmol), N,N-dimethyl-1-(4-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl)methanamine (21 mg, 0.08 mmol), [1,1′-

Tetrakis(triphenylphosphine)palladium(0) (2.8 mg, 0.002 mmol), and cesium carbonate (78.5 mg,

0.241 mmol) were added to a microwave vial that was subsequently sealed and vacuum purged

with nitrogen. A degassed solution of dioxane/water (3:1, 0.8 mL) was added to the vial. The

reaction mixture was microwaved at 150 °C for 15 minutes and monitored by LCMS. The reaction

mixture was filtered through a bed of Celite and concentrated under reduced pressure. The crude

product was used in the subsequent reaction assuming quantitative yield. LCMS [M+H]+ 406.2

m/z, 80% pure by LC trace.

(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-4812 (3.10b) 2-iodo-1-tosyl-

1H-pyrrolo[2,3-b]pyridine (50 mg, 0.126 mmol), (4-(hydroxymethyl)phenyl)boronic acid (23 mg,

0.15 mmol), PdCl2(dppf)·CH2Cl2 (2.0 mg, 0.002 mmol), and cesium carbonate (123 mg, 0.377

203

mmol) were added to a microwave vial that was subsequently sealed and vacuum purged with

nitrogen. A degassed solution of dioxane/water (3:1, 1.2 mL) was added to the vial. The reaction

mixture was microwaved at 150 °C for 10 minutes and monitored by LCMS. The reaction mixture

was filtered through a bed of Celite and concentrated under reduced pressure. The crude product

was purified by silica gel column chromatography using a mobile phase of 0-100% ethyl

acetate/hexanes to afford the title compound as colorless residue (38 mg, 80% yield). LCMS

[M+H]+ 379.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.48 (dd, J=4.9, 1.5 Hz, 1 H),

7.70 - 7.82 (m, 3 H), 7.56 (d, J=8.3 Hz, 2 H), 7.47 (d, J=8.3 Hz, 2 H), 7.19 (dd, J=7.8, 4.9 Hz, 1

H), 7.16 (d, J=8.3 Hz, 2 H), 6.50 (s, 1 H), 4.80 (s, 2 H), 2.33 (s, 3 H).

1-(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine. NEU-4844 (3.11a)

N,N-dimethyl-1-(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (32.6 mg, 0.080

mmol) was dissolved in dioxane (0.8 mL). Sodium hydroxide (3M, 0.281 mmol) was added and

the reaction was microwaved at 150 °C for 15 minutes. The crude product was then purified by

column chromatography eluting with a mobile phase of 0-20% methanol/dichloromethane to

afford the title compound as a light tan solid (11 mg, 54% yield). LCMS [M+H]+ 252.2 m/z; 1H

NMR (500 MHz, METHANOL-d4) ppm 8.21 (dd, J=4.4, 1.5 Hz, 1 H), 7.96 - 8.03 (m, 3 H), 7.59

(d, J=8.3 Hz, 2 H), 7.12 (dd, J=7.8, 4.9 Hz, 1 H), 6.95 (s, 1 H), 4.29 (s, 2 H), 2.84 (s, 6 H).

204

(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-4845 (3.11b) (4-(1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol (83.0 mg, 0.219 mmol) was dissolved in dioxane (2.0

mL). Sodium hydroxide (3M, 0.767 mmol) was added and the reaction was microwaved at 150 °C

for 15 minutes. The crude product was then purified by column chromatography eluting with a

mobile phase of 0-5% methanol/dichloromethane and then recrystallized using methanol to afford

the title compound as yellow needle shaped crystals (6 mg, 12% yield). LCMS [M+H]+ 225.1 m/z;

1H NMR (500 MHz, DMSO-d6) ppm 12.09 (s, 1 H), 8.20 (dd, J=4.4, 1.5 Hz, 1 H), 7.88 - 7.93

(m, 3 H), 7.40 (d, J=8.3 Hz, 2 H), 7.05 (dd, J=7.6, 4.6 Hz, 1 H), 6.89 (d, J=2.4 Hz, 1 H), 5.24 (t,

J=5.6 Hz, 1 H), 4.54 (d, J=5.9 Hz, 2 H).

4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.12) 4-Chloro-1H-pyrrolo[2,3-b]pyridine (2.00 g,

13.1 mmol) and 4-methylbenzenesulfonyl chloride (5.00 g, 26.2 mmol) were dissolved in

dichloromethane (36 mL). Tetrabutylammonium hydrogen sulfate (133 mg, 0.393 mmol) and

sodium hydroxide (3M, 16.2 mL) were added to the reaction mixture. The reaction mixture was

stirred at room temperature for 1 hour, monitoring by TLC, after which the reaction mixture was

quenched with saturated ammonium chloride solution. The organic layer was collected, and the


205

with water and then with brine, dried with sodium sulfate, filtered, and concentrated under reduced

pressure. The reaction mixture was then purified by column chromatography using a mobile phase

of 0-20% ethyl acetate/hexanes to afford the title compound as a white solid (3.25 g, 81% yield).

LCMS [M+H]+ 306.9 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.32 (d, J=4.9 Hz, 1

H), 8.07 (d, J=8.3 Hz, 2 H), 7.78 (d, J=3.9 Hz, 1 H), 7.29 (d, J=7.8 Hz, 2 H), 7.20 (d, J=5.4 Hz, 1

H), 6.70 (d, J=3.9 Hz, 1 H), 2.38 (s, 3 H).

4-Chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.13) To a flame dried flask was added 4-

chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (501 mg, 1.63 mmol). Anhydrous THF (40 mL) was

added and the solution was cooled to -78 °C. Lithium diisopropylamide (1.62M, 1.51 mL) was

then added dropwise to the solution. The reaction mixture was left stirring for 1 hour, after which

iodine (1.24 g, 4.90 mmol) dissolved in anhydrous THF (10 mL) was added dropwise. The reaction

mixture was left stirring overnight and allowed to warm to room temperature. The reaction mixture

was then quenched with saturated aqueous ammonium chloride solution. The organic layer was

collected and the aqueous layer washed 3x with DCM. The combined organic layers were washed

with brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure. The

reaction mixture was then purified by silica gel column chromatography, using a gradient of 0-5%

ethyl acetate/hexanes to afford the title compound as an off-white solid (621 mg, 88% yield).

LCMS [M+H]+ 432.8 m/z (Cl35), 434.8 m/z (Cl37); 1H NMR (400 MHz, CHLOROFORM-d)

206

ppm 8.28 (d, J=5.1 Hz, 1 H), 8.10 (d, J=8.8 Hz, 2 H), 7.29 (d, J=8.1 Hz, 2 H), 7.16 (d, J=5.1 Hz,

1 H), 7.11 (s, 1 H), 2.39 (s, 3 H).

1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine. (3.14)

4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (153 mg, 0.353 mmol), N,N-dimethyl-1-(4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanamine (120 mg, 0.460 mmol),

PdCl2(dppf)·CH2Cl2 (14.4 mg, 0.05 mmol), and sodium carbonate (112 mg, 1.06 mmol) were

added to an oven dried vial that was subsequently sealed and vacuum purged with nitrogen. A

degassed solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was

heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a

bed of Celite and concentrated under reduced pressure and then purified by silica gel column

chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title

compound as an off-white solid (77 mg, 76% yield). LCMS [M+H]+ 439.9 m/z (Cl35), 442.0 m/z

(Cl37); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.36 (d, J=4.9 Hz, 1 H), 7.77 (d, J=8.3 Hz,

2 H), 7.51 (d, J=7.8 Hz, 2 H), 7.40 - 7.44 (m, 2 H), 7.21 (d, J=5.4 Hz, 1 H), 7.19 (d, J=8.3 Hz, 2

H), 6.61 (s, 1 H), 3.53 (s, 2 H), 2.35 (s, 3 H), 2.32 (s, 6 H).

207

N,N-Dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-4877 (3.15a) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)-N,N-dimethylmethanamine (92 mg, 0.21 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-1H-pyrazole (87 mg, 0.42 mmol),

Tetrakis(triphenylphosphine)palladium(0) (12.1 mg, 0.010 mmol), and sodium carbonate (111 mg,

1.05 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.

A degassed solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was

microwaved at 130 °C for 25 minutes and monitored by LCMS. The reaction mixture was filtered

through a bed of Celite and concentrated under reduced pressure and then purified by silica gel

column chromatography using a mobile phase of 1-10% (+ 1% ammonium

hydroxide)/methanol/dichloromethane to afford the title compound as an off white solid (73 mg,

72% yield). LCMS [M+H]+ 486.0 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.29 (d,

J=4.9 Hz, 1 H), 8.25 (s, 1 H), 8.00 (s, 1 H), 7.59 (t, J=8.3 Hz, 4 H), 7.42 - 7.46 (m, 3 H), 7.23 (d,

J=8.3 Hz, 2 H), 6.97 (s, 1 H), 3.94 (s, 3 H), 3.62 (s, 2 H), 2.34 (s, 6 H), 2.31 (s, 3 H).

208

1-(4-(4-(1,3-Dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-

dimethylmethanamine. (3.15b) 1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-

N,N-dimethylmethanamine (50 mg, 0.114 mmol), 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (30.3 mg, 0.136 mmol),

tetrakis(triphenylphosphine)palladium(0) (3.9 mg, 0.003 mmol), and sodium carbonate (36.1 mg,


A degassed solution of dioxane-water (3:1, 1 mL) was added to the vial. The reaction mixture was

heated at 90 °C for 48 h and monitored by LCMS. The reaction mixture was filtered through a bed

of Celite and concentrated under reduced pressure and then purified by silica gel column

chromatography using a mobile phase of 0-20% methanol/dichloromethane to afford the title

compound as a yellow oil (18 mg, 32% yield). LCMS [M+H]+ 500.0 m/z, 81% pure by LC trace.

Used crude in the following reaction.

1-(4-(4-(1H-Pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-

dimethylmethanamine. (3.15c) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-

209

N,N-dimethylmethanamine (50 mg, 0.114 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-1H-pyrazole (26.5 mg, 0.136 mmol), tetrakis(triphenylphosphine)palladium(0) (3.9 mg, 0.003

mmol), and sodium carbonate (72.3 mg, 0.682 mmol) were added to a vial that was subsequently

sealed and vacuum purged with nitrogen. A degassed solution of dioxane-water (3:1, 1 mL) was

added to the vial. The reaction mixture was heated at 90 °C for 48 h and monitored by LCMS. The

reaction mixture was filtered through a bed of Celite and concentrated under reduced pressure and

then purified by silica gel column chromatography using a mobile phase of 0-20% (1% ammonium

hydroxide/methanol)/dichloromethane to afford the title compound as a brown solid (35 mg, 66%

yield). LCMS [M+H]+ 472.0 m/z, 72% pure by LC trace. Used crude in the following reaction.

N,N-Dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. (3.15d) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-

N,N-dimethylmethanamine (50 mg, 0.11 mmol), 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (28 mg, 0.136 mmol), tetrakis(triphenylphosphine)palladium(0)

(3.9 mg, 0.003 mmol), and sodium carbonate (72 mg, 0.68 mmol) were added to a vial that was

subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane-water (3:1,

1 mL) was added to the vial. The reaction mixture was heated at 90 °C for 48 h and monitored by

LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under reduced

pressure and then purified by silica gel column chromatography using a mobile phase of 0-20%

210

(1% ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a brown

solid (16 mg, 28% yield). LCMS [M+H]+ 486.0 m/z. 1H NMR (500 MHz, DMSO-d6) ppm 8.33

(d, J=4.9 Hz, 1 H), 8.16 (d, J=5.4 Hz, 1 H), 7.95 (d, J=8.3 Hz, 2 H), 7.73 (d, J=8.3 Hz, 1 H), 7.55


(d, J=2.4 Hz, 1 H), 3.39 - 3.45 (m, 2 H), 2.29 - 2.34 (m, 3 H), 2.20 (s, 3 H), 2.17 (s, 6 H).

N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. (3.15e) 1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-

N,N-dimethylmethanamine (53.6 mg, 0.122 mmol), (4-(methylsulfonyl)phenyl)boronic acid (29.2

mg, 0.146 mmol), tetrakis(triphenylphosphine)palladium(0) (4.2 mg, 0.003 mmol), and sodium

carbonate (38.7 mg, 0.365 mmol) were added to a vial that was subsequently sealed and vacuum

purged with nitrogen. A degassed solution of dioxane-water (3:1, 2 mL) was added to the vial. The

reaction mixture was heated at 90 °C for 48 h and monitored by LCMS. The reaction mixture was

filtered through a bed of Celite and concentrated under reduced pressure and then purified by silica

gel column chromatography using a mobile phase of 0-10% ethyl acetate/hexanes to afford the

title compound as an off white solid (28 mg, 41% yield). LCMS [M+H]+ 560.0 m/z; 1H NMR (500

MHz, METHANOL-d4) ppm 8.47 (d, J=4.9 Hz, 1 H), 8.09 (d, J=8.8 Hz, 2 H), 7.91 (d, J=8.8 Hz,

211

2 H), 7.65 (d, J=8.3 Hz, 2 H), 7.57 (d, J=8.3 Hz, 2 H), 7.46 (d, J=4.9 Hz, 1 H), 7.44 (d, J=8.3 Hz,

2 H), 7.27 (d, J=7.8 Hz, 2 H), 6.82 (s, 1 H), 3.61 (s, 2 H), 3.16 (s, 3 H), 2.34 (s, 3 H), 2.32 (s, 6 H).

N,N-Dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine.164 NEU-1200 (3.16a) N,N-dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-

yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (67 mg, 0.138 mmol) was

dissolved in dioxane (1.4 mL). Sodium hydroxide (3M, 0.46 mL) was added and the reaction

mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was then concentrated

under reduced pressure. The crude product was purified using column chromatography eluting

with a mobile phase of 1-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford

the title compound as the tosylate salt, yellow solid (46 mg, 66% yield). LCMS [M+H]+ 332.2

m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.13 (s, 1 H), 8.58 (s, 1 H), 8.19 (s, 1 H), 8.14 (d,

J=4.9 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H), 7.38 (d, J=7.8 Hz, 2 H), 7.30 (d, J=2.0 Hz, 1 H), 7.27 (d,

J=4.9 Hz, 1 H), 3.96 (s, 3 H), 3.42 (s, 2 H), 2.17 (s, 6 H). See assignments above.

212

1-(4-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-

dimethylmethanamine. NEU-4927 (3.16b) 1-(4-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine (18.3 mg, 0.037 mmol) was


mixture was microwaved for 30 minutes at 150 °C. The reaction mixture was then concentrated

under reduced pressure. The crude product was purified using column chromatography eluting

with a mobile phase of 1-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford

the title compound a yellow solid (8 mg, 62% yield). 1H NMR (500 MHz, METHANOL-d4)

ppm 8.17 (d, J=4.9 Hz, 1 H), 8.05 (s, 1 H), 7.87 (d, J=8.3 Hz, 2 H), 7.42 (d, J=8.3 Hz, 2 H), 7.10

(d, J=5.4 Hz, 1 H), 6.95 (s, 1 H), 3.94 (s, 3 H), 3.55 (s, 2 H), 2.41 (s, 3 H), 2.30 (s, 6 H). See

assignments above.

213

1-(4-(4-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine.

NEU-4928 (3.16c) 1-(4-(4-(1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-

N,N-dimethylmethanamine (34 mg, 0.072 mmol) was dissolved in dioxane (1.0 mL). Sodium

hydroxide (3M, 0.721 mL) was added and the reaction mixture was microwaved for 15 minutes at

150 °C. The reaction mixture was then concentrated under reduced pressure. The crude product

was purified using column chromatography eluting with a mobile phase of 5-20% ((+1%

ammonium hydroxide)/methanol)/dichloromethane to afford the title compound as the tosylate

salt, yellow solid (10 mg, 45% yield). LCMS [M+H]+ 318.1 m/z; 1H NMR (500 MHz,

METHANOL-d4) ppm 8.33 (br. s., 2 H), 8.18 (d, J=5.4 Hz, 1 H), 8.05 (d, J=8.3 Hz, 2 H), 7.71

(d, J=7.8 Hz, 2 H), 7.61 (d, J=8.3 Hz, 2 H), 7.34 (d, J=5.4 Hz, 1 H), 7.28 (s, 1 H), 7.22 (d, J=7.8

Hz, 2 H), 4.36 (s, 2 H), 2.89 (s, 6 H), 2.35 (s, 3 H). See assignments above.

214

N,N-dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-4929 (3.16d) N,N-dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-

1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (14.7 mg, 0.030 mmol) was dissolved

in dioxane (1.0 mL). Sodium hydroxide (3M, 0.302 mL) was added and the reaction mixture was

microwaved for 15 minutes at 150 °C. The reaction mixture was then concentrated under reduced

pressure. The crude product was purified using column chromatography eluting with a mobile

phase of 5-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford the title


METHANOL-d4) ppm 8.18 (d, J=4.9 Hz, 1 H), 8.00 (s, 1 H), 7.87 (d, J=8.3 Hz, 2 H), 7.44 (d,

J=8.3 Hz, 2 H), 7.10 (d, J=4.9 Hz, 1 H), 6.94 (s, 1 H), 3.61 (s, 2 H), 2.48 (s, 3 H), 2.34 (s, 6 H).

See assignments above.

215

N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-4917 (3.16e) N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1-

tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (28 mg, 0.050 mmol) was dissolved in

dioxane (1.0 mL). Sodium hydroxide (3M, 0.175 mL) was added and the reaction mixture was

microwaved for 30 minutes at 150 °C. The reaction mixture was then concentrated under reduced

pressure. The crude product was purified using column chromatography eluting with a mobile

phase of 0-20% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the title


DMSO-d6) ppm 12.39 (s, 1 H), 8.33 (d, J=4.9 Hz, 1 H), 8.11 (s, 4 H), 7.96 (d, J=8.3 Hz, 2 H),

7.38 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz, 1 H), 7.15 (d, J=2.0 Hz, 1 H), 3.42 (s, 2 H), 3.31 (s, 3

H), 2.16 (s, 6 H). See assignments above.

216

4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.17) 4-Chloro-2-iodo-1-

tosyl-1H-pyrrolo[2,3-b]pyridine (2.32 g, 5.36 mmol), (4-formylphenyl)boronic acid (965 mg, 6.43

mmol), PdCl2(dppf)·CH2Cl2 (43.8 mg, 0.053 mmol), and sodium carbonate (1.70 g, 16.1 mmol)

were added to an oven dried flask that was subsequently sealed and vacuum purged with nitrogen.

A degassed solution of dioxane-water (3:1, 52 mL) was added to the vial. The reaction mixture

was heated at 85 °C overnight and monitored by LCMS. The reaction mixture was filtered through

a bed of Celite and concentrated under reduced pressure, water was added a precipitate formed

and was collected by vacuum filtration to afford the title compound as a tan solid (2.09 g, 95%

yield). LCMS [M+H]+ 411.0 m/z (Cl35), 413.0 m/z (Cl37); 1H NMR (500 MHz, CHLOROFORM-

d) ppm 10.10 (s, 1 H), 8.38 (d, J=5.4 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H),


H).

217

4-(4-(4-(Morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde.

(3.18a) 4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol),

4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine (215 mg, 0.608

mmol), tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol), and sodium carbonate

(129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and vacuum purged with


mixture was heated at 100 °C for 18 h and monitored by LCMS. The reaction mixture was filtered


column chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title

compound as an off white solid (57 mg, 39% yield). LCMS [M+H]+ 602.1 m/z; 1H NMR (500

MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.62 (d, J=4.9 Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H),

7.88 (d, J=6.8 Hz, 4 H), 7.76 (dd, J=9.5, 8.5 Hz, 4 H), 7.32 (d, J=5.4 Hz, 1 H), 7.20 - 7.26 (m, 3

H), 6.74 (s, 1 H), 3.75 - 3.79 (m, 4 H), 3.03 - 3.08 (m, 4 H), 2.38 (s, 2 H).

218

4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde. (3.18b) 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde

(100 mg, 0.243 mmol), 1-methyl-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)sulfonyl)piperazine (223 mg, 0.608 mmol), Tetrakis(triphenylphosphine)palladium(0)

(14.1 mg, 0.012 mmol), and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was


2.5 mL) was added to the vial. The reaction mixture was heated at 100 °C for 18 h and monitored


reduced pressure and then purified by silica gel column chromatography using a mobile phase of

0-100% ethyl acetate/hexanes to afford the title compound as tan-orange solid (82 mg, 55% yield).

LCMS [M+H]+ 615.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.60

(d, J=5.4 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H), 7.87 (d, J=8.3 Hz, 4 H), 7.74 - 7.77 (m, 4 H), 7.29 (d,

J=4.9 Hz, 1 H), 7.25 (d, J=8.3 Hz, 2 H), 6.73 (s, 1 H), 3.09 (br. s., 4 H), 2.56 (br. s., 4 H), 2.38 (s,

3 H), 1.26 (s, 3 H).

219

4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18c) 4-(4-chloro-1-tosyl-

1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), phenylboronic acid (118.7

mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol), and sodium

carbonate (129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and vacuum

purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The

reaction mixture was heated at 90 °C overnight and monitored by LCMS. The reaction mixture

was filtered through a bed of Celite, concentrated under reduced pressure, and then purified by

silica gel column chromatography using a mobile phase of 0-50% ethyl acetate/hexanes to afford

the title compound as a tan solid (90 mg, 82% yield). LCMS [M+H]+ 453.1 m/z; 1H NMR (500

MHz, CHLOROFORM-d) ppm 10.10 (s, 1 H), 8.55 (d, J=4.9 Hz, 1 H), 8.25 (dd, J=7.8, 1.5 Hz,

4 H), 7.97 (d, J=8.3 Hz, 1 H), 7.83 (d, J=8.3 Hz, 1 H), 7.75 (d, J=7.8 Hz, 2 H), 7.57 - 7.60 (m, 2

H), 7.39 - 7.44 (m, 1 H), 7.29 (d, J=5.4 Hz, 1 H), 7.21 (d, J=8.3 Hz, 2 H), 6.80 (s, 1 H), 2.36 (s, 3

H).

220

4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18d) 4-(4-chloro-1-

tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), pyridin-4-ylboronic

acid (119.7 mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol),

and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and

vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the

vial. The reaction mixture was heated at 90 °C overnight and monitored by LCMS. The reaction

mixture was filtered through a bed of Celite, concentrated under reduced pressure, and then

purified by silica gel column chromatography using a mobile phase of 0-100% ethyl

acetate/hexanes to afford the title compound as a off-white solid (65 mg, 59% yield). LCMS

[M+H]+ 454.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.75 (d, J=5.9

Hz, 2 H), 8.61 (d, J=4.9 Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H), 7.87 (d, J=8.3 Hz, 2 H), 7.75 (d, J=7.8

Hz, 2 H), 7.51 (d, J=5.9 Hz, 2 H), 7.32 (d, J=4.9 Hz, 1 H), 7.24 (d, J=8.3 Hz, 2 H), 6.76 (s, 1 H),

2.38 (s, 3 H).

221

4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18e) 4-(4-chloro-1-

tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), (2-methylpyridin-4-

yl)boronic acid (133.3 mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg,

0.012 mmol), and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was


2.5 mL) was added to the vial. The reaction mixture was heated at 90 °C overnight and monitored


pressure, and then purified by silica gel column chromatography using a mobile phase of 0-50%

ethyl acetate/dichloromethane to afford the title compound as a light yellow solid (70 mg, 62%

yield). LCMS [M+H]+ 468.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H),

8.61 (dd, J=13.4, 5.1 Hz, 2 H), 8.00 (d, J=7.8 Hz, 2 H), 7.86 (d, J=8.3 Hz, 2 H), 7.75 (d, J=8.3 Hz,

2 H), 7.34 (s, 1 H), 7.30 (d, J=5.4 Hz, 2 H), 7.24 (d, J=8.3 Hz, 2 H), 6.75 (s, 1 H), 2.64 (s, 3 H),

2.37 (s, 3 H).

222

4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18f) 4-

(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (89 mg, 0.217 mmol), (2,6-

dimethylpyridin-4-yl)boronic acid (130.8 mg, 0.866 mmol),



A degassed solution of dioxane/water (3:1, 2.0 mL) was added to the vial. The reaction mixture


a bed of Celite, concentrated under reduced pressure, and then purified by silica gel column

chromatography using a mobile phase of 0-60% ethyl acetate/dichloromethane to afford the title


DMSO-d6) ppm 10.11 - 10.13 (m, 1 H), 8.49 - 8.54 (m, 1 H), 8.04 (d, J=8.3 Hz, 2 H), 7.88 (d,

J=8.3 Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H), 7.51 (d, J=5.4 Hz, 1 H), 7.38 (d, J=8.3 Hz, 2 H), 7.36 (s,

2 H), 7.15 (s, 1 H), 2.33 (s, 3 H), 1.16 (s, 6 H).

223

N,N-dimethyl-1-(4-(4-(4-(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. (3.19a) 4-(4-(4-(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-2-yl)benzaldehyde (50 mg, 0.083 mmol) was dissolved in dichloroethane (1 mL).

Dimethylamine hydrochloride (33.9 mg, 0.415 mmol), triethylamine (58 μL, 0.415 mmol) were

added and the reaction mixture was left stirring at room temperature for 1 hour, after which sodium

borohydride (15.7 mg, 0.415 mmol) was added. The reaction mixture was left stirring at room

temperature overnight. It was then quenched with sodium hydroxide (3M). The aqueous layer was

extracted 3 times with ethyl acetate. The combined organic layers were washed with water and

brine, dried with sodium sulfate, filtered, and concentrated to afford the title compound as a brown

residue (crude 56 mg). Quantitative yield was assumed. LCMS [M+H]+ 631.2 m/z; 1H NMR (500

MHz, CHLOROFORM-d) ppm 8.55 - 8.61 (m, 1 H), 7.86 (dd, J=7.6, 3.7 Hz, 8 H), 7.77 (d, J=7.3

Hz, 4 H), 7.20 - 7.24 (m, 2 H), 6.65 (s, 1 H), 3.75 - 3.80 (m, 4 H), 3.06 (d, J=4.4 Hz, 4 H), 2.60 (s,

1 H), 2.37 (d, J=3.4 Hz, 3 H), 1.26 (s, 6 H).

224

N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-2-yl)phenyl)methanamine. (3.19b) 4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-

1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (80 mg, 0.130 mmol) was dissolved in

dichloroethane (1 mL). Dimethylamine hydrochloride (53.1 mg, 0.650 mmol), triethylamine (91

μL, 0.650 mmol) were added and the reaction mixture was left stirring at room temperature for 1

hour, after which sodium borohydride (40.9 mg, 0.650 mmol) was added. The reaction mixture

was left stirring at room temperature overnight. It was then quenched with sodium hydroxide (3M).

The aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were

washed with water and brine, dried with sodium sulfate, filtered, and concentrated to afford the

title compound as a brown residue (70 mg, 84% crude yield). LCMS [M+H]+ 644.2 m/z; 1H NMR

(500 MHz, CHLOROFORM-d) ppm 8.58 (t, J=5.1 Hz, 1 H), 7.82 - 7.89 (m, 4 H), 7.75 (dd,

J=14.6, 7.8 Hz, 4 H), 7.58 - 7.63 (m, 2 H), 7.20 - 7.26 (m, 3 H), 6.66 (s, 1 H), 3.06 (br. s., 4 H),

2.57 - 2.61 (m, 2 H), 2.49 (d, J=4.9 Hz, 4 H), 2.27 (s, 6 H), 1.26 (s, 6 H).

225

N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine.

(3.19c) 4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (90 mg, 0.199 mmol)

was dissolved in dichloroethane (1.5 mL) to which dimethylamine (2M in methanol, 1 mL) was

added. The mixture was left stirring at room temperature for 1 hour. Sodium cyanoborohydride

(62.5 mg, 0.994 mmol) was added to the reaction mixture which was left stirring at room

temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide and then

extracted with dichloromethane 3 times. The combined organic layers were washed with water

and brine, collected, and concentrated. The product was purified by column chromatography using

a mobile phase of 0-100% ethyl acetate/dichloromethane to afford the title compound as a yellow

solid (35 mg, 36%). LCMS [M+H]+ 482.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm

8.51 (d, J=4.9 Hz, 1 H), 7.81 (d, J=7.8 Hz, 2 H), 7.38 - 7.61 (m, 9 H), 7.14 - 7.30 (m, 3 H), 6.71

(s, 1 H), 3.55 (br. s., 2 H), 3.48 (s, 3 H), 2.32 (br. s., 6 H).

226

N,N-dimethyl-1-(4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine.

(3.19d) 4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (64 mg, 0.141

mmol) was dissolved in dichloroethane (1.5 mL) to which dimethylamine (2M in methanol, 0.7

mL) was added. The mixture was left stirring at room temperature for 1 hour. Sodium

cyanoborohydride (44.3 mg, 0.706 mmol) was added to the reaction mixture which was left stirring

at room temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide

and then extracted with ethyl acetate 3 times. The combined organic layers were washed with

water and brine, collected, and concentrated. The product was purified by column chromatography

using a mobile phase of 0-100% ethyl acetate/dichloromethane followed by 0-20% methanol/ethyl

acetate to afford the title compound as a white solid (28 mg, 40%). LCMS [M+H]+ 483.2 m/z.

N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. (3.19e) 4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (65 mg, 0.139 mmol) was dissolved in dichloroethane (1.0 mL) to which

dimethylamine hydrochloride (56.7 mg, 0.695 mmol) was added along with triethylamine (97 μL,

0.695 mmol). The mixture was left stirring at room temperature for 1 hour. Sodium



and then extracted with dichloromethane 3 times. The combined organic layers were washed with

227

water and brine, collected, and concentrated to afford the title compound as a brown residue to use

in the next reaction assuming quantitative yield. LCMS [M+H]+ 497.2 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 8.56 (d, J=5.4 Hz, 1 H), 8.48 (d, J=4.9 Hz, 1 H), 7.74 (d, J=8.3 Hz, 2 H), 7.56

(d, J=6.3 Hz, 3 H), 7.48 - 7.52 (m, 2 H), 7.40 (d, J=7.8 Hz, 2 H), 7.36 (d, J=8.3 Hz, 2 H), 6.97 (s,

1 H), 3.48 (s, 2 H), 2.54 (s, 3 H), 2.32 (s, 3 H), 2.20 (s, 6 H).

1-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-

dimethylmethanamine. (3.19f) 4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-

2-yl)benzaldehyde (87 mg, 0.181 mmol) was dissolved in dichloroethane (1.0 mL) to which

dimethylamine hydrochloride (73.7 mg, 0.903 mmol) was added along with triethylamine (126

μL, 0.903 mmol). The mixture was left stirring at room temperature for 1 hour. Sodium



and then extracted with ethyl acetate 3 times. The combined organic layers were washed with

water and brine, collected, and concentrated. The product was purified by silica gel column




228

H), 7.42 (d, J=7.8 Hz, 2 H), 7.25 (d, J=5.9 Hz, 1 H), 7.20 (d, J=8.8 Hz, 2 H), 7.15 (s, 2 H), 6.65

(s, 1 H), 3.52 (s, 2 H), 2.59 (s, 6 H), 2.34 - 2.38 (m, 3 H), 2.31 (s, 6 H).

N,N-dimethyl-1-(4-(4-(4-(morpholinosulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-5828 (3.20a) N,N-dimethyl-1-(4-(4-(4-

(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (55

mg, 0.087 mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide (3M, 0.3 mL) was added

and the reaction mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was

quenched with saturated ammonium chloride solution and diluted with water. A precipitate formed

and was collected by vacuum filtration. The solid crude product was purified by column

chromatography eluting with a mobile phase of 1-20% ((+5% ammonium

hydroxide)/methanol)/dichloromethane to afford the title compound as a yellow solid (26 mg, 63%

yield). LCMS [M+H]+ 477.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.40 (s, 1 H), 8.33 (d,

J=4.9 Hz, 1 H), 8.12 (d, J=8.3 Hz, 2 H), 7.97 (d, J=7.8 Hz, 2 H), 7.91 (d, J=8.3 Hz, 2 H), 7.38 (d,

J=7.8 Hz, 2 H), 7.30 (d, J=4.9 Hz, 1 H), 7.19 (d, J=1.5 Hz, 1 H), 3.64 - 3.72 (m, 4 H), 3.42 (s, 2

H), 2.97 (br. s., 4 H), 2.17 (s, 6 H).

229

N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-5829 (3.20b) N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-

yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (70 mg, 0.109

mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide (3M, 0.3 mL) was added and the

reaction mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was quenched

with saturated ammonium chloride solution and diluted with water. A precipitate formed and was

collected by vacuum filtration. The solid crude product was purified by column chromatography

eluting with a mobile phase of 1-20% ((+5% ammonium hydroxide)/methanol)/dichloromethane

to afford the title compound as a yellow solid (12 mg, 22% yield). LCMS [M+H]+ 490.2 m/z; 1H

NMR (500 MHz, DMSO-d6) ppm 12.39 (br. s., 1 H), 8.33 (d, J=4.9 Hz, 1 H), 8.11 (d, J=8.3 Hz,

2 H), 7.97 (d, J=7.8 Hz, 2 H), 7.90 (d, J=8.3 Hz, 2 H), 7.38 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz,

1 H), 7.18 (s, 1 H), 3.43 (s, 2 H), 2.98 (br. s., 4 H), 2.40 (br. s., 4 H), 2.13 - 2.20 (m, 2 H).

230

N,N-dimethyl-1-(4-(4-phenyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. NEU-5446

(3.20c) N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine (34 mg, 0.071 mmol) was dissolved in dioxane (0.2 mL). Sodium


150 °C. The reaction mixture was quenched with saturated ammonium chloride solution and

concentrated under reduced pressure. The crude product was purified by column chromatography

eluting with a mobile phase of 1-15% methanol/dichloromethane to afford the title compound as

a salt. The salt was dissolved in ethyl acetate which was neutralized using 1M HCl and saturated

sodium bicarbonate solution. The organic layer was collected to afford the title compound as a

yellow solid (2 mg, 10% yield). LCMS [M+H]+ 328.2 m/z; 1H NMR (500 MHz, METHANOL-

d4) ppm 8.24 (d, J=4.9 Hz, 1 H), 7.86 (d, J=8.3 Hz, 2 H), 7.81 (dd, J=8.1, 1.2 Hz, 2 H), 7.56 (t,

J=7.6 Hz, 2 H), 7.41 - 7.49 (m, 3 H), 7.21 (d, J=4.9 Hz, 1 H), 7.02 (s, 1 H), 3.57 (s, 2 H), 2.31 (s,

6 H).

231

N,N-dimethyl-1-(4-(4-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. NEU-

5447 (3.20d) N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine (27 mg, 0.056 mmol) was dissolved in dioxane (0.2 mL). Sodium


150 °C. The reaction mixture was quenched with saturated ammonium chloride solution and

concentrated under reduced pressure. The crude product was purified by column chromatography


a salt. The salt was dissolved in ethyl acetate which was neutralized using 1M HCl and saturated

sodium bicarbonate solution. The organic layer was collected to afford the title compound as a

yellow solid (17 mg, 92% yield). LCMS [M+H]+ 329.2 m/z; 1H NMR (500 MHz, DMSO-d6)

ppm 12.41 (s, 1 H), 8.76 (d, J=5.9 Hz, 2 H), 8.33 (d, J=4.9 Hz, 1 H), 7.97 (d, J=8.3 Hz, 2 H), 7.85


(s, 2 H), 2.17 (s, 6 H).

232

N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-5449 (3.20e) N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1-

tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (69 mg, 0.139 mmol) was dissolved in

dioxane (0.5 mL). Sodium hydroxide (3M, 0.5 mL) was added and the reaction mixture was

microwaved for 20 minutes at 150 °C. The reaction mixture was quenched with saturated

ammonium chloride solution. The aqueous layer was extracted with ethyl acetate 3 times. The

combined organic layers were washed with water and brine, collected, dried with sodium sulfate,

filtered and concentrated under reduced pressure. The crude product was purified by column

chromatography eluting with a mobile phase of 1-20% methanol/dichloromethane to afford the

title compound as a yellow solid (35 mg, 75% yield). LCMS [M+H]+ 343.2 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 12.39 (s, 1 H), 8.62 (d, J=5.4 Hz, 1 H), 8.32 (d, J=4.9 Hz, 1 H), 7.98 (d,

J=8.3 Hz, 2 H), 7.69 (s, 1 H), 7.63 (d, J=5.4 Hz, 1 H), 7.40 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz,

1 H), 7.18 (d, J=1.5 Hz, 1 H), 3.49 (br. s., 2 H), 2.61 (s, 3 H), 2.21 (s, 6 H).

233

1-(4-(4-(2,6-dimethylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-

dimethylmethanamine. NEU-5452 (3.20f) 1-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine (12 mg, 0.023 mmol) was


mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was quenched with

saturated ammonium chloride solution and diluted with water. A precipitate formed and was

collected by vacuum filtration to afford the title compound as a yellow solid (3 mg, 35% yield).

LCMS [M+H]+ 357.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.29 (d, J=4.9 Hz, 1 H),

7.93 (d, J=8.3 Hz, 2 H), 7.51 (s, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.27 (d, J=4.9 Hz, 1 H), 7.06 (s, 1

H), 3.73 (s, 2 H), 2.63 (s, 6 H), 2.43 (s, 6 H).

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-N,N-dimethylaniline. (3.22a) 4-chloro-2-

iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (200 mg, 0.462 mmol), (4-

(dimethylamino)phenyl)boronic acid (152.5 mg, 0.924 mmol), PdCl2(dppf)·CH2Cl2 (18.9 mg,

0.023 mmol), and sodium carbonate (147 mg, 1.39 mmol) were added to a vial that was


234

3 mL) was added to the vial. The reaction mixture was heated at 80 °C overnight and monitored


pressure, and purified by silica gel column chromatography using a mobile phase of 0-50% ethyl

acetate/hexanes to afford the title compound as a yellow crystalline solid (153 mg, 78% yield).

LCMS [M+H]+ 426.2 m/z (Cl35), 428.2 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.29

(d, J=5.4 Hz, 1 H), 7.65 (d, J=8.3 Hz, 2 H), 7.40 - 7.45 (m, 3 H), 7.34 (d, J=8.3 Hz, 2 H), 6.80 (d,

J=8.8 Hz, 2 H), 6.66 (s, 1 H), 3.01 (s, 6 H), 2.32 (s, 3 H).

N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-N-ethylethanamine. (3.22b) 4-

chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (200 mg, 0.462 mmol), N-ethyl-N-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)ethanamine (174 mg, 0.600 mmol),






chromatography using a mobile phase of 1-20% (1% ammonium

hydroxide/methanol)/dichloromethane, followed by 50-100% ethyl acetate/hexanes and 0-20%

methanol/ethyl acetate to afford the title compound as a tan solid (116 mg, 54% yield). LCMS

235

[M+H]+ 468.1 m/z (Cl35), 470.1 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.35 (d, J=5.4

Hz, 1 H), 7.69 (d, J=8.3 Hz, 4 H), 7.49 (d, J=5.4 Hz, 1 H), 7.36 (d, J=8.3 Hz, 4 H), 6.83 - 6.92 (m,

1 H), 2.54 (s, 2 H), 2.32 (s, 9 H), 1.06 (s, 4 H).

4-chloro-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.22c) 4-

chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.347 mmol), (4-(1-(pyrrolidin-1-

yl)ethyl)phenyl)boronic acid (152 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (14.2 mg, 0.017 mmol),

and sodium carbonate (110 mg, 1.04 mmol) were added to an oven dried vial that was subsequently

sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was


The reaction mixture was filtered through a bed of Celite. The crude product was purified by

column chromatography eluting with a mobile phase 0-100% ethyl acetate/hexanes to afford the

title compound as a light brown solid (110 mg, 66% yield). LCMS [M+H]+ 480.1 m/z (Cl35), 482.1

m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.34 (d, J=4.9 Hz, 1 H), 7.68 (d, J=8.3 Hz, 2

H), 7.55 (d, J=7.8 Hz, 2 H), 7.40 - 7.49 (m, 3 H), 7.35 (d, J=8.3 Hz, 2 H), 6.85 (s, 1 H), 2.54 (br.

s., 2 H), 2.36 (br. s., 2 H), 2.32 (s, 3 H), 1.70 (br. s., 5 H), 1.37 (d, J=6.3 Hz, 3 H).

236

4-chloro-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine.

(3.22d) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (214 mg, 0.495 mmol), (4-((4-

methylpiperazin-1-yl)methyl)phenyl)boronic acid (231 mg, 0.989 mmol), PdCl2(dppf)·CH2Cl2

(20.2 mg, 0.025 mmol), and sodium carbonate (157 mg, 1.48 mmol) were added to an oven dried

vial that was subsequently sealed and vacuum purged with nitrogen. A degassed solution of

dioxane/water (3:1, 3.5 mL) was added to the vial. The reaction mixture was heated at 85 °C

overnight and monitored by LCMS. The reaction mixture was filtered through a bed of Celite. The

crude product was purified by column chromatography eluting with a mobile phase 0-20% ethyl

acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as a tan

solid (245 mg, 99% yield). LCMS [M+H]+ 495.2 m/z (Cl35), 497.2 m/z (Cl371H NMR (500 MHz,

DMSO-d6) ppm 8.34 (d, J=5.4 Hz, 1 H), 7.69 (d, J=8.3 Hz, 2 H), 7.56 (d, J=8.3 Hz, 2 H), 7.48

(d, J=5.4 Hz, 1 H), 7.42 (d, J=8.3 Hz, 2 H), 7.35 (d, J=7.8 Hz, 2 H), 6.84 (s, 1 H), 3.57 (s, 2 H),

2.45 (br. s., 8 H), 2.32 (s, 3 H), 2.23 (br. s., 3 H).

tert-butyl 2-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)pyrrolidine-1-

carboxylate. (3.22e) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (250 mg, 0.578 mmol),

237

tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxylate

(259 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (4.7 mg, 0.006 mmol), and sodium carbonate (184

mg, 1.73 mmol) were added to an oven dried vial that was subsequently sealed and vacuum purged

with nitrogen. A degassed solution of dioxane/water (3:1, 4 mL) was added to the vial. The reaction


through a bed of Celite. The crude product was purified by column chromatography eluting with

a mobile phase 0-20% ethyl acetate/hexanes to afford the title compound as a white solid (125 mg,

39% yield). LCMS [M+H]+ 552.1 m/z (Cl35), 554.1 m/z (Cl37); 1H NMR (500 MHz,


H), 7.26 - 7.30 (m, 2 H), 7.15 - 7.23 (m, 3 H), 6.58 (s, 1 H), 3.66 - 3.73 (m, 1 H), 2.38 - 2.46 (m,

1 H), 2.35 (s, 3 H), 1.86 - 2.04 (m, 3 H), 1.44 - 1.53 (m, 2 H), 1.21 - 1.32 (m, 9 H)

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. (3.22f) 4-chloro-2-iodo-1-tosyl-

1H-pyrrolo[2,3-b]pyridine (266 mg, 0.615 mmol), 4-boronobenzoic acid (204 mg, 1.23 mmol),





bed of Celite. The crude product was purified by column chromatography eluting with a mobile

phase 20-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title

238

compound as a white solid (76 mg, 29% yield). LCMS [M+H]+ 427.1 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 8.37 (d, J=5.4 Hz, 1 H), 8.04 (d, J=8.3 Hz, 2 H), 7.87 (q, J=8.1 Hz, 2 H), 7.72


(s, 1 H), 2.32 (s, 3 H).

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. (3.22g) 4-chloro-2-iodo-1-tosyl-

1H-pyrrolo[2,3-b]pyridine (250 mg, 0.578 mmol), (4-carbamoylphenyl)boronic acid (190.6 mg,

1.16 mmol), PdCl2(dppf)·CH2Cl2 (23.6 mg, 0.029 mmol), and sodium carbonate (183.7 mg, 1.73

mmol) were added to a microwave vial that was subsequently sealed and vacuum purged with

nitrogen. A degassed solution of dioxane/water (3:1, 5 mL) was added to the vial. The reaction

mixture was microwaved at 150 °C for 50 minutes and monitored by LCMS. The reaction mixture

was filtered through a bed of Celite and concentrated under reduced pressure to afford the title

compound as a brown solid (75 mg, 31% yield). LCMS [M+H]+ 425.1 m/z (Cl35), 427.1 m/z

(Cl37);; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.39 (d, J=5.4 Hz, 1 H), 7.92 (d, J=8.3

Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H), 7.66 (d, J=8.3 Hz, 2 H), 7.23 (d, J=5.4 Hz, 1 H), 7.20 (d, J=8.8

Hz, 2 H), 6.67 (s, 1 H), 2.36 (s, 3 H)

239

N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide. (3.22h) 4-chloro-2-

iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.347 mmol), (4-acetamidophenyl)boronic acid

(124 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (14.2 mg, 0.017 mmol), and sodium carbonate (110

mg, 1.04 mmol) were added to a microwave vial that was subsequently sealed and vacuum purged

with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The reaction


through a bed of Celite and purified by column chromatography eluting with a mobile phase of 0-

100% ethyl acetate/hexanes to afford the title compound as a white solid (122 mg, 80% yield).

LCMS [M+H]+ 440.1 m/z (Cl35), 442.1 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 10.16

(s, 1 H), 8.33 (d, J=5.4 Hz, 1 H), 7.69 (dd, J=12.4, 8.5 Hz, 4 H), 7.53 (d, J=8.8 Hz, 2 H), 7.46 (d,

J=5.4 Hz, 1 H), 7.35 (d, J=8.3 Hz, 2 H), 6.79 (s, 1 H), 2.32 (s, 3 H), 2.10 (s, 3 H).

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. (3.22i) 4-chloro-2-iodo-1-tosyl-1H-

pyrrolo[2,3-b]pyridine (100 mg, 0.231 mmol), (4-hydroxyphenyl)boronic acid (41.4 mg, 0.300

mmol), PdCl2(dppf)·CH2Cl2 (9.4 mg, 0.01 mmol), and sodium carbonate (73.5 mg, 0.693 mmol)

were added to a vial that was subsequently sealed and vacuum purged with nitrogen. A degassed

solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was heated at

240

90 °C overnight and monitored by LCMS. The reaction mixture was filtered through a bed of

Celite and concentrated under reduced pressure and then purified by silica gel column


compound as a yellow solid (74 mg, 84% yield). LCMS [M+H]+ 399.9 m/z (Cl35), 401.9 m/z (Cl37);

1H NMR (500 MHz, CHLOROFORM-d) ppm 8.35 (d, J=5.4 Hz, 1 H), 7.73 (d, J=8.8 Hz, 2 H),


6.55 (s, 1 H), 2.35 (s, 3 H).

N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline.

(3.23a) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.352 mmol), 1-methyl-4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (219.8 mg, 1.06 mmol),





bed of Celite, diluted with dichloromethane and water. The organic layer was collected and the

aqueous layer extracted 3 times with dichloromethane. The combined organic layers were washed

with water and then dried using sodium sulfate. The compound was purified by silica gel column


241

compound as a bright yellow solid (127 mg, 76% yield). LCMS [M+H]+ 472.1 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 8.50 (s, 1 H), 8.24 (d, J=4.9 Hz, 1 H), 8.10 (s, 1 H), 7.62 - 7.67 (m, 2 H),

7.42 - 7.47 (m, 3 H), 7.31 (d, J=8.8 Hz, 2 H), 6.96 - 6.99 (m, 1 H), 6.78 - 6.84 (m, 2 H), 3.86 - 3.90

(m, 3 H), 2.98 - 3.02 (m, 6 H), 2.29 (s, 3 H).

N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)ethanamine. (3.23b) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-N-

ethylethanamine (116.3 mg, 0.248 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-1H-pyrazole (103.4 mg, 0.497 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.4 mg,

0.012 mmol), and sodium carbonate (131.7 mg, 1.24 mmol) were added to a vial that was


2.5 mL) was added to the vial. The reaction mixture was heated at 90 °C overnight and monitored



2-20% (5% ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a

brown residue (45 mg, 23% yield). LCMS [M+H]+ 514.2 m/z; 1H NMR (500 MHz, DMSO-d6)

ppm 8.55 (s, 1 H), 8.29 (d, J=5.4 Hz, 1 H), 8.14 (s, 1 H), 7.74 - 7.78 (m, 2 H), 7.70 - 7.73 (m, 2

242


(d, J=5.4 Hz, 2 H), 3.86 - 3.91 (m, 4 H), 3.16 (s, 6 H), 1.29 (t, J=7.3 Hz, 6 H).

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridine. (3.23c) 4-chloro-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridine (101 mg, 0.210 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-

pyrazole (56.9 mg, 0.273 mmol), Tetrakis(triphenylphosphine)palladium(0) (12.1 mg, 0.010

mmol), and sodium carbonate (66.9 mg, 0.631 mmol) were added to a vial that was subsequently

sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 2.1 mL) was


The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure,

then purified by silica gel column chromatography using a mobile phase of 50-100% ethyl

acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as a brown

oil (67 mg, 61% yield). LCMS [M+H]+ 526.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.55 (s,

1 H), 8.28 (d, J=4.9 Hz, 1 H), 8.13 (s, 1 H), 7.69 (d, J=8.3 Hz, 2 H), 7.59 (d, J=7.8 Hz, 2 H), 7.48

(d, J=4.9 Hz, 3 H), 7.32 (d, J=8.3 Hz, 2 H), 7.16 (s, 1 H), 3.88 (s, 3 H), 3.56 (s, 6 H), 2.34 - 2.47

(m, 1 H), 2.30 (s, 3 H), 1.72 (br. s., 3 H), 1.40 (br. s., 2 H).

243

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridine. (3.23d) 4-chloro-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-

1H-pyrrolo[2,3-b]pyridine (245 mg, 0.495 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (134 mg, 0.643 mmol),





bed of Celite, concentrated under reduced pressure, then purified by silica gel column

chromatography eluting with a mobile phase of 0-100% ethyl acetate/hexanes to afford the title

compound as a brown flaky solid (192 mg, 72% yield). LCMS [M+H]+ 541.1 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 8.54 (s, 1 H), 8.28 (d, J=4.9 Hz, 1 H), 8.13 (s, 1 H), 7.69 (d, J=8.3 Hz, 2

H), 7.58 (d, J=7.8 Hz, 2 H), 7.45 - 7.50 (m, 1 H), 7.42 (d, J=8.3 Hz, 2 H), 7.33 (d, J=7.8 Hz, 2 H),

7.15 (s, 1 H), 3.88 (s, 3 H), 3.55 (s, 2 H), 2.20 - 2.48 (m, 11 H), 2.16 (s, 3 H).

244

tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)pyrrolidine-1-carboxylate. (3.23e) tert-butyl 2-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-2-yl)phenyl)pyrrolidine-1-carboxylate (125 mg, 0.226 mmol), 1-methyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (61.2 mg, 0.294 mmol),





a bed of Celite, concentrated under reduced pressure, then purified by silica gel column

chromatography eluting with a mobile phase of 20-100% ethyl acetate/hexanes to afford the title

compound as a brown flaky solid (129 mg, 95% yield). LCMS [M+H]+ 598.2 m/z; 1H NMR (500

MHz, CHLOROFORM-d) ppm 8.43 (br. s., 1 H), 7.88 (s, 1 H), 7.75 - 7.83 (m, 4 H), 7.51 (d,


1 H), 3.98 (s, 3 H), 3.66 - 3.73 (m, 1 H), 2.34 (s, 3 H), 2.06 (s, 2 H), 1.75 (s, 1 H), 1.50 (br. s., 2

H), 1.27 (s, 9 H).

245

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.23f) 4-

(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (573.3 mg, 1.40 mmol), 1-methyl-

4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (580.6 mg, 2.79 mmol),

Tetrakis(triphenylphosphine)palladium(0) (80.6 mg, 0.070 mmol), and sodium carbonate (443.7

mg, 4.19 mmol) were added to a vial that was subsequently sealed and vacuum purged with


mixture was heated at 100 °C for 1 hour and monitored by LCMS. The reaction mixture was

filtered through a bed of Celite, concentrated under reduced pressure, and then purified by silica

gel column chromatography using a mobile phase of 30-100% ethyl acetate/hexanes to afford the

title compound as a pale yellow solid (635 mg, 99% yield). LCMS [M+H]+ 457.1 m/z; 1H NMR

(500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.45 (d, J=5.4 Hz, 1 H), 7.97 - 8.03 (m, 2

H), 7.87 (s, 1 H), 7.75 - 7.84 (m, 5 H), 7.23 - 7.27 (m, 1 H), 7.20 (d, J=8.8 Hz, 2 H), 6.80 (s, 1 H),

3.98 (s, 3 H), 2.35 (s, 3 H).

246

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. (3.23g) 4-

(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid (367 mg, 0.860 mmol), 1-methyl-4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (537 mg, 2.58 mmol),




heated at 80 °C for 48 h and monitored by LCMS. The reaction mixture was filtered through a bed

of Celite, concentrated under reduced pressure, and then purified by silica gel column

chromatography using a mobile phase of 3-20% ((+10% ammonium

hydroxide)/methanol)/dichloromethane to afford the title compound as a brown solid (17 mg, 4%

yield). LCMS [M+H]+ 473.1 m/z.

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. (3.23h) 4-(4-

chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide (246 mg, 0.578 mmol), 1-methyl-4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (360.5 mg, 1.73 mmol),

247





through a bed of Celite, concentrated under reduced pressure, and then purified by silica gel

column chromatography using a mobile phase of 1-15% methanol/dichloromethane to afford the

title compound as an off-white solid (114 mg, 42% yield). LCMS [M+H]+ 472.2 m/z; 1H NMR

(500 MHz, DMSO-d6) ppm 8.55 (s, 1 H), 8.31 (d, J=5.4 Hz, 1 H), 8.14 (s, 1 H), 8.11 (s, 1 H),

8.00 (d, J=8.3 Hz, 2 H), 7.72 (dd, J=8.3, 5.9 Hz, 4 H), 7.46 - 7.51 (m, 2 H), 7.34 (d, J=8.3 Hz, 2

H), 7.25 (s, 1 H), 3.89 (s, 3 H), 2.31 (s, 3 H).

N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide.

(3.23i) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide (116 mg, 0.264

mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (71.3 mg, 0.343

mmol), Tetrakis(triphenylphosphine)palladium(0) (15.2 mg, 0.013 mmol), and sodium carbonate

(83.8 mg, 0.791 mmol) were added to a vial that was subsequently sealed and vacuum purged with



through a bed of Celite, concentrated under reduced pressure, and then purified by silica gel

248

column chromatography using a mobile phase of 50-100% ethyl acetate/hexanes to afford the title

compound as an off-white solid (65 mg, 51% yield). LCMS [M+H]+ 486.1 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 10.16 (s, 1 H), 8.33 (d, J=5.4 Hz, 1 H), 7.69 (dd, J=12.4, 8.5 Hz, 4 H),


H), 2.10 (s, 3 H).

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. (3.23j) 4-(4-

chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (50 mg, 0.125 mmol), 1-methyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (78.2 mg, 0.376 mmol),






chromatography using a mobile phase of 0-20% (10% ammonium

hydroxide/methanol)/dichloromethane to afford the title compound as an off white solid (52 mg,

94% yield). LCMS [M+H]+ 445.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 9.77 (s, 1 H), 8.52

(s, 1 H), 8.26 (d, J=5.4 Hz, 1 H), 8.10 (s, 1 H), 7.80 (s, 1 H), 7.67 (d, J=8.3 Hz, 2 H), 7.56 (s, 1 H),

249

7.46 (d, J=4.9 Hz, 1 H), 7.43 (d, J=8.3 Hz, 2 H), 7.32 (d, J=8.3 Hz, 2 H), 7.02 (s, 1 H), 6.88 (d,

J=8.3 Hz, 2 H), 3.93 (s, 1 H), 3.88 (s, 3 H), 3.81 (s, 1 H), 2.29 (s, 3 H).

N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline. NEU-

5323 (3.24a) N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-

2-yl)aniline (122 mg, 0.259 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide was added

(3M, 1 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture

was diluted with water. The precipitate that formed was collected by vacuum filtration to afford

the title compound as a yellow solid (80 mg, 97% yield). LCMS [M+H]+ 318.2 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 11.90 (s, 1 H), 8.53 (s, 1 H), 8.15 (s, 1 H), 8.06 (d, J=5.4 Hz, 1 H), 7.86


(s, 3 H), 2.97 (s, 6 H).

250

N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethanamine.

NEU-4973 (3.24b) N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-2-yl)benzyl)ethanamine (45 mg, 0.088 mmol) was dissolved in dioxane (0.4 mL).

Sodium hydroxide was added (3M, 0.8 mL). The reaction mixture was microwaved at 150 °C for

20 minutes. The reaction mixture was quenched with saturated ammonium chloride solution and

diluted with water. The aqueous mixture was washed 3 times with ethyl acetate. The combined

organic layers were dried with sodium sulfate, filtered, and concentrated to afford the title

compound as a tan solid (15 mg, 47% yield). LCMS [M+H]+ 360.2 m/z; 1H NMR (500 MHz,

METHANOL-d4) ppm 8.35 (s, 1 H), 8.12 (s, 2 H), 7.89 (d, J=8.3 Hz, 2 H), 7.46 (d, J=7.8 Hz, 2

H), 7.26 (d, J=4.9 Hz, 1 H), 7.15 (s, 1 H), 4.01 (s, 4 H), 3.70 (s, 2 H), 2.63 (q, J=6.8 Hz, 3 H), 1.12

(t, J=7.1 Hz, 6 H).

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.

NEU-6072 (3.24c) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-

251

1H-pyrrolo[2,3-b]pyridine (67 mg, 0.127 mmol) was dissolved in dioxane (1 mL). Sodium

hydroxide was added (3M, 0.425 mL). The reaction mixture was microwaved at 150 °C for 15

minutes. The reaction mixture was quenched with saturated ammonium chloride solution and

diluted with water. The aqueous mixture was washed 3 times with ethyl acetate. The combined

organic layers were dried with sodium sulfate, filtered, and concentrated. The crude product was

purified by column chromatography eluting with a mobile phase of 1-20%

methanol/dichloromethane to afford the title compound as an off-white solid (25 mg, 53% yield).

LCMS [M+H]+ 372.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 12.88 (br. s., 1 H),

8.21 (d, J=4.4 Hz, 1 H), 8.00 (s, 1 H), 7.89 - 7.96 (m, 3 H), 7.74 (d, J=8.3 Hz, 2 H), 7.16 (d, J=5.4

Hz, 1 H), 6.94 (s, 1 H), 3.89 - 4.08 (m, 4 H), 3.24 (br. s., 1 H), 2.95 - 3.07 (m, 2 H), 2.04 (br. s., 4

H), 1.80 (d, J=6.3 Hz, 3 H).

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-

b]pyridine. NEU-6076 (3.24d) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-

yl)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (180 mg, 0.333 mmol) was dissolved in

dioxane (0.7 mL). Sodium hydroxide was added (3M, 1.11 mL). The reaction mixture was

microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with saturated

ammonium chloride solution and diluted with water. A precipitate formed and was collected by

vacuum filtration. The crude product was purified by column chromatography eluting with a

252

mobile phase of 1-10% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the

title compound as an off-white solid (27 mg, 21% yield). LCMS [M+H]+ 387.1 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 12.13 (s, 1 H), 8.57 (s, 1 H), 8.19 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.98


(s, 3 H), 3.49 (s, 2 H), 2.36 (br. s., 7 H), 2.15 (s, 3 H).

3.24e - KB2-617-3-1 - tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)pyrrolidine-1-carboxylate. tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)phenyl)pyrrolidine-1-carboxylate (109 mg, 0.182 mmol) was dissolved

in dioxane (1.5 mL). Sodium hydroxide was added (3M, 1.5 mL). The reaction mixture was

microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with saturated

ammonium chloride solution and diluted with water. The aqueous mixture was washed 3 times

with dichloromethane. The combined organic layers were dried with sodium sulfate, filtered, and

concentrated. The crude product was purified by column chromatography eluting with a mobile

phase of 0-100% ethyl acetate/hexanes to afford the title compound as a pale yellow solid (44 mg,

55% yield). LCMS [M+H]+ 444.3 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 12.62 (br.

s., 1 H), 8.27 (d, J=4.9 Hz, 1 H), 8.07 (s, 1 H), 7.96 (br. s., 1 H), 7.87 (d, J=6.8 Hz, 2 H), 7.34 (d,

J=8.3 Hz, 2 H), 7.19 (d, J=4.9 Hz, 1 H), 6.90 - 7.02 (m, 1 H), 4.06 (s, 3 H), 3.70 (br. s., 2 H), 2.41

(br. s., 1 H), 1.76 (br. s., 2 H), 1.51 (br. s., 2 H), 1.18 - 1.33 (m, 9 H).

253

4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. NEU-4974

(3.24f) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde

(320 mg, 0.701 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide was added (3M, 2.34

mL). The reaction mixture was microwaved at 150 °C for 30 minutes. The reaction mixture was

quenched with saturated ammonium chloride solution and diluted with water. A precipitate formed

and was collected by vacuum filtration to afford the title compound as an orange solid (167 mg,

79% yield). LCMS [M+H]+ 303.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 10.02 (s, 1 H), 8.60

(s, 1 H), 8.28 (d, J=8.3 Hz, 2 H), 8.22 (s, 1 H), 8.20 (d, J=4.9 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H),

7.55 (s, 1 H), 7.28 (d, J=4.9 Hz, 1 H), 3.97 (s, 3 H).

4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. NEU-5448 (3.24g)

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid (17 mg,

0.036 mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide was added (3M, 0.12 mL).

The reaction mixture was microwaved at 150 °C for 20 minutes. The reaction mixture was

254

quenched with saturated ammonium chloride solution and diluted with water. The aqueous mixture

was washed 3 times with ethyl acetate. The combined organic layers were dried with sodium

sulfate, filtered, and concentrated. The crude product was purified by column chromatography


a yellow solid (5 mg, 44% yield). LCMS [M+H]+ 319.1 m/z; 1H NMR (500 MHz, METHANOL-

d4) ppm 8.40 (s, 1 H), 8.18 (d, J=4.9 Hz, 1 H), 8.15 (s, 1 H), 8.09 - 8.13 (m, 1 H), 8.02 (d, J=8.3

Hz, 2 H), 7.97 (d, J=8.8 Hz, 1 H), 7.29 - 7.33 (m, 2 H), 4.03 (s, 3 H).

4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. NEU-5306 (3.24h)

4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide (114 mg,

0.242 mmol) was dissolved in dioxane (2 mL). Sodium hydroxide was added (3M, 1 mL). The

reaction mixture was microwaved at 150 °C for 45 minutes. The reaction mixture was quenched

with saturated ammonium chloride solution and diluted with water. A precipitate formed and was

collected by vacuum filtration. The crude product was purified by column chromatography eluting

with a mobile phase of 1-8% methanol/dichloromethane to afford the title compound as a yellow

solid (72 mg, 94% yield). LCMS [M+H]+ 318.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.24

(s, 1 H), 8.60 (s, 1 H), 8.22 (s, 1 H), 8.18 (d, J=4.9 Hz, 1 H), 8.13 (d, J=8.3 Hz, 2 H), 8.03 (s, 1 H),

7.98 (d, J=8.8 Hz, 2 H), 7.46 (d, J=2.4 Hz, 1 H), 7.41 (s, 1 H), 7.29 (d, J=4.9 Hz, 1 H), 3.97 (s, 3

H).

255

N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide. NEU-

6073 (3.24i) N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)acetamide (65 mg, 0.134 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide

was added (3M, 0.446 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The

reaction mixture was quenched with saturated ammonium chloride solution and diluted with water.

The aqueous mixture was washed 3 times with dichloromethane. The combined organic layers

were dried with sodium sulfate, filtered, and concentrated. The crude product was purified by


afford the title compound as a yellow solid (21 mg, 47% yield). LCMS [M+H]+ 332.1 m/z; 1H

NMR (500 MHz, DMSO-d6) ppm 12.05 (s, 1 H), 10.08 (s, 1 H), 8.52 - 8.59 (m, 1 H), 8.16 - 8.19

(m, 1 H), 8.12 (d, J=4.9 Hz, 1 H), 7.97 (s, 2 H), 7.68 (d, J=8.8 Hz, 2 H), 7.25 (d, J=5.4 Hz, 1 H),

7.23 (d, J=1.5 Hz, 1 H), 3.96 (s, 3 H), 2.07 (s, 3 H).

256

4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. NEU-4918 (3.24j) 4-(4-

(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (50 mg, 0.079 mmol)

was dissolved in dioxane (1 mL). Sodium hydroxide was added (3M, 0.276 mL). The reaction

mixture was microwaved at 150 °C for 45 minutes. The reaction mixture was quenched with

saturated ammonium chloride solution and concentrated. The crude product was purified twice by


afford the title compound as a white solid (5 mg, 22% yield). LCMS [M+H]+ 291.2 m/z; 1H NMR

(500 MHz, METHANOL-d4) ppm 8.34 (s, 1 H), 8.11 (d, J=1.0 Hz, 1 H), 8.08 (d, J=5.4 Hz, 1

H), 7.76 (d, J=8.8 Hz, 2 H), 7.25 (d, J=4.9 Hz, 1 H), 6.98 (s, 1 H), 6.90 (d, J=8.8 Hz, 2 H), 4.02

(s, 3 H).

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(pyrrolidin-2-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine. NEU-

6049 (3.24k) tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)pyrrolidine-1-carboxylate (37 mg, 0.083 mmol) was dissolve in dioxane (1 mL).

257

Hydrochloric acid (1M, 0.834 mL) was added and the reaction mixture was stirred at room

temperature overnight. The reaction mixture was neutralized with silicized carbonate. The silica

was filtered off, washed with methanol, and the filtrate was concentrated to afford the title


DMSO-d6) ppm 12.09 (br. s., 1 H), 8.57 (s, 1 H), 8.18 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.96 (d,

J=8.3 Hz, 2 H), 7.53 - 7.65 (m, 1 H), 7.45 (d, J=8.3 Hz, 2 H), 7.24 - 7.28 (m, 2 H), 4.06 (t, J=7.6

Hz, 1 H), 3.96 (s, 3 H), 3.04 (ddd, J=9.8, 7.8, 5.4 Hz, 1 H), 2.89 (td, J=9.0, 6.8 Hz, 1 H), 2.14 (dtd,

J=12.1, 7.6, 7.6, 4.9 Hz, 1 H), 1.69 - 1.84 (m, 2 H), 1.50 (m, J=12.0, 9.0 Hz, 1 H).

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridine. (3.25a) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (50 mg, 0.109 mmol) was dissolved in dichloroethane (0.5 mL). Acetic acid (12.5

μL, 0.219 mmol), piperazine (20.9 μL, 0.219 mmol), and sodium borohydride (8.3 mg, 0.219

mmol) were added. The reaction mixture was left stirring at room temperature overnight. The

reaction mixture was quenched with 3M sodium hydroxide. The aqueous mixture was extracted 3

times with dichloromethane. The combined organic layers were concentrated, and the crude

product was purified by column chromatography using a mobile phase of 50-100% ethyl

acetate/hexanes, followed by 0-20% methanol/ethyl acetate to afford the title compound as an off-

white solid (37 mg, 64% yield). LCMS [M+H]+ 457.1 m/z.

258

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.

NEU-5936 (3.26a) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1-tosyl-

1H-pyrrolo[2,3-b]pyridine was dissolved in dioxane (1 mL). Sodium hydroxide (3M, 1 mL) was

added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture

was quenched with saturated ammonium chloride solution, and then diluted with water. The


with water and brine, then dried with sodium sulfate, filtered, and concentrated under reduced

pressure. The reaction mixture was purified by column chromatography using a mobile phase of

0-20% (1% ammonium hydroxide/methanol)/ethyl acetate to afford the title compound as an

ammonium salt. The free base was yielded by dissolving the salt in water and basifying with

sodium hydroxide. The precipitate that formed was collected by vacuum filtration and washed with

water to afford the title compound as a pale yellow solid (10 mg, 37% yield). LCMS [M+H]+ 372.3

m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s, 1 H), 8.11 - 8.15 (m, 2 H), 7.90 (d,

J=8.3 Hz, 2 H), 7.45 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.17 (s, 1 H), 4.03 (s, 3 H), 3.57

(s, 2 H), 2.49 (br. s., 4 H), 1.59 - 1.67 (m, 4 H), 1.45 - 1.53 (m, 2 H).

259

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.

NEU-4995 (3.26b) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (60 mg, 0.198 mmol) was suspended in dichloroethane (2 mL). Piperidine (34

μL, 0.40 mmol) and sodium triacetoxyborohydride (63.1 mg, 0.298 mmol) were added to the

reaction mixture which was left stirring overnight at room temperature. The reaction mixture was

quenched with saturated ammonium chloride solution, and then diluted with water. The aqueous

layer was extracted 3x with dichloromethane. The combined organic layers were washed with

water and brine, then dried with sodium sulfate, filtered, and concentrated under reduced pressure.

The reaction mixture was purified by column chromatography using a mobile phase of 5-20%

methanol/dichloromethane to afford the title compound as a yellow solid (44 mg, 60% yield).

LCMS [M+H]+ 372.2 m/z; 1H NMR (399 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.57 (s, 1 H),

8.19 (s, 1 H), 8.14 (d, J=5.1 Hz, 1 H), 7.98 (d, J=8.1 Hz, 2 H), 7.38 (d, J=8.1 Hz, 2 H), 7.24 - 7.30

(m, 2 H), 3.96 (s, 3 H), 3.47 (br. s., 2 H), 2.35 (br. s., 4 H), 1.46 - 1.57 (m, 4 H), 1.40 (d, J=4.4 Hz,

2 H).

260

4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.

NEU-5123 (3.26c) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (40 mg, 0.123 mmol) was suspended in methanol (1.3 mL). Pyrrolidine (109 μL,

1.32 mmol) and sodium borohydride (6.0mg, 0.189 mmol) were added to the reaction mixture

which was heated at 70 °C overnight. The reaction mixture was concentrated under reduced

pressure and the crude product was purified by column chromatography using a mobile phase of

1-7% methanol/dichloromethane to afford the title compound as a yellow solid (13 mg, 28% yield).

LCMS [M+H]+ 358.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s, 1 H), 8.12 - 8.17

(m, 2 H), 7.96 (d, J=8.3 Hz, 2 H), 7.53 (d, J=8.3 Hz, 2 H), 7.29 (d, J=5.4 Hz, 1 H), 7.20 (s, 1 H),

4.03 (s, 3 H), 3.95 (s, 2 H), 2.87 (br. s., 4 H), 1.94 (br. s., 4 H).

N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethanamine. NEU-

5124 (3.26d) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (30

mg, 0.099 mmol) was suspended in methanol (1 mL). Ethylamine (64.9 μL, 0.992 mmol) and

261

sodium borohydride (4.5 mg, 0.119 mmol) were added to the reaction mixture which was left

stirring at room temperature overnight. The reaction mixture was concentrated under reduced

pressure and the crude product was purified by column chromatography using a mobile phase of

1-20% methanol/dichloromethane to afford the title compound as a yellow solid (12 mg, 37%

yield). LCMS [M+H]+ 332.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.35 (s, 1 H), 8.14

(d, J=4.9 Hz, 1 H), 8.12 (s, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.56 (d, J=8.3 Hz, 2 H), 7.27 (d, J=5.4

Hz, 1 H), 7.20 (s, 1 H), 4.10 (s, 2 H), 4.01 (s, 3 H), 3.35 (s, 1 H), 3.00 (q, J=7.0 Hz, 2 H), 1.30 (t,

J=7.3 Hz, 3 H).

4-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)morpholine. NEU-

5937 (3.26e) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (25

mg, 0.083 mmol) was suspended in dichloroethane (0.5 mL). Morpholine (21.4 μL, 0.248 mmol),

acetic acid (14.2 μL, 0.248 mmol) and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol) were

added to the reaction mixture which was left stirring overnight at room temperature. The reaction

mixture was quenched with 3M sodium hydroxide, and then diluted with water. The aqueous layer

was extracted 3 times with dichloromethane. The combined organic layers were concentrated and

the crude product was purified by column chromatography using a mobile phase of 0-20% ((+10%

ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a pale yellow solid

(14 mg, 47% yield). LCMS [M+H]+ 374.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37

262

(s, 1 H), 8.09 - 8.16 (m, 2 H), 7.91 (d, J=8.3 Hz, 2 H), 7.47 (d, J=8.3 Hz, 2 H), 7.28 (d, J=5.4 Hz,

1 H), 7.16 (s, 1 H), 4.03 (s, 3 H), 3.72 (t, J=4.6 Hz, 4 H), 3.59 (s, 2 H), 3.35 (s, 1 H), 2.51 (br. s.,

4 H).

N-methyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenyl)methanamine. NEU-5938 (3.26f) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-

b]pyridin-2-yl)benzaldehyde (25 mg, 0.083 mmol) was suspended in dichloroethane (0.5 mL).

Methylamine hydrochloride (16.7 mg, 0.248 mmol), triethylamine (34.6 μL, 0.248), acetic acid

(14.2 μL, 0.248 mmol) and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol) were added to

the reaction mixture which was left stirring overnight at room temperature. The reaction mixture

was quenched with 3M sodium hydroxide, and then diluted with water. The aqueous layer was

extracted 3 times with dichloromethane. The combined organic layers were concentrated, and the

crude product was purified by column chromatography using a mobile phase of 0-20% ((+10%

ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a yellow solid (10

mg, 38% yield). LCMS [M+H]+ 318.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s,

1 H), 8.12 - 8.16 (m, 2 H), 7.94 (d, J=7.8 Hz, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.28 (d, J=5.4 Hz, 1

H), 7.18 (s, 1 H), 4.03 (s, 3 H), 3.85 (s, 2 H), 2.48 (s, 3 H).

263

N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.

NEU-6018 (3.26g) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (74 mg, 0.245 mmol) was dissolved in 10% acetic acid/methanol (2.5 mL).

Isopropylamine (0.1 mL, 1.22 mmol) was added and the reaction mixture was left stirring at room

temperature for 1 hour. Sodium cyanoborohydride (52.6 mg, 0.248 mmol) was added to the

reaction mixture which was heated at 70 °C for 72 h. The reaction mixture concentrated, and the

crude product was purified by column chromatography using a mobile phase of 30-100% ethyl

acetate/hexanes followed by 0-20% methanol/ethyl acetate, and again with 1%


LCMS [M+H]+ 346.3 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.32 (s, 1 H), 8.07 - 8.13

(m, 2 H), 7.89 (d, J=8.3 Hz, 2 H), 7.46 (d, J=8.3 Hz, 2 H), 7.24 (d, J=5.4 Hz, 1 H), 7.12 (s, 1 H),

4.00 (s, 3 H), 3.82 (s, 2 H), 2.92 (spt, J=6.3 Hz, 1 H), 1.15 (d, J=6.3 Hz, 6 H).

264

N1-methyl-N2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethane-

1,2-diamine. NEU-6025 (3.26h) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL).

N1-methylethane-1,2-diamine (43.3 μL, 0.496 mmol) was added and the reaction mixture was left

stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165 mmol)

were added to the reaction mixture which was left stirring overnight at room temperature. The

reaction mixture was diluted with water, concentrated, and the crude product was purified by

column chromatography using a mobile phase of 1-20% ((+1% ammonium

hydroxide)/methanol)/dichloromethane to afford the title compound as a yellow solid (6 mg, 10%

yield). LCMS [M+H]+ 361.3 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.11 (br. s., 1 H), 8.57

(s, 1 H), 8.18 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.42 (d, J=8.3 Hz, 2 H),

7.27 (m, J=8.3 Hz, 3 H), 3.96 (s, 3 H), 3.73 (s, 2 H), 2.56 (dd, J=8.5, 4.1 Hz, 4 H), 2.26 (s, 2 H).

265

N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-2-(pyrrolidin-1-

yl)ethan-1-amine. NEU-6044 (3.26i) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-

2-yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6

mL). 2-(pyrrolidin-1-yl)ethan-1-amine (62.7 μL, 0.496 mmol) was added and the reaction mixture

was left stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165

mmol) were added to the reaction mixture which was left stirring overnight at room temperature.

The reaction mixture was concentrated, and the crude product was purified by column

chromatography using a mobile phase of 1-20% methanol/dichloromethane followed by 1-20%

methanol/ethyl acetate to afford the title compound as a pale yellow solid (9 mg, 14% yield).

LCMS [M+H]+ 401.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.06 - 12.15 (m, 1 H), 8.57 (s,

1 H), 8.18 (s, 1 H), 8.14 (d, J=5.4 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.42 (d, J=7.8 Hz, 2 H), 7.29

(d, J=1.5 Hz, 1 H), 7.26 (d, J=4.9 Hz, 1 H), 3.96 (s, 3 H), 3.73 - 3.77 (m, 2 H), 2.59 - 2.64 (m, 2

H), 2.53 (t, J=6.6 Hz, 2 H), 2.39 - 2.45 (m, 4 H), 1.64 - 1.69 (m, 4 H).

266

(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-6047

(3.26j) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (50 mg,

0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL). Azetidine (33.4 μL, 0.496

mmol) was added and the reaction mixture was left stirring for 20 minutes at room temperature.

Sodium cyanoborohydride (10.4 mg, 0.165 mmol) were added to the reaction mixture which was

left stirring overnight at room temperature. The reaction mixture was concentrated, and the crude

product was purified by column chromatography using a mobile phase of 1-20% methanol/ ethyl

acetate to afford the title compound as a pale yellow solid (13 mg, 18% yield). LCMS [M+H]+

305.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.58 (s, 1 H), 8.19 (s, 1 H), 8.14


(d, J=4.9 Hz, 1 H), 5.14 - 5.36 (m, 1 H), 4.55 (s, 2 H), 3.96 (s, 3 H), 1.65 - 1.84 (m, 1 H).

2,2-difluoro-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethan-1-

amine. NEU-6048 (3.26k) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

267

yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL).

2,2-difluoroethan-1-amine (50.0 μL, 0.496 mmol) was added and the reaction mixture was left

stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165 mmol)

were added to the reaction mixture which was left stirring overnight at room temperature. The

reaction mixture was quenched with saturated sodium bicarbonate. The aqueous solution was

extracted 3 times with dichloromethane. The combined organic layers were dried with sodium

sulfate, filtered, and concentrated. The crude product was purified by column chromatography

using a mobile phase of 1-20% methanol/ethyl acetate followed by 1-5% methanol

dichloromethane to afford the title compound as a yellow solid (10 mg, 17% yield). LCMS [M+H]+

368.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.57 (s, 1 H), 8.19 (s, 1 H), 8.14


(d, J=5.4 Hz, 1 H), 6.03 (m, J=4.1, 4.1 Hz, 1 H), 3.96 (s, 3 H), 3.79 (s, 2 H), 2.87 (td, J=15.9, 4.4

Hz, 2 H).

N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. (3.27a) 4-(4-

chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (2.08 g, 5.06 mmol) was dissolved in

a solution of 10% acetic acid/methanol (50 mL). Isopropylamine (4.14 mL, 50.6 mmol) was added

and the reaction mixture was heated at 50 °C until the solution was clear. Sodium

cyanoborohydride (477 mg, 7.59 mmol) was then added and the reaction mixture was left heating

at 50 °C overnight. The reaction mixture was neutralized with saturated sodium bicarbonate

268

solution. The aqueous mixture was concentrated to remove methanol, then extracted 3 times with

dichloromethane. The combined organic layers were washed with water and then brine, collected,

dried with sodium sulfate, filtered and concentrated to afford the title compound as a tan solid

(2.17 g, 94% yield). LCMS [M+H]+ 454.1 m/z (Cl35), 456.1 m/z (Cl37); 1H NMR (500 MHz,

DMSO-d6) ppm 8.34 (d, J=4.9 Hz, 1 H), 7.70 (d, J=8.3 Hz, 2 H), 7.50 - 7.55 (m, 2 H), 7.43 -

7.49 (m, 3 H), 7.35 (d, J=8.3 Hz, 2 H), 6.80 (s, 1 H), 3.78 (s, 2 H), 2.75 (quin, J=6.2 Hz, 1 H), 2.31

(s, 3 H), 1.92 - 2.19 (m, 1 H), 1.03 (d, J=6.3 Hz, 6 H).

tert-butyl 4-(4-(2-(4-formylphenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-

yl)piperidine-1-carboxylate. (3.27b) 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (150 mg, 0.365 mmol), tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (179 mg, 0.475 mmol),




was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through

a bed of Celite and concentrated under reduced pressure and then purified by silica gel column

269


compound as a tan solid (192 mg, 84% yield). LCMS [M+H]+ 626.2 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 10.12 (s, 1 H), 8.59 (s, 1 H), 8.32 (d, J=5.4 Hz, 1 H), 8.18 (s, 1 H), 8.05 (d,


1 H), 7.33 (d, J=8.3 Hz, 2 H), 4.34 - 4.45 (m, 1 H), 3.98 - 4.13 (m, 2 H), 2.77 - 3.01 (m, 2 H), 2.30

(s, 3 H), 2.00 (br. s., 2 H), 1.79 - 1.92 (m, 2 H), 1.41 (s, 9 H).

4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.27c) 4-

(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (67 mg, 0.163 mmol), (4-

(methylsulfonyl)phenyl)boronic acid (42.4 mg, 0.212 mmol),

Tetrakis(triphenylphosphine)palladium(0) (9.4 mg, 0.008 mmol), and sodium carbonate (51.8 mg,






compound as an off-white solid (36 mg, 42% yield). LCMS [M+H]+ 531.1 m/z; 1H NMR (500

MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.61 (d, J=4.9 Hz, 1 H), 8.08 (d, J=8.3 Hz, 2 H),

270


7.31 (d, J=4.9 Hz, 1 H), 7.25 (d, J=7.8 Hz, 2 H), 6.72 (s, 1 H), 3.11 (s, 3 H), 2.38 (s, 3 H).

N-(4-(4-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. (3.28a) N-

(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (65 mg, 0.143 mmol),

pyridin-3-ylboronic acid (38.2 mg, 0.186 mmol), Tetrakis(triphenylphosphine)palladium(0) (8.3

mg, 0.007 mmol), and sodium carbonate (45.5 mg, 0.429 mmol) were added to a vial that was


1.3 mL) was added to the vial. The reaction mixture was heated at 85 °C for 48 h and monitored



1-20% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the title compound as

a tan solid (50 mg, 70% yield). LCMS [M+H]+ 497.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm

8.90 (d, J=1.5 Hz, 1 H), 8.68 (dd, J=4.9, 1.5 Hz, 1 H), 8.47 (d, J=4.9 Hz, 1 H), 8.15 (dt, J=8.2, 1.8

Hz, 1 H), 7.76 (d, J=8.3 Hz, 2 H), 7.50 - 7.61 (m, 4 H), 7.48 (d, J=7.8 Hz, 2 H), 7.37 (d, J=8.3 Hz,

2 H), 6.91 (s, 1 H), 3.85 (br. s., 2 H), 2.84 (br. s., 1 H), 2.33 (s, 3 H), 1.08 (d, J=5.9 Hz, 6 H).

271

N-(2-hydroxyethyl)-4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-

4-yl)benzenesulfonamide. (3.28b) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (60 mg, 0.132 mmol), N-(2-hydroxyethyl)-4-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)benzenesulfonamide (56.2 mg, 0.172 mmol),






chromatography using a mobile phase of 1-20% ((+10% ammonium

hydroxide)/methanol)/dichloromethane to afford the title compound as a tan solid (67 mg, 81%

yield). LCMS [M+H]+ 619.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.48 (d, J=5.4 Hz, 1 H),


7.47 (s, 2 H), 7.37 (d, J=8.3 Hz, 2 H), 6.93 (s, 1 H), 4.70 (s, 1 H), 3.78 - 3.85 (m, 2 H), 3.38 (d,

J=5.9 Hz, 3 H), 2.76 - 2.86 (m, 3 H), 2.33 (s, 3 H), 1.06 (d, J=6.3 Hz, 6 H).

272

N-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-

amine. (3.28c) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (72

mg, 0.159 mmol), (4-(methylsulfonyl)phenyl)boronic acid (57.1 mg, 0.285 mmol),




mixture was heated at 85 °C for 1 hour and monitored by LCMS. The reaction mixture was filtered


column chromatography using a mobile phase of 50-100% ethyl acetate/hexanes, followed by 0-

20% ((+5% ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a light

tan solid (81 mg, 90% yield). LCMS [M+H]+ 574.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm

8.49 (d, J=4.9 Hz, 1 H), 8.03 - 8.07 (m, 2 H), 7.95 - 7.99 (m, 2 H), 7.76 (d, J=8.3 Hz, 2 H), 7.56


(s, 1 H), 4.05 - 4.21 (m, 1 H), 3.83 (s, 2 H), 3.27 (s, 3 H), 2.76 - 2.85 (m, 1 H), 2.33 (s, 3 H), 1.06

(d, J=6.3 Hz, 6 H).

273

tert-butyl 4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-3,6-

dihydropyridine-1(2H)-carboxylate. (3.28d) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (74 mg, 0.163 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (90.7 mg, 0.293 mmol),




was heated at 85 °C for 1 hour and monitored by LCMS. The reaction mixture was filtered through


chromatography using a mobile phase of 1-5% ((+10% ammonium hydroxide)/methanol)/ethyl

acetate to afford the title compound as a brown residue (97 mg, 99% yield). LCMS [M+H]+ 601.2

m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.28 - 8.32 (m, 1 H), 7.67 - 7.73 (m, 2 H), 7.49 - 7.55

(m, 2 H), 7.41 - 7.48 (m, 2 H), 7.30 - 7.37 (m, 2 H), 7.19 - 7.25 (m, 1 H), 6.93 - 6.98 (m, 1 H), 4.08

- 4.15 (m, 1 H), 3.99 - 4.06 (m, 2 H), 3.75 - 3.81 (m, 2 H), 3.48 - 3.56 (m, 2 H), 3.12 - 3.20 (m, 2

H), 2.70 - 2.79 (m, 1 H), 2.28 - 2.34 (m, 3 H), 1.96 - 2.01 (m, 1 H), 1.41 (s, 9 H), 1.03 (d, J=5.9

Hz, 6 H).

274

N-(4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.

(3.28e) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (70 mg,

0.154 mmol), (2-methylpyridin-4-yl)boronic acid (38.0 mg, 0.277 mmol),






chromatography using a mobile phase of 50-100% ethyl acetate/hexanes, followed by 0-20%

methanol/ethyl acetate to afford the title compound as an off-white solid (53 mg, 67% yield).

LCMS [M+H]+ 511.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.57 (d, J=5.4 Hz, 1 H), 8.48

(d, J=4.9 Hz, 1 H), 7.75 (d, J=8.3 Hz, 2 H), 7.55 - 7.60 (m, 3 H), 7.46 - 7.53 (m, 4 H), 7.37 (d,

J=8.3 Hz, 2 H), 6.95 (s, 1 H), 3.87 (br. s., 2 H), 2.85 (br. s., 1 H), 2.54 (s, 3 H), 2.32 (s, 3 H), 1.91

(s, 1 H), 1.09 (d, J=6.3 Hz, 6 H).

275

N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-

amine. (3.28f) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (70

mg, 0.154 mmol), 2,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (64.7

mg, 0.277 mmol), Tetrakis(triphenylphosphine)palladium(0) (8.9 mg, 0.008 mmol), and sodium

carbonate (49.0 mg, 0.462 mmol) were added to a vial that was subsequently sealed and vacuum

purged with nitrogen. A degassed solution of dioxane/water (3:1, 1.5 mL) was added to the vial.

The reaction mixture was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture

was filtered through a bed of Celite and concentrated under reduced pressure and then purified by

silica gel column chromatography using a mobile phase of 0-20% methanol/ethyl acetate to afford

the title compound as an amber solid (71 mg, 88% yield). LCMS [M+H]+ 525.1 m/z; 1H NMR

(500 MHz, DMSO-d6) ppm 8.47 (d, J=4.9 Hz, 1 H), 7.74 (d, J=8.3 Hz, 2 H), 7.59 (d, J=8.3 Hz,

2 H), 7.51 (d, J=7.8 Hz, 2 H), 7.47 (d, J=5.4 Hz, 1 H), 7.36 (d, J=8.3 Hz, 2 H), 7.33 (s, 2 H), 6.94

(s, 1 H), 4.10 (br. s., 1 H), 3.90 (br. s., 2 H), 2.83 - 2.95 (m, 1 H), 2.49 (s, 6 H), 2.32 (s, 3 H), 1.11

(d, J=5.9 Hz, 6 H).

276

N-(4-(4-(1H-pyrazol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.

(3.28g) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (100 mg,

0.220 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (51.3 mg, 0.264

mmol), ris(dibenzylideneacetone)dipalladium(0) (10.1 mg, 0.011 mmol), tricyclohexylphosphine

(9.3 mg, 0.033 mmol) and potassium carbonate (91.3 mg, 0.660 mmol) were added to a vial that

was subsequently sealed and vacuum purged with nitrogen. A degassed solution of

dimethylformamide/water (3:1, 2.2 mL) was added to the vial. The reaction mixture was heated at

85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through a bed of Celite

and concentrated under reduced pressure and then purified by silica gel column chromatography

using a mobile phase of 1-20% methanol/dichloromethane to afford the title compound as a yellow

solid (101 mg, 94% yield). LCMS [M+H]+ 486.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.37

(d, J=5.4 Hz, 1 H), 7.88 (br. s., 1 H), 7.65 - 7.70 (m, 3 H), 7.56 (d, J=8.3 Hz, 2 H), 7.46 (d, J=7.8

Hz, 2 H), 7.33 (d, J=8.3 Hz, 2 H), 7.29 (br. s., 1 H), 7.01 (s, 1 H), 3.79 (s, 2 H), 2.70 - 2.83 (m, 1

H), 2.29 (s, 3 H), 1.05 (d, J=6.3 Hz, 6 H).

277

N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine. (3.28h) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (100 mg, 0.220 mmol), 1-(tetrahydro-2H-pyran-4-yl)-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (137 mg, 0.492 mmol),






column chromatography using a mobile phase of 1-20% methanol/ethyl acetate to afford the title

compound as an amber solid (90 mg, 72% yield). LCMS [M+H]+ 570.3 m/z; 1H NMR (500 MHz,

DMSO-d6) ppm 8.58 (s, 1 H), 8.29 (d, J=4.9 Hz, 1 H), 8.15 (s, 1 H), 7.68 (d, J=8.3 Hz, 2 H),

7.58 (d, J=7.8 Hz, 2 H), 7.44 - 7.52 (m, 3 H), 7.33 (d, J=8.3 Hz, 2 H), 7.19 (s, 1 H), 4.39 - 4.52

(m, 1 H), 4.10 (d, J=4.4 Hz, 4 H), 3.83 (s, 2 H), 3.38 - 3.52 (m, 4 H), 2.79 (d, J=5.9 Hz, 1 H), 2.30

(s, 3 H), 1.07 (s, 6 H).

278

stert-butyl 4-(4-(2-(4-formylphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-

1-carboxylate. (3.28i) tert-butyl 4-(4-(2-(4-formylphenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-

yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (190 mg, 0.304 mmol) was dissolved in dioxane

(0.735 mL). Sodium hydroxide was added (3M, 1.0 mL). The reaction mixture was microwaved

at 150 °C for 15 minutes. The reaction mixture was quenched with saturated ammonium chloride

solution. A precipitate formed and was collected by vacuum filtration to afford the title compound

as a yellow solid (128 mg, 89% yield). LCMS [M+H]+ 472.2 m/z; 1H NMR (500 MHz, DMSO-

d6) ppm 12.33 - 12.41 (m, 1 H), 10.03 (s, 1 H), 8.65 (s, 1 H), 8.26 - 8.31 (m, 3 H), 8.22 (d, J=4.9

Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H), 7.59 (s, 1 H), 7.33 (d, J=4.9 Hz, 1 H), 4.42 - 4.52 (m, 1 H), 3.99

- 4.20 (m, 2 H), 2.82 - 3.08 (m, 2 H), 2.09 (d, J=12.2 Hz, 2 H), 1.93 (qd, J=12.1, 4.6 Hz, 2 H), 1.43

(s, 9 H).

279

4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridine. (3.28j) 4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzaldehyde (36.4 mg, 0.069 mmol) was dissolved in a solution of 10% acetic acid/methanol

(1 mL). Pyrrolidine (56.3 μL, 0.686 mmol) was added and the reaction mixture was heated at 50

°C until the solution was clear. Sodium cyanoborohydride (6.5 mg, 0.109 mmol) was then added

and the reaction mixture was left heating at 50 °C overnight. The reaction mixture was neutralized

with saturated sodium bicarbonate solution. The aqueous mixture was concentrated to remove

methanol, then extracted 3 times with dichloromethane. The combined organic layers were washed

with water and then brine, collected, dried with sodium sulfate, filtered and concentrated. The

crude product was purified by column chromatography using a mobile phase of 40-100% ethyl

acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as an off

white solid (37.4 mg, 93% yield). LCMS [M+H]+ 586.1 m/z; 1H NMR (500 MHz,


H), 7.76 - 7.83 (m, 4 H), 7.64 (d, J=8.3 Hz, 2 H), 7.30 (d, J=5.4 Hz, 1 H), 7.25 (d, J=8.3 Hz, 2 H),

6.67 (s, 1 H), 4.28 (d, J=5.9 Hz, 2 H), 3.73 (d, J=4.4 Hz, 2 H), 3.11 (s, 3 H), 2.84 - 2.94 (m, 2 H),

2.38 (s, 3 H), 2.26 - 2.34 (m, 2 H), 2.10 (s, 2 H).

280

N-(4-(4-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-6098

(3.29a) N-(4-(4-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (46

mg, 0.093 mmol) was dissolved in dioxane (0.309 mL). Sodium hydroxide was added (3M, 0.309

mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was

quenched with saturated ammonium chloride solution. A precipitate formed and was collected by

vacuum filtration to afford the title compound as a yellow solid (22 mg, 69% yield). LCMS

[M+H]+ 343.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.35 (br. s., 1 H), 9.04 (d, J=1.5 Hz,

1 H), 8.69 (dd, J=4.6, 1.2 Hz, 1 H), 8.31 (d, J=4.9 Hz, 1 H), 8.22 - 8.28 (m, 1 H), 7.95 (d, J=8.3

Hz, 2 H), 7.61 (dd, J=7.3, 4.9 Hz, 1 H), 7.43 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.13 (s,

1 H), 3.75 (s, 2 H), 2.74 (dquin, J=12.0, 5.9, 5.9, 5.9, 5.9 Hz, 1 H), 1.02 (d, J=6.3 Hz, 6 H).

N-(2-hydroxyethyl)-4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-

yl)benzenesulfonamide. NEU-6111 (3.29b) N-(2-hydroxyethyl)-4-(2-(4-

281

((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide

(63 mg, 0.102 mmol) was dissolved in dioxane (0.340 mL). Sodium hydroxide was added (3M,

0.340 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture

was quenched with saturated ammonium chloride solution and concentrated. The crude product

was purified by column chromatography using a mobile phase of 1-20% ((+10% ammonium

hydroxide)/methanol)/dichloromethane followed by 1-20% ((+10% ammonium

hydroxide)/methanol)/ethyl acetate to afford the title compound as a yellow solid (29 mg, 61%

yield). LCMS [M+H]+ 465.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.35 (br. s., 1 H), 8.31

(d, J=5.4 Hz, 1 H), 8.06 (d, J=8.3 Hz, 2 H), 7.92 - 8.00 (m, 4 H), 7.76 (br. s., 1 H), 7.43 (d, J=7.8

Hz, 2 H), 7.27 (d, J=4.9 Hz, 1 H), 7.15 (s, 1 H), 4.74 (t, J=5.6 Hz, 1 H), 3.72 (s, 2 H), 3.43 (q,

J=6.2 Hz, 1 H), 2.88 (t, J=6.1 Hz, 2 H), 2.71 (spt, J=6.0 Hz, 1 H), 1.01 (d, J=6.3 Hz, 6 H).

N-(4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.

NEU-6475 (3.29c) N-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (79 mg, 0.138 mmol) was dissolved in dioxane (0.5 mL). Sodium

hydroxide (3M, 0.5 mL) was added and the reaction mixture was microwaved at 150 °C for 15

minutes. The reaction mixture was quenched with saturated ammonium chloride solution. The

reaction mixture was concentrated and purified by column chromatography using a mobile phase

282

of 1-20% methanol/dichloromethane followed by 0-20% ((+5% ammonium

hydroxide)/methanol)/ethyl acetate to afford the title compound as a pale yellow solid (37.3 mg,

65% yield). LCMS [M+H]+ 420.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.30 (d,

J=4.9 Hz, 1 H), 8.13 - 8.19 (m, 2 H), 8.05 - 8.11 (m, 2 H), 7.89 (d, J=7.8 Hz, 2 H), 7.49 (d, J=7.8

Hz, 2 H), 7.29 (d, J=5.4 Hz, 1 H), 7.05 (s, 1 H), 3.85 (s, 2 H), 3.21 (s, 3 H), 2.93 (spt, J=5.9 Hz, 1

H), 1.16 (d, J=6.3 Hz, 6 H).

N-(4-(4-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.

NEU-6476 (3.29d) tert-butyl 4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (77 mg, 0.13 mmol) was dissolved in

dichloromethane (0.5 mL) and trifluoroacetic acid was added and the reaction mixture stirred at

room temperature until completion as determined by LCMS. The solvent was concentrated to

dryness and the crude mixture then dissolved in dioxane (500 μL). Sodium hydroxide (3M, 426

μL) was added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction

mixture was quenched with saturated ammonium chloride solution. The reaction mixture was

concentrated and purified by column chromatography using a mobile phase of 0-50% (5%

ammonium hydroxide/methanol)/ethyl acetate followed by 1-20% methanol/dichloromethane to

afford the title compound as a yellow solid (14 mg, 31% yield). LCMS [M+H]+ 347.2 m/z; 1H

283

NMR (500 MHz, METHANOL-d4) ppm 8.11 (d, J=5.4 Hz, 1 H), 7.84 (d, J=8.3 Hz, 2 H), 7.45

(d, J=8.3 Hz, 2 H), 6.97 - 7.04 (m, 2 H), 6.43 (br. s., 1 H), 3.82 (s, 2 H), 3.59 (d, J=2.4 Hz, 2 H),

3.13 (t, J=5.6 Hz, 2 H), 2.90 (spt, J=6.3 Hz, 1 H), 2.63 (d, J=2.0 Hz, 2 H), 1.14 (d, J=6.3 Hz, 6 H).

N-(4-(4-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-

6477 (3.29e) N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (50 mg, 0.098 mmol) was dissolved in dioxane (320 μL). Sodium

hydroxide (3M, 320 μL) was added and the reaction mixture was microwaved at 150 °C for 15



of 1-20% methanol/dichloromethane followed by 0-20% methanol/ethyl acetate to afford the title

compound as a yellow residue (20 mg, 57% yield). LCMS [M+H]+ 357.2 m/z; 1H NMR (500 MHz,

METHANOL-d4) ppm 8.58 (d, J=5.4 Hz, 1 H), 8.29 (d, J=4.9 Hz, 1 H), 7.88 (d, J=8.3 Hz, 2 H),

7.72 (s, 1 H), 7.67 (d, J=5.4 Hz, 1 H), 7.47 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.04 (s, 1

H), 3.81 (s, 2 H), 2.88 (spt, J=6.3 Hz, 1 H), 2.67 (s, 3 H), 1.13 (d, J=6.3 Hz, 6 H).

284

N-(4-(4-(2,6-dimethylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-

6478 (3.29f) N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine (70 mg, 0.13 mmol) was dissolved in dioxane (400 μL). Sodium

hydroxide (3M, 445 μL) was added and the reaction mixture was microwaved at 150 °C for 15



of 1-20% methanol/dichloromethane followed by 1-20% (10% ammonium

hydroxide/methanol)/ethyl acetate to afford the title compound as an off-white solid (30 mg, 61%

yield). LCMS [M+H]+ 371.3 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.28 (d, J=4.9

Hz, 1 H), 7.88 (d, J=8.3 Hz, 2 H), 7.51 (s, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.25 (d, J=5.4 Hz, 1 H),

7.02 (s, 1 H), 3.82 (s, 2 H), 2.82 - 2.96 (m, 1 H), 2.63 (s, 6 H), 1.14 (d, J=6.3 Hz, 6 H).

N-(4-(4-(1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-6508

(3.29g) N-(4-(4-(1H-pyrazol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine

(110 mg, 0.23 mmol) was dissolved in dioxane (755 μL). Sodium hydroxide (3M, 755 μL) was

285

added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture

was quenched with saturated ammonium chloride solution. The reaction mixture was concentrated

and purified by column chromatography using a mobile phase of 1-20%


LCMS [M+H]+ 332.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.15 (br. s., 1 H), 8.22 (d,

J=4.9 Hz, 1 H), 7.85 - 8.00 (m, 3 H), 7.36 - 7.54 (m, 4 H), 7.06 (br. s., 1 H), 3.83 (br. s., 2 H), 2.86

(m, J=4.9 Hz, 1 H), 1.08 (d, J=6.3 Hz, 6 H).

N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-

yl)benzyl)propan-2-amine. NEU-6509 (3.29h) N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-

pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (90 mg, 0.16 mmol)

was dissolved in dioxane (300 μL). Sodium hydroxide (3M, 527 μL) was added and the reaction

mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with

saturated ammonium chloride solution. The reaction mixture was concentrated and purified by


title compound as a light tan solid (34.8 mg, 53% yield). LCMS [M+H]+ 416.3 m/z; 1H NMR (500

MHz, DMSO-d6) ppm 12.19 (br. s., 1 H), 8.62 (s, 1 H), 8.25 (s, 1 H), 8.17 (d, J=4.9 Hz, 1 H),

8.10 (d, J=8.3 Hz, 2 H), 7.62 (d, J=7.3 Hz, 2 H), 7.40 (s, 1 H), 7.30 (d, J=5.4 Hz, 1 H), 4.53 (m,

286

J=4.9, 4.9 Hz, 1 H), 4.09 (br. s., 2 H), 4.03 (d, J=10.7 Hz, 2 H), 3.52 (t, J=10.5 Hz, 2 H), 3.13 -

3.26 (m, 1 H), 2.01 - 2.17 (m, 4 H), 1.26 (d, J=4.9 Hz, 6 H).

tert-Butyl 4-(4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-

pyrazol-1-yl)piperidine-1-carboxylate. NEU-6077 (3.29i) tert-Butyl 4-(4-(2-(4-formylphenyl)-

1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (60 mg, 0.13 mmol)

was dissolved in a solution of 10% acetic acid/methanol (1 mL). Isopropylamine (680 μL, 8.3

mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. Sodium

cyanoborohydride (12 mg, 0.19 mmol) was then added and the reaction mixture was left heating

at 70 °C overnight. The reaction mixture was neutralized with saturated sodium bicarbonate

solution. The aqueous mixture was concentrated to remove methanol, then extracted 3 times with

ethyl acetate. The combined organic layers were washed with water and then brine, collected, dried

with sodium sulfate, filtered and concentrated. The crude product was purified by column


compound as a pale yellow solid (29 mg, 44% yield). LCMS [M+H]+ 515.3 m/z; 1H NMR (500

MHz, METHANOL-d4) ppm 8.39 (s, 1 H), 8.09 - 8.15 (m, 2 H), 7.97 (d, J=8.3 Hz, 2 H), 7.55

(d, J=8.3 Hz, 2 H), 7.25 (d, J=4.9 Hz, 1 H), 7.19 (s, 1 H), 4.40 - 4.49 (m, 1 H), 4.23 (d, J=13.2 Hz,

287

2 H), 4.10 (s, 2 H), 3.29 - 3.37 (m, 1 H), 2.86 - 3.04 (m, 2 H), 2.07 - 2.14 (m, 2 H), 2.01 (m, J=12.0,

4.1 Hz, 2 H), 1.46 (s, 9 H), 1.32 (d, J=6.8 Hz, 6 H).

4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.

NEU-6712 (3.29j) 4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-

1H-pyrrolo[2,3-b]pyridine (34 mg, 0.058 mmol) was dissolved in dioxane (500 μL). Sodium

hydroxide (3M, 0.2 mL) was added and the reaction mixture was microwaved at 150 °C for 15

minutes. The reaction mixture was quenched with saturated ammonium chloride solution. A

precipitate formed and was collected by vacuum filtration. The crude product was purified by


title compound as a yellow solid (5 mg, 20% yield). LCMS [M+H]+ 432.2 m/z; 1H NMR (500

MHz, METHANOL-d4) ppm 8.33 (d, J=4.4 Hz, 1 H), 8.12 - 8.19 (m, 2 H), 8.08 (d, J=8.3 Hz, 2


(s, 2 H), 3.24 (br. s., 4 H), 3.22 (s, 3 H), 2.07 (br. s., 4 H).

288

N-(4-(4-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-

amine. NEU-6097 (3.30) tert-butyl 4-(4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-

b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (21 mg, 0.041 mmol) was dissolved in

dichloromethane (0.2 mL). Trifluoroacetic acid (0.12 mL, 1.6 mmol). The reaction mixture was

left stirring overnight at room temperature. The reaction mixture was neutralized using carbonate

on silica gel. The silica was filtered off and washed with methanol. The crude product was purified

by column chromatography using a mobile phase of 1-20% ((+10% ammonium

hydroxide)/methanol)/dichloromethane to afford the title compounds as a yellow solid (10 mg,

59% yield). LCMS [M+H]+ 415.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.38 (s, 1

H), 8.14 (s, 1 H), 8.12 (d, J=4.9 Hz, 1 H), 7.90 (d, J=8.3 Hz, 2 H), 7.46 (d, J=8.3 Hz, 2 H), 7.27

(d, J=4.9 Hz, 1 H), 7.14 (s, 1 H), 4.40 (tt, J=11.6, 4.0 Hz, 1 H), 3.81 (s, 2 H), 3.21 (d, J=13.2 Hz,

2 H), 2.89 (spt, J=6.3 Hz, 1 H), 2.78 (t, J=12.4 Hz, 2 H), 2.12 - 2.19 (m, 2 H), 2.03 (qd, J=12.2,

3.9 Hz, 2 H), 1.14 (d, J=6.3 Hz, 6 H).

289

4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. 4-(4-chloro-1-

tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (74 mg, 0.18 mmol), (4-

(methylsulfonyl)phenyl)boronic acid (44.5 mg, 0.223 mmol),

tetrakis(triphenylphosphine)palladium(0) (6.4 mg, 0.05 mmol), and sodium carbonate (59 mg,


A degassed solution of dioxane-water (3:1, 2 mL) was added to the vial. The reaction mixture was



chromatography using a mobile phase of 0-50% ethyl acetate/hexanes to afford the title compound

as an off white solid (83 mg, 87% yield). LCMS [M+H]+ 519.1 m/z; 1H NMR (500 MHz, DMSO-

d6) ppm 9.80 (s, 1 H), 8.46 (d, J=4.9 Hz, 1 H), 8.05 (d, J=8.8 Hz, 2 H), 7.95 - 7.99 (m, 1 H), 7.73


(d, J=8.8 Hz, 2 H), 6.81 (s, 1 H), 3.27 (s, 3 H), 2.32 (s, 3 H).

290

4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. NEU-1936 4-(4-

(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine

(83 mg, 0.16 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide (3M, 0.56 mL) was added

and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was

quenched with saturated ammonium chloride solution. A precipitate formed and was collected by

vacuum filtration to afford the title compound as an orange solid (53 mg, 90% yield). LCMS

[M+H]+ 365.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.27 (d, J=4.9 Hz, 1 H), 8.09 (s, 4 H),

7.82 (d, J=8.8 Hz, 2 H), 7.25 (d, J=5.4 Hz, 2 H), 6.97 (s, 1 H), 6.86 (d, J=8.3 Hz, 2 H), 3.31 (s, 3

H).

291

Cell and Whole-Organism Assay Protocols

The following assays were performed by members of the Navarro Lab at CSIC.

Strains and media. Bloodstream Trypanosoma brucei brucei Lister 427 was cultured in Hirumi’s

modified Iscove’s medium (HMI-9), supplemented with 10% heat-inactivated FBS, at 37 ºC and

5% CO2 in T-25 vented flask (Corning®). MRC5-SV2 cell line (SV40-transformed human lung

fibroblast cell line) was cultured in DMEM medium supplemented with 10% FBS at 37 ºC and 5%

CO2 in T-75 vented flask (Corning®). The T. cruzi Tulahuen C4 strain, expressing the β-

galactosidase gene (LacZ) and L6 rat skeletal muscle cells, used as host cells, were cultured in

RPMI-1640 supplemented with 10% iFBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100

μg/mL streptomycin at 37 °C and 5% CO2. Leishmania donovani MHOM/ET/67/HU3 cells with

the luciferase gene integrated into the parasite genome169 were grown at 28 °C in RPMI 1640-

modified medium (Invitrogen) supplemented with 20% FBS with 100 mg/ml of hygromycin B.

The Human myelomonocytic cell line THP-1 was grown at 37 °C and 5% CO2 in RPMI-1640

supplemented with 10% iFBS, 2 mM glutamate, 100 U/mL penicillin and 100 mg/mL

streptomycin.

Preparation of compound plates. For dose-response experiments, compound plates were prepared

for each analogue by serial 3-fold dilutions in 100% DMSO. Five concentration points

(mammalian cytotoxicity) or ten concentration points (parasite growth inhibition), were made in

96-well transparent Nunclon plates. Pentamidine was routinely included in compound plates as

internal quality control, and plates were stored sealed at -20 ºC for no more than four weeks.

292

Rate of Action assays. Mid-log T. brucei brucei cultures were diluted to the required cell density,

according to the different incubation time points described. Cultures (90 μL per well) were

dispensed in final assay Nunclon 96-well flat bottom Solid White plates and 10 µL of intermediate

plates were added to each well, as described before. Four sets of assay plates were arranged to

assay in order to be sequentially stopped at each indicated time point. Top and bottom rows were

dismissed for compound assay, to reduce evaporation effects.

Plates were incubated at 37 ºC and 5% CO2 for the indicated time points; incubation was

stopped by addition of 10 μL of prewarmed Cell Titer Glo reagent (Promega®), and after shaking

the plates were incubated at room temperature for 10 min, to allow the signal to settle. Plate

luminescence was read on an Infinite F200 plate reader (Tecan), and raw data were processed and

analyzed as previously described.

Β-D-Galactosidase Transgenic T. cruzi Assay. A Thermo Scientific Multidrop Combi dispenser

(MTX Lab Systems, Vienna, VA) was used to dispense 90 μL of T. cruzi amastigote–infected L6

cell culture (4×103 infected L6 cells per well) into 96-well Corning assay plates (Corning Inc.,

Corning, NY) already containing 10 μL of the compounds to be screened and controls. The plates

were incubated at 37 °C for 96 h. Then, 30 μL of 100 μM CPRG and 0.1% NP40 diluted in PBS

were added to each well, and the plates were incubated for 4 h at 37 °C in the dark. Absorbance at

585 nm was measured in a Vmax kinetic microplate reader (Molecular Probes). Compound

activities were normalized using the in-plate 100% inhibition (benznidazole at 10 μg/mL) and 0%

inhibition (0.2% DMSO) growth controls.

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Cytotoxicity assay in MRC5. Intermediate plates were made as described, adding 95 μL of DMEM

complete media to 5 μL of compound per well setting a 5% DMSO amount. Log-phase MRC5

cells were removed from a T-75 TC flask using TrypLE® Express (Thermo®) and dispersed by

gentle pipetting. Cell density was adjusted to working concentration in prewarmed DMEM

medium: 25,000 cells in 90 μL of culture were plated in 96-well transparent Nunclon plates and

let to settle for 24 h at 37 ºC and 5% CO2. After settling incubation, 10 μL of freshly made

intermediate plate were added per well: final maximal concentration for compounds was 50 μM in

0.5% DMSO per well. Plates were incubated for 48 h at 37 ºC and 5% CO2. At 4 h prior to

fluorescence measurement, 20 μL of 500 μM resazurin solution was added. Fluorescence was read

in an Infinite F200 plate reader (Tecan®) at 550 nm (excitation filter) and 590 nm (emission filter).

A four-parameter equation was used to fit the dose-response curves and determination of

EC50 by SigmaPlot ® 13.0 software. Assays were performed in duplicate at least twice for positive

compounds, to achieve a minimal n=2 per dose response.

Resazurin-Based L6 Assay. One hundred microliters (100 μL) per well of culture medium

containing the compounds and controls were added to L6 cells previously cultured (4×103 L6 cells

per well). After 72 h at 37 °C the medium was exchanged and the viable cell number was

determined by resazurin (Sigma–Aldrich) reduction. 20 μl of resazurin (1.1 mg/ml) was added to

each well and incubated in the dark for 2 h at 37 ºC. Cell viability was estimated by measuring the

final fluorescence at 570-590 nm in an Infinite F200 plate reader (Tecan).

Cytotoxicity assay in THP-1. Cellular toxicity of all compounds was determined using the

colorimetric MTT-based assay after incubation at 37 °C for 72 h in the presence of increasing

294

concentrations of compounds (final maximal concentration was 50 μM in 0.5% DMSO per

well)170. The results are expressed as EC50 values, the concentration of compound that reduces cell

growth by 50% versus untreated control cells. Assays were performed in duplicate at least twice

to achieve a minimal n=3 per dose response.

Determination of EC50 in L. donovani. Macrophage-differentiated THP-1 cells were infected at a

macrophage/parasite ratio of 1:10 with stationary-phase L. donovani promastigotes for 24 h at 35

°C and 5% CO2, and extracellular parasites were removed by washing three times with PBS.

Infected cell cultures were then incubated with different compounds concentrations at 37 °C for

72 h. Luminescence was measured using the Promega kit luciferase assay system (Promega ®,

Madison, WI). Assays were performed in duplicate at least twice, to achieve a minimal n=3 per

dose response.

Cytotoxicity assay in HepG2.171 HepG2 cells were cultured in complete Minimal Essential Medium

prepared by supplementing MEM with 0.19% sodium bicarbonate, 10% heat inactivated FBS, 2

mM L-glutamine, 0.1 mM MEM non-essential amino, 0.009 mg/mL insulin, 1.76 mg/mL bovine

serum albumin, 20 units/mL penicillin–streptomycin, and 0.05 mg/mL gentamycin. HepG2 cells

cultured in complete MEM were first washed with 1x Hank’s Balanced Salt Solution (Invitrogen

#14175095), trypsinized using a 0.25% trypsin/EDTA solution, assessed for viability using trypan

blue, and resuspended at 250,000 cells/mL. Using a Tecan EVO Freedom robot, 38.3 μL of cell

suspension were added to each well of clear, cell culture-treated 384-well microtiter plates for a

final concentration of 9570 liver cells per well, and plated cells were incubated overnight in 5%

CO2 at 37 °C. Drug plates were prepared with the Tecan EVO Freedom using sterile 96 well plates

295

containing twelve duplicate 1.6-fold serial dilutions of each test compound suspended in DMSO.

4.25 μL of diluted test compound was then added to the 38.3 μL of media in each well providing

a 10%-fold final dilution of compound. Compounds were tested from a range of 57 ng/mL to

10,000 ng/mL for all assays. Mefloquine was used as a plate control for all assays with a

concentration ranging from 113 ng/mL to 20,000 ng/mL. After a 48 h incubation period, 8 μL of

a 1.5 mg/mL solution of MTT diluted in complete MEM media was added to each well. All plates

were subsequently incubated in the dark for 1 h at room temperature. After incubation, the media

and drugs in each well was removed by shaking the plate over sink, and the plates were left to dry

in a fume hood for 15 mins. Next, 30 μL of isopropanol acidified by addition of HCl at a final

concentration of 0.36% was added to dissolve the formazan dye crystals created by reduction of

MTT. Plates are put on a 3-D rotator for 15-30 mins. Absorbance was determined in all wells using

a Tecan iControl 1.6 Infinite plate reader. The 50% toxic concentrations (TC50) were then

generated for each toxicity dose response test using GraphPad Prism (GraphPad Software Inc.,

San Diego, CA) using the nonlinear regression (sigmoidal dose-response/variable slope) equation.

ADME Experiment Protocols

All animal studies were performed by AstraZeneca.

Aqueous pH 7.4 Solubility. Compounds were dried down from 10 mM DMSO solutions using

centrifugal evaporation technique. Phosphate buffer (0.1 M pH 7.4) was added and StirStix were

inserted in the glass vials, with shaking then performed at a constant temperature of 25 °C for 20-

24 h. This step was followed by double centrifugation with a tip wash in between, to ensure that

no residues of the dried compounds interfere. The solutions were diluted before analysis and

quantification using LC/MS/MS was performed.

296

Log D7.4. Shake-flask octanol-water distribution coefficient was determined at pH 7.4 (Log D7.4).

The aqueous solution used is 10 mM sodium phosphate pH 7.4 buffer. The method has been

validated for Log D7.4 ranging from -2 to 5.0.

Human Plasma Protein Binding (PPB). PPB was determined using equilibrium dialysis (RED

device) to separate free from bound compound. The amount of compound in plasma (10 µM initial

concentration) and in dialysis buffer (pH 7.4 phosphate buffer) was measured by LC-MS/MS after

equilibration at 37 °C in a dialysis chamber to give the fraction unbound (fu); percent bound is

calculated and reported.

Human Liver Microsomal Clint. In vitro intrinsic clearance was determined from human liver

microsomes using a standard approach.159 Following incubation and preparation, the samples were

analyzed using LC/MS/MS. Refined data were uploaded to IBIS and are displayed as Clint

(intrinsic clearance) in μL/min/mg.

Rat Hepatocyte Clint. In vitro intrinsic clearance was determined from rat hepatocytes using a

standard approach.159 Following incubation and preparation, the samples were analyzed using

LC/MS/MS. Refined data are uploaded to IBIS and are displayed as Clint (intrinsic clearance)

μL/min/1 million cells.

297

Calculated LogP and LogD values. Both LogP and LogD predictions are based on a modified

version of the method160 where the predicted partition coefficients are composed of the

molecules’ atomic increments.

Pharmacokinetics Protocols

All animal studies were performed by GlaxoSmithKline.

Animals and ethical statement: All animal studies were ethically reviewed and carried out in

accordance with European Directive 2010/63/EEC and the GSK Policy on the Care, Welfare and

Treatment of Animals. Compound was administered intraperitoneally (IP) to two groups of female

NMRI mice (Group 1 n=3; Group 2 n=6) supplied by Charles River (Germany) Ltd. The

compound was prepared in 1% (v/v) DMSO:99% (v/v) 20% (w/v) sulfobutyl ether-beta-

cyclodextrin (SBE-β-CD/Captisol®, used as a solubilizing agent) in water and the dosing volume

was 10 mL/kg for a total dose of 10 mg/kg. Food and tap water were available ad libitum.

Following IP dosing, Group 1 blood samples were collected from the tail vein into capillary tubes

containing K2EDTA at the following time-points: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h.

In order to obtain simultaneous blood and brain samples, Group 2 mice were placed under

terminal anaesthetic (isoflurane) and blood samples (0.3 mL) collected from the retro-orbital sinus

into K2EDTA tubes at 0.5 h (n= 3) and 4 h (n=3) after compound administration. Immediately

following blood sample collection, death was confirmed by cervical dislocation and the brain

removed. Aliquots of each blood sample were diluted in an equal volume of water. Mouse brain

samples were weighed, water was added at a ½ (w/v) ratio (brain/water), and then homogenized.

Both blood and brain samples were stored -80 ºC until analysis.

298

Diluted blood and brain homogenates were processed under standard liquid-liquid

extraction procedures using CAN containing an internal standard (Nifedipine) and analysed by

LC-MS/MS. Non-compartmental analysis was performed using the Phoenix pharmacokinetic

software version 1.4 (Certara) and Cmax, tmax, AUClast, AUC, and t1/2 were estimated.

In vivo Efficacy Experiment Protocols

All animal studies were performed by GlaxoSmithKline.

Animals and ethical statement: All procedures were approved by ethical committee of Institute of

Parasitology and Biomedicine Lopez-Neyra (Spanish National Research Council, CSIC), code

MNC.2/2015. This Institute has joined the Agreement on Transparency in Animal

Experimentation, promoted by the Confederation of Scientific Societies of Spain (COSCE), with

the collaboration of the European Association for Animal Research released on September 20,

2016. The Animal Experimentation Unit is under the control of the competent authority, registered

in the national register as Center of Breeding and User of experimental animals code ES-

180210000022, according with the European and Spanish regulations. Female NMRI mice

(Charles River Laboratories) were provided with sterilized water and commercial pellets ad

libitum and maintained under the standard conditions in a conventional room at 20–24 °C with a

12/12-h light/dark cycle. Compound 18 solution for treatment was resuspended in vehicle: 5 %

DMSO in 20 %Captisol® sulfobutyl ether β-cyclodextrin that improves solubility and stability for

drug dosing.

Blood-stage efficacy model. Infection was performed by i.p. injection of 104 bloodstream forms in

0.2 mL TDB glucose of T. brucei brucei (STIB795) from a cryopreserved stock. Three days later,

299

9 infected animals with confirmed parasitemia were divided into two groups: control (infected

mice treated with vehicle, n=4) and treated (infected mice treated with 30 mg/kg/day of 18, n=5).

For treatment, both control and 18 were heated at 50 °C for 10 min to solubilize. Control and drug-

treated mice received a 0.2 mL i.p. injection at the 3rd day from infection during 5 consecutive

days. Parasitemia was individually checked by direct microscopic counting of parasites in a

Neubauer chamber using 2 µL of blood from infected mice tail, diluted in 100 µL of TDB glucose.

Parasitaemia of all mice were periodically checked by tail blood examination up to day 31th. The

day of death was recorded.

CNS efficacy model. Infection was performed by i.p. injection of 2·104 bloodstream forms in 0.2

mL TDB glucose of T. brucei brucei (GVR25 strain) from a cryopreserved stock. Fourteen days

later, 13 infected animals with confirmed parasitemia were divided into three equal groups:

control (infected mice pretreated with a single dose of 40 mg/kg diminazene aceturate -Berenil®-

and treated with vehicle BID, n=5); treatment 1 (infected mice pretreated with 40 mg/kg Berenil®

+ 25 mg/kg /day BID 11, n=4) and treatment 2 (infected mice just treated with 25 mg/kg /day

BID 18, n=4): the total concentration of 18 administered to mice was 50 /mg/kg/day. For treatment,

vehicle and 18 were heated at 50 °C for 10 min to solubilize. Mice received a 0.2 mL i.p. injection

of vehicle or 18 at the 21th day from infection during 5 consecutive days. Parasitemia was

individually checked by direct microscopic counting of parasites in a Neubauer chamber using 2

µL of blood from infected mice tail, diluted in 100 µL of TDB glucose. Parasitaemia of all mice

were checked twice a week by tail blood examination and thereafter mice with parasitaemia

relapses were euthanized and the day of parasitaemia relapse was recorded. Results are expressed

as percentage of accumulative mortality and in Mean Survival Days (MSD).

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