Hit-to-Lead Optimization of Small Molecules for Neglected Tropical Disease Therapeutics
by Dana M. Klug
B.S. in Chemistry, DePaul 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 14, 2018
Dissertation directed by
Michael Pollastri Professor of Chemistry and Chemical Biology
1
Dedication
This dissertation is dedicated to my family – my mother Laura, my father Michael, and my brother Ryan. I am forever grateful for their unconditional love, humor, and support – not only while I pursued my Ph.D., but throughout my life. I am especially grateful to my parents for the
sacrifices that they have made for the sake of my education, and to my brother for being a kind
listener and friend when I needed him. I could not ask for better.
2
Acknowledgements
I am overwhelmingly thankful to so many people for their support over the past five years and throughout my academic career. To my advisor, Dr. Michael Pollastri, my heartfelt thanks for your mentorship and guidance. I truly do not believe that I could have had a better experience while pursuing my Ph.D. than the one I have had working with you. This research group has been an amazing environment in which to learn and grow as a scientist, and I am so grateful to have been given the opportunity.
I am also grateful for the mentoring, patience, and support of senior members of the lab, especially Dr. Lori Ferrins, Dr. Melissa Buskes, and Dr. William Devine. To my many other wonderful labmates and coworkers over the years, especially Dr. Baljinder Singh, Dr. Daniel
Oehme, Dr. Andrew Spaulding, Kelly Bachovchin, Westley Tear, Laura Tschiegg, Jack Fisher, and Max Staab, I am grateful for your advice in the lab, your friendship and support, and most of all for making work a fun and enriching place to be every day.
I would not have pursued my Ph.D. without the incredible mentorship and support of my undergraduate research advisor, Dr. Caitlin Karver. Her patience allowed me to explore my interests in chemistry and build my confidence in the lab, and her encouragement and belief in my abilities were the reasons I chose to pursue this career path.
I would be remiss if I did not acknowledge the hard work and dedication of our many collaborators on the projects described in this dissertation. For the high-throughput screening follow-up work, I would like to thank especially Dr. Miguel Navarro and Dr. Rosario Díaz-
González and their team at CSIC, as well as Dr. Maria Santos and Dr. Pilar Manzano at GSK.
Their expertise, guidance, and contributions to these projects are much appreciated. For the
3 lapatinib project, I am grateful to Dr. Rick Sciotti and his team at WRAIR, Dr. Kojo Mensa-Wilmot and his team at UGA, and Dr. Jair Lage de Siquiera-Neto and his team at UCSD.
I am grateful to my committee members, Dr. George O’Doherty, Dr. Roman Manetsch, and Dr. Penny Beuning, for their helpful feedback and encouragement, which have pushed me to be a better scientist. I am also thankful for the funding support that I received from various organizations, including the ACS MEDI Predoctoral Fellowship, and the Northeastern University
College of Science Graduate Dissertation Research Grant, without which I would not have been able to attain the quality of research presented herein. Finally, I am grateful to the Northeastern
Department of Chemistry and Chemical Biology for allowing to pursue my Ph.D. here and for welcoming me into its rich scientific community.
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Abstract of Dissertation
Neglected tropical diseases (NTDs) are a group of twenty diseases defined by the World
Health Organization and include indications such as sleeping sickness, Chagas disease, and leishmaniasis. These diseases are prevalent in tropical climates and are exacerbated by poor living conditions, and therefore disproportionately affect the world’s poorest populations. Despite representing a significant global health burden, NTDs attract little attention from for-profit pharmaceutical companies because of patients’ inability to pay for treatment. As a result, current treatments are often associated with significant limitations, including severe side effects and complicated administration routes. NTDs therefore represent a significant unmet medical need.
In order to address this need, new treatments that address these limitations must be developed. As most drug discovery for NTDs takes place in resource-limited academic laboratories, repurposing strategies represent an attractive way to find starting points for lead optimization and reduce the drug discovery timeline. These strategies involve identifying homology between human and parasite biology and using chemical matter effective against the human homolog as the basis for anti-parasitic drug discovery.
The work presented in this dissertation describes the results of optimization campaigns against multiple parasites across three different chemotypes. Chapters 2 and 3 describe work on two different chemical series discovered through a high-throughput screen of human kinase inhibitors against Trypanosoma brucei, the causative agent of human African trypanosomiasis
(HAT, also known as sleeping sickness). Chapter 2 outlines work undertaken on a series of benzoxazepinoindazoles identified as potent inhibitors of T. brucei growth. Through systematic exploration of structure-activity relationships (SAR), two compounds were identified with reasonable potency and in vitro absorption, distribution, metabolism, and excretion (ADME)
5 profiles. These compounds were assessed in pharmacokinetic (PK) studies and against a human kinase panel, resulting in the selection of one, NEU-4461, to be tested in a mouse model of HAT.
In a preliminary study, this compound extended the life of infected mice by up to two weeks and is a candidate for further development.
Chapter 3 focuses on a cluster of 3,5-disubstitued-7-azaindoles. Early in the project, metabolism was identified as a major liability of this series and was the focus of initial analog design. Subsequent SAR development identified a set of modifications that reduced clearance and increased solubility, and a second set that improved potency. One analog, NEU-5127, was selected for PK evaluation but did not show sufficient brain penetration to warrant further development (in stage two of HAT, T. brucei crosses the blood-brain barrier). Optimization of this series is ongoing with efforts aimed at using established SAR to develop analogs that are potent, soluble, and metabolically stable.
Chapter 4 is focused on improving the aqueous solubility of compounds derived from lapatinib, a human epidermal growth factor receptor inhibitor. Previous optimization of lapatinib resulted in a compound, NEU-1953, that had good to reasonable activity against multiple parasites, including T. brucei, Trypanosoma cruzi (causative agent of Chagas disease), Leishmania major
(one of the causative agents of leishmaniasis), and Plasmodium falciparum (causative agent of malaria). However, this compound had a poor ADME profile, including low aqueous solubility.
The work presented herein focuses on increasing the sp3-carbon content of subsequent analogs as a way to improve solubility, resulting in compounds that retained anti-parasitic activity and had markedly improved ADME profiles.
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Table of Contents
Dedication ...... 2
Acknowledgements ...... 3
Abstract of Dissertation ...... 5
Table of Contents ...... 7
List of Figures ...... 11
List of Schemes ...... 13
List of Tables ...... 15
List of Abbreviations ...... 17
Chapter 1: Background and Introduction ...... 22
1.1 Neglected tropical diseases ...... 22
1.1.1 Human African trypanosomiasis ...... 23
1.1.2 Other NTDs ...... 26
1.2 NTD drug discovery ...... 29
1.2.1 Target product profile ...... 29
1.2.2 Repurposing strategies for NTD drug discovery ...... 31
1.3 Physicochemical properties and hit-to-lead optimization ...... 33
1.3.1 cLogP and lipophilic ligand efficiency (LLE) ...... 34
1.3.2 CNS multiparameter optimization (CNS-MPO) score ...... 36
1.3.3 Aqueous solubility ...... 38
1.3.4 Other in vitro parameters ...... 39
1.4 High-throughput screening to generate leads for HAT ...... 40
7
1.5 Repurposing lapatinib for neglected diseases ...... 41
Chapter 2: Hit-to-Lead Optimization of Benzoxazepinoindazoles ...... 43
2.1 Characterization of HTS hits ...... 43
2.2 Analog synthesis ...... 45
2.2.1 Synthesis of BOXIs and related cores ...... 45
2.2.2 Oxazepinoindazole synthesis ...... 46
2.2.3 Indazole and aminoindazole synthesis ...... 48
2.3 Structure-activity and -property relationships ...... 50
2.3.1. Tail group analogs ...... 50
2.3.2 Core modifications ...... 55
2.3.3. Ring-opened analogs ...... 58
2.4 Further studies ...... 60
2.4.1 Pharmacokinetic studies ...... 61
2.4.2 Kinase panel assessment ...... 62
2.4.3 In vivo efficacy study ...... 63
2.5 Summary and future work ...... 64
Chapter 3: Hit-to-Lead Optimization of 3,5-Disubstituted-7-Azaindoles ...... 68
3.1 Characterization of HTS hits ...... 68
3.2 Analog synthesis ...... 70
3.2.1 Synthesis of 3-position analogs ...... 70
3.2.2 Synthesis of 5-position analogs ...... 71
8
3.2.3 Core replacement synthesis ...... 73
3.3 Structure-activity and -property relationships ...... 74
3.3.1 Benzonitrile replacements...... 74
3.3.2 Pyrazole replacements ...... 81
3.3.3 Crossover analogs ...... 84
3.3.4 Core replacements ...... 86
3.4 Further characterization of NEU-5127 ...... 87
3.5 Activity against T. cruzi ...... 89
3.6 Summary and future work ...... 91
Chapter 4. Solubility-Driven Optimization of Quinolines for HAT ...... 95
4.1 Discovery and characterization of NEU-1953 ...... 95
4.1.1 Repurposing lapatinib and discovery of NEU-1953...... 95
4.1.2 Strategies to improve the solubility of NEU-1953 ...... 96
4.2 Evaluation of strategy 1: Saturated linkers ...... 100
4.2.1 Synthesis via aryl amination chemistry ...... 100
4.2.2 Synthesis via Suzuki cross-coupling ...... 105
4.3 Evaluation of strategy 2: Piperazine replacements ...... 110
4.3.1 Analog synthesis ...... 111
4.3.2 Structure-activity and structure-property relationships ...... 113
4.4 Summary and future work ...... 116
Chapter 5. Experimental Details ...... 118
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5.1 General methods ...... 118
5.2 Chemical synthesis and characterization ...... 124
5.2.1 Experimental procedures for chapter 2 ...... 124
5.2.2 Experimental procedures for chapter 3 ...... 189
5.2.3 Experimental procedures for chapter 4 ...... 288
References ...... 307
Appendix ...... 313
Appendix 1. Biological activity assay protocols (Chapters 2 and 3) ...... 313
Appendix 2. Biological activity assay protocols (Chapter 4) ...... 316
Appendix 3. ADME and pharmacokinetic experiment protocols ...... 318
Appendix 4. Chapter 2 Spectra ...... 320
Appendix 5. Chapter 3 Spectra ...... 332
Appendix 6. Chapter 4 Spectra ...... 333
References ...... 341
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List of Figures
Chapter 1: Background and Introduction
Figure 1-1. Global distribution of a) T.b. gambiense and b) T.b. rhodesiense. …………………24
Figure 1-2. Life cycle of T. brucei. ……………………………………………………………...25
Figure 1-3. Compounds currently in clinical trials for HAT. …………………………………...26
Figure 1-4. Global distribution of Chagas disease. ……………………………………………...27
Figure 1-5. Global distribution of CL and VL. ………………………………………………….28
Figure 1-6. Common repurposing strategies used for NTD drug discovery. ……………………32
Figure 1-7. LLE in relation to logP and pEC50. …………………………………………………36
Figure 1-8. HTS flow chart. ……………………………………………………………………..40
Figure 1-9. Regions of lapatinib targeted for SAR exploration. ………………………………...42
Chapter 2: Hit-to-Lead Optimization of Benzoxazepinoindazoles
Figure 2-1. Structural features of the BOXI cluster. …………………………………………….43
Figure 2-2. Rate of action of selected analogs. ………………………………………………….53
Figure 2-3. ClogP versus aqueous solubility of BOXI tail group analogs. ……………………...54
Figure 2-4. Brain and blood concentrations of a) NEU-4461 and b) NEU-5388 over time after a
10 mg/kg intraperitoneal dose. …………………………………………………………………...62
Figure 2-5. Percent inhibition of a human kinase panel for NEU-4461 and NEU-5388. …………63
Figure 2-6. % Survival of mice treated with vehicle and NEU-4461 at 10 mg/kg/day ip. ……...... 64
Figure 2-7. SAR summary of the BOXI cluster. …………………………………………………65
Chapter 3: Hit-to-Lead Optimization of 3,5-Disubstituted-7-Azaindoles
Figure 3-1. a) Blood concentration of NEU-1207 over time after a 5 mg/kg oral dose. b) Predicted metabolites of NEU-1207. ……………………………………………………………………….70
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Figure 3-2. Topliss decision tree for 3-position substituents. …………………………………...76
Figure 3-3. Blood and brain concentrations of NEU-5127 over time after a 10 mg/kg intraperitoneal dose. ……………………………………………………………………………..89
Figure 3-4. SAR summary of 7-azaindoles. ……………………………………………………..93
Figure 3-5. Proposed analogs for T. brucei and T. cruzi. ………………………………………..94
Chapter 4: Solubility-Driven Optimization of Quinolines for HAT
Figure 4-1. Hit-to-lead progression of lapatinib derivatives. …………………………………….95
Figure 4-2. Aqueous solubility vs clogP for a selection of NEU-617-derived quinolines. ……….97
Figure 4-3. Summary of all strategies proposed to increase the solubility of NEU-1953. ………..99
Figure 4-4. Retrosynthetic analysis of saturated-linker analogs synthesized via Buchwald coupling, showing both a) the parallel-enabled route, and b) the alternative route. ……………………….101
Figure 4-5. Overlay of NEU-1953 (purple) and NEU-5971 (gray). ………………………….....110
Figure 4-6. SAR summary of linker and tail replacements. …………………………………...... 117
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List of Schemes
Chapter 2: Hit-to-Lead Optimization of Benzoxazepinoindazoles
Scheme 2-1. Synthesis of tail replacement analogs. …………………………………………….46
Scheme 2-2. Synthesis of oxazepinoindazole core. ……………………………………………..48
Scheme 2-3. Synthesis of indazole, aminoindazole, and ring-opened analogs. ………………...49
Scheme 2-4. Synthesis of disubstituted aminoindazoles. ……………………………………….67
Chapter 3: Hit-to-Lead Optimization of 3,5-Disubstituted-7-Azaindoles
Scheme 3-1. Synthesis of 3-position analogs. …………………………………………………..71
Scheme 3-2. Synthesis of 5-position analogs. …………………………………………………..72
Scheme 3-3. Core replacement synthesis. ………………………………………………………74
Chapter 4: Solubility-Driven Optimization of Quinolines for HAT
Scheme 4-1. Synthesis of key intermediate 7-bromo-N-(pyrazin-2-yl)quinoline-4-amine. …….100
Scheme 4-2. Synthesis of pyrrolidinyl-piperazine linker/tail. …………………………………102
Scheme 4-3. Synthesis of saturated-linker analogs using Suzuki coupling. …………………...106
Scheme 4-4. Synthesis of piperazine-replacement analogs. …………………………………...112
Chapter 5: Experimental Details
Scheme 5-1. Synthesis of NEU-4839. …………………………………………………………140
Scheme 5-2. Synthesis of NEU-4461. …………………………………………………………144
Scheme 5-3. Synthesis of NEU-4892. …………………………………………………………155
Scheme 5-4. Synthesis of NEU-4895. …………………………………………………………158
Scheme 5-5. Synthesis of NEU-4933. …………………………………………………………196
Scheme 5-6. Synthesis of NEU-5954, -5976, -5994, -5955, -6016, and -5995. ……………….203
Scheme 5-7. Synthesis of NEU-5421. …………………………………………………………233
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Scheme 5-8. Synthesis of NEU-5422. …………………………………………………………238
Scheme 5-9. Synthesis of NEU-5813, -5814, and -5902. ……………………………………...243
Scheme 5-10. Synthesis of NEU-5127, -5128, -5305, and -5319. …………………………….268
Scheme 5-11. Synthesis of NEU-5903. ………………………………………………………..275
Scheme 5-12. Synthesis of NEU-4411. ………………………………………………………..279
Scheme 5-13. Synthesis of NEU-5398. ………………………………………………………...281
Scheme 5-14. Synthesis of NEU-5004. ………………………………………………………...285
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List of Tables
Chapter 1: Background and Introduction
Table 1-1. Current treatments for HAT. ……………………………………………………….....26
Table 1-2. Summary of DNDi TPP for HAT, Chagas disease, and VL. ………………………….31
Table 1-3. Properties included in CNS-MPO score calculations and desired range for each. …….37
Table 1-4. High, medium, and low bins for HLM Clint and rat hepatocyte clearance. ……………40
Chapter 2: Hit-to-Lead Optimization of Benzoxazepinoindazoles
Table 2-1. Targeted, cluster average, and individual cluster member values for properties of interest. …………………………………………………………………………………………..44
Table 2-2. Kinase activity of NEU-1328. ………………………………………………………..45
Table 2-3. Biological activity and LLE of 2-aminopyrimidine tail replacement analogs. ……...... 51
Table 2-4. Biological activity and LLE of other tail replacement analogs. ……………………….52
Table 2-5. ADME properties of selected analogs. ………………………………………………..55
Table 2-6. Biological activity of N-methylated analogs. …………………………………………56
Table 2-7. Biological activity and selected ADME data for headgroup truncation and replacement analogs. …………………………………………………………………………………………..57
Table 2-8. Biological activity and selected ADME data for ring-opened analogs. ……………….60
Table 2-9. Heat maps of NEU-1117, NEU-4461, NEU-4985, and NEU-5388. …………………61
Table 2-10. PK parameters for NEU-4461 and NEU-5388. …………………………………...... 62
Chapter 3: Hit-to-Lead Optimization of 3,5-Disubstituted-7-Azaindoles
Table 3-1. Targeted, cluster average, and individual cluster member values for properties of interest. …………………………………………………………………………………………..69
15
Table 3-2. Biological activity, LLE, HLM Clint, and aqueous solubility of benzonitrile replacement analogs. …………………………………………………………………………………………..77
Table 3-3. Biological activity, LLE, HLM Clint, and aqueous solubility of second generation benzonitrile replacement analogs. ……………………………………………………………….80
Table 3-4. Biological activity, LLE, HLM Clint, and aqueous solubility of pyrazole replacement analogs. …………………………………………………………………………………………..83
Table 3-5. Biological activity, LLE, HLM Clint, and aqueous solubility of crossover analogs...... 85
Table 3-6. Biological activity of core replacement analogs. ……………………………………..87
Table 3-7. Heat maps of NEU-1207, NEU-5127, NEU-4832, NEU-5423, and NEU-5813. …….88
Table 3-8. PK parameters for NEU-5127. ……………………………………………………….88
Table 3-9. T. cruzi activity, toxicity, and LLE of selected analogs. ………………………………91
Chapter 4: Solubility-Driven Optimization of Quinolines for HAT
Table 4-1. Overall profile of NEU-1953. ………………………………………………………...96
Table 4-2. Results of a Buchwald condition scan using the piperidine model system. ………….103
Table 4-3. Results of a Buchwald condition scan using the desired linker and tail. ……………..104
Table 4-4. Results of a Buchwald condition scan using the quinoline core. …………………….105
Table 4-5. Attempted reductive amination and alkylation conditions. ……………………….....107
Table 4-6. Biological activity of saturated-linker analogs. ……………………………………...108
Table 4-7. ADME properties of saturated-linker analogs. ……………………………………...109
Table 4-8. Biological activity of piperazine-replacement analogs. ……………………………..114
Table 4-9. ADME properties of piperazine replacement analogs. ………………………………115
3 Table 4-10. Fsp , melting points, pKa, and aqueous solubility of selected piperazine replacement analogs. ………………………………………………………………………………………....116
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List of Abbreviations
Ac Acetyl
ACN Acetonitrile
AcOH Acetic acid
ADME Absorption, distribution, metabolism, excretion
Ar Aryl
B2pin2 Bis(pinacolato)diboron
BBB Blood brain barrier
BINAP 2,2’-Bis(diphenylphosphino)-1,1’-binaphthyl
Boc tert-Butoxycarbonyl
Boc2O Di-tert-butyl dicarbonate
BOXI Benzoxazepinoindazole
CAN Cerium ammonium nitrate
CL Cutaneous leishmaniasis
Clint Intrinsic clearance clogP Calculated partition coefficient
Cmax Maximum concentration
CNS Central nervous system
CNS-MPO Central nervous system multiparameter optimization
CSIC Consejo Superior de Investigaciones Cientificas
CYP Cytochrome P450
DALY Disability-adjusted life years dba Dibenzylideneacetone
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DCE 1,2-Dichloroethane
DCM Dichloromethane
DIPEA Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DME 1,2-Dimethoxyethane
DMF N,N’-dimethylformamide
DMSO Dimethyl sulfoxide
DNDi Drugs for Neglected Disease Initiative dppf 1,1′-Bis(diphenylphosphino)ferrocene
EC50 Half maximal effective concentration
EGFR Epidermal growth factor receptor
EtOAc Ethyl acetate equiv Equivalents
EtOH Ethanol
Fsp3 Fraction of sp3-hybridized carbons
GHIT Japanese Global Health Initiative Technology
GSK GlaxoSmithKline h Hours
HAT Human African trypanosomiasis
HBD Hydrogen-bond donor
3-[Bis(dimethylamino)methyliumyl]3H-benzotriazol-1-oxide HBTU hexafluorophosphate
HDAC Histone deacetylase
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HLM Human liver microsomes
HPLC High performance liquid chromatography
HTS High throughput screen ip Intraperitoneal
IV Intravenous
KOAc Potassium acetate
LiHMDS Lithium hexamethyldisilazide
LCMS Liquid chromatograpy/mass spectrometry
LLE Lipophilic ligand efficiency
MDA Mass drug administration
M Molar
MeOH Methanol
MMV Medicines for Malaria Venture
MPO Multiparameter optimization
MW Molecular weight
NECT Nifurtimox-eflornithine combination therapy
NIS N-iodosuccinimide
NMP N-methylpyrrolidine
NMR Nuclear magnetic resonance
NMT N-myristoyltransferase
NTD Neglected tropical disease
PAINS Pan-assay interference compounds pEC50 Negative log of the half maximal effective concentration
19 pIC50 Negative log of the half maximal inhibitory concentration pTC50 Negative log of the half maximal toxic concentration
Ph Phenyl
PK Pharmacokinetic pKa Negative log of the acid dissociation constant
PKIS Published Kinase Inhibitor Set
PMB para-Methoxybenzyl
PPB Plasma protein binding
REU Research Experience for Undergraduates
RuPhos 2-Dicyclohexylphosphino-2’,6’-diisopropoxybiphenyl
RTK Receptor tyrosine kinase
SAR Structure activity relationship
SEM Standard error of the mean
SPR Structure properties relationship
TB Tuberculosis t-Bu tert-Butyl t-BuOH tert-Butanol t-Bu3P Tri-tert-butylphosphine t-BuXPhos 2-Di-tert-butylphosphino-2’,4’,6’-triisopropylbiphenyl
TC50 Half maximal toxic concentration
TEA Triethylamine
TFA 2,2,2-trifluoroacetic acid
THF Tetrahydrofuran
20
TLC Thin layer chromatography tmax Time of maximum concentration
TMS Trimethylsilyl
TPP Target product profile
TPSA Topological polar surface area
Ts Tosyl
TsCl 4-toluenesulfonyl chloride
VL Visceral leishmaniasis
WHO World Health Organization
μM Micromolar
μw Microwave
Xantphos 4,5-Bis(diphenylphosphino)-9,9’-dimethylxanthene
XPhos 2-Dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl
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Chapter 1: Background and Introduction
1.1 Neglected tropical diseases
Neglected tropical diseases (NTDs) are members of a group of twenty communicable diseases, designated by the World Health Organization (WHO), that disproportionately affect some of the poorest people in the world (sometimes referred to as the “bottom billion”).1-3 These diseases represent a significant global health burden, costing over 19 million disability-adjusted life years (DALYs) according to the 2016 Global Burden of Disease study.4 Additionally, they have a substantial economic and social impact due to the effects of time away from work or school, long-term physical disability, and the stigma associated with many NTDs.5, 6 The combined health, economic, and social effects of NTDs perpetuate the cycle of poverty in many developing nations.5
Despite this burden, NTDs have historically failed to attract significant investment in treatment and prevention because the patient population is unable to afford such therapies.1, 2
Recently, however, NTDs have begun to garner more attention from researchers and entities such as the WHO. In 2012, the WHO published a document containing strategies working toward “controlling, eliminating and eradicating” NTDs by 2020.7 These strategies included vector control, sanitation improvement, and preventive chemotherapy, or large-scale administration of preventive drugs.7 In a 2017 report, the WHO assessed progress toward the 2020 targets and found that mass drug administration (MDA) of preventive chemotherapy agents had the greatest impact on NTD control.8 The WHO also reported a 36% increase in the number of people receiving preventive chemotherapy for at least one disease from 2011 to 2015, and an increase in contributions from partners in the pharmaceutical industry following the 2012 London Declaration on NTDs.8
22
While this progress is encouraging, there is a need for continued investment in control and prevention strategies for NTDs. The 2017 WHO report highlighted a number of challenges to be addressed in order to meet the goal of NTD control and elimination by 2020. For example, drugs used for preventive chemotherapy may lose efficacy or become susceptible to resistance mechanisms.8 Vector control efforts have been impacted by insecticide resistance and the effects of global climate change.8 And finally, “a lack of robust, sustained international and domestic financing” impacts all WHO control efforts.8 Thus, there is clearly a need for continued investment in strategies that prevent, manage, or treat NTDs.
1.1.1 Human African trypanosomiasis
Human African trypanosomiasis (HAT), also known as African sleeping sickness, is an
NTD caused by two subspecies of the parasite Trypanosoma brucei, T.b. gambiense and T.b. rhodesiense.9 As illustrated in Figure 1-1, HAT is distributed primarily in sub-Saharan Africa.
T.b. gambiense, the subspecies causing greater than 95% of reported HAT cases in 2015, is concentrated in the eastern region of the continent and is most prevalent in the Democratic
Republic of the Congo.10 T.b. rhodesiense is concentrated in the western region, particularly in
Malawi and Uganda.10 According to the WHO, the prevalence of HAT has been decreasing since
1995 and in 2016 fewer than 3,000 new cases were reported (with 15,000 cases estimated).9
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Figure 1-1. Global distribution of a) T.b. gambiense and b) T.b. rhodesiense. Image from the WHO: http://www.who.int/trypanosomiasis_african/country/en/9 a) b)
The life cycle of T. brucei is illustrated in Figure 1-2. The parasite is transmitted to a human host when the host is bitten by an infected vector, the tsetse fly. Once in the bloodstream, the parasite takes a form (trypomastigote) that can be carried from the blood to other sites in the body, most notably the lymph and spinal fluid. These trypomastigotes remaining in the blood are transferred to an uninfected tsetse fly when the fly takes a blood meal, and parasites undergo maturation and reproduction to end up in the salivary glands of the tsetse fly where they can infect human hosts during the next blood meal, perpetuating the parasitic life cycle.
The human infection of HAT is divided into two stages. In stage one, the parasite resides in the blood and lymph systems and causes mild, flu-like symptoms such as fever, headaches, and muscle and joint aches.11 In T.b. gambiense infections, this stage is chronic and can last for years; in T.b. rhodesiense infections it is acute and lasts only for weeks.11 Eventually both subspecies cross the blood-brain barrier (BBB) and invade the central nervous system (CNS), commencing stage two.11 Stage two is characterized by more severe neurological symptoms, such as psychiatric disorders, sleeping disorders (which give the disease the colloquial name “sleeping sickness”), and coma. HAT is 100% fatal if left untreated.9, 11
24
Figure 1-2. Life cycle of T. brucei. Image from the US Centers for Disease Control & Prevention: https://www.cdc.gov/parasites/sleepingsickness/biology.html11
Shown in Table 1-1 are the drugs currently available to treat HAT and their limitations, which are primarily: lack of efficacy against all stages and subspecies, routes of administration that are problematic in regions where access to health care facilities is poor, and causation of side effects of varying degrees of severity. HAT is “notoriously difficult to treat”9 and new drugs are desperately needed that address these limitations. The Drugs for Neglected Diseases initiative
(DNDi) is a non-profit organization which has developed the two compounds shown in Figure 1-
3, currently in Phase III clinical trials for HAT.12, 13 Both are orally available and expected to be amenable to at-home administration, avoiding the use of melarsoprol and addressing the major limitation of eflornithine.
25
Figure 1-3. Compounds currently in clinical trials for HAT.
Table 1-1. Current treatments for HAT.9, 13-15
Indication Drug Limitations Efficacy: Effective only against stage 1 T.b. rhodesiense. Suramin Administration: IV injections every 3-7 days for 4 weeks. Dosing: 20 mg/kg. Stage 1 Efficacy: Effective only against stage 1 T.b. gambiense. Pentamidine Administration: Daily IM injections for 7-10 days. Dosing: 4 mg/kg. Efficacy: Effective against both stages and subspecies. Side effects: Highly toxic; approx. 5% treatment-related mortality. Melarsoprol Administration: Daily IV injections for 10 days. Dosing: 2.2 mg/kg. Efficacy: Effective only against T.b. gambiense infections. Stage 2 Eflornithine Administration: IV infusions every 6 hours for 14 days (56 total). Dosing: 100 mg/kg/infusion (150 mg/kg for children). Nifurtimox-eflornithine Efficacy: Effective only against T.b. gambiense infections. combination therapy Administration: Twice-daily eflornithine infusions for 7 days, plus 3 doses (NECT) of oral nifurtimox for 10 days.
1.1.2 Other NTDs
As part of an effort to address multiple NTDs, our compounds are routinely screened against parasites other than T. brucei. Trpanosoma cruzi, a related parasite, is the causative agent of Chagas disease. Worldwide, approximately six to seven million people are infected with T. cruzi and another 75 million are at risk of infection.16, 17 As shown in Figure 1-4, Chagas disease is endemic throughout Central and South America and is most prevalent in Bolivia, Argentina, and
Paraguay.18 However, due to migration and other factors, Chagas is spreading beyond this region into the southern United States and globally.18
26
Figure 1-4. Global distribution of Chagas disease. Image from the Drugs for Neglected Diseases initiative: https://www.dndi.org/diseases-projects/chagas/17
Transmitted by the triatomine bug, or “kissing bug,” Chagas disease proceeds in two stages following transmission to a human host.17 The acute infection is typically asymptomatic and lasts four to eight weeks, with a decrease in parasitemia after 90 days.18 This is followed by chronic infection, which can last for decades. Although many chronic Chagas patients do not develop symptoms, roughly 30-40% eventually develop symptoms of organ involvement, of which cardiac complications are the most prevalent and severe.18 Two drugs, nifurtimox and benznidazole, are used to treat both stages of Chagas disease, although their efficacy is known to decrease with time after primary infection.18 Even successful treatment can take up to two months and can cause adverse effects in up to 40% of patients.16
Leishmaniasis is the umbrella term for infection by any one of over twenty species of
Leishmania parasites, which are transmitted by approximately 30 different species of sandflies.19
Clinically, Leishmania infections can take one of four forms, the most common of which are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL).19 Although underreporting makes it difficult to quantify the burden of leishmaniasis, it is estimated that 0.2-0.4 million cases of VL
27 and 0.7-1.2 million cases of CL occur each year, with approximately one billion people worldwide at risk.20, 21 Figure 1-5 shows the global distribution of VL and CL; CL is most common in South
America, west Africa, and the Mediterranean, while over 90% of VL cases occur in India,
Bangladesh, South Sudan, Brazil, and Ethiopia.21
Figure 1-5. Global distribution of CL and VL. Image from DNDi: https://www.dndi.org/diseases- projects/leishmaniasis/ 20
VL, also known as kala-azar, is caused by many Leishmania species, the most common of which are L. donovani (in east Africa and the Indian subcontinent) or L. infantum (in Europe, North
America, and Latin America).19 The early stages of infection are characterized by mild symptoms such as fever, fatigue, and weight loss; later, patients present with abdominal distension and pain.19
The disease is ultimately fatal if left untreated and is estimated to cause 20-40,000 deaths per year.21 CL is caused by over 15 different Leishmania spp. and is characterized by the presence of skin ulcers.20 While these ulcers usually heal spontaneously, they can leave deep scars or lesions which can stigmatize patients even after the infection is cleared.19 This social stigma can lead to
28 ostracization, interrupt education, and cause economic loss; in already resource-limited populations, these consequences can be devastating.20
Several drugs are currently used to treat leishmaniasis, depending on the geographical location and the specific species of Leishmania with which the patient is infected. However, all have serious drawbacks that inhibit their long-term or widespread use. The only orally available drug to treat VL and CL, miltefosine, is not safe for use in pregnant women and is susceptible to resistance due to low compliance with the treatment regimen.20 Other drugs require injections to administer, which can be painful and require hospitalization; some are costly due to expensive formulations; and availability of some treatments is limited in affected areas.20 DNDi has identified the need for a safe, orally available, and low cost treatment for VL; and the need for an orally or topically administered treatment for CL that mitigates the stigmatizing scars left by the disease.20
1.2 NTD drug discovery
1.2.1 Target product profile
The targeted characteristics of new NTD drugs are disease-specific, but all aimed at addressing the limitations of current therapeutics. In 2015, the Japanese Global Health Innovative
Technology Fund (GHIT), a public-private partnership focused on NTDs, partnered with
Medicines for Malaria Venture (MMV), the TB Alliance, DNDi, and the Bill and Melinda Gates
Foundation to publish hit and lead guidelines for malaria, tuberculosis (TB), Chagas disease, and leishmaniasis.22 In general, the GHIT guidelines emphasize the need for high-quality hit and lead compounds in order to increase the likelihood of delivering a clinical candidate. Validation of the activity, selectivity, and in vitro response; a robust chemotype amenable to analog synthesis; and the avoidance of reactive moieties such as pan-assay interference compounds (PAINS)23 are key criteria for hit selection.22 Leads should display high potency, oral efficacy in a disease model, and
29 an acceptable absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic
(PK) profile.22 In addition, safety parameters should be characterized and liabilities of the lead compound identified so that lead optimization is successful.22
Ultimately, lead optimization should result in a compound that fits the target product profile (TPP) for the disease in question. Table 1-2 outlines the general DNDi TPP for HAT,
Chagas disease, and leishmaniasis, the NTDs discussed in this dissertation. For HAT, efficacy against both stages and both subspecies is ideal, with <1% drug-related mortality as the maximum tolerated safety liability. Oral dosing for less than 10 days, compound stability for at least one year, and a low cost are also essential criteria for new HAT therapeutics. The TPP for Chagas disease uses benznidazole, the current treatment, as a benchmark for safety and efficacy. As with HAT, oral dosing, chemical stability, and low cost are also important considerations. Finally, the ideal
VL drug will be effective against all Leishmania spp. in any geographic region. Adverse effects requiring some monitoring are acceptable for the safety profile, as is intravenous (IV) dosing. As with HAT and Chagas disease, chemical stability and treatment cost are also important considerations.
30
Table 1-2. Summary of DNDi TPP for HAT, Chagas disease, and VL.24-26
Acceptable Ideal HAT Stage 1 + stage 2 (used stage 2 only); T.b. Stage 1 + stage 2; T.b. gambiense + T.b. Efficacy gambiense only rhodesiense <0.1% drug-related mortality; safe during Safety <1% drug-related mortality pregnancy Dosing 10 days p.o. (up to three times daily) <7 days p.o. once daily Stability >12 months >3 years Cost <100 €/course <30 €/course Chagas Disease Efficacy Chronic stage; non-inferior to benznidazole Chronic + acute stage; superior to benznidazole Superior to benznidazole (frequency of treatment Superior to benznidazole (frequency of treatment Safety discontinuations) discontinuations) Dosing Oral – any duration Oral – <30 days Stability 3 years 5 years Cost Lowest possible Current treatment VL Efficacy L. donovani only; either India or Africa All species; all areas Adverse effects requiring 1 monitoring visit at the Safety No adverse effects requiring monitoring mid/end-point Twice a day for <10 days p.o. or <3 shots over 10 Once a day for 10 days p.o. or 3 shots over 10 Dosing days days Stability >2 years 3 years Cost <$80/course <$10/course p.o. = oral administration.
1.2.2 Repurposing strategies for NTD drug discovery
Given the non-profitability of NTDs, most private pharmaceutical companies do not make significant investments in NTD drug discovery. This work therefore falls primarily to academic groups and non-profit organizations, which are significantly more resource-limited than large companies. The scarcity of resources available for NTD drug discovery makes repurposing strategies attractive as a way to reduce the time and cost associated with drug development. In general, a repurposing campaign starts with chemical matter with biological activity at a known human target. If the human target has a parasitic homologue, or if the parasite expresses other members of the same target family, the chemical matter is considered suitable for repurposing.
Any compound having anti-parasitic activity is then typically optimized to be selective for the parasitic over the human target.27, 28
31
Figure 1-6 illustrates the relationships between four types of repurposing strategies commonly used for NTD drug discovery. Typically, the repurposed chemical matter is a previously approved drug or compound in clinical trials. If no further optimization of this compound is required, the term “drug repurposing” is used. For example, eflornithine, originally developed as an anti-cancer compound, targets the same biosynthetic pathway in cancer cells and parasites and was found to be safe and efficacious against HAT without further structural modification.29
Figure 1-6. Common repurposing strategies used for NTD drug discovery. Image adapted from Klug et al. Bioorg. Med. Chem. Lett. 2016.27
If optimization is required, the terms “target repurposing” or “target class repurposing” apply. In the case of target repurposing, the known original target of the lead compound has a direct parasitic homologue, which enables target-based optimization strategies and the use of biochemical assays in addition to phenotypic screens. For example, N-myristoyltransferases
(NMTs) are expressed in humans, fungi, and Plasmodium falciparum, the parasite that causes malaria. Repurposing a Roche anti-fungal NMT inhibitor led to a structure-enabled medicinal
32 chemistry campaign which resulted in a compound that was effective against multiple Plasmodium species in vivo and displayed up to ~40-fold selectivity against mammalian cells.30-32
Target class repurposing campaigns, in contrast, do not have the advantages afforded by direct homology, although the general target family and probable mechanism of action are known.
Histone deacetylases (HDACs) comprise a target family that plays a key role in gene regulation, and therefore represent attractive targets for anti-cancer compounds.33 T. brucei expresses known
HDAC homologues which may be susceptible to human HDAC inhibitors.34, 35 Phenotypic screening of human HDAC inhibitors against T. brucei led to the discovery of belinostat, a Phase
III anti-cancer clinical candidate, as a lead anti-trypanosomal compound.34 Although the specific trypanosomal target is unknown, the target class repurposing approach was able to generate leads suitable for further development.
Finally, “lead repurposing” projects typically begin with a phenotypic high-throughput screen (HTS) of biased leads which target the same broad class of enzymes. For example, screening a ~3400-member library of general protease inhibitors led to the discovery of a series of compounds that were potent against T. brucei and selective over mammalian cells in addition to having desirable physicochemical properties.36 Optimization based on microsomal clearance resulted in a compound that was progressed to a murine efficacy model of T. brucei, although this further optimization series was ultimately discontinued due to toxicity concerns.36
1.3 Physicochemical properties and hit-to-lead optimization
Repurposing is an efficient way to generate hits for NTD drug discovery, but often compounds identified via repurposing efforts require further optimization to meet the TPP. Hit-to- lead optimization is the process by which hit compounds, which meet some minimal criteria, are developed into lead compounds, which meet more stringent criteria. One aspect of this is potency
33 optimization. For the projects discussed in this dissertation, percent parasite viability is measured as a function of compound concentration; the lower the concentration required to decrease parasite viability, the more potent the compound. This may be expressed as an EC50, the concentration of the compound required to reduce parasite viability by 50%; or by a pEC50. The relationship between the two is shown in Equation 1 where EC50 is expressed in molarity (M), such that an
EC50 of 1 μM corresponds to a pEC50 of 6. Similar calculations may be performed using mammalian cells to arrive at a TC50 (50% toxic concentration), or pTC50.
pEC50 = -log(EC50) (1)
In addition to biological activity, properties related to the ADME of compounds are monitored throughout the hit-to-lead optimization process. These parameters affect whether the compound gets to its target (absorption and distribution) and how long it remains in the body before it is cleared (metabolism and excretion). In lieu of measuring these parameters in vivo, which can be time-consuming and expensive, calculated physicochemical properties and in vitro assays can be used to indicate whether a compound will have a favorable ADME profile.37, 38 Those properties and assays relevant to the work presented in this dissertation are discussed in more detail below.
1.3.1 cLogP and lipophilic ligand efficiency (LLE)
Lipophilicity is the affinity a particular compound has for nonpolar environments and is usually expressed as a partition coefficient, which describes the extent to which a compound partitions into octanol (a nonpolar solvent) over water (a polar solvent) (Equation 2).
P = [octanol]/[water] (2)
When this parameter is calculated, it is termed clogP; higher clogP indicates a more lipophilic compound that partitions more extensively into octanol than water.39 Lipophilicity is a highly
34 important factor in drug design as it can influence both potency and ADME properties. Because protein binding pockets are typically more lipophilic than the surrounding aqueous environment, binding affinity to a protein target can often be improved by increasing the lipophilicity of the lead compound.40 On the other hand, highly lipophilic compounds can be poorly absorbed, poorly soluble, and highly metabolized; they are also more likely to be promiscuous binders, leading to undesired off-target effects.40 A balance between lipophilicity and potency is therefore desirable.
One way to assess this balance throughout hit-to-lead optimization is to monitor the lipophilic ligand efficiency (LLE, also called LipE)40, 41 of analogs produced. Calculated simply by subtracting a compound’s clogP from its pEC50 or pIC50 (Equation 3), this parameter gives an indication of whether potency is increasing or decreasing proportionally to lipophilicity.
LLE = pEC50 – clogP (3)
For optimization against a protein target, LLE has been shown to correlate with binding enthalpy.41
Even in cases where it cannot be directly related to binding enthalpy to a specific target, such as optimization campaigns based on phenotypic screening results, LLE is a useful metric. Evaluation of LLE helps chemists avoid undue investment in highly potent but highly lipophilic compounds, where potency may be largely due to unspecific hydrophobic interactions; and aids in the identification of compounds that take advantage of specific interactions with a protein target.
The relationship between LLE, logP, and pEC50 is illustrated in Figure 1-7. Compound A is in ideal LLE space, with high potency and low logP (LLE = 5.2). When comparing compounds
B, C, and D, at first glance B may look like the most attractive compound for development because of its high potency. However, when assessed in terms of LLE, compounds D and B are equally viable (LLE = 3.3), and compound C (LLE = 4.3) displays a more favorable LLE than both D and
35
B. As illustrated by this hypothetical example, LLE is quite useful for the simultaneous optimization of potency and properties.
Figure 1-7. LLE in relation to logP and pEC50.
1.3.2 CNS multiparameter optimization (CNS-MPO) score
Given that stage two of HAT is characterized by parasite penetration into the CNS, any successful treatment targeting trypanosomes must also be able to penetrate the CNS. In addition to the standard absorption and distribution challenges, CNS drugs must be able to cross BBB.42, 43
This barrier is formed by a layer of tightly joined cells that strictly regulate the substances that cross into and out of the brain.43 The differences between the BBB and other cell layers that drugs must penetrate (out of the gut and into the bloodstream, for example) mean that the physicochemical properties of orally available brain-penetrant drugs are different than those of drugs that are orally available but not brain-penetrant. Indeed, studies of marketed CNS drugs
36 show that these compounds are generally smaller and have fewer hydrogen bond donors (HBD) and lower topological polar surface areas (TPSA) than oral non-CNS drugs.44
In 2010, medicinal chemists at Pfizer published an analysis of CNS drugs and drug candidates and their physicochemical properties including clogP, clogD (a pH-dependent measure
45 of lipophilicity), molecular weight (MW), TPSA, HBD, and the pKa of the most basic center.
Based on these data, they identified ideal ranges for each parameter and developed a multiparameter optimization (MPO) scoring metric to help others assess whether compounds would be brain-penetrant based on predicted properties.45 Table 1-3 shows the range of values for each property of interest. For each property, a compound is assigned a score between zero (least desirable) and one (most desirable); the scores for each property are then summed for a total CNS-
MPO score between zero and six. According to this retrospective analysis, compounds with MPO scores greater than four are more likely to have high passive permeability across the BBB and low efflux liability; as well as being more likely to be metabolically stable and nontoxic.46
Table 1-3. Properties included in CNS-MPO score calculations and desired range for each. PF-03654746 and PF- 04447943 are Pfizer CNS candidates and are included as examples of how to calculate CNS-MPO scores.46
PF-03654746 PF-04447943 Property Score = 1 Score = 0 Value Score Value Score cLogP clogP ≤3 clogP >5 2.4 1.00 -1.5 1.00 cLogD clogD ≤2 clogD >4 0.0 1.00 -0.7 1.00 MW MW ≤360 MW >500 322.4 1.00 395.4 0.75 TPSA 40
The CNS-MPO metric is designed to allow flexibility in drug design by assigning scores based on a desirable range of values for multiple properties, rather than imposing hard cutoffs. As shown in Table 1-3, neither PF-03654746 nor PF-04447943 have a perfect score for every
37 property, but both have high overall CNS-MPO scores, met preclinical benchmarks, and were progressed to clinical trials. In addition, breakdown of the CNS-MPO score into its component parts can point chemists in the right direction for further optimization. For example, PF-03654746 has a pKa on the high end of the desirable range and modulating the basicity of the compound may be a reasonable strategy for this chemotype, while the MW and TPSA of PF-04447943 are both higher than desirable and tuning the polarity of the compound may be useful. Finally, the CNS-
MPO score is based on calculated or easily measurable properties and may therefore conveniently be used to prioritize chemical series or individual compounds for synthesis.
1.3.3 Aqueous solubility
The aqueous solubility of a small-molecule drug can greatly affect its in vivo performance, and poorly-soluble compounds can cause problems throughout drug development. In the hit-to- lead stage, poor aqueous solubility can lead to inconclusive results in biological assays; later in the process, it can affect the absorption and bioavailability of the compound, which in turn can affect the dosing and formulation.47-49 Solubility is, therefore, an extremely important parameter to consider even at early stages of hit-to-lead and lead optimization programs.
Insoluble compounds are generally grouped into those that display solvation-limited solubility (“greaseballs”) or solid state-limited solubility (“brick dust”).39, 50, 51 “Greaseballs” are hydrophobic compounds which do not interact with water and therefore are not incorporated into the solvent.39 “Brick dust” compounds, on the other hand, may have low clogP but display highly stable crystal structures which are not easily disrupted by solvent.39 In order to address the low solubility of a compound or series, it is important to distinguish between the two.
There are a number of proven strategies available to medicinal chemists wishing to improve the solubility of a compound of interest. The “greaseball” problem may be addressed by the
38 addition of solubilizing or polar groups, or the removal of lipophilic portions of a molecule.47, 51
“Brick dust” compounds may be made more soluble by the addition of functionality that disrupts the planarity of the compound; increasing the fraction of sp3-hybridized carbons (Fsp3); or removing intermolecular hydrogen bonds.47, 51, 52 Prodrugs may also be used to address either category of insoluble compound as they may include the addition of polar or ionizable groups, as well as functioning as a pendant group that could help break up the planarity of the parent compound.47, 50 Examples of these strategies as applied to a compound of interest for HAT, NEU-
1953, may be found in chapter 4 of this dissertation.
1.3.4 Other in vitro parameters
Other ADME assays routinely performed on compounds presented in this dissertation include clearance assays. Compound clearance rate and metabolic stability affect the concentration in the blood over time and the total exposure of the drug in vivo. Metabolic clearance enzymes in the liver decrease the amount of compound in the blood by chemically modifying the compound, marking it for excretion from the body. Cytochrome P450 (CYP) enzymes are responsible for oxidizing the parent drug (Phase I or first-pass metabolism); the oxidized compound is then conjugated with molecules such as glutathione (Phase II metabolism) and excreted.53 In order to predict in vivo clearance, in vitro assays using either human liver microsomes (HLMs) or rat hepatocytes were used. Microsomes are cell extracts that contain only CYPs and give an indication of oxidative metabolism; hepatocytes are whole cells, and as such contain the full complement of
53 metabolic enzymes. Table 1-4 indicates the HLM intrinsic clearance (Clint) and rat hepatocyte values corresponding to high, medium, and low clearance.
39
54 Table 1-4. High, medium, and low bins for HLM Clint and rat hepatocyte clearance.
High Medium Low HLM Clint (μL/min/mg) >47 8.6≤X≤47 <8.6 Rat hepatocyte (μL/min/106 >27.5 5.1≤X≤27.5 <5.1 cells)
1.4 High-throughput screening to generate leads for HAT
Utilizing a lead repurposing approach, and collaborating with GlaxoSmithKline (GSK) and the Consejo Superior de Investigaciones Cientificas (CSIC), our laboratory undertook an HTS of human kinase inhibitors against T. brucei, illustrated in Figure 1-8.55 Known human kinase inhibitors from the Published Kinase Inhibitor Set (PKIS) and GSK’s in-house compound collection were screened against T.b. brucei cells at a 4 μM concentration. Any compound that showed >50% inhibition of parasite growth was then characterized in a dose-response assay and tested against HepG2 cells, a human liver cancer cell line. Any compound with a pEC50 >6 and that was >100× selective over the HepG2 cells was included in the final hit set. These hits were then grouped by structural similarity into 59 clusters, and 53 singleton compounds were also identified.
Figure 1-8. HTS flow chart.
40
The hits were further characterized and prioritized based on multiple criteria. In addition to their performance in potency and toxicity assays, physicochemical properties of these compounds, such as molecular weight and clogP, as well as calculated metrics like LLE and CNS-
MPO scores, were considered. Hit compounds were also assessed in a rate-of-action assay and categorized as either fast-acting (achieved a pEC50 >6 in less than 6 hours), or slow-acting. Fast- acting compounds were progressed into a reversibility assay, which indicated whether they produced a trypanocidal or -static effect. Based on potency, selectivity, physicochemical properties, MPO score, rate of action, and cidality, clusters were assigned an overall score to aid in prioritization for further development. Optimization and follow-up on two high-priority clusters will be discussed in Chapters 2 and 3 of this dissertation.
1.5 Repurposing lapatinib for neglected diseases
We have also used a target class repurposing approach to NTD drug discovery. In 2013, we reported the results of a small screen of nine human epidermal growth factor receptor (EGFR) inhibitors against T. brucei.56 It is known that trypanosomes express essential kinases;57 further, that tyrosine phosphorylation occurs in the cell despite the lack of receptor tyrosine kinases
(RTKs).58-60 Additionally, there is evidence for proteins with “EGFR-like” domains in the parasite.61 We therefore reasoned that human RTKs, including EGFRs, would be a target class amenable to a repurposing strategy.
Lapatinib (Figure 1-9) displayed an EC50 of 1.54 μM against T. brucei and TC50 of >6 μM against HepG2 cells.56 The goal of the initial structure-activity relationship (SAR) campaign was to improve the potency and selectivity of this chemotype, and optimization was focused on exploration of the “head” (red) and “tail” (blue) regions of the molecule.56 Small changes to the headgroup were tolerated; indeed, the SAR around this region produced little variation in either T.
41 brucei potency or HepG2 toxicity. SAR exploration of the tail led to the discovery of NEU-617, a compound with both improved potency and selectivity. Further optimization focused on producing analogs with lower clogP and led to NEU-1953. However, despite its significantly lower clogP
(2.1 vs. 7.3), this compound did not show a similarly dramatic improvement in aqueous solubility.
Alternative strategies to improve the aqueous solubility of these compounds were then pursued; solubility-driven optimization of this series will be discussed in Chapter 4 of this dissertation.
Figure 1-9. Regions of lapatinib targeted for SAR exploration.
42
Chapter 2: Hit-to-Lead Optimization of Benzoxazepinoindazoles
2.1 Characterization of HTS hits
One of the overall highest-scoring clusters identified through a kinase-targeted high- throughput screen against T. brucei55 (described in detail in section 1.4 of this dissertation) comprised a series of substituted benzoxazepinoindazoles (BOXIs). Structural features of interest for this cluster are highlighted in Figure 2-1 and include a benzene head, an oxazepinoindazole core, and a heterocyclic tail. As shown in Table 2-1, the structures of the three HTS hits NEU-
1117, -1118, and -1119 differ only in the substituents on the tail group.
Figure 2-1. Structural features of the BOXI cluster. The headgroup is highlighted in pink, the core in blue, and the tail in yellow.
Table 2-1 shows the targeted property goals, along with the cluster average, and individual values for properties of interest. Fast-acting compounds are defined as those that achieve a pEC50
>6 in less than 18 hours, and cidal compounds are defined as those that kill T.b.b. cells rather than exhibiting a growth-inhibitory effect. Physicochemical properties such as clogP, TPSA, and molecular weight, as well as the LLE and CNS-MPO scores of the hit compounds, are also included (for a more in-depth discussion of physicochemical properties, LLE, and CNS-MPO scores, see section 1.3 of this dissertation).
43
In general, the compounds in this cluster are highly potent against T.b.b. with an average pEC50 of 7.6; all three hits highlighted in Table 2-1 have pEC50s >8 and do not display significant toxicity against HepG2 cells. LLEs and MPO scores are high for this cluster as well. Additionally, all three hits are fast-acting and two of the three exhibited trypanocidal effects. However, these compounds are not without room for optimization. Their physicochemical properties tend to be outside the desired ranges, with high clogP and TPSA. Additionally, ADME properties such as aqueous solubility and plasma protein binding (PPB) need improvement; in particular, the aqueous solubilities of these BOXIs are quite low.
Table 2-1. Targeted, cluster average, and individual cluster member values for properties of interest.
Targeted Value Cluster Average NEU-1117 NEU-1118 NEU-1119
T.b.b. pEC50 ≥7 7.6 8.1 8.9 8.4
HepG2 pTC50 ≤pEC50 – 2 4.8 5.1 5.5 4.0 cLogP ≤3 3.9 2.2 3.7 3.7 TPSA (Å2) 40
44
Given that all the compounds included in the HTS were originally designed to be human kinase inhibitors, we assessed the human kinase activity of a cluster representative. Table 2-2 shows the results of this screen against NEU-1328, a related BOXI, against a small panel of human kinases. This compound shows potent activity against five human kinases included in the panel,
IKK1, SYK, AurB, JAK3, and LRRK2. Although we did not have structure-activity relationship data related to the human kinase activity of this compound, we became aware that human kinase activity was a potential liability of this chemotype.
Table 2-2. Kinase activity of NEU-1328.
Kinase pIC50 Kinase pIC50 IKK1 7.9 ROCK1 6.9 SYK 7.2 AurB 7.5 p38a 1 JAK3 7.4 JNK1 6.4 JAK2 6.3 ITK 1 EGFR 1 LCK 5.5 LRRK2 7.9 BTK 1 PI3K-α 1 IKK2 6.2
2.2 Analog synthesis
2.2.1 Synthesis of BOXIs and related cores
The synthesis of substituted BOXIs is shown in Scheme 2-1. Converting 4-bromo-2,6- difluorobenzoic acid 2-1 to the corresponding acid chloride 2-2 enabled a subsequent amide coupling with 2-aminophenol to provide amide 2-3. This compound was cyclized with potassium carbonate, yielding benzoxazepine 2-4, which was then converted to the thioamide 2-5 using
Lawesson’s reagent. The indazole ring was formed by treatment of 2-5 with hydrazine to yield the
45 brominated BOXI core 2-6. This compound was treated with acetic acid to yield the protected core
2-7, which was converted to the boronic ester 2-8 under Miyaura conditions. Subjecting 2-8 to
Suzuki conditions using various desired aryl halides yielded final compounds 2-9a-s, which were deprotected under the Suzuki conditions.
Scheme 2-1. Synthesis of tail replacement analogs.
Reagents and reaction conditions: a) SOCl2, 75 °C, 4 h. b) 2-aminophenol, TEA, DCM, 0 °C to rt, 12 h (87%). c) K2CO3, DMF, rt, 36 h (72%). d) Lawesson’s reagent, toluene, 100 °C, 12 h (68%). e) Hydrazine, dioxane, 85 °C, 3 h (89%). f) Acetic anhydride, 100 °C, 3 h (93%). g) B2(pin)2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145 °C, μw, 1 h (65%). h) Aryl halide, K2CO3, Pd tetrakis, 3:1 dioxane:water, 100 °C, 4 h (10-74%). Where no yield is reported, crude material was progressed without further purification.
2.2.2 Oxazepinoindazole synthesis
Synthesis of the oxazepinoindazole core (Scheme 2-2) presented a significant synthetic challenge. Reaction of 2-2 with ethanolamine yielded the desired amide 2-10a, but subjection of this intermediate to the same conditions used to make intermediate 2-4 failed to produce the desired oxazepine 2-12, and instead led to the dimer macrocycle 2-18.62 In subsequent reactions, dilution of the reaction mixture disfavored intermolecular macrocyclization but still did not lead to the
46 formation of the desired product, resulting only in recovered starting material. Finally, the incorporation of a para-methoxybenzyl (PMB) protecting group on the amide 2-10b did yield the desired product 2-11, likely because the inclusion of this bulky protecting group forced the molecule into a conformation that favored formation of the oxazepine ring by restricting rotation around the amide bond.
Deprotection of the oxazepine ring directly following cyclization allowed the use of
Lawesson’s reagent to convert amide 2-12 to thioamide 2-13; if the deprotection was performed as the last step the much more aggressive thionating agent P2S5 was required, as Lawesson’s reagent could not access the sterically hindered protected amide. Subsequent transformations proceeded in an analogous manner to those required to make the benzoxazepine core, with thioamide 2-13 converted to oxazepinoindazole 2-14 using hydrazine. This reaction was followed by acetylation and conversion of the bromide 2-15 to the boronic ester 2-16 through a Miyaura borylation using bis(pinacolato)diboron (B2(pin)2). Finally, a Suzuki reaction of this intermediate with the desired chloropyrimidines yielded the desired products 2-17a-b.
47
Scheme 2-2. Synthesis of oxazepinoindazole core.
Reagents and reaction conditions: a) SOCl2, 75 °C, 4 h. b) 2-((4-Methoxy-benzyl)amino)ethan-1-ol or ethanolamine, TEA, DCM, 0 °C to rt, 12 h (54%). c) NaH, DMF, rt, 12 h (96%). d) CAN, 3:1 ACN:water, rt, 3 h (36%). e) Lawesson’s reagent, toluene, 100 °C, 12 h (56%). f) Hydrazine, dioxane, 85 °C, 3 h (95%). g) Acetic anhydride, 100 °C, 3 h (60%). h) B2(pin)2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145 °C, μw, 1 h (79%). i) Aryl halide, K2CO3, Pd(PPh3)4, 3:1 dioxane:water, 100 °C, 4 h 12-(34%). Where no yield is reported, crude material was progressed without further purification.
2.2.3 Indazole and aminoindazole synthesis
The synthesis of indazole and aminoindazole core analogs is shown below in Scheme 2-3.
Indazoles could be readily synthesized by converting 6-bromo-1H-indazole 2-19 to the corresponding boronic acid pinacol ester using Miyaura borylation conditions, followed by a
Suzuki coupling with the desired aryl halide to produce final products 2-26a-c.
Substituted aminoindazoles were constructed from 4-bromo-2,6-difluorobenzonitrile 2-20.
Reaction of this compound with LiHMDS and the desired alcohol produced alkoxy intermediates
2-21a-h, which were then transformed to the aminoindazoles 2-22a-h by reaction with hydrazine.
Before the final Suzuki-Miyaura coupling, these intermediates required protection with either an acyl or a Boc group. In some cases, these intermediates were taken forward as a mixture of the
48 mono- and di-acetylated product; both compounds would undergo the Suzuki-Miyaura sequence and subsequently be fully deprotected. Protected intermediates 2-23a-h were then subjected to
Miyaura conditions to produce boronic esters 2-25a-h. The corresponding boronic acids were also observed under these reactions; therefore, these intermediates were not isolated but taken forward as crude material. This crude material was then coupled with desired aryl halides to produce final products 2-27a-h, which were globally deprotected under the conditions of the Suzuki reaction or using HCl in methanol.
Aminoindazole analogs were synthesized starting a Suzuki coupling between the commercially available (4-cyano-3-fluorophenyl)boronic acid 2-28 and the desired aryl halide.
Reaction of the Suzuki product 2-29a-c with hydrazine closed the indazole ring to successfully produce analogs 2-30a-c.
Scheme 2-3. Synthesis of indazole, aminoindazole, and ring-opened analogs.
Reagents and reaction conditions: a) Alcohol, LiHMDS (1 M in THF), THF, 0 °C to rt, 2 days (55-86%). b) Hydrazine, EtOH, 95 °C, 12 h (71-87%). c) Boc2O, DMAP, DCM, rt, 12 h (75%); or Ac2O, pyridine, 100 C, 3.5 h (24-75%). d) B2(pin)2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145 °C, μw, 30 min – 1.5 h. e) Aryl halide, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 150 °C, μw 30 min, (6-26%). f) Aryl halide, NaHCO3 (sat. aq.), Pd(PPh3)4, dioxane, 95 C, 3 h (43-97%). Where no yield is reported, crude material was progressed without further purification.
49
2.3 Structure-activity and -property relationships
2.3.1. Tail group analogs
The biological activity data of all tail replacement analogs (compounds 2-9a-t) is shown in
Tables 2-3 and 2-4. None of these replacements are as potent as any of the three original hits,
NEU-1117, -1118, or -1119. The most active compounds are those with small aliphatic substituents at the 6-position of the pyrimidine ring of the tail, such as NEU-1329, -1330, -1331, or -4898, which all maintain a pEC50 >7.5. Larger substituents such as those of NEU-1333, -1334, and -4457 are reasonably well-tolerated as long as they include a secondary amine, but no analogs incorporating tertiary amines at this position (NEU-4379, -4380, -4392, -4458, and -4931) achieve a pEC50 >6.3. Replacement of the 2-aminopyrimidine motif with other pyrimidines (NEU-2101,
-2121, -2122, and -2197) results in compounds with acceptable pEC50 values, in the 6-7 range.
Finally, replacement of the heterocyclic tail with a phenyl ring as in NEU-2100 results in a loss of activity, as does excision of the tail altogether (NEU-1336).
50
Table 2-3. Biological activity and LLE of 2-aminopyrimidine tail replacement analogs.
T.b.b. HepG2 T.b.b. HepG2 ID NEU- R LLE ID NEU- R LLE pEC50 pTC50 pEC50 pTC50 2-9a 1330 7.7 5.3 3.2 2-9b 4379‡ 6.3 4.8† 3.2
2-9c 4898‡ 7.8 4.6† 5.1 2-9d 4457‡ 6.9 <4.3† 3.7
2-9e 4392‡ 6.1 4.7† 2.7 2-9f 4458‡ 4.8 <4.3† 0.91
2-9g 4380‡ 6.3 <4.3† 3.4 2-9h 4931‡ 5.7 <4.3† 2.3
2-9i 1329* 7.9 4.8 3.2 2-9j 1331* 7.7 4.6 3.1
2-9k 1334* 7.2 5.0 0.85 2-9l 1333* 7.5 5.3 0.66
*Compound from GSK, not resynthesized. †Data obtained against MRC5 cells, rather than HepG2 cells. ‡Synthesized by L. Tschiegg. All SEM within ± 0.11.
51
Table 2-4. Biological activity and LLE of other tail replacement analogs.
T.b.b. HepG2 ID NEU- R LLE pEC50 pTC50
2-9m 1332 7.2 4.8 3.5
2-9n 2101 6.6 <4.3† 3.5
2-9o 2103 6.9 <4.3† 4.2
2-9p 2122 6.7 <4.3† 3.6
2-9q 2197 6.9 nd 4.8
2-9r 2100 5.7 <4.3† 1.5
2-9s 1336* H 6.2 4.0 2.1 *Compound from GSK, not resynthesized. †Data obtained against MRC5 cells, rather than HepG2 cells. ‡Synthesized by L. Tschiegg. All SEM within ± 0.20.
The rate of action of several tail replacement analogs that achieved a pEC50 >6 was assessed; the results are shown in Figure 2-2. Panel A shows the rate-of-action curves of three slow-acting compounds, NEU-2103, -2123, and -2197, which need >18 hours incubation to achieve a pEC50 >6. Panel B shows the rate-of-action curves for three fast-acting compounds,
NEU-4457, -4460, and -4461. Interestingly, all fast-acting analogs contain the 2-aminopyrimidine tail group, with substitution patterns differing elsewhere in the molecule, while the slow-acting
52 compounds do not. This evidence supports the conclusion that a 2-aminopyrimidine tail group is necessary to achieve a fast rate of action.
Figure 2-2. Rate of action of selected analogs. Slow-acting compounds are shown in red and fast-acting compounds in blue. Dotted gray lines show the cutoff of pEC50 of 6 at 18 hours incubation time. All SEM within ±1.1.
The aqueous solubility and clogP of the tail group analogs are shown in Figure 2-3, with green dots representing highly potent compounds and red dots representing inactive compounds. The targeted aqueous solubility of >10 μM is shown by the dotted gray line. Only two tail group analogs achieve a solubility that exceeds the target; these are NEU-4931, which incorporates a piperidine substituent, and NEU-1336, which excises the tail group altogether. The aqueous solubility of this
53 series does not appear to be correlated with clogP; all other analogs, regardless of lipophilicity, are largely insoluble. Disappointingly, neither of the soluble analogs achieve a pEC50 >7.
Figure 2-3. ClogP versus aqueous solubility of BOXI tail group analogs.
Other ADME properties of NEU-1118, -1336, and -4931, the compounds highlighted in
Figure 2-3, are shown in Table 2-4. Both NEU-1336 and -4391 have higher clogP and LogD than
NEU-1118, yet NEU-1118 is the least soluble of the three compounds. PPB is generally high for the BOXIs and is >95% for all three compounds highlighted in Table 2-4. Human liver microsome
(HLM) and rat hepatocyte clearance (Clint) is in the low to mid-range values for these compounds
(see section 1.3.4 of this dissertation for high/medium/low clearance bins). Therefore, after
54 extensive tail group structure-activity relationship (SAR) exploration, aqueous solubility remained the major problem for this series.
Table 2-5. ADME properties of selected analogs.
T.b.b. Aq. sol. HLM Clint Rat Hepatocyte NEU- cLogP LogD PPB (%) pEC50 (μM) (μg/min/mg) Clint (μg/min/mg) 1118 8.9 2.6 2.1 4† 97 nd nd 1336 6.8 2.9 4.1 19 >99 7.6 47 4931 5.7 2.4 3.4 26 97* 41 16 †Kinetic aqueous solubility. *Predicted PPB. nd = no data.
2.3.2 Core modifications
After establishment of the SAR of the tail region, modifications to the head and core of the
BOXIs were explored. As it is common for kinase inhibitors to rely on a hydrogen bond donor- acceptor-donor motif,63 we reasoned that changing the position of available hydrogen bond donors
(HBDs) was a possible strategy to achieve selectivity over human kinases. Table 2-6 shows the biological activity of a series of N-methylated analogs which systematically eliminate HBDs from the molecule. Methylation of the indazole -NH (NEU-4461) and monomethylation of the tail amine (NEU-5126) are reasonably tolerated with pEC50s >7; however, methylation of the core -
NH (NEU-4389) and demethylation of the tail amine (NEU-4404) are not and lose almost three log units of activity as compared to the parent compound NEU-1119.
55
Table 2-6. Biological activity of N-methylated analogs. Position of methyl group confirmed by NMR spectroscopy when necessary (Appendix 4).
T.b.b. MRC5 NEU- R1 R2 R3 LLE pEC50 pTC50
4389 CH3 H NH2 5.1 <4.3 2.3
4461 H CH3 NH2 7.6 <4.3 4.9
4404 H H N(CH3)2 5.3 <4.3 1.9
5126 H H NHCH3 7.0 <4.3 3.8 All SEM within ± 0.08.
In an attempt to improve the aqueous solubility and clogP of the series, the benzene headgroup was targeted as a probable contributor to the high lipophilicity of these compounds.
Table 2-7 shows the biological activity, aqueous solubility, and clogP for a series of headgroup truncations and replacements. Truncation of the core to an oxazepinoindazole (NEU-2586 and -
2587), aminoindazole (NEU-4390, -4392, and -4361), or indazole (NEU-2209, -2198, and -2208) resulted in a complete loss of activity against T.b.b. However, the solubility of these compounds was consistently improved – drastically so in the case of the indazoles. Additionally, because of their reduced clogP values, these compounds maintained LLE values comparable to those of the parent compounds, despite their greatly reduced activity. These data point to the benzene headgroup as a significant contributor to lipophilicity and poor solubility, without a disproportionately beneficial binding interaction.
In order to marry the high potency of the original hit compounds with the aqueous solubility of the truncated analogs, core and headgroup modifications were made that incorporated elements designed to increase solubility while filling the space occupied by the benzene ring in the BOXI core. Removal of the indazole core (NEU-4892) resulted in a loss of activity, but incorporation of saturated groups (NEU-4895) or heterocycles (NEU-1327) in place of the benzene ring maintained the activity of the original hits. However, none of these compounds resulted in a substantial
56 increase in aqueous solubility. Interestingly, the substituted aminoindazole NEU-1335 showed increased activity over the other truncated analogs we had previously investigated. Taken together, these data reinforce the idea that filling the space occupied by the headgroup of the original BOXIs is a requirement for potent anti-trypanosomal activity.
Table 2-7. Biological activity and selected ADME data for headgroup truncation and replacement analogs.
T.b.b. MRC5 Aq. sol. Core R ID NEU- LLE cLogP pEC50 pTC50 (μM)
2-17a 2586 4.8 <4.3 4.1 65 0.61
2-17b 2587 5.4 <4.3 4.7 nd 0.76
2-30a 4390 4.8 <4.3 4.2 58 0.64
2-30b 4362 4.8 <4.3 4.0 5 0.79
2-30c 4361 5.5 <4.3 4.6 <13 0.87
2-26a 2209 5.3 <4.3 3.9 660 1.4
2-26b 2198 4.3 <4.3 2.8 330 1.5
2-26c 2208 5.2 <4.3 3.6 220 1.6
-- 4892 5.9 <4.3 3.4 0.3 2.6
57
-- 4895 7.1 <4.3 5.3 13 1.8
-- 1327* 8.2 5.9† 5.1 10† 1.4
-- 1335 6.2 4.0† 5.2 >100† 0.71
* Compound from GSK, not resynthesized. †Data provided by GSK; HepG2 toxicity and kinetic aqueous solubility. All SEM within ± 0.19.
2.3.3. Ring-opened analogs
Having shown first, that truncation of the BOXI core to smaller heterocycles or ring- opened forms increased the compounds’ aqueous solubility, and second, that filling the space occupied by the benzene headgroup of the BOXI core led to increased potency, a series of ring- opened analogs were synthesized with various alkyl substituents designed to meet these two requirements simultaneously. While it was not expected that these compounds would be as potent as analogs containing the full head and core motif, this seemed a reasonable strategy to increase the potency of compounds like NEU-1335 while maintaining its favorable ADME properties.
Table 2-8 shows the biological activity and aqueous solubility of these ring-opened analogs. Substituting the amine at R2 with either a phenyl (NEU-4894) or a benzyl (NEU-4962) group marginally improved potency over the unsubstituted NEU-4361; however, these compounds were not soluble and their LLEs were low. Installing small alkoxy groups at R1 consistently improved potency compared to NEU-4361. When considering both potency and LLE, the methoxy
(NEU-1335), ethoxy (NEU-5388), and cyclobutoxy (NEU-5390) substituents performed best, while the phenyl (NEU-5087) and benzyl (NEU-5065) compounds had reduced LLE and the
58 phenyl analog showed some toxicity against mammalian cells. Although none of these compounds recovered the potency observed with the full BOXI core, they were an improvement over the indazoles and aminoindazoles in that respect while maintaining excellent aqueous solubility.
Substituted amines at this position (NEU-5079 and -5068) had a similar effect.
We then wondered if the potency could be further improved by installing R1 substituents that included a hydrogen bond acceptor. We hypothesized that forming an intramolecular hydrogen bond between the R1 substituent and the amine at R2 would favor a conformation that would mimic the orientation of the benzene ring on the BOXI core and provide a further increase in potency. To that end, the tetrahydropyran (NEU-5901 and -5934), tetrahydrofuran (NEU-5899) and oxetane
(NEU-5935) analogs were synthesized. Disappointingly, these compounds did not show improved potency over the alkoxy substituents, although they were highly soluble and had excellent PPB,
HLM Clint, and rat hepatocyte clearance values (data not shown).
59
Table 2-8. Biological activity and selected ADME data for ring-opened analogs.
T.b.b. MRC5 ID NEU- R1 R2 LLE Aq. sol. (μM) cLogP pEC50 pTC50 2-30c 4361 -H -NH2 5.5 <4.3 4.4 <13 0.87 -- 4894 -H -NHPh 5.4 <4.3 2.1 3 3.1 -- 4962 -H -NHBn 6.1 <4.3 2.2 17 2.9 † † -- 5098* -OH -NH2 5.6 <5.0 6.3 400 -0.79 2-27a 1335 -OMe -NH2 6.2 <4.3 5.2 200 0.71 2-27b 5388 -OEt -NH2 6.4 <5.0 5.3 860 1.1 † † 2-27i 5074* -OiPr -NH2 6.4 <5.0 4.9 350 1.5 2-27c 5389 -OtBu -NH2 6.2 4.6 4.4 68 1.8 2-27d 5390 -OcyBu -NH2 6.6 <5.0 5.0 nd 1.6 † † 2-27j 5087* -OPh -NH2 5.7 5.7 3.3 120 2.4 † † 2-27k 5065* -OBn -NH2 5.9 <5.0 3.4 85 2.4 † † -- 5079* -NMe2 -NH2 6.0 <5.0 5.0 310 0.98 † † -- 5068* -NMeEt -NH2 6.0 <5.0 4.6 380 1.3
2-27e 5901 -NH2 5.8 <4.3 5.1 770 0.68
2-27f 5934 -NH2 6.1 <4.3 5.0 1000 1.1
2-27g 5899 -NH2 5.5 <4.3 4.9 750 0.62
2-27h 5935 -NH2 5.7 <43 5.1 380 0.56
*Compound from GSK, not resynthesized. †Data provided by GSK; HepG2 toxicity and kinetic aqueous solubility. All SEM within ± 0.16.
2.4 Further studies
The overall profile of BOXI analogs was continuously assessed in order to identify compounds with an acceptable combination of high activity and a favorable ADME profile. Heat maps including activity, LLE, MPO score, and various ADME parameters of four compounds are shown in Table 2-9. NEU-1117, an original HTS hit, showed high potency, but moderate to poor
ADME properties, including a high logD, low aqueous solubility, high PPB, and high intrinsic
60 clearance in both HLMs and rat hepatocytes. Additionally, it displayed slight toxicity against
MRC5 cells. The structurally-similar NEU-4461 addressed the toxicity issues of NEU-1117 and had a similar overall profile. Although NEU-4985 was slightly less active than either NEU-1117 or NEU-4461, it had improved logD, aqueous solubility, and PPB. However, this compound was so highly metabolized that the parent compound was not detected in the HLM assay. Finally, NEU-
5388 was less active than the other three analogs but had the best ADME profile of the four compounds assessed here. Given the high potency and low toxicity of NEU-4461 and the excellent
ADME profile of NEU-5388, both compounds were advanced into pharmacokinetic (PK) studies; the high in vitro clearance of NEU-4985 (both HLM and rat hepatocyte) eliminated it from consideration for advancement.
Table 2-9. Heat maps of NEU-1117, NEU-4461, NEU-4985, and NEU-5388.
Targeted NEU-1117 NEU-4461 NEU-4985 NEU-5388 Value T.b.b. pEC50 >7.5 8.1 7.6 7.1 6.4 MRC5 pTC50 <5 5.1 4.3 4.3 4.3 MW ≤360 360 330 308 270 cLogP ≤3 2.6 2.7 2.0 1.1 LogD (7.4) ≤2 >4 >4.3 2.8 1.7 LLE ≥4 5.5 4.9 5.1 5.3 MPO Score ≥4 3.9 3.9 4.2 4.1 Aq. sol. (μM) >10 1 3 13 860 HLM Clint <9 24 110 nd* 25 (μg/min/mg) Rat Hepatocyte <5 41 12 52 20 Clint (μg/min/mg) PPB (%) ≤95 >99 nd 88 89 nd = no data. *Compound was too highly cleared for detection.
2.4.1 Pharmacokinetic studies
Shown in Table 2-10 and Figure 2-4 are the results of PK studies of NEU-4461 and NEU-
5388. At a 10 mg/kg dose, both compounds achieve a maximum concentration (Cmax) well in
61 excess of their EC50s (68x and 37x, respectively) within 0.5 hours of dosing. However, their half- lives (t1/2) are short: approximately one hour for both compounds. As shown in Figure 2-4, NEU-
4461 is cleared completely after four hours, while NEU-5388 is cleared within two. In the brain,
NEU-4461 achieves a concentration approximately double that of the blood concentration, while the blood/brain ratio of NEU-5388 is approximately 0.2. Taken together, these data suggest a more favorable PK profile for NEU-4461 than for NEU-5388.
Table 2-10. PK parameters for NEU-4461 and NEU-5388.
T.b.b. EC50 Brain/blood Brain/blood NEU- Cmax (ng/ml) tmax (h) t1/2 (h) (ng/ml) ratio (t = 0.5 h) ratio (t = 4 h) 4461 8.9 615 ± 85.8 0.25-0.5 1.06 ± 0.235 2.39 ± 1.12 2.12 ± 0.52 5388 96.2 3603 ± 636 0.25-0.5 0.893 ± 0.500 0.155 ± 0.021 0.214
Figure 2-4. Brain and blood concentrations of a) NEU-4461 and b) NEU-5388 over time after a 10 mg/kg intraperitoneal (ip) dose. a) b)
Green triangle = Peripheral blood concentration (ng/ml); n = 3 mice. Red circle = blood concentration (ng/ml), blue square = brain concentration (ng/g); n = 3 mice.
2.4.2 Kinase panel assessment
In addition to PK parameters, the human kinase selectivity of NEU-4461 and NEU-5388 was assessed; the results are shown in Figure 2-5. Both compounds were tested at a 1 μM concentration against a panel of human kinases, and the percent inhibition is reported. Kinases that
62 showed >50% inhibition at 1 μM were considered as potently inhibited by the compound, where
>30% inhibition was considered moderate. NEU-5388 shows potent inhibition against 18 kinases and moderate inhibition against an additional 10. In contrast, NEU-4461 potently inhibits only three kinases and moderately inhibits an additional two.
Figure 2-5. Percent inhibition of a human kinase panel for NEU-4461 (blue) and NEU-5388 (green). Compounds were tested at a 1 μM concentration.
2.4.3 In vivo efficacy study
Based on its potency, favorable PK results, and performance against the kinase panel,
NEU-4461 was selected for progression into a murine model of stage 1 HAT. A highest tolerated dose study of NEU-4461 showed no signs of toxicity up to 30 mg/kg/day ip dosing. Figure 2-6 shows the % survival of mice treated with vehicle (closed circles) versus NEU-4461 at 10 mg/kg/day (open circles). As shown, treatment with NEU-4461 once a day for five days effectively
63 extends the life of infected mice for up to two weeks post-infection as compared to vehicle-treated mice. This study is currently being repeated at 30 mg/kg/day with the goal of reducing parasitemia to undetectable levels on day 30.
Figure 2-6. % Survival of mice treated with vehicle and NEU-4461 at 10 mg/kg/day ip.
2.5 Summary and future work
Shown in Figure 2-7 is a summary of the SAR for this chemical series. Through initial exploration of the tail group (yellow), we discovered that 2-aminopyrimidines were the superior heterocycles, consistently resulting in potent, fast-acting compounds. Small aliphatic groups, ethers, and primary amines were tolerated at R1 but larger groups and secondary amines typically resulted in a loss of potency. The benzene head group (pink) contributed to the high potency of the original hits but also contributed to their high lipophilicity. Attempts to replace this moiety with heterocycles or saturated rings resulted in potent but equally insoluble compounds.
64
Finally, the core (blue) was explored. Methylation of R2 showed an approximately 10-fold loss in potency but the resulting compound, NEU-4461, showed improved selectivity over human kinases. Ring-opened cores typically lost 2-3 log units in potency, but consistently displayed improved solubility. Aminoindazoles substituted with various alkoxy groups at the 4-position recovered some activity over their unsubstituted counterparts and resulted in the compounds with the best overall ADME profiles to date for this cluster.
Figure 2-7. SAR summary of the BOXI cluster.
Based on this work, two compounds, NEU-4461 and NEU-5388, were selected for further evaluation. Because of its superior PK profile, including evidence of brain penetration, and cleaner kinase selectivity, NEU-4461 was selected for an in vivo efficacy study. Based on preliminary results dosing with 10 mg/kg/day, this compound is promising as a potential anti-HAT therapeutic.
Further in vivo studies will be undertaken to assess the ability of this compound to cure stage one
HAT, as well as establish its efficacy against stage two. Characterization of NEU-4461 against
CYP450, hERG, and genetic targets should also be undertaken in order to assess its potential for
65 drug-drug interactions, cytotoxicity, and genotoxicity. In addition, the identification of the trypanosomal target of NEU-4461 could be achieved by incorporating click-chemistry-enabled tags onto the molecule.
As a backup series, further development of the ring-opened scaffold should focus on fixing the poor brain penetration and high promiscuity among human kinases of NEU-5388. Both of these problems could potentially be addressed by blocking one or more HBDs of the scaffold.
NEU-5388 has five HBDs; the CNS-MPO calculation gives the highest score to compounds with less than one. Additionally, the kinase selectivity data of NEU-4461 suggests that blocking the
HBD of the indazole core could improve the overall kinase inhibition profile of this scaffold.
Disubstitued aminoindazoles 2-36 (Scheme 2-4) are of interest for a number of reasons.
First, they maintain the 4-alkoxyaminoindazole scaffold which greatly improved solubility and other ADME properties over the original BOXI core. Second, the additional substitution at R2 accomplishes the goal of removing a HBD while installing a second moiety that would better fill the space of the benzene ring of the original core. Ideally, addition of this second substituent would further improve the potency of compounds such as NEU-5934, a moderately potent analog with an excellent ADME profile. Finally, if sufficient activity is achieved with the disubstituted scaffold, a methyl group may be installed at R3 to further mitigate the off-target human kinase activity of this chemotype.
Shown in Scheme 2-4 is a synthetic route to these compounds that we have already begun to explore. Starting with benzoic acid 2-1, an amide coupling using HBTU installs the desired R2 substituent in good yield to afford intermediate 2-31. The R2 alkoxy group is then installed by substitution on one of the fluorines, which is followed by conversion to the thioamide 2-33.
Installing the aminoindazole group by reaction with hydrazine goes smoothly when using the
66
BOXI core; however, on the ring-opened analogs this reaction is extremely slow and does not proceed to completion. Preliminary results suggest that running this reaction under microwave conditions at higher concentrations may push the reaction to completion in a shorter amount of time. Construction of the aminoindazole core 2-34 is followed by a Miyaura borylation and a
Suzuki reaction to afford final compounds 2-36. Desired amines, alcohols, and substituted hydrazines make variation at R1, R2, and R3 possible using this synthetic route, which will likely be of interest for future work on this cluster.
Scheme 2-4. Synthesis of disubstituted aminoindazoles.
67
Chapter 3: Hit-to-Lead Optimization of 3,5-Disubstituted-7-Azaindoles
3.1 Characterization of HTS hits
Our kinase-targeted HTS against T. brucei (discussed in chapter 1 of this dissertation) revealed a group of 3,5-disubstituted-7-azaindoles which had good activity against T. brucei and desirable physicochemical and ADME profiles. Table 3-1 shows the potency and properties of three HTS hits, NEU-1207, -1208, and -1209, as compared to the cluster average and targeted values. NEU-1207 and -1208, with their smaller substituents, have better overall profiles than
NEU-1209, which includes a long aliphatic chain on the 3-position substituent. These two compounds are potent, nontoxic, fast-acting and trypanocidal, and have clogP values, molecular weights, and TPSAs within or just outside of the targeted range. In addition, their CNS-MPO scores indicate they are likely to be brain penetrant. All of these qualities suggested that this cluster would be a good candidate for further optimization.
68
Table 3-1. Targeted, cluster average, and individual cluster member values for properties of interest.
Targeted Value Cluster Average NEU-1207 NEU-1208 NEU-1209
T.b.b. pEC50 ≥7 6.4 7.0 7.2 6.5
HepG2 pTC50 ≤pEC50 – 2 4.2 4.5 5.0 4.0 cLogP ≤3 4.22 2.6 2.8 2.2 TPSA (Å2) 40
Due to its excellent overall profile, as highlighted in Table 3-1, NEU-1207 was progressed to a mouse PK study, the results of which are shown in Figure 3-1a. At a 5 mg/kg oral dose, the compound was cleared from all three mice within six hours. In order to better understand the high clearance of this compound, metabolites of NEU-1207 were predicted computationally by our collaborators at GSK Tres Cantos using MetaSite software. The structures of the two most likely metabolites are shown in Figure 3-1b. Both compounds are the products of oxidation at the substituent on either the 3-position of the 5-position of the 7-azaindole core.
69
Figure 3-1. a) Blood concentrations of NEU-1207 over time after a 5 mg/kg oral dose (n = 3 mice). b) Predicted metabolites of NEU-1207 (Maria Santos, GSK Tres Cantos). a) b)
Thus, although NEU-1207 had a generally favorable overall profile, the PK study revealed
clearance to be a weakness of this compound. In early-stage drug discovery, an in vitro human
liver microsome intrinsic clearance (HLM Clint) is used to predict the likely rate of in vivo clearance
(see section 1.3.4 of this dissertation for a more detailed discussion of clearance). The HLM Clint
of NEU-1207 is extremely high at 191 μg/min/mg; the targeted value for this assay is <9
μg/min/mg and intermediate values are between 9 and 47 μg/min/mg. In optimizing this
chemotype, we therefore looked at both structure-activity relationships and structure-property
relationships, monitoring HLM Clint throughout.
3.2 Analog synthesis
3.2.1 Synthesis of 3-position analogs
We first targeted replacements of the benzonitrile substituent at the 3-position of the 7-
azaindole in an attempt to block the formation of metabolite B (Fig. 3-1). Later, we also sought to
modify the physicochemical properties of the series by changing the substituent at this position.
Synthesis of these analogs (Scheme 3-1) began with a Suzuki coupling between 5-bromo-7-
azaindole 3-1 and 1-methyl-4-pyrazoleboronic acid pinacol ester to produce intermediate 3-2. An
iodide substituent was installed at the 3-position using NIS, yielding intermediate 3-3. Previous
70 efforts at Suzuki couplings at this position using intermediate 3-3 were either unsuccessful or low- yielding;64 however, the incorporation of the electron-withdrawing tosyl group enabled successful
Suzuki couplings using intermediate 3-4.
Installation of the N-methylpyrazole at the 5-position at the beginning of the synthesis enabled late-stage diversification at the 3-position from advanced intermediate 3-4. Using Suzuki conditions in a microwave reactor and a variety of aryl boronic acids and esters, a wide variety of
3-position substituents were synthesized. Tosylated intermediates 3-5 were deprotected using sodium hydroxide to yield final compounds 3-6a-ae.
Scheme 3-1. Synthesis of 3-position analogs.
Reagents and reaction conditions: a) 1-Methyl-4-pyrazoleboronic acid pinacol ester, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 85 °C, 4 h (93%). b) NIS, ACN, 50 °C, 2 h (69%). c) Tosyl chloride, DMAP, TEA, DCM, rt, 12 h (89%). d) Aryl boronic acid or pinacol ester, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 120 °C, μw, 30 min (24-85%). e) NaOH (2M aq), dioxane, 150 °C, μw, 1-10 min (10-83%).
3.2.2 Synthesis of 5-position analogs
As with the 3-position analogs, analogs at the 5-position were prepared in order to block the formation of metabolite A (Figure 3-1). Synthesis of 5-position analogs (Scheme 3-2) was achieved using the same 5-bromo-7-azaindole 3-1 as previously described for the 3-position analogs. In this case, however, the compound was first iodinated using NIS to produce the
71 dihalogenated intermediate 3-7, which was then tosylated to enable a Suzuki coupling at the 3- position of intermediate 3-8. Careful control of reaction time (five minutes in a microwave reactor) enabled a selective Suzuki coupling at the 3-position with (3-cyanophenyl)boronic acid; longer reaction times resulted in an undesired second Suzuki reaction with the benzonitrile substituent installed at both the 3- and 5-positions.
With intermediate 3-9 in hand, diversity at the 5-position was achieved through Suzuki reactions with the requisite aryl boronic esters or acids. Tosylated intermediates 3-10 were then deprotected with sodium hydroxide to produce final compounds 3-11a-i. Again, control of reaction time was key in order to avoid hydrolysis of the benzonitrile to the acid or amide in the presence of aqueous base. Typically, total deprotection without significant hydrolysis was achieved within
2-10 minutes in a microwave reactor.
Scheme 3-2. Synthesis of 5-position analogs.
Reagents and reaction conditions: a) NIS, ACN, 50 °C, 2 h (75%). b) Tosyl chloride, DMAP, TEA, DCM, rt, 12 h (69%). c) (3-Cyanophenyl)boronic acid, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 120 °C, μw, 5 min (60%). d) Aryl boronic acid or pinacol ester, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 85 °C, 4 h (31-84%). e) NaOH (2M aq), dioxane, 150 °C, μw, 1-10 min (10-83%).
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3.2.3 Core replacement synthesis
Further exploration of the structure-activity relationships of this chemotype was accomplished through modifications to the 7-azaindole core, which were synthesized according to
Scheme 3-3. Construction of a 4-methyl-7-azaindole core began with 2-amino-5-bromo-4- methylpyrimidine 3-12, which was iodinated at the 3-position using NIS to yield 2-amino-5- bromo-3-iodo-4-methylpyrimidine 3-13. This enabled a subsequent Sonogashira coupling at the
3-position with TMS-acetylene, affording intermediate 3-14. This compound was then directly cyclized using KOtBu in NMP to afford 5-bromo-4-methyl-7-azaindole 3-15.
With the 5-bromo core in hand, synthesis proceeded as with the synthesis of 3-position analogs of the 7-azaindole core. A Suzuki reaction at the 5-position to afford intermediate 3-16 was followed by iodination and subsequent tosylation to afford the penultimate compound 3-18.
The final compound NEU-5900 was synthesized by the requisite Suzuki reaction to afford 3-19, followed by detosylation.
The furopyridine core replacement was assembled starting with the commercially available bromide 3-20. The Suzuki product 3-21 was successfully brominated to afford 3-22 using bromine and a KOH workup procedure. The furopyridine core was sufficiently electron-deficient at the 3- position to allow the second Suzuki reaction to proceed in reasonable yield to afford the final compound NEU-6017. Substitution at the 3-position was confirmed by NMR (Appendix 5).
73
Scheme 3-3. Core replacement synthesis.
Reagents and conditions: a) NIS, TFA, AcOH, 50 °C, 12h (94%). b) TMS-acetylene, TEA, CuI, PdCl2(PPh3)2, THF, rt, 12 h (85%). c) KOtBu, NMP, 80 °C, 30 min (53%). d) 1-Methyl-4-pyrazoleboronic acid pinacol ester, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 85 °C, 4 h (64-79%). e) NIS, ACN, 50 °C, 2 h (42%). f) Tosyl chloride, DMAP, TEA, DCM, rt, 12 h (25%). g) (3-Cyanophenyl)boronic acid, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, 120 °C, μw, 5 min (59-71%). h) NaOH (2 M aq), dioxane, 150 °C, μw, 3 min (55%). i) NIS, ACN, 90 °C, 4 h (81%). j) TMS-acetylene, n-BuNH2, CuI, Pd(OAc)2, PPh3, THF, 40 °C, 5 h (76%). i) Br2, DCM, KOH/MeOH, 0 °C – rt, 1 h (35%).
3.3 Structure-activity and -property relationships
3.3.1 Benzonitrile replacements.
Because it was predicted that metabolism of NEU-1207 occurred at either the 3- or 5- position substituent of the 7-azaindole core, initial analogs focused on matched pairs to NEU-1207 that explored possible replacements for these substituents. Lisseth Silva, a previous graduate student in the lab, designed and synthesized analogs meant to probe the electronic and steric requirements of the 3-position aromatic ring utilizing a Topliss approach.64, 65 Summarized in
Figure 3-2 with results shown in Table 3-2, this approach starts by comparing the activity of an
74 unsubstituted phenyl ring (NEU-2065) with the 4-chlorophenyl (NEU-2066). Based on whether this compound is more potent (M), equipotent (E), or less potent (L) than the phenyl compound, the next analog is selected for synthesis. As shown, NEU-2066 was almost 10-fold less potent than
NEU-2065, so the 4-methoxyphenyl analog (NEU-2067) is the suggested next step. In this case, the 4-methylphenyl (NEU-2112) and 3,4-dichlorophenyl (NEU-2070) compounds were synthesized as well, and as expected, NEU-2070 is the most potent of the three.
At this point, the next suggested compound on the Topliss tree is the 4-amino-N,N- dimethylphenyl analog. However, analysis of the HLM Clint and aqueous solubility of NEU-2065,
-2066, -2067, -2070, and -2112 shows that analogs that maintain potency do not demonstrate an improvement in ADME properties, and analogs that show a slight improvement in either solubility or HLM Clint are inactive against T. brucei. Therefore, the Topliss approach was discontinued in favor of exploring substituents that would more directly address these issues.
75
Figure 3-2. Topliss decision tree for 3-position substituents.
Lisseth also designed and synthesized three compounds containing a replacement for the nitrile at the meta position of the 3-position phenyl ring. Replacing the nitrile with methoxy (NEU-
2068) or trifluoromethyl (NEU-2069) significantly reduced the potency as compared to NEU-
1207, although the trifluoromethyl analog did show a decrease in HLM Clint. Interestingly, the fluoro compound (NEU-2113) maintained potency as compared to NEU-1207 with a slight decrease in HLM Clint.
Given their potency, further systematic exploration of the 3-cyanophenyl and 3- fluorophenyl analogs was undertaken. A 4-cyanophenyl (NEU-4829) at the 3-position drastically reduced HLM Clint compared to NEU-1207, but lost activity against T. brucei. Conversely, the 4- fluorophenyl analog (NEU-4963) maintained activity but was also highly cleared. Neither a 2-
76 cyanophenyl (NEU-4991) nor a 2-fluorophenyl (NEU-4935) at the 3-position were tolerated in terms of activity or clearance. Disubstituted analogs were also explored; both the 3,4-diflurophenyl
(NEU-4964) and the 3-cyano-4-fluorophenyl (NEU-4938) succeeded in lowering clearance, but neither showed sufficient potency against T. brucei.
Finally, given predicted metabolite B (Figure 3-1b), which suggested that the meta position of the 3-cyanophenyl substituent was susceptible to oxidation, the 3-cyano-5- fluorophenyl analog NEU-5301 was synthesized. This compound was designed to block oxidation at the 5-position of the phenyl ring while taking advantage of the potency of the cyano group at the 3-position of the ring. Of all compounds that showed lower HLM Clint than NEU-1207, NEU-
5301 came closest to maintaining its potency and LLE. However, its HLM Clint of 90.8
(μg/min/mg) remained high compared to the target value of 9 μg/min/mg. Ultimately, no compound synthesized at this stage simultaneously maintained potency and sufficiently lowered clearance, and none showed significantly improved aqueous solubility over NEU-1207.
Table 3-2. Biological activity, LLE, HLM Clint, and aqueous solubility of benzonitrile replacement analogs.
T.b.b MRC5 HLM Clint Aq. sol. ID NEU- R LLE pEC50 pTC50 (μg/min/mg) (μM)
3-6a 1207 7.2 4.3 4.4 190 2
3-6b 2065* 6.7 nd 3.7 200 20
3-6c 2066* 5.8 4.3 2.2 57 0.8
3-6d 2070* 6.0 4.3 1.9 51 nd
77
3-6e 2112* 5.9 4.3 2.4 130 7
3-6f 2067* 6.8 4.3 4.0 180 5
3-6g 2068* 6.6 4.3 3.8 >300 18
3-6h 2069* 6.0 4.3 2.2 66 1
3-6i 2113* 7.1 4.3 4.0 110 3
3-6j 4829† 6.0 4.3 3.3 51 2
3-6k 4991 6.2 4.3 3.4 200 11
3-6l 4963 6.7 4.3 3.6 130 9
3-6m 4935 6.4 4.5 3.3 150 14
3-6n 4964 6.3 4.3 3.1 90 5
3-6o 4938 6.0 4.3 3.1 64 4
3-6p 5301 6.9 nd 4.0 91 0.5
*Designed and synthesized by L. Silva. †Synthesized by K. Forbes. nd = no data. All SEM within ± 0.17.
Further exploration of 3-position substituents focused on N-containing substituents and heterocycles (Table 3-3); a subset of these analogs was synthesized by REU student Katherine
Forbes. Hydrolysis of the nitrile to the amide (NEU-4830) or the acid (NEU-4831) at the para position led to a significant loss of potency, although the clearance improved in both cases and the solubility of NEU-4831 was greatly improved. The aniline (NEU-4827) maintained potency and had improved LLE and solubility as compared to NEU-1207, but significantly increased HLM
78
Clint. Incorporation of a dimethylbenzylamine (NEU-4933) resulted in a loss of activity as well as the introduction of a low level of toxicity against MRC5 cells.
Noting that NEU-1208 (Table 3-1) was approximately equipotent to NEU-1207, the phenol was explored at the 3-position, resulting in a compound (NEU-4833) that was equipotent to NEU-1207 and had significantly lower HLM Clint. However, this compound also showed toxicity against MRC5 cells. Replacement of the nitrile with a nitro group (NEU-4816) led to a slight increase in potency and LLE, but no real improvement to HLM Clint or solubility. The indole
(NEU-5003) and benzoxadiazole (NEU-5007) were used as electronically similar replacements of the phenol and nitro groups, respectively, designed to mitigate toxicity. Unfortunately, neither was as potent against T.b.b. and both were more highly cleared.
Pyridines NEU-4828 and NEU-4832 both showed improved LLE and solubility compared to NEU-1207; NEU-4832 was approximately equipotent and had a lower HLM Clint as well. Given the improved overall profile of NEU-4832, analogs including substituted heterocycles at the 3- position were designed to simultaneously improve the potency (by retaining the nitrile group) and the properties (by incorporating heteroatoms) of this chemotype. Unfortunately, neither NEU-
5054 nor NEU-5125 achieved this goal. Both were at least 10-fold less potent than NEU-1207 and displayed neither improved clearance nor significantly improved solubility.
Finally, saturated groups were incorporated at the 3-position in an attempt to increase the aqueous solubility of this series (for a more thorough discussion of strategies to increase solubility, see Chapter 1 of this dissertation).52 Generally, these compounds were less potent than aromatic groups at the 3-position; NEU-5994 is the most active but does not achieve a pEC50 >6. Boc- protected precursors as well as the free amines were evaluated, and the boc group is detrimental to both. However, the unprotected NEU-5995 and NEU-6016 achieve extremely low clearance and
79 high solubility, suggesting that further exploration of saturated groups at the 3-position may be warranted in order to improve the ADME profile of this series.
Table 3-3. Biological activity, LLE, HLM Clint, and aqueous solubility of second generation benzonitrile replacement analogs.
T.b.b MRC5 HLM Clint Aq. sol. ID NEU- R LLE pEC50 pTC50 (μg/min/mg) (μM)
3-6a 1207 7.2 4.3 4.4 190 2
3-6q 4830* 6.6 4.3 4.8 14 3
3-6r 4831* 4.4 4.3 2.0 <3 850
3-6s 4827* 7.0 4.6 4.8 290 300
3-6t 4933 5.7 4.7 2.9 nd nd
3-6u 4816* 7.6 4.3 4.7 14 2
3-6v 4833 7.2 5.2 4.5 50 <3
3-6w 5003 6.7 5.0 3.6 300 4
3-6x 5007 6.5 4.3 4.1 88 0.3
3-6y 4828* 6.7 4.3 5.0 90 97
3-6z 4832* 7.0 4.3 5.3 39 59
3-6aa 5054 6.0 4.3 4.5 150 17
3-6ab 5125 5.6 4.3 3.6 190 2
80
3-6ac 5954 5.3 4.6 2.7 300 6
-- 5955 5.1 4.3 3.7 3 770
3-6ad 5976 5.7 <4.3 3.4 180 13
-- 6016† 5.2 <4.3 4.1 <3 1000
3-6ae 5994 5.9 4.7 3.1 >300 17
-- 5995 5.5 <4.3 4.0 <3 1000
*Synthesized by K. Forbes. †Tested as HCl salt. nd = no data. All SEM within ± 0.16.
3.3.2 Pyrazole replacements
In addition to analogs at the 3-position, a series of pyrazole-replacement analogs were synthesized in order to explore the SAR at the 5-position; the biological activity, LLE, and ADME properties of these analogs are included in Table 3-4. In general, aromatic replacements of the pyrazole maintained potency as compared to NEU-1207. Incorporation of 1,5-dimethylpyrazole
(NEU-4992) and 1,3,5-trimethylpyrazole (NEU-5512), designed to sterically block the access of metabolic enzymes, did not demonstrate improved HLM Clint. Introduction of a H-bond donor as in the 1H-pyrazole (NEU-4998) and 1-methyl-1H-pyrazol-4-amine (NEU-5423) led to an improvement in aqueous solubility, and in the case of NEU-5423, clearance improved as well.
Installing the 4-(methylsulfonyl)benzene (present in the original hit NEU-1208) resulted in an analog equipotent to NEU-1207 (NEU-4997) which also displayed improved clearance. However, introduction of an -NH linker to this motif (NEU-5421) unfortunately did not lead to the expected improvement in solubility. Finally, a pyrimidine (NEU-5005) and pyridine (NEU-5006) were used as pyrazole replacements, but neither showed an overall improvement over NEU-1207.
81
Again, saturated rings were introduced at the 5-position as a strategy to improve the aqueous solubility of the series. The installation of ring systems that included a basic nitrogen distal to the 7-azaindole core, such as a piperazine (NEU-5318), N-methylpiperazine (NEU-5302), or N-methyl-1,4-diazepane (NEU-5510), did result in compounds with appreciably improved aqueous solubility. In contrast, compounds lacking this nitrogen (NEU-5043 and -5044), or those that included distal amides (NEU-5317) or sulfonamides (NEU-5422), which were less basic in nature, did not show improved solubility. Unfortunately, these saturated analogs were not active against T.b. brucei, with only one, NEU-5422, displaying a pEC50 >6.5.
Finally, substituted pyrazoles were tested as a way of directly addressing the production of predicted metabolite A (Figure 3-1b); we hypothesized that including larger substituents would block the access of oxidizing enzymes to the pyrazole. The tetrahydropyran (NEU-5813), boc- piperidine (NEU-5814), and N-methyl piperidine (NEU-5902) analogs all had lower HLM Clint than NEU-1207; in addition, they are some of the most potent analogs synthesized. NEU-5813 in particular is almost 10-fold more potent than NEU-1207. However, these compounds remain poorly soluble in water.
82
Table 3-4. Biological activity, LLE, HLM Clint, and aqueous solubility of pyrazole replacement analogs.
T.b.b MRC5 HLM Clint Aq. sol. ID NEU- R LLE pEC50 pTC50 (μg/min/mg) (μM)
3-6a 1207 7.2 4.3 4.4 190 2
3-11a 4998 6.9 4.3 4.3 290 26
3-11b 4992 7.3 4.3 4.4 250 2
3-11c 5512 6.4 4.7 3.2 300 5.7
3-11d 4997 7.2 4.3 4.0 30 1
-- 5423 7.2 4.4 4.6 15 31
-- 5421 7.0 4.5 4.0 33 2.1
3-11e 5005 6.5 4.3 4.1 31 3
3-11f 5006 7.3 4.3 4.2 160 0.2
-- 5043 6.1 4.3 2.9 50 0.3
-- 5044 5.4 4.3 1.7 300 4
-- 5302 5.3 4.3 2.6 44 230
-- 5317 5.9 4.3 2.3 94 12
-- 5318 5.3 4.3 3.0 nd 240
-- 5422 6.7 4.3 5.3 32 6.1
83
-- 5510 5.6 4.3 2.9 82 690
3-11g 5813 8.1 4.3 5.3 46 0.6
3-11h 5814 7.3 4.8 3.5 91 1.8
3-11i 5902 7.7 5.1 4.9 17 3.8
nd = no data. All SEM within ± 0.11.
3.3.3 Crossover analogs
Analogs made by combining promising 3- and 5-position substituents are shown in Table
3-5. The pyridyl substituents of NEU-4828 and NEU-4832, which maintained potency and improved solubility, were combined with the low-clearance (methylsulfonyl)benzene of NEU-
4997 to create analogs NEU-5128 and NEU-5127, respectively. While these compounds were potent, they were not highly soluble and only NEU-5127 showed lower clearance.
The incorporation of methyl groups was explored as a way to mitigate the potential CYP liability of unsubstituted pyridyl groups.66 The mono-methylated analogs NEU-5304 and -5305 were both more potent than the di-methylated matched pairs (NEU-5324 and -5319, respectively), although both were more highly cleared than the di-methylated compounds. Similarly, the solubility improvement of the pyridyl groups was only observed for the compounds that included a pyrazole at the 5-position (NEU-5304 and -5324); conversely, analogs with the 4-
(methylsulfonyl)benzene at the 5-position (NEU-5305 and -5319) had consistently lower clearance than the matched pairs containing the pyrazole.
Finally, NEU-5903 was synthesized in an attempt to maintain the potency afforded by incorporation of substituted pyrazoles (as in NEU-5813) while improving the solubility of the
84 compound by including a pyridyl group at the 3-position. Puzzlingly, this compound did not maintain the potency increase of NEU-5813; it is possible that NEU-5813 and NEU-4832 interact with different targets within the parasite and therefore a crossover of the two did not yield the expected synergistic results. While incorporation of the pyridyl moiety did slightly improve the solubility of NEU-5903 as compared to NEU-5813, this compound still does not reach the desired value of >10 μM.
Table 3-5. Biological activity, HLM Clint, and aqueous solubility of crossover analogs.
T.b.b MRC5 HLM Clint Aq. sol. NEU- R1 R2 LLE pEC50 pTC50 (μg/min/mg) (μM)
1207 7.2 4.3 4.4 190 2
5127 7.1 4.3 4.8 17 9
5128 6.6 nd 4.5 190 11
5304 7.1 5.3 5.2 160 150
5305 7.0 4.3 4.8 36 4
5324 6.3 4.3 4.3 96 32
5319 6.1 4.3 3.7 56 6
5903 7.0 5.1 5.3 29 5.9
nd = no data. All SEM within ± 0.15.
85
3.3.4 Core replacements
Finally, the SAR of the 7-azaindole core was explored though a series of core-replacement analogs (Table 3-6). Methylation (NEU-4930) or tosylation (NEU-4796) of the indole -NH resulted in a total loss of activity against T. brucei, as did excision of either the 3- or 5-position substituent (NEU-4463 and NEU-4411, respectively). Replacing the azaindole core with an indole
(NEU-5398) also resulted in an inactive compound. However, methylating the 4-position of the azaindole core (NEU- 5900) or switching the 3- and 5-position substituents (NEU-5004) resulted in minimal or no loss of potency against T. brucei. Lastly, replacing the indole -NH with an oxygen
(NEU-6017) led to a complete loss of anti-trypanosomal activity, confirming the importance of the core HBD. Most core replacements, aside from the tosylated NEU-4796, retained the high clearance of NEU-1207; similarly, the truncated NEU-4463 was the only compound showing an improvement in solubility. In sum, the 7-azaindole core is necessary for anti-trypanosomal activity and does not in itself seem to be a large contributor to low solubility or high clearance.
86
Table 3-6. Biological activity of core replacement analogs.
T.b.b MRC5 HLM Clint Aq. sol. NEU- X Y R LLE pEC50 pTC50 (μg/min/mg) (μM) 1207 N NH H 7.2 4.3 4.4 190 2 4930 N NMe H 5.1 <4.3 2.1 180 3 4796 N NTs H 5.7 <4.3 1.4 15 0.7 6017 N O H <4.3 <4.3 <1.4 130 0.6 5398 CH NH H 5.3 4.3 1.6 190 1.8 5900 N NH Me 7.3 <4.3 4.0 200 0.6
4463 4.7 <4.3 3.4 >300 300
4411 5.3 <4.3 2.6 nd nd
5004 7.0 <4.3 4.2 300 nd
nd = no data. All SEM within ± 0.08.
3.4 Further characterization of NEU-5127
The overall profiles of NEU-1207 and some of the most promising analogs of the series thus far are shown in Table 3-7. All analogs shown are either equipotent or more potent than NEU-
1207, and all have lower HLM Clint and rat hepatocyte clearance. Although it is the most potent analog, NEU-5813 has a high logD, high PPB and low aqueous solubility, which discounted it from consideration for advancement. Both NEU-4832 and NEU-5423 are highly plasma protein bound; with all parameters considered, NEU-5127 had the best overall profile and was selected for further evaluation in a PK study.
87
Table 3-7. Heat maps of NEU-1207, NEU-5127, NEU-4832, NEU-5423, and NEU-5813.
Targeted NEU-1207 NEU-5127 NEU-4832 NEU-5423 NEU-5813 Value T.b.b. pEC50 >7.5 7.2 7.0 7.0 7.2 8.1 MRC5 pTC50 <5 4.5 4.3 4.3 4.4 4.3 MW ≤360 299 349 275 314 369 cLogP ≤3 2.8 2.14 1.7 2.6 2.8 LogD (7.4) ≤2 3.9 2.9 2.8 nd 3.9 LLE ≥4 4.4 4.8 5.3 4.6 5.3 MPO Score ≥4 4.9 5.4 5.4 5.2 5.4 Aq. sol. (μM) >10 2 9 59 31 0.6 HLM Clint <9 190 17 39 15 46 (μg/min/mg) Rat Hepatocyte 6 <5 46 3.9 3.9 20 23 Clint (μg/min/10 ) Low PPB (%) ≤95 99 92 100* 99 recovery nd = no data. *Predicted PPB. Values highlighted in green meet or exceed targeted values; yellow highlighting indicates mid-range values, and red highlighting indicates values that are well outside the target.
The results of the PK study for NEU-5127 are shown in Table 3-8 and Figure 3-3.
Although it has a low tmax at less than one hour, NEU-5127 achieves a Cmax of over 20x its EC50 and remains in the blood longer than NEU-1207 (Figure 3-1a). However, it has a half-life of just over 1 h and is completely cleared from the blood by 8 h post-injection. Most disappointingly,
NEU-5127 is only detected in the brain at very low levels at its tmax and is not detectable in the brain after 4 h. This PK profile therefore makes NEU-5127 unsuitable for further development as an anti-HAT therapeutic.
Table 3-8. PK parameters for NEU-5127
T.b.b. EC50 Brain/blood ratio Brain/blood Cmax (ng/ml) tmax (h) t1/2 (h) (ng/ml) (t = 0.5 h) ratio (t = 4 h) 21 507±277 0.5-1.0 1.23±0.911 0.102 nd* nd = no data. *Brain concentration below the limit of quantitation (25 ng/g).
88
Figure 3-3. Brain and blood concentrations of NEU-5127 over time after a 10 mg/kg intraperitoneal dose.
N E U -5 1 2 7 ; 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 tio n s
/
) )
L 1 0 0 0
g
/
m
/
g
g
n
( n
B lo o d C o n c e n tra tio n (n g /m L )
(
n 7 5 0 n
o B lo o d C o n c e n tra tio n (n g /m L )
i
o
t
i
a t
r B ra in C o n c e n tra tio n (n g /g )
a t
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B 0 2 4 6 8 1 0 T im e (h r)
Green triangle = Peripheral blood concentration (ng/ml); n = 3 mice. Red circle = blood concentration (ng/ml); blue square = brain concentration (ng/g); n = 3 mice.
3.5 Activity against T. cruzi
In addition to activity against T. brucei, some of the compounds in this cluster were active against T. cruzi, the causative agent of Chagas disease. Presented in Table 3-9 are the biological activities and LLEs of all analogs with a T. cruzi pEC50 >6 and some matched pairs useful for comparison. Interestingly, all of these compounds include a 4-pyridyl substituent at either the 3- or the 5-position of the 7-azaindole. This may point to inhibition of T. cruzi CYP51 as the mechanism of action. This enzyme, involved in sterol biosynthesis in T. cruzi and other pathogens, is a validated drug target for Chagas disease.67 Furthermore, its pharmacophore includes a heterocyclic nitrogen atom which coordinates to the iron of the heme moiety of the enzyme, and has been previously targeted with pyridines.66, 68 However, experimental evidence for the mechanism of action of these compounds against T. cruzi is needed to confirm the target.
With a pEC50 of 6.6, NEU-5006 is the most potent analog of this cluster against T. cruzi; the pyridyl at the 5-position is almost 10x more potent than the pyrazole of NEU-1207. The most
89 active compound with a pyrazole at the 5-position is NEU-5304, although it displays some MRC5 and L6 toxicity. Combining the nitrile of NEU-1207 with the pyridyl of NEU-5304 results in
NEU-5125, which was less active than either of the parent compounds. Analogs with a matched (methyl-sulfonyl)benzene at the 5-position of the 7-azaindole allow for a more in-depth analysis of the SAR of the pyridyl at the 3-position. As with the pyrazoles, the most active analog is the 3-methyl-4-pyridyl compound NEU-5305, although the unsubstituted pyridyl (NEU-5127) is essentially equipotent. Interestingly, moving to a 3-pyridyl (NEU-5128) or a 3,5-dimethyl-4- pyridyl (NEU-5319) causes up to a 10-fold loss in activity against T. cruzi.
90
Table 3-9. T. cruzi activity, toxicity, and LLE of selected analogs.
T. cruzi MRC5 L6 NEU- R1 R2 T. cruzi LLE pEC50 pTC50 pTC50
5006 6.6 4.3 >4.3 3.4
5304 6.3 5.3 5.0 4.4
1207 5.8 4.3 5.1 3.0
5125 4.7 4.3 >4.3 2.7
5305 6.2 4.3 5.1 4.0
5127 6.0 4.3 5.3 3.9
5128 5.5 4.3 <4.3 3.4
5319 5.3 4.3 <4.3 2.9
All SEM within ± 0.03.
3.6 Summary and future work
Figure 3-4 summarizes the SAR of this series to date. Substituents at both the 3- and 5- positions of the core are necessary for activity, and blocking the H-bond donor of the core with a tosyl or even a methyl group results in a loss of activity. Modifications to the 7-azaindole structure itself generally resulted in a loss of anti-trypanosomal activity compared to NEU-1207, without any significant improvement to solubility or clearance.
At the 3-position, most aryl groups were either equipotent to NEU-1207 or lost activity, with only a few compounds showing marginal improvement in activity. Pyridyl groups (NEU-
91
4832) generally showed the best overall profile, maintaining potency while showing some improvement in solubility and clearance. While saturated rings (NEU-5955) at the 3-position showed the most promising improvements in both solubility and clearance, they consistently had greatly reduced activity against T. brucei.
At the 5-position, substituted pyrazoles such as that of NEU-5813 improved potency ~10- fold over NEU-1207. However, these analogs remained insoluble. Generally, installing saturated rings containing a basic nitrogen (NEU-5302) were highly soluble, albeit inactive against T. brucei. Finally, both the substituted pyrazoles and saturated rings, as well as an -NH insertion
(NEU-5423) resulted in analogs with improved clearance over NEU-1207.
92
Figure 3-4. SAR summary of 7-azaindoles.
Designing a compound that is highly potent, highly soluble, and has low HLM clearance remains the biggest challenge for this chemotype. Shown in Figure 3-5 are the structures of some proposed analogs based on the SAR which has now been described. Compound 3-23 is based on the highly potent NEU-5813 and its analogs but incorporates an H-bond donor at the 5-position, which has been shown to increase solubility. Similarly, compounds of type 3-24 use a substituted pyrazole at the 5-position and also incorporate an H-bond donor, this time at the 3-position.
Although analogs with this type of group at the 3-position lost activity when paired with the
93 standard N-methylpyrazole, incorporation of the substituted pyrazole will ideally counterbalance this effect and produce compounds that retain reasonable activity.
In conjunction with this work, preparation of proposed metabolite B may provide new directions for the progression of this chemotype. If this compound retains anti-trypanosomal activity, it may be possible to achieve efficacy by dosing with either NEU-1207 or other active compounds containing the benzonitrile at the 3-position. An active metabolite would afford greater flexibility in the design of future compounds by shifting the focus of optimization to just one or two parameters (potency and/or solubility), instead of three simultaneously.
Finally, this chemotype may be explored for further activity against T. cruzi. Compounds
3-25 containing either the pyridyl (R1 = H) or methylpyridyl (R1 = Me) at the 5-position would be useful as matched pairs to NEU-5006; SAR for T. cruzi around the 3-position (R2) could then be established. Core exploration may also be more fruitful against T. cruzi, where the tentative pharmacophore is the pyridyl nitrogen, rather than the putative hinge-binding 7-azaindole.
Figure 3-5. Proposed analogs for T. brucei (top row) and T. cruzi (bottom row).
94
Chapter 4. Solubility-Driven Optimization of Quinolines for HAT
4.1 Discovery and characterization of NEU-1953
4.1.1 Repurposing lapatinib and discovery of NEU-1953
Target class repurposing of human EGFR inhibitors led to the discovery of lapatinib and its derivatives as T. brucei growth inhibitors (for more on target class repurposing, see sections
1.2.2 and 1.5 of this dissertation). Initial SAR exploration of the tail region of this compound
(highlighted in blue in Figure 4-1) led to NEU-617; this compound displayed an almost 40-fold improvement in potency, and >100x selectivity against HepG2 cells.56 However, this compound was poorly water-soluble (<1 μM). In order to address this issue, SAR and structure-properties relationship (SPR) exploration of the lipophilic head region was undertaken; an understanding of the SAR of the core was developed as well.69 This work led to further optimization of a series of quinolines, ultimately yielding NEU-1953 as a potent inhibitor of P. falciparum (the parasite that causes malaria) and a moderate inhibitor of T. brucei and T. cruzi.70 In addition, the solubility of
NEU-1953 was improved over that of NEU-617.
Figure 4-1. Hit-to-lead progression of lapatinib derivatives.
95
The overall profile of NEU-1953 is shown in Table 4-1. Most of its parameters were within the targeted range, aside from its modest potency and high clearance. We therefore felt that NEU-
1953 warranted further profiling and pursued an in vivo efficacy study. Despite its acceptable profile, this compound was only moderately effective at reducing parasitemia in T. brucei infected mice, and extended the life of two mice one day beyond the untreated control group (data not shown). We hypothesized that these results may have been due to the high clearance and moderate aqueous solubility of the compound, in addition to its modest potency, and decided to pursue a properties-focused approach for further optimization.
Table 4-1. Overall profile of NEU-1953.
Targeted Value NEU-1953
T.b.b. EC50 ≤0.10 0.427
HepG2 TC50 ≥100x EC50 >4.0 cLogP ≤3 2.1 TPSA (Å2) 40
HLM Clint (μg/min/mg) <9 179
Rat Hepatocyte Clint <5 127 (μg/min/106) PPB (%) <95 87
4.1.2 Strategies to improve the solubility of NEU-1953
Over the course of optimizing NEU-617, the high lipophilicity of this compound (clogP =
7.3) was considered a major liability. As such, analogs with truncated headgroups or those that incorporated heterocycles into the head or tail of the compound were prioritized for synthesis.
96
Figure 4-2 shows aqueous solubility vs clogP for a selection of quinolines, grouped by whether the headgroup contains a phenyl ring, pyrazine (including NEU-1953), or another heterocycle. As predicted, replacing the large, lipophilic headgroup with a heterocycle successfully reduced the clogP of most analogs synthesized. However, only a few of these compounds showed a corresponding increase in aqueous solubility, and in general we observed little correlation between clogP and aqueous solubility. We concluded that the insolubility of low-clogP compounds such as
NEU-1953 was limited by its strong intramolecular interactions (leading to high crystallinity), rather than its hydrophobicity. For a more in-depth discussion of aqueous solubility, see section
1.3.3 of this dissertation.
Figure 4-2. Aqueous solubility vs clogP for a selection of NEU-617-derived quinolines.
97
Given the limited success of reducing clogP to increase aqueous solubility, alternate strategies for improving the solubility of NEU-1953 were pursued, summarized in Figure 4-3. For highly crystalline insoluble compounds, successful strategies focus on disrupting intramolecular forces that lead to the strong crystal packing of the compound.39, 47 Replacing aromatic rings with saturated rings and increasing the fraction of sp3-hybridized carbons (Fsp3) are two ways to do this;47, 51, 52 a compound with higher three-dimensional character does not stack as easily. We envisioned that these strategies could be applied to the head, linker and tail regions of NEU-1953.
Incorporation of ortho methyl groups can also disrupt packing by forcing the molecule out of a linear, flat conformation;51 this strategy was applied to the core and head of the compound.
In addition to reducing the crystal packing of NEU-1953, we also evaluated strategies that added a pendant water-soluble group or changed the formulation of the compound (Figure 4-3).
The introduction of ionizable groups to the tail, and the use of salt formations, were used to introduce ionic interactions with water. Additionally, prodrug attachment was evaluated as a way to add a solubilizing moiety to the molecule that would be cleaved in the cell. This strategy has the added advantage of being likely to disrupt crystal packing as well.
98
Figure 4-3. Summary of all strategies proposed to increase the solubility of NEU-1953.
The work described in this chapter is focused on the application of strategies designed to increase the Fsp3 and introduce saturated rings to the linker and tail regions of NEU-1953
(highlighted in pink and purple, respectively, in Figure 4-3). Desired analogs could therefore be constructed from the common intermediate 7, the synthesis of which is shown in Scheme 4-1. In the first step, 3-bromoaniline was condensed with diethyl 2-(ethoxymethylene)malonate to afford intermediate 4-2, which was cyclized by refluxing in diphenyl ether to afford the quinolone ester
4-3. This compound was converted to the carboxylic acid 4-4 by refluxing in 2 M NaOH; decarboxylation to afford 7-bromo-4-quinolone 4-5 was achieved by a second reflux in diphenyl ether. This compound could be converted to 7-bromo-4-chloroquinoline 4-6 using POCl3; finally, the headgroup was installed using 2-aminopyrazine and NaH to displace the chlorine, yielding the
99 advanced intermediate 7. Synthesis of the dihalogenated intermediate 4-6 has been previously reported.70
Scheme 4-1. Synthesis of key intermediate 7-bromo-N-(pyrazin-2-yl)quinolin-4-amine.
Reagents and reaction conditions: a) Diethyl 2-(ethoxymethylene)malonate, 100 °C, 3 h (100%). b) Diphenyl ether, 250 °C, 3 h. c) 2 M NaOH, 100 °C, 5 h. d) Diphenyl ether, 250 °C, 6 h. e) POCl3, 100 °C, 3 h (62%). f) 2- Aminopyrazine, NaH, DMF, rt, 12 h (88%). Where no yield is reported, crude material was progressed without further purification.
4.2 Evaluation of strategy 1: Saturated linkers
4.2.1 Synthesis via aryl amination chemistry
Shown in Figure 4-4 is a retrosynthetic analysis of the first proposed saturated-linker analogs. In the first approach (Figure 4-4a), the linker/tail moiety is first assembled via reductive amination, and then coupled to the core using Buchwald aryl amination conditions. This route would potentially allow for a parallel chemistry approach, where the fully constructed core and head could be coupled to various desired linker/tail moieties as the final step of the synthesis. An alternative synthesis was envisioned where the linker/tail could be coupled to the core before the head group was attached (Figure 4-4b), though this approach would not have been as amenable to a rapid generation of analogs, as it requires a longer linear sequence of steps.
100
Figure 4-4. Retrosynthetic analysis of saturated-linker analogs synthesized via Buchwald coupling, showing both a) the parallel-enabled route, and b) the alternative route.
The first linker/tail moiety synthesized incorporated a pyrrolidyl linker coupled to the N- methylpiperazine tail of NEU-1953; the synthesis is shown in Scheme 4-2. A reductive amination was used to couple N-boc-3-pyrrolidinone 4-8 with N-methylpiperazine, yielding the boc- protected pyrrolidinylpiperazine compound 4-9, which was then deprotected with HCl in dioxane to afford the desired product 4-10. The free base of this compound was obtained by stirring with
Si-bound carbonate and washing with methanol; it was not subjected to any further purification. It was envisaged that by using cyclic aminoketones of different ring sizes and varied regiochemistry we could assemble linker variations coupled to the N-methylpiperazine tail that could then be attached to the quinoline core.
101
Scheme 4-2. Synthesis of pyrrolidinyl-piperazine linker/tail.
Reagents and reaction conditions: a) N-methylpiperazine, TEA, AcOH, NaHB(OAc)3, DCM, rt, 12 h (69%). b) 4 M HCl, dioxane, rt, 4 h. Where no yield is reported, crude material was progressed without further purification.
Before coupling the desired tail group to the quinoline core, piperidine was used as a secondary amine model system to determine suitable Buchwald conditions for the reaction. The conditions of entry 1 of Table 4-2 were taken as baseline and various solvents, bases, catalysts, and ligands were scanned. Changing the base from NaOtBu to Cs2CO3 (entry 2) or K2CO3 (entry
3) resulted in a significant decrease in the conversion to the desired product as determined by analytical LCMS (no reaction and 8% conversion, respectively). Likewise, Xantphos was clearly a superior ligand to Xphos (entry 4) and t-Bu3P (entry 5) showing more than twice the conversion as either alternative. Of the three solvents tested, the protic solvent tBuOH (entry 6) gave the worst results, showing only trace conversion, while toluene (entry 7) produced 18% compared with dioxane, 35%. The reaction was less dramatically dependent on catalyst selection, as all three catalysts tested showed at least 20% conversion to product. However, the clear catalyst of choice was Pd(OAc)2 with 56% conversion (entry 9), resulting in the best overall set of conditions shown in entry 9 of Table 4-2.
102
Table 4-2. Results of a Buchwald condition scan using the piperidine model system.
Conversion Entry Base Catalyst Ligand Solvent (LCMS) 1 NaOtBu PdCl2(dppf) Xantphos Dioxane 35% 2 Cs2CO3 PdCl2(dppf) Xantphos Dioxane 0% 3 K2CO3 PdCl2(dppf) Xantphos Dioxane 8% 4 NaOtBu PdCl2(dppf) XPhos Dioxane 5% 5 NaOtBu PdCl2(dppf) t-Bu3P Dioxane 13% 6 NaOtBu PdCl2(dppf) Xantphos t-BuOH Trace 7 NaOtBu PdCl2(dppf) Xantphos Toluene 18% 8 NaOtBu Pd2(dba)3 Xantphos Dioxane 24% 9 NaOtBu Pd(OAc)2 Xantphos Dioxane 56%
Using the results of the model system condition scan, the coupling between the desired tail
(4-10) and the aminoquinoline core was undertaken. Shown in entry 1 of Table 4-3 are the results of the best conditions using the model system (entry 7 in Table 4-2 above). However, when applied to the desired tail, these conditions showed only starting material by LCMS analysis. We reasoned that the tail was sufficiently different from pyrrolidine as to afford completely different results, and therefore undertook a second round of reaction optimization (Table 4-3). In addition to the bases, catalysts, and solvents screened previously we assessed the performance of the RuPhos ligand (entry 7), demonstrated to work well for secondary amines.71 We also assessed the use of a
RuPhos precatalyst (entry 11). As shown, none of these conditions resulted in more than trace conversion to the desired product.
103
Table 4-3. Results of a Buchwald condition scan using the desired linker and tail.
Entry Base Catalyst Ligand Solvent Results (LCMS) 1 NaOtBu Pd(OAc)2 Xantphos Dioxane SM 2 NaOtBu PdCl2(dppf) Xantphos Dioxane SM 3 NaOtBu Pd(OAc)2 tBu3P Dioxane SM 4 NaOtBu Pd(OAc)2 Xantphos Toluene Trace A 5 NaOtBu Pd2(dba)3 Xantphos Toluene Trace A 6 NaOtBu Pd(OAc)2 tBuXPhos Toluene SM 7 NaOtBu Pd(OAc)2 RuPhos Toluene Trace A 8 NaOtBu Pd2(dba)3 Xantphos tBuOH Trace A 9 NaOtBu Pd(OAc)2 Xantphos tBuOH Trace A 10 NaOtBu Tetrakis Xantphos Toluene Trace A 11 NaOtBu RuPhos precat. Ruphos THF Trace A SM: Starting material.
We hypothesized that the nitrogen-rich core and linker could result in chelation to the palladium catalyst, resulting in inhibition of the reaction. As such, we conducted a brief condition scan on the quinolone core, without the pyrazine headgroup installed. The results of this scan are shown in Table 4-4 and the results are similar to those of the conditions scans using the fully elaborated core and headgroup as the substrate. Reactions using pyrrolidine achieved moderate conversion to the desired product by LCMS, but reactions using the desired linker and tail failed.
From this, we concluded that the fully elaborated tail moiety was a likely culprit for palladium chelation and did not pursue this synthetic route further.
104
Table 4-4. Results of a Buchwald condition scan using the quinolone core.
Entry Base Catalyst Ligand Solvent Substrate Results (LCMS) 1 NaOtBu Pd(OAc)2 Xantphos Dioxane 40% A 2 NaOtBu Pd2(dba)3 Xantphos Dioxane 49% A 3 NaOtBu Pd(OAc)2 RuPhos Dioxane 34% A 4 NaOtBu Pd(OAc)2 BINAP Dioxane 37% A
5 NaOtBu Pd(OAc)2 Xantphos Toluene SM
6 NaOtBu Pd2(dba)3 Xantphos Dioxane SM
7 NaOtBu Pd(OAc)2 tBuXPhos Dioxane SM
8 NaOtBu Pd(OAc)2 Xantphos Dioxane SM SM: Starting material.
4.2.2 Synthesis via Suzuki cross-coupling
As the convergent route using the fully assembled linker and tail did not seem amenable to aryl amination via palladium-catalyzed cross-coupling, we developed an alternative route which used Suzuki coupling to attach the linker to the quinoline core (Scheme 4-3). This coupling was undertaken with desired boc-protected boronic ester olefins varying ring size and the position of the nitrogen. The olefins of Suzuki products 4-11 were reduced via transfer hydrogenation using ammonium formate and 10 wt% Pd/C to afford the fully saturated systems 4-12. These compounds were tested for activity and subsequently deprotected using HCl in dioxane to yield free amines 4-
13, which were also evaluated for anti-parasitic activity.
105
Scheme 4-3. Synthesis of saturated-linker analogs using Suzuki coupling.
Reagents and conditions: a) Boronic ester, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, μw, 150 °C, 30 min (32- 53%). b) NH4COO, 10 wt% Pd/C, EtOH, 85 °C, 2 h (15-51%). c) 4M HCl/dioxane, rt, 3 h (69-70%).
The final step to produce analogs of NEU-1953 was envisioned as a reductive amination with N-methyl-4-piperidone. However, this chemistry proved challenging. Entries 1-5 of Table 4-
5 show the results of attempted reductive aminations. Varying the solvent and performing the reaction at room temperature (entries 1-3) did not result in conversion to desired product; neither did performing the reaction refluxing at either 65 or 85 °C (entries 4 and 5, respectively).
In addition to reductive amination, we also examined alkylation conditions using Cs2CO3 as a base (entries 6-11 of Table 4-5). Using the fully elaborated structure 4-13a resulted in product only when using the boc-protected piperidine (entry 7); the desired N-methylpiperidine did not produce any conversion (entry 6). As there are several nucleophilic nitrogens in structure 4-13a, we did not feel that deprotecting the boc-protected product and methylating to produce the desired analog was a viable strategy. We then attempted this chemistry using the desired boronate, with the idea that the products from these reactions could subsequently be coupled to compound 4-7.
Similarly to 4-13a, reactions of B with the N-methylpiperidine did not result in conversion to desired product, either at 90 °C or at room temperature (entries 8 and 9, respectively). Conversion
106 was observed using the boc-piperidine (entry 10); however, this compound proved difficult to isolate and this route was not pursued further.
Table 4-5. Attempted reductive amination and alkylation conditions.
Entry Solvent Temperature Results (LCMS) 1 DCE rt, 3 days SM 2 THF rt, 3 days SM 3 Dioxane rt, 3 days SM 4 THF 65 °C, 2 days SM 5 Dioxane 85 °C, 2 days SM
Entry Starting material Reagent Temperature Results (LCMS)
6 4-13a 90 °C, 12 h SM
7 4-13a 90 °C, 12 h C (R = Boc)
8 B 90 °C, 12 h SM
9 B rt, 12 h SM
10 B 90 °C, 12 h D (R = Boc)
SM: Starting material. rt: room temperature.
4.2.3 Structure-activity and structure-property relationships
The biological activity of compounds 4-12a-b and 4-13a-c are shown in Table 4-6. We assessed the potency of each of these compounds against not only T. brucei, but T. cruzi (Chagas disease), L. major amastigotes (leishmaniasis), and P. falciparum (malaria) as well. In addition,
107 we assessed their activity against a mammalian cell line, HepG2, in order to determine their toxicity. In general, these compounds lost activity compared to NEU-1953. Against T. brucei, the boc-protected analogs NEU-5946, -5979, and -5981 were slightly more potent than the unprotected NEU-5948 and -5980, suggesting that perhaps full elaboration to a complete linker and tail structure would result in the recovery of activity. Likewise, NEU-5948 and -5946 were more than 10-fold less potent against both L. major and P. falciparum than NEU-1953. However, these parasites diverged in the activity of the protected versus deprotected analog. Finally, NEU-
5979 showed some toxicity against HepG2 cells.
Table 4-6. Biological activity of saturated-linker analogs.
T.b.b. EC50 L. major P. falciparum Hep G2 ID NEU- R † ‡ ‡ ‡ (μM) EC50 (μM) D6 EC50 (μM) TC50 (μM)
-- 1953 0.43* 2.5 0.026 >4.0
4-13a 5948 70 28 1.1 >33
4-13b 5980 >20 Pending Pending >6.6
4-12a 5946 6.4 16 24 >25
4-12b 5979 8.1 Pending Pending 8.4
4-12c 5981 10 Pending Pending >26
*Data from NYU assay. †All SEM within ± 4.8. ‡R2 >0.81
The ADME properties of the saturated-linker analogs are shown in Table 4-7. All three of the boc-protected analogs (NEU-5946, -5979, and -5981) have overall ADME profiles similar to
108 that of NEU-1953: low to mid-range solubility, high clearance in both microsomes and hepatocytes, and moderate plasma protein binding. All have much higher Fsp3 than NEU-1953, due to the excision of the pyrimidine ring as well as the addition of the three sp3-hybridized carbons of the boc group; however, their clogP increased as well and they did not display a marked improvement in aqueous solubility. With slightly higher Fsp3 and slightly lower clogP, the unprotected amines NEU-5948 and -5980 showed a dramatic improvement in aqueous solubility, as well as much-improved clearance and plasma protein binding. The effects of saturated linkers on the overall ADME profile of compounds in this series therefore depend on the nature of the
“tail” moiety attached to them.
Table 4-7. ADME properties of saturated-linker analogs.
Aq. sol. HLM Clint Rat Hepatocyte NEU- R Fsp3 clogP PPB (%) (μM) (μL/min/mg) (μL/min/106)
1953 0.23 2.1 44 180 130 87
5948 0.28 1.8 920 3 4.6 86
5980 0.28 1.9 1000 <3 4.5 32
5946 0.39 3.1 15 110 95 100
5979 0.39 3.2 39 150 >300 98
5981 0.36 2.8 22 160 120 97
109
4.3 Evaluation of strategy 2: Piperazine replacements
In addition to increasing the Fsp3 by saturating the linker ring, we considered replacements for the piperazine “tail” ring that would both increase Fsp3 and the overall three-dimensional character of NEU-1953 analogs. The compounds discussed below incorporated either bridged piperazines or spirocycles, both of which introduce a significant degree of three-dimensionality to the structure. Although the spirocycles adopt different conformations as compared to the standard piperazine ring, as shown in Figure 4-5, they maintain a similar positioning for the pendant methyl group relative to the rest of the molecule. Furthermore, it has been demonstrated that the addition of a methylene unit in the form of a bridgehead carbon reduces measured logD compared to the unbridged parent compound and the addition of a non-bridgehead methyl group.72 This therefore seemed like a reasonable strategy to improve the solubility of NEU-1953.
Figure 4-5. Overlay of NEU-1953 (purple) and NEU-5971 (gray). Nitrogen atoms shown in blue.
110
4.3.1 Analog synthesis
The synthesis of both the bridged piperazine and spirocycle tails are shown in Scheme 4-
4. Spirocycles 4-15a-b were commercially available as the boc-protected amines, which were coupled with 5-bromo-2-chloropyrimidine to afford intermediates 4-16a-b. These compounds were deprotected with HCl and subsequently alkylated with methyl iodide, yielding the desired bromopyrimidines 4-18a-b. Similarly, the R,R and S,S enantiomers of the bridged N- methylpiperazines 4-20a-b were commercially available, and the same procedure was used to couple them to 5-bromo-2-chloropyrimidine to afford the bromopyrimidinyl intermediates 4-21a- b.
With the bromopyrimidine compounds in hand, subsequent Miyaura/Suzuki coupling was undertaken to make the desired final compounds. Borylation of the brominated intermediates afforded boronic esters 4-19a-b and 4-22a-b, which were subsequently subjected to Suzuki conditions and coupled to the quinoline core 4-7 to afford final compounds 4-23a-d.
111
Scheme 4-4. Synthesis of piperazine-replacement analogs.
Reagents and conditions: a) 5-Bromo-5-chloropyrimidine, DIPEA, t-butanol, μw, 150 °C, 30 min (57-94%). b) HCl, dioxane, rt, 4 h. c) CH3I, TEA, MeOH, rt, 12 h (10-15%). d) B2(pin)2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, μw, 145 °C, 1 h. e) 7, K2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane:water, μw, 130 °C, 30 min (28-32%). f) 7, K2CO3, PdCl2(dppf), 3:1 dioxane:water, 100 °C, 12 h (21-35%). Where no yield is reported, crude material was progressed without further purification.
112
4.3.2 Structure-activity and structure-property relationships
As with the saturated-linker compounds, we evaluated analogs including piperazine replacements against T. brucei, T. cruzi, L. major amastigotes, P. falciparum, and HepG2 cells; these results are shown in Table 4-8. Also included in this table is a compound incorporating a homopiperazine moiety (NEU-4363), where an additional methylene unit was added to the piperazine tail. As shown, the homopiperazine, both bridged piperazines, and the spirocycles were all slightly (~2-fold) more potent than NEU-1953 against T. brucei. Against T. cruzi, NEU-4363 picked up some activity, while the bridged piperazines remained inactive. Interestingly, all of the piperazine replacements were approximately 10-fold less potent against L. major than NEU-1953, with the exception of NEU-4955, which was equipotent to NEU-1953. Finally, all piperazine replacements showed pEC50 < 100 nM against P. falciparum, with NEU-6000 being the most potent at 11 nM. No compounds showed significant toxicity against HepG2 cells.
113
Table 4-8. Biological activity of piperazine-replacement analogs.
T.b.b. EC50 T. cruzi L. major P. falciparum Hep G2 ID NEU- R † † ‡ ‡ ‡ (μM) EC50 (μM) EC50 (μM) D6 EC50 (μM) TC50 (μM)
-- 1953 0.43** 6.0** 2.5 0.026 >4.00
-- 4363* 0.19 1.2 24 0.030 33
4-21a 5972 0.25 Pending 24 0.058 >24
4-21b 5971 0.15 Pending 23 0.030 16
4-21c 6000 Pending Pending Pending 0.011 Pending
4-21d 4443 0.20 >5** 24 0.035 37
4-21e 4955 0.15 6.1 2.6 0.052 >37
*Synthesis by L. Ferrins. **Data from NYU assay. †All SEM within ± 0.26. ‡R2 >0.81
The ADME properties of the piperazine replacement analogs are summarized in Table 4-
9. Impressively, all compounds displayed at least a 10-fold increase in aqueous solubility over
NEU-1953, with the spirocycles seeming slightly worse than the homo- or bridged piperazines in this respect (although still highly soluble). Additionally, all of these analogs showed improved
HLM Clint, rat hepatocyte clearance, and plasma protein binding. Overall, NEU-6000 has the best
ADME profile, with the lowest HLM Clint values, good rat hepatocyte clearance, excellent solubility and low plasma protein biding. In general, increasing the overall three-dimensional
114 character of NEU-1953 analogs by modifying the piperazine tail was a strategy that was highly successful in improving not only the solubility, but the overall ADME profile of this series.
Table 4-9. ADME properties of piperazine replacement analogs.
Rat Hepatocyte Aq. sol. HLM Clint PPB NEU- R clogP ΔclogP (μL/min/106 (μM) (μL/min/mg) (%) cells)
1953 44 2.1 -- 180 130 87
4363* 990 2.2 0.1 77 54 75
5972 690 2.1 0.0 43 4.0 48
5971 700 2.3 0.2 77 7.5 70
6000 940 2.9 0.8 19 4.3 86
4443 910 2.1 0.0 37 19 51
4955 960 2.1 0.0 41 17 58
*Synthesis by L. Ferrins.
In addition to aqueous solubility, we looked at the effect of increasing Fsp3 on melting point and calculated pKa; these results are presented in Table 4-10 for selected analogs. Generally, these compounds are lower-melting than NEU-1953, although the difference in aqueous solubility is much more dramatic than the difference in melting points. In addition, the compounds presented in Table 4-10 are all predicted to be more basic than NEU-1953 with an average increase in pKa of 1.0. Furthermore, the logD of these compounds decreases by an average of 1.0 compared to that of NEU-1953. LogD is a pH-dependent partition coefficient (analogous to clogP) and is
115 experimentally measured. Interestingly, the clogP of these compounds increased by an average of
0.2 (Table 4-9), indicating that three-dimensional character and basicity influence lipophilicity in ways that are unaccounted for in the logP calculation. It is likely that this combination of decreased crystal packing and increased pKa led to the success of this strategy in increasing the aqueous solubility of this series of compounds.
3 Table 4-10. Fsp , melting points, pKa, and aqueous solubility of selected piperazine replacement analogs.
Melting point Calculated ΔpKa LogD ΔLogD Aq. sol. NEU- Fsp3 range (°C) pKa (7.4) (7.4) (uM) 1953 0.23 240-258 7.4 -- 3.3 -- 44 4363 0.26 205-216 8.3 0.9 2.6 -0.7 990 4955 0.26 92-137 7.6 0.2 2.2 -1.1 960 5972 0.29 192-209 7.8 0.4 1.8 -1.5 690 6000 0.37 214-222 10 2.6 2.5 -0.8 940
4.4 Summary and future work
The work described in this chapter highlights attempts to improve the physicochemical and
ADME properties of NEU-1953, a compound identified after optimization of lapatinib against T. brucei. In particular, this chemistry was focused on decreasing the crystal packing of NEU-1953 by replacing aromatic rings with saturated rings, or by introducing three-dimensional character.
The structure-activity and -property relationships established are summarized in Figure 4-6.
Attempts to replace the pyrimidine “linker” with piperidine and pyrrolidine rings proved unsuccessful. Coupling the quinoline core to the ring via a nitrogen (aryl amination chemistry) was challenging because of the high potential of moieties such as compound 4-10 for chelation to a metal catalyst, such as the palladium required under Buchwald-Hartwig conditions. Suzuki reactions using tetrahydropyridine or dihydropyrrole boronates were more successful, although subsequent reactions to append the piperazine “tail” were not trivial. Analogs without the
116 piperazine were consistently inactive against T. brucei, L. major, and P. falciparum, although incorporating an unprotected secondary amine did result in improved ADME.
Replacing the piperazine “tail” with asymmetric elements such as spirocycles or bridged rings generally resulted in compounds with favorable ADME profiles. In addition, at least one of these compounds retained activity against each parasite; in fact, all of the piperazine replacement compounds showed an ~2-fold improvement in T. brucei activity over NEU-1953. The homopiperazine NEU-4363 was ~5 times more active against T. cruzi than NEU-1953, while the spirocycle NEU-6000 was ~2-fold more active against P. falciparum. Although most compounds lost L. major activity, the bridged piperazine NEU-4955 was equipotent to NEU-1953.
Figure 4-6. SAR summary of linker and tail replacements.
117
Chapter 5. Experimental Details
5.1 General methods
All compounds tested had a purity of > 95% as measured by LCMS, unless otherwise noted. Reagents purchased were used as received, unless otherwise noted. Purification of intermediates was performed using silica gel chromatography using the Biotage® Isolera™One flash purification system. Microwave reactions were performed in either a Biotage® Initiator+
(absorbance was set in accordance with the recommendations set by the manufacturer) or a CEM
Discover®. LCMS analysis was performed using a Waters Alliance reverse phase HPLC using a multi-wavelength photodiode array detector from 210 nm to 600 nm. Preparative HPLC was conducted for final compounds on Waters FractionLynx system using acetonitrile/water and 0.1% formic acid gradient and collected based on UV monitoring at 254 nm.
1H NMR spectra were obtained with Varian NMR systems, operating at either 400 or 500
MHz at room temperature, using solvents from Cambridge Isotope Laboratories. Chemical shifts
1 1 (δ, ppm) are reported relative to the solvent peak (CDCl3: 7.26 [ H]; DMSO-d6: 2.50 [ H];
1 1 Acetone-d6: 2.05; or CD3OD: 3.31 [ H]). Data for H NMR spectra are reported as follows: chemical shift (ppm), multiplicity (s for singlet, d for doublet, t for triplet, dd for doublet of doublet, m for multiplet), coupling constant (Hz), and integration.
General Procedure A (For the synthesis of 6-chloropyrimidine-2,4-diamines, 5-1a-h).
4,6-Dichloropyrimidin-2-amine and the desired amine (10.0 equiv.) were combined in a reaction flask which was filled with nitrogen and evacuated three times. The reaction was stirred and heated at 50 °C for 3 h. Upon completion, the reaction was concentrated under reduced pressure. The resulting solid was dissolved in EtOAc and washed with water. The organic layer was dried with magnesium sulfate and concentrated under reduced pressure to afford the title compound.
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General Procedure B (For the synthesis of substituted benzoxazepinoindazoles, 2-9a-t).
1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-
2(11H)-yl)ethan-1-one 2-8, desired aryl halide (1.5 equiv.), and potassium carbonate (3.5 equiv.) were suspended in 3:1 dioxane:water (0.08 M), and the reaction vial was degassed for 10 min.
Pd(PPh3)4 (5 mol%) was added and the reaction was run under nitrogen at 100 °C until completion as indicated by LCMS analysis (4 h). The reaction mixture was diluted with EtOAc or MeOH, filtered through Celite®, and concentrated under reduced pressure. The resulting crude residue was purified by the stated method to afford the desired product.
General Procedure C (For the synthesis of substituted 1H-indazoles, 2-26a-c). 6-(4,4,5,5-
Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole 2-24, desired aryl halide (1.5 equiv.), and potassium carbonate (2.0 equiv.) were combined and dissolved in 3:1 dioxane:water (0.07 M), and the vial was purged with nitrogen for 10 min. Pd(PPh3)4 (5 mol%) was added and the reaction was purged with nitrogen for an additional 5 min. Finally, tricyclohexylphosphine (10 mol%) was added and the reaction was run for 4.5 h under nitrogen at 100 °C. The reaction was diluted with
EtOAc, filtered through Celite®, and concentrated under reduced pressure. The resulting crude material was purified by the stated method to afford the title compound.
General Procedure D (For the synthesis of 4-bromo-2-alkoxy-6-fluorobenzonitriles, 2-
21a-h). The desired alcohol (1.2 equiv.) was dissolved in THF (0.64 M) and 1 M LiHMDS in THF
(1.2 equiv.) was added dropwise. The reaction was stirred at room temperature for 1 h, then cooled to 0 °C. 4-Bromo-2,6-difluorobenzonitrile 2-20 was added and the reaction was allowed to warm to room temperature and stirred until completion by TLC. The reaction mixture was diluted with
EtOAc, poured over water, and extracted three times. The combined organic layers were washed
119 once with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by the stated method to afford the title compound.
General Procedure E (For the synthesis of 6-bromo-4-alkoxy-1H-indazol-3-amines, 2-
22a-h). The desired 4-bromo-2-alkoxy-6-fluorobenzonitrile 2-21 was dissolved in EtOH (0.10 M) and hydrazine monohydrate (10 equiv.) was added. The reaction was refluxed overnight at 95 °C.
The reaction was cooled to room temperature and the solvent was removed under reduced pressure.
The crude material was purified by the stated method to afford the title compound.
General Procedure F (For the synthesis of 1-(3-amino-6-bromo-4-alkoxy-1H-indazol-1- yl)ethan-1-ones, 2-23a-h). The desired 6-bromo-4-alkoxy-1H-indazol-3-amine 2-22 was dissolved in pyridine (0.09 M) and acetic anhydride (3.5 equiv.) was added. The reaction was refluxed at 100 °C for 4 h. The reaction mixture was diluted with DCM and extracted three times with 1M HCl. The combined aqueous layers were extracted once with DCM, and the combined organic layers were washed once with water, once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was purified by the stated method to afford the title compound.
General Procedure G (For the synthesis of 6-(2-aminopyrimidin-4-yl)-4-alkoxy-1H- indazol-3-amines, 2-27a-h). The desired protected 6-bromo-4-alkoxy-1H-indazol-3-amines 2-23
(1.0 equiv.), potassium acetate (3.5 equiv.), bis(pinacolato)diboron (1.5 equiv.), and
PdCl2(dppf)·CH2Cl2 (5 mol%) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (0.10 M) was added and the reaction was run in the microwave (145 °C) for 0.5 h. The reaction mixture was diluted with EtOAc, filtered through
Celite®, and concentrated under reduced pressure. The crude material was either taken forward
120 without further purification or purified by the stated method and used as a mixture of mono- and di-protected boronic acid and ester.
Potassium carbonate (3.0 equiv.), 2-amino-4-chloropyrimidine (1.2 equiv.), and
PdCl2(dppf)·CH2Cl2 (5 mol%) were combined in a microwave vial that was filled with nitrogen and evacuated three times. The requisite boronic ester 2-25 or mixture of mono- and di-protected boronic acid and ester was dissolved in 3:1 dioxane:water (0.10 M) and added to the reaction mixture. The reaction was degassed for 10 min and run in the microwave (150 °C) for 0.5-1.5 h.
The reaction mixture was diluted with MeOH, filtered through Celite®, and concentrated under reduced pressure. The resulting crude residue was purified by the stated method to afford the desired product.
General Procedure H (For the synthesis of 5-(1-methyl-1H-pyrazol-4-yl)-3-aryl-1-tosyl-
1H-pyrrolo[2,3-b]pyridines, 3-5a-u). 3-Iodo-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H- pyrrolo[2,3-b] pyridine (1.0 equiv), desired boronic acid or ester (2.5 equiv) and
PdCl2(dppf)·CH2Cl2 (10 mol%) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.26 M) and 2M K2CO3 (3.0 equiv) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave at 120 °C for
30 min. The reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure, and the title compound was purified by the stated method to afford the title compounds.
General Procedure I (For the synthesis of 3-(5-aryl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitriles, 3-10a-j). 3-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (1 equiv), the desired boronic acid or ester (1.1 equiv), and PdCl2(dppf)·CH2Cl2 (10 mol%) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (0.17
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M) and 2M K2CO3 (5 equiv) were added and the reaction was degassed. The reaction was heated at 85 °C for 4-5 h, then diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by the stated method to afford the final compounds.
General Procedure J (For the synthesis of 3-(5-amino-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitriles). 3-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (1 equiv),
RuPhos (5 mol%), and RuPhos Pd G1 (5 mol%) were combined in a vial that was filled with nitrogen and evacuated three times. A 1.0 M solution of LiHMDS in THF (2.5 equiv) was added, followed by the addition of the desired amine (1.8 equiv). The reaction was heated at 65 °C for 5 h, then quenched by the addition of 1M HCl, diluted with EtOAc and poured over saturated aqueous NaHCO3. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude residue was purified by the stated method to afford the title compounds.
General Procedure K (For the synthesis of detosylated 3,5-disubstituted-1H-pyrrolo[2,3- b]pyridines). The tosylated azaindole starting material (1.0 equiv) was suspended in dioxane (0.10
M) and 2M NaOH (3.5-5.0 equiv) was added. The reaction was run in the microwave at 150 °C for the specified amount of time. The solvent was removed by rotovap and the crude material was purified by the stated method to afford the title compounds.
General Procedure L (For the synthesis of tert-butyl-(pyrazin-2-ylamino)quinolin-7-yl)- dihydroheterocyclic carboxylates, 4-11a-c). 7-Bromo-N-(pyrazin-2-yl)quinolin-4-amine, desired boronic ester (1.5 equiv), K2CO3 (3.0 equiv), and PdCl2(dppf)·CH2Cl2 (5 mol%) were combined in a microwave vial that was filled with nitrogen and evacuated three times. 3:1 Dioxane:water
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(0.08 M) was added and the reaction mixture was degassed, then run in the microwave (150 °C, H abs) for 30 min. The reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by the stated method to afford the title compound.
General Procedure M (For the synthesis of tert-butyl-(pyrazin-2-ylamino)quinolin-7- yl)piperidine- or pyrrolidine- carboxylates, 4-12a-c). The desired tert-butyl-(pyrazin-2- ylamino)quinolin-7-yl)-dihydroheterocyclic carboxylate was dissolved in EtOH (0.05 M) and 10% wt Pd/C (10 mol%) was added. Ammonium formate (6.5 equiv) was added and the reaction was refluxed at 85 °C for 2 h. After cooling to room temperature, the reaction mixture was diluted with
EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by the stated method to afford the title compound.
General Procedure N (For the synthesis of piperidinyl- or pyrrolidinyl-N-(pyrazin-2- yl)quinolin-4-amines, 4-13a-c). The desired tert-butyl-(pyrazin-2-ylamino)quinolin-7- yl)piperidine- or pyrrolidine- carboxylate was taken up in 4M HCl in dioxane (10 equiv). The reaction was stirred at room temperature for three hours. All volatiles were removed in vacuo. The resulting solid was dissolved in MeOH and Si-carbonate was added. After stirring overnight at room temperature, Si-carbonate was removed by filtration and volatiles were again removed in vacuo. The crude material was purified by the stated method to afford the title compound.
General Procedure O (For the synthesis of 2-amino-5-bromopyrimidines, 4-19a-e). 5-
Bromo-2-chloropyrimidine and the desired amine (1.2 equiv) were suspended in tert-butanol (0.22
M) and DIPEA (3.3 equiv) was added. The reaction was run in the microwave (150 °C) for 30 min. The tert-butanol was removed in vacuo and the reaction mixture was purified by the stated method to afford the title compound.
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General Procedure P (For the synthesis of boronic acid pinacol esters, 4-20a-e). The desired aryl bromide, bis(pinacolato)diboron (1.5 equiv), potassium acetate (3.5 equiv) and
PdCl2(dppf)·CH2Cl2 (5 mol%) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (0.09 M) was added and the reaction was run in the microwave (145 °C) for 1 h. The reaction mixture was diluted with EtOAc and filtered through
Celite®, and all volatiles were removed in vacuo. The crude material was used directly in the next reaction without further purification.
5.2 Chemical synthesis and characterization
5.2.1 Experimental procedures for chapter 2
4-Bromo-2,6-difluorobenzoyl chloride (2-2). 4-Bromo-2,6-difluorobenzoic acid 2-1 (3.00 g,
12.66 mmol) was suspended in thionyl chloride (15 ml, 207 mmol) and the reaction was refluxed at 75 °C for 4 h. Excess thionyl chloride was removed by distillation as the product was azeotroped with toluene three times, affording the title compound as a yellow oil which was used in the next reaction without further purification.
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4-Bromo-2,6-difluoro-N-(2-hydroxyphenyl)benzamide (2-3). 2-Aminophenol (1.38 g, 12.65 mmol) was dissolved in DCM (30 ml, 0.42 M), and TEA (3.5 ml, 25.11 mmol) was added. 4-
Bromo-2,6-difluorobenzoyl chloride 2-2 (3.23 g, 12.64 mmol) was dissolved in DCM (15 ml, 0.22
M), and this solution was added to the reaction mixture dropwise at 0 °C. The reaction was allowed to warm to room temperature and stirred overnight. After pouring the reaction mixture over 1 M
HCl, the title compound was isolated by vacuum filtration as an off-white solid (3.62 g, 87%).
+ 79 81 1 LCMS [M+H] 327.95 m/z ( Br), 329.96 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 10.03
(s, 1 H) 9.88 (br. s., 1 H) 7.88 (dd, J=8.1, 1.2 Hz, 1 H) 7.60 (d, J=7.3 Hz, 2 H) 6.97 - 7.03 (m, 1
H) 6.91 (dd, J=8.1, 1.2 Hz, 1 H) 6.78 - 6.84 (m, 1 H).
3-Bromo-1-fluorodibenzo[b,f][1,4]oxazepin-11(10H)-one (2-4). 4-Bromo-2,6-difluoro-N-(2- hydroxyphenyl)benzamide 2-3 (3.62 g, 11.04 mmol) was dissolved in DMF (30 ml, 0.37 M) and potassium carbonate (3.05 g, 22.06 mmol) was added. The reaction was stirred at room temperature for two days and was then poured over water. The title compound was isolated by vacuum filtration as an off-white solid (2.44 g, 72%). LCMS [M+H]+ 307.99 m/z (79Br), 309.96
125
81 1 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 10.74 (br. s., 1 H) 7.61 (s, 1 H) 7.57 (dd, J=9.8,
1.5 Hz, 1 H) 7.37 - 7.41 (m, 1 H) 7.22 (dd, J=8.3, 1.5 Hz, 1 H) 7.14 - 7.19 (m, 2 H).
3-Bromo-1-fluorodibenzo[b,f][1,4]oxazepine-11(10H)-thione (2-5). 3-Bromo-1-fluorodibenzo-
[b,f][1,4]oxazepin-11(10H)-one 2-4 (2.44 g, 7.91 mmol) was suspended in toluene (160 ml, 0.05
M) and Lawesson's reagent (3.49 g, 8.63 mmol) was added. The reaction was refluxed at 100 °C overnight. Upon cooling to room temperature, a yellow precipitate was observed and collected by vacuum filtration to afford the title compound as a yellow solid (1.75 g) which was carried forward without further purification. LCMS [M+H]+ 323.93 m/z (79Br), 325.95 m/z (81Br).
4-Bromo-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (2-6). 3-Bromo-1- fluorodibenzo[b,f][1,4]oxazepine-11(10H)-thione 2-5 (1.00 g, 3.09 mmol) was suspended in dioxane (10 mL, 0.3 M) and hydrazine monohydrate (0.30 mL, 6.11 mmol) was added. The reaction was refluxed for 3 h at 85 °C. After cooling to room temperature, the reaction mixture was diluted with DCM and concentrated under reduced pressure. The residue was purified by flash chromatography (0-5% MeOH:DCM) to afford the title compound as an off-white solid (833 mg,
+ 79 81 1 89%). LCMS [M+H] 301.97 m/z ( Br), 303.99 m/z ( Br); H NMR (400 MHz, DMSO-d6) δ
126 ppm 12.29 (s, 1 H) 9.52 (s, 1 H) 7.31 (d, J=1.5 Hz, 1 H) 7.26 (dd, J=8.1, 1.5 Hz, 1 H) 7.23 (dd,
J=8.1, 1.5 Hz, 1 H) 7.04 - 7.09 (m, 1 H) 6.85 - 6.91 (m, 2 H).
1-(4-Bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-11(2H)-yl)ethan-1-one and 1-(4- bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one (2-7). Acetic anhydride (12.0 mL, 127.18 mmol) was added to 4-bromo-2,11-dihydrobenzo-
[2,3][1,4]oxazepino[5,6,7-cd]indazole 2-6 (833 mg, 2.76 mmol) and the reaction was refluxed at
100 °C for 3 h. Upon cooling to room temperature, a precipitate was observed and collected by vacuum filtration to afford a mixture of isomers as a bright yellow solid (885 mg, 93%). LCMS
[M+H]+ 343.99 m/z (79Br), 345.96 m/z (81Br).
1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[2,3][1,4]oxazepino[5,6,7- cd]indazol-11(2H)-yl)ethan-1-one (2-8). A mixture of 1-(4- bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-11(2H)-yl)ethan-1-one and 1-(4- bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one 2-7 (401 mg, 1.17 mmol), potassium acetate (405 mg, 4.13 mmol), bis(pinacolato)diboron (443 mg, 1.74 mmol), and
PdCl2(dppf)·CH2Cl2 (46 mg, 0.06 mmol) were combined in a reaction vial that was filled with
127 nitrogen and evacuated three times. Dry, degassed dioxane (8.0 mL, 0.15 M) was added and the reaction was run in the microwave (145 °C) for 1 h. The reaction mixture was then diluted with
EtOAc, filtered through Celite®, and concentrated under reduced pressure. The residue was purified by flash chromatography (0-50% EtOAc:hexanes) to afford the title compound as an off-
+ 1 white solid (297 mg, 65%). LCMS [M+H] 392.13 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
10.03 (s, 1 H) 8.25 (s, 1 H) 7.27 (m, J=7.6, 3.2 Hz, 2 H) 7.26 (s, 1 H) 7.11 (dd, J=7.6 Hz, 1 H)
6.98 (dd, J=7.3 Hz, 1 H) 2.62 (s, 3 H) 1.34 (s, 12 H).
6-Chloro-N4-methylpyrimidine-2,4-diamine (5-1a). The title compound was prepared according to General Procedure A on a 50-mg scale using methylamine (2.0 M in MeOH), purified by preparative HPLC (1-10% ACN:water), and isolated as a solid (15 mg, 31%). LCMS [M+H]+
35 37 1 158.73 m/z ( Cl), 160.71 m/z ( Cl); H NMR (500 MHz, DMSO-d6) δ ppm 6.40 (br. s., 2 H) 5.70
(s, 1 H) 3.16 (s, 1 H) 2.72 (br. s., 3 H).
*Prepared by L. Tschiegg.
6-Chloro-N4,N4-dimethylpyrimidine-2,4-diamine (5-1b). The title compound was prepared according to General Procedure A on a 50-mg scale using dimethylamine (2.0 M in MeOH), and isolated as a solid (39 mg, 74%). LCMS [M+H]+ 172.81 m/z (35Cl), 174.76 m/z (37Cl); 1H NMR
(500 MHz, DMSO-d6) δ ppm 6.43 (br. s., 2 H) 5.92 (s, 1 H) 2.97 (s, 6 H).
*Prepared by L. Tschiegg.
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6-Chloro-N4-ethylpyrimidine-2,4-diamine (5-1c). The title compound was prepared according to General Procedure A on a 51-mg scale using ethylamine (2.0 M in MeOH), and isolated as a solid (43 mg, 80%). LCMS [M+H]+ 172.87 m/z (35Cl), 174.86 m/z (37Cl); 1H NMR (500 MHz, acetone) δ ppm 6.25 (br. s, 1 H) 5.79 (s, 1 H) 5.71 (br. s., 2 H) 3.32 (br. s., 2 H) 1.14 (t, J=7.1 Hz,
3 H).
*Prepared by L. Tschiegg.
6-Chloro-N4-isopropylpyrimidine-2,4-diamine (5-1d). The title compound was prepared according to General Procedure A on a 50-mg scale using isopropylamine (2.0 M in MeOH), purified by preparative HPLC (5-95% ACN:water), and isolated as a solid (18 mg, 32%). LCMS
+ 35 37 1 [M+H] 186.85 m/z ( Cl), 188.81 m/z ( Cl); H NMR (500 MHz, DMSO-d6) δ ppm 8.29 (s, 1 H)
6.96 (br. s, 1 H) 6.35 (br. s, 2 H) 4.04 (br. s, 1 H) 1.09 (d, J=6.8 Hz, 6 H).
*Prepared by L. Tschiegg.
4-Chloro-6-(pyrrolidin-1-yl)pyrimidin-2-amine (5-1e). The title compound was prepared according to General Procedure A on a 50-mg scale using pyrrolidine and isolated as a solid (49
129
+ 35 37 1 mg, 80%). LCMS [M+H] 198.88 m/z ( Cl), 200.84 m/z ( Cl); H NMR (500 MHz, DMSO-d6)
δ ppm 6.41 (s, 2 H) 5.75 (s, 1 H) 3.37 - 3.48 (m, 2 H) 3.19 - 3.29 (m, 2 H) 1.81 - 1.94 (m, 4 H).
*Prepared by L. Tschiegg.
4-Chloro-6-(piperidin-1-yl)pyrimidin-2-amine (5-1f). The title compound was prepared according to General Procedure A on a 75-mg scale using piperidine and isolated as a pale yellow solid (68 mg, 70%). LCMS [M+H]+ 212.98 m/z (35Cl), 214.99 m/z (37Cl); 1H NMR (500 MHz,
DMSO-d6) δ ppm 6.42 (s, 2 H) 6.06 (s, 1 H) 3.52 (br. s., 4 H) 1.56 - 1.64 (m, 2 H) 1.42 - 1.51 (m,
4 H).
4-Chloro-6-morpholinopyrimidin-2-amine (5-1g). The title compound was prepared according to General Procedure A on a 50-mg scale using morpholine and isolated as a solid (25 mg, 39%).
+ 35 37 1 LCMS [M+H] 214.94 m/z ( Cl), 216.95 m/z ( Cl); H NMR (399 MHz, DMSO-d6) δ ppm 6.53
(br. s., 2 H) 6.09 (s, 1 H) 3.61 (m, J=4.4 Hz, 4 H) 3.50 (m, J=5.1 Hz, 4 H).
*Prepared by L. Tschiegg.
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4-Chloro-6-(4-methylpiperazin-1-yl)pyrimidine-2-amine (5-1h). The title compound was prepared according to General Procedure A on a 50-mg scale using N-methylpiperazine (2.0 M in
MeOH), and isolated as a solid (53 mg, 76%). LCMS [M+H]+ 228.04 m/z (35Cl), 230.05 m/z (37Cl);
1 H NMR (500 MHz, DMSO-d6) δ ppm 6.48 (br. s., 2 H) 6.09 (s, 1 H) 3.52 (br. s., 4 H) 2.31 (t,
J=5.1 Hz, 4 H) 2.19 (s, 3 H).
*Prepared by L. Tschiegg.
6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N4-methylpyrimidine-2,4- diamine formate (2-9a/NEU-1330). The title compound was prepared according to General
Procedure B on a 50-mg scale using 6-chloro-N4-methylpyrimidine-2,4-diamine 5-1a. The crude material was purified by flash chromatography (20-100% EtOAc:hexanes – 0-20%
MeOH:EtOAc), then repurified by preparative HPLC (5-95% ACN:water) to afford the formate salt of the title compound as a solid (6 mg, 13%). LCMS [M+H]+ 346.09 m/z; 1H NMR (500 MHz,
METHANOL-d4) δ ppm 8.47 (br. s., 1 H) 7.46 (br. s., 1 H) 7.26 (d, J=7.81 Hz, 1 H) 7.16 (d, J=8.30
Hz, 1 H) 7.01 - 7.11 (m, 2 H) 6.91 (dd, J=7.81 Hz, 1 H) 6.34 (s, 1 H) 3.00 (s, 3 H).
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6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N4,N4-dimethylpyrimidine-
2,4-diamine (2-9b/NEU-4379). The title compound was prepared according to General Procedure
B on a 35-mg scale using 6-chloro-N4,N4-dimethylpyrimidine-2,4-diamine 5-1b. The crude material was purified by flash chromatography (4-20% MeOH:DCM) to afford the title compound
+ 1 as a solid (16 mg, 35%). LCMS [M+H] 360.39 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.35
(s, 1 H) 9.53 (s, 1 H) 7.79 (s, 1 H) 7.41 (s, 1 H) 7.24 - 7.31 (m, 2 H) 7.06 (td, J=6.8, 1.5 Hz, 1 H)
6.90 (td, J=7.8, 1.5 Hz, 1 H) 6.55 (s, 1 H) 6.25 (br. s, 2 H) 3.11 (s, 6 H).
*Prepared by L. Tschiegg.
6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N4-ethylpyrimidine-2,4- diamine (2-9c/NEU-4898). The title compound was prepared according to General Procedure B on a 40-mg scale using 6-chloro-N4-ethylpyrimidine-2,4-diamine 5-1c. The crude material was purified by flash chromatography (0-20% MeOH:DCM) to afford the title compound as a solid
+ 1 (20 mg, 50%). LCMS [M+H] 360.39m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 7.52 (s,
132
1 H) 7.26 (dd, J=7.8, 1.5 Hz, 1 H) 7.10 - 7.17 (m, 2 H) 7.03 (td, J=7.6, 1.5 Hz, 1 H) 6.89 (td, J=7.8,
1.5 Hz, 1 H) 6.26 (s, 1 H) 3.40 (q, J=7.2 Hz, 2 H) 1.24 (t, J=7.3 Hz, 3 H).
*Prepared by L. Tschiegg.
6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N4-isopropylpyrimidine-
2,4-diamine formate (2-9d/NEU-4457). The title compound was prepared according to General
Procedure B on a 50-mg scale using 6-chloro-N4-isopropylpyrimidine-2,4-diamine 5-1d. The crude material was purified by flash chromatography (20-100% EtOAc:hexanes – 0-20%
MeOH:DCM), then repurified by preparative HPLC (5-95% ACN:water) to afford the formate salt of the title compound as a solid (8 mg, 17%). LCMS [M+H]+ 374.09 m/z; 1H NMR (500 MHz,
METHANOL-d4) δ ppm 8.50 (s, 1 H) 7.46 (s, 1 H) 7.27 (dd, J=8.8, 1.46 Hz, 1 H) 7.17 (dd, J=8.3,
1.5 Hz, 1 H) 7.03 - 7.10 (m, 2 H) 6.92 (dd, J=6.4 Hz, 1 H) 6.31 (s, 1 H) 4.21 - 4.39 (m, 1 H) 1.27
(d, J=6.8 Hz, 6 H).
*Prepared by L. Tschiegg.
133
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-6-(pyrrolidin-1- yl)pyrimidin-2-amine formate (2-9e/NEU-4392). The title compound was prepared according to
General Procedure B on a 50-mg scale using 4-chloro-6-(pyrrolidin-1-yl)pyrimidin-2-amine 5-1e.
The crude material was purified by flash chromatography (4-20% MeOH:DCM), then repurified by preparative HPLC (5-95% ACN:water) to afford the formate salt of the title compound as a
+ 1 solid (9 mg, 18%). LCMS [M+H] 386.09 m/z; H NMR (500 MHz, Acetone-d6) δ ppm 11.38 (br. s, 1 H) 8.50 (s, 1 H) 8.12 (s, 1 H) 7.88 (s, 1 H) 7.48 (d, J=0.98 Hz, 1 H) 7.34 (dd, J=7.81 Hz, 2 H)
7.08 (dd, J=7.32 Hz, 1 H) 6.94 (dd, J=6.84 Hz, 1 H) 6.39 (s, 1 H) 5.53 (br. s., 2 H) 3.52 (br. s,
J=5.37 Hz, 4 H) 2.00 (br. s., 4 H).
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-6-(piperidin-1- yl)pyrimidin-2-amine formate (2-9f/NEU-4458). The title compound was prepared according to
General Procedure B on a 50-mg scale using 4-chloro-6-(piperidin-1-yl)pyrimidin-2-amine 5-1f.
The crude material was purified by flash chromatography (20-100% EtOAc:hexanes – 0-20%
MeOH:EtOAc), then repurified by preparative HPLC (5-95% ACN:water) to afford the formate
134 salt of the title compound as a solid (7 mg, 14%). LCMS [M+H]+ 400.13 m/z; 1H NMR (500 MHz,
METHANOL-d4) δ ppm 8.48 - 8.52 (m, 1 H) 7.52 (s, 1 H) 7.28 (dd, J=8.8, 1.46 Hz, 1 H) 7.14 -
7.19 (m, 2 H) 7.06 (td, J=7.3, 1.5 Hz, 1 H) 6.91 (td, J=7.3, 2.0 Hz, 1 H) 6.62 (s, 1 H) 3.80 (m, 4
H) 1.73 - 1.79 (m, 2 H) 1.63 - 1.71 (m, 4 H).
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-6-morpholinopyrimidin-2- amine (2-9g/NEU-4380). The title compound was prepared according to General Procedure B on a 50-mg scale using 4-chloro-6-morpholinopyrimidin-2-amine 5-1g. The crude material was purified by flash chromatography (4-20% MeOH:DCM), then repurified by preparative HPLC (5-
95% ACN:water) to afford the title compound as a solid (5 mg, 10%). LCMS [M+H]+ 402.09 m/z;
1H NMR (500 MHz, Acetone) δ ppm 11.42 (br. s, 1 H) 8.52 (s, 1 H) 7.91 (d, J=1.0 Hz, 1 H) 7.49
(d, J=1.0 Hz, 1 H) 7.33 (m, 2 H) 7.09 (ddd, J=8.3, 7.1, 1.7 Hz, 1 H) 6.93 (ddd, J=7.3, 2.0 Hz, 1 H)
6.71 (s, 1 H) 5.69 (br. s., 2 H) 3.70 - 3.76 (m, 4 H) 3.66 - 3.70 (m, 4 H).
*Prepared by L. Tschiegg.
135
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-6-(4-methylpiperazin-1- yl)pyrimidin-2-amine (2-9h/NEU-4931). The title compound was prepared according to General
Procedure B on a 20-mg scale using 4-chloro-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine 5-1h.
The crude material was purified by flash chromatography (0-60% 10% NH4OH in MeOH:DCM) to afford the title compound as a solid (7 mg, 19%). LCMS [M+H]+ 415.47 m/z; 1H NMR (500
MHz, METHANOL-d4) δ ppm 7.58 (s, 1 H) 7.27 (dd, J=7.8, 1.5 Hz, 1 H) 7.23 (s, 1 H) 7.16 (dd,
J=8.8, 1.5 Hz, 1 H) 7.06 (td, J=8.3, 1.5 Hz, 1 H) 6.92 (td, J=8.8, 1.5 Hz, 1 H) 6.55 (s, 1 H) 3.76
(m, 4 H) 2.54 (m, 4 H) 2.36 (s, 3 H).
*Prepared by L. Tschiegg.
6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-4-amine (2-
9m/NEU-1332). The title compound was prepared according to General Procedure B on a 41-mg scale using 4-amino-6-chloropyrimidine. The crude material was purified preparative HPLC (5-
50% water:ACN) to afford the title compound as a yellow solid (15 mg, 45%). LCMS [M+H]+
1 317.11 m/z; H NMR (400 MHz, DMSO-d6) δ ppm 12.36 (s, 1 H) 9.52 (s, 1 H) 8.46 (s, 1 H) 7.74
136
(s, 1 H) 7.23 - 7.32 (m, 3 H) 7.06 (dd, J=7.3 Hz, 1 H) 6.99 (s, 1 H) 6.92 (br. s, 2 H) 6.89 (dd, J=8.1
Hz, 1 H).
2-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-4-amine (2-
9n/NEU-2101). The title compound was prepared according to General Procedure B on a 41-mg scale using 4-amino-2-chloropyrimidine. The crude material was purified by preparative HPLC
(5-50% ACN:water) to afford the title compound as a yellow solid (17 mg, 51%). LCMS [M+H]+
1 317.11 m/z; H NMR (400 MHz, DMSO-d6) δ ppm 12.32 (s, 1 H) 9.49 (s, 1 H) 8.20 (d, J=5.9 Hz,
1 H) 8.07 (s, 1 H) 7.71 (s, 1 H) 7.28 (d, J=8.1 Hz, 2 H) 7.06 (dd, J=6.6 Hz, 1 H) 6.96 (br. s., 2 H)
6.88 (dd, J=7.3 Hz, 1 H) 6.39 (d, J=5.9 Hz, 1 H).
4-(Pyrimidin-4-yl)-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (2-9o/NEU-
2103). The title compound was prepared according to General Procedure B on a 40-mg scale using
4-chloropyrimidine·HCl. Upon cooling to room temperature, a precipitate was observed and collected by vacuum filtration. The precipitate was suspended in DME (0.75 mL, 0.06 M) and concentrated HCl (0.25 ml, 3.00 mmol) was added. This suspension was stirred at 85 °C for 1 h,
137 then cooled to room temperature. The resulting precipitate was collected to afford the title compound as an orange solid (12 mg, 40%). LCMS [M+H]+ 302.09 m/z; 1H NMR (400 MHz,
DMSO-d6) δ ppm 12.55 (br. s., 1 H) 9.59 (s, 1 H) 9.28 (s, 1 H) 8.89 (d, J=5.9 Hz, 1 H) 8.24 (dd,
J=5.1, 1.5 Hz, 1 H) 8.00 (s, 1 H) 7.58 (s, 1 H) 7.29 (m, J=8.1, 1.5 Hz, 2 H) 7.08 (dd, J=7.7 Hz, 1
H) 6.90 (dd, J=6.6 Hz, 1 H).
6-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyridin-2-amine formate (2-
9p/NEU-2122). The title compound was prepared according to General Procedure B on a 75-mg scale using 2-amino-6-chloropyridine. The crude material was purified by flash chromatography
(50% EtOAc:hexanes – 33%/33%/33% EtOAc:hexanes:acetone – 33% EtOAc:acetone), further purified by flash chromatography (10% MeOH:DCM), and finally purified by preparative HPLC
(5-95% ACN:water) to afford the title compound as a colorless solid (45 mg, 74%). LCMS [M+H]+
1 315.99 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 12.23 (s, 1 H) 9.48 (s, 1 H) 8.15 (s, 1 H) 7.70
(s, 1 H) 7.47 (dd, J=8.1 Hz, 1 H) 7.38 (s, 1 H) 7.27 (m, 2 H) 7.15 (d, J=7.3 Hz, 1 H) 7.05 (dd,
J=7.0 Hz, 1 H) 6.88 (dd, J=8.1 Hz, 1 H) 6.44 (d, J=8.1 Hz, 1 H) 6.06 (s, 2 H).
138
2-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyridin-4-amine (2-9q/NEU-
2197). The title compound was prepared according to General Procedure B on a 51-mg scale using
4-amino-2-bromopyridine. The crude material was dissolved in MeOH (2.0 mL, 0.10 M) and potassium carbonate (54 mg, 0.391 mmol) was added before stirring at room temperature for 1 h.
The reaction mixture was diluted with EtOAc and washed with water; the aqueous layer was then extracted twice with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by flash chromatography (3-20% MeOH:1% TEA/DCM) to afford the title
+ 1 compound as a solid (4.8 mg, 8%). LCMS [M+H] 315.99 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 12.67 (s, 1 H) 9.65 (s, 1 H) 8.14 (d, J=6.8 Hz, 1 H) 7.96 (br. s, 2 H) 7.54 (s, 1 H) 7.25 - 7.33
(m, 2 H) 7.18 (d, J=2.0 Hz, 1 H) 7.11 (s, 1 H) 7.09 (dd, J=8 Hz, 1 H) 6.91 (dd, J=7 Hz, 1 H) 6.82
(dd, J=7, 1.7 Hz, 1 H).
4-Phenyl-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (2-9r/NEU-2100). The title compound was prepared according to General Procedure B on a 40-mg scale using chlorobenzene. The crude material was purified by flash chromatography (40% EtOAc:hexanes)
139 to afford the title compound as a solid (14 mg, 46%). LCMS [M+H]+ 300.06 m/z; 1H NMR (400
MHz, DMSO-d6) δ ppm 12.23 (s, 1 H) 9.48 (s, 1 H) 7.75 (d, J=7.3 Hz, 2 H) 7.49 (m, J=7.3 Hz, 2
H) 7.40 (d, J=7.3 Hz, 1 H) 7.24 - 7.32 (m, 3 H) 7.06 (dd, J=8.1 Hz, 1 H) 7.01 (s, 1 H) 6.88 (dd,
J=8.1, 6.60 Hz, 1 H).
Scheme 5-1. Synthesis of NEU-4389.
3-Bromo-1-fluoro-10-methyldibenzo[b,f][1,4]oxazepin-11(10H)-one (5-2). 3-Bromo-1-fluoro- dibenzo[b,f][1,4]oxazepin-11(10H)-one 2-4 (482 mg, 1.56 mmol) was dissolved in dry DMF (6 ml, 0.27 M). Sodium hydride (129 mg, 3.23 mmol) was added and the reaction was stirred for 30 min at room temperature. Methyl iodide (0.15 mL, 2.41 mmol) was added dropwise at 0 °C and
140 the reaction was stirred for an additional 4 h, warming to room temperature. The reaction was quenched with saturated aqueous ammonium chloride and extracted three times with EtOAc. The combined organic layers were washed once with water and once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by flash chromatography (10-20% EtOAc:hexanes) to afford the title compound as a solid (267 mg, 53%).
+ 79 81 1 LCMS [M+H] 321.98 m/z ( Br), 323.99 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 7.62
(s, 1 H) 7.51 - 7.59 (m, 2 H) 7.43 (d, J=8.3 Hz, 1 H) 7.33 (dd, J=7.8 Hz, 1 H) 7.26 (dd, J=7.8 Hz,
1 H) 3.49 (s, 3 H).
3-Bromo-1-fluoro-10-methyldibenzo[b,f][1,4]oxazepine-11(10H)-thione (5-3). 3-Bromo-1- fluoro-10-methyldibenzo[b,f][1,4]oxazepin-11(10H)-one 5-5 (280 mg, 0.869 mmol) was dissolved in toluene (3.5 ml, 0.25 M) and phosphorus pentasulfide (291 mg, 1.31 mmol) was added. The reaction was refluxed at 100 °C overnight, then additional phosphorus pentasulfide
(256 mg, 1.15 mmol) was added. The reaction continued to reflux at 100 °C for an additional 24 h. The reaction was cooled to room temperature and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (0-25% methyl tert-butyl ether:hexanes) to afford the title compound as a yellow solid (200 mg, 68%). LCMS [M+H]+
79 81 1 337.94 m/z ( Br), 339.93 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 7.66 (dd, J=7.3, 2.0
Hz, 1 H) 7.58 (t, J=1.7 Hz, 1 H) 7.52 (dd, J=10.0, 2.0 Hz, 1 H) 7.44 - 7.48 (m, 1 H) 7.32 - 7.40
(m, 2 H) 3.93 (s, 3 H).
141
4-Bromo-11-methyl-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (5-4). 3-Bromo-
1-fluoro-10-methyldibenzo[b,f][1,4]oxazepine-11(10H)-thione 5-6 (200 mg, 0.620 mmol) was suspended in dioxane (3.1 mL, 0.2 M) and hydrazine monohydrate (0.06 mL, 1.22 mmol) was added. The reaction was refluxed for 3 h at 85 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The resulting crude residue was purified by flash chromatography (20-30% EtOAc:hexanes) to afford the title compound as an orange solid (125
+ 79 81 1 mg, 63%). LCMS [M+H] 315.93 m/z ( Br), 317.92 m/z ( Br); H NMR (500 MHz, DMSO-d6)
δ ppm 12.36 (s, 1 H) 7.31 - 7.35 (m, 3 H) 7.26 (td, J=7.8, 1.5 Hz, 1 H) 7.08 (td, J=6.8, 1.5 Hz, 1
H) 6.91 (d, J=1.0 Hz, 1 H) 3.45 (s, 3 H).
1-(4-Bromo-11-methylbenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one (5-
5). Acetic anhydride (1.1 mL, 11.64 mmol) was added to 4-bromo-11-methyl-2,11- dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole 5-7 (125 mg, 0.395 mmol). The reaction was refluxed at 100 °C for 3 h, then cooled to room temperature. Upon cooling, a white precipitate was observed and collected by vacuum filtration (washed with water) to afford the title compound as a white solid (126 mg, 89%). LCMS [M+H]+ 357.96 m/z (79Br), 359.93 m/z (81Br); 1H NMR (500
142
MHz, DMSO-d6) δ ppm 8.08 (s, 1 H) 7.42 (d, J=7.8 Hz, 1 H) 7.39 (s, 1 H) 7.36 (d, J=7.8 Hz, 1 H)
7.32 (t, J=7.3 Hz, 1 H) 7.18 (t, J=8.3 Hz, 1 H) 3.52 (s, 3 H) 2.63 (s, 3 H).
1-(11-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[2,3][1,4]oxazepino
[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one (5-6). 1-(4-Bromo-11-methylbenzo[2,3][1,4]oxazepine
[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one 5-8 (95 mg, 0.265 mmol), potassium acetate (92 mg,
0.937 mmol), bis(pinacolato)diboron (102 mg, 0.402 mmol), and PdCl2(dppf) (12 mg, 14.7 µmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (2.2 mL, 0.12 M) was added and the reaction was run in the microwave (145
°C) for 1 h. The reaction mixture was diluted with EtOAc and filtered through Celite®, and the crude residue was purified by flash chromatography (0-20% EtOAc:hexanes) to afford the title compound as an off-white solid (90 mg, 83%). LCMS [M+H]+ 406.09 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.24 (s, 1 H) 7.38 - 7.43 (m, 2 H) 7.26 - 7.32 (m, 2 H) 7.16 (td, J=7.6, 1.5 Hz, 1
H) 3.53 (s, 3 H) 2.64 (s, 3 H) 1.16 (s, 12 H).
143
4-(11-Methyl-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-2- amine (NEU-4389). The title compound was prepared according to General Procedure B on a 54- mg scale using 1-(11-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[2,3][1,4] oxazepino[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one 5-9 and 2-amino-4-chloropyrimidne. The crude material was purified twice by flash chromatography (20-100% EtOAc:DCM - 0-5%
MeOH:EtOAc, then 1-5% 1% NH4OH/MeOH:EtOAc) and once by preparative HPLC (5-95% water:ACN) to afford the title compound as a yellow solid (4 mg, 10%). LCMS [M+H]+ 331.07
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.45 (s, 1 H) 8.32 (d, J=5.4 Hz, 1 H) 7.82 (s, 1 H)
7.45 (s, 1 H) 7.32 - 7.38 (m, 2 H) 7.25 (t, J=7.3 Hz, 1 H) 7.22 (d, J=5.4 Hz, 1 H) 7.08 (t, J=8.3 Hz,
1 H) 6.73 (s, 2 H) 3.48 (s, 3 H).
Scheme 5-2. Synthesis of NEU-4461.
144
4-Bromo-2-methyl-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (5-7). 3-Bromo-
1-fluorodibenzo[b,f][1,4]oxazepine-11(10H)-thione 2-5 (199 mg, 0.614 mmol) was suspended in dioxane (2.5 mL, 0.25 M) and methyl hydrazine (0.10 mL, 1.90 mmol) was added. The reaction was refluxed for 4 h at 85 °C, then stopped and cooled to room temperature. Upon cooling, a precipitate was observed and collected by vacuum filtration (washed with water) to afford the title compound as an off-white solid (83 mg, 43%). LCMS [M+H]+ 315.95 m/z (79Br), 317.96 m/z
81 1 ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 9.56 (s, 1 H) 7.55 (s, 1 H) 7.21 - 7.25 (m, 2 H) 7.07
(ddd, J=8.2, 7.0, 1.5 Hz, 1 H) 6.86 - 6.91 (m, 2 H) 3.87 (s, 3 H).
2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,11-dihydrobenzo[2,3][1,4] oxazepino[5,6,7-cd]indazole (5-8). 4-Bromo-2-methyl-2,11-dihydrobenzo[2,3][1,4]oxazepino
[5,6,7-cd]indazole 5-10 (83 mg, 0.262 mmol), potassium acetate (91 mg, 0.927 mmol), bis(pinacolato)diboron (101 mg, 0.398 mmol), and PdCl2(dppf)·CH2Cl2 (12 mg, 0.015 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (2.7 mL, 0.15 M) was added and the reaction was run in the microwave (140
°C) for 30 min. The reaction was diluted with EtOAc, filtered through celite, and concentrated
145 under reduced pressure. The crude residue was purified by flash chromatography (20-60%
EtOAc:hexanes) to afford the title compound as a yellow solid (60 mg, 63%). LCMS [M+H]+
1 364.18 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.49 (s, 1 H) 7.49 (s, 1 H) 7.25 (d, J=7.8 Hz,
2 H) 7.05 (td, J=8.3, 1.5 Hz, 1 H) 6.93 (s, 1 H) 6.87 (td, J=8.3, 1.5 Hz, 1 H) 3.92 (s, 3 H) 1.33 (s,
12 H).
4-(2-Methyl-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-2-amine
(NEU-4461). The title compound was prepared according to General Procedure B on a 60-mg scale using 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,11-dihydrobenzo
[2,3][1,4]oxazepino[5,6,7-cd]indazole 5-8 and 2-amino-4-chloropyrimidine. The crude material was purified by flash chromatography (0-100% EtOAc:hexanes - 0-10% MeOH:EtOAc), then by preparative HPLC (95-30% water:ACN) to afford the title compound as a yellow solid (6 mg,
+ 1 10%). LCMS [M+H] 331.12 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.55 (s, 1 H) 8.35 (d,
J=5.4 Hz, 1 H) 7.95 (s, 1 H) 7.50 (s, 1 H) 7.30 (d, J=4.9 Hz, 1 H) 7.24 - 7.29 (m, 2 H) 7.06 (t,
J=7.8 Hz, 1 H) 6.89 (t, J=7.81 Hz, 1 H) 6.73 (s, 2 H) 3.96 (s, 3 H).
4-Chloro-N,N-dimethylpyrimidin-2-amine (5-9). 4-Chloropyrimidin-2-amine (201 mg, 1.55 mmol) was dissolved in DMF (6.2 mL, 0.25 M) and methyl iodide (250 µL, 4.02 mmol) was added.
146
The reaction was cooled to 0 °C and NaH (170 mg, 4.25 mmol) was added, upon which the reaction turned yellow. The reaction was allowed to warm to room temperature and run overnight, then quenched with water and extracted once with EtOAc. The organic layer was washed three times with water and once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The product was purified by flash chromatography (0-20% EtOAc:hexanes). LCMS
[M+H]+ 156.69 m/z (35Cl), 158.58 m/z (37Cl); 1H NMR (500 MHz, CHLOROFORM-d) δ ppm
8.16 (d, J=4.9 Hz, 1 H) 6.48 (d, J=4.9 Hz, 1 H) 3.19 (s, 6 H).
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N,N-dimethylpyrimidin-2- amine (NEU-4404). The title compound was prepared according to General Procedure B on a
100-mg scale using 4-chloro-N,N-dimethylpyrimidin-2-amine. The crude material was purified by flash chromatography (0-100% EtOAc:hexanes), then repurified by preparative HPLC (95-5% water:ACN) to afford the final compound as a yellow solid (7 mg, 8%). LCMS [M+H]+ 345.00
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.38 (s, 1 H) 9.55 (s, 1 H) 8.43 (d, J=4.9 Hz, 1 H)
7.90 (d, J=1.0 Hz, 1 H) 7.49 (d, J=1.0 Hz, 1 H) 7.25 - 7.32 (m, 3 H) 7.07 (td, J=8.3, 1.5 Hz, 1 H)
6.89 (td, J=8.3, 1.5 Hz, 1 H) 3.23 (s, 6 H).
147
4-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)-N-methylpyrimidin-2- amine (NEU-5126). The title compound was prepared according to General Procedure B on a 75- mg scale using 4-bromo-N-methylpyrimidin-2-amine. The crude material was purified by flash chromatography (70% EtOAc:hexanes) to afford the title compound as a yellow solid (13 mg,
+ 1 21%). LCMS [M+H] 331.12 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.38 (s, 1 H) 9.53 (s,
1 H) 8.36 (s, 1 H) 7.87 (s, 1 H) 7.47 (s, 1 H) 7.28 (dd, J=7.8, 1.5 Hz, 2 H) 7.24 (d, J=4.9 Hz, 1 H)
7.13 - 7.20 (m, 1 H) 7.06 (td, J=8.8, 7.3 Hz, 1 H) 6.88 (td, J=9.3, 6.3 Hz, 1 H) 2.90 (br. s., 3 H).
2-((4-Methoxybenzyl)amino)ethan-1-ol (5-10). 4-Methoxybenzaldehyde (2.50 mL, 20.57 mmol) was dissolved in DCM (60 mL, 0.34 M) and ethanolamine (3.80 mL, 62.84 mmol), TEA
(5.80 mL, 41.55 mmol), and acetic acid (2.50 mL, 43.71 mmol) were added in that order. The reaction was stirred at room temperature for 25 min, followed by the addition of NaBH(OAc)3
(8.75 g, 41.29 mmol). The reaction was stirred overnight at room temperature. The reaction was diluted with DCM and washed twice with 2 M NaOH, once with water, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The title compound was isolated by flash chromatography (0-10% MeOH:DCM) as a yellow oil (2.32 g,
62%). LCMS [M+H]+ 181.96 m/z; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.24 (d, J=8.8
148
Hz, 2 H) 6.87 (d, J=8.8 Hz, 2 H) 3.81 (s, 3 H) 3.72 - 3.77 (m, 2 H) 3.65 (m, J=6.6, 4.4, 2.2 Hz, 2
H) 2.79 (q, J=5.1 Hz, 2 H) 2.45 - 2.54 (m, 2 H).
4-Bromo-2,6-difluoro-N-(2-hydroxyethyl)-N-(4-methoxybenzyl)benzamide (2-10b). 2-((4-
Methoxybenzyl)amino)ethan-1-ol 5-10 (2.32 g, 12.8 mmol) was dissolved in DCM (30 mL, 0.43
M) and TEA (3.80 mL, 27.3 mmol) was added. 4-Bromo-2,6-difluorobenzoyl chloride (2.83 g,
11.1 mmol) was dissolved in DCM (15 mL, 0.74 M) and this solution was added to the reaction mixture dropwise at 0 °C. The reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with DCM and washed once with 1 M HCl, once with water, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The title compound was isolated by flash chromatography (20-50%
EtOAc:hexanes) as a yellow oil (3.81 g, 86%). LCMS [M+H]+ 399.86 m/z (79Br), 401.90 m/z
(81Br).
149
8-Bromo-6-fluoro-4-(4-methoxybenzyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (2-11).
Sodium hydride (766 mg, 19.2 mmol) was suspended in dry DMF (50 mL 0.38 M). 4-Bromo-2,6- difluoro-N-(2-hydroxyethyl)-N-(4-methoxybenzyl)benzamide 2-10b (3.81 g, 9.53 mmol) was dissolved in dry DMF (50 mL, 0.19 M) and added slowly to the sodium hydride suspension. The reaction was stirred at room temperature overnight. The reaction mixture was diluted with DCM and washed three times with saturated aqueous NaHCO3, once with water, and once with brine.
The organic layer was dried with sodium sulfate and concentrated under reduced pressure to afford the title compound as a solid (3.06 g, 84%). LCMS [M+H]+ 379.99 m/z (79Br), 381.99 m/z (81Br);
1 H NMR (500 MHz, DMSO-d6) δ ppm 7.46 (dd, J=9.3, 2.0 Hz, 1 H) 7.29 (d, J=8.3 Hz, 2 H) 7.19
(d, J=1.5 Hz, 1 H) 6.93 (d, J=8.3 Hz, 2 H) 4.66 (s, 2 H) 4.11 (t, J=5.6 Hz, 2 H) 3.74 (s, 3 H) 3.50
(t, J=5.6 Hz, 2 H).
8-Bromo-6-fluoro-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (2-12). 8-Bromo-6-fluoro-4-
(4-methoxybenzyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one 2-11 (1.65 g, 4.33 mmol) was taken up in 3:1 ACN:water (48 mL, 0.09 M) and cerium ammonium nitrate (7.13 g, 13.0 mmol) was added, upon which the reaction mixture turned orange. The reaction was stirred at room
150 temperature and monitored by LCMS. After 3 h, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed once with saturated aqueous NaHCO3 and once with brine. The combined organic layers were dried with sodium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography (20-80% EtOAc:hexanes) to afford the final product as a solid (410 mg, 36%).
+ 79 81 1 LCMS [M+H] 259.96 m/z ( Br), 261.96 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.56
(br. s., 1 H) 7.43 (dd, J=9.5, 1.71 Hz, 1 H) 7.20 (t, J=1.7 Hz, 1 H) 4.21 (t, J=5.6 Hz, 2 H) 3.26 (q,
J=5.5 Hz, 2 H).
8-Bromo-6-fluoro-3,4-dihydrobenzo[f][1,4]oxazepine-5(2H)-thione (2-13). 8-Bromo-6-fluoro-
3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one 2-12 (410 mg, 1.58 mmol) was suspended in toluene
(30 mL, 0.05 M) and Lawesson's reagent (702 mg, 1.74 mmol) was added. The reaction was refluxed overnight at 100 °C. The reaction mixture was concentrated under reduced pressure to a yellow residue, which was then purified by flash chromatography (0-50% EtOAc:hexanes) to afford the title compound as a solid (245 mg, 56%). LCMS [M+H]+ 275.94 m/z (79Br), 277.96 m/z
81 1 ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 10.93 (br. s., 1 H) 7.47 (dd, J=9.3, 1.95 Hz, 1 H)
7.22 (t, J=1.5 Hz, 1 H) 4.27 (t, J=5.9 Hz, 2 H) 3.42 (q, J=5.9 Hz, 2 H).
4-Bromo-2,7,8,9-tetrahydro-[1,4]oxazepino[5,6,7-cd]indazole (2-14). 8-Bromo-6-fluoro-3,4- dihydro-benzo[f][1,4]oxazepine-5(2H)-thione 2-13 (245 mg, 0.887 mmol) was dissolved in
151 dioxane (3.0 mL, 0.30 M) and hydrazine monohydrate (0.10 mL, 2.0 mmol) was added. The reaction was refluxed at 85 °C for 3 h. The reaction mixture was concentrated under reduced pressure, leaving an orange residue. The product was purified by flash chromatography (0-5%
MeOH:DCM) to afford the title compound as a solid (214 mg, 95%). LCMS [M+H]+ 253.96 m/z
79 81 1 ( Br), 255.99 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.75 (s, 1 H) 6.99 (d, J=1.5 Hz,
1 H) 6.46 (br. s., 1 H) 6.45 (d, J=1.5 Hz, 1 H) 4.32 - 4.40 (m, 2 H) 3.39 - 3.45 (m, 2 H).
1-(4-Bromo-8,9-dihydro-[1,4]oxazepino[5,6,7-cd]indazol-2(7H)-yl)ethan-1-one (2-15). Acetic anhydride (2.5 mL, 27 mmol) was added to 4-bromo-2,7,8,9-tetrahydro-[1,4]oxazepino[5,6,7- cd]indazole 2-14 (214 mg, 0.842 mmol) and the reaction was refluxed at 100 °C. After 3 h, the reaction was cooled further to 0 °C and the title compound was isolated as a yellow solid by vacuum filtration (151 mg, 60%). LCMS [M+H]+ 295.99 m/z (79Br), 297.96 m/z (81Br); 1H NMR
(500 MHz, DMSO-d6) δ ppm 7.99 (d, J=1.5 Hz, 1 H) 7.45 (br. s., 1 H) 6.99 (d, J=1.5 Hz, 1 H)
4.41 - 4.45 (m, 2 H) 3.51 - 3.55 (m, 2 H) 3.32 (s, 3 H).
1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-8,9-dihydro-[1,4]oxazepino[5,6,7- cd]indazol-2(7H)-yl)ethan-1-one (2-16). 1-(4-Bromo-8,9-dihydro-[1,4]oxazepino[5,6,7- cd]indazol-2(7H)-yl)ethan-1-one (151 mg, 0.510 mmol), potassium acetate (175 mg, 1.78 mmol),
152 bis(pinacolato)diboron (193 mg, 0.760 mmol), and PdCl2(dppf) (20 mg, 0.06 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (3.4 mL, 0.15 M) was added and the reaction was run in the microwave (145 °C) for 1 h.
The reaction mixture was then diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-75%
EtOAc:hexanes) to afford the title compound as a solid (139 mg, 79%). LCMS [M+H]+ 344.12
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.18 (s, 1 H) 7.40 (br. s., 1 H) 6.97 (s, 1 H) 4.38 - 4.43
(m, 2 H) 3.53 (m, J=4.9 Hz, 2 H) 2.51 (s, 3 H) 1.31 (s, 12 H).
6-(2,7,8,9-Tetrahydro-[1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidine-2,4-diamine (2-
17a/NEU-2586). 1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-8,9-dihydro-[1,4]oxazepino
[5,6,7-cd]indazol-2(7H)-yl)ethan-1-one 2-16 (29 mg, 0.085 mmol), potassium carbonate (25 mg,
0.18 mmol), and 2,6-diamino-4-chloropyrimidine (20 mg, 0.14 mmol) were suspended in 3:1 dioxane:water (1.2 mL, 0.07 M) and the reaction vial was degassed with nitrogen for 10 min.
Pd(PPh3)4 (5 mg, 4 µmol) was added and the reaction was degassed for an additional 5 min.
Tricyclohexylphosphine (3 mg, 10 µmol) was added and the reaction was run at 100 °C under nitrogen for 3 h. Upon completion, the reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (3-10% 15% NH4OH in MeOH:DCM), then repurified by preparative HPLC (5-
95% ACN:water) to afford the title compound as a solid (8 mg, 33%). LCMS [M+H]+ 284.12 m/z;
153
1H NMR (500 MHz, Acetone) δ ppm 11.10 (br. s, 1 H) 8.14 (s, 1 H) 7.60 (s, 1 H) 6.97 (d, J=1.0
Hz, 1 H) 6.41 (s, 1 H) 5.97 (br. s, 2 H) 5.68 (br. s, 2 H) 4.41 - 4.47 (m, 2 H) 3.60 (m, J=6.3 Hz, 2
H).
6-(2,7,8,9-Tetrahydro-[1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-4-amine (2-17b/NEU-
2587). 1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-8,9-dihydro-[1,4]oxazepino[5,6,7- cd]indazol-2(7H)-yl)ethan-1-one 2-16 (29 mg, 0.085 mmol), potassium carbonate (27 mg, 0.20 mmol), and 4-amino-6-chloropyrimidine (20 mg, 0.15 mmol) were suspended in 3:1 dioxane:water
(1.2 mL, 0.07 M) and the reaction vial was degassed with nitrogen for 10 min. Pd(PPh3)4 (5 mg, 4
µmol) was added and the reaction was degassed for an additional 5 min. Tricyclohexylphosphine
(3 mg, 10 µmol) was added and the reaction was run at 100 °C for 3 h. Upon completion, the reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (3-10% MeOH:DCM), then repurified by preparative HPLC (5-95% ACN:water) to afford the title compound as a solid (3 mg,
12%). LCMS [M+H]+ 269.11 m/z; 1H NMR (500 MHz, Acetone) δ ppm 11.03 (br. s, 1 H) 8.47 (s,
1 H) 7.67 (s, 1 H) 7.04 (s, 2 H) 6.21 (br. s, 2 H) 5.71 (br. s, 1 H) 4.44 - 4.48 (m, 2 H) 3.59 - 3.63
(m, 2 H).
154
Scheme 5-3. Synthesis of NEU-4892.
4-Bromo-2-fluorobenzoyl chloride (5-10). To 4-bromo-2-fluorobenzoic acid 5-9 (1.00 g, 4.57 mmol) was added thionyl chloride (3.3 mL, 45.5 mmol). The reaction was refluxed at 75 °C for 5 h. Excess thionyl chloride was removed by distillation and the product was azeotroped with toluene three times to afford the title compound as a tan solid which was used in the next reaction without further purification.
4-Bromo-2-fluoro-N-(2-hydroxyphenyl)benzamide (5-11). 2-Aminophenol (253 mg, 2.32 mmol) was dissolved in DCM (6.0 mL) and TEA (0.400 mL, 2.87 mmol) was added. 4-Bromo-2- fluorobenzoyl chloride 5-10 (500 mg, 2.11 mmol) was dissolved in DCM (3.0 mL) and this
155 solution was added to the reaction mixture dropwise at 0 °C. The reaction was allowed to warm to room temperature and stirred overnight. Upon the addition of 1M HCl, an off-white precipitate was observed; this was collected by vacuum filtration to afford the title compound as an off-white solid (341 mg, 52%). LCMS [M+H]+ 310.03 (79Br) m/z, 311.99 (81Br); 1H NMR (500 MHz,
DMSO-d6) δ ppm 10.04 (s, 1 H) 9.47 (d, J=7.3 Hz, 1 H) 8.01 (d, J=7.8 Hz, 1 H) 7.81 (t, J=8.3 Hz,
1 H) 7.76 (dd, J=10.7, 1.5 Hz, 1 H) 7.59 (dd, J=8.3, 1.5 Hz, 1 H) 7.00 (td, J=8.8, 1.0 Hz, 1 H) 6.92
(d, J=7.8 Hz, 1 H) 6.83 (t, J=7.8 Hz, 1 H).
3-Bromodibenzo[b,f][1,4]oxazepin-11(10H)-one (5-12). 4-Bromo-2-fluoro-N-(2- hydroxyphenyl)benzamide 5-11 (219 mg, 0.938 mmol) was dissolved in DMF (7.2 mL, 0.13 M) and cesium carbonate (457 mg, 1.40 mmol) was added. The reaction was stirred at 50 °C overnight.
The reaction was stopped and cooled to room temperature. Upon addition of water, a gray precipitate was observed; this was collected by vacuum filtration and washed with water to afford the title compound as a gray solid (244 mg, 90%). LCMS [M+H]+ 290.03 m/z (79Br), 219.99 (81Br)
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 10.62 (s, 1 H) 7.67 - 7.72 (m, 2 H) 7.54 (dd, J=8.3,
2.0 Hz, 1 H) 7.37 (dd, J=7.3, 1.5 Hz, 1 H) 7.13 - 7.23 (m, 3 H).
156
3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)dibenzo[b,f][1,4]oxazepin-11(10H)-one (5-
13). 3-Bromodibenzo[b,f][1,4]oxazepin-11(10H)-one 5-12 (244 mg, 0.841 mmol), potassium acetate (290 mg, 2.95 mmol), bis(pinacolato)diboron (322 mg, 1.23 mmol), and
PdCl2(dppf)·CH2Cl2 (33 mg, 0.040 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (9.5 mL, 0.09 M) was added and the reaction was run in the microwave (145 °C) for 30 min. The reaction was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-30% EtOAc:hexanes) to afford the title compound as a tan solid (250
+ 1 mg, 88%). LCMS [M+H] 338.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 10.59 (s, 1 H) 7.78
(d, J=7.3 Hz, 1 H) 7.53 - 7.59 (m, 2 H) 7.41 (d, J=7.8 Hz, 1 H) 7.10 - 7.20 (m, 3 H) 1.30 (s, 12 H).
3-(2-Aminopyrimidin-4-yl)dibenzo[b,f][1,4]oxazepin-11(10H)-one (NEU-4892). 3-(4,4,5,5-
Tetramethyl-1,3,2-dioxaborolan-2-yl)dibenzo[b,f][1,4]oxazepin-11(10H)-one 5-13 (76 mg, 0.225 mmol), K2CO3 (94 mg, 0.680 mmol), and 2-amino-4-chloropyrimidine (48 mg, 0.370 mmol) were
157 combined in a reaction vial and 3:1 dioxane:water (2.2 mL, 0.10 M) was added. The reaction was degassed for 10 min and Pd(PPh3)4 (15 mg, 0.013 mmol) was added. The reaction was refluxed at
100 °C for 3 h, then cooled to room temperature and stirred overnight. The reaction mixture was diluted with EtOAc and the crude material was purified by flash chromatography (50-100%
EtOAc:hexanes), then repurified by preparative HPLC (5-95% water:ACN) to afford the title compound as a white solid (4 mg, 6%). LCMS [M+H]+ 305.11 m/z; 1H NMR (500 MHz, DMSO- d6) δ ppm 10.63 (s, 1 H) 8.54 (s, 1 H) 8.38 (d, J=5.4 Hz, 1 H) 8.02 (d, J=1.5 Hz, 1 H) 7.99 (dd,
J=7.3, 2.0 Hz, 1 H) 7.88 (d, J=8.3 Hz, 1 H) 7.37 (d, J=6.8 Hz, 1 H) 7.13 - 7.25 (m, 4 H) 6.82 (s, 1
H).
Scheme 5-4. Synthesis of NEU-4895.
158
(1R,2R)-2-((4-Methoxybenzyl)amino)cyclopentan-1-ol (5-14). 4-Methoxybenzaldehyde (0.46 mL, 3.78 mmol) was dissolved in DCM (14.5 mL, 0.26 M) and (1R,2R)-2-aminocyclohexan-1-ol hydrochloride 5-18 (701 mg, 5.09 mmol), TEA (1.0 mL, 7.17 mmol), and acetic acid (0.43 mL,
7.52 mmol) were added in that order. The reaction was stirred at room temperature for 30 min.
NaHB(OAc)3 (1.47 g, 6.94 mmol) was added and the reaction was stirred at room temperature overnight. The reaction was diluted with DCM and washed twice with 1M NaOH, once with water, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-100%
EtOAc:hexanes - 0-20% DCM:EtOAc) to afford the title compound as an off-white solid (417 mg,
+ 1 37%). LCMS [M+H] 222.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 7.22 (d, J=8.8 Hz, 2 H)
6.86 (d, J=8.3 Hz, 2 H) 4.47 (d, J=4.4 Hz, 1 H) 3.73 - 3.77 (m, 1 H) 3.72 (s, 3 H) 3.61 (q, J=13.2
Hz, 2 H) 2.72 (q, J=6.3 Hz, 1 H) 1.91 (br. s, 1 H) 1.79 (spt, J=6.3 Hz, 2 H) 1.50 - 1.61 (m, 2 H)
1.34 - 1.42 (m, 1 H) 1.26 (sxt, J=12.7 Hz, 1 H).
159
4-Bromo-2,6-difluoro-N-((1R,2R)-2-hydroxycyclopentyl)-N-(4-methoxybenzyl)benzamide
(5-15). (1R,2R)-2-((4-Methoxybenzyl)amino)cyclopentan-1-ol 5-14 (417 mg, 1.88 mmol) was dissolved in DCM (4.4 mL) and TEA (0.40 mL, 2.87 mmol) was added. 4-Bromo-2,6- difluorobenzoyl chloride 2-2 (526 mg, 2.06 mmol) was dissolved in another DCM (2.2 mL) and this solution was added to the reaction mixture dropwise at 0 °C. The reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with DCM and washed once with 1M HCl, once with water, and once with brine. The combined organic layers were dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:hexanes) to afford the title compound as an orange oil (764 mg) which was used without further purification. LCMS [M+H]+ 440.13 m/z
79 81 1 ( Br), 442.15 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 7.61 (d, J=7.3 Hz, 2 H) 7.25 (d,
J=8.8 Hz, 2 H) 6.88 (d, J=8.8 Hz, 2 H) 4.78 (q, J=4.9 Hz, 2 H) 3.94 (quin, J=7.8 Hz, 1 H) 3.73 (s,
3 H) 3.59 (q, J=8.3 Hz, 1 H) 1.55 - 1.78 (m, 3 H) 1.38 - 1.54 (m, 3 H) 1.26 (td, J=7.8, 4.9 Hz, 1
H).
160
(3aR,10aR)-6-Bromo-8-fluoro-10-(4-methoxybenzyl)-1,2,3,3a,10,10a-hexahydro-9H- benzo[f]cyclopenta[b][1,4]oxazepin-9-one (5-16). 4-Bromo-2,6-difluoro-N-((1R,2R)-2- hydroxycyclopentyl)-N-(4-methoxybenzyl)benzamide 5-15 (764 mg, 1.74 mmol) was dissolved in DMF (12.5 mL, 0.14 M) and cesium carbonate (849 mg, 2.61 mmol) was added. The reaction was stirred at room temperature overnight. The reaction temperature was then increased to 50 °C and the reaction was heated another 24 h. The reaction was stopped and cooled to room temperature. The reaction mixture was diluted with EtOAc and washed three times with water.
The combined aqueous layers were extracted once with EtOAc, and the combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure to afford the title compound as a yellow oil (614 mg, 84%). LCMS [M+H]+ 420.06 m/z (79Br),
81 1 422.04 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 7.38 (dd, J=9.8, 2.0 Hz, 1 H) 7.22 (d,
J=8.3 Hz, 2 H) 7.07 (s, 1 H) 6.88 - 6.93 (m, 2 H) 4.69 (s, 2 H) 4.51 (q, J=9.0 Hz, 1 H) 4.09 (ddd,
J=11.5, 9.8, 7.6 Hz, 1 H) 3.73 (s, 3 H) 1.84 - 1.91 (m, 1 H) 1.75 - 1.84 (m, 1 H) 1.67 - 1.75 (m, 1
H) 1.54 - 1.66 (m, 2 H) 1.44 - 1.54 (m, 1 H).
(3aR,10aR)-6-Bromo-8-fluoro-1,2,3,3a,10,10a-hexahydro-9H-benzo[f]cyclopenta[b][1,4] oxazepin-9-one (5-17). To (3aR,10aR)-6-Bromo-8-fluoro-10-(4-methoxybenzyl)-
161
1,2,3,3a,10,10a-hexahydro-9H-benzo[f]cyclopenta[b][1,4]oxazepin-9-one 5-16 (614 mg, 1.46 mmol) was added TFA (1.1 mL, 14.37 mmol). The reaction was run in the microwave (140 °C) for 30 min. The reaction mixture was then diluted with EtOAc and poured over sat. aq. NaHCO3.
The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:hexanes) to afford the title compound as a pale yellow solid (337 mg, 77%). LCMS [M+H]+ 300.02 m/z (79Br), 302.03 m/z
81 1 ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.57 (d, J=5.4 Hz, 1 H) 7.21 (dd, J=10.3, 2.0 Hz, 1
H) 7.02 (t, J=1.7 Hz, 1 H) 4.52 (q, J=8.0 Hz, 1 H) 3.66 - 3.77 (m, 1 H) 2.06 - 2.16 (m, 1 H) 1.96
(m, J=7.8 Hz, 1 H) 1.62 - 1.77 (m, 4 H).
(3aR,10aR)-6-Bromo-8-fluoro-1,2,3,3a,10,10a-hexahydro-9H-benzo[f]cyclopenta[b][1,4] oxazepine-9-thione (5-18). The (3aR,10aR)-6-bromo-8-fluoro-1,2,3,3a,10,10a-hexahydro-9H- benzo[f]cyclopenta[b][1,4]oxazepin-9-one 5-17 (287 mg, 0.956 mmol) was suspended in toluene
(19 mL, 0.05 M) and Lawesson's reagent (777 mg, 1.92 mmol) was added. The reaction was refluxed at 100 °C overnight. The reaction was stopped and cooled to room temperature and the crude material was purified by flash chromatography (20-50% EtOAc:hexanes) to afford the title compound as a yellow solid (171 mg) which was used in next reaction without further purification.
+ 79 81 1 LCMS [M+H] 315.99 m/z ( Br), 318.03 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.07
(d, J=5.4 Hz, 1 H) 7.33 (dd, J=9.8, 2.0 Hz, 1 H) 7.03 (t, J=1.5 Hz, 1 H) 4.61 (q, J=9.8 Hz, 1 H)
162
3.95 (quint, J=10.3, 10.3, 10.3, 10.3, 8.3, 8.3 Hz, 1 H) 1.91 - 1.97 (m, 1 H) 1.75 - 1.90 (m, 2 H)
1.62 - 1.71 (m, 2 H) 1.51 - 1.61 (m, 1 H).
(6aR,9aR)-4-Bromo-6a,7,8,9,9a,10-hexahydro-2H-cyclopenta[2,3][1,4]oxazepino[5,6,7- cd]indazole (5-19). (3aR,10aR)-6-Bromo-8-fluoro-1,2,3,3a,10,10a-hexahydro-9H-benzo[f] cyclopentab][1,4]oxazepine-9-thione 5-18 (171 mg, 0.540 mmol) was suspended in dioxane (1.8 mL, 0.30 M), and hydrazine monohydrate (0.15 mL, 3.06 mmol) was added. The reaction was refluxed for 3 h at 85 °C. The reaction mixture was concentrated under reduced pressure and the crude material was purified by flash chromatography (50-80% EtOAc:hexanes) to afford the title compound as a tan solid (149 mg, 94%). LCMS [M+H]+ 294.09 m/z (79Br), 296.06 m/z (81Br); 1H
NMR (500 MHz, DMSO-d6) δ ppm 11.80 (s, 1 H) 6.99 (d, J=1.5 Hz, 1 H) 6.58 (s, 1 H) 6.45 (d,
J=1.5 Hz, 1 H) 4.31 (q, J=6.8 Hz, 1 H) 3.48 (q, J=8.8 Hz, 1 H) 2.29 (m, J=8.3, 8.3, 6.3, 6.3, 6.3
Hz, 1 H) 2.18 (m, J=7.8 Hz, 1 H) 1.90 (sxt, J=7.3 Hz, 1 H) 1.75 - 1.84 (m, 1 H) 1.61 - 1.75 (m, 2
H).
1-((6aR,9aR)-4-Bromo-6a,7,8,9,9a,10-hexahydro-2H-cyclopenta[2,3][1,4]oxazepino[5,6,7- cd]indazol-2-yl)ethan-1-one (5-20). Acetic anhydride (2.7 mL, 28.56 mmol) was added to
163
(6aR,9aR)-4-bromo-6a,7,8,9,9a,10-hexahydro-2H-cyclopenta[2,3][1,4]oxazepino[5,6,7- cd]indazole 5-19 (130 mg, 0.442 mmol), upon which the reaction mixture turned bright yellow, and the reaction was refluxed at 100 °C for 3 h. The reaction was kept at room temperature for an additional 12 h, after which a yellow precipitate was observed and collected by vacuum filtration
(washed with water) to afford the title compound as a yellow solid (89 mg, 60%). LCMS [M+H]+
79 81 1 336.06 m/z ( Br), 338.08 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.03 (d, J=1.5 Hz, 1
H) 7.63 (s, 1 H) 6.99 (d, J=1.5 Hz, 1 H) 4.38 (q, J=8.3 Hz, 1 H) 3.62 (q, J=8.8 Hz, 1 H) 2.51 (s, 3
H) 2.27 - 2.39 (m, 1 H) 2.16 - 2.25 (m, 1 H) 1.87 - 1.98 (m, 1 H) 1.77 - 1.85 (m, 1 H) 1.60 - 1.76
(m, 2 H).
1-((6aR,9aR)-4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-6a,7,8,9,9a,10-hexahydro-2H- cyclopenta[2,3][1,4]oxazepino[5,6,7-cd]indazol-2-yl)ethan-1-one (5-21). 1-((6aR,9aR)-4-
Bromo-6a,7,8,9,9a,10-hexahydro-2H-cyclopenta[2,3][1,4]oxazepino[5,6,7-cd]indazol-2- yl)ethan-1-one 5-20 (79 mg, 0.235 mmol), potassium acetate (81 mg, 0.825 mmol), bis(pinacolato)diboron (93 mg, 0.366 mmol), and PdCl2(dppf)·CH2Cl2 (10 mg, 12.3 µmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (2.6 mL, 0.09 M) was added and the reaction was run in the microwave (145 °C) for 20 min. The reaction was diluted with MeOH, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50%
EtOAc:hexanes) to afford the title compound as an off-white solid (56 mg, 62%). LCMS [M+H]+
164
1 384.26 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.20 (s, 1 H) 7.58 (s, 1 H) 6.97 (s, 1 H) 4.33
(q, J=7.8 Hz, 1 H) 3.61 (q, J=8.3 Hz, 1 H) 2.27 - 2.38 (m, 1 H) 2.17 - 2.26 (m, 1 H) 1.88 - 1.97
(m, 1 H) 1.78 - 1.88 (m, 1 H) 1.61 - 1.77 (m, 2 H) 1.34 (s, 3 H) 1.31 (s, 12 H).
1-((6aR,9aR)-4-(2-Aminopyrimidin-4-yl)-6a,7,8,9,9a,10-hexahydro-2H- cyclopenta[2,3][1,4]oxazepino[5,6,7-cd]indazol-2-yl)ethan-1-one (NEU-4895). (6aR,9aR)-4-
(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-6a,7,8,9,9a,10-hexahydro-2H-cyclopenta
[2,3][1,4]oxazepino[5,6,7-cd]indazole 4-21 (56 mg, 0.164 mmol), K2CO3 (70 mg, 0.507 mmol), and 2-amino-4-chloropyrimidine (34 mg, 0.262 mmol) were combined in a reaction vial and 3:1 dioxane:water (1.6 mL, 0.10 M) was added. The reaction was degassed for 10 min and
PdCl2(dppf)·CH2Cl2 (8 mg, 0.010 mmol) was added. The reaction was refluxed at 100 °C overnight. The reaction mixture was diluted with MeOH, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography
(1-15% 10% NH4OH/MeOH:DCM), then repurified by flash chromatography (0-10%
MeOH:EtOAc) to afford the title compound as a yellow solid (25 mg, 56%). LCMS [M+H]+
1 309.17 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.28 (d, J=5.9 Hz, 1 H) 7.64 (s, 1 H)
7.25 (d, J=5.9 Hz, 1 H) 7.12 (s, 1 H) 4.36 - 4.48 (m, 1 H) 3.66 (q, J=8.8 Hz, 1 H) 2.27 - 2.46 (m,
2 H) 2.02 - 2.12 (m, 1 H) 1.89 - 1.98 (m, 1 H) 1.73 - 1.88 (m, 2 H).
165
6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (2-24). 6-Bromo-1H-indazole
(251 mg, 1.27 mmol), bis(pinacolato)diboron (480 mg, 1.89 mmol), potassium acetate (433 mg,
4.41 mmol) and PdCl2(dppf)·CH2Cl2 (53 mg, 0.065 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (10 mL, 0.13 M) was added and the reaction was run in the microwave (145 °C) for 3 h. The reaction was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:hexanes) to afford the title compound as a
+ 1 solid (183 mg, 59%). LCMS [M+H] 245.11 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 13.16
(s, 1 H) 8.09 (s, 1 H) 7.86 (s, 1 H) 7.75 (d, J=7.8 Hz, 1 H) 7.36 (d, J=7.8 Hz, 1 H) 1.32 (s, 12 H).
6-(1H-Indazol-6-yl)pyrimidine-2,4-diamine (2-26a/NEU-2209). The title compound was prepared according to General Procedure C on a 54-mg scale using 2,6-diamino-4- chloropyrimidine. The crude material was purified by flash chromatography (0-10% 10% NH4OH in MeOH:DCM) to afford the title compound as a solid (16 mg, 43%). LCMS [M+H]+ 227.08 m/z;
1 H NMR (399 MHz, DMSO-d6) δ ppm 8.11 (s, 1 H) 8.09 (s, 1 H) 7.79 (d, J=8.8 Hz, 1 H) 7.61 (d,
J=9.5 Hz, 1 H) 6.38 (br. s., 2 H) 6.29 (s, 1 H) 6.00 (br. s., 2 H).
166
6-(1H-Indazol-6-yl)pyrimidin-4-amine (2-26b/NEU-2198). The title compound was prepared according to General Procedure C on a 66-mg scale using 4-amino-6-chloropyrimidine. The crude material was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound
+ 1 as a solid (16 mg, 28%). LCMS [M+H] 212.00 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 13.25
(s, 1 H) 8.46 (s, 1 H) 8.18 (s, 1 H) 8.12 (s, 1 H) 7.85 (d, J=8.3 Hz, 1 H) 7.70 (dd, J=8.6, 1.2 Hz, 1
H) 6.97 (s, 1 H) 6.92 (s, 2 H).
4-(1H-Indazol-6-yl)pyrimidin-2-amine (2-26c/NEU-2208). The title compound was prepared according to General Procedure C on a 44-mg scale using 2-amino-4-chloropyrimidine. The crude material was purified by flash chromatography (0-10% 10% NH4OH in MeOH:DCM) to afford the title compound as a solid. (16 mg, 43%). LCMS [M+H]+ 212.00 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 8.32 (d, J=5.1 Hz, 1 H) 8.27 (s, 1 H) 8.13 (s, 1 H) 8.05 (d, J=2.2 Hz, 1 H) 7.79
- 7.88 (m, 2 H) 7.21 (d, J=5.1 Hz, 1 H) 6.70 (s, 2 H).
4-(2,6-Diaminopyrimidin-4-yl)-2-fluorobenzonitrile (2-29a). (4-Cyano-3-fluorophenyl)boronic acid 2-28 (75 mg, 0.46 mmol) and 6-chloropyrimidin-2,4-diamine (67 mg, 0.46 mmol) were
167 combined in a reaction vial and dissolved in dioxane (3.0 mL, 0.15 M), followed by the addition of saturated aqueous NaHCO3 (0.75 mL). The reaction vial was purged with nitrogen for 10 min and Pd(PPh3)4 (28 mg, 0.024 mmol) was added. The reaction was run at 95 °C for 3 h. Upon completion, the reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated in vacuo. The crude material was purified by flash chromatography (0-7%
MeOH:DCM) to afford the title compound as a yellow solid (75 mg, 72%). LCMS [M+H]+ 230.03
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.00 (t, J=7.3 Hz, 1 H) 7.95 (d, J=11.2 Hz, 1 H) 7.88
(dd, J=8.8, 1.47 Hz, 1 H) 6.54 (br. s., 2 H) 6.29 (s, 1 H) 6.12 (s, 2 H).
4-(6-Aminopyrimidin-4-yl)-2-fluorobenzonitrile (2-29b). (4-Cyano-3-fluorophenyl)boronic acid 2-28 (75 mg, 0.46 mmol) and 6-chloropyrimidin-4-amine (60 mg, 0.46 mmol) were combined in a reaction vial and dissolved in dioxane (3.0 mL, 0.15 M), followed by the addition of saturated aqueous NaHCO3 (0.75 mL). The reaction vial was purged with nitrogen for 10 min and Pd(PPh3)4
(27 mg, 0.023 mmol) was added. The reaction was run at 95 °C for 3 h. Upon completion, the reaction mixture was diluted with EtOAc, filtered through celite, and concentrated in vacuo. The crude material was purified by flash chromatography (70-100% EtOAc:hexanes) to afford the title compound as an off-white solid (42 mg, 43%). LCMS [M+H]+ 214.95 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.49 (s, 1 H) 8.02 - 8.09 (m, 2 H) 7.98 (d, J=7.8 Hz, 1 H) 7.13 (s, 2 H) 6.99 (s,
1 H).
168
4-(2-Aminopyrimidin-4-yl)-2-fluorobenzonitrile (2-29c). (4-Cyano-3-fluorophenyl)boronic acid 2-28 (75 mg, 0.46 mmol) and 4-chloropyrimidin-2-amine (59 mg, 0.46 mmol) were combined in a reaction vial and dissolved in dioxane (3.0 mL, 0.15 M), followed by the addition of saturated aqueous NaHCO3 (0.75 mL). The reaction vial was purged with nitrogen for 10 min and Pd(PPh3)4
(27 mg, 0.023 mmol) was added. The reaction was run at 95 °C for 3 h. Upon completion, the reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated in vacuo.
The crude material was purified by flash chromatography (0-50% EtOAc:hexanes) to afford the title compound as an off-white solid (93 mg, 97%). LCMS [M+H]+ 215.01 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 8.42 (d, J=5.4 Hz, 1 H) 8.17 (d, J=11.7 Hz, 1 H) 8.11 (s, 2 H) 7.29 (d,
J=4.9 Hz, 1 H) 6.88 (s, 2 H).
6-(3-Amino-1H-indazol-6-yl)pyrimidine-2,4-diamine (2-30a/NEU-4390). 4-(2,6-
Diaminopyrimidin-4-yl)-2-fluorobenzonitrile 2-29a (50 mg, 0.218 mmol) was suspended in EtOH
(3.7 mL, 0.06 M) and hydrazine monohydrate (0.900 mL, 18.33 mmol) was added. The reaction was run overnight at 95 °C. Upon cooling, an off-white precipitate was observed and collected by vacuum filtration to afford the title compound as an off-white solid (29 mg, 55%). LCMS [M+H]+
169
1 242.05 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.51 (s, 1 H) 7.81 (s, 1 H) 7.69 (d, J=8.3 Hz,
1 H) 7.39 (d, J=8.8 Hz, 1 H) 6.33 (br. s., 2 H) 6.24 (s, 1 H) 5.95 (br. s., 2 H) 5.37 (s, 2 H).
6-(6-Aminopyrimidin-4-yl)-1H-indazol-3-amine (2-30b/NEU-4362). 4-(6-Aminopyrimidin-4- yl)-2-fluorobenzonitrile 2-29b (42 mg, 0.196 mmol) was suspended in EtOH (3.3 mL, 0.06 M) and hydrazine monohydrate (0.800 mL, 16.30 mmol) was added. The reaction was run overnight at 95 °C. Upon cooling, a yellow precipitate was observed and collected by vacuum filtration to afford the title compound as a yellow solid (25 mg, 57%). LCMS [M+H]+ 227.01 m/z; 1H NMR
(500 MHz, DMSO-d6) δ ppm 11.58 (s, 1 H) 8.44 (d, J=1.0 Hz, 1 H) 7.88 (s, 1 H) 7.75 (d, J=8.3
Hz, 1 H) 7.48 (dd, J=8.6, 1.22 Hz, 1 H) 6.92 (d, J=1.0 Hz, 1 H) 6.89 (s, 2 H) 5.42 (br. s., 2 H).
6-(2-Aminopyrimidin-4-yl)-1H-indazol-3-amine (2-30c/NEU-4361). 4-(2-Aminopyrimidin-4- yl)-2-fluorobenzonitrile 2-29c (50 mg, 0.233 mmol) was suspended in EtOH (3.7 mL, 0.06 M) and hydrazine monohydrate (0.900 mL, 18.33 mmol) was added. The reaction was run overnight at 95
°C. Upon cooling, a yellow precipitate was observed and collected by vacuum filtration to afford the title compound as a yellow solid (44 mg, 83%). LCMS [M+H]+ 227.03 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 11.62 (s, 1 H) 8.29 (d, J=4.9 Hz, 1 H) 7.98 (s, 1 H) 7.76 (d, J=8.3 Hz, 1
H) 7.58 (dd, J=8.6, 1.22 Hz, 1 H) 7.16 (d, J=5.4 Hz, 1 H) 6.66 (s, 2 H) 5.44 (br. s., 2 H).
170
4-Bromo-2-fluoro-6-methoxybenzonitrile (2-21a). The title compound was prepared according to General Procedure D on a 1.0-g scale using MeOH. The reaction was stirred at room temperature for two days and the crude material was purified by flash chromatography (0-20% EtOAc:hexanes)
1 to afford the title compound as a white solid (906 mg, 86%). H NMR (500 MHz, DMSO-d6) δ ppm 7.49 (dd, J=8.8, 1.5 Hz, 1 H) 7.41 (s, 1 H) 3.98 (s, 3 H).
4-Bromo-2-ethoxy-6-fluorobenzonitrile (2-21b). The title compound was prepared according to
General Procedure D on a 302-mg scale using EtOH. The reaction was stirred at room temperature overnight, then heated to 50 °C for another 24 h. The crude material was purified by flash chromatography (0-20% EtOAc:hexanes) to afford the title compound as a white solid (280 mg,
1 83%). H NMR (400 MHz, DMSO-d6) δ ppm 7.48 (d, J=8.8 Hz, 1 H) 7.40 (s, 1 H) 4.26 (q, J=7.3
Hz, 2 H) 1.36 (t, J=7.0 Hz, 3 H).
4-Bromo-2-(tert-butoxy)-6-fluorobenzonitrile (2-21c). The title compound was prepared according to General Procedure D on a 300-mg scale using tert-butanol. The reaction was stirred at room temperature overnight, then heated to 50 °C for another 24 h. The crude material was purified by flash chromatography (0-20% EtOAc:hexanes) to afford the title compound as a yellow
171
1 oil (258 mg, 69%). H NMR (400 MHz, DMSO-d6) δ ppm 7.60 (d, J=8.8 Hz, 1 H) 7.40 (s, 1 H)
1.45 (s, 9 H).
4-Bromo-2-cyclobutoxy-6-fluorobenzonitrile (2-21d). The title compound was prepared according to General Procedure D on a 300-mg scale using cyclobutanol. The reaction was stirred at room temperature overnight, then heated to 50 °C for another 24 h. The crude material was purified by flash chromatography (0-20% EtOAc:Hexanes) to afford the title compound as a
1 yellow oil (259 mg, 70%). H NMR (400 MHz, DMSO-d6) δ ppm 7.48 (d, J=9.5 Hz, 1 H) 7.18 (s,
1 H) 4.96 (quin, J=7.0 Hz, 1 H) 2.46 (br. s., 2 H) 2.09 (quin, J=10.3 Hz, 2 H) 1.82 (q, J=10.3 Hz,
1 H) 1.63 (tq, J=10.3, 8.8 Hz, 1 H).
4-Bromo-2-fluoro-6-((tetrahydro-2H-pyran-4-yl)oxy)benzonitrile (2-21e). The title compound was prepared according to General Procedure D on a 51-mg scale using tetrahydro-2H-pyran-4- ol. The reaction mixture was stirred at room temperature overnight and the crude material was purified by flash chromatography (20-30% EtOAc:hexanes) to afford the title compound as an off-
1 white solid (37 mg, 53%). H NMR (500 MHz, DMSO-d6) δ ppm 7.57 (s, 1 H) 7.47 (dd, J=8.8,
1.5 Hz, 1 H) 4.94 (tt, J=8.2, 4.0 Hz, 1 H) 3.79 - 3.89 (m, 2 H) 3.52 (ddd, J=11.5, 8.5, 2.9 Hz, 2 H)
1.93 - 2.05 (m, 2 H) 1.63 (dtd, J=12.9, 8.6, 8.6, 3.7 Hz, 2 H).
172
4-Bromo-2-fluoro-6-((tetrahydro-2H-pyran-3-yl)oxy)benzonitrile (2-21f). The title compound was prepared according to General Procedure D on a 200-mg scale using tetrahydro-2H-pyran-3- ol. The reaction was stirred at room temperature overnight and the crude material was purified by flash chromatography (0-20% EtOAc:hexanes) to afford the title compound as a yellow solid (143
1 mg, 52%). H NMR (500 MHz, DMSO-d6) δ ppm 7.53 (s, 1 H) 7.47 (dd, J=8.8, 1.5 Hz, 1 H) 4.74
(m, J=2.4 Hz, 1 H) 3.75 (dd, J=12.0, 2.2 Hz, 1 H) 3.53 - 3.65 (m, 3 H) 1.94 - 2.05 (m, 1 H) 1.72 -
1.87 (m, 2 H) 1.49 - 1.58 (m, 1 H).
4-Bromo-2-fluoro-6-((tetrahydrofuran-3-yl)oxy)benzonitrile (2-21g). The title compound was prepared according to General Procedure D on a 204-mg scale using tetrahydrofuran-3-ol. The reaction was stirred at room temperature for two days and the crude material was purified by flash chromatography (0-30% EtOAc:hexanes) to afford the title compound as a white solid (183 mg,
1 68%). H NMR (500 MHz, DMSO-d6) δ ppm 7.50 (dd, J=8.8, 1.5 Hz, 1 H) 7.43 (s, 1 H) 5.32 (t,
J=5.1 Hz, 1 H) 3.82 - 3.92 (m, 3 H) 3.77 (td, J=8.3, 4.4 Hz, 1 H) 2.28 (sxt, J=7.3 Hz, 1 H) 1.95 -
2.04 (m, 1 H).
173
4-Bromo-2-fluoro-6-(oxetan-3-yloxy)benzonitrile (2-21h). The title compound was prepared according to General Procedure D on a 200-mg scale using oxetan-3-ol. The reaction was stirred at room temperature for two days and the crude material was purified by flash chromatography (0-
50% EtOAc:hexanes) to afford the title compound as a beige solid (121 mg, 49%). 1H NMR (500
MHz, DMSO-d6) δ ppm 7.55 (dd, J=8.8, 1.5 Hz, 1 H) 7.07 (s, 1 H) 5.52 (quin, J=5.4 Hz, 1 H) 4.97
(t, J=6.8 Hz, 2 H) 4.58 (dd, J=7.6, 4.6 Hz, 2 H).
6-Bromo-4-methoxy-1H-indazol-3-amine (2-22a). The title compound was prepared according to General Procedure E on a 906-mg scale using 4-bromo-2-fluoro-6-methoxybenzonitrile 2-21a.
The crude material was purified by flash chromatography (20-50% EtOAc:hexanes) to afford the title compound as a white solid (678 mg, 71%). LCMS [M+H]+ 241.91 m/z (79Br), 243.91 m/z
81 1 ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.53 (br. s., 1 H) 6.98 (d, J=1.5 Hz, 1 H) 6.44 (d,
J=1.0 Hz, 1 H) 5.07 (s, 2 H) 3.87 (s, 3 H).
6-Bromo-4-ethoxy-1H-indazol-3-amine (2-22b). The title compound was prepared according to
General Procedure E on a 280-mg scale using 4-bromo-2-ethoxy-6-fluorobenzonitrile 2-21b. The crude material was purified by flash chromatography (0-60% EtOAc:hexanes) to afford the title
174 compound as a yellow solid (240 mg, 82%). LCMS [M+H]+ 256.00 m/z (79Br), 258.01 m/z (81Br);
1 H NMR (500 MHz, DMSO-d6) δ ppm 11.51 (s, 1 H) 6.96 (d, J=1.0 Hz, 1 H) 6.43 (s, 1 H) 5.02
(s, 2 H) 4.13 (q, J=7.0 Hz, 2 H) 1.40 (t, J=6.8 Hz, 3 H).
6-Bromo-4-(tert-butoxy)-1H-indazol-3-amine (2-22c). The title compound was prepared according to General Procedure E on a 258-mg scale using 4-bromo-2-(tert-butoxy)-6- fluorobenzonitrile 2-21c. The crude material was purified by flash chromatography (0-50%
EtOAc:hexanes) to afford the title compound as a red solid (230 mg, 85%). LCMS [M+H]+ 284.08
79 81 1 m/z ( Br), 286.03 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.53 (s, 1 H) 7.03 (s, 1 H)
6.55 (s, 1 H) 4.96 (s, 2 H) 1.45 - 1.48 (m, 9 H).
6-Bromo-4-cyclobutoxy-1H-indazol-3-amine (2-22d). The title compound was prepared according to General Procedure E on a 259-mg scale using 4-bromo-2-cyclobutoxy-6- fluorobenzonitrile 2-21d. The crude material was purified by flash chromatography (0-50%
EtOAc:hexanes) to afford the title compound as a light yellow solid (236 mg, 87%). LCMS
+ 79 81 1 [M+H] 282.07 m/z ( Br), 284.04 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.51 (s, 1
H) 6.95 (s, 1 H) 6.25 (s, 1 H) 5.04 (s, 2 H) 4.83 (quin, J=7.1 Hz, 1 H) 2.41 - 2.49 (m, 2 H) 2.16
(ddd, J=9.8, 7.3, 2.4 Hz, 2 H) 1.82 (q, J=10.7 Hz, 1 H) 1.67 (sxt, J=10.3 Hz, 1 H).
175
6-Bromo-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-3-amine (2-22e). The title compound was prepared according to General Procedure E on a 111-mg scale using 4-bromo-2-fluoro-6-
((tetrahydro-2H-pyran-4-yl)oxy)benzonitrile 2-21e. The crude material was purified by flash chromatography (50-100% EtOAc:hexanes) to afford the title compound as a tan solid (104 mg,
+ 79 81 1 90%). LCMS [M+H] 311.92 m/z ( Br), 313.93 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.52 (s, 1 H) 6.95 (d, J=1.0 Hz, 1 H) 6.57 (s, 1 H) 5.03 (s, 2 H) 4.77 (tt, J=8.2, 4.3 Hz, 1 H)
3.81 - 3.89 (m, 2 H) 3.53 (m, J=2.9 Hz, 2 H) 2.00 (m, J=12.2 Hz, 2 H) 1.66 - 1.76 (m, 2 H).
6-Bromo-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-3-amine (2-22f). The title compound was prepared according to General Procedure E on a 143-mg scale using 4-bromo-2-fluoro-6-
((tetrahydro-2H-pyran-3-yl)oxy)benzonitrile 2-21f. The crude material was purified by flash chromatography (40-70% EtOAc:hexanes) to afford the title compound as a beige solid (123 mg,
+ 79 81 1 82%). LCMS [M+H] 311.98 m/z ( Br), 313.99 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.52 (br. s., 1 H) 6.96 (d, J=1.0 Hz, 1 H) 6.54 (s, 1 H) 5.07 (s, 2 H) 4.59 (tt, J=3.9, 2.4 Hz,
1 H) 3.77 (dd, J=10.7, 2.0 Hz, 1 H) 3.55 - 3.72 (m, 3 H) 1.94 - 2.02 (m, 1 H) 1.83 - 1.90 (m, 1 H)
1.78 (dtd, J=17.3, 8.8, 8.8, 4.6 Hz, 1 H) 1.53 (m, J=10.1, 3.0, 3.0 Hz, 1 H).
176
6-Bromo-4-((tetrahydrofuran-3-yl)oxy)-1H-indazol-3-amine (2-22g). The title compound was prepared according to General Procedure E on a 143-mg scale using 4-bromo-2-fluoro-6-
((tetrahydrofuran-3-yl)oxy)benzonitrile 2-21g. The crude material was purified by flash chromatography (50-100% EtOAc:hexanes) to afford the title compound as an off-white solid (130
+ 79 81 1 mg, 68%). LCMS [M+H] 297.94 m/z ( Br), 299.92 m/z ( Br); H NMR (500 MHz, DMSO-d6)
δ ppm 11.54 (s, 1 H) 6.98 (d, J=1.0 Hz, 1 H) 6.42 (s, 1 H) 5.15 (br. s., 1 H) 5.02 (s, 2 H) 3.92 (d,
J=2.9 Hz, 2 H) 3.88 (q, J=7.3 Hz, 1 H) 3.77 (td, J=8.3, 4.4 Hz, 1 H) 2.25 (m, J=7.3 Hz, 1 H) 2.11
(dt, J=13.3, 6.3 Hz, 1 H)
6-Bromo-4-(oxetan-3-yloxy)-1H-indazol-3-amine (2-22h). The title compound was prepared according to General Procedure E on a 121-mg scale using 4-bromo-2-fluoro-6-(oxetan-3- yloxy)benzonitrile 2-21h. The crude material was purified by flash chromatography (100%
EtOAc) to afford the title compound as a beige solid (82 mg, 65%). LCMS [M+H]+ 283.96 m/z
79 81 1 ( Br), 285.97 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.58 (s, 1 H) 7.01 (d, J=1.0 Hz,
1 H) 6.06 (s, 1 H) 5.39 (quin, J=5.4 Hz, 1 H) 5.20 (s, 2 H) 4.95 (t, J=6.8 Hz, 2 H) 4.70 (dd, J=7.1,
5.1 Hz, 2 H).
177
tert-Butyl 3-amino-6-bromo-4-methoxy-1H-indazole-1-carboxylate and tert-butyl 3-amino-6- bromo-4-methoxy-2H-indazole-2-carboxylate (2-23a). To as suspension of 6-bromo-4- methoxy-1H-indazol-3-amine 2-22a (101 mg, 0.417 mmol) in DCM (2.1 mL, 0.20 M), DMAP (26 mg, 0.212 mmol) was added. Boc anhydride (108 mg, 0.495 mmol) was dissolved in an equal volume of DCM and added to the reaction mixture dropwise, after which the reaction mixture was a clear solution. The reaction was stirred at room temperature overnight. A precipitate was removed by filtration and the filtrate was purified by flash chromatography (20-50%
EtOAc:hexanes) to afford the title compounds as a mixture of isomers (107 mg, 75%). LCMS
[M+H]+ 342.08 m/z (79Br), 344.08 m/z (81Br).
1-(3-Amino-6-bromo-4-ethoxy-1H-indazol-1-yl)ethan-1-one (2-23b). The title compound was prepared according to General Procedure F on a 240-mg scale using 6-bromo-4-ethoxy-1H- indazol-3-amine 2-22b. The crude material was purified by flash chromatography (0-50%
EtOAc:hexanes) to afford the title compound as a light yellow solid (65 mg, 23%). LCMS [M+H]+
79 81 1 298.03 m/z ( Br), 300.01 m/z ( Br). H NMR (500 MHz, DMSO-d6) δ ppm 7.92 (d, J=1.5 Hz, 1
H) 7.00 (d, J=1.0 Hz, 1 H) 5.99 (s, 2 H) 4.23 (q, J=7.2 Hz, 2 H) 2.49 (s, 3 H) 1.41 (t, J=7.1 Hz, 3
H).
178
1-(3-Amino-6-bromo-4-(tert-butoxy)-1H-indazol-1-yl)ethan-1-one (2-23c). The title compound was prepared according to General Procedure F on a 205-mg scale using 6-bromo-4-
(tert-butoxy)-1H-indazol-3-amine 2-22c. The crude material was purified by flash chromatography (0-50% EtOAc:hexanes) to afford the title compound as an orange solid (92 mg,
+ 79 81 1 39%). LCMS [M+H] 326.03 m/z ( Br), 328.04 m/z ( Br). H NMR (500 MHz, DMSO-d6) δ ppm 8.00 (d, J=1.5 Hz, 1 H) 7.07 (d, J=1.5 Hz, 1 H) 5.89 (s, 2 H) 2.50 (s, 3 H) 1.51 (s, 9 H).
1-(3-Amino-6-bromo-4-cyclobutoxy-1H-indazol-1-yl)ethan-1-one (2-23d). The title compound was prepared according to General Procedure F on a 236-mg scale using 6-bromo-4-cyclobutoxy-
1H-indazol-3-amine 2-22d. The crude material was purified by flash chromatography (0-50%
EtOAc:hexanes) to afford the title compound as an off-white solid (65 mg, 24%). LCMS [M+H]+
79 81 1 324.03 m/z ( Br), 326.02 m/z ( Br). H NMR (500 MHz, DMSO-d6) δ ppm 7.92 (d, J=1.5 Hz, 1
H) 6.81 (d, J=1.0 Hz, 1 H) 6.03 (s, 2 H) 4.93 (quin, J=7.1 Hz, 1 H) 2.49 (s, 3 H) 2.41 - 2.48 (m, 2
H) 2.23 (quint, J=9.7, 9.7, 9.7, 9.7, 2.3, 2.3 Hz, 1 H) 1.82 (qt, J=10.3, 2.4 Hz, 1 H) 1.66 (qt, J=10.7,
8.3 Hz, 1 H).
179
1-(3-Amino-6-bromo-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-1-yl)ethan-1-one and
N-(1-acetyl-6-bromo-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-3-yl)acetamide (2-23e).
The title compound was prepared according the General Procedure F on a 104-mg scale using 6- bromo-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-3-amine 2-22e. The crude material was not purified but taken forward as a mixture of mono- and di-acetylated products (148 mg orange solid).
LCMS [M+H]+ 353.92 m/z (79Br mono-aceylated), 355.93 m/z (81Br mono-aceylated), 395.92 m/z
(79Br di-aceylated), 397.87 m/z (81Br di-aceylated).
1-(3-Amino-6-bromo-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-1-yl)ethan-1-one and
N-(1-acetyl-6-bromo-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-3-yl)acetamide (2-23f).
The title compound was prepared according the General Procedure F on a 104-mg scale using 6- bromo-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-3-amine 2-22f. The crude material was not purified but taken forward as a mixture of mono- and di-acetylated products (147 mg orange solid).
LCMS [M+H]+ 353.98 m/z (79Br mono-aceylated), 356.00 m/z (81Br mono-aceylated), 395.98 m/z
(79Br di-aceylated), 398.00 m/z (81Br di-aceylated).
180
1-(3-Amino-6-bromo-4-((tetrahydrofuran-3-yl)oxy)-1H-indazol-1-yl)ethan-1-one and N-(1- acetyl-6-bromo-4-((tetrahydrofuran-3-yl)oxy)-1H-indazol-3-yl)acetamide (2-23h). The title compound was prepared according the General Procedure F on a 130-mg scale using 6-bromo-4-
((tetrahydrofuran-3-yl)oxy)-1H-indazol-3-amine 2-22g. The crude material was not purified but taken forward as a mixture of mono- and di-acetylated products (170 mg orange solid). LCMS
[M+H]+ 339.94 m/z (79Br mono-aceylated), 341.81 m/z (81Br mono-aceylated), 381.88 m/z (79Br di-aceylated), 383.89 m/z (81Br di-aceylated).
1-(3-Amino-6-bromo-4-(oxetan-3-yloxy)-1H-indazol-1-yl)ethan-1-one and N-(1-acetyl-6- bromo-4-(oxetan-3-yloxy)-1H-indazol-3-yl)acetamide (2-23h). The title compound was prepared according the General Procedure F on an 82-mg scale using 6-bromo-4-(oxetan-3-yloxy)-
1H-indazol-3-amine 2-22h. The crude material was not purified but taken forward as a mixture of mono- and di-acetylated products (84 mg yellow solid). LCMS [M+H]+ 325.96 m/z (79Br mono- aceylated), 327.97 m/z (81Br mono-aceylated), 367.96 m/z (79Br di-aceylated), 369.91 m/z (81Br di-aceylated).
181
6-(2-Aminopyrimidin-4-yl)-4-methoxy-1H-indazol-3-amine formate (2-27a/NEU-1335). The title compound was prepared according to General Procedure G on a 107-mg scale using tert-butyl
3-amino-6-bromo-4-methoxy-1H-indazole-1-carboxylate 2-23a. The crude material of the
Miyaura reaction was taken forward without further purification. The crude residue of the Suzuki reaction was first purified by flash chromatography (1-7% 5% NH4OH/MeOH:DCM), then repurified by preparative HPLC (5-30% ACN:water) to afford the title compound as an orange
+ 1 solid (15 mg, 19%). LCMS [M+H] 257.07 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.64 (br. s, 1 H) 8.34 (br. s, 1 H) 8.29 (d, J=5.4 Hz, 1 H) 7.53 (s, 1 H) 7.16 (d, J=4.9 Hz, 1 H) 6.97 (s, 1 H)
6.65 (s, 2 H) 5.06 (s, 2 H) 3.95 (s, 3 H).
6-(2-Aminopyrimidin-4-yl)-4-ethoxy-1H-indazol-3-amine formate (2-27b/NEU-5388). The title compound was prepared according to General Procedure G on a 348-mg scale using 1-(3- amino-6-bromo-4-ethoxy-1H-indazol-1-yl)ethan-1-one 2-23b. The crude material of the Miyaura reaction was taken forward without further purification. The crude residue of the Suzuki reaction was purified by flash chromatography (20-100% EtOAc:hexanes – 0-10% MeOH:DCM), then repurified by preparative HPLC (5-50% ACN:water) to afford the formate salt of the title
182 compound as an orange solid (18 mg, 6%). LCMS [M+H]+ 271.17 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.67 (br. s., 1 H) 8.52 (s, 1 H) 8.28 (d, J=5.4 Hz, 1 H) 7.52 (s, 1 H) 7.14 (d,
J=5.4 Hz, 1 H) 6.96 (s, 1 H) 6.64 (s, 2 H) 5.03 (s, 2 H) 4.21 (q, J=7.2 Hz, 2 H) 1.45 (t, J=7.1 Hz,
3 H).
6-(2-Aminopyrimidin-4-yl)-4-(tert-butoxy)-1H-indazol-3-amine (2-27c/NEU-5389). The title compound was prepared according to General Procedure G on a 92-mg scale using 1-(3-amino-6- bromo-4-(tert-butoxy)-1H-indazol-1-yl)ethan-1-one 2-23c. The product of the Miyaura reaction was isolated by flash chromatography (20% EtOAc:hexanes) to afford 1-(3-amino-4-(tert- butoxy)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-1-yl)ethan-1-one 2-25c as an
+ 1 off-white solid (73 mg, 69%). LCMS [M+H] 374.19 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
8.19 (s, 1 H) 7.12 (s, 1 H) 5.89 (s, 2 H) 2.50 (s, 3 H) 1.48 (s, 9 H) 1.32 (s, 12 H).
The crude residue of the Suzuki reaction was purified by flash chromatography (5%
MeOH:DCM), then repurified by preparative HPLC (5-50% acetronitrile:water) to afford the title compound as a brown solid (8 mg, 14%). LCMS [M+H]+ 299.19 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.68 (s, 1 H) 8.28 (d, J=5.4 Hz, 1 H) 7.58 (d, J=1.0 Hz, 1 H) 7.13 (d, J=1.0 Hz,
1 H) 7.09 (d, J=5.4 Hz, 1 H) 6.64 (s, 2 H) 4.97 (s, 2 H) 1.50 (s, 9 H).
183
6-(2-Aminopyrimidin-4-yl)-4-cyclobutoxy-1H-indazol-3-amine formate (2-27d/NEU-5390).
The title compound was prepared according to General Procedure G on a 65-mg scale using 1-(3- amino-6-bromo-4-cyclobutoxy-1H-indazol-1-yl)ethan-1-one 2-23d. The product of the Miyaura reaction was isolated by flash chromatography (20% EtOAc:hexanes) to afford 1-(3-amino-4- cyclobutoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-1-yl)ethan-1-one 2-25d
+ 1 as an off-white solid (52 mg, 71%). LCMS [M+H] 372.18 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 8.11 (s, 1 H) 6.79 (s, 1 H) 6.00 (s, 2 H) 4.91 (quin, J=7.1 Hz, 1 H) 2.50 (s, 3 H) 2.38 - 2.47
(m, 2 H) 2.17 - 2.29 (m, 2 H) 1.83 (q, J=10.7 Hz, 1 H) 1.72 (q, J=9.8 Hz, 1 H) 1.32 (s, 12 H).
The crude residue of the Suzuki reaction was purified by flash chromatography (5%
MeOH:DCM), then repurified by preparative HPLC (5-50% ACN:water) to afford the title compound as a brown solid (5 mg, 12%). LCMS [M+H]+ 297.18 m/z; 1H NMR (500 MHz, DMSO- d6) δ ppm 11.66 (s, 1 H) 8.52 (s, 1 H) 8.28 (d, J=4.9 Hz, 1 H) 7.52 (s, 1 H) 7.11 (d, J=5.4 Hz, 1 H)
6.80 (s, 1 H) 6.64 (s, 2 H) 5.04 (s, 2 H) 4.91 (quin, J=7.3 Hz, 1 H) 2.53 (m, J=9.8 Hz, 2 H) 2.21
(quint, J=10.3, 10.3, 10.3, 10.3, 2.4, 2.4 Hz, 2 H) 1.84 (q, J=10.3 Hz, 1 H) 1.71 (quin, J=10.7 Hz,
1 H).
184
6-(2-Aminopyrimidin-4-yl)-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-3-amine (2-
27e/NEU-5901). The title compound was prepared according to General Procedure G on a 120- mg scale using 1-(3-amino-6-bromo-4-((tetrahydro-2H-pyran-4-yl)oxy)-1H-indazol-1-yl)ethan-1- one 2-23e. The crude Miyaura reaction mixture was run through a pad of silica using 100% EtOAc as the eluent to afford a mixture of mono-, bi- and tri-acetylated boronic acid and ester which was taken forward to the Suzuki reaction without further purification. The crude residue of the Suzuki reaction was purified by flash chromatography (1-10% 5%NH4OH/MeOH:EtOAc) to afford a mixture of the title compound and the acetylated product. This crude material was suspended in
MeOH (10 mL, 0.04 M) and 12M HCl (1 mL, 12 mmol) was added, upon which the reaction mixture went from a cloudy suspension to a clear solution. The mixture was refluxed at 65 °C overnight, then concentrated under reduced pressure. The resulting residue was redissolved in
EtOAc. The reaction mixture was poured over saturated aqueous NaHCO3 and the aqueous layer was extracted four times with EtOAc. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was repurified by flash chromatography (3% 5%NH4OH/MeOH:EtOAc) to afford the title compound as a yellow-green solid (26 mg, 24%). LCMS [M+H]+ 327.09 m/z; 1H NMR (500 MHz, DMSO- d6) δ ppm 11.65 (s, 1 H) 8.29 (d, J=5.4 Hz, 1 H) 7.54 (s, 1 H) 7.16 (d, J=5.4 Hz, 1 H) 7.02 (s, 1 H)
6.64 (s, 2 H) 5.03 (s, 2 H) 4.87 (m, J=3.9 Hz, 1 H) 3.82 - 3.93 (m, 2 H) 3.57 (td, J=8.8, 2.4 Hz, 2
H) 2.02 - 2.11 (m, 2 H) 1.76 (m, J=8.8 Hz, 2 H).
185
6-(2-Aminopyrimidin-4-yl)-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-3-amine (2-
27f/NEU-5934). The title compound was prepared according to General Procedure G on a 140- mg scale using 1-(3-amino-6-bromo-4-((tetrahydro-2H-pyran-3-yl)oxy)-1H-indazol-1-yl)ethan-1- one 2-23f. The crude Miyaura reaction mixture was run through a pad of silica using 100% EtOAc as the eluent to afford a mixture of mono-, bi- and tri-acetylated boronic acid and ester which was taken forward to the Suzuki reaction without further purification. The crude residue of the Suzuki reaction was purified by flash chromatography (1-10% 5% NH4OH/MeOH:EtOAc) to afford a mixture of the title compound and the acetylated product. This crude material was suspended in
MeOH (10 mL, 0.04 M) and 12M HCl (1 mL, 12 mmol) was added, upon which the reaction mixture went from a cloudy suspension to a clear solution. The mixture was refluxed at 65 °C overnight. The solvent was removed under reduced pressure and the resulting residue was redissolved in EtOAc. The reaction mixture was poured over saturated aqueous NaHCO3 and the aqueous layer was extracted three times with EtOAc. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was then purified by flash chromatography (5% 5% NH4OH/MeOH:EtOAc) to afford the title compound as a yellow solid (12 mg, 9%). LCMS [M+H]+ 327.09 m/z; 1H NMR (500 MHz,
METHANOL-d4) δ ppm 8.27 (d, J=4.9 Hz, 1 H) 7.51 (s, 1 H) 7.12 (d, J=5.4 Hz, 1 H) 7.09 (s, 1
H) 4.66 - 4.72 (m, 1 H) 3.94 (dd, J=10.7, 2.0 Hz, 1 H) 3.84 (dd, J=14.6, 5.4 Hz, 1 H) 3.74 - 3.80
186
(m, 1 H) 3.71 (m, J=8.1, 3.2 Hz, 1 H) 2.03 - 2.15 (m, 2 H) 1.97 (m, J=8.7, 4.2, 4.2 Hz, 1 H) 1.58 -
1.67 (m, 1 H).
6-(2-Aminopyrimidin-4-yl)-4-((tetrahydrofuran-3-yl)oxy)-1H-indazol-3-amine (2-27g/NEU-
5899). The title compound was prepared according to General Procedure G on a 150-mg scale using 1-(3-amino-6-bromo-4-((tetrahydrofuran-3-yl)oxy)-1H-indazol-1-yl)ethan-1-one 2-23g.
The crude Miyaura reaction mixture was run through a pad of silica using 100% EtOAc as the eluent to afford a mixture of mono-, bi- and tri-acetylated boronic acid and ester which was taken forward to the Suzuki reaction without further purification. The crude residue of the Suzuki reaction was purified by flash chromatography (5-10% 5% NH4OH/MeOH:DCM, step gradient) to afford a mixture of the title compound and the acetylated product. This crude material was suspended in MeOH (10 mL, 0.04 M) and 12M HCl (1 mL, 12 mmol) was added, upon which the reaction mixture went from a cloudy suspension to a clear solution. The mixture was refluxed at
65 °C overnight. The solvent was removed under reduced pressure and the resulting residue was redissolved in EtOAc. The reaction mixture was poured over saturated aqueous NaHCO3 and the aqueous layer was extracted four times with EtOAc. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (10% 5%NH4OH/MeOH:EtOAc) to afford the title compound as a yellow solid (26 mg, 19%). LCMS [M+H]+ 313.05 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.67 (s, 1 H) 8.29 (d, J=5.4 Hz, 1 H) 7.56 (s, 1 H) 7.16 (d, J=4.9 Hz, 1 H) 6.91
187
(s, 1 H) 6.64 (s, 2 H) 5.25 (br. s., 1 H) 5.03 (s, 2 H) 3.94 - 4.00 (m, 2 H) 3.91 (q, J=7.8 Hz, 1 H)
3.80 (sxt, J=4.4 Hz, 1 H) 2.30 (quin, J=6.8 Hz, 1 H) 2.13 - 2.21 (m, 1 H).
6-(2-Aminopyrimidin-4-yl)-4-(oxetan-3-yloxy)-1H-indazol-3-amine (2-27h/NEU-5935). The title compound was prepared according to General Procedure G on an 84-mg scale using 1-(3- amino-6-bromo-4-(oxetan-3-yloxy)-1H-indazol-1-yl)ethan-1-one 2-23h. The crude Miyaura reaction mixture was run through a pad of silica using 100% EtOAc as the eluent to afford a mixture of mono-, bi- and tri-acetylated boronic acid and ester which was taken forward to the
Suzuki reaction without further purification. The crude residue of the Suzuki reaction was purified by flash chromatography (1-5-10% 5% NH4OH/MeOH:EtOAc, step gradient) to afford the title compound as a yellow solid (20 mg, 26%). LCMS [M+H]+ 299.07 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.72 (s, 1 H) 8.28 (d, J=5.4 Hz, 1 H) 7.59 (s, 1 H) 7.13 (d, J=4.9 Hz, 1 H) 6.64
(s, 2 H) 6.55 (s, 1 H) 5.48 (quin, J=5.1 Hz, 1 H) 5.18 (s, 2 H) 5.01 (t, J=6.8 Hz, 2 H) 4.75 (dd,
J=7.3, 4.9 Hz, 1 H).
188
5.2.2 Experimental procedures for chapter 3
5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (3-2/NEU-4463). 5-Bromo-1H- pyrrolo[2,3-b]pyridine 3-1 (686 mg, 3.48 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (800 mg, 3.84 mmol), and PdCl2(dppf)·CH2Cl2 (147 mg, 0.180 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times.
Dioxane (20 mL, 0.17 M) and 2M K2CO3 (5.0 mL, 10.00 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85 °C for 4 h. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound as an
+ 1 orange solid (640 mg, 93%). LCMS [M+H] 199.01 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
11.59 (br. s., 1 H) 8.45 (d, J=1.95 Hz, 1 H) 8.13 (s, 1 H) 8.08 (d, J=1.95 Hz, 1 H) 7.88 (s, 1 H)
7.44 (t, J=2.93 Hz, 1 H) 6.41 (dd, J=3.42, 1.95 Hz, 1 H) 3.87 (s, 3 H).
3-Iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (3-3). Intermediate 3-2 (640 mg, 3.23 mmol) was dissolved in ACN (18 mL, 0.18 M) and N-iodosuccinimide (1.09 g, 4.84 mmol) was added. The reaction was stirred at 50 °C for 2 h. Upon cooling to room temperature, a precipitate was observed and collected by vacuum filtration (washed with ACN) to afford the title compound as a light brown solid (730 mg, 69%). LCMS [M+H]+ 324.98 m/z; 1H NMR (500 MHz,
189
DMSO-d6) δ ppm 12.06 (br. s, 1 H) 8.51 (d, J=1.95 Hz, 1 H) 8.25 (s, 1 H) 7.96 (s, 1 H) 7.76 (d,
J=2.44 Hz, 1 H) 7.69 (d, J=2.44 Hz, 1 H) 3.88 (s, 3 H).
3-Iodo-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-4). Intermediate 3-
3 (730 mg, 2.25 mmol) was suspended in DCM (37 mL, 0.06 M) and TEA (1.5 mL, 10.76 mmol),
DMAP (227 mg, 1.86 mmol) and 4-methylbenzenesulfonyl chloride (1.06 g, 5.56 mmol) were added in that order. The reaction was stirred overnight at room temperature. The reaction was washed once with 1M HCl, once with saturated aqueous NaHCO3, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-100% EtOAc:Hex - 0-10% MeOH:DCM) to afford the title compound as a light orange solid (741 mg, 69%). LCMS [M+H]+ 478.95 m/z; 1H
NMR (500 MHz, DMSO-d6) δ ppm 8.66 (d, J=1.95 Hz, 1 H) 8.33 (s, 1 H) 8.12 (s, 1 H) 7.98 - 8.04
(m, 3 H) 7.85 (d, J=2.44 Hz, 1 H) 7.43 (d, J=8.79 Hz, 2 H) 3.87 (s, 3 H) 2.34 (s, 3 H).
190
3-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-
5a/NEU-4796). The title compound was prepared according to General Procedure H on a 199-mg scale using (3-cyanophenyl)boronic acid. The crude material was purified by flash chromatography (20-80% EtOAc:Hexanes) to afford the title compound as a light orange solid
+ 1 (135 mg, 71%). LCMS [M+H] 454.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.70 (d, J=2.0
Hz, 1 H) 8.42 (d, J=2.0 Hz, 1 H) 8.40 (s, 1 H) 8.34 (t, J=1.5 Hz, 1 H) 8.31 (s, 1 H) 8.20 (dt, J=7.8,
1.5 Hz, 1 H) 8.06 (s, 1 H) 8.04 (d, J=3.4 Hz, 2 H) 7.85 (dt, J=7.8, 1.0 Hz, 1 H) 7.70 (t, J=7.8 Hz,
1 H) 7.44 (d, J=8.8 Hz, 2 H) 3.88 (s, 3 H) 2.35 (s, 3 H).
2-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-5b).
The title compound was prepared according to General Procedure H on a 75-mg scale using (2- cyanophenyl)boronic acid. The crude material was purified by flash chromatography (50-100%
EtOAc:hexanes) to afford the title compound as a light orange solid (48 mg, 68%). LCMS [M+H]+
1 454.03 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.72 (d, J=1.95 Hz, 1 H) 8.29 (s, 1 H) 8.27 (s,
191
1 H) 8.16 (d, J=1.95 Hz, 1 H) 8.01 - 8.07 (m, 3 H) 7.98 (s, 1 H) 7.85 (d, J=3.91 Hz, 2 H) 7.64 (m,
J=8.30, 4.20, 4.20 Hz, 1 H) 7.45 (d, J=7.81 Hz, 2 H) 3.85 (s, 3 H) 2.35 (s, 3 H).
*Prepared by K. Forbes.
3-(4-Fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-5c).
The title compound was prepared according to General Procedure H on a 65-mg scale using (4- fluorophenyl)boronic acid. The crude material was purified by flash chromatography (50-100%
EtOAc:hexanes) to afford the title compound as a light orange solid (42 mg, 69%). LCMS [M+H]+
1 447.02 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.68 (d, J=1.95 Hz, 1 H) 8.33 (d, J=1.95 Hz,
1 H) 8.30 (s, 1 H) 8.19 (s, 1 H) 8.04 (d, J=8.30 Hz, 2 H) 8.02 (s, 1 H) 7.84 - 7.90 (m, 2 H) 7.43 (d,
J=7.81 Hz, 2 H) 7.30 - 7.36 (m, 2 H) 3.87 (s, 3 H) 2.34 (s, 3 H).
3-(2-Fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-5d).
The title compound was prepared according to General Procedure H on a 75-mg scale using (2-
192 fluorophenyl)boronic acid. The crude material was purified by flash chromatography (50-100%
EtOAc:Hexanes) to afford the title compound as an off-white solid (55 mg, 78%). LCMS [M+H]+
1 447.08 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.70 (d, J=1.95 Hz, 1 H) 8.28 (s, 1 H) 8.16 (t,
J=1.95 Hz, 1 H) 8.11 (s, 1 H) 8.06 (d, J=8.30 Hz, 2 H) 7.99 (s, 1 H) 7.80 (td, J=7.69, 1.71 Hz, 1
H) 7.46 - 7.52 (m, 1 H) 7.45 (d, J=8.30 Hz, 2 H) 7.34 - 7.43 (m, 2 H) 3.86 (s, 3 H) 2.35 (s, 3 H).
3-(3,4-Difluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-
5e). The title compound was prepared according to General Procedure H on a 69-mg scale using
(3,4-difluorophenyl)boronic acid. The crude material was purified by flash chromatography (50-
100% EtOAc:hexanes) to afford the title compound as a light orange solid (42 mg, 62%). LCMS
+ 1 [M+H] 465.05 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.69 (d, J=1.95 Hz, 1 H) 8.37 (d,
J=1.95 Hz, 1 H) 8.32 (s, 1 H) 8.30 (s, 1 H) 8.02 - 8.07 (m, 3 H) 7.96 (qd, J=8.80, 2.00 Hz, 1 H)
7.68 - 7.73 (m, 1 H) 7.51 - 7.59 (m, 1 H) 7.44 (d, J=8.30 Hz, 2 H) 3.88 (s, 3 H) 2.35 (s, 3 H).
193
2-Fluoro-5-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (3-5f). The title compound was prepared according to General Procedure H on a
74-mg scale using 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile. The crude material was purified by flash chromatography (50-100% EtOAc:Hexanes) to afford the title compound as an off-white solid (48 mg, 66%). LCMS [M+H]+ 472.07 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.69 (d, J=1.95 Hz, 1 H) 8.43 (dd, J=6.35, 2.44 Hz, 1 H) 8.41 (d, J=1.95 Hz, 1
H) 8.39 (s, 1 H) 8.30 (s, 1 H) 8.26 (m, J=2.40, 2.40, 2.40, 2.40, 2.40, 2.40 Hz, 1 H) 8.03 - 8.07 (m,
3 H) 7.65 (t, J=9.03 Hz, 1 H) 7.44 (d, J=8.30 Hz, 2 H) 3.88 (s, 3 H) 2.34 (s, 3 H).
3-Fluoro-5-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (3-5g). The title compound was prepared according to General Procedure H on a
75-mg scale using (3-cyano-5-fluorophenyl)boronic acid. The crude material was purified by flash chromatography (50-100% EtOAc:hexanes) to afford the title compound as an off-white solid (44
194
+ 1 mg, 60%). LCMS [M+H] 472.06 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.71 (d, J=1.95 Hz,
1 H) 8.49 (s, 1 H) 8.45 (d, J=2.44 Hz, 1 H) 8.32 (s, 1 H) 8.23 (s, 1 H) 8.12 (d, J=9.77 Hz, 1 H)
8.04 - 8.08 (m, 3 H) 7.87 (d, J=8.30 Hz, 1 H) 7.44 (d, J=8.79 Hz, 2 H) 3.88 (s, 3 H) 2.35 (s, 3 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(3-nitrophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-5h).
Intermediate 3-4 (38 mg, 0.08 mmol), (3-nitrophenyl)boronic acid (40 mg, 0.238 mmol), and
PdCl2(dppf)·CH2Cl2 (6 mg, 0.007 mmol) were combined in a 8 mL vial. The vial was purged with nitrogen and evacuated three times. Dioxane (0.8 mL, 0.1 M) and 2 M aqueous K2CO3 (0.2 mL,
0.4 mmol) were added and the mixture was degassed for 10 min. The reaction was run at 100 °C for 4 h, then stopped, diluted with EtOAc, and filtered through celite. The crude material was purified by flash chromatography (30-80% EtOAc:hexanes, step gradient) to afford the title
+ 1 compound as a solid (30 mg, 80%). LCMS [M+H] 320.12 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 8.71 (d, J=2.0 Hz, 1 H) 8.60 (t, J=2.4 Hz, 1 H) 8.48 (s, 1 H) 8.41 (d, J=2.0 Hz, 1 H) 8.28 -
8.32 (m, 2 H) 8.24 (dd, J=8.3, 1.5 Hz, 1 H) 8.08 (d, J=8.8 Hz, 2 H) 8.02 (s, 1 H) 7.80 (t, J=7.8 Hz,
1 H) 7.45 (d, J=8.3 Hz, 2 H) 3.87 (s, 3 H) 2.35 (s, 3 H).
*Prepared by K. Forbes.
195
3-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (3-5i).
Intermediate 3-4 (40 mg, 0.083 mmol), (3-aminophenyl)boronic acid (34 mg, 0.251 mmol), and
PdCl2(dppf)·CH2Cl2 (6 mg, 0.007 mmol) were combined in a 8 mL vial. The vial was purged with nitrogen and evacuated three times. Dioxane (0.8 mL, 0.1 M) and 2 M aqueous K2CO3 (0.2 mL,
0.4 mmol) were added and the mixture was degassed for 10 min. The reaction was run at 100 °C for 4 h, then stopped, diluted with EtOAc, and filtered through celite. The crude material was purified by flash chromatography (50-100% EtOAc:hexanes, step gradient) to afford the title
+ 1 compound as a solid (5 mg, 14%). LCMS [M+H] 290.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.66 (d, J=2.0 Hz, 1 H) 8.29 (d, J=2.0 Hz, 1 H) 8.27 (s, 1 H) 8.03 (d, J=8.8 Hz, 2 H) 8.00 (s,
2 H) 7.43 (d, J=8.3 Hz, 2 H) 7.14 (t, J=7.8 Hz, 1 H) 6.96 - 6.98 (m, 1 H) 6.89 - 6.93 (m, 1 H) 6.59
(dd, J=8.1, 2.2 Hz, 1 H) 5.18 - 5.23 (m, 2 H) 3.87 (s, 3 H) 2.34 (s, 3 H).
*Prepared by K. Forbes.
196
Scheme 5-5. Synthesis of NEU-4933.
1-(4-bromophenyl)-N,N-dimethylmethanamine (5-23). 1-Bromo-4-(bromomethyl)benzene 5-
22 (499 mg 2.00 mmol), was suspended in hexanes (2.0 mL, 1.0 M) and the reaction mixture was cooled to 0 °C. Dimethylamine (2 M) in THF (4.00 mL, 8.00 mmol) was added dropwise. The reaction mixture was left stirring overnight and allowed to warm to room temperature. A precipitate was observed and removed by filtration; the filtrate was concentrated to afford the title compound as an orange oil (350 mg, 82%). LCMS [M+H]+ 213.93 m/z (79Br), 215.93 m/z (81Br);
1 H NMR (500 MHz, DMSO-d6) δ ppm 7.50 (d, J=8.30 Hz, 2 H) 7.24 (d, J=8.30 Hz, 2 H) 3.34 (s,
2 H) 2.12 (s, 6 H).
N,N-Dimethyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanamine (5-
24). 1-(4-Bromophenyl)-N,N-dimethylmethanamine 5-23 (350 mg, 1.63 mmol), bis(pinacolato)diboron (622 mg, 2.45 mmol), potassium acetate (480 mg, 4.89 mmol), and
PdCl2(dppf)·CH2Cl2 (71 mg, 0.087 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Anhydrous dioxane (5.0 mL, 0.33 M) was added and the
197 reaction was degassed for 5 mins and run in the microwave (130 °C) for 1.5 h. The reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography (5-20% 5% NH4OH/MeOH:DCM) to afford the title compound as a brown oil (176 mg, 41%). LCMS [M+H]+ 262.22 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 7.62 (d, J=8.30 Hz, 2 H) 7.30 (d, J=8.30 Hz, 2 H) 3.39 (s, 2 H) 2.12 (s, 6 H)
1.28 (s, 12 H).
N,N-Dimethyl-1-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl) phenyl)methanamine (3-5j). The title compound was prepared according to General Procedure
H on a 150-mg scale using N,N-dimethyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenyl)methanamine 5-24. The crude material was purified by flash chromatography (50-100%
EA:H - 0-20% MeOH:EA - 0-10% 5% NH4OH/MeOH:DCM) to afford the title compound as a glassy brown solid (36 mg, 24%). LCMS [M+H]+ 486.23 m/z; 1H NMR (500 MHz, METHANOL- d4) δ ppm 8.59 (d, J=1.95 Hz, 1 H) 8.29 (d, J=1.95 Hz, 1 H) 8.07 (s, 1 H) 8.05 (d, J=8.30 Hz, 2 H)
8.02 (s, 1 H) 7.90 (s, 1 H) 7.70 (d, J=8.30 Hz, 2 H) 7.48 (d, J=8.30 Hz, 2 H) 7.36 (d, J=8.79 Hz, 2
H) 3.94 (s, 3 H) 3.58 (s, 2 H) 2.37 (s, 3 H) 2.32 (s, 6 H).
198
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol (3-5k). The title compound was prepared according to General Procedure H on a 100-mg scale using (4- hydroxyphenyl)boronic acid. The crude material was purified by flash chromatography (50-100%
EtOAc:Hex) to afford the title compound as a tan solid (49 mg, 52%). LCMS [M+H]+ 445.13 m/z;
1 H NMR (500 MHz, DMSO-d6) δ ppm 9.62 (br. s, 1 H) 8.65 (d, J=1.95 Hz, 1 H) 8.30 (s, 1 H) 8.28
(d, J=1.46 Hz, 1 H) 7.98 - 8.04 (m, 4 H) 7.61 (d, J=8.30 Hz, 2 H) 7.42 (d, J=8.30 Hz, 2 H) 6.89
(d, J=8.30 Hz, 2 H) 3.87 (s, 3 H) 2.34 (s, 3 H).
3-(1H-Indol-5-yl)-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-5l).
The title compound was prepared according to General Procedure H on a 76-mg scale using (1H- indol-5-yl)boronic acid. The crude material was purified by flash chromatography (20-100%
EtOAc:hexanes) to afford the title compound as a glassy orange solid (41 mg, 55%). LCMS
199
+ 1 [M+H] 468.007 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 11.21 (br. s., 1 H) 8.67 (d, J=1.47
Hz, 1 H) 8.34 (d, J=2.20 Hz, 1 H) 8.30 (s, 1 H) 8.05 (d, J=8.79 Hz, 2 H) 8.02 (s, 1 H) 8.01 (s, 1 H)
7.95 (s, 1 H) 7.46 - 7.56 (m, 2 H) 7.39 - 7.45 (m, 3 H) 6.52 (br. s., 1 H) 3.87 (s, 3 H) 2.34 (s, 3 H).
5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[c][1,2,5]oxadiazole (5-25). 5-
Chlorobenzo[c][1,2,5]oxadiazole (102 mg, 0.660 mmol), potassium acetate (228 mg, 2.32 mmol), bis(pinacolato)diboron (249 mg, 0.981 mmol), and PdCl2(dppf)·CH2Cl2 (31 mg, 0.038 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (4.3 mL, 0.15 M) was added and the reaction was run in the microwave (145
°C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The dark brown crude material was carried forward without further purification.
*Does not ionize.
5-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzo[c][1,2,5]oxadiazole (3-5m). The title compound was prepared according to General
Procedure H on a 151-mg scale using benzo[c][1,2,5]oxadiazol-5-ylboronic acid 5-25. The crude
200 material was purified by flash chromatography (20-60% EtOAc:hexanes) to afford the title compound as a white solid (109 mg, 73%). LCMS [M+H]+ 471.02 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 8.74 (d, J=2.20 Hz, 1 H) 8.63 (s, 1 H) 8.61 (d, J=2.20 Hz, 1 H) 8.53 (s, 1 H)
8.38 (s, 1 H) 8.21 (dd, J=9.53, 1.47 Hz, 1 H) 8.17 (d, J=8.79 Hz, 1 H) 8.06 - 8.12 (m, 3 H) 7.45
(d, J=8.79 Hz, 2 H) 3.89 (s, 3 H) 2.35 (s, 3 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-5n). The title compound was prepared according to General Procedure H using 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyridine. The crude material was carried forward without further purification. LCMS [M+H]+ 430.11 m/z.
*Prepared by K. Forbes.
5-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinonitrile (3-
5o). The title compound was prepared according to General Procedure H on a 75-mg scale using
201
(5-cyanopyridin-3-yl)boronic acid. The crude material was purified by flash chromatography (50-
100% EtOAc:hexanes) to afford the title compound as a light orange solid (60 mg, 85%). LCMS
+ 1 [M+H] 455.05 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.36 (t, J=2.20 Hz, 1 H) 9.03 (t,
J=1.95 Hz, 1 H) 8.81 (q, J=2.28 Hz, 1 H) 8.72 (t, J=1.95 Hz, 1 H) 8.55 (d, J=1.95 Hz, 1 H) 8.50
(t, J=1.95 Hz, 1 H) 8.33 (d, J=0.98 Hz, 1 H) 8.04 - 8.08 (m, 3 H) 7.44 (d, J=8.30 Hz, 2 H) 3.88 (s,
3 H) 2.35 (s, 3 H).
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)picolinonitrile (5-26). 4-Bromopicolinonitrile
(50 mg, 0.273 mmol), potassium acetate (95 mg, 0.968 mmol), bis(pinacolato)diboron (107 mg,
0.421 mmol), and PdCl2(dppf)·CH2Cl2 (12 mg, 0.015 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (2.1 mL, 0.13 M) was added and the reaction was run in the microwave (145 °C) for 30 min. The reaction was diluted with MeOH, filtered through celite, and concentrated under reduced pressure. The dark brown crude material was carried forward without further purification. LCMS [M+H]+ 148.69 m/z
(boronic acid).
202
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)picolinonitrile (3-
5p). The title compound was prepared according to General Procedure H on a 100-mg scale using crude (2-cyanopyridin-4-yl)boronic acid 5-26. The crude material was purified by flash chromatography (50-100% EtOAc:hexanes) to afford the title compound as a light yellow solid
+ 1 (34 mg, 36%). LCMS [M+H] 455.12 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.80 (d, J=5.37
Hz, 1 H) 8.74 (s, 1 H) 8.73 (d, J=1.95 Hz, 1 H) 8.61 (s, 1 H) 8.57 (d, J=1.95 Hz, 1 H) 8.33 (s, 1 H)
8.27 (dd, J=5.86, 1.95 Hz, 1 H) 8.08 (s, 1 H) 8.06 (d, J=8.30 Hz, 2 H) 7.45 (d, J=8.30 Hz, 2 H)
3.89 (s, 3 H) 2.35 (s, 3 H).
Scheme 5-6. Synthesis of NEU-5954, -5976, -5994, -5955, -6016, and -5995.
203
tert-Butyl 4-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)- carboxylate (5-27a). Intermediate 3-8 (0.323 mg, 0.678 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (272 mg, 0.880 mmol), and
PdCl2(dppf)·CH2Cl2 (28 mg, 0.034 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (3.2 mL, 0.21 M) and 2M K2CO3 (1.0 mL, 2.00 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (80 °C) for 10 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex) to afford the title compound as an off-white solid (200 mg,
56%). LCMS [M+H]+ 532.07 m/z (79Br), 533.96 m/z (81Br); 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 8.46 (d, J=2.0 Hz, 1 H) 8.18 (d, J=2.0 Hz, 1 H) 8.05 (d, J=8.3 Hz, 2
H) 7.67 (s, 1 H) 7.29 (d, J=8.3 Hz, 2 H) 6.11 (br. s, 1 H) 4.13 (br. s., 2 H) 3.67 (t, J=5.4 Hz, 2 H)
2.52 (br. s., 2 H) 2.39 (s, 3 H) 1.51 (s, 9 H).
204
tert-Butyl 4-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6- dihydropyridine-1(2H)-carboxylate (3-5q). tert-Butyl 4-(5-bromo-1-tosyl-1H-pyrrolo[2,3- b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate 5-27a (200 mg, 0.376 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (95 mg, 0.456 mmol), and
PdCl2(dppf)·CH2Cl2 (15 mg, 0.018 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (2.2 mL, 0.17 M) and 2M K2CO3 (0.55 mL, 1.10 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85 °C for
4 h, then the reaction mixture was diluted with EtOAc and filtered through celite. The filtrate was purified by flash chromatography (50% EtOAc:Hex) to afford the title compound as a light orange
+ 1 solid (169 mg, 85%). LCMS [M+H] 534.27 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.64 (d,
J=1.5 Hz, 1 H) 8.38 (d, J=2.0 Hz, 1 H) 8.31 (s, 1 H) 8.04 (s, 1 H) 7.98 (d, J=8.3 Hz, 2 H) 7.87 (s,
1 H) 7.41 (d, J=7.8 Hz, 2 H) 6.45 (s, 1 H) 4.08 (br. s, 2 H) 3.87 (s, 3 H) 3.52 - 3.61 (m, 2 H) 2.55
(br. s, 2 H) 2.33 (s, 3 H) 1.44 (s, 9 H).
205
tert-Butyl 4-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piper- idine-1-carboxylate (5-28a). tert-Butyl 4-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3- b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate 3-5q (169 mg, 0.317 mmol) was dissolved in EtOH (6.3 mL, 0.05 M) and 10% wt Pd/C (35 mg, 0.033 mmol) was added. Ammonium formate
(160 mg, 2.54 mmol) was added and the reaction was refluxed at 85 °C for 1.5 h. The reaction mixture was diluted with EtOAc and filtered through celite. Solids were removed from the filtrate by gravity filtration and the filtrate was concentrated under reduced pressure to afford the title compound as a tan solid (100 mg, 59%). LCMS [M+H]+ 536.16 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 8.54 (s, 1 H) 8.07 (d, J=8.3 Hz, 2 H) 7.83 (s, 1 H) 7.75 (s, 1 H) 7.64
(s, 1 H) 7.46 (s, 1 H) 7.29 (s, 2 H) 4.19 - 4.33 (m, 2 H) 3.98 (s, 3 H) 2.88 (t, J=11.2 Hz, 2 H) 2.38
(s, 3 H) 1.99 (d, J=12.7 Hz, 2 H) 1.66 (qd, J=12.7, 4.4 Hz, 2 H) 1.50 (s, 9 H).
206
tert-Butyl 3-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2,5-dihydro-1H-pyrrole-1- carboxylate (5-27b). Intermediate 3-8 (200 mg, 0.419 mmol), tert-butyl 3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (160 mg, 0.232 mmol), and
PdCl2(dppf)·CH2Cl2 (19 mg, 0.023 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (2.1 mL, 0.21 M) and 2M K2CO3 (0.65 mL,
1.30 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (80 °C) for 15 min. The reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex) to afford the title compound as a yellow solid (159 mg,
+ 79 81 1 73%). LCMS [M+H] 518.09 m/z ( Br), 520.10 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.62 (d, J=8.8 Hz, 1 H) 8.52 (s, 1 H) 8.03 (s, 1 H) 8.00 (d, J=6.8 Hz, 2 H) 7.42 (d, J=7.3 Hz,
2 H) 6.58 (d, J=18.1 Hz, 1 H) 4.48 (br. s., 2 H) 4.22 (br. s., 2 H) 2.34 (s, 3 H) 1.47 (d, J=14.2 Hz,
9 H).
207
tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2,5- dihydro-1H-pyrrole-1-carboxylate (3-5r). tert-Butyl 3-(5-bromo-1-tosyl-1H-pyrrolo[2,3- b]pyridin-3-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 5-27b (159 mg, 0.306 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (77 mg, 0.370 mmol), and
PdCl2(dppf)·CH2Cl2 (14 mg, 0.017 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (1.8 mL, 0.17 M) and 2M K2CO3 (0.45 mL, 0.900 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85 °C for
4 h. The reaction mixture was diluted with EtOAc and filtered through Celite®. The filtrate was purified by flash chromatography (20-50% EtOAc:Hex) to afford the title compound as an off-
+ 1 white solid (132 mg, 83%). LCMS [M+H] 520.17 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
8.67 (d, J=2.0 Hz, 1 H) 8.41 (dd, J=8.8, 2.0 Hz, 1 H) 8.34 (d, J=3.4 Hz, 1 H) 8.06 (d, J=2.4 Hz, 1
H) 8.01 (d, J=6.8 Hz, 2 H) 7.93 (s, 1 H) 7.42 (d, J=7.3 Hz, 2 H) 6.66 (d, J=24.9 Hz, 1 H) 4.50 (br. s., 2 H) 4.27 (br. s., 2 H) 3.88 (s, 3 H) 2.34 (s, 3 H) 1.48 (d, J=14.2 Hz, 9 H).
208
tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)pyrrolidine-1-carboxylate (5-28b). Intermediate 3-5r (132 mg, 0.254 mmol) was dissolved in
EtOH (5.0 mL, 0.05 M) and 10% wt Pd/C (27 mg, 0.025 mmol) was added. Ammonium formate
(126 mg, 2.00 mmol) was added and the reaction was refluxed at 85 °C for 1.5 h. The reaction mixture was diluted with EtOAc and filtered through Celite®. Solids were removed from the filtrate by gravity filtration and the filtrate was purified by flash chromatography (2%
MeOH:DCM). However, separation was not achieved. Impure fractions containing the title compound were combined to afford a yellow oil (109 mg) which was taken forward without further purification. LCMS [M+H]+ 522.18 m/z.
209
tert-Butyl 5-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)- carboxylate (5-27c) Intermediate 3-8 (200 mg, 0.419 mmol), tert-butyl 5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (169 mg, 0.546 mmol), and
PdCl2(dppf)·CH2Cl2 (17 mg, 0.021 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (2.1 mL, 0.20 M) and 2M K2CO3 (0.65 mL, 1.30 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (80 °C) for 15 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex) to afford the title compound as an off-white solid (158 mg,
+ 79 81 1 71%). LCMS [M+H] 532.13 m/z ( Br), 534.09 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.51 (s, 1 H) 8.50 (s, 1 H) 8.00 (d, J=8.8 Hz, 2 H) 7.94 (br. s., 1 H) 7.42 (d, J=8.3 Hz, 2 H)
6.46 (s, 1 H) 4.22 (br. s., 2 H) 3.49 (br. s., 2 H) 2.34 (s, 3 H) 2.28 (br. s., 2 H) 1.45 (s, 9 H).
210
tert-Butyl 5-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3,6- dihydropyridine-1(2H)-carboxylate (3-5s) tert-Butyl 5-(5-bromo-1-tosyl-1H-pyrrolo[2,3- b]pyridin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate 5-27c (158 mg, 0.297 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (75 mg, 0.360 mmol), and
PdCl2(dppf)·CH2Cl2 (12 mg, 0.015 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (1.7 mL, 0.17 M) and 2M K2CO3 (0.50 mL, 1.00 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85 °C for
4 h. The reaction mixture was diluted with EtOAc and filtered through celite. The filtrate was purified by flash chromatography (20-50% EtOAc:Hexanes) to afford the title compound as an
+ 1 off-white solid (93%). LCMS [M+H] 534.21 m/z, H NMR (500 MHz, DMSO-d6) δ ppm 8.63
(d, J=2.0 Hz, 1 H) 8.32 (s, 1 H) 8.28 (s, 1 H) 7.96 - 8.03 (m, 3 H) 7.82 (s, 1 H) 7.40 (d, J=8.3 Hz,
2 H) 6.54 (br. s., 1 H) 4.23 (br. s., 2 H) 3.85 (s, 3 H) 3.50 (br. s., 2 H) 2.28 - 2.34 (m, 5 H) 1.44 (s,
9 H).
211
tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piper- idine-1-carboxylate (5-28c). Intermediate 3-5s (147 mg, 0.275 mmol) was dissolved in EtOH (5.0 mL, 0.05 M) and 10% wt Pd/C (27 mg, 0.025 mmol) was added. Ammonium formate (119 mg,
1.89 mmol) was added and the reaction was refluxed at 85 °C for 1 h. The reaction mixture was diluted with EtOAc and filtered through Celite®. Solids were removed from the filtrate by gravity filtration and the filtrate was purified by flash chromatography (2% MeOH:DCM) to afford the title compound as a white solid (68%). LCMS [M+H]+ 536.23 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 8.54 (s, 1 H) 8.06 (d, J=7.3 Hz, 2 H) 7.88 (br. s., 1 H) 7.75 (s, 1 H)
7.64 (s, 1 H) 7.51 (s, 1 H) 7.29 (s, 2 H) 4.04 - 4.14 (m, 1 H) 3.98 (s, 3 H) 2.91 (m, J=11.7 Hz, 3
H) 2.38 (s, 3 H) 2.16 (d, J=13.2 Hz, 1 H) 1.79 (d, J=12.2 Hz, 1 H) 1.60 - 1.73 (m, 3 H) 1.49 (s, 9
H).
212
5-(1-Methyl-1H-pyrazol-4-yl)-3-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(3-5t). The title compound was prepared according to General Procedure H on a 75-mg scale using
(2-methylpyridin-4-yl)boronic acid. The crude material was purified by flash chromatography (3%
MeOH:DCM) to afford the title compound as a dark orange solid (47 mg, 68%). LCMS [M+H]+
1 444.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.70 (d, J=1.95 Hz, 1 H) 8.52 (d, J=5.37 Hz,
1 H) 8.46 (s, 1 H) 8.45 (d, J=1.95 Hz, 1 H) 8.33 (s, 1 H) 8.03 - 8.08 (m, 3 H) 7.74 (s, 1 H) 7.68 (d,
J=5.86 Hz, 1 H) 7.44 (d, J=8.79 Hz, 2 H) 3.88 (s, 3 H) 2.56 (s, 3 H) 2.35 (s, 3 H).
2,6-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (5-29). 4-Bromo-2,6- di-methylpyridine (150 mg, 0.806 mmol), bis(pinacolato)diboron (312 mg, 1.23 mmol), potassium acetate (276 mg, 2.81 mmol) and PdCl2(dppf)·CH2Cl2 (43 mg, 0.042 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane
(5.0 mL, 0.13 M) was added and the reaction was run in the microwave (145 °C) for 30 min. The reaction mixture was diluted with MeOH, filtered through celite, and concentrated under reduced
213 pressure. The dark brown crude material was carried forward without further purification. LCMS
[M+H]+ 151.78 m/z (boronic acid).
3-(2,6-Dimethylpyridin-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3- b]pyridine (3-5u). The title compound was prepared according to General Procedure H on a 75- mg scale using crude 2,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine 5-29.
The crude material was purified by flash chromatography (5% MeOH:DCM), then repurified by flash chromatography (10-100% EtOAc:DCM) to afford the title compound as a tan solid (43 mg,
+ 1 60%). LCMS [M+H] 458.14 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.69 (d, J=1.95 Hz, 1
H) 8.42 (d, J=1.46 Hz, 1 H) 8.41 (s, 1 H) 8.32 (s, 1 H) 8.05 (m, J=3.90, 3.90 Hz, 3 H) 7.52 (s, 2
H) 7.44 (d, J=8.30 Hz, 2 H) 3.88 (s, 3 H) 2.51 (s, 6 H) 2.35 (s, 3 H).
3-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-1207).
The title compound was prepared according to General Procedure K on a 135-mg scale using 3-
5a. The reaction was run for 1 min, and the crude material was purified by flash chromatography
(1-10% MeOH:DCM) to afford the title compound as an off-white solid (63 mg, 71%). LCMS
214
+ 1 [M+H] 300.12 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.04 - 12.12 (m, 1 H) 8.55 (d, J=2.0
Hz, 1 H) 8.44 (d, J=2.0 Hz, 1 H) 8.27 (s, 1 H) 8.21 (s, 1 H) 8.15 (d, J=7.8 Hz, 1 H) 8.06 (d, J=2.4
Hz, 1 H) 8.01 (s, 1 H) 7.68 (d, J=7.8 Hz, 1 H) 7.65 (t, J=7.8 Hz, 1 H) 3.89 (s, 3 H).
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-4829).
Intermediate 3-4 (90 mg, 0.188 mmol), (4-cyanophenyl)boronic acid (83 mg, 0.565 mmol), and
PdCl2(dppf)·CH2Cl2 were combined in a microwave vial which was then purged with nitrogen and evacuated three times. Dioxane (1.9 mL, 0.1 M) and K2CO3 (0.565 mL, 1.1 mmol) were added and the mixture was then degassed for 10 min. The reaction was then run in the microwave for 30 min at 120 ⁰C. Aqueous 2M NaOH (0.3 mL, 0.6 mmol) was then added to the flask, and the solution was microwaved for 1.5 min at 150 °C. Additional 2M NaOH (0.1 mL, 0.2 mmol) was then added, and the reaction was microwaved for another 2.5 min at 150 °C. Crude product was then diluted with EtOAc, and filtered through Celite® using ethyl acetate to wash. The filtrate was purified by flash chromatography (50-100% EtOAc:hexanes) to afford the title compound as a solid (36 mg,
+ 1 64%). LCMS [M+H] 300.12 m/z, H NMR (500 MHz, METHANOL-d4) δ ppm 8.51 (d, J=2.0
Hz, 1 H) 8.46 (d, J=2.0 Hz, 1 H) 8.08 (s, 1 H) 7.94 (d, J=5.4 Hz, 2 H) 7.93 (s, 1 H) 7.87 (s, 1 H)
7.80 (d, J=8.3 Hz, 2 H) 3.96 (s, 3 H).
*The -NH peak is too rapidly exchanging to be seen in the HNMR.
*Prepared by K. Forbes.
215
2-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-4991).
The title compound was prepared according to General Procedure K on a 48-mg scale using 3-5b.
The reaction was run for 5 min, and the crude material was purified by flash chromatography (1-
5% MeOH:DCM), then by prep HPLC (70-5% water:ACN) to afford the title compound as a formate salt. The resulting solid was dissolved in MeOH and MP-carbonate was added. The suspension was stirred overnight at room temperature, then the solids were removed by filtration and the fitrate was concentrated to afford the title compound as a white solid (9 mg, 28%). LCMS
+ 1 [M+H] 300.06 m/z; H NMR (399 MHz, METHANOL-d4) δ ppm 8.51 (d, J=2.20 Hz, 1 H) 8.22
(d, J=2.20 Hz, 1 H) 8.02 (s, 1 H) 7.84 - 7.89 (m, 2 H) 7.75 - 7.83 (m, 3 H) 7.49 (td, J=8.06, 1.47
Hz, 1 H) 3.94 (s, 3 H).
3-(4-Fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-4963).
The title compound was prepared according to General Procedure K on a 42-mg scale using 3-5c.
The reaction was run for 1.75 h, and the crude material was purified by flash chromatography (1-
5% MeOH:DCM) to afford the title compound as a tan solid (11 mg, 39%). LCMS [M+H]+ 293.17
1 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.45 (d, J=1.46 Hz, 1 H) 8.33 (d, J=1.95 Hz, 1
216
H) 8.03 (s, 1 H) 7.89 (d, J=0.98 Hz, 1 H) 7.67 - 7.72 (m, 2 H) 7.61 (s, 1 H) 7.15 - 7.21 (m, 2 H)
3.95 (s, 3 H).
3-(2-Fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-4935).
The title compound was prepared according to General Procedure K on a 55-mg scale using 3-5d.
The reaction was run for 6 min, and the crude material was purified by flash chromatography (1-
5% MeOH:DCM) to afford the title compound as an off-white solid (23 mg, 64%). LCMS [M+H]+
1 293.11 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 12.02 (br. s., 1 H) 8.54 (d, J=1.47 Hz, 1 H)
8.22 (s, 1 H) 8.18 (s, 1 H) 7.93 (s, 1 H) 7.74 - 7.81 (m, 2 H) 7.28 - 7.37 (m, 3 H) 3.87 (s, 3 H).
3-(3,4-Difluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-
4964). The title compound was prepared according to General Procedure K on a 42-mg scale using
3-5e. The reaction was run for 1.75 h, and the crude material was purified by flash chromatography
(1-5% MeOH:DCM) to afford the title compound as a tan solid (17 mg, 59%). LCMS [M+H]+
1 311.14 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.47 (d, J=1.95 Hz, 1 H) 8.35 (d, J=1.95
Hz, 1 H) 8.06 (s, 1 H) 7.91 (s, 1 H) 7.68 (s, 1 H) 7.59 (ddd, J=11.96, 7.81, 2.20 Hz, 1 H) 7.48 -
7.54 (m, 1 H) 7.34 (m, J=10.50, 8.40, 8.40 Hz, 1 H) 3.94 - 3.99 (m, 3 H).
217
2-Fluoro-5-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile/NEU-
4938. The title compound was prepared according to General Procedure K on a 48-mg scale using
3-5f. The reaction was run for 1 min, and the crude material was purified by flash chromatography
(1-10% MeOH:DCM, then 1-10% MeOH:EtOAc) to afford the title compound as a white solid
+ 1 (11 mg 34%). LCMS [M+H] 318.09 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 12.07 (br. s, 1
H) 8.55 (d, J=2.20 Hz, 1 H) 8.43 (d, J=1.47 Hz, 1 H) 8.28 (dd, J=5.13, 2.20 Hz, 1 H) 8.26 (s, 1 H)
8.16 - 8.23 (m, 1 H) 8.02 (d, J=2.20 Hz, 1 H) 8.01 (s, 1 H) 7.58 (t, J=9.53 Hz, 1 H) 3.89 (s, 3 H).
3-Fluoro-5-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(NEU-5301). The title compound was prepared according to General Procedure K on a 44-mg scale using 3-5g. The reaction was run for 2 min, and the crude material was purified by flash chromatography (5% MeOH:DCM) to afford the title compound as a white solid (22 mg, 73%).
+ 1 LCMS [M+H] 318.09 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.17 (s, 1 H) 8.56 (d, J=1.95
Hz, 1 H) 8.48 (d, J=1.46 Hz, 1 H) 8.29 (s, 1 H) 8.16 (s, 1 H) 8.11 (s, 1 H) 8.03 (s, 1 H) 8.01 (d,
J=10.25 Hz, 1 H) 7.68 (d, J=8.30 Hz, 1 H) 3.90 (s, 3 H).
218
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzamide (NEU-4830). 4-
(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile NEU-4829 (15 mg,
0.05 mmol) was put in a microwave vial and dissolved in dioxane (0.1 M, 0.5 mL). Aqueous 2 M
NaOH was then added (0.25 mL, 0.5 mmol) and the reaction was run in the microwave at 150 °C for 40 min. The crude material was purified by flash chromatography (4-1516%
5%NH4OH/MeOH:DCM) to afford the title compound as a solid (11 mg, 72%). LCMS [M+H]+
1 318.15 m/z, H NMR (500 MHz, DMSO-d6) δ ppm 8.54 (s, 1 H) 8.42 (s, 1 H) 8.26 (s, 1 H) 7.97 -
8.03 (m, 3 H) 7.96 (d, J=8.8 Hz, 2 H) 7.86 (d, J=7.8 Hz, 2 H) 7.32 (s, 1 H) 6.63 (s, 1 H) 3.89 (s, 3
H).
*Prepared by K. Forbes.
4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (NEU-4831). 4-
(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile NEU-4829 (15 mg,
0.05 mmol) was put in a microwave vial and dissolved in dioxane (0.1 M, 0.5 mL). Aqueous 2M
NaOH was then added (1 mL, 2 mmol). The reaction was run in the microwave at 150 °C for 30 mins. The crude material was purified by flash chromatography (4-15%
219
5%NH4OH/MeOH:DCM) to afford the title compound as a solid (16 mg, 100%). LCMS [M+H]+
1 319.13 m/z, H NMR (500 MHz, METHANOL-d4) δ ppm 8.48 - 8.50 (m, 1 H) 8.47 (d, J=2.0 Hz,
1 H) 8.12 (d, J=8.3 Hz, 2 H) 8.08 (s, 1 H) 7.93 (s, 1 H) 7.85 (d, J=8.8 Hz, 2 H) 7.82 (s, 1 H) 3.96
(s, 3 H).
*The -NH and -OH peaks are too rapidly exchanging to be seen in the HNMR.
*Prepared by K. Forbes.
5-(1-Methyl-1H-pyrazol-4-yl)-3-(3-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine (NEU-4816). The title compound was prepared according to General Procedure K on a 30-mg scale for 10 min using
3-5h. The crude material was purified by flash chromatography (3% MeOH:DCM) to afford the title compound as a solid (15 mg, 74%). LCMS [M+H]+ 320.12 m/z, 1H NMR (500 MHz, DMSO- d6) δ ppm 12.13 (br. s, 1 H) 8.57 (d, J=2.0 Hz, 1 H) 8.49 (s, 1 H) 8.42 (d, J=2.0 Hz, 1 H) 8.27 (d,
J=8.3 Hz, 1 H) 8.25 (s, 1 H) 8.14 (d, J=2.4 Hz, 1 H) 8.09 (dd, J=7.8, 2.0 Hz, 1 H) 7.98 - 7.99 (m,
1 H) 7.74 (t, J=8.1 Hz, 1 H) 3.89 (s, 3 H).
*Prepared by K. Forbes.
3-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (NEU-4827). The title compound was prepared according to General Procedure K on a 24-mg scale for 10 min
220 using 3-5i. The crude material was purified by flash chromatography (10% MeOH:DCM) to afford the title compound as a solid (5 mg, 32%). LCMS [M+H]+ 290.19 m/z, 1H NMR (500
MHz, DMSO-d6) δ ppm 8.07 (d, J=2.4 Hz, 1 H) 8.04 (d, J=2.0 Hz, 1 H) 7.96 - 7.97 (m, 1 H)
7.76 (s, 1 H) 7.72 (s, 1 H) 7.47 - 7.49 (m, 1 H) 7.46 (s, 1 H) 7.11 (d, J=7.8 Hz, 2 H) 6.91 (t,
J=7.6 Hz, 1 H) 6.83 (d, J=7.8 Hz, 1 H) 6.14 - 6.20 (m, 1 H) 3.86 (s, 3 H).
*Prepared by K. Forbes.
N,N-Dimethyl-1-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3- yl)phenyl)methanamine (NEU-4933). The title compound was prepared according to General
Procedure K on a 36-mg scale using 3-5j. The reaction was run for 5 min, and the crude material was purified by flash chromatography (1-10% 10% NH4OH/MeOH:DCM), then repurified by preparative HPLC (99-5% water:ACN) to afford the title compound as a colorless oil (6 mg, 25%).
+ 1 LCMS [M+H] 332.12 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.46 (d, J=1.95 Hz, 1
H) 8.39 (d, J=1.95 Hz, 1 H) 8.04 (s, 1 H) 7.89 (d, J=0.98 Hz, 1 H) 7.74 (d, J=8.30 Hz, 2 H) 7.69
(s, 1 H) 7.46 (d, J=8.30 Hz, 2 H) 3.95 (s, 3 H) 3.78 (s, 2 H) 2.48 (s, 6 H).
221
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol (NEU-4833). The title compound was prepared according to General Procedure K on a 50-mg scale using 3-5k. The reaction was run for 15 min, and the crude material was purified by flash chromatography (1-10%
MeOH:DCM) to afford the title compound as a white solid (11 mg, 33%). LCMS [M+H]+ 291.15
1 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.69 (br. s, 1 H) 9.34 (br. s, 1 H) 8.49 (d, J=1.95 Hz,
1 H) 8.28 (d, J=1.95 Hz, 1 H) 8.22 (s, 1 H) 7.94 (d, J=0.98 Hz, 1 H) 7.66 (d, J=2.44 Hz, 1 H) 7.55
(d, J=8.79 Hz, 2 H) 6.85 (d, J=8.30 Hz, 2 H) 3.88 (s, 3 H).
3-(1H-indol-5-yl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-5003). The title compound was prepared according to General Procedure K on a 41-mg scale using 3-5l. The reaction was run for 1 h, and the crude material was purified by flash chromatography (1-10%
MeOH:DCM) to afford the title compound as a white solid (12 mg, 43%). LCMS [M+H]+ 314.09
1 m/z; H NMR (399 MHz, METHANOL-d4) δ ppm 8.42 (br. s., 1 H) 8.37 (s, 1 H) 7.98 (s, 1 H)
7.85 (s, 1 H) 7.83 (s, 1 H) 7.53 (s, 1 H) 7.48 (d, J=8.79 Hz, 1 H) 7.42 (d, J=9.53 Hz, 1 H) 7.26 (d,
J=2.93 Hz, 1 H) 6.51 (d, J=2.93 Hz, 1 H) 3.93 (s, 3 H).
222
5-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c][1,2,5] oxadiazole
(NEU-5007). The title compound was prepared according to General Procedure K on a 109-mg scale using 3-5m. The reaction was run for 5 min, and the crude reaction mixture was neutralized with acetic acid. A precipitate was observed and collected by vacuum filtration to afford the title compound as a yellow-green solid (37 mg, 50%). LCMS [M+H]+ 317.11 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 12.32 (br. s, 1 H) 8.65 (s, 1 H) 8.61 (d, J=1.47 Hz, 1 H) 8.35 (m, J=5.10 Hz, 3
H) 8.16 (d, J=9.53 Hz, 1 H) 8.06 - 8.10 (m, 2 H) 3.91 (s, 3 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-4828).
Intermediate 3-4 (30 mg, 0.0627 mmol) was combined with pyridin-3-ylboronic acid and
PdCl2(dppf)·CH2Cl2 (23 mg, 0.188 mmol) in a microwave vial which was purged with nitrogen and evacuated three times. Dioxane (0.6 mL, 0.1 M) and K2CO3 (0.25 mL, 0.5 mmol) were added and the mixture was degassed for 10 min. The reaction was run in the microwave for 40 min at
120 ⁰C. Aqueous NaOH (2 M, 0.3 mL, 0.6 mmol) added and the solution was then microwaved for 10 min at 150 C. The crude product was then diluted with ethyl acetate, and filtered through
Celite® using ethyl acetate to wash. The filtrate was purified by flash chromatography (5%
MeOH:DCM) to afford the title compound as a solid (13 mg, 73%). LCMS [M+H]+ 276.14 m/z;
223
1 H NMR (500 MHz, METHANOL-d4) δ ppm 8.91 (s, 1 H) 8.51 (d, J=2.0 Hz, 1 H) 8.45 (d, J=4.4
Hz, 1 H) 8.40 (d, J=2.4 Hz, 1 H) 8.21 (dt, J=8.3, 3.4 Hz, 1 H) 8.07 (s, 1 H) 7.92 (s, 1 H) 7.82 (s, 1
H) 7.54 (dd, J=7.8, 4.9 Hz, 1 H) 3.96 (s, 3 H).
*The -NH peak is too rapidly exchanging to be seen in the HNMR.
*Prepared by K. Forbes.
5-(1-Methyl-1H-pyrazol-4-yl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-4832). The title compound was prepared according to General Procedure K on a 40-mg scale for 20 min using crude 3-5n. The crude material was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound as a solid (23 mg, 92%) LCMS [M+H]+ 276.15 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 12.20 (s, 1 H) 8.57 (d, J=2.0 Hz, 1 H) 8.55 (d, J=6.3 Hz, 2 H) 8.51 (d, J=2.0 Hz,
1 H) 8.30 (s, 1 H) 8.21 (d, J=2.9 Hz, 1 H) 8.03 (d, J=1.0 Hz, 1 H) 7.83 (d, J=6.3 Hz, 2 H) 3.89 (s,
3 H).
*Prepared by K. Forbes.
5-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinonitrile (NEU-5054).
The title compound was prepared according to General Procedure K on a 60-mg scale using 3-5o.
The reaction was run for 2 min, and the crude material was purified by flash chromatography (1-
224
10% MeOH:DCM) to afford the title compound as a light pink solid (13 mg, 33%). LCMS [M+H]+
1 301.05 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.22 (br. s, 1 H) 9.33 (d, J=1.95 Hz, 1 H)
8.87 (d, J=1.95 Hz, 1 H) 8.68 (t, J=2.20 Hz, 1 H) 8.58 (d, J=1.46 Hz, 1 H) 8.53 (d, J=1.95 Hz, 1
H) 8.30 (s, 1 H) 8.19 (s, 1 H) 8.05 (s, 1 H) 3.89 (s, 3 H).
4-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)picolinonitrile (NEU-5125).
Intermediate 3-5p (34 mg, 0.075 mmol) was dissolved in dioxane (1.5 mL, 0.05 M) and 2M aq.
NaOH (0.10 mL, 0.200 mmol) was added. The reaction was stirred at 85 °C for 1 h. The reaction mixture was purified directly by flash chromatography (0-10% MeOH:EtOAc) to afford the title compound as an off-white solid (8 mg, 34%). LCMS [M+H]+ 301.08 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 12.41 (br. s, 1 H) 8.68 (d, J=5.37 Hz, 1 H) 8.60 (s, 2 H) 8.46 (d, J=1.46 Hz, 1
H) 8.42 (s, 1 H) 8.31 (s, 1 H) 8.19 (dd, J=4.39, 1.95 Hz, 1 H) 8.06 (s, 1 H) 3.90 (s, 3 H).
tert-Butyl 4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1- carboxylate (NEU-5954). The title compound was prepared according to General Procedure K on a 100-mg scale using 5-28a. The reaction was run for 15 min, and the crude material was purified by flash chromatography (70-80% EtOAc:Hexanes) to afford the title compound as a white solid
225
+ 1 (53 mg, 75%). LCMS [M+H] 382.19 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.30 (s, 1 H)
8.42 (d, J=2.0 Hz, 1 H) 8.16 (s, 1 H) 8.11 (d, J=2.0 Hz, 1 H) 7.89 (s, 1 H) 7.22 (d, J=2.0 Hz, 1 H)
3.98 - 4.16 (m, 2 H) 3.87 (s, 3 H) 2.80 - 3.00 (m, 3 H) 1.97 (d, J=13.7 Hz, 2 H) 1.47 - 1.58 (m, 2
H) 1.42 (s, 9 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(piperidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-5955). tert-Butyl 4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1- carboxylate NEU-5954 (48 mg, 0.125 mmol) was taken up in 4 M HCl in dioxane (0.5 mL, 2.00 mmol). The reaction was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure and the resulting white residue was dissolved in MeOH and stirred at room temperature with Si-carbonate overnight. The Si-carbonate was removed by filtration and the filtrate was concentrated to afford the title compound as a light yellow solid (34
+ 1 mg, 97%). LCMS [M+H] 282.19 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.25 (br. s, 1 H)
8.42 (d, J=2.0 Hz, 1 H) 8.17 (s, 1 H) 8.11 (s, 1 H) 7.90 (s, 1 H) 7.17 (s, 1 H) 3.87 (s, 3 H) 3.05 (d,
J=10.2 Hz, 2 H) 2.86 (t, J=13.2 Hz, 1 H) 2.67 (t, J=11.7 Hz, 2 H) 1.89 (d, J=13.7 Hz, 2 H) 1.52 -
1.66 (m, 2 H).
226
tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrrolidine-1- carboxylate (NEU-5976). The title compound was prepared according to General Procedure K on a 109-mg scale using 5-28b. The reaction was run for 15 min, and the crude material was purified by flash chromatography (2-5% MeOH:DCM, step gradient) to afford the title compound as a
+ 1 white solid (34 mg, 44%). LCMS [M+H] 368.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
11.39 (br. s., 1 H) 8.45 (d, J=2.0 Hz, 1 H) 8.13 - 8.20 (m, 2 H) 7.92 (s, 1 H) 7.30 (br. s., 1 H) 3.87
(s, 3 H) 3.72 - 3.84 (m, 1 H) 3.43 - 3.63 (m, 2 H) 3.33 - 3.36 (m, 1 H) 3.16 - 3.28 (m, 1 H) 2.21 -
2.35 (m, 1 H) 1.99 - 2.14 (m, 1 H) 1.41 (d, J=11.7 Hz, 9 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(pyrrolidin-3-yl)-1H-pyrrolo[2,3-b]pyridine hydrochloride
(NEU-6016). tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3- yl)pyrrolidine-1-carboxylate NEU-5976 (22 mg, 0.060 mmol) was taken up in 4 M HCl in dioxane
(0.1 mL, 0.400 mmol). The reaction was stirred at room temperature for 1 h, then stopped and concentrated under reduced pressure to afford the title compound as a tan solid (14 mg, 78%).
+ 1 LCMS [M+H] 268.19 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.95 (d, J=1.5 Hz, 1 H)
8.68 (d, J=1.5 Hz, 1 H) 8.27 (s, 1 H) 8.06 (s, 1 H) 7.71 (s, 1 H) 3.99 (s, 3 H) 3.94 (q, J=8.8 Hz, 1
227
H) 3.87 (dd, J=11.2, 7.8 Hz, 1 H) 3.59 - 3.67 (m, 1 H) 3.47 (ddd, J=10.7, 9.3, 7.8 Hz, 1 H) 3.41 (t,
J=10.2 Hz, 1 H) 2.64 (m, J=3.9 Hz, 1 H) 2.23 - 2.33 (m, 1 H).
*The -NH peaks are too rapidly exchanging to be seen in the HNMR in CD3OD. 1.0 equiv. HCl salt confirmed by HNMR in DMSO-d6.
tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1- carboxylate (NEU-5994). The title compound was prepared according to General Procedure K on a 109-mg scale using 5-28c. The reaction was run for 15 min, and the crude material was purified by flash chromatography (2-5% MeOH:DCM, step gradient) to afford the title compound as a white solid (38 mg, 54%). LCMS [M+H]+ 382.19 m/z, 1H NMR (500 MHz, CHLOROFORM-d)
δ ppm 8.70 - 8.82 (m, 1 H) 8.43 (s, 1 H) 8.00 - 8.09 (m, 1 H) 7.80 (s, 1 H) 7.67 (s, 1 H) 7.13 (s, 1
H) 4.27 - 4.45 (m, 1 H) 3.99 (s, 3 H) 2.97 - 3.08 (m, 1 H) 2.88 (br. s., 2 H) 2.16 - 2.22 (m, 1 H)
1.69 - 1.85 (m, 4 H) 1.50 (s, 9 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(piperidin-3-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-5995). tert-Butyl 3-(5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1- carboxylate NEU-5994 (34 mg, 0.089 mmol) was taken up in 4M HCl in dioxane (0.25 mL, 1.00 mmol). The reaction was stirred at room temperature for 3 h, after which the solvent was removed
228 under reduced pressure. The resulting yellow solid was dissolved in MeOH and Si-carbonate was added. The mixture was stirred overnight at room temperature. The Si-carbonate was removed by filtration and the filtrate was concentrated to afford the title compound as an off-white solid (20
+ 1 mg, 78%). LCMS [M+H] 282.19 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.40 (d,
J=2.0 Hz, 1 H) 8.19 (d, J=2.0 Hz, 1 H) 8.02 (s, 1 H) 7.88 (s, 1 H) 7.25 (s, 1 H) 3.96 (s, 3 H) 3.44
(d, J=12.7 Hz, 1 H) 3.27 (d, J=9.8 Hz, 1 H) 3.10 - 3.22 (m, 1 H) 2.87 (t, J=12.2 Hz, 2 H) 2.15 -
2.22 (m, 1 H) 1.93 - 1.99 (m, 1 H) 1.76 - 1.88 (m, 2 H).
5-(1-Methyl-1H-pyrazol-4-yl)-3-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine/NEU-
5304. The title compound was prepared according to General Procedure K on a 47-mg scale using
3-5t. The reaction was run for 5 min, and the crude material was purified by flash chromatography
(1-10% 5% NH4OH/MeOH:DCM) to afford the title compound as a brown solid (19 mg, 60%).
+ 1 LCMS [M+H] 290.16 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.45 (d, J=1.95 Hz, 1
H) 8.41 (d, J=1.95 Hz, 1 H) 8.36 (d, J=5.37 Hz, 1 H) 8.03 (s, 1 H) 7.91 (s, 1 H) 7.90 (s, 1 H) 7.60
(s, 1 H) 7.56 (dd, J=5.37, 1.46 Hz, 1 H) 3.94 (s, 3 H) 2.57 (s, 3 H).
3-(2,6-Dimethylpyridin-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine/
NEU-5324. The title compound was prepared according to General Procedure K on a 43-mg scale
229 using 3-5u. The reaction was run for 5 min, and the crude material was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound as a tan solid (24 mg, 83%).
+ 1 LCMS [M+H] 304.21 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.11 (br. s., 1 H) 8.55 (d,
J=1.95 Hz, 1 H) 8.44 (d, J=1.95 Hz, 1 H) 8.27 (s, 1 H) 8.11 (s, 1 H) 8.02 (s, 1 H) 7.45 (s, 2 H) 3.90
(s, 3 H) 2.49 (s, 6 H).
5-Bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (3-7). 5-bBromo-1H-pyrrolo[2,3-b]pyridine 3-1
(1.01 g, 5.13 mmol) was dissolved in ACN (30 mL, 0.17 M) and N-iodosuccinimide (1.71 g, 7.60 mmol) was added. The reaction was stirred at 50 °C for 2 h. Upon cooling to room temperature, a tan precipitate was observed and collected by vacuum filtration (washed with hexanes) to afford the title compound as a pale orange solid (1.38 g, 83%). LCMS [M+H]+ 322.87 m/z (79Br), 324.89
81 1 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 12.35 (br. s., 1 H) 8.32 (d, J=1.95 Hz, 1 H) 7.87
(d, J=2.93 Hz, 1 H) 7.80 (d, J=2.44 Hz, 1 H).
5-Bromo-3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-8). Intermediate 3-7 (1.38 g, 4.27 mmol) was suspended in DCM (20 mL, 0.21 M) and TEA (1.80 mL, 12.91 mmol), DMAP (646 mg, 5.29 mmol) and 4-methylbenzenesulfonyl chloride (2.05 g, 10.75 mmol) were added in that order. The reaction was stirred overnight at room temperature, then washed once with 1M HCl, once with
230 saturated aqueous NaHCO3, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-50% EtOAc:Hex) to afford the title compound as a light orange solid (1.58 g,
+ 79 81 1 78%). LCMS [M+H] 476.90 m/z ( Br), 478.92 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.51 (d, J=1.95 Hz, 1 H) 8.22 (s, 1 H) 7.97 - 8.04 (m, 3 H) 7.43 (d, J=8.79 Hz, 2 H) 2.34 (s,
3 H).
3-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-9). Intermediate 3-8 (575 mg, 1.21 mmol), (3-cyanophenyl)boronic acid (176 mg, 1.20 mmol), and PdCl2(dppf)·CH2Cl2 (88 mg, 0.108 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (8 mL, 0.15 M) and 2M K2CO3 (2.0mL, 4.00 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for 5 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex) to afford the title compound as a light yellow solid (322 mg, 59%). LCMS [M+H]+ 452.03 (79Br),
81 1 454.04 ( Br) m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.65 (d, J=1.95 Hz, 1 H) 8.55 (d, J=1.95
Hz, 1 H) 8.50 (s, 1 H) 8.33 (d, J=1.46 Hz, 1 H) 8.16 (dd, J=8.06, 1.22 Hz, 1 H) 8.04 (d, J=8.30
Hz, 2 H) 7.84 (dd, J=7.81, 0.98 Hz, 1 H) 7.68 (t, J=7.32 Hz, 1 H) 7.44 (d, J=7.81 Hz, 2 H) 2.35 (s,
3 H).
231
3-(5-(1,3-Dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-
10a). The title compound was prepared according to General Procedure I on a 76-mg scale using
1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole. The crude material was purified by flash chromatography (20-100% EtOAc:hexanes) to afford the title compound as
+ 1 a light orange solid (57 mg, 72%). LCMS [M+H] 468.06 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 8.50 (d, J=1.6 Hz, 1 H) 8.43 (s, 1 H) 8.33 (s, 1 H) 8.26 (d, J=1.8 Hz, 1 H) 8.17 (d, J=6.6 Hz,
1 H) 8.07 (d, J=8.4 Hz, 2 H) 8.02 (s, 1 H) 7.84 (d, J=7.7 Hz, 1 H) 7.69 (t, J=8.0 Hz, 1 H) 7.44 (d,
J=8.2 Hz, 2 H) 3.79 (s, 3 H) 2.35 (s, 3 H) 2.29 (s, 3 H).
3-(1-Tosyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(3-10b). The title compound was prepared according to General Procedure I on a 101-mg scale using 1,3,5-trimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole. The crude material was purified by flash chromatography (20-70% EtOAc:Hex) to afford the title compound
+ 1 as a yellow solid (67 mg, 62%). LCMS [M+H] 482.07 m/z; H NMR (500 MHz, DMSO-d6) δ
232 ppm 8.46 (s, 1 H) 8.34 (d, J=2.0 Hz, 1 H) 8.31 (s, 1 H) 8.18 (d, J=2.0 Hz, 1 H) 8.14 (d, J=7.8 Hz,
1 H) 8.10 (d, J=8.3 Hz, 2 H) 7.82 (d, J=7.8 Hz, 1 H) 7.67 (t, J=7.8 Hz, 1 H) 7.46 (d, J=8.3 Hz, 2
H) 3.71 (s, 3 H) 2.36 (s, 3 H) 2.19 (s, 3 H) 2.10 (s, 3 H).
3-(5-(4-(Methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-10c).
The title compound was prepared according to General Procedure I on a 30-mg scale using (4-
(methylsulfonyl)phenyl)boronic acid. The crude material was purified by flash chromatography
(20-70% EtOAc:Hexanes) to afford the title compound as a white solid (11 mg, 31%). LCMS
+ 1 [M+H] 528.07 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.81 (d, J=1.5 Hz, 1 H) 8.63 (d, J=1.5
Hz, 1 H) 8.52 (s, 1 H) 8.41 (s, 1 H) 8.26 (d, J=6.8 Hz, 1 H) 8.07 - 8.13 (m, 4 H) 8.00 - 8.07 (m, 2
H) 7.86 (d, J=7.3 Hz, 1 H) 7.71 (t, J=7.8 Hz, 1 H) 7.46 (d, J=8.8 Hz, 2 H) 3.27 (s, 3 H) 2.36 (s, 3
H).
233
Scheme 5-7. Synthesis of NEU-5421.
N-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-5-amine (5-30). 5-Bromo-1H- pyrrolo[2,3-b]pyridine 3-1 (250 mg, 1.27 mmol), 4-(methylsulfonyl)aniline (264 mg, 1.54 mmol),
BrettPhos (34 mg, 0.063 mmol), and BrettPhos Pd G1 (51 mg, 0.064 mmol) were combined in a vial that was filled with nitrogen and evacuated three times. 1.0 M LiHMDS in THF (3.2 mL, 3.2 mmol) was added and the reaction was heated at 65 °C for 5 h. The reaction was quenched by the addition of 1M HCl, then diluted with EtOAc and poured over saturated aqueous NaHCO3. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hex) to afford the title compound as a light yellow solid (146 mg, 40%). LCMS [M+H]+ 288.08 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.65 (br. s., 1 H) 8.69 (s, 1 H) 8.09 (d, J=2.4 Hz, 1 H) 7.82 (d, J=2.0 Hz, 1 H)
234
7.63 (d, J=8.8 Hz, 2 H) 7.49 (t, J=2.9 Hz, 1 H) 6.91 (d, J=8.8 Hz, 2 H) 6.43 (dd, J=3.2, 1.7 Hz, 1
H) 3.07 (s, 3 H).
3-Iodo-N-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-5-amine (5-31). N-(4-
(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-5-amine 5-30 (146 mg, 0.508 mmol) was dissolved in ACN (2.8 mL, 0.18 M) and N-iodosuccinimide (138 mg, 0.613 mmol) was added.
The reaction was stirred at 50 °C for 5 h. Upon cooling, a dark brown precipitate was observed and collected by vacuum filtration to afford the title compound as a red-brown solid (110 mg,
+ 1 52%). LCMS [M+H] 413.99 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.13 (br. s., 1 H) 8.80
(s, 1 H) 8.15 (d, J=2.4 Hz, 1 H) 7.74 (d, J=2.4 Hz, 1 H) 7.67 (d, J=8.8 Hz, 2 H) 7.47 (d, J=2.0 Hz,
1 H) 6.97 (d, J=8.8 Hz, 2 H) 3.08 (s, 3 H).
3-Iodo-N-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-amine (5-32). 3-
Iodo-N-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-5-amine 5-31 (110 mg, 0.266 mmol) was suspended in DCM (1.3 mL, 0.21 M) and TEA (0.15 mL, 1.08 mmol), DMAP (43 mg,
0.352 mmol) and 4-methylbenzenesulfonyl chloride (129 mg, 0.677 mmol) were added in that order. The reaction was stirred overnight at room temperature. The reaction was washed once with
1M HCl, once with saturated aqueous NaHCO3, and once with brine; the organic layer was dried
235 with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:Hex) to afford the title compound as an off-white solid (66
+ 1 mg, 44%). LCMS [M+H] 567.90 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.03 (s, 1 H) 8.27
(d, J=2.4 Hz, 1 H) 8.13 (s, 1 H) 8.01 (d, J=8.3 Hz, 2 H) 7.71 (d, J=8.8 Hz, 2 H) 7.50 (d, J=2.4 Hz,
1 H) 7.44 (d, J=8.8 Hz, 2 H) 7.10 (d, J=8.8 Hz, 2 H) 3.11 (s, 3 H) 2.35 (s, 3 H).
3-(5-((4-(methylsulfonyl)phenyl)amino)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(3-10d). 3-Iodo-N-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-amine 5-32
(65 mg, 0.114 mmol), (3-cyanophenyl)boronic acid (35 mg, 0.238 mmol), and
PdCl2(dppf)·CH2Cl2 (9 mg, 0.011 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.50 mL, 0.25 M) and 2M K2CO3 (0.20 mL, 0.400 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for five min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude residue was purified by flash chromatography (20-75% EtOAc:Hex) to afford the title compound as a dark orange solid (46 mg,
+ 1 74%). LCMS [M+H] 543.03 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.99 (s, 1 H) 8.42 (s, 1
H) 8.33 (d, J=2.4 Hz, 1 H) 8.29 (t, J=1.5 Hz, 1 H) 8.13 (d, J=2.4 Hz, 1 H) 8.11 (dt, J=8.3, 1.5 Hz,
1 H) 8.06 (d, J=8.3 Hz, 2 H) 7.82 (dt, J=7.8, 1.5 Hz, 1 H) 7.65 - 7.70 (m, 3 H) 7.45 (d, J=8.3 Hz,
2 H) 7.09 (d, J=8.8 Hz, 2 H) 3.09 (s, 3 H) 2.36 (s, 3 H).
236
3-(5-(Pyrimidin-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-10e). The title compound was prepared according to General Procedure I on a 74-mg scale using 5-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine. The crude material was purified by flash chromatography (20-100% EtOAc:Hex) to afford the title compound as an off-white solid (53 mg,
+ 1 72%). LCMS [M+H] 452.06 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 9.29 (s, 2 H) 9.24 (s, 1
H) 8.85 (d, J=2.2 Hz, 1 H) 8.77 (d, J=2.2 Hz, 1 H) 8.55 (s, 1 H) 8.42 (s, 1 H) 8.28 (d, J=8.1 Hz, 1
H) 8.09 (d, J=8.8 Hz, 2 H) 7.86 (d, J=8.1 Hz, 1 H) 7.70 (t, J=8.1 Hz, 1 H) 7.46 (d, J=8.1 Hz, 2 H)
2.35 (s, 3 H).
3-(5-(Pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (3-10f). The title compound was prepared according to General Procedure I on a 74-mg scale using 4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine. The crude material was purified by flash chromatography to afford the title compound as an off-white solid (62 mg, 84%). LCMS [M+H]+
1 451.08 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 8.86 (s, 1 H) 8.67 (m, J=5.9 Hz, 3 H) 8.52 (s,
237
1 H) 8.41 (s, 1 H) 8.26 (d, J=7.3 Hz, 1 H) 8.09 (d, J=8.1 Hz, 2 H) 7.84 - 7.91 (m, 3 H) 7.71 (t,
J=8.1 Hz, 1 H) 7.46 (d, J=8.1 Hz, 2 H) 2.35 (s, 3 H).
Scheme 5-8. Synthesis of NEU-5422.
tert-Butyl 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate (5-33). 5-Bromo-1H- pyrrolo[2,3-b]pyridine 3-1 (250 mg, 1.27 mmol), 1-boc piperazine (286 mg, 1.54 mmol) RuPhos
(30 mg, 0.064 mmol), and RuPhos Pd G1 (51 mg, 0.062 mmol) were combined in a vial that was filled with nitrogen and evacuated three times. 1.0 M LiHMDS in THF (3.2 mL, 3.2 mmol) was added and the reaction was heated at 65 °C for 5 h. The reaction was cooled to room temperature and quenched by the addition of 1M HCl, then diluted with EtOAc and poured over saturated aqueous NaHCO3. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried with sodium sulfate and concentrated under
238 reduced pressure. The crude residue was purified by flash chromatography (50-80%
EtOAc:Hexanes) to afford the title compound as a light yellow solid (350 mg, 91%). LCMS
+ 1 [M+H] 303.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.34 - 11.42 (m, 1 H) 8.06 (d, J=2.4
Hz, 1 H) 7.51 (d, J=2.4 Hz, 1 H) 7.37 (t, J=2.9 Hz, 1 H) 6.32 (dd, J=3.2, 1.7 Hz, 1 H) 3.49 (br. t,
J=4.9, 4.9 Hz, 4 H) 3.01 (br. t, J=4.9, 4.9 Hz, 4 H) 1.43 (s, 9 H).
tert-Butyl 4-(3-iodo-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate (5-34). tert-
Butyl 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate 5-33 (350 mg, 1.16 mmol) was dissolved in ACN (6.5 mL, 0.18 M) and N-iodosuccinimide (314 mg, 1.40 mmol) was added. The reaction was stirred at 50 °C for 2 h. The reaction was cooled to room temperature, diluted with
EtOAc, and washed three times with water and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography (50% EtOAc:Hexanes) to afford the title compound as a yellow solid (230 mg,
+ 1 46%). LCMS [M+H] 429.11 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.83 - 11.93 (m, 1 H)
8.12 (d, J=2.9 Hz, 1 H) 7.61 (d, J=2.9 Hz, 1 H) 7.13 (d, J=2.4 Hz, 1 H) 3.51 (br. s., 1 H) 3.07 (t,
J=4.9 Hz, 4 H) 1.43 (s, 9 H).
239
tert-Butyl 4-(3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate (5-35). tert-Butyl 4-(3-iodo-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate 5-34 (230 mg, 0.537 mmol) was suspended in DCM (2.7 mL, 0.21 M) and TEA (0.30 mL, 2.15 mmol), DMAP (84 mg,
0.688 mmol) and 4-methylbenzenesulfonyl chloride (240 mg, 1.26 mmol) were added in that order.
The reaction was stirred overnight at room temperature. The reaction was washed once with 1M
HCl, once with saturated aqueous NaHCO3, and once with brine; the organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography (20% EtOAc:Hex) to afford the title compound as an orange oil (138 mg, 44%).
+ 1 LCMS [M+H] 583.04 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.22 (t, J=2.4 Hz, 1 H) 8.03
(d, J=2.4 Hz, 1 H) 7.95 (dd, J=8.5, 2.2 Hz, 2 H) 7.41 (d, J=6.8 Hz, 2 H) 7.13 (t, J=2.4 Hz, 1 H)
3.47 (br. s., 4 H) 3.14 (br. s., 4 H) 2.33 (s, 3 H) 1.42 (s, 9 H).
3-Iodo-5-(piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-36). tert-Butyl 4-(3-iodo-1- tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate 5-35 (138 mg, 0.237 mmol) was
240 taken up in 4M HCl in dioxane (0.60 mL, 2.40 mmol) and the reaction was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the yellow residue (HCl salt) was dissolved in water and poured over 1 M NaOH. The aqueous layer (pH 13-14) was extracted three times with DCM. The combined organic layers were dried with sodium sulfate and concentrated under reduced pressure to afford the title compound as a yellow oil (73 mg, 64%).
+ 1 LCMS [M+H] 483.01 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.20 (d, J=2.9 Hz, 1 H) 8.01
(s, 1 H) 7.95 (d, J=8.3 Hz, 2 H) 7.41 (d, J=8.8 Hz, 2 H) 7.05 (d, J=2.9 Hz, 1 H) 3.08 (t, J=4.9 Hz,
4 H) 2.84 (t, J=5.4 Hz, 4 H) 2.34 (s, 3 H).
3-Iodo-5-(4-(methylsulfonyl)piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-37). 3-
Iodo-5-(piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-36 (73 mg, 0.151) was dissolved in
DCM (0.5 mL, 0.32 M) and TEA (42 µl, 0.301 mmol) was added. The reaction was stirred for 10 min at room temperature before the addition of methanesulfonyl chloride (14 µl, 0.181 mmol).
The reaction was stirred overnight at room temperature. The reaction was quenched with water and extracted four times with DCM. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:Hexanes) to afford the title compound as an
+ 1 orange solid (68 mg, 80%). LCMS [M+H] 560.98 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
241
8.25 (d, J=2.4 Hz, 1 H) 8.04 (s, 1 H) 7.96 (d, J=8.3 Hz, 2 H) 7.41 (d, J=8.3 Hz, 2 H) 7.17 (d, J=2.9
Hz, 1 H) 3.28 - 3.32 (m, 4 H) 3.24 - 3.28 (m, 4 H) 2.93 (s, 3 H) 2.34 (s, 3 H).
3-(5-(4-(Methylsulfonyl)piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(3-10g). 3-Iodo-5-(4-(methylsulfonyl)piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (68 mg,
0.121 mmol), (3-cyanophenyl)boronic acid (36 mg, 0.245 mmol), and PdCl2(dppf)·CH2Cl2 (10 mg, 0.012 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.50 mL, 0.25 M) and 2M K2CO3 (0.20 mL, 0.400 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for
5 min, then diluted with EtOAc, filtered through celite, and concentrated under reduced pressure.
The crude residue was purified by flash chromatography (50% EtOAc:Hex) to afford the title compound as a dull red glassy solid (41 mg, 63%). LCMS [M+H]+ 536.10 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 8.31 (s, 1 H) 8.28 (s, 1 H) 8.28 (s, 1 H) 8.10 - 8.15 (m, 1 H) 8.01 (d, J=8.3
Hz, 2 H) 7.83 (d, J=7.8 Hz, 1 H) 7.76 (d, J=2.4 Hz, 1 H) 7.68 (t, J=7.8 Hz, 1 H) 7.42 (d, J=8.8 Hz,
2 H) 3.29 - 3.31 (m, 4 H) 3.26 (m, J=5.9 Hz, 4 H) 2.93 (s, 3 H) 2.34 (s, 3 H).
242
Scheme 5-9. Synthesis of NEU-5813, -5814, and -5902.
Tetrahydro-2H-pyran-4-yl methanesulfonate (5-39a). Tetrahydro-2H-pyran-4-ol 5-38a (0.500 mL, 5.24 mmol) was dissolved in DCM (9.0 mL, 0.60 M) and TEA (0.750 mL, 5.38 mmol) was added, followed by the addition of DMAP (128 mg, 1.05 mmol). The solution was cooled to 0 °C and MsCl (0.410 mL, 5.30 mmol) was added dropwise, upon which the reaction mixture turned opaque. The reaction was run at 0 °C for 4 h, after which water was added and the reaction was transferred to a separatory funnel. The organic layer was washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure to afford the title compound as an off- white solid (770 mg, 82%). 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 4.92 (spt, J=4.2 Hz,
1 H) 3.92 - 4.00 (m, 2 H) 3.56 (td, J=10.2, 8.8 Hz, 2 H) 3.05 (s, 3 H) 2.02 - 2.10 (m, 2 H) 1.84 -
1.95 (m, 2 H).
*Does not ionize.
243
tert-Butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (5-38b). tert-Butyl 4- hydroxypiperidine-1-carboxylate 5-38b (2.00 g, 9.94 mmol) was dissolved in DCM (20 mL, 0.5
M) and the reaction mixture was cooled to 0-5. TEA (3.0 mL, 21.52 mmol) was slowly added followed by the slow addition of methane sulfonyl chloride (1.0 mL, 12.92 mmol). The reaction was stirred for 1 h at room temperature. The reaction mixture was purified by flash chromatography (10% EtOAc:Hexanes) to afford the title compound as a white solid (1.7g, 61%).
+ 1 LCMS [M+H] 223.95 m/z (loss of t-Bu group); H NMR (500 MHz, DMSO-d6) δ ppm 4.82 (tt,
J=8.1, 3.8 Hz, 1 H) 3.60 (tt, J=5.9, 4.4 Hz, 2 H) 3.20 (s, 3 H) 3.12 - 3.19 (m, 2 H) 1.86 - 1.94 (m,
2 H) 1.60 (dtd, J=13.0, 8.8, 8.8, 4.1 Hz, 2 H) 1.40 (s, 9 H).
*Prepared by H. Jalani.
1-Methylpiperidin-4-yl methanesulfonate (5-39c). 1-Methylpiperidin-4-ol 5-38c (0.500 mL,
4.25 mmol) was dissolved in DCM (7.0 mL, 0.60 M) and TEA (0.600 mL, 4.30 mmol) was added, followed by the addition of DMAP (103 mg, 0.843 mmol). The solution was cooled to 0 °C and
MsCl (0.330 mL, 4.26 mmol) was added dropwise, upon which the reaction mixture turned opaque. The reaction was run at 0 °C for 5 h. Water was added and the reaction was transferred to a separatory funnel. The organic layer was washed once more with water, once with brine, dried with sodium sulfate, and concentrated under reduced pressure to afford the title compound as a
244 yellow oil (444 mg, 54%). LCMS [M+H]+ 193.97 m/z; 1H NMR (500 MHz, CHLOROFORM-d)
δ ppm 4.75 (br. s., 1 H) 3.03 (s, 3 H) 2.67 (br. s., 2 H) 2.21 - 2.35 (m, 5 H) 1.99 - 2.07 (m, 2 H)
1.88 - 1.97 (m, 2 H).
4-Bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (5-40a). 4-bromo-1H-pyrazole (200 mg,
1.36 mmol) was dissolved in DMF (1.7 mL, 0.81 M) and cooled to 0 °C. NaH (165 mg, 4.13 mmol) was added portionwise and the reaction was stirred for 1 h at 0 °C. Tetrahydro-2H-pyran-
4-yl methanesulfonate 5-39a (317 mg, 1.76 mmol) was added. The reaction was gradually heated to 100 °C and stirred overnight. The reaction mixture was quenched with water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried with sodium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (2%
5%NH4OH/MeOH:DCM) to afford the title compound as an off-white solid (191 mg, 61%).
+ 79 81 1 LCMS [M+H] 230.93 m/z ( Br), 232.92 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.06
(s, 1 H) 7.55 (s, 1 H) 4.39 (tt, J=10.6, 5.2 Hz, 1 H) 3.94 (d, J=12.7 Hz, 2 H) 3.44 (td, J=11.5, 2.9
Hz, 2 H) 1.85 - 1.98 (m, 4 H).
tert-Butyl 4-(4-bromo-1H-pyrazol-1-yl)piperidine-1-carboxylate (5-40b). 4-Bromo-1H- pyrazole (200 mg, 1.36 mmol) was dissolved in DMF (1.7 mL, 0.81 M) and cooled to 0 °C. NaH
245
(166 mg, 4.15 mmol) was added portionwise and the reaction was stirred for 1 h at 0 °C. tert-Butyl
4-((methylsulfonyl)oxy)piperidine-1-carboxylate 5-39b (497 mg, 1.78 mmol) was added and the reaction was gradually heated to 100 °C and stirred overnight. The reaction mixture was quenched with water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried with sodium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (0-50% EtOAc:Hex) to afford the title compound as a colorless oil (282 mg,
36%). LCMS [M+H]+ 273.94 m/z (79Br), 275.96 m/z (81Br); 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 7.47 (s, 1 H) 7.44 (s, 1 H) 4.16 - 4.41 (m, 3 H) 2.76 - 2.97 (m, 2 H)
2.10 (d, J=12.2 Hz, 2 H) 1.87 (qd, J=12.2, 3.9 Hz, 2 H) 1.47 (s, 9 H).
4-(4-Bromo-1H-pyrazol-1-yl)-1-methylpiperidine (5-40c). 4-bromo-1H-pyrazole (200 mg, 1.36 mmol) was dissolved in dry DMF (1.7 mL, 0.81 M) and cooled to 0 °C. NaH (168 mg, 4.20 mmol) was added portionwise and the reaction was stirred for 1 h at 0 °C, after which 1-methylpiperidin-
4-yl methanesulfonate 5-39c (444 mg, 2.30 mmol) was dissolved in dry DMF (1.4 mL, 1.7 M) added. The reaction was gradually heated to 100 °C and stirred for two days. The reaction mixture was quenched with water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (3% 5%NH4OH/MeOH:DCM) to afford the title compound as an off-white solid (80 mg, 24%). LCMS [M+H]+ 243.97 m/z (79Br), 245.98 m/z
81 1 ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 8.04 (s, 1 H) 7.53 (s, 1 H) 4.09 (dt, J=10.5, 5.0 Hz,
1 H) 2.82 (d, J=11.7 Hz, 2 H) 2.18 (s, 3 H) 1.96 - 2.07 (m, 2 H) 1.85 - 1.95 (m, 4 H).
246
3-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (5-41). 3-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (597 mg,
1.32 mmol), potassium acetate (456 mg, 4.65 mmol), bis(pinacolato)diboron (505 mg, 1.99 mmol), and PdCl2(dppf)·CH2Cl2 (56 mg, 0.069 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (13.0 mL, 0.10 M) was added and the reaction was run in the microwave (145 °C) for 1 h. The reaction was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex), then repurified by flash chromatography (1%
MeOH:DCM) to afford the title compound as a white solid (148 mg, 22%). LCMS [M+H]+ 500.08 m/z; 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.86 (s, 1 H) 8.40 (s, 1 H) 8.15 (d, J=8.3 Hz,
2 H) 7.92 (s, 1 H) 7.89 (s, 1 H) 7.85 (d, J=7.8 Hz, 1 H) 7.67 (d, J=7.8 Hz, 1 H) 7.61 (t, J=7.8 Hz,
1 H) 7.29 (d, J=8.3 Hz, 2 H) 2.39 (s, 3 H) 1.36 (s, 12 H).
247
3-(5-(1-(Tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (3-10h). 4-Bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole 5-40a (44 mg,
0.190 mmol) and PdCl2(dppf)·CH2Cl2 (14 mg, 0.017 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Crude 3-(5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile 5-41 (84 mg, 0.168 mmol) was dissolved in dioxane (1.0 mL, 0.17 M) and added to the reaction mixture, followed by the addition of 2M K2CO3 (0.40 mL, 0.800 mmol). The reaction was degassed and run in the microwave (145 °C) for 5 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-75% EtOAc:Hex) to afford the title compound as a pale yellow solid (37 mg,
+ 1 43%). LCMS [M+H] 524.07 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.72 (s, 1 H) 8.44 (m,
J=6.3 Hz, 2 H) 8.40 (s, 1 H) 8.34 (s, 1 H) 8.19 (d, J=8.3 Hz, 1 H) 8.03 - 8.10 (m, 3 H) 7.86 (d,
J=7.3 Hz, 1 H) 7.71 (t, J=8.3 Hz, 1 H) 7.44 (d, J=7.8 Hz, 2 H) 4.37 - 4.46 (m, 1 H) 3.97 (d, J=12.7
Hz, 2 H) 3.48 (t, J=10.7 Hz, 2 H) 2.35 (s, 3 H) 1.88 - 2.06 (m, 4 H).
248
tert-Butyl 4-(4-(3-(3-cyanophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazol-1- yl)piperidine-1-carboxylate (3-10i). tert-Butyl 4-(4-bromo-1H-pyrazol-1-yl)piperidine-1- carboxylate 5-40b (282 mg, 0.854 mmol) and PdCl2(dppf)·CH2Cl2 (54 mg, 0.066 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Crude 3-(5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
5-41 (332 mg, 0.665 mmol) was dissolved in dioxane (3.5 mL, 0.17 M) and added to the reaction mixture, followed by the additon of 2M K2CO3 (1.3 mL, 2.60 mmol). The reaction was degassed and run in the microwave (145 °C) for 25 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-40-60% EtOAc:Hex, step gradient) to afford the title compound as
+ 1 a pale yellow solid (180 mg, 43%). LCMS [M+H] 623.07 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 8.71 (d, J=1.5 Hz, 1 H) 8.44 (s, 1 H) 8.43 (d, J=1.5 Hz, 1 H) 8.40 (s, 1 H) 8.34 (s, 1 H) 8.18
(d, J=7.8 Hz, 1 H) 8.03 - 8.09 (m, 3 H) 7.85 (d, J=7.8 Hz, 1 H) 7.71 (t, J=8.3 Hz, 1 H) 7.43 (d,
J=7.8 Hz, 2 H) 4.34 - 4.42 (m, 1 H) 4.02 (br. s, 2 H) 2.93 (br. s, 2 H) 2.34 (s, 3 H) 2.04 (d, J=10.7
Hz, 2 H) 1.79 (dd, J=12.2, 3.9 Hz, 2 H) 1.42 (s, 9 H).
249
3-(5-(1-(1-Methylpiperidin-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (3-10j). 3-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H- pyrrolo[2,3-b]pyridin-3-yl)benzonitrile 5-41 (64 mg, 0.128 mmol), 4-(4-bromo-1H-pyrazol-1-yl)-
1-methylpiperidine 5-40c (40 mg, 0.164 mmol) and PdCl2(dppf)·CH2Cl2 (11 mg, 0.013 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane
(1.0 mL, 0.17 M) was added to the reaction mixture, followed by the addition of 2M K2CO3 (0.30 mL, 0.600 mmol). The reaction was degassed and run in the microwave (145 °C) for 5 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The product was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound as an orange residue (45 mg, 65%). LCMS [M+H]+ 537.11 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 8.71 (s, 1 H) 8.43 (s, 2 H) 8.40 (s, 1 H) 8.34 (s, 1 H) 8.19 (d, J=7.3 Hz, 1
H) 8.02 - 8.09 (m, 3 H) 7.86 (d, J=7.8 Hz, 1 H) 7.71 (t, J=7.8 Hz, 1 H) 7.44 (d, J=8.3 Hz, 2 H)
4.05 - 4.17 (m, 1 H) 2.83 - 2.91 (m, 2 H) 2.35 (s, 3 H) 2.20 (s, 3 H) 1.96 - 2.07 (m, 6 H).
250
3-(5-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-4998). 3-(5-Bromo-
1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (18 mg, 0.060 mmol), 4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (49 mg, 0.252 mmol), potassium carbonate (25 mg, 0.181 mmol) and palladium tetrakis (3 mg, 3 µmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. A 4:2:1 mixture of DME:EtOH:H2O (1.5 mL, 0.04M) was added and the reaction was degassed and run in the microwave (175 °C) for 15 min. The reaction mixture was diluted with MeOH, filtered through celite, and concentrated under reduced pressure.
The filtrate was purified by flash chromatography (1-10% 5% NH4OH/MeOH:DCM) to afford the title compound as a white solid (13 mg, 76%). LCMS [M+H]+ 286.09 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 12.98 (br. s, 1 H) 12.06 (br. s., 1 H) 8.60 (d, J=1.5 Hz, 1 H) 8.48 (d, J=1.5 Hz,
1 H) 8.33 (br. s, 1 H) 8.22 (s, 1 H) 8.16 (dt, J=7.5, 1.7 Hz, 1 H) 8.02 - 8.12 (m, 2 H) 7.67 - 7.71
(m, 1 H) 7.64 (t, J=8.1 Hz, 1 H).
3-(5-(1,3-Dimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-
4992). The title compound was prepared according to General Procedure K on a 57-mg scale using
3-(5-(1,3-dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile. The reaction was run for 2 min, and the crude material was purified by flash chromatography (1-10%
MeOH:DCM) to afford the title compound as an off-white solid (31 mg, 81%). LCMS [M+H]+
251
1 314.09 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 12.11 (br. s., 1 H) 8.35 (br. s., 1 H) 8.27 (br. s., 1 H) 8.19 (br. s., 1 H) 8.12 (d, J=8.06 Hz, 1 H) 8.08 (br. s., 1 H) 7.97 (s, 1 H) 7.68 (d, J=7.33
Hz, 1 H) 7.64 (t, J=5.86 Hz, 1 H) 3.81 (s, 3 H) 2.31 (s, 3 H).
3-(5-(1,3,5-Trimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile/NEU-
5512. The title compound was prepared according to General Procedure K on a 67-mg scale using
3-(1-tosyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile. The reaction was run for two min, and the crude material was purified by flash chromatography (5-
10% MeOH:DCM) to afford the title compound as an off-white solid (17 mg, 36%). LCMS
+ 1 [M+H] 328.10 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.14 (s, 1 H) 8.14 - 8.19 (m, 3 H)
8.07 - 8.12 (m, 2 H) 7.67 (d, J=7.8 Hz, 1 H) 7.61 (t, J=8.3 Hz, 1 H) 3.73 (s, 3 H) 2.22 (s, 3 H) 2.13
(s, 3 H).
3-(5-(4-(Methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-4997).
The title compound was prepared according to General Procedure K on an 11-mg scale using 3-
(5-(4-(Methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile. The reaction was run for 2 min, and the crude material was purified by flash chromatography (2-10%
MeOH:DCM) to afford the title compound as an off-white solid (7 mg, 89%). LCMS [M+H]+
1 374.03 m/z; H NMR (399 MHz, DMSO-d6) δ ppm 12.30 (br. s., 1 H) 8.69 (d, J=12.5 Hz, 1 H)
252
8.63 - 8.66 (m, 1 H) 8.28 (s, 1 H) 8.20 (d, J=7.3 Hz, 1 H) 8.16 (s, 1 H) 8.11 (d, J=8.8 Hz, 2 H) 8.03
(d, J=8.1 Hz, 2 H) 7.72 (d, J=8.1 Hz, 1 H) 7.65 (t, J=7.3 Hz, 1 H) 3.27 (s, 3 H).
3-(5-((1-Methyl-1H-pyrazol-4-yl)amino)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-
5423). 3-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (151 mg, 0.334 mmol),
BrettPhos (19 mg, 0.035 mmol), BrettPhos Pd G1 (32 mg, 0.040 mmol), and 4-amino-1- methylpyrazole (69 mg, 0.710 mmol) were combined in a vial that was filled with nitrogen and evacuated three times. 1.0 M LiHMDS in THF (0.800 mL, 0.800 mmol) was added and the reaction was heated at 65 °C overnight. The reaction was quenched by the addition of 1M HCl, then diluted with EtOAc and poured over saturated aqueous NaHCO3. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-100% EtOAc:Hex - 1-5% MeOH:EtOAc), then repurified by preparative
HPLC (95-5% water:ACN) to afford the title compound (0.5 equiv formate salt) as a dark orange
+ 1 residue (5 mg, 8%). LCMS [M+H] 315.19 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm
8.46 - 8.65 (m, 0 H) 7.95 - 7.98 (m, 1 H) 7.93 - 7.95 (m, 1 H) 7.91 (dt, J=6.8, 2.0 Hz, 1 H) 7.71 (d,
J=2.4 Hz, 1 H) 7.69 (s, 1 H) 7.54 - 7.61 (m, 3 H) 7.41 (d, J=1.0 Hz, 1 H) 3.88 (s, 3 H).
253
3-(5-((4-(Methylsulfonyl)phenyl)amino)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-
5421). The title compound was prepared according to General Procedure K on a 46-mg scale using 3-(5-((4-(methylsulfonyl)phenyl)amino)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonit- rile. The reaction was run for 80 min, and the crude material was purified by flash chromatography
(50% EtOAc:Hexanes) to afford the title compound as a white solid (15 mg, 46%). LCMS [M+H]+
1 389.12 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.13 - 12.20 (m, 1 H) 8.79 (s, 1 H) 8.20 (dd,
J=11.5, 2.2 Hz, 2 H) 8.16 (s, 1 H) 8.11 (s, 1 H) 8.06 (d, J=7.8 Hz, 1 H) 7.60 - 7.69 (m, 4 H) 6.97
(d, J=9.3 Hz, 2 H) 3.08 (s, 3 H).
3-(5-(pyrimidin-5-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5005). The title compound was prepared according to General Procedure K on a 53-mg scale using 3-(5-
(pyrimidin-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile. The reaction was run for two min, then diluted with MeOH, upon which a precipitate was observed and removed by gravity filtration. The filtrate was purified by flash chromatography (5% MeOH:DCM) to afford the title compound as an off-white solid (17 mg, 47%). LCMS [M+H]+ 298.03 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 12.34 (br. s, 1 H) 9.31 (s, 2 H) 9.21 (s, 1 H) 8.77 (s, 1 H) 8.71 (s, 1 H) 8.30 (s,
1 H) 8.23 (d, J=8.1 Hz, 1 H) 8.19 (br. s., 1 H) 7.71 (d, J=7.3 Hz, 1 H) 7.65 (t, J=8.1 Hz, 1 H)
254
3-(5-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5006). The title compound was prepared according to General Procedure K on a 62-mg scale using 3-(5-(pyridin-
4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile. The reaction was run for two min, and the crude material was purified by flash chromatography (5% MeOH:DCM) to afford the title compound as an off-white solid (36 mg, 88%). LCMS [M+H]+ 297.04 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 12.34 (br. s., 1 H) 8.74 (s, 1 H) 8.69 (s, 1 H) 8.66 (d, J=5.1 Hz, 2 H) 8.29 (s, 1
H) 8.21 (d, J=8.1 Hz, 1 H) 8.17 (s, 1 H) 7.90 (d, J=5.9 Hz, 2 H) 7.72 (d, J=8.1 Hz, 1 H) 7.65 (t,
J=7.3 Hz, 1 H).
3-(5-(Pyrrolidin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5043). The title compound was prepared according to General Procedure J on a 153-mg scale using pyrrolidine.
The crude material was purified by flash chromatography (50-100% EtOAc:Hexanes) to afford the title compound as a yellow solid (12 mg, 12%). LCMS [M+H]+ 289.12 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 11.68 (br. s., 1 H) 8.11 (s, 1 H) 8.06 (d, J=6.8 Hz, 1 H) 7.91 (d, J=2.4 Hz,
1 H) 7.83 (d, J=2.4 Hz, 1 H) 7.59 - 7.66 (m, 2 H) 7.32 (s, 1 H) 3.29 - 3.32 (m, 4 H) 1.97 - 2.02 (m,
4 H).
255
3-(5-(Piperidin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5044). The title compound was prepared according to General Procedure J on a 152-mg scale using piperidine.
The crude material was purified by flash chromatography (50-100% EtOAc:Hexanes) to afford the title compound as a light orange solid (20 mg, 19%). LCMS [M+H]+ 303.10 m/z; 1H NMR
(500 MHz, DMSO-d6) δ ppm 11.84 (br. s, 1 H) 8.13 (d, J=2.0 Hz, 2 H) 8.06 (d, J=7.3 Hz, 1 H)
7.96 (d, J=2.4 Hz, 1 H) 7.76 (d, J=2.4 Hz, 1 H) 7.60 - 7.67 (m, 2 H) 3.11 (t, J=5.9 Hz, 4 H) 1.70
(quin, J=5.4 Hz, 4 H) 1.54 (quin, J=5.9 Hz, 2 H).
3-(5-(Piperidin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5302). The title compound was prepared according to General Procedure J on a 95-mg scale using N- methylpiperazine. The crude material was purified by flash chromatography (5-10%
MeOH:DCM) to afford the title compound as a light orange solid (12 mg, 18%). LCMS [M+H]+
1 318.15 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.13 (d, J=2.4 Hz, 1 H) 7.95 - 8.00 (m,
2 H) 7.85 (d, J=2.4 Hz, 1 H) 7.73 (s, 1 H) 7.57 - 7.63 (m, 2 H) 3.24 (br. t, J=4.9, 4.9 Hz, 4 H) 2.71
(br. t, J=4.9, 4.9 Hz, 4 H) 2.39 (s, 3 H).
256
tert-Butyl 4-(3-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate
(NEU-5317). The title compound was prepared according to General Procedure J on a 150-mg scale using tert-butyl piperazine-1-carboxylate; the reaction was run overnight. The crude material was purified by flash chromatography (20-100% EtOAc:Hexanes) to afford the title compound as
+ 1 a yellow solid (40 mg, 30%). LCMS [M+H] 404.21 m/z; H NMR (500 MHz, DMSO-d6) δ ppm
11.89 (br. s, 1 H) 8.16 (d, J=2.4 Hz, 1 H) 8.14 (t, J=1.7 Hz, 1 H) 8.08 (dt, J=8.1, 1.6 Hz, 1 H) 7.99
(d, J=2.9 Hz, 1 H) 7.83 (d, J=2.4 Hz, 1 H) 7.64 - 7.68 (m, 1 H) 7.62 (t, J=7.8 Hz, 1 H) 3.52 (m,
J=4.9, 3.4 Hz, 4 H) 3.11 (br. t, J=4.9, 4.9 Hz, 4 H) 1.43 (s, 9 H).
3-(5-(Piperazin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5318). tert-Butyl 4-
(3-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)piperazine-1-carboxylate (40 mg, 0.099 mmol) was taken up in 4M HCl in dioxane (0.25 mL, 1.00 mmol) and the reaction was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the yellow residue
(HCl salt) was dissolved in water and poured over saturated aqueous NaHCO3. The aqueous layer
(pH 8) was extracted three times with DCM. The pH of the aqueous layer was increased to 13-14 by the addition of 1M NaOH and the aqueous layer was extracted once more with DCM. The combined organic layers were dried with sodium sulfate and concentrated under reduced pressure to afford the title compound as a yellow solid (19 mg, 62%). LCMS [M+H]+ 304.17 m/z; 1H NMR
257
(500 MHz, DMSO-d6) δ ppm 11.84 (br. s., 1 H) 8.13 (d, J=2.0 Hz, 2 H) 8.05 - 8.09 (m, 1 H) 7.96
(d, J=2.4 Hz, 1 H) 7.74 (d, J=2.4 Hz, 1 H) 7.60 - 7.68 (m, 2 H) 3.30 (br. s., 1 H) 3.07 (br. t, J=4.4,
4.4 Hz, 4 H) 2.90 (m, J=4.9 Hz, 4 H).
3-(5-(4-(Methylsulfonyl)piperazin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-
5422). The title compound was prepared according to General Procedure K on a 45-mg scale using 3-(5-(4-(methylsulfonyl)piperazin-1-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonit- rile. The reaction was run for 45 min, and the crude material was purified by flash chromatography
(50-100% EtOAc:Hexanes) to afford the title compound as a white solid (15 mg, 53%). LCMS
+ 1 [M+H] 382.13 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.92 (s, 1 H) 8.18 (d, J=2.4 Hz, 1 H)
8.15 (s, 1 H) 8.09 (d, J=7.8 Hz, 1 H) 8.00 (s, 1 H) 7.86 (d, J=2.0 Hz, 1 H) 7.66 (d, J=7.3 Hz, 1 H)
7.63 (t, J=7.8 Hz, 1 H) 3.29 - 3.32 (m, 4 H) 3.27 (m, J=5.9 Hz, 4 H) 2.95 (s, 3 H).
3-(5-(4-Methyl-1,4-diazepan-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-5510).
The title compound was prepared according to General Procedure J on a 70-mg scale using 1- methyl-1,4-diazepane. The crude material was purified by flash chromatography (5-25%
MeOH:DCM) to afford the title compound as a yellow solid (9 mg, 12%). LCMS [M+H]+ 332.16
1 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 7.93 - 8.01 (m, 3 H) 7.67 (s, 1 H) 7.54 - 7.63
258
(m, 3 H) 3.69 (br. t, J=4.4, 4.4 Hz, 2 H) 3.55 (t, J=6.3 Hz, 2 H) 3.04 (br. t, J=4.4, 4.4 Hz, 2 H) 2.90
(br. t, J=4.9, 4.9 Hz, 1 H) 2.57 (s, 3 H) 2.15 (quint, J=11.1, 11.1, 11.1, 11.1, 5.9, 5.9 Hz, 2 H).
3-(5-(1-(Tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (NEU-5813). The title compound was prepared according to General Procedure
K on a 37-mg scale using 3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H- pyrrolo[2,3-b]pyridin-3-yl)benzonitrile 3-10h. The reaction was run for six min, and the crude material was purified by flash chromatography (1-10% MeOH:DCM) to afford the title compound
+ 1 as an off-white solid (13 mg, 49%). LCMS [M+H] 370.10 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 12.07 (br. s., 1 H) 8.58 (d, J=1.5 Hz, 1 H) 8.46 (s, 1 H) 8.40 (s, 1 H) 8.21 (s, 1 H) 8.16 (d,
J=7.8 Hz, 1 H) 8.06 (d, J=2.4 Hz, 1 H) 8.04 (s, 1 H) 7.70 (d, J=7.8 Hz, 1 H) 7.65 (t, J=7.8 Hz, 1
H) 4.43 (tquin, J=6.3, 6.3, 4.9, 4.9, 4.9, 4.9 Hz, 1 H) 3.99 (d, J=11.7 Hz, 2 H) 3.49 (td, J=10.7, 2.4
Hz, 2 H) 1.94 - 2.08 (m, 4 H).
tert-Butyl 4-(4-(3-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazol-1-yl)piper- idine-1-carboxylate (NEU-5814). The title compound was prepared according to General
259
Procedure K on a 50-mg scale using tert-butyl 4-(4-(3-(3-cyanophenyl)-1-tosyl-1H-pyrrolo[2,3- b]pyridin-5-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 3-10i. The reaction was run for 10 min, and the crude material was purified by flash chromatography (3% 5% NH4OH/MeOH:DCM) to afford the title compound as a light yellow solid (21 mg, 55%). LCMS [M+H]+ 469.10 m/z; 1H
NMR (500 MHz, DMSO-d6) δ ppm 12.07 (br. s., 1 H) 8.57 (d, J=1.5 Hz, 1 H) 8.46 (s, 1 H) 8.41
(s, 1 H) 8.21 (s, 1 H) 8.16 (d, J=7.3 Hz, 1 H) 8.06 (d, J=2.4 Hz, 1 H) 8.04 (s, 1 H) 7.69 (d, J=8.3
Hz, 1 H) 7.66 (t, J=7.8 Hz, 1 H) 4.34 - 4.44 (m, 1 H) 4.01 - 4.16 (m, 2 H) 2.81 - 3.06 (m, 2 H) 2.05
(d, J=10.7 Hz, 2 H) 1.83 (qd, J=14.2, 12.2 Hz, 2 H) 1.43 (s, 9 H).
3-(5-(1-(1-Methylpiperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo- nitrile (NEU-5902). The title compound was prepared according to General Procedure K on a 45- mg scale using 3-(5-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3- b]pyridin-3-yl)benzonitrile 3-10j. The reaction was run for five min, and the crude material was purified by flash chromatography (5-10% 5% NH4OH/MeOH:DCM) to afford the title compound
+ 1 as an off-white solid (19 mg, 59%). LCMS [M+H] 383.12 m/z; H NMR (500 MHz, DMSO-d6)
δ ppm 12.06 (br. s., 1 H) 8.57 (d, J=1.5 Hz, 1 H) 8.45 (d, J=1.5 Hz, 1 H) 8.38 (s, 1 H) 8.21 (s, 1
H) 8.16 (d, J=7.8 Hz, 1 H) 8.06 (d, J=2.4 Hz, 1 H) 8.02 (s, 1 H) 7.69 (d, J=7.8 Hz, 1 H) 7.65 (t,
J=7.8 Hz, 1 H) 4.13 (s, 1 H) 2.88 (d, J=10.7 Hz, 2 H) 2.22 (s, 3 H) 1.95 - 2.11 (m, 6 H).
260
5-Bromo-3-iodo-4-methylpyridin-2-amine (3-13). 5-Bromo-4-methylpyridin-2-amine 3-12
(500 mg, 2.67 mmol) was dissolved in acetic acid (3.3 mL, 0.82 M) and N-iodosuccinimide (666 mg, 2.96 mmol) was added, followed by the addition of TFA (41 µL, 0.535 mmol). The reaction was stirred at 50 °C overnight. The reaction solution was poured over ice water and neutralized with 28% aqueous ammonia, upon which a light orange precipitate was observed and collected by vacuum filtration (washed with water) to afford the title compound as an orange solid (784 mg,
+ 79 81 1 94%). LCMS [M+H] 312.80 m/z ( Br), 314.81 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 7.95 (s, 1 H) 6.24 (br. s., 2 H) 2.48 (s, 3 H).
5-Bromo-4-methyl-3-((trimethylsilyl)ethynyl)pyridin-2-amine (3-14). 5-Bromo-3- iodopyridin-2-amine 3-13 (784 mg, 2.51 mmol), copper iodide (26 mg, 0.137 mmol), and
PdCl2(PPh3)2 (37 mg, 0.053 mmol) were combined in a round bottom flask that was filled with nitrogen and evacuted three times. Degassed THF (3.0 mL, 0.84 M) was added, followed by the addition of degassed triethylamine (16.0 mL, 114.79 mmol) and TMS-acetylene (0.450 mL, 3.25 mmol). The reaction was run at room temperature overnight under nitrogen. The reaction mixture was diluted with EtOAc and washed once with water and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20% EtOAc:Hex) to afford the title compound as a tan solid
261
(602 mg, 85%). LCMS [M+H]+ 282.95 m/z (79Br), 284.96 m/z (81Br); 1H NMR (500 MHz, DMSO- d6) δ ppm 8.00 (s, 1 H) 6.26 (br. s., 2 H) 2.35 (s, 3 H) 0.25 (s, 9 H).
5-Bromo-4-methyl-1H-pyrrolo[2,3-b]pyridine (3-15). Potassium tert-butoxide (525 mg, 4.68 mmol) was dissolved in NMP (16.0 mL, 0.30 M) and heated to 80 °C. 5-Bromo-4-methyl-3-
((trimethylsilyl)ethynyl)pyridin-2-amine 3-14 (602 mg, 2.13 mmol) was dissolved in NMP (10 mL, 0.23 M) and added dropwise to the KOtBu solution. The reaction was stirred at 80 °C for 30 min. The reaction mixture was diluted with EtOAc and washed five times with water. The combined organic layers were washed once with brine, dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20%
EtOAc:Hexanes) to afford the title compound as an off-white solid (236 mg, 53%). LCMS [M+H]+
79 81 1 210.84 m/z ( Br), 212.86 m/z ( Br); H NMR (500 MHz, DMSO-d6) δ ppm 11.79 (br. s., 1 H)
8.24 (s, 1 H) 7.48 (d, J=3.4 Hz, 1 H) 6.57 (t, J=2.7 Hz, 1 H) 2.54 (d, J=2.0 Hz, 3 H).
4-Methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (3-16). 5-Bromo-4- methyl-1H-pyrrolo[2,3-b]pyridine 3-15 (236 mg, 1.12 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole (258 mg, 1.24 mmol), and PdCl2(dppf)·CH2Cl2 (46 mg,
0.056 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (6.6 mL, 0.17 M) and 2M K2CO3 (1.7 mL, 3.40 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85 °C for 4 h. The reaction mixture was
262 diluted with MeOH, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (1-10% MeOH:DCM), then repurified by flash chromatography (100% EtOAc) to afford the title compound as a tan solid (152 mg, 64%). LCMS
+ 1 [M+H] 212.99 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.54 (br. s., 1 H) 8.16 (s, 1 H) 7.93
(s, 1 H) 7.65 (s, 1 H) 7.40 (t, J=2.7 Hz, 1 H) 6.52 (dd, J=3.4, 2.0 Hz, 1 H) 3.90 (s, 3 H) 2.52 (s, 3
H).
3-Iodo-4-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (3-17). 4-Methyl-
5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine 3-16 (152 mg, 0.716 mmol) was dissolved in ACN (4.2 mL, 0.17 M) and N-iodosuccinimide (242 mg, 1.08 mmol) was added. The reaction was stirred at 50 °C for 2 h. Upon cooling to room temperature, a dark brown precipitate was observed and collected by vacuum filtration to afford the title compound as a dark brown solid
+ 1 (101 mg, 42%). LCMS [M+H] 338.93 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.02 (br. s,
1 H) 8.16 (s, 1 H) 7.90 (s, 1 H) 7.65 (d, J=2.4 Hz, 1 H) 7.61 (s, 1 H) 3.90 (s, 3 H) 2.79 (s, 3 H).
3-Iodo-4-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3-18). 3-
Iodo-4-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine 3-17 (101 mg, 0.299
263 mmol) was suspended in DCM (1.6 mL, 0.19 M) and TEA (0.150 mL, 1.08 mmol), DMAP (46 mg, 0.377 mmol) and 4-methylbenzenesulfonyl chloride (142 mg, 0.745 mmol) were added in that order. The reaction was stirred overnight at room temperature. The reaction was washed once with
1M HCl, once with saturated aqueous NaHCO3, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-100% EtOAc:Hexanes) to afford the title compound as an orange solid
+ 1 (37 mg, 25%). LCMS [M+H] 492.90 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.30 (s, 1 H)
8.09 (s, 1 H) 8.00 (d, J=8.8 Hz, 2 H) 7.95 (s, 1 H) 7.64 (s, 1 H) 7.43 (d, J=8.3 Hz, 2 H) 3.89 (s, 3
H) 2.76 (s, 3 H) 2.35 (s, 3 H).
3-(4-Methyl-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile (3-19). 3-Iodo-4-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3- b]pyridine 3-18 (37 mg, 0.075 mmol), (3-cyanophenyl)boronic acid (15 mg, 0.102 mmol), and
PdCl2(dppf)·CH2Cl2 (7 mg, 0.009 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.40 mL, 0.21 M) and 2M K2CO3 (0.15 mL, 0.300 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for 5 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hexanes) to afford the title compound as a light yellow solid
264
+ 1 (25 mg, 71%). LCMS [M+H] 468.03 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.34 (s, 1 H)
8.03 - 8.09 (m, 3 H) 7.98 (s, 1 H) 7.95 (s, 1 H) 7.90 (d, J=8.8 Hz, 2 H) 7.64 - 7.69 (m, 2 H) 7.45
(d, J=7.8 Hz, 2 H) 3.88 (s, 3 H) 2.36 (s, 3 H) 2.18 (s, 3 H).
3-(4-Methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(NEU-5900). The title compound was prepared according to General Procedure K on a 25-mg scale using 3-(4-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile 3-19. The reaction was run for three min, and the crude material was purified by flash chromatography (1-5% MeOH:DCM), then repurified by flash chromatography (50-100%
EtOAc:Hexanes) to afford the title compound as a beige solid (9 mg, 55%). LCMS [M+H]+ 314.05 m/z; 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 10.40 (br. s., 1 H) 8.32 (br. s., 1 H) 7.77 (s,
1 H) 7.72 (d, J=7.8 Hz, 1 H) 7.65 (d, J=7.8 Hz, 1 H) 7.61 (s, 1 H) 7.54 (t, J=7.8 Hz, 1 H) 7.48 (s,
1 H) 7.32 (s, 1 H) 4.01 (s, 3 H) 2.32 (s, 3 H).
5-(1-Methyl-1H-pyrazol-4-yl)furo[2,3-b]pyridine (3-21). 5-Bromofuro[2,3-b]pyridine 3-20
(177 mg, 0.893 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(205 mg, 0.985 mmol), and PdCl2(dppf)·CH2Cl2 (37 mg, 0.045 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (5.2 mL, 0.17M) and 2M
K2CO3 (1.3 mL, 2.60 mmol) were added and the reaction was degassed for 10 min. The reaction
265 was heated at 85 °C for 4.5 h. The reaction mixture was diluted with MeOH and filtered through celite. The filtrate was purified by flash chromatography (20-50% EtOAc:Hex) to afford the title compound as an off-white solid (140 mg, 79%). LCMS [M+H]+ 199.95 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 8.46 (s, 1 H) 7.99 (d, J=2.0 Hz, 1 H) 7.79 (s, 1 H) 7.72 (d, J=2.0 Hz, 1
H) 7.67 (s, 1 H) 6.79 (d, J=2.4 Hz, 1 H) 3.99 (s, 3 H).
3-Bromo-5-(1-methyl-1H-pyrazol-4-yl)furo[2,3-b]pyridine (3-22). 5-(1-Methyl-1H-pyrazol-4- yl)furo[2,3-b]pyridine 3-21 (70 mg, 0.351 mmol) was dissolved in DCM (4.2 mL, 0.09 M) and cooled to 0 °C. Bromine (0.4 M in DCM, 0.95 mL, 0.380 mmol) was added dropwise to the reaction mixture. The reaction was stirred at 0 °C for 1 h. The reaction mixture was concentrated, redissolved in THF (5 mL) and treated dropwise with 1M KOH in MeOH (1 mL), upon which the reaction mixture turned cloudy. The reaction was stirred at room temperature for 10 mins, then poured over water and extracted three times with EtOAc. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-40% EtOAc:Hex) to afford the title compound as an off-white solid (34 mg, 35%). 278.04 m/z (79Br), 279.99 m/z (81Br); 1H NMR
(500 MHz, CHLOROFORM-d) δ ppm 8.50 (d, J=2.0 Hz, 1 H) 7.93 (d, J=2.0 Hz, 1 H) 7.83 (s, 1
H) 7.77 (s, 1 H) 7.72 (s, 1 H) 4.00 (s, 3 H).
266
3-(5-(1-Methyl-1H-pyrazol-4-yl)furo[2,3-b]pyridin-3-yl)benzonitrile (NEU-6017). 3-Bromo-
5-(1-methyl-1H-pyrazol-4-yl)furo[2,3-b]pyridine 3-21 (34 mg, 0.122 mmol), (3- cyanophenyl)boronic acid (26 mg, 0.176 mmol), and PdCl2(dppf)·CH2Cl2 (6 mg, 0.007 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times.
Dioxane (0.60 mL, 0.21 M) and 2M K2CO3 (0.30 mL, 0.600 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for 5 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (1% MeOH:DCM), then repurified by flash chromatography (30-50% EtOAc:hexanes) to afford the title compound as an off-white solid (59%). LCMS [M+H]+ 301.15 m/z; 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.53 (d, J=2.0 Hz, 1 H) 8.13 (d, J=2.4 Hz, 1 H) 7.95 (s, 1 H) 7.91 (s, 1 H) 7.86 (d, J=7.8 Hz,
1 H) 7.82 (s, 1 H) 7.73 (s, 1 H) 7.70 (d, J=7.8 Hz, 1 H) 7.63 (t, J=8.3 Hz, 1 H) 4.00 (s, 3 H).
267
Scheme 5-10. Synthesis of NEU-5127, -5128, -5305, and -5319.
5-(4-(Methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (5-42). 5-Bromo-1H-pyrrolo[2,3- b]pyridine 3-1 (1.00 g, 5.08 mmol), (4-(methylsulfonyl)phenyl)boronic acid (1.12 g, 5.60 mmol), and PdCl2(dppf)·CH2Cl2 (414 mg, 0.507 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (32.0 mL, 0.16M) and 2M K2CO3 (14.0 mL,
28.00 mmol) were added and the reaction was degassed for 10 min. The reaction was heated at 85
°C for 4 h. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100%
EtOAc:Hex - 0-10% MeOH:DCM) to afford the title compound as an orange solid (542 mg, 39%).
+ 1 LCMS [M+H] 273.04 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.84 (br. s., 1 H) 8.61 (d,
268
J=2.0 Hz, 1 H) 8.34 (d, J=1.5 Hz, 1 H) 8.01 (s, 4 H) 7.56 (t, J=3.4 Hz, 0 H) 6.55 (dd, J=3.4, 1.5
Hz, 1 H) 3.26 (s, 3 H).
3-Iodo-5-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (5-43). 5-(4-(Methylsulfonyl) phenyl)-1H-pyrrolo[2,3-b]pyridine 5-42 (542 mg, 1.99 mmol) was dissolved in ACN (10.0 mL,
0.20 M) and N-iodosuccinimide (587 mg, 0.261 mmol) was added. The reaction was stirred at 50
°C for 2 h. Upon cooling to room temperature, a precipitate was observed and collected by vacuum filtration (washed with ACN) to afford the title compound as a red-brown solid (568 mg, 72%).
+ 1 LCMS [M+H] 398.88 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.32 (br. s., 1 H) 8.65 (d,
J=2.0 Hz, 1 H) 8.06 (d, J=8.8 Hz, 2 H) 8.02 (d, J=8.8 Hz, 2 H) 7.98 (d, J=2.0 Hz, 1 H) 7.82 (d,
J=2.4 Hz, 1 H) 3.27 (s, 3 H).
3-Iodo-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-44). 3-Iodo-5-(4-
(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine 5-43 (568 mg, 1.43 mmol) was suspended in
DCM (14.0 mL, 0.10 M) and TEA (0.700 mL, 5.02 mmol), DMAP (126 mg, 1.03 mmol) and 4- methylbenzenesulfonyl chloride (818 mg, 4.29 mmol) were added in that order. Upon the addition of 4-methylbenzenesulfonyl chloride, the reaction mixture went from a cloudy suspension to a
269 clear solution. The reaction was stirred overnight at room temperature. The reaction was washed once with 1M HCl, once with saturated aqueous NaHCO3, and once with brine; the organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (0-20% EtOAc:Hex) to afford the title compound as an orange
+ 1 solid (366 mg, 46%). LCMS [M+H] 552.85 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.79 (d,
J=2.4 Hz, 1 H) 8.25 (s, 1 H) 8.00 - 8.09 (m, 7 H) 7.45 (d, J=8.8 Hz, 2 H) 3.27 (s, 3 H) 2.35 (s, 3
H).
5-(4-(Methylsulfonyl)phenyl)-3-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-45a). 3-
Iodo-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-44 (74 mg, 0.134 mmol), pyridin-4-ylboronic acid (26 mg, 0.211 mmol) and PdCl2(dppf)·CH2Cl2 (14 mg, 0.017 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane
(0.60 mL, 0.22 M) was added to the reaction mixture, followed by the addition of 2M K2CO3 (0.20 mL, 0.400 mmol). The reaction mixture was degassed for 10 min and run in the microwave (120
°C) for 30 min, then diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hex) to afford the title compound as an off-white solid (46 mg, 69%). LCMS [M+H]+ 504.05 m/z; 1H
NMR (500 MHz, DMSO-d6) δ ppm 8.83 (d, J=2.0 Hz, 1 H) 8.66 - 8.69 (m, 3 H) 8.62 (s, 1 H) 8.10
270
(dd, J=8.5, 1.7 Hz, 4 H) 8.03 (d, J=7.8 Hz, 1 H) 7.95 (d, J=4.4 Hz, 1 H) 7.46 (d, J=8.3 Hz, 2 H)
3.27 (s, 3 H) 2.36 (s, 3 H).
5-(4-(Methylsulfonyl)phenyl)-3-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-45b). 3-
Iodo-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-44 (76 mg, 0.138 mmol), pyridin-3-ylboronic acid (28 mg, 0.228 mmol) and PdCl2(dppf)·CH2Cl2 (11 mg, 0.013 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane
(0.60 mL, 0.22 M) was added to the reaction mixture, followed by the addition of 2M K2CO3 (0.20 mL, 0.400 mmol). The reaction mixture was degassed for 10 min and run in the microwave (120
°C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography
(50-100% EtOAc:Hex) to afford the title compound as an off-white solid (39 mg, 57%). LCMS
+ 1 [M+H] 504.05 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.10 (s, 1 H) 8.83 (q, J=2.0 Hz, 1 H)
8.61 (dt, J=3.2, 1.8 Hz, 1 H) 8.59 (q, J=1.5 Hz, 1 H) 8.46 - 8.49 (m, 1 H) 8.29 - 8.34 (m, 1 H) 8.10
(d, J=8.3 Hz, 4 H) 8.02 (dt, J=8.3, 1.5 Hz, 2 H) 7.51 - 7.56 (m, 1 H) 7.46 (d, J=7.3 Hz, 2 H) 3.27
(s, 3 H) 2.36 (s, 3 H).
271
3-(2-Methylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(5-45c). 3-Iodo-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-44 (75 mg,
0.136 mmol), (2-methylpyridin-4-yl)boronic acid (31 mg, 0.226 mmol) and PdCl2(dppf)·CH2Cl2
(14 mg, 0.017 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.60 mL, 0.22 M) was added to the reaction mixture, followed by the addition of 2M K2CO3 (0.20 mL, 0.400 mmol). The reaction mixture was degassed for 10 min and run in the microwave (120 °C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hexanes) to afford the title compound as an orange oil
(55 mg, 78%). LCMS [M+H]+ 518.15 m/z; 1H NMR
3-(2,6-Dimethylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3- b]pyridine (5-45d). 3-Iodo-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-44
272
(75 mg, 0.136 mmol) and PdCl2(dppf)·CH2Cl2 (12 mg, 0.015 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. 2,6-Dimethyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.58 M in dioxane, 0.7 mL, 0.406 mmol) was added to the reaction mixture, followed by the addition of 2M aq. K2CO3 (0.20 mL, 0.400 mmol). The reaction mixture was degassed for 10 min and run in the microwave (120 °C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hex) to afford the title compound as a solid (65 mg, 90%). LCMS [M+H]+ 532.13 m/z; 1H NMR (500
MHz, METHANOL-d4) δ ppm 8.73 (d, J=2.0 Hz, 1 H) 8.53 (d, J=2.0 Hz, 1 H) 8.36 (s, 1 H) 8.13
(d, J=8.8 Hz, 2 H) 8.08 (d, J=8.8 Hz, 2 H) 7.98 (d, J=9.3 Hz, 2 H) 7.50 (s, 2 H) 7.40 (d, J=8.3 Hz,
2 H) 3.17 (s, 3 H) 2.58 (s, 6 H) 2.39 (s, 3 H).
5-(4-(Methylsulfonyl)phenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-5127). The title compound was prepared according to General Procedure K on a 46-mg scale using 5-(4-
(methylsulfonyl)phenyl)-3-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-45a. The reaction was run for five min, and the crude material was purified by flash chromatography (0-10%
MeOH:DCM) to afford the title compound as an off-white solid (12 mg, 38%). LCMS [M+H]+
1 350.07 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.39 - 12.47 (m, 1 H) 8.70 (s, 2 H) 8.57 (d,
J=6.3 Hz, 2 H) 8.31 (s, 1 H) 8.12 (d, J=8.3 Hz, 2 H) 8.03 (d, J=8.8 Hz, 2 H) 7.87 (d, J=6.3 Hz, 2
H) 3.27 (s, 3 H).
273
5-(4-(Methylsulfonyl)phenyl)-3-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (NEU-5128). The title compound was prepared according to General Procedure K on a 39-mg scale using 5-(4-
(methylsulfonyl)phenyl)-3-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-45b. The reaction was run for 10min, and the crude material was purified by flash chromatography (50-100%
EtOAc:Hexanes) to afford the title compound as an off-white solid (12 mg,46%). LCMS [M+H]+
1 350.08 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.27 (br. s., 1 H) 9.06 (d, J=2.4 Hz, 1 H) 8.69
(d, J=2.4 Hz, 1 H) 8.60 (d, J=2.4 Hz, 1 H) 8.49 (dd, J=4.9, 1.5 Hz, 1 H) 8.24 (dt, J=8.1, 1.8 Hz, 1
H) 8.10 - 8.14 (m, 3 H) 8.02 (d, J=8.3 Hz, 2 H) 7.47 (dd, J=8.1, 4.6 Hz, 1 H) 3.27 (s, 3 H).
3-(2-Methylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (NEU-
5305). The title compound was prepared according to General Procedure K on a 55-mg scale using
3-(2-methylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-45c.
The reaction was run for five min, and the crude material was purified by flash chromatography
(1-5% 5% NH4OH/MeOH:DCM), then repurified by preparative HPLC (95-5% water:ACN) to afford the title compound as a formate salt. This solid was taken up in DCM and Si-carbonate was added; the mixture was stirred overnight at room temperature. The solids were filtered off and the filtrate was concentrated to afford the title compound (0.67 equiv formate salt) as a white solid.
274
+ 1 LCMS [M+H] 364.11 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.38 (s, 1 H) 8.68 (m, J=2.9
Hz, 2 H) 8.44 (d, J=5.4 Hz, 1 H) 8.25 (s, 1 H) 8.15 (s, 1 H) 8.11 (d, J=8.3 Hz, 2 H) 8.03 (d, J=8.8
Hz, 2 H) 7.70 (s, 1 H) 7.66 (d, J=6.3 Hz, 1 H) 3.27 (s, 3 H) 2.53 (s, 3 H).
3-(2,6-Dimethylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine
(NEU-5319). The title compound was prepared according to General Procedure K on a 65-mg scale using 3-(2,6-dimethylpyridin-4-yl)-5-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3- b]pyridine 5-45d. The reaction was run for five min, and the crude material was purified by flash chromatography (10% MeOH:EtOAc) to afford the title compound as a tan solid (32 mg, 69%).
+ 1 LCMS [M+H] 378.10 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.30 - 12.37 (m, 1 H) 8.66
(dd, J=4.4, 2.0 Hz, 2 H) 8.21 (s, 1 H) 8.09 (d, J=8.3 Hz, 2 H) 8.03 (d, J=8.3 Hz, 2 H) 7.50 (s, 2 H)
3.27 (s, 3 H) 2.48 (s, 6 H).
Scheme 5-11. Synthesis of NEU-5903.
275
5-Bromo-1-(4-(methylsulfonyl)phenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (5-
46). Intermediate 3-8 (200 mg, 0.419 mmol), pyridin-4-ylboronic acid (52 mg, 0.423 mmol), and
PdCl2(dppf)·CH2Cl2 (36 mg, 0.044 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (2.8 mL, 0.15 M) and 2M K2CO3 (0.70 mL, 1.40 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for five min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-60% EtOAc:Hex) to afford the title compound as a light yellow solid (116
+ 79 81 1 mg, 65%). LCMS [M+H] 427.85 m/z ( Br), 429.86 m/z ( Br); H NMR (500 MHz, DMSO-d6)
δ ppm 8.67 (d, J=2.0 Hz, 1 H) 8.65 (d, J=5.9 Hz, 2 H) 8.61 (s, 1 H) 8.57 (d, J=2.0 Hz, 1 H) 8.06
(d, J=8.3 Hz, 2 H) 7.86 (d, J=5.9 Hz, 2 H) 7.45 (d, J=7.8 Hz, 2 H) 2.36 (s, 3 H).
1-(4-(Methylsulfonyl)phenyl)-3-(pyridin-4-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)-1H-pyrrolo[2,3-b]pyridine (5-47). 5-Bromo-3-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-
276 b]pyridine 5-46 (116 mg, 0.271 mmol), potassium acetate (93 mg, 0.947 mmol), bis(pinacolato)diboron (103 mg, 0.405 mmol), and PdCl2(dppf)·CH2Cl2 (10 mg, 0.012 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed dioxane (2.7 mL, 0.10 M) was added and the reaction was run in the microwave (145
°C) for 1 h. The reaction was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure to afford the crude title compound as a dark brown solid; proceeded without further purification. LCMS [M+H]+ 476.09 m/z.
1-(4-(Methylsulfonyl)phenyl)-3-(pyridin-4-yl)-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-
4-yl)-1H-pyrrolo[2,3-b]pyridine (5-48). 4-Bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole
(50 mg, 0.216 mmol) and PdCl2(dppf)·CH2Cl2 (23 mg, 0.028 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Crude 3-(pyridin-4-yl)-5-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-47 (129 mg, 0.271 mmol) was dissolved in dioxane (1.6 mL, 0.17 M) and added to the reaction mixture, followed by the addition of 2M K2CO3 (0.60 mL, 1.20 mmol). The reaction was degassed and run in the microwave (145 °C) for 10 min. The reaction mixture was diluted with EtOAc/MeOH, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (1-10% MeOH:EtOAc) to afford the title compound as a dark red residue (31 mg,
277
+ 1 23%). LCMS [M+H] 500.02 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.74 (d, J=2.0 Hz, 1 H)
8.67 (d, J=6.3 Hz, 2 H) 8.51 (s, 1 H) 8.50 (d, J=2.0 Hz, 1 H) 8.48 (s, 1 H) 8.09 (s, 1 H) 8.07 (d,
J=8.3 Hz, 2 H) 7.90 (d, J=5.9 Hz, 2 H) 7.44 (d, J=8.3 Hz, 2 H) 4.38 - 4.47 (m, 1 H) 3.98 (d, J=10.2
Hz, 2 H) 3.93 (s, 3 H) 3.49 (td, J=11.7, 2.0 Hz, 2 H) 1.93 - 2.07 (m, 4 H).
3-(Pyridin-4-yl)-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3- b]pyridine (NEU-5903). The title compound was prepared according to General Procedure K on a 31-mg scale using 3-(pyridin-4-yl)-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1-tosyl-
1H-pyrrolo[2,3-b]pyridine 5-48. The reaction was run for five min, and the crude material was purified by flash chromatography (5-10% 5% NH4OH/MeOH:DCM) to afford the title compound
+ 1 as an orange solid (12 mg, 57%). LCMS [M+H] 346.11 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.20 (br. s., 1 H) 8.60 (d, J=2.0 Hz, 1 H) 8.56 (d, J=5.9 Hz, 2 H) 8.53 (d, J=2.0 Hz, 1 H)
8.44 (s, 1 H) 8.21 (s, 1 H) 8.06 (s, 1 H) 7.83 (d, J=5.9 Hz, 2 H) 4.43 (spt, J=6.3 Hz, 1 H) 3.99 (d,
J=11.2 Hz, 2 H) 3.50 (td, J=10.7, 2.4 Hz, 2 H) 1.96 - 2.08 (m, 4 H).
3-(1-Methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo-nitrile
(NEU-4930). 3-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile NEU-
1207 (58 mg, 0.194 mmol) was dissolved in dry DMF (0.30 mL, 0.65 M). The reaction was cooled
278 to 0 °C and sodium hydride (81 mg, 0.387 mmol) was added. The reaction was stirred at 0 °C for
1 h before the addition of methyl iodide (25 µl, 0.401 mmol). The reaction was allowed to warm to room temperature and stirred overnight. The reaction was diluted with EtOAc, poured over cold water, and extracted twice. The combined organic layers were washed once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by flash chromatography (0-10% MeOH:EtOAc) to afford the title compound as an off-white solid (41 mg,
+ 1 97%). LCMS [M+H] 314.16 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.60 (d, J=2.0 Hz, 1 H)
8.46 (d, J=2.0 Hz, 1 H) 8.28 (s, 1 H) 8.17 (s, 1 H) 8.11 - 8.14 (m, 2 H) 8.02 (s, 1 H) 7.69 (s, 1 H)
7.66 (d, J=7.8 Hz, 1 H) 3.89 (s, 3 H) 3.88 (s, 3 H).
Scheme 5-12. Synthesis of NEU-4411.
3-Iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-50). 3-Iodo-1H-pyrrolo[2,3-b]pyridine 5-49 (250 mg, 1.002 mmol) was suspended in DCM (10.0 mL, 0.10M) and TEA (0.35 mL, 2.51 mmol),
DMAP (67 mg, 0.548 mmol) and 4-methylbenzenesulfonyl chloride (403 mg, 2.11 mmol) were
279 added in that order. Upon the addition of 4-methylbenzenesulfonyl chloride, the reaction mixture went from a cloudy suspension to a clear solution. The reaction was stirred overnight at room temperature. The reaction was washed once with 1M HCl, once with saturated aqueous NaHCO3, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure to afford the title compound as an orange solid (311 mg, 76%). LCMS [M+H]+
1 398.97 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.41 (dd, J=4.9, 1.5 Hz, 1 H) 8.15 (s, 1 H)
8.02 (d, J=8.3 Hz, 2 H) 7.79 (dd, J=7.8, 1.5 Hz, 1 H) 7.42 (d, J=8.3 Hz, 2 H) 7.39 (dd, J=7.8, 4.9
Hz, 1 H) 2.34 (s, 3 H).
3-(1-Tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (5-51). 3-Iodo-1-tosyl-1H-pyrrolo[2,3- b]pyridine 5-50 (150 mg, 0.377 mmol), (3-cyanophenyl)boronic acid (140 mg, 0.953 mmol), and
PdCl2(dppf)·CH2Cl2 (31 mg, 0.037 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (1.5 mL, 0.25 M) and 2M K2CO3 (0.55 mL, 1.10 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for 30 min, then diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude residue was purified by flash chromatography (20-40%
EtOAc:Hex) to afford the title compound as an off-white solid (67 mg, 48%). LCMS [M+H]+
1 374.13 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.43 - 8.47 (m, 2 H) 8.40 (dd, J=8.3, 1.5 Hz,
280
1 H) 8.31 (s, 1 H) 8.14 (d, J=7.8 Hz, 1 H) 8.06 (d, J=8.8 Hz, 2 H) 7.84 (d, J=8.3 Hz, 1 H) 7.69 (t,
J=7.8 Hz, 1 H) 7.43 (d, J=8.8 Hz, 2 H) 7.38 - 7.42 (m, 1 H) 2.34 (s, 3 H).
3-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (NEU-4411). The title compound was prepared according to General Procedure K on a 67-mg scale using 3-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzonitrile 5-51. The reaction was run for two min, and the crude material was purified by flash chromatography (1-5% MeOH:DCM) to afford the title compound as a white solid (18 mg,
+ 1 45%). LCMS [M+H] 220.03 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 12.09 (br. s., 1 H) 8.38
(d, J=8.3 Hz, 1 H) 8.30 (dd, J=4.6, 1.2 Hz, 1 H) 8.16 - 8.20 (m, 1 H) 8.06 - 8.13 (m, 2 H) 7.67 -
7.72 (m, 1 H) 7.63 (t, J=7.8 Hz, 1 H) 7.19 (dd, J=8.1, 4.6 Hz, 1 H).
Scheme 5-13. Synthesis of NEU-5398.
281
5-(1-Methyl-1H-pyrazol-4-yl)-1H-indole (5-53). 5-Bromo-1H-indole 5-52 (251 mg, 1.28 mmol),
1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (293 mg, 1.41 mmol), and
PdCl2(dppf)·CH2Cl2 (51 mg, 0.062 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dioxane (8.0 mL, 0.16 M) and 2M K2CO3 (2.0 mL, 4.00 mmol) were added and the reaction was degassed for 10 min. The reaction was run in the microwave (145 °C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (10-40% EtOAc:Hex) to afford the title compound as an off-white solid (65 mg,
+ 1 26%). LCMS [M+H] 198.00 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 11.02 (br. s., 1 H) 8.01
(s, 1 H) 7.78 (s, 1 H) 7.68 - 7.71 (m, 1 H) 7.36 (d, J=8.3 Hz, 1 H) 7.31 (t, J=2.7 Hz, 1 H) 7.26 -
7.29 (m, 1 H) 6.38 (dd, J=2.7, 1.7 Hz, 1 H) 3.85 (s, 3 H).
3-Iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-indole (5-54). 5-(1-Methyl-1H-pyrazol-4-yl)-1H- indole 5-53 (65 mg, 0.329 mmol) was dissolved in DCM (6.6 mL, 0.05 M) and KOH (10 mg,
0.178 mmol) was added. The reaction was stirred at room temperature for 30 min, after which NIS
(76 mg, 0.338 mmol) was added. The reaction was stirred overnight at room temperature. The reaction mixture was quenched with Na2S2O3 and extracted twice with DCM. The combined organic layers were dried with sodium sulfate and concentrated under reduced pressure to afford
282 the title compound as a dark purple solid (97 mg, 91%). LCMS [M+H]+ 324.02 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 11.46 - 11.51 (m, 1 H) 8.11 (s, 1 H) 7.82 (d, J=1.0 Hz, 1 H) 7.52 (d, J=2.4
Hz, 1 H) 7.35 - 7.40 (m, 3 H) 3.86 (s, 3 H).
3-Iodo-5-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-indole (5-55). 3-Iodo-5-(1-methyl-1H- pyrazol-4-yl)-1H-indole 5-54 (97 mg, 0.300 mmol) was suspended in DCM (1.5 mL, 0.21 M) and
TEA (0.15 mL, 1.08 mmol), DMAP (46 mg, 0.376 mmol) and 4-methylbenzenesulfonyl chloride
(150 mg, 0.787 mmol) were added in that order. The reaction was stirred overnight at room temperature. The reaction was washed once with 1M HCl, once with saturated aqueous NaHCO3, and once with brine. The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash chromatography (50% EtOAc:Hex) to afford the title compound as a dark orange oil (102 mg, 71%). LCMS [M+H]+ 477.98 m/z; 1H
NMR (500 MHz, DMSO-d6) δ ppm 8.21 (s, 1 H) 8.04 (s, 1 H) 7.89 - 7.93 (m, 3 H) 7.89 (s, 1 H)
7.62 (dd, J=8.8, 1.5 Hz, 1 H) 7.42 (d, J=1.5 Hz, 1 H) 7.40 (d, J=7.8 Hz, 2 H) 3.86 (s, 3 H) 2.32 (s,
3 H).
283
3-(5-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-indol-3-yl)benzonitrile (5-56). 3-Iodo-5-(1- methyl-1H-pyrazol-4-yl)-1-tosyl-1H-indole 5-55 (100 mg, 0.210 mmol), (3-cyanophenyl)boronic acid (62 mg, 0.422 mmol), and PdCl2(dppf)·CH2Cl2 (20 mg, 0.025 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane (0.85 mL, 0.25
M) and 2M K2CO3 (0.40 mL, 0.800 mmol) were added and the reaction mixture was degassed for
10 min. The reaction was run in the microwave (120 °C) for 30 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20-50% EtOAc:Hex) to afford the title compound
+ 1 as an orange solid (81 mg, 86%). LCMS [M+H] 453.14 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 8.25 - 8.28 (m, 2 H) 8.19 (s, 1 H) 8.13 (dt, J=7.8, 1.5 Hz, 1 H) 7.98 (m, J=8.8, 3.4 Hz, 3 H)
7.93 (d, J=1.0 Hz, 1 H) 7.91 (d, J=1.0 Hz, 1 H) 7.85 (dt, J=7.8, 1.5 Hz, 1 H) 7.70 (t, J=7.8 Hz, 1
H) 7.62 (dd, J=8.8, 1.5 Hz, 1 H) 7.41 (d, J=7.8 Hz, 2 H) 3.85 (s, 3 H) 2.32 (s, 3 H).
3-(5-(1-Methyl-1H-pyrazol-4-yl)-1H-indol-3-yl)benzonitrile (NEU-5398). The title compound was prepared according to General Procedure K on an 81-mg scale using 3-(5-(1-methyl-1H- pyrazol-4-yl)-1-tosyl-1H-indol-3-yl)benzonitrile 5-56. The reaction was run for 1 h, and the
284 crude material was purified by flash chromatography (5% EtOAc:DCM) to afford the title compound as an off-white solid (20 mg, 38%). LCMS [M+H]+ 299.13 m/z; 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.53 (br. s, 1 H) 8.09 - 8.14 (m, 3 H) 8.01 (s, 1 H) 7.85 - 7.88 (m, 2 H) 7.61 -
7.68 (m, 2 H) 7.44 (d, J=8.8 Hz, 1 H) 7.39 (dd, J=8.3, 2.0 Hz, 1 H) 3.87 (s, 3 H).
Scheme 5-14. Synthesis of NEU-5004.
5-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (5-57). 5-Bromo-
3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine 3-8 (1502 mg, 0.318 mmol), (1-methyl-1H-pyrazol-4- yl)boronic acid (43 mg, 0.341 mmol), and PdCl2(dppf)·CH2Cl2 (27 mg, 0.033 mmol) were combined in a microwave vial that was purged with nitrogen and evacuated three times. Dioxane
(1.3 mL, 0.24 M) and 2M K2CO3 (0.50 mL, 1.00 mmol) were added and the reaction mixture was degassed for 10 min. The reaction was run in the microwave (120 °C) for 15 min. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure.
The crude material was purified by flash chromatography (20-50% EtOAc:Hex) to afford the title
285 compound as a tan solid (41 mg, 30%). LCMS [M+H]+ 430.95 m/z (79Br), 432.91 m/z (81Br); 1H
NMR (399 MHz, DMSO-d6) δ ppm 8.55 (d, J=2.9 Hz, 1 H) 8.48 - 8.53 (m, 1 H) 8.41 (s, 1 H) 8.24
(s, 1 H) 8.05 (s, 1 H) 7.94 - 8.01 (m, 2 H) 7.38 - 7.46 (m, 2 H) 3.88 (br. s., 3 H) 2.33 (br. s., 3 H).
3-(3-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzonitrile (5-58).
5-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine 5-57 (41 mg, 0.095 mmol), (3-cyanophenyl)boronic acid (24 mg, 0.163 mmol), and PdCl2(dppf)·CH2Cl2 (9 mg, 0.011 mmol) were combined in a reaction vial that was filled with nitrogen and evacuated three times.
Dioxane (0.6 mL, 0.17 M) and 2M K2CO3 (0.20 mL, 0.400 mmol) were added and the reaction was degassed for 10 min. The reaction was run in the microwave (145 °C) for 5 min. The reaction was diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (50-100% EtOAc:Hex) to afford the title compound as a white solid (19 mg, 44%). LCMS [M+H]+ 454.03 m/z; 1H NMR (399 MHz,
DMSO-d6) δ ppm 8.77 (d, J=2.2 Hz, 1 H) 8.54 (d, J=2.2 Hz, 1 H) 8.46 (s, 1 H) 8.35 (s, 1 H) 8.23
(s, 1 H) 8.17 (d, J=7.3 Hz, 1 H) 8.12 (s, 1 H) 8.02 (d, J=8.1 Hz, 2 H) 7.89 (d, J=7.3 Hz, 1 H) 7.71
(t, J=8.1 Hz, 1 H) 7.43 (d, J=8.1 Hz, 2 H) 3.91 (s, 3 H) 2.34 (s, 3 H).
286
3-(3-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzonitrile (NEU-5004).
The title compound was prepared according to General Procedure K on a 19-mg scale using 3-(3-
(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzonitrile 5-58. The reaction was run for two min, and the crude material was purified by flash chromatography (1-5%
MeOH:DCM) to afford the title compound as a white solid (4 mg, 28%). LCMS [M+H]+ 300.12
1 m/z; H NMR (399 MHz, METHANOL-d4) δ ppm 8.51 (d, J=1.5 Hz, 1 H) 8.42 (d, J=2.2 Hz, 1
H) 8.13 (d, J=1.5 Hz, 1 H) 8.09 (s, 1 H) 8.04 (dd, J=8.1, 1.5 Hz, 1 H) 7.86 (s, 1 H) 7.71 - 7.75 (m,
1 H) 7.68 (d, J=7.3 Hz, 1 H) 7.64 (s, 1 H) 3.97 (s, 3 H).
287
5.2.3 Experimental procedures for chapter 4
7-Bromo-N-(pyrazin-2-yl)quinolin-4-amine (4-7). 2-Aminopyrazine (588 mg, 6.19 mmol) was added to a flask under nitrogen to which dry DMF (20 mL, 0.33 M) was added. The flask was cooled to 0 °C and sodium hydride (870 mg, 21.75 mmol) was added slowly. The mixture turned bright yellow upon addition of the sodium hydride. Once addition was complete the mixture was stirred at room temperature. After 30 min, to this suspension 7-bromo-4-chloroquinoline (1.50 g,
6.19 mmol) in dry DMF (20 mL, 0.33 M) was added, and a color change from yellow to brownish yellow was observed. The reaction was stirred under nitrogen at room temperature overnight. The reaction was quenched by addition of water, upon which a precipitate was observed. The precipitate was isolated by vacuum filtration (washed with water) to afford the title compound as a pale orange solid (1.63 g, 88%). LCMS [M+H]+ 301.02 m/z (79Br), 303.04 m/z (81Br); 1H NMR
(500 MHz, DMSO-d6) δ ppm 9.88 (br. s., 1 H) 8.72 (d, J=5.4 Hz, 1 H) 8.71 (s, 1 H) 8.47 (d, J=9.3
Hz, 1 H) 8.37 (d, J=4.9 Hz, 1 H) 8.33 (dd, J=2.9, 1.5 Hz, 1 H) 8.19 (d, J=2.9 Hz, 1 H) 8.16 (d,
J=2.0 Hz, 1 H) 7.79 (dd, J=9.3, 2.0 Hz, 1 H).
288
Tert-butyl 3-(4-methylpiperazin-1-yl)pyrrolidine-1-carboxylate (4-9). N-boc-3-pyrrolidinone
(501 mg, 2.70 mmol) was dissolved in DCM (8 mL, 0.34M) and 1-methyl piperazine (0.9 mL,
8.11 mmol), triethylamine (0.75 mL, 5.37 mmol), and acetic acid (0.3 mL, 5.25 mmol) were added in that order. The reaction was stirred at room temperature for 15 min. NaHB(OAc)3 (2.29 g, 10.80 mmol) was added and the reaction was stirred at room temperature overnight. The reaction mixture was diluted with DCM and washed twice with 2M NaOH, once with water, and once with brine.
The organic layer was dried with sodium sulfate and concentrated under reduced pressure. The crude material was purified by column chromatography (5-15% MeOH/DCM) to afford the title compound as a yellow oil (504 mg, 69%). LCMS [M+H]+ 270.22 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) δ ppm 3.66 - 3.73 (m, 1 H) 3.54 - 3.62 (m, 1 H) 3.47 (d, J=1.5 Hz, 1 H) 3.26
(m, J=10.3 Hz, 1 H) 3.08 (t, J=9.5 Hz, 1 H) 2.71 - 2.85 (m, 1 H) 2.49 (br. s., 7 H) 2.30 (s, 3 H)
2.07 (m, J=6.8 Hz, 1 H) 1.74 (spt, J=9.3 Hz, 1 H) 1.41 - 1.47 (m, 9 H).
1-Methyl-4-(pyrrolidin-3-yl)piperazine (4-10). The tert-butyl 3-(4-methylpiperazin-1- yl)pyrrolidine-1-carboxylate 4-9 (504 mg, 1.87 mmol) was taken up in 4M HCl in dioxane (12 mL, 48 mmol) and the reaction was stirred for 4 h at room temperature. The reaction mixture was
289 concentrated under reduced pressure, leaving the HCl salt. This was taken up in MeOH and resin- bound Si carbonate was added. After stirring overnight at room temperature, the Si-carbonate was removed by filtration and the filtrate was concentrated to afford the crude title compound as a red- brown oil. LCMS [M+H]+ 169.91 m/z.
Tert-butyl 4-(4-(pyrazin-2-ylamino)quinolin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate
(4-11a). The title compound was prepared according to General Procedure L on a 250-mg scale using 3,6-dihydro-2H-pyridin-1-N-boc-4-boronic acid pinacol ester. The crude material was purified by flash chromatography (100% EtOAc) to afford the title compound as a pale yellow
+ 1 solid (179 mg, 53%). LCMS [M+H] 404.17 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.76 (s,
1 H) 8.71 (s, 1 H) 8.69 (d, J=4.9 Hz, 1 H) 8.45 (d, J=8.8 Hz, 1 H) 8.28 - 8.35 (m, 2 H) 8.17 (d,
J=2.4 Hz, 1 H) 7.91 (s, 1 H) 7.83 (s, 1 H) 6.51 (br. s, 1 H) 4.09 (br. s., 2 H) 3.61 (br. s., 2 H) 2.63
(br. s., 2 H) 1.45 (s, 9 H).
Tert-butyl 5-(4-(pyrazin-2-ylamino)quinolin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate
(4-11b). The title compound was prepared according to General Procedure L on a 250-mg scale
290 using tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)- carboxylate. The crude material was purified by flash chromatography (100% EtOAc) to afford the title compound as a light yellow solid (315 mg, 67%). LCMS [M+H]+ 404.23 m/z; 1H NMR
(500 MHz, DMSO-d6) δ ppm 9.78 (s, 1 H) 8.68 - 8.73 (m, 2 H) 8.47 (d, J=8.8 Hz, 1 H) 8.29 - 8.33
(m, 2 H) 8.17 (d, J=2.9 Hz, 1 H) 7.85 (br. s, 1 H) 7.81 (d, J=10.7 Hz, 1 H) 6.64 (br. s., 1 H) 4.38
(br. s., 2 H) 3.53 (br. s., 2 H) 2.35 (br. s., 2 H) 1.45 (s, 9 H).
Tert-butyl 3-(4-(pyrazin-2-ylamino)quinolin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (4-
11c). The title compound was prepared according to General Procedure L on a 150-mg scale using tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate.
The crude material was purified by flash chromatography (1% 5% NH4OH/MeOH:EtOAc), then repurified by flash chromatography (5% MeOH:DCM) to afford the title compound as a yellow
+ 1 solid (62 mg, 32%). LCMS [M+H] 390.13 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.78 (s,
1 H) 8.71 (br. s., 3 H) 8.44 - 8.52 (m, 1 H) 8.29 - 8.36 (m, 1 H) 8.15 - 8.20 (m, 1 H) 7.90 (d, J=8.8
Hz, 1 H) 7.81 (d, J=9.8 Hz, 1 H) 6.69 (d, J=18.1 Hz, 1 H) 4.57 (br. s, 2 H) 4.29 (br. s, 2 H) 1.48
(d, J=10.7 Hz, 9 H).
291
Tert-butyl 4-(4-(pyrazin-2-ylamino)quinolin-7-yl)piperidine-1-carboxylate (4-12a/NEU-
5946). The title compound was prepared according to General Procedure M on a 179-mg scale using tert-butyl 4-(4-(pyrazin-2-ylamino)quinolin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate
4-11a. The crude material was purified by flash chromatography (100% EtOAc - 10% 5%
NH4OH/MeOH:EtOAc, step gradient), then the impure fractions were repurified by flash chromatography (5-10% MeOH:DCM) to afford the title compound as a yellow solid (90 mg,
+ 1 62%). LCMS [M+H] 406.25 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.72 (s, 1 H) 8.70 (d,
J=1.0 Hz, 1 H) 8.67 (d, J=5.4 Hz, 1 H) 8.43 (d, J=8.8 Hz, 1 H) 8.28 - 8.33 (m, 2 H) 8.16 (d, J=2.9
Hz, 1 H) 7.77 (s, 1 H) 7.58 (d, J=8.8 Hz, 1 H) 4.07 - 4.22 (m, 2 H) 2.72 - 2.97 (m, 3 H) 1.89 (d,
J=12.7 Hz, 2 H) 1.63 (qd, J=12.5, 3.9 Hz, 1 H) 1.43 (s, 9 H).
Tert-butyl 3-(4-(pyrazin-2-ylamino)quinolin-7-yl)piperidine-1-carboxylate (4-12b/NEU-
5979). The title compound was prepared according to General Procedure M on a 315-mg scale using tert-butyl 5-(4-(pyrazin-2-ylamino)quinolin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate
4-11b. The crude material was purified by flash chromatography (10% MeOH:DCM), then
292 repurified by preparative HPLC (99-50% ACN:water) to afford the title compound as a yellow
+ 1 solid (54 mg, 17%). LCMS [M+H] 406.25 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm
8.66 (s, 2 H) 8.53 (d, J=5.9 Hz, 1 H) 8.46 (d, J=8.8 Hz, 1 H) 8.39 (d, J=1.0 Hz, 1 H) 8.22 (d, J=2.0
Hz, 1 H) 7.85 (s, 1 H) 7.67 (d, J=5.9 Hz, 1 H) 4.20 - 4.33 (m, 1 H) 4.16 (d, J=15.1 Hz, 1 H) 2.93
(br. s., 3 H) 2.15 (d, J=11.7 Hz, 1 H) 1.87 (m, J=11.2 Hz, 2 H) 1.66 (q, J=13.7 Hz, 1 H) 1.48 (s, 9
H).
Tert-butyl 3-(4-(pyrazin-2-ylamino)quinolin-7-yl)pyrrolidine-1-carboxylate (4-12c/NEU-
5981). The title compound was prepared according to General Procedure M on a 62-mg scale using tert-butyl 3-(4-(pyrazin-2-ylamino)quinolin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 4-11c.
The product was purified by flash chromatography (100% EtOAc), then repurified by preparative
HPLC (99-50% ACN:water) to afford the title compound as a yellow residue (9 mg, 15%). LCMS
+ 1 [M+H] 392.21 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.55 - 8.70 (m, 2 H) 8.46 (d,
J=3.9 Hz, 1 H) 8.40 (d, J=8.8 Hz, 1 H) 8.35 (s, 1 H) 8.16 (br. s., 1 H) 7.85 (br. s., 1 H) 7.61 (d,
J=8.3 Hz, 1 H) 3.90 (br. s., 1 H) 3.64 (br. s., 2 H) 3.39 - 3.53 (m, 2 H) 2.40 (br. s., 1 H) 2.16 (d,
J=9.8 Hz, 1 H) 1.50 (s, 9 H).
293
7-(Piperidin-4-yl)-N-(pyrazin-2-yl)quinolin-4-amine (4-13a/NEU-5948). The title compound was prepared according to General Procedure N on an 80-mg using tert-butyl 4-(4-(pyrazin-2- ylamino)quinolin-7-yl)piperidine-1-carboxylate 4-12a. The crude material was purified by flash chromatography (20% 10% NH4OH/MeOH:DCM) to afford the title compound as a yellow solid
+ 1 (42 mg, 70%). LCMS [M+H] 306.18 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.67 (d,
J=5.9 Hz, 1 H) 8.64 (d, J=1.0 Hz, 1 H) 8.50 (d, J=5.9 Hz, 1 H) 8.46 (d, J=8.8 Hz, 1 H) 8.37 (s, 1
H) 8.20 (d, J=2.4 Hz, 1 H) 7.86 (s, 1 H) 7.64 (d, J=9.3 Hz, 1 H) 3.58 (d, J=13.2 Hz, 2 H) 3.14 -
3.28 (m, 3 H) 2.25 (d, J=14.2 Hz, 2 H) 2.06 (qd, J=13.7, 3.4 Hz, 2 H).
7-(Piperidin-3-yl)-N-(pyrazin-2-yl)quinolin-4-amine (4-13b/NEU-5980). The title compound was prepared according to General Procedure N on an 99-mg using tert-butyl 3-(4-(pyrazin-2- ylamino)quinolin-7-yl)piperidine-1-carboxylate 4-12b. The crude material was purified by flash chromatography (10-20% 10%NH4OH/MeOH:DCM) to afford the title compound as a light
+ 1 yellow solid (52 mg, 69%). LCMS [M+H] 306.18 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.69 (d, J=5.9 Hz, 1 H) 8.66 (s, 1 H) 8.55 (d, J=5.4 Hz, 1 H) 8.52 (d, J=8.8 Hz, 1 H) 8.39 (dd,
J=2.4, 1.5 Hz, 1 H) 8.22 (d, J=2.9 Hz, 1 H) 7.89 (d, J=1.5 Hz, 1 H) 7.69 (dd, J=8.8, 1.5 Hz, 1 H)
294
3.59 (d, J=7.8 Hz, 1 H) 3.50 (d, J=11.2 Hz, 1 H) 3.25 - 3.30 (m, 2 H) 3.12 (td, J=12.7, 2.9 Hz, 1
H) 2.20 (d, J=8.8 Hz, 1 H) 2.15 (d, J=7.3 Hz, 1 H) 1.98 (t, J=10.7 Hz, 2 H).
tert-Butyl 6-(5-bromopyrimidin-2-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (4-16a). The title compound was prepared according to General Procedure O on a 150-mg scale using tert-butyl
2,6-diazaspiro[3.4]octane-2-carboxylate. The crude residue was purified by flash chromatography
(20% EtOAc:Hex) to afford the title compound as an off-white solid (268 mg, 94%). LCMS
[M+H]+ 369.09 m/z (79Br), 371.08 m/z (81Br); 1H NMR (500 MHz, CHLOROFORM-d) δ ppm
8.31 (s, 2 H) 3.93 (d, J=8.3 Hz, 2 H) 3.88 (d, J=8.8 Hz, 2 H) 3.69 (s, 2 H) 3.59 (t, J=6.8 Hz, 2 H)
2.20 (t, J=6.8 Hz, 2 H) 1.46 (s, 9 H).
tert-Butyl 7-(5-bromopyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (4-1b). The title compound was prepared according to General Procedure O on a 300-mg scale using tert-butyl
2,7-diazaspiro[4.4]nonane-2-carboxylate. The crude residue was purified by flash chromatography
(20% EtOAc:Hex) to afford the title compound as a white solid (554 mg, 93%). LCMS [M+H]+
383.07 m/z (79Br), 385.09 m/z (81Br); 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.31 (s, 2
H) 3.58 - 3.71 (m, 2 H) 3.25 - 3.56 (m, 6 H) 1.83 - 2.08 (m, 4 H) 1.47 (s, 9 H).
295
tert-Butyl 9-(5-bromopyrimidin-2-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (4-16c).
The title compound was prepared according to General Procedure O on a 300-mg scale using tert- butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate. The crude residue was purified by flash chromatography (0-10% EtOAc:Hex) to afford the title compound as a white solid (566 mg, 88%).
LCMS [M+H]+ 411.16 m/z (79Br), 413.18 m/z (81Br); 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.28 (s, 2 H) 3.76 (t, J=5.4 Hz, 4 H) 3.42 (t, J=5.4 Hz, 4 H) 1.54 (t, J=5.9 Hz, 4 H) 1.48 - 1.52
(m, 4 H) 1.47 (s, 9 H).
6-(5-Bromopyrimidin-2-yl)-2,6-diazaspiro[3.4]octane (4-17a). Tert-butyl 6-(5- bromopyrimidin-2-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate 4-16a (268 mg, 0.726 mmol) was taken up in 4M HCl in dioxane (1.0 mL, 4.00 mmol). The reaction was stirred at room temperature overnight. All volatiles were removed in vacuo to afford a white solid. The crude material was used directly in the next reaction without further purification. LCMS [M+H]+ 269.03 m/z (79Br),
271.05 m/z (81Br).
296
2-(5-Bromopyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane (4-17b). Tert-butyl 7-(5- bromopyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2-carboxylate 4-16b (554 mg, 1.45 mmol) was taken up in 4M HCl in dioxane (1.8 mL, 7.20 mmol). The reaction was stirred at room temperature overnight. All volatiles were removed in vacuo to afford a white solid. The crude material was used directly in the next reaction without further purification. LCMS [M+H]+ 283.14 m/z (79Br),
285.09 m/z (81Br).
3-(5-Bromopyrimidin-2-yl)-3,9-diazaspiro[5.5]undecane (4-17c). tert-Butyl 9-(5- bromopyrimidin-2-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate 4-16c (566 mg, 1.38 mmol) was taken up in 4M HCl in dioxane (1.7 mL, 6.80 mmol). The reaction was stirred at room temperature overnight. All volatiles were removed in vacuo to afford a light yellow solid. The crude material was used directly in the next reaction without further purification. LCMS [M+H]+
311.10 m/z (79Br), 313.11 m/z (81Br).
297
6-(5-Bromopyrimidin-2-yl)-2-methyl-2,6-diazaspiro[3.4]octane (4-19a). 6-(5-
Bromopyrimidin-2-yl)-2,6-diazaspiro[3.4]octane 4-17a (195 mg, 0.724 mmol) was dissolved in
MeOH (3.0 mL, 0.25 M) and TEA (1.0 mL, 7.17 mmol) was added, followed by the addition of methyl iodide (68 µl, 1.09 mmol). The reaction was stirred overnight at room temperature. All volatiles were removed in vacuo and the resulting residue was dissolved in dichloromethane and washed with a saturated aqueous solution of sodium bicarbonate. The aqueous layer was extracted once more with dichloromethane, then the combined organic layers were washed once with a saturated solution of sodium chloride and dried with sodium sulfate. The crude material was purified by flash chromatography (5-10-20% 10% NH4OH/MeOH:EtOAc) to afford the title compound as an orange oil (20 mg, 10%). LCMS [M+H]+ 283.07 m/z (79Br), 285.09 m/z (81Br);
1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.29 (s, 2 H) 3.61 (s, 2 H) 3.54 (t, J=7.1 Hz, 2 H)
3.30 (d, J=7.8 Hz, 2 H) 3.16 (d, J=7.8 Hz, 2 H) 2.35 (s, 3 H) 2.19 (t, J=6.8 Hz, 2 H).
2-(5-Bromopyrimidin-2-yl)-7-methyl-2,7-diazaspiro[4.4]nonane (4-19b). 2-(5-
Bromopyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane 4-17b (350 mg, 1.24 mmol) was dissolved in
MeOH (5.0 mL, 0.25 M) and TEA (1.7 mL, 12.20 mmol) was added, followed by the addition of methyl iodide (115 µl, 1.85 mmol). The reaction was stirred overnight at room temperature. All
298 volatiles were removed in vacuo and the resulting residue was dissolved in dichloromethane and washed with a saturated aqueous solution of sodium bicarbonate. The aqueous layer was extracted once more with dichloromethane, then the combined organic layers were washed once with a saturated solution of sodium chloride and dried with sodium sulfate. The crude material was purified by flash chromatography (5-10-20% 10% NH4OH/MeOH:EtOAc) to afford the title compound as an orange oil (56 mg, 15%). LCMS [M+H]+ 297.05 m/z (79Br), 299.07 m/z (81Br);
1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.29 (s, 2 H) 3.52 - 3.63 (m, 3 H) 3.46 (d, J=11.2
Hz, 1 H) 2.72 (q, J=7.8 Hz, 1 H) 2.55 - 2.64 (m, 2 H) 2.51 (d, J=9.8 Hz, 1 H) 2.37 (s, 3 H) 2.02
(tdd, J=19.1, 19.1, 12.3, 7.1 Hz, 2 H) 1.87 (sxt, J=6.6 Hz, 2 H).
3-(5-bromopyrimidin-2-yl)-9-methyl-3,9-diazaspiro[5.5]undecane (4-19c). 3-(5-
Bromopyrimidin-2-yl)-3,9-diazaspiro[5.5]undecane hydrochloride 4-17c (411 mg, 1.18 mmol) was dissolved in MeOH (4.7 mL, 0.25 M) and TEA (1.7 mL, 12.20 mmol) was added, followed by the addition of methyl iodide (110 µl, 1.77 mmol). The reaction was stirred overnight at room temperature. All volatiles were removed in vacuo and the resulting residue was dissolved in dichloromethane, then washed with a saturated aqueous solution of sodium bicarbonate. The aqueous layer was extracted once more with dichloromethane, then the combined organic layers were washed once with a saturated solution of sodium chloride and dried with sodium sulfate. The crude material was purified by flash chromatography (5-10-20% 10% NH4OH/MeOH:EtOAc), then repurified by flash chromatography (7% 10% NH4OH/MeOH:DCM) to afford the title compound as a white solid (79 mg, 20%). LCMS [M+H]+ 325.14 m/z (79Br), 327.15 m/z (81Br);
299
1H NMR (500 MHz, CHLOROFORM-d) δ ppm 8.27 (s, 2 H) 3.74 (t, J=5.9 Hz, 4 H) 2.44 (br. s.,
4 H) 2.33 (s, 3 H) 1.62 (t, J=5.6 Hz, 4 H) 1.52 (t, J=5.9 Hz, 4 H).
(1S,4S)-2-(5-bromopyrimidin-2-yl)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (4-19d). The title compound was prepared according to General Procedure O on a 152-mg scale using (1S,4S)-
2-methyl-2,5-diazabicyclo[2.2.1]heptane dihydrobromide. The crude residue was purified by flash chromatography (20-50% EtOAc:Hex – 0-30% MeOH:DCM). The crude material was used directly in the next reaction without further purification. LCMS [M+H]+ 268.98 m/z (79Br), 270.96 m/z (81Br).
(1R,4R)-2-(5-bromopyrimidin-2-yl)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (4-19e). The title compound was prepared according to General Procedure O on a 250-mg scale using (1R,4R)-
2-methyl-2,5-diazabicyclo[2.2.1]heptane dihydrobromide. The crude residue was purified by flash chromatography (20-50% EtOAc:Hex – 0-30% MeOH:DCM). Upon cooling, a precipitate was observed and collected by vacuum filtration (washed with MeOH) to afford the title compound as a tan solid (134 mg, 57%). LCMS [M+H]+ 268.99 m/z (79Br), 271.01 m/z (81Br); 1H NMR (500
MHz, DMSO-d6) δ ppm 9.55 - 9.86 (m, 1 H) 8.54 (s, 2 H) 4.87 (s, 1 H) 4.37 (br. s., 1 H) 3.72 (d,
J=12.70 Hz, 1 H) 3.59 (dd, J=12.21, 1.95 Hz, 2 H) 2.85 (br. s., 3 H) 2.17 (d, J=9.77 Hz, 1 H) 1.31
- 1.38 (m, 2 H).
300
2-Methyl-6-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-2,6- diazaspiro[3.4]octane (4-20a). The title compound was prepared according to General Procedure
P on a 20-mg scale using 4-19a. LCMS [M+H]+ 249.13 m/z (boronic acid).
2-Methyl-7-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-2,7- diazaspiro[4.4]nonane (4-20b). The title compound was prepared according to General Procedure
P on a 56-mg scale using 4-19b. LCMS [M+H]+ 263.17 m/z (boronic acid).
3-methyl-9-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-3,9- diazaspiro[5.5]undecane (4-20c). The title compound was prepared according to General
Procedure P on a 79-mg scale using 4-19c. LCMS [M+H]+ 291.20 m/z (boronic acid).
301
(1S,4S)-2-methyl-5-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-2,5- diazabicyclo[2.2.1]heptane (4-20d). The title compound was prepared according to General
Procedure P on a 56-mg scale using 4-19d. LCMS [M+H]+ 317.11 m/z.
(1R,4R)-2-methyl-5-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-2,5- diazabicyclo[2.2.1]heptane (4-20e). The title compound was prepared according to General
Procedure P on a 134-mg scale using 4-19e. LCMS [M+H]+ 317.24 m/z.
7-(2-(2-Methyl-2,6-diazaspiro[3.4]octan-6-yl)pyrimidin-5-yl)-N-(pyrazin-2-yl)quinolin-4- amine (4-21a/NEU-5972). 7-Bromo-N-(pyrazin-2-yl)quinolin-4-amine (20 mg, 0.066 mmol),
K2CO3 (28 mg, 0.203 mmol), and PdCl2(dppf)·CH2Cl2 (6 mg, 0.007 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Crude (2-(2-methyl-2,6-
302 diazaspiro[3.4]octan-6-yl)pyrimidin-5-yl)boronic acid 4-20a (23 mg, 0.070 mmol) was dissolved in 3:1 dioxane:water (0.7 mL, 0.10 M) and added to the vial. The reaction mixture was degassed for 2 min and run in the microwave (130 °C, H abs) for 30 min. The product was diluted with
MeOH and filtered through celite. The filtrate was purified by flash chromatography (10-20% 10%
NH4OH/MeOH:EtOAc, step gradient) to afford the title compound as a yellow solid (8 mg, 28%).
+ 1 LCMS [M+H] 425.24 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.83 (s, 2 H) 8.69 (d,
J=5.4 Hz, 1 H) 8.62 (d, J=1.0 Hz, 1 H) 8.51 (d, J=8.8 Hz, 1 H) 8.47 (d, J=5.4 Hz, 1 H) 8.36 (s, 1
H) 8.17 (d, J=2.4 Hz, 1 H) 8.14 (d, J=2.0 Hz, 1 H) 7.90 (dd, J=8.8, 2.0 Hz, 1 H) 4.12 (q, J=9.3 Hz,
4 H) 3.91 (s, 2 H) 3.73 (t, J=7.1 Hz, 2 H) 2.91 (s, 3 H) 2.38 (t, J=7.1 Hz, 2 H).
7-(2-(7-Methyl-2,7-diazaspiro[4.4]nonan-2-yl)pyrimidin-5-yl)-N-(pyrazin-2-yl)quinolin-4- amine (4-21b/NEU-5971). 7-Bromo-N-(pyrazin-2-yl)quinolin-4-amine (50 mg, 0.266 mmol),
K2CO3 (72 mg, 0.521 mmol), and PdCl2(dppf)·CH2Cl2 (14 mg, 0.017 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Crude 4-20b (65 mg, 0.189 mmol) was dissolved in 3:1 dioxane:water (1.7 mL, 0.10 M) and added to the vial. The reaction mixture was degassed for 2 min and run in the microwave (130 °C, H abs) for 30 min. The product was diluted with MeOH and filtered through celite. The filtrate was purified by flash chromatography (10-20% 10% NH4OH/MeOH:EtOAc, step gradient) to afford the title compound
303
+ 1 as a tan solid (23 mg, 32%). LCMS [M+H] 439.25 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.79 (s, 2 H) 8.68 (d, J=5.4 Hz, 1 H) 8.61 (s, 1 H) 8.48 (d, J=8.8 Hz, 1 H) 8.45 (d, J=5.4 Hz,
1 H) 8.35 (s, 1 H) 8.15 (d, J=2.4 Hz, 1 H) 8.12 (s, 1 H) 7.87 (dd, J=8.8, 1.5 Hz, 1 H) 3.58 - 3.74
(m, 4 H) 2.76 (t, J=7.1 Hz, 2 H) 2.68 (m, J=4.9 Hz, 2 H) 2.42 (s, 3 H) 2.02 - 2.15 (m, 2 H) 1.91 -
2.00 (m, 2 H).
7-(2-(9-methyl-3,9-diazaspiro[5.5]undecan-3-yl)pyrimidin-5-yl)-N-(pyrazin-2-yl)quinolin-4- amine (4-21c/NEU-6000). 7-Bromo-N-(pyrazin-2-yl)quinolin-4-amine (65 mg, 0.215 mmol),
K2CO3 (89 mg, 0.644 mmol), and PdCl2(dppf)·CH2Cl2 (18 mg, 0.022 mmol) were combined in a microwave vial that was filled with nitrogen and evacuated three times. Crude 4-20c (90 mg, 0.241 mmol) was dissolved in 3:1 dioxane:water (2.2 mL, 0.10 M) and added to the vial. The reaction mixture was degassed for 2 min and run in the microwave (130 °C, H abs) for 30 min. The product was diluted with MeOH, and filtered through celite. The crude material was purified by flash chromatography (10-20% 10% NH4OH/MeOH:EtOAc, step gradient), then repurified by flash chromatography (20% MeOH:DCM, step gradient) to afford the title compound as a bright yellow
+ 1 solid (44 mg, 44%). LCMS [M+H] 467.28 m/z; H NMR (500 MHz, DMSO-d6) δ ppm 9.79 (s,
1 H) 8.92 (s, 2 H) 8.69 - 8.75 (m, 2 H) 8.56 (d, J=8.8 Hz, 1 H) 8.29 - 8.35 (m, 2 H) 8.22 (s, 1 H)
8.17 (d, J=2.9 Hz, 1 H) 7.97 (d, J=8.8 Hz, 1 H) 3.83 (t, J=5.9 Hz, 4 H) 2.35 (br. s., 4 H) 2.20 (s, 3
H) 1.52 (t, J=4.4 Hz, 4 H) 1.47 (t, J=5.4 Hz, 4 H).
304
7-(2-((1S,4S)-5-Methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyrimidin-5-yl)-N-(pyrazin-2- yl)quinolin-4-amine (4-21d/NEU-4443). Crude (1S,4S)-2-Methyl-5-(5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-2,5-diazabicyclo[2.2.1]heptane 4-20d (59 mg, 0.187 mmol), 7-bromo-N-(pyrazin-2-yl)quinolin-4-amine (63 mg, 0.209 mmol), and potassium carbonate (89 mg, 0.644 mmol) were suspended in 3:1 dioxane:water (1.6 mL, 0.12 M) and the reaction vial was purged with nitrogen for 10 min. PdCl2(dppf) (7 mg, 0.010 mmol) was added and the reaction was run overnight under nitrogen at 100 °C. The reaction mixture was diluted with MeOH and filtered through celite. The filtrate was purified by flash chromatography (1-20%
10% NH4OH/MeOH:DCM), the repurified by flash chromatography (20-25% MeOH:DCM, 5-
20% 10% NH4OH/MeOH:DCM) to afford the title compound as a yellow solid (16 mg, 21%).
+ 1 LCMS [M+H] 411.1 m/z; H NMR (500 MHz, METHANOL-d4) δ ppm 8.63 (s, 2 H), 8.54 (d,
J=5.4 Hz, 1 H), 8.51 (d, J=1.5 Hz, 1 H), 8.33 (d, J=5.4 Hz, 1 H), 8.29 (d, J=8.8 Hz, 1 H), 8.25 (dd,
J=2.4, 1.5 Hz, 1 H), 8.06 (d, J=2.9 Hz, 1 H), 7.93 (d, J=2.0 Hz, 1 H), 7.69 (dd, J=8.8, 2.0 Hz, 1
H), 4.82 (s, 1 H), 3.72 (dd, J=10.7, 1.5 Hz, 1 H), 3.58 (s, 1 H), 3.43 (dd, J=11.0, 2.2 Hz, 1 H), 2.87
(dd, J=10.0, 2.2 Hz, 1 H), 2.71 (d, J=10.3 Hz, 1 H), 2.41 (s, 3 H), 1.99 (d, J=10.3 Hz, 1 H), 1.88
(d, J=9.8 Hz, 1 H).
305
7-(2-((1R,4R)-5-Methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyrimidin-5-yl)-N-(pyrazin-2- yl)quinolin-4-amine (4-21e/NEU-4955). (1R,4R)-2-Methyl-5-(5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyrimidin-2-yl)-2,5-diazabicyclo[2.2.1]heptane 4-20e (139 mg, 0.439 mmol),
7-bromo-N-(pyrazin-2-yl)quinolin-4-amine (146 mg, 0.485 mmol), and potassium carbonate (210 mg, 1.52 mmol) were suspended in 3:1 dioxane:water (3.7 mL, 0.12 M) and the reaction vial was degassed for 10 min. PdCl2(dppf) (17 mg, 0.023 mmol) was added and the reaction was run under nitrogen overnight at 100 °C. The reaction mixture was diluted with MeOH and filtered through celite. The filtrate was purified by flash chromatography (50-100% EtOAc:Hexanes – 0-30%
MeOH:DCM), the repurified by flash chromatography (20% 10% NH4OH/MeOH:DCM) to afford the title compound as an orange-yellow solid (63 mg, 35%). LCMS [M+H]+ 411.1 m/z; 1H NMR
(500 MHz, METHANOL-d4) δ ppm 8.82 (s, 2 H) 8.68 (d, J=5.37 Hz, 1 H) 8.61 (s, 1 H) 8.49 (d,
J=8.79 Hz, 1 H) 8.46 (d, J=5.37 Hz, 1 H) 8.35 (dd, J=2.44, 1.46 Hz, 1 H) 8.15 (d, J=2.44 Hz, 1 H)
8.12 (d, J=1.46 Hz, 1 H) 7.88 (d, J=7.81 Hz, 1 H) 5.01 (s, 1 H) 3.91 (br. s., 1 H) 3.84 (d, J=11.72
Hz, 1 H) 3.63 (d, J=12.21 Hz, 1 H) 3.09 (s, 2 H) 2.64 (s, 3 H) 2.18 (d, J=10.74 Hz, 1 H) 2.08 (d,
J=11.23 Hz, 1 H).
306
References
(1) Hotez, P.J.; Fenwick, A.; Savioli, L.; Molyneux, D. Rescuing the bottom billion through control of neglected tropical diseases. Lancet 2009; 373: 1570-1575.
(2) Feasey, N.; Wansbrough-Jones, M.; Mabey, D.C.W.; Solomon, A. Neglected tropical diseases. British Medical Bulletin 2010; 93: 179-200.
(3) World Health Organization. Neglected tropical diseases. http://www.who.int/neglected_diseases/diseases/en/. 13 September 2016.
(4) Global burden of disease study. http://ghdx.healthdata.org/gbd-results-tool. June 8, 2018.
(5) Mitra, A.K.; Mawson, A.R. Neglected tropical diseases: Epidemiology and global burden. Trop. Med. Infect. Dis. 2017; 2: 36.
(6) Hotez, P.J.; Alvarado, M.; Basanez, M.-G.; Bolliger, I.; Bourne, R.; Boussinesq, M.; Brooker, S.J.; Brown, S.A.; Buckle, G.; Budke, C.M.; Carabin, H.; Coffeng, L.E.; Fevre, E.M.; Furst, T.; Halasa, Y.A.; Jasrasaria, R.; Johns, N.E.; Keiser, J.; King, C.H.; Lozano, R.; Murdoch, M.E.; O'Hanlon, S.; Pion, S.D.S.; Pullan, R.L.; Ramaiah, K.D.; Roberts, T.; Shepart, D.S.; Smith, J.L.; Stolk, W.A.; Undurraga, E.A.; Utzinger, J.; Wang, M.; Murray, C.J.L.; Naghavi, M. The global burden of disease study 2010: Interpretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis 2014; 8: e2865.
(7) Accelerating work to overcome the global impact of neglected tropical diseases - a roadmap for implementation, World Health Organization, Geneva, 2012.
(8) Integrating neglected tropical diseases into global health and development: Fourth WHO report on neglected tropical diseases., World Health Organization, Geneva, 2017.
(9) World Health Organization. Human african trypanosomiasis fact sheet. http://www.who.int/mediacentre/factsheets/fs259/en/. November 24, 2015.
(10) Buscher, P.; Cecchi, G.; Jamonneau, V.; Priotto, G. Human African trypanosomiasis. Lancet 2017; 390: 2397-2409.
(11) Centers for Disease Control and Prevention. Parasites - African trypanosomiasis. May 8, 2018.
(12) Jacobs, R.T.; Nare, B.; Wring, S.A.; Orr, M.D.; CHen, D.; Sligar, J.M.; Jenks, M.X.; Noe, R.A.; Bowling, T.S.; Mercer, L.T.; Rewerts, C.; Gaukel, E.; Owens, J.; Parham, R.; Randolph, R.; Beaudet, B.; Bacchi, C.J.; Yarlett, N.; Plattner, J.J.; Freund, Y.; Ding, C.; Akama, T.; Zhang, Y.-K.; Brun, R.; Kaiser, M.; Scandale, I.; Don, R. SCYX-7158, an orally-active benzoxaborole for the treatment of stage 2 human African trypanosomiasis. PLoS Negl Trop Dis 2011; 5: e1151.
307
(13) Torreele, E.; Trunz, B.B.; Tweats, D.; Kaiser, M.; Brun, R.; Mazue, G.; Bray, M.A.; Pecoul, B. Fexinidazole - a new oral nitroimidazole drug candidate entering clinical development for the treatment of sleeping sickness. PLoS Negl Trop Dis 2010; 12: e923.
(14) Drugs for Neglected Diseases initiative. About sleeping sickness. http://www.dndi.org/diseases-projects/hat/. September 1, 2016.
(15) Babokhov, P.; Sanyaolu, A.O.; Oyibo, W.A.; Fagbenro-Beyioku, A.F.; Iriemenam, N.C. A current analysis of chemotherapy strategies for the treatment of human African trypanosomiasis. Pathogens and Global Health 2013; 107: 242-252.
(16) World Health Organization. Chagas disease (American trypanosomiasis). May 20, 2018.
(17) Drugs for Neglected Diseases initiative. About Chagas disease. https://www.dndi.org/diseases-projects/chagas/. May 20, 2018.
(18) Perez-Molina, J.; Molina, I. Chagas disease. Lancet 2018; 391: 82-94.
(19) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R.W.; Alvar, J.; Boelaert, M. Visceral leishmaniasis: What are the needs for diagnosis, treatment and control? Nat. Rev. Microbiol. 2007; 5: 873-882.
(20) Drugs for Neglected Diseases initiative. About leishmaniasis. https://www.dndi.org/diseases-projects/leishmaniasis/. May 25, 2018.
(21) Alvar, J.; Velez, I.D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.; Jannin, J.; den Boer, M.; Team, W.L.C. Leishmaniasis worldwide and global estimates of its incidence. PloS ONE 2012; 7: e35671.
(22) Katsuno, K.; Burrows, J.N.; Duncan, K.; Hooft van Huijsduijnen, R.; Kaneko, T.; Kita, K.; Mowbray, C.E.; Schmatz, D.; Warner, P.; Slingsby, B.T. Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nature Reviews Drug Discovery 2015; 14: 751- 758.
(23) Baell, J.; Walters, M.A. Chemical con artists foil drug discovery. Nature 2014; 513: 481- 483.
(24) Drugs for Neglected Diseases initiative. Target product profile - sleeping sickness. https://www.dndi.org/diseases-projects/hat/hat-target-product-profile/. April 19, 2018.
(25) Drugs for Neglected Diseases initiative. Chagas disease target product profile. https://www.dndi.org/diseases-projects/chagas/chagas-target-product-profile/. May 22, 2018.
(26) Drugs for Neglected Diseases initiative. Target product profile - visceral leishmaniasis. https://www.dndi.org/diseases-projects/leishmaniasis/tpp-vl/. May 22, 2018.
308
(27) Klug, D.M.; Gelb, M.H.; Pollastri, M.P. Repurposing strategies for neglected diseases. Bioorg. Med. Chem. Lett. 2016; 26: 2569-2575.
(28) Njoroge, M.; Njuguna, N.M.; Mutai, P.; Ongarora, D.S.B.; Smith, P.W.; Chibale, K. Recent approaches to chemical discovery and development against malaria and the neglected tropical diseases human African trypanosomiasis and schistosomiasis. Chem. Rev. 2014; 114: 11138- 11163.
(29) Bacchi, C.J.; Nathan, H.C.; Hunter, S.H.; McCann, P.P.; Sjoerdsma, A. Polyamine metabolism: A potential therapeutic target in trypanosomes. Science 1980; 210: 332-334.
(30) Yu, Z.; Brannigan, J.A.; Moss, D.K.; Brzozowski, A.M.; Wilkinson, A.J.; Holder, A.A.; Tate, E.W.; Leatherbarrow, R.J. Design and synthesis of inhibitors of Plasmodium falciparum N- myristoyltransferase, a promising target for antimalarial drug discovery. J. Med. Chem. 2012; 55: 8879-8890.
(31) Rackham, M.D.; Brannigan, J.A.; Moss, D.K.; Yu, Z.; Wilkinson, A.J.; Holder, A.A.; Tate, E.W.; Leatherbarrow, R.J. Discovery of novel and ligand-efficient inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferase. J. Med. Chem. 2013; 56: 371-375.
(32) Rackham, M.D.B., James A.; Rangachari, K.; Meister, S.; Wilkinson, Anthony J.; Holder, Anthony A.; Leatherbarrow, Robin J. and Tate, Edward W. Design and synthesis of high affinity inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferases directed by ligand efficiency dependent lipophilicity (LELP). J. Med. Chem. 2014; 57: 2773-2788.
(33) Minucci, S.P., Pier Giuseppe. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 2006; 6: 38-51.
(34) Kelly, J.M.T., Martin C.; Horn, David; Loza, Elinars; Kalvinsh, Ivars; Bjorkling, Fredrik. Inhibitors of human histone deacetylase with potent activity against the African trypanosome Trypanosoma brucei. Bioorganic Med. Chem. Lett. 2012; 22: 1886-1890.
(35) Carrillo, A.K.G., W. Armand; Guy, R. Kiplin. Evaluation of histone deacetylase inhibitors (HDAcI) as therapeutic leads for human African trypanosomiasis (HAT). Bioorganic Med. Chem. Lett. 2015; 23: 5151-5155.
(36) Cleghorn, L.A.T.; Albrecht, S.; Stojanovski, L.; Simeons, F.R.J.; Norval, S.; Kime, R.; Collie, I.T.; De Rycker, M.; Campbell, L.; Hallyburton, I.; Frearson, J.A.; Wyatt, P.G.; Read, K.D.; Gilbert, I.H. Discovery of indoline-2-carboxamide derivatives as a new class of brain- penetrant inhibitors of Trypanosoma brucei. J. Med. Chem. 2015; 58: 7695-7706.
(37) Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2001; 46: 3-26.
309
(38) Meanwell, N.A. Improving drug candidates by design: A focus on physicochemical properties as a means of improving compound disposition and safety. Chem Res Toxicol 2011; 24: 1420-1456.
(39) Wassvik, C.M.; Holmen, A.G.; Draheim, R.; Artursson, P.; Bergstrom, C.A.S. Molecular characteristics for solid-state limited solubility. J. Med. Chem. 2008; 51: 3035-3039.
(40) Johnson, T.W.; Gallego, R.A.; Edwards, M.P. Lipophilic efficiency as an important metric in drug design. J. Med. Chem. 2018; 61: 6401-6420.
(41) Shultz, M.D. The thermodynamic basis for the use of lipophilic efficiency (LipE) in enthalpic optimizations. Bioorg Med Chem Lett 2013; 23: 5992-6000.
(42) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in central nervous system drug discovery. J. Med. Chem. 2013; 56: 2-12.
(43) Reichel, A. Addressing central nervous system (CNS) penetration in drug discovery: Basics and implications of the evolving new concept. Chemistry and Biodiversity 2009; 6: 2030-2049.
(44) Rankovic, Z. CNS physicochemical property space shaped by a diverse set of molecules with experimentally determined exposure in the mouse brain. J. Med. Chem. 2017; 60: 5943- 5954.
(45) Wager, T.T.; Chandrasekaran, R.Y.; Hou, X.; Troutman, M.D.; Verhoest, P.R.; Villalobos, A.; Will, Y. Defining desirable central nervous system drug space through the alignment of molecular properties, in vitro ADME, and safety attributes. ACS Chem. Neurosci. 2010; 1: 420- 434.
(46) Wager, T.; Hou, X.; Verhoest, P.R.; Villalobos, A. Moving beyond rules: The development of a central nervous system multiparameter optimization (CNS-MPO) approach to enable alignment of druglike properties. ACS Chem. Neurosci. 2010; 1: 435-449.
(47) Ahmad, N.M. Solubility-driven lead optimization: Recent examples and personal perspectives. Bioorg Med Chem Lett 2016; 26: 2975-2979.
(48) Bergstrom, C.A.S.; Wassvik, C.M.; Johannson, K.; Hubatsch, I. Poorly soluble marketed drugs display solvation limited solubility. J. Med. Chem. 2007; 50: 5858-5862.
(49) Savjani, K.T.; Gajjar, A.K.; Savjani, J.K. Drug solubility: Importance and enhancement techniques. ISRN Pharmaceutics 2012; 2012.
(50) Stella, V.J.; Nti-Addae, K.W. Prodrug strategies to overcome poor water solubility. Adv. Drug Deliv. Rev. 2007; 59: 677-694.
(51) Walker, M.A. Improvement in aqueous solubility achieved via small molecular changes. Bioorg Med Chem Lett 2017; 27: 5100-5108.
310
(52) Lovering, F.; Bikker, J.; Humblet, C. Escape from flatland: Increasing saturation as an approach to improving clinical success. J. Med. Chem. 2009; 52: 6752-6756.
(53) Li, A.P. Screening for human ADME/tox drug properties in drug discovery. Drug Discov. Today 2001; 6: 357-366.
(54) Microsomal stability assay. http://www.cyprotex.com/admepk/in-vitro- metabolism/microsomal-stability. August 18, 2018.
(55) Diaz, R.; Luengo-Arratta, S.; Seixas, J.o.D.; Amata, E.; Devine, W.; Cordon-Obras, C.; Rojas-Barros, D.I.; Jimenez, E.; Ortega, F.; Crouch, S.; Colmenarejo, G.; Fiandor, J.M.; Martin, J.J.; Berlanga, M.; Gonzalez, S.; Manzano, P.; Navarro, M.; Pollastri, M.P. Identification and characterization of hundreds of potent and selective inhibitors of Trypanosoma brucei growth from a kinase-targeted library screening campaign. PLoS Negl. Trop. Dis. 2014; 8: e3253.
(56) Patel, G.; Karver, C.E.; Behera, R.; Guyett, P.J.; Sullenberger, C.; Edwards, P.; Roncal, N.E.; Mensa-Wilmot, K.; Pollastri, M.P. Kinase scaffold repurposing for neglected disease drug discovery: Discovery of an efficacious, lapatanib-derived lead compound for trypanosomiasis. J. Med. Chem. 2013; 56: 3820-3832.
(57) Naula, C.; Parsons, M.; Mottram, J.C. Protein kinases as drug targets in trypanosomes and leishmania. Biochimica et Biophysica Acta 2005; 1754: 151-159.
(58) Nett, I.R.E.; Martin, D.M.A.; Miranda-Saavedra, D.; Lamont, D.; Barber, J.D.; Mehlert, A.; Ferguson, M.A.J. The phosphoproteome of bloodstream of Trypanosoma brucei, causative agent of African sleeping sickness. Molecular and Cellular Proteomics 2009; 1527-1538.
(59) Nett, I.R.E.; Davidson, L.; Lamont, D.; Ferguson, M.A.J. Identification and specific localization of tyrosine-phosphorylated proteins in Trypanosoma brucei. Eukaryotic Cell 2009; 617-626.
(60) Parsons, M.; Worthey, E.A.; Ward, P.N.; Mottram, J.C. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. BMC Genomics 2005; 6: 127.
(61) Katiyar, S.; Kufareva, I.; Behera, R.; Thomas, S.M.; Ogata, Y.; Pollastri, M.P.; Abagyan, R.; Mensa-Wilmot, K. Lapatinib-binding protein kinases in the african trypanosome: Identification of cellular targets for kinase-directed chemical scaffolds. PloS ONE 2013; 8: e56150.
(62) Schultz, A.G.; Pinto, D.J.P.; Welch, M. Synthesis of chiral dibenzo-1,8-diaza-14-crown-4, dibenzo-1,9-diaza-16-crown-4, and dibenzo-1,10-diaza-18-crown-4 ethers by aromatic nucleophilic substitution. Application to the preparation of bicyclic chiral crown-lii complexes. J. Org. Chem. 1988; 53: 1372-1380.
311
(63) Xing, L.; Klug-Mcleod, J.; Rai, B.; Lunney, E.A. Kinase hinge binding scaffolds and their hydrogen bond patterns. Bioorg Med Chem 2015; 23: 6520-6527.
(64) Silva, L.E., Hit-to-lead optimization of kinase-targeted inhibitors of Trypanosoma brucei growth, Chemistry and Chemical Biology, Northeastern University, 2015.
(65) Topliss, J.G. Utilization of operational schemes for analog synthesis in drug design. J. Med. Chem. 1972; 15: 1006-1011.
(66) Gunatilleke, S.S.; Calvet, C.M.; Johnston, J.B.; Chen, C.-K.; Erenburg, G.; Gut, J.; Engel, J.C.; Ang, K.K.H.; Mulvaney, J.; Chen, S.; Arkin, M.R.; McKerrow, J.H.; Podust, L.M. Diverse inhibitor chemotypes targeting trypanosoma cruzi cyp51. PLoS Negl. Trop. Dis. 2012; 6: e1736. (67) Choi, J.Y.; Podust, L.M.; Roush, W.R. Drug strategies targeting CYP51 in neglected tropical diseases. Chem Rev 2014; 114: 11242-11271.
(68) Chen, C.-K.; Doyle, P.S.; Yermalitskaya, L.; Mackey, Z.B.; Ang, K.K.H.; McKerrow, J.H.; Podust, L.M. Trypanosoma cruzi cyp51 inhibitor derived from a Mycobacterium tuberculosis screen hit. PLoS Negl Trop Dis 2009; 3: e372.
(69) Devine, W.; Woodring, J.L.; Swaminathan, U.; Amata, E.; Patel, G.; Erath, J.; Roncal, N.E.; Lee, P.J.; Leed, S.E.; Rodriguez, A.; Mensa-Wilmot, K.; Sciotti, R.J.; Pollastri, M.P. Protozoan parasite growth inhibitors discovered by cross-screening yield potent scaffolds for lead discovery. J. Med. Chem. 2015; 58: 5522-5537.
(70) Mehta, N.; Ferrins, L.; Leed, S.E.; Sciotti, R.J.; Pollastri, M.P. Optimization of physicochemical properties for 4-aminoquinoline inhibitors of Plasmodium falciparum proliferation. ACS Infectious Diseases 2018; 4: 577-591.
(71) Maiti, D.; Fors, B.P.; Henderson, J.L.; Nakamura, Y.; Buchwald, S.L. Palladium-catalyzed coupling of functionalized primary and secondary amines with aryl and heteroaryl halides: Two ligands suffice in most cases. Chem Sci 2011; 2: 57-68.
(72) Degorce, S.L.; Bodnarchuk, M.S.; Cumming, I.A.; Scott, J.S. Lowering lipophilicity by adding carbon: One-carbon bridges of morpholines and piperazines. J. Med. Chem. 2018.
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Appendix
Appendix 1. Biological activity assay protocols (Chapters 2 and 3)
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.
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.
Bioactivity assays. In order to determine the T. b. brucei EC50 values, 4 μL per well from compound master plates were dispensed into a new plate and 96 μL of HMI-9 per well were added to generate a 4% DMSO intermediate plate. Mid-log phase growth T. b. brucei was diluted to a working cell density of 2,750 cells/mL and 90 μL/well dispensed into 96-well flat-bottom transparent assay plates (Nunc®). Ten μL/well from intermediate plates were added. The final concentration of compounds was 40 μM in 0.4% DMSO per well.
Assay plates were incubated for 72 h at 37 ºC and 5% CO2. Four hours prior to the end of the incubation, 20 μL of a 440 μM resazurin solution in prewarmed HMI-9 was added to each well
313 and incubated for another 4 h. Fluorescence was then measured in an Infinite F200 plate reader
(Tecan®) at 550 nm (excitation filter) and 590 nm (emission filter). A four-parameter equation was employed to fit the dose-response curves and determine EC50 using the SigmaPlot® 13.0 software. Assays were performed in duplicate at least twice, to achieve a minimal n=2 per dose response.
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
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Probes). Compound activities were normalized using the in-plate negative (benznidazole at 10
μg/mL) and positive (0.2% DMSO) growth controls.
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.
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Appendix 2. Biological activity assay protocols (Chapter 4)
Trypanosoma brucei proliferation (NYU). In a 96-well plate, compounds were added in triplicates at 50 μM and in serial dilutions 1:2 in HMI-9 medium. To each well, 2.5×103 T. b. brucei (strain 427) were added and incubated at 37 °C, 5% CO2 for 48 h. Following incubation,
20 μL of PrestoBlue® were added to each well and incubated for additional 4 h. Fluorescence was read at 530 nm excitation and 590 nm emission. Suramin at 100 μM was used as positive control and reference for calculation of IC50.
Trypanosoma brucei proliferation (UGA). The high-throughput trypanosome proliferation inhibition assay was performed and analyzed as described by Thomas et al. (2016).1
The protocols for the biological assays of Trypanosoma cruzi, Leishmania major amastigotes and promastigotes and Plasmodium falciparum D6 were performed as previously described.2
Drug toxicity to HepG2 cells.3 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 acids, 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 containing twelve duplicate 1.6-fold serial dilutions of each test compound
316 suspended in DMSO. Diluted test compound (4.25 μL) 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 were removed by shaking the plate over sink, and the plates were left to dry in a fume hood for 15 min. 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 were put on a 3-D rotator for 15-30 min. 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.
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Appendix 3. ADME and pharmacokinetic experiment protocols
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. The fraction unbound (fu) is reported.
Human Liver Microsomal Clint. In vitro intrinsic clearance was determined from human liver microsomes using a standard approach.4 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.4 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.
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Calculated LogP and LogD values. Both LogP and LogD predictions are based on a modified version of the method5 where the predicted partition coefficients are composed of the molecules’ atomic increments.
Pharmacokinetics protocols. Compound was administered intraperitoneally (IP) to two groups of female NMRI mice (Group 1 n=3; Group 2 n=6). The compound was prepared in 1%
(v/v) DMSO:99% (v/v) 20% (w/v) sulfobutyl ether-beta-cyclodextrin (SBE-β-CD) (Captisol®) 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 1/2 (w/v) ratio (brain/water), and then homogenized.
Both blood and brain samples were stored -80 ºC until analysis.
Diluted blood and brain homogenates were processed under standard liquid-liquid extraction procedures using ACN containing an internal standard (Nifedipine) and analyzed 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.
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Appendix 4. Chapter 2 Spectra
The placement of the methyl group of DMK-3-441-02 was confirmed by NOE spectroscopy. In order to distinguish DMK-3-441-02 from compound 1, the methyl peak (H B) was irradiated and was found to couple to a singlet at δ7.55 ppm (H C). When this peak was irradiated, it was found to couple to H B. In addition, the -NH A was irradiated and found to couple to a doublet at δ7.23 ppm (H D). The first spectrum shows the 1H NMR of DMK-3-441-02. The second spectrum shows the NOEs described above, in order from top to bottom. The third spectrum shows the 1H NMR of DMK-3-256-03 for comparison, where the methyl peak has a clearly distinct chemical shift (δ3.45 ppm) from that of DMK-3-441-02 (δ3.89 ppm).
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323
The reaction of 2,4-dichloropyrimidine 2 with methylamine resulted in two isolated products, DMK-3-912-02 and DMK-3-912-03. These compounds were distinguished from each other via NOE spectroscopy. In both cases, the methyl peak (H B) was irradiated; this peak was found to couple with a proton at δ7.57 (H A) for DMK-3-912-02, while DMK-3-912-03 did not show any NOE. The 1H NMR and NOE spectra for DMK-3-912-02 are presented first, followed by the 1H NMR and NOE spectra of DMK-3-912-03.
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328
The final spectra of NEU-4461 and NEU-5388, those compounds submitted for PK studies, kinase panel assessment, and in vivo efficacy studies, are presented below.
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Appendix 5. Chapter 3 Spectra
The structure of DMK-4-677-02 was confirmed by 1HNMR spectroscopy. Assignments of the starting material DMK-4-662-02 and product DMK-4-677-00 are shown below.
DMK-4-662-02 DMK-4-677-02 Multiplicity (J Multiplicity (J δ ppm Assignment δ ppm Assignment value) value) 3.99 s, 3H HE 4.00 s, 3H HD 6.79 d, 1H (2.4 Hz) HB 7.72 s, 1H HC 7.67 s, 1H HD 7.77 s, 1H HE 7.72 d, 1H (2.0 Hz) HA 7.83 s, 1H HA 7.79 s, 1H HF 7.93 d, 1H (2.0 Hz) HF 7.99 d, 1H (2.0 Hz) HC 8.50 d, 1H (2.0 Hz) HB 8.46 s, 1H HG
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The 1H NMR spectrum of NEU-5127, the compound submitted for PK studies, is presented below.
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Appendix 6. Chapter 4 Spectra
Included are three representative 1H NMR spectra of final compounds presented in Chapter
4: NEU-4955, NEU-5972, and NEU-6000.
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References
(1) Thomas, S.M.; Purmal, A.; Pollastri, M.; Mensa-Wilmot, K. Discovery of a carbazole- derived lead drug for human African trypanosomiasis. Scientific Reports 2016; 6.
(2) Ochiana, S.O.; Bland, N.D.; Settimo, L.; Campbell, R.K.; Pollastri, M. Repurposing human PDE4 inhibitors for neglected tropical disease. Evaluation of analogs of the human PDE4 inhibitor GSK-256066 as inhibitors of PDEB1 of trypanosoma brucei. Chem Biol Drug Des 2015; 549-564.
(3) Ferrari, M.; Fornasiero, M.C.; Iseta, A.M. MTT coloimetric assay for testing macrophage cytotoxic in vitro. Journal of Immunological Methods 1990; 2: 165-172.
(4) Konsoula, R.; Jung, M. In vitro plasma stability, permeability, and solubility of mercaptoacetamide histone deacetylase inhibitors. International Journal of Pharmaceutics 2008; 361: 19-25.
(5) Viswanadhan, V.N.; Ghose, A.K.; Revankar, G.R.; Robins, R.K. Atomic physicochemical parameters for three dimensional structure directed quantitative structure-activity relationships. 4. Additional parameters for hydrophobic and dispersive interactions and their application for an automated superposition of certain naturally occurring nucleoside antibiotics. Journal of Chemical Information and Computer Sciences 1989; 29: 163-172.
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