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Chemical Modifications for Activity and Property Optimization: Drug Discovery Efforts for
Neglected Tropical Diseases
By Kelly A. Bachovchin
Bachelor of Science in Molecular Biology, Kean University
Master of Science in Biotechnology, Kean University
A dissertation submitted to
The Faculty of
The College of Science of
Northeastern University
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
November 26th, 2019
Dissertation directed by
Michael P. Pollastri
Professor of Chemistry and Chemical Biology
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Acknowledgements
First and foremost, I would like to thank Northeastern University and the Department of
Chemistry and Chemical Biology for allowing me to pursue my doctoral studies. I would
particularly like to thank my committee members, Dr. Robert Hanson, Dr. Roman Manetsch, and
Dr. Leila Deravi for all your insight on this project.
I would like to acknowledge the hard work of our collaborators who, without their efforts,
none of this work would have been possible: Dr. Kojo Mensa-Wilmot, Dr. Miguel Navarro, Dr.
Rosario Diaz, Dr. Gloria Ceballos-Perez, Dr. Domingo Rojas-Barros, Dr. Maria Santos Martinez-
Martinez, Dr. Pilar Manzano, and Dr. Richard Sciotti. Thank you to AstraZeneca for providing all
the in vitro ADME data.
Thank you to my undergraduate advisor Professor James Robert Merritt for introducing
me to life in the lab and mentoring me through my master’s thesis. Without you I would have never
decided to pursue a PhD.
To my Pollastri Lab coworker and labmates, thank you to everyone who has given me
advice and helped me day to day along the way. A special thank you to Dr. Seema Bag who showed
me the ropes in my early days and Dr. Baljinder Singh who picked up the job in her place. To Dr.
Dana Klug for sharing your intermediates and all of the countless hours you spent maintaining the
LCMS. To Dr. Melissa Buskes and Dr. Quillon Simpson for editing and helping with the writing
of this dissertation. A very special thank you to Dr. Lori Ferrins for not only all your edits but for
being a pillar of support over the years, I never would have finished without you.
Thank you to all my “PL2” labmates for keeping the lab a pleasant space to be every day,
including Dr. Meaghan Fallano, Dr. Enrico Mongeau, Dr. Andrew Spaulding, Dr. Hitesh Jalani,
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Dr. Naresh Gunaganti, Dr. Alex Muthengi, and all the wonderful undergraduate researchers. I
would like to especially thank Mitch Rivers who always kept the conversation interesting and for
being the best undergrad mentee I could ask for.
A special thank you goes to my support system outside of Northeastern. To my family,
Mom, Dad, Julie, Cory, and Hailey who supported my decisions even when they took me hundreds
of miles away, who tolerated my absence and stood by me throughout the years, and whose love
and support I’ve cherished for so many years. To Eliza Miller who I started day one with at
Northeastern, and who believed in me and helped me believe in myself even from thousands of
miles away. To my salsa family, especially Andres, Hernan, Amy, Limar, Amelia, Maureen, Celia,
and Beatriz, who have become my family away from home, and given me a community to escape
to when times were hard in the lab.
I want to extend my deepest gratitude to my advisor, Professor Michael Pollastri. Thank
you for always having a box of tissues handy. Most importantly, thank you for believing I could
finish and for all of your help, guidance, and understanding along the way.
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Abstract of Dissertation
Neglected tropical diseases (NTDs) affect the lives of billions of people across the globe,
and disproportionately affect the most poverty-stricken populations. Current chemotherapeutics
present many challenges with difficult treatment regimens and toxic side effects prevalent;
resistance has also been observed for many. It has been shown that repurposing chemical matter
for use in NTD drug development is a successful strategy to discover new drugs, accelerating the
drug discovery process, and requiring less monetary investment. To this end, we have employed
target-class and lead repurposing strategies using human kinase inhibitors to identify inhibitors of
four parasitic diseases: human African trypanosomiasis (HAT, caused by Trypanosoma brucei),
Chagas disease (T. cruzi), leishmaniasis (Leishmania donovani), and malaria (Plasmodium
falciparum).
The first project outlined in this dissertation describes the process of repurposing the drug
lapatinib and subsequent optimizations that led to advanced hits for T. brucei inhibition. Through
the application of known medicinal chemistry strategies we have successfully modulated the
aqueous solubility of the series. This has led to new, potent compounds that inhibit multiple
parasites besides T. brucei, including T. cruzi, L. donovani, and P. falciparum, each with their own
structure-activity relationship optimization pathways.
The second project involves the prioritization of a cluster of 2,4-substituted azaindoles
from a high-throughput screen (HTS) of a kinase-targeted inhibitor library against T. brucei. The
hit compound from this cluster, NEU-1200, showed high in vivo clearance rates. Strategic
modifications were made to NEU-1200 to reduce the clearance and these modifications led to
NEU-5123 which showed improved in vivo clearance rates and good exposure in mice brains, an
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important factor for treating stage II of HAT. NEU-5123 was tested in an acute efficacy model for
HAT and showed an extension of life of 8.8 days when dosed at 10 mg/kg/day, however at higher
doses was highly toxic. Toxicity of this series will be addressed in future work.
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Table of Contents
Acknowledgements ......................................................................................................................... 2
Abstract of Dissertation .................................................................................................................. 4
Table of Contents ............................................................................................................................ 6
List of Figures ............................................................................................................................... 10
List of Schemes ............................................................................................................................. 13
List of Tables ................................................................................................................................ 15
List of Abbreviations .................................................................................................................... 18
Chapter 1: Background and Introduction ................................................................................ 21
1.1 Neglected tropical diseases (NTDs)........................................................................................ 21
1.1.1 Human African Trypanosomiasis (HAT) ............................................................................ 23
1.1.2 Other parasitic diseases ........................................................................................................ 24
1.1.2.1 Chagas disease .................................................................................................................. 25
1.1.2.2 Leishmaniasis .................................................................................................................... 26
1.1.2.3 Malaria .............................................................................................................................. 28
1.2 Current treatments for NTDs .................................................................................................. 29
1.2.1 Current treatments for HAT ................................................................................................. 29
1.2.2 Current treatments for Chagas disease ................................................................................. 31
1.2.3 Current treatments for leishmaniasis .................................................................................... 32
1.2.4 Current treatments for malaria ............................................................................................. 33
1.3 NTD drug development .......................................................................................................... 34
1.3.1 Repurposing chemical matter ............................................................................................... 35
1.3.1.1 Drug repurposing .............................................................................................................. 36
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1.3.1.2 Target repurposing ............................................................................................................ 41
1.3.1.3 Target-class repurposing ................................................................................................... 44
1.3.1.4 Lead repurposing ............................................................................................................... 49
1.4 Compound optimization .......................................................................................................... 52
Chapter 2: Property optimization of lapatinib derived analogs for human African
trypanosomiasis (HAT) drug discovery .................................................................................... 55
2.1 Introduction ............................................................................................................................. 55
2.1.1 Repurposing lapatinib for Trypanosoma brucei inhibition .................................................. 55
2.1.2 Identification of NEU-1912 and NEU-1953 as leads for drug development ....................... 57
2.1.3 Methods for improving aqueous solubility .......................................................................... 61
2.1.3.1 Salt formation.................................................................................................................... 62
2.2 Salt screens.............................................................................................................................. 65
2.2.1 Salt formations of NEU-1953 .............................................................................................. 65
2.2.1.1 Evaluation of NEU-1953 salt forms ................................................................................. 66
2.2.2 Salt screen of NEU-1912 ..................................................................................................... 67
2.2.2.1 Synthesis of NEU-1912 .................................................................................................... 67
2.2.2.2 Salt formations of NEU-1912 ........................................................................................... 69
2.2.2.3 Evaluation of NEU-1912 salt forms ................................................................................. 69
2.3 Increasing Fsp3: Design, synthesis, and evaluation of NEU-1912 and NEU-1953 matched
pairs with saturated head groups ................................................................................................... 70
2.3.1 Selection of saturated primary amines as replacements for aromatic head groups of NEU-
1912 and NEU-1953 ..................................................................................................................... 70
2.3.2 Synthesis of saturated head group replacements, NEU-1953 scaffold ................................ 71
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2.3.3 Synthesis of saturated head group replacements, NEU-1912 scaffold ................................ 75
2.3.4 Evaluation of saturated head group replacements, NEU-1912 and NEU-1953 scaffolds and
matched pairs ................................................................................................................................ 76
2.4 Crossover compounds between NEU-4363 and NEU-1912 ................................................... 79
2.4.1 Synthesis of crossovers ........................................................................................................ 80
2.4.2 Evaluation of crossovers ...................................................................................................... 82
2.5 Evaluation of lapatinib analogs against other parasites .......................................................... 84
2.5.1 Evaluation of lapatinib analogs against Trypanosoma cruzi ............................................... 84
2.5.2 Evaluation of lapatinib analogs against Leishmania ............................................................ 85
2.5.3 Evaluation of lapatinib analogs against P. falciparum ........................................................ 86
2.6 Summary ................................................................................................................................. 87
Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT
drug discovery ............................................................................................................................. 91
3.1 Introduction ............................................................................................................................. 91
3.1.1 High-throughput screen of a kinase targeted library against T. brucei in collaboration with
GSK............................................................................................................................................... 91
3.1.2 Organization of HTS hits and identification of NEU-1200 as a potent inhibitor of T. brucei
growth ........................................................................................................................................... 92
3.2.3 In vivo pharmacokinetic (PK) evaluation of NEU-1200...................................................... 96
3.2 Design, synthesis, and evaluation of NEU-1200 first-generation analogs ............................. 97
3.2.1 Rationale of NEU-1200 first-generation analog design ....................................................... 97
3.2.2 Synthesis of NEU-1200 and first-generation analogs .......................................................... 99
3.2.3 Evaluation of NEU-1200 first-generation analogs ............................................................ 107
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3.2.4 Pharmacokinetic analysis of NEU-5123 ............................................................................ 118
3.2.5 In vivo evaluation of NEU-5123 ........................................................................................ 119
3.2.6 Kinase selectivity evaluation of NEU-5123 and NEU-5449 ............................................. 121
3.3 Design, synthesis, and evaluation of NEU-1200 second-generation analogs ....................... 122
3.3.1 Rationale of NEU-1200 second-generation analog design ................................................ 122
3.3.2 Synthesis of NEU-1200 second-generation analogs .......................................................... 123
3.3.3 Evaluation of NEU-1200 second-generation analogs ........................................................ 124
3.4 Evaluation of cluster compounds against other parasites ..................................................... 128
3.5 Summary ............................................................................................................................... 131
3.6 Future Directions .................................................................................................................. 134
References ................................................................................................................................... 135
Chapter 4: Experimental section ............................................................................................. 149
4.1 General methods ................................................................................................................... 149
4.2 Experimental details.............................................................................................................. 150
4.2.1 Experimental procedures for Chapter 2 ............................................................................. 150
4.2.2 Experimental procedures for Chapter 3 ............................................................................. 187
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List of Figures
Chapter 1: Background and Introduction ................................................................................ 21
Figure 1-1. Life cycle of Trypanosoma brucei. ............................................................................ 24
Figure 1-2. Life cycle of Trypansoma cruzi. ................................................................................ 26
Figure 1-3. Life cycle of Leishmania. ........................................................................................... 27
Figure 1-4. Life cycle of Plasmodium falciparum. ....................................................................... 28
Figure 1-5. Current treatments for HAT. ...................................................................................... 30
Figure 1-6. Drugs in clinical trials for HAT. ................................................................................ 31
Figure 1-7. Current treatments for Chagas disease. ...................................................................... 32
Figure 1-8. Current treatments for leishmaniasis. ......................................................................... 33
Figure 1-9. Current treatments for malaria. .................................................................................. 34
Figure 1-10. The drug development pipeline and different entry points for repurposing
campaigns. .................................................................................................................................... 35
Figure 1-11. Categorization cascade of the different types of repurposing strategies. ................. 36
Figure 1-12. FDA-approved drugs identified as active against T. b. rhodesiense. ....................... 38
Figure 1-13. FDA-approved drugs identified as active against T. cruzi and that could serve as
starting points for further optimization to mitigate toxicity.......................................................... 39
Figure 1-14. FDA-approved drugs shown to inhibit TcPAT12. ................................................... 40
Figure 1-15. Antifungal azoles repurposed for Chagas disease and used in combination with
DB766 to enhance killing of L. donovani. .................................................................................... 41
Figure 1-16. Progression of the diaminothiazole hit to GSK3186899/DDD853651. ................... 43
Figure 1- 17. Human PDE inhibitors that entered medicinal chemistry optimization for T. brucei.
....................................................................................................................................................... 44
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Figure 1-18. Hit to lead compounds resulting from target-guided SAR explorations of
TbrPDEB1..................................................................................................................................... 44
Figure 1-19. The HDAC inhibitor, vorinostat, shows promising in vivo efficacy against L.
donovani when absorbed by gold nanoparticles ........................................................................... 45
Figure 1-20. Anticancer drugs shown to moderately reduce parasitemia in the livers of mice
infected with L. donovani ............................................................................................................. 47
Figure 1-21. Structure of lapatinib-derived analog NEU-4438. ................................................... 48
Figure 1-22. Structures of delaminid and its optimized analogs. ................................................. 49
Figure 1-23. Isatinoid compound NEU-4391 identified from a lead repurposing HTS campaign.
....................................................................................................................................................... 50
Figure 1-24. Most active compound (56) from a kinase library screen and optimized compound
(57) for T. brucei growth inhibition. ............................................................................................. 51
Chapter 2: Property optimization of lapatinib derived analogs for human African
trypanosomiasis (HAT) drug discovery .................................................................................... 55
Figure 2-1. Optimizations of EGFR inhibitor lapatinib to lead compound NEU-617.................. 56
Figure 2-2. Summary of modifications made to NEU-1045. ....................................................... 58
Figure 2-3. Modifications to NEU-961 resulting in NEU-1953. .................................................. 59
Figure 2-4. Comparison of lipophilicity versus aqueous solubility in first and second generation
lapatinib analogs. .......................................................................................................................... 61
Figure 2-5. Clinical candidate AMG-517 and its analog with improved aqueous solubility. ...... 64
Figure 2-6. Comparison of parent compound NEU-1953 and analog NEU-4363. ...................... 80
Figure 2-7. Summary of modifications made to parent compounds NEU-1912 and NEU-1953. 88
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Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT
drug discovery ............................................................................................................................. 91
Figure 3-1. Predicted metabolically labile sites of NEU-1200 ..................................................... 98
Figure 3-2. Summary of analog design plans. .............................................................................. 99
Figure 3-3. Crystal structure of 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b] pyridine. .............. 103
Figure 3-4. Peripheral blood levels of NEU-5123 in mice. ........................................................ 119
Figure 3-5. Efficacy of NEU-5123 in mice after a 10 mg/kg/day dose. ..................................... 120
Figure 3-6. Efficacy of NEU-5123 in mice after a 30 mg/kg/day dose. ..................................... 121
Figure 3-7. Human kinase profiles of NEU-5123 and NEU-5449. ............................................ 122
Figure 3-8. Graph of multiple cell line toxicity values of all synthesized NEU-1200. .............. 128
Figure 3-9. Correlation of T. cruzi potency and L6 host cell toxicity. ....................................... 130
Figure 3-10. SAR/SPR summary for synthesized NEU-1200 analogs. ...................................... 132
Figure 3-11. Compound progressions of the series. ................................................................... 133
Figure 3-12. Summary of future directions for analog design. ................................................... 134
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List of Schemes
Chapter 2: Property optimization of lapatinib derived analogs for human African
trypanosomiasis (HAT) drug discovery .................................................................................... 55
Scheme 2-1. Synthesis of 6-bromoquinolone core ....................................................................... 67
Scheme 2-2. Synthetic route for NEU-1912 tail group................................................................. 68
Scheme 2-3. Synthesis of NEU-1912. .......................................................................................... 68
Scheme 2-4. Synthetic route for NEU-1953 tail group................................................................. 71
Scheme 2-5. Saturated head group matched pairs to NEU-1953 template synthesis. .................. 72
Scheme 2-6. Synthetic attempts via SNAr conditions using NEU-1953 7-bromo-4-
chloroquinoline core for intermediate to final compounds. .......................................................... 75
Scheme 2-7. Synthetic route for various head group analogs. ...................................................... 76
Scheme 2-8. Synthesis of 1-methylhomopiperazine tail group .................................................... 81
Scheme 2-9. Synthesis of 6-position substituted matched pairs of NEU-1912 and NEU-4363. .. 81
Scheme 2-10. Synthesis of 7-position substituted matched pair of NEU-1912 and NEU-4363. . 82
Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT
drug discovery ............................................................................................................................. 91
Scheme 3-1. Synthesis of intermediates 3.2a-c. ......................................................................... 100
Scheme 3-2. Synthesis of analogs truncated at the 2-position. ................................................... 100
Scheme 3-3. Synthesis of boronate coupling partners 3.7a,b. .................................................... 101
Scheme 3-4. Synthesis of analogs truncated at the 4-position. ................................................... 102
Scheme 3-5. Initial synthesis of analogs with modifications at the 4-position. .......................... 103
Scheme 3-6. Modified synthesis of compounds with 4-position modifications. ........................ 104
Scheme 3-7. Attempted synthesis of analogs with modifications at the 2-position. .................. 105
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Scheme 3-8. First successful synthesis of analogs with modifications at the 2-position. .......... 106
Scheme 3-9. Modified synthesis of analogs with modifications at the 2-position. .................... 107
Scheme 3-10. Synthesis of second-generation NEU-1200 analogs. ........................................... 124
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List of Tables
Chapter 1: Background and Introduction ................................................................................ 21
Table 1-1. The 20 neglected tropical diseases as defined by the World Health Organization ..... 21
Table 1-2. Targeted compound criteria for hit and lead compounds. ........................................... 54
Chapter 2: Property optimization of lapatinib derived analogs for human African
trypanosomiasis (HAT) drug discovery .................................................................................... 55
Table 2-1. Comparison of NEU-1045 to NEU-1912 and NEU-961 to NEU-1953. ..................... 58
Table 2-2. Determination of approximate solubility of NEU-1953 in various organic solvents. . 65
Table 2-3. Salts formed of NEU-1953 compared to the free base. ............................................... 66
Table 2-4. Conditions for salt formations of NEU-1912. ............................................................. 69
Table 2-5. Salts formed of NEU-1912 compared to the free base. ............................................... 70
Table 2-6. Synthetic attempts via Buchwald-Hartwig cross coupling using NEU-1953
chlorinated core + tail for final compound synthesis. ................................................................... 73
Table 2-7. Synthetic attempts via SNAr conditions using NEU-1953 chlorinated core + tail for
final compound synthesis. ............................................................................................................. 74
Table 2-8. Saturated head group analogs of NEU-1953, T. brucei activities and physicochemical
properties....................................................................................................................................... 78
Table 2-9. Saturated head group analogs of NEU-1912, T. brucei activities and physicochemical
properties....................................................................................................................................... 79
Table 2-10. Evaluation of NEU-1912 and NEU-4363 matched pairs. ......................................... 83
Table 2-11. Evaluation of lapatinib analogs against Trypanosoma cruzi and T. cruzi host cells
(C2C12)......................................................................................................................................... 85
Table 2-12. Evaluation of lapatinib analogs against Leishmania and B10R host cells. ............... 86
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Table 2-13. Evaluation of lapatinib analogs against common strains of P. falciparum. .............. 87
Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT
drug discovery ............................................................................................................................. 91
Table 3-1. Compound properties for a cluster of 2,4-substituted 1H-pyrrolo[2,3b]pyridines and
selected compound NEU-1200. .................................................................................................... 93
Table 3-2. Multi-parameter optimization (MPO) scoring for predicting CNS exposure. ............ 93
Table 3-3. Subset of cluster 32 compounds from initial HTS typified by a 2,4-substituted
pyrrolo[2,3-b]pyridine core........................................................................................................... 95
Table 3-4. PK evaluation of NEU-1200 following IV administration. ......................................... 97
Table 3-5. PK evaluation of NEU-1200 following IP administration. ......................................... 97
Table 3-6. Activity of truncated analogs of NEU-1200 against T. brucei. ................................. 108
Table 3-7. Activities and clearance rates of 4-position variant first-generation analogs of NEU-
1200............................................................................................................................................. 110
Table 3-8. Activities and clearance rates of 2-position variant first-generation analogs of NEU-
1200............................................................................................................................................. 112
Table 3-9. Other ADME measurements of NEU-1200 and its first-generation analogs. ........... 116
Table 3-10. Toxicity of NEU-1200 and its first-generation analogs against HepG2 and MRC5
cell lines. ..................................................................................................................................... 117
Table 3-11. Individual blood pharmacokinetic parameters of evaluated compounds after
intraperitoneal administration of 10 mg/kg single dose (target dose) to female NMRI mice (n=3).
..................................................................................................................................................... 118
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Table 3-12. Blood and brain levels (ng/mL in blood, ng/g in brain) of NEU-5123, after
intraperitoneal administration of 10 mg/kg single dose (target dose) to female NMRI mice (n=3).
LLOQ=50 ng/mL (blood). .......................................................................................................... 119
Table 3-13. Activities and clearance rates of second-generation analogs of NEU-1200. .......... 125
Table 3-14. Toxicity of second-generation NEU-1200 analogs against HepG2 and MRC5 cell
lines. ............................................................................................................................................ 127
Table 3-15. Potencies and toxicities of NEU-1200 analogs against T. cruzi and Leishmania. .. 129
Table 3-16. Potencies of NEU-1200 analogs against various P. falciparum strains. ................. 131
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List of Abbreviations
Ac Acetyl
ACT Artemisinin-combination therapy
ADME Absorption, distribution, metabolism, excretion
Aq Sol Aqueous solubility
ATP Adenosine triphosphate
AUC Area under the curve
BCS Biopharmaceutical classification system
CDC Centers for disease control
CL Cutaneous leishmaniasis
cLogP Calculated partition coefficient
Cmax Maximum concentration
CNS Central nervous system
CYP51 lanosterol 14α-demethylase
DALY Disability-adjusted life year
DCE Dichloroethane
DCM Dichloromethane
DIPEA Diisopropylethylamine
DNA Deoxyribonucleic acid
DNDi Drugs for neglected diseases initiative
EC50 Half maximal effective concentration
EGFR Epidermal growth factor receptor
eq equivalent
FDA Food and drug administration
Fsp3 Fraction of sp3 hybridized carbon atoms
GI Gastrointestinal
GPCR G-protein coupled receptor
GSK GlaxoSmithKline
GSK3β Glycogen synthase kinase-3 beta
HAT Human African trypanosomiasis
HBD Hydrogen bond donor
HDAC Histone deacetylase
hERG human Ether-a-go-go-Related Gene
HLM Clint Human liver microsomal intrinsic clearance rate
HTS Hight-throughput screen
IP Intraperitoneal
IPA Isopropanol
IRK Insulin receptor tyrosine kinase
IV Intravenous
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KOAc Potassium acetate
Lck Lymphocyte-specific protein tyrosine kinase
LCMS Liquid chromatography mass spectrometry
LDA Lithium diisopropylamide
LE Ligand efficiency
LLE Lipophilic ligand efficiency
M2 Muscarinic receptor 2
M3 Muscarinic receptor 3
MPO Multi-parameter optimization
MTD Maximum tolerated dose
mTOR Mammalian target of rapamycin
MW Molecular weight
NaOH Sodium hydroxide
NECT Nifurtimox eflornithine combination therapy
NIH National institutes of health
NMP N-methylpyrrolidone
NTD Neglected tropical disease
PAT12 Polyamine transporter 12
PdCl2(dppf)·CH2Cl2
[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane
PDE Phosphodiesterase
PDE4 Phosphodiesterase 4
PDEB1 Phosphodiesterase B1
PDEB2 Phosphodiesterase B2
PDGFR Platelet-derived growth factor receptor
PI3K Phosphoinositide kinase-3
PK Pharmacokinetic
PPB Plasma protein binding
RH Clint Rat hepatocyte intrinsic clearance rate
SAR structure-activity relationship
SNAr Nucleophilic aromatic substitution
SPR Structure-property relationship
Src Proto-oncogene tyrosine-protein kinase
t1/2 Time at half the maximum concentration
tBu tert-butyl
tBuOH tert-butanol
tBuOK Potassium tert-butoxide
tbuXPhos 2-Di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl
TC50 Half maximal toxic concentration
THF Tetrahydrofuran
tmax Time at maximum concentration
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TPSA Topological polar surface area
UV Ultraviolet
VEGFR Vascular endothelial growth factor receptor
VL Visceral leishmaniasis
WHO World health organization
Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene
XPhos 2-Dicyclohexylphosphino-2',4',5'-triisopropylbiphenyl
μwave Microwave
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Chapter 1: Background and Introduction
1.1 Neglected tropical diseases (NTDs)
The World Health Organization (WHO) defines the 20 neglected tropical diseases (NTDs)
as communicable diseases prevalent throughout the tropical regions of the world (Table 1-1).
These diseases affect over 1 billion people in 149 countries, and disproportionately affect
impoverished societies, costing their developing economies billions of dollars.1-2 A more
comprehensive assessment of the burden of these diseases is identified by the number of disability-
adjusted life years (DALYs) lost; DALYs are the sum of years of life lost due to premature death
or disability.3 NTDs are commonly caused by parasitic infections, and while some infections can
result in death, it is the chronic, debilitating nature of most of these diseases that accounted for
26.06 million DALYs in 2010.4
Table 1-1. The 20 neglected tropical diseases as defined by the World Health Organization1
Disease Category of infection
• Chagas disease
• Human African trypanosomiasis
• Leishmaniasis
Protozoan
• Dracunculiasis
• Echinococcosis
• Foodborne trematodiases
• Lymphatic filariasis
• Onchocerciasis
• Schistosomiasis
Helminth
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• Soil-transmitted helminthiases
• Taeniasis/Cysticercosis
• Buruli ulcer
• Leprosy
• Trachoma
• Yaws
Bacterial
• Dengue and Chikungunya
• Rabies
Viral
• Mycetoma, chromoblastomycosis and
other deep mycoses
Fungal
• Scabies and other ectoparasites Ectoparasitic
• Snakebite envenoming Venom
There are treatments available for NTDs, but their adverse side effects, poor dosing
regimens, and accessibility to the populations that need them make them non-ideal. Areas of
infection tend to be remote rural or suburban slums where the average person lives on less than
$2.00 US per day.3 Low to no return on investments have caused the pharmaceutical industry to
shy away from neglected disease discovery programs. New treatments are therefore needed that
are safe, orally bioavailable, and have low costs to produce. A target product profile for each of
these diseases was identified by the WHO and Drugs for Neglected Diseases initiative (DNDi) and
will be discussed later in this chapter. With the help of academic institutions and non-profit
organizations in partnership with industry, progress has been made toward eradicating these
diseases.2
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1.1.1 Human African Trypanosomiasis (HAT)
Human African trypanosomiasis (HAT) is an endemic disease in 36 sub-Saharan African
countries. In 2009, continued efforts to eliminate this disease by the year 2020 brought the number
of reported cases below 10,000 for the first time in 50 years. In 2017, there were 1,446 new cases
reported.5 However, poor healthcare infrastructure, indistinguishable symptoms, and co-infections
of other disease cause the number of cases to be underreported.
The disease is caused by an infection of the protozoan parasite Trypanosoma brucei. There
are two subspecies of T. brucei that are infective to humans. T. b. gambiense is predominantly
found in western sub-Saharan Africa and is the cause for 98% of reported cases, where T. b.
rhodesiense is predominantly found in the eastern region. An infection of T. b. gambiense causes
a chronic form of HAT that can take months to years to progress to stage 2, and T. b. rhodesiense
causes an acute version which can progress to stage 2 within a few weeks.6 Both forms of the
disease are fatal if left untreated.
T. brucei belong to the order Kinetoplastida, characterized by the presence of densely
packed DNA called a kinetoplast which can be found in the mitochondria.7 This order is also
characterized by the presence of a flagella, a cellular structure most commonly studied to
determine in which stage of reproduction a parasite may be.7-8 Trypanosomatids characteristically
have four main developmental stages they progress through during proliferation and transmission
between hosts.
Infection begins when a metacyclic trypomastigote enters the blood stream of a host after
the bite from an infected tsetse fly vector (genus Glossina). The hemolymphatic, first stage of
infection begins here as the parasite proliferates generating feverish responses and flu-like
symptoms (Figure 1-1). Antibodies begin to be made by the hosts immune system that eliminates
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most of the parasites present, however some parasites evade this destruction and survive.9-10 Other
symptoms include headache, weakness, and joint stiffness.6 Eventually, the parasites will invade
the central nervous system (CNS), including the brain, thus starting stage 2 of the disease. The
beginning of stage 2 is dependent on the subspecies of infection. More severe symptoms are
experienced during this stage including sleep pattern disruptions, coma, and eventually death.
Figure 1-1. Life cycle of Trypanosoma brucei.
Source: Public Health Image Library, provided by CDC-DPDx, Alexander J. da Silva and
Melanie Mosier. http://phil.cdc.gov/phil/details.asp?pid=3418
1.1.2 Other parasitic diseases
Though the work in the following chapters focuses on developing treatments for HAT,
compounds synthesized were also screened for their potential use against other diseases using a
parasite hopping approach. These other diseases are also caused by protozoan parasites and include
25
Chagas disease, Leishmaniasis, and malaria. Structure-activity optimizations are not performed
specifically for the parasites that cause these diseases, but if cross-screening resulted in interesting
results new potential projects could arise from these data.
1.1.2.1 Chagas disease
Chagas disease is caused by a related kinetoplastid parasite, T. cruzi. There are 65-100
million people are at risk of infection in Mexico, Central, and South America.11 Though the number
of reported cases has dropped from 16-18 million to 6-7 million because of vector control and
blood transfusion programs, there are still 12,000 annual deaths accounted for from this disease.11
There are also about 300,000 documented cases of Chagas disease in the United States.12 Hosts
are most commonly infected after having their blood come into contact with the feces of the
triatomine bug vector. The life cycle of T. cruzi can be seen in Figure 1-2, which coincides with
the two clinically relevant stages of the disease.
First, triatomine bugs become infected with the non-dividing infective trypomastigote form
of the parasite after biting an infected host. The trypomastigotes transform into epimastigotes in
the triatome bug’s midgut. The parasites then develop into metacyclic trypomastigotes which are
excreted through the bug’s feces onto the skin of a human host who will be self-infected after
scratching the skin at the site of the feces, allowing the parasites to enter the blood stream. Thus
begins the first clinical stage of the disease, an acute form where mild symptoms can be
experienced including redness and swelling at the infection site and mild fever.9
Parasites will then go on to invade the human host cells, where they transform into non-
infective amastigotes which multiply by binary fission. Amastigotes develop into trypomastigotes
that can go on to infect other human host cells. Being an intracellular infection is a key factor in
26
the difficulty of killing these parasites; a drug would need to overcome another barrier, the host
cell membrane. It is at this point that patients enter a chronic, asymptomatic form of this disease.
An estimated 70-80% of infected people will live asymptomatically for the rest of their life without
any detectable parasites in their blood stream. The remaining 20-30% of infected people will
experience heart rhythm abnormalities resulting in sudden death, or a dilated heart, esophagus, or
colon resulting in related complications.9
Figure 1-2. Life cycle of Trypansoma cruzi.
Source: Public Health Image Library, provided by CDC-DPDx, Alexander J. da Silva and
Melanie Mosier. http://phil.cdc.gov/phil/details.asp?pid=3384
1.1.2.2 Leishmaniasis
Leishmaniasis is another disease caused by a kinetoplastid containing trypanosomatid.10
There are 21 of 30 species of the Leishmania parasite that are human infective.13 The human
infective species include the L. donovani complex (L. donovani, L. infantum, L. chagasi), L.
27
mexicana complex (L. mexicana, L. amazonensis, L. venezuelensis), the subgenus Vianna (L. [V.]
brazielensis, L. [V.] guyanensis, L. [V.] panamensis, L. [V.] peruviana), and L. tropica, L. major,
and L. aethiopica.10 This disease can be found in 88 countries throughout the Americas and some
parts of Asia, putting about 350 million people at risk for infection.13
Figure 1-3. Life cycle of Leishmania.
Source: Public Health Image Library, provided by CDC-DPDx, Alexander J. da Silva and
Melanie Mosier. http://phil.cdc.gov/phil/details.asp?pid=3400
Infections begin after a person is bitten by an infected female phlebotomine sandfly. The
life cycle of Leishmania can be seen in Figure 1-3. The infective promastigote form of the parasite
becomes phagocytized by host cells where they transform into their dividing amastigote form. The
type of disease that results from infection is dependent on the host, and species of parasite. There
are two major forms of the disease cutaneous (CL) and visceral leishmaniasis (VL). Symptoms of
CL include skin lesions that resemble volcanoes, raised edges with craters in the middle, at the site
28
of the sandfly bite.14 VL symptoms include fever, weight loss, enlarged spleen, and low blood cell
counts, and eventually leads to death.13
1.1.2.3 Malaria
Malaria is another disease cause by a protozoan parasite and is the leading cause of illness
and death in the poor, tropical regions of the world.14 Though it is prevalent in tropical regions, it
is not deemed neglected by the WHO because of the large effort dedicated to combat this disease.
Malaria control efforts have greatly increased in the last decade, yet in 2016 an estimated 445,000
people died of malaria, the major victims being young children in sub-Saharan Africa.15 Young
children and pregnant women are at the highest risk of infection due to underdeveloped, and
decreased immunity to the disease, respectively.15
Figure 1-4. Life cycle of Plasmodium falciparum.
Source: Public Health Image Library, provided by CDC-DPDx, Alexander J. da Silva and
Melanie Mosier. http://phil.cdc.gov/phil/details.asp?pid=3405
29
Plasmodium falciparum, the most common species of the parasite, are transmitted to their
human hosts by the Anopheles mosquito.16 The life cycle for P. falciparum can be seen in Figure
1-4.16 Sporozoites are injected into the hosts blood stream causing flu-like symptoms, the other
stage to this disease is the infection of the liver where parasites can remain dormant for years and
can lead to relapse even after the parasites have been cleared from the blood stream. Depending
on the tissues and organs the parasites invade, severe malaria disease accompanied by more severe
symptoms can be observed. Other Plasmodium species include P. vivax, P. ovale, and P. malariae
which are the species that cause recurring malaria and can stay dormant in the liver, the most
predominantly spread being P. vivax.16
1.2 Current treatments for NTDs
There are no vaccines currently for any of the NTDs. These diseases are managed mainly
through vector control and chemotherapeutics.6, 17-20 Current treatments are limited mainly due to
toxic side effects for patients and grueling dosing regimens.
1.2.1 Current treatments for HAT
Treatments for HAT are specific to the stage the disease is in as well as the subspecies
causing the infection. There are two treatments for stage 1 of the disease (shown in Figure 1-5),
pentamidine and suramin, though the disease has low rates of diagnosis in stage 1 due to the
mildness of symptoms and inaccessibility to clinics. Pentamidine is only used to treat T. b.
gambiense infections and suramin is only effective against T. b. rhodesiense infections.
30
Figure 1-5. Current treatments for HAT.
Eflornithine, originally developed as an anticancer treatment, was a repurposed drug for
treating stage 2 T. b. gambiense infections and is far less toxic than the arsenic based drug,
melarsoprol. Eflornithine was originally used as an intravenous (IV) monotherapy,21 but showed
31
toxicity and evidence of resistance.22-23 With the approval of nifurtimox in 2009, NECT became
the first in class treatment for stage 2; the co-administration of nifurtimox with eflornithine reduces
the amount of eflornithine that needs to be injected into the patient for treatment by 50%.23
However, NECT still requires multiple doses making it logistically challenging to administer in
areas of sub-Saharan Africa. Its position as the first line treatment for stage 2 is expected to change
with the introduction of the oral drug fexinidazole (6), which was recently approved for use by the
European Medical Agency.24-25
Fexinidazole is taken orally once daily for 10 days, and, although somewhat less effective
than the first-line NECT (91.2% vs 97.6% effective, respectively),26-27 fexinidazole is an overall
improvement given that it is an oral drug with shorter treatment course. Another orally available
drug, acoziborole (7, Figure 1-6) is in Phase II/III clinicals trials for treatment of HAT.28
Figure 1-6. Drugs in clinical trials for HAT.
1.2.2 Current treatments for Chagas disease
There are currently only 2 drugs for the treatment of Chagas disease, nifurtimox (3) and
benznidazole (8) (Figure 1-7). Both drugs are orally bioavailable but are more effective on
parasites that have not yet invaded cells and thus are the first in line treatments for the acute phase
of the disease. These treatments are also used to treat the chronic phase (once T. cruzi has invaded
host cells) though they are not as effective and a more rigorous dosing regimen is needed.29-30
32
Benznidazole is more readily used as a treatment for Chagas disease because it has fewer side
effects than nifurtimox.31 Unfortunately, there have been signs of resistance from certain strains
of T. cruzi against both of these treatments.32
Figure 1-7. Current treatments for Chagas disease.
1.2.3 Current treatments for leishmaniasis
Multiple factors must be taken into consideration when treating leishmaniasis, making it
particularly difficult to treat. These factors include the type of infection, availability of the drug,
age of the patient, and whether the patient is pregnant or nursing.20, 33 The first in line treatments
for CL (and alternatives for VL) are injectable pentavalent antimonials pentostam (9) and
meglumine antimoniate (10) (Figure 1-8).18, 20 One of the most common side effects of these drugs
is a change in electrocardiograms of about 50% of patients.18, 34 The 3 other treatments for
leishmaniasis are amphotericin B (11), miltefosine (12), and paromomycin (13) that were
repurposed for their current indications (see 1.3.1 Repurposing chemical matter) (Figure 1-8).
Amphotericin B is a treatment that require intravenous dosing and has a high cost per treatment.18
Miltefosine, the first orally available treatment for leishmaniasis displays toxic side effects.18
Lastly, paromomycin (13) is administered by intramuscular injection to treat VL18 or can be
applied topically as a treatment for CL due to is poor absorption across the GI tract.35
33
Figure 1-8. Current treatments for leishmaniasis.
1.2.4 Current treatments for malaria
The current treatments used to treat uncomplicated malaria are combination therapies of
artemisinin (14) and other antimalarial drugs such as amodiaquine (15), lumefantrine (16),
mefloquine (17), and sulfadoxine(18)/pyrimethamine (19) (Figure 1-9).36-37 These artemisinin-
combination therapies (ACTs) are 90% effective in patients.37-38 Infections caused by P. vivax
34
require treatments to be effective in both blood and liver. P. vivax infections are mainly treated by
using ACT plus primaquine (20) to clear the liver stage parasites.37
One of the biggest challenges to treating malaria is the rapidly evolving strains of these
parasites.36 Partial resistance to artemisinins emerged in southeast Asia in the 2000s causing drug
resistant strains of the parasite.39-41 Chloroquine(21)-resistant P. falciparum has also spread to
most disease prevalent areas.41
Figure 1-9. Current treatments for malaria.
1.3 NTD drug development
The most cost and time efficient methods need to be utilized to develop new treatments for
NTDs. Repurposing chemical matter is one way to cut down on both cost and time. Compounds
are also cross-screened against the various aforementioned parasites due to the organisms’
35
biological similarities.42 Cross-screening can provide new starting points for compound
development against different pathogens, and is a form of repurposing.
1.3.1 Repurposing chemical matter
In classic drug development programs the time between bringing concept to production
takes on average 10-12 years and $1.8 billion.43 The length of stages (Figure 1-10) in the drug
discovery process can vary based on the project and challenges encountered. Late stage failures of
discovery programs can occur, and drug attrition rates are reportedly high.44 NTD drug
development is especially daunting because of the low return on investment when a drug goes to
market. These factors lower the incentive for NTD development by for-profit organizations.
Figure 1-10. The drug development pipeline and different entry points for repurposing
campaigns.
One way to enter the development pipeline later and with less money invested, is through
repurposing chemical matter and utilizing prior knowledge of developed drugs and drug targets.
Repurposing has become an established strategy in NTD drug discovery and is discussed below.
There are four different types of repurposing: drug, target, target-class, and lead repurposing.45 As
shown in Figure 1-10 repurposing allows for different entry points along the development process
ultimately abbreviating the timeline. The type of repurposing strategy utilized in a project depends
36
on the source of the chemical matter and the way in which compounds are optimized. The
categorization of repurposing strategies is summarized in Figure 1-11.
Figure 1-11. Categorization cascade of the different types of repurposing strategies.
The application of repurposing can be powerful due to the often, large knowledge base that
is already in the public domain and that can help illuminate a path along which the desired
selectivity and ADME profiles may be achieved. This can be helpful given the resource-limited
nature of NTD drug discovery programs. Specific examples of each of the repurposing strategies
are discussed herein.
1.3.1.1 Drug repurposing
The most direct and streamlined approach to repurposing is drug repurposing, as it
repositions an already approved drug for a new indication. Directly repurposed drugs do not
require any further chemical optimization and can be tested in Phase II clinical trials (Figure 1-
10). The understanding is that a repurposed approved drug has already been evaluated for safety
and bioavailability. Later stage clinical trials are used to assess the drugs ability to be effective for
37
the new indication. Drug repurposing has a proven track record of identifying successful
treatments for NTDs, including leishmaniasis and HAT, and remains an excellent strategy to
identify new therapies.
Some of the current therapies to treat NTDs have resulted from drug repurposing. For
example, originally developed as an anticancer treatment, eflornithine (4) has been repurposed for
treating Stage 2 of gambiense HAT infections. Amphotericin B (11), was isolated from
Streptomyces nodosus in 1955 and was originally used as an antifungal agent.46 Currently it is used
to treat leishmaniasis as described in Section 1.2.3. Miltefosine (12) is the first available oral
treatment for leishmaniasis and was originally developed as an anticancer drug, though it does
display toxic side effects.18 Lastly, another repurposed treatment for leishmaniasis, paromomycin
(13), was originally developed as an oral antibiotic to treat intestinal bacterial infections.18 Given
its poor absorption across the GI tract, it is administered by intramuscular injection to treat VL,18
or it can be applied topically as a treatment for CL.35
Other drug repurposing campaigns have been pursued in the effort to identify new
treatments for NTDs. One such campaign was pursued by Kaiser and colleagues, which assembled
100 approved drugs with target product profiles (TPP) that matched the requirements as outlined
by the DNDi including oral bioavailability, safety, and moderate price. The list of approved drugs
was also chosen based on their registered indications, mechanisms of action and chemical class.
The drugs were then screened against T. brucei, T. cruzi and L. donovani in parallel with current
treatments for the parasites.47 Two antibiotics, nifuroxazide (22) and nitrofurantoin (23) (Figure
1-12), had better EC50 values (EC50 = 0.03, 0.5 µM respectively) against T. b. rhodesiense than
nifurtimox (EC50 = 1.4 µM). The rheumatoid arthritis drug, auranofin (24) (Figure 1-12), was also
more potent against T. b. rhodesiense (EC50 = 0.01 µM), but when tested in a mouse model was
38
found to be inactive. The antifungal azoles were found to be a promising class of inhibitors against
T. cruzi with EC50 values ranging from 0.003 to 0.3 µM with selectivity index values >100 against
L6 rat myoblast cells. The antibacterial, clofazimine (25) (Figure 1-12), emerged as the only active
compound (EC50 = 0.95 µM) against intracellular L. donovani amastigotes with an acceptable 10-
fold selectivity against mouse peritoneal macrophages. Overall, the study identified several
interesting starting points for future repurposing ventures.47
Figure 1-12. FDA-approved drugs identified as active against T. b. rhodesiense.47
Another screen of FDA approved drugs was reviewed by Hernandez et. al. 48 This screen
utilized 1,148 compounds from the Selleckchem library of FDA-approved drugs and the National
Institutes of Health (NIH) Clinical Collection.49 Promising compounds ifenprodil (26), ziprasidone
(27), clemastine (28), clofibrate (29), and azelastine (30) (Figure 1-13) were more active than
nifurtimox when tested in vitro against T. cruzi. Although the compounds were potent and
bioavailable, the free blood concentrations caused toxicity issues normally associated with their
primary human target in the central nervous system. These compounds were also antagonist of the
39
H1 histamine receptor. Thus, although originally intended for direct repurposing, further
optimization of these drugs is required to mitigate toxicity.
Figure 1-13. FDA-approved drugs identified as active against T. cruzi and that could serve as
starting points for further optimization to mitigate toxicity.48-49
Previously, retinoic acid (31) and retinol acetate (32) (Figure 1-14) were shown to inhibit
L. donovani promastigote growth and drastically lower the amount of polyamines produced.50 T.
cruzi is unable to synthesize polyamines de novo making TcPAT12, a high-affinity spermidine
transporter51, essential to the survival of the parasite.52 With TcPAT12 as the intended target for
inhibition, a drug repurposing program was started using retinol acetate as the template for
desirable ligands. A structural similarity screen of 2,924 commercially available drugs was
performed53 and a virtual screen of these drugs vs. TcPAT12 identified potential inhibitors.
Compounds of interest were subsequently screened in vitro against T. cruzi. Compounds
structurally related to retinoic acid and retinol acetate were hypothesized to inhibit TcPAT12 which
40
was confirmed when isotretinoin (33) (an acne treatment; Figure 1-14) was shown to inhibit
TcPAT12 and kill T. cruzi trypomastigotes (EC50 = 130 nM).53
Figure 1-14. FDA-approved drugs shown to inhibit TcPAT12.50, 53
It has been shown that fungicidal compounds and compounds containing imidazoles,
pyrimidines, and triazoles (compounds 34-36, Figure 1-15) that inhibit lanosterol 14α-
demethylase (CYP51) can also inhibit similar biosynthetic pathways in kinetoplastids.54
Repurposing CYP51 antifungals for kinetoplastid diseases has been widely explored.54
Posaconazole (34) (Figure 1-15), one said antifungal, reached clinical trials for chronic Chagas
disease.55 Patients treated with 100 mg or 400 mg twice daily tested negative for T. cruzi DNA
during the treatment period (with the exception of two patients in the 100 mg/kg twice daily group).
Upon follow-up, however 92% and 81% of patients in the 100 mg/kg and 400 mg/kg twice daily
groups, respectively, tested positive for T. cruzi DNA compared to 38% of the benznidazole-
treated group.55 These results suggest that the TcCYP51 pathway is only essential for rapidly
dividing parasite and not for more dormant forms.56 The lack of clinical efficacy with CYP51
inhibitors has led to their deprioritization by the DNDi.56-57
41
Figure 1-15. Antifungal azoles repurposed for Chagas disease and used in combination with
DB766 to enhance killing of L. donovani.54, 58
Although antifungal azoles as monotherapies have been deprioritized, repurposing them in
combination therapies is prominent in leishmaniasis drug discovery, likely due to the current use
of the repurposed antifungal, amphotericin B. Combinations of the antifungal azoles, posaconazole
(34) and ketoconazole (35) with the arylimidamide, DB766 (36) (Figure 1-15), enhanced the
killing of L. donovani in vitro over the respective monotherapies.54, 58 The most promising
combination, which involved posaconazole, resulted in an 81% reduction of liver parasitemia in
L. donovani-infected mice over either the DB766 (40% reduction) or posaconazole (21%
reduction) monotherapies.59 Posaconazole has also been tested against T. b. brucei with an EC50
of 8.5 µM. Treating mice with a combination of posaconazole and eflornithine extended the
survival of infected mice over either monotherapy, and led to a comparable extension of life to
that of NECT.60
1.3.1.2 Target repurposing
The two factors used to categorize a program as target repurposing are the identification of
a validated parasite target and the need to re-optimize a hit compound. The parasite target also has
42
a homologous mammalian species target for which inhibitors have already been discovered. Using
these known inhibitors narrows the screening scope of chemical matter to be evaluated, speeding
hit identification.
A classic target repurposing program utilized chemical matter to target T. brucei glycogen
synthase kinase-3 (TbGSK3), an enzyme important to parasite development as demonstrated by
knockdown experiments.61 The homologous protein in humans is GSK3β and is implicated in
diabetes and neurodegenerative diseases.62 GSK3β has 52% amino acid identity to TbGSK3 and
was used to generate a homology model.63 A library consisting of kinase inhibitors was docked
into the homology model of TbGSK3 and the resulting hits were screened in vitro against the
enzyme. Hits from the in vitro screen were prioritized based on their ligand efficiencies leading to
the identification of diaminothiazole 37 (Figure 1-16) (EC50 = 2 µM).63 Hit-to-lead optimizations
were carried out on 37, however in vitro results revealed that potency against T. brucei no longer
tracked with TbGSK3 binding, suggesting that the series of compounds had a different molecular
target and that further optimization would need to be phenotypically driven.63
In an extension of this work on TbGSK3β,63 the DDU and GSK identified the hit compound
37 as an inhibitor of growth of L. donovani.64-65 New chemical scaffolds were synthesized and
evaluated, identifying pyrazolopyrimidine 38 that offered improved potency and metabolic
stability.65 Further optimization to improve the aqueous solubility resulted in
GSK3186899/DDD853651 (39, Figure 1-16) which, when dosed at 25 mg/kg orally twice a day,
demonstrated comparable activity to miltefosine and reduced parasitemia by 99%.64 This
compound is currently undergoing further preclinical development for VL.64
43
Figure 1-16. Progression of the diaminothiazole hit to GSK3186899/DDD853651.63-65
Another target repurposing program by Bland et al. focused on T. brucei phosphodiesterase
B1 and B2 (TbrPDEB1 and TbrPDEB2, respectively).66 It has been shown by knockdown of both
genes through RNA interference that together they are essential for parasite survival, but not
individually.67-68 Human PDE inhibitors are shown to be active against both parasite enzymes69,
and TbrPDEB1 and TbrPDEB2 have catalytic domains 30-35% similar in amino acid sequence
to human PDEs.70 A library of compounds with chemotypes known to bind human PDE was
screened against TbrPDEB1, and compounds that had EC50 values <20 µM were subsequently
screened against TbrPDEB2. A human PDE4 inhibitor, Piclamilast (40, Figure 1-17), was
moderately active (TbrPDEB1 EC50 = 4.7 μM, TbrPDEB2 EC50 = 11.4 μM). Analogs were
synthesized as part of a structure-based drug discovery effort and had EC50 values in the
micromolar range.66 Cilomilast (41), rolipram (42) and GSK-256066 (43) (Figure 1-17) were
pursued by Amata et al. 71-72 and Ochiana et al.73 Their optimizations efforts revealed the
challenging nature of decoupling parasite and human enzyme potencies and thus the series was
deprioritized.
44
Figure 1- 17. Human PDE inhibitors that entered medicinal chemistry optimization for T.
brucei.66, 71-73
In parallel, another group pursued TbrPDEB1 as a target for HAT and identified an hPDE4
hit series (typified by 44, NPD-001; Figure 1-18) after a high throughput screen (HTS) of 400,000
compounds.74 The chemical matter used for the HTS was not biased and thus this program is not
strictly speaking “target repurposing”. However, the subsequent medicinal chemistry campaigns
to optimize this series are consider as such and resulted in lead compounds 45 and 46.75-78
Figure 1-18. Hit to lead compounds resulting from target-guided SAR explorations of
TbrPDEB1.74-78
1.3.1.3 Target-class repurposing
The differentiating factor between target-class and target repurposing is whether a specific
human target has homology to a specific parasite target. In target-class repurposing a specific target
is unknown, but a family of targets that are similar in humans and parasites is used to bias the
chemical matter used in hit identification screening. Compounds shown to engage this shared
family of targets are considered. For instance, parasites express essential proteins to their survival,
45
such as histone deacetylases and kinases, for which their homologous human counterparts have
been widely studied. Chemical matter for the human target class is then screened in phenotypic
assays, evaluating for parasite death or growth inhibition. The chemical matter that is screened is
late-stage but requires further optimization for desired effects.
As stated above, histone deacetylases (HDACs) are proteins essential for transcription,
gene expression and the regulation of chromatin structure. 18 HDACs have been categorized into
classes based on genetic sequence similarities and cofactor dependence and have been implicated
as viable drug targets in many human diseases, including cancer,79 neurodegenerative,80 metabolic
and immunological diseases.81 Sirtuins, also known as class III HDACs, function in gene
expression, DNA repair and apoptosis. Kinetoplastid sirtuins have been shown to be essential to
parasite survival.81-84 A review by Hailu summarizes numerous HDAC inhibitors that have been
screened against T. brucei, T. cruzi, L. major and L. donovani.81 Vorinostat (47, Figure 1-19), a
human HDAC inhibitor, and some of its derivatives were screened against L. donovani and L.
infantum. All compounds that advanced to in vivo efficacy studies were unsuccessful.
Interestingly, when the compounds were absorbed by gold nanoparticles and retested, good in vivo
efficacy without toxicity was demonstrated.85
Figure 1-19. The HDAC inhibitor, vorinostat, shows promising in vivo efficacy against L.
donovani when absorbed by gold nanoparticles.81, 85
46
As previously described, trypanosomes express essential protein kinases and they account
for approximately 2% of their respective genomes.86-87 A genomic analysis of T. brucei, T. cruzi,
and L. major identified the presence of 176, 190 and 196 orthologous protein kinases,
respectively.86-87 In 2014, 10 FDA approved anti-cancer drugs, including protein kinase inhibitors
sunitinib (48, EC50 = 1.1 µM), sorafenib (49, EC50 = 3.7 µM) and lapatinib (50, EC50 = 2.5 µM)
(Figure 1-20), were evaluated for their activity against L. donovani amastigotes.88 Each showed a
similar bioactivity to those of the current antileishmanial drugs, amphotericin B (11) (EC50 = 0.02
µM) and miltefosine (12) (EC50 = 1.0 µM).88 Evaluation of the kinase inhibitors, depicted in
Figure 1-20, in a mouse model of Leishmania infection showed moderate reduction in the levels
of liver amastigotes (41%, 36% and 30% respectively). This was the first demonstration that
Leishmania survival in vivo could be challenged by protein kinase inhibitors.88 Sorafenib,
originally designed to target vascular endothelial growth factor receptors (VEGFR) and platelet-
derived growth factor receptors (PDGFR), was unique in that it showed activity across several
Leishmania strains. Although sunitinib, sorafenib and lapatinib were moderately effective in vitro
and in vivo, further optimizations are needed to improve their efficacy.
47
Figure 1-20. Anticancer drugs shown to moderately reduce parasitemia in the livers of mice
infected with L. donovani.88
Lapatinib, originally developed as an epidermal growth factor receptor (EGFR) inhibitor
for breast cancer, was also identified as a micromolar inhibitor of T. b. brucei growth.89 Subsequent
optimizations will be discussed in depth in Chapter 2. Structural modifications designed to
improve the solubility and in vitro clearance of the lapatinib-derived analogs led to NEU-4438
(51, Figure 1-21).90 Undetectable levels of parasitemia were observed in 3 out of 4 mice in an in
vivo efficacy study of 51. The compound was administered orally at 100 mg/kg for six days with
an additional 50 mg/kg given on day 6. However, after dosing was stopped parasitemia rebounded
on day 11. Compound 51 was also tested against a panel of 45 human kinases due to its origins as
a kinase inhibitor. Results showed 51 potently inhibited three kinases: insulin receptor tyrosine
kinase (IRK), lymphocyte-specific protein tyrosine kinase (Lck) and proto-oncogene tyrosine-
protein kinase (Src).90 An Ames test was also performed and 51 and its metabolites were negative
for genotoxicity. One major liability comes with its reactivity to human GPCRs and ion channels
(>50% inhibition at 10 µM). These included muscarinic M2 (85% inhibition), muscarinic M3
48
(52% inhibition), nicotinic acetylcholine α1 receptors (69% inhibition), human ether-a-go-go
(hERG, 94% inhibition), and the norepinephrine transporter (71% inhibition).90
Figure 1-21. Structure of lapatinib-derived analog NEU-4438.
A nitroaromatic structural class of compounds are well-known antiparasitic agents.91-92 The
most successful story for repurposing of a nitroaromatic is the first oral monotherapy for both
stages of T. b. gambiense infections, fexinidazole (6).24 Fexinidazole was first reported as being
effective against T. cruzi, trichomonads and Entamoeba histolytica in 1981,93 and T. brucei in
1983.94 A lack of market incentive caused the discontinuation of development of the drug.26 DNDi
rediscovered the drug in a compound mining campaign that used a targeted library of 700
nitroheterocycles in their screen for inhibition of various parasites.91 Fexinidazole has a good
overall safety profile though nitroaromatic compounds are often found to be toxic.
Another repurposed nitroheterocycle, delaminid (52, Figure 1-22), an approved treatment
for multi-drug resistant tuberculosis, was screened against L. donovani promastigotes and
amastigotes, and identified as a nanomolar inhibitor of growth.95 Delaminid showed similar
clinical efficacy to that of miltefosine when dosed at 30 mg/kg once daily for five days. Further in
vivo efficacy studies showed that decreasing the dosing regimen to 1 mg/kg twice daily increased
49
efficacy. However, because of the complicated pharmacokinetic/pharmacodynamic relationships
human dosing prediction are difficult. Other studies have shown that delaminid meets most of the
TPP criteria (except in cost per patient) and could be considered a good candidate for further
progression as an antileishmanial agent.95
Figure 1-22. Structures of delaminid and its optimized analogs.
Based on the potential of delaminid, Thompson et al. screened other anti-tubercular
nitroimidazoles against L. donovani.96-97 Subsequent optimization studies yielded a preclinical
candidate, 53 (Figure 1-22), which decreased parasitemia by 99% in a chronic leishmaniasis
hamster model.97 During the development of 53, Thompson et al. then sought a backup compound
with a similar scaffold, leading to the identification of 54.98
1.3.1.4 Lead repurposing
Lastly, lead repurposing takes gene-family-targeting (like target-class repurposing)
chemical matter to identify potent and selective hit compounds. The difference from target-class
repurposing is that the chemical matter consists of early stage compounds.
50
Our lab used a lead repurposing approach with the kinase family in mind, beginning with
a biased library of known human kinase inhibitors (and will be discussed in depth in Chapter 3).
This library was screened in collaboration with GlaxoSmithKline (GSK) to identify potential
inhibitors of T. brucei. Inhibitors of parasite growth by 50% in a single concentration assay were
progressed to dose response and selectivity assays.99 Prioritized compounds were then clustered
based on structural similarities.
Various medicinal chemistry hit-to-lead optimization programs have commenced since the
publication of the initial screening results and are in different stages of development.100-102 An
isatinoid-containing series from this screen was pursued by Klug et al. with a focus on improving
potency and aqueous solubility while maintaining its good physicochemical properties.100
Combining potency with a favorable absorption, distribution, metabolism, excretion (ADME)
profile proved challenging as revealed by SARs and structure-property relationships (SPRs).
NEU-4391 (55, Figure 1-23), was progressed to a mouse PK study, however, there was low
exposure of the compound in both the blood and brain. Based on these data, the series was not
worth further pursuit.100
Figure 1-23. Isatinoid compound NEU-4391 identified from a lead repurposing HTS campaign.
51
A similar lead repurposing campaign was pursued by DDU. A kinase inhibitor library was
screened against T. b. rhodesiense identifying 121 compounds that inhibited parasite growth by
50% at 5 µM.103 These hits were subsequently screened in a T. b. brucei proliferation assay to
determine EC50 values. The most active compound 1H-Imidazo[4,5-b]pyrazin2(3H)-one (56,
Figure 1-24, EC50 = 80 nM) had good selectivity over the MRC-5 host cell line (EC50 >50 µM).
Swapping the 1H-imidazo[4,5-b]pyrazin2(3H)-one core, which is associated with static parasite
growth, for a 1H-pyrazolo[3,4-b]pyridine core, seen in compound 57 (Figure 1-24), yielded cidal
compounds. Compound 57 was progressed to an in vivo PK study due to its favorable profile for
potency (EC50 = 0.09 µM), solubility (> 250 µM), metabolic stability (3.6 mL*min-1g-1) and
plasma protein binding (89% bound). However, the molecule had low exposure levels (Cmax = 580
ng*mL-1) and was rapidly cleared (t1/2 = 0.7 h). Thus, further work on this compound series is
required.
Figure 1-24. Most active compound (56) from a kinase library screen and optimized compound
(57) for T. brucei growth inhibition.
In summary, numerous repurposing campaigns have been employed in the effort to find
new therapeutics to treat HAT, Chagas disease and leishmaniasis. Each of the four types of
repurposing strategies has key differences, including whether a specific target is known or
whether optimization of a hit compound is required. Whilst direct drug repurposing is the most
52
efficient repurposing strategy, the most recent example, fexinidazole, came from a target-class
repurposing approach. Fexinidazole stands as an excellent example of the value of repurposing
chemical matter for urgently-needed kinetoplastid drugs.
1.4 Compound optimization
After identifying a hit compound from a repurposing campaign (except direct drug
repurposing) compounds need to be further optimized to eventually become drugs. Not only are
they optimized for potency, but they must have optimal ADME properties in order to become
orally dosed drugs. Recently, development programs have been focused on optimizing compound
properties using different tools as discussed below.
In the 1990s, combinatorial chemistry was widely used to synthesize large libraries of
compounds. These combinatorial libraries allowed for a larger chemical space to be explored,
however the space for clinically relevant drugs were in small clusters. Thus, combinatorial libraries
led to compound with poor ADME properties.104 To filter these large libraries, Lipinski analyzed
the effect of various compound properties on solubility and permeability for published clinical
candidates. This analysis led to a set of “rules” that would help predict drug-likeness.104-105 These
rules stated that a given compound was more likely to be soluble and permeable, and therefore
druglike, if it had fewer than 5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular
weight less than 500 g/mol, and a calculated Log P (cLogP) less than 5.105
Veber et. al. performed a similar analysis to Lipinski utilizing the orally bioavailable
compounds at what is now GSK.106 This analysis took into account the number of rotatable bonds,
total number of hydrogen bonding atoms, and polar surface area as good predictors of whether a
compound would be effectively orally bioavailable.106 The criteria stated that a compound with 10
53
or fewer rotatable bonds and a polar surface area less than 140 Å would have a high probability of
being orally bioavailable in rats.106
In developing a drug, high potency is not the be all and end all for compound progression.
Rather good compounds are ones that make favorable interactions with a target. Calculated
properties for the efficiency of the binding for a compound can help assess hit and early lead
compounds when making go/no-go decisions. Ligand efficiency (LE) characterizes the efficiency
of a compound based on the number of heavy atoms it contains (LE = 1.37×pEC50/# of heavy
atoms).107 The LE essentially helps normalize potency for molecular size. An LE value greater
than 0.3 is a good starting point for optimization efforts. Lipophilic ligand efficiency (LLE) is
another of these calculations which determines a compound’s efficiency based on the lipophilicity
of the molecule (LLE = pEC50-cLogP).108-109 The LLE predicts whether a compound is potent
because it is making a favorable interaction with the target of interest, or it is greasy and interacting
with the intended target to allow it’s escape from water.
Structure-property optimizations are equally as important as structure-activity
optimizations. Compounds that do not reach the intended target due to poor ADME, but still make
specific, favorable interactions are irrelevant regardless of high potency. Table 1-2 shows the
desired properties of compounds pursued as part of discovery and development efforts in the
Pollastri lab.
54
Table 1-2. Targeted compound criteria for hit and lead compounds.
Assay Hit criteria Lead criteria
Potency pEC50 T.b. brucei > 7 > 7.5
T.b. brucei LLE ≥ 4
pEC50 T. cruzi > 5.3 > 6.5
T. cruzi LLE ≥ 4
pEC50 Leish spp > 5 > 6
Leish LLE ≥ 4
pEC50 P. fal > 7 > 7.5
P. fal LLE ≥ 4
Toxicity HepG2 TC50
MRC5 TC50
10 × EC50
10 × EC50
10 × EC50
10 × EC50
ADME &
Phys.
Properties
MW
cLogP
LogD
RH Clint
HLM Clint
Aq Sol (μM)
PPB (%)
≤ 360
≤ 3
≤ 2
≤ 27
≤ 47
> 10
≤ 95
≤ 360
≤ 3
≤ 2
≤ 5
≤ 9
> 100
≤ 95
PK Free plasma
concentration >
10xEC50 for given
parasite
Free plasma
concentration >
EC99 for given
parasite
Efficacy T.b. brucei,
T. cruzi,
Leish spp.,
P. fal
Reduction,
control, or
elimination of
parasitemia
following oral or
IP dosing at
levels that
provide sufficient
exposure (above)
Control, or
elimination of
parasitemia
following oral or
IP dosing at
levels that
provide
sufficient
exposure (above) MW = molecular weight (g/mol); RH Clint = rat hepatocyte clearance rate (μL/min/106 cells); HLM Clint = human liver
microsomal clearance rate (μL/min/mg of protein); PPB = plasma protein binding.
55
Chapter 2: Property optimization of lapatinib derived analogs for human African
trypanosomiasis (HAT) drug discovery
2.1 Introduction
2.1.1 Repurposing lapatinib for Trypanosoma brucei inhibition
T. brucei cells are known to express essential protein kinases. Previous studies have shown
that the parasite growth factor, transferrin, is acquired from host cells through receptor-mediated
endocytosis, and the EGFR inhibitor lapatinib, shown in Figure 2-1 blocks this endocytosis from
occurring.110-111 As a part of a target-class repurposing approach to streamline the drug discovery
process, lapatinib was screened against T. brucei brucei Lister 427112 resulting in a potency in the
low micromolar range (pEC50 = 5.84) and 4-fold selectivity over human HepG2 cells (pTC50 =
5.21).112 With lapatinib as the initial hit molecule, optimizations were then made to improve the
overall potency of the molecule and selectivity for parasite inhibition versus host cells. The
summary of these optimizations can be seen in Figure 2-1.
Chemical modifications were first made to the left-hand side of the molecule, otherwise
referred to as the “tail”. A broad diversity of tail groups was explored, leading to the identification
of NEU-369, a compound equipotent to lapatinib (pEC50 = 5.86) with a larger selectivity over
HepG2 cells (TC50 < 4.82). Head group (shown in green, Figure 2-1) modifications were made
exploring various substitutions on the benzyl group. All head group modifications showed similar
potencies to that of lapatinib, including removal of the benzyl group (NEU-555, Figure 2-1, pEC50
= 6.08, pTC50 < 4.82). Another set of tail group substitutions led to the most potent compound,
NEU-617 (pEC50 = 7.38, pTC50 < 4.70).112 NEU-617 was evaluated in a mouse pharmacokinetic
model, after a single oral 40 mg/kg dose of NEU-617, plasma concentrations of the drug exceeded
56
the EC50 by approximately 4-fold for 10 hours.112 This led to the evaluation of NEU-617 in a mouse
efficacy model as a proof of concept for the further exploration of this chemotype.
Figure 2-1. Optimizations of EGFR inhibitor lapatinib to lead compound NEU-617.
57
The efficacy study of NEU-617 was performed in mice that were infected with 104 T. b.
brucei cells.112 A dose of 40 mg/kg/day was administered orally or intraperitoneally 24 hr after
infection with T. b. brucei.112 Parasitemia was undetectable for 3 days in infected mice, though
drug-related toxicity was observed.112
With NEU-617 in hand, an exploration of the core region of the molecule was undertaken
to determine the importance of nitrogen atom positioning as part of a multi-parasite cross screening
campaign.113 Quinazoline core replacements included quinoline, isoquinoline, cinnoline,
phthalazine, 3-cyanoquinoline, and two thienopyrimidine isomers. Switching from the quinazoline
core to the quinoline resulted in NEU-1045 (Figure 2-1). NEU-1045 had improved potency over
lapatinib (pEC50 = 6.43) with 70-fold selectivity over HepG2 cells. The position of attachment for
the tail group was explored as well as various tail groups as seen in NEU-961 (pEC50 = 7.10, pTC50
< 5.39) (Figure 2-1), which was one of the most potent compounds to date. Perhaps not
unexpectedly based on the analogs’ high molecular weights and lipophilicities, the
physicochemical properties were poor, such as aqueous solubility. These results suggested
improving the pharmacokinetic properties of the compound to be the next step in the hit
development of this series.
2.1.2 Identification of NEU-1912 and NEU-1953 as leads for drug
development
The next round of analog design and synthesis focused on improving the “drug-like”
properties104 and lead quality of the compounds. Lead quality is assessed by calculating lipophilic
ligand efficiencies (LLE = pEC50 - cLogP).114 It was hypothesized that truncation of the large,
lipophilic head group and replacement with small polar heterocycles would lead to an
improvement in the overall ADME profile.115 All compounds synthesized had improved calculated
58
physicochemical properties as compared to parent compound NEU-1045.115 A summary of
modifications to NEU-1045 can be seen in Figure 2-2. From this set of compounds NEU-1912
(Figure 2-2) was synthesized and evaluated. Table 2-1 compares NEU-1045 to analog NEU-
1912.
Figure 2-2. Summary of modifications made to NEU-1045.
Table 2-1. Comparison of NEU-1045 to NEU-1912 and NEU-961 to NEU-1953.
Compound
Hit
Targeted
Values
NEU-1045 NEU-1912 NEU-961 NEU-1953
Tbb pEC50 > 7 6.43 7.70 7.10 6.37
Molecular weight
(g/mol) ≤ 360 569.6 447.5 617.1 398.5
cLogP ≤ 3 5.76 2.72 6.43 2.09
logD (pH 7.4) ≤ 2 5.5 3.3 5.0 1.8
Human plasma
protein binding
(PPB) (%)
≤ 95 100 96 99 88
Aqueous
Solubility (μM) > 10 2 1.4 0.3 44
LLE (pEC50 –
cLogP) ≥ 4 0.67 4.98 0.67 4.28
With improved properties as compared to NEU-1045, NEU-1912 was tested in a mouse
pharmacokinetic study. Female BALB/c mice were dosed intraperitoneally at 10 mg/kg (n=18).
NEU-1912 showed sufficient plasma exposure (>120×EC50 for 4 h) but low brain exposure
59
(brain:plasma = 0.1 at 0.25 hr after dosing). The compound also displayed rapid clearance in
human liver microsomes (HLM) at 207.5 μL/min/mg protein and rat hepatocytes (RH) at 31.5
μL/min/106 cells. Although the properties were improved with the identification of NEU-1912,
ADME optimization was still needed.
Figure 2-3. Modifications to NEU-961 resulting in NEU-1953.
Similar modifications to NEU-961 (Figure 2-3) were made to improve physicochemical
properties of the 7-subsituted quinoline series.116-117 Analogs were synthesized with head group
truncations using small, polar, heterocyclic head groups. Tail group modifications were also made
and crossover compounds combining the most promising head and tail groups were synthesized.
A summary of the modifications made to NEU-961 is depicted in Figure 2-3. Compound NEU-
60
1953116-117 (Figure 2-3) was a result of these modifications. Table 2-1 shows a comparison
between NEU-961 and crossover analog NEU-1953. Although NEU-1953 was a sub-micromolar
inhibitor of T. b. brucei proliferation (pEC50 = 6.37), had an improved LLE as compared to NEU-
961, and was sufficiently water soluble (44 μM), the clearance rates for HLM and RH were still
high (180 μL/min/mg protein and 130 μL/min/106 cells respectively).
NEU-1953 was progressed into a mouse efficacy study. After infection with T. b. brucei,
mice were dosed orally at 60 mg/kg for three days and then at 70 mg/kg for the remaining 3 days
of the study since there was no observable toxicity. Infected control mice were infected and dosed
orally with vehicle only. However, there was no significant difference in the levels of parasitemia
in the treated versus control mice.
The next step in the development process would be to further improve the absorption,
distribution, metabolism, and excretion (ADME) properties of the two advanced hit compounds
NEU-1912 and NEU-1953 to improve the pharmacokinetic properties and in vivo efficacy. It was
originally hypothesized that the lapatinib analogs had poor solubility because they were highly
lipophilic. Truncation of the large, lipophilic head group of lapatinib should therefore have
increased the aqueous solubility. However, an analysis comparing the clogP values versus aqueous
solubilities (Figure 2-4) of the lapatinib analogs synthesized up to this point showed that there
was no correlation between low lipophilicity and increased aqueous solubility.
61
Figure 2-4. Comparison of lipophilicity versus aqueous solubility in first and second generation
lapatinib analogs.
Since the lack of solubility was not due to lipophilicity, a new hypothesis was required:
due to the numerous aromatic rings in the analogs, the compounds would most likely have a flat
3-dimensional shape and multiple hydrogen bond donors/acceptors, allowing the compounds to
stack on top of one another. Solvation by breaking up this crystal packing would be energetically
unfavorable and therefore solubility would be low.118 There are various ways to address low
compound solubility, which are outlined below.
2.1.3 Methods for improving aqueous solubility
In the mid-1990s, the Biopharmaceutical Classification System (BCS) was introduced to
classify drugs based on their aqueous solubility and permeability rates.119 There are four classes
of compounds: class I (soluble, permeable), class II (poorly soluble, permeable), class III (soluble,
poorly permeable), and class IV (poorly soluble, poorly permeable). Improving the solubility of
62
compounds in classes II and IV can help improve oral bioavailability.120 Numerous drug
development failures have been due to poor solubility of drug candidates.121
Drug solubility can be broken down into 3 parts. First, intermolecular forces between
compound molecules must be broken. Second, a void must be formed in the solvent produced by
the breaking of solvent-solvent intramolecular forces. Lastly, the solute must enter the void made
in the solvent. With these steps considered there are two determinants of drug solubility, the energy
needed to break intermolecular forces of the solid being dissolved and the solvation energy.118
Poor aqueous solubility of chemical matter is a consistent problem in the drug discovery
process. Almost 40% of marketed drugs and 75% of drugs in the discovery pipeline have low water
solubility.122-123 The trend towards production of insoluble compounds has caused the
pharmaceutical industry to identify different strategies to circumvent their solubility woes.
Medicinal chemistry strategies used to chemically modify compounds to increase the aqueous
solubility include increasing charge and polarity,124-125 reducing planarity,126-127 or reducing
aromatic ring content.128-129 Two of these strategies were employed.
2.1.3.1 Salt formation
Forming salts of compounds is one of the most common strategies to increase their aqueous
solubility.125 Compounds that are ionizable can be paired with suitable counter ions and potentially
raise their dissolution rates and oral absorption.123 It was demonstrated in the 1950s that the free
acid form of weakly acidic compounds had slower dissolution rates than that of their associated
salts.130-131 One example of using a salt to increase aqueous solubility is that of delveridine. The
initial solubility of the free base of delveridine was 143 μg/mL, however, the mesylate salt of
delveridine was 320 mg/mL, a 2000-fold increase.132 Salt forms are mostly identified and selected
63
through a trial and error process, and using them to enhance aqueous solubility has its advantages
and disadvantages.124, 133
The screening of potential counter ions for salt formation can be performed in a high-
throughput manner where the drug is dissolved in different solvents and precipitated from solvent
by the addition of various counter ions.134-135 This makes salt formation a simple way to drastically
increase solubility without making chemical modifications. However, it has been shown that solids
formed after counter ion screening may not necessarily be salt forms, yet just precipitated parent
compound or counter ion forming agent.125 Partial salt forms can be produced under some
conditions resulting in salts co-crystallized with free base forms of a molecule thus affecting
dissolution rates.136 A potentially useful salt form would need to be confirmed by remaking the hit
on a larger scale and performing a thorough analysis by x-ray diffraction and crystallography.
Remaking a salt form could be made complicated by partial salts and co-crystals.
2.1.3.2 Increasing sp3 hybridized carbon content
Molecules with a planar shape have an increased probability of tightly crystal packing
together and crystal packing has been suggested to increase a compound’s melting point.126-127 It
has been shown that increasing the fraction of sp3 hybridized carbons can decrease the melting
points of compounds and improve solubility.126 Lovering et. al. analyzed the molecular weights
fraction of sp3 hybridized carbon atoms (Fsp3 = number of sp3 hybridized carbon atoms/total
number of carbon atoms) for all compounds published after 1980 from all phases of the discovery
process.126 Their results showed that compounds that reached later stages of the development
process showed higher Fsp3 content.126
Ishikawa and Hashimoto analyzed different examples of reducing molecular planarity that
led to the increase in solubility.127 One such example included clinical candidate, AMG-517
64
(Figure 2-5), produced at Amgen which showed poor thermodynamic aqueous solubility (<1
μg/mL in PBS).137 Chemical modifications to the terminal phenyl ring resulted in the compound
AMG-517b which showed a 13-fold improvement in the thermodynamic aqueous solubility (13
μg/mL in PBS).137
Figure 2-5. Clinical candidate AMG-517 and its analog with improved aqueous solubility.
A study performed by Ritchie et. al. analyzed 280 compounds from the discovery pipeline
for the effect that aromatic ring count had on developability and physicochemical properties.128
The analysis showed that as compounds progressed through the pipeline the number of aromatic
rings decreased and the average number of aromatic rings in oral drugs is 1.6. Approximately
31,000 compounds in the GSK database were used in analyzing the relationship between aqueous
solubility and aromatic ring count.128 It was concluded that solubility decreases dramatically with
the increase in the number of aromatic rings, specifically over a total amount of 2 aromatic rings.
In a follow-up study it was shown that replacement of aromatic rings with heteroaliphatic rings
were beneficial for aqueous solubility.129
We have applied both salt screening and Fsp3 modulation strategies to the problem of
solubility as it applies to NEU-1912 and NEU-1953 described in the sections following.
65
2.2 Salt screens
2.2.1 Salt formations of NEU-1953
The first step of the salt screen began with the qualitative analysis of NEU-1953’s
solubility in various organic solvents (scaled up NEU-1953 provided by Dr. Baljinder Singh)
(Table 2-2). A suitable solvent was identified as one that could dissolve the compound eventually
but not too readily. Approximately 2 mg of NEU-1953 were added to 7 different vials. Seven
different solvents were added to the vials in increments of 200 μL until the compound was
completely dissolved. Solutions that reached 2.5 mL in volume were heated and sonicated; such
was the case for isopropanol, tetrahydrofuran, acetonitrile, ethyl acetate, and water. However, for
these solvents, the compound did not completely dissolve. Both methanol and ethanol successfully
dissolved NEU-1953 with less than 1 mL of solvent. Ethanol was ultimately chosen for the salt
screen of NEU-1953 as the larger volume needed would be better for measuring and handling
when performing the salt screen. Less solid compound would be needed to saturate the solution
and thus require fewer equivalents of acid to be added for a salt form to precipitate.
Table 2-2. Determination of approximate solubility of NEU-1953 in various organic solvents.
Vial Solvent Amount of
NEU-1953
Volume of solvent needed to
dissolve NEU-1953
completely (μL)
1 Methanol 2.0 mg 500
2 Ethanol 2.2 mg 800
3 Isopropanol 2.1 mg 2500
4 Tetrahydrofuran 1.9 mg 2500
5 Acetonitrile 2.5 mg > 2500
6 Ethyl Acetate 1.9 mg > 2500
7 Water 2.5 mg > 2500
66
For the salt formations of NEU-1953, 20 mg of compound was suspended in 4 mL of
ethanol and heated until fully dissolved. Various acids (1 equivalent of 1M acid in ethanol) were
then added to the solution of NEU-1953. If a precipitate formed, it was then collected by vacuum
filtration and washed with ethanol.
2.2.1.1 Evaluation of NEU-1953 salt forms
A common strategy used to increase the kinetic aqueous solubility of a series is through
the formation of salts at acidic or basic centers on compounds.21, 22 A range of salts of NEU-1953
were studied (Table 2-3), taking advantage of the basic nitrogen atoms within the compound. The
hydrochloride (2.1) and sulfate (2.2) salt forms remained unchanged in potency and both
thermodynamic and kinetic solubility, while the citrate salt (2.3) showed a modest increase in
solubility, both thermodynamic (25%) and kinetic. The methanesulfonate salt (2.4) demonstrated
a 4-fold increase in the kinetic solubility which could be the reason for the slight increase in
potency.
Table 2-3. Salts formed of NEU-1953 compared to the free base.
Compound
Salt form (1953) Salt
ratio
T.b.b.
pEC50
(µM)
Thermodynamic
solubility (µM)
Kinetic
solubility
(µM)
HLM Clint
(µL/min/mg)
NEU-1953 Free base - 6.4 44 25 180
2.1 Hydrochloride 4:1 6.5 42 25 160
2.2 Sulfate 1:1 6.3 39 25 170
2.3 Citrate 2:1 6.3 61 50 190
2.4 Methanesulfonate 2:1 6.7 16 100 170
67
2.2.2 Salt screen of NEU-1912
2.2.2.1 Synthesis of NEU-1912
The synthesis of 6-bromoquinolone was carried out via the Gould-Jacobs sequence
following previously reported protocols.115 4-Bromoaniline was coupled through conjugate
addition with ethoxymethylenemalonate to provide diester 2.5. Cyclization was achieved in
refluxing diphenyl ether to generate ester 2.6, followed by hydrolysis, and decarboxylation to give
quinolinone 2.8.
Scheme 2-1. Synthesis of 6-bromoquinolone core.115
Reagents and conditions: a) diethyl 2-(ethoxymethylene)malonate, 100 °C, 2.5 hr (90%); b)
diphenyl ether, reflux 2 hr (91%); c) 2.5 M sodium hydroxide, reflux, 3 hr (97%); d) diphenyl
ether, reflux, 3 hr (97%).
The synthesis of 4-(morpholinosulfonyl)phenyl tail group began first with reaction of 4-
bromophenylsulfonyl chloride with morpholine to give bromide 2.9. The boronic ester 2.10 was
generated via a Miyaura coupling with bis(pinacolato)diboron.
68
Scheme 2-2. Synthetic route for NEU-1912 tail group.
Reagents and conditions: a) morpholine, THF, rt, 12 hr (82%); b) B2pin2, KOAc,
PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 85 °C, 3 hr (assumed quantitative).
With core (2.8) and tail (2.10) groups in hand, the two were coupled using Suzuki
conditions to give 4-((morpholinosulfonyl)phenyl)quinolone 2.11. Deoxychlorination in neat
phosphorus oxychloride provided the chloroquinoline 2.12. Finally, 4-aminopyrimidine was
attached to the core using Buchwald-Hartwig cross coupling conditions, resulting in the final
compound NEU-1912 (2.13).
Scheme 2-3. Synthesis of NEU-1912.
Reagents and conditions: a) (4-(morpholinosulfonyl)phenyl)boronic acid pinacol ester, 6-
bromoquinolin-4(1H)-one, TEA, Pd(OAc)2, 85 °C, 12 h (83%); b) Phosphorus oxychloride, 106
°C, 2 h, (70%); c) pyrimidine-4-amine, tBuOK, xantphos, Pd2(dba)3, 101°C, 24 h (92%).
69
2.2.2.2 Salt formations of NEU-1912
The first step of the salt screen began with the scale up synthesis of NEU-1912 as discussed
in section 2.2.1. A solvent screen similar to the one done for NEU-1953 was performed and
isopropanol was determined to be a suitable solvent for the salt formation of NEU-1912.
Experiments (shown in Table 2-4) that resulted in the precipitation of the salts from the solution,
were deemed successful. Salts were then collected by vacuum filtration and washed with
isopropanol.
Table 2-4. Conditions for salt formations of NEU-1912.
Entry
Mass of
NEU-1912
(mg)
Volume of
isopropanol
(mL)
Acid
(1 eq.) Observation
1 2.3 1.6 Sulfuric ppt
2 2.1 1.46 Hydrochloric crystals after
4 days
3 2.1 1.46 Phosphoric ppt
4 2.4 1.67 Methane
sulfonic acid ppt
5 1.9 1.32 p-toluene
sulfonic acid ppt
6 2.3 1.6 Citric acid No ppt
7 2.0 1.39 Acetic Acid No ppt
8 2.2 1.53 Formic Acid No ppt
2.2.2.3 Evaluation of NEU-1912 salt forms
Various salts forms of NEU-1912 were studied as compared to the free base form of the
compound. In this case, only the thermodynamic solubility was tested. Thermodynamic solubilities
70
were virtually unchanged for all of the salt forms. The T. brucei activities of the salt forms also
remained unchanged.
Table 2-5. Salts formed of NEU-1912 compared to the free base.
Compound
Salt form (1912) Salt
ratio
T.b.b.
pEC50
(µM)
Thermodynamic
solubility (µM)
2.13 Free base - 7.6 1.4
2.14 Phosphate 1:1 7.7 2.0
2.15 Sulfate 1:1 7.6 6.0
2.16 p-toluenesulfonate 1:1 7.6 2.0
2.17 Methanesulfonate 1:1 7.6 3.0
2.3 Increasing Fsp3: Design, synthesis, and evaluation of NEU-1912 and NEU-
1953 matched pairs with saturated head groups
2.3.1 Selection of saturated primary amines as replacements for aromatic
head groups of NEU-1912 and NEU-1953
The second strategy pursued to increase the solubilities of NEU-1953 and NEU-1912 was
to increase the sp3 hybridized atom count and thus reduce the number of aromatic rings in the
structure. The region of focus for modification was the “head-group” since previous SAR studies
showed that changes to this region were highly tolerated in terms of potency. A virtual library of
analogs was enumerated by using JChem reactor (ChemAxon Inc.) and a set of aliphatic, cyclic
primary amines that were available in pre-weighed quantities (Frontier Scientific). Using Vortex
(Dotmatics Inc.) the library was shaped on the basis of clogP (<5) and molecular weight (<500
g/mol). Final compounds containing 6 and 5-membered rings with one heteroatom were selected
from the remaining compounds for synthesis.
71
2.3.2 Synthesis of saturated head group replacements, NEU-1953 scaffold
In an attempt to diversify analogs in the final step of the synthesis, building up the
chlorinated core + tail groups (2.21) was the main priority. First the tail group was synthesized
starting with 5-bromo-2-chloropyrimidine via SNAr displacement of the chlorine with 1-
methylpiperazine (Scheme 2-4). The resulting bromide was then borylated using Miyaura
conditions.
Scheme 2-4. Synthetic route for NEU-1953 tail group.
Reagents and conditions: a) N-methylpiperazine, DIPEA, t-butanol, 90 °C, 12h (%); B2pin2,
KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145 °C, microwave, 1 h (assumed quantitative).
The borylated tail group was subsequently coupled with 7-bromoquinolone (synthesized
and provided by Dr. Baljinder Singh) to give quinolone 2.20 (Scheme 2-5). The core + tail scaffold
was then chlorinated by deoxy-chlorination with neat phosphorus oxychloride.
72
Scheme 2-5. Saturated head group matched pairs to NEU-1953 template synthesis.
Reagents and conditions: a) 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester,
Cs2CO3, Pd(PPh3)4, 3:1 dioxane/H2O, 130 °C μwave, 15 min (96%), or PdCl2(dppf)·CH2Cl2, 90
°C, 12 hr (85%); b) Phosphorus oxychloride, 106 °C, 3 hr (71%); c) 1-methylpiperidin-4-amine or
2-(pyrrolidin-1-yl)ethan-1-amine, Pd2(dba)3, XPhos, KOtBu, 90 °C, 18 hr (47%, 59%
respectively) d) various SNAr conditions (no isolated products).
With desired chlorinated core + tail in hand, we attempted to synthesize final compounds
with saturated head groups determined by the virtual library as previously stated. It has been
previously shown that Buchwald-Hartwig aminations utilizing various ligands and catalysts have
been successful for coupling aliphatic primary amines to aromatic heterocycles.138-142 The results
from attempts to apply Buchwald conditions with aliphatic cyclic amines are shown in Table 2-6.
Although the use of these conditions was successful at converting starting material to product,
hydrolysis of the starting material and dehalogenation were common side products. The production
of these side products, coupled with the high polarity of the final products, made purification of
these compounds by both normal and reverse phase difficult.
73
The most successful set of conditions was the use of XPhos as a ligand with heating at 90
°C overnight. These conditions were successful at converting enough starting material to product
for two of the desired amine substitutions. However, these conditions were not successful for other
amines because of the similarity in polarities of the de-halogenated starting material and products
for that of cyclohexylamine and 4-aminotetrohydropyran. The products and side products were not
separable by column chromatography and therefore other routes to final compounds were
explored.
Table 2-6. Synthetic attempts via Buchwald-Hartwig cross coupling using NEU-1953
chlorinated core + tail for final compound synthesis.
Entry Amine Ligand Catalyst Conditions Conversion1
1
Xantphos Pd2(dba)3
tBuOK,
anhydrous 1,4-
dioxane, 130
°C, 1 h
70%*
2
Xantphos Pd2(dba)3
tBuOK, 1,4-
dioxane, 150
°C, 1 h
65%*
3
Xantphos Pd2(dba)3 tBuOK, tbuOH,
90 °C, 12 h 60%*
4
tbuXPhos Pd2(dba)3
tBuOK, 1,4-
dioxane, 90 °C,
12 h
60%*
5
(2.22a)
XPhos Pd2(dba)3
tBuOK, 1,4-
dioxane, 90 °C,
12 h
60%
(47% isolated)
6
(2.22b) XPhos Pd2(dba)3
tBuOK, 1,4-
dioxane, 85 °C,
12 h
80%
(59% isolated)
7
XPhos Pd2(dba)3
tBuOK, 1,4-
dioxane, 85 °C,
12 h
35%*
8
XPhos Pd2(dba)3
tBuOK, 1,4-
dioxane, 90 °C,
12 h
55%*
1 Monitored by LCMS, conversion based on integration of UV peaks at 254 nm. *0% isolated yield.
74
Since the side products from the Buchwald coupling were difficult to separate from the
products, SNAr conditions were also explored on the core + tail scaffold. Various conditions were
used and are shown in Table 2-7. Conventional heating and microwave irradiation, up to 200 °C,
were both used but only trace amounts of product were observed. A variety of solvents were also
tried, but again there was no significant conversion for any of the solvents tried.
Table 2-7. Synthetic attempts via SNAr conditions using NEU-1953 chlorinated core + tail for
final compound synthesis.
Entry Amine Solvent Conditions Conversion1
1
IPA DIPEA, 90 °C, 48 h 0%
2
IPA DIPEA, 90 °C, 48 h 0%
3
tBuOH DIPEA, μwave 150 °C, 2 h trace
4
nBuOH DIPEA, μwave 180 °C, 1 h 0%
5
NMP DIPEA, μwave 200 °C, 1 h trace
1Monitored by LCMS, conversion based on integration of UV peaks at 254 nm.
With many failed attempts at synthesizing the NEU-1953 analogs with a generalizable
synthetic scheme, a new synthetic route was established and shown in Scheme 2-6. Starting with
7-bromoquinolone, the dihalogenated quinoline 2.23 was synthesized via deoxy-halogenation.
SNAr conditions were then used to selectively add various saturated primary amines at the 4-
position of the quinoline. Bromide intermediates 2.24a-2.24f were subjected to Suzuki conditions
using boronic ester 2.19 as the coupling partner to give final compounds 2.25a-f.
75
Scheme 2-6. Synthetic attempts via SNAr conditions using NEU-1953 7-bromo-4-
chloroquinoline core for intermediate to final compounds.
Reagents and conditions: a) POCl3, reflux, 18 hrs (81%); b) saturated primary amine, DIPEA, n-
butanol, 200 °C, microwave, 3 hrs (22-98%); c) B2pin2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 145
°C, microwave, 1 hr (assumed quantitative); d) Cs2CO3, Pd(PPh3)4, 3:1 dioxane/H2O, 80 °C, 48
hrs (11-44%).
2.3.3 Synthesis of saturated head group replacements, NEU-1912 scaffold
Synthesis of the NEU-1912 matched pairs (Scheme 2-7) began with 6-bromoquinolone
(2.8) which was coupled to 4-(phenylsulfonyl)morpholine boronic acid (2.10) under Suzuki
conditions. Deoxy-halogenation afforded chlorinated compound 2.12. Subsequent chlorine
displacements with various primary aliphatic amines under SNAr conditions provided the desired
final compounds 2.26a-h.
76
Scheme 2-7. Synthetic route for various head group analogs.
Reagents and conditions: a) 4-(phenylsulfonyl)morpholine-boronic acid pinacol ester,
Dichlorobis(triphenylphosphine)palladium(II), TEA, ethanol/water, 100 °C, microwave, 2 h (%);
b) Phosphorus oxychloride, 106 °C, 2 h (82%); c) saturated primary amine, DIPEA, n-butanol,
200 °C, microwave, 3 h (20-80%).
2.3.4 Evaluation of saturated head group replacements, NEU-1912 and
NEU-1953 scaffolds and matched pairs
Comparing the parent compound NEU-1953 to its saturated head group matched pairs, the
analog bearing the cyclohexylamine, NEU-5144 (Table 2-8), showed a modest improvement in
solubility and potency over NEU-1953, but was less metabolically stable. Tertiary amines (NEU-
4920, NEU-5375, and NEU-4987) all exhibited improved solubility with moderate-to-excellent
anti-trypanosomal potencies. Compound NEU-4987 is 20-fold more potent than NEU-1953 with
a 10-fold improvement in aqueous solubility. However, its high plasma protein binding (100%)
and toxicity against HepG2 cells are liabilities that make it less desirable for progression.
Replacement of the headgroup with the tetrahydropyrans (NEU-5139 and NEU-5326) maintained
activity against T. brucei, when compared to NEU-1953, and improved aqueous solubility 15-fold,
77
but increased toxicity against HepG2 cells. The primary alcohol compound NEU-5376, was the
only matched pair to NEU-1953 that showed a loss in potency (4-fold), though the aqueous
solubility was improved.
Saturated head group compounds with the NEU-1912 scaffold (Table 2-9) were similar to
the NEU-1953 analogs in that their aqueous solubilities were improved; however anti-
trypanosomal potencies were all less than that of the parent compound NEU-1912. The most potent
compound of this series, NEU-5536, is 24-fold less potent than NEU-1912, however this is still in
our target range for favorable anti-typanosome activity. Matched pairs in Table 2-8 and Table 2-
9 can also be compared. The NEU-1953 analogs (Table 2-8) have pEC50 values about a log unit
greater than the NEU-1912 analogs (Table 2-9).
78
Table 2-8. Saturated head group analogs of NEU-1953, T. brucei activities and physicochemical
properties.
Compound R T.b.b.
pEC50
Aq. Sol.
(µM)
Human
PPB
(%)
HLM Clint
(µL/min/mg
of protein)
HepG2
pTC50 LLE
NEU-1953
6.37 44 87 180 < 4.52 4.3
NEU-5144
(2.25a)
6.54 98 85 300 ntf 2.8
NEU-4920
(2.22a)
7.27 >1000 ndb ndc ntf 5.3
NEU-5375
(2.25b)
6.77 660 ndd ndd 5.60 3.7
NEU-4987
(2.22b) 7.66 590 100 nde 5.52 5.3
NEU-5139
(2.25c)
6.42 760 50 94 4.82 4.5
NEU-5326
(2.25d) 6.44 760 47 170 4.79 4.0
NEU-5376
(2.25e)
5.74 980 63 160 < 5.44 3.0
NEU-6734
(2.25f)
7.56 836 50 240 pending 4.8
SCYX-7158a 6.54
nt = not tested. nd = not determined. aSCYX-7158 (acoziborole Ch 1. cmpd 7) was used as a control. bCompound
lost, likely due to unspecific binding. cAssay failed due to poor curve fitting. dPoor MS response. eLow recovery. fNot tested initially and compounds with a superior profile overall had already been identified.
79
Table 2-9. Saturated head group analogs of NEU-1912, T. brucei activities and physicochemical
properties.
Compound Head group T.b.b.
pEC50
Aq. Sol.
(µM)
Human
PPB
(%)
HLM Clint
(µL/min/mg
of protein)
HepG2
pTC50 LLE
NEU-1912
7.68 3.7 98 236 < 4.47 4.9
NEU-5533
(2.26a)
5.78 8.1 96 100 4.47 1.9
NEU-5537
(2.26b)
5.43 790 58 23 4.58 3.3
NEU-6735
(2.26c)
5.44 811 69 13 pending 2.2
NEU-5536
(2.26d) 6.30 870 73 28 5.03 3.8
NEU-5825
(2.26e)
6.04 7.0 70 59 4.80 4.0
NEU-5534
(2.26f) 5.60 38 74 70 4.49 3.1
NEU-5535
(2.26g)
5.42 140 72 43 4.49 2.6
NEU-5824
(2.26h)
6.12 200 82 110 4.68 3.3
SCYX-7158a 6.54 aSCYX-7158 (acoziborole Ch 1. cmpd 7) was used as a control.
2.4 Crossover compounds between NEU-4363 and NEU-1912
As part of the optimization of NEU-1953, the tail group region of the molecule was also
explored for increasing the sp3 carbon content.90 For example, the homopiperazine-containing
NEU-4363 (Figure 2-6, Aq. Sol. = 985 μM) displayed improved solubility compared to its parent
80
compound NEU-1953 (Aq. Sol. = 44 μM). With the success of substituting the N-
methylhomopiperazine for N-methylpiperazine, it was hypothesized that the addition of the NEU-
4363 tail group to the NEU-1912 scaffold would also have improved aqueous solubility.
Figure 2-6. Comparison of parent compound NEU-1953 and analog NEU-4363.
2.4.1 Synthesis of crossovers
First the tail group for the crossover compounds was synthesized using a similar scheme
(Scheme 2-8) as those previously mentioned. The chlorine of 5-bromo-2-chloropyrimidine was
displaced using 1-methylhomopiperazine and bromide 2.27 was subjected to Miyaura borylation
conditions to yield boronic acid pinacol ester 2.28. This borylated compound 2.28 was used crude
in subsequent reactions assuming a quantitative yield.
Next, 6-position substituted matched pairs of NEU-1912 and NEU-4363 were synthesized
via Scheme 2-9. Using previously synthesized 6-bromoquinolone 2.8, deoxychlorination in neat
phosphorus oxychloride yielded dihalogenated core 2.29 in 80% yield. Aromatic substitution of
the chloride using sodium hydride and appropriate amine heterocycle resulted in bromide
intermediates 2.30a and 2.30b. Suzuki coupling with previously synthesized boronic acid pinacol
ester 2.28 gave final compounds NEU-5374 (2.31b) and NEU-5377 (2.31a) in moderate yields.
81
Scheme 2-8. Synthesis of 1-methylhomopiperazine tail group.
Reagents and conditions: a) 1-methyl-1,4-diazepane, diisopropylethylamine, THF/IPA (1:1), 80
°C, 18 hr (91%); b) B2pin2, KOAc, PdCl2(dppf)·CH2Cl2, dioxane, 85 °C, 18 hr (assumed
quantitative).
Scheme 2-9. Synthesis of 6-position substituted matched pairs of NEU-1912 and NEU-4363.
Reagents and conditions: a) POCl3, reflux, 18 hr (80%); b) pyrazin-2-amine or pyrimidin-4-
amine, NaH, DMF, rt, 48 hr (36%, 27% respectively); c) 2.28, Pd(PPh3)4, Cs2CO3, EtOH/H2O
(1:1), 85 °C, 12 hr (50%, 42% respectively).
The 7-position matched pair analog NEU-5030 was synthesized via a different route than
the other matched pairs via Scheme 2-10. First, 7-bromoquinolone was coupled with tail group
2.28 using Suzuki conditions. The quinolone 2.32 was then chlorinated using phosphorus
oxychloride in moderate yield. Similar aromatic substitution conditions as above were used to
82
attach the pyrimidine-4-amine head group, which was successful to provide the final compound
NEU-5030 (2.34) but was low yielding.
Scheme 2-10. Synthesis of 7-position substituted matched pair of NEU-1912 and NEU-4363.
Reagents and conditions: a) 2.28, PdCl2(dppf)·CH2Cl2, Cs2CO3, dioxane, 90 °C, 12 hr (77%); b)
POCl3, reflux, 18 hr (69%); c) pyrimidin-4-amine, NaH, DMF, 90 °C, 12 hr (14%).
2.4.2 Evaluation of crossovers
Previously, the replacement of 1-methylpiperazine (NEU-1953) with 1-
methylhomopiperazine (NEU-4363, Aq. Sol. = 985 μM) was shown to significantly increase the
aqueous solubility of the scaffold. It was hypothesized that the tail-group from NEU-4363 could
be used to increase the aqueous solubility of the NEU-1912 scaffold as well, while maintaining
the higher potency against T. brucei of NEU-1912. Matched pairs of NEU-1912 and NEU-4363
were therefore synthesized varying the substitution pattern and head groups. Using the NEU-4363
head group (pyrazine-2-amine) and substitution of the tail group at the 6-position (the substitution
83
pattern of NEU-1912) (NEU-5377) showed almost 1000 times higher solubility than NEU-1912
and was comparable to NEU-4363. However, there was a 2-log unit loss in potency of NEU-5377
as compared to NEU-1912 and 1-log unit loss in potency as compared to NEU-4363. The clearance
rate of NEU-5377 was lower when substitution was at the 6 versus 7 position.
Varying the head group in the matched analog to NEU-4363, compound NEU-5030,
showed similar in vitro results as NEU-4363. Changing both the head group and substitution
position of NEU-4363 as seen in NEU-5374 was the most detrimental to potency but had similar
solubility and clearance rates to that of NEU-4363. All the matched pairs showed an improvement
in aqueous solubility (as compared to NEU-1912) and reduction in potency as compared to both
parent molecules.
Table 2-10. Evaluation of NEU-1912 and NEU-4363 matched pairs.
Compound Tail
group
Tail
pos. R
T.b.b.
pEC50
(µM)
Aq. Sol.
(µM)
Human
PPB
(%)
HLM Clint
(µL/min/mg
of protein)
HepG2
pTC50 LLE
NEU-1912 A 6 C 7.70 1.4 96 210 < 4.47 5.0
NEU-5374
(2.31b) B 6 C 5.20 907 61 87 < 4.44 2.5
NEU-5030
(2.34) B 7 C 6.26 933 66 74 < 4.44 3.6
NEU-5377
(2.31a) B 6 D 5.49 990 81 48 < 4.44 3.4
NEU-4363* B 7 D 6.79 985 75 77 4.48 4.7 *Synthesized by Dr. Lori Ferrins
84
2.5 Evaluation of lapatinib analogs against other parasites
2.5.1 Evaluation of lapatinib analogs against Trypanosoma cruzi
Compounds synthesized and discussed above were also screened against Trypanosoma
cruzi (Table 2-11), the causative agent of Chagas disease. Many of the above synthesized analogs
had favorable hit activity inactive against T. cruzi (see hit criteria Table 1-2). NEU-5535 (pEC50
= 6.48) showed the most promise for development having the highest potency, being selective over
host cells, and showing a moderate LLE value (3.67). As shown in Table 2-9, NEU-5535 has a
moderate in vitro clearance rate of 43 µL/min/mg of protein, and acceptable levels of plasma
protein binding (72%) and aqueous solubility (140 μM). These criteria would make NEU-5535 a
good molecule to pursue for further optimization to increase T. cruzi potency and selectivity and
reduce clearance rates.
85
Table 2-11. Evaluation of lapatinib analogs against Trypanosoma cruzi and T. cruzi host cells
(C2C12).
Compound T. cruzi
pEC50
C2C12
pTC50 T. cruzi LLE
T. cruzi
selectivity
(pEC50-pTC50)
NEU-5144 < 5.00 < 5.00 < 1.24 0
NEU-5375 < 5.00 5.74 < 1.92 < -0.74
NEU-4987 5.68 5.41 3.30 0.27
NEU-5139 < 5.00 5.02 < 3.08 < -0.02
NEU-5326 5.94 5.42 3.55 0.51
NEU-5376 < 5.00 5.09 < 2.31 < -0.09
NEU-6734 pending pending pending pending
NEU-5537 5.89 5.39 3.78 0.50
NEU-6735 pending pending pending pending
NEU-5536 5.73 5.78 3.22 -0.05
NEU-5825 5.02 5.65 2.97 -0.63
NEU-5534 5.78 < 5.00 3.27 > 0.77
NEU-5535 6.48 < 5.00 3.67 > 1.48
NEU-5824 5.33 5.23 2.45 0.09
NEU-5377 < 5.00 5.01 < 2.85 < -0.0
NEU-5374 < 5.00 < 5.00 < 2.34 0
NEU-5030 < 5.00 < 5.00 < 2.34 0
2.5.2 Evaluation of lapatinib analogs against Leishmania
Most compounds synthesized and discussed above were also screened against Leishmania
major and some against L. donovani (Table 2-12), causative agents of Leishmaniasis. All of the
lapatinib analogs that were tested were shown to be inactive against L. major, L. donovani or both
except NEU-5139 which had a favorable potency for L. donovani hit criteria ( hit criteria found in
Table 1-2). Thought NEU-5139 has favorable potency, selectivity for host cells would need to be
addressed if pursued further.
86
Table 2-12. Evaluation of lapatinib analogs against Leishmania and B10R host cells.
Compound L. donovani
pEC50
L. donovani
LLE
B10R
pTC50
L. donovani
selectivity
(pEC50 – pTC50)
L. major
amastigote
pEC50
NEU-5144 < 5.00 < 1.24 5.64 < -0.638 -
NEU-5375 < 5.00 < 1.92 6.00 < -1.00 5.65
NEU-4987 < 5.00 < 2.61 5.41 < -0.407 4.59
NEU-5139 5.39 3.47 5.44 -0.0428 4.61
NEU-5326 5.06 2.68 5.50 -0.438 4.62
NEU-5376 < 5.00 < 2.31 5.01 < -0.006 -
NEU-6734 pending pending pending pending pending
NEU-5533 - - - - 4.65
NEU-5537 - - - - 4.67
NEU-6735 pending pending pending pending pending
NEU-5536 - - - - 4.67
NEU-5825 < 5.00 < 2.95 5.43 < -0.429 4.66
NEU-5534 - - - - 4.67
NEU-5535 - - - - 4.67
NEU-5824 < 5.00 < 2.13 < 5.00 0 4.68
NEU-5377 < 5.00 < 2.85 5.19 < -0.188 4.61
NEU-5374 < 5.00 < 2.34 5.03 < -0.0302 4.61
NEU-5030 < 5.00 < 2.34 < 5.00 0 < 4.43
2.5.3 Evaluation of lapatinib analogs against P. falciparum
Compounds synthesized and discussed above were also screened against 3 strains of
Plasmodium falciparum, the causative agent of malaria (D6, W2, and C235 strains, Table 2-13).
All compounds tested were active against the three strains of P. falciparum. NEU-1953 saturated
head group matched pairs were more potent than the NEU-1912 saturated head group matched
pairs. Matched pairs of NEU-1912 and NEU-4363 were moderately potent against the 3 strains of
P. falciparum. Compounds with the NEU-1953 scaffold and saturated head groups would be good
starting points for further optimization, focusing on the high clearance rates. However, analogs
with the NEU-1912 scaffold and saturated head groups containing nitrogen atoms (NEU-5537,
87
NEU-6735, and NEU-5536) may be a more favorable starting point for P. falciparum optimization
because of their good potencies, high aqueous solubility, and moderate clearance rates.
Table 2-13. Evaluation of lapatinib analogs against common strains of P. falciparum.
Compound P. falciparum
D6 pEC50
P. falciparum
W2 pEC50
P. falciparum
C235 pEC50
NEU-5375 7.28 7.41 7.17
NEU-4987 7.62 7.60 7.60
NEU-5139 7.60 7.32 7.64
NEU-5326 7.58 7.36 7.57
NEU-5376 7.43 7.10 7.39
NEU-6734 pending pending pending
NEU-5533 7.32 6.83 7.30
NEU-5537 7.37 6.98 7.28
NEU-6735 pending pending pending
NEU-5536 7.52 6.90 7.08
NEU-5825 6.69 6.40 6.53
NEU-5534 6.44 6.16 6.45
NEU-5535 6.39 6.15 6.43
NEU-5824 7.12 6.76 6.97
NEU-5377 6.84 6.49 6.74
NEU-5374 6.61 6.06 6.44
NEU-5030 6.79 6.64 6.84
2.6 Summary
Lapatinib was previously shown to be active against T. brucei, the causative agent of
HAT. Several rounds of optimization led to the advanced hit compounds NEU-1912 and NEU-
1953. Though, these compounds had low solubility that was not driven by lipophilicity. Various
strategies were pursued to increase the aqueous solubility of these lead compounds, two of which
are described above, salt formation and increasing sp3 atom content, and summarized in Figure
2-7.
88
Figure 2-7. Summary of modifications made to parent compounds NEU-1912 and NEU-1953.
Salts of basic compounds NEU-1912 and NEU-1953 were formed by mixing the parent
compounds dissolved in an organic solvent with various acids. Forming salts of NEU-1912 and
NEU-1953 were unsuccessful at increasing the thermodynamic aqueous solubilities.
Thermodynamic solubility is important to increase because compounds are tested in vitro and in
vivo after reaching an equilibrium in solution. However, salt formation has a greater effect on
89
increasing dissolution rates (kinetic solubility). In the future, a more in depth look at pH of salt
formation solutions would need to be performed though should not be a high priority for this class
of compounds.
Replacement of the aromatic head groups with various saturated, cyclic amines was carried
out by two synthetic means: using Buchwald coupling chemistry and SNAr conditions. Buchwald
conditions were screened and resulted in two final compounds, but products were difficult to purify
due to the inability to separate the highly soluble final compounds with the de-halogenated starting
material which was a side product of the reaction. SNAr conditions were found to be more
successful and more easily purified using column chromatography.
A small library of NEU-1912 and NEU-1953 matched pair analogs were synthesized and
were successful at improving the aqueous solubility of these compounds. However, none of the
saturated head group analogs were sufficient for further progression as HAT therapies due to low
potency against T. brucei or unfavorable clearance rates. One of the saturated head group analogs,
NEU-5535 did show moderate potency against T. cruzi and could be a good starting point for
further optimization against this parasite. All of the saturated head group analogs were potent
against 3 strains of P. falciparum, NEU-5537, NEU-6735, and NEU-5536 being the most
promising compounds, with moderate clearance rates.
Matched pair analogs of NEU-1912 and advanced lead compound NEU-4363 were also
synthesized to evaluate the effect of the 1-methylhomopiperazine moiety on solubility. All
matched pairs showed improved aqueous solubility but reduced potency against T. brucei as
compared to their parent analogs.
90
The future of this work would likely consist of synthesizing more analogs with increased
sp3 carbon content focusing on improving potency against T. brucei and in vitro clearance rates.
Other work would utilize the above-mentioned compounds for further development against T.
cruzi and P. falciparum. Development of structure-activity relationships for these parasites would
be the first step, utilizing the improved physicochemical properties. More broadly, this work shows
that increasing the sp3 carbon content is beneficial to improving aqueous solubility. This strategy
could therefore be used to improve the aqueous solubility of other previously synthesized lapatinib
analogs with varying scaffolds.
91
Chapter 3: ADME optimization of kinase inhibitor chemotypes from an HTS for HAT
drug discovery
3.1 Introduction
3.1.1 High-throughput screen of a kinase targeted library against T. brucei in
collaboration with GSK
As previously discussed, the pathogenic parasites targeted in the Pollastri lab have similar
enzymes among one another as well as similar enzymes to humans. One such essential target class
is the kinase family of enzymes.86 The overlap of these targets in parasites and humans led to the
idea to use a lead repurposing45 approach to discovering new hit compounds that inhibit T. brucei.
The work was performed in collaboration with GSK and the OpenLab Foundation.
The study began with the selection of a suitable kinase-targeted compound library to test
in the HTS.99 The overall library of 42,444 compounds was composed of three different subsets.
One subset of 2,979 members, was composed of compounds from the GSK screening collection
that had structural similarity with a Tanimoto score greater than 70% to compounds that were
previously reported as inhibitors of PI3K/mTOR.143 The second subset of 367 members, was
composed of compounds from the published kinase inhibitor set from GSK.144 Finally, the last
subset of 39,098 members, were general kinase inhibitors from the GSK screening collection.99
The total compound library was tested in HTS format in a single concentration (4 μM)
whole-cell assay against T. b. brucei Lister 427 strain.143, 145 The growth inhibition of the parasites
was assessed in the log-phase. From the original set, 6,368 compounds showed greater than 50%
growth inhibition. These compounds were retested to confirm their activities resulting in 4,574
compounds that were progressed to dose response studies against T. b. brucei and HepG2 host
92
cells. Compounds that had a pEC50 ≥ 6 and were 100-fold more selective for T. b. brucei inhibition
over HepG2 cells were selected for progression (797 compounds).99
3.1.2 Organization of HTS hits and identification of NEU-1200 as a potent
inhibitor of T. brucei growth
Progressed compounds were grouped into clusters based on structural similarities resulting
in 59 clusters and 53 singleton compounds. These compounds were subjected to cidality and rate
of action assays, and chemical properties were computed to aid in the prioritization of clusters for
advancement.
A cluster of 2,4-subsituted 1H-pyrrolo[2,3b] pyridines, colloquially known as 2,4-
substituted azaindoles (cluster 32), was ultimately chosen for prioritization. Various factors were
considered during prioritization including ligand efficiency (LE),107 lipophilic ligand efficiency
(LLE),146 aqueous solubility, multi-parameter optimization (MPO) score,147 cidality, and speed-
to-kill and are displayed in Table 3-1. A LE > 0.3 is considered a good starting point for chemical
optimization and is the characterization of efficiency based on the heavy-atom count in the
molecule ((1.37*pEC50)/number of heavy atoms).107 LLE (pEC50 – clogP) takes into account
whether favorable potency results are caused by effective lipophilic within the biological target(s)
rather than simply escaping a hydrophilic environment; the targeted LLE value is > 4.109 Lastly,
MPO scores consider multiple chemical properties (shown in Table 3-2) and is thought to be a
good predictor of a compound’s ability to enter the brain.147
93
Table 3-1. Compound properties for a cluster of 2,4-substituted 1H-pyrrolo[2,3b]pyridines and
selected compound NEU-1200.
Property Desired value NEU-1200
value
Cluster average
(23 compounds)
T. b. brucei pEC50 > 7 7.73 6.6
HepG2 pTC50 < 5 4.30 4.2
T. b. gambiense pEC50 > 7 7.93 -
T. b. rhodesiense pEC50 > 7 8.20 -
MW (g/mol) ≤ 360 331 360
clogP ≤ 3 2.81 2.2
logD (7.4) ≤ 2 1.41 2.8
HBD ≤ 0.5 1 -
pKa ≤ 8 8.8 -
TPSA (Å2) 40 < TPSA ≤ 90 49.74 81
MPO score ≥ 5 5.4 3.8
T. b. brucei pEC50
(18h)
≥ 6 6.89 -
T. b. brucei pEC99 ≥ 6 6.57 -
LE ≥ 0.3 0.42 0.32
LLE ≥ 4 4.52 3.2
Human PPB (%) < 90 80 -
Aqueous Solubility
(μM)
> 50 630 >10
Fast Yes Yes 52%
Cidal Yes Yes 13%
Table 3-2. Multi-parameter optimization (MPO) scoring for predicting CNS exposure.
Property Desirable range NEU-1200 value (score)
clogP ≤ 3 2.8 (1)
clogD ≤ 2 1.41 (1)
MW (g/mol) ≤ 360 330 (1)
TPSA (Å2) 40 < TPSA ≤ 90 49.74 (1)
HBD ≤ 0.5 1 (0.833)
pKa ≤ 8 8.8 (0.600)
Score sum 5.4
94
Compound rate-to-kill was determined using an assay that quantified, at increasing
incubation times, living cells (based on ATP content).148 Cell viability was assessed at 6, 12, 18,
and 24 hrs. Compounds tested that showed low cell viability before 18 hours were considered to
be fast acting (pEC50>6 at 18 hr). An additional assay was performed to determine cidality (or
irreversibility), where the compounds were washed out from the cell culture medium after 18 hours
of incubation and cell density data was continued to be collected after washout to see if growth
recovery occurred. A minimum trypanocidal concentration is considered to be the compound
concentration that stops 99% of the growth recovery or pEC99. Compounds were deemed cidal if
their pEC99 > 6 after 72 hrs of incubation.99
The 2,4-substitued azaindole cluster 32 showed good overall properties with compounds
that were both fast-acting and cidal. The cluster has an average MPO score of 3.8, the cluster
average LE is good, and the LLE is modest. Lastly, this cluster had an average aqueous solubility
of 10 μM. Some representative structures from the initial cluster can be seen in Table 3-3. The
best compound from the series was NEU-1200 (Table 3-1) which was progressed to a mouse
pharmacokinetic (PK) study.
95
Table 3-3. Subset of cluster 32 compounds from initial HTS typified by a 2,4-substituted
pyrrolo[2,3-b]pyridine core.
NEU-
#### R1 R2 R3
T.b.b
pEC50 LLE
Aq.
Sol.
(μM)
MPO
score
1200
-H 7.73 4.52 630 5.44
1878
-H 6.93 3.72 >200 4.65
1201
-Cl 7.44 3.71 32 4.34
1705
-H 6.64 1.96 nd 5.22
1700
-H 6.70 4.22 219 4.24
1702
-H 6.77 3.55 286 4.28
1699
-Cl 6.68 4.67 298 4.50
1711
-H 6.53 2.50 273 3.50
1708
-H 7.18 0.94 nd 2.03
96
1202
-H 7.64 4.35 28 3.87
1707
-H 6.13 3.46 24 2.56
1204
-H 6.26 4.05 146 3.54
1205
-H 6.54 5.23 834 4.48
1703
-H 6.04 2.62 >400 4.28
3.2.3 In vivo pharmacokinetic (PK) evaluation of NEU-1200
NEU-1200 had promising properties and was therefore tested in a mouse PK model. The
results obtained for NEU-1200 following intravenous (IV; 1 mg/kg) administration to female
97
NMRI mice (n=3) are shown in Table 3-4. The PK parameters have been obtained from
concentration versus time profiles measured in whole blood. NEU-1200 exhibits moderate to high
clearance, high volume of distribution, and a short half-life.
Table 3-4. PK evaluation of NEU-1200 following IV administration.
Mean Standard Deviation
t1/2 (hr) 1.1 0.4
Cl (mL/min/kg) 101.0 4.8
AUC(0-∞) (hr*ng/mL) 165.2 7.8
Vss (L/kg) 9.0 1.5
The results obtained for NEU-1200 following intraperitoneal (IP; 5 mg/kg, saline, pH 4.8)
administration to female NMRI mice (n=3) are shown in Table 3-5. The PK parameters have been
obtained from concentration versus time profiles measured in whole blood. NEU-1200 exhibits an
average Cmax of 393 ng/ml and an average AUC (0-∞) of 614 hr*ng/mL.
Table 3-5. PK evaluation of NEU-1200 following IP administration.
Mean Standard Deviation
t1/2 (hr) 1.2 0.4
Cmax (ng/mL) 393.0 66.9
AUC(0-∞) (hr*ng/mL) 613.9 50.2
3.2 Design, synthesis, and evaluation of NEU-1200 first-generation analogs
3.2.1 Rationale of NEU-1200 first-generation analog design
Results from the PK study showed that NEU-1200 was highly cleared due to the metabolic
liabilities in the structure. The areas predicted to be most metabolically labile (using MetaSite,
98
predictive metabolism software in use at GSK) are shown in Figure 3-1. Not surprisingly, the most
metabolically labile structure is the methyl on the pyrazole.
Figure 3-1. Predicted metabolically labile sites of NEU-1200. Image generated using MetaSite.
During oxidative metabolism cytochrome P450 enzymes catalyze the oxidation of drugs in
the liver.149 N-demethylation is a well-known metabolic pathway in the breakdown of drugs in the
body.149 Benzylic positions, like the one depicted by a lighter red circle in Figure 3-1, are also
common areas of cytochrome P450 oxidation due to the activation of the carbon by a heteroatom
and sp2 hybridized carbon adjacent to the benzylic position.149 Another oxidative metabolism
product could be the formation of the N-oxide of one or more of the various nitrogen atoms in the
molecule.149
Based on these predictions, compounds were designed to vary the amine pendant at the 2-
position and the pyrazole at the 4-position, in order to block metabolism from occurring at these
sites. Other compounds were synthesized due to the likelihood that they would be metabolites of
the parent compound, and we wished to know whether these metabolites might have biological
activity themselves. Other compounds were synthesized to identify a pharmacophore and to
Dark blue = highest metabolic liability
Dark red = next highest
99
elucidate structure-activity (SAR) and structure-property relationships (SPR). Figure 3-2 shows
the summary for the plan of modifications to be made.
Figure 3-2. Summary of analog design plans.
3.2.2 Synthesis of NEU-1200 and first-generation analogs
The synthesis of boronate coupling partners 3.2a-c for future Suzuki couplings were carried
out via the sequence in Scheme 3-1. Chlorine displacement was carried out using the appropriate
amine. The resulting bromide intermediates 3.1a-c were then borylated using Miyaura borylation
conditions.
100
Scheme 3-1. Synthesis of intermediates 3.2a-c.
Reagents and conditions: a) ethanolamine, triethylamine, anhydrous DCM, -10 °C to rt, 18 hr
(82%); b) secondary amine, THF, rt, 18 hr (82%); c) B2Pin2, PdCl2(dppf)·CH2Cl2, KOAc, dioxane,
80-85 °C, 18 hr (90%).
The first round of analogs synthesized were truncated analogs. The synthesis of analogs
truncated at the 2-postion (Scheme 3-2) started with 4-bromo-7-azaindole or tosyl-protected
bromide 3.3, which were coupled to various boronic acids and boronic acid pinacol esters using
Suzuki conditions in various yields. Tosylated analogs were tested in vitro (Table 3-6) and then
detosylated using 3.0 M aqueous sodium hydroxide at 150 °C in the microwave to give final
compounds 3.5a and 3.5b (Table 3-6).
Scheme 3-2. Synthesis of analogs truncated at the 2-position.
Reagents and conditions: a) various boronic acids/boronic acid pinacol esters, Na2CO3,
PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 130-150 °C μwave, 10-30 min (17-86%).
101
Preparation of benzylamine boronate coupling partners were synthesized according to
Scheme 3-3. Starting with 4-bromobenzyl bromide, the benzyl bromide was substituted with
dimethylamine or diethylamine to give intermediates 3.6a and 3.6b, respectively. The resulting
bromide was then subjected to Miyaura borylation conditions to give the appropriate boronic acid
pinacol ester.
Scheme 3-3. Synthesis of boronate coupling partners 3.7a,b.
Reagents and conditions: a) 4-bromobenzylbromide, dimethylamine or diethylamine, hexanes, 0
°C – rt, 18 hr (92%, 70% respectively); b) B2Pin2, PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 80-85 °C,
18 hr (yield assumed quantitative).
Analogs truncated at the 4-position (Scheme 3-4) were synthesized starting with 7-
azaindole which was protected using tosyl chloride under basic conditions. The 2-postion was
selectively iodinated via deprotonation using LDA and treatment with molecular iodine. Iodinated
compound 3.9 was coupled in good yields to various boronic acids and esters including those
synthesized above according to Scheme 3-3. The resulting tosylated compounds (which were also
tested in vitro, Table 3-6) were subsequently deprotected under basic conditions and are evaluated
in Table 3-6.
102
Scheme 3-4. Synthesis of analogs truncated at the 4-position.
Reagents and conditions: a) 4-methylbenzenesulfonyl chloride, tetrabutylammonium
hydrogensulfate, NaOH, DCM, rt, 12 hr (73%); b) n-butyllithium, diisopropylamine, I2, THF, -78
°C – rt, 12 hr (39%); c) various boronic acids/ pinacol esters, Cs2CO3, PdCl2(dppf)·CH2Cl2, 3:1
dioxane/H2O, 150 °C μwave, 10 min (80%), d) 3M NaOH, dioxane, 150 °C μwave, 15 min.
Compounds modified at the 4-position started with 4-chloro-7-azaindole which was
protected with tosyl chloride as shown in Scheme 3-5. Where previous schemes utilized the
bromo-azaindole (3.3), this scheme utilized the chloride 3.12 to aid in the purification of compound
3.13 (as the corresponding bromide starting material co-eluted with the desired product). The
crystal structure (obtained by Dr. Bo Li at Boston College) of intermediate 3.13 was obtained to
confirm the position of iodination (Figure 3-3). Using compound 3.13 led to an increase in the
regioselectivity of the first Suzuki reaction, occurring first at the 2-postion to give compound 3.14.
With a bromine at the 4-postion, test reactions showed a mixture of products, including reaction
at both the 2- and 4-positions. Chloride 3.14 was subsequently reacted with various boronic acids
and esters to give tosyl protected compounds 3.15a-e. Finally, the tosylated compounds were
deprotected under basic conditions to yield final compounds 3.16a-e which are evaluated in Table
3-7.
103
Scheme 3-5. Initial synthesis of analogs with modifications at the 4-position.
Reagents and conditions: a) 4-methylbenzenesulfonyl chloride, tetrabutylammonium
hydrogensulfate, NaOH, DCM, rt, 12 hr (81%); b) lithium diisopropylamide, I2, THF, -78 °C – rt,
12 hr (88%); c) 3.7a, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 80 °C, 12 hr (76%), d)
various boronic acids/boronic acid pinacol esters, Na2CO3,
tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 90 °C, 48 hr (28-72%), e) 3M NaOH,
dioxane, 150 °C μwave, 15 min (33-63%).
Figure 3-3. Crystal structure of 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b] pyridine. Obtained by
Dr. Bo Li, [Boston College]
104
Other final compounds modified at the 4-position were prepared via the modified synthetic
route shown in Scheme 3-6 which utilized the high-yielding aldehyde intermediate 3.17. Boronic
acids/pinacol esters were coupled at the 4-position in moderate yields to give aldehyde
intermediates 3.18a-f. Reductive amination with dimethylamine hydrochloride salt gave protected
intermediates 3.19a-f. Detosylation under basic conditions afforded the requisite final compounds
in varying yields which are evaluated in Table 3-7.
Scheme 3-6. Modified synthesis of compounds with 4-position modifications.
Reagents and conditions: a) (4-formylphenyl)boronic acid, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1
dioxane/H2O, 85 °C, 12 hr (95%); b) various boronic acids/boronic acid pinacol esters, Na2CO3,
tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 90 °C, 48 hr (39-85%); c)
dimethylamine hydrochloride, triethylamine, NaBH4, DCE, rt, 12 hr (13-84%); d) 3M NaOH,
dioxane, 150 °C μwave, 15 min (10-92%).
The next type of compounds to be synthesized were the 2,4-subsituted analogs that were
modified at the pendant dimethylamine of NEU-1200. The first synthesis attempt (Scheme 3-7)
started with previously-synthesized compound 3.2 and iodination at the 2-position in a similar
fashion as shown in Scheme 3-3. This would have allowed for a handle to perform Suzuki
couplings with various boronic acids/pinacol esters, which would have facilitated parallel
105
synthesis and late stage diversification. Unfortunately, the iodination reaction did not go to
completion and the starting material (3.4a) and product (3.21) were inseparable using
chromatography.
Scheme 3-7. Attempted synthesis of analogs with modifications at the 2-position.
Reagents and conditions: a) n-butyllithium, diisopropylamine, I2, THF, -78 °C – rt, 12 hr; b)
various boronic acids/pinacol esters, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1 dioxane/H2O, 80 °C, 12
hr; c) 3M NaOH, dioxane, 150 °C μwave, 15 min.
The first successful synthesis for analogs with modifications at the 2-position is shown in
Scheme 3-8. Starting with intermediate 3.13, various boronic acids and pinacol esters were used
as coupling partners in Suzuki reactions, after which the resulting chlorides were subjected to a
second Suzuki reaction to yield protected compounds 3.23a-j. Final compounds were elucidated
after tosyl deprotection under basic conditions and are evaluated in Table 3-8.
106
Scheme 3-8. First successful synthesis of analogs with modifications at the 2-position.
Reagents and conditions: a) various boronic acids/pinacol esters, Na2CO3, PdCl2(dppf)·CH2Cl2,
3:1 dioxane/H2O, 80 °C, 12 hr (29-99%); b) (1-methyl-1H-pyrazol-4-yl)boronic acid pinacol
ester, Na2CO3, tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 80 °C, 12 hr (4-
99%), e) 3M NaOH, dioxane, 150 °C μwave, 15 min (21-97%).
Scheme 3-9 shows the modified synthetic route for the 2-position analogs. The
intermediate 3.23f was utilized in two different ways. In the first route, a reductive amination with
piperazine was performed to yield intermediate 3.25. which could then be deprotected under basic
conditions to yield final compound 3.26a. Alternatively as a means to diversify at the last possible
step in the synthesis, 3.23f could be deprotected first followed by various reductive aminations to
yield final compounds 3.26b-k evaluated in Table 3-8.
107
Scheme 3-9. Modified synthesis of analogs with modifications at the 2-position.
Reagents and conditions: a) various amines, NaBH4, DCE, rt, 12 hr (10-64%); b) 3M NaOH,
dioxane, 150 °C μwave, 15 min (37% or 79%).
3.2.3 Evaluation of NEU-1200 first-generation analogs
Truncated matched pair analogs (Table 3-6) were synthesized using similar substituents as
presented in the initial cluster. Tosylated intermediates of these compounds were also tested for T.
brucei inhibition. NEU-4472 is the 2-postion truncated analog and NEU-4844 is the 4-position
truncated analog of parent compound NEU-1200. Truncation in either position results in loss of
activity against T. brucei. Tosylated intermediate (NEU-4877) to NEU-1200 also shows loss of
activity. These data suggest that substitutions at both positions are needed in order to retain activity
against the parasite.
108
Table 3-6. Activity of truncated analogs of NEU-1200 against T. brucei.
Entry R1 R2 R3 T.b.b
pEC50
NEU-1200
-H
7.73
NEU-4472
3.3
-H -H
4.92
NEU-4813
3.2
-Ts -H
<4.40
NEU-4877
-Ts
5.24
NEU-4844
3.17
-H
-H 5.23
NEU-4845
3.18
-H
-H 4.80
NEU-4812
3.16
-Ts
-H 4.40
NEU-4815
3.5
-H -H
<4.40
NEU-4814
3.4
-Ts -H
4.81
109
NEU-4786
3.6
-H -H
4.75
NEU-4471
3.7
-H -H
<4.88
Table 3-7 shows the modifications made to NEU-1200 at the 4-position of the molecule.
NEU-4928 was synthesized as a potential metabolite of NEU-1200 and had similar potency. The
HLM clearance is higher than that of NEU-1200. Methylated pyrazole analogs NEU-4929 and
NEU-4927 have similar activities to NEU-1200. NEU-4927 had a similar clearance rate in rat
hepatocytes as compared to parent compound. The phenyl methyl sulfone compound NEU-4917
showed a slight reduction in potency, though is still acceptable, and also showed a moderate
reduction in clearance rates against both human liver microsomes and rat hepatocytes.
Sulfonamides NEU-5828 and NEU-5829 showed a log unit decrease in potency against T. brucei
and NEU-5828 had increased clearance rates. Pyridyl compound NEU-5446 was less potent and
had higher clearances rates. Methyl substituted pyridines (NEU-5449 and NEU-5452) were
similar in potency but had similar clearance rates to matched analog NEU-5446. The increased
clearance rates of the 4-position modified analogs that do not contain pyrazoles suggest that the
primary metabolism of those compounds occurs at the dimethylamine pendant area of the
molecule.
110
Table 3-7. Activities and clearance rates of 4-position variant first-generation analogs of NEU-
1200.
Entry R T.b.b
pEC50
HLM Clint
(µL/min/mg
of protein)
RH Clint
(µL/min/106
cells)
Hit
Criteria > 7 ≤ 47 ≤ 27
Lead
Criteria > 7.5 ≤ 9 ≤ 5
NEU-1200
7.73 25 25
NEU-4927
7.96 25 19
NEU-4928
7.85 64 26
NEU-4929
7.52 34 34
NEU-4917
7.17 18 13
NEU-5828
6.31 45 37
111
NEU-5829
6.32 nt nt
NEU-5446
6.70 19 38
NEU-5447
6.75 56 64
NEU-5449
6.83 43 38
NEU-5452
6.27 42 19
nt = not tested
The 2-postion of the azaindole was explored through various modifications keeping the
pyrazole in place for matched pair analysis (Table 3-8). Removal of the methylene carbon (NEU-
5323) drastically increased clearance and resulted in a 10x loss in potency. Extension of the
dimethyl to the diethyl (NEU-4973) resulted in similar potency and clearance rates to NEU-1200.
Tying the ethyl chains into a pyrrolidine ring (NEU-5123) was beneficial to reducing clearance
rates in both human liver microsomes and rat hepatocytes. Methylation of the benzyl position
(NEU-6072) yields a similar microsomal clearance rate and reduced rat hepatocyte clearance rate
as compared to NEU-1200 but did not show extra benefit to clearance reduction as compared to
matched pair NEU-5123. NEU-4995 extends the pyrrolidine ring to a piperidine which slightly
increased the potency but also the human liver microsomal clearance (pEC50=7.92, HLM Clint=30
112
μL/min/mg protein). Piperazine analog NEU-5936 was similar to the piperidine in terms of
clearance but showed 10-fold improvement in potency (pEC50=8.70). Though still active
(pEC50=7.70), methylation of the piperazine (NEU-6076) loses the improvement of potency
afforded by NEU-5936 but improves the rat hepatocyte clearance rate to an acceptable level (6.5
μL/min/106 cells). Replacing the nitrogen of the piperazine with an oxygen to form a morpholine
(NEU-5937) increased the clearance rates well above the acceptable level.
Table 3-8. Activities and clearance rates of 2-position variant first-generation analogs of NEU-
1200.
Entry R T.b.b.
pEC50
HLM Clint
(µL/min/mg
of protein)
RH Clint
(µL/min/106
cells)
Hit
Criteria > 7 ≤ 47 ≤ 27
Lead
Criteria > 7.5 ≤ 9 ≤ 5
NEU-5323
6.85 110 300
NEU-1200
7.73 25 25
NEU-4973
7.76 28 17
NEU-5123
7.71 13 20
NEU-6072
7.77 28 15
113
NEU-4995
7.92 30 19
NEU-5936
8.70 35 21
NEU-6076
7.70 27 6.5
NEU-5937
7.48 75 140
NEU-5938
8.16 18 16
NEU-5124
7.62 3.0 17
NEU-6018
8.26 <3.0 14
NEU-6048
7.24 54 >300
NEU-6025
6.82 74 16
NEU-6044
7.69 13 12
NEU-6049
7.78 5.2 9.4
NEU-6047
7.26 92 >300
NEU-4974
7.30 nda nda
NEU-5448
5.26 ndb ndc
NEU-5306
7.23 27 5.3
114
NEU-6073
7.06 46 8.4
NEU-4918
6.73 77 160
nd = not determined; acompound detected only in first sample; bpoor optimization; cfailed to create
analytical method.
The next set of 2-position modifications explored various secondary amines (Table 3-8).
Removal of one of the methyl groups from NEU-1200 resulted in analog NEU-5938 which
showed almost a half of a log unit increase in potency (pEC50 = 8.16) and an acceptable reduction
in intrinsic clearance rates. The best compound of this series was NEU-6018, which extended the
methyl chain of NEU-5124 to an isopropyl substituent. NEU-6018 had an improved pEC50 of 8.26,
human liver microsome clearance rate (< 3.0 µL/min/mg protein), and rat hepatocyte clearance
rate (14 µL/min/106 cells). Though not as potent as NEU-6018, pyrrolidine ethyl amine NEU-
6044, and a pyrrolidine analog attached directly to the phenyl group (NEU-6049) both had very
good clearance rates which were comparable to NEU-6018 and improved over parent compound
NEU-1200.
Compounds NEU-6047, NEU-4974, and NEU-5448 were synthesized as putative
oxidative metabolites of NEU-1200. Benzyl alcohol NEU-6047, not surprisingly, had increased
clearance rates and a reduction in potency. Aldehyde NEU-4974 and carboxylic acid NEU-5448
were both less potent than the parent compound, especially NEU-5448 by two log units. Since the
aldehyde and carboxylic acid would be the subsequent metabolite after the initial metabolism of
NEU-1200 to alcohol NEU-6047, they were not tested in the clearance assays, and assumed the
clearance rates would be lower than NEU-6047.
115
Other ADME properties, including aqueous solubility, human plasma protein binding, and
logD at pH 7.4, were also assessed for all the first-generation analogs and are shown in Table 3-
9. Promising compounds NEU-5123, NEU-5124, NEU-6018, NEU-6044, and NEU-6049 all had
very high solubilities, were not excessively protein bound, and had low logD values.
Host cell toxicities of all first-generation analogs were assessed in MRC5 human lung cell
line. Most compounds were also tested in HepG2 cells, a human liver cancer cell line. All
compounds tested showed no potent activity in these cell lines with pEC50 values less than 5.50,
shown in Table 3-10. Most first-generation compounds were 100 times more selective for T. b.
brucei inhibition versus MRC5 host cells, with 4 exceptions (NEU-5829, NEU-5452, NEU-5306,
and NEU-5448) which had difference values below 2 log units.
116
Table 3-9. Other ADME measurements of NEU-1200 and its first-generation analogs.
Entry Aq. Sol.
(μM)
Human PPB
(%)
LogD 7.4
Hit Criteria > 10 ≤ 95 ≤ 2
Lead Criteria > 100 ≤ 95 ≤ 2
NEU-1200 630 80 2.7
NEU-4917 8 87 2.9
NEU-4918 3 97 3.5
NEU-4927 1000 85 2.9
NEU-4928 483 85 2.0
NEU-4929 68 86 3.0
NEU-4973 832 89 2.9
NEU-4974 < 1 100 3.7
NEU-4995 39 91 3.4
NEU-5123 1 89 2.6
NEU-5124 841 76 1.7
NEU-5306 2 nda 2.8
NEU-5323 4 99 ndb
NEU-5446 2.9 99 3.9
NEU-5447 348 89 3.3
NEU-5449 647 93 3.7
NEU-5452 68 94 3.6
NEU-5828 < 1 94 3.4
NEU-5936 29 91 3.3
NEU-5937 2 99 3.6
NEU-5938 476 nda 1.4
NEU-6018 902 80 1.9
NEU-6025 750 nda 2.5
NEU-6044 580 nda 2.3
NEU-6047 30 nda 3.5
NEU-6048 3 97 3.5
NEU-6049 641 81 2.0
NEU-6072 733 86 3.0
NEU-6073 5 nda 3.1
NEU-6076 60 87 3.0 nd = not determined; alow recovery; bfailed to create analytical method.
117
Table 3-10. Toxicity of NEU-1200 and its first-generation analogs against HepG2 and MRC5
cell lines.
Entry HepG2
pTC50
MRC5
pTC50
Tbb pEC50 – MRC5 pTC50
NEU-1200 4.26 4.98 2.75
NEU-4927 nd 4.86 3.10
NEU-4928 4.69 5.01 2.84
NEU-4929 nd 4.82 2.70
NEU-4917 4.50 4.97 2.20
NEU-5828 5.16 <4.30 >2.01
NEU-5829 4.72 4.58 1.74
NEU-5446 nd <4.30 >2.40
NEU-5447 4.52 <4.30 >2.45
NEU-5449 4.57 <4.30 >2.53
NEU-5452 nd <4.30 >1.97
NEU-5323 4.61 <4.30 >2.55
NEU-4973 4.50 4.81 2.95
NEU-5123 4.72 4.76 2.95
NEU-4995 5.29 5.19 2.73
NEU-5936 4.57 5.79 2.91
NEU-6076 <4.59 4.92 2.78
NEU-5937 4.57 <4.30 >3.18
NEU-5938 4.50 5.46 2.70
NEU-5124 4.66 4.73 2.89
NEU-6018 4.54 5.88 2.38
NEU-6048 nd <4.78 >2.46
NEU-6025 4.56 4.78 2.04
NEU-6044 4.82 5.05 2.64
NEU-6049 nd 5.25 2.53
NEU-6072 4.84 5.05 2.72
NEU-5306 nd <5.26 >1.97
NEU-5448 nd <4.30 >0.96
NEU-4974 <5.30 <4.30 >3.00
NEU-6047 6.13 4.30 2.96
NEU-4918 nd <4.30 >2.43
NEU-6073 4.52 <4.30 2.76
118
3.2.4 Pharmacokinetic analysis of NEU-5123
Compound NEU-5123 was progressed to a mouse PK study due to its favorable in vitro
properties. First, NEU-5123 was dosed intraperitoneally at 10 mg/kg in 3 female NMRI mice.
Blood samples were taken from each of the mice to determine the Cmax, tmax, AUC, and t1/2 shown
in Table 3-11. The average Cmax value was well over 10×EC50 (7.15 ng/mL) and was reached after
30 minutes post injection. Notably, the half-life of NEU-5123 compared to NEU-1200 was
increased from 1.1 hours to 2 hours, showing that the clearance rate of NEU-5123 was improved
over NEU-1200.
Table 3-11. Individual blood pharmacokinetic parameters of evaluated compounds after
intraperitoneal administration of 10 mg/kg single dose (target dose) to female NMRI mice (n=3).
a r< 0.95 and/or only two points to define terminal half life
Measurements were also taken of drug levels in the blood versus the brain in 3 mice (Table
3-12). Measurements were taken at 0.5 hours and 4 hours after an intraperitoneal dose of 10 mg/kg.
After 30 minutes the brain to blood ratio was 0.648 which increased to 1.39 after 4 hours post
injection. Figure 3-3 shows the concentrations of NEU-5123 in the blood and brain of 3 mice.
Results show that exposure levels are well over 10×EC50 (EC50 = 7.61 ng/mL) for the duration of
the study.
Compound Cmax
(ng/mL)
tmax
(h)
AUC0-t
(ng·hr/ml)
AUC
(ng·hr/ml)
t1/2
(hr)
NEU-5123 Mean 520
0.50 1300 1500 2.0
SD 110 190 250 0.78
119
Table 3-12. Blood and brain levels (ng/mL in blood, ng/g in brain) of NEU-5123, after
intraperitoneal administration of 10 mg/kg single dose (target dose) to female NMRI mice (n=3).
LLOQ=50 ng/mL (blood).
Sampling
Time (hr) Matrix Mean SD
0.5
Blood 580 62
Brain 380 83
Brain/Blood
Ratio 0.65 0.11
4
Blood 170 2.8
Brain 230 18
Brain/Blood
Ratio 1.4 0.087
Figure 3-4. Peripheral blood levels of NEU-5123 in mice.
3.2.5 In vivo evaluation of NEU-5123
With decreased in vivo clearance rates and good exposure in the blood and brain, NEU-
5123 was progressed to an acute in vivo efficacy study. First a maximum tolerated dose (MTD)
study was performed. Mouse 1 was dosed with 5+15+30 m/kg to total 50 mg/kg/day, and Mouse
0 2 4 6 8 1 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
N E U -5 1 2 3 ; 1 0 m g /k g ip
M e a n B lo o d / B ra in c o n c e n tra t io n s
T im e (h r )
Blo
od
co
nc
en
tra
tio
n (
ng
/mL
) /
Bra
in c
on
ce
ntr
ati
on
(n
g/g
)
B lo o d C o n c e n tra tio n (n g /m L )
B lo o d C o n c e n tra tio n (n g /m L )
B ra in C o n c e n tra t io n (n g /g )
120
2 was dosed with 50+50 mg/kg to total 100 mg/kg/day. After a dose of 50 mg/kg was achieved the
mice showed toxicity and pain signs, and died within 24 hrs.
For the acute efficacy study NEU-5123 was dosed first at 10 mg/kg/day (Figure 3-4) which
showed an average extension of life of 8.8 days as compared to control mice. The dose of NEU-
5123 was then increased to 30 mg/kg/day (Figure 3-5). This higher dose resulted in the death of
all treated mice by day 5, confirming that this compound is highly toxic. This contradicts the in
vitro data which showed that NEU-5123 did not potently inhibit host cell lines and the selectivity
window was greater than 2 log units.
Figure 3-5. Efficacy of NEU-5123 in mice after a 10 mg/kg/day dose.
Efficacy Model (Acute)
Days
0 5 10 15 20 25 30
% S
urv
iva
l
0
20
40
60
80
100
Control (Vehicle) NEU5123 - 10 mg/kg/day
121
Figure 3-6. Efficacy of NEU-5123 in mice after a 30 mg/kg/day dose.
3.2.6 Kinase selectivity evaluation of NEU-5123 and NEU-5449
Since these compounds came from a kinase targeted library, it was hypothesized that they
could have off-target human kinase activity. Compounds NEU-5123 and NEU-5449 were profiled
at 5 μM for their percent inhibition against a panel of 45 human kinases (Figure 3-6). “Potent”
inhibition was defined as > 50%. NEU-5123 potently inhibited 14 kinases while NEU-5449
potently inhibited 32 kinases. This suggest further optimization is needed to tune selectivity versus
human kinases. The mouse kinome contains 510 orthologs for the 518 human kinases.150 It is
possible that the toxicity observed in the efficacy studies could be due to NEU-5123 hitting
multiple essential host kinases.
Efficacy Model (Acute)
Days
0 5 10 15 20 25 30
% S
urv
ival
0
20
40
60
80
100
Control (Vehicle) NEU5123 - 30 mg/kg/day
122
Figure 3-7. Human kinase profiles of NEU-5123 and NEU-5449.
3.3 Design, synthesis, and evaluation of NEU-1200 second-generation analogs
3.3.1 Rationale of NEU-1200 second-generation analog design
The PK study described above showed that NEU-5123 displayed reduced in vivo clearance
rates relative to NEU-1200. NEU-6018, which was synthesized after the testing of NEU-5123,
showed lower in vitro clearance rates suggesting that it would also be successful in vivo. Second-
generation analogs utilized the benzyl isopropylamine pendant at the 2-position while varying the
4-position to see if further improvements in the clearance could be obtained in combination with
the isopropylamine. NEU-6018 also showed improved activity (pEC50=8.26) over NEU-5123
(pEC50=7.71), and second-generation analogs would probe the activity range of new 4-position
123
substituents. Matched pair analogs with 4-position groups that were correlated with low compound
toxicity were synthesized in an attempt to reduce toxicity level of NEU-6018 while maintaining
its high potency. The next goal for this project would be to increase potency and selectivity to
enable lower doses in the animal studies to see a positive effect.
3.3.2 Synthesis of NEU-1200 second-generation analogs
Second-generation analogs were synthesized in a similar fashion as above (Scheme 3-10).
Starting with dihalide 3.13, Suzuki reaction at the 2-position with 4-formylphenylboronic acid
gave aldehyde 3.17 in 95% yield. Reductive amination with isopropylamine gave chloride 3.27a
in 94% yield. These high yielding reactions allowed for scale-up synthesis of intermediate 3.27a
for subsequent late stage Suzuki reactions at the 4-position (3.28a-h). Detosylation under basic
conditions gave final compounds 3.29a-3.29h. Chloride 3.17 was also coupled with two different
aryl substituents using Suzuki conditions to yield aryl aldehydes 3.27b and 3.27c. Intermediate
3.27b was first deprotected to give 3.28i followed by reductive amination with isopropylamine
(3.29i) and boc-deprotection with trifluoracetic acid to yield final compound 3.30. Aldehyde
intermediate 3.27c was coupled with pyrrolidine using reductive amination conditions. Subsequent
detosylation under basic conditions yielded final compound 3.29j. All second-generation
compounds are evaluated in Table 3-13 for their in vitro T. b. brucei activities.
124
Scheme 3-10. Synthesis of second-generation NEU-1200 analogs.
Reagents and conditions: (4-formylphenyl)boronic acid, Na2CO3, PdCl2(dppf)·CH2Cl2, 3:1
dioxane/H2O, 85 °C, 12 hr (95%); b) isopropylamine or pyrrolidine, NaBH3CN, 10% acetic
acid/methanol, 50 °C, 18 hr (94%, 93%); c) appropriate boronic acids/pinacol esters, Na2CO3,
tetrakis(triphenylphosphine)palladium(0), 3:1 dioxane/H2O, 85-90 °C, 18-48 hr (42-99%); d) 3M
NaOH, dioxane, 150 °C μwave, 15 min (31-69%), e) TFA, DCM, rt, 18 hr (59%).
3.3.3 Evaluation of NEU-1200 second-generation analogs
A library of second-generation 2,4-substituted azaindole analogs were analyzed for their
T. brucei inhibition and in vitro clearance rates (Table 3-13). In general, almost all analogs
maintained or improved clearance relative to NEU-6018. Substituting the methyl on the pyrazole
with a tetrahydropyran (NEU-6509) or piperidine (NEU-6097) further improved the clearance but
potency was reduced by a log unit. Changing the attachment position of the pyrazole to the 5-
position (NEU-6508) reduced the potency two log units (pEC50=6.77) though the clearance rates
were still acceptable. The unsaturated piperidine analog (NEU-6476) had a similar human liver
microsomal clearance rate (<3 μL/min/mg protein) and a 10-fold improved clearance rate of 1.1
125
μL/min/106 cells but had a 2-log unit reduction in potency as compared to NEU-6018. Pyridine
analogs NEU-6098, NEU-6477, and NEU-6478 had moderate clearance rates and reduced
potencies. NEU-6098, which has the nitrogen atom in the 3-position, has a higher pEC50 (7.82)
than the analog with a nitrogen atom in the 4-position (NEU-6477, pEC50=7.32), suggesting that
the position of the nitrogen atom could be beneficial to making a favorable interaction with a
potential target. Finally, sulfonamide NEU-6111 and methyl sulfone analog NEU-6712 lose
potency as compared to their matched pair parent molecules (NEU-6018 and NEU-5123, Table
3-8). NEU-6475 maintains its potency as compared to its matched pair (NEU-4917, Table 3-7).
Table 3-13. Activities and clearance rates of second-generation analogs of NEU-1200.
Entry R1 R2 T.b.b.
pEC50
HLM Clint
(µL/min/mg
protein)
RH Clint
(µL/min/106
cells)
Hit Criteria > 7 ≤ 47 ≤ 27
Lead
Criteria > 7.5 ≤ 9 ≤ 5
NEU-6018
8.26 <3 14
NEU-6077
6.71 59 10
NEU-6097
7.23 <3 1.8
126
NEU-6509
7.28 6.3 7.2
NEU-6508
6.77 17 10
NEU-6476
6.73 <3 1.1
NEU-6098
7.82 27 42
NEU-6477
7.32 16 13
NEU-6478
6.58 15 7.4
NEU-6111
6.64 6.8 8.0
NEU-6475
7.15 <3 5.8
NEU-6712
6.85 pending pending
127
Toxicities for second-generation analogs can be found in Table 3-14. All compounds tested
were found to be minimally active in HepG2 toxicity assays except for parent compound NEU-
6018 (pTC50 = 4.54). Similar results for seen for MRC5 host cell toxicity. Compounds NEU-6508
and NEU-6509 were not active against MRC5 cells with pTC50 values of 4.93 and 4.68
respectively. However, second-generation compounds were less selective than their first-
generation matched pairs. Most second-generation analogs had <100x selectivity windows.
Table 3-14. Toxicity of second-generation NEU-1200 analogs against HepG2 and MRC5 cell
lines.
Entry HepG2 pTC50 MRC5 pTC50 Tbb pEC50 -
MRC5 pTC50*
NEU-6018 4.54 5.88 2.38
NEU-6077 5.06 5.05 1.66
NEU-6097 5.86 5.80 1.43
NEU-6509 - 4.68 2.60
NEU-6508 - 4.93 1.84
NEU-6476 5.06 4.86 1.87
NEU-6098 5.40 5.26 2.56
NEU-6477 5.42 5.48 1.84
NEU-6478 5.32 5.11 1.47
NEU-6111 5.17 - 1.47*
NEU-6475 5.23 5.18 1.97
NEU-6712 - - - *HepG2 pTC50 value used instead of MRC5 pTC50
We note that the mammalian cell toxicity varies significantly based on cell lines. A plot of
the toxicity values for four different host cell lines for all cluster 32 compounds synthesized can
be seen in Figure 3-7. The two alternative host cell line (L6 and THP-1) are the host cells for T.
cruzi and Leishmaniasis assays respectively (data shown in Table 3-15). This plot shows how
widely toxicity varies for most of the compounds. Further exploration of this series is therefore
needed to better understand structure-toxicity relationships.
128
Figure 3-8. Graph of multiple cell line toxicity values of all synthesized NEU-1200.
3.4 Evaluation of cluster compounds against other parasites
A selection of analogs (selected on the basis of available quantities) was screened against
other protozoan parasites when enough material was available. Table 3-15 shows the potencies of
cluster 32 compounds against Trypanosoma cruzi, Leishmania major and L. donovani.
Compounds that were active against T. cruzi showed low selectivity versus L6 host cells. Anti-
trypanosomal activity correlates with host cell toxicity as shown in Figure 3-8. A similar trend
occurs for potency and toxicity when testing compounds against L. major and THP-1 host cells.
Potent compounds against L. major were toxic against host cells, for example NEU-5447
(pEC50=7.43, THP-1 pTC50=5.66). All cluster 32 compounds tested against L. donovani were
inactive.
4
4.5
5
5.5
6
6.5
7
7.5N
EU-1
20
0N
EU-1
93
6N
EU-4
47
1N
EU-4
47
2N
EU-4
78
6N
EU-4
81
2N
EU-4
81
3N
EU-4
81
4N
EU-4
81
5N
EU-4
84
4N
EU-4
84
5N
EU-4
87
7N
EU-4
91
7N
EU-4
91
8N
EU-4
92
7N
EU-4
92
8N
EU-4
92
9N
EU-4
97
3N
EU-4
97
4N
EU-4
99
5N
EU-5
12
3N
EU-5
12
4N
EU-5
30
6N
EU-5
32
3N
EU-5
44
6N
EU-5
44
7N
EU-5
44
8N
EU-5
44
9N
EU-5
45
2N
EU-5
82
8N
EU-5
82
9N
EU-5
93
6N
EU-5
93
7N
EU-5
93
8N
EU-6
01
8N
EU-6
02
5N
EU-6
04
4N
EU-6
04
7N
EU-6
04
8N
EU-6
04
9N
EU-6
07
2N
EU-6
07
3N
EU-6
07
6N
EU-6
07
7N
EU-6
09
7N
EU-6
09
8N
EU-6
11
1N
EU-6
47
5N
EU-6
47
6N
EU-6
47
7N
EU-6
47
8N
EU-6
50
8N
EU-6
50
9N
EU-6
71
2
pTC
50
Toxicity values (in vitro)
L6 THP-1 HepG2 MRC5
129
Table 3-15. Potencies and toxicities of NEU-1200 analogs against T. cruzi and Leishmania.
Entry T. cruzi
pEC50
L6
pTC50
L. major
pEC50
L. donovani
pEC50
THP-1
pTC50
NEU-1200 - >6.21 - - >6.21
NEU-1936 5.00 4.65 - <5.30 4.35
NEU-4471 4.97 4.30 - <5.30 4.51
NEU-4472 <4.70 - - <5.30 4.49
NEU-4786 <4.70 <4.30 - <5.30 <4.30
NEU-4812 <4.70 5.05 - <5.30 <4.30
NEU-4813 <4.70 4.34 - - <4.30
NEU-4814 <4.70 - - <5.30 <4.30
NEU-4815 <4.70 <4.30 - <5.30 <4.30
NEU-4844 <4.70 <4.30 - <5.30 <4.30
NEU-4845 - >6.21 - <5.30 <4.30
NEU-4877 4.77 - - <5.30 4.51
NEU-4917 - 5.73 - - 4.68
NEU-4918 - 5.81 - - 6.01
NEU-4927 7.22 >6.21 - - >6.21
NEU-4928 7.32 >6.21 7.00 - 7.35
NEU-4929 6.62 >6.21 - - >6.21
NEU-4973 6.93 >6.21 - - >6.21
NEU-4974 5.38 4.91 - <5.30 6.03
NEU-4995 7.06 >6.21 - - >6.21
NEU-5123 6.99 >6.21 7.26 - 6.86
NEU-5124 6.83 >6.21 6.65 <5.30 >6.21
NEU-5306 5.78 >6.21 - <5.30 >6.21
NEU-5323 - >6.21 5.77 - 6.03
NEU-5446 5.87 5.39 - - 5.65
NEU-5447 6.03 5.43 7.43 - 5.66
NEU-5448 <4.70 4.46 - <5.30 4.60
NEU-5449 6.05 5.22 7.34 - 5.55
NEU-5452 5.25 4.96 - - 4.80
NEU-5828 - 5.56 5.20 - -
NEU-5829 - 5.71 4.69 - -
NEU-5936 6.92 >6.21 7.27 - >6.21
NEU-5937 6.29 6.03 6.16 - >6.21
NEU-5938 6.75 >6.21 6.82 - >6.21
NEU-6018 7.15 >6.21 7.19 - >6.21
NEU-6025 5.93 5.67 6.28 <5.30 5.74
NEU-6044 6.20 6.14 6.35 <5.30 -
NEU-6047 6.28 >6.21 6.62 <5.30 -
NEU-6048 6.41 >6.21 6.90 - -
NEU-6049 7.09 >6.21 7.49 - -
NEU-6072 7.12 - - <5.30 -
130
NEU-6073 6.40 - - - -
NEU-6076 6.93 - - - -
NEU-6077 5.56 - - - -
NEU-6097 <4.70 5.37 5.21 - 4.66
NEU-6098 - - 6.95 - >6.21
NEU-6111 - - 4.67 - -
Figure 3-9. Correlation of T. cruzi potency and L6 host cell toxicity.
Solid line = 0 selectivity; dotted line = 10x selectivity. Compounds between the two lines are < 10x.
Synthesized compounds from the cluster 32 series were also tested for their activities
against various strains of P. falciparum. The pEC50 values for D6, W2, and C235 strains were
mostly between 6 and 7 (Table 3-16). Compounds with secondary amines in the 2-position showed
slightly higher potencies than tertiary amines.
131
Table 3-16. Potencies of NEU-1200 analogs against various P. falciparum strains.
Entry P. fal D6
pEC50
P. fal W2
pEC50
P. fal C235
pEC50
NEU-4928 6.60 6.96 6.59
NEU-5123 6.83 6.67 6.59
NEU-5124 6.84 6.82 6.56
NEU-5323 6.18 6.60 5.35
NEU-5447 6.18 6.50 5.99
NEU-5449 5.98 6.22 5.72
NEU-5828 6.64 6.45 6.38
NEU-5829 7.00 7.11 6.87
NEU-5936 6.88 6.73 6.70
NEU-5937 6.31 6.25 6.05
NEU-5938 6.57 6.59 6.53
NEU-6018 7.04 6.74 6.68
NEU-6044 7.19 6.80 6.98
NEU-6047 5.90 5.86 5.70
NEU-6048 5.37 5.20 5.12
NEU-6049 6.86 6.50 6.72
NEU-6072 6.23 6.38 6.08
NEU-6073 6.31 6.39 6.24
NEU-6076 6.39 6.44 6.18
NEU-6077 6.59 6.04 6.27
NEU-6097 6.84 6.89 6.69
NEU-6098 6.50 6.59 6.50
NEU-6111 6.49 6.42 6.29
NEU-6475 6.93 6.76 6.42
NEU-6476 6.49 6.23 6.26
NEU-6477 6.89 6.73 6.58
NEU-6478 6.78 6.72 6.45
NEU-6508 6.40 6.18 6.00
NEU-6509 6.78 6.37 6.14
3.5 Summary
A lead repurposing approach was taken to screen a kinase inhibitor library against T.
brucei, the causative agent of HAT. The HTS resulted in 797 primary hit compounds, from which
a cluster of 2,4-substituted azaindoles were chosen to progress. This cluster, deemed cluster 32,
132
was chosen for its good physicochemical properties, potency and selectivity, fast-acting and cidal
nature, and predictability for brain penetration, essential for stage 2 HAT. NEU-1200 was the best
compound from the initial cluster and pursued further in a PK study. The results of the study
showed high clearance rates, most likely due to metabolism of the methyl groups associated with
the pyrazole and pendant dimethylamine.
Structural modifications were made at the 2- and 4-positions of the azaindole core with the
focus on improving the in vitro clearance rates. An SAR summary can be seen in Figure 3-10.
NEU-5123 was shown to have improved in vitro clearance rates and comparable potency with
NEU-1200 and was thus progressed to a PK study. Results showed that NEU-5123 was not rapidly
cleared and showed good exposure in the blood and brain of the mice. Upon dosing in an acute
efficacy model, NEU-5123 was shown to extend mouse survival by 8.8 days when dosed at 10
mg/kg/day, however it was found to be highly toxic when dosed at 30 mg/kg/day. A human kinase
selectivity panel showed that NEU-5123 potently inhibited 14 human kinases.
Figure 3-10. SAR/SPR summary for synthesized NEU-1200 analogs.
133
Second-generation analogs were then synthesized to test the generalizability of using the
isopropylamine pendant to keep clearance rates low, while varying the 4-position substituent in an
attempt to increase the selectivity window. Second-generation compounds were shown to have
lower selectivity windows than their matched pairs and none were progressed further. This
suggests that the substituent at the 2-position may be more important for regulating the toxicity of
these compounds.
Finally, when cross-screening these compounds against other parasites a trend was seen
for correlation of increasing potency and toxicity, especially in T. cruzi and L6 cellular assays.
Although these compounds were also potent against multiple strains of P. falciparum the high
toxicity makes them unideal for further progression. Further work must be done to understand the
structure-toxicity relationship of the compounds in this series. The overall compound progression
can be seen in Figure 3-11.
Figure 3-11. Compound progressions of the series.
134
3.6 Future Directions
The second-generation of analogs resulted in NEU-6097, which showed reduced toxicity
in L6 and THP-1 cell lines. To determine if there is a correlation between toxicity in these cell
lines and in vivo toxicity, a maximum tolerated dose study should be performed. New analogs
should be synthesized to get a better understanding of structure-toxicity relationships suggested
modifications are summarized in Figure 3-12.
Figure 3-12. Summary of future directions for analog design.
135
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Chapter 4: Experimental section
4.1 General methods
All starting materials were commercially procured and were used without further
purification, unless specified. Reaction solvents were purified by passage through alumina
columns on a purification system manufactured by Innovative Technology (Newburyport, MA).
For microwave reactions a Biotage® Initiator+ or CEM Discover® SP was used and the
absorbance was set in accordance with the recommendations set by the manufacturer. NMR
spectra were obtained on Varian NMR systems, operating at 400 or 500 MHz for 1H acquisitions.
LCMS analysis was performed using a Waters Alliance reverse phase HPLC (columns Waters
SunFire C18 4.6 × 50 mm, 3.5 μm, or Waters SunFire C8 4.6 × 50 mm, 3.5 μm), with single-
wavelength UV−visible detector and LCT Premier time-of-flight mass spectrometer (electrospray
ionization) or Waters Micromass ZQ detector (electrospray ionization), or using Agilent 1100
series LCMS instrument, with the binary system water/acetonitrile containing 0.1 % of formic acid
as eluent. Optical rotations were obtained on a Jasco P-2000. Purification of intermediates and
final compounds was performed using silica gel chromatography on a Biotage® IsoleraTM One
Flash purification system. Where required, final compounds were purified by preparative reverse
phase HPLC (columns Waters Symmetry RP8 30 × 50 mm, 5 μm column, or OBD RP18 30 × 50
mm, 5 μm), with a single wavelength UV−visible detector and Waters Micromass ZQ
(electrospray ionization). All final compounds have purities greater than 95% based upon LC/MS
analysis.
150
4.2 Experimental details
4.2.1 Experimental procedures for Chapter 2
General Procedure for Salt Formation of NEU-1953:
The qualitative solubility of 1 was determined in various solvents (methanol, ethanol, isopropanol,
tetrahydrofuran, acetonitrile, ethyl acetate, and water). For each of the solvents, 2.0 mg of 1 as
added to vials. Solvents were added in 200 μL increments until the solid was completely dissolved.
With a solubility of about 2.75 mg/mL, ethanol was chosen as the solvent for the salt screen.
Compound 1 (20 mg, 0.05 mmol) was dissolved in ethanol (4 mL) using sonication and heat. Stock
solutions of various acids were made (1 M in ethanol). Acid solution was added to solution of 1
until a precipitate formed (different amounts needed for each acid). The precipitate was collected
by vacuum filtration and washed with ethanol.
Hydrochloride Salt (2.1).
1H NMR (500 MHz, DMSO-d6) δ ppm 9.04 (s, 2H), 8.91 (s, 1H), 8.81−8.86 (m, 1H), 8.78 (d, J =
5.9 Hz, 1H), 8.38−8.44 (m, 2H), 8.27−8.34 (m, 2H), 8.08−8.13 (m, 1H), 7.52−7.65 (m, 2H), 2.79
(s, 3H).
Sulfate Salt (2.2).
1H NMR (500 MHz, DMSO-d6) δ ppm 10.79−11.01 (m, 1H), 9.07 (s, 2H), 8.86−8.94 (m, 3H),
8.53−8.59 (m, 2H), 8.50 (d, J = 2.9 Hz, 1H), 8.28−8.34 (m, 2H), 3.34−3.40 (m, 2H), 3.17 (s, 3H),
2.88 (s, 3H).
151
Citrate Salt (2.3).
1H NMR (500 MHz, DMSO-d6) δ ppm 9.77−9.85 (m, 1H), 8.98 (s, 2H), 8.73 (s, 2H), 8.57 (d, J =
8.8 Hz, 1H), 8.31−8.35 (m, 2H), 8.23−8.26 (m, 1H), 8.17−8.20 (m, 1H), 7.96−8.02 (m, 1H), 4.34
(br. s., 13H), 3.85−3.96 (m, 4H), 3.72−3.83 (m, 7 H), 3.40−3.48 (m, 8 H), 2.53−2.71 (m,14H),
2.42 (s, 4H).
Methanesulfonate Salt (2.4).
1H NMR (500 MHz, DMSO-d6) δ ppm 10.63−10.88 (m, 1H), 9.62−9.94 (m, 1H), 9.07 (s, 2H),
8.77−8.96 (m, 4H), 8.41−8.62 (m, 3H), 8.30 (d, J= 1.5 Hz, 2H), 4.62−4.98 (m, 2H), 2.87 (s, 4H),
2.32 (s, 6 H).
Diethyl 2-(((4-bromophenyl)amino)methylene)malonate. (2.5) 4-Bromoaniline (5.00 g, 29.1
mmol) and diethyl 2-(ethoxymethylene)malonate (5.87 mL, 29.1 mmol) were combined in a vial
and stirred at room temperature until homogenous. The mixture was then heated at 100 °C for 2.5
h. The mixture was cooled to room temperature and the product was triturated with hexanes, giving
a precipitate. The precipitate was then collected by vacuum filtration, washed with hexanes, and
dried under vacuum overnight to afford the title compound as a white powder (9.47 g, 90% yield).
LCMS rt 3.64 min, [M+H]+ 341.9, 343.9 m/z; 1H NMR (500 MHz, CDCl3) ppm 11.00 (d, J=13.7
Hz, 1 H), 8.46 (d, J=13.7 Hz, 1 H), 7.48 (d, J=8.8 Hz, 2 H), 7.02 (d, J=8.8 Hz, 2 H), 4.28 (dq,
J=28.3, 6.8 Hz, 4 H), 1.36 (dt, J=24.9, 7.3 Hz, 6 H).
152
Ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate. (2.6) In a 3 neck flask with a stir bar,
diethyl 2-(((4-bromophenyl)amino)methylene)malonate (9.47 g, 27.7 mmol) was added with 6 mL
of diphenyl ether. The mixture was heated to reflux for 2 h with nitrogen bubbling into a side neck.
A precipitate formed and was collected by vacuum filtration, washed with hexanes, and dried under
vacuum overnight to afford the title compound as a tan powder (7.47 g, 91% yield). LCMS rt 2.13
min, [M+H]+ 295.9, 297.9 m/z; *Insufficient solubility to obtain an NMR spectrum.
6-Bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.7) In round bottom flask with a stir bar,
ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate (7.47 g, 25.2 mmol) and 2.5 M sodium
hydroxide (200 mL) were added. The suspension was heated to reflux for 3 h. The solution was
cooled to room temperature and acidified with aqueous hydrochloric acid to pH2 (3M, 25 mL).
The precipitate was collected by filtration, washed with water and dried under a vacuum to afford
the title compound as a light tan solid (6.57 g, 97% yield). LCMS rt 2.36 min, [M+H]+ 267.9,
269.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 14.97 (br. s., 1 H), 13.52 (br. s., 1 H), 8.92 (s,
1 H), 8.31 (d, J=2.5 Hz, 1 H), 8.01 (dd, J=8.8, 2.2 Hz, 1 H), 7.75 (d, J=9.1 Hz, 1 H).
153
6-Bromoquinolin-4(1H)-one (2.8) 6-Bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.57
g, 24.5 mmol) was dissolved in 46 mL of diphenyl ether. The reaction mixture was heated to reflux
for 3 h. The reaction mixture was cooled to room temperature, triturated with hexanes, and the
precipitate formed was collected by vacuum filtration to afford the title compound as a light tan
solid (5.32 g, 97% yield). LCMS rt 1.80 min, [M+H]+ 223.9, 225.9 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 11.92 (br. s., 1 H), 8.16 (d, J=2.4 Hz, 1 H), 7.95 (d, J=6.8 Hz, 1 H), 7.79 (dd,
J=8.8, 2.4 Hz, 1 H), 7.52 (d, J=8.8 Hz, 1 H), 6.08 (d, J=7.3 Hz, 1 H).
4-((4-Bromophenyl)sulfonyl)morpholine (2.9, 3.1b) 4-Bromobenzenesulfonyl chloride (200 mg,
0.78 mmol) was dissolved in THF (2 mL). Morpholine (74 μL) was added and the reaction mixture
was left stirring overnight at room temperature. The reaction mixture was neutralized with
saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times. The combined
organic layers were washed with water and with brine, dried using sodium sulfate, filtered, and
concentrated under reduced pressure to afford the title compound as white solid (196 mg, 82%
yield). LCMS [M+H]+ 307.1 m/z (Br79), 309.1 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm
7.89 (d, J=8.3 Hz, 2 H), 7.67 (d, J=8.8 Hz, 2 H), 3.54 - 3.72 (m, 4 H), 2.79 - 2.95 (m, 4 H).
154
4-((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine (2.10, 3.2b) 4-
((4-Bromophenyl)sulfonyl)morpholine (196 mg, 0.640 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-
bi(1,3,2-dioxaborolane) (243 mg, 0.960 mmol), [1,1′-
Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane
(PdCl2(dppf)·CH2Cl2) (26.1 mg, 0.032 mmol), and potassium acetate (188 mg, 1.92 mmol) were
added to a vial that was subsequently sealed and vacuum purged with nitrogen. A degassed solution
of dioxane (4 mL) was added to the vial. The reaction mixture was heated at 85 °C overnight and
monitored by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under
reduced pressure. The resulting residue was used crude in the next reaction. A quantitative yield
was assumed. LCMS [M+H]+ 354.1 m/z. 75% product, 25% boronic acid of product by LC trace.
6-(4-(Morpholinosulfonyl)phenyl)quinolin-4(1H)-one (2.11) 6-Bromoquinolin-4(1H)-one (250
mg, 1.12 mmol), (4-morpholinosulfonyl)phenyl)boronic acid (302 mg, 1.12 mmol), triethylamine
(467 μL, 3.35 mmol), bis(triphenylphosphine)palladium(II) dichloride (10 mg, 0.014 mmol) were
155
dissolved in a 1:1 solution of ethanol/water (10 mL). The reaction mixture was refluxed for 3 h.
The ethanol was removed by rotary evaporation; a precipitate formed and was collected by vacuum
filtration to afford the title compound as an off-white solid (349 mg, 84% yield). LCMS [M+H]+
371.0 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 11.92 (br. s., 1 H), 8.43 (d, J=2.0 Hz, 1 H), 8.08
(dd, J=8.5, 2.2 Hz, 1 H), 8.03 (d, J=8.8 Hz, 2 H), 7.96 (d, J=7.3 Hz, 1 H), 7.83 (d, J=8.8 Hz, 2 H),
7.69 (d, J=8.8 Hz, 1 H), 6.10 (d, J=7.3 Hz, 1 H), 3.61 - 3.69 (m, 4 H), 2.88 - 2.95 (m, 4 H).
4-((4-(4-Chloroquinolin-6-yl)phenyl)sulfonyl)morpholine. (2.12) 6-(4-
(Morpholinosulfonyl)phenyl)quinolin-4(1H)-one (342 mg, 0.942 mmol) was dissolved in neat
phosphorus (V) oxychloride (3 mL) and refluxed for 2.5 h after which the solvent was removed
by vacuum distillation. The crude residue was cooled in an ice water bath, diluted with
dichloromethane and neutralized with saturated sodium bicarbonate solution. The layers were
separated, and the aqueous layer extracted 3 times with dichloromethane. The combined organic
layers were washed with sodium bicarbonate and brine, dried with sodium sulfate, filtered, and
concentrated. The crude product was purified by column chromatography, eluting with a mobile
phase of 2% methanol/dichloromethane to afford the title compound as an off-white solid (249
mg, 69% yield). LCMS [M+H]+ 389.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.90 (d, J=4.4
Hz, 1 H), 8.47 (d, J=2.0 Hz, 1 H), 8.22 - 8.30 (m, 2 H), 8.15 (d, J=8.3 Hz, 2 H), 7.89 (d, J=8.3 Hz,
2 H), 7.85 (d, J=4.9 Hz, 1 H), 3.57 - 3.74 (m, 4 H), 2.82 - 3.07 (m, 4 H).
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6-(4-(Morpholinosulfonyl)phenyl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-1912 (2.13) 4-((4-
(4-Chloroquinolin-6-yl)phenyl)sulfonyl)morpholine (50 mg, 0.128 mmol), pyrimidine-4-amine
(24.5 mg, 0.257 mmol), potassium tert-butoxide (14.4 mg, 0.126 mmol), xantphos (3.0 mg, 0.005
mmol), and tris(dibenzylideneacetone)dipalladium(0) (5.0 mg, 0.005 mmol) were added to a vial
which was vacuum purged with nitrogen, after which 1,4-dioxane (2 mL) was added. The reaction
mixture was heated at 101 °C overnight. The mixture was cooled to room temperature, poured
over water, and extracted with ethyl acetate 3 times. The combined organic layers were washed
with water and brine, dried over sodium sulfate, filtered, and concentrated. The product was
purified by column chromatography, eluting with a mobile phase of 4% methanol/dichloromethane
to afford the title compound as a pale yellow solid (39.4 mg, 68% yield). LCMS [M+H]+ 448.1
m/z; 1H NMR (500 MHz, DMSO-d6) ppm 10.05 (s, 1 H), 8.79 - 8.85 (m, 2 H), 8.76 (d, J=2.0
Hz, 1 H), 8.52 (d, J=5.9 Hz, 1 H), 8.38 (d, J=4.9 Hz, 1 H), 8.14 - 8.21 (m, 3 H), 8.10 - 8.14 (m, 1
H), 7.87 - 7.93 (m, 2 H), 7.30 (dd, J=5.9, 1.0 Hz, 1 H), 3.64 - 3.70 (m, 4 H), 2.89 - 2.97 (m, 4 H).
5-Bromo-2-(4-methylpiperazin-1-yl)pyrimidine. (2.18) 5-Bromo-2-chloropyrimidine (2.00 g, 10.3
mmol), 1-methylpiperazine (4.59 mL, 41.4 mmol), diisopropylethylamine (5.40 mL, 31.0 mmol)
were dissolved in tert-butanol (35 mL). The reaction mixture was heated at 85 °C overnight. Water
157
was added, a precipitate formed and was collected by vacuum filtration to afford the title
compound as a peach colored solid (2.30 g, 86% yield). LCMS [M+H]+ 335.0 m/z (Br79), 337.0
m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.44 (s, 2 H), 3.64 - 3.72 (m, 4 H), 2.30 - 2.37
(m, 4 H), 2.20 (s, 3 H).
2-(4-Methylpiperazin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine. (2.19) 5-
Bromo-2-(4-methylpiperazin-1-yl)pyrimidine (100 mg, 0.389 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (296 mg, 1.17 mmol), PdCl2(dppf)·CH2Cl2 (22.2 mg,
0.027 mmol), and potassium acetate (114 mg, 1.17 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane (5 mL) was
added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure.
The resulting residue was used crude in the next reaction. A quantitative yield was assumed. LCMS
[M+H]+ 301.2 m/z. 50% product, 50% boronic acid of product by LC trace.
158
7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one. (2.20) 2-(4-methylpiperazin-1-
yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (1.2 eq) was dissolved in a 3:1
dioxane/water (0.13 M) mixture. To this was added 7-bromoquinolin-4(1H)-one (1 eq), potassium
carbonate (3 eq) and PdCl2(dppf)·CH2Cl2 (0.05 eq) and the reaction mixture was degassed before
being transferred to the microwave for 30 min at 130 °C (high absorbance). Reaction monitored
by LCMS analysis and once complete, the reaction mixture was filtered through Celite and the
crude material was purified by column chromatography, eluting with the specified mobile phase.
A precipitate was isolated by vacuum filtration and washed with water to afford the title compound
as a tan solid, (411 mg, 85%). LCMS [M+H]+ 322.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm
8.77 (s, 1 H), 8.08 - 8.15 (m, 1 H), 7.87 - 7.92 (m, 1 H), 7.68 - 7.72 (m, 1 H), 7.57 - 7.63 (m, 1 H),
6.31 (s, 2 H), 5.99 - 6.05 (m, 1 H), 3.76 - 3.85 (m, 4 H), 2.34 - 2.42 (m, 4 H), 2.22 (s, 3 H).
4-chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline. (2.21) 7-(2-(4-methylpiperazin-
1-yl)pyrimidin-5-yl)quinolin-4(1H)-one (103 mg, 0.32 mmol) was suspended in neat phosphorus
oxychloride (excess, 1.0 mL). The reaction mixture was refluxed overnight. Phosphorus
oxychloride was then removed by vacuum distillation. The resulting residue was put in an ice bath,
159
diluted with dichloromethane, and quenched using a saturated aqueous sodium bicarbonate
solution. The aqueous layer was extracted 3 times with dichloromethane. The combined organic
layers were washed with water and a saturated sodium chloride solution, before being dried with
sodium sulfate. The volatiles were removed in vacuo to obtain the title compound as a light tan
solid (77.2 mg, 71%). LCMS [M+H]+ 340.1 m/z (Cl35), 342.2 m/z (Cl37); 1H NMR (500 MHz,
CHLOROFORM-d) ppm 8.80 (d, J=4.9 Hz, 1 H), 8.73 (s, 2 H), 8.31 (d, J=8.8 Hz, 1 H), 8.23 (d,
J=1.5 Hz, 1 H), 7.78 - 7.83 (m, 1 H), 7.48 (d, J=4.9 Hz, 1 H), 3.91 - 3.98 (m, 4 H), 2.51 (t, J=4.9
Hz, 4 H), 2.37 (s, 3 H).
7-(2-(4-Methylpiperazin-1-yl)pyrimidin-5-yl)-N-(1-methylpiperidin-4-yl)quinolin-4-amine, tri-
formate salt. NEU-4920 (2.22a) 4-Chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline
(39) (20 mg, 0.059 mmol), 1-methylpiperidin-4-amine (14.8 µL, 0.12 mmol),
tris(dibenzylideneacetone)dipalladium(0) (2.7 mg, 0.003 mmol), 2-dicyclohexylphosphino-
2′,4′,6′-triisopropylbiphenyl (2.8 mg, 0.006 mmol), and potassium tert-butoxide (19.8 mg, 0.177
mmol) were added to a vial that was filled with nitrogen and evacuated three times. Dry, degassed
dioxane (1 mL) was then added. The reaction mixture was heated at 90 °C overnight. LCMS
analysis revealed no remaining starting material and a mass consistent with the desired product.
The reaction mixture was filtered through Celite and purified by column chromatography eluting
160
with 0-20% (10% ammonium hydroxide/methanol)/dichloromethane. The material was further
purified by prep HPLC (method: 99-95_6p5min) to afford the title compound as a yellow solid
(12 mg, 47%). LCMS [M+H]+ 418.1 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.82 (s,
2 H), 8.52 (d, J=8.8 Hz, 1 H), 8.47 (s, 3 H), 8.41 (d, J=6.8 Hz, 1 H), 8.01 (s, 1 H), 7.93 (d, J=8.8
Hz, 1 H), 6.94 (d, J=7.3 Hz, 1 H), 4.06 (br. s., 5 H), 3.50 (br. s., 2 H), 3.06 (br. s., 2 H), 2.95 (t,
J=5.1 Hz, 4 H), 2.62 (s, 6 H), 2.27 (d, J=12.7 Hz, 2 H), 2.03 - 2.17 (m, 2 H).
7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-4-amine.
NEU-4987 (2.22b) 4-chloro-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinoline (39) (20 mg,
0.059 mmol), 2-(pyrrolidin-1-yl)ethan-1-amine (14.8 µL, 0.12 mmol),
Tris(dibenzylideneacetone)dipalladium(0) (2.7 mg, 0.003 mmol), 2-Dicyclohexylphosphino-
2′,4′,6′-triisopropylbiphenyl (2.8 mg, 0.006 mmol), and potassium tert-butoxide (19.8 mg, 0.177
mmol) were added to a vial that was filled with nitrogen and evacuated three times. Dry, degassed
dioxane (1.5 mL) was then added. The reaction mixture was heated at 90 °C overnight. LCMS
analysis revealed no remaining starting material and a mass consistent with the desired product.
The reaction mixture was filtered through Celite and purified by column chromatography eluting
with 2-20% (5% ammonium hydroxide/methanol)/dichloromethane to afford the title compound
as a yellow solid (36 mg, 59%). LCMS [M+H]+ 418.3 m/z; 1H NMR (399 MHz, DMSO-d6) ppm
8.87 (s, 2 H), 8.40 (d, J=5.9 Hz, 1 H), 8.25 (d, J=8.8 Hz, 1 H), 8.01 (s, 1 H), 7.75 (d, J=8.8 Hz, 1
161
H), 7.20 (br. s., 1 H), 6.45 (d, J=5.1 Hz, 1 H), 3.81 (m, J=4.4 Hz, 1 H), 3.38 - 3.47 (m, 1 H), 2.73
(t, J=6.6 Hz, 2 H), 2.55 (br. s., 4 H), 2.38 (t, J=4.8 Hz, 4 H), 2.18 - 2.26 (m, 3 H), 1.70 (br. s., 4
H).
6-Bromo-4-chloroquinoline.151-152 (2.23) 7-Bromoquinolin-4(1H)-one (500 mg, 1.12 mmol) was
dissolved in phosphorus oxychloride (3 mL). The reaction mixture was refluxed overnight. Excess
solvent was removed by vacuum distillation. The remaining brown residue was neutralized using
saturated sodium bicarbonate solution. The resulting precipitate was collected by vacuum filtration
and washed with water to afford the title compound as a light tan solid (440 mg, 81% yield). LCMS
[M+H]+ 241.8 m/z (Br79), 243.9 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.79
(#1, d, J=4.9 Hz, 1 H), 8.32 (#5, s, 1 H), 8.12 (#3, d, J=9.3 Hz, 1 H), 7.75 (#4, dt, J=8.9, 1.6 Hz, 1
H), 7.52 (#2, dd, J=4.6, 1.2 Hz, 1 H).
General Procedure A.
Compound 11 was dissolved in n-butanol (0.1 M). Appropriate saturated primary amine (4 equiv)
and diisopropylethylamine (10 equiv) were added. The reaction was heated in the microwave at
200 °C for 3 h. n-Butanol was removed in vacuo, and the reaction mixture was purified by column
chromatography, eluting with specified mobile phase.
162
7-Bromo-N-cyclohexylquinolin-4-amine. (2.24a) Prepared via General Procedure A. The crude
material was purified by column chromatography eluting with 1-20% methanol/dichloromethane
to afford the title compound as an off-white solid (26 mg, 41%). LCMS [M+H]+ 305.1 m/z (Br79),
307.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.49 (d, J=5.9 Hz, 1 H), 8.12
(d, J=1.5 Hz, 1 H), 7.57 (d, J=8.8 Hz, 1 H), 7.46 (dd, J=9.0, 1.7 Hz, 1 H), 6.44 (d, J=5.4 Hz, 1 H),
4.93 (d, J=6.8 Hz, 1 H), 3.43 - 3.56 (m, 1 H), 2.08 - 2.21 (m, 2 H), 1.78 - 1.89 (m, 2 H), 1.66 - 1.76
(m, 1 H), 1.39 - 1.52 (m, 2 H), 1.22 - 1.38 (m, 3 H).
N1-(7-bromoquinolin-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine. (2.24b) Prepared via
General Procedure A. The crude material was purified by column chromatography, eluting with
1-20% methanol/dichloromethane to afford the title compound as an off-white solid (15.8 mg,
22%). LCMS [M+H]+ 348.1 m/z (Br79), 350.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-
d) ppm 8.51 (d, J=4.9 Hz, 1 H), 8.13 (d, J=2.0 Hz, 1 H), 7.56 (d, J=8.8 Hz, 1 H), 7.48 (dd, J=8.8,
2.0 Hz, 1 H), 6.45 (d, J=5.4 Hz, 1 H), 4.83 (d, J=6.8 Hz, 1 H), 3.39 - 3.49 (m, 1 H), 2.33 (s, 6 H),
2.24 - 2.32 (m, 2 H), 2.12 - 2.22 (m, 1 H), 2.04 (d, J=12.7 Hz, 2 H), 1.27 - 1.51 (m, 4 H).
163
7-bromo-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine. (2.24c) Prepared via General
Procedure A. The crude material was purified by column chromatography eluting with 1-20%
methanol/dichloromethane to afford the title compound as a yellow solid (46 mg, 74%). LCMS
[M+H]+ 307.0 m/z (Br79), 309.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.28
(d, J=5.4 Hz, 1 H), 7.95 (d, J=2.0 Hz, 1 H), 7.73 (d, J=9.3 Hz, 1 H), 7.42 (dd, J=8.8, 2.0 Hz, 1 H),
6.39 (d, J=5.9 Hz, 1 H), 3.98 (dt, J=11.5, 3.3 Hz, 2 H), 3.75 (s, 1 H), 3.66 (tt, J=10.7, 4.2 Hz, 1
H), 3.50 (td, J=11.8, 2.2 Hz, 2 H), 1.98 - 2.05 (m, 2 H), 1.56 - 1.67 (m, 2 H).
7-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-amine. (2.24d) Prepared via General
Procedure A. The crude material was purified by column chromatography eluting with 5-15%
methanol/dichloromethane. The resulting residue was washed with hexanes, after which the title
compound was collected by vacuum filtration as a yellow solid (47 mg, 71%). LCMS [M+H]+
321.1 m/z (Br79), 323.0 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.37 (d, J=5.4 Hz, 1
H), 8.22 (d, J=8.8 Hz, 1 H), 7.93 (d, J=2.0 Hz, 1 H), 7.55 (dd, J=8.8, 2.0 Hz, 1 H), 7.39 (t, J=5.4
Hz, 1 H), 6.52 (d, J=5.4 Hz, 1 H), 3.85 (dd, J=11.2, 2.9 Hz, 2 H), 3.24 - 3.30 (m, 2 H), 1.91 - 2.01
(m, 1 H), 1.68 (d, J=12.7 Hz, 2 H), 1.25 (qd, J=12.2, 4.4 Hz, 2 H), 1.07 (s, 2 H).
164
(1R,2R)-2-((7-bromoquinolin-4-yl)amino)cyclohexan-1-ol. (2.24e) Prepared via General
Procedure A, heating temperature was 180 °C instead of 200 °C. The crude material was purified
by column chromatography eluting with 1-8% methanol (+10% ammonium
hydroxide)/dichloromethane, to afford the product compound as an off-white solid (95 mg, 98%).
LCMS [M+H]+ 321.3 m/z (Br79), 323.1 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm 8.58
(d, J=9.3 Hz, 1 H), 8.41 (d, J=6.8 Hz, 2 H), 8.11 (d, J=2.0 Hz, 1 H), 7.76 (dd, J=9.3, 2.0 Hz, 1 H),
6.86 (d, J=6.3 Hz, 1 H), 5.00 (br. s., 1 H), 3.53 - 3.66 (m, 2 H), 2.84 - 3.06 (m, 1 H), 1.86 - 1.97
(m, 2 H), 1.64 - 1.76 (m, 2 H), 1.28 - 1.51 (m, 3 H).
7-bromo-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinolin-4-amine (2.24f) Prepared via General
Procedure A, heating temperature was 180 °C instead of 200 °C. The crude material was purified
by column chromatography eluting with 1-20% methanol/dichloromethane, to afford the product
compound as a light brown solid (196 mg, 71%).
LCMS [M+H]+ 335.0 m/z (Br79), 337.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-d)
ppm 8.48 (d, J=5.9 Hz, 1 H), 8.15 (d, J=1.5 Hz, 1 H), 7.70 (d, J=8.8 Hz, 1 H), 7.49 (dd, J=8.8, 2.0
Hz, 1 H), 6.42 (d, J=5.4 Hz, 1 H), 5.44 (br. s., 1 H), 4.00 (dd, J=11.2, 3.9 Hz, 2 H), 3.36 - 3.45 (m,
4 H), 2.30 - 2.75 (m, 1 H), 1.72 - 1.80 (m, 2 H), 1.69 (d, J=12.7 Hz, 2 H), 1.34 - 1.48 (m, 2 H).
165
N-cyclohexyl-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-amine, mono-formate salt.
NEU-5144 (2.25a) The appropriate boronate (1.2 equiv) was dissolved in a 3:1 dioxane/water
(0.13 M) mixture. To this was added 7-bromoquinolin-4(1H)-one (2.24a) (1 equiv), potassium
carbonate (3 equiv), and PdCl2(dppf)·CH2Cl2 (0.05 equiv), and the reaction mixture was degassed
before being transferred to the microwave for 30 min at 130 °C (high absorbance). The reaction
was monitored by LCMS analysis and once complete, the reaction mixture was filtered through
Celite and the crude material was purified by column chromatography, eluting with eluting with
1-20% methanol/dichloromethane. The material was further purified by prep HPLC (method: 95-
70_6p5min). The residue was triturated with dichloromethane and the subsequent precipitate was
isolated by vacuum filtration; the title compound was afforded as tan solid (7.3 mg, 11%). LCMS
[M+H]+ 403.3 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.88 (s, 2 H), 8.45 (d, J=9.3 Hz, 1 H),
8.40 (d, J=5.4 Hz, 1 H), 8.15 (s, 2 H), 8.00 (s, 1 H), 7.81 (d, J=8.8 Hz, 1 H), 7.31 - 7.40 (m, 1 H),
6.60 (d, J=5.4 Hz, 1 H), 3.82 (br. s., 4 H), 2.41 (t, J=4.6 Hz, 4 H), 2.24 (s, 3 H), 2.00 (br. s., 2 H),
1.80 (br. s., 2 H), 1.67 (d, J=11.7 Hz, 1 H), 1.35 - 1.48 (m, 4 H), 1.13 - 1.26 (m, 1 H). 1H NMR
(500 MHz, METHANOL-d4) ppm 8.87 (s, 2 H), 8.55 (d, J=8.8 Hz, 1 H), 8.38 (br. s., 2 H), 8.35
(d, J=6.8 Hz, 1 H), 7.99 (br. s., 1 H), 7.96 (d, J=9.3 Hz, 1 H), 6.90 (d, J=6.8 Hz, 1 H), 4.13 (br. s.,
4 H), 3.78 - 3.90 (m, 1 H), 3.08 (br. s., 4 H), 2.74 (s, 3 H), 2.12 (d, J=9.8 Hz, 2 H), 1.92 (d, J=10.7
Hz, 2 H), 1.78 (d, J=12.7 Hz, 1 H), 1.46 - 1.64 (m, 4 H), 1.32 (d, J=10.7 Hz, 1 H).
166
General Procedure B.
The appropriate boronate (1.2 equiv) was dissolved in a 1:2 water/ethanol (0.13 M) mixture. To
this were added 2.24b-f (1 equiv), cesium carbonate (4 equiv), and
tetrakis(triphenylphosphine)palladium(0) (5 mol %), and the reaction mixture was degassed before
being transferred to the microwave for 10 min at 130°C (high absorbance). The reaction was
monitored by LCMS analysis and once complete, the reaction mixture was filtered through Celite
and the crude material was purified by column chromatography, eluting with the specified mobile
phase.
N1,N1-Dimethyl-N4-(7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-yl)cyclohexane-1,4-
diamine. NEU-5375 (2.25b) Prepared via General Procedure B with some exceptions. The
reaction mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction
was heated for 48 h at 80 °C. The crude material was purified by column chromatography eluting
with 4-10% methanol (10% ammonium hydroxide)/dichloromethane to afford the title compound
as an off-white solid, 28 mg (27%). LCMS [M+H]+ 446.3 m/z; 1H NMR (500 MHz, DMSO-d6)
ppm 8.86 (s, 2 H), 8.37 (d, J=5.4 Hz, 1 H), 8.34 (d, J=8.8 Hz, 1 H), 7.99 (d, J=2.0 Hz, 1 H), 7.71
(dd, J=8.8, 2.0 Hz, 1 H), 6.79 (d, J=7.8 Hz, 1 H), 6.48 (d, J=5.4 Hz, 1 H), 3.78 - 3.83 (m, 4 H),
167
3.41 - 3.50 (m, 1 H), 2.39 (t, J=5.1 Hz, 4 H), 2.23 (s, 3 H), 2.20 (s, 7 H), 2.08 (d, J=10.7 Hz, 2 H),
1.87 (d, J=11.2 Hz, 2 H), 1.39 (m, J=14.2 Hz, 4 H).
7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine.
NEU-5139 (2.25c) Prepared via General Procedure B. The crude material was purified by
column chromatography eluting with 1-20% methanol (+50% ammonium
hydroxide)/dichloromethane. The resulting product was triturated with ether and the precipitate
was collected by vacuum filtration to afford the title compound as a light brown solid (22 mg,
36%). LCMS [M+H]+ 405.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.74 (s, 2 H), 8.36
(d, J=5.9 Hz, 1 H), 8.27 (d, J=8.8 Hz, 1 H), 7.92 (s, 1 H), 7.69 (dd, J=8.8, 1.5 Hz, 1 H), 6.65 (d,
J=5.9 Hz, 1 H), 3.99 - 4.08 (m, 2 H), 3.85 - 3.95 (m, 5 H), 3.60 (m, J=11.2, 11.2 Hz, 2 H), 2.52 (t,
J=5.1 Hz, 4 H), 2.34 (s, 3 H), 2.01 - 2.11 (m, 2 H), 1.68 - 1.82 (m, 2 H).
168
7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-
amine. NEU-5326 (2.25d) Prepared via General Procedure B with some exceptions. The reaction
mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction was heated
for 18 h at 80 °C. The crude material was purified by column chromatography eluting with 2-20%
methanol/dichloromethane. The compound was further purified by column chromatography
eluting with 1-8% methanol (+5% ammonium hydroxide)/dichloromethane to afford the title
compound as a colorless solid, (26 mg, 44%). LCMS [M+H]+ 419.2 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 8.86 (s, 2 H), 8.38 (d, J=5.4 Hz, 1 H), 8.31 (d, J=8.8 Hz, 1 H), 8.00 (d, J=1.5
Hz, 1 H), 7.72 (dd, J=8.8, 2.0 Hz, 1 H), 7.25 (t, J=5.6 Hz, 1 H), 6.46 (d, J=5.9 Hz, 1 H), 3.86 (dd,
J=11.2, 2.9 Hz, 2 H), 3.77 - 3.83 (m, 4 H), 3.28 (m, J=10.3 Hz, 2 H), 3.18 (t, J=6.1 Hz, 2 H), 2.38
(t, J=4.9 Hz, 4 H), 2.22 (s, 3 H), 1.93 - 2.03 (m, 1 H), 1.69 (dd, J=12.5, 1.2 Hz, 2 H), 1.26 (qd,
J=12.2, 3.9 Hz, 2 H).
(1R,2R)-2-((7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-yl)amino)cyclohexan-1-ol.
NEU-5376 (2.25e) Prepared via General Procedure B with some exceptions. The reaction
169
mixture was run in a 3:1 mixture of degassed dioxane/water (0.08 M) and the reaction was heated
for 48 h at 80 °C. The crude material was purified by column chromatography eluting with 4-8%
methanol (+10% ammonium hydroxide)/dichloromethane to afford the title compound as a light
brown solid, (46 mg, 38%). [α]D22 + 0.57 ± 0.28 (c 1.1, 1:10 DCM/CH3OH); LCMS [M+H]+ 419.2
m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.87 (s, 2 H), 8.35 (d, J=2.0 Hz, 1 H), 8.34 (s, 1 H),
7.99 (d, J=2.0 Hz, 1 H), 7.72 (dd, J=8.8, 2.0 Hz, 1 H), 6.72 (d, J=7.8 Hz, 1 H), 6.51 (d, J=5.9 Hz,
1 H), 4.74 (br. s., 1 H), 3.92 (br. s., 1 H), 3.74 - 3.85 (m, 4 H), 3.52 - 3.62 (m, 1 H), 2.35 - 2.43 (m,
4 H), 2.23 (s, 3 H), 1.98 (m, J=12.2 Hz, 2 H), 1.68 (m, J=2.0 Hz, 2 H), 1.31 (m, J=2.9 Hz, 4 H).
N-cyclohexyl-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)quinolin-4-amine, mono-formate salt.
NEU-6734 (2.25f) Prepared via General Procedure B. The crude material was purified by column
chromatography, eluting with 1-20% methanol/dichloromethane. The residue was triturated with
dichloromethane and the subsequent precipitate was isolated by vacuum filtration, the title
compound was afforded as a grey solid (93 mg, 44%). LCMS [M+H]+ 433.4 m/z; 1H NMR (500
MHz, METHANOL-d4) δ ppm 8.70 (s, 2 H), 8.34 (d, J=5.4 Hz, 1 H), 8.14 (d, J=8.3 Hz, 1 H), 7.90
(d, J=1.5 Hz, 1 H), 7.61 (dd, J=8.8, 1.5 Hz, 1 H), 6.44 (d, J=5.9 Hz, 1 H), 3.83 - 3.97 (m, 6 H),
3.35 - 3.45 (m, 4 H), 2.49 (t, J=4.9 Hz, 4 H), 2.33 (s, 3 H), 1.65 - 1.74 (m, 5 H), 1.27 - 1.39 (m, 2
H).
170
General procedure C.
4-((4-(4-chloroquinolin-6-yl)phenyl)sulfonyl)morpholine (50 mg, 0.13 mmol) was dissolved in 1
mL of n-butanol. The respective amine (4 eq) and diisopropylethylamine (10 eq) were added to
the solution which was subsequently heated overnight at 180 °C in a sealed vial. The solvent was
removed, and the resulting solid was purified by column chromatography using conditions
described below.
N-cyclohexyl-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine NEU-5533 (2.26a) Prepared via
General Procedure C. The reaction was purified by column chromatography using a mobile
phase of 0-100% ethyl acetate/dichloromethane followed by 0-20% methanol/ethyl acetate, to
afford the title compound as a light tan solid (8 mg, 14% yield). LCMS [M+H]+ 452.2 m/z; 1H
NMR (500 MHz, METHANOL-d4) ppm 8.86 (d, J=1.5 Hz, 1 H), 8.36 (d, J=6.8 Hz, 1 H), 8.26
- 8.32 (m, 1 H), 8.08 (d, J=8.8 Hz, 2 H), 7.96 (d, J=8.8 Hz, 1 H), 7.93 (d, J=8.3 Hz, 2 H), 6.95 (d,
J=7.3 Hz, 1 H), 3.70 - 3.74 (m, 4 H), 3.63 (d, J=11.2 Hz, 2 H), 2.98 - 3.04 (m, 4 H), 2.14 (d, J=12.2
Hz, 2 H), 1.91 (d, J=13.2 Hz, 2 H), 1.78 (d, J=13.2 Hz, 1 H), 1.47 - 1.65 (m, 4 H).
171
N-(1-methylpiperidin-4-yl)-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine NEU-5537
(2.26b) Prepared via General Procedure C. The reaction was purified by column chromatography
using a mobile phase of 0-30% methanol/ethyl acetate, to afford the title compound as an orange
solid (12 mg, 20% yield). LCMS [M+H]+ 467.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66
(s, 1 H), 8.40 (d, J=5.4 Hz, 1 H), 8.13 (d, J=8.3 Hz, 2 H), 7.97 - 8.02 (m, 1 H), 7.87 (dd, J=8.5, 6.1
Hz, 3 H), 7.08 (d, J=7.3 Hz, 1 H), 6.57 (d, J=5.4 Hz, 1 H), 3.62 - 3.69 (m, 4 H), 2.93 (d, J=3.9 Hz,
4 H), 2.84 (d, J=11.2 Hz, 2 H), 2.22 (d, J=8.3 Hz, 4 H), 2.06 (t, J=11.2 Hz, 2 H), 1.99 (d, J=11.7
Hz, 2 H), 1.67 (d, J=11.2 Hz, 2 H).
N1,N1-dimethyl-N4-(6-(4-(morpholinosulfonyl)phenyl)quinolin-4-yl)cyclohexane-1,4-diamine
NEU-6735 (2.26c) Prepared via General Procedure C. The reaction was purified by column
chromatography using a mobile phase of 1-20% methanol/dichloromethane, to afford the title
compound as a light brown solid (64 mg, 50% yield). LCMS [M+H]+ 495.2 m/z; 1H NMR (500
MHz, METHANOL-d4) δ ppm 8.52 (d, J=1.5 Hz, 1 H), 8.35 (d, J=5.9 Hz, 1 H), 8.01 (d, J=8.3 Hz,
2 H), 7.96 (dd, J=8.8, 1.5 Hz, 1 H), 7.87 (d, J=8.3 Hz, 1 H), 7.83 (d, J=8.3 Hz, 2 H), 6.61 (d, J=5.9
172
Hz, 1 H), 3.66 - 3.72 (m, 4 H), 3.58 (br. s., 1 H), 2.93 - 3.01 (m, 4 H), 2.58 (br. s., 1 H), 2.45 (s, 6
H), 2.23 (br. s., 2 H), 2.06 (br. s., 2 H), 1.54 (m, J=9.5, 9.5 Hz, 4 H).
6-(4-(morpholinosulfonyl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-4-amine NEU-5536
(2.26d) Prepared via General Procedure C. The reaction was purified by column chromatography
using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-20% methanol/ethyl
acetate, to afford the title compound as a dark yellow solid (35.3 mg, 59% yield). LCMS [M+H]+
467.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.65 (br. s., 1 H), 8.43 (d, J=5.4 Hz, 1 H), 8.13
(d, J=7.8 Hz, 2 H), 8.03 (d, J=8.8 Hz, 1 H), 7.88 (dd, J=11.7, 8.8 Hz, 3 H), 7.53 (br. s., 1 H), 6.54
(d, J=4.9 Hz, 1 H), 3.65 (d, J=4.4 Hz, 4 H), 3.49 (br. s., 2 H), 2.93 (br. s., 4 H), 2.81 (br. s., 2 H),
2.61 (br. s., 3 H), 2.34 (s, 1 H), 1.72 (br. s., 4 H), 1.23 (br. s., 2 H).
6-(4-(morpholinosulfonyl)phenyl)-N-(tetrahydro-2H-pyran-4-yl)quinolin-4-amine NEU-5825
(2.26e) Prepared via General Procedure C. The reaction was purified by column chromatography
using a mobile phase of 0-10% methanol/ethyl acetate, to afford the title compound as an off white
173
solid (29 mg, 50% yield). LCMS [M+H]+ 454.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66
(s, 1 H), 8.41 (d, J=5.9 Hz, 1 H), 8.13 (d, J=8.8 Hz, 2 H), 8.01 (dd, J=8.5, 1.7 Hz, 1 H), 7.87 (t,
J=8.3 Hz, 3 H), 7.10 (d, J=7.3 Hz, 1 H), 6.65 (d, J=5.9 Hz, 1 H), 3.95 (d, J=9.8 Hz, 2 H), 3.77 -
3.87 (m, 1 H), 3.63 - 3.68 (m, 4 H), 3.50 (t, J=11.5 Hz, 2 H), 2.93 (d, J=4.4 Hz, 4 H), 1.98 (d,
J=14.2 Hz, 2 H), 1.66 (dd, J=11.2, 3.4 Hz, 2 H).
6-(4-(morpholinosulfonyl)phenyl)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinolin-4-amine NEU-
5534 (2.26f) Prepared via General Procedure C. The reaction was purified by column
chromatography using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-
20% methanol/ethyl acetate, to afford the title compound as a light tan solid (11.7 mg, 28% yield).
LCMS [M+H]+ 468.2 m/z; 1H NMR (500 MHz, METHANOL-d4) δ ppm 8.57 (s, 1 H), 8.38 (d,
J=6.3 Hz, 1 H), 8.02 - 8.09 (m, 3 H), 7.93 (d, J=8.8 Hz, 1 H), 7.89 (d, J=8.8 Hz, 2 H), 6.67 (d,
J=6.3 Hz, 1 H), 3.97 (dd, J=11.7, 3.4 Hz, 2 H), 3.69 - 3.76 (m, 4 H), 3.43 (t, J=11.2 Hz, 2 H), 3.37
(d, J=6.8 Hz, 2 H), 2.98 - 3.04 (m, 4 H), 2.07 - 2.18 (m, 1 H), 1.92 (s, 1 H), 1.79 (d, J=13.2 Hz, 2
H), 1.36 - 1.48 (m, 2 H).
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(1R,2R)-2-((6-(4-(morpholinosulfonyl)phenyl)quinolin-4-yl)amino)cyclohexan-1-ol NEU-5535
(2.26g) Prepared via General Procedure C. The reaction was purified by column chromatography
using a mobile phase of 0-100% ethyl acetate/dichloromethane, followed by 0-20% methanol/ethyl
acetate, to afford the title compound as an off white solid (34.5 mg, 57% yield). LCMS [M+H]+
468.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 8.66 (s, 1 H), 8.36 (d, J=5.4 Hz, 1 H), 8.13 (d,
J=8.3 Hz, 2 H), 7.98 (d, J=9.8 Hz, 1 H), 7.82 - 7.89 (m, 3 H), 7.01 (d, J=7.8 Hz, 1 H), 6.59 (d,
J=5.4 Hz, 1 H), 4.78 (br. s., 1 H), 3.66 (t, J=4.4 Hz, 4 H), 3.59 (br. s., 1 H), 2.92 (br. s., 4 H), 1.92
- 2.07 (m, 2 H), 1.64 - 1.77 (m, 2 H), 1.23 - 1.43 (m, 4 H).
6-(4-(morpholinosulfonyl)phenyl)-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinolin-4-amine.
NEU-5824 (2.26h) Prepared via General Procedure C. The reaction was purified by column
chromatography using a mobile phase of 0-20% methanol/ethyl acetate, to afford the title
compound as an off white solid (49.6 mg, 80% yield). LCMS [M+H]+ 482.2 m/z; 1H NMR (500
MHz, DMSO-d6) δ ppm 8.64 (s, 1 H), 8.41 (d, J=5.4 Hz, 1 H), 8.12 (d, J=8.3 Hz, 2 H), 8.01 (dd,
J=8.8, 1.5 Hz, 1 H), 7.83 - 7.91 (m, 3 H), 7.42 (br. s., 1 H), 6.51 (d, J=5.4 Hz, 1 H), 4.10 (d, J=4.9
175
Hz, 1 H), 3.85 (dd, J=11.0, 3.2 Hz, 2 H), 3.66 (t, J=4.4 Hz, 4 H), 3.25 - 3.31 (m, 2 H), 3.17 (d,
J=4.4 Hz, 2 H), 2.93 (br. s., 4 H), 1.61 - 1.72 (m, 4 H), 1.24 (d, J=10.2 Hz, 2 H).
1-(5-bromopyrimidin-2-yl)-4-methyl-1,4-diazepane. (2.27) 5-bromo-2-chloropyrimidine (1.00 g,
5.17 mmol), 1-methyl-1,4-diazepane (771 μL, 6.20 mmol), diisopropylethylamine (1.17 mL, 6.72
mmol) were dissolved in a 1:1 solution of THF/IPA (18 mL). The reaction mixture was heated at
80 °C overnight. A precipitate formed and was collected by vacuum filtration to afford the title
compound as a white solid (1.28 g, 91% yield). LCMS [M+H]+ 271.1 m/z (Br79), 273.0 m/z (Br81);
1H NMR (500 MHz,DMSO-d6) δ ppm 8.50 (s, 2H), 3.95 (br. s., 2H), 3.74 (br. s., 2H), 3.16 (d, J
= 4.9 Hz, 4H), 2.66 (br. s., 3H), 2.13 (br. s., 2H).
1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane. (2.28)
1-(5-bromopyrimidin-2-yl)-4-methyl-1,4-diazepane (250 mg, 0.922 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (351 mg, 1.38 mmol), PdCl2(dppf)·CH2Cl2 (37.6 mg,
176
0.05 mmol), and potassium acetate (271 mg, 2.77 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane (9 mL) was
added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure.
The resulting residue was used crude in the next reaction. A quantitative yield was assumed. LCMS
[M+H]+ 237.1 m/z (mass of boronic acid), 100% by LC trace.
6-bromo-4-chloroquinoline. (2.29) 6-bromoquinolin-4(1H)-one (171 mg, 0.763 mmol) was
dissolved in neat phosphorus oxychloride (3 mL). The reaction mixture was refluxed for 12 h.
Phosphorus oxychloride was removed by vacuum distillation. The remaining brown residue was
cooled to 0 °C and neutralized using saturated sodium bicarbonate solution. A precipitate formed
and was collected by vacuum filtration to afford the title compound as a light tan solid (149 mg,
80% yield). LCMS [M+H]+ 241.9 m/z (Br79), 243.9 m/z (Br81); 1H NMR (500 MHz, DMSO-d6)
ppm 8.89 (d, J=4.4 Hz, 1 H), 8.36 (d, J=1.5 Hz, 1 H), 8.05 - 8.08 (m, 1 H), 8.00 - 8.04 (m, 1 H),
7.84 (d, J=4.9 Hz, 1 H)
177
7-bromo-N-(pyrazin-2-yl)quinolin-4-amine. (2.30a) To a suspension of sodium hydride (40 mg,
0.29 mmol) in dry DMF (1 ml) was added pyrazin-2-amine (68.6 mg, 0.72 mmol) under nitrogen,
upon which the reaction mixture turned bright yellow. To this was added a solution of 6-bromo-4-
chloroquinoline (70 mg, 0.289 mmol) in dry DMF (1 ml), accompanied by a color change from
yellow, to dark orange, to brown. The reaction was stirred under nitrogen at room temperature for
48 h. Upon completion the reaction mixture was quenched with saturated aqueous ammonium
chloride. A precipitate was observed and collected by vacuum filtration and washed with water.
The crude material was purified by column chromatography, eluting with 1-20%
methanol/dichloromethane to afford the title compound as a light-yellow solid (31.2 mg, 36%
yield). LCMS [M+H]+ 301.0 m/z (Br79), 303.1 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm
9.77 - 9.83 (m, 1 H), 8.78 - 8.82 (m, 1 H), 8.73 - 8.77 (m, 1 H), 8.69 - 8.73 (m, 1 H), 8.39 - 8.44
(m, 1 H), 8.32 - 8.36 (m, 1 H), 8.17 - 8.22 (m, 1 H), 7.91 (s, 2 H).
6-bromo-N-(pyrimidin-4-yl)quinolin-4-amine. (2.30b) To a suspension of sodium hydride (40 mg,
0.29 mmol) in dry DMF (1 ml) was added pyrimidin-4-amine (68.6 mg, 0.72 mmol) under
178
nitrogen, upon which the reaction mixture turned bright yellow. To this was added a solution of 6-
bromo-4-chloroquinoline (70 mg, 0.289 mmol) in dry DMF (1 ml), accompanied by a color change
from yellow, to dark orange, to brown. The reaction was stirred under nitrogen at room temperature
for 48 h. Upon completion the reaction mixture was quenched with saturated aqueous ammonium
chloride. A precipitate was observed and collected by vacuum filtration and washed with water.
The crude material was purified by column chromatography, eluting with 1-20%
methanol/dichloromethane to afford the title compound as a light-yellow solid (23.5 mg, 27%
yield). LCMS [M+H]+ 301.1 m/z (Br79), 303.0 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) δ ppm
9.90 (s, 1 H), 8.81 (m, J=3.4 Hz, 2 H), 8.70 (d, J=2.0 Hz, 1 H), 8.51 (d, J=5.9 Hz, 1 H), 8.43 (d,
J=5.4 Hz, 1 H), 7.92 - 7.97 (m, 1 H), 7.89 (m, J=2.0 Hz, 1 H), 7.29 (dd, J=5.9, 1.0 Hz, 1 H).
7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrazin-2-yl)quinolin-4-amine. NEU-5377
(2.31a) 7-bromo-N-(pyrazin-2-yl)quinolin-4-amine (29 mg, 0.096 mmol) was dissolved in a 1:1
mixture of ethanol/water (1 mL). To this was added 1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane (2 equiv), cesium carbonate (3 equiv), and
tetrakis(triphenylphosphine)palladium(0) (5 mol%), and the reaction mixture was degassed before
heating at 85 °C for 12 h. The reaction mixture was monitored by LCMS analysis and, once
complete, the reaction mixture was filtered through Celite and the crude material was purified by
column chromatography, eluting with a mobile phase of 1-20% methanol/dichloromethane,
179
followed by 3-10% (1% ammonium hydroxide/methanol)/dichloromethane. The title compound
was afforded as a yellow solid (19.8 mg, 50% yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 9.71 (s, 1 H), 8.94 (s, 2 H), 8.72 (d, J=1.0 Hz, 1 H), 8.66 - 8.70 (m, 2 H),
8.34 - 8.37 (m, 2 H), 8.19 (d, J=2.9 Hz, 1 H), 8.06 (d, J=1.5 Hz, 1 H), 7.99 - 8.03 (m, 1 H), 3.88 -
3.93 (m, 2 H), 3.83 (t, J=6.1 Hz, 2 H), 2.62 - 2.67 (m, 2 H), 2.47 - 2.50 (m, 2 H), 2.27 (s, 3 H),
1.91 (s, 2 H).
6-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-
5374 (2.31b) 6-bromo-N-(pyrimidin-4-yl)quinolin-4-amine (21 mg, 0.070 mmol) was dissolved
in a 1:1 mixture of ethanol/water (1 mL). To this was added 1-methyl-4-(5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)-1,4-diazepane (2 equiv), cesium carbonate (3 equiv), and
tetrakis(triphenylphosphine)palladium(0) (5 mol%), and the reaction mixture was degassed before
heating at 85 °C for 12 h. The reaction mixture was monitored by LCMS analysis and, once
complete, the reaction mixture was filtered through Celite and the crude material was purified by
column chromatography, eluting with a mobile phase of 7-20% methanol/dichloromethane,
followed by 3-10% (1% ammonium hydroxide/methanol)/dichloromethane. The title compound
was afforded as a yellow solid (12 mg, 42% yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 9.84 (s, 1 H), 8.92 (s, 2 H), 8.82 (d, J=1.0 Hz, 1 H), 8.75 (d, J=4.9 Hz, 1 H),
8.61 (d, J=1.5 Hz, 1 H), 8.52 (d, J=5.9 Hz, 1 H), 8.37 (d, J=5.4 Hz, 1 H), 8.06 - 8.10 (m, 1 H), 8.02
180
- 8.06 (m, 1 H), 7.30 (dd, J=5.9, 1.0 Hz, 1 H), 3.88 - 3.93 (m, 2 H), 3.83 (t, J=6.3 Hz, 2 H), 3.17
(d, J=4.4 Hz, 1 H), 2.62 - 2.67 (m, 2 H), 2.27 (s, 3 H), 1.91 (dt, J=11.7, 5.9 Hz, 2 H).
7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one. (2.32) 7-bromoquinolin-
4(1H)-one (155 mg, 0.691 mmol), 1-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)pyrimidin-2-yl)-1,4-diazepane (1.3 equiv), cesium carbonate (3 equiv), and PdCl2(dppf)CH2Cl2
(3 mol %) were added to a vial that was vacuum purged with nitrogen. A mixture of degassed 3:1
dioxane/water (6 mL) was added to the vial. The reaction mixture was heated at 90 °C for 12 h.
The reaction mixture was monitored by LCMS analysis and, once complete, the reaction mixture
was filtered through Celite and the crude material was purified by column chromatography, eluting
with a mobile phase of 1-20% methanol/dichloromethane. The title compound was afforded as a
tan solid (178 mg, 77% yield). LCMS [M+H]+ 336.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm
8.75 (s, 2 H), 8.10 (d, J=8.3 Hz, 1 H), 7.88 (d, J=7.8 Hz, 1 H), 7.71 (s, 1 H), 7.58 (dd, J=8.3, 1.5
Hz, 1 H), 6.00 (d, J=7.3 Hz, 1 H), 3.87 - 3.97 (m, 2 H), 3.81 (t, J=6.3 Hz, 2 H), 3.05 (t, J=7.1 Hz,
2 H), 2.37 (br. s., 2 H), 1.93 - 2.00 (m, 1 H), 1.89 (s, 3 H), 1.81 (s, 2 H).
181
4-chloro-7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinoline. (2.33) 7-(2-(4-methyl-1,4-
diazepan-1-yl)pyrimidin-5-yl)quinolin-4(1H)-one (178 mg, 0.530 mmol) was dissolved in neat
phosphorus oxychloride (3 mL). The reaction mixture was refluxed for 12 h. Phosphorus
oxychloride was removed by vacuum distillation. The remaining brown residue was cooled to 0
°C and neutralized using saturated sodium bicarbonate solution. The aqueous suspension was
extracted 3 times with dichloromethane. The combined organic layers were dried using sodium
sulfate, filtered, and concentrated under reduced pressure to afford the title compound as a beige
solid (129 mg, 69% yield). LCMS [M+H]+ 354.1 m/z (Cl35), 356.1 m/z (Cl37); 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.98 (s, 2 H), 8.86 (d, J=4.9 Hz, 1 H), 8.39 (s, 1 H), 8.26 (d, J=8.8 Hz, 1 H),
8.13 (d, J=8.8 Hz, 1 H), 7.75 (d, J=4.9 Hz, 1 H), 3.87 (br. s., 2 H), 3.34 (br. s., 3 H), 3.12 (br. s., 2
H), 3.03 (d, J=17.6 Hz, 2 H), 2.61 (br. s., 2 H), 2.11 (br. s., 2 H).
182
7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)-N-(pyrimidin-4-yl)quinolin-4-amine. NEU-
5030 (2.34) To a suspension of sodium hydride (56.5 mg, 1.03 mmol) in dry DMF (1 ml) was
added pyrimidin-4-amine (80.6 mg, 0.848 mmol) under nitrogen. To this was added a solution 4-
chloro-7-(2-(4-methyl-1,4-diazepan-1-yl)pyrimidin-5-yl)quinoline (100 mg, 0.283 mmol) in dry
DMF (1 ml), accompanied by a color change from yellow to dark orange. The reaction was stirred
under nitrogen at 90 °C for 12 h. Upon completion the reaction mixture was quenched with
saturated aqueous ammonium chloride and the resulting solution was lyophilized to dryness. The
crude material was purified by column chromatography, eluting with 0-20%
methanol/dichloromethane, to afford the title compound as a bright-yellow solid (16 mg, 14%
yield). LCMS [M+H]+ 413.2 m/z; 1H NMR (500 MHz, DMSO-d6) δ ppm 9.92 (s, 1 H), 8.92 (s, 2
H), 8.76 - 8.81 (m, 2 H), 8.44 - 8.51 (m, 2 H), 8.31 (d, J=5.1 Hz, 1 H), 8.24 (s, 1 H), 7.98 (d, J=8.8
Hz, 1 H), 7.29 (d, J=5.9 Hz, 1 H), 4.09 (d, J=5.1 Hz, 1 H), 3.87 - 3.94 (m, 2 H), 3.82 (t, J=6.2 Hz,
2 H), 3.17 (d, J=5.1 Hz, 1 H), 2.61 - 2.68 (m, 2 H), 2.27 (s, 3 H), 1.85 - 1.95 (m, 2 H).
183
Cellular biological assays
The protocols for the biological assays of Leishmania major amastigotes, Plasmodium falciparum
D6, W2, C235, and HepG2 cell toxicity were performed as previously described.153-157 The
compounds 1-11 and 35-37 were evaluated against T. cruzi and host cell toxicity (NIH3T3) using
previously reported protocol;153, 158 and rest all of the compounds were tested against T. cruzi and
host cell toxicity (C2C12) as described here.
T. cruzi in vitro assay. C2C12 (ATCC CRL-1772) and T.cruzi (CaI-72) trypomastigotes were
collected from culture flasks and counted. For each assay, a batch infection was prepared by
mixing cells and parasites in solution (DMEM media supplemented with 10% FBS, 1x
penicillin/streptomycin). Multidrop Combi (Thermo Fisher Scientific) was used to dispense 10ul
of that solution in the assay plates containing the compounds. Plates were incubated at 37C, 5%
CO2 incubators for 72h. After incubation, 5ul/well is aspirated using EL-406 (Biotek) and we
dispensed 5ul of 8% paraformaldehyde (PFA) to fix the samples using the Multidrop Combi. After
at least 30 minutes incubation with PFA solution at room temperature, we aspirated the content
and wash one time with isotonic buffer. Next, we added DAPI solution (5ug/ml) for nucleic acid
staining for at least 30 minutes before reading the plates in ImageXpress MicroXL (Molecular
Devices). The images were analyzed by a custom module algorithm in MetaXpress (Molecular
Devices) to determine the number of host cells and number of parasites per well. The averages of
the parasite/cell ratio from the set of controls determined the 100% efficacy normalized activity
(positive control average) and 0% efficacy normalized activity (negative control average).
184
L. donovani in vitro assay. B10R (kindly provided by Martin Olivier, McGill University) and L.
donovani were collected from culture flasks and counted. For each assay, a batch infection was
prepared by mixing cells and parasites in solution (DMEM media supplemented with 10% FBS,
1x penicillin/streptomycin). Multidrop Combi was used to dispense 10ul of that solution in the
assay plates containing the compounds. Plates are incubated at 37C, 5% CO2 incubators for 72h.
After incubation, 5ul/well is aspirated using EL-406 (Biotek) and we dispensed 5ul of 8%
paraformaldehyde (PFA) to fix the samples using the Multidrop Combi. After at least 30 minutes
incubation with PFA solution at room temperature, we aspirated the content and wash one time
with isotonic buffer. Next, we add DAPI solution (5ug/ml) for DNA staining for at least 30 minutes
before reading the plates in ImageXpress MicroXL (Molecular Devices). The images were
analyzed by a custom module algorithm in MetaXpress (Molecular Devices) to determine the
number of host cells and number of parasites per well. Each plate contained wells with positive
controls (non-infected) and wells with negative controls (infected not-treated). The averages of the
parasite/cell ratio from the set of controls determined the 100% efficacy normalized activity
(positive control average) and 0% efficacy normalized activity (negative control average).
Host cell toxicity in vitro assay. To assess the host cell cytotoxicity for both T. cruzi and L.
donovani screening assays, we first determine the average number of host cells in the negative
control wells that are infected and untreated with any compound. To assess the cell viability from
each well with a tested compound, we divided the number of host cells from that well by the
average number of cells from the negative controls from that same plate. The result informed the
percentage of cell relative to an average without any toxicity. Calculating the average number of
cells from negative controls minus three standard deviations from that average it is always around
185
50% cell toxicity. Being that the case, we considered <50% cell viability as a sign of observed
toxicity caused by the tested compound.
ADME Experiment Protocols
Assays were run by AstraZeneca.
Aqueous pH 7.4 Solubility. Compounds were dried down from 10 mM DMSO solutions using
centrifugal evaporation technique. Phosphate buffer (0.1 M pH 7.4) was added and StirStix were
inserted in the glass vials, with shaking then performed at a constant temperature of 25 °C for 20-
24 h. This step was followed by double centrifugation with a tip wash in between, to ensure that
no residues of the dried compounds interfere. The solutions were diluted before analysis and
quantification using LC/MS/MS was performed.
Log D7.4. Shake-flask octanol-water distribution coefficient was determined at pH 7.4 (Log D7.4).
The aqueous solution used is 10 mM sodium phosphate pH 7.4 buffer. The method has been
validated for Log D7.4 ranging from -2 to 5.0.
Human Plasma Protein Binding (PPB). PPB was determined using equilibrium dialysis (RED
device) to separate free from bound compound. The amount of compound in plasma (10 µM initial
concentration) and in dialysis buffer (pH 7.4 phosphate buffer) was measured by LC-MS/MS after
equilibration at 37 °C in a dialysis chamber to give the fraction unbound (fu); percent bound is
calculated and reported.
186
Human Liver Microsomal Clint. In vitro intrinsic clearance was determined from human liver
microsomes using a standard approach.159 Following incubation and preparation, the samples were
analyzed using LC/MS/MS. Refined data were uploaded to IBIS and are displayed as Clint
(intrinsic clearance) in μL/min/mg.
Rat Hepatocyte Clint. In vitro intrinsic clearance was determined from rat hepatocytes using a
standard approach.159 Following incubation and preparation, the samples were analyzed using
LC/MS/MS. Refined data are uploaded to IBIS and are displayed as Clint (intrinsic clearance)
μL/min/1 million cells.
Calculated LogP and LogD values. Both LogP and LogD predictions are based on a modified
version of the method160 where the predicted partition coefficients are composed of the
molecules’ atomic increments.
187
4.2.2 Experimental procedures for Chapter 3
4-Bromo-N-(2-hydroxyethyl)benzenesulfonamide.161-162 (3.1a) Ethanolamine (23.4 μL, 0.391
mmol) and triethylamine (109 μL, 0.783 mmol) were added to a flask containing anhydrous DCM
(1 mL) at -10°C. 4-Bromobenzenesulfonyl chloride (100 mg, 0.391 mmol) was dissolved in
anhydrous DCM (1 mL) and then added dropwise to the stirring solution of amines. The reaction
mixture was left stirring and allowed to warm to room temperature overnight. A white precipitate
formed and filtered off. The filtrate was collected and concentrated to afford the title compound as
white solid (89.5 mg, 82% yield). LCMS [M+H]+ 279.9 m/z (Br79), 281.9 m/z (Br81).
4-((4-bromophenyl)sulfonyl)morpholine.163 (3.1b) 4-bromobenzenesulfonyl chloride (200 mg,
0.78 mmol) was dissolved in THF (2 mL). Morpholine (74 μL) was added and the reaction mixture
was left stirring overnight at room temperature. The reaction mixture was neutralized with
saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times. The combined
organic layers were washed with water and with brine, dried using sodium sulfate, filtered, and
concentrated under reduced pressure to afford the title compound as white solid (196 mg, 82%
yield). LCMS [M+H]+ 307.1 m/z (Br79), 309.1 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-
188
d) ppm 7.71 (d, J=8.3 Hz, 2 H), 7.63 (d, J=8.8 Hz, 2 H), 3.76 (t, J=1.0 Hz, 4 H), 3.01 (t, J=1.0
Hz, 4 H).
1-((4-bromophenyl)sulfonyl)-4-methylpiperazine.163 (3.1c) 4-bromobenzenesulfonyl chloride (200
mg, 0.78 mmol) was dissolved in THF (2 mL). 1-methylpiperazine (95 μL) was added and the
reaction mixture was left stirring overnight at room temperature. The reaction mixture was
neutralized with saturated sodium bicarbonate solution and extracted with ethyl acetate 3 times.
The combined organic layers were washed with water and with brine, dried using sodium sulfate,
filtered, and concentrated under reduced pressure to afford the title compound as light yellow solid
(198 mg, 79% yield). LCMS [M+H]+ 320.0 m/z (Br79), 322.0 m/z (Br81); 1H NMR (500 MHz,
CHLOROFORM-d) ppm 7.66 - 7.70 (m, 2 H), 7.59 - 7.64 (m, 2 H), 3.04 (br. s., 4 H), 2.48 (t,
J=4.6 Hz, 4 H), 2.28 (s, 3 H).
189
N-(2-hydroxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide. (3.2a)
4-bromo-N-(2-hydroxyethyl)benzenesulfonamide (441 mg, 1.57 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (799 mg, 3.15 mmol), PdCl2(dppf)·CH2Cl2 (64.3 mg,
0.08 mmol), and potassium acetate (463 mg, 4.72 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
15 mL) was added to the vial. The reaction mixture was heated at 80 °C overnight and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under reduced
pressure, and then purified by silica gel column chromatography using a mobile phase of 0-100%
ethyl acetate/dichloromethane to afford the title compound as a light orange solid (464 mg, 90%
yield). LCMS [M+H]+ 328.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 7.84 - 7.89 (m, 2 H),
7.78 - 7.82 (m, 2 H), 7.68 (t, J=5.9 Hz, 1 H), 4.68 (t, J=5.6 Hz, 1 H), 3.31 - 3.36 (m, 2 H), 2.77 (q,
J=6.3 Hz, 2 H), 1.31 (s, 12 H), 1.16 (d, J=4.4 Hz, 2 H).
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4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine. (3.2b) 4-((4-
bromophenyl)sulfonyl)morpholine (196 mg, 0.640 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-
bi(1,3,2-dioxaborolane) (243 mg, 0.960 mmol), PdCl2(dppf)·CH2Cl2 (26.1 mg, 0.032 mmol), and
potassium acetate (188 mg, 1.92 mmol) were added to a vial that was subsequently sealed and
vacuum purged with nitrogen. A degassed solution of dioxane (4 mL) was added to the vial. The
reaction mixture was heated at 85 °C overnight and monitored by LCMS. The reaction mixture
was filtered through a bed of Celite, concentrated under reduced pressure. The resulting residue
was used crude in the next reaction. A quantitative yield was assumed. LCMS [M+H]+ 354.1 m/z.
75% product, 25% boronic acid of product by LC trace.
1-methyl-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)piperazine. (3.2c) 1-
((4-bromophenyl)sulfonyl)-4-methylpiperazine (195 mg, 0.611 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (233 mg, 0.916 mmol), PdCl2(dppf)·CH2Cl2 (24.9 mg,
191
0.030 mmol), and potassium acetate (180 mg, 1.83 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane (4 mL) was
added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure.
The resulting residue was used crude in the next reaction. A quantitative yield was assumed. LCMS
[M+H]+ 367.2 m/z, 85% pure by LC trace.
4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine.164 (3.3) 4-bromo-1H-pyrrolo[2,3-b]pyridine (300
mg, 1.52 mmol) and 4-methylbenzenesulfonyl chloride (580 mg, 3.05 mmol) were dissolved in
dichloromethane (3 mL). Tetrabutylammonium hydrogensulfate (15.5 mg, 0.045 mmol) and
sodium hydroxide (3M, 1.9 mL) were added to the reaction mixture. The reaction mixture was
stirred at room temperature for 1 hour, monitoring by TLC, after which the reaction mixture was
quenched with saturated ammonium chloride solution. The organic layer was collected, and the
aqueous layer was extracted 3x with dichloromethane. The combined organic layers were washed
with water and then with brine, dried with sodium sulfate, filtered, and concentrated under reduced
pressure. The reaction mixture was then purified by column chromatogr5aphy using a mobile
phase of 0-10% ethyl acetate/hexanes to afford the title compound as a white, powdery solid (241
g, 45% yield). LCMS [M+H]+ 351.0 m/z (Br79), 353.0 m/z (Br81); 1H NMR (500 MHz,
CHLOROFORM-d) ppm 8.23 (d, J=5.4 Hz, 1 H), 8.07 (d, J=8.3 Hz, 2 H), 7.79 (d, J=3.9 Hz, 1
H), 7.36 (d, J=5.4 Hz, 1 H), 7.29 (d, J=8.3 Hz, 2 H), 6.65 (d, J=3.9 Hz, 1 H), 2.38 (s, 3 H).
192
4-(1-Methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. NEU-4813 (3.4a) 4-Bromo-1-
tosyl-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.285 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan -2-yl)-1H-pyrazole (71.1 mg, 0.342 mmol), sodium carbonate (90.5 mg, 0854
mmol), and PdCl2(dppf)·CH2Cl2 (2.33 mg, 0.002 mmol) were added to a microwave vial that was
vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.8 mL) was added to
the vial. The reaction mixture was microwaved at 150 °C for 10 minutes. The reaction mixture
was monitored by LCMS analysis and, once complete, the reaction mixture was filtered through
Celite and the crude material was purified by column chromatography, eluting with a mobile phase
of 10-100% ethyl acetate/hexanes to afford the title compound as a colorless solid (75 mg, 75%
yield). LCMS [M+H]+ 353.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.37 (d, J=5.4
Hz, 1 H), 8.08 (d, J=8.8 Hz, 2 H), 7.88 (s, 1 H), 7.78 (s, 1 H), 7.76 (d, J=4.4 Hz, 1 H), 7.27 (d,
J=8.3 Hz, 2 H), 7.20 (d, J=4.9 Hz, 1 H), 6.78 (d, J=4.4 Hz, 1 H), 3.99 (s, 3 H), 2.36 (s, 3 H).
193
4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. NEU-4814 (3.4b) 4-bromo-1-
tosyl-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.285 mmol), (4-(methylsulfonyl)phenyl)boronic acid
(68.3 mg, 0.342 mmol), sodium carbonate (90.5 mg, 0.854 mmol), and PdCl2(dppf)·CH2Cl2 (2.33
mg, 0.002 mmol) were added to a microwave vial that was vacuum purged with nitrogen. A
mixture of degassed 3:1 dioxane/water (2.8 mL) was added to the vial. The reaction mixture was
microwaved at 150 °C for 10 minutes. The reaction mixture was monitored by LCMS analysis
and, once complete, the reaction mixture was filtered through Celite and the crude material was
purified by column chromatography, eluting with a mobile phase of 10-60% ethyl acetate/hexanes
to afford the title compound as a colorless solid (104 mg, 86% yield). LCMS [M+H]+ 427.1 m/z;
1H NMR (500 MHz, CHLOROFORM-d) ppm 8.52 (d, J=4.9 Hz, 1 H), 8.11 (d, J=8.3 Hz, 2 H),
8.08 (d, J=8.3 Hz, 2 H), 7.83 (d, J=3.9 Hz, 1 H), 7.78 (d, J=8.3 Hz, 2 H), 7.30 (d, J=8.3 Hz, 2 H),
7.25 (d, J=5.4 Hz, 1 H), 6.71 (d, J=4.4 Hz, 1 H), 3.13 (s, 3 H), 2.38 (s, 3 H).
194
4-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine. NEU-4471 (3.4c) 4-bromo-1H-pyrrolo[2,3-
b]pyridine (50 mg, 0.254 mmol), pyridin-4-ylboronic acid (37.4 mg, 0.304 mmol), sodium
carbonate (80.7 mg, 0.761 mmol), and PdCl2(dppf)·CH2Cl2 (2.1 mg, 0.002 mmol) were added to
a microwave vial that was vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water
(2.5 mL) was added to the vial. The reaction mixture was microwaved at 130 °C for 30 minutes.
The reaction mixture was monitored by LCMS analysis and, once complete, the reaction mixture
was filtered through Celite and the crude material was purified by column chromatography, eluting
with a mobile phase of 0-20% methanol/dichloromethane to afford the title compound as a pale
yellow solid (41 mg, 83% yield). LCMS [M+H]+ 195.9 m/z; 1H NMR (500 MHz, DMSO-d6)
ppm 11.94 (br. s., 1 H), 8.74 (d, J=6.3 Hz, 2 H), 8.34 (d, J=4.9 Hz, 1 H), 7.78 (d, J=5.9 Hz, 2 H),
7.61 - 7.64 (m, 1 H), 7.30 (d, J=4.9 Hz, 1 H), 6.68 (dd, J=3.4, 2.0 Hz, 1 H).
195
N-(2-hydroxyethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide. NEU-4786 (3.4d) 4-
bromo-1H-pyrrolo[2,3-b]pyridine (20 mg, 0.103 mmol), N-(2-hydroxyethyl)-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (42 mg, 0.128 mmol), sodium carbonate
(41 mg, 0.385 mmol), and PdCl2(dppf)·CH2Cl2 (3.1 mg, 0.003 mmol) were added to a microwave
vial that was vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.0 mL)
was added to the vial. The reaction mixture was microwaved at 130 °C for 30 minutes. The reaction
mixture was monitored by LCMS analysis and, once complete, the reaction mixture was filtered
through Celite and the crude material was purified by column chromatography, eluting with a
mobile phase of 40-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to
afford the title compound as a pale yellow solid (7 mg, 17% yield). LCMS [M+H]+ 318.1 m/z; 1H
NMR (500 MHz, DMSO-d6) ppm 11.90 (br. s., 1 H), 8.33 (d, J=4.9 Hz, 1 H), 7.97 - 8.03 (m, 2
H), 7.93 - 7.97 (m, 2 H), 7.74 (t, J=5.9 Hz, 1 H), 7.58 - 7.63 (m, 1 H), 7.27 (d, J=4.9 Hz, 1 H),
6.66 (dd, J=3.4, 1.5 Hz, 1 H), 4.72 (t, J=5.6 Hz, 1 H), 3.41 (m, J=5.4 Hz, 2 H), 2.86 (q, J=6.2 Hz,
2 H).
196
4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine.164 NEU-4472 (3.5a) 4-bromo-1H-
pyrrolo[2,3-b]pyridine (50 mg, 0.254 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (63.3 mg, 0.304 mmol), sodium carbonate (80.7 mg, 0.761
mmol), and PdCl2(dppf)·CH2Cl2 (2.1 mg, 0.002 mmol) were added to a microwave vial that was
vacuum purged with nitrogen. A mixture of degassed 3:1 dioxane/water (2.5 mL) was added to
the vial. The reaction mixture was microwaved at 130 °C for 30 minutes. The reaction mixture
was monitored by LCMS analysis and, once complete, the reaction mixture was filtered through
Celite and the crude material was purified by column chromatography, eluting with a mobile phase
of 70-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title
compound as a yellow solid (47 mg, 93% yield). LCMS [M+H]+ 198.9 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 11.65 (br. s., 1 H), 8.45 (s, 1 H), 8.15 (d, J=4.9 Hz, 1 H), 8.10 (s, 1 H), 7.43 -
7.55 (m, 1 H), 7.24 (d, J=4.9 Hz, 1 H), 6.78 (dd, J=3.4, 2.0 Hz, 1 H), 3.94 (s, 3 H).
197
4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine. NEU-4815 (3.5b) 4-(4-
(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (30.0 mg, 0.070 mmol) was dissolved
in dioxane (0.5 mL). Sodium hydroxide (2.5M, 0.295 mL) was added and the reaction mixture was
microwaved at 150 °C for 15 minutes. The reaction mixture was then concentrated under reduced
pressure and purified by silica gel column chromatography eluting with a mobile phase 0-100%
ethyl acetate/dichloromethane to afford the title compound as a yellow solid (9 mg, 47%). LCMS
[M+H]+ 273.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 11.93 (br. s., 1 H), 8.34 (d, J=4.9 Hz,
1 H), 8.07 - 8.11 (m, 2 H), 8.02 - 8.07 (m, 2 H), 7.62 (t, J=3.2 Hz, 1 H), 7.28 (d, J=4.9 Hz, 1 H),
6.65 (dd, J=3.2, 1.7 Hz, 1 H), 3.30 (s, 3 H).
1-(4-bromophenyl)-N,N-dimethylmethanamine.165 (3.6a) 4-bromobenzylbromide (500 mg, 2.00
mmol) was dissolved in hexanes (2 mL) and the solution then cooled to 0 °C. Dimethylamine (2M,
4.0 mL) was added dropwise. The reaction mixture was left stirring overnight and allowing it to
warm to room temperature. A white precipitate formed and was filtered off. The filtrate was
concentrated under reduced pressure to afford the title compound as a yellow liquid (394 mg, 92%
198
yield). LCMS [M+H]+ 213.9 m/z (Br79), 215.9 m/z (Br81); 1H NMR (500 MHz, DMSO-d6) ppm
7.67 (d, J=8.3 Hz, 2 H), 7.49 (d, J=8.3 Hz, 2 H), 2.91 (s, 2 H), 2.67 (s, 6 H).
N-(4-bromobenzyl)-N-ethylethanamine.166 (3.6b) 4-bromobenzylbromide (200 mg, 0.800 mmol)
was dissolved in hexanes (1 mL) and the solution then cooled to 0 °C. Diethylamine (331 μL, 3.20
mmol) was added dropwise. The reaction mixture was left stirring overnight and allowing it to
warm to room temperature. A white precipitate formed and was filtered off. The filtrate was
concentrated under reduced pressure to afford the title compound as a white solid (137 mg, 70%
yield). LCMS [M+H]+ 243.0 m/z (Br79), 245.0 m/z (Br81); 1H NMR (500 MHz, CHLOROFORM-
d) ppm 7.43 (d, J=8.3 Hz, 2 H), 7.22 (d, J=8.3 Hz, 2 H), 3.51 (s, 2 H), 2.51 (q, J=6.8 Hz, 4 H),
1.03 (t, J=7.1 Hz, 6 H).
N,N-dimethyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanamine.167 (3.7a)
1-(4-bromophenyl)-N,N-dimethylmethanamine (361 mg, 1.69 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (642 mg, 2.53 mmol), PdCl2(dppf)·CH2Cl2 (41.3 mg,
0.05 mmol), and potassium acetate (496 mg, 5.06 mmol) were added to a microwave vial that was
subsequently sealed and vacuum purged with nitrogen. Anhydrous dioxane (10 mL) was added to
199
the flask. The reaction mixture was microwaved at 130 °C for 40 minutes and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite and concentrated under reduced pressure
and then purified by silica gel column chromatography using a mobile phase of 1-20% (10%
ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a brown solid
(325 mg, 74% yield). LCMS [M+H]+ 262.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm
7.78 (d, J=7.8 Hz, 2 H), 7.33 (d, J=7.8 Hz, 2 H), 3.46 (s, 2 H), 2.25 (s, 6 H), 1.35 (s, 12 H).
N-ethyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)ethanamine.168 (3.7b) N-(4-
bromobenzyl)-N-ethylethanamine (124 mg, 0.513 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-
bi(1,3,2-dioxaborolane) (194.5 mg, 0.769 mmol), PdCl2(dppf)·CH2Cl2 (12.6 mg, 0.015 mmol),
and potassium acetate (151.2 mg, 1.54 mmol) were added to a vial that was subsequently sealed
and vacuum purged with nitrogen. Anhydrous dioxane (3 mL) was added to the flask. The reaction
mixture was heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and concentrated under reduced pressure. The product was used crude in
the subsequent reaction, assuming quantitative yield. LCMS [M+H]+ 290.1 m/z.
200
tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxylate.
(3.7c) tert-butyl 2-(4-bromophenyl)pyrrolidine-1-carboxylate (243 mg, 0.74 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (284 mg, 1.12 mmol),
PdCl2(dppf)·CH2Cl2 (30.4 mg, 0.037 mmol), and potassium acetate (219 mg, 2.23 mmol) were
added to a vial that was subsequently sealed and vacuum purged with nitrogen. Anhydrous dioxane
(5 mL) was added to the vial. The reaction mixture was heated at 85 °C overnight and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under
reduced pressure. The product was used crude in the subsequent reaction, assuming quantitative
yield. LCMS [M+H]+ 318.2 m/z (minus t-butyl fragment mass), 90% pure by LC trace.
1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.8) 1H-pyrrolo[2,3-b]pyridine (200 mg, 1.69 mmol) and 4-
methylbenzenesulfonyl chloride (645 mg, 3.39 mmol) were dissolved in dichloromethane (8.6
mL). Tetrabutylammonium hydrogen sulfate (17.2 mg, 0.050 mmol) and sodium hydroxide (3M,
2.1 mL) were added to the reaction mixture. The reaction mixture was stirred at room temperature
for 1 hour, monitoring by TLC, after which the reaction mixture was quenched with saturated
ammonium chloride solution. The organic layer was collected and the aqueous layer was extracted
3x with dichloromethane. The combined organic layers were washed with water and then with
201
brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure. The reaction
mixture was then purified by column chromatography using a mobile phase of 0-70% ethyl
acetate/hexanes to afford the title compound as a white solid (339 mg, 73% yield). LCMS [M+H]+
273.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.43 (dd, J=4.9, 1.5 Hz, 1 H), 8.08 (d,
J=8.3 Hz, 2 H), 7.84 (dd, J=7.8, 1.5 Hz, 1 H), 7.73 (d, J=3.9 Hz, 1 H), 7.27 - 7.29 (m, 2 H), 7.17
(dd, J=7.8, 4.4 Hz, 1 H), 6.59 (d, J=3.9 Hz, 1 H), 1.57 (s, 3 H).
2-Iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.9) To a flame dried flask was added anhydrous THF
(2 mL) and diisopropylamine (178.8 μL, 1.27 mmol). The solution was cooled to 0 °C and n-
butyllithium (505 μL, 1.21 mmol) was added. The solution was then cooled to -78 °C and 1-tosyl-
1H-pyrrolo[2,3-b]pyridine (300 mg, 1.10 mmol) in anhydrous THF (1 mL) was added dropwise.
The reaction mixture was left stirring for 3 h, allowing it to warm to -20 °C after which iodine
(363 mg, 1.43 mmol), dissolved in anhydrous THF (1 mL), was added dropwise. The reaction
mixture was left stirring overnight and allowing it to warm to room temperature. The reaction
mixture was then quenched with saturated aqueous ammonium chloride solution. The organic layer
was collected and the aqueous layer washed 3x with DCM. The combined organic layers were
washed with brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure.
The reaction mixture was then purified by silica gel column chromatography, using a gradient of
0-20% ethyl acetate/hexanes to afford the title compound as tan solid (173 mg, 39% yield). LCMS
[M+H]+ 399.0 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.40 (dd, J=4.9, 1.5 Hz, 1 H),
202
8.10 (d, J=8.3 Hz, 2 H), 7.72 (dd, J=7.8, 2.0 Hz, 1 H), 7.29 (s, 2 H), 7.14 (dd, J=7.8, 4.9 Hz, 1 H),
6.99 (s, 1 H), 2.38 (s, 3 H).
N,N-dimethyl-1-(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. (3.10a) 2-iodo-
1-tosyl-1H-pyrrolo[2,3-b]pyridine (32 mg, 0.08 mmol), N,N-dimethyl-1-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)methanamine (21 mg, 0.08 mmol), [1,1′-
Tetrakis(triphenylphosphine)palladium(0) (2.8 mg, 0.002 mmol), and cesium carbonate (78.5 mg,
0.241 mmol) were added to a microwave vial that was subsequently sealed and vacuum purged
with nitrogen. A degassed solution of dioxane/water (3:1, 0.8 mL) was added to the vial. The
reaction mixture was microwaved at 150 °C for 15 minutes and monitored by LCMS. The reaction
mixture was filtered through a bed of Celite and concentrated under reduced pressure. The crude
product was used in the subsequent reaction assuming quantitative yield. LCMS [M+H]+ 406.2
m/z, 80% pure by LC trace.
(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-4812 (3.10b) 2-iodo-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (50 mg, 0.126 mmol), (4-(hydroxymethyl)phenyl)boronic acid (23 mg,
0.15 mmol), PdCl2(dppf)·CH2Cl2 (2.0 mg, 0.002 mmol), and cesium carbonate (123 mg, 0.377
203
mmol) were added to a microwave vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 1.2 mL) was added to the vial. The reaction
mixture was microwaved at 150 °C for 10 minutes and monitored by LCMS. The reaction mixture
was filtered through a bed of Celite and concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography using a mobile phase of 0-100% ethyl
acetate/hexanes to afford the title compound as colorless residue (38 mg, 80% yield). LCMS
[M+H]+ 379.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.48 (dd, J=4.9, 1.5 Hz, 1 H),
7.70 - 7.82 (m, 3 H), 7.56 (d, J=8.3 Hz, 2 H), 7.47 (d, J=8.3 Hz, 2 H), 7.19 (dd, J=7.8, 4.9 Hz, 1
H), 7.16 (d, J=8.3 Hz, 2 H), 6.50 (s, 1 H), 4.80 (s, 2 H), 2.33 (s, 3 H).
1-(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine. NEU-4844 (3.11a)
N,N-dimethyl-1-(4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (32.6 mg, 0.080
mmol) was dissolved in dioxane (0.8 mL). Sodium hydroxide (3M, 0.281 mmol) was added and
the reaction was microwaved at 150 °C for 15 minutes. The crude product was then purified by
column chromatography eluting with a mobile phase of 0-20% methanol/dichloromethane to
afford the title compound as a light tan solid (11 mg, 54% yield). LCMS [M+H]+ 252.2 m/z; 1H
NMR (500 MHz, METHANOL-d4) ppm 8.21 (dd, J=4.4, 1.5 Hz, 1 H), 7.96 - 8.03 (m, 3 H), 7.59
(d, J=8.3 Hz, 2 H), 7.12 (dd, J=7.8, 4.9 Hz, 1 H), 6.95 (s, 1 H), 4.29 (s, 2 H), 2.84 (s, 6 H).
204
(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-4845 (3.11b) (4-(1-tosyl-1H-
pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol (83.0 mg, 0.219 mmol) was dissolved in dioxane (2.0
mL). Sodium hydroxide (3M, 0.767 mmol) was added and the reaction was microwaved at 150 °C
for 15 minutes. The crude product was then purified by column chromatography eluting with a
mobile phase of 0-5% methanol/dichloromethane and then recrystallized using methanol to afford
the title compound as yellow needle shaped crystals (6 mg, 12% yield). LCMS [M+H]+ 225.1 m/z;
1H NMR (500 MHz, DMSO-d6) ppm 12.09 (s, 1 H), 8.20 (dd, J=4.4, 1.5 Hz, 1 H), 7.88 - 7.93
(m, 3 H), 7.40 (d, J=8.3 Hz, 2 H), 7.05 (dd, J=7.6, 4.6 Hz, 1 H), 6.89 (d, J=2.4 Hz, 1 H), 5.24 (t,
J=5.6 Hz, 1 H), 4.54 (d, J=5.9 Hz, 2 H).
4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.12) 4-Chloro-1H-pyrrolo[2,3-b]pyridine (2.00 g,
13.1 mmol) and 4-methylbenzenesulfonyl chloride (5.00 g, 26.2 mmol) were dissolved in
dichloromethane (36 mL). Tetrabutylammonium hydrogen sulfate (133 mg, 0.393 mmol) and
sodium hydroxide (3M, 16.2 mL) were added to the reaction mixture. The reaction mixture was
stirred at room temperature for 1 hour, monitoring by TLC, after which the reaction mixture was
quenched with saturated ammonium chloride solution. The organic layer was collected, and the
aqueous layer was extracted 3x with dichloromethane. The combined organic layers were washed
205
with water and then with brine, dried with sodium sulfate, filtered, and concentrated under reduced
pressure. The reaction mixture was then purified by column chromatography using a mobile phase
of 0-20% ethyl acetate/hexanes to afford the title compound as a white solid (3.25 g, 81% yield).
LCMS [M+H]+ 306.9 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.32 (d, J=4.9 Hz, 1
H), 8.07 (d, J=8.3 Hz, 2 H), 7.78 (d, J=3.9 Hz, 1 H), 7.29 (d, J=7.8 Hz, 2 H), 7.20 (d, J=5.4 Hz, 1
H), 6.70 (d, J=3.9 Hz, 1 H), 2.38 (s, 3 H).
4-Chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.13) To a flame dried flask was added 4-
chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (501 mg, 1.63 mmol). Anhydrous THF (40 mL) was
added and the solution was cooled to -78 °C. Lithium diisopropylamide (1.62M, 1.51 mL) was
then added dropwise to the solution. The reaction mixture was left stirring for 1 hour, after which
iodine (1.24 g, 4.90 mmol) dissolved in anhydrous THF (10 mL) was added dropwise. The reaction
mixture was left stirring overnight and allowed to warm to room temperature. The reaction mixture
was then quenched with saturated aqueous ammonium chloride solution. The organic layer was
collected and the aqueous layer washed 3x with DCM. The combined organic layers were washed
with brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure. The
reaction mixture was then purified by silica gel column chromatography, using a gradient of 0-5%
ethyl acetate/hexanes to afford the title compound as an off-white solid (621 mg, 88% yield).
LCMS [M+H]+ 432.8 m/z (Cl35), 434.8 m/z (Cl37); 1H NMR (400 MHz, CHLOROFORM-d)
206
ppm 8.28 (d, J=5.1 Hz, 1 H), 8.10 (d, J=8.8 Hz, 2 H), 7.29 (d, J=8.1 Hz, 2 H), 7.16 (d, J=5.1 Hz,
1 H), 7.11 (s, 1 H), 2.39 (s, 3 H).
1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine. (3.14)
4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (153 mg, 0.353 mmol), N,N-dimethyl-1-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanamine (120 mg, 0.460 mmol),
PdCl2(dppf)·CH2Cl2 (14.4 mg, 0.05 mmol), and sodium carbonate (112 mg, 1.06 mmol) were
added to an oven dried vial that was subsequently sealed and vacuum purged with nitrogen. A
degassed solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was
heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title
compound as an off-white solid (77 mg, 76% yield). LCMS [M+H]+ 439.9 m/z (Cl35), 442.0 m/z
(Cl37); 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.36 (d, J=4.9 Hz, 1 H), 7.77 (d, J=8.3 Hz,
2 H), 7.51 (d, J=7.8 Hz, 2 H), 7.40 - 7.44 (m, 2 H), 7.21 (d, J=5.4 Hz, 1 H), 7.19 (d, J=8.3 Hz, 2
H), 6.61 (s, 1 H), 3.53 (s, 2 H), 2.35 (s, 3 H), 2.32 (s, 6 H).
207
N,N-Dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-4877 (3.15a) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)-N,N-dimethylmethanamine (92 mg, 0.21 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole (87 mg, 0.42 mmol),
Tetrakis(triphenylphosphine)palladium(0) (12.1 mg, 0.010 mmol), and sodium carbonate (111 mg,
1.05 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was
microwaved at 130 °C for 25 minutes and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and concentrated under reduced pressure and then purified by silica gel
column chromatography using a mobile phase of 1-10% (+ 1% ammonium
hydroxide)/methanol/dichloromethane to afford the title compound as an off white solid (73 mg,
72% yield). LCMS [M+H]+ 486.0 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.29 (d,
J=4.9 Hz, 1 H), 8.25 (s, 1 H), 8.00 (s, 1 H), 7.59 (t, J=8.3 Hz, 4 H), 7.42 - 7.46 (m, 3 H), 7.23 (d,
J=8.3 Hz, 2 H), 6.97 (s, 1 H), 3.94 (s, 3 H), 3.62 (s, 2 H), 2.34 (s, 6 H), 2.31 (s, 3 H).
208
1-(4-(4-(1,3-Dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-
dimethylmethanamine. (3.15b) 1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
N,N-dimethylmethanamine (50 mg, 0.114 mmol), 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (30.3 mg, 0.136 mmol),
tetrakis(triphenylphosphine)palladium(0) (3.9 mg, 0.003 mmol), and sodium carbonate (36.1 mg,
0.341 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane-water (3:1, 1 mL) was added to the vial. The reaction mixture was
heated at 90 °C for 48 h and monitored by LCMS. The reaction mixture was filtered through a bed
of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 0-20% methanol/dichloromethane to afford the title
compound as a yellow oil (18 mg, 32% yield). LCMS [M+H]+ 500.0 m/z, 81% pure by LC trace.
Used crude in the following reaction.
1-(4-(4-(1H-Pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-
dimethylmethanamine. (3.15c) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
209
N,N-dimethylmethanamine (50 mg, 0.114 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole (26.5 mg, 0.136 mmol), tetrakis(triphenylphosphine)palladium(0) (3.9 mg, 0.003
mmol), and sodium carbonate (72.3 mg, 0.682 mmol) were added to a vial that was subsequently
sealed and vacuum purged with nitrogen. A degassed solution of dioxane-water (3:1, 1 mL) was
added to the vial. The reaction mixture was heated at 90 °C for 48 h and monitored by LCMS. The
reaction mixture was filtered through a bed of Celite and concentrated under reduced pressure and
then purified by silica gel column chromatography using a mobile phase of 0-20% (1% ammonium
hydroxide/methanol)/dichloromethane to afford the title compound as a brown solid (35 mg, 66%
yield). LCMS [M+H]+ 472.0 m/z, 72% pure by LC trace. Used crude in the following reaction.
N,N-Dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. (3.15d) 1-(4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
N,N-dimethylmethanamine (50 mg, 0.11 mmol), 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (28 mg, 0.136 mmol), tetrakis(triphenylphosphine)palladium(0)
(3.9 mg, 0.003 mmol), and sodium carbonate (72 mg, 0.68 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane-water (3:1,
1 mL) was added to the vial. The reaction mixture was heated at 90 °C for 48 h and monitored by
LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under reduced
pressure and then purified by silica gel column chromatography using a mobile phase of 0-20%
210
(1% ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a brown
solid (16 mg, 28% yield). LCMS [M+H]+ 486.0 m/z. 1H NMR (500 MHz, DMSO-d6) ppm 8.33
(d, J=4.9 Hz, 1 H), 8.16 (d, J=5.4 Hz, 1 H), 7.95 (d, J=8.3 Hz, 2 H), 7.73 (d, J=8.3 Hz, 1 H), 7.55
(d, J=8.3 Hz, 1 H), 7.39 (d, J=8.3 Hz, 4 H), 7.35 (d, J=8.3 Hz, 1 H), 7.20 (d, J=5.4 Hz, 1 H), 6.97
(d, J=2.4 Hz, 1 H), 3.39 - 3.45 (m, 2 H), 2.29 - 2.34 (m, 3 H), 2.20 (s, 3 H), 2.17 (s, 6 H).
N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. (3.15e) 1-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
N,N-dimethylmethanamine (53.6 mg, 0.122 mmol), (4-(methylsulfonyl)phenyl)boronic acid (29.2
mg, 0.146 mmol), tetrakis(triphenylphosphine)palladium(0) (4.2 mg, 0.003 mmol), and sodium
carbonate (38.7 mg, 0.365 mmol) were added to a vial that was subsequently sealed and vacuum
purged with nitrogen. A degassed solution of dioxane-water (3:1, 2 mL) was added to the vial. The
reaction mixture was heated at 90 °C for 48 h and monitored by LCMS. The reaction mixture was
filtered through a bed of Celite and concentrated under reduced pressure and then purified by silica
gel column chromatography using a mobile phase of 0-10% ethyl acetate/hexanes to afford the
title compound as an off white solid (28 mg, 41% yield). LCMS [M+H]+ 560.0 m/z; 1H NMR (500
MHz, METHANOL-d4) ppm 8.47 (d, J=4.9 Hz, 1 H), 8.09 (d, J=8.8 Hz, 2 H), 7.91 (d, J=8.8 Hz,
211
2 H), 7.65 (d, J=8.3 Hz, 2 H), 7.57 (d, J=8.3 Hz, 2 H), 7.46 (d, J=4.9 Hz, 1 H), 7.44 (d, J=8.3 Hz,
2 H), 7.27 (d, J=7.8 Hz, 2 H), 6.82 (s, 1 H), 3.61 (s, 2 H), 3.16 (s, 3 H), 2.34 (s, 3 H), 2.32 (s, 6 H).
N,N-Dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine.164 NEU-1200 (3.16a) N,N-dimethyl-1-(4-(4-(1-methyl-1H-pyrazol-4-
yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (67 mg, 0.138 mmol) was
dissolved in dioxane (1.4 mL). Sodium hydroxide (3M, 0.46 mL) was added and the reaction
mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was then concentrated
under reduced pressure. The crude product was purified using column chromatography eluting
with a mobile phase of 1-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford
the title compound as the tosylate salt, yellow solid (46 mg, 66% yield). LCMS [M+H]+ 332.2
m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.13 (s, 1 H), 8.58 (s, 1 H), 8.19 (s, 1 H), 8.14 (d,
J=4.9 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H), 7.38 (d, J=7.8 Hz, 2 H), 7.30 (d, J=2.0 Hz, 1 H), 7.27 (d,
J=4.9 Hz, 1 H), 3.96 (s, 3 H), 3.42 (s, 2 H), 2.17 (s, 6 H). See assignments above.
212
1-(4-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-
dimethylmethanamine. NEU-4927 (3.16b) 1-(4-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-1-tosyl-1H-
pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine (18.3 mg, 0.037 mmol) was
dissolved in dioxane (1.0 mL). Sodium hydroxide (3M, 0.128 mL) was added and the reaction
mixture was microwaved for 30 minutes at 150 °C. The reaction mixture was then concentrated
under reduced pressure. The crude product was purified using column chromatography eluting
with a mobile phase of 1-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford
the title compound a yellow solid (8 mg, 62% yield). 1H NMR (500 MHz, METHANOL-d4)
ppm 8.17 (d, J=4.9 Hz, 1 H), 8.05 (s, 1 H), 7.87 (d, J=8.3 Hz, 2 H), 7.42 (d, J=8.3 Hz, 2 H), 7.10
(d, J=5.4 Hz, 1 H), 6.95 (s, 1 H), 3.94 (s, 3 H), 3.55 (s, 2 H), 2.41 (s, 3 H), 2.30 (s, 6 H). See
assignments above.
213
1-(4-(4-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine.
NEU-4928 (3.16c) 1-(4-(4-(1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
N,N-dimethylmethanamine (34 mg, 0.072 mmol) was dissolved in dioxane (1.0 mL). Sodium
hydroxide (3M, 0.721 mL) was added and the reaction mixture was microwaved for 15 minutes at
150 °C. The reaction mixture was then concentrated under reduced pressure. The crude product
was purified using column chromatography eluting with a mobile phase of 5-20% ((+1%
ammonium hydroxide)/methanol)/dichloromethane to afford the title compound as the tosylate
salt, yellow solid (10 mg, 45% yield). LCMS [M+H]+ 318.1 m/z; 1H NMR (500 MHz,
METHANOL-d4) ppm 8.33 (br. s., 2 H), 8.18 (d, J=5.4 Hz, 1 H), 8.05 (d, J=8.3 Hz, 2 H), 7.71
(d, J=7.8 Hz, 2 H), 7.61 (d, J=8.3 Hz, 2 H), 7.34 (d, J=5.4 Hz, 1 H), 7.28 (s, 1 H), 7.22 (d, J=7.8
Hz, 2 H), 4.36 (s, 2 H), 2.89 (s, 6 H), 2.35 (s, 3 H). See assignments above.
214
N,N-dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-4929 (3.16d) N,N-dimethyl-1-(4-(4-(3-methyl-1H-pyrazol-4-yl)-
1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (14.7 mg, 0.030 mmol) was dissolved
in dioxane (1.0 mL). Sodium hydroxide (3M, 0.302 mL) was added and the reaction mixture was
microwaved for 15 minutes at 150 °C. The reaction mixture was then concentrated under reduced
pressure. The crude product was purified using column chromatography eluting with a mobile
phase of 5-20% ((+1% ammonium hydroxide)/methanol)/dichloromethane to afford the title
compound as a yellow solid (6 mg, 63% yield). LCMS [M+H]+ 332.1 m/z; 1H NMR (500 MHz,
METHANOL-d4) ppm 8.18 (d, J=4.9 Hz, 1 H), 8.00 (s, 1 H), 7.87 (d, J=8.3 Hz, 2 H), 7.44 (d,
J=8.3 Hz, 2 H), 7.10 (d, J=4.9 Hz, 1 H), 6.94 (s, 1 H), 3.61 (s, 2 H), 2.48 (s, 3 H), 2.34 (s, 6 H).
See assignments above.
215
N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-4917 (3.16e) N,N-dimethyl-1-(4-(4-(4-(methylsulfonyl)phenyl)-1-
tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (28 mg, 0.050 mmol) was dissolved in
dioxane (1.0 mL). Sodium hydroxide (3M, 0.175 mL) was added and the reaction mixture was
microwaved for 30 minutes at 150 °C. The reaction mixture was then concentrated under reduced
pressure. The crude product was purified using column chromatography eluting with a mobile
phase of 0-20% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the title
compound as a yellow solid (7 mg, 33% yield). LCMS [M+H]+ 406.0 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 12.39 (s, 1 H), 8.33 (d, J=4.9 Hz, 1 H), 8.11 (s, 4 H), 7.96 (d, J=8.3 Hz, 2 H),
7.38 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz, 1 H), 7.15 (d, J=2.0 Hz, 1 H), 3.42 (s, 2 H), 3.31 (s, 3
H), 2.16 (s, 6 H). See assignments above.
216
4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.17) 4-Chloro-2-iodo-1-
tosyl-1H-pyrrolo[2,3-b]pyridine (2.32 g, 5.36 mmol), (4-formylphenyl)boronic acid (965 mg, 6.43
mmol), PdCl2(dppf)·CH2Cl2 (43.8 mg, 0.053 mmol), and sodium carbonate (1.70 g, 16.1 mmol)
were added to an oven dried flask that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane-water (3:1, 52 mL) was added to the vial. The reaction mixture
was heated at 85 °C overnight and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure, water was added a precipitate formed
and was collected by vacuum filtration to afford the title compound as a tan solid (2.09 g, 95%
yield). LCMS [M+H]+ 411.0 m/z (Cl35), 413.0 m/z (Cl37); 1H NMR (500 MHz, CHLOROFORM-
d) ppm 10.10 (s, 1 H), 8.38 (d, J=5.4 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H),
7.74 (d, J=7.8 Hz, 2 H), 7.23 (d, J=4.9 Hz, 1 H), 7.20 (d, J=8.3 Hz, 2 H), 6.70 (s, 1 H), 2.34 (s, 3
H).
217
4-(4-(4-(Morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde.
(3.18a) 4-(4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol),
4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)morpholine (215 mg, 0.608
mmol), tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol), and sodium carbonate
(129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 2.5 mL) was added to the vial. The reaction
mixture was heated at 100 °C for 18 h and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and concentrated under reduced pressure and then purified by silica gel
column chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title
compound as an off white solid (57 mg, 39% yield). LCMS [M+H]+ 602.1 m/z; 1H NMR (500
MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.62 (d, J=4.9 Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H),
7.88 (d, J=6.8 Hz, 4 H), 7.76 (dd, J=9.5, 8.5 Hz, 4 H), 7.32 (d, J=5.4 Hz, 1 H), 7.20 - 7.26 (m, 3
H), 6.74 (s, 1 H), 3.75 - 3.79 (m, 4 H), 3.03 - 3.08 (m, 4 H), 2.38 (s, 2 H).
218
4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde. (3.18b) 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde
(100 mg, 0.243 mmol), 1-methyl-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)sulfonyl)piperazine (223 mg, 0.608 mmol), Tetrakis(triphenylphosphine)palladium(0)
(14.1 mg, 0.012 mmol), and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
2.5 mL) was added to the vial. The reaction mixture was heated at 100 °C for 18 h and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under
reduced pressure and then purified by silica gel column chromatography using a mobile phase of
0-100% ethyl acetate/hexanes to afford the title compound as tan-orange solid (82 mg, 55% yield).
LCMS [M+H]+ 615.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.60
(d, J=5.4 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H), 7.87 (d, J=8.3 Hz, 4 H), 7.74 - 7.77 (m, 4 H), 7.29 (d,
J=4.9 Hz, 1 H), 7.25 (d, J=8.3 Hz, 2 H), 6.73 (s, 1 H), 3.09 (br. s., 4 H), 2.56 (br. s., 4 H), 2.38 (s,
3 H), 1.26 (s, 3 H).
219
4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18c) 4-(4-chloro-1-tosyl-
1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), phenylboronic acid (118.7
mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol), and sodium
carbonate (129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and vacuum
purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The
reaction mixture was heated at 90 °C overnight and monitored by LCMS. The reaction mixture
was filtered through a bed of Celite, concentrated under reduced pressure, and then purified by
silica gel column chromatography using a mobile phase of 0-50% ethyl acetate/hexanes to afford
the title compound as a tan solid (90 mg, 82% yield). LCMS [M+H]+ 453.1 m/z; 1H NMR (500
MHz, CHLOROFORM-d) ppm 10.10 (s, 1 H), 8.55 (d, J=4.9 Hz, 1 H), 8.25 (dd, J=7.8, 1.5 Hz,
4 H), 7.97 (d, J=8.3 Hz, 1 H), 7.83 (d, J=8.3 Hz, 1 H), 7.75 (d, J=7.8 Hz, 2 H), 7.57 - 7.60 (m, 2
H), 7.39 - 7.44 (m, 1 H), 7.29 (d, J=5.4 Hz, 1 H), 7.21 (d, J=8.3 Hz, 2 H), 6.80 (s, 1 H), 2.36 (s, 3
H).
220
4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18d) 4-(4-chloro-1-
tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), pyridin-4-ylboronic
acid (119.7 mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg, 0.012 mmol),
and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was subsequently sealed and
vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the
vial. The reaction mixture was heated at 90 °C overnight and monitored by LCMS. The reaction
mixture was filtered through a bed of Celite, concentrated under reduced pressure, and then
purified by silica gel column chromatography using a mobile phase of 0-100% ethyl
acetate/hexanes to afford the title compound as a off-white solid (65 mg, 59% yield). LCMS
[M+H]+ 454.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.75 (d, J=5.9
Hz, 2 H), 8.61 (d, J=4.9 Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H), 7.87 (d, J=8.3 Hz, 2 H), 7.75 (d, J=7.8
Hz, 2 H), 7.51 (d, J=5.9 Hz, 2 H), 7.32 (d, J=4.9 Hz, 1 H), 7.24 (d, J=8.3 Hz, 2 H), 6.76 (s, 1 H),
2.38 (s, 3 H).
221
4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18e) 4-(4-chloro-1-
tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (100 mg, 0.243 mmol), (2-methylpyridin-4-
yl)boronic acid (133.3 mg, 0.973 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.1 mg,
0.012 mmol), and sodium carbonate (129 mg, 1.22 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
2.5 mL) was added to the vial. The reaction mixture was heated at 90 °C overnight and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under reduced
pressure, and then purified by silica gel column chromatography using a mobile phase of 0-50%
ethyl acetate/dichloromethane to afford the title compound as a light yellow solid (70 mg, 62%
yield). LCMS [M+H]+ 468.1 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H),
8.61 (dd, J=13.4, 5.1 Hz, 2 H), 8.00 (d, J=7.8 Hz, 2 H), 7.86 (d, J=8.3 Hz, 2 H), 7.75 (d, J=8.3 Hz,
2 H), 7.34 (s, 1 H), 7.30 (d, J=5.4 Hz, 2 H), 7.24 (d, J=8.3 Hz, 2 H), 6.75 (s, 1 H), 2.64 (s, 3 H),
2.37 (s, 3 H).
222
4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.18f) 4-
(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (89 mg, 0.217 mmol), (2,6-
dimethylpyridin-4-yl)boronic acid (130.8 mg, 0.866 mmol),
Tetrakis(triphenylphosphine)palladium(0) (12.5 mg, 0.010 mmol), and sodium carbonate (115 mg,
1.08 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 2.0 mL) was added to the vial. The reaction mixture
was heated at 90 °C overnight and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite, concentrated under reduced pressure, and then purified by silica gel column
chromatography using a mobile phase of 0-60% ethyl acetate/dichloromethane to afford the title
compound as a yellow solid (89 mg, 85% yield). LCMS [M+H]+ 482.1 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 10.11 - 10.13 (m, 1 H), 8.49 - 8.54 (m, 1 H), 8.04 (d, J=8.3 Hz, 2 H), 7.88 (d,
J=8.3 Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H), 7.51 (d, J=5.4 Hz, 1 H), 7.38 (d, J=8.3 Hz, 2 H), 7.36 (s,
2 H), 7.15 (s, 1 H), 2.33 (s, 3 H), 1.16 (s, 6 H).
223
N,N-dimethyl-1-(4-(4-(4-(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. (3.19a) 4-(4-(4-(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridin-2-yl)benzaldehyde (50 mg, 0.083 mmol) was dissolved in dichloroethane (1 mL).
Dimethylamine hydrochloride (33.9 mg, 0.415 mmol), triethylamine (58 μL, 0.415 mmol) were
added and the reaction mixture was left stirring at room temperature for 1 hour, after which sodium
borohydride (15.7 mg, 0.415 mmol) was added. The reaction mixture was left stirring at room
temperature overnight. It was then quenched with sodium hydroxide (3M). The aqueous layer was
extracted 3 times with ethyl acetate. The combined organic layers were washed with water and
brine, dried with sodium sulfate, filtered, and concentrated to afford the title compound as a brown
residue (crude 56 mg). Quantitative yield was assumed. LCMS [M+H]+ 631.2 m/z; 1H NMR (500
MHz, CHLOROFORM-d) ppm 8.55 - 8.61 (m, 1 H), 7.86 (dd, J=7.6, 3.7 Hz, 8 H), 7.77 (d, J=7.3
Hz, 4 H), 7.20 - 7.24 (m, 2 H), 6.65 (s, 1 H), 3.75 - 3.80 (m, 4 H), 3.06 (d, J=4.4 Hz, 4 H), 2.60 (s,
1 H), 2.37 (d, J=3.4 Hz, 3 H), 1.26 (s, 6 H).
224
N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridin-2-yl)phenyl)methanamine. (3.19b) 4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-
1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (80 mg, 0.130 mmol) was dissolved in
dichloroethane (1 mL). Dimethylamine hydrochloride (53.1 mg, 0.650 mmol), triethylamine (91
μL, 0.650 mmol) were added and the reaction mixture was left stirring at room temperature for 1
hour, after which sodium borohydride (40.9 mg, 0.650 mmol) was added. The reaction mixture
was left stirring at room temperature overnight. It was then quenched with sodium hydroxide (3M).
The aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were
washed with water and brine, dried with sodium sulfate, filtered, and concentrated to afford the
title compound as a brown residue (70 mg, 84% crude yield). LCMS [M+H]+ 644.2 m/z; 1H NMR
(500 MHz, CHLOROFORM-d) ppm 8.58 (t, J=5.1 Hz, 1 H), 7.82 - 7.89 (m, 4 H), 7.75 (dd,
J=14.6, 7.8 Hz, 4 H), 7.58 - 7.63 (m, 2 H), 7.20 - 7.26 (m, 3 H), 6.66 (s, 1 H), 3.06 (br. s., 4 H),
2.57 - 2.61 (m, 2 H), 2.49 (d, J=4.9 Hz, 4 H), 2.27 (s, 6 H), 1.26 (s, 6 H).
225
N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine.
(3.19c) 4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (90 mg, 0.199 mmol)
was dissolved in dichloroethane (1.5 mL) to which dimethylamine (2M in methanol, 1 mL) was
added. The mixture was left stirring at room temperature for 1 hour. Sodium cyanoborohydride
(62.5 mg, 0.994 mmol) was added to the reaction mixture which was left stirring at room
temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide and then
extracted with dichloromethane 3 times. The combined organic layers were washed with water
and brine, collected, and concentrated. The product was purified by column chromatography using
a mobile phase of 0-100% ethyl acetate/dichloromethane to afford the title compound as a yellow
solid (35 mg, 36%). LCMS [M+H]+ 482.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm
8.51 (d, J=4.9 Hz, 1 H), 7.81 (d, J=7.8 Hz, 2 H), 7.38 - 7.61 (m, 9 H), 7.14 - 7.30 (m, 3 H), 6.71
(s, 1 H), 3.55 (br. s., 2 H), 3.48 (s, 3 H), 2.32 (br. s., 6 H).
226
N,N-dimethyl-1-(4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine.
(3.19d) 4-(4-(pyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (64 mg, 0.141
mmol) was dissolved in dichloroethane (1.5 mL) to which dimethylamine (2M in methanol, 0.7
mL) was added. The mixture was left stirring at room temperature for 1 hour. Sodium
cyanoborohydride (44.3 mg, 0.706 mmol) was added to the reaction mixture which was left stirring
at room temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide
and then extracted with ethyl acetate 3 times. The combined organic layers were washed with
water and brine, collected, and concentrated. The product was purified by column chromatography
using a mobile phase of 0-100% ethyl acetate/dichloromethane followed by 0-20% methanol/ethyl
acetate to afford the title compound as a white solid (28 mg, 40%). LCMS [M+H]+ 483.2 m/z.
N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. (3.19e) 4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (65 mg, 0.139 mmol) was dissolved in dichloroethane (1.0 mL) to which
dimethylamine hydrochloride (56.7 mg, 0.695 mmol) was added along with triethylamine (97 μL,
0.695 mmol). The mixture was left stirring at room temperature for 1 hour. Sodium
cyanoborohydride (44.7 mg, 0.695 mmol) was added to the reaction mixture which was left stirring
at room temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide
and then extracted with dichloromethane 3 times. The combined organic layers were washed with
227
water and brine, collected, and concentrated to afford the title compound as a brown residue to use
in the next reaction assuming quantitative yield. LCMS [M+H]+ 497.2 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 8.56 (d, J=5.4 Hz, 1 H), 8.48 (d, J=4.9 Hz, 1 H), 7.74 (d, J=8.3 Hz, 2 H), 7.56
(d, J=6.3 Hz, 3 H), 7.48 - 7.52 (m, 2 H), 7.40 (d, J=7.8 Hz, 2 H), 7.36 (d, J=8.3 Hz, 2 H), 6.97 (s,
1 H), 3.48 (s, 2 H), 2.54 (s, 3 H), 2.32 (s, 3 H), 2.20 (s, 6 H).
1-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-
dimethylmethanamine. (3.19f) 4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-
2-yl)benzaldehyde (87 mg, 0.181 mmol) was dissolved in dichloroethane (1.0 mL) to which
dimethylamine hydrochloride (73.7 mg, 0.903 mmol) was added along with triethylamine (126
μL, 0.903 mmol). The mixture was left stirring at room temperature for 1 hour. Sodium
cyanoborohydride (56.8 mg, 0.903 mmol) was added to the reaction mixture which was left stirring
at room temperature overnight. The reaction mixture was quenched with 3M sodium hydroxide
and then extracted with ethyl acetate 3 times. The combined organic layers were washed with
water and brine, collected, and concentrated. The product was purified by silica gel column
chromatography using a mobile phase of 1-5% methanol/dichloromethane to afford the title
compound as a yellow solid (12 mg, 13% yield). LCMS [M+H]+ 511.2 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) ppm 8.54 (d, J=5.9 Hz, 1 H), 7.83 (d, J=8.3 Hz, 2 H), 7.51 (d, J=7.8 Hz, 2
228
H), 7.42 (d, J=7.8 Hz, 2 H), 7.25 (d, J=5.9 Hz, 1 H), 7.20 (d, J=8.8 Hz, 2 H), 7.15 (s, 2 H), 6.65
(s, 1 H), 3.52 (s, 2 H), 2.59 (s, 6 H), 2.34 - 2.38 (m, 3 H), 2.31 (s, 6 H).
N,N-dimethyl-1-(4-(4-(4-(morpholinosulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-5828 (3.20a) N,N-dimethyl-1-(4-(4-(4-
(morpholinosulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (55
mg, 0.087 mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide (3M, 0.3 mL) was added
and the reaction mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was
quenched with saturated ammonium chloride solution and diluted with water. A precipitate formed
and was collected by vacuum filtration. The solid crude product was purified by column
chromatography eluting with a mobile phase of 1-20% ((+5% ammonium
hydroxide)/methanol)/dichloromethane to afford the title compound as a yellow solid (26 mg, 63%
yield). LCMS [M+H]+ 477.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.40 (s, 1 H), 8.33 (d,
J=4.9 Hz, 1 H), 8.12 (d, J=8.3 Hz, 2 H), 7.97 (d, J=7.8 Hz, 2 H), 7.91 (d, J=8.3 Hz, 2 H), 7.38 (d,
J=7.8 Hz, 2 H), 7.30 (d, J=4.9 Hz, 1 H), 7.19 (d, J=1.5 Hz, 1 H), 3.64 - 3.72 (m, 4 H), 3.42 (s, 2
H), 2.97 (br. s., 4 H), 2.17 (s, 6 H).
229
N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-5829 (3.20b) N,N-dimethyl-1-(4-(4-(4-((4-methylpiperazin-1-
yl)sulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (70 mg, 0.109
mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide (3M, 0.3 mL) was added and the
reaction mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was quenched
with saturated ammonium chloride solution and diluted with water. A precipitate formed and was
collected by vacuum filtration. The solid crude product was purified by column chromatography
eluting with a mobile phase of 1-20% ((+5% ammonium hydroxide)/methanol)/dichloromethane
to afford the title compound as a yellow solid (12 mg, 22% yield). LCMS [M+H]+ 490.2 m/z; 1H
NMR (500 MHz, DMSO-d6) ppm 12.39 (br. s., 1 H), 8.33 (d, J=4.9 Hz, 1 H), 8.11 (d, J=8.3 Hz,
2 H), 7.97 (d, J=7.8 Hz, 2 H), 7.90 (d, J=8.3 Hz, 2 H), 7.38 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz,
1 H), 7.18 (s, 1 H), 3.43 (s, 2 H), 2.98 (br. s., 4 H), 2.40 (br. s., 4 H), 2.13 - 2.20 (m, 2 H).
230
N,N-dimethyl-1-(4-(4-phenyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. NEU-5446
(3.20c) N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine (34 mg, 0.071 mmol) was dissolved in dioxane (0.2 mL). Sodium
hydroxide (3M, 0.23 mL) was added and the reaction mixture was microwaved for 20 minutes at
150 °C. The reaction mixture was quenched with saturated ammonium chloride solution and
concentrated under reduced pressure. The crude product was purified by column chromatography
eluting with a mobile phase of 1-15% methanol/dichloromethane to afford the title compound as
a salt. The salt was dissolved in ethyl acetate which was neutralized using 1M HCl and saturated
sodium bicarbonate solution. The organic layer was collected to afford the title compound as a
yellow solid (2 mg, 10% yield). LCMS [M+H]+ 328.2 m/z; 1H NMR (500 MHz, METHANOL-
d4) ppm 8.24 (d, J=4.9 Hz, 1 H), 7.86 (d, J=8.3 Hz, 2 H), 7.81 (dd, J=8.1, 1.2 Hz, 2 H), 7.56 (t,
J=7.6 Hz, 2 H), 7.41 - 7.49 (m, 3 H), 7.21 (d, J=4.9 Hz, 1 H), 7.02 (s, 1 H), 3.57 (s, 2 H), 2.31 (s,
6 H).
231
N,N-dimethyl-1-(4-(4-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine. NEU-
5447 (3.20d) N,N-dimethyl-1-(4-(4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine (27 mg, 0.056 mmol) was dissolved in dioxane (0.2 mL). Sodium
hydroxide (3M, 0.2 mL) was added and the reaction mixture was microwaved for 20 minutes at
150 °C. The reaction mixture was quenched with saturated ammonium chloride solution and
concentrated under reduced pressure. The crude product was purified by column chromatography
eluting with a mobile phase of 1-15% methanol/dichloromethane to afford the title compound as
a salt. The salt was dissolved in ethyl acetate which was neutralized using 1M HCl and saturated
sodium bicarbonate solution. The organic layer was collected to afford the title compound as a
yellow solid (17 mg, 92% yield). LCMS [M+H]+ 329.2 m/z; 1H NMR (500 MHz, DMSO-d6)
ppm 12.41 (s, 1 H), 8.76 (d, J=5.9 Hz, 2 H), 8.33 (d, J=4.9 Hz, 1 H), 7.97 (d, J=8.3 Hz, 2 H), 7.85
(d, J=5.9 Hz, 2 H), 7.38 (d, J=8.3 Hz, 2 H), 7.32 (d, J=4.9 Hz, 1 H), 7.19 (d, J=2.0 Hz, 1 H), 2.26
(s, 2 H), 2.17 (s, 6 H).
232
N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-5449 (3.20e) N,N-dimethyl-1-(4-(4-(2-methylpyridin-4-yl)-1-
tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanamine (69 mg, 0.139 mmol) was dissolved in
dioxane (0.5 mL). Sodium hydroxide (3M, 0.5 mL) was added and the reaction mixture was
microwaved for 20 minutes at 150 °C. The reaction mixture was quenched with saturated
ammonium chloride solution. The aqueous layer was extracted with ethyl acetate 3 times. The
combined organic layers were washed with water and brine, collected, dried with sodium sulfate,
filtered and concentrated under reduced pressure. The crude product was purified by column
chromatography eluting with a mobile phase of 1-20% methanol/dichloromethane to afford the
title compound as a yellow solid (35 mg, 75% yield). LCMS [M+H]+ 343.2 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 12.39 (s, 1 H), 8.62 (d, J=5.4 Hz, 1 H), 8.32 (d, J=4.9 Hz, 1 H), 7.98 (d,
J=8.3 Hz, 2 H), 7.69 (s, 1 H), 7.63 (d, J=5.4 Hz, 1 H), 7.40 (d, J=7.8 Hz, 2 H), 7.29 (d, J=4.9 Hz,
1 H), 7.18 (d, J=1.5 Hz, 1 H), 3.49 (br. s., 2 H), 2.61 (s, 3 H), 2.21 (s, 6 H).
233
1-(4-(4-(2,6-dimethylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-
dimethylmethanamine. NEU-5452 (3.20f) 1-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-
pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N,N-dimethylmethanamine (12 mg, 0.023 mmol) was
dissolved in dioxane (0.1 mL). Sodium hydroxide (3M, 0.1 mL) was added and the reaction
mixture was microwaved for 15 minutes at 150 °C. The reaction mixture was quenched with
saturated ammonium chloride solution and diluted with water. A precipitate formed and was
collected by vacuum filtration to afford the title compound as a yellow solid (3 mg, 35% yield).
LCMS [M+H]+ 357.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.29 (d, J=4.9 Hz, 1 H),
7.93 (d, J=8.3 Hz, 2 H), 7.51 (s, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.27 (d, J=4.9 Hz, 1 H), 7.06 (s, 1
H), 3.73 (s, 2 H), 2.63 (s, 6 H), 2.43 (s, 6 H).
4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-N,N-dimethylaniline. (3.22a) 4-chloro-2-
iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (200 mg, 0.462 mmol), (4-
(dimethylamino)phenyl)boronic acid (152.5 mg, 0.924 mmol), PdCl2(dppf)·CH2Cl2 (18.9 mg,
0.023 mmol), and sodium carbonate (147 mg, 1.39 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
234
3 mL) was added to the vial. The reaction mixture was heated at 80 °C overnight and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite, concentrated under reduced
pressure, and purified by silica gel column chromatography using a mobile phase of 0-50% ethyl
acetate/hexanes to afford the title compound as a yellow crystalline solid (153 mg, 78% yield).
LCMS [M+H]+ 426.2 m/z (Cl35), 428.2 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.29
(d, J=5.4 Hz, 1 H), 7.65 (d, J=8.3 Hz, 2 H), 7.40 - 7.45 (m, 3 H), 7.34 (d, J=8.3 Hz, 2 H), 6.80 (d,
J=8.8 Hz, 2 H), 6.66 (s, 1 H), 3.01 (s, 6 H), 2.32 (s, 3 H).
N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-N-ethylethanamine. (3.22b) 4-
chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (200 mg, 0.462 mmol), N-ethyl-N-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)ethanamine (174 mg, 0.600 mmol),
PdCl2(dppf)·CH2Cl2 (18.9 mg, 0.023 mmol), and sodium carbonate (147 mg, 1.39 mmol) were
added to an oven dried vial that was subsequently sealed and vacuum purged with nitrogen. A
degassed solution of dioxane/water (3:1, 4 mL) was added to the vial. The reaction mixture was
heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 1-20% (1% ammonium
hydroxide/methanol)/dichloromethane, followed by 50-100% ethyl acetate/hexanes and 0-20%
methanol/ethyl acetate to afford the title compound as a tan solid (116 mg, 54% yield). LCMS
235
[M+H]+ 468.1 m/z (Cl35), 470.1 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.35 (d, J=5.4
Hz, 1 H), 7.69 (d, J=8.3 Hz, 4 H), 7.49 (d, J=5.4 Hz, 1 H), 7.36 (d, J=8.3 Hz, 4 H), 6.83 - 6.92 (m,
1 H), 2.54 (s, 2 H), 2.32 (s, 9 H), 1.06 (s, 4 H).
4-chloro-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine. (3.22c) 4-
chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.347 mmol), (4-(1-(pyrrolidin-1-
yl)ethyl)phenyl)boronic acid (152 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (14.2 mg, 0.017 mmol),
and sodium carbonate (110 mg, 1.04 mmol) were added to an oven dried vial that was subsequently
sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was
added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite. The crude product was purified by
column chromatography eluting with a mobile phase 0-100% ethyl acetate/hexanes to afford the
title compound as a light brown solid (110 mg, 66% yield). LCMS [M+H]+ 480.1 m/z (Cl35), 482.1
m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 8.34 (d, J=4.9 Hz, 1 H), 7.68 (d, J=8.3 Hz, 2
H), 7.55 (d, J=7.8 Hz, 2 H), 7.40 - 7.49 (m, 3 H), 7.35 (d, J=8.3 Hz, 2 H), 6.85 (s, 1 H), 2.54 (br.
s., 2 H), 2.36 (br. s., 2 H), 2.32 (s, 3 H), 1.70 (br. s., 5 H), 1.37 (d, J=6.3 Hz, 3 H).
236
4-chloro-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine.
(3.22d) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (214 mg, 0.495 mmol), (4-((4-
methylpiperazin-1-yl)methyl)phenyl)boronic acid (231 mg, 0.989 mmol), PdCl2(dppf)·CH2Cl2
(20.2 mg, 0.025 mmol), and sodium carbonate (157 mg, 1.48 mmol) were added to an oven dried
vial that was subsequently sealed and vacuum purged with nitrogen. A degassed solution of
dioxane/water (3:1, 3.5 mL) was added to the vial. The reaction mixture was heated at 85 °C
overnight and monitored by LCMS. The reaction mixture was filtered through a bed of Celite. The
crude product was purified by column chromatography eluting with a mobile phase 0-20% ethyl
acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as a tan
solid (245 mg, 99% yield). LCMS [M+H]+ 495.2 m/z (Cl35), 497.2 m/z (Cl371H NMR (500 MHz,
DMSO-d6) ppm 8.34 (d, J=5.4 Hz, 1 H), 7.69 (d, J=8.3 Hz, 2 H), 7.56 (d, J=8.3 Hz, 2 H), 7.48
(d, J=5.4 Hz, 1 H), 7.42 (d, J=8.3 Hz, 2 H), 7.35 (d, J=7.8 Hz, 2 H), 6.84 (s, 1 H), 3.57 (s, 2 H),
2.45 (br. s., 8 H), 2.32 (s, 3 H), 2.23 (br. s., 3 H).
tert-butyl 2-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)pyrrolidine-1-
carboxylate. (3.22e) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (250 mg, 0.578 mmol),
237
tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxylate
(259 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (4.7 mg, 0.006 mmol), and sodium carbonate (184
mg, 1.73 mmol) were added to an oven dried vial that was subsequently sealed and vacuum purged
with nitrogen. A degassed solution of dioxane/water (3:1, 4 mL) was added to the vial. The reaction
mixture was heated at 85 °C overnight and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite. The crude product was purified by column chromatography eluting with
a mobile phase 0-20% ethyl acetate/hexanes to afford the title compound as a white solid (125 mg,
39% yield). LCMS [M+H]+ 552.1 m/z (Cl35), 554.1 m/z (Cl37); 1H NMR (500 MHz,
CHLOROFORM-d) ppm 8.36 (d, J=4.9 Hz, 1 H), 7.76 (d, J=7.3 Hz, 2 H), 7.48 (d, J=8.3 Hz, 2
H), 7.26 - 7.30 (m, 2 H), 7.15 - 7.23 (m, 3 H), 6.58 (s, 1 H), 3.66 - 3.73 (m, 1 H), 2.38 - 2.46 (m,
1 H), 2.35 (s, 3 H), 1.86 - 2.04 (m, 3 H), 1.44 - 1.53 (m, 2 H), 1.21 - 1.32 (m, 9 H)
4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. (3.22f) 4-chloro-2-iodo-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (266 mg, 0.615 mmol), 4-boronobenzoic acid (204 mg, 1.23 mmol),
PdCl2(dppf)·CH2Cl2 (25.1 mg, 0.030 mmol), and sodium carbonate (195 mg, 1.84 mmol) were
added to an oven dried vial that was subsequently sealed and vacuum purged with nitrogen. A
degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The reaction mixture was
heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite. The crude product was purified by column chromatography eluting with a mobile
phase 20-100% ethyl acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title
238
compound as a white solid (76 mg, 29% yield). LCMS [M+H]+ 427.1 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 8.37 (d, J=5.4 Hz, 1 H), 8.04 (d, J=8.3 Hz, 2 H), 7.87 (q, J=8.1 Hz, 2 H), 7.72
(d, J=8.3 Hz, 2 H), 7.68 (d, J=8.3 Hz, 1 H), 7.49 (d, J=5.4 Hz, 1 H), 7.37 (d, J=8.3 Hz, 2 H), 6.94
(s, 1 H), 2.32 (s, 3 H).
4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. (3.22g) 4-chloro-2-iodo-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (250 mg, 0.578 mmol), (4-carbamoylphenyl)boronic acid (190.6 mg,
1.16 mmol), PdCl2(dppf)·CH2Cl2 (23.6 mg, 0.029 mmol), and sodium carbonate (183.7 mg, 1.73
mmol) were added to a microwave vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 5 mL) was added to the vial. The reaction
mixture was microwaved at 150 °C for 50 minutes and monitored by LCMS. The reaction mixture
was filtered through a bed of Celite and concentrated under reduced pressure to afford the title
compound as a brown solid (75 mg, 31% yield). LCMS [M+H]+ 425.1 m/z (Cl35), 427.1 m/z
(Cl37);; 1H NMR (500 MHz, CHLOROFORM-d) ppm 8.39 (d, J=5.4 Hz, 1 H), 7.92 (d, J=8.3
Hz, 2 H), 7.77 (d, J=8.3 Hz, 2 H), 7.66 (d, J=8.3 Hz, 2 H), 7.23 (d, J=5.4 Hz, 1 H), 7.20 (d, J=8.8
Hz, 2 H), 6.67 (s, 1 H), 2.36 (s, 3 H)
239
N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide. (3.22h) 4-chloro-2-
iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.347 mmol), (4-acetamidophenyl)boronic acid
(124 mg, 0.693 mmol), PdCl2(dppf)·CH2Cl2 (14.2 mg, 0.017 mmol), and sodium carbonate (110
mg, 1.04 mmol) were added to a microwave vial that was subsequently sealed and vacuum purged
with nitrogen. A degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The reaction
mixture was heated at 85 °C overnight and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and purified by column chromatography eluting with a mobile phase of 0-
100% ethyl acetate/hexanes to afford the title compound as a white solid (122 mg, 80% yield).
LCMS [M+H]+ 440.1 m/z (Cl35), 442.1 m/z (Cl37); 1H NMR (500 MHz, DMSO-d6) ppm 10.16
(s, 1 H), 8.33 (d, J=5.4 Hz, 1 H), 7.69 (dd, J=12.4, 8.5 Hz, 4 H), 7.53 (d, J=8.8 Hz, 2 H), 7.46 (d,
J=5.4 Hz, 1 H), 7.35 (d, J=8.3 Hz, 2 H), 6.79 (s, 1 H), 2.32 (s, 3 H), 2.10 (s, 3 H).
4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. (3.22i) 4-chloro-2-iodo-1-tosyl-1H-
pyrrolo[2,3-b]pyridine (100 mg, 0.231 mmol), (4-hydroxyphenyl)boronic acid (41.4 mg, 0.300
mmol), PdCl2(dppf)·CH2Cl2 (9.4 mg, 0.01 mmol), and sodium carbonate (73.5 mg, 0.693 mmol)
were added to a vial that was subsequently sealed and vacuum purged with nitrogen. A degassed
solution of dioxane/water (3:1, 2 mL) was added to the vial. The reaction mixture was heated at
240
90 °C overnight and monitored by LCMS. The reaction mixture was filtered through a bed of
Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 10-100% ethyl acetate/hexanes to afford the title
compound as a yellow solid (74 mg, 84% yield). LCMS [M+H]+ 399.9 m/z (Cl35), 401.9 m/z (Cl37);
1H NMR (500 MHz, CHLOROFORM-d) ppm 8.35 (d, J=5.4 Hz, 1 H), 7.73 (d, J=8.8 Hz, 2 H),
7.42 (d, J=8.8 Hz, 2 H), 7.21 (d, J=5.4 Hz, 1 H), 7.18 (d, J=8.3 Hz, 2 H), 6.94 (d, J=8.8 Hz, 2 H),
6.55 (s, 1 H), 2.35 (s, 3 H).
N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline.
(3.23a) 4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.352 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (219.8 mg, 1.06 mmol),
Tetrakis(triphenylphosphine)palladium(0) (20.3 mg, 0.018 mmol), and sodium carbonate (112 mg,
1.06 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 3 mL) was added to the vial. The reaction mixture was
heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite, diluted with dichloromethane and water. The organic layer was collected and the
aqueous layer extracted 3 times with dichloromethane. The combined organic layers were washed
with water and then dried using sodium sulfate. The compound was purified by silica gel column
chromatography using a mobile phase of 40-100% ethyl acetate/hexanes to afford the title
241
compound as a bright yellow solid (127 mg, 76% yield). LCMS [M+H]+ 472.1 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 8.50 (s, 1 H), 8.24 (d, J=4.9 Hz, 1 H), 8.10 (s, 1 H), 7.62 - 7.67 (m, 2 H),
7.42 - 7.47 (m, 3 H), 7.31 (d, J=8.8 Hz, 2 H), 6.96 - 6.99 (m, 1 H), 6.78 - 6.84 (m, 2 H), 3.86 - 3.90
(m, 3 H), 2.98 - 3.02 (m, 6 H), 2.29 (s, 3 H).
N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)ethanamine. (3.23b) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-N-
ethylethanamine (116.3 mg, 0.248 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole (103.4 mg, 0.497 mmol), Tetrakis(triphenylphosphine)palladium(0) (14.4 mg,
0.012 mmol), and sodium carbonate (131.7 mg, 1.24 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
2.5 mL) was added to the vial. The reaction mixture was heated at 90 °C overnight and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under
reduced pressure and then purified by silica gel column chromatography using a mobile phase of
2-20% (5% ammonium hydroxide/methanol)/dichloromethane to afford the title compound as a
brown residue (45 mg, 23% yield). LCMS [M+H]+ 514.2 m/z; 1H NMR (500 MHz, DMSO-d6)
ppm 8.55 (s, 1 H), 8.29 (d, J=5.4 Hz, 1 H), 8.14 (s, 1 H), 7.74 - 7.78 (m, 2 H), 7.70 - 7.73 (m, 2
242
H), 7.68 (d, J=8.3 Hz, 2 H), 7.49 (d, J=5.4 Hz, 1 H), 7.33 (d, J=8.3 Hz, 2 H), 7.23 (s, 1 H), 4.41
(d, J=5.4 Hz, 2 H), 3.86 - 3.91 (m, 4 H), 3.16 (s, 6 H), 1.29 (t, J=7.3 Hz, 6 H).
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridine. (3.23c) 4-chloro-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridine (101 mg, 0.210 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (56.9 mg, 0.273 mmol), Tetrakis(triphenylphosphine)palladium(0) (12.1 mg, 0.010
mmol), and sodium carbonate (66.9 mg, 0.631 mmol) were added to a vial that was subsequently
sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1, 2.1 mL) was
added to the vial. The reaction mixture was heated at 85 °C overnight and monitored by LCMS.
The reaction mixture was filtered through a bed of Celite, concentrated under reduced pressure,
then purified by silica gel column chromatography using a mobile phase of 50-100% ethyl
acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as a brown
oil (67 mg, 61% yield). LCMS [M+H]+ 526.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.55 (s,
1 H), 8.28 (d, J=4.9 Hz, 1 H), 8.13 (s, 1 H), 7.69 (d, J=8.3 Hz, 2 H), 7.59 (d, J=7.8 Hz, 2 H), 7.48
(d, J=4.9 Hz, 3 H), 7.32 (d, J=8.3 Hz, 2 H), 7.16 (s, 1 H), 3.88 (s, 3 H), 3.56 (s, 6 H), 2.34 - 2.47
(m, 1 H), 2.30 (s, 3 H), 1.72 (br. s., 3 H), 1.40 (br. s., 2 H).
243
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-1H-
pyrrolo[2,3-b]pyridine. (3.23d) 4-chloro-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (245 mg, 0.495 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (134 mg, 0.643 mmol),
Tetrakis(triphenylphosphine)palladium(0) (28.6 mg, 0.025 mmol), and sodium carbonate (157 mg,
1.48 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 4 mL) was added to the vial. The reaction mixture was
heated at 100 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite, concentrated under reduced pressure, then purified by silica gel column
chromatography eluting with a mobile phase of 0-100% ethyl acetate/hexanes to afford the title
compound as a brown flaky solid (192 mg, 72% yield). LCMS [M+H]+ 541.1 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 8.54 (s, 1 H), 8.28 (d, J=4.9 Hz, 1 H), 8.13 (s, 1 H), 7.69 (d, J=8.3 Hz, 2
H), 7.58 (d, J=7.8 Hz, 2 H), 7.45 - 7.50 (m, 1 H), 7.42 (d, J=8.3 Hz, 2 H), 7.33 (d, J=7.8 Hz, 2 H),
7.15 (s, 1 H), 3.88 (s, 3 H), 3.55 (s, 2 H), 2.20 - 2.48 (m, 11 H), 2.16 (s, 3 H).
244
tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)pyrrolidine-1-carboxylate. (3.23e) tert-butyl 2-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-
b]pyridin-2-yl)phenyl)pyrrolidine-1-carboxylate (125 mg, 0.226 mmol), 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (61.2 mg, 0.294 mmol),
Tetrakis(triphenylphosphine)palladium(0) (13.1 mg, 0.011 mmol), and sodium carbonate (72 mg,
0.679 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 2.5 mL) was added to the vial. The reaction mixture
was heated at 85 °C overnight and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite, concentrated under reduced pressure, then purified by silica gel column
chromatography eluting with a mobile phase of 20-100% ethyl acetate/hexanes to afford the title
compound as a brown flaky solid (129 mg, 95% yield). LCMS [M+H]+ 598.2 m/z; 1H NMR (500
MHz, CHLOROFORM-d) ppm 8.43 (br. s., 1 H), 7.88 (s, 1 H), 7.75 - 7.83 (m, 4 H), 7.51 (d,
J=7.8 Hz, 2 H), 7.28 (d, J=2.9 Hz, 2 H), 7.23 (d, J=4.9 Hz, 1 H), 7.17 (d, J=7.8 Hz, 2 H), 6.68 (s,
1 H), 3.98 (s, 3 H), 3.66 - 3.73 (m, 1 H), 2.34 (s, 3 H), 2.06 (s, 2 H), 1.75 (s, 1 H), 1.50 (br. s., 2
H), 1.27 (s, 9 H).
245
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.23f) 4-
(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (573.3 mg, 1.40 mmol), 1-methyl-
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (580.6 mg, 2.79 mmol),
Tetrakis(triphenylphosphine)palladium(0) (80.6 mg, 0.070 mmol), and sodium carbonate (443.7
mg, 4.19 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 20 mL) was added to the vial. The reaction
mixture was heated at 100 °C for 1 hour and monitored by LCMS. The reaction mixture was
filtered through a bed of Celite, concentrated under reduced pressure, and then purified by silica
gel column chromatography using a mobile phase of 30-100% ethyl acetate/hexanes to afford the
title compound as a pale yellow solid (635 mg, 99% yield). LCMS [M+H]+ 457.1 m/z; 1H NMR
(500 MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.45 (d, J=5.4 Hz, 1 H), 7.97 - 8.03 (m, 2
H), 7.87 (s, 1 H), 7.75 - 7.84 (m, 5 H), 7.23 - 7.27 (m, 1 H), 7.20 (d, J=8.8 Hz, 2 H), 6.80 (s, 1 H),
3.98 (s, 3 H), 2.35 (s, 3 H).
246
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. (3.23g) 4-
(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid (367 mg, 0.860 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (537 mg, 2.58 mmol),
Tetrakis(triphenylphosphine)palladium(0) (49.7 mg, 0.043 mmol), and sodium carbonate (273 mg,
2.58 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 8 mL) was added to the vial. The reaction mixture was
heated at 80 °C for 48 h and monitored by LCMS. The reaction mixture was filtered through a bed
of Celite, concentrated under reduced pressure, and then purified by silica gel column
chromatography using a mobile phase of 3-20% ((+10% ammonium
hydroxide)/methanol)/dichloromethane to afford the title compound as a brown solid (17 mg, 4%
yield). LCMS [M+H]+ 473.1 m/z.
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. (3.23h) 4-(4-
chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide (246 mg, 0.578 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (360.5 mg, 1.73 mmol),
247
Tetrakis(triphenylphosphine)palladium(0) (33.4 mg, 0.029 mmol), and sodium carbonate (183.7
mg, 1.73 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 5 mL) was added to the vial. The reaction
mixture was heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite, concentrated under reduced pressure, and then purified by silica gel
column chromatography using a mobile phase of 1-15% methanol/dichloromethane to afford the
title compound as an off-white solid (114 mg, 42% yield). LCMS [M+H]+ 472.2 m/z; 1H NMR
(500 MHz, DMSO-d6) ppm 8.55 (s, 1 H), 8.31 (d, J=5.4 Hz, 1 H), 8.14 (s, 1 H), 8.11 (s, 1 H),
8.00 (d, J=8.3 Hz, 2 H), 7.72 (dd, J=8.3, 5.9 Hz, 4 H), 7.46 - 7.51 (m, 2 H), 7.34 (d, J=8.3 Hz, 2
H), 7.25 (s, 1 H), 3.89 (s, 3 H), 2.31 (s, 3 H).
N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide.
(3.23i) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide (116 mg, 0.264
mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (71.3 mg, 0.343
mmol), Tetrakis(triphenylphosphine)palladium(0) (15.2 mg, 0.013 mmol), and sodium carbonate
(83.8 mg, 0.791 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 2.1 mL) was added to the vial. The reaction
mixture was heated at 85 °C for 48 h and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite, concentrated under reduced pressure, and then purified by silica gel
248
column chromatography using a mobile phase of 50-100% ethyl acetate/hexanes to afford the title
compound as an off-white solid (65 mg, 51% yield). LCMS [M+H]+ 486.1 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 10.16 (s, 1 H), 8.33 (d, J=5.4 Hz, 1 H), 7.69 (dd, J=12.4, 8.5 Hz, 4 H),
7.53 (d, J=8.8 Hz, 2 H), 7.46 (d, J=5.4 Hz, 1 H), 7.35 (d, J=8.3 Hz, 2 H), 6.79 (s, 1 H), 2.32 (s, 3
H), 2.10 (s, 3 H).
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. (3.23j) 4-(4-
chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (50 mg, 0.125 mmol), 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (78.2 mg, 0.376 mmol),
Tetrakis(triphenylphosphine)palladium(0) (7.2 mg, 0.006 mmol), and sodium carbonate (40 mg,
0.376 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 1 mL) was added to the vial. The reaction mixture was
heated at 90 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 0-20% (10% ammonium
hydroxide/methanol)/dichloromethane to afford the title compound as an off white solid (52 mg,
94% yield). LCMS [M+H]+ 445.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 9.77 (s, 1 H), 8.52
(s, 1 H), 8.26 (d, J=5.4 Hz, 1 H), 8.10 (s, 1 H), 7.80 (s, 1 H), 7.67 (d, J=8.3 Hz, 2 H), 7.56 (s, 1 H),
249
7.46 (d, J=4.9 Hz, 1 H), 7.43 (d, J=8.3 Hz, 2 H), 7.32 (d, J=8.3 Hz, 2 H), 7.02 (s, 1 H), 6.88 (d,
J=8.3 Hz, 2 H), 3.93 (s, 1 H), 3.88 (s, 3 H), 3.81 (s, 1 H), 2.29 (s, 3 H).
N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline. NEU-
5323 (3.24a) N,N-dimethyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-
2-yl)aniline (122 mg, 0.259 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide was added
(3M, 1 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture
was diluted with water. The precipitate that formed was collected by vacuum filtration to afford
the title compound as a yellow solid (80 mg, 97% yield). LCMS [M+H]+ 318.2 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 11.90 (s, 1 H), 8.53 (s, 1 H), 8.15 (s, 1 H), 8.06 (d, J=5.4 Hz, 1 H), 7.86
(d, J=8.8 Hz, 2 H), 7.21 (d, J=4.9 Hz, 1 H), 7.06 (d, J=2.0 Hz, 1 H), 6.80 (d, J=8.8 Hz, 2 H), 3.96
(s, 3 H), 2.97 (s, 6 H).
250
N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethanamine.
NEU-4973 (3.24b) N-ethyl-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridin-2-yl)benzyl)ethanamine (45 mg, 0.088 mmol) was dissolved in dioxane (0.4 mL).
Sodium hydroxide was added (3M, 0.8 mL). The reaction mixture was microwaved at 150 °C for
20 minutes. The reaction mixture was quenched with saturated ammonium chloride solution and
diluted with water. The aqueous mixture was washed 3 times with ethyl acetate. The combined
organic layers were dried with sodium sulfate, filtered, and concentrated to afford the title
compound as a tan solid (15 mg, 47% yield). LCMS [M+H]+ 360.2 m/z; 1H NMR (500 MHz,
METHANOL-d4) ppm 8.35 (s, 1 H), 8.12 (s, 2 H), 7.89 (d, J=8.3 Hz, 2 H), 7.46 (d, J=7.8 Hz, 2
H), 7.26 (d, J=4.9 Hz, 1 H), 7.15 (s, 1 H), 4.01 (s, 4 H), 3.70 (s, 2 H), 2.63 (q, J=6.8 Hz, 3 H), 1.12
(t, J=7.1 Hz, 6 H).
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.
NEU-6072 (3.24c) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(1-(pyrrolidin-1-yl)ethyl)phenyl)-1-tosyl-
251
1H-pyrrolo[2,3-b]pyridine (67 mg, 0.127 mmol) was dissolved in dioxane (1 mL). Sodium
hydroxide was added (3M, 0.425 mL). The reaction mixture was microwaved at 150 °C for 15
minutes. The reaction mixture was quenched with saturated ammonium chloride solution and
diluted with water. The aqueous mixture was washed 3 times with ethyl acetate. The combined
organic layers were dried with sodium sulfate, filtered, and concentrated. The crude product was
purified by column chromatography eluting with a mobile phase of 1-20%
methanol/dichloromethane to afford the title compound as an off-white solid (25 mg, 53% yield).
LCMS [M+H]+ 372.2 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 12.88 (br. s., 1 H),
8.21 (d, J=4.4 Hz, 1 H), 8.00 (s, 1 H), 7.89 - 7.96 (m, 3 H), 7.74 (d, J=8.3 Hz, 2 H), 7.16 (d, J=5.4
Hz, 1 H), 6.94 (s, 1 H), 3.89 - 4.08 (m, 4 H), 3.24 (br. s., 1 H), 2.95 - 3.07 (m, 2 H), 2.04 (br. s., 4
H), 1.80 (d, J=6.3 Hz, 3 H).
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-
b]pyridine. NEU-6076 (3.24d) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-((4-methylpiperazin-1-
yl)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (180 mg, 0.333 mmol) was dissolved in
dioxane (0.7 mL). Sodium hydroxide was added (3M, 1.11 mL). The reaction mixture was
microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with saturated
ammonium chloride solution and diluted with water. A precipitate formed and was collected by
vacuum filtration. The crude product was purified by column chromatography eluting with a
252
mobile phase of 1-10% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the
title compound as an off-white solid (27 mg, 21% yield). LCMS [M+H]+ 387.1 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 12.13 (s, 1 H), 8.57 (s, 1 H), 8.19 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.98
(d, J=8.3 Hz, 2 H), 7.38 (d, J=8.3 Hz, 2 H), 7.29 (d, J=1.5 Hz, 1 H), 7.26 (d, J=4.9 Hz, 1 H), 3.96
(s, 3 H), 3.49 (s, 2 H), 2.36 (br. s., 7 H), 2.15 (s, 3 H).
3.24e - KB2-617-3-1 - tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)pyrrolidine-1-carboxylate. tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-
pyrrolo[2,3-b]pyridin-2-yl)phenyl)pyrrolidine-1-carboxylate (109 mg, 0.182 mmol) was dissolved
in dioxane (1.5 mL). Sodium hydroxide was added (3M, 1.5 mL). The reaction mixture was
microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with saturated
ammonium chloride solution and diluted with water. The aqueous mixture was washed 3 times
with dichloromethane. The combined organic layers were dried with sodium sulfate, filtered, and
concentrated. The crude product was purified by column chromatography eluting with a mobile
phase of 0-100% ethyl acetate/hexanes to afford the title compound as a pale yellow solid (44 mg,
55% yield). LCMS [M+H]+ 444.3 m/z; 1H NMR (500 MHz, CHLOROFORM-d) ppm 12.62 (br.
s., 1 H), 8.27 (d, J=4.9 Hz, 1 H), 8.07 (s, 1 H), 7.96 (br. s., 1 H), 7.87 (d, J=6.8 Hz, 2 H), 7.34 (d,
J=8.3 Hz, 2 H), 7.19 (d, J=4.9 Hz, 1 H), 6.90 - 7.02 (m, 1 H), 4.06 (s, 3 H), 3.70 (br. s., 2 H), 2.41
(br. s., 1 H), 1.76 (br. s., 2 H), 1.51 (br. s., 2 H), 1.18 - 1.33 (m, 9 H).
253
4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. NEU-4974
(3.24f) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde
(320 mg, 0.701 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide was added (3M, 2.34
mL). The reaction mixture was microwaved at 150 °C for 30 minutes. The reaction mixture was
quenched with saturated ammonium chloride solution and diluted with water. A precipitate formed
and was collected by vacuum filtration to afford the title compound as an orange solid (167 mg,
79% yield). LCMS [M+H]+ 303.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 10.02 (s, 1 H), 8.60
(s, 1 H), 8.28 (d, J=8.3 Hz, 2 H), 8.22 (s, 1 H), 8.20 (d, J=4.9 Hz, 1 H), 7.99 (d, J=8.3 Hz, 2 H),
7.55 (s, 1 H), 7.28 (d, J=4.9 Hz, 1 H), 3.97 (s, 3 H).
4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid. NEU-5448 (3.24g)
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzoic acid (17 mg,
0.036 mmol) was dissolved in dioxane (0.3 mL). Sodium hydroxide was added (3M, 0.12 mL).
The reaction mixture was microwaved at 150 °C for 20 minutes. The reaction mixture was
254
quenched with saturated ammonium chloride solution and diluted with water. The aqueous mixture
was washed 3 times with ethyl acetate. The combined organic layers were dried with sodium
sulfate, filtered, and concentrated. The crude product was purified by column chromatography
eluting with a mobile phase of 1-20% methanol/dichloromethane to afford the title compound as
a yellow solid (5 mg, 44% yield). LCMS [M+H]+ 319.1 m/z; 1H NMR (500 MHz, METHANOL-
d4) ppm 8.40 (s, 1 H), 8.18 (d, J=4.9 Hz, 1 H), 8.15 (s, 1 H), 8.09 - 8.13 (m, 1 H), 8.02 (d, J=8.3
Hz, 2 H), 7.97 (d, J=8.8 Hz, 1 H), 7.29 - 7.33 (m, 2 H), 4.03 (s, 3 H).
4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide. NEU-5306 (3.24h)
4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzamide (114 mg,
0.242 mmol) was dissolved in dioxane (2 mL). Sodium hydroxide was added (3M, 1 mL). The
reaction mixture was microwaved at 150 °C for 45 minutes. The reaction mixture was quenched
with saturated ammonium chloride solution and diluted with water. A precipitate formed and was
collected by vacuum filtration. The crude product was purified by column chromatography eluting
with a mobile phase of 1-8% methanol/dichloromethane to afford the title compound as a yellow
solid (72 mg, 94% yield). LCMS [M+H]+ 318.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.24
(s, 1 H), 8.60 (s, 1 H), 8.22 (s, 1 H), 8.18 (d, J=4.9 Hz, 1 H), 8.13 (d, J=8.3 Hz, 2 H), 8.03 (s, 1 H),
7.98 (d, J=8.8 Hz, 2 H), 7.46 (d, J=2.4 Hz, 1 H), 7.41 (s, 1 H), 7.29 (d, J=4.9 Hz, 1 H), 3.97 (s, 3
H).
255
N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)acetamide. NEU-
6073 (3.24i) N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)acetamide (65 mg, 0.134 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide
was added (3M, 0.446 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The
reaction mixture was quenched with saturated ammonium chloride solution and diluted with water.
The aqueous mixture was washed 3 times with dichloromethane. The combined organic layers
were dried with sodium sulfate, filtered, and concentrated. The crude product was purified by
column chromatography eluting with a mobile phase of 1-20% methanol/dichloromethane to
afford the title compound as a yellow solid (21 mg, 47% yield). LCMS [M+H]+ 332.1 m/z; 1H
NMR (500 MHz, DMSO-d6) ppm 12.05 (s, 1 H), 10.08 (s, 1 H), 8.52 - 8.59 (m, 1 H), 8.16 - 8.19
(m, 1 H), 8.12 (d, J=4.9 Hz, 1 H), 7.97 (s, 2 H), 7.68 (d, J=8.8 Hz, 2 H), 7.25 (d, J=5.4 Hz, 1 H),
7.23 (d, J=1.5 Hz, 1 H), 3.96 (s, 3 H), 2.07 (s, 3 H).
256
4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. NEU-4918 (3.24j) 4-(4-
(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (50 mg, 0.079 mmol)
was dissolved in dioxane (1 mL). Sodium hydroxide was added (3M, 0.276 mL). The reaction
mixture was microwaved at 150 °C for 45 minutes. The reaction mixture was quenched with
saturated ammonium chloride solution and concentrated. The crude product was purified twice by
column chromatography eluting with a mobile phase of 0-20% methanol/dichloromethane to
afford the title compound as a white solid (5 mg, 22% yield). LCMS [M+H]+ 291.2 m/z; 1H NMR
(500 MHz, METHANOL-d4) ppm 8.34 (s, 1 H), 8.11 (d, J=1.0 Hz, 1 H), 8.08 (d, J=5.4 Hz, 1
H), 7.76 (d, J=8.8 Hz, 2 H), 7.25 (d, J=4.9 Hz, 1 H), 6.98 (s, 1 H), 6.90 (d, J=8.8 Hz, 2 H), 4.02
(s, 3 H).
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(pyrrolidin-2-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine. NEU-
6049 (3.24k) tert-butyl 2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)pyrrolidine-1-carboxylate (37 mg, 0.083 mmol) was dissolve in dioxane (1 mL).
257
Hydrochloric acid (1M, 0.834 mL) was added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was neutralized with silicized carbonate. The silica
was filtered off, washed with methanol, and the filtrate was concentrated to afford the title
compound as a yellow solid (18 mg, 62% yield). LCMS [M+H]+ 344.2 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 12.09 (br. s., 1 H), 8.57 (s, 1 H), 8.18 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.96 (d,
J=8.3 Hz, 2 H), 7.53 - 7.65 (m, 1 H), 7.45 (d, J=8.3 Hz, 2 H), 7.24 - 7.28 (m, 2 H), 4.06 (t, J=7.6
Hz, 1 H), 3.96 (s, 3 H), 3.04 (ddd, J=9.8, 7.8, 5.4 Hz, 1 H), 2.89 (td, J=9.0, 6.8 Hz, 1 H), 2.14 (dtd,
J=12.1, 7.6, 7.6, 4.9 Hz, 1 H), 1.69 - 1.84 (m, 2 H), 1.50 (m, J=12.0, 9.0 Hz, 1 H).
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridine. (3.25a) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (50 mg, 0.109 mmol) was dissolved in dichloroethane (0.5 mL). Acetic acid (12.5
μL, 0.219 mmol), piperazine (20.9 μL, 0.219 mmol), and sodium borohydride (8.3 mg, 0.219
mmol) were added. The reaction mixture was left stirring at room temperature overnight. The
reaction mixture was quenched with 3M sodium hydroxide. The aqueous mixture was extracted 3
times with dichloromethane. The combined organic layers were concentrated, and the crude
product was purified by column chromatography using a mobile phase of 50-100% ethyl
acetate/hexanes, followed by 0-20% methanol/ethyl acetate to afford the title compound as an off-
white solid (37 mg, 64% yield). LCMS [M+H]+ 457.1 m/z.
258
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.
NEU-5936 (3.26a) 4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperazin-1-ylmethyl)phenyl)-1-tosyl-
1H-pyrrolo[2,3-b]pyridine was dissolved in dioxane (1 mL). Sodium hydroxide (3M, 1 mL) was
added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture
was quenched with saturated ammonium chloride solution, and then diluted with water. The
aqueous layer was extracted 3x with dichloromethane. The combined organic layers were washed
with water and brine, then dried with sodium sulfate, filtered, and concentrated under reduced
pressure. The reaction mixture was purified by column chromatography using a mobile phase of
0-20% (1% ammonium hydroxide/methanol)/ethyl acetate to afford the title compound as an
ammonium salt. The free base was yielded by dissolving the salt in water and basifying with
sodium hydroxide. The precipitate that formed was collected by vacuum filtration and washed with
water to afford the title compound as a pale yellow solid (10 mg, 37% yield). LCMS [M+H]+ 372.3
m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s, 1 H), 8.11 - 8.15 (m, 2 H), 7.90 (d,
J=8.3 Hz, 2 H), 7.45 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.17 (s, 1 H), 4.03 (s, 3 H), 3.57
(s, 2 H), 2.49 (br. s., 4 H), 1.59 - 1.67 (m, 4 H), 1.45 - 1.53 (m, 2 H).
259
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(piperidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.
NEU-4995 (3.26b) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (60 mg, 0.198 mmol) was suspended in dichloroethane (2 mL). Piperidine (34
μL, 0.40 mmol) and sodium triacetoxyborohydride (63.1 mg, 0.298 mmol) were added to the
reaction mixture which was left stirring overnight at room temperature. The reaction mixture was
quenched with saturated ammonium chloride solution, and then diluted with water. The aqueous
layer was extracted 3x with dichloromethane. The combined organic layers were washed with
water and brine, then dried with sodium sulfate, filtered, and concentrated under reduced pressure.
The reaction mixture was purified by column chromatography using a mobile phase of 5-20%
methanol/dichloromethane to afford the title compound as a yellow solid (44 mg, 60% yield).
LCMS [M+H]+ 372.2 m/z; 1H NMR (399 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.57 (s, 1 H),
8.19 (s, 1 H), 8.14 (d, J=5.1 Hz, 1 H), 7.98 (d, J=8.1 Hz, 2 H), 7.38 (d, J=8.1 Hz, 2 H), 7.24 - 7.30
(m, 2 H), 3.96 (s, 3 H), 3.47 (br. s., 2 H), 2.35 (br. s., 4 H), 1.46 - 1.57 (m, 4 H), 1.40 (d, J=4.4 Hz,
2 H).
260
4-(1-methyl-1H-pyrazol-4-yl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.
NEU-5123 (3.26c) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (40 mg, 0.123 mmol) was suspended in methanol (1.3 mL). Pyrrolidine (109 μL,
1.32 mmol) and sodium borohydride (6.0mg, 0.189 mmol) were added to the reaction mixture
which was heated at 70 °C overnight. The reaction mixture was concentrated under reduced
pressure and the crude product was purified by column chromatography using a mobile phase of
1-7% methanol/dichloromethane to afford the title compound as a yellow solid (13 mg, 28% yield).
LCMS [M+H]+ 358.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s, 1 H), 8.12 - 8.17
(m, 2 H), 7.96 (d, J=8.3 Hz, 2 H), 7.53 (d, J=8.3 Hz, 2 H), 7.29 (d, J=5.4 Hz, 1 H), 7.20 (s, 1 H),
4.03 (s, 3 H), 3.95 (s, 2 H), 2.87 (br. s., 4 H), 1.94 (br. s., 4 H).
N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethanamine. NEU-
5124 (3.26d) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (30
mg, 0.099 mmol) was suspended in methanol (1 mL). Ethylamine (64.9 μL, 0.992 mmol) and
261
sodium borohydride (4.5 mg, 0.119 mmol) were added to the reaction mixture which was left
stirring at room temperature overnight. The reaction mixture was concentrated under reduced
pressure and the crude product was purified by column chromatography using a mobile phase of
1-20% methanol/dichloromethane to afford the title compound as a yellow solid (12 mg, 37%
yield). LCMS [M+H]+ 332.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.35 (s, 1 H), 8.14
(d, J=4.9 Hz, 1 H), 8.12 (s, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.56 (d, J=8.3 Hz, 2 H), 7.27 (d, J=5.4
Hz, 1 H), 7.20 (s, 1 H), 4.10 (s, 2 H), 4.01 (s, 3 H), 3.35 (s, 1 H), 3.00 (q, J=7.0 Hz, 2 H), 1.30 (t,
J=7.3 Hz, 3 H).
4-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)morpholine. NEU-
5937 (3.26e) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (25
mg, 0.083 mmol) was suspended in dichloroethane (0.5 mL). Morpholine (21.4 μL, 0.248 mmol),
acetic acid (14.2 μL, 0.248 mmol) and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol) were
added to the reaction mixture which was left stirring overnight at room temperature. The reaction
mixture was quenched with 3M sodium hydroxide, and then diluted with water. The aqueous layer
was extracted 3 times with dichloromethane. The combined organic layers were concentrated and
the crude product was purified by column chromatography using a mobile phase of 0-20% ((+10%
ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a pale yellow solid
(14 mg, 47% yield). LCMS [M+H]+ 374.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37
262
(s, 1 H), 8.09 - 8.16 (m, 2 H), 7.91 (d, J=8.3 Hz, 2 H), 7.47 (d, J=8.3 Hz, 2 H), 7.28 (d, J=5.4 Hz,
1 H), 7.16 (s, 1 H), 4.03 (s, 3 H), 3.72 (t, J=4.6 Hz, 4 H), 3.59 (s, 2 H), 3.35 (s, 1 H), 2.51 (br. s.,
4 H).
N-methyl-1-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)phenyl)methanamine. NEU-5938 (3.26f) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-
b]pyridin-2-yl)benzaldehyde (25 mg, 0.083 mmol) was suspended in dichloroethane (0.5 mL).
Methylamine hydrochloride (16.7 mg, 0.248 mmol), triethylamine (34.6 μL, 0.248), acetic acid
(14.2 μL, 0.248 mmol) and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol) were added to
the reaction mixture which was left stirring overnight at room temperature. The reaction mixture
was quenched with 3M sodium hydroxide, and then diluted with water. The aqueous layer was
extracted 3 times with dichloromethane. The combined organic layers were concentrated, and the
crude product was purified by column chromatography using a mobile phase of 0-20% ((+10%
ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a yellow solid (10
mg, 38% yield). LCMS [M+H]+ 318.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.37 (s,
1 H), 8.12 - 8.16 (m, 2 H), 7.94 (d, J=7.8 Hz, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.28 (d, J=5.4 Hz, 1
H), 7.18 (s, 1 H), 4.03 (s, 3 H), 3.85 (s, 2 H), 2.48 (s, 3 H).
263
N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.
NEU-6018 (3.26g) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (74 mg, 0.245 mmol) was dissolved in 10% acetic acid/methanol (2.5 mL).
Isopropylamine (0.1 mL, 1.22 mmol) was added and the reaction mixture was left stirring at room
temperature for 1 hour. Sodium cyanoborohydride (52.6 mg, 0.248 mmol) was added to the
reaction mixture which was heated at 70 °C for 72 h. The reaction mixture concentrated, and the
crude product was purified by column chromatography using a mobile phase of 30-100% ethyl
acetate/hexanes followed by 0-20% methanol/ethyl acetate, and again with 1%
methanol/dichloromethane to afford the title compound as a yellow solid (15 mg, 18% yield).
LCMS [M+H]+ 346.3 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.32 (s, 1 H), 8.07 - 8.13
(m, 2 H), 7.89 (d, J=8.3 Hz, 2 H), 7.46 (d, J=8.3 Hz, 2 H), 7.24 (d, J=5.4 Hz, 1 H), 7.12 (s, 1 H),
4.00 (s, 3 H), 3.82 (s, 2 H), 2.92 (spt, J=6.3 Hz, 1 H), 1.15 (d, J=6.3 Hz, 6 H).
264
N1-methyl-N2-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethane-
1,2-diamine. NEU-6025 (3.26h) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL).
N1-methylethane-1,2-diamine (43.3 μL, 0.496 mmol) was added and the reaction mixture was left
stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165 mmol)
were added to the reaction mixture which was left stirring overnight at room temperature. The
reaction mixture was diluted with water, concentrated, and the crude product was purified by
column chromatography using a mobile phase of 1-20% ((+1% ammonium
hydroxide)/methanol)/dichloromethane to afford the title compound as a yellow solid (6 mg, 10%
yield). LCMS [M+H]+ 361.3 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.11 (br. s., 1 H), 8.57
(s, 1 H), 8.18 (s, 1 H), 8.14 (d, J=4.9 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.42 (d, J=8.3 Hz, 2 H),
7.27 (m, J=8.3 Hz, 3 H), 3.96 (s, 3 H), 3.73 (s, 2 H), 2.56 (dd, J=8.5, 4.1 Hz, 4 H), 2.26 (s, 2 H).
265
N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)-2-(pyrrolidin-1-
yl)ethan-1-amine. NEU-6044 (3.26i) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-
2-yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6
mL). 2-(pyrrolidin-1-yl)ethan-1-amine (62.7 μL, 0.496 mmol) was added and the reaction mixture
was left stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165
mmol) were added to the reaction mixture which was left stirring overnight at room temperature.
The reaction mixture was concentrated, and the crude product was purified by column
chromatography using a mobile phase of 1-20% methanol/dichloromethane followed by 1-20%
methanol/ethyl acetate to afford the title compound as a pale yellow solid (9 mg, 14% yield).
LCMS [M+H]+ 401.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.06 - 12.15 (m, 1 H), 8.57 (s,
1 H), 8.18 (s, 1 H), 8.14 (d, J=5.4 Hz, 1 H), 7.98 (d, J=8.3 Hz, 2 H), 7.42 (d, J=7.8 Hz, 2 H), 7.29
(d, J=1.5 Hz, 1 H), 7.26 (d, J=4.9 Hz, 1 H), 3.96 (s, 3 H), 3.73 - 3.77 (m, 2 H), 2.59 - 2.64 (m, 2
H), 2.53 (t, J=6.6 Hz, 2 H), 2.39 - 2.45 (m, 4 H), 1.64 - 1.69 (m, 4 H).
266
(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)methanol. NEU-6047
(3.26j) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (50 mg,
0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL). Azetidine (33.4 μL, 0.496
mmol) was added and the reaction mixture was left stirring for 20 minutes at room temperature.
Sodium cyanoborohydride (10.4 mg, 0.165 mmol) were added to the reaction mixture which was
left stirring overnight at room temperature. The reaction mixture was concentrated, and the crude
product was purified by column chromatography using a mobile phase of 1-20% methanol/ ethyl
acetate to afford the title compound as a pale yellow solid (13 mg, 18% yield). LCMS [M+H]+
305.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.58 (s, 1 H), 8.19 (s, 1 H), 8.14
(d, J=4.9 Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H), 7.41 (d, J=8.3 Hz, 2 H), 7.30 (d, J=1.5 Hz, 1 H), 7.26
(d, J=4.9 Hz, 1 H), 5.14 - 5.36 (m, 1 H), 4.55 (s, 2 H), 3.96 (s, 3 H), 1.65 - 1.84 (m, 1 H).
2,2-difluoro-N-(4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)ethan-1-
amine. NEU-6048 (3.26k) 4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
267
yl)benzaldehyde (50 mg, 0.165 mmol) was suspended in 1:1 acetic acid/dichloroethane (0.6 mL).
2,2-difluoroethan-1-amine (50.0 μL, 0.496 mmol) was added and the reaction mixture was left
stirring for 20 minutes at room temperature. Sodium cyanoborohydride (10.4 mg, 0.165 mmol)
were added to the reaction mixture which was left stirring overnight at room temperature. The
reaction mixture was quenched with saturated sodium bicarbonate. The aqueous solution was
extracted 3 times with dichloromethane. The combined organic layers were dried with sodium
sulfate, filtered, and concentrated. The crude product was purified by column chromatography
using a mobile phase of 1-20% methanol/ethyl acetate followed by 1-5% methanol
dichloromethane to afford the title compound as a yellow solid (10 mg, 17% yield). LCMS [M+H]+
368.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.12 (s, 1 H), 8.57 (s, 1 H), 8.19 (s, 1 H), 8.14
(d, J=4.9 Hz, 1 H), 8.00 (d, J=7.8 Hz, 2 H), 7.43 (d, J=7.8 Hz, 2 H), 7.30 (d, J=1.5 Hz, 1 H), 7.26
(d, J=5.4 Hz, 1 H), 6.03 (m, J=4.1, 4.1 Hz, 1 H), 3.96 (s, 3 H), 3.79 (s, 2 H), 2.87 (td, J=15.9, 4.4
Hz, 2 H).
N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. (3.27a) 4-(4-
chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (2.08 g, 5.06 mmol) was dissolved in
a solution of 10% acetic acid/methanol (50 mL). Isopropylamine (4.14 mL, 50.6 mmol) was added
and the reaction mixture was heated at 50 °C until the solution was clear. Sodium
cyanoborohydride (477 mg, 7.59 mmol) was then added and the reaction mixture was left heating
at 50 °C overnight. The reaction mixture was neutralized with saturated sodium bicarbonate
268
solution. The aqueous mixture was concentrated to remove methanol, then extracted 3 times with
dichloromethane. The combined organic layers were washed with water and then brine, collected,
dried with sodium sulfate, filtered and concentrated to afford the title compound as a tan solid
(2.17 g, 94% yield). LCMS [M+H]+ 454.1 m/z (Cl35), 456.1 m/z (Cl37); 1H NMR (500 MHz,
DMSO-d6) ppm 8.34 (d, J=4.9 Hz, 1 H), 7.70 (d, J=8.3 Hz, 2 H), 7.50 - 7.55 (m, 2 H), 7.43 -
7.49 (m, 3 H), 7.35 (d, J=8.3 Hz, 2 H), 6.80 (s, 1 H), 3.78 (s, 2 H), 2.75 (quin, J=6.2 Hz, 1 H), 2.31
(s, 3 H), 1.92 - 2.19 (m, 1 H), 1.03 (d, J=6.3 Hz, 6 H).
tert-butyl 4-(4-(2-(4-formylphenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-
yl)piperidine-1-carboxylate. (3.27b) 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (150 mg, 0.365 mmol), tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (179 mg, 0.475 mmol),
Tetrakis(triphenylphosphine)palladium(0) (21.1 mg, 0.018 mmol), and sodium carbonate (116 mg,
1.10 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 3.5 mL) was added to the vial. The reaction mixture
was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure and then purified by silica gel column
269
chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title
compound as a tan solid (192 mg, 84% yield). LCMS [M+H]+ 626.2 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 10.12 (s, 1 H), 8.59 (s, 1 H), 8.32 (d, J=5.4 Hz, 1 H), 8.18 (s, 1 H), 8.05 (d,
J=8.3 Hz, 2 H), 7.90 (d, J=7.8 Hz, 2 H), 7.70 (d, J=8.3 Hz, 2 H), 7.52 (d, J=4.9 Hz, 1 H), 7.40 (s,
1 H), 7.33 (d, J=8.3 Hz, 2 H), 4.34 - 4.45 (m, 1 H), 3.98 - 4.13 (m, 2 H), 2.77 - 3.01 (m, 2 H), 2.30
(s, 3 H), 2.00 (br. s., 2 H), 1.79 - 1.92 (m, 2 H), 1.41 (s, 9 H).
4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde. (3.27c) 4-
(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (67 mg, 0.163 mmol), (4-
(methylsulfonyl)phenyl)boronic acid (42.4 mg, 0.212 mmol),
Tetrakis(triphenylphosphine)palladium(0) (9.4 mg, 0.008 mmol), and sodium carbonate (51.8 mg,
0.489 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 1.6 mL) was added to the vial. The reaction mixture
was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 0-100% ethyl acetate/hexanes to afford the title
compound as an off-white solid (36 mg, 42% yield). LCMS [M+H]+ 531.1 m/z; 1H NMR (500
MHz, CHLOROFORM-d) ppm 10.12 (s, 1 H), 8.61 (d, J=4.9 Hz, 1 H), 8.08 (d, J=8.3 Hz, 2 H),
270
7.99 (d, J=7.8 Hz, 2 H), 7.88 (d, J=7.8 Hz, 2 H), 7.79 (d, J=8.8 Hz, 2 H), 7.74 (d, J=8.3 Hz, 2 H),
7.31 (d, J=4.9 Hz, 1 H), 7.25 (d, J=7.8 Hz, 2 H), 6.72 (s, 1 H), 3.11 (s, 3 H), 2.38 (s, 3 H).
N-(4-(4-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. (3.28a) N-
(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (65 mg, 0.143 mmol),
pyridin-3-ylboronic acid (38.2 mg, 0.186 mmol), Tetrakis(triphenylphosphine)palladium(0) (8.3
mg, 0.007 mmol), and sodium carbonate (45.5 mg, 0.429 mmol) were added to a vial that was
subsequently sealed and vacuum purged with nitrogen. A degassed solution of dioxane/water (3:1,
1.3 mL) was added to the vial. The reaction mixture was heated at 85 °C for 48 h and monitored
by LCMS. The reaction mixture was filtered through a bed of Celite and concentrated under
reduced pressure and then purified by silica gel column chromatography using a mobile phase of
1-20% ((+10% ammonium hydroxide)/methanol)/dichloromethane to afford the title compound as
a tan solid (50 mg, 70% yield). LCMS [M+H]+ 497.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm
8.90 (d, J=1.5 Hz, 1 H), 8.68 (dd, J=4.9, 1.5 Hz, 1 H), 8.47 (d, J=4.9 Hz, 1 H), 8.15 (dt, J=8.2, 1.8
Hz, 1 H), 7.76 (d, J=8.3 Hz, 2 H), 7.50 - 7.61 (m, 4 H), 7.48 (d, J=7.8 Hz, 2 H), 7.37 (d, J=8.3 Hz,
2 H), 6.91 (s, 1 H), 3.85 (br. s., 2 H), 2.84 (br. s., 1 H), 2.33 (s, 3 H), 1.08 (d, J=5.9 Hz, 6 H).
271
N-(2-hydroxyethyl)-4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-
4-yl)benzenesulfonamide. (3.28b) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (60 mg, 0.132 mmol), N-(2-hydroxyethyl)-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzenesulfonamide (56.2 mg, 0.172 mmol),
Tetrakis(triphenylphosphine)palladium(0) (7.6 mg, 0.007 mmol), and sodium carbonate (42.0 mg,
0.396 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 1.3 mL) was added to the vial. The reaction mixture
was heated at 85 °C for 48 h and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 1-20% ((+10% ammonium
hydroxide)/methanol)/dichloromethane to afford the title compound as a tan solid (67 mg, 81%
yield). LCMS [M+H]+ 619.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.48 (d, J=5.4 Hz, 1 H),
7.92 (d, J=1.5 Hz, 4 H), 7.75 (d, J=8.8 Hz, 3 H), 7.56 (d, J=8.3 Hz, 2 H), 7.51 (d, J=5.4 Hz, 1 H),
7.47 (s, 2 H), 7.37 (d, J=8.3 Hz, 2 H), 6.93 (s, 1 H), 4.70 (s, 1 H), 3.78 - 3.85 (m, 2 H), 3.38 (d,
J=5.9 Hz, 3 H), 2.76 - 2.86 (m, 3 H), 2.33 (s, 3 H), 1.06 (d, J=6.3 Hz, 6 H).
272
N-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-
amine. (3.28c) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (72
mg, 0.159 mmol), (4-(methylsulfonyl)phenyl)boronic acid (57.1 mg, 0.285 mmol),
Tetrakis(triphenylphosphine)palladium(0) (18.3 mg, 0.016 mmol), and sodium carbonate (50.4
mg, 0.476 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 1.6 mL) was added to the vial. The reaction
mixture was heated at 85 °C for 1 hour and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and concentrated under reduced pressure and then purified by silica gel
column chromatography using a mobile phase of 50-100% ethyl acetate/hexanes, followed by 0-
20% ((+5% ammonium hydroxide)/methanol)/ethyl acetate to afford the title compound as a light
tan solid (81 mg, 90% yield). LCMS [M+H]+ 574.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm
8.49 (d, J=4.9 Hz, 1 H), 8.03 - 8.07 (m, 2 H), 7.95 - 7.99 (m, 2 H), 7.76 (d, J=8.3 Hz, 2 H), 7.56
(d, J=7.8 Hz, 2 H), 7.52 (d, J=5.4 Hz, 1 H), 7.47 (d, J=8.3 Hz, 2 H), 7.37 (d, J=8.3 Hz, 2 H), 6.91
(s, 1 H), 4.05 - 4.21 (m, 1 H), 3.83 (s, 2 H), 3.27 (s, 3 H), 2.76 - 2.85 (m, 1 H), 2.33 (s, 3 H), 1.06
(d, J=6.3 Hz, 6 H).
273
tert-butyl 4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-3,6-
dihydropyridine-1(2H)-carboxylate. (3.28d) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (74 mg, 0.163 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (90.7 mg, 0.293 mmol),
Tetrakis(triphenylphosphine)palladium(0) (9.4 mg, 0.008 mmol), and sodium carbonate (51.8 mg,
0.489 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 1.6 mL) was added to the vial. The reaction mixture
was heated at 85 °C for 1 hour and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 1-5% ((+10% ammonium hydroxide)/methanol)/ethyl
acetate to afford the title compound as a brown residue (97 mg, 99% yield). LCMS [M+H]+ 601.2
m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.28 - 8.32 (m, 1 H), 7.67 - 7.73 (m, 2 H), 7.49 - 7.55
(m, 2 H), 7.41 - 7.48 (m, 2 H), 7.30 - 7.37 (m, 2 H), 7.19 - 7.25 (m, 1 H), 6.93 - 6.98 (m, 1 H), 4.08
- 4.15 (m, 1 H), 3.99 - 4.06 (m, 2 H), 3.75 - 3.81 (m, 2 H), 3.48 - 3.56 (m, 2 H), 3.12 - 3.20 (m, 2
H), 2.70 - 2.79 (m, 1 H), 2.28 - 2.34 (m, 3 H), 1.96 - 2.01 (m, 1 H), 1.41 (s, 9 H), 1.03 (d, J=5.9
Hz, 6 H).
274
N-(4-(4-(2-methylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.
(3.28e) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (70 mg,
0.154 mmol), (2-methylpyridin-4-yl)boronic acid (38.0 mg, 0.277 mmol),
Tetrakis(triphenylphosphine)palladium(0) (8.9 mg, 0.008 mmol), and sodium carbonate (49.0 mg,
0.462 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane/water (3:1, 1.5 mL) was added to the vial. The reaction mixture
was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through
a bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 50-100% ethyl acetate/hexanes, followed by 0-20%
methanol/ethyl acetate to afford the title compound as an off-white solid (53 mg, 67% yield).
LCMS [M+H]+ 511.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.57 (d, J=5.4 Hz, 1 H), 8.48
(d, J=4.9 Hz, 1 H), 7.75 (d, J=8.3 Hz, 2 H), 7.55 - 7.60 (m, 3 H), 7.46 - 7.53 (m, 4 H), 7.37 (d,
J=8.3 Hz, 2 H), 6.95 (s, 1 H), 3.87 (br. s., 2 H), 2.85 (br. s., 1 H), 2.54 (s, 3 H), 2.32 (s, 3 H), 1.91
(s, 1 H), 1.09 (d, J=6.3 Hz, 6 H).
275
N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-
amine. (3.28f) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (70
mg, 0.154 mmol), 2,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (64.7
mg, 0.277 mmol), Tetrakis(triphenylphosphine)palladium(0) (8.9 mg, 0.008 mmol), and sodium
carbonate (49.0 mg, 0.462 mmol) were added to a vial that was subsequently sealed and vacuum
purged with nitrogen. A degassed solution of dioxane/water (3:1, 1.5 mL) was added to the vial.
The reaction mixture was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture
was filtered through a bed of Celite and concentrated under reduced pressure and then purified by
silica gel column chromatography using a mobile phase of 0-20% methanol/ethyl acetate to afford
the title compound as an amber solid (71 mg, 88% yield). LCMS [M+H]+ 525.1 m/z; 1H NMR
(500 MHz, DMSO-d6) ppm 8.47 (d, J=4.9 Hz, 1 H), 7.74 (d, J=8.3 Hz, 2 H), 7.59 (d, J=8.3 Hz,
2 H), 7.51 (d, J=7.8 Hz, 2 H), 7.47 (d, J=5.4 Hz, 1 H), 7.36 (d, J=8.3 Hz, 2 H), 7.33 (s, 2 H), 6.94
(s, 1 H), 4.10 (br. s., 1 H), 3.90 (br. s., 2 H), 2.83 - 2.95 (m, 1 H), 2.49 (s, 6 H), 2.32 (s, 3 H), 1.11
(d, J=5.9 Hz, 6 H).
276
N-(4-(4-(1H-pyrazol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.
(3.28g) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (100 mg,
0.220 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (51.3 mg, 0.264
mmol), ris(dibenzylideneacetone)dipalladium(0) (10.1 mg, 0.011 mmol), tricyclohexylphosphine
(9.3 mg, 0.033 mmol) and potassium carbonate (91.3 mg, 0.660 mmol) were added to a vial that
was subsequently sealed and vacuum purged with nitrogen. A degassed solution of
dimethylformamide/water (3:1, 2.2 mL) was added to the vial. The reaction mixture was heated at
85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered through a bed of Celite
and concentrated under reduced pressure and then purified by silica gel column chromatography
using a mobile phase of 1-20% methanol/dichloromethane to afford the title compound as a yellow
solid (101 mg, 94% yield). LCMS [M+H]+ 486.1 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.37
(d, J=5.4 Hz, 1 H), 7.88 (br. s., 1 H), 7.65 - 7.70 (m, 3 H), 7.56 (d, J=8.3 Hz, 2 H), 7.46 (d, J=7.8
Hz, 2 H), 7.33 (d, J=8.3 Hz, 2 H), 7.29 (br. s., 1 H), 7.01 (s, 1 H), 3.79 (s, 2 H), 2.70 - 2.83 (m, 1
H), 2.29 (s, 3 H), 1.05 (d, J=6.3 Hz, 6 H).
277
N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine. (3.28h) N-(4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (100 mg, 0.220 mmol), 1-(tetrahydro-2H-pyran-4-yl)-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (137 mg, 0.492 mmol),
Tetrakis(triphenylphosphine)palladium(0) (12.7 mg, 0.011 mmol), and sodium carbonate (70.0
mg, 0.660 mmol) were added to a vial that was subsequently sealed and vacuum purged with
nitrogen. A degassed solution of dioxane/water (3:1, 2.2 mL) was added to the vial. The reaction
mixture was heated at 85 °C for 18 h and monitored by LCMS. The reaction mixture was filtered
through a bed of Celite and concentrated under reduced pressure and then purified by silica gel
column chromatography using a mobile phase of 1-20% methanol/ethyl acetate to afford the title
compound as an amber solid (90 mg, 72% yield). LCMS [M+H]+ 570.3 m/z; 1H NMR (500 MHz,
DMSO-d6) ppm 8.58 (s, 1 H), 8.29 (d, J=4.9 Hz, 1 H), 8.15 (s, 1 H), 7.68 (d, J=8.3 Hz, 2 H),
7.58 (d, J=7.8 Hz, 2 H), 7.44 - 7.52 (m, 3 H), 7.33 (d, J=8.3 Hz, 2 H), 7.19 (s, 1 H), 4.39 - 4.52
(m, 1 H), 4.10 (d, J=4.4 Hz, 4 H), 3.83 (s, 2 H), 3.38 - 3.52 (m, 4 H), 2.79 (d, J=5.9 Hz, 1 H), 2.30
(s, 3 H), 1.07 (s, 6 H).
278
stert-butyl 4-(4-(2-(4-formylphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-
1-carboxylate. (3.28i) tert-butyl 4-(4-(2-(4-formylphenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-
yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (190 mg, 0.304 mmol) was dissolved in dioxane
(0.735 mL). Sodium hydroxide was added (3M, 1.0 mL). The reaction mixture was microwaved
at 150 °C for 15 minutes. The reaction mixture was quenched with saturated ammonium chloride
solution. A precipitate formed and was collected by vacuum filtration to afford the title compound
as a yellow solid (128 mg, 89% yield). LCMS [M+H]+ 472.2 m/z; 1H NMR (500 MHz, DMSO-
d6) ppm 12.33 - 12.41 (m, 1 H), 10.03 (s, 1 H), 8.65 (s, 1 H), 8.26 - 8.31 (m, 3 H), 8.22 (d, J=4.9
Hz, 1 H), 8.00 (d, J=8.3 Hz, 2 H), 7.59 (s, 1 H), 7.33 (d, J=4.9 Hz, 1 H), 4.42 - 4.52 (m, 1 H), 3.99
- 4.20 (m, 2 H), 2.82 - 3.08 (m, 2 H), 2.09 (d, J=12.2 Hz, 2 H), 1.93 (qd, J=12.1, 4.6 Hz, 2 H), 1.43
(s, 9 H).
279
4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridine. (3.28j) 4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzaldehyde (36.4 mg, 0.069 mmol) was dissolved in a solution of 10% acetic acid/methanol
(1 mL). Pyrrolidine (56.3 μL, 0.686 mmol) was added and the reaction mixture was heated at 50
°C until the solution was clear. Sodium cyanoborohydride (6.5 mg, 0.109 mmol) was then added
and the reaction mixture was left heating at 50 °C overnight. The reaction mixture was neutralized
with saturated sodium bicarbonate solution. The aqueous mixture was concentrated to remove
methanol, then extracted 3 times with dichloromethane. The combined organic layers were washed
with water and then brine, collected, dried with sodium sulfate, filtered and concentrated. The
crude product was purified by column chromatography using a mobile phase of 40-100% ethyl
acetate/hexanes followed by 0-20% methanol/ethyl acetate to afford the title compound as an off
white solid (37.4 mg, 93% yield). LCMS [M+H]+ 586.1 m/z; 1H NMR (500 MHz,
CHLOROFORM-d) ppm 8.59 (d, J=5.4 Hz, 1 H), 8.08 (d, J=8.3 Hz, 2 H), 7.85 (d, J=8.3 Hz, 2
H), 7.76 - 7.83 (m, 4 H), 7.64 (d, J=8.3 Hz, 2 H), 7.30 (d, J=5.4 Hz, 1 H), 7.25 (d, J=8.3 Hz, 2 H),
6.67 (s, 1 H), 4.28 (d, J=5.9 Hz, 2 H), 3.73 (d, J=4.4 Hz, 2 H), 3.11 (s, 3 H), 2.84 - 2.94 (m, 2 H),
2.38 (s, 3 H), 2.26 - 2.34 (m, 2 H), 2.10 (s, 2 H).
280
N-(4-(4-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-6098
(3.29a) N-(4-(4-(pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (46
mg, 0.093 mmol) was dissolved in dioxane (0.309 mL). Sodium hydroxide was added (3M, 0.309
mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was
quenched with saturated ammonium chloride solution. A precipitate formed and was collected by
vacuum filtration to afford the title compound as a yellow solid (22 mg, 69% yield). LCMS
[M+H]+ 343.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.35 (br. s., 1 H), 9.04 (d, J=1.5 Hz,
1 H), 8.69 (dd, J=4.6, 1.2 Hz, 1 H), 8.31 (d, J=4.9 Hz, 1 H), 8.22 - 8.28 (m, 1 H), 7.95 (d, J=8.3
Hz, 2 H), 7.61 (dd, J=7.3, 4.9 Hz, 1 H), 7.43 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.13 (s,
1 H), 3.75 (s, 2 H), 2.74 (dquin, J=12.0, 5.9, 5.9, 5.9, 5.9 Hz, 1 H), 1.02 (d, J=6.3 Hz, 6 H).
N-(2-hydroxyethyl)-4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-
yl)benzenesulfonamide. NEU-6111 (3.29b) N-(2-hydroxyethyl)-4-(2-(4-
281
((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide
(63 mg, 0.102 mmol) was dissolved in dioxane (0.340 mL). Sodium hydroxide was added (3M,
0.340 mL). The reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture
was quenched with saturated ammonium chloride solution and concentrated. The crude product
was purified by column chromatography using a mobile phase of 1-20% ((+10% ammonium
hydroxide)/methanol)/dichloromethane followed by 1-20% ((+10% ammonium
hydroxide)/methanol)/ethyl acetate to afford the title compound as a yellow solid (29 mg, 61%
yield). LCMS [M+H]+ 465.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.35 (br. s., 1 H), 8.31
(d, J=5.4 Hz, 1 H), 8.06 (d, J=8.3 Hz, 2 H), 7.92 - 8.00 (m, 4 H), 7.76 (br. s., 1 H), 7.43 (d, J=7.8
Hz, 2 H), 7.27 (d, J=4.9 Hz, 1 H), 7.15 (s, 1 H), 4.74 (t, J=5.6 Hz, 1 H), 3.72 (s, 2 H), 3.43 (q,
J=6.2 Hz, 1 H), 2.88 (t, J=6.1 Hz, 2 H), 2.71 (spt, J=6.0 Hz, 1 H), 1.01 (d, J=6.3 Hz, 6 H).
N-(4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.
NEU-6475 (3.29c) N-(4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (79 mg, 0.138 mmol) was dissolved in dioxane (0.5 mL). Sodium
hydroxide (3M, 0.5 mL) was added and the reaction mixture was microwaved at 150 °C for 15
minutes. The reaction mixture was quenched with saturated ammonium chloride solution. The
reaction mixture was concentrated and purified by column chromatography using a mobile phase
282
of 1-20% methanol/dichloromethane followed by 0-20% ((+5% ammonium
hydroxide)/methanol)/ethyl acetate to afford the title compound as a pale yellow solid (37.3 mg,
65% yield). LCMS [M+H]+ 420.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.30 (d,
J=4.9 Hz, 1 H), 8.13 - 8.19 (m, 2 H), 8.05 - 8.11 (m, 2 H), 7.89 (d, J=7.8 Hz, 2 H), 7.49 (d, J=7.8
Hz, 2 H), 7.29 (d, J=5.4 Hz, 1 H), 7.05 (s, 1 H), 3.85 (s, 2 H), 3.21 (s, 3 H), 2.93 (spt, J=5.9 Hz, 1
H), 1.16 (d, J=6.3 Hz, 6 H).
N-(4-(4-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine.
NEU-6476 (3.29d) tert-butyl 4-(2-(4-((isopropylamino)methyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (77 mg, 0.13 mmol) was dissolved in
dichloromethane (0.5 mL) and trifluoroacetic acid was added and the reaction mixture stirred at
room temperature until completion as determined by LCMS. The solvent was concentrated to
dryness and the crude mixture then dissolved in dioxane (500 μL). Sodium hydroxide (3M, 426
μL) was added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction
mixture was quenched with saturated ammonium chloride solution. The reaction mixture was
concentrated and purified by column chromatography using a mobile phase of 0-50% (5%
ammonium hydroxide/methanol)/ethyl acetate followed by 1-20% methanol/dichloromethane to
afford the title compound as a yellow solid (14 mg, 31% yield). LCMS [M+H]+ 347.2 m/z; 1H
283
NMR (500 MHz, METHANOL-d4) ppm 8.11 (d, J=5.4 Hz, 1 H), 7.84 (d, J=8.3 Hz, 2 H), 7.45
(d, J=8.3 Hz, 2 H), 6.97 - 7.04 (m, 2 H), 6.43 (br. s., 1 H), 3.82 (s, 2 H), 3.59 (d, J=2.4 Hz, 2 H),
3.13 (t, J=5.6 Hz, 2 H), 2.90 (spt, J=6.3 Hz, 1 H), 2.63 (d, J=2.0 Hz, 2 H), 1.14 (d, J=6.3 Hz, 6 H).
N-(4-(4-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-
6477 (3.29e) N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (50 mg, 0.098 mmol) was dissolved in dioxane (320 μL). Sodium
hydroxide (3M, 320 μL) was added and the reaction mixture was microwaved at 150 °C for 15
minutes. The reaction mixture was quenched with saturated ammonium chloride solution. The
reaction mixture was concentrated and purified by column chromatography using a mobile phase
of 1-20% methanol/dichloromethane followed by 0-20% methanol/ethyl acetate to afford the title
compound as a yellow residue (20 mg, 57% yield). LCMS [M+H]+ 357.2 m/z; 1H NMR (500 MHz,
METHANOL-d4) ppm 8.58 (d, J=5.4 Hz, 1 H), 8.29 (d, J=4.9 Hz, 1 H), 7.88 (d, J=8.3 Hz, 2 H),
7.72 (s, 1 H), 7.67 (d, J=5.4 Hz, 1 H), 7.47 (d, J=8.3 Hz, 2 H), 7.28 (d, J=4.9 Hz, 1 H), 7.04 (s, 1
H), 3.81 (s, 2 H), 2.88 (spt, J=6.3 Hz, 1 H), 2.67 (s, 3 H), 1.13 (d, J=6.3 Hz, 6 H).
284
N-(4-(4-(2,6-dimethylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-
6478 (3.29f) N-(4-(4-(2,6-dimethylpyridin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine (70 mg, 0.13 mmol) was dissolved in dioxane (400 μL). Sodium
hydroxide (3M, 445 μL) was added and the reaction mixture was microwaved at 150 °C for 15
minutes. The reaction mixture was quenched with saturated ammonium chloride solution. The
reaction mixture was concentrated and purified by column chromatography using a mobile phase
of 1-20% methanol/dichloromethane followed by 1-20% (10% ammonium
hydroxide/methanol)/ethyl acetate to afford the title compound as an off-white solid (30 mg, 61%
yield). LCMS [M+H]+ 371.3 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.28 (d, J=4.9
Hz, 1 H), 7.88 (d, J=8.3 Hz, 2 H), 7.51 (s, 2 H), 7.48 (d, J=8.3 Hz, 2 H), 7.25 (d, J=5.4 Hz, 1 H),
7.02 (s, 1 H), 3.82 (s, 2 H), 2.82 - 2.96 (m, 1 H), 2.63 (s, 6 H), 1.14 (d, J=6.3 Hz, 6 H).
N-(4-(4-(1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine. NEU-6508
(3.29g) N-(4-(4-(1H-pyrazol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine
(110 mg, 0.23 mmol) was dissolved in dioxane (755 μL). Sodium hydroxide (3M, 755 μL) was
285
added and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture
was quenched with saturated ammonium chloride solution. The reaction mixture was concentrated
and purified by column chromatography using a mobile phase of 1-20%
methanol/dichloromethane to afford the title compound as a yellow solid (25 mg, 33% yield).
LCMS [M+H]+ 332.2 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 12.15 (br. s., 1 H), 8.22 (d,
J=4.9 Hz, 1 H), 7.85 - 8.00 (m, 3 H), 7.36 - 7.54 (m, 4 H), 7.06 (br. s., 1 H), 3.83 (br. s., 2 H), 2.86
(m, J=4.9 Hz, 1 H), 1.08 (d, J=6.3 Hz, 6 H).
N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-
yl)benzyl)propan-2-amine. NEU-6509 (3.29h) N-(4-(4-(1-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-amine (90 mg, 0.16 mmol)
was dissolved in dioxane (300 μL). Sodium hydroxide (3M, 527 μL) was added and the reaction
mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was quenched with
saturated ammonium chloride solution. The reaction mixture was concentrated and purified by
column chromatography using a mobile phase of 1-20% methanol/dichloromethane to afford the
title compound as a light tan solid (34.8 mg, 53% yield). LCMS [M+H]+ 416.3 m/z; 1H NMR (500
MHz, DMSO-d6) ppm 12.19 (br. s., 1 H), 8.62 (s, 1 H), 8.25 (s, 1 H), 8.17 (d, J=4.9 Hz, 1 H),
8.10 (d, J=8.3 Hz, 2 H), 7.62 (d, J=7.3 Hz, 2 H), 7.40 (s, 1 H), 7.30 (d, J=5.4 Hz, 1 H), 4.53 (m,
286
J=4.9, 4.9 Hz, 1 H), 4.09 (br. s., 2 H), 4.03 (d, J=10.7 Hz, 2 H), 3.52 (t, J=10.5 Hz, 2 H), 3.13 -
3.26 (m, 1 H), 2.01 - 2.17 (m, 4 H), 1.26 (d, J=4.9 Hz, 6 H).
tert-Butyl 4-(4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-
pyrazol-1-yl)piperidine-1-carboxylate. NEU-6077 (3.29i) tert-Butyl 4-(4-(2-(4-formylphenyl)-
1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (60 mg, 0.13 mmol)
was dissolved in a solution of 10% acetic acid/methanol (1 mL). Isopropylamine (680 μL, 8.3
mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. Sodium
cyanoborohydride (12 mg, 0.19 mmol) was then added and the reaction mixture was left heating
at 70 °C overnight. The reaction mixture was neutralized with saturated sodium bicarbonate
solution. The aqueous mixture was concentrated to remove methanol, then extracted 3 times with
ethyl acetate. The combined organic layers were washed with water and then brine, collected, dried
with sodium sulfate, filtered and concentrated. The crude product was purified by column
chromatography using a mobile phase of 1-20% methanol/dichloromethane to afford the title
compound as a pale yellow solid (29 mg, 44% yield). LCMS [M+H]+ 515.3 m/z; 1H NMR (500
MHz, METHANOL-d4) ppm 8.39 (s, 1 H), 8.09 - 8.15 (m, 2 H), 7.97 (d, J=8.3 Hz, 2 H), 7.55
(d, J=8.3 Hz, 2 H), 7.25 (d, J=4.9 Hz, 1 H), 7.19 (s, 1 H), 4.40 - 4.49 (m, 1 H), 4.23 (d, J=13.2 Hz,
287
2 H), 4.10 (s, 2 H), 3.29 - 3.37 (m, 1 H), 2.86 - 3.04 (m, 2 H), 2.07 - 2.14 (m, 2 H), 2.01 (m, J=12.0,
4.1 Hz, 2 H), 1.46 (s, 9 H), 1.32 (d, J=6.8 Hz, 6 H).
4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine.
NEU-6712 (3.29j) 4-(4-(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (34 mg, 0.058 mmol) was dissolved in dioxane (500 μL). Sodium
hydroxide (3M, 0.2 mL) was added and the reaction mixture was microwaved at 150 °C for 15
minutes. The reaction mixture was quenched with saturated ammonium chloride solution. A
precipitate formed and was collected by vacuum filtration. The crude product was purified by
column chromatography using a mobile phase of 1-20% methanol/dichloromethane to afford the
title compound as a yellow solid (5 mg, 20% yield). LCMS [M+H]+ 432.2 m/z; 1H NMR (500
MHz, METHANOL-d4) ppm 8.33 (d, J=4.4 Hz, 1 H), 8.12 - 8.19 (m, 2 H), 8.08 (d, J=8.3 Hz, 2
H), 7.99 (d, J=8.3 Hz, 2 H), 7.63 (d, J=8.3 Hz, 2 H), 7.30 (d, J=4.9 Hz, 1 H), 7.12 (s, 1 H), 4.30
(s, 2 H), 3.24 (br. s., 4 H), 3.22 (s, 3 H), 2.07 (br. s., 4 H).
288
N-(4-(4-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzyl)propan-2-
amine. NEU-6097 (3.30) tert-butyl 4-(4-(2-(4-((isopropylamino)methyl)phenyl)-1H-pyrrolo[2,3-
b]pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (21 mg, 0.041 mmol) was dissolved in
dichloromethane (0.2 mL). Trifluoroacetic acid (0.12 mL, 1.6 mmol). The reaction mixture was
left stirring overnight at room temperature. The reaction mixture was neutralized using carbonate
on silica gel. The silica was filtered off and washed with methanol. The crude product was purified
by column chromatography using a mobile phase of 1-20% ((+10% ammonium
hydroxide)/methanol)/dichloromethane to afford the title compounds as a yellow solid (10 mg,
59% yield). LCMS [M+H]+ 415.2 m/z; 1H NMR (500 MHz, METHANOL-d4) ppm 8.38 (s, 1
H), 8.14 (s, 1 H), 8.12 (d, J=4.9 Hz, 1 H), 7.90 (d, J=8.3 Hz, 2 H), 7.46 (d, J=8.3 Hz, 2 H), 7.27
(d, J=4.9 Hz, 1 H), 7.14 (s, 1 H), 4.40 (tt, J=11.6, 4.0 Hz, 1 H), 3.81 (s, 2 H), 3.21 (d, J=13.2 Hz,
2 H), 2.89 (spt, J=6.3 Hz, 1 H), 2.78 (t, J=12.4 Hz, 2 H), 2.12 - 2.19 (m, 2 H), 2.03 (qd, J=12.2,
3.9 Hz, 2 H), 1.14 (d, J=6.3 Hz, 6 H).
289
4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. 4-(4-chloro-1-
tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (74 mg, 0.18 mmol), (4-
(methylsulfonyl)phenyl)boronic acid (44.5 mg, 0.223 mmol),
tetrakis(triphenylphosphine)palladium(0) (6.4 mg, 0.05 mmol), and sodium carbonate (59 mg,
0.56 mmol) were added to a vial that was subsequently sealed and vacuum purged with nitrogen.
A degassed solution of dioxane-water (3:1, 2 mL) was added to the vial. The reaction mixture was
heated at 80 °C overnight and monitored by LCMS. The reaction mixture was filtered through a
bed of Celite and concentrated under reduced pressure and then purified by silica gel column
chromatography using a mobile phase of 0-50% ethyl acetate/hexanes to afford the title compound
as an off white solid (83 mg, 87% yield). LCMS [M+H]+ 519.1 m/z; 1H NMR (500 MHz, DMSO-
d6) ppm 9.80 (s, 1 H), 8.46 (d, J=4.9 Hz, 1 H), 8.05 (d, J=8.8 Hz, 2 H), 7.95 - 7.99 (m, 1 H), 7.73
(d, J=8.8 Hz, 2 H), 7.50 (d, J=5.4 Hz, 1 H), 7.41 (d, J=8.3 Hz, 2 H), 7.36 (d, J=8.3 Hz, 2 H), 6.86
(d, J=8.8 Hz, 2 H), 6.81 (s, 1 H), 3.27 (s, 3 H), 2.32 (s, 3 H).
290
4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol. NEU-1936 4-(4-
(methylsulfonyl)phenyl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(83 mg, 0.16 mmol) was dissolved in dioxane (1 mL). Sodium hydroxide (3M, 0.56 mL) was added
and the reaction mixture was microwaved at 150 °C for 15 minutes. The reaction mixture was
quenched with saturated ammonium chloride solution. A precipitate formed and was collected by
vacuum filtration to afford the title compound as an orange solid (53 mg, 90% yield). LCMS
[M+H]+ 365.0 m/z; 1H NMR (500 MHz, DMSO-d6) ppm 8.27 (d, J=4.9 Hz, 1 H), 8.09 (s, 4 H),
7.82 (d, J=8.8 Hz, 2 H), 7.25 (d, J=5.4 Hz, 2 H), 6.97 (s, 1 H), 6.86 (d, J=8.3 Hz, 2 H), 3.31 (s, 3
H).
291
Cell and Whole-Organism Assay Protocols
The following assays were performed by members of the Navarro Lab at CSIC.
Strains and media. Bloodstream Trypanosoma brucei brucei Lister 427 was cultured in Hirumi’s
modified Iscove’s medium (HMI-9), supplemented with 10% heat-inactivated FBS, at 37 ºC and
5% CO2 in T-25 vented flask (Corning®). MRC5-SV2 cell line (SV40-transformed human lung
fibroblast cell line) was cultured in DMEM medium supplemented with 10% FBS at 37 ºC and 5%
CO2 in T-75 vented flask (Corning®). The T. cruzi Tulahuen C4 strain, expressing the β-
galactosidase gene (LacZ) and L6 rat skeletal muscle cells, used as host cells, were cultured in
RPMI-1640 supplemented with 10% iFBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100
μg/mL streptomycin at 37 °C and 5% CO2. Leishmania donovani MHOM/ET/67/HU3 cells with
the luciferase gene integrated into the parasite genome169 were grown at 28 °C in RPMI 1640-
modified medium (Invitrogen) supplemented with 20% FBS with 100 mg/ml of hygromycin B.
The Human myelomonocytic cell line THP-1 was grown at 37 °C and 5% CO2 in RPMI-1640
supplemented with 10% iFBS, 2 mM glutamate, 100 U/mL penicillin and 100 mg/mL
streptomycin.
Preparation of compound plates. For dose-response experiments, compound plates were prepared
for each analogue by serial 3-fold dilutions in 100% DMSO. Five concentration points
(mammalian cytotoxicity) or ten concentration points (parasite growth inhibition), were made in
96-well transparent Nunclon plates. Pentamidine was routinely included in compound plates as
internal quality control, and plates were stored sealed at -20 ºC for no more than four weeks.
292
Rate of Action assays. Mid-log T. brucei brucei cultures were diluted to the required cell density,
according to the different incubation time points described. Cultures (90 μL per well) were
dispensed in final assay Nunclon 96-well flat bottom Solid White plates and 10 µL of intermediate
plates were added to each well, as described before. Four sets of assay plates were arranged to
assay in order to be sequentially stopped at each indicated time point. Top and bottom rows were
dismissed for compound assay, to reduce evaporation effects.
Plates were incubated at 37 ºC and 5% CO2 for the indicated time points; incubation was
stopped by addition of 10 μL of prewarmed Cell Titer Glo reagent (Promega®), and after shaking
the plates were incubated at room temperature for 10 min, to allow the signal to settle. Plate
luminescence was read on an Infinite F200 plate reader (Tecan), and raw data were processed and
analyzed as previously described.
Β-D-Galactosidase Transgenic T. cruzi Assay. A Thermo Scientific Multidrop Combi dispenser
(MTX Lab Systems, Vienna, VA) was used to dispense 90 μL of T. cruzi amastigote–infected L6
cell culture (4×103 infected L6 cells per well) into 96-well Corning assay plates (Corning Inc.,
Corning, NY) already containing 10 μL of the compounds to be screened and controls. The plates
were incubated at 37 °C for 96 h. Then, 30 μL of 100 μM CPRG and 0.1% NP40 diluted in PBS
were added to each well, and the plates were incubated for 4 h at 37 °C in the dark. Absorbance at
585 nm was measured in a Vmax kinetic microplate reader (Molecular Probes). Compound
activities were normalized using the in-plate 100% inhibition (benznidazole at 10 μg/mL) and 0%
inhibition (0.2% DMSO) growth controls.
293
Cytotoxicity assay in MRC5. Intermediate plates were made as described, adding 95 μL of DMEM
complete media to 5 μL of compound per well setting a 5% DMSO amount. Log-phase MRC5
cells were removed from a T-75 TC flask using TrypLE® Express (Thermo®) and dispersed by
gentle pipetting. Cell density was adjusted to working concentration in prewarmed DMEM
medium: 25,000 cells in 90 μL of culture were plated in 96-well transparent Nunclon plates and
let to settle for 24 h at 37 ºC and 5% CO2. After settling incubation, 10 μL of freshly made
intermediate plate were added per well: final maximal concentration for compounds was 50 μM in
0.5% DMSO per well. Plates were incubated for 48 h at 37 ºC and 5% CO2. At 4 h prior to
fluorescence measurement, 20 μL of 500 μM resazurin solution was added. Fluorescence was read
in an Infinite F200 plate reader (Tecan®) at 550 nm (excitation filter) and 590 nm (emission filter).
A four-parameter equation was used to fit the dose-response curves and determination of
EC50 by SigmaPlot ® 13.0 software. Assays were performed in duplicate at least twice for positive
compounds, to achieve a minimal n=2 per dose response.
Resazurin-Based L6 Assay. One hundred microliters (100 μL) per well of culture medium
containing the compounds and controls were added to L6 cells previously cultured (4×103 L6 cells
per well). After 72 h at 37 °C the medium was exchanged and the viable cell number was
determined by resazurin (Sigma–Aldrich) reduction. 20 μl of resazurin (1.1 mg/ml) was added to
each well and incubated in the dark for 2 h at 37 ºC. Cell viability was estimated by measuring the
final fluorescence at 570-590 nm in an Infinite F200 plate reader (Tecan).
Cytotoxicity assay in THP-1. Cellular toxicity of all compounds was determined using the
colorimetric MTT-based assay after incubation at 37 °C for 72 h in the presence of increasing
294
concentrations of compounds (final maximal concentration was 50 μM in 0.5% DMSO per
well)170. The results are expressed as EC50 values, the concentration of compound that reduces cell
growth by 50% versus untreated control cells. Assays were performed in duplicate at least twice
to achieve a minimal n=3 per dose response.
Determination of EC50 in L. donovani. Macrophage-differentiated THP-1 cells were infected at a
macrophage/parasite ratio of 1:10 with stationary-phase L. donovani promastigotes for 24 h at 35
°C and 5% CO2, and extracellular parasites were removed by washing three times with PBS.
Infected cell cultures were then incubated with different compounds concentrations at 37 °C for
72 h. Luminescence was measured using the Promega kit luciferase assay system (Promega ®,
Madison, WI). Assays were performed in duplicate at least twice, to achieve a minimal n=3 per
dose response.
Cytotoxicity assay in HepG2.171 HepG2 cells were cultured in complete Minimal Essential Medium
prepared by supplementing MEM with 0.19% sodium bicarbonate, 10% heat inactivated FBS, 2
mM L-glutamine, 0.1 mM MEM non-essential amino, 0.009 mg/mL insulin, 1.76 mg/mL bovine
serum albumin, 20 units/mL penicillin–streptomycin, and 0.05 mg/mL gentamycin. HepG2 cells
cultured in complete MEM were first washed with 1x Hank’s Balanced Salt Solution (Invitrogen
#14175095), trypsinized using a 0.25% trypsin/EDTA solution, assessed for viability using trypan
blue, and resuspended at 250,000 cells/mL. Using a Tecan EVO Freedom robot, 38.3 μL of cell
suspension were added to each well of clear, cell culture-treated 384-well microtiter plates for a
final concentration of 9570 liver cells per well, and plated cells were incubated overnight in 5%
CO2 at 37 °C. Drug plates were prepared with the Tecan EVO Freedom using sterile 96 well plates
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containing twelve duplicate 1.6-fold serial dilutions of each test compound suspended in DMSO.
4.25 μL of diluted test compound was then added to the 38.3 μL of media in each well providing
a 10%-fold final dilution of compound. Compounds were tested from a range of 57 ng/mL to
10,000 ng/mL for all assays. Mefloquine was used as a plate control for all assays with a
concentration ranging from 113 ng/mL to 20,000 ng/mL. After a 48 h incubation period, 8 μL of
a 1.5 mg/mL solution of MTT diluted in complete MEM media was added to each well. All plates
were subsequently incubated in the dark for 1 h at room temperature. After incubation, the media
and drugs in each well was removed by shaking the plate over sink, and the plates were left to dry
in a fume hood for 15 mins. Next, 30 μL of isopropanol acidified by addition of HCl at a final
concentration of 0.36% was added to dissolve the formazan dye crystals created by reduction of
MTT. Plates are put on a 3-D rotator for 15-30 mins. Absorbance was determined in all wells using
a Tecan iControl 1.6 Infinite plate reader. The 50% toxic concentrations (TC50) were then
generated for each toxicity dose response test using GraphPad Prism (GraphPad Software Inc.,
San Diego, CA) using the nonlinear regression (sigmoidal dose-response/variable slope) equation.
ADME Experiment Protocols
All animal studies were performed by AstraZeneca.
Aqueous pH 7.4 Solubility. Compounds were dried down from 10 mM DMSO solutions using
centrifugal evaporation technique. Phosphate buffer (0.1 M pH 7.4) was added and StirStix were
inserted in the glass vials, with shaking then performed at a constant temperature of 25 °C for 20-
24 h. This step was followed by double centrifugation with a tip wash in between, to ensure that
no residues of the dried compounds interfere. The solutions were diluted before analysis and
quantification using LC/MS/MS was performed.
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Log D7.4. Shake-flask octanol-water distribution coefficient was determined at pH 7.4 (Log D7.4).
The aqueous solution used is 10 mM sodium phosphate pH 7.4 buffer. The method has been
validated for Log D7.4 ranging from -2 to 5.0.
Human Plasma Protein Binding (PPB). PPB was determined using equilibrium dialysis (RED
device) to separate free from bound compound. The amount of compound in plasma (10 µM initial
concentration) and in dialysis buffer (pH 7.4 phosphate buffer) was measured by LC-MS/MS after
equilibration at 37 °C in a dialysis chamber to give the fraction unbound (fu); percent bound is
calculated and reported.
Human Liver Microsomal Clint. In vitro intrinsic clearance was determined from human liver
microsomes using a standard approach.159 Following incubation and preparation, the samples were
analyzed using LC/MS/MS. Refined data were uploaded to IBIS and are displayed as Clint
(intrinsic clearance) in μL/min/mg.
Rat Hepatocyte Clint. In vitro intrinsic clearance was determined from rat hepatocytes using a
standard approach.159 Following incubation and preparation, the samples were analyzed using
LC/MS/MS. Refined data are uploaded to IBIS and are displayed as Clint (intrinsic clearance)
μL/min/1 million cells.
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Calculated LogP and LogD values. Both LogP and LogD predictions are based on a modified
version of the method160 where the predicted partition coefficients are composed of the
molecules’ atomic increments.
Pharmacokinetics Protocols
All animal studies were performed by GlaxoSmithKline.
Animals and ethical statement: All animal studies were ethically reviewed and carried out in
accordance with European Directive 2010/63/EEC and the GSK Policy on the Care, Welfare and
Treatment of Animals. Compound was administered intraperitoneally (IP) to two groups of female
NMRI mice (Group 1 n=3; Group 2 n=6) supplied by Charles River (Germany) Ltd. The
compound was prepared in 1% (v/v) DMSO:99% (v/v) 20% (w/v) sulfobutyl ether-beta-
cyclodextrin (SBE-β-CD/Captisol®, used as a solubilizing agent) in water and the dosing volume
was 10 mL/kg for a total dose of 10 mg/kg. Food and tap water were available ad libitum.
Following IP dosing, Group 1 blood samples were collected from the tail vein into capillary tubes
containing K2EDTA at the following time-points: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h.
In order to obtain simultaneous blood and brain samples, Group 2 mice were placed under
terminal anaesthetic (isoflurane) and blood samples (0.3 mL) collected from the retro-orbital sinus
into K2EDTA tubes at 0.5 h (n= 3) and 4 h (n=3) after compound administration. Immediately
following blood sample collection, death was confirmed by cervical dislocation and the brain
removed. Aliquots of each blood sample were diluted in an equal volume of water. Mouse brain
samples were weighed, water was added at a ½ (w/v) ratio (brain/water), and then homogenized.
Both blood and brain samples were stored -80 ºC until analysis.
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Diluted blood and brain homogenates were processed under standard liquid-liquid
extraction procedures using CAN containing an internal standard (Nifedipine) and analysed by
LC-MS/MS. Non-compartmental analysis was performed using the Phoenix pharmacokinetic
software version 1.4 (Certara) and Cmax, tmax, AUClast, AUC, and t1/2 were estimated.
In vivo Efficacy Experiment Protocols
All animal studies were performed by GlaxoSmithKline.
Animals and ethical statement: All procedures were approved by ethical committee of Institute of
Parasitology and Biomedicine Lopez-Neyra (Spanish National Research Council, CSIC), code
MNC.2/2015. This Institute has joined the Agreement on Transparency in Animal
Experimentation, promoted by the Confederation of Scientific Societies of Spain (COSCE), with
the collaboration of the European Association for Animal Research released on September 20,
2016. The Animal Experimentation Unit is under the control of the competent authority, registered
in the national register as Center of Breeding and User of experimental animals code ES-
180210000022, according with the European and Spanish regulations. Female NMRI mice
(Charles River Laboratories) were provided with sterilized water and commercial pellets ad
libitum and maintained under the standard conditions in a conventional room at 20–24 °C with a
12/12-h light/dark cycle. Compound 18 solution for treatment was resuspended in vehicle: 5 %
DMSO in 20 %Captisol® sulfobutyl ether β-cyclodextrin that improves solubility and stability for
drug dosing.
Blood-stage efficacy model. Infection was performed by i.p. injection of 104 bloodstream forms in
0.2 mL TDB glucose of T. brucei brucei (STIB795) from a cryopreserved stock. Three days later,
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9 infected animals with confirmed parasitemia were divided into two groups: control (infected
mice treated with vehicle, n=4) and treated (infected mice treated with 30 mg/kg/day of 18, n=5).
For treatment, both control and 18 were heated at 50 °C for 10 min to solubilize. Control and drug-
treated mice received a 0.2 mL i.p. injection at the 3rd day from infection during 5 consecutive
days. Parasitemia was individually checked by direct microscopic counting of parasites in a
Neubauer chamber using 2 µL of blood from infected mice tail, diluted in 100 µL of TDB glucose.
Parasitaemia of all mice were periodically checked by tail blood examination up to day 31th. The
day of death was recorded.
CNS efficacy model. Infection was performed by i.p. injection of 2·104 bloodstream forms in 0.2
mL TDB glucose of T. brucei brucei (GVR25 strain) from a cryopreserved stock. Fourteen days
later, 13 infected animals with confirmed parasitemia were divided into three equal groups:
control (infected mice pretreated with a single dose of 40 mg/kg diminazene aceturate -Berenil®-
and treated with vehicle BID, n=5); treatment 1 (infected mice pretreated with 40 mg/kg Berenil®
+ 25 mg/kg /day BID 11, n=4) and treatment 2 (infected mice just treated with 25 mg/kg /day
BID 18, n=4): the total concentration of 18 administered to mice was 50 /mg/kg/day. For treatment,
vehicle and 18 were heated at 50 °C for 10 min to solubilize. Mice received a 0.2 mL i.p. injection
of vehicle or 18 at the 21th day from infection during 5 consecutive days. Parasitemia was
individually checked by direct microscopic counting of parasites in a Neubauer chamber using 2
µL of blood from infected mice tail, diluted in 100 µL of TDB glucose. Parasitaemia of all mice
were checked twice a week by tail blood examination and thereafter mice with parasitaemia
relapses were euthanized and the day of parasitaemia relapse was recorded. Results are expressed
as percentage of accumulative mortality and in Mean Survival Days (MSD).