Hit-to-lead optimization of kinase-targeted inhibitors …rx...Hit-to-lead optimization of...

Hit-to-lead optimization of kinase-targeted inhibitors of Trypanosoma brucei growth.

by Lisseth E. Silva

B.S in Chemistry and Biology, Roger Williams University, Bristol RI

A thesis submitted to

The Faculty of

the College of Science of

Northeastern University

in partial fulfillment of the requirements

for the degree of Master of Science

August 17, 2015

A thesis directed by

Michael P. Pollastri

Professor of Chemistry and Chemical Biology

ii

Dedication

This thesis is dedicated to my family, especially my mom and dad who have made so many

sacrifices to get me to where I am today, my younger brother Mauricio, whom I try to be a role

model for, and my older brother Alejandro, who although lives thousands of miles away, has

always been there for me. This thesis is also dedicated to the loving memory of my grandparents,

Mamanita and Papá Matías, and aunt Vicky. Her kindness, unconditional love, and bravery

inspired me to take another look at myself and follow my true passions.

iii

Acknowledgements

First and foremost, I would like to express my gratitude to my advisor Dr. Michael

Pollastri, who provided me with guidance and support throughout my time at Northeastern. Being

part of his lab not only made me grow professionally, but also as a person. He was a great mentor

and a role model, as he always showed genuine love and passion for his work. He was always

there willing to help whenever I encountered a problem with science or life, and showed his

support when I was going through hard times and patience when I was exploring other career

interests, and for that I will be eternally thankful.

I would also like to thank current and past lab members for making my experience in lab a

valuable one, especially Uma Swaminathan for mentoring me during my first year in lab, Will

Devine for teaching me everything about instrumentation and NMR, Seema Bag for providing me

with help when needed, and Naimee Mehta and Dana Klug for not only being great lab buddies but

also wonderful friends. I am also very thankful to have shared this experience with Angela Tanner,

who became such an important person in my life and one of my best friends. I would also like to

thank the new members of the lab, including Lori Ferrins, who is always making me laugh with

her Australian occurrences, Kelly Bachovchin, Takashi Satoh, and Baljinder Singh. These past

couple of months all of you have made my experience in lab truly a blast.

Thank you to my committee members, Dr. Robert Hanson and Dr. Graham Jones, for

taking the time to review my progress reports these past couple of years, and thesis, and for all

your input. I would also like to thank the NSF Graduate Research Fellowship for providing me

with funding to carry on my research, and the Chemistry Department at Northeastern for providing

iv

me with the great opportunity to pursue my studies here as well as for funding me during my first

year.

Lastly, I would like to thank all my family and friends for their unconditional love. I

wouldn’t be here if it weren’t for all the support I’ve gotten throughout the years.

v

Abstract of Thesis

Neglected tropical diseases affect over one billion people worldwide. They are “neglected”

because they affect the poorest parts of the world and do not attract research interest from

pharmaceutical companies as it is not profitable. Human African trypanosomiasis (HAT) is a

neglected disease caused by the parasite Trypanosoma brucei. Current treatments are highly toxic

and inconvenient; therefore new treatments to treat this disease are needed. In collaboration with

GlaxoSmithKline, a high-throughput screen (HTS) of a group of kinase-targeted inhibitors was

performed. Hit-to-lead optimization was initiated to improve the potency and physicochemical

properties of the hit compounds as means to uncover new lead molecules for the treatment of

HAT. The compound triage results and progress in pursuing a cluster of azaindole-derived

Trypanosoma brucei proliferation inhibitors are presented.

vi

Table of Contents

Dedication………………………………………………………………………………………...ii

Acknowledgements……………………………………………………………………………....iii

Abstract…………………………………………………………………………………………....v

List of Abbreviations……………………………………………………………………………viii

List of Figures…………………………………………………………………………………….xi

List of Schemes…………………………………………………………………………………..xii

List of Tables……………………………………………………………………………………xiii

Chapter 1 Introduction

1.1 Neglected Tropical Diseases……………………………………………………….......1

1.2 Human African Trypanosomiasis……………………………………………………...2

1.3 Target Repurposing……………………………………………………………………7

1.4 High-throughput screening………………………………………………………….....9

Chapter 2 Synthesis of Cluster 34 Analogs

2.1 Medicinal Chemistry Approach 1 …………………………………………………...13

2.1.1 Chemistry of Analogs of NEU1936 and NEU1938………………………………..14

2.1.2 Biological Assay and Discussion of Approach 1…………………………………..18

2.2 Medicinal Chemistry Approach 2…………………………………………………….20

2.2.1 Chemistry of Proposed Analogs…………………………………………………....23

2.2.2 Biological Assay and Discussion of Approach 2…………………………………..29

2.3 Future Studies………………………………………………………………………...32

2.3.1 Proposal of Additional Analogs……………………………………………………32

2.3.2 Progress Toward the Synthesis of GSK2280392A Analogs……………………….34

vii

2.4 Conclusion…………………………………………………………………………....36

Chapter 3 Experimental

3.1 General Methods……………………………………………………………………...38

3.2 Experimental Details………………………………………………………………….39

References………………………………………………………………………………………...60

Appendix: Representative NMR Spectra………………………………………………………....64

viii

List of Abbreviations

ACN acetonitrile

ADME absorption distribution metabolism excretion

BBB blood-brain barrier

br broad

CNS central nervous system

CLint intrinsic clearance

ClogD calculated distribution coefficient at pH = 7.4

ClogP calculated partition coefficient

d doublet

DALYs disability-adjusted life years

DCM dichloromethane

dd doublet of doublet

DMAP 4-dimethylaminopyridine

DME dimethoxyethane

DMF dimethylforamide

DMSO dimethylsulfoxide

EC50 half maximal effective concentration

EGFR epidermal growth factor response

GSK GlaxoSmithKline

1H NMR proton nuclear magnetic resonance

HAT human African trypanosomiasis

ix

HBD hydrogen bond donor

HepG2 human liver carcinoma cell line

HLM human liver microsomes

HPLC high-performance liquid chromatography

HTS high-throughput screening

Hz Hertz (coupling constant)

LC-MS liquid chromatography-mass spectrometry

LDA lithium diisopropylamide

LF lymphatic filariasis

m multiplet

mCPBA m-chloroperoxybenzoic acid

MOLT-4 human acute lymphoblastic leukemia cell line

MPO multiparameter optimization

MW molecular weight, microwave

NBS N-bromosuccinimide

NECT nifurtimox-eflornithine combination therapy

NIS N-iodosuccinimide

NTDs neglected tropical diseases

ppm parts per million

PPB protein plasma binding

PTRE post-treatment reactive encephalopathy

rt room temperature

s singlet

x

t triplet

TbAUK1 Trypanosoma brucei aurora kinase 1

TEA triethylamine

THF tetrahydrofuran

TMEDA tetramethylethylenediamine

TMS trimethylsilyl

TPSA topological polar surface area

TsCl 4-toluenesulfonyl chloride

WHO World Health Organization

xi

List of Figures

Figure 1 – Burden map of Neglected Tropical Diseases. Source: Uniting to Combat Neglected

Tropical Diseases…………………………………………………………………………………..1

Figure 2 – HAT distribution for T. brucei gambiense (left) and T. brucei rhodesiense (right).

Source: Global Health Observatory Map Gallery………………………………………………….3

Figure 3 – HAT treatments for stage 1……………………………………………………………..4

Figure 4 – HAT treatments for stage 2. ……………………………………………………………5

Figure 5 – Danusertib analogs for the inhibition of T. b. rhodesiense……………………………..8

Figure 6 – Lapatinib analogs for the inhibition of T. brucei……………………………………….9

Figure 7 – HTS of kinase targeted inhibitors……………………………………………………..10

Figure 8 – Cluster 34 released structures…………………………………………………………10

Figure 9 – Design of regiochemical analogs NEU1936 and NEU1938 from cluster 34 hits….....13

Figure 10 – Concentration of GSK2280392A…………………………………………………….20

Figure 11 – In vitro and in vivo data of GSK2280392A…………………………………………..21

Figure 12 – Analogs designed to address the metabolic stability and structure-activity relationships

of GSK2280392A…………………………………………………………………………………22

Figure 13 – Proposed analogs to explore metabolism of GSK2280392A………………………...32

Figure 14 – Proposed analogs to improve solubility of GSK2280392A………………………….33

Figure 15 – Proposed analogs to explore the structure-activity relationships of GSK2280392A...34

xii

List of Schemes

Scheme 1 – Retrosynthesis of analogs NEU1936 and NEU1938………………………………..14

Scheme 2 – Intermediate 3 synthesis……………………………………………………………..15

Scheme 3 – Synthesis of NEU1935 and NEU1936. …………………………………………….17

Scheme 4 – Synthesis of NEU1937 and NEU1938……………………………………………...18

Scheme 5 – Retrosynthesis of proposed GSK2280392A analogs………………………………..23

Scheme 6 – Synthesis of GSK2280392A analogs………………………………………………..24

Scheme 7 – Proposed synthesis of analogs 13 and 15……………………………………………35

Scheme 8 – Proposed synthesis of boronic acid 40………………………………………………36

Scheme 9 – Proposed synthesis of analog 16…………………………………………………….36

xiii

List of Tables

Table 1 – Ranges of CNS MPO properties.....................................................................................11

Table 2 – Calculated physiochemical properties and MPO scores................…………………….12

Table 3 – Attempted conditions to couple compound 8 to 9……………………………………..16

Table 4 – EC50 values for the original hits and synthesized analogs……………………………..19

Table 5 – Reaction conditions for the synthesis of NEU2068, NEU2069, NEU2070, NEU2112,

and NEU2113……………………………………………………………………………………..25

Table 6 – Reaction conditions for the synthesis of NEU2065, NEU2066, NEU2067, and

NEU2068………………………………………………………………………………………….26

Table 7 – Reaction conditions for the synthesis of analog 13…………………………………….27



Table 10 – Inhibition and ADME data of GSK2280392A analogs……………………………….30

1

Chapter 1

Introduction

1.1 Neglected Tropical Diseases

Neglected tropical diseases (NTDs) are a group of diseases that affect more than 1.4 billion

people that live in the poorest areas in the world (Figure 1), representing a significant challenge

for the medical field.1

Figure 1. Burden map of Neglected Tropical Diseases. Source: Uniting to Combat Neglected

Tropical Diseases.

These NTDs include vector-borne protozoan infections (leishmaniasis, human African

trypanosomiasis (HAT), and Chagas disease), bacterial infections (trachoma, leprosy, and Buruli

ulcer), and parasitic worm infections (hookworm, ascariasis, trichuriasis, lymphatic filariasis

(LF), onchocerciasis, guinea worm, and schistosomiasis). The burden of NTDs is high, not only

causing 534,000 deaths annually, with 5 diseases being responsible for more than 400,000

deaths, but also resulting in healthy years lost due to premature disability, as measured by

2

disability adjusted life years (DALYs). In fact, NTDs result in the loss of 57 million DALYs

annually.2

Treatments for these diseases are limited and highly toxic and draw no commercial interest

as they are not profitable. In fact, between the years of 2000 and 2009, only 26 new drugs were

marketed for NTDs, 21 of which were for the treatment of malaria and HIV.3 Consequently,

most of the drug discovery and development is conducted by academia, public-sector groups,

and public-private partnerships.

NTDs present a global health crisis, and research and development for new and effective

treatments for the eradication of these diseases is desperately needed. This thesis will describe

initial medicinal chemistry efforts targeting human African trypanosomiasis.

1.2 Human African Trypanosomiasis

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a tropical disease

that affects sub-Saharan Africa. It is caused by the parasite Trypanosoma brucei, which is

transmitted through the bite of infected tsetse flies. 4

Two subspecies of T. brucei are pathogenic for humans: T. brucei gambiense, identified

in western and central Africa, and T. brucei rhodesiense, identified in eastern and southern

Africa (Figure 2). T. b. gambiense is responsible for 98% of cases reported in the last decade and

it is characterized by its chronic, long-term infection. T. b. rhodesiense mainly affects animals,

with fewer cases reported in humans, and it is characterized by its acute infection and aggressive

disease progression.5

3

Figure 2. HAT distribution for T. brucei gambiense (left) and T. brucei rhodesiense (right).

Source: Global Health Observatory Map Gallery.6,7

HAT occurs in two disease stages: In the first stage, fever is one of the most common

symptoms observed. In T. b. rhodesiense the infection at this stage can already be severe, and

infected people with no access to treatment will die, often due to a heart attack. In T. b.

gambiense symptoms are more subtle. Some of the signs are an enlarged spleen and liver, a faint

rash, or swelling of the lymph nodes in the back of the neck (also known as Winterbottom’s

sign).8

In the second stage of the disease, which occurs weeks after infection in T. b. rhodesiense

and months in T. b. gambiense, the parasite crosses the blood-brain barrier (BBB) into the central

nervous system (CNS), resulting in chronic encephalopathy linked to behavioral changes and

headaches. Some of the effects that can be observed in patients include a decrease in everyday

4

mental functions, trouble concentrating and coping with surroundings, and a state of lethargy,

which gives the disease its name: sleeping sickness.8

The detection of the disease is also invasive. To detect the parasite, techniques such as

daily lymph node puncture or blood smear examination need to be performed to identify

trypanosomes in the blood. After the parasite has been detected in the blood or lymph, a

cerebrospinal fluid sample is taken through a lumbar puncture to determine the stage of the

disease in order to provide the proper treatment.8 Most of the time, people who have been

infected will die due to the lack of medical resources in their living areas.

Additionally, current drugs to treat HAT are highly toxic and have been found to have

parasite resistance.4 To treat stage 1 of HAT suramin and pentamidine are used (Figure 3).

Figure 3. HAT treatments for stage 1.

5

Suramin is used for T. b. rhodesiense and it is given by slow intravenous injection

throughout a period of 4 weeks, and is thought to act as a glycolytic enzyme inhibitor in the

parasite.9 Side effects include renal failure, severe allergic reactions, and neurologic effects.4

Pentamidine is used to treat T. b. gambiense and it is administered over a course of 7 to

10 days by intramuscular injection rather that intravenous injection because the latter has an

increased risk of hypotension. The mechanism of action of pentamidine is not well known,

however, it appears that the target could be the parasite’s mitochondria. Side effects include

hypotension, and low/high levels of sugar in the blood.9,10

To treat stage 2 of HAT, melarsoprol, eflornithine, and nifurtimox (in combination with

eflornithine) are used (Figure 4).

Figure 4. HAT treatments for stage 2.

Melarsoprol, an organic arsenical, remains the only treatment for against stage 2 T. b.

rhodesiense despite its high toxicity. It is given as an intravenous injection over a course of 3 to

4 days, with 7 to 10 days of resting in between each course. The arsenic in the molecule is

thought to form a complex with trypanothione, a crucial thiol in trypanosomatid cells.9 Side

effects are severe, with 10% patients developing post-treatment reactive encephalopathy (PTRE),

half of whom die. PTRE causes an acute brain inflammation, resulting in seizure, swelling of the

brain, and coma.10,11

6

Eflornithine has a half-life of only 3 hours following intravenous injection, with 80% of

the drug excreted unmetabolized in urine after 24 hours. As a result, the drug is given in large

doses over a long period of time. An inhibitor of ornithine decarboxylase, the mode of action of

this drug is through the inhibition of the parasite’s polyamine biosynthesis. Side effects include

bone marrow toxicity, hair loss throughout the body, and seizures.9,10

Nifurtimox is a tablet that is taken orally in combination with eflornithine to treat T. b.

gambiense. This “nifurtimox-eflornithine combination therapy” (NECT) reduces the required

dosing level of eflorinithine, therefore reducing its toxicity while maintaining good efficacy.

Nifurtimox works by inhibiting the trypanothione metabolism of the parasite. Side effects

include gastrointestinal and neurologic effects.10, 12

Efforts have continued to search for new drugs for HAT. DB289 was an oral prodrug for

early stage disease that reached Phase III studies, however, it was withdrawn due to the

development of renal and liver toxicity. Fexinidazole, an oral nitro-imidazole drug, was observed

to be effective and safe in vivo in late stage HAT. This drug has entered Phase II/III studies

against gambiense HAT. SCYX-7158, an orally active oxaborole, can cross the BBB, can cure

stage 2 HAT in mice model and has entered Phase II studies.11 Even if these drugs are eventually

successful in treating HAT, having more than one effective treatment is ideal. Additionally, even

if one of these drugs could effectively treat the T. b. rhodesiense strain, it doesn’t necessarily

mean that it will effective against the T. b. gambiense strain. More efficient and cost-effective

drugs are still needed for treatment of HAT.

7

1.3 Target Repurposing

New drug candidates can be discovered using three different approaches. The first approach

is the phenotypic-based drug discovery, in which compounds are screened against parasite cells,

and the compounds that show the desired phenotype (typically growth inhibition) are optimized

for improved potency and properties. The second approach is target-based discovery, in which

an essential target is identified and molecules are specifically designed to inhibit that target. The

last approach is target repurposing, in which essential molecular targets in the parasite are

matched to homologous human targets that have been previously pursued for drug discovery.

The advantage of the target repurposing approach is that it can significantly decrease the amount

of time spent in developing a drug as it utilizes an established human inhibitor and re-optimizes

it to specifically target the parasite.

Kinases are by far the biggest group of targets for target-driven discovery approach and

have become a main focus in the pharmaceutical industry due to their druggability and role in

various indications such as cancer and inflammation.3 In T. brucei homologous enzymes have

been found to be involved in cellular signaling, thus making them attractive drug targets. Several

kinases such as hexokinase13, phosphoglycerate kinase14, phosphofructokinase15-17, and glycogen

synthase kinase-3 short18-22, have been investigated in T. brucei to better understand their roles as

possible targets for therapeutic intervention.

An example of target-repurposing can be observed in the case of Danusertib, a phase II

clinical trial Aurora kinase inhibitor against solid tumors. Three Aurora kinases are expressed in

T. brucei, with TbAUK1 being responsible for cytokinesis and growth in the parasite.23

Danusertib was tested against T. b. rhodesiense, and it was shown to inhibit parasite proliferation

8

with an EC50 of 0.15 μM, however, it did not show selectivity over human cells. This compound

was optimized through the design, synthesis, and testing of 19 analogs, culminating in NEU327,

which had an EC50 of 0.61 μM (Figure 5). Though this represents a decrease in potency against

T. brucei, the selectivity over human cells was significantly improved (MOLT-4 EC50 = 14.25

μM, selectivity ratio=23) when compared to danusertib (MOLT-4 EC50 = 0.15 μM, selectivity

ratio = 1).24

Figure 5. Danusertib analogs for the inhibition of T. b. rhodesiense

Lapatinib is an example of repurposing inhibitors of homologous enzymes in which the

target is not known. Tyrphostin is a known tyrosine kinase inhibitor that inhibits parasite growth

by reducing transferrin uptake in the cell. Following these results, lapatinib, a tyrosine kinase

inhibitor that perturbs the epidermal growth factor response (EGFR) pathway and is currently

used to treat breast cancer, was tested against T. brucei. It showed inhibition of parasite

proliferation with an EC50 of 1.54 μM. Lapatinib was then re-optimized, via the synthesis of only

44 analogs, culminating in NEU617, which demonstrates having a 37-fold increase in potency

compared to lapatinib, with and improved selectivity over HepG2 cells (Figure 6).25

9

Figure 6. Lapatinib analogs for the inhibition of T. brucei.

These examples demonstrate how the target repurposing approach as a promising and

efficient method to arrive at starting points for the drug development efforts to treat sleeping

sickness.

1.4 High-throughput screening

On the basis of previous success in repurposing kinase inhibitors, we posited that a broader

collection of investigational human kinase inhibitors could potentially provide large numbers of

compounds that could be repurposed as starting points for sleeping sickness drug discovery. In

collaboration with GlaxoSmithKline, a phenotypic high-throughput screen (HTS) of 42,444

kinase-targeted inhibitors was performed, testing against T. brucei cell cultures and HepG2 cells.

From this set, 797 compounds showed good proliferation inhibition potency (EC50 < 1 μM) and

>100-fold selectivity over HepG2 cells. These compounds were then prioritized resulting in 59

clusters and 53 singletons (Figure 7).26

10

Figure 7. HTS of kinase targeted inhibitors.

Cluster 34 was chosen for lead optimization due to its good potency, desirable

physicochemical properties, and high probability to penetrate the central nervous system (CNS).

GSK released three structures out of the 14 compounds in the cluster. The potency in this cluster

ranges from 0.09 μM to 0.87 μM, sharing a 7-azaindole core with substituents at the 2 and 4

positions as shown in Figure 8.

Figure 8. Cluster 34 released structures.

Screened against T. brucei at

4 μM, >50% inh. selected

Tested against Tbb cells and

HepG2 cells. IC50 < 1 μM and

> 100x selective

Clustering and prioritization

42,444 compounds

4,574 compounds

797 compounds

59 clusters, 53 singletons

11

The physicochemical properties and the CNS multiparameter optimization scores (CNS

MPO) for these compounds were calculated. The CNS MPO score measures the probability of a

compound to penetrate and show activity in the CNS based on a set of six physicochemical

parameters: lipophilicity, calculated partition coefficient (ClogP); calculated distribution

coefficient at pH = 7.4 (ClogD); molecular weight (MW); topological polar surface area (TPSA);

hydrogen bond donors; and most basic center (pKa).27 As shown in Table 1, each

physicochemical property has a score system ranging from less desirable (0) to most desirable

(1).

Properties Score of 1 Score of 0

ClogP ClogP ≤ 3 ClogP > 5

ClogD ClogD ≤ 2 ClogD > 4

MW MW ≤ 360 MW > 500

TPSA 40 < TPSA ≤ 90 TPSA ≤ 20; TPSA > 120

HBD HBD ≤ 0.5 HBD > 3.5

pKa pKa ≤ 8 pKa > 10

Table 1. Ranges of CNS MPO properties.

The summation of each score can vary from 0 to 6, with a desirable CNS MPO score of

≥4. This scoring system prevents a compound from being discarded as a CNS drug candidate if it

only fails to meet one or two of the desired ranges of physicochemical properties. The calculated

physicochemical properties and MPO scores values for the released structures are shown in

Table 2, and all three analogs are predicted to be CNS-active compounds.

12

Properties Desirable

values

GSK2280392A

GSK530672A GSK1992878A

clogP < 5 2.7 2.63 2.22

TPSA ≤ 140 83.05 70.29 125.79

MW < 500 364.42 299.33 422.5

LE ≥ 0.3 0.38 0.42 0.3

LLE ≥ 4 4.4 4.41 4.26

CNS MPO ≥ 4 4.94 5.35 3.09

Table 2. Calculated physicochemical properties and MPO scores.

The compounds GSK530672A and GSK2280392A were selected for improvement of

their potency and to explore the structure-activity relationships of the chemotype. In addition, the

pharmacokinetics of GSK2280392A were evaluated in order to inform further compound

optimization.

In summary, drug research and development to find new treatments for NTDs is crucial

for the eradication of these diseases. Target repurposing has been found to be a promising

approach for the development of new drugs to treat sleeping sickness, with kinases being

identified as attractive drug targets in the parasite. Based on this method, a phenotypic HTS of

known human kinases inhibitors was performed, leading to cluster 34 as a promising starting

point for HAT drug discovery. In the next chapter, hit-to-lead optimization efforts of this cluster

will be presented as means to discover new lead molecules for the treatment of sleeping sickness.

13

Chapter 2

Synthesis of Cluster 34 analogs

2.1 Medicinal Chemistry Approach 1

The compounds GSK530672A and GSK2280392A have desirable drug-like properties and show

good CNS penetration, as predicted by the CNS MPO score.

In an effort to maintain the desirable physicochemical properties while exploring the

structure-activity relationships for antiparasitic potency, the regiochemistry of GSK530672A and

GSK2280392A were altered to give NEU1936 and NEU1938, respectively (Figure 9). The

change in regiochemistry can allow us to see if a molecule’s particular conformation is preferred

for the binding at the active site in the protein.

Figure 9. Design of regiochemical analogs NEU1936 and NEU1938 from cluster 34 hits.

14

A retrosynthesis for these analogs is shown in Scheme 1. Chloroazaindole (4) can be

protected using TsCl and then iodinated with iodine and LDA to give compound 3. To this

compound, the corresponding boronate can be coupled first at the 2-iodo position, followed by

coupling at the 4-chloro position via Suzuki coupling to give the tosyl protected final compound

1, which can then be deprotected to give NEU1936 and NEU1938.

Scheme 1. Retrosynthesis of analogs NEU1936 and NEU1938.

2.1.1 Chemistry of Analogs NEU1936 and NEU1938

The synthesis for the two analogs is shown in Scheme 2. These were synthesized by oxidizing

the starting material, 7-azaindole, with mCPBA28 to give 5 as a m-chlorobenzoate salt in a 83%

yield. Compound 5 was chlorinated using POCl329 giving 4 in a 72% yield. Next, compound 6

15

was tosylated using TsCl, TEA, and DMAP to give 6 in a 79% yield. Subsequently, compound 7

was iodinated using freshly made LDA, TMEDA and iodine30 giving compound 3 in a 98%

yield.

Scheme 2. Reaction conditions: (a) mCPBA, DME/Hexane, 0°C to rt, 26h, 83%; (b) POCl3,

85°C, overnight, 72%; (c) TsCl, TEA, DMAP, DCM, rt, overnight, 79%; (d) LDA, TMEDA, I2,

THF, -70°C, 98%.

Different boronic acids and boronic acid pinacol esters were reacted with 3 using Suzuki

coupling conditions to give the corresponding analogs NEU1935, NEU1936, NEU1937 and

NEU1938. The synthesis of NEU1935 is shown in Scheme 3. Compound 3 was coupled with 7

at the 2-iodo position using Pd(PPh3)4, 2M Na2CO3, DME and ethanol to give compound 8.

Various conditions were then attempted (Table 3, entries 1-5) to couple compound 8 to 9.

16

Entry Catalyst Solvent Temperature,

time

Results

1 Pd(PPh3)4 DME/EtOH 85°C,

overnight

Only 8 by LC

2 Pd(PPh3)4 DME/EtOH 185°C MW,

50 minutes

Only 8 by LC

3 Pd(PPh3)4 1,4-dioxane 100 °C,

24 hrs

Only 8 by LC

4 Pd(PPh3)4 1,4-dioxane 120 °C MW,

2 hrs

Product in 61% yield

5 PdCl2(dppf) 1,4-dioxane 120 °C, 21 hrs 95% 8, 5% product

by LC

Table 3. Attempted conditions to couple compound 8 to 9.

Entry 4 gave the most successful results, in which compound 8 was coupled with 9 at the

4-chloro position using Pd(PPh3)4, 2M Na2CO3, and 1,4-dioxane to give NEU1935. As scheme 3

shows, compound NEU1935 was subsequently deprotected with 2M NaOH to give final

compound NEU1936.

17

Scheme 3. Reaction conditions: (a) Pd(PPh3)4, 2M Na2CO3, DME/EtOH, 85°C, 4h, 64%; (b)

Pd(PPh3)4, 2M Na2CO3, 1,4-dioxane, 120°C MW, 2h, 61%; (c) 2M NaOH, 1,4-dioxane, 110°C,

4h, 34%.

The synthesis of compounds NEU1937 and NEU1938 are shown in Scheme 4,

compound 3 was coupled with 10 at the 2-iodo position using Pd(PPh3)4, 2M Na2CO3, DME and

EtOH to give 11. This compound was then reacted with 12 at the 4-chloro position using

Pd(PPh3)4, 2M Na2CO3, DME and ethanol to give NEU1937. Subsequently, compound

NEU1937 was deprotected using 2M NaOH to give the final compound NEU1938.

18

Scheme 4. Reaction conditions: (d) Pd(PPh3)4, 2M Na2CO3, DME/EtOH, 85°C, 4h, 41%; (e)

Pd(PPh3)4, 2M Na2CO3, DME/EtOH, 85°C, 4h, 61% (f) 2M NaOH, 1,4-dioxane, 110°C, 3h,

21%.

2.1.2 Biological Assay and Discussion of Approach 1

NEU1935, NEU1936, NEU1937 and NEU1938 were screened against T.brucei and the EC50s

for each analog was obtained as shown in Table 4.

19

Compound number Structure EC50 (μM)

GSK530672A

0.067

GSK2280392A

0.09

NEU1935

>5

NEU1936

0.17

NEU1937

2.2

NEU1938

0.16

Table 4. EC50 values for the original hits and synthesized analogs.

20

The high EC50 values of the tosyl protected compounds NEU1935 (>5 μM) and

NEU1937 (2.2 μM) suggests that the free NH could be crucial in the binding at the active site or

that the binding site is too crowded.

Analogs NEU1936 and NEU1938 provided EC50s of 0.17 μM and 0.16 μM,

respectively. The barely discernable decrease in potency of only 2 fold over GSK530672A and

GSK2280392A suggests that the regiochemistry of the compound substituents is not essential for

activity against the parasite. Indeed, the difference in potency is not significant enough to make

this conclusion definitively.

2.2 Medicinal Chemistry Approach 2

Pharmacokinetic data was obtained for GSK2280392A, and the peripheral blood levels of

GSK2280392A after 1mg/kg single dose intravenous (IV) administration were measured for

three mice as shown in Figure 10.

Figure 10. Concentration of GSK2280392A in blood throughout the course of 2 hours.

Additionally, the mean clearance obtained for this compound is very high in vivo

(clearance of 121 mL/min/kg) with a low mean half-life of 0.4 hours, and also in vitro

21

(microsome intrinsic clearance of 6.92 mL/min/g)26 (Figure 11). The high microsomal clearance

suggests that the compound is likely metabolizing quickly via an oxidative pathway.

Figure 11. In vitro and in vivo data of GSK2280392A.

We hypothesized that the high clearance could be due to oxidation occurring on the

cyanophenyl ring, either para to the azaindole, or para to the cyano substituent. Therefore,

analogs that have a substituent at the para position of the 3-azaindole ring and/or blocking groups

para to the cyano group were designed in order to address potential metabolic stability. In

addition, different substituents at the meta position of the benzyl ring were designed to

investigate the importance of the cyano at that position (Figure 12).

22

Figure 12. Analogs designed to address the metabolic stability and structure-activity

relationships of GSK2280392A.

The retrosynthesis of these compounds is shown in Scheme 5. The 7-azaindole can be

brominated to give the corresponding bromo-azaindole (19). The pyrazole boronic acid pinacol

ester can then be reacted via Suzuki coupling to give compound 18. Subsequently, this can be

brominated or iodinated (17) and the corresponding boronic acids can be coupled via Suzuki

coupling to give the corresponding analogs.

23

Scheme 5. Retrosynthesis of proposed GSK2280392A analogs.

2.2.1 Chemistry of Proposed Analogs

The synthesis for these analogs is shown in Scheme 6. The 7-azaindole was brominated using

bromine31 to give compound 20 in a 63% yield. Compound 20 was then treated with zinc31 to

give compound 21 in a 74% yield. A 1M solution of BH3 in THF was then added and the residue

obtained was dissolved in 6N HCl, followed by addition of 6M NaOH. Acetic acid was added to

this residue and this solution was added to a suspension of Mn(OAc)3 H2O in acetic acid to give

compound 19 in a 27% yield.

The boronic acid pinacol ester 12 was then coupled to 18 using PdCl2(dppf)CH2Cl2 and

2M K2CO3 in 1,4-dioxane giving a yield of 76%. Compound 18, was iodinated using NIS32 to

give 23 in a 61% yield. Compound 18 could also be brominated using NBS33 to give 22 in a 49%

yield.

24

Scheme 6. Reaction conditions: (a) Br2, t-butanol/water, rt, 19h, 63%; (b) Zn, AcOH, rt, 4h,

74%; (c) 1. 1M BH3THF, THF, 0°C to rt, 3h; 2. Mn(OAc)3H2O, AcOH, 75°C, 3h, 27%; (d)

PdCl2(dppf)CH2Cl2, 2M K2CO3, 1,4-dioxane, 85°C, 4h, 76%; (e) NIS, acetone, rt, 2.5h, 61%; (f)

NBS, CH3Cl3, 0°C to 50°C, 2.5h, 49% (g) PdCl2(dppf)CH2Cl2, 2M K2CO3, 1,4-dioxane or THF,

120°C MW, 30min.

A variety of Suzuki coupling conditions were attempted either using 22 (Table 5) or 23 (Table

6) as the starting material to obtain some of the desired analogs. Compounds 23 and 22 were

coupled with their respective boronic acids, using PdCl2(dppf)CH2Cl2, 2M K2CO3, and 1,4-

dioxane, with the exception of analog NEU2070, in which THF was used as a solvent.

25

Entry R1 R2 Base Solvent Temperature,

time

Results

1 H OMe 2M

K2CO3

1,4-dioxane 120 °C MW,

30 minutes

NEU2068 (5%)

2 H CF3 2M

K2CO3


30 minutes

NEU2069 (16%)

3 Cl Cl 2M

K2CO3

THF 120 °C MW,

30 minutes

NEU2070 (10%)

2M

K2CO3


30 minutes

50% 18, 20% 22, 30%

NEU2070 by LC

4

Me H 2M

Na2CO3


30 min NEU2112

(7%)

2M

K2CO3

1,4-dioxane 85 °C,

overnight

60% 18, 40% NEU2112

by LC

5 H F 2M

K2CO3


30 min

NEU2113 (9%)

Table 5. Reaction conditions for the synthesis of NEU2068, NEU2069, NEU2070, NEU2112,

and NEU2113.

26

Entry R1 R2 Base Solvent Temperature,

time

Results

1 H H 2M

K2CO3

1,4-dioxane 120 °C MW, 30

minutes

NEU2065 (16%)

2 Cl H 2M

K2CO3


minutes

NEU2066 (42%)

3 OMe H 2M

K2CO3


minutes

NEU2067 (32%)

4 H OMe 2M

K2CO3


2.5 hours

87% 18, 13% NEU2068

by LC

2M

Na2CO3


1 hour

63% 18, 4% NEU2068,

33% 23 by LC

2M

K2CO3

1,4-dioxane

/water

(2.5:1)

120 °C MW,

2.5 hours

70% 18, 30% NEU2068

by LC

2M

KOAc


1 hour

80% 18, 20% 23 by LC

Table 6. Reaction conditions for the synthesis of NEU2065, NEU2066, NEU2067 and

NEU2068.

Additionally, a variety of conditions were attempted for the synthesis of the remaining

proposed analogs 13, 14, and 16, which used different combinations of solvents, bases, and

catalysts, none which provided significant amounts of desired product. The set of conditions

attempted are depicted in Tables 7-9.

27

Entry Catalyst Base Solvent Temperature,

time

Results

1 PdCl2(dppf)CH2Cl2 2M

K2CO3

1,4-dioxane 85 °C, 2 hours Only 22 by

LC


K2CO3

1,4-

dioxane/water

(2.5:1)

120 °C MW,

30 minutes

Only 22 by

LC


K2CO3

THF 120 °C MW,

30 minutes

40% 22, 40%

18, and 20%

product by LC


K2CO3

Toluene 120 °C MW,

30 minutes

Only 22 by

LC


Na2CO3

ACN 120 °C MW,

30 minutes

Only 22 by

LC


K2CO3

ACN 120 °C MW,

30 minutes

Only 22 by

LC


K2CO3

DMF 120 °C MW,

30 minutes

95% 18, and

5% 22 by LC


Cs2CO3

THF 120 °C MW,

30 minutes

52% 22, 28%

18, and 20%

product by LC


K2CO3


30 minutes

Only 18 by

LC


K3PO4

THF 120 °C MW,

30 minutes

63% 22, 29%

18, and 20%

product by LC

11 PdCl2(dppf) 2M

K2CO3

THF 120 °C MW,

30 minutes

58% 22, 26%

18, and 16%

product by LC

12 Pd(PPh3)4 2M

K2CO3


30 minutes

Only 22 by

LC

13 Pd(PPh3)4 2M

Na2CO3

DME/

EtOH (1.4:1)

120 °C MW,

30 minutes

Only 22 by

LC

14 PdCl2(PPh3)2 2M

K2CO3

THF 120 °C MW,

30 minutes

80% 22, and

20% 18.

Table 7. Reaction conditions for the synthesis of analog 13.

28

Entry Catalyst Base Solvent Results


K2CO3

1,4-dioxane 63% 22, 25% 18, 12% product

by LC


K2CO3

THF 64% 22, 23% 18, 13% 14 by

LC


K2CO3

DMF 53% 22, 38% 18, and 9% 14 by

LC


K2CO3

ACN 69% 22, and 31% 18, by LC


Na2CO3

Toluene 50% 22, and 50% 18 by LC

6 PdCl2(dppf)CH2Cl2 K2CO3 1,4-dioxane 77% 22, 12% 18, 11% 14 by LC


K3PO4

1,4-dioxane 74% 22, 19% 18, 7% 14 by LC


Cs2CO3

1,4-dioxane 66% 22, 21% 18, and 13% 14

by LC


K2CO3

Toluene 50% 22, 50% 18 by LC


K2CO3

DME/EtOH

(1.4:1)

67% 22, 27% 18, and 8% 14 by

LC

11 PdCl2(dppf) 2M

K2CO3

1,4-dioxane 76% 22, 18% 18, and 6% 14 by

LC

12 PdCl2(PPh3)2 2M

K2CO3

1,4-dioxane 80% 22, 20% 18 by LC


29

Entry Solvent Results

1 1,4-dioxane Only 22 by LC

2 DMF Only 22 by LC

3 THF Only 22 by LC

4 ACN Only 22 by LC


Debromination was observed in several of these reactions, which could mean that after

the oxidative addition of palladium, displacement of the halogen by an oxygen source, such as

air, is occurring instead of the transmetalation step. This byproduct then undergoes reductive

elimination to provide the dehalogenated product.

2.2.2 Biological Assay and Discussion of Approach 2

Analogs were screened against T. brucei cells and their potency and ADME data were obtained.

The results are summarized in Table 10.

30

EC50

(μM)

Solubility

(μM)

Human

PPB (%)

HLM

CLint(μl/min/mg)

LogD7.4

GSK2280392A

0.09 12 96 6.92 3.8

NEU2065

0.26 20 98 199 3.5

NEU2066

1.18 0.8 >96 56.6 >3.7

NEU2067

0.17 5 99 181 4

NEU2068

0.21 0.18 99 >300 3.8

NEU2069

0.90 1 >9 66.3 >4.2

NEU2070

0.95 No data >9 50.9 >3.8

NEU2112

1.25 7 100 125 >4.1

NEU2113

0.08 3 99 108 4.1

Table 10. Inhibition and in vitro ADME data of GSK2280392A analogs.

31

When looking at the results obtained, analogs NEU2066, NEU2069, NEU2070, and

NEU2112 showed a rather large decrease in potency relative to GSK2280392A ranging from 10

to 14 fold, and a slight improvement in potency with NEU2113, suggesting the need for a more

electronegative substituent at the meta position. In the case of methoxybenze as a substituent,

NEU2067 showed a decrease in potency by 2 fold and NEU2068 showed a decrease in potency

by 3 fold, suggesting that the para position could be slightly more favorable for this particular

substituent. Additionally, the unsubstituted compound, NEU2065, showed a decrease in potency

by 3 fold, suggesting the need of a substituent on this ring. However, these results do not provide

enough information on the structure-activity relationship of these series of analogs and further

studies are needed to provide a definitive conclusion.

In terms of metabolic stability, all analogs showed high clearance in microsomes in vitro.

The analogs with substituents para to the azaindole ring had high clearance rates, with NEU2112

having highest clearance rate, followed by NEU2067, NEU2066, and GSK2280392A suggesting

that clearance may not be due to oxidation at this position but rather to another metabolic

pathway, such as oxidation of the endocyclic nitrogens, or demethylation at the pyrazole ring.

Data for compounds 14 and 15 need to be obtained to have a better understanding of the possible

metabolic pathway.

Moreover, these analogs did not indicate an improvement in terms of solubility or a

significant change in the cLogD value from GSK2280392A. The low solubility in these new

compounds suggests that the compounds may also have low absorption. This data suggests that

GSK2280392A remains to be the best compound from a pharmacokinetic and parasite inhibition

point of view.

32

2.3 Future Studies

2.3.1 Proposal of Additional Analogs

To further investigate the role of the compound’s conformation on inhibition of the parasite, the

2,4-series of the GSK2280392A analogs synthesized previously (Section 2.2.1) can be proposed.

Given that the synthesis of analogs with the 2,4-regiochemistry is straightforward and modest

yields can be obtained (as seen by the analogs synthesized (Section 2.1.1)), more exploration can

be done around this regiochemistry to study the effect of these analogs as antiparasitic agents.

The specific target of action, and the active site of this target are not known and more extensive

research needs to be done in order to understand the mechanism of action of the parasite’s

inhibitors.

To explore the metabolism of GSK2280392A, compound 24 can be synthesized to

explore demethylation at the pyrazole ring as a possible metabolic pathway. Also, compound 25

can be synthesized to investigate oxygenation of the endocyclic nitrogens as a possible metabolic

pathway. The addition of the methyl group at the ortho position could hinder the nitrogen from

possible oxidation (Figure 13).

Figure 13. Proposed analogs to explore metabolism of GSK2280392A.

To address the solubility of GSK2280392A, compound 26 can be proposed along with

compound 16 which would be charged at the physiological pH, improving the solubility.

33

Compound 25 can also be explored since the methyl at the ortho position can increase the torsion

angles in the molecule, resulting in the disruption of the crystal packing to form a higher energy

crystal that is more soluble. A phosphate ester prodrug (27) could also serve as another

alternative for these studies, since prodrugs are well established strategies to address solubility

problems34,35 (Figure 14).

Figure 14. Proposed analogs to improve solubility of GSK2280392A.

To further explore the structure activity relationship, the meta analog 28 of NEU2066 can

be synthesized along with the para analogs 29, and 30 of NEU2069, and NEU2113,

respectively, to investigate the importance of substituents at the meta or para positions in that

ring. Additionally compound 31 can aid in understanding the importance of the pyrazole moiety

in the overall structure of the compound (Figure 15).

34

Figure 15. Proposed analogs to explore the structure-activity relationships of GSK2280392A.

2.3.2 Progress Toward the Synthesis of GSK2280392A Analogs

The synthesis of the analogs 13, 14, 15, and 16 needs to be finalized. In order to synthesize

analog 15, the same synthetic pathway can be followed from Scheme 6. For the synthesis of

analog 13 and 14, different synthetic routes can be attempted (Scheme 7); compound 22 can be

tosylated using TsCl, TEA and DMAP to make compound 32. Bis(pinacolato)diboron (33) can

then be added to compound 32 using PdCl2(dppf)CH2Cl2, 2M KOAc, and 1,4-dioxane to make

compound 34. Compound 35 and 36 can then be reacted with 34 using PdCl2(dppf)CH2Cl2, 2M

K2CO3, and 1,4-dioxane to give compound 37 and 38, respectively. Compounds 37 and 38 can

then be deprotected using 2M NaOH, and 1,4-dioxane to give compound 15 and 13, respectively.

35

Scheme 7. Reaction conditions: (a) TsCl, TEA, DMAP, DCM, rt, overnight; (b)

PdCl2(dppf)CH2Cl2, 2M KOAc, 1,4-dioxane, 120°C MW, 30 min; (c) PdCl2(dppf)CH2Cl2, 2M

K2CO3, 1,4-dioxane, 120°C MW, 30 min; (d) 2M NaOH, 1,4-dioxane, 110°C, 3h.

For the synthesis of compound 16, different catalysts and bases can be used to attempt the

Suzuki coupling, or the 3-carboxylicacid boronic acid (39) can be protected using TMS-

diazomethane in hexanes to give compound 40 (Scheme 8).

36

Scheme 8. Reaction conditions: (a) Toluene/MeOH, rt, overnight.

Compound 40 can then be coupled to 22 using PdCl2(dppf)CH2Cl2, 2M K2CO3, and 1,4-

dioxane to give compound 41 which can then be deprotected using KOH and MeOH36 to give

compound 16 (Scheme 9).

Scheme 9. Reaction conditions: (a) PdCl2(dppf)CH2Cl2, 2M K2CO3, 1,4-dioxane, 120°C MW,

30 min; (b) KOH, MeOH, reflux, 90 min.

Inhibition and ADME data of these analogs will then be obtained to determine their

activity against the parasite as well as their pharmacokinetics to directly compare with

GSK2280392A.

2.4 Conclusion

Various analogs were designed and synthesized from the HTS cluster 34 in search for new lead

compounds for the treatment of HAT. The hit compounds were optimized to improve potency,

while maintaining their physiochemical properties. First, the regiochemistry of the analogs was

changed from a 3,5 series to a 2,4 series, results suggested that the specific regiochemistry is not

37

essential for inhibition. Second, the microsomal stability of the analogs of GSK2280392A was

studied in order to investigate the high clearance of this compound. Though initially thought that

the high clearance could be due to a metabolic oxidative pathway on the cyanophenyl region of

the molecule, our results now suggest otherwise. Though none of the analogs synthesized

showed an improvement in potency, they provided a better understanding of the structure-

activity relationships and likely regions of metabolism leading to rapid in vivo clearance. More

analogs have been proposed for future studies in order to further investigate the role of the

regiochemistry in activity against the parasite and the causes of high clearance in the compound.

38

Chapter 3

Experimental

3.1 General Methods:

Reagents purchased were used as received, unless otherwise noted. Microwave reactions were

run using the Biotage® Initiator Eight automated microwave. Purification of intermediates was

performed using silica gel chromatography on 230-400 mesh silica gel for glass columns, or

using the Biotage® Isolera™One flash purification system. LC-MS analysis was performed

using a Waters Alliance reverse-phase HPLC using a single wavelength UV-visible detector at

254 nm and multi-wavelength photodiode array detector from 210 nm to 600 nm. Preparative

LC-MS was conducted for final compounds on Waters FractionLynx system using

acetonitrile/water and 0.1% formic acid gradient and collected based on UV monitoring at 254

nm. All final compounds submitted for biological testing were determined to be ≥95% pure by

LC-MS. H1 NMR spectra were obtained with Varian NMR systems, operating at 400 MHz or

500 MHz at room temperature. Chemical shifts (δ) are reported in parts per million (ppm) and

are relative to tetramethylsilane (δ=0). Data for H1 NMR spectra are reported as follows:

chemical shift (ppm), multiplicity (s for singlet, d for doublet, t for triplet, dd for doublet of

doublet, m for multiplet), coupling constant (Hz), and integration.

39

3.2 Experimental Details

1H-pyrrolo[2,3-b]pyridine 7-oxide (5)

A mixture of dimethoxyethane and hexane (23 mL, 0.385 molar, 1:2 ratio) was added to 7-

azaindole (1.5 g, 12.70 mmol, 1 eq). This solution was cooled to 0 °C and 3-chlorobenzoperoxic

acid (3.51 g, 15.24 mmol, 1.2 eq) was added portion wise. The reaction was let to warm up to

room temperature and stirred for 26 hours. The reaction mixture was then filtered and washed

with dimethoxyethane and hexane (10 mL, 1:2 ratio) to afford 1H-pyrrolo[2,3-b]pyridine 7-

oxide, 3-chlorobenzoate salt as a white solid (3.05 g, 83% yield). 1H NMR (399 MHz, DMSO-

d6) δ ppm 6.56 (d, J=1.5 Hz, 1 H) 7.01 – 7.07 (m, 1 H) 7.43 (br. s., 1 H) 7.49 – 7.56 (m, 1 H)

7.62 (d, J=8.1 Hz, 1 H) 7.69 (d, J=8.1 Hz, 1 H) 7.85 – 7.91 (m, 2 H) 8.10 (d, J=6.6 Hz, 1 H)

12.43 (br. s., 1 H).

4-chloro-1H-pyrrolo[2,3-b]pyridine (4)

Phosphoryl trichloride (1.75 mL, 18.73 mmol, 4.5 eq) was added to 1H-pyrrolo[2,3-b]pyridine 7-

oxide, 3-chlorobenzoate salt (1.64 g, 5.67 mmol, 1 eq) and the reaction was heated to 85 °C and

run overnight. The reaction mixture was then cooled to room temperature, quenched with sodium

bicarbonate and extracted with dichloromethane. The organic layers were washed with brine,

40

dried over sodium sulfate, and concentrated to give crude product. The crude product was

purified using a glass column (ethyl acetate/hexane with a 50-80% gradient of ethyl acetate) to

afford 4-chloro-1H-pyrrolo[2,3-b]pyridine as a tan solid (0.624 g, 72.1% yield). 1H NMR (399

MHz, DMSO-d6) δ ppm 6.48 (br. s., 1 H) 7.17 (d, J=5.1 Hz, 1 H) 7.57 (br. s., 1 H) 8.15 (d, J=5.1

Hz, 1 H) 12.03 (br. s., 1 H). LCMS found 152.9, [M + H]+

4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (6)

Dichloromethane (50.3 mL, 0.082 molar) was added to 4-chloro-1H-pyrrolo[2,3-b]pyridine

(0.629 g, 4.12 mmol, 1 eq), followed by triethylamine (1.293 mL, 9.28 mmol), N,N-

dimethylpyridin-4-amine (0.151 g, 1.237 mmol) and 4-methylbenzene-1-sulfonyl chloride (1.297

g, 6.80 mmol). The reaction was ran overnight at room temperature and then extracted with 1N

HCl, saturated sodium bicarbonate, and brine. The organic layers were collected, dried over

sodium sulfate, and concentrated to give crude product. The crude product was purified using a

glass column (ethyl acetate/hexane with a 10-50% gradient of ethyl acetate) to afford 4-chloro-1-

tosyl-1H-pyrrolo[2,3-b]pyridine (1.00 g, 79% yield). 1H NMR (399 MHz, chloroform-d) δ ppm

2.38 (s, 3 H) 6.69 – 6.73 (m, 1 H) 7.20 (d, J=5.9 Hz, 1 H) 7.29 (d, J=8.1 Hz, 2 H) 7.78 (d, J=3.7

Hz, 1 H) 8.05 – 8.10 (m, 2 H) 8.32 (d, J=5.1 Hz, 1 H). LCMS found 306.9, [M + H]+

41

4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3)

Diisopropylamine (0.205 mL, 1.441 mmol, 2 eq) was added to tetrahydrofuran (10 mL, 0.051

molar) and this was cooled to -70 °C. n-Butyl lithium (1.201 mL, 1.441 mmol, 2 eq) was then

added dropwise and the solution was stirred for an hour. Separately, THF (4 mL, 0.051molar)

was added to 4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (200 mg, 0.652 mmol, 0.905 eq) and

this was cooled to -70 °C. To this solution, tetramethlyethylenediamine (0.098 mL, 0.652 mmol,

0.905 eq) was added followed by the lithium diisopropylamide solution prepared above. The

solution was stirred for an hour and then iodine (201 mg, 0.792 mmol, 1.1 eq) in THF (1mL) was

added dropwise. The reaction mixture was quenched with ammonium chloride and extracted

with ethyl acetate. The organic layer was collected and washed with brine, dried over sodium

sulfate, filtered and concentrated to give crude product (305 mg, 98% yield). 1H NMR (399

MHz, DMSO-d6) δ ppm 2.32 (s, 3 H) 7.23 – 7.26 (m, 1 H) 7.38 – 7.44 (m, 3 H) 7.92 (d, J=8.1

Hz, 2 H) 8.26 (d, J=5.1 Hz, 1 H). LCMS found 432.9, [M + H]+

42

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (8)

4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (51.4 mg, 0.119 mmol, 1 eq), (4-

hydroxyphenyl)boronic acid (18.02 mg, 0.131 mmol, 1.1 eq), and tetrakis (triphenylphosphine)

palladium(o) (6.86 mg, 5.94 µmol, 0.05 eq) were added to a vial and this was then sealed and

purged with nitrogen three times. Dimethoxyethane (1.5 mL, 0.046 molar), ethanol (1.08 mL,

0.046 molar) and a 2 molar solution of sodium carbonate (0.356 mL, 0.713 mmol, 6 eq) were

then added and this was degassed. The reaction mixture was heated to 85 °C and stirred for 4

hours. The reaction mixture was cooled down, concentrated, and extracted with ethyl acetate.

The organic layers were washed with brine, dried over sodium sulfate and concentrated to give

crude product. The crude product was purified using a glass column (ethyl acetate/hexane with a

30%-60% gradient of ethyl acetate) to afford 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenol as a white solid (23 mg, 64% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 2.34 (s,

3 H) 5.98 (br. s., 1 H) 6.53 – 6.58 (m, 1 H) 6.90 – 6.98 (m, 2 H) 7.17 (d, J=8.1 Hz, 2 H) 7.19 –

7.23 (m, 1 H) 7.39 – 7.45 (m, 2 H) 7.68 – 7.74 (m, 2 H) 8.32 – 8.37 (m, 1 H). LCMS found

398.9, [M + H]+

43

4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (NEU1935)

4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (32.0 mg, 0.080 mmol, 1 eq) was

added to a microwave vial followed by (4-(methylsulfonyl)phenyl)boronic acid (17.65 mg, 0.088

mmol, 1.1 eq) and Tetrakis (triphenylphosphine) palladium(o) (4.64 mg, 4.01 µmol, 0. 05eq).

The vial was sealed and purged with nitrogen three times. 1,4-dioxane (1.744 mL, 0.046 molar)

and a 2 molar solution of sodium carbonate (0.241 mL, 0.481 mmol, 6 eq) were then added and

this was degassed. The reaction was ran in the microwave for 2 hours at 120 °C. The reaction

was then concentrated, and ethyl acetate was added. This solution was then washed with water,

followed by brine. The organic layer was collected, dried over sodium sulfate and concentrated

to give crude product. The crude product was purified on the FractionLynx system (30-70%

ACN/water gradient) to afford 4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-pyrrolo[2,3-

b]pyridin-2-yl)phenol as a white solid (24.5 mg, 60.5% yield). 1H NMR (399 MHz, DMSO-d6) δ

ppm 2.30 (s, 3 H) 3.29 (s, 3 H) 6.80 (s, 1 H) 6.85 (d, J=8.8 Hz, 2 H) 7.34 (d, J=8.1 Hz, 2 H) 7.39

(d, J=8.1 Hz, 2 H) 7.48 (d, J=5.1 Hz, 1 H) 7.72 (d, J=8.1 Hz, 2 H) 7.93 – 7.99 (m, 2 H) 8.01 –

8.06 (m, 2 H) 8.45 (d, J=5.1 Hz, 1 H) 9.83 – 10.03 (br. s., 1 H). LCMS found 518.9, [M + H]+

44

4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (NEU1936)

1,4-Dioxane (2.4 mL, 0.037 molar) was added to 4-(4-(4-(methylsulfonyl)phenyl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)phenol (46.0 mg, 0.089 mmol, 1 eq) followed by a solution of 2 molar

sodium hydroxide (0.118 mL, 0.237 mmol, 2.67 eq ). This reaction mixture was heated to 110 °C

and stirred for 4 hours. The reaction mixture was cooled down and extracted with ethyl acetate.

The orrganic layers were washed with brine, dried over sodium sulfate and concentrated to give

crude product. The crude product was purified on the FractionLynx system (30-70% ACN/water

gradient) to afford 4-(4-(4-(methylsulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)phenol as a

yellow solid (11 mg, 34% yield). 1H NMR (399 MHz, DMSO-d6) δ ppm 3.29 (s, 3 H) 6.83 (d,

J=8.8 Hz, 2 H) 6.95 (s, 1 H) 7.23 (d, J=5.1 Hz, 1 H) 7.80 (d, J=8.8 Hz, 2 H) 8.08 (s, 4 H) 8.25

(br. s., 1 H) 9.79 (br. s., 1 H) 12.21 (br. s., 1 H). LCMS found 365.0, [M + H]+

45

3-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile (11)

4-chloro-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (74.3 mg, 0.172 mmol, 1 eq), (3-

cyanophenyl)boronic acid (18.02 mg, 0.131 mmol, 1.1 eq), and Tetrakis (triphenylphosphine)

palladium(o) (9.92 mg, 8.59 µmol, 0.05 eq) were added to a vial. This was then sealed and

purged with nitrogen three times. Dimethoxyethane (2.2 mL, 0.046 molar), ethanol (1.5 mL,

0.046 molar), and a 2 molar solution of sodium carbonate (0.515 mL, 1.030 mmol, 6 eq) were

then added and this was degassed. The reaction mixture was heated to 85 °C and stirred for 4

hours. The reaction mixture was cooled down, concentrated, and extracted with ethyl acetate.

The organic layers were washed with brine, dried over sodium sulfate and concentrated to give

crude product. The crude product was purified using a glass column (ethyl acetate/hexane with a

20%-50% gradient of ethyl acetate) to afford 4-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-

yl)phenol (25.4 mg, 41.4% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 2.38 (s, 3 H) 6.69

(d, J=1.5 Hz, 1 H) 7.21 – 7.26 (m, 3 H) 7.58 – 7.65 (m, 1 H) 7.76 (s, 1 H) 7.78 (d, J=6.6 Hz, 3 H)

7.85 (dd, J=8.1, 1.5 Hz, 1 H) 8.41 (dd, J=5.1, 1.5 Hz, 1 H). LCMS found 407.9, [M + H]+

46

3-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile

(NEU1937)

3-(4-chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile (34 mg, 0.087 mmol, 1 eq), 1-

methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (19.8 mg, 0.095 mmol, 1.1

eq), and Tetrakis (triphenylphosphine) palladium(o) (5.01 mg, 4.34 µmol, 0.05 eq) were added to

a vial. This was then sealed and purged with nitrogen three times. Dimethoxyethane (1.057 mL,

0.048 molar), ethanol (0.755 mL, 0.048 molar), and a 2 molar solution of sodium carbonate

(0.260 mL, 0.521 mmol, 6 eq) were then added and this was degassed. The reaction mixture was

heated to 85 °C and stirred for 4 hours. The reaction mixture was cooled down, concentrated, and

extracted with ethyl acetate. The organic layers were washed with brine, dried over sodium

sulfate and concentrated to give crude product. The crude product was purified using a glass

column (ethyl acetate/hexane with a 20%-50% gradient of ethyl acetate) to afford 3-(4-(1-

methyl-1H-pyrazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile as a dark brown

solid(24 mg, 61% yield). 1H NMR (500 MHz, chloroform-d) δ ppm 2.36 (s, 3 H) 3.99 (s, 3 H)

6.76 (s, 1 H) 7.22 (d, J=8.3 Hz, 2 H) 7.26 (d, J=5.4 Hz, 1 H) 7.57 – 7.63 (m, 1 H) 7.76 (d, J=7.8

Hz, 1 H) 7.79 (d, J=5.4 Hz, 2 H) 7.81 (d, J=3.4 Hz, 2 H) 7.85 – 7.90 (m, 2 H) 8.46 (d, J=5.4 Hz,

1 H). LCMS found 454.0, [M + H]+

47

3-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile (NEU1938)

1,4-Dioxane (0.89 mL, 0.037 molar) was added to 3-(4-(1-methyl-1H-pyrazol-4-yl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-2-yl)benzonitrile(15.0 mg, 0.033 mmol, 1 eq) followed by a solution of 2

molar sodium hydroxide (0.044 mL, 0.088 mmol, 2.67 eq ). This was heated to 100 °C and

stirred for 3 hours. The reaction mixture was cooled down and extracted with ethyl acetate. The

organic layers were washed with brine, dried over sodium sulfate and concentrated to give crude

product. The crude product was purified on the FractionLynx system (30-70% ACN/water

gradient) to afford 3-(4-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-2-yl)benzonitrile

as a pale yellow solid (2.1 mg, 21.3% yield). 1H NMR (399 MHz, DMSO-d6) δ ppm 3.97 (s, 3 H)

7.31 (d, J=5.1 Hz, 1 H) 7.57 (d, J=1.5 Hz, 1 H) 7.66 – 7.73 (m, 1 H) 7.80 (d, J=8.1 Hz, 1 H) 8.20

(d, J=5.1 Hz, 1 H) 8.26 (s, 1 H) 8.36 (d, J=8.1 Hz, 1 H) 8.58 (d, J=11.7 Hz, 2 H) 12.31 (s, 1 H).

LCMS found 300.0, [M + H]+

3,3,5-tribromo-1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one (20)

1H-pyrrolo[2,3-b]pyridine (100 mg, 0.846 mmol) was dissolved in t-butanol(6.61 mL, 0.064

molar) and water (6.61 mL, 0.064 molar). Bromine (0.540 mL, 10.48 mmol, 12.38 eq) was then

added dropwise and the reaction was stirred for 19 hours at room temperature. The reaction

48

mixture was concentrated and then saturated sodium bicarbonate was added until reaction

mixture reached a pH of 9. The reaction mixture was extracted with ethyl acetate and the organic

layers were collected, washed with brine and dried over sodium sulfate to afford crude product as

a dark brown solid (0.197g, 62.7 % yield). 1H NMR (399 MHz, chloroform-d) δ ppm 8.00 (d,

J=1.5 Hz, 1 H) 8.34 (br. s., 1 H) 10.16 (br. s., 1 H). LCMS found 368.9, [M + H]+

5-bromo-1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one (21)

Zinc (63.3 mg, 0.969 mmol, 9.98 eq) was added to a solution of 3,3,5-tribromo-1H-pyrrolo[2,3-

b]pyridin-2(3H)-one (36.0 mg, 0.097 mmol, 1 eq) in acetic acid (0.719 mL, 0.135 molar) and this

was stirred at room temperature for 4 hours. The reaction mixture was concentrated and water

was added to residue and extracted with ethyl acetate. The organic layers were collected, washed

with brine and concentrated to give crude product. The crude product was purified using a glass

column (methanol/dichloromethane with a 0-5% gradient of methanol) to afford 5-bromo-1,3-

dihydro-2H-pyrrolo[2,3-b]pyridin-2-one (15.2 mg, 73.5 %). 1H NMR (399 MHz, DMSO-d6) δ

ppm 3.59 (s, 2 H) 7.78 (s, 1 H) 8.16 (d, J=2.2 Hz, 1 H) 11.15 (br. s., 1 H). LCMS found 212.9,

[M + H]+

49

5-bromo-1H-pyrrolo[2,3-b]pyridine (19)

Tetrahydrofuran (6.12 mL, 0.536 molar) was added to 5-bromo-1,3-dihydro-2H-pyrrolo[2,3-

b]pyridin-2-one (245mg, 1.149 mmol, 0.250 eq) and this was cooled to 0 °C. A solution of 1

molar borane in tetrahydrofuran (4.60 mL, 4.60 mmol, 1 eq) was then added drop wise. The

reaction mixture was let to warm up to room temperature and stirred for 3 hours. The solvent was

removed under pressure and the residue was diluted with a solution of 6N HCl and heated until

complete dissolution of the solid. After cooling, the mixture was treated with 6M sodium

hydroxide to reach a pH=9 and this solution was extracted with ethyl acetate. The organic layers

were collected, washed with brine, dried over sodium sulfate and concentrated. The residue was

dissolved in acetic acid (2.45 mL) and the resulting solution was added to a suspension of

Mn(OAc)3 H2O (0.501g, 1.87 mmol, 0.407 eq) in acetic acid (2.45 mL). The reaction mixture

was heated to 75 °C and stirred for 3 hours. Toluene was then added and the solution was

concentrated. The residue was diluted with water and extracted with ethyl acetate. The organic

layers were collected, washed with brine, dried over sodium sulfate and concentrated to give

crude product. The crude product was purified using a glass column (ethyl acetate/hexanes with

10-50% gradient of ethyl acetate) to afford 5-bromo-1H-pyrrolo[2,3-b]pyridine as a light pink

solid (60 mg, 27.3% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 6.48 (d, J= 2.9 Hz, 1 H)

7.37 – 7.43 (m, 1 H) 8.11 (d, J=1.5 Hz, 1 H) 8.38 (d, J=2.2 Hz, 1 H) 11.03 (br. s., 1H). LCMS

found 198.9, [M + H]+

50

5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (18)

5-bromo-1H-pyrrolo[2,3-b]pyridine (552 mg, 2.82 mmol, 1 eq) was added to a round bottom

flask followed by 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

(0.644g, 3.09 mmol, 1.098 eq), and [1,1’-bis(diphenylphoshpino)ferrocene]

dichloropalladium(II) dichloromethane adduct (231 mg, 0.282 mmol, 0.101 eq). The round

bottom flask was sealed and purged with nitrogen three times. 1,4-dioxane (17.7 mL, 0.159

molar) and 2 molar solution of potassium carbonate (8.89 mL, 17.77 mmol, 6.31 eq) were then

added and this was degassed. The reaction mixture was heated to 85 °C and stirred for 4 hours.

The reaction was cooled down, concentrated, and then ethyl acetate was added. This solution

was then washed with water, followed by brine. The organic layer was collected, dried over

sodium sulfate and concentrated to give crude product. The crude product was purified using the

Biotage Isolera (ethyl acetate/hexanes with a 90-100% gradient of ethyl acetate) to afford 5-(1-

methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine as a light tan solid (0.427 g, 76% yield). 1H

NMR (399 MHz, chloroform-d) δ ppm 3.89 – 4.09 (m, 3H) 6.53 (d, J=3.7 Hz, 1 H) 7.36 (d,

J=3.7 Hz, 1H) 7.66 (s, 1 H) 7.80 (s, 1 H) 8.04 (s, 1H) 8.50 (br. s., 1 H) 9.43 (br. s. 1 H). LCMS

found 198.9, [M + H]+

51

3-iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (23)

Acetone (8.40 mL, 0.213 molar) was added to 5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-

b]pyridine (355 mg, 1.791 mmol, 1 eq) followed by N-iodosuccinimide (443 mg, 1.970 mmol,

1.1 eq) and this was stirred for 2.5 hours at room temperature. The reaction mixture was filtered

and precipitate was washed with acetone to afford 3-iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-

pyrrolo[2,3-b]pyridine as a crude product (0.353 g, 60.7 % yield). 1H NMR (399 MHz, DMSO-

d6) δ ppm 3.88 (s, 3 H) 7.67 – 7.71 (m, 1 H) 7.77 (s, 1 H) 7.95 (s, 1 H) 8.25 (s, 1 H) 8.51 (s, 1 H)

12.04 – 12.08 (m, 1 H). LCMS found 324.9, [M + H]+

3-bromo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (22)

Chloroform (10.21 mL, 0.211 molar) was added to 5-(1-methyl-1H-pyrazol-4-yl)-1H-

pyrrolo[2,3-b]pyridine (0.427 g, 2.154 mmol, 1 eq) and this solution was cooled to 0 °C. N-

bromosuccinimide was then added portion wise (0.533 g, 3 mmol, 1.392 eq) and the reaction

mixture was stirred for 30 minutes in the dark. The mixture was then heated to 50 °C and stirred

for 2 hours. Upon reaction completion, the mixture was cooled to room temperature. A solution

of 1 molar potassium carbonate was poured in the reaction mixture and this was extracted with

ethyl acetate. The organic layers were washed with brine, dried over sodium sulfate and

52

concentrated to give crude product. The crude product was purified using the Biotage Isolera

(ethyl acetate/hexanes with a 70-90% gradient of ethyl acetate) to afford 3-bromo-5-(1-methyl-

1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine as a light orange solid (0.291 g, 48.7% yield). 1H

NMR (399 MHz, DMSO-d6) δ ppm 3.84 (s, 3H) 7.66 (d, J=2.2 Hz, 1 H) 7.91 (s, 1 H) 7.93 (s, 1

H) 8.22 (s, 1 H) 8.52 (d, J=2.2 Hz, 1 H) 12.00 (br. s., 1 H). LCMS found 276.9, [M + H]+

5-(1-methyl-1H-pyrazol-4-yl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine (NEU2065)

In a microwave tube, 3-iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (37.3 mg,

0.115 mmol, 1 eq) was added followed by phenylboronic acid (41.5 mg, 0.340 mmol, 2.96 eq)

and [1,1’-bis(diphenylphoshpino)ferrocene] dichloropalladium(II) dichloromethane adduct

(12.70 mg, 0.016 mmol, 0.135 eq). The tube was sealed and purged with nitrogen three times.

1,4-Dioxane (0.115 mL, 1 molar), and a 2 molar solution of potassium carbonate (0.517 mL,

1.035 mmol, 9 eq) were then added and the reaction mixture was degassed and ran in the

microwave for 30 minutes at 120 °C. The reaction was then concentrated, and ethyl acetate was

added. This solution was then washed with water, followed by brine. The organic layer was

collected, dried over sodium sulfate and concentrated to give crude product. The crude product

was purified on the FractionLynx system (30-70% ACN/water gradient) to afford 5-(1-methyl-

1H-pyrazol-4-yl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine as a white solid (5 mg, 15.8% yield). 1H

NMR (399 MHz, chloroform-d) δ ppm 3.99 (s, 3 H) 7.30 – 7.38 (m, 1 H) 7.45 – 7.55 (m, 3 H)

7.63 – 7.71 (m, 3 H) 7.82 (s, 1 H) 8.28 (s, 1 H) 8.52 (s, 1 H) 9.49 (br. s., 1 H). LCMS found

53

275.1, [M + H]+

3-(4-chlorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU2066)

In a microwave tube, 3-iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (25 mg,

0.077 mmol, 1 eq) was added followed by (4-chlorophenyl)boronic acid (35.6 mg, 0.228 mmol,

2.96eq) and [1,1’-bis(diphenylphoshpino)ferrocene] dichloropalladium(II) dichloromethane

adduct (8.51 mg, 10.42 μmol, 0.135 eq). The tube was sealed and purged with nitrogen three

times. 1,4-Dioxane (77 μL, 1 molar), and a 2 molar solution of potassium carbonate (0.347 mL,

0.695 mmol, 9.01 eq) were then added and the reaction mixture was degassed and ran in the




was purified on the FractionLynx system (30-70% ACN/water gradient) to afford 3-(4-

chlorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine as a white solid (10 mg,

42.0% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 4.00 (s, 3 H) 7.43 – 7.48 (m, 2 H) 7.50

(d, J=1.5 Hz, 1 H) 7.56 – 7.61 (m, 2 H) 7.68 (s, 1 H) 7.82 (s, 1 H) 8.21 (d, J= 1.5 Hz, 1 H) 8.52

(s, 1 H) 9.17 (br. s., 1 H). LCMS found 309.1, [M + H]+

54

3-(4-methoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU2067)

In a microwave tube, 3-iodo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (21.30

mg, 0.066 mmol, 1 eq) was added followed by (4-methoxyphenyl)boronic acid (28.5 mg, 0.188

mmol, 2.85 eq) and [1,1’-bis(diphenylphoshpino)ferrocene] dichloropalladium(II)

dichloromethane adduct (8 mg, 9.8 μmol, 0.149 eq). The tube was sealed and purged with

nitrogen three times. 1,4-Dioxane (65.7 μL, 1 molar), and a 2 molar solution of potassium

carbonate (0.284 mL, 0.567 mmol, 8.63 eq) were then added and the reaction mixture was

degassed and ran in the microwave for 30 minutes at 120 °C. The reaction was then

concentrated, and ethyl acetate was added. This solution was then washed with water, followed

by brine. The organic layer was collected, dried over sodium sulfate and concentrated to give


gradient) to afford 3-(4-methoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-

b]pyridine as a white solid (6.4 mg, 32% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 3.89

(s, 3 H) 3.99 (s, 3 H) 7.05(d, J=8.8 Hz, 2 H) 7.44 (s, 1 H) 7.58 (d, J=8.1 Hz, 2 H) 7.68 (s, 1 H)

7.81 (s, 1 H) 8.25 (s, 1 H) 8.49 (br. s., 1 H) 9. 17 (br. s., 1 H). LCMS found 305.2, [M + H]+

55

3-(3-methoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU2068)

In a microwave tube, 3-bromo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (30 mg,

0.108 mmol, 1 eq) was added followed by (3-methoxyphenyl)boronic acid (28.1 mg, 0.185


dichloromethane adduct (9.02 mg, 0.011 mmol, 0.102 eq). The tube was sealed and purged with

nitrogen three times. 1,4-Dioxane (0.752 mL, 0.144 molar), and a 2 molar solution of potassium






gradient) to afford 3-(3-methoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-

b]pyridine as a white solid (4.6 mg, 14% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 3.87

– 3.95 (m, 3 H) 3.96 – 4.03 (m, 3 H) 6.90 (d, J=8.8 Hz, 1 H) 7.19 (d, J=1.5 Hz, 1 H) 7.25 (s, 1 H)

7.42 (t, J=8.1 Hz, 1 H) 7.53 (s, 1 H) 7.68 (s, 1 H) 7.81 (s, 1 H) 8.30 (s, 1 H) 8.50 (s, 1 H) 9.60

(br. s., 1 H). LCMS found 305.1, [M + H]+

56

5-(1-methyl-1H-pyrazol-4-yl)-3-(4-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine

(NEU2069)

In a microwave tube, 3-bromo-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (25.3

mg, 0.091 mmol, 1 eq) was added followed by (3-trifluoromethylphenyl)boronic acid (20 mg,

0.105 mmol, 1.153 eq) and [1,1’-bis(diphenylphoshpino)ferrocene] dichloropalladium(II)

dichloromethane adduct (8.9 mg, 10.9 μmol, 0.119 eq). The tube was sealed and purged with







gradient) to afford 5-(1-methyl-1H-pyrazol-4-yl)-3-(4-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-

b]pyridine as a white solid (4.9 mg, 15.7% yield). 1H NMR (399 MHz, chloroform-d) δ ppm

4.01 (s, 3 H) 7.56 – 7.64 (m, 3 H) 7.69 (br. s., 1 H) 7.81 – 7.86 (m, 2 H) 7.88 (s, 1 H) 8.25 (s, 1

H) 8.52- 8.63 (m, 1 H) 9.50 – 9.63 (m, 1 H). LCMS found 343.1, [M + H]+

57

3-(3,4-dichlorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (2070)


0.090 mmol, 1 eq) was added followed by (3,4-dichlorophenyl)boronic acid (19.7 mg, 0.103


dichloromethane adduct (7.51 mg, 9.2 μmol, 0.102 eq). The tube was sealed and purged with

nitrogen three times. Tetrahydrofuran (0.626 mL, 0.144 molar), and a 2 molar solution of

potassium carbonate (0.313 mL, 0.625 mmol, 6.93 eq) were then added and the reaction mixture

was degassed and ran in the microwave for 30 minutes at 120 °C. The reaction was then




gradient) to afford 3-(3,4-dichlorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-

b]pyridine as a white solid (3.2 mg, 10.3% yield). 1H NMR (399 MHz, chloroform-d) δ ppm

4.01 (d, J=1.47 Hz, 3 H) 7.45 - 7.50 (m, 1 H) 7.53 – 7.59 (m, 2 H) 7.71 (d, J= 5.86 Hz, 2 H) 7.82

(s, 1 H) 8.19 (s, 1 H) 8.28 (s, 1 H) 8.46 (br. s., 1 H). LCMS found 343.0, [M + H]+

58

5-(1-methyl-1H-pyrazol-4-yl)-3-(p-tolyl)-1H-pyrrolo[2,3-b]pyridine (NEU2112)


0.253 mmol, 1 eq) was added followed by p-tolylboronic acid (37 mg, 0.275 mmol, 1.089 eq)

and [1,1’-bis(diphenylphoshpino)ferrocene] dichloropalladium(II) dichloromethane adduct (21

mg, 0.026 mmol, 0.102 eq). The tube was sealed and purged with nitrogen three times. 1,4-

Dioxane (1.754 mL, 0.144 molar), and a 2 molar solution of potassium carbonate (0.875 mL,

1.750 mmol, 6.93 eq) were then added and the reaction mixture was degassed and ran in the




was purified on the FractionLynx system (30-70% ACN/water gradient) to afford 5-(1-methyl-

1H-pyrazol-4-yl)-3-(p-tolyl)-1H-pyrrolo[2,3-b]pyridine as a white solid (5.1 mg, 7% yield). 1H

NMR (399 MHz, chloroform-d, D2O) δ ppm 2.36 (s, 3 H) 3.93 (s, 3 H) 7.24 (d, J=8.1 Hz, 2 H)

7.43 (s, 1 H) 7.48 (d, J=8.1 Hz, 2 H) 7.61 (s, 1 H) 7.74 (s, 1 H) 8.23 (s, 1 H) 8.41 (br. s., 1 H).

LCMS found 289.0, [M + H]+

59

3-(3-fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (NEU2113)


0.217 mmol, 1 eq) was added followed by (3-fluoromethylphenyl)boronic acid (33 mg, 0.236


dichloromethane adduct (18 mg, 0.022 mmol, 0.102 eq). The tube was sealed and purged with


carbonate (0.750 mL, 1.5 mmol, 6.93 eq) were then added and the reaction mixture was degassed

and ran in the microwave for 30 minutes at 120 °C. The reaction was then concentrated, and

ethyl acetate was added. This solution was then washed with water, followed by brine. The

organic layer was collected, dried over sodium sulfate and concentrated to give crude product.

The crude product was purified on the FractionLynx system (30-70% ACN/water gradient) to

afford 3-(3-fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine as a white

solid (5.4 mg, 8.5% yield). 1H NMR (399 MHz, chloroform-d) δ ppm 4.01 (s, 3 H) 7.05 (t, J=7.7

Hz, 1 H) 7.34 (d, J=10.3 Hz, 1 H) 7.41 – 7.48 (m, 2 H) 7.58 (s, 1 H) 7.71 (br. s., 1 H) 7.83 (s, 1

H) 8.33 (s, 1 H) 8.62 (br. s., 1 H) 9.97 – 10.24 (m, 1 H). LCMS found 293, [M + H]+

60

References

1. World Health Organization. Neglected Tropical Diseases.

http://www.who.int/neglected_diseases/diseases/en/ (accessed July 15).

2. Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Ottesen, E.; Sachs, S. E.; Sachs, J. D.

Incorporating a rapid-impact package for neglected tropical diseases with programs for

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64

Appendix

Representative H1 NMR Spectra

65

Compound 5

66

Compound 4

67

Compound 6

68

Compound 3

69

Compound 8

70

NEU1935

71

NEU1936

72

Compound 11

73

NEU1937

74

NEU1938

75

Compound 20

76

Compound 21

77

Compound 19

78

Compound 18

79

Compound 23

80

Compound 22

81

NEU2065

82

NEU2066

83

NEU2067

84

NEU2068

85

NEU2069

86

NEU2070

87

NEU2112

88

NEU2113

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