Novel Retinoid Enhancers for Anti- Cancer Therapies

NOVEL RETINOID ENHANCERS FOR ANTI-

CANCER THERAPIES

A thesis submitted in fulfilment of the degree of

Doctor of Philosophy

Christopher R Gardner

Supervisors:

Prof. Naresh Kumar Dr. Belamy Cheung Prof. David StC. Black

School of Chemistry The University of New South Wales Kensington, Australia

July, 2015

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Gardner

First name: Christopher Other namefs: Robert

Abbreviation for degree as given in the University calendar: PhD

School: Chemistry Faculty: Science

Title: Novel retinoid enhancers for anti-cancer therapies

Abstract 350 words maximum: (PLEASE TYPE)

Retinoids play a crucial role in the treatment of multiple human cancers, particularly neuroblastoma and leukaemia. However, their utility is often hampered by the inherent toxicities associated with the concentrations necessary for therapeutic benefit, as well as innate or acquired resistance to treatment. This thesis focuses on the discovery and development of novel scaffolds that possess cytotoxic activity mediated through the retinoid pathways.

A protocol for docking scaffolds into the binding site of the retinoic acid receptor ~ (RAR~) was developed. These docking methodologies were used to identify novel scaffolds as potential RAR~ ligands, as well as probe the mechanisms behind the observed structure activity relationships (SAR) of the prepared compounds.

A wide range of novel indole-benzothiazole acetamides were synthesized via PyBOP amide coupling of 3-indoleacetic acids and 2-aminobenzothiazoles. These scaffolds were investigated for their cytotoxic activity against neuroblastoma cells. Several analogues were found to have low micromolar IC50 values, demonstrated selectivity towards cells with high RAR~ expression and also were selective for cancerous cells over normal cells. Furthermore, the ability of these compounds to modulate RAR protein expression was demonstrated.

Indole and quinoline-oxadiazoles were synthesized by the dehydrocyclization of bis-hydrazides by 4-toluenesulfonyl chloride. The cytotoxic activity of these scaffolds was investigated with their selectivity towards cells overexpressing different RAR subtypes (a, ~ . y) also investigated.

Benzothiazole-thieno[2,3-c]pyrazole amides were also synthesized by the one-pot, single step alkylation-aldol condensation of methyl thioglycolate and formyl-halopyrazoles. The cytotoxicity and SAR of these scaffolds was also explored.

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ABSTRACT

Retinoids play a crucial role in the treatment of multiple human cancers, particularly neuroblaostoma and leukemia. However, their utility is often hampered by the inherent toxicities assosciated with the concentrations necessary for therapeutic benefit, as well as innate or acquired resistance to treatment. This thesis focuses on the discovery and development of novel scaffolds that possess cytotoxic activity mediated through the retinoid pathways.

A protocol for docking scaffolds into the binding site of the retinoic acid receptor β

(RARβ) was developed. These docking methodologies were used to identif y novel scaffolds as potential RARβ ligands, as well as probe the mechanisms behind the observed structure activity relationships (SAR) of the prepared compounds.

A wide range of novel indole-benzothiazole acetamides were synthesized via PyBOP amide coupling of 3-indoleacetic acids and 2-aminobenzothiazoles. These scaffolds were investigated for their cytotoxic activity against neuroblastoma cells. Several analogues were found to have low micromolar IC 50 values, demonstrated selectivity towards cells with h igh RARβ expression and also were selective for cancerous cells over normal cells. Furthermore, the ability of these compounds to modulate RAR protein expression was demonstrated.

Indole and quinoline-oxadiazoles were synthesized by the dehydrocyclization of bis- hydrazides by 4-toluenesulfonyl chloride. The cytotoxic activity of these scaffolds was investigated with their selectivity towards cells overexpressing different RAR subtypes

(α, β, γ) also investigated. iii

Benzothiazole-thieno[2,3-c]pyrazole amides were also synthesized by the one-pot, single step alkylation-aldol condensation of methyl thioglycolate and formyl- halopyrazoles. The cytotoxicity and SAR of these scaffolds was also explored.

ACKNOWLEDGEMENTS

My first words of thanks are offered to my supervisor Prof. Naresh Kumar. Through your guidance, support, encouragement and input, I have been able to overcome the many obstacles of post-graduate research. You have provided me with the opportunity not only to learn and develop as a scientist, but to develop the necessary skills for a career in research and teaching. But most of all, you have given me the opportunity and support to achieve one of my dreams.

I wish to also offer the same sentiment to my co-supervisors Dr. Belamy Cheung and

Prof. David StC. Black. Thank you Belamy for taking me on when I was just a chemist, with no understanding of the intricacies of molecular biology. You gave me every guidance, support and opportunity, allowing me to gain knowledge and skills I never thought I would. Thank you David for your enthusiastic encouragement, immeasurable knowledge, the stories you shared and our discussions about cricket. You have not only set an example, but challenged and encouraged me to reach it.

Also, I must thank my other supervisors; Prof. Glenn Marshall and A/Prof. Renate

Griffith. Thank you for your enthusiasm Glenn, your kindness and insight. You always reiterated the importance of doing good science, the Nature paper question and the reason we do this at all: to make someone’s lif e better. Thank you Renate for all of your assistance with my compoutational studies and the time you spent training me with the software and troubleshooting the issues I had along the way. Thank you also for guiding and supporting me with this new endeavour.

Thanks also to the staff of the UNSW School of Chemistry, the Analytical Centre and

Children’s Cancer Institute . You have taught, helped and befriended me since the time I v

was a first year undergraduate student, providing a safe and enjoyable environment for me to study and work. Without your efforts to maintain the facilities, supply chemicals and equipment, run samples and the countless other duties you perform, there would not be a School of Chemistry for me to be at, let alone this thesis.

To everyone in the Kumar-Black group, both past and present, thank you for your help and friendship. Thank you all for putting up with my questions (particularly in the early days), my OCD behaviour when it comes to random things, playing music (sometimes loudly) in the lab, my strange (possibly offensive) sense of humour and the random things I say and do. But I must single out a few people who deserve special thanks: Dr.

Kasey Wood for being a friend, mentor and gym buddy; Dr. Renxun Chen for his friendship, advice and always wanting to get lunch, or at least coffee; Dr. Samuel Kutty for always providing help with all aspects of my project, being my friend and a fellow cricket enthusiast; Dr. Kitty Ho for her help, particularly in keeping the lab working, and her friendship; and Dr. Murat Bingul for his much appreciated assistance with chemistry, being my friend, gym buddy and coffee lover.

Thank you also to all the members of the Molecular Carcinogenesis program. At some point or other I have come to you all with questions about what I was doing, or more accurately, what should I be doing, which were always met with kindness, understanding and thorough assistance. Without all your help, there would otherwise not be anywhere near the amount of biology that is in this thesis. Special thanks to

Selina, Owen and Patrick for their continued help and friendship. But of course, the biggest thanks here must go to Jess Koach. Thank you for teaching me almost everything I know about how to do molecular biology, for putting up with my countless questions and constant interruptions, and still being my friend after it all. And a special thank you for always having delicious snacks! vi

To all my friends, thank you so much for helping get me through this long journey.

Thank you to my uni friends Michelle, Robbo, Mark and Matt for all your help over the years, your friendly discussions and all the morning teas, meals and times we went for drinks. Thanks to all the guys from cricket for helping take my mind off things and giving me the chance to take out my frustration by bowling a ball or trying to hit and catch one. To my best friends Branden, Ralph, Jayson and Ruben I must offer very special thanks. You guys have always been there to make me laugh and smile, always offered me help, been there to talk to or just listen and agree with me when I rant, and always given me food , caffeine and alcohol. The times we’ve shared, be they real or in

D&D, have helped keep whatever sanity I ever had and gone a long way to helping me achieve this thesis.

To Talissa, mein schatzen, I offer a most sincere danke schön. Your unwavering love and support have helped me to achieve this dream, for which I will always be grateful.

You have put up with my worried frustration, my sleep deprived confusion, general nonsensical behaviour and periods of extended distraction. Yet through all the times I have been far from my best, you have shown me caring and understanding, always helping me through and loving me more for it. Thank you for always being there for me, for always helping me and for sharing in this journey. I also want to express my deepest gratitude to Wendy and Martin for welcoming me into their home, providing me with many dinners, enjoyable conversations, fond memories, and especially for their words of advice and support.

Finally, I must give the biggest thanks to my family, who have given me more love and support than any one man deserves. Thank you to my grandparents for always taking care of me, being supportive and eternally proud. You have taught me many of my most valuable lessons and stood me in good stead for the many challenges I have faced in my vii

PhD studies. To my sister Candice, thank you for always being ready with a hug, for putting up with my loud music and annoying sense of humour, for making me something to eat and above all for being the best friend and sister anyone could ever want. To mum and dad, I cannot thank you enough for everything you have done for me. You were my first teachers and my first friends. No matter what I have done, or what choices I have made, you have stood proudly by, confident in my ability to make my own way in the world. You have always been the first to offer me help and the first to celebrate my success. You have given me everything, even if it meant you had to go without, and done anything you thought might make my today even just a little bit better than my yesterday. Thanks to you, I not only dared to dream, but was able to achieve it. This thesis is as much a testament to your efforts as it is to mine. So for all that and more, I thank you.

viii

TABLE OF CONTENTS

CERTIFICATE OF ORIGINALITY ...... i

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

TABLE OF CONTENTS ...... viii

LIST OF ABBREVIATIONS ...... xiii

PUBLICATIONS AND PRESENTATIONS ...... xvi

CHAPTER 1: INTRODUCTION ...... - 1 -

1.1 Cancer ...... - 1 -

1.1.1 Neuroblastoma ...... - 2 -

1.2 The retinoid signalling pathway ...... - 5 -

1.3 Retinoids and cancer ...... - 8 -

1.3.1 Retinoic acid syndrome ...... - 11 -

1.3.2 Retinoid resistance ...... - 12 -

1.4 Atypical retinoids ...... - 13 -

1.5 Benz-fused heterocycles in medicinal chemistry ...... - 18 -

1.5.1 Indoles as drug scaffolds ...... - 19 -

1.5.2 Benzothiazoles as drug scaffolds ...... - 21 -

1.5.3 Benzimidazoles as drug scaffolds ...... - 23 -

1.5.4 Benzoxazoles as drug scaffolds ...... - 25 - ix

1.5.5 Quinolines in medicinal chemistry ...... - 27 -

1.6 Computer-aided drug design ...... - 31 -

1.6.1 CADD techniques in atypical retinoid design ...... - 31 -

1.7 Thesis aims ...... - 33 -

CHAPTER 2: DEVELOPMENT OF COMPUTATIONAL DOCKING STUDIES FOR

THE IDENTIFICATION OF RAR β LIGANDS ...... - 34 -

2.1 Introduction ...... - 34 -

2.2 Preliminary library screening ...... - 37 -

2.3 The GOLD software package ...... - 44 -

2.4 The RARβ protein ...... - 46 -

2.5 Establishing a docking protocol ...... - 47 -

2.6 Validation of the protocol with the lead compounds as a training set ...... - 51 -

2.7 Conclusions ...... - 55 -

CHAPTER 3: DESIGN, SYNTHESIS AND CHARACTERIZATION OF INDOLE-

BENZOTHIAZOLE ACETAMIDES ...... - 56 -

3.1 Introduction ...... - 56 -

3.2 Identification of indole acetamides as a lead scaffold ...... - 58 -

3.3 Synthesis of indole-benzothiazole amides ...... - 62 -

3.3.1 Synthesis of mixed heterocyclic acetamides ...... - 65 -

3.3.2 Synthesis of indole-benzothiazole amides with modified linkers ...... - 70 -

3.3.3 Synthesis of analogues bearing substituents on indolyl or benzothiazolyl

rings ...... - 74 - x

3.4 Characterization of in vitro activity against neuroblastoma cell lines ...... - 81 -

3.4.1 Screening of selected analogues against normal cells...... - 89 -

3.4.2 Examination of the effect on RAR protein expression ...... - 91 -

3.4.3 Further mechanistic investigations ...... - 95 -

3.5 Conclusions ...... - 97 -

CHAPTER 4: DESIGN, SYNTHESIS AND CHARACTERISATION OF INDOLE

AZOLES ...... - 99 -

4.1 Introduction ...... - 99 -

4.2 Identification of indole azoles as a lead scaffold ...... - 101 -

4.3 Synthesis of indole azoles ...... - 105 -

4.3.1 Synthesis of methylene and ethylene-bridged oxadiazoles ...... - 106 -

4.3.2 Synthesis of analogues with varied linking heterocycles ...... - 116 -

4.3.3 Synthesis of analogues bearing substituents on indolyl or phenyl rings . - 121 -

4.4 Characterization of in vitro activity against neuroblastoma cell lines ...... - 123 -

4.5 Conclusions ...... - 130 -

CHAPTER 5: DESIGN, SYNTHESIS AND CHARACTERIZATION OF

BENZOTHIAZOLE-BASED THIENOPYRAZOLES ...... - 131 -

5.1 Introduction ...... - 131 -

5.2 Identification of benzothiazole-thienopyrazoles as a lead scaffold ...... - 135 -

5.3 Synthesis of benzothiazole-thienopyrazoles ...... - 138 -

5.3.1 Synthesis of starting pyrazolones ...... - 141 -

5.3.2 Attempted synthesis of thienopyrazoles by Sonogashira cross-coupling - 147 - xi

5.3.3 Synthesis of thienopyrazoles from formylpyrazoles ...... - 151 -

5.3.5 Synthesis of furano- and pyrrolopyrazoles ...... - 154 -

5.4 Characterization of in vitro activity against neuroblastoma cell lines ...... - 156 -

5.5 Conclusion ...... - 158 -

CHAPTER 6: 8-ISOPROPYL-4-ARYLQUINOLINES AS RAR α ANTAGONISTS - 159 -

6.1 Introduction ...... - 159 -

6.2 Synthesis of 8-isopropyl-4-arylquinolines ...... - 163 -

6.2.1 Synthesis of quinoline starting materials ...... - 166 -

6.2.2 Synthesis of quinoline oxadiazoles ...... - 173 -

6.2.3 Quinoline-oxadiazoles bearing an o-fluorobenzoic acid ...... - 179 -

6.3.4 Synthesis of quinolines with varied linking regions ...... - 182 -

6.3.5 Synthesis of 4-methoxyquinolines ...... - 186 -

6.4 Characterization of in vitro activity against neuroblastoma cell lines ...... - 189 -

6.5 Conclusions ...... - 191 -

CHAPTER 7: FUTURE DIRECTIONS ...... - 192 -

CHAPTER 8: EXPERIMENTAL ...... - 194 -

8.1 General information ...... - 194 -

8.2 Experimental details ...... - 195 -

8.3 Biological assays ...... - 302 -

8.3.1 Cell culture ...... - 302 -

8.3.2 Cell viability assays ...... - 302 -

8.3.3 Immunoblot analysis ...... - 303 - xii

REFERENCES ...... - 305 -

xiii

LIST OF ABBREVIATIONS

t-RA trans -retinoic acid

13-c-RA 13-cis -retinoic acid

4-TsCl 4-toluenesulfonyl chloride

9-c-RA 9-cis -retinoic acid

AcOH acetic acid

APL acute promyelocytic leukaemia

CADD computer-aided drug design

DCC N,N-dicyclohexylcarbodiimide

DCM dichloromethane

DIPEA N,N-diisopropylethylamine

DMAD dimethyl acetylenedicarboxylate

DMAP N,N -dimethylaminopyridine

DMF N,N-dimethylormamide

DMSO dimethylsulfoxide

EDCI l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

EFS event free survival

ESI electrospray ionization

Et ethyl

Et 2O diethylether

Et 3N triethylamine

HDAC histone deacetylase

HOBt 1-hydroxybenzotriazole xiv

HRMS high resolution mass spectrometry

IAA 3-indoleacetic acid

IC inhibitory concentration

IR infrared spectroscopy

J coupling constant

LBD ligand binding domain lit. literature m.p. melting point

Me methyl mL millilitre(s) mmol milli mol

MYCN myelocytomatosis viral related oncogene, neuroblastoma derived

NMR nuclear magnetic resonance

Pr propyl

PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

RAMBA retinoic acid metabolism blocking agents

RAR retinoic acid receptor

RARE retinoic acid response element

RMSD root-mean-square deviation

RXR retinoid X receptor

SAR structure-activity relationship

SEM standard error of the mean

Tf 2O trifluoromethanesulfonic anhydride

TFA trifluoroacetic acid

THF tetrahydrofuran xv

TTNPB 4-[( E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-

propenyl]benzoic acid

UV ultraviolet spectroscopy

xvi

PUBLICATIONS AND PRESENTATIONS

A part of this research has been submitted for publication as well as presented at the following conferences:

Publications:

Gardner, C. R. ; Cheung, B. B.; Koach, J.; Black, D. S.; Marshall, G. M.; Kumar, N.

Bioorg. Med. Chem. 2012, 20 , 6877-6884.

Presentations:

Gardner, C. R. ; Koach, J.; Marshall, G. M.; Black, D. S.; Kumar, N. Cheung, B. B.;

Identification and characterization of novel small molecules which enhance the retinoid anti-cancer signals, The Lorne Cancer Conference, Lorne, Victoria, 9 th - 11 th February,

2012. (Poster presentation)

Gardner, C. R. ; Marshall, G. M.; Black, D. S.; Kumar, N. Cheung, B. B.; Identification and characterization of novel small molecules which enhance the retinoid anti-cancer signals, The EFMC XXIII International Symposium on Medicinal Chemistry, Lisbon,

Portugal, 7 th - 11 th September, 2014. (Poster presentation)

CHAPTER 1: INTRODUCTION

1.1 Cancer

Cancer is a heterogeneous group of genetic diseases, resulting from the aberration and alteration of key genetic and molecular pathways. 1 There are more than one hundred types of cancer, with each typically identified by the type of cells in which they arise.

However, different types of cancer share some features, commonly referred to as the hallmarks of cancer, outlined in Figure 1.1 below.2

Figure 1.1: The proposed hallmarks of cancer. 2

As a major cause of sickness and death worldwide, we need not look far to see the devastating impact that cancer has had on society. In Australia alone in 2012, approximately 67,260 and 53,460 new cases of the disease were diagnosed in men and women, respectively. Furthermore, in 2012 an estimated 24,328 males died of cancer, 2

along with 18,516 deaths in females.3 It is estimated that in the year 2020, 80,730-

89,530 men and 63,990-66,100 women will be diagnosed with cancer in Australia.4

Importantly, cancer was estimated to be the largest contributor to the burden of disease

(measured as years of healthy life lost) in Australia in 2010, accounting for approximately 19% of the total burden, with cardiovascular disease accounting for

16%.5 Furthermore, cancer was the 9th and 6 th leading cause of death in Australian men and women, respectively, in 2011. 6

1.1.1 Neuroblastoma

Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system, derived from migrating neuroectodermal cells. 7 It is the third most commonly diagnosed children’s cancer, representin g approximately 8-10% of childhood cancer cases. The average age at diagnosis is 1-2 years, with 90% of cases diagnosed before the age of 5, and the disease being very rare in children above the age of 10.

There are two methods for describing the severity and risk associated with a neuroblastoma diagnosis: the International Neuroblastoma Staging System Committee

(INSS) 8 system and the more recent International Neuroblastoma Risk Group Staging

System (INRGSS). 9 Unlike the INSS, the INRGSS uses only the results of imaging tests taken before surgery and does not include surgical results or metastasis to lymph nodes to determine the risk stage (Table 1.1).

Table 1.1: Neuroblastoma risk staging systems

System Stage Description INSS 1 Can be completely surgically removed. Distant lymph nodes may/ may not contain cancer, but nearby lymph nodes do not. 2A The tumour is located only in the area it started and cannot be completely removed during surgery. Nearby lymph nodes do not contain cancer. 2B The tumour is located only in the area where it started and may/ may not be completely removed during surgery, but nearby lymph nodes do contain cancer. 3 The tumour cannot be removed with surgery. It has spread to regional lymph nodes or other areas near the tumour, but not to other parts of the body. 4 The original tumour has spread to distant lymph nodes, bones, bone marrow, liver, skin, and/or other organs, except for those listed in stage 4S. 4S The original tumour is located only where it started (as in stage 1, 2A, or 2B), and it has spread only to the skin, liver, and/or bone marrow, in infants younger than one. The spread to the bone marrow is minimal, usually less than 10%. INRGSS L1 The tumour is located only in the area where it started; no risk factors found on imaging scans, such as CT or MRI. L2 The tumour has not spread beyond the area where it started and the nearby tissue; risk factors are found on imaging scans, such as CT or MRI. M The tumour has spread to other parts of the body (except stage MS) MS The tumour has spread to only the skin, liver, and/or bone marrow.

Due to the highly heterogeneous nature of neuroblastoma,10 as well as its unique propensity for spontaneous regression (typically in stage 4S), 11-13 neuroblastoma risk classification and survival rates depend highly upon the age of the patient, 14 the stage at diagnosis,8 tumour histopathology, 15 DNA index (diploid or hyperploid) 16 and the

MYCN amplification status.17-19 Based on these factors, patients are classified with low, intermediate or high risk neuroblastoma (Table 1.2). 20 Low and intermediate-risk neuroblastoma patients have survival rates above 95% over 5-year and 3-year periods 4

respectively,21,22 while high-risk neuroblastoma patients have much lower survival rates of approximately 40% over 5 years.23 Of all neuroblastoma cases diagnosed, 37% are low-risk, 18% are intermediate-risk and 45% are high-risk. 24 Furthermore, many high- risk patients will relapse after treatment, the 5-year overall survival of which is only

20%.25 As a consequence of these factors, neuroblastoma accounts for 15% of all childhood cancer-related deaths. 26

Table 1.2: Description of the neuroblastoma risk groups.

Risk group Description Low • Stage 1 • Stage 2A/B, more than 50% of the tumour surgically removed, except with MYCN amplification • Stage 4S disease, no MYCN amplification, favourable histopathology, and hyperdiploidy Intermediate • Stage 2A/B, no MYCN amplification, < 50% of the tumour removed with surgery • Stage 3, < 18 months, no MYCN amplification • Stage 3, < 18 months, no MYCN amplification, and favourable histopathology • Stage 4, < 12 months, no MYCN amplification. • Stage 4, 12 -18 months, no MYCN amplification, hyperdiploidy, and favourable histology. • Stage 4S, no MYCN amplification, unfavourable histopathol ogy and/or diploidy High • Stage 2/B and MYCN amplification • Stage 3 and MYCN amplification • Stage 3, ≥ 18 months, no MYCN amplification and unfavourable histopathology • Stage 4, < 12 months and MYCN amplification • Stage 4, 12 -18 months with MYCN amplification, and/or diploidy, and/or unfavourable histology • Stage 4, ≥ 18 months • Stage 4S and MYCN ampl ification

Treatment options for neuroblastoma are tailored to the risk group and associated tumour biology, with more advanced tumours requiring more aggressive therapies and 5

larger combinations of therapies. For low-risk tumours, the approach is either surgery, chemotherapy with agents such as carboplatin, cyclophosphamide, doxorubicin, and etoposide, or a combination of surgery and chemotherapy. 22,27 Intermediate-risk neuroblastoma is often treated with a combination of surgery and chemotherapy, with radiotherapy also administered in progressive tumours or those which do not respond to surgery and chemotherapy. 28,29 For those with high-risk neuroblastoma, treatment is stratified into three phases: induction, consolidation and maintenance. The induction phase involves dose-intensive cycles of chemotherapy, alternating between cisplatin, etoposide, vincristine, cyclophosphamide, doxorubicin, and topotecan, followed by surgery. 30,31 Consolidation first attempts to eradicate the minimal residual disease with myeloablative chemotherapy, using combinations of either carboplatin/etoposide/melphalan or busulfan/melphalan, followed by repopulation of the bone marrow through hematopoietic stem cell transplantation (HSCT). 32-35

Maintenance aims to reduce the risk of recurrence through differentiation therapy, administering 13-cis -retinoic acid (isotretinoin), and immunotherapy, with the monoclonal antibody ch14.18 and immune-activating cytokines (GM-CSF and IL-2). 36-

38 Finally, in the case of recurrent neuroblastoma, treatment involves surgery and/or chemotherapy for patients originally classified as low- and medium-risk, while high-risk patients may receive a combination of chemotherapy, surgery, and radiation therapy followed by HSCT. 39,40

1.2 The retinoid signalling pathway

Retinoids are a class of natural and synthetic derivatives of vitamin A 1, also known as retinol.41 This essential vitamin is procured through the ingestion of foods rich in 6

preformed vitamin A, such as dairy products, fish and meat (particularly liver), or provitamin A 2, also known as β-carotene, which is found in vegetables such as sweet potato, spinach and carrot. 42 However, the oxidised metabolites of vitamin A, primarily trans -retinoic acid ( t-RA, tretinoin) 3, which is capable of performing all cellular functions of vitamin A, and 9-cis -retinoic acid (9-c-RA, alitretinoin) 4, are responsible for the activity of this vitamin. These molecules function as regulatory hormones, similar to the thyroid or steroid hormones, and play an essential role in normal embryonic development and in the maintenance of differentiation in later stages of human lives. 43-46 They are critically involved in the development of the heart, embryonal circulation, central nervous system and normal left-to-right cardiac symmetry, as well as the regulation of eye function, bone growth, spermatogenesis and immune responses. 44,47

The retinoid signalling pathway involves nuclear localised, ligand-activated transcription factors known as retinoic acid receptors (RAR). 48 These receptors are classed in three subtypes encoded by separate genes: RARα (NR1B1), RARβ (NR1B2), and RARγ (NR1B3). 48,49 RARs form heterodimers with retinoid X receptors (RXR), also possessing RXRα, RXRβ and RXRγ subtypes, 50-52 in order to associate with 7

retinoic acid response elements (RARE) in the promoter regions of their target genes. 53

Furthermore, there are at least two isoforms for each subtype, generated by differential promoter usage and alternative splicing that differs only in their N-terminal regions.

RARα, RARγ and t he three RXR subtypes each have two isoforms , while RARβ has four isoforms. 54-56 The variable combination of the eight RAR and six RXR subtypes and isoforms allows the possible formation of forty-eight different heterodimers. 44

Additionally, the retinoid receptors also show differential expression across different cell types, as well as exhibiting changing levels of expression throughout the human life cycle from the foetus, through childhood and into adulthood. 57-60 It is noteworthy that during embryonic development, RARα has near ubiquitous expression throughout the embryo , while RARβ and RARγ have much more localised and specific patterns of expression. 61

The RAR-RXR heterodimer is able to bind to the RARE in the absence of a ligand, but preferentially associates with co-repressors such as the nuclear receptor co-repressor (N-

CoR) proteins, and silencer proteins such as the silencing mediator for retinoid and thyroid receptor (SMRT) (Figure 1.2).62 These co-repressors recruit histone deacetylase

(HDAC) and methyl transferase complexes leading to chromatin condensation and sequestration of promoter elements, thereby inhibiting transcriptional activity. 63 Upon binding of a retinoid agonist, the RAR-RXR heterodimer undergoes a conformational change in its C-terminal helix, which allows for coupling to co-activator proteins (CoA) such as members of the steroid receptor co-activator (SCR-1) family, along with histone acetyl and methyl transferases (HAT), and DNA-dependant ATPases. 64,65 The resulting complex then stimulates the decondensation of the chromatin surrounding the promoter regions of target genes, thereby allowing the binding of the thyroid receptor-associated 8

protein (TRAP) and the recruitment of the transcription machinery, including RNA polymerase II and other general transcription factors. 66,67

Figure 1.2: Transcriptional activation and repression of the retinoid pathway. LBD:

ligand-binding domain. DBD: DNA-binding domain.

1.3 Retinoids and cancer

Due to the essential role of the retinoid pathway in many cellular processes, particularly differentiation and apoptosis, it is not surprising that the deregulation of this pathway has been linked to a range of human diseases, including multiple forms of cancer. In 9

several tissues and organs, the loss of a RAR subtype has been associated with tumourigenesis, suggesting that RARs may be considered as tumour suppressors.

The classical example of RAR dysfunction is in cases of acute promyelocytic leukaemia

(APL), in which chromosomal rearrangements and point mutations of the RARα gene lead to the formation of a RARα fusion protein , which in 95% of cases is the RARα -

PML (promyelocytic leukemia gene) fusion.68-72 This fusion protein has the ability to homodimerize or oligomerize with or without union to RXRα , thus increasing the recruitment of co-repressor complexes and enhancing the repression of target genes.73-75

Following the observation that t-RA 3 promoted the in vitro granulocytic differentiation of APL cells, 76,77 the first clinical trial of retinoids in the treatment of APL was conducted, in which 23 of 24 patients achieved complete remission. 63 Through subsequent trials, the efficacy of t-RA 3 in remission induction and maintenance in APL was confirmed. When used in combination with chemotherapy, t-RA 3 offered complete remission rates of up to 95% and 5 year EFS rates of up to 74%.78,79

The therapeutic benefit of t-RA 3 in APL is mediated through two mechanisms: restoration of retinoid target gene transcription and induction of RARα -PML degradation. Similar to the mechanism for retinoid induction described in Chapter 1.2, pharmacologic doses of 3 induce a conformational change in the RARα -PML protein that results in the dissociation of HDACs and DNA methyl transferases (DNMT). 73

CoA complexes are then recruited and the transcription of target genes, particularly those involved in granulocyte differentiation, is restored. 80 The degradation of the

RARα -PML oncoprotein is achieved through two non-overlapping, co-operating proteolytic mechanisms. In the first mechanism, treatment with 3 results in the targeting of a cleavage site within the PML α -helix, leaving the RARα moiety intact and capable of resuming transcriptional activity. 81 Alternatively, the ubiquitin/proteasome system 10

(UPS) is activated through the phosphorylation of RARα -PML Ser873 and the binding of the ATP-dependent protease SUG-1 to the RARα transactivation domain. 82,83

In neuroblastoma, the dysfunction of multiple RARs has been observed and identified as a key target in disease therapy. Neuroblastoma tumours typically exhibit high levels of RARα, low levels of RARγ and barely detectable levels of RARβ. 84-87 Similar to

APL, pre-clinical experiments have demonstrated that that neuroblastoma cell lines can often be induced to terminally differentiate on exposure to retinoid compounds in vitro .88,89 In the clinical trial that followed, 13-cis -retinoic acid 5 (13-c-RA, isotretinoin), a synthetic retinoid, was administered to patients following myeloablative consolidation therapy. It was found that the 3-year EFS of patients was increased to

46±6% amongst patients who received 13-c-RA 5, compared to 29±5% for those assigned with no further therapy.34 As a result of such trials, together with a better pharmacokinetic profile with higher peak levels and a longer half-life consistent with superior activity against neuroblastoma compared to t-RA 3,90 13-c-RA has become the standard of care for high-risk neuroblastoma.37

The therapeutic benefit of 13-c-RA 5 in neuroblastoma is believed to be predominantly mediated through transcriptional activation of retinoid target genes associated with differentiation. In particular, it has been suggested that 13-c-RA 5 might enhance the activity of RARβ, whose expression has been linked with growth -inhibitory effects.85

This hypothesis is supported by the observation that the loss of RARβ expression has been associated with tumourigenesis in numerous cancer types, such as those of the lung, breast and cervix.91-94 In such cancers, the RARβ gen e is frequently deleted or the

RARβ promoter silenced due to aberrant DNA methylation or repressive histone 11

modifications. 66,67 Furthermore, the restoration of RARβ expression or RARβ promoter activity was shown to decrease the tumourigenicity of the cancer cells. 95-97

Furthermore, in keratinocytes, the decrease , or absence of expression of the RARγ subtype has been linked to a predisposition to tumours. 98 RARγ was found to be absent in oral keratinocytes from head and neck cancers, as well as being dramatically decreased in skin cancers induced by UV radiation. 99,100

1.3.1 Retinoic acid syndrome

Although retinoid therapy has significantly improved long term survival rates for cancers such as neuroblastoma and APL, their use as single agent therapies has been limited by the significant toxicities associated with their use, including teratogenesis and the induction of retinoic acid syndrome. 101

Retinoic acid syndrome is characterized by unexplained fever, weight gain, elevated white blood cells, respiratory distress, interstitial pulmonary infiltrates, pleural and pericardial effusions, dyspnea, episodic hypotension, and acute renal failure. 102-104 The potentially life-threatening syndrome occurs in approximately 25% of APL cases where t-RA 3 is administered, 105 with up to 5% of cases being fatal. 103,105 In neuroblastoma, following treatment with 13-c-RA 5, the syndrome presents with skin toxicity, renal toxicity, transaminase elevation, gastrointestinal toxicity, infection, hypercalcemia, hematologic toxicity, comprised cheilitis and bone pain. 34,106 It has been shown that 13- c-RA 5 has a lower toxicity profile, with its side-effects typically being low to mild in severity. However, its side-effects are also more common, particularly those affecting the skin, and can occur in up to 80% of patients. 107 12

Due to the possible combinations of the RAR and RXR heterodimers, 44 their differential expression across different cell types, 57-60 as well as the different and specific function of each of these heterodimers, it is believed that these toxicities may be related to the non-selective activation of RAR-RXR functions by the natural ligands. 108

1.3.2 Retinoid resistance

Aside from the potential toxicities associated with the use of retinoid therapy, there have been many examples of intrinsic or acquired retinoid resistance.109 This is not surprising, as cancer cells are highly heterogeneous and exhibit deregulation in multiple cellular signalling pathways, leading to the frequent failure of treatments using chemotherapeutic agents specific to a single biological event or pathway.110

One key mechanism of retinoid resistance in both APL and neuroblastoma is the increased metabolism and subsequent reduction in plasma levels of retinoid compounds to suboptimal doses. 111-113 Sustained treatment with retinoids has been shown to induce a catabolic response through cytochrome P450 enzymes, such as P26, 2C8, 3A7, 4A11,

1B1, 2B6, and 2C9. 114-116 Furthermore, the presence of a RARE in the promoter region of the cytoplasmic retinoic acid binding protein (CRABPII) induces CRABPII following treatment with t-RA 3, leading to RA sequestration and subsequent resistance. 117

There have also been reports of retinoid resistance based on genetic mechanisms. In

APL, the rare translocation of the promyelocytic leukaemia zinc finger (PLZF) gene results in a RARα -PLZF fusion protein that is non-responsive to RA treatment. 118

Similarly, mutations in the ligand-binding domain of RARα in the RARα -PML fusion protein generate acquired retinoid resistance in relapsed cases of APL. 119 In 13

neuroblastoma, the mechanisms for retinoid resistance are largely unknown. Several oncogenic events have been examined, such as the deregulation of the MYC pathway, 120 or the failu re to recruit RXRβ to the RARE. 121

The primary method to circumvent retinoid resistance is through intermittent therapy, whereby the patient is administered treatment in the form of on and off cycles.

However, increasing focus has been directed towards the development of combinatorial treatments of retinoids with other drug types,122 especially HDAC inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA),123 as well as retinoic acid metabolism blocking agents (RAMBA) such as liarozole 6 and talarazole 7.124,125

1.4 Atypical retinoids

In addition to the use of natural retinoids for cancer therapies, there has been a great focus on the development of synthetic retinoids, which have shown the potential to target specific retinoid receptors. Hence, these compounds could not only act as tools to investigate the function of specific RAR or RXR subtypes, but may circumvent the toxic effects associated with pan-RAR activation.108 Other than their reduced levels of toxicity, atypical retinoids may also overcome retinoid resistance by simultaneously acting through non-retinoid pathways, sensitizing cell lines to retinoid treatment, or acting synergistically with other retinoids.126,127 14

Collectively termed atypical retinoids, the synthetic retinoids fall into two classes: retinoid related molecules (RRM), such as acyclic retinoid 8 and 4-

(hydroxyphenyl)retinamide (fenretinide) 9; and polyaromatic derivatives (arotinoids), such as the RARα agonist BMS753 10 and the RARβ/γ agonist a dapalene 11 . Both of these classes of compound follow the same general structure of the natural retinoids, being comprised of three units (highlighted in 8): a bulky hydrophobic region, a linker unit and a polar terminus, typically in the form of a carboxylic acid.

Initial developments in the field of synthetic retinoids were directed at producing molecules capable of binding to the RAR protein, which resulted in the discovery of pan-RAR agonists, similar to t-RA 3. Some pan-RAR agonists of note include 13-cis - retinoic acid (13-c-RA) 5, which is now a standard therapy for high-risk neuroblastoma patients with minimal residual disease (MRD),128 TTNPB 12, which was used to obtain the RARβ crystal structure, 129 EC23 13 and CH55 14.130,131 15

Following these discoveries, detailed structure-activity studies enabled the development of arotinoids that were selective for RAR subtypes. The development of these selective atypical retinoids has been aided by the isolation of a number of RAR and RXR crystal structures. Bourget et al . crystallised the RXRα LBD in 1995, while Renaud et al isolated the RARγ LBD in complex with t-RA 3 later that year. 132,133 In 2004, Germain et al . resolved the cr ystal structure of the RARβ LBD bound to TTNPB 12 , which revealed impo rtant features distinguishing the structure of RARβ from the RARα/γ isotypes. 123

RARα selectivity may be achieved by incorporating hydrogen-bond acceptors, such as amides in the linker region, allowing for strong interactions to the hydrogen-bond donor residue Ser232, which replaces the lipophilic alanine residues in RARβ and RARγ. Thi s feature is exemplified in the structures of the RARα agonists AM580 15 and AM555S

16 .134,135 Selectivity may also be improved through the incorporation of halogens in the non-polar region, as well as through the attachment of fluoro substituents ortho to the carboxylic acid group on the benzene ring in the polar region, such as in AGN193836

17 , the first monospecific RARα agonist. 136 16

Conversely, RARγ selectivity may be increased by the presence of hydrogen -bond donors on or adjacent to the non-polar region, which are able to form weak, non- classical hydrogen bonds to the weakly polar Met272 residue.137 Furthermore, RARγ can tolerate larger hydrophobic groups due to the presence of the smaller Ala397 residue in helix 11. These aspects are featured in the RARγ -selective CD666 18 and

138,139 CD437 19 , respectively.

Most importantly, it was found that the RARβ LBD differs from those of RARα and

RARγ in that it has a larger binding cavity, arising from the smaller Ala225 and Ile263 residues in the hydrophobic region. 129 However, although many ligands possess greater sele ctivity for RARβ over RARα and/or RARγ, complete RARβ mono -specificity is difficult to achieve. For example, the RARβ ligand BMS987 20 is also a R ARα agonist, while AGN193174 21 also acts as a RARα and RARγ antagonist due to its inability to dislodge bound co-repressors from RARα and RARγ. 129,140 Interestingly, AC55649 22 and AC261066 23 are isotype-selective agonists that preferentially target the RARβ 2 isoform. 141 17

Additionally, many arotinoids have been developed as RXR-selective ligands by mimicking the bent conformation of 9-c-RA 4. Strategies to achieve this include design of conformationally-restricted analogues of 4 such as AGN194204 24 ,142 the incorporation of ortho -substituents on the hydrophobic unit of RAR agonists giving structures such as 3-Me TTNPB 25 ,140 incorporating rigid single atom linkers such as in bexarotene 26 ,143 or incorporating benzo-fused ring systems such as in HX600 27 .144

A number of atypical retinoids have received FDA approval for the treatment of various conditions. Etretinate 28a received approval for the treatment of psoriasis, but was subsequently replaced by its metabolite acitretin 28b due to its toxic side effects.

Tazarotene 29 is approved for treatment of psoriasis, acne, and sun damaged skin, while 18

adapalene 11 is approved for use in the treatment of acne, but is also used off-label to treat keratosis pilaris. Additionally, bexarotene 26 is approved as a treatment for cutaneous T cell lymphoma (CTCL). 145

1.5 Benz-fused heterocycles in medicinal chemistry

Medicinal chemistry utilises the talents of people from multiple disciplines to discover and develop the next generation of drugs for treating disease. A critical component of this is the development of structure-activity relationships (SAR) and the identification of structures that can reliably act as a pharmacophore in drug development. One such type of pharmacophore is the heterocyclic ring system. Found in approximately half of all therapeutic agents, heterocycles are a key pharmacophore in medicinal chemistry.

Benz fused heterocyclic systems such as indole 30, benzothiazole 31, benzimidazole 32, benzoxazoleǦ 33 and quinolone 34 have been intensely studied for their interesting pharmacological activities. Based on their prevalence in nature and in synthetic drugs, benz-fused heterocycles can be considered as important scaffolds for the construction of molecules with a vast array of biological activities, as will be discussed further in the following sections of this chapter.

1.5.1 Indoles as drug scaffolds

From the amino acid tryptophan 35 to complex alkaloids such as vinblastine 36a and vincristine 36b , the indole motif is an essential component of many biologically active compounds. The spectrum of activity possessed by indole-based compounds includes antitubercular, 146 antimicrobial, 147 antifungal, 148 anti-inflammatory, 149 antihypertensive, 150 antiHIV, 151 antioxidant, 152 antidepressant, 153 tranquilizing and anticonvulsant properties. 154 Examples of drugs possessing some of these activities include etodolac 37 (antiarthritis),155 sumatriptan 38 (antimigraine), pindolol 39

(antihypertensive) and indolmycin 40 (antibiotic).156,157

A number of compounds containing an indole scaffold have also been reported to possess anticancer activity. A series of tetracyclic indoles was developed by Hong et al ., with 41 displaying the highest in vitro activity against human nasopharyngeal carcinoma (HONE-1) and gastric adenocarcinoma (NUGC-3) cell lines. 158 Garcia et al . 20

synthesized pyrrolo[2,3-e] indole derivatives, with 42 displaying in vitro activity against the PC3 human prostate and K562 erythroleukemia cell lines. 159 Also, Queiroz and co- workers developed 3-(dibenzothien-4-yl)indole 43, which showed potent activity in the

MCF-7 breast adenocarcinoma, NCI-H460 non-small cell lung cancer and SF-268 CNS cancer cell lines. 160

Furthermore, the indole scaffold has been utilized in the design of atypical retinoids.

Gernet et al . developed a number of specific RXR modulators based on ( E)-3-(3-(3,5- di-tert -butylphenyl)-1H-indol-5-yl)but-2-enoic acids 44 and ( E)-3-(4-(2-butoxy-3,5-di- tert -butylphenyl)-1H-indol-2-yl)but-2-enoic acid 45.161 These compounds showed improved binding to RXRα compared to their trienoic counterparts.

Yoshimura et al . have also developed a number of RAR antagonists based on 4-

(7,7,10,10-tetramethyl-4,5,7,8,9,10-hexahydro-naphtho[2,3-g]indol-3-yl)benzoic acids

46, 4-(4,5-dihydro-1H-benzo[g]indol-3-yl)benzoic acids 47 and 1,1,4,4,-tetramethyl-

2,3,4,6,7,12-hexahydro-1H-naphtho[2,3-a]carbazole-9-carboxylic acids 48 .162 These compounds were found to inhibit t-RA binding in HL-60 cells at sub-micromolar concentrations. 21

Additionally, the group of Gurkan-Alp et al . synthesized a small series of ( E)-3-(1 H- indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one derivatives. The unsubstituted derivative 49 was found to possess IC 50 values ranging from 3.92 µM to less than 0.01 µM against a range of tumour cell lines. Furthermore, 49 was also shown to influence the levels of RXRα and RXRγ expression in multiple cell lines. 163

1.5.2 Benzothiazoles as drug scaffolds

Benzothiazoles are another medically important scaffold occurring frequently in natural products, such as in luciferin 50 and violatinctamine 51 .164,165 Benzothiazole-based molecules possess a range of biological properties, including antimicrobial, 166 anticonvulsant, 167 antidiabetic, 168 antitubercular, 169 antiviral, 170 anti-inflammatory and antioxidant activities.171,172 Not surprisingly, benzothiazoles also form the basis of pharmaceuticals with various properties, including riluzole 52 (neuroprotective) and phortress 53 (antitumour).173,174 22

Many benzothiazoles, both naturally occurring and designed, have been investigated as potential antitumour agents. The natural product dercitin 54 and its analogues 55, 56a-c, isolated from marine sponges, are examples of benzothiazoles with in vitro and in vivo anti-tumour activity. 175,176 These compounds were shown to inhibit the proliferation of

P388 murine leukaemia, HeLa cervical cancer and MONO-MAC 6 acute monocytic leukemia cells in vitro .177

Yoshida et al. synthesized a series of 2,6-dichloro-N-[2-(cyclopropanecarbonyl-amino) benzothiazol-6-yl]benzamides 57 that were found to possess antitumor activity against the WI-38 VA-13 (2RA) lung cancer cell line.178 Additionally, Besson et al. developed new benzothiazole-2-carbonitrile derivatives, of which 58 displayed the greatest activity against L1210 murine leukaemia cells. 179 Furthermore, Caleta et al. synthesized styrylbenzothiazoles such as 59 and 60 that showed antiproliferative activity against multiple cancer cell lines. 180 23

However, despite their many biological activities, atypical retinoids based on the benzothiazole nucleus have not yet been discovered. The RARβ 2 isotype-selective, thiazole-based 23 and RAMBA 7 do offer some precedent for the investigation of the benzothiazole scaffold in this thesis.

1.5.3 Benzimidazoles as drug scaffolds

Benzimidazole is another important scaffold in medicinal chemistry. However, unlike the simple imidazole motif, benzimidazoles occur quite rarely in natural products, with only a few examples such as kealiiquinone 61 being known.181 In organic synthesis, benzimidazoles are used for the preparation of analogues of natural alkaloids, such as the kealiiquinone regioisomer 62 and analogues of makaluvamines 63 based on imidazoquinoxalinone 64.182,183 24

Despite their relatively rare occurrence in nature, benzimidazoles offer a vast array of biological activities, including antibacterial, 184 antifungal, 185 anthelmintic, 186 antiretroviral, 187 anti-inflammatory, 188 antihepatitic and antihypertensive properties.189,190 Consequently, benzimidazoles form the basis of many pharmaceuticals, such as astemizole 65 (antihistamine) and albendazole 66 (antimicrobial). 191,192

Benzimidazoles have also been widely explored for their anticancer activities, and have shown inhibitory activity against a number of cancer-related pathways. 2-

Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole 67 has the lowest-ever reported

Ki value for a CK2 protein kinase inhibitor at 40 nM, while 3-benzimidazol-2-yl-1H- indazoles 68a and 68b are potent inhibitors of receptor tyrosine kinase (RTK). 193,194 25

Benzimidazoles have also been examined as potential scaffolds for atypical retinoids.

Eyrolles and co-workers synthesized 4-(5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphth[2,3-d]imidazol-2-y1)benzoic acids such as 69 , which were shown to induce differentiation in HL-60 human promyelocytic leukaemia cells through binding to RARs.195 Furthermore, Buyukbingol et al . synthesized a number of 2-(5,5,8,8- tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-benzo[d]imidazole-5-carboxylic acids 70a -70c, of which the methyl ester derivative 70b showed the most potent antiproliferative activity in HL-60 cells. 196,197 Additionally, Hirotaka et al . prepared a series of 2-substituted 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)- benzo[d]imidazole-5-carboxylic acids 71 that demonstrated RXR-modulating activity. 198

1.5.4 Benzoxazoles as drug scaffolds

The benzoxazole is another useful heterocycle in medicinal chemistry. Similar to benzimidazole, it is found infrequently in natural products, being restricted to few 26

examples such as pseudopteroxazole 72 and seco-pseudopteroxazole 73 ,199 ileabethoxazole 74 ,200 or degradation products of cyclic hydroxamic acids in dried corn tissue, such as 6,7-dimethoxy-2-benzoxazolinone 75 .201

Nevertheless, benzoxazoles have been shown to possess various kinds of biological activities including anti-inflammatory, 202 antitubercular, 203 antileishmanial, 204 anti-

HIV, 205 antimicrobial, 206 antifungal, 207 anti-insomnia and anti-Alzheimer properties.208,209 Several examples of benzoxazole-based pharmaceuticals exist, including calcimycin 76 (antibiotic) and flunoxaprofen 77 (anti-inflammatory). 210,211

Benzoxazoles have also been studied for their anticancer activities. Hou et al . developed the hydroxamic acid 78 as a lead HDACi, which possessed potent cytotoxic activity

212 against 56 tumour cell lines with IC 50 values ranging between 10-540 nM.

Additionally, Easmon et al . prepared several series of 2-benzoxazolyl hydrazones, with the pyrimidine-pyridine derivatives 79 and the methyl-pyrimidine derivative 80 displaying antiproliferative activity against 60 cancer cell lines with IC50 values between 12-447 nM. 213 27

In their study to develop potent RXRα agonists, the group of Haffner synthesized the tetrahydrobenzoxazole-based propenoic acid 81. However, 81 was one of the least potent of the 16 compounds tested in the study.

1.5.5 Quinolines in medicinal chemistry

Quinolines are another important scaffold in medicinal chemistry, appearing in numerous alkaloids such as quinine 82 and skimmianine 83 .214 Quinoline based molecules possess many important biological properties including immunosuppressive, 215 antihelmintic, 216 antibacterial, 217 antifungal, 218 anti- inflammatory and antiviral activities. 219,220 Furthermore, quinolone has been incorporated into many pharmaceuticals, including chloroquine 84 (antimalarial) 221 and pitavastatin 85 (cholesterol). 222 28

In addition to these biological activities, quinolines have also been studied for their anticancer properties.223 The group of Koh developed diarlyamides and diarylureas with potent antiproliferative activity against 10 melanoma cell lines. Of the compounds tested, the 2,3-dihydrobenzo[b][1,4]dioxine analogues 86a and 86b were found to be

224 most effective, possessing IC 50 values of 27-460 nM over six of the cell lines.

Furthermore, Croisy-Delcey et al . synthesized diphenyl quinolones 87a and 87b with cytotoxic activity against the L1210 murine leukemic and MCF-7 human mammary carcinoma cell lines. 225

Quinolines have also been intensely investigated in the field of retinoid related molecules for many years. An in vitro screening of the effects of polycyclic aromatic hydrocarbons against the P19 mouse teratocarcinoma cell line identified several 29

quinolone scaffolds as able to inhibit t-RA mediated activity by 30-60%.226

Furthermore, the appendage of a quinolone residue to the dihydro-dimethylnaphthalene core, commonly found in synthetic retinoids, resulted in the generation of BMS614

(88 ), a selective RARα inverse agonist. 227,228

Eyrolles and his group investigated the differentiation activity of multiple classes of quinolone derivatives 89 -92 against the HL-60 human promyelocytic leukaemia cell line. They found that of the quinolones, only 89a displayed activity, with an ED 50 of 2.0

µM. Interestingly, all of the quinoline derivatives not only displayed activity, the ED 50 values were at least an order of magnitude lower than the quinolones. The strongest

229 performers were 90d and 90e, with ED 50 values of 46 and 20 nM, respectively.

Additionally, Beard et al . developed a series of N-aryl tetrahydroquinolines 93 -96 and studied their retinoid agonistic/antagonist properties. Notably, there was little difference 30

in activity between the alkynyl (93 ) and alkenyl (94) derivatives, both demonstrating high affinity for each of the RAR receptors, with K d values ranging between 3-13 and 5-

15 nM respectively. Changing to quinolone 95 had little effect on selectivity relative to

93 , but increased the K d values to 17-59 nM. Interestingly, utilization of the amide linker (96 ) was found to confer extreme selectivity for RARα (K d 27 nM) over RARβ

230 (K d >1000 nM) and RARγ (K d >1000 nM).

Kikuchi et al . designed pyrrole linked quinolines such as 97a-c with potent and selective RARα antagonistic activity. 231 It was found that methoxy derivative 97b had decreased RARα selectivity relative to 97a, as well as inducing RARβ transactivation.

Additionally, the incorporation of a phenyl group in 97c showed high levels of RARα selectivity, potent antagonistic activity against differentiation and no transactivation of other RAR receptors. 231

1.6 Computer-aided drug design

The advent of computer-aided drug design (CADD) techniques has revolutionised drug discovery, as it has allowed for the faster, less expensive and therefore more efficient discovery of bioactive compounds.232 CADD methodologies are frequently applied during the identification and optimisation process of lead compounds, in parallel with experimental techniques. 233-235

CADD methods may be applied to almost any drug design scenario, regardless of whether the three-dimensional structures of a protein target are available or not. 236

Where this structural information is absent, ligand-based approaches such as pharmacophore modelling and quantitative structure-activity relationship (QSAR) methods can be used to predict the activities of compounds that have not been tested experimentally. 233 Where the structure of a known target or a homologous protein is available, CADD methods can be used to predict ligand binding modes (docking) or protein structures (homology modelling), aiding the de novo design of novel compounds. 236 The use of computational techniques allows researchers to focus synthetic efforts, prioritise compounds for biological testing, or visualise possible molecular processes.

1.6.1 CADD techniques in atypical retinoid design

The most common role of computational techniques in retinoid design is in attempts to explain the activity of synthesized compounds, with many groups using computational docking methods to describe the potential binding modes of their ligands to the target

RAR(s). 163,237-239 However, the use of docking in preliminary screening studies or as a predictive technology has had relatively low application to date. 32

Silva and co-workers performed virtual screening of approximately 350,000 molecules from the Available Chemical Directory (ACD) database in order to identify new ligands for RAR isotypes. Using density functional calculations, natural bond orbital charges and multiple docking procedures, they identified three scaffolds 98 -100 as potential

RAR ligands. Additionally, by comparison with the binding modes of other RAR ligands, they proposed that 98 could be a pan-agonist, while 99 and 100 could be

RARγ -selective, as they have an increased number of H-bonds and hydrophobic interactions relative to the control ligands. 240,241

In a similar study, Stebbins et al . used a combination of scoring functions (FlexX,

Screening, Chemscore and DJ score) in a virtual screening campaign of a 50,000- compound subset of the Chem Bridge library to identify novel RXRα inhibitors 101-103.

The 200 top scoring compounds were first verified by competitive ligand binding studies, and subsequent in vitro transcriptional activation assays and receptor expression inhibition assays confirmed the RXRα inhibitory activity of 101-103.242

1.7 Thesis aims

The increasing frequency of cases of intrinsic or acquired retinoid resistance, 109 as well as the significant toxicities associated with conventional retinoid use, 101 highlights the need for the further development of synthetic retinoids that act through novel and selective mechanisms. Considering the limited chemical space that has been explored in the development of these drugs to date, the aim of this thesis is to develop novel atypical retinoids based on scaffolds identified by CADD methods. The new heterocyclic scaffolds that were identified over the course of the studies then became the subject of focus, due to their promising RAR activities.

Chapter 2 describes the identification of novel scaffolds through the in silico screening of a virtual library against the crystal structure of the RARβ -LBD, the subsequent in vitro validation of these scaffolds and the further development of predictive computational docking methods based on these results.

Chapter 3 explores the design, synthesis and in vitro evaluation of indole- benzothiazole acetamides capable of modulating RAR expression.

Chapter 4 covers the design, synthesis and in vitro evaluation of indole azoles as novel chemotherapeutics.

Chapter 5 details the design, synthesis and in vitro evaluation of benzothiazole- thieno[2,3-c]pyrazole carboxamides as RAR modulators.

Chapter 6 investigates the design, synthesis and in vitro evaluation of 8- isopropylquinolines as selective RARα antagonists or dual -action RARα antagonists/

RARβ agonists.

CHAPTER 2: DEVELOPMENT OF COMPUTATIONAL DOCKING

STUDIES FOR THE IDENTIFICATION OF RARβ LIGANDS

2.1 Introduction

The development of a new drug is a very complex process, requiring the collaboration of experts from a range of fields, employing many tools to identify lead candidates for further development. 243 This critical process is therefore very costly in terms of both money; costing in excess of $2.5 billion per drug, and time; typically taking 2-8 years for clinical trials and FDA approval.244 The introduction of computational-based methodology, capable of pre-screening candidate compounds to predict biological activity of compounds prior to their synthesis, has assisted medicinal chemists in streamlining this lengthy and arduous task. With computers rapidly increasing in processing power, the amount of time necessary to perform such preliminary analysis may be reduced, while providing high quality output, thereby reducing the number of unsuccessful hits, speeding up optimization of potential lead compounds and saving both time and money that would otherwise be spent on synthesis and biological assays. 245

Virtual screening is typically implemented in one of two ways: ligand-based or structure-based methods. Ligand-based methods analyse sets of structures with a common receptor target in order to identify common features that may be responsible for the ligand binding and subsequent target activation. The nature and 3-dimensional organisation of these features may then be mapped to generate a pharmacophore, against which novel ligands may be screened to identify ligands that will potentially interact with the target. Additionally, the mapping of such features can also be used in 35

the generation of QSAR models, whereby the mapped features can be correlated to the experimentally determined binding of similar ligands to the target receptor. The main advantages of using ligand-based screening methods is that they do not require any structural information about the target receptor, making them applicable to almost any screening scenario, and that they are a relatively quick and computationally inexpensive method, requiring a fraction of the time and computational cost of many structure-based methods. By comparison, structure-based methods rely on having access to the three- dimensional structure of the target receptor, either through NMR studies, X-ray crystallography or homology modelling of an analogous receptor. These methods then allow the docking of scaffolds or fragments, either as part of an existing or novel library, to an identified or proposed binding site and the fit to be determined by scoring functions based on various levels of theory depending on the method selected. These scoring functions can then be used to compare proposed structures to natural ligands or known binders, allowing for the prioritization of ligands for synthesis and further evaluation. Furthermore, such methods allow researchers to visualize their scaffolds within the binding pocket, allowing them to identify important features such as specific interactions between the ligand and receptor, amino acids that may potentially form interactions with functional groups, additional cavities that may be filled by appending groups and conformational restrictions that may confer more favourable binding modes to the ligand.

With the rapid growth of X-ray crystallography and NMR spectroscopy in generating quality 3D-models of protein structures, the in silico screening of compound collections, such as the National Cancer Institute (NCI) database and commercial compounds databases, represents an increasing trend in academic and industrial research towards the fast and efficient generation of focused compound libraries for specific protein 36

targets. 246 These virtual screening, as well as other molecular modelling techniques, have been widely applied in the pharmaceutical industry, contributing to the development of drugs such as Capoten, 247 Trusopt, 248 Viracept, and Relenza, 249,250 as well as anti-cancer agents Gleevec 104 and Tarceva 105.251,252

A number of different software packages have been developed to perform such virtual screens, relying on different levels of theory (molecular dynamics, molecular mechanics). Typically, such programs consist of two fundamental components: sampling and scoring. Sampling involves the generation of supposed ligand conformations near a protein binding site, and can be further subdivided into two aspects; ligand sampling and protein flexibility, while scoring is the prediction of the binding tightness for individual ligand conformations with a physical or empirical energy function. 253 However, such programs are still limited in their capacity as the output results depend heavily on the quality of the 3D-protein structure, the specific algorithms used to obtain the results and the power of available computational resources. Examples of common protein-ligand docking software packages are detailed below (Table 2.1). 254

Table 2.1: Common docking tools for protein-ligand virtual screening. 254

Method Ligand Sampling Scoring function DOCK incremental build force field or contact score FlexX incremental build empirical score Slide conformational ensembles empirical score FRED conformational ensembles Gaussian or empirical score GOLD genetic algorithm empirical score Glide exhaustive search empirical score Autodock genetic algorithm force field Ligandfit Monte Carlo empirical score ICM Pseudo-Brownian mixed force field & & local minimization empirical score QXP Monte Carlo force field

2.2 Preliminary library screening

In order to efficiently identify novel scaffolds as potential ligands for RARβ, an in silico screening involving computational docking methods and energy calculations was conducted through Quantum Pharmaceuticals. Small molecules from the 1,000,000 compound Enamine Ltd library were used, with all compounds conforming to

Lipinski’s rule of five. 255 3-D structural information of the RARβ -LBD, obtained from

X-ray crystallography, was accessed via the protein data bank (PDB ID: 1XAP). 129 This virtual screen returned a series of 26 compounds proposed to have suitable binding affini ties to RARβ, shown in table 2.2 below.

Table 2.2: The structures and predicted binding affinities of the proposed ligands.

Ligand Structure Predicted

Kd (M)

T0500 -6280 2.1 x 10-07 (L1)

T5302411 7.8 x 10-07 (L2)

T5321905 4.3 x 10-08 (L3)

T5347410 5.1 x 10-07 (L4)

T5392521 7.7 x 10-07 (L5)

T5446398 6.2 x 10-07 (L6)

T5449679 2.1 x 10-07 (L7)

T5460017 6.8 x 10-07 (L8)

T5481961 1.7 x 10-07 (L9)

T5497721 1.9 x 10-08 (L10)

T5514418 2.9 x 10-07 (L11)

T5578882 3.7 x 10-07 (L12)

T5990076 2.1 x 10-08 (L13)

T5628388 5.8 x 10-08 (L14)

T5682216 1.3 x 10-07 (L15)

T5693687 6.6 x 10-07 (L16)

T5729414 6.6 x 10-08 (L17)

T5793743 2.1 x 10-07 (L18)

T5822440 2.7 x 10-07 (L19)

T5950497 5.9 x 10-07 (L20)

T5999398 7.9 x 10-07 (L21)

T6025681 8.6 x 10-07 (L22)

T6028969 2.1 x 10-07 (L23)

T6030571 2.9 x 10-07 (L24)

T6033194 3.8 x 10-07 (L25)

T6043628 2.8 x 10-07 (L26)

In order to determine if these compounds possessed any cytotoxic activity, they were screened against BE(2)-C neuroblastoma cells, stably transfected with the MEP-4

85 expression vector to express either the empty vector (E.V.) or the RARβ 2 gene. The ligands were tested at a fixed concentration of 5 M over 96 h, after which the proportion of viable cells was determined by Alamar blue assay (n = 2) relative to solvent only (DMSO) control. Following a comparison of the effect in the two cell lines, it was observed that of these 26 compounds, 3 appeared to be more effective in the E.V. cell line (L11, L12 and L21), 2 had indistinguishable activity (L1 and L26) and the remaining 21 were more effective in the RARβ cell line (Figure 2.1).

Empty Vector RAR b 1 2 0

1 0 0

8 0

6 0

4 0

Cell Viability2 0 (% )

0 7 D L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 1 0 L 1 1 L 1 2 L 1 3 L 1 4 L 1 5 L 1 6 L 1 L 1 8 L 1 9 L 2 0 L 2 1 L 2 2 L 2 3 L 2 4 L 2 5 L 2 6

Figure 2.1: Cell viability (± SEM) of transfected BE(2)-C cells following 96 h

treatment with 5 M of ligand. 42

Of the 21 ligands that displayed increased cytotoxic activity towards the RARβ overexpressed cell line, it was found that 8 displayed cytotoxic activity with a differential of more than 20% between the RARβ overexpressed and E.V. cell line , indicating that these compounds may indeed exert their activity through the RAR β pathway (Figure 2.2). If we consider these 8 compounds as the leads identified by these studies, we can calculate that the virtual screening provided a hit rate of 31% for compounds displaying cytotoxicity to both cell lines, with at least 20% increased efficacy towards the RARβ overexpressed ce ll line. However, of these 8 ligands only

L10 displays a more marked differential between the two cell lines (>30%), therefore giving a hit rate of only 4% for highly potent and selective compounds.

Empty Vector RAR b 2

1 2 0

1 0 0

8 0

6 0

4 0

Cell Viability2 0 (% )

0 D L7 L9 L10 L13 L14 L15 L19 L23

Figure 2.2: Cell viability (± SEM) of BE(2)-C cells following 96 h treatment with 5 M

of top 8 ligands.

In order to determine if the predicted binding affinity (K d) of these 8 hit ligands could be correlated to their in vitro activity, a comparison was made between the percentage of viable cells following treatment and the K d value (Figure 2.3). Unfortunately, following a Pearson style analysis, it was observed that there was no significant linear correlation (r = 0.73, p = 0.10) between the in vitro activity of the ligands and the affinity predicted by the manufacturer. Furthermore, it was observed that of the five 43

-7 ligands with K d values less than 1.0 x 10 (L3, L10, L13, L14 and L17), two of these,

L3 and L17, did not show sufficient activity to pass the initial validation. Furthermore, this comparison is limited in that the Kd values are only predicted and not experimental, relying solely on the computational methods utilised by the company, as well as only covering a small range, being 2.51 x 10 -7 M for the top 8 hits and 8.41 x 10 -7 M for the entire 26 ligands.

1 0 0

8 0

6 0

4 0

2 0 Cell Viability (% )

0 0 1 .0 0 1 0 -7 2 .0 0 1 0 -7 3 .0 0 1 0 -7 K d (M)

Figure 2.3: Comparison of predicted binding affinity (K d) and observed in vitro

activity.

Considering the structures of these 8 lead molecules (Figure 2.4), it is apparent that due to the large structural differences, comparisons of other physical properties, such as cLog P, would not be able to further enhance an understanding of these compounds ’ activity. Furthermore, none of these compounds bears a carboxylic acid derivative, which is one of the key features of every retinoid derivative reported in the literature thus far. The closest functionality to this is the tetrazole present in L14, which is a common isostere for the carboxylate group. If these compounds, or subsequent analogues, were found to bind to an RAR, they would be the first non-carboxylate derivatives found to do so. However, the available information is unable to explain how these compounds would facilitate such binding and activation of the receptor, as no data 44

regarding the orientations of the ligands, or their interactions with the RARβ -LBD residues was provided. Such information is necessary not only to rationalize these results, but to assist in the development of analogues that are more potent in terms of their cytotoxicity, as well as their selectivity for t he RARβ cell line. Consequently, the development of in-house computational docking methods was investigated as an alternative. For the current work, the commercial protein-ligand docking engine GOLD was accessed through the Discovery Studio software.

Figure 2.4: Structures of the 8 lead molecules

2.3 The GOLD software package

The GOLD software package (Cambridge Crystallographic Data Centre, CCDC) is a popular docking program that employs a genetic algorithm to determine the best 45

binding mode of a ligand. 256 Such genetic algorithms mimic the process of evolution by following the principle of the survival of the fittest.257 A population of possible protein- ligand complex conformations (chromosomes) is initially created randomly and the fitness of each individual is evaluated based on a scoring function. Subsequently, random individuals (biased towards high fitness) are modified to provide new docking solutions through operations including mutations (small modifications to the protein- ligand conformation) and cross-overs (combination of two protein-ligand conformations). The efficiency of this process can be improved through the use of an island model, where the population is divided into sub-populations that develop independently, but are able to migrate to exchange genes. The use of an island model was found to improve the efficiency of the algorithm. 256 New docking solutions

(children) then replace the least-fitting members of the population niche they belong to, which is defined as a cluster of ligand poses where all H-bond donors and acceptors are within a 1.0 Å root-mean-square distance (RMSD). Repetition of this process then progresses the population towards the optimal solution.

The Goldscore fitness function consists of four components: protein-ligand hydrogen bond (external H-bond) and van der Waals (vdW) energies (external vdW), as well as a ligand-internal vdW (internal vdW) and torsional strain energies (internal torsion). 256

When flexible side-chains are used, a protein conformation score is added. The fitness score is the negative sum of the component scores. Weighting factors of the individual components can be applied. By default, the external vdW score is multiplied by 1.375, an empirical value which encourages hydrophobic protein-ligand contacts.

2.4 The RARβ protein

The crystal structure of the RARβ bound to the pan -RAR agonist TTNPB 13 was solved by Germain et al (PDB ID: 1XAP, Figure 2.5), enabling a much more efficient approach to developing computational screening methods towards selective RAR ligands by eliminating the need to construct homology models and develop pharmacophores. 129 The resolution obtained was 2.10 Ǻ, with an R-value of 0.312 and

R-free of 0.253, indicating an accurate representation of the crystal structure, which is necessary to construct accurate computational models.

Figure 2.5: The RARβ – TTNPB complex and the structure of TTNPB 13 . 47

The crystallized protein is truncated from the N-terminus, incorporating residues

Ala177 – Asn409, which make up part of the hinge domain and the entire ligand binding domain. Inspection of the ligand binding reveals two key polar residues involved in binding the deprotonated TTNPB 13 , namely Ser280 and Arg269.

Furthermore, a series of predominantly non-polar amino acids were identified as important residues, namely Phe221, Ala225, Leu259, Leu262, Ile266, Phe295, Arg387,

Val388 and Met406 (Figure 2.6).

Figure 2.6: Reported interactions for the RARβ LBD -TTNPB complex. 129

2.5 Establishing a docking protocol

All structures (protein and ligands) were constructed/edited with DS visualizer

(Discovery studio modelling, Accelrys Software Inc.). The protein was prepared by removing the TTNPB ligand and bound water molecules. Hydrogen atoms were added to the protein and the structure processed using the CHARMm forcefield. Ligand structures were prepared in a similar fashion. 48

The appropriate binding site was determined by detecting cavities, using a site opening of 5 Ǻ and grid resolution of 0.5 Ǻ, which identified an appropriate cavity of size 3138 point units. Based on the observed interactions in the RAR LBD-TTNPB complex, a subset of amino acids was selected to be considered as flexible during the docking protocol, namely Phe221, Ala225, Leu259, Leu262, Ile266, Phe295, Arg387, Val388 and Met406.

The TTNPB ligand was docked into the defined binding site using the Goldscore function, calculating 10 dockings, permitting flipping of ring corners, amide bonds, planar and pyramidal nitrogens and protonated carboxylic acids, and allowing intramolecular hydrogen bonds. The docking process resulted in different poses with the relevant docking scores used for comparison of the different binding poses obtained. It was observed that in this docking (purple), the predicted pose was quite different from that observed in the published complex (yellow). Not only was there significant rotation along multiple bonds, exemplified by heavy atom RMSD values of 0.43 – 2.44 Ǻ (0.95

Ǻ average) , the hydrogen bonding was only observed to one of the carboxyl oxygen atoms, not both as was observed previously (Figure 2.7). 49

Figure 2.7: Preliminary docking of TTNPB (green) results in significant

conformational changes and reduced H-bonds compared to the complex derived from

X-ray crystallography (yellow).

In order to procure a more accurate docking protocol, TTNPB was re-docked using the same protocol with a modified set of flexible amino acids, replacing Ala225 with

Arg269 and Ser280, the pair of residues responsible for the hydrogen bonding between

TTNPB and the protein (Figure 2.8). This set of flexible residues gave much stronger correlation to the literature conformation with highly conserved torsion angles and hydrogen bonding (Table 2.3), as well as RMSD values of 0.23 – 0.72 Ǻ (0.39 Ǻ average) and an average Goldscore of 72.65. 50

Figure 2.8: Modified docking of TTNPB (green) shows consistent binding with the

complex derived from X-ray crystallography (yellow).

Table 2.3: Comparison of torsion angles between the docked TTNPB and X-ray derived structure.

Pose θ1 θ2 Literature 34.65° -158.79° Experimental 34.91° -138.20°

The nature of TTNPB itself was further explored by repeating the docking with the protonated carboxyl group. This returned a greatly diminished hydrogen bonding profile, as well as vastly different conformation with RMSD values of 0.73 – 2.60 Ǻ

(1.86 Ǻ average) and a maximal Goldscore of 50.17, indicating that the deprotonated form is indeed the best ligand for comparison (Figure 2.9). 51

Figure 2.9: Docking of protonated TTNPB (purple) shows significantly altered binding

and reduced H-bonding compared to the complex derived from X-ray crystallography

(yellow).

2.6 Validation of the protocol with the lead compounds as a training set

In order to further validate the docking protocol, it was decided that a comparison of the generated Goldscores and biological activity of the original hit compounds would be the next logical step. Therefore, the eight biologically active hit compounds, along with the two least biologically active compounds, were docked into the RARβ LBD using the established protocol with modified flexible amino acid subset, calculating 100 poses.

Table 2.4 below details the distribution of the poses determined by their similarity within a given RMSD value (Å), the total number of clusters (No.) and the number of poses in the largest cluster. It also describes three potentially useful Goldscore values for analysing the poses; highest individual score of a pose, highest score of the largest cluster, and the average score of the largest cluster.

Table 2.4: Analysis of hit ligand docking results.

Ligand Clustering Goldscore Poses in Highest Cluster Cluster (Å) No. largest overall highest average T5449679 (L7) 1.98 25 11 71.39 63.70 54.46 T5481961 (L9) 1.99 24 22 58.37 54.89 45.34 T5497721 (L10) 1.99 14 19 70.23 63.78 52.89 T5990076 (L13) 2.00 27 17 66.58 66.58 56.19 T5628388 (L14) 2.00 25 15 63.69 57.97 46.80 T5682216 (L15) 2.00 22 17 67.19 67.19 52.61 T5822440 (L19) 1.89 35 12 61.53 54.47 47.69 T6028969 (L23) 2.00 14 37 58.82 58.82 48.09 T6025681 (L22) 1.99 37 9 64.41 53.79 43.88 T6030571 (L24) 2.03 15 49 55.05 55.05 48.26

It is possible to rank the ligands in three ways based on different analysis of the docking outputs. The first would be to rank them based on the overall highest score achieved, the second would be to rank them based on the highest score for the largest cluster, and the third would be to rank them by the average score of the largest cluster. Table 2.5 below details the rankings of each of the compounds based on the biological data and the three computational methods.

Table 2.5: Various ranking methodologies for the hit ligands.

Ligand Ranking Biological Highest Cluster Cluster activity overall highest average T5497721 (L10) 1 2 4 3 T5481961 (L9) 2 9 8 9 T5628388 (L14) 3 6 6 8 T5990076 (L13) 4 4 2 1 T6028969 (L23) 5 8 5 6 T5682216 (L15) 6 3 1 4 T5449679 (L7) 7 1 3 2 T5822440 (L19) 8 7 9 7 T6030571 (L24) 9 10 7 5 T6025681 (L22) 10 5 10 10

Initially, it was assumed that ranking the ligands based on the average score of the largest cluster would most closely reflect the ranking of the biological activity. This assumption was based on the thought that the average of the poses in the largest population would be a better representation of the actual state of the ligand, compared to any single orientation represented by a single score. However, upon comparing the difference between the rankings based on in vitro activity to any of the computational methods, it was found that ranking based on the highest score of the largest pose was marginally better than the other two methods (Table 2.6). On average, this method ranks the ligands closer to the correct rank, having a mean of 2.6 places difference, a median difference of 2.5, being at most 6 ranks off target and having 50% of rankings differing by ≤ 2. Considering this, the ranking method based on highest Goldscore of the largest cluster was selected as the most appropriate method for further investigations in this study. Interestingly, upon a Spearman style analysis of the ranking methods versus the biological activity, it was observed that there was no significant correlation for any 54

method (Table 2.6). However, ranking the ligands based on the highest score of their largest cluster did return the highest r s value (0.37) and lowest p value (0.30), further supporting this as the strongest of the three ranking methods.

Table 2.6: Differences between predicted and observed rankings.

Ligand Absolute Difference Highest Cluster Cluster overall highest average T5449679 (L7) 6 4 5 T5481961 (L9) 7 6 7 T5497721 (L10) 1 3 2 T5990076 (L13) 0 2 3 T5628388 (L14) 3 3 5 T5682216 (L15) 3 5 2 T5822440 (L19) 1 1 1 T6028969 (L23) 3 0 1 T6025681 (L22) 5 0 0 T6030571 (L24) 1 2 4 Mean 3 2.6 3 Median 3 2.5 2.5 Range 7 6 7

rs 0.15 0.37 0.19 p 0.68 0.30 0.61

For the analysis of docking results for novel scaffolds, a cut-off Goldscore of 56.00 for the highest score of the largest cluster has been proposed to differentiate between potentially active and inactive scaffolds (Figure 2.10). This value was selected because it excludes both biologically inactive ligands (L22 and L24), while including six of the eight active ligands in the appropriate region. This cut-off does however exclude L9 and

L19, despite L9 being the second most biologically active compound. Alternatively, a higher cut-off of 60.00 could be employed; however this would then exclude L14 and 55

L23 from the active region, which were the third and fifth most biologically active compounds, respectively.

7 0 Active region

6 0

G o ld5 s c o6 r e

Inactive region 5 0 L7 L9 L10 L13 L14 L15 L19 L23 L22 L24

L ig a n d

Figure 2.10: Proposed separation of active versus inactive Goldscores.

2.7 Conclusions

A comparison of the biological activity and predicted RARβ affinity of the initial 26 ligands demonstrated no significant correlation. A computational docking protocol utilizing the GOLD software package and RARβ crystal data was developed and subsequently employed to generate docking scores for the eight most active and two least active of the ligands. The closest matching between the observed biological data and Goldscore was obtained through comparison of the highest score of the largest cluster. A cut-off Goldscore of 56.00 was proposed to differentiate between potentially active and inactive novel scaffolds in the following chapters.

CHAPTER 3: DESIGN, SYNTHESIS AND CHARACTERIZATION

OF INDOLE-BENZOTHIAZOLE ACETAMIDES

3.1 Introduction

Amides are a common feature of many natural and synthetic compounds. In biology, the amide bond forms the structural link between the amino acids that form proteins. The delocalization of the nitrogen lone pair electrons gives the amide bond partial double- bond character between the nitrogen and carbonyl (Figure 3.1), making amides less basic than amines and also less susceptible to hydrolysis than esters. Furthermore, this partial double-bond character results in a planar configuration around the nitrogen atom, giving rise to cis - and trans -amide geometries, with the trans isomer being the dominant form. This biased geometry, as well as the formation of H-bonds, confers structure to proteins and polypeptides.

Figure 3.1: Delocalization of nitrogen lone pair electrons in amides.

Amide derivatives of 3-indoleacetic acid (IAA) 106 , a naturally occurring plant auxin, are an important class of biologically active molecules. 258 Cysmethynil 107 and its analogues display antitumor activity,259 while N-(pyridin-4-yl)-(indol-3-yl)acetamides

108 are antiallergic agents and 2 (1 H indol 3 yl) N phenylacetamides 109 possess antioxidant activity. 260,261 Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ 57

Derivatives of 2-aminobenzothiazole 110 have also been intensely studied as a particularly important subset of benzothiazoles, with the combination of 110 with other heterocycles being a common approach in the design of new drug-like molecules. A number of amide derivatives with anti-cancer activity include the 1,3,4-thiadiazole-2- thione conjugate 111, and benzothiazolyl urea 112.262,263

The development of indole and benzothiazole conjugates has generated scaffolds with remarkable biological activities. Piperazine linked systems such as 113 have shown selective agonistic activity towards dopamine D2 receptors, with useful antioxidant and anti-Parkinson’s disease activity. 264 Also, the benzyl linked 3-indoleacetamide 114 inhibits macrophage nitric oxide (NO) production by suppression of the iNOS protein and mRNA expression, while also suppressing the expression of COX-2 through NF-κB inactivation. 265 58

Considering the important activities of 3-indoleacetamides, 2-aminobenzothiazoles and their conjugates, this chapter is concerned with the development of amide based indole- benzothiazole hybrids as novel scaffolds with anti-tumour activities.

3.2 Identification of indole acetamides as a lead scaffold

An analysis of the 26 proposed ligands revealed that amide linkages were a common feature, being present in 15 of the ligands, as well as in 4 of the top 8 structures. Benz- fused heterocycles were also a common feature, being present in 10 of the 26 ligands and 3 of the top 8 structures. Interestingly, the most common types of benz-fused heterocycles in the 26 ligands were derivatives of 2-aminobenzimidazoles or 2- aminobenzothiazoles, which were also the moieties present in L10 and L15, the 1 st and

4th most active compounds respectively (Figure 3.2).

Figure 3.2: Lead structures showing amide linkages (blue) and benz-fused heterocycles

(red). 59

Examination of the docking results for L10 and L15 revealed that they displayed a similar orientation within the RARβ -LBD (Figure 3.3). It was observed that both ligands directed the benz-fused heterocyclic moiety towards the Arg269 and Ser280 residues. Furthermore, each ligand displayed several favourable interactions with the receptor residues. In the case of L10, the benzimidazole NH was observed to donate an

H-bond to the backbone carbonyl of the Leu262 residue, while the imidazole and pyrazole rings formed a π -π and π -σ interaction with the Phe221 and Leu259 residues res pectively. Additionally a π -cation interaction was also observed between the benzimidazole phenyl ring and the Arg265 residue. In the case of L15, the benzothiazole heterocycle was observed to form π -σ interactions with the Leu262 residue, while the termin al phenyl ring formed a π -cation interaction with the Arg387 residue. Furthermore, the benzimidazole NH also formed an intramolecular H-bond with the phenylacetyl carbonyl group, generating a 7-membered ring. This result suggests that the aromatic interactions are the dominant contributors to these binding modes.

Figure 3.3: Overlapped docking poses of L10 (green) and L15 (yellow) showing

similar orientations within the RARβ -LBD. 60

In order to determine appropriately-sized starting structures, indole amide scaffolds with linkers of various lengths (Table 3.1) were docked into the RARβ binding pocket following the method detailed previously (Chapter 2). 3-Indolealkylamides were selected as they possessed both the aromatic phenyl moiety of L15, as well as the heterocyclic feature of L10, and also because the 3-indolealkylamides are well established as bioactive moieties in the literature, as discussed previously in Chapters

1.5.1 and 3.1. Additionally, the use of varied alkyl chain lengths may allow the compounds to orientate themselves in a similar manner to L10 or L15, depending upon the length. Furthemore, substitution of the benzothiazole moiety for benzimidazole was also examined.

Table 3.1: Goldscores of the initially proposed scaffolds.

Ligand Goldscore Rank

66.72 2

63.73 3

66.81 1

58.11 4

Analysis of the docking results suggest that the combination of a 3-indoleacetamide moiety with either a benzothiazole (115), or a benzimidazole ( 116), would give ligands 61

with similar affinity for the RARβ -LBD, having docking scores of 66.72 and 63.73, respectively. This agrees with the prevalence of these heterocycles in the initial 26 ligands, with four occurrences of a benzothiazole and one occurrence of a benzimidazole. Gratifyingly, these scores were highly similar to those achieved by the lead compounds L10 and L15, which had docking scores of 63.78 and 67.19 respectively. It was also observed that the binding modes of 115 and 116 were quite similar, with both analogues directing the indole ring towards the hydrophobic RA Rβ cavity, and with the benzothiazole or benzimidazole moiety directed towards the

Arg269 and Ser280 residues (Figure 3.4). Additionally, the docking also suggests that the propionamide 117 is similar to acetamide 115 in terms of its binding orientation and binding affinity (Goldscore 66.81). However, carboxamide 118 showed a marked reduction in its Goldscore (58.11), suggesting that analogues of this size are incapable of filling the large binding cavity of RARβ.

Figure 3.4: Overlapped docking poses of 115 (green), 116 (yellow) and 117 (blue)

showing similar orientation s within the RARβ -LBD.

3.3 Synthesis of indole-benzothiazole amides

Coupling reactions are one of the most commonly used methodologies for the formation of amide bonds. They possess many advantageous properties over other amide forming reactions, such as their high rate of reaction , their ability to achieve near quantitative yield, the use of milder reaction conditions, the reduced incidence of substrate racemization, a lack of side reactions and the formation of easily removable side products. 266 l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is a popular coupling reagent that has been used in the synthesis of both indole and benzothiazole based amides. The Wu group treated a DCM mixture of indole-acid 119 and cyclohexylamine 120 with EDCI and 4-(dimethylamino)pyridine (DMAP), giving the anti-obesity indole-amide 121 in 50% yield (Scheme 3.1). 267

Scheme 3.1: Reagents and conditions: a) EDCI, DMAP, DCM.

Similarly, Kamal et al . treated a mixture of pyrazolo[1,5-a]pyrimidines 122, EDCI and

1-hydroxybenzotriazole (HOBt) with 2-aminobenzothiazoles 123 at 0 °C, followed by stirring at r.t. to give benzothiazole-amides 124 in yields of 72-85% (Scheme 3.2). 268

Scheme 3.2: Reagents and conditions: a) EDCI, HOBt, DCM, 0 °C - r.t. 8 h. 63

N,N-Dicyclohexylcarbodiimide (DCC) is another commonly used coupling reagent that has also been used in the synthesis of indole and benzothiazole amides. Duflos et al . heated a mixture of indole acid 125 and 4-aminopyridine 126 with DCC at reflux in

THF for 48 h. Following work-up, the N-pyridinyl-indole-3-(alkyl)carboxamides 127 were isolated in 58-66% yield (Scheme 3.3).269

Scheme 3.3: Reagents and conditions: a) DCC, THF, reflux, 48 h.

Similarly, Esteves et al . added 2-aminobenzothiazoles 128 to a DMF mixture of aspartic acid ester 129, DCC and HOBt at 5 °C. After 24 h at r.t., amides 130 were isolated in yields of 58 –91% (Scheme 3.4).270

Scheme 3.4: Reagents and conditions: a) DCC, HOBt, DMF, 0 °C – r.t., 24 h.

Aside from carbodiimide-based coupling reagents, the use of (benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) has also seen increased use throughout the literature. PyBOP has several advantages over earlier generation coupling reagents, such as the generation of a non-carcinogenic tripyrrolidinophosphate by-product, compared to the carcinogenic tris(dimethylamino)phosphate by-product produced by the use of the earlier

(benzotriazol-1-yloxy)tris (dimethylamino)phosphonium hexafluorophosphate (BOP) reagent, as well as a simpler work-up procedure and the ability to tolerate more difficult 64

coupling substrates. 271 Thompson et al . coupled 106 with anilines 131 by treating them with PyBOP in the presence of N,N-diisopropylethylamine (DIPEA) in chloroform

(Scheme 3.5). After 18 h, indolyl-phenylacetamides 132 were isolated in yields of 36-

73%.

Scheme 3.5: Reagents and conditions: a) PyBOP, DIPEA, CHCl 3, 18 h.

Similarly, Zhang et al . coupled acids 133 with 110 using PyBOP in the presence of

DIPEA in a DMF solution, giving amides 134 in yields of 65-87% (Scheme3.6).272

Scheme 3.6: Reagents and conditions: a) PyBOP, DIPEA, DMF .

Furthermore, previous work in our group has demonstrated that PyBOP is an effective and efficient reagent for the coupling of IAA 106 and 2-aminobenzothiazole 110, generating acetamide 115 in 53% yield after only 2 h stirring in the presence of DIPEA in DCM (Scheme 3.7).273 Consequently, PyBOP was selected as the primary means for the generation of indole-benzothiazole acetamides in this work.

Scheme 3.7: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h .

3.3.1 Synthesis of mixed heterocyclic acetamides

As the desired benzothiazole-based acetamide 115 was already at hand, it was decided to prepare a series of acetamides with various combinations of heterocycles to determine the SAR for this class of compounds. Firstly, the necessity of the indole heterocycle was investigated by substituting it with a naphthalene moiety. Replacement of the indolyl pyrrole ring with a benzene ring generates a more lipophilic molecule, which may increase bindin g affinity for the hydrophobic RARβ binding cavity.

Additionally, the lack of a hydrogen bond donor may have additional consequences for binding. Finally, by utilizing both the 1-naphthyl and 2-naphthyl acetic acids, the importance of the naphthalene moiet y’s directionality may also be explored. Hence, 2- aminobenzothiazole 110 was added to naphthaleneacetic acids 135a-b and PyBOP in

DCM, and after stirring for for 2h give acetamides 136a-b in yields of 37 and 41% respectively (Scheme 3.8).

Scheme 3.8: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h . 66

As an additional modification to the acid portion of the molecule, 3,4-

(methylenedioxy)phenylacetic acid 137 was also considered. This moiety has the heterocycle directed away from the amide linkage, as well as having two H-bond acceptors, as opposed to the H-bond donor of the indole. Therefore, 110 was coupled to

137 using PyBOP, generating amide 138 in a yield of 46% (Scheme 3.9).

Scheme 3.9: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h .

The identity of 136a, 136b and 138 were confirmed primarily through spectroscopic analysis. The 1H NMR spectra displayed consistent aromatic patterns with respect to the benzothiazole ring, while each aromatic acid displayed the expected splitting patterns

1 and integrations. As an example, the H NMR (d 6-acetone) spectrum of 136a showed the benzothiazole peaks as two doublets of doublets at δ 7.90 and 7.71 ( J = 0.7, 7.9 Hz) corresponding to H4 and H7, and two doublet of doublet of doublets at δ 7.42 and 7.29

(J = 1.3, 7.9, 7.9 Hz) corresponding to H6 and H5. The naphthyl CH 2 was identified as a singlet at δ 4.47, while the naphthyl aromatic peaks were identified as two doublets at δ

7.88 (J = 8.2 Hz, H4′) a nd 7.62 ( J = 6.7 Hz, H7′), three doublet of doublets at δ 8.19 (J

= 0.6, 7.4 Hz, H8′), 7.94 ( J = 2.0, 7.4 Hz, H5′) and 7.49 ( J = 8.3, 8.3 Hz, H3′ and H6′) and a doublet of doublet of doublets at δ 7.55 (J = 1.7, 7.2, 7.2 Hz, H7′).

It was also desired to investigate the SAR of the amine portion of the molecule. As benzimidazoles and benzoxazoles were also present in the initial 26 ligands, these heterocycles were targeted first. Not only would this allow for comparisons back to the lead structures, but it would also investigate the potential importance of hydrogen- bonding by substitution of the sulfur atom with the donor-capable NH group of 67

benzimidazole, or the acceptor-capable oxygen atom of benzoxazole. Furthermore, the resultant changes to polarity may also have an effect on solubility and efficacy of the compound. Therefore, 106 was reacted with 2-aminobenzimidazole 139a or 2- aminobenzoxazole 139b according to the PyBOP procedure developed earlier (Scheme

3.10). In the case of 139b, acetamide 140 was isolated in 62% yield. However, the reaction with 139a did not produce acetamide 116, even after extended durations up to

48 h, possibly due to the low solubility of 139a in DCM. The use of acetonitrile or DMF as solvents also did not afford the desired 116. However, the use of EDCI and HOBt in place of PyBOP generated 116 in a yield of 37% (Scheme 3.10).

Scheme 3.10: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h ; b) EDCI, HOBt,

DMF, 24 h.

The 1H NMR spectra of 116 and 140 both displayed consistent patterns for the indolyl moiety, with different splitting patterns for their benz-fused heterocycles as a result of the electronic effects of their varied heteroatoms. Singlet peaks were observed at δ

1 11.94, 11.63, 10.90 and 3.86 in the H NMR (d 6-DMSO) spectrum of 116, assigned as the benzimidazolyl NH, indolyl NH, amide NH and CH 2 protons respectively. Doublets were observed at δ 7.65 (J = 8.1 Hz), 7.36 ( J = 8.1 Hz) and 7.30 ( J = 2.2 Hz), assigned as H4, H7 and H2 respectively. A doublet of doublet of doublets at δ 6.99 (J = 1.2, 7.2,

7.2 Hz) was assigned as H5, while multiplets at δ 7.41 and 7.05 were assigned as

1 H4′/H7′ and H6/H5′/H6′ respectively. In the H NMR (d 6-DMSO) spectrum of 140 68

singlet resonances were observed at δ 11.80, 10.96 and 3.92, corresponding to the indolyl NH, amide NH and CH 2 protons, respectively. Doublet of doublet of doublets were observed at δ 7.36 (J = 1.2, 1.2, 8.0 Hz), 7.08 ( J = 1.2, 8.0, 8.0 Hz) and 6.99 ( J =

1.2, 8.0, 8.0 Hz) corresponding to H7, H6 and H5 respectively. Multiplet resonances at

δ 7.61-7.55 and 7.30-7.27 were assigned as H4/H4′/H7′ and H5′/H6′ respectively.

Quinoline was also selected as another heterocycle to investigate in the context of the

SAR of these amides. As detailed in Chapter 1.5.5, quinolines possess many important biological properties and have also been explored as retinoid modulators. Subsequently,

106 and 8-aminoquinoline 141 were coupled using PyBOP to generate acetamide 142 in

52% yield (Scheme 3.11).

Scheme 3.11: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h.

1 The H NMR (d 6-acetone) spectrum of 142 displayed characteristic singlet resonances at δ 11.01, 10.16 and 4.01, assigned as the indolyl NH, amide NH and CH 2 protons respectively (Figure 3.5). Doublets were observed at δ 7.63 (J = 7.1 Hz), 7.53 ( J = 3.1

Hz), 7.44 ( J = 2.4 Hz) and 7.40 ( J = 8.1 Hz), assigned as H4, H6′, H2 and H7, respectively. Additionally, doublet of doublet resonances were observed at δ 8.73 (J =

1.7, 4.2 Hz), 8.65 ( J = 1.5, 7.8 Hz), 8.34 ( J = 1.7, 8.3 Hz), 7.60 ( J = 1.5, 7.8 Hz) and

7.57 (J = 1.7, 1.7 Hz), assigned as H4′, H7′, H2′, H5′ and H3′ respectively. Doublet of doublet of doublets were also observed at δ 7.09 (J = 2.5, 7.5, 7.5 Hz) and 6.99 ( J = 1.1,

7.5, 7.5 Hz), assigned as H6 and H5 respectively. 69

1 Figure 3.5: H NMR spectrum of 142 in d 6-acetone.

Finally, 3-indoleacetic acid 106 was coupled to 2-aminothiazole 143 to give acetamide

144 in 62% yield (Scheme 3.12). It was thought that by removing the benzene ring, it would be possible to confirm the importance of steric effects and possible π-interactions in this region of the molecule.

Scheme 3.12: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h.

3.3.2 Synthesis of indole-benzothiazole amides with modified linkers

Further development of the SAR was next targeted towards the linking region of the amides. The preliminary docking studies suggested that increasing the length of the linker by one methylene unit does not affect the predicted affinity of the molecule, as was shown by the similar Goldscores of acetamide 115 (66.72) and propionamide 117

(66.81), allowing different orientations of the ligand within the receptor to be explored.

Additionally, it was also predicted that reducing the length of the linker would dramatically reduce affinity, demonstrated by the relatively low Goldscore of carboxamide 118 (58.11).

As it was predicted to have the most similar binding affinity to acetamide 115, propionamide 117 was first targeted. PyBOP coupling of 3-indolepropionic acid 145 to

110 gave propionamide 117 in a yield of 73% (Scheme 3.13). The 1H NMR (d6-

DMSO) spectrum of propionamide 117 differed from that of acetamide 115 primarily in the alkyl region. Two triplets were observed at δ 3.07 and 2.87 ( J = 7.1 Hz) corresponding to the two CH 2 protons, compared to the singlet in the spectrum of 115, which had only the one CH 2 group. Additionally, the indolyl NH proton was observed to give a resonance slightly upfield at δ 12.36, relative to δ 12.54 for 115.

Scheme 3.13: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h.

As the longer linker was predicted to have slightly higher affinity relative to the acetamide, further analogues with this length were considered attractive targets for synthesis. The replacement of the indolyl methylene group with a sulphur atom was considered a promising option as such linkages were observed throughout many of the 71

initial 26 ligands. Furthermore, the subtle modifications to the hydrophobicity and size of the linker may also have further effects on the affinity of these molecules to the

RA Rβ binding site. In order to achieve this, the method of Levkovskaya was followed, 274 whereby indole 30 was reacted with thiourea in the presence of potassium iodide and iodine in THF over 3 days to generate the intermediate thiouroniumindole iodide 146. Treatment of this intermediate with potassium hydroxide then gave the corresponding thiol 147 in 61% yield. This was then converted to the required (3- indolesulfonyl)acetic acid 148 in 54% yield by treatment with chloroacetic acid in the presence of potassium hydroxide. Finally, PyBOP coupling to 110 gave the desired amide 149 in 87% yield (Scheme 3.14).

Scheme 3.14: Reagents and conditions: a) thiourea, I 2, KI, MeOH/H 2O, 3 days; b) i. 2

M NaOH, 12 h, ii. 2 M HCl; c) chloroacetic acid, KOH, EtOH, 12 h; d) PyBOP,

DIPEA, DCM, 2 h.

As an alternative to extending the length of the acyl chain, the separation of the benzothiazole heterocycle and amide group was considered as another point of modification. It was thought that incorporating a hydrazide moiety, rather than amide, may also confer a more rigid structure through the possible generation of an intramolecular hydrogen bond, as well as again modifying the hydrophobicity of the 72

ligand. However, when 106 was coupled to 2-hydrazinobenzothiazole 150 using PyBOP conditions, the expected hydrazide 151a was not the isolated product, nor was the tautomeric 151b (Scheme 3.15).

Scheme 3.15: Reagents and conditions: a) PyBOP, DIPEA, DCM, 18 h.

1 The H NMR (d 6-DMSO) spectrum of the product showed the expected aromatic peaks for both the indole and benzothiazole moieties (Figure 3.6). The indolyl NH and CH 2 protons appeared as singlets at δ 10.37 and 4.35 respectively, while H4 and H7 gave doublets ( J = 7.9 Hz) at δ 7.55 and 7.36 respectively. The doublet of doublet resonances

(J = 7.9, 7.9 Hz) corresponding to H5 and H6 were observed at δ 6.99 and 7.08 respectively, while H2 overlapped with the be nzothiazole H6′ at δ 7.34-7.29. The remaining benzothiazole protons were observed as doublet of doublets (J = 0.6, 7.9 Hz) at δ 7.95 (H4′) and 7.82 (H7′), with H5′ givng a doublet of doublet of doublets ( J = 1.3,

7.3, 7.3 Hz) at δ 7.46. There was also an additional singlet observed at δ 5.84, integrating for 2 protons, which could not be accounted for by structures 151a or 151b.

A D 2O exchange was also conducted, which resulted in the suppression of this signal in

1 the H NMR (d 6-DMSO + D 2O) spectrum confirming that the protons at δ 5.84 are exchangeable. Additionally, the HRMS (+ESI) identified the [M+H]+ peak at m/z

323.0964, corresponding to the formula of 151a-b: C17 H15 N4OS (required m/z 73

323.0961). Therefore, the structure was assigned as N-(benzo[d]thiazol-2-yl)-2-(1 H- indol-3-yl)acetohydrazide 152, which was isolated in a yield of 89%.

1 Figure 3.6: Comparative H NMR (d 6-DMSO: blue, d 6-DMSO + D 2O: red) spectra of

the unexpected structure 152.

Although unexpected, structures similar to 152 are not entirely unprecedented in the literature. The group of Adam et al . reported hydrazide 153, achieved via a rearrangement of the corresponding hydrazono penicillanate, 275 while Haviv et al . prepared oxazole 154 utilizing a DCC mediated coupling. 276 The benzoxazole 154 is of particular interest as it was also prepared through the use of a peptide coupling reagent, which typically is expected to give the N′-hydrazides ( 151a-b).

Figure 3.7 : Structures of reported hydrazides similar to that of 152. 74

Finally, the synthesis of a carboxamide was targeted in order to determine what effects a reduction in linker length would have on the cytotoxicity of these scaffolds. In order to achieve this, indole-2-carboxylic acid 155 and 110 were coupled under PyBOP conditions to generate carboxamide 156 in 83% yield (Scheme 3.16).

Scheme 3.16: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h .

3.3.3 Synthesis of analogues bearing substituents on indolyl or benzothiazolyl rings

In drug design, substitution about a heterocyclic ring system can have dramatic effects on the biological activity of a molecule. This may be through modulating the hydrophobicity of the compound and increasing its membrane permeability, blocking metabolic sites and increasing half-life, or generating stronger or different interactions with a binding site to improve the efficacy and/or improve the toxicity profile of the compound.

Figure 3.8: Lead structures containing substituted benzothiazoles.

Interestingly, of the four occurrences of the benzothiazole moiety in the initial twenty six ligands, two of these had appended substituents, both of which were methyl groups

(Figure 3.8). In the case of L11, the substituent was located at the benzothiazole C6 position, while in L15 the substituent was located at the benzothiazole C4 position. As 75

the molecular modelling predicted that the benzothiazole moiety would be oriented towards the Arg269 and Ser280 residues responsible for binding to t-RA 3, substitution about this ring, particularly at C6, was considered a crucial aspect of the SAR.

Consequently, 106 was coupled to a range of substituted 2-aminobenzothiazoles 157a-l following the standard PyBOP coupling discussed earlier in this chapter, giving amides

158a-l in good yields of 41-71% (Scheme 3.17).

Scheme 3.17: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h.

The 1H NMR spectral data of amides 158a-l all displayed consistent signals for the indolyl portion, differing only in the splitting patterns and chemical shifts of the substituted benzothiazole protons. The 6-methoxy derivative 158e had representative 1H

NMR (d 6-acetone) spectral data for analogues 158a-g (Figure 3.9). Three singlets were observed at δ 10.93, 10.25 and 3.84, corresponding to the indolyl NH, amide NH and methoxy protons, respectively. Two doublets were observed at δ 7.46 (J = 2.5 Hz) and

4.04 (J = 0.8 Hz), corresponding to H7′ and the CH 2 protons, respectively. Resonances assigned to H7, H4′ and H5′ respectively, were observed as doublet of doublets at δ 7.68

(J = 0.6, 7.9 Hz), 7.56 ( J = 0.4, 8.9 Hz) and 6.99 ( J = 0.6, 8.9 Hz). Two doublet of 76

doublet of doublets resonances were observed at δ 7.12 (J = 1.2, 7.0, 7.0 Hz) and 7.04 ( J

= 1.2, 7.9, 7.9 Hz) assigned as H6 and H5, while H4 was observed as a multiplet at δ

7.41.

1 Figure 3.9: H-NMR (d 6-acetone) spectrum of 158e.

1 Similarly, the H-NMR (d 6-acetone) spectral data of 155k was representative of analogues 158i-k (Figure 3.10). Singlet resonances were observed at δ 12.66, 10.98,

3.91 and 3.89, assigned as the indolyl NH, amide NH, methoxy and CH 2 protons, respectively. One doublet signal was observed at δ 7.31 (J = 2.4 Hz) corresponding to

H2, while two doublet of doublets were observed at δ 7.49 (J = 0.9, 8.0 Hz) and 7.23 ( J

= 8.0, 8.0 Hz), assigned as H7′ and H6′ respectively. The remaining protons ga ve doublet of doublet of doublets at δ 7.60 (J = 1.0, 1.0, 8.0 Hz), 7.36 ( J = 1.0, 1.0, 8.0 Hz),

7.08 (J = 1.0, 8.0, 8.0 Hz) and 6.99 (( J = 1.0, 8.0, 8.0 Hz), assigned as H4, H7, H6 and

H5/H5′ respectively. 77

1 Figure 3.10: H-NMR (d 6-acetone) spectrum of 158k.

The next consideration was to determine if the addition of substituents on the indolyl ring would have any impact upon the SAR. There are many reactions for preparing 3- substituted indoles, such as the Bartoli, Fukuyama, Larock and Madelung methods. 277-

280 However, these methods typically produce indoles that also bear substituents at the

2-position. As it was first desired to produce indoles bearing a single substituent, other methods were investigated.

Initial attempts aimed at producing substituted 3-indoleacetic acids via alkylation of the reactive indole 3-position were unsuccessful. Following the method of Kumar et al. ,281 a methanolic solution 5-methoxyindole 159 was heated at reflux with ethyl chloroacetate in the presence of potassium carbonate for 8 h. However, this did not yield the desired

5-methoxyindole-3-acetic acid 160, returning only unreacted starting material (Scheme

3.18). 78

Scheme 3.18: Reagents and conditions: a) ethyl chloroacetate, MeOH, reflux, 8 h.

The Wolff-Kishner-type reduction of 3-indoleglyoxylic acids reported by Petit et al. was next investigated as a means of generating the desired 3-indoleacetic acids. 282

Treatment of 159 with oxalyl chloride in anhydrous ether at 0 °C, followed by quenching with a saturated solution of aqueous sodium bicarbonate produced glyoxylic acid 161 in a high yield of 91%. However, heating 161 at 150 °C with hydrazine and sodium methoxide in 2-methoxyethanol did not generate the desired 162 (Scheme 3.19).

Scheme 3.19: Reagents and conditions: a) i. oxalyl chloride, Et 2O, 0 °C, 2 h. ii. aq.

NaHCO 3; b) NH 2NH 2, NaOMe, 2-methoxyethanol, 150 °C, 1 h.

Demopoulos reported a catalytic reduction of the glyoxylate esters, which was next investigated. Indole 159 was first treated with monoethyl oxalyl chloride in ether to generate ester 163 in a yield of 69%. The ester 163 was then treated with sodium hypophosphite in the presence of 10% palladium on charcoal and heated at reflux in 1:1 dioxane/water for 48 h. However, this again did not yield the desired ester 160 (Scheme

3.20). 79

Scheme 3.20: Reagents and conditions: a) monoethyl oxalyl chloride, Et 2O, 0 °C, 4 h;

b) NaHPO 4, Pd/C, dioxane/H 2O (1:1), 48 h.

As the required indole acetic acids could not be produced by direct alkylation or reduction of glyoxylates, attention was subsequently turned to the Fischer indole synthesis. Accordingly, phenyl hydrazine 164 and ethyl levulinate 165 were heated at reflux in glacial acetic acid in the presence of sodium acetate for 3 h. The solvent was removed and the crude intermediate 166 heated at reflux in 4 M HCl/dioxane for 15 h to give ester 167 in 25% yield. Hydrolysis of ester 167 with ethanolic sodium hydroxide, followed by acidification with 2 M HCl then gave acid 168 in 90% yield (Scheme 3.21).

Scheme 3.21: Reagents and conditions: a) AcONa, AcOH, reflux, 3 h; b) 4 M

HCl/dioxane, reflux, 15 h; c) i. NaOH, EtOH, 1 h, ii. 2 M HCl. 80

Upon attempting to utilize this procedure with substituted hydrazines, only a thick black residue was obtained, from which no product could be isolated by column chromatography. In order to overcome this, a modified procedure was developed in which hydrazines 169a-b and levulinic acid 170 were heated at reflux in a 1:1 mixture of conc. HCl and toluene for 3 h, generating acids 171a-b in yields of 34 and 67%, respectively (Scheme 3.22).

Scheme 3.22: Reagents and conditions: a) HCl:toluene (1:1), reflux, 3 h.

With the required substituted indoles now in hand, the corresponding acetamides could be prepared. Subsequently, indole acids 168 and 171a-b were coupled to 110 using

PyBOP in the presence of DIPEA in DCM to give acetamides 172a-c in yields of 39-

1 51% (Scheme 3.23). The H NMR (d 6-DMSO) of 172c was representative for these compounds, displaying five singlet resonances at δ 12.51, 10.74, 3.83, 2.38 and 2.34, corresponding to the indolyl NH, amide NH, CH 2, C2 methyl and C5 methyl protons, respectively. Two doublets were observed at 7.31 ( J = 1.3 Hz) and 7.13 ( J = 8.2 Hz), assigned as H4 and H7, respectively, with the doublet of doublets at δ 6.81 ( J = 1.3, 8.2

Hz) assigned as H6. The four doublet of doublet of doublets at δ 7.94 (J = 0.6, 1.2, 7.2

Hz), 7.74 ( J = 0.6, 1.2, 8.0 Hz), 7.43 ( J = 1.3, 7.2, 7.2 Hz) and 7.28 ( J = 1.2, 8.0, 8.0

Hz) were assigned as H4′, H7′, H6′ and H5′ respectively. 81

Scheme 3.23: Reagents and conditions: a) PyBOP, DIPEA, DCM, 2 h.

3.4 Characterization of in vitro activity against neuroblastoma cell lines

In order to determine the cytotoxic activity of the synthesized indole-benzothiazole amides towards neuroblastoma cells in vitro , the compounds were screened against the

BE(2)-C neuroblastoma cell lines stably transfected with the MEP4-empty vector (E.V.) and MEP4-RARβ constructs, which were utilized in the preliminary library screen. The cells were treated with compound at concentrations ranging from 0.1-20 µM over 72 h and the percentage of viable cells was measured by the Alamar blue assay. The treatment was conducted in 6 replicate wells and the experiment repeated three times (n

= 3). Dose response curves were generated and the IC 50 values were determined using the GraphPad Prism 6 software package (Table 3.2).

Table 3.2: Cytotoxicity of indole-benzothiazole amides towards empty vector and

RARβ stably transfected BE(2)-C neuroblastoma cells following 72 h exposure.

Compound IC 50 (M) E.V. RARβ 115 1.5 0.9 136a 33.8 23.5 136b 10.1 8.4 138 >50 31.1 140 >50 >50 116 >50 >50 142 >50 26.2 144 >50 >50 117 15.8 10.8 149 30.5 18.3 152 >50 >50 156 >50 >50 158a 16.1 14.1 158b 23.5 25.7 158c 20.1 23.5 158d 16.5 15.4 158e 37.1 34.2 158f 42.1 41.7 158g 9.0 9.9 158h 6.5 4.1 158i 3.6 2.1 158j 7.5 5.1 158k 14.2 8.3 158l 12.1 10.7 172a 5.4 12.5 172b 3.2 5.0 172c 5.8 5.8

Overall, the cytotoxicity of these compounds was observed to be relatively strong, with the majority of compounds possessing either a low IC 50 value, selectivity towards the

RARβ overexpressing cell s, or both. It was observed that indole-benzothiazole amide

115 possessed the lowest IC 50 values of 1.5 µM for the E.V. and 0.9 µM for the RARβ 83

overexpressing cells. This was a promising result as it showed selectivity towards the

RARβ overexpressing cells (1.7 times) at such a low concentration, similar to what had previously been shown in the SH-SY5Y neuroblastoma cell line where it had an IC 50 value of 1.1 µM. 273

It was also interesting to see that changing either of these heterocycles resulted in a reduction of activity. Replacement of the indolyl moiety in 115 with naphthalene in

136a-b resulted in decreased activity towards both cell lines, with 136a having IC 50 values of 33.8 and 23.5 µM for the E.V. and RARβ overexpressing cells respectively, and 135b having IC 50 values of 10.1 and 8.4 µM for the E.V. and RARβ overexpressing cells respectively. Furthermore, while both 151a and 151b had similar levels of selectivity towards the RARβ cell line (1.4 and 1.2 times, respectively), the 2-napthyl derivative 136b was much more potent (3-fold) than the corresponding 1-napthyl derivative 136a. This suggests that the orientation of the heterocycle is important for both activity and selectivity. Additionally, the methylenedioxy derivative 138 displayed reduced activity compared to 115, possessing IC 50 values of >50 and 31.1 µM for the

E.V. and RARβ overexpressing cells, respectively . Furthermore, upon the change from a phenyl ring in 136b to the methylenedioxy ring in 138, the activity in both cell lines is reduced, suggesting that the presence of a second aromatic ring is favoured to a 5- membered methylenedioxy ring. Substitution of the benzothiazole sulphur atom in 115 with either an NH group ( 116) or oxygen atom ( 140) resulted in a loss of activity, with both 116 and 150 possessing IC 50 values above 50 µM in both cell lines. This is quite an interesting result considering the similar docking scores of 115 and 116 (66.72 and

63.73 respectively). Replacement of the benzothiazole in 115 with quinoline in 142 resulted in decreased cytotoxicity, but increa sed selectivity towards the RARβ cell line

(IC 50 : >50 and 26.2 µM). Additionally, replacing the benzothiazole of 115 with a 84

thiazole in 144 resulted in a loss of cytotoxicity, suggesting that the size of the heterocycle and the ability to form hydrophobic interactions is highly important in this region of the molecule.

With regard to the amide linker, it was observed that increasing the length of the linker by one methylene unit (as in 117) reduced the cytotoxicity relative to acetamide 115, but retained similar selectivity towards the RARβ cell line (IC 50 : 15.8 and 10.8 µM, 1.5 times selectivity). This is despite the near identical Goldscores of 115 and 117 (66.72 and 66.81 respectively). Furthermore, replacement of the additional methylene with a sulfur atom in 149 increased the selectivity towards the RARβ cell line, relative to 117, albeit at the cost of cytotoxicity (IC 50 : 30.5 and 18.3 µM, 1.7 times selectivity).

Additionally, changing the amide of 115 to a hydrazide in 152 resulted in a loss of cytotoxic activity, with 152 possessing IC 50 values above 50 µM in both the E.V. and

RARβ overexpressing cell line s. Similarly, reducing the length of the linker (as in carboxamide 156) also resulted in IC 50 values above 50 µM in both cell lines.

Overall, these results show that the combination of the indole and benzothiazole heterocycles give the most potent scaffold (as in 115). Changes to the type of heterocycle, or the positioning of the amide substituent around the heterocycle result in a loss of cytotoxicity, but may increase the selectivity towards the RARβ overexpressing cell line. Bulkier heterocycles are preferred, particularly those with more hydrophobic character as opposed to the incorporation of electronegative atoms capable of participating in hydrogen bonds. Lengthening the linking region reduces cytotoxicity whilst maintaining selectivity towards the RARβ overexpressing cell line over the E.V. cell line. However, reducing the length resulted in a complete loss of activity. Taken together, these data suggest that both the length of the linker and the orientation of the heterocyclic moieties are important for cytotoxicity. 85

Upon investigation of the biological activity of the benzothiazole substituted analogues, it was observed that 6ɂ -substituted analogues 158a-g generally showed large reductions in activity. The effect also seemed proportional to the size of the substituent, with larger substituents generally resulting in less active compounds. Considering the halogenated analogues 158a-c, the 6-fluoro derivative 158a showed some selectivity towards the

RARβ cell line, with IC 50 values of 16.1 and 14.1 M in the E.V. and RARβ cell lines respectively. Increasing the size of the halogen to chlorine in 158b or bromine in 158c was observed to further reduce the cytotoxic activity, with 158b having IC 50 values of

23.5 and 25.7 M against the E.V. and RARβ overexpressing cell lines, and 158c having IC 50 values of 20.1 and 23.5 µM against the E.V. and RARβ overexpressing cell lines, respectively. Notably, the incorporation of halogens at the benzothiazole C6 led to inversion of the cell line selectivity, with the compounds being more effective in the

E.V. cell line.

Similar trends were observed with the alkyl derivatives 158d-g. Methyl derivative 158d showed similar levels of cytotoxicity and selectivity to fluoro derivative 158a, with IC 50 values of 16.5 and 15.4 M in the E.V. and RARβ cell lines respectively. Subsequent ly increasing the size of this substituent to methoxy ( 158e, IC 50 : 37.1 and 34.2 µM) and ethoxy ( 158f, IC 50 : 42.1 and 41.7 µM) resulted in further decreases to cytotoxicity with no significant impact to selectivity. Interestingly, the large trifluoromethyl group of

158g improved cytotoxicity relative to 158a and 158d, possessing IC 50 values of 9.0 and

9.9 M against the E.V. and RARβ cell lines respectively. Furthermore, thi s analogue was also observed to have inverted cell line selectivity, similar to 158a-c.

The positioning of the substituent about the benzothiazole ring was also found to be an important factor. The 4,6-difluoro analogue 158h was observed to have increased cytotoxicity compared to the 6-fluoro derivative 158a, with IC 50 values of 6.5 and 4.1 86

M in the E.V. and RARβ cell lines respectively. Furthermore, this increased activity was accompanied by an increase in selectivity towards the RARβ cell line, which is opposite to what was observed for 158a. Substituting the benzothiazole C4 position resulted in analogues with increased cytotoxicity compared to the respective C6 analogues, as well as increased selectivity towards the RARβ cell line. The 4-chloro derivative 158i possessed IC 50 values of 3.6 and 2.1 µM against the E.V. and RARβ cell lines respectively, making it more than 6 times as potent as the 6-chloro derivative

158b. Increasing the size of this substituent reduced cytotoxicity but maintained RARβ selectivity for this series of compounds. 4-Methyl derivative 158j possessed IC 50 values of 7.5 and 5.1 M against the E.V. and RARβ cell lines respectively, and was twice as potent as the corresponding 6-methyl analogue 158d. Similarly, 4-methoxy derivative

158k possessed IC 50 values of 14.2 and 8.3 µM against the E.V. and RARβ cell lines respectively, and was also twice as potent as 6-methoxy derivative 158e. Finally, substitution at the benzothiazole C5 position increased cytotoxicity relative to the corresponding C6 analogue, while also maintaining selectivity for the RA Rβ cell line.

5-Bromo derivative 158l possessed IC 50 values of 12.1 and 10.7 µM against the E.V. and RARβ cell lines respectively, making it approximately twice as potent as the 6- bromo analogue 158c. 87

Figure 3.11: Overlapped docking poses of 158b (green) and 158i (yellow) showing

directionality of their chlorine substituents.

In order to better understand these results, computational docking of these substituted benzothiazole scaffolds was performed. The docking results proposed that the 6-chloro and 4-chloro derivatives 158b and 158i would bind to the RARβ binding pocket with a similar affininty, based on their Goldscores of 70.55 and 66.04 respectively. Inspection of the binding poses of these ligands shows that the benzothiazole heterocycle sits in a similar position in both analogues, with the chlorine atom of 158b directed towards the

Ser280 residue, while the chlorine atom of 158i is directed towards a more hydrophobic region near the Leu262 and Ile266 residues (Figure 3.11). Considering that 158b showed selectivity for the E.V. cell line whilst 158i displayed selectivity for the RARβ cell line, the positioning of this substituent may dictate that selectivity.

As the 4-chloro compound 158i had shown good levels of activity, as well as selectivity for the RARβ overexpressing cells over the E.V. transfected cells, it was of interest to determine if this compound acted selectively towards RARβ, compared to the other

RAR family proteins RARα and RARγ . Therefore, 158i was screened against BE(2)-C 88

neuroblastoma cell lines stably transfected with MEP4-RARα an d MEP4-RARγ and the

IC 50 values were then determined (Table 3.3). Interestingly, 158i had an IC 50 value of

3.1 M against the RARα overexpressing cell line, which was similar to the activity it possessed against the E.V. cell line (IC 50 3.6 µM). However, the treatment of the RARγ overexpressing cell line with 158i resulted in an IC 50 value of 2.3 µM, which was more similar to what was observed in the RARβ overexpressing cell line (IC 50 2.1 µM). This result suggests that the cytotoxic activity of these compounds is more specific to the

RARβ and γ subtypes, with the overexpression of these receptors possibly enhancing the cytotoxicity of these compounds in neuroblastoma cells.

Table 3.3: Cytotoxicity of 158i towards transfected BE(2)-C neuroblastoma cells overexpressing three RAR isoforms following 72 h exposure.

Compound IC 50 (M) E.V. RARβ RARα RARγ 158i 3.6 2.1 3.1 2.3

Biological testing of the analogues bearing substituents on the indolyl ring ( 172a-c) revealed that the molecules are sensitive to changes in the substitution pattern of this moiety. Incorporation of the 2-methyl group ( 172a, IC 50 : 5.4 µM in E.V. and 12.5 µM in RARβ ) reduced cytotoxicity compared to 115 (IC 50 : 1.5 µM in E.V. and 0.9 µM in

RARβ ). The incorporation of this group also saw the compound become selective for the E.V. cell line over the RARβ cell line, perhaps due to steric interactions between the

2-methyl group and the amide moiety altering the ligand conformation, or interaction(s) within the RARβ binding pocket. Additional incorporation of a bromine at the indole

C5 ( 172b) did not affect the selectivity relative to 172a, but did improve the cytotoxicity, with IC 50 values of 3.2 M in the E.V. cell line and 5.0 M in the RARβ cell line. Interestingly, dimethyl analogue 172c also had improved cytotoxicity 89

compared to 172a, but showed no cell line selectivity, possessing IC 50 values of 5.8 µM in both cell lines.

3.4.1 Screening of selected analogues against normal cells

Having determined which analogues possessed both cytotoxic activity and selectivity towards the RARβ overexpressing cell line, it was then essential to determine whether these compounds also exhibited selective activity towards cancerous cell lines over normal cells. For these experiments, the MRC-5 and WI-38 normal human lung fibroblasts were employed. Dose response curves were first determined for the seven compounds, as detailed previously, before IC 50 values were determined (Table3.4).

Table 3.4: Cytotoxicity of indole-benzothiazole amides towards transfected BE(2)-C neuroblastoma cells and normal cells following 72 h exposure.

Compound IC 50 (M) E.V. RARβ MRC -5 WI -38 115 1.5 0.9 1.6 n.d. 117 15.8 10.8 47.9 n.d. 158h 6.5 4.1 9.9 8.8 158i 3.6 2.1 10.6 5.4 158j 7.5 5.1 7.2 n.d. 158k 14.2 8.3 12.2 n.d. 158l 12.1 10.7 27.9 5.4 n.d.: not determined

It was found that four derivatives (117, 158h, 158i, 158l) exhibited substantial differences in their IC 50 values in the neuroblastoma cell lines compared to the normal cell lines, while the other three compounds did not. The acetamide scaffold 115 did not show any significant selectivity for the cancerous BE(2)-C cell lines over the normal lung fibroblasts. The propanamide derivative 117, however, did show selectivity, 90

having an IC 50 value of 47.9 µM in the MRC-5 normal fibroblasts, as opposed to 15.8 and 10.8 µM in the E.V. and RARβ overexpressing cancerous cell lines, respectively.

Interestingly, the derivatives with substituents at the benzothiazole 4-position had various levels of selectivity towards the cancerous cell lines. The 4,6-difluoro derivative

158h showed some selectivity towards the BE(2)-C cell lines, with IC50 values of 9.9 and 8.8 µM in the MRC-5 and WI-38 normal fibroblast lines, compared to 6.5 and 4.1

M in the E.V. and RARβ cell lines respectively. The 4 -chloro analogue 158i had the largest difference in cytotoxicity between the cancerous and normal cell lines, possessing IC 50 values of 10.6 and 5.4 µM in the MRC-5 and WI-38 normal fibroblasts, compared to 3.6 and 2.1 M against the E.V. and RARβ cell lines respectively. On the other hand, the 4-methyl ( 158j) and 4-methoxy ( 158k) derivatives, both had lower IC 50 values in the MRC-5 fibroblasts lower than those in the E.V. cell line. Additionally, 5- bromo analogue 158l showed selectivity for neuroblastoma cells (IC 50 : 12.1 µM in E.V. and 10.7 µM in RARβ cells) when compared to the MRC-5 fibroblasts (IC 50 27.9 µM), but not the WI-38 fibroblasts (IC 50 5.4 µM).

Further examination of these results confirmed that the level of selectivity displayed by these compounds was indeed significant (Figure 3.12). At 10 µM, both propionamide

117 and 5-bromo derivative 158l showed a significant difference between their effect on the viability of the RARβ overexpressing cell line and the MRC -5 normal fibroblasts, with p-values less than 0.05 and 0.01 respectively. At 5 µM, difluoro derivative 158h showed a significant difference between its effect on the cell viability of the RARβ overexpressed cell line compared to MRC-5 normal fibroblasts (p<0.01), WI-38 normal fibroblasts (p<0.005) and the E.V. cell line (p<0.05). Furthermore, at 1 µM, chloro derivative 158i showed significantly increased activity in the RARβ overexpressed cell 91

line compared to the MRC-5 (p<0.05) and WI-38 (p<0.005) normal lung fibroblasts, as well as the E.V. cell line (p<0.05).

1 1 7 , 1 0 m M 1 5 8 h , 5 m M

1 2 0 1 2 0

1 0 0 1 0 0 *** * ** 8 0 8 0

6 0 6 0 *

4 0 4 0 Cell Viability (% ) Cell Viability2 0 (% ) 2 0

0 0 E.V.RAR b M R C -5 E.V.RAR b M R C -5 W I-3 8

1 5 8 i, 1 m M 1 5 8 l, 1 0 m M

1 2 0 1 2 0

1 0 0 * *** 1 0 0 ** 8 0 8 0 **

6 0 6 0

4 0 4 0 Cell Viability (% ) Cell Viability2 0 (% ) 2 0

0 0 E.V.RAR M R C -5 E.V.RAR b M R C -5 W I-3 8 b

Figure 3.12: Comparison of the cytotoxicity in cancerous vs. normal cells for 117,

158h, 158i and 158l. * p<0.05, ** p<0.01, *** p<0.005.

3.4.2 Examination of the effect on RAR protein expression

In order to determine whether these novel compounds indeed acted via a mechanism involving the RAR signalling pathways, Western blot analysis of whole cell protein extracts was conducted to quantify the levels of expression of the three RAR protein isotypes (Figure 3.13). The cells were treated with solvent control or 158i at 2 or 5 M 92

for 24, 48 and 72 h. The protein was then isolated from whole cell lysate and the levels of RAR proteins quantified by densitometry using multiple t-tests to determine the statistical significance (Figure 3.14).

E.V. 24 h 48 h 72 h (-) 2 µM 5 µM (-) 2 µM 5 µM (-) 2 µM 5 µM RARα

RARβ

RARγ

GAP

RARβ 24 h 48 h 72 h (-) 2 µM 5 µM (-) 2 µM 5 µM (-) 2 µM 5 µM RARα

RARβ

RARγ

GAP

Figure 3.13: Western blot showing the RAR protein expression of transfected BE(2)-C

cell lines following treatment with 158i at 2 or 5 µM over 24, 48 and 72 h. 93

BE(2)-C-E.V.

2 0 0 ** 2 4 h 4 8 h 7 2 h * 1 5 0 *

1 0 0 ****

5 0 Protein expression (% ) 0 RAR a RAR a RAR a RAR b RAR b RAR b RAR g RAR g RAR g DMSO 2 m M 5 m M DMSO 2 m M 5 m M DMSO 2 m M 5 m M

BE(2)-C-RAR b

2 5 0 2 4 h 4 8 h 7 2 h * 2 0 0 ** * ** ** 1 5 0 **

1 0 0

5 0 Protein expression (% ) 0 RAR a RAR a RAR a RAR b RAR b RAR b RAR g RAR g RAR g DMSO 2 m M 5 m M DMSO 2 m M 5 m M DMSO 2 m M 5 m M

Figure 3.14: Quantification of RAR protein levels following treatment with 158i at 2 or

5 µM over 24, 48 and 72 h. * p<0.05, ** p<0.01.

It was observed that treatment with 158i had an effect on the expression of all three isotypes of the RAR protein. In general, treatment with 158i increased expression of

RAR proteins within 24 h, with the expression then returning to base levels at 48 and 72 h. Furthermore, similar trends were generally observed across both the E.V. and RARβ over-expressed cell lines. 94

RARα expression was found to be increased by 72 % following 24 h treatment of the

E.V. cell line with 5 µM 158i, with the level of expression then being reduced by 23 and

25% at 48 and 72 h, respectively. In the RARβ over -expressed cell line, RARα expression was increased by 85 and 38% at 24 h following treatment with 2 and 5 µM of 158i, respectively. The expression of RARβ was found to be increased by 48 and

19% in the E.V. cell line following 24 and 48 h treatment, respectively, with 2 µM of

158i. In the RARβ over -expressed cell line, the expression of RARβ was also increased by 72 and 44% after 24 h treatment with 2 and 5 µM of 158i, respectively. RARγ expression was found to be quite consistent across all treatments and time points in the

E.V. cell line, however, increases of 60 and 91% were observed after 24 h for 2 and 5

µM treatments of 158i, respectively.

Firstly, the observation that RAR protein levels are modulated by treatment with 158i is an exciting result, suggesting that the mechanism of action of 158i may be related to activation of RAR signalling pathways. This could occur upstream of RARβ, or through binding to RARβ itself, which is ab le to induce transcription of RAR genes due to the presence of RARE target sequences in the RARβ promoter region. 53 However, the selectivity of 158i seems to be low, as it activated all three isotypes. Indeed, the presence of the amide linker is a known enhancer of RARα selectivity, which is likely to be a key factor in this low RAR isotype selectivity. Second, the general trend whereby the RAR expression was increased within the first 24 h and then decreased back to near baseline levels within the next 24 h suggests that the role of the RAR protein is an early response to treatment with 158i, with the downstream targets of RAR transcription potentially responsible for the changes in cell viability observed at 72 h post-treatment. This is in agreement with previous observations in the literature that

RAR is an early response gene. 283 95

3.4.3 Further mechanistic investigations

Having established that RAR protein expression was modulated following treatment with 158i, further insights into the mechanism of these compounds were next sought.

Upon treatment with 13-c-RA 5, neuroblastoma cells undergo RAR-mediated differentiation and growth arrest. The possibility that the synthesized indole- benzothiazole amides acted through a similar mechanism was investigated through an assessment of changes in the cell’s morphology. The transfected neuroblastoma cells were incubated with either solvent control (DMSO), 1 µM 13-c-RA 5, or an appropriate concentration of amide 158h (5 µM), 158i (5 µM) or 158l (10 µM). After 5 days the cells were visualized at 100X magnification using an optical microscope and representative images captured and analysed for neurite outgrowth (Figure 3.15).

Examining the treated cells reveals that the synthesized amides do not significantly alter morphology in either the E.V. or RARβ overexpressed cell line, relative to the untreated control (Figure 3.15). In contrast, however, cells treated with 1 µM 5 showed neurite extension in both cell lines. Interestingly, cells treated with the amides were observed to be dispersed with populations of dead cells, suggesting that the compounds may be acting through the induction of cell death such as apoptosis or necrosis.

E.V. RARβ

DMSO

13 -c-RA

5 (1 µM)

158h (5 µM)

158i (5 µM)

158l (10 µM)

Figure 3.15: Treatment of transfected BE(2)-C neuroblastoma cells over 5 days with

158h, 158i or 158l does not induce differentiation compared to 13-c-RA ( 5) and DMSO. 97

In order to determine if apoptosis was a contributor to the mechanism of action of the compounds, the activation of caspase 3 was investigated by Western blotting. Caspase 3 was selected as it is common to both the intrinsic and extrinsic apoptosis pathways and would therefore give an indication of the involvement of apoptosis. 284 Protein isolates from cells treated with 155i at 2 µM over 24, 48 and 72 h were subsequently analysed for the presence of pro-caspase 3 and active/cleaved caspase 3 (Figure 3.16). It was observed that there was no change in the levels of pro-caspase 3 upon treatment with

155i, as well as no formation of the active form of caspase 3, suggesting that apoptosis is not a likely mechanism of action of the compounds.

24 h 48 h 72 h (-) 2 µM (-) 2 µM (-) 2 µM Pro-Casp 3

Casp 3

GAP

Figure 3.16: Western blot showing the presence of pro-caspase 3, but not formation of

active caspase 3 following treatment with 2 µM 158i.

3.5 Conclusions

A small library of indole-benzothiazole acetamides has been synthesized by the efficient

PyBOP coupling of 3-indoleacetic acids and 2-aminobenzothiazoles. The SAR has been investigated with respect to modifying the heteroatoms, appending substituents about the ring systems and modifying the amide linker. It was found that the acetamide linker conferred the highest cytotoxicity against neuroblastoma cells, while the benzothiazole 98

heterocycle was favoured over the benzimidazole and benzoxazole. Furthermore, derivatives of 2-methylindole-3-acetic acid were less active than the non-substituted indoles. Among all of the compounds tested, the 4-substituted benzothiazoles were the most active compounds. Three analogues: 158h, 158i and 158l, displayed selective cytotoxic activity towards neuroblastoma cells over normal lung fibroblasts. The lead compound 158i also increased expression of all RAR protein subtypes within 24 h of treatment, demonstrating that these compounds may act through an RAR mediated mechanism , likely exerting their influence upstream of, or on RARβ . Further investigations into their mechanism of action suggest that differentiation and apoptosis do not play a significant role in the activity of these acetamides.

CHAPTER 4: DESIGN, SYNTHESIS AND CHARACTERISATION

OF INDOLE AZOLES

4.1 Introduction

Compounds containing heterocycles are some of the most important medical and industrial molecules. One such example is the five-membered oxadiazole motif, which has seen a dramatic increase in its inclusion in molecules described in patents and the wider literature. 285 Raltegravir 173 (antiretroviral) is the first oxadiazole containing pharmaceutical to reach the market, 286 while several examples of oxadiazoles in late stage clinical trials include zibotentan 174 (anticancer) 287 and tiodazosin 175

(antihypertensive).288 Other oxadiazole-containing compounds have been reported to display a broad spectrum of biological activities, including anticancer, 289 antimicrobial, 290 and antifungal properties.291,292

Oxadiazoles possess many properties that medicinal chemists may exploit in order to tune pharmacokinetic properties in the rational design of novel compounds. Firstly, disubstituted oxadiazoles, if asymmetrically substituted, exist in up to four regioisomeric forms, namely the 1,3,4-isomer, 1,3,5-isomer and two 1,2,4-isomers (Figure 4.1). This allows the generation of analogues with similar substituent orientation, particularly with 100

the 1,3,4- and 1,2,4-isomers, which may therefore bind to biomolecules in a similar fashion. 293 This diversity allows the hydrogen bond acceptor properties of the molecule to be tailored.285 Furthermore, oxadiazole heterocycles are very good bioisosteres of carbonyl-containing compounds, such as amides and esters, 294 and so contribute substantially in increasing pharmacological activity by reducing the rate of enzymatic degradation. 295

Figure 4.1: Possible isomers of di-substituted oxadiazoles

The electronic properties of these heterocycles can be further modified by heteroatom replacement. Substitution of the oxygen atom in oxadiazoles with sulfur gives rise to the corresponding thiadiazoles, which are also important components of many biological activity compounds. 296 The more lipophilic nature of the sulphur atom also confers liposolubility, 297 as well as adding to the mesoionic character of the heterocycle and potentially improving membrane permeability. 298 Furthermore, thiadiazoles have also been shown to be able to interfere with the metabolism of nucleic acids, primarily cytosine, thymine and uracil, potentially making them useful for gene-targeted therapies. 299

The incorporation of additional nitrogen atoms to generate triazoles and tetrazoles can further alter the properties of these heterocycles. Triazoles are good amide bioisosteres as they possess similar size, dipole moment and H-bond acceptor capacity, 300 as well as being very stable to metabolic degradation.301 The same is also true for tetrazoles, which 101

also represent a stable bioisostere for amides, while exhibiting a high tendency to form strong H-bonds.302,303

Azole linked indoles have also been identified as biologically active compounds, with some derivatives presenting interesting anti-cancer properties. Formagio et al developed a series of novel 3-(2-substituted-1,3,4-oxadiazol-5-yl) β -carboline derivatives, such as

176, that showed growth inhibitory effects against a panel of eight cancer cell types. 304

Additionally, Kumar et al described the synthesis of some novel 5-substituted-2-(indol-

3-yl)-1,3,4-oxadiazoles, such as 177, with the 3- and 4-pyridyl derivatives displaying broad spectrum activity against a range of cancer cell lines and also performing much better than the analogous phenyl derivatives. 289

Considering the bioisosteric relationship that azoles share with amides, as well as the known biological activities of azoles and their indolyl conjugates, it was envisaged that indole-based 1,3,4-oxadiazoles would provide an interesting complement to the indole amide ligands discussed in Chapter 3. This chapter therefore describes aims to prepare novel indolyl azoles and to investigate their anticancer activity in relation to RARβ.

4.2 Identification of indole azoles as a lead scaffold

Oxadiazoles and triazoles, particularly of the 1, 3, 4 – arrangement, were the most common heterocycles represented in the 26 hit compounds and 8 lead structures. Not only were these moieties present in 7 of the 26 hit ligands, they were also found in 3 of 102

the 8 most effective compounds, namely ligands 7, 9 and 19 (Figure 4.2). Typically, these oxadiazoles contained a hydrophobic aromatic substituent on one end and were connected via a two atom aliphatic or ethereal linker to another bulkier cycle.

Furthermore, the apparent overall length of the molecules appears to be highly conserved.

Figure 4.2: Three lead structures that contain oxadiazoles (red) and triazoles (blue).

A comparison of the docking results for these three lead structures revealed that they adopted two distinct orientations. The aromatic substituent of the oxadiazole ring was directed towards the important Arg269 and Ser280 residues in the binding poses of L7 and L9, with the bulkier substituent attached to the alkyl chain directed towards the large hydrophobic cavity of the RARβ -LBD (Figure 4.3) However, this was reversed in the docking pose of L19, where the aromatic ring appended to the oxadiazole was directed into the hydrophobic cavity, while the triazole moiety was oriented towards the

Arg269 and Ser280 residues. The observed difference in binding mode may be related to the size of the substituents attached to the oxadiazole by the alkyl chain, whereby the

N-phenyl group of L7, being much larger than the corresponding N-methyl of L19, is unable to fit in the region located near Arg 269 and Ser280. Furthermore, the increased hydrophobicity of this group would also favour interactions with the large hydrophobic region of the RARβ -LBD. This result further highlights the importance of controlling the steric bulk and hydrophobicity of potential ligands, as well as the dramatic affects they may have on binding to biomolecules. 103

Figure 4.3: Overlapped docking poses of L7 (green) and L9 (yellow) showing a similar

orientation, but different orientation to L19 (blue).

In order to determine appropriately-sized molecules to bind to the RARβ protein, indole oxadiazole scaffolds with methylene linkers of various lengths (Table 4.1) were docked into the RARβ binding pocket following the procedure detailed previously (Chapter 2) .

Similar to the compounds described in Chapter 3, it was thought that indole oxadiazole derivatives with either a methylene or ethylene linker will possess the most similarity in terms of size and region separation, compared to the hit molecules.

Analysis of the docking results revealed that the methylene ( 179) and ethylene bridged

(180) derivatives bound more favourably than the directly-linked indole-oxadiazole

(178) as they exhibited higher docking scores to the RARβ -LBD. Interestingly, all the scores were within the range of the initial 8 hit compounds, being above the proposed cut-off of 56.00, with very little difference between the methylene and ethylene-linked scaffolds. However, it was observed that these ligands also oriented themselves in significantly different ways when bound to the receptor cavity (Figure 4.4), similarly to the lead structures L7, L9 and L19. With the shorter methylene unit of 179 , the ligand 104

directs the indole substituent towards the important Arg269 and Ser280 residues, allowing for possible π -cation interactions, as well as possible H-bonding between the oxadiazole nitrogen and Arg387. Conversely, with the longer ethylene unit of 180, it is the terminal phenyl ring that is oriented towards these residues. If these compounds do indeed bind in such different modes, but with such similar affinity, as suggested by their comparable docking scores, then the investigation of modifications to both of these scaffolds could yield quite varied and interesting SAR data.

Table 4.1: Goldscores of the initially proposed scaffolds.

Ligand Goldscore Rank

58.16 3

62.51 1

62 .27 2

105

Figure 4.4: Overlapped docking poses of methylene 179 (green) and ethylene 180

(yellow) bridged oxadiazoles.

4.3 Synthesis of indole azoles

There are many methods to synthesize 1,3,4-oxadiazoles, starting from different classes of functionality, but they typically involve the use of acid hydrazides. One such method involves the condensation of indolyl ethyl imido esters 181 with acid hydrazides 182 by heating at reflux in ethanol. It was noted that under these conditions, the carboxylic imido ester 181a was less reactive than the acetic acid derivative 181b, requiring harsher conditions and producing lower yields of oxadiazoles 183 (Scheme 4.1).305

Scheme 4.1: Reagents and conditions: a) EtOH, reflux, 4 h. 106

A more common method involves the formation of imines from hydrazides and aldehydes, followed by oxidative cyclization (Scheme 4.2). Many different oxidants have been reported in the literature, with varying degrees of success. Some of these include lead(IV) species, 306 hypervalent iodine, 307,308 chloramine T, 309 ceric ammonium nitrate, 310 bromine in acetic anhydride, 311 or N-chlorosuccinimide with DBU. 312

Scheme 4.2: Synthesis of 1,3,4-oxadiazoles via oxidative cyclization .

Alternatively, 1,3,4-oxadiazoles may be prepared by cyclodehydration of diacyl hydrazines using an array of dehydrating reagents (Scheme 4.3). Some commonly used

313 314 315 316 examples include thionyl chloride, BF 3-(OEt 2), silyl species, triflic anhydride,

Burgess’s reagent, 317 or the popular phosphoryl chloride.318,319

Scheme 4.3: Synthesis of 1,3,4-oxadiazoles via cyclodehydration .

4.3.1 Synthesis of methylene and ethylene-bridged oxadiazoles

In order to efficiently generate a series of indole-based oxadiazoles, a single-step condensation-cyclization was envisaged as the most convenient method. In order to achieve this, the procedure of Valente et al . was employed, 320 whereby a mixture of 3- indole acetic acid 106 and benzoylhydrazine 184 was heated at reflux in POCl 3 for 5 h, before being poured into ice water (Scheme 4.4). Following basic work-up, a mixture of decomposed materials was obtained, with none of the desired oxadiazole 179 being isolated. 107

Scheme 4.4: Reagents and conditions: a) POCl 3, reflux, 5 h.

It is known that indoles are sensitive to strongly acidic conditions, giving rise to dimers, polymers and decomposition products through protonation of the pyrrole ring and subsequent reaction. 321,322 Whilst this may be remedied by the inclusion of an electron withdrawing substituent, 323 the condensation-cyclization was first attempted using less acidic conditions in order to avoid protection and deprotection steps. Hence, the reaction of 106 and 184 with POCl 3 was carried out in a variety of solvents, such as DCM and

EtOAc, similar to the method of Kandemir et al .324 Unfortunately, these solvents were unable to prevent the continued decomposition of the starting materials.

As the strongly acidic POCl 3 was not suitable for the single-step formation of the desired oxadiazoles, synthetic efforts were then directed towards a two-step procedure involving preparation of the corresponding hydrazide, followed by subsequent cyclodehydration. Therefore, 106 was subjected to treatment with 184 , EDCI and

HOBt, in DMF for 24 h to produce the desired hydrazide 185. Following aqueous work- up, the solid was isolated in 84% yield. 325 However, treatment of 185 with either concentrated or dilute POCl 3 again failed to yield the cyclized product 179. 108

Scheme 4.5: Reagents and conditions: a) EDCI, HOBt, DMF, 24; b) POCl 3, reflux, 4 h.

Confirmation of the structure of hydrazide 185 was obtained from spectral data. The 1H-

NMR spectrum (d 6-DMSO) displayed a broad singlet at δ 10.90, corresponding to the indolyl NH, while two doublets with coupling constants of 1.2 Hz were observed at δ

10.34 and 10.10, confirming the presence of the hydrazide motif. The indolyl protons were observed as doublets at δ 7.64, 7.35 and 7.29 for H4, H7 and H2 respectively, and as doublets of doublets at δ 7.08 and 6.99 for H5 and H6, all with J values of 7.0 Hz.

The methylene unit was also observed as a singlet at δ 3.63. The protons of the terminal phenyl ring gave rise to signals at δ 7.88, 7.57 and 7.48, corresponding to H2/H6, H4, and H3/H5 respectively. Analysis of the 13 C-NMR spectrum also showed the presence of two carbonyl groups at δ 170.1 and 165.5, as well as the methylene group at δ 30.7.

Furthermore, the IR spectrum showed peaks at 1691 and 1639 cm-1, confirming the presence of the carbonyl groups, while the (+ESI) HRMS gave rise to the [M+H] + peak at m/z 294.1244 (C17 H16 N3O2 required 294.1237).

In order to overcome the issues arising from the use of POCl 3, 4-toluenesulfonyl chloride (4-TsCl) was investigated as an alternative reagent for the cyclodehydration of the hydrazide intermediate. Stabile et al have described the use of 4-TsCl to be efficient, high yielding and importantly, tolerant of heterocycles and acid-sensitive groups. 326 109

Hence, the intermediate hydrazide 185 was treated with 4-TsCl in the presence of

Hunig’s base ( N,N-diisopropylethylamine, DIPEA) in acetonitrile (MeCN) for 4 h.

Following basic work-up with ammonia and purification by flash-column chromatography, the desired oxadiazole 179 was isolated in 48% yield.

Scheme 4.6: Reagents and conditions: a) 4-TsCl, DIPEA, MeCN, 4 h.

The structure of oxadiazole 179 was confirmed by the disappearance of the hydrazide

1 NH peaks in the H-NMR spectrum (d 6-DMSO), as well as downfield shifting of the indolyl NH and methylene proton signals to δ 11.05 and 4.43 respectively (Figure 4.5).

Furthermore, analysis of the 13 C-NMR spectrum showed upfield shifting of the hydrazide carbonyl signals, now oxadiazole C2 and C5, to δ 166.1 and 164.0. Final confirmation was observed by (+ESI) HRMS, which exhibited the [M+H] + peak at m/z

276.1122 (C17 H14 N3O required 276.1131).

1 Figure 4.5: H-NMR spectrum of oxadiazole 179 in d 6-DMSO. 110

As the requisite synthetic methodology was now in hand, the first structural modification to be investigated was the effect of substituting various heterocycles at the terminal portion. Thiophene was first selected as it is a known bioisostere for the phenyl ring, 327 capable of maintaining the steric bulk, π -electron cloud and planar structure of the phenyl group, whilst being more polar and therefore hydrophilic. The thiophene motif potentially offers alternative metabolic pathways due to the presence of a heteroatom and the more electron-rich aromatic ring. 328 Additionally, thiophenes were a major substituent of the oxadiazoles identified in the initial lead structures. As a complement to this, furan and pyrrole were also selected in order to investigate the effect of steric bulk, hydrophilicty, polarity and hydrogen bonding ability on biological activity.

In order to achieve this, IAA 106 was first coupled to a range of heterocyclic hydrazides

186a-c following the EDCI procedure established earlier, giving hydrazides 187a-c in yields of 36-46%. This was much lower than the yield of phenyl derivative 179 (84%), with little to no precipitation observed upon addition to ice-water. Consequently, extraction with EtOAc and subsequent flash column chromatography was required to obtain more of the desired hydrazides 187a-c. These were then cyclized with 4-TsCl to give oxadiazoles 188a-c in yields of 23-89% (Scheme 4.7). 111

Scheme 4.7: Reagents and conditions: a) EDCI, HOBt, DMF, 24 h; b) 4-TsCl, DIPEA,

MeCN, 4 h.

Thiophene hydrazide 187a had representative spectral data for this class of analogues.

1 In the H-NMR spectrum (d 6-DMSO) of 187a, the thiophene protons gave rise to a doublet at δ 7.84 corresponding to H3′ and H5′, and a doublet of doublets at δ 7.19 corresponding to H4′, each with a coupling constant of 4.4 Hz. The indolyl protons gave further doublets at δ 7.65 (H4) and 7.37 (H7), two doublet of doublets at δ 7.10 (H6) and 7.01 (H5), all with 7.8 Hz coupling, and two singlets at δ 10.91 (NH) and 3.64

(CH2). Finally, the hydrazide NH protons appeared as singlets at δ 10.38 and 10.12.

Furthermore, carbonyl resonances were observed at δ 170.2 and 160.6 in the 13 C-NMR spectrum, and at 1671 and 1625 cm -1 in the IR spectrum.

Similarly, the spectral data of thiophene oxadiazole 188a is representative of oxadiazoles 188a-c. The disappearance of the hydrazide NH peaks was observed in the

1 H-NMR spectrum (d 6-DMSO) of 188a, while the aromatic thiophene signals were observed to resolve into doublets of doublets at δ 7.89, 7.72 and 7.24. Additionally, there was a corresponding downfield shift in the indolyl NH and methylene peaks to δ 112

11.05 and 4.41 respectively, with the latter also splitting into a doublet with 0.7 Hz coupling (Figure 4.6).

1 Figure 4.6: Comparative H-NMR (d 6-DMSO) spectra of hydrazide 187a (blue) and

oxadiazole 188a (red)

Following a similar synthetic procedure, 3-indolepropionic acid 145 was coupled to acid hydrazides 184 and 186a-c, giving hydrazides 189a-d, respectively, in yields of 28-

88%. Interestingly, these products all generated precipitates upon work-up, most likely due to the increase in hydrophobicity as a result of their increased alkyl chain length.

These hydrazides were then cyclized to oxadiazoles 180 and 190a-c in yields of 30-66%

(Scheme 4.8). The spectroscopic data of the ethyl-bridged derivatives were largely similar to those observed for the corresponding methylene-bridged analogues, with the

1 exception of the linking region. Taking the H-NMR spectrum (d 6-DMSO) of hydrazide

189c as an example, the indolyl protons produced doublets at δ 7.56 and 7.35 corresponding to H4 and H7 respectively, as well as doublet of doublets at δ 7.09 and

7.00 that were assigned as H6 and H5, with coupling of 7.8 Hz. The NH proton gave a singlet at δ 10.81, while the H2 proton gave a doublet at 7.20 ( J = 2.3 Hz) and the 113

ethylene bridge gave rise to two triplets at δ 2.99 and 2.58 ( J = 6.4 Hz). Additionally, the 13 C-NMR spectrum displayed two resonances corresponding to the carbonyl groups at δ 171.3 and 157.1, as well as two CH 2 signals at δ 34.1 and 20.6. Examination of the corresponding data for oxadiazole 190b showed the characteristic downfield shifting of the indolyl NH to δ 10.83, which was less of a downfield shift ( δ 0.02) compared to the methylene-bridged series (δ 0.14-0.15). Downfield shifts were also observed for both CH 2 groups, which gave complex multiplets at δ 3.27 and 3.21, rather than the expected triplets.

Scheme 4.8: Reagents and conditions: a) EDCI, HOBt, DMF, 24 h; b) 4-TsCl, DIPEA,

MeCN, 4 h.

To investigate what other influences the bridging region may have on the activity of this scaffold, the generation of a conformationally-restricted carbonyl bridged analogue was targeted. First, treatment of indole 30 with oxalyl chloride, in anhydrous ether at 0 °C, produced glyoxyl chloride 191 in the high yield of 86%. This was then treated with benzoyl hydrazine 184 in the presence of Et 3N in DCM at r.t. for 2 h to generate the 114

glyoxyl hydrazide 192 in 77% yield. This was followed by cyclodehydration with 4-

TsCl to generate oxadiazole 193 in 82% yield (Scheme 4.9).

Scheme 4.9: Reagents and conditions: a) oxalyl chloride, Et 2O, 0 °C, 2 h; b) Et 3N,

DCM, 2 h; c) 4-TsCl, DIPEA, MeCN, 4 h.

The structure of glyoxyl hydrazide 192 was confirmed by 1H-NMR analysis. The spectrum (d 6-DMSO) showed three singlets at δ 12.32, 10.70 and 10.57, corresponding to the indole NH and two hydrazide NH groups respectively, while the aromatic protons were observed as overlapping multiplets. Furthermore, the three carbonyl groups gave resonances at δ 182.4, 165.7 and 163.8 in the 13 C-NMR spectrum. However, due to the extremely insoluble nature of oxadiazole 193, the 13 C-NMR could not be sufficiently determined.

The next strategy to investigate the consequences of modifications to the ethylene- bridged oxadiazoles involved replacement of the indolyl methylene group with a sulfur atom. This was considered a promising option as similar bridges were observed in several of the oxadiazoles from the initial library screening (L7 and L19), including indole amide 149, which possessed superio r selectivity for the RARβ overexpressing cell line compared to the E.V. To synthesize the targeted compound, indole 148 was 115

coupled with 184, giving hydrazide 194 in 17% yield, followed by cyclodehydration to generate oxadiazole 195 in a yield of 76% (Scheme 4.10).

Scheme 4.10: Reagents and conditions: a) EDCI, HOBt, DMF, 24 h, b) 4-TsCl, DIPEA,

MeCN, 4 h.

Finally, an examination of the linkage between the oxadiazole and the phenyl substituent was considered. As it was observed that all oxadiazole motifs within the initial hit ligands were directly linked to an aromatic feature, the synthesis of a derivative with a methylene spacer was targeted to investigate whether a direct attachment is indeed necessary for biological activity. As the requisite phenylacetic acid hydrazide was not at hand, it was decided that production of the 3-indoleacetic acid hydrazide 196 would be more suitable, as it could then be used for the generation of analogues where other acid hydrazides were not available. Consequently, 106 was converted to the methyl ester 197 in 96% yield by stirring in methanol with H 2SO 4 at ambient temperature over 2.5 h, followed by crystallization from DCM/ hexanes.

Generation of hydrazide 196 was then performed by heating the ester at reflux with hydrazine hydrate in methanol for 4 h, giving the product in 91% yield. Finally, this was converted to oxadiazole 198 in one step by heating with phenylacetic acid at reflux in

POCl 3 for 2 h, with a yield of 23% (Scheme 4.11). 116

Scheme 4.11: Reagents and conditions: a) H 2SO 4, MeOH, 2.5 h, b) NH 2NH 2.H 2O,

MeOH, reflux, 4 h, c) phenylacetic acid, POCl 3, reflux, 2 h.

The analytical data of methyl ester 197 and hydrazide 196 were consistent with the

305,329 1 values reported in the literature. The H-NMR spectrum (d 6-acetone) of oxadiazole

198 displayed three singlets at δ 10.20, 4.31 and 4.16, which correspond to the NH, indolyl methylene and benzylic protons respectively. A multiplet was observed in the range of δ 7.31-7.26 that included the protons for the terminal phenyl ring, as well as the indole H2. The other indole protons were observed at δ 7.57, 7.40, 7.12 and 7.03 as doublet of doublet of doublets, corresponding to H4, H7, H6 and H5 respectively. It was interesting to observe that where the indole acid 106 had readily degraded under strongly acidic conditions with POCl 3, the hydrazide 196 was stable enough to undergo cyclodehydration.

4.3.2 Synthesis of analogues with varied linking heterocycles

In order to further develop the SAR of the linking portion of this class of compounds, the nature of the linking heterocycle was next investigated. By exchanging the heteroatoms, as well as modulating the substitution pattern of the ring, the resultant 117

changes in polarity, hydrophobicity and hydrogen-bonding ability may offer further advantages in terms of ligand binding, receptor selectivity and solubility.

The first modification to the linking heterocycle was to prepare the corresponding thiadiazole 199. Aside from reducing the hydrogen bond accepting ability of the ligand, the substitution of oxygen for sulfur also increases the hydrophobicity of the molecule, which may improve cell membrane permeability and binding to the largely hydrophobic

RARβ protein. 297,298 Initially, hydrazide 185 was heated at reflux with Lawesson’s reagent in toluene, which resulted in the generation of multiple products, as observed by

TLC. Upon work-up and purification by chromatography, none of the desired product was isolated. However, changing the solvent to THF produced thiadiazole 199 in a yield of 7% (Scheme 4.12).

Scheme 4.12: Reagents and conditions: a) Lawesson’s reagent, THF, reflux, 24 h.

1 In the H-NMR spectrum (d 6-acetone) of 199, it was noted that the NH proton gave a singlet at δ 10.38, while the methylene protons also appeared as a doublet at δ 4.62 with

0.7 Hz coupling. The phenyl protons were observed as multiplets at δ7.93-7.90 for H2″ and H6″, and δ 7.51-7.48 for H3″, H4″ and H5″. The indolyl proton signals were more resolved, appearing as doublet of doublet of doublets at δ 7.59, 7.14 and 7.04 for H4′,

H6′ and H5′ respectively, while H2′ and H7′ overlapped as a narrow multiplet at δ 7.45-

7.52. The presence of sulfur in the ring was confirmed by the HRMS (+ESI), showing the [M+H] + peak at m/z 292.0902, matching the calculated m/z of 292.0903 for

C17 H14 N3S. 118

To generate compounds with more soluble end groups, acid 106 was coupled with 4,4- dimethyl-3-thiosemicarbazide 200 to give thiohydrazide 201 in 40% yield. Subsequent cyclization to thiadiazole 202 proceeded in 48% yield (Scheme 4.13). Generation of the thiadiazole, rather than the oxadiazole, was confirmed by HRMS (+ESI) where the

+ [M+Na] peak was observed at m/z 265.1063, matching the expected m/z of 265.1060 calculated for C13 H14 N4SNa.

Scheme 4.13: Reagents and conditions: a) EDCI, HOBt, DMF, 24 h; b) 4-TsCl,

DIPEA, MeCN, 4 h.

The next objective was to generate systems linked by triazoles in order to explore the effects of including a hydrogen bond donor in the linking region. Not only would this modify the overall polarity of the linking heterocycle, the inversion of hydrogen bonding properties might reduce any potential interactions with RARα, which prefers

H-bond acceptors, and induce interactions with RARγ, which prefers H -bond donors.

The preparation of triazole 203 was attempted by heating oxadiazole 179 to 160 °C in a

20% solution of methanolic ammonia, in a sealed tube for 24 h (Scheme 4.14). The reaction generated several products, with the desired 4 H-1,2,4-triazole 203 isolated in a poor yield of 5% following chromatography. 1H-NMR analysis confirmed formation of the product, however, it also revealed isomerization of the 4H-1,2,4-triazole within 119

several hours, most likely to the 2 H-1,2,4-triazoles 204a and 204b. This was further demonstrated by TLC, whereby additional spots were observed to appear from a sample of previously pure product.

Scheme 4.14: Reagents and conditions: a) NH 3, MeOH, 160 °C, 24 h.

As the isomerization of triazole 203 presented a potential problem for the investigation of biological activity, as well as to further develop the SAR, the isomeric 2 H-1,2,4- triazole 204a was targeted. By heating a neat mixture of hydrazide 196 and benzonitrile

205 at 160 °C for 24 h in a sealed tube, the corresponding 2 H-1,2,4-triazole 204a could be generated in 17% yield following chromatography (Scheme 4.15). Examination of the 1H-NMR spectrum of triazole 204a revealed that this product also underwent isomerization over a short period of time, with additional NH peaks observed, as well as further aromatic protons.

Scheme 4.15: Reagents and conditions: a) 160 °C, 24 h. 120

In order to avoid the issues encountered with isomerization of the previously developed triazoles, it was envisaged that 1,2,3-triazole derivatives could be prepared via the popular copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). Not only is this the classic example of “click chemistry”, with the substrates being tolerant of many functional groups, as well as easily introduced and independently stable, 330,331 the reaction itself is typically performed at ambient temperatures and offers complete conversion and selectivity for the 1,4-disubstituted isomer. 332,333 Furthermore, the generated triazoles are highly stable and unreactive, being impervious to hydrolytic cleavage, oxidation and reduction, making them highly suitable for biological applications. 334 Additionally, the lack of an acidic proton from an NH group inhibits isomerization of the triazole, further highlighting the suitability of this linker.

The first target for the generation of 1,2,3-triazole-linked indoles was the prop-2-yn-1- yl-indole 206. This was considered to be an important intermediate as it could be used in the CuAAC reaction, as well as in other cycloadditions to potentially generate a diverse range of heterocycles. Indole 30 was therefore stirred with propargyl bromide and zinc powder in THF for 18 h according to the procedure of Yu et al (Scheme

4.16).335 Consistent with their report, incomplete conversion of the indole starting material was observed, even with the use of freshly activated zinc, and complete separation of the materials by chromatography proved difficult.

Scheme 4.16: Reagents and conditions: a) propargyl bromide, Zn, THF, 18 h. 121

As indole 206 could not be produced, the complementary component; 3-

(azidomethyl)indole 207, was the next target in the synthesis of triazole and tetrazole- linked systems. This was attempted following the procedure of Rogers, 336 in which a solution of indole-3-methanol 208 and diphenyl phosphoryl azide (DPPA), at 0 °C in toluene, was treated with 1,8-diazabycyclo[5.4.0.]undec-7-ene and the resulting mixture stirred at r.t. for 16 h (Scheme 4.17). Unfortunately, following work-up, the desired azide could not be isolated.

Scheme 4.17: Reagents and conditions: a) DPPA, DBU, toluene, 16 h.

4.3.3 Synthesis of analogues bearing substituents on indolyl or phenyl rings

As modifications to the linking portion of the indole and nature of the terminal portion had resulted in dramatic loss of activity, the next phase of the investigation involved appending substituents about the indole and phenyl rings in the hope of improving the pharmacological properties and potential RARβ binding of the molecules. Firstly, substitution about the indole ring was investigated utilizing the Fischer indole derivatives 168 and 171a-b prepared in Chapter 3.5. These were coupled to 184 by standard EDCI coupling, generating hydrazides 209a-c in 47-90% yield, followed by 4-

TsCl cyclodehydration to give oxadiazoles 210a-c in yields of 49-81% (Scheme 4.18). 122

Scheme 4.18: Reagents and conditions: a) EDCI, HOBt, DMF, 24 h; b) 4-TsCl,

DIPEA, MeCN, 4 h.

1 The H-NMR spectrum (d 6-acetone) of 210b showed a singlet at δ 10.33, typical of the indolyl NH, another at δ 4.40, characteristic of the CH 2 group, and a third at δ 2.53 that corresponded to the methyl group. The indolyl protons gave rise to a doublet at δ 7.79 (J

= 1.9 Hz), characteristic of H4′, as well as two do ublet of doublets at δ 7.27 and 7.15 ( J

= 8.5, 8.5 Hz) typical of H6′ and H7′. The phenyl protons appeared as two multiplets in the ranges of δ 7.98-7.94 and 7.58-7.52, corresponding to H3″/H4″/H5″ and H2″ /H6″ respectively.

As the 3-indoleacetic acid hydrazide 196 had been previously isolated, it was envisaged that the coupling of this molecule to 4-substituted benzoic acids via POCl 3, which was the method used to generate oxadiazole 198, would be a more straightforward and time- effective approach than the preparation of numerous hydrazides from their respective benzoic acids and esters. The fluoro and trifluoromethyl substituents were selected due to the known benefits of fluorine in medicinal chemistry as discussed earlier, as well as the success of compounds containing these substituents as described in Chapter 3. 123

Furthermore, many of the other derivatives have previously been synthesized. The methyl-substituted analogue was synthesized mostly as a point of comparison for the trifluoromethyl derivative, as well as for method validation. As such, 196 was heated at reflux in POCl 3 with benzoic acids 211a-e to generate the desired oxadiazoles 212a-e in

22-29% yield. 319

Scheme 4.19: Reagents and conditions: a) POCl 3, reflux, 2 h.

1 Investigation of the H-NMR spectrum (d 6-acetone) of 212c showed a characteristic singlet at δ 10.26 and doublets at δ 7.44 (J = 0.8 Hz) and 4.50 ( J = 0.9 Hz) typical of the

H2′ and CH 2 protons. The para substituted aromatic protons gave signals at δ 8.20 and

7.91 that were split as doublet of doublet of doublets ( J = 0.8, 8.9, 8.9 Hz), corresponding to H2″/H6″ and H3″/H5″ respectively. Other indolyl protons also gave doublet of doublet of doublets ( J = 0.8, 7.8, 7.8 Hz) at δ 7.70, 7.42, 7.14 and 7.07, typical of H4′, H7′, H6′ and H5′ respectively.

4.4 Characterization of in vitro activity against neuroblastoma cell lines

In order to determine the levels of cytotoxic activity towards neuroblastoma cells in vitro , as well as establish an SAR profile for the indole-oxadiazole scaffold, the synthesized oxadiazoles were screened for anti-cancer activity against the transfected

BE(2)-C neuroblastoma cell lines, as described previously in Chapter 3 (Table 4.2). 124

Table 4.2: Cytotoxicity of methylene and ethylene bridged oxadiazoles towards empty vector and RARβ stably transfected BE(2)-C neuroblastoma cells following 72 h exposure.

Compound IC 50 (M) E.V. RARβ 179 >50 23.5 188a >50 >50 188b >50 >50 188c >50 >50 180 >50 >50 190a >50 >50 190b >50 >50 190c >50 >50 193 >50 >50 195 >50 >50 198 >50 >50 199 >50 >50 202 >50 >50 203 >50 >50 204a >50 >50 210a >50 >50 210b >50 >50 210c >50 >50 212a 34.6 33.5 212b 23.2 25.9 212c 13.0 14.5 212d 12.8 19.2 212e >50 >50

The cytotoxic activity of the compounds was observed to be minimal, with the majority not achieving an IC 50 value below 50 µM. With respect to the methylene and ethylene- bridged oxadiazoles synthesized in Section 2.3.1, only oxadiazole 179 possessed an IC 50 value below 50 µM. However, it was encouraging to observe that 179 exhibited a marked difference in activity against the two cell lines, suggesting that its mode of action may be dependent on RARβ. It was also interesting to note that in this case, the 125

replacement of the phenyl ring in 179 with the isosteric thiophene in 188a reduced the activity in both cell lines. The same can also be said for increasing the bridge length from methylene in 179 to ethylene in 180, which was surprising considering their remarkably similar docking scores (62.51 and 62.27 respectively), suggesting the importance of their different binding modes observed during docking. Furthermore, the incorporation of the H-bond capable furan or pyrrole moieties also offered no favourable activity for analogues 188b and 190b, or 188c and 190c, respectively.

Upon further modifications to the methylene-linker, no further enhancement of biological activity was observed. The solubility of methanone derivative 193 was found to be low, resulting in precipitation of the compound upon addition to the cell culture media. Furthermore, the thiomethylene-bridged 195 displayed no enhancement in selectivity or potency relative to the ethylene-bridged 180, also possessing no cytotoxicity towards either cell line, while benzyl derivative 198 also possessed IC 50 values above 50 µM in both cell lines.

Overall, it was found that by altering the length of the bridge portion of the linker, or by changing the end-group of the molecule from phenyl to heterocyclic, the activity of these compounds was dramatically reduced. This suggests that the bridge is highly important, as the influence that it has on the length of the molecule, as well as the orientation of the oxadiazole and end group, dramatically affects the cytotoxicity of the compounds. Additionally, this also suggests that the steric bulk and hydrophobicity of the end-group is more important than its ability to donate or accept hydrogen bonds.

This was demonstrated by changing the phenyl ring ( 179) to the isosteric 5-membered thiophene ( 188a), which has similar steric bulk to benzene whilst being more polar, which resulted in a reduction of cytotoxicity. Furthermore, by introducing more polar 126

heterocycles capable of accepting ( 188b) or donating ( 188c) H-bonds, the cytotoxicity was still reduced compared to phenyl derivative 179 .

Similar results were observed for analogues with modified linking heterocycles. It was observed that the conversion from oxadiazole 179 to the more hydrophobic thiadiazole

199 resulted in a loss of cytotoxic activity against both of the cell lines. This was an unexpected result as the RARβ binding pocket is largely hydrophobic . On the other hand, the change in dipole moment upon substitution of the electronegative oxygen with sulfur may interfere with the hydrogen bonding interactions observed in the molecular model of 179. Furthermore, the dimethylamino derivative 202 was completely inactive, most likely due to the large decrease in steric bulk of the end group compared to the phenyl derivatives, as well as a reduction in the length of the molecule. Additionally, triazoles 203 and 204a both displayed a significant loss of activity compared to oxadiazole 179. Taken together, these results suggest that modifications to the hydrophobicity, dipole moment and hydrogen-bonding capabilities of the linking heterocycle significantly affect the cytotoxic activity of the molecules, possibly as a result of impacting the affinity of these analogues for the RARβ receptor.

An investigation into the predicted binding of triazole 203 returned a Goldscore of

63.23, which was highly similar to that obtained for oxadiazole 179 (62.51). However, it was observed that the predicted pose of 203 did not contain the H-bonding interaction between the Arg269 residue and the nitrogen at the oxadiazole 3-position in 179 (Figure

4.7). On the other hand, thiadiazole 199 displayed an increased Goldscore of 68.52 and no H-bonding interaction, however, the potential formation of a T-shaped π -π interaction between the thiadiazole and the Phe295 residue was observed as a possible cause of this increased Goldscore. 127

Figure 4.7: Overlapped docking poses of thiadiazole 199 (green) and trizole 203

(yellow) showing their similar bind ing modes to the RARβ -LBD.

Biological testing of analogues bearing substituents on the indolyl ring ( 210a-c) revealed that the molecules are highly sensitive to changes in the substitution pattern of the indole moiety . Incorporation of the 2′ -methyl group (210a) again resulted in the compound possessing IC 50 values greater than 50 µM against both cell lines, while subsequent inclusion of a 5′ -bromo (210b) or 5′ -methyl substituent ( 210c) saw no improvement in cytotoxic activity. This reduction in cytotoxic activity could be due to steric interactions between the 2′ -methyl group and the oxadiazole ring, forcing the ligand to adopt a conformation less suitable for binding to the RARβ cavity.

The attachment of substituents at the 4-position of the phenyl ring ( 212a-c) resulted in analogues with cytotoxic activity that was comparable to or better than that of oxadiazole 179 (IC 50 : >50 M in E.V. and 23.5 M in RARβ) . In the case of 4-fluoro derivative 212a, it was observed that the cytotoxicity was less specific than 179, having

IC 50 values of 34.6 and 33.5 M against the E.V. and RARβ cell lines, respectively . The methyl (212b) and trifluoromethyl derivatives (212c) both showed more potent activity 128

than oxadiazole 179, having respective IC 50 values of 23.2 and 13.0 µM in the E.V. cell line as well as 25.9 and 14.5 M in the RARβ over -expressed cell line. Interestingly, both of these derivatives showed an inversion of selectivity relative to oxadiazole 179, having increased cytotoxicity in the E.V. cell line, suggesting that the increased steric bulk and lipophilicity may be improving binding or availability.

Upon relocation of the trifluoromethyl group to the 3-position ( 212d) the level of activity in the E.V. cell line remained similar to that seen in the 4-trifluoromethyl derivative 212c (12.8 ad 13.0 µM, respectively), whilst showing reduced activity in the

RARβ cell line ( 19.2 and 14.5 µM, respectively). This intriguing result suggests that this substitution pattern about the phenyl ring is possibly inducing antagonistic activity towards RARβ, rather than the desired agonistic activity. In stark contrast, however, the

2-trifluoromethyl derivative 212e displayed significantly reduced activity, possessing

IC 50 vales above 50 µM against both cell lines. This suggests that the inclusion of steric bulk so close to the linking portion results in conformational changes that significantly alter receptor binding.

In order to better understand these results, computer docking of these scaffolds was performed. The docking results showed that 4-trifluoromethyl analogue 212c is expected to bind most tightly to RARβ , based on the higher Goldscore of 64.97, followed by 212e (61.20) and 212d (60.40). However, upon examination of the binding modes of each of these ligands, we can see that there are significant differences in their binding modes (Figure 4.8). While 212c and 212d are predicted to have similar orientations within the RARβ binding site , in which the phenyl ring is directed into the cavity, 212e is predicted to have the indolyl motif directed into the cavity. If this is indeed an accurate reflection of the binding modes of these ligands, then this is a significant result as the non-substituted derivative 179, which displayed the most potent 129

measurable selectivity for the RARβ cell line (IC 50 : >50 µM in E.V. and 23.5 µM in

RARβ) was predicted to bind with the indolyl ring directed towards the Arg269 and

Ser280 residues, while 212c and 212d have the phenyl ring oriented towards these residues. This suggests that these two binding modes may be equally valid, differing primarily in their influence on agonistic versus antagonistic effects upon the RARβ protein.

Figure 4.8: Overlay showing similar binding poses of 212c (green) and 212d (yellow),

and the dissimilar pose of 212e (blue).

Further investigation of the predicted binding modes of these varied substitution patterns is the interaction of the trifluoromethyl group with Arg269. This is of particular importance in 212b where the positioning of the functional group directs the fluorine towards the residue, allowing for the potential formation of halogen bonding. However, the Goldscore for this particular orientation is lower than that of 212c, suggesting that the steric crowding is more detrimental than any stability gained from such an interaction. Furthermore, this also results in a significant shift of the indolyl group, 130

reflecting a localization of the aromatic portion in a region of space similar to that occupied by the substituted benzene in the orientation of 212e.

As a rudimentary examination of the RAR selectivity of these compounds, 212c was also screened against BE(2)-C cells t ransfected with RARα and RARγ (Table 4.7). It was interesting to observe that 212c displayed lower IC 50 values in both the RARα and

RARγ cell lines than either of the E.V. and RARβ cell lines. Considering that the oxadiazole was used as an isosteres for an amide, 294 it is not overly surprising that it displays potentially stronger affinity for RARα, which has been previously shown to preferentially bind arotinoids with amide type linkages. A rational explanation for the

RARγ selectivity may be the large steric bulk o f the trifluoromethyl group, which could better fill the larger binding cavity near helix 11.

Table 4.7: Comparative activity of compound 212c against the RAR subtypes.

Compound IC 50 (M) E.V. RARβ RARα RARγ 212c 13.0 14.5 10.2 11.9

4.5 Conclusions

A series of indole azoles has been synthesized, which includes modifications such as different terminal aromatic groups, variable linkers and functional group substitution.

The in vitro cytotoxicity assays suggest that such a scaffold is highly sensitive to modification, preferring the 2-((1 H-indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole core.

Substitution of the terminal aromatic ring may provide increased potency, as well as significantly altering the selectivity for the RARβ over -expressed cell line.

131

CHAPTER 5: DESIGN, SYNTHESIS AND CHARACTERIZATION

OF BENZOTHIAZOLE-BASED THIENOPYRAZOLES

5.1 Introduction

The pyrazole ring is an important structural motif found in many biologically active and pharmaceutical compounds. 337 Both pyrazoles and their derivatives possess a wide spectrum of biological activities, such as antimicrobial,338 anti-inflammatory,339 antiviral 340 and anticancer properties.341 Many pyrazoles have been clinically evaluated as new drugs, including celecoxib 213 (anti-inflammatory),342 fezolamine 214 (antidepressant) 343 and crizotinib 215 (anti-cancer).344

Pyrazoles possess many properties that may be exploited by medicinal chemists to generate new molecules with diverse pharmacophoric features. Firstly, pyrazoles display different reactivity and lower aromatic stability than other azoles.345 This allows for tautomerism and higher reactivity of the heterocycle (Figure 5.1). Furthermore, pyrazoles may contain up to four substituents in various arrangements. By replacing these substituents the electronic properties of the ring may be modified, therefore impacting on the reactivity of the heterocycle, as well as the strength of any intermolecular interactions. Furthermore, the steric bulk and 132

hydrophilicity/hydrophobicity of these substituents will exert further influences upon the overall properties of the molecule.

Figure 5.1: Tautomerism of mono-substituted pyrazoles and the substitution potential

of the pyrazole heterocycle.

Where two adjacent R-groups form a fused ring system, the molecules are another important class of pyrazoles, termed annulated pyrazoles. Annulated pyrazole derivatives also possess many important biological properties, such as antibacterial, 346 insecticidal, 347 anti-HIV, 348 anti-inflammatory 349 and antifungal activities.350

Furthermore, annulated pyrazoles have been incorporated into pharmaceuticals such as apixaban 216 (anticoagulant)351 and allopurinol 217 (anti-hyperuricemic). 352

One particularly interesting sub-class of annulated pyrazoles is the thienopyrazoles

(Figure 5.2). Firstly, the existence of three regioisomers, namely the thieno[2,3- c]pyrazole, thieno[3,2-c]pyrazole and thieno[3,4-c]pyrazole, allows the generation of analogues with diverse substituent orientations. 353 Furthermore, thiophene is a known bioisostere for the phenyl ring, 327 possessing similar steric bulk whilst being more polar and therefore more hydrophilic, as well as potentially offering alternative metabolic 133

pathways due to the presence of a heteroatom and the more electron-rich aromatic ring. 328

Figure 5.2: Isomers of the thienopyrazole scaffold.

Of the three thienopyrazole isomers, the [2,3-c]-regioisomer is the most commonly utilised, being incorporated into many scaffolds with anti-cancer activity. 353 One such example is the work of Akritopoulou-Zanze et al. , who prepared three different classes of thieno[2,3-c]pyrazoles (218-220) as kinase inhibitors with low micromolar potency. 354

Similarly, Carry et al . developed N-phenoxycarboxamide thienopyrazole derivatives such as 221 and 222 that also displayed sub-micromolar inhibition of several kinases.355

Furthermore, Barberic et al . prepared hydrazide derivatives such as 223 for the same purpose. 356 134

Conjugated systems containing benzothiazoles and pyrazoles have also been shown to possess interesting anti-cancer activities. Gabr et al . synthesized a series of amide- linked pyrazoles that displayed potent broad spectrum antitumour activity at 10 µM, of which 224 was the most active analogue.357 Furthermore, El-Hamouly et al . prepared benzothiazole-pyrazole conjugates such as 225 and 226 that possessed cytotoxic activity against the MCF7 breast cancer cell line. 358

Considering the important activities of benzothiazoles, thienopyrazoles and their conjugates, this chapter is concerned with the development of amide-based benzothiazole-thienopyrazole hybrids as novel scaffolds with anti-tumour activities.

135

5.2 Identification of benzothiazole-thienopyrazoles as a lead scaffold

Thienopyrazoles are an interesting scaffold that were present in 3 of the 26 hit compounds identified in the virtual screening (Chapter 2.2), namely ligands 10, 18 and

23 (Figure 5.3). Interestingly, all of these scaffolds contained the 1-isobutyl-3-methyl-

1H-thieno[2,3-c]pyrazole-5-carboxamide moiety, differing only in the amide substituent. Furthermore, both ligands 10 and 23 displayed potent cytotoxic activity against the stably transfected BE(2)-C cell lines, while ligand 18 did not. Furthermore, ligands 10 and 23 also displayed selec tivity towards the RARβ overexpressing cell line, compared to the empty vector cell line.

Figure 5.3: The three lead structures that contain the thienopyrazole scaffold.

A comparison of the docking results for the active lead compounds L10 and L23 revealed that they adopted highly similar geometries within the RARβ binding pocket.

Both ligands had the thienopyrazole moiety oriented towards the hydrophobic pocket, with the isobutyl chain extending farther into this region (Figure 5.4). The benzimidazole of L10 was observed to participate in three non-bonding interactions with the binding pocket. The benzen oid ring engaged in a π -cation interaction with the

Arg265 residue, while the imidazole ring had π -π stacking with the Phe 221 residue and the imidazole NH donated a H-bond to the backbone carbonyl of the Leu262 residue.

Furthermore, a potential σ -π interaction was observed between the pyrazole ring and the 136

Leu259 residue. With L23, the only non-bonding interactions made were two π -cation interactions between the thienopyrazole ring system and the Arg387 residue.

Figure 5.4: Overlapped docking poses of L10 (green) and L23 (yellow) showing a

similar orientation within the RARβ binding pocket .

In order to determine what potential modifications may enhance affinity for the RARβ binding pocket, a series of scaffolds were docked into the cavity following the procedure detailed previously (Chapter 2.6). Modifications to the bulk and hydrophobicity of the pyrazole N-substituent were considered as they may interact more favourably with the hydrophobic pocket. Substitution of the imidazole NH with sulphur was also considered as the benzothiazole amide had proven to be more effective than the corresponding benzimidazole derivative in Chapter 3.

137

Table 5.1: Goldscores of the initially proposed scaffolds.

Ligand Goldscore Rank

65.86 3

73.00 1

70.73 2

Analysis of these docking results suggested that the benzothiazole derivative 227 would possess similar binding affinity to that predicted for L10, with the ligands having similar Goldscores of 65.86 and 63.78, respectively. Interestingly, the dimethyl analogue 229 had a marginally improved Goldscore of 70.73, which is 11% higher than the Goldscore of L10, while the N-phenyl derivative 228 had an even higher Goldscore of 73.00, which is 14% higher than the Goldscore of L10. This suggests that the increased hydrophobicity and aromatic character may interact more favourably with the residues in the binding pocket. Additionally, both 227 and 228 were predicted to bind in the same manner as L10, with the thienopyrazole moiety oriented towards the hydrophobic cavity and the pyrazole N-substituent extending farther into this pocket

(Figure 5.5). Furthermore, it was observed that the N-phenyl ring of 228 overlapped well with the predicted positioning of the N-isobutyl group in 227. On the other hand, the dimethyl analogue 229 was predicted to bind in the reverse orientation compared to

227, 228 and L10, with the thienopyrazole ring of 5-16 directed towards the important 138

Arg269 and Ser280 residues. Despite this reversed orientation, it also had a high

Goldscore of 70.73, indicating that this may still be a valid binding conformation.

Figure 5.5: Overlapped docking poses of 227 (green) and 228 (yellow) showing a

similar orientation within the RARβ binding pocket , but with a different orientation

compared to 229 (blue).

5.3 Synthesis of benzothiazole-thienopyrazoles

The first synthesis of thieno[2,3-c]pyrazoles was reported by Porai-Koshits et al . in

1970.359 By heating a mixture of 5-chloro-4-formylpyrazoles 230a-c with thioglycolic acid 231 in methanolic potassium hydroxide (Scheme 5.1), the carboxylate derivatives

232a-c were isolated in yields of 77-82%. These compounds have been investigated by many groups for medicinal chemistry applications. 139

Scheme 5.1: Reagents and conditions: a) KOH, MeOH, reflux, 4 h.

The group of Akritopoulou-Zanze prepared different classes of thieno[2,3-c]pyrazoles starting from pyrazolone 233a. After formylation under Vilsmeier conditions to give

230d and subsequent N-protection with bis(4-methoxyphenyl)methyl chloride

(BPMPMCl), the intermediate 230e was cyclized with methyl thioglycolate in basic alcohol to give the thienopyrazole 234a (Scheme 5.2). 354 This protected thienopyrazole was then used to prepare derivatives such as 218 .

Scheme 5.2: Reagents and conditions: a) POCl 3, DMF, 0 °C-reflux, 2 h; b) BPMPMCl,

Et 3N, THF, rt, 1 h; c) methyl thioglycolate, Na 2CO 3, MeOH, reflux.

Additionally, the group of Akritopoulou-Zanze also converted aldehyde 230e to oxime

235 by treatment with hydroxylamine hydrochloride, followed by dehydration to the nitrile 236 with acetic anhydride. Cyclization with 2-mercaptoacetamide in basic alcohol then afforded the 4-amino-5-amidothieno[2,3-c]pyrazole 237 (Scheme 5.3), which was used to prepare analogues of 220.354 140

Scheme 5.3: Reagents and conditions: a) NH 2OH .HCl, NaHCO 3, EtOH/H 2O, 7 h; b)

Ac 2O, reflux, 1 h; c) 2-mercaptoacetamide, K 2CO 3, EtOH, reflux, 17 h.

Inoue and co-workers described a similar method, involving a two-step condensation and cyclization for the formation of the thiophene ring. 360 Starting again from the pyrazolones, such as 233b, these were converted to the 4-formyl-5-chloropyrazoles 230f under Vilsmeier formylation conditions before being condensed with ethyl thioglycolate to give 238. The linear precursor was then cyclized to the desired thieno[2,3-c]pyrazoles

239a under thermal conditions with the aid of a crown ether. Subsequent saponification in basic alcohol gave acid 232d, which was converted to amides 240 via generation of the acid chloride and reaction with amines (Scheme 5.4).360

Scheme 5.4: Reagents and conditions: a) DMF, POCl 3, 0 °C-80 °C, 1 h; b) ethyl

thioglycolate, K 2CO 3, MeCN, reflux, 4 h; c) 18-crown-6, K2CO 3, DMF, 130 °C, 2 h; d)

2 3 1 N NaOH/EtOH, 60 °C, 1 h; e) i. SOCl 2, 80 °C, 8 h, ii. NHR R , Et3N, DCM, 1.5 h. 141

Alternatively, the group of Eller et al . designed a synthesis utilizing the popular

Sonogashira coupling reaction. Firstly, the 5-chloropyrazoles 241a-b were halogenated with iodine and iodic acid, giving 242a-b, followed by Sonogashira cross-coupling to phenylacetylene to give 243a-b. Thieno[2,3-c]pyrazoles 244a-b were then generated by reaction with sodium sulphide (Scheme 5.5).361

Scheme 5.5: Reagents and conditions: a) I 2, HIO 3, AcOH, reflux, 4 h; b)

phenylacetylene, Pd(PPh 3)2Cl 2, Et3N, CuI, DMF, Ar (g) , 50 °C, 10 min; c) Na 2S, DMF,

130 °C, o/n.

5.3.1 Synthesis of starting pyrazolones

In this thesis, the Knorr pyrazole synthesis was used as a means of preparing the starting pyrazolones required for the synthesis of the desired thieno[2,3-c]pyrazoles. This reaction involves the condensation and cyclization of a hydrazine, or hydrazine salt, with a β -keto ester using protic solvents such as alcohols or acetic acid.362 This classical method represents one of the simplest and most common methods for the formation of

1,3-disubstituted-pyrazol-5-ones. Therefore, a mixture of hydrazine 164 or 245 and either ethyl acetoacetate 246a or ethyl benzoylacetate 246b were heated at reflux in methanol for 3 h, giving pyrazolones 233c-e in yields of 49-72% (Scheme 5.6). The structures of compounds 233c-e were confirmed by spectral analysis, with the data consistent with those previously reported in the literature. 363-365 142

Scheme 5.6: Reagents and conditions: a) MeOH, reflux, 3 h.

The incorporation of a pyridine moiety was also investigated as a means of increasing the solubility of these compounds, as well as incorporating additional functionality to potentially form further interact ions with the RARβ binding pocket. The inclusion of the electronegative nitrogen atom generates a dipole in the ring system and allows for the donation of the nitrogen lone pair and formation of H-bonds with water. This is also demonstrated in the reported tendency of the pyrazole to exist in the enol tautomer 247a due to the formation of an intramolecular H-bond. 366 Subseqently, 2-hydrazinopyridine

248 was added to a solution of ethylacetoacetate 246a in AcOH and the mixture was heated at reflux for 8 h (Scheme 5.7). Unfortunately, the desired pyrazole 247a was not isolated following work-up. The use of EtOH as the solvent also failed to procure 247a.

Scheme 5.7: Reagents and conditions: a) AcOH, reflux, 8 h.

Therefore, attention was turned towards the generation of N-isobutylpyrazolone 233f.

To achieve this, alkylation of the 3 H-pyrazolone 233a was considered as an attractive procedure as it would eliminate the need to synthesize an isobutyl hydrazine, therefore offering a comparatively shorter synthetic route. First, ethyl acetoacetate 246a was 143

treated with hydrazine monohydrate in EtOH for 16 h to give pyrazolone 233a in 74% yield (Scheme 5.8). Pyrazolone 233a was then treated with KOH in DMSO for 30 minutes, followed by the addition of 1-bromo-2-methylpropane, and the reaction was continued for 2 h. However, upon aqueous work-up the desired product 233f could not be isolated. Other alkylating conditions investigated included the use of tetrabutylammonium bromide (TBAB) and KOH in MeCN with sonication, or CuI,

K2CO 3 and N,N′-dimethylethylenediamine at reflux in MeCN under N 2 for 24 h.

Unfortunately, none of these modified procedures yielded 233f.

Scheme 5.8: Reagents and conditions: a) NH 2NH 2.H 2O, EtOH, 16 h; b) 1-bromo-2-

methylpropane, KOH, DMSO, r.t., 2 h.

As the alkylation of pyrazolone 233a could not be achieved, attention was next turned to preparing a suitable hydrazine for the Knorr-type synthesis of pyrazolone 233f. Initial attempts focused on a reductive amination approach, whereby isobutyraldehyde 249 was treated with tert -butylcarbazate 250 in MeOH and stirred for 20 h to give imine 251 in a yield of 75%. This was then treated with NaCNBH 3 and p-TsOH.H 2O in THF for 5 h to give Boc-hydrazine 252 , which was immediately subjected to deprotection with 4

M HCl/EtOAc to give isobutylhydrazine hydrochloride 253a in 62% yield over two steps (Scheme 5.9). 144

Scheme 5.9: Reagents and conditions: a) tert-butylcarbazate, MeOH, r.t., 20 h; b)

NaCNBH 3, p-TsOH.H 2O, THF, r.t., 5 h; c) 4 M HCl/EtOAc, r.t., 4 h.

Structural confirmation of products 251 -253a was determined primarily by NMR

1 analysis. The H NMR (CDCl 3) spectrum of imine 251 displayed two singlet resonance signals at δ 7.86 (1 H) and 1.46 (9 H), indicating the presence of the imine CH and tert- butyl CH 3 protons, respectively. Two doublet signals were also observed at δ 7.01 (J =

5.0 Hz, 1 H) and 1.05 ( J = 7.0 Hz, 6 H), and were assigned as the NH and isobutyl CH 3 protons, respectively. Further, an octet resonance was observed at δ 2.56 (J = 7.0 Hz, 1

1 H), which was assigned as the isobutyl CH proton. The H NMR (CDCl 3) spectrum of

Boc-hydrazine 252 showed changes in the splitting patterns and chemical shifts of several peaks, indicating that the reaction had occurred. The appearance of an additional singlet at δ 6.45 (1 H) corresponded to the newly introduced alkyl NH proton. The

BocNH proton was observed to shift to δ 6.64, also appearing as a broad singlet, while the Boc CH 3 protons gave another singlet resonance at δ 1.32. The remaining isobutyl peaks were observed as a doublet at δ 0.80 corresponding to the CH 3 protons, a nonet at

δ 1.61 representing the CH proton and a new quartet at δ 2.52 assigned as the CH 2 protons, each with a J value of 7.0 Hz. Finally, deprotection to the hydrochloride salt

253a was demonstrated through disappearance of the resonances corresponding to the

1 Boc group in the H NMR (CDCl 3) spectrum. 145

To improve the yield of the isobutyl hydrazine, the one-pot procedure reported by

Hilpert, 367 in which the intermediate imine 251 is reduced via hydrogenation, was also investigated. Therefore, 249 was treated with tert -butylcarbazate 250 at 0 °C in iPrOH, then at r.t. for 2 h. Platinum on charcoal (5%) was then added and the mixture was heated at 40 °C under a hydrogen atmosphere for 72 h (Scheme 5.10). The intermediate solution of 252 was concentrated and treated for 30 min at 0 °C with sulfuric acid, and then heated at 50 °C for 5 h. Following workup, the hydrogensulfate salt 253b was isolated in 29% yield. Despite the long reaction times, the yield was much lower than reported by Hilpert (88%). It was thought that the use of a balloon to introduce H 2 gas did not supply sufficient pressure, or did not maintain a hydrogen atmosphere for the duration of the reaction. Therefore, the reaction was repeated under higher pressure (5 bar) in a sealed steel reaction vessel for 48 h; however, no improvement in overall yield was obtained.

Scheme 5.10: Reagents and conditions: a) i. tert-butylcarbazate, iPrOH, 0 °C, 2 h, then

r.t., 2 h; ii. Pt/C, H 2, 40 °C, 72 h; b) H 2SO 4, 0 °C, 30 min, then 50 °C, 5 h.

Finally, alkylation was investigated as another means of generating the required isobutylhydrazine. Meyer proposed a simple method of alkylating hydrazones in the presence of a phase-transfer catalyst, which was reportedly robust and tolerant of base- sensitive alkyl chains. 368 Therefore, carbazate 250 was reacted with acetone and a catalytic amount of acetic acid in the presence of magnesium sulfate for 1 h at reflux, giving imine 254 in 98% yield (Scheme 5.11). A mixture of 254, tetrabutylammonium hydrogen sulfate and KOH was then heated to 50 °C in toluene before 1-bromo-2- 146

methylpropane was added. Subsequent heating of this mixture at 80 °C for 3 h gave alkyl hydrazone 255 in a yield of 82% after aqueous work-up. Heating hydrazone 255 at reflux in 2 M HCl/THF for 3 h then afforded the dihydrochloride salt 253c in 73% yield. This procedure was found to be the optimal method for production of the isobutylhydrazine due to its simplicity, lack of purification steps and good yields.

Scheme 5.11: Reagents and conditions: a) AcOH, MgSO 4, acetone, reflux, 1 h; b) 1-

bromo-2-methylpropane, KOH, Bu 4NHSO 4, toluene, 80 °C, 3 h; c) 2 M HCl/THF,

reflux, 3 h.

1 The H NMR (CDCl 3) spectrum of alkyl-hydrazone 255 displayed the isobutyl protons as a doublet at δ 3.32 (CH 2), nonet at δ 1.74 (CH) and doublet at δ 0.85 (2 x CH 3), each with a J value of 7.0 Hz. The Boc methyl groups appeared as a singlet at δ 1.42, while the isopropylidene methyl groups appeared as two singlets at δ 2.03 and 1.85. The spectral data of dihydrochloride 253c was consistent with that observed for the monohydrochloride salt 253a.

Attempts to synthesize the desired pyrazolone 233f by heating hydrochloride salt 253c and ethyl acetoacetate 246a at reflux in MeOH were found to be ineffective.

Consequently, a modified procedure was employed whereby the hydrochloride salt 253c was first treated with KOH in a 10:1 MeOH:H 2O solvent mixture for 15 min to generate the free base. Ethyl acetoacetate 246a was then added and the mixture was heated at 60

°C for 18 h. Purification by Soxhlet extraction using EtOAc for 4 h followed by chromatography then gave pyrazolone 233f in 40% yield (Scheme 5.12). Interestingly, the use of the mono-hydrochloride 253a instead gave the tautomeric pyrazol-5-ol 248b 147

in 79% yield. This is most likely due to the additional equivalent of acid present when using the dihydrochloride 253c, catalysing conversion of the carbonyl form (233f) to the

1 enol form (248b). The identity of 248b was confirmed by analysis of the H NMR (d 6-

DMSO) spectrum, in which the hydroxyl proton gave rise to a singlet at δ 10.61, as did

H4 and the C3-methyl protons at δ 5.09 and 1.99 respectively. The protons of the isobutyl side-chain were also observed at δ 3.51, 2.00 and 0.80 as a doublet (CH 2), nonet (CH) and doublet (2 x CH 3) resonances, respectively.

Scheme 5.12: Reagents and conditions: a) 246a, KOH, MeOH:H 2O (10:1), 60 °C, 18 h.

5.3.2 Attempted synthesis of thienopyrazoles by Sonogashira cross-coupling

In order to synthesize the thieno[2,3-c]pyrazole scaffold, the Sonogashira cross- coupling of 4-iodo-5-chloropyrazoles described by Eller et al . was considered as it offered not only simple and efficient procedures, but also good to high yields for all steps. To investigate this, the prepared pyrazolone starting materials 233c-e were first converted to the corresponding 5-chloropyrazoles 242a-c by treatment with POCl 3 under thermal conditions for 7 h, proceeding in yields of 13-29% (Scheme 5.13).

Iodination was performed by heating chloropyrazoles 242a-c in the presence of I 2 and iodic acid at reflux in acetic acid, giving iodopyrazoles 243a-c in yields of 29-60%. 148

Scheme 5.13: Reagents and conditions: a) POCl 3, reflux, 7 h; b) I 2, HIO 3, AcOH,

reflux, 4 h.

The characteristic physical data of these compounds (M.p., NMR) were consistent with literature reports. The successful conversion of chloropyrazoles 242a-c to iodopyrazoles

243a-c was best demonstrated by the disappearance of the singlet resonance corresponding to the H4 proton in the 1H NMR spectra of pyrazoles 243a-c. For

1 example, in the case of 3-methyl-1-phenylpyrazoles 242b and 243b, the H NMR (d 4-

MeOD) spectra showed the H4 resonance of 242b at δ 6.66, which was no longer present in the spectrum of 243b (Figure 5.6).

149

1 Figure 5.6: Comparative H NMR (d 4-MeOD) spectra of chloropyrazole 242b (blue)

and iodopyrazole 243b (red) showing disappearance of the H4 resonance and downfield

shift of the CH 3 resonance.

The Sonogashira cross-coupling of iodopyrazoles 243a-c proved to be quite problematic. Initially, the cross-coupling reaction was attempted following the protocol outlined by Eller, with ethyl propiolate used in place of phenylacetylene.361 A mixture of the appropriate pyrazole 243a-c and Et 3N in DMF was treated with CuI and

Pd(PPh 3)2Cl 2 under an N 2 atmosphere. After 15 min at 50 °C, ethyl propiolate was added and the mixture was heated at 80 °C for a further 3 h (Scheme 5.14). Upon aqueous work-up, the major component isolated was not the desired alkyne 244a-c, but rather the product of hydrodehalogenation 242a-c. This suggested that the conditions had been unable to sufficiently exclude oxygen and moisture, as well as perhaps being insufficiently energetic for the reaction to proceed. In order to overcome this problem, the reaction conditions were modified in the hope that the desired product would be 150

obtained. The investigated modifications included variation of temperature (80 °C, reflux), exchanging the solvent(s) used (DMF, Et 3N, THF, THF:DMF), the inclusion or exclusion of Et 3N, and the use of Schlenk conditions. Unfortunately, none of these methods were able to yield the desired alkyne.

Scheme 5.14: Reagents and conditions: a) ethyl propiolate, Pd(PPh 3)2Cl 2, DMF, Et 3N,

CuI, N 2, 50 °C, 15 min, then 80 °C, 3 h.

In order to further verify that this method did not produce the desired alkyne, a sample of the crude reaction mixture starting from 242a was carried forward and heated at reflux with sodium sulfide in DMF overnight in an attempt to generate thieno[2,3- c]pyrazole 239b (Scheme 5.15). Examination of the subsequent reaction mixture by

TLC demonstrated no change in the reaction composition, compared to the crude mixture. As a result of this, attention was then turned to alternative procedures.

Scheme 5.15: Reagents and conditions: a) ethyl propiolate, Pd(PPh 3)2Cl 2, DMF, Et 3N,

CuI, N 2, 50 °C, 15 min, then 80 °C, 3 h; b) Na 2S, DMF, reflux, o/n.

151

5.3.3 Synthesis of thienopyrazoles from formylpyrazoles

The reaction of formylpyrazoles with thioglycolates represents a convenient method for the preparation of thienopyrazoles. Furthermore, the method is reportedly simple, mild and efficient. 359 Therefore, pyrazolones 233c-f were treated with a freshly prepared

Vilsmeier complex of POCl 3 in DMF at 0 °C, followed by heating at reflux for 3 h.

Following aqueous work-up and neutralization with sat. K 2CO 3, the desired formylpyrazoles 230a-c and 230g were isolated in 7-46% yield (Scheme 5.16).

Unfortunately, when this procedure was applied to the N-isobutyl analogues 233g and

248b, the desired formyl pyrazoles could not be generated in a sufficiently high yield.

Scheme 5.16: Reagents and conditions: a) POCl 3, DMF, 0 °C, then reflux, 3 h.

Treatment of formylpyrazoles 230a-c with methyl thioglycolate in the presence of

K2CO 3 at reflux in MeCN for 3 h directly afforded the thienopyrazoles 234b-d in yields of 10-83% after purification by flash column chromatography (Scheme 5.17).

Interestingly, the intermediate thioether ( 238) reported by Inoue was not isolated, eliminating an additional step from the overall scheme. 360

152

Scheme 5.17: Reagents and conditions: a) methyl thioglycolate, K 2CO 3, reflux, 3 h.

In the case of the diphenyl analogue 234d , the yield (10%) was much lower than that for the dimethyl ( 234b, 24%) and 3-methyl-1-phenyl derivatives (234c , 83%).

Consequently, an alternate procedure was utilized whereby formylpyrazole 230c was added to a mixture of methyl thioglycolate and NaH in THF at 0 °C. The mixture was stirred at r.t. for 2 h, then treated with a further portion of NaH at 0 °C for 0.5 h.

Aqueous work-up and recrystallization from EtOH then gave 234d in an improved yield of 71% (Scheme 5.18).

Scheme 5.18: Reagents and conditions: a) methyl thioglycolate, NaH, THF, 0 °C, 2 h.

Saponification of esters 234b-d with 2 M KOH in MeOH for 3 h at r.t. afforded the desired carboxylic acids 232a-c in yields of 51-78% following acidic work-up (Scheme

5.19). These were then coupled to 2-aminobenzothiazole 106 and 2- aminobenzimidazole 139a using EDCI and HOBt in DMF for 24 h, as described 153

previously in Chapter 4, generating the desired amides 228, 229 and 256a-b in 14-88% yield.

Scheme 5.19: Reagents and conditions: a) 2 M NaOH, MeOH, 3 h; b) 106 or 139a,

EDCI, HOBt, DMF, 24 h.

Amides 228, 229 and 256a-b were fully characterized by NMR, IR, UV-Vis and HRMS

1 (+ESI) analysis. As a representative example, the H NMR (d 6-DMSO) spectrum of 228 displayed a broad singlet at δ 13.16 corresponding to the amide NH proton, as well as further singlets at δ 8.45 and 2.55 corresponding to the H4 and CH 3 protons, respectively (Figure 5.7). Resonances corresponding to the N-phenyl ring were observed as a multiplet at δ 7.78-7.75, corr esponding to H2′ and H6′, which overlapped with the signal of the benzothiazole H7″. H3′ and H5′ were o bserved as a doublet of doublet of doublets at δ 7.61 (J = 1.3, 7.4, 7.4 Hz), while H4′ was observed as a multiplet at δ 7.38-7.31, which also overlapped with the benzothiazole H5″. The remaining benzothiazole protons gave rise to a doublet of doublets (J = 1.3, 7.4 Hz) at δ

8.00, assigned as H4″, as well as a doublet of doublet of doublets ( J = 1.3, 7.4, 7.4 Hz) at δ 7.47, assigned as H6″. The carbonyl peak also gave rise to a signal at δ 162.2 in the

13 C NMR. Additionally, the mass spectrum displayed a peak at m/z 391.0678 (M+H) +, corresponding to the calculated value of 391.0682 for C 20 H15 N4OS 2. 154

Figure 5.7: Representative numbering of thieno[2,3-c]pyrazoles 228, 229 and 256a-b.

5.3.5 Synthesis of furano- and pyrrolopyrazoles

Despite there being numerous publications on their synthesis, furano[2,3-c]pyrazoles and pyrrolo[2,3-c]pyrazoles have seen limited use in medicinal chemistry compared to the related thieno[2,3-c]pyrazoles. 369-372 The exchange of the S heteroatom for O or NH offers the opportunity for decreased steric bulk and increased hydrophilicity, due to the potential formation of hydrogen bonds, and consequently these heterocycles were explored as potential RARβ ligands.

In order to synthesize furano[2,3-c]pyrazole derivatives, the procedure of Padwa et al . was followed, whereby the furano ring system is introduced via a cycloaddition reaction between a diazopyrazolone and alkyne.369 Pyrazolone 233d was treated with 4- toluenesulfonyl azide (4-TsN 3) and Et 3N in MeOH for 1.5 h to give diazopyrazolone

257 in 78% yield (Scheme 5.20). A mixture of diazopyrazolone 257 and ethyl propiolate was then heated at 165 °C in toluene in a sealed tube for 16 h to give ester

258 in 40% yield after chromatography. Saponification with 2 M NaOH in MeOH at 50

°C for 4 h afforded acid 259 in a yield of 86%, which was followed by subsequent coupling to 106 with EDCI and HOBt in DMF for 24 h to give amide 260 in 34% yield.

The spectral data of compounds 257 and 258 were consistent with that previously 155

reported in the literature, 369 while the data for compounds 259 and 260 was similar to that found for compounds 232b and 228, respectively.

Scheme 5.20: Reagents and conditions: a) 4-TsN 3, Et3N, MeOH, 1.5 h; b) methyl

propiolate, toluene, 160 °C, 16 h; c) 2 M NaOH, MeOH, 50 °C, 4 h; d) 106 , EDCI,

HOBt, DMF, 24 h.

Secondly, pyrrolopyrazoles were targeted following the procedure of El-Saied et al ., in which a Hemmetsberger-type reaction is conducted between an azidoacetate and a formylpyrazole. 371 Phenylhydrazine 164 was reacted with acetone and a catalytic amount of AcOH in water for 2 h to give hydrazone 261 in 37% yield (Scheme 5.21).

Hydrazone 261 was then immediately reacted with two equivalents of POCl 3 in DMF at

-20 °C for 3 h, then at 80 °C for 2 h, to give pyrazole 262 in 29% yield following basic work-up. Pyrazole 262 was then added to a mixture of freshly prepared NaOMe and methyl azidoacetate in MeOH at 0 °C for 2 h. This was then poured over crushed ice and the precipitate was heated at reflux in toluene for 4 h, however this did not result in the desired pyrrolo[2,3-c]pyrazole 263. 156

Scheme 5.21: Reagents and conditions: a) acetone, AcOH, H 2O, 2 h; b) POCl 3, DMF, -

20 °C, 3 h, then 80 °C, 2 h; c) i. methyl azidoacetate, NaOMe, MeOH, 2 h; ii. toluene,

reflux, 4 h.

5.4 Characterization of in vitro activity against neuroblastoma cell lines

In order to determine the levels of cytotoxic activity of the synthesized benzothiazole- thienopyrazoles towards neuroblastoma cells in vitro , the compounds were screened against the BE(2)-C neuroblastoma cell lines stably transfected with the MEP4-empty vector (E.V.) and MEP4-RARβ constructs (Table 5.2), as described previously in

Chapter 3.

Table 5.2: Cytotoxicity of benzothiazole-thienopyrazoles towards empty vector and

RARβ stably transfected BE(2)-C neuroblastoma cells following 72 h exposure.

Compound IC 50 (M) E.V. RARβ L10 7.1 6.6 228 >50 >50 229 21.9 28.9 256a >50 >50 256b >50 >50 260 >50 >50

157

The cytotoxic activity of the synthesized thienopyrazole derivatives was found to be low, with the majority possessing IC 50 values greater than 50 µM. Derivatives 228 and

256a-b, which contained additional phenyl rings, proved to be less active than the lead compound L10. Substitution of the L10 isobutyl group with a phenyl ring in 256a reduced cytotoxic activity, with the compound possessing IC 50 values greater than 50

µM in both cell lines. Although the preliminary modelling suggested that this modification would generate analogues with similar RARβ affinity compared to L10, this reduced activity may be related to the increase in hydrophobicity of the molecule and the resultant impact on solubility and membrane permeability. Additionally, exchanging the benzimidazole in 256a with benzothiazole in 228 was unable to increase the activity, despite this analogue returning a higher Goldscore of 73.00 than that of L10

(63.78) and similar substitutions being favourable in Chapter 3, both in terms of predicted affinity and cytotoxicity. The incorporation of an additional phenyl ring in

256b also resulted in an inactive compound, again with IC 50 values above 50 µM in both cell lines. Docking of this compound returned a high Goldscore of 77.75, suggesting a high affinity for the RARβ LBD. However, other factors such as solubility and membrane permeability, as well as the inherent uncertainty in docking due to the software typically favouring more hydrophobic molecules, may contribute to the observed difference between predicted affinity and actual cytotoxicity.

Interestingly, the dimethyl derivative 229 was the only analogue with activity, possessing IC 50 values of 21.9 M and 28.9 M against the E.V. and RARβ overexpressed cell lines, respectively. However, the IC 50 values of 229 were not only much higher than those of L10, but the cell line selectivity is also inverted, with the IC 50 value lower in the E.V. cell line. It is possible that the alternative conformation of 229 158

proposed in the modelling studies, where the thienopyrazole motif was directed towards the Arg269 and Ser280 residues, is responsible for these changes in activity.

Investigating the activity of 260, in which the thienopyrazole of 228 was substituted with a furanopyrazole, revealed that this heteroatomic substitution did not improve the activity of this scaffold, with IC 50 values greater than 50 µM recorded in both cell lines.

It was felt that any enhanced hydrophilicity from the inclusion of oxygen may have been outweighed by the hydrophobicity of the N-phenyl substituent.

5.5 Conclusion

Several thieno[2,3-c]pyrazole analogues have been synthesized and screened for their anti-cancer activity towards transfected BE(2)-C neuroblastoma cells. The reaction of formylpyrazoles with ethyl thioglycolate was found to be a good method, both it terms of its robust nature and applicability. Derivatives with aryl substituents were found to have low solubility and cytotoxic activity, while heteroatom substitution was also found to have no impact on activity. Analogues bearing alkyl substituents were found to be more active analogues with various selectivities towards the E.V. or RARβ over - expressed cell lines.

159

CHAPTER 6: 8-ISOPROPYL-4-ARYL QUINOLINES AS RARα

ANTAGONISTS

6.1 Introduction

RAR α overexpression plays a crucial role in the progression of multiple diseases, such as chronic obstructive pulmonary disease, 373 and multiple cancer types. Mawson proposed that gliomas may result from an imbalance in retinoid receptor expression, leading to excessive expression of RARα and reduced expression of RARβ. 374 He further postulated that the use of an RARα antagonist and a n RARβ agonist would be expected to inhibit RARα -induced cell proliferation in gliomas, while suppressing tumour growth and possibly contributing to the regeneration of normal glia. 374

Additionally, Khetchoumian et al . has also identified the oncogenic role that RARα plays in the liver, in the absence of co-repressor TRIM24. 375

In order to examine the therapeutic benefits of receptor-specific retinoids, many diffe rent RARα -selective arotinoids have been developed. Of particular note is the

RARα antagonist Ro -41-5253 (264), which has been shown to inhibit proliferation and induce apoptosis in breast cancer cell lines. Compound 264 also exhibits reduced levels of retinoid teratogenicity compared to RARα -selective agonist 15 , as shown by the reduced incidence and severity of malformations in vivo .376,377 However, 264 is a low affinity binder, requiring a 1000-fold molar excess to eliminate the transcriptional response to t-RA 3.376

160

By incorporating the p-tolyl group of the pan-RAR antagonist AGN 193109 (265),378 as well as the amide linkage and o-fluoro substituents of the RARα -selective agonist AGN

193836 (17 ),136 Teng et al . were able to prepare two classes of RARα -selective antagonists (266a-c and 267a-b).379 They found that, in general, increasing the number of o-fluoro substituents increased RARα selectivity , and that the dimethylchromene analogues 267a-b performed far better than the dihydronapthalene equivalents 266a- c.379

Furthermore, as discussed in Chapter 1.5.5, quinolines have also been identified as important heterocycles for the modulation of RAR expression, including RARα. In particular, recalling the structures of RARα inverse agonist s 88 and 96 , and RARα antagonists 97a-c, we can see that their overall design is largely similar. Each of these molecules links a quinoline-containing hydrophobic portion to a para-substituted benzoic acid using an amide, or isostere thereof. 161

The combination of quinolines and oxadiazoles has also generated compounds with exciting anti-cancer properties. Salahudin et al . synthesized several series of 2-

(quinolin-3-yl)-1,3,4-oxadiazoles and screened them against 60 human tumour cell lines from the NCI. 380 Of the synthesized compounds, derivatives 268 and 269 were the most

380 active, displaying GI 50 values of 1.41-15.8 µM and 0.40-14.9 µM, respectively.

Takahiro et al . prepared 2-(quinolin-4-yl)methylthiophenyl derivatives 270a-c that showed tyrosine kinase inhibitory activity at concentrations of 16 ng/mL. 381 Of these derivatives, 270a showed the most potent activity, with 99% inhibition in the ELISA assay, compared to 86% and 93% for 270b and 270c, respectively. 381 162

Additionally, Sun et al . prepared a series of 5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3 H)- thione derivatives, such as 271a-b, and tested their anticancer activity against HepG2,

SGC-7901 and MCF-7 human tumour cell lines, as well as their telomerase inhibitory

382 activity by ELISA. Compounds 271a-b were the most potent analogues, with IC 50 values of 1.2-8.3 µM and 0.8-7.6 µM against the tumour cell lines, respectively, and

IC 50 values of 0.8 µM and 0.9 µM, respectively, for telomerase inhibition.

In order to further develop the field of quinoline-based RARα antagonists, this chapter describes aims to synthesize 8-isopropyl-4-arylquinolines 272 with an oxadiazole linker. The 8-isopropyl-4-aryl quinoline moiety has been selected as it has previously been shown to have RARα selectivity, while the inclusion of bulky substituents at the 4 - position confer RARα antagonistic activity. 231 Oxadiazole heterocycles were selected as they are very good bioisosteres of carbonyl-containing compounds, such as amides, 294 which have been shown to enhance RARα selectivity. Further, the incorporation of a fluorine atom ortho to the benzoic acid will also be investigated as a means of 163

conferring RARα selectivity. This chapter also aims to investigate the SAR of such analogues with respect to their anticancer activity against neuroblastoma cell lines.

6.2 Synthesis of 8-isopropyl-4-arylquinolines

In order to prepare the targeted quinoline oxadiazoles, an approach similar to that implemented in Chapter 4 was proposed (Figure 6.1). The oxadiazole moiety could be prepared by either an oxidative cyclization of a hydrazone, or dehydrocyclization of a hydrazide. Both of these functionalities could be simply prepared from the quinoline hydrazide (A), which could be prepared in turn from the quinoline ester (B).

Figure 6.1: Retrosynthesis of quinoline-oxadiazoles.

The synthesis of 4-arylquinoline esters may be achieved by a number of methods, typically starting from a substituted aniline 273. Several methods have been developed 164

where these anilines have been condensed with ethyl glyoxylate 274 to give an intermediate imine, followed by addition to an alkyne 275 to give the quinoline moiety

276 (Scheme 6.1). Huang et al . achieved this through the use of Cu(OTf) 2 in DCM at r.t. for 16 h, giving yields in the range of 52-92%. 383 Similarly, Li et al . achieved the

384 same scaffolds in yields of 45-82% using I 2 in nitromethane at r.t. for 12 h, while

Bharate et al . obtained yields of 78-92% with the use of 10% formic acid at r.t. for 45 min. 385

Scheme 6.1: Reagents and conditions: a) Cu(OTf) 2, DCM, 16 h; b) I 2, MeNO 2, r.t., 12

h; c) 10% HCO 2H:H 2O, r.t., 45 min.

Wu et al . reported a Skraup-Doebner-Von Miller quinoline synthesis in which the typically observed stereochemistry was reversed. 386 By heating a mixture of aniline derivative 273 and γ -aryl-β,γ -unsaturated α -ketoester 277 at reflux in TFA under nitrogen, the 4-arylquinoline-2-carboxylates 278 were generated in yields of 42-83%

(Scheme 6.2).

Scheme 6.2: Reagents and conditions: a) TFA, N 2, reflux, 8-18 h.

The group of Huo et al . also demonstrated that quinolines 276 may aso be generated by auto-oxidation of N-phenylglycine derivatives 279 with styrenes 280 in a 5:1 mixture of 165

acetonitrile:1,2-dichloroethane (Scheme 6.3). 387 The reaction was performed over 12-

600 h at 40 °C, giving the quinolines in yields of 9-40%. Similarly, Liu et al . reacted iron(III)chloride and di-tert -butyl peroxide in 1,2-dichloroethane for 12 h at 80 °C to give quinolines 276 in 76-82% yield. 388

Scheme 6.3: Reagents and conditions: a) MeCN:DCE, 40 °C, 40-600 h; b) FeCl 3,

(tBuO) 2, DCE, 80 °C, 12 h.

Alternatively, Guillou et al . employed 2-aminoacetophenone 281 as a starting material, which was converted to the 4-hydroxyquinoline 282 by heating at reflux in the presence of dimethyl oxalate and sodium methoxide in methanol for 40 h (Scheme 6.4). This was then converted to the bromoquinoline 283 with phosphoryl bromide and potassium carbonate at reflux in acetonitrile for 3 h, with subsequent Suzuki coupling to aryl boronic acids giving the 4-arylquinolines 284 in 61-81% yield.

Scheme 6.4: Reagents and conditions: a) (CO 2Me) 2, NaOMe, MeOH, reflux, 40 h; b)

POBr 3, K2CO 3, reflux, 3 h; c) ArB(OH) 2, PdCl2dppf, Cs 2CO 3, dioxane/H 2O, 85 °C, 1 h. 166

In their synthesis of quinolines 97a-c, the group of Kikuchi started from 2-isopropyl aniline 273a, treating this with dimethyl acetylenedicarboxylate (DMAD) in a mixture of Triton B and MeOH, followed by thermal cyclization at 250 °C in diphenyl ether

(Ph 2O) to give the 4-quinolone 285 (Scheme 6.5). This was then converted to the triflate

286 by treating with trifluoromethanesulfonyl anhydride (Tf 2O) in the presence of 2,6- lutidine and DMAP in DCM. This was then coupled to phenylboronic acid using

231 Pd(PPh 3)4 with Et 3N in DMF to give quinoline 278a.

Scheme 6.5: Reagents and conditions: a) i.DMAD, Triton B, MeOH; ii. Ph 2O, 250 °C;

b) Tf 2O, 2,6-lutidine, DMAP, DCM; c) phenylboronic acid, Et 3N, Pd(PPh 3)4, DMF.

6.2.1 Synthesis of quinoline starting materials

To synthesize the quinoline esters ( 278) targeted in this project, the method of Wu was first investigated, 386 whereby a mixture of the appropriate benzaldehyde 287a-c and pyruvic acid in MeOH was treated with a methanolic solution of KOH at 0 °C.

Following stirring at r.t. for 1 h and at 0 °C for a further 12 h, the intermediate 167

potassium salts 288a-c were isolated in 90-98% yield following filtration (Scheme 6.6).

These were then added to a methanolic solution of HCl, generated by the addition of

AcCl to MeOH at 0 °C, and the resulting mixture was stirred at r.t. for 2 h and then at reflux for a further 12 h to generate the corresponding esters 277a-c in 21-49% yield.

The melting points and spectral data of esters 277a-c were all consistent with literature reports, confirming formation of the desired products. The methyl substituent (present in 277c) was selected as many arotinoids, such as 265-267, incorporate a p-tolyl ring at the 4-position. Similarly, bromine (present in 277b) is a common atom appended in arotinoids, such as 17 and 267.

Scheme 6.6: Reagents and conditions: a) pyruvic acid, KOH, MeOH, 0 °C, 1 h – r.t., 1

h – 0 °C, 12 h; b) AcCl, MeOH, 0 °C, 0.5 h – r.t., 2 h – reflux, 12 h.

Esters 277a-c were then carried forward and heated at reflux with 2-isopropylaniline

273a in TFA under a nitrogen atmosphere for 24 h. Removal of the solvent and aqueous work-up followed by column chromatography furnished the desired 8-isopropyl-4- arylquinolones 278a-c in 26-39% yield (Scheme 6.7). Treatment of these esters with hydrazine monohydrate under reflux in MeOH for 4 h gave the corresponding hydrazides 289a-c in yields of 84-99%. 168

Scheme 6.7: Reagents and conditions: a) 2-isopropylaniline, TFA, N 2, reflux, 24 h; b)

NH 2NH 2.H 2O, MeOH, reflux, 4 h.

1 The spectral data of 278a was representative of its analogues. The H NMR (CDCl 3) spectrum (Figure 6.2) displayed the quinoline peaks as a singlet at δ 8.09 (H3) and two doublet of doublets at δ 7.79 (J = 1.4, 8.4 Hz, H5) and 7.68 (J = 1.4, 7.4 Hz, H7), with the signal of H6 overlapping with the multiplet signals of the 4-phenyl ring. An additional singlet resonance was observed at δ 4.06 for the methyl ester, while the isopropyl group gave rise to a septet at δ 4.54 (CH) and a doublet at δ 1.43 (2 x CH 3),

13 both with J = 6.9 Hz coupling. Furthermore, the C NMR (CDCl 3) spectrum displayed characteristic resonances at δ 166.6 (C=O), 53.0 (OCH 3), 27.7 (CH) and 23.9 (2 x CH 3), while a single carbonyl stretch was observed at 1734 cm -1 in the IR (ATR) spectrum.

Conversion of the ester 278a to the hydrazide 289a was confirmed by disappearance of

1 the methoxy peak in the H NMR (CDCl 3) spectrum, with the appearance of a broad singlet at δ 9.19 (NH) and a doublet at δ 4.18 ( J = 4.7 Hz, NH 2). Furthermore, the

13 methoxy peak in the C NMR (CDCl 3) spectrum also disappeared, with a resultant shift of the carbonyl resonance to δ 165.6, which was also reflected in the less energetic IR

(ATR) stretch of 1665 cm -1. 169

1 Figure 6.2: H NMR (CDCl 3) spectrum of quinoline ester 278a.

During the synthesis of quinolines 278a-c, upscaling of the reaction from 0.2 mmol as described by Wu proved to be a difficult task. Scaling of this reaction to 2-12 mmol typically resulted in lower yields of 26-39%, compared to 42-83% reported for the 0.2 mmol scale by Wu.386 Furthermore, fresh and dry TFA was required for the reaction in order to avoid the formation of quinoline acids 290a-b, which were observed in 25 and

30% yield, respectively, at 5 mmol scale (Figure 6.3).

Figure 6.3: Quinoline acid side-products isolated during the synthesis of 278a and

278c.

The method of Kikuchi was also investigated as a means of accessing 4-arylquinoline derivatives where the aldehyde starting materials were unavailable.231 Following their procedure, 273a was treated with DMAD in MeOH for 2 h before being heated at 250 170

°C in diphenyl ether. Unfortunately, this method was not ideal as the thermal cyclization proved ineffective, resulting in only partial conversion of the intermediate to the cyclized quinolone system. Alternatively, 273a was stirred in EtOH for 2 h, followed by treatment with freshly prepared Eaton’s reagent according to the procedure of Zewge et al. ,389 giving quinolone 285 in 78% yield (Scheme 6.8). A characteristic NH singlet

1 resonance was observed at δ 9.15 in the H NMR (CDCl 3) spectrum of 285 , as was an

13 additional carbonyl at δ 180.0 in the C NMR (CDCl 3) spectrum. Additionally, two carbonyl stretches were observed at 1734 and 1625 cm -1 in the IR (ATR) spectrum.

Quinolone 285 was then treated with Tf 2O and 2,6-lutidine at 0 °C in DCM for 24 h.

Following acidic work-up and crystallization from DCM/hexanes, the triflate 286 was isolated in 74% yield. This was confirmed by the disappearance of the NH resonance in

1 the H NMR (CDCl 3) spectrum, as well as the appearance of a single carbonyl peak at δ

13 -1 165.0 in the corresponding C NMR (CDCl 3) spectrum and 1722 cm in the IR spectrum. Triflate 285 was then subjected to a Suzuki coupling reaction using pyridine-

4-boronic acid pinacol ester, Pd(PPh 3)4 and Et 3N in DMF at reflux for 48 h.

Unfortunately, the desired quinoline 291 was not isolated on work-up. Modifications to the base (Et 3N, K 3PO 4) and catalyst (Pd(Ph 3)4, Pd(PPh 3)2Cl 2) also failed to achieve the production of 291. 171

Scheme 6.8: Reagents and conditions: a) i. iPrOH, r.t., 2 h; ii. E aton’s reagent, 60 °C,

2 h; b) Tf 2O, 2,6-lutidine, DCM, N 2, 24 h; c) pyridine-4-boronic acid pinacol ester,

Et 3N, Pd(PPh 3)4, DMF, reflux, 48 h.

During the optimization of the synthesis of quinolone 285 an unexpected side product was isolated. This product was much less polar than quinolone 285, as observed by its higher Rf value of 0.60 on TLC (50% EtOAc:hexanes), compared to 0.23 for 285.

1 Furthermore, in the H NMR spectrum (d 6-acetone) of the unknown product, the expected singlet resonance of the NH was not observed, while an additional singlet that integrated for 3 protons had appeared at δ 3.63 (Figure 6.4). The mass spectrum (+ESI) of this unknown compound gave a peak at m/z 324.0893, which corresponded to the formula C15 H18 NO 5S (required 324.0900). In light of this data, the structure was assigned as methyl 8-isopropyl-4-((methylsulfonyl)oxy)quinoline-2-carboxylate 292.

The formation of this product was rationalized by reaction of the desired quinolone 285 with a mixed anhydride formed from methanesulfonic acid and P2O5 in the preparation of Eaton’s reagent (Scheme 6.9 ). 172

1 Figure 6.4: H NMR (d 6-acetone) spectrum of the unexpected methylsulfonyloxy

quinoline 292.

Scheme 6.9: A possible mechanism for the formation of methylsulfonyloxy quinoline

292.

173

6.2.2 Synthesis of quinoline oxadiazoles

Preliminary investigations into the synthesis of quinoline oxadiazoles were focused around employing an oxidative cyclization process. Firstly, hydrazide 289a was condensed with 4-carboxybenzaldehyde by heating the mixture at reflux in EtOH for 48 h. The desired imine 293 precipitated from the reaction mixture and was isolated via filtration in 97% yield (Scheme 6.10). The oxidative cyclization of 293 was then attempted by treatment with iodobenzene diacetate (PhI(OAc) 2) in CHCl 3 for 3 h, however, this did not afford the desired oxadiazole 272a. Several variations to this method were investigated in order to procure oxadiazole 272a. These included the use of PhI(OAc) 2 and Bu 4NCl in DMF, grinding 293 with

[bis(trifluoroacetoxy)iodo]benzene, or using NBS with DBU in CHCl 3. However, none of these methods were found to give 272a, and the reactions returned only unreacted

293.

Scheme 6.10: Reagents and conditions: a) 4-carboxybenzaldehyde, EtOH, reflux, 48 h;

b) PhI(OAc) 2, CHCl 3, r.t., 3 h. 174

In order to overcome the limitations of the oxidative cyclization process, a dehydrocyclization similar to that described in Chapter 4 was considered. Therefore, a mixture of hydrazide 289a and 4-cyanobenzoic acid in DMF was treated with EDCI and

HOBt for 24 h, affording hydrazide 294 in 52% yield following chromatography

(Scheme 6.11). Cyclization of 294 was then achieved using 4-TsCl and DIPEA in

MeCN for 3 h to give oxadiazole 295 in 84% yield.

Scheme 6.11: Reagents and conditions: a) 4-cyanobenzoic acid, EDCI, HOBt, DMF,

24 h; b) 4-TsCl, DIPEA, MeCN, 16 h.

Generation of hydrazide 294 was demonstrated through the inclusion of additional

1 resonances in the H NMR (CDCl 3) spectrum, where H2″/H6″ gave a doublet at δ 8.06 and H3″/H5″ formed part of a multiplet at δ 7.72-7.69, overlapping with H7.

Additionally, the hydrazide NH protons were also observed as two singlets at δ 10.76

13 and 10.16. Furthermore, two carbonyl peaks were observed in the C NMR (CDCl 3) spectrum at δ 162.7 and 162.0, as well as the IR (ATR) spectrum at 1691 and 1637 cm -1.

Cyclization to oxadiazole 295 was confirmed by disappearance of the hydrazide NH

1 peaks in the H NMR (CDCl 3) spectrum (Figure 6.5), downfield shifting of the former 175

13 carbonyl resonances in the C NMR (CDCl 3) spectrum to δ 165.5 and 164.2 and disappearance of the carbonyl stretches in the IR (ATR) spectrum.

1 Figure 6.5: H NMR (CDCl 3) spectrum of oxadiazole 295.

The final phase of this route attempted to generate the carboxylic acid by hydrolysis of the nitrile group. Firstly, oxadiazole 295 was treated with concentrated H 2SO 4 and water in glacial AcOH and the mixture was heated at 120 °C for 20 h (Scheme 6.12).

1 However, upon aqueous work-up and examination of the H NMR (d6-DMSO) spectrum of the product, it was found that the reaction conditions were too harsh. The oxadiazole ring had been hydrolysed along with the nitrile group, giving hydrazide 286a and terephthalic acid 296 . As an alternative approach, a basic method was then investigated, in which oxadiazole 295 was treated with 30% KOH and 30% H2O2 for 3 h. However, upon work-up it was revealed that the nitrile group had not been hydrolysed, while the oxadiazole ring had opened to give hydrazide 294. The spectral data of this compound matched that which had previously been recorded, confirming the undesired outcome of this reaction. 176

Scheme 6.12: Reagents and conditions: a) H 2SO 4, AcOH, H 2O, 120 °C, 20 h; b) KOH,

H2O2, r.t., 3 h.

As the oxadiazole ring was observed to be sensitive to the harsh conditions used to hydrolyse the nitrile group, the method was repeated using hydrazide 294. Treating 294 with concentrated H 2SO4 and water in glacial AcOH at 120 °C for 6 h again led to formation of hydrazide 289a and terephthalic acid 296 (Scheme 6.13). This was again confirmed by comparison of the spectral data to that recorded previously for 6-26a.

Scheme 6.13: Reagents and conditions: a) H 2SO 4, AcOH, H 2O, 120 °C, 6 h.

As the nitrile hydrolysis was proving problematic, the generation of the carboxylic acid was next targeted through ester hydrolysis, which can typically be performed under milder conditions. Therefore, hydrazide 289a was coupled to mono-methyl 177

terephthalate using EDCI and HOBt in DMF for 24 h to generate hydrazide 297a in

85% yield following chromatography (Scheme 6.14). Hydrolysis of the ester with 1:1 2

M NaOH:THF for 2 h then gave acid 298 in a yield of 25%. Finally, cyclization was then achieved through the use of 4-TsCl as described earlier, to afford oxadiazole 272a in 33% yield.

Scheme 6.14: Reagents and conditions: a) mono-methyl terephthalate, EDCI, HOBt,

DMF, 24 h; b) 4-TsCl, DIPEA, MeCN, 16 h; c) 2 M NaOH, THF, 2 h.

As the hydrolysis of 298 was found to proceed in low yield, the cyclization and saponification steps were performed in the reverse order for the synthesis of 272b-c.

Therefore, hydrazides 289b-c were coupled to mono-methyl terephthalate using EDCI and HOBt in DMF for 24 h to generate hydrazides 297b-c in yields of 64-85% following chromatography (Scheme 6.15). Cyclization of these was then achieved through the use of 4-TsCl as described earlier, to afford oxadiazoles 299a-b in 29-70% yield. Finally, saponification of the ester with 0.25 M NaOH/THF for 2 h gave acids

272b-c in yields of 92-97%. 178

Scheme 6.15: Reagents and conditions: a) mono-methyl terephthalate, EDCI, HOBt,

DMF, 24 h; b) 4-TsCl, DIPEA, MeCN, 16 h; c) 2 M NaOH, THF, 2 h.

The identity of carboxylate products 297-299 was established by similar means to the nitrile products 294 and 295. Taking the representative case of 297b, attachment of the methyl terephthalate was shown through the appearance of two doublet of doublet peaks

1 (J = 2.3, 8.7 Hz) in the H NMR (d 6-DMSO) spectrum at δ 8.12 and 8.08, corresponding to H2/H6 and H3/H5 of the terephthalate moiety, respectively. An additional singlet was also observed at δ 3.91, corresponding to the methyl ester protons. Additionally,

13 three carbonyl resonances were observed in the C NMR (d 6-DMSO) spectra at δ

165.7, 164.8 and 163.8. The data for oxadiazole 299b confirmed that the cyclization had

1 occurred, through disappearance of the hydrazide peaks in the H NMR (CDCl 3) spectrum (Figure 6.6), as well as the presence of a single carbonyl resonance in the 13 C

NMR (CDCl 3) spectrum at δ 166.3. Saponification to acid 272b was confirmed by

1 13 disappearance of the methyl ester signals in the H and C NMR (d 6-DMSO) spectra. 179

1 Figure 6.6: H NMR (CDCl 3) spectrum of oxadiazole 299b.

6.2.3 Quinoline-oxadiazoles bearing an o-fluorobenzoic acid

As detailed in Chapter 6.1, the incorporation of o-fluoro substituents on the benzoic acid moiety can dramatically enhance the RARα selectivity of the ligands. 136,379

Incorporating an o-fluoro substituent into the quinoline scaffold was therefore envisaged as a potential strategy to generate a more selective and therefore more potent

RARα ligand. In order to achieve this, the synthesis of the fluorinated carboxylic acid precursor was first undertaken.

To this end, the procedure of Wang was utilized (Scheme 6.16). 390 Firstly, 2-fluoro-4- methylbenzoic acid 300 was esterified using H 2SO 4 in MeOH at reflux for 24 h, giving methyl ester 301 in 84% yield. Ester 301 was then brominated using NBS and benzoylperoxide at reflux in CCl 4 for 48 h. The dibromomethyl intermediate 302 was then immediately oxidized using AgNO 3 in EtOH/H 2O at 50 °C for 1 h, giving aldehyde

303 in 35% yield over the 2 steps. Finally, aldehyde 303 was further oxidized using sulfamic acid and sodium chlorite in MeCN:H 2O for 20 h to give acid 304 in 49% yield. 180

The characteristic physical data of compounds 300-304 was consistent with that reported in the literature.

Scheme 6.16: Reagents and conditions: a) H 2SO 4, MeOH, reflux, 24 h; b) NBS,

(PhCOO) 2, CCl4, reflux, 48 h; c) AgNO 3, EtOH, H 2O, 50 °C, 1 h; d) NaClO 2,

NH 2SO 3H, MeCN:H 2O (2:1), r.t., 20 h.

With the fluorinated acid 304 now in hand, this was coupled to hydrazide 272a using

EDCI and HOBt in DMF for 24 h to give hydrazide 305 in a yield of 73% (Scheme

6.17). Cyclization with 4-TsCl in the presence of DIPEA in MeCN afforded oxadiazole

306 in 43% yield, with subsequent saponification using 0.25 M NaOH/THF for 2 h giving acid 307 in a yield of 77%. 181

Scheme 6.17: Reagents and conditions: a) 3-fluoro-4-(methoxycarbonyl)benzoic acid,

EDCI, HOBt, DMF, 24 h; b) 4-TsCl, DIPEA, MeCN, 16 h; c) 2 M NaOH, THF, 2 h.

As established earlier, hydrazide formation was confirmed through the appearance of additional peaks in the spectra of 305. Peaks corresponding to the fluoro-terephthalate moiety appeared as a doublet at δ 8.07 (J = 8.0 Hz, H6), as part of a multiplet at δ 7.84-

1 7.81 (H3, H5) and as a singlet at δ 3.94 (OCH 3) in the fluorine-decoupled H NMR

13 (d 6-acetone) spectrum. The C NMR spectrum (d 6-acetone) also gave equivalent signals for the two hydrazide carbonyls at δ 163.4, while the ester gave a carbonyl resonance at δ 164.5 and a methoxy resonance at δ 52.9. Cyclization to oxadiazole 306

1 was again confirmed by the loss of hydrazide NH signals in the H NMR (CDCl 3)

13 spectrum, as well as shifting of the former carbonyl peaks in the C NMR (CDCl 3) spectrum. Additionally, ester hydrolysis to acid 307 was demonstrated by loss of the

1 13 methoxy peaks in both the H and C NMR (d 6-DMSO) spectra.

182

6.3.4 Synthesis of quinolines with varied linking regions

Further SAR studies were directed towards the linking region of the quinoline scaffold.

It was envisaged that modifications made to the linking heterocycle may modulate the polarity and therefore hydrogen-bonding capability of the molecule, which may have an effect on t he RARα selectivity. In order to achieve this goal, t he 2-chloroquinoline 308 was viewed as an essential intermediate, as it could then be subsequently converted to other functional groups, such as azides (309), nitriles (310) and alkynes (311 ), for use as substrates in cycloaddition reactions (Figure 6.7).

Figure 6.7: Targeted scaffolds for the synthesis of quinolines with various linkers.

In order to prepare 308, a synthetic route involving chlorination of 2-quinolone 312 was envisaged. Therefore, a mixture of 2-isopropylaniline 273a and ethyl benzoylacetate

246b was heated at 145 °C for 20 min before being treated with 18 M H 2SO 4 at 0 °C for

2 h (Scheme 6.18). Basic work-up then afforded the non-cyclized amide 313 in 4% yield, rather than the desired product 312. Amide 313 was identified by the presence of

1 an NH singlet at δ 9.55 and a CH 2 singlet at δ 4.19 in the H NMR (CDCl 3) spectrum, as 183

13 well as a peak at δ 44.9 in the C NMR and DEPT-135 (CDCl 3) spectra. Furthermore, aromatic peaks totalling 9 protons were also observed in the 1H NMR spectrum, while two carbonyl peaks at δ 197.3 and 164.3 were observed in the 13 C NMR spectrum.

Scheme 6.18: Reagents and conditions: a) i. 145 °C, 20 min, ii. 18 M H 2SO 4, 0°C, 2 h;

b) i. 145 °C, 24 h, ii. Eaton’s reagent , 60 °C, 24 h.

The condensation of 272a and 246b was repeated with an extended duration of 24 h, followed by heating at 60 °C in Eaton’s reagent for a further 24 h. Basic work-up with subsequent chromatography then afforded the desired quinolone 312 in 4% yield

1 (Scheme 6.18). The H NMR (CDCl 3) spectrum displayed quinoline protons as a singlet at δ 8.24 (H3) and three doublet of doublets at δ 7.87 (J = 1.3, 8.4 Hz, H5), 7.75 ( J =

1.3, 7.2 Hz, H7) and 7.62 ( J = 7.2, 8.4 Hz, H6). The 4-phenyl ring gave rise to a multiplet at δ 7.57-7.50, integrating for 5 protons, while the 8-isopropyl group resonated as a septet at δ 4.32 (CH) and a doublet at δ 1.46 (2 x CH 3), each with coupling of 6.9

Hz.

Chlorination of quinolone 312 was next attempted with the use of POCl 3. Heating 312 at reflux in POCl 3 for 3 h did not afford chloroquinoline 308 (Scheme 6.19). Reduction of the reaction temperature to 70 °C, or dilution with a co-solvent such as EtOAc also failed to yield the crucial intermediate 308. Furthermore, extended reaction times also 184

resulted in a complex mixture of baseline impurities, most likely arising from decomposition of the starting material. As the intermediate 2-chloroquinoline 308 could not be produced, the synthesis of analogues with other linkers was next undertaken.

Scheme 6.19: Reagents and conditions: a) POCl 3, reflux, 3 h.

Amide bonds have been used to introduce RARα selectivity into a number of arotinoid scaffolds, such as 17 , 266 and 267. Furthermore, the oxadiazole motif has been frequently used as an isostere for the amide bond, as it has higher levels of metabolic stability and hydrophilicity, while maintaining the substituent directionality and planar character of an amide. In order to determine the necessity of the oxadiazole, a quinoline with an amide linker was next targeted. Therefore, acid 290a was coupled to p- benzocaine utilizing EDCI and HOBt in DMF for 24 h to give amide 314 in a yield of

91% (Scheme 6.20). Saponification of the ester was initially attempted using the 0.5 M

NaOH:THF mixture that had been successful in the generation of oxadiazoles 272 and

307, however this concentration was insufficient to achieve ester hydrolysis. This hurdle was overcome through heating 314 at 50 °C in 2 M NaOH:MeOH for 4 h, giving acid

315 in 96% yield following acidic work-up. 185

Scheme 6.20: Reagents and conditions: a) p-benzocaine, EDCI, HOBt, DMF, 24 h; b)

2 M NaOH:MeOH, 50 °C, 4 h.

1 The H NMR (d 6-acetone) spectrum of amide 314 displayed the quinoline protons as a singlet at δ 8.24, corresponding to H3, as well as a doublet at δ 7.85, corresponding to

H5 and H7, and a doublet of doublets at δ 7.69, corresponding to H6, each with coupling of 7.8 Hz. The 4-phenyl protons gave rise to a multiplet at δ 7.64-7.58, while the isopropyl protons appeared as a septet and doublet at δ 4.62 and 1.49, respectively, with coupling of 6.9 Hz. The remaining aromatic protons were observed as a doublet at

δ 8.07 (J = 1.9 Hz), with the amide proton giving a singlet at δ 10.66 and the ethyl ester giving a quartet (CH 2) and triplet (CH 3) at δ 4.35 and 1.38, respectively, with J = 7.1

Hz. Cleavage of the ester to give acid 315 was confirmed through the disappearance of

1 13 the ester resonances in both the H and C NMR (d 6-DMSO) spectra.

186

6.3.5 Synthesis of 4-methoxyquinolines

In their design of quinoline-based RARα antagonists, Kikuchi et al. discovered that the

8-isopropyl-4-methoxyquinoline scaffold acted as an RARα antagonist, as well as an

RARβ agonist. 231 As overexpression of RARα has been associated with decreased

RARβ expression in cancer, it was thought that such a dual -acting compound may be useful in treating various cancers. Therefore, an analogue based on an 8-isopropyl-4- methoxyquinoline scaffold was targeted as a potential dual-acting RARα antagonist and

RARβ agonist.

Starting from the previously prepared quinolin-4-one 285, methylation was performed according to the procedure of Zhou et al. 391 Compound 285 was heated at 70 °C with

K2CO 3 in DMSO for 1 h, before being cooled to 35 °C and treated with iodomethane for

18 h. Aqueous work-up then afforded 4-methoxyquinoline 316 in 92% yield (Scheme

6.21). Following the established procedure, ester 316 was converted to the corresponding carbohydrazide 317 in 93% yield by heating with hydrazine monohydrate at reflux in MeOH for 4 h. This was then coupled to mono-methyl terephthalate using EDCI and HOBt in DMF for 24 h to give hydrazide 318 in 39% yield following purification. Cyclization using 4-TsCl and DIPEA in MeCN for 3 h then gave oxadiazole 319 in a yield of 71%. Finally, ester saponification with 0.5 M

NaOH/THF for 4 h gave acid 320 in 56% yield. 187

Scheme 6.21: Reagents and conditions: a) MeI, K 2CO 3, DMSO, 35 °C, 18 h; b)

NH 2NH 2.H 2O, MeOH, reflux, 4 h; c) mono-methyl terephthalate, EDCI, HOBt, DMF,

24 h; d) 4-TsCl, DIPEA, 3 h; e) 0.5 M NaOH/THF, 4 h.

Methylation of 285 was confirmed by disappearance of the quinolone NH signal and

1 appearance of the 4-methoxy resonance at δ 4.18 in the H NMR (d 6-acetone) spectrum of 316, with conversion to the hydrazide confirmed by disappearance of the methyl ester

1 peak in the H NMR (d 4-MeOD) spectrum of 317 (Figure 6.8). Derivatives 318-320 gave characteristic data consistent with that for 298a-c, 299a-b and 272a-c respectively

(Figure 6.9). 188

1 Figure 6.8: Comparative H NMR spectra of ester 316 (d 6-acetone, blue) and hydrazide

317 (d 4-MeOD, red).

1 Figure 6.9: Comparative H NMR spectra of ester 319 (d 6-acetone, blue) and acid 320

(d 6-DMSO, red). 189

6.4 Characterization of in vitro activity against neuroblastoma cell lines

In order to determine the levels of cytotoxic activity towards neuroblastoma cells in vitro , as well as establish an SAR profile for the 8-isopropylquinoline scaffold, the synthesized oxadiazoles were screened for anti-cancer activity against the RAR transfected BE(2)-C neuroblastoma cell lines, as described previously in Chapter 3

(Table 6.1). As these compounds were primarily targeted at RARα inhibition, they were primarily screened in the RARα overexpressed cell line , but also in the RARβ and

RARγ over-expressed cell lines.

Table 6.1: Cytotoxicity of 8-isopropylquinolines towards transfected BE(2)-C cell lines following 72 h exposure.

Compound IC 50 (M) E.V. RARα RAR β RARγ 272a 13.4 15.1 19.7 31.1 272b >50 >50 >50 n.d. 272c 18.7 17.1 25.5 n.d. 307 17.4 17.5 19.2 19.1 315 >50 >50 >50 n.d. 320 >50 >50 >50 >50 n.d.: not determined.

The anticancer activity of the synthesized quinolines presented an interesting SAR, with the activity ranging from moderate to low. Oxadiazole 272a displayed cytotoxic activity against all of the BE(2)-C cell lines, with the highest potency against the E.V. transfected cell line, with an an IC 50 value of 13.4 µM. In the RAR over expressing cell lines, 272a displayed greater cytotoxicity where RARα and RARβ were overexpressed, having IC 50 values of 15.1 and 19.7 µM in these cell lines, respectively, compared to

31 .1 M in the RARγ over -expressing cell line. It was interesting to observe that substitution of the 4-phenyl ring had dramatic and diverse effects on the cytotoxicity of 190

these compounds. The inclusion of a 4-bromo substituent in 272b dramatically reduced the cytotoxicity compared to 272a, resulting in IC 50 values greater than 50 µM in all tested cell lines. Meanwhile, the inclusion of a 4-methyl group in 272c slightly reduced cytotoxicity in all cell lines (IC 50 : 18.7, 17.1 and 25.5 µM in E.V., RARα and RARβ overexpressing cell lines, respectively), but increased selectivity for the RARα overexpressing cell line versus both the E.V. and RARβ transfected cell lines. This result suggests that the steric bulk of the 4-aryl substituent may play a key role in the binding of these molecules to RAR subtypes due to the differences in the RAR binding cavity sizes, therefore influencing the cytotoxicity and RAR selectivity of the ligands.

Considering substitution on the benzoic acid moiety, the inclusion of an o-fluoro group in 307 resulted in multiple changes to cytotoxicity and RAR subtype selectivity, relative to 272a. The cytotoxicity was reduced in the E.V. and RARα transfected cell lines, with

IC 50 values of 17.4 and 17.5 µM, compared to 13.4 and 15.1 µM for 272a. However, the cytotoxicity towards the RARβ over -expressed cell line remained consistent, with an

IC 50 value of 19.2 µM compared to 19.7 µM for 272a. The largest difference was the increased cytotoxicity towards the RARγ over -expressed cell line, where 307 displayed an IC 50 value of 19.1 µM, compared to 31.1 µM in the case of 272a. Taken together, the data suggests that compound 307 is relatively non-selective between the RAR subtypes.

The inclusion of the o-fluoro substituent perhaps plays a greater role influencing the pK a of the adjacent carboxylic acid, somewhat like the RARβ 2-selective agonist 23 (Figure

6.10), than it does on other aspe cts of binding to the RARα cavity . 191

Figure 6.10: Comparative structures of the o-fluoro-containing 307 and RARβ 2-

selective agonist 23 .

Further modifications to the quinoline scaffold also resulted in a loss of cytotoxic activity. Screening of the amide linked 315 revealed that the oxadiazole linkage is far superior for cytotoxicity in the context of the quinoline scaffold. The amide modification resulted in a large decrease in cytotoxicity, with 315 possessing IC 50 values above 50 µM in all cell lines tested. Similarly, the 4-methoxyquinoline scaffold

320 presented with IC 50 values greater than 50 µM across all cell lines. This suggests that the phenyl ring is necessary for active binding and the induction of cytotoxic activity.

6.5 Conclusions

A synthetic protocol for the preparation of 4-arylquinoline oxadiazoles has been developed. The synthesized analogues display an interesting SAR with respect to their cytotoxicity towards BE(2)-C neuroblastoma cells, as well as their selectivity towards cell lines over-expressing the various RAR subtypes. It was also found that this selectivity may be modified by substitution about the 4-aryl ring, or by the inclusion of an o-fluoro substituent on the benzoic acid moiety.

192

CHAPTER 7: FUTURE DIRECTIONS

The variety of scaffolds and methods considered in this work has opened up many possibilities for the further study and development of novel retinoid enhancers. Firstly, the computational methods have allowed for the development of a protocol to screen proposed ligands and predict their affinity to RARβ. In order to generate more selective analogues, this protocol can also be applied to crystal structures of RARα and RARγ , with comparisons of the respective affinities able to add another dimension to synthetic planning.

Considering the indole-benzothiazole acetamides, this series offers the greatest promise for further development. The current best performing compound N-(4- chlorobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide ( 158i) requires further mechanistic investigation to determine how it effects the retinoid signalling pathway. A more detailed examination into the exact mechanism of action (apoptosis, necrosis, etc) and the related pathways is necessary before this class of compounds can progress to more rigorous testing. Furthermore, the opportunity still exists to further modify this scaffold, such as investigating the effect of linking the indole acetic acids at other positions ( 321), or indeed doing the same for the benzothiazole (322). Additionally, the inversion of the amide bond by combining a benzothiazole-2-carboxylic acid and an indole-3-methylamine ( 323 ) may also present interesting SAR data. 193

Whilst the in vitro cytotoxicity of the phenyl-substituted benzothiazole-thieno[2,3- c]pyrazoles was not particularly impressive, the N-(benzo[d]thiazol-2-yl)-1,3-dimethyl-

1H-thieno[2,3-c]pyrazole-5-carboxamide ( 229) did display some activity. Furthermore, the targeted benzothiazole derivative ( 227) of the lead compound ( L10 ) was not able to be synthesized within the timeframe of this thesis. Therefore, the further development of alkyl-substituted thieno[2,3-c]pyrazoles is a necessary future direction of this class of compounds. Additionally, the use of substituted benzothiazoles, or indeed other heterocycles ( 324), is also necessary.

Finally, the quinoline-oxadiazoles also offered some interesting SAR data, particularly when substituents were added to the 4-phenyl ring. Therefore, further investigation into the effects of various substituents, as well as their positioning on this ring, is an important future development of these compounds. Additionally, the substitution of the

4-phenyl ring for a heterocyclic moiety was unable to be achieved during this thesis and is also another future goal. Similarly, analogues linked by heterocycles other than an oxadiazole also warrant further investigation.

194

CHAPTER 8: EXPERIMENTAL

8.1 General information

All reactions requiring anhydrous conditions were performed under a nitrogen atmosphere. Methanol (MeOH), ethanol (EtOH), pentane and ethyl acetate were obtained from commercial sources. Anhydrous dichloromethane (DCM), ether (Et2O) and tetrahydrofuran (THF) were obtained using a PureSolv MD Solvent Purification

System. Commercially available reagents were purchased from Fluka, Aldrich, Acros

Organics, Alfa Aesar and Lancaster and used without further purification.

Reactions were monitored using thin layer chromatography, performed on Merck DC aluminium plates coated with silica gel GF 254 . Compounds were detected by short and long wavelength ultraviolet light or using different chemical indicators such as permanganate solutions, iodine vapour, bromocresol green and ninhydrin reagent.

Vacuum column chromatography was carried out using Grace Davison LC60A 6-35 micron silica gel and this method involved the use of vacuum at the base of the column via a vacuum pressure line. Preparative thin layer chromatography was carried out on

3×200×200 mm glass plates coated with Merck 60GF 254 silica gel.

NMR spectra were obtained in the designated solvents on a Bruker DPX 300 or a

Bruker Avance 400 or a Bruker AVANCE DMX 500 spectrometer as designated.

Chemical shifts ( δ) are in parts per million and internally referenced relative to the solvent nuclei. 1H NMR spectral data are reported as follows: chemical shift measured in parts per million (ppm) downfield from TMS ( δ); multiplicity; observed coupling constant ( J) in Hertz (Hz); proton count; assignment. Multiplicities are assigned as singlet (s), doublet (d), doublet of doublet (dd), doublet of triplet (dt), triplet, (t), quartet 195

(q), quintet (p), doublet of doublet of doublets (ddd), multiplet (m) and broad singlet

(bs) where appropriate and the observed coupling constants ( J) are described in Hertz

(Hz). 13 C NMR spectra were recorded in the designated solvents and chemical shifts are reported in ppm downfield from TMS and identifiable carbons are given (where possible).

Melting points were measured using a Mel-Temp melting point apparatus, and are uncorrected. Infrared spectra were recorded on a Perkin Elmer Spotlight 400 FTIR

Microscope. Ultraviolet spectra were measured using a Perkin Elmer Lambda 35 UV-

Visible Spect rometer in the designated solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε) in M -1 cm -1. High-resolution mass spectrometry was performed by the Bioanalytical Mass Spectrometry unit, UNSW. Microanalysis was performed on a Carlo Erba Elemental Analyzer EA 1108 at the Campbell

Microanalytical Laboratory, University of Otago, New Zealand.

8.2 Experimental details

N-(Benzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (115)

3-Indoleacetic acid 106 (0.19 g, 1.12 mmol), 2- aminobenzothiazole 110 (0.15 g, 1.00 mmol) and PyBop

(0.52 g, 1.00 mmol) were dissolved in DCM (2 mL) and

DIPEA (0.20 mL, 1.21 mmol) added dropwise. This mixture was stirred for 2 h before the solvent was removed under reduced pressure, the solid redissolved in EtOAc

(30mL) and washed with water (20mL). The organic phase was concentrated under reduced pressure and the crude solid purified by flash column chromatography (50%

EtOAc:hexanes) to yield the title compound 115 as a pale brown granular solid (0.17 g, 196

1 1 53%). M.p. 209-211 ºC. H NMR (300 MHz, d 6-DMSO): δ 12.54 (s, 1 H, N H), 10.99

(s, 1 H, NH), 7.95 (d, 1 H, J = 7.2 Hz, H4), 7.74 (d, 1 H, J = 7.6 Hz, H7), 7.61 (d, 1 H, J

= 7.8 Hz, H4′), 7.42 (t, 1 H, J = 7.2 Hz, H6), 7.37 (d, 1 H, J = 8.0 Hz, H7′), 7.32 (d, 1 H,

J = 2.4 Hz, H2′), 7.28 (t, 1 H, J = 7.2 Hz, H5), 7.08 (t, 1 H, J = 6.9 Hz, H5′), 7.00 (t, 1

13 H, J = 6.8 Hz, H6′), 3.92 (bs, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 170.7

(C=O), 158.1 (ArC), 148.5 (ArC), 136.1 (ArC), 131.4 (ArC), 127.1 (ArC), 126.1

(ArCH), 124.3 (ArCH), 123.5 (ArCH), 121.7 (ArCH), 121.1 (ArCH), 120.5 (ArCH),

118.6 (ArCH), 111.5 (ArCH), 107.2 (ArC), 32.4 (CH 2). IR (KBr): ν max 3373, 3163,

3062, 2960, 1747, 1693, 1599, 1529, 1450, 1396, 1316, 1261, 752, 727 cm -1. UV-Vis

-1 -1 (MeOH) : λ max 289 nm (ε 17,069 cm M ), 298 (14,413) nm. HRMS (+ESI): Found m/z

+ 308.0849, [M+H] ; C 17 H14 N3OS required 308.0858.

N-(1 H-Benzo[d]imidazol-2-yl)-2-(1 H-indol-3-yl)acetamide (116)

A mixture of IAA 106 (0.24 g, 1.38 mmol), 2- aminobenzimidazole 139a (0.18 g, 1.32 mmol), EDCI

(0.23 g, 1.20 mmol) and HOBt (0.22 g, 1.39 mmol) in

THF (4.0 mL) was stirred for 4 h before being poured into ice-water (20 mL). The precipitate was collected via filtration and purified by flash column chromatography

(20-75% EtOAc:hexanes) to give the title compound 116 as a beige solid (0.13 g, 37%).

1 M.p. 237-239 ºC. H NMR (300 MHz, d 6-DMSO) δ 11.94 (bs, 1 H, N’H), 11.63 (bs, 1

H, N 1H), 10.90 (s, 1 H, CONH), 7.65 (d, J = 8.1 Hz, 1 H, H4), 7.41 (m, 2 H, H4’, H7’),

7.36 (d, J = 8.1 Hz, 1 H, H7), 7.30 (d, J = 2.2 Hz, H2), 7.05 (m, 3 H, H6, H5 ’, H6’),

13 6.99 (ddd, J = 1.2, 7.1 Hz, 1 H, H5), 3.86 (s, 2 H, CH 2). C NMR (75 MHz, d 6-

DMSO): δ 170.9 (C=O), 146.7 (ArC), 136.1 (ArC), 127.1 (ArC), 124.0 (ArCH), 121.0 197

(ArCH), 118.7 (ArCH), 118.4 (ArCH), 111.3 (ArCH), 107.7 (ArC), 32.9 (CH 2). IR

-1 -1 (ATR ): ν max 3405, 3282, 2821, 1629, 1568 cm . UV-Vis : λ max 292 nm (ε 30,000 cm

M-1), 284 (31,000), 251 (17,400), 217 (60,000), 205 (60,200) nm. HRMS (+ESI): Found

+ m/z 291.1241, [M+H] ; C17 H15 N4O required 291.1240.

N-(Benzo[d]thiazol-2-yl)-3-(1H-indol-3-yl)propanamide (117)

This compound was synthesized as described for compound 115, using 3-indolepropionic acid (0.21 g,

1.11 mmol), 2-aminobenzothiazole 110 (0.16 g, 1.06 mmol), PyBOP (0.73 g, 1.40 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM (2.0 mL) for 2 h, to yield the title compound 117 as a white solid (0.15 g, 73%). M.p. 224-

1 1 226 ºC. H NMR (300 MHz, d 6-DMSO): δ 12.36 (s, 1 H, N H), 10.79 (s, 1 H, NɂH),

7.97 (d, J = 7.2 Hz, 1 H, H4), 7.72 (d, J = 7.6 Hz, 1 H, H7), 7.58 (d, J = 7.8 Hz, 1 H, H

4ɂ), 7.42 (dt, J = 1.3, 7.8 Hz, 1 H, H5), 7.31 (dt, J = 1.1, 7.9 Hz, 1 H, H6), 7.28 (d, J =

6.1 Hz, 1 H, H7ɂ), 7.13 (d, J = 2.3 Hz, 1 H, H2ɂ), 7.06 (dt, J = 1.2, 7.5 Hz, 1 H, H6ɂ),

6.97 (dt, J = 1.1, 7.4 Hz, 1 H, H5ɂ), 3.07 (t, J = 7.1 Hz, 2 H, COCH 2), 2.87 (t, J 8.1 Hz,

13 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 172.0 (C=O), 157.9 (C2), 148.5 (C3a),

136.2 (C7a), 131.4 (C7a), 12 6.9 (C3ɂa), 126.0 (C5), 123.4 (C6), 122.3 (C7), 121.7 (C6ɂ),

121.0 (C5ɂ), 120.4 (C4), 118.4 (C4ɂ), 118.2 (C7ɂ), 113.2 (C3ɂ), 111.4(C7ɂ), 36.2

(CH2CO), 20.4(CH 2). IR (KBr): ν max 3374, 3141, 2960, 2922, 1706, 1598, 1541, 1492,

-1 1454, 1444, 1348, 1270, 1232, 1190, 1154, 1106, 754, 735 cm . UV-Vis (MeOH): λ max

295 (ε 73,876 cm -1M-1) 299 (79,028) nm. HRMS (+ESI): Found m/z 322.1004, [M+H] +;

C18 H16 N3OS required 322.1009.

198

N-(Benzo[d]thiazol-2-yl)-2-(naphthalen-1-yl)acetamide (136a)

This compound was synthesized as described for compound 115 using 1-naphthaleneacetic acid 135a (0.20 g, 1.10 mmol), 2-aminobenzothiazole 110 (0.17 g, 1.12 mmol), PyBOP (1.19 g, 2.32 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM (2.0 mL) over 24 h to give the title compound 136a as a white solid (0.13 g, 37%). M.p. 146-

1 148 ºC. H NMR (300 MHz, d 6-acetone): δ 8.19 (dd, J = 0.6, 7.9 Hz, 1 H, H8ɂ), 7.94

(dd, J = 2.0, 7.4 Hz, 1 H, H5ɂ), 7.90 (dd, J = 0.7, 7.9 Hz, 1 H, H4), 7.88 (d, J = 8.2 Hz,

H4ɂ), 7.71 (dd, J = 0.5, 8.1 Hz, 1 H, H7), 7.62 (d, J = 6.7 Hz, 1 H, H2ɂ), 7.55 (ddd , J =

1.7, 7.2 Hz, 1 H, H7ɂ ), 7.49 (dd, J = 8.3, 8.3 Hz, 2 H, H3ɂ, H6ɂ), 7.42 (ddd , J = 1.3, 7.9,

13 7.9 Hz, 1 H, H6), 7.29 (ddd, J = 1.3, 7.9, 7.9 Hz, 1 H, H5), 4.47 (s, 2 H, CH 2). C NMR

(75 MHz, d 6-acetone): δ 170.6 (C=O), 158.7 (C2), 149.9 (C7a), 134.9 (C4aɂ), 133.3

(C8aɂ), 133.1 (C4a), 132.0 (C1ɂ), 129.5 (C5ɂ), 129.2 (C2ɂ), 128.8 (C4ɂ), 127.2 (C7ɂ),

126.9 (C6), 126.7 (C3ɂ), 126.4 (C6ɂ), 125.0 (C8ɂ), 124.5 (C5), 122.2 (C4), 121.7 (C7),

40.8 (CH2). IR (KBr): ν max 3170, 3057, 2959, 1698, 1598, 1534, 1441, 1310, 1274,

-1 -1 -1 1145, 1017, 791, 753 cm . UV-Vis (MeOH): λ max 222 (ε 107,000 cm M ), 281

+ (23,900) nm. HRMS (+ESI): Found m/z 319.0895, [M+H] ; C 19 H15 N2OS required

319.0900.

N-(Benzo[d]thiazol-2-yl)-2-(naphthalen-2-yl)acetamide (136b)

This compound was synthesized as described for compound 115 using 2-naphthaleneacetic acid 135b

(0.22 g, 1.16 mmol), 2-aminobenzothiazole 110 (0.17 g, 1.10 mmol), PyBOP (1.41 g, 2.70 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM 199

(2.0 mL) over 24 h to give the title compound 136b as a white solid (0.14 g, 41%). M.p.

1 173-175 ºC. H NMR (300 MHz, d 6-acetone): δ 7.89 (m, 5 H, H4, H3ɂ, H4ɂ, H6ɂ, H7ɂ),

7.71 (dd, J = 0.5, 8.1 Hz, 1 H, H7), 7.58 (dd, J = 1.7, 8.5 Hz, 1 H, H8ɂ), 7.50 (dd, J =

1.8, 9.3 Hz, 1 H, H5ɂ), 7.49 (s, 1 H, H1ɂ), 7.41 (dt, J = 1.3, 7.2 Hz, 1 H, H6), 7.29 (dt, J

13 = 1.2, 7.4 Hz, 1 H, H5), 4.14 (s, 2 H, CH 2). C NMR (75 MHz, d 6-acetone): δ 170.6

(C=O), 158.7 (C2), 149.9 (C7a), 134.5 (C2ɂ), 133.5 (C8aɂ), 133.2 (C5aɂ), 133.1 (C4a),

129.1 (C7ɂ), 129.0 (C6ɂ), 128.5 (C4ɂ, C8ɂ), 127.0 (C5), 126.8 (C5ɂ), 126.7 (C1ɂ), 124.5

(C4), 122.2 (C3), 121.7 (C7), 43.4 (CH 2). IR (KBr): ν max 3246, 3183, 3056, 2970, 1696,

1598, 1545, 1440, 1455, 1410, 1338, 1265, 1155, 1018, 986, 956, 857, 786 cm -1. UV-

-1 -1 Vis (MeOH): λ max 223 (ε 105,000 cm M ), 275 (22,300) nm. HRMS (+ESI) Found m/z

+ 319.0893, [M+H] ; C19 H15 N2OS required 319.0900.

2-(Benzo[d][1,3]dioxol-5-yl)-N-(benzo[d]thiazol-2-yl)acetamide (138)

This compound was synthesized as described for compound 115 using 3,4-(methylenedioxy)phenylacetic acid 137 (0.26 g, 1.48 mmol), 2-aminobenzothiazole

110 (0.20 g, 1.34 mmol), PyBOP (0.53 g, 1.02 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM (2.0 mL) over 24 h to give the title compound 138 as a white solid (0.19

1 g, 46%). M.p. 187-189 ºC. H NMR (300 MHz, d 6-acetone): δ 7.91 (dd, J = 0.7, 7.3 Hz,

1 H, H4), 7.70 (dd, J = 0.7, 7.7 Hz, 1 H, H7), 7.41 (dt, J = 1.3, 7.8 Hz, 1 H, H6), 7.29

(dt, J = 1.2, 7.4 Hz, 1 H, H5), 6.96 (d, J = 1.7 Hz, 1 H, H4ɂ), 6.89 (dd, J = 1.7, 7.9 Hz, 1

13 H, H7ɂ), 6.81 (d, J = 7.9 Hz, 1 H, H6ɂ). C NMR (75 MHz, d 6-acetone): δ 170.8 (C=O),

158.7 (C2), 149.9 (C4a), 148.8 (C4aɂ), 147.7 (C7aɂ), 133.1 (C5ɂ), 129.1 (C7a), 126.8

(C5), 124.5 (C6ɂ), 123.4 (C6), 122.2 (C7), 121.7 (C4), 110.6 (C4ɂ), 109.0 (C7ɂ), 102.0 200

(C2ɂ), 42.8 (CH2). IR (KBr): ν max 3123, 3063, 3029, 2948, 2897, 2775, 1693, 1597,

1543, 1499, 1429, 1308, 1246, 1131, 1038, 937, 922, 792, 751, 728 cm -1. UV-Vis

-1 -1 (MeOH): λ max 204 (ε 48,100 cm M ), 278 (19,000), 287 (18,500), 297 (15,300) nm;

+ HRMS (+ESI): Found m/z 335.0456, [M+Na] , C16 H12 N2O3SNa requires 335.0461.

N-(Benzo[d]oxazol-2-yl)-2-(1 H-indol-3-yl)acetamide (140)

This compound was prepared as described for compound

115 using IAA 106 (97.4 mg, 0.56 mmol), 2- aminobenzoxazole 139b (70.3 mg, 0.52 mmol), PyBOP

(302.1 mg, 0.58 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 140 as a white solid (94.2 mg, 62%). M.p. 178-180 ºC. 1H

NMR (300 MHz, d 6-DMSO): δ 11.80 (bs, 1 H, NH), 10.96 (bs, 1 H, NH), 7.61-7.55 (m,

3 H, H4, H4ɂ, H7ɂ), 7.36 (ddd, J = 1.2, 8.0 Hz, 1 H, H7), 7.30-7.27 (m, 3 H, H2, H5ɂ,

H6ɂ), 7.08 (ddd, J = 1.2, 8.0 Hz, 1 H, H6), 6.99 (ddd, J = 1.2, 8.0 Hz, 1 H, H5), 3.92 (bs,

13 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 169.2 (C=O), 155.2 (C2ɂ), 147.6 (C7aɂ),

140.7 (C4aɂ), 136.1 (C7a), 127.1 (C4a), 124.5 (C2), 124.3 (C6ɂ), 123.5 (C5ɂ), 121.1

(C6), 118.6 (C5), 118.5 (C4), 118.2 (C4ɂ), 111.4 (C7), 110.0 (C7ɂ), 107.2 (C2), 33.2

(CH2). IR (ATR): ν max 3341, 3032, 2924, 1622, 1576, 1453, 1370, 1237, 1138, 1006,

-1 -1 -1 736 cm . UV-Vis (MeOH): λ max 218 (ε 45,900 cm M ), 246 (19,100), 278(22,200),

+ 284(21,100) nm. HRMS (+ESI): Found m/z 292.1077, [M+H] ; C 17 H14 F3N3O2 required

292.1081.

201

2-(1 H-Indol-3-yl)-N-(quinolin-8-yl)acetamide (142)

This compound was prepared as described for compound

115 using IAA 106 (0.18 g, 1.03 mmol), 8-aminoquinoline

141 (0.30 g, 2.08 mmol), PyBOP (0.53 g, 1.02 mmol) and

DIPEA (0.20 mL, 1.14 mmol) over 18 h to give the title compound 142 as a maroon

1 solid (0.16 g, 52%). M.p. 163-165 ºC. H NMR (300 MHz, d 6-acetone): δ 11.01 (s, 1 H,

N1H), 10.16 (s, 1 H, NH), 8.73 (dd, J = 1.7, 4.2 Hz, 1 H, H4ɂ), 8.65 (dd, J = 1.5, 7.8 Hz,

1 H, H7ɂ), 8.34 (dd, J = 1.7, 8.3 Hz, 1 H, H2ɂ), 7.63 (d, J = 7.1 Hz, 1 H, H4), 7.60 (dd, J

= 1.5, 7.2 Hz, 1 H, H5ɂ), 7.57 (t, J = 1.9 Hz, 1 H, H3ɂ) , 7.53 (d, J = 3.1 Hz, 1 H, H6ɂ),

7.44 (d, J = 2.4 Hz, H2), 7.40 (d, J = 8.1 Hz, 1 H, H7), 7.09 (dt, J = 2.5, 7.5 Hz, 1 H,

13 H6), 6.99 (dt, J = 1.1, 7.4 Hz, 1 H, H5), 4.01 (s, 2 H, CH 2). C NMR (75 MHz, d 6- acetone): δ 170.2 (C=O), 148.7 (C4ɂ), 137.8 (C8ɂ), 136.5 (C2ɂ), 136.4 (C7a), 134.4

(C7aɂ), 127.7 (C4a), 127.1 (C5ɂ), 127.0 (C4), 124.5 (C2), 122.0 (C6ɂ), 121.6 (C3ɂ), 121.2

(C6), 118.6 (C5), 115.9 (C7ɂ), 111.5 (C7), 108.0 (C3), 34.4 (CH 2). IR (KBr): ν max 3359,

3222, 3054, 3011, 2925, 1660, 1618, 1596, 1577, 1527, 1485, 1424, 1386, 1328, 1158,

-1 -1 -1 1107, 1007, 824, 791, 737 cm . UV-Vis (MeOH): λ max 216 (ε 44,600 cm M ), 241

(35,800), 282 (8,300), 289 (8,200) nm. HRMS (+ESI): Found m/z 324.1101, [M+Na] +;

C19 H15 N3ONa required 324.1107.

2-(1 H-Indol-3-yl)-N-(thiazol-2-yl)acetamide (144)

This compound was prepared as described for compound 115 using IAA 106 (24.0 mg, 0.14 mmol), 2-aminothiazole 143

(16.5 mg, 0.17 mmol), PyBOP (88.5 mg, 0.17 mmol) and

DIPEA (0.10 mL, 0.57 mmol) in DCM (1.0 mL) over 12 h to give the title compound 202

1 144 as a pale brown solid (16.0 mg, 62%) M.p. 165-167 ºC. H NMR (300 MHz, d 6-

DMSO): δ 12.26 (s, 1 H, N 1H), 10.94 (s, 1 H, NH), 7.57 (dd, J = 1.2, 7.0 Hz, 1 H, H4),

7.45 (d, J = 3.6 Hz, 1 H, H4′) 7.35 (dd, J = 1.2, 7.0 Hz, 1 H, H7), 7.28 (d, J = 2.4 Hz, 1

H, H2), 7.16 (d, J = 3.6 Hz, 1 H, H5′), 7. 07 (ddd, J = 1.2, 7.0, 7.0 Hz, 1 H, H6), 6.98

13 (ddd, J = 1.2, 7.0, 7.0 Hz, 1 H, H5), 3.85 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO):

δ 169.8 (C=O), 158.2 (ArC), 137.7 (ArCH), 136.1 (ArC), 127.2 (ArC), 124.3 (ArCH),

121.2 (ArCH), 118.7 (ArCH), 118.6 (ArCH), 113.4 (ArCH), 111.5 (ArCH), 107.6

(ArC), 32.2 (CH 2). IR (ATR ): ν max 3355, 3164, 3043, 2927, 2854, 1661, 1570, 1298

-1 -1 -1 1164 cm . UV-Vis (MeOH): λ max 270 (ε 16,100 cm M ), 218 (36,300) nm. HRMS

+ (+ESI): Found m/z 258.0694, [M+H] ; C 12 H12 N2OS required 258.0696.

1H-Indole-3-thiol (147)

To a mixture of indole 30 (3.13 g, 26.17 mmol) and thiourea (2.00 g,

26.27 mmol) in MeOH/ H 2O (4:1, 100 mL) was added I 2 (6.73 g, 26.48 mmol) and KI (4.49 g, 27.05 mmol). After stirring for 72 h, the solvent was removed under reduced pressure and the crude solid washed with H 2O and Et 2O, then recrystallized from acetone/Et 2O to give intermediate 146. This was re-suspended in 2 M NaOH (60 mL) and the suspension heated at 70 °C for 15 min. The mixture was cooled to r.t. and filtered before 10 M HCl (12 mL) was added, giving a white precipitate. The mixture was extracted with DCM (3 x 10 mL), the combined organic phases dried over Na 2SO 4 and the solvent removed under reduced pressure to yield the title compound 147 as an off-white solid (2.39 g, 61%). M.p. 100-102 ºC. Lit. 392 M.p.

1 101-103 °C. H NMR (300 MHz, d 6-DMSO): δ 11.54 (s, 1 H, NH), 7.45 (dd, J = 1.2,

8.0 Hz, 1 H, H4), 7.42 (dd, J = 1.2, 8.0 Hz, 1 H, H7), 7.30 (d, J = 2.7 Hz, 1 H, H2), 7.18 203

(ddd, J = 1.2, 7.0, 7.0 Hz, 1 H, H6), 7.06 (ddd, J = 1.2, 7.0, 7.0 Hz, 1 H, H5), 5.74 (s, 1

13 H, SH). C NMR (75 MHz, d 6-DMSO): δ 136.6 (ArC), 129.8 (ArC), 129.3 (ArCH),

122.1 (ArCH), 119.7 (ArCH), 118.9 (ArCH), 112.2 (ArCH), 95.9 (ArC).

2-((1 H-Indol-3-yl)thio)acetic acid (148)

To a suspension of thiol 147 (1.22 g, 8.19 mmol) in EtOH (5.0 mL) was added a solution of KOH (1.85 g, 32.97 mmol) in

EtOH (15.0 mL), followed by a solution of chloroacetic acid

(0.95 g, 10.05 mmol) in EtOH (5.0 mL). This mixture was heated at reflux for 12 h, cooled to r.t. and the precipitate collected via filtration. The crude solid was dissolved in

H2O (40 mL) and the pH adjusted to 2-3 with 10 M HCl. After cooling in an ice bath, the precipitate was collected via filtration to give the title compound 148 as a pale brown solid (0.91 g, 54%). M.p. 109-111 ºC. Lit. 393 M.p. 110 °C. 1H NMR (300 MHz, d6-DMSO): δ 11.42 (s, 1 H, NH), 7.61 (d, J = 7.6 Hz, 1 H, H4), 7.51 (d, J = 2.6 Hz, 1 H,

H2), 7.41 (d, J = 7.6 Hz, 1 H, H7), 7.15 (ddd, J = 1.2, 7.0, 7.0 Hz, 1 H, H6), 7.08 (ddd, J

13 = 1.2, 7.0, 7.0 Hz, 1 H, H5), 3.37 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 171.3

(C=O), 136.3 (ArC), 131.0 (ArCH), 128.8 (ArC), 121.9 (ArCH), 119.8 (ArCH), 118.5

(ArCH), 112.1 (ArCH), 102.4 (ArC), 38.81 (CH 2).

2-((1 H-Indol-3-yl)thio)-N-(benzothiazol-2-yl)acetamide (149)

This compound was prepared as described for compound 115 using acid 148 (0.21 g,

1.01 mmol), 2-aminobenzothiazole 110 (0.15 g, 1.02 mmol), PyBOP (0.61 g, 1.17 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM (2.0 mL) to give the title compound 204

149 as a white solid (0.30 g, 87%). M.p. 219-221 ºC.

1 H NMR (300 MHz, d 6-DMSO): δ 12.33 (s, 1 H,

N1H), 11.42 (s, 1 H, NɂH), 7.96 (qd, J = 0.7, 7.9 Hz, 1

H, H4ɂ), 7.71 (td, J = 0.5, 7.5 Hz, 1 H, H7ɂ), 7.59 (dd, J = 0.6, 7.3 Hz, 1 H, H4), 7.50 (d,

J = 2.7 Hz, 1 H, H2), 7.41 (m, 2 H, H7, H6ɂ), 7.30 (dt, J = 1.2, 7.6 Hz, 1 H, H5ɂ), 7.12

13 (dt, J = 1.2, 7.5 Hz, 1 H, H6), 7.03 (dt, J = 1.1, 7.4 Hz, 1 H, H5), 3.61 (s, 2 H, CH 2). C

NMR (75.MHz, d 6-DMSO): δ 169.0 (C=O), 157.9 (C2ɂ), 148.5 (C7aɂ), 136.3 (C7a),

131.5 (C4aɂ), 131.4 (C2), 128.9 (C4a) 126.1 (C6ɂ), 123.6 (C5ɂ), 121.9 (C6), 121.7 (C4ɂ ),

120.5 (C7ɂ), 119.7 (C5), 118.4 (C4), 112.1 (C7), 101.7 (C2), 39.7 (CH 2). IR (ATR): ν max

3328, 3132, 3032, 2950, 1710, 1525, 1440, 1367, 1300, 1143, 879, 795 cm -1. UV-Vis

-1 -1 (MeOH): λ max 216 (ε 51,900 cm M ), 279 (19,600) nm. HRMS (+ESI): Found m/z

+ 340.0570, [M+H] ; C17 H14 N3OS 2 required 340.0573.

N-(Benzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetohydrazide (152)

This compound was prepared as described for compound

115, using IAA 106 (0.19 g, 1.08 mmol), 2- hydrazinobenzothiazole 150 (0.17 g, 1.02 mmol), PyBOP

(0.53g, 1.02 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (2.0 mL) for 18 h to yield the title compound 152 as an off-white solid (0.29g, 89%). M.p. 222-224 ºC. 1H

NMR (300 MHz, d 6-DMSO) δ 10.97 (bs, 1 H, NH), 7.95 (dd, J = 0.6, 7.9 Hz, 1 H, H 4′ ),

7.82 (dd, J = 0.6, 7.9 Hz, 1 H, H7′), 7.55 (d, J = 7.9 Hz, 1 H, H4), 7.46 (ddd, J = 1.3,

7.3, 7.3 Hz, 1 H, H5′), 7.36 (d, J = 7.9 Hz, 1 H, H7), 7.34-7.29 (m, 2 H, H2, H6′), 7.08

(dd, J = 7.9, 7.9 Hz, 1 H, H6), 6.99 (dd, J = 7.9, 7.9 Hz, 1 H, H5), 5.84 (s, 2 H, NH 2),

13 4.35 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 173.9 (ArC), 147.9 (ArC), 136.0 205

(ArC), 133.2 (ArC), 127.5 (ArC), 126.1 (ArCH), 124.5 (ArCH), 123.8 (ArCH), 121.7

(ArCH), 121.0 (ArCH), 120.8 (ArCH), 118.6 (ArCH), 118.5 (ArCH), 111.4 (ArCH),

-1 107.3 (ArC), 30.3 (CH 2). IR (ATR): ν max 3424, 3393, 3310, 3213, 3056, 1628 cm . UV-

-1 -1 Vis (DMF) : λ max 299 (ε 17.300 cm M ), 280 (23,200) nm. HRMS (+ESI): Found m/z

+ 323.0964, [M+H] ; C 17 H15 N4OS required 323.0961.

N-(Benzo[d]thiazol-2-yl)-1H-indole-2-carboxamide (156)

This compound was prepared as described for compound

115 using indole-2-carboxylic acid 155 (0.18 g, 1.11 mmol), 2-aminobenzothiazole 110 (0.23 g, 1.50 mmol),

PyBOP (0.57 g, 1.10 mmol) and DIPEA (0.20 mL, 1.14 mmol) in DCM (2.0 mL) to give the title compound 156 as a yellow solid (0.27 g, 83%). M.p. 263-265 ºC. 1H NMR

1 (300 MHz, d 6-DMSO): δ 12.92 (bs, 1 H, N H), 11.99 (s, 1 H, NɂH), 8.03 (d, J = 7.3 Hz,

1 H, H4ɂ), 7.79 (d, J = 7.9 Hz, 1 H, H7ɂ), 7.74 (d, J = 1.4 Hz, 1 H, H3), 7.70 (d, J = 8.1

Hz, 1 H, H7), 7.49 (dd, J = 0.8, 8.3 Hz, 1 H, H4), 7.48 (dt, J = 1.3, 7.1 Hz, 1 H, H6ɂ),

7.34 (dt, J = 1.1, 7.5 Hz, 1 H, H5ɂ), 7.28 (dt, J = 1.1, 7.6 Hz, 1 H, H5), 7.10 (dt, J = 0.9,

13 8.0 Hz, 1 H, H6). C NMR (75 MHz, d 6-DMSO): δ 162.3 (C=O), 137.5 (C4a, C2ɂ),

131.5 (C7aɂ), 129.1 (C2), 127.0 (C7a, C4aɂ), 126.2 (C6ɂ), 124.8 (C5), 123.6 (C5ɂ), 122.3

(C7), 121.7 (C4ɂ), 120.3 (C6, C7ɂ), 112.6 (C4), 106.6 (C3). IR (ATR): ν max 3365, 3228,

-1 -1 -1 1654, 1545 cm . UV-Vis (MeOH): λ max 209 (ε 38,700 cm M ), 322 (36,400) nm;

+ HRMS (+ESI): Found m/z 294.0691, [M+H] ; C 16 H12 N3OS required 294.0696.

206

N-(6-Fluorobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158a)

This compound was prepared as described for compound 115 using IAA 106 (87.6 mg, 0.50 mmol), 2- amino-6-fluorobenzothiazole 157a (97.8 mg, 0.58 mmol), PyBOP (273.9 mg, 0.53 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158a as an off-white solid (54.6 mg, 34%).

1 1 M.p. 230-232 ºC. H NMR (300 MHz, d 6-DMSO): δ 12.57 (s, 1 H, N H), 10.98 (s, 1 H,

NɂH), 7.87 (dd, J = 2.6, 9.0 Hz, 1 H, H7ɂ), 7.74 (dd, J = 4.8, 9.0 Hz, 1 H, H4ɂ), 7.59 (d, J

= 7.7 Hz, 1 H, H4), 7.36 (ddd, J = 1.0, 7.7 Hz, 1 H, H7), 7.31 (dd, J = 1.0, 2.3 Hz, 1 H,

H2), 7.26 (dd, J = 2.3, 9.0 Hz, 1 H , H5ɂ), 7.08 (ddd, J = 1.0, 8.0 Hz, 1 H, H6), 6.99 (ddd,

13 J = 1.0, 8.0 Hz, 1 H, H5), 3.92 (bs, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 170.8

(C=O), 158.2 (C6ɂ) 145.3 (C2ɂ), 139.8 (C7aɂ), 136.1 (C7a), 127.1 (C4a), 124.3 (C2),

121.1 (C6, C4ɂ), 118.6 (C4, C5), 116.0 (C4aɂ), 114.4 (C5ɂ), 111.4 (C7), 108.3 (C7ɂ),

107.1 (C2), 32.3 (CH 2). IR (ATR): ν max 3381, 3190, 3067, 2979, 1698, 1606, 1559,

-1 -1 -1 1455, 1251, 1144, 815, 739 cm . UV-Vis (MeOH): λ max 220 (ε 47,200 cm M ), 278

(17,000), 289 (15,100), 299 (12,100) nm. HRMS (+ESI): Found m/z 326.0752, [M+H] +;

C17 H13 FN 3OS required 326.0758.

N-(6-Chlorobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158b)

This compound was prepared as described for compound 115 using IAA 106 (0.18 g, 1.03 mmol), 2- amino-6-chlorobenzothiazole 157b (0.21 g, 1.14 mmol),

PyBOP (0.52 g, 1.00 mmol) and DIPEA (0.50 mL, 2.87 mmol) in DCM (2.0 mL) over

24 h to give the title compound 158b as an off-white solid (0.26 g, 76%). M.p. 199-201 207

1 ºC. H NMR (300 MHz, d 6-acetone): δ 10.23 (bs, 1 H, NH), 7.98 (dd, J = 0.4, 2.2, Hz, 1

H, H7), 7.69 (s, 1 H, H2’), 7.65 (dd, J = 0.5 8.2 Hz, 1 H, H5) 7.37 (m, 2 H, H4’, H7’),

7.39 (dd, J = 2.2, 6.3 Hz, 1 H, H4), 7.12 (dt, J = 1.3, 7.6 Hz, 1 H, H5’), 7.04 (dt, J = 1.2,

13 6.9 Hz, 1 H, H6’) 4.0 8 (d, J = 0.8 Hz, 2 H, CH 2); C NMR (75 MHz, d 6-acetone): δ

171.4 (C=O), 159.6 (C2), 148.8 (C3a), 137.6 (C7aɂ), 134.7 (C7a), 129.2 (C3aɂ), 127.3

(C5), 125.2 (C3ɂ), 122.7 (C6ɂ), 122.5 (C7), 121.9 (C5ɂ), 119.9 (C4ɂ), 119.5 (C4), 112.3

(C7ɂ), 108.3 (ArC), 33 .7 (CH 2). IR (KBr): ν max 3349, 3177, 3057, 2968, 2914, 1685,

-1 -1 -1 1595, 1533, 1446, 1267, 1099, 743 cm . UV-Vis (MeOH): λ max 291 (ε 25,407 cm M )

+ 304 (17,734) nm. HRMS (+ESI): Found m/z 364.0280, [M+Na] ; C17 H12 ClN 3OSNa required 364.0282.

N-(6-Bromobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158c)

This compound was prepared as described for compound 115 using IAA 106 (92.2 mg, 0.53 mmol),

2-amino-6-bromobenzothiazole 157c (121.1 mg, 0.53 mmol), PyBOP (263.5 mg, 0.51 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158c as a tan solid (94.5 mg, 48%). M.p. 224-

1 226 ºC. H NMR (300 MHz, d 6-acetone): δ 11.11 (bs, 1 H, NH), 10.23 (bs, 1 H, NH),

8.12 (dd, J = 0.5, 2.1 Hz, 1 H, Hɂ), 7.67 (ddd, J = 0.5, 0.7, 7.9 Hz, 1 H, Hɂ), 7.60 (dd, J =

0.5, 8.6 Hz, 1 H, Hɂ), 7.53, (dd, J = 1.9, 8.7 Hz, 1 H, H), 7.43 – 7.40 (m, 2 H, H2, H),

7.12 (ddd, J = 1.2, 7.1 Hz, 1 H, H), 7.04 (ddd, J = 1.2, 7.1 Hz, H), 4.08 (d, J = 0.9 Hz, 2

13 H, CH 2); C NMR (75 MHz, d 6-acetone): δ 171.4 (C=O), 159.6 (C2ɂ), 149.1 (C4aɂ),

135.2 (C7a), 130.0 (C4ɂ), 129.4 (C4a ) 125.2 (C4), 125.1 (C7aɂ), 124.8 (C7ɂ), 123.1

(C5ɂ), 122.5 (C5), 119.9 (C6), 119.5 (C7), 116.6 (C6ɂ), 112.3 (C2), 108.3 (C3), 33.7 208

(CH2). IR (ATR): ν max 3406, 3136, 2952, 2881, 1700, 1519, 1441, 1261, 1149, 1083,

-1 -1 -1 805, 731 cm . UV-Vis (MeOH): λ max 220 (ε 53,800 cm M ), 279 (21,900), 304

+ (13,900) nm. HRMS (+ESI): Found m/z 385.9951, [M+H] ; C17 H13 BrN 3OS required

385.9957.

N-(6-Methylbenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158d)

This compound was prepared as described for compound 115 using IAA 106 (88.7 mg, 0.51 mmol), 2- amino-6-methylbenzothiazole 157d (93.5 mg, 0.57 mmol), PyBOP (280.6 mg, 0.54 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158d as a white solid (98.8 mg, 61%). M.p.

1 1 234-235 ºC. H NMR (300 MHz, d 6-acetone): δ 10.93 (s, 1 H, N H), 10.23 (s, 1 H,

NɂH), 7.68 (m, 2 H, H4, H), 7.54 (d, J = 8.3 Hz, 1 H, H), 7.41 (m, 2 H, H7, H), 7.21

(ddd, J = 0.6, 1.2, 8.3 Hz, 1 H, H), 7.12 (dt, J = 1.0, 6.6 Hz, 1 H, H6), 7.04 (dt, J = 1.1,

13 7.5 Hz, 1 H, H5), 4.06 (s, 2 H, CH 2), 2.41 (s, 3 H, CH 3); C NMR (75 MHz, d 6- acetone): δ 171.0 (C=O), 158.0 (ArC), 147.9 (ArC), 137.6 (ArC), 134.2 (ArC), 133.2

(ArC), 128.2 (ArCH), 125.2 (ArCH), 122.4 (ArCH), 121.9 (ArCH), 121.3 (ArCH),

119.9 (ArCH), 119.5 (ArCH), 112.3 (ArCH), 108.5 (ArC), 33.8 (CH 2), 21.3 (CH3). IR

- (KBr): ν max 3390, 3190, 3053, 2974, 2912, 1697, 1544, 1453, 1261, 1108, 816, 733 cm

1 -1 -1 . UV-Vis (MeOH): λ max 219 (ε 54,900 cm M ), 280 (21,700), 290 (19,700), 301

+ (14,700) nm. HRMS (+ESI): Found m/z 322.1003, [M+H] ; C18 H15 N3OS required

322.1009.

209

N-(6-Methoxybenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158e)

This compound was prepared as described for compound 115 using IAA 106 (94.9 mg, 0.54 mmol),

2-amino-6-methoxybenzothiazole 157e (144.1 mg,

0.80 mmol), PyBOP (280.2 mg, 0.54 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM

(1.2 mL) over 24 h to give the title compound 158e as a white solid (123.4 mg, 68%).

1 M.p. 209-211 ºC. H NMR (300 MHz, d 6-acetone): δ 10.93 (bs, 1 H, NH), 10.25 (bs, 1

H, NH), 7.68 (dd, J = 0.6, 7.9 Hz, 1 H, H7), 7.56 (dd, J = 0.39, 8.9 Hz, 1 H, H4ɂ), 7.46

(d, J = 2.5 Hz, 1 H, H7ɂ), 7.41 (m, 2 H, H2, H4), 7.12 (ddd, J = 1.1, 7.0 Hz, 1 H, H6),

7.04 (ddd, J= 1.2, 7.9 Hz, 1 H, H5), 6.99 (dd, J = 2.6, 8.9 Hz, 1 H, H5ɂ) 4.04 (d, J = 0.8

13 Hz, 2 H, CH 2), 3.84 (s, 3 H, OCH 3). C NMR (75 MHz, d 6-acetone): δ 170.9 (C=O),

157.7 (ArC), 125.2 (ArCH), 122.4 (ArCH), 122.2 (ArCH), 119.9 (ArCH), 119.5

(ArCH), 115.7 (ArCH), 112.3 (ArCH), 108.6 (ArC), 105.1 (ArCH), 56.1 (OCH 3), 33.7

(CH2). IR (ATR): ν max 3392, 3157, 3059, 2931, 1683, 1598, 1550, 1458, 1261, 1218,

-1 -1 -1 816, 749 cm . UV-Vis (MeOH): λ max 219 (ε 70,900 cm M ), 290 (25,200) nm. HRMS

+ (+ESI): Found m/z 338.0954, [M+H] ; C18 H16 N3O2S requires 338.0958.

N-(6-Ethoxybenzo[d]thiazole-2-yl)-2-(1H-indol-3-yl)acetamide (158f)

This compound was prepared as described for compound 115 using IAA 106 (0.18 g, 1.03 mmol), 2- amino-6-ethoxybenzothiazole 157f (0.23 g, 1.18 mmol), PyBOP (0.55 g, 1.06 mmol) and DIPEA (0.50 mL, 2.87 mmol) in DCM (2.0 mL) over 24 h to give the title compound 158f as a white solid (0.33 g, 91%). M.p. 184-

1 1 186 ºC. H NMR (300 MHz, d 6-acetone): δ 10.22 (bs, 1 H, N H), 7.68 (d, J = 7.3 Hz, 1 210

H, H4ɂ), 7.54 (d, J = 8.5 Hz, 1 H, H4), 7.43 (s, 1 H, H7), 7.41 (d, J = 5.3 Hz, 1 H, H7ɂ),

7.12 (dt, J = 1.1, 7.5 Hz, 1 H, H6ɂ), 7.04 (dt, J = 1.1, 7.6 Hz, 1 H, H5ɂ), 6.98 (dd, J =

2.58, 8.84 Hz, 1 H, H5), 4.08 (q, J = 6.9 Hz, 2 H, CH 3CH 2) 4.04 (s, 2 H, CH 2), 1.38 (t, J

13 = 6.9 Hz, 3 H, CH 3CH 2). C NMR (75 MHz, d 6-acetone): δ 169.9 (C=O), 156.4 (C6ɂ),

155.7 (C4aɂ) 136.7 (C7a), 131.1 (C7aɂ) 126.9 (C4a), 124.4 (C6), 123.3 (C5), 121.4 (C4),

120.8 (C5ɂ), 118.5 (C4ɂ), 115.9 (C2), 111.8 (C7), 105.2 (C7ɂ), 64.3 ( CH2CH 3), 33.6

(CH2), 15.0 (CH3). IR (KBr): ν max 3393, 3177, 3048, 2976, 2926, 1688, 1605, 1548,

-1 -1 -1 1458, 1263, 1220, 1152, 1061, 742 cm . UV-Vis (MeOH): λ max 308 (ε 17,906 cm M )

+ nm. HRMS (+ESI): Found m/z 352.1111, [M+H] ; C19 H18 N3O2S required 352.1114.

N-(6-(Trifluoromethyl)benzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158g)

This compound was prepared as described for compound 115 using IAA 106 (96.0 mg, 0.55 mmol),

2-amino-6-(trifluoromethyl)benzothiazole 157g

(136.2 mg, 0.62 mmol), PyBOP (290.6 mg, 0.56 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158g as a white solid (85.3 mg, 41%). M.p. 219-221 ºC. 1H NMR (300 MHz, d6-DMSO): δ 12.80 (bs, 1 H, NH), 10.99 (bs, 1 H, NH), 8.47 (dd, J = 0.7, 1.9 Hz, 1 H,

H7ɂ), 7.90 (dd, J = 1.3, 8.1 Hz, 1 H, H4ɂ), 7.73 (ddd, J = 0.6, 1.9, 8.6 Hz, 1 H, H5ɂ), 7.60

(d, J = 7.7 Hz, 1 H, H4), 7.37 (ddd, J = 1.3, 8.1 Hz, 1 H, H7), 7.32 (d, J = 2.4 Hz, 1 H,

H2), 7.08 (ddd, J = 1.3, 8.1 Hz, 1 H, H6), 7.00 (ddd, J = 1.3, 8.1 Hz, 1 H, H4), 3.96 (bs,

13 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 171.2 (C=O), 161.3 (C2ɂ), 151.5 (C4aɂ),

136.1 (C7a), 132.0 (C7aɂ), 127.1 (C4a), 124.4 (C2, C6ɂ), 123.2 (C5ɂ, CF 3), 121.6 (C6),

121.1 (C4ɂ), 120.9 (C7ɂ), 118.6 (C4, C5), 111.5 (C7), 106.9 (C3), 32.4 (CH 2). IR (ATR): 211

-1 νmax 3388, 3137, 3053, 2960, 1698, 1542, 1310, 1273, 1122, 1084, 830, 746, 682 cm .

-1 -1 UV-Vis (MeOH): λ max 220 (ε 76,000 cm M ), 275 (31,300), 298 (17,100) nm. HRMS

+ (+ESI): Found m/z 376.0720, [M+H] ; C18 H13 F3N3OS required 376.0726.

N-(4, 6-Difluorobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158h)

This compound was prepared as described for compound 115 using IAA 106 (85.2 mg, 0.49 mmol), 2- amino-4,6-difluorobenzothiazole 157h (111.7 mg, 0.60 mmol), PyBOP (292.0 mg, 0.56 mmol) and DIPEA

(0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158h as a

1 19 white solid (82.9 mg, 49%). M.p. 178-180 ºC. H[ F] NMR (300 MHz, d 6-acetone): δ

11.21 (bs, 1 H, CONH), 10.24 (bs, 1 H, NH), 7.67 (d, J = 7.8 Hz, 1 H, H4), 7.61 (d, J =

2.4 Hz, 1 H, H7′), 7.41 (d, J = 7.8 Hz, 1 H, H7), 7.36 (d, J = 2.4 Hz, 1 H, H5′), 7.15 -

13 7.02 (m, 3 H, H2, H5, H6), 4.10 (s, 2 H, CH 2). C NMR (75 MHz, d 6-acetone): δ 171.4

(ArC), 161.2 (ArC), 159.1 (ArC), 137.6 (ArC), 128.4 (ArC), 125.3 (ArCH), 122.5

(ArCH), 119.9 (ArCH), 119.5 (ArCH), 112.3 (ArCH), 108.2 (ArC), 104.6 (ArCH),

102.0 (ArCH), 33.47 (CH 2). IR (ATR): ν max 3468, 3404, 3271, 3081, 1637, 1545, 1463,

-1 -1 -1 1423, 1248, 1108, 987, 835, 732 cm . UV-Vis (MeOH): λ max 220 (ε 53,400 cm M ),

+ 269 (17,700) nm. HRMS (+ESI): Found m/z 344.0661, [M+H] ; C17 H12 F2N3OS required 344.0664.

212

N-(4-Chlorobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158i)

This compound was prepared as described for compound 115 using IAA 106 (89.6 mg, 0.50 mmol), 2- amino-4-chlorobenzothiazole 157i (113.5 mg, 0.61 mmol), PyBOP (283.5 mg, 0.54 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158a as a white solid (84.2 mg, 49%). M.p.

1 1 198-200 ºC. H NMR (300 MHz, d 6-acetone): δ 11.34 (s, 1 H, N H), 10.24 (s, 1 H,

NɂH), 7.85 (dd, J = 1.1, 7.9 Hz, 1 H, H7ɂ), 7.69 (dd, J= 1.3, 7.8 Hz, 1 H, H4), 7.43, (dd,

J = 1.1, 7.8 Hz, 3 H, H5ɂ, H7, H2), 7.25 (dd, J = 7.9 Hz, 1 H, H6′), 7.12 (dd, J = 1.2, 7.1

13 Hz, 1 H, H6), 7.05 (dd, J = 1.2, 7.9 Hz, 1 H, H5), 4.13 (d, J = 0.8 Hz, 2 H, CH 2). C

NMR (75 MHz, d 6-acetone): δ 171.5 (C=O), 159.8 (C2′), 146.8 (C4a′), 137.6 (C7a),

134.6 (C7a′), 127.0 (C4a), 126.6 (C5′), 125.3 (C2), 125.1 (C6′), 122.7 (C4′), 121.1 (C6),

120.4 (C7′), 119.9 (C5), 119.5 (C 4), 112.3 (C7), 108.2 (C3), 33.8 (CH 2). IR (ATR): ν max

3361, 3038, 2962, 1690, 1526, 1455, 1410, 1289, 1261, 1150, 1097, 734 cm -1. UV-Vis

-1 -1 (MeOH): λ max 220 (ε 56,000 cm M ), 247 (15,000), 278 (23,400) nm. HRMS (+ESI):

+ Found m/z 342.0457, [M+H] ; C17 H13 ClN 3OS required 342.0462.

N-(4-Methylbenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158j)

This compound was prepared as described for compound 115 using IAA 106 (87.2 mg, 0.50 mmol),

2-amino-4-methylbenzothiazole 157j (88.8 mg, 0.54 mmol), PyBOP (273.5 mg, 0.53 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (1.2 mL) over 24 h to give the title compound 158j as a white solid (90.1 mg, 56%). M.p.

1 205-207 ºC. H NMR (300 MHz, d 6-acetone): δ 11.12 (bs, 1 H, NH), 10.29 (bs, 1 H, 213

NH), 7.69 (dd, J = 0.8, 8.0 Hz, 2 H, H4, H6′), 7.43 -7.40 (m, 2 H, H2, H7), 7.19-7.16 (m,

2 H, H5′, H7′), 7.12 (ddd, J = 1.1, 7.9 Hz, 1 H, H6), 7.04 (ddd, J = 1.1, 7.9 Hz, 1 H,

13 H5),4.09 (d, J = 0.8 Hz, 2 H, CH 2), 2.51 (s, 3 H, CH 3). C NMR (75 MHz, d 6-acetone):

δ 171.2 (C=O), 157.9 (C2′), 148.9 (ArC), 137.6 (ArC), 132.7 (ArC), 131.2 (ArC), 128.4

(ArC), 127.3 (C5′), 125.2 (C7), 124.3 (C4′), 122.4 (C6), 119.9 (C5), 119.5 (C4, C6′),

112.3 (C2), 108.5 (C3), 33.8 (CH 2), 18.0 (CH 3). IR (ATR): ν max 3400, 3335, 3207,

-1 -1 -1 3053, 1668, 1529, 1454, 1257, 736 cm . UV-Vis (MeOH): λ max 219 (ε 49,900 cm M ),

246 (11,400), 279 (22,000) nm. HRMS (+ESI): Found m/z 322.1003, [M+H] +;

C18 H15 N3OS required 322.1009.

N-(4-Methoxybenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158k)

This compound was prepared as described for compound 115 using IAA 106 (89.7 mg, 0.51 mmol),

2-amino-4-methoxybenzothiazole 157k (106.7 mg,

0.59 mmol), PyBOP (264.2 mg, 0.51 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM

(1.2 mL) over 24 h to give the title compound 158k as an off-white solid (104.3 mg,

1 61%). M.p. 240-242 ºC. H NMR (300 MHz, d 6-acetone): δ 12.66 (bs, 1 H, NH), 10.98

(bs, 1 H, NH), 7.60 (ddd, J = 1.0, 8.0 Hz, 1 H, H4), 7.49 (dd, J = 0.9, 8.0 Hz, 1 H, H7′),

7.36 (ddd, J = 1.0, 8.0 Hz, 1 H, H7), 7.31 (d, J = 2.4 Hz, 1 H, H2), 7.23 (dd, J = 8.0 Hz,

1 H, H6′), 7.08 (ddd, J = 1.0, 8.0 Hz, 1 H, H6), 6.99 (ddd, J = 1.0, 8.0 Hz, 2 H, H5,

13 H5′), 3.91 (s, 3 H, CH 3), 3.89 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 170.5

(C=O), 156.5 (ArC), 151.8 (ArC), 138.4 (ArC), 136.1 (ArC), 132.8 (ArC), 127.1 (ArC),

124.4 (ArCH), 124.3 (ArCH), 121.1 (ArCH), 118.6 (ArCH), 113.5 (ArCH), 111.5

(ArCH), 107.6 (ArCH), 107.2 (ArC), 55.8 (OCH 3), 32.4 (CH 2). IR (ATR): ν max 3394, 214

3317, 3251, 3010, 2929, 1695, 1527, 1423, 1255, 1039, 738 cm -1. UV-Vis (MeOH):

-1 -1 λmax 212 (ε 48,300 cm M ), 247 (12,600), 282 (19,900) nm. HRMS (+ESI): Found m/z

+ 338.0952, [M+H] ; C18 H16 N3O2S required 338.0958.

N-(5-Bromobenzo[d]thiazol-2-yl)-2-(1 H-indol-3-yl)acetamide (158l)

This compound was prepared as described for compound 115 using IAA 106 (112.7 mg, 0.64 mmol), 2-amino-5-bromobenzothiazole 157l (139.0 mg, 0.60 mmol), PyBOP (273.1 mg, 0.52 mmol) and DIPEA (0.20 mL, 1.15 mmol) in

DCM (1.2 mL) over 24 h to give the title compound 158k as a beige solid (82.6 mg,

1 41%). M.p. 196-198 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.30 (bs, 1 H, NH), 7.87

(dd, J = 0.4, 8.5 Hz, 1 H, H7′), 7.83 (dd, J = 0.4, 1.9 Hz, 1 H, H4′), 7.67 (ddd, J = 0.6,

7.9 Hz, 1 H, H4), 7.44-7.40 (m, 3 H, H2, H7, H6′), 7.12 (ddd, J = 1.1, 7.9 Hz, 1 H, H6),

13 7.04 (ddd, J = 1.1, 7.9 Hz, 1 H, H5), 4.08 (d, J = 0.8 Hz, 2 H, CH 2). C NMR (75 MHz, d6-acetone): δ 171.5 (C=O), 160.6 (ArC), 151.4 (ArC), 137.6 (ArC), 132.2 (ArC), 128.3

(ArC), 127.2 (ArCH), 125.2 (ArCH), 124.1 (ArCH), 123.9 (ArCH), 122.4 (ArCH),

119.9 (ArC), 119.8 (ArCH), 119.5 (ArCH), 112.3 (ArCH), 108.2 (ArC), 33.8 (CH 2). IR

-1 (ATR): ν max 3391, 3132, 3036, 2943, 1695, 1526, 1405, 1268, 1143, 894, 733 cm . UV-

-1 -1 Vis (MeOH): λ max 220 (ε 68,900 cm M ), 273 (26,200), 306 (15,200) nm. HRMS

+ (+ESI): Found m/z 385.9951, [M+H] , C17 H13 BrN 3OS required 385.9957.

215

2-(5-Methoxy-1H-indol-3-yl)-2-oxoacetic acid (161)

A solution of 5-methoxyindole 159 (0.32 g, 2.19 mmol) in Et 2O

(3.0 mL) was cooled in an ice bath under an N 2 atmosphere. A solution of oxalyl chloride (0.22 mL, 2.57 mmol) in Et 2O (3.0 mL) was added dropwise and the mixture stirred with cooling for 10 min. Saturated

NaHCO 3 (6.0 mL) was then added dropwise and the mixture heated at reflux for 30 min. The mixture was allowed to cool, acidified with HCl (10 M) and the precipitate collected via filtration to give the title compound as an orange solid (0.44 g, 91%). M.p.

394 1 245-247 ºC. Lit. M.p. 248 °C. H NMR (300 MHz, d 6-acetone): δ 8.70 (s, 1 H, H2),

7.85 (d, J = 2.5 Hz, 1 H, H4), 7.49 (dd, J = 0.5, 8.8 Hz, 1 H, H7), 6.94 (dd, J = 2.5, 8.8

13 Hz, 1 H, H6), 3.86 (s, 3 H, OCH 3). C NMR (75 MHz, d 6-acetone): δ 178.9 (C=O),

163.8 (CO2H), 157.9 (ArC), 139.2 (ArCH), 132.2 (ArC), 128.3 (ArC), 114.6 (ArCH),

114.0 (ArCH), 113.4 (ArC), 104.5 (ArCH), 55.9 (OCH 3).

Ethyl 2-(5-methoxy-1H-indol-3-yl)-2-oxoacetate (163)

A solution of ethyloxalyl chloride (0.4 mL, 3.57 mmol) in Et 2O

(3.0 mL) was added dropwise to 5-methoxyindole 159 (0.36 g,

2.48 mmol) in Et 2O (6.0 mL) at 0 °C. The mixture was stirred at

0 °C for 4 h, the solvent removed under reduced pressure and the crude residue purified by flash column chromatography (1:4 EtOAc:hexanes) to give the title compound 163 as a pale pink solid (0.40 g, 69%). M.p. 215-217 ºC. Lit. 395 M.p. 218 °C. 1H NMR (300

MHz, d 6-acetone): δ 9.97 (1 H, NH), 7.27 (dd, J = 0.5, 8.8 Hz, 1 H, H7), 7.23 (d, J = 2.3

Hz, 1 H, H2), 7.09 (d, J = 2.4 Hz, 1 H, H4), 6.76 (dd, J = 2.4, 8.8 Hz, 1 H, H6), 4.11 (q,

J = 7.2 Hz, 2 H, CH 2), 3.80 (s, 3 H, OCH 3), 1.22 (t, J = 7.2 Hz, 3 H, CH 3). 216

Ethyl 2-(2-methyl-1H-indol-3-yl)acetate (167)

A mixture of phenyl hydrazine 164 (1.20 mL, 12.2 mmol), ethyl levulinate 165 (2.2 mL, mmol) and AcONa (1.11 g, 13.5 mmol) in glacial acetic acid (20.0 mL) was heated at reflux for 5 h and concentrated in vacuo to give 166 as a crude red residue. This was taken up in EtOH (20 mL), treated with 4 M HCl/ dioxane and heated at reflux for 15 h. The mixture was concentrated in vacuo to give a brown residue that was re-dissolved in EtOAc (50.0 mL) and washed with H 2O (50.0 mL), sat. K 2CO 3 (50.0 mL) and brine (50.0 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (1:9 EtOAc:hexanes) to give the title compound 167

396 1 as a yellow oil (0.65 g, 25%). H NMR (300 MHz, CDCl 3): δ 7.96 (bs, 1 H, NH),

7.56 (d, J = 5.3 Hz, 1 H, H4), 7.19 (d, J = 4.8 Hz, 1 H, H7), 7.13-7.10 (m, 2 H, H5, H6),

4.15 (q, J = 7.1 Hz, 2 H, OCH2CH 3), 3.70 (s,2 H, CH 2), 2.34 (s, 3 H, CH 3), 1.26 (t, J =

13 7.1 Hz, 3 H, OCH 2CH3). C NMR (75 MHz, CDCl 3): 172.3 (C=O), 135.2 (ArC), 132.8

(ArC), 128.6 (ArC), 121.2 (ArCH), 119.5 (ArCH), 118.1 (ArCH), 110.4 (ArCH), 104.5

(ArC), 60.8 (OCH2CH 3), 30.6 (CH2), 14.3 (CH3), 11.7 (OCH 2CH3).

2-Methyl-1H-indole-3-acetic acid (168)

Ester 167 (0.65 g, 2.99 mmol) was stirred in 2 M NaOH (15.0 mL) for 18 h before being acidified with 2 M HCl. The precipitate was collected via filtration, washed with H 2O and air dried to give the title compound 168 as an orange solid (0.51 g, 90%). M.p. 195-197 ºC. Lit. 397 M.p. 196-197 °C (dec.). 1H

NMR (300 MHz, d 6-acetone): δ 10.00 (bs, 1 H, NH), 7.48 (dd, J = 0.7, 7.0 Hz, 1 H, H4),

7.26 (dd, J = 0.7, 7.0 Hz, 1 H, H7), 7.00 (ddd, J = 1.5, 7.0, 7.0 Hz, 1 H, H6), 6.96 (ddd, J 217

= 1.5, 7.0, 7.0 Hz, 1 H, H5), 3.65 (s, 2 H, CH2), 2.40 (s, 3 H, CH3). 13 C NMR (75 MHz, d6-acetone): δ 173.4 (C=O), 136.5 (ArC), 133.8 (ArC), 129.7 (ArC), 121.2 (ArCH),

119.5 (ArCH), 118.6 (ArCH), 111.1 (ArCH), 105.2 (ArC), 30.4 (CH 2), 11.5 (CH3).

2-(5-Bromo-2-methyl-1H-indol-3-yl)acetic acid (171a)

A mixture of 4-bromophenylhydrazine hydrochloride 169a (1.0 g,

4.5 mmol) and levulinic acid 170 (0.5 g, 4.9 mmol) in 10 M

HCl:toluene (1:1, 100 mL) was heated at reflux for 3 h. H 2O (100 mL) was added and the mixture extracted with EtOAc (3 x 50 mL), the combined organic extracts dried over Na 2SO 4, concentrated in vacuo and the crude residue purified by flash column chromatography (1:4 EtOAc:hexanes) to give the title compound 171a as a brown solid (0.46 g, 34%). M.p. 186-188 ºC. Lit. 398 M.p. 188-189

1 °C (dec.). H NMR (300 MHz, d 6-DMSO): δ 12.12 (bs, 1 H, CO 2H), 11.06 (bs, 1 H,

NH), 7.54 (d, J = 1.9 Hz, 1 H, H), 7.21 (dd, J = 0.5, 8.5 Hz, 1 H, H), 7.09 (dd, J = 1.9,

13 8.5 Hz, 1 H, H), 3.56 (s, 2 H, CH 2), 2.32 (s, 3 H, CH 3). C NMR (75 MHz, d 6-DMSO):

δ 173.0 (C=O), 134.9 (ArC), 133.7 (ArC), 130.2 (ArC), 122.4 (ArCH), 120.0 (ArCH),

112.3 (ArCH), 110.9 (ArC), 103.9 (ArC), 29.7 (CH 2), 11.3 (CH3).

218

2,5-Dimethyl-1H-indole-3-acetic acid (171b)

This compound was prepared as described for compound 171a using 4-methylphenylhydrazine hydrochloride 169b (0.72 g, 4.54 mmol) and levulinic acid 170 (0.52 mL, 5.08 mmol) in 10 M

HCl:toluene (1:1, 20 mL) over 3 h to give the title compound 171b as a white solid

399 1 (0.59 g, 67%). M.p. 171-173 ºC. Lit. M.p. 172-174 °C. H NMR (300 MHz, d 6-

DMSO): δ 9.81 (bs, 1 H, NH), 7.27 (ddd, J = 0.8 Hz, 1 H, H), 7.14 (d, J = 8.2 Hz, 1 H,

H), 6.84 (dd, J = 1.3, 8.2 H2, 1 H, H), 3.63 (s, 2 H, CH 2), 2.38 (s, 3 H, CH 3), 2.37 (dd, J

= 0.8 Hz, 3 H, CH 3).

N-(Benzo[d]thiazol-2-yl)-2-(2-methyl-1H-indol-3-yl)acetamide (172a)

This compound was prepared as described for compound

115 using acid 168 (0.19 g, 1.00 mmol), 2- aminobenzothiazole 110 (0.21 g, 1.39 mmol), PyBOP

(0.55 g, 1.06 mmol) and DIPEA (0.40 mL, 2.30 mmol) in DCM (2.0 mL) over 24 h to give the title compound 172a as a beige solid (0.13 g, 39%). M.p. 227-229 ºC. 1H NMR

(300 MHz, d 6-DMSO): δ 12.51 (bs, 1 H, NH), 10.86 (bs, 1 H, NH), 7.92 (ddd, J = 0.5,

1.2, 7.8 Hz, 1 H, H), 7.73 (ddd, J = 0.5, 1.2, 7.8 Hz, 1 H, H), 7.49 (dd, J = 1.2, 7.2 Hz, 1

H, H), 7.42 (ddd, J = 1.2, 7.2 Hz, 1 H, H), 7.29 (ddd, J = 1.2, 7.2 Hz, 1 H, H), 7.26 (ddd,

J = 1.0, 7.4 Hz, 1 H, H), 6.98 (ddd, J = 1.2, 7.2 Hz, 1 H, H), 6.92 (ddd, J = 1.2, 7.2 Hz,

13 1 H, H), 3.85 (s, 2 H, CH 2), 2.39 (s, 3 H, CH 3). C NMR (75 MHz, d 6-DMSO): δ 171.4

(C=O), 158.5 (ArC), 148.9 (ArC), 135.5 (ArC), 134.1 (ArC), 131.8 (ArC), 128.7 (ArC),

126.6 (ArCH), 124.0 (ArCH), 122.1 (ArCH), 120.9 (ArCH), 120.7 (ArCH), 118.9

(ArCH), 118.1 (ArCH), 110.9 (ArCH), 103.9 (ArC), 31.6 (CH2), 11.9 (CH 3). IR (ATR): 219

-1 νmax 3308, 3195, 3056, 2920, 1668, 1526, 1448, 1258, 1090, 1013, 795, 745, 667 cm .

-1 -1 UV-Vis (MeOH): λ max 221 (ε 69,200 cm M ), 275 (29,400), 297 (17,900) nm. HRMS

+ (+ESI) Found m/z 322.1003, [M+H] ; C18 H16 N3OS required 322.1009.

N-(Benzo[d]thiazol-2-yl)-2-(5-bromo-2-methyl-1H-indol-3-yl)acetamide (172b)

This compound was prepared as described for compound 115 using acid 171a (0.27 g, 1.01 mmol), 2- aminobenzothiazole 110 (0.16 g, 1.10 mmol), PyBOP

(0.55 g, 1.06 mmol) and DIPEA (0.20 mL, 1.15 mmol) in DCM (2.0 mL) over 24 h to give the title compound 172b as a brown solid (0.19 g, 48%). M.p. 229-231 ºC. 1H

NMR (300 MHz, d 6-acetone): δ 10.39 (bs, 1 H, NH), 7.89 (ddd, J = 0.6, 7.9 Hz, 1 H,

H), 7.78 (d, J = 2.0 Hz, 1 H, H), 7.68 (ddd, J = 0.6, 7.9 Hz, 1 H, H), 7.40 (ddd, J = 1.4,

7.3 Hz, 1 H, H), 7.28 (ddd, J = 1.4, 7.3 Hz, 1 H, H), 7.26 (dd, J = 0.5, 8.6 Hz, 1 H, H),

13 7.13 (dd, J = 1.9, 8.5 Hz, 1 H, H), 4.01 (s, 2 H, CH 2), 2.50 (s, 3 H, CH 3). C NMR (75

MHz, d 6-acetone): δ 170.1 (C=O), 158.0 (ArC), 149.0 (ArC), 135.8 (ArC), 134.4 (ArC),

132.1 (ArC), 130.6 (ArC), 125.9 (ArCH), 123.5 (ArCH), 123.1 (ArCH), 121.3 (ArCH),

120.7 (ArCH), 120.3 (ArCH), 112.2 (ArCH), 111.8 (ArC), 103.5 (ArC), 31.4 (CH 2),

10.9 (CH3). IR (ATR): ν max 3398, 3189, 3054, 2918, 1657, 1528, 1438, 1254, 1159,

-1 -1 -1 1013, 976, 789 cm . UV-Vis (MeOH): λ max 208 (ε 20,000 cm M ), 225 (22,600), 277

+ (9,400), 298 (7,700) nm. HRMS (+ESI): Found m/z 400.0109, [M+H] ; C18 H15 BrN 3OS required 400.0114.

220

N-(Benzo[d]thiazol-2-yl)-2-(2, 5-dimethyl-1H-indol-3-yl)acetamide (172c)

This compound was prepared as described for compound

115 using acid 171b (95.0 mg, 0.47 mmol), 2- aminobenzothiazole 110 (77.0 mg, 0.51 mmol), PyBOP

(271.0 mg, 0.52 mmol) and DIPEA (0.10 mL, 0.58 mmol) in DCM (2.0 mL) over 24 h to give the title compound 172c as an off-white solid (79.6 mg, 51%). M.p. 230-232 ºC.

1 H NMR (300 MHz, d 6-DMSO): δ 12.51 (bs, 1 H, NH), 10.74 (bs, 1 H, NH), 7.94 (ddd,

J = 0.5, 1.2, 7.8 Hz, 1 H, H4ɂ), 7.74 (ddd, J = 0.6, 1.0, 8.0 Hz, 1 H, HH7ɂ), 7.43 (ddd, J

= 1.3, 7.2 Hz, 1 H, HH6ɂ), 7.31 (d, J = 1.1 Hz, 1 H, H4), 7.28 (ddd, J = 1.1, 8.0 Hz, 1 H,

H5ɂ), 7.13 (d, J = 8.1 Hz, 1 H, H7), 6.81 (dd, J = 1.3, 8.2 Hz, 1 H, H6), 3.83 (s, 2 H,

2 5 13 CH 2), 2.38 (s, 3 H, C CH 3), 2.34 (s, 3 H, C CH 3). C NMR (75 MHz, d 6-DMSO): δ

170.8 (C=O), 158.1 (C2ɂ), 148.5 (C4aɂ), 133.6 (C7a), 133.4 (C7a), 131.4 (C2), 128.5

(C5), 126.6 (C4a), 126.1 (C6ɂ), 123.5 (C5ɂ), 121.7 (C4ɂ), 121.6 (C6), 120.4 (C7ɂ), 117.4

5 2 (C4), 110.1 (C7), 102.9 (C3), 31.1 (CH2), 21.4 (C CH 3), 11.5 (C CH 3). IR (ATR): ν max

3392, 3242, 3055, 2908, 1687, 1534, 1444, 1292, 1269, 1146, 1089, 861 cm -1. UV-Vis

-1 -1 (MeOH): λ max 208 (ε 43, 200 cm M ), 222 (45,300), 275 (20,500), 297 (15,400) nm.

+ HRMS (+ESI): Found m/z 336.1164, [M+H] ; C19 H18 N3OS required 336.1165.

2-((1 H-Indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole (179)

A solution of hydrazide 185 (0.24 g, 0.85 mmol) in

MeCN (10 mL) was treated with DIPEA (0.5 mL, 2.91 mmol), followed by 4-TsCl (0.49 g, 2.55 mmol) and the mixture stirred for 1 h. Ammonia solution (14%, 10 mL) was then added and the mixture stirred for a further 15 min. The mixture was extracted with DCM (3 x 20 mL), 221

washed with H 2O, dried over Na 2SO 4 and the solvent removed under reduced pressure.

The crude residue was purified by flash column chromatography with EtOAc:hexanes

(15% - 45%) and crystallized from EtOH to give the title compound 179 as colourless needles (0.11 g, 48%). M.p. 156-157 ºC, Lit. 305 M.p. 110-111 °C. 1H NMR (300 MHz, d6-DMSO): δ 11.05 (bs, 1 H, NH), 7.94 (d, J = 1.7 Hz, 1 H, H6), 7.92 (d, J = , 1 H,

H2),7.58 (m, 4 H, H4ɂ, H3, H4, H5), 7.39 (s, 1 H, H2ɂ), 7.38 (d, J = 8.2 Hz, 1 H,

H7ɂ), 7.10 (dt, J = 1.2, 7.6 Hz, 1 H, H6ɂ), 7.02 (dt, J = 1.1, 7.5 Hz, 1 H, H5ɂ), 4.43 (d, J =

13 0.6 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 166.1 (C2), 164.0 (C5), 136.2

(C7aɂ), 131.8 (C4), 129.4 (C2, C6), 126.6 (C4aɂ), 126.3 (C3, C5), 124.2 (C1),

123.4 (C2ɂ), 121.3 (C6ɂ), 118.8 (C5ɂ), 118.2 (C4ɂ), 111.6 (C7ɂ), 106.8 (C3ɂ), 21.5 (CH 2).

IR (KBr): ν max 3264, 3112, 3068, 3012, 2915, 1567, 1492, 1450, 1353, 1259, 1244,

-1 -1 - 1229, 1090, 1008, 789, 741, 705, 689 cm . UV-Vis (MeOH): λ max 271 (ε 12900 cm M

1 + ), 220 (33600) nm. HRMS (+ESI): Found m/z 276.1132, [M+H] ; C 17 H14 N3O required

276.1131.

2-(2-(1 H-Indol-3-yl)ethyl)-5-phenyl-1,3,4-oxadiazole (180)

This compound was synthesized as described for compound 179 using hydrazide 189a (0.13 g, 0.43 mmol), DIPEA (0.30 mL, 1.75 mmol) and 4-TsCl (0.21 g, 1.08 mmol) in MeCN (6.0 mL) over 1.5 h to give the title compound 180 as an off-

1 white solid (0.08 g, 66%). M.p. 125-127 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.83

(bs, 1 H, NH), 7.97-7.94 (m, 2 H, H2, H6), 7.61 -7.53 (m, 4 H, H4ɂ, H3, H4, H5),

7.33 (ddd, J = 0.8, 8.0 Hz, 1 H, H7ɂ), 7.18 (d, J = 2.4 Hz, 1 H, H2ɂ), 7.06 (ddd, J = 1.1,

8.1 Hz, 1 H, H6ɂ), 6.96 (ddd, J = 1.1, 8.1 Hz, 1 H, H5ɂ), 3.29 (dd, J = 1.0, 6.0 Hz, 2 H, 222

13 CH 2(C=N)O), 3.24 (dd, J = 1.0, 6.0 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ

166.6 (C2), 163.8 (C5), 136.2 (C7aɂ), 131.8 (C4), 129.4 (C3, C5), 126.8 (C4aɂ), 126.4

(C2, C6), 123.5 (C1), 122.8 (C2ɂ), 121.0 (C6ɂ), 118.3 (C5ɂ), 118.1 (C4ɂ), 112.4 (C3ɂ ),

111.4 (C7ɂ), 25.9 (CH2(C=N)O), 21.8 (CH 2). IR (film): ν max 3175, 3053, 2920, 1555,

-1 -1 -1 1445, 1323 cm . UV-Vis (MeOH): λ max 252 (ε 19400 cm M ), 222 (36900), 203

+ (37200) nm. HRMS (+ESI): Found m/z 312.1107, [M+Na] ; C 18 H15 N3ONa required

312.1107.

N'-(2-(1H-Indol-3-yl)acetyl)benzohydrazide (185)

To a mixture of IAA 106 (0.18 g, 1.01 mmol) and benzoyl hydrazine 184 (0.19 g, 1.46 mmol) in DMF

(2.0 mL) were added EDCI (0.22 g, 1.14 mmol) and

HOBt (0.17 g, 1.28 mmol). The mixture was stirred for 24 h before being poured into ice-water and the precipitate collected via filtration. The crude product was purified by flash column chromatography (70% EtOAc:hexanes) to give the title compound 185 as

1 an off-white solid (0.24 g, 84%). M.p. 189-191 ºC. H NMR (300 MHz, d6-DMSO): δ

10.90 (bs, 1 H, N 1H), 10.34 (d, J = 1.2 Hz, 1 H, N 2H), 10.10 (d, J = 1.2 Hz, 1 H, N 3H),

7.88 (td, J = 1.5, 7.7 Hz, 2 H, H2ɂ, H6ɂ), 7.64 (d, J = 7.9 Hz, 1 H, H4), 7.57 (tt, J = 1.4,

7.3 Hz, 1 H, H4ɂ), 7.48 (tt, J = 1.6, 7.0 Hz, 2 H, H3ɂ, H5ɂ), 7.35 (d, J = 8.0 Hz, 1 H, H7),

7.29 (d, J = 2.3 Hz, 1 H, H2), 7.08 (dt, J = 1.2, 7.0 Hz, 1 H, H5), 6.99 (dt, J = 1.1, 7.4

13 Hz, H6), 3.63 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 170.1 (CH 2CO), 165.5

(CO), 136.1 (C7a), 132.5 (C1ɂ), 131.8 (C4ɂ), 128.4 (C3ɂ, C5ɂ), 127.4 (C2ɂ, C6ɂ), 127.2

(C4a), 123.9 (C2), 121.0 (C6), 118.8 (C5), 118.3 (C4), 111.3 (C7), 108.2 (C3), 30.7

(CH2). IR (KBr): ν max 3240, 3056, 3012, 1691, 1639, 1526, 1486, 1458, 1339, 1226, 223

-1 -1 -1 1101, 744, 692 cm . UV-Vis (MeOH): λ max 272 (ε 8,580 cm M ), 220 (48,300) nm.

+ HRMS (+ESI): Found m/z 294.1232, [M+H] ; C 17 H16 N3O2 required 294.1237.

N'-(2-(1 H-Indol-3-yl)acetyl)thiophene-2-carbohydrazide (187a)

This compound was synthesized as described for compound 185 using IAA 106 (0.17 g, 0.97 mmol), hydrazide 186a (0.18 g, 1.28 mmol), EDCI (0.23 g, 1.20 mmol) and HOBt (0.17 g, 1.25 mmol) in DMF (2.0 mL) over 24 h to give the title compound 187a as an off-white solid (0.13 g, 43%). M.p. 164-166 ºC. 1H NMR (300

1 MHz, d 6-DMSO): δ 10.91 (s, 1 H, N H), 10.38 (s, 1 H, NH), 10.12 (s, 1 H, NH), 7.84

(d, J = 4.4 Hz, 2 H, H3ɂ, H5ɂ), 7.65 (d, J = 7.8, 1 H, H4), 7.37 (d, J = 8.0, 1 H, H7), 7.30

(d, J = 2.3 Hz, 1 H, H2), 7.19 (t, J = 4.4 Hz, 1 H, H4ɂ), 7.10 (dt, J = 1.1, 7.0 Hz, 1 H,

13 H6), 7.01 (dt, J = 1.1, 7.8 Hz, H5), 3.64 (s, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO):

δ170.2 (CH2CO), 160.6 (CO), 137.4 (ArC), 136.1 (ArC), 131.6 (ArCH), 128.9 (ArCH),

128.1 (ArCH), 127.2 (ArC), 123.9 (ArCH), 121.0 (ArCH), 118.8 (ArCH), 118.3

(ArCH), 111.3 (ArCH), 108.2 (ArC), 30.7 (CH 2). IR (ATR ): ν max 3386, 3242, 1671,

-1 -1 -1 1625, 1542, 1477, 1413 cm . UV-Vis (MeOH): λ max 271 (ε 14100 cm M ), 220

+ (36500) nm. HRMS (+ESI): Found m/z 300.0796, [M+H] ; C 15 H14 N3O2S required

300.0801.

N'-(2-(1 H-Indol-3-yl)acetyl)furan-2-carbohydrazide (187b)

This compound was synthesized as described for compound 185 using IAA 106 (0.18 g, 1.00 mmol), hydrazide 186b (0.15 g, 1.20 mmol), EDCI (0.27 g, 1.38 mmol) and HOBt (0.17 g, 1.25 mmol) in DMF (2.0 mL) over 24 h. The reaction mixture 224

was partitioned between water (30.0 mL) and EtOAc (20.0 mL), the aqueous phase extracted with EtOAc (2 x 20.0 mL) and the combined organics washed with water

(20.0 mL), dried over Na 2SO 4, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (70% EtOAc:hexanes) to give the title compound 187b as an off-white solid (0.11 g, 36%). M.p. 142-144 ºC. 1H NMR

(300 MHz, d 6-DMSO): δ 10.88 (s, 1 H, N1H), 10.22 (s, 1 H, NH), 10.05 (s, 1 H, NH),

7.88 (dd, J = 0.7, 1.0 Hz , 1 H, H5’), 7.62 (d, J = 7.8 Hz, 1 H, H4), 7.34 (d, J = 7.9 Hz,

H7), 7.27 (d, J = 2.3 Hz, H2), 7.21 (dd, J = 0.7, 3.5 Hz, 1 H, H3ɂ), 7.07 (dt, J = 1.2, 7.0

Hz, 1 H, H6), 6.98 (dt, J = 1.1, 7.9 Hz, 1 H, H5), 6.65 (m, J = 1.7 Hz, 1 H, H4ɂ), 3.61 (s,

13 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 170.5 (CH 2CO), 157.6 (CO), 146.7 (C2ɂ),

146.1 (C5ɂ), 136.5 (C7a), 127.6 (C4a), 124.3 (C2), 121.4 (C6), 119.2 (C4), 118.7 (C5),

114.8 (C3ɂ), 112.2 (C4ɂ), 111.7 (C7), 108.6 (C3), 31.0 (CH 2). IR (KBr): ν max 3253, 3016,

2918, 1659, 1590, 1469, 1301, 1267, 1229, 1166, 1011, 883, 746 cm -1. UV-Vis

-1 -1 (MeOH): λ max 253 (ε 19800 cm M ), 219 (41200) nm. HRMS (+ESI): Found m/z

+ 284.1029, [M+H] ; C 15 H14 N3O3 required 284.1031.

N'-(2-(1 H-Indol-3-yl)acetyl)1 H-pyrrole-2-carbohydrazide (187c)

This compound was synthesized as described for 187b using IAA 106 (0.18 g, 1.01 mmol), hydrazide 186c

(0.17 g, 1.38 mmol), EDCI (0.21 g, 1.08 mmol) and

HOBt (0.18 g, 1.36 mmol) in DMF (2.0 mL) over 24 h to give the title compound 187c as a yellow solid after purification (0.13 g, 46%). M.p. 152-154 ºC. 1H NMR (300 MHz, d6-acetone): δ 10.88 (bs, 1 H, NH), 10.10 (bs, 1 H, NH), 9.36 (s, 1 H, NH), 9.21 (s, 1 H,

NH), 7.61 (ddd, J = 1.6, 1.6, 8.1 Hz, 1 H, H7), 7.34 (ddd, J = 1.6, 1.6, 8.1 Hz, 1 H, H4), 225

7.27 (s, 1 H, H2), 7.06 (ddd, J = 1.6, 8.1, 8.1 Hz, 1 H, H6), 6.99-6.89 (m, 3 H, H5, H3ɂ ,

13 H5ɂ), 6.11 (dd, J = 2.6, 3.8 Hz, 1 H, H4ɂ), 3.68 (d, J = 0.8 Hz, 2 H, CH 2). C NMR (75

MHz, d 6-acetone): δ 171.2 (C=O), 137.4 (ArC), 128.4 (ArC) 124.7 (ArCH), 123.0

(ArCH), 122.2 (ArC) 119.7 (ArCH), 119.6 (ArCH), 112.1 (ArCH), 111.7 (ArCH),

110.0 (ArCH), 109.3 (ArC), 31.8 (CH 2). IR (ATR ): ν max 3232, 3013, 1618, 1552, 1488,

-1 -1 -1 1401, 1331, 1094, 1042, 839, 737 cm . UV-Vis (MeOH): λ max 220 (ε 36900 cm M ),

268 (21600) nm.

2-((1 H-Indol-3-yl)methyl)-5-(thiophen-2-yl)-1,3,4-oxadiazole (188a)

This compound was synthesized as described for compound 179 using hydrazide 187a (0.11 g, 0.36 mmol),

DIPEA (0.30 mL, 1.74 mmol) and 4-TsCl (0.20 g, 1.04 mmol) in MeCN (6.0 mL) over 2 h to give the title compound 188a as an off-white

1 solid after purification (0.09 g, 89%). M.p. 118-120 ºC. H NMR (300 MHz, d 6-

DMSO): δ 11.05 (s, 1 H, NH), 7.89 (dd, J = 1.2, 5.01 Hz, 1 H, H), 7.72 (dd, J = 1.2, 3.7

Hz, 1 H, H), 7.57 (d, J = 7.4 Hz, 1 H, H4), 7.37 (m, 2 H, H2, H7), 7.24 (dd, J = 3.8, 5.0

Hz, 1 H, H4ɂ), 7.10 (dt, J = 1.2, 7.5 Hz, 1 H, H6), 7.01 (dt, J = 1.2, 7.4 Hz, 1 H, H5),

13 4.41 (d, J = 0.7 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 166.4 (ArC), 161.6

(ArC), 137.7 (ArC), 131.1 (ArCH), 130.3 (ArCH), 129.2 (ArCH), 128.0 (ArC), 126.3

(ArC), 124.7 (ArCH), 122.6 (ArCH), 120.0 (ArCH), 119.3 (ArCH), 112.4 (ArCH),

-1 108.5 (ArC), 22.5 (CH2). IR (ATR): ν max 3241, 3094, 2917, 1562, 1429, 1230, 717 cm .

-1 -1 UV-Vis (MeOH): λ max 281 (ε 20900 cm M ), 218 (38400) nm. HRMS (+ESI): Found

+ m/z 304.0516, [M+Na] ; C 15 H11 N3OSNa required 304.0515.

226

2-((1 H-Indol-3-yl)methyl)-5-(furan-2-yl)-1,3,4-oxadiazole (188b)

This compound was synthesized as described for compound 179 using hydrazide 187b (39.1 mg, 0.14 mmol), DIPEA (0.2 mL, 1.16 mmol) and 4-TsCl (0.11 g,

0.57 mmol) in MeCN (3.0 mL) over 2 h to give the title compound 188b as an off-white

1 solid following purification (8.4 mg, 23%). M.p. 159-161 ºC. H NMR (300 MHz, d 6- acetone): δ 10.25 (bs, 1 H, NH), 7.82 (dd, J = 0.6, 1.7 Hz, 1 H, H5ɂ), 7.65 (d, J = 7.9 Hz,

1 H, H4), 7.43-7.40 (m, 2 H, H2, H7), 7.16-7.10 (m, 2 H, H, H6, H3ɂ), 7.05 (ddd, J =

1.1, 7.1, 7.1 Hz, 1 H, H5), 6.68 (dd, J = 1.8, 1.8 Hz, 1 H, H4ɂ), 4.45 (d, J = 0.7 Hz, 2 H,

13 CH 2). C NMR (75 MHz, d 6-acetone): δ 166.3 (ArC), 158.3 (ArC), 146.9 (ArCH),

140.6 (ArC), 137.7 (ArC), 128.0 (ArC), 124.7 (ArCH), 122.6 (ArCH), 120.0 (ArCH),

119.2 (ArCH), 114.3 (ArCH), 113.0 (ArCH), 112.4 (ArCH), 112.3 (ArC), 108.5 (ArC),

-1 22.4 (CH2). IR (ATR): ν max 3252, 1633, 1569, 1519, 1452 cm . UV-Vis (MeOH): λ max

268 (ε 26300 cm -1M-1), 218 (39500) nm. HRMS (+ESI): Found m/z 304.0516,

+ [M+Na] ; C 15 H11 N3O2Na required 304.0515.

2-((1 H-Indol-3-yl)methyl)-5-(1 H-pyrrol-2-yl)-1,3,4-oxadiazole (188c)

This compound was synthesized as described for compound 179 using hydrazide 187c (0.13 g, 0.46 mmol),

DIPEA (0.30 mL, 1.74 mmol) and 4-TsCl (0.30 g, 1.56 mmol) in MeCN (4.0 mL) over 2 h to give the title compound 188c as a pale tan solid

1 after purification (0.05 g, 42%;).M.p. 166-168 ºC. H NMR (300 MHz, d 6-acetone): δ

11.17 (s, 1 H, NH), 10.26 (s, 1 H, NHɂ), 7.65 (dd, J = 0.5, 7.8 Hz, 1 H, H4ɂ), 7.41 (d, J

= 8.0 Hz, 1 H, H7ɂ), 7.38 (d, J = 2.4 Hz, 1 H, H2ɂ), 7.12, (dd, J = 1.2, 7.1 Hz, 1 H, H6ɂ), 227

7.07 (m, 1 H, H5), 7.04 (dd, J = 1.0, 7.1 Hz, 1 H, H5ɂ), 6.74 (m, J = 1.5 Hz, 1 H, H3),

13 6.25 (ddd, J = 2.5, 3.7 Hz, 1 H, H4), 4.39 (d, J = 0.8 Hz, 2 H, CH 2). C NMR (75

MHz, d 6-acetone): δ 165.1 (ArC), 160.6 (ArC), 137.7 (ArC), 128.0 (ArC), 124.6

(ArCH), 123.7 (ArCH), 122.5 (ArCH), 119.9 (ArCH), 119.3 (ArCH), 117.2 (ArC),

112.4 (2xArCH), 110.7 (ArCH), 108.8 (ArC), 22.4 (CH 2). IR (ATR ): ν max 3218, 1623,

-1 -1 -1 1572, 1517 cm . UV-Vis (MeOH): λ max 280 (ε 33500 cm M ), 218 (46900) nm.

+ HRMS (+ESI): Found m/z 287.0901, [M+Na] ; C 15 H12 N4ONa required 287.0903.

N'-(3-(1 H-Indol-3-yl)propanoyl)benzohydrazide (189a)

This compound was synthesized as described for compound 185 using 3-indolepropanoic acid 145

(0.40 g, 2.12 mmol), hydrazide 184 (0.74 g, 5.41 mmol), EDCI (0.90 g, 4.62 mmol) and HOBt (0.60 g, 4.38 mmol) in DMF (4.0 mL) over 24 h to give the title compound 189a as an off-white solid following purification

1 (0.32 g, 50%). M.p. 178-180 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.44 (bs, 1 H,

N1H), 9.72 (bs, 1 H, NH), 8.40 (d, J = 7.1 Hz, 2 H, H2ɂ, H6ɂ), 8.02 (m, 2 H, H4, H4ɂ),

7.93 (t, J = 7.0 Hz, 2 H, H3ɂ, H5ɂ), 7.83 (d, J = 8.0 Hz, 1 H, H7), 7.67 (s, 1 H, H2), 7.54

(dt, J = 1.1, 7.5 Hz, 1 H, H6), 7.46 (dt, J = 1.1, 7.4 Hz, 1 H, H5), 3.57 (t, J = 7.2 Hz, 2

13 H, CH 2), 3.14 (t, J = 7.3 Hz, 2 H, CH 2CO). C NMR (75 MHz, d 6-DMSO): δ 171.6

(CH2CO), 166.1 (CO), 137.3 (C7a), 132.1 ( C1′, C4ɂ), 128.8 (C2ɂ, C6ɂ), 127.9 (C4a),

127.8 (C3ɂ, C5ɂ), 122.6 (C2), 121.6 (C6), 118.9 (C5), 118.8 (C 4), 114.7 (C3), 111.7

(C7), 34.9 (CH2CO), 21.3 (CH 2). IR (KBr): ν max 3350, 3234, 3035, 2926, 1690, 1631,

1503, 1480, 1419, 1340, 1310, 1242, 1137, 1097, 1067, 1009, 744, 714, 690 cm -1. UV- 228

-1 -1 Vis (MeOH): λ max 273 (ε 10,300 cm M ), 223 (63,600) nm. HRMS (+ESI): Found m/z

+ 308.1393, [M+H] ; C 18 H18 N3O2 required 308.1394.

N'-(3-(1 H-Indol-3-yl)propanoyl)thiophene-2-carbohydrazide (189b)

This compound was synthesized as described for compound 185 using 3-indolepropanoic acid 145

(0.19 g, 1.05 mmol), hydrazide 186a (0.19 g, 1.34 mmol), EDCI (0.30 g, 1.58 mmol) and HOBt (0.18 g, 1.30 mmol) in DMF (3.0 mL) over 24 h to give the title compound 189a as an off-white solid following purification

1 1 (0.27 g, 83%). M.p. 164-166 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.83 (s, 1 H, N H),

10.37 (s, 1 H, NH), 9.98 (s, 1 H, NɂH), 7.86 (, J = Hz, 2 H, H3ɂ, H5ɂ), 7.57 (d, J = 7.8

Hz, 1 H, H4), 7.36 (d, J = 8.0 Hz, 1 H, H7), 7.21 (m, 2 H, H2, H4ɂ), 7.09 (dt, J = 1.2, 7.5

Hz, 1 H, H6), 7.01 (dt, J = 1.1, 7.4 Hz, 1 H, H5); 3.00 (t, J = 7.2 Hz, 2 H, CH 2), 2.59 (t,

13 J = 8.2 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 171.4 (CONɂH), 160.5

(CONH), 137.4 (C2ɂ), 136.2 (C4a), 131.5 (C5ɂ), 128.9 (C3ɂ), 128.1 (C4ɂ), 127.0 (C7a),

122.4 (C2), 120.9 (C6), 118.2 (C5, C4), 113.4 (C3), 111.3 (C7), 34.1 (CH2CO), 20.7

-1 (CH2). IR (ATR): ν max 3381, 3227, 1684, 1630, 1567, 1524, 1415 cm . UV-Vis

-1 -1 (MeOH): λ max 271 (ε 15300 cm M ), 222 (41600) nm. HRMS (+ESI): Found m/z

+ 314.0954, [M+H] ; C 16 H16 N3O2S required 314.0958.

229

N'-(3-(1 H-Indol-3-yl)propanoyl)furan-2-carbohydrazide (189c)

This compound was synthesized as described for compound 185 using 3-indolepropanoic acid 145

(0.20 g, 1.04 mmol), hydrazide 186b (0.21 g, 1.61 mmol), EDCI (0.29 g, 1.54 mmol) and HOBt (0.24 g, 1.76 mmol) in DMF (3.0 mL) over 24 h to give the title compound 189c as an off-white solid following purification

1 1 (0.27 g, 88%). M.p. 94-96 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.81 (s, 1 H, N H),

10.23 (s, 1 H, NɂH), 9.92 (s, 1 H, NH), 7.91 (dd, J = 0.7, 1.7 Hz, 1 H, H5ɂ), 7.56 (d, J =

7.8 Hz, 1 H, H4), 7.35 (d, J = 8.0 Hz, 1 H, H7), 7.24 (dd, J = 0.6, 3.5 Hz, 1 H, H3ɂ),

7.20 (d, J = 2.3 Hz, 1 H, H2), 7.09 (dt, J = 1.2, 7.0 Hz, 1 H, H6), 7.00 (dt, J = 1.1, 7.4

Hz, 1 H, H5), 6.68 (m, 1 H, H4ɂ), 2.99 (t, J = 7.1 Hz, 2 H, CH 2), 2.58 (t, J = 6.4 Hz, 2 H,

13 CH 2). C NMR (75 MHz, d 6-DMSO): δ 171.3 (CONɂH), 157.1 (CONH), 146.2 (C1ɂ),

145.7 (C5ɂ), 136.2 (C7a), 127.0 (C3), 122.3 (C2), 1 20.9 (C6), 118.2 (C4, C5), 114.4

(C3ɂ), 113.4 (C4a), 111.8 (C4ɂ), 111.3 (C7), 34.1 (CH 2), 20.6 (CH2). IR (ATR ): ν max

-1 3440, 3227, 3022, 1689, 1646, 1593, 1471 cm . UV-Vis (MeOH): λ max 254 (ε 23000

-1 -1 + cm M ), 221 (54900) nm. HRMS (+ESI): Found m/z 298.1182, [M+H] ; C 16 H16 N3O3 required 298.1186.

N'-(3-(1 H-indol-3-yl)propanoyl)pyrolle-2-carbohydrazide (189d)

This compound was synthesized as described for compound 185 using 3-indolepropanoic acid 145

(0.19 g, 1.01 mmol), hydrazide 186c (0.15 g, 1.20 mmol), EDCI (0.23 g, 1.20 mmol) and HOBt (0.21 g, 1.54 mmol) in DMF (2.0 mL) over 24 h to give the title compound 189d as an off-white solid following purification 230

1 (0.08 g, 28%). M.p. 212-214 ºC. H NMR (300 MHz, d 6-DMSO): δ 11.54 (bs, 1 H,

NH), 10.78 (bs, 1 H, NH), 9.81 (d, J = 8.1 Hz, 2 H, 2xNH), 7.54 (d, J = 7.9 Hz, 1 H,

H4), 7.33 (d, J = 7.9 Hz, 1 H, H7), 7.18 (d, J = 2.2 Hz, 1 H, H2), 7.07 (ddd, J = 1.2, 7.9

Hz, 1 H, H6), 6.98 (ddd, J = 1.2, 7.9 Hz, 1 H, H5), 6.92-6.90 (m, 1 H, Hɂ), 6.88 -6.96 (m,

1 H, H4ɂ), 6.12 -6.09 (m, 1 H, Hɂ), 2.97 (dd, J = 7.0 Hz, 2 H, CH 2), 2.55 (d, J = 7.0 HZ, 2

13 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 171.4 (C=O), 136.2 (ArC), 127.0 (ArC),

124.1 (ArC), 122.3 (ArCH), 122.0 (ArCH), 120.9 (ArCH), 118.2 (ArCH), 113.5 (ArC),

111.3 (ArCH), 110.9 (ArCH), 108.7 (ArCH), 34.1 (CH 2), 20.7 (CH2). IR (ATR ): ν max

-1 3555, 3383, 3254, 1695, 1932, 1579, 1509 cm . UV-Vis (MeOH): λ max 268 (ε 14900 cm -1M-1), 221 (27200) nm. HRMS (+ESI): Found m/z 319.1160, [M+Na] +;

C16 H16N4O2Na required 319.1165.

2-(2-(1 H-Indol-3-yl)ethyl)-5-(thiophen-2-yl)-1,3,4-oxadiazole (190a)

This compound was synthesized as described for compound 179 using hydrazide 189b (0.15 g, 0.47 mmol), DIPEA (0.30 mL, 1.75 mmol) and 4-TsCl (0.30 g, 1.55 mmol) in MeCN (6.0 mL) over 1.5 h to give the title compound 190a as an off- white solid (0.04 g, 30%). M.p. 127-129 ºC, Lit. 308 M.p. 132-134 °C. 1H NMR (300

MHz, d 6-acetone): δ 10.03 (bs, 1 H, NH), 7.78 (dd, J = 1.2, 5.1 Hz, 1 H, H), 7.73 (dd, J

= 1.2, 3.7 Hz, 1 H, H), 7.62 (d, J = 7.9 Hz, 1 H, H4ɂ), 7.38 (d, J = 7.9 Hz, 1 H, H7ɂ),

7.24 (dd, J = 3.7, 5.1 Hz, 1 H, H4), 7.22 (d, J = 2.5 Hz, 1 H, H2ɂ), 7.10 (ddd, J = 1.2,

13 7.9 Hz, 1 H, H6ɂ), 7.02 (ddd, J = 1.2, 7.9 Hz, 1 H, H5ɂ), 3.32 (s, 4 H, 2 x CH 2). C

NMR (75 MHz, d 6-acetone): δ 167.0 (C=N), 161.4 (C=N), 137.7 (ArC), 131.0 (ArCH),

130.2 (ArCH), 129.2 (ArCH), 128.2 (ArC), 126.4 (ArC), 123.3 (ArCH), 122.2 (ArCH),

119.6 (ArCH), 119.1 (ArCH), 114.1 (ArC), 112.2 (ArCH), 27.0 (CH2), 23.0 (CH2). IR 231

-1 (ATR ): ν max 3319, 3234, 2920, 1490, 1319 cm . UV-Vis (MeOH): λ max 281 (ε 21900 cm -1M-1), 221 (48100) nm. HRMS (+ESI): Found m/z 318.0670, [M+Na] +;

C16 H13 N3OSNa required 318.0672.

2-(2-(1 H-Indol-3-yl)ethyl)-5-(furan-2-yl)-1,3,4-oxadiazole (190b)

This compound was synthesized as described for compound 179 using hydrazide 189c (0.13 g, 0.45 mmol), DIPEA (0.30 mL, 1.75 mmol) and 4-TsCl (0.28 g, 1.47 mmol) in MeCN (6.0 mL) over 1.5 h to give the title compound 190b as an off-

1 white solid (0.07 g, 56%). M.p. 130-131 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.83

(s, 1 H, NH), 8.02 (dd, J = 0.7, 1.8 Hz, 1 H, H5), 7.53 (, d, J = 7.9 Hz, 1 H, H4ɂ), 7.34

(d, J = 8.0 Hz, 1 H, H7ɂ), 7.28 (dd, J = 0.7, 3.5 Hz, 1 H, H3), 7.16 (d, J = 2.3 Hz, 1 H,

H2ɂ), 7.07 (dt, J = 1.1, 7.5 Hz, 1 H, H6ɂ), 6.97 (dt, J = 1.0, 7.4 Hz, 1 H, H5ɂ), 6.77 (q, J =

1 2 13 1.8 Hz, 1 H, H 4) 3.27 (m, 2 H, C H2), 3.21 (m, 2 H, C H2). C NMR (75 MHz, d 6-

DMSO): δ 165.9 (C2), 156.8 (C5), 146.7 (C5), 138.8 (C2), 136.2 (C7aɂ), 126.8 (C4aɂ),

122.7 (C2ɂ), 121.0 (C6ɂ), 118.4 (C5ɂ), 118.1 (C4ɂ), 114.1 (C3), 112.5 (C4), 112.3 (C3ɂ),

1 2 111.4 (C7ɂ), 25.8 (C H2), 21.8 (C H2). IR (ATR ): ν max 3179, 3116, 3054, 2923, 1638,

-1 -1 -1 1574, 1521, 1447 cm . UV-Vis (MeOH): λ max 269 (ε 30300 cm M ), 221 (45800) nm.

+ HRMS (+ESI): Found m/z 302.0897, [M+Na] ; C 16 H13 N3O2Na required 302.0900.

232

2-(2-(1 H-Indol-3-yl)ethyl)-5-(pyrrol-2-yl)-1,3,4-oxadiazole (190c)

This compound was synthesized as described for compound 179 using hydrazide 189d (82.1 mg, 0.28 mmol), DIPEA (0.30 mL, 1.75 mmol) and 4-TsCl (118.4 mg, 0.62 mmol) in MeCN (4.0 mL) over 1.5 h to give the title compound 190c as an

1 off-white solid (47.4 mg, 61%). M.p. 160-162 ºC. H NMR (300 MHz, d 6-acetone): δ

11.11 (s, 1 H, NH), 10.03 (s, 1 H, NH), 7.61 (ddd, J = 0.5, 7.2 Hz, 1 H, H4ɂ), 7.37 (ddd,

J = 1.0, 7.9 Hz, 1 H, H7ɂ), 7.20 (d, J = 2.4 Hz, 1 H, H2ɂ), 7.09 (m, 2 H, H6ɂ, H5), 7.02

(ddd, J = 1.1, 7.0 Hz, 1 H, H5ɂ), 6.79 (m, 1 H, H3), 6.28 (m, 1 H, H4), 3.28 (m, J= 2.3

13 Hz, 4 H, CH 2CH 2). C NMR (75 MHz, d 6-acetone): δ 165.7 (ArC), 160.2 (ArC), 137.7

(ArC), 130.2 (ArC), 128.2 (ArC), 126.9 (ArC), 123.6 (ArCH), 123.3 (ArCH), 122.2

(ArCH), 119.6 (ArCH), 119.1 (ArCH), 117.3 (ArC), 114.2 (ArC), 112.3 (ArCH), 112.2

(ArCH), 110.7 (ArCH), 27.0 (CH 2), 23.0 (CH2). IR (ATR): ν max 3389, 3181, 2920,

-1 -1 -1 1620, 1576, 1518 cm . UV-Vis (MeOH): λ max 280 (ε 22200 cm M ), 221 (33300) nm.

+ HRMS (+ESI): Found m/z 301.1059, [M+Na] ; C 16 H14 N4ONa required 301.1060.

α-Oxo-1H-indole-3-acetyl chloride (191)

This compound was synthesized as described for compound 163 using indole 30 (0.62 g, 5.29 mmol) and oxalyl chloride (0.60 mL,

7.00 mmol) in Et 2O (9.0 mL) over 4 h to give the title compound

191 as a yellow solid upon filtration. (0.94 g, 86%). M.p. 116-118 ºC, Lit. 400 117-119

1 ºC. H NMR (300 MHz, CDCl 3): δ 8.93 (bs, 1 H, NH), 8.34 (dd, J = 4.1, 4.1 Hz, 1 H,

H4), 7.74 (d, J = 3.4 Hz, 1 H, H2), 7.41 (dd, J = 4.1 Hz, 1 H H7), 7.32 (m, 2 H, H5,

H6). 233

N'-(2-(1 H-Indol-3-yl)-2-oxoacetyl)benzohydrazide (192)

To a solution of hydrazide 184 (0.35 g, 2.59 mmol) and

Et 3N (0.50 mL, 3.59 mmol) in DCM (5.0 mL) was added glyoxyl chloride 191 (0.16 g, 0.76 mmol). This mixture was stirred for 2 h, the solvent removed under reduced pressure and the crude product recrystallized from MeOH to give the title compound 192 as a white solid (0.18

1 g, 77%). M.p. 296-298 ºC. H NMR (300 MHz, d 6-DMSO): δ 12.32 (bs, 1 H, NH),

10.70 (bs, 1 H, NH), 10.57 (bs, 1 H, NH), 8.72 (s, 1 H, H), 8.28 – 8.24 (m, 1 H, H), 7.95

(ddd, J = 1.6, 8.4 Hz, 2 H, H2ɂ, H6ɂ), 7.63 – 7.52 (m, 4 H, H, H3ɂ, H4ɂ, H5ɂ), 7.32 – 7.29

13 (m, 2 H, H, H). C NMR (75 MHz, d 6-DMSO): δ 182.4 (C=O), 165.7 (CONH), 163.8

(CONH), 138.6 (ArCH), 136.6 (ArC), 132.4 (ArC), 132.1 (ArCH), 128.6 (ArCH), 127.6

(ArCH), 125.9 (ArC), 123.8 (ArCH), 122.8 (ArCH), 121.4 (ArCH), 112.8 (ArCH),

112.7 (ArC). IR (ATR): ν max 3291, 3146, 3054, 1706, 1599, 1470, 1424, 1310, 1233,

-1 -1 -1 1116, 808, 744, 707 cm . UV-Vis (MeOH): λ max 326 (ε 12,700 cm M ), 207 (39,900)

+ nm. HRMS (+ESI): Found m/z 330.0850, [M+Na] ; C 17 H13 N3O3Na required 330.0849.

(1 H-Indol-3-yl)(5-phenyl-1,3,4-oxadiazol-2-yl)methanone (193)

This compound was prepared as described for compound

179 using hydrazide 192 (0.15 g, 0.49 mmol), DIPEA

(0.15 mL, 0.56 mmol) and 4-TsCl (0.21 g, 1.09 mmol) in

MeCN (3.0 mL) over 2 h to give the title compound 193 as colourless crystals following

1 purification (0.12 g, 82%). M.p. 271-273 ºC. H NMR (300 MHz, d 6-DMSO): δ 9.28 (s,

1 H, NH), 8.31 (d, J = 6.5 Hz, 1 H, H4), 8.16 (dd, J = 1.7, 7.7 HZ, 2 H, H2ɂ, H6ɂ), 8.04

(d, J = 6.5 Hz, 1 H, H7), 7.75-7.67 (m, 3 H, H3ɂ, H4ɂ, H5ɂ), 7.53 (ddd, J = 1.7, 7.3, 7.3 234

Hz, 1 H, H6), 7.49 (ddd, J = 1.7, 7.3, 7.3 Hz, 1 H, H5). IR (ATR): νmax 3138, 1648,

-1 + 1602, 1520, 1444 cm . HRMS (+ESI): Found m/z 312.0746, [M+Na] ; C 17 H11 N3O2Na required 312.0744.

N'-(2-((1 H-Indol-3-yl)thio)acetyl)benzohydrazide (194)

This compound was prepared as described for compound 185 using acid 148 (0.22 g, 1.06 mmol), hydrazide 184 (0.15 g, 1.10 mmol), EDCI (0.21 g,

1.10 mmol) and HOBt (0.18 g, 1.13 mmol) in DMF (2.0 mL) over 24 h to give the title compound 194 as a white solid (0.06 g, 17%). M.p. 160-162 ºC. 1H NMR (300 MHz, d6-acetone): δ 10.59 (bs, 1 H, NH), 7.93 (d, J = 7.2 Hz, 2 H, H2ɂ, H6ɂ), 7.74 (d, J = 7.2

Hz, 2 H, H3ɂ, H5ɂ), 7.57 (dd, J = 7.2 Hz, 1 H, H4ɂ), 7.51 -7.44 (m, 3 H, H2, H4, H7),

7.17 (ddd, J = 1.6, 7.0, 7.0 HZ, 1 H, H6), 7.13 (ddd, J = 1.6, 7.0, 7.0 HZ, 1 H, H5), 3.46

13 (s, 2 H, CH 2). C NMR (75 MHz, d 6-acetone): δ 168.7 (C=O), 166.4 (C=O), 137.5

(ArC), 133.7 (ArC), 132.7 (ArCH), 132.2 (ArCH), 130.0 (ArC), 129.3 (ArCH), 128.3

(ArCH), 122.9 (ArCH), 120.8 (ArCH), 119.5 (ArCH), 112.7 (ArCH), 39.4 (CH 2). IR

-1 (ATR ): ν max 3331, 3198, 3012, 1665, 1638, 1559, 1501, 1473, 1270, 1099, 688 cm .

-1 -1 UV-Vis (MeOH): λ max 269 (ε 9,550 cm M ), 219 (46,100) nm. HRMS (+ESI): Found

+ m/z 348.0774, [M+Na] ; C17 H15 N3O2SNa required 348.0777.

235

2-(((1 H-Indol-3-yl)thio)methyl)-5-phenyl-1,3,4-oxadiazole (195)

This compound was synthesized as described for compound 179 using hydrazide 194 (0.13 g, 0.43 mmol),

DIPEA (0.30 mL, 1.75 mmol) and 4-TsCl (0.21 g, 1.08 mmol) in MeCN (6.0 mL) over 1.5 h to give the title compound 195 as an off-white

1 solid (0.10 g, 76%). H NMR (300 MHz, d 6-acetone): δ 10.64 (bs, 1 H, NH), 7.83 (dd, J

= 1.5, 8.0 Hz, 2 H, H2ɂ, H6ɂ), 7.59 -7.50 (m, 5 H, H2, H4, H3ɂ, H4ɂ, H5ɂ), 7.46 (dd, J =

1.6, 8.4 Hz, 1 H, H7), 7.14 (ddd, J = 1.4, 8.0 Hz, 1 H, H6), 7.04 (ddd, J = 1.4, 8.0 Hz, 1

H, H5), 4.15 (s, 2 H, CH 2).

2-(1 H-Indol-3-yl)acetohydrazide (196)

A mixture of ester 197 (0.50 g, 2.66 mmol) and hydrazine monohydrate (2.0 mL, 26.39 mmol) in EtOH (25.0 mL) was heated at reflux for 3 h. The solvent was removed under reduced pressure and the crude product recrystallized from EtOH to give the title compound 196 as colourless prisms (0.60 g, 91%). M.p. 140-142 ºC. Lit. M.p. 305 142-

1 143 ºC. H NMR (300 MHz, CDCl 3): δ 8.18 (bs, 1 H, NH), 7.62 (dd, J = 1.0, 7.4 Hz, 1

H, H4), 7.28 (dd, J = 1.0, 7.4 Hz, 1 H, H7), 7.17 (m, J = 1.3, 7.0 Hz, 2 H, H5, H6), 3.79

(d, J = 0.5 Hz, 2 H, CH 2), 3.71 (s, 3 H, CH 3).

236

Methyl 2-(1 H-indol-3-yl)acetate (197)

Concentrated H 2SO 4 (0.8 mL, 14.4 mmol) was added dropwise to a solution of IAA 106 (2.10 g, 11.96 mmol) in MeOH (20.0 mL) and the mixture stirred at r.t. for 2.5 h. The mixture was cooled on an ice bath before 2 M NaOH (1.8 mL, 3.6 mmol) was added dropwise at 10 °C. The solution was diluted with H 2O (20.0 mL) and the pH adjusted to 7 with the addition of

t sat. K 2CO 3. This was extracted with BuOMe (2 x 25.0 mL) and the combined organic phase washed with H 2O (2 x 15.0 mL) and brine (10.0 mL), dried over Na 2SO 4 and concentrated under reduced pressure. The crude residue was recrystallized from

DCM:hexane to give the title compound 197 as colourless prisms (1.51 g, 96%). M.p.

329 1 48-50 ºC. Lit. M.p. 50-52 ºC. H NMR (300 MHz, CDCl 3): δ 8.18 (bs, 1 H, NH), 7.62

(dd, J = 1.0, 7.4 Hz, 1 H, H4), 7.28 (dd, J = 1.0, 7.4 Hz, 1 H, H7), 7.17 (m, J = 1.3, 7.0

Hz, 2 H, H5, H6), 3.79 (d, J = 0.5 Hz, 2 H, CH 2), 3.71 (s, 3 H, CH3).

2-((1 H-Indol-3-yl)methyl)-5-benzyl-1,3,4-oxadiazole (198)

A mixture of hydrazide 196 (0.20 g, 1.04 mmol) and phenylacetic acid (0.14 g, 1.04 mmol) in POCl 3 (3.0 mL) was heated at reflux over 2 h. The mixture was poured over crushed ice and neutralized with sat. K 2CO 3. The precipitate was collected via filtration and recrystallized from EtOH to give the title compound 198 as an off

1 white solid (0.07 g, 23%). M.p. 106-108 ºC. H NMR (300 MHz, d 6-Acetone): δ 10.20

(bs, 1 H, NH), 7.57 (ddd, J = 0.6, 8.6 Hz, 1 H, H4ɂ), 7.40 (ddd, J = 0.8, 8.0 Hz, 1 H,

H7ɂ), 7.31 -7.26 (m, 6 H, H2ɂ, H2, H3, H4, H5, H6), 7.12 (ddd, J = 1.1, 7.6 Hz, 1 H,

2 H6ɂ), 7.03 (ddd, J = 1.1, 7.6 Hz, 1 H, H5ɂ), 4.31 (d, J = 0.7 Hz, 2 H, C CH 2), 4.16 (s, 2 237

5 13 H, C CH 2). C NMR (75 MHz, d 6-Acetone): δ 167.1 (C2), 166.2 (C5), 137.6 (C7aɂ),

135.7 (C1), 129.6 (C2, C6), 129.5 (C3, C5), 128.0 (C4), 127.9 (C4aɂ), 124.5 (C2ɂ),

5 122.5 (C6ɂ), 119.9 (C5ɂ), 119.3 (C4ɂ), 112.3 (C7ɂ), 108.7 (C3ɂ), 31. 9 (C CH2), 22.5

2 -1 (C CH2). IR (ATR ): ν max 3277, 1563, 1491 cm . UV-Vis (MeOH): λ max 289 (ε 6,200 cm -1 M-1), 279 (7,500), 217 (43,200) nm. HRMS (+ESI): Found m/z 290.1284, [M+H] +;

C18 H16 N3O required 290.1288.

2-((1 H-Indol-3-yl)methyl)-5-phenyl-1,3,4-thiadiazole (199)

A mixture of hydrazide 185 (0.27 g, 0.92 mmol) and

Lawesson’s reagent (0.88 g, 2.18 mmol) was heated at reflux in THF (10.0 mL) for 24 h. The solvent was then removed under reduced pressure and the residue purified by flash column chromatography (20% EtOAc:hexanes) to give the title compound as an off-white solid

1 (0.02 g, 7%). M.p. 197-199 ºC. H NMR (300 MHz, d 6-Acetone): δ 10.28 (bs, 1 H,

NH), 7.93-7.90 (m, 2 H, H2, H6), 7.59 (ddd, J = 1.1, 7.9 Hz, 1 H, H4ɂ), 7.51 -7.48 (m,

3 H, H3, H4, H5), 7.45 -7.42 (m, 2 H, H2ɂ, H7ɂ), 7.14 (ddd, J = 1.3, 7.2 Hz, 1 H, H6ɂ),

13 7.04 (ddd, J = 1.3, 7.2 Hz, 1 H, H5ɂ), 4.62 (d, J = 0.7 Hz, 2 H, CH 2). C NMR (75

MHz, d 6-Acetone): δ 131.7 (C4), 130.1 (C3, C5), 128.4 (C2, C6), 124.6 (C2ɂ),

122.7 (C6ɂ), 120.1 (C5ɂ), 119.3 (C4ɂ), 112.5 (C7ɂ), 27.1 (CH 2). IR (ATR ): ν max 3173,

-1 -1 -1 1455 cm . UV-Vis (MeOH): λ max 271 (ε 19,800 cm M ), 217 (36,600) nm. HRMS

+ (+ESI): Found m/z 292.0902, [M+H] ; C 17 H14 N3S required 292.0903.

238

2-(2-(1 H-Indol-3-yl)acetyl)-N,N-dimethylhydrazinecarbothioamide (201)

This compound was synthesized as described for compound 185 using IAA 106 (0.18 g, 1.03 mmol), 4,4- dimethylthiosemicarbazide 200 (0.13 g, 1.05 mmol),

EDCI (0.23 g, 1.1 mmol) and HOBt (0.20 g, 1.49 mmol) in DMF (2.0 mL) over 24 h to give the title compound 201 as a cream coloured solid after purification (0.11 g, 40%).

1 M.p. 158-160 ºC. H NMR (300 MHz, d 6-acetone): δ 10.13 (bs, 1 H, NH), 9.00 (bs, 1 H,

NH), 7.66 (d, J = 7.8 Hz, 1 H, H), 7.39 (d, J = 7.8 Hz, 1 H, H), 7.37 (d, J = 2.5 Hz, 1 H,

H2), 7.10 (ddd, J = 1.3, 7.2 Hz, 1 H, H), 7.01 (ddd, J = 1.0, 7.8 Hz, 1 H, H), 3.73 (d, J =

13 1.0 Hz, 2 H, CH 2), 3.27 (s, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-acetone): δ 170.1

(ArC), 137.6 (ArC), 128.6 (ArC), 124.9 (ArCH), 122.2 (ArCH), 119.8 (ArCH), 119.6

(ArCH), 112.1 (ArCH), 109.3 (ArC), 40.8 (2 x CH 3), 31.9 (CH2). IR (KBr): ν max 3410,

-1 3250, 3168, 1661, 1557, 1508, 1367, 1303, 1087, 736, 704 cm . UV-Vis (MeOH): λ max

266 (ε 10600 cm -1M-1), 206 (48000) nm. HRMS (+ESI): Found m/z 299.0932,

+ [M+Na] ; C 13 H16 N4OSNa required 299.0937.

5-((1 H-Indol-3-yl)methyl)-N,N-dimethyl-1,3,4-thiadiazol-2-amine (202)

This compound was synthesized as described for compound

179 using hydrazide 201 (0.11 g, 0.41 mmol), DIPEA (0.20 mL, 1.15 mmol) and 4-TsCl (0.20 g, 1.03 mmol) in MeCN

(4.0 mL) over 2 h to give the title compound 202 as a pale tan solid after purification

1 (0.05 g, 48%). M.p. 176-178 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.98 (bs, 1 H,

NH), 7.54 (ddd, J = 0.6, 7.8 Hz, 1 H, H4ɂ), 7.36 (ddd, J = 0.9, 8.0 Hz, 1 H, H7ɂ), 7.29 (d,

J =2.5 Hz, 1 H, H2ɂ), 7.09 (ddd, J = 1.2, 7.0 Hz, 1 H, H6ɂ), 9.66 (ddd, J = 1.2, 7.0 Hz, 1 239

13 H, H5ɂ), 4.14 (d, J = 0.8 Hz, 2 H, CH 2), 2.90 (s, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-

DMSO): δ 164.6 (C5), 159.4 (C2), 136.2 (C7aɂ), 126.7 (C4aɂ), 123.8 (C2ɂ), 121.2 (C6ɂ),

118.6 (C5ɂ), 118.3 (C4ɂ), 111.5 (C7ɂ), 107.5 (C3ɂ), 37.6 (2 x CH 3), 21.6 (CH 2). IR

-1 (ATR ): ν max 3144, 3045, 2922, 1644, 1584, 1433 cm . UV-Vis (MeOH): λ max 289 (ε

7600 cm -1M-1), 279 (9100), 219 (51900) nm. HRMS (+ESI): Found m/z 265.1063,

+ [M+Na] ; C 13 H14 N4SNa required 265.1060.

3-((5-Phenyl-4H-1,2,4-triazol-3-yl)methyl)-1H-indole (203), 3-((5-Phenyl-1H-1,2,4- triazol-3-yl)methyl)-1H-indole (204a) and 3-((3-phenyl-1H-1,2,4-triazol-5- yl)methyl)-1H-indole (204b)

A solution of oxadiazole 179 (0.64 g, 2.33 mmol) in 20%

NH 3/MeOH (6.0 mL) was heated at 160 °C in a pressure tube for 24 h. The mixture was extracted with EtOAc (3 x

10 mL), dried over Na2SO 4 and the solvent removed under reduced pressure. The crude mixture was purified by flash column chromatography (30% EtOAc:hexanes) to give the title copound as a yellow solid (0.03g, 5%).

1 M.p. 246-248 ºC. H NMR (300 MHz, d 6-DMSO): δ 13.76 (bs, 1 H, NHɂ), 10.93 (bs, 1

H, NH), 7.97 (dd, J = 1.5, 8.1 Hz, 2 H, H2, H6), 7.53 (d, J = 8.0 Hz, 1 H, H4), 7.47-

7.39 (m, 3 H, H3, H4, H5), 7.35 (d, J = 8.0 Hz, 1 H, H7), 7.25 (d, J = 1.9 Hz, 1 H,

H2), 7.06 (ddd, J = 1.1, 8.0 Hz, 1 H, H6), 6.96 (ddd, J = 1.1, 8.0 Hz, 1 H, H5), 4.20 (bs,

13 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 159.4 (C3ɂ, C5ɂ), 136.2 (C7a), 132.6

(C1), 128.7 (C3, C4, C5), 126.9 (C4a), 125.7 (C2, C6), 123.6 (C2), 121.1 (C6),

118.4 (C4, C5), 111.4 (C7), 107.7 (C3), 23.0 (CH 2). IR (ATR): ν max 3325, 3141, 3030, 240

-1 -1 -1 2880, 2758, 1618, 1438 cm . UV-Vis (MeOH): λ max 220 (ε 56900 cm M ), 202

+ (59800) nm. HRMS (+ESI): Found m/z 275.1290, [M+H] ; C 17 H15 N4 required

275.1291.

N'-(2-(2-Methyl-1H-indol-3-yl)acetyl)benzohydrazide (209a)

This compound was synthesized as described for compound 185 using acid 168 (0.19 g, 1.00 mmol), hydrazide 184 (0.16 g, 1.20 mmol), EDCI (0.25 g, 1.30 mmol) and HOBt (0.17 g, 1.30 mmol) in DMF (2.0 mL) over 24 h to give the title compound 209a as an off-white solid ( 0.14 g, 47%). M.p. 107-109 ºC. 1H NMR (300

MHz, d 6-Acetone): δ 10.02 (bs, 1 H, NH), 7.91 (dd, J = 1.4, 7.0 Hz, 2 H, H2ɂ, H6ɂ),

7.60-7.43 (m, 4 H, H4, H3ɂ, H4ɂ, H5ɂ), 7.28 (dd, J = 1.4, 7.0 Hz, 1 H, H7), 7.00 (ddd, J =

13 1.8, 7.6 Hz, 2 H, H5, H6), 3.70 (s, 2 H, CH 2), 2.46 (s, 3 H, CH 3). C NMR (75 MHz, d6-Acetone): δ 170.8 (C=O), 136.4 (ArC), 134.2 (ArC), 132.5 (ArCH), 129.7 (ArC),

129.3 (ArCH), 128.3 (ArCH), 121.3 (ArCH), 119.5 (ArCH), 118.8 (ArCH), 111.1

(ArCH), 105.1 (ArC), 30.6 (CH 2), 11.7 (CH 3). IR (ATR): ν max 3224, 3016, 1637, 1509,

-1 -1 -1 1459, 1303, 1237 cm . UV-Vis (MeOH): λ max 272 (ε 8870 cm M ), 223 (46900) nm.

+ HRMS (+ESI): Found m/z 330.1216, [M+Na] ; C 18 H17 N3O2Na required 330.1213.

241

N'-(2-(5-Bromo-2-methyl-1H-indol-3-yl)acetyl)benzohydrazide (209b)

This compound was synthesized as described for compound 185 using acid 169a (0.19 g, 0.71 mmol), hydrazide 184 (0.13 g, 0.96 mmol), EDCI (0.23 g, 1.22 mmol) and HOBt (0.15 g, 1.11 mmol) in DMF (2.0 mL) over 24 h to give the title compound 209b as an off-white solid after purification (0.24 g, 90%). M.p. 252-254 ºC.

1 H NMR (300 MHz, d 6-DMSO): δ 11.07 (bs, 1 H, NH), 10.33 (d, J = 1.0 Hz, 1 H, NH),

10.09 (d, J = 1.0 Hz, 1 H, NH). 7.88 (dd, J = 1.6, 8.5 Hz, 2 H, H), 7.78 (d, J = 1.9, 1 H,

H), 7.57 (ddd, J = 1.5, 7.2 Hz, 1 H, H), 7.51 (ddd, J = 1.6, 6.2 Hz, 2 H, H), 7.22 (d, J =

8.5 Hz, 1 H, H), 7.10 (dd, J = 2.0, 8.5 Hz, 1 H, H), 3.57 (s, 2 H, CH 2), 2.42 (s, 3 H,

13 CH 3). C NMR (75 MHz, d 6-DMSO): δ 169.9 (C=O), 165.5 (C=O), 135.2 (ArC), 133.7

(ArC), 132.5 (ArC), 131.7 (ArCH), 130.3 (ArC), 128.4 (ArCH), 127.4 (ArCH), 122.3

(ArCH), 120.4 (ArCH), 112.2 (ArCH), 111.0 (ArC), 104.3 (ArC), 29.3 (CH 2), 11.5

-1 (CH3). IR (ATR): ν max 3320, 3189, 1665, 1595, 1556, 1459, 1221 cm . UV-Vis

-1 -1 (MeOH): λ max 289 (ε 7800 cm M ), 227 (47200) nm. HRMS (+ESI): Found m/z

+ 408.0313, [M+Na] ; C 18 H15 BrN 3O2Na required 408.0318.

N'-(2-(2, 5-Dimethyl-1H-indol-3-yl)acetyl)benzohydrazide (209c)

This compound was synthesized as described for compound 185 using acid 169b (0.10 g, 0.49 mmol), hydrazide 184 (0.35 g, 2.57 mmol), EDCI (0.23 g, 1.22 mmol) and HOBt (0.15 g, 1.11 mmol) in DMF (2.0 mL) over 24 h to give the title compound 209c as an off-white solid after purification (0.04 g, 25%). 1H NMR (500

MHz, d 6-DMSO): δ 10.64 (s, 1 H, CONH), 10.29 (bs, 1 H, NH), 9.98 (s, 1 H, CONH), 242

7.87 (dd, J = 1.5, 7.1 Hz, 2 H, H2ɂ, H6ɂ), 7.56 (ddd, J = 1.5, 7.1, 7.1 Hz, 1 H, H4ɂ), 7.48

(ddd, J = 1.5, 7.1, 7.1 Hz, 2 H, H3ɂ, H5ɂ), 7.36 (s, 1 H, H4), 7.10 (d, J = 8.2 Hz, H7),

6.80 (dd, J = 1.4, 8.2 Hz, 1 H, H6), 3.53 (s, 2 H, CH 2), 2.37 (s, 3 H, CH 3), 2.36 (s, 3 H,

13 CH 3). C NMR (125 MHz, d 6-DMSO): δ 170.6 (C=O), 165.9 (C=O), 133.8 (ArC),

133.7 (ArC), 133.0 (ArC), 132.2 (ArCH), 129.2 (ArC), 128.9 (ArCH), 127.9 (ArCH),

126.8 (ArC), 121.9 (ArCH), 118.3 (ArCH), 110.4 (ArCH), 104.2 (ArC), 30.0 (CH 2),

21.8 (CH 3), 12.0 (CH3).

2-((2-Methyl-1H-indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole (210a)

This compound was synthesized as described for compound 179 using hydrazide 209a (0.14 g, 0.47 mmol),

DIPEA (0.20 mL, 1.15 mmol) and 4-TsCl (0.28 g, 1.50 mmol) in MeCN (6.0 mL) over 2 h to give the title compound 210a as an off-white

1 solid after purification (0.10 g, 75%). M.p. 125-127 ºC. H NMR (300 MHz, d 6- acetone): δ 10.12 (bs, 1 H, NH), 7.95 (dd, J = 1.7, 8.1 Hz, 2 H, H2ɂ, H6ɂ), 7.59 (dd, J =

2.6, 5.6 Hz, 1 H, H7), 7.56-7.49 (m, 3 H, H3ɂ, H4ɂ, H5ɂ), 7.29 (dd, J = 2.6, 8.3 Hz, 1 H,

13 H4), 7.07-6.98 (m, 2 H, H5, H6), 4.39 (s, 2 H, CH 2), 2.53 (s, 3 H, CH 3). C NMR (75

MHz, d 6-DMSO): δ. 167.1 (C=N), 165.2 (C=N), 143.2 (ArC), 142.4 (ArC), 136.5

(ArC), 134.1 (ArC), 132.3 (ArC), 130.0 (ArCH), 129.2 (ArCH), 127.2 (ArCH), 125.1

(ArC), 121.6 (ArCH), 119.8 (ArCH), 118.3 (ArCH), 111.4 (ArCH), 104.4 (ArC), 32.3

-1 (CH2), 11.5 (CH3). IR (ATR ): ν max 3353, 3256, 1548, 1447 cm . UV-Vis (MeOH): λ max

252 (ε 13900 cm -1M-1), 223 (31900), 201 (32600) nm. HRMS (+ESI): Found m/z

+ 290.1286, [M+H] ; C 18 H16 N3O required 290.1288.

243

2-((5-Bromo-2-methyl-1H-indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole (210b)

This compound was synthesized as described for compound 179 using hydrazide 209b (0.24 g, 0.64 mmol), DIPEA (0.20 mL, 1.15 mmol) and 4-TsCl (0.28 g, 1.47 mmol) in MeCN (6.0 mL) over 2 h to give the title compound 210b as a light

1 brown solid after purification (0.19 g, 81%). M.p. 105-107 ºC. H NMR (300 MHz, d 6- acetone): δ 10.33 (bs, 1 H, NH), 7.98 – 7.94 (m, 2 H, H2ɂ, H6ɂ), 7.79 (d, J = 1.9 Hz, 1 H,

H), 7.58 – 7.52 (m, 3 H, H3ɂ, H4ɂ, H5ɂ), 7.27 (dd, J = 0.3, 8.5 Hz, 1 H, H), 7.15 (dd, J =

13 1.9, 8.5 Hz, 1 H, H), 4.40 (s, 2 H, CH 2), 2.53 (s, 3 H, CH 3). C NMR (75 MHz, d 6- acetone): δ 166.7 (ArC), 165.3 (ArC), 136.2 (ArC), 135.2 (ArC), 132.4 (ArCH), 131.1

(ArC), 130.1 (ArCH), 127.3 (ArCH), 125.1 (ArC), 124.2 (ArCH), 121.0 (ArCH), 113.2

(ArCH), 112.8 (ArC), 104.4 (ArC), 21.1 (CH2), 11.6 (CH3). IR (ATR ): ν max 3295, 3195,

-1 -1 -1 3013, 1707, 1602, 1558, 1475 cm . UV-Vis (MeOH): λ max 252 (ε 31,400 cm M ), 226

(33,400), 202 (56,500) nm. HRMS (+ESI): Found m/z 390.0216, [M+Na] +;

C18 H14 BrN 3ONa required 390.0213.

2-((2, 5-Dimethyl-1H-indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole (210c)

This compound was synthesized as described for compound 179 using hydrazide 209c (30.3 mg, 0.09 mmol), DIPEA (0.05 mL, 0.29 mmol) and 4-TsCl (28.0 mg, 0.15 mmol) in MeCN (2.0 mL) over 2 h to give the title compound 210c as an off-

1 white solid after purification (13.4 mg, 49%). M.p. 200-202 ºC. H NMR (300 MHz, d 6- acetone): δ 9.97 (bs, 1 H, NH), 7.97 – 7.94 (m, 2 H, H2, H6), 7.55 – 7.53 (m, 3 H,

H3, H4, H5), 7.38 (ddd, J = 0.8 Hz, 1 H, H), 7.17 (d, J = 8.2 Hz, 1 H, H), 6.87 (dd, J 244

13 = 1.4, 8.2 Hz, 1 H, H), 4.35 (s, 2 H, CH 2), 2.51 (s, 3 H, CH 3), 2.39 (s, 3 H, CH 3). C

NMR (75 MHz, d 6-acetone): δ 167.1 (C=N), 165.2 (C=N), 134.9 (ArC), 132.2 (ArCH),

130.0 (ArCH), 128.6 (ArC), 127.2 (ArCH), 125.2 (ArC), 123.1 (ArCH), 118.2 (ArCH),

111.1 (ArCH), 104.0 (ArC), 21.7 (CH 3), 21.3 (CH 2), 11.5 (CH 3). IR (ATR ): ν max 3274,

-1 -1 -1 1566, 1448 cm . UV-Vis (MeOH): λ max 250 (ε 41,600 cm M ), 225 (63,400), 204

+ (72,000) nm. HRMS (+ESI): Found m/z 304.1443, [M+H] ; C 19 H18 N3O required

304.1444.

2-((1 H-Indol-3-yl)methyl)-5-(4-fluorophenyl)-1, 3, 4-oxadiazole (212a)

This compound was synthesized as described for compound 198 using hydrazide 196 (0.19 g, 1.01 mmol) and acid 211a (0.15 g, 1.04 mmol) in POCl 3

(3.0 mL) over 2 h to give the title compound 209a as a pale yellow solid after

1 purification (0.09 g, 29%). M.p. 157-159 ºC. H NMR (300 MHz, d 6-Acetone): δ 10.26

(bs, 1 H, NH), 8.03 (dddd, J = 5.3, 9.1 Hz, 2 H, H), 7.69 (ddd, J = 0.6, 7.8 Hz, 1 H, H),

7.43 (m, 1 H, H2), 7.41 (ddd, J = 1.1, 5.0 Hz, 1 H, H), 7.33 (dddd, J = 2.0, 8.9 Hz, 2 H,

H), 7.13 (ddd, J = 1.2, 7.1 Hz, 1 H, H), 7.06 (ddd, J = 1.2, 7.1 Hz, 1 H, H), 4.46 (d, J =

13 0.8 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-Acetone): δ 167.0 (C2, C5), 137.7 (ArC),

129.9 (ArCH), 129.8 (ArCH), 124.7 (ArCH), 122.3 (ArCH), 120.0 (ArCH), 119.3

(ArCH), 117.3 (ArCH), 117.0 (ArCH), 112.4 (ArCH), 108.6 (C3ɂ), 22.6 (CH 2). IR

-1 (ATR): ν max 3383, 3250, 3063, 1601, 1569, 1493 cm . UV-Vis (MeOH): λ max 252 (ε

25,000 cm -1 M-1), 218 (43,700), 204 (43,900) nm. HRMS (+ESI): Found m/z 294.1034,

+ [M+H] ; C 17 H13 FN 3O required 294.1037.

245

2-((1 H-Indol-3-yl)methyl)-5-(p-tolyl)-1, 3, 4-oxadiazole (212b)

This compound was synthesized as described for compound 198 using hydrazide 196 (0.14 g, 0.76 mmol) and acid 211b (0.13 g, 0.93 mmol) in POCl 3 (3.0 mL) over 2 h to give the title compound 212b as an off-white solid following purification (0.08 g, 36%). M.p. 150-

319 1 152 ºC, lit M.p. 147-149 °C. H NMR (300 MHz, d 6-acetone): δ 10.25 (bs, 1 H, NH),

7.86 (dd, J = 1.8, 8.2 Hz, 2 H, H), 7.69 (ddd, J = 0.6, 7.8 Hz, 1 H, H), 7.43 (d, J = 0.7

Hz, 1 H, H2), 7.41 (ddd, J = 0.9, 4.5 Hz, 1 H, H), 7.35 (dd, J = 1.8, 8.2 Hz, 2 H, H),

7.13 (ddd, J = 1.42, 7.1 Hz, 1 H, H), 7.06 (ddd, J = 1.2, 7.1 Hz), 4.45 (d, J = 0.9 Hz, 2

13 H, CH 2), 2.39 (s, 3 H, CH 3). C NMR (75 MHz, d 6-DMSO): δ. IR (ATR ): ν max 3278,

-1 -1 -1 1610, 1571, 1494 cm . UV-Vis (MeOH): λ max 258 (ε 47,700 cm M ), 210 (74,800)

+ nm. HRMS (+ESI): Found m/z 290.1285, [M+H] ; C 18 H16 N3O required 290.1288.

2-((1 H-Indol-3-yl)methyl)-5-(4-(trifluoromethyl)phenyl)-1, 3, 4-oxadiazole (212c)

This compound was synthesized as described for compound 198 using hydrazide 196 (0.20 g, 1.08 mmol) and acid 211c (0.19 g, 1.01 mmol) in POCl 3

(3.0 mL) over 2 h to give the title compound 212c as an off-white solid after

1 purification (0.10 g, 28%). M.p. 181-183 ºC. H NMR (300 MHz, d 6-Acetone): δ 10.26

(bs, 1 H, NH), 8.20 (ddd, J = 0.8, 8.9 Hz, 2 H, H2, H6), 7.91 (ddd, J = 0.7, 8.9 Hz, 2

H, H3, H5), 7.70 (ddd, J = 0.7, 7.8 Hz, 1 H, H4ɂ), 7.44 (d, J = 0.8 Hz, 1 H, H2ɂ), 7.42

(ddd, J = 0.8, 8.1 Hz, 1 H, H7ɂ), 7.14 (ddd, J = 0.4, 1.3, 7.2 Hz, 1 H, H6ɂ), 7.07 (ddd, J =

13 1.2, 7.8 Hz, 1 H, H5ɂ), 4.50 (d, J = 0.9 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-Acetone):

δ 166.8 (C2, C5), 136.8 (C7aɂ, C4), 127.1 (C2, C6), 126.2 (C3, C5, CF 3), 123.9 246

(C2ɂ), 121.7 (C6ɂ), 119.1 (C5ɂ), 118.4 (C4ɂ), 111.5 (C7ɂ), 107.6 (C3ɂ), 21.7 (CH 2). IR

-1 -1 -1 (ATR ): ν max 3307, 1559, 1457 cm . UV-Vis (MeOH): λ max 254 (ε 51,000 cm M ), 218

+ (83,400), 204 (73,800) nm. HRMS (+ESI): Found m/z 344.1001, [M+H] ; C 18 H13 F3N3O required 344.1005.

2-((1 H-Indol-3-yl)methyl)-5-(3-(trifluoromethyl)phenyl)-1, 3, 4-oxadiazole (212d)

This compound was synthesized as described for compound 198 using hydrazide 196 (0.19 g, 1.00 mmol) and acid 211d (0.20 g, 1.03 mmol) in POCl 3

(5.0 mL) at 80 °C over 3 h to give the title compound 212d as an off-white solid after

1 purification (0.07 g, 22%). M.p. 158-160 ºC. H NMR (300 MHz, d 6-DMSO): δ 11.06

(bs, 1 H, NH), 8.23 (d, J = 7.8 Hz, 1 H, H6), 8.17 (s, 1 H, H2), 7.99 (dd, J = 0.9, 7.9

Hz, 1 H, H4), 7.83 (dd, J = 7.9 Hz, 1 H, H5), 7.61 (dd, J = 7.9 Hz, 1 H, H4ɂ), 7.42 (d,

J = 2.7 Hz, 1 H, H2ɂ), 7.38 (dd, J = 0.9, 7.9 Hz, 1 H, H7ɂ), 7.10 (ddd, J = 0.9, 7.9 Hz, 1

13 H, H6ɂ), 7.02 (ddd, J = 0.9, 7.9 Hz, 1 H, H5ɂ), 4.46 (d, J = 0.53 Hz, 2 H, CH 2). C NMR

(75 MHz, d 6-DMSO): δ 166.6 (C2), 162.9 (C5), 136.2 (C7aɂ), 130.9 (C5), 130.3 (C6),

128.3 (C3, C4), 126.7 (C4aɂ), 124.5 (CF 3), 124.3 (C2ɂ), 122.7 (C1, C2), 121.4 (C6ɂ),

118.8 (C5ɂ), 118.3 (C4ɂ), 111.6 (C7ɂ), 106.7 (C3ɂ), 21.3 (CH 2). IR (ATR ): ν max 3223,

-1 -1 -1 1646, 1438 cm . UV-Vis (MeOH): λ max 251 (ε 28,600 cm M ), 207 (54,100) nm.

+ HRMS (+ESI): Found m/z 344.1004, [M+H] ; C 18 H13 F3N3O required 344.1005.

247

2-((1 H-indol-3-yl)methyl)-5-(2-(trifluoromethyl)phenyl)-1, 3, 4-oxadiazole (212e)

This compound was synthesized as described for compound 198 using hydrazide 196 (0.20 g, 1.06 mmol) and acid 211e (0.29 g, 1.53 mmol) in POCl 3 (5.0 mL) at

80 °C over 3 h to give the title compound 212d as an off-white solid after purification

1 (0.08 g, 22%). M.p. 148-150 ºC. H NMR (300 MHz, d 6-DMSO): δ 11.05 (bs, 1 H,

NH), 7.99-7.96 (m, 2 H, H3, H6), 7.88 -7.84 (m, 2 H, H4, H5), 7.55 (ddd, J = 0.9,

8.0 Hz, 1 H, H4ɂ), 7.37 (ddd, J = 0.9, 8.0 Hz, 1 H, H7ɂ), 7.36 (d, J = 2.6 Hz, 1 H, H2ɂ),

7.10 (ddd, J = 1.2, 8.0 Hz, 1 H, H6ɂ), 7.00 (ddd, J = 1.2, 8.0 Hz, 1 H, H5ɂ), 4.45 (d, J =

13 0.7 Hz, 2 H, CH 2). C NMR (75 MHz, d 6-DMSO): δ 167.1 (C=N), 162.1 (C=N), 136.2

(ArC), 133.2 (ArCH), 132.3 (ArCH), 131.8 (ArCH), 127.2 (ArC), 127.1 (ArC), 127.0

(ArC), 126.6 (ArC), 124.1 (ArCH), 121.3 (ArCH), 118.7 (ArCH), 118.2 (ArCH), 111.6

-1 (ArCH), 106.6 (ArC), 21.4 (CH 2). IR (ATR ): ν max 3285, 1569 cm . UV-Vis (MeOH):

-1 -1 λmax 271 (ε 9200 cm M ), 218 (41400), 204 (40000) nm. HRMS (+ESI): Found m/z

+ 366.0819, [M+Na] ; C 18 H12 F3N3ONa required 366.0825.

248

N-(Benzo[d]thiazol-2-yl)-3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5- carboxamide (228)

To a mixture of acid 252b (0.11 g, 0.43 mmol) and 2- aminobenzothiazole 106 0.07 g, 0.48 mmol) in DMF

(2.0 mL) were added EDCI (0.12 g, 0.66 mmol) and

HOBt (0.15 g, 1.09 mmol). The mixture was stirred for

24 h before being poured into ice-water and the precipitate collected via filtration. The crude product was purified by flash column chromatography (70% EtOAc:hexanes) to give the title compound 228 as a white solid (0.05 g, 31%). M.p. 252-253 °C. 1H NMR

(300MHz, d 6-DMSO): δ 13.16 (bs, 1 H, NH), 8.45 (s, 1 H, H4), 8.00 (d, J = 8.0 Hz, 1

H, H4), 7.78 -7.75 (m, 3 H, H2ɂ, H6ɂ, H7), 7.61 (ddd, J = 1.9, 2.1, 7.2 Hz, 2 H, H3ɂ,

H5ɂ), 7.47 (ddd, J = 1.3, 7.4, 7.4 Hz, 1 H, H6), 7.38 -7.31 (m, 2 H, H4ɂ, H5), 2.55 (s, 3

13 H, CH 3). C NMR (75 MHz, d 6-DMSO): δ 162.2 (C=O), 144.5 (ArC), 142.7 (ArC),

138.5 (ArC), 131.1 (ArC), 130.1 (ArCH), 126.3 (ArCH), 125.9 (ArCH), 123.7 (ArCH),

121.9 (ArCH), 121.3 (ArC), 117.1 (ArCH), 12.7 (CH 3). IR (ATR): ν max 3371, 3055,

2920, 2786, 1636, 1538, 1505, 1438, 1375, 1297, 1100, 819, 747, 681 cm -1. UV-Vis

-1 -1 (MeOH): λ max 336 (26,400 M cm ), 270 (21,700), 214 (37,400) nm. HRMS (+ESI) m/z

+ Calcd. for C 20 H15 N4OS 2 (M+H) 391.0682. Found 391.0678.

249

N-(Benzo[d]thiazol-2-yl)-1,3-dimethyl-1H-thieno[2,3-c]pyrazole-5-carboxamide

(229)

This compound was prepared as described for compound

228 using acid 232a (95.4 mg, 0.49 mmol), 2- aminobenzothiazole 106 (96.5 mg, 0.64 mmol), EDCI

(124.1 mg, 0.65 mmol) and HOBt (105.1 mg, 0.66 mmol) in DMF (1.0 mL) over 20 h.

Following recrystallization from MeCN the title compound 229 was obtained as a white

1 solid (142.1 mg, 88%). M.p. 180 °C (dec). H NMR (300MHz, d 6-DMSO): δ 13.38 (bs,

1 H, NH), 7.98 (ddd, J = 0.9, 0.9, 8.5 Hz, 1 H, H4ɂ), 7.75 (s, 1 H, H5), 7.71 (d dd, J =

0.9, 0.9, 8.5 Hz, 1 H, H7ɂ), 7.54 (ddd, J = 0.9, 8.5, 8.5 Hz, 1 H, H6ɂ), 7.41 (ddd, J = 0.9,

13 8.5, 8.5 Hz, 1 H, H5ɂ), 3.86 (s, 3 H, NCH 3), 2.36 (s, 3 H, CH 3). C NMR (75 MHz, d 6- acetone): δ 164.3 (C=O), 146.5 (ArC), 141.6 (ArC), 130.8 (ArC), 128.6 (ArC), 128.1

(ArC), 127.6 (ArCH), 124.8 (ArCH), 123.7 (ArCH), 119.4 (ArCH), 109.9 (ArCH), 38.1

(NCH 3), 12.78 (CH3). IR (ATR): ν max 3124, 3055, 2933, 1651, 1523, 1421, 1379, 1342,

-1 -1 -1 1254, 1008, 789, 767, 697 cm . UV-Vis (MeOH): λ max 297 (24,300 M cm ), 214

+ (48,200) nm. HRMS (+ESI): Found m/z 329.0526, [M+H] ; C 15 H13 N4OS 2 required

329.0525.

5-Chloro-1,3-dimethyl-1H-pyrazole-4-carbaldehyde (230a)

A Vilsmeier complex was prepared by the dropwise addition of POCl 3 (5.0 mL) to DMF (5.0 mL) at 0 °C. This mixture was stirred at 0 °C for 15 min before a solution of dimethyl pyrazolone 233c (3.60 g, 32.12 mmol) in

DMF (2.0 mL) was added dropwise. The mixture was heated at reflux for 3 h, cooled and poured over crushed ice. The solution was neutralized with sat. K 2CO3, the 250

precipitate collected via filtration and recrystallized from EtOH to give the title compound 230a as colourless prisms (0.34 g, 7%). M.p. 79-80 °C. Lit. 359 M.p. 78-79

1 °C. H NMR (300MHz, CDCl 3): δ 9.83 (s, 1 H, CHO), 3.80 (s, 3 H, NCH 3), 2.43 (s, 3

13 H, CH 3). C NMR (75 MHz, CDCl 3): δ 183.5 (CHO), 150.9 (ArC), 133.7 (ArC), 116.4

(ArC), 36.0 (NCH 3), 13.8 (CH3).

5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (230b)

This compound was prepared as described for compound 230a using pyrazolone 233d (1.03 g, 5.92 mmol) and POCl 3 (5.0 mL) in DMF (5.2 mL) over 3 h to give the title compound 230b as colourless prisms after purification (0.60 g, 46%). M.p. 170-172 °C, Lit. 401 M.p. 172-173 °C. 1H

NMR (300 MHz, CDCl 3): δ 9.98 (s, 1 H, CHO), 7.59-7.47 (m, 5 H, ArH), 2.55 (s, 3 H,

13 CH 3). C NMR (75 MHz, CDCl 3): δ 184.0 (CHO), 151.9 (ArC), 137.1 (ArC), 129.4

(ArCH), 129.3 (ArCH), 125.4 (ArCH), 117.6 (ArC), 14.0 (CH 3).

5-Chloro-1,3-diphenyl-1H-pyrazole-4-carbaldehyde (230c)

This compound was prepared as described for compound 233e using pyrazolone 233e (1.60 g, 6.78 mmol) and POCl 3 (5.0 mL) in DMF

(5.0 mL) over 1 h to give the title compound 230c as colourless prisms following purification (0.57 g, 30%). M.p. 107-109 °C, Lit. 402 M.p.

1 107.0-108.5 °C. H NMR (300MHz, CDCl 3): δ 10.07 (s, 1 H, CHO), 251

7.84-7.81 (m, 2 H, H), 7.66-7.62 (m, 2 H, H), 7.56-7.47 (m, 6 H, H); 13 C NMR (75

MHz, CDCl 3): δ 184.1 (CHO), 130.9 (ArC), 129.7 (ArC), 129.7 (ArCH), 129.5 (ArCH),

129.1 (ArCH), 128.7 (ArCH), 125.6 (ArCH), 116.6 (ArC).

1,3-Dimethyl-1H-thieno[2,3-c]pyrazole-5-carboxylic acid (232a)

A solution of ester 234b (0.34 g, 2.16 mmol) in THF (5.0 mL) was treated with 2 M KOH (5.0 mL, 10.0 mmol) and the mixture stirred for 3 h before being concentrated under reduced pressure and acidified with 2 M HCl. The precipitate was collected via filtration to give the title compound 232a as a white solid (0.21 g, 51%). M.p. 314-316 °C. Lit.359 M.p. 316-317

1 °C. H NMR (300MHz, d 6-DMSO): δ 7.73 (s, 1 H, H5), 3.85 (s, 3 H, NCH 3), 2.35 (s, 3

13 H, CH 3). C NMR (75 MHz, d 6-DMSO): δ 164.1 (C=O), 146.3 (ArC), 141.4 (ArC),

130.8 (ArC), 128.4 (ArC), 123.4 (ArCH), 37.82 (NCH 3), 12.55 (CH3).

3-Methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carboxylic acid (232b)

This compound was synthesized as described for compound 232a using ester 234c (1.95 g, 7.17 mmol) and 2 M KOH (25.0 mL) in

THF (25.0 mL) over 2 h to give the title compound 232b as a white solid (1.44 g, 78%). M.p. 241-242 °C. Lit.359 249-250 °C.

1 H NMR (300MHz, d 6-acetone): δ 7.88 (s, 1 H, H5), 7.80 (dd, J = 1.2, 8.8 Hz, 2 H, H2ɂ,

H6ɂ), 7.60 (dd, J = 7.4, 8.8 Hz, 2 H, H3ɂ, H5ɂ), 7.33 (dd, J = 7.4 Hz, 1 H, H4ɂ), 2.55 (s, 3

13 H, CH 3). C NMR (75 MHz, d 6-acetone): δ 164.0 (C=O), 145.3 (ArC), 144.2 (ArC),

140.1 (ArC), 132.1 (ArC), 131.6 (ArC), 130.7 (ArCH), 126.5 (ArCH), 123.8 (ArCH), 252

118.1 (ArCH), 12.9 (CH3). IR (ATR): ν max 2822, 2661, 2538, 1654, 1592, 1502, 1420,

-1 -1 -1 1300, 1277, 1166 cm . UV-Vis (MeOH): λ max 286 (30,100 M cm ), 258 (28,000) nm.

+ HRMS (+ESI): Found m/z 259.0532, [M+H] ; C 13 H11 N2O2S required 259.0536.

1,3-Diphenyl-1H-thieno[2,3-c]pyrazole-5-carboxylic acid (232c)

This compound was synthesized as described for compound 232a using ester 234d (320.1 mg, 0.96 mmol) and 2 M KOH (20.0 mL) in THF (20.0 mL) over 2 h to give the title compound as a white solid (235.2 mg, 77%). M.p. 282-284 °C. Lit.359 M.p. 282-283 °C.

1 H NMR (300 MHz, d 6-DMSO): δ 8.14 (s, 1 H, H4), 8.11 (dd, J =

1.5, 7.1 Hz, 2 H, H2ɂ, H6ɂ), 7.88 (dd, J = 1.2, 8.8 Hz, 2 H , H2, H6), 7.64 (ddd, J = 2.0,

7.6, 7.6 Hz, 2 H, H3ɂ, H5ɂ), 7.54 (ddd, J = 1.6, 7.0, 7.0 Hz, 2 H, H3, H5), 7.45 (ddd, J

= 2.2, 7.3, 7.3 Hz, 1 H, H4ɂ), 7.39 (ddd, J = 1.0, 7.5, 7.5 Hz, 1 H, H4). 13 C NMR (75

MHz, d 6-DMSO): δ 164.1 (C=O), 144.9 (ArC), 143.6 (ArC), 138.7 (ArC), 131.6 (ArC),

130.1 (ArCH), 129.2 (ArCH), 129.0 (ArCH), 128.1 (ArC), 126.4 (ArCH), 126.3

(ArCH), 121.5 (ArCH), 117.7 (ArCH). IR (ATR): ν max 3379, 3053, 1657, 1594, 1506,

-1 -1 -1 1457, 1371, 1297, 1280, 748, 683 cm . UV-Vis (MeOH): λ max 304 (28,200 M cm ),

271 (22,500), 203 (39,100) nm. HRMS (+ESI): Found m/z 321.0691, [M+H] +;

C18 H13 N2O2S required 321.0692.

253

5-Methyl-2,4-dihydro-3H-pyrazol-3-one (233a)

Hydrazine monohydrate (5.0 mL, 65.8 mmol) was added dropwise to a solution of ethyl acetoacetate 246a (6.60 mL, 52.1 mmol) in EtOH (15.0 mL) and the mixture stirred for 16 h. The precipitate was collected via filtration, washed with EtOH and air dried to give the title compound 233a as a white

403 1 solid (3.80 g, 74%). M.p. 222-224 °C. Lit. M.p. 220-224 °C. H NMR (300 MHz, d6-

DMSO): δ 10.29 (bs, 1 H, NH), 5.21 (d, J = 0.7 Hz, 2 H, CH 2), 2.08 (d, J = 0.6 Hz, 3 H,

CH 3).

2,5-Dimethyl-2,4-dihydro-3H-pyrazol-3-one (233c)

Methyl hydrazine 245 (6.20 mL, 117.8 mmol) was added dropwise to ethyl acetoacetate 246a (15.0 mL, 118.5 mmol) and the mixture heated at reflux for 2 h. The mixture was cooled on ice and Et 2O added. The precipitate was collected via filtration, washed with Et 2O and air dried to give the title compound 233c as a yellow solid (9.59 g, 72%). M.p. 115-117 °C. Lit. 363 117 °C. 1H NMR (300 MHz,

13 CDCl 3): δ 3.25 (s, 3 H, NCH 3), 3.16 (s, 2 H, CH 2), 2.07 (s, 3 H, CH 3). C NMR (75

MHz, CDCl 3): δ 172.4 (C=O), 155.7 (C=N), 41.6 (CH 2), 31.14 (NCH 3), 17.0 (CH3).

254

5-Methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (233d)

This compound was prepared as described for compound 233c using phenyl hydrazine 164 (20.0 mL, 0.20 mol) and ethyl acetoacetate 246a (21.0 mL,

0.17 mol) over 2 h to give the title compound 233d as a colourless solid

(12.51 g, 69%). M.p. 122-124 °C. Lit. 365 M.p. 125-126 °C. 1H NMR (300

MHz, CDCl 3): δ 7.85 (dd, J = 1.2, 8.9 Hz, 2 H , H2ɂ, H6ɂ), 7.38 (ddd, J = 2.0, 2.0, 7.9

Hz, 2 H, H3ɂ, H5ɂ), 7.17 (ddd, J = 1.2, 1.2, 7.0 Hz, 1 H, H4ɂ), 3.40 (s, 2 H, CH 2), 2.17 (s,

13 3 H, CH 3). C NMR (75 MHz, CDCl 3): δ 170.7 (C=O), 156.4 (ArC), 138.2 (ArC),

128.9 (ArCH), 125.1 (ArCH), 119.0 (ArCH), 43.2 (CH 2), 17.1 (CH3).

2,5-Diphenyl-2,4-dihydro-3H-pyrazol-3-one (233e)

This compound was prepared as described for compound 233c using phenyl hydrazine 164 (9.1 mL, 0.92 mol) and ethyl benzoylacetate

246b (10.0 mL, 0.93 mol) over 2 h to give the title compound 233e as a colourless solid (10.62 g, 49%). M.p. 127-129 °C. Lit. 364 M.p. 129.5-

1 132 °C. H NMR (300 MHz, CDCl 3): δ 7.98 (ddd, J = 1.0, 1.0, 8.0 Hz,

2 H, H2′, H6′), 7.77 (ddd, J = 1.4, 1.6, 7.4 Hz, 2 H, H2″, H6″), 7.48 -7.40 (m, 5 H, H3′,

13 H4′, H5′, H3″, H5″), 7.22 (ddd, J = 1.6, 7.4, 7.4 Hz, 1 H, H4″), 3.84 (s, 2 H, CH 2). C

NMR (75 MHz, CDCl 3): 170.4 (C=O), 154.8 (C=N), 138.3 (ArC), 131.0 (ArC), 130.8

(ArCH), 129.1 (ArCH), 129.0 (ArCH), 126.1 (ArCH), 125.4 (ArCH), 119.2 (ArCH),

39.8 (CH 2).

255

2-Isobutyl-5-methyl-2,4-dihydro-3H-pyrazol-3-one (233f)

A suspension of hydrochloride 253c (1.25 g, 10.0 mmol) in MeOH:H 2O

(10:1, 11 mL) was treated with KOH (0.59 g, 10.5 mmol) and the mixture stirred for 15 min before ethyl acetoacetate 246a (1.32 mL, 10.42 mmol) was added. This mixture was heated at 60 °C for 18 h, the solvent removed under reduced pressure and the residue extracted with EtOAc by Soxhlet apparatus. The solvent was removed under reduced pressure and the residue purified by flash column chromatography (50% EtOAc:hexanes) to give the title compound 233f as an off-white

1 solid (0.62 g, 40%). M.p. 119-121 °C. H NMR (300 MHz, CDCl 3): δ 3.43 (d, J = 7.2

Hz, 2 H, NCH 2), 3.21 (s, 2 H, CH 2), 2.10 (s, 3 H, CH 3), 2.08 (non, J = 7.2 Hz, 1 H, CH),

0.92 (d, J = 7.2 Hz, 6 H, 2 x CH 3). IR (ATR): ν max 2959, 2869, 1759, 1537, 1446, 1387,

-1 -1 -1 1290, 1170, 1039, 740 cm . UV-Vis (MeOH): λ max 246 (7,760 M cm ) nm. HRMS

+ (+ESI): Found m/z 155.1178, [M+H] ; C 8H15 N2O required 155.1179.

Methyl 1,3-dimethyl-1H-thieno[2,3-c]pyrazole-5-carboxylate (234b)

To a solution of methyl thioglycolate (0.80 mL, 8.95 mmol) and

Na 2CO 3 (139.0 mg, 1.31 mmol) in MeCN (10.0 mL) was added pyrazole-4-carbaldehyde 230a (601.1mg, 3.80 mmol). The mixture was heated at reflux for 3 h, cooled to r.t. and the precipitate collected via filtration. The crude solid obtained was purified by flash column chromatography (25%

EtOAc:hexanes) to give the title compound 234b as a white solid (341.5 mg, 24%).

1 M.p. 97-99 °C. H NMR (300MHz, d 6-DMSO): δ 7.85 (s, 1 H, H4), 3.87 (s, 3 H,

13 NCH 3), 3.82 (s, 3 H, OCH 3), 2.37 (s, 3 H, CH 3). C NMR (75 MHz, d 6-DMSO): δ 162.9

(C=O), 146.4 (ArC), 141.6 (ArC), 128.4 (ArC), 128.3 (ArC), 124.2 (ArCH), 52.3 256

(OCH 3), 37.8 (NCH 3), 12.5 (CH 3). IR (ATR): ν max 3393, 2920, 1710, 1537, 1492, 1422,

-1 1376, 1275, 1240, 1155, 1061, 951, 985, 693 cm . UV-Vis (MeOH): λ max 292 (12,400

M-1cm -1), 232 (6,280), 208 (13,100) nm. HRMS (+ESI): Found m/z 211.0534, [M+H]+;

C9H11 N2O2S required 211.0536.

Methyl 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carboxylate (234c)

This compound was prepared as described for compound 234b using methyl thioglycolate (1.80 mL, 20.10 mmol), Na 2CO 3

(2.30 g, 21.70 mmol) and pyrazole-4-carbaldehyde 230b (1.99 g,

9.04 mmol) in MeCN (30.0 mL) over 3 h. The title compound

234c, was obtained as a white solid (2.04 g, 83%) following purification. M.p. 136-138

1 °C. H NMR (300 MHz, d 6-acetone): δ 7.88 (s, 1 H, H4), 7.80 (dd, J = 1.1, 8.5 Hz, 2 H,

H2ɂ, H6ɂ), 7.60 (ddd, J = 1.1, 8.5 Hz, 2 H, H3ɂ, H5ɂ), 7.33 (ddd, J = 1.1, 8.5 Hz, 1 H,

13 H4ɂ), 2.55 (s, 3 H, CH 3). C NMR (75 MHz, d 6-acetone): δ 164.0 (C=O), 145.3 (ArC),

144.2 (ArC), 140.1 (ArC), 132.1 (ArC), 131.6 (ArC), 130.7 (ArCH), 126.5 (ArCH),

123.8 (ArCH), 118.1 (ArCH), 12.9 (CH 3). IR (ATR): ν max 3401, 2946, 1711, 1591,

-1 1503, 1422, 1373, 1285, 1231, 1157, 1065, 998, 743, 668 cm . UV-Vis (MeOH): λ max

302 (15,100 M-1cm -1), 263 (22,700), 210 (17,700) nm. HRMS (+ESI): Found m/z

+ 273.0690, [M+H] ; C14 H13 N2O2S required 273.0692.

257

Methyl 1,3-diphenyl-1H-thieno[2,3-c]pyrazole-5-carboxylate (234d)

Method A: This compound was synthesized as described for compound 234b using carbaldehyde 230c (0.30 g, 1.06 mmol), methyl thioglycolate (0.50 mL, 5.60 mmol) and K 2CO 3 (0.90 g,

6.50 mmol) in MeCN (10.0 mL) over 48 h to give the title compound as a white solid after purification (0.04 g, 10%).

Method B: To a solution of methyl thioglycolate (0.60 mL, 6.70 mmol) in THF (15.0 mL) at 0 °C was added NaH (0.31 g, 7.70 mmol). The mixture was stirred with cooling for 1 h before a solution of carbaldehyde 230c (1.05 g, 3.70 mmol) in THF (7.0 mL) was added dropwise. The mixture was stirred at r.t. for 2 h before being cooled to 0 °C and an additional portion of NaH (0.36 g, 9.0 mmol) was added. The mixture was stirred with cooling for 30 min and poured into an ice:EtOAc mixture (25.0 mL). The aqueous phase was extracted with EtOAc (20.0 mL) and the combined organic phase washed with H 2O (25.0 mL) and brine (25.0 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure. The crude solid was recrystallized from EtOH to give the title compound 234d as a white solid (1.59 g, 71%). M.p. 204-206 °C. Lit.404 M.p.

1 185-186 °C. H NMR (300 MHz, CDCl 3): δ 8.13 (s, 1 H, H4), 8.06 (dd, J = 1.5, 8.0 Hz,

2 H, H), 7.90 (dd, J = 1.1, 8.8 Hz, 2 H, H), 7.59-7.49 (m, 4 H, H 3ɂ, H5ɂ, H3, H5), 7.43

(ddd, J = 1.6, 7.3 Hz, 1 H, H), 7.33 (ddd, J = 1.3, 7.3 Hz, 1 H, H), 3.96 (s, 3 H, CH 3).

13 C NMR (75 MHz, CDCl 3): δ 163.4 (C=O), 146.2 (C), 144.9 (C), 139.4 (C1ɂ), 132.2

(C), 131.0 (C), 129.8 (CH), 129.1 (C), 128.7 (CH), 126.8 (CH), 126.3 (CH), 123.9 (C4),

118.2 (CH), 52.6 (CH 3). IR (ATR): ν max 3031, 2949, 1706, 1595, 1502, 1455, 1240,

-1 -1 -1 1184, 1070, 992, 744, 680 cm . UV-Vis (MeOH): λ max 305 (33,300 M cm ), 277

(38,400), 203 (47,300) nm. HRMS (+ESI): Found m/z 357.0667, [M+Na] +;

C19 H14 N2O2SNa required 357.0668. 258

5-Chloro-3-methyl-1-phenyl-1H-pyrazole (241b)

Pyrazolone 233d (5.02 g, 28.84 mmol) and POCl 3 (35.5 mL) were heated at reflux over 7 h, cooled and poured over crushed ice. The solution was made alkaline with sat. NaHCO 3, extracted with EtOAc (3 x 20 mL), the organic phase dried over Na 2SO 4 and the solvent removed under reduced pressure to give the title compound 241b as a colourless oil after purification (1.60 g,

361 1 29%). H NMR (300MHz, d 4-MeOD): δ 7.73 (dd, J = 1.5, 8.3 Hz, 2 H, H2ɂ, H6ɂ),

13 7.41-7.33 (m, 3 H, H3ɂ, H4ɂ, H5ɂ), 6.66 (s, 1 H, H4), 3.87 (s, 3 H, CH 3). C NMR (75

MHz, d 4-MeOD): δ 152.4 (ArC), 133.9 (ArC), 129.7 (ArCH), 129.3 (ArCH), 126.4

(ArCH), 102.9 (ArCH), 36.4 (CH 3).

5-Chloro-1,3-diphenyl-1H-pyrazole (242c)

This compound was prepared as described for compound 241b using pyrazolone 233e (7.86 g, 33.3 mmol) and POCl 3 (35.0 mL) to give the title compound 242c as a colourless solid (1.14 g, 13%). M.p. 54-56

405 1 °C. Lit. M.p. 55-57 °C. H NMR (300 MHz, d 6-acetone): δ 7.91

(dd, J = 1.5, 8.4 Hz, 2 H, H), 7.69 (dd, J = 1.5, 8.4 Hz, 2 H, H), 7.60

(ddd, J = 1.5, 7.2 Hz, 2 H, H), 7.52 (dd, J = 1.5, 7.2 Hz, 1 H, H), 7.43 (dd, J = 1.5, 7.2

Hz, 2 H, H), 7.38 (dd, J = 1.5, 7.2 Hz, 1 H, H) 7.02 (s, 1H, CH).

259

5-Chloro-4-iodo-1,3-dimethyl-1H-pyrazole (243a)

To a solution of chloro pyrazole 242a (0.64 g, 4.86 mmol) in glacial AcOH

(5.0 mL) was added iodic acid (0.18 g, 1.01 mmol) and this mixture stirred for 10 min before iodine (1.01 g, 3.97 mmol) was added. The mixture was heated at reflux for 4 h before sat. Na 2SO 3 was added to give a clear yellow solution.

The reaction was extracted with DCM (3 x 10 mL) and the organic phase washed with sat. NaHCO 3 (3 x 10 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure. The residue was recrystallized from MeOH to give the title compound 243a as colourless needles (0.69 g, 55%). M.p. 63-65 °C. Lit. 406 M.p. 64-65 °C. 1H NMR (300

13 MHz, CDCl 3): δ 3.85 (s, 3H, NCH 3), 2.23 (s, 3H, CH 3). C NMR (75 MHz, CDCl 3): δ

150.7 (ArC), 131.6 (ArC), 61.1 (ArC), 37.4 (NCH 3), 14.7 (CH3).

5-Chloro-4-iodo-3-methyl-1-phenyl-1H-pyrazole (243b)

This compound was prepared as described for compound 243a using chloro pyrazole 242b (0.26 g, 1.30 mmol), iodic acid (0.09 g, 0.48 mmol) and iodine (0.27 g, 1.10 mmol) in glacial AcOH (5.0 mL) over 4 h to give the title compound 243b as colourless needles following purification (0.12

361 1 g, 29%). M.p. 59-61 °C. Lit. M.p. 59-60 °C. H NMR (300 MHz, d 4-MeOH): δ 7.76

(dd, J = 1.9, 8.2 Hz, 2 H, H2′, H6′), 7.44 – 7.41 (m, 3 H, H3′, H4′, H5′), 3.97 (s, 3 H,

13 CH 3). C NMR (75 MHz, d 4-MeOD): δ 153.2 (ArC), 133.9 (ArC), 129.7 (ArCH),

129.3 (ArCH), 129.0 (ArCH), 59.6 (ArC), 37.9 (CH3).

260

5-Chloro-4-iodo-1,3-diphenyl-1H-pyrazole (243c)

This compound was prepared as described for compound 243a using chloro pyrazole 242c (1.13 g, 4.48 mmol), iodic acid (0.19 g, 1.09 mmol) and iodine (1.08 g, 4.24 mmol) in glacial AcOH (5.0 mL) over

4 h to give the title compound 243c as colourless needles following purification (1.01 g, 60%). M.p. 114-116 °C. 1H NMR (300MHz,

CDCl 3): δ 7.91 (ddd, J = 1.7, 8.2 Hz, 2 H, H2, H6), 7.62 (ddd, J = 1.6, 8.6 Hz, 2 H, H2ɂ,

13 H6ɂ), 7.52 -7.43 (m, 6 H, H3ɂ, H4ɂ, H5ɂ, H3, H4, H5). C NMR (75 MHz, CDCl 3): δ

62.3 (ArC), 125.1 (ArCH), 128.2 (ArCH), 128.5 (ArCH), 128.9 (ArCH), 129.0 (ArCH),

129.2 (ArCH), 132.4 (ArC), 132.6 (ArC), 138.7 (ArC), 153.1 (ArC). HRMS (+ESI):

+ Found m/z 380.9649, [M+H] ; C 15 H11 ClIN 2 required 380.9650.

1-Isobutyl-3-methyl-1H-pyrazol-5-ol (248b)

This compound was synthesized as described for compound 233f using hydrochloride salt 253b (2.50 g, 20.06 mmol), KOH (1.21g, 21.56 mmol) and ethyl acetoacetate 246a (2.80 mL, 22.18 mmol) in 10:1

MeOH:H 2O (11.0 mL) at 60 C for 24 h to give the title compound 248b as a white solid after purification (1.48 g, 79%). M.p. 121-123 °C. 1H NMR (300 MHz, d 6-DMSO): δ

10.61 (bs, 1 H, OH), 5.09 (s, 1 H, CH), 3.51 (d, J = 7.2 Hz, 2 H, CH 2), 2.00 (non, J =

13 7.2 Hz, 1 H, CH), 1.99 (s, 3 H, CH 3) 0.80 (d, J = 6.7 Hz, 6 H, 2 x CH 3). C NMR (75

MHz, d 6-DMSO): δ 156.3 (ArC), 145.1 (ArC), 85.1 (C4), 52.2 (CH 2) 28.5 (CH), 19.8 (2

x CH 3), 14.0 (CH 3). IR (ATR): ν max 2958, 2870, 2338, 1757, 1537, 1387, 1290, 1170,

-1 -1 -1 1039, 740 cm . UV-Vis (MeOH): λ max 246 (7,490 M cm ) nm. HRMS (+ESI): Found

+ m/z 155.1177, [M+H] ; C 8H15 N2O required 155.1179. 261

tert -Butyl ( E)-2-(2-methylpropylidene)hydrazine-1-carboxylate (251)

A solution of isobutyraldehyde 249 (1.6 mL, 17.5 mmol) in MeOH

(3.0 mL) was treated with tert -butylcarbazate 250 (2.34 g, 17.7 mmol) and the mixture stirred at r.t. for 20 h. The mixture was concentrated under reduced pressure, diluted with H 2O and stood for 5 h. The precipitate was collected via filtration to give the title compound 251 as colourless needles (2.43 g, 75%). The crude product was used in the next step without further purification. 1H NMR (300 MHz,

CDCl 3): δ 7.86 (s, 1 H, NH), 7.01 (d, J = 5.0 Hz, 1 H, C(N)H), 2.56 (oct, J = 7.0 Hz, 1

13 H, CH), 1.46 (s, 9 H, 3 x CH 3), 1.05 (d, J = 7.0 Hz, 2 x CH 3). C NMR (75 MHz,

CDCl 3): δ 152.6 (C=N), 152.4 (C=O), 81.0 ((CH 3)3C), 31.5 (CH), 28.3 ((CH3)3C), 30.0

(2 x CH 3).

Isobutylhydrazine hydrochloride (253a)

A solution of compound 251 (0.75 g, 4.03 mmol) in THF (4.0 mL) was treated with NaCNBH 3 (0.31 g, 4.93 mmol) in THF (4.0 mL) and pTsOH.H 2O (0.92g, 4.84 mmol) in THF (4.0 mL). The mixture was stirred at r.t. for 5 h before being made alkaline by the addition of 1 M NaOH (20.0 mL), extracted with EtOAc (50 mL), washed with sat. NaHCO 3 (20 mL) and brine (20.0 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure to give the intermediate amine 252 as a colourless oil (0.89 g, crude). Compound 252 was stirred in a solution of 4 M HCl in dioxane (15.0 mL) at r.t. for 4 h. The solvent was removed under reduced pressure to give the title compound 253a as a white solid (0.31 g, 62%

1 over 2 steps). H NMR (300 MHz, CDCl 3): δ 2.88 (d, J = 7.0 Hz, 2 H, CH 2), 1.99 (non,

J = 7.0 Hz, 1 H, CH), 1.01 (d, J = 7.0 Hz, 6 H, 2 x CH 3). 262

Isobutylhydrazine sulfate (253b)

Method A: A solution of tert -butylcarbazate 250 (3.94 g, 29.8 mmol) in iPrOH (25.0 mL) was treated with isobutyraldehyde

249 (2.70 mL, 29.6 mmol) in iPrOH (5.0 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, then at 40 °C for 16 h. Platinum on charcoal (5%, 0.20 g) was added and the mixture stirred under a H 2 atmosphere at 35 °C for 48 h. The mixture was cooled, filtered through a pad of Celite and concentrated under reduced pressure to circa 20 mL.

The mixture was cooled to 0 °C and sulfuric acid (18 M, 2.5 mL) added dropwise. The mixture was heated at 50 °C for 20 h, cooled to r.t., diluted with tBuOMe ether (20.0 mL) and stirred in an NH 4Cl salt-ice bath for 1 h. The precipitate was collected via filtration, washed with cold tBuOMe: iPrOH (4:1, 3 x 10 mL) and air dried to give the

1 title compound 253b as a white solid (1.58 g, 29%). H NMR (300 MHz, d 4-MeOD): δ

2.88 (d, J = 7.1 Hz, 2 H, CH 2), 1.98 (non, J = 7.1 Hz, 1 H, CH), 1.01 (d, J = 7.1 Hz, 6

H, 2 x CH 3).

Method B: The title compound 253b was synthesized as described in method A, using tert -butylcarbazate 250 (2.69 g, 21.7 mmol) and isobutyraldehyde 249 (2.20 mL, 24.1

i mmol) in PrOH (20.0 mL) at 0 °C under a H 2 (5 bar) atmosphere in a Parr apparatus over 72 h. Treatment with H 2SO 4 gave a white solid (0.43 g, 10%).

263

Isobutylhydrazine dihydrochloride (253c)

A solution of hydrazine carboxylate 255 (2.72 g, 11.9 mmol) in

THF (23.0 mL) was treated with 10 M HCl (2.5 mL, 25.0 mmol) and heated at reflux for 3 h. The solvent was removed under reduced pressure and the residue azeotroped with toluene (3 x 20 mL) to give the title compound 253c as a white

1 solid (1.40 g, 73%). H NMR (300 MHz, CDCl 3): δ 2.88 (d, J = 7.0 Hz, 2 H, CH 2), 1.99

(non, J = 7.0 Hz, 1 H, CH), 1.01 (d, J = 7.0 Hz, 6 H, 2 x CH 3).

tert -Butyl isopropylidene carbazate (254)

A solution of tert -butyl carbazate 250 (5.00 g, 37.9 mmol) in acetone (35.0 mL) was treated with MgSO 4 (1.10 g) followed by glacial AcOH (3 drops). The mixture was heated at reflux for 1 h, cooled to r.t., filtered and the solvent removed under reduced pressure to give the title compound 254 as a white solid (6.36 g, 98%). M.p. 101-103 °C. Lit. 407 M.p. 103-104 °C. 1H NMR (300

MHz, CDCl 3): δ 7.55 (bs, 1 H, NH), 1.90 (s, 3 H, CH 3), 1.72 (s, 3 H, CH 3), 1.38 (s, 9 H,

13 3 x CH 3). C NMR (75 MHz, CDCl 3): δ 152.9 (QC), 149.8 (QC), 80.6 ((CH 3)3CO),

28.1 (3 x CH 3), 25.2 (CH3), 16.0 (CH3).

tert-Butyl 1-isobutyl-2-(propan-2-ylidene)hydrazine-1-carboxylate (255)

To a solution of carbazate 254 (9.55 g, 55.5 mmol) in toluene (90.0 mL) was added freshly ground KOH (4.20 g, 74.9 mmol), followed by Bu 4NHSO 4 (1.94 g, 5.71 mmol) and the mixture heated to 50

°C. 1-Bromo-2-methylpropane (7.30 mL, 67.1 mmol) was added dropwise and the 264

mixture heated at 80 °C for 24 h, cooled to r.t. and washed with H 2O to give neutral washings. The organic phase was dried over Na 2SO 4 and concentrated under reduced pressure to give the title compound 255 as a yellow oil (9.99 g, 79%). 1H NMR (300

MHz, CDCl 3): δ 3.32 (d, J = 7.1 Hz, 2 H, CH 2), 2.03 (s, 3 H, CH 3), 1.85 (s, 3 H, CH 3),

1.74 (non, J = 6.9 Hz, 1 H, CH), 1.42 (s, 9 H, 3 x CH 3), 0.85 (d, J = 6.9 Hz, 6 H, 2 x

13 CH 3). C NMR (75 MHz, CDCl 3): δ 173.0 (C=N), 152.7 (C=O), 80.0 ((CH 3)3C), 57.7

(CH2), 28.3 (3 x CH 3), 27.4 (CH), 24.8 (CH 3), 20.2 (2 x CH 3), 19.8 (CH3). IR (ATR):

-1 νmax 2959, 1690, 1363, 1247, 1143, 1043, 879, 695 cm . UV-Vis (MeOH): 252 (ε 783

-1 -1 + M cm ) nm. HRMS (+ESI): Found m/z 251.1728, [M+Na] ; C 12 H24 N2O2Na required

251.1730.

N-(1 H-Benzo[d]imidazol-2-yl)-3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5- carboxamide (256a)

This compound was synthesized as described for compound 228 using acid 232a (0.26 g, 1.01 mmol), 2- aminobenzimidazole 139a (0.14 g, 1.07 mmol), EDCI

(0.23 g, 1.07 mmol) and HOBt (0.24 g, 1.55 mmol) in

DMF (4.0 mL) over 18 h to give the title compound 256a as a beige solid (0.09 g, 24%).

1 M.p. 322-324 °C. H NMR (300MHz, d 6-DMSO): δ 13.16 (bs, 1 H, NH), 12.44 (bs, 1

H, NH), 7.94 (s, 1 H, H4), 7.76 (dd, J = 1.1, 7.7 Hz, 2 H, H2ɂ, H6ɂ), 7.61 (ddd, J = 1.1,

7.7, 7.7 Hz, 2 H, H3ɂ, H5ɂ), 7.42 (dd, J = 3.3, 6.1 Hz, 2 H, H4, H7), 7.33 (ddd, J = 1.1,

13 7.7, 7.7 Hz, 1 H, H4ɂ), 7.18 (dd, J = 3.3, 6.1 Hz, 2 H, H5, H6), 2.54 (s, 3 H, CH 3). C

NMR (75 MHz, d 6-DMSO): δ 161.0 (C=O), 144.3 (ArC), 142.5 (ArC), 138.8 (ArC)

131.3 (ArC), 129.9 (ArCH), 125.3 (ArCH), 122.2 (ArCH), 119.1 (ArCH), 116.8 265

(ArCH), 111.8 (ArCH), 12.6 (CH 3). IR (ATR): ν max 3442, 2954, 2868, 2659, 2543,

-1 -1 - 1681, 1572, 1422, 1281, 920, 778, 727 cm . UV-Vis (MeOH): λ max 241 (48,600 M cm

1 + ) nm. HRMS (+ESI): Found m/z 396.0887, [M+Na] ; C 20 H15 N5OSNa required

396.0890.

N-(Benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-thieno[2,3-c]pyrazole-5-carboxamide

(256b)

This compound was synthesized as described for compound 228 using acid 232c (0.24 g, 0.73 mmol),

106 (0.11 g, 0.73 mmol), EDCI (0.15 g, 0.79 mmol) and

HOBt (0.40 g, 2.63 mmol) in DMF (2.0 mL) over 24 h to give the title compound 256b as a white solid (0.05

1 g, 14%). M.p. 299-231 °C. H NMR (300 MHz, d 6-DMSO): δ 13.18 (bs, 1 H, NH), 8.95

(s, 1 H, H4), 8.12 (d, J = 7.3 Hz, 2 H, H2, H6), 8.00 (d, J = 7.5 Hz, 1 H, H4ɂ”), 7.93

(d, J = 7.8 Hz, 2 H, H2ɂ, H6ɂ), 7.77 (d, J = 7.5 Hz, 1 H, H7ɂ”), 7.68 (dd, J = 7.8, 7.8 Hz,

2 H, H3ɂ, H5ɂ), 7.62 (dd, J = 7.3, 7.3 Hz, 2 H, H3, H5), 7.52 (dd, J = 7.3, 7.3 Hz, 1 H,

H4), 7.49 -7.41 (m, 2 H, H4ɂ, H5ɂ), 7.33 (dd, J = 7.5, 7.5 Hz, 1 H, H6ɂ). IR (ATR):

-1 νmax 3055, 2918, 1653, 1594, 1531, 1460, 1269, 1204, 747, 682 cm . UV-Vis (MeOH):

-1 -1 λmax 348 (29,500 M cm ), 301 (31,200), 283 (30,600), 218 (47,600) nm. HRMS (+ESI)

+ m/z Calcd. for C 25 H16 N4OS 2Na (M+Na) 475.0658. Found 475.0660.

266

4-Diazo-3-methyl-1-phenylpyrazole-5-one (257)

A mixture of pyrazole 233d (1.90 g, 10.91 mmol) and 4-toluenesulfonyl azide (3.26 g, 16.55 mmol) in MeOH (100.0 mL) was treated with Et 3N

(3.50 mL, 25.12 mmol) and the solution stirred at r.t. for 1.5 h before being concentrated under reduced pressure and purified by flash column chromatography (10% EtOAc:hexanes) to give the title compound 257 as yellow needles (1.70 g, 78%). M.p. 91-93 °C. Lit. 369 M.p. 93-94 °C. 1H NMR (300 MHz,

CDCl 3): δ 7.88 (dd, J = 1.2, 8.8 Hz, 2 H, H2′, H6′), 7.40 (dd d, J = 1.2, 7.4, 7.4 Hz, 2 H,

13 H3′, H5′), 7.19 (ddd, J = 1.2, 7.4, 7.4 Hz, 1 H, H4′), 2.38 (s, 3 H, CH 3). C NMR (75

MHz, CDCl 3): δ 163.3 (C=O), 142.1 (ArC), 138.6 (ArC), 129.0 (ArCH), 125.4 (ArCH),

119.5 (ArCH), 14.1 (CH 3).

Methyl 3-methyl-1-phenyl-1H-furo[2,3-c]pyrazole-5-carboxylate (258)

A mixture of diazopyrazole 257 (1.69 g, 8.43 mmol) and methyl propiolate (0.80 mL, 9.67 mmol) in toluene (15.0 mL) was heated at 165 °C in a pressure tube for 16 h. After cooling, the solvent was removed under reduced pressure and the residue purified by flash column chromatography (10% DCM:hexanes) and recrystallized from hexane to give the title compound 258 as colourless needles (0.86 g, 40%). M.p. 119-

369 1 120 °C. Lit. M.p. 119-120 °C. H NMR (300 MHz, CDCl 3): δ 7.89 (dd, J = 1.2, 8.8

Hz, 2 H, H2′, H6′), 7.48 (ddd, J = 1.2, 7.4, 7.4 Hz, 2 H, H3′, H5′), 7.33 (s, 1 H, H4),

7.26 (dd, J = 7.4, 7.4 Hz, 1 H, H4′), 3.94 (s, 3 H, OCH 3), 2.47 (s, 3 H, CH3).

267

3-Methyl-1-phenyl-1H-furo[2,3-c]pyrazole-5-carboxylic acid (259)

This compound was synthesized as described for acid 232a using methyl ester 258 (0.86 g, 3.36 mmol) in 1:1 2 M NaOH:THF

(6.0 mL) over 3 h to give the title compound 259 as a white solid after crystallization from MeOH (0.70 g, 86%). M.p. 210-212

1 °C. H NMR (300 MHz, d 6-acetone): δ 7.89 (dd, J = 1.1, 7.7 Hz, 2 H, H2′, H6′), 7.56

(ddd, J = 1.1, 7.7, 7.7 Hz, 2 H, H3′, H5′), 7.50 (s, 1 H, H4), 7.30 (ddd, J = 1.1, 7.4, 7.4

13 Hz, 1 H, H4′), 2.43 (s, 3 H, CH 3). C NMR (75 MHz, d 6-acetone): δ 159.8 (C=O),

156.5 (ArC), 147.8 (ArC), 142.1 (ArC), 130.5 (ArCH), 126.4 (ArCH), 117.7 (ArCH),

113.6 (ArC), 113.0 (ArCH), 13.8 (CH 3).

N-(Benzo[d]thiazol-2-yl)-3-methyl-1-phenyl-1H-furo[2,3-c]pyrazole-5-carboxamide

(260)

This compound was synthesized as described for amide

228 using acid 259 (71.9 mg, 0.29 mmol) and 106 (64.3 mg, 0.43 mmol) in DMF (2.0 mL) over 24 h to give the title compound 260 as solid after purification (37.4 mg,

1 34%). M.p. 231-233 °C. H NMR (300 MHz, d 6-acetone): δ 8.07 (dd, J = Hz, 2 H, H2′,

H6′), 7.98 (dd, J = Hz, 1 H, H4″), 7.78 (dd, J = 1 H, H7″), 7.56 (ddd, J = Hz , 2 H, H3′,

13 H5′), 7.46 (ddd, J = Hz, 1 H, H6″), 7.38 -7.29 (m, 2 H, H4′, H5″), 2.47 (s, 3 H, CH 3). C

NMR (75 MHz, d 6-acetone): δ 180.9 (C=O), 142.2 (ArC), 138.5 (ArC), 133.1 (ArC),

130.4 (ArCH), 127.0 (ArCH), 126.5 (ArCH), 124.7 (ArCH), 122.3 (ArCH), 121.6

(ArCH), 118.0 (ArCH), 114.3 (ArC), 112.3 (ArCH), 13.8 (CH 3). IR (ATR): ν max 3506,

3354, 3103, 2918, 1647, 1572, 1543, 1275, 1186, 859, 748, 685 cm -1. UV-Vis (MeOH): 268

-1 -1 λmax 325 (35,100 M cm ), 262 (26,900), 205 (44,900) nm. HRMS (+ESI): Found m/z

+ 375.0909, [M+H] ; C 20 H15 N4O2S required 375.0910.

1-Phenyl-2-(propan-2-ylidene)hydrazine (261)

To a solution of phenylhydrazine 164 (3.85 g, 35.6 mmol) in water

(29.0 mL) at 5 °C was added acetic acid (0.95 mL, 16.6 mmol), followed by acetone (5.8 mL, 79.0 mmol), dropwise. The solution was stirred for 1.5 h, the precipitate collected via filtration, washed twice with ice water, and dried in vacuo to give the title compound 261 as off-white crystals (2.09 g, 40%). M.p. 39-41 °C.

408 1 Lit. M.p. 42 °C. H NMR (300 MHz, CDCl 3): δ 7.26 (ddd, J = 1.3, 7.3, 7.3 Hz, 2 H,

H3, H5), 7.06 (dd, J = 1.3, 7.3 Hz, 2 H, H2, H6), 6.85 (ddd, J = 1.3, 7.3, 7.3 Hz, 1 H,

H4), 6.84 (bs, 1 H, NH), 2.07 (s, 3 H, CH 3), 1.89 (s, 3 H, CH3)

3-Methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (262)

POCl 3 (5.60 mL, 59.9 mmol) was added dropwise to DMF (40.0 mL) at

0 °C and the mixture stirred for 1 h before the temperature was lowered to -20 °C. A solution of propylidene 261 (2.09 g, 14.1 mmol) in DMF

(4.0 mL) was added dropwise and the mixture stirred at -20 °C for 3 h, then at 80 °C for 2 h. The mixture was allowed to cool before being poured over crushed ice and adjusted to pH 10 with 5 M NaOH. The precipitate was collected via filtration and washed with water to give the title compound 262 as a pale brown solid

409 1 (0.77 g, 29%). M.p. 53-55 °C. Lit. M.p. 53 °C. H NMR (300 MHz, CDCl 3): δ 9.99

(s, 1 H, CHO), 8.34 (s, 1 H, H5), 7.69 (dd, J = 1.7, 8.3 Hz, 2 H, H2′, H6′), 7.48 (ddd, J = 269

1.7, 8.3, 8.3 Hz, 2 H, H3′, H5′), 7.36 (ddd, J = 1.7, 8.3, 8.3 Hz, 1 H, H4′), 2.59 (s, 3 H,

13 CH 3). C NMR (75 MHz, CDCl 3): δ 184.5 (CHO), 152.1 (C3), 139.2 (C1′), 131.9 (C5),

129.8 (C3′, C5′), 127.8 (C4′), 123.1 (C4), 119.7 (C2′, C6′), 13.2 (CH 3).

4-(5-(8-Isopropyl-4-phenylquinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoic acid (272a)

This compound was synthesized as described for compound 179 using hydrazide 298 (320 mg, 0.71 mmol), 4-TsCl (400 mg, 2.00 mmol) and DIPEA

(0.30 mL, 1.73 mmol) in MeCN (4.0 mL) over 3 h to give the title compound 272a as a white solid (101 mg, 33%). M.p. 290-292 ºC. 1H

NMR (400 MHz, 323 K, d 6-DMSO): δ 8.29 (ddd, J = 2.0, 2.0, 8.7 Hz, 2 H, H3, H5),

8.21 (s, 1 H, H3), 8.20 (ddd, J = 2.0, 2.0, 8.7 Hz, 2 H, H2, H6), 7.83 (dd, J = 1.1, 7.1

Hz, 1 H, H5), 7.77 (dd, J = 1.5, 8.4 Hz, 1 H, H7), 7.69 (dd, J = 7.1, 8.4 Hz, 1 H, H6),

7.64-7.58 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.48 (sept, J = 7.0 Hz, 1 H, CH), 1.46 (d, J

13 = 7.0 Hz, 6 H, 2 x CH 3). C NMR (100 MHz, 323 K, d 6-DMSO): δ 166.4 (CO 2H),

164.4 (ArC), 149.9 (ArC), 148.0 (ArC), 147.7 (ArC), 141.0 (ArC), 137.1 (ArCH), 134.0

(ArCH), 130.3 (ArCH), 129.5 (ArCH), 128.9 (ArCH), 127.1 (ArCH), 126.8 (ArCH),

123.2 (ArCH), 27.3 (CH), 23.4 (2 x CH 3). IR (ATR): ν max 3055, 2955, 2689, 1537,

-1 -1 -1 1407, 1226, 1106, 854, 770, 700 cm . UV-Vis (MeOH): λmax 325 (ε 21,300 cm M ),

289 (42,500), 245 (26,200), 211 (32,800) nm. HRMS (+ESI): Found m/z 458.1472,

+ [M+Na] ; C27 H21 N3O3Na required 458.1475.

270

4-(5-(8-Isopropyl-4-(4-bromophenyl)quinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoic acid (272b)

Ester 299a (0.12 g, 0.22 mmol) was stirred in 0.25

M NaOH/THF (6.0 mL) over 4 h before being concentrated in vacuo , acidified with 2 M HCl and the precipitate collected via filtration to give the title compound 272b as a white solid (0.11 g, 97%).

1 M.p. 353-355 ºC. H NMR (300 MHz, d 6-DMSO): δ 8.28 (d, J = 8.5 Hz, 2 H, H3″,

H5″), 8.22 (s, 1 H, H3), 8.19 (d, J = 8.5 Hz, 2 H, H2″, H6″), 7.85 -7.82 (m, 3 H, H5, H3′,

H5′), 7.76 -7.67 (m, 2 H, H6, H7), 7.60 (d, J = 8.5 Hz, 2 H, H2′, H6′), 4.48 (sept, J = 6.7

13 Hz, 1 H, CH), 1.44 (d, J = 6.7 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ

166.6 (C=O), 164.5 (C(=N)O), 164.2 (C(=N)O), 148.6 (ArC), 148.0 (ArC), 145.5

(ArC), 141.1 (ArC), 136.2 (ArC), 131.8 (ArCH), 131.7 (ArCH) 130.4 (ArCH), 129.1

(ArC), 127.1 (ArCH), 126.9 (ArCH), 126.7 (ArCH), 126.5 (ArC), 123.2 (ArC), 122.6

(ArC), 119.4 (ArCH), 27.3 (CH), 23.6 (2 x CH 3). IR (ATR): ν max 2964, 2868, 2665,

-1 2548, 1693, 1538, 1483, 1420, 824, 765, 714 cm . UV-Vis (MeOH): λmax 326 (ε 22,500 cm -1M-1), 290 (47,200), 246 (29,800), 210 (41,400) nm. HRMS (+ESI): Found m/z

+ 536.0579, [M+Na] ; C 27 H20 BrN 3O3Na required 538.0580.

271

4-(5-(8-Isopropyl-4-(p-tolyl)quinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoic acid (272c)

This compound was synthesized as described for compound 272b using ester 299b (77.8 mg, 0.17 mmol), in 0.25 M NaOH/THF (6.0 mL) over 4 h to give the title compound as a white solid (70.1 mg,

1 92%). M.p. 319-321 ºC. H NMR (300 MHz, d 6-

DMSO): δ 8.28 (d, J = 8.6 Hz, 2 H, H3″, H5″), 8.19 (d, J = 8.6 Hz, 2 H, H2″, H6″), 8.17

(s, 1 H, H3) 7.81 (dd, J = 7.1, 8.5 Hz, 2 H, H5, H7), 7.67 (dd, J = 7.1, 8.5 Hz, 1 H, H6),

7.52 (d, J = 8.1 Hz, 2 H, H2′, H6′), 7.43 (d, J = 8.1 Hz, 2 H, H3′, H5′), 4.48 (sept, J =

13 6.9 Hz, 1 H, CH), 2.45 (s, 3 H, CH 3), 1.44 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75

MHz, d 6-DMSO): δ 166.6 (C=O), 164.4 (C=N), 164.2 (C=N), 149.9 (ArC), 147.9

(ArC), 145.6 (ArC), 141.1 (ArC), 138.5 (ArC), 134.2 (ArC), 130.3 ArCH), 129.4

(ArCH), 128.7 (ArCH), 127.1 (ArCH), 126.8 (ArCH), 126.6 (ArCH), 123.4 (ArC),

119.2 (ArCH), 27.2 (CH), 23.5 (2 x CH 3), 20.9 (CH 3). IR (ATR): ν max 2864, 2667,

-1 2547, 1692, 1537, 1497, 1420, 1287, 1083, 816, 764, 712 cm . UV-Vis (MeOH): λmax

325 (ε 23,700 cm -1M-1), 290 (46,500), 245 (29,600), 210 (40,800) nm. HRMS (+ESI):

+ Found m/z 472.1634, [M+Na] ; C28 H23 N3O3Na required 472.1632.

Methyl ( E)-2-oxo-4-phenylbut-3-enoate (277a)

Acetyl chloride (21.0 mL, 0.29 mol) was added dropwise to

MeOH (100.0 mL), at 0 °C to generate HCl. To this was added potassium salt 288a (20.19 g, 0.10 mol) and the mixture stirred at 0 °C for 30 min, r.t. for 2 h and at reflux overnight. The solvent was removed under reduced pressure and the crude residue partitioned between H 2O (50.0 mL) and DCM 272

(50.0 mL). The aqueous phase was extracted with DCM (2 x 50.0 mL) and the combined organics washed with sat. NaHCO 3 (50.0 mL) and H 2O (50.0 mL), dried over

Na 2SO 4 and the solvent removed under reduced pressure. The solid was recrystallized from MeOH to give the title compound 277a as yellow needles (5.59g, 29%). M.p. 70-

410 1 72 ºC. Lit. M.p. 70-71 °C. H NMR (300 MHz, CDCl 3): δ 7.87 (d, J = 16.0 Hz, 1 H,

H), 7.63 (dd, J = 1.6, 7.1 Hz, 2 H, H2, H6), 7.45-7.43 (m, 3 H, H3, H4, H5), 7.37 (d, J =

16.1 Hz, H), 3.93 (s, 3 H, CH 3).

Methyl ( E)-2-oxo-4-(4-bromophenyl)but-3-enoate (277b)

This compound was prepared as described for compound

277a using potassium salt 288b (7.28 g, 24.9 mmol) and

AcCl (20.0 mL, 280.3 mmol) in MeOH (100.0 mL) to give the title compound 277b as yellow needles (3.30 g, 49%). M.p. 111-113 ºC. Lit. 411 M.p.

1 116-118 °C. H NMR (300 MHz, CDCl 3): δ 7.76 (d, J = 16.2 Hz, 1 H, H4), 7.53 (dd, J

= 1.9, 6.5 Hz, 2 H, H2ɂ, H6ɂ), 7.46 (dd, J = 1.9, 6.5 Hz, 2 H, H3ɂ, H5ɂ), 7.33 (d, J = 16.2

Hz, 1 H, H3), 3.91 (s, 3 H, OCH 3).

Methyl ( E)-2-oxo-4-(p-tolyl)but-3-enoate (277c)

This compound was prepared as described for compound

277a using potassium salt 288c (11.60 g, 50.9 mmol) and

AcCl (14.0 mL, 196.9 mmol) in MeOH (50.0 mL) to give the title compound 277c as yellow needles (2.23 g, 21%). M.p. 79-81 ºC. Lit. 412 M.p.

1 81-83 °C. H NMR (300 MHz, CDCl 3): δ 7.86 (d, J = 16.0 Hz, 1 H, H4), 7.54 (d, J = 273

8.2 Hz, 2 H, H2ɂ, H6ɂ), 7.33 (d, J = 16.0 Hz, 1 H, H3), 7.23 (d, J = 8.2 Hz, 2 H, H3ɂ,

H5ɂ), 3.93 (s, 3 H, OCH 3), 2.40 (s, 3 H, CH3).

Methyl 8-isopropyl-4-phenylquinoline-2-carboxylate (278a)

A mixture of ketoester 277a (3.34 g, 17.58 mmol) and 2- isopropylaniline 273a (1.10 mL, 7.86 mmol) in TFA (30.0 mL) was heated at reflux under an N 2 atmosphere for 72 h. The solvent was removed under reduced pressure and the residue taken up in

DCM (50.0 mL), washed with sat. NaHCO 3 (2 x 20.0 mL), H 2O (20.0 mL) and brine

(20.0 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure. The crude residue was purified by flash column chromatography (10% EtOAc:hexanes) to give the title compound 278a as colourless needles (0.90 g, 37%). M.p. 105-107 ºC. 1H

NMR (300 MHz, CDCl 3): δ 8.09 (s, 1 H, H3), 7.79 (dd, J = 1.4, 8.4 Hz, 1 H, H), 7.68

(dd, J = 1.4, 7.4 Hz, 1 H, H), 7.58 – 7.52 (m, 6 H, H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.54

13 (sept, J = 6.9 Hz, 1 H, CH), 4.06 (s, 3 H, OCH 3), 1.43 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C

NMR (75 MHz, CDCl 3): δ 166.6 (C=O), 150.0 (ArC), 149.4 (ArC), 146.2 (ArC), 138.3

(ArC), 129.8 (ArCH), 128.7 (ArCH), 128.6 (ArCH), 128.0 (ArC), 125.9 (ArCH), 123.5

(ArCH), 121.1 (ArCH), 53.0 (OCH 3), 27.7 (CH), 23.9 (2 x CH 3). IR (ATR): ν max 3054,

-1 2954, 1734, 1489, 1432, 1248, 1128, 1040, 987, 767, 701 cm . UV-Vis (MeOH): λ max

306 (ε 8,050 cm -1M-1), 246 (50,800), 211 (31,900) nm. HRMS (+ESI): Found m/z

+ 328.1304, [M+Na] ; C 20 H19 NO 2Na required 328.1308.

274

Methyl 4-(4-bromophenyl)-8-isopropylquinoline-2-carboxylate (278b)

This compound was synthesized as described for compound 278a using ketoester 277b (3.25 g, 12.04 mmol) and 2-isopropylaniline

273a (0.86 mL, 6.07 mmol) at reflux in TFA (10.0 mL) under an

N2 atmosphere for 72 h, to give the title compound 278b as colourless needles (0.60 g, 26%). M.p. 163-165 ºC. 1H NMR (300

MHz, CDCl 3): δ 8.06 (s, 1 H, H3), 7.74-7.65 (m, 4 H, H5, H7, H3′, H5′), 7.56 (dd, J =

7.4, 8.2 Hz, 1 H, H6), 7.39 (d, J = 8.6 Hz, 2 H, H2′, H6′), 4.52 (sept, J = 6.9 Hz, 1 H,

13 CH), 4.06 (s, 3 H, OCH 3), 1.42 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75 MHz,

CDCl 3): δ 166.5 (C=O), 149.6 (ArC), 148.7 (ArC), 146.2 (ArC), 137.2 (ArC), 132.0

(ArCH), 131.3 (ArCH), 128.9 (ArCH), 127.7 (ArC), 126.1 (ArCH), 123.1 (ArCH),

120.9 (ArCH), 53.1 (OCH 3), 27.7 (CH), 23.8 (2 x CH 3). IR (ATR): ν max 3024, 2952,

-1 1731, 1591, 1482, 1245, 1129, 1035, 988, 834, 768 cm . UV-Vis (MeOH): λ max 307 (ε

8,580 cm -1M-1), 246 (48,000), 212 (36,400) nm. HRMS (+ESI): Found m/z 384.0593,

+ [M+H] ; C 20 H19 BrNO 2 required 384.0594.

Methyl 8-isopropyl-4-(p-tolyl)quinoline-2-carboxylate (278c)

This compound was synthesized as described for compound 278a using ketoester 273c (2.23 g, 10.9 mmol) and 2-isopropylaniline

273a (0.71 mL, 5.01 mmol) at reflux in TFA (5.0 mL) under an

N2 atmosphere for 72 h, to give the title compound 278c as colourless needles (0.46 g, 27%). M.p. 106-108 ºC. 1H NMR (300

MHz, CDCl 3): δ 8.09 (s, 1 H, H3), 7.82 (dd, J = 1.4, 8.4 Hz, 1 H, H7), 7.69 (dd, J = 1.2,

7.1 Hz, 1 H, H5), 7.55 (dd, J = 7.1, 8.4 Hz, 1 H, H6), 7.41 (dd, J = 2.0, 6.2 Hz, 2 H, H2′, 275

H6′), 7.34 (dd, J = 2.0, 6.2 Hz, 2 H, H3′, H5′), 4.53 (sept, J = 6.9 Hz, 1 H, CH), 4.07 (s,

13 3 H, OCH 3), 2.47 (s, 3 H, CH 3), 1.42 (d, J = 6.9 Hz, 9 H, 2 x CH 3). C NMR (75 MHz,

CDCl 3): δ 166.7 (C=O), 149.2 (ArC), 146.1 (ArC), 138.7 (ArC), 135.3 (ArC), 129.7

(ArCH), 129.4 (ArCH), 128.6 (ArCH), 128.2 (ArC), 126.1 (ArCH), 123.6 (ArCH),

121.0 (ArCH), 53.1 (OCH 3), 27.7 (CH), 23.9 (2 x CH 3), 21.5 (CH3). IR (ATR): ν max

-1 2947, 1713, 1498, 1442, 1366, 1246, 1108, 1034, 816, 768 cm . UV-Vis (MeOH): λ max

306 (ε 8,510 cm -1M-1), 246 (48,900), 208 (41,000) nm. HRMS (+ESI): Found m/z

+ 342.1463, [M+Na] ; C 21 H21 NO 2Na required 342.1465.

Methyl 8-isopropyl-4-oxo-1,4-dihydroquinoline-2-carboxylate (285)

To a cooled solution of 2-isopropylaniline 273a (1.39 g, 10.29 mmol) in EtOH (10.0 mL) was added dimethyl acetylenedicarboxylate (1.53 mL, 12.49 mmol). This mixture was stirred at r.t. for 2 h before being concentrated under reduced pressure. The residue was taken up in freshly prepared Eaton’s reagent (10.0 mL) and stirred at 50 °C for 4 h. The mixture was cooled in a salt-ice bath before being poured into sat. K2CO 3 (150 mL) at 0

°C. The precipitate was collected via filtration and recrystallized from EtOAc to give the title compound 285 as a white solid (1.98 g, 78%). M.p. 88-90 °C; 1H NMR (300

MHz, CDCl 3): δ 9.15 (bs, 1 H, NH), 8.23 (dd, J = 1.3, 8.1 Hz, 1 H, H5), 7.60 (ddd, J =

0.5, 1.4, 7.4 Hz, 1 H, H7), 7.36 (dd, J = 7.7 Hz, 1 H, H6), 7.02 (s, 1 H, H3), 4.05 (s, 3 H,

13 OCH 3), 3.29 (sept, J = 6.9 Hz, 1 H, CH), 1.41 (d, J = 6.7 Hz, 6 H, 2 x CH 3). C NMR

(300 MHz, CDCl 3): δ 180.0 (C=O), 163.8 (CO2CH 3), 136.5 (C8a), 135.9 (C5a), 129.4

(C7), 126.6 (C8), 124.7 (C6), 124.2 (C5), 111.1 (C3), 54.1 (OCH 3), 27.8 (CH), 22.8 (2 x

CH 3). IR (ATR): ν max 3484, 3423, 3285, 2951, 1734, 1625, 1571, 1521, 1435, 1335, 276

-1 -1 -1 1242, 1128, 1014, 860, 820, 761 cm . UV-Vis (MeOH): λmax 293 (ε 5,580 cm M ),

+ 241 (33,900) nm. HRMS (+ESI): Found m/z 246.1123, [M+H] ; C 14 H16 NO 3 required

246.1125.

Methyl 8-isopropyl-4-(((trifluoromethyl)sulfonyl)oxy)quinoline-2-carboxylate (286)

A mixture of quinolone 285 (0.78 g, 3.18 mmol) and 2,6-lutidine

(1.90 mL, 16.30 mmol) in DCM (15.0 mL), was cooled in an ice- bath under an N 2 atmosphere. To this, a solution of triflic anhydride (1.10 mL, 6.54 mmol) in DCM (10.0 mL) was added dropwise. After 48 h, the mixture was diluted with HCl (2 M, 50.0 mL) and the organic phase collected. The aqueous phase was extracted with DCM (2 x 30.0 mL) and the combined organics dried over Na 2SO 4 and the solvent removed under reduced pressure. The crude product was purified by flash column chromatography (10% EtOAc:hexanes) to give the title compound 286 as colourless needles (0.89 g, 74%). M.p. 84-86 °C. 1H NMR (300 MHz,

CDCl 3): δ 8.13 (s, 1 H, H3), 7.95 (dd, J = 2.5, 7.3 Hz, 1 H, H5), 7.82-7.75 (m, 2 H, H6,

H7), 4.43 (sept, J = 6.9 Hz, 1 H, CH), 4.08 (s, 3 H, OCH 3), 1.39 (d, J = 6.9 Hz, 6 H, 2 x

13 CH 3). C NMR (300 MHz, CDCl 3): δ 165.0 (C=O), 153.8 (C4), 149.7 (C8a), 147.6

(C8), 147.1 (C2), 130.7 (C6), 127.9 (C7), 122.4 (C5a), 118.1 (C5), 116.5 (CF 3), 112.0

(C3), 53.6 (OCH 3), 27.7 (CH), 23.7 (2 x CH 3). IR (ATR): ν max 3092, 2962, 1722, 1428,

-1 1340, 1201, 1132, 1067, 986, 928, 855, 803 cm . UV-Vis (MeOH): λmax 301 (ε 6,020 cm -1M-1), 243 (55,700), 210 (28,400) nm. HRMS (+ESI): Found m/z 400.0434,

+ (M+Na) ; C 15 H14 F3NO 5SNa required 400.0437.

277

Potassium ( E)-2-oxo-4-phenylbut-3-enoate (288a)

To a cooled mixture of pyruvic acid (22.5 mL, 0.32 mol) and benzaldehyde 287a (33.0 mL, 0.32 mol) in MeOH (15.0 mL) was added a solution of methanolic KOH (5.3 M, 50.0 mL), dropwise, so as to maintain the temperature below 25 °C. Following this, an additional portion of methanolic KOH (5.3 M, 25.0 mL) was added at once. The mixture was stirred at r.t. for 1 h, then at 0 °C overnight. The precipitate was collected via filtration, washed twice with cold MeOH, followed by Et 2O and then air dried to give the title compound 288a as a yellow solid (53.29g, 81%). M.p. 243-245 ºC. Lit. 411 M.p. 246-248

1 °C. H NMR (300 MHz, d 6-DMSO): δ 7.63 (dd, J = 4.0, 8.0 Hz, 2 H, H2ɂ, H6ɂ), 7.46 (d,

J = 16.4 Hz, 1 H, H4), 7.42-7.40 (m, 3 H, H3ɂ, H4ɂ, H5ɂ), 6.77 (d, J = 16.4 Hz, 1 H, H3).

13 C NMR (75 MHz, d 6-DMSO): δ 197.4 (C=O), 169.1 (CO 2K), 143.3 (C4), 134.9

(ArCH), 130.1 (ArC), 129.0 (ArCH), 128.1 (ArCH), 125.3 (C3).

Potassium ( E)-2-oxo-4-(4-bromophenyl)but-3-enoate (288b)

This compound was prepared as described for compound

288a using pyruvic acid (2.76 g, 31.3 mmol) and 4- bromobenzaldehyde 287b (4.88 g, 26.4 mmol) to give the title compound 288b as a yellow solid (7.28 g, 94%). M.p. 230-232 ºC (dec). Lit. 411

1 M.p. 233 °C. H NMR (300 MHz, D 2O): δ 7.70-7.60 (m, 5 H, H4, H2ɂ, H3ɂ, H5ɂ, H6ɂ),

6.92 (d, J = 16.5 Hz, 1 H, H3).

278

Potassium ( E)-2-oxo-4-(p-tolyl)but-3-enoate (288c)

This compound was prepared as described for compound

288a using pyruvic acid (4.56 g, 51.8 mmol) and 4- methylbenzaldehyde 287c (6.25 g, 52.0 mmol) to give the

1 title compound 288c as a yellow solid (11.60 g, 98%). H NMR (300 MHz, D 2O): δ

7.67 (d, J = 16.4 Hz, 1 H, H4), 7.58 (d, J = 8.1 Hz, 2 H, H2ɂ, H6ɂ), 7.31 (d, J = 8.1 Hz, 2

H, H3ɂ, H5ɂ), 6.83 (d, J = 16.4 Hz, 1 H, H3), 2.37 (s, 3 H, CH 3).

8-Isopropyl-4-phenylquinoline-2-carbohydrazide (289a)

A solution of ester 278a (0.61 g, 2.00 mmol) in MeOH (10.0 mL) was treated with hydrazine monohydrate (0.50 mL, 9.50 mmol) and heated at reflux for 2 h. The precipitate was collected via filtration and washed with MeOH to give the title compound 289a as colourless needles (0.51 g, 84%). M.p. 161-163 ºC. 1H NMR (300

MHz, CDCl 3): δ 9.19 (bs, 1 H, NH), 8.20 (s, 1 H, H3), 7.81 (dd, J = 1.5, 8.4 Hz, H),

7.67 (dd, J = 1.1, 7.2 Hz, 1 H, H), 7.56 – 7.51 (m, 6 H, H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.35

(sept, J = 6.9 Hz, 1 H, CH), 4.18 (d, J = 4.7 Hz, 2 H, NH 2), 1.43 (d, J = 6.9 Hz, 6 H, 2 x

13 CH 3). C NMR (75 MHz, CDCl 3): δ 165.6 (C=O), 150.7 (ArC), 148.0 (ArC), 146.9

(ArC), 138.3 (ArC), 129.8 (2 x ArCH), 128.7 (3 x ArCH), 127.2 (ArCH), 126.0

(ArCH), 123.8 (ArCH), 118.7 (ArCH), 27.9 (CH), 23.7 (2 x CH 3). IR (ATR): ν max 3304,

-1 3062, 2957, 1665, 1479, 1403, 969, 762, 703 cm . UV-Vis (MeOH): λ max 314 (ε 7,020 cm -1M-1), 245 (44,900), 208 (37,700) nm. HRMS (+ESI): Found m/z 306.1598,

+ [M+H] ; C 19 H20 N3O required 306.1601.

279

8-Isopropyl-4-(4-bromophenyl)quinoline-2-carbohydrazide (289b)

This compound was prepared as described for compound 289a using ester 278b (0.56 g, 1.47 mmol) and hydrazine monohydrate (0.50 mL, 9.50 mmol) in MeOH (10.0 mL) over 4 h to give the title compound 289b as colourless needles (0.51 g,

1 91%). M.p. 181-183 ºC. H NMR (300 MHz, CDCl 3): δ 9.17 (s,

1 H, NH), 8.17 (s, 1 H, H3), 7.74 (dd, J = 1.4, 8.4 Hz, 1 H, H5), 7.69-7.65 (m, 3 H, H7,

H3′, H5′), 7.55 (dd, J = 7.3, 8.4 Hz, 1 H, H6), 7.38 (d, J = 8.9 Hz, 2 H, H2′, H6′), 4.34

(sept, J = 6.9 Hz, 1 H, CH), 4.17 (d, J = 4.0 Hz, 2 H, NH 2), 1.42 (d, J = 6.9 Hz, 6 H, 2 x

13 CH 3). C NMR (75 MHz, CDCl 3): δ 165.4 (C=O), 149.4 (ArC), 148.2 (ArC), 146.9

(ArC), 145.0 (ArC), 137.1 (ArC), 132.0 (ArCH), 131.3 (ArCH), 128.5 (ArCH), 127.9

(ArC), 126.3 (ArCH), 123.4 (ArCH), 123.2 (ArC), 118.6 (ArCH), 27.9 (CH), 23.7 (2 x

CH 3). IR (ATR): ν max 3242, 3057, 2956, 1654, 1545, 1479, 1271, 1006, 959, 902, 829,

-1 -1 -1 773 cm . UV-Vis (MeOH): λ max 245 (ε 44,400 cm M ), 210 (41,200) nm. HRMS

+ (+ESI): Found m/z 384.0704, [M+H] ; C 19 H19 BrN 3O required 384.0706.

8-Isopropyl-4-(p-tolyl)quinoline-2-carbohydrazide (289c)

This compound was prepared as described for compound 289a using ester 278c (0.43 g, 1.35 mmol) and hydrazine monohydrate (0.50 mL, 9.50 mmol) in MeOH (10.0 mL) over 4 h to give the title compound 289c as colourless needles (0.43 g,

1 99%). M.p. 170-172 ºC. H NMR (300 MHz, d 6-DMSO): δ 9.96

(s, 1 H, NH), 7.93, (s, 1 H, H3), 7.75 (ddd, J = 1.8, 1.8, 8.2 Hz, 2 H, H5, H7), 7.62 (dd,

J = 6.8, 8.7 Hz, 1 H, H6), 7.46 (d, J = 8.3 Hz, 2 H, H2′, H6′), 7.39 (d, J = 8.0 Hz, 2 H, 280

H3′, H5′), 4.70 (bs, 2 H, NH 2), 4.63 (sept, J = 6.9 Hz, 1 H, CH), 2.43 (s, 3 H, CH 3), 1.34

13 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ 163.1 (C=O), 149.6

(ArC), 148.2 (ArC), 147.9 (ArC), 144.4 (ArC), 138.2 (ArC), 134.7 (ArC), 129.3

(ArCH), 128.1 (ArCH), 126.9 (ArC), 125.9 (ArCH), 123.0 (ArCH), 118.2 (ArCH), 26.3

(CH), 23.7 (2 x CH 3), 20.9 (CH 3). IR (ATR): ν max 3252, 3021, 2958, 1655, 1524, 1492,

-1 1459, 1411, 1181, 1108, 963, 904, 820, 770 cm . UV-Vis (MeOH): λ max 245 (ε 51,100 cm -1M-1), 209 (48,000) nm. HRMS (+ESI): Found m/z 342.1573, [M+Na] +;

C20 H21 N3ONa required 342.1577.

8-Isopropyl-4-phenylquinoline-2-carboxylic acid (290a)

A mixture of ketoester 277a (1.90 g, 9.99 mmol) and 2- isopropylaniline 273a (0.70 mL, 5.00 mmol) in TFA (15.0 mL) was heated at reflux under an N 2 atmosphere for 72 h. The solvent was removed under reduced pressure and the residue taken up in

DCM (50.0 mL), washed with sat. NaHCO 3 (2 x 20.0 mL), H 2O (20.0 mL) and brine

(20.0 mL), dried over Na 2SO 4 and the solvent removed under reduced pressure. The crude residue was purified by flash column chromatography (40% EtOAc:hexanes) to give the title compound 290a as colourless needles (0.37 g, 25%). M.p. 167-169 ºC. 1H

NMR (300 MHz, d 6-DMSO): δ 13.39 (bs, 1 H, CO 2H), 7.96 (s, 1 H, H3), 7.78, (dd, J =

1.6, 6.9 Hz, 1 H, H5), 7.74 (1.6, 8.5 Hz, 1 H, H7), 7.67 (dd, J = 6.9, 8.5 Hz, 1 H, H6),

7.62-7.55 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ), 4.47 (sept, J = 7.0 Hz, 1 H, CH), 1.37 (d, J = 7.0

13 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ 166.5 (C=O), 149.3 (ArC), 148.3

(ArC), 147.0 (ArC), 145.1 (ArC), 137.4 (ArC), 129.4 (ArCH), 128.8 (ArCH), 128.7

(ArCH), 126.9 (ArC), 126.1 (ArCH), 123.0 (ArCH), 120.4 (ArCH), 26.9 (CH), 23.5 (2 281

-1 x CH 3). IR (ATR): ν max 2955, 2867, 2592, 1683, 1443, 1264, 1135, 918, 769, 702 cm .

-1 -1 UV-Vis (MeOH): λ max 302 (ε 7,280 cm M ), 241 (40,200), 212 (30,100) nm. HRMS

+ (+ESI): Found m/z 314.1151, [M+Na] ; C 19 H17 NO 2Na required 314.1152.

8-Isopropyl-4-(p-tolyl)quinoline-2-carboxylic acid (290b)

This compound was synthesized as described for compound 290a using ketoester 273c (2.23 g, 10.9 mmol) and 2-isopropylaniline

273a (0.71 mL, 5.01 mmol) at reflux in TFA (5.0 mL) under an

N2 atmosphere for 72 h, to give the title compound 290b as colourless needles (0.46 g, 30%) following purification by flash column chromatography (50% EtOAc:hexanes). M.p. 160-162 ºC. 1H NMR (300 MHz,

CDCl 3): δ 8.22 (s, 1 H, H3), 7.90 (dd, J = 1.4, 8.4 Hz, 1 H, H5), 7.74 (dd, J = 0.9, 7.2

Hz, 1 H, H7), 7.61 (dd, J = 7.2, 8.4 Hz, 1 H, H6), 7.42 (dd, J = 1.9 , 6.2 Hz, 2 H, H2′,

H6′), 7.35 (dd, J = 1.9, 6.2 Hz, 2 H, H3′, H5′), 4.31 (sept, J = 7.0 Hz, 1 H, CH), 2.48 (s,

13 3 H, CH 3), 1.45 (d, J = 7.0 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ 164.7

(C=O), 152.5 (ArC), 147.6 (ArC), 144.4 (ArC), 144.0 (ArC), 139.1 (ArC), 134.7 (ArC),

129.6 (ArCH), 129.1 (ArCH), 126.8 (ArCH), 124.2 (ArCH), 119.0 (ArCH), 28.0 (CH),

23.6 (2 x CH 3), 21.5 (CH 3). IR (ATR): ν max 3293, 3046, 2962, 1734, 1379, 1290, 1184,

-1 -1 -1 1125, 1036, 901, 835, 786, 710 cm . UV-Vis (MeOH): λ max 303 (ε 11,300 cm M ),

241 (51,000), 210 (43,600) nm. HRMS (+ESI): Found m/z 328.1304, [M+Na] +;

C20 H19 NO 2Na required 328.1308.

282

Methyl 8-isopropyl-4-((methylsulfonyl)oxy)quinoline-2-carboxylate (292)

This compound was synthesized as described for compound 285 using aniline 273a (1.37 g, 10.13 mmol) and DMAD (1.50 mL,

12.20 mmol) in EtOH (10.0 mL) over 2 h, followed by Eaton’s reagent (10.0 mL) at 50 °C for 4 h, to give the title compound 292

1 as colourless needles (1.21 g, 37%). M.p. 122-124 °C. H NMR (300 MHz, d 6-acetone):

8.19 (s, 1 H, H3), 8.15 (dd, J = 1.8, 8.1 Hz, 1 H, H5), 7.87 (dd, J = 1.8, 7.2 Hz, 1 H,

H7), 7.81 (dd, J = 7.2, 8.1 Hz, 1 H, H6), 4.42 (sept, J = 6.9 Hz, 1 H, CH), 4.02 (s, 3 H,

OCH 3), 3.63 (s, 3 H, SO 2CH 3), 1.39 (d, J = 6.9 Hz, 6 H, 2 x CH 3). IR (ATR): ν max 3020,

-1 2961, 1711, 1341, 1170, 1076, 976, 926, 786 cm . UV-Vis (MeOH): λ max 298 (5,500

-1 -1 + cm M ), 243 (45,700) nm. HRMS (+ESI): Found m/z 324.0893, [M+H] ; C 15 H18 NO 5S required 324.0900.

4-((2-(8-Isopropyl-4-phenylquinoline-2-carbonyl)hydrazono)methyl)benzoic acid

(293)

To a solution of hydrazide 289a (0.10 g, 0.33 mmol) in EtOH (3.4 mL) was added a solution of

4-carboxybenzaldehyde (0.06 g, 0.38 mmol) in

EtOH (2.0 mL), dropwise. This mixture was heated at reflux for 48 h and the precipitate collected via filtration to give the title compound 293 as a white solid (0.15 g, 97%).

1 M.p. 172-175 ºC. H NMR (300 MHz, d 6-DMSO): δ 12.00 (s, 1 H, CHN), 8.78 (s, 1 H,

NH), 8.06 (s, 1 H, H3), 8.03 (d, J = 8.3 Hz, 2 H, H), 7.93 (d, J = 8.3 Hz, 2 H, H), 7.83

(dd, J = 1.3, 7.0 Hz, 1 H, H), 7.78 (dd, J = 1.5, 8.3 Hz, 1 H, H), 7.68 (dd, J = 1.5, 7.0 283

Hz, 1 H, H), 7.63 – 7.57 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.72 (sept, J = 6.9 Hz, 1 H,

13 CH), 1.40 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ 170.5

(CO2H), 167.1 (C=O), 161.3 (CNH), 150.0 (ArC), 148.7 (ArCH), 148.2 (ArC), 147.9

(ArC), 144.5 (ArC), 138.3 (ArC), 137.5 (ArC), 132.1 (ArC), 130.0 (ArCH), 129.5

(ArCH), 129.0 (ArC), 127.4 (ArCH), 127.3 (ArCH), 123.2 (ArCH), 118.9 (ArCH),

26.50 (CH), 23.8 (2 x CH 3). IR (ATR): ν max 3215, 2959, 2863, 2657, 2545, 1684, 1662,

-1 - 1519, 1419, 1288, 1235, 1133, 765, 698 cm . UV-Vis (DMF ): λ max 309 (ε 33,900 cm

1 -1 + M ) nm. HRMS (+ESI): Found m/z 460.1625, [M+Na] ; C 27 H23 N3O3Na required

460.1632.

N'-(4-Cyanobenzoyl)-8-isopropyl-4-phenylquinoline-2-carbohydrazide (294)

This compound was synthesized as described for compound 185 using hydrazide 289a (0.31 g, 1.01 mmol), 4-cyanobenzoic acid (0.15 g, 1.04 mmol),

EDCI (0.25 g, 1.27 mmol) and HOBt (0.20 g, 1.45 mmol) in DMF (2.5 mL) over 24 h to give the title compound 294 as a white solid (0.22 g, 52%) following purification by flash column

1 chromatography (25% EtOAc:hexanes). M.p. 169-171 ºC. H NMR (300 MHz, CDCl 3):

δ 10.76 (bs, 1 H, NH), 10.16 (bs, 1 H, NH), 8.12 (s, 1 H, H3), 8.06 (d, J = 8.6 Hz, 2 H,

H2, H6), 7.83 (dd, J = 1.4, 8.6 Hz, 1 H, H5), 7.72-7.69 (m, 3 H, H7, H3, H5), 7.58

(dd, J = 7.4, 8.5 Hz, 1 H, H6), 7.56-7.48 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.39 (sept, J

13 = 6.9 Hz, 1 H, CH), 1.48 (d, J = 6.9 Hz, 6 H, 2 x CH3). C NMR (75 MHz, CDCl 3): δ

162.7 (C=O), 162.0 (C=O), 151.2 (ArC), 148.1 (ArC), 145.5 (ArC), 145.1 (ArC), 138.0

(ArC), 135.5 (ArC), 132.6 (ArCH), 129.7 (ArCH), 128.9 (ArCH), 128.8 (ArCH), 128.5 284

(ArC), 128.3 (ArCH), 126.5 (ArCH), 123.9 (ArCH), 118.7 (ArCH), 117.9 (ArC), 116.0

(ArC), 28.2 (CH), 23.7 (2 x CH 3). IR (ATR): ν max 3252, 3073, 2964, 2229, 1691, 1637,

-1 -1 -1 1494, 1303, 1255, 768, 698 cm . UV-Vis (MeOH): λmax 247 (ε 44,900 cm M ), 203

+ (43,300) nm. HRMS (+ESI): Found m/z 435.1814, [M+H] ; C27 H23 N4O2 required

435.1816.

4-(5-(8-Isopropyl-4-phenylquinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzonitrile (295)

This compound was synthesized as described for compound 179 using hydrazide 294 (0.22 g, 0.50 mmol), 4-TsCl (0.21 g, 1.13 mmol) and DIPEA (0.30 mL, 1.70 mmol) in MeCN (4.0 mL) over 16 h to give the title compound 295 as a white solid (0.17 g, 84%). M.p. 236-238 ºC. 1H NMR (300

MHz, CDCl 3): δ 8.38 (d, J = 8.3 Hz, 2 H, H2, H6), 8.32 (s, 1 H, H3), 7.89 (d, J = 8.3

Hz, 2 H, H3, H5), 7.83 (dd, J = 1.3, 8.4 Hz, 1 H, H5), 7.73 (dd, J = 1.3, 7.3 Hz, 1 H,

H7), 7.57 (dd, J = 7.3, 8.4 Hz, 1 H, H6), 7.58-7.53 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ),

13 4.54 (sept, J = 7.0 Hz, 1 H, CH), 1.50 (d, J = 7.0 Hz, 6 H, 2 x CH 3). C NMR (CDCl 3):

δ 165.5 (C=N), 164.2 (C=N), 150.5 (ArC), 148.9 (ArC), 146.6 (ArC), 141.1 (ArC),

137.9 (ArC), 133.1 (ArCH), 129.7 (ArCH), 128.9 (ArCH), 128.8 (ArCH), 128.6

(ArCH), 128.0 (ArC), 127.9 (ArCH), 127.7 (ArC), 126.5 (ArCH), 123.8 (ArCH), 119.8

(ArCH), 118.1 (ArC), 115.5 (ArC), 28.1 (CH), 23.8 (2 x CH 3). IR (ATR): ν max 3058,

-1 2960, 2224, 1531, 1485, 1403, 1081, 890, 850, 769, 699 cm . UV-Vis (DMF ): λ max 329

(ε 28,800 cm -1M-1), 291 (53,000) nm. HRMS (+ESI): Found m/z 439.1522, [M+Na] +;

C27 H20 N4ONa required 439.1529.

285

Methyl 4-(2-(8-isopropyl-4-phenylquinoline-2-carbonyl)hydrazine-1-carbonyl) benzoate (297a)

This compound was synthesized as described for compound 185 using hydrazide 289a (100 mg, 0.33 mmol), mono-methyl terephthalate

(67.2 mg, 0.37 mmol), EDCI (86.4 mg, 0.45 mmol) and HOBt (64.9 mg, 0.41 mmol) in

DMF (1.0 mL) over 24 h to give the title compound 297a as a white solid (132.9 mg,

1 85%). M.p. 104-106 ºC. H NMR (300 MHz, CDCl 3): δ 10.81 (bs, 1 H, NH), 9.53 (bs, 1

H, NH), 8.19 (s, 1 H, H3), 8.15 (d, J = 8.4 Hz, 2 H, H2, H6), 8.01 (d, J = 8.4 Hz, 2 H,

H3, H5), 7.83 (dd, J = 1.3, 8.4 Hz, 1 H, H5), 7.72 (dd, J = 1.0, 7.2 Hz, 1 H, H7), 7.60-

7.52 (m, 6 H, H6, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.42 (sept, J = 7.1 Hz, 1 H, CH), 3.95 (s, 3

13 H, OCH 3), 1.49 (d, J = 7.1 Hz, 6 H, 2 x CH 3); C NMR (300 MHz, CDCl3): δ 166.3

(C=O), 148.2 (ArC), 145.2 (ArC), 138.1 (ArC), 135.5 (ArC), 133.7 (ArC), 130.2

(ArCH), 129.8 (ArCH), 128.8 (ArCH), 128.6 (ArCH), 128.5 (ArCH), 127.5 (ArCH),

126.4 (ArCH), 123.9 (ArCH), 118.7 (ArCH), 52.6 (OCH 3), 28.2 (CH), 23.7 (2 x CH 3).

-1 IR (A TR): ν max 3254, 2957, 1720, 1650, 1474, 1275, 1107, 899, 882, 767, 701 cm .

-1 -1 UV-Vis (MeOH): λmax 248 (ε 54,300 cm M ) nm. HRMS (+ESI): Found m/z 490.1739,

+ [M+Na] ; C28 H25 N3O4Na required 490.1737.

286

Methyl 4-(2-(8-isopropyl-4-(4-bromophenyl)quinoline-2-carbonyl)hydrazine-1- carbonyl)benzoate (297b)

This compound was synthesized as described for compound 185 using hydrazide 289b (0.57 g, 1.49 mmol), mono -methyl terephthalate (0.28 g, 1.55 mmol), EDCI (0.34 g, 1.78 mmol) and

HOBt (0.36 g, 2.26 mmol) in DMF (3.0 mL) over 24 h to give the title compound 297b as a

1 white solid (0.53 g, 64%). M.p. 219-221 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.85

(bs, 2 H, 2 x NH), 8.12 (dd, J = 2.3, 8.7 Hz, 2 H, H2, H6), 8.08 (dd, J = 2.3, 8.7 Hz, 2

H, H3, H5), 8.01 (s, 1 H, H3), 7.83-7.79 (m, 3 H, H5, H3ɂ, H5ɂ), 7.74 -7.67 (m, 2 H,

H6), H7), 7.56 (dd, J = 2.0, 8.5 Hz, 2 H, H2ɂ, H6ɂ), 4.76 (sept, J = 6.7 Hz, 1 H, CH),

13 3.91 (s, 3 H, OCH 3), 1.37 (d, J = 6.7 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, CDCl 3): δ

165.7 (C=O), 164.8 (C=O), 163.8 (C=O), 148.6 (ArC), 148.4 (ArC), 147.5 (ArC), 144.4

(ArC), 136.6 (ArC), 132.4 (ArC), 131.8 (ArCH), 131.6 (ArCH), 129.4 (ArCH), 127.9

(ArCH), 126.9 (ArC), 126.4 (ArC), 122.9 (ArC), 122.4 (ArC), 118.5 (ArC), 52.5

(OCH 3), 26.1 (CH), 23.9 (2 x CH 3). IR (ATR): νmax 3319, 3278, 2949, 2935, 2857,

1705, 1696, 1670, 1513, 1438, 1294, 1243, 1107, 1010, 830, 774, 725 cm -1. UV-Vis

-1 -1 (MeOH): λmax 248 (ε 68,500 cm M ) nm. HRMS (+ESI): Found m/z 568.0841,

+ [M+Na] ; C 28 H24 BrN3O4Na required 568.0842.

287

Methyl 4-(2-(8-isopropyl-4-(p-tolyl)quinoline-2-carbonyl)hydrazine-1-carbonyl) benzoate (297c)

This compound was synthesized as described for compound 185 using hydrazide 289c (0.40 g, 1.26 mmol), mono -methyl terephthalate (0.25 g, 1.40 mmol), EDCI (0.29 g, 1.52 mmol) and

HOBt (0.25 g, 1.57 mmol) in DMF (3.0 mL) over 24 h to give the title compound 297c as a

1 white solid (0.49 g, 80%). M.p. 163-165 ºC. H NMR (CDCl 3): δ 10.89 (bs, 1 H, NH),

10.83 (bs, 1 H, NH), 8.12 (d, J = 6.5 Hz, 2 H, H2″, H6″), 8.08 (d, J = 6.5 Hz, 2 H, H3″,

H5″), 7.99 (s, 1 H, H3), 7.80 (dd, J = 7.1, 8.4 Hz, 2 H, H5, H7), 7.68 (dd, J = 7.1, 8.4

Hz, 1 H, H7), 7.49 (d, J = 8.1 Hz, 2 H, H2′, H6′), 7.41 (d, J = 8.1 Hz, 2 H, H3′, H5′),

4.76 (sept, J = 6.8 Hz, 1 H, CH), 3.91 (s, 3 H, OCH 3), 2.44 (s, 3 H, CH 3), 1.37 (d, J =

13 6.8 Hz, 6 H, 2 x CH 3); C NMR (300 MHz, CDCl 3): δ 165.6 (C=O), 164.8 (C=O),

163.5 (C=O), 149.9 (ArC), 148.3 (ArC), 147.4 (ArC), 144.4 (ArC), 138.3 (ArC), 136.7

(ArC), 134.6 (ArC), 132.4 (ArC), 129.4 (ArCH), 129.3 (ArCH), 128.6 (ArCH), 127.9

(ArCH), 127.2 (ArC), 126.2 (ArCH), 123.1 (ArCH), 118.4 (ArCH), 52.4 (OCH 3), 26.1

(CH), 23.9 (2 x CH 3), 20.9 (CH 3); IR (ATR): ν max 3167, 2955, 1725, 1608, 1462, 1273,

-1 -1 -1 1106, 1017, 769, 723 cm . UV-Vis (MeOH): λmax 248 (ε 60,400 cm M ) nm. HRMS

+ (+ESI): Found m/z 504.1891, [M+Na] ; C 29 H27 N3O4Na required 504.1894.

288

4-(2-(8-Isopropyl-4-phenylquinoline-2-carbonyl)hydrazine-1-carbonyl)benzoic acid

(298)

A solution of hydrazide 297a (105.8 mg, 0.22 mmol) in 0.25 M NaOH/THF (4.0 mL) was stirred at r.t. for 3 h. The solution was concentrated in vacuo and acidified with 2 M

HCl. The precipitate was collected via filtration, washed with water and air dried to give the title compound 298 as a white solid (24.6

1 mg, Yield: 25%). M.p. 229-231 ºC. H NMR (300 MHz, d 6-DMSO): δ 10.83 (s, 2 H, 2 x

NH), 8.08 (d, J = 8.6 Hz, 2 H, H3, H5), 8.03 (d, J = 8.5 Hz, 2 H, H2, H6), 8.01 (s, 1

H, H3), 7.83 (dd, J = 1.6, 6.9 Hz, 1 H, H4), 7.76 (dd, J = 1.6, 8.5 Hz, 1 H, H7), 7.69 (dd,

J = 6.9, 8.5 Hz, 1 H, H6), 7.62-7.57 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.77 (sept, J = 6.8

13 Hz, 1 H, CH), 1.37 (d, J = 6.8 HZ, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ

165.1 (CO2H), 163.5 (C=O), 149.8 (ArC), 148.3 (ArC), 147.5 (ArC), 144.4 (ArC),

137.5 (ArC), 129.4 (ArCH), 128.8 (ArCH), 127.6 (ArCH), 127.1 (ArCH), 126.3

(ArCH), 123.1 (ArCH), 118.5 (ArCH), 26.1 (CH), 23.9 (2 x CH 3). IR (ATR): ν max 3174,

3056, 2955, 2637, 2504, 1698, 1635, 1469, 1403, 1262, 866, 767, 698 cm -1. UV-Vis

-1 -1 (MeOH): λmax 248 (ε 46,700 cm M ) nm. HRMS (+ESI): Found m/z 476.1587,

+ [M+Na] ; C 27 H23 N3O4Na required 476.1581.

289

Methyl 4-(5-(8-Isopropyl-4-(4-bromophenyl)quinolin-2-yl)-1,3,4-oxadiazol-2-yl) benzoate (299a)

This compound was synthesized as described for compound 179 using hydrazide 297b (0.51 g, 0.93 mmol), 4-TsCl (0.41 g, 2.15 mmol) and DIPEA

(0.53 mL, 3.04 mmol) in MeCN (8.0 mL) over 3 h to give the title compound 299a as a white solid

1 (0.14 g, 29%). M.p. 241-243 ºC. H NMR (300 MHz, CDCl 3): δ 8.34 (ddd, J = 0.6, 1.3,

8.1 Hz, 2 H, H3″, H5″), 8.29 (s, 1 H, H3), 8.25 (ddd, J = 0.6, 1 .3, 8.1 Hz, 2 H, H2″,

H6″), 7.75 (dd, J = 1.4, 8.3 Hz, 2 H, H5, H7), 7.71 (ddd, J = 2.2, 2.2, 8.5 Hz, 2 H, H3′,

H5′), 7.58 (dd, J = 7.4 Hz, 8.2 Hz, 1 H, H6), 7.44 (ddd, J = 2.0, 2.0, 6.6 Hz, 2 H, H2′,

H6′), 4.54 (sept, J = 6.7 Hz, 1 H, CH), 3.99 (s, 3 H, OCH 3), 1.50 (d, J = 6.7 Hz, 6 H, 2 x

13 CH 3). C NMR (CDCl 3): δ 166.3 (C=O), 165.1 ((C=N)O), 165.0 ((C=N)O), 149.1

(ArC), 146.5 (ArC), 141.4 (ArC), 136.8 (ArC), 133.2 (ArC), 132.1 (ArCH), 131.3

(ArCH), 130.5 (ArCH), 128.8 (ArC), 127.8 (ArC), 127.4 (ArCH), 127.3 (ArC), 126.6

(ArCH), 123.4 (ArCH), 123.3 (ArCH), 119.6 (ArCH), 52.7 (OCH 3), 28.1 (CH), 23.8 (2 x CH 3). IR (ATR): ν max 2949, 1714, 1534, 1481, 1437, 1273, 1105, 1012, 829, 766, 715

-1 -1 -1 cm . UV-Vis (MeOH): λmax 325 (ε 15,000 cm M ), 288 (31,700), 247 (18,500), 214

+ (23,300) nm. HRMS (+ESI): Found m/z 528.0916, [M+H] ; C 28 H23 BrN 3O3 required

528.0917.

290

Methyl 4-(5-(8-isopropyl-4-(p-tolyl)quinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoate

(299b)

This compound was synthesized as described for compound 179 using hydrazide 297c (0.47 g, 0.98 mmol), 4-TsCl (0.40 g, 2.11 mmol) and DIPEA

(0.54 mL, 3.10 mmol) in MeCN (8.0 mL) over 3 h to give the title compound 299b as a white solid

1 (0.32 g, 70%). M.p. 200-202 ºC. H NMR (300 MHz, CDCl 3): δ 8.34 (ddd, J = 1.8, 1.8,

6.7 Hz, 2 H, H2, H6), 8.31 (s, 1 H, H3), 8.25 (ddd, J = 1.8, 1.8, 6.7 Hz, 2 H, H3,

H5), 7.85 (dd, J = 1.3, 8.3 Hz, 1 H, H4), 7.71 (dd, J = 1.3, 7.3 Hz, 1 H, H7), 7.56 (dd, J

= 7.3, 8.3 Hz, 1 H, H6), 7.47 (ddd, J = 1.8, 1.8, 6.3 Hz, 2 H, H2ɂ, H6ɂ), 7.35 (d dd, J =

1.8, 1.8, 6.3 Hz, 2 H, H3ɂ, H5ɂ), 4.55 (sept, J = 6.9 Hz, 1 H, CH), 3.99 (s, 3 H, OCH 3),

13 2.49 (s, 3 H, CH 3), 1.50 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (300 MHz, CDCl 3): δ

166.3 (C=O), 165.3 (C=N), 165.0 (C=N), 150.5 (ArC), 148.9 (ArC), 146.6 (ArC), 141.4

(ArC), 138.8 (ArC), 135.0 (ArC), 133.2 (ArC), 130.5 (ArCH), 129.7 (ArCH), 129.5

(ArCH), 128.4 (ArCH), 127.9 (ArC), 127.8 (ArC), 127.4 (ArCH), 126.4 (ArCH), 123.9

(ArCH), 119.8 (ArCH), 52.7 (OCH 3), 28.1 (CH), 23.8 (2 x CH 3), 21.5 (CH 3). IR (ATR):

-1 νmax 2949, 1716, 1535, 1496, 1437, 1270, 1104, 819, 768, 715 cm . UV-Vis (MeOH):

-1 -1 λmax 325 (ε 22,500 cm M ), 289 (44,500), 246 (27,100), 210 (36,600) nm. HRMS

+ (+ESI): Found m/z 486.1786, [M+Na] ; C 29 H25 N3O3Na required 486.1788.

291

Methyl 2-fluoro-4-methylbenzoate (301)

To a stirred solution of 2-fluoro-4-methylbenzoic acid 300 (7.00 g,

45.4 mmol) in MeOH (100.0 mL) was added H 2SO 4 (18 M, 5.0 mL) dropwise. The reaction mixture was heated at reflux for 24 h, the solvent removed under reduced pressure and the residue taken up in EtOAc (50.0 mL).

This was washed with NaHCO 3 (25.0 mL) and brine (25.0 mL), dried over Na 2SO 4 and concentrated in vacuo to give the title compound 301 as a yellow oil that crystallized on standing (6.43 g, 84%). M.p. 53-55 °C. Lit. 390 M.p. 51-53 °C. 1H NMR (300 MHz,

CDCl 3): δ 7.83 (dd, J = 7.9, 7.9 Hz, 1 H, H6), 7.00 (ddd, J = 0.8, 0.8, 7.9 Hz, 1 H, H5),

6.94 (ddd, J = 0.8, 0.8, 11.8 Hz, 1 H, H3), 3.91 (s, 3 H, OCH 3), 2.39 (s, 3 H, CH3).

Methyl 2-fluoro-4-formylbenzoate (303)

To a stirred solution of methyl 2-fluoro-4-methylbenzoate 301

(6.43 g, 38.2 mmol) in CCl 4 (40.0 mL) was added NBS (17.81 g,

100.06 mmol) and benzoyl peroxide (0.65 g, 2.03 mmol). This mixture was heated at reflux for 48 h, cooled to r.t., the precipitate collected via filtration and washed with CCl 4. The combined filtrates were concentrated under reduced pressure to give methyl 4-(dibromomethyl)-2-fluorobenzoate (13.80 g) 302 as a yellow oil (13.80 g) that was used directly in the next step. The crude 302 was taken up in EtOH (100.0 mL) and heated to 50 °C before a solution of AgNO 3 (14.98 g, 88.19 mmol) in H 2O (20.0 mL) was added dropwise. The mixture was stirred at 50 °C for 1 h, cooled to r.t. and the precipitate collected via filtration. The filtrate was concentrated under reduced pressure and extracted with EtOAc (50.0 mL), washed with H 2O (10.0 mL) and brine (10.0 mL), dried over Na 2SO 4 and purified by flash column 292

chromatography (7% EtOAc:hexanes) to give the title compound 303 as colourless needles (2.20 g, 35% (2 steps)). M.p. 74-76 °C. Lit. 390 M.p. 75-76 °C. 1H NMR (300

MHz, CDCl 3): δ 10.04 (d, J = 1.7 Hz, 1 H, CHO), 8.10 (dd, J = 6.8, 8.0 Hz, 1 H, H6),

7.72 (dd, J = 1.5, 8.0 Hz, 1 H, H5), 7.64 (dd, J = 1.5, 10.1 Hz, 1 H, H3), 3.97 (s, 3 H,

OCH 3).

3-Fluoro-4-(methoxycarbonyl)benzoic acid (304)

To a stirred solution of methyl 2-fluoro-4-formylbenzoate 303

(2.20 g, 13.09 mmol) and sulfamic acid (1.46 g, 15.03 mmol) in a mixture of MeCN:H 2O (2:1, 30.0 mL) was added a solution of

NaClO 2 (0.82 g, 9.07 mmol) in H 2O (10.0 mL) dropwise. The mixture was stirred for 20 h before being poured into a mixture of sat. Na 2SO 3 (20.0 mL) and HCl (1 M, 50.0 mL).

The mixture was extracted with EtOAc (3 x 25.0 mL), the combined extracts washed with brine (20.0 mL), dried over Na 2SO 4 and concentrated under reduced pressure. The solid was collected, washed with Et 2O and air dried to give the title compound 304 as a white powder (1.27 g, 49%). M.p. 212-214 °C. Lit. 390 M.p. 211-212 °C. 1H NMR (300

MHz, d6-DMSO): δ 8.00 (dd, J = 7.7, 7.7 Hz, 1 H, H5), 7.85 (dd, J = 1.6, 8.1 Hz, 1 H,

H2), 7.76 (dd, J = 1.5, 11.3 Hz, 1 H, H6), 3.88 (s, 3 H, OCH 3).

293

Methyl 2-fluoro-4-(2-(8-isopropyl-4-phenylquinoline-2-carbonyl)hydrazine-1- carbonyl)benzoate (305)

This compound was synthesized as described for compound 185 using hydrazide 289a (0.62 g, 2.03 mmol), acid 304 (0.41 g, 2.08 mmol),

EDCI (0.41 g, 2.16 mmol) and HOBt (0.39 g,

2.17 mmol) in DMF (3.0 mL) over 24 h to give the title compound 305 as a white solid (0.58 g, 73%). M.p. 178-180 ºC. 1H[19 F] NMR

(300 MHz, d 6-acetone): δ 10.51 (bs, 2 H, 2 x NH), 8.13 (s, 1 H, H3), 8.07 (d, J = 8.0 Hz,

1 H, H6), 7.94 (dd, J = 1.6, 8.2 Hz, 1 H, H5), 7.84-7.81 (m, 3 H, H7, H3, H5), 7.68

(dd, J = 7.8, 7.8 Hz, 1 H, H6), 7.63-7.57 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4. 62 (sept, J

13 = 6.9 Hz, 1 H, CH), 3.94 (s, 3 H, OCH 3), 1.43 (d, J = 69 Hz, 6 H, 2 x CH 3). C NMR

(75 MHz, CDCl 3): δ 164.5 (CO2CH 3), 163.4 (C=O), 151.5 (ArC), 149.2 (ArC), 148.1

(ArC), 145.8 (ArC), 139.7 (ArC), 139.0 (ArC), 133.3 (ArCH), 130.4 (ArCH), 129.6

(ArCH), 129.5 (ArCH), 128.8 (ArCH), 127.1 (ArCH), 124.4 (ArCH), 124.1 (ArCH),

119.4 (ArCH), 117.2 (ArC), 116.9 (ArCH), 52.9 (OCH 3), 28.0 (CH), 24.0 (2 x CH 3). IR

(ATR): ν max 3316, 3210, 3058, 2958, 1722, 1622, 1556, 1459, 1250, 1073, 845, 764,

-1 -1 -1 699 cm . UV-Vis (MeOH): λmax 300 (ε 11,200 cm M ), 247 (50,400), 210 (35,700)

+ nm. HRMS (+ESI): Found m/z 508.1643, [M+Na] , C28 H24 FN 2O4Na requires 508.1643.

294

Methyl 2-fluoro-4-(5-(8-isopropyl-4-phenylquinolin-2-yl)-1,3,4-oxadiazol-2-yl) benzoate (306)

This compound was synthesized as described for compound 179 using hydrazide 305 (0.58 g, 1.49 mmol), DIPEA (1.0 mL, 5.74 mmol) and 4-TsCl

(0.61 g, 3.20 mmol) in MeCN (8.0 mL) over 3 h to give the title compound 306 as a white solid following crystallization from MeOH

1 19 (0.30 g, 43%). M.p. 201-203 ºC. H[ F] NMR (300 MHz, CDCl 3): δ 8.16 (s, 1 H, H3),

8.04 (d, J = 8.1 Hz, 1 H, H6), 7.98 (d, J = 8.1 Hz, 1 H, H5), 7.90 (s, 1 H, H3), 7.70

(d, J= 8.3 Hz, 1 H, H5), 7.61 (d, J = 7.0 Hz, 1 H, H7), 7.48-7.43 (m, 6 H, H6, H2ɂ, H3ɂ,

H4ɂ, H5ɂ, H6ɂ), 4.41 (sept, J = 7.0 Hz, 1 H, CH), 3.87 (s, 3 H, OCH 3), 1.37 (d, J = 7.0

13 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, CDCl 3): δ 165.5 (C=O), 164.2 (C=N), 150.5

(ArC), 149.0 (ArC), 146.6 (ArC), 141.2 (ArC), 137.9 (ArC), 133.4 (ArCH), 129.8

(ArCH), 129.5 (ArC), 128.8 (ArCH), 128.6 (ArCH), 127.7 (ArC), 126.5 (ArCH), 123.8

(ArCH), 122.7 (ArCH), 121.6 (ArC), 119.8 (ArCH), 116.1 (ArCH), 115.8 (ArCH), 52.9

(OCH 3), 28.1 (CH), 23.8 (2 x CH 3). IR (ATR): ν max 3058, 2950, 1706, 1533, 1486,

-1 -1 - 1436, 1279, 1089, 885, 770, 747, 691 cm . UV-Vis (DMF): λmax 329 (ε 23,100 cm M

1 + ), 288 (40,100) nm. HRMS (+ESI): Found m/z 468.1710, [M+H] , C 28 H23 FN 2O3 requires 468.1718.

295

2-Fluoro-4-(5-(8-isopropyl-4-phenylquinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoic acid

(307)

This compound was synthesized as described for compound 272b using ester 306 (0.28 g, 0.60 mmol), in 0.25 M NaOH/THF (8.0 mL) over 4 h to give the title compound 307 as a white solid

1 following work-up (0.21 g, 77%). M.p. 274-276 ºC. H NMR (300 MHz, d 6-DMSO): δ

8.26 (s, 1 H, H3), 8.16-8.05 (m, 3 H, H3″, H5″, H6″), 7.84 (dd, J = 1.3, 6.9 Hz, 1 H,

H5), 7.77 (dd, J = 1.6, 8.5 Hz, 1 H, H7), 7.70 (dd, J = 6.9, 8.5 Hz, 1 H, H6), 7.65-7.62

(m, 5 H, H2′, H3′, H4′, H5′, H6′), 4.90 (sept, J = 7.3 Hz, 1 H, CH), 1.44 (d, J = 7.3 Hz,

13 6 H, 2 x CH 3). ). C NMR (75 MHz, d 6-DMSO): δ 164.3 (C=O), 163.4 ((C=N)O),

149.9 (ArC), 147.9 (ArC), 145.6 (ArC), 140.9 (ArC), 137.0 (ArC), 133.3 (ArCH), 129.5

(ArCH), 128.8 (ArC), 126.7 (ArCH), 123.3 (ArCH), 122.8 (ArCH), 119.4 (ArCH),

115.1 (ArCH), 27.2 (CH), 23.5 (2 x CH 3). IR (ATR): ν max 3154, 3071, 2961, 1728,

-1 1537, 1489, 1224, 1147, 1088, 894, 749, 702 cm . UV-Vis (DMF): λmax 330 (ε 32,000

-1 -1 + cm M ), 266 (46,300) nm. HRMS (+ESI): Found m/z 454.1559, [M+H] , C27 H21 FN 2O3 requires 454.1561.

8-Isopropyl-4-phenylquinolin-2(1 H)-one (312)

A mixture of aniline 273a (1.36 g, 10.1 mmol) and ethyl benzoylacetate

246b (1.80 mL, 10.4 mmol) was heated at reflux for 48 h before

Eaton’s reagent (15.0 mL) was added. This mixture was heated at 50

°C for 24 h before being poured into ice water and neutralized with

NaHCO 3. The precipitate was collected via filtration to give the title compound 312 as 296

1 colourless prisms (0.11 g, 4%). M.p. 170-172 °C. H-NMR (300 MHz, CDCl 3): δ 8.24

(s, 1 H, H3), 7.87 (dd, J = 1.4, 8.4 Hz, 1 H, H5), 7.75 (dd, J = 1.1, 7.2 Hz, 1 H, H7),

7.62 (dd, J = 7.80 Hz, 1 H, H6), 7.57-7.50 (m, 5 H, H2ɂ, H3ɂ, H4ɂ, H5ɂ, H6ɂ), 4.32 (sept,

13 J = 6.9 Hz, 1 H, CH), 1.46 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C-NMR (300 MHz, CDCl 3):

δ 164.6 (C=O), 152.4 (C 4), 147.6 (), 144.4 (C8a), 144.0 (), 137.7 (C1ɂ), 129.7 (CH),

129.3 (C6), 129.1 (CH), 129.0 (), 128.9 (CH), 126.9 (C7), 124.1 (C5), 119.1 (C3), 28.0

(CH), 23.6 (2 x CH 3). IR (ATR): ν max 2955, 2867, 2591, 1684, 1442, 1264, 1135, 919,

-1 -1 -1 769, 702 cm . UV-Vis (MeOH): λmax 301 ( ε 6,320 cm M ), 242 (34,800) nm. HRMS

+ (+ESI): Found m/z 264.1384, [M+H] ; C 18 H18 NO required 264.1383..

N-(2-Isopropylphenyl)-3-oxo-3-phenylpropanamide (313)

A mixture of ethyl benzoylacetate 246b (4.0 mL, 23.1 mmol) and aniline 273a (2.0 mL, 14.3 mml) was heated at 145 °C for

20 min. The mixture was cooled in an ice bath before H 2SO 4

(18 M, 2.2 mL) was added and the mixture stirred for 2 h. The mixture was then neutralized with NaOH (2 M, 25.0 mL), the precipitate collected via filtration and recrystallized from DMF to give the title compound 313 as colourless prisms (0.17 g,

1 4%). H NMR (300 MHz, CDCl 3): δ 9.55 (s, 1 H, NH), 8.06 (dd, J = 1.6, 8.8 Hz, 2 H,

H2, H6), 7.85 (dd, J = 1.9, 7.8 Hz, 1 H, H3ɂ), 7.67 (ddd, J = 1.6, 7.5, 7.5 Hz, 1 H, H4),

7.54 (ddd, J = 1.6, 7.5, 7.5 Hz, 2 H, H3, H5), 7.32 (dd, J = 2.1, 7.5 Hz, 1 H, H6ɂ), 7.20

(dddd, J = 2.1, 7.2, 7.2, 7.2, 2 H, H4ɂ, H5ɂ), 4.19 (s, 2 H, CH 2), 3.17 (sept, J = 7.0 Hz, 1

13 H, CH), 1.28 (d, J = 7.0 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, CDCl3): δ 197.26

(C=O), 164.3 (CONH), 140.0 (ArC), 136.1 (ArC), 134.6 (ArCH), 134.1 (ArC), 129.1 297

(ArCH), 128.7 (ArCH), 126.5 (ArCH), 125.9 (ArCH), 125.7 (ArCH), 123.9 (ArCH),

44.9 (CH 2), 28.1 (CH), 23.1 (2 x CH 3).

Ethyl 4-(8-isopropyl-4-phenylquinoline-2-carboxamido)benzoate (314)

This compound was synthesized as described for compound 185 using acid 290a (0.29g, 1.00 mmol), p-benzocaine (0.18 g, 1.06 mmol), EDCI (0.29 g, 1.52 mmol) and HOBt (0.17 g, 1.05 mmol) in DMF (4.0 mL) over 24 h to give the title compound 314 as a

1 colourless solid (0.40 g, 91%). M.p. 167-169 ºC. H NMR (300 MHz, d 6-acetone): δ

10.66 (s, 1 H, NH), 8.24 (s, 1 H, H3), 8.07 (d, J = 1.9 Hz, 4 H, H2″, H3″, H5″, H6″),

7.85 (d, J = 7.8 Hz, 2 H, H5, H7), 7.69 (dd, J = 7.8, 7.8 Hz, 1 H, H6), 7.64-7.58 (m, 5

H, H2′, H3′, H4′, H5′, H6′), 4.62 (sept, J = 6.9 Hz, 1 H, CH), 4.35 (q, J = 7.1 Hz, CH 2),

13 1.49 (d, J = 6.9 Hz, 6 H, 2 x CH 3), 1.38 (t, J = 7.1 Hz, CH 2CH 3). C NMR (CDCl 3): δ

166.3 (C=O), 163.6 (C=O), 151.9 (ArC), 148.8 (ArC), 148.5 (ArC), 145.6 (ArC), 143.3

(ArC), 138.9 (ArC), 131.4 (ArCH), 130.4 (ArCH), 129.7 (ArCH), 129.7 (ArCH), 129.6

(ArCH), 128.9 (ArCH), 127.2 (ArCH), 126.8 (ArC), 124.5 (ArCH), 120.0 (ArCH),

119.9 (ArC), 119.2 (ArCH), 61.3 (CH 2), 28.4 (CH), 23.9 (2 x CH 3), 14.6 (CH 2CH 3). IR

- (ATR): ν max 3337, 2340, 1693, 1687, 1519, 1458, 1401, 1271, 1168, 1098, 766, 696 cm

1 -1 -1 . UV-Vis (MeOH): λmax 295 (ε 30,800 cm M ), 248 (36,900), 210 (43,100) nm.

+ HRMS (+ESI): Found m/z 439.2015, [M+H] ; C 28 H27 N2O3 required 439.2016.

298

4-(8-Isopropyl-4-phenylquinoline-2-carboxamido)benzoic acid (315)

Ester 314 (0.37 g, 0.84 mmol) was stirred in 2 M

NaOH/MeOH (1:1, 8.0 mL) at 50 °C for 3 h before being concentrated under reduced pressure and acidified with 2 M HCl. The precipitate was collected via filtration to give the title compound 315 as a

1 colourless solid (0.33 g, 96%). M.p. 289-291 ºC. H NMR (300 MHz, d 6-DMSO): δ

10.73 (s, 1 H, NH), 8.09 (s, 1 H, H3), 8.02 (bs, 4 H, H2″, H3″, H5″, H6″), 7.81 (dd, J =

1.4, 6.9 Hz, 1 H, H5), 7.77 (dd, J = 1.6, 8.5 HZ, 1 H, H7), 7.69 (dd, J = 6.9, 8.5 Hz, 1 H,

H6), 7.63-7.57 (m, 5 H, H2′, H3′, H4′, H5′, H6′), 4.66 (sept, J = 6.9 Hz, 1 H, CH), 1.42

13 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75 MHz, d 6-DMSO): δ 167.0 (COOH), 163.1

(CONH), 150.2 (ArC), 147.9 (ArC), 144.2 (ArC), 141.9 (ArC), 137.4 (ArC), 130.4

(ArCH)m 129.4 (ArCH), 128.8 (ArCH), 127.2 (ArC), 126.5 (ArCH), 126.4 (ArCH),

123.2 (ArCH), 119.7 (ArCH), 118.4 (ArCH), 26.8 (CH), 23.6 (2 x CH 3). IR (ATR): ν max

3339, 2957, 2862, 2663, 2549, 1685, 1604, 1522, 1422, 1290, 1171, 898, 851, 765, 697

-1 -1 -1 cm . UV-Vis (MeOH): λmax 296 (ε 19,200 cm M ), 248 (34,400), 208 (36,900) nm

+ HRMS (+ESI): Found m/z 411.1697, [M+H] ; C 26 H23 N2O3 required 411.1703.

Methyl 8-isopropyl-4-methoxyquinoline-2-carboxylate (316)

A solution of quinolone 285 (0.64 g, 2.61 mmol) in DMSO (15.0 mL) was treated with K 2CO 3 (0.92 g, 6.66 mmol) and heated at 70

°C for 1 h. After cooling to 35 °C, iodomethane (1.0 mL, 16.06 mmol) was added and the mixture stirred for 18 h. The mixture was concentrated under reduced pressure and poured over crushed ice. The precipitate was collected via 299

filtration to give the title compound 316 as a white solid (0.62 g, 92%). M.p. 72-74 °C.

1 H NMR (300 MHz, d 6-acetone): δ 8.08 (dd, J = 1.7, 8.2 Hz, 1 H, H5), 7.73 (dd, J = 1.7,

7.2 Hz, 1 H, H7), 7.62 (dd, J = 7.2, 8.2 Hz, 1 H, H6), 7.58 (s, 1 H, H3), 4.42 (sept, J =

6.9 Hz, 1 H, CH), 4.18 (s, 3 H, OCH 3), 3.98 (s, 3 H, CO 2CH 3), 1.36 (d, J = 6.9 Hz, 6 H,

13 2 x CH 3). C NMR (75 MHz, d 6-acetone): δ 167.0 (ArC), 164.3 (ArC), 149.0 (ArC),

128.4 (ArCH), 127.1 (ArCH), 122.9 (ArC), 120.0 (ArCH), 100.6 (ArCH), 56.7 (OCH 3),

53.0 (CO2CH 3), 28.2 (CH), 23.9 (2 x CH 3). IR (ATR): ν max 2954, 2861, 1719, 1571,

-1 1504, 1461, 1433, 1348, 1241, 1112, 1030, 853, 764 cm . UV-Vis (MeOH): λmax 293 (ε

7,030 cm -1M-1), 240 (43,500), 213 (21,800) nm. HRMS (+ESI): Found m/z 282.1098,

+ (M+Na) ; C15 H17 NO 3Na required 282.1101.

8-Isopropyl-4-methoxyquinoline-2-carbohydrazide (317)

This compound was synthesized as described for compound

289a using ester 316 (0.51 g, 1.98 mmol) and hydrazine monohydrate (0.50 mL, 9.50 mmol) at reflux in MeOH (5.0 mL) over 2 h to give the title compound 317 as colourless prisms (0.48 g, 93%). M.p.

1 142-144 °C. H NMR (300 MHz, d 4-MeOD): δ 8.05 (dd, J = 1.5, 8.4 Hz, 1 H, H5), 7.67

(dd, J = 1.5, 7.4 Hz, 1 H, H7), 7.61 (s, 1 H, H3), 7.54 (dd, J = 7.4, 8.4 Hz, 1 H, H6),

4.38 (sept, J = 7.0 Hz, 1 H, CH), 4.12 (s, 3 H, OCH 3), 1.37 (d, J = 7.0 Hz, 6 H, 2 x

13 CH 3). C NMR (75 MHz, d 4-MeOD): δ 166.1 (ArC), 165.4 (ArC), 150.3 (ArC), 148.6

(ArC), 146.7 (ArC), 128.2 (ArCH), 127.4 (ArCH), 123.3 (ArC), 120.4 (ArCH), 98.2

(ArCH), 56.7 (OCH 3), 28.4 (CH), 24.0 (2 x CH 3). IR (ATR): ν max 3274, 3082, 2940,

-1 1664, 1590, 1476, 1406, 1340, 1024, 980, 871, 772 cm . UV-Vis (MeOH): λ max 293 300

(8,040 cm -1M-1), 240 (46,000) nm. HRMS (+ESI): Found m/z 282.1212, [M+Na] +;

C14 H17 N2O2Na requires 282.1213.

Methyl 4-(2-(8-isopropyl-4-methoxyquinoline-2-carbonyl)hydrazine-1-carbonyl) benzoate (318)

This compound was synthesized as described for compound 185 using hydrazide 317 (0.28 g,

1.09 mmol), mono-methyl terephthalate (0.21 g,

1.17 mmol), EDCI (0.22 g, 1.16 mmol) and

HOBt (0.31 g, 1.95 mmol) in DMF (2.0 mL) over 24 h to give the title compound 318 as a white solid after flash column chromatography (25% EtOAc:hexanes) (0.18 g,

1 39%). M.p. 108-110 ºC. H NMR (300 MHz, d 6-acetone): δ 10.38 (bs, 2 H, 2 x NH),

8.15-8.09 (m, 5 H, H5, H2ɂ, H3ɂ, H5ɂ, H6ɂ), 7.75 (dd, J = 1.6, 7.4 Hz, 1 H, H7), 7.65 (s,

1 H, H3), 7.63 (dd, J = 7.4, 8.2 Hz, 1 H, H6), 4.50 (sept, J = 6.9 Hz, 1 H, CH), 4.19 (s, 3

13 H, OCH 3), 3.93 (s, 3 H, CO 2CH 3), 1.37 (d, J = 6.9 Hz, 6 H, 2 x CH 3). C NMR (75

MHz, d 6-acetone): δ 166.6 (ArC), 165.8 (ArC), 164.9 (ArC), 164.1 (ArC), 150.2 (ArC),

148.4 (Arc), 146.3 (ArC), 137.7 (ArC), 134.0 (ArC), 130.3 (ArCH), 128.6 (ArCH),

128.2 (ArCH), 127.2 (ArCH), 123.1 (ArC), 120.2 (ArCH), 98.4 (ArCH), 56.8 (OCH 3),

52.7 (CO2CH3), 27.9 (CH), 24.0 (2 x CH 3). IR (ATR): ν max 3438, 3314, 2949, 1720,

-1 - 1930, 1475, 1277, 1021, 1107, 767, 713 cm . UV-Vis (MeOH): λmax 244 (ε 43,600 cm

1 -1 + M ) nm. HRMS (+ESI): Found m/z 422.1709, [M+H] , C 23 H24 N2O5 requires

422.1710.

301

Methyl 4-(5-(8-isopropyl-4-methoxyquinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoate

(319)

This compound was synthesized as described for compound 179 using hydrazide 318 (0.19 g, 0.45 mmol), DIPEA (0.30 mL, 1.65 mmol) and 4-TsCl

(0.29 g, 1.52 mmol) in MeCN (5.0 mL) over 4 h to give the title compound 319 as a

1 white solid after purification (0.13 g, 71%). M.p. 192-194 ºC. H NMR (300 MHz, d 6- acetone): δ 8.35 (ddd, J = 1.6, 2.1, 8.2 Hz, 2 H, H 2ɂ, H6ɂ), 8.27 (ddd, J = 1.6, 2.1, 8.2

Hz, 2 H, H3ɂ, H5ɂ), 8.13 (dd, J = 1.5, 8.3 Hz, 1 H, H5), 7.82 (s, 1 H, H3), 7.78 (dd, J =

1.5, 7.2 Hz, 1 H, H7), 7.65 (dd, J = 7.2, 8.3 Hz, 1 H, H6), 4.47 (sept, J = 6.9 Hz, 1 H,

CH), 4.28 (s, 3 H, OCH 3), 3.96 (s, 3 H, CO 2CH 3), 1.44 (d, J = 6.9 Hz, 6 H, 2 x CH 3).

13 C NMR (75 MHz, d 6-acetone): δ 166.4 (C=O), 165.6 (ArC), 164.4 (ArC), 148.6 (ArC),

144.0 (ArC), 134.0 (ArC), 131.1 (ArCH), 128.8 (ArC), 128.4 (ArCH), 127.9 (ArCH),

127.6 (ArCH), 120.3 (ArCH), 99.5 (ArCH), 57.0 (OCH 3), 52.8 (CO 2CH3), 28.5 (CH),

23.8 (2 x CH 3). IR (ATR): ν max 2959, 1718, 1591, 1542, 1508, 1276, 1099, 1024, 890,

-1 -1 -1 761, 710 cm . UV-Vis (DMF): λmax 317 (ε 27,100 cm M ), 288 (45,000) nm. HRMS

+ (+ESI): Found m/z 426.1422, [M+Na] , C23 H21 N2O4Na requires 426.1424.

4-(5-(8-Isopropyl-4-methoxyquinolin-2-yl)-1,3,4-oxadiazol-2-yl)benzoic acid (320)

This compound was synthesized as described for compound 272b using ester 319 (101.7 mg, 0.25 mmol) in 0.25 M NaOH/THF (8.0 mL) over 4 h to give the title compound 320 as a white solid (52.8 mg, 56%). M.p. 273-275 ºC. 1H

NMR (300 MHz, d 6-DMSO): δ 8.27 (ddd, J = 2.1, 2.1 8.4 Hz, 2 H, H2ɂ, H6ɂ), 8.20 (ddd, 302

J = 2.1, 2.1, 8.4 Hz, 2 H, H3ɂ, H5ɂ), 8.07 (dd, J = 1.5, 8.3 Hz, 1 H, H5), 7.78 (s, 1 H,

H3), 7.77 (dd, J = 1.4, 7.2 Hz, 1 H, H7), 7.65 (dd, J = 7.2, 8.3 Hz, 1 H, H6), 4.39 (sept,

13 J = 7.0 Hz, 1 H, CH), 4.19 (s, 3 H, OCH 3), 1.39 (d, J = 7.0 Hz, 6 H, 2 x CH 3). C NMR

(75 MHz, CDCl 3): δ 166.5 (C=O), 164.4 (ArC), 163.0 (ArC), 147.2 (ArC), 145.9 (ArC),

142.6 (ArC), 133.9 (ArC), 130.4 (ArCH), 127.6 (ArCH), 127.1 (ArCH), 126.9 (ArCH),

121.2 (ArC), 119.2 (ArCH), 98.9 (ArCH), 56.7 (OCH 3), 27.1 (CH), 23.5 (2 x CH 3). IR

- (ATR): ν max 2954, 2862, 2668, 2546, 1694, 1589, 1509, 1427, 1289, 1029, 765, 708 cm

1 -1 -1 . UV-Vis (MeOH): λmax 315 (ε 27,400 cm M ), 285 (46,600), 214 (29,900) nm.

+ HRMS (+ESI): Found m/z 390.1442, [M+H] , C 22 H20 N2O4 requires 390.1448.

8.3 Biological assays

8.3.1 Cell culture

All cell lines were cultured under standard conditions at 37 °C in 5% CO2 as an adherent monolayer in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,

USA) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific).

Hygromycin B (1 µL/mL) was added to induce expression of the MEP-4 vector, which had previously been transfected to the BE(2)-C clones. 84

8.3.2 Cell viability assays

Cell viability was measured by the standard Alamar blue assay, as previously described. 413 Briefly, cells were allowed to attach for 24 h in 6 replicate wells of 96-well culture plates. The cells were then continuously exposed to serial dilutions of the compound (0.1-50 µM) for 72 h. Cell viability was determined by addition of 22 µL of 303

Alamar blue (Resazurin) reagent, recording comparative 0 h and 5 h values, using a

Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm. The cell viability of each plate was calculated as a percentage compared to matched DMSO controls (0.5%). GraphPad Prism 6 was used to calculate the IC 50 value from a curve of Log concentration vs percentage of growth inhibition using a least squares fit. The mean (± SEM) is shown for three independent experiments.

8.3.3 Immunoblot analysis

Cells were allowed to attach over 24 h to duplicate T25 culture flasks. Cells were then continuously exposed to the indicated compound at an appropriate concentration in

DMSO, or matched DMSO (0.1%) control, for 24, 48 or 72 h. Cells were harvested and washed twice with cold PBS, then either lysed immediately wherever possible or stored at -70 °C.

Lysis was performed with ice cold RIPA buffer. Cells were incubated in buffer for 30 minutes on ice, with periods of brief (10 sec) vortex every 10 min. Cell lysates were then centrifuged at 16,000 rpm at 4 °C for 20 min and the supernatant transferred to new tubes for storage.

Quantification of proteins was conducted using the Pierce BCA protein Assay Kit

(Thermo) as per the manufacturer’s instructions.

Samples containing 30 µg of protein (or otherwise specified) were mixed with 4x XT protein loading buffer (Bio-Rad) and 20x reducing agent, made to an appropriate volume in RIPA buffer and RNase free H 2O, and boiled at 95 °C for 5 minutes. Samples were quickly spun and placed on ice before loading. The samples were loaded onto 10% polyacrylamide criterion gels manufactured by Bio-Rad. Gels were run for 20 minutes 304

at 80 V and then increased to 150 V until the dye front reached the bottom of the gel in

Tris-Glycine-SDS solution. The proteins on the gel were transferred onto a nitrocellulose membrane at 4 °C, at 30 V for 2 h in 20% methanol Tris-Glycine solution. Membranes were then stained with Ponceau-S and trimmed for particular protein sizes. Membranes were blocked in 10% skim milk in TBST for 2 h, rinsed with

TBST (3 x 5 min), before incubation with 1° antibody for 2 h at r.t. or overnight at 4 °C

(RARα: 1:500, RARβ: 1:1,000, RARγ: 1:500, Caspase 3: 1:1,000, GAPDH: 1:10,000) in 0.5% milk TBST. Membranes were washed in TBST (3 x 5 min) and incubated with

2° antibody for 2 h at r.t. (Rabbit: 1:2,000, Mouse: 1:2,000). Membranes were then washed in TBST (3 x 5 min). Chemiluminescence detection was performed using the

SuperSignal reagents, which were mixed at a 1:1 ratio and poured over membranes and incubated in the dark for 5 minutes, before excess solution was removed and plastic film was used to encase the membrane. X-ray films were exposed to the membrane in the dark and placed in X-Ray film developer for a range of exposures times. QuantityOne software (Bio-Rad) was used to quantify the resulting protein bands. If re-probing of another antibody was required, membranes were striped for 20 min at r.t. and then washed with TBST (2 x 10 min).

305

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