Design, Synthesis and Biological Evaluation of New Molecules To

Design, Synthesis and Biological Evaluation of New

Molecules to Selectively Target Specific Cancers

A Dissertation Submitted to the

Graduate School of the University of Cincinnati

In Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy (Ph.D.)

in the Department of Chemistry

of the College of Arts and Science

Gurdat Premnauth

Bachelor of Science (B.S.), University of Haute Alsace, France, 2013

Master of Science (M.S.), University of Toledo, USA, 2016

Engineer’s degree, National School of Chemistry of Mulhouse, France, 2016

Dissertation Advisor: Edward J. Merino, PhD

University of Cincinnati December 2020

Abstract

Cancer remains one of the leading causes of mortality in the world. Even with the improvement of treatments in multiple type of cancer, current chemotherapies are highly toxic and associated with severe acute side effects due to the low selectivity of the existing drugs. Additionally, multiple cancers develop chemotherapeutic resistance and new targets need to be investigated. Amongst them, Ras is one of the most common aberration found in cancer with approximately 30% of cancer containing the mutated oncogenes. Ras- targeted therapy with small molecule inhibitor could be the key to better and safer treatments. Moreover, the use of targeted therapy with drug conjugate and delivery vehicles such as aptamers, antibodies or targeting peptides could help bring selectivity to treatments and reduce off-target toxicity.

This dissertation focuses on the development of both targeted therapy strategies for cancer treatment. In the first half, we report the identification of IODVA1 as the active compound of NSC124205 for the treatment of Ras-driven cancer models in cellulo and in vivo.

Identification of the IDOVA1 side products led me to synthesize GUPR-195 followed by the synthesis of several related molecules. In the other half, we focus on decreasing toxic off-target inhibitors effect using targeting therapy. Here, we developed two new reactive oxygen species (ROS)-sensitive linkers for selective drug release to leukemic cells. Indeed, cancer cells are known to have higher levels of ROS than their healthy counterparts. By using a ROS-sensitive linker for the conjugation of a cytotoxic payload to a delivery vehicle, it is possible to double the selectivity of drug release. We report in the last chapter of the dissertation the development of a self-cyclizing ROS-sensitive linker and an oxalamide ROS-sensitive linker.

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

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I dedicate this thesis to my parents, Sanmattie and Pascal Renaud. Thank you for inspiring me and encouraging me to pursue my dreams, even the wildest.

Acknowledgements

First, I would like to thank my supervisor Dr. Edward J. Merino for his constant support and guidance, for sharing his knowledge, and for mentoring me into becoming the best scientific version of myself. I would also like to thank my committee members, Dr. David

Smithrud and Dr. In-Kwon Kim, for their questions and advices during my yearly evaluations, and for accepting to be part of my PhD journey.

I would like to thank a few people I worked with during my time at the University of

Cincinnati: Dr. Stephen Macha for helping me solve complex synthetic problems with the help of mass spectrometry. My collaborators Dr. Nicolas Nassar from Cincinnati

Children’s Hospital and Dr. Joo-Youp Lee from the department of Chemical Engineering for helping me to have a better understanding of some aspects of my projects, and Dr. Julio

Landero for teaching me so much about analytical instruments.

I would like to thank my lab members, past and present, from whom I learned so much.

Thank you to Prasadini Sevenerathne for helping me with aspects of my projects, sharing a lab with you was a great experience. Thank you to my other lab members Nazanin

Moktharpour and Alyssa Sterling and to the previous members Dr. Jing Liu, Dr. Haizhou

Zhu, Dr. Purujit Gurjar, Dr. Kay Earnest and Dr. Safnas Abdulsalam for our many scientific discussions and for your help during my early doctorate years. Being in the Merino group with such a diverse and pluridisciplinar group of scientists was a great learning experience.

I am also very grateful to the great mentors throughout my life. Thank you to Dr. Amanda

Bryant-Friedrich for telling me that one day I could become a doctor and do great things if

I decided to. Thank you to Dr. Peter Andreana for teaching me so much about organic chemistry, more specifically about reactions mechanisms which became one of my favorite things, during my Master at the University of Toledo. I would also like to thank my other early life mentors: Mr. Sanguilono and Mr. Rouquas in middle school and Mr. Bossom in high school that taught me that I was allowed to dream big.

Most importantly I would like to thank my parents. Thanks to my mom for encouraging me and showing me what resilience looks like. I could not do this without her constant love and support. I want to thank my dad for everything that I am today. I would not be where

I am today without him, and I hope that he is seeing all of this and is proud.

I want to thank my sister Ronica for always being there for me and for keeping me grounded. I would also like to thank my brother Theophile, my nephew Klayron, and the rest of my family that supported me during the entirety of my journey.

I would also like to thank my support system. My two best friends Charlotte Sorel and

Jason Muller, thank you for our daily conversations about life and science, and for always being there for the important moments of my life. Thank you to my Cincinnati support system, my friends, that made me feel like family and made sure I was never alone here.

Thank you to my other friends across the world in French Guiana, France, Australia,

Canada and other cities in the US, I am very grateful that I have so many of you sticking with me throughout my nomad adventures.

Table of Contents

Abstract ...... ii

Acknowledgements ...... v

Table of Contents ...... vii

List of Figures ...... xvii

List of Abbreviations ...... xxvi

List of Symbols ...... xxix

Chapter 1: Ras inhibition by small molecules for the treatment of cancer ...... 1

1.1 Ras proteins and cancer ...... 1

1.1.1 Ras family ...... 1

1.1.2 Ras overactivation in multiple cancers ...... 2

1.2 Direct inhibition of the Ras proteins ...... 4

1.2.1 Inhibition of nucleotide exchange: GEF binding site ...... 4

1.2.2 Allosteric Inhibition ...... 7

1.2.3 Ras-effector interaction inhibitors ...... 10

1.3 Conclusion and dissertation goal...... 11

vii

Chapter 2: Discovery of IODVA1, the active compound of NSC124205 against RAS- driven cancer model ...... 13

2.1 Introduction ...... 13

2.2 From NSC124205 to IODVA1 ...... 15

2.2.1 NSC124205, a mixture of multiple compounds ...... 15

2.2.2 Literature search reveals possible structures leading to IODVA1 ...... 19

2.3 Results and Discussions ...... 20

2.3.1 Synthesis of hypothetical molecules present in NSC124205 ...... 20

2.3.2 Characterization and identification of synthesized derivatives ...... 23

2.3.3 IODVA1 is active against RAS-driven cancer models ...... 25

2.3.3.1 IODVA1 stops the proliferations of cells harboring activated RAS ...... 25

2.3.3.2 IODVA1 reduces breast cancer and lung cancer in vivo ...... 26

2.4 Experimental section ...... 28

2.4.1 Materials ...... 28

2.4.2 Synthetic methods...... 30

2.4.2.1 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-4,5-di(pyridin-2-yl)-4H-

imidazol-4-ol (1) ...... 30

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2.4.2.2 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5-(pyridin-2-yl)-5-

(pyridin-3-yl)-3,5-dihydro-4H-imidazol-4-one (2) ...... 33

2.4.2.3 Synthesis of N2,N5-bis(1H-benzo[d]imidazol-2-yl)-3a,6a-di(pyridin-2-yl)

1,3a,4,6a-tetrahydroimidazo[4,5-d]imidazole-2,5-diamine (3) ...... 36

Chapter 3: Discovery of GUPR-195 and synthesis of guanidinobenzimidazole derivatives ...... 39

3.1 Discovery of GUPR-195 ...... 39

3.1.1 GUPR-195 was not part of the NSC124205 sample ...... 39

3.1.2 Structural investigation of GUPR-195 ...... 40

3.1.2.1 Proton NMR of GUPR-195 ...... 41

3.1.2.2 Carbon NMR and infrared analysis of GUPR-195 ...... 42

3.1.2.3 Mass Spectrometry...... 43

3.1.2.4 Crystal growth and X-ray crystallography ...... 45

3.1.2.4.1 Different techniques of crystallization ...... 45

3.1.2.4.2 X-ray crystallography characterization ...... 47

3.2 Synthesis and structural identification of benzimidazole derivatives ...... 48

3.2.1 New benzimidazole derivatives synthesized ...... 49

3.2.2 Biological evaluation of synthesized molecules ...... 50

3.3 Experimental section ...... 51

3.3.1 Materials ...... 51

3.3.2 Synthetic methods...... 52

3.3.2.1 Synthesis of 2,2,5-tri(pyridin-2-yl)-2,12-dihydro-3H,5H-benzo[4,5]imidazo

[1,2-a]imidazo[2,1-d][1,3,5]triazin-3-one (4) ...... 52

3.3.2.2 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-diphenyl-1,5-

dihydro-4H-imidazol-4-one (5) ...... 54

3.3.2.3 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-bis(4-

methoxyphenyl)-1,5-dihydro-4H-imidazol-4-one (6) ...... 57

3.3.2.4 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-bis(3-

methoxyphenyl)-1,5-dihydro-4H-imidazol-4-one (7) ...... 60

3.3.2.5 Synthesis of 2-guanidinobenzimidazole (8)...... 63

3.3.2.6 Synthesis of 1-(benzo[d]thiazol-2-yl)guanidine (9)...... 66

3.3.2.7 Synthesis of 2-amino-5,5-di(pyridin-2-yl)-3,5-dihydro-4H-imidazol-4-one

(10) 69

3.3.2.8 Synthesis of 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-

a][1,3,5]triazin-2-amine (11) ...... 72

3.3.2.9 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-1-benzyl-5,5-di(pyridin-

2-yl)-1,5-dihydro-4H-imidazol-4-one (12) ...... 75

3.4 Conclusion ...... 78

Chapter 4: Development of a new ROS-sensitive linker for targeted therapy ...... 79

4.1 Introduction ...... 79

4.2 Background ...... 80

4.2.1 Cleavable linkers ...... 80

4.2.1.1 pH-sensitive linkers ...... 82

4.2.1.2 Redox-sensitive linkers ...... 82

4.2.1.3 Enzyme-cleavable linkers ...... 83

4.2.2 Reactive oxygen species ...... 84

4.2.2.1 What is ROS? ...... 84

4.2.2.2 ROS production ...... 84

4.2.2.2.1 Mitochondria ...... 84

4.2.2.2.2 NADPH oxidase ...... 85

4.2.2.3 ROS levels in cancer ...... 86

4.2.3 Reactive oxygen species linkers ...... 87

4.3 Project idea and hypothesis ...... 90

4.3.1 Double selectivity for drug delivery ...... 90

4.3.2 ROS-sensitive self-cyclizing linker ...... 92

4.3.3 ROS-sensitive oxalamide linker ...... 93

4.4 Results and discussion ...... 95

4.4.1 Choice of drug and synthesis ...... 95

4.4.1.1 NU1025 ...... 96

4.4.1.2 AT7867 ...... 97

4.4.2 Proof of concept: release of a drug from scaffold ...... 98

4.4.2.1 Self-cyclizing scaffold ...... 98

4.4.2.2 Oxalamide scaffold ...... 101

4.4.3 Other linker models to investigate ...... 103

4.4.4 Oxidation of the different linkers and drug ejection...... 104

4.5 Experimental ...... 107

4.5.1 Materials ...... 107

4.5.2 Synthetic methods...... 108

4.5.2.1 Synthesis of NU1025 (13) ...... 108

4.5.2.1.1 Synthesis of 3-methoxy-2-nitrobenzamide (13-b) ...... 109

4.5.2.1.2 Synthesis of 3-methoxy-2-aminobenzamide (13-c) ...... 110

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4.5.2.1.3 Synthesis of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d) ...... 112

4.5.2.1.4 Synthesis of 8-hydroxy-2-methylquinazolin-4(3H)-one (13) ...... 115

4.5.2.2 Synthesis of AT7867 (14) ...... 117

4.5.2.2.1 Synthesis of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b)

118

4.5.2.2.2 Synthesis of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl)

piperidine (14) ...... 120

4.5.2.3 Synthesis of 2-amino-N-(5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl)acetamide (15) ...... 123

4.5.2.3.1 Synthesis of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c) ...... 123

4.5.2.3.2 Synthesis of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl)

amino)-2-oxoethyl)carbamate (15-d) ...... 125

4.5.2.3.3 Synthesis of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl)

amino)-2-oxoethyl)carbamate (15-e) ...... 127

4.5.2.3.4 Synthesis of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxy

phenoxy)phenyl) amino)-2-oxoethyl)carbamate (15-f) ...... 129

4.5.2.3.5 Synthesis of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxy

phenoxy)phenyl) amino)-2-oxoethyl)carbamate (15) ...... 131

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4.5.2.4 Synthesis of 2-(4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chloro-phenyl) piperidin-1-yl)-N-(4-hydroxyphenyl)-2-oxoacetamide (16) ...... 134

4.5.2.5 Synthesis of 2-amino-N-(5-amino-2-(4-methoxyphenoxy) phenyl)pent-4- enamide (17) ...... 137

4.5.2.5.1 Synthesis of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)

amino)-1-oxopent-4-en-2-yl)carbamate (17-a) ...... 137

4.5.2.5.2 Synthesis of tert-butyl (1-((5-amino-2-(4-methoxyphenoxy)

phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b) ...... 139

4.5.2.5.3 Synthesis of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-

4-enamide (17) ...... 141

4.5.2.6 Synthesis of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide

(18) 144

4.5.2.6.1 Synthesis of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide

(18-a) 144

4.5.2.6.2 Synthesis of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-

enamide (18) ...... 146

4.5.2.7 Synthesis of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl) acetamido)benzoic acid (19) ...... 149

4.5.2.8 Synthesis of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1- yl)acetamido)benzoic acid (20) ...... 152

xiv

4.5.2.9 Synthesis of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-

yl)acetamido)benzoic acid (21) ...... 155

4.6 Conclusion ...... 158

References ...... 159

List of Tables

Chapter 2

Table 2. 1: Derivatives hypothetically present in the NSC124205 sample ...... 21

Chapter 3

Table 3. 1: Summary of the molecules synthesized for testing ...... 51

Chapter 4

Table 4. 1: ROS-sensitive cleavable linker existing in literature...... 88

Table 4. 2: Summary of the compound oxidations for ROS-triggered drug release ...... 105

xvi

List of Figures

Chapter 1

Figure 1. 1: Representation of Ras-GDP and Ras-GTP binding with legend for important regions shown by Marin-Ramos and al [5]...... 2

Figure 1. 2: RAS mutations in cancer [7] ...... 3

Figure 1. 3:Small molecules Ras inhibitors binding to the Ras GEF Sos...... 6

Figure 1. 4: Small molecule allosteric Ras inhibitors ...... 9

Figure 1. 5: Ras-effector interaction small molecule inhibitors ...... 11

Chapter 2

Figure 2. 1: MTS cell proliferation assay of 40 best docking candidates in H292 and A549 cell lines...... 14

Figure 2. 2: Image of the structure of NSC124205 listed on Pubchem ...... 15

Figure 2. 3: HPLC chromatogram of the NSC124205 sample from the NCI/DTP ...... 16

Figure 2. 4: High resolution mass spectrometry analysis of NCI/DTP NSC124205 associated with the peaks observed in the HPLC ...... 18

Figure 2. 5: Scheme published by Nishimura and Kitajima in 1979 for reactions leading to structures similar to the structure of 124205 referenced in Pubchem [82] ...... 19

Figure 2. 6: Potential molecules present in the NSC124205 sample following Nishimura and Kitajima’s reaction ...... 20

xvii

Figure 2. 7: Mechanism of formation of derivatives present in NCI/DTP NSC124205 .. 22

Figure 2. 8: IODVA1 cell proliferation assay and colony formation ...... 26

Figure 2. 9: In vivo testing in breast and lung mouse models ...... 28

Figure 2. 11: 1H NMR of 1 ...... 31

Figure 2. 12: 13C NMR of 1...... 31

Figure 2. 13: HRMS of 1 ...... 32

Figure 2. 14: HRMS fragmentation of 1 ...... 32

Figure 2. 15: 1H NMR of 2 ...... 34

Figure 2. 16: 13C NMR of 2...... 34

Figure 2. 17: HRMS of 2 ...... 35

Figure 2. 18: HRMS fragmentation of 2 ...... 35

Figure 2. 19: 1H NMR of 3 ...... 37

Figure 2. 20: 13C NMR of 3...... 37

Figure 2. 21: HRMS of 3 ...... 38

Figure 2. 22: HRMS fragmentation of 3 ...... 38

Chapter 3

Figure 3. 1: HPLC comparison of GUPR-195 and NCI/DT P NSC124205 ...... 40

Figure 3. 2: 1H NMR of GUPR-195 (4) ...... 41

Figure 3. 3: 13C NMR of GUPR-195 (4)...... 42

Figure 3. 4: Infrared spectra of GUPR-195 (4) ...... 43

Figure 3. 5: HRMS of GUPR-195 (4) ...... 44

xviii

Figure 3. 6: MS-MS fragmentation of GUPR-195 (4) ...... 44

Figure 3. 7: crystal cluster obtained by slow cooling of GUPR195 in methanol ...... 45

Figure 3. 8: single crystal obtained by slow evaporation of GUPR195 in methanol-d4 .. 46

Figure 3. 9: X-ray crystallography structural elucidation ...... 48

Figure 3. 10: Benzimidazole derivatives of IODVA1 with replaced pyridine rings ...... 49

Figure 3. 11: Fragments of GUPR-195 and structures to evaluate rings importance ...... 50

Figure 3. 12: 1H NMR of 5 ...... 55

Figure 3. 13: 13C NMR of 5...... 55

Figure 3. 14: HRMS of 5 ...... 56

Figure 3. 15: MS fragmentation of 5 ...... 56

Figure 3. 16: 1H NMR of 6 ...... 58

Figure 3. 17: 13C NMR of 6...... 58

Figure 3. 18: HRMS of 6 ...... 59

Figure 3. 19: MS fragmentation of 6 ...... 59

Figure 3. 20: 1H NMR of 7 ...... 61

Figure 3. 21: 13C NMR of 7...... 61

Figure 3. 22: HRMS of 7 ...... 62

Figure 3. 23: MS fragmentation of 7 ...... 62

Figure 3. 24: 1H NMR of 8 ...... 64

Figure 3. 25: 13C NMR of 8...... 64

Figure 3. 26: HRMS of 8 ...... 65

Figure 3. 27: MS fragmentation of 8 ...... 65

Figure 3. 28: 1H NMR of 9 ...... 67

xix

Figure 3. 29: 13C NMR of 9...... 67

Figure 3. 30: HRMS of 9 ...... 68

Figure 3. 31: MS fragmentation of 9 ...... 68

Figure 3. 32: 1H NMR of 10 ...... 70

Figure 3. 33: 13C NMR of 10...... 70

Figure 3. 34: HRMS of 10 ...... 71

Figure 3. 35: MS fragmentation of 10 ...... 71

Figure 3. 36: 1H NMR of 11 ...... 73

Figure 3. 37: 13C NMR of 11...... 73

Figure 3. 38: HRMS of 11 ...... 74

Figure 3. 39: MS fragmentation of 11 ...... 74

Figure 3. 40: 1H NMR of gupr-254 (12) ...... 76

Figure 3. 41: 13C NMR of GUPR-254 (12) ...... 76

Figure 3. 42: HRMS of GUPR-254 (12) ...... 77

Figure 3. 43: HRMS of GUPR-254 (12) ...... 77

Chapter 4

Figure 4. 1: Schematic structure of selective drug conjugates: carrier, linker, and payload

[67]...... 80

Figure 4. 2: Overview of different cleavable linkers and how they work [67] ...... 81

Figure 4. 3: ROS production and importance in cells [101] ...... 85

Figure 4. 4: ROS-sensitive linker for targeting therapy...... 90

Figure 4. 5: Structure of piperlongumine and MA14 ...... 92

Figure 4. 6: self-cyclizing ROS-sensitive linker skeleton ...... 93

Figure 4. 7: mechanism of drug ejection for ROS-self cyclizing linker ...... 93

Figure 4. 8: Oxalamide ROS-sensitive linker skeleton ...... 94

Figure 4. 9: mechanism of drug ejection for ROS-oxalamide linker ...... 94

Figure 4. 10: Structure of NU1025 and AT7867 ...... 95

Figure 4. 11: Structure of ROS-sensitive prodrugs used for linker design with the drug portion in red...... 98

Figure 4. 12: Oxidation of GUPR98 (15)...... 100

Figure 4. 13: Oxidation of Ox-AT7867 (16)...... 102

Figure 4. 14: Other ROS-responsive self-cyclizing linker models ...... 103

Figure 4. 15: Other ROS-responsive oxalamide linker models ...... 104

Figure 4. 16: 1H NMR of 3-methoxy-2-nitrobenzamide (13-b) ...... 109

Figure 4. 17: 13C NMR of 3-methoxy-2-nitrobenzamide (13-b) ...... 110

Figure 4. 18: 1H NMR of 3-methoxy-2-aminobenzamide (13-c) ...... 111

Figure 4. 19: 13C NMR of 3-methoxy-2-aminobenzamide (13-c) ...... 111

Figure 4. 20: LRMS (ESI) of 3-methoxy-2-aminobenzamide (13-c) ...... 112

Figure 4. 21: 1H NMR of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d) ...... 113

Figure 4. 22: 13C NMR of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d) ...... 114

Figure 4. 23: LRMS of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d) ...... 114

Figure 4. 24: 1H NMR of 8-hydroxy-2-methylquinazolin-4(3H)-one (13) ...... 116

Figure 4. 25: 13C NMR of 8-hydroxy-2-methylquinazolin-4(3H)-one (13) ...... 116

Figure 4. 26: HRMS of NU1025 (13) ...... 117

xxi

Figure 4. 27: 1H NMR of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b) ..... 119

Figure 4. 28: 13C NMR of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b) .... 119

Figure 4. 29: LRMS of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b) ...... 120

Figure 4. 30: 1H NMR of4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine (14)

...... 121

Figure 4. 31: 13C NMR of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine

(14) ...... 122

Figure 4. 32: LRMS of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine (14)

...... 122

Figure 4. 33: 1H NMR of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c) ...... 124

Figure 4. 34: 13C NMR of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c) ...... 125

Figure 4. 35: 1H NMR of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl) amino)-2- oxoethyl)carbamate (15-d)...... 126

Figure 4. 36: 13C NMR of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl) amino)-2- oxoethyl)carbamate (15-d)...... 127

Figure 4. 37: 1H NMR of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-

2-oxoethyl)carbamate (15-e) ...... 128

Figure 4. 38: 13C NMR of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-

2-oxoethyl)carbamate (15-e) ...... 129

Figure 4. 39: 1H NMR of (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15-f) ...... 130

Figure 4. 40: 13C NMR of (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15-f) ...... 131

xxii

Figure 4. 41: 1H NMR of (tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4- methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15) ...... 132

Figure 4. 42: 13C NMR of (tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4- methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15) ...... 133

Figure 4. 43: HRMS of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4- methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15) ...... 133

Figure 4. 44: 1H NMR of ox-AT7867 (16) ...... 135

Figure 4. 45: 13C NMR of ox-AT7867 (16) ...... 136

Figure 4. 46: HRMS of ox-AT7867 (16) ...... 136

Figure 4. 47: 1H NMR of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)amino)-1- oxopent-4-en-2-yl)carbamate (17-a) ...... 138

Figure 4. 48: 13C NMR of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)amino)-1- oxopent-4-en-2-yl)carbamate (17-a) ...... 139

Figure 4. 49: 1H NMR of tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy) phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b) ...... 140

Figure 4. 50: 13C NMR of tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy) phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b) ...... 141

Figure 4. 51: 1H NMR of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4- enamide (17) ...... 142

Figure 4. 52: 13C NMR of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4- enamide (17) ...... 143

Figure 4. 53: HRMS of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4- enamide (17) ...... 143

xxiii

Figure 4. 54: 1H NMR of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18-a)

...... 145

Figure 4. 55: 13C NMR of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18- a) ...... 146

Figure 4. 56: 1H NMR of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18)

...... 147

Figure 4. 57: 13C NMR of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18)

...... 148

Figure 4. 58: HRMS of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18)

...... 148

Figure 4. 59: 1H NMR of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (19) ...... 150

Figure 4. 60: 13C NMR of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (19) ...... 151

Figure 4. 61: HRMS of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (19) ...... 151

Figure 4. 62: 1H NMR of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (20) ...... 153

Figure 4. 63: 13C NMR of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (20) ...... 154

Figure 4. 64: LRMS of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (20) ...... 154

xxiv

Figure 4. 65: 1H NMR of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (21) ...... 156

Figure 4. 66: 13C NMR of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (21) ...... 157

Figure 4. 67: HRMS of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido) benzoic acid (21) ...... 157

xxv

List of Abbreviations

ACN ...... Acetonitrile

ADC ...... Antibody-drug conjugate

AGP...... Andrographolide

ApDC ...... Aptamer-drug conjugate

AML ...... Acute myeloid leukemia

ATP ...... Adenosine triphosphate

DCAI ...... 4,6-dichloro-2-methyl-3-aminoethylindole

DCM ...... Dichloromethane

DMF ...... Dimethylformamide

DMSO ...... Dimethyl sulfoxide

DNA ...... Deoxyribonucleic acid

DTP ...... Developmental therapeutics program

EC50 ...... Half maximal effective concentration

ERK...... Extracellular signal-regulated kinase

FDA...... Food and drug administration

GAP...... GTPase activating protein

GDP...... Guanine diphosphate

xxvi

GEF ...... Guanine nucleotide exchange factor

GPX...... Glutathione peroxidase

GR ...... Glutathione reductase

GSH...... Glutathione

GTP ...... Guanine triphosphate

HD ...... Huntington disease

HPLC ...... High-performance liquid chromatography

HRMS ...... High-resolution mass spectrometry

HRP ...... Horseradish peroxidase

IC50 ...... Half maximal inhibitory concentration

IR ...... Infrared

LCMS ...... Liquid chromatography mass spectrometry

LRMS ...... Low resolution mass spectrometry

MS ...... Mass spectrometry

NADPH ...... Nicotinamide adenine dinucleotide phosphate

NCI ...... National cancer institute

NMR ...... Nuclear magnetic resonance

NOX ...... NADPH oxidase

xxvii

PARP ...... Poly-ADP ribose polymerase

PDC ...... Peptide-drug conjugates

PI3K ...... Phosphatidylinositol 3-kinase

ROS ...... Reactive oxygen species

SAR ...... Structure-activity relationship

SOD...... Superoxide dismutase

THF ...... Tetrahydrofuran

TLC ...... Thin Layer Chromatography

TLR ...... Toll-like receptor

UV ...... Ultraviolet

xxviii

List of Symbols

C ...... Degrees Celcius r.t...... Room Temperature h...... hours

M ...... Molar

xxix

Chapter 1

Ras inhibition by small molecules for the treatment of cancer

1.1 Ras proteins and cancer

1.1.1 Ras family

In cancer research, the Ras family of proteins is one of the most studied with over

40,000 publications back in 2011 due to its involvement in cancer notably [1]. The Ras proteins are small GTP-binding entities around 21 kDa involved in cellular signal transmission. They play an important role in important physiological processes such as cell differentiation, proliferation, and survival. In humans, three forms of the Ras genes can be found: H-Ras, K-Ras and N-Ras. Additionally, K-Ras can be found in two isomeric forms

K-Ras4A and K-Ras4B. The four encoded protein isoforms play the role of molecular switches by wavering between their active guanosine 5’-triphosphate (GTP)-bound form and their inactive guanosine 5’-diphosphate (GDP)-bound form. The strong binding of Ras to the GTP and GDP nucleotides is regulated by components such as guanine nucleotide exchange factiors (GEFs) and GTPase-activating proteins (GAPs). The conformation changes between the two forms affect two molecular switches on the molecular surface, the switch I (SI) and switch II (SII) regions [2, 3]. When in the activated GTP-bound form, Ras isoforms crosstalk with a variety of effector proteins (Raf, PI3K and Ral-GDS). It is important for the equilibrium between the two forms to be maintains as the dysregulation is associated with oncogenic activity [4].

Figure 1. 1: Representation of Ras-GDP and Ras-GTP binding with legend for important

regions shown by Marin-Ramos and al [5].

1.1.2 Ras overactivation in multiple cancers

Point mutations on the Ras genes are often correlated with cancer initiation and progression. K-Ras is found to be the most mutated isoform with 86% of the mutations, followed by N-Ras with 11% and H-Ras with 3% of total Ras mutations. The oncogenic

2 point mutations are found mainly on the codons 12, 13 or 61 which favors the GTP-bound activated form of Ras and its effectors. This contributes to the cancerous cell survival and proliferation [6]. These mutations of RAS oncogenes are found in approximately 30% of all human cancers, especially the most lethal ones, up to 50% in colon cancers and 90% in pancreatic cancers. The mutations of the different forms of Ras can be found in different cancers such as leukemia and others as listed below [7].

Figure 1. 2: RAS mutations in cancer [7]

Consequently, the mutated Ras proteins became widely investigated as potential targets for cancer treatment with the use of small molecule inhibitors. The search for new inhibitors of these oncoproteins is still undergoing, as no inhibitor made it to the market, but past

3 years have shown significant finding in the area. One of the main strategy of Ras inhibition investigated is the direct inhibition of the Ras proteins [5].

1.2 Direct inhibition of the Ras proteins

The inhibition pathway consisting in the inhibition of the Ras oncoprotein is very challenging. Indeed, Ras and GTP/GDP have a very high binding affinity as low a picomolar, the cellular levels of nucleotides are usually high in the micromolar range, and no hydrophobic surface pocket was observed on Ras that would allow the binding of a small molecule [8]. In the past years, the study of the different Ras form has revealed a very dynamic protein surface that can form transient allosteric pockets potentially utilizable for small molecule inhibition [9-11]. The discovery of these pocket led to the finding of a few reversible and irreversible small molecule inhibitors, but the research is still ongoing to find the best inhibitor in term of efficacity, activity and toxicity. They are divided into three main family of direct inhibitors of Ras oncoproteins: nucleotide exchange, allosteric inhibitors and Ras-effector interactions inhibitors [5].

1.2.1 Inhibition of nucleotide exchange: GEF binding site

The oscillation between the active form and inactive form of Ras happens through guanine nucleotide exchange and GTP hydrolysis with the help of guanine nucleotide

4 exchange factors (GEFs) and GTPase activating proteins (GAPs), that regulate and accelerate the processes without covalently modifying Ras [12, 13]. The Ras binding to

GDP is the principal form at the resting state, and GEFs can stimulate and activate the cell to destabilize the nucleotide binding and GDP release. The concentration of GTP is 10- fold higher than GDP, and results in the formation of GTP-bounded Ras which can be disrupted by the hydrolysis of GTP to GDP-bounded species catalyzed by GAPs. In cancer, the mutations on the Ras oncoproteins usually cause the protein to be blocked into the active GTP-bound form [14].

One of the first strategy for nucleotide exchange inhibitors investigated was the direct inhibition of the nucleotide site with small molecules. These inhibitors can be nucleotide analogues or different type of molecules [5, 15-22]. As previously mentioned, due to the high binding affinity between that particular site and the nucleotides it is hard to find good competitive inhibitors. The research in this area suggest reversibility of the found inhibitors does not allow for good inhibitory potency. Consequently, other nucleotide exchange inhibitors were investigated such as ones blocking the binding between Ras and

GEFs [5].

Guanine nucleotide exchange factors (GEFs) play an important role in the nucleotide exchange process as it allows the GDP-bounded form to be switched into the

GTP-bounded form. Consequently, the inhibition of the GEF binding would result in the

Ras to stay bounded to the GDP into its inactive form and cause a decrease Ras activity.

There are three classes of GEF specific to Ras: Sos, RasGRF1/Cdc25Mm and GRP/Cal-

DAG-GEF [23]. Sos is the most important Ras GEF and has been actively studied with the development of small molecule modulators for the Ras-Sos complex. The molecular

5 switches SI and SII are two important Ras regions to target in order to block Sos interactions [24].

Figure 1. 3:Small molecules Ras inhibitors binding to the Ras GEF Sos

It has been studied by several researchers using NMR techniques to identify small molecules that can bind to those domains. The first found inhibitors decreasing the Ras activation by blocking the interaction between Ras and Sos with activity in the hundreds of micromolar range were small molecules such as SCH-53873, bisphenol A, IND, andrographolide (AGP) and 4,6-dichloro-2-methyl-3-aminoethylindole (DCAI) (Figure

1.3) [25-29]. Further other molecules such as Kobe-0065 were found for the inhibition of

Ras proteins with moderate potency (46 +/- 13 μM) and show the deactivation of Ras in multiple cancer and in vivo at high concentrations [30]. More research needs to be done in this area to find more potent Sos Ras inhibitors, but structure activity relationship (SAR) studies could lead the more potent small molecules inhibitors. Additionally, the inhibitors need to be more selective, as the previously mentioned small molecules did not differentiate the wild-type Ras and mutant Ras such as K-Ras. Some recent research published in 2017 reported the finding of 4-AM for the targeting of mutated K-RasG12C.

The molecule belongs to a new class of bivalent inhibitors that targets both the SII pocket and the nucleotide binding site, and can inhibit the specific Ras signaling and cancer proliferation at a low-micromolar ranges [31]. Currently, BI-1701963 and BI-3406, two

Sos inhibitor are in phase I clinical development in combination therapy. BI-1701963 is evaluated as a single agent inhibitor or combination with the mitogen-activated protein kinase kinase (MEK) inhibitor Trametinib [32] while BI-3406 is evaluated for combination therapy with the latter [33, 34]. Another strategy for Ras inhibition is the use of Ras surface dynamics with the use of allosteric inhibitors.

1.2.2 Allosteric Inhibition

Ras is characterized as an allosteric enzyme which is defined by being enzymes that change their conformations when they bind to an effector or allosteric modulator generating a change in the 3D structure of the proteins [11, 35]. This change consequently creates new pockets and potential for Ras small molecule inhibition. The search for new inhibitors was done using K-Ras molecular dynamics stimulation and computational

7 docking. In 2013, Ostream and al. identified the SII pocket, a novel allosteric pocket in K-

RasG12C found under the switch II region [36]. The inhibition showed selectivity for the mutant K-Ras over the wild-type K-Ras as the small molecule inhibitors contained strong acrylamide and vinyl sulphonamide electrophile that can selectively and irreversibly react with the cysteine thiols in the mutated SII pocket for binding. One of the potent inhibitors found was the acrylamide ACR (Figure 1.4) that is able to block the nucleotide exchanged catalyzed by Sos in G12C lung cancer cells. The inhibitor was able to reduce cell viability as well as increment apoptosis in those cells but did not show promising results in the

G12C-containing H358 cell line [36].

Other inhibitors of the SII pocket were investigated via a SAR study and led to the discovery of the selective ARS-853 that showed good activity at a low micromolar range

(Figure 1.4). The crystal of the protein-ligand complex revealed that the binding happens with a cysteine of the GDP-bound mutated K-RasG12C extending into the SII pocket[37].

The SAR studies on these molecules is still undergoing to find potential candidates for in vivo studies, but the K-RasG12C inhibition through this pathway seem to be promising.

Multiple single-agent inhibitors are currently in clinical development for K-RasG12C inhibition such as AMG510 [38-40], MRTX849 [41-43], ARS-3428 [44-46] and

LY3499446 [47, 48]

Figure 1. 4: Small molecule allosteric Ras inhibitors

The previously mentioned inhibitors targeted the G12C mutation of K-Ras that usually is blocked into the GDP-bound form due to the mutation. They are thereby not efficient for other mutations of K-Ras that lock the confirmation into the GTP-bound conformation such as G12V and G12D [49]. Inhibitors of the GTP state were investigated, and the SII pocket allosteric pocket in the GTP-bound protein could be inhibited with the

9 disulfide 2C07 (Figure 1.4). The compound was able to inhibit the catalytic activation of the SII region in both GTP and GDP states [50]. Other bigger size reversible allosteric inhibitors were investigated such as the cyclic peptide KRpep-2d. This peptide can selectively inhibit K-RasG12D by binding noncovalently near the GDP-bound SII pocket

[51]. This type of inhibition show promise as the IC50 was around 1.6 nM in the cell-free enzyme assay [52] and further investigation needs to be done on this class of inhibitors to find more clinical suitable inhibitors.

1.2.3 Ras-effector interaction inhibitors

The last class of direct Ras inhibitors are focusing on the inhibition of the interactions between Ras and the Ras-effector proteins such as Raf serine/threonine kinases playing an important role in the initiation and maintenance of Ras-mediated tumors [14].

The strategy focuses on both the inhibition of the Ras binding domain on the effector protein and the inhibition of the effector binding site on Ras. One of the recently found

Ras-effector interaction inhibitor uses the cyclic peptide cyclorasin 9A5 that can bind to

Ras-GTP and block the effector protein interaction causing a decrease in cancer growth and an increase apoptosis of the cancerous cells. This compound has good activity with an

IC50 of 0.12 μM but work still needs to be done in this area [53]. Indeed, the existing inhibitors of Ras-effector interaction usually lack potency, selectivity and/or membrane permeability, except for cyclorasin 9A5 [5, 14, 54]. This is seen with the finding of a few

Ras-effector interaction small molecule inhibitors, that can target the downstream PI3K or

Raf kinases, such as the early found sulindac sulfide [55, 56]. More recently other molecules such as MCP110 were found, showing the ability to block the Ras-Raf interactions and reduce ERK phosphorylation. These molecules target proteins are still unknown and further investigation need to be done [57-60]. Other NMR in silico and NMR studies led to the discovery of derivatives of Kobe2602, a derivative of Kobe0065, that is able to inhibit the H-Ras-GTP-Raf1 interaction with the IC50 in various cancer in the low micromolar range [29]. More work needs to be done to find more potent and selective Ras- effector interaction small molecule inhibitors, but the recent advancement in the field in the past decade is very promising.

Figure 1. 5: Ras-effector interaction small molecule inhibitors

1.3 Conclusion and dissertation goal

Ras-targeted therapy with small molecule inhibitors for direct inhibition of mutant

Ras protein is promising for the finding of new selective therapies to treat cancer. The mutations, most specifically the K-Ras mutations, are present in approximately 30% of cancer and are found amongst the most virulent ones such as pancreatic and colon cancers.

In the past decade, the research has significantly improved to find successful Ras direct inhibitors targeting the nucleotide sites, guanine nucleotide exchange factors (GEFs), allosteric sites and Ras-effector interaction. Multiple promising leads have been recently found and are in clinical trials, with one of the best strategies currently under investigation being the targeting of KRASG12C with small allosteric inhibitors. This could be combined with therapies targeting Ras effector interactions for the effective treatment of Ras-driven cancers.

In this dissertation, the first two chapter focus on the finding of new mutant Ras small molecule inhibitors. Previous work was done by computational docking and cell testing to identify NSC124205 as an active sample against Ras-driven cancers. Since the sample was a mixture of several components, the goal was first to identify the active compound. Further objective in this project was to ameliorate the activity of the active compound through structure-based drug design.

Chapter 2

Discovery of IODVA1, the active compound of NSC124205 against RAS-driven cancer model

2.1 Introduction

This chapter reference the discovery of a new molecule, IODVA1, a small potent small molecule that shows activity against Ras-driven lung and breast cancer.

This project started at the Cincinnati Children’s Hospital in Dr. Nicolas Nassar’s with a virtual screening of 118,500 compounds from the National Cancer

Institute/Developmental Therapeutics Program (NCI/DTP) Open Chemical Repository using the program Autodock in order to identify potential binders to an interface pocket of a small GTP-binding protein Ras with the ultimate goal of reducing its signaling in disease.

Ras is a target in several human cancers and in a set of genetic diseases and a small molecule that keeps Ras in the open conformation would inhibit its signaling. Here, the virtual screening was done targeting the GTP bound form of the G60A point mutant. Small molecules were docked by investigating the conformational space of each possible compound against a chosen pocket on a receptor’s surface in order to give the binding energy between the protein and the ligand. The 40 best candidates with the highest Ki were ordered from the NCI Developmental Therapeutics Program (NCI/DTP) and screened doing a cellular and biochemical screening assay. The best molecules are tested in vitro and cellular assays for the desired effect before ultimately testing the most efficient non- toxic compound(s) in a mouse model of disease for in vivo efficacy [61].

The 40 best candidates were tested using an MTS cell proliferation assay in human lung mucoepidermoid carcinoma cell line H292 and human lung adenocarcinoma cell line

A549, both encoding for wile-type-RAS and KRas respectively. They were plated at 500 cells in 96 well-plates and treated in triplicate with vehicle control at a concentration of 10 micromolar. The proliferation was monitored doing an MTS assay after 4 days (figure 1).

Figure 2. 1: MTS cell proliferation assay of 40 best docking candidates in H292 and

A549 cell lines. Data from [61] accomplished by Anjelika Gasilina and colleagues in Dr.

Nicolas Nassar laboratory

The figure show that in the A549 cell line only two molecules had an effect on the cell survival percentage, on being NSC124505 with an average percent survival of A549 cells compared to vehicle control of 32.9%. In H292 cell line, more compound had effects on the cell survival but NSC124205 also showed decreased survival of the cell line with an average percent survival of H292 cells compare to vehicle control of 41.6% [61].

In this project we focused on sample NSC124205 in order to further analyze the molecule, its activity and mechanism of action.

2.2 From NSC124205 to IODVA1

2.2.1 NSC124205, a mixture of multiple compounds

The first step before moving on to the next step of biological studies was to validate the structure of NSC124205 listed on Pubchem (figure 2.2) [62] and the purity of the sample used for the MTS cell proliferation assay.

Figure 2. 2: Image of the structure of NSC124205 listed on Pubchem [62]

The sample was submitted to High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS) analysis in order to both determine the purity by looking to visualize a single peak in the chromatogram, and structure by comparing the found mass to the mass corresponding to the structure of NSC124205 listed on pubchem of chemical formula C20H13N7 and exact mass of 351.1232 Da.

The HPLC analysis of NSC124205 was done using a C18 column and an acetonitrile/water gradient as a mobile phase and the resulting chromatogram showed that the sample had three peaks suggesting the presence of multiple molecules in the sample from the NCI Developmental Therapeutics Program (figure 2.3). Our next goal was to determine if any of the molecules present in the NCI/DTP NSC124205 sample corresponded to the structure listed on Pubchem so we used a Liquid Chromatography-

Mass Spectrometry (LCMS) analysis in order to determine the mass of the different compounds present in the sample.

Figure 2. 3: HPLC chromatogram of the NSC124205 sample from the NCI/DTP [61]

The mixture was analyzed by High Resolution LCMS using a similar column and conditions. The first peak at 11.6 min (peak 1a) had a mass-to-charge ratio m/z of

527.21607 [M+H]+ and 264.11178 [M+2H+], corresponding to an elemental composition of C28H23N12+ (theoretical: 527.21632). This mass difference could here correspond to

16 the product of a guanidinobenzimidazole addition on the NSC124205 structure. This correlates with the observation that its spectrum lacks red-shifted absorbance maxima due to a possible absence of the extended aromatic system. The other two peaks at 12.8 and

13.4 min have nearly the same m/z 370.14096 [M+H]+ and 370.19097 [M+H]+ respectively, both corresponding to elemental composition, C20H16N7O+ (theoretical:

370.14108). The minor peaks at m/z 392.12286 and 392.12289 respectively, correspond to

[M’ + Na+] (Figure 2.4 B and C). Both peaks were observed to possess similar absorbance spectra that would suggest that both species could be isomers or have some relationship to each other. Neither peak gave a mass corresponding to the structure of NSC124205 as reported by the NCI (Figure 1B), which has a calculated mass of 352.1305 Da. The mass difference of 18 Da suggests the presence of additional (O + 2 x H) in the structure of

NSC124205 [61].

The mass fragmentation of both molecules showed that the additional H2O were most likely bonded to the molecule. Our next goal was to unfold the mystery of the structure of the different derivatives as well as to identify the active molecule in the

NSC124205 sample. In order to do that, we looked into the literature to better understand the process of making molecules as referenced on Pubchem and what would the potential side products be with an additional H2O.

Figure 2. 4: High resolution mass spectrometry analysis of NCI/DTP NSC124205 associated with the peaks observed in the HPLC at A) 11.6 min B) 12.8 min and C) 13.4

min. [61]

2.2.2 Literature search reveals possible structures leading to IODVA1

A paper published by Nishimura and Kitajima in the journal of organic chemistry discussed reactions between guanidine derivatives and alpha-diketones leading to the formation of different types of substituted imidazole [63]. As seen in the manuscript, many things can influence those type of reactions such as the solvent nature and the type of acid/base leading to multiple derivatives, one being of nature of the structure of 124205 referenced (figure 2.5, molecule 8).

Figure 2. 5: Scheme published by Nishimura and Kitajima in 1979 for reactions leading

to structures similar to the structure of 124205 referenced in Pubchem [63]

Two of the conditions led to molecules with a mass difference of 18 Da or and additional H2O (figure 2.5, molecule 4 and 5) which could explain the potential molecule present in the initial sample from NCI/DTP. We consequently adapted the chemistry to our

19 molecules and hypothesized that the molecules present in the initial sample with the mass seen in the HRMS analysis are the one below:

Figure 2. 6: Potential molecules present in the NSC124205 sample following Nishimura

and Kitajima’s reaction

The next goal of my project was to synthesize the three derivatives potentially present in the active sample and to investigate which one was the possible active molecule targeting the Ras-driven cell lines.

2.3 Results and Discussions

2.3.1 Synthesis of hypothetical molecules present in NSC124205

The three molecules hypothetically present in the NSC124205 were then synthesized following the reaction dictated by Nishimura and Kitajima in their 1979 paper by reacting a guanidine derivative with a diketone species.

Compound 1 2 3

Structure

Table 2. 1: Derivatives hypothetically present in the NSC124205 sample

In our case we reacted 2-guanidinobenzimidazole with 2,2’-pyridil and modulated the reactions conditions as shown in figure 2.5 in order to obtain the targeted molecule.

The first derivative (table 2.1, 1), described as an hemiaminal derivative, can be obtain by reacting the two reagents in dimethyl sulfoxide at room temperature. The reaction of guanidine derivatives with 1,2 diketone lead to both nitrogen nucleophiles of the guanidine react with both cabonyls to create a new cyclized 4,5-dihydroxyimidazolines. When the two position of the imidazole is an electron donating group such as an amino in our case,

a dehydration takes place easily to form the 4H-imidazole generating the 4-hydroxy-4H- imidazole 1. Under alkaline conditions the molecule can undergo a pinacol rearrangement

to give the derived imidazolinone derivatives. For the synthesis of our targeted molecule we reacted 2-guanidinobenzimidazole, 2,2’-pyridil and sodium hydroxide at equimolar ratio in dimethyl sulfoxide at room temperature to generate 24% of compound 2 (table 2.1, 2). The 4-hydroxy-4H-imidazole 1 can also react with 2-guanidinobenzimidazole if it is in excess and then undergo intramolecular cyclization and dehydration for form the imidazoimadazole 3 containing two benzimidazole molecule. A two equivalent of 2-

21 guanidinobenzimidazole in our case can lead us to the synthesis of compound 3 (table 2.1, 3) with a 70% yield. Furthermore compound 1 can also be reduced in order to form the antiaromatic molecule referenced for NSC124205 on PubChem but that molecule is most likely hard to isolate and unstable in presence of water.

Figure 2. 7: Mechanism of formation of derivatives present in NCI/DTP NSC124205

2.3.2 Characterization and identification of synthesized derivatives

To confirm the identity of each synthesized molecule and validate their purity before testing we utilized high resolution mass spectroscopy, nuclear magnetic resonance spectroscopy and infrared spectroscopy since some of the molecules have similar mass and features. Each of the molecule structure was validated as followed.

Compound 1 had a protonated mass of 370.1411 which is C20H15N7O. Both compound 1 and 2 had those mass so we looked at the other characterization spectra to distinguish both. The proton NMR showed 12 aromatic protons with no evidence of symmetry. Compound 1 theoretically has no molecular symmetry whereas compound 2 contain molecular symmetry. Two doublets at 8.2 and 8.5 ppm corresponding to the C6- proton of the pyridine rings from the 2,2-pyridil showed that they were not equivalent. The aromatic region looked crowded with multiple multiplets integrating for one suggesting no equivalents hydrogens. Additionally, the IR showed no evidence of CO stretch at 1600-

1780 cm-1 and showed what seems to be a OH stretch around 3200 cm-1. The MS-MS fragmentation showed two main peaks at 327.10 and 264.08. The first fragment showed a loss of 42.97 or CN2H3 corresponding to a portion from the benzimidazole ring. The second fragment correspond to a loss of 106.05 or C6H6N2 which is characteristic of the cleavage of benzimidazole rings. No evident CO loss was showed in the MS-MS which would have showed a loss of 27.99 Da. Here, the instrumental analysis showed the structure of 1 to be the 4-hydroxy-4H-imidazole 1 (table 2.1, 1).

Compound 2 also have a protonated mass of 370.1411 corresponding to

1 C20H15N7O. which again could have been either molecule 1 or 2. The H NMR show the compound has a symmetric character. Indeed, the peak at 8.6 ppm corresponding to the

C6-proton of the pyridine integrated for 2H which suggest that the pyridine rings in the molecule are equivalents. The benzimidazole peaks integrated for 4 protons at 7.39 ppm and the remaining peaks, all three integrating for 2, matched the peaks found in remaining pyridine, showing that both pyridines in the molecule were equivalent as their shift and multiplicity were the same. The MS-MS fragmentation of the molecule showed a loss of

43.01 corresponding to the loss of an amide group CONH, which was confirmed by IR spectroscopy with 2 showing a CO stretch at 1707 cm-1 characteristic of amides. All the data showed evidence that the molecule underwent a pinacol rearrangement (Nishimura,

JOC 1979) to give the symmetric imidazolinone derivative 2 (table 2.1, 2).

Compound 3 has a protonated mass of 527.3914 of chemical formula C28H22N12.

The 1H NMR showed that the compound was also symmetric. The C6-proton of the pyridine rings showed a doublet at 8.4 ppm integrating for 2 showing their equivalence and the aromatic peaks of the benzimidazole around 7.2 ppm both integrated for 4 protons suggesting the presence of two benzimidazole in the structure. The MS-MS fragmentation showed one main peak at 352.1305 showing a loss of 175.0856 or C8H9N5 which corresponds to the 2-guanidinobenzimidazole also suggesting the addition of a second benzimidazole group in the structure. The data here showed that the compound was a di- benzimidazoimadazole derivative 3 (table 2.1, 3).

All the compound hypothetically present in the initial NCI/DTP NSC124205 sample were synthesized and the structures and purity were confirmed using the available

24 characterization and analysis technique, then tested for activity. This revealed IODVA1 (1) to be the active component of the NSC124205.

2.3.3 IODVA1 is active against RAS-driven cancer models

The work showed below was done in collaboration with Dr. Nicolas Nassar laboratory at Cincinnati Children’s at the University of Cincinnati Medical campus and was published in March 2020 in PlosONE [61].

2.3.3.1 IODVA1 stops the proliferations of cells harboring activated RAS

IODVA1 potential to inhibit the proliferation of cells containing active RAS as the initial NCI/DTP NSC124205 initial sample. The cell lines chosen for this study were triple negative breast cancer cell line MDA-MB-231 containing the G13D K-Ras mutation, the wide-type Ras containing breast cell line MCF10A, the wild-type p53 breast cancer cell line MCF7 and the mutant p53 T47D breast cancer cell line. The cells were cultivated in complete growth media in presence of IODVA1 from 0.1 to 1 μM or vehicle control and the results were collected by counting the cells using the trypan blue exclusion method.

Increasing concentration of IODVA1 inhibits the RAS mutant-containing MCF7,

MDA-MB-231, and T47D cells proliferation with estimated 50% growth inhibitory concentration (GI50) below or equal to 1 μM whereas no noticeable proliferation difference was observed in the wide-type containing MCF10A cells. IODVA1 also reduces the number of colonies of the breast cancer cells in soft agar at 1 and 3 μM which is in correlation with the cell proliferation results (figure 2.8) [61].

Figure 2. 8: IODVA1 cell proliferation assay and colony formation (A) MCF10A, MCF7,

MDA-MB-231, and T47D cells counted in presence of fluctuating concentrations of

IODVA1. (B) Number of colonies made by MCF7, T47D, and MDA-MB-231 cells in presence of IODVA1. Data from [61] accomplished by Anjelika Gasilina and colleagues in Dr. Nicolas Nassar laboratory

2.3.3.2 IODVA1 reduces breast cancer and lung cancer in vivo

IODVA1 was then tested in vivo against xenograft mouse model of breast cancer and lung cancer to prove that the results observed in cells can be translated in vivo. To do so, MDA-MB-2311 cells were used for the breast cancer, where cells were orthotopically

26 injected into the right and left inguinal mammary fat pads of female nu/nu (nude) mice.

The tumor bearing-mice then were administered an intraperitoneal 250 μL of 1 mM

IODVA1 every other day at an average dose of 3.5mg/kg. After four weeks of treatment it was observed that the tumor volumes decreased by more than fifty percent compared to the control vehicle-treated mice. The mice treated with IODVA1 saw their tumor stop growing after the beginning of the treatment. The cleaved caspase-3 stained revealed that there were more cells undergoing apoptosis after IODVA1 treatment compared to the vehicle control, leading to the limitation of tumor growth in vivo [61].

IODVA1 was also tested in lung cancer mouse models. The in vivo mouse model was done by generating xenograft where the mouse model was injected subcutaneously with H2122 lung cancer cells at the right and left flanks of the NSG mice. H2122 cells contain KRAS G12C mutation and are known to form aggressive tumors. In this study, the mice are received seven 250 µL intraperitoneal injections of 1mM IODVA1 or control vehicle every other day. The tumor volumes were also significantly smaller here for the mice treated with IODVA1 in comparison to the vehicle control. Further studies showed that the H2122 treated with IODVA1 had a smaller frequency of mitotic cells and increased infiltration of lymphoid and stromal cells compared to the vehicle control indicating that cells had a therapy-induced immune and fibrotic response, and IODVA1 seem to decrease cell proliferation in lung cancer in vivo [61].

Both in vivo testing suggesting that IODVA1 can be an efficient treatment against solid tumors such as RAS-driven solid tumors, seemingly increasing tumor cells apoptosis and decreasing cell proliferation. Additionally, IODVA1 does not affect the bone marrow function in vivo since no peripheral blood count change was observed [61].

Figure 2. 9: In vivo testing in breast and lung mouse models (A) MDA-MB-231 triple negative breast cancer cells xenografts (B) H2122 mouse xenografts treated with IODVA1 injected every other day with 250 μl of 1 mM IODVA1 (average dose of 3.5 mg/kg). Data from [61] accomplished by Anjelika Gasilina and colleagues in Dr. Nicolas Nassar laboratory

The found 4-hydroxy-4H-imidazole derivative showed activity in vitro and in vivo against cancer cell lines and mouse xenograft models. The compound reduces cell proliferation of multiple cancer cell models at a micromolar range. It shows promising results in vivo by stopping tumor growth by apoptosis and/or cell proliferation inhibition.

2.4 Experimental section

2.4.1 Materials

All chemicals, reagents, and solvents were purchased from Fisher Scientific,

Sigma-Aldrich Inc., Ambeed or other suppliers through Quartzy and used as received

28 unless stated otherwise. All reactions were carried out under argon in clean oven-dried glassware and stirred with a magnetic stir bar. All the indicated reaction temperatures correspond to the temperature of the metallic heating block used and indicated as room temperature (r.t.) for any reaction from 22-25 degree Celsius. The thin layer chromatography (TLC) used to monitor the reactions were all glass backed silica plates (20 x 20 cm, 60 Å, 250 μm) cut into smaller plates to monitor individual reactions. The visualization of the TLC plates was done by using a 254 nm UV lamp or TLC stains such as ninhydrin, permanganate, or iodine. The proton 1H and carbon 13C nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Advance 400 MHz spectrometer and the samples were dissolved in deuterated solvents such as chloroform-d (CDCl3), methanol-d4 (CD3OD), dimethylsulfoxide-d6 (DMSO-d6), or water-d2 (D2O). The analysis of the NMR was done using Mestrenova and the chemical shifts on the spectra are shown in ppm with trimethylsilane as standard. Data analysis are shown as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, b = broad, m = multiplet, abq = ab quartet), and coupling constants. High resolution mass spectral data were collected on a Orbitrap Fusion Lumos

Tribrid Mass Spectrometer (Thermo Fisher Scientific). All novel compounds were characterized by 1H, 13C and high-resolution mass spectrometry. HPLC analysis of final products was performed on a Beckman system Gold HPLC. They were ran using a Venusil

AQ C18 column (5Å, 7.5cm x 4.6mm) with an acetonitrile/water linear gradient and detected by UV with a scanning wavelength range from 200 to 500 nm.

2.4.2 Synthetic methods

2.4.2.1 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-4,5-di(pyridin-2-yl)-4H-

imidazol-4-ol (1)

2-guanidinobenzimidazole (1-a, 512.75 mg, 2.93 mmol) and 2,2’-pyridil (1-b, 725 mg,

2.93 mmol) were mixed in dimethyl sulfoxide (3ml) and stirred at room temperature for

15h. The reaction was quenched with water, and the aqueous layer was extracted 3 times with methylene chloride. The organic layers were combined and washed with water and brine, dried over sodium sulfate anhydrous and filtered using filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography with 10% methanol/methylene chloride. IODVA1 was isolated as a single compound a yellow powder (1, 421 mg, 39 %).

1H NMR (400 MHz, MeOD-d6): : δ = 7.05 (m, 3H), 7.23 (m, 1H), 7.32 (m, 2H), 7.42 (m,

2H), 7.83 (m, 1H), 7.89 (m, 1H), 8.37 (d, 1H), 8.53 (d, 1H).

13C NMR (400 MHz, MeOD-d6): δ = 113.89, 122.04, 122.19, 123.16, 124.17, 124.74,

128.85, 129.53, 137.93, 138.93, 148.65, 149.81, 150.36, 150.60, 152.89, 156.43, 157.79,

161.23.

HRMS-ESI: [M+H] + (C20H16N7O): calculated: m/z =370.1411. Found: m/z=370.1418

Figure 2. 10: 1H NMR of 1

Figure 2. 11: 13C NMR of 1

370.1411 100 90 80 70

50 40

30 Abundance Relative Relative 20 10

0 300 400 500 600 700 800 900 1000 1100 1200 m/z

Figure 2. 12: HRMS of 1

264.0333 100

60 370.0667 50 40 30

Relative Abundance 20

10 327.1000

0 180 200 220 240 260 280 300 320 340 360 380 400 m/z

Figure 2. 13: HRMS fragmentation of 1

2.4.2.2 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5-(pyridin-2-yl)-5-

(pyridin-3-yl)-3,5-dihydro-4H-imidazol-4-one (2)

2-guanidinobenzimidazole (1-a, 175 mg, 1.00 mmol) and 2,2’-pyridil (1-b, 215 mg, 1.01 mmol) were dissolved in dimethyl sulfoxide (3ml). After 30 minutes sodium hydroxide (40 mg, 1.0 mmol) was added to the reaction mixture and it was carried at room temperature for 10h. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with methylene chloride. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography with 10% methanol/methylene chloride. The resulting compound was isolated as a single compound a white powder (2, 152 mg, 0.41 mmol, 41 %).

1H NMR (400 MHz, MeOD-d4): δ = 7.21 (s, 2H), 7.39 (m, 4H), 7.67 (d, 2H), 7.85 (t, 2H),

8.56 (d, 2H).

13 C NMR (400 MHz, CDCl3): δ = 77.23, 99.99, 121.50, 123.05, 123.09, 137.66, 148.72,

154.10, 154.13, 158.17, 184.81

HRMS-ESI: [M+H] + (C20H16N7O): calculated: m/z =370.1411. Found: m/z=370.1418

Figure 2. 14: 1H NMR of 2

Figure 2. 15: 13C NMR of 2

370.1418 100

Abundance Relative 20 281.1153 392.1239

0 200 300 400 500 600 700 800 900 1000 1200 m/z

Figure 2. 16: HRMS of 2

352.1305 100 527.2161 90 80 70

60 50 40 30

Abundance Relative 20 369.1572 10

0100 200 300 400 500 600 700

m/z

Figure 2. 17: HRMS fragmentation of 2

2.4.2.3 Synthesis of N2,N5-bis(1H-benzo[d]imidazol-2-yl)-3a,6a-di(pyridin-2-yl)

1,3a,4,6a-tetrahydroimidazo[4,5-d]imidazole-2,5-diamine (3)

2-guanidinobenzimidazole (1-a, 349 mg, 1.99 mmol) and 2,2’-pyridil (1-b, 215 mg, 1.01 mmol) were mixed methanol (40ml) with sodium hydroxide (80 mg, 2.0 mmol) . The reaction was carried rt for 12h. The white suspension was filtered under vacuum and the cake was washed several times with cold methanol. The white powder obtained was dried in the oven at 80oC to give the final compound (3, 379 mg, 0.72 mmol, 72 %).

1H NMR (400 MHz, DMSO-d6): δ = 6.96 (m, 4H), 7.15 (m, 4H), 7.28 (m, 4H), 7.54 (t,

2H), 8.37 (s, 2H), 8.55 (s, 2H), 10.01 (s, 2H), 11.46 (s, 2H).

13 C NMR (400 MHz, DMSO-d6): δ = 66.70, 108.05, 115.84, 119.03, 120.64, 120.87,

124.20, 131.21, 137.60, 143.00, 149.65, 153.32, 155.59, 158.20

+ HRMS-ESI: [M+H] + (C28H23N12) : calculated: m/z =527.2163 Found: m/z=527.2166

Figure 2. 18: 1H NMR of 3

Figure 2. 19: 13C NMR of 3

527.2166 100 90 80 70

60 352.1308 50 40 264.1121 30 20

Relative Relative Abundance 1053.4256 10 0 200 300 400 500 600 700 800 900 1000

m/z

Figure 2. 20: HRMS of 3

352.1305 100 527.2161 90

70 60 50

Abundance 40

30 Relative Relative 20 369.1572 10

0100 200 300 400 500 600 700 m/z

Figure 2. 21: HRMS fragmentation of 3

Chapter 3

Discovery of GUPR-195 and synthesis of guanidinobenzimidazole derivatives

3.1 Discovery of GUPR-195

The conditions detailed in the last chapter in the synthesis of the previous compounds led to the discovery of an uncommon massed compound that did not correlate with the theoretical compound obtained reacting diketones and guanidine derivatives as referenced by Kitajima and Nishimura. Indeed, while reacting 2.2’-pyridil with the 2- guanidinobenzimidazole in dimethyl formamide (DMF) at high temperature a mass of 459

Da was observed while the same conditions were used to synthesize IODVA1 of mass 370.

This compound, termed GUPR-195 had a molecular structure that was not at first obvious looking at the different characterization data. It was then important to solve the structure of this molecule before moving to the in vitro and in vivo study of the derivative.

3.1.1 GUPR-195 was not part of the NSC124205 sample

The novel compound was first compared to the NSC124205 sample received from

NCI to investigate if some trace amount was present in the sample, increasing the activity of the first screening. To do that high-performance liquid chromatography (HPLC) was used where the NSC124205 sample was run along with GUPR195 and a 1:1 ratio mixture of both samples. The comparison was done using a C18 column along with an acetonitrile/water/0.05% formic acid linear gradient. The NSC124205 showed the 3 peaks initially observed belonging to compounds 1 to 3 at 14.4 min, 16.9 and 18.4 minutes, respectively. GUPR-195 (4) showed a peak eluting at 14.2 minutes seemingly different from the NSC124205 component. This was then confirmed with the chromatogram of the mixture showing 4 distinct peaks for compounds 1 through 4.

Figure 3. 1: HPLC comparison of GUPR-195 and NCI/DT P NSC124205

3.1.2 Structural investigation of GUPR-195

In this section, the analytical spectra (1H NMR, 13C NMR, IR, and mass spectroscopy) were analyzed to have structural insights for GUPR-195 to determine the molecular structure of the compound.

3.1.2.1 Proton NMR of GUPR-195

Initially, we analyzed the 1H NMR. 17 aromatic protons were found, showing that the compound have 5 additional protons compared to IODVA1. There was no evidence of structural symmetry in the molecule, like it was observed for compound 2 and 3. One interesting detail was the three doublets observed at greater than 8.5 ppm that typically are characteristic of the C6-proton of the pyridine. One of the doublets observed was more downfield and integrated for one and the other two were close to each other and integrated for two. This suggest that there might be 3 pyridine rings in the molecule and that two out of three of the pyridines were very similar, maybe due to symmetry in that portion of the molecule. This type of symmetry was previously observed in the molecules undergoing a pinacol rearrangement, which will lead to the formation of a carbonyl which can usually be identified by 13C carbon NMR and infrared spectroscopy.

Figure 3. 2: 1H NMR of GUPR-195 (4)

3.1.2.2 Carbon NMR and infrared analysis of GUPR-195

To confirm the theory that there was the presence of a carbonyl the carbon an infrared spectrum of compound 4 was done. The carbon NMR (figure 3.3) showed a total of 24 peaks which shows more carbons than the previously found IODVA1. Interestingly the carbon shows 2 nonaromatic peaks, which was not observed for the previous compounds. No evident carbonyl peak was observed that will show from 170-190 ppm for amides, but the peak around 160 ppm could be a nonaromatic carbonyl with alpha carbons.

Figure 3. 3: 13C NMR of GUPR-195 (4)

The infrared spectrum showed an important peak around 1644 cm-1 that could be characteristic of a carbonyl in the molecular structure. It also shows evident N-H stretching at 1759 cm-1 in total correlation the previous molecule backbones.

Figure 3. 4: Infrared spectra of GUPR-195 (4)

3.1.2.3 Mass Spectrometry

The mass spectroscopy analysis of GUPR-195 (figure 3.5) was done and compared to the mass of the previously made compound. Compared to IODVA1 (1) and molecule 2 that had masses of 370.1411 Da and a molecular formula of C20H15N7O, GUPR-195 (4) has a mass of 459.1679 Da and a molecular formula of C26H18N8O. This difference of

89.0268 Da is characteristic of C6H3N. This would mean that if there is an extra pyridine ring as previously hypothetised it would be linked to a CH. This CH could be the explanation of the non-aromatic carbon observed in the carbon NMR.

The MS-MS fragmentation showed three main fragments (Figure 3.6). They first one at 471.1722 Da showed a loss of 27.99 Da or CO which shows an evident presence of

459.1679 100

80 + (C26H18N8O)H 60

Relative Abundance Relative

20 917.3285

0 400 600 800 1000 1200 1400 1600 m/z

Figure 3. 5: HRMS of GUPR-195 (4) a carbonyl group in the structure. The second fragment given from the MS-MS at 326.1270

Da, showing a loss of 133.04 or C6H5N2-CO corresponding to the loss of a pyridine-CH and an amide. The third MS-MS peak was found at 273.1131 with a loss of 186.05 or

C9H6N4O where the fragment seems to be a tripyridine substituted aziridine ring.

326.1270 100

459.1670 60 431.1722

273.1132 Relative Abundance Relative 20

0 200 250 300 350 400 450 500

m/z Figure 3. 6: MS-MS fragmentation of GUPR-195 (4)

3.1.2.4 Crystal growth and X-ray crystallography

3.1.2.4.1 Different techniques of crystallization

To determine the exact structure of GUPR-195 and if the structural insights and hypothesis were correct, crystal of the synthesized derivative was grown in organic solvents. Multiple techniques such as solvent evaporation, slow cooling and solvent diffusion were used in order to grow crystals and obtain a promising transparent sharped- edged crystal from 0.1 to 0.4 mm for X-ray analysis.

Slow cooling was done using organic solvents where GUPR195 was not fully soluble such as ethyl acetate, THF and methanol. To do that, a near-saturated solution of

32 mg of GUPR-195 was prepared in the different solvents in a 20 ml scintillating vial and heated to near ebullition. Once the solid was totally dissolved the liquid was filtrated using a pipet loaded with cotton into a clean scintillating vial and was left to cool by adding a triple layered rubber attached Kim wipe on top. None of the solvent here gave good quality crystals for X-ray analysis. Methanol gave me clusters.

Figure 3. 7: crystal cluster obtained by slow cooling of GUPR195 in methanol

Another technique tried was solvent diffusion. Here, we dissolved 32 mg of GUPR-

195 in a solvent where it was fully soluble such as chloroform, methylene chloride, acetonitrile to near saturation level and then a layer a less dense solvent such as hexane was deposited on top using a pipet in a scintillating vial. The vial was closed with a triple layered rubbered Kim wipe. Usually with this technique crystals are formed at the boundary where the solvent slowly diffuse. It our case no crystal was observed with this technique.

The last technique used to grow GUPR-195 crystals was slow evaporation. It is known to be the simplest technique for air stable samples. Here, a near saturated solution of GUPR195 was prepared in a wide range of solvents: THF, acetonitrile, methanol, and chloroform. Once dissolved and closed with the Kim wipes like previously, the vials were put on a stable shelf at room temperature for days to let the solvent slowly evaporate to form crystals. The methanol here again was the one with the best results but was not able to form nice single crystals usable for the X-ray crystallography. Nevertheless, the use of deuterated methanol was able to give usable crystals with this technique.

Figure 3. 8: single crystal obtained by slow evaporation of GUPR195 in methanol-d4

3.1.2.4.2 X-ray crystallography characterization

The slow evaporation of GUPR-195 in methanol-d6 at room temperature with an initial concentration close to saturation led us to the growth of a colorless plate-shaped crystal of approximate dimensions 0.216 x 0.181 x 0.078 mm for X-ray examination and data collection. It was mounted in a loop with Paratone-N oil and transferred to the goniostat bathed in a cold stream. Intensity data were collected at 120K on a Bruker DUO

Photon-II diffractometer, Cu Kα radiation, λ=1.54178 Å. Data collection frames were measured in shutterless mode. The data frames were processed using the program SAINT.

The data were corrected for decay, Lorentz and polarization effects as well as absorption and beam corrections based on the multi-scan technique. The structure was solved by a combination of direct methods and the difference Fourier technique as implemented in the

SHELX suite of programs and refined by full-matrix least squares on F2 to give the crystal structure of GUPR195 (figure 3.9).

The structural elucidation of GUPR-195 by X-ray crystallography revealed that it indeed contained 3 pyridine rings in the structure as well as a carbonyl. The molecule seems like it underwent a pinacol rearrangement similarly to compound 2 and reacted with a pyridine derivative. One option could be that it reacted with residual 2-formylpyridine present in the mixture, which is a reagent to make one of the commercially purchased starting material 2,2’-pyridil. In that case it would show that the rearranged species reacted with the formylpyridine and underwent a dehydration to yield the found molecule with the

47 mass of 459 Da and high activity. The new potent compound was found to be a racemic mixture of both enantiomers.

Figure 3. 9: X-ray crystallography structural elucidation (A) structure of GUPR-195(4) (B)

2D representation of GUPR-195 ORTEP drawn at 50% probability ellipsoids. Complex crystallizes as a hydrate (1/4 molecule crystallized in the unit cell) (C) Packing diagram ac plane. For clarity, only H-atoms involved in H-bonding interactions (dashed lines) are shown. (D) Submitted crystalline sample

3.2 Synthesis and structural identification of benzimidazole

derivatives

3.2.1 New benzimidazole derivatives synthesized

This section started at the discovery of IODVA1 as the active component of

NSC124205 with the synthesis of multiple derivatives of the NSC124205 components. I then continued after the identification of GUPR-195 with the synthesis of several fragments of the molecule and similar derivatives. The goal was to make molecules that could identify the important features of the molecular series. I decided to investigate the pyridine portion of the molecule first by trying alternate group and replacing the 2’2’-pyridil with several benzil derivatives.

Figure 3. 10: Benzimidazole derivatives of IODVA1 with replaced pyridine rings

Interestingly, when the 2-guanidinobenzimidazole and the benzoin derivatives were put to react together the molecules rearranged via pinacol-rearrangement without the need of any base or temperature, in contrary to what was previously observed with the pyridines. It was consequently not possible to synthesize derivatives of IODVA1 via this route but nonetheless these targets were interesting for investigation. It was also important to evaluate the necessity of having seven rings in the structure of GUPR-195 . The previous structures (5-8) had 5 rings, so two other molecules were made with six and seven rings.

Other structures were then synthesized to evaluate the importance of the rings in the

49 structure. First it was important to evaluate the importance of the benzimidazole ring by synthesizing and testing a pure 2-guanidinobenzimidazole (9). It was then important to test another type of benzimidazole ring by synthesizing 2-guanidinobenzothiol (10) . The other portion of the IODVA1 derivatives was then synthesized by making the pinacol-rearranged

3-membered portion of the molecule (11). The additional ring in GUPR-195 was then investigated by making the molecule containing the important benzimidazole and additional pyridine molecule (12). Finally, a molecule containing 6 rings, structurally similar to GUPR-195 was synthesized by reacting the pinacol rearranged IODVA1 (2) with benzyl bromide to have a 6-ring structure (13).

Figure 3. 11: Fragments of GUPR-195 and structures to evaluate rings importance

3.2.2 Biological evaluation of synthesized molecules

The derivatives then needed to be evaluated in vitro against Ras-diven cancer models similarly to IODVA1. As the NSC124205 sample was a mixture, it was important

50 for us to see if any other possible molecules/fragments were also active. This is future work in the Nassar Lab.

Table 3. 1: Summary of the molecules synthesized for testing

3.3 Experimental section

3.3.1 Materials

All chemicals, reagents, and solvents were purchased from Fisher Scientific, Sigma-

Aldrich Inc., Ambeed or other suppliers through Quartzy and used as received unless stated otherwise. All reactions were carried out under argon in clean oven-dried glassware and stirred with a magnetic stir bar. All the indicated reaction temperatures correspond to the temperature of the metallic heating block used and indicated as room temperature (r.t.) for

51 any reaction from 22-25 degree Celsius. The thin layer chromatography (TLC) used to monitor the reactions were all glass backed silica plates (20 x 20 cm, 60 Å, 250 μm) cut into smaller plates to monitor individual reactions. The visualization of the TLC plates was done by using a 254 nm UV lamp or TLC stains such as ninhydrin, permanganate, or iodine. The proton 1H and carbon 13C nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Advance 400 MHz spectrometer and the samples were dissolved in deuterated solvents such as chloroform-d (CDCl3), methanol-d4 (CD3OD), dimethylsulfoxide-d6 (DMSO-d6), or water-d2 (D2O). The analysis of the NMR was done using Mestrenova and the chemical shifts on the spectra are shown in ppm with trimethylsilane as standard. Data analysis are shown as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, b = broad, m = multiplet, abq = ab quartet), and coupling constants. High resolution mass spectral data were collected on a Orbitrap Fusion Lumos Tribrid Mass Spectrometer

(Thermo Fisher Scientific). All novel compounds were characterized by 1H, 13C and high- resolution mass spectrometry. HPLC analysis of final products was performed on a

Beckman system Gold HPLC. They were ran using a Venusil AQ C18 column (5Å, 7.5cm x 4.6mm) with an acetonitrile/water linear gradient and detected by UV with a scanning wavelength range from 200 to 500 nm.

3.3.2 Synthetic methods

3.3.2.1 Synthesis of 2,2,5-tri(pyridin-2-yl)-2,12-dihydro-3H,5H-benzo[4,5]imidazo

[1,2-a]imidazo[2,1-d][1,3,5]triazin-3-one (4)

2-guanidinobenzimidazole (1-a, 170 mg, 0.97 mmol) and 2,2’-pyridil (1-b, 210 mg, 0.99 mmol) were dissolved in dimethyl sulfoxide (3ml). The reaction was carried at 115oC for

48h. The reaction was cooled then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with methylene chloride. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a methanol: methylene chloride slow gradient from 5 to 15%. The resulting compound was isolated as a single compound as a gold brown solid (4, 36.8 mg,0.08 mmol,

8.3 % yield).

1H NMR (400 MHz, DMSO-d6): δ = 10.47 (s, 1H), 8.61 (d, J= 4 Hz, 1H), 8.52 (d, J= 4

Hz, 1H), 8.41 (d, J= 4 Hz, 1H), 7.88 (dt, J=24 Hz, 8Hz, 4H), 7.69 (s, 1H), 7.44 (t, J= 8 Hz,

2H,), 7.35(m, 5H), 7.00 (td, J= 16Hz, 8Hz, 2H)

13 C NMR (400 MHz, CDCl3): δ = 68.30, 77.22, 108.58, 118.66, 121.34, 121.69, 121.74,

121.93, 122.41, 123.48, 123.82, 124.74, 131.97, 137.00, 137.09, 137.24, 149.59, 149.73,

149.96, 152.93, 153.93, 155.22, 156.39

+ + HRMS-ESI: [M+H] (C26H19N8O) : calculated: m/z =459.1676. Found: m/z=459.1679

3.3.2.2 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-diphenyl-1,5-

dihydro-4H-imidazol-4-one (5)

2-guanidinobenzimidazole (1-a, 175 mg, 1.00 mmol) and benzil (5-a, 215 mg, 1.01 mmol) were dissolved in dimethyl sulfoxide (3ml). The reaction was carried at 115oC for 12h. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper.

Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a ethyl acetate/hexanes gradient. The resulting compound was isolated as a single compound a white powder (5,

108 mg, 0.29 mmol, 29 %).

1 H NMR (400 MHz, CDCl3): δ = 7.55 (m, 5H), 7.42 (m, 8H), 7.17 (m, 2H)

13 C NMR (400 MHz, CDCl3): δ =157.11, 141.53, 138.48, 131.72, 129.94, 128.97, 128.88,

128.77, 127.15, 126.92, 121.83, 117.95, 110.37, 73.43

HRMS-ESI: [M+H] + (C22H17N5O): calculated: m/z =368.1506. Found: m/z=368.1498

Figure 3. 12: 1H NMR of 5

Figure 3. 13: 13C NMR of 5

[GUPR-186-benzil] #122-180 RT: 0.27-0.57 AV: 59 NL: 3.46E8 F: FTMS + p ESI Full ms [150.0000-2000.0000] 368.1498 100

85 80 + 75 (C H N O)H 22 17 5 70

45 RelativeAbundance 40 35

30 25 20

5 380.1497 338.3412 526.2091 735.2932 456.1812 564.5536 649.4218 796.2070 856.2099 904.3681 0 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z Figure 3. 14: HRMS of 5

Figure 3. 15: MS fragmentation of 5

3.3.2.3 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-bis(4-

methoxyphenyl)-1,5-dihydro-4H-imidazol-4-one (6)

2-guanidinobenzimidazole (1-a, 177 mg, 1.02 mmol) and 4-methoxybenzil (6-a, 240 mg,

1.01 mmol) were dissolved in dimethyl sulfoxide (3ml). The reaction was carried at 115oC for 12h. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a ethyl acetate/hexanes gradient. The resulting compound was isolated as a single compound a white powder (115 mg, 0.27 mmol, 26 %).

1 H NMR (400 MHz, CDCl3): δ = 9.84 (s, 2H), 7.54 (dd, J= 8 Hz, 1H), 7.45 (m, 4H), 7.35

(d, J=4 Hz, 1H), 7.17 (m, 2H), 6.95 (m, 4H), 3.82 (s, 6H)

13 C NMR (400 MHz, CDCl3): δ =178.93, 159.84, 141.69, 131.78, 130.61, 128.46, 121.91,

121.76, 117.47, 114.26, 110.29, 99.99, 72.69, 55.39

HRMS-ESI: [M+H] + (C22H17N5O): calculated: m/z =428.1717 Found: m/z=428.1706

Figure 3. 16: 1H NMR of 6

Figure 3. 17: 13C NMR of 6

[GUPR-188-methoxybenzil] #98-169 RT: 0.23-0.54 AV: 60 NL: 1.64E9 F: FTMS + p ESI Full ms [150.0000-2000.0000] 428.1706 100 95 90

75 + 70 (C24H21N5O3)H 65 60

55 50 45

RelativeAbundance 40 35 30

5 855.3345 282.2785 368.1498 488.1743 586.2300 757.4273 874.8097 1088.3912 1220.4756 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

Figure 3. 18: HRMS of 6

Figure 3. 19: MS fragmentation of 6

3.3.2.4 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-5,5-bis(3-

methoxyphenyl)-1,5-dihydro-4H-imidazol-4-one (7)

2-guanidinobenzimidazole (1-a 177 mg, 1.02 mmol) and 2-methoxybenzil (7-a, 240 mg,

1.01 mmol) were dissolved in dimethyl sulfoxide (3ml). T he reaction was carried at 115oC for 12h. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a ethyl acetate/hexanes gradient. The resulting compound was isolated as a single compound a white powder (115 mg, 0.27 mmol, 26 %).

1 H NMR (400 MHz, CDCl3): δ = 7.55 (d, J=8 Hz, 1H), 7.35 (t, J=8 Hz, 3H), 7.18 (m, 7H),

6.92 (dd, J= 8 Hz, 2H), 3.80 (s, 6H)

13 C NMR (400 MHz, CDCl3): δ =159.88, 141.54, 139.81, 131.71, 130.02, 121.81, 119.39,

117.48, 113.73, 113.50, 110.31, 73.20, 55.38

HRMS-ESI: [M+H] + (C22H17N5O): calculated: m/z =428.1717 Found: m/z=428.1706

Figure 3. 20: 1H NMR of 7

Figure 3. 21: 13C NMR of 7

[GUPR-190-metamethoxy] #78-172 RT: 0.23-0.53 AV: 57 NL: 3.06E8 F: FTMS + p ESI Full ms [150.0000-2000.0000] 428.1718 100

RelativeAbundance 40

5 293.6192 373.2352 458.6289 586.2313 641.2544 771.3655 855.3369 916.2511 991.3775 1099.1600 0 300 400 500 600 700 800 900 1000 1100 m/z

Figure 3. 22: HRMS of 7

Figure 3. 23: MS fragmentation of 7

3.3.2.5 Synthesis of 2-guanidinobenzimidazole (8)

2-aminoaniline (8-a 5.4 g, 50 mmol) was dissolved in 50 ml of 10% HCl. Dicyandiamide

(10-b, 6.3 g, 75 mmol) was added to the mixture and put to reflux for one hour. The reaction was cooled at room temperature and 10 ml of 50% NaOH was added to the mixture and put under reflux for an additional 20 minutes. Cool down the reaction and filter the gray solid using vacuum filtration. The solid was then recrystallized in ethanol to yield pure off- white crystals of compound 8 (2.57 g, 29%).

1H NMR (400 MHz, MeOD-d4): δ = 7.58 (ddd, J= 28 Hz, 8 Hz, 2H), 7.27 (m, 1H), 7.12

(m, 1H)

13 C NMR (400 MHz, MeOD-d4): δ = 174.60, 160.09, 153.23, 132.34, 126.48, 123,47,

121.69, 120.33

+ + HRMS-ESI: [M+H] (C8H9N4S) : calculated: m/z =176.0931. Found: m/z=176.0931

Figure 3. 24: 1H NMR of 8

Figure 3. 25: 13C NMR of 8

Figure 3. 26: HRMS of 8

[GUPR-199-2] 176 MS2 #20 RT: 0.11 AV: 1 NL: 8.34E8 T: FTMS + p ESI Full ms2 [email protected] [100.0000-400.0000] 176.0931 100

70 134.0714 65

RelativeAbundance 40

35 159.0666

5 101.9184 118.0522 125.5276 142.2200 150.3821 171.7723 178.2550 201.0952 0 100 110 120 130 140 150 160 170 180 190 200 m/z

Figure 3. 27: MS fragmentation of 8

3.3.2.6 Synthesis of 1-(benzo[d]thiazol-2-yl)guanidine (9)

2-aminobenzenethiol (9-a 5.4 ml, 50 mmol) was dissolved in 50 ml of 10% HCl.

Dicyandiamide (8-b, 6.3 g, 75 mmol) was added to the mixture and put to reflux for one hour. The reaction was cooled at room temperature and 10 ml of 50% NaOH was added to the mixture and put under reflux for an additional 20 minutes. Cool down the reaction and filter the gray solid using vacuum filtration. The solid was then recrystallized in chloroform to yield pure gray crystals of compound 9 (6.1 g, 64%).

1H NMR (400 MHz, MeOD-d4): δ = 7.58 (ddd, J= 28 Hz, 8 Hz, 2H), 7.27 (m, 1H), 7.12

(m, 1H)

13 C NMR (400 MHz, MeOD-d4): δ = 174.60, 160.09, 153.23, 132.34, 126.48, 123,47,

121.69, 120.33

+ + HRMS-ESI: [M+H] (C8H9N4S) : calculated: m/z =193.0553. Found: m/z=193.0539

Figure 3. 28: 1H NMR of 9

Figure 3. 29: 13C NMR of 9

[GUPR-230] #161-216 RT: 0.22-0.52 AV: 56 NL: 6.95E8 T: FTMS + p ESI cv=0.00 Full ms [50.0000-1000.0000] 193.0539 100

RelativeAbundance 40

15 151.0323 10

5 125.9861 236.0713 304.8957 447.0223 531.8345 592.2118 677.1331 760.5339 886.4556 974.7719 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure 3. 30: HRMS of 9

Figure 3. 31: MS fragmentation of 9

3.3.2.7 Synthesis of 2-amino-5,5-di(pyridin-2-yl)-3,5-dihydro-4H-imidazol-4-one

(10)

2-guanidine hydrochloride (10-a, 552 mg, 5.78 mmol) in 15 ml absolute ethanol was slowly added to a solution of NaOH (229.2 mg, 5.73 mmol) in 15 ml absolute ethanol. The mixture was stirred under reflux for 1.5 hours then cooled down to filter out the solid that formed. The mixture filtrate was then heat to reflux again and a solution of 2,2’-pyridil (1- b, 500 mg, 2.35 mmol) in 15ml absolute ethanol was added dropwise to it over 15 minutes.

The reflux was carried for an additional 2 hours and then cooled in an ice bath. The white solid formed was filtered using vacuum filtration and the filtrate was washed with cold absolute ethanol to yield a pure white solid (10, 292.5 mg, 1.15 mmol, 49.0 % yield).

1H NMR (400 MHz, DMSO-d6): δ = 8.50 (d, J= 4Hz, 2H), 7.79 (dt, J= 8Hz, 2H), 7.55 (d,

J= 8Hz, 2H), 7.30 (dt, J= 8Hz, 4Hz, 2H)

13 C NMR (400 MHz, DMSO-d6): δ = 75.26, 121.42, 122.67, 136.52, 148.66, 158.31,

172.12, 185.24

+ + HRMS-ESI: [M+H] (C13H12N5O) : calculated: m/z =254.1036. Found: m/z=254.1038

Figure 3. 32: 1H NMR of 10

Figure 3. 33: 13C NMR of 10

[GUPR-312] #200 RT: 0.61 AV: 1 NL: 3.71E9 T: FTMS + p ESI cv=0.00 Full ms [150.0000-2000.0000] 254.1038 100

80 75 70 65 60 55 50

RelativeAbundance 40

25 169.0760 20

15 507.2003 10 5 281.0635 561.1198 659.1406 760.2972 0 200 300 400 500 600 700 800 900 1000 m/z Figure 3. 34: HRMS of 10

Figure 3. 35: MS fragmentation of 10

3.3.2.8 Synthesis of 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-

a][1,3,5]triazin-2-amine (11)

2-guanidinobenzimidazole (1-a, 300 mg, 1.71 mmol), 2-formylpyridine (4-a, 0.16 mg, 1.50 mmol) and piperidine (0.15 ml, 1.52 mmol) in 10 ml absolute ethanol was refluxed for 4 hours and then cooled in an ice bath. The white solid formed was filtered using vacuum filtration and the filtrate was washed with cold absolute ethanol to yield a pure white solid

(11, 159.2 mg, 0.61 mmol, 40.4 % yield).

1H NMR (400 MHz, DMSO-d6): δ = 8.57 (d, J= 4 Hz, 1H), 8.11 (d, J= 12 Hz, 1H), 7.81

(td, J=8 Hz, 2H), 7.36 (td, J=8 Hz, 1H), 7.24 (dd, J= 16 Hz, 8 Hz, 2H), 6.91 (m, 2H), 6.81

(m, 2H), 6.47 (s, 2H)

13 C NMR (400 MHz, DMSO-d6): δ = 66.66, 107.88, 115.90, 118.85, 120.51, 120.73,

124.15, 131.32, 137.58, 143.35, 143.35, 149.60, 153.29, 155.35, 158.26

+ + HRMS-ESI: [M+H] (C14H13N6) : calculated: m/z =265.1196. Found: m/z=265.1206

Figure 3. 36: 1H NMR of 11

Figure 3. 37: 13C NMR of 11

[GUPR-311] #137-174 RT: 0.36-0.57 AV: 38 NL: 3.07E8 T: FTMS + p ESI cv=0.00 Full ms [150.0000-2000.0000] 265.1206 100

70 + (C H N )H 65 14 12 6

RelativeAbundance 40 35 30

25 20 15

10 529.2340 5 186.0781 248.0941 279.1361 354.1476 551.2163 627.2021 0 200 250 300 350 400 450 500 550 600 650 700 m/z Figure 3. 38: HRMS of 11

Figure 3. 39: MS fragmentation of 11

3.3.2.9 Synthesis of 2-((1H-benzo[d]imidazol-2-yl)amino)-1-benzyl-5,5-

di(pyridin-2-yl)-1,5-dihydro-4H-imidazol-4-one (12)

Compound 2 (30 mg, 0.08 mmol), benzyl bromide (12-a, 10 μl, 0.08 mmol) and potassium carbonate (12.3 mg, 0.09 mmol) in 1 ml dimethyl formamide was stirred at 62 degree

Celsius for 12 hours. The reaction was cooled then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with methylene chloride. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. Silica (100 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a methanol: methylene chloride gradient to yield GUPR-254

(12, 17 mg, 0.05 mmol, 59%) as a pure white solid.

1H NMR (400 MHz, MeOD-d4): δ = 8.56 (d, J=4 Hz, 2H), 7.77 (td, J= 8Hz, 2H), δ = 7.56

(d, J= 8 Hz, 2H), 7.43 (d, J= 8 Hz, 2H), 7.35 (m, 4H), 7.25 (m, 4H), 7.08 (m, 2H)

13 C NMR (400 MHz, MeOD-d4): δ = 43.92, 75.07, 114.23, 122.41, 123.17, 124.85,

128.58, 128.85, 129.48, 137.65, 138.59, 150.68, 156.05, 156.59, 158.17, 173.65

+ + HRMS-ESI: [M+H] (C27H22N7O) : calculated: m/z =360.1880. Found: m/z=360.1874

Figure 3. 40: 1H NMR of gupr-254 (12)

Figure 3. 41: 13C NMR of GUPR-254 (12)

[GUPR-254-2] #147-201 RT: 0.34-0.62 AV: 55 NL: 1.78E9 T: FTMS + p ESI cv=0.00 Full ms [150.0000-2000.0000] 460.1874 100 95 90 85 80 75

RelativeAbundance 40

15 919.3677 10 972.2793 386.1358 5 867.2989 1009.4144 281.1143 550.2344 793.2069 1096.4847 1378.5482 1556.6690 0 200 400 600 800 1000 1200 1400 1600 m/z

Figure 3. 42: HRMS of GUPR-254 (12)

Figure 3. 43: HRMS of GUPR-254 (12)

3.4 Conclusion

In these two chapters we were able to identify IODVA1 as the active component of

NSC124205. Biological testing of the molecule showed that it was active against RAS- driven cancer models in vitro and in vivo and was able to inhibit the proliferation in the lung and breast cancer. Additionally, the synthesis of analogs led to the accidental discovery of GUPR-195, a more potent molecule. The unknown structure was solved using

X-ray crystallography and this led to the synthesis of more derivatives for a structure activity study that need to be tested in vitro.

Chapter 4

Development of a new ROS-sensitive linker for targeted therapy

4.1 Introduction

Cancer treatment is still very limited when it comes to off-target inhibitor effects.

Despite achieving tremendous effect in growth inhibition and the elimination of tumors, they are a usually associated with severe side effects due to poor selectivity. [64]

Consequently, for the past years treatments have been focused around targeting therapies.

Those therapies often use the overexpression of cell surface receptors in cancer cells [65] to bring selectivity to treatments using peptides, aptamers, antibodies, proteins, or small molecules [66]. Those delivery vehicles are conjugated with a drug using different type of linkers to form the commonly used peptide-drug conjugates (PDC) [67], aptamer-drug conjugates (ApDC) [68] or antibody-drug conjugates (ADC) [69]. Once internalized by the targeted cancerous cell these conjugates will be cleaved at the linker junction to release the payloads that will lead to the cancer cell death [70]. Those payloads can be drugs such as toxophores, transcription factors, non-radioactive effectors, or nucleotides [67]. Current strategies for payload release include acid-lability, reduction, enzyme-assisted release, and other non-cleavable stable linkers that require internalization [67, 70].

Figure 4. 1: Schematic structure of selective drug conjugates: carrier, linker, and payload

[67].

This research is focused on the design of a cleavable release strategy dependent on reactive oxygen species (ROS). These entities are common at cancer microenvironments sites at high levels and thus can lead to selective release of a payload. Using a delivery vehicle such as an aptamer, antibody, or short targeting peptide, it is possible to target a particular type of cancer for the delivery, doubling the specificity features for a selective drug release at tumor site. Here, we focus on the development of a two novel Reactive

Oxygen Species (ROS)- sensitive linkers for peptide-drug conjugates: a ROS-self- cyclizing linker that was previously developed in the Merino lab and a newly investigated oxalamide linker.

4.2 Background

4.2.1 Cleavable linkers

An important feature in the development of targeting therapies conjugates is the selection of the linker technology that will be connecting the delivery vehicle with the cytotoxic

80 payload. The linker is crucial for the development of the technology as it should bring selectivity and ensure biological efficacy of the payload. Most of the drug used are not toxic while being coupled to the carriers but once it arrives to the tumor site, it will be cleaved and release the activated payload. Therefore, it is essential for the conjugate to be stable in plasma, until it arrives to the tumor site, to avoid premature release of the toxophore and unwanted cytotoxicity around healthy tissues [71]. To guarantee that stability, it is essential to develop the good linker strategies that will utilize the proprieties within the cancerous cells that differ from healthier cells to guarantee selective release.

Figure 4. 2: Overview of different cleavable linkers and how they work [67]

81 4.2.1.1 pH-sensitive linkers

The first type of linkers used for selective release of toxophores are pH-sensitive linkers. Tumor cells are known to be more acidic than healthy cells with pH around 6.2-

6.8 and 7.4 respectively. This is due to the fast growth and metabolism of cancerous cells that are often unable to get enough nutrients and oxygen causing anaerobic glycolysis, the generation of excess lactic acid and the acidification of tumor tissues (Warburg effect) [72].

Additionally, these linkers can be cleaved once they are transported in the endosomes

(pH~5.0-6.0) and lysosomes (pH~4.8) [73]. A widely used pH-sensitive linkers are hydrazone used for their ability to be easily hydrolyzed at weakly acidic conditions [74,

75]. Other pH-sensitive linkers used are oximes, imines, acetals and cis-aconityls [76].

Nevertheless, those linkers can undergo slow hydrolysis at physiological conditions (pH

7.4, 37oC) that can lead to early release in plasma and undesired peripheral toxicity [77].

4.2.1.2 Redox-sensitive linkers

Other type of linkers commonly used are redox-sensitive linkers. It relies on the high antioxidant concentrations in the cytoplasm such as glutathione (GSH). GSH is known to be thousand-fold higher in cells compared to plasma with concentrations of 15 mM and

15 μM respectively [78]. This concentration is even higher in cancerous cells caused by hypoxia due to the abnormal blood flow in the tumor microenvironment [79].

Consequently, GSH-sensitive linkers such as disulfide bonds have been widely used for targeted therapy [80], and lately other reduceable linkers such as thioesters and azo-bond derivatives have been studied [81, 82].

82 4.2.1.3 Enzyme-cleavable linkers

The last big category of widely used linkers are enzyme-cleavable linkers. They can be cleaved by specific linkers such as esterases and cytochrome P450 [83, 84].

Structure such as esters and carbamates can be found during peptide synthesis, but they can be hydrolyzed in serum at physiological conditions which lead focus to shorter amino acid sequence linkers [85]. Some of those short sequences can be recognized by proteases overexpressed in tumor tissues such as cathepsin B or legumain. Cathepsin B is a lysosomal protease that is part of multiple human oncologic process and is overexpressed in cancer

[86]. The enzyme is able to recognize certain sequences such as phenylalanine-lysine (Phe-

Lys), valine-citrulline (Val-Cit) and Glycine-Phenylalanine-Leucine-Glycine (Gly-Phe-

Leu-Gly) and is able to cleave the peptide bond on the C-terminal of the sequence. When it is coupled with p-aminobenzyloxycarbonyl the linker can be cleaved even easily for Val-

Cit (Val-Cit-PABC) and Val-Ala (Val-Ala-PABC) linkers and is the most successfully used enzymatic linkers for ADCs [87, 88]. Legumain is the only mammalian asparaginyl endopeptidase and can recognize Alanine-Alanine-Asparagine (Ala-Ala-Asn) and cleave the C-terminal after Asn where the cytotoxic payload will be conjugated [89-93] .

Reactive-oxygen species have been utilized for targeted therapy in the past years in order to develop the same kind of technology to selectively target cancerous cells, using the overexpression of cell oxidants at tumor microenvironment and their relatively low levels in plasma for selectivity, similarly to the GSH, cathepsin B and legumain sensitive linkers.

83 4.2.2 Reactive oxygen species

4.2.2.1 What is ROS?

Reactive oxygen species or ROS are relative chemical molecules containing oxygen. They are byproduct of oxygen metabolism and can be found under many highly

- reactive forms such as hydrogen peroxide (H2O2). superoxide (O2 ), hydroxyl (•OH), hypochlorous acid (HOCl), alkoxyl (RO•) and peroxyl (ROO•) radicals. In the past they were considered mostly toxic to the body due to its presence notably in cancer [94] but presently ROS are also seen for their positive role as a signaling molecule for its regulations of multiple physiological and biological responses such as growth factor signaling, proliferation, angiogenesis, and adaptation to hypoxia [95, 96].

4.2.2.2 ROS production

4.2.2.2.1 Mitochondria

The mitochondria, known to be the energy producer of the body, is for many cells the main source of ROS [97]. A majority of the oxygen in the body is consumed by the mitochondria in order to produce adenosine triphosphate (ATP) as energy source. A series of mitochondrial complexes with electrons are part of normal oxidative phosphorylation with O2 leading to production of energy and water. Sometimes electrons “leaked” leading

- to reduction of O2 to produce superoxide anion O2 . Mitochondria complexes I, II and III have shown to play a role in the redox signaling of up to 10 sites of superoxide mitochondrial production [97, 98]. The superoxide anion can be released in the mitochondrial matrix by complexes (I-III) where it will be converted to H2O2 by superoxide

84 .- dismutase 2 (SOD2) [98]. O2 can also be release out of the mitochondria with the help of complex III, passing through voltage-dependent anion channels and released into the cytosol where it is converted into H2O2 by SOD1 [99, 100].

Figure 4. 3: ROS production and importance in cells [101]

4.2.2.2.2 NADPH oxidase

The second source of ROS in the body are NOX enzymes promoting its production.

NADPH oxidase (NOX) is a membrane-bound enzyme complex with multiple components: the catalytic membrane component cytochrome b558 (p22phox and gp91phox heterodimer) and the cytosolic subunit proteins (p40phox, p47phox, p67phox and RAC1) [102].

85 .- These enzymes produce O2 anion intracellularly and extracellularly depending on how they are oriented on the membrane. These enzymes oxidize a substrate, like NADPH, and reduce O2. There are NADPH oxidases in the plasma membrane, in the nucleus, the mitochondria, and the endoplasmic reticulum [103]. SOD1 and SOD3 then convert rapidly the formed superoxide anion into H2O2 that can then react with protein-bound thiols. The peroxide will create a sulfenic oxide (charged in water) or a disulfide. These lead to large scale changes in the proteins structure and can change its activity [104]. In cellular signaling these protein modifications can shut down activity, like phosphatase activity, that block growth-related kinases. Many of these pathways are active in leukemia and other cancers [105].

4.2.2.3 ROS levels in cancer

It is important for the ROS levels to be regulated. At moderate levels it causes the cells to differentiate and proliferate and at high levels it can cause the death of the cells

[106]. Therefore, they are multiple existing systems to scavenge ROS in eukaryote cells such as superoxide dismutase (SODs) in the cytoplasm, mitochondria and extracellular matrix, glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin, catalase and peroxiredoxin that are used to convert the superoxide anions into water and are converted into their recyclable reduced state [107]. ROS homeostasis is essential for the cell function as oxidative damage can be caused to multiples biomolecules if not regulated such as lipids, proteins, and DNA [108]. If it not properly regulated the cells get into an oxidative stress state that can cause irreversible damage and/or decrease cell function. That state has been linked to many diseases[109] such as cancer [110-112], autoimmune

86 disorders[113, 114], neurodegenerative diseases [115, 116], cardiovascular diseases [117], and inflammations [118-120].

Each healthy cell is exposed daily to approximately 1.5 x 105 oxidative hits and this number is even higher in oxidative stress condition accompanied with a decrease in ROS scavenging. In cancerous cells, the fast growth and metabolism cause the cells to lack nutrients and oxygen, causing an ongoing high rate of anaerobic glycolysis followed by pyruvate oxidation in the mitochondria causing the generation of high levels of ROS through this pathway [121, 122]. In the case of hydrogen peroxide (H2O2), healthy cells have an extracellular concentration ranging from 0.5-7 μM whereas pathological conditions usually have concentrations 100-fold higher and can go up to 1.0 mM [123-

126]. It is then possible to target the cancerous cells using this high level of ROS to enhance drug selectivity. One of the strategies used it to utilize the high levels of ROS in order to be used in targeted therapy and develop efficient ROS linkers.

4.2.3 Reactive oxygen species linkers

Reactive oxygen species linkers have been widely investigated lately in the field of targeted therapy but more specifically in the case of polymers. One of the most popular existing ROS-prodrug used is (aryl)boronic acids or esters [127-129], where in presence of hydrogen peroxide the B-C bond can be oxidized then hydrolyzed to give the self- immolative phenol derivative that will trigger the release of the active drug in benzylic position and a the corresponding boronic acid/ester [130]. In targeted therapy, they have been used with polymer nanoparticles for ROS sensitive release. An example of existing strategies used a solubility switch after H2O2 oxidation. In 2011, Broaders and al. showed

87 the modification of the hydroxyls of the water-soluble polymer dextran with arylboronic

esters yielding the water insoluble ROS-sensitive dextran (Oxi-DEX). After the emulsion-

created nanoparticle was exposed to H2O2 for 2 hours, they gave back the parent

hydrophilic polymer that then degraded and released the payload [128].

ROS-responsive linker Chemical structures and ROS cleavage

Arylboronic ester

Thioketal

Sulfides, selenide, and telluride

Diselenide

Aminoacrylate

Oligoproline

Peroxalate ester

Table 4. 1: ROS-sensitive cleavable linker existing in literature

88 Other linkers such as thioketals can be rapidly cleaved when exposed to reactive oxygen species and give the corresponding acetone and thiols [131]. They are stable at acidic and basic conditions and can be linked to a targeting device on one end and a cytotoxic payload on the other [132-137]. Additional linkers utilizing sulfurs are thioesters, which is one of the most popular ROS-sensitive system investigated for biomedical research in polymer science [138, 139]. In presence of ROS it usually oxidizes from sulfide to hydrophilic sulfoxide or sulfone. The similar process happens to selenide or telluride containing linker that undergo oxidation in presence of ROS to form hydrophilic selenenyl/selenonyl and tellurinyl/telluronyl respectively. The similar three linkers (thioester, selenide, and telluride) after switching solubility post-oxidation have the ability for a release at a specific site [140, 141]. Indeed, after the oxidation to the hydrophilic form, the group’s proximal ester is easier hydrolysable and can be used for drug release [142-144]. Another selenide-containing linker are diselenide. Selenium have been popularly used as antioxidant and selenides are oxidizable by ROS and cleaved into selenic acid and further reduced into selenol by GSH [145, 146].

Other few linkers subjected to cell oxidants are developed and can be applied to targeted therapy for selective drug release. Aminoacrylate are very stable molecules that can be cleaved in presence of ROS in order to release the alcohol derivative and the formamide portion with the release of carbon dioxide. This can be used to release an alcohol-based drug [147-151]. Oligoproline can be used in order to attach an amine-derived drug to the C-end of proline. In presence of ROS the linker is oxidized to form the corresponding pyrrodilone, the free amino-drug and carbon dioxide [152-154]. Peroxalate ester are ROS-sensitive linkers used to couple alcohol-based drug with delivery vehicles

89 with terminal alcohol as well. In presence of hydrogen peroxide, the peroxalate ester can be oxidized in order to release the free alcohol-based drug and the other side along with carbon dioxide [155, 156].

4.3 Project idea and hypothesis

4.3.1 Double selectivity for drug delivery

This project focuses on the development of a new cleavable linker for targeted therapy that will be utilizing reactive oxygen species for drug release. This technology has two selectivity features for treatment in order to avoid off-site drug release and the associated side effects: the use of a delivery vehicle (targeting peptide, aptamer, antibody...) and the high levels of reactive oxygen species (ROS) in cancer cells.

Figure 4. 4: ROS-sensitive linker for targeting therapy. The delivery vehicle recognizes a

biomarker on the cancer cell surface and binds to it followed by internalization. The

90 cytotoxic payload is then cleaved using the high levels of ROS in the cancerous cell.

A delivery vehicle such as an aptamer, targeting peptide or antibody is used that will target a specific cancerous cell surface biomarker prior to being internalized. Some receptors are overexpressed on cancerous cells and are targetable using those delivery vehicles. An example in AML cells is the overexpression of toll-like receptor-2 (TLR) in some cell lines such as U937, THP1 or HL-60 that can be targeted using a targeted peptide pep2 (HLYVSPW). By functionalizing the N-terminus of that peptide, it is possible to attach a linker with a drug for selective drug release to those cells. Using the cell oxidants for drug release is still novel for peptide-drug conjugates (PDC) and can bring higher selectivity for drug release and avoid premature release of the drug due to the low levels of

ROS in plasma. Additionally, the levels of ROS are 100-fold higher in cancerous cells compared to healthy cells, helping to reduce peripheral toxicity in this drug release technology. Here, we are investigating the development of two different type of ROS- sensitive linkers. The first technology will trigger the cyclization of the linker in presence of cell oxidants that will cause drug release. The second technology uses an oxalamide linker that can react with hydrogen peroxide and trigger the release of an amine-containing drug at the oxalamide junction. Both linkers are investigated to show that they are stable at physiological conditions and that they can release a drug in presence of cancerous cells levels of different types of ROS at a significant rate for targeted therapies.

91 4.3.2 ROS-sensitive self-cyclizing linker

The Merino lab had been focused on the use of ROS antioxidant inhibitors for the past years. One of the molecular designs found by the group was inspired by piperlongumine that had the ability to increase the generation of ROS and inhibit the glutathione synthesis leading to cancer cell death. One of the potent analogues found was MA14 that contained

3 essential parts for ROS-related activity: a reactive quinone, a linker arm and a portion that has the potential to be kicked out once cyclization occurred.

Figure 4. 5: Structure of piperlongumine and MA14

Using this design, we have developed a linker with the hydroquinone-derived species where in presence of ROS it is possible to activate the self-cyclization of the molecule that will lead to the ejection of a potential drug. For the purpose of targeted therapy, we decided to add a functional group that would allow us to add the delivery vehicle to the scaffold.

Here, we chose to use an alkene group that would allow us to do thiol-ene click chemistry with a terminal thiol on an aptamer, peptide or other delivery vehicles. As shown in the figure below, the alkene can be put either in para position of the linker or on the linker arm.

In presence of a thiolated delivery vehicle it is possible to click the two entities together by using a 365 nm wavelength to catalyze the formation of the covalent stable thioether bond.

Figure 4. 6: self-cyclizing ROS-sensitive linker skeleton

The drug can be attached to this scaffold at the para position of the linker in order to be ejected when the molecule cyclizes in presence of cell oxidants. It has been proved that amine and alcohol-based drugs can be ejected from this position. In presence of ROS, the linker aminophenol is activated similarly to the quinone derivatives which triggers the

6-membered ring cyclization with the amine on the glycine arm of the linker. Once cyclized the molecule releases the portion in para position as shown in the figure below.

Figure 4. 7: mechanism of drug ejection for ROS-self cyclizing linker

4.3.3 ROS-sensitive oxalamide linker

The second type of linkers developed are oxalamide linkers. Those linkers very similar to the existing peroxalate linkers bring additional stability since the amide groups are less likely to be hydrolyzed or cleaved at physiological conditions in the plasma.

Additionally, we added a quinone-like group to the design in order to make it more sensitive to the reactive oxygen species in tumor cells and to enhance the drug release

93 through that pathway. On one side of the oxalamide it is possible to add an amine- containing drug and on the other side will be the ROS-sensitive aminophenol group. We then add a functional group in order to attach the delivery vehicle to the linker. Here we chose to add a carboxylic acid for possible coupling with a amine terminal vehicle.

Figure 4. 8: Oxalamide ROS-sensitive linker skeleton

In this design, the proposed mechanism of ejection is very similar to what occurs in glow sticks with the oxidation of diphenyl oxalate in presence of the hydrogen peroxide present in the glass vial. Here, the ROS will first activate the quinone portion of the linker making it easier to react with hydrogen peroxide and eject that portion of the linker. The peroxoate portion will then react with the vicinal carbonyl in order to release the drug and form a 4-membered 1,2-dioxetanedione that will then decompose into two molecules of carbon dioxide.

Figure 4. 9: mechanism of drug ejection for ROS-oxalamide linker

94 In this project it is then essential to validate our hypothesis for the designs: 1) we are able to release a potential drug from the two designs in presence of ROS 2) It is possible to attach a delivery vehicle to the linkers to use it for targeted therapy 3) The release can happen in cellulo using the amount of cell oxidants in cancerous cells.

4.4 Results and discussion

4.4.1 Choice of drug and synthesis

The first step before looking into proving that it was possible to release a drug from the different linkers was for us to select two drugs with available alcohols and amines for the proof-of-concept studies. We then decided to select a poly-ADP ribose polymerase

(PARP) inhibitor NU1025 with an available phenol, and an inhibitor of the serine/threonine protein kinase Akt (protein kinase B) AT7867 with an available amine.

Figure 4. 10: Structure of NU1025 and AT7867

95 4.4.1.1 NU1025

PARP inhibitor NU1025 is a good model drug for the development of our first technology. Poly (ADP-ribose) polymerases (PARPs) are enzymes used for cellular homeostatis involved in DNA transcription, cell-cycle regulation and in DNA repair. PARP inhibitors cancel the activity of PARPs which leads to cell instability, high degradation and sometimes cellular apoptosis [157]. Many PARP inhibitors have been synthesized as a target for multiple cancers (ovarian, breast, pancreatic, melanoma, ...) and many are undergoing clinical trials for cancer therapy [158]. For the development of our biotechnique NU1025 was chosen for the available phenol group compatible with our self- cyclizing strategy and for the easy chemistry behind its synthesis [159]. This compound has already shown very good cytotoxicity for human breast cancer [160] and has shown the ability to increase the cytotoxicity of diverse anti-cancer agents evaluation in L1210 cells [161]. The drug was then synthesized in a 4-step synthesis as shown below.

Scheme 4.1: Synthesis route for NU1025 (13)

96 4.4.1.2 AT7867

The serine/threonine kinase AKT (also called protein kinase B, PKB) is found downstream of the phosphatidylinositol 3-kinase (PI3K). The AKT and AKT-regulated pathways are often overexpressed and/or hyper-activated in some cancers such as human colorectal cancer (CRC). It usually helps with cancerous cell growth, proliferation and metabolism additionally to cell survival and resistance to apoptosis [162, 163]. The inhibition of AKT with small molecules has been investigated and often lead to cancerous cell death, and multiple inhibitors are investigated in pre-clinical and clinical stages [164-

169].

In 2010, Grimshaw and al. reported the identification of AT7867 as a new potent inhibitor of AKT as well as the downstream kinase p70S6K and PKA. The ATP- competitive small molecule was shown to inhibit the cellular activity of AKT and p70S6K leading to the growth inhibition and apoptosis in multiple CRC cell lines [170, 171].

In our design, we chose AT7867 for our amine-containing drug for the known correlation between ROS and AKT/PI3K pathway, as well as the ability to synthesize it in a simple 2-step route as shown below [172].

Scheme 4.2: Synthesis route for AT7867 (14)

97 4.4.2 Proof of concept: release of a drug from scaffold

The first goal in the development of the technology was to prove that it was possible to eject the drug portion (in red) of the two linkers in presence of reactive oxygen species while proving that no drug release was occurring at physiological conditions. To do that two simple prodrugs were synthesized using the ROS-sensitive designs in order to be exposed to reactive stress to evaluate the drug release by HPLC.

15 16

Figure 4. 11: Structure of ROS-sensitive prodrugs used for linker design with the drug

portion in red.

4.4.2.1 Self-cyclizing scaffold

The self-cyclizing scaffold was developed in the Merino lab for the past years and it has been proven that it is possible to release an alcohol or amine-containing drug portion in presence of reactive oxygen species when there is an OH or NH2 in para position of the scaffold and with a glycine arm in ortho position. Here the goal is to prove that it is possible

98 to alkylate the NH2 in para in order to have the available alkene for click chemistry with the delivery vehicle. Compound 15 was then synthetized using a 5-step route shown below, where a methoxyphenol was used for the drug portion, mimicking the phenol present on

NU1025.

Scheme 4.3: Synthesis route for 15

The compound was dissolved in PBS buffer pH 7.4 and multiple conditions were tested. First the compound stability at physiological conditions (pH 7.4 and 37 degrees

Celcius). Then it was exposed to different type of reactive oxygen species: 5-10 equivalents of hydrogen peroxide and 5-10 equivalents of hydrogen peroxide/peroxidase (Figure 4.10).

The oxidation was monitored using a HPLC, with a C18 column and an acetonitrile/water linear gradient. HPLC analysis of the controls revealed that the GUPR98 (15) eluted at

11.9 minutes while the methoxyphenol eluted at 12.3 minutes. The study showed that the compound is stable at physiological conditions and in presence of hydrogen peroxide as no peak decrease was observed nor no new peaks. Nevertheless, once exposed to 6.5

99 equivalents of hydrogen peroxide in presence of peroxidase, the compound oxidizes as the peak at 11.9 minutes decreased and the HPLC chromatogram revealed a new peak at 12.3 minutes corresponding to the release of methoxyphenol (drug portion) and another new peak at 14.6 minutes. The mass spectrometry analysis of that new peak at 14.6 revealed that it was the self-cyclized molecule with a mass of 244 Da. The half-life observed for the release of the drug portion was 28 minutes. This here confirms that it is possible to release the drug portion on the ROS-sensitive self-cyclizing linker in presence of cell oxidants while maintaining stability in plasma at physiological conditions with this technology.

Figure 4. 12: Oxidation of GUPR98 (15) A) HPLC chromatograms of a) controls b)

GUPR98 with or without ROS; B) structure and concentrations used in the study; C)

Mass spectrometry of the new major product.

100 4.4.2.2 Oxalamide scaffold

The oxalamide scaffold is very novel in the Merino lab and has been investigated for the past year for its potential to be used as a prodrug or linker for drug delivery due to its sensitivity to reactive oxygen species, more specifically hydrogen peroxide. In order to confirm the effectiveness of our linker design, a compound was synthesized with AT7867 on one side of the oxalamide and with the ROS-activatable aminophenol on the other side, ox-AT7867 (16). Here, this prodrug is then exposed to reactive oxygen species to show that it is possible to eject AT7867 in those conditions.

Scheme 4.4: Synthetic route of 16

The HPLC monitoring of the oxidation using a C-18 column and a acetonitrile/water gradient. Th prodrug ox-AT7867 (16) eluted at 17.8 minutes while

AT7867 eluted at 12.4 minutes. The study showed that the molecule was stable at physiological conditions for a long time as no peak decreased was observed nor new peaks.

Once exposed to five equivalents of H2O2, the compound showed a reduced peak and multiple oxidation products, one being at 12.4 minutes characteristic of AT7867. The UV

101 trace confirmed that the peak had the same UV absorbance spectra than AT7867. After 48 hours, almost one hundred percent of the drug was ejected from the prodrug in presence of hydrogen peroxide. This result along with the stability at physiological conditions show that this design is usable for the development of a ROS-responsive linker for drug delivery.

Figure 4. 13: Oxidation of Ox-AT7867 (16) A) HPLC chromatograms B) structure and

conditions of oxidation (C) UV trace of AT7867 and new oxidation compound.

The next step was to then add a functional group for the attachment of the delivery vehicle. Here, we chose a carboxylic acid functional group that will be attached on the

ROS-activatable portion of the linker.

102 4.4.3 Other linker models to investigate

As shown in figure 4.6, it is possible to add the available alkene at two different positions for the development of the self-cyclizing model. It was then essential for us to look at the two possibilities in order to see which one would be better for the development of the linker and that would work with the different forms of ROS present in cancerous cells. We consequently designed two additional linker models with an allyl glycine in para- position (17) or a 4-pentenoic (18) that would allow to investigate the importance of the self-cyclizing mechanism for the drug ejection.

Figure 4. 14: Other ROS-responsive self-cyclizing linker models

In the oxalamide linker it was necessary to add a carboxylic acid group to the ROS- activatable portion of the design. In order to test the release once we added the linker, a series of oxalamide linkers was synthesized with 1-phenylpiperazine on one end mimicking the AT7867 portion for release and with derivatives of aminosalicylic acid on the other side of the oxalamide (Figure 4.13).

103

Figure 4. 15: Other ROS-responsive oxalamide linker models

The first designed used an aminocatechol design for the ROS-activated portion with the carboxylic acid group in para position for drug attachment (19). The next two designs used 5-aminosalicylic acid for the quinone like-oxidation of that structure. The carboxylic acid was then changed between the meta position (20) and the ortho position (21). In the last design it was important to investigate the oxidation when the withdrawing carboxylic acid was not attached directly to the oxidizable ring.

4.4.4 Oxidation of the different linkers and drug ejection

The different synthesized linkers were then exposed to physiological conditions

(pH 7.4, 37 °C) to make sure that the linker will be stable in the plasma and no trigger early drug release. They then were exposed to reactive oxygen species (H2O2 and H2O2 + HRP) to show that the drug portion (4-methoxyphenol and 1-phenylpiperazine) can be released under those conditions.

104 ROS Physiological Compound % oxidation Time Observations conditions Number of Type equivalents

t is around 30 minutes. 4- H O 5. 0 % Hours 1/2 2 2 methoxyphenol released 15 Stable observed along with two H O + 5 + 1μl of other oxidation products 2 2 82 % 2 h HRP 1g/l peaks

. When using 1 equiv. of H2O2 5 0 % Hours ROS 82% was oxidized after 2 hours. 8 oxidation peaks 17 Stable observed, with poor C18 H O + 5 + 1μl of 2 2 83% 30 min separation. Need to further HRP 1g/l confirm drug release Almost all the compound is H2O2 5 0 % Hours oxidized at 5 eq. of ROS after 30 minutes. When using 1 equiv. of ROS 69% 18 Stable was oxidized after 2 hours. H O + 5 + 1μl of 5 main new oxidation 2 2 95% 45 min HRP 1g/l product peaks. Drug release need to be confirmed by LC- MS.

H2O2 5 62 % 48 h The compound is not stable at physiological conditions 19 Unstable H O + 5 + 1μl of hence not viable for the linker 2 2 72 % 48 h HRP 1g/l development.

t1/2 is around 24 hours for H2O2 5 87 % 48 h these linkers. The release of 1-phenylpiperazine was not 20 Stable obvious. New product peak H O + 5 + 1μl of 2 2 78 % 48 h observed. Need to run LC- HRP 1g/l MS.

H2O2 5 77% 48 h Same observation than previous molecule. HRP 21 Stable H O + 5 + 1μl of conditions seem better for the 2 2 90 % 48 h HRP 1g/l potential drug release.

Table 4. 2: Summary of the compound oxidations for ROS-triggered drug release

105 The self-cyclizing molecules oxidizes within minutes. Compound 15 was stable at physiological conditions and can be oxidized in presence of hydrogen peroxide and peroxidase with a half time of 28 minutes. The release of 4-methoxyphenol could be observed by HPLC, as well as the self-cyclized molecule. Compound 17 was also stable at physiological conditions and oxidized faster than the previous molecule. After 30 most of the compound was oxidized, and further studies showed that lower levels of ROS could be use for the oxidation of the molecule. The release of the drug-portion still needs to be confirmed by LC-MS. Compound 18 is stable at physiological conditions and also oxidized quickly as most of the compound is oxidized after 30 minutes and it can be oxidized at a quick rate with low levels of ROS as well. LC-MS can be used to confirm the release of the drug-portion and identify the nature of the oxidation products.

The oxalamide models oxidizes in hours. Compound 19 was revealed to be unstable at physiological conditions which made it unusable for the development of a linker.

Compounds 20 and 21 both showed similar oxidation profile. The half-life for these molecules seems to be around 24 hours. One main more polar oxidation peak is observed for both compounds. When using hydrogen peroxide and peroxidase more oxidations products can be observed with the potential release of the drug portion, 1-phenylpiperazine at low levels. Further LC-MS studies need to be done to confirm the release is possible from these scaffolds and identify the main oxidation products.

106 4.5 Experimental

4.5.1 Materials

All chemicals, reagents, and solvents were purchased from Fisher Scientific,

Sigma-Aldrich Inc., Ambeed or other suppliers through Quartzy and used as received unless stated otherwise. All reactions were carried out under argon in clean oven-dried glassware and stirred with a magnetic stir bar. All the indicated reaction temperatures correspond to the temperature of the metallic heating block used and indicated as room temperature (r.t.) for any reaction from 22-25 degree Celsius. The thin layer chromatography (TLC) used to monitor the reactions were all glass backed silica plates (20 x 20 cm, 60 Å, 250 μm) cut into smaller plates to monitor individual reactions. The visualization of the TLC plates was done by using a 254 nm UV lamp or TLC stains such as ninhydrin, permanganate, or iodine. The proton 1H and carbon 13C nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Advance 400 MHz spectrometer and the samples were dissolved in deuterated solvents such as chloroform-d (CDCl3), methanol-d4 (CD3OD), dimethylsulfoxide-d6 (DMSO-d6), or water-d2 (D2O). The analysis of the NMR was done using Mestrenova and the chemical shifts on the spectra are shown in ppm with trimethylsilane as standard. Data analysis are shown as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, b = broad, m = multiplet, abq = ab quartet), and coupling constants. High resolution mass spectral data were collected on a Orbitrap Fusion Lumos

Tribrid Mass Spectrometer (Thermo Fisher Scientific). All novel compounds were characterized by 1H, 13C and high-resolution mass spectrometry. HPLC analysis of final

107 products was performed on a Beckman system Gold HPLC. They were ran using a Venusil

AQ C18 column (5Å, 7.5cm x 4.6mm) with an acetonitrile/water linear gradient and detected by UV with a scanning wavelength range from 200 to 500 nm.

4.5.2 Synthetic methods

4.5.2.1 Synthesis of NU1025 (13)

108 4.5.2.1.1 Synthesis of 3-methoxy-2-nitrobenzamide (13-b)

3-methoxy-2-nitrobenoic acid (13-a, 1.0 g, 5.1 mmol) and thionyl chloride (0.6 ml, 7.6 mmol) were both mixed in 20 ml dry tetrahydrofuran with 2 drops of N,N- dimethylformamide under argon. The reaction was carried at room temperature for 24h.

The resulting reaction mixture was added to an excess of aqueous ammonia (8 ml) dropwise. The solvent was removed using the rotavap and the remaining white solid was washed with iced-cold water and filtered. 3-methoxy-2-nitrobenzamide (13-b) was obtained as a white solid (0.90 g, 4.6 mmol, 90%).

1 H NMR (MeOD): δ 3.95 (3H, s), 7.29 (1H, d, J=10 Hz), 7.41 (1H, d, J= 10 Hz), 7.58 (1H, t, J=10 Hz)

13 C NMR (MeOD): 57.33, 116.76, 120.61, 131.16, 132.59, 152.45, 169.85

Figure 4. 16: 1H NMR of 3-methoxy-2-nitrobenzamide (13-b)

109

Figure 4. 17: 13C NMR of 3-methoxy-2-nitrobenzamide (13-b)

4.5.2.1.2 Synthesis of 3-methoxy-2-aminobenzamide (13-c)

3-methoxy-2-nitrobenzamide (13-b, 0.85 g, 4.3 mmol) and triethylsilane (2.8 mL, 18 mmol) was dissolved in dry methanol (50 ml) in presence of 10% palladium on carbon

(0.2g). The reaction was carried at room temperature for 2h. After filtrating the reaction mixture through celite and removing the methanol using the rotavap, 3-methoxy-2- aminobenzamide (13-c) was obtained as a beige solid (0.65g, 3.9 mmol, 91%).

1 H NMR (DMSO): δ 3.88 (3H, s), 7.31 (1H, d, J=10 Hz), 7.45 (1H, d, J= 10 Hz), 7.60 (1H, t, J=10 Hz), 7.70 (2H, s), 8.17 (2H, s)

110 13 C NMR (DMSO): 56.80, 115.85, 119.59, 129.43, 131.33, 139.12, 150.39, 165.39

+ LRMS [M+Na] : calc. for C8H10N2O2, 189.0640; found 189.3

Figure 4. 18: 1H NMR of 3-methoxy-2-aminobenzamide (13-c)

Figure 4. 19: 13C NMR of 3-methoxy-2-aminobenzamide (13-c)

111

Figure 4. 20: LRMS (ESI) of 3-methoxy-2-aminobenzamide (13-c)

4.5.2.1.3 Synthesis of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d)

3-methoxy-2-aminobenzamide (13-c, 0.60 g, 4.5 mmol) was dissolved in dry THF with dry pyridine (0.5 mL, 6.0 mmol). Acetyl chloride (0.7 ml, 10.0 mmol) in 2 ml dry THF was slowly added to the mixture under argon. The reaction was carried at room temperature for

12h. The solvent was removed using the rotavap and the solid was resuspended in 2% aqueous NaOH. The solid was then recrystallized from methanol/water. 8-methoxy-2- methylquinazolin-4-[3H]-one (13-d) was obtained as a white crystal (0.50 g, 2.6 mmol,

68%).

1 H NMR (DMSO): δ 2.34 (3H, s), 3.87 (3H, s), 7.30 (1H, d, J=10 Hz), 7.37 (1H, t, J= 10

Hz), 7.61 (1H, d, J=10 Hz), 12.22 (1H, s)

112 13 C NMR (DMSO): 21.47, 55.70, 114.64, 116.60, 121.57, 125.96, 139.39, 152.79,

153.82, 161.66

+ LRMS [MH] : calc. for C10H10N2O2, 191.0821; found 191.08

Figure 4. 21: 1H NMR of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d)

113

Figure 4. 22: 13C NMR of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d)

Premnauth_GUPR-NU1025-3_20170420-R01 # 1-371 RT: 0.00-3.38 AV: 130 NL: 5.64E4 F: ITMS + p ESI Full ms [100.00-1000.00] 191.08 100

75 70

50 RelativeAbundance

30 25 20

10 213.08

5 471.25 130.08 229.00 403.17 537.50 619.50 663.50 727.17 0 100 200 300 400 500 600 700 800 900 1000 m/z Figure 4. 23: LRMS of 8-methoxy-2-methylquinazolin-4-[3H]-one (13-d)

114 4.5.2.1.4 Synthesis of 8-hydroxy-2-methylquinazolin-4(3H)-one (13)

8-methoxy-2-methylquinazolin-4-[3H]-one (13-d, 350 mg, 1.85 mmol) was dissolved in a

1M solution of boron tribromide in dichloromethane (4.2 mL, 4.2 mmol) and put to reflux under nitrogen for 24 hours. The solution was then distilled to get rid of the remaining boron tribromide and HBr gas. It was then quenched with 5 ml of 5% sodium hydroxide and neutralized using a 1M hydrochloride acid solution. A white solid formed after cooling the mixture in an ice bath and was filtered using vacuum filtration. The cake was washed with water several times to yield a pure 8-hydroxy-2-methylquinazolin-4(3H)-one (13) as a white solid (156 mg, 0.89 mmol, 48%).

1 H NMR (DMSO): δ 2.44 (3H, s), 7.21 (1H, d, J=4 Hz, J=8 Hz), 7.31 (1H, t, J= 8 Hz),

7.55 (1H, dd, J=8 Hz), 9.51 (1H, s), 12.23 (1H, s)

13 C NMR (DMSO): 21.38, 99.48, 115.38, 118.09, 121.39, 126.18, 137.90, 152.23,

152.46, 161.67

+ HRMS [MH] : calc. for C9H9N2O2, 177.0659; found 177.0657

115

Figure 4. 24: 1H NMR of 8-hydroxy-2-methylquinazolin-4(3H)-one (13)

Figure 4. 25: 13C NMR of 8-hydroxy-2-methylquinazolin-4(3H)-one (13)

116 [GP-NU1025-1] #156-226 RT: 0.22-0.49 AV: 54 NL: 2.78E9 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 177.0657 100

RelativeAbundance 40

5 406.0363 141.9585 199.0477 261.0183 335.1141 466.9838 514.4002 582.0945 630.1258 0 100 150 200 250 300 350 400 450 500 550 600 650 m/z

Figure 4. 26: HRMS of NU1025 (13)

4.5.2.2 Synthesis of AT7867 (14)

117 4.5.2.2.1 Synthesis of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b)

Anhydrous aluminum chloride (1.83g, 13.73 mmol) was suspended in 10 ml of chlorobenzene at 0oC. A suspension of 4-(4-bromo-phenyl)-piperidin-4-ol (14-a, 1.01 g,

3.93 mmol) in 10 ml of chlorobenzene is slowly added to the mixture. The reaction is stirred in an ice bath for 2 hours then quenched using ice then 10ml of methyl-t-butyl ether.

The white suspension is then stirred an additional hour then filter using vacuum filtration.

The cake is washed with water and methyl-t-butyl ether to give a pure 4-(4-bromophenyl)-

4-(4-chlorophenyl)piperidine (14-b) as a white solid (1.34g, 3.81mmol, 97%).

1 H NMR (MeOD): δ 2.68 (4H, t, J=8Hz), 3.22 (4H, t, J=8Hz), 7.30 (2H, d, J=8 Hz), 7.37

(4H, m), 7.52 (2H, d, J=8 Hz)

13 C NMR (DMSO): 33.44, 42,48, 44.51, 121.22, 129.46, 129.80, 130.11, 133.14, 133.75

+ LRMS [MH] : calc. for C17H18NBrCl, 350.0306; found 350.0616

118

Figure 4. 27: 1H NMR of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b)

Figure 4. 28: 13C NMR of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b)

119

Figure 4. 29: LRMS of 4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b)

4.5.2.2.2 Synthesis of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl)

piperidine (14)

4-(4-bromophenyl)-4-(4-chlorophenyl)piperidine (14-b, 426 mg, 1.22 mmol) was dissolved in 10 ml of 1,2-methoxyethane and 1 ml of water and purged with argon.

Tetrakis(triphenylphosphine)palladium(0) (46mg, 3 mol%) was added to the reaction mixture with pyrazole-4-boronic acid pinacol ester (14-c, 306 mg, 1.58 mmol) and potassium carbonate (580.5 mg, 4.20 mmol). The reaction was refluxed at 100oC for 15 hours. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using

120 filter paper. Silica (500 mg) was added before removing the solvent by rotavap. The solid was loaded to a silicas column then purified by flash chromatography using a 1M triethylamine in MeOH/DCM gradient. The resulting 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4- chlorophenyl) piperidine (14) was isolated as a single compound a white powder (62 mg,

0.18 mmol, 15 %).

1 H NMR (MeOD): δ 2.43 (4H, m), 2.90 (4H, m), 7.29 (6H, m), 7.51 (2H, d, J=8 Hz), 7.91

(2H, s)

13 C NMR (MeOD): δ 37.03. 43.54, 45,44, 123.21, 126.79, 128.57, 129.49, 129.70, 131.89,

132.72

+ LRMS [MH] : calc. for C20H21N3Cl, 338.1419; found 338.1609

Figure 4. 30: 1H NMR of4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine

(14)

121

Figure 4. 31: 13C NMR of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine

(14)

Sample ID: GP2-14-4 (C20 H20 N3 Cl) 337 Da (in MeOH) PREMNAUTH-G-062320-[GP2-14-4] 3 (0.059) Cm (2:57) TOF MS ES+ 338.1609 100 3.17e4

340.1691 %

341.1755

194.0907 364.1995 150.1201 532.2517 196.0890 152.1207 390.2154

0 m/z 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925

Figure 4. 32: LRMS of 4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl) piperidine (14)

122 4.5.2.3 Synthesis of 2-amino-N-(5-(hex-5-en-1-ylamino)-2-(4-methoxy-

phenoxy)phenyl)acetamide (15)

4.5.2.3.1 Synthesis of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c)

4-methoxyphenol (15-b, 3.84 g, 31.0 mmol) was dissolved in 5ml of dimethylformamide and sodium hydride (0.75g, 31.0 mmol) was slowly added in an iced bath. The mixture was stirred for 30 minutes at room temperature then 2-Fluoro-5-nitroaniline (15-a, 4 g,

25.6 mmol). The reaction was carried for 15 hours at 80 oC. The reaction cooled then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. The solvent was removed using rotary evaporator to obtain 2-(4-methoxyphenoxy)-5-nitroaniline (15- c, 6.56g, 25.2 mmol, 81%).

123 1 H NMR (MeOD): δ 3.82 (3H, s), 6.63 (1H, d, J=8 Hz), 7.02 (4H, q, H=8 Hz, J=28 Hz),

7.46 (1H, m), 7.68 (1H, s)

13 C NMR (CDCl3): δ 55.71, 110.05, 114.35, 114.82, 115.21, 121.32, 137.39, 143.00,

148.11, 151.06, 156.86

Figure 4. 33: 1H NMR of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c)

124

Figure 4. 34: 13C NMR of 2-(4-methoxyphenoxy)-5-nitroaniline (15-c)

4.5.2.3.2 Synthesis of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl)

amino)-2-oxoethyl)carbamate (15-d)

Boc-glycine (660 mg, 3.77 mmol) and HATU (1.90 g, 5 mmol) were dissolved in dimethylformamide and stirred for 30 minutes at room temperature. 2-(4- methoxyphenoxy)-5-nitroaniline (15-c, 655 mg, 2.5 mmol) was then added to the mixture and stirred for an additional 30 minutes before adding N,N-diisopropylethylamine (1.1 ml,

6.25 mmol). The reaction was carried for an additional 12 hours at room temperature then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 1g of silica was

125 added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using 1:2 ethyl acetate/hexanes mobile phase. The fractions were collected then dried to yield the pure tert-butyl (2-((2-(4- methoxyphenoxy)-5-nitrophenyl) amino)-2-oxoethyl)carbamate (15-d, 834 mg, 2.0 mmol, 80 %) as a brown oil solid.

1 H NMR (CDCl3): δ 1.40 (9H, s), 3.49 (1H, S), 3.85 (3H, s), 4.01 (2H, d, J=4 Hz), 5.21

(1H, s), 6.70 (1H, d, J=32 Hz), 6.99 (4H, m), 7.86 (1H, dd, J= 4 Hz, J=8 Hz), 8.80 (1H, s),

9.36 (1H, d, J=4 Hz)

13 C NMR (CDCl3): δ 34.94, 35.84, 38.65, 43.49, 44.55, 115.01, 121.72, 121.86, 125.53,

127.20, 128.24, 128.30, 129.06, 130.82, 131.64, 143.39, 145.52, 154.68, 162.13, 163.44

Figure 4. 35: 1H NMR of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl) amino)-2-

oxoethyl)carbamate (15-d)

126

Figure 4. 36: 13C NMR of tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl) amino)-

2-oxoethyl)carbamate (15-d)

4.5.2.3.3 Synthesis of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl)

amino)-2-oxoethyl)carbamate (15-e)

tert-butyl (2-((2-(4-methoxyphenoxy)-5-nitrophenyl) amino)-2-oxoethyl)carbamate (15-d,

595 mg, 1.44 mmol) and tin chloride (785 mg, 7.2 mmol) were dissolved in methanol and stirred at room temperature for 5 hours. The reaction was then neutralized with sodium bicarbonate, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. The solvent was removed using rotary

127 evaporator to obtain tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-2- oxoethyl)carbamate (15-e, 406 mg, 1.05 mmol, 73%)

1 H NMR (CDCl3): δ 1.32 (9H, s), 3.71 (3H, s), 3.81 (2H, s), 5.03 (1H, s), 6.27 (1H, dd,

J=4 Hz, J=8 Hz), 6.61 (1H, d, J=8 Hz), 6.79 (4H, m), 7.80 (1H, d, J=4 Hz), 8.29 (1H, s)

13 C NMR (CDCl3): δ 28.20, 28.31, 37.99, 55.69, 91.99, 107.30, 110.51, 114.83, 118.59,

119.32, 130.07, 138.28, 142.92, 151.22, 155.47, 167.62

Figure 4. 37: 1H NMR of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-

2-oxoethyl)carbamate (15-e)

128

Figure 4. 38: 13C NMR of tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-

2-oxoethyl)carbamate (15-e)

4.5.2.3.4 Synthesis of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxy

phenoxy)phenyl) amino)-2-oxoethyl)carbamate (15-f)

tert-butyl (2-((5-amino-2-(4-methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15- e, 400 mg, 1.03 mmol) was dissolved in 2 ml of absolute ethanol. 6-bromohex-1-ene (550

μl, 4.12 mmol) was slowly added to the mixture and the reaction was carried at 69oC for

48 hours. The mixture was then quenched and neutralized with 1M sodium hydroxide and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 1g of silica was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography

129 using 1:4 ethyl acetate/hexanes mobile phase to yield tert-butyl (2-((5-(hex-5-en-1- ylamino)-2-(4-methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15-f, 193 mg,

0.41 mmol, 40%).

1 H NMR (CDCl3): δ 1.39 (9H, s), 1.50 (2H, m), 1.61 (2H, m), 2.09 (2H, m), 3.10 (2H, t,

J=8 Hz), 3.77 (3H, s), 3.88 (2H, d, J=4 Hz), 5.00 (2H, m), 5.81 (1H, m), 6.25 (1H, m), 6.72

(1H, d, J=8 Hz), 6.80 (4H, m), 7.78 (1H, d, J=4 Hz), 8.29 (1H, s)

13 C NMR (CDCl3): δ 26.39, 28.21, 29.00, 29.71, 33.49, 44.25, 45.32, 55.68, 80.43, 105.22,

207.68, 114.71, 114.79, 118.28, 119.63, 130.30, 137.07, 138.55, 145.38, 151.55, 155.32,

167.52

Figure 4. 39: 1H NMR of (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl)

amino)-2-oxoethyl)carbamate (15-f)

130

Figure 4. 40: 13C NMR of (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl)

amino)-2-oxoethyl)carbamate (15-f)

4.5.2.3.5 Synthesis of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxy

phenoxy)phenyl) amino)-2-oxoethyl)carbamate (15)

tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl) amino)-2- oxoethyl)carbamate (15-f, 170 mg, 0.36 mmol) was dissolved in 1ml of a 1:1 trifluoro acetic acid/dichloromethane and stirred at room temperature for an hour. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with 1 ml of dichloromethane each time. The organic layers were combined and washed with water and brine then filtered using a glass piper loaded with cotton and sodium sulfate anydrous. The solvent was removed using rotary evaporator to obtain tert-butyl (2-((5-

131 (hex-5-en-1-ylamino)-2-(4-methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15,

109 mg, 0.30 mmol, 82 %)

1 H NMR (CDCl3): δ 1.42 (2H, m), 1.58 (2H, m), 2.05 (2H, m), 3.61 (2H, S), 3.75 (3H, s),

4.96 (2H, m), 5.77 (1H, m), 6.31 (1H, m), 6.69 (1H, d, J=8 Hz), 6.79 (4H, m), 7.74 (1H, d,

J=4 Hz), 9.05 (1H, s)

13 C NMR (CDCl3): δ 26.40, 29.03, 33.51, 44.27, 44.46, 55.69, 104.97, 10.7.38, 114.66,

114.70, 117.84, 120.31, 130.76, 136.95, 138.59, 145.61, 151.99, 155.03, 170.90

+ HRMS [MH] : calc. for C21H28N3O3, 370.2125; found 370.2126

Figure 4. 41: 1H NMR of (tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-

methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15)

132

Figure 4. 42: 13C NMR of (tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-

methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15)

[GP2-GUPR-89] #167-221 RT: 0.22-0.47 AV: 50 NL: 3.92E9 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 370.2126 100

RelativeAbundance 40

10 185.6098 739.4173 5 313.1911 121.0759 247.1678 392.1947 488.2404 573.7883 707.5470 761.3994 884.6175 951.4822 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure 4. 43: HRMS of tert-butyl (2-((5-(hex-5-en-1-ylamino)-2-(4-

methoxyphenoxy)phenyl) amino)-2-oxoethyl)carbamate (15)

133 4.5.2.4 Synthesis of 2-(4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chloro-

phenyl) piperidin-1-yl)-N-(4-hydroxyphenyl)-2-oxoacetamide

(16)

4-aminophenol (16-a, 12.5 mg, 0.115 mmol) and diphenyl oxalate (16-b, 28.0 mg,0.12 mmol) were dissolved in dimethylformamide in presence of N,N-diisopropylethylamine

(19μl, 0.1115 mmol) and reacted together for 4 hours at 60 °C. The reaction progress was monitored by thin layer chromatography (90% conversion for 16-c) and the next step was carried on-pot by adding a mixture of AT7867 (14, 39 mg, 0.115 mmol) in dimethylformamide was added slowly and the reaction was carried for an additional 10 hours at 60 °C. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 300 mg of silica was added to the flask before removing the solvent by rotavap.

The solid was loaded to a silica column then purified by flash chromatography using methanol/dichloromethane mobile to give 2-(4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chloro-

134 phenyl) piperidin-1-yl)-N-(4-hydroxyphenyl)-2-oxoacetamide (16, 36 mg, 0.0.72 mmol, overall 63%).

1 H NMR (MeOD): δ 2.53 (4H, m), 3.70 (4H, m), 6.78 (2H, m), 7.37 (6H, m), 7.44 (2H, m), 7.56 (2H, m), 7.94 (2H, s)

13 C NMR (MeOD): 34.94, 3584, 38.65, 43.49, 44.55, 115.01, 121.72, 121.86, 125.53,

127.20, 128.24, 128.30, 129.06, 130.82, 131.64, 143.39, 145.52, 154.68, 162.13, 163.44

+ LRMS [MH] : calc. for C28H26N4O3Cl, 501.1688; found 501.1687

Figure 4. 44: 1H NMR of ox-AT7867 (16)

135

Figure 4. 45: 13C NMR of ox-AT7867 (16)

[AT7867-OX] #1-241 RT: 0.25-0.77 AV: 101 NL: 8.55E7 T: FTMS + p ESI cv=0.00 Full ms [100.0000-2000.0000] 501.1687 100

RelativeAbundance 40

10 473.1737 536.6127 360.3236 619.6135 5 675.6763 429.2401 563.5509 723.2581 782.4408 838.5032 0 350 400 450 500 550 600 650 700 750 800 850 900 m/z

Figure 4. 46: HRMS of ox-AT7867 (16)

136 4.5.2.5 Synthesis of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)

phenyl)pent-4-enamide (17)

4.5.2.5.1 Synthesis of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)

amino)-1-oxopent-4-en-2-yl)carbamate (17-a)

Boc-allylglycine (255 mg, 0.95 mmol) and HATU (380 mg, 1.51 mmol) were dissolved in dimethylformamide and stirred for 30 minutes at room temperature. 2-(4- methoxyphenoxy)-5-nitroaniline (15-c, 221 mg, 0.85 mmol) was then added to the mixture and stirred for an additional 30 minutes before adding N,N-diisopropylethylamine (296 μl,

1.7 mmol). The reaction was carried for an additional 12 hours at room temperature then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine

137 then dried over sodium sulfate anhydrous and filtered using filter paper. 1 grams of silica was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using 1:2 ethyl acetate/hexanes mobile phase. The fractions were collected then dried to yield the pure tert-butyl (1-((2-(4- methoxyphenoxy)-5-nitrophenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-a, 133 mg,

0.30 mmol, 35 %) as a brown oil solid.

1 H NMR (CDCl3): δ 1.39 (9H, s), 2.65 (2H, m), 3.85 (3H, s), 4.37 (1H, s), 5.05 (1H, s),

5.23 (2H, m), 5.84 (1H, m), 6.69 (1H, d, J=12 Hz), 6.99 (4H, m), 7.85 (1H, dd, J= 4 Hz),

8.92 (1H, s), 9.38 (1H, s)

13 C NMR (CDCl3): δ 28.20, 55.71, 113.68, 115.35, 115.90, 119.78, 121.81, 142.99,

152.48, 157.43, 170.12

Figure 4. 47: 1H NMR of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)amino)-1-

oxopent-4-en-2-yl)carbamate (17-a)

138

Figure 4. 48: 13C NMR of tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)amino)-1-

oxopent-4-en-2-yl)carbamate (17-a)

4.5.2.5.2 Synthesis of tert-butyl (1-((5-amino-2-(4-methoxyphenoxy)

phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b)

tert-butyl (1-((2-(4-methoxyphenoxy)-5-nitrophenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-a, 133 mg, 0.30 mmol) and tin chloride (365 mg, 1.93 mmol) were dissolved in methanol and stirred at room temperature for 5 hours. The reaction was then neutralized with sodium bicarbonate, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. The solvent was removed

139 using rotary evaporator to obtain tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy) phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b, 100 mg, 0.23 mmol, 78%)

1 H NMR (CDCl3): δ 1.31 (9H, s), 2.45 (2H, m), 3.70 (3H, s), 4.18 (1H, s), 4.88 (1H, s),

5.03 (2H, m), 5.62 (1H, m), 6.28 (1H, d, J=12 Hz), 6.62 (1H, d, J=8 Hz), 6.78 (4H, m),

7.80 (1H, d, J= 4 Hz), 8.36 (1H, s), 9.85 (1H, s)

13 C NMR (CDCl3): δ 28.20, 36.53, 54.63, 55.69, 107.38, 110.51, 114.68, 114.81, 118.01,

118.44, 119.52, 130.16, 132.78, 138.28, 142.99, 151.34, 155.43, 169.61

Figure 4. 49: 1H NMR of tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy)

phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b)

140

Figure 4. 50: 13C NMR of tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy)

phenyl)amino)-1-oxopent-4-en-2-yl)carbamate (17-b)

4.5.2.5.3 Synthesis of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-

4-enamide (17)

Tert-butyl tert-butyl (1-((5-amino-2-(4-methoxyphenoxy) phenyl)amino)-1-oxopent-4-en-

2-yl)carbamate (17-b, 50 mg, 0.12 mmol) was dissolved in 1ml of a 1:1 trifluoro acetic acid/dichloromethane and stirred at room temperature for one hour. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with 1 ml of dichloromethane each time. The organic layers were combined and washed with water and brine then filtered using a glass piper loaded with cotton and sodium sulfate anhydrous. The solvent was removed using rotary evaporator to obtain 2-amino-N-(5- amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (17, 24 mg, 0.073 mmol, 62 %)

141 1 H NMR (CDCl3): δ 2.26 (1H, m), 2.62 (1H, m), 3.46 (1H, m), 3.77 (3H, s), 5.10 (2H, m), 5.69 (1H, m), 6.35 (1H, dd, J=4 Hz), 6.74 (1H, d, J=8 Hz), 6.85 (4H, m), 7.92 (1H, d,

J= 4 Hz), 9.91 (1H, s)

13 C NMR (CDCl3): δ 39.28, 54.63, 55.69, 56.28, 107.27, 110.30, 114.68, 118.01, 119.00,

120.23, 130.61, 134.19, 138.17, 143.25, 151.75, 155.14, 172.74

+ HRMS [MH] : calc. for C18H22N3O3, 328.1656; found 328.1656

Figure 4. 51: 1H NMR of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-

enamide (17)

142

Figure 4. 52: 13C NMR of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-

enamide (17)

[GP2-71] #9-215 RT: 0.19-0.54 AV: 70 NL: 1.21E9 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 328.1656 100

RelativeAbundance 40

15 231.1127 10

5 196.0967 123.0552 282.2792 340.1656 655.3236 424.1482 523.2549 609.4374 714.3383 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 m/z

Figure 4. 53: HRMS of 2-amino-N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-

enamide (17)

143 4.5.2.6 Synthesis of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-

enamide (18)

4.5.2.6.1 Synthesis of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide

(18-a)

4-pentenoic acid (255 μl, 2.5 mmol) and HATU (1.14 g, 3 mmol) were dissolved in dimethylformamide and stirred for 30 minutes at room temperature. 2-(4- methoxyphenoxy)-5-nitroaniline (15-c, 650 mg, 2.5 mmol) was then added to the mixture and stirred for an additional 30 minutes before adding N,N-diisopropylethylamine (650 μl,

3.75 mmol). The reaction was carried for an additional 14 hours at room temperature then quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 1 grams of silica

144 was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using 1:2 ethyl acetate/hexanes mobile phase. The fractions were collected then dried to yield the pure N-(2-(4- methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18-a, 564 mg, 1.65 mmol, 66 %) as a white oil.

1 H NMR (MeOD): δ 2.46 (2H, m), 2.63 (2H, m), 3.83 (3H, s), 5.07 (2H, m), 5.91 (1H, m), 6.77 (1H, dd, J=8 Hz), 7.04 (4H, m), 7.89 (1H, m), 9.06 (1H, s)

13 C NMR (MeOD): δ 30.63, 36.96, 56.17, 115.62, 116.09, 116.41, 119.00, 121.39, 129.61,

138.18, 143.35, 149.10, 155.55, 158.89, 174.00

Figure 4. 54: 1H NMR of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18-a)

145

Figure 4. 55: 13C NMR of N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18-a)

4.5.2.6.2 Synthesis of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide

(18)

N-(2-(4-methoxyphenoxy)-5-nitrophenyl)pent-4-enamide (18-a, 564 mg, 1.65 mmol and tin chloride (365 mg, 1.93 mmol) were dissolved in methanol and stirred at room temperature for 5 hours. The reaction was then neutralized with sodium bicarbonate, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. The solvent was removed using rotary evaporator to obtain N-

(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18, 396 mg, 1.27 mmol, 77%)

146 1 H NMR (MeOD): δ 2.29 (2H, m), 2.38 (2H, m), 3.76 (3H, s), 5.01 (2H, m), 5.77 (1H, m), 6.52 (1H, m), 6.71(1H, m), 6.85 (4H, m), 7.36 (1H, m), 9.06 (1H, s)

13 C NMR (MeOD): δ 30.81, 37.03, 56.11, 112.48, 113.68, 115.75, 115.89, 119.04, 121.54,

131.30, 138.12, 141.57, 145.17, 153.46, 156.64

+ LRMS [MH] : calc. for C18H2N2O3, 313.1547; found 313.1545

Figure 4. 56: 1H NMR of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18)

147

Figure 4. 57: 13C NMR of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide

(18)

[GP-67] #164-229 RT: 0.23-0.48 AV: 50 NL: 3.28E9 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 313.1545 100

RelativeAbundance 40

15 231.1126 10

625.3017 5 123.0551 190.1099 141.9584 281.1284 335.1366 397.1073 488.2018 594.4263 647.2838 698.4738 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 m/z

Figure 4. 58: HRMS of N-(5-amino-2-(4-methoxyphenoxy)phenyl)pent-4-enamide (18)

148 4.5.2.7 Synthesis of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)

acetamido)benzoic acid (19)

4-amino-3-hydroxybenzoic acid (19-a, 316 mg, 2.06 mmol) and diphenyl oxalate (16-b,

500 mg, 2.06 mmol) were dissolved in dimethylformamide in presence of N,N- diisopropylethylamine (358 μl, 2.06 mmol) and reacted together for 4 hours at 60 °C. The reaction progress was monitored by thin layer chromatography (90% conversion for 19-b) and the next step was carried on-pot by adding 1-phenylpiperazine (19-c, 334 μl, 2.06 mmol) in dimethylformamide was added slowly and the reaction was carried for an additional 10 hours at 60 °C. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 300 mg of silica was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using methanol/dichloromethane mobile to give 3-hydroxy-4-(2-oxo-2-

(4-phenylpiperazin-1-yl)acetamido)benzoic acid (19, 90 mg, 0.24 mmol, overall 12 %) as a white powder.

149 1 H NMR (MeOD): δ 3.29 (4H, m), 3.86 (2H, m), 4.11 (2H, m), 6.89 (1H, t, J=4 Hz), 7.03

(2H, d, J=8 Hz), 7.27 (2H, t, J=4 Hz), 7.57 (2H, d, J=4 Hz), 8.26 (1H, d, J=8 Hz)

13 C NMR (MeOD): 43.75, 47.55, 50.47, 51.23, 116.67, 118.02, 121.07, 121.64, 122.59,

130.16, 148.11, 148.39, 152.50, 160.35, 163.07, 169.66

+ HRMS [MH] : calc. for C19H20N3O5, 370.1397; found 370.1399

Figure 4. 59: 1H NMR of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (19)

150

Figure 4. 60: 13C NMR of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (19)

[GP2-oxalink 1] #125-224 RT: 0.23-0.45 AV: 44 NL: 2.55E8 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 370.1399 100

RelativeAbundance 40

10 163.1229 344.1244 191.1178 395.2080 5 207.1127 271.1513 413.2122 560.2503 651.4116 739.2722 793.1910 943.3130 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure 4. 61: HRMS of 3-hydroxy-4-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (19)

151 4.5.2.8 Synthesis of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-

yl)acetamido)benzoic acid (20)

5-amino-sallicylic acid (20-a, 250 mg, 1.63 mmol) and diphenyl oxalate (16-b, 395 mg,

1.63 mmol) were dissolved in dimethylformamide in presence of N,N- diisopropylethylamine (283 μl, 1.63 mmol) and reacted together for 4 hours at 60 °C. The reaction progress was monitored by thin layer chromatography (90% conversion for 20-b) and the next step was carried on-pot by adding 1-phenylpiperazine (19-c, 253 μl, 1.63 mmol) in dimethylformamide was added slowly and the reaction was carried for an additional 10 hours at 60 °C. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 300 mg of silica was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using methanol/dichloromethane mobile to give 2-hydroxy-5-(2-oxo-2-

(4-phenylpiperazin-1-yl)acetamido)benzoic acid (20, 101 mg, 0.27 mmol, overall 17 %) as white powder.

152

1 H NMR (MeOD): δ 3.20 (4H, m), 3.66 (4H, m), 6.66 (1H, d, J=12 Hz), 6.83 (1H, t, J=8

Hz), 6.98 (2H, t, J=8 Hz), 7.24 (2H, t, J=8 Hz), 7.44 (1H, d, J=8 Hz), 8.00 (1H, s)

13 C NMR (MeOD): 42.90, 47.48, 50.48, 51.23, 117.67, 118.00, 118.10, 121.72, 122.28,

124.23, 127.51, 129.23, 130.17, 130.32, 152.48, 160.16, 163.10, 164.69

+ LRMS [MH] : calc. for C19H20N3O5, 370.3845; found 370.1218

Figure 4. 62: 1H NMR of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (20)

153

Figure 4. 63: 13C NMR of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (20)

Sample ID: [GP2-24] (C19 H19 N3 O5) 369 Da (in MeOH)/ESIB PREMNAUTH-G-071320-[GP2-24]-R2-CV10 54 (0.938) Cm (1:57) TOF MS ES+ 370.1218

100 1.99e4 %

177.1325 371.1240

372.1433 178.1377 344.1067

0 m/z 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925

Figure 4. 64: LRMS of 2-hydroxy-5-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (20)

154 4.5.2.9 Synthesis of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-

yl)acetamido)benzoic acid (21)

2-amino-5-hydroxy benzoic acid (21-a, 513 mg, 3.27 mmol) and diphenyl oxalate (16-b,

799 mg, 3.27 mmol) were dissolved in dimethylformamide in presence of N,N- diisopropylethylamine (570 μl, 3.27 mmol) and reacted together for 4 hours at 60 °C. The reaction progress was monitored by thin layer chromatography (90% conversion for 21-b) and the next step was carried on-pot by adding 1-phenylpiperazine (19-c, 213 μl, 3.27 mmol) in dimethylformamide was added slowly and the reaction was carried for an additional 6 hours at 60 °C. The reaction was quenched with water, the pH neutralized, and the aqueous layer was then extracted 3 times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over sodium sulfate anhydrous and filtered using filter paper. 300 mg of silica was added to the flask before removing the solvent by rotavap. The solid was loaded to a silica column then purified by flash chromatography using methanol/dichloromethane mobile to give 5-hydroxy-2-(2-oxo-2-

(4-phenylpiperazin-1-yl)acetamido)benzoic acid (21, 325 mg, 0.88 mmol, overall 27 %) as brown-gold powder.

155 1 H NMR (MeOD): δ 3.26 (4H, m), 3.85 (2H, m), 4.13 (2H, m), 6.89 (1H, d, J=8 Hz),

7.04 (3H, m), 7.27 (2H, t, J=8 Hz), 7.54 (1H, d, J=4 Hz), 8.46 (1H, d, J=8 Hz)

13 C NMR (MeOD): 43.93, 47.54, 50.56, 51.39, 117.99, 118.47, 121.62, 123.21, 130.14,

133.09, 152.47, 154.98, 161.11, 163.40

+ HRMS [MH] : calc. for C19H20N3O5, 370.1397; found 370.1397

Figure 4. 65: 1H NMR of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (21)

156

Figure 4. 66: 13C NMR of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (21)

[GP2-oxalink 3] #56-179 RT: 0.17-0.42 AV: 49 NL: 2.77E8 T: FTMS + p ESI cv=0.00 Full ms [100.0000-1000.0000] 370.1397 100

RelativeAbundance 40

15 269.1647

5 165.1020 352.1293 248.1181 739.2719 282.2790 392.1219 497.3022 543.2500 651.4113 793.1912 0 200 300 400 500 600 700 800 m/z Figure 4. 67: HRMS of 5-hydroxy-2-(2-oxo-2-(4-phenylpiperazin-1-yl)acetamido)

benzoic acid (21)

157 4.6 Conclusion

We developed two ROS-sensitive linkers that can be used for targeted therapy. The in vitro studies are showing promising results so far, as most of the developed scaffold can be oxidized and show potential for drug release. The self-cyclizing linker show a potential release in less than an hour whereas the oxalamide linker can trigger a release over days.

Further LC-MS studies need to be done with both designs to confirm the release before using NU1025 and AT7867. The self-cyclizing model will then be clicked to pep2 for selective release of NU1025 in AML cells. The oxalamide linker will be attached to polymers amine terminus for the creation of polymeric nanoparticles for selective AT7867 release.

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