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Home , Cyclopropenone

Design and synthesis of anti-cancer agents that inhibit cysteine proteases, limit oxidative stress or terminate proliferation of BCR-

ABL expressing cells.

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 McMicken College of Arts and Sciences by

Purujit N. Gurjar

Bachelor of Technology,

Institute of Chemical Technology, Mumbai, India, 2012

Dissertation Advisor: Edward J. Merino, Ph.D.

Abstract

Malignant diseases or in common terms Cancer is one of the major causes of death in the world.

Cancer patients are treated using several advanced techniques including surgery, radiation and chemotherapy. Chemotherapeutic treatment involves the use of anticancer agents or drug to fight against cancer. It is used to decrease the tumor burden and to eliminate malignant cells. However, in most cases, resistance against chemotherapy develops. Therefore, there is a permanent need for new additional treatment strategies and chemotherapeutic agents.

In this thesis I have explained my work on three different projects targeting different mechanisms to stop the growth of cancer cells. The first project is focused on inhibition of cysteine proteases which are overexpressed in many types of cancers. By inhibiting these enzymes, we believe that the malignant capacity of a cancer tumor will be arrested. We have tried to impart a solution to a common problem in enzyme inhibition world: can a reversible inhibitor be converted to an irreversible inhibitor?

Apart from cysteine proteases, there are another molecular species which are found expressed in large amounts in cancerous tumors. They are known as Reactive Oxygen Species (ROS). There are several types of ROS and each can have lethal effects on humans depending upon their concentrations. In the second chapter we have tried to selectively inhibit one of these ROS species: hydroxyl radical. Hydroxyl radicals are known to form DNA lesions and protein degradation inside human cells and their inhibition can be part of the solution to stop malignancy.

In the third project I got an opportunity to work on a di-pyridine derivative which shows in vivo against many cancer models in mice. This molecule was obtained from NCI in an impure form.

We were able to purify it and synthesize it on large scale. Upon testing the pure product, we

i achieved more potency and further experiments are still ongoing. Furthermore, we immobilized this active molecule on agarose-based microspheres. These microspheres will be used to investigate protein and enzyme targets of the active compound inside the cell.

Overall, I worked on 3 different strategies towards development of new chemotherapeutic drugs.

Although targets and mechanism of these molecules are different, they all works towards a common goal of making chemotherapy more efficient than it is today.

iii

Acknowledgements

Firstly, I would like to express my sincere gratitude to my advisor Prof. Edward Merino for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis.

I could not have imagined having a better advisor and mentor for my Ph.D. study. Besides my advisor, I would like to thank the rest of my thesis committee: Prof. David Smithrud and Prof. In-

Kwon Kim, for their insightful comments and encouragement, but also for the hard question which incented me to widen my research from various perspectives. Thank you, University of Cincinnati department of Chemistry, for giving me a chance to pursue my doctoral studies here. My sincere thanks also goes to Dr. Stephen Macha, Dr. Larry Sallans, and Dr. Keyang Ding, who gave access to the laboratory and research facilities. Without they precious support it would not be possible to conduct this research. I was extremely fortunate to work in collaboration with Dr. Nicolas Nassar at Cincinnati children’s hospital. I am thankful to him and his entire team for giving me the opportunity to work on a fantastic research project.

I thank my present and past labmates; Anish Vadukoot, Shazna Nusair, Safnas Abdulsalam, Kaylin

Earnest, Haizhou Zhu, Jing Liu. Gurdat Premnauth and Priyangika Senevirathne for the stimulating discussions, for the sleepless nights we were working together before deadlines, and for all the fun we have had in the last six years. Also, I thank all my friends from University of

Cincinnati, in particular, I am grateful to Jessica Ringo and Kayla Borland for always motivating me when I needed it the most.

Last but not the least I thank my family: my father Dr. Narendra and my mother Mrs. Anita and my sister Poorva for supporting me spiritually all my life. You have been pillars of support,

iv guidance and love in my life since the day I was born. I am eternally grateful for all the sacrifices you have made and all the love you have always bestowed upon me.

Table of Contents

Chapter 1: Role of anticancer agents in cancer therapy ...... 1

1.1 History of anticancer agents ...... 2

1.2 Classification of anticancer agents ...... 3

1.2.1 Alkylating agents ...... 3

1.2.2 Antimetabolites ...... 3

1.2.3 Microtubule targeting agents ...... 4

1.2.4 Topoisomerase inhibitors ...... 4

1.2.5 Anthracyclines ...... 5

1.2.6 Monoclonal antibodies ...... 5

1.2.7 ROS-activated anticancer prodrugs ...... 5

1.2.8 Protease inhibitors: Aid to an antineoplastic agent ...... 6

1.2.9 Kinase inhibitors ...... 7

1.3 Goals of dissertation ...... 8

1.4 Overview of Chapter 2 ...... 9

1.4.1 Cysteine Protease Inhibitors: Research Strategy ...... 10

1.4.2 Hypothesis ...... 11

1.4.3 Methods to Synthesize Cyclopropene moiety ...... 13

1.4.4 Example of application ...... 14

1.5 Overview of Chapter 3 ...... 16

1.5.1 Free radicals: introduction ...... 16

1.5.2 How are free radicals generated? ...... 16

1.5.3 How do free radicals damage the cells: ...... 20

1.5.4 ROS-targeted anticancer agents ...... 23

1.6 Overview of Chapter 4 ...... 24

Chapter 2: Cyclopropenes as Potential Warheads for Inhibitors of Cysteine Proteases ...... 25

2.1 Introduction ...... 26

2.2 Experimental ...... 27

2.2.1 Materials ...... 27

2.2.2 Synthetic Methods ...... 28

2.3 Results and discussion ...... 37

2.3.1 Synthesis of cyclopropenes ...... 37

2.3.2. Stability studies...... 38

2.3.3 Kinetic studies ...... 39

2.4 Conclusion ...... 41

Chapter 3: Synthesis and Characterization of Self-Cyclizing Antioxidants as Scavengers of

Hydroxyl Radicals ...... 42

3.1 Introduction ...... 43

3.2 Origin of the Idea ...... 46

vii

3.2.1 Quinone-like oxidation of the core ...... 47

3.2.2 Self-cyclization via intramolecular nucleophilic substitution reaction ...... 48

3.3 Strategy to optimize self-cyclizing scaffold to selectively inhibit HO• ...... 49

3.4 Experimental ...... 51

3.4.1 Materials ...... 51

3.4.2 Synthetic methods...... 52

3.4.3 Oxidation studies of synthesized molecules ...... 86

3.5 Data and results ...... 94

3.5.1 Calculation for t1/2 values for compounds 3a-3e ...... 94

3.5.2 Analysis of oxidation trends ...... 95

3.6 Discussion ...... 96

3.7 Conclusion ...... 97

Chapter 4: Synthesis of a di-pyridine derivative with in vivo activity against cancer models and its immobilization on agarose beads using click chemistry...... 98

4.1 Introduction ...... 99

4.2 Identification and characterization of NSC-124205...... 100

4.3 NSC124205 reduces tumor burden of Ras-driven solid tumors in vivo ...... 103

4.4 Immobilization of NSC-124205 on agarose based fluorescent beads using click chemistry

...... 104

4.5 Experimental ...... 105

viii

4.5.1 Materials ...... 105

4.5.2 Synthesis of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine (NSC-

124205) ...... 106

4.5.3 Synthesis of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

...... 112

4.6 Immobilization of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-

imine on agarose based microspheres ...... 116

4.6.1 Idea of the experiment ...... 116

4.6.2 Design and synthesis of the negative control ...... 116

4.6.3 Immobilization of target molecule on azide beads ...... 118

4.6.4 Calculation of the concentration of 124205-clicked sites on agarose beads ...... 121

4.7 Synthesis of Fluorescent and 124205 clicked beads ...... 122

4.7.1 Synthesis of alkyne linked fluorescein ...... 122

4.7.2 Click reaction general procedure ...... 124

4.7.3 Storage of synthesized clicked beads ...... 125

4.8 Conclusion ...... 125

References: ...... 127

List of Figures

Figure 1.1: Different warheads used for Cysteine Protease inhibitors ...... 10

Figure 1.2: Potent Cysteine Protease Inhibitor BDA-410 by Li et al (2007) ...... 11

Figure 1.3: Irreversible binding of cysteine proteases to inhibitors bearing cyclopropene

“warhead” ...... 12

Figure 1.4: Problems with cyclopropenone warhead in existing inhibitor and ways to improve it

...... 12

Figure 1.5: Elimination reaction of 1,2-disubstituted Cyclopropane ...... 13

Figure 1.6: Rearrangement of Vinyl Carbenes ...... 13

Figure 1.7: Carbene attack on an Alkyne system ...... 14

Figure 1.8: Reference molecule by Ando et al...... 15

Figure 1.9: Formation of ROS from Oxygen ...... 18

Figure 1.10: Mechanism of Lipid peroxidation ...... 21

Figure 1.11: Sulfhydryl containing compounds as antioxidants ...... 23

Figure 2.2: 1HNMR of tosyl azide ...... 29

Figure 2.3: 1HNMR of dimethyldiazomalonate ...... 30

Figure 2.4: 1HNMR of 1-Phenyl-3,3-dicarboxymethylcyclopropene ...... 31

Figure 2.5: 1HNMR of 1-Trimethylsilyl-3,3-dicarboxymethylcyclopropene ...... 32

Figure 2.6: 1HNMR of 3,3-dicarboxymethylcyclopropene ...... 33

Figure 2.7: 1HNMR of 1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene ...... 34

Figure 2.8: 13CNMR of 1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene ...... 35

Figure 2.9: 1HNMR of Methyl N-acetyl-S-(2-phenyl-3,3-dicarboxymethylcyclopropan-1- yl)cysteinate ...... 36

Figure 2.10: HRMS of Methyl N-acetyl-S-(2-phenyl-3,3-dicarboxymethylcyclopropan-1- yl)cysteinate ...... 37

Figure 2.11: Reactivity of cyclopropenes toward SH - reaction completion time...... 39

Figure 2.12. HPLC profile of the reaction of 1-phenyl-3,3-dicarboxymethylcyclopropene with methyl N-acetylcysteinate ...... 40

Figure 2.13: Proposed novel irreversible cysteine protease inhibitor with a cyclopropene warhead...... 41

Figure 3.1: 1HNMR of Compound 3a ...... 55

Figure 3.2: 13CNMR of Compound 3a ...... 55

Figure 3.3: HRMS of Compound 3a ...... 56

Figure 3.4: 1HNMR of Compound 3b...... 59

1 Figure 3.5: HNMR of Compound I1 ...... 60

Figure 3.6: 1HNMR of Compound 3c ...... 63

Figure 3.7: 1HNMR of Compound 3d...... 65

Figure 3.8: 1HNMR of Compound A ...... 67

Figure 3.9: 13CNMR of Compound A...... 67

Figure 3.10: 1HNMR of Compound 3e ...... 69

Figure 3.11: 13CNMR of Compound 3e ...... 69

Figure 3.12: HRMS of Compound 3e ...... 70

Figure 3.13: 1HNMR of Compound 3f ...... 73

Figure 3.14: 1HNMR of Compound 3g ...... 76

Figure 3.15: 1HNMR of Compound 3h ...... 78

Figure 3.16: 1HNMR of Compound 3i ...... 81

Figure 3.17: 1HNMR of N1-Methyl-4-nitro-1,2-phenylenediamine ...... 83

Figure 3.18: 1HNMR of 1-Methyl-5-nitro-2,3-dihydro-1H-1,3-benzodiazol-2-one ...... 84

Figure 3.19: 1HNMR of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one ...... 85

Figure 3.20: HRMS of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one ...... 86

Figure 21: Oxidation of compound 3a in presence of hydrogen peroxide over time monitored by

HPLC ...... 91

Figure 3.22: Plot of natural logarithm of area under the curve (concentration) against time of the reaction in minutes ...... 92

Figure 3.23: Oxidation of compound 3a in presence of hydrogen peroxide over time monitored by UV-Vis spectroscopy ...... 94

Figure 4.1: Tautomerism in NSC-124205 ...... 101

Figure 4.2: (A) Method for HPLC- Solvent A: 95% Water, 5% ACN, 0.5% Formic acid; Solvent

B: 95% ACN, 5% Water, 0.1% Formic acid; Method: 1 mM compound, 20 μL injection; 100% solvent A for 3 min followed by gradient to 100% solvent B over 20 min. (B) LCMS of the sample. (C) HPLC trace of the synthesized NSC124205. Same method as (A) was used. (D)

HRMS of synthesized NSC124205 (E) Stability studies of the compound NSC124205 ...... 102

Figure 4.3 In vivo survival potential of 124205: Mouse models of Breast and Lung Cancers. . 104

Figure 4.4: Design of agarose beads based experiment ...... 105

Figure 4.5: 1HNMR of 2-Guanidinobenzimidazole ...... 108

Figure 4.7: 1HNMR of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine ...... 109

(NSC 124205) Solvent: Methanol-d4 ...... 109

Figure 4.8: 13CNMR of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine ...... 109

(NSC 124205) Solvent: Methanol-d4 ...... 110

xii

Figure 4.9: HRMS of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine ...... 110

(NSC 124205) Solvent: Methanol-d4 ...... 110

Figure 4.10: HPLC trace of Synthesized N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-

2-imine (NSC 124205) ...... 111

1 Figure 4.11: HNMR of compound I1 ...... 113

Figure 4.12: 1HNMR of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2- imine ...... 115

Figure 4.13: Mass spectrum of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-

2-imine ...... 115

Figure 4.14: Design of a click chemistry based experiment between azide beads and alkyne linked fluorescein molecule...... 116

Figure 4.15: Fluorescence microscope images indicate that as the equivalents of alkyne- fluorescein increase, the mean fluorescence of the clicked beads increases...... 117

Figure 4.16: Plot of mean fluorescence vs Equivalents of alkyne-fluorescein used can be used to predict concentration of the fluorescent sites at any given fluorescence value...... 118

Figure 4.17: Immobilization of alkyne-124205 on agarose beads ...... 119

Figure 4.18: As the concentration of alkyne-124205 increases (reaction 1 to reaction 5), fluorescence of the agarose beads decreases...... 120

Figure 4.19: Mean fluorescence decreases as concentration of alkyne-124205 increases in the reaction environment...... 121

Figure 4.20: Synthesis of alkyne-fluorescein...... 122

Figure 4.21: 13CNMR of alkyne linked fluorescein ...... 123

Figure 4.22: HRMS of alkyne-linked fluorescein...... 124

xiii

Figure 4.23: Filter frits after water workup. Fluorescent beads are on the top and clear filtrate is at the bottom...... 125

xiv

Chapter 1: Role of anticancer agents in cancer therapy

1.1 History of anticancer agents

An anticancer agent, also known as an antineoplastic agent, is any drug molecule which is potent in the treatment of different types of cancers. There are a few major classes of anticancer drugs; enzyme inhibitors, antimetabolites, ROS-targeted compounds or angiogenesis inhibitors. In addition, there are a number of drugs that do not fall within those classes but that demonstrate anticancer activity and thus are used in the treatment of malignant disease. The term chemotherapy frequently is associated with the use of anticancer agents, although it more appropriately is assigned to the use of all the chemical compounds to treat the disease in general.

When we look at the history of anticancer agents, it can be noticed that mechlorethamine (nitrogen mustard) was the first molecule clinically used to treat cancer. It is a vesicant and an alkylating agent which is destructive to mucous membranes. Formerly used as a war gas, this compound is used as an antineoplastic in Hodgkin's disease and lymphomas.1 However, it has excessive side effects because of its hyper-reactive nature, which includes DNA cross-linking. In 1956,

Methotrexate (MTX), formerly known as amethopterin was accepted as a ‘antimetabolite’ anticancer agent which was a primary attempt to cure a solid tumor.2 It was effective against numerous types of cancers including breast cancer, leukemia, lung cancer, lymphoma, and osteosarcoma. This was followed by discovery of a vital breakthrough drug 5-Fluorouracil. It is a pyrimidine analog that is an antineoplastic antimetabolite. It interferes with DNA synthesis by blocking the Thymidylate synthetase conversion of deoxyuridylic acid to thymidylic acid.3 Since then. We have come a long way in design and development of novel anticancer agents.

The selection process of ‘choice of drug’ in a particular case of cancer depends on various factors.

Type, location, progress and stage of the cancer play important role in the decision. Side effects of the drug and existing pathological conditions of the patient should also be in agreement before the

2 chemotherapy formula is finalized. Most anticancer drugs are administered intravenously; however, some can be taken orally, and others can be injected intramuscularly or intrathecally

(within the spinal cord).

1.2 Classification of anticancer agents

1.2.1 Alkylating agents

Alkylating agents in general attach an alkyl group to DNA, allowing cross-linking of the base pairs. This induces DNA damage that inhibits the replication process resulting in the cell death.

They are not specific to any phase of the cell cycle. Alkylating agents are further classified in two categories: typical and atypical alkylating agents. Cyclophosphamide and Ifosfamide are examples of typical alkylating agents which add an actual alkyl moiety to DNA. On the contrary, compounds like cisplatin, carboplatin and nitrosoureas do not add an actual alkyl group but still damage the

DNA in a similar way. Hence, they are known as atypical alkylating agents

1.2.2 Antimetabolites

Antimetabolites inhibit DNA replication by mimicking normal cell compounds. They are specific to S phase of cell cycle. Antimetabolites are further classified in 3 groups:

i. Folate inhibitors: They inhibit an enzyme known as dihydrofolate reductase (DHFR) which

prevents the conversion of folate (a dietary supplement) into tetrahydrofolate (THF). THF

is essential for purine and pyrimidine base biosynthesis, hence inhibition of DHFR stops

the synthesis of DNA, RNA, thymidylates and proteins.4 Example: Methotrexate.

ii. Pyrimidine inhibitors: Most widely used example of the subcategory is 5-Fluorouracil (5-

FU). 5-FU acts in several ways, but principally as a thymidylate synthase (TS) inhibitor.

Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which

is a nucleoside required for DNA replication. Other examples include capecitabine and

cytarabine (AraC). iii. Purine analogs: These agents affect purine nucleotide synthesis and metabolism by

inhibiting an enzyme called phosphoribosyl pyrophosphate amidotransferase (PRPP

amidotransferase). Since this enzyme is the rate limiting factor for purine synthesis, it alters

the synthesis and function of RNA and DNA. Most widely used example is 6-

Mercaptopurine (6-MP)

1.2.3 Microtubule targeting agents

These agents inhibit mitosis during the M phase of cell cycle by inhibiting the microtubule activity in the cell. More specifically, they interfere with tubulin, a protein required for the synthesis of microtubules. Major category of microtubule targeting agents is Vinca alkaloids, naturally obtained from plant called as the Madagascan periwinkle or Catharanthus roseus L. Examples include vincristine, vinblastine and vinorelbine which destroy microtubules preventing the progression of mitosis. Another subcategory of microtubule targeting agents is Taxanes. These molecules stabilize the microtubules, arresting the mitosis in metaphase. Examples include paclitaxol and docetaxol.

1.2.4 Topoisomerase inhibitors

Topoisomerase I and topoisomerase II are enzymes that participate in the overwinding or underwinding of DNA. Topoisomerase I inhibitors prevent the relaxation of super-coiled DNA.

Examples include topotecan and irinotecan. Topoisomerase II inhibitors prevent the recoiling of

DNA post transcription. Etoposide and teniposide are the examples of this category.

1.2.5 Anthracyclines

Anthracyclines is a multi-functional class of anticancer agents. They can intercalate DNAby inserting themselves in the DNA and thus preventing the replication of cancer cells. They can also inhibit the topoisomerase II preventing the relaxation of supercoiled DNA and thus blocking

DNA transcription and replication. They are also known to generate reactive oxygen species

(ROS) which damages the cancer cells by oxidation reactions. Most widely administered subcategory in this class is the rubicins which include examples such as doxorubicin, daunorubicin and idarubicin. Major side effects of this category include biventricular heart failure and necrosis with extravasation.

1.2.6 Monoclonal antibodies

Majority of monoclonal antibodies are the antibodies that are synthesized to a specific target that has been known to inhibit the progression of cancer. For instance, Rituximab, a chimeric antibody involving mice and humans, targets CD20 which is a receptor common to b-cells which is used to treat lymphomas. Another example includes Cetuximab which targets the epithelial growth factor receptor (EGFR). It is used to treat solid tumor and was initially developed to cure colorectal cancer. Origin of these antibodies is determined from its suffixes: -omab (100% mouse, 0% human), -ximab (33% mice, 67% human), -umab (10% mice, 90% human) and -mumab (0% mice,

100% human).

1.2.7 ROS-activated anticancer prodrugs

Cancer cells undergo increased oxidative stress, compared to normal cells, caused by oncogenic transformations and changes in metabolism. Consequently, this leads to over production of reactive oxygen species (ROS). These ROS contribute to cell proliferation, metastasis, and angiogenesis in addition to DNA alterations and damage, which effectively increase mutation rate

5 within cells directly promoting oncogenic transformations. Therefore, ROS are important metabolites found in high concentrations in cancerous cells.

This difference in concentration of ROS between healthy and cancerous cells can be used to develop a strategy to selectively kill cancer cells. A ROS-activated prodrug is such type of strategy where a prodrug molecule reacts with higher concentration of ROS to release an antineoplastic agent. These prodrugs are extremely effective in selectively targeting cancer cells, thus they have less side-effects.

1.2.8 Protease inhibitors: Aid to an antineoplastic agent

A protease, also known as peptidase or proteinase, is an enzyme that is responsible for proteolysis: protein catabolism by hydrolysis of peptide bonds. There are 3 different types of proteases which are common: serine proteases, cysteine proteases and aspartic acid proteases based on the residue present at the catalytic site. In cancer, proteases are overexpressed in solid tumors which have the role of degrading surrounding tissue to create space for the tumor to grow. These proteases such as cathepsins (a class of cysteine proteases) attack and hydrolyze the cell membranes of tumor- surrounding cells, destroying them to help the progression of tumor.

For instance, Cathepsin B was the first lysosomal protease to be associated with breast carcinoma.5

Increased expression and/or activity of cathepsin B is seen in breast5-6, colorectal7, lung8, and prostate carcinomas9, gliomas10, melanomas11, and osteoclastomas12, suggesting that this protease might be involved in the development, invasion and metastasis of more than one type of tumor.

Protease inhibitors are thus an important aid to stop the metastasis of tumor and as a result, stop the rapid progression of cancer. Combined with antineoplastic agent, they can be very effective to confine and kill cancer cells.

1.2.9 Kinase inhibitors

A protein kinase inhibitor is a class of enzyme inhibitors that can block the action of protein kinases. Protein kinases add a phosphate group to a protein in a process called phosphorylation, which can turn the activity of a protein on or off and therefore affect its level of activity and function.

Protein kinase inhibitors can be subdivided according to the amino acid on a protein that they add the phosphate to (e.g serine, threonine or tyrosine) in order to inhibit phosphorylation of that amino acid. Kinases mostly act on both serine and threonine, but tyrosine kinase acts on tyrosine only and some dual-specificity kinases act on all three of these amino acid residues. Some protein kinases also phosphorylate other amino acids, such as histidine kinases that act on histidine residues.

Phosphorylation is often a required step in the growth of some cancers and inflammatory disorders, meaning inhibition of the enzymes that trigger phosphorylation provides an approach to treating such diseases. One example of a drug being used in this way is the tyrosine kinase inhibitor dasatinib, which is used as an anticancer therapy in several forms of leukemia. Another agent currently being tested in clinical trials for polycystic kidney disease is PLX5568.

Tyrosine kinase inhibitors are particularly important agents because these high-affinity cell surface receptors play a critical role in the progression of many cancers.

Tyrosine kinases are involved in various cell functions including cell signaling, cell growth and cell division. In some forms of cancer, these enzymes are present in high levels or overactive and

7 inhibiting them can prevent the proliferation of cancer cells. Tyrosine kinase inhibitors therefore provide an important form of targeted therapy in the fight against cancer.

1.3 Goals of dissertation

This dissertation explains the work on 3 different projects. Each project focuses a particular class of anticancer agents. These agents inhibit the progress of cancer in different mechanisms. As a result, this dissertation is divided into 3 chapters:

Chapter 2: This chapter explains design and synthesis of cysteine protease inhibitors. These enzyme inhibitors play a crucial role in inhibiting the progress of breast and lung cancers where cysteine proteases (mainly cathepsins) are overexpressed by tumors for the function of metastasis.

Chapter 3: This chapter focuses on design and synthesis of ROS active small molecules which selectively inhibit hydroxyl radicals. These molecules are designed in such a way that they are stable and unreactive in presence of hydrogen peroxide, however get oxidized in presence of ROS with higher oxidation potential such as hydroxyl radicals.

Chapter 4: In this chapter, design and synthesis of a dipyridine derivative is explained which is active against many in vivo cancer models. In addition to that, immobilization of active molecule on agarose beads is achieved with the calculation of approximate active sites.

1.4 Overview of Chapter 2

Cysteine proteases are enzymes that destroy proteins by hydrolysis, turning them back into individual amino acids. They play very important role in programmed cell death (apoptosis). They are classified into various families such as Calpains, Cathepsins, Papains, Caspases etc.

Overexpression of these enzymes in the human body leads to severe pathological conditions. For instance, calpains (having calcium-dependent proteolytic activities), are involved in variety of neurodegenerative diseases including Alzheimer disease, cataract formation, stroke as well as diseases like myocardial infarcts, multiple sclerosis etc.13 Destruction of cartilage tissue and bone atrophy is the result of overexpressed cathepsins,14 another family of cysteine proteases.

Destruction of cartilage tissue in rheumatoid arthritis is a consequence of overexpressed cathepsins

B, L, and K supported also by metallo proteases. In carcinoma progression and metastasis, elevated proteolytic activities of cathepsins L, B, H and S are needed for the creation of space for the tumor tissue. Cathepsin S has been proposed to be involved in angiogenesis. Cathepsins play important roles in autoimmune diseases associated with a malfunction in regulation of antigen presentation.

15 The protozoan parasite Plasmodium falciparum, causing malaria in human, expresses three papain-like cysteine proteases. They are falcipains 1, 2 and 3 playing critical role in hemoglobin proteolysis to provide amino acids for the parasites progression.16 Leishmania species which cause e.g. Kala Azar as the most dangerous disease are also known for abundant expression of various cathepsin L- and B-like proteases.17 Some viruses are entirely dependent on cysteine proteases.

These enzymes in viruses are involved in the viral life cycles by generating mature viral proteins from their precursor polyproteins (e.g. coronavirus).18

Thus, developing inhibitors of cysteine proteases is an important target for organic and medicinal chemists. The goal of this research project is to develop efficient and specific inhibitors of cysteine proteases exploiting the high reactivity of the thiol group toward a cyclopropene system as a novel

“warhead”, a reactive group capable of covalently binding active site cysteine residues.

1.4.1 Cysteine Protease Inhibitors: Research Strategy

One strategy to create Cysteine Protease inhibitors is the use of electrophilic moieties, which covalently bind to the cysteine residue of the active site of the target protease. Thus, because of this irreversible covalent bonding, concentration of corresponding cysteine protease is reduced, and its activity is restricted.

Many of the electrophilic inhibitors have already been reported. Vicik et al have shown use of

Michael Acceptors, heterocyclic epoxides and aziridines for irreversibly binding with cysteine proteases.19 Their activity, however, is strongly pH dependent, and the inhibition is not always restricted to cysteine proteases. It often affects aspartate and serine proteases as well.

Michael Acceptors

O 2 O R - R' NHR' Ar R' S RHN O O O

Methylene Ketones Vinyl Sulfones Fumaryl diamides

Strain Ring Species G O N R' R R' R

Epoxides Aziridines

Figure 1.1: Different warheads used for Cysteine Protease inhibitors (R= alkyl group or H)

Li et al used Cyclopropene moiety in their molecule and synthesized a very potent molecule with an excellent IC50 value of 0.534 µM.20

O O O NH S NH O OH

Figure 1.2: Potent Cysteine Protease Inhibitor BDA-410 by Li et al (2007)

Among carbocyclic unsaturated three-membered ring compounds, this is the only moiety used for binding of cysteine proteases.21 Furthermore, these cyclopropenones bind to the cysteine proteases reversibly. This suggests the addition of the thiol group of the enzyme to the 3-carbonyl, rather than to the cyclopropene double bond. To the contrary, 1,2-cyclopropene moiety irreversibly inhibits acyl desaturases by reaction with the cysteine residue of the active site, leaving other amino acid chemical probes unaffected.22

1.4.2 Hypothesis

It can, therefore, be hypothesized that inhibitors bearing a cyclopropene “warhead” will provide excellent drug candidates that will inhibit cysteine proteases selectively and irreversibly. (Scheme

1), which is particularly advantageous for targeting enzymes expressed in foreign organisms (e.g. falcipains in malaria parasite) or those that are overexpressed in neoplastic tissues (e.g. cathepsins

L, B, H, and S).

Falcipains, 3CLpro,

3 4 4 3 Cathepsins B,L,H,S R R R R 1. Cysteine Selective 2. Irreversible addition S 1 2 1 2 R R R R

Figure 1.3: Irreversible binding of cysteine proteases to inhibitors bearing cyclopropene

“warhead”

Various Cyclopropene systems including alkyl cyclopropenes, cyclopropanones, difluorocyclopropenes, nitrocyclopropenes, carboxycyclopropenes may have the potential to provide an excellent site of binding for cysteine proteases (Figure 1.4).

Figure 1.4: Problems with cyclopropenone warhead in existing inhibitor and ways to improve it

1.4.3 Methods to Synthesize Cyclopropene moiety

There are three main methods to synthesize cyclopropene rings.23

I. Elimination reaction of 1,2-disubstituted Cyclopropane:

Figure 1.5: Elimination reaction of 1,2-disubstituted Cyclopropane

Elimination reaction of 1,2-disubstitued cyclopropane such as 1,2-dichlorocyclopropane

gives cyclopropene ring. Harsh conditions are required to eliminate a molecule from

cyclopropane in solution, which severely limits product yield. Another limitation is the

ring strain of the cyclopropene formed, which makes it thermodynamically unstable. Gas

phase elimination of trimethylsolylhalide23 opens the door to a variety of strained ring

compounds that are otherwise inaccessible, but this method is limited by the ability of

precursors to evaporate.

II. Rearrangement of Vinyl Carbenes

Vinyl carbenes rearrange to give cyclopropene system if the controlled reaction is carried out.

Carbenes in general are highly unstable molecules, thus these reactions give very low yields.

3 4 1 3 R R R R C 4 R 1 2 2 R R R Figure 1.6: Rearrangement of Vinyl Carbenes

III. Carbene attack on an Alkyne system

Carbene addition to a triple bond results in formation of a cyclopropene system by

simultaneous formation of two sigma bonds. This method is not limited to free carbenes;

metallocarbenes are also capable of addition to alkynes.

Figure 1.7: Carbene attack on an alkyne system

(TM = Transition metal such as Cu or Rh)

1.4.4 Example of application

In 1999 Ando et al. designed cysteine protease inhibitors using cyclopropenone moiety. They showed strong inhibitory activities only to cysteine proteases such as calpain, papain, cathepsin B and cathepsin L and not to serine proteases (e.g. thrombin and cathepsin G) or aspartic proteases

(e.g. cathepsin D).20 They analyzed the m-Calpain inhibitory activities of these molecules.

O 1 O R O NH 2 NH R O OH

Figure 1.8: Reference molecule by Ando et al.

Some of the important results of the assay are as follows:

1 2 R R IC50 (µM)

CH(CH3)2 Phenyl 1.62

CH2CH2CH2CH3 CH3 40.00

CH2CH2CH2CH3 Phenyl 2.70

Table 1.1: SAR studies of reference molecule by Ando et al.

Taking into consideration the IC50 value and the synthetic feasibilities of all the molecules, the molecule with R1 as i-Pr and R2 as phenyl was selected as a reference molecule.

O O O NH NH O OH

Reference Molecule

This molecule can be synthesized and used as a reference molecule to compare potency of other

3 3 3 3 new molecules that instead of C =O have C H2, C HCOOEt, and C F2 groups, all of which are expected to make the novel inhibitors more potent and irreversible. The reference compound

3 3 (C =O) and C H2 can be synthesized in the following way: The cyclopropene ring and then the peptidomimetics will be synthesized independently, and then coupled to each other. The synthesis of the other two will start with making the precursor with the alkyne in place of the cyclopropene group, followed by cyclopropenation by ethyl diazoacetate in the presence of CuOTf to make

3 22 3 C HCOOEt , and heating with Me3SiCF3 to achieve the addition of difluorocarbene to form C F2 cyclopropene.24

1.5 Overview of Chapter 3

1.5.1 Free radicals: introduction

Free radicals are the chemical species/atoms or group of atoms that have single or more unpaired electrons in their outermost orbit. The presence of these unpaired electrons makes them highly reactive and unstable chemical species. Because of their unstable nature they get degraded spontaneously unless and until they are able to donate a free electron to an oxidant or accept a free electron form a reducing agent.

1.5.2 How are free radicals generated?

There are two ways to generate a free radical inside human body: Physiological and Pathological.

Physiological ways are characteristic of or appropriate to an organism's healthy or normal functioning. One of the examples of physiological way to generate a free radical is through

Oxidative phosphorylation. The NADH and FADH2 formed in glycolysis, fatty acid oxidation,

16 and the citric acid cycle are energy-rich molecules because each contains a pair of electrons having a high transfer potential. When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from

NADH or FADH 2 to O2 by a series of electron carriers. This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.

Process of reduction of Oxygen to form different ROS

In the process of oxidative phosphorylation in mitochondria, 4 electrons are utilized for the oxygen to get reduced to water. However, when the process does not involve consumption of all 4 electrons, free radicals are generated. This process is also called as the partial reduction of oxygen.

Several physiological enzymes are involved in this process. One such enzyme NADPH oxidase is abundantly found in neutrophils. With acceptance of only 1 electron, a molecule of oxygen can be converted to a superoxide anion under the action of NADPH oxidase. This is the primary ROS derived directly from molecular oxygen.

Superoxide dismutase or SOD is an enzyme that converts superoxide to hydrogen peroxide. It is the process in which a free radical is transformed back to a relatively stable molecule. Hydrogen peroxide is the most abundant ROS form found inside the cell. It can yield different products when interacted with different enzymes. For instance, when hydrogen peroxide acts a substrate to the enzyme Myeloperoxidase or MPO, it gets converted to Hypochlorous acid (HOCl). Since myeloperoxidase is also abundant in neutrophils, we find a relatively high concentration of HOCl radicals in white blood cells. However, hydrogen peroxide in a cellular environment has other fate.

In presence of iron, peroxide undergoes homolytic fission to form Hydroxyl radicals. This reaction discovered by scientists Haber and Weiss which follows Fenton chemistry, is the major mechanism

17 by which hydroxyl radicals are generated inside the cells. Hydrogen peroxide can also be converted directly into water by the action of an enzyme known as catalase. This is one of the most common antioxidative controls that are found inside a cell. If concentration of peroxide inside the cell is elevated beyond the safety limit, by converting it into water catalase prevents the oxidative damage that might potentially occur.

Glutathione is another such example of antioxidant enzyme which converts hydroxyl radical into a much safer product water. Ionizing radiation, commonly found in UV rays can convert the water back to hydroxyl radicals which are highly unstable and reactive.

Figure 1.9: Formation of ROS from Oxygen

Pathological ways

1. Ionizing radiation: It predominantly converts when water inside the tissue is exposed to a

source carrying ionizing radiation such as UV light. Ionizing radiation is capable to knock

off an electron from water resulting in the formation of hydroxyl radicals.

2. Inflammation: As a first response to the infection in human body is the line of defense

involving neutrophil cells. Neutrophils use two mechanisms to eliminate the foreign

microbe causing infection. One of those mechanisms is oxygen dependent. This

mechanism starts with an oxidative burst, where the enzyme NADPH oxidase converts

molecular oxygen to superoxide radical. The role of superoxide radical is vital in the

antimicrobial activity of neutrophils.

Defect in NADPH oxidase can cause chronic granulomatous disease or CJD. This is an

immunodeficiency due to the neutrophils inability to generate free radicals needed to

destroy foreign materials. Specifically, this disease can make individuals susceptible to

reoccurring infections by catalase positive organisms.

3. Exposure to metals: There are two common metals found in human body which play a key

role in the formation of free radicals: iron and copper. Iron binds to proteins called

transferrins which regulate the amount of iron in the blood. However, if transferrins are

inefficient due to pathological conditions in controlling iron levels, the excess of iron

allows cells to undergo fenton reactions to generate hydroxyl radicals. One of such

examples is hemochromatosis where iron builds up inside human body to cause serious

tissue damage such as liver cirrhosis. Primary mechanism behind this damage is generation

of hydroxyl radicals. Similarly, in Wilson’s disease, presence of excess unbound copper is

found which also generates free radicals resulting in oxidative damage.

4. Drugs and chemicals: Several drugs and chemicals are responsible in generating high

amounts of free radicals due to the metabolism path they undergo. For instance,

acetaminophen, a commonly used analgesic drug is metabolized in liver. In the liver, P450

system which is a group of drug metabolizing enzymes interact and oxidize acetaminophen.

This process of metabolizing generates significant concentration of free radicals. Hence

when high doses of acetaminophen are administered, it can cause massive death of tissue

in the liver from free radical damage.

Another such example of a chemical which causes free radical damage is Carbon

tetrachloride (CCl4). CCl4 is used in dry cleaning industry and is very dangerous if enters

the blood stream of a human. If it gets into the blood, it is converted to trichloromethyl

. radical (CCl3 ), which is a free radical. Formation of this radical is mainly seen in the P450

system of the liver. Since it is a free radical, it can destroy the hepatocytes by damaging

proteins, DNA and cell membranes. In the early stages, this damage is reversible but in the

advanced conditions, it causes the swelling of endoplasmic reticulum (ER) of the

hepatocytes. This swelling causes the release of ribosomes from ER resulting in inhibition

of protein synthesis such as apolipoproteins. Lower level of apolipoproteins results in the

massive deposition of fats in the liver affecting its function.

1.5.3 How do free radicals damage the cells:

1. Lipid peroxidation: Lipid peroxidation is the oxidative degradation of lipids. It is the

process in which free radicals abstract an electron from the lipids in cell membrane to

satisfy the valency of oxygen, resulting in the formation of a free radical in the lipid

molecule. Free radical in the lipid membrane then propagates in search of the missing

electron and thus this chain reaction proceeds by a free radical chain reaction mechanism.

This propagation of free radical damages the cell membrane severely. (Figure 1.10)

2. DNA and protein oxidation: There are two sites on the DNA where ROS react

predominantly: Nucleobases and ribose sugar. All the nucleobases can be easily oxidized

by a high potential ROS such as hydroxyl radical, however, guanine is the primary target

of these oxidation reactions because it has lowest oxidation potential (-1.3 V) which forms

8-oxo-guanine (dG) upon oxidation.25. This kind of DNA oxidation can be mutagenic or

lethal.25-29 Furthermore, DNA-protein crosslinks formed by radical coupling between

lysine or tyrosine side chains and dG is another type of oxidative damage to DNA.

Figure 1.10: Mechanism of Lipid peroxidation

Defense against damaging free radicals: Antioxidant mechanisms

Free radicals are extremely unstable and are degraded spontaneously once are formed. However, even in their short time of existence they can cause severe damage to cellular environment. To prevent that, there are antioxidant mechanism found in human body. These mechanisms are broadly divided in two types: enzymatic and non-enzymatic mechanisms. Role of these mechanisms is to either prevent the formation of free radicals or degrade them before they could cause any damage.

1. Non- enzymatic antioxidants:

i. Metal carrier proteins: Metal carrier proteins such as transferrins and ceruloplasmin,

which respectively bind or carry iron and copper in the blood are responsible for

controlling the levels of these metals in blood. Transferrin carries and delivers iron to

the liver and macrophages. When bound in macrophages or the liver by a molecule

called ferritin, it is unavailable for getting involved in fenton chemistry inside the cell.

Thus, formation of hydroxyl radicals due to excess iron is prevented.

ii. Vitamins: Vitamins A, C and E are extremely effective naturally found antioxidants.

They act as electron donors which fulfill the valency of a free radical. Thus, they

degrade the highly reactive species such as hydroxyl radical and prevent any cellular

damage.

iii. Sulfhydryl containing compounds: This category mainly comprises of naturally

occurring thiol molecules such as cysteine and glutathione which react with

superoxide and hydroxyl radicals and thus stop any subsequent oxidative damage.

Figure 1.11: Sulfhydryl containing compounds as antioxidants

2. Enzymatic antioxidants: There are three very important enzymes which have a vital role in

free radical control: superoxide dismutase (SOD), catalase and glutathione peroxidase.

These enzymes are responsible for controlling the levels of superoxide anion, hydrogen

peroxide and hydroxyl radical respectively in the cellular environment.

1.5.4 ROS-targeted anticancer agents

In this chapter, we designed a novel class of self-cyclizing antioxidant agents to remove lethal

ROS molecules, such as HO•, by altering our previous design of ROS activated pro-drug agent.30

Merino lab has previously reported that our lead prodrug agent which has a hydroquinone moiety, an amide containing linker arm and a cyclohexylamine nucleophile, self cyclizes into a bicyclic ring that equilibrate between an electrophilic, oxidized and stable, reduced state upon ROS activation.30 We pursued further mechanistic study of this agent in the presence of Fenton reaction

23 conditions to show formation of novel product in further oxidation. This altered the structure of this agent, eliminating the cytotoxicity by removal of the cyclohexane ring and stabilizing it by replacing the hydroxy group with an amine at position 5 at the quinone ring. The idea is to make antioxidants with poor tendency to be reduced, so that these molecules remain in the cells requiring the presence of strong oxidant such as HO•.

1.6 Overview of Chapter 4

In this chapter, we describe the identification of a potent small molecule compound, NSC-124205, that is active in xenograft mouse models of breast and lung cancers. This molecule was found by an in-silico screen against a Ras conformation that mimics the structure of nucleotide-free Ras in complex with Sos.31-32 Top scoring candidates identified in silico were screened for (i) Ras binding in vitro, and for inhibition of (ii) 2D-proliferation and (iii) 3D-colony formation of oncogenic-Ras driven NIH-3T3 cells. NSC-124205 was among the top candidates that fulfilled all three criteria.

Compared to vehicle treated, tumors of xenograft mice generated with Ras-driven MDA-MB-231 and H2122 cells significantly decreased when treated with NSC-124205. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis.

Merino lab’s contribution to this chapter is identification, synthesis and purification of compound

NSC-124205. We were also able to immobilize this active molecule on agarose based microspheres using copper-catalyzed click chemistry.

Chapter 2: Cyclopropenes as Potential

Warheads for Inhibitors of Cysteine Proteases

2.1 Introduction

Cysteine proteases are protein processing and protein degrading enzymes whose overexpression in human body may result in serious pathological changes. For instance, calpains, one family of cysteine proteases, are involved in Alzheimer disease, multiple sclerosis, stroke, myocardial infarcts, cataract formation, etc.13 Destruction of cartilage tissue and bone atrophy is the result of overexpressed cathepsins,14 which are also involved in carcinoma progression and metastasis.15

Other cysteine proteases play an essential role in life cycles of some viruses (e.g. coronavirus18) and parasites (e.g. malaria16). Therefore, developing inhibitors of cysteine proteases is an important target for organic and medicinal chemists.

The most efficient inhibitors of cysteine proteases bear an electrophilic “warhead” capable of covalently binding active site cysteine residues. Examples include species with an activated carbon-carbon double bonds,33 acyloxymethyl ketones,34 or a three-membered ring heterocycle

(e.g. epoxide35, aziridine36), the latter generally having greater potency.19, 36-37

Their activity, however, is strongly pH dependent, and the inhibition is not always restricted to cysteine proteases, often affecting aspartate and serine proteases as well.19 Surprisingly, among carbocyclic unsaturated three-membered ring compounds, only cyclopropenone derivatives have

26 been used as cysteine protease inhibitors,20-21 and their binding appears to be reversible (Figure

1).21 This suggests the addition of thiol group to the 3-carbonyl, rather than to the cyclopropene double bond. To the contrary, 1,2-cyclopropene moiety irreversibly inhibits acyl desaturases by reaction with the cysteine residue of the active site, leaving other amino acid chemical probes unaffected.22 It can, therefore, be hypothesized that inhibitors bearing a cyclopropene “warhead” will provide excellent drug candidates that will inhibit cysteine proteases selectively and irreversibly, which is particularly advantageous for targeting enzymes expressed in foreign organisms (e.g. falcipains in malaria parasite or C3-like protease in coronavirus) or those that are overexpressed in neoplastic tissues (e.g. cathepsins L, B, H, and S).

In this chapter, we report the synthesis of model cyclopropenes and their evaluation as potential warheads that could be incorporated into the existing cysteine protease inhibitors in order to increase their selectivity and potency.

2.2 Experimental

2.2.1 Materials

All chemicals, reagents, and solvents were purchased from Sigma-Aldrich Inc., TCI, and Fisher

Scientific, Inc., and used as received unless stated otherwise. All reactions were carried out under an atmosphere of dry argon in oven-dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25°C. Analytical thin layer chromatography (TLC) was performed with glass backed silica plates (5 x 20 cm, 60 Å, 250 μm).

Visualization was accomplished using a 254 nm UV lamp. 1H and 13C NMR spectra were recorded on either a Bruker Avance 400 MHz spectrometer or Bruker DPX 500 MHz spectrophotometer using solutions of samples in either of the deturated solvents: chloroform, methanol, acetonitrile, or water. Chemical shifts are reported in ppm with tetramethylsilane as standard. Data are reported

27 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 Shimadzu Q-TOF 6500. All novel compounds were characterized by 1H, 13C, DEPT 13C NMR spectroscopy and high- resolution mass spectrometry. The identity of previously made cyclopropenes was confirmed comparison of their 1H NMR to the published data (reference provided). HPLC analysis of final products was performed on an Agilent 1200 HPLC with UV detection.

2.2.2 Synthetic Methods

2.2.2.1 Tosyl azide synthesis38

To a solution of sodium azide (11.9 g, 62.5 mmol) in the mixture of water (15 mL) and 95% ethanol

(25 mL) a solution of p-toluenesulfonyl chloride (4.475 g, 70 mmol) in 95% ethanol (125 mL) was added. After stirring at 40oC for 3 hours, the solvent was removed in vacuo. The oily crude product was dissolved in diethyl ether, washed with water, dried using sodium sulfate and purified with

1 hexane/ethyl acetate (6:1) to yield 11.6 g (84%) of product. H NMR (CDCl3, 400 MHz): δ 7.83

(d, J = 8.0 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 2.47 (s, 3 H).

Figure 2.2: 1HNMR of tosyl azide

2.2.2.2 Dimethyl diazomalonate synthesis39

Dimethylmalonate (1.05 mL, 1.0 eq), triethylamine (1.4 mL, 1.1 eq) and tosyl azide (2 g, 1.0 eq) were dissolved in acetonitrile (20 mL). The solution was stirred at room temperature overnight.

The reaction mixture was concentrated under reduced pressure and partitioned between CH2Cl2 and water. The resulting solution was stirred for 1 hour at room temperature. The organic layer was collected, dried over MgSO4 and concentrated. Crude mixture was first filtered over a plug of silica gel (Pet ether/diethyl ether 1:1) to remove most of the tosylamide. The purification by silica gel chromatography with Pet ether/diethyl ether 1:1 afforded product as a yellow oil. 1H

NMR (CDCl3, 400 MHz): δ 3.84 (s, 6 H).

Figure 2.3: 1HNMR of dimethyldiazomalonate

2.2.2.3 General procedure for rhodium acetate mediated cyclopropenation40

Into a 50 mL round bottom flask was added anhydrous CH2Cl2, alkyne (3.0 eq) and Rh2(OAc)4

(0.01 eq) under Argon atmosphere. Solution of diazoacetate (1.0 eq) in anhydrous CH2Cl2 was added to it dropwise using a syringe pump with the rate of 0.7 mL/hour at room temperature. After the addition was complete, reaction was stirred for additional 3 hours followed by filtration through celite and purification using silica gel chromatography with hexane-ethyl acetate eluent system.

40 1 1-phenyl-3,3-dicarboxymethylcyclopropene. (Yield: 81%) H NMR (CDCl3, 400 MHz): δ 7.64

(m, 2 H), 7.45 (m, 3 H), 6.90 (s, 1 H), 3.72 (s, 6 H)

41 1 1-trimethylsilyl-3,3-dicarboxymethylcyclopropene. (Yield: 92%) H NMR (CDCl3, 400 MHz):

δ 7.04 (s, 1 H), 3.69 (s, 6 H), 0.24 (s, 9H)

42 1 1-methoxymethyl-3,3-dicarboxymethylcyclopropene. (Yield: 23%) H NMR (CDCl3, 400 MHz):

δ 6.64 (s, 1 H), 4.45 (s, 2 H), 3.66 (s, 6 H), 3.36 (s, 3 H)

14 1 1-acetoxymethyl-3,3-dicarboxymethylcyclopropene. (Yield: 46%) H NMR (CDCl3, 400 MHz):

δ 6.69 (s, 1 H), 4.93 (s, 2 H), 3.68 (s, 6 H), 1.21 (s, 3H)

Figure 2.4: 1HNMR of 1-Phenyl-3,3-dicarboxymethylcyclopropene

Figure 2.5: 1HNMR of 1-Trimethylsilyl-3,3-dicarboxymethylcyclopropene

2.2.2.4 General procedure for TMS deprotection.

1-(TMS)-3,3-disubstituted cyclopropene (1 g) was dissolved in 20 mL of regular THF. The

o resulting solution was cooled to 0 C and 10% aq. K2CO3 was added dropwise. After the addition was complete, it was stirred for another 10 min in ice bath. The reaction was then slowly warmed up to room temperature and stirred for 24 hours. The solvent was removed under reduced pressure and crude mixture was purified using silica gel chromatography with hexane-ethyl acetate system.

43 1 3,3-dicarboxymethylcyclopropene. (Yield: 95%) H NMR (CDCl3, 400 MHz): δ 6.89 (s, 2 H),

3.70 (s, 6 H).

Figure 2.6: 1HNMR of 3,3-dicarboxymethylcyclopropene

2.2.2.5 General procedure for difluorocarbene addition to a carbon-carbon triple bond.24

To a high pressure sealed tube with a magnetic stir bar was added anhydrous NaI (2.2 eq), TMSCF3

(2 eq), alkyne (1 eq) and anhydrous THF. The reaction mixture was heated at 110oC overnight.

Reaction was quenched with saturated Na2CO3 solution, followed by extraction with diethyl ether.

The organic phase was dried over anhydrous K2CO3. After removal of solvent under vacuum, the residue was subjected to silica gel chromatography with petroleum ether / triethylamine (40:1, v/v) as eluent.

1-phenyl-3,3-difluorocyclopropene.44 This compound was not stable enough to obtain a clean

NMR spectrum of, let alone introduce into subsequent studies.

1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene. (Yield: 43%) 1H NMR

(CDCl3, 400 MHz): δ 7.63 (m, 2 H), 7.48 (m, 3 H), 5.76 (m, 1 H), 2.20 (s, 3 H), 2.22 (m, 1 H),

13 1.08 (d, 3 H, J = 6.9 Hz), 1.04 (d, 3 H, J = 6.8Hz). C NMR (CDCl3, 400 MHz): δ 169.97 (s),

131.23 (s), 130.34 (s), 129.10 (s), 128.57 (t, J = 10.8 Hz), 124.33 (t, J = 11.9 Hz), 123.36 (s),

102.09 (t, J = 273 Hz), 73.19 (s), 31.62 (s), 20.93 (s), 17.83 (s), 17.48 (s).

Figure 2.7: 1HNMR of 1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene

Figure 2.8: 13CNMR of 1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene

2.2.2.6 General procedure for reaction of modified cysteine with cyclopropenes.

To a NMR tube was added 500 µL acetonitrile-D3, cyclopropene (1 eq.), methyl N- acetylcysteinate (2 eq.) and DBU (2 mol%). The mixture was stirred using a vortex and NMR was taken at regular time intervals. The rate of the reaction was calculated using disappearance of the characteristic cyclopropene peak.

Methyl N-acetyl-S-(2-phenyl-3,3-dicarboxymethylcyclopropan-1-yl)cysteinate: The adduct between N-acetyl-cysteine methyl ester and 1-phenyl-3,3-dicarboxymethylcyclopropene was isolated by preparative HPLC using reverse-phase C18 column and acetonitrile/water = 5:95 to

1 50:50 as a mixture of stereoisomers (Yield 15%). H NMR (CDCl3, 400 MHz): for major diastereomer δ 7.28 (m, 3H), 7.20 (m, 2H), 6.53 (d, 1H, J = 6.9 Hz), 4.93 (m, 1H), 3.85 (d, 3H, J

= 11.5 Hz), 3.78 (s, 3H), 3.45 (d, 3H, J= 5.6 Hz), 3.32 (t, 1H, J= 8.3 Hz), 3.20 (m, 1H), 3.07 (m,

13 1H), 2.03 (d, 3H, J= 8.2 Hz). C NMR (CDCl3, 400 MHz): for major diastereomer δ 170.85,

169.95, 166.17, 166.17, 133.22, 128.69, 128.40, 127.77, 53.16, 52.81, 52.36, 46.13, 37.02, 35.78,

32.37, 31.20, 22.99. HRMS (ESI): m/z calculated for [M+Na]+ 432.1021, observed 432.1087.

Figure 2.9: 1HNMR of Methyl N-acetyl-S-(2-phenyl-3,3-dicarboxymethylcyclopropan-1-

yl)cysteinate

Figure 2.10: HRMS of Methyl N-acetyl-S-(2-phenyl-3,3-dicarboxymethylcyclopropan-1-

yl)cysteinate

2.3 Results and discussion

2.3.1 Synthesis of cyclopropenes

Scheme 2.1. Synthesis of cyclopropenes by carbene addition to a triple bond. (i) R2R3C=N2,

Rh2(OAc)4, CH2Cl2, r.t.; (ii) K2CO3 , THF (wet), r.t.; (iii) TMSCF3, NaI, 110°C, 2h

There are three main routes23 to access cyclopropenes by synthesis: 1,2-elimination of cyclopropanes, cyclization of vinylcarbenes, and addition of carbenes to carbon-carbon triple bonds. The latter method represents the most plausible way to access cyclopropene, as it constructs the three-membered ring by simultaneous formation of the two sigma bonds. Most importantly, it is not limited to free carbenes; metallocarbenoids also readily undergo reaction with alkynes, with dirhodium(II) catalyst being the most efficient for cyclopropenation of terminal alkynes using the corresponding diazo compounds.45-47 to form the cyclopropene with the desired C3-substitution pattern. This method, however, does not work well for halocarbenes, as their metallocarbenoids react differently with terminal alkynes,44 so 3,3-difluorocyclopropenes were synthesized by addition of the in situ24 generated difluorocarbene to the alkynes (Scheme 1). Furthermore, 1-

(trimethylsilyl)cyclopropenes41 can be easily converted into 1,2-non-substituted cyclopropenes by treatment with potassium carbonate.

2.3.2. Stability studies

Our initial stability studies revealed that 3-monosubstituted cyclopropenes (R2 = H) and 1-phenyl-

3,3-difluorocyclopropene were not stable for an extended period of time even in chloroform-D.

The other halogenated analog, 1-phenyl-2-(1-acetoxy-2-methyl)butyl-3,3-difluorocyclopropene, albeit stable at room temperature in an organic solvent, rapidly decomposed upon the contact with a phosphate aqueous buffer with pH 7.4. Other cyclopropene derivatives turned out to be sufficiently stable in such conditions, so we proceeded with studies of their reactivity toward N- acetyl cysteine methyl ester.

2.3.3 Kinetic studies

-4 R1 0 200 400 600 800 1000 1200 -4.5 Phenyl

-5 TMS -5.5 CH2OMe

-6 ln[cyclopropene] CH2OAc -6.5

H -7 Time (s)

Figure 2.11: Reactivity of cyclopropenes toward SH - reaction completion time.

Due to the limited aqueous solubility of both the cyclopropenes and derivatized cysteine, studies were performed in acetonitrile-D3 in the presence of DBU needed to ionize the SH (without a base no reaction is observed) at 37oC. As thiols in cysteine proteases exist in ionized form due to catalytic triads, this was the approximate simulation of the environment in vitro. The reaction progress was monitored by disappearance of the cyclopropene hydrogen signal in 1H NMR. The results (Figure 2) indicate that the fastest reacting cyclopropenes had a substituent at one of the olefin atoms bearing a group capable of stabilizing the intermediate carboanion (Scheme 2), which is consistent with the previously published work on reactivity of 1-(trimethylsilyl)cyclopropene.

More detailed studies were performed on the reaction 1-phenyl-3,3-dicarboxymethylcyclopropene with methyl N-acetylcysteinate, since this derivative appears to be reacting the fastest, and the reaction could also be profiled using HPLC with UV detection (Figure 3).

Scheme 2.2: Proposed mechanism of the thiol addition to cyclopropene (B = Base)

The adduct

Figure 2.12. HPLC profile of the reaction of 1-phenyl-3,3-dicarboxymethylcyclopropene with

methyl N-acetylcysteinate

The addition product was isolated as a diastereomeric mixture and identified. There was no reaction between either cyclopropene derivative in Table 1 with similarly derivatized serine and aspartic acid.

Given that Ph-C bond cannot possibly undergo hydrolytic cleavage, contrary to Si-C bond, 1- phenyl-3,3-dicarbonylcyclopropene appears to be the warhead of choice for the contemplated synthesis of the proposed irreversible inhibitor analogous to BDA-410 (Figure 4), as it is most reactive toward cysteine among all the reasonably stable cyclopropene derivatives identified in these studies.

Figure 2.13: Proposed novel irreversible cysteine protease inhibitor with a cyclopropene

warhead.

2.4 Conclusion

We have successfully demonstrated that 1,3,3-trisubstituted cyclopropenes are sufficiently stable, reactive, and selective toward cysteine residues. Nucleophilic thiols can attack electrophilic cyclopropenes to form stable adducts via covalent bonding which can be isolated. Attachment of a substituent at C-1 capable of stabilizing an intermediate carboanion enhances the reactivity of the cyclopropene moiety toward cysteine residue, thereby making cyclopropenes promising warheads for potential cysteine protease inhibitors. The substitution is limited to carbon atoms, as

3,3-dihalo derivatives appear to be excessively reactive to be stable in aqueous media. They undergo dihydrolysis reactions to form a carbonyl carbon at position 3, which loses the activity of irreversible inhibition.

Chapter 3: Synthesis and Characterization of

Self-Cyclizing Antioxidants as Scavengers of

Hydroxyl Radicals

3.1 Introduction

Reactive oxygen species (ROS) are derivatives of molecular oxygen that are more reactive

- than molecular oxygen. A primary ROS is superoxide anion (O2• ), which is formed by one- electron reduction of molecular oxygen. Hydrogen peroxide (H2O2) is produced by reduction of superoxide by the action of dismutase.48 Hydroxyl radical (HO•) arises from electron exchange

- between O2• and H2O2 via the Haber–Weiss reaction or it can also be generated by the reduction

49-50 of H2O2 by the Fenton reaction. Due to the difference in oxidation state of the reactive oxygen, these species show variation in reactivity, stability and subsequently toxicity in a cellular environment.

For instance, hydroxyl radical (HO•) is the least stable and most reactive of all ROS in the biological environment (table 3.1).25, 49, 51 Consequently, it is the most dangerous type of oxidative species and can be extremely harmful to human cells at a very low concentration. On the other hand, hydrogen peroxide is relatively less reactive, more stable, and thus can be tolerated by cells at a much higher concentration than hydroxyl radicals.51

ROS Redox potential (V) Approx. half-life in cell

Hydrogen peroxide 1.80 8 hours – 20 days

Superoxide 1.0 5 seconds

Hydroxyl radical 2.40 10-9 seconds

Hypochlorite 1.0 4-10 weeks

Table 3.1: Comparison of oxidation potentials and half-lives of different ROS

Being extremely unstable and highly reactive, ROS like hydroxyl radical and superoxide anions cannot be transported for long distances in the human body. Hence, they react with the molecules available in proximity of their site of formation. These targets include but are not only limited to nucleic acids, proteins and lipids. The target molecules upon interaction with highly reactive ROS are damaged often to an extent where their function is altered. This is the cause for many pathological conditions such as neurodegeneration, cancer and cardiovascular diseases.

Among these ROS, hydroxyl radical (HO•) is the most potent oxidant and one of the most reactive natural species known to us.52 It rapidly reacts with biological molecules after its formation. In some cases, the reaction rates involving HO• are as high as 109 to 1010 M-1 x s-1.53

HO• is not only formed in biological systems but it is also found to be formed in the atmosphere.

In the air, it is primarily formed by the reaction of water molecules with singlet oxygen species which are formed via photolysis of ozone by UV radiation from sunlight.54 Hydroxyl radicals thus formed are responsible to alter the chemical composition of a variety of atmospheric gases including methane, chlorofluorocarbons, carbon dioxide any many more.55

There are 3 main types of reactions HO• is involved in:

i. Hydrogen abstraction: It can readily abstract H• or hydrogen radical from molecules having

relatively acidic hydrogens to form a water molecule. In biological systems, HO• is

powerful enough to abstract a hydrogen from a guanine base or from the ribose C-H bond

and cause severe DNA damage as a result. HO• attacks a guanine base at position 8 and

converts it into a known product 8-oxoguanine.56 However, another uncommon reaction of

HO• with guanine results in a ring-opening reaction to yield 2,6-diamino-4-hydroxy-5-

formamidopyrimidine. These DNA modifications when go untreated by DNA

polymerases, can lead to the onset of cancer.

ii. Addition reaction: This is the type of reaction where HO• would typically insert itself in a

reactive molecule and form a OH-adduct. This can completely alter the function of a

protein or other biomolecule. Paracetamol, an analgesic may undergo such type of reaction

when it is metabolized in the liver due to the action of P450 system. iii. Electron transfer: In the case of lipid oxidation, hydroxyl radical interacts with a lipid

subunit and donates one electron to the polyunsaturated fatty acids in the cell membrane.

This newly formed radical can undergo a propagation reaction and cause destruction of the

entire lipid membrane (Figure 3.2).57

HO• can also react with various proteins present nearby. Irreversible oxidative damage caused by

HO• to these proteins can impair functioning of antibodies, enzymes, receptors and signal cascades inside the cell. Just like proteins, hydroxide radicals are known to react with amino acids to oxidize them into different products. Methionine can be oxidized easily by HO• to form methionine sulfoxide or metO. Tyrosine can undergo an addition reaction with HO• to form a catechol-like product. All these alterations can be disruptive in the case of a 3-dimensional protein structure.58

Since hydroxyl radical is the most reactive ROS, efforts were made to design a molecule which can be an excellent hydroxyl radical scavenger, is stable in physiological environment and is soluble in the aqueous phase. We were successfully able to synthesize and characterize a series of self-cyclizing antioxidants which are efficient, flexible, and tunable to limit highly reactive hydroxyl radicals.

3.2 Origin of the Idea

Mechanistic studies of a previous self-cyclizing ROS inhibitor (1) revealed the opportunities for the modification of its scaffold in terms of changing oxidation potential, reactivity and stability.30

Compound 1 is found to be oxidized in all ROS environments including hydrogen peroxide, hydroxyl radical and superoxide anion. Furthermore, oxidation of compound 1 in the presence of hydroxyl radical revealed formation of a major product 1-ox which was identified and characterized successfully using HRMS and NMR (scheme 3.1). Identification and characterization of all the intermediates in the proposed mechanism is under progress. Compound

1 undergoes a 6-electron oxidation path to form a self-cyclized final product 1-ox.59

Scheme 3.1: Oxidation of compound 1 under Fenton conditions revealed formation of the major

product 1-ox along with the release of 6 electrons from the molecule.

Discovery of this mechanism indicated that multiple positions of the scaffold can be tuned to optimize oxidation rate, reactivity and stability of the molecule. There are two distinct steps in the proposed mechanism: quinone-like oxidation of the phenyl ring of the scaffold and self- cyclization of the molecule.

Scheme 3.2: Designed scaffold comprises 3 important functional groups with distinctive role in oxidation mechanism. X and Y moieties are responsible for initial quinone-like oxidation of core

whereas, Z moiety is the required nucleophile for self-cyclization.

3.2.1 Quinone-like oxidation of the core

It can be safely predicted that the X and Y pair of the molecule (usually a pair of heteroatoms such as oxygen or nitrogen) is the most important part in the very first step of quinone-like oxidation of the core. There are 4 different variations possible with change in the X and Y atoms during the process of optimization:

Scheme 3.3: Possible variations of core part of the designed scaffold

All of these variations of the core have different oxidation potentials. For instance, we found that with both X and Y as oxygens, the resulting molecules are highly unstable and oxidize rapidly upon exposure to any ROS. Hence, changing X and Y with desired substituents proved to be the correct strategy to control the overall oxidation rate of the scaffold.

3.2.2 Self-cyclization via intramolecular nucleophilic substitution reaction

After initial oxidation step occurs via 2-electron oxidation, the resulting oxidized species has electrophilic nature. The oxidized quinone-like product is sufficiently electron deprived to undergo a thermodynamically driven intramolecular nucleophilic attack from the Z part of the scaffold.

After the attack of Z moiety, the resulting adduct contains a 6-membered ring imparting thermodynamic stability to the self-cyclized product. Rate of nucleophilic attack is governed by the sterics and the electronics of Z. For instance, we have found that Z needs to be a primary or secondary amine. When Z part of the scaffold was replaced by a tertiary amine, the oxidation pathway was terminated at the 2-electron oxidation of core. This indicates that tertiary amines are not nucleophilic enough to promote the self-cyclization step.

3.3 Strategy to optimize self-cyclizing scaffold to selectively inhibit HO•

With the knowledge of the oxidation mechanism, we decided to tune the oxidation potential of our scaffold in such a way that it will be oxidized faster only in presence of hydroxyl radicals and will be relatively stable in other ROS environments. For this purpose, we capitalized on the fact that

HO• has very high oxidation potential (2.40 V) which is more than double of the superoxide anion and significantly higher than hydrogen peroxide.48

We designed and synthesized a series of molecules which included all 4 variations on the core of the scaffold. We also changed the Z moiety to a primary amine or secondary amine. The idea was to explore the effect of these modifications on the rate of oxidation.

Scheme 3.4: Designed series of molecules for the optimization of oxidation rate of the scaffold

3.4 Experimental

3.4.1 Materials

All the chemicals, reagents, and solvents were purchased from Sigma-Aldrich Inc., TCI, and Fisher

Scientific, Inc., and used as received unless stated otherwise. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (RT) is noted as 25°C. Analytical thin layer chromatography (TLC) was performed with glass backed silica plates (5 x 20 cm, 60 Å, 250 μm).

Visualization was accomplished using a 254 nm UV lamp. 1H and 13C NMR spectra were recorded on either a Bruker Avance 400 MHz spectrometer or Bruker DPX 500 MHz spectrophotometer using solutions of samples in either of the deuterated solvents: chloroform, methanol, acetonitrile,

DMSO or water. Chemical shifts are reported in ppm with tetramethylsilane as standard. Data are reported 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 Shimadzu Q-TOF 6500.

All novel compounds were characterized by 1H, 13C, DEPT 13C NMR spectroscopy and high- resolution mass spectrometry. The identity of previously made self-cyclizing scaffolds was confirmed comparison of their 1H NMR to the published data (reference provided). HPLC analysis of final products was performed on an Agilent 1200 HPLC with UV detection.

3.4.2 Synthetic methods

3.4.2.1 Synthesis of N-(5-hydroxy-2-piperidinophenyl)-2-(methylamino)acetamide (3a)

Scheme 3.5: Synthesis of N-(5-hydroxy-2-piperidinophenyl)-2-(methylamino)acetamide (3a)

Step 1: Synthesis of compound A60 (Yield: 96%)

A solution of 500 mg (1 eq) of 4-Fluoro-3-nitrophenol in 15 mL dichloromethane was cooled in an ice bath for 10 min. 1.5 mL (2.8 eq) of diisopropylethylamine (DIPEA) was added to it dropwise. Reaction mixture was stirred for 10 min followed by addition of 484 µL (2 eq) MOM chloride. Reaction was monitored using TLC and upon completion after 4 hours, reaction mixture was evaporated in vacuo and resuspended in Ethyl acetate (50mL). Organic layer was washed with

25 mL saturated NaHCO3, 50 x 2 mL water, 20 mL brine, dried over sodium sulfate and

1 concentrated on rotavapor to yield pure compound A. H NMR (CDCl3, 400 MHz): δ 3.45 (s, 3H),

5.23 (s, 2H), 7.20 (t,1H), 7.30 (dd, 1H), 7.75 (dd, 1H).

Step 2: Synthesis of compound B (Yield: 95%)

To 100 mg compound A (1 eq) was added 172 µL (3.5 eq) of piperidine. The reaction was stirred neat overnight at room temperature.61 Upon completion of the reaction monitored by TLC, reaction mixture was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. On drying the organic layer in vacuo, compound B was obtained as

1 orange-yellow oil. H NMR (CDCl3, 400 MHz): δ 1.51 (m, 2H), 1.72 (m, 4H), 2.94 (m, 4H), 3.45

(s, 3H), 5.23 (s, 2H), 7.12 (d, 1H), 7.20 (d, 1H), 7.45 (s, 1H).

Step 3: Synthesis of compound C

In a 50 mL round bottom flask flushed with argon, 100 mg compound B was dissolved in 10 mL methanol. 15 mg Palladium over dry carbon (15% w/w) was added to it and flask was sealed with a septum. Hydrogen gas was introduced to the reaction mixture via balloon and reaction was stirred overnight.62 Upon completion of reaction monitored by TLC, reaction mixture was filtered through celite to remove Palladium-carbon, concentrated in vacuo to obtain compound C which was used in step 4 without purification due to its unstable nature.

Step 4: Synthesis of compound D (Yield: 70%)

In a 25 mL round bottom flask, 488 mg (1.5 eq) Boc-sarcosine was dissolved in 10 mL DMF. 1.3 g (2 eq) HBTU was added to it and the resulting mixture was stirred for 15 min. It was followed by the addition of 330 mg (1 eq) compound C and the reaction mixture was stirred for 30 min.

Finally, 1.2 mL (4 eq) DIPEA was added and the reaction was stirred at room temperature overnight. It was then dissolved in 50 mL ethyl acetate, washed with 4 x 50 mL water, 20 mL brine and dried over sodium sulfate. After concentration the organic layer on rotavapor, crude mixture was obtained which was purified with silica gel chromatography (Hexane:Ethyl acetate / 5:1). 1H

NMR (CDCl3, 400 MHz): δ 1.49 (s, 9H), 1.52 (m, 2H), 1.72 (m, 4H), 2.58 (s, 3H), 2.94 (broad s,

4H), 3.45 (s, 3H), 3.49 (s, 2H), 5.23 (s, 2H), 6.77 (d, 1H), 7.10 (d, 1H), 8.51 (s, 1H), 10.50 (broad s, 1H).

Step 5: Synthesis of N-(5-hydroxy-2-piperidinophenyl)-2-(methylamino)acetamide (3a)

In a 50 mL round bottom flask, 100 mg of compound D was dissolved in 20 mL dichloromethane.

3 mL (15% v/v) Trifluoroacetic acid was added to it dropwise and the resulting mixture was stirred overnight. Upon completion of reaction monitored by TLC, both the phenol group and amine group were deprotected. The reaction mixture was neutralized with solid NaHCO3, filtered, concentrated in vacuo and purified with silica gel chromatography. (Dichloromethane:methanol /

1 20:1) to obtain compound 3a. H NMR (CDCl3, 400 MHz): δ 1.52 (m,2H), 1.72 (m, 4H), 2.58 (s,

3H), 2.94 (broad s, 4H), 3.49 (s, 2H), 6.62 (d, 1H), 7.01 (d, 1H), 8.48 (s, 1H), 10.50 (broad s, 1H).

13 C NMR (CDCl3, 400 MHz): δ 24.17, 26.89, 36.90, 54.32, 55.37, 106.44, 110.69, 121.68, 133.35,

+ + 135.18, 154.69, 177.10. HRMS (ESI ) for C14H22N3O2 : Calculated: 264.3485. Observed:

264.3857

Figure 3.1: 1HNMR of Compound 3a

Figure 3.2: 13CNMR of Compound 3a

Sample ID: PG-2-97 (C14 H21 N3 O2) 263.16 Da (in MeOH) PURUJIT-G-060116-PG-2-97 (0.025) Is (1.00,1.00) C14 H21 N3 O2 H TOF MS ES+ 264.1712

100 8.40e12 %

265.1742

266.1768 0

PURUJIT-G-060116-PG-2-97 33 (0.576) AM (Cen,4, 80.00, Ar,5000.0,360.32,1.00); Sm (Mn, 2x3.00); Sb (1,40.00 ); Cm (2:58) TOF MS ES+ 264.1665

100 2.05e4 %

265.1718

260.1497 261.1562 262.1539 263.1470 266.1710 267.1006 269.1319 0 m/z 260 261 262 263 264 265 266 267 268 269 270

Figure 3.3: HRMS of Compound 3a

3.4.2.2 Synthesis of N-(5-methoxy-2-piperidinophenyl)-2-(methylamino)acetamide (3b)

Scheme 3.6: Synthesis of N-(5-methoxy-2-piperidinophenyl)-2-(methylamino)acetamide (3b)

Step 1: Synthesis of compound A (Yield: 99%)

To a solution of 1 g (1 eq) 4-Fluoro-3-nitrophenol in 25 mL acetone at 0ºC was added 2.2 g (2.5 eq) potassium carbonate. Reaction mixture was stirred for 10 min followed by dropwise addition of 793 µL (2 eq) of methyl iodide. Reaction was allowed to warm up to room temperature and was stirred overnight. Precipitated solid was then filtered and reaction mixture was concentrated in vacuo. It was purified using silica gel chromatography (Hexane:Ethyl acetate/ 10:1). 1H NMR

(CDCl3, 400 MHz): δ 3.89 (s, 3H), 7.21-7.26 (m, 2H), 7.53 (d,1H).

Step 2: Synthesis of compound B (Yield: 97%)

To 100 mg compound A (1 eq) was added 173 µL (3 eq) of piperidine. The reaction was stirred neat overnight at room temperature. Upon completion of the reaction monitored by TLC, reaction mixture was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. On drying the organic layer in vacuo, compound B was obtained as

1 orange-yellow solid. H NMR (CDCl3, 400 MHz): δ 1.54 (m, 2H), 1.75 (m, 4H), 2.96 (m, 4H),

3.84 (s, 3H), 7.05 (d, 1H), 7.15 (d, 1H), 7.41 (s, 1H).

Step 3: Synthesis of compound C

138 mg (1 eq) of compound B was dissolved in 10 mL 1:1 mixture of dichloromethane and methanol. 396 mg (3eq) anhydrous SnCl2 was added to it and the resulting mixture was stirred overnight. Reaction mixture was monitored using TLC and upon consumption of all starting compound was concentrated in vacuo and dissolved in 50 mL ethyl acetate. This organic layer was washed with 50 mL saturated Na2CO3 solution, 2 x 50 mL water, 20 mL brine and dried over sodium sulfate. Concentrating the organic layer on rotavapor yielded crude product which was used for next reaction without any further purification.

Step 4: Synthesis of compound D

In a 25 mL round bottom flask, 111 mg (1 eq) Boc-sarcosine was dissolved in 10 mL DMF. 267 mg (1.2 eq) HATU was added to it and resulting mixture was stirred for 15 min. Crude compound

C obtained from step 3 was dissolved in 5 mL DMF and slowly added to this reaction mixture followed by addition of 204 µL (2 eq) DIPEA. This reaction mixture was then stirred at room temperature. After 16 hours, reaction was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. The compound was purified using silica gel

1 chromatography (Hexane:Ethyl acetate/ 4:1). H NMR (CDCl3, 400 MHz): δ 1.50 (s,9H), 1.69 (m,

4H), 2.18 (s, 2H), 2.46 (s, 3H), 2.70 (m, 4H), 3.30 (s, 2H), 3.76 (s, 3H), 6.55 (d, 1H), 7.01 (d,1H),

8.21 (s,1H), 10.33 (broad s, 1H).

Step 5: Synthesis of N-(5-methoxy-2-piperidinophenyl)-2-(methylamino)acetamide (3b)

In a 50 mL round bottom flask, 100 mg of compound D was dissolved in 20 mL dichloromethane.

3 mL (15% v/v) Trifluoroacetic acid was added to it dropwise and the resulting mixture was allowed to stir overnight. Upon completion of reaction which monitored by TLC, the reaction mixture was neutralized with solid NaHCO3, concentrated in vacuo and purified with silica gel

1 chromatography. H NMR (CDCl3, 400 MHz): δ 1.69 (m, 4H), 2.18 (s, 2H), 2.46 (s, 3H), 2.70 (m,

4H), 3.30 (s, 2H), 3.76 (s, 3H), 6.55 (d, 1H), 7.01 (d,1H), 8.21 (s,1H), 10.33 (broad s, 1H).

Figure 3.4: 1HNMR of Compound 3b

For synthesis of compounds 3c-3f, a common intermediate compound I was used.

3.4.2.3 Synthesis of tert-butyl 2-(2-fluoro-5-nitrophenylamino)-2-oxoethyl(methyl) carbamate (compound I). (Yield: 70%)

Scheme 3.7: Synthesis of Compound I1

To a solution of 0.95 g Boc-sarcosine (1 eq) in 5 mL DMF at room temperature, 2.1 g HATU (1.1 eq) was added. Resulting mixture was stirred for 15 min and 0.78 g of 2-fluoro-5-nitroaniline (1 eq) was added to it, followed by the addition of 1.8 mL DIPEA (2 eq). The reaction mixture was then stirred at room temperature for 4 h, and was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. The compound was purified using silica gel chromatography (Hexane:Ethyl acetate/ 2:1) to obtain the product compound I as

1 yellowish solid. H NMR (CDCl3, 400 MHz): δ 9.30 (dd, J = 6.8 Hz, 1H), 9.06 (s, 1H), 8.13 – 7.81

13 (m, 1H), 7.23 (d, J = 9.4 Hz, 1H), 4.04 (s, 2H), 3.03 (s, 3H), 1.51 (s, 9H). C NMR (CDCl3, 400

MHz): δ 168.40, 156.63, 154.10, 144.45, 119.91, 117.08, 115.53, 115.27, 54.58, 33.95, 28.24.

1 Figure 3.5: HNMR of Compound I1

3.4.2.4 Synthesis of N-(5-amino-2-phenoxyphenyl)-2-(methylamino)acetamide (3c)

Scheme 3.8: Synthesis of Compound 3c

Step 1: Synthesis of compound A (Yield: 78 %)

In a 25 mL round bottom flask, 250 mg (1 eq) compound I1 was dissolved in 10 mL DMSO. 80 mg (1.1 eq) Phenol and 116 mg (1.1 eq) K2CO3 were added to it and the reaction mixture was heated to 80ºC for 12 hours. Upon disappearance of starting compound I1 monitored by TLC, reaction was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. The compound was purified using silica gel chromatography

1 (Hexane:Ethyl acetate/ 4:1). H NMR (CDCl3, 400 MHz): δ 1.45 (s, 9H), 2.99 (s, 3H), 4.12 (s, 2H),

6.75 (d, 1H), 7.15 (d, 2H), 7.25 (m, 2H), 7.46 (m,2H), 7.85 (s, 1H), 9.45 (s, 1H).

Step 2: Synthesis of compound B (Yield: 85%)

In a 25 mL round bottom flask, 60 mg (1 eq) compound A was dissolved in 10 mL methanol. 142 mg (5 eq) anhydrous SnCl2 was added to it and the reaction mixture was heated to 55ºC for 4

61 hours. Upon completion of reaction monitored by TLC, reaction was concentrated in vacuo and dissolved in 30 mL ethyl acetate. This organic layer was washed with 50 mL saturated Na2CO3 solution, 2 x 30 mL water, 15 mL brine and dried over sodium sulfate. Concentrating the organic layer on rotavapor yielded crude product which was used for next reaction without any further purification.

Step 3: Synthesis of N-(5-amino-2-phenoxyphenyl)-2-(methylamino)acetamide (3c)

Crude mixture obtained from step 3 was dissolved in 20 mL dichloromethane. 3 mL (15% v/v) trifluoroacetic acid was then added to it dropwise. The reaction mixture was then stirred overnight.

Upon completion of reaction monitored by TLC, reaction was concentrated in vacuo and dissolved in 30 mL ethyl acetate. This organic layer was washed with 30 mL saturated NaHCO3 solution, 2 x 30 mL water, 15 mL brine and dried over sodium sulfate. Product was purified using silica gel

1 chromatography (Dichloromethane:methanol / 20:1). H NMR (CDCl3, 400 MHz): δ 2.08 (s, 3H),

3.17 (s, 2H), 6.39 (dd, 1H), 6.84 (d, 1H), 6.92 (d, 2H), 7.01 (t, 1H), 7.22-7.29 (m, 2H), 7.95 (d,

1H), 9.61 (broad s, 1H).

Figure 3.6: 1HNMR of Compound 3c

3.4.2.5 Synthesis of N-(5-amino-2-ethoxyphenyl)-2-(methylamino)acetamide (3d)

Scheme 3.9: Synthesis of N-(5-amino-2-ethoxyphenyl)-2-(methylamino)acetamide (3d)

Step 1: Synthesis of compound A (Yield: 80%)

A 25 mL round bottom flask containing 10 mL ethanol was cooled to 0ºC using ice bath. 500 mg of the 60% suspension of NaH in paraffins was slowly added to it and the reaction was stirred for

30 min at the same temperature. 100 mg of compound I1 was added to the reaction mixture in 4 portions which were added in 5 min intervals. Final reaction mixture was allowed to warm up to room temperature and was stirred for 4 hours. Upon completion of reaction confirmed by TLC, reaction mixture was evaporated and dissolved in 30 mL ethyl acetate. washed with 3 x 30 mL water, 15 mL brine and dried over sodium sulfate. The compound was purified using silica gel

1 chromatography (Hexane:Ethyl acetate/ 10:1). H NMR (CDCl3, 400 MHz): δ 1.45 (s, 9H), 1.52

(t, 3H), 3.03 (s, 3H), 4.02 (s, 2H), 4.22 (q, 2H), 6.92 (d, 1H), 8.00 (d, 1H), 8.77 (s, 1H), 9.31 (d,

13 1H). C NMR (CDCl3, 400 MHz): δ 14.55, 28.29, 33,96, 54.26, 109.86, 114.99, 116.28, 120.16,

141.20, 151.83, 156.28, 156.58.

Step 2: Synthesis of compound B (Yield: 95%)

In a 50 mL round bottom flask, 100 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 228

µL (5 eq) of triethylsilane was added dropwise. After all the bubbling was allowed to subside, reaction was stirred for 1 hour. Upon completion of reaction confirmed by TLC, reaction mixture was filtered through celite to remove palladium, and concentrated on rotavapor to yield crude dark brown oil. It was purified with silica gel chromatography to obtain a yellow solid. (Hexane:Ethyl

1 acetate/ 4:1). H NMR (CDCl3, 400 MHz): δ 1.33 (t, 3H), 1.45 (s, 9H), 2.94 (s, 3H), 3.44 (s, 2H),

13 3.92 (q, 2H), 6.29 (d, 1H), 6.62 (d, 1H), 8.39 (s, 1H). C NMR (CDCl3, 400 MHz): δ 14.99, 28.27,

35.70, 53.97, 64.98, 107.29, 109.83, 112.60, 112.79, 128.04, 140.36, 140.47, 167.14.

Step 3: Synthesis of N-(5-amino-2-ethoxyphenyl)-2-(methylamino)acetamide (3d) (Yield: 75%)

Crude mixture obtained from step 3 was dissolved in 20 mL dichloromethane. 3 mL (15% v/v) trifluoroacetic acid was then added to it dropwise. The reaction mixture was then stirred overnight.

1 chromatography (Dichloromethane:methanol / 20:1) H NMR (CDCl3, 400 MHz): δ 1.41 (t, 3H),

2.51 (s, 3H), 3.36 (s, 2H), 4.00 (q, 2H), 6.36 (d, 1H), 6.71 (d, 1H), 7.91 (s, 1H), 9.61 (broad s, 1H).

13 C NMR (CDCl3, 400 MHz): δ 15.34, 36.58, 55.69, 66.39, 109.90, 112.57, 114.71, 129.09, 142.13,

142.97, 171.88. HRMS (ESI) m/z for C11H17N3O2: calculated [M + H] 224.1394, observed [M + H]

224.1395.

Figure 3.7: 1HNMR of Compound 3d

3.4.2.6 Synthesis of N-(5-amino-2-piperidinophenyl)-2-(methylamino)acetamide (3e)

Scheme 3.10: Synthesis of N-(5-amino-2-piperidinophenyl)-2-(methylamino)acetamide (3e)

Step 1: Synthesis of compound A (Yield: 82%)

In a 50 mL round bottom flask, 100 mg (1 eq) compound I1 was mixed with 5 mL (excess) piperidine neat. This reaction mixture was stirred overnight. Upon completion of reaction monitored by TLC, 30 mL of ethyl acetate was then added to it followed by 3x30 mL water washes.

Finally, the organic layer was washed with brine, dried over Na2SO4, evaporated and purified by silica gel chromatography (hexane/ethyl acetate = 10/1) to give an orange solid product. 1H NMR

(CDCl3, 400 MHz): δ 1.46 (s, 9H), 1.62 (m, 2H), 1.76 (m, 4H), 2.83 (m, 4H), 3.03 (s, 3H), 4.08

(s, 2H), 7.2 (s, 1H), 7.95 (d, J=8.76 Hz, 1H), 8.88 (d, J=14.42 Hz, 1H), 9.3 (s, 1H). 13C NMR

(CDCl3, 400 MHz): δ 23.83, 26.28, 28.27, 35.95, 53.38, 81.13, 119.42, 120.30, 132,99, 167.90.

Figure 3.8: 1HNMR of Compound A

Figure 3.9: 13CNMR of Compound A

Step 2 and step 3: One-pot synthesis of N-(5-amino-2-piperidinophenyl)-2-

(methylamino)acetamide (3e) from compound A (Overall yield: 60%)

In a 50 mL round bottom flask, 100 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 204

This oil was dissolved in 20 mL dichloromethane and to it 3 mL trifluoroacetic acid (15% by vol) was added. Reaction mixture was stirred for 4 h and then evaporated. Resulting crude solid was dissolved in 30 mL ethyl acetate and washed with 2x20 mL sat NaHCO3 solution, 20 mL water and 10mL brine. Organic layer was dried over Na2SO4, evaporated and purified by silica gel chromatography (dichloromethane:methanol / 10:1) to give a yellow solid.

1 H NMR (CDCl3, 400 MHz): δ 1.70 (m, 4H), 2.11 (m, 2H), 2.51 (s, 3H), 2.96 (m, 4H), 3.4 (m,

2H), 3.78 (m, 2H), 6.38 (dd, J=8.38, 2.65 Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 7.90 (d, J=2.62 Hz,

13 1H), 10.22 (broad S, 1H). C NMR (CDCl3, 400 MHz): δ 24.19, 26.89, 36.89, 53.41, 54.27, 55.79,

+ + 105.90, 109.69, 121.40, 134.10, 134.54, 143.66. HRMS (ESI ) calculated for C14H23N4O :

263.1866 (MH+); Observed: 263.1863.

Figure 3.10: 1HNMR of Compound 3e

Figure 3.11: 13CNMR of Compound 3e

+ M+H

+ M+Na

Figure 3.12: HRMS of Compound 3e

3.4.2.7 Synthesis of N-(5-amino-2-N-acetylpiperizinophenyl)-2-(methylamino)acetamide

(3f)

Scheme 3.11: Synthesis of N-(5-amino-2-N-acetylpiperizinophenyl)-2-(methylamino)acetamide

Step 1: Synthesis of compound A (Yield: 70%)

In a 50 mL round bottom flask, 100 mg (1 eq) compound I1 was dissolved in 5 mL DMF. To it

100 mg (excess) of N-acetylpiperizine was added by portions. This reaction mixture was stirred overnight. Upon completion of reaction monitored by TLC, it was dissolved in 30 mL of ethyl acetate followed by 3x30 mL water washes. Finally, the organic layer was washed with brine, dried over Na2SO4, evaporated and purified by silica gel chromatography (hexane/ethyl acetate =

1 10/1) to give an orange solid product. H NMR (CDCl3, 400 MHz): δ 1.46 (s, 9H), 2.21 (s, 3H),

2.53 (s, 3H), 2.92 (m, 4H), 3.44 (s, 2H), 3.69 (m, 2H), 3.84 (m, 2H), 7.13 (d, 1H), 7.92 (d, 1H),

9.35 (d, 1H), 10.16 (broad s, 1H).

Step 2: One-pot synthesis of N-(5-amino-2-N-acetylpiperizinophenyl)-2-(methylamino) acetamide (3f) starting from compound A

In a 50 mL round bottom flask, 104 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 215

NMR (CDCl3, 400 MHz): δ 2.12 (s, 3H), 2.48 (s, 3H), 2.74-2.79 (m, 5H), 3.36 (s, 2H), 3.60 (m,

3H), 6.33 (d, 1H), 6.88 (d, 1H), 7.92 (d, 1H), 10.33 (broad s, 1H).

Figure 3.13: 1HNMR of Compound 3f

For the synthesis of compounds 3g-3i, a common intermediate 5-Nitro-2-piperidinoaniline

(Compound I2) was synthesized.

3.4.2.8 Synthesis of 5-Nitro-2-piperidinoaniline (Compound I2) (Yield: 96%)

Scheme 3.12: Synthesis of compound I2

1 g of 2-Fluro-5-nitroaniline (1 eq) and piperidine (1mL, excess) were mixed together and reaction mixture was heated at 50o C overnight. 30 mL of ethyl acetate was then added to it and the organic layer was washed with 3x30 mL water, 15 mL brine and drier over Na2SO4. Upon evaporation,

1 the product was obtained as an orange solid. H NMR (CDCl3, 400 MHz): δ 1.61 (m, 2H), 1.72 (m,

4H), 2.9 (m, 4H), 6.96 (d, 1H), 7.54 (s, 1H), 7.60 (d, 1H).

3.4.2.9 Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminoacetamide (3g)

Scheme 3.13: Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminoacetamide (3g)

Step 1: Synthesis of compound A (Yield: 65%)

In a 50 mL round bottom flask, 99 mg of Boc-glycine (1 eq) was dissolved in 5 mL DMF. With the constant stirring, 215 mg (1 eq) of HATU was added to it and the resulting mixture was stirred for 30 min. 250 mg of compound I2 (2 eq) was then added to it followed by the addition of 150 µL

(1.5 eq) of DIPEA. Reaction mixture was stirred at room temperature. After 16 hours, reaction was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. The compound was purified using silica gel chromatography (Hexane:Ethyl acetate/ 6:1).

1 H NMR (CDCl3, 400 MHz): δ 1.50 (s, 9H), 1.62 (m, 2H), 1.76 (m, 4H), 2.83 (m, 4H), 4.12 (s,

2H), 7.2 (s, 1H), 7.95 (d, 1H), 8.88 (d, 1H), 9.3 (s, 1H).

Step 2: One-pot synthesis of N-(5-amino-2-piperidinophenyl)-2-aminoacetamide (3g) starting from compound A

In a 50 mL round bottom flask, 100 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 211

1 chromatography (dichloromethane:methanol / 10:1) to give a yellow solid. H NMR (CDCl3, 400

MHz): δ 2.18 (m, 2H), 2.81 (s, 4H), 3.45 (s, 2H), 3.83 (s, 2H), 6.33 (d, 1H), 6.94 (d, 1H), 7.92 (d,

1H), 10.25 (broad s, 1H).

The NMR indicated presence of some grease impurity at 1-2 ppm. However, since it would not interfere with further experiment, decision was made to not purify it again.

Figure 3.14: 1HNMR of Compound 3g

3.4.2.10 Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminopropanamide (3h)

Scheme 3.14: Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminopropanamide (3h)

Step 1: Synthesis of compound A (Yield: 60%)

In a 50 mL round bottom flask, 342 mg of Boc-alanine (2 eq) was dissolved in 5 mL DMF. With the constant stirring, 344 mg (1 eq) of HATU was added to it and the resulting mixture was stirred for 30 min. 200 mg of compound I2 (1 eq) was then added to it followed by the addition of 158 µL

(1 eq) of DIPEA. Reaction mixture was stirred at room temperature. After 16 hours, reaction was dissolved in 50 mL ethyl acetate, washed with 3 x 50 mL water, 20 mL brine and dried over sodium sulfate. The compound was purified using silica gel chromatography (Hexane:Ethyl acetate/ 6:1).

1 H NMR (CDCl3, 400 MHz): δ 1.49 (d, 3H), 1.53 (s, 9H), 1.70 (m, 4H), 2.71 (m, 6H), 3.63 (m,

1H), 7.22 (s, 1H), 7.95 (d, 1H), 8.88 (d, 1H), 9.3 (s, 1H).

Step 2: One-pot synthesis of N-(5-amino-2-piperidinophenyl)-2-aminopropanamide (3h) starting from compound A

In a 50 mL round bottom flask, 100 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 203

1 chromatography (dichloromethane:methanol / 10:1) to give a yellow solid. H NMR (CDCl3, 400

MHz): δ 1.42 (d, 3H), 1.70 (m, 4H), 2.71 (m, 6H), 3.63 (m, 1H), 6.36 (d, 1H), 6.91 (d, 1H), 7.87

(d, 1H), 10.29 (broad s, 1H).

Figure 3.15: 1HNMR of Compound 3h

3.4.2.11 Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminohexanamide (3i)

Scheme 3.15: Synthesis of N-(5-amino-2-piperidinophenyl)-2-aminohexanamide (3i)

Step 1: Synthesis of compound A (Yield: 60%)

In a 50 mL round bottom flask, 419 mg of Boc-leucine (2 eq) was dissolved in 5 mL DMF. With the constant stirring, 344 mg (1 eq) of HATU was added to it and the resulting mixture was stirred for 30 min. 200 mg of compound I2 (1 eq) was then added to it followed by the addition of 158 µL

1 H NMR (CDCl3, 400 MHz): δ 0.97 (m, 6H), 1.49 (m, 1H), 1.53 (s, 9H), 1.70 (m, 4H), 2.71 (m,

8H), 3.56 (m, 1H), 7.22 (s, 1H), 7.95 (d, 1H), 8.88 (d, 1H), 9.3 (s, 1H).

Step 2: One-pot synthesis of N-(5-amino-2-piperidinophenyl)-2-aminopropanamide (3h) starting from compound A

In a 50 mL round bottom flask, 100 mg (1 eq) of compound A was dissolved in 15 mL methanol.

15 mg Palladium over dry carbon (15% w/w) was added to it. To this resulting suspension, 203

1 chromatography (dichloromethane:methanol / 10:1) to give a yellow solid. H NMR (CDCl3, 400

MHz): δ 0.97 (m, 6H), 1.49 (m, 1H), 1.70 (m, 4H), 2.71 (m, 8H), 3.56 (m, 1H), 6.36 (s, 1H), 6.91

(d, 1H), 7.87 (d, 1H), 10.20 (broad s, 1H).

Figure 3.16: 1HNMR of Compound 3i

3.4.2.11 Synthesis of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one (ox-P)

Scheme 3.16: Synthesis of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one (ox-P)

Step 1: Synthesis of N1-Methyl-4-nitro-1,2-phenylenediamine (A)

2-Fluoro-5-nitroaniline (1g, 6.4 mmol), methylamine hydrochloride (520 mg, 7.7 mmol) and potassium carbonate (2.6 g, 19.2 mmol) were dissolved in 5 mL of DMSO. The mixture was stirred overnight at 60o C. Then the reaction mixture was dissolved in 50 mL ethyl acetate and was washed with 3x50 mL water. The organic layer was then washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified by silica gel chromatography (n-Hexane/ethyl acetate =

1 10/1) to give an orange solid. (950 mg, y. 89%). H NMR (400 MHz, CDCl3): δ 2.99 (s, 3H), 3.28

(broad s, 2H), 4.30 (broad s, 1H), 6.54 (d, J=8.8 Hz, 1H), 7.62 (s, 1H), 7.88 (d, J=11.33 Hz, 1H).

Step 2: Synthesis of 1-Methyl-5-nitro-2,3-dihydro-1H-1,3-benzodiazol-2-one (B)

N1-Methyl-4-nitro-1,2-phenylenediamine (50 mg, 0.3 mmol) was dissolved in 3 mL of ethyl acetate. CDI (55 mg, 0.33 mmol) was added to it and reaction mixture was stirred overnight.

Product was formed as white solid precipitate which was filtered and washed with ethyl acetate

1 multiple times. (37 mg, y. 64%). H NMR (400 MHz, DMSO-D6): δ 3.32 (s, 3H), 7.28 (d, J=8.69

Hz, 1H), 7.74 (s, 1H), 8.01 (dd, J=8.69, 1.9 Hz, 1H), 11.43 (broad s, 1H).

Step 3: Synthesis of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one (ox-P)

1-Methyl-5-nitro-2,3-dihydro-1H-1,3-benzodiazol-2-one (30 mg, 0.15 mmol) was dissolved in 5 mL of methanol. Palladium over carbon (4.5 mg, 15% by wt) was added to it. To this suspension triethylsilane (0.124 mL, 0.77 mmol) was added dropwise. Reaction was stirred for 1 hour and then filtered through a celite column. Resulting filtrate was evaporated and purified using silica gel chromatography (dichloromethane/methanol = 10/1) to give a dark orange solid. (23 mg, y.

91%). 1H NMR (400 MHz, MeOD): δ 3.27 (s, 3H), 6.50 (m, 2H), 6.79, (d, J=8.86 Hz, 1H). 13C

NMR (400 MHz, MeOD): δ 26.94, 98.86, 109.41, 110.74, 125.28, 130.25, 143.80, 157.03. HRMS

+ + + (ESI ) calculated for C8H10N3O : 164.08184 (MH ); Observed: 164.08185.

Figure 3.17: 1HNMR of N1-Methyl-4-nitro-1,2-phenylenediamine

Figure 3.18: 1HNMR of 1-Methyl-5-nitro-2,3-dihydro-1H-1,3-benzodiazol-2-one

Figure 3.19: 1HNMR of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one

Purujit_PG-2-221_20170503-R02 #66-151 RT: 0.58-1.03 AV: 30 NL: 2.28E6 F: FTMS + p ESI Full ms [50.00-500.00] 164.08185 100 + 95 M+H

45 RelativeAbundance

35 + 30 M+Na

25 186.06382

0 110 120 130 140 150 160 170 180 190 200 210 220 230 240 m/z Figure 3.20: HRMS of 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one

3.4.3 Oxidation studies of synthesized molecules

3.4.3.1 Strategy of oxidation experiment

The reason for synthesizing this series of molecules was to explore and investigate the oxidation

potential of the scaffold with various substituents. For this purpose, the first consideration was to

investigate the molecules with different substituents on the core phenyl ring. To start the

experiment of oxidation, molecules 3a-3e were taken as representatives. It can be noticed that

86 molecules 3a-3e show 5 different combinations of functional groups which have different oxidation and reduction potentials.

Scheme 3.17: Representative compounds selected for oxidation studies

These compounds were subjected to different ROS including hydrogen peroxide, superoxide anion and hydroxyl radical. The oxidation reactions were either monitored by HPLC or UV-vis spectroscopy. For the initial investigation, disappearance of the starting compound due to oxidation was monitored. Assuming the reaction kinetics to be pseudo-unimolecular, the rate of decrease of concentration over time was used to find the rate of the oxidation reaction. The rate was then applied to estimate the half-life of the molecule in the respective environment. Half-life can safely be considered as a standard of stability of the molecule in the given ROS environment.

3.4.3.2 Preparation for in-vitro generation of ROS for oxidation experiment

Compound stock solution: 250 mM stock solutions of compounds 3a-3e were made in HPLC grade acetonitrile solvent (ACN).

Buffer: 20 mM phosphate buffer was adjusted to obtain final pH of 7.6.

Hydrogen peroxide: 10 M hydrogen peroxide (H2O2) was bought from Fischer scientific. It was used as it is without any dilution.

Superoxide anion: 0.7 mg of KO2 was used as a solid in 1 mL reaction mixture to make 10 mM superoxide solution.

Hydroxyl radical: Fenton chemistry is used to generate hydroxyl radicals in situ. Two separate solutions A and B were synthesized and mixed with hydrogen peroxide to yield HO•

Solution A: 3.9 mg of Ammonium iron (II) sulfate [(NH4)2Fe(SO4)2.6H2O] was dissolved in 1 mL of water. 20 µL of 0.5 M EDTA solution was added to it. This gives 10 mM final concentration for iron (II) solution.

Solution B: 3.4 mg of ascorbic acid was dissolved fresh in 1 mL water to give 20 mM of ascorbate solution.

3.4.3.3 HPLC conditions

A Beckman Coulter’s HPLC system consisting of a dual pump Model 126 with 32 Karat Software, a System Gold 168 detector and an System Gold 508 Auto Sampler was used. A reversed-phase

C-18 column (Synergi™ 4 μm Hydro-RP 80 Å, LC Column 150 × 4.6 mm, Ea) was used. The mobile phase consisted of HPLC grade Acetonitrile (Fisher Scientific), LCMS grade formic acid

(Fisher scientific) and distilled water filtered through a Millipore Milli-Q water purification

88 system. Solvent A for the mobile phase was 95% water, 5% acetonitrile with 0.1% formic acid for oxidation study of 3a-3e. Solvent B is 95% acetonitrile and 5% water. The gradient was 0% B for

4 minute and 95% B over 15 minutes. Flow was 1 ml/min. A detection wavelength of 250 nm was used for oxidation of 3a-3e.

3.4.3.4 Reactions performed for oxidation experiment

Reaction 1: Control

10 µL compound stock solution and 990 µL buffer solution were mixed together.

Reaction 2: Hydrogen peroxide mediated oxidation

10 µL compound stock solution, 990 µL buffer solution and 1 µL hydrogen peroxide solution (10

M) were mixed together to generate 2.5 mM final concentration of compound in 10 mM final concentration of hydrogen peroxide.

Reaction 3: Superoxide anion medicated oxidation

10 µL compound stock solution and 990 µL of buffer solution were introduced in a vial containing

0.7 mg potassium superoxide (KO2) to generate 2.5 mM final concentration of compound in 10 mM final concentration of superoxide anion.

Reaction 4: Hydroxyl radical mediated oxidation (Fenton chemistry)

10 µL compound stock solution, 970 µL buffer solution, 10 µL solution A and 10 µL solution B were mixed together. 1 µL hydrogen peroxide solution (10 M) was added to it immediately to

89 generate 2.5 mM final concentration of compound in 10 mM final concentration of hydroxyl radicals.

3.4.3.5 HPLC and UV-vis analysis

For the HPLC analysis, 20 µL of these reactions were injected and HPLC trace was generated over time using the protocol mentioned above. Area under the curve was used as a direct correlation of the concentration of compound present unreacted in the reaction environment. For HPLC monitored reactions, it was observed that monitoring area under the curve of disappearance of a peak was beneficial. This is mainly because of the inability to monitor absorbances at different wavelengths at the same time.

On the other hand, in case of UV-vis spectroscopy, the same reaction can be observed at a very low concentration (~ 1mM) and absorbance at different wavelengths can be monitored at the same time. In case of UV-vis spectroscopy, formation of the new product peak was observed and the rate of reaction was determined by rate of increase in concentration of the product formed.

Example 1: Hydrogen peroxide mediated oxidation of compound 3a using HPLC

Det 168-250 nm Det 168-250nm Det 168-250nm Det 168-250nm Det 168-250nm 1201_PG243_0_H2O2_20 1201_PG243_0_H2O2_60 1201_PG243_0_H2O2_100 1201_PG243_0_H2O2_140 1201_PG243_0_H2O2_180

400

mAU 200

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Minutes

Figure 21: Oxidation of compound 3a in presence of hydrogen peroxide over time monitored by

HPLC. Black trace is after 20 min of reaction, red trace is 60 min of reaction, green trace is 100

min of reaction, blue trace is 140 min of reaction and magenta trace is 180 min of reaction.

Based on these traces, area under the curve of each trace was calculated. The following table

represents area under the curve for each peak and its natural logarithmic value.

t A ln(A) 20 10564916 16.17305 60 6255115 15.64891 100 2938512 14.89341 140 1384276 14.14069 180 685745 13.43826

Where A is area under the curve and t is time in minutes

Table 3.2: Time, area under the curve (A) and the natural logarithm of area under the curve (ln

A) for the oxidation reaction of compound 3a

Assuming pseudo-unimolecular reaction, a first order kinetics plot can be drawn where y-axis is the natural log of the concentration and x-axis is time of the reaction.

ln[A] vs time 16.5 16 15.5 15 14.5 y = -0.0174x + 16.603 R² = 0.9964

ln [A} ln 14 13.5 13 12.5 12 0 50 100 150 200 time (minutes)

Figure 3.22: Plot of natural logarithm of area under the curve (concentration) against time of the

reaction in minutes

The curve with a R2 value of 0.9964 gives us slope of the linear line passing through all the data points. In first order kinetics, negative of the slope is the reaction constant ‘k’. In this case, we found the value of k = 0.0174 s-1.

According to first order kinetics,

ln 2 푡1 = ⁄2 k

Therefore, in case of hydrogen peroxide based oxidation of compound 3a,

0.693 푡1 = ⁄2 0.0174

And hence,

푡1 = 39.83 푚푖푛. ⁄2

Example 2: Hydrogen peroxide (H2O2) mediated oxidation of compound 3b using UV-vis spectroscopy.

2.877

2.000 Abs.

1.000

0.000

-0.298 250.00 300.00 350.00 400.00 450.00 500.00 nm.

Oxidation of compound 3b 1

0.9

0.8

0.7

0.6

0.5

0.4

Absorbance (mAU) 0.3

0.2

0.1

0 0 20 40 60 80 100 120 140 160 180 200 Time (minutes)

Figure 3.23: Oxidation of compound 3a in presence of hydrogen peroxide over time monitored by

UV-Vis spectroscopy. Top part of the figure shows no change in starting compound in presence of hydrogen peroxide even after 24 hours of incubation. There was no formation of any new compound observed. The fact is supported by the bottom plot where no degradation of the starting compound is observed over time. In fact, concentration of compound 3b was unchanged over time, indicating its stable nature in H2O2 environment.

In this experiment, compound 3b was introduced to 10 mM hydrogen peroxide for 24 hours and the reaction mixture was monitored constantly using UV-vis spectroscopy. Absorbance reading at

300 nm wavelength every 15 min was plotted against time. A linear line parallel to the x-axis indicated that compound 3b had not oxidized at all. Furthermore, there was no degradation of the compound observed showing that compound 3b is stable in H2O2 environment for up to 24 hours.

3.5 Data and results

3.5.1 Calculation for t1/2 values for compounds 3a-3e

Synthesized compounds 3a-3e were subjected to different forms of ROS and analysis of stability was performed based on two methods previously explained. For hydrogen peroxide reaction, protocol from reaction 2 was used. For superoxide anion mediated oxidation, reaction 3 protocol was administered. For hydroxyl radical, Fenton chemistry was applied according to reaction 4. All data points were then evaluated using Microsoft excel and based on the concentration vs time plots as previously shown, half-lives of the compounds were predicted in all the ROS environments.

- t1/2 in H2O2 Compound t1/2 in H2O2 t1/2 in O2• t1/2 in HO• t1/2 in HO •

3a 40 min N/A < 15 min < 2.66

3b ˃24 hours ˃24 hours ˃24 hours N/A

3c 40 min 28 min 15 min 2.66

3d 88 min 123 min < 15 min < 5.86

3e 160 min N/A 27 min 5.92

Table 3.3: Half-lives for the compounds 3a-3e in different ROS environments

3.5.2 Analysis of oxidation trends

3.5.2.1 Hydrogen peroxide (H2O2) oxidation

Compilation of the data revealed the trends in the oxidation profiles of the representative molecules

3a-3e. Since hydrogen peroxide is the weakest oxidant amongst all ROS, the molecules were relatively stable in this environment compared to others. This is validated by the higher half-lives of the compounds in 10 mM H2O2.

It should be noted that half-lives of compounds 3a, 3c and 3d were lower compared to 3b and 3e.

In the case of compound 3b, a methoxy group prevents the formation of the quinone-like intermediate after oxidation (scheme 3.1), preventing further oxidation. Hence compound 3b was found to be stable in all ROS environments. But among the rest of the compounds, compounds containing O-N pair on the core phenyl ring were easily oxidized by peroxide compared to compound 3e which consisted of a N-N pair (scheme 3.3). Hence, it can be concluded that

95 compounds with an oxygen and nitrogen pair on core ring have lower oxidation potential than a nitrogen-nitrogen pair.

- 3.5.2.2 Superoxide anion (O2• ) oxidation

Some experiments of superoxide anion oxidations were conducted by my colleagues, and are not included in my thesis. However, a similar trend was observed as peroxide oxidation in this experiment. Compound 3b was not oxidized at all in 1 day. Compounds 3a-3b were generally oxidized faster than peroxide reaction except for compound 3d. Further investigation needs to be conducted to understand this anomaly.

3.5.2.3 Hydroxyl radical (HO•) oxidation

It was observed that in hydroxyl radical (HO•) environment, all of the compounds except for 3b were rapidly oxidized. Oxidation of compounds 3a and 3d was so fast in hydroxyl radical environment, that correct values of their half-lives could not be calculated. Compound 3e was harder to oxidize compared to rest of the compounds with a half-life of 27 min in Fenton reaction conditions. It can be concluded that compounds 3d and 3e show more selectivity in reacting with hydroxyl radicals HO• than hydrogen peroxide (H2O2), with a ratio of half-lives ~ 6.

3.6 Discussion

With the help of my colleagues, it was proved that the oxidation product of compounds 3c, 3d and

3e is the same. The oxidation reaction under Fenton conditions follows the proposed mechanism

(scheme 3.1) and forms 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one (ox-P). The

96 reaction product spectra (HNMR and HRMS) exactly match the characterization spectra of synthesized compound 1-Methyl-5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one.

This overall reaction of oxidation of compounds 3a-3e under Fenton conditions to yield 1-Methyl-

5-amino-2,3-dihydro-1H-1,3-benzodiazol-2-one was found to be a 6-electron oxidation reaction.

In other words, one molecule of 3a-3e reacts with 3 hydroxyl radicals. Therefore, if the proposed scaffold is used as a hydroxyl radical scavenger, we have proved that a single molecule can scavenge 3 hydroxyl radicals. In the case of compounds 3d and 3e, this scavenging is relatively more selective towards hydroxyl radicals compared to hydrogen peroxide.

3.7 Conclusion

In this chapter, we were able to successfully design and synthesize a series of hydroxyl radical

(HO•) selective scavengers of ROS. Compounds we synthesized (3a-3e) undergo a self-cyclizing mechanism consuming 6 electrons to be oxidized in the presence of hydroxyl radical. These compounds show variation in selectivity of reactivity towards different kinds of ROS. It is due to the fact that compounds that are relatively easy to oxidize such as 3a, show less resistance in oxidation even in the presence of low potential oxidant hydrogen peroxide. Compounds 3d and 3e having higher oxidation potential, are difficult to oxidize by hydrogen peroxide. However, in presence of an oxidant such as hydroxyl radical with higher oxidation potential (2.40 V), they undergo oxidation easily to form a proposed oxidation product. Thus, compounds 3d and 3e show better selectivity in reactivity (~6 folds more) with hydroxyl radical over hydrogen peroxide. These non-toxic, long acting, self-cyclizing molecules could be used to inhibit highly reactive ROS such as hydroxyl radical and therefore can be used to limit oxidative stress.

Chapter 4: Synthesis of a di-pyridine derivative with in vivo activity against cancer models and its immobilization on agarose beads using click chemistry.

4.1 Introduction

With the increasing wealth in 3D structural information of biological targets, docking- or structure-based virtual screening is becoming the go to technique to identify small molecules that bind to a specific pocket on a given biomolecule target to induce a specific biological outcome. In such screen, a library of small molecules is docked computationally by exploring the conformational space of each tested compound in a docking program against a chosen pocket on a receptor’s surface. A scoring function, typically optimized to provide an approximation to the free-energy of binding over different classes of compounds, is used in conjunction with a conformational sampling algorithm to allow for compound ranking and docking pose prediction

63-65. Top compounds are then tested in 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.

We used virtual screening to identify small molecules that bind to the 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 termed RASopathies 66-69. We targeted the GTP- bound form of the G60A point mutant we previously described 31. We justify targeting the

RasG60A structure by virtual screening as follows. First, a cleft situated between the switch 1 and the triphosphate nucleotide large enough to accommodate a small molecule can be identified in this structure. These cleft results from the adoption of the switch 1 region in the GTP- but not

GDP-bound structure of this mutant of a conformation that is different from wild-type Ras but similar to nucleotide-free Ras in complex with guanine nucleotide exchange factor Sos 70 we term

‘the open conformation’ of Ras. Switch 1 corresponds to residues 24-40 and is responsible for

GTP-/Mg2+-coordination and binding to effectors and regulators. Second, this mutation severely

99 attenuates the binding of Ras to its effector Raf kinase in vitro 31 and reverts the transforming ability of constitutively active Ras in cells 71-73. Third and most importantly, it was previously shown using solution 31P-NMR spectroscopy that GTP-bound Ras adopts two conformations in equilibrium 74-76: one is capable of effector binding and therefore signaling, while the other is non- signaling and is mimicked by the G60A and T35S mutants 77-79. Taken together, we argue that a small molecule that keeps Ras in the open conformation would inhibit its signaling. A similar approach has previously led to the discovery of the anti-Ras ‘Kobe’ compounds 80.

Combined data from virtual screening and cell growth and colony formation assays identifies a small molecule with cellular activity against several transformed cell lines including

Ras-driven cells. Further, we show that our compound has in vivo activity against the patient- derived MDA-MB-231 triple negative breast cancer (TNBC) and H2122 non-small cell lung cancer cell lines xenograft mouse models.

4.2 Identification and characterization of NSC-124205

Before entering in vivo studies, we experimentally checked a sample of the compound NSC124205 obtained from NCI for its identity and purity. We tested a freshly made DMSO solution of

NSC124205 by high-performance liquid chromatography-mass spectroscopy (HPLC). Initial reverse-phase HPLC analysis (Figure 4.2-A) on the authentic sample found that the vial of

NSC124205 was impure with the presence of 3 bands eluting at 11.6 min, 12.8 min and 13.4 min

(Figure 4.2-A). Inspection of the UV profile of each peak indicated that peaks at 12.8 min and 13.4 min had a similar absorbance profile with maxima close to 250 nm and 300 nm. The mixture was analyzed by LCMS (Figure 4.2-B) using the same column and conditions. The first band at 11.6

+ min has an elemental composition of C28H23N12 which likely corresponds to two guanidinobenzimidazole additions. This is in line with the observation that its spectrum lacks red-

100 shifted absorbance maxima due to lack of the extended aromatic system. The other two bands at

+ 12.8 and 13.4 min have the same elemental composition, C20H14N7 . It was unclear if the two bands were tautomers since several can be drawn and they possessed a similar absorbance spectrum. Importantly, HRMS indicated that water and methanol additions were possible, though

NMR analysis confirmed the structure was as drawn. We therefore further explored the stability of NSC124205. (Figure 4.2-E). We synthesized NSC124205 according to scheme 1 and prepared

5 mM solution of synthesized NSC124205 with 10% DMSO in water and incubated it at 50°C and

90°C for 30 min. We found that only 40% of the compound survived at 50°C and all the compound degraded at 90°C. Furthermore, when the same solution was introduced to 10 mM H2O2 none of it survived the oxidative environment. Thus, it was observed that the compound NSC124205 is unstable at high temperatures and in presence of an oxidant. We guess that the sample received from NSC had 3 peaks, possibly because of storage of the compound for several days in DMSO solution; causing its degradation or in this case formation of a tautomer. (Figure 4.1)

Figure 4.1: Tautomerism in NSC-124205

101

Figure 4.2: (A) Method for HPLC-

Solvent A: 95% Water, 5% ACN,

0.5% Formic acid; Solvent B: 95%

ACN, 5% Water, 0.1% Formic acid;

Method: 1 mM compound, 20 μL

injection; 100% solvent A for 3 min

followed by gradient to 100%

solvent B over 20 min. (B) LCMS

of the sample. (C) HPLC trace of

the synthesized NSC124205. Same

method as (A) was used. (D) HRMS

of synthesized NSC124205 (E)

Stability studies of the compound

NSC124205

As previously mentioned, we synthesized NSC124205 according to Scheme 1.1. The synthesis is derived from analogous molecules in literature.81-82 We heated 2-Guanidinobenzimidazole and α- pyridoin in presence of acetic acid, which formed our target molecule after a double dehydration reaction. Low yield of the product formed may be because of its unstable nature at the reaction temperature. However, no reaction between starting compounds was observed at lower temperatures. HPLC analysis of the synthesized compound shows that a single band is observed at 12.8 min (Figure 4.2-C). This band corresponds to the fraction B observed in the sample provided by NSC. The UV-profile of the synthesized compound matches with the fraction B of

102 the provided sample (Figure 4.2-A). Additionally, the same water (m/z 370.14) and methanol (m/z

384.15) additions were found to occur during MS analysis (Figure 4.2-D). It follows the similar pattern observed in previous mass spec analysis. 1H-NMR analysis shows 12 protons in the aromatic region indicative of NSC124205. Analysis of the compound shows it has some favorable drug-like properties despite the large nitrogen content and aromatic character. We performed several calculations to predict the druggability of NSC124205. Our calculations show that the target molecule is non-planar, reasonably soluble in aqueous phase with a log P value of 3.25, has

1 hydrogen bond donor and 4 hydrogen bond acceptors. Because of these chemical properties,

NSC124205 follows the Lipinski’s rule of 5. We then examined synthesized NSC124205 in biological assays.

4.3 NSC124205 reduces tumor burden of Ras-driven solid tumors in vivo

This part of the experiment was conducted in collaboration with Dr. Nicolas Nassar at Cincinnati children’s hospital. To examine if the ability of NSC124205 to reduce oncogene-driven cell proliferation can be translated in vivo, we tested its efficacy on one breast cancer and one lung cancer mouse model. The breast cancer model utilizes the human triple negative breast cancer

MDA-MB-231 cells harboring the KRASG13D mutation. Cells were orthotopically injected into the right and left inguinal mammary glands of immunodeficient female mice. Tumor-bearing mice then received an intraperitoneal (IP) injection of 20 uL of 1 mM NSC124205 every other day. Four weeks post treatment a significant decrease of ≥ 50% of tumor volume compared to vehicle-treated control mice was observed (Figure 4.3A-4.3C). While vehicle treated tumors doubled in volume,

NSC124205-treated tumors increased by ~25% at the end of the 4-week treatment (Figure 4.3D).

Tumors were then excised, sliced, fixed, and stained for a proliferation marker (Ki-67), an apoptosis marker (cleaved Caspase-3), and with DAPI. Comparison of Ki-67 stained tumors

103 treated with vehicle control and with NSC124205 did not reveal a statistical change in cell proliferation. However, cleaved caspase-3 stained tumor sections showed significant increase in apoptosis for cells treated with NSC124205 compared to vehicle control.

A- RON-driven BC B- MDA-MB-321 C- H2122

D- Cleaved Caspase 3

DMSO 124205

Figure 4.3 In vivo survival potential of 124205: Mouse models of Breast and Lung Cancers.

4.4 Immobilization of NSC-124205 on agarose based fluorescent beads using click chemistry

After successfully synthesizing NSC-124205 and confirming that it is active in Ras-driven solid tumors, we decided to design an agarose bead based experiment to investigate the protein targets of the molecule in-vivo. In this experiment, an alkyne version of NSC-124205 was immobilized on agarose beads using copper-catalyzed click chemistry. These beads will be then subjected to a mixture of potential target proteins and based on the extent of binding between proteins and the

104 beads, potential biological targets of NSC-124205 will be determined. (Figure 4.4) Our role in this experiment is to design and synthesize agarose beads with NSC-124205 immobilized on it.

Figure 4.4: Design of agarose beads based experiment

4.5 Experimental

4.5.1 Materials

All chemicals, reagents and solvents were purchased from Sigma-Aldrich, Ark Pharm Inc., and

Fisher Scientific. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (RT) is noted as 25oC. Analytical thin layer chromatography (TLC) was performed with glass backed silica plates (5 x 20 cm, 60 Å, 250 μm).Visualization was accomplished using a

254 nm UV lamp. 1H- and 13C-NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer using solutions of samples in deuterated Chloroform, Methanol, or Dimethyl sulfoxide. Chemical shifts are reported in ppm with tetramethylsilane as standard. Data are reported as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, b = broad, m = multiplet). All novel compounds were characterized by 1H-NMR, 13C-NMR and high resolution mass spectroscopy (HRMS). Previously

105 synthesized compounds were identified by comparison of their 1H -NMR and 13C-NMR to published data.

4.5.2 Synthesis of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine (NSC-

124205)

Scheme 1.1: Synthesis of 2-guanidinobenzimidazole (1) and NSC-124205 (2).

2-Guanidinobenzimidazole (1)

2-Guanidinobenzimidazole was prepared by the condensation reaction between o- phenylenediamine and cyanoguanidine.81 A mixture of o-phenylenediamine (1 mmol), cyanoguanidine (1 mmol) and concentrated HCl (2 mmol) in water (50 mL) was heated to reflux for 3 h, cooled to 0oC and KOH (10% 15 mL) was added slowly. Precipitated 2- guanidinobenzimadole was filtered, washed with water and recrystallized in ethanol to yield pure product. (%yield: 64%)

1H NMR (400 MHz): δ = 6.92 (m, 2H), 7.16 (s, 2H), 11.06 (s, 1H)

13C NMR (400 MHz): δ = 119.41, 158.82, 159.10

106

N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine (NSC 124205) (2)

A mixture of 2-guanidinobenzimidazole (1 mmol), α-pyridoin (5 mmol) and glacial acetic acid (2 mmol) was heated to 90oC for 8 h. The mixture was then cooled, dissolved in ethyl acetate (30 mL), washed with sat NaHCO3 solution (30 mL) and water (2 x 30 mL). The organic layer was then evaporated and subjected to flash column chromatography. Compound 124205 is with Rf value 0.5 in 10% Methanol in DCM. (%yield: 14 %)

1H NMR (400 MHz): δ = 7.05 (m, 2H), 7.33 (m, 2H), 7.49 (m, 2H), 7.61 (m, 1H), 7.86 (m, 1H),

8.04 (m, 1H), 8.16 (m, 1H), 8.31 (s, 1H), 8.53 (m, 1H).

13C NMR (400 MHz): δ = 113.89, 122.04, 123.16, 123.32, 124.17, 124.74, 124.94, 126.42, 129.53,

137.93, 138.93, 148.65, 148.94, 149.81, 150.36, 150.60, 152.89, 156.40, 157.79, 161.23.

HRMS: Expected: m/z 352.1305 (C20H14N7) Observed: 352.1297 (C20H14N7)

107

Figure 4.5: 1HNMR of 2-Guanidinobenzimidazole (1) Solvent: DMSO-d6

13 Figure 4.6: CNMR of 2-Guanidinobenzimidazole (1) Solvent: DMSO-d6

108

Figure 4.7: 1HNMR of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

(NSC 124205) Solvent: Methanol-d4

Figure 4.8: 13CNMR of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

109

(NSC 124205) Solvent: Methanol-d4

Figure 4.9: HRMS of N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

(NSC 124205) Solvent: Methanol-d4

110

Figure 4.10: HPLC trace of Synthesized N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-

2-imine (NSC 124205) Solvent: Methanol-d4

111

4.5.3 Synthesis of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

Scheme 1.2: Synthesis of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-

imine (3)

Step 1: Synthesis of compound I1

A 50 ml round bottom flask was purged with Argon. 20 ml anhydrous THF was added to it followed by addition of 2g (1 eq) of 4-Iodo-2-nitroaniline. After the nitroaniline is completely dissolved, 58.5 mg (0.011 eq) of PdCl2(PPh3)2, 36 mg (0.025 eq) of CuI and 4 ml (3 eq) of DIPEA was added to it. Reaction was stirred for 15 min followed by addition of 1.15 ml (1.1 eq) of TMS acetylene. Resulting reaction mixture was then stirred at room temperature for 3 h. After the completion of reaction monitored by TLC, 30 ml water was added and resulting solution was extracted with ethyl acetate (3 x 50 ml). Organic layer was washed with (2 x 25 mL) water, 20 mL

112 brine, dried over sodium sulfate and concentrated on rotavapor to yield crude compound I1. It was the purified using silica gel chromatography. (Hexane:Ethyl acetate / 10:1).

1 H NMR (CDCl3, 400 MHz): δ 0.21 (s, 9H), 6.32 (broad s, 2H), 6.75 (d, 1H), 7.35 (d, 1H), 8.21

(s, 1H).

1 Figure 4.11: HNMR of compound I1

Step 2: Synthesis of Compound 1

In a 50 ml round bottom flask, 250 mg (1 eq) of compound I1 was dissolved in a mixture of 20 ml water and 10 ml methanol. 2.7 g (10 eq) of Na2S2O4 and 2.1 g (10 eq) of K2CO3 was added to it.

The resulting mixture was for 2 h. Upon completion of the reaction monitored by TLC, reaction

113 mixture was extracted using ethyl acetate (3 x 20 ml). The organic layer was evaporated in vaccuo and crude product obtained was used without purification for the next step because of its unstable nature.

Step 3: Synthesis of compound 2

A mixture of Compound 1 (1 eq), cyanoguanidine (1 eq) and conc HCl (2 eq) in water (50 mL) was heated to reflux for 3 h, cooled to 0oC and KOH (10% solution, 15 mL) was added slowly.

Precipitated solid containing mixture of compound 2 and TMS attached compound 2 was filtered and washed with 2 x 10 ml of water. Obtained solids were used in the next reaction without any purification. It was assumed that TMS will not survive harsh conditions of next reaction and thus purification was delayed until next reaction.

Step 4: Synthesis of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-imine

(compound 3)

A mixture of compound 2 (1 mmol), α-pyridoin (5 mmol) and glacial acetic acid (2 mmol) was heated to 90oC for 8 h. The mixture was then cooled, dissolved in ethyl acetate (30 mL), washed with sat NaHCO3 solution (30 mL) and water (2 x 30 mL). The organic layer was then evaporated and subjected to flash column chromatography. Compound 3 is with Rf value 0.6 in 10% Methanol in DCM.

1H NMR (400 MHz): δ = 4.11 (s, 1H), 7.05 (m, 1H), 7.33 (m, 3H), 7.69 (m, 2H), 7.81 (m, 1H),

8.01 (m, 3H), 8.53 (m, 1H).

MS: Expected: m/z 393.7(C22H13N7) Observed: 393.7 (C22H13N7)

114

Figure 4.12: 1HNMR of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-2-

imine

Intens. +MS, 0.1-2.0min #(14-154) x108 393.7

2.0 437.6

349.7 1.5 481.6

1.0 304.2 525.5

0.5 569.5 257.4 613.5 685.6 0.0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Intens. +MS2(394.0), 2.0min #152 x107 393.7 8

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 4.13: Mass spectrum of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2-ylimidazol-

2-imine

115

4.6 Immobilization of 6-Ethynyl-N-(1H-benzimidazol-2-yl)-4,5-dipyridin-2- ylimidazol-2-imine on agarose based microspheres

4.6.1 Idea of the experiment

As mentioned previously, the goal of this experiment was to immobilize 124205 on agarose beads using click chemistry. However, to monitor the concentration of the active sites clicked on beads, a negative control was necessary. We designed a clickable fluorescent probe which when clicked to the beads made them fluorescent. That was a classic negative control which we utilized to determine approximate number of clicked 124205 sites.

4.6.2 Design and synthesis of the negative control

We purchased agarose beads with the concentration of 0.55 µmol azide groups. A fluorescein derivative with alkyne group attached to the fluorescent core via alkyl linker was synthesized.

Using copper based click chemistry, several experiments were conducted with different molar ratios of azide beads and alkyne-fluorescein. (Figure 4. 14)

Figure 4.14: Design of a click chemistry based experiment between azide beads and alkyne

linked fluorescein molecule.

116

As the ratio of alkyne-fluorescein compared to azide sites increased in the reaction mixture, it was observed that fluorescence of the clicked beads increased in a non-linear way. This mean fluorescence was calculated using fluorescence microscopy (table 4.1)

Eq of azide beads Eq of alkyne-fluorescein Mean fluorescence of clicked beads

1 0 0

1 0.5 2.36

1 1.5 7.72

1 3 14.40

1 8 24.64

Table 4.1: Mean fluorescence of clicked beads based on equivalents of alkyne-fluorescein used

R1: 0 eq R2: 0.5 eq

R3: 1.5 eq R4: 3 eq R5: 8 eq

Figure 4.15: Fluorescence microscope images indicate that as the equivalents of alkyne-

fluorescein increase, the mean fluorescence of the clicked beads increases.

117

These different molar ratios provided vital information about saturation point of fluorescence. This indirectly indicated the amount of azide sites clicked when excess of fluorescein was used. Based on this graph, it was now possible to predict the extent of click reaction at any given fluorescence.

(Figure 4.16)

Fluorescent beads 30

10 Integrated density Integrated mean density

0 0 1 2 3 4 5 6 7 8 9 Eq of Fluorescein-alkyne used

Figure 4.16: Plot of mean fluorescence vs Equivalents of alkyne-fluorescein used can be used to

predict concentration of the fluorescent sites at any given fluorescence value.

Based on Figure 4.16, it can be predicted that if 0.1 eq Fluorescein-alkyne concentration is introduced to the reaction, 0.0122 µmol of the azide sites out of 0.55 µmol will get clicked to alkyne. With this useful information we planned the next experiment.

4.6.3 Immobilization of target molecule on azide beads

After establishing a negative control in the form of alkyne-fluorescein clicked beads, we decided to click alkyne-124205 on the agarose beads. Alkyne-fluorescein and alkyne-124205 both were

118 reacted with azide agarose beads at the same time under click conditions. (Figure 4.17)

Figure 4.17: Immobilization of alkyne-124205 on agarose beads

Different alkynes when present in the same click reaction environment compete to react with azides. The ratio of the clicked products is proportional to the ratio of alkynes used. Thus, decrease in fluorescence was assumed to provide indirect concentration of 124205 immobilized beads. For instance, if fluorescence drops by 50% it could be assumed that half the sites on the beads are occupied by 124205.

Reaction number Eq. of azide beads Eq. of alkyne-fluorescein Eq. of alkyne-124205

1 1 0.1 0

2 1 0.1 0.5

3 1 0.1 1

4 1 0.1 2

5 1 0.1 3

Table 4.2: Ratios of alkyne-fluorescein and alkyne-124205 used in different reactions

119

In reaction 1, none of the alkyne-124205 was present to compete with alkyne-fluorescein under clicked conditions. Hence, it can be safely assumed that all the alkyne-fluorescein was clicked on azide beads. This provided the base fluorescence where 0.0122 µmol of the azide sites out of 0.55

µmol was clicked to fluorescein-alkyne.

In subsequent reactions, concentration of alkyne-124205 was increased. As a result, there was more competition for alkyne-fluorescein to be clicked on the beads. This resulted in decrease of fluorescence as the reaction sequence proceeded. (Figure 4.18)

Reaction 1 Reaction 2 Reaction 3 Reaction 4 Reaction 5

Transmittance

Fluorescence

Figure 4.18: As the concentration of alkyne-124205 increases (reaction 1 to reaction 5),

fluorescence of the agarose beads decreases.

Constant decrease in fluorescence was plotted against equivalents of alkyne-124205 used. It was noticed that, at 0 eq of 124205-alkyne, all the 0.1 eq fluorescein-alkynes are clicked resulting in

100% possible Fluorescence. This drops to 3.38% when 3 eq 124205-alkyne (30 times the fluorescent alkyne) is used. For every molecule of Fluorescein-alkyne, 30 molecules of 124205- alkynes are competing to be clicked. (Figure 4.19)

120

20 Integrated density Integrated density mean 10

0 0 0.5 1 2 3 Eq of 124205-alkyne used

Figure 4.19: Mean fluorescence decreases as concentration of alkyne-124205 increases in the

reaction environment.

4.6.4 Calculation of the concentration of 124205-clicked sites on agarose beads

Assuming the yield of click reaction between azide agarose beads and alkyne to be 100% we calculated approximate number of 124205 clicked sites.

Reaction Eq. of alkyne-124205 used Number of Number of 124205

number per 0.1 eq alkyne- fluorescein clicked clicked sites (µmol)

fluorescein sites (µmol)

1 0 0.0122 0

2 0.5 0.002469 0.2725

3 1 0.00087 0.5491

4 2 0.000676 0.5493

5 3 0.000413 0.5496

Table 4.3: Calculation of number of 124205 clicked sites in different reactions

121

As it can be observed from table 4.3, after 1 eq of alkyne -124205 was used, the fluorescence curve showed a saturation. This is the indication of high efficiency in the clicked reaction. Now these number of clicked sites can be used to abstract target proteins from either cell matrix or from mixture of target proteins. This experiment will be designed in the future.

4.7 Synthesis of Fluorescent and 124205 clicked beads

4.7.1 Synthesis of alkyne linked fluorescein

As shown in figure 4.20, alkyne linked fluorescein was synthesized by coupling 1-Amino-3-butyne with 5(6)-carboxyfluorescein in presence of HATU and DIPEA.

Figure 4.20: Synthesis of alkyne-fluorescein

General method: In a 10 ml round bottom flask, 100 mg 5(6)-carboxyfluorescein was dissolved in

2 ml DMF. 121 mg of HATU and 111 µL of DIPEA were added to it and the resulting mixture was stirred at room temperature for 30 min. 52 µL of 1-Amino-3-butyne was then added to it and the reaction mixture was stirred overnight. It was then dissolved in 15 mL ethyl acetate, washed with 4 x 15 mL water, 10 mL brine and dried over sodium sulfate. After concentration the organic layer on rotavapor, crude mixture was obtained which was purified with silica gel chromatography

(Dichloromethane:Methanol / 10:1).

122

1 H NMR (CDCl3, 400 MHz): δ 2.84 (m, 2H), 3.16 (s, 1H), 3.36 (m, 2H), 6.48 (m, 2H), 7.53 (m,

13 3H), 8.58 (m, 2H), 8.84 (m, 2H), 13.81 (broad s, 1H). C NMR (CDCl3, 400 MHz): δ 19.95, 30.96,

40.45, 71.15, 82.35, 103.80, 111.18, 114.05, 124.29, 125.16, 125.67, 126.04, 130.42, 135.61,

- - 137.98, 142.34, 154.37, 191.79, 161.84, 168.64, 170.67, 173.18. HRMS (ESI ) for C25H16NO6 :

Calculated: 426.0983. Observed: 426.0976

Figure 4.21: 13CNMR of alkyne linked fluorescein

123

Figure 4.22: HRMS of alkyne-linked fluorescein

4.7.2 Click reaction general procedure

Stock solutions (50 mM) of the following reagents were prepared: 2,6- lutidine (DMF), 2,2’ bipyridine (DMF), CuBr (DMF), and sodium ascorbate (water). A slurry of agarose-azide/ alkyne beads (1.0 equiv) in DMF in a disposable frit was treated with the molecule to be attached bearing the complementary (alkyne or azide) group (4.0 equiv), followed by 2,6-lutidine (8.0 equiv), 2,2¢- bipyridine (8.0 equiv), cuprous bromide (4.0 equiv), and finally sodium ascorbate (8.0 equiv). The resulting suspension was bubbled with a gentle flow of argon for 1 min, capped, and rotated at room temperature for 12-18 h. The reaction mixture was drained and washed sequentially in a filter frit with approximately 5 column volumes each of DMF, H2O, MeOH, 0.1 M aq EDTA, H2O, and

DMF. (Figure 4.23)

124

Beads

Filtrate

Figure 4.23: Filter frits after water workup. Fluorescent beads are on the top and clear filtrate is

at the bottom.

4.7.3 Storage of synthesized clicked beads

After the workup explained in the previous section, the beads are suspended in 80:20/

Water:ethanol solution and stored at 2-8ºC in an air tight vial. The suspension is to be shaken before each use.

4.8 Conclusion

From the crude mixture provided to us by NCI containing 3 different compounds, we have identified a di-pyridine derivative small molecule with in vitro and in vivo activity against models of Ras-driven cancers. We were able to successfully synthesize and purify the active molecule

NSC-124205. We have optimized the synthetic conditions which are suitable for large scale synthesis of compound NSC-124205. Furthermore, we immobilized the active molecule on

125 agarose microspheres using click chemistry. We also determined the approximate concentration of 124205-clicked sites on these beads which will help us identify the protein targets on this molecule in vivo. Experiments to identify the mechanism of action of NSC124205 using synthesized agarose beads and other targeted approaches are under way.

126

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