Based HDAC-Inhibitors As Anti-Cancer Agents

A Thesis

entitled

Guanidine- Based HDAC-Inhibitors as Anti-Cancer Agents

Shaimaa Hesham Sindi

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Medicinal Chemistry

______L.M. VIRANGA TILLEKERATNE, D.Phil, Committee Chair

______JAMES T. SLAMA, Ph.D, Committee Member

______KATHERINE A. WALL, Ph.D., Committee Member

______ZAHOOR SHAH, Ph.D, Committee Member

______Dr. Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo

August 2019

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

An Abstract of

Guanidine- Based HDAC-Inhibitors as Anti-Cancer Agents

Shaimaa Hesham Sindi

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Masters of Science Degree in Medicinal Chemistry

The University of Toledo August 2019

Abstract.

It is evident that epigenetics plays an important role in gene regulation.

Consequently, any disruption in epigenetic memory will manifest into gene expression and lead to long-established transformation events. Covalent modification of histone is one of the epigenetic regulation mechanisms, and includes acetylation, methylation, phosphorylation, and ubiquitylation among others. Combination of two or more of these modifications acts like a sensor for gene expression or gene repression through highly condensed or uncondensed chromatin structures. The cancer epigenome is specifically marked with global DNA methylation and histone alteration patterns. The gene regulation is controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation through HATs results in expansion of chromatin, and thus increases the accessibility of transcription factors to DNA. On the other hand, the deacetylation with HDACs leads to very condensed chromatin structures and consequently, to transcription repression. Owing to their specificity for cancer cells, HDAC inhibitors have now become a powerful target in anticancer drug development. The pharmacophore

iii of HDAC inhibitors consists of a zinc-binding group, a linker and a capping group. These drugs exert their effect on cancer cells by arresting them in G1 or G2-M phases of the cell cycle, or by induction of differentiation and apoptosis. They can also inhibit angiogenesis and metastasis and regulate the host immune response.

Generally, HDACs can be classified into four different classes (class I, II, III, and

IV). It is well established that most HDAC inhibitors in clinical use lack specificity. It is believed that variations in the nature of the capping group can lead to variations in HDAC inhibitory activity and isoform selectivity. Our group has successfully developed a new class of HDAC inhibitors incorporating an imidazole ketone moiety as the capping group.

Cytotoxicity studies of these molecules have shown promising activity. Efforts are underway in our laboratory to modulate the activity and selectivity of these compounds by changing the capping group.

Guanidines have been reported to possess a wide range of biological activity. The biochemical and biophysical properties of guanidine are partly attributed to their specific hydrogen bonding, Y aromaticity, and stacking properties. A molecule with such properties is able to bind to carboxylates, phosphates and metals. In this study, several

HDAC inhibitors with imidazole ketone metal-binding group and guanidine moieties as the capping group were designed for synthesis and methods for making versatile cyclic guanidine molecules to be used in the synthesis of HDAC inhibitors were developed. In addition, two HDAC inhibitors incorporating guanidine moieties were synthesized. One of the analogues showed potent cytotoxic properties in the NCI 60 cell line assay with a mean growth inhibition of 64.5% and GI50 values in the low micro molar range against some cell lines. The cytotoxic activity of the compound was confirmed in studies

performed in the laboratory of Dr. William Taylor of the biological Sciences

Department. The other compound did not show activity in studies carried out in Dr.

Taylor’s lab and is yet to be tested at NCI for its cytotoxic activity. The incorporation of other guanidine moieties in to target molecules proved problematic, probably due to poor nucleophilicity of their amine groups. However, synthesis of these target molecules using different reaction conditions and changing synthetic design is currently in progress.

In memory of

Hesham Bakour Sindi & Asmaa Yehya Aladdin

With all love to Eman Sindi, Hani Sindi, Bakour Sindi, Elaf Sindi, Mohammed

Abdulghani Sindi, and Ghena Mohammed Sindi and Endri Karaj

Acknowledgements

I would like to express my deepest gratitude for my supervisor, Dr. Tillekeratne, without whom my scientific career would have never taken off. Dr. Tillekeratne not only took a chance on me when no one else would, but he honed my skills in the lab and helped me identify my true passion. He gave me his time, guided me and helped me obtain a well- rounded training during the course of these two years. I am fortunate for receiving my training from such a revered scientist so early in my career. I would like to thank my committee members for the support and guidance of Dr. Slama, Dr. Shah, and Dr. Wall. I am so honored to work with person whose life is all about chemistry, his passion about chemistry push me to explore the same feeling about it. To be very grateful, this work is collaboration between Endri Karaj, and me, and it would not have been done without his effort. I would like to thank my friend who is family to me Endri Karaj. I would like to thank my colleagues, for the lab guidance, and support. I would never find better chance than educational platform to thank my sister Eman Sindi appropriately. After my parents passed away, she was emotionally and financially support for us, despite that she is on the same age of me, she gave up many things in her life for us, she is equal to life for me, and without her, I will not be writing these words. I would like to thank Hani Sindi, Bakour

Sindi, Elaf Sindi, and Mohammed Abdulghani Sindi, Ghena Mohammed Sindi, my family for all support and love.

vii

Table of Contents

Abstract ...... iii Acknowledgements...... vii Table of Contents ...... viii List of Tables ...... ix List of Figures ...... x List of Schemes ...... xi List of Spectra ...... xiii List of Abbreviations and Definitions ...... xv Introduction...... 1 Epigenetics in cancer ...... 1 Chromatin structure and histone post- translational modifications ...... 2 Impact of histone acetylation and deacetylation on cancer ...... 5 Isoforms of histone deacetylases ...... 6 The molecular mechanism of HDAC inhibitors ...... 8 Direct effect on gene regulation ...... 9 Effects of HDAC inhibitors on non-histone proteins ...... 13 Guanidines in HDAC inhibitors ...... 15 Results and Discussion ...... 22 Chemistry ...... 24 Biological studies ...... 44 Conclusion and future directions ...... 49 Experimental and Materials ...... 50 Experimental ...... 51 Spectroscopic data ...... 71 References ...... 107

viii

List of Tables

1.1 Table 1: Histone post-translation modification ...... 4

List of Figures

Figure 1: Composition of nucleosome ...... 3

Figure 2: Global post-translation modification in primary tumor ...... 5

Figure 3: Histone deacetylase isoforms ...... 7

Figure 4: (a) Chemical decomposition of guanine. (b) examples of Superbases ...... 15

Figure 5: (a) Conjugate acid of guanidine stabilized by resonance. (b) proton sponge ...16

Figure 6: Guanidine bonding molecular orbitals and pi-stacking interactions ...... 17

Figure 7: HDAC inhibitor with guanidine as zinc-binding group ...... 18

Figure 8: HDAC inhibitors with guanidine as capping group ...... 19

Figure 9: HDAC pharmacophore ...... 21

Figure 10: Catalytic site ...... 21

Figure 11: HDAC inhibitors with imidazole ketone as metal-binding group ...... 22

Figure 12: The target molecules ...... 23

Figure 13: Relation between absorption and wavelength for compound (65) ...... 33

Figure 14: Relationship between max and equivalents of zinc ...... 33

Figure 15: Guanidine di-ketone condensation ...... 35

Figure 16: Analogue (15) ...... 38

Figure 17: Transition state of HATU coupling ...... 40

Figure 18: Results of NCI 60 cell line assay at 10 M ...... 46

Figure 19: Five dose response graphs of compound (15)...... 47

Figure 20: Growth inhibition of different cancer cell lines by compound (15) ...... 48

List of Schemes

Scheme 1: Retrosynthetic approach to HDAC inhibitors with guanidine-based capping groups...... 24

Scheme 2: Retrosynthesis of Guanidine capping group…………………………………………………….25

Scheme 3: Zinc binding group, and linker synthesis ...... 26

Scheme 4: Synthesis of 4,5-Diphenyl-1H-imidazol-2-amine (47) ...... 27

Scheme 5: Grignard reactions ...... 27

Scheme 6: Synthesis of 5-Phenyl-1H-imidazol-2-amine (53) ...... 29

Scheme 7: Synthesis of 5-(3-Nitrophenyl)-1H-imidazol-2-amine (56-b)...... 30

Scheme 8: Synthesis of 5-(2,3-Dimethoxyphenyl)-1H-imidazol-2-amine (62) ...... 30

Scheme 9: Synthesis of 1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66) ...... 31

Scheme 10: Synthesis of 1H-Phenanthro[9,10-d]imidazol-2-amine (68) ...... 34

Scheme 11: Alternative synthesis of 1H-phenanthro[9,10-d]imidazol-2-amine

(68)…………...... 36

Scheme 12: Synthesis of 1H-imidazo[4,5-f][1,10]phenanthrolin-2-amine

(87)………………… ...... 37

Scheme 13: 4-Methyl-5-phenyl-1H-imidazol-2-amine (91) ...... 37

Scheme 14: EDC coupling of amino-benzoimidazole (92) ...... 38

Scheme 15: Screening of coupling conditions with benzoic acid (94) ...... 39

Scheme 16. Amide coupling before the addition of zinc-binding group.……………….41

Scheme 17. Successful coupling and isolation of target molecule (15) ...... 42

Scheme 18. Successful coupling and isolation of target molecule (16) ...... 42

Scheme 19. Unsuccessful coupling of acid (28) with different guanidines ...... 43

xii

List of Spectra

1-Methyl 4-formylbenzoate (30) ...... 72

2-Methyl (E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoate

(29) ...... 73

3-(E)-4-(3-(1-Methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoic acid

(28)...... 74

4-1,2-Diphenylethan-1-ol (43) ...... 75

5-1,2-Diphenylethan-1-one (44) ...... 76

6-2-Bromo-1,2-diphenylethan-1-one (45) ...... 77

7-N-(4,5-diphenyl-1H-imidazol-2-yl)acetamide (46) ...... 78

8-4,5-Diphenyl-1H-imidazol-2-amine (47) ...... 79

9-1-Phenylethan-1-ol (49) ...... 80

10-Acetophenone (50) ...... 81

11-2-Bromo-1-phenylethan-1-one (51) ...... 82

12-N-(5-phenyl-1H-imidazol-2-yl)acetamide (52) ...... 83

13-5-Phenyl-1H-imidazol-2-amine (53) ...... 84

14-2-Bromo-1-(3-nitrophenyl)ethan-1-one (55) ...... 85

15-N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)acetamide (56-a) ...... 86

16-5-(3-Nitrophenyl)-1H-imidazol-2-amine (56-b) ...... 87

17-1-(2,3-Dimethoxyphenyl)ethan-1-ol (58) ...... 88

xiii

18-1-(2,3-Dimethoxyphenyl)ethan-1-one (59) ...... 89

19-2-Bromo-1-(2,3-dimethoxyphenyl)ethan-1-one (60) ...... 90

20-N-(5-(2,3-dimethoxyphenyl)-1H-imidazol-2-yl)acetamide (61) ...... 91

21-5-(2,3-Dimethoxyphenyl)-1H-imidazol-2-amine (62) ...... 92

22-1-(1-Methyl-1H-imidazol-2-yl)ethan-1-one (32) ...... 93

23-2-Bromo-1-(1-methyl-1H-imidazol-2-yl)ethan-1-one (64) ...... 94

24-N-(1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-yl)acetamide (65) ...... 95

25-1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66) ...... 96

26-1H-Phenanthro[9,10-d]imidazol-2-amine room temperature synthesis (68) ..97

27-1H-Phenanthro[9,10-d]imidazole (78) ...... 98

28-1-Benzyl-1H-phenanthro[9,10-d]imidazole (79) ...... 99

29-2-Azido-1-benzyl-1H-phenanthro[9,10-d]imidazole (80) ...... 100

30- 1,10-Phenanthroline-5,6-dione (83-b) ...... 101

31-1H-imidazo[4,5-f][1,10]phenanthroline (84) ...... 102

32-N-(4-methyl-5-phenyl-1H-imidazol-2-yl)acetamide (90) ...... 103

33-4-Methyl-5-phenyl-1H-imidazol-2-amine (91) ...... 104

34-(E)-N-(1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3- oxoprop-1-en-1-yl)benzamide (15) ...... 105

35-(E)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2- yl)-3 oxoprop-1-en-1-yl)benzamide (16) ...... 106

xiv

List of Abbreviations and Definitions

ABHA Azelaic bishydroxamic acid AcOH Acetic acid Akt Protein kinase B- anti-apoptotic protein AML Acute myeloid leukemia AML1–ETO Fusion protein promotes the expansion of human hematopoietic stem cells. BCR–ABL The breakpoint cluster region protein (BCR bFGF Basic fibroblast growth factor BH3 BH3-only proteins are proapoptotic members of the broader Bcl-2family, which promote cell death by directly or indirectly activating Bax and Bak. Bid The BH3 interacting-domain death agonist, or BID, gene is a pro- apoptotic member of the Bcl-2 protein family. Bik Bcl-2-interacting killer is a protein that in humans is encoded by the BIK gene, it is the founding member of the BH3-only family pro-apoptotic proteins Bim Bcl-2-like protein 11, is a protein that in humans is encoded by the BCL2L11 gene, act as anti- or pro-apoptotic regulators Bmf Bcl-2-modifying factor is a protein that in humans is encoded by the BMF gene, act as anti- or pro-apoptotic regulators bp base pair c-MYC a family of regulator genes and proto-oncogenes that code for transcription factors. c-Raf RAF proto-oncogene serine/threonine-protein kinase, CD40 Cluster of differentiation 40, CD40 is a costimulatory protein found on antigen presenting cells and is required for their activation. The binding of CD154 (CD40L) on TH cells to CD40activates antigen presenting cells and induces a variety of downstream effects. CD8 T Cluster of differentiation 8, is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. CD80 A costimulatory molecule known for its role in T-cell activation and also in regulating normal and malignant B cells activity CD86 Cluster of Differentiation 86 is a protein expressed on antigen-presenting cells that provides costimulatory signals necessary for T cell activation and survival. CDK2 Cyclin-dependent kinase 2 CDK4 Cyclin-dependent kinase 4 CDKN1A Cyclin Dependent Kinase Inhibitor 1A CREBBP CREB-binding protein CTP cytidine triphosphate xv

CXCR4 C-X-C chemokine receptor type 4 (CXCR-4)  Heat DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N, N'-Dicyclohexylcarbodiimide DCM Dichloromethane DLBCLs Diffuse B-cell lymphomas DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMP Dess–Martin periodinane DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide eNOS Endothelial nitric oxise synthase equiv Equivalent Et3N Triethylamine EtOAC Ethyl acetate EtOH Ethanol H1 Histone 1 H2A Histone 2 A H2B Histone 2 B H3 Histone 3 H3K18 Histone 3 lysine 18 H4 Histone 4 H4K16 Histone 4 lysine 16 H4K16Ac Histone 4 acetylated lysine 16 H4K20 Histone 4 lysine 20 H4K20me3 Histone 4 trimethylated lysine 20 HATs Histone acetyltransferases HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate HBTU 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate HDACS Histone deacetylases HIF1α Hypoxia-inducible factor 1-alpha HOBt Hydroxybenzotriazole HRMS High-resolution mass spectrometry Hsp70 Heat shock protein 70 Hsp90 Heat shock protein 90 is a chaperone protein that assists other proteins to fold properly, stabilizes proteins against heat stress, and aids in protein degradation. IAPs Inhibitors of apoptosis proteins, are a family of functionally and structurally related proteins that serve as endogenous inhibitors of programmed cell death (apoptosis). ICAM1 Intercellular Adhesion Molecule 1 IL-1 Interleukin-1 xvi

INF Υ Interferon gamma K-ac Lysine acetylation K-me1 Lysine mono methylated K-me2 Lysine di methylated K-me3 Lysine tri methylated K-su Lysine Sumoylation K-ub Lysine-Ubiquitylation LRMS Low resolution mass spectrometry MeCN Acetonitrile MeI Methyl iodide MeOH Methanol MHC Major histocompatibility complex MMP2 Matrix Metallopeptidase 2 MMP9 Matrix Metallopeptidase 9) is a Protein Coding gene. MMPs Matrix metalloproteinases W Microwave n-BuLi n-Butyllithium NAD+ Nicotinamide adenine dinucleotide NBS N-Bromosuccinimide NCOA2 nuclear receptor coactivator 2 NMM N-Methylmorpholine NMR Nuclear magnetic resonance p-TsOH p-Toluenesulfonic acid PP1 Protein Phosphatase 1 Puma p53 upregulated modulator of apoptosis R > Cit Arginine deamination R-me Arginine methylation r.t Room temperature Rf Retention factor RNA Ribonucleic acid ROS Reactive oxygen species S-ph Serine phosphorylation SAHA Suberoylanilide hydroxamic acid SAR Structure–activity relationship siRNA Small interfering RNA. SMAC Second mitochondria-derived activator of caspase STAT1 Signal transducer and activator of transcription 1 STAT3 Signal transducer and activator of transcription 3. T-ph Threonine phosphorylation TDAC Tubulin deacetylase THF Tetrahydrofuran TLC Thin-layer chromatography TNF α Tumor necrosis factor a TNFR The tumor necrosis factor receptor

xvii

TRADD Tumor necrosis factor receptor type 1-associated DEATH domain protein is a protein that in humans is encoded by the TRADD gene. TRAIL TNF-related apoptosis-inducing ligand VEGF Vascular endothelial growth factor

xviii

Chapter 1

Introduction.

Epigenetics in cancer.

Mutations in certain genes can lead to uncontrolled proliferation of cells leading to cancer, which remains one of the leading causes of death in the USA. Apart from such direct mutations, epigenetics that control gene expression without a change in DNA sequence can lead to development of abnormal conditions like cancer. Epigenetics used to refer to the interactions of genes with their environment that results in certain phenotypic changes or changes in the genotype that did not lead to any phenotypic changes1. However, a more recent definition of epigenetics refers to the heritable changes in gene expression without altering DNA sequence2. There are many gene regulation mechanisms that contribute to compactly controlled gene expression. Generally, the reciprocity between all gene regulation processes shape the epigenome. The level of epigenetic regulations decides how the genome is designed across different cell types throughout the development of the eukaryotic species. Depending on the manifestation of the genomic distribution, cancer and other diseases might arise upon diversion3.

Changes in epigenetic machineries due to mutations (deletion or the distorted expression of any of their components) are known to incite aberrant gene expression patterns, that in turn can lead to long-established transformation events. Such 1

classical preliminary events include, but are not limited to mutations in tumor suppressors, proto-oncogenes, or both, and genomic instability in general4. There are many different ways of epigenetic gene expression control which work on different cell components like

RNA, prions, nucleosome positioning etc., but our focus will be on covalent modifications which include modifications either of DNA or histone proteins leading to chromatin remodeling.

Chromatin structure and histone post- translational modifications.

Histones are the main proteins in the chromatin construction. The organization of eukaryotic chromatin structure under the electron microscope shows that 147 bp DNA stands are associated with small globule shaped histone proteins. DNA and histones together form the nucleosome, the basic unit of chromatin. They appear like small beads on a string (Figure 1). The DNA double helix is wrapped approximately 1.7 times around each nucleosome, which consists of a core (yellow) octamer of two copies each of four different histone molecules H2A, H2B, H3 and H4 often associated with an additional histone H1 (green) located outside. X-ray crystallography shows that the core is disc- shaped and the N-terminal (green) of each of the four histones extends outside the core.

Histone H1 and other non-histone proteins link and interact with the nucleosomes to pack the nucleosomes to form a complex order of chromatin structures (Figure1).

Histone N- terminus tails, and distinct loci in the globular domain have the ability to undergo post-translational modifications (Table1).

Figure 1: Composition of nucleosome. (Adapted from Weinberg, R. A. (2014). The biology of cancer. New York, NY, US: Garland Science.)

Table 1: Histone post-translation modification.5 (Adopted from Cell. 2007. 02.005.)

Combination of two or more of these modifications acts like a sensor for gene expression or gene repression through highly condensed or uncondensed chromatin structures. The fundamental mechanism for this lies on electrostatic interactions between the positively charged histones and the negatively charged DNA. Histones contain many residues (e.g. lysine) that can get protonated at physiological pH and gain positive charge that will make the interaction with the negatively charged phosphate groups of the DNA greater. However, when covalent modifications like acetylation take place, these residues lose the ability to ionize, thus making the interaction between the histones and the DNA weaker leading to loosened form of chromatin. Any disruption in epigenetic memory will manifest into gene expression and may lead to different disease states.

In cancer, epigenome is specifically marked with global DNA methylation and histone alteration patterns (Figure 2). Global acetylation of H4K16 (H4K16Ac) and the trimethylation of H4K20 (H4K20me3), along with DNA hypomethylation at repeating sequences is one of the hallmarks in many primary tumors6.

Figure 2: Global post-translation modification in primary tumor6. Reprinted with permission from Nat. Med. 2011, 17 (3), 330–339.

Impact of histone acetylation and deacetylation on cancer.

Hematological and solid cancers result from cellular transformations and genome instability through hyperacetylation in H3K18 regulating genes EP300, CREBBP,

NCOA2, MYST3 and MYST4 resulting in abnormal cell growth and divisions78. Missense mutation of EP300 and monoallelic loss of KAT5 are associated with malignant transformation in colorectal, gastric, breast and pancreatic tumor910. The overexpression of

many isoforms of HDACs has been observed to be associated with cancer. Gene regulation is controlled by the opposing activities of histone acetyltransferases (HATs), which acetylate the -nitrogen of lysine side chains of histones and histone deacetylases

(HDACs), which hydrolyze the acetyl groups. HATs- mediated acyl transfer takes place at lysine residue and results in expansion of chromatin, and thus increases the accessibility of transcription factors to DNA. On the other hand, the deacetylation with HDACs leads to very condensed structure of chromatin and consequently, to transcription repression1112.

Owing to their overexpression in cancer cells HDACs have now become a powerful target in anticancer drug development. HDAC inhibitors have shown promising potency in checking cancer progression. For all the above reasons, there is an urgent need for profound understanding of the molecular mechanisms of HDAC inhibitors. Understanding how exactly they induce cell death can help in designing drugs that will function as anticancer agents.

Isoforms of histone deacetylases.

Based on homology to yeast HDACs, cellular sublocalization and their activities, eighteen HDACs have been recognized in humans. They fall into four categories (Figure

3)12.

Class I HDACs (HDAC 1, 2, 3 and 8) are yeast RPD3 protein homologues, their sublocalization in general is in the nucleus, and they exhibit global expression in diverse human cell lines and tissues.

Class IIa: HDACs (4, 5, 7, and 9) are homologous to yeast Hda1 protein; they shuttle back and forth between the nucleus and cytoplasm.

Class IIb: HDAC6 and HDAC10 are mainly found in the cytoplasm, and contain two deacetylase catalytic domains, HDAC6 with its α-tubulin deacetylase (TDAC) domain has a unique substrate specificity for the cytoskeletal protein α-tubulin13.

Class III: HDACs (SIRT1, 2, 3, 4, 5, 6, 7) are yeast protein Sir2 homologues. They regulate gene expression based on changes of the cellular redox status through deacetylation of p53.

Class IV: HDAC11 is the only member of Class IV. Although that has similar sequence in the catalytic domain as in class I and class II, it does not have sufficient characteristics to be placed in either class I or II14. Class I, II and IV are zinc-dependent enzymes as they have a Zn2+ ion in their active site. Class III HDACs requires NAD+ for their activity.

Figure 3: Histone deacetylase isoforms.15 Reprinted with permission from Trends

Microbiol. 2013, 21 (6), 277–285.

The molecular mechanism of HDAC inhibitors.

The main molecular mechanisms associated with HDAC inhibitors are their aberrant recruitment to the promoters, and binding to oncogenic DNA-binding fusion proteins following chromosomal translocations. The other mechanisms that studies have been focusing on is overexpression of repressive transcription factors that interact with

HDACs. For instance, in acute promyelocytic leukemia (APL) and acute myeloid leukemia

(AML), oncogenic PML–RARα, PLZF–RARα and AML1–ETO fusion proteins recruit

HDAC-containing repressor complexes to repress expression of certain genes. Hence,

HDAC inhibitors have been used in combination with retinoids to treat AML and APL1617.

Another example is the overexpression of B-cell lymphoma 6 transcription factor (BCL6) due to hypo-acetylation, it recruits HDAC2. HDAC2 will repress CDKN1A (growth regulatory target genes) that encodes p21WAF1/CIP1.This phenomenon is observed in around 40% of diffuse B-cell lymphomas (DLBCLs). Treatment with HDAC inhibitors leads to hyper-acetylation of BCL6 and liberate HDAC2, resulting in reactivation of suppressed genes, and apoptosis18. In addition to aberrant recruitment, it recruits HDACs to specific loci and altered expression has been observed in many tumor samples. For example, overexpression of HDAC1 is observed in prostate, gastric, colon and breast carcinomas; HDAC2 overexpression in colorectal, cervical and gastric cancer; HDAC3 overexpression in colon tumors; and HDAC6 overexpression in breast cancer1920212223242526. siRNA-mediated knockdown of different HDACs that are overexpressed in certain tumor cell lines suppresses tumor cell growth and survival indicating a possible pharmacological target27. There are principle epigenetic variations between cancer cells and normal cells 8

that could modify the transcriptional response to HDAC inhibitors. For example, recent findings show global hypo-acetylation of histone H4, which is common in human tumors.

These findings highlight the importance of HDAC inhibitors in reversing the aberrant modification of H4, or by selective induction of TRAIL (tumor-suppressive gene) 28.

Direct effect on gene regulation.

Direct evidence supported by in vitro studies shows that HDAC inhibitors have higher sensitivity (around 10-fold) for tumor cells than for normal cells. The molecular pathway that is involved in HDAC inhibitor-mediated killing of tumor cells remains to be elucidated. HDAC inhibitors do not take advantage of pleiotropic biological effects of the target genes to kill the tumor cells. It is possible that their activities are cell-type dependent.

There is strong evidence that the diversity in their structure gives rise to different activities within the same cell type. This could be related to the fact that some HDAC inhibitors like suberoylanilide hydroxamic acid (SAHA) has more prevalent biological activities than selective inhibitors like Tubacin. Apoptotic cell death pathway involved includes both extrinsic and intrinsic pathways. Many studies have investigated the HDAC inhibitor- mediated cell death receptors (extrinsic pathway) that are induced when a particular ligand binds its respective death receptor. This results in the binding of adaptor proteins (Fas- associated death domain (FADD) and TNFR-associated death domain (TRADD)) and recruitment and activation of membrane proximal caspases like, caspases 8 and 10.2930.

Intrinsic pathway can be initiated by stress stimuli; for example, oncoproteins, ionizing radiation, chemotherapeutic agents and withdrawal of growth factors. These stimuli disturb the mitochondrial membrane and provoke the release of cytochrome C, which, together with APAF1 (apoptotic peptidase activating factor), stimulates the assembly of the 9

apoptosome, HTRA2 (High-temperature requirement protein A2) and SMAC (Second mitochondria-derived activator of caspase), thus activating caspase 9 which neutralizes the caspase inhibitory actions of apoptosis proteins (IAPs). These signal transduction pathways activate other downstream caspases (caspase 3, 6, 7) causing cleavage of a number of nuclear and cytoplasmic substrates to induce morphological changes that lead to and appear in apoptosis31. Several proteins tightly control mitochondrial membrane integrity. BCL-2

(B-cell lymphoma2) is a family of apoptotic regulators, which includes anti-apoptotic multi-domain members (BCL2, BCL-XL, and MCL1) and pro-apoptotic members (Bak,

Bax) and BH3 domain-only pro-apoptotic members (Bid, Bad, Bik, Bim, Noxa, Puma, Hrk and Bmf). BH3 only leads to inactivation of pro-survival BCL2-like proteins through direct protein interactions. Bim and Puma bind to all pro-survival proteins, and inhibit them, whereas other pro-apoptotic regulators selectively inhibit BCL2 proteins32. Many independent studies support that HDAC inhibitors induce tumor cell death through mitochondrial apoptotic pathway. It is possible that HDAC inhibitors induce global changes in gene expression that change the balance of expression of pro- and anti- apoptotic genes, preferentially favoring apoptosis. Two models have been established to investigate these pathways.

1) It has been shown that the activities of BH3-only protein are regulated transcriptionally through post-translational modifications.

2) It has been observed that elevation of ROS (reactive oxygen species) upon treatment with HDAC inhibitors alters the mitochondrial membrane potential.

Remarkably, ROS production results in the transcriptional induction and activation of Bim.

This finding raised the fascinating correlation between treatment with HDAC inhibitors, elevation of ROS and BH3-only protein apoptotic pathway33.

Cell Cycle arrest, HDAC inhibitors were first recognized because of their ability to initiate cellular differentiation. Cell-cycle arrest at G1/S phase is closely related with cellular differentiation, and this association is mediated by pRb (retinoblastoma protein) and related proteins. To date all HDAC inhibitors can induce cycle arrest at G1/S point except tubacin. pRb Hypophosphorylation due to HDAC inhibitors, and thus cell cycle arrest takes place through two major pathways. First, HDAC inhibitors induced p53- independent CDKN1A (encoding p21WAF1/CIP1) which promotes pRb hypophosphorylation. Secondly, HDAC inhibitors repress the transcription of cyclin D and cyclin A genes, which contribute to the loss of CDK2 and CDK4 kinase activities, and hypophosphorylation of pRb343536. CTP synthase and thymidylate synthetase

are genes that are involved in DNA synthesis. HDAC inhibitors repress their transcription, thus inhibiting the cell cycle in G/S phase, similar to the effect of anti- metabolite treatment. Other mechanistic tools that HDAC inhibitors used to induce cell cycle arrest are through up-regulation of cell cycle regulatory genes such as GADD45 and

TGFβreceptor signaling, resulting in repression of c-MYC, and subsequently cell-cycle arrest37. HDAC inhibitors have limited ability to block the cell cycle in G2/M phase. Many tumor cells have defects at G2- phase checkpoint, and most HDAC inhibitor treated cells pile up at G2/M phase. They then go through this malfunctioned checkpoint, and undergo apoptosis38. In contrast, a study shows that cells that preserve functional checkpoint are resistant to HDAC inhibitors; for example azelaic bishydroxamic acid (ABHA) 39.

The anti-angiogenic properties of HDAC inhibitors result from repressing the transcription and expression of angiogenic genes such as bFGF (basic fibroblast growth factor), HIF1α (hypoxia-inducible factor-1), eNOS (endothelial nitric oxide synthase), angiopoietin, VEGF (vascular endothelial growth factor) and TIE2 (tunica intima endothelial kinases 2)404142. Down regulation of expression of CXCR4 ((C-X-C motif) receptor 4) that is necessary for the recruiting bone marrow progenitor and circulating endothelial cells to the location of angiogenesis is an effect associated with HDAC inhibitors. The alleged anti- metastatic mechanism of HDAC inhibitors is through suppression of MMPs (matrix metalloproteinases). This might be accomplished firstly by transcriptional repression of MMP2 and MMP9 and upregulation of TIMP1 and TIPMP2, and secondly by inducing the expression of RECK (a membrane glycoprotein that negatively regulates MMP2 activity)434445.

Immuno-modulatory effect of HDAC inhibitors have the ability to upregulate the expression of MHC (major histocompatibility) class I, and class II, and co-stimulatory receptor such as CD40, CD80, CD86 and intercellular ICAM1 (adhesion molecule 1)4647.

As has been illustrated by many studies there is upregulation of MHC class I chain-related molecules MICA and MICB on the surface of tumor cells. These molecules act as ligand for NKG2D immunoreceptor (natural killer cell protein group 2D) on the surface of natural killer cells, γδT cells and CD8 T cells. Tumor cells that express MICA and MICB on their surface are targets for NKG2D restricted cytotoxicity4849. Some of HDAC inhibitors

(SAHA) show suppression effect on cytokines TNF α (tumor-necrosis factor α), IL-1

(interleukin-1) and INF (interferon-). The mechanism of the repression effect is either direct on the cytokines genes, or indirect and is secondary to other effects of HDAC 12

inhibitors and either effects are poorly understood50. However, what has been proven so far is that immune regulatory transcription factors such as STAT1 (signal transducer and activator of transcription 1), STAT3 and NF-κB (nuclear factor-κB) are directly regulated by acetylation, which raises the possibility that acylation of these factors upon HDAC blockade treatments causes the indirect effect of regulating the cytokines515253.

Effects of HDAC inhibitors on non-histone proteins.

HDAC inhibitors induced apoptosis through ‘indirect’ regulation of gene expression. Direct acetylation due to HDAC inhibition of E2F1, p53, STAT1, STAT3 and

NF-B is observed with HDAC inhibitors with subsequent expression of downstream target genes51535455. In addition, they induced apoptosis independent of altered gene expression. HDAC inhibitors -induced apoptosis independent of altered gene expression.

Anti-cancer effects of HDAC inhibitors might also from their transcription-independent effects. For example, Bax is bound and sequestered in the cytoplasm by Ku70 (DNA end- joining protein). Acetylation of Ku70 in response to inhibition by class III HDAC inhibitors leads to initiation of apoptosis by causing translocation of Bax to the outer membrane of the mitochondria5657. HDAC inhibitors also have activities on many kinases that mediate signal transduction pathways and dysregulate many downstream substrate phosphorylation pathways. For example, HDAC1, 6 and 10 directly interact with the PP1 (protein phosphatase). Treatment with HDAC inhibitors block HDAC-PP1 binding which leads to dephosphorylation, and thus deactivation of anti-apoptotic proteins like Akt and sensitization of cells to apoptosis585960. Another example is the effect on the abundant cellular chaperone Hsp90 (heat shock protein 90). Overexpression of this chaperone in cancer cells results in poor prognosis and outcomes. Hsp70 and Hsp90 form multi protein 13

complexes with many oncogenic and antiapoptotic proteins. This interaction is concomitant with prevention of their ubiquitinoylation and proteasomal breakdown. Hsp90 is deacetylated by HDAC6. Thus using HDAC 6 inhibitors results in hyperacetylation of

Hsp90 and proteasomal degradation of Hsp90 associated proteins HER2/neu, ERBB1,

ERBB2, Akt, c-Raf, BCR–ABL and FLT36162636465.

Guanidines in HDAC inhibitors

Several studies have discussed guanidine from chemical as well as biological point of view. Guanine (nitrogenous base in nucleic acid) and arginine amino acid) are examples of guanidine-based compounds. These compounds, upon identification and chemical accessibility, gained interest for both chemical and biological applications due to their unique nature. Guanidine (3) a white crystalline solid, was first synthesized in 1861 by Adolph Strecker from the chemical decomposition of guanine (1) (Figure 4a) 66.

Guanidine has interesting chemical properties. It is strongly basic with pKa around

13.6. Some guanidine derivatives are called superbases with pKa values higher than that of hydroxide ion (Figure 4b)67 .

Figure 4: (a) chemical decomposition of guanine. (b)examples of

Superbases70

Some derivatives are even stronger than classical proton sponges (9) (Figure 5).

Therefore, they possess a wide range of basicity that can contribute to high levels of ionization and thus varying levels of electrostatic interactions. The basic reason for this basicity is the fact that their conjugate acid (guanidinium ion) (8) is stabilized by resonance67.

Figurer 5: (a) conjugate acid of guanidine stabilized by resonance67. (b) proton sponge.

One of the most intriguing characteristics of guanidines, which also make them unique is the so-called Y aromaticity. At a first look, it appears to be counterintuitive to the common definition of aromaticity, but the resonance in guanidine group and molecular orbital analysis show that Y aromaticity is possible. Experimentally, it is evident from the fact that many protein structure surveys revealed that the guanidinium group of arginine interacts with nearby aromatic side chains. In fact, guanidine/ guanidinium has 6

electrons, all of which are in the bonding molecular orbitals (Figure 6). While the delocalization energy for benzene is 33 kcal/mol, the delocalization energies of guanidine and the guanidinium ion are 19.8 and 26.4 kcal/mol, respectively, indicating the highly

aromatic nature of these groups. Consequently, guanidine shows the ability to undergo

stacking interactions (Figure 6), as well as prefers substitution over addition68.

Figure 6: Guanidine bonding molecular orbitals and pi-stacking interactions69 . Reprinted with permission from Org. Lett. 2008.

Another factor that grasped our attention in using guanidine moiety as a part of

HDAC inhibitor pharmacophore is their strong hydrogen bond donor nature. This is an important factor as hydrogen bonding is one of the most common types of interactions of a drug molecule with its target. The stronger the nature of the hydrogen bond interaction, the stronger the drug will bind to the protein.

Guanidines are naturally occurring molecules. Compounds with guanidine group are extensively found in marine organisms due to their special living environments. The marine environment has been considered one of the most multifaceted environments on earth due to its huge arrays of light spectra that range from full darkness to extensive brightness, temperatures that extend from −2 ◦C to over 300 ◦C, pressures starting from 1 atmosphere and increasing to more than 1000 atmospheres with depth, nutrient conditions varying from sparse to rich in nutrients70. Such diverse conditions enable the existence of plentiful species that produce diverse and complex secondary metabolites with a wide array of biological activities. An example of a guanidine–based marine natural product possessing cytotoxic effect is monanchocidin, a novel pentacyclic guanidine derivative 17

isolated from the sponge M. pulchra in 201070. Monanchocidin displayed cytotoxicity against human leukemia THP-1 cell line with an IC50 of 5.1 M, human cervix epithelioid carcinoma HeLa cell line with an IC50 11.8M, and mouse epidermal JB6 Cl41 cell line with an IC50 of 12.3 μM. It exhibited 66% apoptosis-inducing cytotoxicity in THP-1 cells

7172 with an IC50 value of 3.0 μM . Many natural products contain amino acid arginine as part of polypeptides produced by bacteria73. The biochemical and biophysical properties of guanidine are attributed to specific hydrogen bonding, Y aromaticity, and stacking. A molecule with such basicity is able to bind to carboxylates, phosphates and metals and may be exploited for its cytotoxic activity

The variation of HDAC inhibitory activity, and isoform selectivity of HDAC inhibitors is attributed to variations in the nature of the capping group. The wide range of chemical activity that coexists in one single moiety like guanidine grabbed our attention for use in medicinal chemistry applications. In fact, guanidine group has been used in

HDAC inhibitors for its ability to bind to metal ions (Figure 7).

Figure 7: HDAC inhibitor with guanidine as zinc-binding group74

This compound (10) displayed some potency at IC50 200 μM concentration, with

83 ± 1% deacetylase activity towards endogenous HDACs from HeLa cell lysates. The amidine of the guanidine group chelates with zinc 74. The low level of activity observed with compound (10) may be due to the formation of energetically less favorable four- membered metallocycle upon coordination with metal ions. This may be overcome by making guanidine a part of a larger zinc-binding group. Alternatively, it may constitute a part of the capping group. The later approach can probably be used as a mean to achieve isoform selectivity or to enhance the activity of other binding groups.

Examples of HDAC inhibitors containing guanidine in the capping group are shown in Figure 8.

Figure 8: HDAC inhibitors with guanidine as capping group7576

Mocetinostat (MGCD0103) (11) is a benzamide class HDAC inhibitor in phase II clinical trial78. It acts by inhibiting mainly HDAC 1, but the selectivity is not high as it

inhibits other HDAC subtypes such as HDAC 2,3 and 11. Severe adverse side effects like cardiac problems associated with this drug can be attributed to this lack of selectivity.

The two compounds (12) and (13) may be anticipated to be more effective in clinical trials as they combine both the selectivity advantages of MGCD0103 derived from the guanidine based capping group and the high efficacy of SAHA which contains a hydroxamic acid based zinc binding group.

The pharmacophore model of HDAC inhibitors consists of three major parts, a zinc binding group, a linker and a capping group (Figure 9) . The zinc binding group, which is responsible for binding to the zinc ion in the HDAC active site through chelation, is considered to be an essential part of any HDAC inhibitor, and is a major contributor to potency. It does not usually contribute to selectivity since zinc is a common component of all zinc-dependent HDACs. However, it is believed to be necessary for activity, although some zinc binding group free HDAC inhibitors have been reported, but these are a few and a small exception to the rule. Until recently, five classes of zinc binding groups have been identified, they being hydroxamic acid derivatives, short chain fatty acids, benzamides, electrophilic ketones, and cyclic tetrapeptides12. The linker is usually an aliphatic chain or a phenyl ring. It is responsible for connecting the zinc binding group with the capping group, but in addition to that, it helps binding to HDAC through pi-stacking. It has been reported in the literature that a 4 or 5 carbon spacer between the zinc binding group and the capping group is the optimal condition for zinc binding74. The capping group, shows the biggest structural diversity among HDAC inhibitors. Many different groups, including bulky groups, are tolerated at this position. Usually the capping group is responsible for both potency and selectivity. In fact, it is believed to be the major part of the molecule that 20

can give rise to selectivity due to the fact it binds to a region in which the amino acid residues are less conserved in different HDAC isoforms.

Zinc binding Capping group group Linker

Figure 9: HDAC inhibitor pharmacophore.

It is well established that the capping group or the surface recognition unit of

HDAC inhibitors bind to the region at the rim at the entrance to the active site of HDAC proteins , thus delivering the metal-binding group specifically to the Zn2+ ion at the end of the 11 Å channel (Figure 10).

Figure 10: catalytic site 77. Reprinted with permission from Handb. Exp. Pharmacol.

2011, 206, 103–123.

Chapter 2

Results and Discussion

In a program to develop selective HDAC inhibitors with new metal-binding groups, our laboratory has designed and synthesized a new class of HDAC inhibitors with an imidazole ketone moiety as the metal-binding group (Figure 11)78.

Figure 11. HDAC inhibitors with an imidazole ketone as the metal-binding group78.

In order to modulate the activity and the selectivity of this class of HDAC inhibitors, we designed new analogues incorporating guanidine moieties as the capping group (Figure

12).

Figure 12. The target molecules consisting of imidazole based zinc-binding group and guanidine-based capping groups designed for synthesis.

Chemistry Synthesis of analogues containing guanidine groups is challenging due to the highly basic nature of guanidines. Therefore, our strategy was to incorporate guanidine group in the final step of the synthesis. The general synthetic approach used is depicted in the

retrosynthetic analysis shown in scheme 1.

Scheme 1: Retrosynthetic approach to HDAC inhibitors with guanidine-based capping groups.

We planned to make the final products through coupling of the carboxylic acid intermediate (28) with the guanidines. The carboxylic acid (28) results from simple hydrolysis of the ester (29), which can be obtained through aldol reaction between the aldehyde (30) and the imidazole ketone (32). Some of the guanidines required for synthesis were commercially available, while the others had to be obtained by de novo synthesis. The general synthetic approach used to synthesize guanidines is shown in scheme 2.

Scheme 2: Retrosynthesis of Guanidine capping group.

The final guanidine product79 (34) can result upon removal of the amine protecting group (R3) of the guanidine intermediate (35). There are many different protecting groups that can be useful in the synthesis of guanidines. We chose acetyl as the protecting group as it can be easily introduced and removed under relatively mild conditions. The cyclization of linear protected guanidine to obtain the cyclic guanidine (35) can be achieved using the alpha-bromoketone compounds80(36), which can easily be synthesized by -bromination of the ketone80 (37). The ketone (37) can be obtained by Grignard reaction81, followed by oxidation. This approach allows us to synthesize substituted

guanidines by choosing appropriately substituted aldehydes and alkyl halides as the starting materials. As previous structural-activity relationship, (SAR) studies in our laboratory have shown that aromatic capping groups contribute to higher activity, in most cases aldehydes and alkyl halide carrying aromatic substituents were chosen for synthesis.

Scheme 3: Zinc binding group, and linker synthesis.

The synthesis of the carboxylic acid intermediate (28) is shown in scheme 3. As our previous attempts at direct aldol coupling of the aldehyde (31) carrying an unprotected carboxylic acid moiety with the imidazole ketone (32) have not been successful, carboxylic acid group was first protected as an ester. Esterification of (31) using DBU as a base and methyl iodide as a source of electrophilic methyl group yielded the methyl ester (30) in excellent yield, without the need for any purification. Aldol coupling between the aldehyde

(30) and the imidazole ketone (32) was carried out using the Stork enamine approach assisted by microwave irradiation. Although this was a clean reaction giving only the aldol 26

product, it gave relatively poor yields. The final step is the hydrolysis of the ester group of

(29) to give the acid (28) in excellent yield through precipitation. With the acid in our hands, the next task was the synthesis of the guanidines.

Scheme 4: Synthesis of 4,5-diphenyl-1H-imidazol-2-amine (47).

The synthesis of the 4,5-diphenyl-1H-imidazol-2-amine capping group is shown in scheme 4. The Grignard reaction between benzaldehyde (42) and benzyl bromide (41) was found to be challenging

Scheme 5: Grignard reactions

Classical Grignard conditions where the reaction was initiated by heat or by chemical activation using iodine, failed to give the required product. Multiple attempts using different reaction conditions and reaction times and a wide range of temperatures were unsuccessful, yielding either multiple products or the starting material. A common side reaction observed was the homo coupling of the benzyl bromide to yield a biphenyl system as a side product, which has no synthetic usefulness at this point for us. This is probably due to the nucleophilic attack on carbonyl is taking place slower than the homo coupling reaction leading to the side product. Therefore, activation of the aldehyde carbonyl group by the addition of a Lewis acid was attempted. The first Lewis acid used was cerium chloride in complex with lithium bromide. However, the reaction did not yield any addition product. We then turned to bismuth trichloride, which is an excellent Lewis acid, but has the drawback of being really sensitive to moisture, making it difficult to handle it. To our delight, by careful handling and carrying out the reaction under scrupulously anhydrous conditions, we were able to obtain the desired product successfully(Scheme 4). Besides, the reaction was highly reproducible and gave good yields. The next step in the synthesis is the oxidation of the alcohol (43). Jones oxidation resulted in poor yields, probably due to elimination of the alcohol under strong acidic conditions used in Jones oxidation. Changing the oxidizing agent to milder Dess-Martin

Periodinane (DMP) resulted in a clean and reproducible reaction. -Bromination of this ketone was carried out using N-bromo succinimide in the presence of p-toluenesulfonic acid in acetonitrile. It is worth mentioning here that the time and the concentration of the reaction mixture are crucial in controlling the ratio between mono bromination and di bromination products. We were able to optimize the conditions and obtain the mono bromo 28

compound (45) in a reproducible way and in excellent yields. The cyclic guanidine moiety(46) was made by cyclization of the -bromo compound (45) with N-acetyl guanidine. The reaction was firstly tried using guanidine itself, but it yielded multiple spots in TLC. It may be that upon cyclization, the product with a free amine group is reactive and may be involved in reactions leading to side products. This was overcome by using N- acetylguanidine to obtain the cyclized guanidine (46) as the major product. After that, the acetyl protecting group was removed using strong acid conditions, but short reaction times, yielding the final biphenyl guanidine (47) in pure form. It is worth mentioning here that we changed the hydrolysis conditions from sulfuric acid reported in literature to hydrochloric acid, because it allowed easier precipitation and isolation of the hydrochloric salt under such conditions. Using the same synthetic strategy, guanidine (53) was synthesized

(Scheme 6).

Scheme 6: Synthesis of 5-phenyl-1H-imidazol-2-amine (53) capping group.

In order to study the efficacy of the coupling reaction using the hydrochloride salt versus the free amine, a part of the product was neutralized with 5M KOH solution.

Although the isolation of the neutralized compound proved cumbersome, probably due to its basic nature, it was isolated in pure form and in sufficient amounts for the next step. 29

Scheme 7: Synthesis of 5-(3-nitrophenyl)-1H-imidazol-2-amine (56-b) capping group.

The synthesis of the amine 5-(3-nitrophenyl)-1H-imidazol-2-amine (56-b) commenced with thebromination of the methyl ketone (54) (Scheme 7). It was observed that di-bromo derivative was the main product when the reaction was carried out in low volumes of acetonitrile and therefore, high concentration of the reagents. Using 1 mmol of (54) per 50 mL of acetonitrile gave the best yields of the monobrominated product. It was observed that yield of this reaction was high when compounds with electron withdrawing groups are present because of their ability to activate the carbonyl group, compared to electron donating groups.

Scheme 8: Synthesis of 5-(2,3-dimethoxyphenyl)-1H-imidazol-2-amine (62) capping group.

In our attempt to make 5-(2,3-dimethoxyphenyl)-1H-imidazol-2-amine (62), we started with Grignard reaction of 2,3 dimethoxybenzaldehyde (57) with methylmagnesium chloride. The reaction was fast and gave quantitative yields. This can be attributed to the chelation of magnesium with the oxygen atom of the carbonyl group and the oxygen atom of the adjoining methoxy group, facilitating the reaction. As with the nitro-analogue, - bromiantion of the dimethoxy compound gave two products, depending on the concentration, the mono bromo being the predominant product at lower concentrations.

Scheme 9: 1-methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66) capping group.

In our attempts to synthesize 1-methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66), our group has tried varying conditions for -bromaination of the imidazole methyl ketone (32).

All attempts have failed and led to electrophilic aromatic substitution products. We assumed this can be overcome by using strong acidic conditions to protonate the imidazole ring, making it resistant to electrophilic aromatic substitution, and at the same time activating the carbonyl group to form the enolate. As speculated carrying out bromination using bromine in HBr-acetic acid gave the required product (64). Making the guanidine imidazole (65) form the bromo derivative required much experimentation. The reaction

was monitored by low-resolution mass spectrometry for product formation at varying time intervals. No product formation was observed after 10 minutes. The optimum time was found to be over six hours. This probably due to the delocalization lone pair of electrons of imidazole nitrogen over the ketone carbonyl, making it less electrophilic and less susceptible to the nucleophilic attack. The other challenging issues were both the workup and the separation of the product, which is very basic, and highly polar. Eventually, we successfully overcame these difficulties and isolated the product (65) with the acetylated amine group. Hydrolytic removal of the acetyl protecting group proved equally problematic due to very polar nature of the compound and the free amine (66) was obtained in low yields. It is worth mentioning that this compound not only is a good capping group but potentially has the ability to chelate with zinc. Therefore, the zinc binding studies using

(65) were carried out. This was done by recording the UV absorption spectra of (65) with different concentration of zinc acetate (Figure 13). No linear relationship was observed between absorption and the equivalents of zinc ion. However, a small change in max was observed with increasing zinc equivalents. This is believed to be due to chelation of zinc with the imidazole nitrogens. The relationship between max and zinc equivalents is shown in figure 14. The relationship is not linear, but it follows a more complex correlation where after some point we see saturation of chelation.

2 0 equivalents 1.8 of zinc 0.1 1.6 equivalents of zinc 1.4 0.2 equivalents 1.2 of zinc 0.3 1 equivalents

Absorption 0.8 of zinc 0.4 0.6 equivalents of zinc 0.4 0.5 equivalents 0.2 of zinc 0.5 0 equivalents 180 230 280 330 380 430 of zinc Wavelength

Figure 13: UV absorption of (65) with different equivalents of zinc ion.

280 y = -2986.1x6 + 10316x5 - 13705x4 + 8734.8x3 - 2733.5x2 + 410.23x + 241.77 278 R² = 0.9698

276

274

272 max

 270

268

266

264

262 0 0.2 0.4 0.6 0.8 1 1.2 Zn equivalents

Figure 14: Relationship between max and zinc ion concentration.

Scheme 10: Synthesis of 1H-phenanthro[9,10-d]imidazol-2-amine (68) capping group82.

As per reports from many studies that highlight the importance of the capping group for stabilization of the interaction between the zinc-binding group and the catalytic site, we desired chemical versatility as well as a large capping groups. Accordingly, we designed the 2-aminobenzimidazole analogue (68). The synthesis is shown in scheme 10.

Unfortunately, this reaction proved highly problematic and was not reproducible., It has been previously reported by Itano et al. in 1973 for analytical purposes82. However, in our view, the compound has not been fully characterized. In one study, they provided only the mass spectra data82.The same group, in a subsequent publication, reported a similar compound with NMR data, but there were missing signals without proper explanation85

.The previous group had used acidic work up conditions. We have found that the usage of acid was not necessary and we were able to isolate the product without using acid. The mechanism of this reaction, whether in the presence or the absence of acid, seems intriguing. An extensive literature survey revealed a similar reaction using a di-ketone as starting material (Figure 15). According to this mechanism, a role for an acid in generating the imidazole amine product is not obvious. Whether in the presence or the absence of acid, there appears to be a need for a reducing agent to generate the final imidazole amine product. Therefore, ethanol used as the solvent playing the role of reducing agent cannot

be ruled out. In this context, it needs to be pointed out that Itano et al. later reported that using a reducing agent such as NaBH4 in this condensation reaction gave better results. In view of the low yields of this reaction, we developed another methodology for approaching this product (68) (Scheme 11).

Figure 15: Guanidine di-ketone condensation. Nishimura, T., Kitajima, K. (1979)83

Scheme 11: Alternative synthesis of 1H-phenanthro[9,10-d]imidazol-2-amine (68) synthesis.

The alternative way to access compound (68) proved to be more reliable.

Condensation of compound84 (67) with in situ generated formaldehyde from chemical decomposition of hexamethyl tetramine (77) yielded the imidazole (78) in excellent yields.

Imidazole of nitrogen (78) was protected through benzylation85 as it could be removed along with the reduction of the azide group in the final step. Lithiation of imidazole at 2- postion86 using n-butyl lithium allowed the installation the azide by using tosyl azide87 (82) which was easily synthesized from tosyl-chloride and sodium azide87. Catalytic hydrogenation in the final step reduced the azide group to amine, simultaneously removing the benzyl protecting group to give (68). This synthetic approach is much more elegant and more reliable than the previously described method.

Driven by the success of this method, we decided to make the corresponding bipyridine analogue (87) of the triphenyl guanidine (68) using the same strategy. We have successfully synthesized the benzyl protected intermediate (85) and expect to obtain the final product (87) by catalytic hydrogenation.

Scheme 12: Synthesis of 1H-imidazo[4,5-f][1,10]phenanthrolin-2-amine (87) capping group.

Scheme 13: Synthesis of 4-methyl-5-phenyl-1H-imidazol-2-amine (91) capping group.

The synthesis of 4-methyl-5-phenyl-1H-imidazol-2-amine (91) is shown in scheme

13. Working with 2-bromo-1-phenylpropan-1-one (88) was challenging as it was a strong lachrymator and eye irritant. The yield of the cyclized product was low, probably due to

-bromo derivative (88) undergoing elimination in the presence of basic guanidine

Coupling of carboxylic acid (28) with guanidines to obtain the final products.

Having synthesized the carboxylixc acid (28) and the required guanidine intermediates, we then explored the coupling of the two components to derive the final products as potential HDAC inhibitors.

Synthesis of (E)-N-(1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3- oxoprop-1-en-1-yl)benzamide (15).

Figure 16: Analogue (15); (E)-N-(1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H- imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzamide.

We first attempted EDC coupling using standard conditions (Scheme 14).

Scheme 14: EDC coupling of amino-benzoimidazole (92) with acid (28) resulting in anhydride formation rather than the coupling product.

Although EDC coupling is widely used in amide formation, no product amide was obtained in this instance. Repeating the reaction in the presence of triethyl amine made no

difference. The acid anhydride (93) was isolated rather than the product. The structure of the anhydride was confirmed by NMR spectroscopy.Treating the isolated anhydride with excess of the guanidine amine (92) in DCM too failed to give the coupling product. The failure may be due to the low nucleophilicity of guanidine amine.

In view of the failure of the above EDC coupling reaction, we investigated different coupling conditions using our carboxylic acid as well as benzoic acid as a model reaction.

HATU has been proven to be a very efficient amide coupling agent even in difficult sterically hindered couplings and usually gives negligible level of racemization. Therefore, we decided to screen different coupling conditions using HATU, HBTU, and DCC.

Scheme 15: Screening of coupling conditions with benzoic acid (94) and guanidine (92) as model reaction.

The reaction was monitored by TLC. The formation of a new product was observed with both HATU and HBTU in the presence of HOBt, A similar product was not observed with DCC. However, a draw back in the methods using HATU and HBTU was the difficulty in working up the reaction and isolating and purifying the product. The product was never isolated when HBTU was used. Reaction gave a more complex product mixture when compared to using HATU. The product was isolated with HATU by crystallization, though over a long time period. The success of the coupling reaction when HATU was used as the coupling agent can be due to the stabilization of the transition state by hydrogen bonding of amine N-H bond with nitrogen of the pyridine ring of HATU forming a 7- membered ring (Figure17).

Figure 17: Transition state of HATU coupling.

After the optimization of the reaction conditions, the next difficulty was the isolation of the compound by chromatography on silica gel. The product isolated had a more complex NMR spectrum than the crude reaction product, probably due to the acidity of silica. Reversed phase chromatography was not a good choice either due to low solubility of the product in water and methanol, which could lead to precipitation of the compound. 40

Scheme 16. Amide coupling before the addition of zinc-binding group

As the isolation of the product proved difficult, we explored another route for making the final product by carrying out the amide coupling first, prior to the incorporation of the zinc-binding group (Scheme 16). A complex product mixture was obtained and the amide (96) was isolated in low yields by silica gel chromatography. The crude reaction product was used in the next step as purification by column chromatography in ethyl acetate/DCM gave low yield, probably due to decomposition of the product on silica. The final product (15) was not isolated.

At this stage, we took advantage of the low solubility of the compound (15) in water and most of the organic solvents. The coupling reaction was repeated using the smallest possible volume of DMF (Scheme 17). Addition of deionized water after the completion of the reaction resulted in the precipitation of the product. The product (15) was obtained by filtration in remarkable purity.

Scheme 17. Successful coupling and isolation of target molecule (15).

Driven by the success of these conditions we tried to apply them on all of the guanidines we designed and synthesized previously. The first choice was a similar compound with guanidine (92) with the difference being presence of methyl group on imidazole nitrogen. This reaction described in scheme 18 was successful leading to the target molecule (16).

Scheme 18. Successful coupling and isolation of target molecule (16).

After the second successful coupling and isolation of product, an attempt was made to carry out coupling of several guanidines made de novo, as well as commercially available ones (Scheme 18). Unfortunately all guanidines that did not have the benzyimidazole structure did not couple with (28). What was even more intriguing was the fact that the same product was isolated when different guanidine were used. It was identified as the product (99) formed by coupling of acid (28) with HOBt. This is the activated ester intermediate formed during the course of the coupling reaction. The reaction appears to stop here, without it being attacked by guanidine amine acting as nucleophile.

We assume this is happening due to poor nucleophilcity of the guanidine amine groups used.

Scheme 19. Unsuccessful coupling of acid (28) with different guanidines.

Biological studies

Having successfully synthesized two HDAC inhibitors (15) and (16) by incorporating two guanidine moieties, aminobenzimidazole and 2-amino-1-methylbenzimidazole, respectively, they were tested for cytotoxic activity on HeLa cells in the laboratory of Dr.

William Taylor in the Department of Biological Sciences, University of Toledo. The 2- aminobenzimidazole, analogue (15) showed cytotoxic activity at 20 M concentration. It was also tested at the NCI in the 60 cell line assay at 10 M , The compound showed a mean growth inhibition of 64.5% on the 60 cell lines (Figure 18) at this concentration. It was then tested in the dose response assay at the NCI (Figures 19 and 20). The GI50 values are shown in figure 20. The leukemia cell lines are the most sensitive to the compound. For example, the cell lines K-562, RPMI-8226 and SR had GI50 values around 0.1 M. In contrast, melanoma cell lines were the least sensitive exhibiting growth inhibition with average GI50 of 15.6 M. Among colon cancer cell lines, HCT-15 and SW-620 showed the highest activity with GI50 of 0.1 M. Similar to melanoma, renal cancer and non-small cell lung cancer cell lines showed minimal response with average inhibition concentration of 6.54 M and 6 M, respectively. On average CNS cancer cell lines had GI50 of 7 M,, except for U251 cell line which had a GI50 of 0.916 M. The ovarian, prostate and breast cancer cell lines had average IG50 values of 5.1 M, 2.3 M,

and 2.7 M, respectively. The isoform selectivity of this compound is yet to be determined. The analogue (16) was tested in the laboratory of Dr.William Taylor in the

Department of Biological Sciences, University of Toledo, against HeLa cell lines at 100

M concentration and was found to be inactive. This result shows that imidazole nitrogen has to be unsubstituted for activity. This can be due to either the involvement of the imidazole nitrogen in hydrogen bonding with the receptor, or the steric interference caused by the methyl group during receptor binding.

Developmental Therapeutics Program NSC: D-813835 / 1 Conc: 1.00E-5 Molar Test Date: Apr 08, 2019

One Dose Mean Graph Experiment ID: 1904OS92 Report Date: Apr 30, 2019

Panel/Cell Line Growth Percent Mean Growth Percent - Growth Percent Leukemia CCRF-CEM 15.37 HL-60(TB) 26.71 K-562 2.24 MOLT-4 45.19 RPMI-8226 10.66 SR 11.15 Non-Small Cell Lung Cancer EKVX 62.29 HOP-62 34.12 HOP-92 52.75 NCI-H226 82.40 NCI-H23 23.32 NCI-H322M 43.81 NCI-H460 10.89 NCI-H522 58.64 Colon Cancer COLO 205 55.07 HCC-2998 45.40 HCT-116 23.13 HCT-15 24.30 HT29 8.78 KM12 14.36 SW-620 5.35 CNS Cancer SF-268 40.25 SF-295 64.91 SF-539 9.47 SNB-19 41.14 SNB-75 -16.97 Melanoma LOX IMVI 11.48 MALME-3M 72.68 M14 71.05 MDA-MB-435 18.85 SK-MEL-2 85.38 SK-MEL-28 89.65 SK-MEL-5 83.10 UACC-62 74.69 Ovarian Cancer IGROV1 35.96 OVCAR-3 7.54 OVCAR-4 36.69 OVCAR-5 59.92 NCI/ADR-RES 24.56 SK-OV-3 77.21 Renal Cancer 786-0 23.64 A498 51.07 ACHN 49.27 CAKI-1 18.55 RXF 393 2.67 SN12C 51.71 TK-10 66.48 UO-31 28.30 Prostate Cancer PC-3 38.10 DU-145 -13.69 Breast Cancer MCF7 19.47 MDA-MB-231/ATCC 19.31 HS 578T 40.20 BT-549 5.05 T-47D 49.61 MDA-MB-468 -3.55 Mean 35.53 Delta 52.50 Range 106.62

150 100 50 0 -50 -100 -150

Figure 18. Results of NCI 60 cell line assay at 10 M

Figure 19: Five dose response graphs of compound (15) in different cancer cell lines.

Figure 20. Growth inhibition of different cancer cell lines by compound (15)

Conclusion and future directions

In conclusion, seven guanidine based capping groups were successfully synthesized. Two

HDAC inhibitors incorporating guanidine as the capping group were synthesized. One of the analogues showed promising cytotoxic activity while the other was not active.

The future plans consist of completing the synthesis of target molecules (17-26), and evaluation of their cytotoxic activity ad isoform selectivity. The direct amide coupling of some of the guanidines with carboxylic acid (28) was not successful and this may be due to poor nucleophilicty of the guanidine amine function. A way to overcome this difficulty may be by treating the guanidine with a strong base such as n-BuLi and converting the amine to the amide anion, which will be more nucleophilic. Alternatively, a strongly electron withdrawing group like nosyl group can be attached to the amine group. This will facilitate the deprotonation of the amine under basic conditions to generate the corresponding anion, which will be more nucleophilic.

Chapter 3

Experimental and Materials

All solvents and chemicals where purchased from commercial sources and used as they were without additional purification. Ether based solvents like tetrahydrofuran and diethyl ether where distilled upon usage. 1H and 13C NMR spectra were recoreded on

Brucker Avance 600MHz, INOVA 600 MHz and Varian VXRS 400 MHz NMR spectrometers in deuterated solvents using residual undeuterated solvents as internal standard. High-resolution mass spectra (HRMS) were recorded. Melting points were determined using a Fisher-Johns melting point apparatus. Mixtures of products were purified by chromatography using silica gel, standard grade from Sorbent Technologies

(catalog number 060219N, 230 x 400 mash size, pH 6.5-7.5). Thin layer chromatography

(TLC) plates (20 cm x 20 cm) were purchased from Sorbent Technologies (catalog number

4115126) and were viewed under Model UVG-54 mineral light lamp UV-254 nm. For preparative thin layer chromatography purposes, Uniplates (1000 μm) were purchased from Analtech Inc. w. A Biotage initiator was used for all microwave reactions in recommended vials of 0.5 mL, 5 mL, and the 20 mL capacity using compatible caps.

Experimental

Methyl 4-formylbenzoate (30)

To a stirred solution of 4-formylbenzoic acid (1.0 g ,6.67 mmol) was added DBU (1mL,

6.67 mmol, 1 equiv.) followed by Iodomethane (1.25 mL 20 mmol, 3 equiv.). The reaction mixture was stirred overnight at room temperature. After the addition of aqueous sodium bicarbonate solution to basify, the mixture was extracted with ethyl acetate. The combined ethyl acetate extract was washed with 1N HCl and brine. The organic extract was dried over sodium sulfate, filtrated and concentrated under reduced pressure to yield the ester

1 (30) (1.06 g, 97%. H NMR (600 MHz, CDCl3) δ 9.94 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H),

13 7.78 (d, J = 8.4 Hz, 2H), 3.79 (d, J = 9.9 Hz, 3H). C NMR (151 MHz, CDCl3) δ 191.76

(s), 168.28 – 163.31 (m), 139.19 (s), 135.02 (s), 130.28 – 130.08 (m), 129.53 (s), 52.61 (s).

Methyl (E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoate (29)

A mixture of methyl 4-formylbenzoate (30) (1.00 g, 6.4 mmol), N-methyl imidazole ketone

(806 mg ,6.5 mmol, 1.1 equiv.) , piperidine (2.37 mL, 24 mmol, 3.75 equiv.) and anhydrous methanol (10 mL) in a 20 mL microwave vial was heated in the microwave synthesizer at

85 oC for 6 hours. The crude reaction mixture was concentrated and purified by flash chromatography on silica in ethyl acetate/ hexanes (20 %-> 70% ethyl acetate), followed by recrystallization from ethyl acetate yielding compound (29) (542 mg, 31%) as a pure 51

1 product. H NMR (600 MHz, CDCl3) δ 8.02 (d, J = 16.0 Hz, 1H), 7.93 (dd, J = 10.6, 4.7

Hz, 2H), 7.68 (d, J = 16.1 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.11 (s, 1H), 7.00 (s, 1H), 3.97

13 (s, 3H), 3.81 (s, 3H). C NMR (151 MHz, CDCl3) δ 180.05 (s), 166.58 (s), 143.96 (s),

141.68 (s), 139.28 (s), 131.44 (s), 130.15 (s), 129.64 (s), 128.61 (s), 127.73 (s), 125.11 (s),

52.40 (s), 36.50 (s).

(E)-4-(3-(1-Methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoic acid (28)

To a stirred solution of methyl (E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1- yl)benzoate (542 mg, 2 mmol) in 20 mL MeOH/H2O (1.5:1) was added potassium carbonate (829.2 mg , 6 mmol , 3 equiv.) and the reaction mixture was stirred at room temperature overnight. Methanol was evaporated, and 5 mL of sodium bicarbonate was added, and the mixture was extracted with ethyl acetate. The aqueous layer was collected and acidified with concentrated HCl to pH 5, when the product precipitated as a yellowish solid. The solid was collected by filtration, washed with cold water and dissolved in a mixture of methanol and ethyl acetate (1:4). The solution was dried over sodium sulfate filtrated, and concentrated under reduced pressure to yield compound (28) (475 mg, 93 % yield) as pure product. 1H NMR (400 MHz, DMSO) δ 7.99 (d, J = 16.1 Hz, 1H), 7.86 (d, J

= 8.0 Hz, 2H), 7.70 (d, J = 16.1 Hz, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.55 (s, 1H), 7.18 (s,

1H), 3.99 (s, 3H). 13C NMR (151 MHz, DMSO) δ 179.50, 167.26, 143.59, 141.31, 139.02,

132.54, 130.38, 129.63, 129.38, 129.07, 125.52, 36.35.

1,2-Diphenylethan-1-ol (43)

To a mixture of BiCl3 (47.3 mg, 0.15 mmol, 15 mol %), Mg turnings (72.9 mg, 3 mmol,

, 3 equiv.) and PPh3 (39.3 mg , 0.15 mmol, 15 mol %) and anhydrous THF (10 mL) heated under reflux under nitrogen was added benzyl bromide (0.36 mL, 3 mmol, 3 equiv.) via 52

syringe. The reaction mixture was heated under reflux for 10 min. Benzaldehyde (0.10 mL,

1 mmol, 1 equiv.) was added and heating was continued for 4 h. when the reaction was found to be completed using TLC. The reaction was quenched with 1 M aq HCl solution

(15 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate. After removal of solvent under reduced pressure on a rotary evaporator, the residue was subjected to column chromatography on silica in 10% ethyl acetate in hexanes to obtain 1,2-diphenylethan-1- ol (43)(147 mg, 74.2%). 1H NMR (600 MHz, CDCl3) δ 7.43 – 7.37 (m, 1H), 7.37 – 7.31

(m, 1H), 7.32 – 7.27 (m, 1H), 7.24 (d, J = 7.1 Hz, 1H), 4.93 (dd, J = 8.4, 4.9 Hz, 1H), 3.06

(qd, J = 13.7, 6.6 Hz, 1H), 2.25 – 2.07 (m, 1H).13C NMR (151 MHz, CDCl3) δ 143.85 (s),

138.10 (s), 129.58 (s), 128.56 (s), 128.50 (s), 127.67 (s), 126.65 (s), 125.98 (s), 75.39 (s),

46.30 (s).

1,2-Diphenylethan-1-one (44)

To a stirred solution of 1,2-diphenylethan-1-ol (43) (91 mg, 0.5 mmol, 1 equiv.) in DCM

(20 mL) cooled to 0 oC. was slowly added DMP (214.2 mg, 0.5 mmol, 1.1 equiv.). The reaction mixture was stirred at room temperature overnight. When the reaction was found to be completed) TLC), aqueous NaHCO3 solution was added and the mixture was extracted with ethyl acetate (3 x 30 mL), The combined organic extract was dried over anhydrous sodium sulfate and removal of solvent under reduced pressure gave (44) (80

1 mg, 90%) . H NMR (400 MHz, CDCl3) δ 8.06 – 7.98 (m, 2H), 7.56 (t, J = 7.4 Hz, 1H),

7.45 (dd, J = 15.3, 7.9 Hz, 2H), 7.37 – 7.22 (m, 5H), 4.30 (s, 2H). 13C NMR (151 MHz,

CDCl3) δ 197.65 (s), 136.58 (s), 134.53 (s), 129.48 (s), 128.69 (s), 128.66 (s), 128.63 (s),

126.91 (s), 45.52 (s). 53

2-Bromo-1,2-diphenylethan-1-one (45)

To a stirred solution of 1,2-diphenylethan-1-one (44) (1,96 g, 10 mmol, 1 equiv.) and p- toluene sulfonic acid mono monohydrate (2,85 g, 15 mmol, 1.5 equiv.) in MeCN (300 mL).

Was slowly added NBS (1,96 g, 10 mmol, 1 equiv.) The reaction mixture was refluxed for

2 h when the reaction was found to be complete (TLC). MeCN was removed in vacuo and the residue was partitioned between brine, and DCM. The combined organic extract was dried over anhydrous sodium sulfate and removal of solvent under reduced pressure to give

1 2-bromo-1,2-diphenylethan-1-one (45) (2,75 g,100%). H NMR (400 MHz, CDCl3) δ 8.01

(dd, J = 8.4, 1.1 Hz, 2H), 7.60 – 7.50 (m, 3H), 7.48 – 7.28 (m, 5H), 6.43 (s, 1H).

N-(4,5-diphenyl-1H-imidazol-2-yl)acetamide (46)

A mixture of 2-bromo-1,2-diphenylethan-1-one (45) (272.4 mg, 0.99 mmol, 1 equiv.), 1- acetylguanidine (300.3 mg, 2.9 mmol, 3 equiv.) and MeCN (20 mL) in a 20 mL microwave vial was heated in the microwave synthesizer at 100 oC for 10 minutes. The reaction mixture was concentrated under reduced pressure and the residue was treated with water and filtered. Repeated crystallization of the solid product collected from MeOH/DCM gave

(46) (205.6 mg 74.6%)1H NMR (600 MHz, DMSO) δ 11.61 (s, 1H), 11.19 (s, 1H), 7.48 –

7.16 (m, J = 55.8, 48.0, 19.8, 7.3 Hz, 10H), 2.09 (s, 3H). 13C NMR (151 MHz, DMSO) δ

169.40 (s), 141.51 (s), 135.55 (s), 133.06 (s), 131.44 (s), 129.04 (s), 128.65 (s), 128.52 (s),

127.82 (s), 127.30 (s), 126.84 (s), 123.43 (s), 23.28 (s).

4,5-Diphenyl-1H-imidazol-2-amine (47)

N-(4,5-Diphenyl-1H-imidazol-2-yl)acetamide (46) (277.3 mg, 1 mmol, 1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O (1:1) in a 20 mL microwave vial with a final volume of 20 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 min.

The reaction mixture was concentrated under reduced pressure to remove methanol and filtered. Repeated crystallization of the solid product collected from MeOH/DCM gave

(47) (239 mg 88 %) 1H NMR (600 MHz, MeOD) δ 7.42 (s, 10H). 13C NMR (151 MHz,

MeOD) δ 147.27 (s), 128.74 (s), 127.89 (d, J = 18.5 Hz), 127.56 (s), 122.44 (s).

1-Phenylethan-1-ol (49)

To solution of benzaldehyde (42) (1.02 mL, 10 mmol, 1 equiv.) in THF (30 mL), CH3MgCl

(3.3 mL, 10 mmol, 1equiv.) was slowly added under nitrogen. The reaction mixture was stirred for 4 hrs. at room temperature, when the reaction found to be complete by TLC. 1M

HCl was added to quench the reaction. Brine was added and the reaction mixture was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give compound (49) (725.68 mg 59.4%

1 yield). H NMR (600 MHz, CDCl3) δ 7.38 – 7.30 (m, 4H), 7.29 – 7.21 (m, 1H), 4.85 (dd,

13 J = 12.4, 6.1 Hz, 1H), 2.58 (br, 1H), 1.46 (d, J = 6.4 Hz, 3H). C NMR (151 MHz, CDCl3)

δ 145.74 (s), 128.54 (s), 127.52 (s), 125.40 (s), 70.47 (d, J = 3.0 Hz), 25.08 (s).

Acetophenone (50)

To stirred solution of 1-phenylethan-1-ol (49) (314 mg, 2.57 mmol, 1 equiv.) in DCM

20 mL. cooled to 0 oC, DMP (1199.131 mg, 2.82 mmol, 1.1 equiv.) was slowly added. The reaction mixture was stirred at room temperature overnight. It was found to be complete by TLC. Aqueous NaHCO3 was added, and the mixture was extracted with ethyl acetate 55

(3X 10 mL), The combined organic extract was dried over anhydrous sodium sulfate and removal of solvent under reduced pressure to give compound (50) (193 mg , 63%). 1H

NMR (600 MHz, CDCl3) δ 7.87 – 7.71 (m, 2H), 7.45 – 7.18 (m, 3H), 2.51 (s, 3H). 13C

NMR (151 MHz, CDCl3) δ 197.72 (s), 136.95 (s), 132.94 (s), 128.44 (s), 128.17 (s), 26.39

(s).

2-Bromo-1-phenylethan-1-one (51)

To stirred solution of acetophenone (50) (1.165 mL 10 mmol, 1 equiv.), para toluene sulfonic acid mono monohydrate (2.85 g, 15 mmol, 1.5 equiv.) in MeCN 100 mL. N- bromosuccinimide (1.7 g, 10 mmol, 1 equiv.) was added slowly. The reaction mixture was refluxed for 1- 2 hours. The reaction was found to be completed after 2 hrs. using

TLC. The reaction mixture was concentrated to get rid of MeCN, and worked up with brine, extracted with DCM. The collected organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain compound (51) (1,9 g 97 %). 1H NMR

(600 MHz, CDCl3) δ 8.01 (dd, J = 5.3, 2.9 Hz, 2H), 7.66 – 7.60 (m, 1H), 7.52 (dd, J =

11.1, 4.2 Hz, 2H), 4.49 (s, 2H).13C NMR (151 MHz, CDCl3) δ 191.31 (s), 134.00 (s),

133.95 (s), 128.94 (s), 128.89 (s), 31.07 (s).

N-(5-phenyl-1H-imidazol-2-yl)acetamide (52)

In 20 mL microwave vial, 2-bromo-1-phenylethan-1-one (51) (197 mg, 0.99 mmol, 1 equiv.) and 1-acetylgunidine (300.3 mg, 3 mmol, 3 equiv.) were dissolved in MeCN (20 mL). The vial was heated in the microwave synthesizer at 100 oC for 10 minutes. The reaction mixture was concentrated under reduced pressure, washed with H2O, and filtered. The collected solid mixture was crystallized from MeOH/DCM. Multiple

crystallization gave compound (52) (165.3 mg 83%). 1H NMR (600 MHz, DMSO) δ

11.63 (s, 1H), 11.25 (s, 1H), 7.75 – 7.68 (m, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.26 (s, 1H),

7.16 (t, J = 7.3 Hz, 1H), 2.08 (s, 3H). 13C NMR (151 MHz, DMSO) δ 168.94 (s), 141.70

(s), 136.56 (s), 135.16 (s), 128.87 (s), 126.33 (s), 124.48 (s), 109.65 (s), 23.27 (s).

5-Phenyl-1H-imidazol-2-amine (53)

In 20 mL microwave vial N-(5-phenyl-1H-imidazol-2-yl)acetamide (52) (201.23 mg, 1 mmol,1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O (1:1) with final volume of 20 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 minutes. The reaction mixture was concentrated under reduced pressure to get rid of methanol, and filtered. The collected solid mixture was crystallized from MeOH/DCM. The product (53)

(142.8 mg 74%) was obtained after multiple crystallization. The product was used as salt

1 for the next step. . H NMR (600 MHz, CD3OD) δ 7.57 – 7.52 (m, 2H), 7.41 (t, J = 7.7 Hz,

2H), 7.33 (t, J = 7.4 Hz, 1H), 7.14 (s, 1H). 13C NMR (151 MHz, MeOD) δ 147.88 (s),

128.82 (s), 128.20 (s), 127.62 (s), 127.47 (s), 124.20 (s), 108.39 (s).

2-Bromo-1-(3-nitrophenyl)ethan-1-one (55)

To a stirred solution of 1-(3-nitrophenyl)ethan-1-one (54) (660.6 mg, 4 mmol, 1 equiv.) and p-toluenesulfonic acid mono monohydrate (1.141.32 mg, 6 mmol, 1.5 equiv.) in MeCN

(200 mL) was slowly added NBS (711.9 mg, 4 mmol, 1 equiv.) and the reaction mixture was refluxed for 1- 2 h when the reaction was found to be complete (TLC). MeCN was removed under reduced pressure and the residue was treated with brine and extracted with

DCM. The combined organic layer was dried over anhydrous sodium sulfate, and

concentrated under reduced pressure to give (55) (927.4 mg 95.4%). NMR (600 MHz,

CDCl3) δ 8.75 (d, J = 19.3 Hz, 1H), 8.36 (m, 2H), 7.82 – 7.67 (m, 1H), 2.69 (s,2H).13C

NMR (151 MHz, CDCl3) δ 189.52 (s), 148.36 (s), 135.07 (s), 134.54 (s), 130.30 (s), 128.12

(s), 123.75 (s), 30.48 (s).

N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)acetamide (56-a)

A solution of, 2-bromo-1-(3-nitrophenyl)ethan-1-one (55) (272.4 mg, 0.99 mmol, 1 equiv.) and 1-acetylguanidine (303.3 mg, 3 mmol, 3 equiv. in MeCN (20 mL) in a 20 mL microwave vial was heated in the microwave synthesizer at 100 oC for 10 min. The reaction mixture was concentrated under reduced pressure, the residue was treated with water, and filtered. Repeated crystallization of the solid product collected from MeOH/DCM gave

(56-a) (152.8 mg, 62.72%) as yellow crystals. 1H NMR (600 MHz, DMSO) δ 11.85 (s,

1H), 11.32 (s, 1H), 8.55 – 8.52 (m, 1H), 8.17 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 8.01 (ddd, J

= 8.1, 2.4, 1.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 1.4 Hz, 1H), 6.94 (s, 1H), 2.10

(s, 3H).13C NMR (151 MHz, DMSO) δ 169.10 (s), 148.80 (s), 142.20 (s), 136.93 (s),

134.47 (s), 130.63 (s), 130.46 (s), 120.80 (s), 118.50 (s), 111.92 (s), 23.30 (s).

5-(3-Nitrophenyl)-1H-imidazol-2-amine (56-b)

N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)acetamide (56-a) (70 mg, 0.3 mmol, 1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O (1:1) In a 10 mL microwave vial with a final volume of 6 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 min.

The reaction mixture was concentrated under reduced pressure, and filtered. Repeated crystallization of the solid product from MeOH/DCM gave (56-b) (56 mg, 93%).1H NMR

(600 MHz, DMSO) δ 9.32 (s, 1H), 8.89 (dd, J = 42.4, 7.4 Hz, 2H), 8.47 (t, J = 7.9 Hz, 1H),

8.33 (s, 1H), 8.03 (s, 1H), 7.38 (s, 2H). 58

13C NMR (151 MHz, DMSO) δ 158.66 (s), 150.00 (s), 148.78 (s), 134.05 (s), 130.46 (s),

130.37 (s), 121.05 (s), 118.34 (s), 112.03 (s).

1-(2,3-Dimethoxyphenyl)ethan-1-ol (58)

To mixture of 2,3-dimethoxybenzaldehyde (57) (3 g, 20 mmol, 1 equiv.) in THF (40 mL), methylmagnesium chloride (7.3 mL, 22 mmol, 1.1 equiv.) was injected via syringe under nitrogen. The reaction mixture was refluxed for 2-3 hours, till precipitate was formed. The reaction mixture then was filtered to obtain a solid which later was treated with 1 M HCl to give pure product 1-(2,3-dimethoxyphenyl)ethan-1-ol. The filtrate was worked up with

1 M HCl, and brine, extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give (58) (3.2 g,

1 88.5%). H NMR (600 MHz, CDCl3) δ 7.06 – 7.01 (m, 1H), 7.00 – 6.97 (m, 1H), 6.82 (dd,

J = 8.0, 1.5 Hz, 1H), 5.13 (q, J = 6.5 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 1.46 (d, J = 6.5

13 Hz, 3H). C NMR (151 MHz, CDCl3) δ 152.38 (s), 145.86 (s), 139.28 (s), 124.23 (s),

117.97 (s), 111.36 (s), 65.56 (s), 60.87 (d, J = 4.6 Hz), 55.73 (s), 24.11 (s).

1-(2,3-Dimethoxyphenyl)ethan-1-one (59)

To stirred solution of 1-(2,3-dimethoxyphenyl)ethan-1-ol (58) (8,9 g, 28.98 mmol, 1 equiv.) in DCM (50 mL) cooled to 0 oC, DMP (13 g, 31.8 mmol, 1.1 equiv.) was slowly added. The reaction mixture was stirred at room temperature overnight. It was found to be complete by TLC. Aqueous NaHCO3 was added, and extracted with ethyl acetate (3X 50 mL), The combined organic extract, was dried over anhydrous sodium sulfate and solvent removed under reduced pressure. The crude mixture was then subjected to purification by column chromatography using ethyl acetate and hexane as eluent to get (59) (4,3 g,

1 84.87%). H NMR (600 MHz, CDCl3) δ 7.25 – 7.20 (m, 1H), 7.12 – 7.03 (m, 2H), 3.91 (dt, 59

J = 2.3, 0.7 Hz, 3H), 3.90 (dd, J = 3.6, 1.0 Hz, 3H), 2.65 – 2.63 (m, 3H). 13C NMR (151

MHz, CDCl3) δ 200.43 (s), 153.08 (s), 148.67 (s), 133.70 (s), 124.07 (s), 120.89 (s), 115.81

(s), 61.40 (s), 55.85 (d, J = 57.1 Hz), 31.28 (s).

2-Bromo-1-(2,3-dimethoxyphenyl)ethan-1-one (60)

To stirred solution of, 1-(2,3-dimethoxyphenyl)ethan-1-one (59) (2,7 g, 15 mmol, 1 equiv.), para-toluene sulfonic acid mono monohydrate (4,2 g, 22.6 mmol, 1.5 equiv.) in

MeCN (300 mL) was added slowly NBS (2,6 g, 15 mmol, 1 equiv.). The reaction mixture was refluxed for 1- 2 hours. The reaction was found to be completed after 2 hrs. by TLC.

The reaction mixture was concentrated to remove MeCN, and worked up with brine, extracted with DCM. The collected organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was then subjected to column chromatography using ethyl acetate and DCM as eluent to obtain (60) (1,6 g 42.6 %). 1H

NMR (600 MHz, CDCl3) δ 7.27 – 7.22 (m, 1H), 7.10 – 7.05 (m, 2H), 4.58 (s, 1H), 3.94 (s,

2H), 3.87 (s, 2H).13C NMR (151 MHz, CDCl3) δ 194.28 (s), 153.65 (s), 148.60 (s), 129.80

(s), 124.29 (s), 121.75 (s), 117.22 (s), 61.81 (s), 55.85 (s), 37.07 (s).

N-(5-(2,3-dimethoxyphenyl)-1H-imidazol-2-yl)acetamide (61)

In a 20 mL microwave vial, 2-bromo-1-(2,3-dimethoxyphenyl)ethan-1-one (60) (256.5 mg,

0.99 mmol, 1 equiv.) and 1-acetylguanidine (300.2 mg, 2.9 mmol, 3 equiv.) were dissolved in MeCN (20 mL). The vial was heated in the microwave synthesizer at 100 oC for 10 minutes. The reaction mixture was concentrated under reduced pressure, the residue washed with water, and filtered. The collected solid product was crystallized from

MeOH/DCM. Multiple crystallizations gave (61) (569 mg 55 %)1H NMR (600 MHz,

CDCl3) δ 7.25 – 7.21 (m, 2H), 7.11 (dd, J = 10.1, 6.0 Hz, 1H), 6.89 – 6.86 (m, 1H), 3.94 60

13 (s, 3H), 3.93 (s, 3H), 2.32 (s, 3H). C NMR (151 MHz, CDCl3) δ 169.85 (s), 153.57 (s), 144.84 (s),

142.22 (s), 124.76 (s), 124.43 (s), 120.53 (s), 117.89 (s), 112.64 (s), 111.26 (s), 60.68 (s), 56.07 (s),

23.65 (s).

5-(2,3-Dimethoxyphenyl)-1H-imidazol-2-amine (62)

N-(5-(2,3-dimethoxyphenyl)-1H-imidazol-2-yl)acetamide (61) (216.3mg, 1 mmol, 1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O (1:1) in a 10 mL microwave vial to a final volume of 6 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 min. The reaction mixture was concentrated under reduced pressure and filtered. Repeated crystallization of the solid product from MeOH/DCM gave (62) (217 mg, 99%).1H NMR

(600 MHz, MeOD) δ 7.22 (s, 1H), 7.14 (q, J = 7.7 Hz, 2H), 7.05 (d, J = 6.9 Hz, 1H), 3.90

(s, 3H), 3.85 (s, 3H).13C NMR (151 MHz, MeOD) δ 153.36 (s), 147.32 (s), 145.65 (s),

124.55 (s), 123.81 (s), 120.77 (s), 117.48 (s), 112.57 (s), 111.10 (s), 59.38 (s), 55.20 (s).

1-(1-Methyl-1H-imidazol-2-yl)ethan-1-one (32)

To a solution of 1-methyl-1H-imidazole (33) (8 mL, 110 mmol, 1.1 equiv.) in THF (75 mL) maintained at 0 oC was added n-BuLi (44 mL of , 110 mmol, 1.1 equiv.) over 5 min.

The reaction mixture was stirred for 10 min, after which it was cannulated in to a solution of N-acetyl morpholine (11.6 mL, 100 mmol, 1 equiv.) in THF (100 mL) maintained at

78 oC over 15 min The reaction mixture was stirred for 1 h at -78 oC, after which it was brought to room temperature and stirred for 15 min. 1 M HCl was added and the mixture was stirred for 10 minutes, followed by addition of brine, and NaHCO3. It was extracted with ethyl acetate (3x 50 mL) and the combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give (32) (1,35 g 99%).

1H NMR (600 MHz, CDCl3) δ 6.77 (s, 1H), 6.74 (s, 1H), 3.63 (s, 3H), 2.26 (s, 3H). 61

13C NMR (151 MHz, CDCl3) δ 189.95 (s), 142.76 (s), 128.57 (s), 126.89 (s), 35.79 (s),

26.74 (s).

2-Bromo-1-(1-methyl-1H-imidazol-2-yl)ethan-1-one (64)

Bromine, (457 mg, 2.8 mmol, 1.1 equiv.) was added dropwise to a stirred solution of 1-(1- methyl-1H-imidazol-2-yl)ethan-1-one (32) (322.7 mg, 2.7 mmol, 1 equiv.) in 25% HBr-

AcOH. cooled to 0 oC. The reaction mixture was stirred for 30 min at 0 oC, followed by 1 h at room temperature. The reaction mixture was cooled to 0 oC and anhydrous diethyl ether was poured in to the mixture. The solid precipitate was collected by filtration to obtain

2-bromo-1-(1-methyl-1H-imidazol-2-yl)ethan-1-one (64) as the hydrobromic salt (360 mg,

66%). 1H NMR (600 MHz, DMSO) δ 7.60 (s, 1H), 7.19 (s, 1H), 4.78 (s, 2H), 3.91 (s, 3H).

13C NMR (151 MHz, MeOD) δ 144.18 (s), 125.62 (s), 118.34 (s), 35.16 (s), 34.58 (s).

N-(1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-yl)acetamide (65)

2-Bromo-1-(1-methyl-1H-imidazol-2-yl)ethan-1-one (64) (280 mg, 0.99 mmol, 1 equiv.) and 1-acetylguanidine (400.3 mg, 3.96 mmol, 4 equiv.) were dissolved in MeCN (20 mL) in a 20 mL microwave vial. The vial was heated in the microwave synthesizer at 100 oC for 12 h. The reaction mixture was filtered and MeCN was removed under reduced pressure. Repeated crystallization from ethyl acetate/ hexanes. gave (65) (184.6 mg of

90%). 1H NMR (600 MHz, MeOD) δ 7.29 (s, 1H), 7.12 (s, 1H), 7.01 (s, 1H), 3.93 (s, 4H),

2.18 (s, 4H).13C NMR (151 MHz, MeOD) δ 170.17 (s), 141.66 (s), 125.37 (s), 122.00 (s),

33.64 (s), 21.46 (s).

Zinc binding study using compound (65)

1mM stock solutions of compound (65) and zinc acetate in methanol were made in two 50 mL volumetric flasks. Mixtures of 2.5 mL of the test solution 65 and different volumes of the zinc acetate solutions ranging from 0 mL – 2.5 mL (increasing by 0.25 mL each time) were made in 25 mL volumetric flasks. These solutions were diluted to 25 mL, giving final test solutions with 1/10th the concentration of the stock solution of test compound (65) and concentrations of the zinc acetate varying from 0.1 – 1.0 molar equivalents. After at least

1 hour, the UV spectra of the final test solutions were recorded using a UV spectrophotometer.

1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66)

N-(1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-yl)acetamide (65) (184.7mg, 0.99 mmol, 1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O In a 20 mL microwave vial with a final volume of 20 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 min. The reaction mixture was concentrated under reduced pressure and filtered. Repeated crystallization of the solid product from MeOH/DCM gave compound (66) (42 mg,

1 21%) H NMR (600 MHz, CD3OD) δ 7.72 (s, 1H), 7.69 (s, 1H), 7.62 (s, 1H), 3.94 (s, 3H).

13C NMR (151 MHz, MeOD) δ 158.51 (s), 148.98 (s), 133.92 (s), 125.41 (s), 119.69 (s),

119.07 (s), 35.32 (s).

1H-Phenanthro[9,10-d]imidazol-2-amine room temperature synthesis (68)

Phenanthrene-9,10-dione (67) (208 mg, 1 mmol, 1 equiv.) and guanidinium chloride (95.5 mg, 1 mmol, 1 equiv.) were dissolved in a 0.25 M solution of NaOH in a mixture of ethanol and H2O (4:1). The reaction mixture was stirred at room temperature for 3 h. The pH of the reaction mixture was adjusted to 3 and Norite was added to quench the color. After filtration and concentration under reduced pressure, the solid formed was collected by 63

filtration. Repeated crystallizations from ethanol gave (68) as a solid (29.5 mg, 11%). 1H

NMR (600 MHz, MeOD) δ 8.86 (d, J = 8.2 Hz, 1H), 8.24 – 8.18 (m, 2H), 7.81 (t, J = 9.0

Hz, 1H), 7.74 (tdd, J = 8.3, 7.5, 1.2 Hz, 2H), 7.61 (dqd, J = 14.7, 7.4, 1.3 Hz, 2H). 13C

NMR (151 MHz, MeOD) δ 157.88 (s), 130.75 (s), 129.77 (s), 128.53 (s), 128.05 (s), 127.66

(s), 127.52 (s), 126.58 (s), 126.04 (s), 124.00 (s), 123.78 (s), 122.06 (s), 120.59 (s), 120.47

(s).

1H-Phenanthro[9,10-d]imidazole (78)

To phenanthrene-9,10-dione (67) (1 g, 5 mmol, 1 eqiuv.), urotropine (140.19 mg, 1 mmol,

0.2 equiv.) , and NH4OAc (3 g, 43 mmol, 8.6 equiv.) were mixed in a 20 mL microwave vial. A few drops of AcOH were added, till it became paste. The mixture was heated in a microwave synthesizer at 112 oC four minutes. The mixture was poured in to water (75 mL) and aq. NH3 was added until the pH reached 8. Products was collected by filtration to obtain (78) (1.0 g, 99%).1H NMR (600 MHz, DMSO) δ 13.44 (s, 1H), 8.85 (dd, J = 22.2,

8.2 Hz, 2H), 8.51 (t, J = 11.4 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.32 (s, 1H), 7.72 (q, J =

7.7 Hz, 2H), 7.61 (dd, J = 15.9, 8.5 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 139.71 (s),

136.43 (s), 128.01 (s), 127.79 (s), 127.57 (s), 127.52 (s), 126.45 (s), 125.69 (s), 125.39 (s),

124.54 (s), 124.17 (s), 123.09 (s), 122.21 (s), 122.07 (s).

1-Benzyl-1H-phenanthro[9,10-d]imidazole (79)

NaH (60% in oil, 204.7 mg, 5.1 mmol, 1.05 equiv.) was added to a DMF (5 mL) solution of 1H-phenanthro[9,10-d]imidazole (78) (1 g, 4.9 mmol, 1 equiv.) at 0 oC, and the resulting suspension was stirred at room temperature for 2 h . Benzyl bromide (0.6 mL, 4.9 mmol,

1equiv.) was added and the mixture was stirred at room temperature for another 4 h . The solvent was removed under reduced pressure and the residue was partitioned between brine 64

and DCM. Concentration of the organic layer after drying over sodium sulfate gave (79)

(1,5g 96%).1H NMR (600 MHz, CDCl3) δ 8.81 – 8.76 (m, 2H), 8.71 (d, J = 8.3 Hz, 1H),

8.07 – 8.00 (m, 2H), 7.79 – 7.74 (m, 1H), 7.67 (ddd, J = 8.3, 7.1, 1.4 Hz, 1H), 7.58 (ddd, J

= 8.3, 7.1, 1.2 Hz, 1H), 7.51 – 7.45 (m, 1H), 7.39 – 7.25 (m, 3H), 7.18 (d, J = 7.2 Hz, 2H),

5.88 (s, 2H).

13C NMR (151 MHz, CDCl3) δ 142.13 (s), 138.46 (s), 135.63 (s), 129.31 (s), 129.15 (s),

128.22 (s), 127.48 (s), 127.19 (s), 126.76 (s), 126.19 (s), 125.74 (s), 125.16 (s), 124.30 (s),

123.12 (s), 122.81 (s), 122.51 (s), 121.21 (s), 51.43 (s).

2-Azido-1-benzyl-1H-phenanthro[9,10-d]imidazole (80)

A solution of 1-benzyl-1H-phenanthro[9,10-d]imidazole (79) (1,5 g, 4.7 mmol, 1 equiv.) in dry THF (20 mL) was cooled to -78 °C, followed by addition of n-BuLi (2.1 equiv. , 1.6

M solution in hexane). The cooling bath was removed for 5 minutes. The mixture was re- cooled again to -75 °C, tosyl azide (1,4 g, 7 mmol, 1.5 equiv.) in THF (5 mL) was added slowly to the stirred solution. After 10 min, water was added, and the reaction mixture was extracted three times with DCM. The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography using Hexane/DCM to obtain (80) (788 mg, 48%). 1H NMR (600 MHz,

CDCl3) δ 8.79 (d, J = 8.2 Hz, 1H), 8.73 – 8.65 (m, 2H), 7.97 (dd, J = 8.2, 0.7 Hz, 1H), 7.76

– 7.72 (m, 1H), 7.66 (ddd, J = 7.0, 6.1, 1.4 Hz, 1H), 7.54 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H),

7.46 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.36 – 7.33 (m, 2H), 7.32 – 7.28 (m, 3H), 7.18 (d, J =

7.2 Hz, 2H), 5.71 (s, 2H). 13C NMR (151 MHz, CDCl3) δ 145.45 (s), 136.18 (s), 135.72

(s), 129.21 (s), 128.44 (s), 127.86 (s), 127.86 (s), 127.28 (s), 126.77 (s), 126.48 (s), 125.71

(s), 125.56 (s), 125.66 (s), 124.65 (s), 124.15 (s), 123.09 (s), 122.47 (s), 122.36 (s), 120.17

(s), 48.57 (s).

1H-Phenanthro[9,10-d]imidazol-2-amine (68)

A mixture of 2-azido-1-benzyl-1H-phenanthro[9,10-d]imidazole (212 mg, 0.6 mmol, 1 equiv.), and palladium on carbon (21 mg, 10% wt. ) in ethyl-acetate (20 mL) was stirred under hydrogen at 2 atmospheric pressure for 12 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The product obtained is currently under spectroscopic confirmation.

4-Methylbenzenesulfonyl azide (82)

To solution of tosyl chloride (81) (10.0 g, 52.46 mmol, 1.0 eq) in acetone (100 mL) and water (50 mL) maintained at 0 oC was added NaN3 (3.58 g, 55.09 mmol, 1.05 eq) and the reaction mixture was stirred at room temperature over 4 hrs. The reaction mixture was concentrated under reduced pressure to remove acetone. The aqueous layer was washed with DCM (3×50 mL). The combined organic extract was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain tosyl azide as a transparent oil

(82) (10 g, 98%). The NMR spectral data are consistent with those reported in the is literature87

1,10-Phenanthroline-5,6-dione (83-b)

A mixture of 1,10-phenanthroline(83-a), (1.0 g, 5.5 mmol, 1 equiv.), and KBr (6.56 g , 55 mmol was added slowly to concentrated sulfuric acid (20 mL) cooled to 0 o`C.) . Nitric acid

(10 mL) was added and the reaction mixture was heated at 90 o`C for 2 h. The reaction mixture was allowed to cool to room temperature and poured onto ice, and NaHCO3 was added to bring it to pH 6-7. It was extracted with DCM (3 x 40mL) and the combined 66

organic layer was concentrated under reduced pressure. The solid formed was recrystallized from ethanol and dichloromethane to give (83-b) (936 mg, 81%).1H NMR

(600 MHz, CDCl3) δ 9.16 (dd, J = 4.7, 1.8 Hz, 2H), 8.55 (dd, J = 7.9, 1.8 Hz, 2H), 7.63

13 (dd, J = 7.8, 4.7 Hz, 2H). C NMR (151 MHz, CDCl3) δ 178.71 (s), 156.50 (s), 152.94 (s),

137.35 (s), 128.11 (s), 125.69 (s).

1H-imidazo[4,5-f][1,10]phenanthroline (84)

In a round bottom flask 1,10-phenanthroline-5,6-dione (83-b), (700 mg 3.5 mmol, 1 equiv.)

, urotropin ( 981.302 mg, 7 mmol, 2 equiv.) and of ammoniumacetate (1,4 gm, 17 mmol,

5 equiv.) were suspended in 10 mL of glacial acetic acid. After refluxing the resulting mixture for two hours, the solvent was removed under reduced pressure. The resulting solid was dissolved in water and neutralized with ammonia. Stirring this mixture over night yielded an precipitate. The solid was filtered off, and washed twice with water and dried under vacuum to give (84) (560 mg 77%). 1H NMR (600 MHz, DMSO) δ 9.05 – 9.02 (m,

1H), 8.87 (dd, J = 8.0, 1.6 Hz, 1H), 8.78 (t, J = 7.9 Hz, 1H), 8.47 (s, 1H), 7.83 (ddd, J =

17.9, 8.0, 4.3 Hz, 1H).13C NMR (151 MHz, DMSO) δ 148.15 (s), 144.43 (s), 135.4 (s),

129.70 (s), 123.91 (s), 120. (s).

1-Benzyl-1H-imidazo[4,5-f][1,10]phenanthroline (85)

1H-imidazo[4,5-f][1,10]phenanthroline (84) (560 mg, 2.5 mmol, 1equiv.) was treated with sodium hydride (83.9 mg of 60% dispersion in paraffin, 3.5 mmol, 1.4 equiv.) under nitrogen atmosphere in DMF (4 mL). Benzyl bromide (0.35 mL, 2.9 mmol, 1.19 equiv.) was added to the stirred reaction mixture sodium. After stirring for three hours at room temperature, the solvent was removed under vacuum and the residue was redissolved in 67

chloroform and washed with water. The organic phase was dried over Na2SO4, and concentrated to give (85).

N-(4-methyl-5-phenyl-1H-imidazol-2-yl)acetamide (90)

A mixture of 20 mL, 2-bromo-1-phenylpropan-1-one (88) (0.156 mL, 0.99 mmol, 1 equiv.) and 1-acetylguanidine (300.3mg, 2.9 mmol, 3 equiv.) and MeCN (20 mL) in a microwave vial was heated in the microwave synthesizer at 100 oC for 10 min. The reaction mixture was concentrated under reduced pressure and the residue treated with water and filtered.

Repeated crystallization of the solid product collected from MeOH/DCM gave (90) (16.9 mg, 8%.). 1H NMR (600 MHz, CDCl3) δ 7.56 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H),

7.29 (dd, J = 9.0, 6.9 Hz, 1H), 2.44 (s, 2H), 1.82 (s, 2H). 13C NMR (151 MHz, DMSO) δ

168.66 (s), 139.74 (s), 136.24 (s), 131.34 (s), 128.73 (s), 126.03 (s), 125.65 (s), 119.49 (s),

23.25 (s), 11.61 (s).

4-Methyl-5-phenyl-1H-imidazol-2-amine (91)

N-(4-methyl-5-phenyl-1H-imidazol-2-yl)acetamide (90) (62 mg, 0.3 mmol, 1 equiv.) was dissolved in 20% of HCl in MeOH/ H2O (1:1) in a 10 mL microwave vial with a final volume of 6 mL. The vial was heated in the microwave synthesizer at 100 oC for 10 min.

The reaction mixture was concentrated under reduced pressure, and filtered. Repeated crystallization of the solid product from MeOH/DCM gave (91) (50 mg, 97%).1H NMR

(600 MHz, MeOD) δ 7.44 (d, J = 51.2 Hz, 5H), 2.31 (s, 3H). 13C NMR (151 MHz, MeOD)

δ 146.52 (s), 128.32 (s), 127.87 (s), 126.41 – 125.77 (m), 122.00 (s), 118.57 (s), 8.61 (s).

(E)-N-(1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1- en-1-yl)benzamide. 15

(E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoic acid (28)(100 mg, 0.4 mmol, 1 equiv.) was dissolved in DMF (0.7 mL under nitrogen). HATU (177.8 mg, 0.5 mmol, 1.2 equiv.) , and HOBt (68.50 mg , 0.56 mmol, 1.3 equiv.) were dissolved in DMF

(0.9mL), and transferred to the reaction flask via syringe, followed by a solution of 1H- benzo[d]imidazol-2-amine (57.4 mg, 0.4 mmol, 1 equiv.) in DMF (0.9mL). The reaction mixture was stirred for 20 minutes at room temperature before NMM (0.23 mL, 2.9 mmol,

3 equiv.) was added via syringe. The mixture was stirred overnight at room temperature.

Water (1.5-2 mL) was added and the solid product formed, collected by filtration, to give

(15) (126 mg, 34%). 1H NMR (600 MHz, DMSO) δ 12.38 (s, 1H), 8.22 (d, J = 8.2 Hz, 2H),

8.15 (d, J = 16.1 Hz, 1H), 7.92 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 16.1 Hz, 1H), 7.61 (s, 1H),

7.46 (dd, J = 5.7, 3.2 Hz, 2H), 7.24 (s, 1H), 7.16 (dd, J = 5.8, 3.1 Hz, 2H), 4.04 (s, 3H). 13C

NMR (151 MHz, DMSO) δ 179.65 (s), 143.74 (s), 141.43 (s), 137.95 (s), 130.23 (s), 129.66

(s), 129.48 (s), 129.34 (s), 129.21 (s), 128.87 (s), 125.00 (s), 122.26 (s), 36.34 (s).

(E)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3- oxoprop-1-en-1-yl)benzamide (16)

To a solution of (E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoic acid

(28) (100 mg, 0.4 mmol, 1 equiv.) in DMF (0.7 mL) under nitrogen was added a solution of . HATU (177.8 mg, 0.5 mmol, 1.2 equiv.) and HOBt (68.5 mg , 0.56 mmol, 1.3 equiv.) in DMF (0.9mL via syringe , followed by a solution of 1-methyl-1H-benzo[d]imidazol-2- amine (147.2 mg, 0.4 mmol, 1equiv.) in DMF (0.9mL) . The reaction mixture was stirred for 20 min at room temperature before NMM (0.13 mL, 1.2 mmol, 3 equiv.) was added via syringe. The mixture was stirred overnight at room temperature. Water (0.6-1 mL) was added and the solid product formed was collected by filtration, to give (16) (34. mg, 22.6 69

%). 1H NMR (600 MHz, DMSO) δ 8.32 (d, J = 8.3 Hz, 1=2H), 8.12 (d, J = 16.1 Hz, 1H),

7.89 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 16.0 Hz, 1H), 7.62 (s, 1H), 7.54 (dd, J = 22.8, 7.6 Hz,

2H), 7.33 – 7.18 (m, 3H), 4.04 (s, 3H), 3.77 (d, J = 2.7 Hz, 3H). 13C NMR (151 MHz,

DMSO) δ 179.59 (s), 143.55 (s), 142.18 (s), 137.33 (s), 129.87 (s), 129.78 (s), 129.72 –

129.70 (m), 129.34 (s), 129.25 (s), 128.75 (s), 124.44 (s), 123.25 (s), 110.17 (s, J = 10.5

Hz), 40.45 (s, J = 16.1 Hz), 36.39 (s).

Chapter 9

Spectroscopic data

Methyl 4-formylbenzoate (30)

Methyl (E)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoate (29)

(E)-4-(3-(1-Methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)benzoic acid (28)

1,2-Diphenylethan-1-ol (43)

5-1,2-Diphenylethan-1-one (44)

2-Bromo-1,2-diphenylethan-1-one (45)

N-(4,5-diphenyl-1H-imidazol-2-yl)acetamide (46)

4,5-Diphenyl-1H-imidazol-2-amine (47)

1-Phenylethan-1-ol (49)

Acetophenone (50)

2-Bromo-1-phenylethan-1-one (51)

N-(5-phenyl-1H-imidazol-2-yl)acetamide (52)

5-Phenyl-1H-imidazol-2-amine (53)

2-Bromo-1-(3-nitrophenyl)ethan-1-one (55)

N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)acetamide (56-a)

5-(3-Nitrophenyl)-1H-imidazol-2-amine : (56-b)

1-(2,3-Dimethoxyphenyl)ethan-1-ol (58)

1-(2,3-Dimethoxyphenyl)ethan-1-one (59)

2-Bromo-1-(2,3-dimethoxyphenyl)ethan-1-one (60)

N-(5-(2,3-dimethoxyphenyl)-1H-imidazol-2-yl)acetamide (61)

5-(2,3-Dimethoxyphenyl)-1H-imidazol-2-amine (62)

1-(1-Methyl-1H-imidazol-2-yl)ethan-1-one (32)

2-Bromo-1-(1-methyl-1H-imidazol-2-yl)ethan-1-one (64)

N-(1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-yl)acetamide (65)

1-Methyl-1H,3'H-[2,4'-biimidazol]-2'-amine (66)

1H-Phenanthro[9,10-d]imidazol-2-amine room temperature synthesis (68)

1H-Phenanthro[9,10-d]imidazole (78)

1-Benzyl-1H-phenanthro[9,10-d]imidazole (79)

2-Azido-1-benzyl-1H-phenanthro[9,10-d]imidazole (80)

100

1,10-Phenanthroline-5,6-dione (83-b)

101

1H-imidazo[4,5-f][1,10]phenanthroline (84)

102

N-(4-methyl-5-phenyl-1H-imidazol-2-yl)acetamide (90)

103

4-Methyl-5-phenyl-1H-imidazol-2-amine (91)

104

(E)-N-(1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1- yl)benzamide (15)

105

(E)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)-4-(3-(1-methyl-1H-imidazol-2-yl)-3 oxoprop-1-en-1-yl)benzamide (16)

106

References

(1) Waddington, C. H. Reprints and Reflections. The Epigenotype. Int. J. Epidemiol. 2012. https://doi.org/10.1093/ije/dyr184. (2) Berger, S. L.; Kouzarides, T.; Shiekhattar, R.; Shilatifard, A. An Operational Definition of Epigenetics. Genes Dev. 2009. https://doi.org/10.1101/gad.1787609. (3) Rodríguez-Paredes, M.; Esteller, M. Cancer Epigenetics Reaches Mainstream Oncology. Nat. Med. 2011, 17 (3), 330–339. https://doi.org/10.1038/nm.2305. (4) Feinberg, A. P. Cancer Epigenetics Is No Mickey Mouse. Cancer Cell. 2005. https://doi.org/10.1016/j.ccr.2005.09.014. (5) Kouzarides, T. Chromatin Modifications and Their Function. Cell. 2007. https://doi.org/10.1016/j.cell.2007.02.005. (6) Fraga, M. F.; Ballestar, E.; Villar-Garea, A.; Boix-Chornet, M.; Espada, J.; Schotta, G.; Bonaldi, T.; Haydon, C.; Ropero, S.; Petrie, K.; et al. Loss of Acetylation at Lys16 and Trimethylation at Lys20 of Histone H4 Is a Common Hallmark of Human Cancer. Nat. Genet. 2005, 37 (4), 391–400. https://doi.org/10.1038/ng1531. (7) Chan, E. M.; Chan, R. J.; Comer, E. M.; Goulet, R. J.; Crean, C. D.; Brown, Z. D.; Fruehwald, A. M.; Yang, Z.; Boswell, H. S.; Nakshatri, H.; et al. MOZ and MOZ- CBP Cooperate with NF-ΚB to Activate Transcription from NF-ΚB-Dependent Promoters. Exp. Hematol. 2007. https://doi.org/10.1016/j.exphem.2007.07.015. (8) Yang, X. J. The Diverse Superfamily of Lysine Acetyltransferases and Their Roles in Leukemia and Other Diseases. Nucleic Acids Research. 2004. https://doi.org/10.1093/nar/gkh252. (9) Gayther, S. A.; Batley, S. J.; Linger, L.; Bannister, A.; Thorpe, K.; Chin, S. F.; Daigo, Y.; Russell, P.; Wilson, A.; Sowter, H. M.; et al. Mutations Truncating the EP300 Acetylase in Human Cancers. Nat. Genet. 2000, 24 (3), 300–303. https://doi.org/10.1038/73536. (10) Gorrini, C.; Squatrito, M.; Luise, C.; Syed, N.; Perna, D.; Wark, L.; Martinato, F.; Sardella, D.; Verrecchia, A.; Bennett, S.; et al. Tip60 Is a Haplo-Insufficient Tumour Suppressor Required for an Oncogene-Induced DNA Damage Response. Nature 2007. https://doi.org/10.1038/nature06055. (11) Roth, S. Y.; Denu, J. M.; Allis, C. D. Histone Acetyltransferases. Annu. Rev. Biochem. 2001. https://doi.org/10.1146/annurev.biochem.70.1.81. (12) Shirakawa, K.; Chavez, L.; Hakre, S.; Calvanese, V.; Verdin, E. Reactivation of Latent HIV by Histone Deacetylase Inhibitors. Trends Microbiol. 2013, 21 (6), 277–285. https://doi.org/10.1016/j.tim.2013.02.005. (13) Liu, N.; Xiong, Y.; Li, S.; Ren, Y.; He, Q.; Gao, S.; Zhou, J.; Shui, W. New HDAC6-Mediated Deacetylation Sites of Tubulin in the Mouse Brain Identified by 107

Quantitative Mass Spectrometry. Sci. Rep. 2015. https://doi.org/10.1038/srep16869. (14) Gao, L.; Cueto, M. A.; Asselbergs, F.; Atadja, P. Cloning and Functional Characterization of HDAC11, a Novel Member of the Human Histone Deacetylase Family. J. Biol. Chem. 2002. https://doi.org/10.1074/jbc.M111871200. (15) Shirakawa, K.; Chavez, L.; Hakre, S.; Calvanese, V.; Verdin, E. Reactivation of Latent HIV by Histone Deacetylase Inhibitors. Trends Microbiol. 2013, 21 (6), 277–285. https://doi.org/10.1016/j.tim.2013.02.005. (16) Pandolfi, P. P. Histone Deacetylases and Transcriptional Therapy with Their Inhibitors. Cancer Chemother. Pharmacol. 2001. https://doi.org/10.1007/s002800100322. (17) Lin, R. J.; Sternsdorf, T.; Tini, M.; Evans, R. M. Transcriptional Regulation in Acute Promyelocytic Leukemia. Oncogene. 2001. https://doi.org/10.1038/sj.onc.1204853. (18) Pasqualucci, L.; Bereschenko, O.; Niu, H.; Klein, U.; Basso, K.; Guglielmino, R.; Cattoretti, G.; Dalla-Favera, R. Molecular Pathogenesis of Non-Hodgkin’s Lymphoma: The Role of Bcl-6. Leukemia and Lymphoma. 2003. https://doi.org/10.1080/10428190310001621588. (19) Halkidou, K.; Gaughan, L.; Cook, S.; Leung, H. Y.; Neal, D. E.; Robson, C. N. Upregulation and Nuclear Recruitment of HDAC1 in Hormone Refractory Prostate Cancer. Prostate 2004, 59 (2), 177–189. https://doi.org/10.1002/pros.20022. (20) Choi, J. H.; Kwon, H. J.; Yoon, B. I.; Kim, J. H.; Han, S. U.; Joo, H. J.; Kim, D. Y. Expression Profile of Histone Deacetylase 1 in Gastric Cancer Tissues. Japanese J. Cancer Res. 2001. https://doi.org/10.1111/j.1349-7006.2001.tb02153.x. (21) Mariadason, J. M.; Wilson, A. J.; L’Italien, K.; Velcich, A.; Murray, L. B.; Arango, D.; Sowa, Y.; Augenlicht, L. H.; Byun, D.-S.; Popova, N. Histone Deacetylase 3 (HDAC3) and Other Class I HDACs Regulate Colon Cell Maturation and P21 Expression and Are Deregulated in Human Colon Cancer. J. Biol. Chem. 2006. https://doi.org/10.1074/jbc.m510023200. (22) Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Ando, Y.; Mita, K.; Hamaguchi, M.; Hara, Y.; Kobayashi, S.; Iwase, H. Quantitation of HDAC1 MRNA Expression in Invasive Carcinoma of the Breast. Breast Cancer Res. Treat. 2005. https://doi.org/10.1007/s10549-005-6001-1. (23) Zhu, P.; Martin, E.; Mengwasser, J.; Schlag, P.; Janssen, K. P.; Göttlicher, M. Induction of HDAC2 Expression upon Loss of APC in Colorectal Tumorigenesis. Cancer Cell 2004. https://doi.org/10.1016/S1535-6108(04)00114-X. (24) Huang, B. H.; Laban, M.; Leung, C. H. W.; Lee, L.; Lee, C. K.; Salto-Tellez, M.; Raju, G. C.; Hooi, S. C. Inhibition of Histone Deacetylase 2 Increases Apoptosis and P21 Cip1/WAF1 Expression, Independent of Histone Deacetylase 1. Cell Death Differ. 2005. https://doi.org/10.1038/sj.cdd.4401567. (25) Song, J.; Noh, J. H.; Lee, J. H.; Eun, J. W.; Ahn, Y. M.; Kim, S. Y.; Hyung Lee, S.; Park, W. S.; Yoo, N. J.; Lee, J. Y.; et al. Increased Expression of Histone Deacetylase 2 Is Found in Human Gastric Cancer. APMIS 2005. https://doi.org/10.1111/j.1600-0463.2005.apm_04.x. (26) Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Omoto, Y.; Ando, Y.; Mita, 108

K.; Hamaguchi, M.; Hayashi, S. I.; Iwase, H. HDAC6 Expression Is Correlated with Better Survival in Breast Cancer. Clin. Cancer Res. 2004. https://doi.org/10.1158/1078-0432.CCR-04-0455. (27) Glaser, K. B.; Li, J.; Staver, M. J.; Wei, R. Q.; Albert, D. H.; Davidsen, S. K. Role of Class I and Class II Histone Deacetylases in Carcinoma Cells Using SiRNA. Biochem. Biophys. Res. Commun. 2003. https://doi.org/10.1016/j.bbrc.2003.09.043. (28) Fraga, M. F.; Ballestar, E.; Villar-Garea, A.; Boix-Chornet, M.; Espada, J.; Schotta, G.; Bonaldi, T.; Haydon, C.; Ropero, S.; Petrie, K.; et al. Loss of Acetylation at Lys16 and Trimethylation at Lys20 of Histone H4 Is a Common Hallmark of Human Cancer. Nat. Genet. 2005. https://doi.org/10.1038/ng1531. (29) Minucci, S.; Pelicci, P. G. Histone Deacetylase Inhibitors and the Promise of Epigenetic (and More) Treatments for Cancer. Nature Reviews Cancer. 2006. https://doi.org/10.1038/nrc1779. (30) Johnstone, R. W. Histone-Deacetylase Inhibitors: Novel Drugs for the Treatment of Cancer. Nat. Rev. Drug Discov. 2002, 1 (4), 287–299. https://doi.org/10.1038/nrd772. (31) Cory, S.; Adams, J. M. The BCL2 Family: Regulators of the Cellular Life-or- Death Switch. Nature Reviews Cancer. 2002. https://doi.org/10.1038/nrc883. (32) van Delft, M. F.; Wei, A. H.; Mason, K. D.; Vandenberg, C. J.; Chen, L.; Czabotar, P. E.; Willis, S. N.; Scott, C. L.; Day, C. L.; Cory, S.; et al. The BH3 Mimetic ABT-737 Targets Selective Bcl-2 Proteins and Efficiently Induces Apoptosis via Bak/Bax If Mcl-1 Is Neutralized. Cancer Cell 2006. https://doi.org/10.1016/j.ccr.2006.08.027. (33) Glick, R. D.; Swendeman, S. L.; Coffey, D. C.; Rifkind, R. A.; Marks, P. A.; Richon, V. M.; La Quaglia, M. P. Hybrid Polar Histone Deacetylase Inhibitor Induces Apoptosis and CD95/CD95 Ligand Expression in Human Neuroblastoma. Cancer Res. 1999, 59 (17), 4392–4399. (34) Kim, Y. B.; Lee, K. H.; Sugita, K.; Yoshida, M.; Horinouchi, S. Oxamflatin Is a Novel Antitumor Compound That Inhibits Mammalian Histone Deacetylase. Oncogene 1999. https://doi.org/10.1038/sj.onc.1202564. (35) Sandor, V.; Senderowicz, A.; Mertins, S.; Sackett, D.; Sausville, E.; Blagosklonny, M. V; Bates, S. E. P21-Dependent g(1)Arrest with Downregulation of Cyclin D1 and Upregulation of Cyclin E by the Histone Deacetylase Inhibitor FR901228. Br. J. Cancer 2000, 83 (6), 817–825. https://doi.org/10.1054/bjoc.2000.1327. (36) Chen, Z.; Clark, S.; Birkeland, M.; Sung, C. M.; Lago, A.; Liu, R.; Kirkpatrick, R.; Johanson, K.; Winkler, J. D.; Hu, E. Induction and Superinduction of Growth Arrest and DNA Damage Gene 45 (GADD45) α and β Messenger RNAs by Histone Deacetylase Inhibitors Trichostatin A (TSA) and Butyrate in SW620 Human Colon Carcinoma Cells. Cancer Lett. 2002. https://doi.org/10.1016/S0304- 3835(02)00322-1. (37) Jaboin, J.; Wild, J.; Hamidi, H.; Khanna, C.; Kim, C. J.; Robey, R.; Bates, S. E.; Thiele, C. J. MS-27-275, an Inhibitor of Histone Deacetylase, Has Marked in Vitro and in Vivo Antitumor Activity against Pediatric Solid Tumors. Cancer Res. 2002, 62 (21), 6108–6115. 109

(38) Peart, M. J.; Tainton, K. M.; Ruefli, A. A.; Dear, A. E.; Sedelies, K. A.; O’Reilly, L. A.; Waterhouse, N. J.; Trapani, J. A.; Johnstone, R. W. Novel Mechanisms of Apoptosis Induced by Histone Deacetylase Inhibitors. Cancer Res. 2003, 63 (15), 4460–4471. (39) Qiu, L.; Burgess, A.; Fairlie, D. P.; Leonard, H.; Parsons, P. G.; Gabrielli, B. G. Histone Deacetylase Inhibitors Trigger a G2 Checkpoint in Normal Cells That Is Defective in Tumor Cells. Mol. Biol. Cell 2000. https://doi.org/10.1091/mbc.11.6.2069. (40) Qian, D. Z.; Kato, Y.; Shabbeer, S.; Wei, Y.; Verheul, H. M. W.; Salumbides, B.; Sanni, T.; Atadja, P.; Pili, R. Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: The Hydroxamic Acid Derivative LBH589. Clin. Cancer Res. 2006. https://doi.org/10.1158/1078-0432.CCR-05-1132. (41) Shalinsky, D. R.; Bischoff, E. D.; Gregory, M. L.; Gottardis, M. M.; Hayes, J. S.; Lamph, W. W.; Heyman, R. A.; Shirley, M. A.; Cooke, T. A.; Davies, P. J. Retinoid-Induced Suppression of Squamous Cell Differentiation in Human Oral Squamous Cell Carcinoma Xenografts (Line 1483) in Athymic Nude Mice. Cancer Res. 1995, 55 (14), 3183–3191. (42) Deroanne, C. F.; Bonjean, K.; Servotte, S.; Devy, L.; Colige, A.; Clausse, N.; Blacher, S.; Verdin, E.; Foidart, J. M.; Nusgens, B. V.; et al. Histone Deacetylases Inhibitors as Anti-Angiogenic Agents Altering Vascular Endothelial Growth Factor Signaling. Oncogene 2002. https://doi.org/10.1038/sj.onc.1205108. (43) Kim, S. H.; Ahn, S.; Han, J. W.; Lee, H. W.; Lee, H. Y.; Lee, Y. W.; Kim, M. R.; Kim, K. W.; Kim, W. B.; Hong, S. Apicidin Is a Histone Deacetylase Inhibitor with Anti-Invasive and Anti-Angiogenic Potentials. Biochem. Biophys. Res. Commun. 2004. https://doi.org/10.1016/j.bbrc.2004.01.149. (44) Liu, L.-T.; Chang, H.-C.; Chiang, L.-C.; Hung, W.-C. Histone Deacetylase Inhibitor Up-Regulates RECK to Inhibit MMP-2 Activation and Cancer Cell Invasion. Cancer Res. 2003, 63 (12), 3069–3072. (45) Klisovic, D. D.; Klisovic, M. I.; Effron, D.; Liu, S.; Marcucci, G.; Katz, S. E. Depsipeptide Inhibits Migration of Primary and Metastatic Uveal Melanoma Cell Lines in Vitro: A Potential Strategy for Uveal Melanoma. Melanoma Res. 2005, 15 (3), 147–153. (46) Maeda, T.; Towatari, M.; Kosugi, H.; Saito, H. Up-Regulation of Costimulatory/Adhesion Molecules by Histone Deacetylase Inhibitors in Acute Myeloid Leukemia Cells. Blood 2000. (47) Magner, W. J.; Kazim, A. L.; Stewart, C.; Romano, M. A.; Catalano, G.; Grande, C.; Keiser, N.; Santaniello, F.; Tomasi, T. B. Activation of MHC Class I, II, and CD40 Gene Expression by Histone Deacetylase Inhibitors. J. Immunol. 2000, 165 (12), 7017–7024. https://doi.org/10.4049/jimmunol.165.12.7017. (48) Armeanu, S.; Bitzer, M.; Lauer, U. M.; Venturelli, S.; Pathil, A.; Krusch, M.; Kaiser, S.; Jobst, J.; Smirnow, I.; Wagner, A.; et al. Natural Killer Cell-Mediated Lysis of Hepatoma Cells via Specific Induction of NKG2D Ligands by the Histone Deacetylase Inhibitor Sodium Valproate. Cancer Res. 2005. https://doi.org/10.1158/0008-5472.CAN-04-4252. (49) Skov, S.; Pedersen, M. T.; Andresen, L.; Straten, P. T.; Woetmann, A.; Ødum, N. 110

Cancer Cells Become Susceptible to Natural Killer Cell Killing after Exposure to Histone Deacetylase Inhibitors Due to Glycogen Synthase Kinase-3-Dependent Expression of MHC Class I-Related Chain A and B. Cancer Res. 2005. https://doi.org/10.1158/0008-5472.CAN-05-0599. (50) Reddy, P.; Maeda, Y.; Hotary, K.; Liu, C.; Reznikov, L. L.; Dinarello, C. A.; Ferrara, J. L. M. Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Reduces Acute Graft-versus-Host Disease and Preserves Graft-versus-Leukemia Effect. Proc. Natl. Acad. Sci. 2004. https://doi.org/10.1073/pnas.0400380101. (51) Nusinzon, I.; Horvath, C. M. Interferon-Stimulated Transcription and Innate Antiviral Immunity Require Deacetylase Activity and Histone Deacetylase 1. Proc. Natl. Acad. Sci. 2003. https://doi.org/10.1073/pnas.2433987100. (52) Tang, X.; Gao, J. S.; Guan, Y. jie; McLane, K. E.; Yuan, Z. L.; Ramratnam, B.; Chin, Y. E. Acetylation-Dependent Signal Transduction for Type I Interferon Receptor. Cell 2007. https://doi.org/10.1016/j.cell.2007.07.034. (53) Chen, L. -f.; Fischle, W.; Verdin, E.; Greene, W. C. Stat3 Dimerization Regulated by Reversible Acetylation of a Single Lysine Residue. Science (80-. ). 2001. https://doi.org/10.1126/science.1062374. (54) Martínez-Balbás, M. A.; Bauer, U. M.; Nielsen, S. J.; Brehm, A.; Kouzarides, T. Regulation of E2F1 Activity by Acetylation. EMBO J. 2000. https://doi.org/10.1093/emboj/19.4.662. (55) Luo, J.; Su, F.; Chen, D.; Shiloh, A.; Gu, W. Deacetylation of P53 Modulates Its Effect on Cell Growth and Apoptosis. Nature 2000, 408 (6810), 377–381. https://doi.org/10.1038/35042612. (56) Cohen, H. Y.; Lavu, S.; Bitterman, K. J.; Hekking, B.; Imahiyerobo, T. A.; Miller, C.; Frye, R.; Ploegh, H.; Kessler, B. M.; Sinclair, D. A. Acetylation of the C Terminus of Ku70 by CBP and PCAF Controls Bax-Mediated Apoptosis. Mol. Cell 2004. https://doi.org/10.1016/S1097-2765(04)00094-2. (57) De Zio, D.; Bordi, M.; Cecconi, F. Oxidative DNA Damage in Neurons: Implication of Ku in Neuronal Homeostasis and Survival. International Journal of Cell Biology. 2012. https://doi.org/10.1155/2012/752420. (58) Canettieri, G.; Morantte, I.; Guzmán, E.; Asahara, H.; Herzig, S.; Anderson, S. D.; Yates, J. R.; Montminy, M. Attenuation of a Phosphorylation-Dependent Activator by an HDAC-PP1 Complex. Nat. Struct. Biol. 2003. https://doi.org/10.1038/nsb895. (59) Brush, M. H.; Guardiola, A.; Connor, J. H.; Yao, T. P.; Shenolikar, S. Deactylase Inhibitors Disrupt Cellular Complexes Containing Protein Phosphatases and Deacetylases. J. Biol. Chem. 2004. https://doi.org/10.1074/jbc.M310997200. (60) Chen, C. S.; Weng, S. C.; Tseng, P. H.; Lin, H. P.; Chen, C. S. Histone Acetylation-Independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes. J. Biol. Chem. 2005. https://doi.org/10.1074/jbc.M505733200. (61) Whitesell, L.; Lindquist, S. L. HSP90 and the Chaperoning of Cancer. Nature Reviews Cancer. 2005. https://doi.org/10.1038/nrc1716. (62) Budillon, A.; Bruzzese, F.; Gennaro, E.; Caraglia, M. Multiple-Target Drugs: Inhibitors of Heat Shock Protein 90 and of Histone Deacetylase. Curr. Drug 111

Targets 2005. https://doi.org/10.2174/1389450053765905. (63) Fuino, L.; Bali, P.; Wittmann, S.; Donapaty, S.; Guo, F.; Yamaguchi, H.; Wang, H.-G.; Atadja, P.; Bhalla, K. Histone Deacetylase Inhibitor LAQ824 Down- Regulates Her-2 and Sensitizes Human Breast Cancer Cells to Trastuzumab, Taxotere, Gemcitabine, and Epothilone B. Mol. Cancer Ther. 2003, 2 (10), 971– 984. (64) Yu, C.; Krystal, G.; Varticovksi, L.; McKinstry, R.; Rahmani, M.; Dent, P.; Grant, S. Pharmacologic Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Mitogen-Activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-Expressing Human Leukemia Cells. Cancer Res. 2002, 62 (1), 188–199. (65) Bali, P.; George, P.; Cohen, P.; Tao, J.; Guo, F.; Sigua, C.; Vishvanath, A.; Scuto, A.; Annavarapu, S.; Fiskus, W.; et al. Superior Activity of the Combination of Histone Deacetylase Inhibitor LAQ824 and the FLT-3 Kinase Inhibitor PKC412 against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3. Clin. Cancer Res. 2004. https://doi.org/10.1158/1078-0432.CCR-04-0210. (66) Strecker, A. Ueber Das Lecithin. Justus Liebigs Ann. Chem. 1868. https://doi.org/10.1002/jlac.18681480107. (67) Ishikawa, T. Superbases for Organic Synthesis: Guanidines, Amidines, Phosphazenes and Related Organocatalysts; 2009. https://doi.org/10.1002/9780470740859. (68) Wang, X.; Sarycheva, O. V.; Koivisto, B. D.; McKie, A. H.; Hof, F. A Terphenyl Scaffold for π-Stacked Guanidinium Recognition Elements. Org. Lett. 2008. https://doi.org/10.1021/ol7027042. (69) Gund, P. Guanidine, Trimethylenemethane, and “Y-Delocalization.” Can Acyclic Compounds Have “Aromatic” Stability? J. Chem. Educ. 2009. https://doi.org/10.1021/ed049p100. (70) Liu, J.; Li, X. W.; Guo, Y. W. Recent Advances in the Isolation, Synthesis and Biological Activity of Marine Guanidine Alkaloids. Marine Drugs. 2017. https://doi.org/10.3390/md15100324. (71) Guzii, A. G.; Makarieva, T. N.; Denisenko, V. A.; Dmitrenok, P. S.; Kuzmich, A. S.; Dyshlovoy, S. A.; Krasokhin, V. B.; Stonik, V. A. Monanchocidin: A New Apoptosis-Inducing Polycyclic Guanidine Alkaloid from the Marine Sponge Monanchora Pulchra. Org. Lett. 2010. https://doi.org/10.1021/ol101716x. (72) Makarieva, T. N.; Tabakmaher, K. M.; Guzii, A. G.; Denisenko, V. A.; Dmitrenok, P. S.; Shubina, L. K.; Kuzmich, A. S.; Lee, H. S.; Stonik, V. A. Monanchocidins B-E: Polycyclic Guanidine Alkaloids with Potent Antileukemic Activities from the Sponge Monanchora Pulchra. J. Nat. Prod. 2011. https://doi.org/10.1021/np200452m. (73) Rauf, M. K.; Imtiaz-ud-Din; Badshah, A. Novel Approaches to Screening Guanidine Derivatives. Expert Opin. Drug Discov. 2014, 9 (1), 39–53. https://doi.org/10.1517/17460441.2013.857308. (74) Singh, E. K.; Ravula, S.; Pan, C. M.; Pan, P. S.; Vasko, R. C.; Lapera, S. A.; Weerasinghe, S. V. W.; Pflum, M. K. H.; McAlpine, S. R. Synthesis and Biological Evaluation of Histone Deacetylase Inhibitors That Are Based on 112

FR235222: A Cyclic Tetrapeptide Scaffold. Bioorganic Med. Chem. Lett. 2008. https://doi.org/10.1016/j.bmcl.2008.03.047. (75) Ourailidou, M. E.; Leus, N. G. J.; Krist, K.; Lenoci, A.; Mai, A.; Dekker, F. J. Chemical Epigenetics to Assess the Role of HDAC1-3 Inhibition in Macrophage pro-Inflammatory Gene Expression. Medchemcomm 2016. https://doi.org/10.1039/c6md00375c. (76) Zhang, X.; Lv, P. C.; Li, D. D.; Zhang, W. M.; Zhu, H. L. Navigating into the Chemical Space between MGCD0103 and SAHA: Novel Histone Deacetylase Inhibitors as a Promising Lead. Medchemcomm 2015. https://doi.org/10.1039/c5md00247h. (77) Hancock, W. W. Rationale for HDAC Inhibitor Therapy in Autoimmunity and Transplantation. Handb. Exp. Pharmacol. 2011, 206, 103–123. https://doi.org/10.1007/978-3-642-21631-2_6. (78) TILLEKERATNE, V.; AL-HAMASHI, A.; DLAMINI, S.; ALQAHTANI, A. S.; KARAJ, E. IMIDAZOLE-BASED ANTICANCER AGENTS AND DERIVATIVES THEREOF, AND METHODS OF MAKING AND USING SAME. 2019. (79) Soh, C. H.; Chui, W. K.; Lam, Y. An Efficient and Expeditious Synthesis of Di- And Monosubstituted 2-Aminoimidazoles. J. Comb. Chem. 2008. https://doi.org/10.1021/cc700143n. (80) Lee, J. C.; Bae, Y. H.; Chang, S. K. Efficient α-Halogenation of Carbonyl Compounds by N-Bromosuccinimide and N-Chlorosuccinimde. Bull. Korean Chem. Soc. 2003. https://doi.org/10.5012/bkcs.2003.24.4.407. (81) Wen, Y.; Chen, G.; Tang, Y.; Chen, J.; Yang, J.; Zhang, Y. Effective Bismuth(III)- Promoted Barbier-Grignard-Type Reactions of Bromides with Aldehydes. Chinese J. Org. Chem. 2016. https://doi.org/10.6023/cjoc201506025. (82) Itano, H. A.; Hirota, K.; Kawasaki, I.; Yamada, S. Mechanism and Specificity of the Phenanthrenequinone Test for Monosubstituted Guanidines. Anal. Biochem. 1976. https://doi.org/10.1016/0003-2697(76)90271-2. (83) Guo, X.; Chen, W.; Chen, B.; Huang, W.; Qi, W.; Zhang, G.; Yu, Y. One-Pot Three-Component Strategy for Functionalized 2-Aminoimidazoles via Ring Opening of α-Nitro Epoxides. Org. Lett. 2015. https://doi.org/10.1021/acs.orglett.5b00289. (84) Bratulescu, G. Synthesis of 4,5-Substituted Imidazoles by a Fast Condensation of 1,2-Diketones and Urotropine in Heterogeneous Medium. Synthesis (Stuttg). 2009. https://doi.org/10.1055/s-0029-1216841. (85) ZHUO, L.; ZHANG, C.; ZHANG, Y.; YING, J. Y.; GOPALAN, B.; KE, Z.; DING, Z. METHOD FOR TREATING FIBROSIS AND CANCER WITH IMIDAZOLIUM AND IMIDAZOLINIUM COMPOUNDS. 2009. (86) Lindel, T.; Hochgürtel, M. Synthesis of the Marine Natural Product Oroidin and Its Z-Isomer. J. Org. Chem. 2000. https://doi.org/10.1021/jo991395b. (87) Kumar, P.; Jiang, T.; Li, S.; Zainul, O.; Laughlin, S. T. Caged Cyclopropenes for Controlling Bioorthogonal Reactivity. Org. Biomol. Chem. 2018. https://doi.org/10.1039/c8ob01076e.

113