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Adventures in medicinal chemistry: design and synthesis of small molecule biological modulators

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Adventures in medicinal chemistry: design and synthesis of small molecule biological modulators

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Content ADVENTURES IN MEDICINAL CHEMISTRY: DESIGN AND SYNTHESIS OF SMALL MOLECULE BIOLOGICAL MODULATORS by Kevin J. Gaffney A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPY (CHEMISTRY) August 2013 Copyright 2013 Kevin J. Gaffney i Dedication To Mom, Dad, Michael, and Dorthy ii Acknowledgements I would like to thank my advisor Dr. Nicos Petasis for his constant support, encouragement, and patience that helped turn a psychology major into a chemist. I will never forget our marathon meetings in your office that concluded with pages and pages of yellow legal pad paper filled with red scribbling foretelling many hours at the bench. I can never thank him enough for all of time and effort he put in to teach me many great lessons of chemistry, but more importantly lessons of life. I would like to additionally thank my qualifying committee members Professors G.K. Surya Prakash, Travis Williams, Axel Schönthal, and Barry Thompson. A special thanks to my thesis committee members Professor Stan Louie for excellent work as a collaborator as well as his mentorship and constant faith in me and Professor Pratt for his guidance, perspective, encouragement, and CH3CH2OH-fueled scientific discussions. A huge thank you goes out to all past and present member of the Petasis group for making the lab an amazing experience: Dr. Kalyan Nagulapalli Ventkata for being my mentor from day one, Jeremey Winkler for being my sounding board and occasional boxing partner, Dr. Jamie Jarusiewicz for showing me what it looks like to love science, Marcos Sainz for always believing in my crazy ideas, Leslie Batemen for the love and support, as well as Min Zhu, Anne-Marie Finaldi, Charles Arden, Dr. Kenny Young, Dr. Jasim Uddin, Dr. Malgorzata Myslinska, Dr. Alex Butkevich, and Dr. Rong Yang. Additionally, I would like to the teachers that helped guide me to chemistry, especially Brother Tim for being the first teacher to get me interested in science, Professor Anne Wilson for making organic chemistry fun and exciting and Professors Dana Spence and Stephen Johnson for seeing my potential and allowing me to do research in your labs. I thank the staff of the Loker Hydrocarbon Institute and the Department of Chemistry at USC: Carole Phillips, Jessy May, David Hunter, Dr. Robert Anizfeld, Michele Dea, and Kati McKissick. Lastly, I would like to thank my friends and family for their unwavering encouragement and support without whom this would not have been possible. In particular, I am greatful for the love, support, and encouragement of my mother over all these years, for teaching me that I can do anything, and for allowing me to find myself as I wandered from psychology to medicine to djing to chemistry. iii Table of Contents Dedication i Acknowledgements ii List of Tables vi List of Figures vii Abstract xi Chapter 1. Introduction 1 1.1 Hallmarks of Cancer 1 1.2 Theraputics 4 1.3 Chapter 1. References 7 Chapter 2. Design and synthesis of dUTPase inhibitors 9 2.1. Introduction 9 2.2 Analysis of dUTPase 12 2.3 dUTPase inhibitor design 13 2.4 dUTPase inhibitor synthesis 17 2.5 Biological activity of dUTPase inhibitors 19 2.6 Conclusion 23 2.7 Experimental 24 2.7.1 Structure-based design 24 3.7.2 Synthetic chemistry 25 2.8 Chapter 3. References 32 Chapter 3. Design and synthesis of CXCR2 allosteric antagonist 36 3.1 Introduction 36 3.2 CXCR2 antagonist design 36 3.3 Structure-based design of new CXCR2 Antagonists 41 3.4 CXCR2 antagonist synthesis 43 3.5 CXCR2 antagonist evaluation 45 3.6 Conclusion 46 3.7 Experimental 47 3.7.1 PDB functional group search 47 3.7.2 Homology model docking 47 3.7.3 Synthetic chemistry 48 3.8 References 60 iv Chapter 4. HDAC inhibition via MEF2 binding. 64 4.1 Introduction 64 4.2 Chemistry 67 4.3 Biological activity 69 4.4 Crystal structure reevaluation 74 4.5 Design of new MEF2-inhibitors 76 4.6 Conclusion 77 4.7 Experimental 78 4.7.1 19 F NMR 78 4.7.2 Synthetic Chemistry 78 4.7.3 Molecular modeling studies 97 4.8 Chapter 4. References 99 Chapter 5. Synthesis and anti-cancer activity of non-COX-2-inhibiting Analogs of celecoxib 102 5.1 Introduction. 102 5.2 Experimental Design 103 5.3 Synthesis 104 5.4 In vivo activity 106 5.5 Applications 109 5.5.1 COX-2 inhibition is neither necessary nor sufficient for celecoxib to suppress tumor cell proliferation and focus formation in vitro 109 5.5.2 Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress 110 5.5.3 Antiangiogenic activities of 2,5-dimethyl-celecoxib on the tumor vasculature 112 5.5.4 Enhancement of photodynamic therapy by 2,5-dimethyl celecoxib, a non-cyclooxygenase-2 inhibitor analog of celecoxib 113 5.5.5 Preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors 115 5.5.6 Cytotoxic effects of celecoxib on Raji lymphoma cells correlate with aggravated endoplasmic reticulum stress but not with inhibition of cyclooxygenase-2 116 5.5.7 Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors 118 5.6 Conclusion 119 5.7 Experimental 120 5.8 Chapter 5. References 136 v Bibliography 139 Appendix: Selected spectra 152 vi List of Tables Table 3.1 Summary of interactions of Ar-NHCONH-R from the PDB 39 Table 3.2 List of analogs 11a-b, 13a-b, 15a-b, 17-18b, 19a-b, 46 and 4 and their inhibitory activity in vitro tumor cell proliferation assay Table 4.1 Optimization of linker length of trifluoromethyl MEF2 70 inhibitors at 10µM in luciferase assay Table 4.2 Activity of ortho-aminoanilide replacement MEF2 71 inhibitors at 10µM in luciferase assay Table 4.3 Activity of MEF2 inhibitor ortho-aminoanilide 72 replacements at 10µM in luciferase assay Table 4.4 Activity of MEF2 inhibitor linker replacements at 76 10µM in luciferase assay. Table 5.1 COX-2 and COX-2 activity of structural analogs of 104 celecoxib and rofecoxib Table 5.2 Structure-activity relationship between celecoxib and 108 rofecoxib analogs Table 5.3 Celcoxib analogs and their activity 108 vii List of Figures Figure 1.1 Schematic of the Hallmarks of Cancer targeted in this thesis 2 Figure 2.1 List of FDA-approved drugs for colorectal cancer 9 Figure 2.2 Biological Role of dUTPase 11 Figure 2.3 Crystal structure of dUpNHpp-bound dUTPase 12 Figure 2.4 Structural analysis of dUTPase active site 13 Figure 2.5 Structural analysis of known dUTPase inhibitors 14 Figure 2.6 Fragment docking studies for inhibitor design 15 Figure 2.7 Comparison of hydrogen bonding interactions of dUpNHpp 16 and designed inhibitor Figure 2.8 Structural progression in the development of dUTPase 17 inhibitors and the strategy for the addition of the cyclopropylmethyl ether (orange) to our benzimidazole-based inhibitors Figure 2.9 Structural overlay of the predicted binding orientations of 6b 17 and 13 Figure 2.10 Structure of 2a and preparation of compound 2b 18 Figure 2.11 Preparation of dUTPase inhibitors 6a-d and 9a-f 18 Figure 2.12 Preparation of dUTPase inhibitor 13 19 Figure 2.13 Growth inhibition of human colorectal cancer HCT-116 cells 20 in response to 1 µM FUdR and increasing concentrations of dUTPase inhibitors Figure 2.14 Growth inhibition of human cervical cancer HeLa cells 21 in response to 1 µM FUdR and increasing concentrations of dUTPase inhibitors Figure 2.15 A comparison of the growth inhibition activity of 6a and 6b 21 of HCT and HeLa cells in combination with 1 µM FUdR viii Figure 2.16 A comparison of the growth inhibition activity of 6b and 6c 22 of HCT and HeLa cells in combination with 1 µM FUdR Figure 2.17 The effect of FUdR on the growth inhibition activity of 6b 22 in HeLa cells Figure 3.1 Allosteric CXCR2 antagonists 37 Figure 3.2 Effect of methylation of the 3,4-diaminocyclobut-3-ene- 37 1,2-dione on CXCR2 antagonist activity Figure 3.3 Workflow for the acquisition, processing, and analysis of 38 interactions of protein-aryl ureas crystal structures Figure 3.4 Examples of binding orientations of ureas forming two 40 hydrogen bonds to Asp Figure 3.5 Overlay of top scoring GPCR templates for the CXCR2 41 homology model Figure 3.6 Sequence alignment of human CXCR2 (target) and human 42 µ-opiod receptor from crystal structure PDB 4DKL (template) used to construct the homology model. Figure 3.7 Analysis of Induced-Fit Docking result 43 Figure 3.8 Preparation of anilines 8-9 44 Figure 3.9 Preparation of CXCR2 antagonists 11a-b 44 Figure 3.10 Preparation of CXCR2 antagonists 13a-b 45 Figure 3.11 Preparation of CXCR2 antagonists 15a-b 45 Figure 3.12 Preparation of CXCR2 antagonist 17 45 Figure 3.13 Preparation of CXCR2 antagonists 18a-b 45 ix Figure 4.1 Histone lysine residues are acetylated by the histone 64 acetyltransferases enzymes (HATs) and the acetyl-donar acetyl-CoA and deacetylated by histone deacetylase enzymes (HDACs) Figure 4.2 HDAC inhibitors 65 Figure 4.3 Structureal comparison of binding sites for HDAC9 and 66 BML-210 on MEF2 Figure 4.4 Preparation of MEF2 inhibitors 8a-c and 48 68 Figure 4.6 Preparation of MEF2 inhibitors 41a-b 68 Figure 4.7 Preparation of MEF2 inhibitors 19, 21a-b, 24, and 26 68 Figure 4.8 Preparation of MEF2 inhibitors 37 and 42 69 Figure 4.9 Preparation of MEF2 inhibitor 32 69 Figure 4.10 Preparation of MEF2 inhibitor 33 69 Figure 4.11 Detecting the binding of a fluorinated analog of BML-210 70 to MEF2 by 19F NMR Figure 4.12 Crystallographic understanding of analog activity and design 73 Figure 4.13 Induced-Fit Docking studies to reevaluate small molecule- 74 MEF2 binding interactions Figure 4.14 PDB example of the aniline of KNI-1689 forming two 75 hydrogen bonds two aspartic acids of HIV-1 protease Figure 5.1 Structures of selective COX-2-inhibitors 102 Figure 5.2 Structural analogs of celecoxib and rofecoxib and the 104 COX-1 and COX-2 activity Figure 5.3 Synthesis of analogs 1 and 3-7 105 Figure 5.4 Synthesis of analog 8 105 x Figure 5.5 Synthesis of analogs 10-11 and 13-16 106 Figure 5.6 Synthesis of analog 12 106 Figure 5.7 Synthesis of analogs 30-31 106 Figure 5.8 MTT activity of analogs 1, 4, and 6 107 Figure 5.9 Altered chemosensitivity after knock-down of GRP78 111 or CHOP Figure 5.10 Cytotoxic effects of DMC on TuBECs. TuBECs and 113 subconfluent BECs were treated with DMC, celecoxib (CXB), and UMC for 72 h Figure 5.11 DMC and celecoxib increase phototoxicity in PDT-treated 114 BA cells Figure 5.12 Colony formation after combination treatment 116 Figure 5.13 Enhancement of ER stress and cell death by combination 117 drug treatment Figure 5.13 1 H NMR spectra and 13 C NMR spectra of combinations 119 of BZM and EGCG in 20% D2O in CD3CN xi Abstract This dissertation details my efforts towards the design and development of novel small molecules anti-cancer agents including joint efforts with a host of colleagues and collaborators. The introduction, Chapter 1, provides a brief overview of the current “Hallmarks of Cancer.” This is a list of characteristics acquired during tumorigenesis, which provide the targets for drug development. In this sojourn, we have utilized a wide spectrum of strategies and technigues to develop specific drugs. These efforts are described in this Thesis. Chapter 2 describes the computational fragment-based design, synthesis, and biological validation of a series of novel inhibitors of the enzyme dUTPase as potential anti-cancer agents. Chapter 3 details the design, synthesis, biological evaluation, and molecular modeling studies of novel CXCR2 antagonists. Chapter 4 reports the synthesis and structure-activity relationship of a series of small molecules capable of disrupting the protein-protein interaction between transcription factor MEF2 and class IIa HDACs. Chapter 5 outlines our efforts to optimize a series of analogs of the FDA-approved cyclooxygenase-2 (COX-2) inhibitor Celebrex ® (Celecoxib), that have anti-cancer properties an devoid of COX-2 activity and the efforts of our collaborators to characterize the efficacy of these agents in a variety of models and therapeutic combinations are also summarized. 1 Chapter 1: Introduction 1.1 Hallmarks of Cancer Cancer is a catch-all term used to describe a collection of phenotypically related diseases marked by several common features. A huge breakthrough in the treatment of cancer came in 1965 when, lessons learned from the successful treatment of tuberculosis, led to the use of existing chemotherapies in combination. Over the next decade, this led to development of treatments capable of inducing the complete remission in Hodgkin’s and non-Hodgkin’s lymphoma and acute lymphoblastic leukemia in children. 1 The success of the combination drug strategy points to the inherent similarities between cancer and bacteria; both are highly mutable cells fighting selective pressures to replicate and survive. By combining multiple therapies, mutations are unlikely to arise that impart survival fitness to all modulated pathways simultaneously. While these early success were encouraging, the ever increasing complexity genetic and clinical data on cancer requires a holistic understanding of the capabilities normal cells must acquire to become malignant tumors. In an attempt to codify the principles of tumorigenesis, Hanahan and Weinberg developed the “Hallmarks of Cancer.” However, this list has significantly expanded to reflect the advances in our understanding of tumorigenesis and cancer survival. In the latest iteration these hallmarks are: 1) sustaining proliferative signaling, 2) evading growth suppressors, 3) resisting cell death, 4) enabling replicative immortality, 5) inducing angiogenesis 6) activating invasion and metastasis, 7) genome instability and mutation, 8) tumor-promoting inflammation, 9) reprogramming energy metabolism, and 10) evading immune destruction. 2 While the aforementioned list is a useful mental framework for evaluating therapeutic targets and developing better combination strategies, the demarcations do not always coincide directly with their underpinning biological components. A single pathway or target can be implicated in inducing multiple hallmark capabilities. Therefore, the biological outcome any drug or target is dependent on the polypharmacology of both the target and the drug which can result in a multitude of outcomes. Expanded explanations 2 of the hallmarks targeted by the medicinal chemistry efforts described in this thesis are given below (Figure 1.1). Figure 1.1. Schematic of the Hallmarks of Cancer targeted in this thesis. Resisting Cell Death: Programmed cell death, or apoptosis, is an essential process for the maintenance of cellular homeostasis in both embryonic and adult tissues. The apoptotic pathway is composed of regulators, responsible for monitoring the intra- and extracellular milieu for signals of cellular or environmental distress, and effectors, which upon regulator activation carry out cellular degradation. As genetically encoded machinery, components of the apoptotic pathway can be deactivated by mutations allowing cancer cells to avoid death. 3 Inactivation of the key tumor suppressor p53, termed the “guardian of the genome,” is an important mechanism by which cancer is able to circumvent apoptosis. In normal cells, wild type p53 has an array of function including regulating the expression of the anti- 3 apoptotic protein survivin. 4,5,6 Mutations to p53, seen in over 50% of tumors, cause the overexpression of survivin the central suppressor the apoptotic effector capsases. Genome Instability & Mutation: The ability of neoplastic cells to acquire the hallmarks of cancer is largely the result of changes to the function and expression of proteins at the genomic level. The microenvironment of the tumor places selective pressure on cells to express phenotypes that impart survival fitness. While gene mutation has long been shown to facilitate the metamorphosis to malignancy, recently, epigenetic modifications have been shown as an alternative pathway for cells to vary gene expression. Through the reversible modifications of DNA methylation and histone remodeling, cancer cells are able to modulate protein levels in a heritable, mutation-independent manner. Tumor-Promoting Inflammation: For some time, it has been known that tumors are significantly populated with cells from both the adaptive and innate branches of the immune system. 7 Traditionally, these cells were believed to be present to help destroy the tumor, or at least antagonize tumor growth. 2 However, new research has shown that these cells have the opposite effect of promoting tumorigenesis. These disparate effects are each carried out by separate two arms of the immune system with the adaptive immunity as the antagonist and the innate immunity agonizing tumorigenesis. Through the release of growth, proangiogenic, and survival factors and matrix-degrading enzymes, the innate immune response and the resulting inflammation, all aspects of tumor development are assisted from facilitating cell proliferation and angiogenesis to preventing cell death to facilitating metastasis and invasion. 8-11 Inducing Angiogenesis: Following the vasculature requirements of embryogenesis, the angiogenic pathway lays mostly dormant, only activating temporarily during menstruation and wound healing. 12 Cancer hijacks this process to meet the needs of tumorigenesis and metastasis to supply oxygen and nutrients and remove waste and CO2. Angiogenesis is controlled by countervailing proangiogenic and antiangiogenic signals. As is a potent inducer of angiogenesis, vascular endothelial growth factor-A (VEGF-A) and its downstream targets have been successfully targeted by a number of FDA- approved drugs including the monoclonal antibody bevacizumab (Avastin ® ) used in the 4 treatment of metastatic colorectal cancer. 13 These therapies work in large part by blocking the VEGF-induced chemotaxis and proliferation of endothelial cells and thus preventing vasculogenesis. Sustaining Proliferation: The ability to undergo persistent proliferation is the cardinal characteristic of cancer. In the evolution from single to multicellular organisms, controls governing cellular division were developed to allow super cellular organization. 14 As a result, growth-promoting signals responsible for the initiation of proliferation are tightly regulated in normal cells. Cancer is able to overcome the growth suppression by either producing its own growth signal factors allowing for autocrine activation or upregulating the growth factor receptors to sensitize the cell to external signals. 1.2 New Therapeutics for Cancer Considering the importance of the aforementioned hallmarks, the projects described in the following pages sought to address therapeutic needs in cancer by targeting the pathways that allow the acquisition of the hallmark characteristics. Chapter 2 highlights our efforts to decrease cellular proliferation through the inhibition of deoxyuridine 5'-triphosphate nucleotidehydrolase (dUTPase). The first line therapy for colorectal cancer is a combination drug regimen centered on thymidylate synthase (TS) inhibitors. TS-inhibitors impart their cytotoxicity by covalently binding to TS which prevents the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). The results accumulation of deoxyuridine triphosphate (dUTP) which, in turn, leads to the misincorporation of the RNA base uracil into DNA causing DNA damage and apoptosis. Unfortunately, even the most highly optimized therapeutic combinations including TS-inhibitors have rates of resistance greater than 50% in colorectal cancer. 15 This resistance has been positively correlated with both p53 mutations 16 and dUTPase overexpression. 17 Additionally, the dysregulation of p53 has also been shown shown to upregulate dUTPase in neoplastic cells. Therefore, dUTPase presents itself as an interesting target for overcoming chemotherapeutic resistance in colorectal cancer. This chapter outlines our efforts to 5 design and synthesize inhibitors of dUTPase and show their efficacy as a stand-alone treatment or as a combination therapy with the TS inhibitor 5-FUdR. Chapter 3 details our efforts to address tumor-promoting inflammation as well as sustained proliferation and angiogenesis through the antagonism of the GPCR recptor CXCR2. We set out to target the innate immune system to block its protumoral inflammatory mechanism. Leukocytes, including neutrophils, are crucial to maintaining the inflammatory environment that allows tumor development. The recruitment of proinflammatory neutrophils has been shown in many disease states to be dependent on the expression of the receptor CXCR2 and its ligand IL-8 a proinflammatory chemokine upregulated in a variety of cancers. Additionally, IL-8 has been shown to be the primary angiogenic activator in various tumors. 18 Recent studies have shown the effectiveness of existing CXCR2 antagonists in inhibiting cell viability and angiogenesis in vivo and in vitro. This chapter summerizes our efforts to design and develop novel CXCR2 antagonists as anti-inflammatory, and anti-tumoral agents. Chapter 4 highlights our efforts to decrease cellular proliferation through the inhibition of Histone deactylases (HDACs), which are a class of enzymes responsible for the removal of acetyl groups from lysine residues of histones causing a decrease in gene expression. Tumors have recently been shown to suffer from lower acetylation levels which, it is believed, allows them to down regulate the expression of tumor suppressor genes. 19 While HDAC inhibitors have been the subject of a great deal of effort from the pharmaceutical industry, these drugs suffer from unacceptable side effects. 20 To avoid these problems, we set out to develop a class of inhibitors that will phenotypically decrease HDAC activity, but without direct HDAC inhibition. As described in this chapter, we were able to achieve this effect through the synthesis and optimization of small molecules capable of disrupting of the DNA-specific binding of class II HDACs to their transcription factor MEF2. 21 Chapter 5 highlights our efforts involved the anti-cancer properties of celecoxib to decrease proliferation and angiogenesis. Celecoxib (Celebrex ® ) is a selective COX-2 inhibitor approved for rheumatoid arthritis. In 1999, celecoxib received an additional 6 indication for the preventative treatment of the precancerous condition familial adenomatous polyposis. In recent years, as a result of the promiscuity of celecoxib, an array of additional targets have been implicated as sources of the anti-tumoral activity of celecoxib. This chapter highlights our efforts to delineate celecoxibs COX-2 activity from its anti-cancer activity. We were able to successfully eliminate COX-2 activity in a series of new celecoxib analogs while optimizing their anti-tumoral activity. Further studies showed the ability of the non-COX-2 inhibitor 2,5-dimethyl celecoxib (DMC) to induce apoptosis through a series of pathways including decreasing survivin and blocking angiogenesis by preventing ednothelin-1 (EC-1) secretion. 22 7 References 1.3 (1) Leukemia and Related Disorders; Estey, E. H.; Appelbaum, F. R., Eds.; Springer New York: New York, NY, 2012; pp. 37–66. (2) Hanahan, D.; Weinberg, R. A. Cell 2011, 144, 646–74. (3) Lowe, S. W. Carcinogenesis 2000, 21, 485–495. (4) Hoffman, W. H.; Biade, S.; Zilfou, J. T.; Chen, J.; Murphy, M. J Biol Chem 2002, 277, 3247–57. (5) Mirza, A.; McGuirk, M.; Hockenberry, T. N.; Wu, Q.; Ashar, H.; Black, S.; Wen, S. F.; Wang, L.; Kirschmeier, P.; Bishop, W. R.; Nielsen, L. L.; Pickett, C. B.; Liu, S. Oncogene 2002, 21, 2613–22. (6) Zhou, M.; Gu, L.; Li, F.; Zhu, Y.; Woods, W. G.; Findley, H. W. J Pharmacol Exp Ther 2002, 303, 124–31. (7) Dvorak, H. F. New Engl J Med 1986, 315, 1650–9. (8) DeNardo, D. G.; Andreu, P.; Coussens, L. M. Cancer Metast Rev 2010, 29, 309– 16. (9) Grivennikov, S. I.; Greten, F. R.; Karin, M. Cell 2010, 140, 883–99. (10) Qian, B.-Z.; Pollard, J. W. Cell 2010, 141, 39–51. (11) Karnoub, A. E.; Dash, A. B.; Vo, A. P.; Sullivan, A.; Brooks, M. W.; Bell, G. W.; Richardson, A. L.; Polyak, K.; Tubo, R.; Weinberg, R. A. Nature 2007, 449, 557– 63. (12) Hoeben, A.; Landuyt, B.; Highley, M. S.; Wildiers, H.; Van Oosterom, A. T.; De Bruijn, E. A. Pharmacol Rev 2004, 56, 549–80. (13) Ellis, L. M.; Hicklin, D. J. Nat Rev Cancer 2008, 8, 579–91. (14) Andreeff M, Goodrich DW, Weichselbaum, R.R. et al., Cell Proliferation, Differentiation, and Apoptosis. Cancer Medicine; Kufe, D.W., Pollock, R.E., et al. ed; BC Decker: Hamilton, Canada, 2000. (15) Galvani, E.; Peters, G. J.; Giovannetti, E. Expert Opin Inv Drug 2011, 20, 1343– 56. 8 (16) Giovannetti, E.; Backus, H. H. J.; Wouters, D.; Peters, G. J. Nucleos Nucleot Nucl 2008, 27, 740–5. (17) Ladner, R. D.; Lynch, F. J.; Groshen, S.; Xiong, Y. P.; Sherrod, A.; Caradonna, S. J.; Stoehlmacher, J.; Lenz, H.-J. Cancer Res. 2000, 60, 3493–3503. (18) Smith, D. R. J Exp Med 1994, 179, 1409–1415. (19) Xiao, L.; Huang, Y.; Zhen, R.; Chiao, J. W.; Liu, D.; Ma, X. Acta Haematol 2010, 123, 71–6. (20) Bruserud, Ø.; Stapnes, C.; Ersvaer, E.; Gjertsen, B. T.; Ryningen, A. Curr Pharm Biotechno 2007, 8, 388–400. (21) Jayathilaka, N.; Han, A.; Gaffney, K. J.; Dey, R.; Jarusiewicz, J. a; Noridomi, K.; Philips, M. a; Lei, X.; He, J.; Ye, J.; Gao, T.; Petasis, N. a; Chen, L. Nucleic Acids Res 2012, 40, 5378–88. (22) Virrey, J. J.; Liu, Z.; Cho, H.-Y.; Kardosh, A.; Golden, E. B.; Louie, S. G.; Gaffney, K. J.; Petasis, N. A.; Schönthal, A. H.; Chen, T. C.; Hofman, F. M. Mol Can Ther 2010, 9, 631–41. 9 Chapter 2: Design and Synthesis of dUTPase Inhibitors 2.1 Introduction Colorectal cancer (CRC) is the leading killer in cancer-related deaths worldwide. In 2010 alone, there were 142,000 new cases and 51,000 deaths in the US, making it the US's second deadliest cancer after the preventable cancers of the lung and bronchus. 1 Of these new cases, 20% will clinically present with metastastic disease at the time of diagnosis. 2 For 35 years, the standard of care for CRC was 5-fluoro-1H-pyrimidine-2,4-dione (5- FU). 3 5-FU, the fluorinated derivative of the RNA base uracil, acts as an irreversible inhibitor of thymidylate synthase (TS), a crucial enzyme in DNA synthesis and cell division, and induces cell death. The addition of leucovorin, approved by the FDA in 1997, to the regimen has improved the overall response rate (RR) of 5-FU therapy from 10% to 23% indicating the benefits of a synergistic approach in the treatment of CRC. 3,4,5 In the past 10 years, the addition of oxaliplatin and irinotecan to the 5-FU regimen has pushed the RR even further to 45% and 20 month overall survival (OS), compared to 10 months with 5-FU alone (Figure 2.1). Figure 2.1: List of FDA-approved drugs for colorectal cancer. Despite these advances, it appears as though this 50% RR is the maximum efficacy that can be reached with this optimized synergistic-5-FU-based therapeutic regimen. For many patients the lack of response is due to acquired or intrinsic drug resistance gained through the over expression dUTPase, the non-redundant enzyme responsible for the conversion of the deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate 10 (dUMP). In the enzymatic pathway, this dUMP is converted by TS to thymidine monophosphate (TMP), the biological precursor to thymidine triphosphate (TTP) the nucleoside triphosphate of DNA synthesis and repair (Figure 2.2a). 6 DNA synthase, the enzyme responsible for DNA replication and repair, is unable to distinguish between the DNA base TTP and the RNA base dUTP and indiscriminately incorporates both into DNA. Under normal physiological conditions, this errant incorporation is prevented because dUTP is kept at undetectable levels by dUTPase. Inhibition of TS, the key step in the conversion of uracil the RNA base uracil to thymidine prevents the conversion of dUMP to TMP. This shifts the equilibrium causing a severe reduction in TTP and an increase in dUTP resulting in high levels of dUTP-substitutions in the DNA. These misincorporations are recognized by the uracil-base excision pathway and repeated attempts are made to repair the DNA by replacing the dUTP with TTP. However, with persistent low TTP levels, dUTP is reincorporated (Figure 2.2b). Successive cycles of excisions and repairs cause severe breaks in the cell's DNA and results in apoptosis. 6 11 Figure 2.2. Biological Role of dUTPase. (a) Normal Thymidylate Metabolism; (b) Thymidylate Metabolism Under TS-inhibition. Resistance to TS-inhibitor therapy is the result of high levels of dUTPase activity that decreases levels of dUTP; (c) Mechanism of overcoming TS-resistance by dUTPase inhibition. C B A 12 This dUTPase-based resistance has been supported by an array of data. Cancers cells with intrinsic or acquired resistances to TS-inhibitors have corresponding high levels of dUTPase. 6-10 Expression levels of dUTPase correlate with preclinical and clinical efficacy of TS-inhibitor therapy and have been shown to be the mechanism of 5-FU resistance in non-responding tumors. 11,12 By over-expressing dUTPase, resistant cells are able to reduce the dUTP levels and prevent its concentration-dependent incorporation into DNA. Therefore, the addition of a dUTPase-inhibitor to a TS-inhibitor therapeutic regimen would block this resistance pathway, elevate levels of dUTP and induce cell death in 5-FU resistant tumors thus overcoming resistance (Figure 2.2c). 2.2 Structureal Analysis of dUTPase Human dUTPase is a homotrimeric enzyme that converts dUTP to dUMP and pyrophosphate by Mg 2+ -mediated catalytic hydrolysis (Figure 2.3). Access to active site is mediated by the flexible C-terminus. dUTPase has high catalytic activity with a kcat ~7, the hydrolysis of 7 molecules of dUTP per second. 13 As a result, attempts to co- crystallize dUTPase with dUTP have instead resulted in a dUMP bound active site due to the fast kinetics of this reaction. Two crystal structures of human dUTPase have been solved. The initial structure, PDB:1Q5H, overcame the issues of reactivity by co- crystallizing with deoxyuridine diphosphate (dUDP). 14 However, this technique did not resolve the structure of the flexible C-terminus. A second structure, PDB:2HQU, employed a dUTP mimetic α,β,-imino-dUTP, (dUpNHpp) which replaced the oxygen of the phosphate-phosphate ester with a nitrogen and prevented hydrolysis. 15 By using the hydrolytically stable dUTP-mimetic, this second crystal structure gave a more complete picture of the role and orientation of interactions in the active site prior to the hydrolytic event. Interestingly, this resulted in visibility of the C-termini of two of the three active sites indicating that the third phosphate group is important for immobilizing this flexible C-terminus. Mechanistically, the action of dUTPase can be understood as a five step process: dUTP binds to the active-site, the C-terminus closes the active site to solvent and additional dUTPs, phosphate ester undergoes hydrolysis, the C-terminus opens, and 13 finally dUMP and pyrophosphate are released from the active site starting the cycle again. 14,16 Figure 2.3. Crystal structure of dUpNHpp-bound dUTPase (PDB:2HQU). (a) Top view with dUpNHpp (outlined in black) bound to the active site of dUTPase; (b) side view of active site. (Image generated with PyMol) 2.3 dUTPase Inhibitor Design With this mechanistic understanding of dUTPase activity, we sought to use a rational, or structure-based, design to develop dUTPase inhibitors that mimic the important interactions of dUTP. The bound dUpNHpp (PDB: 2HQU) was used as the starting point of design (Figure 2.4a). 15 The structure of dUTP can be broken down into three parts: the uracil-binding site (U), the sugar component (S), and the phosphate-binding site (P) (Figure 2.4b-c). Figure 2.4. Structural analysis of dUTPase active site. (a) dUpNHpp bound to the open active site A of dUTPase; (b) Functional group analysis of dUTPase binding site: uracil-binding site U, sugar linker S, and phosphate-binding site P; (c) Overlay of functional groups onto dUTP (PDB:2HQU). (Image generated with UCSF Chimeral) B A C 14 A survey of the literature yielded a several series of dUTPase inhibitors. Interestingly, an overlay of all the existing, crystallographically solved inhibitors revealed that they completely avoided the P-site of the dUTPase. Instead, these molecules access a non- essential, hydrophobic binding site above the uracil pocket (Figure 2.5b). 17-21 Considering these molecules were developed as potential anti-bacterials, the targeting of this hydrophobic pocket seems like a strategy prone to the development of resistance. Similarly in cancer, the selective pressure provided by chemotherapeutics has been shown to result in resistance. 22 Therefore, we believed that our strategy of mimicking the native substrate would help to decrease our inhibitors resistance profile. Figure 2.5. Structural analysis of known dUTPase inhibitors. (a) dUpNHpp (blue) bound to the active site of dUTPase (PDB:2HQU); (b) An overlay of the all the crystallographically determined inhibitors with dUpNHpp (wire); (c) structure of 3’-Deoxy-5’-O-trityl Uridine 1, the lead for the development of all inhibitors accessing the hydrophobic pocket. To interrogate the active site for potential binding functionalities, the crystal structure (PDB:3ARA) was virtually screened with ~350,000 “Clean Fragments” (MW≤ 250, logP≤ 3.5, # rotatable bonds≤ 5) from the ZINC Database using Schrodinger’s Glide. 23-27 Unremarkably, the top hit from the first screen was a carbocyclic analogs of 5- aminouradine (frag 1) (Figure 2.6a, aqua). An analysis of the top 100 fragments showed exclusive occupancy of the U-site by largely, cytosine, uracil, and thymine analogs. This was the result of the high hydrogen bond density of the U-site. To decrease these interactions, the conserved uracil-binding H2O was deleted and a strategy of increasing computational alkylation of the backbone -NH of G99 and G110 was A B C 1 15 employed. After several iterations of increasing alkyl length, successful population of the S and P-sites was obtained by connecting the two residues with a nonyl linker (Figure 2.6b, yellow). A manual analysis of the screening results biasing for lipophilic molecules hydrogen bonding to D102 yielded the 2-(p-tolyl)-1H-imidazole (frag 2) (Figure 2.6b). An overlay of the two fragments shows the methyl group of 2-(p-tolyl)-1H-imidazole in the same position as the 1’ carbon of dUTP, or the analogous position on frag 1, and indicates fragment linking strategy (Figure 2.6c). With synthetic accessibility in mind, the 2-substitued imidazole of frag 1 was converted to a 2-substitued benzimidazole. Figure 2.6. Fragment docking studies for inhibitor design. (a) An overlay of the dock orientation of a top scoring fragment, frag 1 (aqua), from the ZINC “Clean Fragments” library in the U-site and dUpNHpp (wire); (b) An overlay of an optimized P-site accessing fragment, frag 2 (periwinkle), in the modified (yellow) U-site blocking structure; (c) An overlay of the U-site and the P-site fragments with dUpNHpp (wire); (d) An overlay of the two fragment linking strategies: a 6-carbon alkyl linker 9b (magenta) and the benzyl-aryl 6b (green). (Image generated with PyMol) dUTPase hydrolyzes dUTP to dUMP and pyrophosphate with a catalytic H2O (Figure 2.7a, red circle) stabilized in the active site by D102 and, therefore, this interaction was 16 specifically targeted in the inhibitor design. 15 The hydrogen bond of H2O in the dUTPase active site is replaced in the designed inhibitors with an –NH hydrogen bond or an –NH + cationic interaction of the benzimidazole. Due to the catalytic importance of D102, it is highly unlikely that a mutation to this residue could exist in catalytically active enzyme therefore limiting the possibility of acquired resistance. Additionally, this synthetically beneficial switch from imidazole to benzimidazole also increased hydrophobic and π- interactions with A45, R85, and Q131 (Figure 2.7b) and thus increase binding affinity. Figure 2.7. Comparison of hydrogen bonding interactions of dUpNHpp and designed inhibitor. (a) Shows the hydrogen bonding network between dUpNHpp and dUTPase. Circled in red is the catalytic water which is hydrogen bonded to D102 and used by the dUTPase to hydrolyze dUTP into dUMP showing the hydrogen bonding network (PDB:2HQU); (b) Predicted binding of compound 6b show it maintaining the hydrogen bonds to D102 and G110 of dUpNHpp and adding hydrophobic and π-interactions to A45, R85, and Q131. Following our initial design work described above, a research group at Taiho Pharmaceuticals published a series of papers detailing the development of their own dUTPase inhibitors based on 1. 28 Though the removal of the sugar and the addition of N- sulfonylpyrrolidine compound 2 is able to better bind at the hydrophobic pocket resulting in its increased potency (Figure 2.8). 20 While a significant number of modifications were made during the optimization of compound 3, the most important were the benzylic linking strategy, the opening of the pyrrolidine to the mono-substituted sulfonamide, and the addition of the cyclopropylmethyl ether group. 29 Due to the conformational rigidity of the benzylic linker and the ability of the sulfonamide –NH to hydrogen bond to D102, the potency of 3 strongly implies a reorientation of the molecule from the hydrophobic pocket above uracil to the P-site, which was further supported by a docking study of a 17 simplified analog 13 (Figure 2.9). Therefore, compound 6a was designed to place the cyclopropylmethyl ether in the corresponding position on our benzimidazole ring four carbons from the –NH. Interestingly, the symmetry of our benzimidazole allows only two possible substitution positions and simplifies optimization. Figure 2.8. Structural progression in the development of dUTPase inhibitors and the strategy for the addition of the cyclopropylmethyl ether (orange) to our benzimidazole-based inhibitors. Figure 2.9. Structural overlay of the predicted binding orientations of 6b and 13. The flexibility of the sulfonamide allows the benzyl to access the same A45, R85, and Q131 pocket. 2.4 dUTPase Inhibitor Synthesis Considering the synthetic tractability of the inhibitors throughout the design process led to the facile synthesis of analogs. Selected analogs were prepared jointly with my collegue Marcos Sainz. The substitued 1,2-phenylene diamines 2b were synthesized in a two-steps process of selective phenolic alkylation followed by Pd-mediated hydrogenation (Figure 2.10). 30,31 Initially, we were concerned that the alkylation of uracil could result in functionalization of the 3 position of uracil. To test this, compound 4b was synthesized with and without the TMS protection. 32,33 Since this resulted in identical NMRs, the remaining analogs were synthesized without protection. Following, D102 G110 18 the alkylation of the bromo-esters 3a-b and 7a-b, the esters were hydrolyzed to the free acids 4a-b and 8a-b with LiOH in THF, H2O, and MeOH (Figure 2.11). 34 Despite the literature precedence, attempts to convert these free acids directly to the benzimidazole were unsuccessful. 35 Figure 2.10. Structure of 2a and preparation of compound 2b,. Reagents and conditions: (a) (Bromomethyl) cyclopropane, K 2CO 3, KI, MeCN, 70°C, 13hr; (b) H 2, Pd/C, MeOH, 2.5 hr Figure 2.11. Preparation of dUTPase inhibitors 6a-d and 9a-f,. Reagents and conditions: (a) uracil, Cs 2CO 3, DMSO; (b) LiOH·H 2O, THF, H 2O, MeOH, 3 hr; (c) 2a-c, HATU, DIEA, DMF, rt, 12-16 hr; (d) AcOH, 90-105°C. Due to the polarity of the final product, a two-step process of amide coupling followed by AcOH-catalyzed cyclization allowing for chromatographic purification of the less polar o-aminoanilides in the first step and then a simple recrystallization in the second to yield the desired benzimidazoles 6a-c and 9a-b. 36,37 With compounds 8a-b on hand and the desire for hydrogen bond donation to D102, the amides 10a-b were also synthesized with 19 the same HATU-coupling conditions used to access o-aminoanilides 4a-b and 8a-b. 36 Compound 13 was synthesized in a two step process of nucleophillic addition of benzylamine to compound 11 in the presences of Hunig’s base followed by uracil alkylation (Figure 2.12). 32 Figure 2.12. Preparation of dUTPase inhibitor 13. Reagents and conditions: (a) benzylamine, DIEA, ether, 0°C, 2hr; (b) uracil, TMSCl, HMDS, reflux, 2hr, then TBAI, 11, DCM. 2.5 Biological Activity of dUTPase Inhibitors Though in vitro assays are the workhorse of drug discovery screening efforts, recently there has been a rediscovery of the benefit of phenotypic assays as a means of decreasing clinical attrition rates. 38 Therefore, we sought to determine the efficacy of our designed inhibitors in an MTT cell viability assay in collaboration with the group of Prof. Stan Louie (USC School of Pharmacy). Recent studies have shown that the silencing of dUTPase expression with siRNA sensitized cells in vitro to 5-fluoro deoxyuridine (FUdR). 39 Researchers at Taiho then extended this work by adding 1 µM of FUdR to a standard MTT-assay to test the ability of the combination of TS and dUTPase inhibition to decrease cell viability. 29 Our assay data for all the analogs are summarized in Figure 2.13 for the human colorectal cancer HCT-116 cells and Figure 2.14 for the human cervical cancer HeLa cells. The general activity trend was: aryl benzimidazole > arylsulfonamide > alkylbenzimidazole > alkylamides. A further comparison of benzyl linker substitution pattern shows greater potency with para 6b compared to the meta 6a in both HCT-116 and HeLa cells (Figure 2.15). As a result, the para substituted 6c was synthesized and, as predicted, the addition of the cyclopropylmethyl ether did result in an increase in efficacy in both cell lines compared to the unsubstituted analog 6b (Figure 2.16). Finally, while great lengths were taken to design these compounds to inhibit dUTPase, by treating HeLa cells with 6b alone and in combination with 1 µM of FUdR, we were able to see ~4 fold 20 increase in growth inhibition with the addition FUdR indicating that we were, in fact, targeting this pathway with our inhibitors (Figure 2.17). Figure 2.13. Growth inhibition of human colorectal cancer HCT-116 cells in response to 1 µM FUdR and increasing concentrations of dUTPase inhibitors. 0% 20% 40% 60% 80% 100% 0 5 10 20 40 80 160 % Inhibitions Concentration (µM) HCT-116 6a 6b 6c 9a 9b 10a 10b 13 21 Figure 2.14. Growth inhibition of human cervical cancer HeLa cells in response to 1 µM FUdR and increasing concentrations of dUTPase inhibitors. Figure 2.15. A comparison of the growth inhibition activity of 6a and 6b of HCT and HeLa cells in combination with 1 µM FUdR. 0% 20% 40% 60% 80% 100% 0 5 10 20 40 80 160 % Inhibition Concentration (µM) Hela 6a 6b 6c 9a 9b 10a 10b 13 0% 20% 40% 60% 80% 100% 0 5 10 20 40 80 160 % Inhibtion Concentration (µM) 6a-b 6a HCT-119 6a Hela 6b HCT-119 6b Hela 22 Figure 2.16. A comparison of the growth inhibition activity of 6b and 6c of HCT and HeLa cells in combination with 1 µM FUdR. Figure 2.17. The effect of FUdR on the growth inhibition activity of 6b in HeLa cells. 0% 20% 40% 60% 80% 100% 0 5 10 20 40 80 160 % Inhibitions Concentration (µM) 6b & 6c 6b HTC 6b Hela 6c HTC 6c Hela 0% 20% 40% 60% 80% 100% 0 5 10 20 40 80 160 % Inhibitions Concentration (µM) 6b in HeLa 6b + No FUdR 6b + 1 µM FUdR 23 2.6 Conclusion The overexpression of dUTPase in colorectal cancer has been tied to resistance to chemotherapeutic intervention. Therefore, we set out to develop novel, drug-like inhibitor leads. Through the utilization of virtual fragment screening and synthesis-based design, we were able to develop novel, potent, and synthetically accessible small molecules capable of growth inhibition in both the HCT colon cancer and HeLa cervical cancer cell lines. 24 2.7 Experimental Solvents and reagents were used as is from their commercial sources. Mercury 400, Varian 400-MR (400 MHz), Varian VNMRS-500 (500 MHz), or Varian VNMRS-600 (600 MHz) spectrometers were used to record 1 H, 13 C, and 19 F spectra. All flash chromatography columns were run with silica gel (60 Å, 40-63 μm; Sorbent Technologies). The Isolera One flash purification system (Biotage) was used for automated flash chromatography. Sample mass was determined using the on the Agilent 1260 Infinity LC/MS. 2.7.1 Structure-based Design While the dUpNHpp-bound dUTPase crystal structure (PDB:2HQU) contains a fully intact active site, the differing degrees of C-terminal coverage for each of the active sites did not give a clear picture of the actual size and shape of the site. Therefore, after a review of the available structures, PDB:3ARA was chosen for its uniform active sites and high resolution. The protein was prepared for docking using Schrodinger’s Protein Preparation Wizard tool to add hydrogens, correct bond orders, delete non-essential waters, predict side-chain protonation states, tautomers, and polar hydrogen orientations, and minimize the energy of the protein structure. The receptor grid was prepared as a 20- Å box centered around G110, A45, R85, and Q131. The ZINC Database was sorted for subsets by property and 354,309 “Clean Fragments” (MW≤ 250, logP≤ 3.5, # rotatable bonds≤ 5) were downloaded as .sdf files in Linux. These molecules were prepared for docking with LigPrep using the OPLS_2005 force field to generate protonation states between pH 5-9 using Epik. Initial studies showed identical results for all three active sites, so the active site between chain A and B that would be covered by the C-terminus of chain C was chosen for the remaining docking studies. The fragments were initially docked and scored using the HTVS setting in Glide 5.8 and then the top docking were redocked using the SP setting. The U-site was blocked manually in Maestro 9.3 by converting the –NH of the G99 and G110 to –NC with the “Set element” feature in the Build toolbar, hydrogens were added to the carbon with the “Add hydrogens” feature of the Edit toolbar. A cycle of converting 25 one of the hydrogens to a carbon, adding hydrogens, and adjusting the bond angles using the “Quick Torsion” tool in the “Adjust” feature in the Edit toolbar was repeated until the chain from G99 was connected to G110 with the “Draw structure” feature of the Build toolbar. A grid file was set up using the same parameters as above and docked with the fragments using the same HTVS to SP protocol. The top docking fragments for the U- and P-sites were overlay in the Maestro 9.3 window and the proposed linking strategies were drawn in ChemDraw, saved as .sdf file, prepared with LigPrep, docked into the original grid using the SP setting, and selected for synthesis based on GlideScore. 2.7.2 Synthetic Chemistry. 4-(cyclopropylmethoxy)-2-nitroaniline: To a solution of 4-amino-3-nitrophenol (1) (2.00g, 12.977 mmol) in CH3CN (30 mL) was added K2CO3 (3.587g, 25.953 mmol), (bromomethyl)cyclopropane (1.89 mL, 19.645 mmol), and KI (0.215g, 1.298 mmol). The mixture was stirred at 70 o C for 13 hours. The reaction mixture was poured into H2O (100 mL) and extracted with EtOAc (3 x 125 mL). The organic layers were combined, washed with brine, dried over Na2SO4, and concentrated in vacuo onto Celite. The residue was purified by flash column chromatography (15% EtOAc/hexanes). The relevant fractions were combined and concentrated under reduced pressure to yield 5a as a bright red-orange solid (2.814g, 81% yield). 1 H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 3.0 Hz, 1H), 7.10 (ddd, J = 9.1, 2.9, 1.4 Hz, 1H), 6.76 (d, J = 9.1 Hz, 1H), 5.02 (brs, 2H), 3.77 (d, J = 7.0 Hz, 2H), 1.44 – 1.12 (m, 1H), 0.65 (d, J = 8.2 Hz, 2H), 0.35 (d, J = 4.9 Hz, 2H). 13 C NMR (100 MHz, CDCl3) δ 150.33, 139.83, 127.38, 127.37, 120.23, 120.20, 107.50, 73.77, 10.26, 3.35. 26 4-(cyclopropylmethoxy)benzene-1,2-diamine (2b): A flask containing a solution of 4- (cyclopropylmethoxy)-2-nitroaniline (1.00g, 4.803 mmol) in methanol (30 mL) was purged several times with argon. Palladium on carbon (5 wt%, 0.200g) was added, and the flask was quickly re-purged with argon, and then several times with hydrogen. The mixture was allowed to stir at room temperature under a hydrogen atmosphere for 2 hours. The reaction mixture was subsequently filtered over a pad of Celite, and the cake was washed with copious amounts of methanol. The filtrate was concentrated under reduced pressure to yield a deep purple solid (0.828g, 97% yield). 1 H NMR (600 MHz, CDCl3) δ 6.61 (d, J = 8.3 Hz, 1H), 6.33 (d, J = 2.7 Hz, 1H), 6.25 (d, J = 5.8 Hz, 1H), 3.70 (d, J = 6.9 Hz, 2H), 3.50 (brs, 2H), 3.07 (brs, 2H), 1.39 – 1.13 (m, 1H), 0.60 (d, J = 8.9 Hz, 2H), 0.31 (d, J = 5.1 Hz, 2H). 13 C NMR (150 MHz, CDCl3) δ 154.09, 137.09, 127.50, 118.38, 105.23, 103.96, 73.37, 10.56, 3.25. Methyl 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate: 1.16g (10.3mmol) of uracil, 3.11 (10.3mmol) of Cs2CO3, and 2.36g (10.3mmol) of methyl 3- (bromomethyl)benzoate (3a) were combined in an oven dried RBF under a N2 atmosphere outfitted with a rubber septa and 10mL of DMSO was added. The reaction was stirred at room temperature for 3hrs, then dissolved in EtOAc. The organic was washed with brine 2X, then 2N NaOH. The aqueous layer was acidified with 6N HCl, extracted with EtOAc, dried with MgSO4, filtered through cotton, and concentrated in vacuo to yield 680mg of product (25% yield). 1 H NMR (400 MHz, cd3od) δ 7.97 (d, J = 11.4 Hz, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.61 – 7.57 (m, 1H), 7.49 (t, J = 7.7 Hz, 1H), 5.68 (d, J = 7.9 Hz, 1H), 4.99 (s, 2H), 3.90 (s, 3H). 13 C NMR (101 MHz, cd3od) δ 168.06, 27 166.55, 152.85, 146.94, 138.31, 133.59, 132.00, 130.23, 130.19, 129.77, 102.82, 52.74, 51.96. 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (4a): To a stirring solution of 680mg (2.6mmol) of methyl 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzoate in 10mL of H2O and 10mL of THF was added 436mg (10.4mmol, 4eq) of LiOH·H2O. The reaction was stirred overnight, concentrated in vacuo, the solid was dissolved in H2O, the solution was acidified with 6N HCl to ~pH3 (pink by Precision Labs, Inc.’s “Universal pH Indicator paper”) upon which a white precipitate formed. This was filtered, washed with H2O, and placed on high vacuum to yield 158mg of a white powder (25% yield). 1 H NMR (400 MHz, dmso) δ 13.04 (s, 1H), 11.35 (s, 1H), 7.89 – 7.85 (m, 2H), 7.81 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.52 – 7.47 (m, 1H), 5.62 (dd, J = 7.8, 2.2 Hz, 1H), 4.94 (s, 2H). 13 C NMR (101 MHz, dmso) δ 167.45, 164.05, 151.44, 146.06, 137.84, 132.37, 129.41, 129.01, 128.63, 101.89, 50.48. N-(2-aminophenyl)-3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide: 158mg of (0.64mmol) 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (4a), 90mg (0.83mmol, 1.3eq) of o-phenylenediamine, 315mg (0.83mmol, 1.3eq) of HATU, and 144µL of DIEA were dissolved in 4mL of DMF and stirred at room temperature for overnight. The reaction was concentrated in vacuo, dissolved in EtOAc, extracted with 2N NaOH. The aqueous layer was then neutralized with 6N HCl, extracted with EtOAc, the organic layer was then dried with MgSO4, concentrated in 28 vacuo onto Celite, and purified by silica flash column with a gradient from 5% MeOH in DCM to 8% MeOH in DCM (Rf=0.2 in 5%MeOH in DCM) to yield 93mg of the desired product (43% yield). 1 H NMR (400 MHz, cd3od) δ 7.94 (d, J = 8.9 Hz, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.54 (dt, J = 15.1, 7.7 Hz, 2H), 7.18 (dd, J = 7.9, 1.3 Hz, 1H), 7.12 – 7.04 (m, 1H), 6.90 (dd, J = 8.0, 1.2 Hz, 1H), 6.77 (td, J = 7.7, 1.3 Hz, 1H), 5.69 (d, J = 7.9 Hz, 1H), 5.02 (s, 2H). 13 C NMR (101 MHz, cd3od) δ 165.14, 145.52, 136.77, 134.86, 130.88, 128.79, 127.18, 127.08, 126.90, 126.21, 118.19, 117.26, 101.41, 50.68. 1-(3-(1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (6a): 93mg (0.36mmol) of N-(2-aminophenyl)-3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide was dissolved in 10mL of glacial acetic acid and 1mL of 12N HCl, stirred at 120°C for 72hrs, and concentrated in vacuo. The resulting residue was dissolved in a minimal amount of MeOH and the product was crashed out of solution with EtOAc to yield 68mg of a light brown power (59% yield, the product appeared fluorescently purple under a 254nm UV lamp, Rf=0.2 in 5% MeOH in DCM). 1 H NMR (400 MHz, cd3od) δ 8.11 (d, J = 9.0 Hz, 2H), 7.84 (dd, J = 6.2, 3.1 Hz, 2H), 7.77 (t, J = 7.2 Hz, 3H), 7.64 (dd, J = 6.2, 3.1 Hz, 2H), 5.74 (d, J = 7.9 Hz, 1H), 5.12 (s, 2H). 13 C NMR (101 MHz, cd3od) δ 147.01, 140.10, 134.19, 133.23, 131.68, 128.62, 128.25, 127.89, 124.80, 115.00, 103.10, 52.11. LC-MS: calcd. [M+H] + 319.11 (100%), 320.12 (19.7%), & 321.12 (2.2%); found 319.1 (100%), 320.1 (18.26%), & 321.1 (2.4%) 29 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H) -yl)methyl)benzoic acid (4b): 560mg (5mmol) of uracil and 1.5g (5mmol) of Cs2CO3 were combined in an oven dried RBF under a N2 atmosphere outfitted with a rubber septa and 10mL of DMSO was added. 1.15g (5mmol) of methyl 4-(bromomethyl)benzoate was dissolved in 3mL of DMSO, added dropwise to the uracil/Cs2CO3 solution, and stirred at room temperature for 3hrs. The reaction mixture was then dissolved in brine with a small portion of 6N HCl and extracted with EtOAc. The organic layer was then washed with 2N NaOH. Remarkably, acidification of the aqueous layer with 6N HCl yielded 519mg of the hydrolyzed acid (42% yield). 1H NMR (400 MHz, dmso) δ 12.93 (s, 1H), 11.35 (d, J = 1.8 Hz, 1H), 7.96 – 7.89 (m, 2H), 7.77 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 5.62 (dd, J = 7.8, 2.3 Hz, 1H), 4.95 (s, 2H). 13 C NMR (101 MHz, dmso) δ 166.98, 163.66, 151.00, 145.65, 141.83, 130.03, 129.66, 127.30, 101.50, 50.12. 1-(4-(1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (6b): 158mg of (0.64mmol) 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (4b), 90mg (0.83mmol, 1.3eq) of o-phenylenediamine, 315mg (0.83mmol, 1.3eq) of HATU, and 144µL of DIEA were dissolved in 4mL of DMSO and stirred at room temperature for 240hrs. The reaction was dissolved in EtOAc and the organic was extracted with brine 3X. The organic layer was then dried with MgSO4, concentrated in vacuo onto Celite, and purified by silica flash column with a gradient from 2% MeOH in DCM to 5% 30 MeOH in DCM to yield 114mg of the desired product (56% yield). 1 H NMR (400 MHz, cd3od) δ 8.00 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 7.7 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.78 (t, J = 7.6 Hz, 1H), 5.71 (d, J = 7.9 Hz, 1H), 5.04 (s, 2H). 114mg (0.36mmol) of N-(2-aminophenyl)-4- ((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide from the previous step was dissolved in 10mL of glacial acetic acid and 1mL of 12N HCl, stirred at 120°C overnight, and concentrated in vacuo. The resulting residue was dissolved in a minimal amount of MeOH and the product was crashed out of solution with EtOAc to yield 81mg of a light brown powder (70% yield, the product appeared fluorescently purple under a 254nm UV lamp). 1 H NMR (400 MHz, dmso) δ 11.38 (s, 1H), 8.29 (d, J = 8.4 Hz, 2H), 7.89 – 7.77 (m, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (dd, J = 6.1, 3.1 Hz, 2H), 5.65 (dd, J = 7.9, 2.2 Hz, 1H), 5.01 (s, 1H). 13C NMR (101 MHz, dmso) δ 163.72, 151.05, 148.86, 145.67, 141.92, 128.30, 128.14, 125.45, 123.49, 114.19, 109.56, 101.62, 50.23. LC-MS: calcd. [M+H] + 319.11 (100%), 320.12 (19.7%), & 321.12 (2.2%); found 319.1 (100%), 320.2 (20.45%), & 321.1 (249%). N-benzyl-4-(bromomethyl)benzenesulfonamide (12): 1.19 g (4.4mmol) of 4- (Bromomethyl) benzenesulfonyl chloride (11) was placed in an oven-dried RBF under N2 atmosphere, sealed with a rubber septa, dissolved in 2mL of anhydrous ether, cooled to 0°C, 782µL (4.5mmol) of DIEA added, then 483µL (4.42mmol) of benzylamine. The reaction was allowed to warm to room temperature, stirred for 2hrs, concentrated in vacuo, and purified by automated flask chromatography (Rf=0.25 in 20% EtOAc in hexanes) to yield 227mg of product (15% yield). 1 H NMR (400 MHz, cd3od) δ 7.79 (dd, J = 11.2, 8.5 Hz, 2H), 7.56 (dd, J = 8.6, 2.1 Hz, 2H), 7.26 – 7.16 (m, 5H), 4.70 (s, 1H), 4.61 (s, 1H), 4.07 (s, 2H). 31 N-benzyl-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzenesulfonamide (13): 74mg (0.66mmol) of uracil, 170µL (1.34mmol, 2eq) of chlorotrimethylsilane, and 1.38mL (6.6mmol, 10eq) of hexamethyldisilazane were refluxed for 2hrs. 227mg (0.67mmol) N-benzyl-4-(bromomethyl) benzenesulfonamide (12) and 16mg (0.04mmol, 0.06eq) of tetrabutylammonium iodide were dissolved in 1.4mL of dry DCM and added to the uracil solution and stirred at room temperature for 96hrs. H2O was then added and the precipitate was filtered to yield 7.9mg of an off-white solid (3% yield). 1 H NMR (400 MHz, cd3od) δ 8.08 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 – 7.43 (m, 5H), 6.02 (d, J = 7.8 Hz, 1H), 5.29 (s, 2H), 4.37 (s, 2H). 13 C NMR (101 MHz, cd3od) δ 146.99, 142.24, 142.02, 138.47, 129.38, 129.36, 128.90, 128.53, 128.41, 102.90, 51.87, 47.90. LC-MS: calcd. [M+H] + 372.09 (100.0%), 373.10 (19.8%), & 374.09 (4.7%); found 372.1 (100%), 373.1 (21.42%), & 374.1 (7.31%). 32 2.8 References (1) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Thun, M. J. CA A Cancer Journal for Clinicians. 2010, 59, 225-249. (2) Ng, K.; Zhu, A.X. Crit Rev Oncol Hematol, 2008, 65, 8-20. (3) Poon, M.A.: O'Connell, M.J.; Moertel, C.G.; Wieand,H.S.; Cullinan, S.A.; Everson, L.K.; Krook, J.E.; Mailliard, J.A.; Laurie, J.A.; Tschetter, L.K. et al. J Clin Oncol, 1989, 7, 1407-1418. (4) Petrelli, N.; Douglass, H. O.; Herrera, L.; Russell, D.; Stablein, D. M.; Bruckner, H. W.; Mayer, R. J.; Schinella, R.; Green, M. D.; Muggia, F. M. Gastrointestinal Tumor Study Group.; 1989; Vol. 7, pp. 1419-1426. (5) Piedbois, P.; Buyse, M. Ann Oncol 4 Suppl, 1993, 2, 15-19. (6) Ladner, R.D. Current Protein Pept Sci 2001, 2, 361-370. (7) Koehler, S. E.; Ladner, R. D. Mol Pharmacol. 2004, 66, 620-626. (8) Tinkelenberg, B. A.; Hansbury, M. J.; Ladner, R. D. Cancer Res. 2002, 62, 4909- 4915. (9) Adlard, J.W.; Richman, S.D.; Royston, P.; Allan, J.M.; Meade, A.; Parmar, M.; Shelby, P.; Quirke, P.; Seymour, M.T.; J Clin Oncol. 2004, 22, 9506. (10) Wilson, P. M.; Fazzone, W.; LaBonte, M. J.; Deng, J.; Neamati, N.; Ladner, R. D. Mol Cancer Ther. 2008, 7, 3029-3037. (11) Takatori, H.; Yamashita, T.; Honda, M.; Nishino, R.; Arai, K.; Takamura, H.; Ohta, T.; Zen, y.; Kaneko, S.; Liver Int. 2010, 30, 438-446. (12) Nobili, S.; Napoli, C.; Landini, I.; Morganti, M.; Cianchi, F.; Valanzano, R.; Tonelli, F.; Cortesini, C.; Mazzei, T.; Mini, E. Int J Cancer, 2011, 128, 1935- 1945. (13) Tóth, J.; Varga, B.; Kovács, M.; Málnási-Csizmadia, A.; Vértessy, B. G. J Biol Chem. 2007, 282, 33572–82. (14) Mol, C. D.; Harris, J. M.; McIntosh, E. M.; Tainer, J. A. Structure 1996, 4, 1077– 1092. 33 (15) Varga, B.; Barabás, O.; Kovári, J.; Tóth, J.; Hunyadi-Gulyás, E.; Klement, E.; Medzihradszky, K. F.; Tölgyesi, F.; Fidy, J.; Vértessy, B. G. FEBS letters 2007, 581, 4783–8. (16) Barabas, O.; Pongracz, V.; Kovari, J.; Wilmanns, M.; Vertessy, B.G. J. Biol. Chem., 2004, 279, 42907–42915. (17) Whittingham, J. L.; Leal, I.; Nguyen, C.; Kasinathan, G.; Bell, E.; Jones, A. F.; Berry, C.; Benito, A.; Turkenburg, J. P.; Dodson, E. J.; Ruiz Perez, L. M.; Wilkinson, A. J.; Johansson, N. G.; Brun, R.; Gilbert, I. H.; Gonzalez Pacanowska, D.; Wilson, K. S. Structure. 2005, 13, 329–38. (18) Baragaña, B.; McCarthy, O.; Sánchez, P.; Bosch-Navarrete, C.; Kaiser, M.; Brun, R.; Whittingham, J. L.; Roberts, S. M.; Zhou, X.-X.; Wilson, K. S.; Johansson, N. G.; González-Pacanowska, D.; Gilbert, I. H. Bioorg Med Chem 2011, 19, 2378– 91. (19) Hampton, S. E.; Baragaña, B.; Schipani, A.; Bosch-Navarrete, C.; Musso- Buendía, J. A.; Recio, E.; Kaiser, M.; Whittingham, J. L.; Roberts, S. M.; Shevtsov, M.; Brannigan, J. A.; Kahnberg, P.; Brun, R.; Wilson, K. S.; González- Pacanowska, D.; Johansson, N. G.; Gilbert, I. H. ChemMedChem 2011, 6, 1816– 1831. (20) Miyakoshi, H.; Miyahara, S.; Yokogawa, T.; Chong, K. T.; Taguchi, J.; Endoh, K.; Yano, W.; Wakasa, T.; Ueno, H.; Takao, Y.; Nomura, M.; Shuto, S.; Nagasawa, H.; Fukuoka, M. J Med Chem 2012, 55, 2960–9. (21) Miyahara, S.; Miyakoshi, H.; Yokogawa, T.; Chong, K. T.; Taguchi, J.; Muto, T.; Endoh, K.; Yano, W.; Wakasa, T.; Ueno, H.; Takao, Y.; Fujioka, A.; Hashimoto, A.; Itou, K.; Yamamura, K.; Nomura, M.; Nagasawa, H.; Shuto, S.; Fukuoka, M. J Med Chem 2012, 55, 2970–80. (22) Anderson, A. C.; Pollastri, M. P.; Schiffer, C. A.; Peet, N. P. Drug Discov Today 2011, 16, 755–61. (23) Irwin, J. J.; Sterling, T.; Mysinger, M. M.; Bolstad, E. S.; Coleman, R. G. J Chem Inf Model 2012, 52, 1757–1768. 34 (24) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J Med Chem 2004, 47, 1739–49. (25) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. J Med Chem 2004, 47, 1750–9. (26) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J Med Chem 2006, 49, 6177–96. (27) Suite 2012: Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012. (28) Nguyen, C.; Kasinathan, G.; Leal-Cortijo, I.; Musso-Buendia, A.; Kaiser, M.; Brun, R.; Ruiz-Pérez, L. M.; Johansson, N. G.; González-Pacanowska, D.; Gilbert, I. H. J Med Chem 2005, 48, 5942–54. (29) Miyahara, S.; Miyakoshi, H.; Yokogawa, T.; Chong, K. T.; Taguchi, J.; Muto, T.; Endoh, K.; Yano, W.; Wakasa, T.; Ueno, H.; Takao, Y.; Fujioka, A.; Hashimoto, A.; Itou, K.; Yamamura, K.; Nomura, M.; Nagasawa, H.; Shuto, S.; Fukuoka, M. J Med Chem 2012, 55, 5483–96. (30) Madsen, P.; Knudsen, L. B.; Wiberg, F. C.; Carr, R. D. J Med Chem 1998, 41, 5150–7. (31) Zhu, Y.; Skupinska, K.; McEachern, E. Heterocycles 2006, 67, 769–775. (32) Shelke, S. A.; Sigurdsson, S. T. Angew Chem 2010, 49, 7984–6. (33) Nguyen, C.; Ruda, G. F.; Schipani, A.; Kasinathan, G.; Leal, I.; Musso-Buendia, A.; Kaiser, M.; Brun, R.; Ruiz-Pérez, L. M.; Sahlberg, B.-L.; Johansson, N. G.; Gonzalez-Pacanowska, D.; Gilbert, I. H. J Med Chem 2006, 49, 4183–95. (34) Brown, F. J.; Cronk, L. A.; Aharony, D.; Snyder, D. W. J Med Chem 1992, 35, 2419–2439. (35) Lim, C. J.; Kim, N.; Lee, E. K.; Lee, B. H.; Oh, K.-S.; Yoo, S.; Yi, K. Y. Bioorg Med Chem Lett 2011, 21, 2309–12. (36) Lamblin, M.; Dabbas, B.; Spingarn, R.; Mendoza-Sanchez, R.; Wang, T.-T.; An, B.-S.; Huang, D. C.; Kremer, R.; White, J. H.; Gleason, J. L. Bioorg Med Chem 2010, 18, 4119–37. 35 (37) Huang, S.-T.; Hsei, I.-J.; Chen, C. Bioorg Med Chem 2006, 14, 6106–19. (38) Kotz, J. SciBX 2012, 5. (39) Wilson, P. M.; LaBonte, M. J.; Lenz, H.-J.; Mack, P. C.; Ladner, R. D. Mol Cancer Ther 2012, 11, 616–28. 36 Chapter 3: Design and Synthesis of CXCR2 Allosteric Antagonist 3.1 Introduction Interleukin-8 (IL-8) is a potent proinflammatory chemokine of the CXC family. 1 IL-8 imparts its chemotactic activity through the activation of two evolutionarily related GPCRs CXCR1 and CXCR2 causing the cell to migrate towards increasing concentration of IL-8. While IL-8 is important for wound healing and embryogenesis, the IL-8- CXCR1/2 pathway has been implicated in a variety of diseases including psoriasis, bronchitis, and COPD. 2,3 As a result, the development of CXCR2-antagonsists has been the focus of a large degree of pharmaceutical efforts resulting in several clinical candidates. 4,5 Recently, this IL-8/CXCR2 signaling process has been implicated as an important factor in tumor metastasis and the maintenance of the tumor microenvironment. 1 A large number of studies have found IL-8 to be overexpressed in a variety of tumors. 6 Several groups, including our collaborators in the lab of Dr. Heinz-Josef Lenz (USC Keck School of Medicine), have shown the efficacy of CXCR2 antagonists in the inhibition of tumor growth and angiogenesis. 7-9 As a result, we set to better understand the structural necessities of these molecules through SAR analysis and structure-based design to develop our own structurally novel molecules. 3.2 CXCR2 Antagonist Design We began our work with a structural analysis of known CXCR2 allosteric antagonists (Figure 3.1). Reported in 1998, compound 1 (SB-225002) 10 , a HTS hit analog, was the first, selective small molecule CXCR2 antagonist. Following this paper, a large number of structurally related urea analogs were developed including 2 (SB-332235) 11 and 3 (SB- 656933), 5 currently a stage II clinical candidate at GSK. Each of these structurally related compounds contains three conserved structural motifs: an acidic phenol activated by and electron withdrawing group, a halogen substituted arene, and a urea linker. 12 The original –NO2 of 1, generally believed to be a structurally liable functional group was replaced with the more “tolerable” sulfonamide in 2 and 3. 13 Researchers at Schering- Plough were able to replace the ureas of 1-3 with the bioisosteric 3,4-diaminocyclobut-3- 37 ene-1,2-dione and the sulfonamide with a dimethylamide to develop the novel antagonist 4 (SCH 527123). 14,15 Figure 3.1. Allosteric CXCR2 antagonists. Due the lack of structural diversity in allosteric CXCR2 antagonists 1-4, it is unclear whether the role of the urea and 3,4-diaminocyclobut-3-ene-1,2-dione is as a binding motif or simply a linker placing the acidic phenol and the arene in the correct spatial position. Some insight is gained through the comparison of 5 and 6 where the methylation of the 3,4-diaminocyclobut-3-ene-1,2-dione causes a significant decrease in activity (Figure 3.2). 14 Unfortunately, no comparable data was available for the urea based antagonist. Figure 3.2. Effect of methylation of the 3,4-diaminocyclobut-3-ene-1,2-dione on CXCR2 antagonist activity. With the wealth of structural data contained in the over 85,000 structures in the Protein Database (PDB), we set out to develop a procedure to screen these structures for urea containing ligands to better understand their interactions. A simple three step process was developed to search the PDB for any functional group (Figure 3.3). 1) Employing the “Chemical component search” tool at rscb.org, a list of x-ray and NMR structures containing the desired functional group could be downloaded. 2) These files served as the input for a short KNIME 16 workflow of Schrodinger modules that reads the PDB files, refines and optimizes the hydrogen bonding interactions with the “Protein 38 Preparation Tool,” and opens the functional group-containing structures in Maestro, 17 the GUI for the Schrodinger Suite. 3) Finally, the protein-ligand interactions are evaluated manually in 3D in Maestro or 2D via the “Ligand Interaction Diagram” function. 17 Figure 3.3. Workflow for the acquisition, processing, and analysis of interactions of protein-aryl ureas crystal structures. With the exhaustive distribution of ureas, the PDB was searched for substructures of 1- methyl-3-phenylurea and 1,3-diphenylurea with two defined urea hydrogens to limit the results to relevant urea examples. Twenty-two structures from eleven unrelated targets were found containing the 1-methyl-3-phenylurea moiety. Four structures, each from their own protein target contained “linker” ureas spatially connecting two functionally important groups. The remaining eighteen, from seven different proteins, were shown to engage in some hydrogen bonding interaction with their protein partner. These interactions are summarized in Table 3.1. 39 Table 3.1. Summary of interactions of Ar-NHCONH-R from the PDB. a Side chain amide NH H-bond donator to urea carbonyl. b Urea carbonyl H-bond acceptor of OH of both tyrosines. The urea appears to provide two different interactions either bonding with carboxylates of aspartic acids or with back bone carbonyls. Two structures showing the two binding orientations of a urea with a carboxylate either with both urea hydrogens binding to one oxygen of the acid or each urea hydrogen binding to a different oxygen are highlighted in Figure 3.4. Taken together, ureas serve to hydrogen bond with acidic amino acids (5/11 protein targets), as a linker (4/11), or to hydrogen bond to the protein backbone (2/11). 18- 23 Urea Target PDB ID -NH -NH =O Arginine Methyltransferase 3 3SMQ D422 D422 R396 BACE 3L5C K168 (=O) F169 (=O) - DOT1L 4EQZ,4EQ7 D161 D161 N241 (NH 2) a 4ERO,4EQ6 D161 - - 4EQ3,4EQ5 D161 D161 - Epoxide Hydrolase 1VJ5 D333 D333 Y381, Y465 b Methionyl-tRNA Synthetase 3TUN,3U1E, 3U1F,3U1G D287 D287 - Peptide Deformylase 3U7K, S57 (=O) S57 (=O) - 3U7L S57 (=O) - - ROCK1 3V8S D216 D216 - 40 Figure 3.4. Examples of binding orientations of ureas forming two hydrogen bonds to Asp. Top panels show each nitrogen binding to different oxygen of the carboxylate (a) ribbon, (b) Ligand Interaction Diagram (PDB 4EQZ); bottom panels show both nitrogens binding to the same oxygen (c) ribbon, (d) Ligand Interaction Diagram (PDB 1VJ5). 18 With an understanding of the functional relevance of urea, we set out to make a series of analogs to test which of these bipartite roles the ureas of antagonists 1-3, and by extension the bioisosteric 3,4-diaminocyclobut-3-ene-1,2-dione of antagonist 4, play in binding to CXCR2. During the development of the urea-based antagonist, much of the focus was placed on optimization of the EWG of the acidic phenol moiety. This group went through several structural iterations from the -NO2 of the original hit 1 to sulfonamide 2 to finally the sulfonyl piperazine of clinical candidate 3. 5,10,11 It is unclear whether this was the result of toxicity, sulfonamide-related allergies, or pharmacokinetics issues. In contrast, the 3-amino-2-hydroxy-N,N-dimethyl benzamide 8 employed by the Merck researchers was conserved throughout their optimization process which allowed 4, despite its late start, to progress through the clinic faster. 4,14,15 Additionally, with its lack of free, chemically reactive –NH bonds, the dimethylamide is considerably more A B D C 41 chemically inert than the sulfonamide and sulfonyl piperazine allowing for the more facile synthesis of analogs. Considering these factors together, we settled on 8 as the acidic phenol moiety for our analogs. 3.3 Structure-based Design of New CXCR2 Antagonists To better understand, the SAR of CXCR2 antagonists a homology model of CXCR2 was constructed. Based on BLAST bit score, 24 a statistical analysis of sequence similarity, CXCR4 was, not surprisingly, the highest rated template. 25 However, as can be seen in Figure 3.5a, the CXCR4 crystal structures are poorly resolved at the end of the 7 th transmembrane helix (TM7) and are missing helix 8. Previous mutagenic studies have shown the importance of D97 of TM2 and K320 at the beginning of helix 8 for the binding of urea based antagonists and 4. 26,27 Therefore, the next ranking structures, the opiod receptors, were evaluated. 28 All showed structurally intact TM7 and helix 8 (Figure 3.5b) and, based on resolution, the morphinan antagonist-bound µ-opiod receptor structure (PDB:4DKL) was selected as the template. 28 Figure 3.5. Overlay of top scoring GPCR templates for the CXCR2 homology model. (a) Highlighted in periwinkle are the 7 th transmembrane (TM7) from CXCR4 crystal structures 28 (PBD:3OE0, 3OE8, 3OE8, 3OE9) showing the lack of helix 8 (red circle). (b) Illustrates the fully intact TM7 and helix 8 from the µ- opiod receptor 31 (PDB:4DKL magenta) which presents a better template for the modeling of allosteric CXCR2 antagonists than CXCR4 (periwinkle). A B 42 While class A GPCRs are involved in a wide array of biological processes from sight, to taste, to inflammation, to neurotransmission, they all contain a series of evolutionarily conserved transmembrane structural motifs. 29,30 Using these motifs the sequences of CXCR2 and µ-opiod receptor were manually aligned placing gaps in the loops where the most sequence diversity occurs (Figure 3.6). With this alignment in hand, Prime (Prime, v3.1, Schrödinger, LLC, New York, NY) was used to construct a homology model using a knowledge-based building method to account for sequence gaps. The new analogs 11b and 18b were then docked into the CXCR2 homology model using Induced Fit Docking (IFD). 31-34 This workflow combines static Glide docking with the protein structure refinement of Prime allowing both protein flexibility and ligand-induced protein reorientation to be considered in ligand docking. Studies comparing apo (ligand-free) and holo (ligand-bound) structures have shown the importance of considering protein conformational changes in the accurate docking of a ligand from the later into the former to predict the later. 35-37 To account this induced fit and the inherent inaccuracies of the homology models, IFD is the recommended for homology model docking. Figure 3.6. Sequence alignment of human CXCR2 (target) and human µ-opiod receptor from crystal structure PDB 4DKL (template) used to construct the homology model. Docking results show the urea of 11b binding to D84, in the same configuration as the urea in the DOT1L complex (PDB:4EQZ) where both urea hydrogens binding to one oxygen of the acid side-chain, and the phenolate binding to K320 (Figure 3.7b-c). Both of these residues were shown in mutagenic studies to be important or required for the activity urea-based antatgonist and compound 4. Compound 18b showed the ability to produce the same interactions as 11b with D84 and K320 (Figure 3.7b). While these docking results are encouraging, a PDB substructure search was unable to duplicate this 43 binding interaction though hydrogen bonds to aspartic acids from the 4-amino were seen in crystal structures of histone lysine methyltransferases (PDB:3MO0, 3MO2, 3MO5) and methionyl-tRNA synthetase (PDB:4EG6). 38,39 Figure 3.7. Analysis of Induced-Fit Docking results. (a) Overlay of 11b (yellow) and 18b (magenta) into the allosteric site CXCR2 homology model; (b) Key mutagenically-determined residues D84 and K320 for the binding of 2 to CXCR2 are highlighted. Both 11b and 18b donate two hydrogen bonds to D84 and have an anionic interaction with K320; (c) Ligand Interaction Diagram for the predicted binding of 11b to CXCR2; (d) Ligand Interaction Diagram for the predicted binding of 18b to CXCR2. 3.4 CXCR2 Antagonist Synthesis We began our work with the synthesis of a series of simple urea analog containing phenol 8 (Figure 3.8). A comparison of 2 and 4 left some structural questions about antagonist requirements. Each of the ureas contains a halogenated arene while antagonists based on phenol 8 show a high degree of potency either with benzylic substituents, as in 4, or phenyl, as in 5. To answer this question, urea analogs were synthesized with brominated and unsubstituted anilines and benzylamines. While the aryl-aryl analogs 11a-b were synthesized in two steps in one-pot using CDI (Figure 3.9), the aryl-benzyl analogs 13a- b could not be accessed under these conditions, so a 4-nitrophenyl chloroformate-based route was employed (Figure 3.10). Of these initial compounds, 11b showed the greatest B A D C 44 activity, so some remaining analogs were synthesized with various functional groups linking 8 to a 4-bromo benzene. In 1990, Damon and Hoover showed that a ketodifluoromethylene could serve as an amide bioisostere. 40 Therefore, we postulated that an α-difluoromethylene amide could serve as a urea mimetic. Compounds 13a and 19a-b were synthesized by my colleague Kalyan Nagulapalli Venkata. Analogs 19a-b were accessed in two steps starting from iodobenzene or 4-iodobromobenzene and ethyl bromo(difluoro) acetate. 41 Initial attempts to convert to 8 to the corresponding azide using imidazole-1-sulfonyl azide·HCl were unsuccessful, likely the result of the proximal acidic, so the methyl- protected phenol 9 synthesized and was easily converted to the azide 14. Azide 14 was converted by Cu-mediated 3+2 cycloaddition with phenyl acetylene or bromo-4- ethynylbenzene 42 and deprotected with BBr3 to yield 15a-b (Figure 3.11). Due to the robust nature of the methyl ether, this protected molecule 9 was used throughout the synthesis of the remaining analogs to prevent any untoward side-reactions. Compound 17 was synthesized in four steps starting with 10b and ethyl chlorooxoacetate followed by saponification, HATU coupling of 9, and BBr3 deprotection (Figure 3.12). Finally, pyrimidines 18a-b were synthesized with two sequential SNAr reactions first at the 4 position and second at the 2 followed by BBr3 deprotection (Figure 3.13). Figure 3.8. Preparation of anilines 8-9. Reagents and conditions: (a) oxalyl chloride, DCM, 0°C, then (CH 3) 2NH; (b) 5% Pd/C, H 2, MeOH; (c) (i) MeI, K 2CO 3, acetone, 60°C; (ii) 5% Pd/C, H 2, MeOH. Figure 3.9. Preparation of CXCR2 antagonists 11a-b. Reagents and conditions: (a) CDI, DCM, 2hr, then 3. 45 Figure 3.10. Preparation of CXCR2 antagonists 13a-b. Reagents and conditions: (a) benzyl amine or 4- bromo benzylamine·HCl, 4-nitrophenyl chloroformate, TEA, DCM, 0°; (b) 3, TEA, DCM. Figure 3.11. Preparation of CXCR2 antagonists 15a-b. Reagents and conditions: (a) 1H-imidazole-1- sulfonyl azide, K 2CO 3, CuSO4·5H2O, MeOH; (b) phenyl acetylene or bromo-4-ethynylbenzene, CuSO4•5H2O, sodium ascorbate, tBuOH, H 2O; (c) 1M BBr 3, DMC, 0°C. Figure 3.12. Preparation of CXCR2 antagonist 17. Reagents and conditions: (a) ethyl chlorooxoacetate, TEA, DCM, 0°C; (b) NaOH,, EtOH, H 2O; (c) 9, HATU, DIEA, DMF; (d) 1M BBr 3, DMC, 0°C. Figure 3.13. Preparation of CXCR2 antagonists 18a-b. Reagents and conditions: (a) TEA, n-BuOH, 80°C; (b) PTSA, 10b or 9, DMF, 80°C; (c) 1M BBr 3, DMC, 0°C. 3.5 CXCR2 Antagonist Evaluation Recently, several research groups have shown anti-metastatic and anti-tumoral activity of compound 4 in melanoma and colon cancer via a variety of techniques including in vitro tumor cell proliferation MTT assay. 7-9 Therefore, our collaborators in the Louie and Lenz laboratories used this assay as our initial screen for compound activity. (Additionally, I would like to thank Dr. Pratt’s helping to evaluate previous generation of compounds). The compounds were tested at 24-, 48-, and 72-hour time points in two cell lines: HCT-116 and the IL-8 overexpressing E2. While the majority of the 46 compounds showed little activity, 11b, 18a, and 18b showed comparable or greater activity than 4 in both cell lines, with compound 18b as the most potent (Table 3.2). Table 3.2. List of analogs 11a-b, 13a-b, 15a-b, 17-18b, 19a-b, and 4 and their inhibitory activity in vitro tumor cell proliferation assay. nr= IC 50 not reached 3.6 Conclusion Following a detailed understanding of the existing CXCR2 antagonists, we set out to develop our own novel antagonist by varying the linker moiety. A series of synthetically accessible molecules were designed and synthesized, linking the acidic phenol of compounds 4-6 with the arene of 5-6 or the halogenated arene of 1-3. While the majority of these molecules were inactive, compounds 11b and 18a-b showed significant growth inhibition activity with the latter two having an IC50 greater than compound 4. Based on homology modeling of CXCR2, the binding orientations were predicted which could allow for further structure-based optimization of these novel scaffolds. HCT-116 (IC 50) E 2 (IC 50) HCT-116 (IC 50) E 2 (IC 50) Analog Linker X 24h 48h 72h 24h 48h 72h Analog Linker X 24h 48h 72h 24h 48h 72h 11a -H nr nr nr nr nr nr 17 -H nr nr nr nr nr nr 11b - -Br nr 108 140 182 171 117 18a -Br 108 64 63 58 57 53 13a -H nr nr nr nr nr nr 18b -Br 73 35 42 43 53 45 13b - -Br nr nr nr nr nr nr 19a -H nr nr nr nr nr nr 15a -H nr nr nr nr nr nr 19b - -Br nr nr nr nr nr nr 15b - -Br nr nr nr nr nr nr 4 145 142 141 178 158 144 47 3.7 Experimental 3.7.1 PDB Functional Group Search The PDB was searched for the desired urea moiety using "Search by ligand" and "additional ligand options" functions at http://www.rcsb.org/pdb/home/home.do. The substructure was drawn in the "Structure" tab specifically drawing the urea N-H bonds. The PDB was searched for the structure as a "Substructure" with the "Submit Query" button. The "Structure Hits" tab was selected in the "Ligand results" page, the "Filter" dropdown menu was set to "Download Checked," and the structures were downloaded as uncompressed PDB files. KNIME Workflow was constructed by connecting in series the "PDB Reader" module to the "Protein Preparation" module to the "Open Maestro" and the "Write Files" modules which wrote the files as .mae. The default setting for "Protein Prep" module were used except the "Minimization" function was turned off. The PDB files downloaded in the prior step were opened in the "PDB Reader" module and the workflow started with the BLANK button. The output process structures were opened in Maestro and the bonding interactions were evaluated using manually using either the 3D Maestro workspace or the Ligand Interaction Diagram. 3.7.2 Homology Model Docking The CXCR2 homology model was constructed using the “Homology Modeling” workflow of Prime. A list of potential templates was populated in the Prime workflow using the BLAST Homology Search feature. Templates were evaluated for their fitness with based on BLAST bit score and visual inspection of TM7 and helix 8. The opiod receptor structures were the highest ranking structures with intact TM7 and helix 8. The µ-opiod structures (PDB:4DKL) was selected from the other opiod structures for its high resolution 2.8 Å. The CXCR2 and opiod sequences were aligned based on the conserved TM residues using the extensive alignment at http://swift.cmbi.kun.nl/euroschool/gpcr_intro/MVIEW.mview.html as a guide. Gaps were placed in the middle position of each loop to minimize perturbations to the TM helices. The model was constructed using the "Knowledge-based" model building method. 48 Compounds 11b and 18b were draw in separate ChemDraw, saved as .sdf file, and prepared for docking with LigPrep by generating ionization states at pH 5 to 9 using Epik. The output structures were opened in Maestro and the structure with a phenolate was saved for docking. The compound 11b was docked first using the Induced Fit Docking workflow at the standard precision setting. The receptor grid was set up as a 20 Å box centered on residues D84 and K320. H-bond constraints were setup to the –O - of the carboxylate of D84 and the –NH of the side-chain amine of K320. The output structures were evaluated based on their ability of replicate the hydrogen bonding interactions seen in the PDB search. Compound 18b was then docked using the same procedure as 11b into the best 11b-bound structure. 3.7.3 Synthetic Chemistry 2-hydroxy-N,N-dimethyl-3-nitrobenzamide (7): To a stirring room temperature solution of 1g (5.56mmol) of 3-Nitrosalicylic acid in 20mL of dry DCM was added 3mL (16.7mmol, 3eq) of oxalyl chloride and 6 drops of dry DMF. The reaction was monitored by removing an aliquot through the rubber septa with a syringe, dissolving the sample in MeOH, and checking for the methyl ester by TLC which took 3hrs. The reaction was concentrated in vacuo while minimizing exposure to atmospheric H2O, placed on high vacuum for 30min, 20mL of dry DCM added, followed by the dropwise addition of 1.3mL of a 2M dimethylamine in THF solution. The reaction was stirred overnight, concentrated in vacuo, dissolved in EtOAc, extracted with a saturated solution of NaHCO3 and brine, dried with Na2SO4, and concentrated in vacuo to yield 924mg of a light yellow solid (quantitative). 1 H NMR (400 MHz, CD3OD) δ 8.19 (dd, J = 9.2, 1.6 Hz, 1H), 7.62 (dd, J = 8.2, 1.7 Hz, 1H), 7.13 (t, J = 8.3 Hz, 1H), 3.12 (s, 1H), 2.95 (s, 1H). 13C NMR (101 MHz, CD3OD) δ 169.10, 151.42, 136.40, 129.65, 127.20, 121.35, 38.64, 35.18. 49 3-amino-2-hydroxy-N,N-dimethylbenzamide (8): 924mg (4.4mmol) of 2-hydroxy-N,N- dimethyl-3-nitrobenzamide was dissolved in 30mL of MeOH and 30mL of EtOH and 500mg of Pd/C 5% wetted. The flask was sealed with a rubber stopper, evacuated and filled with a H2 balloon 3X, and stirred at room temp overnight. The reaction was filtered through a plug of Celite, concentrated in vacuo, and purified by automated flash chromatography (Rf=0.30 in 30:70:3 EtOAC:hexanes:MeOH) to yield 595mg of a off- white powder. 1 H NMR (400 MHz, CD3OD) δ 6.80 (dd, J = 7.8, 1.4 Hz, 1H), 6.72 (t, J = 7.7 Hz, 1H), 6.59 (dd, J = 7.6, 1.4 Hz, 1H), 3.01 (s, 6H). 13 C NMR (101 MHz, CD3OD) δ 171.44, 142.23, 136.96, 122.18, 119.96, 116.93, 116.77, 36.05. LC-MS: calcd. [M+H] + 179.08; found 179.1. 2-methoxy-N,N-dimethyl-3-nitrobenzamide: To a stirring solution of 500mg (2.38mmol) of 2-hydroxy-N,N-dimethyl-3-nitrobenzamide # and 1.64g (11.85mmol, 5eq) of K2CO3 in 10mL of acetone was added 1.47mL (23.7mmol, 10eq) of methyl iodine and the reaction was heated 60°C overnight. The reaction was concentrated in vacuo, dissolved in EtOAc, the organic layer was extracted 3X with 1N NaOH, dried with MgSO4, concentrated in vacuo to yield 530mg of product (quantitative). 1 H NMR (400 MHz, CDCl3) δ 7.76 (dd, J = 8.1, 1.7 Hz, 1H), 7.43 (dd, J = 7.6, 1.7 Hz, 1H), 7.20 (t, 1H), 3.85 (s, 3H), 3.07 (s, 3H), 2.80 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 166.90, 149.40, 133.52, 132.56, 125.65, 124.33, 63.21, 38.19, 34.83. 3-amino-2-methoxy-N,N-dimethylbenzamide (9): 530mg (2.37mmol) of 2-methoxy- N,N-dimethyl-3-nitrobenzamide # was dissolved in 20mL of MeOH. A large scoop of 50 Pd/C 5% wetted was added. The flask was sealed with a rubber stopper, evacuated and filled with a H2 balloon 3X, and stirred at room temp overnight. The reaction was filtered through a plug of Celite and concentrated in vacuo to yield 297mg of product (64% yield). 1 H NMR (400 MHz, CDCl3) δ 6.93 (t, J = 7.7 Hz, 1H), 6.77 (dd, J = 7.9, 1.5 Hz, 1H), 6.62 (dd, J = 7.5, 1.5 Hz, 1H), 3.79 (s, 3H), 3.12 (s, 3H), 2.87 (s, 3H). 2-hydroxy-N,N-dimethyl-3-(3-phenylureido)benzamide (11a): To a solution of 23 µL of aniline (0.25 mmol) in 3mL of dry DCM at room temperature was added 42mg of CDI (0.25 mmol, 1eq). After stirring at room temperature for 2 h, 45mg of 3-amino-2- hydroxy-N,N’-dimethylbenzamide (0.25 mmol, eq) was added to the reaction mixture and refluxed for 72hrs. The reaction mixture was then concentrated in vacuo. The residue was dissolved in ethyl acetate and then washed with brine. The organic phase was dried over sodium sulfate, concentrated in vacuo, and purified by manual flash chromatography with a gradient from DCM to 2% MeOH in DCM to provide 14 mg of a brown oil (19%). 1H NMR (400 MHz, CD3OD) δ 7.75 (d, J = 6.1 Hz, 1H), 7.44 (d, J = 7.5 Hz, 2H), 7.32 – 7.26 (m, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.94 (dd, J = 14.1, 6.8 Hz, 2H), 3.06 (d, J = 7.1 Hz, 6H). LC-MS: calcd. [M-H] - 29f8.12; found 298.1. 3-(3-(4-bromophenyl)ureido)-2-hydroxy-N,N-dimethylbenzamide (11b): To a solution of 4-bromoaniline (0.072 g, 0.42 mmol) in CH2Cl2 (5 mL) at room temperature was added CDI (0.067 g, 0.42 mmol). After stirring at room temperature for 2 h, 3- amino-2-hydroxy-N,N’-dimethylbenzamide (0.075 g, 0.42 mmol) was added to the reaction mixture and the temperature was increased to 30°C for 48 h. The reaction mixture was then concentrated. The residue was dissolved in ethyl acetate and then washed sequentially with 10% aq. HCl solution, 20% aq. NaOH solution, and brine. The organic phase was dried over sodium sulfate and concentrated onto celite. Purification by 51 automated flash column chromatography gave a brown oil which was then triturated with hexanes to provide 7 mg of a pale yellow solid (4%). Rf = 0.2 (5% MeOH/CH2Cl2). 1 H NMR (CD3OD): δ 7.78 (d, J = 6 Hz, 1H), 7.38-7.43 (m, 4H), 6.89-6.95 (m, 2H), 3.06 (s, 6H). 13 C NMR (CD3OD): δ 154.28, 131.39, 121.91, 120.51, 72.41, 62.96. 3-(3-(4-bromobenzyl)ureido)-2-hydroxy-N,N-dimethylbenzamide (13b): To a solution of 108mg (0.5mmol) of 4-Nitrophenyl chloroformate and 111mg (0.5mmol) 4- Bromobenzylamine hydrochloride in 5mL of dry DCM at 0°C was added 209µL of TEA. The reaction was allowed to warm to room temperature, stirred for 4hrs, concentrated in vacuo, washed with H2O, the organic was dried with Na2SO4, filtered, and concentrated in vacuo to yield a light yellow solid that carried on the next step without further purification. 1-(4-bromobenzyl)-3-(4-nitrophenyl)urea from the previous step was dissolved in 6mL of dry DCM, 50mg (0.28mmol) of 3-amino-2-hydroxy-N,N- dimethylbenzamide and 77µL (0.54mmol) of TEA were added, and stirred at room temperature overnight. The reaction was dissolved in EtOAc and extracted with brine. The organic was concentrated in vacuo and purified by preparative thin-layer chromatography (100:100:3 ratio of EtOAc:hexanes;MeOH) to yield 8mg of product. 1 H NMR (400 MHz, CD3OD) δ 7.51 – 7.44 (m, 3H), 7.26 (d, J = 8.4 Hz, 2H), 6.93 (dd, J = 7.6, 1.8 Hz, 1H), 6.88 (t, J = 7.7 Hz, 1H), 4.37 (s, 2H), 3.04 (s, 6H). 3-azido-2-methoxy-N,N-dimethylbenzamide (14): To a stirring solution of 102mg (0.52mmol) of 3-amino-2-methoxy-N,N-dimethylbenzamide, 287mg (2.08mmol) of K2CO3, and 12.5mg (0.05mmol) of CuSO4 · 5H2O in 3mL of DriSolv ® MeOH was added 218mg (1.04mmol, 2eq) 1H-imidazole-1-sulfonyl azide. The reaction was stirred overnight, concentrated in vacuo, dissolved in EtOAc, the organic was extracted with 52 brine, dried with MgSO4, and purified by automated flash column chromatography Rf=0.40 in 60% EtOAc in hexanes to yield 57mg of product (50% yiel d). 1 H NMR (400 MHz, CD3OD) δ 7.13 – 7.06 (m, 1H), 6.93 (dd, J = 6.2, 3.0 Hz, 1H), 3.73 (s, 1H), 3.01 (s, 1H), 2.78 (s, 1H). 13 C NMR (101 MHz, CD3OD) δ 170.60, 149.45, 134.67, 133.05, 126.34, 125.02, 122.79, 62.76, 38.92, 35.13. 2-hydroxy-N,N-dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)benzamide (15a): 57mg (0.3mmol) of 3-azido-2-methoxy-N,N-dimethylbenzamide, 31µL (0.286mmol, 1.1eq) of phenylacetylene, 23mg (0.11mmol, 0.4eq) of sodium ascorbate, and 13mg (0.052mmol, 0.2eq) of CuSO4·5H2O were dissolved in 1ml of tBuOH and 1ml H2O and stirred overnight at room temperature. The reaction mixture was dissolved in EtOAc, extracted with brine, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography with a gradient from 10% EtOAc in hexanes to 100% EtOAc in hexanes with a 1% MeOH additive to yield 59mg of product (58% yield, Rf=0.20 in 60% EtOAc in hexanes). 1 H NMR (400 MHz, CD3OD) δ 8.73 (s, 1H), 7.93 (dd, J = 8.4, 1.3 Hz, 1H), 7.80 (dd, J = 7.8, 1.9 Hz, 1H), 7.50 – 7.36 (m, 1H), 3.63 (s, 1H), 3.16 (s, 1H), 3.00 (s, 1H). 31 mg (0.10mmol) of the previously obtained 2- methoxy-N,N-dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)benzamide # was placed in an oven-dried RBF sealed with a rubber septa, placed under vacuum for 30min, the flask filled with and Ar balloon, 1mL of dry DCM was added, the reaction was cooled to - 78°C, 700µL (0.7mmol) of a 1M BBr3 solution in DCM was added dropwise, the reaction was allowed to warm to room temp, and stirred overnight. The reaction was quenched with an ice pellet, water was added, then extracted with EtOAc, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography Rf=0.30 in 60% EtOAc in hexanes (the product appeared fluorescently purple under a 254nm UV lamp) to yield 9mg of product (29% yield). 1 H NMR (400 MHz, MeCN) δ 10.74 (s, 1H), 8.67 (s, 1H), 7.95 (d, J = 7.2 Hz, 2H), 7.80 (dd, J = 8.0, 1.2 Hz, 1H), 7.55 – 7.45 (m, 3H), 7.40 (t, J = 7.4 Hz, 1H), 7.12 (t, J = 7.9 Hz, 1H), 3.10 (s, 53 6H). 13 C NMR (101 MHz, MeCN) δ 170.48, 150.65, 147.95, 131.47, 130.23, 129.97, 129.29, 126.75, 126.54, 126.26, 123.07, 122.62, 120.15, 30.31, 23.36. LC-MS: calcd. [M+H] + 309.13; found 309.2. 3-(4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-N,N-dimethylbenzamide (15b): 29 mg (0.13mmol) of 3-azido-2-methoxy-N,N-dimethylbenzamide, 35mg (0.20mmol, 1.5eq) of 1-Bromo-4-ethynylbenzene, 10mg (0.05mmol, 0.4eq) of sodium ascorbate, and 7mg (0.03mmol, 0.2eq) of CuSO4·5H2O were dissolved in 1ml of tBuOH and 1ml H2O and stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, dissolved in EtOAc, extracted with brine, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography with a gradient from 10% to 100% EtOAc in hexanes. 1 H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.69 (dd, J = 7.8, 1.7 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.38 (dd, J = 7.7, 1.7 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 3.53 (s, 3H), 3.06 (s, 3H), 2.89 (s, 3H). 13 C NMR (101 MHz, CD3OD) δ 170.03, 150.63, 148.05, 133.20, 132.84, 131.81, 130.63, 128.55, 128.45, 126.07, 124.23, 123.26, 111.41, 62.53, 39.09, 35.29. The previously obtained 3-(4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl)-2- methoxy-N,N-dimethylbenzamide then was placed in an oven-dried RBF sealed with a rubber septa, placed under vacuum for 30min, the flask filled with an Ar balloon, 3mL of dry DCM was added, the reaction was cooled to 0°C, 2mL of a freshly made solution BBr3 (5g of BBr3 in 10ml of DCM) was added dropwise, the reaction was allowed to warm to room temp, and stirred overnight. The reaction was quenched with an ice pellet, water was added, then extracted with EtOAc, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography (the product appeared fluorescently purple under a 254nm UV lamp) to yield 5mg of a white powder (10% yield over two steps). 1 H NMR (500 MHz, CD3OD) δ 9.69 (s, 1H), 8.68 (d, J = 5.4 Hz, 2H), 8.56 (s, 1H), 8.46 (d, J = 5.7 Hz, 2H), 8.23 (s, 1H), 7.95 (s, 1H), 3.93 54 (s, 6H). LC-MS: calcd. [M+H] + 387.04 (100%) & 389.04 (99.6%); found 387.0 (100%) & 389.0 (92.54%). Ethyl 2-((4-bromophenyl)amino)-2-oxoacetate: 443mg (2.58mmol) of 4-Bromoaniline was added to an oven-dried RBF outfitted with a magnetic stir bar. The flask was sealed with a rubber septa and placed under vacuum for 15min. The flask placed under an Ar atmosphere and 5mL of dry DCM added. The stirring solution was cooled to 0°C, 400µL (2.9mmol, 1.1eq) of TEA, then 288µL (2.58mmol) of ethyl chlorooxoacetate dropwise. The reaction was held at 0°C for 1hr, allowed to warm to room temperature, stirred overnight, dissolved in EtOAc. The organic was extracted with H2O, twice with 25mL of a 25% K2CO3 aqueous solution, then H2O. The organic was the dried with Na2SO4 and filtered through cotton to yield 695mg of a white solid (quantitative). 1 H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 160.86, 154.01, 135.52, 132.33, 132.30, 121.48, 118.42, 63.97, 14.08. 2-((4-bromophenyl)amino)-2-oxoacetic acid (16): To a stirring solution of 695mg (2.6mmol) of ethyl 2-((4-bromophenyl)amino)-2-oxoacetate # in 8mL of EtOH and 8mL of H2O was added 204mg (5.1mmol, 2eq) of NaOH. The reaction was stirred overnight at room temperature and acidified with 2N HCl upon. The resulting precipitate was washed with H2O and high vacuumed to afford 117mg of product (18% yield). 1 H NMR (400 MHz, CD3OD) δ 7.66 (d, J = 8.9 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 161.25, 156.62, 136.39, 131.47, 121.99, 117.39. 55 N1-(4-bromophenyl)-N2-(3-(dimethylcarbamoyl)-2-methoxyphenyl)oxalamide: 91mg (0.37mmol) of 2-((4-bromophenyl)amino)-2-oxoacetic acid #, 52mg (0.27mmol) of 3-amino-2-methoxy-N,N-dimethylbenzamide, 155mg (41mmol, 1.1eq) of HATU, 71µL (0.41mmol, 1.1eq) of DIEA were dissolved in 3mL of DMF and stirred at room temperature overnight. The reaction was concentrated in vacuo, and purified by automated flash column chromatography with a gradient from 0%-80% EtOAc in hexanes (Rf=0.30 in 50% EtOAc in hexanes) to yield 53mg of product (35% yield). 1 H NMR (400 MHz, CD3OD/CDCl3) δ 8.41 (dd, J = 8.1, 1.4 Hz, 1H), 7.66 (t, J = 7.5 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 7.20 (t, J = 7.9 Hz, 1H), 7.06 (dd, J = 7.7, 1.5 Hz, 1H), 3.87 (s, 3H), 3.14 (s, 3H), 2.92 (s, 3H). 13C NMR (101 MHz, CD3OD/CDCl3) δ 170.19, 158.86, 158.26, 146.65, 136.81, 132.65, 130.76, 129.82, 125.31, 124.68, 122.84, 122.02, 118.79, 62.34, 39.00, 35.36. LC-MS: calcd. [M+H] + 420.05 (99.9%) & 422.05 (100%).; found 420.0 (95.60%) & 422.0 (100%). N1-(4-bromophenyl)-N2-(3-(dimethylcarbamoyl)-2-hydroxyphenyl)oxalamide (17): 53mg of (0.13mmol) of N1-(4-bromophenyl)-N2-(3-(dimethylcarbamoyl)-2- methoxyphenyl)oxalamide # was placed in an oven-dried RBF sealed with a rubber septa, placed under vacuum for 30min, the flask filled with an Ar balloon, 3mL of dry DCM was added, the reaction was cooled to 0°C, 2mL of a freshly made solution BBr3 (5g of BBr3 in 10ml of DCM) was added dropwise, the reaction was allowed to warm to room temp, and stirred overnight. The reaction was quenched with an ice pellet, water was added, then extracted with EtOAc, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography to yield 14mg off-white solid (27% yield). 1 H NMR (400 MHz, CDCl3) δ 10.85 (s, 1H), 9.96 (s, 56 1H), 9.30 (s, 1H), 8.41 (dd, J = 8.0, 1.4 Hz, 1H), 7.60 (d, J = 8.9 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.19 (dd, J = 8.0, 1.4 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 3.20 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 171.55, 157.46, 149.80, 135.59, 132.45, 126.46, 124.74, 122.62, 121.53, 121.42, 118.40, 118.38, 116.85, 29.86. LC-MS: calcd. [M+H] + 406.03 (100%) & 408.03 (97.5%); found 406.0 (93.03%) & 408.0 (100%). 3-((2-chloropyrimidin-4-yl)amino)-2-methoxy-N,N-dimethylbenzamide To a mixture of 3-amino-2-methoxy-N,N’-dimethylbenzamide (0.050 g, 0.26 mmol) and 2,4- dichloropyrimidine (0.077 g, 0.52 mmol), in butanol (2 mL) was added triethylamine (0.144 mL, 1.04 mmol). After stirring at 80°C for 36 h the reaction mixture was concentrated to remove butanol. The resulting yellow oil was redissolved in ethyl acetate and filtered. The filtrate was concentrated and purified by automated flash chromatography to give a yellow oil which was triturated with hexanes to provide 36 mg of a yellow solid (45%). Rf = 0.2 (ethyl acetate). 1 H NMR (CD3OD): δ 8.10 (d, J = 5.6 Hz, 1H), 7.97 (d, J = 6.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.10 (d, J = 9.2 Hz, 1H), 6.77 (d, J = 5.6 Hz, 1H), 3.76 (s, 3H), 3.14 (s, 3H), 2.95 (s, 3H). 13 C NMR (CD3OD): δ 169.74, 162.52, 159.89, 156.26, 131.14, 130.16, 125.32, 124.05, 123.75, 60.53, 37.65, 33.69. LC-MS: calcd. [M+H] + 307.1 (100%) & 309.09 (32%); found 307.1 (100%) & 309.1 (30.98%). 3-((2-((4-bromophenyl)amino)pyrimidin-4-yl)amino)-2-methoxy-N,N- dimethylbenzamide: A mixture of 3-((2-chloropyrimidin-4-yl)amino)-2-methoxy-N,N- dimethylbenzamide (0.035 g, 0.11 mmol), para-toluene sulphonic acid monohydrate (0.042 g, 0.22 mmol), and 4-bromoaniline (0.020 g, 0.11 mmol) was stirred at 80°C in DMF (0.5 mL) for 5 h. The reaction mixture was cooled to room temperature then diluted with water (15 mL) and extracted into ethyl acetate (20 mL). The organic phase 57 was washed with brine, then dried over sodium sulfate and concentrated. Purification by automated flash chromatography gave 8 mg of a white solid (16%). Rf = 0.4 (50% ethyl acetate/hexanes). 1 H NMR (CD3OD): δ 8.11 (d, J = 6.4 Hz, 1H), 7.98 (d, J = 4.4 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.19 (t, J = 6.4 Hz, 1H), 7.02 (d, J = 6.4 Hz, 1H), 6.34 (d, J = 4.8 Hz, 1H), 3.78 (s, 3H), 3.14 (s, 3H), 2.92 (s, 3H). 13 C NMR (CD3OD): δ 170.04, 161.62, 155.74, 148.07, 143.00, 139.69, 132.61, 131.04, 129.93, 125.60, 123.77, 122.31, 121.29, 113.59, 98.36, 60.53, 37.63, 33.94. 3-((2-((4-bromophenyl)amino)pyrimidin-4-yl)amino)-2-hydroxy-N,N- dimethylbenzamide (18a): Cooled a mixture of 3-((2-((4-bromophenyl)amino)pyrimin- 4-yl)amino)-2-methoxy-N,N-dimethylbenzamide (0.008 g, 0.018 mmol) in CH2Cl2 (3 mL) to 0°C then added solution of boron tribromide (1 M in CH2Cl2, 0.9 mL, 0.9 mmol) and stirred while warming to room temperature for 16 h. The reaction was quenched with addition of cold water while stirring, then extracted into ethyl acetate (3 x 10 mL). The organic phase was dried over sodium sulfate and concentrated onto celite. Purification by flash chromatography gave 2 mg colorless oil (26%). Rf = 0.2 (5% MeOH/CH2Cl2). 1 H NMR (CD3OD): δ 8.22 (br s, 1H), 7.47-7.51 (m, 3H), 7.35 (d, J = 9.0 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.96 (t, J = 8.4 Hz, 1H), 6.26 (d, J = 6.0 Hz, 1H), 3.04 (br s, 6H). 13 C NMR (CD3OD): δ 170.99, 131.17, 129.77, 121.51, 119.79, 113.94, 28.39, 22.18. LC-MS: calcd. [M+H] + 428.28; found 428. N-(4-bromophenyl)-2-chloropyrimidin-4-amine: To a stirring solution of 149mg (1mmol) of 2,4-dichloropyrimidine and 172mg (1mmol) of 4-Bromoaniline in 3mL of n- butanol was added 417µL (3mmol, 3eq) of TEA. The reaction was heated at 80°C overnight, then concencentrated in vacuo onto Celite ® , and purified by automated flash column chromatography to yield 177mg of a white powder (63% yield, Rf=0.3 in 30% 58 EtOAc in hexane). 1 H NMR (400 MHz, CD3OD) δ 8.10 (d, J = 6.0 Hz, 1H), 7.60 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 9.0 Hz, 2H), 6.71 (d, J = 6.0 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 161.73, 159.84, 156.01, 137.86, 131.48, 122.02, 105.28. LC-MS: calcd. [M+H] + 283.96 (77.3%) & 285.96 (100%); found 284.0 (77.12%) & 286.0 (100%). 3-((4-((4-bromophenyl)amino)pyrimidin-2-yl)amino)-2-methoxy-N,N- dimethylbenzamide: 41mg (0.14mmol) of N-(4-bromophenyl)-2-chloropyrimidin-4- amine, 28mg (0.14mmol) of 3-amino-2-methoxy-N,N-dimethylbenzamide, and 56mg (0.30mmol, 2.3eq) of p-toluenesulfonic acid monohydrate were dissolved in 0.5mL of DMF and heated at 60°C for 5hrs. The reaction was concentrated in vacuo, the residue was dissolved in EtOAc and extracted with H2O then brine, dried with MgSO4, filtered, concentrated in vacuo, and purified by automated flash column chromatography to yield 61mg of product (quantitative). 1 H NMR (400 MHz, CD3OD) δ 8.26 (d, J = 7.9 Hz, 1H), 7.97 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.10 (t, J = 7.9 Hz, 1H), 6.90 (d, J = 6.9 Hz, 1H), 6.23 (s, 1H), 3.80 (s, 3H), 3.12 (s, 3H), 2.90 (s, 3H). 13 C NMR (101 MHz, CD3OD) δ 171.47, 162.54, 156.76, 147.26, 140.07, 134.38, 132.63, 130.70, 125.22, 123.97, 123.87, 122.04, 116.40, 100.11, 62.10, 39.03, 35.21. 3-((4-((4-bromophenyl)amino)pyrimidin-2-yl)amino)-2-hydroxy-N,N- dimethylbenzamide (18b): 20 mg (0.05mmol) of 3-((4-((4- bromophenyl)amino)pyrimidin-2-yl)amino)-2-methoxy-N,N-dimethylbenzamide # was placed in an oven-dried RBF sealed with a rubber septa, placed under vacuum for 30min, the flask filled with an Ar balloon, 3mL of dry DCM was added, the reaction was cooled to 0°C, 1mL (1mmol) of a 1M BBr3 solution in DCM was added dropwise, the reaction 59 was allowed to warm to room temp, and stirred overnight. The reaction was quenched with an ice pellet, water was added, then extracted with EtOAc, dried with MgSO4, filtered through cotton, concentrated in vacuo, and purified by automated flash column chromatography to yield 15mg of a white solid (29% yield). 1H NMR (400 MHz, CD3OD/CDCl3) δ 7.83 (d, J = 6.1 Hz, 1H), 7.52 (d, J = 0.7 Hz, 1H), 7.47 (t, J = 8.6 Hz, 3H), 7.43 – 7.38 (m, 2H), 6.97 (dd, J = 7.6, 1.6 Hz, 1H), 6.86 (t, J = 7.8 Hz, 1H), 6.16 (d, J = 6.1 Hz, 1H), 3.05 (s, 6H). 13C NMR (101 MHz, CD3OD/CDCl3) δ 171.74, 161.92, 159.59, 153.48, 146.31, 138.64, 132.25, 129.36, 125.45, 124.17, 123.38, 120.28, 116.66, 99.16, 30.13. 60 3.8 References (1) Waugh, D. J. J.; Wilson, C. Clin Can Res 2008, 14, 6735–41.Minucci, S.; Pelicci, P. G. Nat Rev Cancer 2006, 6, 38–51. (2) Li Jeon, N.; Baskaran, H.; Dertinger, S. K. W.; Whitesides, G. M.; Van de Water, L.; Toner, M. Nature Biotechnol 2002, 20, 826–30. (3) Chapman, R. W.; Phillips, J. E.; Hipkin, R. W.; Curran, A. K.; Lundell, D.; Fine, J. 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V Org Lett 2007, 9, 3797–800. 64 Chapter 4: HDAC Inhibition Via MEF2 Binding 4.1 Introduction Abnormal levels of gene expression are a trademark of many diseases including cancer. 1-3 Epigenetic modifications to DNA and its associated proteins alter levels of gene expression without permanently modifying the DNA sequence. 2,3 Unlike DNA mutations, epigenetic changes are reversible. 4 Remarkably, by targeting the enzymes responsible for these epigenetic modifications, gene and protein expression can be returned to pre-disease levels. 5 A majority of these modifications are achieved through chromatin remodeling: the phosphorylation, ubiquitinylation, methylation, or acetylation of the nucleosomal histone tail. 6-8 In the nucleus, acetylation the histone’s lysines, by histone acetyltransferases enzymes (HATs), causes the histone-associated DNA to dissociate from the histone protein (Figure 4.1). 8 This allows transcription factors access to the now free DNA sequence resulting in increased transcription and translation of the gene. 9 Histone deacetylase enzymes (HDACs) are responsible for reversing this process through the deacetylation of the histone which causes gene silencing. 9 Recently, a variety of cancers, including leukemia, have been shown to suffer from low levels of histone acetylation. 10 As a result, it was strategized that the HDACs could serve as therapeutic target for cancer by restoring acetylation and, potentially, return cellular homeostasis. 11,12 Figure 4.1. Histone lysine residues are acetylated by the histone acetyltransferases enzymes (HATs) and the acetyl-donor acetyl-CoA and deacetylated by histone deacetylase enzymes (HDACs). 65 In recent years, a large number of small molecule inhibitors of HDACs have been developed. With its 2006 approval for advanced cutaneous T-cell lymphoma, Vorinostat (Zolinza ® ) became the first FDA-approved HDAC inhibitor approved for of cancer. 13 Additionally, LBH-589, 14 CI-994, 15 BML-210, and MS-275 16,17 are or have been the subject of clinical studies (Figure 4.2). Figure 4.2. HDAC inhibitors. As a family of enzymes, HDACs are divided into four classes based upon sequence homology: Class I HDACs are 1,2,3, and 8 and Class II HDACs are 4, 5, 6, 7, 9, and 10. 11 Isoform selectivity has emerged as a significant issue in the of development of HDAC inhibitors 18 in order to mitigate side-effects associated with pan-HDAC inhibition, such as cardiac arrhythmias; bone marrow depression; weight loss; and fatigue. 19 An important subclass of HDACs are the Class IIa HDACs, which play vital roles in the developmental processes related to the expression of heart, brain, and smooth muscle tissue. 20 Class IIa HDACs have also recently been shown to be disregulated or mutated in leukemia and lymphoma. 21-23 Unfortunately, many of the known HDAC inhibitors exhibit diminished potency against class IIa HDACs. 24 As a class, these HDACs have a unique regulatory domain for binding to transcription factors prior to deacetylation of the histone. 25 Prior work has demonstrated that levels of expression of the FXN gene, relevant in the occurrence of the neurodegenerative disease Friedreich’s ataxia, after treatment with small molecule HDAC inhibitors did not correlate well with HDAC inhibition data. 26 While it was suggested that these compounds can interact with HDAC3, 27 we considered that rather than binding to an HDAC, it could also be possible that compounds which display phenotypic effects relevant to HDAC inhibitory activity could bind to 66 transcription factors. We have therefore pursued a unique strategy to achieve class IIa specificity by investigating the interaction of the class IIa HDACs’ unique regulatory domain with an associated transcription factor, Myocyte Enhancer Factor-2 (MEF2) and inhibiting the binding of HDAC to MEF2 at the protein-protein interface. Previous studies of MEF2 have shown a hydrophobic groove exists on the surface of the MADS/MEF2 domain of MEF2 28 and crystallographic data demonstrated MEF2 was capable of binding the α-helix of HDAC9 (Figure 4.3a). 29 The development of small molecules that could bind to this hydrophobic region of MEF2 could thus potentially block the interaction of MEF2 with class IIa HDACs and impact the expression of MEF2-dependent genes To this end, our collaborators in the group of Prof. Lin Chen (USC) discovered, remarkably, that the known HDAC inhibitor BML-210 was capable of disrupting HDAC9-MEF2 binding and were able to solve the BML-210-MEF2 crystal structure (Figure 4.3b). 25 Figure 4.3: Structureal comparison of binding sites for HDAC9 and BML-210 on MEF2. (a) X-ray crystal structure of the α-helix of HDAC9 (purple helix) bound to MEF2 (grey surface) (PDB:1TQE); (b) X-ray crystal structure of small molecule BML-210 (yellow) bound to MEF2 in the same cleft as the α- helix of HDAC9 (PDB:3MU6). A B 67 4.2 Chemistry The development and optimization of MEF2-binding molecules started with the structure of BML-210. Though the structure was informative, however, as a result the disorder in this structure, a detailed understanding of the molecular interactions between BML-210 and MEF2 were not clear. Therefore, our first approach was to make iterative structural modifications to establish structural-activity relationships (SAR). Together with my collegue Jamie Jarusiewicz, we prepared several series of compounds that can serve as protein-protein interaction (PPI) inhibitors for MEF2. The compounds are shown in Table 4.1-4.4 and whose synthesis is outlined in Figures 4.4-4.10. In order to quickly develop an SAR, several synthetic routes were developed. The o-anilidine moiety was optimized by reacting equal equivalents of the dicarboxylic acids with the anilines via neat heating 30 or HBTU coupling 31,32 to access the mono acid 7 or 46 which could be coupled with various anilines to allow the facile synthesis of ortho, meta, and para analogs 9a-c and 48 (Figure 4.4). To vary the position anilidine, two synthetic routes were utilized. Symmetrical analogs 10 and 27a-31b were easily accessed using the previously mentioned HBTU coupling with an excess of the aniline (Figure 4.5). Tert-butyl (2-aminophenyl)carbamate was synthesized as previously described 33 and served as a useful reagent for the adding the o-anilidine moiety. Using tert-butyl (2- aminophenyl)carbamate compounds 17a-b (Figure 4.5) and 41a-b (Figure 4.6) could be easily accessed in two steps of HBTU coupling and TFA deprotection. Alternatively, the o-anilidine functionality could be first installed via an amide coupling reaction of pimelic acid or suberic acid to tert-butyl (2-aminophenyl)carbamate with HBTU. The monoacid 18 was then subjected to a second HBTU coupling with a variety of anilines and, if the aniline was Boc-protected, deprotected with TFA to produce analogs 19, 21a-b, 24, and 26 (Figure 4.7). While this route proved useful for easy access to the o-anilidine analogs, it suffered from to unreliable yields and required tedious purification. The analogs reversing the benzamide to 2-amino-N-methylbenzamide were synthesized by reacting isotonic anhydride with cadaverine (37) or m-xylylenediamine (42) (Figure 68 4.8). 34 Due to the proximity of additional aspartic acids to the binding of the o-anilidine, two pyridine analogs were synthesized. Compound 32 was synthesized in five steps from pimelic acid according to Figure 4.9 and 33 was access via reductive amination of 7 with 2-pyridine-carboxaldehyde (Figure 4.10). 34 Figure 4.4: Preparation of MEF2 inhibitors 8a-c and 48. (a) aniline, HBTU, DIPEA, DMSO, rt 12h or aniline, 110°C 24 h (b) tert-butyl (3-aminophenyl)carbamate or tert-butyl (4-aminophenyl)carbamate, HBTU, DIPEA, DMSO, rt 3-12h; (c) TFA, CH 2Cl 2, 0°C, 3 h. Figure 4.5: Preparation of MEF2 inhibitors 10, 17a-b, and 29-31b. (a) HBTU, DIPEA, DMSO, RT, 12h; (b) TFA, CH 2Cl 2, 0°C, 3 h. Figure 4.6: Preparation of MEF2 inhibitors 41a-b. (a) HBTU, DIPEA, DMSO, RT, 12h; (b) TFA, CH 2Cl 2, 0°C, 3 h. Figure 4.7: Preparation of MEF2 inhibitors 19, 21a-b, 24, and 26. (a) tert-butyl (2-aminophenyl) carbamate, HBTU, DIPEA, DMSO, rt 12h; (b) aniline, HBTU, DIPEA, DMSO, rt 3-12h; (c) TFA, CH 2Cl 2, 0°C, 3 h. 69 Figure 4.8: Preparation of MEF2 inhibitors 37 and 42. (a) isatoic anhydride, EtOH, reflux, overnight. Figure 4.9: Preparation of MEF2 inhibitor 32. (a) HBTU, DIPEA, DMSO, rt 12h; (b) HBTU, DIPEA, DMSO, rt 12h; (c) LiOH, H 2O, THF; (d) HBTU, DIPEA, DMSO, rt 12h; (e) TFA, CH 2Cl 2, 0°C, 3 h. Figure 4.10: Preparation of MEF2 inhibitor 33. (a) 2-pyridine-carboxaldehyde, MeOH, 4hr, reflux; (b) 0°C, NaBH 4 to rt overnight. 4.3 Biological Activity Despite our collaborators in the laboratory of Dr. Lin Chen showing that BML-210 was able to disrupt HDAC-MEF2 protein-protein interaction in vitro 25 and having a crystal structure of it binding to MEF2, it was important to obtain more evidence of direct binding between BML-210 and MEF2. Since 19 F NMR is routinely used to show small molecule-protein interactions, we postulated that a trifluoromethylated analog of BML- 210, with its molecular abundance of 19 F, could be used to detect direct binding at low concentrations. 35 In order to see this interaction a such low concentrations, we sought to increase the activity of out –CF3 analog by varying the distance between the ortho- aminoanilide moiety and the meta-CF3 substituted benzamide (Table 4.1). Compounds were tested by the Chen lab. While linkers of four (1), six (2), and seven (4) carbons had similar activity, the five carbon linker (3) proved to be the optimal length (Table 4.1) with greater activity that BML-210 (Figure 4.11b). 25 Further increasing of the linker eight carbons (5) abolished the molecule’s activity, potentially due to entropic reasons. 70 Table 4.1: Optimization of linker length of trifluoromethyl MEF2 inhibitors at 10µM in luciferase assay. 19 F NMR studies of compound 2 with MEF2 showed a new, higher field signal (Figure 4.11c), representing the bound ligand. Increasing concentrations of MEF2 caused an increase in the up field signal and a decrease in the down field signal (Figure 4.11d). The change in chemical shift of the ligand's 19 F signal in the presence of different MEF2 concentrations indicates a change from the free to the bound state of the ligand. This direct binding data was further confirmed with Surface Plasmon Resonance (SPR). 25 Figure 4.11: Detecting the binding of a fluorinated analog of BML-210 to MEF2 by 19 F NMR. (a) Structure of the fluorinated analog 2; (b) Similar to BML-210, 2 inhibits the HDAC4-VP16-driven reporter signal; (c) 19 F NMR spectrum when the free fluorinated compound is in excess; (d) 19 F NMR spectrum when the MEF2 protein is in excess. 25 Entry Analog % Inhibition Entry Analog % Inhibition 1 66 4 61 2 98 5 0 3 64 A B C D 71 Table 4.2: Activity of ortho-aminoanilide replacement MEF2 inhibitors at 10µM in luciferase assay. Entry Analog % Inhibition Entry Analog % Inhibition 6 87 11 58 9a 98 12 38 9b 21 13 17 9c 19 14 76 10 0 15 0 Our next goal was to improve upon or replace the ortho-aminoanilide functionality. As shown in Table 4.2, in comparison with BML-210 (6), we again observed the same trend of increased potency when the linker length was reduced to five carbons (9a). Shifting the position of the ortho-aniline to meta or para resulted in a sequential decrease in activity (9a-b) and its removal resulted in an inactive compound (10). We also synthesized a series of isosteric pyridine, ether, and quinoline derivatives which would maintain the same heteroatom position of the ortho-aminoanilides. While the quinolinyl- (14) and pyridinyl- nitrogens (11) and, to a lesser extent, the ortho-ether (12) were able to provide the necessary interaction for binding, none improved upon the potency of 9a. However, the modest activity produced by the quinoline moiety of 14 confirmed that additional steric bulk may be accommodated for in the ortho-aminoanilide binding site. Having determined the optimal linker distance and with the ortho-aminoanilide established as a potent binding moiety, we next turned our focus to the benzamide ring (Table 4.3). It was found that activity improved in the order meta>para>ortho for both amine (22a>21a>17a) and benzene (22c>21c>20) substituents. Detrimental steric interactions of the ortho-substituents may cause the reduction in activity of the ortho- substituted compounds relative to the meta- and para- substituted compounds in the luciferase assay. 25 This can be seen in the BML-210-MEF2 crystal structure where the benzamide ring occupies a hydrophobic pocket lined by the D63A, L67A, and T70B 72 (Figure 4.12a). 25 While the ortho positions are in close contact with the side chains of these residues, space for the para-substituents is offered by a gap between D63 and L67 (Figure 4.12a, blue arrow) and large channel is provided for the meta-substituents by the continuation of the binding cleft between chain A and B (Figure 4.12a, red arrow). Table 4.3: Activity of MEF2 inhibitor ortho-aminoanilide replacements at 10µM in luciferase assay. Entry Analog % Inhibition Entry Analog % Inhibition 17a 69 27a 78 17b 40 27b 0 19 41 29 50 20 46 30a 0 21a 91 30b 0 21b 94 31a 0 21c 94 31b 0 22a 94 35 30 22b 96 22c 98 36 37 24 0 26 8 73 Additionally, an overlay of the BML-210-MEF2 and HDAC-MEF2 crystal structures showed an overlap of the benzamide ring of BML-210 with the Leu117 of HDAC. These two moieties were combined into 24, but unfortunately this molecule showed no activity (Figure 4.12b). Figure 4.12: Crystallographic understanding of analog activity and design. (a) Structural explanation of the activity ortho<meta<para activity trend. While the ortho positions make direct contact with the protein preventing the addition of substituents, the blue arrow shows a small gap between L67A and D63A for para substituents and the red arrow shows a larger channel between D63A and T70B to accommodate meta substituents; (b) An overlay of the HDAC9 (periwinkle helix)-MEF2 (PDB:1TQE) and BML-210(yellow)- MEF2 (PDB:3MU6) crystal structures indicates the possibility of replacing the benzamide with an isopropylamide to mimic L117 of HDAC9. Additionally, with the near symmetry of the binding site, we thought we would be able to develop a complementary symmetrical ligand. While 17a-b and 27a-b showed reasonably activity, none of these showed inhibition on the level of the non-symmetrical molecules. This is likely the result of a failure of these compounds to span the appropriate distance between the D61 and D63 of chains A and B. Interestingly, compound 27a has the highest activity of non-ortho-aminoanilide inhibitors, but none of the structural hybrids, analogs, and extensions of the pyridine moieties 11, 26, 27a, and 35-36 were able to maintain this potency. D63 A L117 B T70B L67A A 74 4.4 Crystal Structure Reevaluation Given our inability to further optimize the MEF2-inhibitors based on the BML-210 structure, we pursued a detailed analysis of all MEF2-related structures. While X-ray crystal structures are presented as a static structure, the dynamic nature of the protein needs to be considered in assigning residue orientations. 35 At resolutions greater than 1.6 Å, residues cannot be resolved for their absolute position, so they positioned at the average of their conformational positions, a process that poorly represents each of the average’s constituent rotameric state. 36 An analysis of the assigned orientations of the residues about the active site in the BML-210-MEF2 crystal structure brings into question the deposited assignments. While we showed that the ortho-aminoanilide was required for activity our MEF2 inhibitors (Table 4.2), no hydrogen or ionic interactions are made between the molecule and protein despite the close proximity of two anionic residues, D61 and D63, which would logically interact with our molecules ortho-aminoanilide (Figure 4.13a). As an inhibitor of the protein-protein interaction between MEF2 and HDAC, the binding of BML-210 occurs on the protein surface. It has been shown that surface side-chain, especially polar residues, can be reoriented by crystal packing. 37 Figure 4.13: Induced-Fit Docking studies to reevaluate small molecule-MEF2 binding interactions. (a) The x-ray crystallographically determined structure of BML-210 bound to MEF2; (b) predicted binding of BML-210 using Induced Fit Docking shows a reorientation of D61 and D63 to hydrogen bond the ortho- aminoanilide; (c) predicted binding of 9a using Induced Fit Docking shows the same reorientation of D61 and D63 to hydrogen bond the ortho-aminoanilide and the additional hydrogen bond of the benzamide –NH to T70. A B C D63 63 D63 63 D63 63 D61 63 D61 63 D61 636 T70 75 Throughout this project, many unsuccessful attempts were made to dock our active molecules into the BML-210-MEF2 crystal structure. With the 2.43 Å of this structure, the surface binding pocket, our docking failures, and the proximity of the two anionic residues, we sought to consider other orientations of the aspartic acid residues. Recently, we were able to overcome our previous docking failures using Induced Fit Docking, a protocol that considers the flexibility of both the ligand and the protein, through sampling of side-chain rotomers. 38-41 The results propose possible alternative binding interactions for BML-210 (Figure 4.13b). The bridging hydrogen bond network the aniline forms to both D61 and D63 of the ortho-aminoanilide explains why no other group was able to replace this group since none of the synthesized replacements had the ability to interact with both residues simultaneously. While this is structurally interesting, we wanted evidence that this interaction was possible. A scan of the PDB for aniline-containing ligands found a similar hydrogen bound network in the crystal structure of HIV-1 protease with the aniline of inhibitor KNI-1689 hydrogen bonding to D129 and D130 (Figure 4.14). 42 With confidence that this was a “real” interaction, the same technique was then applied to compound 9a which predicted the same hydrogen bonds to D61 and D63, but, interestingly, showed an additional hydrogen bond to T70 by the benzamide not seen in BML-210 (Figure 4.13c). This hydrogen bond to T70 could account for the greater activity of the five carbon linker since it puts the –NH of the benzamide in the perfect position for this interaction. Figure 4.14. PDB example of the aniline of KNI-1689 forming two hydrogen bonds two aspartic acids of HIV-1 protease. (a) ribbon; (b) Ligand Interaction Diagram (PDB:3A2O). A B 76 4.5 Design of New MEF2-Inhibitors Using these structural insights, we set out depart from our original scaffold. Our docking studies indicated that through modifications to the alkyl linker, we could gain additional interactions to MEF2. Some of these structures are outlined in Table 4.4. We reversed the amide bond the ortho-aminoanilide side (37), the benzamide side (38), and on both sides of the molecule (39). Interestingly, 37 showed the highest activity of all the molecules synthesized. We predict that the increase in activity of 37 results from the ability of this molecule to gain an additional hydrogen bond to the backbone carbonyl of L66 (structure not shown). We also sought to employ a more conformationally restrained functionality specifically tailored to the MEF2 biding site which could decrease possible off-target activity stemming from the flexibility of the alkyl linker and decrease the entropic cost of binding. Although a piperazinyl moiety (45), potentially as a result of its polar nature, and an aromatic linker (41a-b, 42) did not exhibit high levels of inhibitory activity, the alkynyl-containing analog (44) and a meta-substituted aryl-alkyoxy (43) linker were tolerated. These modifications could offer new scaffolds upon which to develop novel molecules for further investigation of inhibition of the protein-protein interaction between class IIa HDACs and MEF2. Table 4.4: Structures of selected MEF2 inhibitor linker replacements. Entry Analog Entry Analog 37 42 38 43 39 44 41a 45 41b 48 77 4.6 Conclusion We have designed, synthesized, and evaluated a series of compounds for their potential ability to function as modulators of epigenetic activity through inhibition of the interaction of class IIa HDACs and the associated transcription factor MEF2. Many of these novel analogs exhibit more potent inhibition than the parent analog BML-210. In particular, we have determined that the presence of an ortho-aniline on one end of an aliphatic spacer is important for biological activity whereas a wide range of functionality located on the distal side of the molecule, particularly with substitution at the meta position, is amenable to inhibitory activity. Additionally, we were able to refine the BML-210-MEF2 crystal structure to make it amenable to future structure-based design efforts. 78 4.7 Experimental 4.7.1 19 F NMR. NKL-54 at 0.5-5 µM was incubated with MEF2A (1-95) at 0.5-10µM for 30 min, and the 19 F NMR spectra were acquired on a Varian VNMRS 500 Spectrometer at 25 °C using a 54° pulse with total acquisition and delay time of 1.6 s for 3000 scans (80 min. acquisition time). An internal capillary containing DMSO-d6 was used to obtain a lock signal. A spectral width of 10 ppm was used and the data were multiplied with an exponential function of 0.2 Hz prior to Fourier transformation. The CF3 resonance of the free NKL-54 was at -62.86 ppm and that of the compound bound to MEF2 was at -63.23 ppm. The CF3 resonance of the free compound without any added protein was also observed at -62.86 ppm. Chemical shifts are referenced to trifluoroacetic acid. The final DMSO concentration in the samples was below 0.05%. 4.7.2 Synthetic Chemistry General Procedure 1: The reaction was diluted with EtOAc, extracted with brine (3x), 5% NaOH solution, and brine, dried with MgSO4, filtered, concentrated in vacuo, and purified via automatic chromatography. The reaction mixture was dissolved in EtOAc and extracted brine (3x), 5% NaOH solution, and brine. 7-oxo-7-(phenylamino)heptanoic acid (7): 5g (31.3mmol) of pimelic acid and 2.85ml (31.3mmol) of aniline were heated at 138°C for 4hrs. The reaction mixture was extracted with EtOAc and with 5% NaOH solution. The aqueous layer was then acidified and extracted with EtOAc. The organic layer was dried with MgSO4, filtered, concentrated in vacuo, and recrystalized with CH3CN and H2O to yield 3g (40%) of white powder. 1 H NMR (400 MHz, CD3OD) δ 7.56 (ddd, J = 3.9, 3.1, 1.6 Hz, 1H), 7.33 (ddd, J = 8.5, 5.8, 1.8 Hz, 1H), 7.17 – 7.03 (m, 1H), 2.43 – 2.38 (m, 1H), 2.34 (t, J = 7.4 Hz, 1H), 1.72 (dtd, J = 22.8, 15.1, 7.5 Hz, 2H), 1.46 (tt, J = 7.3, 5.3 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 176.11, 173.11, 138.50, 128.36, 123.72, 119.86, 36.38, 33.36, 28.40, 25.22, 24.41. 79 N 1 -(2-aminophenyl)-N 7 -phenylheptanediamide (9a): 243mg (1mmol) of monoacid 7, 379 mg (1mmol) of HBTU, 173uL (0.23mmol) of DIEA, and 208mg (1mmol) of BOC- 1,2-phenylenediamine in 4mL of DMSO. The reaction was worked up according to General Procedure 1 and automatic chromatography to yield 383mg (90%). Rf=0.45 in 70% ethyl acetate in hexanes. The product was dissolved in 20mL of DCM at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 210mg (72%).Rf=0.30 in 7% MeOH in DCM. 1 H NMR (600 MHz, CD3OD) δ 7.60 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.08 (t, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.76 (t, J = 7.6 Hz, 1H), 2.51 (t, J = 7.5 Hz, 1H), 2.47 (t, J = 7.4 Hz, 1H), 1.84 (dq, J = 14.6, 7.2 Hz, 2H), 1.62 – 1.51 (m, 1H). 13 C NMR (151 MHz, CD3OD) δ 174.97, 174.51, 143.28, 139.88, 129.74, 128.22, 127.14, 125.16, 125.11, 121.32, 119.50, 118.52, 106.43, 37.74, 37.02, 29.81, 26.68, 26.55. N 1 -(2-aminophenyl)-N 7 -phenylheptanediamide (8b): 100mg of 7 (0.42mmol), 161mg (0.42mmol) of HBTU, 73uL (0.42mmol) DIEA, and 87mg (tert-butyl (2- aminophenyl)carbamate 2mL of DMSO.The reaction was worked up according to General Procedure 1 to yield 112mg (62%). Rf=0.30 in 100% EtOAc. 1 H NMR (400 MHz,CD3OD) δ 7.69 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 8.6, 1.0 Hz, 2H), 7.29 – 7.23 (m, 2H), 7.20 – 7.01 (m, 4H), 2.36 (td, J = 7.4, 3.9 Hz, 4H), 1.79 – 1.62 (m, 4H), 1.49 (s, 8H), 1.44 (dd, J = 8.8, 6.2 Hz, 2H). 13 C NMR (101 MHz, CD3OD) δ 174.32, 174.30, 155.00, 140.84, 140.05, 139.65, 129.74, 129.57, 124.93, 121.10, 115.50, 37.57, 37.55, 29.60, 28.53, 26.41, 26.40. 80 N 1 -(3-aminophenyl)-N 7 -phenylheptanediamide (9b): 109mg (0.25mmol) of 7 was dissolved in 10mL of DCM at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 71mg (86%). Rf=0.5 in 80% ethyl acetate in hexanes. 1 H NMR (600 MHz, CD3OD) δ 7.55 (d, J = 8.5, 0.8 Hz, 1H), 7.30 (t, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.06 – 6.99 (m, 1H), 6.82 (d, J = 7.8, 1.6 Hz, 1H), 6.48 (d, J = 7.9, 2.0 Hz, 1H), 2.43 – 2.31 (m, 2H), 1.81 – 1.66 (m, 2H), 1.52 – 1.40 (m, 1H). 13 C NMR (151 MHz, CD3OD) δ 173.09, 172.97, 147.84, 139.10, 138.43, 128.86, 128.33, 123.70, 119.89, 111.17, 109.87, 107.10, 36.38, 36.33, 28.37, 25.21, 25.16. N 1 -(4-aminophenyl)-N 7 -phenylheptanediamide (9c): 100mg (0.43mmol) of 7, 161mg (0.43mmol) of HBTU, 73.8uL (0.43mmol) of DIEA, and 46mg (0.43mmol) of 1,4- phenylenediamine in 1.5mL of DMSO. The reaction was worked up according to General Procedure 1. Rf=0.45 in 100%. 1 H NMR (600 MHz, CD3OD) δ 7.55 (d, J = 8.0 Hz, 1H), 7.30 (t, 1H), 7.25 (d, J = 8.7, 1.7 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.70 (d, J = 8.7, 1.7 Hz, 1H), 2.40 (t, J = 11.0, 3.9 Hz, 1H), 2.36 (t, J = 11.0, 3.8 Hz, 1H), 1.82 – 1.69 (m, 2H), 1.53 – 1.39 (m, 1H). 13 C NMR (151 MHz, CD3OD) δ 174.51, 174.13, 145.51, 139.85, 130.67, 129.77, 125.09, 123.29, 123.23, 121.32, 121.26, 116.67, 116.65, 37.74, 37.55, 29.77, 26.70, 26.58. N 1 ,N 7 -diphenylheptanediamide (10): 80mg (0.50mmol) of pimelic acid, 174uL (1mmol) of DIEA, 417mg (1.1mmol) of HBTU, 91uL (1mmol) of aniline were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 160mg 81 (52%). 1 H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.3 Hz, 1H), 7.21 (dd, J = 12.9, 4.4 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 2.32 (t, J = 7.1 Hz, 1H), 1.69 (dd, J = 14.5, 7.2 Hz, 1H), 1.44 – 1.34 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 171.51, 156.43, 137.92, 128.98, 124.24, 119.92, 77.38, 77.06, 76.74, 38.66, 37.10, 28.28, 24.75. Di-tert-butyl ((heptanedioylbis(azanediyl))bis(2,1-phenylene))dicarbamate (16a): 160mg (1mmol) of pimelic acid, 418uL (2.4mmol) of DIEA, 910mg (2.4mmol) of HBTU, 500mg (2.4mmol) of BOC-1,2-phenylenediamine were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 489mg (90%). 1 H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.40 (t, J = 6.9 Hz, 2H), 7.25 – 7.05 (m, 3H), 2.35 (t, J = 6.9 Hz, 2H), 1.53 (s, 9H), 1.42 (d, J = 6.3 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ 172.62, 156.53, 154.40, 130.92, 129.94, 126.25, 125.40, 124.64, 80.96, 70.06, 38.74, 36.76, 28.46, 25.07. N 1 ,N 7 -bis(2-aminophenyl)heptanediamide (17a): 491g (0.92mmol) compound 28 was dissolved in dissolved in 15mL at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatography to yield 296mg (95%). 1 H NMR (400 MHz, CD3OD) δ 7.14 – 7.01 (m, 1H), 6.87 (dd, J = 8.0, 1.3 Hz, 1H), 6.79 – 6.69 (m, 1H), 2.49 (t, J = 7.4 Hz, 1H), 1.88 – 1.76 (m, 1H), 1.64 – 1.48 (m, 1H). 13 C NMR (101 MHz, CD3OD) δ 173.53, 141.84, 126.79, 125.69, 123.72, 118.08, 117.10, 35.57, 28.41, 25.24. 82 Di-tert-butyl ((octanedioylbis(azanediyl))bis(2,1-phenylene))dicarbamate (16b): 174mg (1mmol) of suberic acid, 418uL (2.4mmol) of DIEA, 910mg (2.4mmol) of HBTU, 500mg (2.4mmol) of BOC-1,2-phenylenediamine were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 493mg (89%). 1 H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 7.46 – 7.31 (m, 1H), 7.11 (dt, J = 15.2, 7.4 Hz, 2H), 2.40 – 2.30 (m, 1H), 1.78 – 1.67 (m, 1H), 1.50 (s, 4H), 1.44 – 1.37 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 172.54, 156.54, 154.40, 130.83, 130.10, 126.30, 125.54, 125.44, 124.70, 81.04, 70.19, 37.01, 28.46, 25.48. N 1 ,N 8 -bis(2-aminophenyl)octanediamide (17b): 27mg (0.076mmol) of the 16b was dissolved in 15mL at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatography to yield the product. 1 H NMR (400 MHz, CD3OD) δ 7.44 (dd, J = 6.0, 3.2 Hz, 1H), 7.15 (dd, J = 6.0, 3.2 Hz, 1H), 7.06 – 6.96 (m, 2H), 6.81 (d, J = 8.0, 1.2 Hz, 1H), 6.68 (t, J = 7.7, 1.4 Hz, 1H), 2.92 – 2.79 (m, 1H), 2.44 – 2.32 (m, 2H), 1.85 (s, 1H), 1.79 – 1.64 (m, 2H). 13 C NMR (101 MHz, CD3OD) δ 171.61, 153.88, 128.23, 123.13, 119.53, 96.38, 67.13, 41.81, 37.13, 37.09, 34.34, 29.90, 29.64, 26.87. 83 7-((2-((tert-butoxycarbonyl)amino)phenyl)amino)-7-oxoheptanoic acid (18): 960 mg (6mmol) of pimelic acid, 418mg (2mmol), 1.164g of EDC, and 834uL of TEA were dissolved in 10mL of DCM and stired over night at room temperature. The solvent was removed invacu, the residue taken up in EtOAc and extracted with 5% NaOH solution. The aqueous layer was then acidified, extracted with EtOAc, the EtOAc layer was washed with NaHCO3, dried with MgSO4, and purified by automated chromatography to yield 145mg (20%). Rf=0.50 in 100% EtOAC. 1 H NMR (600 MHz, acetone) δ 7.67 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.22 – 7.17 (m, 1H), 7.11 (td, J = 7.8, 1.5 Hz, 1H), 2.47 (t, J = 7.4 Hz, 1H), 2.34 (t, J = 7.4 Hz, 1H), 1.80 – 1.72 (m, 1H), 1.68 (dt, J = 15.2, 7.5 Hz, 1H), 1.51 (s, 4H), 1.47 (tt, J = 9.4, 4.8 Hz, 1H). 13 C NMR (151 MHz, acetone) δ 173.62, 171.93, 153.39, 131.96, 125.36, 124.69, 123.99, 79.31, 36.13, 33.16, 28.39, 27.60, 25.18, 24.45. tert-butyl (2-(7-((2-aminophenyl)amino)-7-oxoheptanamido)phenyl)carbamate (19): 271mg (0.77mmol) of 18, 439 mg (1.16mmol) of HBTU, 202uL (1.16mmol) of DIEA, and 125mg (1.16mmol) of 1,2-phenylenediamine were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 to yield 116mg (34%). 1 H NMR (600 MHz, CD3OD) δ 7.51 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.17 (t, J = 7.7 Hz, 1H), 7.13 – 7.04 (m, 2H), 7.00 (t, J = 7.6 Hz, 1H), 6.68 (t, J = 7.6 Hz, 1H), 2.42 (t, J = 7.3 Hz, 3H), 1.80 – 1.71 (m, 3H), 1.48 (d, J = 8.5 Hz, 9H). 13 C NMR (151 MHz, CD3OD) δ 173.66, 173.44, 154.35, 141.83, 131.67, 126.78, 125.85, 125.68, 125.13, 124.44, 123.73, 118.06, 117.10, 80.05, 35.86, 35.57, 28.35, 27.25, 25.21, 25.10. 84 N 1 -(2-aminophenyl)-N 7 -(4-aminophenyl)heptanediamide (21a): 55mg (0.157mmol) of 18, 194.8 mg (0.51mmol) of HBTU, 39.8uL (0.23mmol) of DIEA, and 18.4mg (0.17mmol) of 1,4-phenylenediamine in 1.5mL of DMSO. The reaction was worked up according to General Procedure 1. Rf=0.20 in 10% MeOH in DCM. The product was dissolved in 5mL of DCM at 0°C, 1mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 31.5mg (59%).Rf=0.10 in 10% MeOH in DCM. 1 H NMR (400 MHz, CD3OD) δ 7.30 – 7.24 (m, 1H), 7.12 – 7.02 (m, 1H), 6.87 (dd, J = 8.0, 1.4 Hz, 1H), 6.76 – 6.67 (m, 1H), 2.47 (t, J = 7.4 Hz, 1H), 2.38 (t, J = 7.4 Hz, 1H), 1.85 – 1.72 (m, 2H), 1.57 – 1.46 (m, 1H). 13 C NMR (151 MHz, CD3OD) δ 173.54, 172.71, 144.09, 141.84, 129.27, 126.78, 125.71, 123.72, 121.84, 118.06, 117.07, 115.22, 36.12, 35.59, 28.37, 25.24. N 1 -(2-aminophenyl)-N 7 -(4-bromophenyl)heptanediamide (21b): 86mg (0.24mmol) of 18, 63uL (0.35mmol) of DIEA, 302mg (0.79mmol) of HBTU, 51mg (0.295mmol) of p- bromoaniline were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 130mg (quantitative). Rf=0.60 in 60% ethyl acetate in hexanes. The BOC- protected product was dissolved in 10mL of DCM at 0°C, 1mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatography to produce 92mg (93%). 1 H NMR (400 MHz, CD3OD) δ 7.89 (s, 1H), 7.46 – 7.39 (m, 1H), 7.22 – 7.14 (m, 2H), 7.07 – 6.97 (m, 2H), 6.82 (d, J = 8.0, 1.2 Hz, 1H), 6.68 (t, J = 7.8, 4.4, 0.9 Hz, 1H), 2.45 – 2.34 (m, 4H), 1.79 – 1.69 (m, 4H), 1.51 – 1.41 (m, 2H). 13 C NMR (101 MHz, CD3OD) δ 85 174.94, 174.59, 143.25, 141.49, 131.33, 128.24, 127.74, 127.11, 125.09, 123.74, 123.25, 119.51, 119.45, 118.52, 49.64, 37.71, 36.97, 29.76, 26.67, 26.41. N 1 -(2-aminophenyl)-N 7 -(3-aminophenyl)heptanediamide (22a): Compound 22a was prepared by the same procedure as 21a using 1,3-phenylenediamine. 1 H NMR (400 MHz, CD3OD) δ 7.11 – 7.02 (m, 1H), 6.89 – 6.81 (m, 1H), 6.76 – 6.70 (m, 1H), 6.52 – 6.48 (m, 1H), 2.47 (t, J = 7.4 Hz, 1H), 2.40 (t, J = 7.4 Hz, 1H), 1.85 – 1.73 (m, 1H), 1.57 – 1.47 (m, 1H). 13 C NMR (101 MHz, CD3OD) δ 173.59, 173.02, 147.84, 128.89, 126.85, 125.74, 118.15, 117.15, 111.20, 109.87, 107.12, 36.40, 35.61, 28.42, 25.33, 25.24. N1-(2-aminophenyl)-N7-(3-bromophenyl)heptanediamide (22b): Compound 22b was prepared by the same procedure as 21b using m-bromoaniline. 1 H NMR (400 MHz, CD3OD) δ 7.94 (t, J = 1.8 Hz, 1H), 7.48 (dt, J = 7.1, 1.9 Hz, 1H), 7.31 – 7.16 (m, 2H), 7.13 – 7.00 (m, 2H), 6.87 (dd, J = 8.0, 1.2 Hz, 1H), 6.72 (ddd, J = 7.8, 4.4, 0.9 Hz, 1H), 2.53 – 2.33 (m, 4H), 1.79 (dq, J = 14.8, 7.4 Hz, 4H), 1.58 – 1.45 (m, 2H). 13 C NMR (101 MHz, CD3OD) δ 173.58, 173.23, 141.89, 140.12, 129.96, 126.88, 126.38, 125.75, 123.73, 122.38, 121.89, 118.15, 118.08, 117.16, 36.35, 35.61, 28.40, 25.31, 25.05. Tert-butyl (2-(7-(isopropylamino)-7-oxoheptanamido)phenyl)carbamate (23): 55mg (0.157mmol) of 18, 194.8 mg (0.51mmol) of HBTU, 39.8uL (0.23mmol) of DIEA, and 18.4mg (0.17mmol) of 1,4-phenylenediamine in 1.5mL of DMSO. The reaction was worked up according to General Procedure 1. Rf=0.30 in 60% ethyl acetate in hexanes. 86 1 H NMR (400 MHz, CD3OD) δ 7.55 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 3.98 (td, J = 13.1, 6.7 Hz, 1H), 2.45 (t, J = 7.4 Hz, 2H), 1.82 – 1.63 (m, 4H), 1.54 (d, J = 0.9 Hz, 8H), 1.50 – 1.41 (m, 2H), 1.15 (dd, J = 6.6, 1.1 Hz, 5H). 13 C NMR (101 MHz, CD3OD) δ 173.70, 154.38, 131.68, 125.89, 125.13, 124.45, 80.04, 35.88, 35.56, 28.30, 27.27, 25.34, 25.15, 21.23. N1-(2-aminophenyl)-N7-isopropylheptanediamide (24): Compound 23 was dissolved in 5mL of DCM at 0°C, 1mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 15mg (32% over 2 steps). 1 H NMR (400 MHz, CD3OD) δ 7.89 (s, 1H), 7.14 – 7.01 (m, 2H), 6.88 (dd, J = 8.0, 1.3 Hz, 1H), 6.74 (td, J = 7.6, 1.4 Hz, 1H), 3.99 (qd, J = 13.2, 6.6 Hz, 1H), 2.45 (t, J = 7.5 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.84 – 1.62 (m, 5H), 1.52 – 1.40 (m, 2H), 1.20 – 1.10 (m, 7H). 13 C NMR (101 MHz, CD3OD) δ 173.55, 141.80, 126.83, 125.69, 123.80, 118.15, 117.18, 94.99, 40.93, 35.60, 28.37, 25.37, 25.28, 21.26, 21.22. Tert-butyl (2-(7-oxo-7-((pyridin-2-ylmethyl)amino)heptanamido)phenyl)carbamate (25): 55mg (0.157mmol) of 18, 194.8 mg (0.51mmol) of HBTU, 39.8uL (0.23mmol) of DIEA, and 18mg (0.17mmol) of 3-(Aminomethyl)pyridine were dissolved in 1.5mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 to yield 60mg (80%). Rf=0.30 in 10% MeOH in DCM. 1 H NMR (600 MHz, CD3OD) δ 8.44 (d, J = 4.7 Hz, 2H), 7.74 (t, J = 7.7 Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 7.34 (dd, J = 17.0, 7.9 Hz, 4H), 7.29 – 7.20 (m, 2H), 7.17 (t, J = 7.6 Hz, 2H), 7.10 (t, J = 7.6 Hz, 3H), 4.46 (s, 4H), 2.41 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.80 – 87 1.53 (m, 8H), 1.51 – 1.20 (m, 25H). 13 C NMR (151 MHz, CD3OD) δ 174.73, 173.59, 157.90, 154.32, 148.29, 137.37, 131.66, 125.84, 125.10, 124.42, 122.29, 121.44, 80.03, 44.07, 35.86, 35.34, 28.33, 27.26, 25.10, 25.07. N 1 -(2-aminophenyl)-N 7 -(pyridin-2-ylmethyl)heptanediamide (26): 56mg (0.13mmol) of 11was dissolved in 10mL of DCM at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 24mg (54%). 1 H NMR (600 MHz, CD3OD) δ 8.44 (d, J = 4.4 Hz, 1H), 7.75 (t, J = 7.7 Hz 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.28 – 7.23 (m, 1H), 7.09 – 7.04 (m, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 6.68 (t, J = 7.6 Hz, 1H), 4.46 (s, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.78 – 1.66 (m, 4H), 1.45 (dd, J = 15.2, 7.7 Hz, 2H). 13 C NMR (151 MHz, CD3OD) δ 174.82, 173.48, 157.91, 148.27, 141.86, 137.40, 126.78, 125.69, 123.73, 122.28, 121.44, 118.03, 117.07, 44.07, 35.58, 35.35, 28.38, 25.19, 25.12. N1,N7-bis(pyridin-2-ylmethyl)heptanediamide (27a): 80mg (0.5mmol) of pimelic acid, 500mg (1.3mmol) of HBTU, 204uL (1.2mmol) of DIEA, and 130mg (1.2mmol) of 3-(aminomethyl)pyridine were dissolved in 1.5mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 to yield 139mg (82%). 1 H NMR (400 MHz, DMSO) δ 9.15 (s, 1H), 7.20 (dd, J = 7.8, 1.3 Hz, 1H), 6.97 – 6.87 (m, 1H), 6.75 (dd, J = 8.0, 1.3 Hz, 1H), 6.58 (td, J = 7.6, 1.5 Hz, 1H), 3.39 (s, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1.72 – 1.62 (m, 2H), 1.42 (dd, J = 15.1, 8.3 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 173.59, 141.89, 126.85, 125.73, 123.76, 118.14, 117.16, 66.12, 35.61, 28.45, 25.30. 88 N 1 ,N 8 -bis(pyridin-2-ylmethyl)octanediamide (27b): 87mg (0.5mmol) of suberic acid, 500mg (1.3mmol) of HBTU, 204uL (1.2mmol) of DIEA, and 130mg (1.2mmol) of 3- (aminomethyl) pyridine were dissolved in 1.5mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 to yield 119mg (67%). 1 H NMR (400 MHz, CD3OD) δ 7.05 (d, J = 7.8, 1.4 Hz, 1H), 7.00 (t, 130aH), 6.81 (d, J = 8.0, 1.3 Hz, 1H), 6.68 (t, J = 7.6, 1.3 Hz, 1H), 3.28 (s, 3H), 2.42 (t, J = 7.4 Hz, 2H), 1.82 – 1.70 (m, 2H), 1.50 (dt, J = 18.2, 7.6 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 174.98, 153.88, 143.28, 128.24, 127.12, 125.16, 119.53, 118.55, 67.51, 37.00, 29.84, 26.69. Di-tert-butyl ((heptanedioylbis(azanediyl))bis(3,1-phenylene))dicarbamate (28): 53mg (0.33mmol) of pimelic acid, 138uL (0.79mmol) of DIEA, 300mg (0.79mmol) of HBTU, 165mg (0.79mmol) of Boc-1,3-phenylenediamine were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 97mg (54%). 1 H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 20.3 Hz, 1H), 7.27 – 7.17 (m, 1H), 7.11 (d, J = 7.9 Hz, 1H), 6.58 (s, 1H), 2.37 (t, J = 7.2 Hz, 1H), 1.81 – 1.70 (m, 1H), 1.54 (s, 4H), 1.48 – 1.40 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 171.56, 156.43, 152.78, 138.88, 138.63, 129.45, 114.52, 110.00, 69.59, 41.04, 38.64, 37.16, 28.36, 24.82. N 1 ,N 7 -bis(3-aminophenyl)heptanediamide (29): Compound 28 was deprotected with the same procedure as compound 19. 1 H NMR (400 MHz, CD3OD) δ 7.04 – 6.96 (m, 2H), 6.80 (d, J = 8.0, 1.0 Hz, 1H), 6.45 (d, J = 7.9, 1.3 Hz, 1H), 2.33 (t, J = 7.4 Hz, 2H), 1.76 – 1.64 (m, 2H), 1.47 – 1.35 (m, 1H). 13 C NMR (101 MHz, CD3OD) δ 174.37, 149.17, 140.48, 130.27, 112.57, 111.26, 108.48, 66.96, 37.77, 30.68, 29.75, 26.60. 89 N 1 ,N 7 -bis(2-isopropylphenyl)heptanediamide (30a): 80mg (0.50mmol) of pimelic acid, 174uL (1mmol) of DIEA, 417mg (1.1mmol) of HBTU, 141.5uL (1mmol) of 2- isopropylaniline were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 166mg (84%). 1 H NMR (400 MHz, CD3OD) δ 7.33 (d, J = 7.9 Hz, 1H), 7.25 – 7.11 (m, 3H), 3.14 (dt, J = 13.8, 6.9 Hz, 1H), 2.44 (t, J = 7.4 Hz, 2H), 1.79 (dt, J = 15.1, 7.5 Hz, 2H), 1.53 (dt, J = 15.1, 7.4 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, CD3OD) δ 174.17, 144.33, 133.95, 127.28, 126.91, 125.70, 125.43, 35.57, 28.49, 27.68, 25.30, 22.29. N 1 ,N 7 -bis(4-isopropylphenyl)heptanediamide (30b): 80mg (0.50mmol) of pimelic acid, 174uL (1mmol) of DIEA, 417mg (1.1mmol) of HBTU, 139uL (1mmol) of 4- isopropylaniline were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 90mg (46%). 1 H NMR (400 MHz, CD3OD) δ 7.46 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 2.94 – 2.86 (m, 1H), 2.41 (t, J = 7.4 Hz, 1H), 1.78 (dt, J = 15.1, 7.5 Hz, 1H), 1.49 (dt, J = 15.1, 7.7 Hz, 1H), 1.26 (d, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, CD3OD) δ 173.03, 144.62, 136.13, 126.19, 120.07, 94.99, 37.49, 36.29, 33.51, 25.26, 23.10. 90 N 1 ,N 7 -bis(3-methoxyphenyl)heptanediamide (31a): 80mg (0.50mmol) of pimelic acid, 174uL (1mmol) of DIEA, 417mg (1.1mmol) of HBTU, 113uL (1mmol) of m-anisidine were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 139mg (75%).Rf=0.40 in 60% ethyl acetate in hexanes. 1 H NMR (400 MHz, CD3OD) δ 7.25 (t, J = 2.2 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 7.05 – 7.00 (m, 1H), 6.62 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 2.34 (t, J = 7.5 Hz, 1H), 1.76 – 1.66 (m, 1H), 1.46 – 1.39 (m, 1H). 13 C NMR (101 MHz, CD3OD) δ 173.08, 160.04, 139.59, 129.07, 111.98, 109.19, 105.66, 54.24, 36.40, 28.38, 25.14. N 1 ,N 7 -bis(3,5-dimethoxyphenyl)heptanediamide (31b): 80mg (0.50mmol) of pimelic acid, 174uL (1mmol) of DIEA, 417mg (1.1mmol) of HBTU, 153uL (1mmol) of 3,5- dimethoxyaniline were dissolved in 3mL of DMSO and stirred overnight. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 142mg (66%). Rf=0.30 in 60% ethyl acetate in hexanes. 1 H NMR (400 MHz, CD3OD) δ 6.75 (d, J = 2.2 Hz, 1H), 6.18 (t, J = 2.3 Hz, 1H), 3.70 (s, 3H), 2.32 (t, J = 7.4 Hz, 1H), 1.78 – 1.59 (m, 1H), 1.47 – 1.34 (m, 1H). 13 C NMR (101 MHz, DMSO) δ 171.26, 160.40, 140.96, 97.29, 94.98, 55.01, 36.35, 28.28, 24.81. 7-((3-bromophenyl)amino)-7-oxoheptanoic acid (32): To pimelic acid (5.00 g, 31.2 mmol) was added 3- bromoaniline (3.4 mL, 31.2 mmol) and the mixture was stirred at 140⁰C for 24 h. The reaction mixture was cooled to room temperature, taken up in ethyl acetate (200 mL) and extracted into 5% aqueous KOH solution (250 mL). The aqueous 91 phase was acidified to pH 2 with concentrated HCl then extracted into ethyl acetate (3 x 300 mL). The organic phase was washed with saturated NaCl solution (200 mL), dried over sodium sulfate and evaporated. The residue was recrystallized from water/acetonitrile to yield 5.11g (52%). Rf = 0.10 in 60% ethyl acetate in hexanes. HRMS (ESI): m/z 314.0383, [M+H] + , calc. 314.0392). 1 H-NMR (600 MHz, MeOD): δ 7.87 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.17-7.21 (m, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.8 Hz, 2H), 1.59-1.72 (m, 4H), 1.37-1.42 (m, 2H). 13 C-NMR (150 MHz, MeOD): δ 176.1, 173.2, 140.1, 129.9, 126.4, 122.4, 121.8, 118.1, 36.3, 33.3, 28.3, 25.0, 24.3. Methyl 3-(7-((3-bromophenyl)amino)-7-oxoheptanamido)-4-((tert- butoxycarbonyl)amino) benzoate (33): 472mg (1.5mmol) compound 32, 567mg (1.5mmol) of HBTU, 261uL (1.5mmol) of DIEA, and 266mg (1mmol) of methyl 3- amino-4-((tert-butoxycarbonyl)amino)benzoate in 4.5mL of DMSO. The reaction was worked up according to General Procedure 1 and automatic chromatography to yield 151mg (27%). Rf=0.30 in 55% ethyl acetate in hexanes. 1 H NMR (600 MHz, cd3cn) δ 8.51 (d, J = 52.8 Hz, 1H), 7.89 (dd, J = 28.3, 1.8 Hz, 1H), 7.84 – 7.74 (m, 1H), 7.71 (s, 1H), 7.40 (dt, J = 7.5, 1.8 Hz, 1H), 7.21 – 7.13 (m, 1H), 2.40 (t, J = 7.4 Hz, 1H), 2.32 (t, J = 7.4 Hz, 1H), 1.68 (qd, J = 15.0, 7.5 Hz, 2H), 1.48 (s, 3H), 1.43 – 1.36 (m, 1H). 13 C NMR (151 MHz, cd3cn) δ 173.30, 172.05, 165.96, 153.01, 140.53, 136.92, 130.45, 127.33, 126.91, 126.23, 125.21, 122.08, 121.85, 118.09, 80.62, 51.78, 36.57, 36.07, 28.37, 27.58, 24.98, 24.86. 92 Tert-butyl (2-(7-((3-bromophenyl)amino)-7-oxoheptanamido)-4-((pyridin-2- ylmethyl) carbamoyl)phenyl)carbamate (34): 151mg (0.2mmol) of compound 33 was dissolved in 10mL of H2O and 10mL of THF, 26.4mg (0.69mmol) of LiOH was added, and stirred over night at room temperature. The reaction was neutralized with saturated solution of 2N HCl solution, extracted with EtOAc, saturated NaCl solution, and purified by column chromatography to produce 102mg (quantitative). 55mg (0.1mmol) of the acquired hydrolyzed carboxylic acid, 46 mg (0.12mmol) of HBTU, 21uL (0.12mmol) of DIEA, and 16mg (0.15mmol) of 3-(aminomethyl)pyridinein 4mL of DMSO. The reaction was worked up according to General Procedure 1 and automatic chromatography to yield 51mg (80%). Rf=0.25 in 10% MeOH in DCM. 1 H NMR (600 MHz, CD3OD) δ 8.37 (d, J = 4.7 Hz, 1H), 7.84 – 7.74 (m, 2H), 7.71 – 7.65 (m, 2H), 7.63 (d, J = 8.6, 2.1 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.21 – 7.15 (m, 1H), 7.11 – 7.02 (m, 2H), 4.56 (s, 2H), 2.36 (t, J = 7.4 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.65 (tt, J = 15.1, 7.5 Hz, 4H), 1.40 (s, 8H), 1.40 – 1.32 (m, 2H). 13 C NMR (151 MHz, CD3OD) δ 175.37, 174.47, 169.19, 159.38, 155.05, 149.99, 149.41, 141.44, 138.76, 136.92, 130.77, 129.75, 127.22, 126.57, 126.22, 124.25, 123.86, 123.69, 123.21, 122.34, 81.87, 38.15, 37.66, 37.15, 28.83, 28.38, 26.33. 93 N 1 -(2-amino-5-((pyridin-2-ylmethyl)carbamoyl)phenyl)-N 7 -(3-bromophenyl) heptanediamide (35): 51mg (0.079mmol) of compound 34 was dissolved in 15mL at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatagraphy to produce 28mg (66%). Rf=0.50 in 10% MeOH in DCM. 1 H NMR (600 MHz, CD3OD) δ 8.46 (s, 1H), 7.86 (d, J = 1.9 Hz, 1H), 7.76 (t, 1H), 7.68 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 8.5, 2.1 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.29 – 7.24 (m, 1H), 7.17 (dt, J = 15.7, 8.0 Hz, 2H), 6.81 (d, J = 8.5 Hz, 1H), 2.44 (t, J = 7.4 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 1.79 – 1.70 (m, 4H), 1.51 – 1.44 (m, 2H). 13 C NMR (151 MHz, CD3OD) δ 175.39, 174.58, 169.88, 159.78, 149.91, 149.32, 147.92, 141.45, 138.74, 130.78, 128.27, 127.56, 127.38, 123.41, 123.27, 119.89, 117.17, 116.90, 116.11, 111.42, 45.83, 44.98, 37.71, 37.57, 36.97, 29.80, 26.28. N 1 -phenyl-N 7 -(2-((pyridin-2-ylmethyl)amino)phenyl)heptanediamide (36): 35mg (0.10mmol) of compound 8a and 9uL (0.10mmol) of 2-pyridine-carboxaldehyde were dissolved in 5mL of MeOH and refluxed for 4 hours. After cooling the solution to 0°C, 50mg (1.3mmol) of NaBH4 and stirred at room temperature overnight. The reaction was concentrated in vacuo, taken up in EtOAc, extracted with NaCl (sat.) then 3 times with a 3N KOH solution. The organic layer was dried with MgSO4, filtered, and purified by automatic chromatography to yield a sufficient quantity for testing (>5mg). Rf= 0.45 in 94 100% ethyl acetate. 1 H NMR (600 MHz, CD3OD) δ 8.50 – 8.45 (m, 1H), 7.71 (td, J = 7.7, 1.8 Hz, 1H), 7.51 (dt, J = 8.7, 1.6 Hz, 1H), 7.28 – 7.21 (m, 1H), 7.10 – 7.03 (m, 1H), 6.98 (ddd, J = 8.1, 7.5, 1.5 Hz, 1H), 6.61 (td, J = 7.6, 1.3 Hz, 1H), 6.51 (dd, J = 8.2, 1.1 Hz, 1H), 4.46 (s, 1H), 1.85 – 1.69 (m, 1H), 1.50 (ddd, J = 18.1, 8.8, 6.5 Hz, 1H). 13 C NMR (151 MHz, CD3OD) δ 175.65, 174.46, 160.81, 149.63, 144.27, 139.88, 138.74, 129.74, 128.64, 128.09, 125.08, 123.55, 123.06, 121.25, 118.07, 112.92, 37.71, 37.05, 29.82, 26.62, 26.53. N,N'-(pentane-1,5-diyl)bis(2-aminobenzamide) (37): 81.5mg(0.5mmol) of isatoic anhydride and 118uL (1mmol) were added to 3mL of EtOH and stirred for 6hrs. The solvent was removed in vacuo and purified by automatic chromatography to yield 71mg (41%) of product. Rf=0.3 in 60% ethyl acetate in hexanes. 1 H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 7.9, 1.4 Hz, 1H), 7.34 (s, 1H), 7.31 – 7.20 (m, 1H), 7.16 (t, 1H), 6.73 (d, J = 8.2, 1.0 Hz, 1H), 6.59 (t, J = 8.1, 7.2, 1.2 Hz, 1H), 4.51 (s, 1H). 13 C NMR (151 MHz, CD3OD) δ 171.98, 171.96, 149.98, 149.95, 133.45, 133.41, 132.67, 132.49, 132.45, 129.43, 129.29, 128.58, 118.61, 118.23, 118.19, 118.12, 118.01, 117.99, 117.63, 117.59, 117.18, 116.92, 39.97, 39.50, 30.53, 30.24, 25.43. di-tert-butyl (((2,2'-(1,4-phenylene)bis(acetyl))bis(azanediyl))bis(2,1-phenylene)) dicarbamate (40): 108mg (0.55mmol) of 1,3-phenylenediacetic acid, 288mg (1.4mmol) of N-BOC-1,2-phenelyenediamine, 531mg (1.4mmol) of HBTU, and 200uL (1.2mmol) of DIEA in 3mL of DMSO. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography. Rf=0.50 in 7% MeOH in DCM. 1 H NMR 95 (600 MHz, DMSO) δ 9.61 (s, 1H), 8.27 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.28 (s, 2H), 7.10 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 3.64 (s, 2H), 1.42 (s, 9H). 13 C NMR (151 MHz, DMSO) δ 169.61, 153.00, 134.14, 131.13, 128.95, 125.11, 124.80, 123.81, 79.29, 42.48, 28.01. 2,2'-(1,4-phenylene)bis(N-(2-aminophenyl)acetamide) (41a): Compound 40 was dissolve in 10mL of DCM at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, filtered, and washed with an excess of H2O and MeOH to yield 90mg (43% over 2 steps). 1 H NMR (600 MHz, DMSO) δ 9.35 (s, 1H), 7.33 (s, 2H), 7.18 (d, J = 7.8 Hz, 1H), 6.94 – 6.89 (m, 1H), 6.74 (d, J = 7.9 Hz, 1H), 6.58 – 6.53 (m, 1H), 4.83 (s, 2H), 3.65 (s, 2H). 13 C NMR (151 MHz, DMSO) δ 169.08, 141.86, 134.40, 128.92, 125.81, 125.23, 123.31, 116.13, 115.82, 42.29. 2,2'-(1,3-phenylene)bis(N-(2-aminophenyl)acetamide) (41b): 97mg (0.5mmol) of 1,3- phenylenediacetic acid, 239mg (1.2mmol) of N-Boc-1,2-phenelyenediamine, 178mg (1.2mmol) of EDC, and 160uL (1.2mmol) of TEA in 5mL of DCM. The reaction was worked up according to General Procedure 1 and purified by automatic chromatography to yield 85mg (72%) of product. Rf=0.50 in 7% MeOH in DCM. The Boc-protected product was deprotected with the same procedure as19. 1 H NMR (400 MHz, CD3OD) δ 7.45 (dd, J = 7.9, 1.4 Hz, 1H), 7.34 (s, 1H), 7.25 (dt, J = 7.9, 4.8 Hz, 2H), 7.19 – 7.12 (m, 1H), 7.19 – 7.12 (m, 1H), 6.73 (dd, J = 8.2, 1.0 Hz, 1H), 6.59 (ddd, J = 8.1, 7.2, 1.2 Hz, 1H), 4.51 (s, 2H). 13 C NMR (101 MHz, CD3OD) δ 171.88, 150.27, 140.78, 133.15, 129.66, 129.02, 127.14, 127.11, 118.17, 117.60, 117.45, 43.94. 96 N,N'-(1,3-phenylenebis(methylene))bis(2-aminobenzamide) (42): 114uL (0.5mmol) m-xylylenediamine was added dropwise to a stirring of 195mg (1mmol) of isatoic anhydride in 6mL of EtOH and the reaction was refluxed overnight. The solvent was removed in vacuo and purified by automatic chromatography to yield 180mg (quantitative). 1 H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 7.9, 1.4 Hz, 1H), 7.34 (s, 1H), 7.31 – 7.20 (m, 1H), 7.16 (t, 1H), 6.73 (d, J = 8.2, 1.0 Hz, 1H), 6.59 (t, J = 8.1, 7.2, 1.2 Hz, 1H), 4.51 (s, 1H). 13 C NMR (101 MHz, CD3OD) δ 171.88, 150.27, 140.78, 133.15, 129.66, 129.02, 127.14, 127.11, 118.17, 117.60, 117.45, 43.94. 3-((3-oxo-3-(phenylamino)propyl)thio)propanoic acid (46): 356mg (2mmol) of 3,3′- thiodipropionic acid and 182uL (2mmol) of aniline were heated with stirring at 120°C for 3 hours. The reaction was worked up and purified via the same protocol as compound 32 to yield 67mg (13%) of product. Rf=0.15 in 60% ethyl acetate in hexanes. 1 H NMR (400 MHz, CD3OD) δ 7.53 (dd, J = 8.6, 0.9 Hz, 1H), 7.31 – 7.24 (m, 1H), 7.07 (dd, J = 10.6, 4.2 Hz, 1H), 2.87 (t, J = 7.2 Hz, 1H), 2.80 (t, J = 7.2 Hz, 1H), 2.65 (t, J = 7.2 Hz, 1H), 2.60 (t, J = 7.2 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 175.65, 172.45, 139.69, 129.75, 125.21, 121.29, 38.24, 35.61, 28.55, 27.89. Tert-butyl (2-(3-((3-oxo-3- (phenylamino)propyl)thio)propanamido)phenyl)carbamate (47): 67mg (0.26mmol) of 46, 110 mg (0.29mmol) of HBTU, 50uL (0.29mmol) of DIEA, and 55mg (0.264mmol) of 1,2-phenylenediamine in 1.5mL of DMSO. The reaction was worked up according to 97 General Procedure 1 and purified by automatic chromatography to yield 85mg (72%) of product. Rf=0.25 in 50% ethyl acetate in hexanes. 1 H NMR (400 MHz, CD3OD) δ 7.61 (d, J = 8.0 Hz, 1H), 7.51 (dd, J = 6.9, 1.2 Hz, 2H), 7.32 (dd, J = 7.9, 1.4 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.19 (td, J = 8.1, 1.5 Hz, 1H), 7.10 – 7.02 (m, 2H), 2.92 (t, J = 7.0 Hz, 4H), 2.75 – 2.61 (m, 4H), 1.48 (s, 8H). 13 C NMR (101 MHz, CD3OD) δ 173.34, 172.37, 155.49, 139.67, 133.73, 129.74, 127.63, 127.01, 125.35, 125.19, 121.24, 81.43, 38.12, 37.41, 28.67, 28.60, 28.33. N-(2-aminophenyl)-3-((3-oxo-3-(phenylamino)propyl)thio)propanamide (48): 85mg (19mmol) of compound 47 was dissolved in 10mL of DCM at 0°C, 1.5mL of TFA was added, and stirred overnight. The reaction was neutralized with saturated solution of NaHCO3, extracted with EtOAc, purified by column chromatography to produce 53mg (81%). 1 H NMR (400 MHz, CD3OD) δ 7.52 (d, J = 8.0 Hz, 2H), 7.27 (t, J = 7.8 Hz, 2H), 7.11 – 6.97 (m, 3H), 6.81 (d, J = 8.0 Hz, 1H), 6.70 – 6.63 (m, 1H), 2.97 – 2.90 (m, 3H), 2.70 (dt, J = 14.0, 7.1 Hz, 4H). 13 C NMR (101 MHz, CD3OD) δ 173.12, 172.50, 143.69, 139.73, 129.77, 128.51, 127.53, 125.23, 124.62, 121.31, 119.27, 118.19, 49.64, 49.43, 49.21, 38.13, 37.19, 28.75, 28.44. 4.7.3 Molecular Modeling Studies The x-ray crystallographically determined structure of BML-210 bound to MEF2 (PDB:3MU6) was prepared for docking using Schrodinger’s Protein Preparation Wizard tool to add hydrogens, correct bond orders, delete non-essential waters, predict side-chain protonation states, tautomers, and polar hydrogen orientations, and minimize the energy of the protein structure. Compounds BML-210 and 9a were draw in ChemDraw, saved 98 as .sdf file, and prepared for docking with LigPrep by generating ionization states at pH 5 to 9 using Epik. The compounds were docked using the Induced Fit Docking workflow at the standard precision setting with a 20-Å box centered around D61A, D63A, D61B, and D63B and H-bond constraints to the –O - of the carboxylate of D63A. The output structures were evaluated based on their docking scores. 99 4.8 References (1) Jones, P. A.; Baylin, S. B. Nat Rev Genet 2002, 3, 415 –28. (2) Minucci, S.; Pelicci, P. G. Nat Rev Cancer 2006, 6, 38–51. (3) Moradei, O.; Maroun, C. R.; Paquin, I.; Vaisburg, A. Curr Med Chem Anti-cancer Agent 2005, 5, 529–60. (4) Holliday, R. Dev Genet 1994, 15, 453 –7. (5) Boumber, Y.; Issa, J.-P. J. Oncology 2011, 25, 220 –6, 228. (6) Biel, M.; Wascholowski, V.; Giannis, A. Angew Chem Int Edit 2005, 44, 3186 – 216. (7) Yoo, C. B.; Jones, P. A. Nat Rev Drug Discov 2006, 5, 37–50. (8) Kouzarides, T. 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S.; Farid, R. Chem Biol Drug Des 2006, 67, 83-84 (41) Suite 2012: Schrödinger Suite 2012 Induced Fit Docking protocol; Glide version 5.8, Schrödinger, LLC, New York, NY, 2012; Prime version 3.1, Schrödinger, LLC, New York, NY, 2012. (42) Hidaka, K.; Kimura, T.; Abdel-Rahman, H. M.; Nguyen, J.-T.; McDaniel, K. F.; Kohlbrenner, W. E.; Molla, A.; Adachi, M.; Tamada, T.; Kuroki, R.; Katsuki, N.; Tanaka, Y.; Matsumoto, H.; Wang, J.; Hayashi, Y.; Kempf, D. J.; Kiso, Y. J Med Chem 2009, 52, 7604–17. 102 Chapter 5: Synthesis and Anti-Cancer Activity of Non-COX-2-Inhibiting Analogs of Celecoxib 5.1 Introduction Celecoxib (Celebrex ® ) is an FDA-approved drug for the treatment of inflammation associated with osteo- and rheumatoid arthritis (Figure 5.1). Traditional non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen act through the inhibition of two enzymes, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Celecoxib selectively inhibits COX-2 to avoid the adverse gastrointestinal issues associated with inhibition COX-1. Due to the tumor-promoting effects of inflammation, celecoxib was the subject of pre-clinical studies where it showed chemopreventive properties in several forms of cancer including colon cancer and non-melanoma skin cancer. As a result, celecoxib received a second indication for the preventative treatment of familial adenomatous polyposis (FAP). 1 In the intervening years, the antitumoral activity of celecoxib has been demonstrated a wide array of cancers both in cellular assays and in animal tumor models 2-4 and been the subject of further human clinical studies for colorectal polyps and adenomas. 1,5,6 Unfortunately, with the withdrawal of rofecoxib (Vioxx ® ) over its link to increased risk of heart attack and stroke at long-term high-doses, a question of safety has been cast over the whole class of COX-2 inhibitors. 7 Figure 5.1: Structures of selective COX-2-inhibitors. The anti-tumor mechanism of action celecoxib is a highly debated topic. As a result of increasing evidence of celecoxib’s diverse bioactivities, a growing number of COX-2- independent pathways have emmegred. 8-10 Among these pathways the involvement of the sarcoplasmic/endoplasmic reticulum (ER) calcium ATPase (SERCA) is the most extensively studied. 11-13 SERCA is a transmembrane calcium-pump of the endoplasmic 103 reticulum ER responsible for maintaining a high gradient of Ca 2+ in the lumen of the ER. The first cellular event upon administration of celecoxib is a sharp increase in the levels of Ca 2+ in the cytosol. This influx of Ca 2+ is characteristic of SERCA inhibition. 11-15 The loss of the calcium gradient in the ER triggers ER-stress. Celecoxib has been shown to cause this ER stress-instigated cell death in cancer cells and animal tumor models. 12,16-19 ER stress response induces apoptosis through the expression of CHOP/GADD153 and caspase 7 and celecoxib induces the expression of both of these. 16,17,19-21 While SERCA- mediated ER stress has been implicated, 8 COX-2-inhibition has thus far not been completely ruled out as a likely mechanism responsible for at least some of the antitumor activity of celecoxib. 10,22 5.2 Experimental Design In order to elucidate the relevant pathways for the antitumor activity of celecoxib, we have investigated several structurally related molecules with the goal of identifying new and improved anti-cancer agents (Figure 5.2). We postulated that through simple structural modification to celecoxib we might be able to eliminate COX-2 inhibition while maintaining the anti-cancer properties. A survey of the literature provides insights into the structure activity relationship for COX-1 and COX-2 inhibition (Table 5.1). 23,24,25 A comparison of compounds 1 and 4 shows that the replacement of the methyl group from celecoxib with a hydrogen decreases COX-1 inhibition 3.5 fold and slightly increased COX-2 inhibition. 2,5-substitutions of methyl groups, 6, or chlorides on ring A completely removes any COX inhibition activity. 1 Compounds 4 and 5 illuminate the role of the sulfonyl-groups. The change from a sulfonamide to a sulfone decreased COX-2 inhibition only 4 fold removes any measureable binding to COX-1. Additionally, this analysis yielded two interesting modifications to the celecoxib core that eliminated COX-2 activity: para sulfone 11 and para phenol 13. 1 COX-1 activity of 5 was estimated from 4-(3-(difluoromethyl)-5-(2,5-dimethylphenyl)-1H-pyrazol-1- yl)benzenesulfonamide and 4-(5-(2,5-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1- yl)benzenesulfonamide analogs with COX-1 IC 50 >100. 23 104 Figure 5.2: Structural analogs of celecoxib and rofecoxib and the COX-1 and COX-2 activity. IC 50 (µM) IC 50 (µM) COX-1 COX-2 COX-1 COX-2 1 15 0.043 11 >100 >100 2 >500 0.43 12 13.6 1.1 4 55.1 0.032 13 >100 >100 5* >1000 0.10 14 2.58 0.008 6 >100 >100 15 1.32 0.34 10 1.19 0.009 Table 5.1: COX-2 and COX-2 activity of structural analogs of celecoxib and rofecoxib. *Activities for –CF 2H analog, not –CF 3 5.3 Synthesis Following our analysis, this series of structurally related analogs of celecoxib was synthesized according to Figures 5.3-5.7. Celecoxib analogs were synthesized in a one- pot procedure utilizing either NaOMe or, more optimally, NaH to form the enolate of the desired acetophenone, 17a-c, which condenses with ethyl trifluororaceate to produce the intermediate 1,3-ketoenols, 18a-c (Figure 5.3). An acid catalyzed, regio-selective cyclization with 4-hydrazinobenzenesulfonamide (19a) to produce the sulfonamide analogs 1, 4, 5, and 12 or 4-(methylsulfonyl) phenylhydrazine (19b) to produce the sulfone analogs 3 and 6. 23,26 Compound 8 was synthesized according to Figure 5.4. 105 Alkylation of 2,5-phenylacetic acid with ethyl bromoacetate followed by KOtBu- mediated cyclization produced the desired lactone 21. While attempts to covert the lactone hydroxyl to the triflate proved unsuccessful, bromination with oxalyl bromide proceeded smoothly to produce the corresponding bromide 23. 27,28 This set the stage for Suzuki-coupling with benzenesulfonamide-4-boronic acid pinacol ester 24 to yield the rofecoxib analog 8. Para-substituted analogs 10, 12, 13, and 14 were synthesized using the same two step protocol as 1, 4, and 6 (Figure 5.5-5.6). Compound 10 was subsequently oxidized with mCPBA to sulfone 11 which was shown in the literature to have poor COX-2 activity. 23 Methyl ether 13 was converted to the corresponding phenol 14 with BBr3. Aniline 15 was reacted with benzoyl chloride to yield benzamide 16 (Figure 5.5). Additionally, modifications to the sulfonamide were made through reactions of various amines with sulfonyl chloride intermediate 29 to yield analogs 30-35 (Figure 5.7). Figure 5.3: Synthesis of analogs 1 and 3-7. (a) NaH, THF, ethyl trifluoroacetate; (b) 19a or 19b, EtOH, reflux. Figure 5.4: Synthesis of analog 8 (a) NaOMe, MeOH, then ethyl bromoacetate; (b) KOtBu, THF; (c) DCM, DMF, oxalyl bromide; (d) 24, Na 2CO 3, Pd(PPh 3) 4, C 6H 6, toluene, H 2O:EtOH (2:1). 106 Figure 5.5: Synthesis of analogs 10-11 and 13-16. (a) NaH, THF, ethyl trifluoroacetate; (b) 19a or 19b, EtOH, reflux; (c) mCPBA, DCM; (d) DCM, BBr 3; (e) benzoyl chloride, pyridine, DCM. Figure 5.6: Synthesis of analogs 12. (a) NaH, THF, ethyl trifluoroacetate; (b) 19a, EtOH, reflux. Figure 5.7: Synthesis of analogs 30-31. (a) 28, AcOH, EtOH, reflux; (b) thionyl chloride, DMF; (c) amine, DIEA, DCM, then TFA, DCM if Boc-protected. 5.4 In Vivo Activity Our collaborators in the labs of Dr. Stan Louie (USC School of Pharmacy) and Drs. Axel Schönthal and Florence Hofman (USC Keck School of Medicine) evaluated our analogs in an array of models. Initially, the ability of the analogs to inhibit cellular growth was determined by MTT assay against three cell lines. The role of substituents on ring A was directly compared, as shown in Figure 5.8. Compound 6 is the most potent of the three analogs at inducing cell death with an IC50 of 30 µM followed by compound 1 and then compound 4. Overall, the inhibition of cellular growth follows the order 6 > 1 > 4. This 107 trend is opposite that of COX-2 inhibition where compound 4 was the most potent followed by compound 1 then compound 6, which had no measurable activity against COX-1 or COX-2, or summarized as the trend 4 > 1 > 6 for COX-2. From these trends, it is apparent that the anticancer properties of celecoxib and related compounds are independent of inhibition of COX-2. Figure 5.8: MTT activity of analogs 1, 4, and 6. Three glioblastoma cell lines (LN229, U251, T98G) were treated with increasing concentrations of 1, 4, or 6. After 48 hours, MTT assays were performed to indicate the overall viability in response drug treatment. The charts on the left present the quantitative readout of the optical density of the 96-well plates (average of two wells each). The right panels show representative 96- well plates of treated cells. Deep purple indicates fully viable cell cultures, whereas yellow color indicates cell death. Further analysis of the data yields a complete view of the structural components of celecoxib that are required for induction of apoptosis (Table 5.2). A comparison of compounds 1, 3, 6, and 7 illustrates the requirement of the sulfonamide for activity. Interestingly, rofecoxib was able to be turned into a relatively potent anticancer agent, compound 8, by the addition of the 2,5-dimethyl substitutions on ring A and the conversion of the sulfone of ring C to a sulfonamide indicating that ring B is not important for activity. An additional series of compounds were synthesized in an attempt to maximize potency of the celecoxib scaffold (Table 5.3). Ring A was tolerant of hydrophobic groups 13 and large functional groups 15 and 16. Due to the inactivity of the sulfone derivatives, we realized that the sulfonamide played an important role in binding to its target. Functionalization of the sulfonamide led to 108 increased potency in the MTT assay. Increased in affinity was seen with extended amine, 29. This trend suggests better binding to their likely target, SERCA. Table 5.2: Structure-activity relationship between celecoxib and rofecoxib analogs. Analog R X B Analog R X B 1 4-CH 3 -NH 2 6 2,5-CH 3 -NH 2 2 - -CH 3 7 2,5-CH 3 -CH 3 3 4-CH 3 -CH 3 8 - -NH 2 4 - -NH 2 9 2,5-CH 3 -NH 2 Table 5.3. Celcoxib analogs and their activity. Analog R X Analog R X 11 4-SMe -NH 2 16 12 4-SO 2Me -NH 2 13 4-OMe -NH 2 29 2,5-CH 3 14 4-NH 2 -NH 2 30 2,5-CH 3 15 4-NHCOPh -NH 2 31 2,5-CH 3 B 109 5.5 Applications Through a series of collaborations, we were able to use compounds 4 (unmethylated celecoxib or UMC) and 6 (dimethyl celecoxib or DMC) to better understand the therapeutic extensions of the non-COX-2 activity of celecoxib. The work is summarized below with the abstracts from the published papers. The abstracts and figures are from the papers referenced at the opening of each subsection. 5.5.1 COX-2 inhibition is neither necessary nor sufficient for celecoxib to suppress tumor cell proliferation and focus formation in vitro 29 Chuang, H.-C.; Kardosh, A.; Gaffney, K. J.; Petasis, N. A.; Schönthal, A. H. Mol Cancer 2008, 7, 38. “BACKGROUND: An increasing number of reports is challenging the notion that the antitumor potential of the selective COX-2 inhibitor celecoxib (Celebrex) is mediated primarily via the inhibition of COX-2. We have investigated this issue by applying two different analogs of celecoxib that differentially display COX-2-inhibitory activity: the first analog, called unmethylated celecoxib (UMC), inhibits COX-2 slightly more potently than its parental compound, whereas the second analog, 2,5- dimethyl-celecoxib (DMC), has lost the ability to inhibit COX-2. RESULTS: With the use of glioblastoma and pancreatic carcinoma cell lines, we comparatively analyzed the effects of celecoxib, UMC, and DMC in various short-term (< or =48 hours) cellular and molecular studies, as well as in long-term (< or =3 months) focus formation assays. We found that DMC exhibited the most potent antitumor activity; celecoxib was somewhat less effective, and UMC clearly displayed the overall weakest antitumor potential in all aspects. The differential growth- inhibitory and apoptosis-stimulatory potency of these compounds in short- term assays did not at all correlate with their capacity to inhibit COX-2, but was closely aligned with their ability to trigger endoplasmic reticulum stress (ERS), as indicated by the induction of the ERS marker 110 CHOP/GADD153 and activation of the ERS-associated caspase 7. In addition, we found that these compounds were able to restore contact inhibition and block focus formation during long-term, chronic drug exposure of tumor cells, and this was achieved at sub-toxic concentrations in the absence of ERS or inhibition of COX-2. CONCLUSION: The antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2. Rather, the drug's ability to trigger ERS, a known effector of cell death, might provide an alternative explanation for its acute cytotoxicity. In addition, the newly discovered ability of this drug to restore contact inhibition and block focus formation during chronic drug exposure, which involved neither ERS nor COX-2, suggests a novel, as yet unrecognized mechanism of celecoxib action.” 5.5.2 Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress 30 Ferrario, A.; Lim, S.; Xu, F.; Luna, M.; Gaffney, K. J.; Petasis, N. A.; Schönthal, A. H.; Gomer, C. J. Cancer Lett. 2011, 304, 33–40. “Moderate activity of the endoplasmic reticulum (ER) stress response system exerts anti-apoptotic function and supports tumor cell survival and chemoresistance, whereas its more severe aggravation may exceed the protective capacity of this system and turn on its pro-apoptotic module. In this study, we investigated whether the combination of two pharmacologic agents with known ability to trigger ER stress via different mechanisms would synergize and lead to enhanced tumor cell death. We combined the HIV protease inhibitor nelfinavir (Viracept) and the cyclooxygenase 2 (COX-2) inhibitor celecoxib (Celebrex) and investigated their combined effect on ER stress and on the viability of breast cancer cells. We found that this drug combination aggravated ER stress and caused pronounced toxicity in human breast cancer cell lines, inclusive of variants that were 111 highly resistant to other therapeutic treatments, such as doxorubicin, paclitaxel, or trastuzumab. The anti-tumor effects of celecoxib were mimicked at increased potency by its non-coxib analog, 2,5-dimethyl- celecoxib (DMC), but were substantially weaker in the case of unmethylated-celecoxib (UMC), a derivative with superior COX-2 inhibitory efficacy. We conclude that the anti-tumor effects of nelfinavir can be enhanced by celecoxib analogs in a COX-2 independent fashion via the aggravation of ER stress, and such drug combinations should be considered as a beneficial adjunct to the treatment of drug-resistant breast cancers.” Fig. 5.9. Altered chemosensitivity after knock-down of GRP78 or CHOP. MCF7/Dox cells were transfected with siRNA directed at CHOP or GFP and treated with 15 μM NVF and 30 μM DMC alone or in combination for 48 h. In all cases, cell survival was determined by colony formation assay. Shown is percent colony formation (mean ± SD, n ⩾ 3), where the number of colonies in control cultures was set to 100%. Asterisk ( ∗) indicates statistically significant (p < 0.05) difference between the number of colonies obtained from drug-treated cells transfected with siGRP78 or CHOP as compared to siGFP (control). 112 5.5.3 Antiangiogenic activities of 2,5-dimethyl-celecoxib on the tumor vasculature 31 Virrey, J. J.; Liu, Z.; Cho, H.-Y.; Kardosh, A.; Golden, E. B.; Louie, S. G.; Gaffney, K. J.; Petasis, N. A.; Schönthal, A. H.; Chen, T. C.; Hofman, F. M. Mol Cancer Ther. 2010, 9, 631–41. “Our laboratory has previously shown that a novel compound, 2,5- dimethyl-celecoxib (DMC), which is structurally similar to the cyclooxygenase-2 (COX-2) inhibitor celecoxib but lacks the COX-2- inhibitory function, mimics the antitumor effects of celecoxib. Most studies on DMC, however, focused on its effects on tumor cells. Here, we investigated the activities of DMC as an antiangiogenic agent in both in vitro and in vivo systems. Using primary cultures of human glioma specimens, we found that DMC treatment was cytotoxic to tumor- associated brain endothelial cells (TuBEC), which was mediated through the endoplasmic reticulum stress pathway. In contrast, confluent cultures of quiescent human BEC did not undergo cell death. DMC potently suppressed the proliferation and migration of the TuBEC. DMC caused no apparent effects on the secretion of vascular endothelial growth factor and interleukin-8 but inhibited the secretion of endothelin-1 in tumor- associated EC. DMC treatment of glioma xenografts in mice resulted in smaller tumors with a pronounced reduction in microvessel density compared with untreated mice. In vitro and in vivo analyses confirmed that DMC has antivascular activity. Considering that DMC targets both tumor cells and tumor-associated ECs, this agent is a promising anticancer drug.” 113 Figure 5.10. Cytotoxic effects of DMC on TuBECs. TuBECs and subconfluent BECs were treated with DMC, celecoxib (CXB), and UMC for 72 h. Cell viability was assessed using the MTT assay (A) and the cell death ELISA (B). Statistical comparisons were made from the DMC treatments to the celecoxib and UMC treatments. C, photographs of subconfluent and confluent BECs were taken after treatment with 60 μmol/L DMC for 72 h. D, TUNEL assay was done on untreated (Untr) and DMC-treated (60 μmol/L, 48 h) cells. The staining denotes TUNEL-positive cells. E, confluent and subconfluent BECs were treated with DMC for 72 h and analyzed with the MTT assay. Untreated cells were incubated with vehicle. *, P < 0.02. 5.5.4 Enhancement of photodynamic therapy by 2,5-dimethyl celecoxib, a non- cyclooxygenase-2 inhibitor analog of celecoxib 32 Ferrario, A.; Lim, S.; Xu, F.; Luna, M.; Gaffney, K. J.; Petasis, N. A.; Schönthal, A. H.; Gomer, C. J. Cancer Lett. 2011, 304, 33–40. “Photodynamic therapy (PDT) effectiveness can be improved by employing combined modality approaches involving pharmaceuticals targeting the tumor microenvironment and/or tumor cell death pathways. In one approach, combining PDT with celecoxib improves long-term tumoricidal activity without increasing normal tissue photosensitization. However, side effects arising from the use of coxib based cyclooxygenase- 2 (COX-2) inhibitors, including cardiovascular injury, decreases the 114 clinical applications of this class of compounds. A growing number of studies demonstrate that the tumoricidal actions of coxibs such as celecoxib involve non-COX-2 mediated mechanisms. The celecoxib analog, 2,5-dimethyl celecoxib (DMC), lacks COX-2 inhibitory activity but exhibits cytotoxic properties comparable to the COX-2 inhibitor celecoxib. We compared the effectiveness of DMC and celecoxib in modulating PDT response at both the in vitro and in vivo level using a C3H/BA murine mammary carcinoma model. Both DMC and celecoxib blocked PDT induced expression of the pro-survival protein survivin, enhanced the endoplasmic reticulum stress (ERS) response of PDT, and increased both apoptosis and cytotoxicity in BA cells exposed to combination protocols. DMC enhanced the in vivo tumoricidal responsiveness of PDT without altering PGE2 levels. Our data demonstrates that DMC improved PDT by increasing apoptosis and tumoricidal activity without modulating COX-2 catalytic activity. Our results also suggest that celecoxib mediated enhancement of PDT may involve both COX-2 dependent and independent mechanisms.” Figure 5.11. DMC and celecoxib increase phototoxicity in PDT-treated BA cells. Clonogenic survival of BA cells exposed to single PDT and combination treatments using PDT together with either celecoxib or DMC at a doses of 40 μM. Cells were incubated with PH (25 μg/ml) in the dark for 16 h and exposed to red light (600 J/m 2 ). Celecoxib and DMC were added to the culture medium immediately after PDT and left in the medium for the duration of the experiment. Columns, mean; bars, ±SE of three separate experiments; * , p < 0.05 (PDT versus either PDT + celecoxib or PDT + DMC); ** ,p < 0.05 (PDT + celecoxib versus PDT + DMC). 115 5.5.5 Preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors 33 Thomas, S.; Sharma, N.; Golden, E. B.; Cho, H.; Agarwal, P.; Gaffney, K. J.; Petasis, N. A.; Chen, T. C.; Hofman, F. M.; Louie, S. G.; Schönthal, A. H. Cancer Lett. 2012, 325, 63–71. “The cellular processes of autophagy and endoplasmic reticulum stress (ERS) appear to be interconnected, and it has been proposed that autophagy may serve to reduce ERS via removal of terminally misfolded and aggregated proteins. Conversely, there are indications that blockage of autophagy may increase ERS. Based on earlier work demonstrating that pharmacologically aggravated ERS can result in tumor cell killing, we investigated whether blockage of autophagy would enhance this effect in a therapeutically useful manner. We therefore combined chloroquine (CQ), a pharmacological inhibitor of autophagy, with other drugs known to act as ERS aggravators (ERSA), namely nelfinavir (an HIV protease inhibitor) and celecoxib (a cyclooxygenase-2 inhibitor) or its non-coxib analog 2,5-dimethyl-celecoxib (DMC), and investigated combination drug effects in a variety of breast cancer cell lines. We found that the addition of CQ resulted in synergistic enhancement of tumor cell killing by ERSA compounds, particularly in triple-negative breast cancer (TNBC) cells. This combination effect could also be confirmed in an in vivo model, where CQ boosted low-dose ERSA effects, resulting in rapid deterioration of xenografted tumors in mice. Altogether, our results indicate that combinations of an autophagy inhibitor with pharmacological ERSA (i.e. compounds that lead to ER stress aggravation) should be further explored for potential therapy of otherwise difficult-to-treat TNBC.” 116 Figure 5.12. Colony formation after combination treatment. Two hundred (a) MDA- MB-231 or (b) MDA-MB-468 cells were seeded into 6-well plates and treated with nelfinavir (N), DMC (D), or chloroquine (Q) alone and in combination for 48 h. (Numbers after letters represent concentrations in l M.) Thereafter, medium was replaced with fresh medium without drugs, and emerging colonies were counted 2 weeks later. Shown are representative photos of colonies; the charts display the average number of colonies from two independent experiments (with n P 3 each), where colony formation by untreated cells (0) was set to 100%. Tumor volume after treatment. Shown are results from four animals harboring subcutaneous MDA-MB-468 tumors. (c) Animals with pea- sized tumors remained untreated or received three-drug treatment (nelfinavir + DMC + chloroquine) for 5 consecutive days. Shown is tumor volume 4 weeks later, where tumor volume in two untreated animals had increased an average of threefold, whereas tumor size in the treated animal had shrunk to below 20%. 5.5.6 Cytotoxic effects of celecoxib on Raji lymphoma cells correlate with aggravated endoplasmic reticulum stress but not with inhibition of cyclooxygenase- 2 34 Chen, S. T.; Thomas, S.; Gaffney, K. J.; Louie, S. G.; Petasis, N. A.; Schönthal, A. H. Leukemia Res. 2010, 34, 250–3. “Inhibition of cyclooxygenase 2 (COX-2) by the selective COX-2 inhibitor celecoxib has been suggested as potentially useful for B-cell lymphoma therapy. However, additional pharmacological activities of celecoxib have been discovered and have challenged the notion that its antitumor effects are mediated primarily via the inhibition of COX-2. To A B C D 117 shed light on this issue, we have investigated the effects of different pharmacological agents with greatly varying COX-2 inhibitory potency in Raji lymphoma cells in vitro. We found that cytotoxic potency of these compounds did not at all correlate with their COX-2 inhibitory activity; in fact, the most potent COX-2 inhibitors lacked the ability to kill Raji cells. Instead, the cytotoxic outcome was closely aligned with these agents’ ability to trigger endoplasmic reticulum (ER) stress, which could be further enhanced by bortezomib, an agent with known ER stress-inducing potency. Together, these results indicate that celecoxib's cytotoxic effects on Raji lymphoma cells do not involve the inhibition of COX-2.” Figure 5.13. Enhancement of ER stress and cell death by combination drug treatment. Raji cells were cultured in the presence of celecoxib (CXB), UMC, or DMC individually or in combination with bortezomib (BZM). Top part: Cell survival was determined by MTT assay after 48 h of drug treatment. Asterisks indicate statistically significant differences between combination drug treatments and the respective individual drug treatments: * p < 0.01; ** p < 0.001. Bottom part: Expression of the ER stress marker CHOP and the apoptosis indicator PARP was determined by Western blot after 24 h of drug treatment. Actin was used as a loading control to verify equal amounts of lysate in each lane. f.l.: full length PARP protein; cl.: cleaved PARP protein (indicating the initiation of apoptosis). 118 5.5.7 Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors 35 Interestingly, during the exploration of synergistic combinations of various ER-stressors, a practice that led to a large portion of the preceding papers, it was found that the cytotoxicity of bortozomib was completely inactivated by the green tea polyphenol EGCG. As an extension of our work on celecoxib, this work has also been included. Golden, E. B.; Lam, P. Y.; Kardosh, A.; Gaffney, K. J.; Cadenas, E.; Louie, S. G.; Petasis, N. A; Chen, T. C.; Schönthal, A. H. Blood 2009, 113, 5927–37. “The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self- medicate with this "miracle herb" in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid-based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non- boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of 119 green tea products may be contraindicated during cancer therapy with BZM.” Figure 5.14. 1 H NMR spectra (400 MHz; left) and 13 C NMR spectra (100 MHz; right) of combinations of BZM and EGCG in 20% D 2O in CD 3CN. (i) Pure BZM, (ii) pure EGCG, (iii) a 2:1 mixture of BZM and EGCG, and (iv) a 1:1 mixture of BZM and EGCG. Selected peaks from the NMR spectra indicate the presence of the new adduct from the reaction of a 1,2-diol unit of EGCG with the boronic acid group of BZM. By increasing the amount of EGCG relative to BZM, from 2:1 in panel iii to 1:1 in panel iv, the product peaks (red arrows in the 1 H NMR and red dots in the 13 C NMR) increase relative to the BZM peaks (black arrows in the 1 H NMR and black dots in the 13 C NMR). 5.6 Conclusion Through a series of targeted modifications to the structures of both celecoxib and rofecoxib, we were able to identify the key moieties for apoptotic activity and also establish a COX-2-independent pathway for this activity. Additionally, through a series of structural analogs with variations on both ring Aand ring C we were able to develop more potent analogs and to establish structure-activity relationships. Finally, DMC served as a useful model compound for illustrating the effectiveness of the non-COX-2 anti-tumoral celecoxib activity in combination with nelfinavir, chloroquine, or bortozomib, as an antiangiogenic agent and potent ER-stressor, and as an enhancer of photodynamic therapy. 120 5.7 Experimental General Procedure for the synthesis of 4,4,4-trifluoro-1-phenylbutane-1,3-diones: 1.6g (40mmol, 10eq) of sodium hydride in a 60 % dispersion in mineral oil was added to an oven-dried RBF and the flask was sealed with a rubber septa under a N2 atmosphere. 10mL of DriSolv ® THF and then 4mmol of the corresponding acetophenone were added to the flask fitted with an Ar balloon and the solution was stirred at room temperature for 30min. 2.86mL (24mmol, 6eq) of ethyl trifluoroacetate were then added and stirred overnight at room temperature. The reaction was concentrated in vacuo, dissolved in EtOAc, extracted with 2M HCl, then H2O, dried with MgSO4, filtered, concentrated onto Celite ® , and purified by automated chromatography with a gradient of 0-40% of a 9:1 EtOAc:hexanes solution. 4,4,4-trifluoro-1-phenylbutane-1,3-dione (18a): 495uL (4mmol) of acetophenone yielded 835 mg of a white solid (85% yield). 1 H NMR (400 MHz, acetone) δ 8.06 – 7.87 (m, 2H), 7.53 – 7.40 (m, 3H), 6.37 (s, 1H), 4.77 (s, 2H). 13 C NMR (101 MHz, acetone) δ 188.79, 171.68 (d, J = 28.8 Hz), 139.37, 131.54, 128.22, 127.39, 119.44 (q, J = 286.6 Hz), 89.78. 19 F NMR (376 MHz, acetone) δ -76.12. LC-MS: calcd. [M-H] - 215.03; found 215.1 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione (18b): 553uL (4mmol) of 4’- methylacetophenone yielded 701mg of an off-white solid (76% yield). 1 H NMR (600 MHz, cd3od) δ 7.69 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 2.34 (s, 3H). 13 C NMR (151 MHz, cd3od) δ 190.41, 171.45 (q, J = 29.3 Hz), 141.96, 140.43, 129.72, 128.16, 121.02 (q, J = 288.9 Hz), δ 89.99 (q, J = 43.8, 19.7 Hz), 21.38. 19 F NMR (564 MHz, cd3od) δ -77.20, -83.55. LC-MS: calcd. [M-H] - 229.05; found 229.1. 121 1-(2,5-dimethylphenyl)-4,4,4-trifluorobutane-1,3-dione (18c): 610uL (4mmol) of 2′,5′- dimethyl acetophenone yielded 852 mg of a white solid (quantiative 99% yield). 1 H NMR (500 MHz, acetone) δ 7.31 (s, 1H), 7.09 (dd, J = 20.2, 7.6 Hz, 2H), 5.93 (s, 1H), 4.34 (s, 2H), 2.37 (s, 3H), 2.30 (s, 3H). 13 C NMR (126 MHz, acetone) δ 194.38, 171.16 (q, J = 32.6 Hz), 140.50, 134.75, 133.57, 130.99, 130.38, 128.15, 119.37 (q, J = 286.3 Hz), 93.91, 19.96, 19.37. 19 F NMR (470 MHz, acetone) δ -76.31. LC-MS: calcd. [M-H] - 243.06; found 243.1. General Procedure for the Synthesis of 4-(5-phenyl-3-(trifluoromethyl)-1H-pyrazol- 1-yl) benzenesulfonamides: The corresponding 4,4,4-trifluoro-1-phenylbutane-1,3- dione (18a-c) and 4-hydrazinobenzenesulfonamide ·HCl (19a) (1.1eq) were dissolved in 10mL of 200 proof EtOH and stirred at 80°C overnight. The reaction mixture was concentrated onto Celite ® and purified by automated flash chromatography (Biotage ® ) with a gradient of 0-40% of EtOAc in hexanes. The fractions containing the product were concentrated in vacuo. The residue was dissolved in a minimal amount of EtOAc and hexanes was added dropwise until the solution became cloudy. The flask was allowed to stand at room temperature for 3hrs during this time a white precipitate formed which was filtered and washed with hexanes to yield the product. 122 4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (1): 213mg (0.93mmol) of 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione (18b) and 228mg (1.02mmol, 1.1eq) of 4-hydrazinobenzenesulfonamide ·HCl (19a) yielded 106mg of a fluffy white powder (30% yield. 1 H NMR (400 MHz, cd3od) δ 7.93 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.24 – 7.13 (m, 4H), 6.91 (s, 1H), 2.35 (s, 3H). 13 C NMR (101 MHz, cd3od) δ 145.65, 143.65, 143.37 (q, J = 38.3 Hz), 141.83, 139.56, 129.17, 128.63, 126.84, 126.57, 125.57, 121.25 (q, J = 268.1 Hz), 105.44, 105.42, 19.82. 19 F NMR (376 MHz, cd3od) δ -63.95. LC-MS: calcd. [M+H] + 382.08; found 382.1. 4-(5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (4): 182mg (0.84mmol) of 4,4,4-trifluoro-1-phenylbutane-1,3-dione (18a) and 207mg (0.92 mmol, 1.1eq) of 4-hydrazinobenzene sulfonamide ·HCl (19a) yielded 156mg of a tan crystalline solid (47% yield, Rf= 0.3 in 30% EtOAc in hexane). 1 H NMR (400 MHz, cd3od) δ 7.93 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 7.43 – 7.33 (m, 3H), 7.30 – 7.26 (m, 2H), 6.93 (s, 1H). 13 C NMR (101 MHz, cd3od) δ 146.90, 145.00, 144.81 (q, J = 39.39), 143.11, 130.52, 130.12, 130.02, 129.96, 128.27, 126.97, 122.60 (q, J = 536.5, 268.2 Hz), 107.14 (d, J = 2.0 Hz). 19 F NMR (376 MHz, cd3od) δ -63.77. LC-MS: calcd. [M-H] - 366.05; found 366.1. 123 4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (5): 205mg (0.84mmol) of 1-(2,5-dimethylphenyl)-4,4,4-trifluorobutane-1,3-dione (18c) and 27mg (0.92mmol, 1.1eq) of 4-hydrazinobenzenesulfonamide·HCl (19a) yielded 187mg of a white powder (56% yield). 1 H NMR (500 MHz, cd3od) δ 7.86 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 7.9 Hz, 2H), 6.82 (s, 1H), 2.30 (s, 3H), 1.91 (s, 3H). 13 C NMR (126 MHz, cd3od) δ 146.41, 144.72 (q, J = 38.5 Hz), 144.60, 143.28, 137.36, 135.16, 132.08, 131.93, 131.70, 129.92, 128.18, 125.43, 122.71 (q, J = 268.1 Hz), 108.04 (d, J = 1.9 Hz), 20.79, 19.38. 19 F NMR (376 MHz, cd3od) δ -63.88. LC-MS: calcd. [M+H] + 396.1; found 396.1. General Procedure for the Synthesis of 1-(4-(methylsulfonyl)phenyl)-5-phenyl-3- (trifluoromethyl)-1H-pyrazoles: The corresponding 4,4,4-trifluoro-1-phenylbutane- 1,3-dione (18a-c) and 4-(methylsulfonyl) phenylhydrazine·HCl (19b) (1.1eq) were dissolved in 10mL of 200 proof EtOH and stirred at 80°C overnight. The reaction mixture was concentrated onto Celite ® and purified by automated flash chromatography (Biotage ® ) with a gradient of 0-20% of EtOAc in hexanes. The fractions containing the product were concentrated in vacuo, and pentane was added. The solution was sonicated 124 for 30min to produce a precipitate, the supernatant was pipetted off. This process of pentane-sonication was repeated to yield the desired product. 1-(4-(methylsulfonyl)phenyl)-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole (3): 171mg (0.74mmol) of 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione (18b) and 181mg (0.81mmol, 1.1eq) of 4-(methylsulfonyl) phenylhydrazine·HCl (19b) yielded 98mg of a white powder (35% yield). 1 H NMR (400 MHz, acetone) δ 8.03 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 1.1 Hz, 4H), 7.01 (s, 1H), 3.18 (s, 3H), 2.35 (s, 3H). 13 C NMR (101 MHz, acetone) δ 146.54, 144.17 (q, J = 38.0 Hz), 144.11, 141.94, 140.46, 130.41, 129.81, 129.34, 126.89, 126.75, 122.40 (q, J = 268.1 Hz), 106.95 (d, J = 2.0 Hz), 44.21, 21.25. 19 F NMR (376 MHz, acetone) δ -62.89. LC-MS: calcd. [M+H] + 381.09; found 381.1. 5-(2,5-dimethylphenyl)-1-(4-(methylsulfonyl)phenyl)-3-(trifluoromethyl)-1H- pyrazole (6): 200mg (0.82mmol) of 1-(2,5-dimethylphenyl)-4,4,4-trifluorobutane-1,3- dione (18c) and 200mg (0.9mmol, 1.1eq) of 4-(methylsulfonyl) phenylhydrazine·HCl (19b) yielded 155 mg of a white powder (48% yield, Rf=0.3 in 20% EtOAc in hexanes). 1 H NMR (400 MHz, acetone) δ 7.97 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.26 – 7.16 (m, 3H), 6.96 (s, 1H), 3.16 (s, 3H), 2.30 (s, 3H), 1.98 (s, 3H). 13 C NMR (101 MHz, acetone) δ 145.80, 144.07 (q, J = 38.2 Hz), 144.04, 141.44, 136.78, 134.70, 131.86, 125 131.65, 131.48, 129.46, 129.26, 125.24, 122.37 (q, J = 268.3 Hz), 108.07 (d, J = 1.9 Hz), 44.15, 20.77, 19.38. 19 F NMR (376 MHz, acetone) δ -62.74. LC-MS: calcd. [M+H] + 295.10; found 295.1. 2-ethoxy-2-oxoethyl 2-(2,5-dimethylphenyl)acetate (21): To a stirring solution of 1.642g (10mmol) of 2,5-dimethylphenyl acetic acid in 17mL of MeOH was added 2.80 mL (13 mmol) of NaOMe (25% wt), the reaction was stirred for 30 min, and then 1.10 mL (10 mmol) of ethyl bromoacetate was added. After stirring overnight, the reaction was concentrated in vacuo, dissolved in EtOAc, washed with NH4Cl and brine, dried with MgSO4, and purified by automated chromotagraphy to yield 724 mg (29% for ethyl). Rf= 0.30 in 10% ethyl acetate in hexanes. 1 H NMR (600 MHz, cd3od) δ 7.39 (dd, J = 7.9, 1.1 Hz, 1H), 7.14 (t, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.62 – 6.55 (m, 1H), 3.33 (t, J = 7.1 Hz, 1H), 1.69 – 1.57 (m, 1H), 1.44 (t, J = 7.6 Hz, 1H). 13 C NMR (101 MHz, Acetone) δ 171.39, 168.33, 135.89, 134.67, 133.42, 131.91, 130.81, 128.69, 61.57, 61.54, 38.97, 20.88, 19.14, 14.36. 3-(2,5-dimethylphenyl)-4-hydroxyfuran-2(5H)-one (22): 3.07 mL (3.07 mmol) of a 1M solution of KOtBu in THF was added to a flame-dried and sealed flask. 724 mg (2.89 mmol) of 21 was dissolved in 0.5 mL of THF and the solution added drop wise to the sealed flask. The flask was washed again with 0.5 mL of THF and added to ensure complete transfer. After flask was refluxed overnight, H2O was added, and extracted 2 times with Et2O. The H2O was acidified with 10% HCl to pH 2, extracted with EtOAc, dried with MgSO4, and concentrated in vacuo to yield 417 mg of product (71%). 1 H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 7.8 Hz, 1H), 7.08 (dd, J = 7.8, 1.3 Hz, 1H), 6.98 (s, 1H), 4.69 (s, 2H), 3.33 (s, 1H), 2.32 (s, 3H), 2.19 (s, 3H). 13 C NMR (101 MHz, 126 CDCl3) δ 176.38, 174.33, 135.19, 134.70, 130.86, 130.21, 129.42, 126.96, 102.44, 67.68, 50.67, 20.86, 19.41. 4-bromo-3-(2,5-dimethylphenyl)furan-2(5H)-one (23): To as stirring solution of 382 mg (1.87 mmol) of 22 dissolved in 1.5 mL of DCM was added first 188 uL (2.43 mmol) of DMF and then 1.22 mL (2.25 mmol) of 2M solution of oxalyl bromide in DCM at 0°C. The reaction was allowed to warm to room temperature and stirred overnight. H2O was added to the reaction and extracted with Et2O. The organic layer was washed with brine, dried with Na2SO4, filtered, concentrated in vacuo, and triturated with ether to produce 161 mg (32%). 1 H NMR (400 MHz, cdcl3) δ 7.16 (dd, J = 8.7, 4.5 Hz, 1H), 6.97 (s, 1H), 4.97 (d, J = 3.6 Hz, 1H), 2.33 (s, 1H), 2.21 (s, 1H). 13 C NMR (101 MHz, cdcl3) δ 169.58, 141.37, 135.24, 133.64, 132.87, 130.30, 130.26, 129.71, 127.42, 73.18, 20.68, 19.16. 4-(4-(2,5-dimethylphenyl)-5-oxo-2,5-dihydrofuran-3-yl)benzenesulfonamide (8): 61.7 mg (0.23 mmol) of 23, 26.56 mg (0.02 mmol) of Pd(PPh3)4, 121 mg (1.15 mmol) of Na2CO3, and 130 mg (0.40 mmol) benzenesulfonamide-4-boronic acid pinacol ester (24) were added to a flasked under nitrogen, sealed, and 3mL of benzene, 500 uL of H2O, 250 uL of EtOH, and 1.25 mL of toluene were added. The reaction was heated at 100°C and stirred overnight. The reaction was concentrated in vacuo, taken up in EtOAc, extracted with brine, dried with MgSO4, filtered, and purified by preperative TLC to yield 67 mg (85%). Rf=0.40 in 60% ethyl acetate in hexanes. 1 H NMR (400 MHz, cdcl3) δ 7.79 (d, J = 8.7 Hz, 1H), 7.29 (d, J = 8.7 Hz, 1H), 7.14 – 7.07 (m, 1H), 6.89 (s, 1H), 2.25 (s, 1H), 127 1.99 (s, 1H). 13 C NMR (101 MHz, cdcl3) δ 173.08, 153.70, 143.68, 136.40, 134.99, 133.48, 130.99, 130.50, 129.83, 129.45, 127.84, 127.26, 77.48, 77.16, 76.84, 70.37, 25.00, 21.10, 19.38. 4-(5-(4-(methylthio)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1- yl)benzenesulfonamide (10): 479 mg (2.89 mmol) of 4-(methylthio)acetophenone and 173 mg (4.32 mmol) of NaH (60% dispersion in mineral oil) were dissolved in 4 mL of THF and stirred at room temperature for 4 hours. 447 uL of ethyl trifluoroacetate was added and stirred at room temperature overnight. The reaction was then concentrated in vacuo, diluted in 10 mL of EtOH, 644 mg (2.88 mmol) of 4-hydrazino benzenesulfonamide hydrochloride (19a) was added, and refluxed overnight. The reaction was concentrated in vacuo, dissolved in EtOAc, extracted with saturated NaHCO3. extracted three times with brine, dried with MgSO4, filtered, and purified by silca gel chromatography to yield 889mg (74%). 1 H NMR (400 MHz, CD3OD) δ 7.97 (q, 1H), 7.61 – 7.45 (m, 1H), 7.30 – 7.14 (m, 2H), 6.94 (s, 1H), 2.47 (s, 2H). 13 C NMR (101 MHz, CD3OD) δ 145.18, 143.68, 143.28, 141.80, 141.38, 129.05, 127.01, 125.96, 125.81, 125.68, 125.53, 124.64, 122.62, 119.95, 105.60, 13.55. 19 F NMR (376 MHz, CD3OD) δ -63.69. 128 4-(5-(4-(methylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1- yl)benzenesulfonamide (11): 883 mg (2.14 mmol) of 10 were dissolved in 30 mL of DCM and 1 g (5.80 mmol) of mCPBA was added. After the reaction was stirred at room temperature for 3 hours, 537 uL (3.78 mmol) of TEA was added. The reaction mixture was extracted with saturated NaHCO3 and purified by silica gel column chromotagraphy. 1 H NMR (400 MHz, CD3OD) δ 7.99 (t, J = 7.8 Hz, 1H), 7.58 (dd, J = 15.1, 8.5 Hz, 1H), 7.17 (s, 1H), 3.18 (s, 1H). 13 C NMR (101 MHz, CD3OD) δ 144.14, 143.89, 143.67, 143.50, 141.40, 141.38, 133.87, 129.79, 127.64, 127.21, 125.88, 106.92, 42.78. 19 F NMR (376 MHz, CD3OD) δ -63.78. 4-(5-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (13): 600 mg (4 mmol) of 4'-methoxy acetophenone and 160 mg (4 mmol) of NaH (60% dispersion in mineral oil) were dissolved in 2.5 mL of DME and stirred at room temperature for an hours. 1 mL (8.40 mmol) of ethyl trifluoroacetate was added and stirred at room temperature overnight. The reaction was then concentrated in vacuo, diluted in 6mL of EtOH, 537 mg (2.4 mmol) of 4-hydrazino benzenesulfonamide hydrochloride (19a) was added, and refluxed overnight. The reaction was concentrated in vacuo, dissolved in EtOAc, extracted with saturated NaHCO3. extracted three times with brine, dried with MgSO4, filtered, and purified by silca gel chromatography to yield 770 mg (48%). 1 H NMR (400 MHz, CD3OD) δ 7.94 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 1.9 Hz, 1H), 7.66 (dd, J = 8.3, 1.9 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.45 (d, J = 8.2 Hz, 1H), 6.99 (s, 1H). 13 C NMR (101 MHz, CD3OD) δ 160.62, 145.51, 143.58, 143.49, 143.20, 142.82, 141.93, 130.20, 126.97, 125.65, 122.67, 120.73, 120.01, 114.06, 105.34, 54.55. 19 F NMR (376 MHz, CD3OD) δ -63.56. 129 4-(5-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (14): 508 mg (1.27 mmol) of 13 was dissolved in 4 mL of DriSolv ® DCM and cooled to -78°C. 191 µL of BBr3 was added dropwise. The reaction was stirred for 1hr at -78°C, warmed to room temperature, and monitored by TLC. The reaction was quenched with a saturated solution of NaHCO3, dissolved in EtOAc, and extracted with brine. The organic layer was dried with MgSO4 and purified by column chromatography. 1 H NMR (400 MHz, CD3OD) δ 7.92 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 6.82 (s, 1H), 6.75 (d, J = 8.7 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 160.00, 147.29, 144.92, 144.52, 143.38, 131.61, 128.26, 126.98, 120.88, 116.73, 106.40. 4-(5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (15): 1.27 g (9.36 mmol) of 4-amino acetophenone and 1.20 g (30 mmol) of NaH (60% dispersion in mineral oil) were dissolved in 20 mL of THF and stirred at room temperature for an hours. 1 mL (8.40 mmol) of ethyl trifluoroacetate was added and stirred at room temperature overnight. The reaction was then concentrated in vacuo, diluted in 20 mL of EtOH, 2.24 g (10mmol) of 4-hydrazino benzenesulfonamide 130 hydrochloride (19a) was added, and refluxed overnight. The reaction was concentrated in vacuo, dissolved in EtOAc, extracted with saturated NaHCO3. extracted three times with brine, dried with MgSO4, filtered, and purified by silca gel chromatography to yield 2.38 g (64%). Rf=0.50 in 60% ethyl acetate in hexanes and was fluorecensent blue under UV-lamp. 1 H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.9 Hz, 1H), 7.56 – 7.51 (m, 1H), 7.00 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 0.4 Hz, 1H), 6.66 (d, J = 8.8 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 149.41, 146.51, 143.48, 143.39, 143.10, 142.21, 129.70, 126.84, 125.58, 116.74, 114.35, 104.50, 48.27, 48.06, 47.85, 47.63, 47.42, 47.21, 46.99. 19 F NMR (376 MHz, CD3OD) δ -63.85. N-(4-(1-(4-sulfamoylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)benzamide (16): To a stirring solution of 80 mg (0.21 mmol) of 15 in 2mL of pyridine at 0°C was added 20uL (0.17 mmol) of benzoyl chloride in 1 mL of DCM. After stirring overnight, the reaction was concentrated in vacuo, taken up in EtOAc, washed with NaHCO3, dried with MgSO4, and purified by automated chromotagraphy. 1 H NMR (400 MHz, CD3OD) δ 7.97 (dd, J = 18.0, 7.4 Hz, 1H), 7.79 (d, J = 7.1 Hz, 1H), 7.65 – 7.49 (m, 1H), 7.32 (d, J = 8.5 Hz, 1H), 6.99 (s, 1H). 13 C NMR (101 MHz, CD3OD) δ 168.95, 146.65, 145.13, 143.24, 141.27, 136.03, 133.08, 130.70, 129.66, 128.65, 128.37, 127.09, 125.67, 121.97, 106.97. 19 F NMR (376 MHz, CD3OD) δ -63.82. 131 4-(3-(trifluoromethyl)-4,5-dihydro-1H-benzo[g]indazol-1-yl)benzenesulfonamide (12): 160 mg (4 mmol) of NaH (60% dispersion in mineral oil) was added to 270 uL (4mmol) of tetralone in 4 mL of THF and stirred overnight. 480 uL of ethyl trifluoroacete was added and stirred overnight. The reaction was concentrated in vacuo, dissolved in 20 mL of ethanol, 537 mg (2.4 mmol) of 4-hydrazino benzenesulfonamide hydrochloride (19a) and 3.5 mL of HCl were added, and refluxed overnight. The reaction was then cooled to room temperature, concentrated in vacuo, dissolved in EtOAc, extracted with brine, dried with MgSO4, purified by column chromotagraphy, and recrystallized with EtOAc in hexanes to yield 283mg (18%). 1 H NMR (400 MHz, dmso) δ 8.00 (t, J = 8.8 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.25 (t, 1H), 7.10 (t, J = 8.8, 4.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 3.01 (t, J = 7.4 Hz, 1H), 2.80 (t, J = 7.3 Hz, 1H). 13 C NMR (101 MHz, dmso) δ 145.17, 142.18, 140.18, 139.16, 138.79, 137.66, 129.52, 129.26, 127.70, 127.07, 126.70, 125.25, 123.02, 118.66, 40.59, 40.38, 29.44, 18.89. 4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonic acid (29): 6 g (25.6 mmol) of 18c and 4.87g (25.9 mmol) of 4-hydrazinobenzenesulfonic acid (28) were dissolved in 42 mL of EtOH and 7 mL of 6N HCl and stirred at reflux overnight. The reaction was cooled to room temperature, concentrated in vacuo, dissolved in EtOAc, and extracted with water then brine. The organic layer was dried 132 with MgSO4, concentrated in vacuo, and recrystallized with ether to yield 3.9 g of a white solid (38%). 1 H NMR (400 MHz, DMSO) δ 7.52 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.15 – 7.07 (m, 3H), 7.02 (s, 1H), 2.21 (s, 3H), 1.85 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 148.10, 144.34, 138.61, 135.19, 133.49, 131.03, 130.48, 130.33, 128.45, 126.38, 123.68, 106.72, 20.43, 19.02. 19 F NMR (376 MHz, DMSO) δ -60.55. 4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzene-1-sulfonyl chloride (30): To a stirring solution of 2.0 g (5.04 mmol) of 29 in 5 mL of DMF was added 0.7 mL of thionyl chloride drop wise . The mixture was stirred at room temperature for 2hrs. The reaction was dried under high vacuum and quenched with ice water. The aqueous solution was extracted with ice-cold DCM, dried with MgSO4, and concentrated in vacuo to yield a yellowish oil which was carried on to the next step without further purification. N-(2-aminoethyl)-4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl) benzenesulfonamide (31): To a stirring solution of 4g (10mmol) of 29 in 10ml of DMF was added 2mL (27mmol) of thionyl chloride drop wise and stirred at room temperature. After 4 hours, the reaction was concentrated in vacuo, placed on high vacuum to remove excess thionyl chloride. The resulting oil was dissolved in 10mL of DCM. The solution was cooled to 0°C and 2.23mL (14mmol) of N-Boc-ethylenediamine and 3ml (17mmol) 133 of DIEA in 20ml of DCM were added. After stirring overnight, the reaction was concentrated in vacuo, dissolved in EtOAc, extracted with saturated NaHCO3. extracted three times with brine, dried with MgSO4, filtered, and purified by silca gel chromatography at 30% ethyl acetate in hexanes to produce 800mg (14%). 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.09 (s, 1H), 6.70 (s, 1H), 5.46 (s, 1H), 4.84 (s, 1H), 3.23 (dd, J = 11.3, 5.8 Hz, 1H), 3.06 (dd, J = 11.0, 5.6 Hz, 1H), 2.35 (s, 1H), 1.94 (s, 1H), 1.45 (s, 4H). 13 C NMR (101 MHz, CDCl3) δ 144.66, 143.74, 142.64, 138.78, 136.14, 133.76, 131.01, 130.85, 130.77, 128.54, 107.37, 70.22, 28.32, 20.88, 19.33, 14.24. 19 F NMR (376 MHz, CDCl3) δ -62.35. 100mg (0.19mmol) of the product from the previous step was dissolved in 5mL of DCM, cooled to 0°C, and 0.5mL of TFA were added. After 2 hours, the reaction was neutralized with saturated NaHCO3 and the DCM was removed in vacuo. Ethyl acetate was then added and extracted. The organic layer was extracted with brine, dried with MgSO4, filtered, concentrated in vacuo, and purified by automated chromatography at 15% MeOH in DCM to yield the product. 1 H NMR (400 MHz, CD3OD) δ 7.84 – 7.66 (m, 1H), 7.50 – 7.31 (m, 1H), 7.10 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 6.9 Hz, 1H), 6.74 (s, 1H), 2.91 (t, J = 6.1 Hz, 1H), 2.74 (t, J = 6.0 Hz, 1H), 2.21 (s, 1H), 1.82 (s, 2H). 13 C NMR (101 MHz, CD3OD) δ 145.01, 143.63, 143.25, 142.39, 139.57, 136.03, 133.74, 130.69, 130.65, 130.41, 128.47, 127.72, 124.18, 122.65, 106.83, 43.09, 40.22, 19.50, 18.07. 19 F NMR (376 MHz, CD3OD) δ -63.63, -76.77. 4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-(2-hydroxyethyl) benzenesulfonamide (32): 600mg (1.52mmol) of 29 was dissolved in 3mL of thionyl 134 chloride. After refluxing at 80°C overnight, the reaction was concentrated in vacuo and placed on high vacuum. The residue was then dissolved in 5mL of DCM, 274uL (4.5mmol) of ethanolamine and 531uL (3.30mmol) of DIEA were added at 0°C. After stirring overnight, the reaction was concentrated in vacuo, dissolved in EtOAc, extracted with brine and saturated NH4Cl, dried with MgSO4, filtered, concentrated in vacuo, and purified in by automated chromatography. 1 H NMR (400 MHz, CD3OD) δ 7.82 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 9.3 Hz, 1H), 6.81 (s, 1H), 4.88 (s, 1H), 3.72 (dt, J = 13.0, 6.5 Hz, 1H), 3.22 (q, J = 7.3 Hz, 1H), 2.32 (s, 1H), 1.93 (s, 2H). 13 C NMR (101 MHz, CD3OD) δ 146.18, 146.15, 144.82, 144.05, 143.67, 141.83, 137.14, 135.08, 132.05, 131.78, 131.58, 129.95, 127.84, 125.01, 124.08, 121.41, 107.68, 55.78, 43.80, 20.83, 19.43, 18.72, 17.26, 13.20. 19 F NMR (376 MHz, CD3OD) δ -63.39. 4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-(2- morpholinoethyl)benzenesulfonamide (33): To a stirring solution of 41uL (0.30mmol) of 4-(2-aminoethyl)morpholine and 42uL (0.24mmol) of DIEA were dissolved in 1mL of DCM was added 0.24mmol 30 from a stock solution in DCM drop wise over 15 minutes. After stirring for 2 days, the reactions were concentrated in vacuo, dissolved in EtOAc, extracted with saturated NaHCO3, extracted three times with brine, dried with MgSO4, filtered, and purified by silca gel chromatography to produce 110mg (90%). 1 H NMR (400 MHz, CDCl3) δ 7.87 – 7.79 (m, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.10 (s, 1H), 6.71 (s, 1H), 5.28 (s, 1H), 3.72 – 3.57 (m, 2H), 3.01 (t, J = 5.4 Hz, 1H), 2.46 – 2.39 (m, 1H), 2.36 (s, 1H), 2.32 (t, 2H), 1.94 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 144.77, 144.28, 143.89, 142.79, 138.66, 136.26, 133.81, 135 131.10, 130.91, 130.88, 128.63, 128.17, 123.95, 122.50, 119.82, 107.50, 66.88, 56.22, 53.03, 38.97, 20.98, 19.41. 19 F NMR (376 MHz, CDCl3) δ -62.32. 136 5.8 References (1) Steinbach, G.; Lynch, P. M.; Phillips, R. K.; Wallace, M. H.; Hawk, E.; Gordon, G. B.; Wakabayashi, N.; Saunders, B.; Shen, Y.; Fujimura, T.; Su, L. K.; Levin, B. New Engl J Med. 2000, 342, 1946-52. 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J Med Chem 2010, 53, 951–65. 152 Appendix: Selected Spectra 1 H NMR 13 C NMR 153 1 H NMR 13 C NMR 154 1 H NMR 13 C NMR 155 1 H NMR 13 C NMR 156 1 H NMR 13 C NMR 157 1 H NMR 158 1 H NMR 13 C NMR 159 1 H NMR 13 C NMR 160 1 H NMR 13 C NMR 161 1 H NMR 13 C NMR 162 1 H NMR 13 C NMR 163 1 H NMR 164 1 H NMR 165 1 H NMR 13 C NMR 166 1 H NMR 167 13 C NMR 1 H NMR 168 13 C NMR 1 H NMR 169 1 H NMR 13 C NMR 170 1 H NMR 171 1 H NMR 13 C NMR 172 1 H NMR 13 C NMR 173 1 H NMR 13 C NMR 174 1 H NMR 13 C NMR 175 1 H NMR 13 C NMR 176 1 H NMR 13 C NMR 177 1 H NMR 13 C NMR 178 1 H NMR 13 C NMR 179 1 H NMR 13 C NMR 180 1 H NMR 13 C NMR 181 1 H NMR 13 C NMR 182 1 H NMR 13 C NMR 183 1 H NMR 13 C NMR 184 1 H NMR 13 C NMR 185 1 H NMR 13 C NMR 186 1 H NMR 13 C NMR 187 1 H NMR 13 C NMR 188 1 H NMR 13 C NMR 189 1 H NMR 13 C NMR 190 13 C NMR 1 H NMR 191 13 C NMR 1 H NMR 192 1 H NMR 13 C NMR 193 1 H NMR 13 C NMR 194 1 H NMR 13 C NMR 195 1 H NMR 13 C NMR 196 1 H NMR 13 C NMR 197 1 H NMR 13 C NMR 198 1 H NMR 13 C NMR 199 1 H NMR 13 C NMR 200 1 H NMR 13 C NMR 201 1 H NMR 13 C NMR 202 1 H NMR 13 C NMR 203 1 H NMR 13 C NMR 204 1 H NMR 13 C NMR 205 1 H NMR 13 C NMR 206 1 H NMR 13 C NMR 207 1 H NMR 13 C NMR 208 1 H NMR 13 C NMR 209 1 H NMR 13 C NMR 210 1 H NMR 13 C NMR 211 1 H NMR 13 C NMR 212 19 F NMR 213 1 H NMR 13 C NMR 214 19 F NMR 215 1 H NMR 13 C NMR 216 19 F NMR 217 1 H NMR 13 C NMR 218 19 F NMR 219 1 H NMR 13 C NMR 220 19 F NMR 221 1 H NMR 13 C NMR 222 19 F NMR 223 1 H NMR 13 C NMR 224 1 H NMR 225 1 H NMR 13 C NMR 226 19 F NMR 227 1 H NMR 13 C NMR 228 1 H NMR 13 C NMR 229 1 H NMR 13 C NMR 230 1 H NMR 13 C NMR 231 1 H NMR 13 C NMR 232 19 F NMR 233 1 H NMR 13 C NMR 234 19 F NMR 235 1 H NMR 13 C NMR 236 19 F NMR 237 1 H NMR 13 C NMR 238 19 F NMR 239 1 H NMR 13 C NMR 240 19 F NMR 241 1 H NMR 13 C NMR 242 1 H NMR 13 C NMR 243 19 F NMR 244 1 H NMR 13 C NMR 245 19 F NMR 246 1 H NMR 13 C NMR 247 19 F NMR

Abstract (if available)

Abstract This dissertation details my efforts towards the design and development of novel small molecules anti-cancer agents including joint efforts with a host of colleagues and collaborators. ❧ The introduction, Chapter 1, provides a brief overview of the current "Hallmarks of Cancer." This is a list of characteristics acquired during tumorigenesis, which provide the targets for drug development. In this sojourn, we have utilized a wide spectrum of strategies and techniques to develop specific drugs. These efforts are described in this Thesis. ❧ Chapter 2 describes the computational fragment-based design, synthesis, and biological validation of a series of novel inhibitors of the enzyme dUTPase as potential anti-cancer agents. ❧ Chapter 3 details the design, synthesis, biological evaluation, and molecular modeling studies of novel CXCR2 antagonists. ❧ Chapter 4 reports the synthesis and structure-activity relationship of a series of small molecules capable of disrupting the protein-protein interaction between transcription factor MEF2 and class IIa HDACs. ❧ Chapter 5 outlines our efforts to optimize a series of analogs of the FDA-approved cyclooxygenase-2 (COX-2) inhibitor Celebrex® (Celecoxib), that have anti-cancer properties an devoid of COX-2 activity and the efforts of our collaborators to characterize the efficacy of these agents in a variety of models and therapeutic combinations are also summarized.

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Asset Metadata

Creator Gaffney, Kevin J. (author)

Core Title Adventures in medicinal chemistry: design and synthesis of small molecule biological modulators

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 01/28/2015

Defense Date 12/14/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag medicinal chemistry,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Petasis, Nicos A. (committee chair), Louie, Stan G. (committee member), Pratt, Matthew R. (committee member)

Creator Email kevinjohngaffney@gmail.com,vinylrichie@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-303233

Unique identifier UC11293437

Identifier etd-GaffneyKev-1864.pdf (filename),usctheses-c3-303233 (legacy record id)

Legacy Identifier etd-GaffneyKev-1864-0.pdf

Dmrecord 303233

Document Type Dissertation

Format application/pdf (imt)

Rights Gaffney, Kevin J.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA