Towards Novel Small Molecule Epigenetic Inhibitors
Total Page：16
File Type：pdf, Size：1020Kb
TOWARDS NOVEL SMALL MOLECULE EPIGENETIC INHIBITORS A Thesis Submitted by Matthias G. J. Baud In partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry November 2011 Imperial College London South Kensington Campus London SW7 2AZ DECLARATION OF ORIGINALITY I, Matthias Baud, hereby confirm that I solely produced the presented thesis under the supervision of Doctor Matthew J. Fuchter at the Department of Chemistry, Imperial College London, and that I did not use any other material than cited or known to the public domain. London, 14th October 2011 Matthias Baud Towards novel small molecule epigenetic inhibitors ABSTRACT Studies during the last decades have shown the cellular epigenetic machinery to be of major importance in the control of gene expression in living organisms. Misregulation of these mechanisms has been described in a variety of cancers, and small molecule inhibitors of enzymes involved in epigenetic gene regulation have emerged as promising agents for the development of new anticancer therapies. Naturally isolated products continue to play a significant role in anticancer drug discovery. We are interested in the structural and biological properties of the natural product Psammaplin A. Recent studies highlighted its extreme potency against both Histone Deacetylases (HDACs) and DNA Methyltransferase 1 (DNMT1) enzymes, which both play a central role in the regulation of gene expression and tumour development. In enzyme-based assays, Psammaplin A displayed potent activity (IC50s) against an HDAC extract (4.2 nM) and Dnmt1 (18.6 nM). Subsequent studies have shown Psammaplin A to exhibit significant cytotoxicity against a number of cancer cell lines. In vivo, studies showed inhibition of tumour growth in the A549 lung xenograph mouse model while maintaining low toxicity. The goal of our project is to establish a structure-activity relationship (SAR) of Psammaplin A, in order to understand the structural features responsible for such HDAC and DNMT inhibition and help us in the design of new generations of drug-like molecules for epigenetic anticancer therapies. An important number of Psammaplin A analogues could be obtained in few steps from versatile reagents. We will present our synthetic routes towards libraries of Psammaplin A analogues, results on their biological activity, and recent developments towards novel epigenetic modulators. Based on our high interest in HDACs, results of a number of other novel and early stage projects aimed at dissecting the chemical biology of HDACs will be presented. Towards novel small molecule epigenetic inhibitors ACKNOWLEDGEMENTS I would like to thank Dr. Matthew Fuchter for giving me the opportunity to perform my PhD within his group, and for his availability, enthusiasm, help and guidance during these three years, while giving me freedom in the pursuit of my research. I would like to express my gratitude to the analytical staff: Peter Haycock and Richard Sheppard (NMR), Jonathan Barton (mass spectrometry) and Andrew White (crystallography) for their help. Many thanks also to Katie Mertens, and more recently Graeme Cleugh and Rachael Youren for their kindness and for sorting out all the “scary administrative stuff” surrounding my studies here. A great thank to all the people of the Fuchter group, past and present, for their help, support and all the good moments spent in and outside the lab. Thanks to Jean-Noel Levy the “carbene guy”. Thanks to Paolo Di Fruscia for his eternal good mood and enthusiasm. Thanks also to Fanny Cherblanc and Robert Davidson for all the funny moments and for their help. Last but not least, a great thank to Marko, Nildo, Angela, Dil, Nina, Fu-Howe aka. “Jezz”, Nitipol aka. “Ken”, Tom, Loic, Zain and Nadine for their advice, good mood, and for making working in this group more than just enjoyable. It has also been a real pleasure to supervise and work with Mr Tomohiro Takeichi, Miss Yah-Ting Koh and Miss Alison Keating during their undergraduate projects within the group. Many thanks also to all the collaborators I have worked with during my PhD research. A special thank to all my crazy lab mates and friends, past and present, especially Jullien Rey and Andreas (aka. Germain), Georg Bender, Robin Aldworth (the “loser with at least a pretty face”) and Sylvain Laclef, for all the chemistry and non chemistry chats and for making the Owen Lab the best place to work at Imperial. I will also miss the people from the Barrett group, past and present, who contributed to make my time here unforgettable. I am also very grateful to Matt, Marko, Paul, Andreas, Fanny, Rob, Paul and Aniello for proof reading this thesis. Finally, I would like to thank particularly Miss Keren Abecassis, my parents, my brother and all my family for their invaluable presence and support in all situations. Towards novel small molecule epigenetic inhibitors ABBREVIATIONS °C Degree Celsius Å Angström δ Chemical shift Ac Acetyl AcOEt Ethyl acetate AcOH Acetic acid Anal. Analysis app Apparent aq. Aqueous Ar Aryl Bn Benzyl Boc tert-Butoxycarbonyl BOP Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate br Broad brsm Based on the recovery of starting material Bu Butyl Bz Benzoyl C Concentration ca. Circa Calc. Calculated Cat. Catalytic CDI Carbonyldiimidazole CI Chemical ionisation Cy Cyclohexyl d Doublet d Deuterated DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC 1,3-Dicyclohexylcarbodiimide dd Doublet of doublets DIAD Diisopropyl azodicarboxylate Towards novel small molecule epigenetic inhibitors DIPEA Diisopropylethylamine DMF Dimethylformamide DMAP 4-(Dimethylamino)pyridine DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNMT DNA methyltransferase EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide e.g. Exempli gratia EI Electron ionisation Enz Enzyme eq. Equivalent ESI Electron spray ionisation Et Ethyl FDA Food and Drug Administration g Gram h Hour HAT Histone acetyl transferase HDAC Histone deacetylase HDAH Histone deacetylase-like amidohydrolase HOBt 1-Hydroxybenzotriazole HRMS High resolution mass spectrometry Hz Hertz i Iso i.e. Id est IR Infrared J Coupling constant K Kelvin Lit. Literature m Multiplet μ Micro (10-6) M Molar or mega (106) MD Molecular dynamics Me Methyl min Minute Towards novel small molecule epigenetic inhibitors MOM Methoxymethyl mp Melting point MS Mass spectrometry or molecular sieves m/z Mass to charge ratio NBS N-Bromosuccinimide NHS N-Hydroxysuccinimide nm Nanometer nM Nanomolar NMO 4-Methylmorpholine N-oxide NMR Nuclear Magnetic Resonnance NOE Nuclear Overhauser effect NOHP N-hydroxyphthalimide pdb Protein data bank PDT Photodynamic therapy PE Petroleum ether Ph Phenyl pm Part per million Pr Propyl PS Photosensitiser PTM Post translational modification Py Pyridine q Quartet Rf Retention factor RNA Ribonucleic acid rt Room temperature s Singlet SAH S-adenosyl homocysteine SAHA Suberoylanilide hydroxamic acid SAM S-adenosyl methionine SAR Structure-activity relationship t Triplet T Temperature TBS tert-Butyldimethylsilyl TCEP Tris(2-carboxyethyl)phosphine hydrochloride Towards novel small molecule epigenetic inhibitors TFA Trifluoroacetic acid THF Tetrahydrofuran THP Tetrahydropyran TLC Thin layer chromatography TMS Trimethylsilyl Ts Toluene sulfonyl TSA Trichostatin A TSG Tumour suppressor gene UV Ultra violet VDW van der Waals ZBG Zinc binding group Towards novel small molecule epigenetic inhibitors Table of Contents INTRODUCTION 1. The genetic basis of cancer 1 2. Epigenetics 4 2.1. Overview 4 2.2. DNA methylation and histone acetylation/deacetylation 8 2.3. Aberrant DNA methylation and histone deacetylation in cancer 17 2.4. HDAC inhibitors 19 2.5. DNMT inhibitors 23 RESULTS AND DISCUSSION 1. Design and synthesis of Psammaplin A analogue libraries 26 1.1. Introduction 26 1.2. Results and discussion 34 1.21. Library design 34 1.22. Previous syntheses of Psammaplin A and other bromotyrosine metabolites 35 1.23. Synthesis of oxime and O-methyloxime containing analogues 40 1.24. New synthetic route towards Psammaplin A analogues bearing electron rich aromatic rings 46 1.25. Synthesis of ketoamide derivatives 50 1.26. Synthesis of hydrazonoamide derivatives 57 1.27. Synthesis of a Psammaplin A analogue lacking the oxime unit 58 1.28. Synthesis of hydroxamic acid analogues of Psammaplin A 59 1.29. Synthesis of acetate protected thiol analogues of Psammaplin A 61 1.3. Conclusions 61 2. Biological evaluation 62 2.1. Introduction 62 2.2. Results and discussion 62 2.21. HDAC inhibition 62 2.22. DNMT inhibition 73 2.23. Cell studies 77 2.24. Discussion 80 Towards novel small molecule epigenetic inhibitors 2.3. Conclusions 90 3. Deeper investigation of the role of the oxime unit in Psammaplin A and design of novel lead compounds 91 3.1. Introduction 91 3.2. Results and discussion 92 3.21. Computational studies 92 3.22. Synthesis of (hetero)aromatic amides as potential oxime mimics 103 3.23. HDAC inhibition 105 3.24. Further SAR around N-(2-mercaptoethyl)picolinamide 156’ 108 3.25. HDAC inhibition 113 3.3. Curren/future work and conclusions 115 4. Towards photoswitchable HDAC inhibitors 117 4.1. Introduction 117 4.2. Results and discussion 120 4.21. Design of photochromic HDAC inhibitors 120 4.22. Synthesis of azobenzene-hydroxamic acid conjugates 122 4.23. HDAC inhibition 125 4.24. Photophysical study of 201 and 202 125 4.25. Current and future work 130 4.3. Conclusions 135 5. Development of new probes for a Fluorescence Resonance Energy Transfer (FRET) for histone deacetylases 136 5.1. Introduction 136 5.2. Results and discussion 139 5.21. Design of the fluorescent conjugates 139 5.22. Synthesis of target compounds 141 5.3. Preliminary results and conclusions 141 6. Chemoproteomics of HDAC8 143 6.1. Introduction 143 6.2. Synthesis of a polymer supported PCI-34051 derivative 145 Towards novel small molecule epigenetic inhibitors EXPERIMENTAL 1. Design and synthesis of Psammaplin A analogue libraries 149 2. Biological evaluation 196 3. Deeper investigation of the role of the oxime unit in Psammaplin A and design of novel lead compounds 201 4.