The Design and Synthesis of Duocarmycin Prodrugs
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The Design and Synthesis of Duocarmycin Prodrugs By Natalia Ortuzar Kerr University of London School of Pharmacy Ph.D. May 2006 The School of Pharmacy University of London 29-39 Brunswick Square London, WCIN lAX ProQuest Number: 10104888 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104888 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Plagiarism Statement This thesis describes research conducted in the School of Pharmacy, University of London between December 2002 and November 2005 under the supervision of Dr Mark Searcey and Prof Laurence Patterson. I certity that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this dissertation that has already appeared in publication. q S" /cX o ^ o ib Signature Date 11 Abstract Abstract The design of targeted prodrugs has become a viable aim in cancer chemotherapy, as research moves away from classical cytotoxic agents. The development of various compounds that exploit tumour physiological and protein expression profiles for targeted activation is reviewed. Cytochrome P450 enzymes, either overexpressed intratumourally or introduced by gene therapy, have potential for activation of deshydroxy prodrug analogues of the duocarmycins via bioxidation of the pharmacophore. The duocarmycins are potent antitumour antibiotics capable of sequence-selective alkylation of DNA. They bind in the minor groove and exhibit high selectivity towards AT rich sequences. Herein, the synthesis of deshydroxy-seco-CI-MI 59 and the fluorinated derivative 105 by two complementary routes is described. These prodrugs are based on the m inim um potent pharmacophore of the duocarmycins (Cl). Initial investigations of the 5-exo-trig free radical cyclisation with catalytic BugSnH, using polymethylhydrosiloxane (PMHS) as a hydride source gave good yields. This was further improved by the use of tris(trimethylsilyl)silane (TTMSS) in stoichiometric quantities. An alternative route involving alkylation of a 2 -fluoronitrobenzene with dimethyl malonate was also developed. Functional group interconversions allowed a 5-exo-tet nucleophilic substitution to form the A ring. This synthesis allows the application of an enzyme- based resolution of an early intermediate without downstream racémisation. Deprotection and coupling to 5 -methoxyindole-2-carboxylic acid 60 gave an extended prodrug in good yield. Duocarmycin SA 19 is among the most potent compounds in this class of natural products. Synthesis of the deshydroxy-^eco-DSA pharmacophore was also achieved using the 5-exo-trig free radical cyclisation. A methoxy prodrug 126, where the activity is removed by ether formation rather than removal of the hydroxyl group, was designed based on known substrates of CYPIBI. Based on the seco-CQ pharmacophore of natural products duocarmycin B] 21 and C 2 2 2 , the novel synthesis was achieved via direct allylation of an aniline derivative followed by dihydroxylation to give a 1,2-diol. Selective tosylation of the primary alcohol and protection of the secondary hydroxyl group allowed cyclisation to the 6 - membered ring exclusively. Preliminary biological results are also given. Ill Acknowledgements Acknowledgements I would like to acknowledge my supervisors Dr Mark Searcey and Prof. Laurence Patterson for their assistance and guidance throughout my PhD. Special thanks to Mark for all of his words of wisdom and encouragement; the unwavering support was greatly appreciated. Thanks to all of the lab members past and present for their help, advice and friendships. Thank you finally to my family and friends for their continued support and patience these past 3 years. I would also like to acknowledge the EPSRC and the School of Pharmacy for funding. IV Contents Table of Contents Title page i Plagiarism statement ii. Abstract iii. Acknowledgements iv. Table of contents v List of figures viii. List of schemes xii. List of tables xv. Abbreviations xvi. Chapter 1 Literature review 1. 1.1 Cancer chemotherapy 2. 1.2 Development of antitumour prodrugs 4. 1.2.1 Nitrogen mustard prodrugs 4. 1.2.2 Antbracyclme prodrugs 9. 1.3 CC-1065 and the duocarmycins 12. 1.3.1 The DNA alkylation reaction 15. 1.4 Clinical candidates of the duocarmycins 26. 1.4.1 Duocarmycin drug candidates 26. 1.4.2 Duocarmycin prodrug candidates 28. 1.5 Conclusions 35. Chapter 2 The design of a duocarmycin prodrug 36. 2.1 Introduction 37. 2.2 Cytochrome P450 38. 2.3 The CYP activated duocarmycin prodrug rationale 39. 2.4 The design of a CYP activated duocarmycin prodrug 41. 2.4.1 Deshydroxy prodrugs 41. 2.4.2 Phenolic ether prodrugs 42. 2.5 Preliminary results and biological proof of concept 43. 2.6 Thesis aims 44. Contents Chapter 3 The synthesis of deshydroxy-sec<?-CI-MI and a fluoro 47. derivative: A ring formation using 5-exo-triga radical cyclisation 3.1 Introduction 48. 3.2 Synthesis of deshydroxy-^gco-CI analogues via an intramolecular 54. radical cyclisation of a tethered vinyl chloride 3.2.1 The synthesis of the 7V-aIkylated precursors 55. 3.2.2 The radical ring closure reaction 59. 3.2.3 Synthesis of the extended analogues 64. 3.3 Conclusions 69. Chapter 4 The synthesis of deshydroxy-^gco-CI and a fluoro 70. derivative: A ring formation using 6-exo-teta nucleophilic substitution reaction 4.1 The design of a novel synthetic route to A^-boc-deshydroxy-s^co-CI 71. 4.2 Synthesis of 7V-boc-deshydroxy-^gco-CI derivatives 72. 4.2.1 Synthesis of A^-boc-deshydroxy-^eco-CI and the fluoro 73. derivative 4.2.2 Synthesis of the extended analogue, deshydroxy-^eco-CI-MI 84. 4.2.3 Enzymatic induction of stereospecificity 89. 4.3 Conclusions 90. Chapter 5 Synthesis of duocarmycin derived quinoline prodrug 91. 5.1 Introduction 92. 5.2 The design of a quinoline prodrug -discussion of the disconnection 94. rationale and stereo synthetic control 53 The synthesis of a quinoline prodrug 96. 5.3.1 Synthesis of the acyclic precursor 97. 5.3.2 Intramolecular ring closure to the seco-CQ methoxy prodrug 113. 5.4 The synthesis of the active drug 117. 5.4.1 Strategic redesign of the synthetic route 117. 5.4.2 Synthesis of an active seco-CQ drug 119. 5.5 Preliminary biological results 121. VI Contents 5.6 Conclusions 129. Chapter 6 Synthesis of deshydroxy-sec<>-DSA, a duocarmycin SA 130. prodrug analogue 6.1 Introduction 131. 6.2 Review of synthetic routes to duocarmycin SA 131. 6.3 Design of a duocarmycin SA prodrug-disconnection rationale 138. 6.4 The synthesis of A-boc-deshydroxy-seco-DSA 140. 6.4.1 Initial synthesis 140. 6.4.2 Revised synthesis 147. 6.5 Conclusions 152. Chapter 7 Conclusions and future work 153. 7.1 The deshydroxy-^gco-CI prodrugs 154. 7.2 The CQ prodrug 154. 7.3 The deshydroxy-^gco-DSA prodrug 155 7.4 Stereospecificity 155. Chapter 8 Experimental 156. 8.1 General experimental information 157. 8.1.1 Physical characterisation and spectroscopic techniques 157. 8.1.2 Chromatographic techniques 157. 8.1.3 Reagent, solvent and apparatus preparation 157. 8.2 Experimental: Chemistry 159. 8.3 Experimental: Biology 194. References 195. Quick Reference Sheet Back pocket YU List o f Figure, Schemes and Tables List of Figures Chapter One 1.1 The nitrogen mustards 5 1.2 The alkylation of DNA by mechlorethamine. 5 13 In vivo metabolism of cyclophosphamide 6 1.4 Examples of hypoxia activated nitrogen mustard prodrugs 7 1.5 The activation of a prodrug in a hypoxic cell 8 1.6 The activation of PR-104 9 1.7 The activation of AQ4N to AQ4 by cytochrome P450 11 1.8 CC-1065 and the duocarmycins 12 1.9 Natural products, year of isolation and streptomyces sp. 13 1.10 Alkylation of an adenine residue by a duocarmycin 13 1.11 Examples of minor groove binders 16 1.12 The amphipathic nature of CC-1065 16 1.13 Derivatives incorporating the minor-groove binding subunits of the 17 natural products 1.14 The co m m on pharmacophores of CC-1065 and the duocarmycins 18 1.15 Normal, reversed and sandwiched duocarmycin SA analogues 21 1.16 Key structures in the shape-dependant catalysis studies 24 1.17 The Fukuda Library of A-methylpiperazirylcarbamoyl prodrugs 31 Chapter Two 2.1 Structure/activity series 39 2.2 The activating positions of a deshydroxy- 5eco-CI prodrug 40 2.3 Design of a simplified duocarmycin derived prodrug 42 2.4 A structural comparison of oestradiol, ethoxyresorufin and the phenohc 43 ether prodrug 2.5 The observed metabolites of deshydroxy- 5eco-CI-MI after incubation 44 with human hver microsomes Chapter Three 3.1 Disconnection routes to 5eco-CI 48 Vlll List o f Figure, Schemes and Tables 3.2 Retrosynthesis of the prodrug deshydroxy- 5eco-CI-MI 54 3.3 Retrosynthetic analysis of the iV-Boc-deshydroxy-seco-CI derivatives 55 3.4 Mass spectrometry fragmentation of the A^-alkylated derivatives 58 3.5 a. The formation of the tributyl tin radical; b. The mechanism of the 60 radical ring closure 3.6 The catalytic cycle of BusSnH and PMHS: halogen abstraction and 62 intramolecular cyclisation followed by hydrogen abstraction and radical termination 3.7 The numbering system of the indoline analogues 64 3.8 The mechanism of amide coupling using EDC 65 3.9 The numbering system of the extended analogues including the 65 inequivalent methylene protons of the pharmacophore’s heterocycle.