Design, synthesis and testing of β-strand mimics as protease inhibitors . A thesis submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Biochemistry at the University of Canterbury by Steve Aitken . University of Canterbury 2006 i TABLE OF CONTENTS TABLE OF CONTENTS i PUBLICATION LIST v ABSTRACT vi ABBREVIATIONS viii ACKNOWLEDGEMENTS xi CHAPTER ONE: INTRODUCTION 1.1: Introduction to peptidomimetics and proteases 1 1.2: Protease inhibitors in the clinic 2 1.3: Importance of the β-strand in protease inhibitor design 5 1.4: Calpain as the prototype protease for the testing of β-strand mimics 8 CHAPTER TWO: DESIGN AND SYNTHESIS OF ACYCLIC β-STRAND MIMICS 2.1: Current calpain inhibitors in the scientific literature 15 2.2: Use of molecular modelling to design β-strand calpain inhibitors 19 2.3: Patentability evaluation 23 2.4: Design, synthesis and testing of N-heterocyclic dipeptide aldehyde 2.5: calpain inhibitors 26 2.5: Conclusions and future work 30 CHAPTER THREE: DESIGN AND SYNTHESIS OF CONFORMATIONALLY CONSTRAINED β-STRAND MIMICS 3.1: Conformational constraint of peptidomimetics 35 3.2: History of RCM 36 3.3: Mechanism elucidation and metathesis catalyst development 37 3.4: The advent of well defined single-component catalysts 39 3.5: Design and synthesis of N-N conformationally constrained SJA analogues 48 3.6: Design and synthesis of β-amino acid C-N carbocycles 53 ii 3.7: Conclusions and future work 60 CHAPTER FOUR: DESIGN AND SYNTHESIS OF MACROCYCLIC β-STRAND MIMICS 4.1: Use of macrocyclisation to constraint a peptidomimetic in a 68 4.2: β-strand conformation 4.2: Generation of an in-silico library of macrocyclic β-strand templates 69 4.3: Conformational analysis of the in-silico library 73 4.4: Synthesis of 17-membered Tyr-aa-Gly based macrocycles 75 4.5: Microwave assisted RCM 76 4.6: Effect of microwave irradiation on the E/Z isomer ratios 78 4.7: Synthesis of 16-membered ring Tyr-Val-Gly macrocycle 79 4.8: Attempted synthesis of 18-membered ring Tyr-Leu-Gly macrocycle of using 81 homo-allyl glycine 4.9: Synthesis of 18-membered ring Tyr-Leu-Gly macrocycle 82 4.10: Synthesis of 17-membered ring Tyr-Leu-Gly, Tyr-Phe-Gly, 84 4.11: Tyr-cyclohexane-Gly macrocycles 4.11: Synthesis of 17-membered ring Tyr-Val-β-Gly macrocycle 85 4.12: Synthesis of 19-membered ring Tyr-Val-Cys-macrocycle 86 4.13: Synthesis of 19-membered ring Tyr-Val-Ser-macrocycle 87 4.14: Attempted synthesis of 23-membered ring Tyr-Val-Tyr-macrocycle 88 4.15: Attempted synthesis of 21-membered ring Tyr-Val-Gln-macrocycle 89 4.16: Effect of substrate chelation on RCM 90 4.17: Design and synthesis of a model system to understand chelation 93 4.18: effects in RCM 4.18: Strategies to avoid catalyst deactivation from substrate chelation 95 4.19: Use of a Lewis acid as an additive in RCM reactions 95 4.20: Chain length alteration to avoid catalyst deactivation from substrate chelation 97 4.21: Conclusions and further work 100 iii CHAPTER FIVE: DESIGN AND SYNTHESIS OF MACROCYCLIC CALPAIN INHIBITORS 5.1: Use macrocyclic calpain inhibitors as anti-cataract agents 106 5.2: Use of molecular modelling to design macrocyclic calpain inhibitors 108 5.3: Physicochemical analysis of the in-silico library 111 5.4: Synthesis of 17-membered ring Tyr-xx-Gly macrocyclic calpain inhibitors 114 5.5: Synthesis of 16 and 18-membered Tyr-xx-Gly macrocyclic calpain inhibitors 116 5.6: Synthesis of various N-substituted 17-membered Tyr-xx-Gly macrocycles 118 5.7: Synthesis of unsaturated 17-membered Tyr-Val-Gly macrocycle 119 5.8: Synthesis of 17-membered ring Tyr-Val-Gly β-amino acid macrocycle 120 5.9: Synthesis of 19-membered ring Tyr-Val-Cys macrocyclic calpain inhibitors 120 5.10: Calpain inhibition results of the macrocyclic β-strand mimics 122 5.11: Conclusions and future work 127 CHAPTER SIX: SYNTHETIC OPTIMISATION AND IN-VIVO EVALUATION OF THE BEST CALPAIN INHIBITORS 6.1: Ovine in-vivo cataract model 131 6.2: Synthetic optimisation of 5.17 (Cat-812) 133 6.3: Synthesis of 2.13 (CAT-0059) for in-vivo evaluation 135 6.4: Results of intravitreal trial of 2.13 (Cat 0059) 136 6.5: In-vivo sheep trial results of 2.13 (CAT-0059) 137 6.6: Synthetic optimisation of 5.14 (CAT-811) 141 6.7: In-vivo sheep trial results of 5.14 (CAT-811) 144 6.8: Conclusions and further work 147 CHAPTER SEVEN: APPLICATION OF RING CLOSING METATHESIS TO THE SYNTHESIS OF PEPTIDOMIMETICS 7.1: Synthesis of α-α conformationally constrained peptidomimetic lactams 150 7.2: Synthesis of C-N conformationally constrained β-amino acid lactams 156 7.3: Conclusions and future work 157 iv CHAPTER EIGHT: EXPERIMENTAL 8.1: General Methods and Experimental Procedures 161 8.2: Experimental work described in chapter two 171 8.3: Experimental work described in chapter three 181 8.4: Experimental work described in chapter four 202 8.5: Experimental work described in chapter five 242 8.6: Experimental work described in chapter six 257 8.7: Experimental work described in chapter seven 260 APPENDICES APPENDIX 1: Calpain inhibition assay 270 APPENDIX 2: In-vivo sheep trial drug formulations 273 v Publication list Work in this thesis has been published in the following articles; Abell, A.D; Coxon, J.M; Jones, M.A; Neffe, A.T; Aitken, S.G; Nikkel, J.M; Jones, S; McNabb, S.B; PCT-550631 Aitken, S.G; Chemistry in New Zealand, October 2006, 88-92 (Invited review) Abell, A.D; Aitken, S.G; McNabb, J.B; Gardiner, J; J.Organometallic.Chem, 2006, in press Jones, M.A; Neffe, A.T; Stuart, B.G; Cain, T.P; Coxon, J.M; Aitken, S.G; Nikkel, J.M; Payne, R.J; Lee, H.Y. Y; Morton, J.D; Andrew, A.D; J.Med.Chem, 2006, submitted Abell, A.D; Coxon, J.M; Miyamoto, S; Jones, M.A; Neffe, A.T; Aitken, S.G; Nikkel, J.M; Lee, H.Y. Y; Morton, J.D; Robertson, L. J. G; Muir, M.S; PCT NZ 547303 Aitken, S.G, ed. Happer, A.R; Chemistry in New Zealand, September 2005 Aitken, S.G, Abell. A.D; Aus.J.Chem, 2005, 58, 3-13 vi Abstract Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC50 of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC50 against calpain II of 27 nM. The best non- electrophilic warhead macrocycle (5.13) had an IC50 against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.