ASPECTS OF THIONITRITES AND NITRIC OXIDE IN CHEMISTRY AND BIOLOGY A Thesis Presented by Marta Cavero Tomas In Partial Fulfilment of the Requirements for the Award of the Degree of DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF LONDON Christopher Ingold Laboratories, Department of Chemistry, University College London, London WC IN OAJ October 1999 ProQuest Number: 10797749 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 com plete 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 10797749 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT This thesis is divided into three parts: Part one is comprised of six chapters and provides a topical review of the main aspects of the chemistry and biology of nitric oxide and of thionitrites. The first chapter is a general introduction to the topic. The second chapter reviews the biology of nitric oxide. The third chapter provides a survey of some of the known chemistry of nitric oxide, with particular emphasis on those aspects which might be relevant in biological systems. The fourth chapter describes the biology of thionitrites in relation to NO. The fifth chapter describes in detail the chemistry of thionitrites, emphasising both those aspects which might be of biological importance and those which make the compounds of potential use in organic synthesis. The sixth chapter states the aims of the present project in the context of the current state of the field, as discussed in the preceding sections of the review. Part two describes the work that has been carried out during this thesis and is divided into 6 chapters. The first three chapters describe the initial results of our studies aimed at finding novel potential uses of thionitrites in organic synthesis, focusing on the thermal or photochemically induced addition of thionitrites onto olefins. Chapter 1 describes the synthesis of unsaturated thionitrites. Chapter 2 reports on the intra­ molecular cyclisation of thionitrites onto alkenes and its applications for the synthesis of episulfides and thiofiiran compounds. Chapter 3 examines the inter-molecular version of this reaction, with particular emphasis on reactions involving trityl thionitrite, which was chosen as a model of a stable tertiary thionitrite. This thionitrite has been shown to add very efficiently to alkenes which contain an electron withdrawing or aromatic group and to conjugated dienes. The products obtained are the corresponding a-oximino sulfides. Chapters 4 and 5 describe the results obtained in studies towards achieving a better understanding of the reactivity of thionitrites in biological systems. Chapter 4 provides additional insight into the possible mechanism of NO release from bio-active thionitrites in vivo, by comparison of the biological activity of enantiomeric pairs of thionitrites in simple rat-artery models. On the other hand, the results discussed in Chapter 5 seem to indicate that a direct thionitrite-disulfide exchange reaction in solution systems which model physiological conditions does not take place. Finally, in Chapter 6 , a series of overall conclusions have been drawn. Part three provides a formal description of the experimental results and procedures employed throughout this work and is divided into 6 chapters. The first chapter describes the general procedures. The remaining five chapters describe the experimental details corresponding to the work described in Chapters 1 to 5 of the second part. 1 CONTENTS Page Abstract. 1 Contents. 2 Acknowledgements. 5 Index of abbreviations and acronyms. 6 Part one: Introduction. 10 Chapter 1: General Introduction. 11 Chapter 2: Nitric Oxide in Biology. 12 2. 1 . The nitric oxide story: an overview. 12 2. 2. NO biosynthesis, NO Synthases and the detection of NO. 1 2 2. 3. The biological actions of NO. 16 2. 4. Non-enzymatic production of NO in humans. 18 2. 5. Soluble Guanylate Cyclase: the NO receptor. 19 2. 6 . The clinical aspects of NO. 20 Chapter 3: Nitric Oxide in Chemistry. 2 1 3. 1. Introduction. 21 3. 2 . Physico-chemical properties and synthesis. 2 1 3. 3. NO as a radical. 2 1 3. 4. NO as a nucleophile or electrophile. 25 3.5. Reaction with alkenes and dienes. 26 3. 6 . Reactions with metals: co-ordination chemistry of NO. 31 3. 7. Redox chemistry of NO. 32 3. 8 . The chemistry of the redox activated forms of NO:NO+ and NO”. 34 Chapter 4: Thionitrites in Biology. 36 4. 1. Introduction. 36 4. 2. Thionitrites as endogeneous NO carriers in vivo. 36 4. 3. Thionitrites in medicine: exogenous NO-releasing drugs. 37 4. 4. Thionitrites as intermediates in regulatory processes in vivo. 40 2 Chapter 5: Thionitrites in Chemistry. 41 5. 1 . General properties. 41 5. 2. Reactivity of thionitrites: introduction and generalities. 42 5.3. Preparation of thionitrites. 45 5. 4. Thermal decomposition of thionitrites. 48 5.5. Photochemistry of thionitrites. 50 5.6. Metal-catalysed reactions of thionitrites. 53 5. 7. Thionitrites, thiols, sulfides, disulfides and other sulfur derivatives. 63 Chapter 6 : Conclusive Remarks and Aims. 75 Part two: Results and Discussion. 76 Chapter 1: Synthesis of Allylic Tertiary Thionitrites and of Primary and Secondary Thiols for the In Situ Preparation of Primary and Secondary Thionitrites. 77 1.1. Introduction. 77 1.2. Preparation of tertiary allylic thionitrites. 79 1.3. Preparation of primary and secondary thiols for the in-situ. preparation of primary and secondary unsaturated thionitrites. 97 Chapter 2: Thermal Decomposition of Alkene-Containing Thionitrites. 99 2. 1. Introduction. 99 2. 2. Thermal decomposition of the 6 -membered cyclic thionitrite series 158-161. 100 2. 3. Decomposition of 5-membered cyclic thionitrites 162 and 163. 108 2. 4. Decomposition of cyclic thionitrite 167, containing. an endocyclic double bond. 109 2. 5. Decomposition of aromatic ring-containing thionitrites 166, 185 and 186. 110 2. 6 . Decomposition of linear thionitrites 164, 165 and 192. 114 2. 7. Photochemical vs. thermally induced decomposition of unsaturated thionitrites. 118 Chapter 3: Inter-molecular Addition of Thionitrites onto Alkenes. 119 3. 1. Introduction. 119 3. 2. Preliminary studies. 119 3.3. Substrate scope: initial studies. 1 2 2 3. 4. The possible reaction mechanism and further optimisation. 124 3 3.5. Extending the applicability of the reaction. 139 3. 6 . A case study: the reaction of trityl thionitrite with A,A-diallylacrylamide. 145 3. 7. Extension of the limits of the reaction to other thionitrites. 146 Chapter 4: Synthesis and Biological Evaluation of Enantiopure Thionitrites. 149 4. 1. Introduction. 149 4. 2 . Substrate preparation. 149 4. 3. Biological tests. 153 Chapter 5: A Study of the Thionitrite-Disulfide Exchange Reaction. 156 5. 1. Introduction. 156 5. 2 . Generalities of our work and choice of substrates. 158 5. 3. Substrate preparation. 159 5. 4. Initial studies: exchange processes between SNAP and GSSG. 161 5.5. Exchange studies with other substrates. 167 Chapter 6 : Conclusions. 170 Part three: Experimental section. 173 Chapter 1: General procedures. 174 Chapter 2: Synthesis of Allylic Tertiary Thionitrites and of Primary and Secondary Thiols for the In Situ Preparation of Primary and Secondary Thionitrites. 176 Chapter 3: Thermal Decomposition of Alkene-Containing Thionitrites. 214 Chapter 4: Inter-molecular Addition of Thionitrites onto Alkenes. 229 Chapter 5: Synthesis and Biological Evaluation of Enantiopure Thionitrites. 248 Chapter 6 : A Study of the Thionitrite-Disulfide Exchange Reaction. 267 Appendices. 282 Appendix 1 : X-ray Crystallographic Data. 283 Appendix 2: Structure Index. 312 References. 317 4 ACKNOWLEDGEMENTS Firstly, thanks must go to my supervisor, Willie Motherwell, for his guidance and encouragement over the last three years and for giving me the opportunity of completing my PhD in London. Thanks to Santi for patiently reading and correcting this thesis, for constant advice and, above all, for his friendship. I would also like to thank all past and present members of the WBM research group for creating such an inspiring atmosphere. Thanks to all for being so special in your own individual ways; I have learned something from each of you. Special thanks go to Pierre and Yvan for their friendship; to Ray for being such a patient and tolerant benchmate and to Tilly for all the moments shared, in and outside the lab, and for teaching me the logic of the English language. This work would have not been possible without the assistance of the very dedicated technical staff at University College London: Steve Corker for his patience and advice with HPLC and HPLC/MS and for mass spectra, John Hill for mass spectra, Alan Stones and Jill Maxwell for microanalyses, Jorge Outerino for NMR. Thanks also to M. Cocksedge and his staff at the School of Pharmacy for mass spectra and accurate mass measurements and to Dr. D. Tocher (UCL) and Ms A. M. Z. Slawin (Loughborough University) for the X-Ray structure determinations. Thanks to the chemists at the Wolfson Institute of Biomedical Research for their help and ideas; particular thanks go to Dr. David Selwood and Dr. David Madge for listening; to Dana and Marianne for their help with the prep-HPLC. Thanks to Patricia for her friendship; to Dr. Adrian Hobbs, at the Rayne Institute, for the biological work. For funding and ideas I am grateful to Professor Pierre Potier at the CNRS, Paris. Above all, I thank Gabriel and my parents and brothers, for their love and unconditional support.