A Fragment Based Approach to the Development of Novel Antibacterial Agents Inspired by the Natural Product Simocyclinone D8
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A fragment based approach to the development of novel antibacterial agents inspired by the natural product simocyclinone D8 Michael Jonathan Austin School of Pharmacy University of East Anglia A thesis submitted for the degree of Doctor of Philosophy August 2015 © “This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution.” Declaration This thesis is submitted to the University of East Anglia for the Degree of Doctor of Philosophy and has not been previously submitted at this or any University for assessment or for any other degree. Except where stated, and reference and acknowledgment is given, this work is original and has been carried out by the author alone. Michael Jonathan Austin 2 Abstract The novel mechanism of simocyclinone D8 (SD8) against DNA gyrase has inspired medicinal chemists for over a decade. The search for antibiotics with new mechanisms of action has never been more important with ever increasing prevalence of resistance. This project had three objectives. Firstly to contribute towards the total synthesis of SD8, by exploring chlorination reactions of dihydroxycoumarins and their corresponding starting reagents. Additionally, there was a need to establish a viable route towards the complex polyketide scaffold. Secondly, to generate a library of coumarins and screen for a low molecular weight fragment that could be taken forward as a lead compound. Thirdly, inspired by the bi-functional mode of action of SD8, the project sought to design and synthesise a coumarin-quinolone hybrid via a fragment-based approach. Herein we exploit ortho and para directing effects of phenolic OH groups to selectively chlorinate starting materials. However, we show chlorination of reagents prior to forming a coumarin is not a viable synthetic strategy. This is due to the reduced nucleophilicity imparted by the halogen on adjacent atoms. Consequently, this abrogates any ring closure reaction. We illustrate that a previously established Diels-Alder route can furnish a novel isomerised pericyclic adduct. This provides a good starting point for reaction optimisation and the onward enantioselective synthesis of the complex polyketide scaffold. Lastly, 27 different coumarin fragments were synthesised and assessed for biological activity. We illustrate that simple coumarins lack inhibition in supercoiling assays against DNA gyrase. Furthermore, when ciprofloxacin is attached to a linker there is an attenuation of activity. Strikingly, when a simple coumarin is joined to ciprofloxacin, via a linker there is a restoration of activity. We show DNA gyrase inhibition is via cleavage stabilisation. Moreover, we demonstrate activity is related to the coumarin structure and not the presence of an aromatic ring. 3 Acknowledgments ‘When I was a child, I spoke like a child; I thought like a child, I reasoned like a child: but when I became a man, I put away childish things’ (King James 2000 bible, 1 Corinthians 13:11) I would like to thank Professor Mark Searcey and Dr Lesley Howell for giving me the opportunity to do a PhD. Their passion for science and medicinal chemistry inspired me to embark on this scientific adventure. No one else would have put up with my musical choices in the lab! Without their expertise and guidance this wouldn't have been possible. I would also like to thank Professor Tony Maxwell for allowing me to work in his lab and for his guidance with the biological assays performed in this thesis. I also thank Steve Hearnshaw and Lesley Mitchenell for their help with the biological assays, and their unwavering patience when training me. I would like to dedicate this thesis to my mother Suzanne Austin, her continued belief; support and encouragement sustained me over the years it took to complete. I also thank my Grandmother for helping with my early years of education. I want to thank my brothers and sisters in Christ, and the fellowship I have had from them. I am grateful for God’s provision of joys, challenges and grace for growth. Lastly, but not least I want to thank my lab buddies from the Medicinal Chemistry group. The banter, music and tears have all been worth it. 4 Table of Contents Contents Declaration ......................................................................................................................................... 2 Abstract .............................................................................................................................................. 3 Acknowledgments .............................................................................................................................. 4 Contents ............................................................................................................................................. 5 Tables ................................................................................................................................................ 8 Figures ............................................................................................................................................... 8 Schemes .......................................................................................................................................... 11 Abbreviations ................................................................................................................................... 16 Chapter 1. Introduction .................................................................................................................... 20 1.0 Gram-positive and Gram-negative bacteria ............................................................................ 21 1.01 Antimicrobial resistance and mechanisms of resistance ......................................................... 22 1.02 Current clinical targets for antibiotics ....................................................................................... 27 1.03 DNA ......................................................................................................................................... 31 1.04 DNA higher order structures .................................................................................................... 34 1.05 Topology and geometry of DNA ............................................................................................... 35 1.06 DNA replication in Prokaryotes ................................................................................................ 36 1.07 Topoisomerase enzymes ......................................................................................................... 37 1.08 Type IA ..................................................................................................................................... 37 1.09 Type IB ..................................................................................................................................... 37 1.10 Type IC .................................................................................................................................... 38 1.11 Type IIA (DNA gyrase) ............................................................................................................. 39 1.12 Type IIB .................................................................................................................................... 39 1.13 Mechanism of DNA gyrase supercoiling .................................................................................. 39 1.14 Aminocoumarin antibiotics ....................................................................................................... 41 1.15 Quinolone antibiotics history and SAR ..................................................................................... 43 1.16 Quinolone mechanism of action ............................................................................................... 45 1.17 Cellular response to quinolones ............................................................................................... 49 1.18 Contribution of ROS to cell death ............................................................................................. 50 1.19 Isolation of the simocyclinone antibiotics ................................................................................. 51 1.20 Fermentation and classification of simocyclinones .................................................................. 52 1.21 Structural elucidation of SD4 and SD8 ..................................................................................... 53 1.22 Radiolabelled feeding experiments .......................................................................................... 54 1.23 SD8 a novel mode of action ..................................................................................................... 54 1.24 Crystal structure of SD8 bound to N-terminal GyrA59 ............................................................. 55 1.25 Mass spectrometry studies into SD8 binding ........................................................................... 58 1.26 Evidence of binding to Gyrase B .............................................................................................. 59 1.27 A new crystal structure gives fresh insight into SD8 binding ................................................... 60 1.28 Significance of the