W LIVERPOOL Synthesis and Structure-Metabolism Relationships of Halogenated Carbamazepine Analogues Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Emma-Claire Elliott 30th September 2011 Acknowledgem en ts I would like to begin by thanking my supervisors, Dr, Andrew V. Stachulski and Prof. B. Kevin Park, for giving me the opportunity to study with them. I am also indebted to Prof. Rick Cosstick for all his assistance, support, and encouragement in the final years of the PhD. I also express gratitude to Dr. Sophie L. Regan and Dr. James L. Maggs of the MRC Centre for Drug Safety Science for their close collaboration with the structure-metabolism work, and for their assistance in determining the purity of the carbamazepine analogues. Thanks go to the members of the Stachulski group, both past and present, and to the members of the Aspinall and Greeves group for their excellent company in the lab and for making Liverpool a thoroughly enjoyable place to work for the past for years. Special thanks go to Laura, Helen, Xiao Li, and Chandra for all their help, friendly discussions and camaraderie To all my friends, old and new, in no particular order: Jacqui, Ev, Oliver, Matt, Neil, Lyndsey, Ste, Robin and Michelle for always being on hand with a cup of tea or a pint on a Friday night. I am extremely thankful to my family, to whom I dedicate this thesis. My parents Alex and Jean, and my brother Adam, for not disowning me. For their love, support, inspiration and encouragement through good times and bad. Finally I thank Tom for always going the extra mile and especially for his forbearance over the late nights, early starts, and long weekends. Funding from the EPSRC is also gratefully acknowledged. i Abstract Adverse drug reactions are a significant burden on industry and healthcare providers. They account for approximately 5 % of hospital admissions and are a considerable cause of morbidity and mortality in patients. While it is widely considered that an individual's susceptibility to idiosyncratic reactions is caused by a variety of factors; ADRs are thought to be linked to the formation and accumulation of reactive drug metabolites rather than the parent drug. Of the patients administered carbamazepine 30-50 % are subject to the development of some form of adverse drug reaction. Carbamazepine is metabolized extensively in vivo and hypersensitivity reactions have been hypothetically linked to the formation of chemically reactive arene oxide or iminoquinone metabolites. In order to understand the mechanisms behind such ADRs it is important to synthesize a variety of chemical probes to observe changes in pharmacokinetic and pharmacodynamic properties of the compounds. The overall theme of this thesis is the synthesis and the examination of the structure-metabolism relationships for halogenated analogues of carbamazepine, It is divided into three main sections; a general introduction introduces the reader to the basics of drug metabolism from how drugs are metabolized to the implications of halogen substitution on the metabolism of drugs. It next covers the synthetic strategies employed in the formation of halogenated carbamazepine analogues before ending on a discussion of the key findings of the structure-metabolism relationships that may be derived from in vitro investigations 11 Publications Elliott, E.-C.; Bowkett, E. R.; Maggs, J. L.; Bacsa, J.; Park, B. K,; Regan, S. L; O'Neill, P. M.; Stachulski, A. V., Convenient Syntheses of Benzo-Fluorinated Dibenz[b,f]azepines: Rearrangements of Isatins, Acridines, and Indoles. Organic Letters 2011. Paper in press Emma-Claire Elliott, Sophie L. Regan, James L. Maggs, Elizabeth R. Bowkett, Laura Parry, Dominic P. Williams, B. Kevin Park, and Andrew V. Stachulski: Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives. Manuscript in Preparation Abbreviations 9-AC 9-Acridine carbaldehyde ABT 1-Aminobenzotriazole AC2O Acetic anhydride ACBN l,r-Azobis[cyclohexanecarbonitrile) AcCl Acetyl chloride Acetone-dfi Deuterated acetone ADR Adverse Drug Reaction AI Acridine AO Acridone ATR-IR Attenuated Total Reflection Infrared Spectroscopy br. s. Broad singlet CBZ Carbamazepine CBZ-2,8-Br 2,8-DiBromo carbamazepine CBZ-2,8-Br E 2,8-DiBromo carbamazepine-10,11-epoxide CBZ-2,8-Br -A-gluc 2,8-DiBromo carbamazepine-iV-glucuronide CBZ-2,8-BrE-A-gluc 2.8-DiBromo carbamazepine-10,11-epoxide-IV-glucuronide CBZ-2,8-Cl 2.8-DiChloro carbamazepine CBZ-2,8-Cl E 2,8-DiChloro carbamazepine-10,11-epoxide CBZ-2,8-Cl E-A-gluc 2,8-DiChloro carbamazepine-10,11-epoxide-N-glucuronide CBZ-2,8-Cl -JV-gluc 2.8- DiChloro carbamazepine-iV-glucuronide CBZ-2.8-F 2.8- DiFluoro carbamazepine CBZ-2,8-F -iV-gluc 2,8-DiFluoro carbamazepine-Af-glucuronide CBZ-2.8-FE 2,8-DiFluoro carbamazepine-10,11-epoxide CBZ-2,8-FE-JV-gluc 2,8-DiFluoro carbamazepine-10(ll-epoxide-lV-glucuronide CBZ-2-Br 2-Bromo carbamazepine CBZ-2-BrE 2-Bromo carbamazepine-10,11-epoxide CBZ-2BrE-A-gluc 2-Bromo carbamazepine-10,11-epoxide-Af-glucuronide CBZ-2-Br-JV-gluc 2-Bromo carbamazepine-iV-glucuronide CBZ-2-C1 2-Chloro carbamazepine CBZ-2-C1E 2-Chloro carbamazepine-10,11-epoxide CBZ-2-ClE-iV-gluc 2 -Chloro carbamazepine-10,1 l-epoxide-N-glucuronide CBZ-2-Cl-iV-gluc 2-Chloro carbamazepine-Af-glucuronide CBZ-2-F 2-Fluoro carbamazepine CBZ-2-F E 2-Fluoro carbamazepine-10,11-epoxide CBZ-2-F E-iV-gluc 2-Fluoro carbamazepine-10,11-epoxide-Af-glucuronide iv CBZ-2-F -W-gluc 2-Fluoro carbaraazepine-W-glucuronide CBZ-2-F.OSO3 2-Fluoro carabamazepine O-sulfonate CBZ-2-0H 2-Hydroxy carbamazepine CBZ-3-0H 3-Hydroxy carbamazepine CBZDHD Carbamazepine-10,ll-dihydrodiol CBZE Carbamazepine-10,11-epoxide CBZ-JV-Gluc Carbamazepine-N-glucuronide CBZ-SG Carbamazepine-glutathione conjugate CC14 Carbon tetrachloride CDCIs Deuterated chloroform CF3SO3H Trifluromethyl sulfonic acid CH2CI2 Dichloromethane (methylene chloride) CHCls Chloroform Cl Chemical Ionisation d Doublet dd Doublet of doublets ddd Doublet of doublets of doublets DHD Di-hydrodiol DHOH Dihydroxylated DMAP Dimethyl amino pyridine DMSO Dimethylsulfoxide DMSO-f/g Deuterated dimethylsulfoxide dt Doublet of triplets El Electron Ionisation eq Equivalents Et20 Diethyl ether EtOAc Ethyl acetate EtOH Ethanol GSH Glutathione GST Glutathione-S-transferase h Hours HBr Hydrogen bromide HC1 Hydrochloric Acid HLM Human Liver Microsomes HPLC High-Pressure Liquid Chromatography I2 Iodine V IDB Iminodibenzyl IR Infra-Red spectroscopy ISB Iminostilbene ISB-2-0H 2-Hydroxy iminostilbene K2CO3 Potassium carbonate KOCN Potassium isocyanate KOH Potassium Hydroxide LC-MS High Pressure Liquid Chromatography-Mass Spectrometry LC-UV High Pressure Liquid Chromatography (with UV detection) m (NMR) multiplet; (IR) medium MeCN Acetonitrile min Minutes mp Melting Point MPO Myeloperoxidase Na2S04 Sodium sulfate NaOCN Sodium isocyanate NaOH Sodium Hydroxide NAPQI IV-acetyl para-benzo quinoneimine NBS N-bromo succinimide NCS IV-chloro succinimide NEt3 Triethylamine NH4CI Ammonium chloride NIH National Institutes of Health NMR Nuclear Magnetic Resonance P450 Cytochrome P450 PEG 400 Polyethylene glycol (molecular weight = 400) PI1CF3 Trifluoromethyl toluene pKa acid dissociation constant PPA Polyphosphoric acid P£-Bu3 Tri-tert-butyl phosphine q Quartet s (NMR) singlet (IR) strong SAR Structure-Activity Relationship Si02 Silica Gel SKF Proadifen hydrochloride t Triplet VI TFA Trifluoroacetic acid THF Tetrahydro furan TIC Total Ion Current TLC Thin layer chromatography w Weak w/v Weight to volume WHO World Health Organisation XIC Extracted Ion Current Structural Abbreviations Abbreviation Structure CBZ-2-OH CBZ-3-OH AI AO CBZ CBZE CBZE-JV-gluc OH OH vm CBZ-W-gluc Glucuronic acid GSH (glutathione] ix Contents Acknowledgements i Abstract ii Publications iii Abbreviations iv Structural Abbreviations viii Contents x 1. General Introduction 2 1.1 Drug metabolism 2 1.1.1 Cytochrome P450 and oxidative metabolism 5 1.1.2 Cytochrome P450 Catalytic cycle 6 1.1.3 Other forms of oxidative metabolism 9 1.2 Carbamazepine 13 1.2.1 Carbamazepine Metabolism 14 1.3 Implications of Halogen Substitution 20 1.3.1 Physicochemical properties of Carbon-Halogen bonds 20 1.3.2 Metabolic dehalogenation 27 1.3.3 Halogens and the National Institutes of Health (NIH) shift 31 1.4 Conclusions 35 1.5 References 37 2. Synthesis of Halogenated Carbamazepine Derivatives 48 2.1. Introduction: Dibenz[/E>,/|azepines and related ring systems 48 2.2 Synthesis of dibenzfh/Jazepines by ring closing reactions 50 2.3 Synthesis of dibenz[hj]azepines by ring expansion reactions 56 2.4 Synthetic project aims 61 2.5 Direct Electrophilic Halogenation of Iminodibenzyl 62 2.5.1 Direct electrophilic bromination with N-halo succinimides 62 2.5.2 Incorporation of the 10,11-double bond 69 2.5.3 Radical Bromination of the 10-11 bond 72 2.6 Synthesis from Halogenated Building Blocks 80 2.6.1 Synthesis of N-aryl Indoles 80 2.6.2 Polyphosphoric acid cyclisation 88 2.7 Incorporation of the carboxamide moiety 96 2.7.1 Phosgenation 96 x 2.7.2 Isocyanates 100 2.8 Conclusions 108 2.9 References 109 3. Results and Discussion: Structure-Metabolism Relationships of Halogenated Carbamazepine Analogues 117 3.1 Introduction 117 3.2 Project Aims 118 3.3 Identification of CBZ Metabolites in freshly isolated hepatocyte suspensions 119 3.3.1 Rat vs. Mouse metabolism 119 3.3.2 Role of Cytochromes P450 in CBZ metabolism in hepatocyte suspensions 127 3.3.3 Structure-metabolism relationships