Novel fenofibrate derivatives as cannabinoid receptor ligands Sarah Jane Spencer B.Sc School of Pharmacy The University of Nottingham Nottingham UK Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy May 2011 Abstract Fenofibrate is a PPAR α agonist, used to treat dyslipidemia. Unpublished work that has been previously carried out in our group has identified that fenofibrate also displays affinity for the cannabinoid receptors, CB1 and CB 2. A dual receptor ligand, with the PPAR α agonist activity of fenofibrate, combined with antagonist activity at the CB 1 receptor, or agonist activity at the CB 2 receptor, could reduce appetite, decrease plasma triglyceride levels, increase high density lipoprotein (HDL) levels, lower low density lipoprotein (LDL) levels and reduce atherosclerosis. This could enable the multi- symptomatic treatment of obesity with the advantage of avoiding side effects associated with taking multiple medications. However, whilst fenofibrate has affinity for the cannabinoid receptors, only its active metabolite; fenofibric acid (10b ) activates PPAR α. This project sought to develop novel ligands for the cannabinoid receptors that retain activity at PPAR α. A series of amide derivatives of fenofibrate were synthesised, and pharmacological testing revealed that the piperidinyl (48g) and morpholino (48h) derivatives had agonist activity and a higher affinity for the cannabinoid receptors than fenofibrate. However these derivatives failed to bind and activate PPAR α. Although a dual PPAR α / cannabinoid receptor ligand has not been found with these amide derivatives, the compounds synthesised could provide a platform for the further development of cannabinoid receptor ligands of this novel class. This was demonstrated by further modifications to compounds (48g) and (48h) which indicated that activity at the cannabinoid receptors is tuneable. i Acknowledgements Firstly I would like to thank my two supervisors Dr Barrie Kellam and Professor David Kendall for their support and guidance throughout this project, Dr Andrew Bennett for help and guidance with the PPAR α pharmacology and also Monika Owen for her instruction in cell culture techniques. I would like to thank the C-floor chemists, for their help and advice throughout my time working on this project, and also technicians Lee Hibbett and Graeme Parry. Finally I would like to thank Dr Shailesh Mistry for his support, especially during the writing of this thesis, and my family who have always encouraged and supported me. This work was funded by the BBSRC. ii Abbreviations Acetyl-CoA Acetyl Coenzyme A AIDS Acquired immunodeficiency syndrome Apo-AI Apolipoprotein AI Apo-AII Apolipoprotein AII ATP Adenosine triphosphate BSA Bovine Serum Albumin CB 1 Cannabinoid receptor 1 CB 2 Cannabinoid receptor 2 CBP CRE binding protein CDCl 3 Deuterated Chloroform cDNA Complementary DNA CHO Chinese Hamster Ovary Cyclic AMP Cyclic adenosine monophosphate DCM Dichloromethane DIAD Diisopropyl azodicarboxylate Dpm Disintegrations per minute E. coli Escherica coli EDTA Ethylenediaminetetraacetic acid EtOAc Ethyl Acetate FBS Fetal Bovine Serum TR-FRET Time resolved fluorescence resonance energy transfer FT-IR Fourier Transform Infra-Red GABA Gamma aminobutyric acid GTP γS Guanosine 5’-[γ-thio]-triphosphate HBTU 2-(1H -benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate hCB 2 human Cannabinoid Receptor 2 HPLC High Performance Liquid Chromatography IC 50 Median Inhibition Concentration IL-6 Interleukin 6 Ki Equilibrium affinity constant LB Luria Bertani M+ Molecular ion (positively charged) MAP Kinase Mitogen Activated Protein Kinase iii MeCN Acetonitrile MeOH Methanol NMR Nuclear Magnetic Resonance OSu Succinimide ester p300 Binding protein p300 PE Petroleum ether PBS Phosphate Buffered Saline PEI Polyethylenimine ppm Parts per million RGS Regulator of G protein signalling RLU Relative light units SD Standard Deviation SE Standard Error SRC-1 Steroid Coactivator 1 protein TBME tert-Butyl methyl ether TH1 Type 1 helper T cells THF Tetrahydrofuran TLC Thin Layer Chromatography TMS Tetramethylsilane TOF-ES Time of Flight-Electrospray TR-FRET Time Resolved-Fluorescence Resonance Energy Transfer UV Ultra Violet iv Table of Contents Abstract .................................................................................................................................. i Acknowledgements ................................................................................................................ ii Table of Contents ................................................................................................................... v 1. Introduction ....................................................................................................................... 1 1.1. Potential Dual PPAR/ Cannabinoid Receptor Ligands ................................................. 1 1.2. Peroxisome Proliferator Activated Receptors ............................................................... 2 1.2.1. PPAR Structure ................................................................................................... 2 1.2.2. Activation Mechanism ......................................................................................... 3 1.2.3. PPAR α ................................................................................................................ 4 1.2.4. Ligands for PPAR α ............................................................................................. 5 1.3. G-Protein Coupled Receptors .................................................................................... 12 1.3.1. GPCR Structure ................................................................................................ 12 1.4. Cannabinoid Receptors ............................................................................................. 16 1.4.1. CB1 Receptor ..................................................................................................... 17 1.4.2. CB 2 Receptor ..................................................................................................... 17 1.4.3. GPR55 .............................................................................................................. 18 1.4.4. Ligands for Cannabinoid Receptors ................................................................... 20 1.4.5. CB 1 as a therapeutic target ................................................................................ 30 1.4.6. CB 2 as a therapeutic target ................................................................................ 33 1.5. Aims ........................................................................................................................ 35 1.5.1. Design of fenofibrate analogues......................................................................... 36 2. Amide derivatives of fenofibrate....................................................................................... 38 2.1. Synthesis .................................................................................................................. 38 2.2. Synthetic Route ........................................................................................................ 38 2.3. Pharmacology results ................................................................................................ 41 2.3.1. Activity at the Cannabinoid Receptors ............................................................... 41 2.3.2. Activity of the analogues at PPAR α .................................................................... 48 2.4. Conclusions .............................................................................................................. 53 3. Substituted benzyl and phenyl derivatives ......................................................................... 55 3.1. Synthesis of derivatives ............................................................................................ 55 3.1.1. Synthetic route .................................................................................................. 55 3.2. Pharmacology ........................................................................................................... 58 3.2.1. Activity at the cannabinoid receptors ................................................................. 58 3.2.2. Activity at PPAR α .............................................................................................. 63 3.3. Conclusions .............................................................................................................. 63 4. N-Morpholino-2-(4-(4-chlorobenzoyl)-phenoxy)-2-methylpropanamide (48h) and N- piperidinyl-2-(4-(4-chlorobenzoyl)-phenoxy)-2-methylpropanamide (48g) derivatives .......... 65 4.1. Synthesis of derivatives ............................................................................................ 65 4.1.1. Synthetic route .................................................................................................. 66 4.1.2. Pharmacology ................................................................................................... 69 4.2. Unbranched and Tertiary Amide Derivatives ............................................................. 81 4.2.1. Synthetic routes ................................................................................................. 81 4.2.2. Pharmacology ................................................................................................... 84 4.3. Conclusions .............................................................................................................