Fluorescent cannabinoids: Strategies towards the synthesis of fluorescently labelled CB2 receptor ligands Andrew Stephen Yates BPharm MRPharmS AMRSC School of Pharmacy, The University of Nottingham Nottingham UK Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy November 2004 1 “A chemist dabbling in his lab Felt his project was a little drab So embarked to discover fluorescent weed Citing a greater scientific need Soon had friends with glowing lips Funded his life on their grateful tips Checked his spectra, in disbelief When his friends got no relief He made an error in his reaction That gave his friends no satisfaction In desperation he hid away Improving his creation day by day His project done, his girlfriend’s plea Was to write it up as a PhD This is what I submit to you To read, to marvel, to hold as true” - A. S. Yates 2 Table of contents Abstract ...........................................................................................................................i Acknowledgements ........................................................................................................ii Abbreviations ................................................................................................................iii 1. Introduction............................................................................................................1 1.1 Fluorescent labelled ligands as a tool to study G-protein coupled receptors.2 1.1.1 Principles of fluorescence ......................................................................4 1.1.2 Fluorescent techniques ...........................................................................5 1.1.2.1 Confocal microscopy..........................................................................5 1.1.2.2 Single molecule detection..................................................................6 1.1.2.2.1 Fluorescence correlation spectroscopy.........................................7 1.1.2.2.2 Other single molecule detection techniques.................................8 1.1.2.3 High throughput screening applications.............................................9 1.2 Cannabis.......................................................................................................10 1.2.1 Historical overview of cannabis...........................................................10 1.2.2 Therapeutic use of cannabis .................................................................14 1.2.2.1 Multiple Sclerosis.............................................................................15 1.2.2.2 Analgesia..........................................................................................16 1.2.2.3 Nausea and vomiting........................................................................17 1.2.2.4 Appetite modification.......................................................................17 1.2.2.5 Other therapeutic areas.....................................................................18 1.3 The cannabis receptors.................................................................................20 1.3.1 GPCR receptors....................................................................................20 1.3.2 Gi/o cell signalling.................................................................................23 1.3.3 The CB1 receptor system......................................................................25 1.3.4 The CB2 receptor system......................................................................28 1.3.4.1 The distribution of the CB2 receptor................................................29 1.3.4.2 Immune functions of the CB2 receptor.............................................32 1.3.4.3 In-vitro bioassay systems .................................................................33 1.3.4.3.1 Binding assays ............................................................................33 1.3.4.3.2 [35S] Guanosine-5’-O-(3-thiotriphosphate) ([35H] GTP-g-S) binding........................................................................................34 1.3.4.3.3 Inhibition of cyclic AMP production.........................................34 1.3.4.3.4 Practical difficulties....................................................................34 1.3.4.4 Therapeutic indications of the CB2 receptor....................................35 1.3.4.4.1 Pain.............................................................................................35 1.3.4.4.2 Multiple Sclerosis.......................................................................35 1.3.4.4.3 Immunomodulation....................................................................36 1.3.4.4.4 Cancer.........................................................................................36 1.4 Ligands for the cannabis receptors...............................................................38 1.4.1 Nomenclature .......................................................................................39 1.4.2 Classical cannabinoids .........................................................................41 1.4.2.1 The side chain...................................................................................42 1.4.2.2 The phenolic group...........................................................................43 1.4.2.3 The 9-methyl group ..........................................................................44 1.4.2.4 CB2 selective classical cannabinoids................................................46 1.4.3 Non-classical cannabinoids..................................................................47 1.4.3.1 CB2 selective Non-Classical Cannabinoids......................................50 1.4.4 Arachidonic acid derivatives................................................................52 3 1.4.5 Pyrazole cannabinoids..........................................................................53 1.4.5.1 CB2 selective pyrazoles....................................................................54 1.4.6 Indole based cannabinoids ...................................................................56 1.4.6.1 Modeling studies on indoles.............................................................58 1.4.6.2 CB2 selective indoles........................................................................60 1.4.7 Japan Tobacco ligands .........................................................................63 1.5 Research Aims..............................................................................................66 2 Fluorescent CB2 ligand based on a modification to the benzamide ring of a Japan Tobacco compound. .....................................................................................................67 2.1 Background to designing a GPCR fluorescent ligand..................................67 2.1.1 Fluorescent adenosine agonist ligands .................................................67 2.1.1.1 Synthetic strategy used for fluorescent adenosine agonists .............71 2.1.2 Fluorescent serotonin ligands...............................................................73 2.1.1 Summary of the design and synthesis requirements of fluorescent ligands ..................................................................................................75 2.2 Designing a fluorescent cannabinoid CB2 ligand.........................................76 2.2.1 Selection of a suitable CB2 ligand........................................................77 2.2.2 Selection of a suitable site upon JTE2-6 for conjugation.....................77 2.2.3 Determining a suitable linker ...............................................................78 2.2.4 Selection of a fluorophore....................................................................79 2.2.5 Synthetic strategy.................................................................................81 2.3 Chemistry results..........................................................................................84 2.3.1 Conjugation with amine reactive dyes.................................................87 2.4 Pharmacology results ...................................................................................90 2.5 Use of Dansyl-JTE2-6 and BODIPY-JTE2-6 in fluorescence confocal microscopy. ......................................................................................................91 2.6 Discussion....................................................................................................92 2.7 Conclusions ..................................................................................................96 3 Molecular modelling of the CB2 receptor and docking of JTE2-6 ......................97 3.1 Computational modelling a of G-protein coupled receptor .........................97 3.1.1 Crystal structure template.....................................................................98 3.1.2 Sequence alignment..............................................................................98 3.1.3 Energy minimisation and molecular dynamics ..................................100 3.1.4 Ligand docking...................................................................................103 3.2 Previous modelling of the CB2 receptor.....................................................104 3.2.1 Mutational analysis ............................................................................104 3.2.2 Molecular modelling studies ..............................................................108 3.2.2.1 Results from previous CB2 models ................................................108