A Click-Chemistry Based Approach for the Synthesis of New BODIPY
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A Click-Chemistry based approach for the synthesis of new BODIPY-labelled fluorescent ligands Daniel Speed BSc. MSc Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy July 2013 Abstract Fluorescent ligands have found numerous applications for studying interactions of drug molecules with their target and as a probe of biological systems. A common approach when designing and synthesising a fluorescent ligand is to separate the fluorophore and pharmacophore via a linker. One novel approach is to utilise click chemistry to allow the coupling of fluorophore to a pharmacophore. This thesis reports the results of an investigation into utilising click chemistry, specifically the alkyne-azide copper (I) cycloaddition to synthesis novel fluorescent GPCR ligands. Targets included the β1, β2 adrenoceptor and the muscarinic M3 receptor. Investigations into the introduction of a 1,2,3-triazole within the linker to the fluorophore resulted in 14 novel fluorescent antagonists active at the β1 and β2 adrenoceptor. The most promising ligand had log Ki values of -6.77 ± 0.20 (β1) and -7.32 ± 0.05 (β2). These ligands were used in a confocal microscopy studies to visualise the β1 and β2 adrenoceptors on the surface of CHO cells. However the ligands internalistion, and receptor visualisation was not possible. A range of structural modifications were made to reduce this with the introduction of a polar linker but this did not reduce the intracellular accumulation. The change to a longer wavelength fluorophore stopped intracellular accumulation but reduced the binding log Ki to - 5.16 ± 0.06 (β1) -5.96 ± 0.20 (β2). Twenty two novel fluorescent M3 ligands were synthesised and their inhibitory properties were investigated. An initial screen showed four promising ligands and further study into the binding affinities showed the ligands to have high potency (log Kb -7.97 ± 0.07 to -8.89 ± 0.11). These ligands were studied with confocal microscopy and intracellular accumulation did not occur. Structural changes to include a polar side chain or a sulfonic acid onto the fluorophore were investigated and led to three novel fluorescent ligands that had reduced lipophilicity. With this reduced lipophilicity, binding affinities were also reduced by ten fold compared to the original fluorescent ligand. The seven ligands were fully profiled physiochemically and kinetically. The physioschemical properties of these seven ligands gave a wide variety of lipophilic values. The kinetic profiles of the ligands exhibited very similar dissociation properties to those of the parent ligand with varying association rates. i The Muscarinic M3 ligands synthesised show great binding affinities for fluorescent ligands and kinetic profiles that are extremely similar to the parent ligand. These fluorescent ligands hold characteristics that can be used to further examine the pharmacology of muscarinic receptors and be used to replace radioligands for binding studies. ii Acknowledgements Many thanks to my supervisors Barrie Kellam, Stephen Hill, Stephen Charlton and Robin Fairhurst for their excellent guidance and knowledge throughout my PhD. I would also like to thank Tim Self, David Sykes and John Reilly for their direction and support in confocal microscopy, kinetic experiments and physchem HPLC analysis. I would like to acknowledge the help and time given by all the people in chemistry, and at Novartis. In particular I would like to thank Austin, Jim, Leigh, and Shailesh for their continuous advice and patience. Jack and Jo I thank you for the distraction of a coffee every now and again. Finally I would like to thank Mum, Dad, Paul and Kayleigh for endless support and for listening to me as I ramble on about chemistry and pharmacology when they have absolutely no idea what I was talking about. I am extremely grateful to the BBSRC and Novartis for funding my work. iii Abbreviations 5-TAMRA 5-carboxytetramethylrhodamine 6-TAMRA 6-carboxytetramethylrhodamine ATP adenosine triphosphate βAR β-adrenoceptor β1AR β1-adrenoceptor β2AR β2-adrenoceptor β3AR β3-adrenoceptor BBSRC Biotechnology & biological science research council Boc tert-Butoxycarbonyl BODIPY boron-dipyrromethene br broad Calc. calculated cAMP cyclic adenosine monophosphate CHI chromatographic hydrophobicity Index CHIIAM 7.4 chromatographic hydrophobicity Index at pH 7.4 CHO chinese hamster ovary CGP 12177 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H- benzimidazol-2-one hydrochloride CNS central nervous system COPD chronic obstructive pulmonary disease d doublet Dansyl chloride 5-(dimethylamino)naphthalene-1-sulfonyl chloride dd doublet of doublets DCE 1,2 – dichloroethane DCM dichloromethane DDQ 2,3-dichoro-5,6-dicyano-1,4-benzoquinone DIAD diisopropyl azodicarboxylate DIPEA N,N,-diisopropylethylamine DMF N,N,- dimethylformamide DMSO dimethyl sulfoxide DMSO-d6 dueterated dimethyl sulfoxide DR dose ratio iv ES electrospray FCS fluorescence correlation spectroscopy FP fluorescence polarisation FRET fluorescence resonance energy transfer FT-IR fourier transform -infrared Fura-2 acetoxymethyl 2-[5-[bis[(acetoxymethoxy-oxo- methyl)methyl]amino]-4-[2- [2-[bis[(acetoxymethoxy-oxo- methyl)methyl]amino]-5-methyl- phenoxy]ethoxy]benzofuran-2-yl]oxazole-5-carboxylate GABA γ-amino butyric acid GDP guanosine diphosphate GPCR G-protein coupled receptor GTP guanosine triphosphate HBSS Hank’s buffered saline solution HBTU O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HPLC high performance liquid chromatography h hours HSQC heteronuclear single-quantum correlation spectroscopy IC50 molar concentration which inhibits 50% of the maximal stimulatory response. IAM immobilised artificial membrane ICI 118,551 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol IP3 inositol 1,4,5-triphosphate IR infrared J coupling constant k3 calculated associated rate k4 calculated dissociation rate kD dissociation constant ki equilibrium association constant kob observed rate constant kon dissociation rate koff association rate Log D partition coefficient Log D 7.4 partition coefficient at pH 7.4 M molar v m multiplet MeCN acetonitrile MeOH methanol Min minutes MS mass spectroscopy MW microwave NBD nitrobenzofuran NMR nuclear magnetic resonance spectroscopy q quartet QNB 3-quinuclidinyl-benzilate RLB radio ligand binding Rt retention time s singlet SAR structure activity relationships SBD benzo-2-oxa-1,3-diazole-4-sulfonate t triplet TAMRA carboxytetramethylrhodamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TM transmembrane TOF time of flight s.e.m standard error of the mean W Watt XAC xanthine amine congener vi Table of Contents 1. Introduction 1 1.1. Gprotein coupled receptors 1 1.1.1 G‐proteins 2 1.2 Studying GPCRs as drug targets 3 1.2.1 Molecular modelling 4 1.2.2 X‐ray crystallography 4 1.2.3.GPCR Pharmacology 5 1.2.3.1.Radioligand binding experiments 6 1.2.3.2. GTPγS binding assays 7 1.2.3.3. cAMP assays 7 1.2.3.4. Reporter assays 7 1.2.3.5. Ca2+ assay 8 1.2.4 Fluorescence 8 1.2.4.1 Principles of fluorescence 9 1.2.4.2 Fluorescence techniques 10 1.2.4.2.1 Confocal microscopy 10 1.2.4.2.2 Fluorescence polarisation 10 1.2.4.2.3 Fluorescence correlation spectroscopy 11 1.2.4.2.4 Fluorescence resonance energy transfer 11 1.3 Current GPCR fluorescent ligands 11 1.4 Click chemistry 17 1.5 Research aims 20 2. Design and synthesis of fluorescent betaadrenoceptor ligands 22 2.1 Adrenoceptors 22 2.1.1 β –Adrenoceptor 22 2.1.1.1 β –Adrenoceptor structure 23 2.2 β –Adrenoceptors as drug targets 23 2.2.1 Cardiovascular targets 23 2.2.1.1 Hypertension 24 2.2.1.2 Angina pectoris 24 2.2.1.3 Myocardial infarction 25 2.2.1.4 Arrhythmia 25 2.2.2 Respiratory targets 25 2.2.2.1 Asthma 26 2.2.2.2 Chronic obstructive pulmonary disease 26 2.2.3 Aims 27 2.3. Design and synthesis of fluorescent ligands 27 2.3.1. Retrosynthesis of fluorescent ligands 29 2.3.2 Synthesis of pharmacophore and linker 30 2.3.3 Fluorophore synthesis 32 2.3.4 Fluorescent spectroscopy 35 2.3.5 Final pharmacophore fluorophore coupling reaction 36 2.3.6 Pharmacology 39 2.4 Synthesis of secondgeneration ligands 42 2.4.1 Linker synthesis 42 2.4.2 Pharmacology of second‐generation ligands 46 2.4.3 Confocal imaging studies 49 2.4.4 Lipophilicity evaluation of fluorescent ligands 51 2.5. Amide linked ligands 53 2.5.1 Amide linker synthesis 53 2.5.2 Amide linker pharmacology 54 vii 2.5.3 Lipophilic study of amide linker fluorescent ligands 56 2.5.4 Amide linker confocal imaging 57 2.6 Longer wavelength fluorophores 59 2.6.1 Longer wavelength fluorescence ligand synthesis 59 2.6.2 Red‐shifted fluorescent ligand pharmacology 60 2.6.3 Lipophilic study of longer wavelength fluorescent ligands 61 2.6.4 Long wavelength fluorescent ligand confocal imaging 62 2.7 Synthesis of longer wavelength fluorophore 63 2.7.1 Retrosynthesis of a long wavelength fluorophore 64 2.7.1.1 Synthesis of red shifted ligands 64 2.7.1.2 Pharmacology of long wavelength fluorescent ligand 68 2.7.2.1 Confocal imaging of red‐shifted ligands 69 2.8 Triazole bearing fluorophores and pharmacophores 70 2.8.1 Capped molecule synthesis 70 2.8.2 Pharmacology of triazole pharmacophores and capped fluorophores 71 2.8.3 Stability assays 72 2.9 Conclusions and future work 73 3. Design and synthesis of fluorescent Muscarinic M3 antagonists 76 3.1 Muscarinic receptors 76 3.1.1 Muscarinic receptor structure 76 3.1.2 Muscarinic M3 receptor 77 3.2 Muscarinic M3 receptor as drug targets 78 3.2.1 Glaucoma 78 3.2.2 Urinary incontinence 79 3.2.3 Chronic obstructive pulmonary disease 79 3.2.4 Aim 80 3.3 Design and synthesis of fluorescent M3 Ligands 80 3.3.1 Retrosynthetic analysis 81 3.3.2 Synthesis of pharmacophore and linker 82 3.3.3 Synthesis of fluorescent ligands 84 3.4 Muscarinic antagonist pharmacology 86 3.4.1 Antagonist confirmation and initial screen 86 3.4.1.1 Counteracting extracellular fluorescence 87 3.4.2 Percentage inhibition assay 88 3.4.3 Dose response competition assays 90 3.4.4 Confocal imaging 92 3.4.5.