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Synthesis and Structure-Activity Studies of Novel Potassium Ion SYNTHESIS AND STRUCTURE - ACTIVITY STUDIES OF NOVEL POTASSIUM ION CHANNEL BLOCKERS A thesis presented In partial fulfilment of the requirements for the Doctor of Philosophy Degree of the University of London DAVID FLETCHER Department of Chemistry University College London March 1997 Attention is drawn to the fact that the copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it, is understood to recognise that the copyright rests with its author and no quotation from the thesis and no information derived from it may be published without prior consent of the author. ProQuest Number: 10046053 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10046053 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Small conductance Ca^'^ activated potassium channels (SKca) occur in many cells but, until recently, have been relatively little studied. Interest in this field has been generated by the discovery of agents which selectively block the channel, notably several insect and anthropod toxins with high potency, and a variety of smaller molecules, the most active of which is dequalinium. This thesis describes the synthesis of novel compounds as potential blocking agents of the SKca channel, in order to examine the nature of the interaction with the channel protein, and in particular to identify any structural features additional to the two arginine residues in the peptide toxins which have been proposed to participate in binding. Each compound was assayed in vitro for its ability to block the after­ hyperpolarisation (mediated by the opening of the SKca channels) that follows the action potential in rat sympathetic neurones. An initial series of compounds was prepared comprising various elaborations on a fumaric acid-based bis-guanidinium compound, which had possessed weak activity. Incorporation of an additional aminoalkyl substituent resulted in a significant increase in potency. Replacement of the guanidine groups in this compound by 4-aminoquinoline increased the activity 100 fold to a level greater than dequalinium. A variety of derivatives where three and four aminoquinolinyl groups are attached by alkyl chains to a meta-substituted aromatic central unit was also prepared, based on a 1 ,6- disubstituted indane as a mimic for an a-helix. All were found to be more potent than dequalinium. The relative potencies are discussed in terms of the energy changes of the binding process, and it is concluded that only two of the electropositive groups interact with the channel protein. A proposal for their relative spatial relationship is discussed, and is consistent with current hypotheses concerning the dependence on charge delocalisation, and the involvement of a hydrophobic contribution, probably operating at short range, in addition to the primary electrostatic interaction of two charged groups separated by approximately 11 A. Quaternisation of the basic residues is unnecessary, provided that the groups are protonated at physiological pH; the linkage position of the chains through the exocyclic or endocyclic nitrogen of each aminoquinoline is also not critical. Acknowledgements I would like to thank Professor C.R Ganellin for giving me the opportunity to work on this project, and for his advice, support and encouragement. Special thanks are due to colleagues at Wyeth Research, U.K, in particular Mr George Weston for giving me the impetus to begin the work, and to Dr Ken Heatherington for his assistance in obtaining and interpreting analytical and spectroscopic data. I am grateful to Dr Phil Dunn of the Department of Pharmacology for performing the electrophysiological testing of the compounds and to Professor D.H Jenkinson for helpful discussions. I would like to thank Wyeth Research for providing financial support during the majority of the study period. Finally, but by no means least, I would like to thank all members of the Ganellin group for their support and comraderie during my visits. To my parents, my wife and 'the tribe'. 'Patience achieves more than force' CONTENTS Page Chapter 1. Introduction. 10 1.1. Ion channels 10 1.1.1. Membranes and cell resting potential 10 1.1.2. Receptor and action potentials 11 1.1.3. Gating 13 1.1.4. Ion selectivity and the pore 15 1.1.5. Modulation and second messengers 19 1.2. Potassium channels 21 1.2.1. Voltage regulated channels 22 1.2.2. Receptor regulated channels 23 1.2.3. Miscellaneous potassium channels 24 1.2.4. Ca2+ activated channels 24 1.3. Apamin 27 1.3.1. Isolation and characteristics 27 1.3.2. Location of binding sites 27 1.3.3. Structure 28 1.3.4. Structure of the apamin binding site 30 1.3.5. Structure-activity relationships 32 1.3.6. Mode of action 32 1.4. Leiurotoxin 33 1.5. PO 5 36 1.6. Binding and toxicity 37 1.7. Other scorpion toxins 38 1.7.1. A common structure 38 1.7.2. Charybdotoxin 39 1.7.3. Structure of the charybdotoxin-binding protein 42 1.7.4. Iberiotoxin 43 1.7.5. Channel selectivity 44 1.8 . Other blockers of the SKqq channel 45 1.9. Drug design 46 1.9.1. Peptidomimetics 47 1.9.2. General considerations 49 Chapter 2. Selection of compounds 51 2.1. Fumaric/ maleic acid based compounds 52 2.2. Replacement of guanidinium groups by aminoquinolinium 56 2.3. Trisubstituted aryl centred compounds 59 2.4. Tetrasubstituted aryl centred compounds 66 Chapter 3. Synthesis of compounds 68 3.1. UCL1190 68 3.2. UCL1641 75 3.3. UCL1597 78 3.4. UCL1716 81 3.5. UCL 1714 87 3.6. UCL1714 ethyl analogue 93 3.7. Other 1,2,4-trisubstituted compounds 94 3.8. 1,3,5-Trisubstituted benzenes 96 3.9. UCL1735 102 3.10. Pyrogallol based compounds 106 3.11. UCL1821 110 3.12. UCL1823 113 4. Biological method 116 5. Results and discussion 118 5.1. Fumaric and maleic acid based compounds 119 5.1.1. UCL1190 and UCL1641 120 5.1.2. UCL1597 123 5.1.3. UCL1716 124 5.2. Trisubstituted aryl centred compounds 127 5.3. Tetrasubstituted aryl centred compounds 130 5.4. Conclusion ' 131 6 . Experimental 133 7. References 183 Abbreviations abs EtOH absolute ethanol Ac acetyl ACh acetylcholine ADP adenosine 5'-diphosphate AHP after-hyperpolarisation Ala alanine 4-AP 4-aminopyridine Ar aryl Arg arginine Asn asparagine Asp aspartic acid ATP adenosine 5'-triphosphate Boo t-butoxycarbonyl br broad B K ca high conductance Ca 2 + -activated channel Bu t-butyl lCa2+]j intracellular concentration of Ca 2 + cAMP cyclic adenosine -3',5'-monophosphate CBZ benzyloxycarbonyl CDCI3 deuterated chloroform ChTX charybdotoxin CM A 100 chloroform/methanol/0.91 NH 3 ,100/10/1 CMA 200 chloroform/methanol/0.91 NH 3 , 200/30/5 CNS central nervous system Cys cysteine d doublet Da Dalton (molecular mass unit) DCCU 1,3-Dicyclohexylurea DMF dimethylformamide DM SO -de deuterated dimethylsulphoxide ED 50 dose (of drug) that causes 50% of maximum response El electron impact EMR equiffective molar ratio Et ethyl FAB fast atom bombardment fmol femtomole ( 10''!^) g gramme G guanidine Gin glutamine Glu glutamic acid Gly glycine h hour H5 putative pore forming region of channel proteins HOBT 1-hydroxybenzotriazole Har homoarginine His histidine HPLC high performance liquid chromatography 5-HT 5-hydroxytryptamine Hz hertz IbTX iberiotoxin icv intracerebroventricularly IKca intermediate conductance Ca2+-activated channel IR infra red IRK1 cloned K|r channel J coupling constant (NMR) K/\ A-channel (transient outward, channel subtype) ^ATP ATP-senstive K'*' channel Kd dissociation constant kDa kilodalton (10^ daltons) Leu leucine Lys lysine M1 ,M2 segments of internally rectifying/ ATP activated channel channel proteins MIBK methyl isobutyl ketone nM nanomolar NMR nuclear magnetic resonance NIX noxiustoxin P l,P 2 protecting groups (general) Ph phenyl Phe phenylalanine pKg -logiKa pM picomolar molar) PO 5 peptide toxin isolated from the venom of the scorpion Androctonus mauretanecus mauretanicus Pro proline pS picosiemens (10"^2 siemens) q quartet R0MK1 cloned ATP-regulated channel RP reverse phase RT room temperature s singlet (NMR), strong (IR) S serotonin S1-S6 segments of voltage-dependent channel proteins SCyTX leiurotoxin I SKQa small conductance Ca2+-activated channel t triplet TEA triethylammonium; toluene/abs ethanol/0.91 NH3 , 80/20/1 TFA trifluoroacetic acid THF tetra hydrofu ran Thr threonine TLC thin layer chromatgraphy TMS trimethylsilane Trp tryptophan TS thermospray TsOH p-toluenejBulphonic acid Tyr tyrosine UCL identification tag (University College London) UV ultraviolet V.I.P vasoactive intestinal peptide Val valine w weak Thesis compounds UCL No Page for discussion Page of experimental 1190 68 134 1597 78 149 1641 75 141 1714 87 158 1715 98 162 1716 81 152 1735 102 167 1821 110 175 1822 106 171 1823 113 180 1836 109 174 Thesis tables Table No Title Page 1 Structure & biological results for compounds in this study 8 2 SKca blocking agents 46 3 Biological results for compounds in series 1 119 4 Biological results for compounds in series 2 128 5 Biological results for compounds in series 3 130 Table 1 - Structure and biological results for the compounds in this study UCL No Co. Ref Notebook Structure EMR No No NH CON NH.
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