P^ge Urn Replace with 30 8 ...its responce being... ...and its response is... 31 1 Dispite... Despite... 32 10 These channels were... This channel was... 33 5 ...and can be envisaged... ...and it can be envisaged... 38 13 ...with rise... ...with a rise... 42 11 etal etal. 42 13 ...of Lys^... ...or Lys^... 43 15 etal etal. 44 16 etal etal. 45 7 etal etal. 48 13 Concequently... Consequently... 49 1 etal etal. 52 14 etal etal. 52 25 ...suggeted... ...suggested... 60 2 etal etal. 77 14 ...quatermaiy... ...quaternary... 79 3 ...whch... ...which... 82 9 ...appart... ...apart... 99 6 ...excert... ...exert... 104 entry 14 26.0 + 14 26 ±14 109 9 ...propeiies... ...properties... 122 7 ...accosiated... ...associated... 123 4 —Elumo consists... -E lumo provides... 123 5 ...ralationships... ...relationships... 124 12 etal etal. 131 4 ...section 2.3... ...section 2.4... 133 12 ...IC-jS... ...Ki values... 133 13 ...500 times... ...50 times... 135 Table Kcal/raol kcal/mol 135 11 Kcal/mol kcal/mol 136 6 Kcal/mol kcal/mol 136 23 Kcal/mol kcal/mol 136 26 Kcal/mol kcal/mol 136 27 Kcal/mol kcal/mol 137 13 Kcal/mol kcal/mol 137 14 Kcal/mol kcal/mol 137 15 Kcal/mol kcal/mol 137 16 Kcal/mol kcal/mol 138 6 Kcal/mol kcal/mol 138 Table KcalAnol kcal/mol 139 9 Kcal/mol kcal/mol 139 Table Kcal/mol kcal/mol 140 Table Kcal/mol kcal/mol 141 10 Kcal/mol kcal/mol 141 12 Kcal/mol kcal/mol 143 6 ...space i.e.... ...space, i.e.... 152 15 ...comparable but not identical... ...comparable, but not identical,... 155 26 etal etal. 164 1 4-chloroquinoline 4-ChloroquinolLne 164 3 4-hydroxyquinoline 4-Hydroxyquinoline 184 4 Genaral... General... 184 7 4-chloroquinaldine... 4-Chloroquinaldine 216 3 4-Bromobut-l-yl 4-bromobut-l-yl 233 17 1 g of epoxy-activated Sepharose 6B Epoxy-activated Sepharose 6B (Pharmacia) (Pharmacia)... (1 g)- 238 6 etal etal. 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 DIMITRIOS GALANAKIS Department of Chemistry University College London April 1995 Attention is drawn to the fact that 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 that no quotation from the thesis and no information derived from it may be published without prior consent of the author. Signature : Date : [ 1 - S - / 9 33“ ProQuest Number: 10106673 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 10106673 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 Acknowledgements I am indebted to Professor C.R. Ganellin for giving me the opportunity to work on this project, for the award of a grant, which enabled me to carry out the work and for his advice, insight and encouragement. I am grateful to Dr Phil Dunn of the Department of Pharmacology for performing the electrophysiological testing of the compounds and to Professor Donald Jenkinson for helpful discussions. I would also like to thank Dr Peter Strong of the Neuromuscular Unit of Hanunersmith Hospital for offering me the chance to perform radioligand binding studies in his laboratory and Dr Jonathan Wadsworth for help with the practical aspects of binding experiments. Compounds 32 - 35 were synthesised by Mr Sajeed Malik as part of his 3^^ year undergraduate project, working under the supervision of Prof. Ganellin and myself. Special thanks are due to Drs Ramesh Bambal, Paul Bishop and Andrew Moore for their assistance during my first months in the lab; Dr Julie Calder for introducing me to molecular modelling techniques and Drs Keith Wibley and Andy Vinter for helpful discussions on the modelling. I thank Alan Stones, Steve Corker, Jill Maxwell and Dr Margaret Mruzek for providing analytical and spectroscopic data. Finally, but by no means least, I would like to thank Mr Wasyl Tertiuk for his help and support and all members of the Ganellin group for making my stay enjoyable. The Upjohn Company is acknowledged for the purchase of HPLC equipment and a Silicon Graphics IRIS 4D workstation. I am grateful to the Wellcome Trust for the purchase of a Silicon Graphics Indigo/2 workstation and for a grant for consumables. This work has been accomplished thanks to the devotion, love and support of my parents. To them this thesis is dedicated. Abstract K+ channels are found in all animal cells where they play a key role in controlling the excitability of the cell. They exist as multiple subtypes most of which have yet to be exploited for therapeutic use. An area in need of pharmacological exploration is that of Ca^+ - activated K+ channels. One of the subtypes, the small conductance (apamin - sensitive) Ca^+ - activated K+ (SKca) channel is blocked by dequalinium (I, = CH3, R2 = NH2, R^ = H) at micromolar concentrations. ^ W V +N — (CH2>io — ]S ^ — N+ +N — R II Dequalinium is not adequately potent. Having this as the lead structure, novel analogues of the general type I have been synthesised and submitted for testing for their ability to block the slow after - hyperpolarisation that follows the action potential measured on rat sympathetic neurones. This series was designed to explore whether particular chemical properties of the substituents might affect activity and quantitative structure - activity correlations were sought. Good correlations were obtained between blocking potency and the energies of either of the frontier orbitals (HOMO and LUMO) for the compounds. Furthermore, compounds of the general structure II, where the aminoquinoline rings have been “inverted”, have been synthesised and submitted for testing. Again, good correlations were obtained between blocking potency and the energies of either of the frontier orbitals for the compounds. It was also possible to combine the results for both series I and II (i.e. 24 compounds) into a single correlation of potency against the energies of either of the frontier orbitals. Rigidification of the alkyl chain of dequalinium did not alter potency significantly. Furthermore, compounds belonging to series I with Rl = R^ = H, R4 = NH 2 having 5, 6 , 8__________________________________________________________________________ 8 , 10 and 12 methylene groups in the alkyl chain had similar activities. The above suggest that the conformational mobility of the alkyl chain as well as the maximum distance between the quinolinium groups are not critical for SKca channel blockade. Compounds in which the quinolinium groups of dequalinium have been replaced by other charged heterocycles, have also been synthesised and submitted for testing. The aim has been to reveal any special features associated with the quinolinium group. It was shown that compounds with different heterocycles are able to block the SKca channel but that quinoline was the best heterocycle. NH. CH IV: n = 3 V : n = 9 III NH Finally, to examine whether both quinolinium groups of dequalinium contribute to SKca channel blockade and to investigate the nature of their contribution, compound III (having three quinolinium groups) was synthesised and compared with compounds IV and V (having one quinolinium group, previously made) and dequalinium. The affinities of the compounds were determined from inhibition of the specific - apamin binding to SKca channels of rat brain synaptic plasma membranes. Increasing the number of quinolinium groups increased potency and it is suggested that the contribution of the second and third quinolinium groups in the molecules of dequalinium and III may not be the consequence of direct binding to the channel but may arise from a statistical effect. Compound III is the most potent non - peptidic blocker of the SKca channel on this assay so far reported. CONTENTS PAGE Abbreviations .......................................................................................................................11 Thesis compounds ...............................................................................................................16 Thesis tables .........................................................................................................................18 Table 1 ..................................................................................................................................21 CHAPTER 1 INTRODUCTION.................................................................................. 27 1.1 Voltage - dependent K+ channels 1.1.1 Delayed rectifier (Ky)...................................................................28 1.1.2 Rapid delayed rectifier (K vr) ...................................................... 29 1.1.3 Slow delayed rectifier (Kvs) ........................................................29 1.1.4 A - channel (Ka ).................................................................... 29 1.1.5 Inward rectifier (Kjr) ...................................................................31 1. 1.6 Sarcoplasmic reticulum channel