Potassium Channel Blockers as Potential Antisickling Agents A thesis presented to the University of London in fulfilment of the requirements for the degree of Doctor of Philosophy by Zena Miscony University College London Chemistry Department Christopher Ingold Laboratories 20 Gordon Street London WCIH OAJ January 1998 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. ProQuest Number: U641953 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 U641953 Published by ProQuest LLC(2015). 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 This thesis is dedicated to my parents for their endless love, support, encouragement and enduring patience. "If he is indeed wise he does not bid you enter the house of his wisdom, but rather leads you to the threshold of your own mind. " Kahlil Gibran Abstract K+ channels play an important role in controlling membrane potential and hence excitability of cells. They are present in all cells and exist as many types, several often being present on the same cell. An area in need of pharmacological exploration is that of calcium- activated potassium channels, one of the subtypes is the intermediate-conductance, voltage insensitive calcium activated K+ channel (IKca) found in red blood cells. Cetiedil (±2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1 H-azepin-1 -yl) ethyl ester) has been shown to be an inhibitor, with moderate activity, (IC 50 = 24.5 |xM) of the IKca channel found in erythrocytes. Cetiedil, a known drug originally found to be a peripheral vasodilator has also been reported to exhibit antisickling properties in erythrocytes. This thesis describes the synthesis and biological activity of a series of analogues based on the lead compound, cetiedil, with the aim of designing a more potent and selective blocker. All the compounds synthesised were submitted for pharmacological testing for their ability to inhibit K+ efflux from rabbit erythrocytes. Previous results obtained on the channel had suggested that drug potency correlates well with drug lipophilicity. A series of novel analogues were synthesised for this thesis using established chemical routes to investigate the critical features present in the cetiedil analogue, 2-[N-(5-ethyl-2- methylpiperidino)]ethyl triphenylethanoate (IC5o=1.16|xM). Exploration of the pharmacophore of 2-[N-(5-ethyl-2-methylpiperidino)]ethyl triphenylethanoate led to a structure-activity analysis designing more potent blockers which were more lipophilic and stereoisomeric. In general, less of a correlation between drug potency and drug lipophilicity was found in comparison to the previous results obtained. This was because certain structural features now seemed to be influencing activity and not only increases in lipophilicity. The presence of an acetylenic moiety was found to be important for activity. The most potent blockers of the series were l-[9-(benzyl)fluoren-9-yl)]-4-(N-5-ethyl-2- methylpiperidino)-but-2-yne and the corresponding open chain analogue, l-[(9-benzyl)- fluoren-9-yl)]-4-(N, N-dibutylamino)-but-2-yne with IC50 values of 0.21 jiM and 0.13 pM respectively. Clotrimazole (diphenyl-(2-chlorophenyl)-N-imidazolyl methane), a known antifungal drug has also recently been shown to be an inhibitor of IKca in red blood cells with an IC50 value of 0.94 |iM. Based on clotrimazole, a series of heterocyclic and open chain analogues and a series of non-amino analogues were synthesised. The main structural feature of clotrimazole, the trityl group, was retained and investigations into the role of the imidazole moiety, and importance of the substituents and positions of substitution on the aromatic rings was carried out. Pharmacological results obtained indicated that the imidazole ring was not essential for activity. The most active compound found in the heterocyclic and open chain series besides clotrimazole was 3-{N-[l-(2-chlorophenyl)-l,l- diphenyl]methyl} aminopyridine, IC 50 = 1.95 |iM. In the non-amino analogue series, the most active compound found was also calculated to be the most lipophilic of the series, tris-(4-chlorophenyl)-methanol, IC 50 = 0.56 pM. In general, less of a correlation between drug potency and drug lipophilicity was observed for the clotrimazole analogues in comparison to the cetiedil analogues synthesised for this thesis. Stereochemical assignments have been carried out for this thesis using 2D NMR techniques namely one bond heteronuclear shift correlations and nuclear Overhauser enhancements. Lipophilicity throughout the thesis has been expressed as calculated log P values using the manual fragmental method, (X/) of Rekker and the computerised CLOG? method of Leo and Hansch. A drug solubility analysis involving UV spectroscopy was also carried out. Acknowledgements I am indebted to my supervisor, Professor C.R. Ganellin for his support, encouragement and insight throughout the course of this work. Thanks also to Mr Wasyl Tertiuk for his help, support and friendship. To my friends in the Chemistry Department, past and present who have made the whole experience so enjoyable. A special thankyou to Nicola, Kit, Antonia, Karen, Tiz, Ashley, Simon, Bruce, Paul, Dimitrios and Salah. I wish to specially thank Professor D.H.Jenkinson and various members of his group, Ms M. Malik, and Dr. D.C.H. Benton for carrying out the pharmacological testing, and their helpful discussions. Thanks also to Dr. D.G. Haylett for his helpful discussions. I am extremely grateful to the technical staff at UCL - Steve Corker, Alan Stone, John Hill and Jill Maxwell. Thanks also to Dr. L. Zhao and Dr. D. Yang for their helpful discussions. I would also like to thank Professor J.G. Vinter for the molecular modelling. I am grateful to Jane Hawks at the Department of Chemistry, Kings College for carrying out the 2D NMR experiments. Special thankyou to dear Roshanak and Mary, for their love and support. Finally, last but certainly not least, to my dear uncle Lutfi for having inspired me as my first teacher of organic chemistry. Contents Abstract 4 Acknowledgements 6 Contents 7 Glossary 11 1. Chapter 1 Introduction 15 1.1 Historic Perspective 15 1.2 Voltage-dependent Channels 17 1.2.1 Delayed Rectifiers (Kv) 18 1.2.2 (Kv) channel structure 19 1.2.3 Rapid delayed rectifier (Kvr) 19 1 .2 .4 Slow delayed rectifier (Kvs) 2 0 1.2.5 Transient K+ channels (Ka) 20 1.2.6 (Ka) channel structure 2 2 1.2.7 Inward rectifier channels (Kir ) 24 1.2.8 (Kir ) channel structure 25 1.2.9 Sarcoplasmic reticulum channel (Ksr ) 25 1.3 Calcium-activated potassium channels (Kca) 2 6 1.3.1 Large conductance Ca^+-activated K+ channels (BKca) 28 1.3.2 BKca channel structure 30 1.3.3 Intermediate conductance Ca^+-activated K+channels (IKca) 30 1.3.4 Voltage-sensitive IKca channels 31 1.3.5 Voltage insensitive IKca channels 32 1.3.6 Sickle cell anaemia 35 1.3.7 Potential antisickling agents 36 1.3.8 Antifungal drugs as potential antisickling agents 42 1 .3 .9 Small conductance Ca^+-activated K+channels (SKca) 46 1.4 SKca channel structure 48 1.5 Cell volume-sensitive K+ channel (Kvol) 48 1.6 ATP-sensitive K+ channels (Katp) 48 1.6.1 K atp channels in pancreatic 6 -cells 49 1 .6 .2 Katp channel in heart cells 50 1.6.3 Katp channel in skeletal muscle 51 1.6.4 Katp channel in neurones 53 1.6 .6 Blockers of the Katp channels 53 1.7 G-protein gated K+ channels 55 1.7.1 Muscarinic-Inactivated K+channel (Km) 56 1.7.2 Atrial muscarinic-activated K+ channel (KACh) 57 1.7.3 5-HT-inactivated K+channel (K5 _ht) 58 1.7.4 Na+-activated K+ channel (K^a) 59 1.8 Potassium channel modulators 60 1.8.1 K+ channel openers 60 1.8.2 K+ channel blockers 64 Chapter 2 Previous compounds synthesised and tested 67 2.1 Introductory remarks 67 2.2 Cetiedil, the IKca (or Gàrdos channel) blocker 6 8 2.3 Lipophilicity 72 2.3.1 The partition coefficient (P) 72 2.3.2 Hydrophobicity 75 2.3.3 Methods of calculating partition coefficients 77 2.3.4 Calculation procedures for molecular lipophilicity 78 2.3.5 The X/ system of Rekker 78 2.3.6 CLOGP system of Hansch and Leo 80 2.3.7 Limits of fragmental systems 83 2.4 UCL 1274 84 2.5 UCL 1495 85 2.6 UCL 1422 88 2.7 UCL 1608 90 Chapter 3 Selection of compounds 93 3.1 Introductory remarks 93 3.2 Compounds synthesised for this study 93 3.2.1 The UCL 1495 series 93 3.2.2 The UCL 1710 series 96 3.2.3 The UCL 1608 series 99 3.2.4 The clotrimazole heterocyclic and open chain series 103 3.2.5 The clotrimazole non-amino series 105 Chapter 4 Discussion of synthesis and characterisation of compounds 107 4.1 Introductory remarks 107 4.2 Synthesis of the UCL 1495 series 107 4.2.1 Synthesis of compounds UCL 1644 and UCL 1645 108 4.2.2 Synthesis of triphenylacetyl chloride 110 4.2.3 Synthesis of UCL 1631 111 4.2.4 Synthesis of UCL 1617 111 4.3 Synthesis of the acetylene analogues