INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly fi^om the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter 6ce, \riiile others may be from aity type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e g., maps, drawings, charts) are reproduced by sectioning the original, b%inning at the upper left-hand comer and continuing fi-om left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & HowellInformation Conqtany 300 NorthZeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 PHARMACOLOGICAL CHARACTERIZATION OF THE CALCIUM CHANNEL MEDIATED PROPERTIES OF 2-(4 •-ISOTHIOCYANATOBENZYL) IMIDAZOLINE ANALOGS IN SMOOTH AND SKELETAL MUSCLES A DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Longping Lei, M.D., M.S. ***** The Ohio State University 1997 Dissertation Committee: Approved by Dr. Dennis R. Feller, Advisor Dr. Dennis B. McKay îdvisor Dr. Norman J. Uretsky College of Pharmacy UMI Number: 9721125 UMI Microform 9721125 Copyright 1997, by UMI Company. Ail rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Studies investigated the pharmacological mechanism of 2- (4 '-isothiocyanatobenzyl) imidazoline [IBI] and analogs for interaction with imidazoline preferring receptors (IPRs), a- adrenoceptors (a-ARs) and calcium channels in cardiovascular and skeletal muscle systems. IBI differs from tolazoline by attachment of an electrophilic isothiocyanato (NCS) group. IBI produced an irreversible, progressive and sustained contraction of rat aorta with a maximal contraction greater than that of tolazoline. The IBI-induced contractions were dependent upon calcium channels (inhibited by verapamil and nifedipine) , and independent of a-ARs (not inhibited by phenoxybenzamine ) or IPRs (not inhibited by efaroxan, moxonidine, idazoxan and cirazoline) . In rabbit diaphragm skeletal muscle, IBI produced contractile responses which were inhibited by dantrolene, an inhibitor of calcium channels present in the sarcoplasmic reticulum. In binding assays, IBI was an inhibitor of dihydropyridine (DHP) binding to calcium 1 1 channels of t-tubule membranes in rabbit skeletal muscle. In contrast to the complete displacement by DHP compounds (nifedipine), IBI and NCS derivatives partially displaced specifically bound radioligand and gave Hill slope values which differed from unity. These results indicate that these NCS derivatives interact allosterically with the DHP binding site. In structure activity relationship studies, IBI analogs were tested to examine their mechanisms of contractile response in rat aorta. Only NCS containing derivatives produced phenoxybenz amine-insensit ive, calcium channel dependent contractions similar to that of IBI. The data indicate that the isothiocyanato (NCS) group is responsible for the calcium channel dependent contractile properties of IBI and its analogs in smooth and skeletal muscles. Thus, IBI and analogs may be useful as affinity probes to characterize sites of interaction on L type calcium channels. Xll Dedicated to Ling, Gracia and my family ACKNOWLEIXaœNTS I sincerely appreciate my academic advisor Dr. Dennis Feller, for his invaluable guidance, assistance and understanding throughout the course of this work; and Dr. Dennis McKay and Dr. Norman Uretsky for serving on the dissertation committee and providing me assistance. I also wish to thank Dr. Duane Miller, Dr. Meri Slavica and Dr. De Los Angeles (Department of Pharmaceutical Sciences, College of Pharmacy, the University of Tennessee, Memphis, TN) for synthesizing the compounds used in this dissertation ; to Dr. Pal Vaghy and Dr. David Johnson (Department of Medical Biochemistry at the Ohio State University) for allowing me generous access to their laboratories; to Dr. Yeindong Jiang (Dr. Johnson's laboratory) and Ms. Sherri Mazetis (Dr. Vaghy's laboratory) for co-performing some experiments; and to the faculty and the graduate fellows of Division of Pharmacology for making my studying here an enjoyable and unforgettable one. I am deeply indebted to my family with which I should have spent more time, and from which I have been constantly recharged with unending encouragement and resources during this research period and hereto journeys. V VITA August 25, 1958......... B o m in Hunan, The People's Republic of China 1977-1982...............M.D., Hunan College of Chinese Medicine, Changsha, China 1982-1984...............Resident Physician, Jiahe Hospital, Hunan, China 1984-1987...............M.S., Hunan College of Chinese Medi c ine, Changsha, China 1987-1990...............Physician, Department of Heart & Kidney, China-Japan Friendship Hospital, Beijing, China 1990-1992...............M.S., Division of Pharmacology, College of Pharmacy, The Ohio State University 1992-present............Research Assistant, Division of Pharmacology, College of Pharmacy, The Ohio State University PUBLICATIONS X. Zhang, X. Yao, J.T. Dalton, G. Shams, L. Lei, P. Patil, D. Feller, F. Hsu, C. George and D.D. Miller: Medetomidine analogs as ttj- adrenergic ligands. 2 . design, synthesis and biological activities of conformationally restricted naphthalene derivatives of Medetomidine, Journal of Medicinal Chemistry 39: 3001-3013, 1996. K.J. Romstedt, L. Lei, D.R. Feller, D.T. Witiak, F . Loiodice and V. Tortorella: Differential eudismic ratios in the antagonism of human platelet function by phenoxy- and thiophenoxyacetic acids. II Farmaco 51: 107-14, 1996. VI L. Lei, M. Slavica, L. De Los Angeles, P.N. Patil, D.D. Miller and D.R. Feller: a-Adrenoceptor and imidazoline receptor- independent contractions of rat aorta : role of the 4'- isothiocyanate group on tolazoline (abstract) . The FASEB Journal 9: 3, A398, March 9, 1995. L. Lei, M. Slavica, P. Emsberger, M.E. Graves, P.N. Patil, D.D. Miller and D.R. Feller : Calcium channel-dependent and I^- imidazoline receptor binding properties of 2 -(4'- isothiocyanatobenzyl) imidazoline analogs in vascular and brain tissues. The Annals of the New York Academy of Sciences 763: 283-286, July 12, 1995. M. Slavica, L. Lei, P.N. Patil, A. Kerezy, D.R. Feller and D.D. Miller: Synthesis and biological activities of a new set of irreversibly acting 2-(4 '-isothiocyanatobenzyl) imidazoline analogs in rat thoracic aorta. Journal of Medicinal Chemistry 37: 1874-81, 1994. L. Lei : Some pharmacological activities of 2- (4 ' - isothiocyanatobenzyl) imidazoline in rat aorta, guinea pig ileum and human platelets, thesis, in the pharmacy library, the Ohio State University, 1993 . L. Lei, M. Slavica, K.J. Romstedt, P.N. Patil, D.D. Miller and D.R. Feller: Non-a-adrenergic actions of 2-(4'-isothiocyanato­ benzyl) imidazoline [IBI] analogs in rat aorta (abstract). The Pharmacologist 34: 148, 1992. L. Lei: Case report on treatment of a patient with congestive cardiomyopathy. The Chinese Journal of Chinese Medicine 10: 60, 1988. L. Lei : Anti-arrhythmic effect of Ping Lu on chloroform- induced arrhythmia in rabbits and epinephrine-induced arrhythmia in guinea pig. thesis, the library of Hunan College of Chinese Medicine, Changsha, PRC, 1985. FIELDS OF STUDIES Major fields : Pharmacy Specific field: Pharmacology V I 1 CONTENT Page Abstract............................................... ii Dedication............................................. iv Acknowledgments........................................ v V i t a ................................................... vi List of Tables......................................... x List of Figures......................................... xii Chapter 1. Introduction 1.1 Fundamentals of drug-receptor theory........... 1 1.2 Calcium channels............................... 12 1.3 a-Adrenergic receptor.......................... 30 1.4 Imidazoline preferring receptors................ 37 1.5 2-(4'-Isothiocyanatobenzyl) imidazoline [IBI] Analog ............................................ 55 1.6 Statement of the problems...................... 60 2. Mechanism of 2-(4'-isothiocyanatobenzyl) imidazoline [IBI] action: calcium channel dependent properties of the contractile responses in smooth and skeletal muscles of rat and rabbit 2.1 Introduction.................................. 65 2.2 Materials and methods.......................... 6 6 2.3 Result........................................ 81 2.4 Discussion.................................... 95 2 .5 Summary and conclusions........................ 110 3. Structure activity relationship