INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be fiom any 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 affect 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, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one eq)osure 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 & Howell Information Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 PHARMACOLOGICAL EVALUATION OF TRIMETOQUINOL ANALOGS AS AFFINITY LIGANDS FOR S-ADRENERGIC RECEPTOR SYSTEMS A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Ratnavali C. Mehta, B. Pharm., D.M.S., M.S.(Pharm). ***** The Ohio State University 1997 Dissertation Committee: Approved by Dennis R, Feller, Ph.D., Advisor Advisor Popat N. Patil, Ph.D. Norman J. Uretsky, Ph.D. Co- isor Lane J. Wallace, Ph.D., Co-advisor College of Pharmacy UMI Number: 9731678 UMI Microform 9731678 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arhor, M l 48103 ABSTRACT Analogs of the S-adrenoceptor (S-AR) agonist, trlmetoquinol (TMQ) possessing potent agonist activity and selectivity at the S 3 -AR subtype, may have use in the treatment of obesity, non-insulin-dependent diabetes and gastrointestinal hypermotility disorders. While the catechol portion of the compound is essential for potent agonist activities at the B,- and Sj-ARs, the 1-benzyl side-chain of TMQ is important for selective activity on the S 3 -AR subtype. Characterization of the interactive site for the 1-benzyl ring of TMQ is necessary for rational design of potent and selective B 3 -AR agonist analogs. Hence, 4'-substituted analogs including affinity (a-haloacetamido and isothiocyanato) and photoaffinity (azido) analogs of 3'-iodo-5'-demethoxy- or 3',5'-diiodo-TMQ were evaluated for their binding affinities and biochemical potencies ( cAMP accumulations) on rat S 3 -AR and the three human B-AR subtypes expressed in Chinese hamster ovary (CMC) cells, and for functional activities on isolated rat tissues. These TMQ analogs possessed higher affinities and agonist potencies than (-)-isoproterenol on the B-AR subtypes, and the biochemical potencies were in agreement with values obtained in functional assays. Agonist potencies were comparable to that of the B 3 -AR-selective agonist, BRL 37344; however, unlike the latter compound, the TMQ analogs IX are full agonists on the hum an IÎ3-AR. 4 ’-Acetamido-3',5’-dliodoTMQ and 4'- azido-3’-lodo-5'-demethoxyTMQ are among the most potent S-AR agonists currently known. Activities of the TMQ derivatives and isoproterenol on human B^-AR were blocked by propranolol in a concentration-dependent manner. However, the agonist-dependent discordance in the pA^ values of propranolol on human S ;AR implies that sites of interaction of the 4'-acetamido- and 4'-a-chloroacetamido- 3 ,5 -diiodoTMO analogs with this receptor differ from that of isoproterenol. Additionally, 4 ’-a-chloroacetamido-3',5'-diiodoTMG and 4'-isothiocyanato-3'- iodo-5 -demethoxyTMQ demonstrated significant concentration-dependent irreversible binding to the rat S 3 - and human Sg-AR, respectively. However, the failure of reversible S-AR ligands to protect against this irreversible binding indicates that the lipophilic 1 -benzyl ring of these affinity TMQ analogs interacts with a hydrophobic region of the S-AR that may represent an "exo-site" or allosteric binding site on the receptor. By contrast, the photoaffinity (azido) TMQ analog demonstrated concentration- and time-dependent Irreversible binding to the human Sg-AR. This w as protected by com peting S-AR ligands, implying an overlap of interaction sites of these compounds within the receptor binding domains. These studies confirm the importance of the 1-benzyl ring of TMQ in agonist interactions with the S-ARs. Affinity and photoaffinity labels of the 1 - benzyl substituted analogs of TMQ may serve as useful tools for characterizing the receptor binding domains of the S-AR, and thereby provide clues for the design of potent and selective S 3 -AR agonist analogs of TMQ. XXI Dedicated to my mother IV ACKNOWLEDGMENTS I wish to express my sincere gratitude to: Drs. Popat Patil, Norman Uretsky and Lane Wallace for their participation on my research committee and for their tremendous support and guidance. I really appreciate the academic discussions related to my research with Dr. Patil. These enhanced my understanding of the field. Dr. Paul Fraundorfer, Edwin Lezama and Anish Konkar for all their help and support during my initiation into research in the Feller laboratory and for their input in occasional trouble-shooting. Shamina Rangwala, Dr.Gamal Shams and Longping Lei for their friendliness and for the fun times we shared as members of the Feller group. Dr. Richard Fertel, Dr. Dan Mullet and Nina Zaveri, for their help with cAMP radioimmunoassays; Dr. Margarita Salazar-Bookaman and Dr. Asad Dalia, for their technical support with animal studies; Dr. Duane Miller and his group, for synthesizing the TMQ analogs. Rose Smith, for her cheerful smile, significant help and moral support. Kathy Brooks, for her excellent support throughout the duration of my stay in the college. All the faculty, staff and student members of the division of pharmacology and college of pharmacy, who accepted me as a member of the group and helped me along the way. Marina Bergman, Shridhar Narayan, Virunya Singhatat and Janette Loch, who made life in the division especially enjoyable. Thank you for your friendship and for great support through the times. Dr. Dennis Feller, who has been a great advisor and a very considerate person. Thank you for all your understanding, patience, guidance and support throughout the time I have known you. I consider myself very fortunate to have had the opportunity to work with you. National Institutes of Health (USPHS HL-22533) for financial support of this research project. VI VITA Jan 16, 1962 ...................................... Born, Bombay, India 1982 ....................................................... 8 . Pharm., Bombay Univ. Dept. Of Chemical Technology, India 1988 .......................................................Diploma in Management Studies, Bombay University, India 1991-9 2 ................................................ University Fellow, The Ohio State University, USA 1992-9 4 ................................................ Graduate Teaching Associate, The Ohio State University, USA 1994 .......................................................M.S. Pharmacy, The Ohio State University, USA 1994-1997 ............................................Graduate Teaching/Research Associate, The Ohio State University, USA PUBLICATIONS Miller DD, Christoff J J , De Los Angeles J , Nikulin V, Konkar A, M ehta R, Fraundorfer PF and Feller DR, Sites of interaction of trimetoquinol and related analogs with S-adrenergic receptors. Pharmaceut Res 11(10): SI 23, 1994. V l l Sham s G, Mehta R, Romstedt KJ, De Los Angeles J , Nikulin VI, Miller DD and Feller DR, Affinity labels of trimetoquinol: Interaction with thromboxane A;/ prostaglandin endoperixide and f^-adrenergic receptors. FASEB J 9{2): A398, 1995. Nikulin VI, De Los Angeles J, Sham s G, M ehta R, Feller DR and Miller DD, Synthesis of antithromboxane A; and R-adrenergic activity of iododerivatives of trimetoquinol (TMQ). National ACS Meeting, Anaheim, CA, MEDI188, 1995. De Los Angeles J, Nikulin VI, Shams G, Konkar A, Mehta R, Feller DR and Miller DD, lodinated analogs of trimetoquinol (TMQ) as highly potent and selective Bz-adrenergic Ligands. J Med Chem 39(19): 3701-11, 1996. Mehta R, Salazar-Bookaman MM, Fertel RH, De Los Angeles J , Miller DD and Feller DR, Biochemical and functional activities of affinity trimetoquinol (TMQ) analogs at rat B-adrenoceptors. The Pharmacologist 39{^): A282, 1997. FIELD OF STUDY Major Field: Pharmacy Studies in: B-Adrenergic Pharmacology V l l l TABLE OF CONTENTS P age ABSTRACT........................................................................................................................ ii DEDICATION.................................................................................................................. iv ACKNOWLEDGMENTS.................................................................................................. v VITA..................................................................................................................................vii