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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. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI* STRUCTURAL PROBES OF RETINOID ACTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Kevin L. Weiss, B.S., R.Ph. ***** The Ohio State University 2001 Dissertation Committee: Approved by Robert W. Curley, Jr., Ph.D., Adviser Robert W. Brueggemeier, Ph.D. Adviser Larry W. Robertson, Ph.D. College of Pharrfiacy UMI Number 3011160 __ ® UMI UMI Microform 3011160 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and teaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT In cancer chemoprevention studies, 4-hydroxyphenylretlnamide (4-HPR) has been shown to be a less toxic, more effective analog of retinoic acid (RA). In tumor cells, research has shown that the biological effects of 4-HPR differ in some ways from retinoic acid. Most investigators find that 4-HPR does not bind to retinoic acid receptors (RARs) and that it induces apoptosis while RA induces differentiation. In order to explore the possibility that the RA-like effects of 4-HPR may be in part due to hydrolysis, we have synthesized 4-hydroxybenzylretinone (4-HBR). Furthermore, previous studies have shown that 4-HPR-O-glucuronide is more potent and less toxic than 4-HPR. The higher potency and lower toxicity of this glucuronide suggests that 4-HBR-O-glucuronide may also provide an in vivo chemotherapeutic advantage. The synthesis of this derivative is also presented. Retinoic acid is thought to support animal growth and cellular differentiation by binding to the RARs. The precursor to RA, retinol (ROL), is not believed to directly activate the RARs. However, our collaborators have previously reported that ROL is only 4 to 7 fold less potent than RA in competing with [^H]-RA for binding to the RARs. In order to further examine the structure activity relationships of such RAR-binding, we have prepared a series of ROL analogs that cannot be readily oxidized to retinoic acid. In addition, another group has recently reported that 4-oxoretinol also binds to the retinoic acid receptors. An improved synthesis of this material is presented. The C-linked analog of retinoyl-p-glucuronide has previously been synthesized in our laboratory. Unfortunately, the known route to this analog is lengthy and suffers from poor isomeric purity of the final product. It has been envisioned that this analog may be synthesized by alkylation of a retinoid acyl anion equivalent that was utilized in the synthesis of 4-HBR and its O- glucuronide. Preliminary investigations toward the newly proposed synthesis are described. Ill DEDICATION ca. 1946 Dedicated to my family IV ACKNOWLEDGMENTS The author wishes to acknowledge his family, loved ones, friends, collaborators and co-workers. Thank you for your guidance and support prior to and throughout graduate school. Mom and Dad Joan Dandrea Kristin Jack Fowble Grandparents Dr. Witiak Kim Dr. Curley Jon Dr. Brueggemeier Tim and Michele Dr. Robertson Sherry and Mike Dr. Abou-lssa and co-workers Tim and Stacy Dr. Clagett-Dame and co-workers L.V. teachers and friends Dr. Albert Awad Susan Dr. Stephen Cutler Kalyan Dr. Michael Milks Venki Maureen Gearhart, PharmD Derek Dr. Michael Panigot Joel Dr. Steve Wong Kathy Brooks Dr. Judy Amburgey-Peters VITA September 20, 1969 ........................... Born - Newark, Ohio May 1992 ..............................................B.S. in Pharmacy, Ohio Northern University 1996....................................................... Chairman, Graduate Student Symposium in Medicinal Chemistry The Ohio State University Spring 1999 ...........................................Lecturer, Medicinal Chemistry The Ohio State University 1992-1997 .............................................Graduate Teaching Assistant The Ohio State University 1992-2000............................................. Staff Pharmacist, Larry’s Drug Inc. 1995-2001............................................. Graduate Research Assistant The Ohio State University PUBLICATIONS Wong, M. P.; Weiss, K. L.; Curley, R. W., Jr. Recent Improvements Towards the Synthesis of the C-glucuronosyl Cancer Chemopreventive (P-D- Glucopyranosyluronate)-4-retinamidophenylmethane. J. Carbohydr. Chem. 1996, 15, 763-768. Clagett-Dame, M.; Curley, R. W., Jr.; Weiss, K. L.; Tephly, L. A.; Sikri, V. “C- Linked Analogs of A/-(4-Hydroxyphenyl)retinamide”, U.S. Patent No. 6,117,845. Weiss, K.L.; Aishafie, G.; Chapman, J.S.; Abou-lssa, H.; Clagett-Dame, M.; Curley, R.W., Jr., An Unhydrolyzable Analog of /V-(4-Hydroxyphenyl)retinamide; Synthesis and Preliminary Biological Studies, Bioorg. Med. Chem. Lett., 2001, 11, in press. VI Abou-lssa, H.; Curley, R.W., Jr.; Aishafie, G.A.; Weiss, K.L.; Clagett-Dame, M.; Chapman, J.S.; Mershon, S.M., "Chemotherapeutic Evaluation of 4-Hydroxybenzylretinone (4-HBR), a Nonhydrolyzable C-Linked Analog of A/-(4-Hydroxyphenyl)retinamide (4-HPR) Against Mammary Carcinogenesis", Anticancer Res., manuscript submitted. Weiss, K. L.; Curley, R. W., Jr., “Synthesis and Evaluation of 4- Hydroxybenzylretinone: A Structural Probe of Retinoid Action”,(manuscript in preparation) FIELDS OF STUDY Major Field: Pharmacy Specialization: Medicinal Chemistry VII TABLE OF CONTENTS Page ABSTRACT........................................................................................................................ Il DEDICATION....................................................................................................................IV ACKNOWLEDGMENTS.................................................................................................. V V IT A ................................................................................................................................... VI LIST OF TABLES........................................................................................................... XII LIST OF FIGURES........................................................................................................XIII LIST OF ABBREVIATIONS........................................................................................XVII CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION...........................................................................................................1 1.1 The Discovery of Vitamin A ...........................................................................1 1.2 Retinoid Nomenclature ...................................................................................2 1.3 Properties of the Naturally Occurring Retinoids ........................................5 1.3.1 Stability, Handling, and Storage .......................................................... 5 1.3.2 Solubility ...................................................................................................6 1.3.3 Ultraviolet-Visible Spectrophotometry ................................................ 6 1.3.4 Nuclear Magnetic Resonance Spectroscopy of Retinoids ..............7 1.4 Retinol Processing ......................................................................................... 8 1.4.1 Intestinal Absorption of Dietary Vitamin A ..........................................8 1.4.2 Transport and Storage of Retinol ......................................................12 1.4.3 Retinoic Acid Biosynthesis ................................................................. 14 1.5 Biological Functions of Retinoids .............................................................. 14 1.5.1 G row th...................................................................................................16 1.5.2 Differentiation ........................................................................................16 1.5.3 Vision ..................................................................................................... 17 1.5.4 Reproduction .........................................................................................20 1.5.5