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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 ilustrations appearing in this copy for an additional charge. Contact UMI direclly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 \M IDENTIFICATION OF SYNTHETIC BENZOPYRANONES AS SELECTIVE AGENTS FOR MOLECULAR TARGETS IN BREAST CANCER DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jennifer Lynn Whetstone, B.S. Biochemistry ***** The Ohio State University 2001 Dissertation Committee: Approved by Professor Robert W. Brueggemeier, Adviser Professor Larry W. Robertson a / AdviserAHvicer Professor Pui-Kai Li Pharmacy UMI Number 3031287 UMI* UMI Microfbrm 3031287 Copyright 2002 by Beil & HoweH Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United Stales Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 ABSTRACT Currently, one outof eight American women will develop breast cancer in her lifetime, making breast cancer the second highest cause of mortality of all female cancers. The majority of breast cancer cases (60-70%) are hormone-dependent, meaning that estrogens are needed for the growth of the tumor. Various antiestrogens, including tamoxifen, are widely used for the treatment of hormone-dependent breast cancer. However, the need for newer antiestrogens with greater specificity and reduced side effects exists. The hypothesis of this research is that the design, synthesis, and screening of substituted benzopyranone libraries would allow us to utilize the biological potential of these molecules and develop more selective therapeutic agents for molecular targets in breast cancer. The benzopyranone ring system is the core structure found in a number of natural products such as the flavonoids and isofiavonoids. Substituted 4/f-l-benzopyran-4- ones have shown activity as protein tyrosine kinase inhibitors, estrogen receptor agonists or antagonists, or inhibitors of steroidogenic enzymes. The prevalent literature methods for constructing benzopyianones were not ideally suited for making libraries as these methods suffer from harsh reactkm conditions, poor substituent tolerance, and low yields. Initial synthetic chemistry produced a novel synthetic route utilizing readily ii available salicylic acids and terminal alkynes as starting materials to construct the benzopyranone nucleus. This approach is characterized by mild and high yielding reactions with good functional groiq* tolerance, and it is ideal for developing combinatorial libraries centered around the benzopyranone ring system. The novel solution-phase chemistry developed to synthesize the benzopyranones can be accomplished in several steps. Retrosynthetically, it was envisioned to make the benzopyranones by the cyclization of alkynones. Substituted bis-TBDMS-salicylic acids underwent a one-pot acid chlorination-Sonogashira coupling resulting in the synthesis of the critical intermediate, alkynone, in excellent yields. To date, electronic and steric requirements for these coupling reactions have been determined. Substitutions at the 3-, 4-, and 5-position of salicylic acid, including halogens, aromatic, and methoxy functionalities, have been used in coupling and result in yields ranging from 40-96%. For the Sonogashira coupling, various terminal alkynes were used (aromatic, alkyl, acetal). The one-pot acid chlorination-Sonogashira coupling, key for introducing diversity, displays a wide substituent tolerance in both of the coupling partners. Michael addition of the secondary amine to the alkynone, followed by a 6-endo-trig cyclization results in the formation of the six-membered benzopyranone with yields from 70-96%. By using a secondary amine addition to the alkynone, the synthetic strategy prevents the cyclization of the competing five-membered benzofuranone and thus resolves the regioselectivity problem encountered by previous efforts. Synthetic fii approaches for diversifying the benzopyranone skeleton have also been pursued; substituents at the 3-position on the ring system would dramatically increase the diversity of our library. Evaluation of a library with more than forty synthetic benzopyranones in initial bioassays (cell proliferation, estrogen receptor binding, and aromatase) using human breast cancer cell lines has resulted in agents exhibiting enhanced and differential activities on breast cancer cell growth and on aromatase inhibition. Continued synthetic efforts will concentrate on development of more selective agents for molecular targets in breast cancer based upon the benzopyranone nucleus. IV Dedicated to Shawn Mom, Dad, Beth, & Matt ACKNOWLEDGMENTS My graduate school career is a culmination of not only my individual accomplishments, but also the contributions of many individuals, both in my personal and professional life. I cannot begin to thank everyone for their siq)port and guidance throughout this journey. I would like to specifically recognize and thank the following individuals: Traci Peters Abercrombie, who is responsible for introducing me to my husband and my move to Columbus to become a “true buckeye.” Mindy Gerdeman, thank you for being there on my first and last day of graduate school and everyday in between. Susan Fundy Donaldson, for what doin’? and listening for the answer. Abhijit Bhat, for teaching me the art and skill of chemistry and Indian vodoo magic. Joan Dandrea and Kathy Brooks, for being able to solve any problem. The Whetstone Family, for boating distractions and wedding planning. The Windholtz Family, for always loving me and knowing when to change the subject. Mr. Moo and Izzy, for the countless hours of therapy and love. VI Shawn, for giving me a life outside of graduate school. Dr. Robert Brueggemeier, for allowing me to start my career as a chemist in your laboratory. Thank you for the respect and support you have shown me. vu VTTA November 6,1973 .......................... 3otn-Cincinnati, Ohio 1996 ................................................3.S. Biochemistry, University of Dayton 1996 - present ................................ .Graduate Teaching and Research Associate, The Ohio State University PUBUCATIONS Research Publications 1. R.W. Brueggemeier, J.A. Richards; S. Joomprabutra; A.S. Bhat; JX. Whetstone. Molecular Pharmacology of Aromatase and its Regulation by Endogenous and Exogenous Agents. J. Steroid Biochem. Mol. BioL, 2001,1603, 1-10. 2. R.W. Brueggemeier, X. Gu; J.A. Mobley; S. Joomprabutra; A S. Bhat; J.L. Whetstone. Effects of Phytoestrogens and Synthetic Combinatorial Libraries on Aromatase, Estrogen Biosynthesis, and Metabolism. In Annals o f the New York Academy o f Sciences, Environmenttd Hormones: The Scientific Basis o f Endocrine Disruption; J.A. McLachlan, L J. Guillette, T. Iguchi, Eds.; New York Academy of Sciences, New York,Volume 948,2001, in press. 3. A.S. Bhat; J.L. Whetstone; R.W. Brueggemeier. A Method for the R ^id Synthesis of Benzopyrone Libraries Employing a Resin Capture Strategy. J. Comb. Chem., 2000,2 ,597-599. 4. A.S. Bhat; J.L. Whetstone; R.W. Brueggemeier. Novel Synthetic Routes Suitable for Constructing Benzopyrone Combinatorial Libraries. Tetrahedron Lett., 1999, 40,2469-2472. 5. D. Stewart; J.A. Trauth, JX. Windholtz; O f. Church. Adenosine Nucleoside Analog Synthesis Via Electrophilic Activation of Electron Rich Alkenes. Syn. Commun, 1996, 2 6 , 4279-4288. vm MELDS OF STUDY Major Field: Pharmacy Medicinal Chemistry IX TABLE OF CONTENTS Pagg Abstract........................................................................................................................... ü Dedication ....................................................................................................................... v Acknowledgments ...........................................................................................................vi V ita............................................................................................................................................................................VÜ1 List of Tables............................................................................................................... xiv List of Figures..............................................................................................................