DESIGN, SYNTHESIS, AND EVALUATION OF THIAZOLIDINEDIONE DERIVATIVES INHIBITING BCL-2/BCL-XL OR ABLATING ANDROGEN RECEPTOR FOR THE TREATMENT OF PROSTATE 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 Jian Yang, M.S. * * * * * The Ohio State University 2009 Dissertation Committee: Approved by Professor Ching-Shih Chen, Advisor Professor Pui-Kai (Tom) Li Adviser Professor Werner Tjarks Pharmacy Gradate Program Professor Dale Hoyt COPYRIGHT BY JIAN YANG 2009 ABSTRACT As of 2008, prostate cancer remains the number one cause of estimated new cancer cases in American men. Although early prostate cancer responds to androgen ablation, most tumors eventually recur as hormone-refractory prostate cancer (HRPC). Recent advances have identified many abnormal signaling pathways that contribute to the development of HRPC, mainly including dysregulation of androgen receptor (AR) function and the over- expression of the anti-apoptotic proteins, Bcl-2/Bcl-xL. It was found that thiazolidinediones (TZD), which include the anti-diabetic agents troglitazone, rosiglitazone, pioglitazone and ciglitazone, possess anti-tumor activity. It has been revealed that the anti-tumor activity of the TZDs could be attributed to PPARγ- independent mechanisms including Bcl-2/Bcl-xL inhibition and AR transcriptional repression. The objective of this dissertation is to further modify the TZD structures to enhance anti-prostate cancer activity by targeting the dysregulated Bcl-2/Bcl-xL and AR signaling pathways. Directed by method of molecular modeling, over 50 troglitazone derivatives have been designed and synthesized in two stages, yielding the optimal compound HepCNCF3, which showed two orders of magnitude improvement in Bcl-xL/Bcl-2 inhibition ii compared to troglitazone. Results from molecular modeling, Western blotting assay and co-immunoprecipitation assay confirmed the inhibitory effects of HepCNCF3 on Bcl- 2/Bcl-xL and its function of apoptosis induction at low micromolar level in prostate cancer cells. The effects of ciglitazone on transcriptional repression of AR have been verified, and as a lead compound, ciglitazone possessed advantages of simpler molecular and stronger potency compared to troglitazone. Partly aided by method of focused-library solid-phase combinatorial chemistry, three stages of optimization have been performed based on ciglitazone. Out of totally approximately 150 new derivatives, the optimal compounds such as CG12, CC5 and CC22 showed one order of magnitude improvement in AR repression and cytotoxicity in LNCaP cells than ciglitazone. The optimized activites of these compounds have been confirmed in AR reporter gene luciferase assay, Western blotting assay and flow-cytometry analysis. Overall, through comprehensive methods of computer-based design, ligand-based design and solid-phase combinatorial chemistry, several promising agents targeting on the abnormalities in prostate cancer, particularly in HRPC, have been successfully developed and their effects of Bcl-2/Bcl-xL inhibition or AR ablation have been validated. iii Dedicated to my grandparents, my parents, and especially my wife Yanhui and my daughter Alisa iv ACKNOWLEDGEMENTS I would like to give special thanks to my advisor, Dr. Ching-shih Chen for his guidance, encouragement, support and patience. I could never finish my Ph.D training without his consistent kind help. I would also like to express my sincere appreciation for the faculty in college of pharmacy, especially Dr. Pui-Kai (Tom) Li, Dr. Werner Tjarks, Dr. Dale Hoyt, Dr. Kenneth Chan and Dr. Duxin Sun for all their help and advice. I am grateful to Dr. Da-sheng Wang for his advices in chemistry and Dr. Samuel Kulp for his assistance with animal studies and proofreading. I would like to thank Wei (Dennis) Shuo, Dr. Hao-Chieh(Jay) Chiu, Errica and other labmates in helping biological evaluation. I would like to thank Hsiao-Ching Yang and Su-Lin (Jack) Lee in helping molecular modeling. I would like thank Ms Judith Gallucci for analyzing the crystal structure and providing the experimental details. I want to thank Dr. Pui-Kai (Tom) Li, Ms Kathy Brooks, Ms Beth Bucher and other staff in my gradate program, office of international affairs and the graduate school for their support to make my graduate studies going smoothly. I would like give thanks to all the members in Dr. Chen’s lab and all my friends including Ye’s family, Yunlong’s family, Mathew, Xiaobing’s family, Mingxuan’s family, Dennis’ family and so on during my Ph.D study. Particularly, I would like present many thanks to my wife, Yanhui Lu, my daughter, Alisa Yang, my parents and my parents-in-law. v VITA 1994-1998……………………………………………….. B.S., Medicinal Chemistry College of Pharmacy, Beijing Medical University, Beijing, China 1998-2000………………. Professional Teacher Assistant and Research Assistant College of Pharmacy, Beijing Medical University, Beijing, China 2000-2003………………………………………………. M.S., Medicinal Chemistry College of Pharmacy, Peking University, Beijing, China 2003-2004……………………………………………………………. Group Leader Haimen Wisdom Pharmaceutical Co., Haimen, Jiangsu, China 2004-present……………. Ph.D. Candidate, Medicinal Chemistry & Pharmacognosy The Ohio State University, Columbus, OH, USA PUBLICATIONS 1. PPARγ-Independent Antitumor Effects of Thiazolidinediones. Shuo Wei, Jian Yang, Su-Lin Lee, Samuel K. Kulp, and Ching-Shih Chen. Cancer Letters (2009) Cancer Letters 276, 119–124. 2. Pharmacological Exploitation of the PPARγ Agonist Ciglitazone to Develop a Novel Class of Androgen Receptor-Ablative Agents Jian Yang, Shuo Wei, Dasheng Wang, Yu-Chieh Wang, Samuel K. Kulp, and Ching-Shih Chen. Journal of Medicinal Chemistry (2008) 51, 2100–2107. 3. A Novel Mechanism by Which Thiazolidinediones Facilitate the Proteasomal Degradation of Cyclin D1 in Cancer Cells. Shuo Wei, Hsiao-Ching Yang, Hsiao- Ching Chuang, Jian Yang, Samuel K. Kulp, Pei-Jung Lu, Ming-Derg Lai, and Ching-Shih Chen. Journal of Biological Chemistry (2008) 283, 26759-70. 4. Bioassay-directed Purification of an Acidic Phospholipase A2 from Agkistrodon halys pallas Venom. Yuwei Wang, Guohui Cui, Ming Zhao, Jian Yang, Chao Wang, Roger W. Giese, and Shiqi Peng. Toxicon (2008) 51, 1131–1139. 5. Development of Small-Molecule Cyclin D1-Ablative Agents. Jui-Wen Huang, Chung-Wai Shiau, Jian Yang, Dasheng Wang, Hao-Chieh Chiu, Ching-Yu Chen, and Ching-Shih Chen. Journal of Medicinal Chemistry (2006) 49, 4684-4689. 6. Synthesis and Evaluations of Pentahydroxylhexyl-L-Cysteine and Its Dimer as Chelating Agents for Cadmium or Lead Decorporation. Chao Wang, Ming Zhao, Jian vi Yang, Xingwei Li, and Shiqi Peng. Toxicology and Applied Pharmacology (2004) 200, 229-236. 7. Identification, Synthesis and Bioassay for the Metabolites of P6A. Ming Zhao, Chao Wang, Jian Yang, Jiangyuan Liu, Youxuan Xu, Yanfen Wu and Shiqi Peng. Bioorganic & Medicinal Chemistry (2003) 11, 4913-4920. 8. Studies on the Synthesis of Estrogen-GHRPS. Chao Wang, Ming Zhao, Weina Cui, Jian Yang, and Shiqi Peng. Synthetic Communication (2003) 33, 1633-1641. 9. Studies on the Synthesis and Anti-osteoporosis of Estrogen-GHRPs Linkers. Chao Wang, Weina Cui, Ming Zhao, Jian Yang, and Shiqi Peng. Bioorganic and Medicinal Chemistry Letters (2003) 13, 143-146. 10. Synthesis and Analgesic Effects of Kyotorphin - Steroid Linkers. Chao Wang, Ming Zhao, Jian Yang, and Shiqi Peng. Steroids (2001) 66, 811-815. 11. Synthesis of RGD-Containing Peptides and Their Vasodilation Effect. Ming Zhao, Chao Wang, Jian Yang, and Shiqi Peng. Preparative Biochemistry & Biotechnology (2000) 30, 247-256. 12. The Establishment of a Rat Model of Thrombolysis and Its Application. Na Lin, Jian Yang, Chao Wang, Ming Zhao, and Shiqi Peng. Journal of Beijing Medical University (2000) 32, 283-284. 13. Androgen Receptor-Ablative Agents. Ching-Shih Chen, Dasheng Wang, Jian Yang. United States Provisional Patent, Serial No. 61/030,860; filed on February 22, 2008. 14. Imidazoline Substituted Phenyloxyacetyl Oligopeptide Compounds: Their Synthesis and Applications in Medicine. Shiqi Peng, Ming Zhao, Chao Wang, Junling Liu, Jian Yang. Chinese Patent, Application No. 02134992.4, 2003. 15. Imidazoline Substituted Phenyloxyacetyl Oligopeptide Derivatives: Their Synthesis and Applications in Medicine. Shiqi Peng, Ming Zhao, Chao Wang, Junling Liu, Jian Yang. Chinese Patent, Application No. 02134994.0, 2003. FIELDS OF STUDY Major Field: Pharmacy Specification: Medicinal Chemistry vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii Dedication..……………………………………………………………………………….iv Acknowledgements………………………………………………………………..………v Vita………………………………………………………………………………………..vi List of tables………………………………………………………………………………xi List of figures……………………………………………………………………………xiv Chapters: 1. Introduction………………………………………………………………………..……1 1.1 Prostate cancer, current treatments and agents in development…………….………1 1.2 Mechanisms of castration resistant prostate cancer (CRPC) …………..…………...5 1.3 AR as a good target for treatment in CRPC……………………………………..…..7 1.4 Anti-apopotic proteins Bcl-2/Bcl-xL as targets for treatment of CRPC……….……9 1.5 Thiazolidinediones, mechanisms in anti-cancer and structure optimization………13 2. Development of troglitazone derivatives targeting on Bcl-2/Bcl-xL directed by molecular modeling…………………………………………………………………...…17 2.1 Design of ∆2TG derivatives directed by molecular modeling ……………………17 2.2 ∆2TG derivatives with an n-alkyl side chain …………………………………...…21 2.3 ∆2TG