IN VITROO AND IN VIVO PHARMACOLOGY OF 4-SUBSTITUTED METHOXYBENZOYL-ARYL-THIAZOLES (SMART) AND 2- ARYLTHIAZOLIDINE-4-CARBOXYLIC ACID AMIDES (ATCAA) Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Chien-Ming Li, M.S. Graduate Program in Pharmacy The Ohio State University 2010 Dissertation Committee: Dr. James T. Dalton, Advisor Dr. Robert W. Brueggemeier Dr. Thomas D. Schmittgen Dr. Mitch A. Phelps Copyright by Chien-Ming Li 2010 ABSTRACT Formation of microtubules is a dynamic process that involves polymerization and depolymerization of the αβ-tubulin heterodimers. Drugs that enhance or inhibit tubulin polymerization can destroy this dynamic process, arresting cells in the G2/M phase of the cell cycle. Although drugs that target tubulin generally demonstrate cytotoxic potency in sub-nanomolar range, resistance due to drug efflux is a common phenomenon among the antitubulin agents. We recently reported a class of 4-Substituted Methoxybenzoyl-Aryl- Thiazoles (SMART), which exhibited great in vitro and in vivo potency. SMART compounds effectively inhibited tubulin polymerization in dose dependent manner, suggesting that SMART compounds may bind to tubulin and subsequently hamper the polymerization. To date the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a novel non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using a highly sensitive and specific LC-MS/MS method. The method does not require the use of radiolabeled ligands, can be applied to a wide variety of drugs that either inhibit or promote tubulin polymerization, and allows for studies to define the reversibility of the interaction with ii tubulin. This method was subsequently applied to determine the tubulin binding site of 4- (3,4,5-trimethoxylbenzoyl)-2-phenyl-thiazole (SMART-H). The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel-sites. In metabolic stability studies, SMART-H was used to examine in the liver microsomes of four species (mouse, rat, dog, and human) revealed half-lives between < 5 and 30 min, demonstrating the inter-species variability in the metabolism of SMART-H. The clearance predictions based on in vitro data correspond well with in vivo clearance obtained from mouse, rat, and dog in vivo pharmacokinetic studies. Four major metabolic processes were found in human liver microsomes. We designed and tested four derivatives by blocking labile sites to improve metabolic stability. The oxime and hydrazide derivatives, replacing the ketone site, demonstrated 2-3-fold improved half-life in human liver microsomes, indicating that metabolic stability of SMART-H can be extended by blocking ketone reduction. These studies led us to the next generation of SMART compounds with greater metabolic stability. Evaluation of the in vivo anti-cancer activities of three SMART compounds, SMART-H (H), SMART-F (F) and SMART-OH (OH) with varying substituents at the 4- position of aryl ring, were used to perform in vivo anti-tumor efficacy in human prostate (PC-3) and melanoma (A375) cancer xenograft models. These data indicated that SMART-H and SMART-F showed greater than 70% tumor growth inhibition (TGI), while SMART-OH failed to inhibit tumor growth. In addition, higher dose of SMART-H (15 mg/kg) exhibited 96% PC-3 xenograft TGI after 21-days of treatment without any apparent neurotoxicity. iii In conclusion, these studies provide the first in vivo evidence and proof-of- concept that SMART compounds represent a potent and broad spectrum class of antitubulin agents for the treatment of cancer. iv Dedicated to my parents and my wife v ACKNOWLEDGMENTS I would like to express my deeply appreciation to my advisor, Dr. James T. Dalton, for his guidance, advice and also appreciate the opportunity to be at GTx Inc. for continuing my training in anticancer drug discovery and development. I thank my committee members, Dr. Robert W. Brueggemeier, Dr. Thomas D. Schmittgen, and Dr. Mitch A. Phelps, for their valuable advice. I truly thank Dr. Duane D. Miller, Dr. Wei Li, Yan Lu and Jianjin Chen for helping design all the compounds and helpful discussions. I also thank Dr Eunju Hurh to help me continue ATCAA project. I thank Amanda Jones and Sunjoo Ahn to be my classmates for five years. I would like to express my thanks to my lovely wife, Huei-Ling Chang, for her encouragement and support. I would like to thank my parents for their support. vi VITA March 13, 1976…………………………Born-Tainan, Taiwan 1998……………………………………..B.S. Chemistry National Cheng Kung University, Taiwan 1998-2000………………………………M.S. Analytical Chemistry National Cheng Kung University, Taiwan Advisor: Shu-Hui Chen Thesis: Quantitative detection of N7-(2- hydroxyethyl)guanine adducts in DNA using LC-MS/MS 2001-2005………………………………Research Associate National Health Research Institutes, Taiwan 2005-2008………………………………Graduate Teaching Associate The Ohio State University 2008-present…………………………….Research Associate II GTx Inc. PUBLICATIONS 1. Liao PC, Li CM, Hung CW, Chen SH. Quantitative detection of N(7)-(2- hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Journal of Mass Spectrometry, 36(3): 336-343, 2001. vii 2. Liao PC, Li CM, Lin LC, Hung CW, Shih TS. An online automatic sample cleanup system for the quantitative detection of the benzene exposure biomarker S- phenylmercapturic acid in human urine by electrospray ionization tandem mass spectrometry. Journal of Analytical Toxicology, 26(4): 205-210, 2002. 3. Chang YC, Li CM, Li LA, Jong SB, Liao PC, Chang LW. Quantitative measurement of male steroid hormones using automated on-line solid phase extraction-liquid chromatography-tandem mass spectrometry and comparison with radioimmunoassay. Analyst, 128(4): 363-368, 2003. 4. Hsieh PW., Chang FR., Wu CC., Wu KY., Li CM., Wang WY., Gu LC, Wu YC. Selective Inhibition of collagen-induced platelet aggregation by a cyclic peptide from drymaria diandra. Helvetica Chimica Acta, 87(1): 57-66, 2004. 5. Hsieh PW, Chang FR, Wu CC, Wu KY, Li CM, Chen SL, Wu YC. New cytotoxic cyclic peptides and dianthramide from Dianthus superbus. Journal of Natural Products, 67(9): 1522-1527, 2004 6. Hsieh PW, Chang FR, Wu CC, Li CM, Wu KY, Chen SL, Yen HF, Wu YC. Longicalycinin A, a new cytotoxic cyclic peptide from Dianthus superbus var. longicalycinus (MAXIM.) WILL. Chemical & Pharmaceutical Bulletin (Tokyo), 53(3): 336-338, 2005. 7. Li CM, Hu CW, Wu KY. Quantification of urinary N-acetyl-S- (propionamide)cysteine using an on-line clean-up system coupled with liquid chromatography/tandem mass spectrometry. Journal of Mass Spectrometry, 40(4): 511-515, 2005. 8. Li CM, Chu RY, Hsientang Hsieh DP. An enhanced LC-MS/MS method for microcystin-LR in lake water. Journal of Mass Spectrometry, 41(2): 169-174, 2006. 9. Wang SL, Chang YC, Chao HR, Li CM, Li LA, Lin LY, Papke O. Body burdens of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls and their relations to estrogen metabolism in pregnant women. Environmental Health Perspectives, 114(5): 740-745, 2006. viii 10. Huang CC, Li CM, Wu CF, Jao SP, Wu KY. Analysis of Urinary N-acetyl-S- (propionamide)-cysteine as a biomarker for the assessment of acrylamide exposure in smokers. Environmental Research. 104(3): 346-51, 2007. 11. Lu Y, Li CM, Wang Z, Ross CR, Chen J, Dalton JT, Li W, Miller DD. Discovery of 4-substituted methoxybenzoyl-aryl-thiazole as novel anticancer agents: synthesis, biological evaluation, and structure-activity relationships. J Med Chem. 52(6):1701- 11, 2009 12. Li CM, Yeh TK, Chen CP, Chuu JJ, Huang CL, Wang HS, Shen CC, Lee TY, Chang CY, Chang CM, Chao YS, Lin CT, Chang JY, Chen CT. Antitumor activities and pharmacokinetics of silatecans DB-67 and DB-91. Pharmacol Res. 61(2):108-115, 2010. 13. Lu Y, Wang Z, Li CM, Chen J, Dalton JT, Li W, Miller DD. Synthesis, in vitro structure-activity relationship, and in vivo studies of 2-arylthiazolidine-4-carboxylic acid amides as anticancer agents. Bioorg Med Chem. 18(2): 477-495, 2010. FIELD OF STUDY Major Field: Pharmacy ix TABLE OF CONTENTS Abstract.............................................................................................................................. ii Dedication.......................................................................................................................... v Acknowledgments ............................................................................................................ vi Vita................................................................................................................................... vii Table of Contents...............................................................................................................x List of Tables ................................................................................................................... xv List of Figures................................................................................................................ xvii 1. Introduction....................................................................................................................