Luận án tiến sĩ: Design, Synthesis and Bio-Evaluation of New Curcumin Analogs as Potential Drug Candidates for The Treatment of Prostate Cancer

DESIGN, SYNTHESIS AND BIO-EVALUATION OF NEW CURCUMIN ANALOGS AS POTENTIAL DRUG CANDIDATES FOR THE TREATMENT OF PROSTATE CANCER by Li Lin A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in School of Pharmacy Chapel Hill 2005 Approved by: Advisor: Dr. Kuo-Hsiun Lee Yk) - Reader: Dir. Bastow ¬ > } fe ont Reader: Dr. Arnold Brossi Reader: Dr.

Jian Liu Rehder: Dr. Alexandra Tropsha UMI Number: 3212470 INFORMATION TO USERS 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 bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted.

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ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT Li Lin: Design, Synthesis and Bio-evaluation of New Curcumin Analgos as Drug Candidates for the Treatment of Prostate Cancer (Under the direction of Kenan Professor Kuo-Hsiung Lee) Curcumin is the major yellow pigment isolated from the rhizome of Curcuma longa, known as turmeric. Over a long period of study, curcumin has been found to possess a wide range of bioactivities including anti-prostate cancer activity in vitro and in vivo. Based on these observations, our laboratory has been using curcumin as the lead compound to develop various analogs as potential anti-prostate cancer agents.

Two curcumin analogs, dimethyl curcumin (DMC, 4) and 4-ethoxycarbonylethyl curcumin (ECECur, 5) developed previously were found to be active anti-androgen receptor agents, which showed greater potency than hydroxyflutamide, an anti-androgen currently used clinically. However, the tautomerism of ECECur, which causes it to exist in both enol-keto and di-keto forms, may hinder its potential as a clinically useful drug. To solve the problem inherent in the tautomerism of ECECur, I subsequently designed, synthesized and evaluated several new curcumin analogs for the anti-androgen receptor activity as well as cytotoxicity 1n prostate cancer cell lines. To establish an extensive structure-activity relationship (SAR) for curcumin analogs as anti- prostate cancer agents, I designed and synthesized four series of new curcumin analogs having various structural features.

Based on the structures and the bioactivities of these new il compounds, I studied the structure-activity relationship of curcumin analogs in an extensive level and discovered several structural features of curcumin analogs responsible for their anti-prostate cancer activity including 3’,4’-dimethoxy phenyl rings or 3’-methoxy-4’- hydroxy phenyl rings, unsaturated and conjugated linker, proper substitution at C4 position of the linker and so on. This new information will guide us for the further optimization of curcumin analogs as anti-prostate cancer agents. Besides three conjugates were designed and synthesized in this work. Through this study, the problem of tautomerism of ECECur has been solved successfully and sixteen new potent curcumin analogs (11, 12, 13, 14, 15, 16, 31, 34, 35, 37, 41, 43, 44, 50 and 52) were developed as promising anti-prostate cancer drug candidates for further investigation in vivo.

1H ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Kuo-Hsiung Lee, and my committee members, Dr. Jian Liu, Dr. Alexandra Tropsha and Dr.

Arnold Brossi, for their guidance through my Ph. I would also like to give great appreciation to Dr. Qian Shi, Dr. Ching-yuan Su and Dr.

Charles Shih in Androscience Incorporation for their help in my research. I am especially grateful to my parents Zhiguo Lin and Jianping Luo, my boyfriend Michael Chou, and my roommate Di Hu for their heartfelt support and encouragement. iv Dedicated to My Grandfather, Rongfa Lin TABLE OF CONTENTS Page LIST OF TABLPES. HH TK eee eee ebe eee ea teen neeaeeaeeateneneeenees X LIST OF FIGURES.

ST nnn ĐK ĐK KT kh kg xi LIST OF SCHEMES. HH HH nnn Eee Ki ko KĐT nếp xI LIST OF ABBREVIATIONS. SH nh ĐK Bà nh XI CHAPTERS 1. Introduction—Prostate Cancer and Clinical Antiandrogenic Agents 1.1 Prostate Cancer and Risk Factors .ccesceeee eee e eee e eee een ee nee nas 1 1.2 Androgens and androgen receptor (AR) in the prostate.3 Treatment of Prostate CanC€T.

nhe nhe nh se 3 1.4 Clinical Antiandrogenic Agents for the Treatment of Prostate CATC€T. eee en EE EEE EEE EE EEE He 4 1.5 Antiandrogen Withdrawal Syndrome. Background—Curcumin and Curcumin Analogs 2.1 Drug Discovery from Natural Products.2 The Origin of CurCUIH.3 The Bioactivities of CurcumIn.4 The Anti-Prostate Cancer Activity of CurcumIn.5 The Anti-Prostate Cancer Activity of Curcumin Analogs. Research Part I—The Resolution of the Tautomerism of 4-Ethoxycarbonylethyl Curcumin (ECECur) 3.

ee nn ne nr rennet nner tenets 14 3. ch nà Ti n kh tk BH 15 3.3 Synthesis of New ECECur Analogs.4 Biological Results and DDIscussion. nen nhe21 XO k ©0900) 106°) (0) 0 Oe 27 3.ccceecee cece eee e eee ene eee nh ne ene nn nh hiện 27 4. Research Part II—Design, Synthesis and Bio-evaluation of Four Series of Curcumin Analogs 4.c en ee ence nn renner renee reas 38 4,2 Monophenyl Curcumin Analogs 4.

nh nh nh nh kh k ket 39 4.cn nền nh nh nh nh nh 39 4. 39 cọ nhe nhe nhe 4.4 Discussion and conclusion.3 Heterocyclic Curcumin Analogs 4. kh nh nh ng 41 con nh nh. nh nh nh be nhà 41 cọ HH nh nh.

43 on nh nh nh nh heo 4. Discussion and conclusion.4 New Curcumin Analogs Bearing Various Substituents on Phenyl Rings 44. ch ern nh nhe nở 44 vil 4. cece cece cece seen tenet eee nh nh nh nh kh 44 4.

Hs n nen nen nh re, 45 4.4 Discussion and conclusion.5 New Curcumin Analogs with Various Linkers ““¬. uc nh nh nh TK kh khe 51 4.4 Discussion and conclusion.6 Antiandrogenic Acitivity of the Four Series of New Curcumin AnaÌOgS.‹ en ene nner nh nh eer EEE nh trên 57 4.7 Conclusion in the SARs of Curcumin Analogs. ST nen nh rere 60 5. Research Part II—Design, Synthesis and Bio-evaluation of Curcumin Analogs Conjugated with Anti-Prostate Cancer Drugs 5.

eee HH nh enn EEE nh 76 5. cece cece ee EE EE tenes 77 5. eee BE BE rene nnn eee 79 2à nh am.5 Discussion and ConcÏuslon. HH nh nh nh kh nhe eae 85 6.

Conclusions and Future Studies 6. en ence teens ee eet eee eet ng ng kh vn EEE EEE te 91 6. ccc cece c ccc cece teen cece seen eens eens setae este nese beeen kg 95 Vill REFERENCES. HH EEE EEE Kế ng rene nề ha APPENDIX I: The Cytotoxicity of Selected Analogs Against A Panel Of Cell LANES.

ccc enn EEE ng EEE EEE EE EE EEE Enea 1X LIST OF TABLES Table 4.1 The chemical structures of curcumin analogs 17-23 and their cytotoxicity against LNCaP and PC-3 human prostate cancer Cell ÏIn€§. cece cee nsennveseececeeeteterenrnrnnnes Table 4.2 The chemical structures and cytotoxicity of compounds 24-26 against LNCaP and PC-3 human prostate cancer cell lines.3 The chemical structures of series C of curcumin analogs and their cytotoxicity against LNCaP and PC-3 human prostate cancer cell lines.-ccccc cv bàn bà.4 The chemical structures of curcumin analogs with various linkers and their cytotoxicity against LNCaP and PC-3 human prostate cancer cell lines.5 The antiandrogenic activity of compound 5, 11, 12 and 15.1 The cytotoxicity of starting materials, intermediates and target conjugates against LNCaP and PC-3 human prostate cancer cell lines.‹‹-c cà S2 nhe Table 6.1 The cytotoxicity of the active curcumin analogs towards - LNCaP and PC-3 human prostate cancer cell lines.I The cytotoxicity of selected compounds against a panel Of CELL ]1TIỂS. cece cece e eee eee cee te een ng ng ng seen nh khu LIST OF FIGURES Figure 1.1 Androgen receptor signaling pathWay. ch nhe ee ke 3 Figure 1.2 The chemical structures of some clinical antiandrogenic agents.1 Curcuminoids from Curcumad longa.cccccccccc cece ccc ce eee ene enter etn se, 10 Figure 2.2 Anti-AR curcumin analogs DMC (4) and ECECur (5).1 The design of di-keto ECECur analog 6 and enol-keto analog 7.2 Anti-AR activity of compound 4-11 (3 uM) in prostate cancer cells and their cytotoxicity against the growth Of LNCaP celÌS.

con HH TT nee nee entre tk xa24 Figure 3.3 The conformations of ECECur (Š). con nh nhe ho 25 Figure 3.4 Anti-AR activity of compound 4, 5, 12-16 (5 uM) in prostate cancer cells and their cytotoxicity against the growth of LNCaP cells. uc nh nh khe 26 Figure 4.1 Monophenyl curcumin analOgØS.c con nhe39 Figure 4.2 Curcumin analogs with various lInK€TS. cà cà 50 Figure 5.1 Structures of antiandrogens used 1n the cÌinIc.2 The design of curcumin analogs conjugated with N-arylmethacrylamide mOI€fy.

co cọ nh nh een ke nh 79 Figure 5.3 The antiandrogenic activity of 54-60 in LNCaP cells and PC-3 cells transfected with wild-type andrOgen LECEPLOL. cee EEE EER EE EEE EEE 84 Figure 6.1 The pharmacophores derived from SAR of curcumin analogs for inhibition against the growth of human prostate cancer cells 1n VITO.ị e tte sen nhe 94 Figure 6.2 Optimization of C-4 side chain of curcumin analogs.3 Some proposed novel curcumin conJugafes. 99 XI LIST OF SCHEMES Scheme 3.1 Synthesis of compound 6. c2» nh hề nh 18 Scheme 3.2 Synthesis of compounds 7, 10-13 and 16.3 Synthesis of compounds 14 and 15.1 The general synthetic method of monophenyl curcumin analogs.2 The general synthetic methods of heterocyclic nh kh se 42 en nhe Qnn curcumin analogs 24 — 27 .3 The general synthetic strategy of some symmetric Curcumin analOgÿS.

ba 45 EE EEE nh hà kh .4 The general synthetic scheme of some asymmetric CULCUMIN analOgS. nh nh hệ 45 enn nh nh nh EE nh.5 The general synthetic method of 1, 5-diphenyl-1,4-pentadiene-3-ONnes. cece cece tenes 52 Scheme 4.6 The synthesis of curcumin analogs 46 and 47 having .ch te 52 EEE kh kh.7 The hydrogenation of DMC to 48. 52 co cà nhe hen Scheme 4.8 The synthesis of analog 49.

nh nh kh 53 cà eee eee nh nh kh.9 The synthesis of imide analog 5Ũ. cành ee 53 Scheme 4.10 nen nnn The synthesis of 52 and 53.1 cành nena es 81 The synthesis of conjugates 55 and 56.2 The synthesis of conjugate 60. cere ne nh nhe ch: 81 xi LIST OF ABBREVIATIONS acetyl androgen receptor ARE androgen receptor binding element CPA cyproterone acetate d Doublet dd doublet of doublet DHP Dihydropyran DHT 5a-dihydrotestosterone Dibal-H diisobutylaluminum hydride DMC dimethyl curcumin DMF N, N-dimethylformamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid ECECur 4-ethoxycarbonylethyl curcumin EGF epidermal growth factor HF hydroxyflutamide HIV human immunodeficiency virus HMDA 1,1,1,3,3,3-Hexamethy! disilazane Hsp heat-shock proteins IC50 inhibitory concentration that is toxic to 50% of the cells Xiil LDA lithium diisopropylamide Multiplet m/z ratio of mass by an ion's charge number MAPK mitogen-activated protein kinase MMTV mouse mammalian tumor virus NF-KB nuclear factor kappa B PPTS pyridinium toluene 4-sulfonate PSA prostate-specific antigen Singlet SAR Structure-activity relationship Triplet THF tetrahydrofuran THP tetrohydropyranyl TNF tumor necrosis factor XIV CHAPTER 1 INTRODUCTION — PROSTATE CANCER AND CLINICAL ANTIANDROGENIC AGENTS 1.1 Prostate Cancer and Risk Factors Prostate cancer is the most prevalent cancer in American men and the second leading cause of cancer-related death among men in the United States.' The well- established risk factors for prostate cancer include age, race, family history, diet, and environmental agents.? Age is the most important factor in prostate cancer development. A man under the age of 40 rarely develops detectable prostate cancer, while about 80% of prostate cancer is diagnosed in men over 65.

A wide variation in incidence has been reported among different races and ethnic groups.