Design, Synthesis, and Evaluation of Thiazolidinedione Derivatives Inhibiting Bcl-2/Bcl-Xl Or Ablating Androgen Receptor for the Treatment of Prostate Cancer

Home , Ciglitazone, Rivoglitazone

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

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

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 overexpression 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

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.

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Dedicated to my grandparents, my parents, and especially my wife Yanhui and my daughter Alisa

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.

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

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

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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 derivatives with an unsatuated n-alkyl side chain…………………………..22

2.4 ∆2TG derivatives with a branched alkyl side chain……………………………….23

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2.5 ∆2TG derivatives with a CN substituted alkyl side chain…………………………24

2.6 ∆2TG derivatives with side chain of various H-bonding acceptors at the

tail……………………………………………………………………………….…25

2.7 ∆2TG derivatives with OMe or CF3 substitution at the phenyl group……………28

2.8 Reduced forms and R-forms of optimized ∆2TG derivatives………………..……29

2.9 Detailed mechanistic evidences of targeting on Bcl-2/Bcl-xL for optimized ∆2TG

derivatives………………………………………………………………………….30

2.10 Molecular modeling evidence of the binding between HepCNCF3 and Bcl-xL

protein……………………………………………………………………………...36

2.11 Conclusion of troglitazone derivatives targeting on Bcl-2/Bcl-xL…………….…38

2.12 Experimental section for troglitazone derivatives…………………………….….39

3. Development of AR ablating agents based on ciglitazone partly aided by

combinatorialchemistry……………,,,,,,,,,,,,,,…………………….…………………...69

3.1 Strategies for lead optimization of ciglitazone………………………………….…69

3.2 Validation of AR suppressing activity of ciglitazone and ∆2CG…….……….…...71

3.3 Regioisomerization of the (1-methylcyclohexyl)-methyl moiety of ∆2CG…….…74

3.4 Phenyl ring substitutions of ∆2CG…………………………………………….…..76

3.5 Permutational rearrangement of the (1-methylcyclohexyl)-methyl group (CG9)…77

3.6 Regioisomerization of the phenolic group in CG9……………...…………………79

3.7 Phenyl ring substitutions (X) of CG9………….…………………….……………80

3.8 Phenolic group (R1) modification on CG9.…………………………..……………88

3.9 Substitution of 4’ phenolic group in CG9 by amides or sulfonamides…….………90

3.10 Substitution of 4’ phenolic group in CG12 by selected amides or sulfonamides..93

3.11 Substitution of hydroxylphenyl group by various aromatic rings…………….…96

3.12 Modification of the (1-methylcyclohexyl)-methyl group (R2) on Compound CG12

aided by solid-phase combinatorial chemistry………………………………..…98

3.13 Stereochemistry conformation of double bond in structure unit of 5-benylidene-

2,4-thiazolidinedione…………………………………………………………...104

3.14 Conclusions on ciglitazone derivatives ablating AR…………………………....105

3.15 Experimental section of ciglitazone derivatives………………………………...106

List of references...……………………………………………………………………165

LIST OF TABLES

Table Page

1.1 Agent in development for hormone refractory prostate cancer……………………….4

2.1 ∆2TG derivatives with an n-alkyl side chain and their IC50 values of individual agents

in inhibiting the cell viability of PC3 cells……………………………...…….…………21

2.2 ∆2TG derivatives with an unsatuated n-alkyl side chain and their IC50 values of

individual agents in inhibiting the cell viability of PC3 cells……………………………22

2.3 ∆2TG derivatives with a branched alkyl side chain and their IC50 values of individual

agents in inhibiting the cell viability of PC3 cells.………………………………………23

2.4 ∆2TG derivatives with a CN substituted alkyl side chain and their IC50 values of

individual agents in inhibiting the cell viability of PC3 cells.……………………….…..24

2.5 ∆2TG derivatives with side chain of various H-bonding acceptors at the end and their

IC50 values of individual agents in inhibiting the cell viability of PC3 cells.…………..27

2.6 ∆2TG derivatives with OMe or CF3 substitution at the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells.…………...……28

2.7 Reduced optimized ∆2TG derivatives at the linkage between TZD ring the phenyl ring and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells………………………………………………………………………………………29

2.8 R-form of optimized ∆2TG derivatives at the linkage between TZD ring the phenyl

ring and their IC50 values in inhibiting the cell viability of PC3 cells.…………………29

2.9 IC50 values of the inhibition of protein interactions between Bak BH3 Fluorescence -

peptide and Bcl-xL by optimized ∆2TG-derivatives……………………………………31

3.1 Isomerized compound (3’-∆2CG ) with (1-methylcyclohexyl)-methoxy group at 3’ position instead of 4’ position in ∆2CG and its IC50 values of individual agents in

inhibiting the cell viability of LNCaP cells ……………………………………….……75

3.2 ∆2CG derivatives with different substitution on phenyl group and their IC50 values of

individual agents in inhibiting the cell viability of LNCaP cells……………..….…….76

3.3 Compound (CG9) and its IC50 values of individual agents in inhibiting the cell

viability of LNCaP cells ……………………………………………………..………….77

3.4 CG9 derivatives with phenolic group at various positions and multiple hydroxyl

groups on phenyl group and their IC50 values of individual agents in inhibiting the cell

viability of LNCaP cells…………………………………..………………………….….79

3.5 CG9 derivatives with different substitution on phenyl group and their IC50 values of

individual agents in inhibiting the cell viability of LNCaP cells……………….…….….81

3.6 CG9 derivatives with different substitution on 4’ position of phenyl group and their

IC50 values of individual agents in inhibiting the cell viability of LNCaP cells……..…..89

3.7 CG9 derivatives with different amide group at 4’ position of the phenyl group and

their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells……….……….………………………………………………….……….……...….91

3.8 CG9 derivatives with different sulfonamide group on the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells………...……92

3.9 CG12 derivatives with different sulfonamide group at 4’ position of the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP

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cells. …….……….………………………………………………….………….…………..

….95

3.10 CG9 derivatives with heterocyclic group to substitute 4’ hydroxyphenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells…...97

3.11 CG12 derivatives with various substitutions at R2 and their IC50 values of individual

agents in inhibiting the cell viability of LNCaP cell….….……….……………..…..….100

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LIST OF FIGURES

Figures Page

1.1 Statistics of male cancer in 2008……………………………………….…….……….1

1.2 Mechanisms of castration resistant prostate cancer………………………..…………7

1.3 Structure of androgen receptor ……………………….…………….………….…….8

1.4 Pathways of apoptosis……………………………………………………………….12

1.5 Development of thiazolidinedione derivatives………………………………………16

2.1 Molecular modeling analysis to show the binding between ∆2TG and Bcl-xL protein…………………………………………………….………….………….……….19

2.2 Overview of the strategies for structure modification based on ∆2TG ……………..20

2.3 General synthetic procedure for ∆2TG derivatives………………………...………..20

2.4 Representative synthetic routes for acid and amide derivatives……………………..26

2.5 Effects of HepCNCF3 on the dynamics of Bcl-2/Bak and Bcl-xL/Bak interactions in

PC-3 cells………………………………………………….……….……….……….…..32

2.6 Effect of HepCNCF3 on the expression levels of Bcl-2 family members in PC-3 cells………………………………………………….……………….…………………..33

2.7 Dose-dependent effect of HepCNCF3 on PARP cleavage and caspase-9 activation in

LNCaP cells………………………………………………….………….………….……34

2.8 Dose-dependent effect of HepCNCF3 on PARP cleavage and caspase-9 activation in

PC-3 cells………………………………………………….…………….……………….34

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2.9 Cell viability assay of HepCNCF3 on PC-3, LNCaP and Bcl-xL over-expression

LNCaP/B3 cell lines (serum free) ……………………………………………………….35

2.10 Cell viability assay of HepCNCF3 on PC-3, LNCaP and Bcl-xL over-expression

LNCaP/B3 cell lines (5% serum) ………………………………………………,.……..35

2.11 Upper panel: Binding of HepCNCF3 to Bcl-xl highlighting the surrounding residues; down panel: Two-dimensional representation of the binding mode of HepCNCF3 to Bcl-

xL highlighting the surrounding residues and involved interactions……………….……37

3.1 Strategy of structure modification based on ciglitazone………………..……………70

3.2 Effect of ciglitazone (CG) and ∆2CG on AR ablation in LNCaP cells ………….….72

3.3 Time-dependent effect of CG and ∆2CG on suppressing the mRNA level of AR…..73

3.4 Synthetic route for the isomerized compound 3’-∆2CG …………………...……….75

3.5 Synthetic routes for ∆2CG derivatives with different substitution on phenyl group..76

3.6 Synthetic routes for CG9 ……………………………………………………………77

3.7 Analysis of the effects of individual compounds on the transcriptional repression of

the AR gene by the AR promoter-luciferase reporter assay.…………………………….78

3.8 Western blot analysis of the dose-dependent effect of compounds CG1, CG6, and

CG9 on reducing AR protein levels……………………………………………………...78

3.9 Synthetic route for CG9 derivatives with phenolic group at various positions……...79

3.10 Synthetic routes for CG9 derivatives with different substitution on phenyl group..80

3.11 Analysis of the effects of individual compounds on the transcriptional repression of the AR gene by the AR promoter-luciferase reporter assay………………………..……82

3.12 Western blot analysis of the dose-dependent effect of compounds CG12 and CG16, on reducing AR protein levels………………………………………………….….…….83

3.13 Western blot analysis of the dose-dependent effect of compounds CG12, on PARP cleavage, and the proteolytic activation of caspase 3 and caspase 7…………….………84

3.14 Dose-dependent effect of ciglitazone (CG), ∆2CG, and compounds 12 and 16,

relative to that of 10 µM troglitazone (TG), on PPARγ activation in PC-3 cells………..85

3.15 Antitumor effects of OSU-CG12 in LNCaP cells…………………………………87

3.16 Synthetic routes for CG9 derivatives with various substitutions on 4’ position of

phenyl ring……………………………………………………………………………….88

3.17 Synthetic routes for amide and sulfonamide derivatives of CG9……..……….…...90

3.18 Synthetic routes for optimized amide and sulfonamide derivatives of CG12…...…94

3.19 Synthetic route for CG9 derivatives with various heterocyclic groups……..….…..96

3.20 Solid-phase synthetic routes for combinatorial library with various R2…………...99

3.21 Analysis of the effects of selected compounds with optimized different R2 groups on

the transcriptional repression of the AR gene by the AR promoter-luciferase reporter assay.…………………………………………………. ………………….…………….101

3.22 Western blot analysis of the dose-dependent effect of selected R2 position optimized compounds on cleavage of PARP protein levels………………………………..……...102

3.23 Western blot analysis of the dose-dependent effect of compounds CG12 and CC5,

PARP cleavage, protein levels of AR and β-TrCP……………….…………………….103

3.24 Z-form of double bond in structure unit of 5-benylidene-2,4-thiazolidinedione.

Upper panel, crystal structure; down panel, schematic illustration…………………….104

3.25 Proposed interactions between CG derivatives and their receptor…………….….105

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CHAPTER 1

INTRODUCTION

1. 1 Prostate cancer, current treatments and agents in development

Prostate cancer is one of the most prevalent male malignancies in the world.

Advances in biochemically diagnostic methods, particularly wide application of the prostate specific antigen (PSA) screening, have resulted in an earlier stage diagnosis of prostate cancer [1-2] and also have provided better standards to evaluate the progression of this disease. Since 1975, the incidence of prostate cancer has surpassed that of all other cancers in the United States. The estimated number of new cases of prostate cancer for 2008 exceeded that of lung and bronchus cancer, the second most common cancer type, by more than 1.5-fold, and is expected to account for 25% of new cancer cases in 2008 [3] (Figure 1.1, left panel). Although the death of most patients with prostate cancer is not directly related to this disease, it still represents

Estimated new male cancer cases Estimated deaths by cancer in male

186320 90,810 Prostate Lung&bronchus Lung&bronchus Prostate 315690 Colon&rectum 132,890 Colon&rectum Urinary bladder Pancreas others others

114690 28,660

51230 77250 17,500 24,260

Figure 1.1 Statistics of male cancer in 2008 [3] the second leading cause of cancer related deaths with a number of 28, 660 [3]

(Figure 1.1, right panel).

The growth and proliferation of both normal and tumor prostate cells are dependent

on androgen hormones and androgen receptor passway [reviews, 4-7]. Two major

biological androgen forms are testosterone and dihydrotestosterone (DHT).

Testosterone is synthesized in and secreted by Leydig cells of testis, which is regulated by the pituitary hormone luteinizing hormone (LH). The satuation of 4,5 double bond in testosterone by 5α-reductase will give DHT, a more potent and more stable form of androgen in terms of prostate cells. Upon binding with androgens, AR will be phosphorylated, be freed from chaperone proteins and dimerize. The dimerized AR will be translocated into cell nucleus, facilitating the growth and proliferation signal transductions after DNA binding and transcription regulation.

Treatment strategies for prostate cancer vary a lot depending on different patient conditions and different physicians’ consideration [reviews, 8-10]. For prostate tumors at early stages, patients are frequently subjected to active surveillance, radical prostatectomy i.e., surgical removal of the tumor, radiotherapy, cryotherapy or a combination of these therapeutic methods [11-12]. Along with the progression of the disease, patients will be treated by surgical castration (bilateral orchiectomy) or medical castration by using estrogen compounds, luteinizing hormone-releasing hormone (LHRH) agonists like leuprolide and goserelin or LHRH antagonists like abarelix, degarelix [13]. The hormone therapy treatment method is based on the discovery by Dr. Huggins in 1940s [14], who first reported the efficacy of surgical orchiectomy and/or estrogen on prostate cancer. Androgen receptor antagonist, which

is also called antiandrogens, are usually applied after castration or combined with castration. Clinically used antiandrogens include one form of steroidal antiandrogen like cyproterone acetate and megestrol acetate, or another form of non-steroidal antiandrogen such as flutamide, nilutamide and bicalutamide [9].

Although most of the patients show good response to hormone therapy, progression to castration resistant prostate cancer (CRPC) or hormone refractory prostate cancer

(HRPC) usually occurs within 2 to 5 years [7], when androgen deprivation therapy does not show effects any longer. For CRPC, it has been considered incurable for many years until 2004, when docetaxel-based chemotherapy demonstrated survival benefit (about two months) in addition to its palliative effects in two phase III clinical trials (TAX 327 and SWOG 9916) [15-16] and subsequently was approved by the

FDA. Docetaxel and prednisone are now considered to be the standard care for treatment of hormone therapy resistant metastatic prostate cancer. However, these improvements of survival benefit by docetaxel were so moderate that novel therapeutic strategies targeting the molecular basis of androgen therapy chemoresistance are still urgently needed.

Many agents and therapeutic methods undergoing pre-clinical and clinical investigation rationally target the molecular alternations and abnormities during prostate cancer progression and metastasis (Table 1.1) [17-19]. These agents include angiogenesis inhibitors (VEGF antibody Bevacizumab and VEGFR inhibitors thalidomide analogs, Sorafenib, Sunitinib and Vatalinib) [20-22], PTEN/PI3 kinase

mTOR pathway inhibitors (rapamycin, temsirolimus, Everolimus and AP23573) [23-

24], EGFR/HER2 inhibitors (Gefitinib, Lapatinib) [25], endothelin subtype A receptor antagonist (Atrasentan, ZD4054) [26], Vitamin D (DN-101) [27], histone deacetylase (HDAC) inhibitors (SAHA, LBH 589B) [28], Bcl-2 antisense oligodexoynucleotide (oblimersen) [29-30], new antiandrogens (Abiraterone acetate

Target Agent Phase of Clinical test VEGF Bevacizumab (Avastin®) III VEGF receptor Sorafenib (Nexavar®) II Sunitinib (Sutent®) II Vatalinib II PTEN/PI3 kinase Everolimus (RAD 001) I/II mTOR pathway EGFR/HER2 AP233573 II Lapatinib (Tykerb®) II Bone interface Atrasentan (Xinlay) III ET-A receptor ZD4054 RANK ligand Denosumab (AMG 162) III Immunotherapy Sipuleucel-T (Provenge®) III Prostate GVAX® III Vitamin D DN-101 III Histone deacetylase SAHA (Vorinostat, Zolinza®) II (HDAC) LBH 589B II Androgen Receptor Abiraterone acetate III MDV-3100 I/II

Table 1.1 Agent in development for hormone refractory prostate cancer. Re-typed from [26] with permission from Elsevier.

and MDV-3100) and immunotherapeutic agents (autologous dendritic cell Provenge,

allogenic whole cell Prostate GVAX and Anti-CTLA4 antibody MDX-010) [32-33].

Most of these agents failed to show significant improvements in clinical trials when

they were used as single agent and some of them are now under investigation being

combined with docetaxel [26].

1.2 Mechanisms of castration resistant prostate cancer (CRPC)

A variety of molecular alterations in prostate cancer cells have been demonstrated to explain why prostate cancer cells become resistant to hormone therapy [reviews, 34-

37]. These mechanisms (Figure 1.2) include: 1) AR hypersensitivity because of AR

amplification, 2) AR ligands de-selectivity because of point mutation, 3) ligand

independent AR activation because of altered expression of AR co-regulatory

proteins or formation of androgen-independent isoforms by proteolytic processing and 4) activation of alternate cancer cell growth and/or anti-apoptosis pathways.

1) AR amplification [38-42]

Over-expression of AR protein level by amplification of AR gene, increased AR transcription and translation and enhanced AR protein stability facilitate prostate cancer cells to respond to relatively low level of androgen. AR gene amplification has been reported in 25–30% of androgen-independent prostate cancer patients and the

AR amplification was not found in any untreated prostate cancer samples.

2) AR mutation [43-47]

LNCaP is the first reported cell line containing a point mutation, which is at codon

877 of the hormone-binding domain. This mutation will allow the AR to acquire capability to use certain anitandrogens as ligands. The majority of AR point mutations in prostate cancer have clustered in areas of the hormone-binding domain.

Mutations in these areas flank the ligand-binding pocket and alter this pocket to allow

the binding of ligands other than androgens.

3) AR activation independent of ligands [48-53]

The overexpression of AR coactivators may enhance AR response to low levels of

androgen or broaden ligand specificity similar to amplification and mutation of AR. It has been reported that AR could be activated without ligands by protein kinase A

(PKA) signaling pathway, tyrosin kinase pathways including Her2/Neu (erbB2), insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Other proteins that have been shown an ability to activate AR in CRPC were cytokines like interleukin-6 (IL-6) and other factors such as β-catenin, caveolin-1, cyclin E, tumor susceptibility gene 101, cyclin D1, Rb, p53, c-jun, and the Smad3 pathway.

4) Other signaling pathways independent of AR [54-55]

Apart from these pathways that act through the AR, other signaling pathways have been postulated, including the upregulation of antiapototic proteins and epigenetic changes. The antiapoptotic genes Bcl-2/Bcl-xL were found to be overexpressed in

CRPC. Deregulation of epigenetic pathways, such as DNA methylation, histone methylation and deacetylation have been shown to be involved in both AR dependent and AR independent CRPC mechanisms.

Figure 1.2 Mechanisms of castration resistant prostate cancer. Reprint from [55] –

thanks to permission by Nature Publishing Group (http://www.nature.com/nrc/index.html).

1.3 AR, a good target for treatment of CRPC [reviews, 6, 56-57]

Androgen receptor [58], a 110 kDa nuclear receptor containing 919 amino acids, is located on charomatin Xq11–12. AR protein is a member of ligand-activated transcription factors belonging to the steroid hormone superfamily. Like most nuclear factor superfamily members, AR is composed of a central DNA-binding domain

(DBD) containing two zinc-finger motifs, a ligand binding domain (LBD) at the C- terminal and a hinge domain in between them (Figure 1.3). It also contains two regulation domains, one in the N-terminal (transcriptional activation function site 1,

AF-1) and one near the C-terminal (transcription activation function site 2, AF-2)

(Figure 1.3). There are repeats of poly-glutamine, poly-glycine, and poly-proline near the N-terminal. In the absence of hormone binding, AR is primarily bound to the cytoplasm heat shock proteins (HSPs).

PloyQ PolyPPolyG AF1 DBDH LBD AF2

1 919

Figure 1.3 Structure of androgen receptor [7]

Androgen receptor pathway is crucial for development and growth of benign and

malignant prostate cells [59]. It may be hypothesized that the prostate cancer cells

depend on androgen receptor no longer when they don’t respond to androgen ablation

therapy. However, there are many evidences showing that it is not the case. Firstly, as

mentioned above, AR amplification was observed in about 20-30% of CRPC patients

[60-61]; secondly, the androgen downstream pathway proteins like PSA is

constitutively expressed and positively correlated with disease progression; thirdly,

although the circular androgens decreased dramatically by surgical castration and/or

medical castration, the levels of androgens measured in tumor cells remain at similar

levels as those in the benign cells and castration responding cells [62-63]. All these

phenomena clearly show that the prostate cancer never develops its independency to

androgen receptor pathway so that androgen receptor is still a very important target

for development of new therapeutic agents and methods against CRPC.

1.4 Anti-apopotic proteins Bcl-2/Bcl-xL as targets for treatment of CRPC

[reviews, 64- 68]

Apoptosis, also referred to as programmed cell death, is an active brake for abnormal

or damaged cells, which is characterized by chromatin condensation, nuclear DNA

fragmentation and formation of apoptotic bodies. In contrast to necrotic cell death,

apoptotic cell death actively utilizes ATP and lacks inflammatory responses. The rate

of cell survival and cell proliferation is always delicately balanced by the rate of

apoptosis in normal cells and dysregulation of apoptosis accounts for one of the six

hallmarks of cancer. Prostate cancer progression and the development of hormone

therapy resistant prostate cancer have been linked to a number of genetic and

epigenetic abnormalities, among which, inhibition of apoptotic pathways, particularly due to overexpression of anti-apoptotic proteins Bcl-2 and Bcl-xL, plays an important role in addition to androgen receptor amplification and mutations.

Two main apoptotic pathways (Figure 1.4) are the extrinsic pathway [69-70] (death receptor-mediated pathway or cytoplasmic pathway) and intrinsic pathway

(mitochondrial-mediated pathway). Both pathways share later steps of the effector caspases cascade activation which are responsible for the morphological changes characterizing apoptotic cells. Caspases are cysteine proteases that cleave substrate proteins at the peptide bonds after specific aspartic acid residues and not all caspases are involved in apoptosis. The extrinsic pathway involves binding of natural death receptor ligands family with death receptors [71]. Death receptor ligands belong to tumour-necrosis factor (TNF) and the death receptors are members of TNF receptor superfamily, which consist of Fas/CD95, TNF-related apoptosis-inducing ligand-R1

(TRAIL-R1), death receptor 4 (DR4), TRAIL-R2, DR5), DR3 (Apo 2) and DR6.

The activated death receptors trigger the ligation of adaptor protein Fas-associated death domain protein (FADD) and then recruit and activate caspase-8 and caspase-10

[72]. Capase-8 will activate the first effector caspase, caspase-3, which could also be activated by caspase-9, the product of intrinsic pathway. In certain cell types, caspase

8 could activate the intrinsic apoptotic pathway as well by cleaving and activating Bid and leading to the subsequent release of cytochrome c. Cytochrome c and Bcl-2 family members [73-75] are the major players in the intrinsic apoptotic pathway. The

Bcl-2 family members consist of proapoptotic proteins (such as Bax, Bak, Bad, Bcl-

Xs, Bid, Bik, Bim, and Hrk), and antiapoptotic proteins (such Bcl-2, Bcl-XL, Bcl-W,

Bfl-1, and Mcl-1), both of which are localized on the outer mitochondrial membrane and they compete to regulate the release of cytochrome c [76]. In response to

apoptotic stimuli, cytochrome c is released to the cytosol and leads to association of

Apaf-1 and activation of caspase-9 which will activate caspase-3 [77]. Other effector

caspases, such as caspase-6 and caspase-7 will also activated by caspase-3, and all

these effector caspases are responsible for breaking down of DNA repair proteins

(such as Poly ADP ribose polymerase (PARP)), protein kinases and cytoskeletal proteins and resulting in the morphologic manifestations of apoptosis (such as DNA fragmentation and membrane blebbing).

Bcl-2 is an anti-apoptotic mediator that has been found to be involved in the molecular biology of a wide range of human cancers since its discovery as an oncoprotein in human follicular lymphoma fifteen years ago [78]. Bcl-xL, which displays remarkable amino acid and overall structural homology to Bcl-2, functions similarly as Bcl-2. These two proteins can switch expression in tumor cells under certain conditions [79]. Bcl-2 and Bcl-xL, are often involved in the progression of prostate cancer [67, 80]. Bcl-2 expression is restricted to the basal cells of the glandular epithelium in normal prostate tissue. These cells are resistant to the cell killing effect from treatment of androgen deprivation, while the secretory epithelial cells with undetectable Bcl-2 experession are sensitive to androgen ablation [81]. The upregulation of Bcl-2 has been observed in a male Sprague-Dawley rat model 10 days after castration and this effect was prevented by the co-administration of testosterone.

Furthermore, castrated mice have been shown a dramatic tumor volume reduction after treatment with antisense bcl-2 oligodeoxynucleotides (ODN) when compared with control mice [82].

Immunohistochemical analysis of clinical samples showed that high incidences of

Bcl-2/Bcl-xL expression have been observed in precursor of prostate cancer, prostatic

intraepithelial neoplasia (PIN), [83] and hormone therapy resistant carcinomas [84],

while low incidences have been shown in androgen-dependent samples or benign

hyperplasia [85-86]. Furthermore, high Bcl-2 expression and low levels of Bax

expression have been correlated with the poor therapeutic response of prostate cancer

to hormone therapy or radiation therapy [87-88]. Thus, Bcl-2 and Bcl-xL appear to be

attractive targets for hormone therapy resistant prostate cander patients.

Figure 1.4 Two major pathways of apoptosis. Reprint from [69] -- thanks to permission

by Nature Publishing Group (http://www.nature.com/nrc/index.html).

1.5 Thiazolidinediones, mechanisms in anti-cancer and structure optimization

[reviews: 89-93]

Thiazolidinediones (TZDs) [94], are a group of synthetic compounds with a

thiazolidinedione ring, including marketed drugs rosiglitazone (Avandia),

pioglitazone (Actos), and troglitazone (TG, Rezulin) (troglitazone was withdrawn

from the market due to its hepatotoxicity) [95] and experimental agents such as

ciglitazone (CG), rivoglitazone and MCC-555. The marketed agents are clinically

used as for type II (insulin resistant) diabetic patients to sensitize insulin response by

activating peroxisome proliferator-activated receptor γ (PPARγ) [96].

PPAR is a member of nuclear receptor superfamily. There are three subtypes of

PPAR proteins: PPARα, PPARβ/δ and PPARγ [97]. The name of PPAR came from the first reported subtype, PPARα, which could be activated by peroxisome proliferator such as long-chain polyunsaturated fatty acids and its natural derivatives.

Four isoforms of PPARγ genes [98] encode and express two isoforms of PPARγ

proteins [99], either of which contains a DNA binding domain, a ligand binding domain and two transcriptional activation function domains (AF-1 and AF-2) [100-

101]. After activation, PPARγ will form heterodimer with the retinoid X receptor

(RXR) and then binds to the peroxisome proliferator response elements (PPREs) to

regulate transcription of target genes [102]. PPARγ is expressed most abundantly in adipose tissue but is also found in pancreatic beta cells, vascular endothelium, and macrophages [103]. Mechanisms of TZDs’ insulin sensitizing effects [104] including

the hypotheses that thiazolidinediones directly promote fatty acid uptake and storage

in adipose tissue from other insulin sensitive tissues [105] or indirectly, by increasing

adiponectin level which will sensitize the insulin effects [106-107].

Although the functions of PPARγ have been mostly investigated in insulin

sensitization, metabolism of lipid and glucose [108], inflammation [109] and

atherosclerosis [110], more and more evidences have been shown the relationship

between PPARγ and carcinogenesis. The inhibitory effects of TZDs on a variety of

cancers in vitro and in vivo, which includes many solid cancer cell lines including

prostate [111], breast [112], colon [113], thyroid [114], lung [115], bladder [116], and

pituitary carcinoma [117] and also hematological malignancies [118] such as myeloid

cells (U937 and HL-60) [119] and lymphoid cells [120] (Su-DHL, Sup-M2, Ramos,

Raji, Hodgkin’s cell lines and primary chroniclymphocytic leukemia (CLL)) [121]. In

addition to adipocytes, PPARγ were also reported expressed in many human cancer

cell lines and PPARγ ligands were found to be potent inducers of differentiation and

apoptosis in many cancer cells. PPARγ regulated genes have been linked to tumor suppressing mechanisms of anti-proliferation (CDK inhibitor upregulation, PI3K/Akt

inhibition)[122], inducing differentiation (upregulation of carcinoembryonic antigen,

E-cadherin and alkaline phosphatas) [123-124], inducing apoptosis (Bcl-2

downregulation and BAX/BAD upregulation, FLICE-inhibitory protein (FLIP) down

regulation) [125], angiogenesis inhibition (endothelial cells inhibition and VEGF

downregulation) [126-127] and metastasis inhibition (inhibition of intercellular adhesion) [128-129].

However, more and more evidence supports another school of scientists who hold the

viewpoint that the TZDs exert their anti-cancer activities through PPARγ independent mechanisms [89-90, 130-132]. Firstly, the antitumor effects of these TZDs do not show a positive correlation with their potencies on PPARγ activation, for example troglitazone and ciglitazone are more active in inducing apoptosis in prostate cancer cells compared to rosiglitazone and pioglitazone; secondly, there lacks a correlation between the sensitivities of cancer cells to TZDs and the expression levels of PPARγ; thirdly, a series of PPARγ-inactive TZD analogues including ∆2TG and ∆2CG have shown more potency than their parent compounds (troglitazone and ciglitazone) in suppressing cell proliferation in cancer cells; and finally, siRNA mediated knockdown of PPARγ does not affect the apoptotic death of PC-3 cells induced by troglitazone or ∆2TG (Chen lab, unpublished data). Several lines of PPARγ independent mechanisms have been revealed, including the inhibition of Bcl-2/Bcl- xL function, and transcriptional repression of AR through Sp1 degradation.

Dissociation of the antitumor effects of TZDs from their PPARγ activity provides a rationale for structure optimization to develop novel agents for cancer therapy [130,

133]. The proof-of-principle of this hypothesis was demonstrated by STG28 and

TG88, PPARγ-inactive derivatives of ∆2TG, with improved antitumor potency.

STG28 has been shown five times higher potency in suppressing the expression of cyclin D1, Sp1, and AR through proteasomal degradation or transcriptional repression in prostate cancer cells than the parent molecules troglitazone and ∆2TG. TG88 has

improved the inhibitory potency of troglitazone and ∆2TG on Bcl-2/Bcl-xL of about

10 times.

O O

NH NH O S S O O O O HO Ciglitazone Troglitazone O O

NH NH N N S S O N O O O

Rosiglitazone Pioglitazone

O O

NH NH O S S O O O O HO Delta2-CG Delta2-TG

O O

NH NH N N S S O N O O O Delta2-RG Delta2-PG

O O Br MeO NH O S NH O O S O O O O O STG28 TG88

Figure 1.5 Development of thiazolidinedione derivatives

CHAPTER 2

DEVELOPMENT OF TROGLITAZONE DERIVATIVES TARGETING ON

BCL-2/BCL-XL DIRECTED BY MOLECULAR MODELING

2.1 Design of ∆2TG derivatives directed by molecular modeling

Recent research has provided several lines of evidence and suggested that the effects of troglitazone and its PPARγ independent troglitazone derivative ∆2TG on apoptosis in prostate cancer cells be attributable in part to the inhibition of Bcl-xL/Bcl-2 functions, by inhibiting Bak-BH3 peptide binding to Bcl-xL/ Bcl-2 [130].

∆2TG was used as a lead compound in the computer aided molecular modeling to study the binding with Bcl-2/Bcl-xL. It was done by applying a representative docking method of CDOCKER 2.0, a molecular dynamics (MD) simulated- annealing-based algorithm combined with Monte Carlo (MC) to perform the structure-based design. Bcl-2 and Bcl-xL are highly homologous proteins in both amino acid sequence and structure, and each of them contains four conserved regions denoted as BH1, BH2, BH3, and BH4 domains. Human Bcl-xL structure with PDB id,

1BXL, in the complex of Bcl-xL and Bak BH3 peptide was used for molecular modeling analysis.

It could be found that (Figure 2.1), the double bond neighboring the TZD ring made molecule more rigid so that the TZD ring inserts more deeply into the crevice between BH3 domain and trans-membrane domain of Bcl-xL. The amino (NH) group of TZD ring acted as a hydrogen bond acceptor with the Glu96 (heavy atom distance of 2.8 and 3.0 Å, respectively), and the TZD ring formed a π-π interaction of 3.8 Å

with Tyr195. In consequence, the following benzene interacted with Phe97 through a

T shape π-π interaction around 3.0 Å. It has been reported an importantly

hydrophobic interactions between the side chain of Leu578 of Bak and the

hydrophobic residues Tyr101, Val126, and Phe146 of Bcl-xL, however, the chroman

ring moiety of ∆2TG turned aside this Leu578-binding subpocket and the hydroxyl

group interacted with Glu 129 in close distance with 2.0 Å.

The first stage of our optimization for ∆2TG was to introduce side chain (R1) at

hydroxyl group to block the polar hydroxyl group and support more hydrophobic

interaction (Figure 2.2). Three kinds of alkyl group, including n-alkyl, n-alkenyl and

branched alkyl groups were introduced; moreover, alkyl chains with a cyano group

were also tried at R1 to facilitate polar interaction or hydrogen bonding out of the

hydrophobic subpocket. Bromo substitution was chosen on phenyl group neighboring

thiazolindinedione ring based on formerly optimized compound TG88. The second

stage of optimization involved the substituting group on phenyl ring. Trifluoromethyl

group and methoxyl group were introduced to compare with the bromo group to

exploit possibilities of potentiating the aromatic interaction between phenyl ring and

side chain in residue Phe97 (Figure 2.3).

To obtain these ∆2TG derivatives, a general synthetic procedure was applied (Figure

2.3). In brief, 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid was reduced to alcohol by lithium aluminum hydride and the R1 side chain was then introduced by reaction between alkyl bromide and phenolic group on chroman ring. The methylene- hydroxyl group on alkyl substituted compound was activated into its corresponding triflate which then was attacked by hydroxyl benzaldehyde to provide the ether benzaldehyde. At the final step, these benzaldehydes were condensed with thiazolidinedione ring to form the objective troglitazone derivatives. The objective compounds were subjected to assessment of PC3 cancer cell viability inhibition to provide information on the structure-activity relationship (SAR). Detailed mechanisms of Bcl-2/Bcl-xL inhibition were evaluated for the most optimized compounds.

Figure 2.1 Molecular modeling analysis to show the binding between ∆2TG and Bcl2-xL protein.

NH O S O O HO O Delta2-TG Br NH O S O O O i-Alkyl-O Br NH O O S Br O O O NH O S n-Alkyl-O Br O NH O O S CNAlkyl-O O O Alkenyl-O

O Br NH O S O O O R2 NH R3OCAlkyl-O O S O O R1Opti

O R O 2 NH R2 O S NH O O S O O R Opti O 1 R1Opti

Figure 2.2 Overview of the strategies for structure modification based on ∆2TG

O COOH LiAlH4 O O OH R Br/K CO 1 2 3 OH (CF3SO2)2O O OSO2CF3 HO THF HO Acetone, reflux R1O R O Pyridine/CH2Cl2 1

CHO O R 2 O H N R2 R2 CHO O NH O S S O OH O O O R O AcOH/piperidine 1 K2CO3, DMF R O 1 ethanol/reflux

Figure 2.3 General synthetic procedure for ∆2TG derivatives

2.2 ∆2TG derivatives with an n-alkyl side chain

It could be found that the length of four carbons was the most optimized choice for n- alkyl group substitutions (Table 2.1). Decreased length and particularly increased length of the side chain will negatively affect the activity of PC-3 cell viability inhibition.

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 (µM)

1 EthTGBr n-ethyl Br 5

2 ProTGBr n-propyl Br 3.6

3 ButTGBr n-butyl Br 2.3

4 PenTGBr n-pentyl Br 3.75

5 HexTGBr n-hexyl Br 6.84

6 HepTGBr n-heptyl Br 15.67

Table 2.1 ∆2TG derivatives with an n-alkyl side chain and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells. Cells were exposed to individual agents at various concentrations in serum free RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

2.3 ∆2TG derivatives with an unsatuated n-alkyl side chain

Introduction of a double bond would change the steric posing of the side chain, so

that three of these compounds with carbon-carbon double bond on the side chain were

designed and synthesized. It was shown a trend that C5 was more potent than C4 and

C3 for this series of compounds (Table 2.2).

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 (µM)

7 AllTGBr Br 7.5

8 BueTGBr Br 4.2

9 PeneTGBr Br 2.9

Table 2.2 ∆2TG derivatives with an unsatuated n-alkyl side chain and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells. Cells were exposed to individual agents at various concentrations in serum free RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

2.4 ∆2TG derivatives with a branched alkyl side chain

It could be observed that the increase of IC50 values by introducing branched side chain on chroman ring (Table 2.3). The SAR for this series of derivatives was

consistent with the series with n-alky substitutions, which showed that the activity

decreased dramatically when the side chain became longer and more bulky. This may

attribute to the narrow space near the hydrophobic subpocket in the binding pocket of

Bcl-xL.

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 (µM)

10 IproTGBr Br 7.1

11 IpenTGBr Br 8.4

12 EBTGBr Br 9.1

13 EHTGBr Br > 20

14 CHETGBr Br > 20

15 DMOTGBr Br > 20

Table 2.3 ∆2TG derivatives with a branched alkyl side chain and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells. Cells were exposed to individual agents at various concentrations in serum free RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

2.5 ∆2TG derivatives with a CN substituted alkyl side chain

Surprisingly, when we introduce a cyano group at the end of the alkyl side chain, the

SAR results between the efficacy and the side chain length was totally reversed,

which implicates that the Bcl-2/Bcl-xL protein prefer longer chain with a CN tail

(Table 2.4). Another interesting finding from compound 19 (DMHCNBr) was that

branches in this series of compounds seemed not to negatively affect the activity

(Table 2.4).

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 (µM) CN 16 BuCNBr Br 5.3 CN 17 HexCNBr Br 4.2 CN 18 HepCNBr Br 1.5 19 DMHCNBr CN Br 2.6

20 BnCNBr Br 3.0 CN

Table 2.4 ∆2TG derivatives with a CN substituted alkyl side chain and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells. Cells were exposed to individual agents at various concentrations in serum free RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

2.6 ∆2TG derivatives with side chain of various H-bonding acceptors at the end

Based on the finding on ∆2TG analogues with a CN tail, a series of derivatives with

various H-bonding acceptors were designed and synthesized. Out of our expectation,

more bulky group with cyano group such as cyano biphenyl group lost most of PC3

cell viability inhibition activity, and the same result has been shown in another bulky

group, 4-Benzoyl-benzyl group which also contained a carbonyl group as H-bonding

acceptor (Table 2.5). To further exploit derivatives of HepCN-Br, the most potent compound so far, compounds with ester end, carboxylic acid end, or amide end to replace the cyano end were synthesized. Ester compounds were synthesized according to the general synthetic methods, while acid compounds were obtained by hydrolysis of ester compounds. Following activation of the acid into its corresponding

ester of N-Hydroxy-succinimide, it was ready to react with ammonia gas to provide

its corresponding amide (Figure 2.4). It could be observed that ester or acid

compounds were a little bit less active than their corresponding nitrile compounds,

however, the anti-prostate cancer cell activity were completely removed in amide

compound, which may be led by the higher polarity of amide or the disturbance of amide bond in peptide-peptide reaction (Table 2.5).

O Br NH O S O O O O O NaOH/EtOH

O Br NH O S O O O HO O SuNOH/EDC

O Br NH O S O O O O O O N O NH 3 /THF

O Br NH O S O O O H2N O

Figure 2.4 Representative synthetic routes for acid and amide derivatives

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 (µM)

21 PPhenoeBr Br 20.2

O 22 BPCNBr NC Br 25.6

23 PenECNBr O Br 2.7

O 24 HepECNBr O Br 4.5

O 25 PenABr OH Br 7.8 O 26 HepABr OH Br 3.5 O 27 HepNH2Br NH2 Br >20 O

Table 2.5 ∆2TG derivatives with side chain of various H-bonding acceptors at the end and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells.

2.7 ∆2TG derivatives with OMe or CF3 substitution at the phenyl group

To potentiate the aromatic interaction between the phenyl ring in ∆2TG derivatives and side residues in Bcl-2/Bcl-xL, methoxyl group and trifluoromethyl group were used to in place of bromo group in selected optimized compounds. For most of these derivatives, CF3 showed an additive effect while MeO showed subtracting effects, which may because of their different electronegativity (Table 2.6). The most potent compound, HepCNCF3 was achieved by this stage of optimization.

O R2 NH O S O O R O 1

Entry Compounds R1 R2 IC50 ((µM)) 28 ButTGOMe OMe 4.2

CN 29 HepCNOMe OMe 2.5 30 ButTGCF3 CF3 1.3 CN 31 HepCNCF3 CF3 0.8 32 PenTGCF3 CF3 4 33 HexTGCF3 CF3 11 34 HepTGCF3 CF3 14.4 CN 35 ButCNCF3 CF3 2.7 CN 36 HexCNCF3 CF3 3.8 37 DMACF3 CF 3.7 3 38 BenCNCF CF 2.6 3 CN 3

Table 2.6 ∆2TG derivatives with OMe or CF3 substitution at the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells.

2.8 Reduced forms and R-forms of optimized ∆2TG derivatives

When double bond between the phenyl ring and the TZD ring in the optimized

compounds were reduced, anti-prostate cancer of these derivatives were kept to

relatively decreased levels (Table 2.7), which was consistent with the performances

of lead compounds troglitazone and ∆2TG. R-forms of these optimized compounds

were also synthesized by their corresponding optically active raw material, which

showed less active results compared to their corresponding S-form compounds

(Table 2.8).

O R2 NH O S O O R O 1 Entry Compounds R1 R2 IC50 ((µM)) 39 2H-ButTGCF3 CF3 2.6 CN 40 2H-HepCNCF3 CF3 3.1

Table 2.7 Reduced optimized ∆2TG derivatives at the linkage between TZD ring the phenyl ring and their IC50 values of individual agents in inhibiting the cell viability of PC3 cells.

O R2 NH O S O O R O 1 Entry Compounds R1 R2 IC50 (µM))

41 R-TG88 Br 4.05

42 R-ButTGCF3 CF3 3.9 CN 43 R-HepCNCF3 CF3 3.0

Table 2.8 R-form of optimized ∆2TG derivatives at the linkage between TZD ring the phenyl ring and their IC50 values in inhibiting the cell viability of PC3 cells.

2.9 Detailed mechanistic evidences of targeting Bcl-2/Bcl-xL for optimized ∆2TG

derivatives

The capability of selected optimized compounds to inhibit binding of fluorescein-

labeled Bak BH3 domain peptide to Bcl-xL protein have been investigated and the

most optimized compound, HepCNCF3, was about one order of magnitude more

potent than TG88 and two orders of magnitude more potent than troglitazone and

∆2TG (Table 2.9).

The effects of HepCNCF3 on the dynamics of Bcl-2/Bak and Bcl-xL/Bak interactions

in PC-3 cells have been assessed. Probing of the immunoprecipitates with anti-Bak

antibodies by Western blotting indicated that the level of Bak associated with Bcl-2

and Bcl-xL was greatly reduced after treatment of low micromolar concentration of

HepCNCF3 as compared to the DMSO control (Figure 2.5). Western blotting assay showed that the total protein levels of neither anti-apoptotic proteins Bcl-2 and Bcl- xL nor antiapoptotic proteins such as Bak, Bax, Bad or Bid did not significantly change after treatment by HepCNCF3 (Figure 2.6), which implicated that the

HepCNCF3 directly target the interaction between Bak and Bcl-2/Bcl-xL.

Assessment of activated caspase 9 and PARP cleavage as indicators of apoptotic signaling downstream of cytochrome c release revealed a dose-dependent increase in activated caspase 9 and PARP cleavage after treatment of PC-3 cells with HepCNCF3

(Figures 2.7 & 2.8).

Overexpression of ectopic Bcl-xL in LNCaP cells (LNCaP/B3) conferred partial protection to the cytotoxic effects of HepCNCF3, which further demonstrated the effects of HepCNCF3 on Bcl-2/Bcl-xL (Figures 2.9 & 2.10).

Compound Structure IC50 (µM) for FP

assay

O TG88 F3C 2.33+1.53 NH O S O O O (N = 3)

O BuCF3 F3C 1.08+0.38 NH O S O O (N = 3) O

O HepCNCF3 F3C 0.35+0.14 NH O S O O O NC (N = 5)

Table 2.9 IC50 values of the inhibition of protein interactions between Bak BH3 fluorescein peptide and Bcl-xL by optimized ∆2TG-derivatives.

Figure 2.5 Effects of HepCNCF3 on the dynamics of Bcl-2/Bak and Bcl-xL/Bak interactions in PC-3 cells

Figure 2.6 Effect of HepCNCF3 on the expression levels of Bcl-2 family members in PC-3 cells.

Figure 2.7 Dose-dependent effect of HepCNCF3 on PARP cleavage and caspase-9 activation in LNCaP cells.

Figure 2.8 Dose-dependent effect of HepCNCF3 on PARP cleavage and caspase-9 activation in PC-3 cells.

MTT assay for HepCN-CF3 (3)1 day treatment in 0% serum

110 100 90 80 PC-3 70 LNCaP LNCaP/B3 60 50

Cell Viability (%) Cell Viability 40 30 20 10 0 0246810 HepCN-CF3 (uM)

Figure 2.9 Cell viability assay of HepCNCF3 on PC-3, LNCaP and Bcl-xL overexpression LNCaP/B3 cell lines (24 hr, serum free)

MTT assay for HepCN-CF3 (3)1 day treatment in 5% serum

110 100 90

80 PC-3 70 LNCaP LNCaP/B3 60 50

Cell Viability (%) 40 30

0 0 1020304050 HepCN-CF3 (uM)

Figure 2.10 Cell viability assay of HepCNCF3 on PC-3, LNCaP and Bcl-xL overexpression LNCaP/B3 cell lines (24 hr, 5% serum)

2.10 Molecular modeling evidence of the binding between HepCNCF3 and Bcl- xL protein

The same molecular modeling method was used for exploiting the binding mode

HepCNCF3 and Bcl-xL protein as that for binding between ∆2TG and Bcl-xL protein.

It could be found (Figure 2.11) that structure unit of benzylidene-thiazolidinedione possessed a similar posing and interaction in Bak-Bcl-xL binding pocket as ∆2TG did, in which the phenyl ring of HepCNCF3 interacts with Phe97 through a T shape π-π interaction around 3.0 Å, in addition, the substituent CF3 on trifluoromethyl-benzene was close to the charged residue Arg139 and the electrostatic interactions were expected between each other. Furthermore, the importantly hydrophobic interactions between the side chain of Leu578 of Bak and the residues Tyr101, Val126, and

Phe146 of Bcl-xL, was effectively replaced by a new π-π interactions between the chroman ring moiety of the HepCN-CF3 and Try101, Phe146 after introducing the heptanenitrile group. It was also observed that the heptanenitrile side chain of

HepCN-CF3 anchors the narrow hydrophobic cleft with the terminal CN group extending to the Gln125 of Bcl-xL; Such ligand anchoring resulted in the better orientation and tight biding of HepCN-CF3 with Bcl-xL than that of ∆2TG where the hydroxyl group interacted with Glu 129 and the chroman ring moiety strayed from the hydrophobic cleft of Bak binding. Overall, the binding energy with Bcl-xL protein was -54.9, -43.8 and -39.2 kcal/mol for HepCNCF3, TG88 and ∆2TG individually, which was fit well with experimental data on Bcl-xL binding inhibition and PC-3 cell viability inhibition.

Figure 2.11 Upper panel: binding of HepCNCF3 to Bcl-xL highlighting the surrounding residues; down panel: two-dimensional representation of the binding mode of HepCNCF3 to Bcl-xL highlighting the surrounding residues and involved interactions

2.11 Conclusion of troglitazone derivatives targeting Bcl-2/Bcl-xL Directed by method of molecular modeling, more than 50 troglitazone derivatives have been designed and synthesized in two stages of structure modification based on troglitazone and its PPARγ-independent derivative ∆2TG. The optimal compound,

HepCNCF3, was obtained with an optimized side chain on chroman ring and preferred substitution on phenyl group adjacent the thiazolidinedione ring. This optimized compound showed two orders of magnitude improvement in Bcl-xL/Bcl-2 inhibition compared to troglitazone and one order of magnitude improvement compared to former optimal compound TG88. Binding mode between compound

HepCNCF3 and Bcl-xL predicted by molecular modeling have explained improvement on Bcl-2/Bcl-xL inhibition with lower binding energy. Western blotting assay and Co-immunoprecipitation (Co-IP) 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.

2.12 Experimental section of troglitazone derivatives

Chemical reagents and organic solvents were purchased from Sigma-Aldrich unless otherwise mentioned. Rabbit antibodies against Bcl-xL, Bax, Bak, Bid, PARP, and cleaved caspase-9 were purchased from Cell Signaling Technology, Inc. (Beverly,

MA). Rabbit antibodies against Bad, and mouse anti-Bcl-2 were from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-actin was from ICN

Biomedicals, Inc. (Costa Mesa, CA). Goat anti-rabbit IgG-horseradish peroxidase conjugates and rabbit antimouse IgG horseradish peroxidase conjugates were from

Jackson Immuno-Research Laboratories (West Grove, PA). Hamster anti-human Bcl-

2 antibody for immunoprecipitation was purchased from PharMingen (San Diego,

CA).

Nuclear magnetic resonance spectra (1H NMR) were measured on a Bruker DPX 300, model spectrometer. Chemical shifts (δ) were reported in parts per million (ppm) relative to the TMS peak. Electrospray ionization (ESI) mass spectrometry analyses were performed with a Micromass Micromass Q-Tof II High-Resolution electrospray mass spectrometer. Elemental analyses were performed by the Atlantic Microlab, Inc.

(Norcross, GA), and were within 0.4% of calculated values. Flash column chromatography was performed with silica gel (230-400 mesh).

General synthetic procedure for TG derivatives. Compounds were synthesized

according to general methods decribed below,

O OH

2-hydroxymethyl-2, 5, 7, 8-tetramethyl-chroman-6-ol (2): 1.0 g of LiAlH4 (26 mmol) was added in 100 mL of THF at 4 oC and stirred for half an hour, and then 5 g

(20 mmol) of 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (1) in 250

mL of anhydrous THF was titrated dropwise. The solution was reflux overnight. After

o cooling to 4 C, 1 mL of H2O, 1 mL of 1 N NaOH, and 2 mL of H2O was slowly

added to the solution to quench the reaction. The solution was stirred at room

temperature for 2 more hours, filtered, and concentrated, giving the product 2 in 85%

yield.

O OH

R1O

General procedure for compound 3 (Ether): A solution of 2.0 mmol of 2-

hydroxymethyl-2, 5, 7, 8-tetramethyl-chroman-6-ol (2), 4.0 mmol of bromide and 5.0

mmol of K2CO3 in 20 mL of acetone was refluxed for 48 hrs. The solution was

filtered and concentrated. The residue was re-suspended in ethyl acetate and purified

by column chromatography (ethyl acetate/hexanes = 1/3).

O OSO2CF3

R1O

General procedure for compound 4 (Triflates): A solution of compound 3 (1mmol)

and 1.5 mmol pyridine in dry CH2Cl2 (5mL) was stirred in ice bath, and 1.2mmol

triflate anhydride was added to the solution. After 2hr, the solution was concentrated

and the residue was purified by column chromatography (ethyl acetate/hexanes = 1/4).

R2 CHO O O

R O 1

General procedure for compound 5 (Aldehydes): A mixture of compound 4 (0.5

mmol), benzaldehyde (0.55 mmol) and K2CO3 were dissolved in 5 mL DMF. The

solution was heated to 60oC overnight. After reaction, the solution was poured into

water (10ml), extracted with ethyl acetate (30 ml), washed with saturated saline and

dried with anhydrous sodium sulfate. The solution was filtered and concentrated and

the residue was purified by column chromatography (ethyl acetate/hexanes = 1/6).

O R2 NH O S O O R O 1

General procedure for Compound 6 (TG derivatives): A mixture of aldehyde 5

(0.3 mmol), 2,4-thiazolidinedione (0.33 mmol), catalytic amount of piperidine was

refluxed in 5 mL EtOH for 48 hr and then concentrated. The oil product was acidified

with acetic acid and purified by chromatography (ethyl acetate/hexanes = 1/4) and

followed by re-crystallization.

O F3C NH O S O O O

5-[4-(6-Ethoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl-

benzylidene]-thiazolidine-2,4-dione (TGEtCF3) 1H NMR (300 MHz, CDCl3), δ

0.96 (t, J = 9.0 Hz, 3H), 1.57(s, 3H), 1.90-2.03(m, 1H), 2.05-2.23 (m, 10H), 2.58-2.72

(m, 2H), 3.73 (q, J = 6.6 Hz, 2H), 4.10 (q, J = 9.0 Hz, 2H), 7.05 (d, J = 9.0 Hz, 1H),

7.63 (dd, J = 9.0, 2.1 Hz, 1H), 7.74 (d, J = 2.1, 1H Hz), 7.92 (s, 1H), 8.64 (s, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(6-ethoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-

benzylidene]-thiazolidine-2,4-dione (TGEtBr) 1H NMR (300 MHz, CDCl3), δ 1.42

(t, J = 7.2 Hz, 3H), 1.51(s, 3H), 1.92-2.03(m, 1H), 2.05-2.23 (m, 10H), 2.58-2.72 (m,

2H), 3.74 (q, J = 7.2 Hz, 2H), 4.14 (q, J = 9.3 Hz, 2H), 7.00 (d, J = 8.4 Hz, 1H), 7.41

(dd, J = 8.4, 2.4 Hz, 1H), 7. 71 (d, J = 2.4 Hz, 1H), 7.75 (s, 1H), 8.65 (s, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(2,5,7,8-tetramethyl-6-propoxy-chroman-2-ylmethoxy)-

benzylidene]-thiazolidine-2,4-dione (TGProBr) 1H NMR (300 MHz, CDCl3), δ

1.08 (t, J = 9.0 Hz, 3H), 1.52 (s, 3H), 1.76-1.85(m, 3H), 1.92-1.99(s, 1H), 2.05-2.23

(m, 10H), 2.55-2.64 (m, 2H), 3.64 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 9.3 Hz, 2H), 6.97

(d, J = 8.4 Hz, 1H), 7.37 (dd, J = 8.4, 2.1 Hz, 1H), 7. 69 (d, J = 2.1 Hz, 1H), 7.74 (s,

1H), 8.45 (br, 1H).

O F3C NH O S O O O

5-[4-(6-Butoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl-

benzylidene]-thiazolidine-2,4-dione (TGBuCF3) 1H NMR (300 MHz, CDCl3), δ

0.92 (t, J = 7.0 Hz, 3H), 1.54-1.31 (m, 5H), 1.83-1.73(m, 2H), 1.95-1.87 (m, 1H),

2.18-1.95 (m, 10H), 2.60 -2.64 (m, 2H), 3.61 (t, J = 6.7 Hz, 2H), 4.03 (q, J = 9.0 Hz,

1H), 4.08 (q, J = 9.3 Hz, 1H), 7.08(d, J = 9.0 Hz, 1H), 7.58 (dd, J = 9.0 Hz, 2.1, 1H),

7.70 (d, J = 2.1 Hz, 1H), 7.78 (s, 1H), 8.83 (s, 1H), MS (ESI, m/z) calcd for

C29H32F3NO5SNa (M+Na): 586.1851, found: 586.1848. Anal. (C29H32F3NO5S)

C, H, F, N, O, S.

O Br NH O S O O O

5-[3-Bromo-4-(6-butoxy-2,7,8-trimethyl-1,2,3,4-tetrahydro-naphthalen-2- ylmethoxy)-benzylidene]-thiazolidine-2,4-dione (TGBuBr). 1H NMR (300 MHz,

CDCl3), δ 0.92 (t, J = 7.0 Hz, 3H), 1.58-1.46 (m, 5H), 1.77-1.68(m, 2H), 1.98-1.90

(m, 1H), 2.17-2.02 (m, 10H), 2.60 (m, 2H), 3.62 (t, J = 7.0 Hz, 2H), 4.05 (d, J = 8.7,

1H), 4.08 (d, J = 9.3 Hz, 1H), 6.67 (d, J = 1.5 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 7.37

(dd, J = 9.0 Hz, 1.5Hz, 1H), 7.70 (s, 1H), 8.44 (s, 1H), MS (ESI, m/z) calcd for

C28H32BrNO5SNa (M+Na): 569.1082, found: 569.1075. Anal. (C28H32BrNO5S) C,

H, Br, N, O, S.

O MeO NH O S O O O

5-[4-(6-Butoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-methoxy- benzylidene]-thiazolidine-2,4-dione (TGBuOMe) 1H NMR (300 MHz, CDCl3), δ

0.99 (t, J = 7.2 Hz, 3H), 1.45 (s, 3H), 1.52-1.66 (m, 2H), 1.73-1.85(m, 2H), 1.89-1.99

(m, 1H), 2.02-2.23 (m, 10H), 2.60-2.69 (m, 2H), 3.63 (t, J = 6.6 Hz, 2H), 3.90 (s, 3H),

4.04 (d, J = 9.3, 1H), 4.12 (d, J = 9.3, 1H), 6.96-7.10 (m, 3H), 7.80 (s, 1H), 8.58 (br,

1H).

O F3C NH O S O O O

5-[4-(2,5,7,8-Tetramethyl-6-pentyloxy-chroman-2-ylmethoxy)-3-trifluoromethyl- benzylidene]-thiazolidine-2,4-dione (TGPenCF3) 1H NMR (300 MHz, CDCl3), δ

0.93 (t, J = 6.9 Hz, 3H), 1.42-1.53 (m, 7H), 1.78(p, J = 7.2 Hz, 2H), 1.89-1.99 (m,

1H), 2.02-2.21 (m, 10H), 2.50-2.69 (m, 2H), 3.62 (t, J = 6.9 Hz, 2H), 4.05 (q, J = 9.3

Hz, 2H), 7.09 (d, J = 9.0 Hz, 1H), 7.62 (dd, J = 9.0, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz,

1H), 7.79 (s, 1H), 8.83 (s, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(2,5,7,8-tetramethyl-6-pentyloxy-chroman-2-ylmethoxy)- benzylidene]-thiazolidine-2,4-dione (TGPenBr) 1H NMR (300 MHz, CDCl3), δ

0.93 (t, J = 7.2Hz, 3H), 1.42-1.51 (m, 7H), 1.76-1.85(m, 2H), 1.89-1.99 (m, 1H),

2.02-2.21 (m, 10H), 2.52-2.69 (m, 2H), 3.62 (t, J = 6.7 Hz, 2H), 4.04 (q, J = 9.0 Hz,

2H), 6.96 (d, J = 8.7 Hz, 1H), 7.37 (dd, J = 8.7, 2.1 Hz, 1H), 7.67 (d, J = 2.1 Hz, 1H),

7.72 (s, 1H), 9.11 (s, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(6-hexyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-

benzylidene]-thiazolidine-2,4-dione (TGHexBr) 1H NMR (300 MHz, CDCl3), δ

0.92 (t, J = 6.9 Hz, 3H), 1.33-1.43 (m, 4H), 1.46-1.59(m, 5H), 1.74-1.83 (m, 2H),

1.94-2.03 (m, 1H), 2.05-2.24 (m, 10H), 2.57-2.69 (m, 2H), 3.65 (t, J = 6.6 Hz, 2H),

4.07 (q, J = 9.3 Hz, 2H), 6.99 (d, J = 8.7 Hz, 1H), 7.38 (dd, J = 8.7, 2.1 Hz, 1H), 7.69

(d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 8.79 (br, 1H).

O F3C NH O S O O O

5-[4-(6-Hexyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl-

benzylidene]-thiazolidine-2,4-dione (TGHepCF3) 1H NMR (300 MHz, CDCl3), δ

0.92 (t, J = 6.9 Hz, 3H), 1.28-1.57 (m, 9H), 1.83 (p, J = 6.6 Hz, 2H), 1.94-2.03 (m,

1H), 2.05-2.24 (m, 10H), 2.53-2.75 (m, 2H), 3.64 (t, J = 6.6 Hz, 2H), 4.10 (q, J = 9.0

Hz, 2H), 7.10 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.7, Hz, 1H), 7.73 (s, 1H), 7.82 (s, 1H),

8.97 (br, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(6-heptyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)- benzylidene]-thiazolidine-2,4-dione (TGHepBr) 1H NMR (300 MHz, CDCl3), δ

0.91 (t, J = 6.6 Hz, 3H), 1.27-1.59 (m, 11H), 1.81 (p, J = 6.9 Hz, 2H), 1.90-2.00 (m,

1H), 2.05-2.24 (m, 10H), 2.53-2.75 (m, 2H), 3.64 (t, J = 6.6 Hz, 2H), 4.06 (q, J = 9.3

Hz, 2H), 6.97 (d, J = 8.7 Hz, 1H), 7.37 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz,

1H), 7.74 (s, 1H), 9.07 (br, 1H).

O F3C NH O S O O O

5-[4-(6-Heptyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl- benzylidene]-thiazolidine-2,4-dione (TGHepBr) 1H NMR (300 MHz, CDCl3), δ

0.91 (t, J = 6.9 Hz, 3H), 1.24-1.59 (m, 11H), 1.81 (p, J = 6.9 Hz, 2H), 1.90-2.00 (m,

1H), 2.05-2.24 (m, 10H), 2.53-2.75 (m, 2H), 3.64 (t, J = 6.6 Hz, 2H), 4.08 (dd, J =

15.3, 9.0 Hz, 2H), 7.10 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.7, Hz, 1H), 7.73 (s, 1H),

7.90 (s, 1H), 8.78 (s, 1H)

O Br NH O S O O O

5-[3-Bromo-4-(6-isopropoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)- benzylidene]-thiazolidine-2,4-dione (TGIPBr) 1H NMR (300 MHz, CDCl3), δ

1.19 (s, 3H), 1.22 (s, 3H), 1.50 (s, 3H), 1.94-2.25 (m, 11H), 2.58-2.70 (m, 2H), 3.90-

4.42 (m, 3H), 4.52 (s, 2H), 6.99 (d, J = 9.0 Hz, 1H), 7.39 (dd, J = 9.0, 2.4 Hz, 1H),

7.70 (d, J = 2.4 Hz, 1H), 7.73 (s, 1H), 8.27 (br, 1H).

O Br NH O S O O O

5-{3-Bromo-4-[2,5,7,8-tetramethyl-6-(3-methyl-butoxy)-chroman-2-ylmethoxy]- benzylidene}-thiazolidine-2,4-dione (TGIPBr) 1H NMR (300 MHz, CDCl3), δ 0.97

(s, 3H), 0.99 (s, 3H), 1.50 (s, 3H), 1.72 (q, J = 6.9 Hz, 2H), 1.82-2.02 (m, 2H), 2.07 (s,

3H), 2.11-2.25 (m, 7H), 2.58-2.72 (m, 2H), 3.64 (t, J = 6.9 Hz, 2H), 4.02 (d, J = 9.3,

2H), 4.08 (d, J = 9.3, 2H), 6.97 (d, J = 8.7 Hz, 1H), 7.38 (dd, J = 8.7, 2.1 Hz, 1H),

7.70 (d, J = 2.1 Hz, 1H), 7.73 (s, 1H), 8.60 (br, 1H).

O Br NH O S O O O

5-{3-Bromo-4-[2,5,7,8-tetramethyl-6-(4-methyl-pentyloxy)-chroman-2-

ylmethoxy]-benzylidene}-thiazolidine-2,4-dione (TGMPBr) 1H NMR (300 MHz,

CDCl3), δ 0.93 (s, 3H), 0.95 (s, 3H), 1.34-1.41(m, 2H), 1.50 (s, 3H), 1.57-1.68 (m,

2H), 1.72-2.03 (m, 2H), 2.07 (s, 3H), 2.10-2.20 (m, 7H), 2.58-2.69 (m, 2H), 3.63 (t, J

= 6.9 Hz, 2H), 4.01 (d, J = 9.0, 2H), 4.08 (d, J = 9.0, 2H), 6.99 (d, J = 8.4 Hz, 1H),

7.38 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 7.73 (s, 1H), 8.57 (br, 1H).

O Br NH O S O O O

5-{3-Bromo-4-[6-(2-ethyl-butoxy)-2,5,7,8-tetramethyl-chroman-2-ylmethoxy]- benzylidene}-thiazolidine-2,4-dione (TGEBBr) 1H NMR (300 MHz, CDCl3), δ

0.96 (t, J = 7.5 Hz, 6H), 1.40-1.73 (m, 8H), 1.89-2.02 (m, 2H), 2.08 (s, 3H), 2.10-2.24

(m, 7H), 2.58-2.72 (m, 2H), 3.50 (d, J = 5.7 Hz, 2H), 4.04 (d, J = 9.3, 2H), 4.09 (d, J

= 9.3, 2H), 6.99 (d, J = 8.4 Hz, 1H), 7.38 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz,

1H), 7.74 (s, 1H), 8.43 (br, 1H).

O Br NH O S O O O

5-{3-Bromo-4-[6-(4-ethyl-hexyloxy)-2,5,7,8-tetramethyl-chroman-2-ylmethoxy]- benzylidene}-thiazolidine-2,4-dione (TGEHBr) 1H NMR (300 MHz, CDCl3), δ

0.96 (t, J = 6.9 Hz, 6H), 1.28-1.73 (m, 12H), 1.93-2.04 (m, 2H), 2.07 (s, 3H), 2.10-

2.24 (m, 7H), 2.54-2.70 (m, 2H), 3.50 (d, J = 6.0 Hz, 2H), 4.04 (d, J = 9.3, 2H), 4.11

(d, J = 9.3, 2H), 6.96 (d, J = 8.4 Hz, 1H), 7.38 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (d, J =

2.1 Hz, 1H), 7.74 (s, 1H), 8.89 (br, 1H).

O Br NH O S O O O

5-{3-Bromo-4-[6-(3,5-dimethyl-decyloxy)-2,5,7,8-tetramethyl-chroman-2-

ylmethoxy]-benzylidene}-thiazolidine-2,4-dione (TGDMOBr) 1H NMR (300 MHz,

CDCl3), δ 0.88 (d, J = 6.6 Hz, 6H), 0.93 (d, J = 6.3 Hz, 3H), 1.17-1.40 (m, 6H), 1.50

(s, 3H), 155-2.04 (m, 7H), 2.07 (s, 3H), 2.10-2.24 (m, 9H), 2.54-2.70 (m, 2H), 3.67 (d,

J = 6.9 Hz, 2H), 4.04 (d, J = 9.3, 2H), 4.11 (d, J = 9.3, 2H), 6.96 (d, J = 8.7 Hz, 1H),

7.38 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 8.99 (br, 1H).

O F3C NH O S O O O

5-{4-[2,5,7,8-Tetramethyl-6-(3-methyl-but-2-enyloxy)-chroman-2-ylmethoxy]-3-

trifluoromethyl-benzylidene}-thiazolidine-2,4-dione (TGDMACF3) 1H NMR (300

MHz, CDCl3), δ 1.47 (s, 3H), 1.72 (s, 3H), 1.81 (s, 3H), 1.90-2.01 (m, 1H), 2.04-

2.25 (m, 10H), 2.58-2.73 (m, 2H), 4.07 (d, J = 9.3 Hz, 2H), 4.10 (d, J = 9.3 Hz, 2H),

4.16 (d, J = 6.9 Hz, 2H), 5.60 (t, J = 6.9 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 7.61 (dd, J

= 8.7, 2.1 Hz, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.81 (s, 1H), 8.94 (br, 1H).

O F3C NH O S O O O

5-[4-(6-Allyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl- benzylidene]-thiazolidine-2,4-dione (TGAllBr) 1H NMR (300 MHz, CDCl3), δ

1.49 (s, 3H), 1.90-2.01 (m, 1H), 2.04-2.20 (m, 10H), 2.58-2.68 (m, 2H), 4.07 (d, J =

9.0 Hz, 2H), 4.10 (d, J = 9.0 Hz, 2H), 4.18 (d, J = 5.4 Hz, 2H), 5.25 (d, J = 9.6 Hz,

2H), 4.48 (d, J = 12.3 Hz, 2H), 6.05-6.14 (m, 1H), 7.11 (d, J = 8.7 Hz, 1H), 7.61 (d, J

= 8.7 Hz, 1H), 7.73 (s, 1H), 7.80 (s, 1H), 8.23 (br, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(6-but-2-enyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-

benzylidene]-thiazolidine-2,4-dione (TGBueBr) 1H NMR (300 MHz, CDCl3),

δ1.50 (s, 3H), 1.76 (d, J = 7.5, 3H), 1.93-2.05 (m, 1H), 2.04-2.26 (m, 10H), 2.58-2.68

(m, 2H), 4.01-4.14 (m, 4H), 5.79-5.92 (m, 2H), 7.00 (d, J = 8.7 Hz, 1H), 7.39 (dd, J =

8.7, 2.4 Hz, 1H), 7.71 (d, J = 2.4 Hz, 1H), 7.73 (s, 1H), 8.31 (br, 1H).

O Br NH O S O O O

5-[3-Bromo-4-(2,5,7,8-tetramethyl-6-pent-2-enyloxy-chroman-2-ylmethoxy)-

benzylidene]-thiazolidine-2,4-dione (TGPeneBr) 1H NMR (300 MHz, CDCl3), δ

1.04 (t, J = 7.5, 3H), 1.50 (s, 3H), 1.92-2.05 (m, 1H), 2.04-2.23 (m, 12H), 2.58-2.68

(m, 2H), 4.02-4.18 (m, 4H), 5.74-5.92 (m, 2H), 6.99 (d, J = 8.7 Hz, 1H), 7.39 (dd, J =

8.7, 2.1 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 8.70 (br, 1H).

O Br NH O S O O NC O

{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-2,5,7,8-

tetramethyl-chroman-6-yloxy}-acetonitrile (TGEtCNBr) 1H NMR (300 MHz,

CDCl3), δ 1.50 (s, 3H), 1.94-2.25 (m, 11H), 2.58-2.70 (m, 2H), 4.30 (q, J = 9.0 Hz,

2H), 4.52 (s, 2H), 6.97 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (d, J =

2.1 Hz, 1H), 7.74 (s, 1H), 8.55 (br, 1H), MS (m/z).

O Br NH O S O O NC O

4-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-butyronitrile (TGBuCNBr) 1H NMR (300

MHz, CDCl3), δ 1.49 (s, 3H), 1.94-2.23 (m, 13H), 2.58-2.74 (m, 4H), 3.76 (t, J = 5.7

Hz, 2H), 4.00-4.16 (m, 2H), 6.97 (d, J = 8.7 Hz, 1H), 7.40 (dd, J = 8.7, 2.1 Hz, 1H),

7.70 (d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 8.36 (br, 1H).

O F3C NH O S O O NC O

4-{2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-trifluoromethyl-

phenoxymethyl]-2,5,7,8-tetramethyl-chroman-6-yloxy}-butyronitrile

(TGBuCNCF3) 1H NMR (300 MHz, CDCl3), δ 1.47 (s, 3H), 1.94-2.01 (m, 1H),

2.02-2.20 (m, 12H), 2.55-2.64 (m, 2H), 2.68 (t, J = 6.9 Hz, 2H), 3.77 (t, J = 5.7 Hz,

2H), 4.03-4.12 (m, 2H), 7.10 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.74 (s,

1H), 7.80 (s, 1H), 8.56 (br, 1H), MS (m/z).

O F3C NH O S O O NC O

6-{2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-trifluoromethyl-

phenoxymethyl]-2,5,7,8-tetramethyl-chroman-6-yloxy}-hexanenitrile

(TGHexCNBr) 1H NMR (300 MHz, CDCl3), δ 1.44 (s, 3H), 1.62-1.84 (m, 6H),

1.90-1.98 (m, 1H), 2.02-2.20 (m, 10H), 2.38 (t, J = 6.6Hz, 2H), 2.51-2.68 (m, 2H),

3.63 (t, J = 6.0 Hz, 2H), 3.99-4.12 (m, 2H), 7.10 (d, J = 8.4Hz, 1H), 7.60 (d, J = 8.4Hz,

1H), 7.72 (s, 1H), 7.79 (s, 1H), 8.26 (br, 1H).

O Br NH O S O O NC O

6-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-2,2-dimethyl-hexanenitrile (TGDMHBr)

1H NMR (300 MHz, CDCl3), δ 1.37 (s, 6H), 1.49 (s, 3H), 1.59-1.89 (m, 6H), 1.99-

1.90 (m, 1H), 2.20-2.01(m, 10H), 2.66-2.69 (m, 2H), 3.67 (t, J = 6.3Hz, 2H), 4.04-

4.15 (m, 2H), 6.97(d, J = 9.0 Hz, 1H), 7.38 (dd, J = 8.7, 2.1Hz, 1H), 7.68 (d, J =

2.1Hz, 1H), 7.74 (s, 1H), 9.04 (s, 1H).

O Br NH O S O O NC O

7-{6-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

1,3,4,6-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yloxy}-heptanenitrile

(HepCN-Br) 1H NMR (300 MHz, CDCl3), δ 1.37(s, 6H), 1.49 (s, 3H), 1.50-1.58 (m,

4H), 1.65-1.82 (m, 4H), 1.90-1.99 (m, 1H), 2.01-2.20 (m, 10H), 2.36 (t, J = 6.9Hz,

2H), 2.61 (m, 2H), 3.62 (t, J = 6.3Hz, 2H), 4.04 (dd, J = 9.0, 10.2Hz, 2H), 6.95(d, J =

8.7Hz, 1H), 7.36 (dd, J = 8.7, 2.1Hz, 1H), 7.67(d, J = 2.1Hz, 1H), 7.71 (s, 1H), 8.59 (s,

1H), HRMS (ESI, m/z) calcd for C31H35BrN2O5SNa (M+Na): 649.1338, found:

649.1334. Anal. (C31H35BrN2O5S) C, H, Br, N, O, S.

O F3C NH O S O O NC O

7-{6-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-trifluoromethyl-

phenoxymethyl]-1,3,4,6-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yloxy}-

heptanenitrile (HepCN-CF3) 1H NMR (300 MHz, CDCl3), δ 1.47(s, 3H), 1.50-

1.59 (m, 4H), 1.68- 1.84 (m, 4H), 1.89-1.95 (m, 1H), 1.98-2.18 (m, 10H), 2.39 (t, J =

7.5Hz, 2H), 2.62 (m, 2H), 3.63 (t, J = 6.0Hz, 2H), 4.04 (d, J = 9.6 Hz, 1H), 4.08 (d, J

= 9.6 Hz, 1H), 7.08 (d, J = 8.4Hz, 1H), 7.59(d, J = 8.4Hz, 1H), 7.69 (s, 1H), 7.78 (s,

1H), 8.23 (br, 1H), HRMS (ESI, m/z), calcd for C32H35F3N2O5SNa (M+Na):

639.2116, found: 639.2113. Anal. (C32H35F3N2O5S) C, H, F, N, O, S.

O H3CO NH O S O O NC O

7-{2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-methoxy-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanenitrile (HepCN-OMe) 1H NMR

(300 MHz, CDCl3), δ 1.44(s, 3H), 1.51-1.59 (m, 4H), 1.68-1.84 (m, 4H), 1.90-1.99

(m, 1H), 2.01- 2.22 (m, 10H), 2.38 (t, J = 6.9Hz, 2H), 2.61-2.71 (m, 2H), 3.64 (t, J =

6.0 Hz, 2H), 3.90 (s, 3H), 4.03 (d, J = 9.6, 1H), 4.12 (d, J = 9.6, 1H), 6.96-7.10 (m,

3H), 7.80 (s, 1H), 8.70 (br, 1H).

O F3C NH O S O O O

4-{6-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-trifluoromethyl-

phenoxymethyl]-1,3,4,6-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-

yloxymethyl}-benzonitrile (BenzylCN-CF3) 1H NMR (300 MHz, CDCl3), δ 1.47(s,

3H), 2.24-1.90 (m, 11H), 2.61-2.72 (m, 2H), 4.04-4.12 (m, 2H), 4.76 (s, 2H), 7.12 (d,

J = 7.8 Hz, 1H), 7.58-7.72 (m, 6H), 7.80 (s, 1H), 8.49 (br, 1H), HRMS (ESI, m/z)

calcd for C33H29F3N2O5SNa (M+Na): 655.0878, found: 655.0877.

O Br NH O S O O O

4-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxymethyl}-benzonitrile (BenzylCN-Br) 1H

NMR (300 MHz, CDCl3), δ 1.52 (s, 3H), 1.95-2.28 (m, 11H), 2.60-2.74 (m, 2H),

4.03-4.17 (m, 2H), 4.77 (s, 2H), 6.99 (d, J = 8.7 Hz, 1H), 7.40 (dd, J = 7.5, 2.1 Hz,

1H), 7.76 (d, J = 8.1Hz, 2H), 7.68-7.76 (m, 4H), 8.56 (s, 1H).

O Br NH O S O O O O O

5-{6-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

1,3,4,6-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yloxy}-pentanoic acid ethyl

ester (PenE-Br) 1H NMR (300 MHz, CDCl3), δ 1.25 (t, J = 7.5 Hz, 1H), 1.47(s, 3H),

1.88-1.75 (m, 4H), 2.18-1.95(m, 11H), 2.39(t, J = 7.5Hz, 2H), 2.64 (m, 2H), 3.67 (t, J

= 6.6 Hz, 2H), 4.18-3.95 (m, 4H), 6.95 (d, J = 7.5Hz, 1H), 7.36 (d, J = 7.5Hz, 1H),

7.76 (m, 2H), 8.38 (br, 1H), HRMS (ESI, m/z) calcd for C31H36BrNO7SNa (M+Na):

668.1294, found: 668.1290.

O Br NH O S O O O O O

7-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanoic acid ethyl ester (TGHepEBr) 1H

NMR (300 MHz, CDCl3), δ 1.27 (t, J = 7.2 Hz, 1H), 1.36-1.59 (m, 7H), 1.62-1.87

(m, 4H), 1.92-2.03 (m, 1H), 2.06 (s, 3H), 2.12-2.20 (m, 7H), 2.33 (t, J = 7.5Hz, 2H),

2.58-2.70 (m, 2H), 3.63 (t, J = 6.6 Hz, 2H), 4.05 (t, J = 10.3 Hz, 2H), 4.14 (q, J = 7.5

Hz, 2H), 6.98 (d, J = 8.7 Hz, 1H), 7.39 (dd, J = 8.7, 1.8 Hz, 1H), 7.70 (d, J = 1.8 Hz,

1H), 7.74 (s, 2H), 8.52 (br, 1H).

O Br NH O S O O O HO O

5-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-pentanoic acid (TGPenABr) 1H NMR (300

MHz, CDCl3), δ 1.49 (s, 3H), 1.78-1.90 (m, 4H), 1.96-2.20 (m, 11H), 2.48 (t, J =

7.5Hz, 2H), 2.58-2.70 (m, 2H), 3.65 (t, J = 6.6 Hz, 2H), 4.05 (t, J = 10.3 Hz, 2H),

6.97 (d, J = 8.7 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.69 (s, 1H), 7.73 (s, 2H), 8.84 (br,

1H).

O Br NH O S O O O HO O

7-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanoic acid (TGHepABr) 1H NMR

(300 MHz, DMSO-D6), δ 1.36-1.59 (m, 7H), 1.62-1.73 (m, 2H), 1.79-1.95 (m, 4H),

1.98-2.08 (m, 7H), 2.16 (t, J = 7.2Hz, 2H), 2.54-2.62 (m, 2H), 3.63 (t, J = 6.6 Hz,

2H?), 4.06 (s, 2H), 7.16-7.23 (d+s, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.71 (s,

1H), 8.52 (br, 1H).

O Br NH O S O O O O O O N O

7-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanoic acid 2,5-dioxo-pyrrolidin-1-yl

ester (TGHepASucBr) 1H NMR (300 MHz, CDCl3), δ 1.39-1.59 (m, 7H), 1.62-1.87

(m, 4H), 1.92-2.03 (m, 1H), 2.06 -2.20 (m, 10H), 2.39 (t, J = 7.5Hz, 2H), 2.58-2.85

(m, 4H), 3.64 (t, J = 6.3 Hz, 2H), 4.07 (q, J = 9.0 Hz, 2H), 6.98 (d, J = 8.7 Hz, 1H),

7.39 (d, J = 8.7Hz, 1H), 7.70 (s, 1H), 7.73 (s, 2H), 9.35 (br, 1H).

O Br NH O S O O O H2N O

7-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanoic acid amide (TGHepAmBr) 1H

NMR (300 MHz, DMSO-D6), δ 1.28-1.59 (m, 7H), 1.62-1.73 (m, 2H), 1.79-1.89 (m,

1H), 1.93 (s, 1H), 2.05 (m + s, 7H), 2.21 (t, J = 6.9 Hz, 2H), 2.56-2.62 (m, 2H), 3.50-

3.64 (m, 4H?), 4.09 (s, 2H), 7.17-7.22 (d+s, J = 6.6 Hz, 2H), 7.44 (dd, J = 6.9, 2.1 Hz,

1H), 7.71 (d, J = 2.1Hz, 1H), 8.52 (br, 1H).

O Br NH O S O O O O

5-{4-[6-(4-Benzoyl-benzyloxy)-2,5,7,8-tetramethyl-chroman-2-ylmethoxy]-3-

bromo-benzylidene}-thiazolidine-2,4-dione (TG-BenzoPh-Br) 1H NMR (300 MHz,

CDCl3), δ 1.52 (s, 3H), 1.95-2.06 (m, 1H), 2.10 (s, 3H), 2.15-2.28 (m, 7H), 2.62-

2.74 (m, 2H), 4.11 (q, J = 9.0 Hz, 2H), 4.82 (s, 2H), 6.99 (d, J = 8.7 Hz, 1H), 7.41 (dd,

J = 8.7, 2.1 Hz, 1H), 7.52 (d, J = 7.8Hz, 2H), 7.59-7.64 (m, 3H), 7.71 (d, J = 2.1Hz,

1H), 7.74 (s, 1H), 7.85 (t, J = 8.1 Hz, 4H), 8.51 (s, 1H).

O Br NH O S O O O

4'-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxymethyl}-biphenyl-2-carbonitrile (TG-

BPCN-Br) 1H NMR (300 MHz, CDCl3), δ 1.52 (s, 3H), 1.95-2.06 (m, 1H), 2.11 (s,

3H), 2.15-2.28 (m, 7H), 2.62-2.72 (m, 2H), 4.05-4.18 (m, 2H), 4.78 (s, 2H), 7.00 (d, J

= 8.7 Hz, 1H), 7.38-7.82 (m, 11H), 8.47 (s, 1H).

O Br NH O S O O O

R-5-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-me- thylbut-2-enyloxy)chroman-2-

yl)methoxy)benzylidene)thiazolidine-2,4-dione (R-TG-88) 1H NMR (250 MHz,

CDCl3) δ 1.46 (s, 3H), 1.72 (s, 3H), 1.82 (s, 3H), 1.99-1.87 (m, 1H), 2.01- 2.22 (m,

10H), 2.63 (m, 2H), 4.10 (d, 2H, J =9.3 Hz), 4.18 (d, 2H, J = 6.7 Hz), 5.58 (t, 1H, J =

7.2 Hz), 6.95-7.10 (m, 3H), 7.78 (s,1H), 8.27 (brs, 1H), MS (ESI, m/z) calcd for

C29H32BrNO5SNa (M+Na): 608.1076, found: 608.1094.

O F3C NH O S O O O

R-5-[4-(6-Butoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl-

benzylidene]-thiazolidine-2,4-dione (R-TGBuCF3). 1H NMR (300 MHz, CDCl3),

δ 0.92 (t, J = 7.0 Hz, 3H), 1.47(s, 3H), 1.48 - 1.58 (m, 2H), 1.73-1.83 (m, 2H), 1.89-

2.01 (m, 1H), 2.02- 2.18 (m, 10H), 2.60-2.64 (m, 2H), 3.61 (t, J = 6.7 Hz, 2H), 4.06

(q, J = 9.3 Hz, 2H), 7.12(d, J = 9.3 Hz, 1H), 7.60 (dd, J = 9.3, 1.2 Hz, 1H), 7.72 (d, J

= 1.2 Hz, 1H), 7.79 (s, 1H), 8.22 (br, 1H), HRMS (ESI, m/z), calcd for

C29H32F3NO4SNa (M+Na): 586.1851, found: 586.1848.

O F3C NH O S O O NC O

R-7-{6-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-2-trifluoromethyl-

phenoxymethyl]-1,3,4,6-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yloxy}-

heptanenitrile (HepCN-CF3) 1H NMR (300 MHz, CDCl3), δ 1.47(s, 3H), 1.59-

1.47 (m, 4H), 1.85-1.68(m, 4H), 1.98-1.89 (m, 2H), 2.18-1.98(m, 9H), 2.36(t, J =

7.5Hz, 2H), 2.62 (m, 2H), 3.63 (t, J = 6.0Hz, 2H),4.16 (q, J = 9.6, 2H), 7.10 (d, J =

8.4Hz, 1H), 7.66 (s, 1H), 7.61(d, J = 8.4Hz, 1H), 7.78 (s, 1H), 8.40 (br, 1H), MS (ESI, m/z), calcd for C32H35F3N2O5SNa (M+Na): 639.2116, found: 639.2113.

Procedure to reduce the double bond of ∆2TG derivatives. A mixture of

individual ∆2TG derivative (1mmol) and Pd-C (150 mg) in methanol (5 mL) was

stirred under hydrogen (50 psi) overnight, filtered, and concentrated to dryness under

vacuum. The residue was purified by silica gel flash chromatography (ethyl

acetate/hexanes system), giving reduced objective compound.

O F3C NH O S O O O

5-[4-(6-Butoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-trifluoromethyl-

benzyl]-thiazolidine-2,4-dione (2H-Bu-CF3). 1H NMR (300 MHz, CDCl3), δ 0.98

(t, J = 7.2 Hz, 3H), 1.34-1.68 (m, 5H), 1.68-1.85 (m, 2H), 1.86-1.98 (m, 1H), 1.98-

2.22 (m, 10H), 2.62-2.65 (m, 1H), 3.15-3.21 (m, 1H), 3.40-3.52 (m, 1H), 3.63 (t, J =

6.9 Hz, 2H), 4.26 (q, J = 10.0 Hz, 2H), 4.50 (m, 1H), 6.95 (d, J = 6.6 Hz, 1H), 7.25 (d,

J = 6.6 Hz, 1H), 7.44 (s, 1H), 8.16(s, 1H).

O F3C NH O S O O NC O

7-{2-[4-(2,4-Dioxo-thiazolidin-5-ylmethyl)-2-trifluoromethyl-phenoxymethyl]-

2,5,7,8-tetramethyl-chroman-6-yloxy}-heptanenitrile (2H-HepCN-CF3) 1H NMR

(300 MHz, CDCl3), δ 1.46(s, 3H), 1.52-1.63 (m, 4H), 1.67-1.84 (m, 4H), 1.88-1.99

(m, 2H), 2.03-2.21 (m, 10H), 2.39 (t, J = 6.9 Hz, 2H), 2.55-2.69 (m, 2H), 3.10-3.21(m,

1H), 3.42-3.53(m, 1H), 3.64 (t, J = 6.0Hz, 2H), 3.97 (q, J = 9.6, 2H), ), 4.43-4.57 (m,

1H), 6.94 (d, J = 8.4 1H), 7.32 (d, J = 6.6, 1H), 7.44 (s, 1H), 8.22 (s, 1H).

Molecular modeling. It was adopted a representative docking method of CDOCKER

2.0, a molecular dynamics (MD) simulated-annealing-based algorithm combined with

Monte Carlo (MC) to perform the structure-based screening. Human Bcl-xL structure

with PDB id 1BXL in the complex of Bcl-xL and BakBH3 peptide was used for

virtual screening of new proposed molecules, taking the corresponding BakBH3

peptide binding pocket on the Bcl-2 protein as the target (90 × 90 × 90 3-D affinity

grids centered on the Bak peptide binding site with 0.375 Å spacing were chosen as the target pocket). Bak peptide and all water molecules were deleted. For both Bcl-xL and screening compound, all hydrogen was added and all atoms were assigned with

CHARMm force field potential.

In the process of this flexible docking calculation, the interaction with the Bcl-xL surface pocket of the molecule in different orientations was scored by a shape complementarity scoring function that resembles the nonbonding interaction energy.

As a result, the screening molecule in the best orientation was saved and the binding

energy between molecule and Bcl-xL protein was evaluated.

Cell culture. LNCaP androgen-dependent (p53+/+), and PC-3 androgennonresponsive

(P53-/-) prostate cancer cells were maintained in RPMI 1640 supplemented with 10%

fetal bovine serum at 37 °C in a humidified incubator containing 5% carbon dioxide.

Preparation of the stable Bcl-xL-overexpressing LNCaP clones B3 were previously

described [130].

Cell viability analysis. The effect of individual test agents on cell viability was

assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay in 6 to 12 replicates. Cells were seeded and incubated in 96-well, flat-

bottomed plates in serum-free media for 24 hours and were exposed to various

concentrations of test agents dissolved in DMSO ( final concentration, 0.1%) in serum-free RPMI 1640 for different time intervals. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. The medium was removed, replaced by 200 µL of 0.5 mg/mL MTT in 10% fetal bovine serum–containing RPMI

1640, and cells were incubated in the carbon dioxide incubator at 37 °C for 2 hours.

Supernatants were removed from the wells and the reduced MTT dye was solubilized in 200 µL/well DMSO. Absorbance at 570 nm was determined on a plate reader.

Immunoblotting. Cells in T-75 flasks were collected by scraping and suspended in

60 µL of PBS. Two microliters of the suspension was taken for protein analysis using the Bradford assay kit (Bio-Rad, Hercules, CA). The same volume of 2 × SDS-PAGE sample loading buffer [100 mmol/L Tris-HCl (pH 6.8), 4% SDS, 5% h- mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue] was added to the remaining solution. The mixture was sonicated briefly and then boiled for 5 minutes.

Equal amounts of proteins were loaded onto 10% SDS-PAGE gels. After electrophoresis, protein bands were transferred to nitrocellulose membranes in a semi-dry transfer cell. The transblotted membrane was washed twice with TBS containing 0.1% Tween 20 (TBST). After blocking with TBST containing 5% nonfat milk for 40 minutes, the membrane was incubated with the appropriate primary antibody in TBST-1% nonfat milk at 4 °C overnight. All primary antibodies were diluted 1:1,000 in 1% nonfat milk–containing TBST. After treatment with the primary antibody, the membrane was washed three times with TBST for a total of 15 minutes followed by incubation with goat anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugates (diluted 1:5,000) for 1 hour at room temperature and three washes with TBST for a total of 1 hour. The immunoblots were visualized by enhanced chemiluminescence.

Competitive fluorescence polarization assay. The binding affinity of the test agent to Bcl-xL was analyzed by a competitive fluorescence polarization assay in which the ability of the agent to displace the binding of a Bak BH3–domain peptide to either

Bcl-xL was determined. Flu-BakBH3, a Bak-BH3 peptide labeled at the NH2 terminus with fluorescein, was purchased from Genemed Synthesis (San Francisco,

CA). COOH-terminal-truncated, His-tagged Bcl-xL was purchased from EMD

Biosciences (San Diego, CA). The KD determination was carried out in a dual–path

length quartz cell with readings taken at λem 480 nm and λex 530 nm at room temperature.

Determination of IC50 values. Data from cell viability and fluorescence polarization

assays were analyzed by using the CalcuSyn software (Biosoft, Ferguson, MO) to

determine IC50 values, in which the calculation was based on the medium-effect

equation [i.e., log(fa/fu) = mlog(D) - mlog(Dm), where fa and fu denote fraction affected

and unaffected, respectively; m represents the Hill-type coefficient signifying the sigmoidicity of the doseeffect curve; and D and Dm are the dose used and IC50.

Co-immunoprecipitation. PC3 cells treated with HepCNCF3 for 12 hours were

collected and lysed by NP40 isotonic lysis buffer with freshly added protease

inhibitors [142 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L HEPES (pH 7.2), 1

mmol/L EGTA, 0.2% NP40, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 1

µg/mL each aprotinin, leupeptin, and pepstatin]. After centrifugation at 13,000 × g for

15 minutes, the supernatants were collected, preincubated with protein A-Sepharose

(Sigma) for 15 minutes, and centrifuged at 1,000 × g for 5 minutes. The supernatants were exposed to Bcl-2 or Bcl-xL antibodies in the presence of protein A-Sepharose at

4 °C for 2 hours. After brief centrifugation, protein A-Sepharose were collected,

washed with the aforementioned lysis buffer twice, suspended in 2 × SDS sample buffer, and subjected to Western blot analysis with antibodies against Bak.

CHAPTER 3

DEVELOPMENT OF AR ABLATING AGENTS BASED ON

CIGLITAZONE PARTLY AIDED BY COMBINATORIAL CHEMISTRY

3.1 Strategies for lead optimization of ciglitazone

Early studies in our group have shown the effects of troglitazone and its PPARγ

independent counterpart ∆2TG on PSA repression [131] and down-regulation of AR

expression [132] in LNCaP prostate cancer cells. STG28, a compound with higher

potency in AR repression, was obtained after strcture modification and optimization based on troglitazone. IC50 of STG28 on AR ablation was about 10 µM, while about

80 µM for troglitazone and 40 µM for ∆2TG. Since ciglitazone was demonstrated

similar anti-prostate cancer activity as troglitazone with simpler structure and lower

molecular weight than troglitazone, it was chosen as lead compound to do a series of

ligand based design and synthesis to exploit the underlying relationships between the

structures of derivatives and their activities on androgen receptor ablation.

The lead optimization of ciglitazone to develop more potent compounds consisted of

three stages (Figure 3.1). Firstly, the activities on AR of ciglitazone and its PPARγ

independent counterpart ∆2CG have been confirmed; secondly, structural

modification on ∆2CG have been done via three distinct strategies: (a) phenyl ring

substitutions, (b) regioisomerization of the (1-methylcyclohexyl)-methyl moiety, and

from oxygen atom of phenol group to nitrogen atom of imide group to generate compound CG9; thirdly, by using CG9 as a new lead structure scaffold, functional group modification at phenolic group (R1), phenyl ring substitutions (X), and modification on the (1-methylcyclohexyl)-methyl group (R2) have been done. For the last stage, optimization of the substituting group on the nitrogen of thiazolidinedione

(R2), a strategy of solid-phase focused library synthesis was introduced. These obtained ∆2CG derivatives and CG9 derivatives were screened by cell viability assay and AR promoter-luciferase reporter gene assay on LNCaP cells. The abilities to suppress AR expression of selected compounds were assessed by the followed by

Western blotting analysis.

O O

NH NH S S O O O O

Ciglitazone ∆ 2CG O NH S O O O O

NH N S S O X O HO O CG9

O O O Ar N N N S S S O R1 O HO X O

O O O

O N O N N S S R2 S S R N O R N O HO O O H H CF3

Figure 3.1 Strategy of structure modification based on ciglitazone

3.2 Validation of AR suppressing activity of ciglitazone and ∆2CG

Dose-dependent and time-dependent effects of CG and ∆2CG on suppressing AR expression were assessed in LNCaP cells by Western blotting and and reverse transcriptase-polymerase chain reaction (RT-PCR) (Figure 3.2 & 3.3). These results indicated that ∆2CG, although lacking PPARγ agonist activity, exhibit modestly higher potency than ciglitazone in mediating repression of AR. The concentrations required for complete suppression of AR expression were approximately 60 µM for ciglitazone and 30 µM for ∆2CG respectively. Time-dependent effects of CG (60 µM) and ∆2CG (30 µM) on suppressing the mRNA level of AR have suggested that the

AR repression by CG and ∆2CG be at transcriptional stage. With more compact structures and more potent activities compared to troglitazone and ∆2TG, ciglitazone and ∆2CG were good lead compounds for further modification on their AR transcriptional repression activities. To expedite the screening of AR-ablative agents, we used a luciferase reporter assay in place of RT-PCR to analyze the effect of individual derivatives on suppressing AR transcription in addition to cell viability assays on LNCaP cells. The luciferase reporter assay was fulfilled by transiently transfected the AR promoter-linked luciferase reporter plasmid LNCaP cells.

Figure 3.2 Effect of ciglitazone (CG) and ∆2CG on AR ablation in LNCaP cells – Western blotting. Dose- and time- dependent effects of CG and ∆2CG on suppressing AR protein expression levels.

Figure 3.3 Time-dependent effect of CG (60 µM) and ∆2CG (30 µM) on suppressing the mRNA level of AR. Cells were treated with either agent in 10% FBS- supplemented medium for the indicated times. Total RNA was isolated and subjected to RT-PCR analysis as described in the experimental part.

3.3 Regioisomerization of the (1-methylcyclohexyl)-methyl moiety of ∆2CG

Several strategies have been used to exploit relationship between the potency of

LNCaP cancer cell viability inhibition and ∆2CG structure modification. The first strategy is to transfer the bulky group moiety, (1-methylcyclohexyl)-methyl group, from 4’ position to 3’ position. The obtained compound 3’-∆2CG showed moderately better activity than ∆2CG (Table 3.1), which suggested the position of (1- methylcyclohexyl)-methyl group be flexible. Another compound (2’-∆2CG) with the bulky group on 2’ position, has been tried using similar synthetic method, which failed to yield any objective compound, mostly because of strong steric hindrance between the neighboring (1-methylcyclohexyl)-methyl group and the thiazolidinedione ring.

To synthesize ∆2CG analogs, general reaction procedure was applied (Figure 3.4). In brief, the starting material (1-methylcyclohexyl)-carboxylic acid was reduced to alcohol by lithium aluminum hydride, and the alcohol was transformed into its active form triflate by reaction with trifluoromethylsulfonyl anhydride. The triflate was coupled to the phenolic group of the benzaldehyde aided by the base of potassium carbonate to form etherized benzaldehyde which was condensed with thiazolidinedione ring to provide our objective compound 3’-∆2CG.

CHO O H N O O (CF SO ) O OH O CHO O COOH LiAlH4 OH 3 2 2 OTfl S NH THF Pyridine/CH2Cl2 K2CO3, DMF AcOH/piperidine S ethanol/reflux O

Figure 3.4 Synthetic route for the isomerized compound 3’-∆2CG

O O S NH O

Entry Compounds IC50 of MTT (µM) 1 3’-∆2CG 12.7 (CG1)

Table 3.1 Isomerized compound (3’-∆2CG ) with (1-methylcyclohexyl)-methoxy group at 3’ position instead of 4’ position in ∆2CG and its IC50 values of individual agents in inhibiting the cell viability of LNCaP cells. Cells were exposed to individual agents at various concentrations in 5% FBS-supplemented RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

3.4 Phenyl ring substitutions of ∆2CG

The second strategy to exploit SAR studies on ∆2CG derivatives was to introduce various substitutions on phenyl group in ∆2CG with different size, polarity and electronegativity. Results showed a moderate optimization for compounds with both electron-withdrawing groups of bromo and trifluoromethyl and electron pushing methoxyl and ethoxyl groups on 3’ position, however, a hydrophilic nitro group at 3’ position or bulky di-methyl groups on the phenyl ring will slightly decrease the potency (Table 3.2). To synthesize these derivatives, above mentioned general procedure to synthesize 3’-∆2CG was applied (Figure 3.5).

O H O H CHO N NH HO O O S (CF3SO2)2O COOH LiAlH4 OH OTfl X S O X O O X THF Pyridine/CH2Cl2 K2CO3, DMF AcOH/piperidine ethanol/reflux

Figure 3.5 Synthetic routes for ∆2CG derivatives with different substitution on phenyl group

NH S O X O

Entry Compounds X IC50 of MTT (µM) 2 ∆2CG-Br 3’-Br 16.9 3 ∆2CG-CF3 3’-CF3 17.2 4 ∆2CG-NO2 3’-NO3 40.0 5 ∆2CG-OMe 3’-OMe 19.2 6 ∆2CG-OEt (CG6) 3’-OEt 21.8 7 ∆2CG-DiMe 3’5’-DiMe 43.8 8 ∆2CG-Naph 2’3’-benzo 24.8

Table 3.2 ∆2CG derivatives with different substitution on phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

3.5 Permutational rearrangement of the (1-methylcyclohexyl)-methyl group (CG9)

Former findings in our group showed that when the bulky substituted chroman group

in STG28 was transferred from the phenolic oxygen atom to imidic nitrogen atom, the

activities of anti-prostate cancer and AR repression have been kept to some extent

(data not published). Thereby, the third strategy used was to design permutational

compound CG9 to further exploit the position flexibility of the (1-methylcyclohexyl)-

methyl group in the structure of ∆2CG. To synthesize CG9 (Figure 3.6), the above

mentioned triflate from (1-methylcyclohexyl)-carboxylic acid by reduction and activation was used to couple on the nitrogen of thiazolidinedione ring, the obtained substituted thiazolidinedione was condensed with hydroxyl benzaldehyde to yield

objective compound CG9. Cell viability assay and luciferase assay showed that CG9

doubled the potency of ∆2CG in terms of AR repression and LNCaP cell killing

effects. Although the optimization of CG9 was still moderate (Table 3.3), it provides

a good new structural scaffold for further modification.

COOH O Piperidine N OH Pyridine OSO2CF3 N S O LiAlH /THF HO O 4 O H S (CF3SO2)2O N HO CHO O S

Figure 3.6 Synthetic routes for CG9

N S HO O Entry Compounds IC50 of MTT (µM) 9 CG-OH-H (CG9) 18

Table 3.3 Compound (CG9) and its IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

Results from the AR promoter-luciferase reporter gene assay and Western blotting

assay for AR protein level confirmed our screening data in cell viability assay

(Figure 3.7 & 3.8).

Figure 3.7 Analysis of the effects of individual compounds on the transcriptional repression of the AR gene by the AR promoter-luciferase reporter assay. LNCaP cells were transiently transfected with an AR promoter-linked luciferase reporter plasmid and exposed to DMSO vehicle (D), ciglitazone (CG, 20 µM), ∆2CG (∆2, 20 µM), or compounds 1–9 (10 µM) in 10% FBSsupplemented RPMI 1640 medium for 48 h. Analysis of luciferase activity was carried out as described in the Experimental section:(columns) mean; (n ) 3); (bars) standard deviation (SD).

Figure 3.8 Western blotting analysis of the dose-dependent effect of compounds CG1, CG6, and CG9 on reducing AR protein levels.

3.6 Regioisomerization of the phenolic group in CG9

First strategy to exploit CG9 analogs includes synthesis of three derivatives, with

hydroxyl group(s) at 2’ position, 3’ position or 2’, 3’, 4’ – tri positions. Results

showed that moving the terminal para OH group to the ortho or meta position

(compounds CG10 and CG11) abolished the ability to suppress AR promoter- luciferase activity and cell viability (Figure 3.11 & Table 3.4), indicating its

important role in interacting with the target protein. Multiple hydroxyl group

substitution did not provide better optimization either. These compounds were

synthesized by almost the same procedure as that for CG9 (Figure 3.9).

COOH O Piperidine N OH Pyridine OSO2CF3 N S O LiAlH /THF HO O 4 O H S (CF3SO2)2O N CHO O HO S

Figure 3.9 Synthetic routes for CG9 derivatives with phenolic group at various positions

N S R1 O Entry Compounds R1 IC50 of MTT (µM) 9 CG-OH-H (CG9) 4’ OH 18 10 CG-2’OH 2’ OH 62 11 CG-3’OH 3’ OH 44 20 CG-tri-OH 3’,4’,5’-tri-OH 32

Table 3.4 CG9 derivatives with phenolic group at various positions and multiple hydroxyl groups on phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

3.7 Phenyl ring substitutions (X) of CG9

Further modification on CG9 was performed by introducing individual substitutions

on phenyl group with different steric effects and electronic effects. Of these derivatives, compounds CG12 and CG16 represented the optimal agents in inhibiting

LNCaP cell viability (Table 3.5). Both optimized compounds were with hydrophobic and electron-withdrawing group, which may be beneficial to the neighboring para-

hydroxyl group required by the binding pocket of targeted protein. To synthesize

these compounds, fragment coupling method was applied to improve the reaction

yield (Figure 3.10).

Luciferase assay also showed that compounds CG12 and CG16 account for the most

potent derivatives so far in AR m-RNA repression (Figure 3.11). Western blotting

analysis indicates that the IC50 values for suppressing AR expression by compounds

12 and 16 after 72 h of exposure were approximately 2 and 4 µM, respectively

(Figure 3.12).

COOH O OH Pyridine OSO2CF3

LiAlH4/THF (CF SO ) O N 3 2 2 S K2CO3/DMF HO X O Piperidine HO CHO HO O O H N X X O S NH S O

Figure 3.10 Synthetic routes for CG9 derivatives with different substitution on phenyl group

N S X O HO

Entry Compounds X IC50 of MTT (µM) 12 CG-OH-CF3 3’-CF3 8.2 (CG12) 13 CG-OH-NO2 3’-NO2 25.4 14 CG-OH-Br 3’-Br 18 15 CG-OH-OMe 3’-OMe 31 16 CG-OH-DiBr 3’5’-DiBr 9 17 CG-OH-IOMe 3’-I, 5’-MeO 27 18 CG-OH-DiMe 3’5’-DiMe >50 19 CG-OH-Naph 2’3’-benzo 17.5

Table 3.5 CG9 derivatives with different substitution on phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells. Cells were exposed to individual agents at various concentrations in 5% FBS-supplemented RPMI 1640 medium for 48 h, and cell viability was assessed by MTT assays.

Figure 3.11 Analysis of the effects of individual compounds on the transcriptional repression of the AR gene by the AR promoter-luciferase reporter assay. LNCaP cells were transiently transfected with an AR promoter-linked luciferase reporter plasmid and exposed to DMSO vehicle (D), or compounds 10 – 19 (10 µM) in 10% FBSsupplemented RPMI 1640 medium for 48 h. Analysis of luciferase activity was carried out as described in the Experimental section: (columns) mean (n ) 3); (bars) standard deviation (SD).

Figure 3.12 Western blotting analysis of the dose-dependent effect of compounds CG12 and CG16, on reducing AR protein levels. Cells were exposed to individual agents at the indicated concentrations in 10% FBS-supplemented medium for 72 h, and the lysates were subjected to Western blotting analysis.

Furthermore, the abilities of compounds 12 and 16 to induce apoptosis in LNCaP

cells were demonstrated by their dose-dependent effects on modulating various

apoptotic biomarkers, including PARP cleavage, and the proteolytic activation of

caspase 3 and caspase 7 (Figure 3.13).

Figure 3.13 Western blotting analysis of the dose-dependent effect of compounds CG12, on PARP cleavage, and the proteolytic activation of caspase 3 and caspase 7. Cells were exposed to individual agents at the indicated concentrations in 10% FBS- supplemented medium for 72 h, and the lysates were subjected to Western blotting analysis.

To demonstrate that transcriptional repression effects of AR by these derivatives were

independent of PPARγ, we examined the ability of compounds 12 and 16 versus

troglitazone, ciglitazone, and ∆2CG to transactivate PPARγ by using the PPAR

response element (PPRE) luciferase reporter assay (Figure 3.14). In PC-3 cells

transfected with a reporter construct (PPRE-x3-TK-Luc), troglitazone and ciglitazone

at 10 µM exhibited a significant effect on increasing luciferase activity, ranging from

2.5-fold to 4-fold (P < 0.05). As we expectated, CG12 and CG16, lacked appreciable

activity in PPARγ activation at up to 10 µM just as their parent compounds ∆2CG.

Figure 3.14 Dose-dependent effect of ciglitazone (CG), ∆2CG, and CG12 and CG16, relative to that of 10 µM troglitazone (TG), on PPARγ activation in PC-3 cells. PC-3 cells were transiently transfected with PPRE-x3-TK-Luc reporter vector and then exposed to individual agents or DMSO vehicle (D) in 10% FBS-supplemented RPMI 1640 medium for 48 h. Analysis of luciferase activity was carried out as described in the Experimental section: (columns) mean (n = 6); (bars) SD.

The antitumor effects of CG12 were evaluated in both androgen-responsive LNCaP

and androgen-nonresponsive PC-3 prostate cancer cells via three different methods,

including the MTT assay for cell viability (Figure 3.15-A), cell counting for cell

proliferation (Figure 3.15-B), and flow cytometric analysis for cell cycle distribution

(Figure 3.15-C).

Mostly because of the lack of AR expression, PC-3 cells exhibited substantially lower

sensitivity to the cell viability inhibition by CG12 compared to the androgen

expressing LNCaP cells. The IC50 values for suppressing cell viability were 15 and 12

µM, respectively, in PC-3 cells and were 8 and 3 µM at 48 and 72 h of drug treatment, respectively, in LNCaP cells. This differential susceptibility was also manifest in the cell counting assay, in which compound CG12 exhibited at least 2-fold less potency

in inhibiting the proliferation of PC-3 cells compared to LNCaP cells.

Cell cycle analysis was carried out after exposing LNCaP cells to different doses of

compound 12 for 72 h. As shown, CG12 caused a dose-dependent increase in the sub-

G1 population, accompanied by decreases in the G2/M phase, which also indicated

the apoptotic effects induced by CG12.

Figure 3.15 Antitumor effects of CG12 in LNCaP cells. (A) Differential dose- dependent effects of CG12 on the inhibition of cell viability of LNCaP versus PC-3 cells at 48 h (inset) and 72 h of treatment. Points, mean (n = 6); bars, SD. (B) Dose- and time-dependent antiproliferative effects of CG12 in LNCaP (left panel) and PC-3 (right panel) cells. (C) Flow cytometric analysis of LNCaP cells after treatment with DMSO or the indicated concentrations of CG12 for 72 h. Each data point represents the mean of two independent determinations

3.8 Phenolic group (R1) modification on CG9

Former structure activity relationship (SAR) studies have demonstrated that the para

position of hydroxyl group on phenyl ring was important to keep the activities of AR

repression and LNCaP cell viability inhibition. Here we synthesized compounds

CG21 to CG33 with a series of bioisosteric substitutions of hydroxyl group. All the

compounds were synthesized by the same reaction procedure in synthesizing CG9

(Figure 3.16), except compound CG24, which were synthesized by reduction of the

nitro group in compound CG32.

Most of the derivative totally lost the potency (Table 3.6), some of the derivatives

dissolve neither in DMSO nor in water because of their hydrophobicity, which made

the evaluation impossible. However, compounds with para-amino or para-acetamide

groups kept most of the activity, which suggests a potential hydrogen bonding effects

between the effective compounds and the receptor. The observed potency of the

compound with acetamide opened another door for further modification with

substitutions of various amides and sulfonamides on 4’ position of the phenyl ring.

COOH O Piperidine N OH Pyridine OSO2CF3 N S O LiAlH /THF R1 O 4 O H S (CF3SO2)2O N R1 CHO O S

Figure 3.16 Synthetic routes for CG9 derivatives with various substitutions on 4’ position of phenyl ring

N S R1 O

Entry Compounds R1 IC50 of MTT (µM) 9 CGOH-H (CG9) -OH 18 21 CG-Ph-F -F 90 22 CG-Ph-Cl -Cl > 100 23 CG-Ph-Br -Br N.D. 24 CG-Ph-NH2 -NH2 30 25 CG-Ph-DMN -N(CH3)2 > 100 26 CG-Ph-DEN -N(CH2CH3)2 N.D. 27 CG-Ph-COOMe -COOMe N.D 28 CG-Ph-COOH -COOH > 100 29 CG-Ph-CH2OH -CH2OH > 100 30 CG-Ph-CN -CN >100 31 CG-Ph-AcN -NHCOCH3 20 32 CG-Ph-NO2 -NO2 > 100 33 CG-Ph-OCF3 -OCF3 N.D.

Table 3.6 CG9 derivatives with different substitution on 4’ position of phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells. N.D. , not dissolved in Water or DMSO.

3.9 Substitution of 4’ phenolic group in CG9 by amides or sulfonamides

On the basis of our finding that substitution of the hydroxyl group in CG9 by the

acetamide group showed similar activity on LNCaP cell as CG9 did, a library of

amide compounds and a library of sulfonamide compounds were synthesized and

their inhibitory effects on viability of LNCaP cells were evaluated. Interestingly, 3-

chloro-4-nitro-benzyl group accounts for the best substitution in both cases of the

amides and the sulfonamides (Tables 3.7 & 3.8). Overall, compounds with

sulfonamide substitutions were more potent than compounds with amide substitutions and compounds with electron-withdrawing groups were more potent than those with

electron-pushing groups.

All these compounds were synthesized from amine compound CG-Ph-NH2 (Figure

3.17), with reaction with acyl chlorides or sulphonyl chlorides in water-miscible

solvent dioxane or acetone. Upon TLC confirmation of the reactions, water was

added and objective compounds were obtained after filtering and drying.

N Zn/FeCl3 COOH O Piperidine OH OSO2CF3 N S Pyridine O O2N O DMF/H2O LiAlH4/THF O H S O2N CHO (CF3SO2)2O N O S O

O N S O CO3 R N O l/K2 OC H RC N S H2N O R O SO 2 C l/P N yri O din S e S R N O O H

Figure 3.17 Synthetic routes for amide and sulfonamide derivatives of CG9

R N S O N H O

Compounds R IC50 Compounds R IC50 (Entry) (µM) (Entry) (µM) CGPhAcN -CH3 20 CGAm4BrPh >50 Br (31) (43) CGAmPro -C2H5 27 CGAm4NO2Ph 12.5 NO2 (34) (44) CGAmPalm -n-C15H31 >50 CGAm4CNPh >50 CN (35) (45) CGAmCCl3 -CCl3 17.3 CGAm2,4DCPh >50 Cl (36) (46) Cl CGAmcHex 19 CGAm4Cl3NO Ph 9.5 2 Cl (37) (47)

NO2 CGAmPh 38 CGAm3,4DCPh >50 Cl (38) (48)

Cl CGAmPyr 18 CGAmPiperonyl >50 O (39) N (49) O CGAm3NO2Ph 20.5 CGAm2Naph >50 (40) (50) NO2 CGAm3CF3Ph >50 CGAmTriMeOPh OMe 15 (41) (51) OMe CF3 OMe CGAm3CNPh >50 CGAmBiPh >50 (42) (52) CN

Table 3.7 CG9 derivatives with different amide group at 4’ position of the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

O N s S O N H R O Entry Compounds R IC50 (µM) 53 CGSmCH3 CH3 20 54 CGSmNH2 NH2 18.1 55 CGSm2NO2 10

O2N

56 CGSm3NO2Ph 18.3 O2N

57 CGSm4AcNPh 35.3 H3COCHN 58 CGSm4OMePh >50 H3CO

59 CGSm4MePh 25.3 H3C

60 CGSm4ClPh 15.8 Cl

61 CGSm4AcPh 18.8 H3COC

62 CGSm4NO2 10.2 O2N

63 CGSm4Cl3NO2Ph Cl 5.3

O2N 64 CGSm2,3OMePh H3CO 29

H3CO

65 CGSm4NO2,3CF3 8.2 O2N

F3C 66 CGSm4NO2,2OMe OMe 27.6

O2N

Table 3.8 CG9 derivatives with different sulfonamide group on the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

3.10 Substitution of 4’ phenolic group in CG12 by selected amides or

sulfonamides

In efforts of combining the advantages of X substitution (CF3 group) and R1

substitution group (amide or sulfonamide with 3-chloro-4-nitro-benzyl group) in one

molecule, we synthesized compounds 67 – 71. Because neither 4-nitro-3-

trifluoromeyl-benzaldehyde nor 4-amino-3-trifluoromethyl-benzaldehyde was

available commercially, we purchased the precursor compound 4-nitro-3-

trifluoromeyl-benzonitrile which was conveniently reduced to 4-nitro-3-

trifluoromeyl-benzaldehyde. Considering lower yield and dirty workup by using

Zn/FeCl3 system in reducing the nitro group, we tried system of pressured hydrogen

under catalysts of palladium-carbon in shorter reaction time (1-2 hours) and this

procedure successfully provided amino compound while the carbon-carbon double

bond was not affected (Figure 3.18).

Out of our expectation (Table 3.9), only compound 67 showed slightly optimization while the activity of most designed compounds was decreased significantly by introducing a CF3 group in the amide compound and especially the sulfonamide compounds.

N COOH O Piperidine OH Pyridine OSO2CF3 N S O H /Pd-C O2N O 2 LiAlH4/THF O H S O2N CHO (CF3SO2)2O N CF3 O F3C S F H /T AL IB D O2N CN

F3C

O N S O N O O K C 3 R Cl/ 2 H CF RCO 3 F3C N S H2N O R O SO 2 C l/P N yri O din S e S R N O O H CF3

Figure 3.18 Synthetic routes for optimized amide and sulfonamide derivatives of

CG12

O F3C N S RHN O Entry Compounds R IC50 of MTT (µM) 67 CGAmF4Cl3NO2 O 5.6

Cl NO2 68 CGAmF4NO2 O 18.7

O2N 69 CGSmF4Cl3NO2 O > 20 S O Cl NO2 70 CGSmF4NO2 O 19.2 S O

O2N 71 CGSmF3CF34NO2 O 8.3 S O

F3C NO2

Table 3.9 CG12 derivatives with different sulfonamide group at 4’ position of the phenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

3.11 Substitution of hydroxylphenyl group by various aromatic rings

Another strategy we used in optimizing the compound CG9 is to replace the whole hydroxyl-phenyl group with heterocyclic groups such as furan, pyridine, indo, quinoline or isoquinoline. Synthesis of these compounds (Figure 3.19) involved condensation between the corresponding aromatic aldehyde and the 1-methylcyclohexyl methyl thiazolidinedione. The results biological evaluation (Table 3.10) showed that replacement of 4-hydroxylphenyl ring with a 4’pyridine ring or a 5’ indo ring slightly optimized the activity. However, introduction of a furan ring would completed deprive the activity of CG9, mostly because no effective hydrogen bond could be formed between the compound and the targeted receptor. For 3’-indo derivatives, an electron-withdrawing group Cl at 5’ position potentiated its activity while a electron-pushing group MeO at 5’ position dramatically decreased the activity.

COOH O Piperidine OH Pyridine OSO2CF3 N Ar N O S LiAlH /THF 4 O H S Ar CHO O (CF3SO2)2O N O S

Figure 3.19 Synthetic routes for CG9 derivatives with various heterocyclic groups

Ar N S O Entry Compounds Ar IC50 of MTT (µM) 72 CG-4’-Pyr 15.0

N 73 CG-3’-Pyr 60.0

N H 74 CG-3’-Indo N 26.0

H 75 CG-5’-Cl-3’- N 12.5 Indo Cl

H 76 CG-5’-MeO-3’- N >100 Indo MeO H 77 CG-5’-Indo N 11.2

78 CG-3’-Fur O >100

79 CG-4’-Q 23.7

N 80 CG-3’-Q 31.6

Table 3.10 CG9 derivatives with heterocyclic group to substitute 4’ hydroxyphenyl group and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

3.12 Modification of the (1-methylcyclohexyl)-methyl group (R2) on compound

CG12 aided by solid-phase combinatorial chemistry

Focused library solid-phase combinatorial chemistry method was applied when we

were trying to optimize the (1-methylcyclohexyl)-methyl group (R2) on the nitrogen

of thiazolidinedione ring (Figure 3.20). The hydroxy group on the phenyl ring

provides a convenient nucleophilie for coupling with electrophilic resin. The most popular and traditional electrophilic resin, Merifield resin, was chosen and reacted with 4-hydroxyl-3-trifluoro-benzaldehyde. However, reaction didn’t happen at the second step (condensing with thiazolidinedione ring) when similar condition in liquid reactions were used. This problem was overcome by replacing the solvent of ethanol

with toluene, increasing the amount of catalyst piperidine and prolonging the reaction

time. Two major commercial sources for R2 group were available, bromides and

alcohols. For bromide raw material, it could be easily reacted with nitrogen on

thiazolidinedione ring under basic condition (potassium carbonate). When alcohols

were used as the source of R2, we introduced Misunobu reaction in place of by activating alcohol into triflate which was the original method we had used. One of the main reasons is that triflate was rather unstable and must be freshly made, which was hardly feasible when we needed to stock many triflates and did more than ten solid- phase reactions simultaneously. 10% trimetylsilyl trifluoromethane sulfonate

(TMSOTfl) in dichloromethane (DCM) was chosen as decoupling agent after we tried

20% trifluoroacetic acid in DCM without yielding any objective product.

SAR studies (Table 3.11) showed that a hydrophobic group was preferred on R2

position, when introducing a hydrophilic group such as hydroxyl group (CC30) or

cyano group (CC34), the activity decreased dramatically. Various sizes of cycloalkyl methyl group have been introduced and it showed that the activity decreased along the decrease of ring size (CC19, 43, 32 and 33). Generally, substitutions other than on position 1 or introduction of double bond on cyclohexylmetyl group would lead less potent compound and the same phenomenon was observed when a planar phenyl group was used to substitute the cyclohexyl ring (CC4, 6 & 9). However, a bulky group or a hydrophobic, electron-withdrawing group on the phenyl ring may increase the potency compared to benzyl group alone (CC15, 16 & 17). Ethylbutyl group

(CC5), ethylhexyl group (CC22) or (2’-methyl)-ethylbenzyl group (CC15) showed similarly optimized cytotoxic activities on LNCaP cells, implicating that the linker for hydrophobic groups is flexible.

Results from the AR promoter-luciferase reporter gene assay and Western blotting assay of AR level for selected optimized compounds were consistent with our screening data from the cell viability assay (Figures 3.21 & 3.22) and confirmed the increased potency on AR ablating effects by our optimized compounds. PARP cleavage showed by Western blotting assay of selected optimized compounds also indicated that the inhibitory activities on LNCaP cell viability was due to their apoptotic effects (Figure 3.23).

N S R2 HO O CF3 Compounds R2 IC50 Compounds R2 IC50 (Entry) (µM) (Entry) (µM) CG12 6.8 CC23 >20 (12) (96) CC0 H >20 CC24 9.7 (81) (97) CC4 16.0 CC25 17.2 (82) (98) CC5 3.7 CC26 12.6 (83) (99) CC6 16.4 CC27 9.4 (84) (100) CC9 13.8 CC28 17.4 (85) (101) CC12 O O 11.5 CC30 CH2OH >21 (86) O (102)

CC14 15.6 CC31 9.3 (87) (103) CC15 4.0 CC32 18.3 (88) (104) CC16 4.9 CC33 16.2

(89) (105) CC17 4.5 CC43 14.3 (90) (106) F3C CC18 12.2 CC39 >20 (91) (107) O CC19 7.9 CC34 CN >20 (92) (108) CC20 11.8 CC35 CN 16.8 (93) (109) CC21 10.7 CC36 18.3 (94) (110) CC22 4.4 CC38 O >20 (95) (111) O

Table 3.11 CG9 derivatives with various substitutions at R2 and their IC50 values of individual agents in inhibiting the cell viability of LNCaP cells.

100

CHO NH S Cl O TMSOTfl CF3 O R OH O CHO O 2 O O HO O DCM K CO /DMF F3C S NH PPh /DIAD 2 3 F3C Piperidine/EtOH 3 F3C S N R2 F3C S N R2 OH O O O RBr/K2CO3

Figure 3.20 Solid-phase synthetic routes for combinatorial library with various R2

Luc

160

140

120

100

60 Relative Luc 40

O 2 5 4 5 6 7 9 0 2 4 6 7 1 2 3 yr o o 3 m 4 7 S 3 P Q A CC C1 C1 C1 C1 C21 C2 C2 C2 C3 4 'In d 'In d g CC CC Cg1 C C CC1 C C CC2 C C CC2 C C CC CC3 C g CC4 C CgSm DM C Cg4' Cg5 Cg3 Compound(5uM)

Figure 3.21 Analysis of the effects of selected compounds with optimized different R2 groups on the transcriptional repression of the AR gene by the AR promoter- luciferase reporter assay.

101

Figure 3.22 Western blotting analysis of the dose-dependent effect of selected optimized compounds on R2 position, on cleavage of PARP protein levels. Cells were exposed to individual agents at the indicated concentrations in 10% FBS- supplemented medium for 72 h, and the lysates were subjected to Western blotting analysis.

102

Figure 3.23 Western blotting analysis of the dose-dependent effect of compounds CG12 and CC5, protein levels of PARP cleavage, AR, and β-TrCP. Cells were exposed to individual agents at the indicated concentrations in 10% FBS- supplemented medium for 72 h, and the lysates were subjected to Western blot analysis.

103

3.13 Stereochemistry conformation of double bond in structure unit of 5- benylidene-2,4-thiazolidinedione

Crystal structure by X-ray diffraction of one of our TZD derivatives, IP-NH, has

confirmed the Z-form conformation of this double bond in structure unit of 5-

benylidene-2,4-thiazolidinedione (Figure 3.24). The N-H bonds were involved in an

intermolecular hydrogen bonding network with O in the carbonyl group. This finding

was consistent with the results by Okazaki and colleagues [134], who reported the Z

form of the double bond in the crystal structure of compound 5-benylidene-2,4-

thiazolidinedione and that there were two intermolecular H-bond between between two thiazolidinedione rings.

Br O O H N S

O O

N H O S O Br

Figure 3-24 Z-form of double bond in5-benylidene-2,4-thiazolidinedione. Upper panel, crystal structure; down panel, schematic illustration

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3.13 Conclusions on ciglitazone derivatives ablating AR

The effects of ciglitazone and its PPARγ-independent derivative ∆2CG on

transcriptional repression of AR have been confirmed by Western blotting and RT-

PCR. By using ciglitazone and ∆2CG as lead compounds, they possessed advantages

of simpler molecular and stronger potency compared to troglitazone and ∆2TG. Three

stages of optimization have been performed based on ciglitazone and ∆2CG. The

method of focused-library solid-phase combinatorial chemistry has been introduced at

later stage of the optimization. Out of a total of about 150 new derivatives, some of

the optimal compounds such as CG12, CC5 and CC22 showed one order of

magnitude improvement in AR repression and in viability inhibition of LNCaP cells compared to ciglitazone and ∆2CG. Their effects have been confirmed in AR reporter gene luciferase assay, Western blotting assay and flow cytometric analysis. Detailed structure-activity relationships have been investigated (Figure 3.25), which showed the importance of the para-phenolic group, orthor-substitution of the CF3 group and a flexible hydrophobic group on the nitrogen atom of the thiazolidinedione ring. The thiazolidinedione ring may play an important role in supporting the backbone conformation of the entire compound and in providing hydrogen bonding aceptors.

X: Hydrophobic Interaction Aromatic Stacking or Polar-polar interaction X O R2 R2: Flexible Hydrophobic Pocket N S R1 O

R1: H-bond donor/acceptor TZD ring: Molecular conformation & H bonding Electrostatic Interaction

Figure 3-25 Proposed interactions between CG derivatives and their receptor

105

3.14 Experimental section for ciglitazone derivatives

Chemical reagents and organic solvents were purchased from Sigma-Aldrich unless

otherwise mentioned. Nuclear magnetic resonance spectra (1H NMR) were measured

on a Bruker DPX 300, or Bruker DRX 400 model spectrometer. Chemical shifts (δ)

were reported in parts per million (ppm) relative to the TMS peak. Electrospray

ionization (ESI) mass spectrometry analyses were performed with a Micromass

Micromass Q-Tof II High-Resolution electrospray mass spectrometer. Elemental

analyses were performed by the Atlantic Microlab, Inc. (Norcross, GA), and were within 0.4% of calculated values. Flash column chromatography was performed with silica gel (230-400 mesh).

∆2CG derivatives in talbes 3.1 and 3.2 were synthesized according to general methods in Figure 3.4, which are illustrated by the synthesis of 3’-∆2-CG, described as follows.

Procedure a: To a stirring solution of 0.27 g LiAlH4 (20 mmol) in 10 mL of THF at

0 oC was added 1 g (7.0 mmol) of 1-methyl-cyclohexanecarboxylic acid in 50 mL of

THF dropwise over a period of 1 hour. The solution was stirred at room temperature

under N2 for 6 hours. After 6 hours, 1 mL of H2O, 1 mL of 1 N NaOH and 2 mL of

H2O was slowly added to the solution to quench the reaction. The solution was stirred

at room temperature for another hour and then filtered out of solid. The solution was

concentrated. Purification by flash silica gel chromatography (ethyl acetate/hexanes =

1/2) gave the product, (1-methyl-cyclohexyl)-methanol (ii), with 82% yield.

106

OSO2CF3

Procedure b: A solution of compound ii (1mmol) in dry CH2Cl2 (5mL) was cooled

to 0oC. 1.1 mmol of pyridine was added to the solution. 1.1mmol triflate anhydride

was added to the solution slowly to the solution. After 2 hours reaction at 4oC, the solution was concerned and the residue was purified by flash silica gel column chromatography (ethyl acetate/hexanes = 1/10), affording the triflate, Trifluoro- methanesulfonic acid 1-methyl-cyclohexylmethyl ester (iii), with a yield of 35%.

O O

Procedure c for 3-(1-Methyl-cyclohexylmethoxy)-benzaldehyde (iv): A mixture of compound iii (0.5 mmol), 3-hydoxyl-benzaldehyde (0.6 mmol) and K2CO3 (0.7 mmol)

were dissolved in 3 mL DMF. The solution was heated to 80 oC for 4 hr. the solution

was poured into water, extracted with ethyl acetate (10 ml X 3), and concentrated.

The residue was purified by chromatography (ethyl acetate/hexanes = 1/6) and

resulted 0.22 mmol of 3-(1-Methyl-cyclohexylmethyl)-benzaldehyde with a 44% yield.

NH S O

Procedure d for (Z)-5-[3-(1-Methyl-cyclohexylmethyl)-benzylidene]-thiazolidine-

2,4-dione (3’-∆2-CG) : A mixture of compound iv (0.5 mmol), 2,4-

107

thiazolidinedione(0.6 mmol) and catalytic amount of piperidine was refluxed in 5 mL

EtOH for 24 h and then concentrated. The oil product was dissolved in ethyl acetate

and poured into water and acidified with AcOH. The solution was extracted with

ethyl acetate, dried and concentrated. The residue was purified by silica gel

chromatography (ethyl acetate/hexanes = 1/3), providing a yield of 67% compound

1 3’-∆2-CG. H NMR (300 MHz, CDCl3) 1.04 (s, 3H), 1.46-1.56 (m, 10H), 3.69 (s,

2H), 6.78-7.28(m, 2H), 7.08 (d, 1H, J=8.4 Hz), 7.39(dt, 1H, J= 2.1, 8.4 Hz), 7.84 (s,

+ 1H), 8.21-8.78(br, 1H), HRMS (ESI, m/z) calcd for, C18H21NO2S, (M Na): 354.1140,

found: 354.1136

O Br NH S O O

(Z)-5-[3-Bromo-4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-

1 dione (∆2-CG-Br). H NMR (300 MHz, CDCl3) 1.11 (s, 3H), 1.40-1.61 (m, 10H),

3.77 (s, 2H,), 6.95 (d, J = 8.4 Hz, 1H), 7.41 (dd, J=2.1, 8.4 Hz, 1H), 7.69 (d, J=2.1Hz,

+ 1H), 7.74 (s, 1H), 8.38 (s, 1H), HRMS (ESI, m/z) calcd for, C18H20BrNO3S, (M Na):

432.0245, found: 432.0247

O O2N NH S O O

(Z)-5-[4-(1-Methyl-cyclohexylmethoxy)-3-nitro-benzylidene]-thiazolidine-2,4-

1 dione (∆2-CG-NO2). H NMR (300 MHz, CDCl3) 1.08 (s, 3H), 1.42-1.59 (m, 10H),

108

3.79 (s, 2H,), 7.23 (d, 1H, J = 8.4 Hz) , 7.85 (d, J = 8.4 Hz, 1H), 7.92 (1H, s), 8.10 (s,

+ 1H), 8.22 (1H,s), 8.33 (s, 1H), calcd for C18H20N2O5S, (M Na): 399.0991, found:

399.0995.

O F3C NH S O O

(Z)-5-[4-(1-Methyl-cyclohexylmethoxy)-3-trifluoromethyl-benzylidene]-

thiazolidine-

1 2,4-dione (∆2-CG-CF3). H NMR (300 MHz, CDCl3) 1.08 (s, 3H), 1.42-1.59 (m,

10H), 3.79 (s, 2H,), 7.10 (d, J = 8.4 Hz, 1H) , 7.63 (d, J = 8.4 Hz, 1H), 7.71(1H, s),

7.80 (s, 1H), 7.73 (1H,s), 8.09-8.12 (br, 1H), HRMS (ESI, m/z), calcd for

+ C19H20F3NO3S, (M Na): 422.1014, found: 422.1019

O MeO NH S O O

(Z)-5-[3-Methoxy-4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-

1 dione (∆2-CG-OMe). H NMR (300 MHz, CDCl3) 1.09 (s, 3H), 1.40-1.58 (m, 10H),

3.75 (s, 2H,), 3.96 (s, 3H), 6.96 (d, J = 8.4 Hz, 1H), 7.00 (1H, s), 7.11 (d, J=8.4, 1H),

+ 7.79 (s, 1H), 8.55 (s, 1H), HRMS (ESI, m/z) calcd for, C19H23NO4S, (M Na):

384.1245, found: 384.1239

109

O EtO NH S O O

(Z)-5-[3-Ethoxy-4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-

1 dione (∆2-CG-OEt). H NMR (300 MHz, CDCl3) 1.08 (s, 3H), 1.40-1.58 (m, 13H),

3.74 (s, 2H,), 4.10 (q, J=6.9Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 7.01 (d, J=2.1 Hz, 1H),

7.11 (dd, J=8.4, 2.1 Hz, 1H), 7.79 (s, 1H), 8.42 (s, 1H), HRMS (ESI, m/z) calcd for,

+ C20H25NO4S, (M Na): 398.1402 , found: 398.1402

NH S O O

(Z)-5-[3,5-Dimethyl-4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-

1 2,4-dione (∆2-CG-DiMe). H NMR (300 MHz, CDCl3) 1.13 (s, 3H), 1.32-1.59 (m,

10H), 2.42 (s, 6H), 3.48 (s, 2H), 7.17 (s, 2H), 7.76 (1H), 8.26 (s, 1H), HRMS (ESI,

+ m/z) calcd for, C20H25NO3S, (M Na): 382.1453, found: 382.1448

NH S O O

(Z)-5-[4-(1-Methyl-cyclohexylmethoxy)-naphthalen-1-ylmethylene]-thiazolidine-

1 2,4-dione (∆2-CG-Naph). H NMR (300 MHz, CDCl3) 1.18 (s, 3H), 1.51-1.59 (m,

10H), 3.91 (s, 2H), 6.915 (d, J=8.7Hz, 1H), 7.55-7.69(m, 3H), 8.12 (d, J=8.7, 1H),

110

8.39 (d, J=8.4Hz, 1H), 8.59 (s, 1H), HRMS (ESI, m/z) calcd for, C22H23NO3S,

(M+Na): 404.1296, found: 404.1299.

Compounds in talbes 3.5 were synthesized according to general methods in Figure 3.6, which are illustrated by the synthesis of CG-OH-Br, described as follows.

HO O

Br S NH

General procedure for 5-(3-Bromo-4-hydroxy-benzylidene)-thiazolidine-2,4- dione (vii) : A mixture of 3-bromo-4-hydroxyl-aldehydes (vi) (0.5 mmol), 2,4- thiazolidinedione, (0.6 mmol), catalytic amount of piperidine and acetic acid was refluxed in 5 mL Toluene for 24. The precipitated product was filtered, washed with

10 ml of toluene for three times and then dried in 60 oC vacuum oven overnight, affording 5-(4-Hydroxy-benzylidene)-thiazolidine-2,4-dione (compound vii) in a 85% yield.

N S HO O Br

(Z)-5-(3-Bromo-4-hydroxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)- thiazolidine-2,4-dione (14, CG-OH-Br)

General procedure for compound CG-OH-Br: A mixture of compound vii (0.5 mmol), compound iii (0.6 mmol) and K2CO3 (0.65 mmol) were stirred in 3 mL DMF.

The solution was heated to 80 oC for 4 hr. the solution was poured into water,

111

extracted with ethyl acetate (3 times, totally 30ml), dried and concentrated. The

residue was purified by chromatography (ethyl acetate/hexanes = 1/3), resulting in a

1 42% yield of compound (CG-OH-Br). H NMR (300 MHz, CDCl3) 0.79 (s, 3H),

1.17-1.46 (m, 10H), 3.36 (s, 2H), 7.01 (d, J=8.4Hz, 1H), 7.37(d, J= 8.4Hz, 1H), 7.73

+ (s, 2H), HRMS (ESI, m/z) calcd for, C18H20BrNO3S, (M Na): 432.0245, found:

432.0245. Anal. (C18H20BrNO3S) C, H, N, O.

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(1-methyl-

1 cyclohexylmethyl)-thiazolidine-2,4-dione (CG12, CG-OH-CF3) H NMR (300

MHz, CDCl3) 0.95 (s, 3H), 1.46-1.56 (m, 10H), 3.64 (s, 2H), 6.08-6.38(br, 1H), 7.09

(d, J=8.4Hz, 1H), 7.59(d, J= 8.4Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H), HRMS (ESI, m/z)

calcd for, C19H20F3NO3S, (M+Na): 422.1014, found: 422.1012. Anal.

(C19H20F3NO3S) C, H, N, S, O, F.

O O2N N S HO O

(Z)-5-(4-Hydroxy-3-nitro-benzylidene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-OH-NO2) H NMR (300 MHz, CDCl3) 0.96 (s, 3H),

1.23-1.57 (m, 10H), 3.68 (s, 2H), 7.31(d, J=8.4Hz, 1H), 7.74 (dd, J=8.4, 2.1Hz, 1H),

7.81 (s, 1H), 8.29(d, J= 2.1Hz, 1H), HRMS (ESI, m/z) calcd for C18H20N2O5S,

(M+Na): 399.0991, found: 399.0991.

112

N S HO O OCH3

(Z)-5-(4-Hydroxy-3-methoxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione , (CG-OH-OMe) H NMR (300 MHz, CDCl3) 0.92 (s, 3H),

1.21-1.58 (m, 10H), 3.62 (s, 2H), 3.97 (s, 3H), 5.95 (br, 1H), 6.90-7.03 (m, 2H), 7.10

+ (d, J= 7.8Hz, 1H), 7.82 (s, 1H), HRMS (ESI, m/z) calcd for, C19H23NO4S, (M Na):

384.1245, found: 384.1245 Anal. (C19H23NO4S) C, H, N, O.

O Br N S HO O Br

(Z)-5-(3,5-Dibromo-4-hydroxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG16, CG-OH-DiBr) H NMR (300 MHz, CDCl3) 0.94 (s,

3H), 1.32-1.56 (m, 10H), 3.63 (s, 2H), 6.22(s, 1H), 7.62 (s, 2H), 7.68(s, 1H), HRMS

+ (ESI, m/z) calcd for, C18H19Br2NO3S, (M Na): 511.9330, found: 511.9329. Anal.

(C18H19Br2NO3S) C, H, N, S, O, Br.

O I N S HO O OCH3

113

(Z)-5-(4-Hydroxy-3-iodo-5-methoxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)

1 -thiazolidine-2,4-dione (CG-OH-IOMe) H NMR (300 MHz, CDCl3) 0.94 (s, 3H),

1.22-1.62 (m, 10H), 3.63 (s, 2H), 3.96 (s, 3H), 6.44 (s, 1H), 6.97 (s, 1H), 7.50(s, 1H),

+ 7.73(s, 1H), HRMS (ESI, m/z) calcd for, C19H22INO4S, (M Na): 510.0212, found:

510.0213

O H3C N S HO O CH3

(Z)-5-(4-Hydroxy-3,5-dimethyl-benzylidene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-OH-DiMe) H NMR (300 MHz, CDCl3) 0.94 (s, 3H),

1.22-1.66 (m, 10H), 2.30 (s, 6H), 3.62 (s, 2H), 5.06 (s, 1H), 7.17 (s, 2H), 7.78(s, 1H),

+ HRMS (ESI, m/z) calcd for, C20H25NO3S, , (M Na): 382.1453, found: 382.1454

N S HO O

(Z)-5-(4-Hydroxy-naphthalen-1-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-OH-Naph) H NMR (300 MHz, CDCl3) 0.98 (s, 3H),

1.20-1.66 (m, 10H), 3.67 (s, 2H), 5.91 (s, 1H), 6.91 (d, J = 7.8Hz, 1H), 7.56-7.67(m,

3H), 8.15(d, J = 8.4Hz, 1H), 8.29 (d, J = 7.2 Hz, 1H), 8.60 (s, 1H), HRMS (ESI, m/z)

+ calcd for, C22H23NO3S, (M Na): 404.1296, found:404.1296

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General procedure for synthesis of compounds in Tables 3.3, 3.4, 3.6 and 3.10 was

decrided as following showed in figure 3.9, 3.10, 3.16 and 3.19 by using compound

CG-OH-H as an example.

O N O S

Procedure for 3-(1-Methyl-cyclohexylmethyl)-thiazolidine-2,4-dione (viii) : A

mixture of trifluoro-methanesulfonic acid 1-methyl-cyclohexylmethyl ester (iii) (0.5

mmol), 2,4-thiazolidinedione(0.6 mmol) and K2CO3 (0.7 mmol) were dissolved in 3

mL DMF. The solution was heated to 80 oC for 4 hr and was poured into water, extracted with ethyl acetate (10 ml X 3), and concentrated. The residue was purified

by chromatography (ethyl acetate/hexanes = 1/6) and resulted 0.22 mmol of 3-(1-

Methyl-cyclohexylmethyl)-2,4-thiazolidinedione (compound viii) with a 50 % yield.

N S HO O

Procedure for compound (Z)-5-(4-Hydroxy-benzylidene)-3-(1-methyl-

cyclohexylmethyl)-thiazolidine-2,4-dione (CG-OH-H) : A mixture of compound

viii (0.5 mmol), and 3-hydoxyl-benzaldehyde (0.6 mmol) catalytic amount of piperidine was refluxed in 5 mL EtOH for 24 h and then concentrated. The oil product was dissolved in ethyl acetate and poured into water and acidified with

AcOH. The solution was extracted with ethyl acetate, dried and concentrated. The

115

residue was purified by silica gel chromatography (ethyl acetate/hexanes = 1/3),

providing a yield of 47 % compound CG-OH-H.

1 H NMR (300 MHz, CDCl3) 0.94 (s, 3H), 1.14-1.86 (m, 10H), 3.63 (s, 2H), 5.69 (s,

1H), 6.94(d, J = 8.40Hz, 2H), 7.43(d, J = 8.40Hz, 2H), 7.83 (s, 1H), HRMS (ESI, m/z)

+ calcd for, C18H21NO3S, (M Na): 354.1140, found: 354.1141.

OH O

N S O

(Z)-5-(2-Hydroxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

dione (CG-2’-OH) 1H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.22-1.65 (m, 10H),

3.66 (s, 2H), 6.44 (d, J = 0.9, 1H), 6.91 (dd, J = 8.1, 0.9 Hz, 1H), 7.04 (td, J = 7.2,

0.6Hz, 1H), 7.32(tdd, J = 7.5, 1.5, 0.6Hz, 1H), 7.46 (dd, J = 7.80, 1.5Hz, 1H), 8.42 (s,

+ 1H), HRMS (ESI, m/z) calcd for, C18H21NO3S, (M Na): 354.1140, found: 354.1145

O HO N S O

(Z)-5-(3-Hydroxy-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (CG-3’-OH) H NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.24-1.67 (m, 10H),

3.65 (s, 2H), 5.24 (s, 1H), 6.93(dd, J = 8.1, 1.5 Hz, 1H), 6.70 (dd, J = 7.5, 1.5Hz, 1H),

7.10(d, J = 7.8Hz, 1H), 7.36 (dd, J = 7.8, 7.5Hz, 1H), 7.84 (s, 1H), HRMS (ESI, m/z)

+ calcd for, C18H21NO3S, (M Na): 354.1140, found: 354.1143

116

O HO N S HO O OH

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-(3,4,5-trihydroxy-benzylidene)-

1 thiazolidine-2,4-dione (CG-triOH) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.30-

1.68 (m, 10H), 3.63 (s, 2H), 5.45 (s, 2H), 5.67 (s, 1H), 6.72(s, 2H), 7.72 (s, 1H).

N S Cl O

(Z)-5-(4-Chloro-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (22, CG-Ph-Cl) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.22-1.74 (m, 10H),

3.64 (s, 2H), 5.69 (s, 1H), 7.46 (s, 4H), 7.84 (s, 1H).

N S Br O

(Z)-5-(4-Bromo-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (23, CG-Ph-Br) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.20-1.72 (m, 10H),

3.64 (s, 2H), 7.38 (d, J = 8.4Hz, 2H), 7.63(d, J = 8.4Hz, 2H), 7.82 (s, 1H).

117

N S NC O

(Z)-4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 benzonitrile (CG-Ph-CN) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.28-1.65 (m,

10H), 3.66 (s, 2H), 7.61 (d, J = 8.4Hz, 2H), 7.79(d, J = 8.4Hz, 2H), 7.87 (s, 1H).

N S O2N O

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-(4-nitro-benzylidene)-thiazolidine-2,4-

1 dione (CG-Ph-NO2) H NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.21-1.68 (m, 10H),

3.66 (s, 2H), 7.67 (d, J = 8.4Hz, 2H), 7.91 (s, 1H), 8.34(d, J = 8.4Hz, 2H).

N S F3CO O

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-(4-trifluoromethoxy-benzylidene)-

1 thiazolidine-2,4-dione (CG-Ph-OCF3) H NMR (300 MHz, CDCl3) 0.90 (s, 3H),

1.11-1.54 (m, 10H), 3.59 (s, 2H), 7.23 (d, J = 8.4Hz, 2H), 7.49(d, J = 8.4Hz, 2H),

7.82 (s, 1H).

118

N O S O OCH3

(Z)-4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 benzoic acid methyl ester (CG-Ph-COOMe) H NMR (300 MHz, CDCl3) 0.95 (s,

3H), 1.21-1.62 (m, 10H), 3.65 (s, 2H), 3.96 (s, 3H), 7.58 (d, J = 8.4Hz, 2H), 7.91 (s,

1H), 8.14(d, J = 8.4Hz, 2H).

N S N O

(Z)-5-(4-Dimethylamino-benzylidene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-Ph-DMN) H NMR (300 MHz, CDCl3) 0.95 (s, 3H),

1.15-1.62 (m, 10H), 3.07 (s, 6H), 3.62 (s, 2H), 6.74 (dd, J = 9.0, 3.0Hz, 2H), 7.43(dd,

J = 9.0, 3.0Hz, 2H), 7.82 (s, 1H).

N S N O

(Z)-5-(4-Diethylamino-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-

1 2,4-dione (CG-Ph-DEN) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.23(t, J = 7.2Hz,

6H), 1.29-1.62 (m, 10H), 3.42(q, J = 7.2Hz, 4H), 3.62 (s, 2H), 6.71 (d, J = 8.7Hz, 2H),

7.39(d, J = 8.7Hz, 2H), 7.80 (s, 1H).

119

N S O N H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 phenyl}-acetamide (CG-Ph-AcN) H NMR (300 MHz, CDCl3) 0.89 (s, 3H), 1.26-

1.62 (m, 10H), 2.18 (s, 3H), 3.63 (s, 2H), 7.31(s,1H), 7.49 (d, J = 8.4Hz, 2H), 7.65(d,

J = 8.4Hz, 2H), 7.84 (s, 1H).

N O S O H

(Z)-4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- benzaldehyde (CG-Ph-CHO) 1H NMR (300 MHz, DMSO-D6) 0.91 (s, 3H), 1.20-

1.59 (m, 10H), 3.57 (s, 2H), 7.87 (d, J = 8.4Hz, 2H), 7.91-8.13(m, 3H), 10.09 (s, 1H).

O N S O

(Z)-5-Furan-3-ylmethylene-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-dione

1 (CG-2’Furan) H NMR (300 MHz, CDCl3) 0.88 (s, 3H), 1.13-1.57 (m, 10H), 3.56 (s,

2H), 6.58(s, 1H), 7.48 (s, 1H), 7.71(d, J = 10.5Hz, 2H).

120

N N S O

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-pyridin-4-ylmethylene-thiazolidine-2,4-

1 dione (CG-4-Pyr) H NMR (300 MHz, CDCl3) 0.89 (s, 3H), 1.22-1.63 (m, 10H),

3.65 (s, 2H), 7.36 (dd, J = 7.5, 1.5Hz, 2H), 7.91 (s, 1H), 8.76(dd, J = 7.5, 1.5Hz, 2H).

N S N O

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-pyridin-3-ylmethylene-thiazolidine-2,4-

1 dione (CG-3-Pyr) H NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.24-1.67 (m, 10H),

3.66 (s, 2H), 7.43(dd, J = 7.8, 4.8 Hz, 1H), 7.82 (ddd, J = 7.6, 1.5, 2.1Hz, 1H), 7.87 (s,

1H), 8.65(dd, J = 4.8, 1.5Hz, 1H), 8.79 (d, J = 2.1Hz, 1H).

N S N O

(Z)-5-Isoquinolin-4-ylmethylene-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (CG-3-Q) H NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.21-1.68 (m, 10H), 3.67

(s, 2H), 7.64(td, J = 7.5, 1.2 Hz, 1H), 7.81 (td, J = 7.5, 1.5Hz, 1H), 7.91 (d, J = 7.5,

1H), 8.04 (s, 1H), 8.13 (d, J = 7.5, 1H), 8.27(d, J = 1.5Hz, 1H), 9.04 (d, J = 1.2Hz,

1H).

121

N N S O

(Z)-3-(1-Methyl-cyclohexylmethyl)-5-quinolin-4-ylmethylene-thiazolidine-2,4-

1 dione (CG-4-Q) H NMR (300 MHz, CDCl3) 0.98 (s, 3H), 1.23-1.67 (m, 10H), 3.68

(s, 2H), 7.49 (d, J = 4.5Hz, 1H), 7.67 (dd, J = 6.9, 7.8Hz, 1H), 7.81 (dd, J = 6.9, 8.1Hz,

1H), 8.12 (d, J = 7.8, 1H), 8.19 (d, J = 8.1, 1H), 8.51 (s, 1H), 8.76(d, J = 4.5Hz, 1H).

N S N H O

(Z)-5-(1H-Indol-5-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (CG-5’-Indo) H NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.20-1.67 (m, 10H),

3.64 (s, 2H), 6.66 (m, 1H), 7.29 (dd, J = 2.7, 3.0Hz, 1H), 7.37 (dd, J = 8.4, 1.5, 1H),

7.47 (d, J = 8.4, 1H), 7.84 (s, 1H), 8.05 (s, 1H), 8.38 (br, 1H).

N HN S O

(Z)-5-(1H-Indol-3-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4-

1 dione (CG-3’-Indo) H NMR (300 MHz, CDCl3) 0.97 (s, 3H), 1.22-1.66 (m, 10H),

3.66 (s, 2H), 7.33 (m, 2H), 7.47 (d, J = 7.5Hz, 1H), 7.55 (d, J = 2.4, 1H), 7.86 (d, J =

7.2, 1H), 8.26 (s, 1H), 8.76 (br, 1H).

122

Cl O

N HN S O

5-(5-Chloro-1H-indol-3-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-5’-Cl-3-Indo) H NMR (300 MHz, CDCl3) 0.92 (s, 3H),

1.22-1.64 (m, 10H), 3.65 (s, 2H), 7.30 (dd, J = 8.7, 2.4Hz, 1H), 7.38 (d, J = 8.7Hz,

1H), 7.55 (d, J = 2.4Hz, 1H), 7.80 (s, 1H), 8.14 (s, 1H), 8.20 (br, 1H).

O O

N HN S O

5-(5-Methoxy-1H-indol-3-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-5’-MeO-3-Indo) H NMR (300 MHz, CDCl3) 0.97 (s,

3H), 1.22-1.65 (m, 10H), 3.64 (s, 2H), 3.90 (s, 3H), 6.97 (dd, J = 8.7, 2.4Hz, 1H),

7.28(s, 1H?), 7.33 (d, J = 8.7Hz, 1H), 7.50 (d, J = 2.4Hz, 1H), 8.22 (s, 1H), 8.66 (br,

1H).

N N S O Cl

(Z)-5-(2-Chloro-pyridin-4-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-3-Cl-4-Pyr) H NMR (300 MHz, CDCl3) 0.95 (s, 3H),

1.20-1.67 (m, 10H), 3.65 (s, 2H), 7.40 (d, J = 5.1Hz, 1H), 8.09 (s, 1H), 8.61(d, J =

5.1Hz, 1H), 8.72 (s, 1H).

123

N N S O F

(Z)-5-(2-Fluoro-pyridin-4-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-3’F-4’-Pyr) H NMR (300 MHz, CDCl3) 0.94 (s, 3H),

1.25-1.64 (m, 10H), 3.65 (s, 2H), 7.39 (dd, J = 4.8, 1.8Hz, 1H), 8.00 (s, 1H), 8.55(d, J

= 4.8Hz, 1H), 8.59 (d, J = 1.8Hz, 1H).

F O

N N S O

(Z)-5-(3-Fluoro-pyridin-4-ylmethylene)-3-(1-methyl-cyclohexylmethyl)-

1 thiazolidine-2,4-dione (CG-3’F-4’Pyr) H NMR (300 MHz, CDCl3) 0.95 (s, 3H),

1.25-1.64 (m, 10H), 3.65 (s, 2H), 7.39 (dd, 1H, J = 4.8, 2.1Hz), 8.00 (s, 1H), 8.55(d, J

= 4.8Hz, 1H), 8.59 (d, J = 2.1Hz, 1H).

N S H2N O

(Z)-5-(4-Amino-benzylidene)-3-(1-methyl-cyclohexylmethyl)-thiazolidine-2,4- dione

Procedure of reducing CG-Ph-NH2 from CGPh-NO2 showed in figure 3.17 was decribed as following. A mixture of compound CG-Ph-NO2 (0.5 mmol), and metal

Zn (4 mmol), iron chloride (3 mmol) was suspended in 10 mL DMF, then 1 ml of

124

water was poured into the mixture and stirred for 2 h which was cooled with cold water when necessary. The reaction mixture was filtered and washed with ethyl acetate (30 mL in three times). The combined filtrates were washed with water, satuated brine, dried and concentrated. The residue was purified by silica gel chromatography (ethyl acetate/hexanes = 1/3), providing compound CG-Ph-NH2 in a

1 yield of 37 %. CG-Ph-NH2 H NMR (300 MHz, CDCl3) 0.94 (s, 3H), 1.21-1.65 (m,

10H), 3.62 (s, 2H), 4.10 (s, 2H), 6.73 (d, J = 8.4Hz, 2H), 7.35(d, J = 8.4Hz, 2H), 7.80

(s, 1H).

General procedure of synthesizing amide from amine showed in figure 3.17 was decribed as following by using CGAm-CCl3 as an example. A mixture of compound

CG-Ph-NH2 (0.1 mmol), trichloroacetyl chloride (0.12 mmol) and potassium carbonate (0.2 mmol) was suspended in 5 mL 1,4-dixoane, and the mixture was stirred for 2 h at room temperature. The reaction mixture was poured into water (15 mL) and filtered and washed with 2mLof mixture of ethyl acetate/hexane (1:5). The obtained solid was dried and confirmed by NRM and was purified by silica gel chromatography (ethyl acetate/hexanes system) when necessary, providing a yield of

60 % compound CGAm-CCl3.

Cl O Cl Cl N S O N H O

(Z)-2,2,2-Trichloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

1 ylidenemethyl]-phenyl}-acetamide (CGAm-CCl3) H NMR (300 MHz, CDCl3)

125

0.96 (s, 3H), 1.21-1.64 (m, 10H), 3.66 (s, 2H), 7.58 (d, J = 8.7Hz, 2H), 7.74(d, J =

8.7Hz, 2H), 7.84 (s, 1H), 8.44(br,1H).

O O N N S O N H O

(Z)-Morpholine-4-carboxylic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-

thiazolidin-5-ylidenemethyl]-phenyl}-amide

1 H NMR (300 MHz, CDCl3) 0.92 (s, 3H), 1.18-1.62 (m, 10H), 3.45-3.78 (m, 4H),

4.07 (s, 2H), 6.71 (d, J = 8.1Hz, 2H), 7.32(d, J = 8.1Hz, 2H), 7.46 (br, H), 7.78(s, 1H).

O O OH N S O N H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-oxalamic acid (CGAmOx) 1H NMR (300 MHz, DMSO-D6) 0.86 (s, 3H),

1.19-1.58 (m, 10H), 3.52 (s, 2H), 7.55 (d, J = 8.7Hz, 2H), 7.85 (s, 1H), 7.94(d, J =

8.7Hz, 2H), 10.38(s,1H).

O O N S N H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 phenyl}-propionamide (CGAmPro) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.19-

126

1.62 (m, 13H), 2.46 (q, J = 7.2 Hz, 2H), 3.63 (s, 2H), 7.31(s,1H), 7.49 (d, J = 8.4Hz,

2H), 7.66(d, J = 8.4Hz, 2H), 7.84 (s, 1H).

O O N S N H O

(Z)-Hexadecanoic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-

1 5-ylidenemethyl]-phenyl}-amide (CGAmPal) H NMR (300 MHz, CDCl3) 0.87 (t,

J = 6.6Hz, 3H), 0.94 (s, 3H), 1.18-1.77 (m, 36H), 2.39 (q, J = 7.5 Hz, 2H), 3.63 (s,

2H), 7.30(s,1H), 7.48 (d, J = 8.4Hz, 2H), 7.66(d, J = 8.4Hz, 2H), 7.84 (s, 1H).

O O N S N H O

(Z)-Cyclohexanecarboxylic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo- thiazolidin-5-ylidenemethyl]-phenyl}-amide (CGAmCH) 1H NMR (300 MHz,

CDCl3) 0.95 (s, 3H), 1.22-1.75 (m, 16H), 1.89 (d, J = 8.7 Hz, 2H), 1.98 (d, J = 8.7 Hz,

2H), 2.27 (m, 1H), 3.64 (s, 2H), 7.48 (d, J = 7.8Hz, 2H), 7.67(d, J = 7.8Hz, 2H), 7.84

(s, 1H).

O O N S N H O

127

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 phenyl}-benzamide (CGAmPh) H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.21-1.67

(m, 10H), 3.68 (s, 2H), 7.48-7.62 (m, 6H), 7.80 (d, J = 8.7Hz, 2H), 7.85-7.92 (m, 3H),

8.04(s, 1H).

O O N MeO S N H O MeO OMe

(Z)-3,4,5-Trimethoxy-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-

5-ylidenemethyl]-phenyl}-benzamide (CGAmTriMeOPh) 1H NMR (300 MHz,

CDCl3) 0.96 (s, 3H), 1.22-1.62 (m, 10H), 3.72 (s, 2H), 3.92 (s, 3H), 3.95 (s, 6H), 7.09

(s, 2H), 7.54 (d, J = 8.7Hz, 2H), 7.81 (s, 1H), 7.89(d, J = 8.7Hz, 2H).

O O N S N H O Br

(Z)-4-Bromo-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzamide (CGAmPh4Br) 1H NMR (300 MHz, CDCl3)

0.95 (s, 3H), 1.24-1.58 (m, 10H), 3.64 (s, 2H), 7.55 (d, J = 8.7Hz, 2H), 7.67(d, J =

8.4Hz, 2H), 7.75-7.82(m, 4H), 7.87 (s, 1H).

128

O O N S N H O O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-4-nitro-benzamide (CGAmPh4NO2) 1H NMR (300 MHz, DMSO-D6) 0.86

(s, 3H), 1.12-1.52 (m, 10H), 3.52 (s, 2H), 7.66 (d, J = 7.5Hz, 2H), 7.89 (s, 1H), 7.98(d,

J = 6.9Hz, 2H), 8.20 (d, J = 6.9Hz, 2H), 8.38(d, J = 7.5Hz, 2H), 10.87(s,1H).

O O N S N H O NC

(Z)-4-Cyano-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

1 ylidenemethyl]-phenyl}-benzamide(CGAmPh4CN) H NMR (300 MHz, CDCl3)

0.95 (s, 3H), 1.22-1.62 (m, 10H), 3.64 (s, 2H), 7.55 (d, J = 8.4Hz, 2H), 7.75-7.87(m,

5H), 8.02(d, J = 8.4Hz, 2H).

O O N S N H O Cl Cl

(Z)-2,4-Dichloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzamide(CGAmPh2,4DiCl) (300 MHz, CDCl3) 0.95 (s,

3H), 1.23-1.67 (m, 10H), 3.64 (s, 2H), 7.41 (dd, J = 8.4, 1.5Hz, 1H),7.51 (d, J =

129

1.5Hz, 1H), 7.55(d, J = 8.7Hz, 2H), 7.78(d, J = 8.7Hz, 2H), 7.87 (s, 1H), 8.08 (d, J =

8.4Hz, 1H).

O O N S N H O Cl Cl

(Z)-3,4-Dichloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5- ylidenemethyl]-phenyl}-benzamide(CGAmPh34DiCl) 1H NMR (300 MHz,

DMSO-D6) 0.86 (s, 3H), 1.13-1.54 (m, 10H), 3.52 (s, 2H), 7.65 (d, J = 8.7Hz,

2H),7.86 (d, J = 8.7Hz, 1H), 7.95(d, J = 9.0Hz, 2H), 8.05(s, 1H), 8.22 (s, 1H), 10.68

(s, 1H).

O O N S N H O Cl

NO2

(Z)-4-Chloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5- ylidenemethyl]-phenyl}-3-nitro-benzamide(CGAmPh4Cl3NO2) 1H NMR (300

MHz, DMSO-D6) 0.86 (s, 3H), 1.15-1.53 (m, 10H), 3.52 (s, 2H), 7.66 (d, J = 8.7Hz,

2H),7.87-8.02 (m, 4H), 8.26(s, 1H), 8.64(s, 1H), 10.85 (s, 1H).

130

O O N S N H O O O

(Z)-Benzo[1,3]dioxole-5-carboxylic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-

dioxo-thiazolidin-5-ylidenemethyl]-phenyl}-amide (CGAmPiperonyl) 1H NMR

(300 MHz, CDCl3) 0.95 (s, 3H), 1.15-1.63 (m, 10H), 3.64 (s, 2H), 6.08-6.11 (m, 3H),

6.90 (d, J = 8.1Hz, 1H), 7.38(s, 1H), 7.42(d, J = 8.1Hz, 1H), 7.53 (d, J = 8.4Hz, 2H),

7.76 (d, J = 8.4Hz, 2H), 7.86(s, 1H).

O O N S N H O

(Z)-3-Cyano-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

1 ylidenemethyl]-phenyl}-benzamide(CGAmPh3CN) H NMR (300 MHz, CDCl3)

0.96 (s, 3H), 1.22-1.63 (m, 10H), 3.65 (s, 2H), 7.56 (d, J = 8.7Hz, 2H), 7.68 (t, 1H, J

= 7.8Hz), 7.81(d, J = 8.7Hz, 2H), 7.87(s, 1H), 7.88(d, J = 7.8Hz, 1H), 8.01 (s, 1H),

8.15 (d, J = 7.8Hz, 1H), 8.21(s, 1H).

131

O O N S N H O

NO2

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 phenyl}-3-nitro-benzamide (CGAmPh3NO2) H NMR (300 MHz, CDCl3) 0.96 (s,

3H), 1.22-1.63 (m, 10H), 3.65 (s, 2H), 7.57 (d, J = 8.4Hz, 2H), 7.76 (t, J = 8.1Hz, 1H),

7.82(d, J = 8.4Hz, 2H), 7.88(s, 1H), 8.09 (s, 1H), 8.31(d, J = 8.1Hz, 1H), 8.45 (d, J =

8.1Hz, 1H), 8.73(s, 1H).

O O N S N H O

CF3

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-3-trifluoromethyl-benzamide (CGAmPh3CF3) 1H NMR (300 MHz,

CDCl3) 0.96 (s, 3H), 1.22-1.68 (m, 10H), 3.65 (s, 2H), 7.57 (d, J = 8.7Hz, 2H), 7.68 (t,

J = 8.1Hz, 1H), 7.80(d, J = 8.7Hz, 2H), 7.86(d, J = 8.1Hz, 1H), 7.88(s, 1H), 7.95(s,

1H), 8.09 (d, J = 8.1Hz, 1H), 8.15(s, 1H).

132

O O N S N H O N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

1 phenyl}-nicotinamide (CGAm3Pyr) H NMR (300 MHz, CDCl3) 0.95 (s, 3H),

1.21-1.67 (m, 10H), 3.64 (s, 2H), 7.48 (dd, J = 8.4, 3.9Hz, 1H), 7.56 (d, J = 8.4Hz,

2H),7.81(d, J = 8.4Hz, 2H), 7.87(s, 1H), 8.01 (s, 1H), 8.24(d, J = 8.4Hz, 1H), 8.82 (d,

J = 3.9Hz, 1H), 9.12(s, 1H).

O O N S N H O

(Z)-Biphenyl-4-carboxylic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-

thiazolidin-5-ylidenemethyl]-phenyl}-amide (CGAmBiPh) 1H NMR (300 MHz,

CDCl3) 0.95 (s, 3H), 1.18-1.68 (m, 10H), 3.64 (s, 2H), 7.36-8.07 (m, 15H).

O O N S N H O

(Z)-Naphthalene-1-carboxylic acid {4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo- thiazolidin-5-ylidenemethyl]-phenyl}-amide (CGAmNaPh) 1H NMR (300 MHz,

133

DMSO-D6) 0.87 (s, 3H), 1.18-1.56 (m, 10H), 3.52 (s, 2H), 7.58-7.72(m, 4H), 7.90(s,

1H), 7.97-8.18 (m, 6H), 8.61(s, 1H), 10.76 (s, 1H).

O O N S S N O H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-methanesulfonamide (CGSmCH3)

General procedure of synthesizing sulfonamide from amine showed in figure 3.17

was decribed as following by using CGSmCH3 as an example. A mixture of

compound CG-Ph-NH2 (0.1 mmol), methyl sulfonyl chloride (0.12 mmol) and

pyridine (0.2 mmol) was dissolved in 5 mL acetone, and the mixture was stirred for 2

h at room temperature. The reaction mixture was poured into water (15 mL) and

filtered and washed with 2mLof mixture of ethyl acetate/hexane (1:3). The obtained

solid was dried and confirmed by NRM and was purified by silica gel

chromatography (ethyl acetate/hexanes system) when necessary, providing a yield of

60 % compound CGSmCH3.

1H NMR (300 MHz, MeOD-D4) 0.96 (s, 3H), 1.23-1.62 (m, 10H), 3.12 (s, 3H), 3.64

(s, 2H), 6.78 (s, 1H), 7.32 (d, J = 8.7Hz, 2H), 7.53(d, J = 8.7Hz, 2H), 7.85 (s, 1H).

O O N S S F3C N O H O

134

(Z)-C,C,C-Trifluoro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-

5-ylidenemethyl]-phenyl}-methanesulfonamide (CGSmCF3) 1H NMR (300 MHz,

DMSO-D6) 0.88 (s, 3H), 1.17-1.56 (m, 10H), 3.46 (s, 2H), 7.59 (d, J = 8.7Hz, 2H),

7.76(d, J = 8.7Hz, 2H), 7.94 (s, 1H).

O O N S S H2N N O H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-aminosulfonamide (CGSmNH2) 1H NMR (300 MHz, CDCl3/MeOD-D4)

0.87 (s, 3H), 1.15-1.57 (m, 10H), 3.55 (s, 2H), 7.22 (d, J = 8.4Hz, 2H), 7.41(d, J =

8.4Hz, 2H), 7.76 (s, 1H), MS (m/z).

O O N S S N O H O MeO

(Z)-4-Methoxy-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzenesulfonamide (CGSmPh4MeO) 1H NMR (300

MHz, CDCl3/MeOD-D4) 0.96 (s, 3H), 1.22-1.62 (m, 10H), 3.66 (s, 2H), 3.91 (s, 3H),

7.02 (d, J = 8.7Hz, 2H), 7.55 (d, J = 8.7Hz, 2H), 7.79 (d, J = 8.1Hz, 2H), 7.87(d, J =

8.1Hz, 2H), 7.88 (s, 1H).

135

O O N S S O N O H O N H

(Z)-N-(4-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenylsulfamoyl}-phenyl)-acetamide (CGSmPh4NHAc) 1H

NMR (300 MHz, CDCl3/MeOD-D4) 0.78 (s, 3H), 1.05-1.51 (m, 10H), 1.99 (s, 3H),

3.47 (s, 2H), 7.07 (d, J = 8.4Hz, 2H), 7.23 (d, J = 8.4Hz, 2H), 7.53 (d, J = 8.1Hz, 2H),

7.61(d, J = 6.0Hz, 3H).

O O N S S N O H O O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-4-nitro-benzenesulfonamide (CGSmPh4NO2) 1H NMR (300 MHz,

CDCl3) 0.93 (s, 3H), 1.21-1.62 (m, 10H), 3.63 (s, 2H), 7.20 (d, J = 8.1Hz, 2H), 7.44

(d, J = 8.7Hz, 2H), 7.79 (s, 1H), 8.01 (d, J = 8.7Hz, 2H), 8.33(d, J = 8.1Hz, 2H).

136

O O N S S N O H O Cl

(Z)-4-Chloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzenesulfonamide (CGSmPh4Cl) 1H NMR (300 MHz,

CDCl3) 0.93 (s, 3H), 1.21-1.62 (m, 10H), 3.63 (s, 2H), 7.18 (d, J = 8.4Hz, 2H), 7.44

(m, 4H), 7.76 (d, J = 8.4Hz, 2H), 7.79 (s, 1H).

O O N S S N O H O O

(Z)-4-Acetyl-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzenesulfonamide (CGSmPh4Ac) 1H NMR (300 MHz,

CDCl3/MeOD-D4) 0.94 (s, 3H), 1.20-1.71 (m, 10H), 2.63 (s, 3H), 3.63 (s, 2H), 7.11

(s, 1H), 7.21 (d, J = 8.4Hz, 2H), 7.42 (d, J = 8.4Hz, 2H), 7.79 (s, 1H), 7.94 (d, J =

8.1Hz, 2H), 8.04(d, J = 8.1Hz, 3H).

O O N O N S S 2 N O H O Cl

(Z)-4-Chloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5- ylidenemethyl]-phenyl}-3-nitro-benzenesulfonamide (CGSmPh4Cl3NO2) 1H

137

NMR (300 MHz, DMSO-D6) 0.84 (s, 3H), 1.18-1.54 (m, 10H), 3.51 (s, 2H), 7.28 (d,

J = 8.7Hz, 2H), 7.56 (d, J = 8.4Hz, 2H), 7.97-8.07 (m, 2H), 8.03 (s, 1H), 8.50 (s, 1H).

O O N F C S S 3 N O H O O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide (CGSmPh4NO23CF3) 1H

NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.23-1.62 (m, 10H), 3.65 (s, 2H), 7.25 (d, J =

8.7Hz, 2H), 7.49 (d, J = 8.7Hz, 2H), 7.82 (s, 1H), 7.95 (d, J = 8.4Hz, 1H), 8.15 (d, J =

8.4Hz, 1H), 8.27 (s, 1H).

O O O N S S N O H O O2N

(Z)-2-Methoxy-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5- ylidenemethyl]-phenyl}-4-nitro-benzenesulfonamide (CGSmPh2MeO4NO2) 1H

NMR (300 MHz, CDCl3) 0.92 (s, 3H), 1.23-1.62 (m, 10H), 3.62 (s, 2H), 4.17 (s, 3H),

7.21 (d, J = 8.7Hz, 2H), 7.39 (d, J = 8.7Hz, 2H), 7.53 (s, 1H), 7.75 (s, 1H), 7.85-7.92

(m, 2H), 8.12 (d, J = 8.4Hz, 1H), MS (m/z).

138

O O N S S N O H O O O

(Z)-3,4-Dimethoxy-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-phenyl}-benzenesulfonamide (CGSmPh34DiMeO) 1H NMR (300

MHz, CDCl3) 0.95 (s, 3H), 1.21-1.68 (m, 10H), 3.63 (s, 2H), 3.85 (s, 3H), 3.92 (s,

2H), 6.89 (d, J = 8.7Hz, 1H), 6.95 (s, 1H), 7.20 (d, J = 8.7Hz, 2H), 7.42 (d, J = 8.7Hz,

2H), 7.48 (d, J = 8.7Hz, 1H), 7.80 (s, 1H).

O NO O 2 N S S N O H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-2-nitro-benzenesulfonamide (CGSmPh2NO2) 1H NMR (300 MHz,

CDCl3) 0.94 (s, 3H), 1.21-1.68 (m, 10H), 3.63 (s, 2H), 7.35 (d, J = 8.1Hz, 2H), 7.40

(d, J = 8.1Hz, 2H), 7.65 (t, J = 7.5Hz, 1H), 7.74 (t, J = 7.5Hz, 1H), 7.80 (s, 1H), 7.90

(d, J = 7.5Hz, 1H), 7.96 (d, J = 7.5Hz, 1H).

139

O O N O N S S 2 N O H O

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]- phenyl}-3-nitro-benzenesulfonamide (CGSmPh3NO2) 1H NMR (300 MHz,

CDCl3) 0.94 (s, 3H), 1.17- 1.67 (m, 10H), 3.64 (s, 2H), 7.24 (d, J = 8.4Hz, 2H), 7.45

(d, J = 8.4Hz, 2H), 7.72 (t, J = 7.5Hz, 1H), 7.80(s, 1H), 8.15 (d, J = 7.5Hz, 1H), 8.44

(d, J = 7.5Hz, 1H), 8.71(s, 1H).

O2N CHO

F3C

Procedure for synthesizing 4-Nitro-3-trifluoromethyl-benzaldehyde

4-Nitro-3-trifluoromethyl-benzonitrile (5g, 23mmol) was dissolved in 20 ml anhydrous toluene and cooled at – 70 ° C, followed by titration of diisobutylaluminum hydride (DIBAL, 40mmol in hexane). The reaction mixture was stirred at – 70 ° C for 1 hour, and then the dry-ice acetone cooling bath was removed and the reaction mixuture was allowed to increase to room temperature, followed by 1 hour of reaction. Methanol (2 mL) was added slowly to quench the reaction and the reaction mixture was poured into diluted sulfuric acid and extracted with ethyl acetate

(200 mL for two times). The organic layers were combined, washed with water until neutral and then washed with satuated brine. After drying with anhydrous sodium sulfate, the solvents were removed and the obtained solide was subjected to purification by silica gel comlumn.

140

N S O2N O CF3

Procedure to synthesize Z-3-(1-Methyl-cyclohexylmethyl)-5-(4-nitro-3- trifluoromethyl-benzylidene)-thiazolidine-2,4-dione (CGPhFNO2). A mixture of compound 3-(1-Methyl-cyclohexylmethyl)-thiazolidine-2,4-dione viii (2.5 mmol), and 3-hydoxyl-benzaldehyde (2 mmol) catalytic amount of piperidine was refluxed in

5 mL EtOH for 24 h and then concentrated. The oil product was dissolved in ethyl acetate and poured into water and acidified with AcOH. The solution was extracted with ethyl acetate, dried and concentrated. The residue was purified by silica gel chromatography (ethyl acetate/hexanes = 1/7), providing a yield of 44 %

CGPhFNO2.

1 H NMR (300 MHz, CDCl3) 0.94 (s, 3H), 1.20-1.58 (m, 10H), 3.65 (s, 2H), 7.81 (d, J

= 8.7Hz, 1H), 7.87 (s, 1H), 7.91 (s, 1H), 8.01 (d, J = 8.7Hz, 1H).

F3C N S O H2N

Procedure to synthesize Z-5-(4-Amino-3-trifluoromethyl-benzylidene)-3-(1- methyl-cyclohexylmethyl)-thiazolidine-2,4-dione (CGPhFNH2)

A mixture of CGPhFNO2 (2 g) and Pd-C (150 mg) in methanol-ethyl acetate (15mL -

15 mL) was stirred under hydrogen (50 psi) for 1.5 hr, filtered, and concentrated to dryness under vacuum. The residue was purified by silica gel flash chromatography with ethyl acetate–hexane (1:8), giving compound CGPhFNH2 (1.2g, 67% yield).

141

1H NMR (300 MHz, CDCl3) 0.94 (s, 3H), 1.22-1.60 (m, 10H), 3.65 (s, 2H), 5.37 (s,

1H), 7.47 (s, 1H), 7.58 (m, 3H), 7.80 (s, 1H).

O O N S N H O CF3 Cl

NO2

(Z)-4-Chloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-2-trifluoromethyl-phenyl}-3-nitro-benzamide

(CGAmFPh4Cl3NO2) 1H NMR (300 MHz, CDCl3) 0.97 (s, 3H), 1.21-1.67 (m,

10H), 3.68 (s, 2H), 7.70-7.90 (m, 3H), 7.96 (d, J = 8.7Hz, 1H), 8.27 (s, 1H), 8.43 (s,

1H), 8.54 (s, 1H), 8.61 (d, J = 8.4Hz, 1H).

O O N S N H O CF3 O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

2-trifluoromethyl-phenyl}-4-nitro-benzamide (CGAmFPh4NO2) 1H NMR (300

MHz, CDCl3) 0.97 (s, 3H), 1.23-1.67 (m, 10H), 3.67 (s, 2H), 7.80-7.92 (m, 3H), 8.07

(d, J = 8.7Hz, 1H), 8.22 (d, J = 8.1Hz, 1H), 8.33 (d, J = 8.1Hz, 2H), 8.42 (d, J = 8.7Hz,

1H).

142

O O N F C S S 3 N O H O CF3 O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

2-trifluoromethyl-phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide

(CGAmFPh4NO23CF3) 1H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.21-1.64 (m,

10H), 3.66 (s, 2H), 7.35 (s, 1H), 7.48 (d, J = 8.7Hz, 1H), 7.60 (d, J = 8.7Hz, 1H), 7.71

(s, 1H), 7.79 (s, 1H), 8.08 (d, J = 8.1Hz, 1H), 8.37 (d, J = 8.1Hz, 1H).

O O N S S N O H O CF3 O2N

(Z)-N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidenemethyl]-

2-trifluoromethyl-phenyl}-4-nitro-benzenesulfonamide (CGSmFPh4NO2) 1H

NMR (300 MHz, CDCl3) 0.96 (s, 3H), 1.23-1.62 (m, 10H), 3.65 (s, 2H), 7.30 (d, J =

8.7Hz, 2H), 7.55 (s, 1H), 7.68 (s, 1H), 8.18 (d, J = 8.7Hz, 1H), 8.48 (d, J = 8.7Hz,

1H).

143

O O N S S N O H O CF3 Cl

NO2

(Z)-4-Chloro-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-

ylidenemethyl]-2-trifluoromethyl-phenyl}-3-nitro-benzenesulfonamide

(CGSmFPh4Cl3NO2) 1H NMR (300 MHz, CDCl3) 0.95 (s, 3H), 1.21-1.64 (m, 10H),

3.66 (s, 2H), 7.58 (s, 1H), 7.78 (d, J = 8.7Hz, 1H), 7.96 (d, J = 8.7Hz, 1H), 7.90 (s,

1H), 8.21 (d, J = 8.1Hz, 1H), 8.28 (d, J = 8.1Hz, 1H), 8.51 (s, 1H).

General procedure of focused library solid phase combinatorial chemistry synthesis

showed in figure 3.20 was decribed below.

O CHO

F3C

Synthesis of 4-resin-oxy-3-trifluoromethyl-benzaldehyde: A mixture of Merrifield resin (3 g, 100-200 mesh, >4.0 mmol chloride/g resin), 4-hydroxyl-3-trifluoromethyl- benzaldehyde (3 g, 15.8mmol) and potassium carbonate (4g, 28.9mmol) were suspended in DMF (40 mL) and gently stirred at 70 ° C for 2 days. The resin was filtered, followed by subsequent wash with water, DMF/H2O 2:1, DMF, THF, methanol and CH2Cl2. The resin was dried at the aspirator pressures and finally under

high vacuum.

144

O O

F3C S NH

Synthesis of 5-(4-resin-oxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione:

A mixture of above mentioned 4-resin-oxy-3-trifluoromethyl-benzaldehyde (4g),

thiazolidine-2,4-dione (3g, 25.6 mmol) and catalytic amound of piperidine (0.2 mL) were suspended in Toluene (40 mL) and gently stirred at 90 ° C for 3 days. The resin was filtered, followed by subsequent wash with toluene, ethyl acetate, THF, methanol and CH2Cl2. The resin was dried at the aspirator pressures and finally under high vacuum.

O O

F3C S N R2

Synthesis of resin coupled objective compound from bromides: A mixture of 5-

(4-resin-oxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione (150mg), bromide (1 mmol), potassium carbonate (300mg, 2.2mmol) were suspended in DMF

(4 mL) were suspended in a reaction vial of the Bohdan MinoBlock pararell reaction station and shaked (500rpm) at 60 ° C for 3 days. The resin was filtered, followed by subsequent wash with water, DMF/H2O 2:1, DMF, THF, methanol and CH2Cl2. The resin was dried at the aspirator pressures and kept in the vial for decoupling.

145

O O

F3C S N R2

Synthesis of resin coupled objective compound from alcohols: A mixture of 5-(4- resin-oxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione (150mg), individual alcohol (1 mmol), triphenylphosphine (1.2 mmol) and

Diisopropylazocarboxylate (DIAD, 1.4mmol) were suspended in THF (4 mL) at 0 ° C in a reaction vial of the Bohdan MinoBlock pararell reaction station and shaked

(500rpm) for 1 hr and then at 60 ° C for 3 days. The resin was filtered, followed by subsequent wash with THF, ethyl acetate, methanol and CH2Cl2. The resin was dried

at the aspirator pressures and kept in the vial for decoupling.

HO O

F3C S N R2

Decoupling of the resin: In each vial containing the dried resin with R2 attached,

trimetylsilyl trifluoromethane sulfonate (TMSOTfl, 100mg) in dichloromethane

(DMC, 5mL) was added and the vials were shaked at 400 rpm for two hours. After

filtering, the decoupled resins were washed by 9 mL DMC for three times and all the

filtrate combined, concentrated in vacuum. Water was added to the residue and the

solid was obtained after filtering and was subjected to TLC and NMR confirmation.

Purification by silica gel column was done when necessary.

146

N S HO O CF3

(Z)-3-(1,4-Dimethyl-cyclohexylmethyl)-5-(4-hydroxy-3-trifluoromethyl-

1 benzylidene)-thiazolidine-2,4-dione (CC4) H NMR (300 MHz, CDCl3) 0.89 (s,

3H), 0.96 (s, 3H), 1.22-1.46 (m, 9H), 3.61 (s, 2H), 5.95 (br, 1H), 7.09 (d, J=8.4Hz,

1H), 7.60 (d, J= 8.4Hz, 1H), 7.69 (s, 1H), 7.82 (s, 1H).

O F3C N S HO O

(Z)-3-(2-Ethyl-butyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-thiazolidine-

1 2,4-dione (CC5) H NMR (300 MHz, CDCl3) 0.94 (t, 6H, J = 7.5Hz), 1.35 (p, 4H, J

= 7.5), 1.80 (p, J =7.5, 1H), 3.69 (d, J = 7.5Hz, 2H), 6.00(br, 1H), 7.10 (d, J=8.7Hz,

1H), 7.61 (d, J= 8.7Hz, 1H), 7.70 (s, 1H), 7.84 (s, 1H).

N S HO O CF3

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(4-methyl-cyclohex-3-

1 enylmethyl)-thiazolidine-2,4-dione(CC6) H NMR (300 MHz, CDCl3) 1.56-2.10

(m, 11H), 5.35(br, 1H), 5.46(t, J = 7.2Hz, 1H), 7.10 (d, J=8.7Hz, 1H), 7.61 (d, J=

8.7Hz, 1H), 7.70 (s, 1H), 7.84 (s, 1H).

147

N S HO O CF3

(Z)-3-Bicyclo[2.2.2]oct-5-en-2-ylmethyl-5-(4-hydroxy-3-trifluoromethyl-

1 benzylidene)-thiazolidine-2,4-dione (CC9) H NMR (300 MHz, CDCl3) 0.84 (d, J =

7.5 Hz, 2H), 1.16-1.53 (m, 8H), 1.75 (d, J = 7.5 Hz, 2H), 2.12-2.27 (m, 1H), 2.32 (s,

1H), 2.53 (s, 1H), 3.31-3.62 (m, 2H), 6.21 (t, J = 7.5Hz, 1H), 6.35 (t, J = 7.5Hz, 1H),

6.55 (br, 1H), 7.10 (d, J=8.7Hz, 1H), 7.60 (dd, J= 8.7, 2.1Hz, 1H), 7.69 (d, J = 2.1 Hz,

1H), 7.84 (s, 1H).

O O

N S HO O CF3

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(3,4,5-trimethoxy-benzyl)-

1 thiazolidine-2,4-dione (CC12) H NMR (300 MHz, CDCl3) 3.74 (s, 3H), 3.85(s, 6H),

4.07 (s, 2H), 6.45 (s, 2H), 7.10 (d, J=8.4Hz, 1H), 7.58 (d, J= 8.4Hz, 1H), 7.67 (s, 1H),

7.80 (s, 1H).

148

O F3C N S HO O

(Z)-3-Benzyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione

(CC14)

1 H NMR (300 MHz, CDCl3) 4.92 (s, 2H), 7.06 (d, J=8.1Hz, 1H), 7.34 (d, J= 6.3Hz,

3H), 7.40 (d, J=6.3Hz, 2H), 7.56 (d, J= 8.1Hz, 1H), 7.67 (s, 1H), 7.84 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(4-isopropyl-benzyl)-

1 thiazolidine-2,4-dione (CC15) H NMR (300 MHz, CDCl3) 1.23 (d, J = 6.9Hz, 6H),

2.84-2.95 (m, 1H), 4.88 (s, 2H), 7.08 (d, J=8.7Hz, 1H), 7.20 (d, J= 7.8Hz, 2H), 7.38

(d, J=7.8Hz, 2H), 7.56 (d, J= 8.7Hz, 1H), 7.66 (s, 1H), 7.83 (s, 1H).

O F3C N S HO O

(Z)-3-(4-tert-Butyl-benzyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC16) H NMR (300 MHz, CDCl3) 1.31 (s, 9H), 4.87 (s,

149

2H), 7.07 (d, J=8.7Hz, 1H), 7.36 (s, 4H), 7.57 (d, J= 8.7Hz, 1H), 7.67 (s, 1H), 7.83 (s,

1H).

O F3C N CF3 S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(2-trifluoromethyl-benzyl)-

1 thiazolidine-2,4-dione (CC17) H NMR (300 MHz, CDCl3) 5.17 (s, 2H), 7.12 (dd,

J=8.1, 7.2Hz, 2H), 7.41 (d, J= 8.1Hz, 1H), 7.49 (d, J= 7.2Hz, 1H), 7.61 (d, J= 8.7Hz,

1H), 7.67-7.74(m, 2H), 7.87 (s, 1H).

O F3C N S HO O

(Z)-3-(4-Benzoyl-benzyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC18) H NMR (300 MHz, CDCl3) 4.99 (s, 2H), 7.09 (d,

J=8.7Hz, 1H), 7.43-7.63 (m, 6H), 7.68 (s, 1H), 7.78 (d, J= 7.8Hz, 4H), 7.92 (s, 1H).

O F3C N S HO O

(Z)-3-Cyclohexylmethyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione(CC19) H NMR (300 MHz, CDCl3) 0.93-1.27 (m, 5H), 1.62-

150

1.83 (m, 6H), 3.61 (d, J = 7.5Hz, 2H), 7.07 (d, J=9.0Hz, 1H), 7.59 (d, J= 9.0Hz, 1H),

7.68 (s, 1H), 7.81 (s, 1H).

O F3C N S HO O

(Z)-3-(2-Cyclohexyl-ethyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC20) H NMR (300 MHz, CDCl3) 0.87-1.03 (m, 2H),

1.15-1.43 (m, 4H), 1.54 (q, J=7.5Hz, 2H), 1.61-1.85 (m, 5H), 3.78 (t, J = 7.5Hz, 2H),

7.08 (d, J=8.7Hz, 1H), 7.59 (d, J= 8.7Hz, 1H), 7.68 (s, 1H), 7.82 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(4-methyl-pentyl)-

1 thiazolidine-2,4-dione (CC21) H NMR (300 MHz, CDCl3) 0.88 (d, J = 6.6Hz, 6H),

1.15-1.24 (m, 2H), 1.55-1.78 (m, 3H), 3.74 (t, J = 10.2Hz, 2H), 7.08 (d, J=8.7Hz, 1H),

7.59 (d, J= 8.7Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H).

O F3C N S HO O

(Z)-3-(4-Ethyl-hexyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-thiazolidine-

1 2,4-dione (CC22) H NMR (300 MHz, CDCl3) 0.86 -0.96 (t, J = 7.2Hz, 6H), 1.23-

151

1.42 (m, 8H), 1.78-1.81 (m, 1H), 3.66 (d, J = 7.5Hz, 2H), 7.08 (d, J=8.7Hz, 1H), 7.59

(d, J= 8.7Hz, 1H), 7.68 (s, 1H), 7.82 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(3-methyl-but-2-enyl)-

1 thiazolidine-2,4-dione (CC23) H NMR (300 MHz, CDCl3) 1.64 (s, 6H), 3.94 (t, J =

7.5Hz, 2H), 6.08-6.12(m, 1H), 7.09 (d, J=8.7Hz, 1H), 7.55 (d, J= 8.7Hz, 1H), 7.67 (s,

1H), 7.80 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-isobutyl-thiazolidine-2,4-

1 dione(CC24) H NMR (300 MHz, CDCl3) 0.94 (d, 6H, J = 6.6Hz), 2.08-2.19 (m, 1H),

3.59 (d, J = 7.5Hz, 2H), 7.08 (d, J=8.7Hz, 1H), 7.59 (d, J= 8.7Hz, 1H), 7.68 (s, 1H),

7.83 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-propyl-thiazolidine-2,4-

1 dione (CC25) H NMR (300 MHz, CDCl3) 0.97 (t, J = 7.5Hz, 3H), 1.60-1.78 (m, 2H),

152

3.74 (t, J = 7.5Hz, 2H), 6.19(br, 1H), 7.09 (d, J=8.4Hz, 1H), 7.59 (d, J= 8.4Hz, 1H),

7.69 (s, 1H), 7.83 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-pentyl-thiazolidine-2,4-dione

1 (CC26) H NMR (300 MHz, CDCl3) 0.97 (t, J = 6.9Hz, 3H), 1.22-1.38 (m, 4H), 1.68

(p, J = 7.5Hz, 2H), 3.74 (t, J = 7.5Hz, 2H), 6.19(br, 1H), 7.09 (d, J=8.4Hz, 1H), 7.59

(d, J= 8.4Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(3-methyl-butyl)-

1 thiazolidine-2,4-dione (CC27) H NMR (300 MHz, CDCl3) 0.94 (d, 6H, J = 6.0Hz),

1.51-1.63 (m, 3H), 3.59 (d, J = 7.2Hz, 2H), 6.19(br, 1H), 7.10 (d, J=8.1Hz, 1H), 7.59

(d, J= 8.1Hz, 1H), 7.68 (s, 1H), 7.83 (s, 1H).

O F3C N S HO O

(Z)-3-Heptyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione

1 (CC28) H NMR (300 MHz, CDCl3) 0.97 (t, J = 6.0Hz, 3H), 1.19-1.42 (m, 8H), 1.61-

153

1.72 (m, 2H), 3.77 (t, J = 7.5Hz, 2H), 6.84 (br, 1H), 7.09 (d, J=8.7Hz, 1H), 7.58 (d, J=

8.7Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H).

O OH F3C N S HO O

(Z)-3-(3-Hydroxy-propyl)-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC30) H NMR (300 MHz, CDCl3) 2.12-2.25 (m, 2H), 3.65

(t, J = 6.9Hz, 2H), 3.96 (t, J = 7.5Hz, 2H), 7.11 (d, J=9.0Hz, 1H), 7.58 (d, J= 9.0Hz,

1H), 7.70 (s, 1H), 7.86 (s, 1H).

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-phenethyl-thiazolidine-2,4-

1 dione (CC31) H NMR (300 MHz, CDCl3) 3.00 (t, J = 7.5Hz, 2H), 4.00 (t, J = 7.5Hz,

2H), 7.11 (d, J=7.8Hz, 1H), 7.28-7.33(m, 5H), 7.58 (d, J= 7.8Hz, 1H), 7.68 (s, 1H),

7.81 (s, 1H).

O F3C N S HO O

(Z)-3-Cyclobutylmethyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC32) H NMR (300 MHz, CDCl3) 1.75-1.92 (m, 4H), 1.97-

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2.12 (m, 2H), 2.74 (p, J = 7.5Hz, 1H), 3.81 (d, J = 7.5Hz, 2H), 5.25(br, 1H), 7.11 (d,

J=8.4Hz, 1H), 7.58 (d, J= 8.4Hz, 1H), 7.68 (s, 1H), 7.82 (s, 1H).

O F3C N S HO O

(Z)-3-Cyclopropylmethyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC33) H NMR (300 MHz, CDCl3) 1.65-1.82 (m, 4H), 2.44

(m, 1H), 3.69 (d, J = 7.2Hz, 2H), 5.25(br, 1H), 7.11 (d, J=8.4Hz, 1H), 7.58 (d, J=

8.4Hz, 1H), 7.68 (s, 1H), 7.82 (s, 1H).

O CN F3C N S HO O

(Z)-4-[5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-2,4-dioxo-thiazolidin-3-yl]-

1 butyronitrile (CC34) H NMR (300 MHz, CDCl3) 1.95-2.06 (m, 2H), 2.38 (t, 2H, J

= 7.2Hz), 3.82 (t, J = 7.5Hz, 2H), 6.98 (d, J=8.7Hz, 1H), 7.46 (d, J= 8.7Hz, 1H), 7.61

(s, 1H), 7.78 (s, 1H).

CN O F3C N S HO O

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(Z)-6-[5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-2,4-dioxo-thiazolidin-3-yl]-

1 2,2-dimethyl-hexanenitrile (CC35) NMR (300 MHz, CDCl3) H NMR (300 MHz,

CDCl3) 1.36 (s, 6H), 1.51-1.67 (m, 4H), 1.68-1.78 (m, 2H), 3.79 (t, J = 7.2Hz, 2H),

6.34(br, 1H), 7.10 (d, J=8.7 Hz, 1H), 7.59 (d, J= 8.7Hz, 1H), 7.69 (s, 1H), 7.84 (s,

1H).

O F3C N S HO O

(Z)-3-Allyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-thiazolidine-2,4-dione

1 (CC36) H NMR (300 MHz, CDCl3) 4.37 (d, J = 6.0Hz, 2H), 5.28 (dd, J = 8.7, 10.8,

2H), 5.88 (m, 1H), 7.11 (d, J=8.7Hz, 1H), 7.59 (d, J= 8.7Hz, 1H), 7.69 (s, 1H), 7.85 (s,

1H).

O O F3C O N S HO O

(Z)-3-[1,3]Dioxan-2-ylmethyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione(CC38) H NMR (300 MHz, CDCl3) 2.25-2.46 (m, 2H),

3.78(d, 2H, J = 7.5Hz), 4.55(t, J = 7.5Hz, 4H), 5.95 (t, J = 7.5Hz, 1H), 7.23(d,

J=8.7Hz, 1H), 7.66 (d, J= 8.7Hz, 1H), 7.66 (s, 1H), 7.85 (s, 1H).

156

O F3C N S HO O

(Z)-5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-naphthalen-2-ylmethyl-

1 thiazolidine-2,4-dione (CC39) H NMR (300 MHz, CDCl3) 5.08 (s, 2H), 7.06 (d,

J=7.8Hz, 1H), 7.47-7.52 (m, 2H), 7.55 (d, J=9.0Hz, 2H), 7.67 (s, 1H), 7.78-7.86(m,

4H), 7.92 (s, 1H).

O F3C N S HO O

(Z)-3-Cyclopentylmethyl-5-(4-hydroxy-3-trifluoromethyl-benzylidene)-

1 thiazolidine-2,4-dione (CC43) H NMR (300 MHz, CDCl3) 1.50-1.82 (m, 8H), 2.35-

2.46 (m, 1H), 3.73 (d, J = 7.5Hz, 2H), 7.11(d, J=8.7Hz, 1H), 7.61 (d, J= 8.7Hz, 1H),

7.70 (s, 1H), 7.84 (s, 1H).

N S HO O

(Z)-5-(3,5-Di-tert-butyl-4-hydroxy-benzylidene)-3-(2-ethyl-butyl)-thiazolidine-

1 2,4-dione (CC51) H NMR (300 MHz, CDCl3) 0.90 (t, J = 6.9Hz, 6H), 1.32 (p, J =

6.9, 4H), 1.46 (s, 18H), 1.81 (sep, J =6.9, 1H), 3.64 (d, J = 7.2Hz, 2H), 5.64(s, 1H),

7.36(s, 2H), 7.84 (s, 1H).

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O O N S HO O O

(Z)-3-(2-Ethyl-butyl)-5-(4-hydroxy-3,5-dimethoxy-benzylidene)-thiazolidine-2,4-

1 dione (CC52) H NMR (300 MHz, CDCl3) 0.91 (t, J = 7.2Hz, 6H), 1.33 (p, J = 7.2,

4H), 1.79 (sep, J = 6.6, 1H), 3.63 (d, J = 7.5Hz, 2H), 3.94(s, 6H), 5.85(s, 1H), 6.76 (s,

2H), 7.78 (s, 1H).

O O N S HO O Br

(Z)-5-(3-Bromo-4-hydroxy-5-methoxy-benzylidene)-3-(2-ethyl-butyl)-

1 thiazolidine-2,4-dione (CC53) H NMR (300 MHz, CDCl3) 0.90 (t, J = 7.2Hz, 6H),

1.32 (p, J = 7.2, 4H), 1.78 (sep, J = 7.2, 1H), 3.65 (d, J = 7.2Hz, 2H), 3.96(s, 3H),

6.94 (s, 1H), 7.30 (s, 1H), 7.72 (s, 1H).

NH O S O Br

5-(4-Bromo-3-isopropoxy-benzylidene)-thiazolidine-2,4-dione (IP-NH) The

compound was synthesized according to general synthetic route shown for CG-OH-

Br (Figure 3-10) except by using isopropyl bromide instead of triflate to react with 5-

158

(4-Bromo-3-hydroxy-benzylidene)-thiazolidine-2,4-dione, three products were obtained with isopropyl group on O (IP-OH, 42% yield) , on N (IP-NH, 32% yield) or

1 on both O and N (IP-noH, 10% yield). For IP-NH, H NMR (300 MHz, CDCl3) 0.97

(d, 6H, J = 6.9Hz), 3.97 (sep, J = 6.9Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 7.43 (dd, J=2.1,

8.7 Hz, 1H), 7.70 (d, J=2.1Hz, 1H), 7.74 (s, 1H), 8.38 (s, 1H).

X-ray diffraction for crystal structure of IP-NH

The data collection crystal was a clear, colorless rectangular block. Examination of the diffraction pattern on a Nonius Kappa CCD diffractometer indicated a monoclinic crystal system. All work was done at 150 K using an Oxford Cryosystems

Cryostream Cooler. The data collection strategy was set up to measure a quadrant of reciprocal space with a redundancy factor of 4.5, which means that 90% of these reflections were measured at least 4.5 times. Phi and omega scans with a frame width of 1.0˚ were used.

Data integration was done with Denzo, and an absorption correction and merging of

the data was done with Sortav. Merging the data and averaging the symmetry

equivalent reflections (for the Laue group m) resulted in an Rint value of 0.044. The structure was solved in Cc by the direct methods procedure in SHELXS-97. Full- matrix least-squares refinements based on F2 were performed in SHELXL-97, as incorporated in the WinGX package. There are two molecules in the asymmetric unit and these are labeled as A and B. For each methyl group, the hydrogen atoms were added at calculated positions using a riding model with U (H) = 1.5*Ueq (bonded carbon atom). The torsion angle, which defines the orientation of the methyl group

159

about the C-C bond, was refined. The hydrogen atoms bonded to N (1A) and N (1B)

were refined isotropically. The rest of the hydrogen atoms were included in the

model at calculated positions using a riding model with U (H) = 1.2*Ueq (bonded atom).

The final refinement cycle was based on 6186 intensities and 355 variables and resulted in agreement factors of R1 (F) = 0.037 and wR2 (F2) = 0.051. For the subset

of data with I > 2*sigma (I), the R1 (F) value is 0.028 for 5429 reflections. The final

difference electron density map contains maximum and minimum peak heights of

0.26 and -0.35 e/Ǻ. Neutral atom scattering factors were used and include terms for

anomalous dispersion. The N-H bonds are involved in an intermolecular hydrogen

bonding network with O (2) atoms.

Cell culture. LNCaP androgen-dependent (p53+/+), and PC-3 androgen-

nonresponsive (p53-/-) prostate cancer cells were obtained from the American Type

Culture Collection (Manassas, VA), and were maintained in RPMI 1640

supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator

containing 5% carbon dioxide.

Cell counting and cell viability assay. PC3 and LNCaP cells were placed in six-

well plates (2.5 x 105 cells/well) in 10% FBS-supplemented RPMI 1640 for 24 h, and

treated with various concentrations of OSU-CG12 for additional 24, 48 or 72 h. Cells

were then trypsinized and subjected to numeration by using a Coulter counter (Model

Z1 D/T, Beckman Coulter, Fullerton, CA). Cell viability was assessed by using the 3-

160

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (TCI

America, Inc., Portland, OR). The assay was done in six replicates (96-well format).

LNCaP cells were seeded at 6000 cells per well plates in 10% FBS-supplemented

RPMI 1640 for 24 h, followed by treatments with various compounds in 5% FBS-

supplemented RPMI 1640 at concentrations indicated. Controls received DMSO at a

concentration equal to that in drug-treated cells. After indicated incubation times,

MTT (0.5 mg/ml) in 10% FBS-supplemented RPMI 1640 was added to each well, and cells were incubated at 37°C for 2 hours. Medium was removed and the reduced

MTT dye was solubilized in 200 µl/well DMSO. Absorbance was determined at 570 nm.

Transfection and luciferase assay. The 3.6-kilobase AR promoter-linked reporter plasmid p-3600ARCAT was kindly provided by Dr. Chawnshang Chang (University of Rochester Medical Center, Rochester, NY). The AR promoter gene (-3600 to +550) encompassing the transcription start site was isolated by using PCR to generate hAR- luc with the following primers: 5′- ACAGGTACCGGTATCTCGACCTGCAGGTC-

3′ and 5′-TGTTAGATCTTGCTGAAGCCGCTCCCCAGT-3′. The fragment was subcloned into the pGL3 luciferase reporter vector (Promega, Madison, WI) at KpnI and BglII in the multiple cloning sites. The PPRE-x3-TKLuc reporter vector contains three copies of the PPAR-response element (PPRE) upstream of the thymidine kinase promoter luciferase fusion gene and was kindly provided by Dr. Bruce Spiegelman

(Harvard University, Cambridge, MA). The pCMVSp1 plasmid was purchased from

Origene Technologies, Inc. (Rockville, MD). LNCaP or PC3 cells were transfected

161

with 5 µg of individual plasmids in an Amaxa Nucleofector using a cell-line-specific

Nucleofector kit according to the manufacturer’s protocol (Amaxa Biosystems,

Cologne, Germany) and were then seeded in six-well plates at 5 × 105 cells per well

for 48 hr. The transfection efficiency was determined to be 70–80% by transfecting

cells with 2 µg of pmaxGFP plasmid, followed by fluorescence microscopy to measure GFP expression. For each transfection, herpes simplex virus thymidine kinase promoter-driven Renilla reniformis luciferase was used as an internal control for normalization.

For the reporter gene assay, after transfection, cells were cultured in 24-well plates in

10% FBS-supplemented RPMI 1640 medium for 48 hr, subject to different treatments for the indicated times, collected, and lysed with passive lysis buffer (Promega). Then

50µL aliquots of the lysates were added to 96-well plates, and luciferase activity was monitored after adding 100 µL of luciferase substrate (Promega) to each well by using a MicroLumatPlus LB96V luminometer (Berthold Technologies, Oak Ridge,

TN) withthe WinGlow software package. All transfection experiments were carried out in six replicates.

Cell cycle analysis. LNCaP cells were seeded in 6-well plates in three replicates (2.5 x 106 cells/well) and treated with different concentrations of CG12 for 72 h. After extensively PBS wash, cells were trypsinized followed by fixation in ice-cold 80% ethanol at 4oC overnight. Cells were then centrifuged for 5 min at 1500 X g at room

temperature, and stained with propidium iodide (50 µg/ml) and RNase A (100

162

units/ml) in PBS. Cell cycle phase distributions were determined on a FACScort flow cytometer and analyzed by the ModFitLT V3.0 program.

Analysis of PPARγ activation. The analysis was carried out by using a PPARγ

transcription factor ELISA kit (Active Motif) in which an oligonucleotide containing

the peroxisome proliferator response element was immobilized onto a 96-well plate.

PPARs contained in nuclear extracts bind specifically to this oligonucleotide and are

detected through an antibody directed against PPARγ. In brief, PC-3 cells were

cultured in RPMI 1640 supplemented with 10% fetal bovine serum and treated with

DMSO vehicle or individual test agents, 10 µmol/L each, for 48 hours. Cells were

collected and nuclear extracts were prepared with a Nuclear Extract kit (Active Motif).

Nuclear extracts of the same protein concentration from individual treatments were

subject to the PPARγ transcription factor ELISA according to the manufacturer’s

instruction.

RT-PCR and immunoblotting. LNCaP cells were cultured in T25 flasks at the

1x106 cells/flask density. After exposure to various compounds at indicated

conditions, cells were subject to total RNA isolation by using an RNeasy mini kit

(QIAGEN, Valencia, CA). RNA concentrations were determined by measuring

absorption at 260 nm in a spectrophotometer. Aliquots of 6 mg of total RNA from

each sample were reverse-transcribed to cDNA using an Omniscript RT Kit

(QIAGEN) according to the manufacturer’s instructions. PCR primers: AR, 5’-

ACACATTGAAGGCTATGAATGTC-3’ and 5’- TCACTGGGTGTGGAAA

163

TAGATGGG-3’; and β-actin, 5’-TCTACAATGAGCTGCGTGTG-3’ and 5’-GGTC

AGGATCTTCATGAGGT-3’. PCR reaction products were separated

electrophoretically in 1.5% agarose gels. For immunoblotting, protein extracts were

prepared by M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL)

with freshly added 1% phosphatase and protease inhibitor cocktails (Calbiochem) followed by centrifugation at 13,000 x g for 10 min. Supernatant was collected and

protein concentration was determined by protein assay reagent (Bio-Rad, CA).

Protein extracts were then suspended in 2x SDS sample buffer, and subject to 10%

SDS-polyacrylamide gels. After electrophoresis, gel was transferred to nitrocellulose

membranes using a semidry transfer cell. The transblotted membrane was washed

twice with Tris-buffered saline containing 0.1% Tween 20 (TBST). After blocking

with TBST containing 5% nonfat milk for 1 hr, the membrane was incubated with

mouse monoclonal anti-AR (Santa cruz) or anti-β-actin (MP Biomedicals) antibodies

(diluted 1:1000) in 1% TBST nonfat milk at 4°C overnight. After incubation with the

primary antibody, the membrane was washed three times with TBST for a total of 30

min, followed by incubation with horseradish peroxidase conjugated goat anti-mouse

IgG (diluted 1:2500) for 1 hr at room temperature. After three times of extensive

wash with TBST for a total of 30 min, the immunoblots were visualized by enhanced

chemiluminescence.

164

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