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

To solve the problem inherent in the tautomerism of ECECur, I subsequently designed, synthesized and evaluated several new curcumin analogs for the anti-androgen receptor activity as wel

DESIGN, SYNTHESIS AND BIO-EVALUATION OF NEW CURCUMIN ANALOGS

AS POTENTIAL DRUG CANDIDATES FOR THE TREATMENT OF PROSTATE

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Reader: Dr Arnold Brossi

Reader: Dr Jian Liu

Rehder: Dr Alexandra Tropsha

UMI Number: 3212470

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Li Lin: Design, Synthesis and Bio-evaluation of New Curcumin Analgos as Drug

Candidates for the Treatment of Prostate Cancer(Under the direction of Kenan Professor Kuo-Hsiung Lee)

Curcumin is the major yellow pigment isolated from the rhizome of Curcuma longa,known as turmeric Over a long period of study, curcumin has been found to possess a widerange of bioactivities including anti-prostate cancer activity in vitro and in vivo Based on these observations, our laboratory has been using curcumin as the lead compound to develop various analogs as potential anti-prostate cancer agents Two curcumin analogs, dimethyl curcumin (DMC, 4) and 4-ethoxycarbonylethyl curcumin (ECECur, 5) developed previously were found to be active anti-androgen receptor agents, which showed greater potency than hydroxyflutamide, an anti-androgen currently used clinically However, the tautomerism of ECECur, which causes it to exist in both enol-keto and di-keto forms, may hinder its potential as a clinically useful drug To solve the problem inherent in the tautomerism of ECECur, I subsequently designed, synthesized and evaluated several new curcumin analogs for the anti-androgen receptor activity as well as cytotoxicity 1n prostate cancer cell lines To establish an extensive structure-activity relationship (SAR) for curcumin analogs as anti- prostate cancer agents, I designed and synthesized four series of new curcumin analogshaving various structural features Based on the structures and the bioactivities of these new

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compounds, I studied the structure-activity relationship of curcumin analogs in an extensivelevel and discovered several structural features of curcumin analogs responsible for theiranti-prostate cancer activity including 3’,4’-dimethoxy phenyl rings or 3’-methoxy-4’-hydroxy phenyl rings, unsaturated and conjugated linker, proper substitution at C4 position

of the linker and so on This new information will guide us for the further optimization ofcurcumin analogs as anti-prostate cancer agents Besides three conjugates were designed andsynthesized in this work Through this study, the problem of tautomerism of ECECur hasbeen solved successfully and sixteen new potent curcumin analogs (11, 12, 13, 14, 15, 16,

31, 34, 35, 37, 41, 43, 44, 50 and 52) were developed as promising anti-prostate cancer drugcandidates for further investigation in vivo

1H

I would like to thank my advisor, Dr Kuo-Hsiung Lee, and my committee members, Dr.

Kenneth F Bastow, Dr Jian Liu, Dr Alexandra Tropsha and Dr Arnold Brossi, for their

guidance through my Ph D study I would also like to give great appreciation to Dr Qian

Shi, Dr Ching-yuan Su and Dr Charles Shih in Androscience Incorporation for their help in

my research.

I am especially grateful to my parents Zhiguo Lin and Jianping Luo, my boyfriend MichaelChou, and my roommate Di Hu for their heartfelt support and encouragement

iv

Dedicated to

My Grandfather, Rongfa Lin

TABLE OF CONTENTS

Page

LIST OF TABLPES HH TK eee eee ebe eee ea teen neeaeeaeeateneneeenees X

LIST OF FIGURES ST nnn ĐK ĐK KT kh kg xiLIST OF SCHEMES HH HH nnn Eee Ki ko KĐT nếp xILIST OF ABBREVIATIONS SH nh ĐK Bà nh XI

CHAPTERS

1 Introduction—Prostate Cancer and Clinical Antiandrogenic Agents

1.1 Prostate Cancer and Risk Factors ccesceeee eee e eee e eee een ee nee nas 11.2 Androgens and androgen receptor (AR) in the prostate 21.3 Treatment of Prostate CanC€T nhe nhe nh se 3

1.4 Clinical Antiandrogenic Agents for the Treatment ofProstate CATC€T eee en EE EEE EEE EE EEE He 41.5 Antiandrogen Withdrawal Syndrome c.c 6

2 Background—Curcumin and Curcumin Analogs

2.1 Drug Discovery from Natural Products 8

2.2 The Origin of CurCUIH chen bào 92.3 The Bioactivities of CurcumIn - c che 102.4 The Anti-Prostate Cancer Activity of CurcumIn 11

vi

2.5 The Anti-Prostate Cancer Activity of Curcumin Analogs L2

3 Research Part I—The Resolution of the Tautomerism of

4-Ethoxycarbonylethyl Curcumin (ECECur)

3.1 Introduction aa ee nn ne nr rennet nner tenets 14

3.2 RatlOnÌ€ ch nà Ti n kh tk BH 15

3.3 Synthesis of New ECECur Analogs c 16

3.4 Biological Results and DDIscussion nen nhe 21

3.6 Experimental Section ccceecee cece eee e eee ene eee nh ne ene nn nh hiện 27

4 Research Part II—Design, Synthesis and Bio-evaluation of

Four Series of Curcumin Analogs

4.1 IntrOduCfiOn c en ee ence nn renner renee reas 384,2 Monophenyl Curcumin Analogs

4.2.1 7 RatiOnale nh nh nh nh kh k ket 394.2.2 SVnth€SIS cn nền nh nh nh nh nh 394.2.3 Bioassay r€SUÏ{S cọ nhe nhe nhe 394.2.4 Discussion and conclusion 414.3 Heterocyclic Curcumin Analogs

4.3.1 RatiOnale con nh nh kh nh nh ng 414.3.2 SyntheSiS cọ HH nh nh nh nh nh be nhà 414.3.3 Bioassay r€SUÏ{S on nh nh nh nh heo 434.3.4 Discussion and conclusion - - ees 44

4.4 New Curcumin Analogs Bearing Various Substituents

on Phenyl Rings

44.1 RatiOnaÌ€ ch ern nh nhe nở 44

vil

4.4.2 Synthesis cece cece cece seen tenet eee nh nh nh nh kh 44

4.4.3 Bioassay T€SUÏ(S Hs n nen nen nh re, 45

4.4.4 Discussion and conclusion 46

4.5 New Curcumin Analogs with Various Linkers

““¬.t:Uua:IAẠỤỌIẠIỤẠIỢIiiiađađ 49

4.5.2 Synthe€$lS uc nh nh nh TK kh khe 51

4.5.3 Bioassay r€SUÏS c2 vn cà 54

4.5.4 Discussion and conclusion 54

4.6 Antiandrogenic Acitivity of the Four Series of New

Curcumin AnaÌOgS ‹ en ene nner nh nh eer EEE nh trên 57

4.7 Conclusion in the SARs of Curcumin Analogs 59

4.8 Experimental SeCfIOn ST nen nh rere 60

5 Research Part II—Design, Synthesis and Bio-evaluation of

Curcumin Analogs Conjugated with Anti-Prostate Cancer Drugs

5.1 Introducfiom eee HH nh enn EEE nh 76

5.2 Rationale 0 cece cece ee EE EE tenes 775.3 SyntH€SIS eee BE BE rene nnn eee 792à nh am 825.5 Discussion and ConcÏuslon - c cà 845.6 Experimental Secfion - HH nh nh nh kh nhe eae 85

6 Conclusions and Future Studies

6.1 COnCÏUSIOTS en ence teens ee eet eee eet ng ng kh vn EEE EEE te 916.2 Future Studies ccc cece c ccc cece teen cece seen eens eens setae este nese beeen kg 95

Vill

REFERENCES HH EEE EEE Kế ng rene nề ha

APPENDIX I: The Cytotoxicity of Selected Analogs Against A Panel

Of Cell LANES ccc enn EEE ng EEE EEE EE EE EEE Enea

1X

The chemical structures and cytotoxicity of compounds 24-26 against LNCaP and PC-3 human prostate cancer cell lines

The chemical structures of series C of curcumin analogs and their cytotoxicity against LNCaP and PC-3 human prostate cancer cell lines - -ccccc cv bàn bà.

The chemical structures of curcumin analogs with various linkers and their cytotoxicity against LNCaP and PC-3 human prostate cancer cell lines - ‹- c

The antiandrogenic activity of compound 5, 11, 12 and 15

The cytotoxicity of starting materials, intermediates and target conjugates against LNCaP and PC-3 human prostate cancer cell lines ‹‹-c cà S2 nhe

The cytotoxicity of the active curcumin analogs towards

LNCaP and PC-3 human prostate cancer cell lines The cytotoxicity of selected compounds against a panel

Of CELL ]1TIỂS cece cece e eee eee cee te een ng ng ng seen nh khu

Androgen receptor signaling pathWay ch nhe ee ke 3

The chemical structures of some clinical antiandrogenic agents 4

Curcuminoids from Curcumad longa cccccccccc cece ccc ce eee ene enter etn se, 10

Anti-AR curcumin analogs DMC (4) and ECECur (5) 13

The design of di-keto ECECur analog 6 and enol-keto analog 7 15

Anti-AR activity of compound 4-11 (3 uM) in prostate

cancer cells and their cytotoxicity against the growth

Of LNCaP celÌS con HH TT nee nee entre tk xa 24

The conformations of ECECur (Š) con nh nhe ho 25

Anti-AR activity of compound 4, 5, 12-16 (5 uM)

in prostate cancer cells and their cytotoxicity againstthe growth of LNCaP cells uc nh nh khe 26Monophenyl curcumin analOgØS c.c con nhe 39Curcumin analogs with various lInK€TS cà cà 50Structures of antiandrogens used 1n the cÌinIc 78

The design of curcumin analogs conjugated withN-arylmethacrylamide mOI€fy co cọ nh nh een ke nh 79

The antiandrogenic activity of 54-60 in LNCaPcells and PC-3 cells transfected with wild-typeandrOgen LECEPLOL 0 cee EEE EER EE EEE EEE 84

The pharmacophores derived from SAR ofcurcumin analogs for inhibition against the growth

of human prostate cancer cells 1n VITO ị e tte sen nhe 94Optimization of C-4 side chain of curcumin analogs - 96Some proposed novel curcumin conJugafes 99

XI

The general synthetic methods of heterocycliccurcumin analogs 24 — 27 -Qnn en nhe nh kh se 42

The general synthetic strategy of some symmetricCurcumin analOgÿS cọ EE EEE nh hà kh ba 45

The general synthetic scheme of some asymmetricCULCUMIN analOgS ‹.‹ án enn nh nh nh EE nh nh nh hệ 45

The general synthetic method of

1, 5-diphenyl-1,4-pentadiene-3-ONnes cece cece tenes 52

The synthesis of curcumin analogs 46 and 47 havinglonger linkers ch EEE kh kh te 52The hydrogenation of DMC to 48 co cà nhe hen 52The synthesis of analog 49 cà eee eee nh nh kh nh nh kh 53The synthesis of imide analog 5Ũ cành ee 53The synthesis of 52 and 53 - nen nnn nhớ 53The synthesis of conjugates 55 and 56 c cành nena es 81The synthesis of conjugate 60 cere ne nh nhe ch: 81

xi

N, N-dimethylformamidedimethyl sulfoxidedeoxyribonucleic acid4-ethoxycarbonylethyl curcuminepidermal growth factor

hydroxyflutamidehuman immunodeficiency virus1,1,1,3,3,3-Hexamethy! disilazaneheat-shock proteins

inhibitory concentration that is toxic to 50% of the cells

Xiil

nuclear factor kappa B

pyridinium toluene 4-sulfonateprostate-specific antigen

Singlet

Structure-activity relationship

Triplet

tetrahydrofurantetrohydropyranyltumor necrosis factor

XIV

CHAPTER 1

INTRODUCTION —

PROSTATE CANCER AND CLINICAL ANTIANDROGENIC AGENTS

Prostate Cancer and Risk Factors

Prostate cancer is the most prevalent cancer in American men and the second

leading cause of cancer-related death among men in the United States.' The

well-established risk factors for prostate cancer include age, race, family history, diet, andenvironmental agents.? Age is the most important factor in prostate cancerdevelopment A man under the age of 40 rarely develops detectable prostate cancer,while about 80% of prostate cancer is diagnosed in men over 65 A wide variation inincidence has been reported among different races and ethnic groups Prostate cancerincidence is lower in Asia than in western countries (U.S rates were 50-60 times

higher than those in China and Japan in 1999) African-Americans are the most

likely to develop prostate cancer, black men have 60% higher incidence than whitemen, and Asian men living in Asia have the lowest incidence However, theincidence and mortality of prostate cancer are rising rapidly in most countries,

including in low-risk populations

1.2 Androgen and androgen receptor (AR) in the prostate

Androgens, acting through the androgen receptor, are required for normal

prostate cell growth and function The endogenous androgens include testosterone

and its more active metabolite 5a-dihydrotestosterone (DHT), formed by action of

5ơ-reductase on testosterone Although the real cause of prostate cancer is still

unknown, androgens and the androgen receptor (AR) are documented to play

important roles in the development and progression of prostate cancer.” Constitutive

activation of the AR by a high level of androgen is often detected in prostate cancerpatients The AR belongs to the nuclear receptor superfamily It is a ligand-inducibletranscription factor regulating the expression of target genes such as prostate-specificantigen (PSA) PSA is considered as the most sensitive biochemical marker availablefor monitoring the presence of prostatic diseases, such as prostate cancer, and theresponse to therapy The AR signaling pathway is illustrated in Figure 1.1 Theinactive AR is usually present in the cytoplasm, associated with heat shock proteinssuch as Hsp70, Hsp90, Hsp54 and Hsp56 Upon binding to the androgen testosterone

or DHT, AR undergoes a conformational change and releases the heat-shockproteins The androgen-bound AR is then phosphorylated and translocated into thenucleus In the presence of AR coactivators such as ARA 70 and ARA 55, thephosphorylated AR is then homodimerized The homodimer complex binds to theandrogen receptor binding elements (AREs) and turns on the transcription of targetgenes including the expression of PSA Other ways to activate the AR are throughcertain growth signaling pathways such as the Smad/MAPK/Pyk2 kinase pathway,bypassing the requirement for androgens The growth receptor signaling pathways

are possible mechanisms through which androgen-independent prostate cancer cells

bypass the AR pathway with the AR remaining active.

Figure 1.1 Androgen receptor signaling pathway

from Endoncrine Reviews 25 (2), 276-308, by courtesy of Dr Chawnshang Chang

Treatment of Prostate Cancer

Prostate cancer is a complex heterogeneous disease that acts differently in different men Over the years, many various treatments for prostate cancer have been

developed including surgery, radiation, chemotherapy, dietary changes and the use of

various herbal supplements.”

The chemotherapeutic agents currently used in androgen deprivation therapy are

steroidal antiandrogens (e.g cyproterone acetate and megestrol acetate) and

nonsteroidal antiandrogens (e.g flutamide, nilutamide and bicalutamide) (Figure

1.2) These antiandrogens block the AR signaling pathway and therefore inhibit thetranscriptional activity of the AR

Figure 1.2 The chemical structures of some clinical antiandrogenic agents

CF3 NO

1.4 — Clinica] Antiandrogenic Agents for the Treatment of Prostate Cancer

Cyproterone acetate (CPA) is the first antiandrogen used in Europe to treat

advanced prostate cancer.” The structure of this steroid mimics that of DHT In

addition to blocking androgen action, CPA induces many complications including

severe cardiovascular problems,’ gynecomastia, loss of libido, erectile dysfunction

and central nervous system effects such as headache, fatigue, and weakness.°® These

side effects may be attributed to CPA’s partial agonistic activity and overlapping

effects with other hormonal systems

Flutamide was the first nonsteroidal antiandrogen approved in 1989 for clinical

use to treat prostate cancer.’ Hydroxyflutamide is the active metabolite of flutamide.

It was thought to be a pure androgen antagonist Hepatotoxicity, asymptomaticelevations in aminotransferases, diarrhea, and gynecomastia have been observed in

patients treated with flutamide.”!2 Flutamide withdrawal syndrome has also been

observed in patients taking flutamide for several months

Bicalutamide was approved for clinical use in 1995.'° Thus far, it is the most

preferred nonsteroidal antiandrogen available and has a better side effect profile thanflutamide and nilutamide as well as the highest effectiveness of the nonsteroidalantiandrogens.’* One proposed action mechanism is that bicalutamide binds to the

AR and thereby prevents it from binding to DNA.”° Another action mechanism is

that bicalutamide functions as an AR antagonist not by preventing the AR frombinding to DNA, but by causing assembly of a transcriptionally inactive AR onDNA.'° Clinically used bicalutamide is a mixture of R and S isomers R-

bicalutamide is about 30-fold more potent than the S isomer.'’

Nilutamide was approved in 1996.'? A high incidence of visual problems

(adverse light-dark adaptation) was observed in patients taking nilutamide.Nilutamide has other side effects such as alcohol intolerance, respiratory disturbance,

nausea and vomiting.!"! It is considered as a second line hormonal treatment for

patients with advanced prostate cancer in whom androgen ablation fails.!”

Nonsteroidal antiandrogens are more favorable than steroidal antiandrogens,because they induce fewer side effects

Antiandrogen Withdrawal Syndrome

Some patients experience a decline in PSA level after discontinuation ofantiandrogen treatment, and antiandrogen withdrawal syndrome has been discovered

in some patients after several months of antiandrogen administration.”??? This

phenomenon has been observed more often with nonsteroidal than steroidalantiandrogens Molecular mechanisms responsible for the antiandrogen withdrawalsyndrome have not been fully determined The first possible mechanism is amutation of AR that alters its binding sensitivity and specificity to ligands and

thereby enables antiandrogens to function as AR agonists.”!?? Another possibility is

that some AR coactivators, such as ARA70 and ARASS, are over-expressed afterantiandrogen treatment and thereby enhance the agonistic effect of some

antiandrogens including hydroxyflutamide, bicalutamide and CPA.”” A third possible

mechanism is that some antiandrogens such as hydroxyflutamide may turn on themitogen-activated protein kinase (MAPK) pathway The activated MAPK may

specifically bind to and phosphorylate the AR in the absence of androgens.”° This

androgen-independent activation of the AR could also be a potential mechanism forthe androgen-independent growth of prostate cancer So far the clinically availableantiandrogens are unable to kill prostate cancer cells allowing the cancer to enter an

androgen-independent stage, usually within one to three years of administration ofcurrently available antiandrogens Therefore it is necessary to improve the currenttreatment of prostate cancer by developing new anti-prostate cancer drugs or/andaltering the treatment plan for prostate cancer patients.

CHAPTER 2

BACKGROUND

-CURCUMIN AND -CURCUMIN ANALOGS

2.1 Drug Discovery from Natural Products

Natural products have been major sources of new drugs Approximatelyone-third of the top-selling drugs worldwide are natural products or their

derivatives, and they often originate from uses as folk medicines.”’ The

prevailing approach to drug discovery from natural products takes advantage ofmodern high-throughput screening bioassay techniques Using bioactivity-guidedfractionation, crude plant solvent fractions are initially examined for the targetedbioactivity, and the bioactive fractions are further fractionated and tested foractivity until the active pure natural products are isolated and identified Theisolated active natural product leads are then synthetically modified to elucidatethe structure-activity relationship (SAR) and other drug-related properties Theoptimized drug candidates are designed and developed based on the SAR

correlations derived from the modification of the natural product leads.”*”°

2.2 The Origin of Curcumin

Plants of the Zingiberaceae family have been used as spices and indigenousmedicine in Asia for thousands of years The rhizome of Curcuma longa(Zingiberaceae), which is commonly named turmeric, is used as a spice (e.g., curry),flavoring agent, food preservative, coloring agent, and medicine to treatinflammation and sprains in Asian countries such as India, China and Japan.Recently, turmeric has been found to have anticancer, chemopreventive andhepatoprotective effects Curcumin (1) [diferuloyl methane; 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is the major constituent of the yellowpigments isolated from Curcuma longa (Zingiberaceae) and other Curcuma species.The main components of turmeric include curcumin (1), demethoxycurcumin (2),and bisdemethoxycurcumin (3), together referred to as curcuminoids (Figure 2.1)

Curcumin was first isolated in 1870 Its chemical structure was determined in 1910°°

and subsequently confirmed by synthesis Curcumin has a unique conjugatedstructure including two methoxylated phenols and the enol form of a heptadiene-3, 5-diketone linking the two phenols, giving a bright yellow color

Figure 2.1 Curcuminoids from Curcuma longa

+ OHO

i 7 1HạCO 3 WES ZA 3 0CH3

The Bioactivities of Curcumin

Over a long period of study, curcumin has been found to possess various

31,32 33-35

beneficial biological activities, such as inflammation, antioxidation,

anti-HIV,°°3” chemoprevention,’ 8 anti-angiogenesis' ? and anticancer in several cell

types.ˆ” Curcumin reportedly modulates many signal transduction pathways and has

multiple biological targets such as HIV-1 integrase,” NF-kB, COX2, and TNF.”°

Although it has numerous reported therapeutic effects, curcumin is non-toxic even athigh dosages It has been classified as “generally recognized as safe” (GRAS) by the

National Cancer Institute.*° Currently various groups worldwide have been using

curcumin as a lead compound to develop numerous analogs for different bioactivities

32,42

including anti-inflammation, chemo-prevention, anti-angiogenesis,ˆ“

anti-HTV-41,4 ar 4° and anti-prostate cancer.*°*’ Curcumin is currently in phase II

1 integrase,

clinical trials for advanced pancreatic cancer,*® Alzheimer Disease,” and the

chemoprevention of colorectal cancer.""

Most cancer biologists suggest that, because tumor cells always have multiplepathways to escape the host defense mechanisms, a drug that is specific for

10

modulation of only one signal transduction pathway in tumor cells may not be

adequate.” Curcumin is an ideal anti-cancer drug candidate that affects multiplepathways and yet is still pharmacologically safe However, due to its naturaloccurrence and long history of dietary use, curcumin cannot be patented Hence,curcumin is a good lead compound for the development of better analogs that are

patentable and more potent in the targeted activities.’ >’ The current SAR studies of

curcumin and various curcumin analogs relating to different bioactivities and distinct

biological targets of interest were review by us.

The Anti-Prostate Cancer Activity of Curcumin

Dorai et al first explored the effect of curcumin on prostate cancer cells in vitro.”

They reported that, at a range between 20 and 50 uM, curcumin strongly inhibitedthe growth of both androgen-dependent LNCaP and androgen-independent PC-3human prostate cancer cell lines by 60-80% They also observed that curcumindecreased the proliferative potential and induced the apoptosis potential of bothhuman prostate cancer cell lines by down-regulating the level of apoptosissuppressor proteins, modulating the bax/bcl2 ratio and interfering with the growth

factor receptor signaling pathways as exemplified by the EGF-receptor.””” To

extend their in vitro observations, they also studied the in vivo effects of curcumin onLNCaP cells and reported that a synthetic diet containing 2% curcumin caused anapparent decrease in the extent of LNCaP cell proliferation, a significant increase inthe extent of apoptosis of the tumor cells as well as a significant decrease in the

microvessel density around the tumor."'! Androgen-dependent LNCaP cells are only

lãi

slightly susceptible to tumor necrosis-factor-related apoptosis-inducing ligand(TRAIL), a member of the tumor necrosis factor family of cell death-inducingligands Curcumin reportedly able to induce TRAIL-induced apoptosis in LNCaP

cells in vitro However, no study was reported on whether curcumin interferes with

AR signaling pathways in human prostate cancer cells

The Anti-Prostate Cancer Activity of Curcumin Analogs

Based on the observed anti-prostate cancer effects of curcumin, we selectedcurcumin as a lead to develop curcumin-analogs as anti-prostate cancer agents Overthe past few years, a number of curcumin analogs were synthesized or isolated from natural sources and evaluated first for cytotoxic activity against a panel of cancer cell

lines, including prostate cancer cell lines, and then for antiandrogenic activity

against androgen-dependent LNCaP cells and an androgen-independent PC-3 human

prostate cancer cell line transfected with wild-type androgen receptor.ˆ“*” The

anti-AR bioassay model was developed by Chang et al and briefly described as

follows The androgen-dependent LNCaP cell line expresses mutant AR, which

remains functional The androgen-independent PC-3 cell line, which does notexpress functional AR, is transfected with wild-type AR gene to produce wild-type

AR Both LNCaP and PC-3 human prostate cancer cell lines are transfected with amouse mammalian tumor virus (MMTV) luciferase gene This luciferase genecontains a luciferase promoter and the AR binding element (ARE) Activation of AR

by DHT turns on both the transcription of AR target genes and the expression of luciferase The visible luciferase activity reflects the AR transcriptional activity The

12

human prostate cancer cells are incubated with test compounds and DHT The

antiandrogenic activity (the inhibitory activity against the transcription of androgen

receptor) of each tested compound is recorded according to the relative luciferase

activity In this anti-AR bioassay, curcumin did not exhibit antiandrogenic activity;

however, dimethyl curcumin (DMC, 4) and 4-ethoxycarbonylethyl curcumin

(ECECur, 5), two curcumin analogs developed in our laboratory, were found to be

potent anti-AR agents These two compounds are currently being evaluated for in

vivo activity ECECur is a mixture of two tautomers: enol-keto and di-keto (Figure

2.2) In our preliminary antiandrogenic SAR study of curcumin analogs, substitutions

on the C-4 and C-4’ positions were important and the acyclic structure of the

diarylheptanoid was required for activity.

Figure 2.2 Anti-AR curcumin analogs DMC (4) and ECECur (5)

HO OH

O” 'OEt

ECECur (5)

13

CHAPTER 3

RESEARCH PART

I-THE RESOLUTION OF I-THE TAUTOMERISM OF

4-ETHOXYCARBONYLETHYL CURCUMIN (ECECur)

Introduction

In the preliminary development of curcumin analogs as potential anti-prostate

cancer agents, we reported that 4-ethoxycarbonylethyl curcumin (ECECur) (5),showed greater potency in anti-AR assays than hydroxyflutamide, an anti-androgen

currently used clinically.“°*’ ECECur has also exhibited interesting biologicalactivities other than potent anti-AR activity (unpublished data) It is regarded as apromising drug candidate for the treatment of prostate cancer, as well as certain otherdiseases However, the tautomerism of ECECur, which causes it to exist in bothenol-keto and di-keto forms, may hinder its potential as a clinically useful drug Here

a resolution to the tautomerism of ECECur is presented In addition, the investigation

of which form of ECECur is responsible for its anti-AR activity was conducted ThisSAR study was used to guide the further design of five new analogs, among whichcompound 12 exhibited great potential as a drug candidate to treat prostate cancer

An extensive structure-activity relationship (SAR) regarding the antiandrogenicactivity was established based on this study

3.2 Rationale

To circumvent the tautomerism of ECECur and determine the needed structural

features for anti-AR activity, two target compounds 6 and 7 was designed (Figure

3.1) Ideally, target compounds should be stable in only one form as well as have

minimal structural change from the parent compound ECECur in order to most likely

retain similar potency

Figure 3.1 The design of di-keto ECECur analog 6 and enol-keto analog 7

Compound 7 was proposed as the enol-keto ECECur analog The only structuraldifference between 7 and its parent compound 5 occurs in the C-4 side chain The

15

ethoxycarbonylethyl side chain in ECECur contains a saturated single (C-C) bond,

while the ethoxycarbonylethylenyl side chain of 7 contains an unsaturated double

(C=C) bond Compound 7 exists exclusively in an enol-keto form due to the highly

conjugated polyene.

Synthesis of New ECECur Analogs

Compound 6, the C-4 fluorinated analog of 5, was modified from ECECur as

shown in Scheme 3.1 Commercially available vanillin was condensed with ethyl

4-acetyl-5-oxo-hexanoate using the method of Pedersen et al” to obtain compound 5.

Boric anhydride was used to form a boron complex with ethyl

4-acetyl-5-oxo-hexanoate in order to prevent Knoevenagel condensation Compound 5 was then

protected as its tetrahydropyranyl (THP) ether by using DHP in the presence of

PPTS in dry dichloromethane.TM The THP ether 8 was fluorinated with

1-alkyl-4-fluoro-1, 4-diazabicyclo[2,2,2]octane salt (SelectFluorTM) in the presence of sodium

hydride in DMF.°’ Deprotection of compound 9 with PPTS in ethanol afforded the

target compound 6

The preparation of analog 7 (Scheme 3.2) started from curcumin 1, which wasobtained by recrystallization of commercially available curcumin (Aldrich, Inc.).After the two phenoxy groups of 1 were protected as the THP ethers, the resultingcompound 10 was alkylated at the C-4 position using sodium hydride in anhydroustetrahydrofuran, followed by the addition of ethyl propiolate to affordmonosubstituted compound 11 The target compound 7 was obtained by cleavage ofthe THP ethers as described above for 6

16

Compounds 12 and 13 were derived directly from dimethyl curcumin (4), which

was prepared from commercially available 3,4-dimethoxybenzaldehyde as described

above for compound 5 Dimethyl curcumin reacted in a Michael addition with ethyl

propiolate to give compound 12 and with methyl propiolate to give compound 13

(Scheme 3.2).

As shown in Scheme 3.3, compound 14 was obtained by reacting dimethyl

curcumin with N-ethylpropiolamide, which was prepared from ethylamine by the

slow addition of methyl propiolate at -30 °C.°* Compound 15 was obtained by thereaction of dimethyl curcumin with propiolic acid amide under reflux Propiolic acid

amide was prepared from concentrated aqueous ammonia and methyl propiolate at

-30 °C.

Compound 12 was reduced to the allylic alcohol 16 with Dibal-H at -78 °C(Scheme 3.2)

17

(%6L) 9t eb

3.4 Biological Results and Discussion

The target ECECur analogs (6 and 7) and their synthetic intermediates (8 ~ 11)

were tested for inhibitory activity against AR transcription in the

androgen-dependent LNCaP cell line and in the androgen-inandrogen-dependent PC-3 cell line

transfected with wild-type AR Cytotoxicity was also examined in the LNCaP cell

line The bioassay results are shown in Figure 3.2 and generally were consistent overthe three models The parent compound ECECur (5) showed the highest activity,followed by compound 11 Compounds 7 and 8 showed weak activity, and di-ketocompounds 6, 9 and 10 were inactive Compounds 7 and 11 exist exclusively in theenol-keto form; thus, the enol-keto form is likely the active form for anti-ARactivity Because fluorination of ECECur at the C-4 position (6) abolished activity inall bioassay models, we propose that the di-keto form may not contribute to the anti-

AR activity of ECECur In a previous paper, we also reported that a di-ketocurcumin analog with di-methyl substitution at the C-4 position did not exhibit anti-

AR activity in either human prostate cancer cell line.“® The large conformational

difference between the di-keto and enol-keto forms of ECECur may help to explainthe different anti-AR activity of these two analog types (Figure 3.3) The electron-withdrawing effect of fluorine may add to the cause of anti-AR activity loss In theenol-keto conformation, the two phenyl rings and the unsaturated linker are in thesame plane, because of the high degree of conjugation as well as the formation of astrong hydrogen bond between the enol proton and the carbonyl oxygen However,

in the di-keto conformation, ECECur does not stabilize in a planar structure, becausethe two electron-rich carbonyl oxygen atoms repel each other Compound 8, the THP

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ether of ECECur (5), showed decreased activity, while compound 11, the equivalent

analog of 7, showed greater anti-AR activity Thus, the THP protecting group has a

dissimilar influence on the anti-AR activity of different compounds With

ethoxycarbonylethyl substitution at C-4, tetrahydropyranylation of both phenols had

a negative impact on the anti-AR activity However, with an

ethoxycarbonylethylenyl group at C-4, it had a positive influence on the anti-AR

activity Therefore, both the C-4 position and the C-4’ moieties on the phenyl rings

of curcumin analogs are important pharmacophores with respect to the anti-AR

activity In order to obtain an optimal anti-AR agent, we next focused on

modification of the substituents at these positions

Based on the preceding SAR information, I designed and synthesized compounds

12 - 16, which have C-4’ methoxy groups on both phenyl rings and various

ethylenyl side chains at C-4 As shown in Figure 3.4, among the five new analogs,compound 12 exhibited the highest potency in all three bioassay models Compound

15 showed moderate anti-AR activity in LNCaP cells, weak anti-AR activity in PC-3cells transfected with wild type androgen receptor, and weak inhibitory activityagainst the growth of LNCaP cells Compounds 13, 14 and 16 showed either weak or

no activity in the prostate cancer cells Compared with ECECur (5), compound 12showed similar anti-AR potency in the LNCaP cell line, was slightly less potent inPC-3 cells transfected with wild type androgen receptor and was more cytotoxic inthe LNCaP cell growth assay (Figure 3.4) Since compound 12 showed significantactivity and does not undergo or be limited by tautomerism, it is regarded as a verypromising drug candidate for in vivo investigation The SAR conclusions were

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extended on the basis of the structural features and bioactivity of these five new

analogs Although the structures of these analogs vary only in the side chain at C-4,

the replacement of ethoxy (compound 12) with methoxy (compound 13) or

N-ethylamino (compound 14) resulted in loss of activity, which implies that a long

chain ester may be more favorable The reduction of ester to alcohol also led to loss

of activity, emphasizing the necessity for an ester in the side chain From comparing

compound 12 and 15, the nitrile functional group does not enhance the anti-AR

activity or the cytotoxicity in LNCaP cells Therefore, in the future study, more

attention should put on the optimization of the ester length.

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Figure 3.2 — Antiandrogenic activity of compounds 4 - 11 (3 uM) in LNCaP and

PC-3 human prostate cancer cells and their cytotoxicity in LNCaP cells.

A) and B) LNCaP and PC-3 human prostate cell lines were seeded and cotransfected with reporter

MMTV-luciferase (both cell lines), wild-type AR expression plasmid (PC-3) using SuperFect Subsequently, the transfected cells were harvested and re-plated in 10% charcoal-stripped fetal bovine serum DMEM medium The cells were then treated with dehydrotestosterone (DHT, 1 nM), and test compounds (3 uM) and harvested for detection of the luciferase activity (cf Experimental).

| DHT (1 nM) + tested compound (3 uM)

DHT (1 nM) + tested compound (3 uM)

C) LNCaP cell growth assay was used to further confirm the antiandrogenic activity detected by the

above-mentioned AR tranactivation assay An MTT assay was used to measure cell growth (cf Experimental) Compounds 11 and 5 showed comparable potency to inhibit the growth of LNCaP

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