A Study on Novel 2-Substituted Isoflavones As Therapeutic Agents

Home , Chrysin, Ribitol

NOVEL 2-SUBSTITUTED ISOFLAVONES: A PRIVILEGED STRUCTURE APPROACH TO NEW AGENTS FOR HORMONE-DEPENDENT BREAST CANCER

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Young-Woo Kim, M.S.

* * * * *

The Ohio State University 2003

Dissertation Committee: Approved by Professor Robert W. Brueggemeier, Adviser

Professor Robert W. Curley, Jr. ______Adviser Professor Pui-Kai Li College of Pharmacy

ABSTRACT

Natural isoflavones are well known as phytoestrogens due to their biological activities

that mimic endogenous estrogens. Their structural similarity to 17β-estradiol, the most

potent endogenous estrogen, is believed to be responsible for their ability to interact with

many molecular targets of estrogens. For this reason, we recognized the isoflavone ring

system as a promising privileged structure for the development of new therapeutics for

hormone-dependent breast cancer, in which estrogens play a key role in growth and

development of tumor. More importantly, we envisioned that a specific activity could be

achieved by introducing proper functional groups into the isoflavone nucleus, and we

designed a library of novel 2-substituted isoflavones.

For the library synthesis, we developed efficient synthetic routes, in which α-oxoketene

dithioacetals are employed as key intermediates. Various 2-(alkylthio)isoflavones were

obtained directly from readily available deoxybenzoins and electrophiles using a phase

transfer catalysis procedure. Alternatively, 2-substituted isoflavones were prepared

through a 1,4-conjugate addition-elimination reaction of 2-(methylsulfonyl)isoflavones

using a variety of commercially available nucleophiles.

ii With efficient synthetic routes developed, we examined the potential utility of this

approach for the discovery of new leads towards specific molecular targets in the breast

cancer. Initially, we have focused on two major classes of therapeutic agents, aromatase

inhibitors and selective estrogen receptor modulators (SERMs). As potential aromatase

inhibitors, we prepared various isoflavones possessing a nitrogen-containing heterocyclic

moiety, which is known to interfere with the activity of aromatase by coordinating with

its heme iron. As a result, several compounds were identified with potent aromatase

inhibitory activities. As potential SERMs, we prepared a series of isoflavones containing

an amine-bearing side chain, which is known to be essential for the tissue selectivity of

many known SERMs. Several compounds in this series were highly potent in inhibiting

proliferation of human breast cancer cells. However, their low binding affinities for

estrogen receptors suggest that these compounds may not be SERMs. Therefore,

extended studies are currently underway to elucidate their mechanisms of action.

Consequently, our approach using isoflavone as a privileged structure proved useful to

identify new leads for breast cancer treatment. In addition, with a growing number of

newly identified proteins in this post-genome era, the number of potential targets for

therapeutic intervention has also increased. Therefore, our approach could also be useful in identifying new chemical probes that can provide insight into biological problems presented by breast cancer.

iii

Dedicated to my parents

iv ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Robert Brueggemeier, for allowing me full, unconditional liberty to pursue and materialize my research ideas and providing consistent encouragement and unrelenting support.

I would also like to thank the previous and current faculty members of the Division for their excellent teaching. Special thanks must go to Dr. Robert Curley for his concern, support, advice, and encouragement.

I am thankful to Kathy Brooks, Joan Dandrea, and Kelly Ballouz for making my graduate school life much easier.

I wish to express my most heartfelt thanks to my classmates, Jon Baker, Linda Tran

(Rakes), Serena Mershon, and John Means for helping me get through my tumultuous first year in this program, The Ohio State University, Columbus, and this country.

I also deeply thank the former lab colleagues, Jen Whetstone, Trevor Petrel, Daniele

Pellegata, Jon Baker, Holly Coughenour, Shaw Joomprabutra, Abhijit Bhat, and Jim

Mobley, and the current lab mates, John Hackett, Edgar Diaz-Cruz, Jeanette Richards,

Danyetta Davis, Serena Landini Bin Su, Nancy Gilbert, and Dr. Mike Darby, for their every word of friendship and motivation. I am especially grateful to Jim Mobley not only for running ER binding assay for me but also for being my mentor. I am also thankful to John Hackett, undoubtedly the most genuinely helpful person I have ever met,

v for having been not only a great research partner but also graciously and unselfishly

devoted many hours of his own time to help me out with my scientific and personal

matters.

I sincerely thank the triumvirate of Dr. Curley’s lab, Joe Walker, Derek Barnett, and

Kevin Weiss, for having been my friends by sharing the “happiest time” in my graduate school life.

I am forever grateful to my lifelong friends, Woon-Joo, Hong-Min, Young-Jin, Boo-Il,

Sung-Hwan, and June-Seop, for their unchanged friendship despite my long “physical” absence from them.

I will always be indebted to my brother and sister and their spouses for willingly covering my contributions to family matters while I have been studing.

Most of my thanks are reserved for my parents, who have encouraged me in my endeavors despite the hardships of geographical separation and enabled me to fulfill my dream referred to as “higher education.” I also wish to extend my heartfelt thanks to my parents-in-law whose unfaltering love, support, and encouragement have helped me persevere throughout it all.

Special mention must go to my precious little girl, Yoon Jae, for being healthy physically as well as mentally despite the sudden environmental change that might have been too much for a three-year-old kid to overcome and for providing consistent joy and motivation. Finally but most importantly, words cannot express my gratitude to my love,

Hye Weon, for willingly giving up our seemingly stable situation back in Korea and sharing an adventurous life with me.

vi VITA

July 30, 1966...... Born – Seoul, Korea

1990...... B.S. Pharmacy, Seoul National University

1990 – 1992...... M.S. Medicinal Chemistry, Seoul National University Seoul, Korea

1992 – 1998...... Associate and Senior Researcher, Life Science R&D Center, SK Chemicals Co., Ltd. Suwon, Korea

1998 – present...... Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

Research Publications

1. Kim, Y.-W.; Mobley, J. A.; Brueggemeier, R. W. Synthesis and estrogen receptor binding affinities of 7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-ones containing a basic side chain. Bioorg. & Med. Chem. Lett. 2003, 13, 1475−1478.

2. Kim, Y.-W.; Brueggemeier, R. W. A convenient one-pot synthesis of 2-(alkylthio)- isoflavones from deoxybenzoins using a phase transfer catalyst. Tetrahedron Lett. 2002, 43, 6113−6115. 3. Kim, D.-K.; Ryu, D. H.; Lee, J. Y.; Lee, N.; Kim, Y.-W.; Kim, J.-S.; Chang, K.; Im, G.-J.; Kim, T.-K.; Choi, W.-S. Synthesis and biological evaluation of novel A-ring modified hexacyclic camptothecin analogues. J. Med. Chem. 2001, 44, 1594−1602. 4. Choi, W.-S.; Im, G.-J.; Kim, D.-K .; Kim, T.-K.; Jung, I.; Kim, T.-S.; Lee, S.-H.; Lee, N.; Kim, Y.-W.; Kim, J.-S.; Chang, K. Pharmacokinetic studies of 2-amino-9- (3-acetoxymethyl-4-isopropoxycarbonyl-oxybut-1-yl)purine, an oral prodrug for the antiviral agent penciclovir. Drug Metab. Dispos. 2001, 29, 945−949.

vii 5. Kim, D.-K.; Lee, N.; Lee, J. Y.; Ryu, D. H.; Kim, J.-S.; Lee, S.-H.; Choi, J.-Y.; Chang, K.; Kim, Y.-W.; Im, G.-J.; Choi, W.-S.; Kim, T.-K.; Ryu, J.-H.; Kim, N.-H.; Lee, K. Synthesis and phosphodiesterase 5 inhibitory activity of novel phenyl ring modified sildenafil analogues. Bioorg. Med. Chem. 2001, 9, 1609−1616. 6. Kim, D.-K.; Kim, Y.-W.; Lee, N. Synthesis of 5-[2-(guanin-9-yl)- and 5-[2-(2- aminopurin-9-yl)ethyl]-2-D-ribo-(1’,2’,3’,4’-tetrahydroxybutyl)-1,3-dioxane. J. Heterocyclic Chem. 2001, 38, 45−51. 7. Kim, D.-K.; Lee, N.; Kim, Y.-W. Synthesis of 9-[2-(2-hydroxymethyl-2-methyl-, - (2-acetoxy methyl-2-methyl-, -(2,2-di(hydroxymethyl)-, and -(2,2-di(acetoxymethyl)-1,3-dioxan-5-yl)ethyl) derivatives of guanine and 2-aminopurine. J. Heterocyclic Chem. 2000, 37, 1113−1119. 8. Ryu, K. H.; Rhee, H. I.; Jung, I.; Kim, T.-S.; Lee, S. J.; Im, G.-J.; Lee, N.; Ryu, D. H.; Kim, Y.-W.; Kim, J.- S.; Chang, K.; Lee, B. H.; Shin, H. S.; Kim, E.-J.; Kim, K. H.; Kim, D.-K. General pharmacology of the new antiviral agent SK 1899. Arzneim.-Forsch. Drug Res. 2000, 50(I), 4, 395−403. 9. Kim, D.-K.; Lee, J.; Kim, Y.; Lee, N.; Kim, Y.-W.; Chang, K.; Kim, J.-S.; Lee, K.; Kim, K. H Synthesis of carbon-14 labelled 2-amino-9-(3-hydroxymethyl-4- isopropoxycarbonylbut-1-yl)purine (SK 1875), a potential prodrug of penciclovir. J. Labelled Cpd. Radiopharm. 1999, 42, 597−604. 10. Kim, D.-K.; Lee, N.; Rhu, D. H.; Kim, Y.-W.; Kim, J.-S.; Chang, K.; Im, G. J.; Choi, W.-S.; Cho, Y.-B.; Kim, K. H Synthesis and evaluation of 2-amino-9-(3-acyloxy- methyl-4-alkoxycarbonylbut-1-yl)purines and 2-amino-9-(3-alkoxycarbonylmethyl- 4-alkoxycarbonylbut-1-yl)purines as potential prodrugs of penciclovir. Bioorg. & Med. Chem. 1999, 7, 1715−1725. 11. Kim, D.-K.; Lee, N.; Kim, Y.-W.; Chang, K.; Im, G. J.; Choi, W.-S.; Kim, K. H Synthesis and evaluation of amino acid esters of 6-deoxypenciclovir as potential prodrugs of penciclovir. Bioorg. & Med. Chem. 1999, 7, 419−424. 12. Kim, D.-K.; Lee, N.; Kim, Y.-W.; Chang, K.; Kim, J.-S.; Im, G. J.; Choi, W.-S.; Jung, I.; Kim, T.-S.; Hwang, Y.-Y.; Min, D.-S.; Um, K. A.; Cho, Y.-B.; Kim, K. H Synthesis and evaluation of 2-Amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut- 1-tyl)purines as potential prodrugs of penciclovir. J. Med. Chem. 1998, 41, 3435−3441. 13. Kim, D.-K.; Gam, J.; Kim, Y.-W.; Lim, J.; Kim, H.-T.; Kim, H. Synthesis and anti- HIV-1 activity of a series of 1-alkoxy-5-alkyl-6-(arylthio)uracils. J. Med. Chem. 1997, 40, 2363−2373.

viii 14. Lee, N.; Kim, Y.-W.; Kim, K. H.; Kim, D.-K. A new route to the improved synthesis of 1-(alkoxymethyl)-5-alkyl-6-(arylselenenyl)uracils. J. Heterocyclic Chem. 1997, 34, 659−663. 15. Kim, D.-K.; Kim, Y.-W.; Kim, K. H. Synthesis of 6-substituted 1-alkoxy-5- alkyluracils. J. Heterocyclic Chem. 1997, 34, 311−314. 16. Kim, D.-K.; Kim, Y.-W.; Gam, J.; Kim, G.; Lim, J.; Lee, N.; Kim, H.-T.; Kim, K. H. Synthesis of anti-HIV activity of a series of 1-(alkoxymethyl)-5-alkyl-6-(arylselenenyl)uracils and -2-thiouracils. J. Heterocyclic Chem. 1996, 33, 1275−1283. 17. Kim, D.-K.; Lee, N.; Im, G. J.; Kim, Y.-W.; Chang, K.; Kim, H.-T.; Cho, Y.-B.; Choi, W.-S.; Jung, I.; Kim, K. H. Synthesis and evaluation of amino acid ester prodrugs of penciclovir. Bioorg. & Med. Chem. Lett. 1996, 6, 1849−1854. 18. Kim, D.-K.; Kim, H.-T.; Lim, J.; Gam, J.; Kim, Y.-W.; Kim, K.H.; Shin, Y. O. Synthesis and anti-HIV activity of 1,5-dialkyl-6-(arylselenenyl)uracils and -2- thiouracils. J. Heterocyclic Chem. 1996, 33, 885−894. 19. Kim, D.-K.; Kim, Y.-W.; Kim, H.-T.; Kim, K. H. Synthesis and in vitro cytotoxicity of cis-dichloro[(2S,3R,4S)-2-aminomethyl-3,4-(O-isopropylidene)dihydroxy- or -3,4-dihydroxypyrrolidine]platinum(II). Bioorg. & Med. Chem. Lett. 1996, 6, 643−646. 20. Kim, D.-K.; Kim, G.; Kim, Y.-W. Preparation of optically active 3-substituted piperidines via ring expansion: Synthesis of 4-amino- and 4-fluoro-1,4,5-trideoxy- 1,5-imino-D-ribitol and 1,5-dideoxy-1,5-imino-D-ribitol. J. Chem. Soc., Perkin Trans. 1 1996, 803−808. 21. Lee, N.; Kim, Y.-W.; Chang, K.; Kim, K. H.; Jew, S.-S.; Kim, D.-K. Enantioselective synthesis of (R)- and (S)-2-alkyl-1,4-butanediols via enantiomerically pure 3-alkyl-5-(menthyloxy)butyrolactones. Tetrahedron Lett. 1996, 37, 2429−2432. 22. Kim, H.-T.; Kim, D.-K.; Kim, Y.-W.; Kim, K. H.; Sugiyama, Y.; Kikuchi, M. Antiviral activity of 9-[[(ethoxyhydroxyphosphinyl)methoxy]methoxy]guanine against cytomegalovirus and herpes simplex virus, Antiviral Res. 1995, 28, 243−251. 23. Kim, D.-K. ; Kim, Y.-W.; Gam, J.; Lim, J.; Kim, K. H. A convenient approach to the synthesis of 6-substituted 1,5-dialkyluracils and 2-thiouracils. Tetrahedron Lett. 1995, 36, 6257−6260.

24. Kim, D.-K.; Kim, Y.-W.; Kim, K. H. Synthesis and anti-HCMV activity oix 9- [[(ethoxy-hydroxyphosphinyl)methoxy]methoxy]guanine, an isostere of PMEG monoethyl ester, Bioorg. & Med. Chem. Lett. 1994, 4, 2241−2244.

ix FIELDS OF STUDY

Major Field: Pharmacy

Medicinal Chemistry

x TABLE OF CONTENTS

Page

Abstract...... ii

Dedication...... iv

Acknowledgments...... v

Vita...... vii

List of Tables ...... xv

List of Figures...... xvi

List of Abbreviations ...... xxi

Chapters:

1. Estrogens and breast cancer...... 1

1.1 Breast cancer...... 1 1.2 Estrogens...... 2 1.3 Estrogen biosynthesis...... 3 1.4 Mechanisms of estrogen signaling ...... 5 1.5 Estrogen receptors...... 7 1.5.1 Functional structures...... 8 1.5.2 Differences between ERα and ERβ ...... 9 1.5.3 Estrogen receptors and breast cancer...... 10 1.6 Aromatase...... 11 1.6.1 Aromatization reaction...... 12 1.6.2 Aromatase and breast cancer ...... 13 1.7 Other therapeutic strategies for breast cancer ...... 14 1.8 A “privileged structure” approach...... 14 1.9 References...... 18

2. Isoflavone library design ...... 22

xi 2.1 Flavonoids...... 22 2.1.1 Structures of flavonoids...... 24 2.1.2 Biological activities of flavonoids...... 25 2.2 Isoflavones...... 25 2.2.1 Genistein...... 27 2.2.2 Daidzein...... 28 2.2.3 Biochanin A...... 28 2.2.4 Other natural isoflavones...... 29 2.2.5 Synthetic isoflavones...... 29 2.3 Isoflavone synthesis...... 30 2.4 Isoflavone as a privileged structure...... 34 2.5 Isoflavone library design...... 35 2.5.1 7-Position...... 35 2.5.2 4’-Position...... 36 2.5.3 2-Position...... 36 2.5.4 Other positions...... 37 2.5.5 2,4’7-Trisubstituted isoflavone library...... 37 2.6 References...... 38

3. Chemistry development ...... 43

3.1 New synthetic methods ...... 43 3.2 Synthetic strategy...... 44 3.3 α-Oxoketene dithioacetals...... 46 3.4 Synthesis of 2’-hydroxydeoxybenzoins ...... 50 3.5 Construction of isoflavone scaffold ...... 51 3.5.1 Initial study...... 51 3.5.2 Application to 4’-alkoxy-2’-hydroxydeoxybenzoins ...... 55 3.5.3 A new approach: Phase transfer catalysis procedure...... 60 3.5.4 Direct synthesis using sodium hydride as a base...... 64 3.6 Introduction of substituents...... 66 3.6.1 Oxidation...... 66 3.6.2 Substitution...... 67 3.7 References...... 68

4. Aromatase inhibitors...... 71

4.1 Aromatase inhibitors...... 71 4.2 Steroidal aromatase inhibitors...... 73 4.3 Nonsteroidal aromatase inhibitors...... 74 4.3.1 Triazole-containing nonsteroidal aromatase inhibitors ...... 76 4.3.2 Imidazole-containing nonsteroidal aromatase inhibitors...... 77 4.3.3 Pyridine-containing nonsteroidal aromatase inhibitors...... 79

xii 4.3.4 Other nonsteroidal aromatase inhibitors...... 80 4.4 Isoflavones as aromatase inhibitors: Inhibitor design ...... 82 4.5 First set of inhibitors...... 85 4.5.1 Synthesis using phase transfer catalysis procedure ...... 86 4.5.2 Human placental microsome aromatase assay ...... 89 4.5.3 Kinetic studies...... 95 4.5.4 Further studies...... 98 4.6 Second set of inhibitors ...... 99 4.7 References...... 104

5. Antiproliferative agents...... 110

5.1 Selective estrogen receptor modulators...... 110 5.2 Tamoxifen and raloxifene ...... 111 5.3 Molecular mechanisms of SERM action...... 113 5.4 New SERMs...... 117 5.5 Genistein as a SERM...... 117 5.6 Design of isoflavone-based SERMs...... 118 5.7 First set of target compounds ...... 120 5.7.1 Synthesis...... 121 5.7.2 Biological evaluation...... 125 5.7.2.1 Proliferation assay...... 125 5.7.2.2 ER binding assay...... 129 5.7.2.3 Further studies...... 133 5.8 Second set of compounds ...... 133 5.8.1 Synthesis...... 134 5.8.2 Biological evaluation...... 136 5.9 References...... 136

6. Conclusions...... 139

7. Experimental methods ...... 146

7.1 General methods...... 146 7.2 Synthetic methods ...... 147 7.2.1 Preparation of deoxybenzoins...... 147 7.2.2 Protection of deoxybenzoins ...... 150 7.2.3 Preparation of α-oxoketene dithioacetals...... 157 7.2.4 Construction of isoflavone scaffold...... 163 7.2.5 Oxidation...... 182 7.2.6 Substitution...... 187 7.2.7 Alkylation...... 206

xiii 7.2.8 Deprotection ...... 212 7.2.9 Selective debenzylation...... 226 7.3 References ...... 232

Bibliography ...... 234

xiv LIST OF TABLES

Table Page

1.1 New anti-cancer treatment under development in breast cancer...... 15

4.1 Advantages and disadvantages of the two major classes of aromatase chemical inhibitors...... 75

4.2 Aromatase inhibitory activities of isoflavones 9a−l and 12a−h ...... 92

4.3 Aromatase inhibitory activities of three potent isoflavones, 9k, 12g, and 12h..... 96

4.4 Aromatase inhibitory activities of isoflavones 11d and 13a−f ...... 101

4.5 Aromatase inhibitory activities of isoflavones 13f−i in comparison with aminoglutethimide...... 104

5.1 Antiproliferative activity of the selected isoflavones in MCF-7 human breast cancer cell line...... 126

5.2 Binding affinities of compounds 15a−d and 16a−b for ERα ...... 130

xv LIST OF FIGURES

Figure Page

1.1 Structures of three endogenous estrogens ...... 2

1.2 Estrogen biosynthesis in the human ovary. LH and FSH sites of action are also indicated ...... 4

1.3 Traditional model of estrogen action for transcriptional activation via interaction between E2-bound ER and ERE of target genes...... 6

1.4 Schematic comparison of two estrogen receptors. Numbers above each diagram represent the amino acid position for the boundaries of each receptor structural domain ...... 7

1.5 Mechanism of aromatization reaction by aromatase...... 12

1.6 Benzodiazepine nucleus as a privileged structure and examples of its biologically active derivatives...... 16

1.7 4H-1-Benzopyran-4-one nucleus as a privileged structure and examples of its biologically active derivatives...... 17

2.1 Basic structures of flavonoids ...... 23

2.2 Examples of simple natural isoflavones...... 27

2.3 Examples of synthetic isoflavones ...... 30

2.4 Deoxybenzoin approach using various one-carbon unit agents ...... 31

2.5 Direct synthesis of isoflavones from phenols and arylacetic acids ...... 31

2.6 Synthesis of 2-alkylisoflavones by Baker-Venkataraman rearrangement followed by intramolecular condensation...... 32

2.7 Synthesis of 2-alkylisoflavones using amide acetals...... 32

2.8 Synthesis of isoflavones by oxidative rearrangement of 2’-hydroxychalcones .... 33

xvi 2.9 Synthesis of isoflavones by Suzuki cross coupling of 3-halochromone with aryl boronic acids using palladium(0) catalysts...... 33

2.10 Synthesis of isoflavones by rearrangement and cyclization of chalcone epoxides using borontrifluoride diethyl etherate ...... 33

2.11 Synthesis of isoflavones by rearrangement of flavanones using iodobenzene...... 34

2.12 Library of 2-substituted isoflavones...... 37

3.1 Retrosynthetic analysis...... 45

3.2 An application of α-oxoketene dithioacetals as key intermediates for the synthesis of 2,6-substituted pyrimidines...... 45

3.3 Retrosynthesis...... 46

3.4 Two principle reactions of α-oxoketene dithioacetals ...... 47

3.5 Synthetic utility of α-oxoketene dithioacetals...... 48

3.6 Synthesis of D-ring functionalized and annulated estrone derivatives via α- oxoketene dithioacetal...... 49

3.7 One-pot synthesis of α-oxoketene dithioacetals ...... 49

3.8 Synthesis of deoxybenzoins using boron trifluoride diethyl etherate (BF3·OEt2). 51

3.9 Protection of hydroxyl groups with tert-butyldimethylsilyl (TBS) group ...... 52

3.10 Synthesis of α-oxoketene dithioacetal using lithium diisopropylamide (LDA) as a base...... 53

3.11 Expected (I, concerted) and observed (II, stepwise) mechanisms of cyclization reaction ...... 54

3.12 Selective alkylation of 4’-hydroxyl group using a Mitsunobu condition...... 56

3.13 Synthesis of 2-(alkylthio)isoflavones via α-oxoketene dithioacetals...... 57

3.14 Plausible explanation on the fast cyclization of compounds 6b−e...... 59

3.15 Plausible explanation on the slow cyclization of compound 6a...... 59

3.16 One-pot synthesis of O-alkyl-S-methyl dithiocarbonates (xanthates) using a phase transfer catalyst...... 61

xvii 3.17 O-alkyl-S-methyl dithiocarbonates proposed as potential intermediates for the synthesis of 2-(methylthio)isoflavones...... 61

3.18 α-Oxoketene dithioacetal intermediate (6f) in the transformation of 2’- hydroxydeoxybenzoin 4b into 2-(methylthio)isoflavone 8a in a phase transfer catalysis reaction ...... 62

3.19 One-pot synthesis of various 2-(alkylthio)isoflavones via a phase transfer catalysis procedure ...... 64

3.20 One-pot synthesis of various 2-(alkylthio)isoflavones using sodium hydride as a base...... 65

3.21 Oxidation of sulfides (8a−e) to sulfones (10a−e) and sulfoxide (10f) using 3- chloroperoxybenzoic acid (mCPBA)...... 67

3.22 Displacement reactions of sulfones with various nucleophiles...... 68

4.1 Androstenedione and selected steroidal aromatase inhibitors...... 74

4.2 Aminoglutethimide and triazole-containing nonsteroidal aromatase inhibitors ... 76

4.3 Imidazole-containing nonsteroidal aromatase inhibitors...... 77

4.4 Pyridine-containing nonsteroidal aromatase inhibitors...... 79

4.5 A sesquiterpene lactone as a selective aromatase inhibitor...... 80

4.6 Flavonoids as aromatase inhibitors ...... 81

4.7 Two synthetic approaches to 2-substituted isoflavones targeting aromatase...... 84

4.8 Synthesis of 2-substituted isoflavones as aromatase inhibitors using phase transfer catalysis procedure ...... 86

4.9 Deprotection reactions using boron tribromide...... 87

4.10 Failure in selective debenzylation using hydrogenation in the presence of palladium due to catalyst poisoning ...... 87

4.11 Selective debenzylation using boron trifluoride diethyl ether with dimethyl sulfide ...... 88

4.12 Tritiated water release aromatase assay ...... 89

xviii 4.13 Screening results of compounds 9a−l and 12a−h for aromatase inhibitory activity at 1 µM concentration of each compound (10 µM for 9h and 50 µM for AG)..... 90

4.14 Dose-response curves of compounds 9k, 12g, and 12h in inhibiting aromatase activity in comparison with aminoglutethimide (AG)...... 95

4.15 Lineweaver-Burk plots from kinetic study on the inhibition of aromatase activity by compounds 9k, 12g, and 12h ...... 97

4.16 Synthesis of the second set of aromatase inhibitors via a 1,4-conjugate addition- elimination process...... 99

4.17 Screening results of compounds 11d and 13a−f for aromatase inhibitory activity at 1 µM concentration of each compound...... 100

4.18 Synthesis of 2-imidazolyl isoflavones 13f−i ...... 102

4.19 Dose-response curves of 2-imidazolyl isoflavones 13f−i in inhibiting aromatase activity ...... 103

5.1 Structures of representative SERMs (tamoxifen and raloxifene) and a pure entiestrogen (ICI 182,780)...... 112

5.2 Schematic representations of the interactions between E2 and ERα (A), RAL and ERα (B), and GEN and ERβ (C) ...... 115

5.3 Selected SERMs under the clinical trials ...... 117

5.4 Library of isoflavones containing a basic side chain as potential SERMs...... 119

5.5 Initial targets of interest...... 120

5.6 Synthesis of 2-(4-hydroxyphenoxy)- and 2-(4-hydroxyphenylthio)-isoflavones from 2-(methylsulfonyl)isoflavone ...... 121

5.7 Dealkylation reactions for the preparation of target compounds 15a and 15b ... 122

5.8 Selective removal of benzyl protecting group...... 123

5.9 Deprotection reactions for the preparation of triphenolic (16a and 16b) and diphenolic (16c and 16d) isoflavones...... 124

5.10 Unexpected product from catalyst transfer hydrogenation of compound 11h.... 125

5.11 Antiproliferative activity of target isoflavones (5 µM) in MCF-7 human breast cancer cell line...... 127

xix 5.12 Anti-proliferative activity of target isoflavones (5 µM) in MCF-7 in the presence of E2 (10 nM) ...... 127

5.13 Anti-proliferative activity of the selected isoflavones in MDA-MB-231 human breast cancer cell line ...... 129

5.14 Dose-response curves of compounds 15a−d and 16a−b for ERα binding affinity in comparison with 17β-estradiol (E2)...... 131

5.15 Second set of target isoflavones ...... 134

5.16 Synthesis of second set of target isoflavones ...... 135

6.1 Two isoflavones as new potential lead compounds identified in the present study...... 142

6.2 Focused libraries for lead optimization ...... 143

xx LIST OF ABBREVIATIONS

4-OHT 4-Hydroxytamoxifen

7α-APTA 7α-[(4-Aminophenyl)thio]androst-4-ene-3,17-dione

7α-APTADD 7α-[(4-Aminophenyl)thio]androsta-1,4-diene-3,17-dione

9-OH-ANF 9-Hydroxy-α-Naphthoflavone

Ac Acetyl, or Acetate

AF Transcriptional activation function

AG Aminoglutethimide

ANF α-Naphthoflavone

BCA Biochanin A (5,7-dihydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran- 4-one)

Bn Benzyl

Bu Butyl

CNS Central nervous system

DAI Daidzein (7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one)

DBD DNA binding domain

DIAD Diisopropyl azodicarboxylate

DMF N,N-Dimethylformamide

DMSO Dimethyl sulfoxide

DNA Dioxyribonucleic acid

E2 17β-Estradiol

xxi EC50 Median effective concentration

EGF Epidermal growth factor

ER Estrogen receptor

ERE Estrogen responsive element

Et Ethyl

FOR Formononetin (7-hydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4- one)

FSH Follicle stimulating hormone

GEN Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4- one)

GnRH Gonadotropin releasing hormone

HRMS High resolution mass spectroscopy

HRT Hormone replacement therapy

IC50 Inhibitory concentration for half of maximal activity

IGF Insulin growth factor

IR Infrared

LBD Ligand binding domain

LCA Lithium dicyclohexylamide

LDA Lithium diisopropylamide

LG Leaving group

LH Leutinizing hormone

LHMDS Lithium hexamethyldisilazide

LRMS Low resolution mass spectroscopy

m.p. Melting point

MAPK Mitogen-activated protein kinase

xxii mCPBA m-Chloroperoxybenzoic acid

Me Methyl

RNA Ribonucleic acid

MsCl Mesyl chloride, Methanesulfonyl chloride

MsOH Methanesulfonic acid

NADPH β-Nicotinamide adenine dinucleotide phosphate

NMR Nuclear magnetic resonance

Nu Nucleophile

Ph Phenyl

PKC Protein kinases C

R Alkyl

RAL Raloxifene

RBA Relative binding affinity rt room temperature

SERM Selective estrogen receptor modulator

TAM Tamoxifen

TBAF Tetrabutylammonium fluoride

TBS tert-Butyldimethylsilyl

THF Tetrahydrofuran

TLC Thin layer chromatography

TsOH p-Toluenesulfonic acid

TTN Thallium(III) nitrate trihydrate

TxA2 Thromboxane A2 synthase

UV Ultraviolet

xxiii CHAPTER 1

ESTROGENS AND BREAST CANCER

1.1 BREAST CANCER

Breast cancer is the most common cancer diagnosed in American women and an

estimated 211,300 new cases of female breast cancer are expected to occur among

women in the United States alone during 2003 [1]. Breast cancer also represents the

second leading cause of cancer-related deaths for all women. In the United States, it was

predicted that 39,800 women were expected to die from the disease during 2003. In

addition, the lifetime risk of developing breast cancer in North American women is one

in eight [2]. Despite the decrease in mortality due to early detection and improved

treatment, breast cancer still remains a major public health concern.

There are a number of factors that are known to increase the risk of developing breast cancer. The most obvious risk factors are gender and age: female breast cancers account for over 99% of all breast cancer cases and the risk dramatically increases with age. In addition, it is well established that women at higher risk are those who have family history of the disease, increased breast density, a long menstrual history, obesity after

1 menopause, recently used oral contraceptive or postmenopausal estrogens and progestin,

never had children or had their first child after age 30, or who consume one or more

alcoholic beverages per day [1].

Figure 1.1: Structures of three endogenous estrogens.

1.2 ESTROGENS

Estrogens, including 17β-estradiol, estrone, and estriol (Figure 1.1), are steroid hormones that play a significant role in the normal development and growth of many different tissues within the body, most notably the female reproductive system and secondary sexual organs such as mammary glands, ovaries, vagina, and uterus. In addition, estrogens are known to exert numerous biological activities in nonreproductive tissues including the brain, bone, cardiovascular system, kidney, immune system, and liver.

However, their precise mechanisms of action are not yet fully defined. Nonetheless, given the obvious role of estrogens, it is not surprising that estrogen deficiency has been involved in many pathological processes like arteriosclerosis, osteoporosis, and degenerative processes in the central nervous system (CNS), whereas excessive exposure

2 to, or inappropriate stimulation by estrogens has been linked to aberrant development

and growth of tissues including breast cancer. In fact, approximately 60-70% of all

breast cancer patients have hormone-dependent breast cancer, in which estrogens play a

predominant role in development and growth of tumor [3].

1.3 ESTROGEN BIOSYNTHESIS

The biosynthesis of estrogens occurs primarily in the ovaries in mature, premenopausal

women and is highly regulated by hormones. The release of gonadotropin releasing

hormone (GnRH) from hypothalamus stimulates the anterior pituitary to release follicle

stimulating hormone (FSH) and leutinizing hormone (LH). FSH and LH in turn bind to

cell membrane receptors on the granulosa and theca cells of the ovary, thereby

promoting estrogen synthesis in the ovary [4]. Shown in Figure 1.2 is the biosynthesis of

estrogens from cholesterol, a C27 precursor to the estrogens. In the theca cells, LH

promotes the release of cholesterol from its storage, which is converted into

pregnenolone (C21) through an enzymatic cleavage of the side chain. Pregnenolone is

then converted into a C19 steroid, androstenedione, which is transferred to the granulosa

cells. In the granulosa cells, FSH promotes the final steps of estrogen synthesis by

stimulating aromatase, the cytochrome P450 enzyme complex responsible for the

conversion of androgen to estrogens (C18). The detailed mechanism of aromatase reaction will be discussed in Section 1.6.1.

Figure 1.2: Estrogen biosynthesis in the human ovary [4]. LH and FSH sites of action are also indicated.

4 Biosynthesis of estrogens also occurs in various extragonadal sites including placenta,

brain, adipose tissue, muscle, skin, and breast. After menopause, this extragonadal

estrogen synthesis is increased while ovarian estrogen synthesis significantly declines.

In a postmenopausal woman, therefore, adipose tissue is known to be the main source of

circulating estrogens, predominantly estrone [5].

1.4 MECHANISMS OF ESTROGEN SIGNALING

Estrogens are transported to target tissues through the blood stream. Upon arrival at a

target tissue, estrogens diffuse freely across the cell membrane and translocate across the

nuclear membrane (Figure 1.3). In the nucleus, estrogens bind to their specific receptors

termed estrogen receptors (ER), resulting in dissociation of heat-shock proteins that keep

the receptors in an inactive state by preventing them from dimerization. Binding of

estrogens induces a conformational change in ERs, which allows dimerization with

another estrogen-bound ER. The dimerized estrogen-ER complex then binds to a

specific sequence called estrogen responsive element (ERE) within the promoter region

of the target gene (Figure 1.3). The DNA-bound receptor complex releases corepressors

and subsequently recruits various necessary coactivators through the transcriptional

functions (AF) of the receptor, thereby initiating transcription in conjunction with the basal transcriptional complex. Coactivators are believed to act through their histone acetyltransferase activity, chromatin remodeling, and transcription reinitiation activities

[6]. In addition to EREs, recent studies have shown that the ERs can indirectly induce gene expression through alternative sites on the DNA such as AP-1 [7] or Sp-1 [8]. The

5 mRNA produced by transcription activity undergoes processing and translocation to the cytoplasm, followed by translation to form a protein which can alter cellular function by endocrine, paracrine, or autocrine interactions.

While it is clear that estrogens regulate transcription via a nuclear interaction after binding their receptors, nongenomic actions of estrogens in various organs have also been demonstrated, which might be mediated through cell-surface receptors [9].

Cytoplasm

Nucleus ER dimerization conformational change ER E2 HSP90

ER ER DNA binding

ER ER mRNA Target gene translation ERE mRNA coactivators proteins processing

effects Coactivators transcription

ER ER

Figure 1.3: Traditional model of estrogen action for transcriptional activation via interaction between E2-bound ER and ERE of target genes.

6 1.5 ESTROGEN RECEPTORS

The ER is a member of the nuclear receptor superfamily that includes receptors for

progestins, glucocorticoids, androgens, thyroid hormones, vitamin D, retinoids, and bile acids as well as a number of orphan receptors for which no known ligand has been identified. Members of this superfamily share many common structural features, possessing discrete domains that function in specific roles required for their activities as ligand-regulated transcription factors [10]. Recent studies have revealed the existence of two distinct estrogen receptors in humans: ERα and ERβ [11,12]. These two ER subtypes exhibit substantial similarities as well as distinct differences in both their structures and functions, which allows for a wide range of diverse and complex processes to take place.

1 180 263 302 553 595

H2N A/B C D E F COOH ERα

AF-1 DBD LBD AF-2

1 144 227 255 504 530

H2N A/B C D E F COOH ERβ

Amino acid 26% 84% 12% 58% 12% homology

Figure 1.4: Schematic comparison of two estrogen receptors [13]. Numbers above each diagram represent the amino acid position for the boundaries of each receptor’s structural domain.

7 1.5.1 FUNCTIONAL STRUCTURES

A schematic representation of the protein sequences of human ERα and ERβ is shown in

Figure 1.4. Both subtypes have the modular structure that is characteristic for members of the nuclear receptor superfamily with the different functional domains termed A through F [14]. The A/B-domain contains a transcriptional activation function (AF-1), which can be modulated in a ligand-independent manner via the mitogen-activated protein kinase (MAPK) pathway [ 15 ]. In this mechanism, growth factors such as epidermal growth factor (EGF) and insulin growth factor (IGF) trigger growth hormone signaling pathways, in which the MAPK phosphorylates a serine residue (Ser118) in the

AF-1 domain, thereby enhancing the transcriptional activity of the domain. The centrally located C-domain is the most conserved among the members of the nuclear receptor superfamily, and is the DNA-binding domain (DBD). This domain contains two zinc-finger motifs, each of which consists of four cysteine residues and is followed by an extended α-helix [16]. There are also other elements in this domain such as P-box, which provides the recognition site for the EREs, and D-box, which is essential for receptor dimerization. The domain D is believed to function as a hinge region, and was proposed as an important binding site for coaccessory proteins such as coactivators and corepressors [17]. The domain E is the ligand-binding domain (LBD), which consists of twelve α-helices and two very short β-pleated sheets. This LBD is multifunctional: it contains a second transcriptional activation function (AF-2) and another site for receptor dimerization as well as provides a ligand recognition site. The AF-2 resides in the helix

12 located in the carboxy terminus of the domain and can be activated as a result of

8 conformational change of the receptor upon the ligand binding, thereby providing an

interaction interface for transcriptional coactivators [18]. The domain F is located in the carboxy terminus of the receptor and is not well conserved among family members.

1.5.2 DIFFERENCES BETWEEN ERα AND ERβ

ERα is a 65 kDa protein consisting of 595 amino acids while ERβ is a 59 kDa protein

containing 530 amino acids [19]. The N-terminal A/B-domain is poorly conserved

between the two subtypes and is somewhat shorter in length in ERβ, indicating that their

AF-1 functions may differ. However, the consensus sequence for MAPK-mediated

serine phosphorylation is well conserved. The DBD of human ERα and ERβ shows the

highest identity, which is not surprising since this domain is the most conserved region

among the members of the nuclear receptor superfamily. In addition, all eight cysteine

residues that comprise the zinc fingers are fully conserved, and both the P-box and D-

box in this domain display complete conservation, indicating both subtypes may bind

DNA response elements in a very similar manner. The LBDs of the receptors show only

58% homology, however, all the residues involved in the interaction with ligand are

conserved, which may explain why both subtypes show similar affinities for 17β-

estradiol despite the relatively low degree of homology within the LBD [19]. The

carboxy terminus of the LBD, helix 12, is well conserved in both subtypes, indicating

their AF-2 functions may be very similar. The hinge region and F-domain are not well

conserved. These structural differences between ERα and ERβ are thought to contribute

9 to a number of their distinct responses to certain ligands, most possibly by recruiting

different transcriptional coactivators [20].

Both ERα and ERβ are widely expressed in the body with quite different tissue distributions, suggesting that these two receptor subtypes may have different physiological roles. ERβ transcripts have been identified predominantly in the brain, thymus, bladder, prostate, lung, and bone. ERα, on the other hand, is exclusively detected in the testis, pituitary gland, ovary, uterus, kidney, adrenal, and mammary gland

[21, 22]. However, distinct localizations of each receptor subtype have been observed within a single tissue, which reflects the different functional requirements of cells within a tissue [19]. For instance, ERα expression is highly concentrated in the stromal compartment and theca cells whereas the expression of ERβ is restricted to the granulosa

cells of developing follicles [23].

1.5.3 ESTROGEN RECEPTORS AND BREAST CANCER

As previously described, estrogens elicit most of their biological activities as a consequence of their interaction with the estrogen receptor. Since hormone-dependent breast cancer is characterized by high expression levels of ERs in tumor cells, the use of antiestrogens that block the interaction of estrogens with ERs has been one of the most

promising and logical strategies for the treatment of the disease. However, in order to

selectively repress the growth of breast tumor while maintaining the beneficial effects of

10 estrogens on other tissues, a great deal of research effort has been currently focused on alternative ligands that exert interesting tissue-specific profiles. Such ligands are termed selective estrogen receptor modulators (SERMs), and will be discussed in detail in

Chapter 5.

1.6 AROMATASE

Aromatase is a microsomal enzyme complex bound in the endoplasmic reticulum of the cell. The enzyme complex is comprised of a cytochrome P450 hemoprotein (P450arom, a

55 kDa protein of 503 amino acids) and a ubiquitous flavoprotein NADPH (β- nicotinamide adenine dinucleotide phosphate) cytochrome P450 reductase [24]. It is involved in many important biological processes such as reproduction, development, sexual differentiation and behavior, and some brain functions. The enzyme presents in various tissues in both males and females, including the brain, especially in the hypothalamus, amygdala, and hippocampus. The enzyme presents in highest levels in the ovaries of premenopausal women, in the placenta of pregnant women, and in the peripheral adipose tissues of postmenopausal women and of men. Although our knowledge of the biochemistry, molecular biology, and regulation of aromatase has greatly increased over the past two decades [25, 26], many aspects of the enzyme still remain to be fully elucidated.

Figure 1.5. Mechanism of aromatization reaction by aromatase [27].

1.6.1 AROMATIZATION REACTION

Aromatase is responsible for estrogen biosynthesis by conversion of androgens into

estrogens. It catalyzes aromatization of the A ring of androgens by removing their C-19

methyl group via three sequential hydroxylations (Figure 1.5) [27]. The first two steps

are oxidation processes resulting in conversion of the angular C-19 methyl group into an

aldehyde after loss of a water molecule. The aromatization of the A ring of the steroid is

completed by loss of the C-19 carbon atom as formic acid [28], the precise mechanism of

which is still unclear. Therefore, three moles of NADPH and three moles of oxygen are

utilized for the synthesis of one mole of estrogen product. Flavoprotein NADPH-

cytochrome P450 reductase mediates the sequential transfer of two electrons from

NADPH to the heme-iron of aromatase in this process.

12 1.6.2 AROMATASE AND BREAST CANCER

In premenopausal women, the granulosa cells in the ovary are the main source of

estrogen biosynthesis [29]. The placenta also produces high levels of estrogens during pregnancy [ 30 ]. In postmenopausal women, however, ovarian estrogen synthesis significantly declines, and extragonadal tissues such as adipose and muscle are the main source of endogenous estrogens [5]. Although serum levels of estrogens in postmenopausal women are typically low in comparison with those in premenopausal women, the levels in breast tissue have been found to be several times higher than in serum, which is similar to those in premenopausal women [31]. Furthermore, estrogen concentrations in breast tumors are higher than those in breast fat, suggesting that local production of estrogens within the breast may play a key role in tumor proliferation [18].

Several studies have shown that aromatase is highly expressed near breast tumor sites and its expression and activity are precisely regulated [32, 33, 34, 35]. Therefore, effective inhibition of breast aromatase is believed to be a promising strategy for the treatment of hormone-dependent breast cancer. Aromatase is a particularly attractive

target for inhibition because the aromatization is the rate-limiting step as well as the last

step in steroid biosynthesis, thereby its blockade should not interfere with the production

of other steroids. Aromatase inhibitors will be discussed in detail in Chapter 4.

13 1.7 OTHER THERAPEUTIC STRATEGIES FOR BREAST CANCER

As discussed above, estrogen receptors and aromatase have been the major targets for the therapeutic intervention in the treatment of hormone-dependent breast cancer. Recently, expanded progress in understanding of the biology of breast carcinoma as well as other types of cancer led to the development of various rational therapeutic interventions as summarized in Table 1.1 [36]. Many of these new approaches are currently under active clinical development. Among those, promising approaches currently under development include inhibition of signal transduction pathways, interference with tumor angiogenesis, inhibition of growth factor receptors, and modulation of apoptosis. Other productive directions of therapeutic research include cell cycle regulation, modulation of immune systems, inhibition of invasion and metastasis, and development of vaccines.

1.8 A “PRIVILEGED STRUCTURE” APPROACH

Over the past decades, we have witnessed great advances in genomics and proteomics as well as in traditional biological research, which have led to new prospects in therapeutic approaches to many serious diseases including cancer. As the number of newly identified proteins has dramatically increased, our attempts to understand the precise molecular mechanisms of those diseases have become seemingly more complicated. In breast cancer biology, a number of new proteins and their related novel pathways have been also identified in the past few years [37], which has widened our therapeutic options by providing new potential targets that are currently not exploited.

Cytotoxic drug New analogs New molecular/mechanistic classes

Modulators of drug resistance

Immunologic approaches Antibodies: monoclonal or polyclonal Immunoconjugates: With cytotoxic drugs With radioactive substances With toxins ADEPT systems Fusion proteins Vaccines

Growth factor or growth factor receptor directed HER-2/neu EGFR IGF-I Steroid hormones and their receptors Osteoclast activating factors Mammastatin

Signal transduction inhibitors Tyrosine kinase inhibitors Farnesyl protein transferase inhibitors Grb2 inhibitors

Angiogenesis inhibitors Anti-VEGF agents Inhibitors of endothelial proliferation

Inhibitors of tissue invasion and metastasis Inhibitors of adhesion molecules, integrins Matrix metalloprotease inhibitors

Modulators of apoptosis

Telomerase inhibitors

Table 1.1: New anti-cancer treatment under development in breast cancer [36].

Figure 1.6: Benzodiazepine nucleus as a privileged structure and examples of its biologically active derivatives.

While the traditional medicinal chemistry approach including rational drug design is still valuable in many aspects, it appears to be not efficient enough to fulfill such increasing demands in this post-genome era, primarily due to its time-consuming and complicated process, low diversity, and high costs. With a growing number of potential targets for therapeutic intervention, there is a need to have more efficient approaches to enhance the drug discovery process. In this context, combinatorial approaches and high throughput screening technologies have provided powerful tools in the development of ligands that can be useful not only as potential therapeutic agents but also as probes for studying the newly identified proteins. In these approaches, it is often very important to choose appropriate templates for library design since certain types of molecular frameworks are

16 found more frequently associated with high biological activity than others. The term

“privileged structures” was coined by Evans et al. [38] to describe such molecular

frameworks that confer biological activities to a diverse collection of pharmacological

targets. The benzodiazepine ring system, for instance, is one of the most well known privileged structures and exerts a wide variety of biological activities depending on functional groups around the system (Figure 1.6) [39].

The main idea behind the privileged structure approach is that specificity for a particular target can be modulated by substitution patterns on the privileged structure. Given this rationale, our laboratory has been interested in 4H-1-benzopyran-4-one scaffold as a promising privileged structure. The ring system is present in a number of natural products termed flavonoids, which have demonstrated numerous biological activities of pharmacological importance by interacting with various proteins (Figure 1.7).

Figure 1.7: 4H-1-Benzopyran-4-one nucleus as a privileged structure and examples of its biologically active derivatives.

17 In this study, we have paid special attention to isoflavone (3-phenyl-4H-1-benzopyran-4-

one) scaffold for the library design, which will be discussed in detail in Chapter 2. It was

also envisioned that a reliable synthetic route for libraries of structurally diverse

isoflavones would be a powerful tool to identify novel molecules that can selectively

modulate various molecular targets in breast cancer biology. The detailed processes in

development of synthetic routes will be described in chapter 3.

1.9 REFERENCES

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21 CHAPTER 2

ISOFLAVONE LIBRARY DESIGN

2.1 FLAVONOIDS

Flavonoids are a diverse group of plant-derived chemicals that are produced by various

higher plants [40], which can therefore be found in numerous food sources such as fruits,

vegetables, whole grains, and tea leaves [41]. To date, over 8,000 individual compounds

in this class have been identified [42]. Many of these natural products are believed to be

involved in significant biological processes in plants such as plant growth, development,

and defense of plants against microorganisms and pests by exerting a wide range of

biochemical and physiological activities toward other life forms such as viruses, fungi,

bacteria, and insects [43]. Due to their exceptional variety, wide distribution, and

numerous interesting properties, flavonoids have attracted a great deal of attention from scientists in many areas such as chemistry, genetics, taxonomy, and biology. Therefore, literature dealing with flavonoids on their chemistry, biochemistry, and other related aspects is seemingly countless.

Figure 2.1: Basic structures of flavonoids.

23 2.1.1 STRUCTURES OF FLAVONOIDS

Although flavonoids comprise a huge number of individual compounds, they share only a limited number of basic structures (Figure 2.1). All the flavonoids are based on the C15

skeleton consisting of two benzene rings joined by a linear three-carbon chain (C6-C3-C6

system). The chroman ring is the basic scaffold of flavonoid compounds, which

comprises a pyran ring fused with a benzene ring that are labeled as ring A and C,

respectively. Various subgroups of flavonoids are classified according to the substitution

patterns and oxidation state of the ring C. The position of ring B, a second aromatic ring,

is important to classify the compounds into two major subgroups: flavonoids (2-phenyl

substituted) and isoflavonoids (3-phenyl substituted). Thus, the chromans containing a phenyl group at the 2-position are called flavans, and those with 3-phenyl group are termed isoflavans. Oxidation state of the ring C is also another important factor in the classification. For example, flavans containing an oxo group at the 4-position on the ring

C are assigned as flavanones, and those containing a hydroxyl group at the 3-position is called flavanols. Flavanones containing a double bond between C2-C3 are classified as flavones, 3-hydroxy analogs of which are thus called flavonols. The flavonoids possessing two conjugated double bonds in the C-ring belong to a well-known group of anthocyanidins, which are natural pigments responsible for various colors in plants. In a few cases, the six-membered heterocyclic ring C occurs in an isomeric open form resulting in chalcones or is replaced by a five-membered ring resulting in aurones. There are several subgroups that are structurally more complicated in this class, including dimerized (biflavones) or cyclized flavonoids (e.g. coumarins), structures of which are

24 not shown in Figure 2.1. With these basic structures, various substitution combinations

of multiple hydroxyl and methoxy groups give rise to the enormous number of individual

compounds in each subgroup.

2.1.2 BIOLOGICAL ACTIVITIES OF FLAVONOIDS

A wide variety of physiological and biochemical activities of flavonoids have been discovered in living systems including most plants and animals since the beneficial action of citrus flavonoids on capillary function was revealed by Rusznyák and Szent-

Györgyi in 1936 [44]. The antioxidant activity is one of the fundamental properties of flavonoids, which protects the plants from UV radiation and free radicals formed during photosynthesis. Indeed, flavonoids are shown to have the ability of scavenging hydroxy radicals, superoxide anion radicals, and lipid peroxyradicals, which may be useful for prevention of diseases associated with an oxidative damage of membranes, proteins and

DNA [42]. In addition, compounds in this class have demonstrated a variety of other important biological activities, including antiviral [ 45 ], antiinflammatory [ 46 ], antibacterial [47], antifungal [48], and anticancer activities [49].

2.2 ISOFLAVONES

Hormonal activity is another fundamental property of flavonoids; especially many of the

compounds in this class have been shown to exert estrogenic and/or antiestrogenic

activities [ 50 ]. For this reason, flavonoids, along with several other classes of

25 compounds such as stilbenes and mycotoxins, are often called phytoestrogens, which refer to naturally occurring plant-derived chemicals that elicit estrogenic or antiestrogenic activity. Among the various phytoestrogens known, special interest has been drawn to isoflavones, 3-phenyl-4H-1-benzopyran-4-ones, although only a few compounds in this class have been shown to elicit estrogenic activity. In addition, like other flavonoid compounds, isoflavones have demonstrated a variety of biological activities as discussed in the following sections. Examples of such isoflavones include genistein (GEN), daidzein (DAI), biochanin A (BCA), and formononetin (FOR) (Figure

2.2). These isoflavones are present in a significant amount in plants such as beans, cabbage, sprouts, spinach, grains and legumes. GEN and DAI are found most abundantly in soybeans, whereas BCA and FOR are rich in animal feeds such as clover or alfalfa. These isoflavones are therefore believed to contribute to the many beneficial effects of soy products on human health. Indeed, it has been proposed that these isoflavones, especially GEN, may have protective effects against development of hormone-dependent cancers. Some researchers suggest that these protective effects may be responsible for the relatively low incidence of such diseases in certain regions with high consumption of soy foods [51]. Given their close relevance to estrogens both functionally and structurally, it is not surprising that great interest has been paid to isoflavones for their potential role in many estrogen-dependent diseases including breast cancer.

Figure 2.2: Examples of simple natural isoflavones.

2.2.1 GENISTEIN

Genistein (GEN), alternatively known as 4’,5,7-trihydroxyisoflavone or 5,7-dihydroxy-

3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, is one of the simplest flavonoids and a key intermediate in the biosynthesis of other complex flavonoids. It is probably the most extensively studied flavonoid on which over 3,600 papers have been published during the last decade [52]. GEN displays structural similarities to endogenous estrogens; two phenolic groups separated by an approximately 11-12 Å planar core [53]. Thus, it is not surprising that GEN displays moderate binding affinities to estrogen receptors [54].

GEN has been shown to inhibit proliferation of various human breast cancer cell lines by several mechanisms of action [55, 56]. However, it appears to maintain many beneficial effects of endogenous estrogens on several tissues such as bone [ 57 , 58 ] and cardiovascular system [59].

In addition to estrogenic activities, GEN has also demonstrated a variety of other interesting biological activities. It exerts antioxidant activity and is a potent scavenger of hydrogen peroxide [60]. In addition, GEN has shown inhibitory abilities against various

27 enzymes involved in tumor development and growth such as protein tyrosine kinases

[61], DNA topoisomerases [62], and protein kinase C (PKC) [63]. It has also shown to

inhibit angiogenesis [64] and to induce apoptosis and cell cycle arrest in the G2-phase

[65].

2.2.2 DAIDZEIN

Daidzein (DAI), also known as 4',7-dihydroxyisoflavone or 7-hydroxy-3-(4-

hydroxyphenyl)-4H-1-benzopyran-4-one, is the second most abundant isoflavone in

soybeans and soy foods following GEN. DAI shares most of GEN’s activities including

estrogenic effects [66], stimulation activity on bone formation and mineralization in vitro

[58], and inhibitory activity against the growth of breast cancer cells [56]. One of the

most interesting non-estrogenic activities of DAI is its ability to suppress ethanol intake

that may be useful for the treatment of alcohol-dependent patients [67]. A recent study

showed that daidzin, the major delivery form of DAI, inhibits serotonin and dopamine

metabolism in isolated mitochondria, which is believed to be one of the mechanisms by

which DAI suppresses ethanol intake [68].

2.2.3 BIOCHANIN A

Biochanin A (BCA), also known as 5,7-dihydroxy-4'-methoxyisoflavone or 5,7-

dihydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one, is the 4'-methyl ether of

genistein. BCA has shown estrogenic activity [66] and antiproliferative activity against

28 human breast cancer cells [56]; however, these activities are much weaker than those observed from GEN presumably because its 4’-hydroxyl group is masked. However,

BCA exhibits about 100 times more potent activity in inhibiting aromatase than that of

GEN [69]. Other notable activities of BCA include anticarcinogenic [70], hypolipidemic

[71], and antitumor activities [72].

2.2.4 OTHER NATURAL ISOFLAVONES

In addition to these simple isoflavones, a number of “complex” isoflavones have also been shown to possess numerous biological activities. For example, prenylated isoflavones and their cyclized derivatives are found in many plants such as Lupin, and the prenylation is known to increase the antifungal activity of isoflavones [73]. While hormonal activities from these compounds are not very common, some cyclized isoflavones such as coumestrol have also been known to exhibit estrogenic activities [66].

2.2.5 SYNTHETIC ISOFLAVONES

Despite a number of interesting and promising biological activities of natural isoflavones, there have been only a few research efforts for novel synthetic isoflavones as potential therapeutic agents. MD 831 (3,9,11-trihydroxy-5,6-dihydrobenzo[a]xanthone), for example, is a GEN derivative with a conformationally confined B-ring (Figure 2.3), and has been shown to inhibit the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) as efficiently as GEN [74]. Among those published

29 synthetic isoflavones, however, the most notable is ipriflavone (7-isopropoxy-3-phenyl-

4H-1-benzopyran-4-one), which is a 7-isopropyl ether analog of DAI (Figure 2.3). This

drug has been reported to prevent bone loss predominantly by inhibiting bone resorption

[75] and is currently used in the clinic for the prevention and treatment of osteoporosis in

some countries. Furthermore, ipriflavone itself has been a lead for the development of

new synthetic isoflavones with higher potency in inhibiting bone resorption [76].

Figure 2.3: Examples of synthetic isoflavones.

2.3 ISOFLAVONE SYNTHESIS

Due to their extreme diversity in structures and biological activities, synthesis of natural

flavonoids has been important as a traditional tool for structural elucidation of natural analogs as well as for the development of new drugs with more desired therapeutic profiles. Accordingly, a number of synthetic routes for isoflavones have been previously developed.

30 The deoxybenzoin (phenyl benzyl ketone) approach is the most frequently employed

method, which involves the condensation of 2-hydroxydeoxybenzoins with a reagent

containing an activated one-carbon unit for C-2 of the isoflavone scaffold leading to

isoflavones containing no substituents at the 2-position (Figure 2.4). Various reagents

have been used to provide the necessary one carbon fragment under a variety of reaction

conditions including N,N-dimethylformamide (DMF) followed by mesyl chloride

(MsCl) [77], acetic formic anhydride [78], N,N-dimethylformamide dimethyl acetal in refluxing DMF [ 79 ] or in THF with a microwave-assist [ 80 ], and N,N- dimethyl(chloromethylene)ammonium chloride in the presence of boron trifluoride diethyl etherate [81]. A direct, one-pot procedure for various polyhydroxyisoflavones from unprotected phenols and arylacetic acids was recently described using boron trifluoride diethyl etherate followed by DMF and MsCl (Figure 2.5) [82].

Figure 2.4: Deoxybenzoin approach using various one-carbon unit agents.

Figure 2.5: Direct synthesis of isoflavones from phenols and arylacetic acids.

Figure 2.6: Synthesis of 2-alkylisoflavones by Baker-Venkataraman rearrangement followed by intramolecular condensation.

Figure 2.7: Synthesis of 2-alkylisoflavones using amide acetals.

2-Alkylisoflavones are traditionally synthesized by intramolecular condensation of α- acyldeoxybenzoins that are usually obtained by Baker-Venkataraman rearrangement of deoxybenzoins esters (Figure 2.6) [83]. This approach usually requires drastic reaction conditions such as long reaction time or high temperature under acidic or basic conditions. Recently, a mild synthetic method has been reported involving reaction of deoxybenzoins with amide acetals, which was also demonstrated in solid-phase chemistry (Figure 2.7) [84].

Another major approach to prepare isoflavones is the oxidative rearrangement of 2’- hydroxychalcones using thallium(III) nitrate trihydrate (TTN) (Figure 2.8) [85]. A recent synthetic strategy involves Suzuki palladium(0)-catalyzed cross coupling of 3-

32 halochromone with aryl boronic acids (Figure 2.9) [ 86 ], which appears to be not

appropriate for the synthesis of 2-substituted isoflavones due to steric hindrance caused

by the 2-substitutent. Other less general approaches to the synthesis of isoflavones

include rearrangement and cyclization of chalcone epoxides using borontrifluoride

diethyl etherate (Figure 2.10) [87], and rearrangement of flavanones using iodobenzene

in the presence of methanesulfonic acid (MsOH) or p-toluenesulfonic acid (TsOH)

(Figure 2.11) [88].

Figure 2.8: Synthesis of isoflavones by oxidative rearrangement of 2’-hydroxychalcones.

Figure 2.9: Synthesis of isoflavones by Suzuki cross coupling of 3-halochromone with aryl boronic acids using palladium(0) catalysts.

Figure 2.10: Synthesis of isoflavones by rearrangement and cyclization of chalcone epoxides using borontrifluoride diethyl etherate.

Figure 2.11: Synthesis of isoflavones by rearrangement of flavanones using iodobenzene.

2.4 ISOFLAVONE AS A PRIVILEGED STRUCTURE

As described thus far, flavonoids have been shown to exhibit a variety of pharmacologically important biological activities. Among many subcategories of flavonoids, special attention should be paid to isoflavones with regard to their estrogenic and antiestrogenic activities. Furthermore, several natural isoflavones have demonstrated numerous biological activities that may potentially be useful for the treatment of hormone-dependent breast cancer. Given their versatile abilities in interacting with a variety of proteins of pharmacological importance in cancer biology, we envisioned that isoflavone ring system is a promising “privileged structure,” especially for the hormone-dependent breast cancer. It was therefore our hypothesis that chemical modification of functionalities on the isoflavone nucleus would provide novel isoflavone derivatives that would specifically interact with various target proteins in breast cancer cells, thereby allowing us to harvest new leads for the development of highly potent and selective agents for the treatment of breast cancer.

34 2.5 ISOFLAVONE LIBRARY DESIGN

As an initial step to determine the feasibility of our hypothesis, we designed an isoflavone library focused on being amenable to solid-phase synthesis. The isoflavone nucleus is an appropriate scaffold for solid-phase combinatorial synthesis since it represents a fairly rigid molecular framework and contains multiple sites for potential diversity elements. Although the isoflavone nucleus offers many potential sites for elaboration and diversification, special attention in the library design was given to the following positions.

2.5.1 7-POSITION

Almost all natural isoflavones possess a 7-hydroxy group in their structures, which seems to be essential for their estrogenic and antiestrogenic activity. For the library design, we therefore decided to retain this 7-hydroxyl group and considered this moiety as a potential site to anchor the isoflavone scaffold to solid supports. The benzyl group was chosen as a surrogate for hydroxymethyl polystyrene resin (Wang resin) because of its structural and chemical similarities to the resin. It was also anticipated that it would provide a good strategy to prepare 7-hydroxy analogs by selective debenzylation with other alkoxy groups (e.g. methoxy group) remaining intact on the ring system. The methoxy group was also chosen as a variation at the 7-position in order to investigate any possible role of a free hydroxyl group at this position.

35 2.5.2 4’-POSITION

A substituent, a hydroxyl group in most cases, at the 4’-position also appears to be one of

the common structural features in many biologically active isoflavones. Accordingly, we

were interested in the isoflavones possessing a 4’-hydroxyl group, but also we were

interested in those containing no or masked 4’-hydroxyl group to investigate any

possible roles of the 4’-subtituent on biological activities. For the initial study, several

variations were chosen for the 4’-position such as hydroxyl, methoxy, and methyl group as well as no substituent (proton).

2.5.3 2-POSITION

Among all the possible and potential sites around the isoflavone nucleus, the 2-postion is

expected to be an ideal site for the introduction of major diversity in the library. Most natural isoflavones possess no substituent but proton or rarely a hydroxyl group at this position. In addition, this position is chemically reactive since it is a conjugate acceptor

(Michael acceptor); therefore, a substituent should be easily introduced as a nucleophile via 1,4-addition (Michael addition), which can be also further modulated if necessary.

Therefore it was envisioned that this potential reactivity at the 2-position of the isoflavone scaffold would provide efficient strategies not only for the diversity-oriented synthesis by using a number of easily available nucleophiles but also for the target- oriented synthesis aiming at a specific activity of interest by introducing appropriate functional groups at this site.

36 2.5.4 OTHER POSITIONS

As previously described, various substitution combinations of multiple hydroxyl and methoxy groups at various sites on the isoflavone nucleus provide the incredibly large number of individual isoflavones. However, in the present study, we decided to focus only on at the 2, 4’, and 7-positions. Substitutions at other sites are relatively less common in the natural isoflavones. More importantly, it was believed that minimizing the introduction of polar functionalities, such as hydroxyl or methoxy group, would be beneficial to maintain a certain level of lipophilicity of the library. Therefore, chemical modulations at other positions were not pursued in this study.

Figure 2.12: Library of 2-substituted isoflavones.

2.5.5 2,4’,7-TRISUBSTITUTED ISOFLAVONE LIBRARY

Shown in Figure 2.12 is the target library of 2,4’,7-trisubstituted isoflavones, in which special focus is placed on the diversity of the substituents at the 2-position. Potential substituents for the 2-position may include all the nucleophiles that can carry out the

37 conjugate addition to a Michael acceptor such as enolates, imines, enol ethers, and

organo cuprates for 2-alkyl substituents. However, we initially focused on

heteronucleophiles such as oxygen, sulfur, and nitrogen nucleophiles, which are easily

accessible or commercially available.

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42 CHAPTER 3

CHEMISTRY DEVELOPMENT

3.1 NEW SYNTHETIC METHODS

There are a number of methods known for the synthesis of isoflavones; however, they

are not ideally suited for the preparation of our library. These previously developed

methods usually require harsh reaction conditions, such as high temperatures, highly

acidic conditions, and long reaction times, which are not amenable to solid-phase

synthesis. Furthermore, most of them only aim for the generation of simple isoflavones

that contain no substituent at the 2-position. Even the known methods for the 2-

substituted isoflavones are not quite suitable for the synthesis of our library since their

reaction conditions are often too drastic to apply to the solid-phase synthesis. There are

several milder reactions reported, which are known to be amenable to solid-phase

synthesis. However, these methods still have some limitations. Suzuki coupling [86],

for example, showed limitation in its reaction scope for the synthesis of 2-substituted isoflavones. In addition, the recently developed method using amide acetals [84] also suffers from some limitations such as limited availability of the amide acetal reagents as the 2-substituent unit and its unproven reaction scope for more diverse substituents.

43 For these reasons, there was an obvious need to develop alternative synthetic methods

for the preparation of our isoflavone library. Our initial study therefore focused on the development of an efficient synthetic route that can be accomplished under mild reaction conditions, allow for flexibility in substitutions at the 2-position, and provide for moderate to high yields. Such methods would conceivably be suitable for adaptation to

the generation of synthetic combinatorial libraries and be a useful tool to identify novel

molecules that can selectively modulate various molecular targets in breast cancer

biology.

3.2 SYNTHETIC STRATEGY

A retrosynthetic analysis for the preparation of 2-substituted isoflavones is shown in

Figure 3.1. As previously described, since the 2-postion of isoflavone is a conjugate

acceptor, various nucleophiles (Nu) can be introduced by conjugate addition. However,

it was envisioned that the presence of a leaving group (LG) at the 2-position (3B) would

be beneficial in order to retain the conjugated double bond between C-2 and C-3 after

introduction of the substituent by subsequent elimination of the leaving group. In

addition, disconnection of the bond between O-1 and C-2 of the intermediate 3B leads to

an α,β-unsaturated ketone intermediate 3C whose β-carbon is highly functionalized with two leaving groups. In other words, the intermediate 3C would undergo an intramolecular 1,4-conjugate addition of 2’-alkoxy group to the β-carbon followed by

removal of one of the leaving groups furnishing the C-ring of the isoflavone ring.

Figure 3.1: Retrosynthetic analysis.

Figure 3.2: An application of α-oxoketene dithioacetals as key intermediates for the synthesis of 2,6-substituted pyrimidines.

The intermediate 3C is reminiscent of a versatile functionality in organic synthesis, α- oxoketene dithioacetals. Shown in Figure 3.2 is a synthetic approach that employed a similar strategy for the synthesis of 6-substituted pyrimidines [89]. In this study, the ureas containing α-oxoketene dithioacetal functionality, prepared from simple aliphatic esters, were used as key intermediates for the construction of a pyrimidine ring via an intramolecular 1,4-conjugate addition-elimination pathway. Furthermore, it was

45 demonstrated that the resulting 6-methylthio group, upon oxidation to a methylsulfonyl group, could serve as a leaving group to introduce various substituents via an intermolecular 1,4-conjugate addition-elimination reaction. We speculated that this strategy would be suitable for the synthesis of our 2-substituted isoflavone library. The retrosynthesis using α-oxoketene dithioacetal intermediates is outlined in Figure 3.3, in which 2’-hydroxydeoxybenzoins would be appropriate starting materials.

Figure 3.3: Retrosynthesis.

3.3 α-OXOKETENE DITHIOACETALS

α-Oxoketene dithioacetals are highly functionalized α,β-unsaturated carbonyl compounds that can undergo a variety of transformations and have demonstrated their utilities as an extremely versatile three-carbon synthon in organic synthesis [90]. The α- oxoketene dithioacetal functionality can be considered as a β-keto ester with its ester group protected as a ketene dithioacetal or as an α,β-unsaturated ketone with a highly

46 functionalized β-carbon atom. The versatility of α-oxoketene dithioacetals stems from their intrinsic reactivity as an electrophile, thereby offering considerable potential for new carbon-carbon or carbon-heteroatom bond formations. In such transformations, two main reactions are typically involved: 1,2-addition of a nucleophile to the carbonyl group or 1,4-conjugate addition of a nucleophile followed by elimination of an alkylthio group

(Figure 3.4). The resulting allylic alcohols or enones are, in turn, often further manipulated for the synthesis of complex molecules. Based on the α-oxoketene dithioacetal chemistry, a variety of benzene derivatives and heterocyclic compounds, such as pyridines, pyrazoles, pyrimidines, thiophenes, 3-thione-1,2-dithiols, isoxazoles, furans, and lactones, have been prepared as briefly described in Figure 3.5.

Figure 3.4: Two principle reactions of α-oxoketene dithioacetals.

In addition, shown in Figure 3.6 is an interesting example in which a number of such transformations have been collectively demonstrated on the D-ring of estrone. In this study, a variety of novel estrone derivatives containing an additional cyclic system on the

D-ring, which may possess interesting biological activities, were synthesized via the α- oxoketene dithioacetal chemistry [91].

Figure 3.5: Synthetic utility of α-oxoketene dithioacetals.

Figure 3.6: Synthesis of D-ring functionalized and annulated estrone derivatives via α- oxoketene dithioacetal.

Figure 3.7: One-pot synthesis of α-oxoketene dithioacetals.

α-Oxoketene dithioacetals were prepared in a stepwise manner in early syntheses, usually via β-oxo dithiolic acids, until it was found that they could be synthesized directly from ketones, thereby avoiding the usually unstable β-oxo dithiolic acid

intermediates [92]. They are now typically prepared in a one-pot procedure from corresponding ketones by condensation with carbon disulfide (CS2), followed by

49 alkylation of the intermediate dithiolate species (Figure 3.7). There have been many

different reaction conditions known, most of which only differ in the bases employed.

Various bases have been utilized to generate enolates from ketones including sodium or sodium t-amylate [92], 2,6-di-tert-butyl-4-methylphenoxide [93], tert-butoxide [94], and sodium hydride (NaH) [95]. The use of strong but non-nucleophilic bases such as lithium diisopropylamide (LDA), lithium dicyclohexylamide (LCA), and lithium hexamethyldisilazide (LHMDS) has been also reported, which has opened the way for the synthesis of α-oxoketene dithioacetals from various enolates such as α,β-unsaturated enones, esters , α,β-unsaturated esters, lactones, hydrazones, and nitriles [96].

3.4 SYNTHESIS OF 2’-HYDROXYDEOXYBENZOINS

A coupling reaction of organometallic compounds and acyl halides may be the most

fundamental method for the synthesis of ketones. However, this strategy would not be

proper for the synthesis of 2’-hydroxydeoxybenzoins since it would require relatively too many synthetic steps, e.g. protection and deprotection of the 2-hydroxyl group of the starting benzoyl chloride. A reductive coupling of acyl chlorides and benzyl bromides

by means of zinc and a palladium catalyst has been reported; unfortunately, its reaction

scope is still quite limited [97]. The Houben-Hoesch reaction has been a traditional

method for the synthesis of phenolic deoxybenzoins, in which phenols are acylated with

arylacetonitriles in the presence of zinc chloride [98]. However, low synthetic yields and

time- and labor-consuming processes have been the major disadvantages of this method.

Figure 3.8: Synthesis of deoxybenzoins using boron trifluoride diethyl etherate (BF3·OEt2).

A study showed that phenolic deoxybenzoins can be prepared from phenols and

arylacetic acids in the presence of boron trifluoride in high yields [99]. This method has

been employed in direct conversions of phenols and arylacetic acids into

polyhydroxyisoflavones [100]. We utilized this method to prepare the necessary starting

deoxybenzoins since this procedure appears to provide many advantages over other

methods, such as high yields, short reaction times, and easiness to perform. As a result,

the deoxybenzoins 3a−c were obtained in high yields from resorcinol (1) and

corresponding arylacetic acids (2a−c) using boron trifluoride diethyl etherate (BF3·OEt2) as a solvent (Figure 3.8).

3.5 CONSTRUCTION OF ISOFLAVONE SCAFFOLD

3.5.1 INITIAL STUDY

We investigated the feasibility of the α-oxoketene dithioacetal functionality for the

construction of the isoflavone scaffold using deoxybenzoin 3c as a starting material.

First, in order to prevent any possible undesired reactions in the next synthetic step, two

51 phenolic hydroxyl groups of deoxybenzoin 3c were masked with tert-butyldimethylsilyl

(TBS) group. The mixture of 3c and tert-butyldimethylsilyl chloride (TBSCl) in dichloromethane (CH2Cl2) in the presence of triethylamine (Et3N) was heated at reflux overnight affording the desired 2’,4’-bisTBS deoxybenzoin 5a in a quantitative yield

(Figure 3.9). When the reaction was carried out at room temperature, much longer reaction time was required (up to 5 days depending on the reaction scale).

Figure 3.9: Protection of hydroxyl groups with tert-butyldimethylsilyl (TBS) group.

In this reaction, it was observed that the silylation of 4’-hydroxyl group seemed to be much faster than that of 2’-hydroxyl group. Therefore, when 3c was treated with 1.0 equivalent of TBSCl at 0 ºC, 4’-monoTBS deoxybenzoin 4a was obtained as an exclusive product within 5 minutes in 94% yield. Participation of the 2’-hydroxyl group in a hydrogen bond with the carbonyl oxygen may explain its low reactivity.

Figure 3.10: Synthesis of α-oxoketene dithioacetal using lithium diisopropylamide (LDA) as a base.

Treatment of 5a with LDA followed by CS2 and then iodomethane gave the desired α- oxoketene dithioacetal 6a (Figure 3.10). Unfortunately, this reaction suffered from low and inconsistent synthetic yields, which varied from poor to moderate (20~52%) depending mostly on reaction temperatures. Further optimization attempts to improve synthetic yields, however, were not made at this point.

It was anticipated that, upon removal of silyl groups of 6a, a ring closure reaction would simultaneously occur, resulting in the complete isoflavone skeleton by an intramolecular

1,4-conjugate addition of the oxygen anion at the 2’-position and release of a methylthio group (Figure 3.11). When compound 6a was treated with tetrabutylammonium fluoride

(TBAF) at 0 oC, all the starting material disappeared within 5 minutes and a more polar compound was observed as a major product, with a trace amount of a minor one, as monitored by thin layer chromatography (TLC). Even after allowing the reaction to run overnight at room temperature, there was no significant change on the TLC pattern.

However, the 1H-NMR spectrum of the crude products after typical work-up indicated

53 that the major compound was not the desired product. Instead, it appeared that a dihydroxy intermediate 7’ was a major product and the desired compound 7 was

observed as a minor product (the ratio was about 1/4 based on the integration). This

result suggests that deprotection of the TBS group and the cyclization reaction did not

occur simultaneously and that more energy may be required for the latter reaction to

occur. Indeed, when we used higher temperature (75 ºC), it was observed that all the

intermediate 7’ disappeared within an hour, and the cyclized compound 7 was obtained

as a sole product in 73% yield.

Figure 3.11: Expected (I, concerted) and observed (II, stepwise) mechanisms of cyclization reaction.

54 3.5.2 APPLICATION TO 4’-ALKOXY-2-HYDROXYDEOXYBENZOINS

In the initial study, it was demonstrated that α-oxoketene dithioacetals could be efficient intermediates for the construction of the isoflavone scaffold containing a highly functionalized C-2 carbon. Although 7-methoxy- or 7-benzyloxyisoflavones can be easily prepared by alkylating the 7-hydroxyl group of compound 7, it was decided to

carry out the alkylation on the starting deoxybenzoins. Benzylation of starting material

would be more beneficial than benzylation of an intermediate because the benzyl group

in this synthesis can be considered as a surrogate for the solid support and it is ideal to

anchor a template to the solid support in the very early stage. Furthermore, this approach

would also allow us to use a reduced amount of the expensive protecting reagent

(TBSCl).

As previously described, the 2’- and 4’-hydroxyl groups exhibit a considerable

difference in their reactivity; therefore, selective alkylations of 4’-hydroxyl group of

2’,4’-dihydroxydeoxybenzoins 3a−c were easily carried out. 4’-Alkoxy-2’-

hydroxydeoxybenzoins 4b−f were prepared in a Mitsunobu reaction condition [101]

using corresponding alcohols (methanol or benzyl alcohol), triphenylphosphine (Ph3P), and diisopropyl azodicarboxylate (DIAD) (Figure 3.12). This method was so efficient that all the reactions were completed within 5 minutes at 0 ºC and the yields were excellent. Furthermore, this method is expected to be a suitable protocol for the attachment of deoxybenzoin templates to a solid support such as Wang resin.

Figure 3.12: Selective alkylation of 4’-hydroxyl group using a Mitsunobu condition.

Compound 4f, which contains a benzyl group as a surrogate for a solid support, was

chosen for the further study (Figure 3.13). The 2-hydroxyl group of 4f was protected with 1.2 equivalents of TBSCl under the same reaction conditions as previously described, affording deoxybenzoin 5b in a quantitative yield. For the preparation of α- oxoketene dithioacetals from 5b, we searched for an alternative base since LDA provided inconsistent yields in the initial study. Our literature survey revealed that sodium hydride usually affords good yields for aromatic ketones [102], whereas it is not an appropriate base for aliphatic ketones or esters due to the undesired Claisen condensation of products [95]. Since our substrate 5b was as an aromatic ketone, we anticipated that sodium hydride would be a proper choice of base. Indeed, treatment of

5b with sodium hydride in tetrahydrofuran (THF) followed by sequential addition of carbon disulfide and iodomethane offered α-oxoketene dithioacetal 6b in a very high

yield (93%).

Figure 3.13: Synthesis of 2-(alkylthio)isoflavones via α-oxoketene dithioacetals.

In addition to iodomethane, we investigated the reaction scope using several different

alkyl halides such as allyl bromide, benzyl bromide, and 4-nitrophenethyl bromide.

Surprisingly, those bulky alkyl groups were well tolerated in this reaction, providing

corresponding α-oxoketene dithioacetals 6c−e in good yields. However, it should be noted that alkylation using 4-nitrophenethyl bromide, a relatively less reactive agent,

required much longer reaction time to be completed (more than two days) compared with

others (within a day), and its synthetic yield was relatively low.

Finally, treatment of compounds 6b−e with TBAF gave the cyclized products 8e and

9a−c. Another interesting observation was significant difference between compound 6a

and 6b−e in their reaction rate. As discussed in the previous section, cyclization of

57 bisTBS compound 6a appeared to proceed in a stepwise manner, requiring at least 30

minutes at a high temperature. On the other hand, the cyclization reactions of 4’-alkoxy analogs 6b−e were considerably faster under the same reaction conditions compared with

6a. All the reactions of 6b−e seemed to be completed as soon as TBAF was added at

0 ºC and no intermediate was detected on TLC.

Extensive kinetic studies may be required to elucidate the exact mechanisms for these different reaction profiles. However, aware that the only difference between compounds

6a and 6b−e is their protecting group at the 4’-position, silyl group versus alkyl group, their different behaviors in the same reaction conditions might be explained by any possible role of these protecting groups.

In case of compounds 6b−e, the mechanism of cyclization appears to be straightforward.

After the TBS protecting group is removed by fluoride anion, the resulting phenoxide species would have two major resonance contributors, A and B (Figure 3.14). It is obvious that resonance B cannot undergo the necessary 1,4-conjugate addition since the

formation of enolate disrupts both the reactivity of 2’-oxygen as a nucleophile and the

electrophilicity of β-carbon as a conjugate acceptor. In contrast, the 2’-alkoxide in state

A would undergo a typical 1,4-conjugate addition to the β-carbon followed by sequential

elimination of one of alkylthio groups, resulting in the cyclized compounds.

Furthermore, this reaction would drive the equilibrium in favor of the A form until all the

intermediates are depleted.

Figure 3.14: Plausible explanation on the fast cyclization of compounds 6b−e.

Figure 3.15: Plausible explanation on the slow cyclization of compound 6a.

59 In case of compound 6a, treatment with TBAF would produce a diphenoxide species that

might have three major resonance contributors designated as C, D and E in Figure 3.15.

Resonance C would not undergo the 1,4-conjugate addition for the same reason

described for resonance B. In the state of resonance D, despite the nucleophilicity on the

2’-oxygen, its β-carbon still suffers from the lack of electrophilicity as a conjugate

acceptor. Therefore, resonance E is the only form that can undergo the necessary 1,4-

conjugate addition. However, resonance E is believed to be much less stable than the

others due to an additional anionic species on the phenyl ring; therefore, there would be

much less chance for the intermediate to exist in this form. Consequently, this may

explain why a higher temperature is needed for the cyclization of compound 6a.

3.5.3 A NEW APPROACH: PHASE TRANSFER CATALYSIS PROCEDURE

As part of our continuous efforts to optimize the synthetic route, we investigated the

possibility to avoid the use of the TBS group since it causes several disadvantages such

as high molecular weights and high lipophilicity of intermediates. Instead of searching

for alternative protecting groups, we were interested in xanthates (O,S-dialkyl dithiocarbonates) as potential alternative intermediates. Several years ago a highly efficient one-pot procedure for the synthesis of O-alkyl-S-methyl dithiocarbonates was reported (Figure 3.16) [103]. They demonstrated high-yielding syntheses of O-alkyl-S-

methyl dithiocarbonates from various alcohols using carbon disulfide and iodomethane

in the presence of tetrabutylammonium hydrogensulfate as a phase transfer catalyst.

Figure 3.16: One-pot synthesis of O-alkyl-S-methyl dithiocarbonates (xanthates) using a phase transfer catalyst.

We chose this procedure to prepare O-aryl-S-alkyl dithiocarbonate intermediates from deoxybenzoins. Furthermore, we anticipated that these O-aryl-S-alkyl dithiocarbonate

intermediates might not have to be isolated. The resulting intermediates would undergo

further cyclization reaction in this reaction condition due to the presence of an active

methylene moiety in their structures, thereby directly generating the desired 2-

(alkylthio)isoflavones from deoxybenzoins in a single step (Figure 3.17).

Figure 3.17: O-alkyl-S-methyl dithiocarbonates proposed as potential intermediates for the synthesis of 2-(methylthio)isoflavones.

Although the reactions in the original paper employed carbon disulfide as a solvent as

well as a reagent, we modified the reaction conditions by using THF as a solvent and

reducing the amount of carbon disulfide. As expected, when the deoxybenzoin 4b,

carbon disulfide, and methyl iodide in a THF-water two-phase system were treated with

61 aqueous NaOH solution at room temperature in the presence of 10 mole % of tetrabutylammonium hydrogensulfate (n-Bu4N·HSO4), the desired 2-

(methylthio)isoflavone 8a was obtained in 87% yield after 3 h at room temperature

(Figure 3.18). The reaction apparently occurred in a stepwise manner as monitored by

TLC. The intermediate was isolable in a high yield (>90%) when the reaction was quenched after 5 minutes at 0 ºC. Interestingly, however, the isolated intermediate was not the expected O-alkyl-S-methyl dithiocarbonate but α-oxoketene dithioacetal 6f according to the spectral data (Figure 3.18). It is even more interesting to note that the formation of the intermediate 6f was extremely fast even at a low temperature (5 min, 0

ºC), whereas its cyclization to isoflavone 8a required a much longer time (34 h, room temperature) or higher temperature (1 h, 50 ºC).

Figure 3.18: α-Oxoketene dithioacetal intermediate (6f) in the transformation of 2’- hydroxydeoxybenzoin 4b into 2-(methylthio)isoflavone 8a in a phase transfer catalysis reaction.

62 These interesting results may be explained by the intramolecular hydrogen bond of the

starting deoxybenzoin 4b. In general, phenolic alcohols are more acidic than the active

methylene group in typical benzyl phenyl ketones: the approximate pKa of a typical

phenolic alcohol is approximately 10, whereas that of methylene in a typical benzyl phenyl ketone is about 13. For this reason, it was initially expected that 2’-hydroxyl

group would react with carbon disulfide followed by iodomethane to give a xanthate

intermediate. However, in 2’-hydroxydeoxybenzoins such as 4b, the hydroxyl group at

the ortho-position to the carbonyl group appears to form a hydrogen bond with an

electron pair on carbonyl oxygen, which can be confirmed by the appearance of its signal

relatively down field in the 1H-NMR spectra (~12.7 ppm). This might result in

considerable change in their acidity: the partial protonation of carbonyl oxygen by

hydrogen bonding would result in an increase in electron density on this atom, thereby

weakening the bond between C-2 and its hydrogens. As a result, the pKa of methylene

groups in such compounds should be lower than that of typical benzyl phenyl ketones.

Furthermore, the phenolic hydrogen involved in a hydrogen bond is believed to be harder

to remove than typical phenolic hydrogens: its removal would require more energy since

it would disrupt the hydrogen bond that contributes to the stability of the molecule. In

other words, the pKa of phenolic alcohols in such compounds would be higher than that

of typical ones. Consequently, in 2’-hydroxydeoxybenzoins, the methylene protons

appear to be more acidic than their phenolic proton at the 2’-position, thereby generating

α-oxoketene dithioacetals instead of xanthates as intermediates under the given reaction

conditions. Indeed, the slow progress of the intermediate 6f toward cyclization, more

63 specifically 1,4-conjugate addition of its phenoxide anion, might be attributed to its rigid hydrogen bond.

Similar results were obtained when other starting materials and alkylating agents were used (Figure 3.19). Therefore, this method proved to be a very efficient synthetic tool for the preparation of a number of structurally diverse 2-(alkylthio)isoflavones in a single synthetic step at the ambient reaction conditions from various readily available deoxybenzoins and alkylating agents [104].

Figure 3.19: One-pot synthesis of various 2-(alkylthio)isoflavones via a phase transfer catalysis procedure.

3.5.4 DIRECT SYNTHESIS USING SODIUM HYDRIDE AS A BASE

Given the finding that the 2’-hydroxyl group of deoxybenzoins 4b−f is much less acidic

than their methylene group, it was concluded that protection of 2’-hydroxyl group is not

64 necessary to prepare the α-oxoketene dithioacetal intermediates. This led us to

reevaluate the reaction conditions that had been employed for the preparation of α-

oxoketene dithioacetals (6b−e) from TBS-protected deoxybenzoin (5b) (Figure 3.13). It

was anticipated that the application of starting 4’-alkoxy-2’-hydroxydeoxybenzoins to such reaction conditions would directly provide the 2-(alkylthio)isoflavones. As expected, when deoxybenzoins 4b−f were treated with sodium hydride in DMF followed by sequential addition of carbon disulfide and iodomethane, α-oxoketene dithioacetal

8a−e were obtained in high yields (Figure 3.20). This method, indeed, would offer an alternative route to prepare the 2-(alkylthio)isoflavones and, more importantly, it is expected to be readily amenable to the solid-phase synthesis.

Figure 3.20: One-pot synthesis of various 2-(alkylthio)isoflavones using sodium hydride as a base.

65 3.6 INTRODUCTION OF SUBSTITUENTS

As described thus far, efficient synthetic methods were successfully developed for the preparation of 2-(alkylthio)isoflavones from readily accessible deoxybenzoins through

α-oxoketene dithioacetal intermediates. While a number of structurally varied

isoflavones can be directly generated using these methods, displacement of the alkylthio

group by a variety of nucleophiles would be a useful addition to further increase the

diversity. Therefore, we investigated the potentiality of this strategy using 2-

(methylthio)isoflavones as substrates for nucleophilic substitution.

3.6.1 OXIDATION

As previously mentioned, such displacement reactions would involve 1,4-conjugate

addition-elimination process. Although the methylthio group itself is a good leaving

group, its oxidized forms such as sulfinyl and sulfonyl group have been known to be

more efficient for substitution reactions [105]. While a sulfinyl group may be as

efficient as a sulfonyl group as a leaving group, its synthesis is in general more difficult.

Although one equivalent of an oxidizing agent would give a sulfinyl compound as a

major product, it is usually accompanied by sulfonyl compound and unreacted starting

material, which causes time- and labor-consuming processes for purification. Therefore,

it was speculated that the full oxidation of sulfide to sulfonyl group would offer much more efficient synthetic route.

Figure 3.21: Oxidation of sulfides (8a−e) to sulfones (10a−e) and sulfoxide (10f) using 3-chloroperoxybenzoic acid (mCPBA).

A number of oxidizing agents have been used for the oxidation of sufides to sulfones, among which m-chloroperoxybenzoic acid (mCPBA) is the most frequently employed reagent. Treatment of compounds 8a−e with an excess amount of mCPBA in refluxing

dichloromethane for 2 h gave 2-(methylsulfonyl)isoflavones 10a−e in excellent yields.

When the reactions were carried out at room temperature, a much longer reaction time

was required to complete the transformation (>2 days). In addition, when sulfide 8e was

treated with mCPBA at 0 ºC, sulfoxide 10f was obtained as a major product (78%) along

with small amount of sulfone 10e.

3.6.2 SUBSTITUTION

Substitution of the 2-methylsulfonyl group by nucleophiles was investigated. Treatment of compound 10a with sodium salts of 4-methylbenzenethiol and p-cresol gave desired

2-(arylthio)isoflavone 11a and 2-(arylalkoxy)isoflavone 11b, respectively, in high yields

(Figure 3.22). In addition, treatment of sulfone 10e with a sodium salt of benzenethiol

67 also provided the desired 2-(phenylthio)isoflavone 11f. Several aryl compounds containing an additional functional group, such as an amino or hydroxy group, were also successfully introduced as nucleophiles, affording the corresponding 2-substituted isoflavones 11c−d and 11f−h in excellent yields. This would allow us to further manipulate the introduced substituents by e.g. alkylation or oxidation

Figure 3.22: Displacement reactions of sulfones with various nucleophiles.

3.7 REFERENCES

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95. Shahak, I.; Sasson, Y. Preparation and reactions of α-keto ketene mercaptals. Tetrahedron Lett. 1973, 4207−4210.

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70 CHAPTER 4

AROMATASE INHIBITORS

4.1 AROMATASE INHIBITORS

As the role of endogenous estrogens in the development of hormone-dependent breast cancer has been widely recognized, blocking the biosynthesis of estrogens has been one of the most promising and logical therapeutic strategies for the disease. Among a number of enzymes involved in estrogen biosynthesis, aromatase is a particularly attractive target for inhibition because aromatization is the final, rate-limiting step in estrogen biosynthesis; therefore, its blockade should not interfere with the production of other steroids. For this reason, a great deal of research effort has focused on the development of new aromatase inhibitors [106, 107, 108].

In general, aromatase inhibitors have been used clinically as a second line treatment for hormone-dependent breast cancer, following antiestrogens such as tamoxifen. Although tamoxifen (TAM) is currently the most widely used endocrine therapy for hormone- dependent breast cancer, some concerns remain about its long-term use. For instance, resistance to TAM can eventually develop [109] and it is known to increase the risk of

71 secondary tumors of endometrium [110, 111]. Therefore, aromatase inhibitors could be safer and more effective than antiestrogens in breast cancer treatment and could provide an alternative treatment when antiestrogens become ineffective or intolerable.

Along with the inhibitory potency against aromatase, another critical challenge in developing new aromatase inhibitors is to improve their selectivity for aromatase with respect to other cytochrome P450 enzymes. Fortunately, aromatase has distinct features by which it can be distinguished from other cytochrome P450 enzymes. For example, although there are a number of members in the cytochrome P450 family, aromatase shares only ~30% identical amino acid sequences with other cytochrome P450 enzymes

[112]. Due to its low sequence homology with other cytochrome P450 enzymes, aromatase has been assigned to a separate gene family termed CYP19 [ 113 ].

Accordingly, it has been suggested that it is possible to develop selective inhibitors for aromatase, which would not disturb other steroid biosynthesis in the pathway.

Ever since the aromatase inhibitory activity of the known drug aminoglutethimide (AG) was discovered [114], a number of compounds have been developed using different approaches to inhibit the enzyme [108]. By chronology of clinical trials, these aromatase inhibitors are classified into four generations. For instance, with AG as the first generation of aromatase inhibitors, formestane (4-hydroxyandrostenedione) and fadrozole are assigned to the second generation, and vorozole belongs to the third generation. Recently anastrozole and letrozole have been marketed and generally

72 classified as fourth generation inhibitors. Aromatase inhibitors can be also divided by their modes of action, which is reflected in the changes they cause to the UV absorption spectrum Soret’s band of the heme. Suicide inhibitors, exemestane and formestane, are classified as type I inhibitors, which induce a hypsochromic shift of Soret’s band from about 420 to 390 nm. The type II inhibitors cause a bathochromic shift of Soret’s band originated from the coordination of a heteroatom, such as nitrogen, sulfur, or oxygen, of the inhibitors to the heme iron of the enzyme [112]. However, aromatase inhibitors are typically classified by their chemical structures: steroidal and nonsteroidal compounds.

4.2 STEROIDAL AROMATASE INHIBITORS

Inhibitors in this class are aromatase substrate analogs in which chemical substituents are incorporated at varying positions on the steroid skeleton (Figure 4.1). Thus, these inhibitors bind to the aromatase cytochrome P450 enzyme in the same manner as the substrate androstenedione and usually present high affinities for the enzyme. Among the steroidal inhibitors developed thus far, formestane has been the most selective and effective for the treating breast cancer [115, 116, 117]. This molecule behaves as a suicide inhibitor, which binds irreversibly to the active site of the enzyme [ 118 ].

Formestane also displays prolonged action because estrogen synthesis is blocked until new enzyme is produced. However, androgen effects at high doses and poor oral bioavailability of formestane are its major drawbacks.

Figure 4.1: Androstenedione and selected steroidal aromatase inhibitors.

In addition to formestane, a number of other potent steroidal inhibitors have been identified including exemestane [119], atemestane [120], and 10-propargylestr-4-ene-

3,17-dione (MDL 18,962) [121]. However, only exemestane remains currently available in clinic and is known to have several advantages over formestane including its higher potency and orally availability [122]. Our laboratory has also identified several 7α- substituted androstenedione analogs as potent aromatase inhibitors, e.g. 7α-APTA [123],

7α-APTADD [ 124 ], 7-phenethyl-1,4,6-androstatriene-3,17-diones [ 125 ], and 7α- arylaliphatic androgens [126, 127].

4.3 NONSTEROIDAL AROMATASE INHIBITORS

After AG was found to be effective in inhibition of aromatase, a number of nonsteroidal

inhibitors have been identified (Figure 4.2.) [108]. All these nonsteroidal compounds are

74 reversible inhibitors that compete with the natural substrates, androstenedione and testosterone, for binding to the active site of aromatase. These nonsteroidal inhibitors have several advantages over steroidal inhibitors such as good bioavailability due to their higher degree of hydrophilicity. A brief comparison of nonsteroidal and steroidal aromatase inhibitors is shown in Table 4.1.

Many of these nonsteroidal inhibitors have been derived from the structures of other nonspecific cytochrome P450 enzyme inhibitors, such as antifungal agents, with the aim of improving their selectivity for aromatase. Indeed, the selectivity issue is extremely important in the development of nonsteroidal inhibitors. However, due to the distinct features of aromatase, high selectivities for aromatase have been achieved in many nonsteroidal inhibitors, as will be discussed later in this chapter.

Steroidal Nonsteroidal

Advantages More selective Oral bioavailability

Longer half-life (inactivation Less expected hormonal feature) properties

Disadvantages Poor oral bioavailability in Less selective (inhibition of or general (except exemestane) metabolism by other cytochrome P450 enzymes) Hormonal properties (androgenic effects at high Shorter half-life (daily doses) administration)

Possible metabolism by cytochrome P450 enzymes

Table 4.1: Advantages and disadvantages of the two major classes of aromatase chemical inhibitors [108].

75 Compounds in this class are type II competitive inhibitors and possess a heteroatom that

interferes with hydroxylation of steroids by coordinating with the heme iron of P450arom.

Although several heteroatoms, such as sulfur, oxygen, and nitrogen, are known to show

abilities to bind to heme iron, the majority of compounds in this class possess a nitrogen- containing heterocyclic moiety including imidazole, triazole, pyrimidine, and pyridine.

4.3.1 TRIAZOLE-CONTAINING NONSTEROIDAL AROMATASE INHIBITORS

The triazole ring has been identified as one of the most effective heterocycles in the development of new nonsteroidal aromatase inhibitors. Several triazole compounds such as anastrozole [128], and letrozole [129], have been identified as highly potent and selective aromatase inhibitors (Figure 4.2). Both inhibitors have proven to be well

tolerated by patients due to their favorable therapeutic indices [130,131] and have been

approved recently for treatment of postmenopausal breast cancer. Vorozole is another

newer triazole-containing aromatase inhibitor under investigation [132].

Figure 4.2: Aminoglutethimide and triazole-containing nonsteroidal aromatase inhibitors.

Figure 4.3: Imidazole-containing nonsteroidal aromatase inhibitors.

4.3.2 IMIDAZOLE-CONTAINING NONSTEROIDAL AROMATASE INHIBITORS

The imidazole moiety has proven to be a more effective coordinating functional group in a certain series of inhibitors (Figure 4.3). Fadrozole is a representative imidazole- containing inhibitor and known to be a potent and selective inhibitor [133]. Molecular modeling studies comparing fadrozole with androstenedione suggests that its cyano group aligns with the C-17 ketone of androstenedione, the aryl ring aligns with the C ring, and the saturated piperidine ring has some molecular volume matching the A ring of androstenedione [134]. These structural similarities of fadrozole to androstenedione could allow interactions with the natural binding site of aromatase. Liarozole is an indole derivative possessing an imidazole moiety and has been also shown to exhibit a

77 potent activity in inhibiting aromatase [135]. A number of liarozole analogs bearing the

azolylbenzyl fragment in different positions of the indole nucleus have been synthesized, among which 4A (IC50 = 0.10 µM) [136] and 4B (IC50 = 0.04 µM) [137] have been found to be the most effective compounds. Replacement of indole ring by benzofuran nucleus also results in potent aromatase inhibitors such as compound 4C [138]. In these types of compounds, the imidazole moiety seems to perform better than the triazole moiety. Similar trend has been observed in a series of xanthone derivatives, in which a dramatic loss of activity was observed upon replacement of the imidazole in compound

4D with a triazole [139]. It is interesting to note that several simple para-substituted phenylalkylimidazoles such as compound 4E and 4F exhibit a potent inhibitory activity against aromatase, in which two key functionalities that are critical for binding are separated by a two- or three-carbon unit [ 140 ]. Compound 4G is an imidazole-

containing quinoline derivative that elicits an interesting dual inhibitory activity against

aromatase (IC50 = 0.50 µM) as well as thromboxane A2 synthase (TxA2) (IC50 = 0.50

µM), an enzyme involved in the arachidonic acid metabolism converting prostaglandin

H2 into thromboxane A2 [141]. Since TxA2 is believed to play a key role in tumor

metastasis, such a molecule could be more effective to treat advanced breast cancer.

Tetralone derivative 4H is another example of a potent imidazole-containing aromatase

inhibitor [142].

Figure 4.4: Pyridine-containing nonsteroidal aromatase inhibitors.

4.3.3 PYRIDINE-CONTAINING NONSTEROIDAL AROMATASE INHIBITORS

Another coordinating potential that is often introduced in aromatase inhibitors is the

pyridyl moiety (Figure 4.4). Medicinal chemistry efforts, prompted from the promising

results obtained with (E)-2-(4-pyridylmethylene)-1-tetralone (4I, IC50 = 4.6 µM) [143], have led to identification of many related pyridine-containing aromatase inhibitors.

Compound 4J (IC50 = 0.061 µM), for instance, is a tetralin analog possessing a potent

aromatase inhibitory activity, in which its pyridyl moiety is constrained by a fused

cyclopropane ring [144]. Indanone derivative 4K has also shown similar potency (IC50 =

3.5 µM) to compound 4I [145]. Other examples of pyridine-containing nonsteroidal aromatase inhibitors include indolizinone 4L [146] and its ring contracted analog 4M

[147], a pyrrolizinone, both of which show submicromolar aromatase inhibiting potency.

79 4.3.4 OTHER NONSTEROIDAL AROMATASE INHIBITORS

All the compounds described above possess a heterocyclic ring containing a heme-

coordinating nitrogen atom. However, there are several compounds devoid of nitrogen

atoms that can inhibit aromatase, most of which are natural compounds. One interesting

series of compounds is plant-derived sesquiterpene lactones, among which compound 4N

(Figure 4.5) shows the most potent inhibitory activity (IC50 = 7 µM) and selectivity for

aromatase [148]. This compound has been a new lead for the development of novel

nonsteroidal aromatase inhibitors [149].

Figure 4.5: A sesquiterpene lactone as a selective aromatase inhibitor.

Among the non-nitrogenated aromatase inhibitors, flavonoids may be the best known and the most studied group of compounds (Figure 4.6) [150, 151]. Flavones and flavanones have demonstrated higher aromatase inhibitory activity than any other members of the flavonoids. For example, among the natural flavonoids tested so far, 7- hydroxyflavone (IC50 = 0.21 µM) is the most potent aromatase inhibitor, followed by

80 chrysin (5,7-dihydroxyflavone, IC50 = 0.7 µM) and apigenin (5,7,4’-trihydroxyflavone,

IC50 = 2.9 µM) [152]. It is interesting to note that even nonsubstituted flavone 4O was found to exert a moderate potency (IC50 = 48 µM), but it was much less potent than 7-

hydroxyflavone. Flavanones that have shown potent activity in inhibiting aromatase

include naringenin (5,7,4’-trihydroxyflavanone, IC50 = 9.2 µM) and 7-methoxyflavanone

(IC50 = 2.6 µM) [152].

Figure 4.6: Flavonoids as aromatase inhibitors.

81 Several synthetic flavones and flavanones have been identified as potent aromatase

inhibitors. α-Naphthoflavone (7,8-benzoflavone, ANF) is the first synthetic flavone

found to be highly potent in aromatase inhibition (IC50 = 0.07 µM) [153]. Its 9-hydroxy

derivative (9-OH-ANF) is even more potent (IC50 = 0.02 µM), which is indeed the most potent aromatase inhibitors among all the flavonoids tested to date [154]. Several B-ring modified 7-methoxyflavanone derivatives have been synthesized and shown to exhibit higher inhibitory effects on aromatase activity than their parent compound, among which compound 4P, 3’4’-dihydroxy-7-methoxyflavanone, was found to be the most potent

(IC50 = 2.5 µM) [155]. Compound 4Q is another interesting synthetic flavanone that

contains a pyridine moiety, and is known to be very potent in inhibiting aromatase (IC50

= 0.62 µM) [156]. Other classes of flavonoids including chalcones [157] and isoflavones

[151] have also demonstrated inhibitory activity against aromatase, which is however

much weaker than those observed from flavones or flavanones.

4.4 ISOFLAVONES AS AROMATASE INHIBITORS: INHIBITOR DESIGN

Contrary to flavones and flavanones, only a few isoflavones have been known to elicit

aromatase inhibitory activity. GEN, the most extensively studied flavonoid for its

versatile biological activities, has been reported to exert no [152, 158] or very weak

[151] aromatase inhibition. DAI is also known to exert no effect on aromatase activity

[158]. The most potent isoflavone aromatase inhibitor is BCA, which is the 4’-methyl

ether analog of GEN. Nonetheless, the aromatase inhibitory potency of BCA is not very

82 significant in comparison to flavones or flavanones. BCA’s reported IC50 value is 113

µM [158] and Ki value is 49 [158] or 12 ± 5 µM [151].

Due to their relatively weak aromatase inhibitory activities with respect to flavones or isoflavanones, isoflavones have not been considered as a promising class of compounds

for aromatase inhibitors. Nevertheless, we have perceived that isoflavones might have

considerable potential as aromatase inhibitors. It should be noted that BCA, the 4’-

methyl ether analog of GEN, possesses a significantly higher activity (approximate 10-

fold) than that of GEN (12 ± 5 µM versus 123 ± 8 µM as Ki value) [151]. The higher

activity of BCA with respect to that of GEN indicates that the isoflavone ring can be

tolerated in the active site of aromatase and, more importantly, it is a good example

showing that inhibitory potency can be improved by chemical modification of functional

groups around the ring system.

To the best of our knowledge, there has been no medicinal chemistry effort to utilize the

isoflavone ring as a core scaffold for the development of new aromatase inhibitors.

Therefore, with efficient synthetic methods established for the construction of isoflavone

libraries, our research group has investigated the feasibility of isoflavone analogs as new

nonsteroidal aromatase inhibitors. We hypothesized that the desired degree of inhibitory

activity against aromatase could be achieved by chemical modification of functional groups around the isoflavone nucleus, especially by introducing an appropriate nitrogen- containing heterocycle at the 2-position as a heme-coordinating functionality.

Figure 4.7: Two synthetic approaches to 2-substituted isoflavones targeting aromatase.

We expected that such compounds would be easily prepared via the phase transfer catalysis procedure, which is described in Chapter 3, using a number of commercially available or readily accessible electrophiles that contain a nitrogen-containing heterocycle. In addition, the nucleophilic 1,4-conjugate addition-elimination process would offer an additional option to introduce more diversity using various nucleophiles containing such a heterocycle (Figure 4.7).

The functional variation at other positions on the nucleus remains the same as described in chapter 3, which also seems to be appropriate for the design of aromatase inhibitors.

For instance, the 4-oxo group of flavonoids is known to play a role in binding to aromatase; reduction of this 4-oxo group to a hydroxyl group greatly decreased the inhibitory potency [159]. The presence of a 5-hydroxyl group is known to have little effects on aromatase inhibition; instead, it has been proposed it could interfere with electron-donating ability of the 4-oxo group by forming an intramolecular hydrogen bond. Therefore, we concluded that a 5-hydroxyl group is not necessary. In addition, since the 4’-methoxy group of BCA appears to play a role in binding ability presumably

84 by providing favorable interactions in the active site of the enzyme, we have been

initially interested in a methoxy substituent at the 4’-position (R1 = MeO). However, we

were also interested in other simple substituents such as hydrogen, methyl, and hydroxy

group at this position for a brief structure-activity relationship study. The 7-hydroxyl

group seems to be essential for aromatase inhibition in most flavonoids, but in some

cases the 7-methoxy group has been found to be more effective. Therefore, it would be

also interesting to investigate the effects of substitution pattern at the 7-position,

resulting in several variations such as methoxy, benzyloxy, and hydroxy group at the

position.

4.5 FIRST SET OF INHIBITORS

The first set of inhibitors was designed to introduce a heme-coordinating functionality

using various corresponding electrophiles, e.g. alkyl halides, via the phase transfer

catalysis procedure described in the previous chapter. We were initially interested in the

pyridyl moiety since it has proven to be effective in flavonoid derivatives [156], and

alkyl halides containing such a moiety are easily available. Since we were also

interested in the effects of nitrogen’s position on the activity, we chose 2-, 3-, and 4-

(bromomethyl)pyridine as alkylating agents. Other non-nitrogenated alkyl halides, such as benzyl bromide, were also used to examine the effects of the absence of nitrogen on the activity.

85 4.5.1 SYNTHESIS USING PHASE TRANSFER CATALYSIS PROCEDURE

The phase transfer catalysis procedure proved to also be efficient with pyridine-

containing alkyl halides. Treatment of a mixture of a deoxybenzoin (4b−f), carbon

disulfide, and methyl iodide with aqueous NaOH solution in a THF-water two-phase

system in the presence of 10 mole % of tetrabutylammonium hydrogensulfate (n-

Bu4N·HSO4) gave the desired 2-substituted isoflavones 9a−l in good yields (Figure 4.8).

Figure 4.8: Synthesis of 2-substituted isoflavones as aromatase inhibitors using phase transfer catalysis procedure.

Non-selective dealkylation reactions of selected compounds were carried out under a typical reaction condition using boron tribromide to offer the hydroxy compounds 12a−g

(Figure 4.9). Several analogs showed relatively low synthetic yields; however, no attempts were made to improve the yields at the moment.

Figure 4.9: Deprotection reactions using boron tribromide.

In order to prepare 7-hydroxy-4’-methoxyisoflavone 12h, the benzyl group should be selectively removed leaving the 4’-methoxy group intact. Among a number of methods available for the dealkylation, the most useful and extensively used method for debenzylation might be hydrogenolysis, in which benzyl ethers are reductively cleaved by hydrogen into toluene and corresponding alcohols in the presence of a transition metal catalyst such as palladium (Pd). Nevertheless, we were reluctant to use these methods because of catalyst poisoning that would be likely caused by our compounds: it is well known that substrates containing a thioether often cause inactivation of metal catalyst.

Figure 4.10: Failure in selective debenzylation using hydrogenation in the presence of palladium due to catalyst poisoning.

87 Indeed, when compound 9l was treated under typical reaction conditions for catalytic

hydrogenation (hydrogen gas in the presence of palladium on carbon), only starting

material was recovered. Catalyst transfer hydrogenation using various hydrogen sources,

such as ammonium formate or cyclohexene, in the presence of palladium on carbon also

proved to be unsuccessful. Alternatively, in order to take advantage of the different

reactivity between methoxy and benzyloxy groups, we tried using only one equivalent of

boron tribromide at 0 ºC. However, this method gave only a complex mixture with

unreacted starting material being the major component.

Fortunately, we found that the benzyl group could be selectively removed when treated

with boron trifluoride diethyl etherate and dimethyl sulfide in dichloromethane. This

reaction condition was originally reported as a mild alternative debenzylation method in

order to avoid undesirable 1,4-conjugate addition of ethanethiol to substrates containing

a Michael acceptor [160]. This reaction proved to be mild enough to leave the methoxy

group intact, providing the desired 7-hydroxy-4’-methoxyisoflavone 12h in 84% yield after overnight stirring at room temperature (Figure 4.11).

Figure 4.11: Selective debenzylation using boron trifluoride diethyl ether with dimethyl sulfide.

Figure 4.12: Tritiated water release aromatase assay.

4.5.2 HUMAN PLACENTAL MICROSOME AROMATASE ASSAY

Biological evaluation of synthesized isoflavones 9a−l and 12a−h was performed by John

Hackett to determine their ability to inhibit aromatase activity. AG was used as a

positive control. The aromatase assay was performed according to the modified method

of the procedure previously reported by our laboratory [126], in which human placental

microsomes were used as the aromatase source. In this assay, the aromatase enzyme

catalyzes the conversion of [1β-3H]-androst-4-ene-3,17-dione to estrone and tritiated

3 water ( H2O) in the presence of NADPH and oxygen (O2) (Figure 4.12). The tritiated

water released is counted via liquid scintillation as an index of estrogen formation.

NADPH is continuously provided by a regenerating system, in which NADP+ is reduced

89 to NADPH by glucose-6-phosphate dehydrogenase while it catalyzes the conversion of glucose-6-phosphate into 6-phosphoglucono-δ-lactone.

120

100

% Control Activity Control % 20

0 9i 9j 9f 9a 9c 9e 9k 9b 9g 12f 12a 12c 12b 12d 12g 12h Control 10 uM 9h 50 uM AG

Figure 4.13: Screening results of compounds 9a−l and 12a−h for aromatase inhibitory activity at 1 µM concentration of each compound (10 µM for 9h and 50 µM for AG).

The synthesized compounds were screened for their aromatase inhibitory activity. Each compound was tested at 1 µM concentration except for 9h (10 µM), and 50 µM AG was used as a positive control. The results are shown in Figure 4.13. The screening assay showed that the compounds lacking the pyridyl group, such as 9a−e and their unprotected derivatives 12a−d, showed no significant inhibition at 1 µM concentration.

Some of these compounds did not show any significant inhibitory activity even at 100

µM concentration (data not shown). On the other hand, most of compounds containing

90 a pyridyl moiety showed inhibitory activity, suggesting that the pyridyl group might play

a key role in inhibiting aromatase, presumably via coordinating with the heme iron of the

enzyme.

The compounds that had shown reasonable inhibitory activity at 1 µM concentration in the screening assay were chosen for the further study. In order to determine their IC50

values for their aromatase inhibitory activity, each compound was tested in ten

appropriate concentrations with each experiment performed in triplicate. Except for

compound 9h, all the pyridine-containing compounds showed dose-dependent inhibition

of aromatase. Log IC50 values were calculated by a nonlinear regression analysis using

GraphPad Prism software (version 3.0) and resulting IC50 values are summarized in

Table 4.2

Compound R1 R2 R3 IC50 (µM) Log IC50 (nM) (± S.E.)

9a OMe OBn >100 −

9b OMe OBn >100 − 9c OMe OBn >100 −

9d H OMe >100 −

9e H OMe >100 −

9f H OMe 1.6 3.21 ± 0.11

9g H OMe 9.2 3.96 ± 0.16

9h H OMe >100 −

9i Me OMe 3.0 3.48 ± 0.05

9j OMe OMe 2.0 3.36 ± 0.30

9k H OBn 0.21 2.33 ± 0.03

9l OMe OBn 0.53 2.72 ± 0.11 12a OH OH Not determined −

12b OH OH Not determined − 12c OH OH Not determined −

12d H OH Not determined −

12e H OH 0.61 2.79 ± 0.11

12f H OH 3.6 3.56 ± 0.06

12g OH OH 0.28 2.44 ± 0.07

12h OMe OH 0.22 2.34 ± 0.04 Aminoglutethimide (AG) 2.8 3.45 ± 0.05

Table 4.2: Aromatase inhibitory activities of isoflavones 9a−l and 12a−h.

92 Compounds 9f−h were designed to examine the effects of the position of the nitrogen

atom on the inhibitory activity. The assay results clearly indicate that the position of the

nitrogen atom of pyridyl moiety is important for the inhibitory activity. 4-Pyridyl analog

9f (IC50 = 1.6 µM) exerted more potent inhibitory activity (5.8-fold) than 3-pyridyl

analog 9g (IC50 = 9.2 µM), whereas 2-pyridyl moiety showed no inhibitory effect even at

100 µM concentration. This trend can be also observed in their 7-hydroxy analogs: 4-

pyridyl analog 12e is 5.9-fold more potent than 3-pyridyl analog 12f. Although the

precise mechanisms of action of these compounds are not clear at present, the pyridyl moiety is thought to be involved in coordination with the heme iron of aromatase. Based on this hypothesis, it may be postulated that the nitrogen atom of the 4-pyridyl group may be in the more favorable position to coordinate to the heme iron than that of the 3- or 2-pyridyl group.

In addition, the 7-hydroxy compounds, 12e (IC50 = 0.61 µM) and 12f (IC50 = 3.6 µM),

exhibited 2.6-fold greater inhibitory activity than their 7-methoxy analogs, 9f (IC50 = 1.6

µM) and 9h (IC50 = 9.2 µM), respectively. This result suggests that the presence of

hydrogen donor at the 7-position might provide a better interaction with the enzyme. As

far as 4’-substituents concern, the methyl group (9i, IC50 = 3.0 µM) appears to be inferior to hydrogen and the methoxy group, while 4’-hydrogen analog (9f, IC50 = 1.6 µM) showed a slightly higher activity than 4’-methoxy analog (9j, IC50 = 2.0 µM). However,

the relatively small difference in their activities may indicate that the effects of 4’- substitutent may not be critical for the activity. Interestingly, this trend was not observed

93 in the 7-hydroxy analogs, 12h and 12e. In the presence of 7-hydroxy group, 4’-methoxy

analog 12h (IC50 = 0.22 µM) is more potent than 4’-hydrogen analog 12e (IC50 = 0.61

µM). Furthermore, the difference in their potency between 4’-methoxy and 4’-hydrogen

group in the 7-hydroxy analogs appeared to be much greater (3-fold) than that observed in the 7-methoxy analogs (1.25-fold). In addition, the potency of 4’,7-dihydroxy analog

12g (IC50 = 0.28 µM) was comparable to 12h (7-hydroxy-4’-methoxy analog), but

greater than that of 12e (7-hydroxy-4’-hydrogen analog). This result indicates that the

presence of a proton acceptor at the 4’-position in the 7-hydroxy analogs might be

beneficial for the aromatase inhibitory activity.

To our surprise, 7-benzyloxy analog 9l, originally prepared as a precursor of compound

12h, turned out to be a potent aromatase inhibitor with an IC50 value of 0.53 µM, which

is one of the lowest in this series. This result was interesting but unexpected because it

was believed that compounds possessing a benzyl protecting group would be too bulky to fit in the active site of the enzyme. This interesting result led us to synthesize its 4’-

hydrogen analog 9k since, in the 7-methoxy series, the 4’-hydrogen analog showed

relatively greater potency than others. As expected, 9k (7-benzyloxy-4’-hydrogen

analog) showed 2.5-fold greater potency (IC50 = 0.21 µM) than that of 9l (7-benzyloxy-

4’-methoxy analog), and 7.6-fold greater than that of 9f (4’-hydrogen-7-methoxy analog),

being the most potent analog in this series. The dose-response curves of three of the

most potent compounds (9k, 12g, and 12h) in this set are shown in comparison with AG

in Figure 4.14.

94 120

100

80 12g 12h 60 9k

40 AG

% Control Activity Control % 20

0 -2 -1 0 1 2 3 4 5 log[nM]

Figure 4.14: Dose-response curves of compounds 9k, 12g, and 12h in inhibiting aromatase activity in comparison with aminoglutethimide (AG).

4.5.3 KINETIC STUDIES

Kinetic studies were also performed to examine the mode of aromatase inhibition. The

selected compounds (9k, 12g, and 12h) were evaluated for their aromatase inhibitory

activity at concentrations ranging from 0 to 2 µM. The enzyme assays were run under initial velocity conditions of low product formation by limiting the enzyme concentration.

During these kinetic assays, the inhibitor concentration was held constant while the concentration of androstenedione was varied from 50 to 300 nM. Each substrate concentration was run in triplicate, and the results were plotted in a typical Lineweaver-

Burk plot (Figure 4.15). Analysis of the data by weighted regression analysis [161] provided apparent Ki values for the compounds, which are listed in Table 4.3 with

apparent Km values and Ki/Km ratios.

95 The Lineweaver-Burk plots showed typical competitive type of inhibition, suggesting

that compounds 9k, 12g, and 12h may inhibit aromatase activity by competing with

androstenedione for the substrate binding site of the enzyme. Compounds 9k, 12g, and

12h exhibited inhibition constants (Ki) of 0.22 ± 0.02, 0.31 ± 0.02, and 0.26 ± 0.02 µM, respectively. These Ki values may be directly used for evaluation of their activities since

this assay produced reasonably consistent corresponding Km values (0.13 ± 0.01, 0.11 ±

0.07, and 0.10 ± 0.02 µM, respectively). However, their Ki/Km ratios were also

calculated as a relative inhibitory potency. As reflected by their Ki/Km ratios, compounds

9k, 12g, and 12h are apparently very potent inhibitors with compound 9k being the most

potent in this series.

Apparent K (µM) Apparent K (µM) Compound IC (µM) i m K /K 50 (± S.E.) (± S.E.) i m

9k 0.21 0.22 ± 0.02 0.13 ± 0.01 1.69

12g 0.28 0.31 ± 0.02 0.11 ± 0.07 2.82

12h 0.22 0.26 ± 0.02 0.10 ± 0.02 2.60

Table 4.3: Aromatase inhibitory activities of three potent isoflavones, 9k, 12g, and 12h.

96 30000 ) duct o in 20000 0 nM 9k m / pr 100 nM 9k d s e e

l 500 nM 9k m o r

m 2000 nM 9k

fo 10000 n ( / 1

0 0.000 0.025 0.050 0.075 0.100 1/[Androstenedione (nM)]

15000

12500 uct ) n od i 10000 0 nM 12g m / d s pr 100 nM 12g e e 7500 l m o 500 nM 12g r m

fo 5000

n 2000 nM 12g ( / 1 2500

0 0.000 0.025 0.050 0.075 0.100 0.125 1/[Androstenedione (nM)]

12500

10000 ) duct o in 0 nM 12h m /

pr 7500 d

s 100 nM 12h e e l

m 500 nM 12h o

r 5000 m

fo 2000 nM 12h n ( /

1 2500

0 0.000 0.025 0.050 0.075 0.100 0.125 1/[Androstenedione/nM]

Figure 4.15: Lineweaver-Burk plots from kinetic study on the inhibition of aromatase activity by compounds 9k, 12g, and 12h.

97 4.5.4 FURTHER STUDIES

This study has shown that, by introducing proper functionalities such as a heme- coordination potential, aromatase inhibitory activity can be embodied in the isoflavone nucleus, which has been considered as an inappropriate scaffold for aromatase inhibitors.

The structure-activity relationships show that the binding modes of 7-protected analogs

(9f−l) might be different from those of 7-hydroxy analogs (12e−h). Hydrogen-bonding potential seemed to be important in the 7-hydroxy analogs (12e−h) for their aromatase inhibitory activity, whereas hydrophobic interactions might play a role in 7-protected analogs (9f−l). Among the tested isoflavones, compounds 9k, 12g, and 12h were identified as potent aromatase inhibitors. While detailed studies of their mechanisms of action, including the binding modes, are currently underway, these compounds would be new leads for the development of more potent inhibitors. Especially of great interest as a new lead is compound 9k, containing a benzyl group that seems to be a key feature for the inhibitory activity. Therefore, introduction of a proper functionality on the phenyl ring of the 7-benzyloxy group of 9k may confer more potent aromatase inhibitory activity. Based on this hypothesis, it would be worth generating a library of 9k derivatives containing diverse functional groups on the phenyl ring of its 7-benzyloxy group.

98 4.6 SECOND SET OF INHIBITORS

The second set of target compounds was designed aimed at investigating the fidelity of

the intermolecular conjugate addition-elimination pathway for the generation of

isoflavone library containing a heme-coordinating potential. As previously described, we envisioned that this method would allow us to use various readily available nucleophiles as sources for 2-substituents. Initially we chose 2-

(methylsulfonyl)isoflavone 10a as a substrate and several commercially available nucleophiles possessing a pyridine or azole type functionality. Isoflavones 11d and

13a−f were prepared in a single step from 2-(methylsulfonyl)isoflavone 10a in moderate to good yields by displacing the 2-methylsulfonyl group by various nucleophiles (Figure

4.16).

Figure 4.16: Synthesis of the second set of aromatase inhibitors via a 1,4-conjugate addition-elimination process.

99 150

100

50 % Control Activity % Control

0 13f 13a 13c 13e 11d 13b 13d Control

Figure 4.17: Screening results of compounds 11d and 13a−f for aromatase inhibitory activity at 1 µM concentration of each compound.

The inhibitor screening assay was performed using the same method described in the

previous section and the amount of inhibition was compared to a control in which there

was no inhibitor present. The results of the assay are shown in Figure 4.17 and Table 4.4.

While studies to determine their IC50 values are underway, these screening results

provide some interesting structure-activity relationships. First, compound 11d

possessing a 4-aminophenylthio group, which is reminiscent of the side chain of AG or

7α-APTA, did not show any significant inhibition at 1 µM concentration. Compound

13a, a homologous derivative of 9f, showed a promising inhibitory activity in the screening assay; it inhibited 75% of the aromatase activity at 1 µM concentration. 13a might be more potent than 9f according to the screening data; however, further examination should be carried out, i.e. to determine its IC50 value to confirm this issue.

Compounds 13b−d comprise azole type functionalities such as imidazole or triazole instead of pyridine. Since these azoles have proven to be effective in their heme-

100 coordinating ability in many nonsteroidal aromatase inhibitors, reasonable inhibitory activities were anticipated from 13b−d. However, to our disappointment, none of these compounds showed significant activities in inhibiting aromatase as shown in Figure 4.17.

Compound R3 % inhibition (1µM)

11d 2.81 ± 2.34

13a 75.09 ± 1.91

13b 12.58 ± 12.41

13c 10.58 ± 4.49

13d 15.23 ± 2.67

13e 9.66 ± 16.74

13f 56.40 ± 10.43

Table 4.4: Aromatase inhibitory activities of isoflavones 11d and 13a−f.

Compounds 13e and 13f also contain the triazolyl or imidazolyl moieties, respectively, but are unique in their structures since their heme-coordination functionalities are directly connected to the core ring without sulfide linkage. Interestingly, the imidazolyl derivative, 13f, exhibited a considerable inhibitory activity, whereas the triazolyl derivative, 13e, was much less active. The imidazole ring has often shown to be superior

101 to triazole ring in some structures in inhibiting cytochrome P450 enzymes as previously discussed. This trend has been explained by the different degree in localization of molecular orbitals between two the azole types: a more highly localized orbital has been observed on the imidazole ring than triazole [139].

Figure 4.18: Synthesis of 2-imidazolyl isoflavones 13f−i.

The promising screening result of 13f and its unique structure prompted us to synthesize its derivatives 13g−i, which vary at the 4’- and 7-positions (Figure 4.18). Compounds

13f−i also produced dose-dependent inhibition as seen in their dose-response curves in

Figure 4.19.

102 100

13f 13g

50 13h 13i % Control Activity Control %

0 -2 -1 0 1 2 3 4 5 log[nM]

Figure 4.19: Dose-response curves of 2-imidazolyl isoflavones 13f−i in inhibiting aromatase activity.

IC50 values of these compounds are summarized in Table 4.5. Compound 13f showed 2-

fold greater inhibitory activity (IC50 = 0.77 µM) than that of its (4-pyridyl)methanethio analog 9f (IC50 = 1.6 µM). Therefore, based on the structure-activity relationship studies

on the pyridylmethanethio analogs, we anticipated that compound 13h might exhibit the

greatest potency in this series: its (4-pyridyl)methanethio analog 9k (IC50 = 0.21 µM)

showed about 8-fold greater activity than 9f. As it turned out, 13h was more potent than

13f but only by a slight difference (IC50 = 0.52 µM), which was 2.5-fold less potent than

9k and compatible to 9l. Compound 13g showed a comparable inhibitory activity (IC50

= 2.0 µM) to its (4-pyridyl)methanethio analog 9j. Compound 13i, on the other hand,

was 8.8-fold less potent (IC50 = 4.7 µM) than its (4-pyridyl)methanethio analog 9j.

Further examination on the biological activities of these compounds is currently underway by John Hackett.

103

Compound R1 R2 IC50 (µM) Log IC50 (nM) (± S.E.)

13f H OMe 0.77 2.89 ± 0.042 13g OMe OMe 2.0 3.29 ± 0.04 13h H OBn 0.52 2.71 ± 0.03 13i OMe OBn 4.7 3.67 ± 0.09 AG 2.8 3.45 ± 0.05

Table 4.5: Aromatase inhibitory activities of isoflavones 13f−i in comparison with aminoglutethimide.

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109 CHAPTER 5

ANTIPROLIFERATIVE AGENTS

5.1 SELECTIVE ESTROGEN RECEPTOR MODULATORS

Estrogens play an important role in various tissues of the body, such as reproductive, central nervous, cardiovascular, and skeletal systems. Accordingly, estrogen deficiency has been implicated in many pathologies of those tissues. Estrogen-deprived conditions in postmenopausal women have been correlated with a variety of menopausal symptoms including mood swings, hot flashes, irritability, increased risk for osteoporosis, and various cardiovascular diseases including arteriosclerosis. Therefore, the use of estrogens, such as hormone replacement therapy (HRT), has been a choice of the treatment or prevention of postmenopausal complications, mainly osteoporosis. Despite their many beneficial effects, estrogens have been notorious for their ability to promote the development and growth of cancer in the reproductive tissues. It is therefore not

surprising that the use of estrogens in HRT has been associated with an increased risk of

endometrial or breast cancer [162].

110 Estrogens have been recognized as predominant players in the development of hormone- dependent breast cancer [163]. Although many aspects of estrogen’s action still remain unclear, a great deal of progress has been made in understanding the molecular biology of estrogen action. It is well established that the majority of biological effects of estrogens are initiated by binding to their specific receptors, estrogen receptors (ERs).

Therefore, the use of antiestrogens, which can block the interaction of estrogens with

ERs, is a rational approach for the treatment of hormone-dependent breast cancer. Due to the multifaceted effects of estrogens in various tissues in the body, however, there has been a need for the antiestrogens that exert tissue specific activity. Ideally such agents would act as antiestrogens in the reproductive tissues while maintaining beneficial effects of estrogens in other tissues. The term selective estrogen receptor modulator

(SERM) is used to describe such molecules that exhibit such tissue-specific estrogenic/antiestrogenic activities. Tamoxifen and raloxifene are representative SERMs and have been shown to be effective in the treatment or prevention of breast cancer as well as osteoporosis.

5.2 TAMOXIFEN AND RALOXIFENE

Tamoxifen (TAM), a triphenylethylene derivative (Figure 5.1), was first used clinically in the early 1970s for the treatment of metastatic breast cancer [164], and is currently the most widely prescribed anticancer drug for the treatment of hormone-dependent breast cancer. The drug has been also used for the prevention of breast cancer in high-risk, pre- and postmenopausal women. TAM is considered the first example of SERMs. It has

111 shown antiestrogenic effects in breast cancer while displaying estrogenic effects in other

tissues. While the estrogen-like properties of TAM in cardiovascular and skeletal systems are considered to be beneficial, its estrogenic effects on endometrial

proliferation have been the major concern for long-term use of this drug as it

significantly increases the risk of endometrial cancer [165]. This undesirable tissue selectivity of TAM has led researchers to search for new SERMs with superior profiles

in appropriate tissues.

Figure 5.1: Structures of representative SERMs (tamoxifen and raloxifene) and a pure antiestrogen (ICI 182,780).

112 Raloxifene (RAL) is a benzothiophene analog (Figure 5.1) that was originally developed

as an antiestrogen for the treatment of breast cancer. However, preclinical studies found

that its antitumor activity was not as effective as that of TAM in the treatment of breast

cancer [164]. Instead, later studies showed RAL increased bone mineral density and

lowered LDL and, more importantly, it did not show stimulating effects on endometrium

[166]. RAL is currently being used clinically for the treatment and prevention of

postmenopausal osteoporosis.

5.3 MOLECULAR MECHANISMS OF SERM ACTION

Like estrogens, it is believed that most of the biological activities of SERMs are

mediated by the ERs. Binding of estrogens to the ERs initiates a series of events for the

expression of target genes involved in the proliferation and growth of breast tumor. As

previously discussed, there are two isoforms of estrogen receptors, ERα and ERβ, which

are different in their ligand selectivity, tissue distribution patterns, and transcriptional

properties [167]. In addition, studies with receptor subtype-specific knockout mice

substantiate that these two ER subtypes are not functionally identical [168]. Therefore,

the intriguing tissue selectivity of SERMs has been attributed to the ligand specificity or

tissue distribution of the ER subtypes. However, there are still many aspects of the unique tissue specificity of several SERMs that cannot be explained by the presence of two ER subtypes. For instance, there are some examples of SERMs that bind to both

ERs with similar affinities but elicit significantly different biological results. In addition, some compounds in this class are known to exhibit different biological effects among

113 tissues in which the same ER subtype is expressed, indicating that there might be other

mechanisms of action by which cells in such tissues can recognize the same ER-ligand

complex in a different manner.

The tissue-selective expression of transcriptional cofactors, coactivators and corepressors,

might be another explanation for the tissue selective activity of SERMs. For example,

SRC-1, SRC-2, and SRC-3 are the steroid receptor coactivators that interact with ligand-

activated receptors to activate gene transcription by stabilizing the transcription initiation

complex. These proteins are functionally different from each other and expressed at

different levels in different cells, which may explain how some compounds exhibit

different biological effects among tissues in which the same ER subtype is expressed.

These coactivators interact with the receptors via the two transcriptional activation

functions, AF-1 and AF-2, using their repeated LXXLL motif. AF-1 is the ligand-

independent transcriptional activation function located near the amino terminus and AF-

2 is the ligand-dependent function within the LBD of ERs. In most cells, both activation

functions are essential for the maximal transcriptional activity, while there are some cells

that require only one of them [169]. TAM, for instance, is known to block cofactors

from interacting with the AF-2 [170], thereby acting as an ER antagonist in those cells requiring AF-2 function. In those cells in which coactivators interact with only the AF-1 domain, on the other hand, TAM exerts its agonist activities [ 171 ]. In addition, compounds blocking both AF-1 and AF-2, such as ICI 182,780 (Figure 5.1), act as an antagonist regardless of cell types and are classified as pure antiestrogens [172].

114

Figure 5.2: Schematic representations of the interactions between E2 and ERα (A) [174], RAL and ERα (B) [174], and GEN and ERβ (C) [175].

115 Recent studies have shown that the unique conformation of the ER induced by the ligand

binding is critical for the tissue selective activity of SERMs [173]. Different SERMs

induce different conformational changes of the receptors, thereby resulting in different

biological consequences by recruiting different set of cofactor proteins. A crystallographic study of the LBD of ER with 17β-estradiol (E2) and RAL provided

structure-based evidence of the mechanism of antagonism of RAL. E2 and RAL bind at

the same site within the LBD, but their binding modes are different [174]. This study

suggested that the basic side chain of RAL plays a critical role for tissue selective

activity by inducing a unique conformational change of the ER. The key event is the

repositioning of helix 12 after ligand binding, which contains the ligand dependent

transcriptional function, AF-2. When E2 binds to the ER, helix 12 seals the ligand binding pocket, revealing a groove formed by residues from helices 3,4,5, and 12 of the

receptor. This groove provides an interface for interaction with the LXXLL motif of

various coactivators that are necessary for estrogenic activity. On the other hand, when

RAL binds to the ER, its long side chain protrudes from the binding pocket, thereby

preventing helix 12 from positioning properly over the ligand binding pocket (Figure

5.2). The resulting unique conformation of ER-RAL does not provide the correct

binding interface for the recruitment of the coactivators that are normally involved in

estrogenic activity [174]. In addition to those described above, there seem to be more

factors involved in the tissue selective mechanisms of SERMs. Emerging data from

numerous ongoing studies will continue to provide more precise insight into the

molecular basis of mechanism of SERMs actions.

116

Figure 5.3: Selected SERMs under the clinical trials.

5.4 NEW SERMS

The favorable tissue selectivity profile of RAL has prompted a vigorous search for new

SERMs that would exert more ideal tissue selective profiles with higher potency. As the

ERs have demonstrated their capability to bind a wide range of nonsteroidal ligands, various ring systems have been employed as a non-steroidal core template of SERMs including benzothiophene, indole, naphthalene, benzopyran, indene, furan, and pyrazole rings (Figure 5.3). However, these compounds share several common structural features: two phenolic groups separated by two atoms (usually a stilbene type arrangement) and, most notably, an amine-bearing side chain.

5.5 GENISTEIN AS A SERM

GEN, the representative phytoestrogen, has been considered a naturally occurring selective estrogen receptor modulator. As previously discussed, GEN binds to ERs with moderate affinities and exhibits estrogenic or antiestrogenic activities in a tissue-specific

117 manner. It inhibits proliferation of breast cancer cells (antiestrogenic activity) while

displaying beneficial effects on bone and cardiovascular system (estrogenic activity). It

is interesting to note that GEN displays ER subtype selectivity, it binds to ERβ with 20-

fold greater affinity than ERα. Thus, the tissue specific estrogenic/anti-estrogenic

effects of GEN may be partly attributed to this receptor selectivity [167]. A

crystallographic study with GEN-bound LBD of human ERβ has shown that GEN binds

in a similar manner to that observed for E2 [175]. The phenol group (B-ring) of GEN

mimics the A-ring of E2 while the C- and A-rings occupy a position similar to that

adopted by the C- and D-rings of E2 (Figure 5.2). Nevertheless, the conformation of the

resulting ERβ-GEN complex is not identical to that induced by E2. Furthermore, the

study has shown that the repositioning of helix 12 in ERβ-GEN complex differs from

that induced by RAL, indicating that estrogenic and antiestrogenic activity of GEN may be attributed to different molecular mechanisms.

5.6 DESIGN OF ISOFLAVONE-BASED SERMS

Estrogen receptors have been one of the major targets for the pharmaceutical

intervention in the treatment of breast cancer, and SERMs have been the most promising

class of compounds. Since the present research has focused on exploitation of the

isoflavone scaffold as a privileged structure aimed at developing new agents that

specifically disrupt various molecular targets in breast cancer biology. We have been

interested in identifying a new series of SERMs constructed on an isoflavone scaffold.

118 As GEN has proven to be a good ligand for the ERs and displays SERM-like tissue

selectivity, we envisioned that the isoflavone ring system would provide an appropriate

nonsteroidal template for the development of new SERMs. Since the basic side chain of many typical SERMs is known to be responsible for the tissue selective activity, we introduced such a basic side chain at the 2-position of the ring system, affording a library of 2-substituted isoflavones (Figure 5.4). Especially, we were interested in the effects of different linkages that connect the side chain to the isoflavone core, which are known to play an important role in SERMs by directing the basic side chain to a proper space in the binding pocket. For example, in the ER-RAL complex, the carbonyl hinge of RAL is known to induce an orthogonal orientation of the side chain [174]. In fact, a study has shown displacement of the carbonyl group of RAL with an alternative linkage such as an oxygen, sulfur, or nitrogen atom, enhances the potency with the oxygen analog being the most potent [176], substantiating the importance of the role of the hinge group.

Figure 5.4: Library of isoflavones containing a basic side chain as potential SERMs.

119

Figure 5.5: Initial targets of interest.

5.7 FIRST SET OF TARGET COMPOUNDS

Figure 5.5 shows three isoflavone analogs that we were initially interested in, which are

4’,7-dihydroxyisoflavones containing a 4-(piperidinylethoxy)phenyl group connected via

a carbonyl (5A), sulfide (15a), or ether (15b) linkage. The rationale for this set of

compounds is as follows: first, the presence of both 4’- and 7-hydroxyl groups may be

critical for binding the receptors, which is often the case in many SERMs. In fact, both

3- and 17-hydroxyl groups of E2 are known to participate in critical interactions in the

binding pocket of the ERs (Figure 5.2). Second, the 4-(piperidinylethoxy)phenyl group

is known to exhibit the best results in many non-steroidal ring systems including

benzothiophene (RAL); therefore, it is the most typically employed in the development

of new SERMs. Finally, in order to maintain the basic side chain perpendicular to the

plane of benzopyran-4-one core, the presence of a hinge group would be critical. Thus, carbonyl, sulfide, and ether linkages were chosen. The carbonyl group is the most frequently employed hinge in SERMs, while oxygen and sulfur atoms proved to be better hinges in some cases. Furthermore, it has been demonstrated that the sulfide and oxygen

120 linkages can be easily introduced using our synthetic methods. However, our literature

survey revealed that the compound 5A, containing a carbonyl linkage, has been already

synthesized by another research group [177]. Therefore, our initial effort has focused on

the synthesis of compounds 15a and 15b.

Figure 5.6: Synthesis of 2-(4-hydroxyphenoxy)- and 2-(4-hydroxyphenylthio)- isoflavones from 2-(methylsulfonyl)isoflavone.

5.7.1 SYNTHESIS

The displacement of the methylsulfonyl group with a phenol or thiophenol via a 1,4-

conjugate addition-elimination pathway was described in Chapter 3. Using the same

reaction procedure, 2-(4-hydroxythiophenoxy)isoflavone 11g and 2-(4-

hydroxyphenoxy)isoflavone 11h were synthesized from sulfone 10e by the treatment of

sodium salt of 4-mercaptophenol or hydroquinone, respectively (Figure 5.6). The

resultant phenolic hydroxy groups of compound 11g and 11h were alkylated with 1-(2-

chloroethyl)piperidine using cesium carbonate as a base to give compound 14a and 14b,

respectively.

121

Figure 5.7: Dealkylation reactions for the preparation of target compounds 15a and 15b.

Initial attempts to generate the target molecule 15a using typical reaction conditions for demethylation, such as boron tribromide in dichloromethane, were unsuccessful; the competitive cleavage of the piperidinyl ethoxy group led to compound 16a as a major product with the desired compound 15a being a minor product (Figure 5.7). This partial cleavage of the basic side chain has been previously noted by Katzenellenbogen and coworkers with their pyrazole derivatives [178]. They have successfully overcome this problem using the milder reaction conditions in which aluminum chloride (AlCl3) as a

Lewis acid and ethanethiol (EtSH) as a deprotecting reagent. However, the application of this reagent to our compounds, which contain a Michael acceptor in the C-ring of the structures, was considered to be inappropriate because of the intrinsic nucleophilicity of ethanethiol [164]. We believed that the basic amine in the side chain was responsible for the susceptibility of the side chain, presumably by coordinating with the Lewis acid and thereby allowing access of the Lewis acid to the susceptible ethoxy group. Based on this

122 explanation, we speculated that the undesired cleavage of the side chain might be

avoided by converting the amino group of the side chain into an positively charged

ammonium salt, which might prevent the access of Lewis acid to the ethoxy group in the

side chain. Thus, the hydrochloride salts of 14a and 14b were prepared by treatment of

hydrochloride gas in ethyl acetate or dichloromethane. As expected, the treatment of the

salt forms of 14a and 14b with boron tribromide gave the desired targets 15a and 15b,

respectively, as exclusive products in excellent yields (Figure 5.7).

Figure 5.8: Selective removal of benzyl protecting group.

Although we anticipated that both 4’- and 7-hydroxyl groups of compound 15a or 15b would be essential for the binding ability to the ERs, we were also interested in the preparation of their 7-hydroxy-4’-methoxy analogs. Since the 3-hydroxyl group of E2 is

known to be more critical for the binding affinity than the 17-hydroxyl group, these 4’-

methoxy analogs were expected to provide insight into the binding modes of compounds

in this series. In order to prepare these 4’-methoxy analogs from compounds 14a and

14b, the benzyl group at the 7-position should be selectively removed. Selective debenzylation of compound 14b was successfully accomplished by catalyst transfer

123 hydrogenation using ammonium formate as a hydrogen source in the presence of

palladium on carbon to afford compound 15d (Figure 5.8). However, as discussed in

Chapter 4, this method is not applicable to the sulfide-containing compound 14a due to

the catalyst poisoning. Therefore, we used the same reaction condition employed for the

preparation of compound 12h, thereby obtaining the desired 4’-methoxy analog 15c in a

good yield (Figure 5.8).

Figure 5.9: Deprotection reactions for the preparation of triphenolic (16a and 16b) and diphenolic (16c and 16d) isoflavones.

In addition, triphenolic compounds 16a and 16b were prepared from compounds 11g and

11h, respectively, using boron tribromide (Figure 5.9). These compounds were expected to offer important information about the role of the basic piperidinyl group in the biological activity by being compared with compound 15a and 15b. In addition, 4’- methoxy analog 16c and 16d were also prepared using boron trifluoride diethyl etherate and dimethyl sulfide from compound 11g and 11h (Figure 5.9). Interestingly, catalyst transfer hydrogenation of compound 11h gave compound 17 as a sole product, which appears to be a hydrolyzed product. In this case, not even a trace amount of compound

124 16d was detected in this reaction condition (Figure 5.10). Further studies of this

interesting result are not currently being pursued.

Figure 5.10: Unexpected product from catalyst transfer hydrogenation of compound 11h.

5.7.2 BIOLOGICAL EVALUATION

5.7.2.1 PROLIFERATION ASSAY

The growth inhibitory activities of the synthesized compounds were determined in the

breast cancer cell lines in collaboration with Edgar Diaz-Cruz. Two different human

breast cancer cell lines, hormone-responsive MCF-7 and hormone-non-responsive MDA-

MB-231, were used in a preliminary screening. The results of the antiproliferation

screening at 5 µM concentration of each compound in the MCF-7 human breast cancer

cell line are shown in Figure 5.11 (in the absence of E2) and in Figure 5.12 (in the

presence of E2 [10 nM]). Results were expressed as percentage of control (untreated

cells) and 4-hydroxytamoxifen (4-OHT), the active form of TAM, was used as a positive

control. Data points represent the mean values of three independent experiments. To

125 determine IC50 values, compounds were tested in eight appropriate concentrations with each experiment performed in triplicate and the results are listed in Table 5.1.

Compound X R1 R2 R3 IC50, µM 11g S OMe OBn OH 4.70 14a·HCl S OMe OBn 2-(1-piperidin-1-yl)ethoxy 4.17 15c S OMe OH 2-(1-piperidin-1-yl)ethoxy 0.058 16a S OH OH OH 11.12 14b·HCl O OMe OBn 2-(1-piperidin-1-yl)ethoxy 3.67 15d O OMe OH 2-(1-piperidin-1-yl)ethoxy 6.31 4-OHT 4.22

Table 5.1: Antiproliferative activity of the selected isoflavones in MCF-7 human breast cancer cell line.

It was disappointing that both compound 15a and 15b, from which the most potent inhibitory activities were expected, were almost inactive in inhibiting proliferation at the given concentration (Figure 5.11). On the other hand, the highly protected, bulky compounds 14a and 14b showed the most potent inhibition at 5 µM concentration. It is also interesting to note that compound 11g was also a potent inhibitor, whereas its ether- linked analog 11h appeared to elicit a stimulatory activity. However, based on IC50 data,

126 125

100

50 % Control Activity Control %

0 15a 15c 14a 16a 11g 15b 15d 11h 14b 16b 4-OHT Control

Figure 5.11: Antiproliferative activity of target isoflavones (5 µM) in MCF-7 human breast cancer cell line.

150

100

50 % Control Activity Control %

0 E2 15a 15c 14a 16a 11g 15b 15d 11h 14b 16b 4-OHT Control

Figure 5.12: Antiproliferative activity of target isoflavones (5 µM) in MCF-7 in the presence of E2 (10 nM).

127 compound 15c is the most potent inhibitor of cell proliferation in this series (Table 5.1).

Antiproliferative activity of compound 15c is 72-fold and 63-fold greater than that of compound 14a and 14b, respectively, and 81-fold greater that that of compound 11g.

However, its ether-linked analog 15d is 108-fold less potent than compound 15c. In the screening assay in the presence of E2, these four compounds (11g, 14a, 14b, and 15c) also showed potent inhibitory activities, indicating these molecules suppressed the stimulating effect of E2 in the growth of breast cancer cells. However, it should be noted that their potency seems to remain the same regardless of the presence of E2 while the potency of the positive control, 4-OHT, appears to be decreased in the presence of E2.

The fact that their inhibitory potencies are not affected by addition of E2 suggests that the antiproliferative activities of these compounds may not be mediated by estrogen receptor signaling pathways. This possibility was also substantiated by the proliferation assay in

MDA-MB-231, which is a hormone-non-responsive human breast cancer cell line. As shown in Figure 5.13, compound 15c also showed a potent anti-proliferative activity in this cell line, indicating this molecule might exert its activity in an ER-independent manner.

128 on theERsubtypeselectivity, asprelim on thedevelopm ERs isconsideredasap us toinvestigatetheirbindingabilitieses features ofERligands.Noneth benzyloxy analogssuchas independent. Furthermore,thestructures series arep As describedabove,theproliferationassay 5.7.2.2 ERB hum Figure 5.13: an breastcancercellline. o tent an Antiprolif INDING

ent of % Control Activity 10 12 15 25 50 75 0 5 0 0 tiproliferative agents, A newSERMsreportb SSAY erative activ r erequ

14a Control

i and site tobea ele s s, theinte 14b ity oftheselectedis 15a inary biologicaldata. , seem 129 whose m SERM; the ofthoseactivecom trog i ndi res indicates thatsevera nottoagreewiththeco 15b ting resultsinthep ng af en recepto echanism f i r nities ef 15c ore rs oflavones inMDA-MB-231 toERs,especiallyf , . Indeed,bindingabilityto m s ofactionm o

st of 16a pounds, especially7- l compoundsinthis r olif thecurren e mmon structural r ation i ght beER- ass t o stu cusing a y led d ies

Binding affinities of the selected compounds for human ERα and ERβ were determined in collaboration with Dr. James Mobley by fluorescence polarization (FP) using a modified protocol [179] of the previously reported procedure [180]. The FP-based ER assay is based on the competition of a fluorescent estrogen ligand (Fluormone™ ES2) and estrogen competitors for binding to ER. In brief, when ER is added to ES2, an ER/ES2 complex is formed with high fluorescence polarization, which is in turn added to a compound of interest. The shift in polarization in the presence of test compounds is used to determine the relative affinity of test compounds for ERs.

Compound X R1 R2 R3 EC50, µM RBA 15a S OH OH 2-(1-piperidin-1-yl)ethoxy 0.77 0.51 15b O OH OH 2-(1-piperidin-1-yl)ethoxy 0.20 1.95 15c S OMe OH 2-(1-piperidin-1-yl)ethoxy 1.48 0.32 15d O OMe OH 2-(1-piperidin-1-yl)ethoxy 1.35 0.35 16a S OH OH OH 1.86 0.21 16b O OH OH OH 1.74 0.22 4-OHT 0.0157 26

E2 0.005 100

Table 5.2: Binding affinities of compounds 15a−d and 16a−b for ERα.

130 Binding results for ERα are listed in Table 5.2 as EC50, the concentration resulting in a half-maximum shift in polarization, and relative binding affinity (RBA), which for E2 is

100. E2 was used as a control, and 4-OHT was also included as a positive control, for which the published RBA value is 26 using this fluorescence polarization assay [180].

Dose-response curves of compounds 15a−d and 16a−b for ERα binding affinity are shown in Figure 5.14 in comparison with E2.

120

100 15a 80 15b

60 15c 15d 40

% Relative FP Relative % 16a 20 16b E2 0 -11 -10 -9 -8 -7 -6 -5 log[M]

Figure 5.14: Dose-response curves of compounds 15a−d and 16a−b for ERα binding affinity in comparison with 17β-estradiol (E2).

No compound showed significant binding to ERβ below 5 µM (data not shown), whereas they showed binding affinities for ERα. This is opposite to our expectation that our compounds would be rather ERβ-selective because GEN, our lead compound, is known to bind to ERβ with higher affinity than to the other subtype. Albeit with a preference for ERα, binding affinities of the compounds in this series are still low. However, some

131 structure activity relationships for binding affinity are apparent in this series. As expected, 4’,7-dihydroxy analogs (15a and 15b) bind to the ER with higher affinities

than 7-hydroxy-4’-methoxy analogs (15c and 15d). In addition, the oxygen linkage

seems to be a more favorable hinge than sulfur, which appears to be more obvious in the

4’,7-dihydroxy analogs: the binding affinity of 15b is about 4-fold greater than that of

15a. A similar trend was observed in a study with RAL analogs, in which an oxygen

linkage provides an enhanced affinity for ER relative to carbonyl or sulfur group [181].

It is interesting to note that the structurally related compound 5A has been reported to

bind well to estrogen receptors with a Ki value of 3.9 ± 0.3 nM (IC50 value was not

reported) [177]. While further investigation is needed, these results indicated that both sulfur and oxygen atoms in this series may significantly alter the orientation of the basic side chain, which may not be well tolerated by the receptors.

Another interesting observation is that both compound 16a and 16b, which lack the bulky side chain, bind to ERα with even lower affinities than compound 15a and 15b.

Their binding affinities are even lower that those of 7-hydroxy-4’-methoxy analogs.

This was unexpected because most triphenolic compounds tend to bind reasonably well to the estrogen receptors [182, 183]. It is plausible that, in this series of compounds, the basic side chain may provide an additional interaction with ER in the binding pocket, which can compensate for its bulkiness. Overall, the binding affinities of tested compounds do not correlate with their antiproliferative activities: the most potent inhibitor, 15c, showed no significant affinity for ERs, whereas 15b, one of the weakest

132 inhibitors, exhibited the highest binding affinity for the ER in this series. This might be another evidence for the non-ER-mediated antiproliferative activity of 15c.

5.7.2.3 FURTHER STUDIES

We have identified compound 15c as a potent inhibitor of proliferation of breast cancer cells. However, all the biological data described above suggest its potent antiproliferative activity may not be mediated by ERs. Therefore, while more studies may be required to further confirm its non-ER-mediated actions, research efforts are focusing on elucidation of its precise mechanisms of action. In addition, proliferation studies using different cancer cell lines including endometrial and prostate cancer cell lines are currently underway.

5.8 SECOND SET OF TARGET COMPOUNDS

Although the mechanism of action of compound 15c for its potent antiproliferative activity is not clear at this point, the compound might serve as a new lead for the development of more potent analogs. Thus, based on the structure activity relationships from the preliminary biological evaluation, we designed another set of target isoflavones shown in Figure 5.15.

133

Figure 5.15: Second set of target isoflavones.

First, we chose the sulfide linkage as the hinge group, which has proven to confer greater

potency than ether linkage in this series. Second, we were interested in replacement of

4’-substituent with small nonpolar ones such as hydrogen or a methyl group since the

free hydroxyl group at this position proved to be unfavorable for the antiproliferative

activity: protection of 4’-hydroxyl group of inactive 15a with a methyl group provided a

highly potent inhibitor, 15c. Finally, although 7-hydroxy analog 15c (IC50 = 0.058 µM)

is more potent than 7-benzyloxy analog 14a (IC50 = 4.17 µM), compound 14a is still a potent inhibitor. This led us to investigate the effects of a smaller protecting group (i.e. methyl) rather than benzyl group at the 7-position. We were also interested in the analogs that lack the basic side chain.

5.8.1 SYNTHESIS

The synthetic route for the second set of isoflavones is similar to that for the first set and

is summarized in Figure 5.16. Treatment of sulfones 10a−c with the sodium salt of 4-

mercaptophenol provided 2-(4-hydroxythiophenoxy)isoflavone 11c, 11i, and 11j,

134 respectively, in good yields. Alkylation of their phenolic hydroxy groups with 1-(2- chloroethyl)piperidine gave corresponding isoflavones possessing a full basic side chain, which were then treated with hydrogen chloride gas to form hydrochloride salts. The methyl group of compounds 14c·HCl and 14d·HCl was removed by boron tribromide to afford 7-hydroxyisoflavone 15e and 15f, respectively, which were also converted into hydrochloride salt forms using hydrogen chloride gas. In addition, demethylation of compound 11c and 11i using boron tribromide provided corresponding 7- hydroxyisoflavone 16e and 16f, respectively.

Figure 5.16: Synthesis of second set of target isoflavones.

135 5.8.2 BIOLOGICAL EVALUATION

Biological evaluation of the second set of compounds is currently under way to evaluate

antiproliferative activities and binding affinities for ERs. We envision that the results

from these studies would provide valuable insight into structure activity relationships that would be critical for the development of more potent inhibitors. However, the key to further development would be to decipher the mechanisms of action of these compounds to enhance or amplify specific properties.

5.9 REFERENCES

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173. Paige, L. A.; Christensen, D. J.; Gron, H.; Norris, J. D.; Gottlin, E. B.; Padilla, K. M.; Chang, C. Y.; Ballas, L. M.; Hamilton, P. T.; McDonnell, D. P.; Fowlkes, D. M. Estrogen receptor (ER) modulators each induce distinct conformational changes in ERα and ERβ. Proc. Natl. Acad. Sci. USA 1999, 96, 3999−4004.

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138 CHAPTER 6

CONCLUSIONS

As part of our ongoing research projects on discovery and development of new agents for the treatment of hormone dependent breast cancer, our laboratory has recognized a great deal of potential in the isoflavone ring system as a promising privileged structure.

In this study we hypothesized that chemical modification of functionalities upon the isoflavone nucleus would provide novel isoflavone derivatives that would specifically interact with various target proteins in breast cancer biology, thereby allowing us to harvest new leads for the development of highly potent and selective agents for the treatment of hormone-dependent breast cancer.

For the library design, the 2-, 4’-, and 7-positions have been chosen as potential sites for chemical modulation among others on the isoflavone nucleus. We have particularly focused on the functional diversification at the 2-position of the ring system to endow isoflavone derivatives with a specific activity.

139 For the synthesis of the target library, we developed efficient synthetic routes in which

α-oxoketene dithioacetals are employed as key intermediates for the construction of the

isoflavone skeleton. As one of those methods, we developed a convenient phase transfer

catalysis procedure that allows the efficient conversion of deoxybenzoins into 2-

(alkylthio)isoflavones in a single step at the ambient reaction conditions. This method

may be an efficient synthetic tool for the generation of a number of drug-like compounds

from easily available deoxybenzoins using numerous electrophiles in a short period of

time. Alternatively, various substituents can be introduced as nucleophiles through a

1,4-conjugate addition-elimination process using 2-(methylsulfonyl)isoflavones as

substrates. This method provides us with the opportunity to utilize a number of

commercially available or readily accessible nucleophiles for diverse substitution. In

addition, further diversification can be accomplished by chemical modification, such as

alkylation or oxidation reactions, of the introduced substituents.

With efficient synthetic routes established, we examined the feasibility of the isoflavone

nucleus as a privileged structure to achieve specific activities toward molecular targets

by introducing proper functional groups onto the ring. The first molecular target described was aromatase, the enzyme responsible for estrogen biosynthesis from androgen substrates. The majority of nonsteroidal inhibitors identified to date possess a nitrogen-containing heterocyclic moiety such as imidazole, triazole, pyrimidine, or pyridine, which is known to interfere with the activity of aromatase by coordinating with the heme iron. Therefore, it was envisioned that an isoflavone possessing such a heme-

140 coordinating functionality would have a great deal of potential as an aromatase inhibitor.

Using our developed synthetic methods, we performed parallel syntheses of targeted 2- substituted isoflavone libraries that contain such a nitrogen-containing heterocyclic moiety. As a result, several compounds were identified with potent aromatase inhibitory activities, which, to the best of our knowledge, represent the first examples of synthetic isoflavones as aromatase inhibitors.

Another set of 2-substituted isoflavones was prepared by targeting ERs. Especially, we were interested in a specific group of ER ligands, SERMs, which represent a structurally diverse group of compounds that bind to ERs and elicit agonistic and antagonistic activity in a tissue-specific manner. Most of the known SERMs share common structural features such as a flat nonsteroidal heterocycle and an amine-bearing side chain. Aware that the amine-bearing side chain of SERMs is known to play a key role in their tissue- selective activities, we speculated that isoflavone derivatives equipped with such a basic side chain might exhibit potential SERM activities. Thus, we have designed and synthesized a series of isoflavones possessing the same basic side chain as that of raloxifene, a representative SERM. Preliminary biological evaluation has revealed that several compounds exhibit highly potent activities in suppressing proliferation of breast cancer cells, which however may not be mediated by ERs. Although their mechanisms of action are unclear at present, these compounds might serve as lead compounds for the development of a new type of anticancer agents. Currently, intensive studies are underway to elucidate their precise mechanisms of action.

141 Promising results obtained from the present study has encouraged us to pursue more

extended studies on this project. Several subjects that should be included are as follows:

ELUCIDATION OF MECHANISMS OF ACTION: The potent antiproliferative activity of

compound 15c in human breast cancer cells appears to be a non-ER-mediated effect.

Therefore, extended studies should be made to elucidate its precise mechanisms of action,

which would be useful for the further development of more potent analogs. In addition,

studies on the modes of action of aromatase inhibitors should also be pursued.

Compound 9k is of special interest since its benzyloxy group at the 7-position seems to

play a role in its potent activity in inhibiting aromatase.

Figure 6.1: Two isoflavones as new potential lead compounds identified in the present study.

LEAD OPTIMIZATION: In this study, we have identified several novel isoflavones with promising biological activities. Especially, the most notable were compounds 9k and

15c (Figure 6.1), which might serve as new lead compounds for the development of

142 more potent analogs. For example, based on the preliminary biological data, the 7-

benzyloxy group of compound 9k seems to play a role in its potent aromatase inhibitory

activity. Therefore, it might be worth conducting a SAR study by introducing various

functional groups onto the phenyl ring of the 7-benzyloxy group (Figure 6.2, Library A).

Figure 6.2: Focused libraries for lead optimization.

In case of compound 15c, the biological data indicate that its potent antiproliferative activity may not be mediated by estrogen receptors, although it was originally designed as a potential SERM. Despite its unclear modes of action, biological evaluation provided some important information on the structure activity relationships. First, the substitution patterns at the 4’- and 7-position seem to have considerable effects on the antiproliferative activity. For this reason, several derivatives of compound 15c containing different substituents at the 4’- and 7-positions were already synthesized and are currently under biological evaluation (Figure 5.15). In addition, the basic side chain of

15c seems to be essential for its potent anti-proliferative activity. However, since 15c

143 appears not to be a SERM, its side chain does not have to mimic those of other SERMs.

Therefore, it would be also worthy investigating the structure activity relationship with

varied side chains, e.g. its homologous analogs or alternative amine groups (Figure 6.2,

Library B).

TARGETING OTHER PROTEINS: In this study, we have demonstrated the potential of

the isoflavone ring system as a privileged structure in the identification of new leads for

specific molecular targets. Aromatase and estrogen receptors were chosen as molecular

targets for intervention since the inhibitors of these two proteins have been the main

approaches to the treatment of hormone-dependent breast cancer. However, there are a

number of other molecular targets in breast cancer biology that can be targeted in this

approach. Especially, those proteins that are known to interact with genistein would be reasonable targets such as protein tyrosine kinases, DNA topoisomerases, and protein

kinase C as discussed in Chapter 2.

SOLID PHASE SYNTHESIS: Our synthetic routes were developed aimed at being

applicable to solid-phase synthesis. For this reason, the benzyloxy group has been

employed as a surrogate for hydroxymethyl polystyrene resin (Wang resin) in order to

examine the feasibility of our synthesis for solid-phase synthesis. Reaction conditions in

our synthetic route are compatible with solid phase synthesis: high synthetic yields, high

degrees of conversions, no highly acidic conditions, and no extreme reaction

temperatures. Therefore, application of this synthesis to the solid-phase should be

pursued, which would significantly enhance the drug discovery and development process.

144 In this case, a higher degree of chemical diversity in the target libraries can be pursued by utilizing a number of commercially available electrophiles and nucleophiles.

Furthermore, the resulting isoflavone libraries could be used not only for the development of new therapeutic agents but also for the identification of chemical probes that would be useful in the study of the newly identified proteins.

145 CHAPTER 7

EXPERIMENTAL METHODS

7.1 GENERAL METHODS

Chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Lancaster

Chemical Inc. (Windham, NH) and were used as received unless otherwise indicated.

Reactions involving moisture-sensitive reagents were carried out under an inert atmosphere of dry argon. All glassware was dried prior to use, and all liquid transfers were performed using dry syringes and needles. Anhydrous solvents were dried by standard procedures: Tetrahydrofuran (THF) was distilled from sodium metal in the presence of benzophenone under argon. Dichloromethane (CH2Cl2) was distilled from calcium hydride under argon. Methanol was distilled from sodium metal under argon.

N,N-Dimethylformamide (DMF) was distilled under reduced pressure from calcium hydride and stored over 4 Å molecular sieve under argon. Thin layer chromatography was performed on precoated silica gel F254 plates (Whatman). Silica gel column chromatography was performed using silica gel 60A (Merck, 230-400 Mesh). Melting points were determined in open glass capillaries using a Thomas Hoover apparatus and

146 are uncorrected. Infrared spectra were recorded on a Nicolet Protégé 460 spectrometer

using KBr pellets or neat samples. High-resolution electrospray ionization mass spectra

were obtained on the Micromass QTOF Electrospray mass spectrometer at The Ohio

State Chemical Instrumentation Center. All the NMR spectra were recorded on a Bruker

AC 250, Bruker DPX 250, or Bruker DRX 400 model spectrometer in either DMSO-d6

or CDCl3 solution. The abbreviations s, d, t, q, m, dd, dt, and br are used for singlet,

doublet, triplet, quartet, doublet of doublets, doublet of triplets, and broad, respectively.

Chemical shifts (δ) for 1H NMR spectra are reported in ppm relative to residual solvent protons. Chemical shifts (δ) for 13C NMR spectra are reported in ppm relative to residual solvent carbons.

7.2 SYNTHETIC METHODS

7.2.1 PREPARATION OF DEOXYBENZOINS

GENERAL METHOD FOR PREPARATION OF DEOXYBENZOINS (3a−c).

A mixture of resorcinol (6.67 g, 60.3 mmol) and an appropriate phenylacetic acid (60.3

mmol) in boron trifluoride diethyl etherate (BF3·OEt2) (150 mL) was heated at 95−100

ºC overnight. The reaction mixture was poured into ice-cold water (300 mL) and

extracted with EtOAc twice (300 mL and then 150 mL). The combined organic layer

was washed with water (3 × 200 mL) and then brine, dried over magnesium sulfate

(MgSO4), and concentrated under reduced pressure. The oily residue was dried in vacuo

147 overnight to give orange-colored solid, which was dispersed in an EtOAc/hexane (1/9)

mixture. After ultrasonicated, the insoluble solid was collected by filtration to give

desired product. The filtrate was concentrated under reduced pressure and purified by

silica gel column chromatography (eluting with 3% methanol MeOH in CHCl3 or 25% of

EtOAc in hexane) to give more product. The resulting compound was further purified by

recrystallization (EtOAc and hexane).

1-(2,4-Dihydroxyphenyl)-2-phenylethanone (3a):

Using the previous procedure and starting from phenylacetic acid (8.29 g, 60.3 mmol),

10.59 g (77%) of the title compound was obtained as a white solid: mp 116−118 ºC (lit.

115 ºC) [184]; IR (KBr) 3165, 1622, 1587, 1579, 1498, 1454, 1328, 1263, 1173, 1139,

-1 1 974, 799, 777, 736, 723, 698, 611 cm ; H NMR (250 MHz, CDCl3) δ 12.71 (br s, 1H),

7.72 (d, J = 8.7 Hz, 1H), 7.26−7.32 (m, 5H), 6.34−6.37 (m, 3H), 4.19 (s, 2H); 13C NMR

(62.9 MHz, CDCl3) δ 202.84, 165.91, 163.45, 134.64, 133.35, 129.76, 129.23, 127.60,

113.88, 108.60, 104.07, 45.25.

148

1-(2,4-Dihydroxyphenyl)-2-(4-methylphenyl)ethanone (3b):

Using the previous procedure and starting from 4-methylphenylacetic acid (9.14 g, 60.3

mmol), 12.11 g (83%) of the title compound was obtained as a white solid: mp 115−116

ºC (lit. 114 ºC) [184]; IR (KBr) 3215, 1623, 1604, 1586, 1509, 1456, 1327, 1172, 1139,

-1 1 974, 801, 784 cm ; H NMR (250 MHz, CDCl3) δ 12.70 (br s, 1H), 7.73 (d, J = 8.4 Hz,

1H), 7.13 (m, 4H), 6.33−6.37(m, 2H), 6.90 (br s, 1H), 4.15 (s, 2H), 2.30 (s, 3H); 13C

NMR (62.9 MHz, CDCl3) δ 202.98, 165.95, 163.25, 137.25, 133.30, 131.54, 129.93,

129.60, 113.91, 108.44, 104.06, 44.89, 21.48.

1-(2,4-Dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (3c):

Using the previous procedure and starting from 4-methylphenylacetic acid (10.02 g, 60.3

mmol), 14.0 g (85%) of the title compound was obtained as a white solid: mp 158−159

ºC (lit. 163 ºC) [185]; IR (KBr) 3359, 1617, 1511, 1437, 1412, 1352, 1299, 1242, 1175,

-1 1 1130, 1024, 996, 967, 851, 806, 792, 617, 540, 512 cm ; H NMR (400 MHz, CDCl3) δ

12.54 (br s, 1H), 10.64 (br s, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 8.5 Hz, 2H), 6.83

149 (d, J = 8.5 Hz, 2H), 6.36 (dd, J = 8.9, 2.2 Hz, 1H), 6.34 (d, J = 2.2 Hz, 1H), 4.16 (s, 2H),

13 3.68 (s, 3H); C NMR (100 MHz, CDCl3) δ 203.33, 165.81, 165.56, 158.90, 134.41,

131.36, 127.84, 114.70, 112.95, 109.11, 103.36, 55.85, 44.07.

7.2.2 PROTECTION OF DEOXYBENZOINS

1-[4-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-2-hydroxyphenyl]-2-(4- methoxyphenyl)ethanone (4a):

To a stirred suspension of 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (1.49

g, 5.77 mmol) in CH2Cl2 (27 mL) was added Et3N (1.72 mL, 12.7 mmol) at 0 ºC. After

the mixture became homogeneous, the resulting solution was slowly treated with a

solution of tert-butyldimethylsilyl chloride (0.844 g, 5.59 mmol) in CH2Cl2 (7 mL) at 0

ºC. After stirring at 0 ºC for 10 min, the solvent was removed by evaporation. The

residue was dissolved in ether, and the insoluble salt was removed by filtration. The

filtrate was concentrated under reduced pressure and then purified by silica gel column

chromatography (eluting with 10% ether in hexane) to yield a colorless oil, which was

solidified by vacuum drying to give 1.93 g (94%) of desired product as a white solid: mp

53−54 ºC; IR (neat) 1633, 1513, 1464, 1421, 1347, 1250, 1179, 1131, 1037, 987, 851,

150 -1 1 711, 696, 672 cm ; H NMR (250 MHz, CDCl3) δ 12.55 (br s, 1H), 7.71 (d, J = 9.4 Hz,

1H), 7.17 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.34−6.38 (m, 2H), 4.14 (s, 2H),

13 3.77 (s, 3H), 0.97 (s, 9H), 0.23 (s, 6H); C NMR (62.9 MHz, CDCl3) δ 202.84, 165.80,

163.48, 159.09, 132.52, 130.81, 126.71, 114.61, 114.29, 112.72, 108.64, 55.66, 44.39,

+ 25.95, 18.64, −3.93; HRMS calculated for C21H28NaO4Si (M + Na) 395.1655, found

395.1652.

1-[2,4-bis[[(1,1-Dimethylethyl)dimethylsilyl]oxy]phenyl]-2-(4- methoxyphenyl)ethanone (5a):

To a stirred suspension of 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (0.52

g, 2.0 mmol) in CH2Cl2 (8 mL) was added Et3N (1.12 mL, 8.0 mmol) at room

temperature. After the mixture became homogeneous, the resulting solution was treated

with tert-butyldimethylsilyl chloride (0.70 g, 4.6 mmol). After stirring at room

temperature for 10 min, the reaction mixture was refluxed overnight. After cooling to

room temperature, the volatile material was removed by evaporation. The residue was

dissolved in ether, and the insoluble salt was removed by filtration. The filtrate was

concentrated under reduced pressure and then purified by silica gel column

chromatography (eluting with 10% ether in hexane) to yield a colorless oil, which was solidified by vacuum drying to give 0.96 g (99%) of desired product as a white solid: 1H

151 NMR (250 MHz, CDCl3) δ 7.48 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.81 (d, J

= 8.7 Hz, 2H), 6.44 (dd, J = 8.6, 2.2 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 4.20 (s, 2H), 3.76

(s, 3H), 1.00 (s, 9H), 0.96 (s, 9H), 0.28 (s, 6H), 0.2 (s, 6H); LRMS calculated for

+ C27H42NaO4Si2 (M + Na) 509.2519 found 509.24 (100%), 487.28 (14%), 427.10 (4%).

GENERAL METHOD FOR ALKYLATION OF 4-HYDROXYL GROUP OF DEOXYBENZOINS (4b−f).

To a solution of 2-aryl-1-(2,4-dihydroxyphenyl)ethanone (31.0 mmol) and alcohol

(32.55 mmol) in THF (150 mL) was added triphenylphosphine (8.538 g, 32.55 mmol),

followed by diisopropyl azodicarboxylate (6.41 mL, 32.55 mmol) at 0 ºC, and the

resulting yellow solution was stirred at 0 ºC for 10 min. The solvent was removed under reduced pressure, and the oily residue was directly purified by silica gel column chromatography (eluting with EtOAc:hexane, 1:4) and recrystallization (EtOAc and hexane) to yield desired product.

1-(2-Hydroxy-4-methoxyphenyl)-2-phenylethanone (4b):

Using the previous procedure and starting from 1-(2,4-dihydroxyphenyl)-2- phenylethanone (7.08 g, 31.0 mmol) and methanol (1.32 mL, 32.55 mmol), 6.60 g (88%)

152 of the title compound was obtained as a white solid: mp 87−88 ºC (lit. 92 ºC) [186]; IR

(KBr) 1635, 1620, 1589, 1437, 1350, 1291, 1230, 1206, 1127, 1021, 958, 802, 739, 729,

-1 1 551 cm ; H NMR (400 MHz, CDCl3) δ 12.71 (br s, 1H), 7.74 (d, J = 8.7 Hz, 1H),

7.31−7.34 (m, 2H), 7.23−7.26 (m, 3H), 6.40−6.44 (m, 2H), 4.20 (s, 2H), 3.81 (s, 3H);

13 C NMR (100 MHz, CDCl3) δ 202.38, 166.61, 166.30, 134.82, 132.47, 129.76, 129.16,

127.50, 113.58, 108.27, 101.44, 56.00, 45.27.

1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (4c):

Using the previous procedure and starting from 1-(2,4-dihydroxyphenyl)-2-(4-

methylphenyl)ethanone (7.51 g, 31.0 mmol) and methanol (1.32 mL, 32.55 mmol), 7.38

g (93%) of the title compound was obtained as a white solid: mp (EtOAc/hexane) 71−72

ºC; IR (KBr) 1639, 1623, 1590, 1516, 1508, 1439, 1388, 1355, 1268, 1231, 1205, 1131,

-1 1 1033, 1010, 957, 800, 780, 571, 504, 491 cm ; H NMR (400 MHz, CDCl3) δ 12.72 (br

s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.24−7.14 (m, 4H), 6.43−6.40 (m, 2H), 4.15 (s, 2H),

13 3.81 (s, 3H), 2.31 (s, 3H); C NMR (100 MHz, CDCl3) δ 202.63, 166.56, 166.28,

137.14, 132.47, 131.71, 129.87, 129.61, 113.60, 108.20, 101.43, 55.98, 44.89, 21.48;

+ HRMS calculated for C16H16NaO3 (M + Na) 279.0997, found 279.0989.

153

1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methoxylphenyl)ethanone (4d):

Using the previous procedure and starting from 1-(2,4-dihydroxyphenyl)-2-(4-

methoxyphenyl)ethanone (8.0 g, 31.0 mmol) and methanol (1.32 mL, 32.55 mmol), 6.85

g (81%) of the title compound was obtained as a white solid: mp 101−102 ºC (lit. 104

ºC) [187]; IR (KBr) 1637, 1611, 1513, 1459, 1346, 1301, 1237, 1174, 1148, 1026, 797,

-1 1 787, 626, 522 cm ; H NMR (400 MHz, CDCl3) δ 12.72 (br s, 1H), 7.73 (d, J = 8.8 Hz,

1H), 7.17 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.40−6.44 (m, 2H), 4.13 (s, 2H),

13 3.81 (s, 3H), 3.77 (s, 3H); C NMR (100 MHz, CDCl3) δ202.73, 166.55, 166.27, 159.07,

132.42, 130.78, 126.73, 114.61, 113.53, 108.21, 101.42, 55.99, 55.67, 44.37.

1-[2-Hydroxy-4-(phenylmethoxy)phenyl]-2-phenylethanone (4e):

Using the previous procedure and starting from 1-(2,4-dihydroxyphenyl)-2-

phenylethanone (7.08 g, 31.0 mmol) and benzyl alcohol (3.37 mL, 32.55 mmol), 8.62 g

(87%) of the title compound was obtained as a white solid: mp 106−108 ºC (lit. 104−105

ºC) [188]; IR (KBr) 1620, 1572, 1500, 1389, 1352, 1270, 1230, 1192, 1134, 1000, 974,

154 -1 1 830, 760, 729, 697, 628, 561 cm ; H NMR (400 MHz, CDCl3) δ 12.69 (br s, 1H), 7.48

(d, J = 8.7 Hz, 1H), 7.25−7.39 (m, 10H), 6.49−6.51(m, 2H), 5.07 (s, 2H), 4.20 (s, 2H);

13 C NMR (100 MHz, CDCl3) δ 202.41, 166.20, 165.68, 136.22, 134.79, 132.53, 129.76,

129.17, 129.14, 128.76, 127.95, 127.51, 113.77, 108.75, 102.47, 70.66, 45.28; HRMS

+ calculated for C21H18NaO3 (M + Na) 341.1154, found 341.1136.

1-[2-Hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (4f):

Using the previous procedure and starting from 1-(2,4-dihydroxyphenyl)-2-(4-

methoxyphenyl)ethanone (8.0 g, 31.0 mmol) and benzyl alcohol (3.37 mL, 32.55 mmol),

10.23g (95%) of the title compound was obtained as a white solid: mp 97−98 ºC (lit.

93−95 ºC) [189]; IR (KBr) 1635, 1611, 1512, 1496, 1387, 1351, 1289, 1227, 1173, 1131,

-1 1 1029, 994, 948, 842, 830, 791, 745, 725, 695, 535 cm ; H NMR (250 MHz, CDCl3) δ

12.73 (s, 1H), 7.75 (d, J = 9.6 Hz, 1H), 7.33−7.40 (m, 5H), 7.18 (d, J = 8.5 Hz, 2H), 6.87

(d, J = 8.5 Hz, 2H), 6.53−6.49 (m, 2H), 5.07 (s, 2H), 4.14 (s, 2H), 3.78 (s, 3H); 13C NMR

(69.3 MHz, CDCl3) δ 202.77, 166.19, 165.63, 159.10, 136.26, 132.51, 130.82, 129.15,

128.77, 127.97, 126.71, 114.63, 113.73, 108.71, 102.48, 70.65, 55.69, 44.38; HRMS

+ calculated for C22H20NaO4 (M + Na) 371.1259, found 371.1265.

155

1-[2-[(1,1-Dimethylethyl)dimethylsilyl]oxy-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)ethanone (5b):

To a stirred suspension 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)ethanone (3.05 g, 8.75 mmol) in CH2Cl2 (35 mL) was added Et3N (3.05

mL, 21.9 mmol) at room temperature. After the mixture became homogeneous, the

resulting solution was treated with tert-butyldimethylsilyl chloride (1.58 g, 10.5 mmol).

After stirring at room temperature for 10 min, the reaction mixture was refluxed for 4 h.

After cooling to room temperature, the volatile material was removed by evaporation.

The residue was dissolved in ether, and the insoluble salt was removed by filtration. The

filtrate was concentrated under reduced pressure and then purified by silica gel column

chromatography (eluting with EtOAc/hexane = 1/7) to give a colorless oil, which was

solidified after dried using a vacuum pump to give 4.01 g (99%) of desired product as a

white solid: mp 59.5−60.5 ºC; IR (KBr) 1689, 1670, 1599, 1514, 1422, 1338, 1247, 1209,

-1 1 1197, 1176, 1168, 1122, 1032, 991, 855, 836, 783 cm ; H NMR (250 MHz, CDCl3) δ

7.59 (d, J = 8.7 Hz, 1H), 7.36−7.41 (m, 5H), 7.13 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz,

2H), 6.61 (dd, J = 8.7, 2.2 Hz, 1H), 6.43 (d, J = 2.2 Hz, 1H), 5.07 (s, 2H), 4.23 (s, 2H),

13 3.77 (s, 3H), 1.00 (s, 9H), 0.25 (s, 6H); C NMR (69.3 MHz, CDCl3) δ 199.71, 162.97,

158.78, 156.81, 136.73, 132.73, 131.06, 129.15, 128.64, 127.82, 127.75, 124.48, 114.32,

156 108.51, 107.13, 70.57, 55.65, 49.17, 26.37, 18.95, −3.52; LRMS calculated for

+ C28H34NaO4Si (M + Na) 485.2124, found 485.20 (100%), 463.23 (8%).

7.2.3 PREPARATION OF α-OXOKETENE DITHIOACETALS

1-[2,4-bis[[(1,1-Dimethylethyl)dimethylsilyl]oxy]phenyl]-2-(4-methoxyphenyl)-3,3- bis(methylthio)-2-propen-1-one (6a):

To a stirred solution of n-butyllithium (0.052 mL of 1.6 M solution in hexane, 0.88 mmol) in THF (2.32 mL) was slowly added diisopropylamine (0.13 mL, 0.93 mmol) over 3 min at 0 ºC. After the mixture was stirred at 0 ºC for 0.5 h, a solution of 1-[2,4- bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]phenyl]-2-(4-methoxyphenyl)ethanone (0.43 g,

0.88 mmol) in THF (3 mL) was added over 5 min, and the stirred solution was maintained was at 0 ºC for 0.5 h. The contents were then cooled to −78 ºC, and carbon disulfide was added over 7 min. The mixture was further stirred at 0 ºC for 0.5 h, and then treated with iodomethane (0.22 mL, 3.53 mmol). After stirring at room temperature for 17 h, the reaction mixture was quenched with saturated aqueous NH4Cl solution. The mixture was extracted with EtOAc twice (2 × 20 mL), and the combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The remnant was purified by silica gel column chromatography (eluting with

157 ether/hexane = 1/9) to give 0.27 g (52%) of the title compound as a yellowish oil: 1H

NMR (250 MHz, CDCl3) δ 7.50 (d, J = 8.5 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 6.83 (d, J

= 8.8 Hz, 2H), 6.39 (dd, J = 8.5, 2.3 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 3.78 (s, 3H), 2.25

(s, 3H), 2.16 (s, 3H), 0.99 (s, 9H), 0.94 (s, 9H), 0.24 (s, 6H), 0.20 (s, 6H).

GENERAL METHOD FOR PREPARATION OF α-OXOKETENE DITHIOACETALS (6b−e).

To a stirred suspension of sodium hydride (0.053 g, 2.2 mmol) in DMF (3 mL) was dropwise added a solution of an appropriate deoxybenzoin (1.0 mmol) in DMF via a cannula at 0 ºC under argon atmosphere. The resulting mixture was allowed to warm to room temperature over 40 min and then carbon disulfide (0.09 mL, 1.5 mmol) was dropwise added. After stirring at room temperature for 30 min, the resulting orange- colored suspension was treated with appropriate alkyl halide (3.0 mmol) at room temperature. After stirring at room temperature overnight, the resulting yellow suspension was cooled to 0 ºC, and quenched with saturated aqueous NH4Cl solution.

The reaction mixture was extracted with EtOAc twice (2 × 20 mL), and the combined

organic layer was washed with brine, dried over MgSO4, filtered, and concentrated under

reduced pressure. The remnant was purified by silica gel column chromatography

(eluting with EtOAc/hexane = 1/9) to give desired product.

158

1-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)-3,3-bis(methylthio)-2-propen-1-one (6b):

Using the previous procedure and starting from 1-[2-[(1,1-

dimethylethyl)dimethylsilyl]oxy-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)ethanone (0.46 g, 1.0 mmol) and iodomethane (0.19 mL, 3.0 mmol),

0.52 g (93%) of the title compound was obtained as a yellow oil: IR (neat) 1660, 1603,

1558, 1506, 1464, 1427, 1331, 1295, 1252, 1208, 1178, 1145, 1127, 1027, 910, 839, 782,

-1 1 734, 697 cm ; H NMR (250 MHz, CDCl3) δ 7.58 (d, J = 8.8 Hz, 1H), 7.33−7.40 (m,

7H), 6.83 (d, J = 8.8 Hz, 2H), 6.52 (dd, J = 8.8, 2.3 Hz, 1H), 6.40 (d, J = 2.3 Hz, 1H),

5.03 (s, 2H), 3.76 (s, 3H), 2.24 (s, 3H), 2.17 (s, 3H), 1.01 (s, 9H), 0.19 (s, 6H); 13C NMR

(69.3 MHz, CDCl3) δ 193.06, 163.15, 159.65, 158.62, 147.67, 136.63, 134.61, 130.86,

129.37, 129.12, 128.64, 127.82, 122.77, 113.93, 108.40, 107.64, 70.54, 55.60, 26.35,

+ 18.93, 17.89, 17.37, −3.65; LRMS calculated for C31H38NaO4S2Si (M + Na) 589.1878,

found 589.18 (100%), 567.21 (22%), 475.13 (20%).

159

1-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)-3,3-bis[(propen-2-yl)thio]-2-propen-1-one (6c):

Using the previous procedure and starting from 1-[2-[(1,1-

dimethylethyl)dimethylsilyl]oxy-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)ethanone (0.46 g, 1.0 mmol) and allyl bromide (0.26 mL, 3.0 mmol),

0.55 g (89%) of the title compound was obtained as a yellow oil: IR (neat) 1662, 1603,

-1 1 1558, 1505, 1251, 1178, 1027, 840, 782 cm ; H NMR (250 MHz, CDCl3) δ 7.61 (d, J =

8.8 Hz, 1H), 7.30−7.40 (m, 7H), 6.82 (d, J = 8.8 Hz, 2H), 6.50 (dd, J = 8.8, 2.3 Hz, 1H),

6.40 (d, J = 2.3 Hz, 1H), 5.78 (m, 1H), 5.58 (m 1H), 5.18−4.97 (m, 4H), 5.04 (s, 2H),

3.76 (s, 3H), 3.35−3.40 (m, 4H), 1.00 (s, 9H), 0.19 (s, 6H); 13C NMR (69.3 MHz,

CDCl3) δ 192.86, 163.21, 159.72, 158.70, 150.98, 136.66, 135.22, 134.26, 133.96,

131.05, 131.00, 129.36, 129.12, 128.62, 127.77, 122.62, 118.33, 118.21, 113.88, 108.34,

107.45, 70.53, 55.60, 37.64, 36.84, 26.33, 18.89, −3.64.

160

1-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)-3,3-bis[(phenylmethyl)thio]-2-propen-1-one (6d):

Using the previous procedure and starting from 1-[2-[(1,1-

dimethylethyl)dimethylsilyl]oxy-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)ethanone (0.46 g, 1.0 mmol) and benzyl bromide (0.36 mL, 3.0 mmol),

0.65 g (91%) of the title compound was obtained as a yellow oil: IR (neat) 1660, 1598,

-1 1 1557, 1248, 1208, 1027, 839 cm ; H NMR (250 MHz, CDCl3) δ 7.15−7.39 (m, 15H),

7.05 (d, J = 8.6 Hz, 3H), 6.73 (d, J = 8.6 Hz, 2H), 6.36 (d, J = 2.4 Hz, 1H), 6.31 (dd, J =

8.7, 2.4 Hz, 1H), 5.03 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.75 (s, 3H), 0.98 (s, 9H), 0.14

13 (s, 6H); C NMR (69.3 MHz, CDCl3) δ 192.85, 163.05, 159.66, 158.59, 151.30, 138.35,

137.42, 136.68, 134.86, 131.16, 130.87, 129.69, 129.65, 129.32, 129.16, 128.93, 128.85,

128.66, 127.79, 127.67, 127.53, 122.42, 113.81, 108.23, 107.79, 70.53, 55.59, 39.44,

+ 38.37, 26.29, 18.87, −3.71; LRMS calculated for C43H46NaO4S2Si (M + Na) 741.2504,

found 741.4 (100%), 719.4 (10%).

161

1-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)-3,3-bis[[2-(4-nitrophenyl)ethyl]thio]-2-propen-1-one (6e):

Using the previous procedure and starting from 1-[2-[(1,1- dimethylethyl)dimethylsilyl]oxy-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)ethanone (0.46 g, 1.0 mmol) and 4-nitrophenethyl bromide (0.58 g, 2.5 mmol), 0.59 g (71%) of the title compound was obtained as a yellow oil: IR (neat) 1658,

-1 1 1601, 1518, 1345, 1251, 1178, 839 cm ; H NMR (250 MHz, CDCl3) δ 8.06 (d, J = 8.4

Hz, 2H), 8.01 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.31−7.39 (m, 7H), 7.22 (d, J

= 8.7 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.48 (dd, J = 8.7, 2.0

Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 5.03 (s, 2H), 3.79 (s, 3H), 2.74−2.96 (m, 4H), 1.02 (s,

13 9H), 0.20 (s, 6H); C NMR (69.3 MHz, CDCl3) δ 192.29, 163.51, 160.08, 159.00,

152.36, 148.18, 147.82, 147.07, 136.46, 134.75, 130.92, 129.95, 129.73, 129.14, 129.08,

128.69, 128.57, 127.74, 124.07, 123.94, 121.97, 113.96, 108.41, 107.78, 70.60, 55.69,

36.00, 35.87, 34.89, 34.54, 26.32, 18.92, −3.62; HRMS calculated for

+ C45H48N2NaO8S2Si (M + Na) 859.2519, found 859.2537.

162 7.2.4 CONSTRUCTION OF ISOFLAVONE SCAFFOLD

7-Hydroxy-3-(4-methoxyphenyl)-2-(methylthio)-4H-1-benzopyran-4-one (7):

To a stirred solution of 1-[2,4-bis[[(1,1-Dimethylethyl)dimethylsilyl]oxy]phenyl]-2-(4-

methoxyphenyl)-3,3-bis(methylthio)-2-propen-1-one (0.14 g, 0.24 mmol) in THF (4 mL)

was added a 1.0 M solution of tetrabutylammonium fluoride in THF (0.96 mL, 0.96 mmol) at room temperature, and the resulting mixture was heated at 75 ºC for 1 h. After

cooling to room temperature, the reaction mixture was quenched with acetic acid and

concentrated under reduced pressure. The residue was suspended in EtOAc and the

precipitated solid was collected by filtration to yield desired compound. The filtrate was

concentrated and purified by silica gel column chromatography (eluting with

MeOH/CHCl3 = 4/96). The combined solid was further purified by recrystallization

from EtOAc to give 0.055 g (73%) of the title compound as a white solid: mp 263−264

ºC; IR (KBr) 3423, 1623, 1603, 1509, 1447, 1377, 1293, 1273, 1248, 1193, 1180, 1108,

-1 1 1030, 964, 947, 843, 820, 780, 541 cm ; H NMR (250 MHz, DMSO-d6) δ 10.66 (br s,

1H), 7.86 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H),

6.90−6.93 (m, 2H), 3.79 (s, 3H), 2.55 (s, 3H); LRMS calculated for C17H14NaO4S (M +

Na)+ 337.0510, found 337.04 (32%), 315.05 (100%).

163 GENERAL METHOD FOR PREPARATION OF 2-(ALKYLTHIO)ISOFLAVONES (8a−e and 9a−l).

Method A: To a stirred solution of α-oxoketene dithioacetal (0.5 mmol) in THF (5 mL)

was added a 1.0 M solution of tetrabutylammonium fluoride in THF (0.6 mL, 0.6 mmol)

at 0 ºC. After stirring at 0 ºC for 10 min, the reaction mixture was concentrated under

reduced pressure, and the residue was directly purified by silica gel column

chromatography (eluting with MeOH/CHCl3 or EtOAc/hexane) and recrystallization

(EtOAc/hexane) to yield desired product.

Method B: To a stirred mixture of a deoxybenzoin (1 mmol), carbon disulfide (0.6 mL,

10 mmol), alkyl halide (2.2 mmol), and tetrabutylammonium hydrogensulfate (34 mg,

0.1 mmol) in THF (3 mL) and water (1 mL) was slowly added 10 M solution of NaOH in water (1.2 mL, 12 mmol) at room temperature. A slight exothermic reaction and a color

change of the mixture were observed. The resulting mixture was vigorously stirred at room temperature for several hours, and the product was extracted with ethyl acetate (2 ×

10 mL). The combined organic layer was washed with water (10 mL) and then with brine (10 mL), dried over MgSO4, and filtered. The filtrate was concentrated under

reduced pressure, and the residue was purified by silica gel column chromatography

(eluting with MeOH/CHCl3 or EtOAc/hexane) and recrystallization (EtOAc/hexane) to yield desired product.

164 Method C: To a stirred solution of a deoxybenzoin (10 mmol) and carbon disulfide (1.2

mL, 20 mmol) in DMF (50 mL) was added sodium hydride (1.01 g, 40 mmol, 95%

powder). After vigorously stirring at 0 oC for 5 min, the reaction mixture was treated with alkyl halide (50 mmol) and stirred at 0 oC for 5 min. After warming to room

temperature over 1 h, the reaction mixture was quenched with saturated NH4Cl solution

at 0 oC and extracted with EtOAc twice (2 × 100 mL). The combined organic layer was

washed several times with H2O and then brine, dried over MgSO4, filtered, and

concentrated in vacuo. The residue was triturated in EtOAc/hexane, and the precipitated

solid was collected by filtration. The collected solid was washed several times with

EtOAc/hexane and recrystallized (EtOAc/hexane) to give desired product.

7-Methoxy-2-(methylthio)-3-phenyl-4H-1-benzopyran-4-one (8a):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a

starting deoxybenzoin and iodomethane (0.12 mL, 2.4 mmol) as an alkyl halide, 0.260 g

(87%) of the title compound was obtained as a white solid (Method B); Using 1-(2-

hydroxy-4-methoxyphenyl)-2-phenylethanone (2.42 g, 10 mmol) as a starting deoxybenzoin and iodomethane (2.52 mL, 50 mmol) as an alkyl halide, 2.61 g (87%) of the title compound was obtained as a white solid (Method C): mp 140−142 ºC (lit.

165 136−139 ºC) [190]; IR (KBr) 1629, 1619, 1584, 1541, 1499, 1432, 1373, 1349, 1319,

1252, 1201, 1098, 1017, 942, 835, 776, 757, 703, 661, 597, 544 cm-1; 1H NMR (400

MHz, CDCl3) δ 8.13 (d, J = 8.9 Hz, 1H), 7.32−7.44 (m, 5H), 6.96 (dd, J = 8.9, 2.4 Hz,

13 1H), 6.84 (d, J = 2.3 Hz, 1H), 3.91 (s, 3H), 2.53 (s, 3H); C NMR (62.9 MHz, CDCl3) δ

173.96, 164.41, 164.01, 158.52, 132.64, 131.05, 128.88, 128.68, 128.34, 122.29, 117.66,

+ 114.62, 100.10, 56.30, 14.12; HRMS calculated for C17H14NaO3S (M + Na) 321.0561, found 321.0551.

7-Methoxy-3-(4-methylphenyl)-2-(methylthio)-4H-1-benzopyran-4-one (8b):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (0.256 g, 1.0 mmol) as a starting deoxybenzoin and iodomethane (0.12 mL, 2.4 mmol) as an alkyl halide,

0.281 g (90%) of the title compound was obtained as a white solid (Method B); Using 1-

(2-hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (2.56 g, 10 mmol) as a starting deoxybenzoin and iodomethane (2.52 mL, 50 mmol) as an alkyl halide, 2.63 g

(84%) of the title compound was obtained as a white solid (Method C): mp 188−189.5

ºC; IR (KBr) 1632, 1612, 1544, 1510, 1500, 1433, 1368, 1334, 1287, 1265, 1196, 1149,

1116, 1067, 1025, 951, 841, 813, 776, 744, 594, 553, 521, 507 cm-1; 1H NMR (400 MHz,

CDCl3) δ 8.14 (d, J = 8.9 Hz, 1H), 7.20−7.26 (m, 4H), 6.95 (dd, J = 8.9, 2.3 Hz, 1H),

166 6.83 (d, J = 2.3 Hz, 1H), 3.90 (s, 3H), 2.53 (s, 3H), 2.37 (s, 3H); 13C NMR (62.9 MHz,

CDCl3) δ 174.08, 164.20, 163.96, 158.51, 138.50, 130.84, 129.68, 129.58, 128.36,

122.24, 117.66, 114.55, 100.08, 56.28, 21.85, 14.14; HRMS calculated for C18H16NaO3S

(M + Na)+ 335.0718, found 335.0721.

7-Methoxy-3-(4-methoxyphenyl)-2-(methylthio)-4H-1-benzopyran-4-one (8c):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (0.272 g, 1.0

mmol) as a starting deoxybenzoin and iodomethane (0.12 mL, 2.4 mmol) as an alkyl

halide, 0.280 g (85%) of the title compound was obtained as a white solid (Method B);

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (2.72 g, 10 mmol) as a starting deoxybenzoin and iodomethane (2.52 mL, 50 mmol) as an alkyl halide,

2068 g (82%) of the title compound was obtained as a white solid (Method C): mp

169−170 ºC; IR (KBr) 1616, 1585, 1540, 1508, 1437, 1374, 1348, 1286, 1252, 1198,

1176, 1103, 1022, 943, 844, 823, 783, 659, 594, 542, 467 cm-1; 1H NMR (400 MHz,

CDCl3) δ 8.12 (d, J = 8.8 Hz, 1H), 7.24−7.27 (m, 2H), 6.93−6.96 (m, 3H), 6.83 (d, J =

13 2.3 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 2.52 (s, 3H); C NMR (62.9 MHz, CDCl3) δ

174.15, 164.31, 163.95, 159.90, 158.50, 132.24, 128.32, 124.69, 121.82, 117.61, 114.57,

167 + 114.40, 100.07, 56.28, 55.65, 14.15; HRMS calculated for C18H16NaO4S (M + Na)

351.0667, found 351.0668.

2-(Methylthio)-3-phenyl-7-(phenylmethoxy)-4H-1-benzopyran-4-one (8d):

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-phenylethanone (3.18 g, 10 mmol) as

a starting deoxybenzoin and iodomethane (2.52 mL, 50 mmol) as an alkyl halide, 3.25 g

(87%) of the title compound was obtained as a white solid (Method C): mp 130−131.5

ºC; IR (KBr) 1613, 1541, 1502, 1458, 1439, 1364, 1267, 1196, 1154, 1120, 1030, 954,

-1 1 943, 774, 752, 727, 700 cm ; H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.8 Hz, 1H),

7.32−7.45 (m, 10H), 7.04 (dd, J = 8.8, 2.3 Hz, 1H), 6.93 (d, J = 2.3 Hz, 2H), 5.16 (s, 2H),

13 2.52 (s, 3H); C NMR (62.9 MHz, CDCl3) δ 173.92, 164.39, 163.05, 158.42, 136.15,

132.61, 131.04, 129.19, 128.89, 128.85, 128.69, 128.45, 127.94, 122.36, 117.90, 115.07,

+ 101.22, 70.99, 14.12; HRMS calculated for C23H18NaO3S (M + Na) 397.0874, found

397.0874.

168

3-(4-Methoxyphenyl)-2-(methylthio)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (8e):

Using 1-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)-3,3-bis(methylthio)-2-propen-1-one (0.283 g, 0.5 mmol) as a substrate,

0.184 g (91%) of the title compound was obtained as a white solid (Method A); Using 1-

[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (0.348 g, 1.0

mmol) as a starting deoxybenzoin and iodomethane (0.12 mL, 2.4 mmol) as an alkyl

halide, 0.350 g (87%) of the title compound was obtained as a white solid (Method B);

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (3.48 g,

10 mmol) as a starting deoxybenzoin and iodomethane (2.52 mL, 50 mmol) as an alkyl halide, 3.56 g (88%) of the title compound was obtained as a white solid (Method C):

mp 134−135 ºC; IR (KBr) 1620, 1610, 1546, 1511, 1439, 1367, 1282, 1250, 1194, 1177,

-1 1 1151, 1113, 1027, 1003, 952, 836, 744, 703, 607, 531 cm ; H NMR (250 MHz, CDCl3)

δ 8.15 (d, J = 8.9 Hz, 1H), 7.33−7.46 (m, 5H), 7.26 (d, J = 8.7 Hz, 2H), 7.03 (dd, J = 8.9,

2.2 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 2.2 Hz, 1H), 5.15 (s, 2H), 3.82 (s, 3H),

13 2.52 (s, 3H); C NMR (62.9 MHz, CDCl3) δ 174.08, 164.47, 163.04, 159.93, 158.43,

136.18, 132.25, 129.19, 128.84, 128.45, 127.96, 124.65, 121.85, 117.80, 115.07, 114.42,

+ 101.22, 70.99, 55.66, 14.16; HRMS calculated for C24H20NaO4S (M + Na) 427.0980, found 427.0917.

169

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4- one (9a):

Using 1-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)-3,3-bis[(propen-2-yl)thio]-2-propen-1-one (0.309 g, 0.5 mmol) as a substrate, 0.195 g (91%) of the title compound was obtained as a white solid (Method

A); Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone

(0.348 g, 1.0 mmol) as a starting deoxybenzoin and ally bromide (0.208 mL, 2.4 mmol)

as an alkyl halide, 0.396 g (92%) of the title compound was obtained as a white solid

(Method B): mp 101−102 ºC; IR (KBr) 1618, 1509, 1438, 1363, 1342, 1291, 1247, 1176,

-1 1 1152, 1101, 1030, 944, 821, 740, 696 cm ; H NMR (250 MHz, CDCl3) δ 8.13 (d, J =

8.8 Hz, 1H), 7.33−7.46 (m, 5H), 7.25 (d, J = 8.7 Hz, 2H), 7.02 (dd, J = 8.9, 2.3 Hz, 1H),

6.95 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 2.3 Hz, 1H), 5.87 (ddt, J = 16.9, 10.0, 6.9 Hz, 1H),

5.25 (dd, J = 16.9, 1.2 Hz, 1H), 5.11−5.06 (m, 3H), 3.82 (s, 3H), 3.69 (d, J = 6.9 Hz,

13 2H); C NMR (62.9 MHz, CDCl3) δ 174.34, 163.40, 163.10, 159.93, 158.38, 136.17,

133.30, 132.30, 129.20, 128.84, 128.48, 127.95, 124.64, 122.88, 119.22, 117.86, 114.96,

+ 114.36, 101.29, 71.01, 55.65, 34.59; HRMS calculated for C26H22NaO4S (M + Na)

453.1137, found 453.1123.

170

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(phenylmethyl)thio]-4H-1-benzopyran- 4-one (9b):

Using 1-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)-3,3-bis[(phenylmethyl)thio]-2-propen-1-one (0.360 g, 0.5 mmol) as a

substrate, 0.233 g (97%) of the title compound was obtained as a white solid (Method

A); Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone

(0.348 g, 1.0 mmol) as a starting deoxybenzoin and benzyl bromide (0.274 mL, 2.3 mmol) as an alkyl halide, 0.427 g (89%) of the title compound was obtained as a white solid (Method B): mp 131−132 ºC; IR (KBr) 1618, 1508, 1438, 1364, 1247, 1176, 1029,

-1 1 822, 697 cm ; H NMR (250 MHz, CDCl3) δ 8.14 (d, J = 8.9 Hz, 1H), 7.22−7.48 (m,

10H), 7.21 (d, J = 8.7 Hz, 2H), 7.03 (dd, J = 8.9, 2.3 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H),

6.89 (d, J = 2.3 Hz, 1H), 5.17 (s, 2H), 4.28 (s, 2H), 3.81 (s, 3H); 13C NMR (62.9 MHz,

CDCl3) δ 174.33, 163.61, 163.09, 159.91, 158.38, 136.61, 136.17, 132.25, 129.33,

129.25, 129.18, 128.88, 128.49, 128.15, 127.96, 124.52, 122.42, 117.82, 115.03, 114.36,

+ 101.25, 71.01, 55.65, 36.17; HRMS calculated for C30H24NaO4S (M + Na) 503.1293, found 503.1258.

171

3-(4-Methoxyphenyl)-2-[[2-(4-nitrophenyl)ethyl]thio]-7-(phenylmethoxy)-4H-1- benzopyran-4-one (9c):

Using 1-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4-(phenylmethoxy)phenyl]-2-(4-

methoxyphenyl)-3,3-bis[[2-(4-nitrophenyl)ethyl]thio]-2-propen-1-one (0.419 g, 0.5 mmol) as a substrate, 0.238 g (88%) of the title compound was obtained as a pale yellow solid (Method A); Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4- methoxyphenyl)ethanone (0.348 g, 1.0 mmol) as a starting deoxybenzoin and 4- nitrophenethyl bromide (0.506 g, 2.2 mmol) as an alkyl halide, 0.440 g (82%) of the title compound was obtained as a pale yellow solid (Method B): mp 149−150 ºC; IR (KBr)

-1 1 1618, 1509, 1438, 1344, 1247, 1176, 822 cm ; H NMR (250 MHz, CDCl3) δ 8.12 (d, J

= 8.9 Hz, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.36−7.46 (m, 5H), 7.30 (d, J = 8.6 Hz, 2H),

7.22 (d, J = 8.6 Hz, 2H), 7.03 (dd, J = 8.9, 2.0 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 6.82 (d,

J = 2.0 Hz, 1H), 5.17 (s, 2H), 3.82 (s, 3H), 3.31 (t, J = 7.3 Hz, 2H), ), 3.07 (t, J = 7.3 Hz,

13 2H); C NMR (100 MHz, CDCl3) δ 174.17, 163.13, 162.53, 160.00, 158.15, 147.22,

147.18, 136.11, 132.18, 129.89, 129.23, 128.89, 128.53, 127.92, 124.46, 124.21, 123.11,

117.76, 114.87, 114.36, 101.34, 71.02, 55.66, 36.66, 32.11; HRMS calculated for

+ C31H25NaO6S (M + Na) 562.1300, found 562.1304.

172

7-Methoxy-3-phenyl-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one (9d):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a starting deoxybenzoin and ally bromide (0.190 mL, 2.2 mmol) as an alkyl halide, 0.314 g

(96%) of the title compound was obtained as a white solid (Method B): mp 117−118 ºC;

IR (KBr) 1635, 1615, 1585, 1546, 1503, 1435, 1373, 1345, 1252, 1197, 1108, 1017, 942,

-1 1 922, 831, 753, 701, 662 cm ; H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 8.9 Hz, 1H),

7.30−7.44 (m, 5H), 6.96 (dd, J = 8.9, 2.4 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 5.83−5.94 (m,

1H), 5.27 (dd, J = 16.9, 1.2 Hz, 1H), 5.14 (dd, J = 10.1, 0.8 Hz, 1H), 3.91 (s, 3H), 3.70 (d,

13 J = 6.9 Hz, 2H); C NMR (62.9 MHz, CDCl3) δ 174.21, 164.12, 163.40, 158.50, 133.20,

132.61, 131.08, 128.82, 128.68, 128.39, 123.35, 119.27, 117.69, 114.51, 100.13, 56.31,

+ 34.54; HRMS calculated for C19H16NaO3S (M + Na) 347.0718, found 347.0705.

7-Methoxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (9e):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a

starting deoxybenzoin and benzyl bromide (0.262 mL, 2.2 mmol) as an alkyl halide,

173 0.365 g (97%) of the title compound was obtained as a white solid (Method B): mp

153−154 °C; IR (KBr) 1636, 1617, 1586, 1546, 1502, 1438, 1373, 1341, 1252, 1205,

-1 1 1106, 1016, 942, 831, 699, 661 cm ; H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 8.9 Hz,

1H), 7.22−7.41 (m, 10H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 4.30 (s,

13 2H), 3.91 (s, 3H); C NMR (62.9 MHz, CDCl3) δ 174.22, 164.10, 163.54, 158.48,

136.51, 132.52, 131.04, 129.32, 129.17, 128.81, 128.66, 128.39, 128.17, 122.97, 117.70,

+ 114.49, 100.19, 56.31, 36.17; HRMS calculated for C23H18NaO3S (M + Na) 397.0874,

found 397.0856.

7-Methoxy-3-phenyl-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9f):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a

starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.305 g (81%) of the title compound was obtained as a white solid

(Method B): mp 136−137 °C; IR (KBr) 1634, 1622, 1600, 1549, 1497, 1433, 1369, 1257,

1200, 1098, 1067, 1013, 943, 831, 775, 756, 703, 658, 570 cm-1; 1H NMR (400 MHz,

CDCl3) δ 8.53 (dd, J = 4.5, 1.5 Hz, 2H), 8.09 (d, J = 8.9 Hz, 1H), 7.33−7.42 (m, 3H),

7.24−7.27 (m, 4H), 6.93 (dd, J = 8.9, 2.4 Hz, 1H), 6.70 (d, J = 2.3 Hz, 1H), 4.21 (s, 2H),

13 3.87 (s, 3H); C NMR (100 MHz, CDCl3) δ 174.09, 164.21, 161.95, 158.33, 150.45,

174 146.38, 132.22, 130.95, 128.89, 128.86, 128.46, 124.04, 123.58, 117.59, 114.50, 100.16,

+ 56.31, 34.79; HRMS calculated for C22H17NNaO3S (M + Na) 398.0827, found 398.0818.

7-Methoxy-3-phenyl-2-[(3-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9g):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a

starting deoxybenzoin and 3-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.334 g (89%) of the title compound was obtained as a white solid

(Method B): mp 151.5−152 °C; IR (KBr) 1635, 1617, 1585, 1547, 1503, 1438, 1427,

1373, 1344, 1253, 1203, 1108, 1017, 943, 831, 754, 701, 662 cm-1; 1H NMR (400 MHz,

CDCl3) δ 8.62 (br s, 1H), 8.49 (d, J = 4.3 Hz, 1H), 8.10 (d, J = 8.9 Hz, 1H), 7.69 (d, J =

7.9 Hz, 1H), 7.35−7.41 (m, 3H), 7.23−7.28 (m, 3H), 6.95 (dd, J = 8.9, 2.1 Hz, 1H), 6.79

13 (d, J = 2.1 Hz, 1H), 4.27 (s, 2H), 3.91 (s, 3H); C NMR (100 MHz, CDCl3) δ 174.11,

164.24, 162.30, 158.42, 150.24, 149.35, 136.81, 132.93, 132.29, 130.96, 128.86, 128.80,

128.41, 124.11, 123.46, 117.60, 114.66, 100.10, 56.34, 33.21; HRMS calculated for

+ C22H17NNaO3S (M + Na) 398.0827, found 398.0840.

175

7-Methoxy-3-phenyl-2-[(2-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9h):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol) as a

starting deoxybenzoin and 2-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.345 g (92%) of the title compound was obtained as a white solid

(Method B): mp 168.5−169 °C; IR (KBr) 1634, 1617, 1586, 1546, 1502, 1431, 1373,

1344, 1252, 1202, 1153, 1106, 1016, 943, 831, 782, 752, 698, 661 cm-1; 1H NMR (400

MHz, DMSO-d6) δ 8.47 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.74 (dt,

J = 7.7, 1.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.30−7.39 (m, 3H), 7.34 (ddd, J = 7.6, 4.9,

0.9 Hz, 1H), 7.17−7.19 (m, 3H), 7.03 (dd, J = 8.8, 2.4 Hz, 1H), 4.54 (s, 2H), 3.88 (s,

13 3H); C NMR (100 MHz, DMSO-d6) δ 173.33, 164.34, 163.86, 158.54, 157.30, 150.20,

137.88, 133.14, 131.46, 129.03, 128.86, 127.75, 124.04, 123.43, 122.40, 117.23, 115.46,

+ 101.23, 57.02, 37.39; HRMS calculated for C22H17NNaO3S (M + Na) 398.0827, found

398.0819.

176

7-Methoxy-3-(4-methylphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9i):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (0.256 g, 1.0 mmol)

as a starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.335 g (86%) of the title compound was obtained as a white solid (Method B): mp 157−160 °C; IR (KBr) 1628, 1598, 1585, 1543, 1497, 1434, 1373,

-1 1 1343, 1254, 1198, 1182, 1099, 1016, 936, 837, 814 cm ; H NMR (400 MHz, CDCl3) δ

8.52 (d, J = 5.6 Hz, 2H), 8.08 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 5.5 Hz, 2H), 7.21 (d, J =

7.8 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 6.92 (dd, J = 8.9, 2.1 Hz, 1H), 6.69 (d, J = 2.0 Hz,

13 1H), 4.20 (s, 2H), 3.87 (s, 3H), 2.35 (s, 3H); C NMR (100 MHz, CDCl3) δ 174.20,

164.15, 161.79, 158.32, 150.46, 146.37, 138.72, 130.75, 129.67, 129.17, 128.46, 124.05,

123.46, 117.58, 114.44, 100.14, 56.29, 34.79, 21.85; HRMS calculated for

+ C23H19NNaO3S (M + Na) 412.0983, found 398.1004.

177

7-Methoxy-3-(4-methoxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9j):

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (0.272 g, 1.0

mmol) as a starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide (0.557 g,

2.2 mmol) as an alkyl halide, 0.332 g (82%) of the title compound was obtained as a white solid (Method B): mp 140−141 °C; IR (KBr) 1622, 1609, 1549, 1510, 1434, 1369,

1343, 1288, 1250, 1199, 1180, 1099, 1024, 961, 945, 835, 821, 778 cm-1; 1H NMR (400

MHz, CDCl3) δ 8.53 (d, J = 5.8 Hz, 2H), 8.08 (d, J = 8.9 Hz, 1H), 7.27 (d, J = 5.7 Hz,

2H), 7.18 (d, J = 8.6 Hz, 2H), 6.92−6.94 (m, 3H), 6.69 (d, J = 2.1 Hz, 1H), 4.21 (s, 2H),

13 3.88 (s, 3H), 3.80 (s, 3H); C NMR (100 MHz, CDCl3) δ 174.29, 164.14, 161.82,

160.05, 158.32, 150.42, 146.46, 132.16, 128.48, 124.24, 124.06, 123.11, 117.57, 114.42,

+ 114.40, 100.13, 56.29, 55.65, 34.82; HRMS calculated for C23H19NNaO4S (M + Na)

428.0932, found 428.0949.

178

3-Phenyl-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (9k):

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-phenylethanone (0.318 g, 1.0 mmol)

as a starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.386 g (86%) of the title compound was obtained as a pale yellow solid (Method B): mp 169−170 °C; IR (KBr) 1619, 1599, 1584, 1540, 1491,

1440, 1372, 1344, 1259, 1196, 1157, 1099, 991, 943, 836, 819, 781, 747, 695 cm-1; 1H

NMR (400 MHz, CDCl3) δ 8.51 (dd, J = 4.5, 1.6 Hz, 2H), 8.11 (d, J = 8.9 Hz, 1H),

7.34−7.45 (m, 8H), 7.22−7.26 (m, 4H), 7.03 (dd, J = 8.9, 2.3 Hz, 1H), 6.78 (d, J = 2.3 Hz,

13 1H), 5.15 (s, 2H), 4.19 (s, 2H); C NMR (100 MHz, CDCl3) δ 174.03, 163.22, 161.99,

158.23, 150.61, 146.23, 136.06, 132.19, 130.94, 129.25, 128.90, 128.88, 128.57, 127.88,

123.94, 123.59, 117.80, 115.11, 101.25, 71.03, 34.78; HRMS calculated for

+ C28H21NNaO3S (M + Na) 474.1140, found 474.1136.

179

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1- benzopyran-4-one (9l):

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (0.348 g,

1.0 mmol) as a starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide

(0.557 g, 2.2 mmol) as an alkyl halide, 0.402 g (84%) of the title compound was obtained

as a white solid (Method B): mp 169.5−170.5 °C; IR (KBr) 1616, 1539, 1509, 1440,

1414, 1371, 1342, 1294, 1251, 1197, 1173, 1156, 1100, 1029, 991, 943, 824, 735, 697,

-1 1 665 cm ; H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 5.8 Hz, 2H), 7.87 (d, J = 8.8 Hz,

1H), 7.35−7.49 (m, 7H), 7.30 (d, J = 2.3 Hz, 1H), 7.08−7.12 (m, 3H), 6.93 (d, J = 8.7 Hz,

13 2H), 5.26 (s, 2H), 4.39 (s, 2H), 3.75 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 173.39,

163.30, 162.91, 159.90, 158.31, 150.68, 147.69, 136.95, 132.64, 129.46, 129.08, 128.82,

127.86, 124.79, 124.70, 122.37, 117.32, 115.95, 114.55, 102.25, 70.95, 55.96, 34.03;

+ HRMS calculated for C29H23NNaO4S (M + Na) 504.1245, found 504.1238.

180

1-(2-Hydroxy-4-methoxyphenyl)-3,3-bis(methylthio)-2-phenyl-2-propen-1-one (6f):

To a stirred mixture of a 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.121 g,

0.5 mmol), carbon disulfide (0.3 mL, 5.0 mmol), iodomethane (0.06 mL, 2.4 mmol), and

tetrabutylammonium hydrogensulfate (17 mg, 0.05 mmol) in THF (1.5 mL) and water (1

mL) was slowly added 10 M solution of NaOH in water (0.6 mL, 6 mmol) at 0 ºC. The

resulting mixture was vigorously stirred at 0 ºC for 5 min, and the product was extracted

with ethyl acetate (2 × 10 mL). The combined organic layer was washed with water (10 mL) and then with brine (10 mL), dried over MgSO4, and filtered. The filtrate was

concentrated under reduced pressure, and the residue was purified by silica gel column

chromatography (eluting with EtOAc/hexane=1/4) to yield 0.158 g (91%) of the title

1 compound as a yellow gel: H NMR (400 MHz, CDCl3) δ 12.37 (br s, 1H), 7.56 (d, J =

8.6 Hz, 1H), 7.43−7.45 (m, 2H), 7.27−7.35 (m, 3H), 6.39−6.43 (m, 2H), 3.80 (s, 3H),

13 2.27 (s, 3H), 2.25 (s, 3H); C NMR (100 MHz, CDCl3) δ 199.21, 166.71, 166.46,

143.82, 137.06, 136.34, 134.11, 129.21, 128.96, 128.83, 113.88, 108.40, 101.43, 56.03,

18.05, 17.28.

181 7.2.5 OXIDATION

GENERAL METHOD FOR PREPARATION OF 2-(ALKYLSULFONYL)ISOFLAVONES (10a−e).

A mixture of 7-alkoxy-3-aryl-2-(methylthio)-4H-1-benzopyran-4-one (8.0 mmol) and 3-

chloroperoxybenzoic acid (mCPBA) (4.14 g, 24 mmol) in CH2Cl2 (80 mL) was stirred

under reflux for 2 h. After the solvent was removed under reduced pressure, the residue

was suspended in diethyl ether and hexane (1:1, 100 mL), sonicated, and placed in a

refrigerator overnight. The insoluble solid was collected by filtration, washed with

diethyl ether and hexane (1:1) several times, and recrystallized from ethyl acetate to give

desired product.

7-Methoxy-2-(methylsulfonyl)-3-phenyl-4H-1-benzopyran-4-one (10a):

Using the previous procedure and starting from 7-methoxy-2-(methylthio)-3-phenyl-4H-

1-benzopyran-4-one (2.39 g, 8.0 mmol), 2.59 g (98%) of the title compound was

obtained as a pale yellow solid: mp 208−210 ºC; IR (KBr) 1648, 1622, 1604, 1569, 1436,

1326, 1256, 1203, 1155, 1134, 1100, 1052, 1029, 962, 825, 772, 755, 583 cm-1; 1H NMR

(400 MHz, DMSO-d6) δ 7.94 (d, J = 8.9 Hz, 1H), 7.36−7.38 (m, 3H), 7.34 (d, J = 2.3 Hz,

1H), 7.25−7.27 (m, 2H), 7.12 (dd, J = 8.9, 2.3 Hz, 1H), 3.91 (s, 3H), 3.30 (s, 3H); 13C

182 NMR (100 MHz, DMSO-d6) δ 176.39, 165.70, 157.72, 157.47, 131.39, 129.98, 129.26,

128.41, 127.86, 124.68, 117.28, 117.03, 101.73, 57.30, 42.46; HRMS calculated for

+ C17H14NaO5S (M + Na) 353.0460, found 353.0459.

7-Methoxy-3-(4-methylphenyl)-2-(methylsulfonyl)-4H-1-benzopyran-4-one (10b):

Using the previous procedure and starting from 7-methoxy-3-(4-methylphenyl)-2-

(methylthio)-4H-1-benzopyran-4-one (2.50 g, 8.0 mmol), 2.62 g (95%) of the title

compound was obtained as a pale yellow solid: mp 221−222 ºC; IR (KBr) 1686, 1654,

1611, 1571, 1509, 1428, 1329, 1275, 1259, 1202, 1157, 1139, 1103, 997, 854, 842, 823

-1 1 cm ; H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 8.9 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H),

7.10−7.19 (m, 5H), 3.91 (s, 3H), 3.28 (s, 3H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-

d6) δ 176.47, 165.67, 157.72, 157.44, 138.63, 131.31, 129.06, 127.87, 126.88, 124.65,

117.25, 116.99, 101.70, 57.27, 42.48, 21.80; HRMS calculated for C18H16NaO5S (M +

Na)+ 367.0616, found 367.0621.

183

7-Methoxy-3-(4-methoxyphenyl)-2-(methylsulfonyl)-4H-1-benzopyran-4-one (10c):

Using the previous procedure and starting from 7-methoxy-3-(4-methoxyphenyl)-2-

(methylthio)-4H-1-benzopyran-4-one (2.63 g, 8.0 mmol), 2.68 g (93%) of the title

compound was obtained as a pale yellow solid: mp 227−228.5 ºC; IR (KBr) 1646, 1618,

1610, 1570, 1509, 1438, 1337, 1311, 1261, 1246, 1203, 1176, 1141, 1100, 1024, 973,

-1 1 955, 849, 773, 588, 539, 520, 482 cm ; H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J =

8.9 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 7.11 (dd, J = 8.9, 2.3 Hz,

1H), 6.94 (d, J = 8.7 Hz, 2H), 3.91 (s, 3H), 3.77 (s, 3H), 3.28 (s, 3H); 13C NMR (100

MHz, DMSO-d6) δ 176.59, 165.65, 160.31, 157.75, 157.43, 132.81, 127.88, 124.37,

121.66, 117.24, 116.96, 113.96, 101.69, 57.27, 55.96, 42.44; HRMS calculated for

+ C18H16NaO6S (M + Na) 383.0565, found 383.0569.

2-(Methylsulfonyl)-3-phenyl-7-(phenylmethoxy)-4H-1-benzopyran-4-one (10d):

Using the previous procedure and starting from 2-(methylthio)-3-phenyl-7-

(phenylmethoxy)-4H-1-benzopyran-4-one (3.0 g, 8.0 mmol), 3.12 g (96%) of the title

184 compound was obtained as a white solid: mp 198−199 ºC; IR (KBr) 1651, 1620, 1568,

1499, 1440, 1332, 1256, 1161, 1140, 1113, 1027, 1005, 975, 832, 775, 744, 700, 616 cm-

1 1 ; H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.9 Hz, 1H), 7.26-7.48 (m, 11H), 7.19

13 (dd, J = 8.9, 2.0 Hz, 1H), 5.28 (s, 2H), 3.29 (s, 3H); C NMR (100 MHz, DMSO-d6) δ

176.39, 164.66, 157.72, 157.37, 136.77, 131.94, 131.40, 129.99, 129.46, 129.27, 129.10,

128.77, 128.42, 127.94, 124.77, 117.45, 102.71, 71.16, 42.53; HRMS calculated for

+ C23H18NaO5S (M + Na) 429.0773, found 429.0761.

3-(4-Methoxyphenyl)-2-(methylsulfonyl)-7-(phenylmethoxy)-4H-1-benzopyran-4- one (10e):

Using the previous procedure and starting from 2-(methylthio)-3-phenyl-7-

(phenylmethoxy)-4H-1-benzopyran-4-one (3.24 g, 8.0 mmol), 3.39 g (97%) of the title

compound was obtained as a white solid: mp 226−227 ºC; IR (KBr) 1648, 1624, 1609,

1578, 1569, 1510, 1439, 1332, 1292, 1248, 1162, 1138, 1101, 1030, 972, 840, 827, 774,

-1 1 698, 613, 536, 525 cm ; H NMR (250 MHz, DMSO-d6) δ 7.95 (d, J = 8.9 Hz, 1H),

7.33−7.49 (m, 6H), 7.17−7.21 (m, 3H), 6.94 (d, J = 8.7 Hz, 2H), 5.29(s, 2H), 3.77 (s,

13 3H), 3.27 (s, 3H); C NMR (62.9 MHz, DMSO-d6) δ 176.57, 164.60, 160.32, 157.75,

157.32, 136.79, 132.83, 129.46, 129.10, 128.77, 127.95, 124.45, 121.67, 117.41, 117.38,

185 + 113.97, 101.67, 71.14, 55.96, 42.51; HRMS calculated for C24H20NaO6S (M + Na)

459.0878, found 459.0868.

3-(4-Methoxyphenyl)-2-(methylsulfinyl)-7-(phenylmethoxy)-4H-1-benzopyran-4- one (10f):

A mixture of 3-(4-methoxyphenyl)-2-(methylthio)-7-(phenylmethoxy)-4H-1- benzopyran-4-one (0.324 g, 0.8 mmol) and mCPBA (0.195 g, 1.12 mmol) in CHCl3 (4 mL) was stirred at 0 ºC for 0.5 h. After the solvent was removed under reduced pressure, the residue was suspended in diethyl ether and hexane (1:1, 10 mL), sonicated, and placed in a refrigerator overnight. The insoluble solid was collected by filtration and washed with diethyl ether and hexane (1:1) several times. The solid was further purified by silica gel column chromatography (eluting with MeOH/ CHCl3=3/97) and

recrystallization (MeOH/CHCl3) to yield 0.262 g (78%) of the title compound as a white

solid: mp 212−213 ºC; IR (KBr) 1647, 1619, 1610, 1512, 1438, 1341, 1293, 1252, 1188,

1172, 1101, 1068, 1028, 986, 970, 922, 839, 767, 700, 612 cm-1; 1H NMR (400 MHz,

DMSO-d6) δ 7.97 (d, J = 8.9 Hz, 1H), 7.31−7.48 (m, 5H), 7.43 (d, J = 2.4 Hz, 1H), 7.23

(d, J = 8.7 Hz, 2H), 7.18 (dd, J = 8.9, 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 5.30 (s, 2H),

13 3.77 (s, 3H), 2.95 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 175.70, 164.38, 163.01,

160.46, 158.46, 136.87, 133.05, 129.45, 129.08, 128.77, 128.02, 125.53, 122.19, 117.69,

186 117.08, 114.42, 102.58, 71.10, 56.05, 38.14; HRMS calculated for C24H20NaO5S (M +

Na)+ 443.0929, found 443.0916.

7.2.6 SUBSTITUTION

GENERAL METHOD FOR NUCLEOPHILIC SUBSTITUTION REACTIONS OF 2- (ALKYLSULFONYL)ISOFLAVONES (11a−j and 13a−i).

To a stirred solution of a nucleophile (as a sodium salt purchased or generated in situ by

treatment of sodium hydride) in DMF (3 mL) at 0 ºC was added a solution of 7-alkoxy-3-

aryl-2-(methylsulfonyl)-4H-1-benzopyran-4-one (1.0 mmol) in DMF (1 mL). After

stirring at 0 ºC for 0.5 h, most reactions were completed according to TLC. The reaction

mixture was allowed to warm to room temperature over 1 h, then cooled to 0 ºC, and

quenched with saturated aqueous NH4Cl solution. After the volatile solvents were

removed in vacuo, the residual solid was suspended in a mixture of water and EtOAc,

ultrasonicated for 5 min, and cooled to 0 ºC. Insoluble solid was collected by filtration

and the filter cake was washed with EtOAc/hexane mixture to give desire product. The

filtrate was extracted with EtOAc twice (2 × 10 mL), and the combined organic layer

was washed with brine, dried over MgSO4, filtered, and concentrated under reduced

pressure. The remnant was purified by silica gel column chromatography (eluting with

MeOH/CHCl3) to collect additional product. The combined solid was further purified by

recrystallization.

187

7-Methoxy-2-(4-methylphenylthio)-3-phenyl-4H-1-benzopyran-4-one (11a):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.20 g, 0.6 mmol) and sodium salt of 4-methylbenzenethiol

(0.177 g, 1.21 mmol, purchased), 0.204 g (90%) of the title compound was obtained as a pale yellow solid: mp 187−188 ºC; IR (KBr) 1633, 1615, 1585, 1540, 1489, 1435, 1371,

1344, 1251, 1198, 1100, 1017, 939, 837, 812, 781, 759, 703 cm-1; 1H NMR (400 MHz,

DMSO-d6) δ 7.90 (d, J = 8.9 Hz, 1H), 7.31−7.44 (m, 7H), 7.24 (d, J = 8.1 Hz, 2H), 7.01

(dd, J = 8.9, 2.3 Hz, 1H), 6.62 (d, J = 2.3 Hz, 1H), 3.79 (s, 3H), 2.31 (s, 3H); 13C NMR

(100 MHz, DMSO-d6) δ 174.04, 164.58, 161.72, 158.59, 140.03, 134.21, 133.28, 131.54,

131.14, 129.02, 128.95, 127.89, 125.38, 124.58, 117.25, 115.61, 100.77, 57.01, 21.68;

+ HRMS calculated for C23H18NaO3S (M + Na) 397.0874, found 397.0896.

7-Methoxy-2-(4-methylphenoxy)-3-phenyl-4H-1-benzopyran-4-one (11b):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.15 g, 0.46 mmol), p-cresol (0.10 g, 0.92 mmol), and sodium

188 hydride (0.02g, 0.92 mmol), 0.141 g (86%) of the title compound was obtained as a pale

yellow solid: mp 186−188 ºC; IR (KBr) 1627, 1563, 1498, 1439, 1385, 1355, 1257, 1195,

-1 1 1158, 1101, 1024, 960, 846, 783, 699, 504 cm ; H NMR (400 MHz, DMSO-d6) δ 7.95

(d, J = 8.8 Hz, 1H), 7.25−7.40 (m, 5H), 7.19 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.6 Hz,

2H),7.04 (dd, J = 8.8, 2.3 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 3.81 (s, 3H), 2.26 (s, 3H);

13 C NMR (100 MHz, DMSO-d6) δ 177.12, 164.67, 160.61, 155.33, 152.11, 135.30,

131.37, 131.32, 131.29, 128.65, 128.25, 127.75, 119.19, 116.94, 115.84, 108.71, 101.09,

+ 57.08, 21.14; HRMS calculated for C23H18NaO4 (M + Na) 381.1103, found 381.1118.

2-(4-Hydroxyphenylthio)-7-methoxy-3-phenyl-4H-1-benzopyran-4-one (11c):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.83 g, 2.50 mmol), 4-mercaptophenol (0.63 g, 5.0 mmol), and

sodium hydride (0.12g, 5.0 mmol), 0.85 g (90%) of the title compound was obtained as a

pale yellow solid (recrystallized from EtOAc/EtOH): mp 242−243 ºC; IR (KBr) 3170,

1625, 1606, 1574, 1535, 1496, 1436, 1380, 1350, 1259, 1204, 1170, 1103, 1016, 943,

-1 1 836, 781, 755, 698, 663, 520 cm ; H NMR (400 MHz, DMSO-d6) δ 9.98 (br s, 1H),

7.88 (d, J = 8.9 Hz, 1H), 7.30−7.44 (m, 7H), 6.99 (dd, J = 8.9, 2.3 Hz, 1H), 6.82 (d, J =

13 8.6 Hz, 2H), 6.55 (d, J = 2.3 Hz, 1H), 3.78 (s, 3H); C NMR (100 MHz, DMSO-d6) δ

189 173.80, 164.43, 163.02, 159.88, 158.46, 137.31, 133.22, 131.62, 129.04, 128.90, 127.85,

122.90, 117.44, 117.18, 116.07, 115.51, 100.61, 56.93; HRMS calculated for

+ C22H16NaO4S (M + Na) 399.0667, found 399.0671.

2-(4-Aminophenylthio)-7-methoxy-3-phenyl-4H-1-benzopyran-4-one (11d):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 4-aminothiophenol (0.125 g, 1.0 mmol),

and sodium hydride (0.024 g, 1.0 mmol), 0.157 g (84%) of the title compound was obtained as a pale yellow solid (recrystallized from EtOAc/hexane): mp 215−216.5 ºC;

IR (KBr) 3430, 3331, 3228, 1609, 1538, 1494, 1435, 1375, 1348, 1256, 1201, 1179,

-1 1 1100, 1070, 1021, 943, 924, 835, 822, 764 cm ; H NMR (400 MHz, DMSO-d6) δ 7.88

(d, J = 8.9 Hz, 1H), 7.29−7.44 (m, 5H), 7.19 (d, J = 8.5 Hz, 2H), 7.00 (dd, J = 8.9, 2.3

Hz, 1H), 6.58 (d, J = 8.5 Hz, 2H), 6.54 (d, J = 2.3 Hz, 1H), 5.59 (br s, 2H), 3.80 (s, 3H);

13 C NMR (100 MHz, DMSO-d6) δ 173.93, 164.41, 164.24, 158.44, 151.23, 137.25,

133.14, 131.59, 129.11, 128.93, 127.91, 122.14, 117.08, 115.48, 115.36, 110.54, 100.52,

+ 56.89; HRMS calculated for C22H17NNaO3S (M + Na) 398.0827, found 398.0831.

190

2-(4-Hydroxyphenylthio)-3-phenyl-7-(phenylmethoxy)-4H-1-benzopyran-4-one (11e):

Using the previous procedure and starting from 2-(methylsulfonyl)-3-phenyl-7-

(phenylmethoxy)-4H-1-benzopyran-4-one (0.15 g, 0.37 mmol), 4-mercaptophenol (0.093 g, 0.74 mmol), and sodium hydride (0.018 g, 0.74 mmol), 0.152 g (91%) of the title compound was obtained as a pale yellow solid: mp 230−231.5 ºC; IR (KBr) 3422, 1615,

1577, 1496, 1439, 1374, 1349, 1253, 1222, 1102, 834, 697 cm-1; 1H NMR (400 MHz,

DMSO-d6) δ 10.00 (br s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.30−7.44 (m, 12H), 7.07 (dd, J

= 8.9, 2.3 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 2.3 Hz, 1H), 5.15 (s, 2H); 13C

NMR (100 MHz, DMSO-d6) δ 173.76, 163.41, 163.08, 159.96, 158.32, 137.37, 136.92,

133.18, 131.61, 129.37, 129.04, 129.00, 128.91, 128.81, 127.87, 122.82, 117.44, 117.35,

+ 117.31, 116.00, 101.79, 70.93; HRMS calculated for C28H20NaO4S (M + Na) 475.0980,

found 475.0957.

191

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-(phenylthio)-4H-1-benzopyran-4-one (11f):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-

(methylsulfonyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.185 g, 0.424 mmol) and

sodium salt of benzenethiol (0.124 g, 0.85 mmol, purchased), 0.184 g (93%) of the title

compound was obtained as a white solid: mp 154.5−157.5 ºC; IR (KBr) 1633, 1618,

1610, 1511, 1438, 1367, 1338, 1289, 1250, 1191, 1177, 1153, 1098, 1033, 1012, 941,

-1 1 833, 751, 740, 690 cm ; H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 8.9 Hz, 1H),

7.50−7.52 (m, 2H), 7.30−7.43 (m, 8H), 7.24 (d, J = 8.7 Hz, 2H), 7.08 (dd, J = 8.9, 2.3 Hz,

1H), 6.97 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 2.3 Hz, 1H), 5.14 (s, 2H), 3.76 (s, 3H); 13C

NMR (100 MHz, DMSO-d6) δ 174.23, 163.51, 161.20, 159.97, 158.45, 136.89, 133.91,

132.76, 130.46, 130.02, 129.37, 129.29, 128.99, 128.77, 127.93, 125.11, 124.60, 117.38,

+ 116.06, 114.48, 101.81, 70.93, 55.99; HRMS calculated for C29H22NaO4S (M + Na)

489.1137, found 489.1098.

192

2-(4-Hydroxyphenylthio)-3-(4-methoxyphenyl)-7-(phenylmethoxy)-4H-1- benzopyran-4-one (11g):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-

(methylsulfonyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (7.50 g, 17.183 mmol), 4-

mercaptophenol (4.40 g, 34.37 mmol), and sodium hydride (0.825 g, 34.37 mmol), 7.97

g (96%) of the title compound was obtained as a white solid (recrystallized from EtOH):

mp 228−229 ºC; IR (KBr) 3163, 1623, 1601, 1577, 1538, 1508, 1493, 1440, 1369, 1345,

1275, 1247, 1205, 1178, 1103, 1029, 947, 835, 734, 696 cm-1; 1H NMR (250 MHz,

DMSO-d6) δ 9.98 (br s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.42−7.30 (m, 7H), 7.23 (d, J =

8.7 Hz, 2H), 7.06 (dd, J = 8.9, 2.2 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.6 Hz,

2H), 6.65 (d, J = 2.2 Hz, 1H), 5.15 (s, 2H), 3.77 (s, 3H); 13C NMR (62.9 MHz, DMSO-

d6) δ 173.90, 163.33, 162.99, 159.90, 158.27, 137.36, 136.92, 132.84, 129.37, 129.90,

128.81, 127.87, 125.05, 122.34, 117.38, 117.29, 116.19, 115.91, 114.51, 101.75, 70.91,

+ 55.98; HRMS calculated for C29H23O5S (M + H) 483.1266, found 483.1260.

193

2-(4-Hydroxyphenoxy)-3-(4-methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran- 4-one (11h):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-

(methylsulfonyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.96 g, 2.20 mmol),

hydroquinone (0.55 g, 5.0 mmol), and sodium hydride (0.12 g, 5.0 mmol), 0.958 g (93%)

of the title compound was obtained as a white solid (recrystallized from EtOAc/hexane):

mp 218−220 ºC; IR (KBr) 3183 1631, 1614, 1561, 1513, 1444, 1387, 1357, 1292, 1248,

1192, 1178, 1104, 1041, 1026, 1011, 970, 868, 833, 780, 750, 672, 543, 523 cm-1; 1H

NMR (250 MHz, DMSO-d6) δ 9.48 (br s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.43−7.30 (m,

7H), 7.10 (dd, J = 8.8, 2.3 Hz, 1H), 7.05 (d, J = 8.9, Hz, 2H), 7.00 (d, J = 2.3 Hz, 1H),

6.91 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.9 Hz, 2H), 5.16 (s, 2H), 3.73 (s, 3H); 13C NMR

(DMSO-d6) δ 177.15, 163.51, 161.07, 159.32, 155.64, 155.03, 146.28, 137.01, 132.58,

129.36, 128.96, 128.71, 127.77, 123.33, 120.84, 117.07, 116.97, 116.17, 114.14, 107.35,

+ 101.95, 70.97, 55.91; HRMS calculated for C29H23O6 (M + H) 467.1494, found

467.1488.

194

2-(4-Hydroxyphenylthio)-7-methoxy-3-(4-methylphenyl)-4H-1-benzopyran-4-one (11i):

Using the previous procedure and starting from 7-methoxy-3-(4-methylphenyl)-2-

(methylsulfonyl)-4H-1-benzopyran-4-one (0.86 g, 2.50 mmol), 4-mercaptophenol (0.63

g, 5.0 mmol), and sodium hydride (0.12g, 5.0 mmol), 0.81 g (83%) of the title compound

was obtained as a white solid (recrystallized from EtOAc/EtOH): mp 241−242 ºC; IR

(KBr) 3134, 1623, 1602, 1575, 1540, 1494, 1437, 1378, 1348, 1276, 1258, 1224, 1204,

1168, 1104, 1022, 947, 936, 834, 809, 781, 697, 663, 596, 521, 475 cm-1; 1H NMR (400

MHz, DMSO-d6) δ 9.97 (br s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H),

7.22 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.99 (dd, J = 8.9, 2.3 Hz, 1H), 6.82 (d,

J = 8.6 Hz, 2H), 6.54 (d, J = 2.3 Hz, 1H), 3.78 (s, 3H), 2.33 (s, 3H); 13C NMR (100 MHz,

DMSO-d6) δ 173.85, 164.39, 162.90, 159.89, 158.44, 138.21, 137.31, 131.46, 130.17,

129.64, 127.86, 122.75, 117.43, 117.18, 116.17, 115.47, 100.58, 56.92, 21.79; HRMS

+ calculated for C23H18NaO4S (M + Na) 413.0824, found 413.0811.

195

2-(4-Hydroxyphenylthio)-7-methoxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one (11j):

Using the previous procedure and starting from 7-methoxy-3-(4-methoxyphenyl)-2-

(methylsulfonyl)-4H-1-benzopyran-4-one (0.63 g, 1.75 mmol), 4-mercaptophenol (0.44

g, 3.5 mmol), and sodium hydride (0.084 g, 3.5 mmol), 0.67 g (94%) of the title

compound was obtained as a white solid (recrystallized from EtOAc/EtOH): mp

234−235.5 ºC; IR (KBr) 3105, 1623, 1600, 1570, 1535, 1509, 1493, 1434, 1380, 1350,

1283, 1245, 1219, 1200, 1169, 1101, 1028, 941, 832, 775, 696, 663, 597 cm-1; 1H NMR

(400 MHz, DMSO-d6) δ 9.97 (br s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 8.6 Hz,

2H), 7.23 (d, J = 8.6 Hz, 2H), 6.96−7.00 (m, 3H), 6.82 (d, J = 8.6 Hz, 2H), 6.53 (d, J =

13 2.3 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 173.94,

164.35, 162.93, 159.89, 159.83, 158.42, 137.31, 132.84, 127.87, 125.08, 122.41, 117.41,

117.16, 116.24, 115.45, 114.50, 100.56, 56.91, 55.97; HRMS calculated for

+ C23H18NaO5S (M + Na) 429.0773, found 439.0767.

196

7-Methoxy-3-phenyl-2-(4-pyridylthio)-4H-1-benzopyran-4-one hydrochloride (13a):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 4-mercaptopyridine (0.111 g, 1.0 mmol), and sodium hydride (0.024 g, 1.0 mmol), 0.141 g (78%) of the title compound was obtained as a yellow solid. Its HCl salt was prepared by acidification with HCl gas in

dichloromethane: mp >215 ºC (salt form, decomposed); IR (salt form, KBr) 3055, 1627,

1596, 1555, 1489, 1442, 1416, 1342, 1271, 1183, 1102, 1075, 1020, 821, 698 cm-1; 1H

NMR (free form, 400 MHz, DMSO-d6) δ 8.47 (dd, J = 4.6, 1.5 Hz, 2H), 7.95 (d, J = 8.9

Hz, 1H), 7.42 (dd, J = 4.6, 1.5 Hz, 2H), 7.29−7.40 (m, 5H), 7.07 (dd, J = 8.9, 2.3 Hz,

13 1H), 6.96 (d, J = 2.3 Hz, 1H), 3.83 (s, 3H); C NMR (free form, 100 MHz, DMSO-d6) δ

174.97, 165.05, 159.07, 157.45, 150.88, 142.68, 133.09, 131.16, 129.65, 129.21, 129.00,

127.97, 124.66, 117.34, 116.20, 101.03, 57.10; HRMS calculated for C21H16NO3S (M +

H)+ 362.0851 found 362.0843.

197

2-(1H-Imidazolyl-2-thio)-7-methoxy-3-phenyl-4H-1-benzopyran-4-one (13b):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 2-mercaptoimidazole (0.153 g, 1.5 mmol),

and sodium hydride (0.036 g, 1.5 mmol), 0.156 g (89%) of the title compound was obtained as a white solid (recrystallized from EtOH): mp 248−250 ºC (decomposed); IR

(KBr) 3434,1622, 1589, 1537, 1437, 1376, 1349, 1331, 1260, 1205, 1104, 1028, 939,

-1 1 831, 784, 760, 711 cm ; H NMR (400 MHz, DMSO-d6) δ 12.95 (br s, 1H), 7.91 (d, J =

8.9 Hz, 1H), 7.38−7.47 (m, 4H), 7.33−7.35 (m, 2H), 7.11 (s, 1H), 7.04 (dd, J = 8.9, 2.3

13 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H), 3.81 (s, 3H); C NMR (100 MHz, DMSO-d6) δ

173.85, 164.59, 160.91, 158.42, 132.57, 131.69, 131.48, 130.14, 129.27, 129.19, 127.97,

123.41, 122.38, 117.12, 115.75, 100.79, 56.99; HRMS calculated for C19H14N2NaO3S

(M + Na)+ 373.0623, found 373.0640.

198

7-Methoxy-2-(1-methyl-1H-imidazolyl-2-thio)-3-phenyl-4H-1-benzopyran-4-one (13c):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 2-mercapto-1-methylimidazole (0.171 g,

1.5 mmol), and sodium hydride (0.036 g, 1.5 mmol), 0.175 g (96%) of the title compound was obtained as a white solid (recrystallized from EtOAc): 172−173 ºC; IR

(KBr) 1640, 1618, 1590, 1499, 1437, 1368, 1344, 1253, 1198, 1099, 1016, 939, 783, 756,

-1 1 698 cm ; H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 1.0 Hz,

1H), 7.40−7.48 (m, 3H), 7.34−7.36 (m, 2H), 7.11 (d, J = 1.2 Hz, 1H), 7.03 (dd, J = 9.0,

2.3 Hz, 1H), 6.57 (d, J = 2.3 Hz, 1H), 3.82 (s, 3H), 3.68 (s, 3H); 13C NMR (100 MHz,

DMSO-d6) δ 173.88, 164.60, 160.85, 158.42, 132.59, 131.89, 131.46, 131.06, 129.30,

129.22, 127.98, 126.94, 123.53, 117.15, 115.70, 100.96, 57.02, 34.68; HRMS calculated

+ for C20H17N2O3S (M + H) 365.0960, found 365.0955.

199

7-Methoxy-3-phenyl-2-(1H-1,2,4-triazolyl-3-thio)-4H-1-benzopyran-4-one (13d):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 1H-1,2,4-triazole-3-thiol (0.152 g, 1.5 mmol), and sodium hydride (0.036 g, 1.5 mmol), 0.075 g (43%) of the title compound was obtained as a pale yellow solid (recrystallized from EtOH): mp 246−247 ºC; IR

(KBr) 1612, 1582, 1502, 1440, 1379, 1349, 1262, 1201, 1185, 1104, 1014, 969, 939, 830,

-1 1 783, 758, 700 cm ; H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.91 (d, J = 8.9 Hz,

1H), 7.36−7.45 (m, 3H), 7.32−7.34 (m, 2H), 7.04 (dd, J = 8.9, 2.3 Hz, 1H), 6.73 (d, J =

13 2.3 Hz, 1H), 3.81 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 174.51, 164.83, 159.47,

158.67, 146.80, 132.74, 131.27, 129.33, 129.17, 127.96, 126.01, 117.02, 116.01, 100.81,

+ 56.99; HRMS calculated for C18H13N3NaO3S (M + Na) 374.0575, found 374.0586.

200

7-Methoxy-3-phenyl-2-(2H-1,2,4-triazol-2-yl)-4H-1-benzopyran-4-one (13e):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.165 g, 0.5 mmol) and sodium salt of 1,2,4-triazole (0.091 g,

1.0 mmol, purchased), 0.144 g (90%) of the title compound was obtained as a white solid

(recrystallized from EtOAc): mp 188.5−189.5 ºC; IR (KBr) 1643, 1619, 1575, 1502,

1429, 1337, 1262, 1217, 1203, 1124, 1104, 1060, 993, 920, 837, 831, 785, 755, 705, 662

-1 1 cm ; H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.21 (s, 1H), 8.01 (d, J = 8.9 Hz,

1H), 7.28−7.29 (m, 4H), 7.11−7.16 (m, 3H), 3.89 (s, 3H); 13C NMR (100 MHz, DMSO- d6) δ 176.56, 165.48, 156.70, 153.62, 150.08, 147.29, 130.79, 130.67, 129.01, 128.92,

127.98, 119.39, 117.14, 116.57, 101.69, 57.25; HRMS calculated for C18H13N3NaO3 (M

+ Na)+ 342.0855, found 342.0843.

201

2-(1H-Imidazol-1-yl)-7-methoxy-3-phenyl-4H-1-benzopyran-4-one (13f):

Using the previous procedure and starting from 7-methoxy-2-(methylsulfonyl)-3-phenyl-

4H-1-benzopyran-4-one (0.330 g, 1.0 mmol) and sodium salt of imidazole (0.180 g, 2.0

mmol, purchased), 0.231 g (73%) of the title compound was obtained as a white solid

(recrystallized from EtOAc): mp 216−217 ºC; IR (KBr) 1640, 1619, 1575, 1492, 1441,

1404, 1342, 1266, 1255, 1201, 1104, 1053, 1016, 953, 908, 834, 784, 754, 738, 703, 648

-1 1 cm ; H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.9 Hz, 1H), 7.71 (s, 1H), 7.31−7.33

(m, 3H), 7.28 (d, J = 2.4 Hz, 1H), 7.17−7.20 (m, 3H), 7.12 (dd, J = 8.9, 2.4 Hz, 1H), 6.93

13 (s, 1H), 3.89 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 176.84, 165.15, 156.61, 151.04,

138.23, 131.39, 131.04, 129.92, 129.21, 129.00, 127.81, 120.31, 116.99, 166.20, 116.18,

+ 101.65, 57.14; HRMS calculated for C19H15N2O3 (M + H) 319.1082, found 319.1060.

202

2-(1H-Imidazol-1-yl)-7-methoxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one (13g):

Using the previous procedure and starting from 7-methoxy-3-(4-methoxyphenyl)-2-

(methylsulfonyl)-4H-1-benzopyran-4-one (0.36 g, 1.0 mmol) and sodium salt of imidazole (0.180 g, 2.0 mmol, purchased), 0.324 g (93%) of the title compound was obtained as a white solid (recrystallized from EtOH): mp 232−233 ºC; IR (KBr) 1639,

1611, 1578, 1515, 1441, 1405, 1342, 1291, 1253, 1201, 1174, 1054, 1024, 831, 816, 747

-1 1 cm ; H NMR (400 MHz, CDCl3) δ 8015 (d, J = 8.9 Hz, 1H), 7.62 (s, 1H), 7.12 (d, J =

8.7 Hz, 2H), 7.00−7.03 (m, 2H), 6.96 (s, 1H), 6.89−6.91 (m, 3H), 3.92 (s, 3H), 3.80 (s,

13 3H); C NMR (100 MHz, CDCl3) δ 177.20, 164.98, 160.18, 156.18, 150.21, 137.22,

131.71, 130.38, 128.41, 122.28, 118.77, 117.12, 115.46, 115.01, 114.43, 100.46, 56.41,

+ 55.67; HRMS calculated for C20H16N2NaO4 (M + Na) 371.1008, found 371.1015.

203

2-(1H-Imidazol-1-yl)-3-phenyl-7-(phenylmethoxy)-4H-1-benzopyran-4-one (13h):

Using the previous procedure and starting from 2-(methylsulfonyl)-3-phenyl-7-

(phenylmethoxy)-4H-1-benzopyran-4-one (0.41 g, 1.0 mmol) and sodium salt of

imidazole (0.180 g, 2.0 mmol, purchased), 0.323 g (82%) of the title compound was

obtained as a white solid (recrystallized from EtOAc): mp 152−153 ºC; IR (KBr) 1633,

1610, 1572, 1494, 1441, 1402, 1389, 1336, 1251, 1193, 1150, 1103, 1094, 1051, 1017,

-1 1 991, 908, 839, 790, 740, 699 cm ; H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.9 Hz,

1H), 7.58 (s, 1H), 7.35−7.45 (m, 8H), 7.19−7.21 (m, 2H), 7.10 (dd, J = 8.9, 2.3 Hz, 1H),

6.99 (s, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.92 (s, 1H), 5.19 (s, 2H); 13C NMR (100 MHz,

CDCl3) δ 176.90, 164.06, 156.12, 150.35, 137.19, 135.85, 130.53, 130.41, 129.47,

129.26, 129.13, 128.96, 128.49, 127.91, 118.77, 117.32, 116.08, 114.82, 101.58, 71.14;

+ HRMS calculated for C25H19N2O3 (M + H) 395.1395, found 395.1396.

204

2-(1H-Imidazol-1-yl)-3-(4-methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4- one (13i):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-

(methylsulfonyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.71 g, 1.63 mmol) and

sodium salt of imidazole (0.30 g, 3.25 mmol, purchased), 0.604 g (87%) of the title

compound was obtained as a white solid (recrystallized from EtOAc): mp 160−161 ºC;

IR (KBr) 1637, 1613, 1577, 1512, 1439, 1403, 1339, 1293, 1245, 1194, 1175, 1098,

-1 1 1055, 1018, 908, 823, 748, 700, 654 cm ; H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.9

Hz, 1H), 7.61 (s, 1H), 7.36−7.45 (m, 5H), 7.12 (d, J = 8.7 Hz, 2H), 7.09 (dd, J = 8.9, 2.3

Hz, 1H), 7.00 (s, 1H), 6.96 (d, J = 2.3 Hz, 1H), 6.95 (s, 1H), 6.90 (d, J = 8.7 Hz, 2H),

13 5.18 (s, 2H), 3.80 (s, 3H); C NMR (100 MHz, CDCl3) δ 177.17, 163.99, 160.19,

156.09, 150.23, 137.22, 135.87, 131.70, 130.38, 129.25, 128.94, 128.49, 127.90, 122.25,

118.77, 117.31, 116.01, 115.02, 114.47, 101.54, 71.12, 55.67; HRMS calculated for

+ C26H21N2O4 (M + H) 425.1501, found 425.1511.

205 7.2.7 ALKYLATION

GENERAL METHOD FOR INTRODUCTION OF 2-(PIPERIDIN-1-YL)ETHYL GROUP (14a−e).

To a stirred suspension of cesium carbonate (1.30 g, 4.0 mmol) in DMF (12 mL) at 0 oC was added 1-(2-chloroethyl)piperidine monohydrochloride (0.368 g, 2.0 mmol) in one portion, and the resulting mixture was allowed to warm to room temperature over 15 min.

A solution of 2-(4-hydroxyphenoxy)- or of 2-(4-hydroxyphenylthio)-7-alkoxy-3-aryl-

4H-1-benzopyran-4-one (1.0 mmol) in DMF (8 mL) was slowly added at room temperature, and the resulting suspension was stirred overnight. After removal of the insoluble solid by filtration, the filtrate was concentrated in vacuo, and the residue was distributed between H2O and EtOAc. The organic layer was washed several times with

H2O and then brine, dried over MgSO4, and concentrated in vacuo. The resulting solid

was purified by silica gel column chromatography (eluting with MeOH in CHCl3) and

then treated with HCl in EtOAc or CH2Cl2. The precipitated salt was collected by

filtration, washed with ether and EtOAc, and dried in vacuo to give the desired product as a white solid.

206

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[4-[2-(piperidin-1- yl)ethoxy]phenylthio]-4H-1-benzopyran-4-one hydrochloride (14a):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (1.0 g, 2.07 mmol), 1.08 g

(83%) of the title compound was obtained as a white solid: mp 197−199.5 ºC

(decomposed); IR (KBr) 3448, 2949, 1619, 1595, 1509, 1493, 1438, 1342, 1291, 1248,

-1 1 1175, 1107, 1025, 933, 826 cm ; H NMR (400 MHz, DMSO-d6) δ 10.79 (br s, 1H),

7.90 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H), 7.30−7.42 (m, 5H), 7.24 (d, J = 8.6 Hz,

2H), 7.05−7.10 (m, 3H), 6.98 (d, J = 8.6 Hz, 2H), 6.68 (d, J = 2.1 Hz, 1H), 5.15 (s, 2H),

4.46 (m, 2H), 3.77 (s, 3H), 3.44 (m 4H), 2.96 (m, 2H), 1.74 (m, 4H), 1.63 (m, 1H), 1.32

13 (m, 1H); C NMR (62.9 MHz, DMSO-d6) δ 173.97, 163.39, 162.33, 159.95, 159.55,

158.29, 136.92, 136.89, 132.83, 129.37, 129.04, 128.85, 127.95, 124.99, 122.86, 119.56,

117.32, 116.72, 115.79, 114.53, 101.93, 70.96, 63.41, 56.00, 55.41, 53.45, 53.40, 23.15,

+ 22.05; HRMS calculated for C36H36NO5S (M + H) 594.2314, found 594.2289.

207

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]- 4H-1-benzopyran-4-one hydrochloride (14b):

Using the previous procedure and starting from 2-(4-hydroxyphenoxy)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.584 g, 1.25 mmol),

0.656 g (89%) of the title compound was obtained as a white solid: mp 221−222 ºC

(decomposed); IR (KBr) 3436, 2941, 1633, 1614, 1569, 1514, 1505, 1440, 1373, 1352,

1292, 1247, 1198, 1179, 1100, 1025, 867, 835, 733, 696 cm-1; 1H NMR (250 MHz,

DMSO-d6) δ 10.92 (br s, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.31−7.42 (m, 7H), 7.22 (d, J =

8.9 Hz, 2H), 7.11 (dd, J = 8.8, 2.0 Hz, 1H), 7.03−6.99 (m, 3H), 6.91 (d, J = 8.5 Hz, 2H),

5.17 (s, 2H), 4.40 (m, 2H), 3.73 (s, 3H), 3.42 (m 4H), 2.96 (m, 2H), 1.76 (m, 4H), 1.63

13 (m, 1H), 1.36 (m, 1H); C NMR (62.9 MHz, DMSO-d6) δ 177.24, 163.57, 160.69,

159.38, 155.61, 155.12, 148.21, 136.99, 132.53, 129.37, 128.98, 128.71, 127.81, 123.18,

120.74, 117.10, 116.75, 116.20, 114.16, 107.97, 102.03, 70.98, 63.67, 55.93, 55.47,

+ 53.45, 23.13, 22.08; HRMS calculated for C36H36NO6 (M + H) 578.2542, found

578.2525.

208

7-Methoxy-3-phenyl-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4- one hydrochloride (14c):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-7-methoxy-3-

phenyl-4H-1-benzopyran-4-one (0.414 g, 1.10 mmol), 0.483 g (84%) of the title compound was obtained as a white solid: mp 188.5−189.5 ºC; IR (KBr) 3433, 2946,

1637, 1619, 1590, 1491, 1437, 1371, 1343, 1255, 1200, 1175, 1101, 1028, 1013, 942,

-1 1 841, 783, 760, 705 cm ; H NMR (400 MHz, DMSO-d6) δ 10.78 (br s, 1H), 7.90 (d, J =

8.9 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.31−7.45 (m, 5H), 7.06 (d, J = 8.8 Hz, 2H), 7.02

(dd, J = 8.9, 2.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 4.43 (m, 2H), 3.80 (s, 3H), 3.42 (m

4H), 2.94 (m, 2H), 1.74 (m, 4H), 1.63 (m, 1H), 1.35 (m, 1H); 13C NMR (100 MHz,

DMSO-d6) δ 173.88, 164.50, 162.33, 159.61, 158.49, 136.80, 133.16, 131.58, 129.06,

128.97, 127.91, 123.52, 119.45, 117.21, 116.76, 115.49, 100.82, 63.50, 57.01, 55.48,

+ 53.53, 23.24, 22.16; HRMS calculated for C29H30NO4S (M + H) 488.1895, found

488.1871.

209

7-Methoxy-3-(4-methylphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1- benzopyran-4-one hydrochloride (14d):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-7-methoxy-3-

(4-methylphenyl)-4H-1-benzopyran-4-one (0.430 g, 1.10 mmol), 0.395 g (73%) of the

title compound was obtained as a white solid: mp 197−199 ºC; IR (KBr) 3433, 2944,

1639, 1613, 1588, 1494, 1438, 1340, 1252, 1190, 1171, 1104, 1051, 931, 833 cm-1; 1H

NMR (400 MHz, DMSO-d6) δ 10.49 (br s, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 8.8

Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 7.02

(dd, J = 8.9, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 4.43 (m, 2H), 3.80 (s, 3H), 3.44 (m

4H), 3.00 (m, 2H), 2.33 (s, 3H), 1.76 (m, 4H), 1.64 (m, 1H), 1.34 (m, 1H); 13C NMR

(100 MHz, DMSO-d6) δ 173.94, 164.46, 162.17, 159.53, 158.46, 138.30, 136.79, 131.43,

130.12, 129.66, 127.93, 123.42, 119.67, 117.22, 116.76, 115.42, 100.83, 63.37, 56.99,

+ 55.45, 53.49, 23.18, 22.06, 21.80; HRMS calculated for C30H32NO4S (M + H) 502.2052,

found 502.2034.

210

7-Methoxy-3-(4-methoxyphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1- benzopyran-4-one hydrochloride (14e):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-7-methoxy-3-

(4-methoxyphenyl)-4H-1-benzopyran-4-one (0.203 g, 0.5 mmol), 0.216 g (78%) of the

title compound was obtained as a white solid: mp 161−161.5 ºC; IR (KBr) 3431, 2943,

1639, 1607, 1511, 1493, 1434, 1371, 1341, 1292, 1251, 1190, 1171, 1103, 1055, 1034,

-1 1 939, 928, 839, 815 cm ; H NMR (400 MHz, DMSO-d6) δ 10.69 (br s, 1H), 7.89 (d, J =

8.9 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H),

7.01 (dd, J = 8.9, 2.3 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.58 (d, J = 2.2 Hz, 1H), 4.44 (m,

2H), 3.79 (s, 3H), 3.77 (s, 3H), 3.44 (m 4H), 2.96 (m, 2H), 1.76 (m, 4H), 1.65 (m, 1H),

13 1.33 (m, 1H); C NMR (100 MHz, DMSO-d6) δ 174.03, 164.43, 162.22, 159.95, 159.52,

158.45, 136.78, 132.83, 127.93, 125.03, 123.07, 119.70, 117.20, 116.75, 115.40, 114.52,

100.80, 63.39, 56.99, 56.00, 55.42, 53.47, 23.16, 22.06; HRMS calculated for

+ C30H32NO5S (M + H) 518.2001, found 518.1965.

211 7.2.8 DEPROTECTION

GENERAL METHOD FOR DEPROTECTION USING BORON TRIBROMIDE (12a−g, 15a−b, 15e−f, 16a−b, and 16e−f).

To a stirred solution of 2-substituted 7-alkoxy-3-aryl-4H-1-benzopyran-4-one (0.5

mmol) in CH2Cl2 (10 mL) was slowly added a 1.0 M solution of BBr3 (2.0 mL, 2 mmol) at 0 oC, and the resulting suspension was allowed to warm to room temperature and

stirred overnight. After cooling to 0 oC, the reaction mixture was quenched with water

and concentrated under reduced pressure. The residue was suspended in a mixture of

water and EtOAc, and the insoluble product was collected by filtration. The filtrate was extracted with EtOAc twice (2 × 20 mL), and the combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give

additional product. The combined solid was purified by silica gel column

chromatography (eluting with MeOH/CHCl3) and/or directly applied to recrystallization.

7-Hydroxy-3-(4-hydroxyphenyl)-2-(methylthio)-4H-1-benzopyran-4-one:

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-(methylthio)-7-

(phenylmethoxy)-4H-1-benzopyran-4-one (0.102 g, 0.25 mmol), 0.066 g (87%) of the

title compound was obtained as a white solid: mp 296−298 ºC (decomposed); IR (KBr)

212 3254, 1618, 1603, 1577, 1513, 1498, 1451, 1381, 1251, 1220, 1199, 1174, 1110, 949,

-1 1 830 cm ; H NMR (250 MHz, DMSO-d6) δ 10.70 (br s, 1H), 9.48 (br s, 1H), 7.82 (d, J =

9.2 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 6.85−6.89 (m, 2H), 6.75 (d, J = 8.6 Hz, 2H), 2.50

13 (s, 3H); C NMR (62.9 MHz, DMSO-d6) δ 173.35, 164.21, 162.89, 158.51, 157.96,

132.66, 128.05, 123.57, 121.40, 116.26, 115.83, 115.78, 102.68, 14.02; HRMS

+ calculated for C16H12NaO4S (M + Na) 323.0354, found 323.0342.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one (12a):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one (0.179 g, 0.416 mmol),

0.123 g (91%) of the title compound was obtained as a pale yellow solid: mp 230−232 ºC

(decomposed); IR (KBr) 3231, 1610, 1561, 1539, 1512, 1497, 1439, 1376, 1245, 1220,

-1 1 1194, 1174, 1103, 972, 949, 928, 846, 827, 809 cm ; H NMR (400 MHz, DMSO-d6) δ

10.72 (br s, 1H), 9.50 (br s, 1H), 7.81 (d, J = 9.3 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H),

6.85−6.88 (m, 2H), 6.74 (d, J = 8.6 Hz, 2H), 5.84 (ddt, J = 16.9, 9.9, 6.9 Hz, 1H), 5.27

(dd, J = 16.9, 1.4 Hz, 1H), 5.09 (dd, J = 9.9, 1.4 Hz, 1H), 3.75 (d, J = 6.9 Hz, 2H),; 13C

NMR (100 MHz, DMSO-d6) δ 173.61, 163.10, 163.07, 158.51, 158.01, 134.75, 132.67,

213 128.07, 123.49, 122.50, 119.27, 116.24, 115.83, 115.80, 102.70, 34.24; HRMS

+ calculated for C18H14NaO4S (M + Na) 349.0511, found 349.0529.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (12b):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (0.147 g, 0.305 mmol),

0.106 g (92%) of the title compound was obtained as a white solid: mp 261−264 ºC

(decomposed); IR (KBr) 3280, 1625, 1605, 1592, 1508, 1459, 1375, 1246, 1228, 1189,

-1 1 1174, 1111, 966, 948, 843, 821, 694 cm ; H NMR (400 MHz, DMSO-d6) δ 10.75 (br s,

1H), 9.49 (br s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.35−7.37 (m, 2H), 7.18−7.29 (m, 3H),

6.94 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 2.2 Hz, 1H), 6.86 (dd, J = 8.7, 2.2 Hz, 1H), 6.72 (d,

13 J = 8.5 Hz, 2H), 4.36 (s, 2H); C NMR (100 MHz, DMSO-d6) δ 173.51, 163.25, 163.06,

158.48, 158.01, 138.18, 132.61, 129.75, 129.40, 128.20, 128.06, 123.38, 121.96, 116.23,

+ 115.86, 115.81, 102.73, 35.38; HRMS calculated for C22H16NaO4S (M + Na) 399.0667,

found 399.0656.

214

7-Hydroxy-3-(4-hydroxyphenyl)-2-[[2-(4-nitrophenyl)ethyl]thio]-4H-1-benzopyran- 4-one (12c):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-2-[[2-(4-

nitrophenyl)ethyl]thio]-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.203 g, 0.376 mmol), 0.141 g (86%) of the title compound was obtained as a pale yellow solid: mp

250−253 ºC (decomposed); IR (KBr) 3230, 1625, 1607, 1561, 1543, 1519, 1543, 1374,

-1 1 1349, 1236, 1220, 1198, 1104, 946, 854, 817 cm ; H NMR (400 MHz, DMSO-d6) δ

10.67 (br s, 1H), 9.47 (br s, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 1H), 7.45

(d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.82 (dd, J = 8.7, 2.2 Hz, 1H), 6.79(d, J =

2.2 Hz, 1H), 6.72 (d, J = 8.5 Hz, 2H), 3.37 (t, J = 7.0 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H);

13 C NMR (100 MHz, DMSO-d6) δ173.52, 162.97, 162.83, 158.33, 157.98, 148.63,

146.91, 132.63, 130.95, 127.92, 124.14, 123.58, 122.42, 116.19, 115.78, 115.69, 102.81,

+ 36.57, 31.82; HRMS calculated for C23H17NNaO6S (M + Na) 458.0675, found 458.0670.

215

7-Hydroxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (12d):

Using the previous procedure and starting from 7-methoxy-3-phenyl-2-

[(phenylmethyl)thio]-4H-1-benzopyran-4-one (0.223 g, 0.596 mmol), 0.189 g (88%) of the title compound was obtained as a white solid: mp 215−217 ºC; IR (KBr) 3413, 1610,

1559, 1486, 1453, 1375, 1269, 1250, 1219, 1194, 1105, 970, 947, 848, 699 cm-1; 1H

NMR (400 MHz, DMSO-d6) δ 10.80 (br s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.20−7.37 (m,

8H), 7.13−7.19 (m, 2H), 6.94 (d, J = 2.2 Hz, 1H), 6.87 (dd, J = 8.7, 2.2 Hz, 1H), 4.38 (s,

13 2H); C NMR (100 MHz, DMSO-d6) δ 173.31, 163.47, 163.20, 158.54, 138.11, 133.26,

131.46, 129.76, 129.41, 128.99, 128.77, 128.24, 128.08, 122.21, 116.20, 115.97, 102.79,

+ 35.39; HRMS calculated for C22H16NaO3S (M + Na) 383.0718, found 383.0710.

7-Hydroxy-3-phenyl-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (12e):

Using the previous procedure and starting from 7-methoxy-3-phenyl-2-[(4-

pyridylmethyl)thio]-4H-1-benzopyran-4-one (0.165 g, 0.439 mmol), 0.098 g (62%) of

216 the title compound was obtained as a pale yellow solid: mp 229−230 ºC (decomposed);

IR (KBr) 3427, 1617, 1584, 1544, 1504, 1417, 1366, 1260, 1191, 1102, 1015, 969, 945,

-1 1 843, 701 cm ; H NMR (400 MHz, DMSO-d6) δ 10.80 (br s, 1H), 8.48 (d, J = 4.0 Hz,

2H), 7.80 (d, J = 8.4 Hz, 1H), 7.33−7.40 (m, 5H), 7.16 (d, J = 7.0 Hz, 2H), 6.86−6.88 (m,

13 2H), 4.38 (s, 2H); C NMR (100 MHz, DMSO-d6) δ 173.28, 163.19, 162.72, 158.48,

150.65, 147.82, 133.12, 131.45, 129.06, 128.87, 128.09, 124.61, 122.50, 116.14, 116.01,

+ 102.71, 34.05; HRMS calculated for C21H15NNaO3S (M + Na) 384.0670, found

384.0667.

7-Hydroxy-3-phenyl-2-[(3-pyridylmethyl)thio]-4H-1-benzopyran-4-one (12f):

Using the previous procedure and starting from 7-methoxy-3-phenyl-2-[(3-

pyridylmethyl)thio]-4H-1-benzopyran-4-one (0.181 g, 0.482 mmol), 0.082 g (47%) of

the title compound was obtained as a yellow solid: mp 194−197 ºC (decomposed); IR

(KBr) 3053, 1618, 1584, 1542, 1502, 1462, 1366, 1268, 1219, 1187, 1102, 970, 946, 843,

-1 1 782, 754, 701 cm ; H NMR (400 MHz, DMSO-d6) δ 10.82 (br s, 1H), 8.73 (br s, 1H),

8.53 (d, J = 4.4 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.53 (dd, J =

7.8, 5.1 Hz, 1H), 7.30−7.39 (m, 3H), 7.13−7.15 (m, 2H), 6.95 (d, J = 2.1 Hz, 1H), 6.89

13 (dd, J = 8.7, 2.1 Hz, 1H), 4.46 (s, 2H); C NMR (100 MHz, DMSO-d6) δ 173.51, 163.12,

217 162.83, 158.55, 148.07, 147.00, 140.30, 136.09, 132.92, 131.36, 129.15, 128.99, 128.13,

125.64, 122.61, 116.10, 116.04, 102.79, 32.23; HRMS calculated for C21H15NNaO3S (M

+ Na)+ 384.0670, found 384.0674.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (12g):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (0.173 g, 0.36

mmol), 0.117 g (86%) of the title compound was obtained as a pale yellow solid: mp

>240 ºC (decomposed); IR (KBr) 3430, 1621, 1607, 1560, 1513, 1499, 1369, 1263, 1234,

-1 1 1194, 1171, 1107, 950, 807 cm ; H NMR (400 MHz, DMSO-d6) δ 10.74 (br s, 1H),

9.52 (br s, 1H), 8.76 (d, J = 6.4 Hz, 2H), 7.94 (d, J = 6.4 Hz, 2H), 7.78 (d, J = 9.0 Hz,

1H), 6.97 (d, J = 8.5 Hz, 2H), 6.86−6.88 (m, 2H), 6.76 (d, J = 8.5 Hz, 2H), 4.58 (s, 2H);

13 C NMR (100 MHz, DMSO-d6) δ 173.49, 163.15, 161.71, 158.42, 158.17, 157.23,

144.66, 132.61, 128.07, 126.94, 123.07, 122.47, 116.14, 115.91, 115.89, 102.83, 34.15;

+ HRMS calculated for C21H15NNaO4S (M + Na) 400.0619, found 400.0627.

218

7-Hydroxy-3-(4-hydroxyphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1- benzopyran-4-one (15a):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4-one

hydrochloride (0.630 g, 1.0 mmol), 0.455 g (93%) of the title compound was obtained as

a pale yellow solid (recrystallized from EtOH): mp 172−173 ºC (decomposed); IR (KBr)

3243, 2940, 1593, 1508, 1493, 1444, 1371, 1276, 1248, 1196, 1171, 1110, 944, 828 cm-1;

1 H NMR (250 MHz, DMSO-d6) δ 7.79 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.10

(d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.78−6.83 (m, 3H), 6.32 (d, J = 2.0 Hz, 1H),

4.07 (t, J = 5.8 Hz, 2H), 2.63 (t, J = 5.8 Hz, 2H), 2.25-2.42 (m, 4H), 1.25−1.53 (m, 6H);

13 C NMR (62.9 MHz, DMSO-d6) δ 173.94, 163.12, 162.13, 160.65, 158.29, 158.08,

137.39, 132.78, 128.13, 123.34, 122.10, 118.47, 116.42, 116.07, 115.85, 115.83, 102.13,

+ 66.71, 58.13, 55.27, 26.41, 24.76; HRMS calculated for C28H28NO5S (M + H) 490.1688,

found 490.1669.

219

7-Hydroxy-3-(4-hydroxyphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-4H-1- benzopyran-4-one (15b):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-4H-1-benzopyran-4-one hydrochloride (0.154 g, 0.25 mmol), 0.119 g (92%) of the title compound was obtained as a white solid (recrystallized from EtOAc/EtOH): mp 188−190 ºC (decomposed); IR

(KBr) 3401, 2938, 1610, 1558, 1500, 1385, 1270, 1243, 1194, 1170, 1106, 1032, 1010,

-1 1 836 cm ; H NMR (250 MHz, DMSO-d6) δ 7.86 (d, J = 8.7 Hz, 1H), 7.17 (d, J = 8.4 Hz,

2H), 7.11 (d, J = 9.0 Hz, 2H), 6.85−6.93 (m, 3H), 6.72 (d, J = 8.4 Hz, 2H), 6.56 (d, J =

1.7 Hz, 1H), 4.01 (t, J = 5.7 Hz, 2H), 2.60 (t, J = 5.7 Hz, 2H), 2.28−2.46 (m, 4H),

13 1.28−1.55 (m, 6H); C NMR (62.9 MHz, DMSO-d6) δ 177.33, 163.38, 160.52, 157.43,

156.54, 155.07, 147.64, 132.50, 128.11, 121.60, 120.61, 116.39, 115.86, 115.84, 115.44,

107.70, 102.60, 66.86, 58.22, 55.26, 26.41, 24.77; HRMS calculated for C28H28NO6 (M

+ H)+ 474.1916, found 474.1900.

220

7-Hydroxy-3-phenyl-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4- one hydrochloride (15e):

Using the previous procedure and starting from 7-methoxy-3-phenyl-2-[4-[2-(piperidin-

1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4-one hydrochloride (0.133 g, 0.254 mmol),

0.110 g (85%) of the title compound was obtained as a white solid: mp 214−217 ºC; IR

(KBr) 3429, 2946, 1620, 1591, 1492, 1452, 1362, 1249, 1176, 1104, 942cm-1; 1H NMR

(400 MHz, DMSO-d6) δ 10.81 (br s, 1H), 38 (br s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.53 (d,

J = 8.8 Hz, 2H), 7.31−7.52 (m, 5H), 7.07 (d, J = 8.8 Hz, 2H), 6.86 (dd, J = 8.7, 2.1 Hz,

1H), 6.41 (d, J = 2.1 Hz, 1H), 4.43 (m, 2H), 3.46 (m 4H), 2.98 (m, 2H), 1.76 (m, 4H),

13 1.65 (m, 1H), 1.36 (m, 1H); C NMR (100 MHz, DMSO-d6) δ 173.79, 163.41, 162.01,

159.64, 158.38, 137.27, 133.22, 131.61, 129.05, 128.92, 128.10, 122.66, 119.37, 116.65,

116.11, 116.03, 102.26, 63.36, 55.48, 53.50, 23.20, 22.05; HRMS calculated for

+ C28H28NO4S (M + H) 474.1739, found 474.1705.

221

7-Hydroxy-3-(4-methylphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1- benzopyran-4-one hydrochloride (15f):

Using the previous procedure and starting from 7-methoxy-3-(4-methylphenyl)-2-[4-[2-

(piperidin-1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4-one hydrochloride (0.454 g,

0.844 mmol), 0.360 g (81%) of the title compound was obtained as a white solid: mp

>210 ºC (decomposed); IR (KBr) 3433, 2945, 1638, 1620, 1592, 1494, 1453, 1361, 1251,

-1 1 1179, 1102, 967, 938, 847, 808 cm ; H NMR (400 MHz, DMSO-d6) δ 10.72 (br s, 1H),

9.96 (br s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.2 Hz,

2H), 7.19 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.85 (dd, J = 8.7, 2.0 Hz, 1H),

6.39 (d, J = 2.0 Hz, 1H), 4.42 (m, 2H), 3.47 (m 4H), 3.00 (m, 2H), 2.32 (s, 3H), 1.76 (m,

13 4H), 1.65 (m, 1H), 1.37 (m, 1H); C NMR (100 MHz, DMSO-d6) δ 173.85, 163.29,

161.93, 159.62, 158.36, 138.23, 137.25, 131.46, 130.18, 129.65, 128.13, 122.53, 119.50,

116.64, 116.07, 116.03, 102.25, 63.30, 55.53, 53.54, 23.23, 22.00, 21.80; HRMS

+ calculated for C29H29NO4S (M + H) 488.1895, found 488.1883.

222

7-Hydroxy-3-(4-hydroxyphenyl)-2-(4-hydroxyphenylthio)-4H-1-benzopyran-4-one (16a):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-3-(4- methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.097 g, 0.20 mmol),

0.067 g (89%) of the title compound was obtained as a white solid: mp 287−289 ºC

(decomposed); IR (KBr) 3405, 3236, 1624, 1607, 1590, 1509, 1495, 1470, 1367, 1231,

-1 1 1177, 1170, 945, 829, 819 cm ; H NMR (250 MHz, DMSO-d6) δ 10.51 (br s, 1H),

10.00 (br s, 1H), 9.52 (br s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.10

(d, J = 8.3 Hz, 2H), 6.78−6.84 (m, 5H), 6.32 (d, J = 2.2 Hz, 1H); 13C NMR (62.9 MHz,

DMSO-d6) δ 173.90, 163.04, 162.61, 159.94, 158.25, 158.07, 137.79, 132.79, 128.12,

123.36, 121.70, 117.25, 116.19, 116.08, 115.86, 115.84, 102.10; HRMS calculated for

+ C21H14NaO5S (M + Na) 401.0460, found 401.0436.

223

7-Hydroxy-3-(4-hydroxyphenyl)-2-(4-Hydroxyphenoxy)-4H-1-benzopyran-4-one (16b):

Using the previous procedure and starting from 2-(4-hydroxyphenoxy)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.125 g, 0.268 mmol),

0.073 g (75%) of the title compound was obtained as a white solid: mp 287−289 ºC; IR

(KBr) 3311, 3225, 1626, 1544, 1499, 1455, 1391, 1255, 1187, 1032, 836 cm-1; 1H NMR

(250 MHz, DMSO-d6) δ 10.66 (br s, 1H), 9.45 (br s, 1H), 9.37 (br s, 1H), 7.85 (d, J = 8.7

Hz, 1H), 7.17 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.87 (dd, J = 8.7, 2.0 Hz, 1H),

13 6.70−6.74 (m, 4H), 6.56 (d, J = 2.0 Hz, 1H); C NMR (62.9 MHz, DMSO-d6) δ 177.29,

163.24, 160.78, 157.41, 155.51, 155.00, 146.38, 132.52, 128.10, 121.68, 120.72, 116.90,

+ 115.90, 115.80, 115.45, 107.36, 102.60; HRMS calculated for C21H14NaO6 (M + Na)

385.0688, found 385.0688.

224

7-Hydroxy-2-(4-hydroxyphenylthio)-3-phenyl-4H-1-benzopyran-4-one (16e):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-7-methoxy-3-

phenyl-4H-1-benzopyran-4-one (0.132 g, 0.35 mmol), 0.099 g (78%) of the title

compound was obtained as a pale yellow solid: mp >257 ºC (decomposed); IR (KBr)

3344, 3260, 3168, 1617, 1600, 1578, 1494, 1458, 1370, 1253, 1190, 1107, 945, 831, 775,

-1 1 693 cm ; H NMR (400 MHz, DMSO-d6) δ 10.62 (br s, 1H), 10.00 (br s, 1H), 7.81 (d, J

= 8.7 Hz, 1H), 7.30−7.44 (m, 7H), 6.81−6.84 (m, 3H), 6.35 (d, J = 2.1 Hz, 1H); 13C

NMR (100 MHz, DMSO-d6) δ 173.69, 163.18, 162.83, 160.01, 158.31, 137.81, 133.22,

131.63, 129.04, 128.86, 128.13, 121.94, 117.29, 116.07, 115.97, 115.86, 102.16; HRMS

+ calculated for C21H14NaO4S (M + Na) 385.0511, found 385.0489.

7-Hydroxy-2-(4-hydroxyphenylthio)-3-(4-methylphenyl)-4H-1-benzopyran-4-one (16f):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-7-methoxy-3-

(4-methylphenyl)-4H-1-benzopyran-4-one (0.137 g, 0.35 mmol), 0.097 g (73%) of the

225 title compound was obtained as a pale yellow solid: mp 266−269 ºC (decomposed); IR

(KBr) 3441, 3128, 1624, 1606, 1585, 1509, 1493, 1454, 1371, 1323, 1273, 1258, 1222,

-1 1 1200, 1174, 1114, 939, 844, 829, 811, 600 cm ; H NMR (400 MHz, DMSO-d6) δ 10.61

(br s, 1H), 10.01 (br s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.22 (d, J

= 7.8 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 6.81−6.83 (m, 3H), 6.34 (d, J = 2.0 Hz, 1H),

13 2.32 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 173.75, 163.12, 162.71, 159.99, 158.29,

138.15, 137.82, 131.47, 130.17, 129.64, 128.13, 121.77, 117.26, 116.06, 115.97, 115.92,

+ 102.13, 21.79; HRMS calculated for C22H16NaO4S (M + Na) 399.0667, found 399.0682.

7.2.9 SELECTIVE DEBENZYLATION

GENERAL METHOD FOR THE SELECTIVE DEBENZYLATION USING BORON TRIFLUORIDE DIETHYL ETHERATE AND METHYL SULFIDE (12h, 15c, and 16c−d).

To a stirred suspension of 2-substituted 7-benzyloxy-3-aryl-4H-1-benzopyran-4-one (0.2 mmol) in Me2S (1.5 mL) and CH2Cl2 (1.5 mL) was slowly added boron trifluoride diethyl etherate (0.76 mL, 6 mmol) at room temperature. The resulting yellow solution

was vigorously stirred at room temperature overnight. After cooling to 0 oC, the reaction

mixture was quenched with water and concentrated under reduced pressure. The residue

was suspended in a mixture of water and EtOAc, and the insoluble product was collected

by filtration. The filtrate was extracted with EtOAc twice (2 × 15 mL), and the

combined organic layer was washed with brine, dried over MgSO4, filtered, and

concentrated under reduced pressure to give additional product. The combined solid was

226 purified by silica gel column chromatography (eluting with MeOH/CHCl3) and/or

directly applied to recrystallization.

7-Hydroxy-3-(4-methoxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (12h):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (0.200 g, 0.415

mmol), 0.137 g (84%) of the title compound was obtained as a white solid (recrystallized from EtOH): mp 197−200 °C; IR (KBr) 3400, 1623, 1606, 1561, 1512, 1455, 1363, 1293,

1247, 1219, 1180, 1106, 1076, 1023, 1003, 968, 856, 822 cm-1; 1H NMR (400 MHz,

DMSO-d6) δ 10.76 (br s, 1H), 8.79 (d, J = 6.4 Hz, 2H), 8.01 (d, J = 6.5 Hz, 2H), 7.80 (d,

J = 8.5 Hz, 1H), 7.11 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.86−6.89 (m, 2H),

13 4.56 (s, 2H), 3.73 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 173.45, 163.22, 161.74,

159.96, 158.48, 158.09, 144.07, 132.68, 128.10, 127.18, 124.80, 122.28, 116.13, 115.98,

+ 114.57, 102.87, 56.00, 34.12; HRMS calculated for C22H18NO4S (M + H) 392.0956,

found 392.0962.

227

7-Hydroxy-3-(4-methoxyphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1- benzopyran-4-one (15c):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[4-[2-(piperidin-1-yl)ethoxy]phenylthio]-4H-1-benzopyran-4-one

hydrochloride (0.126 g, 0.20 mmol), 0.086 g (86%) of the title compound was obtained

as a white solid: mp >218 ºC (decomposed); IR (KBr) 3434, 2933, 1619, 1595, 1509,

1494, 1449, 1371, 1293, 1246, 1175, 1108, 1028, 945, 820 cm-1; 1H NMR (250 MHz,

DMSO-d6) δ 7.80 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H),

7.02−6.95 (m, 4H), 6.82 (dd, J = 8.7, 2.0 Hz, 1H), 6.34 (d, J = 2.0 Hz, 1H), 4.07 (t, J =

5.8 Hz, 2H), 3.77 (s, 3H), 2.63 (t, J = 5.7 Hz, 2H), 2.28-2.43 (m, 4H), 1.29−1.51 (m,

13 6H); C NMR (62.9 MHz, DMSO-d6) δ 173.89, 163.15, 162.26, 160.69, 159.89, 158.32,

137.39, 132.84, 128.14, 125.09, 121.90, 118.34, 116.44, 116.06, 115.92, 114.51, 102.13,

+ 66.75; HRMS calculated for C29H30NO5S (M + H) 526.1664, found 526.1663.

228

7-Hydroxy-3-(4-methoxyphenyl)-2-(4-hydroxyphenylthio)-4H-1-benzopyran-4-one (16c):

Using the previous procedure and starting from 2-(4-hydroxyphenylthio)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.110 g, 0.228 mmol),

0.080 g (89%) of the title compound was obtained as a white solid: mp >253 °C

(decomposed); IR (KBr) 3424, 1623, 1604, 1583, 1510, 1494, 1451, 1373, 1323, 1293,

-1 1 1275, 1252, 1177, 1110, 1034, 945, 842, 830 cm ; H NMR (400 MHz, DMSO-d6) δ

10.59 (br s, 1H), 10.01 (br s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.22

(d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 6.80−6.83 (m, 3H), 6.33 (d, J = 2.1 Hz, 1H),

13 3.77 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 173.84, 163.08, 162.73, 159.96, 159.86,

158.27, 137.80, 132.84, 128.13, 125.09, 121.47, 117.25, 116.04, 116.02, 115.90, 114.50,

+ 102.11, 55.97; HRMS calculated for C22H16NaO5S (M + Na) 415.0616, found 415.0586.

229

7-Hydroxy-3-(4-methoxyphenyl)-2-(4-hydroxyphenoxy)-4H-1-benzopyran-4-one (16d):

Using the previous procedure and starting from 2-(4-hydroxyphenoxy)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.080 g, 0.171 mmol),

0.057 g (88%) of the title compound was obtained as a white solid: mp >210 °C

(decomposed); IR (KBr) 3269, 1626, 1597, 1568, 1515, 1505, 1461, 1388, 1297, 1250,

-1 1 1195, 1177, 1162, 1105, 1027, 1012, 843 cm ; H NMR (400 MHz, DMSO-d6) δ 10.68

(br s, 1H), 9.47 (br s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.02 (d, J =

8.9 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.88 (dd, J = 8.7, 2.3 Hz, 1H), 6.73 (d, J = 8.9 Hz,

13 2H), 6.56 (d, J = 2.2 Hz, 1H), 3.72 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 177.18,

163.28, 160.86, 159.26, 155.57, 155.01, 146.24, 132.58, 128.11, 123.45, 120.81, 116.90,

116.82, 115.85, 114.10, 106.93, 102.62, 55.90; HRMS calculated for C22H16NaO6 (M +

Na)+ 399.0845, found 399.0858.

GENERAL METHOD FOR SELECTIVE DEBENZYLATION USING CATALYTIC HYDROGENATION (15d and 17).

A solution of 2-substituted 7-benzyloxy-3-aryl-4H-1-benzopyran-4-one (0.5 mmol) and

ammonium formate (2.5 mmol) in anhydrous MeOH was vigorously stirred in the

230 presence of 10 % palladium on activated carbon (100% w/w) at room temperature for 10

min. The reaction mixture was filtered through a Celite pad and the filtrate was

concentrated under reduced pressure. The residue was purified by silica gel column

chromatography (eluting with MeOH/CHCl3) to give the product as a white solid.

7-Hydroxy-3-(4-methoxyphenyl)-2-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-4H-1- benzopyran-4-one (15d):

Using the previous procedure and starting from 3-(4-methoxyphenyl)-7-

(phenylmethoxy)-2-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-4H-1-benzopyran-4-one

hydrochloride (0.327 g, 0.566 mmol), 0.241 g (87%) of the title compound was obtained

as a white solid: mp 189−192 ºC; IR (KBr) 3408, 2935, 1630, 1608, 1570, 1514, 1501,

1456, 1378, 1293, 1246, 1195, 1160, 1101, 1024, 1009, 833 cm-1; 1H NMR (250 MHz,

DMSO-d6) δ 7.87 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.9 Hz, 2H),

6.88−6.95 (m, 5H), 6.60 (d, J = 1.9 Hz, 1H), 4.06 (t, J = 5.4 Hz, 2H), 3.72 (s, 3H), 2.72 (t,

J = 5.4 Hz, 2H), 2.40-2.58 (m, 4H), 1.30−1.54 (m, 6H); 13C NMR (62.9 MHz, DMSO-

d6) δ 177.23, 163.43, 160.61, 159.30, 156.45, 155.07, 147.62, 132.56, 128.09, 123.38,

120.71, 116.44, 115.91, 115.81, 114.11, 107.31, 102.63, 66.36, 57.82, 55.89, 54.99,

+ 25.94, 24.36; HRMS calculated for C29H30NO5S (M + H) 488.2073, found 488.2052.

231

2,7-Dihydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one (17):

Using the previous procedure and starting from 2-(4-hydroxyphenoxy)-3-(4-

methoxyphenyl)-7-(phenylmethoxy)-4H-1-benzopyran-4-one (0.203 g, 0.435 mmol),

0.107 g (87%) of the title compound was obtained as a white solid: mp 266-269 °C

(decomposed); IR (KBr) 3362, 1617, 1574, 1560, 1512, 1456, 1439, 1293, 1253, 1218,

-1 1 1173, 1142, 1101, 1022, 856, 840, 776, 536 cm ; H NMR (400 MHz, DMSO-d6) δ

10.91 (br s, 1H), 10.47 (br s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.25 (d, J = 8.7 Hz, 2H), 6.92

(d, J = 8.7 Hz, 2H), 6.76 (dd, J = 8.7, 2.2 Hz, 1H), 6.67 (d, J = 2.2 Hz, 1H), 3.75 (s, 3H);

13 C NMR (100 MHz, DMSO-d6) δ 163.35, 162.08, 161.55, 159.23, 154.88, 133.09,

125.86, 125.16, 114.29, 113.42, 109.18, 103.39, 102.66.

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