Natural products have played an important role in anticancer drug development for many decades. A recent review analyzing clinically approved anticancer drugs in Western countries and Japan during a twenty-five year period from 1981 to 2006 showed that, under the class of ―non-biologicals/vaccines‖, 63 of 81 (77.8%) anticancer drugs were either natural products or their derivatives, or synthesized molecules based on natural product pharmacophores. As part of a collaborative, multi-disciplinary approach to the discovery of new naturally occurring anticancer drugs, two medicinal plants, namely,

Hyptis brevipes and Vitex quinata, collected in Indonesia, were selected for further investigation.

The chloroform-soluble extract of a sample of the entire plant of H. brevipes showed activity against the MCF-7 human breast cancer cell line. Bioassay-guided

fractionation and purification of the CHCl3-soluble extract of H. brevipes led to the isolation of six new 5,6-dihydropyrone derivatives, namely, brevipolides A-F (342-346), together with seven known compounds. Brevipolides A-F (342-346), and a previously

ii ii known 5,6-dihydropyrone derivative (347), were assigned with the absolute configuration,

5R, 6S, 7S, and 9S, as elucidated by analysis of data obtained from their CD spectra and by Mosher ester reactions. Brevipolides B and D, as well as compound 347 exhibited

ED50 values of 6.1, 6.7 and 3.6 M against MCF-7 cells. Brevipolides A, B, and F, and

compound 349 (the known 5,6,3-trihydroxy-3,7,4-trimethoxyflavone) gave ED50 values of 5.8, 6.1 7.5, and 3.6 M against HT-29 cells, respectively. However, no significant cytotoxicity was found against Lu1 cells for any of the compounds isolated. When these compounds were subjected to evaluation in a panel of mechanism-based in vitro assays, compound 347 were found to be active in an enzyme-based ELISA NF-B p50 assay,

with an ED50 value of 15.3 M. In a mitochondrial transmembrane potential assay,

brevioplide C, compounds 348 and 349 showed ED50 values of 8.5, 75, and 310 nM, respectively. However, no potent activity was found in a proteasome inhibition assay for any of the isolated compounds.

A phytochemical investigation of the chloroform-soluble and ethyl acetate-soluble extracts of the leaves of V. quinata led to the isolation of a new -truxinate derivative

(378) and a new prostaglandin-like octadecanoid (379), together with 12 known compounds including flavonoids and quinic acid derivatives. All the isolates were tested

iii iii in a panel of human cancer cells, and (S)-5-hydroxy-7,4-dimethoxyflavanone (384) was

found to be an active principle with ED50 values of 6.7, 4.7, and 1.1 M against LNCaP

(hormone-dependent prostate cancer), Lu1 (human lung cancer), and MCF-7 (human breast cancer) cell lines, respectively. Compounds 382 and 384-390 were tested in an enzyme-based ELISA NF-B p65 assay to evaluate their potential in inhibiting the binding of NF-B to DNA. Compounds 382, 384, 385, and 390 showed activity, with

ED50 values of 22.8, 54.3, 17.8, 10.3 M, respectively.

iv iv

This dissertation is dedicated to my parents, my wife and son, and all of my family

members who have encouraged and supported me to accomplish this work.

v v

Acknowledgments

First of all, I would like to sincerely thank my advisor, Dr. A. Douglas Kinghorn, for his encouragement, generosity, guidance and support during the course of this study. I really appreciate for all the advice, help, and opportunities he has given to me.

I also would like to thank Drs. Esperanza J. Carcache de Blanco, James R. Fuchs, and Pui-Kai Li for serving as members of my dissertation defense committee and for reviewing this dissertation.

I would like to express my gratitude to Drs. Esperanza Carcache de Blanco and

Heebyung Chai for providing data related to the NF-κB, mitochondrial transmembrane potential, and cytotoxicity assays used, as well as helping me to learn related bioassay techniques. I am grateful to Dr. Steven M. Swanson of the University of Illinois at

Chicago for the result using the proteasomal inhibition assay.

I wish to thank Drs. Young-Won Chin and Bao-Ning Su for training on the various instrumentation or bioassays, as well as guiding research and discussing problems. I also thank Drs. Angela A. Salim and Soyoung Kim who spent a lot of time to teach me the

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NMR instrumentation methods.

One of the plants studied in this dissertation research was transferred from Dr. Marcy

J. Balunas, formerly of the University of Illinois at Chicago. I am grateful to her for providing me the valuable sample and for completing some initial phytochemical work.

This dissertation study was supported by grants U19 CA52956 and P01 CA125066 awarded to Dr. A Douglas Kinghorn, from the National Cancer Institute, National

Institutes of Health (NCI, NIH), Bethesda, MD. The plant Hyptis brevipes was collected through grant P01 CA48112 awarded to Dr. John M. Pezzuto, formerly of the University of Illinois at Chicago, by NCI, NIH. All plant collections were supported through formal befefit sharing agreements that were approved by the U.S. National Cancer Institute. This research was also supported by the Raymond W. Doskotch Graduate Fellowship Fund in

Medicinal Chemistry and Pharmacognosy from The Ohio State University. Other funding sources throughout my graduate work include teaching and research assistantships from the Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University.

I thank Mr. J. Fowble, College of Pharmacy, The Ohio State University, for facilitating the acquisition of the 300 and 400 MHz NMR spectra. We are grateful to the

Mass Spectrometry and Proteomics Facility of the OSU CCIC, for the mass spectra.

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I am grateful to my friends and colleagues in the College of Pharmacy, especially former and present members of Dr. Kinghorn’s lab, Drs. Ah-Reum Han, William Jones, and Alison Pawlus, and Ms. Lynette Bueno, Mr. Mark Bahar, Mr. Josh Fletcher, Mr. Jie

Li, and Mr. Patrick Still.

My deepest gratitude and appreciation go to my parents for their love, inspiration, encouragement, and always supporting me. Words can not adequately express my gratitude towards my wife, Dr. Li Pan, who endured many sacrifices during my graduate study period and helped as a colleague in resolving various research problems together. I am grateful to my son, Yihan (Russell), for filling my life with joy and motivation.

Furthermore, I wish to thank my brother, sister, and the rest of my family for their support and encouragement.

viii viii

Vita

EDUCATION 1991-1994…………. A.E. Chemical Engineering, Hunan University of Science and Technology, Xiangtan, People’s Republic of China 1998-2001…………. M.S. Chengdu Institute of Biology, The Chinese Academy of Sciences, Chengdu, People’s Republic of China 2002-2004…………. Graduate student, Hong Kong Baptist University, Hong Kong SAR, People’s Republic of China 2004-2010…………. Graduate student, College of Pharmacy, The Ohio State University

CAREER EXPERIENCE: 1994-1997…… Researcher, Guangli Resin Chemical Co. Ltd., Guang Dong, People’s Republic of China 1997-1998…… Teacher, the Affiliated Middle School of Hunan University of Science and Technology, Xiangtan, People’s Republic of China 2001-2002…… Associate Lecturer, Hunan University of Science and Technology, Xiangtan, People’s Republic of China.

AWARDS AND HONORS 1. General Travel Grant, 2009, American Society of Pharmacognosy. 2. Raymond W. Doskotch Fellowship, 2007-2008, College of Pharmacy, The Ohio State University. 3. Nature Sunshine Travel Grant, 2007, American Society of Pharmacognosy. 4. Jack L. Beal Graduate Student Award, 2006, College of Pharmacy, The Ohio State University. 5. Liu Yongling Award, 2001, Chinese Academy of Sciences. 6. Excellent Student Award, 1994, Hunan University of Science and Technology. 7. Excellent Student Award, 1993, Hunan University of Science and Technology. 8. Excellent Student Award, 1992, Hunan University of Science and Technology.

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NATIONAL MEETING PRESENTATIONS 1. Y. Deng, M.J. Balunas, J.-A. Kim, D.D. Lantvit, Y.-W. Chin, H. Chai, S. Sugiarso, L.B.S. Kardono, H.H.S. Fong, J.M. Pezzuto, S.M. Swanson, E.J. Carcache de Blanco, A.D. Kinghorn. ―Bioactive 5,6-dihydropyrone derivatives from Hyptis brevipes‖. 50th Anniversary Meeting of American Society of Pharmacognosy; Honolulu, Hawaii, June 2009; oral presentation. 2. Y. Deng, Y.-W. Chin, H. Chai, W.J. Keller, A.D. Kinghorn. ―New benzophenone derivatives from Morinda citrifolia (Noni) roots‖. 48th Annual Meeting of American Society of Pharmacognosy; Portland, Maine, July 2007; poster presentation. 3. Y. Deng, H. Chai, Y.-W. Chin, L.B.S. Kardono, S.Riswan, N.R. Farnsworth, A.D. Kinghorn. ―New prostaglandin-like octadecanoid and cytotoxic flavonoids from the leaves of Vitex quinata‖. 47th Annual Meeting of American Society of Pharmacognosy; Arlington, VA, July 2006; poster presentation.

Publications 1. Y. Deng, M.J. Balunas, J.-A. Kim, D.D. Lantvit, Y.-W. Chin, H. Chai, S. Sugiarso, L.B.S. Kardono, H.H.S. Fong, J.M. Pezzuto, S.M. Swanson, E.J. Carcache de Blanco, A.D. Kinghorn. Bioactive 5,6-dihydropyrone derivatives from Hyptis brevipes. Journal of Natural Products 2009, 72, 1165-1169. 2. M. Bahar, Y. Deng, J.N. Fletcher, A.D. Kinghorn. Plant-derived natural products. In drug discovery and development: an overview. In Selected Topics in the Chemistry of Natural Products. R. Ikan, Ed.; World Scientific Publishing, Singapore, 2008; pp 11-48. 3. Y. Deng, Y.-W. Chin, H.-B. Chai, W.J. Keller, A.D. Kinghorn. Anthraquinones with quinone reductase-inducing activity and benzophenones from Morinda citrifolia (Noni) roots. Journal of Natural Products 2007, 70, 2049-2052. 4. C. Dan, Y. Zhou, Y. Deng, S.L. Peng, L.S. Ding, M.L. Gross, S.X. Qiu. Cimicifugadine from Cimicifuga foetida, a new class of triterpene alkaloids with novel reactivity. Organic Letters 2007, 9, 1813-1816. 5. L. Pan, X.F. Zhang, Y. Deng, H. Wang, D.G. Wu, and X.D. Luo. Chemical constituents from the whole plant of Euphorbia altotibetica. Helvetica Chimica Acta 2003, 86, 2525-2532. 6. Y. Deng, S.L. Peng, Q. Zhan, X. Liao, and L.S. Ding. Clerodane diterpenoids from

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Kinostemon alborubrum. Helvetica Chimica Acta 2002, 85, 2547-2552. 7. Y. Deng, X.R. Zhan, L.S. Ding, and M.K. Wang. Triterpenoids from Rubus buergeri. Acta Botanica Sinica 2001, 43, 644-646. 8. Y. Deng, X.X. Ye, S.L. Peng, J. Liang, and L.S. Ding. 2001. Spectral analysis of aromatic acyl monosaccharoses. Chinese Journal of Analytic Laboratory 2001, 20 (Suppl.), 177. 9. Y. Deng, R. Liu, S.L. Peng, X.M. Zhu, and M.K. Wang. Chemical constituents of two species of Rubus. Chinese Journal of Analytic Laboratory 2001, 20 (Suppl.), 405-406.

Fields of Study

Major Field: Pharmacy

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Table of Contents

Abstract……………………………………………………………………………………ii Dedication………………………………………………...………………………………iv Acknowledgements…………………………………………………………………..…..vi Vita……………………………………………………………………………….…..…...ix Table of Contents……………………………………………………………....…...……xii List of Tables……………………………………………….………………….…….…xvii List of Figures………………………………………………………………...………xviii List of Abbreviations……………………………………………………………………xxi

Chapter 1: Plant-Derived Anticancer Drugs and Plant-Derived NF-B Inhibitors…...….1 A. Plant Derived Anticancer Drugs…………………………………………..…………1 1. Vinca alkaloids……………………………………………………………….……3 2. Podophyllotoxin derivatives……………………………………………...….…….4 3. Paclitaxel and derivatives………………………………………………..……..…8 4. Camptothecin derivatives………………………………………………….………..9

B. Plant Derived Nuclear Factor-B inhibitors………………………………………….12 1. Nuclear Factor B (NF-B) pathway as an anticancer drug target………………12 2. Plant-derived NF-B Inhibitors…………………………………………………16 2.1. Plant-derived inhibitor B kinase (IKK) inhibitors………………...………16 2.2. Plant-derived inhibitor B (B) phosphorylation suppressors……..………18 2.3. Plant-derived IB degradation blockers………...…………………………31 2.4. Plant-derived NF-B translocation inhibitors……...………………………33 2.5 Plant-derived NF-B DNA-binding inhibitors……………………………44 2.6. NF-B transactivation inhibitors…………………………..………………45

Chapter 2: Bioactive dihyro--pyrone derivatives and flavonoids from Hyptis brevipes…………………………….………………………….…………57

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A. Background on Hyptis brevipes………………………...………………….…………57 1. Lamiaceae………………………………………………………….……………...57 2. Genus Hyptis and Hyptis brevipes Piot.……………………………….…….…….58 3. Constituents of the genus Hyptis………………………………………..…………60 3.1. Pyrone and furanone derivatives…………………….….…………….….….60 3.2. Lignans……...………….….……………………………….……………..…61 3.3. Flavonoids……………………………………….………….……….………67 3.4. Diterpenoids………………………………………………...….……..……..68 3.5. Triterpenoids………….………………………..……………....….….……..76 3.6. Other constituents………………………………………………….………..81

B. Statement of problem………………………………………………..…………...….82

C. Experimental………………………………………………………………………...83 1. General experimental procedures…………………………………………….....84 2. Plant material…………………………………………………………..………..84 3. Solvent extraction of Hyptis brevipes…………………...…………….…..……84 4. Initial cytotoxicity assays on Hyptis brevipes extracts and fractions……..….…84 5. Column chromatography of the chloroform-soluble extract of Hyptis brevipes..85 6. Characterization of brevipolide A (342)…………………………………….…..87 7. Characterization of brevipolide B (343)…………………………………...…..88 8. Characterization of brevipolide C (344)…………………………………...…..88 8.1. Brevipolide C (344)……………………..………………………...…….89 8.2. Preparation of R- and S-MTPA ester derivatives of brevipolide C (344).89 8.3. R-MTPA ester of brevipolide C (344)……………………….………….89 8.4. S-MTPA ester of brevipolide C (344)……………………….………….89 9. Characterization of brevipolide D (345)……………………….…………...….89 10. Characterization of brevipolide E (346)…………………….…………….….91 11. Characterization of brevipolide F (347)……………………………….……..91 12. Characterization of (E)-1-{(1S,2S)-2-[(S)-hydroxy(R)-6-oxo-3,6-dihydro-2H- pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate (348)….………………………………………………………….………….….92 13. Characterization of 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone (349)……….92 14. Characterization of ayanin (350)………………………………………….….92

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15. Characterization of ombuin (351)…………………………………………….95 16. Biological activities of isolates from H. brevipes……………………………...95

D. Discussion………………………………………………………………………….....96 1. Structure elucidation and identification of compounds isolated from H. brevipes……………………………………………………………..…….……96 1.1. Structure elucidation of brevipolide A (342)……….….…………....96 1.2. Structure elucidation of brevipolide B (343)……………….………107 1.3. Structure elucidation of brevipolide C (344)………..………....….107 1.4. Structure elucidation of brevipolide D (345)………..………..…...114 1.5. Structure elucidation of brevipolide E (346)………………..………117 1.6. Structure elucidation of brevipolide F (347)………….……………117 1.7. Identification of (E)-1-{(1S,2S)-2-[(S)-hydroxy(R)-6-oxo-3,6- dihydro-2H- pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4- hydroxyphenyl)acrylate (348)……………………………………..122 1.8. Absolute configuration of compounds 342-348…………………..122 1.9. Identification of 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone (349)..123 1.10. Identification of ayanin (350)…………………………..…..…123 1.11. Identification of ombuin (351)………..…………………………124 2. Conclusions…………………………………..……………….……...………124

Chapter 3: Phytochemical and Bioactive Studies on the Leaves of Vitex quinata……..127 A. Background on Vitex quinata (Lour.) Williams……………………………………..127 1. Verbenaceae………………………………………………………………...……128 2. Genus Vitex and Vitex quinata (Lour.) Williams……………………….………..127 3. Phytochemical constituents of the genus Vitex……………………………………….130 3.1. Diterpenoids……………………………………………………..…………132 3.2. Flavonoids…………………………………………………………...……..133 3.3. Iridoids and iridoid glycosides……………………………………………..134 3.4. Ecdysteroids……………………….……………………………….………135

B. Statement of Problem……………………………………………………..………..136

C. Experimental……………………………………………………………….………..136

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1. General experimental procedures………………………………………………..136 2. Plant material…………………………………………………………...………..138 3. Solvent extraction and partitioning of Vitex quinata…………………………..138 4. Column chromatography of the chloroform-soluble and ethyl acetate-soluble extracts of Vitex quinata………………………………………………………..138 5. Characterization of dimethyl 3,3,4,4-tetrahydroxy--truxinate (378)………….142 6. Characterization of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5-methoxy-3- oxocyclopent-1-enyl}octanoate (379)……………………………..……………141 7. Characterization of (S)-5-hydroxy-7,4'-dimethoxyflavanone (380)...... 144 8. Characterization of (S)-isosakuranetin (381)...... 144 9. Characterization of 2-hydroxy-4,4,6-trimethoxychalcone (382)….…………144 10. Characterization of 2',6'-dihydroxy-4,4'-dimethoxychalcone (383)……………144 11. Characterization of 3,5-dihydroxy-7,4-dimethoxyflavonone (384)...... 146 12. Characterization of rhamnocitrin (385)…………………….…………………..146 13. Characterization of (-)-loliolide (386)...……...………………………...………146 14. Characterization of methyl 3,4-O-dicaffeoyl quinate (387)……...…………….147 15. Characterization of methyl 3,5-O-dicaffeoyl quinate (388)……………………147 16. Characterization of methyl 4,5-O-dicaffeoyl quinate (389)……………………148 17. Characterization of methyl 3,4,5-O-tricaffeoyl quinate (390)…………………149 18. Characterization of -sitosterol (339)…………………………………………..150 19. Biological activities of isolates from Vitex quinata……………….………….150

D. Discussion…………………………………………………………………………151 1. Structure elucidation of new compounds isolated from V. quinata…151 1.1. Structure elucidation of dimethyl 3,3,4,4-tetrahydroxy--truxinate (378)…………………………………………………………………….…..151 1.2. Structure elucidation of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]- 5-methoxy-3-oxocyclopent-1-enyl}octanoate (379)………………………..152 2. Identification of known compounds……………………………………………..161 2.1. Identification of (S)-5-hydroxy-7,4'-dimethoxyflavanone (380)...……….…161 2.2. Identification of (S)-isosakuranetin (381)………………..…………………162 2.3. Identification of 2-hydroxy-4,4,6-trimethoxychalcone (382)...... 171 2.4. Identification of 2',6'-dihydroxy-4,4'-dimethoxychalcone (383)……………172

xv xv

2.5. Identification of compound 3,5-dihydroxy-7,4-dimethoxyflavonone (384)………………………………………………………………………...172 2.6. Identification of rhamnocitrin (385)………………………………………..173 2.7. Identification of (-)-loliolide (386)……………………...……………….…174 2.8. Identification of methyl 3,4-O-dicaffeoyl quinate (387)………………...…175 2.9. Identification of methyl 3,5-O-dicaffeoyl quinate (388)……………...……176 2.10. Identification of methyl 4,5-O-dicaffeoyl quinate (389)………….………176 2.11. Identification of methyl 3,4,5-O-tricaffeoyl quinate (390)……….………177 2.12. Identification of -sitosterol (339)……………………………….……….178 3. Conclusions……………………………………………………...……………….178

References……………………………………………………………...……………….180

Appendix A……………………………………………………………………….….210 1. Cytotoxicity assay…………….………………………...…….……...……..….210 2. Proteasome fraction preparation and proteasome inhibition assay…………….212 3. Enzyme-based ELISA NF-B assay…………………………………………...212 4. Mitochondrial transmembrane potential assay (MTP assay)………………...213 5. Characterization of ursolic acid (56)………………………...…………………214 6. Characterization of maslinic acid (320)………………………………………214 7. Characterization of daucosterol (340)……………………….…………...…….215

Appendix B…………………………………………………………………….…….217 1. Initial cytotoxicity assays of Vitex quinata extracts and fractions…………..217

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List of Tables

Table 1.1. Plant derived IKK activity inhibitors ……………………………….………..19 Table 1.2. Plant-derived IB phosphorylation inhibitors………………….……………..27 Table 1.3. Plant-derived IB degradation inhibitors…………………………...………..34 Table 1.4. Plant-derived NF-B translocation inhibitors………………………..………39 Table 1.5. Plant-derived NF-B DNA-binding inhibitors………………………….……46 Table 1.6. Plant-derived NF-κB transactivation inhibitors………………………………55

Table 2.1. Trivial names and plant sources of pyrone and furanone derivatives isolated from the genus Hyptis……………………………………………………………………62 Table 2.2. Trivial names and plant sources of lignans isolated from the genus Hyptis….65 Table 2.3. Trivial names and plant sources of flavonoids isolated from the genus Hyptis……………………………………………………………………...……………..68 Table 2.4. Trivial names and plant sources of diterpenoids isolated from the genus Hyptis………………………………………………………………….……….…..…….70 Table 2.5. Trivial names and plant sources of triterpenoids isolated from the genus Hyptis…………………………………………………………………………………….77 Table 2.6. 1H NMR chemical shifts of compounds 342-348…………………………….93 Table 2.7. 13C NMR chemical shifts of compounds 342-348……………………………94

Table 3.1. Selected examples of natural products found in the genus Vitex…..………..131

Table A.1. Initial cytotoxicity assay results of H. brevipes extracts and fractions……..211 Table B.1. Cytotoxicity assay results of extracts and fractions prepared from V. quinata………………………………………………………………………………….217

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List of Figures

Figure 1.1. Structures of vinca alkaloids used in cancer therapy………………………..5 Figure 1.2. Structures of podophyllotoxin and epipodophyllotoxin analogs used in cancer chemotherapy……………………………………………………………………….……7 Figure 1.3. Structures of paclitaxel, 10-deacetylbaccatin III and docetaxel…………….10 Figure 1.4. Structures of camptothecin, topotecan, irinotecan and SN-38………………12 Figure 1.5. NF-B signal transduction pathway…………………………………………15 Figure 1.6. Structures of plant-derived IKK inhibitors…………………………………..22 Figure 1.7. Structures of plant-derived IB phosphorylation inhibitors…………………29 Figure 1.8. Structures of plant-derived IB inhibitors…………………………………..36 Figure 1.9. Structures of plant-derived NF-B translocation inhibitors…………………41 Figure 1.10. Structures of plant-derived NF-κB DNA binding inhibitors……………….50 Figure 1.11. Structures of plant-derived NF-κB transactivation inhibitors……………...55

Figure 2.1. Illustration of Hyptis brevipes Poit…………………………………………..59 Figure 2.2. Structures of pyrone and furanone derivatives isolated from the genus Hyptis…………………………………………………………………………………….63 Figure 2.3. Structures of lignans isolated from the genus Hyptis………………………..66 Figure 2.4. Structures of flavonoids isolated from the genus Hyptis……………………69 Figure 2.5. Structures of diterpenoids isolated from the genus Hyptis………………….73 Figure 2.6. Structures of triterpenoids isolated from the genus Hyptis………………….78 Figure 2.7. Structures of sterols and a sesquiterpene isolated from the genus Hyptis…..82 Figure 2.8. Solvent extraction scheme used for the plant material of H. brevipes………85 Figure 2.9. Structures of compounds isolated from H. brevipes in the present investigation…………………………………………………………………90 1 Figure 2.10. H NMR spectrum of brevipolide A (342) in CDCl3 (400 MHz)……….....99 1 1 Figure 2.11. H- H COSY NMR spectrum of brevipolide A (342) in CDCl3…..……...100 13 Figure 2.12. C NMR spectrum of brevipolide A (342) in CDCl3 (100 MHz).…….…101 13 Figure 2.13. C DEPT135 NMR spectrum of brevipolide A (342) in CDCl3…………102

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Figure 2.14. HMBC NMR spectrum of brevipolide A (342) in CDCl3…..…...……..…103

Figure 2.15. HSQC NMR spectrum of brevipolide A (342) in CDCl3……...…….……104

Figure 2.16. NOESY NMR spectrum of brevipolide A (342) in CDCl3………….....…105 Figure 2.17. Selected 1H-1H COSY, HMBC, and NOESY NMR spectroscopic correlations observed for brevipolide A (342)…..………………………………...……106 Figure 2.18. Circular dichroism spectrum of brevipolide A (342) in MeOH………..…106 1 Figure 2.19. H NMR spectrum of brevipolide B (343) in CDCl3 (400 MHz)…..……108 13 Figure 2.20. C NMR spectrum of brevipolide B (343) in CDCl3 (100 MHz)……..…109 1 Figure 2.21. H NMR spectrum of brevipolide C (344) in acetone-d6 (400 MHz)….…110 13 Figure 2.22. C NMR spectrum of brevipolide C (344) in acetone-d6 (100 MHz)...….111 Figure 2.23. 1H NMR spectra of (S)-MTPA and (R)-MTPA ester of brevipolide C (344) in pyridine-d5 (400 MHz)………………………………...………………………………..112 Figure 2.24. Observed 1H NMR chemical shift difference values between S- and R-MTPA esters of brevipolide C (344, S-R)…………….……………………..….…113 Figure 2.25. Mosher models (a, b) for the assignment of absolute configuration (c, d) by 1H NMR spectroscopy and the expected sign of SR …...………………….……..….114 1 Figure 2.26. H NMR spectrum of brevipolide D (345) in acetone-d6 (400 MHz)….…115 13 Figure 2.27. C NMR spectrum of brevipolide D (345) in acetone-d6 (400 MHz)....…116 1 Figure 2.28. H NMR spectrum of brevipolide E (346) in CD3OD (400 MHz)……..…118 13 Figure 2.29. C NMR spectrum of brevipolide E (346) in CD3OD (100 MHz)..…..…119 1 Figure 2.30. H NMR spectrum of brevipolide F (347) in acetone-d6 (400 MHz)….…120 13 Figure 2.31. C NMR spectrum of brevipolide F (347) in acetone-d6 (100 MHz)....…121

Figure 3.1. Illustration of Vitex quinata (Lour.) Williams…………………………...…129 Figure 3.2. Photography of Vitex quinata collected from Kego Nature Reserve, Hatinh Province, Vietnam……………………………………………………………….…...…130 Figure 3.3. Structures of selected diterpenoids isolated from the genus Vitex……...….132 Figure 3.4. Structures of selected flavonoids isolated from the genus Vitex………...…134 Figure 3.5. Structures of an iridoid (371) and an iridoid glycoside (372) isolated from the genus Vitex……………………………...……………………………………....….…...135 Figure 3.6. Structures of selected ecdysteroids (373 and 374) isolated from the genus Vitex………………………………………………………………………………....….135 Figure 3.7. Structures of compounds isolated from V. quinata reported in the literature.………………………………………………………………………….….…137

xix xix

Figure 3.8. Solvent extraction scheme used for the leaves of V. quinata………………139 Figure 3.9. Structures of compounds isolated from the leaves of Vitex quinata in the present investigation ……………………………………………………………..….…143 13 Figure 3.10. C NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 (100 MHz)…………………………...……………………………………………….…153 1 Figure 3.11. H NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6

(400 MHz)………………………………………………………………………….…154 Figure 3.12. 13C DEPT NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 (100 MHz)…………….………………………………………………….…155 Figure 3.13. HSQC NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6…………………………………………………………………………….…156 Figure 3.14. 1H-1H COSY NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6……………………………………………………………………………….157 Figure 3.15. HMBC NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6………………………………………………………………………………156 Figure 3.16. NOESY NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6……………………………………………………………………….………158 Figure 3.17. 13C NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3 (100 MHz)……...……….163 Figure 3.18. 1H NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3 (400 MHz)……………....164 Figure 3.19. HMBC NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3…………………………...165 Figure 3.20. 13C DEPT135 NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-

3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3 (100 MHz)...….166 Figure 3.21. HMQC NMR spectrum of methyl 8-{(5R)-2-[(E)-2- hydroxypent-

3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3 (100 MHz)…....167 Figure 3.22. 1H-1H COSY NMR spectrum methyl 8-{(5R)-2-[(E)-2-hydroxypent-

3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3………………...168 Figure 3.23. NOESY NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-

3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379) in CDCl3………………...169 Figure 3.24. Conformation analysis (a and b), octant rule analysis (c and d), and circular dichroism (CD) spectrum (e) of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3- enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379)…………………….……….170

xx xx

List of Abbreviations

1D, 2D: one- or two-dimensional

ACN: acetonitrile

[α]D: Specific optical rotation

Calcd: calculated

CD: circular dichroism spectroscopy

CDCl3: deuterated chloroform

CHCl3: chloroform

COSY: correlation spectroscopy

δ (ppm): chemical shift in parts per million

δC: carbon-13 chemical shift

δH: proton chemical shift

DEPT: distortionless enchancement by polarization transfer

DMSO: dimethyl sulfoxide

EC50: effective concentration that inhibits a response by 50% relative to a control

Gal: β-D-galactopyranose

xxi xxi

Glc: β-D-glucopyranose

GlcA: β-D-gluconic acid

HMBC: heteronuclear multiple bond correlation spectroscopy

HPLC: high-performance liquid chromatography

HRESIMS: high-resolution electrospray ionization mass spectroscopy

HSQC: heteronuclear single-quantum coherence spectroscopy

Hz: hertz

IC50: sample concentration that inhibits cell growth by 50% compared to untreated control

IR: infrared spectroscopy

J: coupling constant

: wavelength in nanometer

M: molar concentration

MeOH: methanol min: minute m.p.: melting point

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

xxii xxii m/z: mass to charge ratio

NEMO: NF-B essential modifier

NF-κB: nuclear factor kappa B

NMR: nuclear magnetic resonance

NOESY: nuclear Overhauser enhancement spectroscopy

 (cm-1): infrared absorption frequency in reciprocal centimeter

tR: retention time

TLC: thin-layer chromatography

UV: ultraviolet

xxiii xxiii

Chapter 1

PLANT-DERIVED ANTICANCER DRUGS AND

PLANT-DERIVED NF-B INHIBITORS

A. Plant-Derived Anticancer drugs

The purification and discovery of drug molecules such as atropine, digitoxin, and morphine from plants in the 19th century opened a new era in human medicinal history.

Since then, natural products have played an irreplaceable role as single new chemical entities as drugs and as pharmacological tools (Newman et al., 2000; Li and Vederas,

2009). Despite competition from other drug discovery methods, natural products are still providing a fair proportion of new drugs and clinical candidates in western medicine.

This status was reviewed by Newman and Cragg from the U.S. National Cancer Institute

(NCI), who analyzed the number of natural product-derived drugs represented among the total single chemical entity launches in the 25.5 years from January 1981 to June 2006 for all diseases worldwide (Newman and Cragg, 2007). Structurally modified natural product

1 1 molecules alone accounted for about one fourth of the total number of these drugs, including 43 entities such as artemisinin and paclitaxel. Additionally, there have been natural product derivatives, mimics, and totally synthetic compounds based on natural product pharmacophores introduced into therapy. In particular, natural products have played an important role in anticancer drug development for nearly four decades (Cragg et al., 2009). A recent review analyzing clinically approved anticancer drugs in Western countries and Japan during a twenty-seven year period from 1981 to 2008 showed that, under the class of ―non-biologicals/vaccines‖, 62.9% of anticancer drugs were either natural products or their derivatives, or synthetic molecules based on natural product pharmacophores (Cragg et al., 2009). Currently, there are ten officially approved plant-derived anticancer drugs used clinically in the United States, and they can be classified into four groups: the vinca alkaloids, the podophyllotoxin derivatives, taxanes, and the camptothecin drivatives (Cragg et al., 2005). The importance of plant natural products and derivatives in cancer therapy can be demonstrated by their recent overall market share. In 2002, derivatives of paclitaxel and camptothecin represented nearly one-third of all anticancer medication in this respect (Oberlies and Kroll, 2004). The history, human clinical applications, and mechanisms of action of these plant-derived

2 2 drugs will be discussed briefly in the following paragraphs, with their structures shown in

Figures 1.1 through 1.4.

1. Vinca alkaloids

Vinblastine (1, VLB, formerly vincaleukoblastine) and vincristine (2, VCR, formerly leurocristine) are bisindole alkaloids found in the plant Catharanthus roseus (L.)

G. Don (Apocynaceae), previously known as Vinca rosea L. This species is cultivated widely all over the world as an ornamental flower, and is known commonly as

―Madagascar periwinkle‖ or ―rosy periwinkle‖. The first vinca alkaloid, vinblastine (then known as ―vincaleukoblastine‖), was separately isolated by a group at the University of

Western Ontario and by researchers at Eli Lilly Co. (Indianapolis, IN), in the late 1950s

(Noble et al., 1958; Svoboda et al., 1959). Later, vincristine (then known as

―leurocristine‖) was isolated by chemists at Eli Lilly Co. (Svoboda et al., 1959, 1961;

Johnson et al., 1960; Noble, 1990). Vincristine (Oncovin®) and vinblastine (Velban®) were approved by the United States Food and Drug Administration (U.S. FDA) to Eli

Lilly in 1963 and 1965, respectively. Clinically, vinblastine is used mainly to treat

Hodgkin’s disease, whereas vincristine is used for the treatment of Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, and nephroblastoma.

3 3

Vindesine (3, Eldisine®) is a semi-synthetic drug derived from vinblastine, approved for clinical use in France, the UK, and several other countries, but not in the

U.S. The drug is used clinically for the treatment of several different types of cancer, including acute lymphocytic leukemia, breast cancer, chronic myelocytic leukemia, colorectal cancer, non-small cell lung cancer, and renal cell cancer (Dancey and Steward,

1995). Vinorelbine (4, Navelbine®) is a semi-synthetic anhydro derivative of

8-norvinblastine, obtained from anhydrovinblastine. The drug was first approved to treat bronchial cancer in France in 1989, and was later approved for the therapy of non-small cell lung cancer in 1991. In Europe, vinorelbine is also approved for breast cancer and prostate cancer treatment. GlaxoSmithKline received approval from the U.S. FDA. in

1994 for the use of vinorelbine for the treatment of advanced non-small cell lung cancer

(Gregory and Smith, 2000). The vinca alkaloids bind to microtubulin at the ―vinca domain‖ site in the -subunit, and therefore interrupt the formation of microtubules in metaphase during mitosis, leading to the apoptosis of cancer cells (Jordan and Wilson,

2004; Kingston, 2009).

4 4

1 R1 = CH3 R2 = CH3 Vinblastine 4 Vinorelbine

2 R1 = CHO R2 = CH3 Vincristine

3 R1 = CH3 R2 = NH2 Vindesine

Figure 1.1. Structures of vinca alkaloids used in cancer therapy

2. Podophyllotoxin derivatives

Podophyllotoxin (5) is a lignan that was first isolated by Podwyssotzki in 1880 from the plant Podophyllum peltatum L. (Berberidaceae), commonly known as American mayapple or mandrake, and it was structurally characterized in 1951 (Hartwell and

Schrecker, 1951). An impure extractive of P. peltatum, commonly known as

―podophyllin‖, has been used topically in the treatment of genital warts and hairy leukoplakia. Mayapple extract had been listed in the U.S. Pharmacopoeia since the First

Edition in 1820, but was withdrawn in 1840 due to its associated toxicity. The plant regained the interest of the pharmaceutical industry when it afforded a curative effect for

5 5 condylomata acuminata in the 1940s (Canel et al., 2000). Presently, plant extracts containing podophyllins are prepared from P. peltatum and P. hexandrum. The latter species is known as ―Indian mayapple‖ or ―Himalaya mayapple‖, and has a higher content of podophyllins than American mayapple. Two drugs derived from the podophyllins, namely, SP-G (Proresid oral®), the condensation product of Podophyllum glucoside fraction with benzaldehyde, and SP-I (Proresid i.v.®), podophyllinic acid ethyl hydrazide, were first commercialized for the treatment of systematic cancers by Sandoz,

Ltd. (Basel, Switzerland) in 1963 (Stahelin and von Wartburg, 1991). Further studies of

SP-G in Sandoz in the 1960s led to the development of etoposide (6, VePesid®) and teniposide (7, Vumon®), which were produced by condensation with acetaldehyde and thiophene, respectively. Even though Sandoz had commercialized both drugs in some countries, etoposide and teniposide were licensed to Bristol-Myers in 1978 for commercialization in the U.S. market. Etoposide (Vepesid®, VP-16®) was approved in

1983 by the U.S. FDA for the treatment of refractory testicular tumors, and in 1996 for small-cell lung cancer therapy. Etoposide phosphate salt, etopophos (8, Eposin®,

Etopophos®) was developed as a prodrug to resolve the water solubility problem encountered in drug formulation and administration of the natural drug. Studies have

6 6 shown that etopophos is less toxic and more active in comparison with etoposide, with increased bioavailability from only 0.04% for etoposide to over 50%. The U.S. FDA approved the intravenous use of etopophos for small-cell lung cancer in 1996 (Bohlin and

Rosen, 2006). Teniposide was approved in 1992 for the treatment of childhood acute lymphoblastic leukemia. In contrast to the mechanism of podophyllotoxin, which binds to tubulin and inhibits the assembly of microtubules to form mitotic spindles in mitosis, etoposide and teniposide are topoisomerase II inhibitors. They stabilize the covalent

DNA-enzyme cleavable complex, and induce topoisomerase II-mediated DNA breakage, leading to the arrest of the cell cycle in metaphase (Hande, 1998).

5 Podophyllotoxin 6 R = OH Etoposide 7 Teniposide

8 R = H2PO4 Etophos

Figure 1.2. Structures of podophyllotoxin and epipodophyllotoxin analogs used in cancer chemotherapy

7 7

3. Paclitaxel and derivatives

Paclitaxel (9) is a nitrogen-containing diterpenoid discovered from the bark of the

Pacific yew tree, Taxus brevifolia Nutt. (Taxaceae), reported in 1971 by Monroe E. Wall and Mansukh C. Wani, of Research Triangle Institute, and by Andrew McPhail, of Duke

University, in a study sponsored by the U.S. NCI. The compound was originally given the trivial name ―taxol‖, but the generic name was changed to ―paclitaxel‖ when

Bristol-Myers Squibb (B-MS) acquired the trade mark name ―Taxol‖ from a French company, who had used the name for an unrelated laxative product before the anticancer compound was published (Wani et al., 1971). Taxol was approved in 1992 by the U.S.

FDA for the treatment of patients with refractory metastatic carcinoma of the ovary after the failure of first-line or subsequent chemotherapy. The drug is currently used to treat patients with lung, ovarian, breast cancer, head and neck cancer, and advanced forms of

Kaposi's sarcoma. With the rapid clinical application of the drug in the early 1990s, the increasing demand for paclitaxel presented a stress on the available resources of the

Pacific yew tree, for which the bark was the only commercial source of the drug at the time. This costly and environmentally challenging method was soon replaced by a semi-synthetic process using 10-deacetylbaccatin III (10), which is found abundantly in

8 8 the leaves of the European yew tree, Taxus baccata L., a renewable resourse. Presently, paclitaxel is also successfully produced by B-MS by plant tissue culture in large scale for commercial purposes (Kingston, 2009). Abraxane® is an albumin-covered paclitaxel injectable suspension, using ―nanoparticle albumin bound‖ (nab™) technology to facilitate drug delivery. Abraxane was approved by U.S. FDA in 2005 for the treatment of breast cancer. Docetaxel (11, Taxotere®) is a semi-synthetic paclitaxel analogue produced from 10-deacetylbaccatin III, in which an N-tert-butoxycarbonyl group is substituted for the N-benzoyl group in the side chain and the C-10 hydroxy group is free.

Docetaxel was approved in 1996 by U.S. FDA and licensed to the forerunner of Aventis, and is presently used for the treatment of breast, non-small cell lung, gastric, prostate, as well as neck and head cancers (Montero et al., 2005). In contrast to the mechanism of the vinca alkaloids, which disrupt the assembly of microtubules in mitosis, paclitaxel and docetaxel stabilize microtubules by binding to the taxane site, and as a result, interfere with the normal breakdown of microtubules during cell division (Schiff and Horwitz,

1980; Kingston, 2009).

9 9

9 R1 = OAc R2 = Ph Paclitaxel 10 10-Deacetylbaccatin III

11 R1 = OH R2 = t-Bu Docetaxel

Figure 1.3. Structures of paclitaxel, 10-deacetylbaccatin III and docetaxel

4. Camptothecin derivatives

Camptothecin (12) is a quinoline alkaloid isolated from the bark and stems of

Camptotheca acuminata Decne. (Cornaceae), a tree native to mainland China that was introduced to the U.S. in 1911 as an ornamental. Camptothecin was discovered in the mid-1960s by Monroe E. Wall and Mansukh C. Wani, of Research Triangle Institute, collaborating with Andrew T. McPhail and George A. Sim at the University of Illinois at

Urbana, as part of a systematic screening of anticancer plant natural products carried out with support from the U.S. NCI (Wall et al., 1966; Wall and Wani, 1994). Although a

Phase II study of camptothecin sodium salt in treating gastrointestinal cancer was suspended in 1972 due to poor efficacy and toxic side effects, interest in camptothecin

10 10 was regained after its unique mechanism of action was elucidated (Oberlies and Kroll,

2004). Studies have shown that camptothecin arrests the cell cycle at the S-phase by binding to topoisomerase I, leading to the inhibition of DNA replication and transcription

(Hsiang, et al., 1985; 1989). Among numerous semi-synthetic camptothecin derivatives aimed at improving on the solubility and activity of the parent compound, as well as to decrease side effects, two analogues, topotecan (13, Hycamtin®, GlaxoSmithKline) and irinotecan (14, Camptosar®, Pfizer), were eventually launched onto the cancer chemotherapy market. The substitution of a N,N-dimethylaminomethyl group at the C-9 position and the insertion of a hydroxy group at the C-10 position in topotecan significantly increases water solubility and bioavailability, when compared to camptothecin. GlaxoSmithKline (GSK) first received approval of topotecan from the U.S.

FDA for treating ovarian cancer in 1996. Later, it was approved for the treatment of stage

IVB recurrent or persistent cervical carcinoma, and for the treatment of relapsed small-cell lung cancer (SCLC) in 2007. Therefore, this camptothecin derivative is the only oral single-agent chemotherapy approved for the treatment of SCLC after the failure of first-line therapies. Irinotecan is a prodrug of the potent 7-ethyl-10-hydroxy- camptothecin analog, SN-38 (15), with the substitution of a bis-piperidine functionality at

11 11

12 R1 = H R2 = H R3 = H Camptothecin

13 R1 = H R2 = R3 = Topotecan

14 R1 = Et R2 = H R3 = H Irinotecan

15 R1 = Et R2 = H R3 = OH SN-38

Figure 1.4. Structures of camptothecin, topotecan, irinotecan and SN-38

the C-10 position. The drug was first developed in Japan by Yakult Pharmaceutical

Industries. In the human body, the prodrug irinotecan is hydrolyzed by liver carboxylesterases into its active metabolite, SN-38 (Ma and McLeod, 2003). Irinotecan was approved by the U.S. FDA in 2000 for the treatment of metastatic colorectal cancer, in combination with 5-fluorouracil and leucovorin (5-FU/LV).

B. Plant Derived Nuclear Factor-B Inhibitors

1. Nuclear Factor B (NF-B) pathway as an anticancer drug target

Although mortality rates of cancer have declined in recent years owing to earlier detection programs and the application of new chemotherapy, radiology, immunotherapy,

12 12 and surgical methods, most cancers, such as lung and bronchial cancers, are still difficult to treat (Jemal et al., 2009). Therefore, the demand for new anticancer drugs has not yet been satisfied. Specific signal transduction pathways involved in abnormal cell growth and survival have been studied extensively for the understanding of the mechanism of basic cancer biology and for the development of new anticancer drugs. One of these important pathways is the nuclear factor kappa B (NF-B) signaling cascade (Shen and

Tergaonkar, 2009). NF-B is a family of structurally related eukaryotic protein complexes that control DNA transcription and is therefore related to the expression of hundreds of genes. NF-B is found almost in all animal cells and is involved in the control of a large number of normal cellular and organism processes, such as apoptosis, cellular growth, and immune and inflammatory responses. It was found that constant activation of these transcription factors is related to a number of disease states, including arthritis, asthma, autoimmune diseases, cancer, chronic inflammation, neurodegenerative diseases, and heart disease (Wong and Tergaonkar, 2009). Abnormal NF-B activation has been observed in many solid and hematopoietic malignancies, and the function of

NF-B is involved in all six hallmarks of cancer progression: self-sufficiency of growth signals, insensitivity to growth inhibition signals, evasion of apoptosis, acquisition of

13 13 limitless explicative potentials, angiogenesis, and metastasis (Shen and Tergaonkar, 2009).

With an important role in carcinogenesis, NF-B is regarded as one of the promising molecular targets for future anticancer drug development (Baud and Karin, 2009).

In mammals, active NF-B transcription factors are either homo- or hetero-dimers composed of combinations of five proteins: RelA (p65), RelB, c-rel, NF-B1 (p50 and its precursor p105), and NF-B2 (p52 and its precursor p100). Upon an upstream stimulus signal, a dominant NF-B dimer sequestered in the cytoplasm by interaction with inhibitor B (IB) complex is activated and then binding occurs to the B site of DNA to activate transcription. The most common signal transduction routes found to be involved in NF-B activation are the classical (or canonical) and the alternative (or non-canonical) pathways. The classical pathway is more likely related to the activation of RelA

(p65)-p50 dimers and the control of innate immunity and inflammation, whereas the alternative pathway is mainly for the activation of RelB-p50/52 dimers and for the modulation of NF-B during B and T-cell organ development (Gilmore, 2006). In the classical pathway (Figure 1.5), under a broad range of stimuli, such as tumor necrosis factor  (TNF), viruses, and ionizing radiation, the IB that binds to an NF-B dimer, such as RelA-p50, is phosphorylated by an inhibitor B kinase (IKK) complex and then

14 14 hydrolyzed by the 26S proteosome, leading to the release of activated free NF-B dimer.

The IKK complex contains an  and a  catalytic subunit, as well as two molecules of the regulatory NF-B essential modifier (NEMO, also known as IKK). The alternative pathway is stimulated by the CD40 ligand, lymphotoxin , and a more restricted set of

Figure 1.5. NF-B signal transduction pathway. NEMO: NF-B essential modifier; IKK: inhibitor B kinase; IB: inhibitor B (adapted from Gilmore and Herscovitch, 2006) 15 15 cytokines belonging to the TNF family. The upstream NF-B inducing kinase (NIK) activates IKK, which in turn phosphorylates the p100 subunit of the RelB-p100 dimer, leading to the release of active RelB-p52/50 NF-B dimer after the processing of the p100 subunit by the proteasome. Both pathways result in NF-B nuclear translocation and DNA binding. Therefore, the steps involved in the signal transduction cascade of

NF-B activation provide several targets for specific inhibition of NF-B activity: inhibition of inhibitor B kinase (IKK) activity; stabilization of IB, prevention of inhibition of NF-B nuclear translocation; inhibition NF-B DNA binding; and inhibition of NF-B transactivation (Gilmore and Herscovitch, 2006).

2. Plant-derived NF-B Inhibitors

2.1. Plant-derived inhibitor B kinase (IKK) inhibitors

IKK has been one of the pivotal targets in blocking the NF-B signal transduction pathway, especially in the upstream part. Of the over 150 IKK inhibitory agents reported until 2006, more than 40 were plant-derived compounds, with, in addition, 14 plant extracts being found to be active (Gilmore and Herscovitch, 2006). The IKK complex consists of two subunits with kinase activity, IKK and IKK, as well as an associated regulatory protein generally known as NEMO, or IKK (Hacker and Karin, 2007). The 16 16 molecular mechanisms of small-molecular weight compounds can be classified generally into the following four categories: ATP analogs, allosteric regulators, Cys-179 binders

(which interrupt the activation loop of IKK), and compounds targeting NEMO. Among the plant-derived IKK inhibitors listed in Table 1.1, berberine (22) and butein (24) were found to interrupt the activation loop of IKK, whereas the detailed mechanism of the remaining compounds has not been revealed yet (Pandey et al., 2007, 2008). Pandey and coauthors provided direct proof that berberine (22) inhibited IKK-β activity through the modification of the Cys-179 residue when treated at a concentration range of 10 to 50 M

(Pandey et al., 2007). 17-Acetoxyjolkinolide B (16), an abietane diterpenoid isolated from the traditional Chinese medicinal herb, Euphorbia fischeriana, interacts with IKK

directly, with an IC50 of 0.3 M using human hepatocellular carcinoma HepG2 cells. The mechanism was proposed theoretically as direct binding to IKK-β by a Michael addition between cysteine and α,β-unsaturated lactone or epoxy groups (Yan et al., 2008).

In addition to those in vitro studies conducted, an in vivo experiment found humulone (34) to significantly inhibit TPA (12-O-tetradecanoylphorbol-13-acetate)- induced IKK activity, and to a lesser extent IKK activity, in mouse skin (Lee et al.,

2007).

17 17

The purified plant-derived natural IKK inhibitors, including diterpenoids, flavonoids, triterpenoids, and miscellaneous compounds, are listed in Table 1.1, and their structures are shown in Figure 1.6.

2.2. Plant-derived inhibitor B (B) phosphorylation suppressors

The inhibitor B (IB) proteins are associated with NF-B and inhibit its activity in non-stimulated cells. To date, eight structurally related members of the mammalian IB family have been discovered, including BCL-3, IB, IB, IB, IB, IB, p100, and p105 (Gilmore and Herscovitch, 2006). Studies have shown these different IB molecules inhibit distinct subsets of NF-B/Rel dimers. In the process of NF-B activation, initially the IB protein is specifically phosphorylated by activated IKK, then recognized and ubiquitinated by ubiquitin ligases. The ubiquitinated IB proteins are then degraded by the proteasome and lead to the release of activated NF-B dimers. Therefore, IB phosphorylation has also been proposed as a target of NF-B pathway inhibition (Moorthy et al., 2006).

Similar to IKK inhibitors, plant-derived natural products also represent a significant proportion of the known IB phosphorylation inhibitors (Gilmore and Herscovitch, 2006).

18 18

Compound Name Plant of Origin Phytochemical Category References 17-Acetoxyjolkinolide B (16) Euphorbia fischeriana Abietane diterpenoid Yan et al., 2008 1'-Acetoxychavicol acetate Languas galangal Phenypropanoid Ichikawa et al., 2005; Ito et al., (17) 2005 1-O-Acetylbritannilactone (18) Inula Britannica Sesquiterpenoid Liu et al., 2007 Apigenin (19) Not stated Flavone Shukla and Gupta, 2004; Yoon et al., 2006 Asiatic acid (20) Centella asiatica Ursane triterpenoid Yun et al., 2008 Azorellane (21) Laretia acaulis Azorellane diterpenoid Borquez et al., 2007 Berberine (22) Not stated Benzylisoquinoline alkaloid Hu et al., 2008; Yi et al., 2008; Pandey et al., 2008 Betulinic acid (23) Betula pubescens Lupane triterpenoid Takada and Aggarwal, 2003; Rabi et al., 2008 19 Butein (24) Dalbergia odorifera Chalcone Pandey et al., 2007

Rhus verniciflua Cardamonin (25) Alpinia conchigera Chalcone Lee et al., 2006 7-Deacetylazorellanol (26) Laretia acaulis Azorellane diterpenoid Borquez et al., 2007 Denbinobin (27) Cannabis sativa 4-Phenanthrenequinone Sanchez-Duffhues et al., 2008, 2009 3,4-Dihydroxybenzalacetone Inonotus obliquus Phenylpropanoid Sung et al., 2008b (28) 5,7-Dihydroxy-3,4,6- Isoetes delilei and I. durieui Flavone Kim et al., 2009 trimethoxyflavone (29) Diosgenin (30) Not stated Steroid Shishodia and Aggarwal, 2005; Liagre et al., 2005 Garcinone B (31) Hypericum patulum Xanthone Yamakuni et al., 2005 Table 1.1. Plant-derived IKK activity inhibitors

Table 1.1. continued Compound Name Plant of Origin Phytochemical Category References Guggulsterone (32) Commiphora mukul Steroid Ichikawa and Aggarwal, 2006; Deng, 2007; Lv et al., 2008; Lee et al., 2008 Honokiol (33) Magnolia officinalis Lignan Tse et al., 2005 Humulone (34) Humulus lupulus Phloroglucinol derivative Lee et al., 2007 Hypoestoxide (35) Hypoestes rosea Diterpenoid Ojo-Amaize et al., 2001 Isorhapontigenin (36) Rhei rhapontici Stilbenoid Li et al., 2005 Kahweol (37) Coffea arabica ent-Kaurane diterpenoid Kim et al., 2004 Licochalcone A (38) Glycyrrhiza glabra Chalcone Funakoshi-Tago et al., 2009 -Mangostin (39) Garcinia mangostana Xanthone Nakatani et al., 2004 Morin (40) Not stated Flavonol Manna et al., 2007 20 Morusin (41) Morus australis Flavone Lee et al., 2008

6-Nonadecylsalicylic acid (42) Anacardium occidentale Anacardic acid Sung et al., 2008a Oleic acid (43) Not stated Fatty acid Oh et al., 2009 Piceatannol (44) Not stated Stilbene Islam et al., 2004 Pinitol (45) Abies pindrow Inositol isomer Sethi et al., 2008 Plumbagin (46) Plumbago zeylanica Naphthoquinone Sandur et al., 2006 Pristimerin (47) Pristimera indica Oleanane triterpenoid Tiedemann et al., 2009 Quercetin (48) Not stated Flavonol Peet and Li, 1999 Rosmarinic acid (49) Rosmarinus officinalis Phenylpropanoid Lee et al., 2006 continued

Table 1.1. continued Compound Name Plant of Origin Phytochemical Category References Silibinin (50) Silybum marianum Flavonolignan Dhanalakshmi et al., 2002; Singh et al., 2004; Min et al., 2007 Sulforaphane (51) Not stated Organosulfur compound Xu et al., 2005; Murakami et al., 2007; Liu et al., 2008: Hayes et al., 2008 Tetrandrine (52) Stephania tetrandra Benzyisoquinoline Ho et al., 2004; Xue et al., 2008; Lin et alkaloid al., 2008 Theaflavin-3,3'-digallate (53) Camellia sinensis Theaflavin Aneja et al., 2004; Ukil et al., 2006; Kalra et al., 2007; Adhikary et al., 2009 Tilianin (54) Tilia japonica Flavone glycoside Nam et al., 2005 -Tocotrienol (55) Elaeis guineensis Vitamin E derivative Shah and Sylvester, 2005 Ursolic acid (56) Not stated Ursane triterpene Shishodia et al., 2003; Manu and Kuttan, 21 2008

Wedelolactone (57) Eclipta alba Coumestan Kobori et al., 2004 Zerumbone (58) Zingiber zerumbet Sesquiterpene Takada et al., 2005

17-Acetoxyjolkinolide B 1'-Acetoxychavicol acetate 1-O-Acetylbritannilactone (16) (17) (18)

Apigenin (19) Asiatic acid (20) Azorellane (21)

Berberine (22) Betulinic acid (23)

Butein (24) Cardamonin (25) 7-Deacetylazorellanol (26)

Figure 1.6. Structures of plant-derived IKK inhibitors

22 22

Figure 1.6. continued

Denbinobin (27) 3,4-Dihydroxybenzal- 5,7-Dihydroxy-3,4,6-tri- acetone (28) methoxyflavone (29)

Diosgenin (30) Garcinone B (31)

Guggulsterone (32) Honokiol (33) Humulone (34)

Hypoestoxide (35) Isorhapontigenin (36) Kahweol (37)

Continued

23 23

Figure 1.6. continued

Licochalcone A (38) -Mangostin (39)

Morin (40) Morusin (41) 6-Nonadecyl-salicylic acid (42)

Oleic acid (43)

Piceatannol (44) Pinitol (45) Plumbagin (46)

Pristimerin (47) Quercetin (48) continued

24 24

Figure 1.6. continued

Rosmarinic acid (49) Silibinin (50)

Sulforaphane (51)

Tetrandrine (52) Theaflavin-3,3'-digallate (53)

Tilianin (54) -Tocotrienol (55)

Ursolic acid (56) Wedelolactone (57) Zerumbone (58)

25 25

The potency of most of these compounds has been found to be in the micromolar level.

For example, one study found LPS (lipopolysaccharide)-induced IκB-α degradation to be significantly blocked by pretreatment with 10 M buddlejasaponin IV (60). Further experiments revealed that the reduction of IκB-α phosphorylation by buddlejasaponin IV

(60) occurred in a time-dependent manner (Won et al., 2006). Another study demonstrated that the chalcone, cardamonin (25), at a concentration range of 1-10 M, can suppress phosphorylation of IB. In a mechanism-based study, sanguinarine (77), a quaternary benzylisoquinoline alkaloid, blocked NF-B activation by inhibiting IB phosphorylation at a 5 M concentration (Chaturvedi et al., 1997).

An in vivo study reported the inhibition of IB phosphorylation by 5-hydroxy-

3,6,7,8,3',4'-hexamethoxyflavone (65), a flavone found thus far exclusively in the genus

Citrus. By topical application of this substance to mouse skin prior to the treatment of

12-O-tetradecanoylphorbol-13-acetate (TPA), it was found that phosphorylation of p65/RelA at serine 536 was inhibited by such pre-treated animal groups in a dose- dependent manner (Lai et al., 2007).

The plant-derived natural IB phosphorylation inhibitors, including diterpenoids, flavonoids, sesquiterpenoids, triterpene glycosides, as well as miscellaneous compounds are listed in Table 1.2, and their structures are shown in Figure 1.7. 26 26

Compound Name Plant of origin Phytochemical References category Anethole (59) Not stated Phenylpropanoid Chainy et al., 2000 Buddlejasaponin IV (60) Buddleja japonica Oleanane triterpene Won et al., 2006 glycoside Cardamonin (25) Alpinia rafflesiana Chalcone Israf et al, 2006 Decursin (61) Angelica gigas Coumarin Kim et al., 2006 Delphinidin (62) Not stated Anthocyanin Syed et al., 2008 Digitoxin (63) Digitalis lanata Steroid glycoside Srivastava et al., 2004 (6)-Gingerol (64) Zingiber officinale Benzenoid Kim et al., 2005j; Aktan et al., 2006 Guggulsterone (32) Commiphora mukul Steroid Shishodia and Aggarwal, 2004 5-Hydroxy-3,6,7,8,3',4'- Not stated Flavone Lai et al., 2007 27 hexamethoxyflavone (65) Indirubin-3'-oxime (66) Isatis indigotica; Semi-synthetic indole Mak et al., 2004 Strobilanthes cusia alkaloid Kaempferol (67) Not stated Flavonol Garcia-Mediavilla et al., 2006; Kim et al., 2007 Kurarinone (68) Sophora flavescens Flavanone Han et al., 2006 Obovatol (69) Magnolia obovata Phenylpropanoid Lee et al., 2008 Oleandrin (70) Nerium oleander Steroid glycoside Manna et al., 2000b; Sreeivasan et al., 2003 continued Table 1.2. Plant-derived IB phosphorylation inhibitors

Table 1.2. continued Compound Name Plant of origin Phytochemcial category References Oleanolic acid 3-O--D- Aralia elata Oleanane triterpene glycoside Suh et al., 2007 glucopyranosyl(13)--L- rhamnopyranosyl (12)--L- arabinopyranoside (71) Panduratin A (72) Kaempferia pandurata Chalcone Yun et al., 2003 Parthenolide (73) Tanacetum parthenium Sesquiterpenoid Hehner et al., 1998 Pinosylvin (74) Pinus densiflora Stilbenoid Lee et al., 2006

28 Rengyolone (75) Forsythia koreana Benzofuranone Kim et al., 2006 Saikosaponin D (76) Bupleurum falcatum Oleanane triterpene glycoside Leung et al., 2005 Sanguinarine (77) Sanguinaria canadensis Benzylisoquinoline alkaloid Chaturvedi et al., 1997 Scoparone (78) Artemisia capillaris Coumarin Jang et al., 2005 Xanthoangelol D (79) Angelica keiskei Chalcone Sugii et al., 2005

Anethole (59) Buddlejasaponin IV (60)

Decursin (61) Delphinidin (62)

Digitoxin (63)

(6)-Gingerol (64) continued Figure 1.7. Structures of plant-derived IB phosphorylation inhibitors

Figure 1.7. continued

5-Hydroxy-3,6,7,8,3',4'- Indirubin-3'-oxime (66) hexamethoxyflavone (65)

Kaempferol (67) Kurarinone (68)

Obovatol (69) Oleandrin (70)

Oleanolic acid 3-O--D-glucopyranosyl(13)--L-rhamnopyranosyl (12)--L- arabinopyranoside (71) continued

Figure 1.7. continued

Panduratin A (72) Parthenolide (73) Pinosylvin (74)

Rengyolone (75) Saikosaponin D (76)

Sanguinarine (77) Scoparone (78) Xanthoangelol D (79)

2.3. Plant-derived IB degradation blockers

The degradation of IB is the last step in the upstream regulation of the NF-B activation process. A phosphorylated IB is labeled by the SCF--TrCP ubiquitin ligase complex, then quickly recognized and hydrolyzed by the 26S proteasome. Therefore,

interruption of either step in the ubiquitin-proteasome pathway will block the activation of NF-B dimer. There are more than 30 plant natural products that have been reported to inhibit the degradation of IB, but for most of these compounds the detailed mechanism remains unclear. For example, one study demonstrated the amide capsaicin (84) to inhibit tumor necrosis factor--stimulated degradation of IB in PC3 human prostate cancer cells, which was associated with the inhibition of proteasome activity. Further in vivo experiments in the same study showed that when capsaicin (84) was given orally, it slowed the growth of PC3 prostate cancer xenografts significantly, as measured by size and weight (Mori et al., 2006). RANKL was found to induce degradation of IκB within

30 min in an in vitro study using RAW264.7 cells, but the process was largely inhibited when treated with 1-5 M momordin I (97), suggesting that this oleanane triterpene glycoside inhibits RANKL-induced NF-κB activity by interfering with the degradation of

IκB (Hwang et al., 2005). Aquila and associates showed that the three ent-kauranoid diterpenoids xerophilusin A (105), xerophilusin B (106), and xerophilusin F (107), isolated from Isodon xerophylus, can reduce IκB degradation, all at a concentration of 1

μM (Aquila et al., 2009).

Also, a number of plant extracts have been found with similar activities (Gilmore

and Herscovitch, 2006). Flavonoids and diterpenoids are the two major groups of compounds that have been reported to show IB degradation inhibiting activities. These compounds, together with other miscellaneous natural products, are summarized in Table

1.3, and their structures are shown in Figure 1.8.

2.4. Plant-derived NF-B translocation inhibitors

After the removal of IB in cytoplasm, the activated NF-B dimer can then translocate into the cell nucleus through nuclear pores. Approximately 30 plant-derived natural products have been found, often with other NF-B inhibition activities, to be able to inhibit NF-B translocation. When compared to other NF-B inhibitors, this group of natural products includes more polar compounds, such as glycosides and tannins.

A study reported that when murine melanoma B16F10 cells were treated with 10

μg/ml of the piperidine alkaloid, piperine (125), for two hours, the translocation of

NF-B p65, p50, and c-Rel was all significantly reduced, with percentages of 69.64%,

69.83%, and 88.25%, respectively (Pradeep and Kuttan, 2004). The stilbenoid, trans-resveratrol (126), was reported to suppress lipopolysaccharide (LPS)-stimulated nuclear translocation of the p65 subunit at a concentration of 10 μM (Park et al., 2009).

In another study, immunoblot analysis indicated cells treated with 50 mM of the

Compound Plant of origin Phytochemical category References Amentoflavone (ginkgetin, 80) Ginkgo biloba Biflavone Banerjee et al., 2002 Andrographolide (81) Andrographis paniculata Labdane diterpenoid Bao et al., 2009 Aucubin (82) Aucuba japonica Iridoid glycoside Jeong et al., 2002 Baicalein (83) Scutellaria baicalensis Flavonone Ma et al., 2004 Capsaicin (84) Capsicum spp. Capsaicinoid Singh et al., 1996; Mori et al., 2006 -Caryophyllene (85) Not stated Sesquiterpene Hehner et al., 1998 Catalposide (86) Catalpa ovata Iridoid glycoside Kim et al., 2004 Delphinidin (62) Not stated Anthocyanidin Yun et al., 2009

34 Emodin (87) Polygonum cuspidatum Anthraquinone Kumar et al., 1998; Huang et

al., 2004 Genistein (88) Glycine max Isoflavone Natarajan et al., 1998; Baxa and Yoshimura, 2003 Glabridin (89) Glycyrrhiza glabra Flavonoid Kang et al., 2004 α-Humulene (90) Humulus lupulus Sesquiterpene Hehner et al., 1998 Incensole acetate (91) Not stated Diterpenoid Moussaieff et al., 2007 Isomallotochromanol (92) Mallotus japonicus Phloroglucinol derivative Ishii et al., 2003 Isomallotochromene (93) Mallotus japonicus Phloroglucinol derivative Ishii et al., 2003 β-Lapachone (94) Tabebuia avellanedae Sesquiterpenoid Manna et al., 1999 continued

Table 1.3. Plant-derived IB degradation inhibitors

Table 1.3. continued

Compound Plant of origin Phytochemical category References Longikaurin B (95) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Lycopene (96) Solanum lycopersicum Carotenoid Feng et al., 2009 Momordin I (97) Ampelopsis radix Oleanane triterpene glycoside Hwang et al., 2005 Parthenolide (73) Tanacetum parthenium Sesquiterpenoid Hehner et al., 1998 Plumbagin (46) Plumbago zeylanica Naphthoquinone Checker et al., 2009 Pseudolaric acid B (98) Pseudolarix kaempferi Diterpenoid Li et al., 2009 Resiniferatoxin (99) Euphorbia resinifera Diterpenoid Singh et al., 1996

Scutellarin (100) Scutellaria barbata Flavonone glycoside Tan et al., 2007 Siegeskaurolic acid (101) Siegesbeckia pubescens ent-Kaurane diterpenoid Parker et al., 2007

1,2,6-Tri-O-galloyl--D-allose (102) Not stated Gallotannin Kim et al., 2009 Tussilagone (103) Tussilago farfara Sesquiterpenoid Lim et al., 2008 Vanillin (104) Not stated Benzenoid Liang et al., 2009 Xerophilusin A (105) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Xerophilusin B (106) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Xerophilusin F (107) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Zedoarondiol (108) Curcuma heyneana Sesquiterpenoid Cho et al., 2009 continued

Amentoflavone (80) Andrographolide (81) Aucubin (82)

Baicalein (83) Capsaicin (84)

-Caryophyllene (85) Catalposide (86) Emodin (87)

Genistein (88) Glabridin (89)

α-Humulene (90) Incensole acetate (91) continued

Figure 1.8. Structures of plant-derived IB inhibitors

Figure 1.8. continued

Isomallotochromanol (92) Isomallotochromene (93) β-Lapachone (94)

Longikaurin B (95) Momordin I (97)

Lycopene (96)

Pseudolaric acid B (98) Resiniferatoxin (99)

Scutellarin (100) Siegeskaurolic acid (101) continued

Figure 1.8. continued

1,2,6-Tri-O-galloyl--D-allose (102) Tussilagone (103)

Vanillin (104) Xerophilusin A (105) Xerophilusin B (106)

Xerophilusin F (107) Zedoarondiol (108)

coumarin sphondin (127) can inhibit interleukin 1-induced translocation of p65 from the cytosol to the nucleus (Yang et al., 2002).

The plant-derived natural NF-B translocation inhibitors are summarized in

Table 1.4, and their structures are shown in Figure 1.9.

Compound Plant of Origin Phytochemical Category References Andrographolide (81) Andrographis paniculata ent-Labdane diterpenoid Bao et al., 2009 Anemarsaponin B (109) Anemarrhena asphodeloides Steroid glycoside Kim et al., 2009 Astragaloside IV (110) Astragalus membranaceus Cycloartane triterpene Zhang et al., 2003 glycoside 2',8"-Biapigenin (111) Selaginella tamariscina Biflavonone Woo et al., 2006 Carnostic acid (112) Not stated Abietane diterpenoid Yu et al., 2008 α-Chaconine (113) Solanum tuberosum Steroidal glycoalkaloid Shih et al., 2007 Chiisanoside (114) Acanthopanax chiisanensis Lupane triterpene glycoside Won et al., 2005 Corilagin (115) Caesalpinia coriaria Polyphenol Zhao et al., 2008 Cryptotanshinone (116) Salvia miltiorrhiza Abietane diterpenoid Jin et al., 2009 Delphinidin (62) Not stated Anthocyanidin Yun et al., 2009

(-)-Epigallocatechin gallate (117) Camellia sinensis Polyphenol Lin et al., 2009 Eriocalyxin B (118) Isodon eriocalyx ent-Kaurane diterpenoid Wang et al., 2006 Eutigoside C (119) Eurya emarginata Phenonic glucoside Lee et al., 2008 Hesperidin (120) Not stated Anthocyanidin Yeh et al., 2009 Hirsutenone (121) Alnus hirsute Diarylheptanoid Kim et al., 2005 Longikaurin B (95) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Lycopene (96) Solanum lycopersicum Carotenoid Feng et al., 2009 Oregonin (122) Alnus formosana Diarylheptanoid derivative Lee et al., 2005 continued

Table 1.4. Plant-derived NF-B translocation inhibitors

Table 1.4. continued

Compound Plant of Origin Phytochemical Category References Paeonol (123) Paeonia suffruticosa Phenol Li et al., 2009 1,2,3,4,6-Penta-O-galloyl-β- Paeonia suffruticosa Polyphenol Kang et al., 2005 D-glucose (PGG) (124) Piperine (125) Piper nigrum Piperidine alkaloid Pradeep and Kuttan, 2004 Pseudolaric acid B (98) Pseudolarix kaempferi Diterpenoid Li et al., 2009 trans-Resveratrol (126) Not stated Stilbenoid Park et al., 2009 Sphondin (127) Heracleum laciniatum Coumarin Yang et al., 2002 Sulforaphane (51) Brassica oleracea Organosulfur compound Moon et al., 2009 -Tocotrienol (128) Not stated Vitamin E derivative Xu et al., 2009

40 1,2,6-Tri-O-galloyl--D- Not stated Polyphenol Kim et al., 2009 allose (102) Xerophilusin A (105) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Xerophilusin B (106) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009 Xerophilusin F (107) Isodon xerophylus ent-Kaurane diterpenoid Aquila et al., 2009

Anemarsaponin B (109) 2',8"-Biapigenin (111)

Astragaloside IV (110) Carnostic acid (112)

α-Chaconine (113) Corilagin (115)

Chiisanoside (114) continued Figure 1.9. Structures of plant-derived NF-B translocation inhibitors

Figure 1.9. continued

Cryptotanshinone (116) Eriocalyxin B (117)

(-)-Epigallocatechin gallate (118) Eutigoside C (119)

Hesperidin (120) Hirsutenone (121)

continued

Figure 1.9. continued

Oregonin (122) Paeonol (123)

Piperine (125)

1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) trans-Resveratrol (126) (124)

Sphondin (127) -Tocotrienol (128)

2.5. Plant-derived NF-B DNA-binding inhibitors

NF-B DNA-binding inhibitors are the largest group of non-specified NF-B signal transduction supressors. These plant-derived DNA-binding inhibitors are mainly composed of diterpenoids, flavonoids, lignans, and triterpene glycosides. It is notable that more than 25 diterpenoids with abietane or ent-kaurane skeletons have been reported as active principles, accounting for more than one third of all the reported plant-derived

DNA binding inhibitors up to date. An ent-kaurane diterpenoid, namely, ent-18-acetoxy-

7,14-dihydroxykaur-16-en-15-one (145), exhibited significant activity in murine

macrophage RAW264.7 cells, with an IC50 value of 0.07 M (Giang et al., 2003).

Among these natural products, the rocaglate derivatives isolated from Aglaia species

(Meliaceae) have been attracted special interest due to their potent activities as NF-B

DNA binding inhibitors (Baumann et al., 2002). The parent compound, rocaglamide

(182), has also been used as positive control in some NF-B binding studies (Salim et al.,

2007; Han et al., 2009; Pan et al., 2010). A recent study reported that ponapensin (178), a rocaglamide derivative isolated from the leaves of Aglaia ponapensis, showed a potent

NF-κB inhibitory activity with an IC50 value of 0.06 M, even less than the value of 2.0

M of rocaglamide (positive control), in an enzyme-based Elisa assay NF-κB assay

(Salim et al., 2007). Deoxypodophyllotoxin (142) is a naturally occurring lignan isolated

from Anthriscus sylvestris, with reported potency in the inhibition of DNA binding activity at 1.0 M in RAW264.7 cells (Jin et al., 2008).

The plant-derived natural NF-B DNA-binding inhibitors are summarized in the

Table 1.5, and their structures are shown in Figure 1.10.

2.6. NF-B transactivation inhibitors

Transactivation is the last step in NF-B pathway available for specific blockage.

One study showed that 4-hydroxykobusin (199) acted mainly on the transcriptional process of the iNOS (inducible nitric oxide synthase) gene, when using 100 M

4-hydroxykobusin (199) to treat LPS (lipopolysaccharide)-induced RAW264.7 cells.

NF-B is believed to be an essential component of iNOS gene transcription (Pokharel et al., 2007). Mesuol (202), a coumarin isolated from Marila pluricostata, can specifically block RelA phosphorylation, a step required for optimal RelA-mediated transactivation

(Marquez et al., 2005).

The plant-derived NF-B transactivation inhibitors, mostly lignans, as well flavones and terpenoids, are listed in Table 1.6, and their structures are shown in Figure 1.11.

Compound Plant of Origin Phytochemical Category References 25-Acetylcimigenol Cimicifuga racemosa Cycloartane triterpene Qiu et al., 2007 xylopyranoside (129) glycoside Aglain (130) Aglaia elliptifolia Rocaglamide derivative Baumann et al., 2002 Anemarsaponin B (131) Anemarrhena asphodeloides Steroid glycoside Kim et al., 2009 (S)-Armepavine (132) Nelumbo nucifera Benzyltetrahydro- Liu et al., 2007 isoquinoline alkaloid Artemisinin (133) Artemisia annua Sesquiterpenoid Aldieri et al., 2003; Wang et al., 2006 Arzanol (134) Helichrysum italicum -Pyrone Appendino et al., 2007 Baicalein (135) Scutellaria baicalensis Flavonone Suk et al., 2003 Brazilin (136) Caesalpinia sappan Polyphenol Bae et al., 2005

46 Campthothecin (7) Camptotheca acuminata Pyrroloquinoline alkaloid Hentze et al., 2004 Capsiate (137) Capsicum annuum Capsinoid Sancho et al., 2002 Catalposide (138) Catalpa ovata Iridoid glycoside Oh et al., 2002 Cinnamaldehyde (139) Cinnamomum cassia Phenylpropanoid Reddy et al., 2004; Lee et al., 2005; Kim et al., 2007; Liao et al., 2008 Cryptotanshinone (140) Salvia miltiorrhiza Abietane diterpenoid Zhou et al., 2006; Jeon et al., 2008 Danshensu (Salvianic acid A, Salvia miltiorrhiza Phenylpropanoid Jiang et al., 2001; Luo et al., 2008 141) Delphinidin (62) Not stated Anthocyanidin Yun et al., 2009 Deoxypodophyllotoxin (142) Podophyllum peltatum Lignan Jin et al., 2008 Ellagic acid (143) Not stated Polyphenol Edderkaoui et al., 2008 continued Table 1.5. Plant-derived NF-B DNA-binding inhibitors

Table 1.5. continued Compound Plant of Origin Phytochemical Category References ent-18-Acetoxy-7,14- Croton tonkinensis ent-Kaurane diterpenoid Giang et al., 2003 dihydroxykaur-16-en-15-one (144) ent-1-Acetoxy-7,14-dihydroxy Croton tonkinensis ent-Kaurane diterpenoid Giang et al., 2003 kaur-16-en-15-one (145) ent-18-Acetoxy-7-hydroxykaur- Croton tonkinensis ent-Kaurane diterpenoid Giang et al., 2003 16-en-5-one (146) ent-7,14-Dihydroxykaur-16-en- Croton tonkinensis ent-Kaurane diterpenoid Giang et al., 2003 15-one (147) Evodiamine (148) Evodia rutaecarpa Indole alkaloid Choi et al., 2006b Excisusins A, B, D, and E Isodon excisus ent-Kaurane diterpenoids Hong et al., 2007

47 (149-152) Gallic acid (153) Not stated Polyphenol Kim et al., 2005f Garcinol (154) Not stated Diphenyl Hong et al., 2005 Ginkgolide B (155) Ginkgo biloba Diterpenoid Nie et al., 2004 Glycyrrhizin (156) Glycyrrhiza glabra Oleanane triterpene glycoside Wang et al., 1998 Hematein (157) Caesalpinia sappan Polyphenol Oh et al., 2001 2-Hydroxycinnamaldehyde (158) Cinnamomum cassia Phenylpropanoid Reddy et al., 2004; Lee et al., 2005; Kim et al., 2007; Liao et al., 2008 Hypericin (159) Hypericum perforatum Dianthrone Bork et al., 1999 Inflexanins A and B (160 and 161) Isodon excisus ent-Kaurane diterpenoids Hong et al., 2007

continued

Table 1.5. continued Compound Plant of Origin Phytochemical Category References Iinflexarabdonins G and I Isodon excisus ent-Kaurane diterpenoids Hong et al., 2007 (162 and 163) Inflexin (164) Isodon excisus ent-Kaurane diterpenoid Hong et al., 2007 Inflexinol (165) Isodon excisus ent-Kaurane diterpenoid Hong et al., 2007 Jungermannenones A-D Jungermannia spp. ent-Kaurene diterpenoids Kondoh et al., 2005 (166-169) Kamebakaurin (170) Isodon kameba Kaurane diterpenoid Lee et al., 2002b Licochalcone E (171) Glycyrrhiza inflata Chalcone Chang et al., 2007 2-Methoxycinnamaldehyde Cinnamomum cassia Phenylpropanoid Reddy et al., 2004; Lee et al., 2005; (172) Kim et al., 2007; Liao et al., 2008 4-O-Methylhonokiol (173) Magnolia officinalis Benzenoid Oh et al., 2009 Myricetin (174) Not stated Flavonol Kang et al., 2005; Lee et al., 2007 48 Oridonin (175) Isodon rubescens ent-Kaurane diterpenoid Leung et al., 2005; Ikezoe et al.,

2005 1,2,3,4,6-Penta-O-galloyl- Paeonia suffruticosa Polyphenol Oh et al., 2001 -D-glucose (124) Pepluanone (176) Euphorbia peplus Pepluane deterpenoid Corea et al., 2005 Phylligenin (177) Forsythia koreana Lignan Lim et al., 2008 Ponapensin (178) Aglaia ponapensis Rocaglate derivative Salim et al., 2007 Prenylbisabolane (179) Croton eluteria Bisabolane diterpenoid Campagnuoloe et al., 2005 Pseudocoptisine (180) Coptis groenlandica Benzyltetrahydroisoquinoline Yun et al., 2009 alkaloid Quinic acid (181) Uncaria tomentosa Shikimic acid derivative Akesson et al., 2005 Rocaglamide (182) Aglaia elliptifolia Rocaglate derivatives Baumann et al., 2002 Resveratrol (126) Polygonum cuspidatum Stilbene Bentez et al., Saikosaponin D (183) Bupleurum falcatum Oleanane triterpene glycoside Wong et al., 2009

Continued

Table 1.5. continued Compound Plant of Origin Phytochemical Category References Silibinin (184) Not stated Flavonol Schumann et al., 2003 Sinomenine (185) Silybum marianum Benzyltetrahydroisoquinoline Chen et al., 2004; Yamazaki et al., alkaloid 2007: Zhao et al., 2007 Syringaresinol-di-O--D- Acanthopanax senticosus Lignan Yamazaki et al., 2007 glucoside (186) Talosin A (187) Kitasatospora kifunensis Flavonol glycoside Hwang et al., 2009 Triptolide (188) Tripterygium wilfordii Abietane diterpenoid Qiu et al., 1999; Kim et al., 2004; Yinjun et al., 2005; Jang et al., 2007; Chang et al., 2007; He et al., 2007; Liou et al., 2008 Ursolic acid (56) Not stated Ursane diterpene Hsu et al., 2004 Withaferin A (189) Withania somnifera Steroid Mohan et al., 2004; Kaileh et al., 49 2007; Yang et al., 2007; Singh et

al., 2007 Xanthohumol (190) Humulus lupulus Chalcone Colgate et al., 2006 Xanthorrhizol (191) Curcuma xanthorrhiza Sesquiterpene Chung et al., 2007 Xindongnins A and B (192 Isodon rubescens ent-Kaurane diterpenoid Leung et al., 2005 and 193) Zapotin (194) Casimiroa edulis Flavonone Maiti et al., 2007 Zedoarondiol (195) Curcuma heyneana Sesquiterpene Cho et al., 2009

25-Acetylcimigenol xylopyranoside Aglain (130) (129)

Anemarsaponin B (131) (S)-Armepavine (132) Artemisinin (133)

Arzanol (134) Baicalein (135) Brazilin (136)

Capsiate (137)

Catalposide (138) Cinnamaldehyde (139) Cryptotanshinone (140) continued Figure 1.10. Structures of plant-derived NF-κB DNA binding inhibitors 50 50 Figure 1.10. continued

Danshensu Deoxypodophyllotoxin (142) Ellagic acid (143) (Salvianic acid A, 141) R1 R2 R3 144 H OAc OH ent-18-Acetoxy-7,14- dihydroxykaur-16-en-15-one 145 OAc H OH ent-1-Acetoxy-7,14- dihydroxykaur-16-en-15-one 146 H OAc H ent-18-Acetoxy-7-hydroxykaur- 16-en-15-one 147 H H OH ent-7,14-Dihydroxykaur-16-en- 15-one

R1 R2 R3 R4 R5 149 H OAc OH H OH Excisusin A (149) 150 OH OAc H H OH Excisusin B (150) 151 OH OH H OH OAc Excisusin D (151) 152 H =O OAc H OH Excisusin E (152)

Evodiamine (148) Gallic acid (153)

Glycyrrhizin (156) Garcinol (154) Ginkgolide B (155)

continued 51 51 Figure 1.10. continued

Hematein (157) 2-Hydroxycinnamaldehyde Hypericin (159) (158)

R1 R2 R3 R4 R5 OH β-OAc OH OAc β-OH Inflexanin A (160) OH β-OAc OH OH =O Inflexanin B (161) OH β-OAc H OAc =O Iinflexarabdonin G (162) H =O OH OH =O Iinflexarabdonin I (163) OH β-OAc =O OAc =O Inflexin (164) OH β-OAc OH OAc =O Inflexinol (165)

R1 R2 OH H Jungermannenone A (166) H H Jungermannenone B (167) H OH Jungermannenone C (168) OH OH Jungermannenone D (169)

Kamebakaurin (170) Licochalcone E (171) 4-O-Methylhonokiol (173)

Myricetin (174) Oridonin (175) Pepluanone (176) continued

52 52 Figure 1.10. continued

Phylligenin (177) Ponapensin (178)

Prenylbisabolane III (179) Pseudocoptisine (180) Quinic acid (181)

Rocaglamide (182) Saikosaponin D (183)

Silibinin (184) Sinomenine (185)

Syringaresinol-di-O--D-glucoside (186) Talosin A (187)

continued 53 53 Figure 1.10. continued

Triptolide (188) Withaferin A (189) Xanthohumol (190)

Xanthorrhizol (191) Xindongnin A (192) Xindongnin B (193)

Zapotin (194) Zedoarondiol (195)

54 54

Compound Origin of Plant Phytochemical References Category Artemisolide (196) Artemisia asiatica Sesterterpenoid Reddy et al., 2006; Kim et al., 2007 Curcumin (197) Curcuma longa Curcuminoid Ghosh et al., 2009 4'-Demethyl-6-methoxy- Linum tauricum Lignan Vailev et al., 2005 podophyllotoxin (198) Hesperidin (120) Not stated Flavanone Yeh et al., 2007 glycoside 4-Hydroxykobusin (199) Geranium Lignan Pokharel et al., thunbergii 2007 Manassantins A and B Saururus chinensis Lignans Lee et al., 2003; (200 and 201) Son et al., 2005 Mesuol (202) Marila pluricostata Coumarin Marquez et al., 2005 Nobiletin (203) Citrus sunki Polyphenol Murakami et al., derivative 2005; Choi et al., 2007 Paeonol (123) Paeonia suffruticosa Benzoid Ishiguro et al., 2006;Tsai et al., 2008 Saucerneols D and E Saururus chinensis Lignans Hwang et al., 2003 (204 and 205) Table 1.6. Plant-derived NF-κB transactivation inhibitors

Artemisolide (196) Curcumin (197)

continued Figure 1.11. Structures of plant-derived NF-κB transactivation inhibitors

55 55 Figure 1.11. continued

4'-Demethyl-6-methoxypodophyllotoxin (198) 4-Hydroxykobusin (199)

Manassantin A (200)

Manassantin B (201)

Mesuol (202) Nobiletin (203)

R = OMe Saucerneol D (204)

R = OH Saucerneol E (205)

56 56

Chapter 2

BIOACTIVE DIHYDRO--PYRONE DERIVATIVES AND FLAVONOIDS FROM

HYPTIS BREVIPES

A. Background on Hyptis brevipes

1. Lamiaceae

The Lamiaceae, formerly known as the Labiatae, is generally known as the mint family and contains 250 genera and 6,700 species (Woodland, 2000). Most plants in the family are aromatic and therefore some have been used as culinary spices, such as basil, lavender, marjoram, mint, oregano, perilla, rosemary, sage, savory, and thyme. Certain plants from the genus Pogostemon are cultured in Southeast Asia as sources of perfumes

(Woodland, 2000).

The botanical diagnostic characteristics of the plants in the family Lamiaceae and their distribution are described as follows (Woodland, 2000):

General Description: herbs or shrubs (rarely mid-sized trees); often covered with hairs and aromatic glands containing ethereal oils; usually young stems 4 sided with well-developed collenchyma in the angles. Leaves: opposite (rarely alternate or whorled), simple to pinnately compound; no stipules. Flowers: irregular, often 2 lipped (bilabiate), perfect (rarely unisexual); hypogynous, usually having bracts; inflorescence of solitary flower or flowers borne in axillary cymes or verticils, head, panicles, or racemes. Sepals: 5, connate. Petals: 5, 57 57 sympetalous, the corolla usually bilabiate. Stamens: 2 or 4, filaments distinct (rarely connate), sometimes didynamous and adnate to the corolla tube; anthers opening by longitudinal slits; nectary disk present; many specialized types of pollination occur. Pistil: compounds of 2 united carpels; locules 4; ovules 1 per locule and borne on a basal-axile placenta; ovary superior; style 1, gynobasal (rarely terminal); stigma 2 lobed. Fruit: of 4 (rarely 1-3), 1-seeded nutlets. Seeds: with straight embryo; endosperm scanty or absent. Distribution: 250 genera and 6700 species worldwide in all climates, with many taxa in the Mediterranean region. The largest genera are Salvia (ca. 900 species), Hyptis (ca. 300 species), Scutellaria (ca. 350 species), Coleus (ca. 200 species), Plectranthus (ca. 200 species), and Stachys (ca. 300 species). (Woodland, 2000)

2. Genus Hyptis and Hyptis brevipes Piot.

Hyptis is one of the largest genera in the Lamiaceae, with about 300 species distributed from tropic to temperate regions. Selected botanical diagnostic characteristics of plants of the genus Hyptis are described as follows (Cheng, 1978):

Herbs or suffruticose plants; stems stout, square. Inflorescence in verticillate cymes, or clustered axillary or terminal racemes or rarely forming heads. Calyx campanulate to tubular, prominently 10-nerved, 5-toothed, the teeth spine-like, equal or nearly so; corolla tubular, 5-lobed, the lowest lobe abruptly deflexed, the edge thickened, the others erect or spreading, flat. Fruitlets ellipsoidal, smooth or slightly reticulate (Cheng, 1978).

Botanical diagnostic characteristics of Hyptis brevipes Piot. include the following

(Cheng, 1978):

A suffruticose plant, up to 100 cm high, covered with separate hairs; stems square, the angles prominent, puberulent or villous, densely so at nodes. Leaves sessile or with very short petioles; blades 3-8 cm long, 1-2 cm wide, narrowly ovate, the base cuneate, the apex acute, both surfaces puberulent or villous, glandularly dotted beneath. Inflorescence in cymes forming capitate clusters, axillary or terminal, sessile or with peduncles up to 1 cm long, the peduncles puberulent or villous; bracteoles linear, puberulent on margin. Flowers subsessile; calyx campanulate, minutely hairy, 5-toothed, 2-3 mm long in anthesis, 4-5 mm long in fruit, the tube short, the teeth spine-like, equal, minutely hairy; corolla tubular, bilabiate, glabrous or minutely hairy, 2-3 mm long, the upper lip broadly ovate, the apex emarginated, the lower lip 3-lobed;

58 58 stamens 4, didynamous, exserted, the anthers 2-celled, the filaments glabrous; stigma bifid. Fruitlets ellipsoidal, glabrous, about 0.5 mm long. Distributed in tropical America, Asia and Africa (Cheng, 1978)

Figure 2.1. Illustration of Hyptis brevipes Poit. – 1, top of flowering and fruiting stem; 2, flower in lateral view; 3, flower in front view; 4, fruiting calyx; 5, fruit after removal of calyx (from Lemmens et al., 2003)

59 59 3. Constituents of the genus Hyptis

Previous investigations have led to reports of flavonoids, lignans, monoterpenoids, pyrone and furanone derivatives, sesquiterpenoids, sterols, as well as triterpenoids from plants of the genus Hyptis. Since monoterpenoids and sesquiterpenoids have been found normally in essential oils of these plants, the following brief review will focus on the other types of natural products found typically in non-volatile extractives.

3.1. Pyrone and furanone derivatives

The 6-heptylpyrone derivatives are a unique group of natural products found only in the Lamiaceae family thus far, such as in the genera Hyptis, Syncolostemon, and

Tetradenia. Up to the present time, there have been 17 different 6-heptylpyrone derivatives (206-217, 220-224) and two closely related 5-octylfuranone derivatives (218 and 219) isolated from five different Hyptis species. Two 6-heptylpyrone derivatives, anamarine (206) and olguine (211), first isolated from the genus Hyptis, were reported in

1979, and their structures were determined by NMR experiments and X-ray crystallographic analysis. Structural studies showed that the conformation of the pyrone ring is in the form of a distorted envelope, with C-5 being at the flap (Alemany et al.,

1979a,b). Later studies employed circular dichroism spectroscopy to determine the absolute configuration of the C-5 stereogenic center, as in the case of hyptolide (209,

Achmad et al., 1987). However, the establishment of the relative configuration of the highly flexible heptyl side chain of acylated pyrones has proved to be a great challenge to investigators if a suitable crystal was not available for X-ray crystallographic analysis. In

60 60 many cases, research papers on 6-heptylpyranone derivatives were published without full elucidation of the configuration, such as that of hypurticin (210, Vivar et al., 1991). A recent study afforded a feasible methodology to resolve this problem, by a comparison of the measured 1H NMR spectrum of hypurticin with a calculated 1H NMR spectrum, obtained as a result of DFT B3LYP/DGDZVP molecular modeling calculations running on a supercomputer (Mendoza-Espinoza et al., 2009).

In the few bioactivity-related studies that have been reported to date, some

6-heptylpyrone and 5-octylfuranone derivatives have shown cytotoxic or antimicrobial activities (Pereda-Miranda et al., 1993; Fargoso-Serrano et al., 2005). In a cytotoxicity assay, pectinolides A-C (212-214) showed ED50 values in the range 0.7-3.8 g/mL against a panel of cancer cell lines, inclusive of BC1 human breast cancer, HT-1080 human fibrosarcoma, Lu1 human lung cancer, Mel2 human melanoma, Col2 human colon cancer, KB human nasopharyngeal carcinoma, KB-V vinblastine-resistant KB cells,

P388 murine lymphocytic leukemia, A431 human epidermoid carcinoma, LNCaP hormone-dependent human prostate cancer, and ZR75-1 hormone dependent human breast cancer (Pereda-Miranda et al., 1993).

The trivial names, plant sources, and references for pyrone and furanone derivatives isolated from the genus Hyptis are summarized in Table 2.1, and their structures are presented in Figure 2.2 following the table.

3.2. Lignans

To date, there have been 20 different lignans isolated from five different Hyptis species. Interestingly, these lignans include the well-known anticancer lead compound, 61 61 Code Trivial name Source Reference 206 Anamarine Unspecified Alemany et al., 1979b 207 4-Deacetoxy- Hyptis oblongifolia Delgado et al., 1985; 10-epi-olguine Peranda-Miranda et al., 1990 208 10-epi-Olguine Hyptis capitata Almtorp et al., 1991 209 Hyptolide Hyptis pectinata Achmad et al., 1987 210 Hypurticin Hyptis urticoides Romo de Vivar et al., 1991 211 Olguine Unspecified Alemany et al., 1979a 212 Pectinolide A Hyptis pectinata Peranda-Miranda et al., 1993; Fragoso-Serrano et al., 2005 213 Pectinolide B Hyptis pectinata Peranda-Miranda et al., 1993; Fragoso-Serrano et al., 2005 214 Pectinolide C Hyptis pectinata Peranda-Miranda et al., 1993; Fragoso-Serrano et al., 2005 215 Pectinolide D Hyptis pectinata Boalino et al., 2003 216 Pectinolide E Hyptis pectinata Boalino et al., 2003 217 Pectinolide F Hyptis pectinata Boalino et al., 2003 218 Pectinolide G Hyptis pectinata Boalino et al., 2003 219 Pectinolide H Hyptis pectinata Fragoso-Serrano et al., 2005 220 Spicigera Hyptis pectinata Aycard et al., 1993 -lactone 221 Spicigerolide Hyptis pectinata Peranda-Miranda et al., 2001 222 Unnamed Hyptis oblongifolia Peranda-Miranda et al., 1990 223 Unnamed Hyptis oblongifolia Peranda-Miranda et al., 1990 224 Unnamed Hyptis oblongifolia Peranda-Miranda et al., 1990

Table 2.1. Trivial names and plant sources of pyrone and furanone derivatives isolated from the genus Hyptis

62 62 O O O

OAc OAc O OAc OAc O OAc O

OAc OAc OAc OAc OAc O OAc 206 Anamarine 210 Hypurticin 211 Olguine O R OAc O H 207 4-Deacetoxy-10-epi-olguine OAc 208 10-epi-Olguine OAc O R

R3 R2 O

R4 R1

R1 R2 R3 R4 209 H OAc OAc OAc Hyptolide 212 OAc OAc H H Pectinolide A 213 OAc OH H H Pectinolide B 214 OH OAc H H Pectinolide C 215 OH OAc OH OAc Pectinolide D 216 OAc OAc OAc OAc Pectinolide E

O OAc R2 OAc O O O OMe OAc OAc OAc OAc 217 Pectinolide F 218 Pectinolide G continued

Figure 2.2. Structures of pyrone and furanone derivatives isolated from the genus Hyptis

63 63

Figure 2.2. continued

O H O OAc O O OAc OH OAc O 219 Pectinolide H 220 Spicigera -lactone

O R1 R2 O OAc OAc O 222 OAc OAc Unnamed OAc R2 O 223 OH OMe Unnamed OAc OAc OAc R1 221 Spicigerolide 224 OH OH Unnamed

podophyllotoxin (239), and several analogs. Desoxypodophyllotoxin (233), isolated from

H. tomentosa, showed potent cytotoxicity with an ED50 value of 0.032 g/mL in a KB cell culture system (Kingston et al., 1979). In another study, -apopicropodophyllin (225) and demethyldeoxypodophyllotoxin (227), isolated from H. verticillata, exhibited significant cytotoxicity against P-338 murine lymphocytic leukemia cells, with ED50 values of 0.002 and 0.005 g/mL, respectively, when compared with the ED50 value of the positive control agent, podophyllotoxin, of 0.003 g/mL. The activities of these two compounds (225 and 227) were equivalent to that of podophyllotoxin in the brine shrimp lethality test, with LD50 values of 0.2 g/mL (Novelo et al., 1993).

64 64 The trivial names, and plant sources, and references for lignans isolated from the genus Hyptis are summarized in Table 2.2, and their structures are presented in the following Figure 2.3.

Code Trivial name Source Reference 225 -Apopicropodophyllin Hyptis verticillata Novelo et al., 1993 226 (-)-Cubebin Hyptis salzmanii Messana et al., 1990 227 4-Demethyldeoxypodophyllo- H. verticillata German et al., 1971; toxin 228 4'-Demethylpodophyllotoxin H. verticillata Novelo et al., 1993; Kuhnt et al., 1994 229 Dehydro--peltatin methyl ether H. verticillata Novelo et al., 1993 230 Dehydropodophyllotoxin H. verticillata Novelo et al., 1993 231 Deoxydehydropodophyllotoxin H. verticillata Novelo et al., 1993 232 Deoxypicropodophyllin H. verticillata Novelo et al., 1993 233 Desoxypodophyllotoxin Hyptis tomentosa Kingston et al., 1979 234 Epipodorhizol H. verticillata Kuhnt et al., 1994 235 (-)-Hinokinin H. salzmanii Messana et al., 1990 236 Hyptinin H. verticillata Kuhnt et al., 1994 237 Isodeoxypodophyllotoxin H. verticillata Novelo et al., 1993 238 -Peltatin H. verticillata German et al., 1971; 239 Podophyllotoxin H. verticillata Kuhnt et al., 1994 240 Podorhizol H. verticillata Kuhnt et al., 1994 241 (+)-Sesamin H. tomentosa Kingston et al., H. salzmanii 1979; Messana et al., 1990 242 (-)-Yatein H. verticillata Novelo et al., 1993 243 Unnamed Hyptis capitata Almtorp et al., 1991 244 Unnamed H. verticillata Novelo et al., 1993; Kuhnt et al., 1994

Table 2.2. Trivial names and plant sources of lignans isolated from the genus Hyptis

65 65

R1 R2 R3 R2 R1 H 225 H H Me -Apopicropodophyllin O H O 227 H H H 4-Demethyldeoxypodophyllotoxin O H H O 228 OH H H 4'-Demethylpodophyllotoxin 233 H H Me Desoxypodophyllotoxin MeO OMe 238 H OH Me -Peltatin OR3 239 OH H Me Podophyllotoxin

R2 R1

O R1 R2 O O 229 H OMe Dehydro--peltatin methyl ether O 230 OH H Dehydropodophyllotoxin 231 H H Deoxydehydropodophyllotoxin MeO OMe OMe 244 OH OMe Unnamed H O R1 R2 R1 O O 234 H OH Epipodorhizol R2 H O 240 OH H Podorhizol

MeO OMe 242 H H (-)-Yatein OMe H O H O O O O O O H O O O MeO OMe OMe 226 (-)-Cubebin 232 Deoxypicropodophyllin

continued

Figure 2.3. Structures of lignans isolated from the genus Hyptis

66 66 Figure 2.3. continued O O H O O O O O O H H O O

MeO OMe OMe 235 (-)-Hinokinin 236 Hyptinin H O H O O O O H H H O O O H O H O MeO OMe OMe 237 Isodeoxypodophyllotoxin 241 (+)-Sesamin O HO O O H H O O

Unnamed (243)

3.3. Flavonoids

Flavones (245, 250, 252-254, and 257), a flavonol (247), flavanones (246, 251,

256), a chalcone (255), and flavone glycosides (248 and 249) have been reported from

Hyptis species in the published literature. The flavone, sideritoflavone (250), was found to be active against the KB cancer cell line with an ED50 value of 1.6 g/mL (Novelo et al., 1993). The trivial names, plant sources, and references for flavonoids isolated from 67 67 the genus Hyptis are presented in Table 2.3, and their structures are provided in the following Figure 2.4.

Code Trivial name Source Reference 245 Eupatorin Hyptis tomentosa Kingston et al., 1979 246 (-)-Isosakuranetin Hyptis salzmanii Messana et al., 1990 247 Kaempferol Hyptis rhomboides Lin et al., 1993 248 Kaempferol-3-O-rutinoside H. rhomboides Lin et al., 1993 249 Rutin H. rhomboides Lin et al., 1993; Hyptis suaveolens Indane et al., 2007 250 Sideritoflavone Hyptis verticillata Novelo et al., 1993; Kuhnt et al., 1995 251 ()-Sakuranetin H. salzmanii Messana et al., 1990 252 Unnamed H. tomentosa Kingston et al., 1979 253 Unnamed H. tomentosa Kingston et al., 1979 254 Unnamed H. tomentosa Kingston et al., 1979 255 Unnamed H. salzmanii Messana et al., 1990 256 Unnamed H. salzmanii Messana et al., 1990 257 Unnamed Hyptis capitana Almtorp et al., 1991

Table 2.3. Trivial names and plant sources of flavonoids isolated from the genus Hyptis

3.4. Diterpenoids

Diterpenoids isolated from the genus Hyptis and their bioactivity studies have been reviewed recently (Piozzi et al., 2009). Since the first report of two diterpenoids isolated 68 68

R1 R2 R3 R4 R5 R6 R7 245 H OH OMe OMe H OH OMe Eupatorin 247 OH OH H OH H H OH Kaempferol 250 H OH OMe OMe OMe OH OH Sideritoflavone 252 H OH OMe OMe OMe OH OMe Unnamed 253 OMe OH OMe OMe OMe OH OMe Unnamed 254 H OMe OMe H H OMe OMe Unnamed 257 H OH H OMe H H OMe Unnamed OH CH3 OMe HO O OH HO O O O O OH OH O OH HO OH OH O OH 246 (-)-Isosakuranetin 248 Kaempferol-3-O-rutinoside OH OH OH CH3 HO O OH MeO O

O O O OH OH O OH HO OH OH O OH 249 Rutin 251 ()-Sakuranetin OMe OH MeO O MeO OH

OH O OH O 255 Unnamed 256 Unnamed

Figure 2.4. Structures of flavonoids isolated from the genus Hyptis

69 69 from H. suaveolens in 1974, 66 different diterpenoids altogether have been isolated from different Hyptis species. Among these diterpenoids, 61 were isolated from non-volatile extracts. Most of these 61 compounds belong to three groups according to their structures: the largest group, the abietane type, such as diacetylcarnosic acid (269) and carnosol

(265); the isopimarane type, such as salzol (301); and the labdane type, such as compounds 258 and 259. The abietane diterpenoid, carnosol (265), isolated from H. martiusii, showed cytotoxicity against several tumor cell lines (Costa-Lotufo et al., 2004).

In another report, another abietane diterpenoid, 7-acetoxyroyleanone (262), isolated from the same plant, was found to be active against several cancer cell lines with IC50 values in the range 0.9 to 7.6 g/mL (Araujo et al., 2006). The trivial names, plant sources, and references for selected Hyptis diterpenoids are shown Table 2.4, and their structures are presented in the following Figure 2.5.

Code Trivial or semi-systematic name Source Reference 258 19-Acetoxy-2α,7α-dihydroxylabda- Hyptis Fragoso-Serrano et 8(17),(13Z)-diene-15-al spicigera al., 1999 259 19-Acetoxy-7α,15-dihydroxylabda- H. spicigera Fragoso-Serrano et 8(17),(13Z)-dien-2-one al., 1999 260 19-Acetoxy-2α,7α-dihydroxylabda- H. spicigera Fragoso-Serrano et 14,15-dinor-labd-8(17)-en-13-one al., 1999 261 19-Acetoxy-2α,7α,15-trihydroxy- H. spicigera Fragoso-Serrano et labda-8(17),(13Z)-diene al., 1999 262 7α-Acetoxyroyleanone Hyptis Araujo et al., 2006 martiusii 263 Acetylpisiferic acid methyl ester Hyptis dilatata Urones et al., 1998 264 Carnosic acid H. dilatata Urones et al., 1998 continued Table 2.4. Trivial names and plant sources of diterpenoids isolated from the genus Hyptis

70 70 Table 2.4. continued Code Trivial or semi-systematic name Source Reference 265 Carnosol H. dilatata; Urones et al., 1998; H. martiusii Costa-Lotufo et al., 2004 266 Coulterone Hyptis Araujo et al., 2005 platanifolia 267 15,19-Diacetoxy-2α,7α-dihydroxy- H. spicigera Fragoso-Serrano et labda-8(17),(13Z)-diene al., 1999 268 Diacetylepiethylrosmanol H. dilatata Urones et al., 1998 269 Diacetylcarnosic acid H. dilatata Urones et al., 1998 270 Diacetylcarnosic acid methyl ester H. dilatata Urones et al., 1998 271 Diacetylcarnosol H. dilatata Urones et al., 1998 272 Diacetylepimethylrosmanol H. dilatata Urones et al., 1998 273 Diacetylesquirolin B H. dilatata Urones et al., 1998 274 Diacetylethylrosmanol H. dilatata Urones et al., 1998 275 Diacetylmethylrosmanol H. dilatata Urones et al., 1998 276 11,14-Dihydroxy-8,11,13-abietatrien- H. Araujo et al., 2005 7-one platanifolia 277 11,14-Dihydroxy-12-methoxy-7-oxo- H. martiusii Costa-Lotufo et al., 8,11,13-abietatrien-19,20-β-olide 2004 278 9α,13α-epi-Dioxiabiet-8(14)-en-18-ol Hyptis. Chukwujekwu et suaveolens al., 2005 279 Epiethylrosmanol H. dilatata Urones et al. 280 Epimethylrosmanol H. dilatata Urones et al. 281 Epirosmanol H. dilatata Urones et al. 282 19,20-Epoxy-12-methoxy-11,14,19- H. Araujo et al., 2005 trihydroxy-7-oxo-8,11,13-abietatriene platanifolia 283 Esquirolin B H. dilatata Urones et al., 1998 284 Ethylrosmanol H. dilatata Urones et al., 1998 285 Horminone H. fruticosa Marletti et al. 1976 286 7β-Hydroxy-11,14-dioxoabieta-8,12- H. martiusii Araujo et al., 2006 diene 287 Hyptol H. fruticosa Delle Monache et al., 1977 288 Inuroyleanol H. Araujo et al., 2005 platanifolia 289 Isorosmanol H. dilatata Urones et al., 1998 continued

71 71 Table 2.4. continued Code Trivial or semi-systematic name Source Reference 290 5βΗ,8βΗ,9βΗ,10α-Labd-14-ene H. suaveolens Iwu et al., 1990 291 Martiusane H. martiusii Araujo et al., 2004 292 15α-Methoxyfasciculatin H. fasciculata Ohsaki et al., 2005 293 15β-Methoxyfasciculatin H. fasciculata Ohsaki et al., 2005 294 14-Methoxytaxodione H. fruticosa Marletti et al., 1976 295 12-O-Methylcarnosic acid H. martiusii Araujo et al., 2004 296 Methylnepetaefolin H. fasciculata Ohsaki et al., 2005 297 Methylrosmanol H. dilatata Urones et al., 1998 298 19-Oxoinuroyleanol H. platanifolia Araujo et al., 2005 299 Pisiferic acid H. dilatata Urones et al., 1998 300 Rosmanol H. dilatata Urones et al., 1998 301 Salzol H. salzmanii Messana et al. 1990 302 7-Seco-7(20),11(20)-diepoxy-7,14- Hyptis martiusii Araujo et al., 2004 dihydroxyabieta-8,11,13-triene 303 Suavelol H. suaveolens Manchand et al., 1974 304 Suaveolic acid H. suaveolens Manchand et al., 1974 305 7α, 15,19-Triacetoxy-2α-hydroxy- H. spicigera Fragoso-Serrano et labda-8(17),(13Z)-diene al., 1999 306 2α,7α,15,19-Tetrahydroxy-ent- H. spicigera Fragoso-Serrano et labda-8(17),(13Z)-diene al., 1999 307 Triacetyl epirosmanol H. dilatata Urones et al., 1998 308 Triacetyl isorosmanol H. dilatata Urones et al., 1998 309 Triacetyl rosmanol H. dilatata Urones et al., 1998 310 Umbrosone Hyptis umbrosa Delle Monache et al., 1990 311 Unnamed H. dilatata Urones et al., 1998

72 72

R1 R2 258 OH CHO 19-Acetoxy-2α,7α-dihydroxylabda- 8(17),(13Z)-diene-15-al 261 OH CH2OH 19-Acetoxy-2α,7α,15-trihydroxy- labda-8(17),(13Z)-diene 267 OH CH2OAc 15,19-Diacetoxy-2α,7α-dihydroxylabda-8(17),(13Z)-diene 305 OAc CH2OAc 7α, 15,19-Triacetoxy-2α-hydroxy- labda-8(17),(13Z)-diene

259 19-Acetoxy-7α,15-dihydroxy- 260 19-Acetoxy-2α,7α-dihydroxylabda-8(17),(13Z)-dien-2-one labda-14,15-dinor-labd-8(17)-en- 13-one

R1 R2 262 -OAc OH 7α-Acetoxyroyleanone 286 -OH H 7β-Hydroxy-11,14-dioxoabieta- 8,12-diene

R1 R2 R3 263 COOMe H OAc Acetylpisiferic acid methyl ester 264 COOH OH OH Carnosic acid 269 COOH OAc OAc Diacetylcarnosic acid 270 COOMe OAc OAc Diacetylcarnosic acid methyl ester 299 COOH H OH Pisiferic acid

R1 R2 265 H OH Carnosol 271 H OAc Diacetylcarnosol 289 OH OH Isorosmanol 308 OAc OAc Triacetyl isorosmanol

continued Figure 2.5. Structures of diterpenoids isolated from the genus Hyptis

73 73 Figure 2.5. continued

266 Coulterone

R1 R2 268 -OEt OAc Diacetylepiethylrosmanol 272 -OMe OAc Diacetylepimethylrosmanol 274 -OEt OAc Diacetylethylrosmanol 275 -OEt OAc Diacetylmethylrosmanol 279 -OEt OH Epiethylrosmanol 280 -OMe OH Epimethylrosmanol 281 -OH OH Epirosmanol 284 -OEt OH Ethylrosmanol

297 -OMe OH Methylrosmanol 300 -OH OH Rosmanol 307 -OAc OAc Triacetyl epirosmanol 309 -OAc OAc Triacetyl rosmanol R 273 Ac Diacetylesquirolin B 283 H Diacetylmethylrosmanol

R1 R2 276 Me H 11,14-Dihydroxy-8,11,13-abietatrien-7- one 288 Me OMe Inuroyleanol 298 CHO OMe 19-Oxoinuroyleanol R 277 =O 11,14-Dihydroxy-12-methoxy-7-oxo-8,11,13- abietatrien-19,20-β-olide 282 O 19,20-Epoxy-12-methoxy-11,14,19-trihydroxy- H 7-oxo-8,11,13-abietatriene

continued

74 74 Figure 2.5. continued

278 9α,13α-epi- 285 Horminone 287 Hyptol 290 Dioxiabiet-8(14)- 5βΗ,8βΗ,9βΗ, en-18-ol 10α-Labd-14-ene

R1 R2 292 H OMe 15α-Methoxy- fasciculatin

291 Martiusane 293 OMe H 15β-Methoxy- fasciculatin

294 295 296 301 14-Methoxytaxo- 12-O-Methylcar- Methylnepetaefolin Salzol dione nosic acid

R 303 CH2OH Suavelol 304 COOH Suaveolic acid

302 7-Seco-7(20),11(20)-diepoxy- 7,14-dihydroxy-abieta-8,11,13- triene

continued

75 75 Figure 2.5. continued

306 310 311 2α,7α,15,19-Tetrahydroxy- Umbrosone Unnamed ent-labda-8(17),(13Z)-diene

3.5. Triterpenoids

More than 25 different triterpenoids have been isolated from the genus Hyptis.

According to their skeletons, these compounds can be classified mainly into lupane (314,

321, 328, and 329), oleanane (312, 315, 318, 320, 322, 323, and 325), and ursane (313,

316, 317, 319, 324-327, and 330) types. Hyptadienic acid (317) was the first A-ring

contracted natural triterpene outside the lupane series (Rao et al., 1990).

2-Hydroxyursolic acid (316) and hyptatic acid A (318) demonstrated in vitro

cytotoxicity against HCT-8 human colon tumor cells with ED50 values of 2.7 and 4.2

g/mL, respectively (Yamagishi et al., 1988). The trivial names, plant sources, and

references for triterpenoids isolated from the genus Hyptis are summarized in Table 2.5,

and their structures are presented in the following Figure 2.6.

76 76 Code Trivial name Source Reference 312 -Amyrin H. suaveolens; Misra et al., 1983a; Biggs et al., H. verticillata 2008 313 -Amyrin H. suaveolens; Misra et al., 1983a; Biggs et al., H. verticillata 2008 314 Betulinic acid H. albida; Sheth et al., 1972; Misra et al., H. emoryi; 1983b; Pereda-Miranda et al., H. rhomboides; 1990; Lin et al., 1993; Biggs et H. suaaveolens; al., 2008 H. verticillata 315 2,3-Dihydroxy- H. oblongifolia Pereda-Miranda et al., 1990b oleanolic acid 316 2-Hydroxyursolic H. capitana; Yamagishi et al., 1988; acid H. oblongifolia; Pereda-Miranda et al., 1990b; H. rhomboides Lin et al., 1993 317 Hyptadienic acid H. suaveolens Rao et al., 1990 318 Hyptatic acid A H. capitana Yamagishi et al., 1988 319 Hyptatic acid B H. capitana Yamagishi et al., 1988 320 Maslinic acid H. capitana; Yamagishi et al., 1988; H. mutabilis; Pereda-Miranda et al., 1988; H. oblongifolia Pereda-Miranda et al., 1990b 321 Methyl betulinate H. mutabilis Pereda-Miranda et al., 1988 322 Oleanolic acid H. albida; Misra et al., 1981; H. suaveolens; Pereda-Miranda et al., 1990; H. verticillata Biggs et al., 2008 323 Oleanolic acid acetate H. albida; Pereda-Miranda et al., 1988 H. mutabilis Pereda-Miranda et al., 1990a 324 -Peltoboykinolic acid H. suaveolens Misra et al., 1981 325 Pomolic acid H. oblongifolia Pereda-Miranda et al., 1990b 326 Tormentic acid H. capitana Yamagishi et al., 1988 327 Ursolic acid H. albida; Misra et al., 1983b; H. mutabilis; Pereda-Miranda et al., 1988; H. oblongifolia; Pereda-Miranda et al., 1990a,b; H. rhomboides; Lin et al., 1993; Biggs et al., H. suaveolens; 2008 H. verticillata 328 Unnamed H. suaveolens Misra et al., 1983a 329 Unnamed H. suaveolens Misra et al., 1983b 330 Unnamed H. mutabilis Pereda-Miranda et al., 1988 331 Unnamed H. mutabilis Pereda-Miranda et al., 1988 continued Table 2.5. Trivial names and plant sources of triterpenoids isolated from the genus Hyptis

77 77 Table 2.5. continued

Code Trivial name Source Reference 332-334 Unnamed H. albida Pereda-Miranda et al., 1990a 335 Unnamed H. rhomboides Lin et al, 1993 336 Unnamed H. mutabilis Biggs et al., 2008

HO HO

312 -Amyrin 313 -Amyrin

COOH COOH HO

HO HO 314 Betulinic acid 315 2,3-Dihydroxyoleanolic acid

COOH COOH HOH C HO 2

316 2-Hydroxyursolic acid 317 Hyptadienic acid continued Figure 2.6. Structures of triterpenoids isolated from the genus Hyptis

78 78 Figure 2.6. continued

COOH COOH HO HO

HO HO HOH2C HOH2C 318 Hyptatic acid A 319 Hyptatic acid B

COOH HO COOMe

HO HO

320 Maslinic acid 321 Methyl betulinate

COOH COOH HO O

O 322 Oleanolic acid 323 Oleanolic acid acetate

COOH

COOH HO HO

324 -Peltoboykinolic acid 325 Pomolic acid continued

79 79 Figure 2.6. continued

COOH COOH HO

HO HO

326 Tormentic acid 327 Ursolic acid

COOH COOH HO

328 Unnamed 329 Unnamed

OH CO COOH

AcO 330 Unnamed 331 Unnamed

CO CO

HO HO 332 Unnamed 333 Unnamed

continued

80 80 Figure 2.6. continued

O CO

COOH HO

HO HO HOH2C 334 Unnamed 335 Unnamed

COOH O

336 Unnamed

3.6. Other constituents

In addition to the aforementioned constituents, plant sterols and sesquiterpenoids have also been found in Hyptis species. Stigmasterol (337), stigmasterol glucoside (338),

-sitosterol (339), and -sitosterol glucoside (daucosterol, 340) are the sterols most frequently encountered in the genus (Misra et al., 1981, 1983; Lin et al., 1993; Araujo et al. 2005). An acaricidal and insecticidal sesquiterpenoid, cadina-4,10(15)-dien-3-one

(341), was isolated from Hyptis vertiicillata (Porter et al., 1995).

81 81 R O R O

337 Stigmasterol R = OH 339 -Sitosterol R = OH 338 Stigmasterol glucoside R = Glucose 340 Daucosterol R = Glucose

O H 341 Cadina-4,10(15)-dien-3-one Figure 2.7. Structures of sterols and a sesquiterpene isolated from the genus Hyptis

B. Statement of Problem

Biological studies on crude extracts of Hyptis brevipes have shown inhibitory activities against bacterial and fungal growth, as well as DNA intercalation inhibition activity (Gupta et al., 1996; Goun et al., 2003). However, before the initiation of this dissertation study, there have been no reports on the phytochemical or biological evaluation of H. brevipes.

As part of a collaborative, multi-disciplinary approach to the discovery of new naturally occurring anticancer drugs (Kinghorn et al., 2003 and 2009), the entire plant of

H. brevipes, collected in central Java, Indonesia, was selected for further investigation after a chloroform extract of the plant was found active in an initial cytotoxicity screening 82 82 using the MCF-7 human breast cancer cell line, with an ED50 value of 11.7 g/mL. This plant was collected initially for a NCI/NIH-founded program project directed towards the discovery of plant-derived cancer chemopreventive agents, directed by Dr. John M.

Pezzuto, then at the University of Illinois at Chicago (P01 CA48112; Kinghorn et al.,

2004).

Accordingly, the investigation described in the second chapter of this dissertation consists of the bioassay-guided isolation, structure elucidation, and biological evaluation of the constituents obtained from H. brevipes.

C. Experimental

1. General experimental procedures

Optical rotations were obtained on a Perkin-Elmer 343 automatic polarimeter. UV spectra were measured with a Perkin-Elmer Lambda 10 UV/vis spectrometer. CD spectra were measured on a JASCO J-810 spectrometer. IR spectra were run on a Thermo

Scientific Nicolet 6700 FT-IR spectrometer. NMR spectroscopic data were recorded at room temperature on a Bruker Avance DPX-300 or DRX-400 spectrometer. Column chromatography was performed with 65-250 or 230-400 mesh silica gel (Sorbent

Technologies, Atlanta, GA). Analytical thin-layer chromatography was conducted on 250

 m thickness Partisil silica gel 60 F254 glass plates (Whatman, Clifton, NJ). Analytical and semi-preparative HPLC were carried out on a Waters system composed of a 600 controller, a 717 plus autosampler, and a 2487 dual wavelength absorbance detector, with

83 83 Waters Sunfire analytical (4.6  150 mm) and preparative (19  150 mm) C18 columns.

All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

2. Plant material

The entire plants of H. brevipes were collected from Tawangmangu village,

Karanganyar, Central Java, Indonesia, in February 2003. The plant was identified by Dr.

Sugeng Sugiarso, Research Center for Indonesian Medicinal Plants, Ministry of Health,

Indonesia. A voucher specimen (accession number P4046) has been deposited at the Field

Museum of Natural History, Chicago, Illinois.

3. Solvent extraction on Hyptis brevipes

The air-dried whole plants of H. brevipes (716 g) were ground and extracted with methanol overnight (3 × 3 L). The macerate was concentrated in vacuo (46.5 g) and partitioned to afford a hexane extract (12.0 g), a chloroform extract (10.3 g), and an aqueous extract (21.9 g).

4. Initial cytotoxicity assays of Hyptis brevipes extracts and fractions

Extracts and fractions of the aerial parts of H. brevipes were subjected to initial cytotoxicity assays for the purposes of bioactivity-guided isolation. The results are shown in Table A.1 in Appendix A. Fractions F07-F11 showed ED50 values of less than 20

g/mL against MCF-7 cells and were regarded as active and selected for phytochemical investigation.

84 84

Figure 2.8. Solvent extraction scheme used for the plant material of H. brevipes

5. Column chromatography of the chloroform-soluble extract of Hyptis brevipes

The chloroform extract was fractionated using silica gel vacuum-liquid chromatography (Aldrich, Si gel 60, 63-200 mesh, 8.5 × 19 cm) using initially pure hexane, followed, in turn, by a gradient of increasing polarity composed of hexane-ethyl acetate and ethyl acetate-methanol mixtures. The column was washed finally with 100% methanol. Altogether, ten pooled fractions (F06-F15) were collected. Ursolic acid (56, 15 mg) was obtained as a precipitate from F07. A yellow solid precipitated from fraction F08, which was chromatographed further using silica gel (Aldrich, Si gel 60, 230-400 mesh,

3.0 × 52 cm), beginning with 5:1 hexane-acetone, followed by a gradient of increasing

85 85 polarity, and washed with 100% methanol, to afford purified ayanin (350, 30 mg).

Daucosterol (340, 11 mg) was obtained as a precipitate from F12.

Fraction F08 (1.03 g) was found to be the most active for the MCF-7 breast cancer cell line (ED50 6.0 µg/mL) and was fractionated by passage over a silica gel column (5.0

× 58 cm, 230-400 mesh), using the following solvent systems for elution: 100% CH2Cl2

(1.5 L), 2% MeOH-CH2Cl2 (1 L); 3% MeOH-CH2Cl2 (1 L), 5% MeOH-CH2Cl2 (500 mL),

15% MeOH-CH2Cl2 (1 L), 50% MeOH-CH2Cl2 (1 L), and 100% MeOH (1.5 L), with eleven pooled fractions obtained (F08F1–F08F11). Ombuin (13 mg) was isolated from the mother liquor of F08F5. Maslinic acid (320, 20 mg) was obtained as a precipitate from F08F9. Fraction F09 was partitioned on a Sephadex LH-20 column (4  50 cm), using pure MeOH, to give three subfractions (F0901-F0903). A mixture (60 mg) obtained from subfraction F0902 (1.2 g) by repeated silica gel chromatography was purified by preparative HPLC to give the new compounds 342 (tR 26.1 min, 34 mg) and 343 (tR 27.7 min, 10 mg), using MeCN-H2O (28:72, 10 mL/min) as eluting solvent.

Fraction F10 (1.27 g) was fractionated using a Sephadex LH-20 column (230-400 mesh, 3.0 × 100 cm) eluted with pure MeOH, and seven pooled fractions (F10F1–F10F7) were obtained. Fraction F10F2 (560 mg) was chromatographed over a fine silica gel column (2.0 × 45 cm), eluted with CHCl3-acetone from 10:1 to 6:1, to give a mixture

(110 mg). This mixture was subjected to preparative HPLC, with MeCN-H2O (30:70, 10 mL/min) as solvent, to give compound 348 (tR 21.2 min, 28 mg), 347 (tR 24.1 min, 7 mg

F10F2K2), and 346 (tR 36.3 min, 4 mg). 5,6,3'-Trihydroxy-2,7,4'-trimethoxyflavone (349,

108 mg) was purified from F10F4 by repeated silica gel column chromatography

86 86 (230-400 mesh, 4  50 cm), eluted with CHCl3-MeOH (from 10:1 to 5:1).

Fractions F12 and F13 were combined and the new bulked fraction F12 (1.8 g) was chromatographed over a Sephadex LH-20 column (4.5  50 cm) using pure MeOH as eluting solvent to give five sub-fractions (F12F1-F12F5). Sub-fraction F12F4 (460 mg) was passaged over a silica gel column (230-400 mesh, 1  40 cm), eluted with

CHCl3-acetone (4:1), to give a mixture (60 mg). This mixture was separated by preparative HPLC, using MeCN-0.5% formic acid water solution (25:75, 10 mL/min) as solvent system, to afford the new compounds 344 (tR 21.8 min, 42 mg) and 345 (tR 23.2 min, 11 mg).

These isolated compounds were identified as six new 5,6-dihydro-- pyrone derivatives (342-347) and one known 5,6-dihydro--pyrone derivative (348), as well as five other known compounds, namely, 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone (349),

5,3'- dihydroxy-3,7,4'- trimethoxyflavone (ayanin, 350), 3,5,3'-trihydroxy-7,4'- dimethoxyflavone (ombuin, 351), ursolic acid (56), 2-hydroxyursolic acid (maslinic acid, 320), and sitosterol-3-O--D-glucopyranoside (340). The six new compounds

(342-347) were named brevipolides A-F. Ursolic acid (56), 2-hydroxyursolic acid

(maslinic acid, 320), and sitosterol-3-O--D-glucopyranoside (340) were isolated and identified by Marcy J. Balunas (University of Illinois at Chicago) from the same plant sample in a preliminary study (see Appendix B5-B7).

6. Characterization of brevipolide A (342)

20 -5 Colorless gum; [] D +6.3 (c 0.2, MeOH); CD (c 1.1×10 M, MeOH) λmax (Δε) 232

(-0.75), 259.5 (+2.27), 279 (+0.95), 300 (+2.14) nm; UV (MeOH) max (log ) 208 (3.93), 87 87 316 (3.90) nm; IR (film) max 3355 (br), 3018, 2938, 1703, 1623, 1605, 1582, 1514, 1438,

-1 1 1370, 1229, 1165, 1066, 1039, 816, 752 cm ; H NMR (400 MHz, CDCl3) data, see

13 Table 2.6; C NMR (100 MHz, CDCl3) data, see Table 2.7; HRESIMS m/z 451.1354

+ [M+Na] (calcd for C23H24O8Na, 451.1369).

7. Characterization of brevipolide B (343)

20 -5 Colorless gum; [] D +3.7 (c 0.2, MeOH); CD (c 1.1×10 M, MeOH) λmax (Δε) 230

(-0.86), 160 (+2.82), 280 (+1.02), 300 (+0.55) nm; UV (MeOH) max (log ) 213 (4.23),

315 (4.18) nm; IR (film) max 3360 (br), 3015, 2933, 1704, 1605, 1512, 1446, 1372, 1234,

-1 1 13 1160, 1046, 817, 756 cm ; H NMR (400 MHz, CDCl3) data, see Table 2.6; C NMR

+ (100 MHz, CDCl3) data, see Table 2.7; HRESIMS m/z 451.1343 [M+Na] (calcd for

C23H24O8Na, 451.1369).

8. Characterization of brevipolide C (344)

20 -5 8.1. Brevipolide C (344): colorless gum; [] D +9.4 (c 0.1, MeOH); CD (c 1.2×10

M, MeOH) λmax (Δε) 228 (-1.00), 261 (+3.6), 280 (+2.40), 300 (+3.41) nm; UV (MeOH)

max (log ) 208 (4.17), 252 (4.00) nm; IR (film) max 3408 (br), 3018, 2919, 1703, 1631,

1600, 1517, 1445, 1385, 1263, 1161, 1074, 812, 756 cm-1; 1H NMR (400 MHz,

13 acetone-d6) data, see Table 2.6; C NMR (100 MHz, acetone-d6) data, see Table 2.7;

+ HRESIMS m/z 425.1216 [M+Na] (calcd for C21H22O8Na, 425.1212).

88 88 8.2. Preparation of R- and S-MTPA ester derivatives of brevipolide C (344)

Compound 344 (1.5 mg) was added to two NMR tubes each and was dried under a vacuum overnight. Deuterated pyridine (0.4 mL) was transferred to each tube under a N2 flow, followed by injection of (S)-(+)--methoxy--(trifluoromethyl)phenylacetyl

(MTPA) chloride (5 L), or (R)-MTPA chloride (5 L), to give the (R)-MTPA ester and the (S)-MTPA ester of 344, respectively. The tubes were stored overnight at room temperature to allow completion of each reaction before NMR measurements were taken.

8.3. R-MTPA ester of brevipolide C (344): H 6.75 (1H, m, H-3), 6.06 (1H, d, J =

9.9 Hz, H-2), 5.55 (1H, q, J = 7.0 Hz, H-11), 5.38 (1H, dd, J = 9.7, 2.7 Hz, H-6), 4.82

(1H, m, H-5), 2.77 (1H, m, H-9), 2.32 (2H, m, H-4), 2.13 (1H, m, H-7), 1.56 (3H, d, J =

7.0 Hz, H-12), 1.47 (1H, m, H-8), 1.45 (1H, m, H-8).

8.4. S-MTPA ester of brevipolide C (344): H 6.86 (1H, m, H-3), 6.12 (1H, dd, J =

9.7, 1.2 Hz, H-2), 5.51 (1H, q, J = 7.1 Hz, H-11), 5.39 (1H, dd, J = 8.8, 2.3 Hz, H-6),

4.93 (1H, dt, J = 12.0, 2.5, 2.5 Hz, H-5), 2.74 (1H, m, H-9), 2.52 (2H, m, H-4), 2.02 (1H, m, H-7), 1.56 (3H, d, J = 7.1 Hz, H-12), 1.41(1H, m, H-8), 1.34 (1H, m, H-8).

9. Characterization of brevipolide D (345)

20 Colorless gum; [] D +8.3 (c 0.1, MeOH); UV (MeOH) max (log ) 208 (3.97), 333

-5 (3.77) nm; CD (c 1.2×10 M, MeOH) λmax (Δε) 229 (-1.11), 261 (+2.46), 279 (+1.10),

301 (+2.15) nm; IR (film) max 3410 (br), 2953, 2927, 1722, 1703, 1631, 1604, 1517, 89 89

Figure 2.9. Structures of compounds isolated from Hyptis brevipes in the present study. [Ursolic acid (56), 2-hydroxyursolic acid (maslinic acid, 320), and sitosterol-3-O--D- glucopyranoside (340) were isolated by Marcy J. Balunas from the same plant sample in a preliminary study]

90 90 -1 1 1445, 1389, 1263 1165, 820, 756 cm ; H NMR (400 MHz, acetone-d6) data, see Table

13 2.6; C NMR (100 MHz, acetone-d6) data, see Table 2.7; HRESIMS m/z 425.1187

+ [M+Na] (calcd for C21H22O8Na, 425.1212).

10. Characterization of brevipolide E (346)

20 Colorless gum; [] D +10.0 (c 0.1, MeOH); UV (MeOH) max (log ) 208 (4.22),

-5 333 (4.07) nm; CD (c 1.1×10 M, MeOH) λmax (Δε) 230 (-0.95), 260 (+2.74), 280

(+0.99), 300 (+2.53) nm; IR (film) max 3390 (br), 3018, 2919, 2843, 1714, 1631, 1597,

-1 1 1514, 1442, 1245, 1157, 816, 756 cm ; H NMR (400 MHz, CD3OD) data, see Table 2.6;

13 + C NMR (100 MHz, CD3OD) data, see Table 2.7; HRESIMS m/z 467.1293 [M+Na]

(calcd for C23H24O9Na, 467.1318).

11. Characterization of brevipolide F (347)

20 Colorless gum; [] D +7.0 (c 0.2, MeOH); UV (MeOH) max (log ) 210 (4.08), 316

-5 (4.22) nm; CD (c 1.2×10 M, MeOH) λmax (Δε) 231 (-0.83), 261 (+1.94), 282 (+0.88),

301 (+1.83) nm; IR (film) max 3397 (br), 3010, 2930, 1703, 1605, 1514, 1438, 1385,

-1 1 1264, 1169, 1070, 1036, 816, 756 cm ; H NMR (400 MHz, acetone-d6) data, see Table

13 2.6; C NMR (100 MHz, acetone-d6) data, see Table 2.7; HRESIMS m/z 409.1263

+ [M+Na] (calcd for C21H22O7Na, 409.1263).

91 91 12. Characterization of (E)-1-{(1S,2S)-2-[(S)-hydroxy(R)-6-oxo-3,6-dihydro-2H-

pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate

(348)

20 Colorless gum; [] D +7.2 (c 0.2, MeOH); UV (MeOH) max (log ) 209 (4.17),

-5 316 (4.20) nm; CD (c 1.2×10 M, MeOH) λmax (Δε) 232 (-0.84), 261 (+2.02), 283

(+0.78), 303 (+1.63) nm; IR (film) max 3397 (br), 3010, 2928, 1705, 1605, 1510, 1435,

-1 1 1380, 1260, 1170, 1075, 1032, 818, 754 cm ; H NMR (400 MHz, CDCl3) data, see

13 Table 2.6; C NMR (100 MHz, CDCl3) data, see Table 2.7; HRESIMS m/z 409.1265

+ [M+Na] (calcd for C21H22O7Na, 409.1263).

13. Characterization of 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone (349)

1 H NMR (400 MHz, CDCl3) H 12.41 (1H, s, OH-5), 7.73 (1H, dd, J = 8.4, 1.8 Hz,

H-6), 7.68 (1H, d, J = 1.8 Hz, H-2), 6.97 (1H, d, J = 8.4 Hz, H-5), 6.56 (1H, s, H-8),

13 4.00 (3H, s, C-OCH3), 3.99 (3H, s, C-OCH3), 3.88 (3H, s, C-OCH3); C NMR (100 MHz,

CDCl3) C 178.8 (C-4), 155.9 (C-7), 152.9 (C-5), 149.7 (C-4), 148.7 (C-3), 145.5 (C-9),

145.2 (C-2), 139.0 (C-3), 123.7 (C-1), 121.6 (C-6), 114.3 (C-2), 110.4 (C-5), 106.4

(C-10), 129.1 (C-6), 90.2 (C-8), 59.9 (C-3-OCH3), 56.3 (C-OCH3), 55.8 (C-OCH3);

ESIMS m/z 383 [M+Na]+.

14. Characterization of ayanin (350)

1 H NMR (300 MHz, DMSO-d6) H 12.56 (1H, s, OH-5), 9.44 (1H, s, OH-3), 7.58

(1H, overlapped with H-6, H-2,), 7.58 (1H, overlapped with H-2, H-6), 7.11 (1H, d, J =

9.0 Hz, H-5), 6.73 (1H, d, J = 2.1 Hz, H-8), 6.38 (1H, d, J = 2.1 Hz, H-6), 3.87 (6H, s,

13 C-7-OCH3 and C-4-OCH3), 3.81 (3H, s, C-3-OCH3); C NMR (75 MHz, DMSO-d6) C 92 92 Position 342a 343a 344b 345b 346c 347b 348a 2 6.05, dd, 9.4, 6.03, dd, 7.9, 5.92, dd, 9.9, 5.92, ddd, 5.99, d, 7.9 5.92, ddd, 9.5, 5.94, ddd, 9.6, 2.6 1.2 1.5 10.8, 1.7, 1.7 1.9, 1.9 1.9, 1.9 3 6.95, ddd, 9.4, 6.93, m 7.08 7.06, ddd, 7.07 7.05, dt, 9.5, 7.05, dt, 9.5, 6.6, 2.3 (overlapped 10.2, 4.5, 4.5 (overlappped 4.3 4.4 with H-5) with H-5) 4 2.48, m; 2.65, 2.39, m; 2.62, 2.61, m (2H) 2.62, m (2H) 2.60, m (2H) 2.63, m (2H) 2.55, m (2H) m m 5 4.61, dt, 12.2, 4.59, dt, 12.2, 4.50, ddd, m 4.49, ddd, 8.0, 4.59, dt, 10.8, 4.50, ddd, 8.0, 4.45, ddd, 8.2, 3.7 3.7 8.0, 4.5 3.9 8.0, 4.3 8.2, 4.2 6 4.70, dd, 8.7, 4.68, dd, 8.6, 3.72, t, 4.7 3.73, t, 4.5 4.77, dd, 8.4, 3.74, t, 4.3 3.64, t, 4.6 3.7 3.7 3.9 7 1.69, m 1.68, m 1.61, m 1.62, m 1.64, m 1.61, m 1.62, m 8 1.08, m; 1.41, 1.03, m; 1.41, 1.14, m (2H) 1.15, m (2H) 1.16, m; 1.31, 1.13, m (2H) 1.14, m (2H)

93 m m m

9 2.41, m 2.35, m 2.36, m 2.36, m 2.35, m 2.36, m 2.36, m 11 5.25, q, 7.0 5.16, q, 7.0 5.30, q, 7.0 5.26, q, 7.1 5.21, q, 7.1 5.28, q, 7.1 5.27, q, 7.0 12 1.50, d, 7.0 1.43, d, 7.0 1.49, d, 7.0 1.46, d, 7.1 1.46, d, 7.1 1.46, d, 7.1 1.45, d, 7.0 2 7.35, d, 8.5 7.57, d, 8.2 7.19, br. s 7.64, d, 1.8 7.07, br. s 7.77, d, 8.5 7.67, d, 8.5 3 6.83, d, 8.5 6.79, d, 8.2 — — — 6.85, d, 8.5 6.90, d, 8.5 5 7.35, d, 8.5 7.57, d, 8.2 6.88, d, 8.2 6.82,d, 8.2 6.80, d, 8.1 7.77, d, 8.5 7.71, d, 8.5 6 6.83, d, 8.5 6.79, d, 8.2 7.08, d, 8.2 7.16, dd, 8.2, 6.97, dd, 8.1, 6.85, d, 8.5 7.60, d, 8.5 1.8 1.6 7 7.62, d, 16.0 6.89, d, 12.7 7.60, d, 15.9 6.89, d, 12.9 7.60, d, 16.0 6.97, d, 12.9 6.90, d, 12.9 8 6.27, d, 16.0 5.86, d, 12.7 6.36, d, 15.9 5.85, d, 12.9 6.32, d, 16.0 5.87, d, 12.9 6.43, d, 12.9 2 2.14, s 2.06, s — — 2.13, s — — 1 a b c Table 2.6. H NMR chemical shifts of compounds 342-348 (δH, mult., J in Hz). In CDCl3. In acetone-d6. In CD3OD.

position 342a 343a 344b 345b 346c 347b 348a 1 163.7 163.9 164.4 164.2 166.0 164.1 163.6 2 121.0 121.1 121.5 121.2 121.4 121.2 120.1 3 145.1 145.1 147.4 147.3 148.0d 147.1 147.3 4 24.3 24.6 25.2 24.9 25.7 24.8 24.1 5 77.9 78.1 81.8 81.5 79.8 81.6 80.4 6 74.5 74.5 72.1 71.6 76.0 71.6 69.8 7 25.2 25.2 28.0 27.6 26.6 27.7 27.1 8 14.4 14.7 14.5 14.3 15.1 14.2 13.7 9 22.5 22.6 21.4 21.0 23.5 21.1 20.1 10 207.3 207.3 207.3 207.1 208.9 207.1 207.0 11 75.0 75.0 76.1 75.9 76.6 75.8 74.9 12 15.9 15.8 16.8 16.4 16.4 16.4 16.1

1 126.1 126.8 127.8 127.8 127.7 131.1 125.5 2 130.1 132.3 115.6 115.6 115.3 133.9 130.6 3 116.0 115.1 149.3 148.4 150.0 115.7 115.8 4 158.9 157.6 146.6 145.1 147.0 159.9 160.1 5 116.0 115.1 116.7 118.5 116.7 115.7 115.9 6 130.1 132.3 123.1 125.7 123.3 133.9 130.6 7 146.0 145.2 147.0 145.9 148.1d 145.6 145.8 8 113.6 115.6 115.1 115.5 114.4 115.7 113.5 9 168.8 165.9 167.2 166.2 168.5 166.3 166.1 1 170.5 170.6 172.1 2 20.9 20.9 21.0 13 a b c d Table 2.7. C NMR chemical shifts of compounds 342-348. In CDCl3. In acetone-d6. In CD3OD. Data interchangeable.

178.9 (C-4), 165.7 (C-7), 161.3 (C-9), 156.6 (C-5), 156.6 (C-4), 150.7 (C-2), 146.7

(C-3), 138.5 (C-3), 122.9 (C-1), 120.6 (C-6), 112.1 (C-5), 105.7 (C-10), 98.2 (C-6),

92.6 (C-8), 60.1 (C-3-OCH3), 56.4 (C-7-OCH3), 56.0 (C-4-OCH3); HRESIMS m/z

+ 367.0788 [M+Na] (calcd for C18H16O7Na, 367.0788).

15. Characterization of ombuin (351)

1 H NMR (400 MHz, DMSO-d6) H 7.82 (1H, d, J = 8.2, 2.0 Hz, H-6), 7.80 (1H, d, J

= 2.0 Hz, H-2), 7.13 (1H, d, J = 8.2 Hz, H-5), 6.73 (1H, d, J = 2.2 Hz, H-8), 6.32 (1H, d,

13 J = 2.2 Hz, H-6), 3.94 (3H, s, C-OCH3), 3.93 (3H, s, C-OCH3); C NMR (100 MHz,

DMSO-d6) C 176.6 (C-4), 166.7 (C-7), 161.6 (C-9), 157.7 (C-5), 150.2 (C-4), 147.2

(C-3), 146.8 (C-2), 137.2 (C-3), 124.7 (C-1), 121.2 (C-6), 115.1 (C-2), 112.1 (C-5),

104.8 (C-10), 98.4 (C-6), 92.7 (C-8), 56.4 (C-3-OCH3), 56.3 (C-7-OCH3); ESIMS m/z

353 [M+Na]+.

16. Biological activities of isolates from H. brevipes

All the isolated compounds obtained from the aerial parts of H. brevipes were evaluated for their cytotoxicity against MCF-7 human breast cancer cells, HT-29 human colon cancer cells, and Lu1 human lung cancer cells. Of these thirteen isolates, compounds 343, 347, and 348 were found to be active principles, with ED50 values of 6.1,

6.7, and 3.6 M, respectively, against MCF-7 cells, as were compounds 342, 343, 347, and as were 349 with ED50 values of 5.8, 6.1, 7.5, and 3.6 M, respectively, against

HT-29 cells. No significant activity was found against Lu1 cells for any of the 95 95 compounds tested, with ED50 values of compounds 342 and 344-346 against MCF-7 cells being greater than 10 M in each case. These data suggest that the cis isomers are more cytotoxic than their trans counterparts for this cell line among compounds 342-348, and esterification at the C-6 position as in compounds 342 and 343, as well as hydroxy substitution at the C-3 position in compounds 344 and 345, led to a decrease of activity when compared to compounds 347 and 348. It is also apparent that hydroxy group substitution at the C-3 position resulted in decreased cytotoxicity for these

5,6-dihydro--pyrones against HT-29 cells.

All compounds were subjected to evaluation in a small panel of mechanism-based in vitro assays (Appendix A.2-A.4). However, only compounds 344, 345 and 349 were found to show any discernible activity in a proteasome inhibition assay, with ED50 values of 38.0, 44.5 and 17 µM, respectively. In an enzyme-based ELISA NF-B p50 assay, compound 348 demonstrated an ED50 value of 15.3 M, compared with ED50 values of >50 M for the remaining compounds. In a mitochondrial transmembrane potential

(MTP) assay, only compounds 344, 348, and 349 showed activity, with ED50 values of

8.5, 75, and 310 nM, respectively.

D. Discussion

1. Structure elucidation and identification of compounds isolated from H. brevipes

1.1. Structure elucidation of brevipolide A (342)

Compound 342 was obtained as a colorless gum and afforded a sodiated molecular

1 ion peak at m/z 451.1354 (calcd for C23H24O8Na, 451.1369) in the HRESIMS. The H

96 96

NMR spectrum of 342 (Table 2.6 and Figure 2.10) showed a characteristic AABB system for a para-substituted benzene ring at δH 7.35 (2H, d, J = 8.5 Hz, H-2, H-6) and

6.83 (2H, d, J = 8.5 Hz, H-3, H-5). Resonance signals at δH 7.62 (1H, d, J = 16.0 Hz,

H-7) and 6.27 (1H, d, J = 16.0 Hz, H-8) suggested the presence of a trans double bond.

Signals of a cis double bond adjacent to a methylene group were also observed at δH 6.95

(1H, ddd, J = 9.4, 6.6, 2.3 Hz, H-3), 6.05 (1H, dd, J = 9.4, 2.6 Hz, H-2), 2.65 (1H, m,

H-4), and 2.48 (1H, m, H-4). In the COSY NMR spectrum (Figure 2.11), two oxygenated methine groups were found to be connected to one another, and their proximity was substantiated by analyzing coupling constants of the resonance signals at

δH 4.70 (1H, dd, J = 8.7, 3.7 Hz, H-6) and 4.61 (1H, dt, J = 12.2, 3.7 Hz, H-5), Also, using the COSY spectrum, another oxygenated methine group was determined to be connected to a methyl functionality from correlations between signals at δH 5.25 (1H, q, J

= 7.0 Hz, H-11) and 1.50 (3H, d, J = 7.0 Hz, H-12). The methylene signals occurring in the upfield region at δH 1.41 (1H, m, H-8) and 1.08 (1H, m, H-8), together with signals at δH 2.33 (1H, m, H-9) and 1.69 (1H, m, H-7), were attributed to the presence of a cyclopropane ring (Wilberg et al., 1973). A singlet signal at δH 2.14 (3H, s, OAc) suggested the presence of an acetyl group. Consistent with the 1H NMR data, the 13C NMR (Table 2.7 and Figure 2.12) and DEPT135 (Figure 2.13) spectroscopic data of 342 also showed signals of a para-substituted benzene ring at δC 158.9 (C-4), 126.1 (C-1), 130.1 (C-2,

6), and 116.0 (C-3, 5), two double bonds at δC 146.0 (C-7), 145.1 (C-3), 121.0 (C-2), and 113.6 (C-8), three oxygenated methine carbons at δC 77.9 (C-5), 75.0 (C-11), and

74.5 (C-6), two methine carbons at C 25.2 (C-7) and 22.5 (C-9), two methylene carbons 97 97 at δC 24.3 (C-4) and 14.4 (C-8), as well as two methyl carbons at 20.9 (OAc) and 15.9

(C-12). Furthermore, four carbonyl carbon signals at C 207.3 (C-10), 170.5 (OAc), 168.8

(C-9), and 163.7 (C-1), could be assigned, in turn, to ketone, acetyl, ,-unsaturated ester, and ,-unsaturated lactone functionalities. In the 1H-1H COSY spectrum of compound 342 (Figure 2.11), correlations from H-5 and H-7 to H-6 indicated the lactone and cyclopropane moieties to be connected through C-6. Other 2D NMR spectroscopic correlations observed in the HMBC NMR experiment (Figure 2.14) were supportive of the planar structure proposed for compound 342. HSQC NMR spectroscopy (Figure 2.15) was used to finalize the assignment of proton and carbon signals.

The relative configuration of compound 342 at the C-5, C-6 and C-7 positions was determined by correlations from H-7 to H-4, H-5 and H-8, as well as from H-6 to

H-8 and H-9 in the NOESY NMR spectrum (Figure 2.16), and by comparison with the data of reported analogues (Hedge et al., 2004). Selected signal correlations observed in

2D NMR spectroscopic methods are summarized in Figure 2.17.

The CD spectrum (Figure 2.18) of compound 342 exhibited a positive Cotton effect at

259.5 nm. According to the published observations between chirality in ,-unsaturated

-lactones and the observed Cotton effect around 260 nm, the absolute configuration of compound 342 at the C-5 position could be determined as R (Davies-Coleman, 1987).

Thus, the structure of the new compound 342 was proposed as

(E)-1-{(1S,2S)-2-[(S)-acetoxy((R)-6-oxo-3,6-dihydro- 2H-pyran-2-yl) methyl] cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate, and it was assigned the trivial name, brevipolide A.

98 98

H-2

H-12

H-2 and 6 H-3 and 5 O 1'' 2'' O 12 H-7 H-8 8 O O 1 O 5 H 7' 2' H-8a 6 10 1' H-4b H-11 7 9 11 O 9' 3' H 8' H-2 2 4 4' H-9 H-3 H-6 H-5 O 6' 3 OH H-7 H-8b 5' H-4a

1 Figure 2.10. H NMR spectrum of brevipolide A (342) in CDCl3 (400 MHz) 99

H-7/H-8a

H-9/H-8a H-11/H-12 H-9/H-8b H-6/H-7 H-3/H-4 H-7/H-8b H-4a/H-4b

H-5/H-4

H-7/H-8

H-3/H-2

H-2 and 5/H-3 and 6

O 1'' 2'' O 12 1 1 8 O O 1 O 5 H 7' 2' Figure 2.11. H- H COSY NMR spectrum of brevipolide A (342) in CDCl6 3 10 1' 7 9 11 O 9' 3' H 8' 2 4 4' O 6' 3 OH 100 5'

C-2 and 6 C-3 and 5

C-12 C-1 C-11 C-2

1 C-5 O C-9 1'' C-9 2'' O 12 8 O C-4 O 1 O 5 H 7' 2' C-3 C-4 6 10 1' 7 9 11 O 9' 3' C-6 H 8' 2 4 4' C-1 C-2 O 6' C-7 3 OH C-7 5' C-8 C-10 C-1 C-8

13 Figure 2.12. C NMR spectrum of brevipolide A (342) in CDCl3 (100 MHz)

101

C-2 and 6 C-3 and 5

C-11 C-2 C-5 C-6 C-9 C-12 C-8 C-3 C-7 C-2 C-7

C-8 C-4

O 1'' ` 2'' O 12 8 O O 1 O 5 H 7' 2' 6 10 1' 13 7 9 11 O 9' 3' H 8' Figure 2.13. C DEPT135 NMR spectrum of brevipolide2 A4 (342) in CDCl3 4' O 6' 3 OH 5' 102

C-12/H-11 C-4/H-2

C-5/H-3

C-2/H-6 and C-6/H-2 C-11/H-12

10 3 C-8/H-7

C-2 and 6/H-3 and 5

C-3/H-5 and C-5/H-3 C-1/H-8 C-1/H-3 and 5 C-7/H-2 and 6 C-4/H-3 and 5 C-1/H-2 C-9/H-11 C-1/H-2 C-1/H-3 C-9/H-8 C-4/H-2 and 6 O 1'' C-9/H-7 2'' O 12 C-10/H-11 C-10/H8 -12 O O 1 O 5 H 7' 2' 6 10 1' 7 9 11 O 9' 3' H 8' 2 4 4' O 6' 3 OH 5'

Figure 2.14. HMBC NMR spectrum of brevipolide A (342) in CDCl3 103

C-8/H-8a and 8b C-2/H-2 C-9/H-9 C-12/H-12 C-7/H-7 C-4/H-4a and 4b

C-11/H-11 C-6/H-6

C-5/H-5

C-3 and 5/H-3 and 5

C-8/H-8 C-2/H-2 C-2 and 6/H-2 and 6

C-7/H-7 C-3/H-3

O 1'' 2'' O 8 12 H O Figure 2.15. HSQC NMR spectrum of brevipolide A (342) in CDCl3 O 1 O 5 7' 2' 6 10 1' 7 9 11 O 9' 3' H 8' 2 4 4' O 6' 3 OH 104 5'

H-8/H-9 H-6/H-8b H-11/H-12 H-6/H-7

H-3/H-4 H-6/H-9 H-5/H-4

H-11/H-9

H-7/H-8

Figure 2.16. NOESY NMR spectrum of brevipolide A (342) in CDCl3 O 1'' 2'' O 12 8 O 105 O 1 O 5 H 7' 2' 6 10 1' 7 9 11 O 9' 3' H 8' 2 4 4' O 6' 3 OH 5'

Figure 2.17. Selected 1H-1H COSY ( ), HMBC ( ), and NOESY ( ) NMR spectroscopic correlations observed for brevipolide A (342).

CD/mdeg 4

-2 200 250 300 350 400 Wavelength (nm)

Figure 2.18. Circular dichroism spectrum of brevipolide A (342) in MeOH

106 106

1.2. Structure elucidation of brevipolide B (343) The HRESIMS of compound 343 provided a sodiated molecular ion peak at m/z

451.1343, corresponding to an elemental formula of C23H24O8Na (calcd for 451.1369), the same as that of compound 342. In addition, the UV, CD, IR, and NMR spectra

(Figures 2.19 and 2.20) of compound 343 were found to be closely comparable to those of compound 342, indicating that these two compounds are geometrical isomers. The coupling constant values of the resonance signals at δH 6.89 (1H, d, J = 12.7 Hz, H-7) and 5.86 (1H, d, J = 12.7 Hz, H-8) suggested that 343 is a Z isomer of compound 342 at the phenylpropenoid double bond position. Accordingly, the new compound, brevipolide

B (343), was assigned as (Z)-1-{(1S,2S)-2-[(S)-acetoxy(R)-6-oxo-3,6-dihydro-2H- pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate.

1.3. Structure elucidation of brevipolide C (344) Compound 344 (brevipolide C) was obtained as a colorless gum. Its HRESIMS exhibited a sodiated molecular ion peak at m/z 425.1216 (calcd for C21H22O8Na,

425.1212). The structure of compound 344 was found to be similar to that of compound

342. However, the 1H NMR spectrum of 344 (Table 2.4 and Figure 2.23) showed 1,3,4- trisubstitued aromatic proton signals at H 7.19 (1H, br s, H-2), 7.08 (1H, d, J = 8.2 Hz,

H-6) and 6.88 (1H, d, J = 8.2 Hz, H-5), indicating the presence of a caffeoyl moiety. The absence of acetyl functionality signals in both the 13C and 1H NMR spectra (Figures 2.21

1 and 2-22), as well as an upfield shift of the H-6 signal in the H NMR spectrum to δH

3.72 in compound 344, when compared with δH 4.70 for compound 342, suggested the occurrence of a free hydroxy group at the C-6 position. Therefore, the planar structure could be proposed for 344. 107 107

H-2

H-12

H-3 and 5 H-8a H-3 O H-2 and 6 H-6 1'' H-7 H-8 3' 2'' O OH H-11 H-5 8 12 H-2 H-O4b H-92' 4' O 1 O 5 H H-7 H-8b 6 10 5' 7 9 11 OH-94a' 1' 4 H 8' 7' 6' 2 O 3

1 Figure 2.19. H NMR spectrum of brevipolide B (343) in CDCl3 (400 MHz) 108

C-11 C-2 C-2 and 6 C-9 C-12 C-4 C-5 C-1

10 C-9 9 C-3 and 5 C-6 C-1 C-7 C-10 C-2 C-3 O 1'' C-7 3' C-8 2'' C-8 O 8 12 OH O 2' 4' O 1 O 5 H C-1 6 10 5' 7 9 11 O 9' 1' 4 H 8' 7' 6' 2 O 3 C-4

13 Figure 2.20. C NMR spectrum of brevipolide B (343) in CDCl3 (100 MHz) 109

H-12

H-3 and 5 H-8 H-8

0 H-7 H-4 H-2 H-11 H-6 Acetone-d6 H-6 H-2 H-5 H-9 OH 12 H-7 8 O O 1 O 5 H 7' 2' 6 10 1' 3' OH 7 9 11 O 9' H 8' 2 4 4' O 6' 3 OH 5'

1 Figure 2.21. H NMR spectrum of brevipolide C (344) in acetone-d6 (400 MHz)

110

C-11

11 C-2 C-7

C-5 C-5 C-4 C-9 C-7 C-6 C-6 C-2 C-12 C-4 C-3 C-8O H 12 C-10 8 O O 1 O 5 H 7' 2' 6 10 1' 3' OH 7 9 11 O 9' C-8 C-9 H 8' C-1 2 4 4' O 6' 3 OH C-3 5' C-1

13 Figure 2.22. C NMR spectrum of brevipolide C (344) in acetone-d6 (100 MHz)

111

(S)-MTPA ester

H-11 H-8 H-2 H-6 H-4 H-5 H-3 H-9 H-7

(R)-MTPA ester

H-4 H-8 H-11 H-2 H-6 H-9 H-3 H-5 H-7

1 Figure 2.23. H NMR spectra of (S)-MTPA and (R)-MTPA ester of brevipolide C (344) in pyridine-d5 (400 MHz)

112

The absolute configuration at C-6 of compound 344 was determined using the

Mosher ester procedure (Dale et al., 1973; Sullivan et al., 1973), by measurement in

NMR tubes (Su et al., 2002). The chemical shift difference values [Figures 2.24 and 2.25,

S-R, H-2 (+0.06), H-3 (+0.11), H-4 (+0.2), H-5 (+0.11), H-6 (+0.01), H-7 (-0.11), H-8

(-0.06 and -0.11), H-9 (-0.03), H-11 (-0.04)] obtained by comparing the relevant 1H NMR data of the R- and S-MTPA esters of compound 344, indicated the absolute configuration of C-6 to be S (Figure 2.25). Hydrolysis reactions catalyzed by different acids and bases, as well as the esterase enzyme, were carried out in an attempt to produce a derivative of

344 with a free C-11 hydroxy group, so that this could be used for a Mosher ester reaction to determine the configuration of this position. However, all the hydrolysis products obtained racemized at the C-11 position, probably due to the formation of enol and enolate intermediates during the reactions. Therefore, the structure of compound 344 was determined as (E)-1-{(1S,2S)-2-[(S)-hydroxy((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl) methyl] cyclopropyl}-1-oxopropan-2-yl 3-(3,4-dihydroxyphenyl)acrylate.

Figure 2.24. Observed 1H NMR chemical shift difference values between S- and R-MTPA esters of brevipolide C (344, S-R)

113 113

Figure 2.25. Mosher models (a, b) for the assignment of absolute configuration (c, d) by 1H NMR spectroscopy and the expected sign of SR (from Seco et al., 2004)

1.4. Structure elucidation of brevipolide D (345)

The molecular formula of compound 345 was determined as C21H22O8, from the sodiated molecular ion peak at m/z 425.1187 (calcd for C21H22O8Na, 425.1212) in the

HRESIMS. Its 1H (Figure 2.26) and 13C (Figure 2.27) NMR spectra were similar to those of compound 344 except for a difference due to the double bond in the caffeoyl moiety. A cis double bond in compound 345 was concluded according to the coupling constant values of the resonance signals at H 6.89 (1H, d, J = 12.9 Hz, H-7) and 5.85 (1H, d, J =

12.9 Hz, H-8), compared to the value of J78 = 15.9 Hz in the trans form in compound

344. Therefore, the structure of the new compound brevipolide D was assigned as (Z)-1-

{(1S,2S)-2-[(S)-hydroxy((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl)methyl] cyclopropyl}-1- oxopropan-2-yl 3-(3,4-dihydroxyphenyl)acrylate.

114 114

H-12

Acetone-d6

H-8 H-7 H-8 H-2 H-3 H-11 H-4 H-5 H-6 H-6 H-2 H-5 H-9 5H' -7 OH 12 4' OH 8 O 6' O 1 O 5 H 6 10 7 9 11 O 9' 1' 3' OH 4 H 8' 7' 2' 2 O 3

1 Figure 2.26. H NMR spectrum of brevipolide D (345) in acetone-d6 (400 MHz) 115

C-6 C-4 C-2 C-5 C-12 C-3 C-5' C-7 C-6' C-11 C-1 C-2' C-8 C-8' C-9' C-7' C-9 C-10 5' C-1' OH 12 4' OH 8 O 6' C-3' O 1 O 5 H 6 10 C-4' 7 9 11 O 9' 1' 3' OH 4 H 8' 7' 2' 2 O 3

13 Figure 2.27. C NMR spectrum of brevipolide D (345) in acetone-d6 (100 MHz) 116

1.5. Structure elucidation of brevipolide E (346)

Compound 346 was obtained as a colorless gum. The HRESIMS showed a sodiated molecular ion peak at m/z 467.1293, consistent with an elemental formula of C23H24O9Na

(calcd 467.1318). The 1H and 13C NMR (Figures 2.28 and 2.29) spectra of compound 346 exhibited closely related signals to those of compound 344, but with signals of an additional acetyl group at H 2.13 (3H, s) and C 172.1 and 21.0. The downfield shift of the proton resonance at H 4.77 (1H, dd, J = 8.4, 3.9 Hz, H-6), when compared with that in compound 344, suggested that this acetyl group is substituted at the C-6 hydroxy functionality. Thus, the new compound, brevipolide E was assigned with the structure

(E)-1-{(1S,2S)-2-[(S)-acetoxy((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl)methyl] cyclopropyl}-1-oxopropan-2-yl 3-(3,4-dihydroxyphenyl)acrylate.

1.6. Structure elucidation of brevipolide F (347)

A sodiated molecular ion peak for compound 347 was observed at m/z 409.1263

1 13 (calcd for C21H22O7Na, 409.1263) in the HRESIMS. The H and C NMR spectra

(Figures 2.30 and 2.31) of compound 347 were closely comparable to those of compound

343. However, the absence of acetyl functionality signals, and the migration of H-6

1 signals from δH 4.68 of compound 343 to δH 3.74 in the H NMR spectrum indicated the presence of a free hydroxy group at the C-6 position. Therefore, the new compound, brevipolide F was established structurally as (Z)-1-((1S,2S)-2-{(S)-hydroxy[(R)-6-oxo-

3,6-dihydro-2H- pyran-2-yl)methyl] cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl) acrylate.

117 117

H-12

H-2 and 5

H-6 H-8 H-7 H-3 H-4 H-11 H-6 O H-8 H1-'1' 2'' H-9 O 12 8 O H-5 H-7 H-8 O 1 O 5 H 7' 2' 6 10 1' 3' OH 7 9 11 O 9' H 8' 2 4 4' O 6' 3 OH 5'

1 Figure 2.28. H NMR spectrum of brevipolide E (346) in CD3OD (400 MHz) 118

C-4

C-2 C-11 C-7 C-6 C-9 C-12

11 9 C-9' C-3 and 7' C-5'

C-10 C-5 C-2'' C-2' C-1'' C-1 C-6' C-8 O C-8' 1''C-4' 2'' O 12 C-3' 8 O O 1 O 5 H 7' 2' 6 C10-1' 1' 3' OH 7 9 11 O 9' H 8' 2 4 4' O 6' 3 OH 5'

13 Figure 2.29. C NMR spectrum of brevipolide E (346) in CD3OD (100 MHz) 119

H-12

Acetone-d6

H-3' and 5' H-8 H-2' and 6' H-8'

0 H-11 H-7' H-4 H-2 H-6 H-7 H-3 H-5 H-9

1 Figure 2.30. H NMR spectrum of brevipolide F (347) in acetone-d6 (400 MHz) 120

C-3', 5', and 9'

C-4

1 C-2' and 9' C-12 C-5 C-7 C-1 C-7' C-2 C-4' C-11 C-9 C-3 C-8 C-9' C-10 C-6 C-1'

13 Figure 2.31. C NMR spectrum of brevipolide F (347) in acetone-d6 (100 MHz) 121

1.7. Identification of (E)-1-{(1S,2S)-2-[(S)-hydroxy(R)-6-oxo-3,6-dihydro-2H-

pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate

(348)

The molecular formula of compound 348 was determined as C21H22O7 from a sodiated molecular ion peak at m/z 409.1266 (calcd for C21H22O7Na, 409.1263) in the

HRESIMS, the same molecular formula as that of compound 347. Its 1H and 13C NMR spectra were similar to those of compound 347 except for a variation due to different geometrical double bonds in the caffeoyl moiety. A trans double bond in compound 348 was concluded according to the coupling constant values of the resonance signals at δH

6.90 (1H, d, J = 16.0 Hz, H-7) and 6.43 (1H, d, J = 16.0 Hz, H-8), when compared to the value of J78 = 12.9 Hz in the cis form in compound 347. Therefore, the structure of compound 348 was assigned as (E)-1-{(1S,2S)-2-[(S)-hydroxy((R)-6-oxo-3,6-dihydro-

2H-pyran-2-yl)methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate, comparable to the reported data for this substance as a constituent of Lippia alba (Hegde et al., 2004).

1.8. Absolute configuration of compounds 342-348

Based on the similar CD spectroscopic profiles and NOESY NMR correlations of all the new compounds and of the known compound 348, it could be assumed that all of these substances have the same absolute configuration. Therefore, the absolute configuration of compounds 342-348 was determined as 5R, 6S, 7S, and 9S, with the

C-11 position unresolved, in each case.

122 122

1.9. Identification of 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone (349)

A sodiated molecular ion at m/z 383 was observed from the ESIMS of compound 349, and was consistent with a molecular formula of C18H18O8 (calcd for C18H18O8Na, 383). In

1 its H NMR spectrum, three protons of a 1,3,4-trisubstituted phenyl ring resonating at H

7.73 (1H, dd, J = 8.4, 1.8 Hz, H-6), 7.68 (1H, d, J = 1.8 Hz, H-2), and 6.97 (1H, d, J =

8.4 Hz, H-5), as well as a singlet proton signal belonging to another benzyl ring resonating at δH 6.56 (1H, s, H-8), were observed. Furthermore, taking consideration of the molecular formula and three groups of methoxy protons present at δH 4.00 (3H, s,

C-OCH3), 3.99 (3H, s, C-OCH3), and 3.88 (3H, s, C-OCH3), the structure of compound

349 could be proposed as a trihydroxy- and trimethoxy-substituted flavonone. A careful analysis of the 2D NMR spectra of compound 349 indicated the respective positions of these functional groups. The identification as 5,6,3'-trihydroxy-3,7,4'- trimethoxyflavone could be proposed for compound 349, and was supported by comparison with NMR data of published data for this substance (Priestap et al., 1977).

1.10. Identification of ayanin (350)

The HRESIMS of compound 350 showed a sodiated molecular ion peak at m/z

367.0788, corresponding to an elemental formula of C18H16O7Na (calcd for C18H16O7Na,

367.0788). Its 1H and 13C NMR spectra were similar to those of compound 349 except that one of the hydroxy groups was missing. The doublet proton signal of H-8 at δH 6.73

(1H, d, J = 2.1 Hz) suggested that the unsubstituted proton is located at the C-6 position, corresponding to δH 6.38 (1H, d, J = 2.1 Hz, H-6). The proposed structure was supported

123 123

by 2D NMR experiments and by comparison with reported NMR data of ayanin

(Matsuda et al., 2002). Therefore, compound 350 was identified as 5,4-dihydroxy-

3,7,4'-trimethoxyflavone (ayanin).

1.11. Identification of ombuin (351)

The ESIMS of compound 351 provided a sodiated molecular ion peak at m/z 353,

1 13 suggesting an elemental formula of C17H14O7 (calcd for C17H14O7Na, 353). Its H and C

NMR spectra were similar to those of compound 350 except that a resonance signal of one of the methoxy groups was missing. An analysis of its 2D NMR spectra indicated that the C-3 position is substitued with a hydroxy group, instead of a methoxy group as in compound 350. Therefore, the structure 3,5,4-trihydroxy-7,4'-dimethoxyflavone was determined as the identity of compound 351, which is in agreement with reported NMR data of ombuin (Skibinski et al., 1994).

2. Conclusions

Cytotoxicity-guided isolation of Hyptis brevipes using the MCF-7 human breast cancer cell line led to the isolation of six new 5,6-dihydro-α-pyrone derivatives (342-347) and one known analog (348), as well as six known compounds. The new compounds were given the trivial names brevipolides A-F (342-347), and the known analogs were identified as (E)-1-{(1S,2S)-2-[(S)-hydroxy((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl) methyl]cyclopropyl}-1-oxopropan-2-yl 3-(4-hydroxyphenyl)acrylate (348), 5,6,3'-

124 124

trihydroxy-3,7,4'-trimethoxyflavone (349), 5,3'-dihydroxy-3,7,4'-trimethoxyflavone

(ayanin, 350), 3,5,3'-trihydroxy-7,4'-dimethoxyflavone (ombuin, 351), ursolic acid (56),

2-hydroxyursolic acid (maslinic acid, 320), and sitosterol-3--D-glucoside (daucosterol,

340). (The latter three compounds were isolated and identified by Marcy J. Balunas,

University of Illinois at Chicago, in a preliminary study on the same sample investigated in greater detail in this dissertation work.)

These new compounds bearing a cyclopropane moiety (342-347), belong to a novel skeletal class that was first reported in 2004 without determination of the absolute configuration (Hedge et al., 2004). This is therefore the second report of this class of natural products, and an absolute configuration of 5R, 6S, 7S, and 9S was elucidated in the present work by the analysis of data obtained from 2D NMR and CD spectra, as well as Mosher ester derivatizations, but leaving the C-11 position unresolved.

All the isolated compounds were evaluated for their cytotoxicity against MCF-7 human breast cancer cells, HT-29 human colon cancer cells, and Lu1 human lung cancer cells. Brevipolides B (343) and F (347), as well as compound 348, exhibited ED50 values of 6.1, 6.7 and 3.6 M against MCF-7 cells, and brevipolides A (342), B (343), F (347), and 5,6,3-trihydroxy-3,7,4-trimethoxyflavone (349) gave ED50 values of 5.8, 6.1, 7.5, and 3.6 M against HT-29 cells, respectively. However, no significant cytotoxicity was found against Lu1 cells for any of the compounds isolated. From these data, it can be proposed that the presence of a free hydroxy group at C-6 and a cis configuration in the phenylpropanoid moiety of the 5,6-dihydro--pyrone compounds are required for activity in these cell-based assays.

125 125

When these compounds were subjected to evaluation in a panel of mechanism- based in vitro assays, compound 348 were found to be active in an enzyme-based ELISA

NF-B p50 assay, with an ED50 value of 15.3 M. In a mitochondrial transmembrane potential assay, brevipolide C (344), compound 348, and 5,3'-dihydroxy-3,7,4'- trimethoxyflavone (ayanin, 350) showed ED50 values of 8.5, 75, and 310 nM, respectively. However, no potent activity was found in a proteasome inhibition assay for any of the isolated compounds.

126 126

Chapter 3

PHYTOCHEMICAL AND BIOACTIVE STUDIES ON THE LEAVES OF

VITEX QUINATA

A. Background on Vitex quinata (Lour.) Williams

1. Verbenaceae

The plant family Verbenaceae is closely related to the family Lamiaceae from the taxonomic point of view. Plant taxonomists have suggested that some genera of

Verbenaceae should be classified in the Lamiaceae (Cantino et al., 1992). The botanical diagnostic characteristics of the plants in the family Verbenaceae and their distribution are described as follows:

Shrubs or trees, sometimes climbing shrubs, rarely herbs. Indumentum of simple, stellate, and/or other complex hairs. Leaves opposite or rarely whorled, without stipules, simple or 3-foliolate, less often palmately (or pinnately) compound. Inflorescences terminal or axillary, racemose, cymose, spicate, or thyrses. Flowers bisexual or polygamous by abortion, zygomorphic or rarely actinomorphic. Calyx persistent. Corolla 4- or 5- or more lobed; lobes usually spreading, aestivation overlapping. Fertile stamens inserted on corolla tube, alternate with lobes; filaments free; anthers dorsifixed, 1- or 2-locular, dehiscing by longitudinal slits or sometimes a circular pore. Ovary entire or 4-grooved, 2-8-locular; ovules 1 or 2 per locule, erect or pendulous. Style terminal, simple, entire or 2-cleft. Fruit a drupe or indehiscent capsule, sometimes breaking up into nutlets. Seeds (1 or) 2-4, endosperm usually absent, seed coat thin; embryo straight, as long as seed; radicle short, inferior. Some 91 genera and ca. 2000 species: primarily tropical and subtropical. (Chen and Gilbert, 1994)

127 127

2. Genus Vitex and Vitex quinata (Lour.) Williams

Vitex is one of the largest genera in the Verbenaceae, with about 250 species distributed mainly in tropical regions of the world. Some species of the genus have been used as folk medicines to treat various diseases. Vitex agnus-castus, commonly known as

―vitex‖, ―chaste tree‖, ―chasteberry‖, or ―monk’s pepper‖, is currently used as a botanical dietary supplement in the U.S., and has purported actions in the alleviation of premenstrual symtoms. The botanical diagnostic characteristics of the genus Vitex may be described as follows:

Trees or shrubs. Branches glabrous or sparsely pubescent. Leaves opposite, palmately (1-) 3-8-foliolate; leaflets petiolulate, margin entire, dentate, serrate, or incised. Inflorescences terminal or axillary cymes, thyrses, or panicles; bracts usually small, often early deciduous. Calyx campanulate, tubular, or funnelform, sometimes 2-lipped, usually truncate or shortly 5-dentate. Corolla blue, white, or yellow, 2-lipped, lower lip 3-lobed with middle lobe greatly elongated, upper lip usually 2-lobed. Stamens 4, didynamous, sometimes exserted; anther locules attached only at tip, becoming divaricate. Ovary 2-4-locular; ovules 1 or 2 per locule. Style filiform; stigma 2-cleft. Drupes subtended by enlarged calyx, globose, ovoid, or obovoid, normally 4-locular and 4-seeded but often some locules suppressed and base of pyrene forming a hollowed cavity, endocarp a bony pyrene, mesocarp generally fleshy. Seeds obovoid or oblong, endosperm absent; cotyledons usually fleshy. About 250 species: chiefly tropical, few in temperate regions of both hemispheres. (Chen and Gilbert, 1994)

The botanical diagnostic characteristics of Vitex quinata (Figures 3.1 and 3.2) include the following:

Tree 4-12 m tall, evergreen; bark brown. Branchlets pubescent and glandular when young, glabrescent. Leaves 3-5-foliolate; petioles 2.5-6 cm; petiolules 0.5-2 cm; leaflets obovate-elliptic to obovate or oblong to elliptic, thickly papery, both surfaces shiny, abaxially yellow glandular, base cuneate, margin entire or sometimes apically crenulate dentate, apex acuminate, acute, or obtuse; central leaflet 5-20, 2.5-8.5 cm. Panicles terminal, lax, 9-18 cm, densely yellowish brown pubescent. Calyx 2-3 mm, rudimentarily dentate, densely yellowish brown pubescent, glandular. Corolla yellowish, 6-8 mm, 2-lipped, 5-lobed, outside pubescent and glandular. Stamens exserted. Ovary glandular. 128 128

Fruiting calyx truncate. Fruit black, obovoid to globose, ca. 8 mm in diam. Fl. May-Jul, fr. Aug-Sep. (Chen and Gilbert, 1994)

Figure 3.1. Illustration of Vitex quinata (Lour.) Williams: 1, fruiting branch; 2 and 5,

opened corolla showing stamens; 3, fruiting branch; 4, leaves. (From Wu et

al., 1992)

129 129

Figure 3.2. Photograph of Vitex quinata collected from Kego Nature Reserve, Hatinh Province, Vietnam. (Taken by Dr. D. D. Soejarto, University of Illinois at Chicago)

3. Phytochemical constituents of the genus Vitex

Phytochemical investigations on about 30 species from the genus Vitex have revealed that the major secondary metabolites of these plants are diterpenoids, flavonoids, iridoid glycosides, and ecdysteroids (Table 3.1 and Figures 3.3 to 3.6). Other components, such as lignans, phenylpropanoids, sesquiterpenoids, and triterpenoids have been found in some Vitex plants, but with less frequency. Among these plants, the species V. agnus-castus, V. negundo, V. rotundifolia, and V. trifolia are the most thoroughly investigated phytochemically. Chemical constituents and related biological studies on

Vitex species have been reviewed in several different journals published in recent years

130 130

(Ganapaty and Vidyadhar, 2005; Li et al., 2005; Filho et al., 2008), so a complete review

is not provided in this dissertation.

Code Trivial name Source Reference 352 Vitetrifolin A V. trifolia Ono et al., 2000 353 Vitexifolin C V. rotundifolia Ono et al., 2002 354 Vitetrifolin D V. trifolia Ono et al., 2001 355 Vitetrifolin I V. trifolia Wu et al., 2009 356 Vitexifolin A V. rotundifolia Ono et al., 2002 357 Dihydrosolidagenone V. trifolia Ono et al., 2000 358 Limonidilactone V. leucoxylon Krishina, et al., 1997 359 Viteoside A V. rotundifolia Ono et al., 1998 360 Prerotundifuran V. rotundifolia Asaka et al., 1973 361 Previtexlactone V. rotundifolia Kondo et al., 1986 362 Vitexifolin D V. rotundifolia Ono et al., 2002 363 Vitexifolin E V. rotundifolia Ono et al., 2002 364 Unamed V. leptobotrys Trinh et al., 1998 365 Cardamomin V. leptobotrys Trinh et al., 1998 366 Unamed V. negundo, V. rotundifolia Achari et al, 1984 367 Luteolin V. agnus-castus, V. negundo, Suksamran et al., 2002 V. rotundifolia, V. scabra 368 Unamed V. negundo, V. rotundifolia Kobayakawa et al, 2004 369 Isoorietin V. lucens, V. negundo, V. Suzana and Franco, polygama, V. trifolia 1998 370 Casticin V. agnus-castus, V. negundo, Diaz et al., 2003 (Vitexicarpin) V. rotundifolia 371 Viteoid II V. cymosa Dos Santos et al., 2001 372 Agnuside V. agnus-castus, V. cymosa, Dos Santos et al., 2001 V. limonifolia, V. negundo, V. polygama, V. trifolia 373 20-Hydroxyecdysone V. agnus-castus, V. cooperi, Trinh et al., 1998 V. cymosa, V. fisherii, V. gardneriana, V. glabrata, V. madiensis, V. megapotamica, V. pinata, V. poligama, V. rehmanni, V. scabra, V. sereti, V. strickeri, V. thyrsiflora 374 Makisterone A V. leptobotrys Trinh et al., 1998 Table 3.1. Selected examples of natural products found in the genus Vitex. 131 131

3.1. Diterpenoids

More than fifty diterpenoids have been isolated from plants of the genus Vitex, with

over forty of these representing the labdane or nor-labdane skeletal classes, and others

belonging to the abietane, halimane, and linear structure groups. Selected examples of

diterpenoids with abietane (352 and 353), halimane (354 and 355), labdane (356-361),

and nor-labdane (362 and 363) skeletons, isolated from plants of the genus Vitex, are

presented in Figure 3.3. A recent study showed that the halimane-type diterpenoid,

vitetrifolin I (355), a constituent of the fruits of Vitex trifolia L., inhibited Hela cell

proliferation by both induction of G0/G1 cell cycle phase arrest and apoptosis, with an

IC50 value of 4.9 M (Wu et al., 2009).

O 15 11 17 20 13 1 O

10 8 3 5 HO 18 19 HO 352 353 354 15 O 16

20 11 1 17 8 10

3 5

18 19 O 355 356 357 continued Figure 3.3. Structures of selected diterpenoids isolated from the genus Vitex

132 132

Figure 3.3. continued

O MeO 15 O O O O 13 11 16 20 O 17 O 1 10 8 3 O 5

GlcO 18 19 OAc OAc 358 359 360

O O 13 O 14 O 11 11 17 18 O O 14 15 1 1 O 10 8 10 8

3 3 5 5

15 16 OAc 16 17 OAc 361 362 363

3.2. Flavonoids

Flavonoids found in the plants of genus Vitex comprise several different classes,

such as the dihydrochalcone (364), chalcone (365), flavanone (366), flavone (367 and

368), flavone glycosides (369), and flavonol (370) types (Figure 3.4). Vitexicarpin (371)

isolated from the leaves of V. negundo was reported to exhibit broad cytotoxicity against

KB, LNCaP, and Lu1 cells with ED50 values of 0.5, 0.5, and 0.7 g/mL, respectively

(Diaz et al., 2003).

133 133

4 OH

1 HO OH HO 4' OH 7 1' OH 9

OMe O OMe O 364 365

4' OMe OH

MeO 7 8 O HO O 1 1' OH OH 4 9 MeO 5 OH O OH O 366 367

OMe OH

MeO O HO O OH OH

MeO GlcO OH O OH O 368 369

OMe

MeO O OH

MeO OMe OH O 370

Figure 3.4. Structures of selected flavonoids isolated from the genus Vitex

3.3. Iridoids and iridoid glycosides

More than 14 iridoids and iridoid glycosides have been isolated from the genus

Vitex, with nine of them bearing a 4-demethyl structure. A 4-demethyl iridoid, namely, viteoid II (371), and the iridoid glycoside, agunside (372) have been selected as examples, and are shown in Figure 3.5.

134 134

371 372

Figure 3.5. Structures an iridoid (371) and an iridoid glycoside (372) isolated from the

genus Vitex

3.4. Ecdysteroids

In addition to very common sterols found in the plant kingdom, such as β-sitosterol

(339), a number of ecdysteroids have been reported from the plants of the genus Vitex.

The distribution, structures, sources, and 13C NMR spectroscopic data of 24 different ecdysteroids isolated from Vitex species were recently reviewed (Filho et al., 2008).

Representive examples of ecdysteroids are given in Figure 3.5.

373 374

Figure 3.6. Structures of selected ecdysteroids (373 and 374) isolated from the genus

Vitex

135 135

B. Statement of Problem

A previous study on Vitex quinata reported the isolation of five compounds (Figure

3.7), namely, daucosterol (340), 3,5-O-dicaffeoyl quinic acid (375), 20-hydroxyecdysone

20,22-monoacetonide (376), β-sitosterol (339), and vitexin (377), without including any biological data (Cheng et al., 2007). Furthermore, none of these isolated compounds was found to correlate with either cytotoxicity or any anticancer potential by searching the

Scifinder and NAPRALERT databases, and no biological activity study has been reported for the constituents of this plant.

As part of a collaborative, multi-disciplinary approach to the discovery of new naturally occurring anticancer drugs, as described in Chapter 2, the leaves of V. quinata collected in Indonesia, were selected for further study, after a chloroform extract was found to be active in an initial cytotoxicity screening procedure using the MCF-7 human breast cancer cell line, with an ED50 value of 3.1 g/mL (Appendix B, Table 1).

The present study, comprising Chapter 3 of this dissertation, consists of the isolation, structure elucidation, and cytotoxicity and mechanism biological evaluation of the constituents obtained from the leaves of Vitex quinata, collected from West Java,

Indonesia.

C. Experimental

1. General experimental procedures

Optical rotations were obtained on a Perkin-Elmer 343 automatic polarimeter. UV spectra were measured with a Perkin-Elmer Lambda 10 UV/vis spectrometer. CD spectra

136 136

OH H O OH

O O H OH OH HO OH O OH RO O

340 R = glucose 375

342 R = H

O O OH OGlc OH HO O H HO OH OH O HO O

376 377

Figure 3.7. Structures of compounds isolated from V. quinata reported in the literature

were measured on a JASCO J-810 spectrometer. IR spectra were run on a Thermo

Scientific Nicolet 6700 FT-IR spectrometer. NMR spectroscopic data were recorded at room temperature on a Bruker Avance DPX-300 or Bruker Avance DRX-400 spectrometer. Column chromatography was performed with 65-250 or 230-400 mesh silica gel (Sorbent Technologies, Atlanta, GA). Analytical thin-layer chromatography was

 conducted on 250 m thickness Partisil silica gel 60 F254 glass plates (Whatman, Clifton,

NJ). Analytical and semi-preparative HPLC were carried out on a Waters system

137 137

composed of a 600 controller, a 717 plus autosampler, and a 2487 dual wavelength absorbance detector, with Waters Sunfire analytical (4.6  150 mm) and preparative (19 

150 mm) C18 columns. All chemicals and reagents were purchased from Sigma-Aldrich

(St. Louis, MO).

2. Plant material

The leaves of Vitex quinata (Lour.) Williams (Verbenaceae) were collected from

West Java, Indonesia, at geographic coordinates -81758.62 and +1162431.32, altitude 750 m, on August 13, 2003, by Dr. Soedarsano Riswan from the Herbarium

Bogoriense, Research Center for Biology, Indonesian Institute of Science, Bogor,

Indonesia. The plant was identified by Dr. Soedarsano Riswan. A voucher specimen

(A05789) has been deposited at the Field Museum of Natural History, Chicago, Illinois.

3. Solvent extraction and partitioning of Vitex quinata

The dried, powdered leaves of V. quinata (830 g) were extracted with methanol at room temperature. The methanol extract (125 g) was suspended in a mixture of methanol-water (9:1) and then partitioned between hexanes, chloroform, and ethyl acetate, successively, as summarized in Figure 3.8.

4. Column chromatography of the chloroform-soluble and ethyl acetate-soluble

extracts of Vitex quinata

The chloroform extract (3.2 g) was chromatographed over a silica gel column

(65-250 mesh, 2.2  47 cm), eluted with a gradient solvent system of increasing polarity

138 138

Figure 3.8. Solvent extraction scheme used for the leaves of V. quinata

(chloroform-methanol, 50:1 to 5:1, then pure methanol) to give nine fractions

(D2F1-D2F9).

A white solid precipitate that formed from fraction D2F2 was washed with hexane to give -sitosterol (339). The remainder of fraction D2F2 (100 mg) was subjected to silica gel chromatography (230-400 mesh, 1  40 cm), using chloroform-acetone (10:1) for elution, to afford 15 subfractions (D3F2F1-D3F2F15). Further purification of subfraction

D3F2F11, using a silica gel column (230-400 mesh, 1  30 cm) eluted with hexane-ethyl acetate (3:1), led to the isolation of the new compound, methyl 8-{(5R)-2-[(E)-2- hydroxypent-3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379, 6 mg).

139 139

Subfraction D3F2F14 was purified on a silica gel column (230-400 mesh, 1  30 cm), using hexane-ethyl acetate (3:1) as eluting solvent, to give 2-hydroxy-4,4,6- trimethoxychalcone (382, 2 mg).

Fraction D2F3 (180 mg) was subjected to silica gel column chromatography

(230-400 mesh, 1  40 cm), eluted with a gradient solvent system of increasing polarity

(hexanes-acetone, 5:1 to 3:1), to give eight subfractions (D2F3F1-D2F3F8). Purification of subfraction D2F3F3 (40 mg) using silica gel column chromatography (230-400 mesh,

1  20 cm), eluted with hexane-ethyl acetate (5:1), led to the isolation of (-)-loliolide (386,

15 mg). Subfraction D2F3F5 (33 mg) was separated using a silica gel column (230-400 mesh, 1  35 cm) to give (S)-5-hydroxy-7,4'-dimethoxyflavanone (380, 3 mg).

Subfraction D2F3F8 (20 mg) was chromatographed over a silica gel column (230-400 mesh, 1  20 cm), using hexane-ethyl acetate (5:1) as eluting solvent, to afford

2',6'-dihydroxy-4,4'-dimethoxychalcone (383, 2 mg).

Fraction D2F5 (80 mg) was chromatographed over a Sephadex LH-20 column (4 

50 cm), using pure methanol as eluting solvent, to give five subfractions

(D2F5F1-D2F5F5). Sufraction D2F5F4 (36 mg) was separated by preparative thin-layer chromatography (PTLC), using chloroform-methanol (10:1) as developing solvent, to give compound 384 (3 mg, Rf = 0.45).

The ethyl acetate extract (16 g) was fractionated over a silica gel column (63-200 mesh, 8.5 × 19 cm), eluted with a gradient of increasing polarity containing chloroform-methanol (from 20:1 to 2:1), and was finally washed with pure methanol.

Altogether, six pooled fractions (D3F1-D3F6) were collected. 140 140

Fraction D3F2 (270 mg) was chromatographed over a Sephadex LH-20 column (4 

50 cm), using pure methanol as eluting solvent, to give four subfractions

(D3F2F1-D3F2F4). Subfraction D3F2F2 (40 mg) was subjected to passage over a silica gel column (230-400 mesh, 1  40 cm), eluted with chloroform-methanol (30:1), to give rhamnocitrin (385, 2 mg).

Fraction D3F3 (1.6 g) was subjected to passage over a Sephadex LH-20 column (4 

50 cm), eluted with pure methanol, to give six subfractions (D3F3F1-D3F3F6).

Subfraction D3F4F2 (290 mg) was separated using a silica gel column (230-400 mesh, 20

 1.5 cm) to afford methyl 3,4-dicaffeoyl quinic acid ester (387, 30 mg) and methyl

4,5-dicaffeoyl quinic acid ester (389, 4 mg). Subfraction D3F3F3 (80 mg) was further fractionated by silica gel column chromatography, using chloroform-methanol (15:1), to give a mixture containing compound 378. The mixture was further purified by semi-preparative HPLC, using acetonitrile-0.5% formic acid water solution (30:70, 8 mL/min) as eluting solvent, to give the new compound dimethyl 3,3,4,4-tetrahydroxy-

-truxinate (378, 4 mg, tR = 11.4 min). Another subfraction, D3F3F5 (230 mg), was subjected to passage over a silica gel column (1.0  40 cm), eluted with chloroform-acetone (8:1), to furnish compound 381 (8 mg).

Fraction D3F4 (2.5 g) was chromatographed over a Sephadex LH-20 column (4  50 cm), eluted with pure methanol, to afford five subfractions (D3F4F1-D3F4F5).

Subfraction D3F4F2 (1 g) was purified over a silica gel column (230-400 mesh, 1.8  35 cm), using chloroform-acetone (6:1) as solvent, to give methyl 3,4,5-O-tricaffeoyl quinate (390, 500 mg). A mixture obtained from subfraction D3F4F4 was further purified 141 141

using semi-preparative HPLC, eluted with methanol-water (45:55, 8 mL/min), to afford methyl 3,5-dicaffeoyl quinic acid ester (388, 65 mg, tR = 13 min).

5. Characterization of dimethyl 3,3,4,4-tetrahydroxy- -truxinate (378)

20 Light yellow gum; [] D +5.4 (c 0.1, MeOH); UV (MeOH) max (log ) 215 (4.07),

345 (3.88) nm; IR (film) max 3350 (br), 3100, 2956, 1710, 1624, 1514, 1440, 1165, 825

-1 1 cm ; H NMR (400 MHz, acetone-d6) δH 6.80 (2H, d, J = 2.1 Hz, H-2, -2), 6.77 (2H, d, J

= 8.1 Hz, H-5, -5), 6.65 (2H, dd, J = 8.1, 2.1 Hz, H-6, -6), 3.68 (6H, s, H-10, -10), 3.46

13 (2H, dd like, H-7, -7), 3.30 (2H, dd like, H-8, -8); C NMR (100 MHz, acetone-d6), δC

173.6 (C-9, -9), 145.9 (C-3, -3), 145.0 (C-4, -4), 134.2 (C-4, -4), 119.1 (C-6, -6), 116.1

(C5, -5), 114.8 (C-2, -2), 52.3 (C-10, -10), 48.5 (C-7, -7), 45.6 (C-8, -8); HRESIMS

+ m/z 411.1061 [M + Na] (calcd for C20H20O8Na, 411.1057).

6. Characterization of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5-methoxy-

3-oxocyclopent-1-enyl}octanoate (379)

20 Colorless gum; [] D -19.8 (c 0.1, MeOH); UV (MeOH) max (log ) 208 (3.87),

-1 310 (3.72) nm; IR (film) max 3300, 2944, 2925, 1725, 1672, 1543, 1432, 1375, 1244 cm ;

1 H NMR (400 MHz, CDCl3) H 5.65 (1H, ddq, J = 15.2, 6.4, 0.4 Hz, H-17), 5.43 (1H, ddd, J = 15.2, 6.8, 1.5 Hz, H-16), 4.46 (1H, br d, J = 5.2, Hz, H-10), 4.22 (1H, q like, J =

142 142

Figure 3.9. Structures of compounds isolated from the leaves of Vitex quinata in the

present investigation 143 143

5.8 Hz, H-15), 2.64 (1H, dd, J = 18.2, 5.8 Hz, H-11), 2.44 (2H, d, J = 6.3 Hz, H-14), 2.42

(2H, m, H-7), 2.32 (1H, dd, J = 18.2, 1.9 Hz, H-11), 2.31 (2H, t, J = 7.5 Hz, H-2), 1.66

(3H, d, J = 6.5 Hz, H-18), 1.63 (2H, m, H-3), 1.60 (1H, m, H-8), 1.47 (1H, m, H-8), 1.34

13 (2H, m, H-6), 1.33 (4H, m, H-4 and 5); C NMR (100 MHz, CDCl3) δC 207.0 (C-12),

(C-15), 57.1 (C-1), 51.5 (C-2), 40.5 (C-11), 34.0 (C-2), 32.1 (C-14), 29.7 (C-6), 29.0

(C-4 and 5), 28.1 (C-7), 27.4 (C-8), 24.9 (C-3), 17.6 (C-18); HRESIMS m/z 375.2136 [M

+ + Na] (calcd for C20H32O5Na, 375.2142).

7. Characterization of (S)-5-hydroxy-7,4'-dimethoxyflavanone (380)

20 1 Colorless gum; [] D -73 (c 0.1, MeOH); H NMR (400 MHz, MeOD) δH 7.39

(2H, d, J = 8.6 Hz, H-2', H-6'), 6.92 (2H, d, J = 8.6 Hz, H-3', H-5'), 5.98 (1H, d, J = 2.1

Hz, H-8), 5.92 (1H, d, J = 2.1 Hz, H-6), 5.29 (1H, dd, J = 12.6, 2.8 Hz, H-2), 3.80 (6H, s,

C-7-OMe, C-4'-OMe), 2.96 (1H, dd, J = 16.7, 12.6 Hz, H-3b), 2.62 (1H, dd, J = 16.7, 2.8

13 Hz, H-3a); C NMR (100 MHz, MeOD) δC 190.6 (C-4), 165.8 (C-7), 163.2 (C-5), 163.0

(C-8a), 160.3 (C-4), 130.5 (C-1), 127.8 (C-2, -6), 113.9 (C-3, -5), 102.5 (C-4a), 97.2

(C-8), 94.5 (C-6), 78.2 (C-2), 54.7 (C-7-OMe, C-4-OMe), 45.3 (C-3); ESIMS m/z 323

[M+Na]+.

8. Characterization of (S)-isosakuranetin (381)

20 1 Colorless needles; mp 187-179 C; [] D -57 (c 0.1, MeOH); H NMR (300 MHz,

CDCl3) δH 7.36 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.95 (2H, d, J = 8.8 Hz, H-3', H-5'), 6.02

(1H, d, J = 2.0 Hz, H-8), 5.96 (1H, d, J = 2.0 Hz, H-6), 5.34 (1H, dd, J = 13.0, 3.0 Hz,

144 144

H-2), 3.81(3H, s, C-4-OMe), 3.09 (1H, dd, J = 17.1, 13.0 Hz, H-3b), 2.77 (1H, dd, J =

13 17.1, 3.0 Hz, H-3a); C NMR (100 MHz, MeOD) δC 190.4 (C-4), 164.5 (C-7), 162.9

(C-5), 163.0 (C-8a), 160.2 (C-4), 130.4 (C-1), 127.6 (C-2, -6), 113.7 (C-3, -5), 102.9

(C-4a), 96.7 (C-8), 94.1 (C-6), 78.3 (C-2), 54.7 (C-4-OMe), 45.1 (C-3); ESIMS m/z 309

[M+Na]+.

9. Characterization of 2-hydroxy-4,4,6-trimethoxychalcone (382)

1 Yellow needles; mp 114-116 C; H NMR (300 MHz, CDCl3) δH 7.77 (1H, d, J =

16.2 Hz, H-7), 7.76 (1H, d, J = 16.2 Hz, H-8), 7.54 (2H, d, J = 9.0 Hz, H-2, -6), 6.90 (2H, d, J = 9.0 Hz, H-3, -5), 6.07 (1H, d, J = 2.4 Hz, H-3), 5.92 (1H, d, J = 2.4 Hz, H-5), 3.88

13 (3H, s, OMe), 3.82 (3H, s, OMe),  3.80 (3H, s, OMe); C NMR (75 MHz, CDCl3) δC

192.6 (C-9), 168.5 (C-4), 166.1 (C-6), 162.8 (C-4), 161.6 (C-2), 142.7 (C-7), 130.2

(C-2, -6), 128.5 (C-1), 125.3 (C-8), 114.6 (C-3, -5), 55.8 (C-4-OMe), 106.4 (C-1), 93.9

(C-3), 91.4 (C-5), 56.1 (C-6-OMe), 55.6 (C-6-OMe); ESIMS m/z 337 [M+Na]+.

10. Characterization of 2',6'-dihydroxy-4,4'-dimethoxychalcone (383)

1 Yellow gum; H NMR (300 MHz, CDCl3) δH 8.15 (1H, d, J = 15.5 Hz, H-8), 7.75

(1H, d, J = 15.5 Hz, H-7), 7.62 (2H, d, J = 9.0 Hz, H-2, -6), 6.90 (2H, d, J = 9.0 Hz, H-3,

-5), 6.07 (1H, d, J = 2.4 Hz, H-8), 5.92 (1H, d, J = 2.4 Hz, H-6), 3.88 (3H, s, OMe), 3.82

13 (3H, s, OMe), 3.80 (3H, s, OMe); C NMR (75 MHz, CDCl3) δC 192.6 (C-9), 168.5

(C-4), 166.1 (C-6), 162.8 (C-4), 161.6 (C-2), 142.7 (C-7), 130.2 (C-2, -6), 128.5 (C-1),

125.3 (C-8), 114.6 (C-3, -5), 55.8 (C-4-OMe), 106.4 (C-1), 93.9 (C-3), 91.4 (C-5), 56.1 145 145

(C-6-OMe), 55.6 (C-6-OMe); ESIMS m/z 323 [M+Na]+.

11. Characterization of 3,5-dihydroxy-7,4-dimethoxyflavonone (384)

1 Yellow crystals; mp 180-182C; H NMR (300 MHz, CDCl3) δH 11.7 (1H, s, OH-5),

8.14 (2H, d, J = 9.0, H-2, H-6), 7.00 (2H, d, J = 9.0, H-3, -5), 6.58 (1H, s, OH-3), 6.46

(1H, d, J = 2.1 Hz, H-8), 6.35 (1H, d, J = 2.1 Hz, H-6), 3.87 (6H, s, OMe); 13C NMR

(100 MHz, CDCl3) δC 175.1 (C-4), 165.7 (C-7), 161.1 (C-4), 160.8 (C-5), 156.8 (C-9),

145.7 (C-2), 135.6 (C-3), 128.8 (C-2, 6), 123.1 (C-1), 114.0 (C-3, 5), 103.9 (C-10),

97.9 (C-6), 92.2 (C-8), 55.8, (Me-7), 55.4 (OMe-4); ESIMS m/z 337.

12. Characterization of rhamnocitrin (385)

1 Yellow needle crystals, mp 202–203 C; H NMR (300 MHz, MeOD) δH 8.12 (2H, d, J = 9.1 Hz, H-2', -6'), 6.92 (2H, d, J = 9.1 Hz, H-3', 5'), 6.58 (1H, d, J = 2.1 Hz, H-8),

13 6.30 (1H, d, J = 2.1 Hz, H-6), 3.87 (3H, s, C-7-OMe); C NMR (75 MHz, MeOD) δC

176.2 (C-4), 166.2 (C-7), 161.4 (C-5), 159.2 (C-4′), 156.3 (C-8a), 147.4 (C-2), 136.4

(C-3), 129.8 (C-2′, 6′), 121.7 (C-1′), 115.7 (C-3′, 5′), 104.2 (C-4a), 97.6 (C-6), 92.2 (C-8),

56.1 (Ome-7); ESIMS m/z 323 [M+Na]+.

13. Characterization of (-)-loliolide (386)

20 1 White needles; mp 151-153 C; [] D -80 (c 0.1, MeOH); H NMR (400 MHz,

CDCl3) δH 5.70 (1H, s, H-7), 4.34 (1H, br s, H-3), 2.47 (1H, ddd, J = 2.6, 2.6, 14.0 Hz,

H-4), 1.98 (1H, ddd, J = 14.0, 2.6, 2.6 Hz, H-2), 1.79 (1H, dd, J = 3.6, 14.2 Hz, H-4), 146 146

1.79 (3H, s, Me-5), 1.62 (1H, br s, OH-3), 1.55 (1H, dd, J = 14.6, 3.6 Hz, H-2), 1.48

13 (3H, s, Me-1), 1.28 (3H, s, CH3-1); C NMR (100 MHz, CDCl3) δC 182.6 (C-8),

172.3 (C-6), 112.7 (C-7), 87.4 (C-5), 66.8 (C-3), 45.5 (C-2), 46.2 (C-4), 35.6 (C-1), 29.2

(Me-1), 26.9 (Me-7), 19.8 (Me-1); ESIMS 219 [M+Na]+.

14. Characterization of methyl 3,4-O-dicaffeoyl quinate (387)

20 1 Pale yellow gum; []D -190  (c 0.1, MeOH); H NMR (DMSO-d6, 300 MHz) δH

7.56 (1H, d, J = 15.9 Hz, H-7), 7.47 (1H, d, J =15.9 Hz, H-7), 7.07 (2H, d, J = 2.1 Hz,

H-2, -2), 7.03 (2H, d, J = 8.1, 2.1 Hz, H-6, -6), 6.82 (2H, br d, J = 8.1 Hz, H-5, -5),

6.32 (1H, d, J =15.9 Hz, H-8), 6.19 (1H, d, J = 15.9 Hz, H-8), 5.32 (1H, m, H-3), 5.02

13 (1H, m, H-4), 4.20 (1H, m, H-5), 3.65 (3H, s, OCH3), 2.27 (2H, m, H-2, -6); C NMR

(DMSO-d6, 75 MHz) δC 173.5 (C-7), 166.6 (C-9), 165.3 (C-9), 148.7 (C-4), 148.1

(C-4), 147.6 (C-7), 147.1 (C-7), 145.7 (C-3), 145.5 (C-3), 125.6 (C-1), 125.1 (C-1),

121.5 (C-6), 121.0 (C-6), 115.9 (C-5), 114.9 (C-5), 114.6 (C-2), 114.5 (C-2), 113.9

(C-8), 113.4 (C-8), 73.2 (C-1), 72.4 (C-4), 67.9 (C-3), 65.5 (C-5), 52.1 (OCH3), 37.8

(C-2), 36.8 (C-6); ESIMS m/z 531 [M+H]+.

15. Characterization of methyl 3,5-O-dicaffeoyl quinate (388)

20 1 Pale yellow gum; []D -188 (c 0.20, MeOH); H NMR (400 MHz, MeOD) δH

7.48 (1H, d, J = 16.0 Hz, H-3), 7.42 (1H, d, J = 16.0 Hz, H-3), 7.04 (1H, d, J = 2.5 Hz,

H-5), 7.04 (1H, d, J = 2.5 Hz, H-5), 6.99 (1H, d, J = 8.0 Hz, H-9), 6.99 (1H, d, J = 8.0

147 147

Hz, H-9), 6.77 (1H, d, J = 8.0 Hz, H-8), 6.77 (1H, d, J = 8.0 Hz, H-8), 6.23 (1H, d, J =

16.0 Hz, H-2), 6.14 (1H, d, J = 16.0 Hz, H-2), 5.38 (1H, br dd, J = 9.5, 4.5 Hz, H-5),

5.29 (1H, dt, J = 7.0, 3.5 Hz, H-3), 3.98 (1H, dd, J = 9.5, 3.0 Hz, H-4), 3.59 (3H, s, OMe),

2.21 (1H, br d, J = 12.5 Hz, H-2a), 1.99 (1H, br d, J = 12.5 Hz, H-2b), 1.98 (1H, br d, J =

13 12.5 Hz, H-6); C NMR (100 MHz, MeOD) δC 174.7 (C-7), 167.3 (C-1'), 166.8 (C-1"),

149.8 (C-7'), 149.5 (C-7"), 146.7 (C-3'), 146.6 (C-3"), 146.3 (C-6'), 146.1 (C-6"), 126.7

(C-4'), 126.4 (C-4"), 122.4 (C-9'), 122.3 (C-9"), 117.0 (C-8'), 116.9 (C-8"), 115.9 (C-2'),

115.8 (C-2"), 115.8 (C-5'), 114.8 (C-5"), 73.8 (C-1), 72.1 (C-3), 71.1 (C-5), 67.9 (C-4),

+ 53.4 (OCH3-7),35.9 (C-6), 35.6 (C-2); ESIMS m/z 531 [M+H] .

16. Characterization of methyl 4,5-O-dicaffeoyl quinate (389)

20 1 Pale yellow gum; []D -179 (c 0.20, MeOH); H NMR (400 MHz, MeOD) δH

2.32 (1H, br d, J = 12.4 Hz, H-2a), 2.16 (1H, br d, J = 12.4 Hz, H-2b), 5.63 (1H, dt, J =

9.4, 3.0 Hz, H-3), 5.09 (1H, dd, J = 9.4, 3.0 Hz, H-4), 4.39 (1H, br dd, J = 9.4, 4.6 Hz,

H-5), 2.18 (1H, br d, J = 12.5 Hz, H-6a), 2.28 (1H, br d, J = 12.5 Hz, H-6b), 3.68 (3H, s,

OMe-7), 6.21 (1H, d, J = 15.8 Hz, H-2), 6.27 (1H, d, J = 15.8 Hz, H-2), 7.56 (1H, d, J

= 15.8 Hz, H-3), 7.48 (1H, d, J = 15.8 Hz, H-3), 7.14 (1H, d, J = 2.5 Hz, H-5), 7.16

(1H, d, J = 2.5 Hz, H-5), 6.84 (1H, d, J = 8.1 Hz, H-8), 6.85 (1H, d, J = 8.1 Hz, H-8),

7.02 (1H, d, J = 8.1 Hz, H-9), 7.03 (1H, d, J = 8.1 Hz, H-9); 13C NMR (100 MHz,

MeOD) δC 75.8 (C-1), 36.9 (C-2), 67.8 (C-3), 74.9 (C-4), 68.5 (C-5), 38.9 (C-6), 52.3

(OCH3-7), 174.1 (C-7), 166.2 (C-1'), 166.5 (C-1"), 115.0 (C-2'), 115.1 (C-2"), 146.1

(C-3'), 146.1 (C-3"), 122.4 (C-4'), 122.4 (C-4"), 116.1 (C-5'), 116.1 (C-5"), 148.7 (C-6'), 148 148

148.8 (C-6"), 148.9.8 (C-7'), 148.9 (C-7"), 115.2 (C-8'), 115.2 (C-8"), 126.3 (C-9'), 126.3

(C-9"); ESIMS m/z 531 [M+H]+.

17. Characterization of methyl 3,4,5-O-tricaffeoyl quinate (390)

20 1 Pale yellow gum; []D -180 (c 0.1, MeOH); H NMR (MeOD, 400 MHz) δH 7.64

(1H, d, J = 15.9 Hz, H-7), 7.55 (1H, d, J = 15.9 Hz, H-7), 7.55 (1H, d, J = 15.9 Hz,

H-7), 7.13 (1H, d, J = 2.1 Hz, H-2), 7.03 (1H, dd, J = 8.2, 2.1 Hz, H-6), 7.00 (1H, d, J

= 2.1 Hz, H-2), 7.00 (1H, d, J = 2.1 Hz, H-2), 6.91 (1H, dd, J = 8.2, 2.1 Hz, H-6),

6.91 (1H, dd, J = 8.2, 2.1 Hz, H-6), 6.85 (1H, d, J = 8.2 Hz, H-5), 6.74 (1H, d, J = 8.2

Hz, H-5), 6.73 (1H, d, J = 8.2 Hz, H-5), 6.42 (1H, d, J = 15.9 Hz, H-8), 6.42 (1H, d, J

= 15.9 Hz, H-8), 6.16 (1H, d, J = 15.9 Hz, H-8), 5.73 (1H, m, H-5), 5.68 (1H, m, H-3),

5.30 (1H, dd, J = 10.1, 3.4 Hz, H-4), 3.65 (3H, s, OCH3), 2.67 (1H, dd, J = 16.1, 3.4 Hz,

H-2), 2.85 (1H, dd, J = 12.8, 3.8 Hz, H-6), 2.59 (1H, dd, J = 16.1, 3.2 Hz, H-2), 2.15

13 (1H, dd, J = 12.8, 10.3 Hz, H-6); C NMR (MeOD, 100 MHz) δC 175.5 (C-7), 165.8

(C-9), 165.6 (C-9), 165.2 (C-9), 148.7 (C-7), 148.6 (C-7), 148.4 (C-7), 146.0

(C-4), 146.0 (C-4), 145.9 (C-4), 145.6 (C-3), 145.6 (C-3), 145.5 (C-3), 125.5

(C-1), 125.3 (C-1), 125.2 (C-1), 121.6 (C-6), 121.5 (C-6), 121.4 (C-6), 115.8

(C-5), 115.8 (C-5), 115.7 (C-5), 115.0 (C-2), 114.9 (C-2), 114.9 (C-2), 113.9

(C-8), 113.2 (C-8), 113.2 (C-8), 74.7 (C-1), 73.6 (C-4), 69.6 (C-3), 69.2 (C-5), 53.1

(OMe-7), 36.5 (C-6), 32.8 (C-2); ESIMS m/z 715 [M+Na]+.

149 149

18. Characterization of -sitosterol (339)

20 1 White crystals; mp 147-149C; []D -36 (c 0.1, MeOH); H NMR (400 MHz,

CDCl3) δH 5.35 (1H, d, J = 4.6 Hz, H-6), 3.51 (1H, m, H-3), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.84 (3H, t, J = 7.7 Hz, H-29), 0.83 (3H, d, J = 7.7 Hz, H-27), 0.81

13 (3H, d, J = 8.2 Hz, H-26), 0.68 (s, H-18); C NMR (100 MHz, CDCl3) δC 141.0 (C-5),

121.9 (C-6), 72.1 (C-3), 56.8 (C-14), 56.2 (C-17), 50.3 (C-9), 46.0 (C-24), 42.5 (C-13),

42.5 (C-4), 40.0 (C-12), 37.5 (C-1), 36.7 (C-10), 36.4 (C-20), 34.1 (C-22), 32.1 (C-7),

32.1 (C-8), 31.9 (C-2), 29.3 (C-25), 28.5 (C-16), 26.2 (C-23), 24.5 (C-15), 23.3 (C-28),

21.3 (C-11), 20.0 (C-19), 19.6 (C-27), 19.2 (C-26), 19.0 (C-21), 12.2 (C-18), 12.1 (C-29);

ESIMS m/z 437 [M+Na]+.

19. Biological activities of isolates from Vitex quinata

All the isolated compounds isolated from V. quinata were evaluated for their cytotoxicity against LNCaP hormone-dependent prostate cancer cells, Lu1 human lung cancer cells, and MCF-7 human breast cancer cells. Of these isolates, only

(S)-5-hydroxy-7,4-dimethoxyflavanone (380) was found to be an active principle, with

ED50 values of 6.7, 4.7, and 1.1 M, respectively, against LNCaP, Lu1, and MCF-7 cells.

Compounds 382 and 384-390 were tested in an enzyme-based ELISA NF-B p65 assay to evaluate their potential in inhibiting the binding of NF-B to DNA. Compounds

382, 384, 385, and 390 showed activity, with ED50 values of 22.8, 54.3, 17.8, 10.3 M, respectively.

150 150

D. Discussion

1. Structure elucidation of new compounds isolated from V. quinata

1.1. Structure elucidation of dimethyl 3,3,4,4-tetrahydroxy--truxinate (378)

Compound 378 (3.0 mg) was isolated from the ethyl acetate-soluble extract of Vitex quinata leaves as a light yellow gum. Its molecular formula, C20H20O8, was determined by the observation of a sodiated molecular ion peak at m/z 411.1061 (calcd for

C20H20O8Na, 411.1057) in the HRESIMS, with a calculated unsaturation number of 11.

However, its 13C NMR spectrum (Figure 3.10) showed only ten resonance signals, which suggested that the molecule is composed of two identical units. These carbon resonance signals were assignable to two such units, with each composed of an ester functional group [C 173.6 (C-9 and 9) and 52.3 (C-10 and 10)], an aromatic ring with substitution by two hydroxy groups [C 145.9 (C-3 and 3), 145.0 (C-4 and 4), 134.2 (C-4 and 4),

119.1 (C-6 and 6), 116.1 (C-5 and 5), 114.8 (C-2 and 2)], and two methine carbons [C

48.5 (C-7 and 7) and 45.6 (C-8 and 8)]. By deduction of the unsaturation number of 10 due to the aromatic rings and ester functionalities, it was evident that another ring is present in the molecule. Further analysis of the 1H NMR spectrum of 378 (Figure 3.11) suggested the molecule to be a modified lignan with two phenylpropenoid units coupled at the C-7 (C-7) and C-8 (C-8) positions, from the observation of an AABB system of methine signals at H 3.46 (2H, dd like, H-7 and H-7) and 3.30 ppm (2H, dd like, H-8 and H-8). Other resonance signals observed in the 1H NMR spectrum were assignable to two aromatic rings at δH 6.80 (2H, d, J = 2.1 Hz, H-2 and H-2), 6.77 (2H, dd, J = 2.1, 8.1

Hz, H-5 and H-5), and 6.65 (2H, dd, J = 2.1, 8.1 Hz, H-6 and H-6), as well as 151 151

carboxymethyl groups at H 3.68 (6H, s, H-10 and 10). The shape of the AABB system of methine signals at δH 3.46 and 3.30 ppm implied a chemically equivalent but magnetically non-equivalent environment for the cyclobutanoid proton set, and the relative configuration of the cyclobutyl ring was determined to be the same as that of -truxinic acid, based on a comparison of the 1H NMR chemical shifts of compound 1 with reported data of various truxillic and truxinic acid derivatives (Montaudo and Caccamese, 1973;

Ben-Efraim and Green, 1974). This conclusion was supported by a further comparison of the NMR data of compound 378 with those of -truxinic acid derivatives reported in recent years (Ito et al., 2003; Saito et al., 2004). The assignments of the 1H and 13C NMR signals of compound 378 were finalized with DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY experiments (Figures 3.12 to 3.16). Therefore, the structure of compound 378 was determined as dimethyl 3,3,4,4-tetrahydroxy--truxinate, which has not been reported in the literature previously.

1.2. Structure elucidation of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5-

methoxy-3-oxocyclopent-1-enyl}octanoate (379)

Compound 379 (6.0 mg) was obtained as a colorless amorphous powder and provided a sodiated molecular ion peak at m/z 375.2136 (calculated for C20H32O5Na,

375.2142) in the HRESIMS. An unusual double bond carbon signal observed in the

13 downfield region at δC 174.0 (C-13) of the C NMR spectrum (Figure 3.17), together with signals at δC 207.0 (C-12), 174.0 (C-9), 78.0 (C-10), and 40.5 (C-11), suggested the presence of a conjugated cyclopentenone structure, which was supported by comparison

152 152

15 3 C-6 and 6

C-5 and 5 C-1 and 1 C-8 and 8 C-4 and 4 C-2 and 2 C-7 and 7 C-3 and 3

C-9 and 9 Two methoxy carbons

13 Figure 3.10. C NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 (100 MHz) 153

H-6 and 6 H-2 and 2 Two methoxy H-7 and 7 H-8 and 8 H-5 and 5 group protons

Acetone

1 Figure 3.11. H NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 (400 MHz)

154

C-6 and 6 C-2 and 2 C-7 and 7

C-8 and 8 C-5 and 5

Two methoxy carbons

13 Figure 3.12. C DEPT NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 155

C-8 and 8 with H-2 and 2 C-7 and 7 with H-7 and 7

Methoxy carbons with methoxy protons

C-2 and 2 with H-2 and 2 C-6 and 6 with H-6 and 6

C-5 and 5 with H-5 and 5

Figure 3.13. HSQC NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 156

H-7 with H-8 and H-7 with H-8

H-5 with H-6 and H-5 with H-6

1 1 Figure 3.14. H- H COSY NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6 157

C-7 with H-6 and C-7 with H-6

C-7 with H-2 and C-7 with H-2

C-2 with H-5 and C-2 with H-5 C-5 with H-2 and C-5 with H-2 C-1 with H-5 and C-1 with H-5 C-4 with H-2 and C-4 with H-2 C-4 with H-5 and C-4 with H-5 C-1 with H-7 and C-1 with H-7 C-3 with H-2 and C-3 with H-2 C-9 with H-8 and C-9 with H-8 C-9 and 9 with methoxy protons

Figure 3.15. HMBC NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6

158

H-7 with H-8 and H-7 with H-8

Figure 3.16. NOESY NMR spectrum of 3,3,4,4-tetrahydroxy--truxinate (378) in acetone-d6

159

with NMR data of a similar ring system reported (Challener, 1993). The carbonyl carbon signal at C 174.1 (C-1) and seven methylene signals, at C 34.0 (C-2), 29.7 (C-6), 29.0

(C-4 and 5), 28.1 (C-7), 27.4 (C-8), and 24.9 (C-3), were assignable to a fatty acid chain moiety. Another chain containing a terminal methyl (C 17.6, C-18), a double bond (C

133.3 and 126.6, C-16 and C-17), an oxymethine (C 71.2, C-15), and a methylene (C

32.1, C-14) functionality was also inferred from the 13C NMR spectrum. The 1H NMR spectrum of 379 (Figure 3.18) showed signals at δH 4.46 (1H, br d, J = 5.2, Hz, H-10),

2.64 (1H, dd, J = 18.2, 5.8 Hz, H-11), and 2.32 (1H, dd, J = 18.2, 1.9 Hz, H-11), assignable to the above-mentioned cyclopentenone ring. Also, resonance signals belonging to the fatty acid chain moiety were observed in the range δH 1.34 to 2.33. The planar structure of another five-membered chain was determined by analyzing chemical shifts and coupling constants of the remaining signals in the 1H NMR spectrum of 379.

From the terminus of this chain, the connectivity was established in the following sequence: a methyl group (δH 1.66, 3H, d, J = 6.5 Hz, H-18), a trans-double bond [δH

5.65 (1H, ddq, J = 15.2, 6.4, 0.4 Hz, H-17) and 5.43 (1H, ddd, J = 15.2, 6.8, 1.5 Hz,

H-16)], an oxymethine functionality (δH 4.22, 1H, q like, J = 5.8 Hz, H-15), and a methylene group (δH 2.44, 2H, d, J = 6.3 Hz, H-14). The linkage of this short chain to the cyclopentanone ring at C-13 position was established by the correlation of H-14 to the

C-13 observed in the HMBC spectrum (Figure 3.19). DEPT and 2D NMR techniques were used to finalize the planar structure of 379 as depicted from Figures 3.20 to 3.23.

The absolute configuration at the C-10 position in this compound was assigned according to a circular dichroism (CD) spectroscopic measurement. Analysis of the conformation of the unsaturated cyclopentanone ring present in the molecule suggested

160160

that a pseudoaxial orientation of the C-10 methoxy group was favored over a pseudoequatorial orientation, because of reduced repulsive interactions with the H-11 protons, as depicted in Figure 3.24 (a and b) (Bifulco et al., 2007). Further investigation of the doublet 1H NMR resonance signal of H-10 revealed that this proton only coupled with one of the two H-11 protons, with a coupling constant of 5.2 Hz. This value is corresponds to a 30-40 dihedral angle between these two coupled protons, and an 80-90 dihedral angle between H-10 and the uncoupled H-11 proton, consistent with the aforementioned conformational analysis. Therefore, projection diagrams for the two possible configurations of C-10 can be drawn from the application of octant rule, as shown in Figure 3.24 (c and d). The CD spectrum of compound 379 (Figure 3.24, e) exhibited a strong negative Cotton effect around 315 nm, resulting from the n* transition in the cyclopentanone ketone double bond. In the case of unsaturated cyclopentanones, it has been suggested that an ―inverse‖ octant rule may be applied to determine absolute configuration (Lavie et al., 1971). Accordingly, the observed negative n* Cotton effect is related to a perturber located in the positive region in the projection diagram (Figure 3.24, c). Thus, the absolute configuration of C-10 could be determined as S, and the new compound 379 was identified as methyl 8-{(5R)-2-[(E)-

2-hydroxypent-3-enyl]-5-methoxy-3- oxocyclopent-1-enyl}octanoate. The absolute configuration of the C-15 hydroxy group remains unresolved.

2. Identification of known compounds

2.1. Identification of (S)-5-hydroxy-7,4'-dimethoxyflavanone (380)

Compound 380 was obtained as a colorless gum. The molecular formula was

1 determined as C17H16O5 by a combined analysis of its NMR and MS data. Its H NMR

161161

spectrum showed typical flavanone ring-C signals at H 5.29 (1H, dd, J = 12.6, 2.8 Hz,

H-2), 2.96 (1H, dd, J = 16.7, 12.6 Hz, H-3b), and 2.62 (1H, dd, J = 16.7, 2.8 Hz, H-3a).

Two doublet peaks resonating at H 5.98 and 5.92 with a meta-coupling constant value of

2.1 Hz could be assigned to H-8 and H-6 of ring A, respectively. Two peaks present in the downfield region at H 7.39 (2H, d, J = 8.6 Hz, H-2', H-6') and 6.92 (2H, d, J = 8.6 Hz,

H-3', H-5') showed an ortho coupling constant of 8.6 Hz, indicating the protons to be located in the 4-substituted benzyl ring B. The positions of two methoxy groups were determined by the observation of correlations of H 3.80 (6H, s, OMe-7 and 4') with C

165.8 (C-7) and 160.3 (C-4) in the HMBC spectrum. The NMR data and optical rotation measurement of compound 380 were comparable with reported values for this known compound (Kamperdick, 2002; Li et al., 2005). Therefore, the structure of compound 380 was established as (S)-5-hydroxy-7,4'-dimethoxyflavanone.

2.2. Identification of (S)-isosakuranetin (381)

Compound 381 was obtained as a colorless gum. The molecular formula was

1 determined as C16H14O5 by a combined analysis of its NMR and MS data. Its H NMR spectrum was similar to that of (S)-5-hydroxy-7,4'-dimethoxyflavanone (380) except for the absence of one methoxy group. Its 1H NMR spectrum also displayed typical flavanone ring C signals at H 5.34 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.09 (1H, dd, J = 17.1,

13.0 Hz, H-3b) and 2.77 (1H, dd, J = 17.1, 3.0 Hz, H-3a). Two doublet peaks present at

H 6.02 (1H, d, J = 2.0 Hz, H-8) and 5.96 (1H, d, J = 2.0 Hz, H-6) were assignable to ring

A protons. Aromatic proton resonance signals at H 7.36 (2H, d, J = 8.8 Hz, H-2', H-6') and 6.95 (2H, d, J = 8.8 Hz, H-3', H-5') were attributed to protons in ring B.

162162

 31.0: acetone  29.7: C-6  29.0: C-4  29.0: C-5  28.1: C-7  27.4: C-8

C-2

16 C-3

C-1 C-14 C-17 C-15 C-10 C-18 C-16 C-2 C-12 C-11 and acetone C-1 C-9 C-13

Figure 3.17. 13C NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5-methoxy- 3-oxocyclopent-1-enyl} octanoate (379) in CDCl3 (100 MHz) 163

5.690 5.674 5.673 5.656 5.636 5.635 5.620 5.619 5.604 5.465 5.461 5.448 5.444 5.427 5.423 5.410 5.406 4.465 4.452 4.243 4.228 4.214 4.199 3.671 3.381 2.698 2.683 2.652 2.638 2.464 2.454 2.438 2.348 2.344 2.332 2.314 2.303 2.295 2.176 1.669 1.652 1.346 1.337

H-1 acetone H-2

H-11

H-17 H-16 H-10 H-15 H-18

1.00 0.98 1.00 0.99 3.20 3.11 1.10 4.23 3.40 5.47 9.18 7.28

Figure 3.18. 1H NMR spectrum6.0 of methyl 85.-0{(5R)-2-[(E)-2-hydroxypent4.0 -3-enyl]-35.0-methoxy-3-oxocyclopent2.0 - 1.0 ppm (t1) 1-enyl} octanoate (379) in CDCl3 (400 MHz) 164

C-18 and H-17 C-18 and H-16

C-2 and H-10

C-15 and H-17

5 C-15 and H-16

C-10 and H-2

C-13 and H-15 C-17 and H-16 C-17 and H-18 C-16 and H-18 C-13 and H-10 C-13 and H-11 C-1 and H-1

C-9 and H-10 C-9 and H-11

C-12 and H-11

Figure 3.19. HMBC NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl} octanoate (379) in CDCl3 165

C-10 C-15 C-1‘ C-16 C-2‘ C-17 C-18 Acetone

C-11 C-3 C-2

C-8 C-14 C-7 C-4 and 5 C-6

Figure 3.20. 13C DEPT135 NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5-methoxy-3- oxocyclopent-1-enyl} octanoate (379) in CDCl3 166

C-18 and H-18 C-7 and H-7 C-3 and H-3 C-14 and H-14 C-8 and H-8 C-2 and H-2 C-4 and H-4 C-11 and H-11 C-5 and H-5 C-2 and H-2 C-6 and H-6

C-1 and H-1

C-2 and H-2

C-15 and H-15

C-17 and H-17

C-16 and H-16

Figure 3.21. HMQC NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]- 5-methoxy-3-oxocyclopent-1-enyl} octanoate (379) in CDCl3 167

H-17 and H-18

H-14 and H-15

H-10 and H-11

H-16 and H-17

Figure 3.22. 1H-1H COSY NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl} octanoate (379) in CDCl3

168

9 H-10 and H-11

H-10 and H-2

H-16 and H-17

Figure 3.23. NOESY NMR spectrum of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3-enyl]-5- methoxy-3-oxocyclopent-1-enyl} octanoate (379) in CDCl3

169

  

H O H OCH3    12 H 9 13 13 11 9 12 H OCH H O 3 H (a) (b)

    13 12 11 OCH3 O 10 9 9 10 O 13 12 11 OCH3  +  + (c) (d)

-3 20

] x 10 x ]



[ 10

0 200 220 240 260 280 300 320 340 360 380 400 -10

-20 Wavelength (nm) (e) Figure 3.24. Conformation analysis (a and b), octant rule analysis (c and d), and circular dichroism (CD) spectrum (e) of methyl 8-{(5R)-2-[(E)-2-hydroxypent-3- enyl]-5- methoxy-3-oxocyclopent-1-enyl} octanoate (379)

170170

The correlation of H 3.81(3H, s, OMe-4') with C 160.2 (C-4) observed in the HMBC spectrum suggested the only methoxy group present to be substituted at the C-4 position.

A full assignment of the signals of the 13C NMR spectrum was achieved using 2D NMR experiments. The optical rotation of 381 was comparable with that reported for

(S)-isosakuranetin (Wagner et al., 1976). Thus, the structure of compound 381 was established as (S)-5,7-dihydroxy-4'-methoxyflavanone [(S)-isosakuranetin].

2.3. Identification of 2-hydroxy-4,4,6-trimethoxychalcone (382)

Compound 382 exhibited a sodiated molecular ion in its ESIMS at m/z 337,

1 consistent with an elemental formula of C18H18O5. In the H NMR spectrum of compound

382, matching doublets (J = 16.2 Hz) at H 7.77 (H-7) and 7.76 (H-8) suggested that these protons were related to one another in a trans fashion. Two doublet peaks

integrating as two protons each, resonating at H 7.54 (J = 9.0 Hz, H-2 and -6) and 6.90 (J

= 9.0 Hz, H-3 and -5), were assigned as ortho-coupled protons of a 1,4-disubstituted

phenyl ring. Two further doublet signals observed at H 6.07 (1H, d, J = 2.4 Hz, H-8) and

5.92 (1H, d, J = 2.4 Hz, H-6) were assigned as meta-coupled aromatic protons. Three

13 methoxy singlet peaks occurred at H 3.88, 3.82, and 3.80. The C NMR spectrum exhibited 16 signals, of which two resonances at C 130.2 (C-2 and 6) and 114.6 (C-3 and

5), together with those at C 162.8 (C-4) and 128.5 (C-1), were assignable to the aforementioned 1,4-disubstituted phenyl ring. The remaining 13C NMR signals could be attributed to an ,-unsaturated ketone moiety, a phenyl group, and three methoxy

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functionalities. The presence of an ,-unsaturated ketone in a trans double bond, together with substitutions of two aromatic rings is characteristic for the structure of a chalcone. The NMR spectroscopic and physical data obtained for compound 382 were in close agreement with literature values for 2-hydroxy-4,4,6-trimethoxychalcone (Aponte et al., 2008). Confirmation of this identification was carried out via analysis of the 2D

NMR spectra for compound 382.

2.4. Identification of 2',6'-dihydroxy-4,4'-dimethoxychalcone (383)

The ESIMS of compound 383 showed a sodiated molecular ion peak at m/z 323, in

agreement with a sodiated elemental formula of C16H12O6Na. The chemical shifts of its

1H and 13C NMR spectra were similar to those of compound 382 except for the replacement of one methoxy group with a hydroxy functionality. The HMBC NMR

spectrum showed that the signal at H 3.87 (3H, s, OMe-7) was correlated to the resonance at C 166.2 (C-7), so the only methoxy group could be located at the C-7 position, and the C-3, 5, and 4 positions were substituted therefore by free hydroxy groups. Therefore, the identity of compound 383 was determined as

2',6'-dihydroxy-4,4'-dimethoxychalcone (383), by comparison with literature data for this compound (Zhang et al., 2006; Tu et al., 2007).

2.5. Identification of 3,5-dihydroxy-7,4-dimethoxyflavonone (384)

A sodiated molecular ion at m/z 337 was observed in the ESIMS of compound 384,

1 and was consistent with a molecular formula of C17H14O6. In the H NMR spectrum of 172172

this compound, four protons of a 1,4-distustitued phenyl ring were found to resonate at H

8.14 (2H, d, J = 9.0 Hz, H-2, H-6) and 7.00 (2H, d, J = 9.0 Hz, H-3, 5), with two meta-coupled protons belonging to another benzyl ring resonating at δH 6.46 (1H, d, J =

2.1 Hz, H-8) and 6.35 (1H, d, J =2.1 Hz, H-6). Furthermore, a typical C-5 hydroxy proton

of a flavonoid occurred at H 11.70 (1H, s, OH-5) and C-3 hydroxy proton of hydroxyflavonoid at 6.58 (1H, s, OH-3) and together implied that compound 384 is a hydroxyflavonone, with two methoxy groups ( 3.87 6H, s, OMe) substituted at the C-7 and C-4 positions. Its 13C NMR spectrum displayed characteristic 5,7-substituted ring A

signals at C 165.7 (C-7), 160.8 (C-5), 156.8 (C-9), 103.9 (C-10), 97.9 (C-6), and 92.2

(C-8), and ring B signals at C 161.1 (C-4), 128.8 (C-2, 6), 123.1 (C-1), and 114.0 (C-3,

5). The signals present at C 175.1 (C-4), 145.7 (C-2), and 135.6 (C-3) are typical chemical shifts of a hydroxyflavonone. The identification as 3,5-dihydroxy-7,4- dimethoxyflavonone for 384 was supported by spectroscopic data comparison with published values (Rossi et al., 1997).

2.6. Identification of rhamnocitrin (385)

The ESIMS of compound 385 showed a sodiated molecular ion peak at m/z 323,

1 13 corresponding to an elemental formula of C16H12O6. Its H and C NMR spectra were similar to those of compound 384 except that one of the methoxy groups was replaced by

a hydroxy functionality. The HMBC spectrum showed that the signal resonating at H

3.87 (3H, s, OMe-7) was correlated to the resonance at C 166.2 (C-7), implying the only

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methoxy group to be located at the C-7 position, with the C-3, 5, and 4 positions substituted with free hydroxy groups. Therefore, the structure of compound 385 was determined as 3,5,4-trihydroxy-7-methoxyhydroxyflavonone, which is in agreement with reported NMR data of rhamnocitrin (Zhang et al., 2006; Tu et al., 2007).

2.7. Identification of (-)-loliolide (386)

20 Compound 386 was isolated as white needle crystals (mp 151-153C; [α]D -80).

The molecular formula was assigned as C11H16O2 by the observation of a molecular ion peak at m/z 219 ([M+Na]+) in the ESIMS and 11 carbon signals in the 13C NMR spectrum, indicating four degrees of unsaturation for the molecule. Its 1H NMR spectrum displayed

a singlet double bond proton signal at H 5.70 assignable to H-7, an oxymethine proton

peak at H 4.34 and a hydroxy proton peak at H 1.62 assignable to H-3 and OH-3, as well

as signals of three methyl groups at H 1.79 (3H, s, Me-5), 1.48 (3H, s, α-Me-1), and 1.28

(3H, s, β-Me-3). The signals at C 182.6 (C-8), 172.3 (C-6), 112.7 (C-7), 87.4 (C-5) in the

13C NMR spectrum could be assigned to an ,-unsaturated lactone ring, which

accounted for three degrees of unsaturation. Carbon signals resonating at C 66.8 (C-3),

45.5 (C-2), 46.2 (C-4), and 35.6 (C-1) were the components of another ring in the molecule according to the one degree of unsaturation remaining. The positions of the

methyl groups were determined by the HMBC correlations of H 1.79 (s, 3H, Me at C-5)

with C 87.4 (C-5), as well as H 1.48 (3H, s, α-Me-1) and 1.28 (3H, s, β-Me-1) with C

35.6 (C-1). The identification of compound 386 was finalized with 2D NMR experiments,

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and the 1H and 13C NMR data were in agreement with published data of the known compound, (-)-loliolide (Valdes, 1986; Chavez et al., 1997).

2.8. Identification of methyl 3,4-O-dicaffeoyl quinate (387)

Compound 387 was obtained as light yellow gum. Its ESIMS gave a protonated

molecular ion peak at m/z 531, in agreement with an elemental formula of C26H26O12. The

1 H NMR spectrum exhibited signals of two trans-caffeoyl groups at H each, d, J = 16.0 Hz), 7.04/7.04 (1H each, d, J = 2.5 Hz), 6.99/6.99 (1H each, d, J = 8.0

Hz), 6.77/ 6.77 (1H each, d, J = 8.0 Hz), and 6.23/6.14 (1H each, d, J = 16.0 Hz), as well

as three oxymethine protons at H 5.38 (1H, br dd, J = 9.5, 4.5 Hz), 5.29 (1H, dt, J = 7.0,

J = 9.5, 3.0 Hz). Signals for the two caffeoyl groups were also

13 found in its C NMR spectrum in the range C 166.6 to 114.5, together with two

methylene carbons at C 37.8 (C-2) and 36.8 (C-6), four oxygenated carbons at δC 73.2

(C-1), 67.9 (C-3), 72.4 (C-4), and 65.5 (C-5), a carbonyl carbon signal at C 173.5 (C-7),

1 13 as well as a methoxy carbon at C 52.1. The H and C NMR chemical shifts were found to be typical for a dicaffeoyl quinic acid derivative (Clifford et al., 1986; Pauli et al.,

1998). The positions of the two caffeoyl groups were established by the correlations of H

5.32 (1H, m, H-3) with C 166.6 (C-9) and H 5.02 (1H, m, H-4) with C 165.3 (C-9) in the HMBC spectrum. Thus, the structure of compound 387 was determined as 3,4-O- dicaffeoyl quinic acid methyl ester. The physical and spectroscopic data of compound 387 were in agreement with those reported in the literature for 3,4-O-dicaffeoyl quinic acid

175175

methyl ester (Ma et al., 2009).

2.9. Identification of methyl 3,5-O-dicaffeoyl quinate (388)

Compound 388 was isolated as a light yellow gum. Its ESIMS gave a protonated

molecular ion peak at m/z 531, suggesting a molecular formula of C26H26O12, the same as that of compound 387. Its 1H and 13C NMR spectrum showed many similarities to those of 3,4-O-dicaffeoyl quinic acid methyl ester (Ma et al., 2009). This information implied that compound 388 is a regioisomer of 3,4-O-dicaffeoyl quinate (387), with the substitution of caffeoyl units at different positions. Analysis of the 2D NMR spectra of compound 388 suggested that the two caffeoyl functionalities are connected to the C-3 and C-5 postions. Therefore, the structure of compound 388 was identified as methyl

3,5-O-dicaffeoyl quinate. The assignments made for its 1H and 13C NMR spectroscopic data, and the other physical and spectroscopic data measured for this compound, were in agreement with previously reported data for this substance (Choi et al., 2004).

2.10. Identification of methyl 4,5-O-dicaffeoyl quinate (389)

Compound 389 was isolated as light yellow gum. Its ESIMS gave a protonated

molecular ion peak at m/z 531, suggesting a molecular formula of C26H26O12, the same as those of compounds 387 and 388. Its 1H and 13C NMR spectra showed close crorrelations to analogous data for 3,4-O-dicaffeoyl quinic acid methyl ester. This information implied that compound 389 is an isomer of 3,4-O-dicaffeoyl quinate (387) and 3,5-O-dicaffeoyl

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quinate (388), with the differential substitution of the caffeoyl units present. Analysis of the 2D NMR spectra of compound 390 suggested that the two caffeoyl functionalities are connected to the C-4 and -5 postions. Therefore, the structure of compound 389 was determined as methyl 3,5-O-dicaffeoyl quinate. The assignments of its 1H and 13C NMR signals were in agreement with previously reported data for this compound (Jiang et al.,

2002).

2.11. Identification of methyl 3,4,5-O-tricaffeoyl quinate (390)

Compound 390 was obtained as a colorless gum. The molecular formula was

1 determined as C17H16O5 by combined analysis of its NMR and MS data. Its H NMR

spectrum showed signals for three caffeoyl moieties. Six doublets at H 7.64 (1H, H-7),

7.55 (1H, H-7), 7.55 (1H, H-7), 6.42 (1H, H-8), 6.42 (1H, H-8), and 6.16 (1H,

H-8), each with a coupling constant of 15.9 Hz appeared for the trans olefinic protons of the caffeoyl units. Three sets of aromatic protons in an ABX coupling pattern around

H 7.0 (d, J = 2.1 Hz, H-2, 2, 2), 6.7 (d, J = 8.2 Hz, H-5, 5, 5), and 6.9 (dd, J = 8.2,

2.1 Hz, H-6, 6, 6), were assignable to the phenyl rings of the caffeoyl moieties. The remaining signals of four methines, three oxymethines, and one methoxy group were comparable to those found for compounds 387-389. Therefore, compound 390 could be deduced as a quinic acid methyl ester substituted with three caffeoyl units. The positions of caffeoyl substitution on the quinic acid ring were determined from the correlations between H-3, -4, -5 with the caffeoyl carbonyl carbons observed in the HMBC spectrum.

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The full assignment of all signals in 1H and 13C NMR spectra was performed by additional 2D NMR experiments. Thus, the structure of compound 390 was elucidated as methyl 3,4,5-O-tricaffeoyl quinate, and comparable physical and spectroscopic data to published values were obtained for this compound (Merfort et al., 1992).

2.12. Identification of -sitosterol (339)

Compound 342 showed a molecular ion at m/z 414 in its EIMS, consistent with an

1 elemental formula of C29H50O, indicating five degrees of unsaturation. Its H NMR

spectrum exhibited a double bond proton signal at H 5.35 (1H, d, J = 4.6 Hz, H-6), an

oxymethine proton peak at H 3.51 (1H, m, H-3), as well as two singlets, three doublets,

13 13 and a triplet methyl signal in the range H 1.01 to 0.68. The C NMR and C DEPT

spectra revealed a double bond resonating at C 141.0 (C-5) and 121.9 (C-6), an

oxymethine at C 56.8 (C-14), as well as seven methine, eleven methylene, six methyl,

and two quaternary carbon signals between C 57.2 and 12.1. This information suggested the compound to be a plant sterol with four rings occurring in the molecule. A comparison of the physical and spectroscopic data obtained from compound 342 with literature values

(Kovganko et al., 1999), established the identity of the isolate as -sitosterol.

Conclusions

An initial bioassay showed that the chloroform soluble extract of a small sample of

Vitex quinata was active against against MCF-7 human breast cancer cells, LNCaP

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hormone-dependent prostate cancer cells, and Lu1 human lung cancer cells, with ED50 values of 3.1, 3.1 and 2.5 g/mL. A phytochemical investigation of the chloroform- soluble and ethyl acetate-soluble extracts of the leaves of V. quinata led to the isolation of a new -truxinate derivative (379) and a new prostaglandin-like octadecanoid derivative

(380), together with 12 known compounds. The structures of these compounds were determined, by spectrometric and spectroscopic methods, as methyl 8-{(5R)-2-[(E)-2- hydroxypent-3-enyl]-5-methoxy-3-oxocyclopent-1-enyl}octanoate (379), (S)-5-hydroxy-

7,4'-dimethoxyflavanone (380), (S)-isosakuranetin (381), 2-hydroxy-4,4,6-trimethoxychalcone (382), 2',6'-dihydroxy-4,4'-dimethoxychalcone (383), 3,5-dihydroxy-7,4- dimethoxy-hydroxy-flavonone (384), rhamnocitrin (385), (-)-loliolide (386), methyl

3,4-O-dicaffeoyl quinate (387), methyl 3,5-O-dicaffeoyl quinate (388), methyl 4,5-O- dicaffeoyl quinate (389), methyl 3,4,5-O-tricaffeoyl quinate (390), and -sitosterol (339).

All the isolated compounds isolated from V. quinata were evaluated for their cytotoxicity against LNCaP, Lu1, and MCF-7 cancer cells. Of these isolates, only

(S)-5-hydroxy-7,4-dimethoxyflavanone (380) was found to be an active principle, with

ED50 values of 6.7, 4.7, and 1.1 M, respectively, against LNCaP, Lu1, and MCF-7 cells.

Compounds 382, and 384-390 were tested in an enzyme-based ELISA NF-B p65 assay to evaluate their potential in inhibiting the binding of NF-B to DNA. Compounds

382, 384, 385, and 390 showed activity, with ED50 values of 22.8, 54.3, 17.8, 10.3 M, respectively.

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Appendix A

Lu1 LNCaP MCF-7 HUVEC Methanol extract >20 >20 >20 >20 Hexane-soluble extract >20 >20 >20 >20 Chloroform-soluble extract >20 8.9 11.7 14.7 F01 >20 F02 >20 F03 >20 F04 >20 F05 >20 F06 >20 F07 9.8 F08 6.0 F09 10.1 F10 8.7 F11 11.3 F12 >20 F13 >20 F14 >20 F15 >20

Table A.1. Initial cytotoxicity assay results (ED50 values, μg/mL) of Hyptis brevipes extracts and fractions; Lu1: human lung cancer cells; LNCaP: human prostate adenocarcinoma cells; MCF-7: human breast cancer cells; HUVEC: human umbilical vein endothelial cells. Active extracts and fractions are those with ED50  20 μg/mL.

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1. Cytotoxicity assay

Initial cytotoxicity assays on extracts and chromatographic fractions were tested against Lu1 human lung cancer cells, LNCaP human prostate adenocarcinoma cells

MCF-7 human breast cancer cells and HUVEC human umbilical vein endothelial cells.

The isolated compounds from Hyptis brevipes were tested against MCF-7 human breast cancer cells, HT-29 human colon cancer cells, and Lu1 human lung cancer cells, using an established protocol (Seo et al., 2001). These assays were based on an in vitro anticancer drug screening method developed by the National Cancer Institute (NCI) (Skehan et al.,

1990). The method determines cytotoxicity by measuring the cellular protein content of adherent cell cultures in 96-well microtiter plates. Cells were first fixed with trichloroacetic acid (TCA) and then stained with 0.4% SRB, and protein-bound dye extracted with 10 mM unbuffered tris base [tris(hydroxymethyl)aminomethane] for the determination of optical obsorption at 515 nm with a 96-well Bio-Tek uQuant® microtiter plate reader (Skehan et al., 1990).

The calibrated absorption values were obtained by subtracting zero day control data from the averaged readout absorption values. These final values are then expressed as a

percentage, relative to the solvent-treated control incubation, and ED50 values are calculated using nonlinear regression analysis (percent survival versus concentration).

Extracts and fractions showing an ED50 value smaller than 20 g/mL and pure isolates less than 5 g/mL are considered active. Paclitaxel was used as positive control in 211211

all the cytotoxicity assays, with ED50 values of 1.0, 5.0, 2.0, and 0.4 ng/mL against HT-29,

LNCaP, Lu1, and MCF-7 cells, respectively.

2. Proteasome fraction preparation and proteasome inhibition assay

A proteasome fraction was prepared from a HL-60 human keukemic cell line as described previously (Vinitsky et al., 1997; Su et al., 2004). The assay buffer (50 mM

Tris, pH 7.5, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.03% SDS) was added to blank and control wells. Dilutions of the tested compounds were prepared in assay buffer and added to the appropriate wells. The enriched proteasome fraction was diluted to a final assay concentration of 4 g/mL using assay buffer, and this dilution was then added to each well. The microtiter plates were then pre-incubated for 10 min at 37 C to facilitate inhibitor-enzyme interactions. The enzyme reaction was initiated by adding substrate

[N-succinyl-Leu-Leu-Val-Tyr-AMC (7-amino-4-methylcoumarin; abbreviated

Suc-LLVY-AMC)] to a final concentration of 75 M and fluorescence measurements commenced immediately. The chymotrypsin-like proteasome activity was determined by measuring the generation of free AMC using and excitation at a wavelength of 360 nm and detection of emitted light at 460 nm. The Suc-LLVY-AMC substrate was obtained from Biomol International (Plymouth Meeting, PA). The proteasome inhibitor bortezomib

was used as positive control with an ED50 value of 2.5 nM.

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3. Enzyme-based ELISA NF-B assay

The NF-B assay was carried out according to an established protocol (Renard et al.,

2001; Salim et al., 2007). A nuclear extract was prepared from Hela cells purchased from the American Type Culture Collection. An EZ-DetectTM Transcription Factor Assay

System ELISA kit (Pierce Biotechnology, Rockford, IL) was used to assess the specific binding ability of activated NF-B to the biotinylated-consensus sequence under the presence of test compounds. The activity of the p50 and p65 subunits (the latter was used only in Chapter 3 of this dissertation) of NF-B was measured by detecting the chemiluminescent signal in a Fluostar Optima plate reader (BMG Labtech Inc., Durham,

NC). Rocaglamide was used as a positive control with an ED50 values of 4.0 and 0.075

M in the NF-κB p50 and p65 assays, respectively.

4. Mitochondrial transmembrane potential assay (MTP Assay)

Changes on the mitochondrial transmembrane potential were detected and quantified by a fluorescence cell-based assay. In brief, HT-29 cells cultured in black 96-well plates or black clear bottom 96-well plates at a density of 6  104 were incubated overnight at 37 oC

in a CO2 incubator. Cells were then treated with the test compounds or staurosporine

(positive control, ED50 2.6 nM) for 2 h. Immediately afterwards, cells were incubated with the lipophilic cationic dye 5,5,6,6-tetrachloro-1,1,3,3- tetraethylbenzymidazolyl- carbocyanide (JC-1) (Cayman Chemical Company, Ann Arbor, MI) for 30 min. After incubation, cells were washed with a wash buffer to remove unbound staining reagent. The

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clear bottom plates were then scanned with fluorescent imaging microscope (Axiovert 40

CFL, Carl Zeiss Microimaging, Inc., Thornwood, NY). The 96-well plates were analyzed by a FLUOstar Optima fluorescence plate reader (BMG Labtech, Inc.) with an excitation wavelength of 485 nm and emission wavelength of 530 nm for JC-1 monomers and an excitation wavelength of 560 nm and emission wavelength of 595 nm for J-aggregates.

Measurements were performed in duplicate and are representative of at least two independent experiments (Salvioli et al., 1997).

5. Characterization of ursolic acid (56)

20 1 White powder; []D +6.8 (c 0.2; CHCl3); H NMR (400 MHz, pyridine-d5)  5.48

(1H, br s, H-12), 3.42 (1H, dd, J = 11.5, 9.3, H-3), 2.70 (1H, d, J = 13.6 Hz, H-18), 1.24

(3H, s, H-23), 1.24 (3H, s, H-27), 1.07 (3H, s, H-26), 1.02 (3H, s, H-24), 1.01 (3H, s,

13 H-29), 0.98 (3H, s, H-30), 0.92 (3H, s, H-25); C NMR (100 MHz, pyridine-d5)  179.8

(C-28), 139.4 (C-13), 125.8 (C-12), 68.3 (C-3), 55.9 (C-5), 53.7 (C-18), 48.1 (C-9), 48.1

(C-17), 42.7 (C-14), 40.3 (C-8), 39.6 (C-4), 39.5 (C-19), 39.4 (C-20), 39.3 (C-1), 37.7

(C-22), 37.6 (C-10), 33.8 (C-7), 31.1 (C-21), 28.8 (C-15), 28.8 (C-23), 28.2 (C-2), 25.0

(C-16), 24.0 (C-27), 23.7 (C-11), 21.5 (C-30), 18.9 (C-6), 17.6 (C-29), 17.5 (C-26), 16.6

(C-24), 15.8 (C-25); ESIMS m/z 480 [M+Na]+.

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6. Characterization of maslinic acid (2-hydroxyursolic acid, 320)

20 1 White powder; []D + 10.7 (c 0.2; CHCl3); H NMR (300 MHz, pyridine-d5)  5.46

(1H, t, J = 3.5 Hz, H-12), 4.09 (1H, ddd, J = 11.2, 9.6, 4.4 Hz, H-2), 3.39 (1H, d, J = 9.6

Hz, H-3), 3.29 (1H, dd, J = 13.5, 3.8 Hz, H-18), 1.28 (3H, s, H-23), 1.25 (3H, s, H-27),

1.08 (3H, s, H-26), 1.02 (3H, s, H-24), 0.99 (3H, s, H-29), 0.98 (3H, s, H-30), 0.93 (3H, s,

13 H-25); C NMR (75 MHz, pyridine-d5)  180.2 (C-28), 144.9 (C-13), 122.5 (C-12), 83.8

(C-3), 68.6 (C-2), 55.9 (C-5), 48.2 (C-8), 48.2 (C-9), 47.8 (C-1), 46.7 (C-17), 46.4 (C-18),

42.2 (C-14), 42.0 (C-19), 39.8 (C-4), 38.5 (C-10), 34.2 (C-21), 33.2 (C-6), 33.2 (C-22),

33.2 (C-29), 30.9 (C-20), 29.3 (C-23), 28.3 (C-15), 26.2 (C-26), 23.9 (C-11), 23.7 (C-30),

23.6 (C-16), 18.9 (C-6), 17.7 (C-26), 17.5 (C-25), 16.9 (C-23); ESIMS m/z 495 [M+Na]+.

7. Characterization of daucosterol (-sitosterol-3-β-D-glycoside, 340)

20 1 White powder; []D -21 (c 0.2; MeOH); H NMR (400 MHz, pyridine-d5)  5.33

(1H, t, J = 3.0 Hz, H-6), 5.02 (1H, d, J = 7.8 Hz, H-1), 4.52 (1H, dd, J = 12.0, 2.5 Hz,

H-6a), 4.38 (1H, dd, J = 12.0, 4.9 Hz, H-6b), 4.27 (1H, t, J = 7.6 Hz, H-2), 4.27 (1H, t,

J = 7.6 Hz, H-3), 4.02 (1H, dd, J = 7.8, 7.6 Hz, H-2), 3.94 ((1H, m, H-5), 3.95 (1H, m,

H-3), 0.96 (3H, d, J = 6.6 Hz, H-21), 0.92 (3H, s, H-19), 0.86 (3H, d, J = 7.6 Hz, H-29),

0.84 (3H, d, J = 7.1 Hz, H-27), 0.82 (3H, d, J = 7.1 Hz, H-26), 0.65 (3H, s, H-18); 13C

NMR (100 MHz, pyridine-d5)  141.3 (C-5), 122.2 (C-6), 102.8 (C-1), 78.7 (C-3), 78.6

(C-3), 78.3 (C-5), 75.5 (C-2), 71.9 (C-4), 62.9 (C-6), 56.9 (C-14), 56.4 (C-17), 50.4

(C-9), 46.1 (C-24), 42.7 (C-13), 40.1 (C-12), 39.4 (C-4), 37.6 (C-1), 36.5 (C-20), 36.2

215215

(C-10), 34.3 (C-22), 32.2 (C-7), 32.2 (C-8), 30.3 (C-2), 29.76 (C-25), 28.6 (C-16), 26.5

(C-23), 24.6 (C-15), 23.6 (C-28), 21.5 (C-11), 20.1 (C-27), 19.6 (C-19), 19.3 (C-26), 19.1

(C-21), 12.3 (C-29), 12.1 (C-18); ESIMS m/z 599 [M+Na]+.

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Appendix B

1. Initial cytotoxicity assays of Vitex quinata extracts and fractions

Extracts and fractions of V. quinata were subjected to initial cytotoxicity assays for the purpose of bioactivity-guided isolation. The results are shown in the following Table

B.1.

Extracts and fractions MCF-7 Lu1 LNCaP Hexane-soluble Extract (D1) >20 >20 >20 Chloroform-soluble Extract (D2) 3.1 2.5 3.1 EtOAc-soluble Extract (D3) >20 >20 >20

H2O Layer (D4) >20 >20 >20 D2F1 4.5 D2F2 4.1 D2F3 2.1 D2F4 2.1 D2F5 0.6 D2F6 4.1 D2F7 2.1 D2F8 10.9 D2F9 15.7 Table B.1. Cytotoxicity assay results of extracts and fractions prepared from V. quinata

(ED50 values, μg/mL); MCF-7: human breast cancer cells; Lu1: human lung cancer cells; LNCaP: human prostate adenocarcinoma cells. Active extracts and fractions are those with ED50  20 μg/mL.

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