Drug lead discovery through plant bioprospecting in Latin America

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Authors Khera, Smriti

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Link to Item http://hdl.handle.net/10150/282898 DRUG LEAD DISCOVERY THROUGH PLANT BIOPROSPECTING IN LATIN AMERICA

by

Smriti Khera

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF PHARMACEUTICAL SCIENCES

In Partial Fulfillment of the Requirements For the degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2005 UMI Number: 3177540

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As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Smritl Khera entitled Drug Lead Discovery through Plant Bloprospecting

in Latin America

and recommend that it be accepted as fulfilling the dissertation requirement for the

Degree of Doctor of Philosophy

Karl H.Schram, Ph.D. date

'A 11' s. Moore, Ph.D.

ie Gunatilaka, Ph.D. date

Robert B. Bates, Ph.D. f ( I I ^ Barbara N. Timmermann, Ph.D. date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this disseitation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Q I | j ^ |J. 00-^ Dissertation Director; Barbara N.Timmermann, Ph.D. date 3

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SIGNED: 4

ACKNOWLEDGEMENTS

I would like to thank my dissertation advisor Dr. Barbara Timmermann for her support and encouragement throughout the course of this research. I thank her for always standing by my side through my accomplishments and failures alike. She believed in me even at times when I was unsure of myself. She has been an enormous source of strength, encouragement, scientific insight, and emotional support for me throughout the duration of this research.

I would like to thank Dr. Brad Moore for his scientific vigilance and commitment to my success. His suggestions, advice and help have been invaluable to this research. I would also like to thank Dr. Moore for being extremely accessible and approachable even on matters unrelated to research. Discussions with him have been invaluable in terms of career planning and personal growth.

I would also like to thank Drs. Karl Schram, Leslie Gunatilaka, and Robert Bates for their generous help, advice and consultations that have been critical to this work. I thank them for being extremely accessible and for sharing their expertise for the benefit of this research.

I would like to thank Dr. Neil Jacobsen and Dr. Michael Carducci for teaching me everything I know about NMR spectroscopy and X-ray crystallography respectively. Over the years I have spent and enormous amount of time with them and without their support, advice and training this research would not have been possible. I would also like to thank Dr. Arpad Samyogyi for extremely helpful consultations about mass spectrometry and for being an excellent resource throughout this study.

I would also like to thank past and present members of Dr. Timmermann's laboratory, particulary, Drs. Girma Woldemichael, Shivanand Jolad, Jianqiao Gu, Aniko Solyom, along with Veronica Rodriguez and Katy Prudic for their training, help and guidance.

At a personal level, I would also like to acknowledge my grandparents- Vidya Rani and Thakar Das Kalra, late Rattan Kumari and late Tilak Raj Khera for their vision and values that have helped shape my life. Their support and encouragement was invaluable throughout my graduate career. I would also like to thank Abhishek Gupta, who has been a home away from home for me for the past several years. His support and friendship have been very critical to the successful completion of this work.

Finally, I would like to thank my wonderful parents Manmohan Krishna and Manju Khera for supporting me every step of the way through graduate school. If not for their encouragement, and love it would not have been possible to pursue my education so far away from home and accomplish my goals. DEDICATION

To my mom, Manju, for her many sacrifices in raising me, and her tireless strength, love, patience and wisdom that has always guided me through the most challenging of times.

And to my papa, Manmohan Krishna, for his belief in me that has always bolstered my own belief in myself. 6

TABLE OF CONTENTS

Page

LIST OF FIGURES 12

LIST OF TABLES 18

ABSTRACT 20

CHAPTER I - INTRODUCTION 22

1.1 Natural Products in the Post-Genomic Era 22

1.2 Plants as a Source of Pharmaceuticals and Lead Compounds 25

1.3 The International Cooperative Biodiversity Group (ICBG) Program 29

1.4 Collection and Investigation Stragies 31

1.5 Ahead 34

CHAPTER II- A NEW ANTIBACTERIAL TRITERPENE AND A NEW IRIDOID

FROM CAIOPHORA CORONATA 36

2.1 Introduction 36

2.2 Distribution and Botanical Features 37

2.3 Systematics and Phytochemistry in Loasaceae 37

2.4 Ecological Significance of Iridoids in the Loasaceae 40

2.5 Statement of Purpose 42

2.6 Bioassay Guided Fractionation 43

2.7 Structure Elucidations and Identification 47

2.7.1 Structure Elucidation of ip,3P-Dihydroxyurs-12-en-27-oic Acid (1)... .47

2.7.2 Structure Elucidation of Caiophoraenin (2) 53 7

TABLE OF CONTENTS - Continued

Page

2.7.3 Identification of Isoboonein (3) 61

2.8 Antibacterial Activity 63

2.9 LC/MS Survey 65

2.10 Experimental Section 76

2.10.1 NMR Experiments 76

2.10.2 Plant Material 76

2.10.3 In Vitro Antimicrobial Activity Testing 77

2.10.4 LC/MS Instrumentation and Technique 78

Sample Preparation 78

Standard Preparaton 79

HPLC/DAD 79

2.10.5 Spectroscopic Data 79

ip,3P-Dihydroxyurs-12-en-27-oic Acid (1) 79

Caiophoraenin (2) 79

CHAPTER III - GROWTH INHIBITION OF MYCOBACTERIUM TUBERCULOSIS

BY THE CONSTITUENTS 0¥ MYRCIANTHES COQUIMBENSIS 80

3.1 Introduction 80

3.2 Distribution and Botanical Features 81

3.3 Taxonomy and Nomenclature 82 8

TABLE OF CONTENTS - Continued

Page

3.4 Chemical Composition of the Volatile Leaf Oil 84

3.5 Statement of Purpose 89

3.6 Bioassay Guided Fractionation 90

3.7 Structure Elucidation and Identifications 97

3.7.1 Structure Elucidation of (15,3'S',4/?)-l-Methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (3p-Hydroxy-c/5-p-terpineol, 10) 97

3.7.2 Identification of Oleanolic Acid (11) 107

3.7.3 Identification of 3p-Caffeoyl-12-oleanen-28-oic Acid (12) 107

3.7.4 Idetification of Trans (+)-sobrerol (13) 109

3.7.5 Identification of Epi-catechin (14) 110

3.7.6 Identification of Catechin (15) 110

3.8 AntitubercularActivity Ill

3.9 LC/MS Survey 113

3.10 Experimental Section 129

3.10.1 NMR Experiments 129

3.10.2 Preparation of the (R)- and (5)-MTPA Derivatives of 10 129

3.10.3 Plant Material 130

3.10.4 Microplate Alamar Blue Assay 131

3.10.5 Vero Cell Cytotoxicity Assay 133 9

TABLE OF CONTENTS - Continued

Page

3.10.6 GC/MS 134

3.10.7 LC/MS Instrumentation and Technique 134

Sample Preparation 134

Standard Preparation 135

HPLC/DAD 135

3.10.8 Spectroscopic Data 136

(1S,3S,4R)- 1-Methyl-4-( 1 -methylethenyl)-1,3-cyclohexanediol (10)....136

CHAPTER IV - CHLOROFORM SOLVATE OF (47?,5/?,75,85,95)-7-HYDROXY-8-

HYDROXYMETHYL-4-METHYLPERHYDROCYCLOPENTA[C]PYRAN-1-ONE

FROM VALERIANA LAXIFLORA 137

4.1 Introduction 137

4.2 Results and Discussion 139

4.3 Experimental Section 145

4.3.1 Plant Material 145

4.3.2 Isolation 145

4.3.3 Refinement 146

CHAPTER V - (3/?,55,9/?,105,135,145,17/?,18/?,21/?)-(-)-FERN-7-EN-3a-OLFROM

SEBASTIANIA BRASILIENSIS 148

5.1 Introduction 148

5.2 Results and Discussion 150 10

TABLE OF CONTENTS - Continued

Page

5.3 Experimental Section 151

5.3.1 Plant Material 151

5.3.2 Isolation 157

5.3.3 Preparation of the {R)- and (5)-MTPA Derivatives of 30 157

5.3.4 Refinement 158

CHAPTER VI - SUMMARY AND CONCLUSIONS 160

6.1 Summary 160

6.2 Antitubercular Triterpenoids 162

6.3 Structure-Activity Relationships in Antitubercular Triterpenoids 164

6.3.1 The Oleananes 164

6.3.2 TheUrsanes 169

6.3.3 The Lupanes 172

6.3.4 The Hopanes and a Femane 175

6.3.5 The Steroids 178

CHAPTER VII - GENERAL EXPERIMENTAL 183

7.1 General Instrumentation 183

7.2 HPLC/DAD/MS/MS 184

7.3 X-ray Crystallography Data Collection 184

APPENDIX A 185 11

TABLE OF CONTENTS - Continued

Page

APPENDIX B 189

REFERENCES 205 12

LIST OF FIGURES

Page

Figure 2.1. Known iridoids from the genus Caiophora 39

Figure 2.2. Examples of bitter-tasting iridoids 41

Figure 2.3. Isolates (1-3) from C. coronata 45

Figure 2.4. l(3,3p-Dihydroxyurs-12-en-27-oic acid (1), a-peltoboykinolic acid (4) and

related natural products 49

Figure 2.5. Significant DQF-COSY correlations of l(3,3P-dihydroxyurs-12-en-27-oic

acid (1) 51

Figure 2.6. Significant HMBC correlations of l[3,3|3-dihydrox}airs-12-en-27-oic acid

(1) 51

Figure 2.7. Significant ID NOE correlations of l(3,3p-dihydroxjairs-12-en-27-oic

acid (1) 52

Figure 2.8. Significant HMBC correlations of caiophoraenin (2) 55

Figure 2.9. Significant DQF-COSY correlations of caiophoraenin (2) 55

Figure 2.10. Caiophoraenin (2) and gibboside aglucone (5) 56

Figure 2.11. Iridomyrmecin (6), isoiridomyrmecin (7), epiiridomyrmecin (8), and

isoepiiridomyrmecin (9) 58

Figure 2.12. Significant ID NOE correlations of caiophoraenin (2) 60

Figure 2.13. Compound 1 from C. coronata. Samples A-C: HPLC chromatograms at

200 nm 68 13

LIST OF FIGURES - Continued

Page

Figure 2.14. Compound 1 from C. coronata. Samples A-C: MS spectra 69

Figure 2.15. Sample A: MS/MS spectrum at 54.9 min 70

Figure 2.16. Compound 2 from C. coronata. Samples A-C: MS spectra 71

Figure 2.17. Compound 2 from C. coronata. Samples A-C: MS/MS spectra 72

Figure 2.18. Samples A-C: MS spectra 73

Figure 2.19. Sample A: MS/MS spectrum at 12.7 min 74

Figure 3.1. Volatile oil components of M. coquimbensis 85-87

Figure 3.2. Isolates (10, 12) from M. coquimbensis 95

Figure 3.2. (contd) Isolates (11,13-15) fromM. coquimbensis 96

Figure 3.3. ElMS spectrum of 3p-hydroxy-cw-P-terpineol (10) 99

Figure 3.4. 3P-Hydroxy-a'5-p-terpineol (10) fromM. coquimbensis and cw-p-

terpineol (16) 100

Figure 3.5. Significant HMBC correlations of 3P-hydroxy-cjs-P-terpineol (10) 102

Figure 3.6 Significant DQF-COSY correlations of 3|3-hydroxy-d5'-P-terpineol

(10) 102

Figure 3.7. Significant ID NOE correlations of 3P-hydroxy-d5-P-terpineol (10)... 104

Figure 3.8. 'H NMR A6(5-/?) values obtained for the MTPA esters 10s and lOr.. ..104

Figure 3.9. Synthetic stereoisomers of 3p-hydroxy-cw-p-terpineol (10) 106 14

LIST OF FIGURES - Continued

Page

Figure 3.10. Compound 11 fromM. coquimbensis. Samples D-F: HPLC

chromatograms at 200nm 117

Figure 3.11. Compound 11 from M. coquimbensis. Samples D-F: MS spectra 118

Figure 3.12. Compound 11 from M coquimbensis. Samples D-F: MS/MS spectra.119

Figure 3.13. Compound 13 from M. coquimbensis, HPLC chromatograms at 200

nm 120

Figure 3.14. Compound 13 fromM. coquimbensis. Samples D-F: MS spectra 121

Figure 3.15. Compound 13 fromM. coquimbensis. Samples D-F: MS/MS

spectra 122

Figure 3.16. Compound 14 fromM. coquimbensis. Samples D-F: HPLC

chromatograms at 200nm 123

Figure 3.17. Compound 14 from M. coquimbensis. Samples D-F: MS spectra 124

Figure 3.18. Compound 14 from M. coquimbensis. Samples D-F: MS/MS spectra.125

Figure 3.19. Compound 10 from M. coquimbensis. Samples D-F: HPLC

chromatograms at 200nm 126

Figure 3.20. Compound 10 fromM. coquimbensis. Samples D-F: MS spectra 127

Figure 3.21. Compound 10 from M. coquimbensis and Sample E: MS/MS spectra.128

Figure 4.1. (4/?,5i?,75,85,95)-7-Hydroxy-8-hydroxymethyl-4-

methylperhydrocyclopenta[c]pyran-1-one (18) 138 15

LIST OF FIGURES - Continued

Page

Figure 4.2. A displacement ellipsoid plot (50% probability level) of (4/?,5/?,75,85,95)-

7-hydroxy-8-hydroxymethyl-4-methylperhydrocyclopenta[c]pyran-l-one

(18) 140

Figure 4.3 Crystal packing geometry of 18 looking at the face of the hydrogen bonded

tape 143

Figure 4.4. Crystal packing geometry of 18 along the ^-axis, colinear with the

hydrogen bonded tape 144

Figure 5.1. (37?,55,9/?,105,135,145,17/?,18^,21/?)-(-)-fem-7-en-3a-ol (30) 149

Figure 5.2. A displacement ellipsoid plot (50% probability level) of (-)-Fem-7-en-3a-

ol (30) 152

Figure 5.3. Crystal packing geometry of (-)-Fem-7-en-3a-ol (30) perpendicular to b-

axis 153

Figure 5.4. Crystal packing geometry of (-)-Fem-7-en-3a-ol (30) along &-axis 154

Figure 5.5. 'H NMR A6 (S-R) values obtained for the MTPA esters 30s and 30r...l56

Figure 6.1. Antitubercular oleananes 166-167

Figure 6.2. Antitubercular ursanes 170

Figure 6.3. Antitubercular lupanes 173

Figure 6.4. Antitubercular hopanes and a femane 176

Figure 6.5. Antitubercular steroids 179-181 16

LIST OF FIGURES - Continued

Page

Figure B.l. 'H NMR spectrum of lp,3P-ciihydroxyurs-12-en-27-oic acid (1) in

CD30Dat600 MHz 190

Figure B.2. '"^C NMR spectrum of lp,3p-dihydroxjairs-12-en-27-oic acid (1) in

CD30Datl25 MHz 191

Figure B.3. HMBC spectrum of ip,3P-dihydroxyurs-12-en-27-oic acid (1) 192

Figure B.4 DQF-COSY spectrum of lp,3(3-dihydroxyurs-12-en-27-oic acid (1)....193

Figure B.5. ID NOE spectrum of l|3,3P-dihydroxyurs-12-en-27-oic acid (1) in

CD30Dat600 MHz 194

Figure B.6. 'H NMR spectrum of caiophoraenin (2) in CD3OD at 600 MHz 195

Figure B.7. '^C NMR spectrum of caiophoraenin (2) in CD3OD at 125 MHz 196

Figure B.8. HMBC spectrum of caiophoraenin (2) 197

Figure B.9. DQF-COSY spectrum of caiophoraenin (2) 198

Figure B.IO. ROESY spectrum of caiophoraenin (2) 199

Figure B.ll. 'H NMR spectrum of (15',35',4^)-l-methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (10) in CD3OD at 600 MHz 200

Figure B.12. ''^C NMR spectrum of (15',3'S',4/?)-l-methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (10) in CDCI3 at 125 MHz 201

Figure B.13. HMBC spectrum of (15,3'S',4^)-l-methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (10) 202 17

LIST OF FIGURES - Continued

Page

Figure B.14. DQF-COSY spectrum of (15',3'S',4/?)-l-methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (10) 203

Figure B.15. ID NOE of (15",35,47?)-l-methyl-4-(l-methylethenyl)-1,3-

cyclohexanediol (10) in pyridine-dj at 600 MHz 204 18

LIST OF TABLES

Page

Table 2.1. C. coronata chromatographic fractions: Yields and Antimicrobial activity

profiles 44

Table 2.2. '^C and 'H NMR data for ip,3p-dihydroxyurs-12-en-27-oic acid (1) and

a-peltoboykinolic acid (4) 50

Table 2.3. 13" C and I H NMR data for caiophoraenin (2) and gibboside aglucone (5).57

Table 2.4. Antibacterial activity of isoboonein from C. mongolica 62

Table 2.5. Antibacterial activity of isolates from C. coronata 64

Table 3.1. Volatile oil components of other Myrcianthes species 88

Table 3.2. M. coquimbensis extracts and fractions: Yields and Antitubercular activity

profile 91

Table 3.3. M. coquimbensis chromatographic fractions; Yields and Antitubercular

activity profile 93

Table 3.4. '^C and 'h NMR data for 3P-hydroxy-cw-(3-terpineol (10), cw-P-terpineol

(16) and (li?,37?,47?)-l-hydroxy neoisopulegol (17) 101

Table 3.5 'H NMR data for 10s and lOr and A5 values obtained for the MTPA

esters 105

Table 3.6. Antitubercular activity of M. coquimbensis isolates 112

Table 4.1. Hydrogen-bonding and short-contact geometry of 18 142

Table 4.2. Experimental Data for (4/?,5-/?,75',85',95)-7-hydroxy-8-hydroxymethyl-4-

methylperhydrocyclopenta[c]pyran-l-one (18) 147 19

LIST OF TABLES - Continued

Page

Table 5.1. Short contact geometry of (-)-fem-7-en-3a-ol (30) 155

Table 5.2. and 'H NMR data for select protonated signals of (-)-fem-7-en-3a-ol

(30) 155

Table 5.3. 'H NMR data for 30s and 30r and A5 values obtained for the MTPA esters

156

Table 5.4 Experimental Data for (-)-fem-7-en-3a-ol (30) 159

Table 6.1. Antitubercular oleananes and their minimum inhibitory concentration

(MIC) values against MTB 168

Table 6.2. Antitubercular ursanes and their minimum inhibitory concentration (MIC)

values against MTB 171

Table 6.3. Antitubercular lupanes and their minimum inhibitory concentration (MIC)

values against MTB 174

Table 6.4. Antitubercular hopanes and femane and their minimum inhibitory

concentration (MIC) values against MTB 177

Table 6.5. Antitubercular steroids and their minimum inhibitory concentration (MIC)

values against MTB 182 20

ABSTRACT

The bioassay guided fractionation of two Latin American plants, structure elucidation of pure isolates, and LC/MS studies of six plant extracts is presented here along with the structure determination of two compounds using X-ray diffraction. The bioassay guided fractionation of the antibacterial and antitubercular CH2Cl2-MeOH extracts of the

Argentinean plant Caiophora coronata Hook, et Am. (Loasaceae) and the Chilean plant

Myrcianthes coquimbensis (Bam.) Landmm et Grifo (Myrtaceae) respectively led to the isolation and complete structure elucidation of nine compounds from the active fractions.

Three of these isolates were determined to be new. Namely, a new triterpene, lp,3p- dihydroxyurs-12-en-27-oic acid, anew iridoid, la-methoxy-6a,10- dihydroxyisoepiiridomyrmecin (caiophoraenin) from C. coronata and a new monoterpene (15,35,4/?)-l-methyl-4-(l-methylethenyl)-l,3-cyclohexanediol from M. coqumibensis. All chemical structures were established unequivocally by physical and spectroscopic techniques including-melting point, optical rotation, ID and 2D NMR, HR-

FABMS, and FT-IR. Absolute configuration of the new monoterpene was established by

Mosher's esterification. The antibacterial activity of all isolates from C. coronata were determined against methicillin-sensitive (MSSA) and -resistant (MRSA) strains of

Staphylococcus aureus, Bacillus subtilis (BS), vancomycin-resistant Enterococcus faecium (VREF), Escherichia coli (EC), E. coli imp (ECimp), and Candida albicans

(CA). ip,3(3-Dihydroxyurs-12-en-27-oic acid was found active against BS, MSSA,

MRSA, VREF, and ECimp with MIC values of 2, 4, 4, 4 and 16 ^ig/ml respectively. 21 whereas, other isolates were essentially inactive. The antitubercular activity of all isolates from M. coquimbensis was evaluated against M. tuberculosis using the microplate alamar blue assay. Oleanolic acid was determined to be the active principle of the extract with an

MIC value of 29.66 |Xg/mL whereas other isolates were regarded as inactive (MIC > 128

|a,g/mL). Chemical investigations by LC/MS of species closely related to C. coronata and

M. coquimbensis were also conducted.

Structure solutions by single crystal X-ray crystallography, of an iridoid

(4/?,5R,7S,8S,9S)-(-)-7-hydroxy-8-hydroxymethyl-4-methyl-

perhydrocyclopenta[c]pyran-l-one, and a fernane (3i?,55,9/?,105,135,145,17i?,18/?,21/?)-

(-)-fem-7-ene-3a-ol, isolated from the antitubercular methanolic extracts of Valeriana laxiflora DC (Valerianaceae) and Sebastiania brasiliensis Spreng. (Euphorbiaceae) respectively is also presented. The absolute configuration of these compounds was also determined. 22

CHAPTER I

INTRODUCTION

1.1 Natural Products in the Post-Genomic Era

The shift of pharmaceutical drug discovery away from natural products comes at a most inopportune time in scientific history when the need for chemical diversity is paramount.

The unraveling of a majority (93%) of the human genomic sequence in February 2001 by two different groups,1 ' 2 fifty years after the monumental publication of the double helical structure of DNA by Watson and Crick, was recorded as a landmark discovery. It is now known that only about -2% of the human genome encodes somewhere between 26,000-

40,000 genes. While this translates to at least as many protein products, protein-protein interactions and post-transcriptional modifications are likely to increase this number considerably. However, it remains to be seen how many of those protein products will qualify as molecular drug targets."

So far more than 600 different molecular targets have been identified and this number is likely to increase enormously.' Additionally, the genomes of certain pathogens like the bacterium responsible for tuberculosis (TB), Mycobacterium tuberculosis (MTB ),'^ the malarial parasite Plasmodium falciparum,^ and the mosquito vector Anopheles gambiae^ have also been sequenced. Thus, it can be predicted that with the identification and 23 validation of new targets arising from genomic research, greater demands will be placed on the identification of new leads that can affect a desired physiological response for each target. The ever-increasing efficiencies and capacities of High-Throughput Screens

(HTS) further necessitate vast and diverse chemical libraries to screen against.

The promise of combinatorial chemistry to provide such vast chemical libraries, after its birth in the 1980s, has yet to bear fruit. The shift of focus of the pharmaceutical companies from natural products research to combinatorial chemistry over the past twenty years has led to a dramatic decrease in the number of New Chemical Entities

(NCEs), with a twenty year low of thirty-seven NCEs being reported in the year 2001.

One reason for the downsizing of natural products drug discovery was the expense related to these programs. But, although combinatorial chemistry is cheaper per molecule tested, since millions of compounds need to be tested each time, the overall demand for resources is very high.^ In fact, despite the commonly held assumption, natural products can be a more economical source of chemical diversity than the synthesis of equivalent numbers of diverse chemicals.'^

Natural products chemistry is still rich with drug discovery opportunities. The chemical novelty associated with natural products is unparalleled and significantly higher than that of any other source: 40% of the chemical scaffolds in a published database of natural products (Dictionary of Natural Products, Chapman and Hall) are absent from synthetic 24 chemistry." This is particularly important when searching for lead molecules against newly discovered targets for which there are no known small-molecule leads.

Additionally, biologically active natural products are generally small molecules with drug-like properties, capable of being absorbed and metabolized by the body. They thus have a cost advantage over other biotechnological products or compounds produced by combinatorial chemistry that are associated with high development costs.'" Some natural products including taxol, mescaline, the cannabinoids, and yohimbine have also been critical pharmacological probes to characterize novel disease pathways and targets and have thus, been indispensable tools for the molecular and cell biologist.

Through the ages, natural sources have been the most reliable and successful sources of pharmaceuticals and pharmaceutical leads. According to Newman et al. -50% of all small molecule NCEs in the years 2000 and 2001 were from the field of natural products.

In fact, 60% of all present day anticancer drugs, 75% of antiinfectives, 70% of antimigraine drugs, and 48 out of 74 of all currently used antihypertensives are either natural products or derived from natural products. Also, nine of the twenty best-selling drugs in 1999 were either natural products or derived from natural products, namely, simvastatin, lovastatin, enalapril, pravastatin, atorvastatin, augmentin, ciprofloxacin, clarithromycin, and cyclosporin, with a combined annual sales of > US $ 16 billion.

Thus, the contribution of natural products to the pharmaceutical armamentarium, not just historically but also in the present scenario, is beyond doubt very significant. 25

There are other additional incentives natural products offer. For example, pharmacologically active natural products often bring about more than one important biological activity. Aspirin, whose origin can be traced back to the natural substance salicin from the bark of the willow tree Salix alba, is used not only for its analgesic effect but also, to prevent strokes in patients with hyperlipidemia. Similary, caffeic acid phenethyl ether from propolis is not only a potent antiinflammatory agent but also, has differential cytotoxicity observed in melanoma and breast carcinoma in both transformed rat and human cell lines.The active ingredient in Neem seed oil, azadirachtin, is associated with many different mechanisms to deter over 200 insect species. It inhibits chitin synthesis and thus prevents the formation of the exoskeleton, disrupts mating and sexual communication, causes sterility, decreases gut motility, and prevents swallowing,

thus acting as a feeding-deterrent. I "3 In fact, synthetic derivatives of lead compound khellin, from the Egyptian medicinal plant Ammi visnaga, led to the development of two

important medicines with different pharmacological activities, namely the antiasthmatic drug cromoglycate, and the antiarrhythmic agent amiodarone.'^

1.2 Plants as a Source of Pharmaceuticals and Lead Compounds

The potential of plants, especially those utilized in traditional medicine, for drug

discovery has been known for more than 200 years. The curare alkaloids, cardiac

glycosides, physostigmine, A^-tetrahydrocannabinol, the opiates- morphine and codeine,

the Cinchona alkaloids- quinine and quinidine, the solanaceous tropane alkaloids- 26 atropine and /-hyoscyamine, scopolamine, pilocarpine, ephedrine, cocaine, theophylline, vincristine, vinblastine, taxol, artemisin, forskolin, and caffeine are just some of the most popular plant-derived drugs.

In an analysis of the top 150 'most-prescribed' drugs in the US during the year 1993, it was found that 84 of them were of natural origin and of these, 34 were of plant origin.'^

Moreover, at least 80% of the population in developing countries depends on traditional medicines, which are mostly plant-based, for their healthcare needs.This translates to

-3.2 billion people that depend on their local flora to be their pharmacy.'^

But, only a fraction of the total plant biodiversity has systematically been studied for biological activity. There are ca. 287,655 species of plants on earth'^ that have been identified and approximately another 10,000 as yet undescribed species, most of which, it I R is estimated represent tropical flora. Out of the known plant species, only 5-15% have been systematically surveyed for biological activity. Even the plants that have been surveyed for activity are usually screened for only a single or at best a few types of activities, and in many cases using simple, bench-top bioassays.'^

Moreover, ethnobotanical data is continuously being lost as the field of traditional medicine fades. Seventy-four percent of the total 119 plant-derived drugs in 1985 were isolated based on ethnobotanical data regarding their traditional use.'^ Eighty six percent of the plant species used in Samoan traditional medicine show pharmacological activity 27 in broad in vitro and in vivo screens.20 Prostatin, an anti-HIV drug candidate about to enter clinical trials, was isolated from the bark of the mamala tree {Homalanthus nutans) as a result of its use in traditional Samoan plant medicine. But, with the death of the old medicine men, and the lack of interest in the young of the community to learn from them, the science is in want of comprehensive documentation by ethnobotanists and 9n 99 pharmacognosists if we hope to use this knowledge for modem drug discovery.

Alarmingly, most biologists now agree that mankind is documenting its sixth major extinction. Unlike previous extinctions, the present one is due to uncontrolled and disruptive human activity. " Out of a currently described plant biodiversity of 287,655 species as of year 2003,'^ an estimated 12% are threatened with extinction in the wild in the next 50 years.94

Tropical forests, which contain about two-thirds of the total eukaryotic biodiversity, originally covered between 14 and 18 million square kilometers of the earth's surface, of which only half now remains. 24 It has been calculated that 44% of all the species of vascular plants are now confined to 25 'hotspots' or areas featuring exceptional concentrations of endemic species and experiencing exceptional loss of habitat- 17 of

9c which are in tropical forests. And these hotspots in total occupy just 2.1 million square kilometers or 1.4% of the Earth's total ice-free land surface. 28

In fact, it is predicted that if current rates of habitat destruction continue, 50% of all eukaryotic species will be eliminated by the year 2060.^^ This estimate is exponentially greater than the overall rate of natural extinction that has been calculated by some to be of the order of 10" species, per species per year. These estimates however do not account for the adverse effects on biodiversity by global warming, introduction of alien species, and overkill of wild species for resources.

This massive loss in species richness is occurring in a malevolent 'cookie cutter' fashion, by stamping out through habitat destruction, certain areas that ironically are the most specious. From a pharmacognosy viewpoint, these stamped-out areas represent the most interesting of all biodiversities, based on the ecological competition that exists within the natural habitat. Thus, loss in biodiversity potentially corresponds to an even greater loss of chemical diversity.

Since a large part of the current pharmaceutical armamentarium includes natural products

and natural product derived molecules, and relies heavily on knowledge obtained from

natural products, it is imperative that every effort be made to conserve the remaining

biodiversity. Several proposals for conservation have been put forth including one that

indicates that by concentrating conservation efforts on the 25 hotspots with an annual

expenditure of $500 million we can preserve to a great extent the biodiversity they

contain. It seems only obvious that the pharmaceutical industry and the entire global

community who stand to gain directly from biodiversity take an active role in its preservation. Thus, it is logical to try and build conservation incentives into the pharmaceutical research and development process. It is within this context that the research of this dissertation takes place, as a part of the larger effort known as the

International Cooperative Biodiversity Group (ICBG) Program. The research described herein demonstrates how pharmaceutical discovery can stimulate biodiversity conservation and economic development.

1.3 The International Cooperative Biodiversity Group (ICBG) Program

In 1992 The National Institutes of Health (NIH), the National Science Foundation (NSF) and the US Agency for International Development (USAID), jointly created the ICBG program, which is a multidisciplinary program for drug discovery involving cooperation between academic institutions in the USA, pharmaceutical companies and a number of 97 98 host countries. The main tenet of the ICBG program is that natural products research and development can lead not only to new pharmaceuticals, but can also foster economic development and biodiversity conservation in the countries that supply the genetic resources.

The research described in this dissertation is part of the larger research effort of the Latin

American ICBG program headed by Dr. Barbara N. Timmermann entitled, "Bioactive

Agents from Dryland Biodiversity of Latin America". The specific goals of this program are to discover and develop pharmaceuticals, veterinary therapeutics and crop-protection 30 agents from plants and microbes growing in the arid and semi-arid ecosystems of

Argentina, Chile, and Mexico, and to promote sustainable economic activity and biodiversity conservation in these regions. 29" 30

The project involves collaborations in the US with Wyeth, American Cyanamid

Company, the G.W.L Hansen's Disease Center in Louisiana, Fort Dodge Animal Health, and the University of Illinois at Chicago to perform the biological assays. In Latin

America, collaborations that participate in the collection, identification, cataloguing and conservation are the National Institute of Agricultural Technology of Argentina, the

National University of Patagonia, the National Center for Research in Patagonia, the

Pontifical Catholic University of Chile, and the National Autonomous University of

Mexico. The extraction, isolation and structure elucidation work is done at the College of

Pharmacy, University of Arizona. The University of Arizona has established a set of multilateral, contractual agreements with all participating institutions that define work and funding commitments, ownership of materials, licensing rights and distribution of future benefits, if any.

The Latin American ICBG targets xerophytic plants for investigation as they are known to possess potent chemical defenses against the drought, radiation and herbivory that dominates their environment. Thus, the secondary metabolites that afford an ecological advantage in the defense of these plants may also have the potential to act as therapeutic agents. Thus, the systematic screening of Latin America's arid-land plants in a battery of 31 high-throughput biological assays might lead to the identification of active lead compounds that can be further developed as drugs for a number of therapeutic areas.

An important aspect of the ICBG is to facilitate economic development and environmental awareness in the host countries where collections are made. Through the promise of equitable distribution of profits and scientific information to the host-country regarding new commercial agents, the ICBG hopes to add value to the local biodiversity and encourages its conservation. Additionally, several well-illustrated accounts of the local flora and its medicinal value have been published during the course of the Latin

American ICBG to spread awareness about the value of local biodiversity in host countries."^

1.4 Collection and Investigation Strategies

A rational and all-inclusive collection strategy is usually a very important part of any successful natural products drug discovery program. Since bioassay guided isolation and subsequent structure determination are labor intensive and fairly expensive steps, the process is most likely to bear positive result if the source of the plant material was rationally selected. Procurement strategies usually employed in the collection of plant and other biological materials include the ethnobotanical, ecological, biomedical, taxonomic and random approaches. 32

The ethnobotanical approach relies on traditional knowledge of therapeutic properties to suggest potential sources of new drugs. This strategy has historically been the most I f\ '?n 00 successful and has been adequately discussed elsewhere. '

Another strategy to optimize a natural products collection is the ecological approach.

This involves the careful observation of biochemical interactions of organisms in an ecosystem to suggest potential sources. This approach has also been discussed in the literature. 18

Random collection is the third approach that involves sampling of diverse flora systematically from a particular area. This approach involves the most complete study of the local biodiversity through the analysis of all available common and endemic species.

Because of the large number of samples generated by random collections, this approach is well suited to high-throughput screening. 22

Finally, the biomedical and taxonomic collection strategies use existing knowledge about active chemical structures and the taxonomic group they are commonly isolated from to suggest other logical sources.

The Latin American ICBG procures plants from the dry lands of Latin America based on a combination of the ethnobotanical, ecological, random, biomedical and taxonomic collection strategies. Plants are collected and identified by the host country botanists who 33 have experience with the local flora and have access to local 'medicine' men and women.

A voucher specimen of each collected species is deposited at herbaria of collaborating host-country institutions as well as at the herbaria at the University of Arizona, Royal

Botanic Gardens at Kew, New York Botanic Gardens, and Geneva Botanical Garden.

Once properly identified and cataloged enough biomass to give about 1 kg of dry material is harvested, dried at ambient temperature and ground in the host country. Properly identified biomass is shipped to the University of Arizona for chemical analysis and submission for bioassays to collaborating US institutions.

All plant material is subjected to a standardized extraction procedure involving soaking in

CH2Cl2-MeoH (1:1), followed by filtration and solvent evaporation under vacuum to generate the organic extract. This process has over the past eleven years resulted in a library of 10,028 extracts from the dry-land flora of Latin America. The extracts are permanently cataloged and stored, so as to be available for screening in various biological assays to determine which ones are active and thus deserve further study.

Extracts found to be active in initial screens may be subjected to bioassay guided fractionation to isolate the active constituents without having to examine all of the components of the mixture. Before an extract is selected for fractionation, however, a comprehensive literature survey is conducted to determine the likelihood of finding new chemical entities as well as to check whether the plant was previously evaluated for a similar biological activity. In cases where compounds previously isolated from the same 34 or related species, have elsewhere displayed the same biological activity we are looking for, the extract is not further studied. Also, when possible dereplication of organic extracts is conducted to identify possible active ingredients and minimize isolation effort.

Bioassay guided fractionation relies on chromatographic fractionation of a mixture, followed by testing of each fraction to determine the location of any bioactive compounds. This process of separation followed by bioassay is repeated in an iterative fashion until a pure active compound is isolated. Once a single chemical compound has been isolated and purified it is progressed to the structure determination stage utilizing a battery of available spectroscopic methods such as NMR, IR, mass spectrometry, and X- ray crystallography.

1.5 Ahead

The following two chapters (Chapters 2 and 3) give a detailed account of the bioassay directed isolation and structure determination process as it was applied to the CH2CI2-

MeOH (1:1) extracts of the Argentinean plant Caiophora coronata Hook et Am. and the

Chilean Myrcianthes coquimbensis (Bam.) Landrum et Grifo respectively. While C. coronata was evaluated for its antibacterial activity, M. coquimbensis was evaluated for antitubercular activity. Chapters 2 and 3 begin with some background about the botanical features, taxonomic classification and known chemistry about the plant. In case of C. coronata, ecological significance of iridoids in the family Loasaceae is also presented.

This is followed by a detailed account of the bioassay directed fractionation. 35 spectroscopic identification of known isolates, structure elucidation of the new isolates, and bioassay results obtained for all pure compounds. LC/MS studies on related plants under the same genus or different acquisitions of the same species were also conducted and results from these studies are also presented.

Chapters 4 and 5 describe structure solutions of a femane and an iridolactone respectively using single crystal X-ray crystallography. In the case of the femane absolute stereochemistry was determned by Mosher's esterification technique. For the iridolactone, the anomalous dispersion of X-rays by a heavy-atom was used to arrive at the absolute stereochemistry due to the inclusion of a chloroform molecule in the crystal lattice.

Chapter 6 presents a brief summary of the results obtained in Chapters 2-5 and a discussion of some overall conclusions of this research. This is followed by a brief discussion on the findings from the bioassay-directed isolation of antitubercular plant extracts from the Latin American ICBG as well as from other studies. Some empirical structure-activity relationships are then discussed for six triterpene skeletal types, namely, ursanes, oleananes, hopanes, femanes, lupanes and steroids. 36

CHAPTER II

A NEW ANTIBACTERIAL TRITERPENE AND A NEW IRIDOID FROM

CAIOPHORA CORONATA

2.1 Introduction

Bacterial diseases continue to present a major threat to human health. Emerging new strains of pathogenic bacteria"^' as well as escalating resistance to current drugs'^^ forms the incentive for our search for new antibacterial leads in the Latin American biodiversity. To this end, our ICBG program routinely screens plant extracts for antibacterial activity. The extract of the Argentinean plant Caiophora coronata Hook et

Am., collected using our bioprospecting approach to drug lead discovery, was observed to have activity against both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S. aureus in an agar diffusion test. Based on this important activity C. coronata was selected for further chemical investigation.

The bioassay guided isolation of this plant led to the isolation of a new triterpene, ip,3p- dihydroxyurs-12-en-27-oic acid (1), and a new iridoid, la-methoxy-6a,10- dihydroxyisoepiiridomyrmecin (caiophoraenin) (2), along with the known iridoid isoboonein (3). The structure elucidations of 1 and 2 and the identification of 3 is 37 presented. All isolates were assayed against a host of clinically relevant gram- positive and gram-negative bacteria and the results from these assays are presented.

2.2 Distribution and Botanical Features

The genus Caiophora (Loasaceae: Loasoideae) contains 65 species distributed in the high

Andes ranging from Central Peru to Northern Argentina and Chile between 2800-3600 meters above sea level.The generic name is derived from the greek kaios (to bum) and phoreo (to bear) referring to the stinging hairs characteristic of these plants.

The plant is a perennial herb about 30 cm in height. It is covered with hard and irritant trichomas or hairs. It bears compound leaves developed in rosettes with long petioles, and solitary flowers about four cm wide developed from buds located in the axils of the leaves, with five sepals and five white petals. The fruit is a capsule with many wingy seeds.This plant, known locally as charrua, ortiguilla, and mpa chica, is locally used

in Chile to treat colds.'^^

2.3 Systematics and Phytochemistry in the Loasaceae

The family Loasaceae contains ~315 species distributed in two subfamilies, the

Loasoideae (-225 species) and Mentzelioideae (-90 species)."^"^ The genus Caiophora is

one of the largest genera in Loasoideae, containing -65 species.''^ Other genera are: Aosa 38

(7 species), Blumenbachia (12 species), Chichicaste (1 species), Huidobria (2 species),

Kissenia (2 species), Loasa (36 species), Nasa (-100 species), Presliophytum (3 species), and Scyphanthus (1-2 species).''^

Iridoids have been determined to be present throughout the genus Caiophora and in other congenerics of Loasoideae. In fact, the discovery of iridoids in the Loasaceae has led to the inclusion of this family in the order Comales in the Asterids. Loasaceae is now considered closely related to families such as Dipsacaceae, Ericaceae, Comaceae,

Gentianaceae, Loganiaceae, Verbenacea, and Rubiaceae, which were previously considered to be an exclusive group based on the presence of iridoids. Other families,

Passifloraceae and Tumeraceae, traditionally believed to be related to the Loasaceae have now been placed in the Rosids."^^

It has also been observed that dimeric iridoids and oleosides in Loasaceae are restricted to the sub-family Loasoideae and within this family, oleosides and hetero-oligomeric iridoids are restricted to Caiophora and Loasa.'^

10-hydroxyoleoside dimethyl ester (Fig. 2.1), and a lectin have previously been reported in C. coronata Hook et Additionally, pentlandioside I (Fig. 2.1), a bis- secoiridoid, was isolated from Cajophora pentlandii (Paxt) G. Don48 The iridoid, 10- hydroxyoleoside dimethyl ester is used as a chemotaxonomic marker for this genus."^^ COOCHa

COOCH3

pentlandioside I

OH

10-hydroxyoleoside dimethyl ester

Figure 2.1. Known iridoids from the genus Caiophora46 40

2.4 Ecological Significance of Iridoids in the Loasaceae

It has been indicated that iridoids play an ecological role in the deterrence of vertebrate and invertebrate herbivores in dry desert climates Specifically in Loasoideae, these iridoids protect the seeds in the absence of stinging hairs and/or harsh indument, which cover all other aerial portions of the plant. Secoiridoids are known to be bitter tasting. For example, gentiopicrin (Fig. 2.2) is the bitter principle of gentian roots (Gentianae lutea

L.: Gentianaceae) and tricolorside methyl ester (Fig. 2.2) isolated from the seeds of a plant belonging to Loasa ser. Macrospermae as well as the parent seed tastes bitter to the human palate. Rodents and presumably seed-feeding insects would otherwise consume these seeds with high oil and starch content. The seeds of Loasoideae also have a higher content of iridoids (12%) than any other species in Loasaceae.'^'^

Since these seeds remain dormant for very long periods of time, there should be selection on the individual plant to decrease seed predation. Thus, it is believed that the presence of high concentrations of bitter-tasting iridoids offers defense for these seeds in the absence of stinging hairs

Also, plants belonging to other genera such as Nasa (Loasaceae) have very small

quantities of common iridoids such as loganin and no novel chemical structures and these

plants are found to inhabit more mesic habitats, where invertebrate pests are more

important for predation than mammalian grazers. o Gentiopicroside

.OCH3 HgC

HO

HO' HO HO OH

HO OH HO'

Tricolors ide methyl ester

Figure 2.2. Examples of bitter-tasting iridoids 42

Even some species of Caiophora such as C. lateritia Klotzsch and C. buaraevii Urb. et

Gilg. that inhabit humid climates contain very small quantities of common iridoids. In these plants predation of leaves by caterpillars is considered to be important. Thus, some species under Nasa have different adaptive strategies such as evolution of widely diverse leaf shape. It is believed that the diversification of leaf shapes might play a deterring role in host recognition by the predator, in addition to the stinging hairs

2.5 Statement of Purpose

Aerial parts of C. coronata were collected and identified by Renee H. Fortunato and R.

Kiesling on January 22, 1997, 28 kilometers west of Puesto de Gendarmeria in the

province of San Juan, Argentina (30° 21' 49" S; 69° 41' 30" W).

The CH2Cl2-MeOH (1:1) extract of the air-dried and powdered aerial parts of C

coronata exhibited activity against both the methicillin-sensitive (MSSA) and -resistant

(MRSA) strains of S. aureus in an agar diffusion test with zones of growth inhibition of

11 and 12 mm, respectively. Based on these important biological activities, we proposed

to carry out a bioassay directed fractionation of the active extract to isolate the

antibacterial principle(s) and establish the chemical structure of all isolates using IR, MS,

and ID and 2D NMR techniques. We also proposed to assay all isolates against an array

of gram-positive and gram-negative bacteria including- methicillin-sensitive and resistant

Staphylococcus aureus (MSSA,and MRSA respectively). Bacillus subtilis (BS), 43 vancomycin-resistant Enterococcus faecium (VREF), Escherichia coli (EC), E. coli imp

(ECimp), and Candida albicans (CA).

2.6 Bioassay Guided Fractionation

Air-dried and powdered aerial parts of C. coronata (684 g) were extracted with 9L

CH2Cl2-MeOH (1:1) to give 31.1 g of crude extract. This extract exhibited activity against both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S. aureus in an agar diffusion test with zones of growth inhibition of 11 and 12 mm, respectively.

Thirty grams of this extract was fractionated by column chromatography (Si gel, 600 g,

63-200 |J,, Scientific Adsorbents Incorporated) using an EtOAc in n-hexane gradient (0-

80% EtOAc in n-hexane) followed by a MeOH wash. The chromatographic fractions were pooled based on their TLC profile to generate 27 fractions (F001-F026) and evaluated for their antimicrobial activity. Fractions F020 and F023 were selected for further fractionation based on their activity against B. subtilis (Table 2.1).

Fraction F020, eluting with 49-55% EtOAc in n-hexane (0.15 g, 0.5%), showed one major pink spot (compound 1, Fig. 2.3) on the TLC. Further fractionation using Sephadex

LH-20 (column length = 220 mm, column width = 20 mm) and eluting with MeOH afforded a sub-fraction containing compound 1 (0.00056 g) which was further purified using RPHPLC (Varian C18 5 |J, column; 40% ACN in H2O to 100% ACN in 30 min,

100% ACN 5 min) leading to the isolation of 1 (Rt = 22.4 min, 0.002 g, 0.007%). 44

Table 2.1. C. coronata chromatographic fractions: Yields and Antimicrobial activity profiles"

Fraction Wt. (in g) % Yield" Antimicrobial activity (as zones of inhibition, in mm)'--'' FOOl 1.99 6.6 0 F002 0.72 2.4 8 VH F003 2.26 7.5 8 VH F004 3.58 11.9 8H F005 0.99 3.3 9H F006 2.89 9.6 9H F007 3.54 11.8 7H F008 1.82 6.1 8H F009 0.19 0.63 7H FOlO 1.87 6.2 8H FOll 0.43 1.4 8H F012 0.64 2.1 8H F013 0.06 0.2 8H F014 0.3 1 8:9 H F015 0.70 2.3 8:10H F016 0.05 0.17 8:10H F017 0.21 0.7 8:10H F018 0.03 0.1 8:10H F019 0.002 0.0067 8:10H F020 0.15 0.5 12 F021 0.05 0.17 8:10H F022 0.08 0.27 9:10H F023 0.07 0.23 9 F024 0.05 0.17 8:9H F025 0.03 0.1 8 VH F026 6.9 23 0

" Fractions with zones of inhibition > 8 mm were selected for further fractionation '' As a percentage of the CH2Cl2-MeOH (1:1) extract (30 g) ' Antimicrobial activity of all fractions determined against B. subtilis at a concentration of 200 |j.g/spot ''H; Hazy, VH: Very Hazy 29

COOH

Me HO

10 OMe

OH 2

HO

Me 10 3

Figure 2.3. Isolates (1-3) from C. coronata 46

Fraction F023, eluting with 63-65% EtOAc in n-hexane (0.07 g, 0.23%) was subjected to preparative TLC (Silica gel 60, 20x20 cm, 0.5 mm; 80% EtOAc in n-hexane) resulting in the isolation of 2 (RF = 0.54, 0.0081 g, 0.027%) and 3 (RF = 0.36, 0.0088 g, 0.029%) (Fig.

2.3). 47

2.7 Structure Elucidations and Identiilcation

2.7.1 Structure Elucidation of ip,3p-Dihydroxy-urs-12-en-27-oic Acid (1)

Compound 1 (Fig. 2.3) was isolated as a white powder. Its HR-FABMS showed an

[M+H]"^ ion at m/z 473.3629 (Obs) corresponding to the molecular formula C30H48O4

(Calc, 473.3618). The IR spectrum suggested the presence of hydroxyl (3360 cm ') and carbonyl (1686 cm"') groups. The '^C and DEPT spectra indicated seven methyls, eight methylenes, eight methines, and seven quaternary carbons. Of the seven methyls, two were identified as secondary methyls based on HSQC and 'h spectra, indicating an ursane skeleton.

The quaternary carbon at 5179.3 was assigned to a free carboxylic group (C27). The methine at 5130.6 and quaternary carbon at 5133.9 were assigned to a trisubstituted double bond (C12 and C13 respectively). Two downfield methines at 580.9 and 76.8 were assigned to hydroxyl bearing carbons (CI and C3 respectively). The presence of two hydroxyl groups was in addition confirmed by FARMS, due to two sequential losses of water giving rise to fragment ions at m/z 455 and m/z 437.

A close analog of 1, a-peltoboykinolic acid (4, urs-12-ene-3P-ol-27-oic acid. Fig. 2.4) has previously been isolated from Peltoboykinia tellimoides (Maxim.) Hara.,''^ P. watanabei (Yatabe) Hara, Boykinia lycoctonifolia (Maxim.) Engl.,^° and Hyptis suaveolens (L.) Poit. Recently, 4 has been isolated from Astilbe chinensis (Maxim.) 48

Franch. et Savat. and its complete stmcture elucidation by ID and 2D NMR has been described.^^ Compound 1 is a CI P-hydroxy derivative of 4 and its ''^C and 'H data is comparable to the latter. (Fig. 2.4 and Table 2.2).

The hydroxyl group in 1 was assigned to CI based on its chemical shift value of 680.9.

Also, HI and H3 had DQF-COSY crosspeaks to H2, (Fig. 2.5) and were a part of the same spin-system in a ID-TOCSY experiment. HI had HMBC correlations with C2, C9 and C25 (Fig. 2.6). This confirmed the position of the hydroxyl bearing carbons in the A- ring. The carboxylic acid was located at C27 based on the downfield chemical shifts of

C12, C13, C14 and CI8 (Table 2.2). A similar trend in chemical shifts has been noted in case of 4^^. Additionally, the HMBC correlation of the H15 multiple! at 51.74 with the carboxyl group further supported its assignment at C27. Other DQF-COSY and HMBC data were used to further assign the '^C and 'H spectrum as shown in Figure 2.5 and 2.6 respectively.

Relative stereochemistry at CI in 1 was established based on observed coupling constants and ID-NOE data (Fig. 2.7). HI at 63.38 had a diaxial coupling associated with it. Also,

Hi, H3, H5 and H9 had ID NOE correlations to each other establishing that 1 was a lp,3p diol. The a- stereochemistry at C27 was determined by comparison of the chemical shifts of C12, C13, C14 and C18 with related compounds.^^"^'^ 49

29

OH

28

COOH COOH

HO' HO' 4

COOH

HO'

Plectranthoic acid B

•C C—c •OH

COOH

HO'

2',3' = cis = c/5-karenin 2'3' = trans - trans-kaxcnm

Figure 2.4. ip, 3(3-Dihydroxy-urs-12-en-27-oic acid (1), a-peltoboykinolic acid^^ (4) and related natural products 50

Table 2.2. '^C and 'H NMR data for lp,3p-dihydroxyurs-12-en-27-oic acid (1) and a- peltoboykinolic acid (4)" ''

Position Compound 1 Compound 4 I^C 'H multiplicity (J in Hz) ^^C 'H multiplicity (J in Hz) 1 80.9 3.38 dd (6.65, 9.39) 38.7 1.70, 1.07 2 38.3 1.74 ov 27.0 1.65 2.03 3 76.8 3.14dd(6.65, 10.08) 79.1 3.21 dd (6.5, 14) 4 39.8 38.6 5 54.5 0.57 d (10.54) 55.1 0.71 d(14.5) 6 18.9 1.56 dt (3.66, 13.29) 18.2 1.56 m 1.47 qd (2.29,13.29) 7 38.0 1.67 dt (2.98, 12.83) 36.6 1.67 1.19 td (3.67, 13.29) 1.15 8 41.2 39.8 9 48.9 2.48dd (5.04, 11.46) 46.8 2.21 dd (6.5, 14.5) 10 44.4 36.9 11 27.3 2.75 dt(5.27, 18.79) 22.7 2.03 2.09 m 1.92 12 130.6 5.52 dd (1.83, 5.27) 128.5 5.55 br s 13 133.9 133.2 14 57.3 55.9 15 23.5 1.95 dt(3.15, 13.35) 22.4 1.98 1.74 ov 1.76 16 30.3 0.84 ov 28.9 2.08 0.9 17 34.7 33.7 18 61.6 1.33 ov 60.2 1.34 m 19 38.8 0.97 m 39.8 0.86 m 20 40.9 0.87 ov 37.5 0.85 m 21 317 1.34 0V 30.4 1.34 1.24 m 1.24 22 42.2 1.41 m 40.9 1.41 1.26 m 1.18 23 28.6 0.91 s 28.1 0.96 s 24 16.0 0.74 s 15.7 0.78 s 25 12.9 1.02 s 16.5 0.96 s 26 19.4 1.06 s 18.2 1.02 s 27 179.3 179.8 28 29.7 0.83 s 29.0 0.80 s 29 21.7 0.89 d (5.04) 21.3 0.86 s 30 18.5 0.84 d (5.96) 17.8 0.79 s " ov = overlapped '' ''^C and 'H NMR for 1 and 4 were recorded in CD3OD at 125 and 500 MHz respectively; NMR data for 4 reproduced from Sun et al. 2003^^ 29

OH

COOH

HO

Figure 2.5. Significant DQF-COSY correlations of lp,3p- dihydroxyursl2-en-27-oic acid (1)

29

OH

COOH

HO

Figure 2.6. Significant HMBC correlations of ip,3P-dihydroxjairs- 12-en-27-oic acid (1) Figure 2.7. Significant ID NOE correlations of ip,3P-dihydroxyurs-12-en-27-oic acid (1) 53

Thus, the structure of 1 was established as ip,3p-dihydrox5airs-12-en-27-oic acid. This is the first report of compound 1. Related compounds with a carboxylic acid group at C27 have previously been isolated from nature and are presented in Figure 2.4, but are not

ubiquitous like their C28 counterparts.

2.7.2 Structure Elucidation of Caiophoraenin (2)

Caiophoraenin 2 (Fig. 2.3) was obtained as a pale yellow oil. Its HR-FABMS gave the

[M+Cs]"^ ion at m/z 363.0214 (Obs) suggesting a molecular formula of CnHigOs (Calc,

363.0203) corresponding to three degrees of unsaturation. The IR spectra indicated the

presence of a hydroxyl group (3388 cm"') and a 6-lactone group (1731 cm"'). The and

DEPT spectra indicated two methyls, two methylenes, six methines, and one quaternary

carbon. The quaternary carbon at 6175.3 indicated the presence of a lactone carbonyl.

Thus, an iridolactone skeleton was suggested for 2.

The carbon at 6175.3 could be assigned to the lactone carbonyl at C3. Also, observed in

the '^C spectrum were a methine at 675.0 and a methylene at 665.2 that could be

attributed to hydroxyl bearing carbons. The highly downfield methine signal at 696.9

indicated a dioxy substituted carbon. This signal was correlated in the HMBC to H9, and

was thus assigned as CI (Fig. 2.8). Also, HI and H9 had a coupling of 8.6 Hz between

them according to a DQF-COSY experiment (Fig. 2.9). The methyl signal at 652.3 was

identified as a methoxyl group, based on its correlation in the HSQC spectrum to a three 54

proton singlet at 53.68 and was assigned to the C12 methoxyl because of its ^/ch

correlation to CI in the HMBC. The oxymethine at 575.0 was assigned to C6 based on

the HMBC correlation of the H6 signal at 54.16 to C5 and C7. The hydroxy methylene at

565.2 could be assigned to CIO based on the HMBC correlation of H10a,b to C8 and CI.

The methyl doublet at 50.98 (Cll) was correlated to C5 in the HMBC experiment.

The '^C NMR data of 2 were comparable to gibboside aglucone (5, CioH|604)^^ (Fig. 2.10

and Table 2.3), the hydolysis product of gibboside isolated from Patrinia gibbosa

Maxim., except that 2 was a C9 epimer of 5, the hydroxyl group in 5 was located at C7

and there was a methoxyl group at CI in 2.

The four stereoisomers of irido-3-lactones encountered in nature are iridomyrmecin 6,

isoiridomyrmecin 7, epiiridomyrmecin 8, and isoepiiridomyrmecin 9 as shown in Figure

2.11. Iridomyrmecin (6) and isoiridomyrmecin (7) are both 5,9-c/5-irido-3-lactones but

differ in the configuration at C4. Epiiridomyrmecin (8) and isoepiiridomyrmecin (9) are

both 5,9-fran5-irido-3-lactones and also differ in the configuration about C4. Sisido et al.

established empirical rules to distinguish 5,9-cis from 5,9-trans lactones, and within 5,9-

cis lactones, between 6 or 7 type lactones based on coupling constants.^^ They reported

that in the case of 5,9-^ran5-irido-3-lactones, 7 and 8 both have a coupling constant of 9

Hz between H9 and HI ax and 4-5 Hz between H9 and Hleq, but differ with each other in

the orientation of the methyl group at C4. II HO CH

OCH OH

Figure 2.8. Significant HMBC correlations of caiophoraenin (2)

Figure 2.9. Significant DQF-COSY correlations of caiophoraenin (2) CH HO

10 OCH

2

CH

HO

OH

Figure 2.10. Caiophoraenin (2), and gibboside aglucone^^ 57

Table 2.3. '^C and 'H NMR data for caiophoraenin (2) and gibboside aglucone (Sf

Position Compound 2 Compound 5 '^C 'H multiplicity(7 in '^C Hz) 1 96.9 4.71 d (8.6) 69.5

3 175.3 176.4 4 39.9 1.88 ov 37.4 5 43.5 1.72 ov 41.1 6 75.0 4.16 dt (2.75, 5.96) 41.9 7 40.4 1.88 dd (8.8, 14)^ 75.6 1.72 dd (5.3, 14)" 8 39.7 2.4 m 49.4 9 54.2 2.16 dd (8.6, 11.85) 39.2 10 65.2 3.85 dd (4.12, 12.37)'' 61.5 3.35 dd (2.75, 11.91)'' 11 12.2 0.98 d (6.89) 13.9 12 52.3 3.68 s

" "C and 'h spectra of 2 recorded in CD3OD at 125 and 500 MHz respectively; '^C spectra of 5 was recorded in CDCI3 at 25 MHz [Uesato et ai, 1987]^' '' ov = overlapped 'fromHSQC '' from ID TOCSY 58

Figure 2.11. Iridomyrmecin (6), isoiridomyrmecin (7), epiiridomyrmecin (8), and isoepiiridomyrmecin (9) It has also been determined that 5-lactones can be assigned a boat or a half-chair conformation based on their carbonyl absorptions in the IR.57 A carbonyl stretching frequency of 1750-1765 cm ' indicates a boat form and a carbonyl stretching frequency at

1730-1740 cm ' indicates a half-chair conformation. It has further been shown that a half-chair 5-lactone must have a rigid 5,9-trans configuration, whereas, a boat 6-lactone must have a flexible 5,9-cis configuration.

A close scrutiny of ID NOE patterns and coupling constants, in conjunction with the above rules allowed the determination of relative stereochemistry of 2. The 6-lactone carbonyl at 1731 cm"' was characteristic for a half-chair conformation for the lactone ring and suggested a 5,9-trans ring junction as detailed above.^^'^^ The coupling constant of 8.6 Hz between the HI doublet and H9 indicated a diaxial relationship between the two and further confirmed the trans-fusion of the ring junction.^^

The H9 double-doublet possessed an additional diaxial coupling of 11.85 Hz, indicating a diaxial relationship with H5. NOE enhancement of H5 was not observed when H9 was irradiated in a selective ID NOE experiment. H6 was determined to be cis to H5 based on its coupling constants (Table 2.3). Also, H6 had ID NOE peaks to the Hll methyl and the H10a,b hydroxymethylene protons (Fig. 2.12). H8 and H9 were also related in a ID

NOE experiment. Thus, it was concluded that 2 possessed a similar conformation and relative configuration as that of isoepiiridomyrmecin 9.^^ Therefore, the structure of 2 was established as la-methoxy-6a,10-dihydroxy-isoepiiridomyrmecin. This is the first report of compound 2. 60

Figure 2.12. Significant ID NOE correlations of caiophoraenin (2) 61

2.7.3 Identification of Isoboonein (3)

Compound 3 was isolated as a pale yellow oil. HR-FABMS resulted in the detection of the [M+H]"^ ion at 171.1027 (Obs) suggested a molecular formula of C9H14O3 (Calc,

171.1017). A quaternary carbon in the '^C spectrum at 5176.82 indicated a lactone group.

A methylene at 570.12 that correlated in the HSQC to two downfield proton signals at

54.38 and 4.16 indicated the attachment of the -0C(0). A methine at 576.25 that correlated in the HSQC to the signal at 54.03 indicated a free hydroxyl group. The methyl doublet at 51.05 correlated in the HMBC to C8 as well as through a V coupling to the methine multiplet at 51.8. These findings established the structure of compound 3 as shown in Figure 2.3. The I H and 13" C NMR and MS data of compound 3 were similar to those reported in the literature for isoboonein previously isolated from Rauwolfia grandiflora Mart.^'' This is the first report of 3 from the genus Caiophora.

Isoboonein has also been isolated from the Chinese medicinal plant Cymbaria mongolica

Maxim. (Scrophulariaceae), the rhizome of which is used for the treatment of rheumatism

and dermatosis, and tested for cytotoxicity and antibacterial activity.^^ Isoboonein had

some activity against BS, EC, and S. aureus as shown in Table 2.4 in comparison to the control chloramphenicol. The activity was measured as zones of inhibition (in mm), or

the diameter of circles around a well containing the test compound where there was no 62

Table 2.4. Antibacterial activity of isoboonein from C. mongolica (as zones of inhibition in mm)"'''"^

B. subtilis E. coli S. aureus Isoboonein 13.510.6 12.011.5 10.111.4 Chloramphenicor 14.5±1.1 14.911.3 15.111.2 "Data reproduced from Dai et al. (2002)''°, source of bacterial strains was not reported ''Data expressed as mean ± standard deviations of triplicate determinations ' Control 63 growth of the test bacterium when the concentration of the compound was 10 )J,g/mL in the wells.^''

2.8 Antibacterial Activity

The antimicrobial activities of all isolates from C. coronata were determined against a variety of clinically relevant pathogens, namely, methicillin-sensitive and resistant S. aureus (MSSA and MRSA respectively), B. subtilis (BS), vancomycin-resistant E. faecium (VREF), E. coli (EC), E. coli imp (ECimp), and Candida albicans (CA) (Table

2.5). Compound 1 showed activity against MSSA and BS with MIC values of 4 and 2

|ig/mL, respectively. Interestingly, 1 was more potent against MRSA and VREF, with

MIC values of 4 jig/mL each, then the control Penicillin G. Also, 1 exhibited moderate activity against ECimp with MIC of 16 [Xg/mL. Compounds 2 and 3 were essentially inactive in our assay against all the bacterial strains tested with MIC values >128 |J.g/mL.

None of the compounds tested was active against EC or CA.

The bioassay directed isolation of C. coronata, structure elucidation of 1 and 2, and the biological activity of all isolates as presented here have been published in the Journal of

Matured Products, 2003, 65, 1628-1631 (See Appendix A). 64

Table 2.5. Antibacterial activity (MIC values in |ig/ml) of isolates from C. coronata

1 2 3 Penicillin Bacterial strain Bacillus subtilis 327 2 >128 >128 0.25 Staphylococcus aureus 375 4 >128 >128 0.06 S. aureus 310" 4 >128 >128 128 Enteroccocus faecium 319^ 4 >128 >128 32 Escherichia coli imp 389 16 >128 >128 4 E. coli 442 >128 >128 >128 32 Candida albicans 54 >128 >128 >128 >128 "methicillin-resistant ''vancomycin-resistant 'Control 65

2.9 LC/MS Survey

Inter-species differences in the phytochemical composition of plants from the same genus are well known. Even within a species, differences in the chemical compositions of different lines and acquisitions have been observed.^"^ The secondary metabolism of a plant depends not only on its evolutionary history and genetic makeup but also on environmental factors such as geography, soil, altitude, season of collection, part of plant collected, diseases or bacterial infections, external chemicals (fertilizers/pollutants) in the environment, and local predation.

In order to detect the compounds 1-3 isolated in this study from C. coronata (collected in

Argentina, Plant A) and to possibly identify an additional source for 1, an LC/MS study was initiated on the organic extracts of two other plants belonging to Caiophora in our collection- C. sepiaria (R. et Pav. ex G. Don) Macbr. (collected in Chile, Plant B) and C. coronata (Gillies ex Am.) (collected in Chile, Plant C), alongside standards for 1 and 2.

The CH2Cl2-MeOH (1:1) of Plant A (Sample A) was also included in the study as reference. Each of these plants (200 g dry weight) were pulverized and extracted with

CH2Cl2-MeOH (1:1). The TLC profiles (normal phase silica gel, 40% EtOAc in Hex) of the CH2Cl2-MeOH (1:1) extracts of Plant B (Sample B) and Plant C (Sample C) were very similar and less complicated when compared to the TLC profile of Sample A. 66

RPHPLC of 1 resulted in its elution at Rt= 54.9 min while detecting at 200 nm.

Compound 1 was detected as a cluster of ions with m/z 473.3 [M+H]"^, 455.3, and 437.4

in the ion trap at 54.9 min. This cluster was also accompanied with an ion at m/z 490.3,

which suggested an ammonium adduct of compound 1 [M+NH4]"^. MS/MS of the

[M+H]"^ ion also gave fragments at m/z 455.2 [(M+H)-H20]"^ and 437.2 [(M+H -HaO)-

H20]^. The UV spectrum of compound 1 and Samples A-C is shown in Figure 2.13. The

MS spectrum (Fig. 2.14) suggested the presence of compound 1 in Sample A, but was

indeterminate. When the peak at m/z 490.4 [M+NH4]'^ in Sample A was fragmented (Fig.

2.15), the fragmentation pattern resembled very closely the fragmentation of compound 1

in the MS spectrum (Fig. 2.14). This established the presence of compound 1 in Sample

A from which it was originally isolated. But, it was not detected in either Sample B or C

(Fig 2.13-2.14).

Compound 2 was not detected in the UV chromatogram at 200 nm, but, was detected in

the ion trap using a positive ESI source at m/z 252.9 [M+Na]"^ and 213.0 [M+H-H20]^

between 12.0-13.0 min. The m/z 213.0 ion was fragmented by MS/MS to fragments at

m/z 194.7 [(M+H-H20)-H20]^ 180.7 [(M+H-H20)-CH30H]^ and 162.7 [(M+H-H2O -

H20)-CH30H]'^. Since, 2 was not visible in the UV spectrum and did not seem to have a

sharp retention time, it was directly infused to the positive ESI source. Compound 2 was

detected again at m/z 213.0 [M+H-HjO]^ and 253.4 [M+Na]^ The MS/MS of the ion at

m/z 213.3 was the same as discussed above with major fragments at m/z 194.7, 180.7, and

162.7. Additionally, the MS/MS of m/z 252.9 gave fragments at m/z 234.8 [(M+Na )- 67

HaO]"^ and m/z 220.7 [(M+Na )-CH30H]'^. Thus, confirming that the peaks at m/z 213.0 and 252.9 were indeed from compound 2.

As shown in Figure 2.16 the [M+H-HaO]"^ fragment of compound 2 was detected in

Samples A-C at around 12.5 min. This ion was fragmented in samples A and C, and the fragmentation pattern matched that of the standard as shown in Figure 2.17.

In the absence of a standard, compound 3 was first identified in Sample A, from which it was originally isolated based on LC/MS/MS fragmentation pattern and then detected in

Samples B and C based on retention time and the observed [M+H]"^ ion. In Sample A an ion at m/z 171.6 corresponding to the [M+H]"^ ion was observed at Rt=12.7 min (Fig.

2.18). This ion was selected for MS/MS and resulted in a most abundant fragment at m/z

152.7 corresponding to the loss of a water molecule by the parent ion at m/z 171.6 (Fig.

2.19). Samples B and C also indicated ions at m/z 171.9 (Rt= 12.5 min) and 171.6 (Rt=

12.6 min) respectively (Fig. 2.18).

Thus, 2 and 3 were detected in all three samples whereas, 1 was detected only in sample

A from which it had been originally isolated. This suggests that either compound 1 is not accumulated by C. coronata (Chile, Plant B) and C. sepiaria (Chile, Plant C) or that it is present in very low concentrations and went undetected in the present study. Compound 1

DA01 ,A, Stg-2Ml nm Sample A

•.A01 A, S«g=2S''0 nm Sample B

DAD1 A, Srg-200 nrr. Sample C

u.n

Figure 2.13 Compound 1 from C. coronata. Samples A-C: HPLC chromatograms at 200nm. 69

iftf" 455,3 +MS. 54.Smin 0,8-

o.e- 409.5 0,4- 391.7 437.4 490.3 0,2- 0,0- 40G

Compound 1 at 54.8 min

Inlsjis. " xlSV "i-MS, 54,9min 1,0- 454.1 ^''5.5 0,S ae- 0,^ - 49(3.4 0,2- 47^ O.D 10?5 ••1'—'300 X5DD m^2

Sample A at 54.9 min

•n^|igr +MS. 54.9mln 1 454.0475.4 o.s-

0.6 D.4 0.2- 0. mQ 400 m

Sample B at 54.9 mm

ntferssrxtw- +MS, 64 9min 1.,e- '""•irsA 0.^- dx 0.4- U2 •n-.* 2W m 4DD .5S0 •"•T—'T—'r""!800 i"'mli I*

Sample C at 54.9 nun

Figure 2.14 Compound 1 from C. coronata. Samples A-C: MS spectra 70

+MS2(490.4), 54.9mtn 473.2

4Q9,2 455.2

391,2 319.2

Sample A at 54.9 mia

Figure 2.15 Sample A: MS/MS spectra at 54.9 min mTiens 213.0 +MS. 12.5min

Compouad 2 at 12.5 inin

214.0 •••MS, I2.4tnin 245,7 391,4

284,0 1:09,5 135,4

IDO 150 Sample A at 12.4 nnii

taSefis +MS, 12.7min

391.4 3715

283 7 316,3 339,7 155.3 186.7 244.2

0^ ID

Sample B at 12.7 mm

+MS, 12,5min

391.4 245.4 349,1 360,6 381,3 2306 258,5 171.8 267.0 287.7 121.6 139.2

Saiiqjie C at 12.5 mm

Figure 2.16 Compound 2 from C coronata. Samples A-C: MS spectra 72

inse?$s xiO^ -PvlS2(213.0), 12.5min n0.7

110.7 f'4.7 « 2.7 166.7 92.7 102.6 1 116.7 J 194 J 144.6150 7 1 176.6 . .1 1. HI I 1 I.I 1. ilr . 1 1 1 1. 1. *

Compoimd 2 at 12.5 mm

+MS2C214.0), 12.6min

Sample A at 12.6 imii

h-MS2{213.8), n.Smm 1807

134.8 162.8 92.9102.8 38.ff®p8 1166,8 194.7 •••I. • I- I.I'- • "r- 60 80

Sample C ai 12.6 min

Figure 2.17. Compound 2 from C. coronata. Samples A, C: MS/MS spectra 73

171,5

153.7 176.3 ie6„9 213.5 160.1 243.6

180 20D mlz

Sample A at 12.6 mis

240.0

208,5 214.,6 245.1 0.6 153.6 171.9 196,9 229

0,D 140 1M ISO 260

Saiaple B at 12,6 min

+MS, 12.6min 258,5 2,0 171.6 ie2,3 f.D 141,3

14D 180 240 mfz

Sample C at 12.6 mm

Figure 2.18. Samples A-C: MS spectra 74

+MS2(1716), 12,7min 152.7 ^ '

134 S 923 ^ j 141,0 ) M+H".• _^j y- ^ " 1 •' ^ '""t" —"—"t—''' 'v'" T 1 f*- r" 60 SO 1DD 12D 140 160 180

Figure 2.19. Sample A: MS/MS spectrum at 12.7 min 75

Compound 1, was isolated as the active principle from the antibacterial extract of Plant A with moderate activity against MSSA, and BS and potent activity against MRSA and

VREF (Table 2.5) as already discussed. Interestingly, the extract of Plant A was active in the initial survey against MSSA and MRSA but neither Plant B nor Plant C extracts were active, suggesting that compound 1 in Plant A was being produced either exclusively or in higher concentrations by the Argentinean plant.

The reason(s) for the specific or elevated accumulation of compound 1 by Plant A remains to be uncovered. The selective biosynthesis of 1 may be in response to bacterial infection faced by the plant or it may be due to the fact that the region where the particular collection was made is extremely arid. This is a good example of plant-to-plant differences in secondary metabolism within a species (C. coronata in this case) depending on the location of the plant and perhaps in response to local predation.

Similar plant-to-plant differences in chemistry have been seen before as in the case of anti-HIV calanolides isolated from Calophyllum lanigerum var. austrocoriaceum collected in Sarawak, Malaysia. When additional trees of the same species were explored to identify a possible source for the pre-clinical development of calanolides A and B, it was found that not a single tree produced the desired compounds. Consequently, the pre­ clinical development of this compound that is very potent against drug resistant strains of

HIV-1 now awaits the resolution of the supply problem.^' 76

The LC/MS data presented here also lend further support to the claim that iridoids are chemotaxonomic markers in the genus Caiophora and more generally in Loasoideae."^^

2.10 Experimental Section

All general instrumentation and experimentation is described in Chapter 7. Experimental conditions and techniques unique to the C. coronata work are presented here.

2.10.1 NMR Experiments

Chemical shifts were expressed in ppm (6) using partially deuterated solvent chemical shifts at 549 (''^C) and 63.31 ('H) as reference. The mixing times used in recording selective ID NOE were 400 ms for 1 and 700 ms for 2. Mixing times for selective ID

TOCSY spectra were 60 ms for 1 and 20 ms for 2. ID and 2D NMR spectra for 1 and 2 are included in Appendix B.

2.10.2 Plant Material

Plant A: Aerial parts of C. coronata were collected and identified by Renee H. Fortunato

and R. Kiesling on January 22, 1997, 28 kilometers west of Puesto de Gendarmeria in the

province of San Juan, Argentina (30° 21' 49" S; 69° 41' 30" W) at an altitude of 3550 m.

A voucher specimen (RHF 5574) has been deposited at the Instituto Nacional de 77

Tecnologia Agropecuaria (INTA), Buenos Aires, Argentina. Intellectual Property Rights

Agreements for plant collections and collaborative research have been fully executed between the University of Arizona and INTA.

Plant B: The whole plant C. coronata was collected by Luis Gonzalez on January 15,

1995, 550 km north of La Serena in the district of La Serena, Chile (29° 48' 3" S; 70° 2'

7" W) at an altitude of 3600 m. The area where the collection was made was rocky. This

plant is locally called charrua and is used as a stimulant by the local people.

Plant C: The leaves, flowers and stem of C. sepiaria were collected by Gloria

Montenegro, L. Iturriaga, and Luis Gonzalez on December 13, 1995, 7 km from Camino

a Putre in the District of Arica, Chile (18° 20' 31" S; 69° 50' 59" W) at an altitude of

1895 m. This collection was made from a region with a desert plain topography. The

plant is locally called atapilla or ortiga maQho and it has a variety of local medicinal uses

including antidiarrhea, anticonvulsive, fever reducing, contraceptive, and purgative.

2.10.3 In Vitro Antimicrobial Activity Testing

The in vitro antibacterial activities were determined by the agar diffusion on microbroth

dilution method as previously described.^^ The minimum inhibitory concentrations

(MICs) were determined by the microbroth dilution method as previously described.^^

Briefly, microtiter plates containing 2.5 |iL per well of 2-fold serial dilutions of each antimicrobial agent were inoculated with 100 |xL of bacterial suspension in Mueler-

Hinton II broth to yield the appropriate density (1-5 x 10^ CFU/mL). The plates were incubated for 18 h at 35° C in ambient air. The MIC was defined as the lowest concentration of a compound that completely inhibited the growth of the organism as determined by the unaided eye.

A modified version of the agar diffusion method was used to evaluate the activities of the crude extract, fractions and pure compounds against selected bacterial isolates. Assay plates (12 X 12 in Sumilon) were prepared by pouring 125 mL of agar medium (tempered at 50° C) inoculated with an overnight broth culture of the test organisms (adjusted to a final inoculum density of approximately 10^ cells per mL). The medium was allowed to solidify, and 144 wells (5 mm diameter) were bored into the agar layer using an automated plate-welling machine. Twenty microliters (20 |xL) of the test compound (10 mg/mL in DMSO) was dispensed into agar wells, and the plates were incubated at 37° C for 18 h. The zones of growth inhibition were measured using a hand-held digital caliper.

Fractions showing zones of inhibition of 8 mm or greater were selected for further

purification.

2.10.4 LC/MS Instrumentation and Technique

Sample Preparation. 0.0074 and 0.0072 g of CH2Cl2-MeOH (1:1) extracts of C. coronata (Chile, Plant B) and of C. sepiaria (Chile, Plant C) respectively were each 79 dissolved in 1 mL MeOH. The resultant solution was filtered through a 0.45 fi PTFE syringe filter (Whatman, Clifton, NJ) and transferred into 2 mL HPLC vials.

Standard Preparation. A solution of 0.00141 g/mL MeOH of compound 1 and 0.00209 g/mL MeOH of compound 2 was prepared. Each HPLC injection utilized 20 |J.L of these stock solutions.

HPLC/DAD. The mobile phase consisted of acetic acid (0.05%) in water (solvent A) and acetic acid (0.05%) in acetonitrile (solvent B) gradient, starting at 100% solvent A and going to 100% solvent B in 60 minutes, and holding at 100% solvent B for 20 minutes.

The chromatograms were recorded at 200, 220 and 280 nm and the spectral data for standards 1-2 and samples A-C were accumulated in the range of 190-450 nm.

2.10.5 Spectroscopic Data

ip,3p-Dihydroxyurs-12-en-27-oic Acid (1): white powder; m. p. 173-174 °C; [a]^^D

+62° (CH3OH, c 0.045); IR (KBr) Vn,ax 3360, 2947, 2183, 1686, 788 cm"'; 'H and '^C data in Table 2.2; For C30H49O4 HRFAB^ exact mass: Calc, 473.3619; Obs, 473.3629, m/z 455 and 437.

Caiophoraenin (2): pale yellow oil; [a]^^D +58° (CH3OH, c 0.31); IR (KBr) Vmax 3388,

2954, 1731, 1437, 1261, 783 cm '; 'h and ''^C data in Table 2.4; For CnHigOsCs

HRFAB^ exact mass: Calc, 363.0203; Obs, 363.0214. 80

CHAPTER HI

GROWH INHIBITION OF MYCOBACTERIUM TUBERCULOSIS BY THE

CONSTITUENTS OF MYRCIANTHES COQUIMBENSIS

3.1 Introduction

The alarming increase in the number of deaths due to tuberculosis (TB) and growing resistance to existing drugs has created an urgent need for the identification of leads for new antimycobacterial drugs. Our Latin American ICBG program routinely screens plant extracts collected in the semi-arid regions of Argentina, Chile and Mexico for their activity against Mycobacterium tuberculosis (MTB). Using this bioprospecting approach to drug discovery, an extract of Myrcianthes coquimbensis (Bameoud) Landrum et Grifo

(Myrtaceae) was found to inhibit the growth of MTB by 73% at a concentration of 50 jXg/mL. Based on this important activity this plant was selected for further investigation.

M. coquimbensis was collected in Coquimbo, Chile, which is one of the 25 remaining

'hotspots' of biodiversity^^'^"^ and is used by the indigenous people as a stimulant and digestive and locally called mirto.

The bioassay guided isolation of this plant led to the isolation of a new monoterpene,

(15,35,4/?)-1-methyl-4-( 1 -methylethenyl)-1,3-cyclohexanediol (3 p-hydroxy-cw- P- 81 terpineol, 10), along with oleanolic acid (11), 3p-caffeoyl-12-oleanen-28-oic acid (12), trans-(+)-sohrcm\ (13), epi-catechin (14), and catechin (15). The structure elucidation of

10 as well as the identifications of 11-15 is presented. All isolates were assayed against

MTB in vitro and evaluated for their cytotoxicity and the results from these biological assays are presented.

3.2 Distribution and Botanical Features

The Chilean Myrtaceae are endemic to coastal Chile from Coquimbo to the southern tip of South America. They grow in the humid temperate forests of southern Chile and adjacent Argentina and in Mediterranean sclerophyllous vegetation of central Chile, one of the 25 remaining 'hotspots' of biodiversity or areas featuring exceptional concentrations of endemic species and experiencing exceptional loss of habitat.

They can be distinguished from other families based on their non-deciduous leaves bearing entire margins, whitish flowers bearing inferior ovaries and numerous stamens, unlignified portions of the plant dotted with glands containing viscous aromatics, and opposite leaves.^^

Myrcianthes is a genus of about thirty species, ranging from Mexico and the Caribbean to northern Argentina, Uruguay and north central Chile.^^ However, Myrcianthes coqumibensis has a fairly restricted distribution and is found from about twenty kilometers south (Tortoralillo) to forty kilometers north (Punta Calavera) of the city of La 82

Serena in Chile. It usually grows among large boulders on sea-facing slopes that receive a constant cool Pacific breeze.

The plant is a dense, rounded shrub bearing distinctive pentamerous flowers and strong

coriaceous leaves with revolute margins. The plant usually grows to be around 1.5 m in

height and bears 0.3 mm long hairs. The leaves are 1-3.4 cm long and 0.6-3 cm wide,

elliptic, ovate, obovate, or suborbicular in shape densely covered with hairs when young

and become glabrous with age. The flowers are pentamerous and have a petiole and a

solitary peduncle. The ovary is bi-locular bearing 5-11 ovules per locule. The fruit is a

berry, 1-2 cm in diameter, subglobose and bears a single seed.^^

3.3 Taxonomy and Nomenclature

The plant family Myrtaceae consists of two sub-families Leptospermoideae (containing

15 genera) and Myrtoideae (containing 16 genera) based on fruit structure. Myrtoideae

contains several genera including Myrcianthes, Austromyrtus, Blepharocalyx,

Calyptranthes, Campomanesia, Decaspermum, Eugenia, Marlierea, Myrceugenia,

Myrcia, Myrcianthes, Myrciaria, Plinia, Psidium, Rhodamnia, Syzygium, and

Xanthomyrtus.^^'^^

Until the 1988 clarification by Landrum, Myrcianthes coquimbensis had a certain degree

of confusion associated with its classification.®^ Originally described as Myrtus 83 coquimbensis by Bameoud in 1844, it was placed in a new genus Reichea by Kausel in

1940, who at the time also suggested Myrcianthes coquimbensis as a synonym. In 1941,

Burret suggested a new genus Aspidogenia for the same species based on the peltate placental shape being distinct from that of o\h&x Myrcianthes. In 1948 Kausel claimed that Reichea had priority over Aspidogenia because of his 1940 report where he also mentioned the peltate placental shape.

In 1988, Landrum and Grifo detailed the morphological similarities between

Myrtus/Reichea/Aspidogenia with those of Myrcianthes and acknowledged the variation in placental shape. They placed this plant as a distinct species under Myrcianthes while pointing out that placental condition alone was insufficient evidence for recognizing a separate genus.

The plant in this study was identified as Reichea coquimbensis (Bam.) Kausel after collection, but has been corrected to Myrcianthes coquimbensis (Bameoud) Landmm et

Grifo. 84

3.4 Chemical Composition of the Volatile Leaf Oil

The volatile leaf oil components of Myrcianthes coquimbensis have previously been evaluated by GC/MS and quantified (Fig. 3.1). Limonene was found to be a major component of the volatile fraction (14.54 ± 2.45%), followed by carvone (8.67 ± 1.09%) and a and (3-pinenes (7.20 ± 0.95 and 5.67 ± 3.35% respectively).

The volatiles of four other species of Myrcianthes have also been studied. The results from these studies are presented in Table 3.1.

This to our knowledge is the first effort to study the non-volatiles in the genus

Myrcianthes and the first evaluation of this plant in terms of the antitubercular activity of its organic extract. HaC" a-pmene 2-plieiiykAyt acetate fr£3«5-p-menrha-2,S-di«s-l-oi

HoC

H,C

-puifiie 2-phenyletIiyl alcohol carcacrol

CH, / H,C HjC OAc Imah'l acetate A

p-tlmjoae CK-Careeol

CH3 CHj H3C--Ti %'OH H,C OH CH2

luialool

CK-pinocarveol c'«-l{7),8-p-mentliadiea-2-ol

Figure 3.1. Volatile oil components of M coquimbensis [Tucker et al, 2002^^] 86

HsC

CHO -OH H,C HjC

H,C

H,C myrtenai m^Ttenol cMyophylleiie oxide

OH r^'

OH

p-cjmen-S-ol p-ciesol perilla alcohol

CH3 HsC-

V

CH2

p-meiitlia-1,3 ,S-ti pmocsTvone sabiaene

\ •OH

H,C

' //

veroenoiie irfiHS-carveoi sfjattoleaoi

Figure 3.1 (contd) Volatile oil components of M. coquimbensis [Tucker et al, 2002^^] 87

"3^- ^QH

H-X CH, HsC.

CHO H3C'-'^~==5 A a-eanipholejisl r«-p-meiitha-2,8-ciie!i-l-ol

p-isoprc^ylphenol liiiioHene terpinolene

HO CHj HO CH,

•CH, O

CTi-tiJialooI oside ^rflNi-imalool oxide 1,8-cmeoIe

H,C,

HO CH,

eletnol

Figure 3.1 (contd) Volatile oil components of M. coquimbensis [Tucker et al, 2002^^] 88

Table 3.1. Volatile oil components of oxhex Myrcianthes species

Plant Compound Percent Reference composition M. fragrans (Sw.) limonene 56% Tucker et al, J. Essent. Oil. McVaugh Res. 1992*^^ (Jamaica) a-terpineol 10.9%

M. callicoma a-pmene Carmen et al, Essenze McVaugh Deriv. Agrum. 1972^° (Argentina) limonene 1,8-cineole cedrol eugenol o-cresol p-cresol acetic acid butyric acid

M. cisplatensis limonene 6.5-22.1% Taher et al., Essenze Deriv. (Camb.) Berg. Agrum. 1983^'; (Argentina) Zygadlo et al., J. Essent. Oil. Res. I99f^ 1,8-cineole 13.5-40.7% geraniol 0-8.4%

M. pungens 1,8-cineole 13.5-45.9% Ubiergo et al., An. Assoc. (Berg.) Legrand Quim. Argent. 1986^^; (Argentina) Zygadlo et al. J. Essent. Oil. Res. 199772 limonene 1.8-17.3% pulegone 1-9.4% famesol 0-9% nerol 0-5.4% geraniol 0-4.5%

Myrcianthes sp. 1,8-cineole 52.8% Setzer et al, Planta Med. (Costa Rica) 1999^3 a-pinene 11.8% a-terpineol 11.7% 2-heptanol 11.1% 89

3.5 Statement of Purpose

Leaves, stems and fruits of Myrcianthes coquimbensis (Bameoud) Landrum et Grifo

(identified at the time as Reichea coquimbensis (Bam.) Kausel) were collected by Gloria

Montenegro and Luis Gonzalez on July 22, 2002, in Quebrada Honda in the district of

Coquimbo, 30 km north of La Serena, Chile at an altitude of 558 m (29° 38' 24" S; 71°

17' 13" W).

A CH2Cl2-MeOH (1:1) extract of the dried and ground plant material was observed to inhibit the growth of MTB by 73% at a concentration of 50 )J,g/mL. Since the CH2CI2-

MeOH extraction utilized dried plant parts, we proposed that non-volatile component(s) in the plant might be responsible for the antitubercular activity of the plant extract.

Although the volatile contents of this plant have been investigated, there is no previous report on non-volatile components of any plant belonging to the genus Myrcianthes. To this end, we proposed to carry out an antitubercular assay-guided fractionation of the

CH2Cl2-MeOH (1:1) extract in order to isolate the active principle(s), establish the chemical stmcture of all isolates using IR, MS, and ID and 2D NMR techniques as needed, and evaluate the activity of all the isolates against M. tuberculosis. 90

3.6 Bioassay Guided Fractionation

Dried and ground leaves, stem, and fruit of M. coquimbensis (490.7 g) were triturated with 5L n-hexane. The residual plant material (481.2 g) was dried completely and then extracted with CH2Cl2-MeOH (1:1, 3 L x 2). The filtrate was dried under vacuum to yield

100 g of the extract. This extract was partitioned between «-hexane (1 L x 2) and

Me0H/H20 (90:1, 1 L). The Me0H/H20 (90:1) soluble portion was adjusted to

Me0H/H20 (1:1, 1.8 L) and subsequently partitioned with CH2CI2 (1 L, 0.5 L, and 0.5

L). The CH2CI2 soluble filtrate was concentrated under vacuum to yield 19.1 g of the

CH2CI2 fraction. The Me0H/H20 (1:1) soluble filtrate was then partitioned between saturated BuOH (2 L) and H2O (1:1, 2 L). The BuOH soluble filtrate was concentrated under vacuum to give 38.6 gm of BuOH fraction. The n-hexane (FOOl), CH2CI2 (F002),

BuOH (F003) and H2O (F004) soluble fractions were evaluated for biological activity against M. tuberculosis (MTB, Table 3.2).

The activity was found concentrated in F002 (98% inhibition) and F003 showed only moderate activity (51% inhibition). Although their activities differed, F002 and F003 had very similar TLC profiles (Si gel, 50% EtOAc in n-hexane) and were thus combined. The combined F002 + F003 (56.7 g) was fractionated by column chromatography (Si gel,

1200 g, 63-200 |.i. Scientific Adsorbents Incorporated) employing a gradient of EtOAc in n-hexane (0-90%) followed by a MeOH wash. 91

Table 3.2. M. coquimbensis extracts and fractions; Yields and Antitubercular activity profile

Extract/Fraction Wt. (in g) % Yield" Activity against M. tuberculosis^ (as % inhibition)

Hexane extract 9.5 - 32

CH2Cl2-MeOH(l:l) 100 - 73 extract FOOl (Hexane soluble) 6.7 6.7 49 F002 (CH2CI2 soluble) 19.1 19.1 98 F003 (BuOH soluble) 38.6 38.6 51 F004 (H2O soluble) 34.9 34.9 19 "As a percentage of the CHaCla-MeOH (1:1) extract ''Tested concentration of extract/fraction = 50|ig/mL 92

Chromatographic fractions were pooled based on their TLCs into 44 fractions (F005-

F048) and all fractions were tested for MTB growth inhibition as shown in Table 3.3.

All fractions were tested for activity against MTB. F028 (0.3 g, 0.3%) eluting with 40%

EtOAc in n-hexane, was moderately active in its growth inhibition of MTB. It led to 70% growth inhibition at a stock concentration of 50 |a,g/mL. F028 was fractionated using preparative Si gel plates (Si gel 60, 0.5 mm, 20 x 20 cm, concentration zone 20 x 4 cm, F

254 |i, Merck; 50% EtOAc in Hex; Rf = 0.36). The PLC fraction containing compound

10 (0.031 g, 0.031%) was purified over a Sephadex LH-20 column (length = 250 mm, width = 20 mm) leading to the isolation of compound 10 as a colorless oil (0.0127 g,

0.013%) (Fig. 3.2).

Fraction eluting with 30% EtOAc in Hex (F015, 2 g, 2%) was the most active fraction inhibiting the MTB growth by 91% at a concentration of 50 ^ig/mL. F015 had one major spot on the TLC (Si gel, 50% EtOAc in n-hexane). It was dissolved in MeOH and centrifuged. The filtrate was decanted and concentrated. On slow evaporation of solvent white crystals of compound 11 (0.08 g, 0.08%) resembling cotton puffs were formed

(Fig. 3.2). In order to remove any impurities, these were washed repeatedly with MeOH, I 11 and finally analyzed by H and " C NMR for identification. 93

Table 3.3. M. coquimbensis chromatographic fractions: Yields and Antitubercular activity profile" Fraction Wt. (in g) % Yield'' Activity against M. tuberculosis (as % inhibition)'"'' F005 0.87 0.87 8 F006 0.81 0.81 16 F007 0.33 0.33 30 F008 0.31 0.31 50 F009 0.21 0.21 40 FOlO 0.47 0.47 41 FOl 1 0.43 0.43 40 F012 0.57 0.57 23 F013 0.97 0.97 53 F014 0.83 0.83 68 F015 2.0 2.0 91 F016 0.85 0.85 65 F017 0.24 0.24 54 FOl 8 0.80 0.80 NA F019 0.73 0.73 53 F020 0.18 0.18 NA F021 0.24 0.24 47 F022 0.14 0.14 58 F023 0.11 0.11 42 F024 0.05 0.05 65 F025 0.15 0.15 36 F026 0.50 0.50 53 F027 0.92 0.92 58 F028 0.30 0.30 70 F029 0.13 0.13 50 F030 0.30 0.30 60 F031 0.33 0.33 55 F032 0.80 0.80 60 F033 0.91 0.91 54 F034 0.45 0.45 53 F035 0.29 0.29 55 F036 0.43 0.43 NA F037 0.52 0.52 43 F038 0.53 0.53 73 F039 0.70 0.70 58 F040 0.64 0.64 50 F041 0.21 0.21 62 F042 0.06 0.06 56 F043 0.74 0.74 NA F044 0.51 0.51 7 F045 0.59 0.59 50 F046 0.17 0.17 50 F047 0.41 0.41 59 F048 32.12 32.12 10 "Fractions with % growth inhibition of M tuberculosis > 70% were selected for fractionation ''As a percentage of the CH2Cl2-MeOH (1:1) extract (100 g) 'Tested concentration of fractions = SOpg/mL ''NA = Not Active 94

F038 (0.53 g, 0.53%), eluting with 60% EtOAc in n-hexane, led to a 73% growth inhibition of MTB at a concentration of 50 |J,g/mL. F038 was fractionated on a Sephadex

LH-20 column (length = 250 mm, width = 20 mm), eluting with MeOH, and monitored by TLC. Further RPHPLC purification (CI8, 5|x; 80% MeOH in HjO - 100% MeOH, 12 min, 254 nm) resulted in the isolation of compound 12 (Rt= 9.3 min) as a yellow powder

(0.0018 g, 0.0018%) (Fig. 3.2).

Although F043 (0.74 g, 0.74%), eluting with 70% EtOAc in n-hexane, was ineffective in inhibiting the growth of MTB at the tested concentration of 50 |^g/mL, it had bright orange and a pink spots on the TLC and was further fractionated to isolate these components (Si gel, 13 g, 62-120 |j,, SAI; 0%- 80% Ace in n-hexane gradient). Fractions eluting with 15% Ace in n-hexane were further purified using RPHPLC (C18, 10 (X,

Econosphere; 30% MeOH in H2O - 50% MeOH in H2O, 15 min, 200 nm) leading to the isolation of compound 13 (Rt = 7 min, 0.0029 g, 0.0029%) (Fig. 3.2). Fractions eluting between 35-40% Ace in n-hexane were further purified using RPHPLC (C18, 10 |i,

Econosphere; 5% MeOH in H2O - 20% MeOH in H2O, 10 min, 200 nm) to isolate compounds 14 (R, = 2.2 min, 0.019 g, 0.019%) and 15 (Rt = 7.7 min, 0.0054 g, 0.0054%)

(Fig. 3.2). ^3% .OH

H3C 10 10 R = H 10s R = 5-MTPA lOr R = /?-MTPA

Figure 3.2. Isolates (10,12) from M. coquimbensis Figure 3.2. (contd) Isolates (11,13-15) from M. coquimbensis 97

3.7 Structure Elucidation and IdentiHcations

3.7.1 Structure Elucidation of (lS',35,4/?)-l-Methyl-4-(l-methylethenyl)-l,3-

cyclohexanediol (3P-Hydroxy-cis'-P-terpineol, 10)

Compound 10 was isolated as colorless oil, which on the GC/MS had a retention time of

12.5 min and gave a molecular ion peak at m/z 170, as well as fragments at m/z 152, 137,

119, 109, and 67 (Fig. 3.3). Fragments at m/z 152 and 137 can be easily explained as a result of the loss of a H2O molecule [M-HaO]"^ followed by the loss of a methyl [(M -

H20)-CH3] respectively. The peak at m/z 119 can be explained as resulting from further loss of H2O from the m/z 137 peak [(M-H20-CH3)-H20] ^ . Allylic cleavage of the fragment at m/z 137 resulted in fragment at m/z 109 (m/z 137-28) and a further loss of -

C(CH3)CH2 {m/z 108-41) explains the base peak at m/z 67.

The 13" C and DEPT experiments indicated the presence of ten carbon atoms out of which two were methyls, four methylenes, two methines, and two quaternaries. The downfield methylene and quaternary signals at 6108.98 (C9) and 148.56 (C8) respectively, indicated the presence of a disubstituted double bond (Table 3.4). Two other downfield signals- the methine at 674.40 (C3) and the quaternary at 671.70 (CI) - along with the two losses of water observed by GC/MS suggested the presence of two free hydroxyl groups. The I 3 C data along with the observed molecular ion peak at m/z 170 and fragmentation pattern in the GC/MS supported a molecular formula of C10H18O2 and was in accord with the proposed structure for compound 10 (Fig. 3.2). 98

Compound 10 is a C3 p-hydroxy derivative of cw-P-terpineol (16) and its 'H and '^C

NMR data is comparable to the latter (Fig. 3.4 and Table 3.4).^^

The hydroxyl group was assigned to C3 based on HMBC correlations between the methine proton at 53.53 and C4 (/^ch) and CI (Z'^ch) (Fig. 3.5). Additionally, ID-TOCSY experiments identified C2-C3-C4-C5-C6 [CH2CH(OH)CH(C(CH3)CH2)CH2CH2] as a single spin system. Excitation of the two methyl singlets or the methylene at 64.67 and

4.70 did not spread to other signals indicating their direct attachment to quaternary carbons. The methylene signals at 54.67 and 4.70 were correlated in the HMBC to C4

(7"CH) and CIO (/CH), confirming a terminal isopropylene group at C4 (Fig. 3.4). The methyl singlet at 51.7 had a f'cw correlation to the quaternary carbon at 5148.56 further

confirming this assignment. The other methyl singlet at 51.20 was correlated to CI (/^ch)

and C3 (7^ch) as well as to the methylene at 534.36 (C2). The 'H spectrum was further assigned based on the DQF-COSY experiment (Fig. 3.6). CO

Figure 3.3. EIMS spectrum of 3 P-hydroxy-c/.v-P-terpineol (10) 100

DH

HO'

IscA 10 10

PH

,.A 16

Figure 3.4. 3(3-hydroxy-c/s-|3-terpineol (10) from M coquimbensis, and cw-|3- terpineol (16) [Brecknell et al.,\99li'^ 101

Table 3.4. '^C and 'H NMR data for 3(3-hydroxy-cw-[3-terpineol (10), cw-p-terpineol (16) and (l/?,3/?,4/?)-l-hydroxy neoisopulegol (17)

Position 10" 16" 17c I3c 'H multiplicity (7 13c 'H I3c 'H in Hz) mult.

1 71.7 70.7 - 70.5

2 35.1 1.89 dt (2.29, 40.1 - 42.9 2.09 td (3, 12.83) 14.5,)

1.61 dt (2.29, - 1.51 dd (3, 12.83) 14.5)

3 74.4 3.53 m 28.9 - 68.5 4.09 m

4 38.8 2.26 m 44.5 - 48.5 1.99 m

5 34.4 1.72 ov 28.9 - 39.4 1.78 m 1.48 ov 1.45 m

6 27.6 1.61 ov 40.1 - 20.3 1.99 m

1.47 ov - 1.45 m 7 27.4 1.20 s 25.6 1.19 s 30.9 1.18s 8 148.6 149.6 147.3

9 108.9 4.67 s 108.5 - 112.0 4.97 m 4.70 s 10 21.0 1.72 s 21.0 1.67 s 23.1 1.78 m "ov= overlapped; H and C data for 10 recorded in CD3OD at 500 and 125 MHz respectively '' 'H and '^C data for 16 recorded in CDCI3 at 400 and 100 MHz respectively [Brecknell et a/.,1997]^' ''Hand '^C data for 17 recorded in CDCI3 at 500 and 125 MHz respectively [Fkyerat era/., 1997]^® 102

Figure 3.5. Significant HMBC correlations of 3P-hydroxy-c/5-(3-terpineol (10)

PH

HO

10 /8 HgC

Figure 3.6. Significant DQF-COSY correlations of 3|3-hydroxy-c/5-p-terpineol (10). 103

The relative stereochemistry of compound 10 was assigned based on ID NOE data (Fig.

3.7). The methyl singlet H7 was enhanced when the methine proton H3 at 53.53 was excited, allowing for the hydroxy at CI as well as C3 to be assigned as p. Additionally,

H9a,b were also enhanced when H3 was irradiated, suggesting that the isopropylene group was a. The absolute configuration of 10 was determined by comparing the 'H

NMR spectrum of the S and i?-MTPA esters, 10s and lOr respectively as shown in Fig.

3.8 and Table 3.5. Thus, compound 10 was assigned as (15',35',47?)-l-methyl-4-(l- methylethenyl)-1,3-cyclohexanediol.

Stereoisomers of compound 10 have been reported previously by synthetic methods (Fig.

3.9).'^ The difference between compound 10 and its stereoisomer (I/?,3/?,4/?)-l-hydroxy

I neoisopulegol (17, Fig. 3.9) is evident on comparison of the ID " C chemical shift data as shown in Table 3.4, in particular the upfield shifted axial methyl (C7) at 627.4 in the former is a characteristic of the naturally occuring cw-P-terpineol (16).^^'^^ Figure 3.7. Significant ID NOE correlations of 3(3-hydroxy-cw-P-terpineol (10)

0. ov

ov, -0.92

'3 +9.62 ^10.0 MO.O

lOs R = 5-MTPA lOr R = /?-MTPA

Figure 3.8 'h NMR Ad(S-R) values obtained for the MTPA esters 10s and lOr 105

Table 3.5 'H NMR data for 10s and lOr and A6 values obtained for the MTPA esters"'''

Position 10s 'H (in Hz) lOr 'H (in Hz) A6 (S-R) (in Hz) 2a 1418.85 1444.06 -25.20 2b 1223.19 1226.40 -3.21 3 2857.67 2848.05 +9.62

4 ov ov -

5a ov ov - 5b 1071.98 1072.90 -0.92 6a 1263.52 1263.52 0

6b ov ov - 7 1003.25 1001.88 +1.37 9a 2903.95 2894.33 +9.62 9b 2876.92 2866.84 +10.08 10 1025.24 1015.16 +10.08 "ov = overlapped '' Data recorded at 600 MHz in pyridine-<^5 106

10

-hydroxy neoisopulegol (1i?, 3 S,4R)- 1-hydroxy isopiilegol 17 '

H,Q .OH

HO'

H3C (1S,3S,4S)-1 -hydi'oxy neoisopulegol (1S.sRASy 1-hydroxy isopulegol

Figure 3.9. Synthetic stereoisomers of 3p-hydroxy, c/5-p-terpineoI (10) [Fkyerat et al, 19971^6 107

3.7.2 Identification of Oleanolic Acid (11)

Compound 11 (Fig. 3.2) was isolated as colorless crystals. The '"^C spectrum indicated thirty carbons: seven methyls, ten methylenes, five methines, and eight quaternaries, suggesting an oleanane skeleton. The presence of a carbonyl carbon, a downfield shifted methine along with a trisubstituted double bond indicated an oleanolic acid structure.

This was confirmed by comparison of the 13" C and I H chemical shifts with those reported in the literature.78

Oleanolic acid has been isolated as the active ingredient of many antitubercular plant extracts due to its moderate activity against MTB.^^"^^ However, this is the first report of its isolation from M. coquimbensis. Structure-activity relationships within oleananes and other triterpenoids are dealt with in Chapter 6.

3.7.3 Identification of 3p-Caffeoyl-12-oleanen-28-oic Acid (12)

Compound 12 was isolated as a white powder. The 1 3C and DEPT experiments indicated the presence of thirty-nine carbons: seven methyls, ten methylenes, ten methines and twelve quaternaries. Downfield quaternary carbons at 5169.05 and 175.07 indicated acid/ester carbonyls. The seven methyl singlets and a trisubstituted double bond between the methine at 6123.08 and the quaternary at 6145.39 indicated an olean-12(13)-ene skeleton for compound 12. 108

The three downfield aromatic methines with 'H signals at 57.0, 6.9 and 6.7, and the two downfield aromatic quaternary signals at 5149.2 and 146.6 suggested a caffeoyl side chain. Further, the methine at 54.56 was correlated in the HSQC to the signal at 682.26 and in the HMBC to the two geminal methyls at C4 (C23 and C24), suggesting a C3 attachment of the caffeoyl side chain. These and other 'H and ' "^C data were consistent with those reported in the literature for 3P-caffeoyl-12-oleanen-28-oic acid (Fig. 3.2).^"^

Thus, compound 12 was identified as 3P-caffeoyl-12-oleanen-28-oic acid.

This compound has previously been isolated from the bark of several birch trees belonging to the genus Betula (Betulaceae): B. schmidtii Regel., B. davurica Pall., B. maximowicziana Sieb. & Zucc., B. ermanii Cham., and B. pubescens Ehrh. 83'" 89 It has also been isolated from Geranium niveum S. Watson^° and Celastrus stephanotiifolius

(Makino) Makino.^' This is the first report of the isolation of 12 from M. coquimbensis.

Compound 12 is associated in the literature with a wide range of biological activities. It has been isolated from evening primrose oil and determined to be a potent inhibitor of neutrophil elastase and cyclooxygenase-1 and 2. It was also determined in the same study to have pronounced radical scavenging activity against 2,2-diphenyl-l- picrylhydrazyl radical. Compound 12 from Shorea wangtianshuea Yang et Wu has been reported to afford significant liver protection on CCU-induced hepatic injury in mice.^^

Additionally, it has been reported to have cytotoxicity against several human tumor cell 109 lines; A549 (non small cell lung), SK-OV-3 (ovarian cancer), SK-MEL-2 (melanoma),

XF498 (central nervous system), and HCT-15 (colon).^"^

3.7.4 Identincation of Trans-i+)-sobrero\ (13)

Compound 13 (Fig. 3.2) was isolated as colorless needles. The I '3 C and DEPT spectra showed ten carbons- three methyl, two methylene, three methine and two quaternary carbons. The methine signal at 5125.15 and the quaternary at 5134.39 suggested a

trisubstituted double bond. The downfield methine at 568.26 (C6) and quaternary at

571.78 (C8) indicated two free hydroxyl groups. A slightly downfield methyl singlet at

51.78 was assigned to the methyl (C7) next to the double bonded quaternary carbon (CI).

The other two methyl singlets at 51.18 (6H) were assigned to H9 and HIO, attached to

C8, the quaternary at 571.78. A ID NOE experiment irradiating H6 did not enhance H4

and vice-versa, suggesting a trans relationship between the two signals. Thus, compound

13 was identified as ?ran5-(-i-)-sobrerol and its 'H and '"^C were comparable to those

reported in the literature.^^ This is the first report of the isolation of 13 from M.

coquimbensis.

Compound 13 is presently used as a muco-modifying drug Sobrepin® (Camillo Corvi

S.p.A., Piacenza, Italy) in the treatment of respiratory diseases with bronchial obstruction

caused by mucus hypersecretion.^^ 110

3.7.5 Identification of Epi-catechin (14)

Compound 14 (Fig. 3.2) was isolated as an orange powder. The '^C and DEPT spectra indicated the presence of sixteen carbons: one methylene, eight methines and seven quaternary carbons. The absence of a carbonyl group, and the presence of one methylene signal and two downfield methines indicating oxygenation at 682.85 and 68.81, indicated a flavanol skeleton.

As expected for flavan-3-ols, the methylene signals at 62.86 (IH; dd, 7= 5.5, 16.0) and

62.52 (IH: dd, J = 8.2, 16.0) were observed as double doublets. The H3 methine was

observed at 63.99 (IH: dt like, J= 5.5, 7.6, 7.8) and the H2 methine was observed as a

doublet at 64.58 (IH: d, 7 = 7.6).

Additionally, in the ID NOE experiment, when the 64.58 doublet was irradiated the

signal at 63.99 was enhanced. Thus, compound 14 was identified as epZ-catechin (Fig.

3.2). Its '^C data were comparable to those reported in the literature.^^ This is the first

report of the isolation of 14 from M. coquimbensis.

3.7.6 Identification of Catechin (15)

Compound 15 (Fig. 3.2) was isolated as an orange powder. It was similar to compound

14 in the 'H experiment. But, in a ID NOE experiment, when the 64.58 doublet was Ill irradiated the signal at 63.99 was not enhanced. Thus 15 was identified as catechin (Fig.

3.2). Its '"^C data were comparable to those reported in the literature.^^ This is the first report of the isolation of 15 from M. coquimbensis.

Flavonoids are associated with a wide spectrum of pharmacological activities and catechchin and ep«-catechin are well-known antioxidants and are present in large amounts in green tea.^^''"®

3.8 Antitubercular Activity

All isolates obtained from M. coquimbensis were evaluated for their potential to inhibit

the growth of M. tuberculosis and African green monkey Vero cells according to

established protocols as described in the Experimental Section (Section 3.10.4). The

results as shown in Table 3.6 suggest that compound 11 afforded growth inhibition with

an MIC value of 29.66 jig/mL whereas the other isolates were virtually inactive. When

tested for cytotoxicity against a mammalian cell line, 11 was less cytotoxic than the

positive control rifampin.

Compound 11 has been isolated as the active ingredient of many antitubercular plant

extracts due to its moderate activity against MTB.^^'^^ Structure activity relationships

within oleananes and other triterpenoids are dealt with in Chapter 6. 112

Table 3.6 Antitubercular activity of M. coquimbensis isolates"

10 11 12 13 14 15 Rifampin MIC" >128 29.66 >128 >128 >128 >128 0.049 (^ig/ml) IC50' ND >102.4 ND ND ND ND 90.64 (|ig/ml) sr' ND 3.45 ND ND ND ND 1850 " ND= not determined ''Minimum inhibitory concentration (MIC) against M. tuberculosis H37RV ' Cytotoxicity against African green monkey Vero cells '' Selectivity Index (SI) = IC50/MIC. Compounds with SI > 10 are selected as drug leads. 113

3.9 LC/MS survey

In order to determine the phytochemical compositions of different accessions of M. coquimbensis in our collection and to identify potential sources for the compounds isolated in this study from the antitubercular extract of M. coquimbensis, an LC/MS study on their organic extracts was initiated. Two different accessions of M. coquimbensis, besides the one reported here to carry out the bioassay directed fractionation (Plant D), were available in our collection. All of these plants were collected from the biodiversity

'hotspot' in Coquimbo, Chile^^'^'^ but from different locations, altitudes and at different times over a six-year period. In each collection different plant parts were included based on the developmental stage of the plant at the time of collection. The LC/MS survey was conducted on the CHiCla-MeOH (1:1) extracts of these plants alongside standards for 3(3- hydroxy-cw-P-terpineol (10), oleanolic acid (11), rran5-(-i-)-sobrerol (13), and epi- catechin (14).

M. coquimbensis (Plant D) was collected by Gloria Montenegro and Luis Gonzalez on

October 31, 2001 from Quebrada Honda in the district of La Serena, 30 km north of

Caleta Homo, Chile at 558 m above sea level (29° 38' 24" S; 71° 17' 13" W). When the collection was made, the plant was fruiting and the fruit was included in the collection besides leaves and stem. 114

M.coquimbensis (Plant E) was collected by Gloria Montenegro and Luis Gonzalez on

August 31, 2000 15 km north of La Serena in the district of Coquimbo, Chile. This collection included leaves and stems only.

M.coquimbensis (Plant F) was collected by Luis Gonzalez on October 10, 1994 from 48 km north of La Serena in the district of Coquimbo, Chile at 170 m above sea level (29°

59' 52" S; 71° 22' 29" W). This collection also included leaves and stems only. The soil from which it was collected was rocky and the area was hilly.

One hundred grams each of dried and pulverized Plants E and F were further powdered and extracted with CH2Cl2-MeOH (1:1). The crude extract was then partitioned to effect a polarity-based fractionation and facilitate detection of minor components on LC/MS.

The extract was first partitioned between MeOH and n-hexane and subsequently the

MeOH soluble portion was partitioned between CH2CI2 and water. The CH2CI2 fractions of Plants E and F (Samples E and F), along with the CH2CI2 soluble fraction (F002) of

Plant D (Sample D, fractionation described earlier), were used for the LC/MS study.

Samples D-F were analyzed by LC/MS/MS using electrospray ionization (ESI) in positive ion mode coupled to an ion trap detector. These were run alongside standards to facilitate detection based on retention time, molecular weight as well as observed fragmentation. Oleanolic acid 11 was detected at 200 nm, using a DAD coupled to the HPLC, at Rt=58.9 min and observed by MS at m/z 439.3 [M+H-H2O]'*". It was further fragmented to give ions at m/z 421.2 [(M+H-H20)-H20]^ 393.1 [(M+H-H20)-C02H-H]^ and other lower mass fragments.

As seen in Figures 3.10 and 3.11, compound 11 was identified at Rt ~ 58.9 min and m/z

439 in all three samples. The fragmentation patterns of all these ions matched the expected fragmentation for compound 11 as seen in Figure 3.12.

rran5-(+)-Sobrerol 13 was observed at Rt=19.9 min detected at 200 nm on the HPLC using a DAD detector, and at m/z 170.4 [M+H]"^ by MS. This ion was further fragmented to ions at m/z 152.7 [M+H-H2O]'", 134.7[(M+H-H20)-H20]^, and 94.8[(M+H-H20)-

CsHyO+H]^. As seen in Figures 3.13, 3.14, and 3.15, compound 13 was detected in all three samples based on retention time, observed molecular ion and fragmentation pattern.

£/7/-catechin 14 was eluted from the HPLC at Rt= 13.9 min, detecting at 280 nm. On the

MS this corresponded with m/z 291.1 [M+H]^. This ion was fragmented by MS/MS to ions at m/z 272.8 [(M+H)-H20]'^, and other fragment ions. As seen in Figures 3.16, and

3.17, compound 14 was detected in all three samples based on retention time and observed [M+H]^ ion. Comparable MS/MS fragmentation of the m/z 291.1 ion was observed for Samples E and F as seen in Figure 3.18. 116 sp-Hydroxy-cw-P-terpineol 10 was eluted from the HPLC at Rt= 20.6 min, detecting at

200 nm. On the MS this corresponded with m/z 193.3 [M+Na]"^. This ion was fragmented

to a major fragment at m/z 174.7 [(M+Na)-H20]"^, and other minor fragments. As seen in

Figures 3.19, and 3.20 compound 10 was detected in Samples D-F based on retention

time and observed [M+Na]"^ ion. The MS/MS of m/z 193.3 in the standard and Sample E

observed a similar fragmentation pattern as seen in Figure 3.21. Due to its low

abundance, 10 was not further fragmented in Sample D and F and was identified in these

based on retention time and observed [M+Na]^ ion. DADf A, Stg=20G- nm Compound 11

DAD1 A, Sig=2DD nm Sample D

DAD 1 A, Sig=200 nm Sample E

DA01 A. Sig=20D nm Sample F

1GCH

55.S

Figure 3.10. Compound 11 from M. coquimbensis. Samples D-F: HPLC chromatograms at 200nm 118

xio" • l-Q- 439.3 +MS, 59.0min <

S;6' QA'

0.2' 411.4 O.Q m 2m m 4m 60Q 7^ ^0 m mtt

Coiiipomid 11 at 59.0 mm intens 439.3 •^mS. oB.Bmm

411.4

200 3D m/z Sample D at 58.9 mm

4; l9.3 +IV1S, ss.Mn

411,3 1—1 "r'H "v 1 • 'i-'-r T" "i- —1' ••T"i "f f 1 1—1—r- 1 1 1 1 • \ 1 1 1 ^ t 1 V 1 1 I 1G0 200 300 400 .^0 600^ im 800 500 miz

Sample E at 59,0 mui

439.3 +MS, 58.9min

T SOGT"

Sample F at 58.9 nisn

Figure 3.11. Compound 11 fromM coquimbensis, Samples D-F: MS spectra 119

Eflfens . •xAD*. +MS2(439.3), 59.0min 190.8

202.8 393,1 216.8 134.7 160.6176. I 248.9 315.0 4212^ . •» ''I I ' . -LI • j ' . ^ • I I. • 50 150 200 250 300 3-50 4C0 450 mj'z

Cornpoimd 11 at 59.0 min

Intern x\Q*- +MS:2(439.3), SB.Smin 3 - 190.8

202.i 393.2 132^g 162.6174^5 1216 8 248.9 351.2 3S8.9 421. Q ' r • I »—I—'—r I I V* I"'!—1 r f'-f 'f 'I r"I' ^ 'I'—R"-!—1—t I" H—R- r—T—1—'—I—1—T— 50 m 150 2G0 300 350 4m 4&0 irifz

Sample D at 58.9 miii miefss - +MS2|439.3). 59-Ornin

202.9 421,2 1—I—1—^ m!z

Sample E at 59.0 mm

x1Cf 15- +MS2(439.3). 59.0min 190.8

t.D- 393.2 OJ- 2 32.9 216.^3Q g24g Q .,34 gl62.6174.pl 315.0 351.1 421.2 o.§- • ,il . Ill .III , h , • 1^1/, -LJ ,-L, ,1 J I V . / ^ t —i—I 5G MO 200 zfe ' 400 44O

Sample F at 58.9 ima

Figure 3.12. Compound 11 from M. coquimbensis, Samples D-F: MS/MS spectra >1 A. Sja==2D0 nm Compound 13

DA01 A, Sample D rx-

DA01 A. $ig=200 nm Sample E

DA01 A, Sig=20B nm Sample F

Figure 3.13. Compound 13 from M. coquimhensis. Samples D-F: HPLC chromatograms at 200nm

oto 121

+MS, I9.9mln 135.4 170.4

153.4 193.3 ,391.6 95.I°PI23 1 227.4 371.7' II. ,..L J ' 11.J^ ^iftiii|ii Im "f'-r ^lULof '•1' "T 100 150 200 2S0 3DS

Cotiipoimd 13 at 19,9 mm

mterra +MS, 20 0min 303.4

235,7'^¥' 316.6 107,4^3 2 170.4184,5

5 ICS 150 Sample D at 20,0 min

»10' 5®.0min e,e- 253,8 303,5 236,1 270,8 391.4 93,4109,2^^ 6155-6 ^ 6

100 1SJ Sample E at 20.0 min

*10'' +MS. 20.0min .303,3

235,8 253,9 293,7 391,3

Sample F at 20.0 mm

Figure 3.14. Compound 13 from M. coquimbensis. Samples D-F: MS spectra 122 lc»te>ns +MS2( 170-4), 20.0min U32.7 e - 9 t,3 4.7 4 -

2 106,8 1 p ..1.. 1, • efa ' m ' 100 • tio ' 140 ' im ' iso ' miz

Confound 13 at 19.90 mm l^ntens; +MS2C170,4), 20.1min 1500": 15 2.6

9^t ..8 4,7 500 •: 1 106,7 124,7 1 1. 82,8 , 1 0 1. . [ II ll.. Li.i . 1 . 11 1. 1 , _IL sb ' sb ' i3o ' tio ' i4o ' tte ' ite ' n>fz'

Sample D at 20.0 mm interns +MS2( 170,6), 2Q,0m:ln 1! .2,6 4.7 9 4.7

; 82.8 106,7 -TN , -N R-" ill-, Luj ^ Ji I—T 1^——, R—

Sample E at 20,0 nua

2^ +MS2(170,4),20.1min 13 4.7 AWz &18

2^z 106.7 82.7 1 122.7 L,—^^ , 1 .. ., 1 AI , P , 1 R—

Sample F at 20.0 mm

Figure 3.15. Compound 13 from M. coquimbensis. Samples D-F: MS/MS spectra DAD1 A, Sig=2eD nm Compound 14

DAD1 A. nm Sample D

2-5-

DA01 A. 5i'g=28D nrr Sample E

DA0-1 A, Shg'=28D nm Sample F

Figure 3.16. Compound 14 from M. coquimhensis. Samples D-F: HPLC chromatograms at 200nm

K) 124

+;lvlS. 13.9mtn

475.1 391.2 602.7

Campoimd 14 at 13.9 mm

m'' +ys. 13 9fnirt s- 248,3

235.2 253.2 311.2 283.2 291.3 337.1

I; II I I I I I I I. I I I 1 I I [ I I I I ll: I j II III I I ll I ) llI I Kl I I 1 I I I J r N I ll f II I l| Ill-- I I I I- Ill Ili-lll IIj I I 240 280 2B0 300 320 IT^Z

Sample D at 13.9 mio

xi# i +MS, M.lmin 1.25^ 1.00^ 227.6 Q,75 j O.SOj 249.5 291.5 0.251 237.5 I 259.5 • 31,7.2 333..3 g QQ I III. II III h IIII, f I I.. I.. III! I,)I J .I,II11 .1II .i-^..i-^.'j < IM.iriVi I .t 11111, I I I, IIII. . ii11 ,1.1 I111 I I I.I .11 aj. I I I. Ml II t < • I ^ i.ii f. I.I . 240 260 330 nv'z Sample E at 13.6 mm

291.4 +MS. 14.2min

227.6 317.3

301.i' lii< mil III

Sample F at 14.2 min

Figure 3.17. Compound 14 fromM. coquimbensis. Samples D-F: MS spectra 125

Intens +!v1S2(291.1), M.Ornin 15 122.7138.6

10 ,164.7 0.6 150.6 272.8

0.0 4 I'j' 60 !«) 150

Cmnpousid 14 at 13.9 min

inters. = +MS2(291.5), 14.2min SOOD| 4000i soooi 2000 = 1000^ s - 50 t» m 2® 250 .300 mfz

Sample E at 13.6 nan

+MS2(291.4), 14,3min 122.8 138.7 236.9

164 7178.8 272.0 150.S 218.9 • li'Ji'if I.IL • 255,0.iL. • 200 3^

Sample F at 14.2 inin

Figure 3.18. Compound 14 from M. coquimbensis. Samples E and F: MS/MS spectra DADt A, S!fl=2GD nm Compound 10

21.5

DAD1 A, Sig=20Q mm Sample D

^.D1 A. S^g=20D nm Sample E

DA.01 A. S?g=20G n.m Sample F

fnAul DAD1 A, Sjg-=20G nm Blank

Figure 3.19. Compound 10 from M. coquimhensis. Samples D-F: HPLC chromatograms at 200nm 127

xto®: +MS, 20.7min 109,3

153,3 193.3

12.3.l''^P I 171..3 I I

m 75 1G0^ 125- 150 m 225

Coiiipoimd 10 at 20.7 mm

p +MS, 20.6min a - 235.8 6 - 4 - 153.3 • 203.6 217.7 121 2 2- " 137.5 ...II I.W.I ULI -V-J LLILI I ii flillll ill III lliiiil'll liiUl 60 100 12S

Sample D at 20.6 mui iinlens . *10": +MS, 20.emio 4 •: 109.2 235.7 135,4 153,5 3: 197,5 I25..4 193.5 171? 207,8 221.7 79,4 99.3 163.1 -.1—1-JjJl ill 11.1 ll llill'l ll m ao IQi 12Q 140 m m 200 220 mi(z

Sample E at 20.8 miB

.r- xfQ*" +MS. 20-6min i9fl.e 197.2 1.! 191 6 195,9 1.0- 183.9 192,6 186,9 190,4 l|^4 194,8 0.5- 181.4 .1 I. 1,1 ll O.D-*- m 184 lUiiS, ?» 162 18^ isa 108 mfz

Sample F at 20.6 iiim

Figure 3.20. Compound 10 from M. coquimbensis. Samples D-F: MS spectra 128

InSesis- +MS2(193.3), 20.aTOn 174.7

30.6

Compound 10 at 20.7 miii

intesis- +MS2(193.5), 20.9min 176.7 174. f 134.6 148.7 132 J3S.7 104 7 120.6 S50.I 160.5 I 108 8 116.8 1 ' 164,6 . .1.1.1I' 7,r I . ,ii, i.lir-T—r- -Ui, IIIil l ^1 ISO 120 m 140 160 180 miz Sample E at 20.8 nan

Figure 3.21. Compound 10 from M coquimbensis and Sample E: MS/MS spectra 129

3.10 Experimental Section

General instrumentation and experimentation is described in Chapter 7. Experimental conditions and techniques unique to the M. coquimbensis work are presented here.

3.10.1 NMR Experiments

DEPT spectra were recorded in CDCI3; '^C , 'H, ID NOE and 2D spectra were recorded in CD3OD. Chemical shifts were expressed in ppm (5) using partially deuterated solvent chemical shifts at 577 ('^C) and 67.24 ('H) as reference in the case of CDCI3, and 649

('•^C) and 63.30 ('H) in the case of CD3OD. The mixing time used in recording selective

ID NOE for 10 was 100 ms and that used for recording selective ID TOCSY spectra was

20 ms. ID and 2D NMR spectra for 10 are included in Appendix B.

3.10.2 Preparation of the (/?)- and (S)- MTPA ester Derivatives of 10

Compound 10 (0.0006 g) was dried in the fume hood and transferred into a clean and vacuum dried vial. Deuterated pyridine-Js (0.5mL) and a catalytic amount of dimethyl-4-aminopyridine (DMAP, 0.0002 g), also completely dried under vacuum, was added to the vial and dissolved. The esterification was initiated by adding (5')-(+)-a- methoxy-a(trifluoromethyl)phenylacetyl (MTPA)chloride (0.001 g) into the vial under a continuous N2 stream. The vial was then sealed and shaken to mix the reactants. The reaction was complete in 2 hrs at room temperature as evidenced by TLC and 'H NMR and yielded in the (7?)-MTPA ester derivative lOr of compound 10. The (5')-MTPA ester,

10s, was similarly prepared in another vacuum dried vial using (/?)-(+)-a-methoxy-a-

(trifluoromethyl)phenylacetyl chloride (0.001 g) and keeping all other reaction conditions same as those mentioned above. The reaction was seen to be complete after 2 hrs as monitored by 'H NMR. The 'H NMR data (pyridine-J5, 600 MHz) for 10s and lOr are shown in Table 3.5.

3.10.3 Plant Material

Plant D: Leaves, stem and fruit of Myrcianthes coquimbensis (Bameoud) Landrum &

Grifo, comb. nov. (identified at the time as Reichea coquimbensis (Bam.) Kausel) were collected by Gloria Montenegro and Luis Gonzalez on October 31, 2001, in Quebrada

Honda in the district of La Serena, 30 km north of Caieta Homo, Chile at an altitude of

558 m (29° 38' 24" S; 71° 17' 13" W). A voucher specimen (No. 1076) has been

deposited at the Pontificia Universidad Catolica de Chile, Santiago, Chile. Intellectual

Property Rights Agreements for plant collections and collaborative research have been

fully executed between the University of Arizona and Pontificia Universidad Catolica de

Chile. 131

Plant E: M.coquimbensis (Bameoud) Landrum & Grifo, comb. nov. was collected by

Gloria Montenegro and Luis Gonzalez on August 31, 2000 15 km north of La Serena in the district of Coquimbo, Chile. This collection included leaves and stems only. A voucher specimen (No. 1017) has been deposited at the Pontificia Universidad Catolica de Chile, Santiago, Chile.

Plant F: M.coquimbensis (Bameoud) Landmm & Grifo, comb. nov. was collected by Luis

Gonzalez on October 10, 1994 from 48 km north of La Serena in the district of

Coquimbo, Chile at 170 m above sea level level (29° 59' 52" S; 71° 22' 29" W). This collection also included leaves and stems only. The soil from which it was collected was rocky and the area was hilly. A voucher specimen (No. 468) has been deposited at the

Pontificia Universidad Catolica de Chile, Santiago, Chile.

3.10.4 Microplate Alamar Blue Assay

The biological activities of the cmde extract, chromatographic fractions and pure compounds were determined against M. tuberculosis H37RV (ATCC 27294) in the

Microplate Alamar Blue Assay (MABA)'°' described briefly as follows:

Antimicrobial susceptibility testing was performed in black, clear-bottomed, 96-well microplates (black view plates; Packard Instmment Company, Meriden, Conn.) in order to minimize background fluorescence. Outer perimeter wells were filled with sterile 132 water to prevent dehydration in experimental wells. Initial drug dilutions were prepared in either dimethyl sulfoxide or distilled deionized water, and subsequent two-fold dilutions were performed in 0.1 mL of 7H9GC (no Tween 80) in the microplates.

BACTEC 12B-passaged inocula were initially diluted 1:2 in 7H8GC, and 0.1 mL was added to wells. Subsequent determination of bacterial titers yielded 1 x 10^ CFU/mL in plate wells for H37RV. Frozen inocula were initially diluted 1:20 in BACTEC 12B medium followed by a 1:50 dilution in 7H9GC. Addition of 1/10 mL to wells resulted in final bacterial titers of 2.0 x 10^ CFU/mL for H37RV. Wells containing drug only were used to detect autofluorescence of compounds. Additional control wells consisted of bacteria only (B) and medium only (M). Plates were incubated at 37 °C. Starting at day 4 of incubation, 20 fxL of 10 x AlamarBlue solution (Alamar Biosciences/Accumed,

Westlake, Ohio) and 12.5 |J,L of 20% Tween 80 were added to one B well and one M well, and plates were re-incubated at 37 °C. Wells were observed at 12 and 24 h for a color change from blue to pink and for a reading of > 50,000 fluorescence units (FU).

Flurescence was measured in a Cytofluor II microplate fluorometer (PerSeptive

Biosystems, Framingham, Mass.) in bottom-reading mode with excitation at 530 nm and emission at 590 nm. If the B wells became pink by 24 h, reagent was added to the entire plate. If the well remained blue or < 50,000 FU was measured, additional M and B wells were tested daily until a color change occurred, at which time reagents were added to all remaining wells. Plates were then incubated at 37 °C, and results were recorded at 24 h post-reagent addition. Visual MICs were defined as the lowest concentration of drug that prevented a color change. For fluorometric MICs, a background subtraction was 133 performed on all wells with a mean of triplicate M wells. Percent inhibition was defined as 1 - (test well FU/mean FU of triplicate B wells) x 100. The lowest drug concentration effecting an inhibition of > 90% was considered the MIC.

3.10.5 Vero Cell Cytotoxicity Assay

Test compounds were dissolved at 20-40 mg/mL in DMSO. Geometric three-fold dilutions were performed in growth medium Ml99 (Gibco, Grand Island, NY) -1-5% fetal bovine serum (HyClone, Logan, UT) +25 mM A^-(2-hydroxyethyl)-piperazine-A'^'-2- ethanesulfonic acid (HEPES, Gibco) -f- 0.2% NaHC03 (Gibco) -f- 2 mM glutamine (Irvine

Scientific, Santa Ana, CA) to achieve final concentrations ranging from 4.2 to 400

[tg/mL. Final ethanol concentrations did not exceed 1% v/v. Drug dilutions were distrubuted in duplicate in 96-well tissue culture plates (Becton Dickinson Labware,

Lincoln Park, NJ) at a volume of 50 |iL/well. An equal volume containing 5 x 10 log phase Vero cells (CCL-81; American Type Culture Collection, Rockville, MD) was added to each well, and the cultures were incubated at 37 °C in an atmosphere of 5% CO2 in air. After 72 h, cell viability was measured using the CellTiter 96 aqueous non­ radioactive cell proliferation assay (Promega Corp., Madison Wl) according to the manufacturer's instructions. Absorbance at 490 nm was read in a BioRad Model 3550 microplate reader (Hercules, CA). The IC50 is defined as the reciprocal dilution resulting in 50% inhibition of Vero cells. Maximum cytotoxicity (100%) was determined by lysing the cells with sodium dodecyl sulfate (Sigma Chemical Co., St. Louis, MO).'°^ 134

3.10.6 GC/MS

GC/MS data were recorded with a Varian Saturn 2100T. The gas chromatograph was fitted with a Chromapack capillary column (CP Sil 8 CB; 30m x 0.25 mm). Operating

conditions: column oven temperature programmed at 80 °C for 5 min and then to 280 °C

at 10 °C/min; injector/transfer line/trap temperatures 250/250/200 °C respectively;

electron voltage, 50-80 eV. UHP helium was used as the carrier gas at a flow rate of 1.2

mlVmin.

3.10.7 LC/MS Instrumentation and Technique

Sample Preparation. 200 g of the dried and powdered plant material of M. coquimbensis

(Plant E) and M. coquimbensis (Plant F) were extracted with CH2Cl2-MeOH (1:1). The

extracts thus obtained were dried under vacuum and partitioned between MeOH (200

mL) and n-hexane (100 mL x 2). The MeOH soluble fraction was concentrated under

vacuum and partitioned between H2O (100 mL) and CH2CI2 (200, 100 mL). The CH2CI2

fraction thus obtained from Plant E (Sample E, 3.9 g), and Plant F (Sample F, 3.6 g) were

used for the LC/MS study.

The CH2CI2 fraction (as described in the bioassay-guided isolation) of M. coquimbensis

(Sample D, 0.0086 g), 0.0076 g of Sample E, and 0.0083 g of Sample F were each 135

dissolved in 1 mL MeOH. The resultant solution was filtered through a 0.45 |lI PTFE syringe filter (Whatman, Clifton, NJ) and transferred into 2 mL HPLC vials.

Standard Preparation. A 0.00144 g/mL solution of 11 in MeOH was adjusted to

0.000101 g/mL and transferred to a 2 mL HPLC vial. Similarly, 1 mL of 0.000106 g/mL and 0.0000994 g/mL of 13, and 14 respectively were prepared and transferred to 2 mL

HPLC vials. 0.00023 g of 10 was dissolved in 500 |a,L MeOH, transferred to a 2 mL

HPLC vial (at a concentration of 0.00046 g/mL). Each HPLC injection utilized 20 |liL of these stock solutions.

HPLC/DAD. The mobile phase consisted of acetic acid (0.05%) in water (solvent A) and acetic acid (0.05%) in MeOH (solvent B) gradient; starting at 100% solvent A and going to 100% solvent B in 60 min, and holding at 100% solvent B for 5 min. The same gradient with acetic acid (0.05%) in acetonitrile as solvent B was first tried for the standards as well as the three samples but, results with acetic acid (0.05%) in MeoH were superior. The chromatograms were recorded at 200, 220, 254 and 280 nm and the spectral data for samples (D-F) and standards were accumulated in the range of 190-450 nm. 136

3.10.8 Spectroscopic Data

(lS,3S',4/f)-l-Methyl-4-(l-methylethenyl)-l,3-cyclohexanediol (10). [a]^^D -1-2

(CH3OH, c 0.60); IR (KBr) v^ax 3402, 2933, 1707, 1660, 1034 cm '; 'H and '^C data in

Table 3.4; EIMS m/z 170, 152 137, 119, 109, and 67. 137

CHAPTER IV

CHLOROFORM SOLVATE OF (4/?,5/?,7S,85,95)-7-HYDROXY-8-

HYDR0XYMETHYL-4-METHYLPERHYDR0CYCL0PENTA[C]PYRAN-l-

ONE FROM VALERIANA LAXIFLORA

4.1 Introduction

The methanolic extract of the Chilean plant Valeriana laxiflora DC (Valerianaceae), was found to inhibit the growth of Mycobacterium tuberculosis H37RV by 53% at a concentration of 50 |a.g/mL. Bioassay guided fractionation of V. laxiflora led to the isolation of the title compound, 18 (Fig. 4.1) in addition to (+)-l-hydroxy-2,6-bis- epipinoresinol (19), betulin (20), betulinic acid (21), 5,7-dihydroxy-3,6,4'- trimethoxyflavone (22), 23-hydroxy ursolic acid (23), oleanolic acid (11), tricin (24), ursolic acid (25), ferulic acid (26), (+)-l-hydroxypinoresinol (27), prinsepiol (28), and

5,7,3'-trihydroxy-4'-methoxyflavone (29) as previously described. Compounds 20-

23,11, 24-25 were found to inhibit the growth of M. tuberculosis with MIC values of

30.0, 62.1, 46.2, 15.5, 28.7, 58.5, and 41.9 )a,g/mL respectively, whereas compounds 18,

19,26-29 were found to be essentially inactive with an MIC of > 128 |a,g/mL. However,

18 was a new chemical compound and single crystal X-ray crystallographic analysis of

18 was initiated to unequivocally establish its chemical structure and to determine its absolute and relative stereochemistry. CH

HO

HO

Figure 4.1. (4/?,5/?,7iS',85',95)-7-Hydroxy-8-hydroxymethyl-4- methylperhydrocyclopenta[c]pyran-1-one (18) 139

4.2 Results and Discussion

The two rings are cw-fused with H5 and H9 with p orientation as shown in Figure 4.2.

The 6-lactone ring adopts a slightly twisted half-chair conformation and the cyclopentane ring adopts an envelope conformation. The hydroxyl group at CI, hydroxymethylene group at C8 and the C4 methyl group all have [3 orientation.

The unusually long bond distance of 1.572 (7) A between the bridge carbons C5-C9 suggests strain in the fused system. This observation has been made previously for a closely related iridolactone. " As expected in esters, differences in the two C-(Ol) bond

o distances were observed, with the C3-01 bond being longer (1.453 (7) A) then the Cl-01 bond (1.336(6) A)."

The lactone group C9-C1(02)-01-C3 was observed to be almost planar with a relative

o o mean deviation of 0.0208 A and a maximum deviation of 0.352 (4) A for atom Ol. The torsional angle between C3-01-C1-02 was observed to be +177.3 (5)° showing only a slight aberration from planarity. This concurred with the observation made for (+)- nepetalic acid and an iridane-derived lactone, boonein."^^"'®^ Figure 4.2. A displacement ellipsoid plot (50% probability level) of (4i?,5^,75',8>S',95')-7-hydroxy-8-hydroxymethyl-4- methylperhydrocyclopenta[c]pyran-l-one (18) 141

The packing is dominated by hydrogen bonding which creates an infinite tape of molecules coincident with the 2| axis (Table 4.1 and Fig. 4.3). The C7 hydroxyl group of each molecule is H-bonded through 03-H3 group to the /7-translated neighbouring hydroxymethylene 04 to form a chain. Each chain faces another chain of molecules related by the 2i axis, so that the cyclopentane rings of the first chain are facing the cyclopentane rings of the next. Each C7 hydroxyl group is then also hydrogen bonded through atom 03 to the H4-04 hydroxymethylene group of the molecule facing it, forming a molecular tape. A solvent-accessible channel along each side of the tape allows for H-bonding between chloroform and the tape (Fig. 4.4). Each molecule of chloroform is hydrogen bonded through atom H20A to atom 04 of the hydroxymethylene group. In addition, each carbonyl oxygen 02 is makes short contacts with atom H5 of an adjacent molecule in the same row as well as to atom H9 of a molecule in an adjoinging tape

(Table 4.1).

Due to the inclusion of a chloroform solvent molecule in the crystal lattice, the anomalous dispersion data was used to arrive at the absolute configuration of 18 as

4R,5R,7S,SS,9S. The same absolute configuration has been determined by the Mosher's 8"? esterification, which also unequivocally proves enantiomeric purity.

The crystals were isolated as very thin plates and crystal decomposition due to evaporation of solvent at room temperature posed considerable problems in the quick mounting of a suitable crystal. Nevertheless, we were able to get important information Table 4.1. Hydrogen-bonding and short-contact geometry of 18 (A, °)

D-H- • -A D—H H- • A D- • -A D—H- • A

03—H3- • 04' 0.84 1.88 2.651 (5) 152 6 0 0.84 1.87 2.654 (4) 156 C20—H20A- • -04 1.00 2.42 3.402 (6) 166 C5-H5- • 02' 1.00 2.70 3.575(7) 146 C9-H9 • • •02"' 1.00 2.49 3.188(6) 127 Symmetry codes: (i) jc, j - 1, z; (ii) -x, ¥2+ y,2- z\ (iii) -x,y-V2,l-z 143

Cft

ef

OS^A 02

'fmA %

Figure 4.3. Crystal packing geometry of 18 looking at the face of the hydrogen bonded tape; A = -x, y+Vi, -z+2; B = x, y-1, z H20A A

Figure 4.4. Crystal packing geometry of 18 along the fe-axis, colinear with the hydrogen bonded tape; A = -X, y+V2, -z+2 145 about the constitution, conformation and configuration of 18 from this study. This information can now be used to predict the three-dimensional structure of closely related iridolactones isolated from nature but which do not crystallize. This to our knowledge is the first report of the absolute configuration of a 1-iridolactone based on X-ray data.

4.3 Experimental Section

4.3.1 Plant Material

Valeriana laxiflora DC, was collected in February 2002 in Termas de Chillian, VIII

Region, Chile (36° 54' S; 71° 2' W) by Gloria Montenegro. A voucher specimen (No.

1098) has been deposited in the herbarium at the Pontificia Universidad Catolica de

Chile, Santiago, Chile. Intellectual Property Rights Agreements for plant collections and

collaborative research have been fully executed between University of Arizona and

Pontificia Universidad Catolica de Chile.

4.3.2 Isolation

Dried and powdered aerial parts as well as roots of V. laxiflora (770 g) were extracted

repeatedly with MeOH. This MeOH extract was filtered and the solvent was evaporated

under vacuum and the resultant extract was partitioned between 90% aqueous MeOH and

n-hexane. The 90% aqueous MeOH soluble portion was dried under vacuum and 146 subsequently partitioned between CH2CI2 and H2O (1:1). The CH2CI2 soluble extract was further fractionated by column chromatography on a silica gel column using a step gradient of CHCl^/MeOH. Fraction eluting with CHCls/MeOH (20:1, 2.2 g) was chromatographed on a silica gel column using n-hexane/iPrOH/MeOH step gradient. The fraction eluting with n-hexane/iPrOH/MeOH (18:1:1, 0.37 g) was applied to a Sephadex

LH-20 column, using MeOH as the mobile phase. The fraction containing 18 (0.060 g)

was purified by RP-PLC using CH3CN/H2O (1:1). Compound 18 (0.014 g) was isolated at Rf = 0.62. Crystals were obtained as very thin colorless rectangular plates using CHCI3.

4.3.3 Refinement

The best available crystal, which was larger than the beam in one dimension, was used to

enhance diffraction."'^ Despite this, the observed diffraction was weak and was further

complicated by solvent loss during mounting. The structure was solved using direct

methods. Hydrogen positions were easily visible in difference Fourier maps, but all

hydrogens were placed at ideal geometric positions and constrained to ride on the atoms

to which they were bonded, except the hydroxyl protons, which were constrained to an

0 0-H distance of 0.84 A and a C-O-H angle of 109.5°.Due to the fortuitous inclusion of a

heavy atom solvent molecule (CHCI3) in the crystal lattice, we were able to use the

anomalous dispersion data to arrive at the absolute configuration of 18.

The structure solution of 18 has been published in Acta Crystallographica, 2004, C60,

o773-o775 (See Appendix A). 147

Table 4.2. Experimental Data for (4/?,5/?,75,85,95)-7-hydroxy-8-hydroxymethyl-4- methylperhydrocyclopenta[c]pyran-l-one (18)

Crystal data P = 94.478 (5)° Cell parameters from 794 C,oH,604.CHCl3 V= 711.7 (3)A-^ reflections M,= 319.6

Z = 2 ^=2.5-18.76° Monoclinic, P2|

£>^=1.491 Mg m"-^ a = 10.523 (3) A // = 0.647 mm"'

b = 6.0404 (15) A Dm not measured T= 173 (2)K

c = 11.231 (3) A Mo Ka radiation Parallelepiped, colorless

X = 0.71073 °A 0.78 X 0.09 X 0.02 mm

Refinement

Refinement on w = l/[a^(Fo^) + (0.0327P)^]

R[F^ > 2a(F^)] = 0.062 where F = (Fo^+ 2Fc^)/3

wR(F^) = 0.104 (A/a)max = < 0.001

S = 0.98 AyOmax = 0.30 e A

2420 reflections AyOmin = "0.26 e A

161 parameters Absolute structure: Flack (1983),

H-atom parameters constrained 1038 Friedel pairs

Flack parameter = 0.11 (12) 148

CHAPTER V

(3/?,5S,9/?,105,135,145,17/?,18/?,21/?)-(-)-FERN-7-EN-3a-OL FROM

SEBASTIANIA BRASILIENSIS

5.1 Introduction

Bioassay guided fractionation of the antitubercular CH2Cl2-MeOH (1:1) extract of S. brasiliensis Spreng. (Euphorbiaceae) found to inhibit the growth of H37RV strain of

Mycobacterium tuberculosis (MTB) by 94% at a concentration of 50 |j,g/mL, is ongoing in our laboratories. So far the fractionation of S. brasiliensis has led to the isolation of the title compound 30 (Fig. 5.1). Microplate Alamar Blue Assay of 30 has indicated that it has an MIC value >128 [Xg/mL when assayed against MTB, and thus has been regarded as inactive.

X-ray crystallographic analysis of 30 was undertaken to establish its chemical structure and assign the relative and absolute stereochemistry at C3. The absolute configuration was determined by 'H NMR comparison of the R- and S- MTPA esters of 30,30r and 30s respectively. Although 30 has previously been isolated from Adiantum capillus-veneris

this is the first report of its isolation from S. brasiliensis and of its crystal structure.

The X-ray structure of 30 can now serve as a standard for comparison of related femanes. o

o "o sI cn c\ idI r-

(n cn 0^ 00 r-H as < < r- DH di h

.. ^ (d oi oi Pi U o o q? o o^ co­ in «< m

m lU s ec

DC 150

5.2 Results and Discussion

As shown in Figure 5.2, rings A and D assume chair conformations, while rings B and C adopt twist-boat conformations. The A^fi, C/D, and D/E ring junctions are fran^-fused about the C5-C10, C13-C14 and C17-C18 bonds, respectively. The hydroxyl group at

C3 and the isopropyl group at C21 are a. No hydrogen bonds were detected between the hydroxyl groups of screw-related molecules. However, the molecules pack such that the hydroxyls of adjacent molecules are as close to each other as possible [5.075(2) A]. The distance between hydroxyls related by unit translation along the b axis is 7.5526(11) A. A short contact is observed between the C24 methyl atom H24 and the hydroxyl oxygen of a screw-related molecule with a bond length of 2.72(3) A and a bond angle of 162 (2)

(Fig. 5.3, 5.4, and Table 5.1).

Due to the absence of any heavy atoms in the crystal structure, anomalous dispersion data could not be used to arrive at an absolute configuration. Thus, the absolute configuration was determined by preparing R and 5-MTPA esters and noting the differences in chemical shift between 'H signals in the two esters as described by Ohtani et al.

Although the 'h and ' ^C NMR data have been reported for 30,'°^ these data were recorded in CDCI3 and were not directly comparable to the data we obtained in pyridine.

To assign our data, a 2D HSQC experiment was carried out. The chemical shifts of the

13 protonated " C signals observed in the HSQC experiment were comparable with those 151 reported previously, and the 'H signals could then be assigned by correlation. The signals that were assigned in this fashion are presented in Table 5.2.

R and 5-MTPA esters were prepared conveniently in the NMR tube as detailed in the experimental section. The difference in the 'H chemical shifts of the S and R esters [ASCS-

R)] was in direct proportion to their distance from the ester moiety as expected. The A5

values, shown in Table 5.3 and Figure 5.5, were used to assign the absolute configuration of C3 as R. The absolute configuration of 30 was determined to be

3R,5S,9R,10S,135,145,17/?, 1 8R,2 IR.

The structure solution of 30 was published in Acta Crystallographica 2003, E59, ol403-

1404 (See Appendix A).

5.3 Experimental Section

5.3.1 Plant Material

Aerial parts of Sebastiania brasiliensis were collected and identified by Renee H.

Fortunato and A. D'Agostini on July 12, 1998, 21 kilometers south of Campo Silvo

Pastoril in the province of Chaco, Argentina (27° 18' 0" S; 59° 24' 0" W). A voucher specimen (RHF 6145) has been deposited at the Institute Nacional de Tecnologia

Agropecuaria (INTA), Buenos Aires, Argentina. Intellectual Property Rights Agreements rnci! \ / V K

I 3 J :

Figure 5.2. A displacement ellipsoid plot (50% probability level) of (-)-Fern-7-en-3a-ol (30)

U\ K) Vi

to /7-axis. ^30) perpendicular -3a-ol ofO-Fem-l-en-

Ei8»r«®-'-^^^3-x,%+y,2-' H24 A ^ I

iss-An^j^ «, \C34 01*

Figure 5.4. Crystal packing geometry of (-)-Fem-7-en-3a-ol (30) along ^-axis. A = 3-x, Vz+y, 2-z 155

Table 5.1. Short contact geometry (A, ) of (-)-fem-7-en-3a-ol (30)

D-n A D-H HA DA D-H A C24-H24C...01' 0.99(3) 2.72(3) 3.677(3) 162(3)

Symmetry code: (i) 3-x, V2+3;, 2-z.

Table 5.2. '^C and 'H NMR data for select protonated signals of (-)-fem-7- en-3a-ol (30)"

Position ' •^C (ppm) 'H (ppm) 1 31.73 1.94 1.39 2 26.61 2.02 1.83 3 75.36 3.67 5 48.28 2.65 6 24.71 2.01 2.18 7 117.09 5.47 9 45.06 2.25 15 30.71 1.52 1.64 18 54.43 1.42 20 28.66 1.75 21 59.70 0.86 22 30.00 1.35 23 28.81 1.15 24 22.22 0.97 25 13.29 0.87 26 24.56 1.08 27 21.34 0.89 28 14.46 0.69 29 22.36 0.89 30 23.39 0.82 " "'^C and 'H NMR data recorded in pyridine-J5 at 125 and 500 MHz respectively 156

Table 5.3. 'H NMR data for 30s and 30r and A5 values obtained for the MTPA esters"'''

Position 30s 'H(in Hz) 30r 'H (in Hz) A5 {S-R) (in Hz)

1 ov ov -

2 ov ov - 5 1479.34 3228.37 -78.36 7 3232.96 3228.37 +4.59 9 1352.42 1323.09 +29.33 23 518.45 536.78 -18.33 24 484.09 528.07 -43.98 25 468.51 436.89 +31.62 26 627.05 617.88 +9.17 27 556.03 543.19 +12.84 28 416.27 425.43 -9.16 29 536.78 545.49 -8.71 30 498.29 501.95 -3.66 "o\ - overlapped ''Data recorded in pyridine-i/s at 600 MHz

+ 12.84 -9.16

+31.62

-3.66 ov +29.33

ov

+9.17

+4.59

RQ ov -78.36

-43.98 -18.33

30s R=5-MTPA 30r R=/?-MTPA

Figure 5.5. 'H NMR A6 {S-R) values obtained for the MTPA esters 30s and 30r 157

for plant collections and collaborative research have been fully executed between the

University of Arizona and INT A.

5.3.2 Isolation

The antitubercular CH2Cl2-MeOH extract of S. brasiliensis was chromatographed on a silica gel column with increasing concentrations of EtOAc in n-hexane. The fraction eluting with 10% EtOAc in n-hexane was found to inhibit the growth of M. tuberculosis

H37RV (ATCC 27294) by 90% at 50 mg/mL concentration. This fraction, when treated

with ether, gave a crystalline substance which on thin-layer chromatography (TLC)

showed essentially one major spot. Preparative silica gel TLC of this fraction followed by crystallization led to the isolation of 30. Crystals were obtained by slow evaporation of n- hexane/acetone. Colorless shining transparent rectangular rods were formed after three days.

5.3.3 Preparation of the (/?)- and (5)- MTPA ester derivatives of 30

The R- and S'-MTPA derivatives were prepared according to the method described by

Ohtani et al. Compound 30 (0.003 g) was dried under vacuum and transferred into a

clean NMR tube. Pyridine-Jj (0.5 mL) and a catalytic amount of A',A^-dimethyl-4-

aminopyridine (DMAP, 0.0002 g), also dried under vacuum, were added to the NMR tube and dissolved. The esterification was initiated by adding (5)-(+)-a-methoxy- a(trifluoromethyl)phenylacetyl (MTPA) chloride (0.01 g) into the NMR tube under a N2 stream. The NMR tube was then sealed and shaken to mix the reactants. The reaction was complete in 2 hrs at room temperature as evidenced by 'H NMR and yielded in the R-

MTPA ester derivative 30r of compound 30. The iS-MTPA ester, 30s, was similarly prepared in another NMR tube using (/?)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (0.01 g) and keeping all other reaction conditions the same as those mentioned above. The reaction was seen to be complete after 2 hrs as monitored by 'H NMR. The

'H NMR data for 30s and 30r (pyridine-Jj, 600 MHz) is shown in Table 5.3.

5.3.4 Refinement

H-atom positions were easily visible in difference Fourier maps and all H atoms were allowed to refine freely, except for the hydroxyl H atom, which was constrained to an O-

H distance of 0.84 A and a C-O-H angle of 109.47°, with Uiso(H) = 1.5Ueq(0). In the final cycles of refinement, all Friedel pairs were merged. The enantiomer was selected based on the observed negative optical rotation of the compound in solution and the fact

I OS that this is the most common enantiomer observed in nature . The absolute configuration was subsequently established unequivocally by synthesizing R and 5-

MTPA esters of 30, 30r and 30s respectively as already described. 159

Table 5.4. Experimental Data for (-)-fem-7-en-3a-ol (30)

Crystal data

C30H50O p= 101.320 (3)° ^=2.5±23.4

Mr = 426.70 V= 1253.8 (3) // = 0.07 mm '

Monoclinic, P2i Z = 2 T= 170 (2)K a = 12.4234 (18) A £>^=1.130 Mgm -^ Parallelepiped, colorless

Z? = 7.5526 (11) A Mo Ka radiation 0.35 X 0.27 X 0.19 mm c = 13.628 (2) A Cell parameters from 4025

reflections

Refinement

Refinement on F' w = l/[o^(Fo^) + (0.0564P)^

R[F^ > 2a(F^)] = 0.044 + 0.2128F] wR(F') = 0.103 where P = (Fo^ 2Fc^)/3

5= 1.06 (A/a)max = 0.001

3278 reflections A/?max 0.29 e A -3

427 parameters A/?min ~ -0.21 e A -3

H atoms treated by a mixture of independent and constrained refinement 160

CHAPTER VI

SUMMARY AND CONCLUSIONS

6.1 Summary

The research described here illustrates the fundamental philosophy of the ICBG as detailed in Chapter 1. Briefly, it illustrates how plant bioprospecting can lead to pharmaceutical lead discovery while encouraging conservation and facilitating economic development in the countries where the collections are made. Chapters 2 and 3 illustrate the bioassay directed isolation approach to drug lead discovery as applied to the

Argentinean plant C. coronata, and the Chilean plant M coquimbensis. Chapter 4 and 5 present structure elucidations of a new iridoid and a triterpene isolated using the bioassay guided isolation of the antitubercular extracts of the Chilean V. laxiflora, and the

Argentinean S. brasiliensis respectively.

The bioassay directed isolation of the antibacterial CH2Cl2-MeOH (1:1) extract of C. coronata as described in Chapter 2, led to the isolation of a new antibacterial triterpenoid, namely ip,3P-dihydroxy-urs-12-en-27-oic acid 1 in addition to a new iridoid caiophoraenin 2, along with the known iridoid isoboonein 3. The iridoids isolated in this study were found to be inactive in the bacterial assays whereas, 1 was found to be more potent against methicillin-resistant S. aureus and vancomycin-resistant E. faecium than the control. Penicillin G, along with significant activity against methicillin-sensitive 5. 161 aureus, B. subtilis, and E. coli imp. Our pharmaceutical collaborators at Wyeth are interested in conducting further biological testing to determine the mechanism of action of 1 as well as to determine its effects in vivo. Further research in this regard awaits re­ collection of C. coronata from San Juan, Argentina and isolation of 1 to support the biological testing. If found to be a viable drug lead, chemical synthesis routes to 1 will be explored to address the supply issue and analogs of 1 will be prepared to determine structure-activity relationships.

The bioassay directed isolation of the antitubercular CH2Cl2-MeOH (1:1) extract of M. coquimbensis as detailed in Chapter 3, led to the isolation of six compounds- namely, the new monoterpene (15',35',4i?)-l-methyl-4-(l-methylethenyl)-l,3-cyclohexanediol 10, oleanolic acid 11,3P-caffeoyl-12-oleanen-28-oic acid 12, trans (-i-)-sobrerol 13, epi- catechin 14, and catechin 15. Compound 11 was found to inhibit the growth of M. tuberculosis (MTB) moderately with an MIC of 29.66 |Lig/mL whereas all other isolates were regarded as inactive (MIC 128 |a.g/mL). Compound 11 was tested for its cytotoxicity against African green monkey Vero cells in order to evaluate its selectivity potential and was found to have very low cytotoxicity with an IC50 value of >102.4 )Xg/mL.

Chapter 4 describes the structure elucidation of (4/?,5/?,75,85,95)-7-hydroxy-8- hydroxymethyl-4-methyl-perhydrocyclopenta[c]pyran-l-one 18 using single crystal X- ray crystallography. Compound 18 was isolated from the antitubercular methanolic extract of V. laxiflora but was found to be essentially inactive with an MIC of >128 162

Hg/mL. In fact pentacyclic triterpenoids- betulin, betulinic acid, ursolic acid, oleanolic acid, and 23-hydroxy ursolic acid along with 5,7-dihydrox-3,6,4'-trimethoxyflavone were found to be responsible for the antitubercular activity of this plant extract as described elsewhere.^^

Chapter 5 describes the structure solution of (3/?,55,9/?,105,135,145,17/?,18i?,21/?)-(-)- fem-7-ene-3a-ol 30 using single crystal X-ray crystallography. Compound 30 was isolated from the antitubercular methanolic extract of the Argentinean plant S. brasiliensis but was found to be essentially inactive with an MIC of > 128 |J,g/mL.

6.2 Antitubercular Triterpenoids

Results presented here and from other plants investigated under the Latin American

ICBG suggest that pentacyclic triterpenoids are a pharmacologically important class of natural products, particularly against tuberculosis. An overview of some recent literature describing antitubercular plant isolates reiterates the potential of pentacyclic triterpenoids against

Triterpenoids have also been isolated as the active constituents of some plants employed for treatment of tuberculosis in traditional medicine, e.g. stigmasta-4-en-3-one and stigmasta-4,22-dien-3-one from Morinda citrifolia (Rubiaceae) (Section 6.3.5, compounds 93 and 94 respectively), as well as from antitubercular plant extracts.'"^ Since 163 many ubiquitous triterpenoids including oleanolic acid, ursolic acid, and betulinic acid, have moderate to high levels MTB growth inhibition, it is imperative that active plant extracts be subjected to dereplication studies to minimize isolation effort as shown recently."^

A closer look at the activities of compounds belonging to different triterpenoid skeletal

classes and their relationship to structure is presented here. These include compounds that

have been isolated in this study from C. coronata, M. coquimbensis, and S. brasiliensis as

well as those reported in the literature. Some general trends in activities were observed

leading to some empirical rules for structure requirements for minimum activity.

However, the structure-activity relationship observations described here are purely

empirical and based on the data of this study and some reported in the literature. They do

not take into account probable differences in assay conditions, degree of compound

purity and other factors that might affect reproducibility of results across different

studies. 164

6.3 Structure-Activity Relationships in Antitubercular Triterpenoids

6.3.1 The Oleananes

Oleanolic acid 11 seems to have the minimum structure requirements for activity against

MTB (Fig. 6.1, Table 6.1). The reported MIC values for 11 are indicative of moderate inhibition of MTB growth. If the C28 acid moiety in 11 is replaced by a hydroxymethylene as in the case of erythrodiol 32, the activity is not affected. But, if it is substituted with a methyl as in the case of 35, the activity is adversely affected.

If the 3P-hydroxyl group is substituted with a 3a-hydroxyl, the activity improves as in case of e/?/-oleanolic acid 31. A similar improvement in activity is seen in the case of oleanonic acid 33, by the oxidation of the 3p-hydroxyl group. An oleanane derivative with a 3a-hydroxyl and a C28 methyl, 34, is reported to have far superior activity when compared with its 3p isomer, 35. Thus, a keto at C3 or a 3a-hydroxyl seem to be beneficial for activity. The substitution of the free hydroxyl for a caffeoyl moiety as seen in case of 12 abolishes activity.

MTB inhibition of several 3a-hydroxyl derivatives with an ether linkage between C3 and

C25 and different substitutions in the E-ring have been reported (39-40, 42-45).""^ Within the group, those with a C22 angeloyloxy group (39-40) are the most active. The same 165 study reported a C22 angeloyloxy and a C3 keto derivative, 41, as the most potent compound, reinstating the importance of a ketone functionality at C3 for MTB inhibition.

But friedelinol 36, despite its 3a-hydroxyl group is inactive, suggesting that some other features of the oleanane skeleton, or perhaps a CI 2(13) double bond are just as important for activity as the 3a-hydroxyl group. Thus, some other oleananes with a reduced

CI2(13) double bond need to be studied for a more complete understanding of this functionality for MTB inhibition. 166 V "N

4-MJi

11 R = p-Ott a-H; Rj = COOH I ' 36 V 31 R =a;-.OH, P-H:Ri = COOH

32 R = p-OH, a-K; R:= CHjOH

33 R = O, Rj = COOH

34 R = a-OH. e-H; S, = CHj Hd^ -g-OH, a-KRj-CHj 35 37 V I.. >'COOH

39 R = OH 40 R = H

•-V \

A.'o •S^ R :ooH ^rriAi.'COOH •"X. / T I J ^ ,..l^ i A 41 u A 42 o 1 43 S= o"

Figure 6.1. Antitubercular oleananes 167

-OH *COOH

HO^

O

HO­

MO

Figure 6.1. (contd) Antitubercular oleananes 168

Table 6.1. Antitubercular oleananes and their minimum inhibitory concentration (MIC) values against MTB

Compound Activity as MIC vs. Reference Number Mycobacterium tuberculosis Reported (|iM Normalized to or )Xg/mL) ^iM

11 64 ^M - Cantrell et al., 2001''' U 28.7 |J,g/mL 62.94 from V. laxiflora^^ 44 29.66 |Xg/mL 65.04 from M. coquimbensis 31 16 iig/mL 35.09 Copp, 2003"°

32 64|iM - Wachter et at., 1999^" 33 16 ng/mL 35.24 Copp, 2003"° 34 12.2 |ig/mL 28.64 Woldemichael et al, 2004' 35 64 ng/mL 150.23

36 > 128 [iM - Wachter era/., 1999^° 37 64 |lg/mL 140.97 Copp, 2003"° 38 64 |J,g/mL 140.97 (4 39 38 lilg/mL 65.18 Wachter et a/., 2001 40 36 )J.g/mL 63.49 (4 41 18 |lg/mL 32.61 44 42 >75 |Lig/mL >131.58 44 43 75 )ig/mL 131.58 44 44 73 (ig/mL 128.52 44 45 73 (ig/mL 125.00 44 12 >128 |j,g/mL 211.22 from M. coquimbensis

rifampin" 0.3 |XM - " First-line drug used for TB therapy for comparison 169

6.3.2 The Ursanes

Although, some disparity is observed in the MIC values of 25 reported by different studies, all reported MIC values are indicative of moderate inhibition of MTB growth

(Fig. 6.2, Table 6.2). Ursolic acid, 25, has comparable MTB inhibitory activity with

Oleanolic acid 11, suggesting that the E-ring functionalities can be slightly altered without compromising potency appreciably. When the acid group in 25 is substituted by a

C28 hydroxymethylene in 46, no change in activity is observed as in the case of 11. But, when the acid group in 25 is replaced by a methyl in 47, a significant loss in activity is observed, as in the case of 35.

An additional p-hydroxyl group at C2 in 52 when compared with 25 abolishes activity in the former. A similar lack of activity was observed in the case of the 2a,3a,19a- trihydroxyl derivative 54, or the 2a,3a,23- trihydroxyl derivative 53. The lp,3p- dihydroxy-urs-12-en-27-oic acid 1 isolated from C. coronata was only marginally active.

And, the 3[3,19a-dihydroxyl derivative, 50, is reported to have moderate activity.

Although 50 is only about half as potent when compared with 25, its 3(3-acetylated derivative 51 is as potent as 25. Also, a C23 hydroxymethylene group does not seem to bode well for MTB inhibition as seen when comparing activities of 48 and 49. However, in this case the C2 a-hydroxyl does not seem to affect activity. Thus, it can be extrapolated that hydroxylations in the A-ring at CI, C2, or C23 are not favorable for

MTB inhibition. However, C24 hydroxylation seems to favor activity as seen for 23. 170

Y^ ^ "'KI ^ri I V x/" I

HO 25 R = COOH Hd^ '^"''' 23 XH^OH 46 R = C:HjOH

47 R^CHj OH S OH i N--

~rCOOH 1 w/k

HO' XN 50 R = OH 48 R = CHj 51 R = OAc

49 R = CHiOH

COOH 'COOH

HOH,C

OH 5 > S ^..'' COOH I .z" Ha

54

Figure 6.2. Antitubercular ursanes 171

Table 6.2. Antitubercular ursanes and their minimum inhibitory concentration (MIC) values against MTB

Compound Activity as MIC vs. Mycobacterium Reference Number tuberculosis Reported (fJ,M or Normalized to |Xg/mL) ^iM

25 32|iM - Wachter efa/., 1999**" 41.9 |iig/mL 91.89 from V. laxiflora^^ (6 46 32|iM - 47 >128 |ig/mL >300.47 Woldemichael et ai. 2004"^ 23 15.5 |lg/mL 32.84 from V. laxiflora82

48 32|iM - Wachter et al, 1999^°

49 128 |iM - (( 50 64|xM - (( 51 32 ^iM - (6 52 128 ^iM - (( 53 128|iiM - (( 54 128 )XM - 1 101.32 jag/mL 214.66 from C. coronata

rifampin" 0.3 iiM - " First-line drug used for TB therapy for comparison 172

6.3.3 The Lupanes

Betulinic acid 21 also has activity comparable to 25 and 11. Substitution at C28 as in case of betulin 20 (32 |J,M) and lupeol 56 (64 |i,M) did not seem to change this activity, suggesting a considerable flexiblity for substitution at C28, and in the E-ring (Fig. 6.3,

Table 6.3).

Although, ep/-betulinic acid 55 is not as active as 21, the 3a-hydroxyl isomer 57 of lupeol 56 is more then ten times as active as 56. A complete loss in activity is observed in the case of 58, the 3p-acetylated derivative of 56. A similar loss in activity is seen for

59, the C3 keto derivative.

A complete loss in activity was seen for 60 and 61 suggesting that the isopropyl group in lupanes is also important for activity.

*Two different activities were reported for lupeol 56: in one study it had moderate activity^" and in the other it had none at all. ' Results from each study have been considered individually to evaluate relative activities of compounds within each study. 173

21 Rj = P-OH.R, = COOH

55 R,=a-OH,R, = a>OH

20 R^ = P-OH.R2 = CHJOH

56 Rj= P-0H,R2 = CH3

57 R,= a-0H, R,=CH3

58 Ri= P-0Ac,R, = CH3

59 R,=0,Rj = CHj

m R = OH

Figure 6.3. Antitubercular lupanes 174

Table 6.3. Antitubercular lupanes and their minimum inhibitory concentration (MIC) values against MTB

Compound Activity as MIC vs. Mycobacterium Reference Number tuberculosis Reported (|iM or Normalized to |lg/mL) }iM

21 32|xM - Wachter et ai, 1999*^" 62.1 |j.g/mL 140.50 from V. laxiflora^

55 64|iM - Wachter et al., 1999^"

20 32|iM - 30 |i.g/mL 70.09 from V. laxiflora^^

56 64|lM - Wachter et al, 1999^'' a >128 [Xg/mL 310.68 Woldemichael et al. 2004"^ 57 13.4 iig/mL 32.52 (4

58 >128 ^iM - Wachter et al., 1999^*^

59 >128 ^iM -

60 >128 [iM -

61 >128 |XM -

rifampin" 0.3 |XM - " First-line drug used for TB therapy for comparison 175

6.3.4 The Hopanes and a Fernane

Zeorin 64 has significant activity against MTB growth with its 6a,29 dihydroxy substitution (Fig. 6.4, Table 6.4). The C6 reduced and C7 oxygenated derivatives 65 and

66 lose this activity entirely. (-)-Fem-7-ene-3a-ol (30) isolated from S. brasiliensis was found inactive.

In order to derive any structure-activity relationships for hopanes and femanes many more derivatives with different functionalities will need to be evaluated for MTB inhibition. 176

"'"mf-—OH

62 R = OH

63 R = OAc

""'%Z OH

64 Ri = OH, R, = H

65 Ri = H, R, = OAc

66 Ri = H, R3=0H

Figure 6.4. Antitubercular hopanes and a femane 177

Table 6.4. Antitubercular hopanes and femane and their minimum inhibitory concentration (MIC) values against MTB

Compound Number Activity as MIC vs. Reference Mycobacterium tuberculosis Reported (|iM Normalized to or |ig/mL) [jM 62 12.5 iig/mL 28.03 Okunade et al. 2004'°^ 63 12.5 )xg/mL 25.61

64 - Wachter et al, 1999^° 8 |ig/mL 18.02 Konig et al., 2000 (4 65 >128 [iM -

66 >128 ^M - 30 >128 |Lig/mL 300.47 from S. brasiliensis

rifampin" 0.3 uM - " First-line drug used for TB therapy for comparison 178

6.3.5 The Steroids

Steroids are a class of natural products closely related in terms of their biogenesis and structure to the pentacyclic triterpenoids, which have yielded several potent antiinfective molecules. The bacterial steroid fusidic acid (79, Fig. 6.5) is used clinically against Staph infections. Two different reports present two different activities for fusidic acid against

MTB and these range from fair to moderate activity (Table 6.5). Structures and reported

activities of some antitubercular steroids are presented in Figure 6.5 and Table 6.5

respectively. The most promising of the sterols are the 24 R and S epimers of

saringosterol, 67 and 68 respectively, of which 67 is more potent with an MIC of 0.125

|a,g/mL. It also has no appreciable toxicity against mammalian cells as indicated by an

IC50 >128 |a.g/mL in the Vero cell assay.Saringosterol did not exhibit activity towards

other mycobacteria including M africanum, M. bovis, M. chelonei, M. intracellulare, M.

marinum and M. avium, or for other gram positive and negative bacteria - B. subtilis, S.

aureus, S. aureus methcillin-resistant, E. faecium, E. coli and C. albicans. However, this

compound was only marginally active in vivo. Its remarkable in vitro activity, low

toxicity, and exceptional specificity qualify saringosterol as a good lead structure for the

development of antitubercular agents. To this end, work is ongoing at Mycosynthetix to

produce analogs of saringosterol using biotransformations and these will be evaluated in

vitro and in vivo for their activity and bioavailability. 179

VL/'

r!^

61 MJ? 69

68 MS

V-- < \ P k-

70 R=H 73 R = o 71 R=OH 74 R = P-ORa-H 72 R=OAc 75 R = P-OAc, a-H

V R< 1^' ij / / COOH w / 4^ iO/te

/Ct

= 76 Rj = OH, R, = H

77 TS-i = H,Rj = OH 79

78 Rs = R: = H

Figure 6.5. Antitubercular steroids 180

lOH

HO'

82 83 R = OH

84 R=OAc

Figure 6.5. (contd) Antitubercular steroids 181

87 dmydfo

g§ 4,5 4&hydic

I .^OH

H,COCQ

fCH2)sis

92 93 2-. 23 iiiliyto

94 22.25 deliyiro

4-^

\ V

95

Figure 6.5. (contd) Antitubercular steroids 182

Table 6.5. Antitubercular steroids and their minimum inhibitory concentration (MIC) values against MTB

Compound Activity as MIC vs. Mycobacterium Reference Number tuberculosis" Reported (iliM Normalized to |j,M or |ig/mL) 67 0.125 |Xg/mL 0.29 Wachter et a/., 2001"^ 68 1 )j.g/mL 2.34 69 32 [j-g/mL 75.12 Okunade et ah, 2004"^^ 70 3.13 |ig/mL 9.21 Copp, 2003'"^ u 71 NR - 72 12.5 jig/mL 31.41 (4 73 8 iig/mL 18.26 Cantrell a/., 2001^^ 74 8 |j,g/mL 18.22 (4 75 >128 |lg/mL >266.11 (4 76 16 |j.g/mL 34.93 44 77 4 iig/mL 8.73 44 44 78 NR - 79 4 |lg/mL 9.03 Cantrell, etal, 1996'°^ 32-64 la^g/mL 87.43-161.62 Copp, 2003"" 80 16 |j,g/mL 43.72 Cantrell et al, 2001^^ 81 >128 |ig/mL >323.23 (4 82 >128 |ig/mL >323.23 44 83 1 |ig/mL 2.34 Copp, 2003'"^ 84 8 )j,g/mL 17.02 44 85 32 |xg/mL 74.77 Okunade ef a/., 2004'°'^ 86 >128 |ig/mL 281.94 44 87 64-128 |Xg/mL 150.0-290.91 Cantrell et al, 2001^*^ 88'' Mixture- 2 4.88 Okunade et al, 2004'°^ 89^^ fXg/mL 44 90 >128 |j.g/mL > 280.09 Cantrell, etal, 1996'°^ 91 <128 )ig/mL <286.35 Konig et al, 2000''' 92 >128 |ig/mL >205.13 Woldemichael et al. 2004'"^ 93 128 ixg/mL 310.68 Okunade et al, 2004'°^ 94 32 |Xg/mL 77.67 44 95 >128 i^g/mL >227.35 Woldemichael et al. 2004'"^

rifampin^ 0.3 |XM - " NR = not reported ' Unresolved Mixture ' First-line drug used for TB therapy for comparison 183

CHAPTER VII

GENERAL EXPERIMENTAL

7.1 General Instrumentation

Optical rotations of compounds isolated from C. coronata were measured on JASCO P-

1020 polarimeter, and of compounds isolated from M. coquimbensis were measured on

JASCO P-1010 polarimeter. IR (as a film on a diamond cell) was measured on a Thermo

Nicolet Avatar 360 FT-IR spectrometer. NMR spectra were recorded in CD3OD, CDCI3, or pyridine-cfs on a Bruker Avance DRX-500, or a Bruker Avance DRX-600 spectrometer at the Department of Chemistry, University of Arizona, with residual solvent as reference. HR-FABMS with m-NBA as matrix on a JEOL HXllOA mass spectrometer.

Melting points were determined on Fisher-Johns melting point apparatus. HPLC was carried out using a Varian Prostar pump, and a Varian Prostar UV-Vis detector. Column and solvent conditions for each HPLC isolation and purification were selected by method development to optimize conditions, and the conditions finally used are described in the text discussing the bioassay guided isolation of each compound. TLC plates were viewed using Spectroline Model CM-10 fluorescent analysis cabinet detecting both short wave

(254 nm) and long wave (356 nm). TLC plates were sprayed with a solution of 0.5% anisaldehyde, 10% glacial acetic acid, and 5% concentrated sulphuric acid in methanol followed by gentle heating. 184

7.2 HPLC/DAD/MS/MS

All LC/MS analyses were performed using an Agilent 1100 HPLC system consisting of a quaternary gradient pump, a thermostatted column compartment, an autosampler, and a degasser coupled with a diode array detector (DAD) tandem with an Agilent LC-MSD-

Trap-SL ion trap mass spectrometer interfaced to a ChemStation operation software

(Agilent Technologies, Palo Alto, USA). HPLC separation was achieved using a Synergi

Hydro-RP 80A C18 reversed phase column (4 |a,; 4.6 x 250 mm) (Phenomenex, Torrance,

CA, USA) and protected by a SecurityGuard AJO-4287 (Phenomenex, Torrance, CA,

USA) cartridge (4.0 x 3.0 mm i.d.). The column temperature was set at 30°C. Samples of

20 |J,L were injected. For MS detection in the positive ionization mode, the optimized conditions were: ESI conducted at 350 °C; nebulizer pressure, 60 psi; drying nitrogen flow rate, 10 L/min; capillary voltage, 4000 V. The MS range was measured at m/z 50-

1000 in an automatic detection mode. The isolation width for MS(2) was 4.0 m/z, and the collision energy used was 1.5 V.

7.3 X-Ray Crystallography Data Collection.

SMART (Bruker, 1997); cell refinement: SMART; data reduction: SAINT (Bruker,

1997); program(s) used to solve structure: SHELXS97 (Bruker, 1997); program(s) used to refine structure: SHELXL97 (Bruker, 1997); molecular graphics: SHELXTL (Bruker,

1997); Mercury; software used to prepare material for publication: SHELXTL. 185

APPENDIX A

Peer-reviewed publications of results obtained from the research presented in this dissertation. 186

organic papers

Actii S

S. Khera/ S. D. Jolad," The structure of a fernane feolated from S. hrasilw.mis was 21 liiV estaWisfied as fern-7en-:to-ol,Cx)HjaO. Rings/t at»d D asBiume ! 4 AUKIJST 2<'|t 170.'i I. St., iso|wopyl group is ui. Tiic.?on, Al USA, artJ «.n' Chi?mis?f jf, LJnivsssitv !"rtl Xmt aiC-C) » 0,00,^ A R iM'tof « every country (Cohn et al, 1997), and a percentage of these tv/; tf^cKsr ~ 0.1 Cli are resistant to multiple drugs mating effective treattnent Diit4-R>?Mram«?t

X-ray crystallographic analysis of (1) was undertaken to unequivocally establi.sh the structure and to assign the relative stereochemi.stry at O. Although fl) has previotely been reported in the literature (Nakane 0 al,. 19<>>)), this is the first report of its isdation from S. hrasiUatsis and of its cry.stal structure. The X-ray structure of (1) can now .serve as a standard for comparison of related f'ernane.s With reference to :C IH'iO.'i kev^nvilkuuil L'riion ot Cry^^alb^fi^ihy the Scheme, the aiwolute configuration at all chiral centers is in ffei'.aiji - .ijit iiiiiifs 5S. 9/?, 10.9, 13.S, MS. 17/f, 1S«. 21«.

Acu Cfyst. (2tt'5JL E59, fiJ-WiJ-ol-IM iX3l; 10.1 TF)7;'SIM05.M>8F);I01S2:I:L O1403 187

l«28 J. Nat. Prod. 2003, SB. 1628-1631

A Novel Antibacterial It icioid and Triterpeiie from Cuiophora coronata

Snirltl Kltera,' Glrnia M. WoWenilchaeL* Maya P. Singh,' Enrique Suarcx,* find Barbara N, 'i intniei tnanii'- De/xiititifjit ofFh4Ujnacohgs' of Medkimil NatwssI Paxh^rt.^ Chemistt v. of Unhvi sify t)f'i 703 B. Tuc^fti. Arti^oii^i 85721. Wyi*th Rese;m:h. Natuiul Prmhiirts .Soummg. 401 N. Mkhlk'tovin Road, PearJ River. New York 10965. mid fistiruto de Reairsm Bioic^km, Las Cabimm y Los Resems s/n. illi^ Villa Udaondor- Casrelar, Pvovinciit ik' Buenos Aij-m. .'hj^eiidna

Rmvival ./tdy 17. 2m3

Bloassay-jjuided fiactlonation of t he antibacterial CHjClj MeOH ext ract obtafripd from the aerial paits of the Argent inean plant Catqjhoa} coi-mMa led to the isolatloti of a new tiit erpeiH>. l/i3/,i-dihydroxyiirs- 12-en-27-oic acid. 1, aiui a new iridoid, la-melhoxy-fia.lO-dihydroxylstwpliridorayrnietiii {caiophoraeiiinl, 2, along with the Icnown irkloid fsobwjneiii 3 Their stniatires were {••stai.Jllslted by sfMarost-opif techniques |iD and 2D NMR. IIRFABMS. FIIR). 'liie MfC; valiifs of Isolated coiiijwtmtis were detertiilrjecl against tiiethicillin-sensitive (MSS<\| and -resistant (MRSA) strains of Staphyiiicxcus aiwms, fkicllhis siihtilis {BS), vanconiyciii-reslstant Eitterococcm faecium (VREF). Escimtdiia mil (EC). E. mil imp (ECinipj. and Candida ^ilblairis (CAJ. (Compound 1 was found active against BS, MSS.A. MRSA, Vf-JEF, and fif'imp with MIC values of 2. 4, '1. 4, and 16 /«g/mL. rps(mtlvely.

As a part of the Intprnatiotiaf Ccxjpt'raOve Biodiversity Group, our research pragi ara entitled 'Bioactivc Agents frnrti Dryland Biodlver.slty of f .atin America" explores the dry land vegetation of South America for the purpose of drug lead dlsc-ovei^'. We routinely screett plant extracts for their antibacterial activity against a variety of ciitiically OH significant bacterial straln.s. The CTl2t;b-MeC)H extraa of aerial parts of the Aigeiitinean plant Caiophoia comnata Hook, et .Arn. (f^oasaceat'l was found t.a be aclive against COOH both the (nethicilllii-,s(;nsitive atul -resistant bacteiial strains of Slaphylocoecus aitnnis in an agar diffusion test. On the tHsis of this .Important activity, the extract was selected for blonssay-guided fractionation.

Prior clieniica) studies on CaiOf.ilmra have led to the 11 Me isolation of numerous Irldoids. An utiidentlfied irldlod, 10 HO hydroxyoieosldff dimethyl ester, as well as a iortiti fiave previously been reported iti C, Additionally, pentlandloslde f. a t.)is-se(.-oiridoicl. was isolated from Cn- Ji^ilmra iM'ntlandii} The iridoid lO-hydroxyoleoside di- metliyl ester Is used as a cliemotaxonomic niaricer for this genus.' Here we report the isolation, cluuacleiization. and antimicrobial Ktivities of a new Iridolactone. a new ursanc- OH type triterpeiie, and tiie known iridoid isatoonein. 2 'I'he crude ('HjCi?-MeOI i (1:1) extract of the aerial parts of C. vmniuiia exhibited activity against both tiie methl- e;illir)-.sen,sit.{ve and -r«istant bacterial strains of S. auimis NOJ it! an agar diffusloti test with zones of growth inhitjition of 11 and 12 ram. respeaively. The extract mis then fraction­ ated on a silica gei txilumn using ethyl acetate (0 to (KRu Me gradienti in w hexane. Ftsrther ft aeUotMlJoti of the active fractions afforded two nc>vv compounds, ttie ursane-lype spnndlng to the niolwiilar formula Ci^ofiinOd. Thp IR triterjxine l/i',3//-dihydroxyi!rs-I2-en-27-oic acid. 1, and tiie specu'ijin stigg»?steti tiie of hydroxyl (3360 rnr hidold cftiophoraenln. 2. as well as isolxKMieln 3, Isotooneln arici cafJ,x}tiyi (UaSO rriV"^) groujis. flu,i and DEPT was Identlfieit by compiuison of its sf»ctral data with tliose sppcT.ra jiidi€at

* To whom corr^sporidenfje shoulirJ acldresspfl. Tel: (5'^0) G26-2481. The quaternaiv carbon at d 170.3 w^is assigned to a free Fax: (520) (i26-25l5. Email: l:)t:!tmTipf<®ph',13, respea,ively). 10,1021/ni3£00314a CCC: $25.00 €' 2(X13 Aiiwlraii Chenilaii Socleiiy and Anierfcan Scictetv of Pliarinac

organic compounds

As a part of the International Co-operative BiiMSversity Crystal Structure Group, our program focuses on the seareh for novel anti- Coiranuni cations Euberculiif principles of plant or microbial origins from the ISSN 0106-270! dry-land biodive rsity of i jitin America. It is in this regard lhat the bioassay-guided chemical invesligatbn of the Chilean plant Vak'riaim laxiftora DC (Valerianaceae) was initialed. (4/?,4aff,6S,7S,7aS)-6-Hydroxy-7-hy- The methanol extract of V. hxifiora was found to I* active droxymethyl-4-itielhylperhydrocyclo- against the i-l.wRv strain (ATCC 27294) irf Mycuhacterimn luhercithtis, and the bioassay-directed fractionation led to the pentafc]pyran-1-one chloroform isolation of the title compound, (I), an iridcilactone, as solvate from Valeriann iaxifhra previou-sly dejscritied (Gu

Smriti Khcra/ MichacI D. Carducci,** jian-Qiao Gu" and H I Barbara N. TimnKrmann^* *Unfv<5fsity i«^y I JOi f. .S<., Tuc-i

X-Ray crystalbgrapltic analysis of (1) was initiated to Receivitd J \urw 2s establi.shed as (•4/?.4a/?,M.75.7a.S)-<)-hydroxy-7- laxifiura /^-oriented, as sliown in Fig. 1. The e cItKely related iridolactone (Eisenbraun «*/ al. 19S1). As adventitious inclusion of a chloroform solvent molecule. expected in the case of ^-lactones, differences in the two Hydrogen boiwling, crystal packing and ring ctaiformations C—01 bond distances are (jbserved, the C.?—01 bond are discu^cd in detail. [1.451 (7) A) being longer than the CI—Ot bond [1335 (6) A| (Cheung et ai. 1965). Corement The 09—€1 (02)—01 —C.3 lactone group is almist planar, with an r.m.si deviali(*i of 0.0208 A and a maximum deviation The alarming increase in the number of deaths due to tuber­ of 0..152 (4) A for atom Ol. T'he C3—Ol—CM—02 torsion culosis (TB) and growing resi.«anee to e.visiing drugs has created an urgent need for the identification of leads for new antimycobacterial dri^s. There were H.i million new case.s of fulicrcuksis in tlie year 2000 alone, and this numter i.s on fl>e I jcn ri.se. Appro.\imately one death every 15 s is due to TB, resulting itt a total of about two million IB deaths annually (Corbett et al., 20fB). .Strains of TB resistant to e.xisting drugs r arc found in nearly every ajunlry (Ciihn el a!., 1997), and a percentage of these strains are resistant to multiple drug.s. I making effective treatment extremely expen.sive and, in many cases, impossible. Most patients in developing countries, where TH is an even bigger problem than elsewhere in the world, cannot afford expensive drug treatments. Tliere have been no 7 " new drugs developed for TB in over W years. Current IB therapy relies on drugs thai are 50 years ok! and lake six to Figure I nine months to complete, making patient compliance very A vkw of 0). shown wtth 5tl% pfohijbrtity displac^mcnl eUipsiikls. H difficult (Workl Heahh Organization, 200.?). atijRjs arc shown as

AcU mm). C6(l, o7?:i-i>?75 [XH: 10. i 511 i|" iy 077S 189

APPENDIX B

ID and 2D NMR spectra of new compounds 1 and 2 isolated from C. coronata, and 10 isolated from M. coquimbensis as described in Chapters 2 and 3 respectively. COOH

Figure B.l. 'h NMR spectrum of lp,3|3-dihydroxyurs-12-en-27-oic acid (1) in CD3OD at 600 MHz 3 =

.Sloiid^id i^iC'Specl.i'uni C.Hinl IW—IjC ^1CT>E

Figure B.2. '^C NMR spectrum of ip,3P-dihydroxyurs-12-en-27-oic acid (1) in CD3OD at 125 MHz 192

^ujcidj uoc|vJO 3 O'Ob 0'08 0'0^^ 0'09f i

-4- "' ^ Ab. . .a«»s..

... -

*• • i' i t•' 1 • • t t t

*• ' . • »

11

^ k t t i - 1 1

1 t i 1

, 1

» 1 « *

Figure B.3. HMBC spectrum of lp,3p-ciihydroxyurs-12-en-27-oic acid (1) 193

( uudd _) I J

Figure B.4. DQF-COSY spectrum of l(3,3P-dihydroxyurs-12-en-27-oic acid (1) H2a.b H9

-7 ^ ; ? ! % 5 ' ^ ^ ^ ^ I ' ^ ^ ' 3.5 3C 4.5 4.0 13 3,0 3.3 lO U- l.O

Figure B.5. ID NOE spectrum of lp,3P-dihydroxyurs-12-en-27-oic acid (1) in CDaODat 600 MHz cni<3pii(?V3fin IIill

Me HO

10 OMe OH

Figure B.6. 'H NMR spectrum of caiophoraenin (2) in CD3OD at 600 MHz 3R SS is Sn 53 fi S? S i:

^IstKlnid. 13C Spfdmin '^ir r'Tiai iH—i3c pt«iw

Me HO

10 OMe

ii»(jlll|bpi»t»iiti»it«l)i>^iifWi*i4)»»W[iii>i|Miiyiii>i!Wt#i#i^(fc »m0»iillwji# g

ISO 17S 170 165 160 155 ISO l.."5 t,"0 12.5 liO ll.S l.IO ICB .tOO •K' S.'j SO T.'? TO 65 60 .55 SO 4S 40 ."1 JO 2.5 33 15 lO T^pni

Figure B.7. ' "^C NMR spectrum of caiophoraenin (2) in CD3OD at 125 MHz 197

^ uidd ^ UOC|UDQ

O'Ob 0'09 0*08 O'OS 0'09/

fM

- tllM)- < /M-- I i f\i 6 a a 00 • • c fM 0 0

'Kh-

T

Figure B.8. HMBC spectrum of caiophoraenin (2) 198

(•^dd) [J

* c 0*2 8'2 9•£ I? ' k

w

m

M ife • «• ''V*i% , 4lt - ii Ip.

m m

• # m •»

c » •

<» m m m

% ^ a «>« • m 5' m HI

- " «>

Figure B.9. DQF-COSY spectrum of caiophoraenin (2) 199

(li^dd) IJ

\

Figure B.IO. ROESY spectrum of caiophoraenin (2) Figure B.ll. 'H NMR spectrum of (15',35',4/?)-l-methyl-4-(l-methylethenyl)-l,3-cyclohexanediol (10) in CD3OD at 600 MHz Hfilp.fif.f 3 ^ 3^ STUIDAD 13C \ f I'Tial IH—t3C pic-te m; I M i

7 PH

HO

bcA, 10

I > • I. ). . I J: li III, ml •iiWi.iu

130 143 140 135 130 123 120 113 110 103 ICO «»3 <10 35 SO 73 70 feJ 60 33 30 45 40 3S 30 25 X*

Figure B.12. '"^C NMR spectrum of (15,35,4/?)-l-methyl-4-(l-methylethenyl)-!,3-cyclohexanediol (10) in CDCI3 at 125 MHz 202

( Uidd ^ uoc|JO J 0*02 O'Ob 0'09 0*08 O'OO O'Ob

Figure B.13. HMBC spectrum of (15,3'S',47?)-l-methyl-4-(l-methylethenyl)-l,3- cyclohexanediol (10) 203

f Ludd ) I J 91 ^'£ O'P 8>

o

Figure B.14. DQF-COSY spectrum of (15',3>S',4i?)-l-methyl-4-(l-methylethenyl)-l,3- cyclohexanediol (10) H9a

5,0 4.S -4.0 3 O 2-S

Figure B.15. ID NOE of (15,35,4/?)-l-methyl-4-(l-methylethenyl)-l,3-cyclohexanediol (10) in pyridine-ds at 600 205

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