Antimycobacterial Natural Products from Higher Plants. Charles Lowell Cantrell Louisiana State University and Agricultural & Mechanical College

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1998 Antimycobacterial Natural Products From Higher Plants. Charles Lowell Cantrell Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Cantrell, Charles Lowell, "Antimycobacterial Natural Products From Higher Plants." (1998). LSU Historical Dissertations and Theses. 6660. https://digitalcommons.lsu.edu/gradschool_disstheses/6660

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UMI

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Chemistry

by Charles L. Cantrell B. S., Louisiana State University, 1994 May 1998

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UMI 300 North Zeeb Road Ann Arbor, MI 48103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

I wish to express my sincere appreciation to my major professor, Dr. Nikolaus H.

Fischer, for his scientific as well as personal guidance, for being a wonderful professor

and mentor, and a great friend. I also wish to thank Mrs. Helga Fischer for her assistance

on numerous research projects and for being a true friend.

I am also thankful for the support and encouragement provided to me by Dr. Scott

Franzblau of the G.W.L. Hansen’s Disease Center at LSU School of Veterinary

Medicine. Additionally, I wish to thank Anita N. Biswas and Ken E. White for technical

support which they provided to me in performing radiorespirometric bioassays. Financial

support from the National Institute of Allergy and Infectious Diseases, National Institutes

of Health under contract No. Y02-AI-30123 is gratefully acknowledged.

I would like to acknowledge Dale Traleaven and Marcus Nauman for their patient

assistance in operating the NMR instruments. I am grateful to Dr. Frank Fronczek for

providing X-ray structures of numerous compounds presented in this dissertation. I thank

Dr. Lowell Urbatsch for his assistance in plant identifications and collections.

Thanks to my research group, the faculty, and fellow graduate students for their

support and friendship. I wish to especially thank Dr. Tiansheng Lu for his patient

assistance and guidance as I began my graduate career.

Lastly, I would like to thank my wife, Julie Cantrell, for her patient support and

understanding throughout these years. She has been a constant source of encouragement

and an inspiration to me.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGMENTS ......

LIST OF TABLES ......

LIST OF FIGURES ......

ABSTRACT ......

CHAPTER 1. INTRODUCTION AND REVIEW ...... 1.1 Epidemiology of Tuberculosis ...... 1.2 Current Treatment Methodology ...... 1.3 Review of Antituberculosis Terpenoids ...... 1.3.1 Monoterpenes ...... 1.3.2 Sesquiterpenes ...... 1.3.3 Diterpenes ...... 1.3.4 Triterpenes ...... 1.3.5 Phytol, derivatives, and analogs ...... 1.4 Conclusions ...... 1.5 References ......

2. ANTIMYCOBACTERIAL CRUDE PLANT EXTRACTS FROM SOUTH, CENTRAL, AND NORTH AMERICA 2.1 Introduction ...... 2.2 Experimental ...... 2.3 Results ...... 2.4 Discussion ...... 2.5 References ......

3. ANTIMYCOBACTERIAL CYCLOARTANES FROM BORRICHIA FRUTESCENS...... 3.1 Introduction ...... 3.2 Results and Discussion ...... 3.3 Experimental ...... 3.4 References ......

4. ANTIMYCOBACTER1AL ACTIVITIES OF DEHYDROCOSTUS LACTONE AND ITS OXIDATION PRODUCTS ...... 4.1 Introduction ...... 4.2 Results and Discussion ...... 4.3 Experimental ...... 4.4 References ......

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. ANTIMYCOBACTERIAL SESQUITERPENE LACTONES FROM INULA HELENIUM AND RUDBECKIA SUBTOMENTOSA ...... 112 5.1 Introduction ...... 113 5.2 Results and Discussion ...... 113 5.3 Experimental ...... 116 5.4 References ...... 137

6. TWO NEW ANTIMYCOBACTERIAL TRITERPENES FROM MELIA VOLKENSII ...... 139 6.1 Introduction ...... 140 6.2 Results and Discussion ...... 141 6.3 Experimental ...... 144 6.4 References ...... 163

7. SUMMARY AND CONCLUSIONS...... 165

APPENDIX: LETTERS OF PERMISSION ...... 171

VITA ...... 173

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

1.1 Estimated Global TB Incidence and Mortality in 1992 ...... 2

1.2 Terpenoid Minimum Inhibitory Concentrations (MICs) Against M. tuberculosis (H37R.V) 7

2.1 Antimycobacterial Activity of Various Crude Plant Extracts 36

3.1 Inhibitory Activity of Borrichia frutescens Fractions (Flowers, CH2CI2 Extract) Against Mycobacterium tuberculosis (H37R.V) 64

3.2 'H-NMR Spectral Data of Compounds 3.1,33a. 3.3b,3.4, and 3.5 (250 MHz, CDCI3) ...... 65

3.3 13C-NMR Spectral Data of Compounds 3.1,33a, 33b, 3.4, and 3.5 (62.5 MHz, CDCI3) ...... 66

3.4 Crystallographic Data of Triterpenes 3.1 and 3.4 67

3.5 Coordinates and Equivalent Isotropic Thermal Parameters for (24/?)-24,25-Epoxycycloartan-3-one (3.1) 68

3.6 Coordinates and Equivalent Isotropic Thermal Parameters for (23/?)-3-Oxolanosta-8,24-dien-23-ol (3.4) 69

3.7 Minimum Inhibitory Concentrations Against M. tuberculosis and IC50 Values Against Vero cells ...... 70

4.1 300 MHz 'H-NMR Spectral Data for Compounds 4.2-4.4c Performed in CDC1 3 ...... 92

4.2. 75.4 MHz l3C-NMR Spectral Data for Compounds 4.2-4.4c Performed in CDCI 3 ...... 93

4.3 Crystal Data and X-ray Collection Parameters o f 4.1 b, 4.4a, and 4.4c 94

4.4 Coordinates and Equivalent Isotropic Thermal Parameters for 4.1b 95

4.5 Coordinates and Equivalent Isotropic Thermal Parameters for 4.4a 96

4.6 Coordinates and Equivalent Isotropic Thermal Parameters for 4.4c 97

4.7 Minimum Inhibitory Concentrations of Compounds 4.la-4.6 Against Mycobacterium tuberculosis ...... 98

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1 Percent Inhibition of Crude Plant Extracts of Inula helemum and Rudbeckia subtomentosa Against Mycobacterium tuberculosis at 1000 andlOOng/ml 120

5.2 Percent Inhibition of Inula helenium and Rudbeckia subtomentosa Fractions Against M. tuberculosis ...... 121

5.3 'H NMR (300 MHz) and 13C NMR (75.4 MHz) Spectral Data of Compounds 7 and 8 (CDCh) ...... 122

5.4 Minimum Inhibitory Concentrations of Compounds 1-8 Against M. tuberculosis ...... 123

5.5 Coordinates and Equivalent Isotropic Thermal Parameters for 7 124

6 .1 Inhibitory Activity of Melia volkensii Fractions (Fruit, MeOH Extract)

Against Mycobacterium tuberculosis (H 3 7 R.V) 146

6.2 1 H-NMR (400 MHz, CDCI3) and HMBC (300 MHz, CDCI3) Spectral Data of Compounds 6.1a and 6.1b 147

6.3 I3C-NMR Spectral Data of Compounds 6.1a and 6.1b (62.5 MHz, CDCI3 ) ...... 148

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

1.1 First and second line antimycobacterial drugs ...... 15

1.2 Monoterpenes tested against M. tuberculosis...... 16

1.3 Costunolide, parthenolide, and derivatives tested against M. tuberculosis 17

1.4 Additional sesquiterpenes tested against M tuberculosis ...... 18

1.5 Diterpenes tested against M. tuberculosis ...... 23

1.6 Triterpenes tested against M. tuberculosis ...... 26

1.7 Phytol, derivatives, and structural analogs tested against M. tuberculosis 27

3.1 Triterpenes isolated from B. frutescens ...... 71

3.2 Molecular structures of compounds 3.1 and 3.4 ...... 72

3.3 250 MHz 'H-NMR spectrum of (24/?)-24,25-epoxycycIoartan-3-one (3.1) in CDCI3 ...... 73

3.4 62.5 MHz DEPT 90°, DEPT 135°, and broad band , 3C-NMR spectra of (24/?)-24,25-epoxycycloartan-3-one (3.1)...... 74

3.5 250 MHz 'H-NMR spectrum of (3(3,24/?>-24, 25-epoxycycloartan-3-ol (3.3a) in CDC13 ...... 75

3.6 62.5 MHz DEPT 90°, DEPT 135°, and broad band UC-NMR spectra of (3(3,24/?)-24, 25-epoxycycloartan-3-ol (3,3a) ...... 76

3.7 250 MHz 1 H-NMR spectrum of compound 33b in CDCI3 ...... 77

3.8 250 MHz 1 H-NMR spectrum of (23R)-3-oxolanosta-8,24-dien-23-ol (3.4) in CDCI3 ...... 78

4.1 Dehydrocostus lactone and derivatives ...... 99

4.2 Molecular structures of compounds 4.1b, 4.4a, and 4.4c ...... 100

4.3 300 MHz 1 H-NMR spectrum of 4a( 15 )-epoxydehydrocostus lactone (4.2) in CDCI3 ...... 101

4.4 75.4 MHz DEPT 90°, DEPT 135°, and broad band UC-NMR spectra of 4a( 15)-epoxydehydrocostus lactone (4 .2 ) ...... 102

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.5 300 MHz 'H-NMR spectrum of 10P( 14)-epoxydehvdrocostus lactone (4.3a) in CDCI3 ...... ’...... 103

4.6 75.4 MHz DEPT 90°, DEPT 135°, and broad band l 3C-NMR spectra of 10(3(14 )-epoxydehydrocostus lactone (4.3a) 104

4.7 300 MHz 'H-NMR spectrum of 10a( 14)-epoxvdehydrocostus lactone (4.3b) in CDCb ...... '...... 105

4.8 75.4 MHz broad band I 3C-NMR spectra of 10a( 14)- epoxydehydrocostus lactone (4.3 b) ...... 106

4.9 300 MHz 1 H-NMR spectrum of 4P( 15), 10a( 14)-diepoxydehvdrocostus lactone (4.4a) in CDCI3 ...... 107

4.10 75.4 MHz DEPT 90°, DEPT 135°, and BB l 3C-NMR spectra of 4P( 15), 10a( 14)-diepoxydehydrocostus lactone (4.4a) 108

4.11 400 MHz lH-NMR spectrum of4a(15),10a(14)-diepoxydehydrocostus lactone (4.4b) in CDCI3 ...... 109

4.12 400 MHz 'H-NMR spectrum of 4a( 15), 10P( 14)-diepoxydehydrocostus lactone (4.4c) in CDCI3 ...... 110

5.1 Sesquiterpene lactones isolated from I. helemum and R. subtomentosa 125

5.2 Molecular structure of 5.7 at 100° K ...... 126

5.3 300 MHz 1 H-NMR spectrum of alantolactone (5.1 ) in CDCh 127

5.4 75.4 MHz broad band I 3C-NMR spectrum of alantolactone (5.1 ) in CDCI3 ...... 128

5.5 300 MHz 'H-NMR spectrum of isoalantolactone (5.2a) in CDCI3 129

5.6 300 MHz 'H-NMR spectrum of dihydroisoalantolactone (5.3) in CDCI3 130

5.7 300 MHz 1 H-NMR spectrum of 3-oxoalloalantolactone (5.5b) in CDCI3 131

5.8 300 MHz 'H-NMR spectrum of a-epoxyisoalantolactone (5.6) in CDCI3 132

5.9 300 MHz lH-NMR spectrum of a-epoxyalantolactone (5.7) in CDCI3 133

5.10 62.5 MHz DEPT 90°, DEPT 135°, and broad band 13C-NMR spectra of a-epoxyalantolactone (5.7) 134

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.11 300 MHz 'H-NMR spectrum of 11, 13 dihydroxvalantolactone (5.8) in CDCH...... ' ...... 135

5.12 75.4 MHz broad band l 3C-NMR spectrum of 11, 13 dihydroxvalantolactone (5.8) in CDCH ...... 136

6.1 Triterpenes isolated from M. volkensu ...... 149

6.2 !H-13C HMBC correlations for 6.1a and 6.1b ...... 150

6.3 300 MHz ‘H-NMR spectrum of 12-P-hydroxykulactone (6.1a) in CDCH 151

6.4 75.4 MHz DEPT 90°, DEPT 135°, and broad band I 3C-NMR spectra of 12-P-hydroxykulactone (6.1a) 152

6.5 300 MHz ‘H^H COSY spectrum of 12-P-hydroxykulactone (6.1a) 153

6.6 300 MHz 'H -13C correlation (HMQC) spectrum of 12-P-hydroxykulactone (6.1a) 154

6.7 300 MHz ‘H-i3C correlation (HMBC) spectrum of 12-P-hydroxykulactone (6.1 a) ...... 155

6.8 300 MHz lH-'H NOESY spectrum of 12-P-hydroxykulactone (6.1a) 156

6.9 300 MHz ‘H-NMR spectrum of 6-P-hydroxykulactone (6.1b) in CDCH 157

6.10 75.4 MHz broad band l 3C-NMR spectra of 6-P-hydroxykulactone (6.1b) 158

6.11 300 MHz 'H -’H COSY spectrum of 6-P-hydroxykulactone (6.1b) 159

6.12 300 MHz ‘H-^C correlation (HMQC) spectrum of 6-P-hydroxykulactone (6.1b) ...... 160

6.13 300 MHz ’H-^C correlation (HMBC) spectrum of 6-P-hydroxykulactone (6.1b) ...... 161

6.14 300 MHz 'H -’H NOESY spectrum of 6-P-hydroxykulactone (6.1b) 162

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Two-hundred and thirty crude organic extracts from 118 plant species distributed

among ten families of higher plants have been evaluated for antimycobacterial activity

against Mycobacterium tuberculosis (H37R.V) and M. avium using the BACTEC 460

radiorespirometric assay. At 100 pg/ml, twenty-four and ten of the extracts caused more

than 95% inhibition of growth of M. tuberculosis and M. avium, respectively.

Bioactive chromatographic fractions of Borrichia frutescens (Asteraceae)

provided two new triterpenes and one known cycloartenol. In radiorespirometric

bioassays against M. tuberculosis, the new (24/?}-24,25-epoxycycloartan-3-one and the

known (24j'?)-24,25-epoxycycloartan-3(3-ol exhibited minimum inhibitory concentrations

(MICs) of 8 gg/ml. In contrast, the new (23/?)-3-oxolanosta-8,24-dien-23-ol showed no

significant inhibition at 128 pg/ml.

In an attempt to study the structural dependence of antimycobacterial activity of

the highly active guaianolide dehydrocostus lactone (MICs of 2 and 16 pg/ml against

Mycobacterium tuberculosis and M. avium, respectively) and its derivatives, and to

determine configurational and possible conformational effects upon activity, m-

chloroperoxybenzoic acid oxidations of dehydrocostus lactone were performed. Three

new monoepoxides, one previously synthesized diepoxide, and two new diepoxides were

obtained, all of which showed strongly reduced antimycobacterial activity.

Antimycobacterial root extracts of Inula helenium and Rudbeckia subtomentosa

were chemically investigated. Chromatographic fractions of active root extracts of /.

helenium provided the known eudesmanolides alantolactone, isoalantolactone, and 1 lotH,

13-dihydroisoaIantolactone. Active fractions from root extracts o f R. subtomentosa gave

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the known alloalantolactone and 3-oxoalloalantolactone. All natural and semisynthetic

eudesmanolides were tested for biological activity against XI. tuberculosis and

demonstrated MICs ranging from 8 pg/ml for 5,6a-epoxyalantolactone to >128 pg/ml for

11,13-dihydroisoalantolactone and 11,13-dihydroxyalantolactone.

Methanol extracts of fruits of Melia volkensii were chemically investigated and

shown to contain two new triterpenes and the known triterpenoid kulonate. The

structures of the new 12[}-hydroxykulactone and 6|3-hydroxykulactone were elucidated

by ID and 2D NMR experiments and FABMS studies. All three triterpenes exhibited

significant activity against M tuberculosis with MICs ranging from 4 pg/ml to 16 pg/ml.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

INTRODUCTION AND REVIEW

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1 Epidemiology of Tuberculosis

According to the World Health Organization (WHO), the global prevalence of

tuberculosis (TB) was estimated to be 1.7 billion persons, or approximately one-third of

the world’s population (Kochi, 1991). Active disease develops in 8 to 10 million people

per year, and TB is responsible for 3 million deaths per year, making it the leading

infectious cause of death in the world, far surpassing measles (2 million deaths per year)

and malaria (1 million) (Raviglione et al., 1995). Table 1.1 shows the worldwide

distribution of TB divided amongst seven regions (Snider et al., 1994).

Table 1.1 Estimated global TB incidence and mortality in 1992

Incidence Mortality Area No. of cases Rate* No. of cases Rate0

Southeast Asia 3,263,000 240 1,142,000 84

Western Pacific* 1,921,000 136 672,000 48

Africa 1,182,000 214 468,000 85

Eastern Mediterranean 683,000 166 266,000 65

Americas0 584,000 128 117,000 26

Eastern Europe 197,000 47 29,000 7

Industrialized countries'* 199,000 22 14,000 2

All regions 8,029,000 146 2,708,000 49

° Per 100,000 population. b All countries of the region except Australia, Japan, and New Zealand. 0 All countries of the region except Canada and the United States. d Western Europe plus Australia, Canada, Japan, New Zealand, and the United States.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3

Such dramatically high morbidity and mortality rates are in contrast to the

experiences in the United States, even at the height of the problem. Beginning in the

1920’s, annual rates of TB in the United States declined. In 1953, when official annual

reporting began, there were 84,304 reported cases of tuberculosis; by 1984, reported

cases had dropped to 22,255, a decrease of about 5% per year. However, in 1984 the

trend reversed, and by 1988 the annual rate of TB in the United States began to rise. In

1992, 26,673 cases, or about 10/100,000 persons, were reported (Sepkowitz et al., 1995).

1.2 Current Treatment Methodology

The current regimen for the treatment of pulmonary TB evolved over many years

and is based on dozens of clinical trials involving thousands of patients worldwide

(Grosset et al., 1980). In 1990, the U. S. Public Health Service evaluated short course

chemotherapy (6 versus 9 month) for pulmonary TB (Inderlied et al., 1996). On the basis

of this study, a 6-month course of isoniazid (1.1), rifampin (1.2), and pyrazinamide (1.3)

for 2 months followed by isoniazid (1.1) and rifampin (1.2) for an additional 4 months

was recommended for uncomplicated susceptible TB (Inderlied et al., 1996).

More recent problems in the treatment o f pulmonary TB are exacerbated by the

AIDS pandemic and also by resistance to existing anti-TB drugs such as isoniazid,

rifampin and streptomycin (Cohn et al., 1997). In general there are currently two key

drugs in the treatment of TB: isoniazid and rifampin. Susceptibility to both allows 6- to

9-month regimens; susceptibility to rifampin but not isoniazid allows 9- to 12- month

regimens; and susceptibility to isoniazid but not rifampin allows 12- to 18-month

regimens. However, when the isolate is resistant to both isoniazid and rifampin, the

outcome is uncertain (Sepkowitz et al., 1995).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since the introduction of rifampin in 1966, there have been no new drugs for the

treatment of TB. The combined effects of the current pandemic AIDS and multi-drug

resistant TB strains have created a need for the development and implementation of new

TB drugs.

13 Review of Antituberculosis Terpenoids

Presented below is a review of terpenoids and other selected structural types of

natural products that have been tested for biological activity against Mycobacterium

tuberculosis, the gram-positive bacterium species primarily responsible for the disease

TB. Over 75 % of the compounds discussed below have been evaluated for biological

activity by our research group, the remaining compounds being tested by various other

research groups. Their structural types have been divided into six categories; first and

second line drugs (Figure 1.1), monoterpenes (Figure 1.2), sesquiterpenes (Figures 1.3

and 1.4), diterpenes (Figure 1.5), triterpenes (Figures 1.6), and phytol derivatives and

analogs (Figure 1.7). The biological activities of each of the 145 compounds, which have

been tested against M. tuberculosis, are presented in Table 1.2. Minimum inhibitory

concentrations ranged from 2 pg/ml to >128 pg/ml.

13.1 Monoterpenes

The relatively inactive monoterpenes demonstrated MICs ranging from 50 pg/ml

to 128 pg/ml. The lack of an adequate pool of monoterpenes tested against M

tuberculosis makes it unreliable to speculate on conformational requirements for

biological activity.

13.2 Sesquiterpenes

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Over 70 sesquiterpene lactones (SL) have been tested resulting in MICs ranging

from 2 pg/ml to >128 pg/ml. In the discussion below, the SL were divided into two

categories; the germacrolide-type SL represented by costunolide, parthenolide, and

derivatives (Figure 1.3), and other SL shown in Figure 1.4.

Costunolide (1.14), the most lipophilic lactone among this category of

sesquiterpenes, gave an MIC of 32 pg/ml. Its 4,5-epoxide derivative, parthenolide (1.16),

was the most active germacrolide against M. tuberculosis with an MIC of 16 pg/ml. The

1( lOVepoxycostunolide (1.18) was less active than 1.14 and 1.16 with an MIC of 64

pg/ml. With the exception of 1,10-epoxyparthenolide (1.20), the sesquiterpene lactones

in this series bearing a a-methylene-y-lactone moiety (1.14, 1.16, 1.18, 1.22, 1.24, and

1.29) are active against M. tuberculosis with MICs at concentrations of 64 gg'ml or

below. In contrast, their lipH, 13-dihydroderivatives (1.15, 1.17, 1.19, 1.23, and 1.25)

as well as the sesquiterpenes obtained by reductive opening of the lactone ring (1.26,

1.27, and 1.28) (Lu et al., 1996) showed no activity against M. tuberculosis at

concentrations below 128 pg/ml, suggesting that the presence of the alkylating exocyclic

a-methylene-y-lactone moiety is essential for activity.

Examination of the biological data for the additional sesquiterpenes (Figure 1.4),

indicated that only 10 compounds out of the 61 in this category possessed MICs of 32

pg/ml or lower. The active compounds include damsin (1.39), dehydrocostuslactone

(1.45), aromaticin (1.51), l,2-dehydro-3-epi-isotelekin (1.74), encelin (1.76), 4,5-epoxy-

6-epidesacetyl-laurenobiolide (1.77), 6-epi-desacetyl laurenobiolide (1.78), curcuphenol

(1.89), nerolidol (1.90), and famesol (1.91). The most active of these compounds is

dehydrocostuslactone (1.45) with an MIC of 2 pg/ml.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6

1 3 3 Diterpenes

Thirty-seven diterpenes have been tested against M. tuberculosis and seven of

them possessed MIC less than 8 pg/ml (Figure 1.5). The seven active compounds,

multicaulin (1.123), 12-demethylmulticauline (1.124), multiorthoquinone (1.125), 12-

demethylmultiortho quinone (1.126), 12-methyl-5-dehydrohorminone (1.127), 12-

methyl-5-dehydroacetylhorminone (1.128), and salvipimarone (1.129) were all isolated

from Salvia multicaulis (Ulubelen et al., 1997). The most active of these compounds was

12-demethylmulticauline (1.124) which demonstrated a remarkable MIC of 0.46 pg/ml.

1.3.4 Triterpenes

Ten triterpenes have been tested against M. tuberculosis with only two

demonstrating biological activities at 32 pg/ml (Table 1.2). One of these, fusidic acid

(1.134), was isolated from a fermentation broth of Fusidium coccineumhas in 1962

(Godtffedsen et al., 1962) and has been thoroughly investigated for clinical treatment

against both M leprae and M. tuberculosis (Hoffner et al., 1990 and references therein).

The other active triterpene is cholesterol (1.136), which has an MIC of 32 pg/ml.

13.5 Phytol, derivatives, and analogs

The last set of compounds tested for biological activities against M. tuberculosis

are the phytol, its derivatives, and structural analogs (Table 1.2) (Figure 1.7). Thirteen

compounds were tested and all but three compounds demonstrated MICs at or below 32

pg/ml. Three compounds, (£>phytol (1.140), (Z)-phyto! (1.145), and (3R£, 1R, 11R)-

phytanol (1.146), demonstrated MICs of 2 pg/ml (Rajab et al., 1998). (£>phytyl acetate

(1.141) and (£>phytol methyl ether (1.142) however, showed MICs of 16 pg/ml

implying that a free hydroxyl group, as present in 1.140, is required for significant

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity. Additionally, the equipotency of (£>phytol (1.140), (Z)-phytol (1.145), and

(3RJ>, 1R, 1 l/?)-phytanol (1.146) suggests that the 2,3-double bond may not be essential

for bioactivity.

Table 1.2 Terpenoid Minimum Inhibitory Concentrations (MICs) Against M. tuberculosis (H3 7 R.V)

Compound Class Source MIC Reference Name (pig/ml)*

First and Second Line Drugs isoniazid ( 1.1) 0.05 Heifets etal., 1991 rifampin ( 1.2 ) 0.25 Heifets et al., 1991 streptomycin (1.3) 2.0 Heifetsetal., 1991 ethambutol (1.4) 3.8 Heifets et al., 1991 pyrazinamide (1.5) 100 Heifetsetal., 1991 thiacetazone ( 1.6) 1.5 Heifetsetal., 1991 ethionamide (1.7) 1.25 Heifetsetal., 1991 ciprofloxacin ( 1.8 ) 1.0 Heifetsetal., 1991

Monoterpenes dolabrin (1.9) 50 Ito et al., 1959 hinokitiol ( 1.10) 50 Ito etal., 1959 citronellol ( 1.11) Aldrich 64 Rajah et al., unpublished data nerol( 1.12) Aldrich 128 Rajab et al., unpublished data geraniol (1.13) Sigma 64 Rajab et al., in press

Sesquiterpenes costunolide (1.14) Saussurea lappa 32 Lu et al., 1996 1 ipH-dihydrocostunoIide synth. der. 128 Joshi etal., 1966 (1.15) parthenolide (1.16) Magnolia 16 Lu et al., 1996 grandiflora 1 ipH-dihydroparthenolide Ambrosia 128 Lu et al., 1996 (1.17) artemisiifolia 1,10-epoxycostunolide synth. der. 64 Lu, et al., 1996 (1.18) 1, 10-epoxy dihydro synth. der. 128 costunolide (1.19) (table continued)

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Compound Class i Source ! MIC i Reference Name 1 i 1, 10-epoxvparthenolide ! synth. der. ! 128 ; Fischer et al., 1990 * (1.2 0 ) j 1, 10-epoxydihydro synth. der. | 128 1 Pentes, 1991 1 I parthenolide ( 1.21) i santamarine (1.2 2 ) Ambrosia 64 ' Yoshioka et al, 1970 confertiflora 1 1 ipH, 13-dihvdro Sonchus hierrensis | >128 j Bermejo Barrera et al., santamarine (1.23) i ; 1968 reynosin (1.24) Ambrosia 64 | Yoshioka et al, 1970 confertiflora i 11 pH, 13-dihydroreynosin synth. der. ! >128 Lu et al., 1996 (1.25) reynosin triol derivative >128 Luetal., 1996 (1.26) 11,13-dihydro triol of >128 Lu et al., 1996 reynosin (1.27) santamarine triol derivative >128 Lu etal.. 1996 (1.28) micheliolide (1.29) 50 costunolide diepoxide synth. der. 128 (1.30) 11 P-hydroxydihydro synth. der. 128 Pentes, 1991 parthenolide (131) 14-hydroxydihydro synth. der. 128 Pentes, 1991 parthenolide (1.32) glaucolide A (133) Vemonia glauca 128 Padolina et al., 1974 melfiisin (134) Melampodium 128 Quijano et al., 1981 diffusum montafrusin A (1.35) Montanoa >128 Quijano et al., 1979a frutescens glaucolide D (136) Vemonia uniflora >128 Betkouski et al., 1975 11 P-hydroxydihydro synth. der. 128 costunolide (1.37) pumilin (138) Berlandiera texana 128 Korpetal., 1982 damsin (139) Ambrosia maritima 32 Abu-Shady et al., 1953 desacetylconfertiflorin Ambrosia 128 Fischer et al., 1967 (1.40) confertiflora confertiflorin (1.41) Ambrosia 128 Fischer et al., 1967 confertiflora desacetylisoconfertiflorin synth. der. 128 (1.42) parthenin (1.43) Parthenium 64 Herz et al., 1962b hysterophorus tenulin (1.44) Helenium amarum 128 Herz et al., 1962a (table continued)

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Compound Class 1 Source j MIC ! Reference Name i (pg/ml)* i dehydrocostuslactone ; Saussurea lappa i 21 Mathuretal., 1965 (1.45) i i 7-hydroxy : Podachernum | >128 j Fronczek et al., 1984 i dehydrocostuslactone ! eminens (1-46) zaluzaninC (1.47) j Zaluzania triloba >128 | Yabutaetal.. 1978 peruvin (1.48) ; Ambrosia peruvianaj 128 Joseph-Nathan et al., j I j 1966 peruvin acetate (1.49) j synth. der. | >128 l Joseph-Nathan et al., | 1966 burrodin (1.50) Ambrosia dumosa i 128 i Geissman et al., 1968a aromaticin (1.51) Helenium 1 16 j Roma etal., 1964 aromaticin ! j ■ la-angeloxycarotol (1.52) Ambrosia trifida ! >ioo 1 Luetal., 1993b 1 a -(2 -methylbutyroyloxy) Ambrosia trifida ! >50 | Lu et al., 1993b 1 carotol (1.53) 1 melampodinin A (1.54) Melampodium 1 >128 j Fischer et al., 1976 americanum i | melampodinin B (1.55) Melampodium j >128 1 Fischer et al., 1975 americanum i enhydrin (1.56) Polymnia uvedalia j 128 j Fischer et al., 1978a melampolidin (1.57) Melampodium j >128 j Fischer et al., 1976 1 leucanthum ! 1 calein C (1.58) Calea zacatechichi >128 Quijano et al., 1979b neurolenin B (1.59) Neurolaena lobata >128 Manchand et al., 1978 zoapatanolide A (1.60) Montanoa >128 Quijano etal., 1982 lomentosa melcanthin A (1.61) Melampodium 128 Fischer et al., 1978b leucanthum melcanthin B (1.62) Melampodium 128 Fischer et al., 1978b leucanthum melampodin B hexanoate synth. der. 128 Walachv-Klimash, (1.63) 1980 melampodin B palmitate synth. der. 128 Walachy-Klimash, (1.64) 1980 melampodin B isobutyrate synth. der. 128 Walachy-Klimash, (1.65) 1980 cinerenin hexanoate ( 1.66) synth. der. 128 Walachy-Klimash, 1980 cinerenin palmitate (1.67) synth. der. 128 Walachy-Klimash, 1980 cinerenin isobutyrate ( 1.68 ) synth. der. 128 Walachy-Klimash, 1980 cinerenin (1.69) synth. der. 128 Walachy-Klimash, (table continued)

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Compound Class j Source j MIC ! Reference Name | j (jig/ml)* ! 1980 cinerenin acetate (1.70) ' synth. der. i 128 ' Walachy-Klimash. ! 1 1980 cinerenin cinnamate (1.71) ! synth. der. : >128 ! Walachy-Klimash, i ; 1980 melampodin C acetate synth. der. | >128 ! Perry et al., 1975 j 1 *" (1.72) ! i i liscunditrin (1.73) Liatris ohlingerae j >128 j Lu et al., 1994 1,2-dehydro-3-epi- Leo i 32 i1 isotelekin (1.74) | j i i valin (1.75) Iva imbricata I 64 j Herzetal., 1964 encelin (1.76) Encelia farinosa 1 16 1I Geissman et al.,7 196! 4,5-epoxy-6-epidesacetyl- 16 laurenobiolide (1.77) 6-epi-desacetyl Montanoa 16 Quijano et al., 1984 laurenobiolide (1.78) grandiflora a-santonin (1.79) Artemisia ramosa >128 Gonzalez Gonzalez e al., 1975 a-cyclocostunolide (1.80) synth. der. 64 ! (3-cyclocostunolide (1.81) synth. der. 64 3,11(13 )-eudesmadien-12- 5 mg/mlc Shtacher et al., 1970 oic acid (1.82) 1,2-dehydro-3-oxo-costic Leo >128 acid (1.83) 6p-hydroxygermacradien- Leo 7aH,8pH-olide (1.84) psilostachyin A (1.85) Ambrosia 128 Herz et al., 1969 confertrflora dihydropsilostachyin A synth. der. 128 (1.86) (3aR)-(+)-sclareolide Sigma >100 (1.87) fusarenone X (1.88) Fusariian nivale Ib Ueno et al., 1971 curcuphenol (1.89) Euthamia 16 leptocephela nerolidol (1.90) Magnolia 32 Cantrell et al., acuminata unpublished data famesol (1.91) Sigma 8 Rajab et al., 1998

Diterpenes (+) 3P-hydroxymanool Solidago rugosa >100 Lu et al., 1995 (1.92) (+)-3 P-acetoxymanool Solidago rugosa 128 Lu et al., 1995 (1.93) (table continued)

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Compound Class Source ! MIC j Reference Name i (ug/ml)* j (+)-3p, I3-diacetoxymanool Solidago rugosa ! >128 1 Lu et al., 1995 ! i (1.94) i abietic acid (1.95) Solidago rugosa !, >100 Lu et al., 1995 kolavenic acid (1.96) Solidago canadensis >100 Lu et al., 1993a kolavenol (1.97) Solidago canadensis >100 Luetal., 1993a 6 p-angeloyloxy kolavenic Solidago canadensis >100 Luetal., 1993a acid (1.98) 6 P-tigloyloxy kolavenic Solidago canadensis >100 Lu et al., 1993a acid (1.99) solidago lactone ( 1.100) Solidago canadensis >100 Lu et al., 1993a 15,17-lactone grindelic S. pauciflosculosa >100 Menelaou et al., 1993 acid (1.101) grindelic acid ( 1.102) >100 Menelaou et al., 1993 17-hydroxy grindelic acid S. pauciflosculosa >100 Menelaou et al., 1993 (1.103) 17-acetoxy grindelic acid S. pauciflosculosa >100 Menelaou et al., 1993 (1.104) 17-hydroxy grindelic acid S. pauciflosculosa >100 Menelaou etal., 1993 der. methoxy ester (1.105) grindelic acid der. diacid S. pauciflosculosa >100 Menelaou et al., 1993 (1.106) hardwickiic acid (1.107) Solidago rugosa >128 Lu et al., 1995 13£'-7a-acetoxy kolavenic Solidago canadensis >100 Lu et al., 1993a acid (1.108) 13Z-7a-acetoxy kolavenic Solidago canadensis >100 Lu et al., 1993a acid (1.109) cryptanol ( 1.110) Salvia pisidica Ic Topcu etal., 1987 horminone ( 1.111) Salvia pisidica Ic Topcu etal., 1987 7a-acetylhorminone Salvia pisidica Ic Topcu etal., 1987 (1.112) femiginol (1.113) Salvia pisidica f Topcu etal., 1987 pisiferal (1.114) Salvia pisidica r Topcu etal., 1987 danshenxinkun D (1.115) Salvia miltiorrhiza Ac Luo et al., 1985 hypargenin A (1.116) Salvia hypargeia Ulubelen et al., 1988 hypargenin B (1.117) Salvia hypargeia Ib Ulubelen et al., 1988 hypargenin C (1.118) Salvia hypargeia Ulubelen et al., 1988 hypargenin D (1.119) Salvia hypargeia TJ bb Ulubelen et al., 1988 rb hypargenin E (1.120) Salvia hypargeia Ulubelen et al., 1988 hypargenin F (1.121) Salvia hypargeia yb Ulubelen et al., 1988 sandaracopimara- 8 ( 14)-15- Tetradenia riparia 25-100° Puyvelde et al., 1994 diene-7-a,l8-diol (1.122) multicaulin (1.123) Salvia multicaulis 5.6 Ulubelen et al., 1997 12-demethylmulticauline Salvia multicaulis 0.46 Ulubelen et al., 1997 (table continued)

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Compound Class i Source ! MIC i Reference Name (pg/ml)‘ 1 1 (1.124) [ multiorthoquinone (1.125) J Salvia multicaulis 1 2.0 ' Ulubelen et al., 1997 12-demethylmultiortho Salvia multicaulis ! 1.2 ' Ulubelen et al., 1997 j i quinone (1.126) j 12-methyl-5- i Salvia multicaulis 1.2 Ulubelen et al., 1997 1 dehydrohorminone \ 1 (1.127) j 12-methyl-5- j Salvia multicaulis | 0.89 Ulubelen et al., 1997 1 dehydroacetvlhorminone \ i 1 (1.128) i salvipimarone (1.129) Salvia multicaulis 7.3 i Ulubelen et al., 1997

T riterpenes I epifriedelinol (1.130) Chrysoma 1 >100 j Menelaou et al., 1992 pauciflosculosa 1 1 chondrillasterol (1.131) Heterotheca j >128 1i subaxillaris baccharis oxide (1.132) Baccharis >128 hahmifolia betulin (1.133) Betula sp. 128 fusidic acid (1.134) .32 Hoffner et al., 1990 (3-sitosterol (1.135) Aldrich >128 cholesterol (1.136) Aldrich 32 harrisonin (1.137) H. abyssinica >128 Rajab et al., 1997 12(3-acetoxyharrisonin H. abyssinica >128 Rajabetal., 1997 (1.138) vitamin D3 (1.139) Aldrich 128

Phytol, derivatives, and analogs (£>phytol (1.140) Lucas volkensii 2 Rajab et al., 1998 (£>phytyl acetate (1.141) synth. der. 16 Rajabetal., 1998 (£>phytol methvl ether synth. der. 16 Rajab et al., (1.142) unpublished data phytyl amine (1.143) synth. der. 32 Rajab et al., unpublished data phytyldiisopropylamine synth. der. 64 Rajab et al., (1.144) unpublished data (Z>phytol (1.145) Aldrich 2 Rajabetal., 1998 (3R£, 7R, 1 l/?>phytanol synth. der. 2 Rajab et al., 1998 (1.146) (£>phytol epoxide (1.147) synth. der. 8 Rajab et al., 1998 (3RJS, 7R, 1 lR)-phytanic synth. der. >128 Rajab etal., 1998 acid (1.148) (table continued)

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Compound Class Source ! MIC 1 Reference Name | (pg/ml)* i 2-phytolphenol (1.149) synth. der. t 32 ! Rajab et al., 1 ! unpublished data phytantriol (1.150) Aldrich i j Rajab et al., 1 unpublished data diphytylsulfide (1.151) synth. der. 1 >128 ! Rajab et al., i j I unpublished data 2-hexadecanol (1.152) Aldrich i 8 Rajab et al., 1I ! unpublished data 1 a Biological activity determined using BACTEC radiorespirometric bioassay except where noted otherwise. I=Inactive; A=Active. b Biological activity determined using agar plate. c Biological activity determined by unreported method.

1.4 Conclusions

It has been demonstrated above that natural products are an excellent source for

the identification of new structural types of antimycobacterial compounds. These

investigations have led to the isolation and structural elucidation of over 27 compounds

with MICs at or below 32 pg/ml. Eleven compounds, dehydrocostuslactone (1.45),

multicaulin (1.123), 12-demethylmulticauline (1.124), multiorthoquinone (1.125), 12-

demethylmultiortho quinone (1.126), 12-methyl-5-dehydrohorminone (1.127), 12-

methyI-5-dehydroacetylhorminone (1.128), and salvipimarone (1.129) (£>phytol (1.140),

(Z)-phytol (1.145), and (3 RJS, 7R, 1 l/?)-phytanol (1.146), showed remarkable MICs of 2

pg/ml or lower. These data indicate that the sesquiterpene, diterpene, and phytol classes

of molecules may be good sources of chemicals or synthetic models for future

investigations. However, this observation can only be substantiated by additional testing

of other types of terpenoids. All data discussing preliminary structure activity

relationships will require additional biological testing of structurally similar compounds.

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c h 3 c h 3

CH3 COO

H3 CO CONHNH

1.1 N— CH3

1.2

NH NH HN NH NH OH

O O

OH 1 J 1.4

Figure 1.1. First and second line antimycobacterial drugs (figure continued)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 r HN N CONH2 NH2

N . 4 NH (JN 1.5 1.6

OH 1.7 1.8

(figure continued)

OH OH 1.11

1.9 R=methylethenyl 1.12 1.10 R=methylethyl OH

1.13

Figure 1.2. Monoterpenes tested against M tuberculosis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.14 1.16 1.15=11 pH, 13-dihydro 1.17=11 pH, 13-dihvdro

1.18 1.20 1.19=11PH, 13-dihydro 1.21=1 lpH, 13-dihydro

OH OH

1.22 1.24 1.23=11PH, 13-dihydro 1.25=1 ipH, 13-dihydro

OH OH OH

OH OH OH OH OH OH 26 27 28 29 O

Figure 1.3. Costunolide, parthenolide, and derivatives tested against M. tuberculosis

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1.30 1.32

O OH

OMac OEpoxyang OAc OAc

1.33

OH HO,

OAc

1.35

1.37

Figure 1.4. Additional sesquiterpenes tested against M tuberculosis (figure continued)

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pA ng

•■"OH R

1.38 1.39 R=H \ \ 1.42 1.40 R=OH O O O 1.41 R=OAc

O 1.43 1.44 1.45 R*=H, R2=H 1.46 R*=OH, R2=H 1.47 R1=H, R2=OH

O 1.51 1.48 R1=OH, R2=H 1.52 R=Ang 1.49 R'=OA c, R2=H 1.53 R=2-Mebut 1.50 R*=H, R2=OH

(figure continued)

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,OAc .OEpoxyang .OEpoxyang AcO' HO :-Mebut-0

1.54 1.55 1.56

OH PH OH .OAc ^OAng

1.57 1.58 R=2-methylpropenovl 1.60 1.59 R=i-Val

O R O.

OAng

AcO

HO 1.61 R=H O O 1.62 R=OH 1.63 RI=Ac, R2=hexanoate 1.64 R 1=Ac, R2=palmitate H 1.65 R l=Ac, R2=isobutyrate 1.66 RI=Et, R2=hexanoate 1.67 R1=Et, R2=palmitate rv 1.68 R1=Et, R2=isobutyrate OAc 1.69 RI=Et, R2=OH OH 1.70 R1=Et, R2=OAc O 1.71 R l=Et, R2=cinnamate 1.73 1.72 R 1=isobutyl, R2=acetate

(figure continued)

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1.74

OH 1.76 1.77

i\0, O

OH 1.78 1.79

1.80 1.81

(figure continued)

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OH COOH 1.83 COOH 1.82 1.84

O O O"' 0'“'

OH OH CH 1.85 1.86 1.87

<10? ..

OH e CH20H J)Ac 1.88

1.90

1.91

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R 0 CO,H 1.92 R*=H, R2=H 1.93 R1=Ac, R2=H 1.95 1.94 Rl=Ac, R2=Ac

-O

1.100

1.96 R=H, R1=COOH 1.97 R=H, Rl=CH2OH VO 1.98 R=OAng, R'COOH 1.99 R=OTig, R‘=COOH

V r f 9 1.102 R=COOH, R1=H 1.103 R=COOH, Ri=CH2OH 1.104 R=COOH, R1=CH2OAc 1.105 R=C02CH3, R'KTHjOH 1.106 R=COOH, R1=COOH

COOH

OAc

c o 2h 1.108 13£ 1.107 1.109 13 Z

Figure 1.5. Diterpenes tested against M tuberculosis (figure continued)

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OH OH HO

1.110 1.111 R=OH 1.112 R=OAc

CH3 c h 2o h

1.113 R=CH, 13 1.115 1.114 R=CHO

OH R3

1.116 r ‘=r 4=o , r 2=r 3=h , R5=P-OH 1.117 r ‘=R2=R5=H, R3=OH, R4=0 1.118 R*=R2=R3=H, R4=R5=0 1.119 R1=R3=R4=R5=H, R2=0,6,7-dehydro

(figure continued)

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OH F"HII\

OH OH

1.121 1.122

1.123 R=Me 1.125 R=Me 1.124 R=H 1.126 R=H

OMe

OR OH

1.129 1.127 R=H 1.128 R=Ac

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HO HO' 1.131 1.130

1.132 1.133

COOH

OAc

HO'

I. IJJ R -V il 1.134 1.136 R=H

OH OMe

1.137 R=H 1.139 1.138 R=OAc

Figure 1.6. Triterpenes tested against M. tuberculosis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.140 R=OH 1.141 R=OAc 1.142 R=OCH3 1.143 R=NH2 1.144 R=N( isopropyl )2

1.145 OH

OH 1.146

OH 1.147 O H .H

OH 1.148 OH

1.149

OH OH 1.150 OH

1.151

Figure 1.7. Phytol, derivatives, and structural analogs tested against M. tuberculosis

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1.5 References

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Sepkowitz, K. A.; Raffalli, J.; Riley, L.; Kiehn, T. E.; Armstrong, D. Clinical Microbiol. Rev. 1995, 8 ,180-199.

Shtacher, G.; Kashman, Y. J. Med Chem. 1970, 13, 1221.

Snider, Jr, D. E.; Raviglione, M.; Kochi, A. In Tuberculosis: Pathogenesis, Protection, and Control', Bloom, B. R., Ed.; ASM Press: Washington, D. C., 1994; pp 3-11.

Topcu, G.; Ulubelen, A.; Terem, B. Fitoterapia 1987, 58, 281.

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Ueno, Y.; Ueno, I.; Iitoi, Y.; Tsunoda, H.; Enomoto, M.; Ohtsubo, K. Japan J. Exp. Med. 1971, 41, 521.

Ulubelen, A.; Evren, N.; Tuzlaci, E.; Johansson, C. J. Mat. Prod. 1988, 51, 1178.

Ulubelen, A.; Topcu, G.; Johansson, C. B. J. Nat. Prod. 1997, 60, 1275.

Walachy-Klimash, J. The Chemistry and Biological Activity of Sesquiterpene Lactones of Heliantheae Ph.D. Dissertation, Louisiana State University, 1980.

Yabuta, G.; Olsen, J. S.; Mabry, T. J. Rev. Latinoamer. Quim. 1978, 9, 83.

Yoshioka, H.; Renold, W.; Fischer, N. H.; Higo, A.; Mabry, T. J. Phvtochemistrv 1970, 9, 823.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2

ANTIMYCOBACTERIAL CRUDE PLANT EXTRACTS FROM SOUTH, CENTRAL, AND NORTH AMERICA*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1 Introduction

It is estimated that annually 8 to 10 million individuals fall victim to tuberculosis

and about 3 million die (Raviglione, et al., 1995). The problem is exacerbated by the

AIDS pandemic and also by resistance to existing anti-TB drugs such as isoniazid,

rifampin and streptomycin (Cohn et al., 1997). Since the introduction of rifampin in

1966, no new drugs have been made available for the treatment of this disease.

Mycobacterium avium is a more recently recognized opportunistic pathogen, causing

disease almost exclusively in immunosuppressed patients. Although considerably less

virulent than M tuberculosis, it has a greater innate resistance to most available

antimicrobics including the anti-tuberculosis drugs.

Although there are many reports of primary screenings of crude plant extracts with

the intent of identifying new TB-active compounds, relatively few have employed

authentic M. tuberculosis and used quantitative dilution assays (as opposed to diffusion

assays which are affected by other factors such as molecular weight and hydrophobicity).

In an effort to identify sources for bioassay-directed isolation of novel compounds

active against M. tuberculosis and M avium, extracts of 118 plant species from South,

Central and North America were screened using a procedure which allows activity to be

quantitated in such a way that the extracts can be prioritized for further study.

2.2 Experimental

Plant material and extract preparation. Two-hundred and thirty different

crude extracts were prepared from 118 different plant species collected in various

locations in North, Central, and South America. All voucher specimens were deposited

at the Louisiana State University Herbarium in Baton Rouge, Louisiana. Nomenclature

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for the North American taxa follows Kartesz (Kartesz, 1994). Approximately 500 g of

plant material of each species were allowed to air dry and subsequently ground in a

Thomas Wiley Mill (model 4). Plant material was soaked in the appropriate solvent(s) for

24-48 hours. The solvent was removed using a rotary evaporator followed by high

vacuum for 18 hours.

Anti-mycobacterial testing. All extracts were solubilized at 80 mg/ml in

dimethylsulfoxide (DMSO), sterilized by passage through a 0.22 pm PFTE filter (Millex-

FG, Millipore, Bedford, MA) and stored at -80°C until used. A 1:10 dilution was made

in DMSO and 50 pi of both stocks and dilutions added to 4 ml 7H12 medium (BACTEC

12B; Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) to achieve the

desired final concentrations of 1000 and 100 pg/ml. Controls received 50 pi DMSO.

Positive drug controls for M. tuberculosis and M. avium received, respectively, either

fixed concentrations of 2 pg/ml rifampin (Sigma Chemical Co., St. Louis, MO) and 32

pg/ml clarithromycin (Abbott Labs, North Chicago, EL) or a range of concentrations to

determine the minimum inhibitory concentrations (MIC).

Anti-mycobacterial bioassays were performed using the BACTEC 460 system

(Collins and Franzblau, 1997) as modified below to determine percent inhibitions with

plant extracts. M tuberculosis H37Rv ATCC 27294 (American Type Culture Collection,

Rockville, MD) was cultured in BACTEC 12B broth until the daily growth index (GI)

reached 400-999. One-tenth ml of this culture was used to inoculate 4 ml fresh BACTEC

12B media containing the plant extracts.

M. avium ATCC 25291 was cultured in BACTEC 12B broth until the daily GI

reached 999. Cultures were then diluted 1:25 in BACTEC 12B broth and frozen at -80°C

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until needed. One-tenth ml of the thawed culture was used to inoculate 4 ml fresh

BACTEC 12B media containing the plant extracts.

For both M. tuberculosis and M avium, additional control vials were included

which received a further l:lOO-diluted inoculum for use in calculating the MIC of

rifampin and clarithromycin, respectively, by established procedures (Inderlied and

Salfinger, 1995; Inderlied and Nash, 1996).

Cultures were incubated at 37°C and the GI determined daily (starting on the third

day of incubation) until (solvent) control cultures achieved a GI of 999. Assays were

completed in 10 days for M. tuberculosis and 5 days for M. avium. Percent inhibition

was defined as 1-(GI of test sample/GI of control) x 100.

2.3 Results

The results from screening organic extracts of 118 different plant species against

M. tuberculosis and M. avium at 1000 pg/ml and 100 pg/ml are shown in Table 2.1.

Some extracts were only screened at 1000 pg/ml and due to lack of activity or sample

were not tested at 100 pg/ml. Other extracts were only tested at the lower concentration

due to limited sample availability.

Extracts, which demonstrated high activity (>90% inhibition) against both

mycobacteria at 100 pg/ml included: Chrysacthimia mexicana, Erigeron annua, E.

strigosus, Euthamia leptocephala, Rudbeckia subtomentosa, Solidago petiolaris,

Magnolia acuminata and M. grandiflora.

Extracts which demonstrated high activity against M. tuberculosis and moderate

or weak activity against M. avium included: Aster praeltus, Borrichia frutescens, Calea

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Jivaricata , Celtis laevigata , Echinacea artrorubens, Gaillardia aestivalis, Heterotheca

subaxillaris, Liatris ohlingerei, Solidago arguta and Polygonella fimbriata.

Extracts with high activity against M. tuberculosis and little to no activity against

M. avium at 100 pig/ml included: Ambrosia artemisiifolia, and Rudbeckia lacmiata. No

extracts demonstrated significantly greater activity against M. avium compared to M

tuberculosis.

Roots had greater activity than aerial parts (flowers, leaves or stems) in

dichloromethane extracts of Ambrosia artemisiifolia, Aster praeltus, Echinacea

artrorubens, Erigeron philadelphicus, E. strigosus, Euthamia leptocephala, Gaillardia

aestivalis , Liatris acidota, Rudbeckia laciniata, R. subtomentosa, and Solidago

semperivens as well as in methanol extracts of Solidago rugosa, hexane extracts of

Echinacea artrorubens and acetone extracts of Camptotheca acuminata.

In plants which demonstrated significant activity against one or more

mycobacteria and for which the same plant part was extracted with more than one

solvent, activities of different solvent extracts could be compared. Dichloromethane

extracts had higher activities than methanol extracts in 8 species. Hexane extracts were

more active than dichloromethane in 3 species and hexane extracts were more active than

methanol extracts in 2 species. In no cases were the reverse relative activities observed.

2.4 Discussion

To our knowledge this is the first large-scale examination of the anti-

mvcobacterial activity of crude plant extracts using the BACTEC 460 radiorespirometric

assay, the clinical TB drug susceptibility method of choice for most of the past 10 years

(Inderlied and Nash, 1996; Inderlied and Salfinger, 1995). Instead of performing a

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Table 2.1. Antimycobacterial Activity of Various Crude Plant Extracts

Family Plant Location Exa Percent Inhibition Part Species M - M . tuberculosis avium 1000 100 1000 100 pg/ml tig/ml pg/ml ng/ml Apocynaceae Vinca rosea L. aerial Louisiana D 100 40 0 0

aerial Louisiana M 100 - 0 - Asteraceae

Ageratina altissima (L.) aerial Louisiana D 100 3 - 0 King & H. E. Robins Alloispermum aerial Venezuala D 100 81 100 79 caracasanum (H. B. K.) H. Robinson Ambrosia artemisiifolia L. aerial Louisiana H 98 79 91 0

aerial Louisiana D 97 58 - 0

aerial Louisiana W 6 - - -

roots Louisiana D 99 94 - 0 Ambrosia confertiflora aerial Texas H 69 35 99 0 DC.

aerial Texas C 100 34 - 60

aerial Texas D -42 - 0 -

Ambrosia trifida L. aerial Louisiana M 3 - 0 -

Artemisia annua L. aerial Oregon D - 77 - 0

Artemisia douglasiana aerial Oregon D - 22 - 0 Bess.

Artemisia ludoviciana ssp. aerial Oregon D - 4 - 0 estesii Chambers

Artemisia ludoviciana var. aerial Oregon D - 29 - 0 vatiloba Nutt.

Aster adnatus Nutt. aerial Louisiana D 64 - - -

Aster praealtus Poir. aerial Louisiana D 35 - 0 -

flower Louisiana D 53 - 0 - roots Louisiana D 97 93 94 43 (table continued)

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Family Plant Location Ex8 Percent Inhibition Part Species M M tuberculosis avium 1000 100 1000 100 pg/ml pg/ml pg/ml pg/ml Baccharis halimifolia L. roots Louisiana D 100 58 98 87 Balsamorhiza hookeri aerial Colorado D 98 30 61 0 Nutt.

Balsamorhiza sagittata aerial Wyoming D 81 - 0 - (Pursh) Nutt.

Berlandiera lyatra Benth. aerial Texas D 31 - 48 -

Berlandiera pumila aerial Texas D 81 - 1 - (Michx.) Nutt.

Berlandiera texana DC. aerial Texas D 46 - 48 - Bigelowia nuttallii L. C. aerial Louisiana D 100 75 68 6 Anders aerial Louisiana A 95 49 48 0 roots Louisiana D 99 50 0 0

Borrichia frutescens (L.) flower Louisiana H 78 - - 71 DC. flower Louisiana D 99 100 99 52 leaves Louisiana D 100 90 100 72

roots Louisiana H 99 - - -

roots Louisiana D 94 -- - roots Louisiana M 84 23 33 1 Calea berteriana DC. aerial Venezuela D 100 54 100 64 Calea cardonae Maguire aerial Venezuela D 100 89 100 97 & Wurdack Calea divaricata Benth. aerial Venezuela D 100 98 100 85 Calea orizabaensis Klatt aerial Mexico D 99 26 99 0 var. websteri Wussow & Urbatsch Calea prunifolia Kunth aerial Venezuela D 100 52 100 41 Calea ternifolia Kunth aerial Chiapas, D 100 100 var. calyculata Mexico (table continued)

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Family Plant Location Exa Percent Inhibition Part Species A /. A /. tuberculosis avium 1000 100 1000 100 Ug/ml pg/ml pg/ml pg/ml Calea temifolia Kunth aerial Chiapas, D 99 89 0 var. temifolia Mexico Chloracantha spinosa aerial Texas D 100 21 100 0 (Benth.) Nesom aerial Texas M 13 0 Chrysacthinia mexicana aerial Nuevo D 99 94 99 Gray Leon, Mexico Chrysogonum virginianum aerial North D 99 69 81 L. Carolina Chrysoma pauciflosculosa aerial Florida H 100 (Michx.) Greene leaves Florida D 100 56 13 stems Florida D 100 80 61 Croptilon divaricatum roots North D -37 (Nutt.) Raf. Carolina Desmanthodium aerial Honduras D 76 hondurense A. Molina Desmanthodium aerial Chiapas, PE 38 perfohatum Benth. Mexico aerial Chiapas, D 27 0 Mexico aerial Chiapas, M 2 Mexico Dracopis amplexicaulis aerial Louisiana D -45 (Vahl) Cass. aerial Louisiana M -20 Echinacea atrorubens flower Texas H 100 92 34 Nutt. flower Texas D 100 58 76 leaves Texas H 61 (table continued)

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Family Plant Location Exa Percent Inhibition Part Species M. M tuberculosis avium 1000 100 1000 100 pg/ml pg/ml pg/ml pg/ml leaves Texas D -13 --- roots Texas H 100 - 100 - roots Texas D 100 - 100 - stems Texas D 100 94 100 36 Elephantopus sp. aerial Venezuala D 100 67 99 41 Elephantopus carolinianusroots Louisiana D 98 35 - 0 Raeusch. Elephantopus tomentosusaerial Louisiana D 100 100 L & roots Engelmannia pinnatifida aerial Texas D 96 68 _ 17 Gray ex Nutt. flower Texas D 100 -3 - 0 flower Texas M 2 --- Ericameria arborescens aerial California C 97 61 58 24 (Gray) Greene Ericameria cuneata (Gray) aerial California C 68 - -- McClatchie Ericameria ericoides aerial California D 72 - 51 - (Less.) Jepson Ericameria nana Nutt. aerial California D 5 - 0 - Erigeron annuus (L.) Pers. roots Louisiana D 100 99 - 99 Erigeron philadelphicus L. aerial Louisiana D -38 --- aerial Louisiana W 26 - 0 - roots Louisiana D 100 -18 - 0 Erigeron strigosus Muhl. aerial Louisiana D 100 56 - 55 ex Willd. roots Louisiana D 100 100 - 100 Erigeron tenuis Torr. & aerial Louisiana D -46 - - - Gray Espeletia sp. aerial Merida, D 100 77 100 38 Venezuela (table continue<

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Family Plant Location Ex'* Percent Inhibition Part Species M M. tuberculosis avium 1000 100 1000 100

Espeletia schultzii Wedd. aerial Mugubaji, D 100 23 0 0 Venezuela Eupatorium altissimum L. aerial Louisiana D -47 - -- Eupatorium havanense H. aerial Tamaulipas D 100 83 - 73 B. K. , Mexico Euthamia leptocephala aerial Louisiana D 100 22 - 19 (Torr. & Gray) Greene flower Louisiana D 100 -11 - 0 leaves Louisiana D 99 -31 - 67 roots Louisiana D 100 73 100 94 stems Louisiana D 83 -17 0 0 Gaillardia aestivalis flower Louisiana D 97 40 8 0 (Walt.) H. Rock leaves Louisiana D 98 52 78 0 roots Louisiana D 100 99 100 58 stems Louisiana D -37 - 0 - Gaillardia pulchella Foug. leaves Louisiana D 100 65 0 0 Garberia heterophylla leaves Florida H 100 _2 50 0 (Bartr.) Merr. & F. Harper Gundlachia corymbosa leaves Bahamas D 99 61 98 56 (Urban) Britt, ex Boldingh Heleniwn amarum (Raf.) aerial Louisiana D 100 - 100 - H. Rock Helianthus angustifolius buds Louisiana D 89 - 20 - L. leaves Louisiana D 54 _ 0 _ Heterotheca camporum aerial Tennessee D -35 -- - (Greene) Shinners (table continued)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41

Family Plant Location Exa Percent Inhibition Part Species M. M. tuberculosis avium 1000 100 1000 100 pg/ml pg/rnl pg/ml pg/ml Heterotheca subaxillaris roots Louisiana H 100 98 99 44 (Lam.) Britt. & Rusby roots Louisiana D 100 71 55 0 Hymenoxys scaposa (DC.) aerial Texas H 70 - -- K. F. Parker aerial Texas D 100 59 - 0 aerial Texas M 100 15 100 0 Inula helenium L. roots Louisiana H 100 100 - - roots Louisiana D 100 100 -- roots Louisiana M 100 83 - - Liatris acidota Engelm. & aerial Texas D 12 14 0 0 Gray aerial Texas M 18 20 0 0 flower Texas D 73 13 0 0 flower Texas M 5 25 0 0 roots Texas D 100 87 99 61 roots Texas M 27 13 0 0 Liatris ohlingerae (Blake) aerial Florida D - 96 - 75 B. L. Robins Melampodium cinereum aerial Texas D 100 25 35 0 DC. Melampodium aerial Costa Rica D 100 - 0 - costaricense Stuessy Melampodium leucanthumaerial Texas D 81 - 88 - Torr. & Gray leaves Texas D 66 - 76 - Melampodium longicome aerial Arizona D 53 - 0 - Gray roots Arizona M -11 - - -

Montanoa tomentosa aerial Mexico D 100 - • _ Cerv. (table continued)

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Family Plant Location Exa Percent Inhibition Part Species M .V /. tuberculosis avium 1000 100 1000 100

Parthenium argentatum aerial Mexico M 99 - 59 - Gray Parthenium hysterophorus aerial Louisiana D 100 82 100 43 L. Parthenium integrifolium aerial Arkansas D 99 41 0 0 L. aerial Arkansas M 23 9 0 0 Pluchea foetida (L.) DC. aerial Texas D 84 5 0 0 aerial Texas M 43 11 0 0 Podachaenium eminens aerial Mexico 1st -47 - - - Baill. D aerial Mexico 2nd -29 - - - D aerial Mexico E 99 18 - 0 Pseudoconyza sp. aerial Tamaulipas D 100 94 99 52 , Mexico Pyrrhopappus flower Louisiana D -10 - -- carolinianus (Walt.) DC. flower Louisiana EE -3 ___ leaves Louisiana D -41 --- leaves Louisiana EE -35 --- roots Louisiana D 19 --- roots Louisiana EE -16 - -- Rudbeckia grandiflora (D. flower Louisiana D -29 - - - Don) J. F. Gmel. ex DC. leaves Louisiana D 93 _ __ roots Louisiana D 100 70 - 80 roots Louisiana M -1 - 0 - stems Louisiana D -47 - - - Rudbeckia laciniata L. flower Mississippi D -47 - -- roots Mississippi D 99 99 - 0 (table continued)

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Family Plant Location Exa Percent Inhibition Part Species A/. M tuberculosis avium 1000 100 1000 100 ______M-g/ml pg/ml ug/ml pg/ml

stems Mississippi D 79 - -

Rudbeckia missouriensis flower Arkansas H 100 - 97 Engelm. ex C. L. Boynt. & Beadle flower Arkansas D 100 - 0 - flower Arkansas M -15 - 0 - roots Arkansas H 100 - 97 - roots Arkansas M -47 - 0 - stems Arkansas M -22 - 0 - Rudbeckia subtomentosa flower Louisiana D 95 30 - 22 Pursh leaves Louisiana D 96 43 0 roots Louisiana D 100 99 - 98 stems Louisiana D 70 - - - Silphium terebinthinaceum aerial Ohio H 98 65 - 6 Jacq. aerial Ohio D 95 34 _ 0 aerial Ohio M 21 - --

Smallanthus uvedaha (L.) aerial Louisiana D 64 - 0 _ Mackenzie ex Small aerial Louisiana M -27 - 0 - Solidago arguta Ait. roots Louisiana D 100 100 99 80 Solidago canadensis L. aerial Louisiana D -29 - -- Solidago nemoralis Ait. aerial Louisiana D 100 82 98 19 aerial Louisiana M 26 3 0 0 Solidago odor Ait. a aerial Louisiana D 100 36 100 11 aerial Louisiana M 21 14 19 0 Solidago petiolaris Ait. aerial Louisiana D 100 93 99 94 aerial Louisiana A 100 99 100 98 Solidago rugosa P. Mill. flower Louisiana D 100 35 - 0 roots Louisiana D 100 10 80 0 (table continued)

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Family Plant Location Ex8 Percent Inhibition Part Species M. M. tuberculosis avium 1000 100 1000 100

roots Louisiana M 86 25 0 0 stems Louisiana D 100 2 23 0 stems Louisiana M 22 16 0 0 Solidago sempervirens L. aerial Louisiana D 100 20 89 0 roots Louisiana D 100 63 100 59 Tetrachyron brandegei aerial Mexico D 100 65 99 59 (Greenman in Brandegee) Wussow & Urbatsch Tetrachyron grayi (Klatt) aerial Mexico D 99 88 96 23 Wussow & Urbatsch Tetrachyron manicata aerial Mexico D 82 - 42 - (Schlechtendal) Bentham & Hooker ex Hemsley Tetragonotheca repanda aerial Texas D 96 - - - (Buckl.) Small Trigonospermum annuumaerial Guatemala D 100 35 0 0 McVaugh & Laskowski Verbesina virginica L. aerial Louisiana D 63 - 0 - Viguiera cordifolia Gray aerial Costa Rica D -9 -- - aerial Costa Rica M -36 - 0 - Viguiera dentata (Cav.) leaves Mexico D 100 7 62 0 Spreng. leaves Mexico M 100 17 - 0 stems Mexico D 99 - -- Xanthium strumarium L. aerial Louisiana PE 70 - 0 - aerial Louisiana D -12 - - - aerial Louisiana M 12 - 0 - Youngia japonica (L.) DC. aerial Louisiana D 100 62 0 0 Empetraceae (table continued)

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Family Plant Location Exa Percent Inhibition Part Species M. M. tuberculosis avium 1000 100 1000 100 ______Hg/ml M-g/ml p-g/ml ug.ml

Ceratiola ericoides aerial Florida D 100 - 100 - Michx. Lamiaceae Calamintha ashei aerial Florida 1st 100 - 99 (Weatherby) Shinners D aerial Florida 2nd 98 - 99 “ D aerial Florida M 38 - 55 - Calamintha coccinea aerial Mississippi D 100 76 0 9 (Nutt, ex Hook.) Benth. Conradina canescens Gray leaves Florida D 100 - 99 - Lauraceae Per sea humilis Nash leaves Florida D 99 - 97 - stems Florida E 9 - - - Magnoliaceae Liriodendron tulipifera L. leaves Louisiana D 100 50 - 0 Magnolia acuminata (L.) aerial Louisiana D 99 100 100 99 L. bark Louisiana D 100 100 100 100 Magnolia grandiflora L. flower Louisiana D 100 99 - 91 petals fruit Louisiana D 100 80 - 84 leaves Louisiana D 100 88 - 92 Magnolia macrophylla leaves Mississippi D 20 - - - Michx. Magnolia pyramidata leaves Mississippi D - 33 - 0 Bartr. Magnolia virginiana L. aerial Mississippi D 100 - - - flower Mississippi D 100 - -- leaves Mississippi D 100 -- - Nyssaceae (table continued)

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Family Plant Location Exa Percent Inhibition Part Species M. M tuberculosis avium 1000 100 1000 100 pg/ml pg/ml pg/ml pg/ml Camptotheca acuminata leaves Louisiana PE -35 20 0 0 Decne. leaves Louisiana D -24 7 0 0 leaves Louisiana A 2 4 0 0 roots Louisiana A 72 2 0 0 Nyssa sylvatica Marsh. leaves Mississippi H 100 - 85 - leaves Mississippi D 100 - -25 -

leaves Mississippi A -36 - 15 -

leaves Mississippi M -41 - -6 - Polygonaceae

Polygonella fimbriata leaves Florida D -9 - -- (Ell.) Horton stems Florida D 100 100 100 0 Polygonella robusta aerial Florida H 10 1 26 0 (Small) Nesom & Bates aerial Florida D 34 -6 18 0 aerial Florida M 6 -18 - 0 roots Florida H -5 -13 24 0 roots Florida D -9 -30 40 30 roots Florida M 16 -4 57 0 Selaginellaceae

Selaginella arenicola L. leaves Florida H 58 - -- Underwood

leaves Florida D 10 --- Ulmaceae

Celtis laevigata Willd. berries Mississippi D 99 99 39 a Ex = Extraction Solvent, D = Dichloromethane, E = Ethanol, M = Methanol, C = Chloroform, W = Water, H = Hexane, A = Acetone, EE = Ethyl Ether, PE = Petroleum ether. Plant material within the same species and plant part was extracted consecutively in the order indicated in the table using least polar to most polar solvents.

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classical determination of the MIC by testing at multiple concentrations, a ranking of

activity (and examination of dose response) was performed by determining the percent

inhibition effected by various extracts at only two concentrations; 1000 and 100 pg/ml.

The quantitative nature of the BACTEC 460 system, yielding a “growth index” (or

measure of l4CO: evolution from i 4C-palmitic acid) lends itself well to this type of

analysis. Nonetheless, the BACTEC is quite expensive, and requires the use of

radioisotopes and is not considered a high-throughput format. The recently described

rapid, inexpensive, microplate-based assays utilizing reporter genes such as firefly

luciferase (Shawar et al., 1997), green fluorescent protein (Collins et al., submitted) or

redox dyes such as Alamar Blue (Collins and Franzblau, 1997) should facilitate the

future high-throughput screening of plant extracts for anti-mycobacterial activity.

A detailed literature search of the species (or genera) tested in this study revealed

that several have ethnomedical documentation of use in tuberculosis or respiratory

disorders in general (which may have included tuberculosis) and some of the collections

were in fact based on ethnobotanical leads.

Cherokee, Mohicans, and Lriquois used decoctions and infusions of Inula

helenium roots to treat respiratory disorders which could have been the manifestations of

tuberculosis (Moerman, 1986). Both fresh leaf juice (Fitzpatrick, 1954) as well as

ethanolic extracts (Grange and Davey, 1990) of I. helenium were shown to inhibit growth

of M tuberculosis H37Rv. In the present study, the crude organic extract of I. helenium

was among the most active, effecting 100 percent inhibition against M tuberculosis at

100 pg/ml. I. helenium contains the well known eudesmanolides, alantolactone and

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isoalantolactone (Milman, 1990) and the MIC of a mixture of these compounds was

reported to be 31.2 pg/ml (Bichkanova et al., 1977).

A decoction of the roots of Elephantopus scaber was used by natives of the

Lamjung district of Nepal for treating hemoptysis, a symptom (but not exclusively) of

tuberculosis (Manandhar, 1987). Both Artemisia abrotanum and A. vulgaris (Duke,

1985) were used for treating tuberculosis among other ailments as were several species of

Baccharis.

Fitzpatrick (1954) examined the fresh juices of many plant species for the

presence of anti-TB activity, expressed as the lowest dilution of the plant juice which

prevented visible growth of M. tuberculosis on solid medium. Active species from

genera which were also assessed in the current study included Inula helenium (>1:80) and

Nyssa sylvatica (>1:160, after tannin removal, 1:80). Inactive species (at 1:20 or 1:40)

included Ageratina altissima, Magnolia macrophylla, and Magnolia virginiana.

The data obtained here provide a valuable, quantitative basis on which to

prioritize plants for further study. A case in point is Borrichia frutescens which was

recently shown to contain various cycloartane derivatives, one of which had an MIC of 8

pg/ml against M. tuberculosis (Cantrell et al., 1996). Further investigation of other plants

with high activity is currently in progress.

2.5 References

Bichkanova, S. A.; Adgina, V. V.; Izosimova, S. B. Rast. Res. 1977, 13,428.

Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams, L. B.; Franzblau, S. G. J. Nat. Prod. 1996, 59, 1131.

Cohn, D. L.; Bustreo, F.; Raviglione, M. C.; Clin. Infect. Dis. 1997, 24 (Suppl. 1), S121.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49

Collins, L. S.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.

Duke, J. A. CRC Handbook of Medicinal Herbs', CRC Press, Boca Raton, 1985; p. 65-70.

Fitzpatrick, F. K. Antibiot. Chemother. 1954, 4, 528.

Grange, J. M.; Davey, R. W. J. Appl. Bacteriol. 1990, 68, 587.

Hirschhom, H. H. J. Ethnopharmacol. 1981, 4, 129.

Inderlied, C. B.; Nash, K. A. In: Antibiotics in Laboratory Medicine, 4th ed; Lorian, V., Ed.; Williams and Wilkins: Baltimore, 1996; pp 127-175.

Inderlied, C. B., Salfinger, M..\ In: Manual of Clinical Microbiology, 6th ed; Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. and Yolken, R. H., Eds.; ASM Press: Washington D.C., 1995; pp 1385-1404.

Kartesz, J. T. A synonymized checklist o f the vascular flora o f the United States, Canada, and Greenland, 2nd ed; Vol 1-2, Timber Press, Portland, Oregon, 1994.

Manandhar, N. P. Int. J. Crude Drug. Res. 1987, 25, 236.

Milman, I. A. Khimiya Prirodnykh Soedinenii 1990, 3, 307.

Moerman, D. E. Medicinal plants of Native America, vol. 1. Research Reports in Ethnobotany, contribution 2, University of Michigan Museum of Anthropology Technical Reports, Number 19, Ann Arbor. P., 1986; pp 234-235.

Raviglione, M. C.; Snider, D. E.; Kochi, A. JAMA 1995, 273, 220.

Shawar, R. M.; Humble, D. J.; Van Dalfsen, J. M.; Stover, C. K.; Hickey, M. J.; Steele, S.; Mitscher, L. A.; Baker, W. Antimicrob. Agents Chemother. 1997, 41, 570.

Siddiqi, S. H. In: Clinical Microbiology Procedures Handbook., Vol. 1., Isenberg, H. D., Ed.; American Society for Microbiology, Washington, D. C., 1992; pp. 5.14.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHA PTER3

ANTIMYCOBACTERIAL CYCLOARTANES FROM BORRICHIA FRUTESCENS *

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3.1 Introduction

In our continued search for biologically active natural products from higher plants

of the southeastern USA, we investigated the aerial parts of the sea daisy, Borrichia

firutescens (L). DC. This monotypic genus of the family Asteraceae, tribe Heliantheae, is

a widely distributed halophyte in the saline and brackish coastal marshes of Louisiana

and other neighboring Gulf Coast states. A previous chemical study of B. frutescens

from Veracruz, Mexico has afforded the triterpenes stigmastanol, stigmasterol, and

oleanolic acid as well as the heliangolide-type sesquiterpene lactone zoapatanoiide A

(Delgado et al., 1992; Quijano et al., 1982). We describe below the structures of two

new triterpenes, one cycloartanone and one lanostadiene, from the flowers of B.

frutescens collected near Grand Isle, Louisiana.

3.2 Results and Discussion

Crude dichloromethane extracts of the flowers, leaves, and stems of B. frutescens

were tested by a radiorespirometric method for activity against M. tuberculosis (H37Rv)

(Heifets et al., 1991). The highest level of antimycobacterial activity was found in the

flower extract, which was separated into eight fractions with increasing solvent polarity

by a standard VLC procedure using silica gel (Table 3.1). Activities of the eight fractions

against M tuberculosis, which are also listed in Table 3.1, indicated that the nonpolar

fractions 2-4 of the crude flower extract exhibited the highest inhibitory activity. At 33

(ig/ml, all three fractions showed inhibitions of 95% or higher, while all other fractions

gave values near 30% or below. Chemical investigation of fractions 2-4 led to the

isolation of two new triterpenes (3.1 and 3.4) and one known triterpene (3.3a), the

structures of which were elucidated as described below.

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Compound 3.1, C30H48 O2, mp 119-122°, gave a strong IR absorption at 1708

cm-1, suggesting the presence of ketone group(s). The 'H-NMR spectrum of 3.1 (Table

3.2) exhibited seven methyl signals and two upfield, mutually coupled one-proton

doublets at 8 0.57 and 0.78, which indicated the presence of a cyclopropane methylene

group in the molecule. The above JH-NMR data together with a mass spectral peak at

m z 440 lead us to suggest a cycloartanone-type triterpene skeleton. The absence of an IR

OH absorption in 3.1 together with the NMR spectral comparison o f 3.1 with the known

cycloartanone desoxypreffutecin B (3.2) (Jakupovic et al., 1987) strongly suggested that

compound 3.1 differed from 3.2 only in the absence of a C-16 P-hydroxyl group in 3.1.

This was supported by the lack of the downfield multiplet (H-16) in 3.1, which appeared

at 8 4.44 in 3.2 (Jakupovic et al., 1991). Also, the methyl absorption at 8 1.19 in 3.2 (C-

13 methyl) was shifted upfield in 3.1 (8 0.99), supporting the absence of the deshielding

P-hydroxyl group at C-16 in 3.1.

The 13C-NMR spectrum of 3.1 (Table 3.3) and DEPT experiments confirmed the

presence of seven methyls, a ketone (8 216.5), a cyclopropane methylene (8 29.5, t), and

an epoxide group at C-24-25 (8 64.7 and 58.3). 13C-NMR assignments were based on

DEPT experiments as well as spectral comparison with a previously reported structural

analogue, which differed from 3.1 only in the position of the epoxide function (C-23-C-

24) in the cycloartenone side chain and the presence of a P-hydroxyl group at C-16 as in

3.2 (Herz et al., 1985). Differences observed included the absence of the C-16 oxygen-

bearing carbon signal in 3.1, and the presence of epoxide carbon absorptions in

agreement with a C-24-C-25 epoxide group as shown in 3.1, exclusive of

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stereochemistry. The mass spectrum confirmed the molecular weight of 3.1 with a parent

peak m z 440.7 and a strong peak at m z 313 corresponding to the fragment of the

tetracyclic ring system by loss of the side-chain. Crystallization of 3.1 from hexane-

EtOAc (19:1) provided crystals suitable for single-crystal X-ray diffraction analysis,

which unambiguously established the molecular structure and relative stereochemistry of

3.1 as shown in Figure 3.1. Table 3.4 lists its crystallographic data and the coordinates

are given in Table 3.5. Details of the X-ray data of 3.1 will be discussed at the end of

this section. A negative CD Cotton effect near 295 nm was observed for 3.1 which

confirmed the absolute stereochemistry as that shown in structural formula 3.5.

From a more polar VLC fraction (hexane-EtOAc, 9:1), a colorless crystalline

compound (3.3a, mp 101-103 °C) was obtained, which differed from 3.1 only in the

presence of a P-hydroxyl group at C-3 instead of the ketone group in 3.1, as indicated by

the lack of a carbonyl absorption and the presence of a hydroxyl IR band at 3387 cm*1.

The empirical formula, C 30H50O2, was initially derived from 'H- and 13C-NMR spectral

data including DEPT experiments as well as mass spectral values. The mass spectrum

gave a parent peak at m z 442, which indicated a molecular weight two mass units higher

than 3.1, and supported the presence of a hydroxyl at C-3 in 3.3a instead of the C-3

ketone moiety in 3.1. The ^-N M R spectrum of 3.3a (Table 3.2) gave a signal at 5 3.28

(dd, 7=10.1, 4.7 Hz) which was in agreement with a P-oriented hydroxyl group at C-3

(Herz et al., 1985). Nearly identical proton signals due to the side-chain o f 3.3a, in

particular the triplet due to H-24 at 8 2.69 (7=6 .1 Hz), indicated that 3.1 and 3.3a must

have the same side-chain. As in the case of compound 3.1, 3.3a exhibited a 'H-NMR

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spectrum with seven methyl absorptions between 8 0.80 and 1.30, and cyclopropane

doublets appeared at 8 0.33 and 0.55 (J=4.2 Hz). l3C-NMR data obtained at 62.5 MHz

(Table 3.3) and DEPT experiments confirmed the presence of seven methyls, and instead

of the C-3 carbonyl absorption at 8 216.5 in 3.1, in 33a a signal typical of a hydroxyl-

bearing carbon absorption appeared at 8 78.8 (C-3). Further lH-and 13C-NMR signals of

3.3a were assigned by inspection and spectral comparison with 3.1 and a previously

isolated mixture of epimers (De Pascual Teresa et al., 1987), which differed only in the

configuration of the epoxide ring at C-24.

Acetylation of 33a gave the monoacetate 3.3b, C32H52O3, which was indicated

by a three-proton methyl singlet at 8 2.05 in the 1H-NMR spectrum. In addition, H-3 was

shifted from 8 3.28 in 33a to 4!56 in 33b, supporting the presence of an acetate group at

C-3 in 3.3b. The 13C-NMR spectrum (Table 3.2) of 33b differed from 33a only in the

presence of two additional signals, a singlet at 8 170.9 and a quartet at 8 21.3, which

correspond to the acetate carbonyl and methyl, respectively. All other carbon signals

were very similar to those of 33a and peak assignments of 33b were made by

correlation with 3.3a and related compounds reported in the literature (De Pascual Teresa

et al., 1987; Anjaneyulu et al., 1985; Barik et al., 1994; Takaishi et al., 1987).

Chemical correlation of alcohol 33a with ketone 3.1 was first attempted by

oxidation of 33a with pyridinium chlorochromate (PCC) (Mendes et al., 1994).

However, this reagent not only oxidized the C-3 hydroxyl group of 33a but also affected

the C24-C-25-epoxide function leading to the fragmentation product 3.5, C27H42O2, the

structure of which was supported by mass spectral, l 3C-NMR, and DEPT data. The mass

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spectrum of 3.5 gave a parent peak at m r 398 and, as in 3.1, loss of the side-chain gave a

peak at m z 313. The compound showed IR bands at 1704 and 1726 (sh) cn r 1

corresponding to two carbonyl absorptions. The !H-NMR spectrum of 3.5 indicated the

presence of only five methyl groups, with the methyl absorptions corresponding to H-26

and H-27 in 3.1 being absent in 3.5 (Table 3.2). A triplet at S 9.78 (7=1.8 Hz) was

assigned to an aldehyde proton (H-24) and the two diagnostic cyclopropane doublets

appeared at 8 0.59 and 0.79 (7=4.2 Hz). When compared with data for compound 3.1,

the 13C-NMR spectrum of 3.5 indicated the loss of three carbons (C-25, C-26, and C-27).

Instead, an aldehyde signal appeared at 5 203.3, which was assigned to C-24. Also,

carbon signals of the adjacent positions C-22 (8 41.4) and C-23 (8 28.5) were shifted

downfield in 3.5 relative to 3.1 due to the deshielding effect of the aldehyde carbonyl (C-

24). All other 13C-NMR spectral signals of 3.5 were nearly identical to those of 3.1, with

assignments being based on comparison with the analog absorptions of 3.1 and literature

values reported for structurally related compounds (Jakupovic et al., 1991; De Pascual

Teresa et al., 1987). Selective oxidation of 3.3a with RUCI 3 and NaI04 (Ashby et al.,

1981) provided 3.1 which confirmed that the stereogenic center C-24 in 3.3a also has the

R configuration. Therefore 3.3a differs from 3.1 only by the presence of a 3 p-hydroxyl

moiety instead of a keto group (Jakupovic et al., 1991; Herz et al., 1985).

Compound 3.4, C 30H48 O2, showed IR absorptions at 1630 (ketone) and 3435

(hydroxyl) cm"1. When compared with 3.1, its ^-N M R spectrum (Table 3.2) lacked the

diagnostic triplet at 8 2.69 corresponding to H-24 of the epoxide moiety. Instead, two

additional signals were present in 3.4: a broadened doublet at 8 4.48 (H-23) and a doublet

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of doublets at 8 5.19 (H-24), which were allylically coupled to two broadened methyl

singlets absorbing at 8 1.69 and 1.71. This indicated the absence of an epoxide and

strongly suggested the presence of a double bond in the side-chain of 3.4. The diagnostic

cyclopropane methylene proton doublets at 8 0.57 and 0.78 in 3.1 were also absent in 3.4.

Instead, compound 3.4 contained eight methyl signals, indicating one additional methyl

group, when compared with 3.1. The l3C-NMR spectrum of 3.4 (Table 3.2) and DEPT

experiments supported the absence of the cyclopropane ring at C-19. Furthermore, the

presence of a C-3 keto group (5 217.7), eight methyl quartets and four olefinic carbons

was established. Spectral comparison with literature values suggested a lanostadiene

type skeleton (Takaishi et al., 1987). The mass spectrum gave a molecular ion peak at

m z 440 and the typical [M-side-chain]+ peak at m z 313. NMR spectral assignments of

3.4 were made by spectral comparison with data described in the literature for

structurally closely related compounds (De Pascual Teresa et al., 1987; Bank et al.,

1994). The molecular structure of 3.4 (Figure 3.1) was determined by single-crystal X-

ray diffraction techniques. Table 3.4 summarizes the crystallographic data and its

coordinates are listed in Table 3.6. A negative Cotton effect in the CD spectrum was

observed near 295 nm, which confirmed the absolute stereochemistry shown in structural

formula 3.4 (Herz et al., 1985).

Details of data collection and refinements made in the X-ray diffraction analysis

of 3.1 and 3.4 are given in Table 3.4. Figure 3.1 illustrates the a-oriented epoxide at C-

24-C-25 and a P-cyclopropane ring fused at C-9-C-10 in 3.1 and the OH group at C-23

and C-8=C-9 double bond in 3.4. The C8=C9 distance in 3.4 is 1.352(3) A. The quality

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of the crystal of 3.4 was much higher than that of 3.1, and thus the precision of the

determination was also much higher. This is apparently a result of hydrogen bonding in

3.4. The OH group 0-2 is engaged in a linear, intermolecular hydrogen bond with

carbonyl oxygen 0-1 (at 1+x, y, 1+r) as acceptor. The 0 —0 distance is 2 .886 (2 ) A, and

the angle about the H atom is 173(3).

A search of the Cambridge Crystallographic Database (Allen et al., 1983) yielded

no previous crystal structure determinations for triterpenes with the cycloartane or

lanostadiene skeletons having an epoxide at C24-C25. The compounds most closely

related to 3.1 for which crystal structures have been determined are 3-oxo-24-cycloarten-

21-oic acid (Nishizawa et al., 1989) and argentatin C, (Romo de Vivar et al., 1990),

which differ from 3.1 only by having a glycol at C-24-C-25 rather than an epoxide, and

by having a P-OH group at C-16. The compounds most closely related to 3.4 for which

crystal structures have been determined are euphyl acetate and tirucallyl acetate (Nes et

al., 1984). Both have acetate substituents at C-3 and lack the OH group at C-23.

In a radiorespirometric bioassay against M. tuberculosis (H37Rv) (Heifets et al.,

1991), both triterpenes 3.1 and 33a showed minimum inhibitory concentrations (MICs)

of 8 pg/ml while compounds 3.4 and 33b had MIC values of 64-128 pg/'ml and >128

pg/ml, respectively (Table 3.7). Correlations of structural features and the MICs of the

four triterpenes suggest that the presence of the C-3 keto and/or P-hydroxy group, the

cyclopropane ring and the epoxide moieties as in 3.1 and 33a seem to play a major role

in the in vitro antituberculosis activity. Both the cyclopropane and epoxide functions are

absent in triterpene 3.4, resulting in its loss of activity (MIC 64-128 pg/ml). Also, the

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loss of activity by the presence of a C-3 acetoxv group (MIC of 3.3b >128 pg/ml)

strongly supports our suggestion that either a free hydroxyl or a keto group at C-3 in 3.1

or 3.3a is required for significant activity. Argentatine A (3.6) and B (3.7) were also

tested against M. tuberculosis due to their structural and biosynthetic similarities to the

bioactive natural triterpenes (Jakupovic et al., 1991). The lack of significant activity of

compounds 3.6 and 3.7 with MIC’s >128 pg/ml (Table 3.7) allows us to suggest that the

epoxide ring present in the side-chain of the active triterpenes 3.1 and 3.3a appears to be

essential for the in vitro antituberculosis activity. These preliminary structure-activity

data require further verification by testing structurally related triterpenes to leam about

the essential active regions necessary for significant antituberculosis activities. The

clinically active triterpene fusidic acid was also tested against M. tuberculosis for

comparison. In our radiorespirometric bioassay, its MIC of 4 pg/ml was lower than the

previously reported value of 32-64 pg/ml (Hoffner et al., 1990). Cytotoxicity results

(Table 3.7) for a mammalian cell line indicate that the active triterpenes 3.1 and 3.3a

have IC50 values of 71.8 and 39.8 M-g/ml, respectively, while the inactive triterpene 3.4

has an IC50 of 103.6 pg/ml, suggesting some degree of selective toxicity for M.

tuberculosis.

3 J Experimental

General Experimental Procedures. *H- and I 3C-NMR spectra were recorded in

CDCI3 on a Bruker AM 250 MHz spectrometer. Mass spectra were obtained on a

Hewlett-Packard 5971A GC-MS, or a TSQ70 FAB mass spectrometer. IR spectra were

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run on a Perkin-Elmer 1760X spectrometer as a film on KBr plates. Vacuum-liquid

chromatographic (VLC) separations were carried out on silica gel (MN Kieselgel).

Plant Material. B. frutescens was collected in May, 1994 in a brackish

environment about one mile inland from the Gulf of Mexico at Grand Isle, Louisiana

(N.H. Fischer No. 501; voucher deposited at LSU Herbarium).

Extraction and Isolation. Air-dried flowers (910 g) of B. frutescens were

extracted at room temperature with 1700 ml of hexane. Evaporation of the solvent in

vacuo provided 10.1 g of crude extract. The plant residue was then extracted with

CH2C12, (2x1800 ml for 24 h) to yield, after removal of solvent, 19.8 g of crude CH 2C12

extract. Biological screening of the extracts led to the investigation of the CH 2C12

extract, 9.8 g of which was adsorbed on 8 g of Si gel and placed onto a VLC column (4

cm in diameter and 30 cm long) packed with 70 g of Si gel (Coll et al., 1986). The

extract was separated into eight fractions using a gradient of hexane-EtOAc-MeOH of

increasing polarity. Table 3.1 lists the amounts and proportions of solvents used in the

fractionations as well as the percent inhibition of each fraction against M. tuberculosis.

Based on these results, fractions 2-4 were used for further isolation of the active

constituents.

Fraction 3 (1.8 g) was adsorbed on 3 g of silica gel and placed onto a VLC

column (3 cm in diameter and 25 cm long), packed with 40 g of Si gel and

chromatographed using 11x100 ml hexane-EtOAc mixtures of increasing polarity.

Altogether 53 fractions (20 ml each) were collected. Fractions 11-14 (hexane-EtOAc,

95:5) were evaporated to provide 145 mg of crystalline 3.1. Fractions 21-23

(hexane:EtOAc, 92:8) gave 30 mg of crystalline 3.4. Fractions 28-29 (hexane:EtOAc,

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9:1) slowly crystallized from a mixture of hexane and EtOAc (85:5) to yield 41 mg of

3.3a.

(24/?)-24,25-Epoxycycloartan-3-one (3.1). colorless crystals; C 30H48 O2; mol wt

440.717; mp 119-122 °C; IR o max (KBr), 1708 (C=0) cm 1; CD nm (e) 212 (-1.8), 225

(-18.2), 236 (-0.5), 298 (-32.6), 401 (-0.1) (c 0.0019; MeOH); lH-NMR spectral data see

Table 3.2; 13C-NMR spectral data see Table 3.3; EIMS (70 eV) m z [M]' 440 (7), [M -

Me]" 425 (4), [M - H20]" 422 (1), [M - side-chain]" 313 (40), 302 (16), 175 (35), 163 (24),

[side-chain]" 127 (13), 121 (50), [127-H20]+ 109 (53), 107 (56), [127-M e-OHf 95 (100),

81 (52), 69 (60), 55 (76), 43 (74).

(3(5r24/?)-24, 25-Epoxycycloartan-3-ol (3.3a). colorless crystals; C 3oH5o0 2; mol

wt 442.732; mp 101-103 °C; IR u max (KBr), 3387 (OH) cm-1; CD nm (e) 201 (-0.06),

222 (+19.1), 244 (-0.01), 307 (-2.9), 396 (-0.03) (c 0.0005; MeOH); 'H-NMR spectral

data see Table 3.2; l3C-NMR spectral data see Table 3.3; EEMS (70 eV) m z [M-H20]"r

424 (8 ), [M-Me-OHf 410 ( 10), [424-M ef 409 (18), [M-side-chain]+ 315 (9), 311 ( 20),

[315-H2O f 297 (47), 260 (10), 258 (17), 241 (14), 227 (14), 203 (48), [side-chain-H.O]^

109 (64), 107 (100), 91 (79), 81 (91), 79 (78), 69 (69), 55 (98.6), 43 (99); FABMS m z

[M]+ 442.6, [M-H]+ 441.7, [M-OH]+ 425.5, [M-Me-H2OJ+ 409.7.

(23/?)-3-Oxolanosta-8,24-dien-23-oI (3.4). colorless crystals; C 3oH 4s0 2; mol wt

440.717; mp 73-75 °C; IR u max (KBr), 1630 (C=0), 3435 (OH) cm*1; CD nm (e) 215

(0.0), 227 (-4.4), 275 (0.0), 311 (-3.7), 369 (-0.1), (c 0.0005; MeOH); *H-NMR spectral

data see Table 3.2; 13C-NMR spectral data see Table 3.3; EIMS (70 eV) m z [M-OH]+

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423 (3), [M-H2O f 422 (8 ), [423-Mf 408 (4), [422-M]+ 407 (14), [M-side-chain]* 313

(16), 271 (13), 257 (13), [side-chainf 127 (1), [127-OH]+ 110 (12), [127-H20 f 109

(100), 81 (50), 69 (29), 67 (35), 55 (41), 43 (31), 41 (31).

Acetylation of 3.3a. Compound 3.3a (20 mg) was dissolved in 1 ml of pyridine.

After addition of 1 ml of Ac20 the mixture was allowed to react at room temperature for

12 h. After removal of the reagents in vacuo the residue was separated by VLC (2 cm

inner diameter column, 8 g of silica gel) using a gradient elution with hexane and

hexane-EtOAc mixtures of increasing polarity. This yielded 11 mg of pure crystalline

33b: colorless crystals; C 32Hs20 3 ; mol wt 484.37; mp 161-163 °C; IR u max (KBr),

1726 (C=0) cm '1; CD nm (e) 207 (0.4), 226 (-6.56), 257 (0.0), (c 0.0012; MeOH); lH-

NMR spectral data see Table 3.2; 13C-NMR spectral data see Table 3.3; EIMS (70 eV)

m z [M f 484 (1), [M-AcOH]+ 424 (2), [424-Me]+ 409 (2), 302 (4), [424-side-chain]+

297 (3), 175 (20), 161 (13), 135 (25), [side-chainf 127 (10), 107 (35), 95 (39), 69 (42),

59(15), [Ac]+ 43 (100).

Pyridinium Chlorochromate (PCC) Oxidation of 33a to 3.5. Compound 33a

(48 mg), dissolved in 3 ml of CH 2C12, was added to a solution of PCC (100 mg) in

CH2C12 (25 ml) and the mixture was allowed to react for 1 h. After addition of 10 ml of

Et20 , the reaction mixture was adsorbed onto silica gel and separated by VLC (2.3 cm

inner diameter column, 6 g of silica gel) using a gradient elution of hexane or hexane-

EtOAc of increasing polarity to yield 9 mg of pure, crystalline 3.5: gum; C 27Hs20 2; mol

wt 389.63; IR u max (KBr) 1704 (C=0), 1726 (C=0 sh) cm '1; 'H-NMR spectral data see

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Table 3.2; 13C-NMR spectral data see Table 3.3; EIMS (70 eV) m z [M]~ 398 (11), [M-

CH3]+ 383 (7), [M-H 20 ]+ 380 (7), [380-M ef 365 (3), [M-side-chainf 313 (27), 175

(39), 161 (49), 147(36), 133 (49), 121 (62), 107 (71), 95 (98), [side-chainf 85 (37), 81

(73), 67 (74), 55(100).

RuCl3/NaI0 4 Oxidation of 3.3a to 3.1. To a solution of 47.4 mg of 33a in 2 ml

CH3CN, 2 ml CCI4 and 3 ml H20 , 85.6 mg (4 equivalents) of sodium metaperiodate

were added (Ashby et al., 1981). To this biphasic solution l.l mg (4.1 mol %) o f

ruthenium trichloride hydrate was added and stirred magnetically for 8 h while

monitoring the reaction by TLC. At completion of the reaction, 10 ml of CH 2CI2 were

added to this solution and the phases separated. The aqueous layer was extracted three

times with a total of 30 ml of CH 2C12. The organic extracts were combined, dried

(MgSC> 4), and concentrated. The residue was diluted with 20 ml of ether and filtered

through Celite (Ashby et al., 1981). The remaining material was adsorbed onto silica gel

and separated by VLC, to yield 11.3 mg of pure 3.1.

X-Ray Crystallographic Analysis. (Atomic coordinates reference) Intensity

data for 3.1 and 3.4 were collected on an Enraf-Nonius CAD4 diffractometer equipped

with CuKa radiation (A.=l .54184 A), and a graphite monochromator, by ©-20 scans o f

variable rate. Data reduction included corrections for background, Lorentz, polarization,

decay, and absorption effects. Absorption corrections were based on vp scans, and linear

corrections were made for decay. The structures were solved by direct methods and

refined by full-matrix least squares, treating non-hydrogen atoms anisotropically, using

the Enraf-Nonius MolEN programs (Fair, 1990). Hydrogen atoms were placed in

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calculated positions, except for that of the OH group of 3.4, which was refined

isotropically. Details of data collections and refinements are given in Table 3 .4.

Radiorespirometric Bioassays. All compounds were solubilized at 10.24 mg'ml

in DMSO, filter sterilized and stored at -80 °C until used. Subsequent dilution was done

in DMSO. Fifty microliters of each solution were added to 4 ml BACTEC 12B broth

(Becton Dickinson, Towson, MD) to achieve the desired final concentrations.

MIC’s were performed in the BACTEC 460 essentially as described by Heifets

(Heifets et al., 1991). M tuberculosis H37R.V was cultured in 4 ml BACTEC 12B broth

until a daily growth index (GI) of 400-999 was reached. One tenth ml of this was used to

inoculate 4 ml fresh BACTEC 12B medium containing test compounds. Additional

controls diluted 1:100 were also included. Cultures were incubated at 37 °C and the GI

determined daily starting on the third day of incubation. Percent inhibition of fractions

was calculated as (1-GI test sampIe/GI undiluted control) x 100 on the day which the

undiluted controls reached peak values of 999. The minimum inhibitory concentration

(MIC) of pure compounds was defined as the lowest concentration of drug which

effected a daily GI increase and final GI lower than the 1:100 diluted control vial

readings when the 1:100 GI was >30. This corresponds to the concentration which

inhibited the growth of 99% of the organisms. Experiments were usually completed

within 10 days.

Cytotoxicity Assay. Test compounds were dissolved at 20-40 mg/ml in ethanol.

Geometric three-fold dilutions were performed in growth medium M l99 [Gibco, Grand

Island, NY] + 5% fetal bovine serum [HyClone, Logan, UT] +25 mM N-2-

hydroxyethylpiperazine-jV’-2-ethanesulfonic acid [HEPES, Gibco] + 0.2% NaHCOj

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[Gibco] -j- 2 mM glutamine [Irvine Scientific, Santa Ana, CA] to achieve final

concentrations ranging from 4.2 to 400 pig/ml. Final ethanol concentrations did not

exceed 1% v/v. Drug dilutions were distributed in duplicate in 96-well tissue culture

plates (Becton Dickinson Labware, Lincoln Park, NJ) at a volume of 50 nL/well. An

equal volume containing 5 x 10 3 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% CCL in air. After 72 h, cell viability was measured using the

CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corp., Madison,

WI) according to the manufacturer’s instructions. Absorbance at 490 nm was read in a

BioRad Model 3550 microplate reader (Hercules, CA). The IC 50 is defined as the

reciprocal dilution resulting in 50% inhibition o f the Vero cells. Maximum cytotoxicity

(100%) was determined by lysing the cells with sodium dodecyl sulfate (Sigma Chemical

Co., St. Louis, MO).

Table 3.1. Inhibitory Activity of Borrichia frutescens Fractions (Flowers, CHiCL

Extract) against Mycobacterium tuberculosis (H3 7 RV)

percent inhibition fraction 0 % hexane % EtOAc % MeOH 33 pg/ml 100 ng/ml

1...... 100 _ __ 31 23 2...... 95 5 — 95 95 3...... 80 20 — 97 97 4...... 50 50 — 95 96 5...... 20 80 — 27 48 6 ...... — 100 — 26 58 7...... — 50 50 21 19 8 ...... — 100 24 19

°100 ml of solvent.

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Table 3.2. 'H-NMR Spectral Data of Compounds 3.1, 3.3a, 3.3b,3.4, and 3.5 (250 MHz, CDCljf

proton compound

3.1 3.3a 33b 3.4 3.5

H-2a...... 2.26 m 2.39 ddd 2.31m (3.8,6.8, 15) H-2b...... 2.32 m —— 2.59 ddd 2.72ddd (7.0,11,15) (6.7, 14, 14) H-3...... — 3.28 dd 4.56 dd — — (10.1,4.7) (10.1,5.2) H-18 ...... 0.89 s 0.80 s 0.84 s 0.74 s 0.91s

H-19a ...... 0.57 d 0.33 d 0.36 d 1.06 s 0.59d (4.4) (4.3) (4.3) (4.2) H-19b ...... 0.78 d 0.55 d 0.57 d — 0.79d (3.6) (4.1) (4.1) (4.2) H-21...... 0.89 d 0.88 d 0.87 d 0.98 d 0.89d (5.5) (6.2) (6.8) (6.4) (6.2) H-23...... — — — 4.48 ddd — (2.7,9.1,9.1) H-24...... 2.69 dd 2.69 dd 2.69 dd 5.19 dd 9.78dd (6.2) (6.1) (5.9) (2.2, 9.1) (1.8,1.8) H-26...... 1.26 s 1.26 s 1.27 s 1.69 s —

H-27...... 1.30 s 1.30 s 1.31 s 1.71 s —

H-28 ...... 1.04 s 0.96 s 0.88 s 1.09 s 1.05s

H-29 ...... 1.09 s 0.96 s 0.89 s 1.12s 1.1 Is

H-30...... 0.90 s 0.89 s 0.96 s 0.88 s 1.00s

— H-2'...... — — 2.05 s —

a Expressed as 8 values in ppm, with J values in Hz in parentheses.

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Table 3.3. 13C-NMR Spectral Data of Compounds 3.1,3.3a, 33b, 3.4, and 3.5 (62.5 MHz, CDCl3f

carbon compound 3.1 33a 33b 3.4 3.5

C -l...... 33.41 32.01 31.61 36.1 t 33.61 C-2...... 37.4 t 30.41 26.5 t 34.6 t 37.71 C-3...... 216.5 s 78.8 d 80.7 d 217.7 s 216.7 s C-4...... 50.2 s 40.5 s 39.5 s 47.4 s 50.5 s C-5...... 48.4 d 48.0 d 47.8 d 50.9 d 48.6 d C-6...... 21.51 21.11 20.91 19.4 t 21.71 C-7...... 28.11 28.2 t 28.2 t 28.3 t 28.3 t C-8 ...... 47.9 d 47.1 d 47.2 d 133.2 s 48.1 d C-9 ...... 21.1 s 20.0 s 20.1 s 133.8 s 21.3 s C-10...... 26.0 s 26.0 s 26.0 s 36.9 s 26.2 s C -ll...... 25.81 26.11 25.81 21.1 t 26.11 C-l 2...... 35.51 35.5 t 35.5 t 26.3 t 35.71 C-13...... 45.3 s 45.3 s 45.3 s 44.6 s 45.6 s C-14...... 48.7 s 48.8 s 48.9 s 50.0 s 49.0 s C-15...... 32.81 32.91 32.9 t 31.01 33.01 C-16...... 26.71 26.41 26.8 s 30.91 26.91 C-l 7...... 52.2 d 52.1 d 52.2 d 51.3 d 52.4 d C-l 8 ...... 18.1 q 18.0 q 18.0 q 18.1 q 18.2 q C-l 9 ...... 29.51 29.91 29.81 18.7 q 29.71 C-20...... 35.8 d 35.8 d 35.9 d 33.1 d 35.9 d C-21...... 18.3 q 18.3 q 18.3 q 18.7 q 18.3 q C-22...... 32.61 32.91 32.61 44.4 t 41.4 t C-23...... 25.61 25.61 25.71 66.0 d 28.5 t C-24...... 64.7 d 64.8 d 64.8 d 129.1 d 203.3 d C-25...... 58.3 s 58.4 s 58.4 s 135.3 s — C-26...... 18.7 q 18.7 q 18.8 q 24.2 q — C-27...... 24.9 q 24.9 q 24.9 q 26.2 q — C-28 ...... 22.2 q 25.4 q 25.4 q 25.7 q 22.4 q C-29 ...... 20.7 q 14.0 q 15.1 q 15.9 q 21.0 q C-30...... 19.2 q 19.3 q 19.3 q 21.3 q 19.5 q C -l'...... — — 170.9 s — — C-2'...... ---- — 21.3 q ----

“Peak multiplicities were determined by heteronuclear multipulse programs (DEPT); s = singlet, d = doublet t = triplet, q = quartet.

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Table 3.4. Crystallographic Data of Triterpenes 3.1 and 3.4

compound 3.1 compound 3.4

Formula C30H48 O2 C30H48 O2 Molecular weight 440.7 440.7 Space group Monoclinic P2\ Monoclinic P2i a (A) 7.527(1) 11.926(1) b (A) 9.127(1) 7.479(1) c (A) 19.664(3) 16.203(2) P(°) 97.60(1) 112.25(1) V(A3) 1339.0(6) 1337.6(6) Dc (g cm3) 1.093 1.094 Z 2 2 u CuKa (cm 1) 4.5 4.7 Temperature (°C) 23 22 Crystal dimensions (mm) 0.25x0.22x0.05 0.60x0.40x0.12 Crystal Colorless plate Colorless lath 0 range (°) 2-75 2-75 (hemisphere) Unique data 2935 5440 Observed data 1610 5202 Criterion for observed I>lo(I) I>3ct(I) Refined variables 289 293 Intensity decay (%) 14.0 4.3 Min. rel. tranmission (%) 80.9 93.7 R 0.100 0.055 R-w 0.077 0.075 Max. resid. density (eA*3) 0.31 0.64 Min. resid. density (eA-3) -0.10 -0.13

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Table 3.5. Coordinates and Equivalent Isotropic Thermal Parameters for (24/?)-24,25- Epoxycycloartan-3-one (3.1 )a

atom .r y z 5eq(A2)

O -l...... 0.5791(6) 0 1.0479(2) 10.4(2) 0-2...... 0.0685(9) -0.105(1) 0.2595(3) 12.5(3) C -l...... 0.3169(8) 0.026(1) 0.8864(3) 6.7(3) C-2...... 0.3696(9) 0.089(1) 0.9576(3) 6.8(2) C-3...... 0.5535(9) 0.040(1) 0.9902(3) 6.6(2) C-4...... 0.7034(8) 0.053(1) 0.9443(3) 5.8(2) C-5...... 0.6380(8) 0.005(1) 0.8711(3) 6.4(2) C-6...... 0.7789(8) 0.019(1) 0.8223(3) 7.4(3) C-7...... 0.7061(9) -0.048(1) 0.7531(3) 7.4(3) C-8 ...... 0.5513(8) 0.043(1) 0.7178(3) 5.3(2) C-9 ...... 0.4072(7) 0.077(1) 0.7630(3) 4.7(2) C-10...... 0.4564(8) 0.0646(9) 0.8411(3) 4.3(2) C -ll...... 0.2101(8) 0.040(1) 0.7336(3) 5.6(2) C-l 2...... 0.1564(8) 0.051(1) 0.6570(3) 5.5(2) C-13...... 0.3146(7) 0.0814(9) 0.6143(3) 3.6(2) C-l 4...... 0.4720(8) -0.0134(9) 0.6471(3) 4.3(2) C-l 5...... 0.6052(9) 0.004(1) 0.5925(3) 6.8(2) C-16...... 0.4874(9) 0.020(1) 0.5241(3) 6-9(3) C-17...... 0.2878(7) 0.0259(9) 0.5392(3) 4.2(2) C-l 8 ...... 0.3560(8) 0.244(1) 0.6195(3) 4.9(2) C-l 9 ...... 0.4355(8) 0.209(1) 0.8070(3) 5.7(2) C-20...... 0.1757(9) 0.112(1) 0.4842(3) 5.5(2) C-21...... -0.023(1) 0.114(1) 0.4964(4) 7.0(3) C-22...... 0.1805(9) 0.028(2) 0.4118(3) 11.3(4) C-23...... 0.091(1) 0.091(2) 0.3577(4) 12.4(5) C-24...... 0.147(1) 0.018(2) 0.2911(4) 12.3(4) C-25...... 0.038(1) 0.043(1) 0.2229(4) 11.4(4) C-26...... -0.146(2) 0.088(2) 0.2174(7) 17.0(5) C-27...... 0.140(2) 0.048(2) 0.1638(5) 16.7(5) C-2 8 ...... 0.426(1) -0.173(1) 0.6496(4) 6.8(2) C-29 ...... 0.857(1) -0.042(1) 0.9754(4) 9.0(3) C-30...... 0.760(1) 0.212(1) 0.9493(4) 8.1(3)

a Figures in parentheses are ESD.

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Table 3.6. Coordinates and Equivalent Isotropic Thermal Parameters for (23/?)-3- Oxolanosta-8,24-dien-23-ol (3.4)a

atom .T y - #eq(A2)

0-1...... 0.1260(2) 0 0.4697(1) 5.32(4) 0-2...... 0.9858(1) -0.1580(3) 1.29915(9) 4.74(4) C -l...... 0.3570(2) 0.1313(3) 0.6674(1) 3.91(4) C-2...... 0.2277(2) 0.1657(3) 0.6007(1) 4.38(5) C-3...... 0.1714(2) -0.0016(3) 0.5509(1) 3.55(4) C-4...... 0.1745(2) -0.1674(3) 0.6049(1) 3.38(4) C-5...... 0.3047(2) -0.1875(3) 0.6787(1) 3.09(3) C-6...... 0.3180(2) -0.3464(3) 0.7402(2) 4.41(5) C-7...... 0.4512(2) -0.3891(3) 0.7886(2) 4.48(5) C-8 ...... 0.5303(2) -0.2287(3) 0.8222(1) 3.13(4) C-9 ...... 0.4920(2) -0.0602(3) 0.7960(1) 2.96(3) C-10...... 0.3615(2) -0.0184(3) 0.7335(1) 3.10(3) C-l 1...... 0.5769(2) 0.0979(3) 0.8297(1) 3.91(4) C-l 2...... 0.7006(2) 0.0658(3) 0.9056(1) 4.15(5) C-13...... 0.7062(1) -0.1103(3) 0.9542(1) 2.95(3) C-l 4...... 0.6613(2) -0.2593(3) 0.8818(1) 3.14(4) C-15...... 0.6918(2) -0.4288(3) 0.9391(2) 5.08(6) C-l 6...... 0.8132(2) -0.3864(3) 1.0152(1) 4.17(5) C-l 7...... 0.8335(2) -0.1816(3) 1.0160(1) 3.23(4) C-l 8 ...... 0.6243(2) -0.0952(4) 1.0071(1) 4.29(4) C-l 9 ...... 0.2983(2) 0.0462(4) 0.7962(2) 5.37(6) C-20...... 0.8933(2) -0.1043(3) 1.1105(1) 3.68(4) C-21...... 0.9174(2) 0.0952(4) 1.1108(2) 5.05(5) C-22...... 1.0110(2) -0.2084(4) 1.1615(1) 4.90(5) C-23...... 1.0750(2) -0.1614(4) 1.2601(1) 4.88(5) C-24...... 1.1665(2) -0.3097(5) 1.3066(2) 6.22(7) C-25...... 1.2792(2) -0.2982(7) 1.3619(2) 7.68(9) C-26...... 1.3437(3) -0.1212(9) 1.3866(2) 10.9(1) C-27...... 1.3502(3) -0.4605(8) 1.4067(2) 11.1(1) C-28 ...... 0.7346(2) -0.2646(4) 0.8211(1) 5.28(5) C-29 ...... 0.1461(2) -0.3306(3) 0.5435(2) 4.72(5) C-30...... 0.0731(2) -0.1495(4) 0.6413(2) 4.88(5)

a Figures in parentheses are ESD.

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Table 3.7. Minimum Inhibitory Concentrations Against M. tuberculosis and IC50 Values Against Vero cells

compound MIC (jig/ml) IC50 (pg/ml)

3.1 8 71.8

3.3a 8 39.8

3.3b >128 -

3.4 64-128 103.6

3.6 >128 -

3.7 >128 -

fusidic acid 4 105.8

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R1 R*

3.1 =0 H

3.2 =0 OH

3.3a P-OH, a-H H

3.3b P-OAc, a-H H

3.5 =o H H

3.6 =o ‘OH

HO h

3.4 3.7

Figure 3.1. Triterpenes isolated from B. frutescens

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 Figure Figure 3.2. structures Molecular of compounds 3.1 and 3.4

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73

O CU CM

O CD

O PPM

O in

ID Figure3.3. 250 MHz 'll-NMR spectrum of (24/0-24,25-epoxycyc!oartan-3-one (3.1) CDCIj in o

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 74

u c o 01 ' cu

OS3 O o >> ' V u X Q.0 U1 o

3- o o CD C3 o 0 a . K o QC ■ o 2 z1 o £ CL O CL 53 OJ J3 T358 O TJ o C ■ T 53 3vn m H O a . •CO urs o o L- o LUCL •CD Q Zu 2 o • o CN ru 'O o O' H CL L4 ) lu 3 Q 00 cZ

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75

O'! m CDCI3 C'lsC

o OJ

o m

o PPM 'T

o in

o in Figure Figure 3.5. 250 MHz 'H-NMR spectrum (30,24/0-24, of 25-epoxyeycloartan-3-ol (3.3a) in o r-

DEPT 90 m 'to 'in ' ’T 'cn 'OJ 03 o r^ o o O O o o PPM Figure 3.6. 62.5 MHz DEPT 90\ DEPT 135°, and broad band nC-NMR spectra of (3p,24K)-24, 25-epoxycycloartan-3-ol (3.3a) 76 77

oo oo CN rn

o cu

o cn

o PPM •*r

o in

o Figure Figure 3.7. 250 MHz l-NMR 'l spectrum of compound 3.3b CDCI in CD

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oo CDCI3

o cu

o cn

■

o in

o cn Figure Figure 3.8, 250 MHz 'H-NMR spectrum (3.4)in of (23/

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3.4 References

Allen. F. H.; Kennard, O.; Taylor, R. Acc. Chem. Res. 1983, 16, 146.

Anjanevulu, V.; Harischandra Prasad, K.; Ravi, K.; Connollv, J. D.Phvtochemistrx' 1985, 24, 2359.

Ashby, E. C.; Goel, A. B. J. Org. Chem. 1981, 4 6 ,3936.

Atomic coordinates, thermal parameters, bond distances and angles, and observed and calculated structure parameters have been deposited with the Cambridge Crystallographic Data Centre and can be obtained upon request from Dr. Olga Kennard, University Chemical Laboratory, 12 Union Road, Cambridge CB2 IEZ, UK.

Barik, B. R.; Bhaumik, T.; Dey, A. K.; Kundu, A. B. Phytochemistry 1994, 35, 1001.

Coll, J. C.; Bowden, B. T. J. Mat. Prod. 1986, 49, 934.

De Pascual Teresa, J.; Urones, J. G.; Marcos, I. S.; Basare, P.; Sexmero Cuadrado, M. L.; Fernandez Moro, R. Phytochemistry 1987, 26, 1767.

Delgado, G.; Rios, M. Y.; Colin, L. P.; Garcia E.; Alvarez, L. Fitoterapia 1992, 63, 273.

Fair, C. K.. MolEN. An Interactive System for Crystal Structure Analysis, Enraf-Nonius: Delft, The Netherlands; 1990.

Heifets, L. B. Drugs Susceptibility in the Chemotherapy of Mycobacterial Infections.; CRC Press, Inc.: Boca Raton, FL, 1991; Chapter 3, pp 89-121.

Herz, W.; Watanabe, K.; Palaniappan, K.; Blount, J. F. Phytochemistry 1985, 24, 2645.

Hoffner, S. E.; Olsson-Liljequist, B.; Rydgard, K. J.; Svenson, S. B.; Kallenius, G. Eur. J. Clin. Microbiol. Infect. Dis. 1990, 9,294.

Jakupovic, J.; Pathak, V. P.; Bohlmann, F.; Dominguez, X. A. Phytochemistry, 1987 26, 761.

Mendes, F. N. P.; Silveira, E. R. Phytochemistry 1994, 35, 1499.

Nes, W. D.; R. Wong, Y.; Benson, M.; Landrey, J. R.; Nes, W. R. Proc. Nat. Acad. Sci. U.S.A. 1984, 81, 5896.

Nishizawa, M.; Emura, M.; Yamada, H.; Shiro, M.; Chairul; Hayashi, Y.; Tokuda, H. Tetrahedron Letters 1989, 30, 5615.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80

Quijano, L.; Calderon, J. S.; Gomez, F.; Rios, T. Phytochemistry 1982, 21, 2041.

Romo de Vivar, A.; Martinez-Vazquez, M.; Matsubara, C.; Perez-Sanchez, G.; Joseph- Nathan, P. Phytochemistry 1990, 29, 915.

Takaishi, Y.; Murakami, Y.; Ohashi, T.; Nakano, K..; Murakami, K.; Tomimatsu, T. Phytochemistry 1987, 26, 2341.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4

ANTEVTYCOBACTERIAL ACTIVITIES OF DEHYDROCOSTUS LACTONE AND ITS OXIDATION PRODUCTS *

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4.1 Introduction

In a search for new structural types of antimycobacterial natural products, in vitro

tests against M. tuberculosis of a series of natural and semisynthetic guaianolide-tvpe

sesquiterpene lactones were performed. Dehydrocostus lactone (4.1a), a major

constituent of Saussurea lappa Clark (Asteraceae) (Romanuk et al., 1956; Lu et al.,

1996), gave MICs of 2 and 16 pg/ml against M. tuberculosis and M. avium, respectively.

It was therefore of interest to leam whether epoxidation of the nonlactonic double bonds

of 4.1a would result in derivatives with increased activity, since antimycobacterial

triterpenes from B. frutescens correlated well with the presence of an epoxyprenyl group

(Cantrell et al., 1996). The presence of epoxides could possibly increase activity due to

enzyme alkylation(s) of the epoxide(s) by essential nucleophiles (thiols and/or amino

groups) at enzyme active sites o f the mycobacterium. Therefore, 4.1a was used as a

starting compound for various oxidative modifications in an attempt to find sesquiterpene

lactones with increased antimycobacterial activity.

4.2 Results and Discussion

Dehydrocostus lactone (4.1a) was previously isolated from costus root oil in our

laboratory as described earlier (Lu et al., 1996). The reaction of a solution of 4.1a in

CH2CI2 with 1.5 molar equivalents of m-chloroperbenzoic acid (m-CPBA) gave a mixture

of reaction products, which were separated by vacuum-liquid chromatography (VLC),

providing the monoepoxides 43a, 4.2, and 43b in the less polar fractions, followed by

the more polar diepoxides 4.4a, 4.4b, and 4.4c.

Inspection of the ‘H-NMR spectrum of 43a indicated a monoepoxide that

differed from 4.1a in the absence of the two olefinic methylene protons at C-l4 with

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singlets at 8 4.82 and 8 4.90. Instead, two mutually coupled doublets appeared at 5 2.75

and 3.10 (J=4.9 Hz), suggesting epoxidation at the C-10( 14) position. This was

supported by the 13C-NMR spectrum of 4.3a, in which the two olefinic carbon signals (C-

10 and C-14) in 4.1a were replaced by signals corresponding to the oxygen-bearing C-10

(5 65.1, s) and C-14 (8 48.4, t) in 43a. The stereochemistry of the epoxide ring was

determined by NOESY spectroscopy with correlations being observed between H-14b

and H-5a, suggesting a C-10 (i-epoxide. Further lH- and l3C- NMR assignments were

made by inspection of COSY, HMQC, and HMBC spectra, and the chemical shifts

obtained are summarized in Tables 1 and 2, respectively.

*H- and 13C-NMR analysis of epoxide 43b also indicated epoxidation at C-l 0(14)

based on similar reasoning to that described above for 4.3a. The stereochemistry of 43b

was confirmed by NOESY spectroscopy, which showed interactions between H-6(3 and

both epoxide protons at C-14, suggesting a C-10 a-epoxide. The application of 2D

COSY, HMQC, and HMBC permitted all assignments in the lH- and 13C-NMR spectra,

which are summarized in Tables 1 and 2, respectively.

The ’H-NMR. spectrum of compound 4.2 indicated that it differed from 4.1a in

the absence of the olefinic methylene protons at C -l5. Instead, 4.2 exhibited mutually

coupled oxirane methylene doublets at 8 2.84 and 3.36 (.7=4.5 Hz) which were absent in

4.1a. The 13C-NMR data also indicated that the epoxide group in 4.2 could be placed

between the C-4(15) positions. The stereochemistry at C-4 was determined by NOESY

spectroscopy which indicated a strong NOE correlation between H-15b and H-6(3.

Further support for a C-4a-epoxide was provided by reaction of 4.2 with excess m-CPBA

to produce diepoxides 4.4b and 4.4c. Since the stereochemistry of 4.4c was determined

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by single crystal X-ray diffraction, this conversion unambiguously established the oc-

epoxide stereochemistry at C-4(15) in 4.2. *H- and I3C-NMR assignments of 4.2 were

based on inspection of COSY, HMQC, and HMBC spectra and are summarized in Tables

1 and 2, respectively.

In the ’H- NMR spectrum of compound 4.4a, the olefinic H-14 and H-15 signals

present in 4.1a were replaced by two sets of mutually coupled oxirane proton doublets at

5 2.55 and 2.85 (.7=4.3 Hz) corresponding to H-14a and H-I4b and doublets (H-15a and

H-15b) at 8 2.80 and 3.27 (.7=4.4 Hz), suggesting a C-10(14), C-4(15) diepoxide. The

relative stereochemistry of the epoxide groups was unambiguously determined by single

crystal X-ray diffraction. Its molecular structure is shown in Figure 4.1 and the X-ray

crystallographic atomic coordinates are given in Table 4.5. The molecular structure

indicated a C-4(15) P-epoxide while the C-10(14) epoxide oxygen is a-oriented.

Compound 4.4a has been previously prepared from 4.1a as a mixture of C-10(14)

epoxide epimers with an undefined stereochemistry of the products (Kalsi et al., 1979).

‘H- and l3C-NMR spectra of pure 4.4a were assigned by various 2D NMR techniques

and the spectral data are reported in Tables 1 and 2, respectively.

Examination of the *H- and I3C-NMR spectra of diepoxides 4.4b and 4.4c

indicated that they represented C-l0(14), C-4(15) isomers based on arguments made

above for 4.4a. The epoxide stereochemistry of compound 4.4b was determined by

NOESY spectroscopy with correlations observed between H-6P and H-14b as well as

between H-6P and H-15b, both indicating an a-orientation for the two epoxide oxygens.

The relative stereochemistry of compound 4.4c was determined by single-crystal X-ray

diffraction (Figure 4.1), which showed a C-4(15) a-epoxide and a C-10(14) p-epoxide.

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Therefore, the epimers 4.4b and 4.4c differed only in the configuration of the C-10(14)

epoxide group. X-ray coordinates for 4.4c are listed in Table 4.6 and lH- and nC-NMR

spectral assignments by various 2D NMR techniques for 4.4b and 4.4c are summarized in

Tables 1 and 2, respectively.

3-Epizaluzanin C (4.1b) was prepared from 4.1a by a previously described

method (Kalsi et al., 1984) using Se02 and /-BuOOH in CTLCL. The ‘H- and 13ONMR

spectral data of 4.1b were in full agreement with reported values (Kalsi et al., 1983).

Single-crystal X-ray crystallographic analysis of 4.1b (Figure 4.1) was performed and its

atomic coordinates are listed in Table 4.4.

The crystal structures of 4.1b, 4.4a, and 4.4c are illustrated in Figure 4.1. Lactone

4.1b is essentially isomorphous with dehydrocostus lactone (Fronczek et al., 1989),

which lacks the OH group at 0 3 , despite the intermolecular hydrogen bonding by the

OH group. That hydrogen bond is linear, and involves the lactone carbonyl oxygen 0-2

as acceptor, having an 0 ...0 distance of 2.858(2) A and an angle about H of 179(3)°.

While the conformations of the seven-membered and lactone rings in 4.1b and

dehydrocostus lactone (4.1a) are nearly identical, the conformations of their other 5-

membered rings differ as a result of the C-3-OH substitution. While this ring in

dehydrocostus lactone has a distorted half-chair conformation, it has the envelope

conformation with C-5 in the flap position in 4.1b.

The conformation of 4.4c is similar to that of 4.1b, with a twist-chair seven-

membered ring having C-8 on the twist axis, and a half-chair lactone. Diepoxide 4.4a has

two independent molecules in the asymmetric unit. Their conformations are nearly

identical, but differ markedly from that of 4.4c. Lactone 4.4a has its seven-membered

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ring in a distorted twist-chair with C-5 on the twist axis, and an envelope cyclopentane

ring with C-4 at the flap position.

The MICs of the guaianolides were determined in radiorespirometric bioassays

against Mycobacterium tuberculosis (H 3 7 R.V) and 4.1a and 4.1b were also tested against

M. avium (Cantrell et al., 1996; Connell et al., 1994). The MICs against M. tuberculosis

of dehydrocostus lactone (4.1a), its oxidative derivatives (4.2, 4.3a, 4.3b, and 4.4a-4.4c)

and hydroxylated analogs (4.1b, 4.1c, 4.5, and 4.6) are listed in Table 4.7. The most

active lactone 4.1a, with an MIC of 2 pg/ml, is also the most lipophilic among this group

of guaianolides. It is evident that the presence of a hydroxyl group at different positions

of the guaianolide skeleton, as shown for 4.1b, 4.1c, 4.5, and 4.6, significantly reduces

activity against M. tuberculosis. This might be due to the increase in polarity which

possibly reduces transport through the outer lipid layer of the mycobacterium (Connell et

al., 1994).

Since, in our previous study on cycloartane triterpenes from Borrichia frutescens,

the presence of an epoxide group in the molecule significantly augmented the

antituberculosis activity (Cantrell et al., 1996), mono- and diepoxide derivatives of

dehydrocostus lactone (4.1a) were tested for their activity. Compared to 4.1a, the

monoepoxides 4.2, 4.3a, and 43b showed significantly decreased activity against M.

tuberculosis with MICs of 32, 32, and 64 jig/ml, respectively. The more polar diepoxides

4.4a, 4.4b, and 4.4c exhibited MICs of 64, 128, and 128 pg/ml, respectively, supporting

the correlation between lipophilicity and antituberculosis activity within this guaianolide

series.

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In addition to the correlation between lipophilicity and antituberculosis activity,

there may also be a correlation of ring conformation with antituberculosis activity. It

appears that diepoxides 4.4a and 4.4c not only differ in relative configuration of their

epoxide rings, but also in their ring conformations, as indicated by their molecular

structures based on X-ray data (Figure 4.1). Compound 4.4a has a one-fold lower MIC

of 64 fig/ml and contains a seven-membered ring in a distorted twist-chair with C-5 on

the twist axis. Compound 4.4c with an MIC of 128 pg/ml however contains a twist-chair

seven-membered ring having C-8 on the twist axis.

In conclusion, the above structure-activity data permit us to suggest that the

antimycobacterial activity of the tested guaianolides is not as strongly determined by the

exocyclic methylene lactone moiety, as by polarity factors. Activity is most dramatically

reduced when at least one hydroxyl group is present in the molecule (Compounds 4.1b,

4.1c, 4.5, and 4.6). The successive replacement of double bonds with epoxide groups also

caused a significant loss of activity with the more polar diepoxides being less active than

the monoepoxide analogues. As contrasted by the activity of 4.4a and 4.4c,

configurational and conformational influences seem to exist but appear to be of lesser

importance. Since the most lipophilic guaianolde 4.1a gives the highest antimycobacterial

activity, it can be concluded that activity is most strongly influenced by the polarity of a

given molecule. Therefore, within this structurally related group of sesquiterpene lactones

antimycobacterial activity is most likely controlled by the transport through the outer

lipid layer of mycobacteria.

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43 Experimental

General Experimental Procedures. IH- and 13C-NMR spectra were recorded in

CDCI3 on either a Bruker ARX 300 MHz spectrometer, or a Bruker AM 400 MHz

spectrometer. Mass spectra were obtained on a Hewlett-Packard 5971A GC-MS. IR

spectra were run on a Perkin-Elmer 1760X spectrometer as a film on KBr plates.

Vacuum-liquid chromatographic (VLC) separations were earned out on TLC grade silica

gel (MN Kieselgel) (Coll et al., 1986).

m-CPBA Oxidation of Dehydrocostus Lactone (4.1a). A solution of 980 mg of

dehydrocostus lactone (4.1a) in 20 ml CH2CI; was added to a solution of 1.5 molar

equivalents m-CPBA in 20 ml CH 2CI2 and stirred in an ice bath for 2 h. The reaction

mixture was washed twice with 40 ml 0.01 M NaOH soln and once with 40 ml of

distilled H2O. TLC of the reaction mixture revealed at least five products. Accordingly,

the crude mixture was adsorbed onto silica gel and chromatographed on a VLC column

(3.5 cm i. d.) using solvent mixtures of hexane and EtOAc of increasing polarity.

Fraction 7 (100 ml hexane-EtOAc, 88:12) contained 70 mg of 43a and fraction 8

(hexane-EtOAc, 86:14) provided 138 mg of 4.2. Fraction 9 (hexane-EtOAc, 84:16) gave

18 mg 43b, and fraction 11 (hexane-EtOAc, 4:1) contained 160 mg of crystalline 4.4a.

Fraction 14 (hexane-EtOAc, 3:2) contained a mixture of two diepoxides which required

further purification. Crude fraction 14 was adsorbed onto silica gel and

rechromatographed on a VLC column (2.3 cm i.d.) using hexane or hexane-EtOAc

mixtures of increasing polarity. Fraction 14-10 (50 ml hexane-EtOAc, 77:23) contained

33 mg of 4.4b and fraction 14-11 (hexane-EtOAc , 76:24) gave 20 mg of crystalline 4.4c.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89

Dehydrocostus lactone, 4ot(15)-epoxide (4.2). Gum; IR 1764 (C=0), 1637,

1258, 1138 cm'1; ‘H-NMR, see Table 4.1; 13C-NMR, see Table 4.2; EIMS 7 0 e V m : 246

[M f (12), 228 [M-H20r (5 ), 217 (10), 188 (20), 161 (24), 150 (37), 124 (64), 91 (100),

79 (67), 67 (43), 53 (81).

m-CPBA Oxidation of 4.2. Compound 4.2 (40 mg) was dissolved in 5 ml

CH2C12 and added to 5 ml CH2C12 solution containing 4 equivalents m-CPBA. The

reaction mixture was placed in an ice bath and stirred until TLC indicated that all 4.2 had

reacted. The reaction was stopped after 2 h and worked up as described previously.

Crude reaction products were chromatographed as indicated above to yield 16 mg of 4.4b

and 11 mg of 4.4c.

Dehydrocostus Lactone, 10P(14)-epoxide (4.3a). Gum; IR v ^ 1763 (C=0),

1255, 996 cm'1; ‘H-NMR, see Table 4.1; 13C-NMR, see Table 4.2; EIMS 70 eV m : 246

[M f (2), 228 [M-HiOf (7), 215 (44), 199 (9), 187 (12), 173 (14), 159 (21), 150 (99),

123 (41), 105 (45), 91 (100), 79 (76), 53 (84).

Dehydrocostus Lactone, 10a(14)-epoxide (4.3b). Gum; IR v ^ 1764 (C=0),

1254, 995 c m 1; ‘H-NMR, see Table 4.1; I3C-NMR, see Table 4.2; EIMS 70 eV m : 246

[M]+ (2), 228 [M-H2O f (3), 216 (13), 200 (8), 171 (13), 159(12), 120 (36), 105 (40), 91

(69), 80(100), 53 (47).

Dehydrocostus Lactone, 4P( 15), 10a( 14)-diepoxide (4.4a). Colorless crystals

(hexane-EtOAc); mp 97 °C; IR vr a 1765 (C=0), 1254, 996, 867 c m 1; ‘H-NMR, see

Table 4.1; 13C-NMR, see Table 4.2; EIMS 70 eV m z 262 [M]+ (1), 244 [M-H2O f (2),

231 (35), 214 (27), 203 (13), 185 (26), 173 (17), 159 (24), 105 (47), 91 (100), 79 (79), 67

(59), 53 (80).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90

Dehydrocostus Lactone, 4a(15),10a(14)-diepoxide (4.4b). Gum; IR 1763

(C=0), 1258, 732 cm*1; ‘H-NMR, see Table 4.1; 13C-NMR, see Table 4.2; EIMS 70 eV

m z 262 [M f (1), 261 (3), 247 [M-CH^f (D, 244 [M -H.Of (2), 231 (14), 215 (7), 203

(19), 187 (13), 175 (20), 151 (29), 131 (33), 117 (44), 105 (47), 91 (100), 79 (67), 67

(54), 53 (58).

Dehydrocostus Lactone, 4a(15),10f)(14)-diepoxide (4.4c). Colorless crystals

(hexane-EtOAc); mp 133-135 °C; IR vmx 1765 (C=0), 1258, 999 cm*1; ‘H-NMR, see

Table 4.1; 13C-NMR, see Table 4.2; EIMS 70 eV m z 262 [M f (1), 244 [M-H2O f (2),

231 (4), 215 (5), 203 (9), 151 (19), 131 (32), 117 (34), 105 (41), 91 (100), 79 (80), 67

(76), 53 (99).

Selenium Dioxide Oxidation of Dehydrocostus Lactone (4.1a). Oxidation was

performed as previously described and the crude product was purified by preparative

TLC to yield pure 4.1b (Kalsi et al., 1984). The NMR data were essentially identical

with values reported in the literature (Kalsi et al., 1984) and the molecular structure was

unambiguously established by single-crystal X-ray diffraction.

Compounds 4.1c, 4.5, and 4.6. The natural sesquiterpene lactones 7a-

hydroxydehydrocostus lactone (4.1c) (Fronczek et al., 1984), micheliolide (4.5)

(Castaneda-Acosta et al., 1993), and pumilin (4.6)(Korp et al., 1982) have been obtained

previously in our laboratory.

X-ray Crystallographic Analyses. Intensity data were collected for 4.1b, 4.4a,

and 4.4c on an Enraf-Nonius CAD4 diffractometer with graphite-monochromated CuKa

radiation (A.=1.54184 A) by o)-20 scans. Crystals of 4.4a were of considerably lower

quality than those of the other two compounds, yielding broad diffraction peaks. The

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91

resulting structure determination is of lower precision, but is sufficient to establish the

molecular structure. Two octants of data were collected for 4.4a and five octants for

4.4c. Data reduction included corrections for background, Lorentz, polarization, decay

(for 4.4a, 19%), and absorption (for 4.4c) effects. Absorption corrections were based on

iy scans, vy Scans for 4.4c indicated no change in intensity with rotation about the

diffraction vector. Crystals of 4.1b are isomorphous with dehydrocostus lactone; all

atoms but C-3 of that structure were used as a beginning refinement model. Structures

4.4a and 4.4c were solved by direct methods. Refinement was by full-matrix least

squares, with neutral-atom scattering factors and anomalous dispersion corrections. All

non-hydrogen atoms were refined anisotropically, while H atoms were treated as

specified in Table 4.3. Refinement of the absolute structures of 4.1b and 4.4c is

consistent with the expected absolute configurations. Crystal data, details of data

collection and refinement, and final agreement indices for the three structures are given in

Table 4.3.

Radiorespirometric Bioassays. Bioassays were performed essentially as

described previously (Cantrell et al., 1996; Inderleid et al., 1996; Inderleid et al., 1995;

Siddiqi et al., 1992). Experiments for A/, avium were usually completed within five days

and those for M. tuberculosis in ten days.

4.4 References

Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams, L. B.; Franzblau, S. G. J. Nat. Prod. 1996, 59, 1131.

Castaneda-Acosta, J.; Fischer, N. H.; Vargas, D. J. Nat. Prod. 1993, 56, 90.

Cohn, D. L.; Bustreo, F.; Raviglione, M. C. Clin. Infectious Dis. 1997, 24 (Suppl. 1). S 121.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92

Table 4.1. 300 MHz 'H-NMR Spectral Data for Compounds 4.2-4.4c Performed in CDCl3a

proton 4.2 4 Ja 4.3b 4.4a 4.4b 4.4c 1 3.21 ddd 2.93 ddd 2.43 ddd 2.85 m 2.45 ddd 2.74 ddd (8.1, 8.1, (4.0,9.0, (8.1,8.1, (8.7, 8.7, (8.1,8.1, 8.1) 9.0) 8.1) 8.7) 8.1) 2a 1.91 m 2.05 m 1.6-1.85* 1.66 m 1.70 m 1.68 m 2b 2.11 m 2.05 m 2.37 m 1.78 m 2.02 m 1.92 m 3a 1.69 m 1.86 ddd 1.6-1.85* 1.49 m 1.60-1.78* 1.55-1.70* (6.5,9.0, 14.5) 3b 2.25 m 2.26 m 2.37 m 2.14 m 2.11-2.25* 2.02-2.12* 5 2.13 dd 2.64 dd 2.88 dd 2.69 dd 2.12m 2.22 dd (8.1,11.0) (9.6,9.6) (9.3,9.3) (9.9, 9.9) (8.8, 10.7) 6 4.04 dd 4.08 dd 4.12 dd 4.10 dd 4.13 dd 4.23 dd (8.8, 11.0) (9.6,9.6) (9.0, 10.2) (9.7, 9.8) (8.8, 11.2) (9.4, 9.4) 7 2.74 m 2.86 m 2.98 m 2.85 m 2.99 m 2.74 m 8a 1.45 m I.42 dddd I.48 dddd 2.23 dddd 1.52 m 1.61 m (5.0,11.9, (6.1, 8.6, (3.0, 3.0, II.9, 13.0) II.3, 13.6) 4.7, 13.7) 8b 2.25 m 2.22 m 2.27 dddd 1.45 m 2.29 m 2.11 m (4.3,6.0, 6.0, 13.6) 9a 2.19 m 2.09 m 1.78 m 1.62 m 1.70 m 1.55-1.70* 9b 2.46 dd 2.56 ddd 1.91 m 2.10 m 2.02 m 2.02-2.12* (6.5, 12.7) (3.5, 5.0, 14.6) 13a 5.46 d (3.4) 5.48 d (3.1) 5.53 d (3.1) 5.53 d (3.1) 5.51 d (3.1) 5.50 d (2.9) 13b 6.17 d (3.6) 6.19 d (3.6) 6.26 d (3.1) 6.24 d (3.5) 6.22 d (3.6) 6.22 d (3.67) 14a 4.94 s 2.75 d (4.9) 2.54 d (4.7) 2.55 d (4.3) 2.63 d (4.8) 2.60 d (4.8) 14b 4.97 s 3.10 d (4.9) 2.62 dd 2.85 d (4.3) 2.68 d (4.8) 2.78 d (4.7) (1.2,4.5) 15a 2.84 d (4.4) 5.00 s 5.02 d ( 1.7) 2.80 d (4.4) 2.87 d (4.6) 2.86 d (4.4) 15b 3.36 d (4.5) 5.00 s 5.26 d( 1.8) 3.27 d (4.5) 3.33 d (4.4) 3.32 d (4.7) a Coupling constants (Hz) are given in parentheses. * Observed due to overlap with other signals.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93

Table 4.2. 75.4 MHz 13C-NMR Spectral Data for Compounds 4.2-4.4c Performed in CDCha

Carbon 4.2 43a 43b 4.4a 4.4b 4.4c

1 46.8 d 46.6 d 46.5 d 43.8 d* 45.1 db 44.4 d

2 28.4 th 29.5 t* 25.5 t* 22.4 t 25.4 t 24.5 i*

3 31.4 tc 33.5 tc 32.2 tc 32.61 29.6 t 31.11

4 66.4 s 149.1 s 150.9 s 65.1 s 66.3 s 66.8 s

5 53.2 d 49.2 d 51.0 d 47.4 d* 52.8 d 52.8 d

6 81.8 d 82.0 d 84.1 d 81.1 d 81.1 d 81.6 d

7 45.8 d 44.9 d 44.7 d 44.5 d* 46.4 d* 47.4 d

8 29.8 tb 31.51* 28.81* 27.5 t 25.6 t 24.71*

9 33.1 tc 38.5 tc 33.4 tc 38.11 30.71 32.61

10 148.1 s 65.1 s 58.1 s 56.8 s 58.3 s 58.7 s

It 139.3 s 139.3 s 139.7 s 138.5 s 139.6 s 139.4 s

12 169.8 s 170.4 s 170.2 s 169.7 s 169.8 s 169.8 s

13 120.2 t 120.6 t 120.8 t 120.9 t 121.01 120.5 t

14 114.1 t 48.41 51.6 t 48.1 tc 50.3 tc 50.71

15 50.1 t 113.2 t 109.8 t 48.9 tc 54.6 tc 53.81

a Peak multiplicities were determined by DEPT experiments. b,c Data in same column are interchangeable.

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Table 4.3. Crystal Data and X-ray Collection Parameters of 4.1b, 4.4a, and 4.4c

compound ______4.1b ______4.4a ______4.4c

Formula C 1 5 H 1 8 O 3 C 15H18 O4 C 15H18 O4 FW 246.3 262.3 262.3 Crystal System Orthorhombic Orthorhombic Orthorhombic Space group P2i2i2i P2i2i2i P21212 1 Cell constants a, A 7.650(1) 6.7140(5) 8.3193(6) b, A 11.8057(7) 15.980(2) 10.1704(8) c, A 14.2153(6) 24.931(3) 15.919(3) V, A3 1283.8(4) 2674.9(9) 1346.9(5) z 4 8 4 Dc, g cm-3 1.274 1.303 1.293 ji(CuKa ), cm_1 6.7 7.3 7.3 Temp., °C 24 24 22 Crystal size, mm 0.11 x 0.13 x 0.50 0.18x0.38x0.60 0.15 x 0.27x0.55 0 limits, deg. 2-75 2-75 2-75 Octants collected ±h, k, ±1 h, k, 1 ±h, ±k, i l Unique data 2603 3224 2763 Observed data 2427 2509 2427 Criterion I>3a(I) I>3o(I) I>lo(I) Variables 236 344 245 R 0.031 0.091 0.045 Rw 0.040 0.107 0.049 Resid. density, e' 0.23 0.66 0.27 A-3 Extinction 3.8(3) x 10-6 1.7(2) x 10-6 2.7(1) x 10-6 Hydrogen atoms Refined iso Calculated Refined iso

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Table 4.4. Coordinates and Equivalent Isotropic Thermal Parameters for 4.1b

atom x V Bea'A Y

0-1 0.0798(1) 0.94141(7) 0.92791(7) 3.69(2)

0-2 0.2231(1) 1.09870(8) 0.96659(8) 4.89(2)

0-3 -0.0907(2) 0.51947(9) 0.8894(1) 6.45(3)

C-l -0.2437(2) 0.7666(1) 0.78196(9) 3.57(2)

C-2 -0.2973(2) 0.6747(1) 0.8548(2) 6.06(4)

C-3 -0.1310(2) 0.6345(1) 0.9026(1) 3.97(3)

0.0138(2) 0.70896(9) 0.86393(9) 3.24(2)

C-5 -0.0674(2) 0.81357(9) 0.82126(8) 2.94(2)

C-6 -0.0930(2) 0.90910(9) 0.89195(8) 2.91(2)

C-l -0.1695(2) 1.01729(9) 0.84877(9) 3.04(2)

C-8 -0.3672(2) 1.0285(1) 0.8588(1) 4.59(3)

C-9 -0.4654(2) 0.9187(1) 0.8368(1) 4.77(3)

C-10 -0.3871(2) 0.8494(1) 0.7588(1) 3.62(2)

C-l 1 -0.0640(2) 1.1091(1) 0.89462(8) 3.29(2)

C-12 0.0949(2) 1.0548(1) 0.93343(9) 3.54(2)

C-13 -0.0961(2) 1.2180(1) 0.9046(1) 4.38(3)

C-14 -0.4460(3) 0.8583(2) 0.6717(1) 5.79(4)

C-15 0.1812(2) 0.6811(1) 0.8619(1) 4.12(3)

° Beq — (8 7t^/3)Zi£jUijai aj ai*aj.

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Table 4.5. Coordinates and Equivalent Isotropic Thermal Parameters for 4.4a

atom X V r Bea (A*)0

0-1A 0.2190(6) 0.4585(3) 0.6081(1) 4.90(9) 0-2A 0.2238(8) 0.3525(3) 0.6657(2) 7.6(1) 0-3 A 0.3605(8) 0.6486(3) 0.5576(2) 6-7(1) 0-4A 0.3271(9) 0.5685(3) 0.3922(2) 7.1(1) C-l A 0.094(1) 0.5504(3) 0.4695(2) 4.6(1) C-2A 0.052(1) 0.6452(4) 0.4611(3) 6.9(2) C-3A 0.030(1) 0.6832(4) 0.51-55(3) 6.8(2) C-4A 0.155(1) 0.6263(4) 0.5512(2) 5.6(2) C-5A 0.1081(9) 0.5395(3) 0.5322(2) 4.0(1) C-6A 0.2498(9) 0.4712(3) 0.5502(2) 3.8(1) C-7A 0.2090(9) 0.3858(3) 0.5241(2) 4.1(1) C-8A 0.3402(9) 0.3713(4) 0.4751(2) 4.5(1) C-9A 0.278(1) 0.4257(4) 0.4279(2) 5.5(2) C-lOA 0.271(1) 0.5183(4) 0.4392(2) 4.5(1) C-l 1A 0.2355(9) 0.3269(4) 0.5693(3) 4.9(1) C-12A 0.2267(9) 0.3743(4) 0.6191(2) 5.3(1) C-13A 0.265(1) 0.2452(4) 0.5686(3) 7.2(2) C-14A 0.459(1) 0.5651(5) 0.4375(3) 7.0(2) C-l 5 A 0.229(1) 0.6496(4) 0.6047(3) 6.6(2) 0-1B 0.7720(7) 0.5216(3) 0.1331(1) 5.26(9) 0-2B 0.7719(8) 0.6165(4) 0.0675(2) 7.8(1) 0-3 B 0.9224(9) 0.3467(3) 0.1984(2) 7.8(1) 0-4B 0.838(1) 0.4558(4) 0.3558(2) 8.9(2) C-1B 0.621(1) 0.4560(4) 0.2752(2) 5.6(2) C-2B 0.595(2) 0.3635(5) 0.2893(3) 9.5(3) C-3B 0.591(1) 0.3129(5) 0.2402(3) 8.5(2) C-4B 0.713(1) 0.3648(4) 0.2018(2) 6.2(2) C-5B 0.6541(9) 0.4543(4) 0.2123(2) 4.4(1) C-6B 0.7904(9) 0.5206(4) 0.1919(2) 4.2(1) C-7B 0.7388(9) 0.6105(4) 0.2097(2) 4.3(1) C-8B 0.855(1) 0.6368(4) 0.2599(3) 5.8(2) C-9B 0.785(1) 0.5910(5) 0.3107(3) 6.9(2) C-l OB 0.787(1) 0.4988(5) 0.3064(2) 5-6(2) C-l IB 0.7772(9) 0.6588(4) 0.1601(3) 4.9(1) C-12B 0.7745(9) 0.6023(4) 0.1156(2) 5-5(1) C-13B 0.813(1) 0.7399(5) 0.1558(4) 8.7(2) C-14B 0.983(1) 0.4563(7) 0.3123(3) 8.6(2) C-15B 0.794(2) 0.3340(5) 0.1524(3) 8.3(2)

a Beq — (8 7r,-/3)!£jZjUjjaj aj &i*aj

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Table 4.6. Coordinates and Equivalent Isotropic Thermal Parameters for 4.4c

atom X V - B«(Azr

01 0.5714(2) 0.7098(1) 0.39907(9) 4.80(3)

02 0.7116(2) 0.8958(2) 0.3942(1) 7.04(4)

03 0.5560(2) 0.3325(2) 0.3468(1) 8.42(4)

04 -0.0431(2) 0.5139(2) 0.2787(1) 9.48(5)

Cl 0.2248(3) 0.4807(2) 0.3402(1) 5.54(5)

C2 0.2003(3) 0.3974(3) 0.4198(2) 7.53(6)

C3 0.3615(3) 0.3791(2) 0.4603(2) 6.49(6)

C4 0.4800(3) 0.4308(2) 0.3980(2) 5.58(5)

C5 0.3959(2) 0.5394(2) 0.3495(1) 4.35(4)

C6 0.4015(2) 0.6694(2) 0.3953(1) 3.75(3)

C l 0.3146(2) 0.7833(2) 0.3526(1) 4.07(3)

C8 0.1412(3) 0.8038(2) 0.3835(2) 5.25(4)

C9 0.0429(3) 0.6768(3) 0.3853(2) ' 6.40(6)

CIO 0.0917(3) 0.5773(2) 0.3197(2) 5.99(5)

C ll 0.4261(3) 0.8959(2) 0.3666(1) 4.46(4)

C12 0.5842(2) 0.8408(2) 0.3868(1) 4.86(4)

C13 0.3982(3) 1.0239(2) 0.3637(2) 6.55(6)

C14 0.0568(3) 0.6065(4) 0.2316(2) 8.23(8)

C15 0.6526(4) 0.4132(3) 0.4015(2) 8.58(7)

a Beq — (8 7r^/3)Xj£jUjjaj aj aj*aj

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.7. Minimum Inhibitory Concentrations of Compounds 4.1a-4.6 Against Mycobacterium tuberculosis

compound MIC (pg/ml)

4.1aa 2

4.1b >128

4.1c >128

4.2 32

4.3a 32

4.3b 64

4.4a 64

4.4b 128

4.4c 128

4.5 50

4.6* 128

a Lactones 4.1a and 4.6 were also tested against M. avium and gave MICs of 16 and 128 pg/ml, respectively.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99

0 O 4.1a R = R' = H 4.2 4.1b R = OH; R' = H 4.1c R = H; R' = OH

O O 4.3a R = P-epoxide 4.4a R[ = a-epoxide; R2 = P-epoxide 4.3b R = a-epoxide 4-4b Ri = a-epoxide; R2 = a-epoxide 4.4c R] = P-epoxide; R2 = a-epoxide

pA ng

HO OH

4.5 4.6 O Figure 4.1. Dehydrocostus lactone and derivatives

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100

tj u CD < \i

LJ o

c _ j m

CM LJ

T. co LJ Figure Figure 4.2. Molecular structures of compounds 4.1b, 4.4a, and 4.4c a LJ

m LJ

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101

■oa

‘IIIIIO

-in

rnas

c*\-O Figure 4.3 Figure 4.3 H-NMR 300 Mil? spectrum of 4a(l5)-epoxydehydrocostus 1 lactone (4.2) CDClj in

ao .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 102 Figure Figure 4.4. and broad band DEPT 75.4 nC-NMR MHz DEPT 90°, 135°, spectra ol'4a(15)-epoxydehydrocostus lactone (4.2)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 103 Figure Figure 4.5, 300 MHz 'H-NMR spectrum of I0p(14)-epoxydehydrocostus lactone (4.3a) in CDCIj

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission 104 Figure Figure 4.6. 75.4 MHz DliPT and DHPT broad 90°, band 135°, IJC-NMR spectra of 10p(14)-epoxydehydrocostus lactone (4.3a)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 107

■HIIIO

XIIIIM •MIIIX

cm oo

~co Figure Figure 4.9. 300 MHz 'H-NMR spectrum of4P(l5),IOa(l4)-diepoxydehydrocostus lactone (4.4a) CDCI? in

a.a>

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108 Figure Figure 4.10. 75.4 spectra DEPT MHz 90 of 4P(15),10a(14)-diepoxydehydrocostus , DEPT and , BB 1JC-NMR 135 lactone (4.4a)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 109

• UIIIO

XIIIII. (••'UIIIX

o cu

o cn

o T PPM

O in

o CD

O Figure Figure 4.11. 400 MHz 'll-NMR !4)-diepoxydehydrocostus 10u( lactone spectrum (4.4b) of 4a( CDCIi in 15),

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110

o c

O "IIIO y Xllli. T

u O oc m % o cu _ c n C/3 2 O o Tk3 . o _Co cn T3 X> . uc. •5

CL O X O ■ -C L X ' T CL ITi

a T c_ O £ 2 o k. 0 in u cr,a. A/ 1 z1 X o N X CO 2 o o T fN -T U o Im s r-~ M

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ill

Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934.

Connell, N. D.; Nikaido, H. In Tuberculosis: Pathogenesis, Protection, and Control, Bloom, B. R., Ed.; ASM Press: Washington D. C., 1994; pp 333-352.

Fronczek, F. R.; Vargas, D.; Fischer, N. H.; Hostettmann, K. J. Nat. Prod. 1984, 47, 1036.

Fronczek, F. R.; Vargas, D.; Parodi, F. J.; Fischer, N. H. Acta. Cryst. 1989, C45, 1829.

Inderleid, C. B.; Nash, K. A. In Antibiotics in Laboratory Medicine-, Lorian, V., Ed.; Williams and Wilkins: Baltimore, 1996; pp 127-175.

Inderleid, C. B.; Sal finger, M. In Manual o f Clinical Microbiology, Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., and Yolken, R. H., Eds.; ASM Press: Washington D. C., 1995; pp 1385-1404.

Kalsi, P. S.; Kaur, P.; Chhabra, B. R. Phytochemistry 1979, 18, 1877.

Kalsi, P. S.; Kaur, G.; Sharma, S.; Talwar, K. K. Phytochemistry 1984, 23, 2855.

Kalsi, P. S.; Sharma, S.; Kaur, G. Phytochemistry 1983, 2 2 , 1993.

Korp, J. D.; Bernal, I.; Fischer, N. H.; Leonard, C.; Lee, I. Y.; LeVan, N. J. Heterocvclic Chem. 1982, 19, 181.

Lu, T.; Fischer, N. H. Spectroscopy Lett. 1996, 29,437.

Romanuk, M.; Herout, V.; Sorm, F. Coll. Czech. Chem. Commun. 1956, 21, 894.

Sepkowitz, K. A.; Raffalli, J.; Riley, L.; Kiehn, T. E.; Armstrong, D. Clinical Microbiol. Rev. 1995, 8, 180.

Siddiqi, S. H. In Clinical Microbiology Procedures Handbook, Isenberg, H. D., Ed.; American Society for Microbiology: Washington, D. C., 1992; pp 5.14.2-5.14.25.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5

ANTIMYCOBACTERIAL SESQUITERPENE LACTONES FROM INULA HELENIUM AND RUDBECKIA SUBTOMENTOSA

112

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5.1 Introduction

In continuation of our search for new structural types of antimycobacterial natural

products, active root extracts of Inula helenium and Rudbeckia subtomentosa (Cantrell et

al., 1998) were chemically investigated. Native Americans (Iroquois, Cherokees, and

Mohegans) used infusions and decoctions o f I. helenium roots for the treatment of lung

disorders and against TB (Moerman, 1986). The crude dichloromethane extract of roots

of /. helenium gave 100 percent inhibition against M tuberculosis at 100 ng/ml.

Therefore, a detailed chemical investigation of its active fractions was performed. Since

a bioassay-guided fractionation of the crude extract of R subtomentosa had also

demonstrated a 99 percent inhibition against M tuberculosis at 100 pg/ml, the active

fractions were also chemically investigated for their constituents. The chemical studies

and their biological activities will be discussed below.

5.2 Results and Discussion

The results of radiorespiratory antituberculosis bioassays of crude extracts of /.

helenium and R. subtomentosa, using solvents of increasing polarity, are presented in

Table 5.1. The hexane and dichloromethane extracts of /. helenium roots demonstrated

100 percent inhibition at 100 pg/ml.

VLC chromatography of the dichloromethane root extract of I. helenium and

subsequent bioassay of the fractions against M. tuberculosis indicated that the nonpolar

fractions 2, 3, and 4 contain the biologically active constituents responsible for the high

active of the crude extract (Table 5.2). All three fractions gave 99 percent inhibitions at

33 pg/ml against M tuberculosis. Chemical investigation of these fractions resulted in

the isolation of the known eudesmanolides, alantolactone (5.1), isoalantolactone (5.2a),

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and 11, 13-dihydroisoalantolactone (53), as shown by spectroscopic comparison with

data previously reported for these three sesquiterpene lactones (Kashman et al., 1967;

Bohlmann et al., 1978; Milman, 1990; Marshal et al., 1964).

Bioassay of crude extracts of various plant parts of R. subtomentosa indicated that

root extracts were most active against M. tuberculosis with a 99 percent inhibition at 100

Hg/ml. VLC fractionation results of the root extract of R. subtomentosa are summarized

in Table 5.2. Further VLC separation of the most active fractions 2 and 3 provided the

known alloalantolactone (53a) and fraction 4 afforded the known 3-oxoalloalantolactone

(5.5b) as the major component and 5.5a as a minor constituent. Both compounds 5.5a

and 5.5b had been previously isolated from Eupatorium quadrangularae (Okunade et al.,

1985), but this is the first report of their isolation from R. subtomentosa. The structures

of 5.5a and 5.5b were determined by spectral comparison with previously reported data

(Bohlmann et al., 1978; Okunade et al., 1985).

The eudesmanoiides isolated from /. helenium and R. subtomentosa were tested

for their biological activities against M. tuberculosis. Lactones 5.1, 5.2a, and 5.5a had

MICs of 32 pg/ml while compounds 53 and 5.5b showed MICs of >128 pg/ml and 128

pg/ml, respectively (Table 5.4). Various eudesmanoiides of structures similar to those

isolated from /. helenium and R. subtomentosa were also evaluated for their in vitro

activities against M. tuberculosis. Encelin (5.2b) was the most active with an MIC of 16

pg/ml and l,2-dehydro-3-epi-isotelekin (53c) and isoalloalantolactone (5.4) showed

values of 32 jig/ml and 128 ng/ml, respectively (Table 5.4).

Therefore, a preliminary evaluation of structure activity relationships within this

series of biologically active compounds suggested that epoxidation of the C-5 double

permission of the copyright owner. Further reproduction prohibited without permission. 115

bond of 5.1 or the C-4(14) double bond of 5.2a may result in derivatives with lower

MICs. In our previous study of cycloartane-type triterpenes from Borrichm fruiescens

(Cantrell et al., 1996), epoxides were more active against M. tuberculosis than their

corresponding alkene analogs.

Epoxidation of 5.2a provided epoxide 5.6, which had been previously isolated

(Jakupovic et al., 1988) and synthesized (Chen et al., 1994). Its structure was determined

by spectral comparison, mainly of lH NMR values with those previously reported

(Jakupovic et al., 1988) and was shown to be identical in all respects.

Epoxidation of 5.1 gave epoxide 5.7, which was identified by 'H, 13C NMR,

including 90° and 135° DEPT methods and mass spectroscopy as well as comparison of

spectroscopic data with values previously reported for similar lactones (Bohlmann et al.,

1978; Marshal et al., 1964). The molecular structure of 5.7 was confirmed by single

crystal X-ray crystallography.

In an attempt to synthesize a 5,6-diol derivative, lactone 5.1 was reacted with

0 s04 Dihydroxylation occurred exclusively at the lactonic 11,13-double bond to provide

diol 5.8 which gave a MS parent peak at m z 266. The *H NMR spectrum indicated that

the H-6 doublet of 5.1 (8 5.11) was nearly unchanged in 5.8 (8 5.03, d) enabling us to

suggest that dihydroxylation had occurred at the 11,13- rather than at the 5,6- position.

Instead, the two diagnostic methylene lactone signals (H-13) were missing in 5.8. 'H

NMR values are reported in Table 5.3 and peak assignment of diol 5.8 were made by

spectroscopic comparison with data of structural analogs (Kashman et al., 1967;

Bohlmann et al., 1978; Milman, 1990; Marshal et al., 1964).

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Oxidative modifications by epoxidation of 5.1 produced a-epoxyalantolactone

(5.7), with a MIC of 8 pg/ml, a value significantly lower than observed for 5.1 with an

MIC of 32 pg/ml. In contrast, epoxide 5.6 gave an MIC of 32 pg/ml which is the same

value as that of its starting molecule (5.2a). This suggests that the 4(15) double bond in

5.2a may be less important for biological activity, which could only be confirmed with

further modifications. Oxidation of compound 5.1 to diol 5.8 resulted in complete loss of

biological activity. Compound 5.8 gave a MIC of >128 pg/ml in contrast to the MIC of

5.1 of 32 pg/ml. This strongly supports the necessity of the presence of the a, (3-

unsaturated lactone group, which is quite likely acting as a site of alkylation. Further

support for the necessity of the lactonic double bond is seen when observing the high

activity of compound 5.2a (32 pg/ml) as compared to the inactivity of its 11(13)

dihydroderivative 5 3 (>128 pg/ml).

5.3 Experimental

General Experimental Procedures. 'H- and l 3C-NMR spectra were recorded in

CDCI3 on a Bruker ARX 300 MHz spectrometer. Mass spectra were obtained on a

Hewlett-Packard 5971A GC-MS instrument. IR spectra were run on a Perkin-Elmer

1760X spectrometer as a film on KBr plates. Vacuum-liquid chromatographic (VLC)

separations were carried out on silica gel (MN Kieselgel) (Coll et al., 1986).

Plant material. Roots of I. helenium were collected on June 25, 1995 and

obtained from Mr. George Sturtz of Aromagen, 31787 Peoria Road, Albany, Oregon

97321, U.S.A.. A voucher (Sturtz-Fischer number 570) is deposited at the Louisiana

State University Herbarium. The crude plant extract of R. subtomentosa was obtained

from our repository, which we had previously chemically investigated (Vasquez et al,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 117

1990). The voucher is deposited at the Herbarium of Louisiana State University, U.S.A..

Cox No 4923.

Extraction and isolation. Root material (4.5 kg) of I. helenium was cut into

small pieces and dried at room temperature for two weeks. After soaking in hexane for

24 hours the solvent was drained from the plant residue and evaporated in vacuo to yield

15.16 g of crude extract. The remaining plant material was re-soaked for 24 hours in

CHiCL and subsequently for 24 hours in MeOH, yielding 23.3 g and 85.2 g, respectively.

The CH2CI2 extract was absorbed onto silica gel (15.2 g) and separated by VLC (Coll et

al., 1986) into 11 fractions using solvents of increasing polarity (hexane, ethyl acetate,

and MeOH) as summarized in Table 5.2. The most active fractions 2-4 were further

separated by repeated VLC over silica gel, essentially as described previously, to yield

pure compounds 5.1,5.2a, and S3 (Kashman et al., 1967; Bohlmann et al., 1978).

Previously obtained CH 2CI2 root extract (1.75 g) o f R. subtomentosa (Vasquez et

al., 1990) was adsorbed onto 1.6 g silica gel and placed onto a 2.3 cm i. d. VLC column

packed with 25 g of silica gel. The extract was separated into 8 fractions of increasing

polarity using hexane, EtOAc, and MeOH or mixtures thereof as listed in Table 5.2. The

most active fraction 2 (120 mg) was adsorbed onto 200 mg o f silica gel and placed onto a

1.3 cm i. d. VLC column containing 5 g of silica gel. Subsequent elution using 10 ml

fractions of hexane:EtOAc of increasing polarity afforded 17 mg of alloalantolactone

(5.5a). Fraction 3 (88 mg) was separated as described above for fraction 2 to afford 7 mg

of compound 5.5a. Fraction 4 (167 mg) was adsorbed onto 240 mg of silica gel and

placed onto a 1.3 cm i. d. VLC column that was packed with 6 g of silica gel. Elution of

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the column with 20 ml fractions of hexane:EtOAc of increasing polarity afforded 19 mg

of pure 5.5b.

Radiorespirometric Bioassays. Bioassays were performed essentially as

described previously (Cantrell et al., 1996; Inderleid et al., 1996; Inderleid et al., 1995;

Siddiqi et al., 1992). Experiments for M tuberculosis were usually completed within ten

days.

Epoxidations of 5.1. Lactone 5.1 (101 mg) was dissolved in 10 ml of CLLCL

and added to a solution of 10 ml of CH 2CI2 containing 1.2 equivalents mCPBA. Reaction

mixture was kept in an ice bath and stirred until TLC indicated all of 5.1 had reacted.

Reaction was stopped at 1.5 hours and washed with 10 ml of 10 % aqueous NaHCC >3 and

subsequently with 10 ml H 2O. Organic phase was dried with anhydrous MgS0 4 and

crude reaction products were separated by prep TLC (Si 0 2 ) using hexane/EtOAc (5:2) as

the mobile phase to yield 103 mg of 5.7. Lactone 5.2a was converted to epoxide 5.6

essentially as described previously (Jakupovic et al., 1988).

5 ,6a-Epoxyalantolactone (5.7). C 15H20O3 (Mr! 248) colorless crystals in

EtOAc; m. p. 164-166 °C; IR u max (KBr), 1741 (C=0) cm-1; EI-MS (70 eV): m = (%

rel. int.) = 248 (M+, 9), 233 (Nf-Me, 7), 204 (14), 192 ( 6 ), 159 ( 6), 149 (28), 133 (11),

126 (30), 123 (34), 109 ( 86 ), 95 (44), 81 (100), 67 (74), 55 (73); 'H-NMR and 13C-NMR

see Table 5.3.

Os04 oxidation of 5.1. To an ice-cooled solution of 134.9 mg o f a 50 % solution

of 4-methylmorpholine //-oxide in water was added a solution of 0.33 mg Os0 4 dissolved

in 0.33 ml r-BuOH. Lactone 5.1 (125.7 mg) dissolved in 3 ml of acetone was added and

the reaction mixture stirred for 18 hours (Renound-Grappin et al., 1994). A solution of

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aqueous 10 % NaHSOj (3 ml) was added and all solvents evaporated on rotary

evaporator. Crude mixture was absorbed onto silica gel and separated by VLC (2.3 cm i.

d. column) to provide fractions of increasing polarity using hexane and hexane:EtOAc

mixtures resulting in pure 5.8 (102 mg).

11, 13-Dihydroxyalantolactone (5.8). CisH^rOi (Mr: 266) colorless oil; ER u

max (KBr), 3388 (OH) cm*1; EI-MS (70 eV): m:(% rel. int.) = 266 (M+, 16), 251 (NT-

Me, 1), 235 (10), 221 (6), 215 (19), 207 (3), 191 (12), 162 (100), 147 (33), 133 (15), 119

(25), 105 (96), 91 (89), 77 (30), 67 (18), 55 (37); 1 H-NMR and I3C-NMR see Table 5.3.

Compounds 5.2b, 5.2c, and 5.4. Other sesquiterpene lactones used for bioassays

had been previously isolated in our laboratory. Isoalloalantolactone (5.4) had been

obtained from Rudbeckia mollis (Vasquez et al., 1992). Encelin (5.2b) and 1,2-dehydro-

3-epi-isotelekin (5.2c) (Geissman et al., 1968) were provided by Professor Leovigildo

Quijano, UNAM, Mexico City.

X-ray Crystallographic analysis of epoxide 5.7. A colorless fragment of

dimensions 0.62x0.35x0.35mm was used for data collection at T=100K on an Enraf-

Nonius CAD4 diffractometer equipped with MoKa radiation (>.=0.71073 A) and a

graphite monochromator. The temperature of the sample was maintained by an Oxford

Cryostream device. Crystal data are: C)5H20O3) Mr=248 .3, monoclinic space group

P2i, a=7.968(6), b=6.181(2), c=13.132(4) A, 0= 105.58(6)°, V=623.0(8) A3, Z=2,

dc=1.324 g cm-3. Intensity data were measured by co-20 scans of variable rate. Two

quadrants of data were collected within the limits 2<9<30°. Data reduction included

corrections for background, Lorentz, and polarization effects. 3938 intensities were

averaged to yield 1974 unique data (Rint=0.019), of which 1932 had I>0 and were used

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in the refinement. The structure was solved by direct methods using SER92 (Altomare et

al., 1994) and refined by fiill-matrix least squares, treating nonhydrogen atoms

anisotropically. Hydrogen atoms were located in difference maps and refined

isotropically. Convergence was achieved with R=0.031, Rw=0036, and GOF= 1.687.

The Enraf-Nonius MolEN programs (Fair, 1990) were used for all computations. The

structure is illustrated in Figure 5.1, and fractional coordinates are given in Table 5.5.

Table 5.1. Percent Inhibition of Crude Plant Extracts of Inula helenium and Rudbeckia subtomentosa Against Mycobacterium tuberculosis at 1000 and 100 pg/ml

species plant part extract" 1000 pg/ml 100 pg/ml o /. helenium roots o 100 H

roots «—*o o 100 D roots M 100 83

R. subtomentosa roots D 100 99

leaves D 96 43 i i stems D 70 ' - ! flowers D 95 30

* H=hexane; D=dichloromethane; Vl=methanol

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Table 5.2. Percent Inhibition of Inula helenium and Rudbeckia subtomentosa Fractions Against M. tuberculosis

fraction solvent percentages / helenium R. subtomentosa

(Hexane:EtOAc:MeOH)

^ -> I. heleniuma R. subtomentosa b 100 33 100

pg/ml Hg/ml Hg/ml lig/ml

1 100:0:0 100:0:0 100 94 79 43

2 95:5:0 95:5:0 100 99 100 97

«■* J 90:10:0 75:25:0 100 99 99 91

4 85:15:0 50:50:0 99 99 99 67

5 80:20:0 25:75:0 99 96 53 26

6 70:30:0 0:100:0 92 84 26 24

7 50:50:0 0:50:50 90 80 28 23

8 0:100:0 0:0:100 -20 -9 70 44

9 0:50:50 - -14 -10

10 0:50:50 - -3 10 _ -

11 0:0:100 - -19 -12 - -

a Using 6.5 cm i.d. column with 200 ml of solvent for each fraction. b Using 2.3 cm i.d. column with 100 ml of solvent for each fraction.

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Table 5.3. 'H NMR (300 MHz) and !3C NMR (75.4 MHz) Spectral Data of Compounds 5.7 and 5.8 (CDC13)a

atom 1 5.7 ! 5.8 ‘H NMR I3c n m r ‘H NMR I 13c n m r

1 37.71* 42.3 t* 2 16.5 t - 16.9 t 3a . 29.5 t 1.54 m 32.9 t 3p -- 1.54 m - 4 1.34 m 37.1 d** 2.47 m 38.6 d 5 - 67.5 s - 152.9 s 6P 2.89 d (2.6) 61.2 d 5.03 d (3.5) 112.5 d 7a 3.66 dddd (2.5, 37.4 d** 2.98 dd (3.5, 44.9 d 2.6, 2.9, 8.8) 5.6) 8a 4.66 ddd (1.9, 75.2 d 5.12 ddd (3.0. 78.0 d 4.5, 8.8) 3.0, 5.6) 9a 1.55 dd (1.9, 39.6 t * 1.54 dd (3.0, 42.6 t* 15.0) 15.0)

9P 1.87 dd (4.5, - 2.14 dd (3.0, - 15.0) 15.0) 10 - 32.6 s - 33.0 s 11 - 136.7 s - 77.4 s 12 - 169.7 s - 177.9 s 13a 5.77 d (2.5) 123.8 t 3.72 br d 64.2 t (12.0) 13b 6.39 d (2.9) - 3.93 d (12.0) - 14 1.10s 24.0 q 1.22 s 28.8 q 15 1.03 d (2.7) 18.1 q 1.12 d (7.6) 23.1 q 11-OH - - 3.77 brs - 13-OH - 2.93 br d (9.9) -

a Expressed as 8 values in ppm, with J values in Hz in parentheses. * Data in same column are interchangeable.

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Table 5.4. Minimum Inhibitory Concentrations of Compounds 5.1-5.8 Against A/. tuberculosis

compound MIC (pg/ml)

5.1 32

5.2a 32

5.2b 16

5.2c 32

5 J >128

5.4 128

5.5a 32

5.5b 128

5.6 32

5.7 8

5.8 >128

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Table 5.5. Coordinates and Equivalent Isotropic Thermal Parameters for 5.7 a i atom .r y - Bq q(A-) 01 0.2578(1) 0 0.52446(7) 1.32(2) 02 0.1467(1) -0.3326(2) 0.49271(7) 1.59(2) 03 0.4076(1) 0.2927(2) 0.83350(7) 1.35(2) Cl 0.7548(2) 0.2840(3) 0.75276(9) 1.22(2) C2 0.8863(2) 0.1986(3) 0.8518(1) 1.39(2) C3 0.8052(2) 0.1817(3) 0.9450(1) 1.40(2) C4 0.6416(2) 0.0379(3) 0.91.896(9) 1-16(2) C5 0.5173(2) 0.1137(2) 0.81574(9) 1.02(2) C6 0.3296(2) 0.0850(2) 0.79999(9) 1-14(2) C7 0.2001(2) 0.0807(2) 0.69209(9) 1.12(2) C8 0.2689(2) 0.1766(3) 0.60147(9) 116(2) C9 0.4526(2) 0.2687(2) 0.63338(9) 1.19(2) CIO 0.5876(2) 0.1449(2) 0.71898(9) 0.98(2) C ll 0.1473(2) -0.1459(2) 0.6558(1) 1.15(2) C12 0.1798(2) -0.1769(3) 0.5500(1) 1.21(2) C13 0.0856(2) -0.3044(3) 0.7032(1) 1.78(3) C14 0.6279(2) -0.0748(3) 0.67540(9) 1.20(2) C15 0.6857(2) -0.2046(3) 0.9212(1) 1.45(2) Hla 0.806(2) 0.287(3) 0.692(1) 1-1(3)* Hlb 0.720(2) 0.423(3) 0.768(1) 1-5(3)* H2a 0.989(2) 0.297(3) 0.869(1) 2.3(4)* H2b 0.936(2) 0.058(3) 0.838(1) 1.8(4)* H3a 0.777(2) 0.329(4) 0.964(1) 2.7(4)* H3b 0.896(2) 0.131(3) 1.008(1) 1-6(3)* H4 0.580(2) 0.061(3) 0.973(1) 0.6(3)* H6 0.290(2) 0.017(3) 0.853(1) 1.3(3)* H7 0.097(2) 0.157(3) 0.694(1) 1.3(3)* H8 0.184(2) 0.291(3) 0.562(1) 1.4(3)* H9a 0.442(2) 0.416(3) 0.659(1) 1.5(3)* H9b 0.495(2) 0.271(3) 0.570(1) 1.3(3)* H13a 0.051(2) -0.440(3) 0.665(1) 2.2(4)* H13b 0.060(2) -0.284(4) 0.772(1) 2.1(4)* H14a 0.643(2) -0.056(4) 0.604(1) 1.9(4)* H14b 0.529(2) -0.174(3) 0.667(1) 1.4(3)* H14c 0.730(2) -0.139(3) 0.722(1) 1.3(3)* HI 5a 0.775(2) -0.231(3) 0.886(1) 1.4(3)* HI 5b 0.578(2) -0.278(3) 0.887(1) 1.7(4)* HI 5c 0.732(2) -0.250(3) 0.993(1) 2.2(4)* a Figures in parentheses are ESD.

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5.5a R = H 5.1 5.5b R = 0=

5.2a R = H 5.6 5.2b R = 0=, A 1, 2-d.b. 5.2c R = p-OH, A 1, 2-d.b.

5.3 5.7

c h 2o h 5.4 5.8 Figure 5.1 Sesquiterpene lactones isolated from I. helenium and R. subtomentosa

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CD CD

CM ID Figure 5.2. Molecular structure of 5.7 at 100K CO

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m Figure 5.3. 300 MHz 'H-NMR spectrum of alantolactone (5.1) in C D C lj

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 128

. O OJ u u c

Xies -3 C5

PM o X ■OJ N

c~-

in 4>1_ BA3 is.

o -1C

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m O'c a

o •tiuix

m Figure 5.5. 300 MHz 'H-NMR spectrum of isoalantolactone (5.2a) in CDCI3

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oo 00 o

rr ••mix Figure 5.6. 300 MHz 1 H-NMR spectrum Figure spectrum H-NMR of dihydroisoalantolactone 5.6. (5.3) CDCIj in 300 MHz 1

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-co

_D rn Figure 5.7. 300 MHz 1 H-NMR spectrum Figure H-NMR spectrum of 3-oxoalloalantolactone 5.7. (5.5b) CDC1 in 300 MHz 1

a . a

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CO.

oo •m ix Figure Figure 5.8. 300 MHz 'H-NMR spectrum of a-epoxyisoalantolactone (5.6)in CDCh

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 ^ •> 1->J 3

00 o

m ilO 00 Figure Figure 5.9. 300 MHz 'H-NMR spectrum of u-epoxyalantolactone (5.7) CDCI in m

6 OLOl

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 134 Figure Figure 5.10. and broad band DEPT 62.5 nC-NMR DEPT MHz 90°, spectra 135°, of a-epoxyalantolactone (5.7)

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o 0 C Figure Figure 5.11. 300 MHz 'il-NMR spectrum of 11,13 dihydroxyalantolactone (5.8) CDCIj in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 136 Figure Figure 5.12. broad 75.4 MHz band spectrum |}C-NMR of dihydroxyalantolactone (5.8) 11, 13 CDCI in

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5.4 References

Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M. C.; Polidori, G.; and Camalli, M. J. Appl. Cryst. 1994,27,435.

Bohlmann, F.; Mahanta, P. K..; Jakupovic, J.; Rastogi, R. C.; Natu, A. A. Phvtochemistrv 1978, 77,1165.

Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams, L. B.; Franzblau, S. G. J. Nat. Prod. 1996, 59, 1131.

Cantrell, C. L.; Fischer, N. H.; Urbatsch, L.; Franzblau, S. G. Phytomedicine 1998, in press.

Chen, C.; Yang, L.; Lee, T. T.; Shen, Y.; Zhang, D.; Pan, D.; McPhail, A. T.; Liu, S.; Li, D.; Cheng, Y.; Lee, K. Bioorganic & Medicinal Chemistry 1994, 2, 137.

Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934.

Fair, C. K. MolEN. An Interactive System for Crystal Structure Analysis', Enraf-Nonius, Delft, The Netherlands, 1990.

Geissman, T. A.; Murkheijee, R. J. Organ. Chem. (U.S.A.) 1968, 33, 656.

Inderleid, C. B.; Nash, K. A In Antibiotics in Laboratory Medicine', Lorian, V., Ed.; Williams and Wilkins: Baltimore, 1996; pp 127-175.

Inderleid, C. B.; Salfinger, M. In Manual o f Clinical Microbiology, Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., and Yolken, R. H., Eds.; ASM Press: Washington D. C., 1995; pp 1385-1404.

Jakupovic, J.; Jaensch, M.; Bohlmann, F.; Dillon, M. O. Phytochemistry 1988, 27, 3551.

Kashman, Y.; Lavie, D.; and Glotter, E. Israel Journal o f Chemistry 1967, 5, 23.

Marshal, J. A.; Cohen, N. J. Org. Chem. 1964, 29, 12, 3727.

Milman, A. Khimiya Prirodnykh Soedinenu 1990, 3, 307.

Moerman, D. E. In Medicinal Plants o f Native America, vol II, Ann Arbor, Michigan, USA, 1986; p 642.

Okunade, A. L.; Wiemer, D. F. Phytochemistry 1985, 2 4 ,1199.

Renoud-Grappin, M.; Vanucci, C.; Lhommet, G. J. Org. Chem. 1994, 59, 3902.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 138

Siddiqi, S. H. In Clinical Microbiology Procedures Handbook; Isenberg, H. D., Ed.: American Society for Microbiology, Washington, D. C., 1992; pp 5.14.2-5.14.25.

Vasquez, M.; Quijano, L.; Fronczek, F. R.; Macias, F. A.; Urbatsch, L. E.; Cox, P. B.: Fischer, N. H. Phytochemistry 1990, 29, 561.

Vasquez, M.; Quijano, L.; Urbatsch, L. E.; Fischer, N. H. Phytochemistry 1992, 31, 2051.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6

TWO NEW ANTIMYCOBACTERIAL TRITERPENES FROM MELIA VOLKENSII

139

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6.1 Introduction

Melia volkensii Gurke (Meliaceae) is a subtropical tree which is found in dry

areas of East Africa. It has been reported that a tea prepared from the bark is used in

local folk medicine to alleviate pain and is poisonous in overdose (Kokwaro, 1976). It

has recently been shown that an extract of the seed kernels possess potent antifeedent

activity against the desert locust, Schistocerca gregaria (Nasseh et al., 1993; Mwangi,

1982). Fruit kernel extracts have also demonstrated growth-inhibiting activity against

larvae of the mosquitoes Aedes aegypti and Anopheles arabiensis (Mwangi et al., 1988a;

Mwangi et al., 1988b). Chemical investigations of the fruits have resulted in the

isolation of the insect antifeedants volkensin, salannin, 1-cinnamoyltrichilinin, 1-

tigloyltrichilinin, and 1-acetyltrichilinin (Rajab et al., 1988a; Rajab et al., 1988b). More

recent investigations of M volkensii have focused on the investigation leading to

compounds with activity against the human breast tumor cell line (MCF-7). These

chemical investigations resulted in the isolation of meliavolin, meliavolkin, meliavolen,

melianinone, 3-episapelin A, nimbolin B, meliavolkensins A and B, melianin A. and

meliavolkenin (Zeng et al., 1995a; Zeng et al., 1995b; Zeng et al., 1995c; Zeng et al.,

1995d).

Described below is the chemical investigation of the methanol extract of the

seeds of M volkensii, which resulted in the isolation of two new and one previously

isolated triterpenes. Their structure elucidation and spectroscopic characterization as

well as results of bioassays against Mycobacterium tuberculosis are presented.

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6.2 Results and Discussion

In a screening of crude plant extracts for biological activity against M

tuberculosis (Cantrell et al., 1998), the methanol extract of the seeds of Melia volkensii

was chosen for investigation because it demonstrated 99 percent inhibition against M

tuberculosis at 100 pg/ml. From this crude extract, the known kulonate (6.2) and the

new 12 [5-hydroxy kulac tone (6.1a) and 6 (5-hydroxy kulactone (6.1b) were isolated.

Kulonate (6.2) had been previously isolated from Melia azedarach (Chiang et al., 1973).

Compounds 6.1a and 6.1b are hydroxyl derivatives of kulactone (6.1c), which had also

been previously isolated from M. azedarach (Chiang et al., 1973; Ochi et al., 1977).

The crude methanol extract of the seeds of M. volkensii was fractionated using

vacuum liquid chromatography (VLC) into 10 fractions of increasing polarity as

indicated in Table 6.1. Each fraction was subsequently tested for its biological activity

against M. tuberculosis at 100 pg/ml and 33 pg/ml as summarised in Table 6.1.

Fractions 3, 4, and 5 were most active with percent inhibitions of 82, 96, and 95 at 33

pg/ml, respectively. This suggested that the active principles responsible for the high

inhibition of this crude extract are likely to be present in these three fractions.

Consequently, chemical investigation began with the most active fraction 4.

Multiple vacuum liquid chromatographic (VLC) investigations of fraction 4 (refer

to Experimental) resulted in the isolation of compound 6 . 1 a, 6.1b, and 6.2. The IR data

for 6.1a indicated the presence of hydroxyl(s) (3516 cm'1) and carbonyls (1781 cm'1,

1705 cm'1), the peak at 1781 cm'1 strongly suggesting the presence of a y-Iactone moiety.

FAB mass spectral data indicated a parent molecular ion at m z 469.2 corresponding to

[M+H]+. Inspection of the lH NMR spectral data (Table 6.2) for 6.1a indicated the

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presence of seven methyl singlets, two one-proton olefinic signals at 8 5.39 and 8 5.11,

and two deshielded one-proton signals at 8 4.19 and 5 4.03. The 'H-NMR data combined

with IR and mass spectral data suggested that 6.1a is structurally closely related to and is

most likely a hydroxylated derivative of kulactone (6.1c), a compound which had been

previously isolated from M azedarach (Chiang et al., 1973; Ochi et al., 1977). This was

supported by the presence of a proton absorption at 8 4.03 (dd, 7=4.6, 9.3) in 6.1a

corresponding to H-12, which was the only proton absorption not present in the spectrum

of 6 .1c.

The 13C-NMR experiments confirmed the presence of seven methyls, and

indicated that ketone (5 216.3 s), lactone (8 180.6 s. 8 82.3 d), and hydroxyl (8 72.2 d)

groups must be present. I 3C-NMR assignments were based on DEPT, HMQC, and

HMBC spectral data (Tables 6.2 and 6.3). The location of the hydroxyl group was

assigned by HMBC experiments (Table 6.2), which indicated the presence o f a long

range ( 3J) coupling between C-12 and H-18 in the HMBC experiment (Figure 6.1).

Additional spectral assignments and HMBC couplings are listed in Table 6.2 and are

summarized by arrows in Figure 6.1. An NOE correlation between H-12 and H-18

suggested that by analogy with 6.1c, the relative configuration of the hydroxyl group at

C-12 had to be p.

Further investigations of fraction 4 resulted in the isolation of compound 6.1b

(C30H44O4), which gave a parent peak of me 469.5 in the FABMS corresponding to

[M+H]+. IR spectral data indicated that all absorptions were very similar to those

observed for 6.1a with a hydroxyl absorption at 3504 cm'1. ‘H-NMR chemical shifts and

splitting patterns of 6.1b (Table 6.2) were very similar to those of 6.1a. However, instead

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of the resonance corresponding to H-12 (5 4.03 dd) in 6.1a a farther down field broad

singlet appeared at 8 4.49 in 6.1b. Examination of the COSY spectrum of 6.1b indicated

a strong coupling between the olefinic proton H-7 and the broad singlet at 5 4.49 which

suggested that 6.1a and 6.1b are constitutional isomers differing only in the position of

the hydroxyl group, which must be at C-6 in 6.1b. In addition to the COSY couplings

shown above, examination of HMQC and HMBC spectral data for 6.1b (Table 6.2)

confirmed that the hydroxyl group was located on C-6 due to a strong coupling between

H-5 (8 1.49 d) and C-6 (8 67.3), which had to be an oxygen-bearing carbon based on

chemical shift considerations. A strong coupling observed between H-6 and H-29 in the

NOESY spectrum revealed that the relative configuration of the OH group at C-6 had to

be |3. All ‘H-NMR and I3C-NMR spectral peak assignments were based on DEPT,

HMQC, and HMBC experiments and the data is summarised in Table 6.2 and Table 6.3,

respectively. A summary of HMBC correlations of 6.1b is presented in Figure 6.1.

Also isolated from fraction 4 was kulonate (6.2), which had been previously

obtained from M azedarach (Chiang et al., 1973) and represents the methyl ester

solvolysis product of kulactone (6.1c). Identification of 6.2 was done by comparison of

spectroscopic data (IR, lH-NMR, and l3C-NMR) with previously reported values (Chiang

et al., 1973).

All compounds isolated from M volkensii were evaluated for biological activity

against M. tuberculosis (H37Rv) using a radiorespirometric bioassay (Cantrell et al.,

1996; Inderleid et al., 1996; Inderleid et al., 1995; Siddiqi et al., 1992). Triterpenes 6.1a

and 6.2 showed MICs of 16 pg/ml, while 6.1b had an MIC of 4 pg/ml.

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6.3 Experimental

General Experimental Procedures. Mass spectra were obtained on a Hewlett-

Packard 5971A GC-MS or a TSQ70 FAB mass spectrometer. IR spectra were run on a

Perkin-Elmer 1760X spectrometer as a film on KBr plates. Vacuum-liquid

chromatographic (VLC) separations were carried out on TLC grade silica gel (MN

Kieselgel) (Coll et al., 1986). lH- and l3C-NMR spectra were recorded in CDC1? on

either a Bruker AM 400 MHz spectrometer or a Bruker ARX 300 MHz spectrometer. 2D

NMR were recorded using a Bruker ARX 300 MHz spectrometer utilising Bruker’s

standard pulse programs. In the Heteronuclear Multiple Bond Correlation (HMBC)

experiments (Bax et al., 1986) the following parameters were used: relaxation delay of 1

s, 60 ms delay for evolution of long-range couplings, coupling constant of 145 Hz. In the

Heteronuclear Multiple Quantum Coherence (HMQC) experiments (Bax et al., 1983) a

relaxation delay of 1 s was used with a coupling constant of 145 Hz. In the NOESY

experiments, a relaxation delay of 2 s was used with a mixing time of 0.8 s. In HMBC

and HMQC experiments, 2048 data points were collected in t2, with 256 FED’s in ti.

Typical sweep widths were 6 ppm in F2 and 220 ppm in FI. The spectra were

transformed into a 2048 x 512 matrix by zero filling.

Plant Material. Seeds of M volkensii were obtained from ripening fruits

collected in November 1995 in Voi, Kenya. A voucher specimen (Voucher No.

MU/BOT/75M) has been deposited at the Department of Botany Herbarium, Moi

University, Kenya.

Extraction and Isolation. Crushed seeds (1 kg) were allowed to stand in 2 L of

MeOH for 1 week. The extract was decanted and the residual pulp re-extracted under

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similar conditions. The combined extracts were evaporated under vacuum to yield 18.6 g

of a thick brown oil. Part of the of the crude MeOH extract (8.0 g) was adsorbed onto 8.0

g of silica gel and packed onto a 6.5 cm i. d. column containing 160.0 g o f silica gel. The

crude extract was separated into 10 fractions using an increasing polarity gradient of

hexane, EtOAc, acetone, and methanol as summarized in Table 6.1. After evaluation of

each fraction for its antimycobacterial activity, the most active fraction 4 was chosen for

further chromatographic investigation.

Fraction 4 (410 mg) was adsorbed on 360 mg of reverse phase C-l 8 silica gel (40-

63 pm) and placed onto a VLC column (2.3 cm in diameter) packed with 8.1 g of C-l 8

silica gel and chromatographed using 20x50 ml H 20:MeOH mixtures of decreasing

polarity. 61 fractions (20 ml each) being collected. Subfractions 46-50 (88 mg) were

combined as well as subfractions 51-54 (53 mg) due to similar TLC and NMR patterns

and each were further separated.

The mixture of compounds in subfractions 46-50 were adsorbed onto 112 mg of

silica gel and placed onto a VLC column (2.3 cm i. d.) packed with 7.5 g o f silica gel and

chromatographed using 18x50 ml hexanerEtOAc mixtures of increasing polarity, 58

fractions (20 ml) being collected. Subfractions 19-22 contained 8 mg of pure 6.1a and

fractions 23-27 provided 12 mg of 6.1b.

The mixture of compounds in subfractions 51-54 were separated in a manner

similar to that described above for subfractions 46-50, providing 69 fractions (20 ml

each) with fractions 27-29 providing 6 mg of pure 6.2.

12P-hydroxykulactone (6.1a). colorless oil; C 30H44O4; mol wt 468; IR u max

(KBr), 3516 (OH), 2979, 1781 (y-lactone), 1705 (ketone), 1452, 1365 cm-l; 1 H-NMR

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spectral data see Table 6.2; 13C-NMR spectral data see Table 6.3; EIMS (70 eV) m z 444

(5), 296 (9), 148 (72), 120(28), 107 (100), 91 (17), 77 (11), 65 (6), 55 (6); FABMS m z

[M -H f 469.2, [469-H20]~ 451.2.

6f3-hydroxykuiactooe (6.1b). colorless oil; C 30H44O4; mol wt 468; IR u max

(KBr), 3504 (OH), 1781 (y-lactone), 1705 (ketone), 1452, 1365 cm'1; 1H-NMR spectral

data see Table 6.2; 13C-NMR spectral data see Table 6.3; EIMS (70 eV) m z [M-HiO]^

444(11), 393 (4), 227(28), 185 (18), 171 (16), 157(16), 145 (18), 119(18), 107 (28), 91

(26), 81 (26), 69 (100), 55 (54); FABMS m z [M +H f 469.5, [469-H20 f 451.2.

Radiorespirometric Bioassays. Bioassays were performed essentially as

described previously (Cantrell et al., 1996; Inderleid et al., 1996; Inderleid et al., 1995;

Siddiqi et al., 1992) using BACTEC radiorespirometry. Experiments were usually

completed within 10 days.

Table 6.1. Inhibitory Activity of Fractions of a Crude Methanol Extract of Fruits of

Melia volkensii Against Mycobacterium tuberculosis (H3 7 RV)

fraction 3 % hexane % EtOAc % acetone % MeOH percent inhibition 100 pg/ml 33 pg/ml

1 100 0 0 0 2 95 5 0 0 -- j 80 20 0 0 95 82 4 50 50 0 0 97 96 5 20 80 0 0 97 95 6 0 100 0 0 57 0 7 0 50 50 0 0 0 8 0 0 100 0 0 0 9 0 0 50 50 0 0 10 0 0 0 100 0 0

“200 ml of solvent.

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Table 6.2. 'H-NMR (400 MHz, CDCl,) and HMBC (300 MHz, CDC13) Spectral Data of Compounds 6.1a and 6.1b

6.1a 6.1b proton 5h multi, J (Hz)a HMBC 8 h multi, J (Hz)a HMBC 1 b 1.97 m - - -

~ P 2.78 ddd 1,3 2.86 ddd 1,3 (5.5, 14.6, 14.6) (5.5,14.1, 14.1) 5 1.72 m 6, 9, 10, 19, 28, 1.49 d 4,6, 10 29 (4.4)

6 - - 4.49 brs -

7 5.39 brd - 5.46 dd - (2.4) (3.5, 3.5)

12 a 4.03 dd * - ** (4.6, 9.3) 15a 1.75 m 13, 14, 16, 30 1.76 dd 13, 14, 16, 30 (7.2, 13.6)

15b ” - 2.34 dd - (10.2, 13.6) 16 a 4.19 ddd 20 4.15 ddd - (7.3, 10.4, 12.0) (7.2, 10.2, 12.0) 17 P 2.53 dd 14, 16, 18,20 2.14 dd 13, 16,18 (12.0, 12.0) (12.0, 12.0) 18 0.83 s 12, 13, 14, 17 0.91 s 12, 13, 14, 17

19 1.04 s 1,5, 9, 10 1.26 s 1,5, 9, 10

23 2.18 m 20, 22, 24, 25 - -

24 5.11 dddd - 5.10 dddd - (1.1, 1.1, 7.2, (1.3, 1.3, 6.9, 7.2) 6.9) 26 1.69 s 24, 25, 27 1.69 s 24, 25, 27

27 1.63 s 24, 25, 26 1.62 s 24, 25, 26

28 1.13s 3,4, 5,29 1.52 s 3,4, 5, 29

29 1.05 s 3, 4, 5, 28 1.23 s 3, 4, 5, 28

30 1.39 s 8, 13, 14,15 1.31 s 8, 13, 14, 15 a Expressed as 8 values in ppm, with J values in Hz in parentheses.

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Table 6.3. ,3C-NMR Spectral Data of Compounds 6.1a and 6.1b (75.4 MHz. CDCI 3)"

Compound b carbon 6.1a 6.1b

C-l 38.6 t 40.2 t C-2 34.91 34.9 t C-3 216.3 s 216.0 s C-4 48.1 s 49.2 s C-5 52.6 d 56.9 d C-6 24.61 67.3 d C -l 119.4 d 122.2 d C-8 143.2 s 146.3 s C-9 48.2 d 49.0 d C-10 35.5 s 35.7 s C-l 1 30.3 t 17.01 * C-12 72.2 d 29.5 t C-13 55.2 s *** 39.6 s C-14 44.9 s *** 55.4 s C-l 5 36.5 t 35.71 C-16 82.3 d 82.4 d C-l 7 53.5 d 58.3 d C-l 8 20.1 q 21.6 q C-19 12.8 q 15.3 q C-20 45.7 d 45.6 d C-21 180.6 s 180.6 s C-22 29.3 t 29.71* C-23 26.2 t 26.3 t * C-24 123.8 d 123.5 d C-25 133.0 s 133.0 s C-26 26.0 q * 25.9 q C-27 18.1 q* 18.1 q C-28 21.6 q** 24.9 q C-29 24.6 q ** 24.1 q C-30 34.0 q 31.6 q

a Peak multiplicities were determined by heteronuclear multipulse programs (DEPT); s = singlet, d = doublet, t = triplet, q = quartet. b Interchangeable peaks are represented by *, **, or ***.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 149

6.1a R,=OH; R2=H 6.1b R[=H; R2=OH 6.1c R,=H; R2=H

OCH ,OH

6.2

Figure 6.1. Triterpenes isolated from M. volkensii

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6.1a

6.1b

Figure 6.2. ‘H-I3C HMBC correlations for 6.1a and 6.1b

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33 Figure Figure 6.3. 3 z *H-NMR spectrum of 12-fl-liydroxykulactone (6.1a) CDCIj in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 152

^_ cuin

Hoo

tn f— cu

-tno

-r*»tn

o • o CM o ro On H H CL a* m Figure 6.4. 75.4 MHz DEPT DEPT 90°, and broad band 135°, nC-NMR spectra of 12-f3-hydroxykulactone (6.1a) Q aCL

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 153

° ! t 9 t

@ B

I o m

as m a — 6.5. 6.5. 300 MHz 'H-'H COSY spectrum of !2-(}-hydroxykulactone (6.1a) Figure Figure

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 154 . . 300 MHz ‘H-nC correlation (HMQC) spectrum of 12-(l-hydroxykuIactone (6.1a) 6 . 6 Figure Figure

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 155

rr^:::= j- • ; r . r ' ... . . 1 : 3 -L - > • T #»T • p » 4- (6.1a) ' S. t

. :t

I )■ i I- ! P nr j i MHz MHz ’H-n C correlation (HMBC) spectrum of 12-()-hydroxykulactone Figure Figure 6.7. 300 o

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u i

iL -«n

T^vywii(rvywnr-^r “vm r " V r

/° Figure6.8. 300 MHz 'll-’ll spectrum of NOHSY (6.1a) 12-(l-hydroxykulactone _lll*»

•mux

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 157

r'lO'

c m fNo -(}-hydroxykulactone -(}-hydroxykulactone (6.1b) CDClj in 6

-in

(-mill Figure Figure 6.9. 300 MlIz 'H-NMR spectrum of

. Q. K a.

« $ e * a L o — CM i-

< Figure Figure 6.11. 300 MHz 'H-'H COSY spectrum of 6-|Chydroxykulactone (6.1b)

; i i ! . c c • a o ► • • O ’ • o ’ •

• i 1 7 » • : : * O > « 0 « Hr { i t 1

i ! i »i • ! i |

i ■ f . 1

■ nr m 'rp rm -T ir ~T ~--1 n '

O Figure 6.13. 300 MHz 'H-13C Figure 6.13. (6.1b) correlation (HMBC) spectrum of 6-(}-hydroxykulactone 300 MHz 'H-13C

o •mix

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 162 Figure Figure 6.14. 300 MHz ‘H-'H NOESY spectrum of6-p-hydroxykulactone (6.1b)

MIIIX

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6.4 References

Bax, A.; Griffey, R. H.; Hawkins, B. L. J. Magn. Reson. 1983, 55, 301.

Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093.

Cantrell, C. L.; Fischer, N. H.; Urbatsch, L.; Franzblau, S. G. Phytomedicine, 1998, in press.

Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams, L. B.; Franzblau, S. G. J. Nat. Prod. 1996 ,59, 1131.

Chiang, C.; Chang, F. C. Tetrahedron 1973, 29, 1911.

Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934.

Inderleid, C. B.; Nash, K. A In Antibiotics in Laboratory Medicine', Lorian, V., Ed.; Williams and Wilkins, Baltimore, 1996; pp 127-175.

Inderleid, C. B.; Salfinger, M. In Manual o f Clinical Microbiology-, Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., and Yolken, R. H., Eds.; ASM Press, Washington D. C., 1995; pp 1385-1404.

Kokwaro, J. O. Medicinal Plants o f East Africa', East African Literature Bureau: Nairobi, Kenya, 1976, p. 157.

Mwangi, R. W. Entomol. Exp. Appl. 1982, 32, 277.

Mwangi, R. W.; Mukiama, T. K. J. Am. Mosq. Control Assoc. 1988a, 4, 442.

Mwangi, R. W.; Rembold, H. Entomol. Exp. Appl. 1988b, 46, 103.

Nasseh, O.; Wilps, H.; Rembold, H.; Krall, S. J. Appl. Entomol. 1993, 116, 1.

Ochi, M.; Kotsuki, H.; Tokoroyama, T.; Kubota, T. Bull. Chem. Soc. Jpn. 1977, 50, 2499.

Rajab, M. S.; Bentley, M. D. J. Nat. Prod. 1988a, 51, 840.

Rajab, M. S.; Bentley, M. D.; Alford, A. R.; Mendel, M. J. J. Nat. Prod. 1988b, 51, 168.

Siddiqi, S. H. In Clinical Microbiology Procedures Handbook, Isenberg, H. D., Ed.; American Society for Microbiology, Washington, D. C„ 1992; pp 5.14.2-5.14.25.

Zeng, L.; Gu, Z.; Chang, C.; Smith, D. L.; McLaughlin, J. L. Bioorg. Med. Chem. Lett. 1995a, 5, 181

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 164

Zeng, L.; Gu, Z.; Chang, C.; Wood, K. V.; McLaughlin, J. L. Bioorg. Med. Chem. Lett. 1995b, 3, 383.

Zeng, L.; Gu, Z.; Fang, X.; Fanwick, P. E.; Chang, C.; Smith, D. L.; McLaughlin, J. L. Heterocycles 1995c, 41, 741.

Zeng, L.; Gu, Z.; Fang, X.; Fanwick, P. E.; Chang, C.; Smith, D. L.; McLaughlin, J. L. Tetrahedron 1995d, 51, 2477.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER7

SUMMARY AND CONCLUSIONS

165

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Two-hundred and thirty crude extracts from 118 plant species distributed among

ten families were evaluated for antimycobacterial activity, which was determined against

Mycobacterium tuberculosis (H37R.V) and M. avium, using the BACTEC 460

radiorespirometric assay. At 100 Mg/ml, twenty-four and ten of the extracts, respectively,

caused more than 95% inhibition of growth of M. tuberculosis and M. avium. The most

active plant extracts with 100% inhibition were obtained from Borrichia frutescens,

Solidago arguta, and Inula helenium against M. tuberculosis. Euthamia leptocephala

was active against M. avium, and Erigeron strigosus and Magnolia acuminata were

active against both mycobacteria. Extracts possessing high biological activities were

chemically investigated in an effort to isolate the active principles.

The sea daisy (Borrichia frutescens, Asteraceae) collected from coastal marshes

of Louisiana, was chemically investigated for its active constituents due to the high

activity of its crude extracts against M. tuberculosis. Bioactive chromatographic

fractions provided two new triterpenes, (24/?)-24,25-epoxycycloartan-3-one and (23/?)-3-

oxolanosta-8,24-dien-23-ol, and (24/?)-24,25-epoxvcycloartan-3£-ol, which had been

previously isolated as a mixture of C-24 epimers. The structures of these three

compounds were established by spectroscopic methods and chemical transformations and

the molecular structures of (24/?)-24,25-epoxycycIoartan-3-one and (23/?)-3-oxolanosta-

8,24-dien-23-ol were determined by single-crystal X-ray diffraction. In a

radiorespirometric bioassay against M. tuberculosis, the epoxycycloartanes (24/?)-24,25-

epoxycycloartan-3-one and (24/?)-24,25-epoxycycloartan-3p-ol exhibited minimum

inhibitory concentrations of 8 pg/ml. In contrast, the lanostadiene-type triterpene (23/?)-

3-oxolanosta-8,24-dien-23-ol showed no significant inhibition at 128 pg/ml, as did the

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acetate of (24/?)-24,25-epoxycycloartan-3(3-ol. Correlations of structural features and the

MICs of the four triterpenes suggest that the presence of the C-3 keto and/or (3-hydroxy

group, the cyclopropane ring and the epoxide moieties as in (24/? >-24.25-

epoxycycloartan-3-one and (24/?)-24,25-epoxycycloartan-3|3-ol seem to play a major role

in the in vitro antituberculosis activity. Both the cyclopropane and epoxide functions are

absent in triterpene (23/?)-3-oxolanosta-8,24-dien-23-ol, resulting in its loss of activity

(MIC 64-128 pg/ml). Also, the loss of activity by introduction of a C-3 acetoxv group

(MIC >128 pg/ml) strongly suggests that either a free hydroxyl or a keto group at C-3 in

(24/?)-24,25-epoxycycloartan-3-one or (24/?)-24,25-epoxycycloartan-3(3-oI is required for

significant activity. Cytotoxicity for Vero cells gave IC 50 values of 71.8 pg/ml, 39.8

pg/ml, and 103.6 pg/ml for triterpenes (24/?)-24,25-epoxycycloartan-3-one, (24/?)-24,25-

epoxycycloartan-3(3-ol and (23/?)-3-oxolanosta-8,24-dien-23-ol, respectively.

In an attempt to study the structural dependence of antimycobacterial activity of

the guaianolide dehydrocostus lactone and its derivatives, and to determine

configurational and conformational effects on activity, m-chloroperoxybenzoic acid

oxidations of dehydrocostus lactone were performed. Three new monoepoxides, one

previously synthesized diepoxide, and two new diepoxides were obtained. Two of the

monoepoxides are C-10 epimers, while the 4(15)-monoepoxide has the 4 a-0 -

configuration. The known 4(15),10(14)-diepoxide contains an C-10-a-epoxide and a 13-

epoxide at C-4. Both new diepoxides contain a C-4 a-epoxy group and differ in the

configuration of the epoxide ring at C-10. Allylic oxidation of dehydrocostus lactone

with selenium dioxide//-butyl hydroperoxide afforded the known 3-epizaluzanin C. The

relative configurations of the above compounds were established by one- and two

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dimensional NMR techniques (‘H, i3C, COSY, NOESY, HMQC, and HMBC) as well as

comparison with literature data. The molecular structures of 3-epizaluzanin C, the

4P(15),10a(14)-diepoxide, and the 4a(15),10P(14)-diepoxide were determined by single

crystal X-ray diffraction.

In radiorespirometric bioassays against Mycobacterium tuberculosis and M.

avium, dehydrocostus lactone exhibited MICs of 2 and 16 pg/ml, respectively. In

contrast, its monoepoxides and diepoxides as well as its hydrogenated derivatives and

other analogues (3-epizaluzanin C, 7a-hydroxydehydrocostus lactone, micheliolide, and

pumilin) showed significantly lower activities against M. tuberculosis. The above data

allowed us to suggest that the antimycobacterial activity of the tested guaianolides is not

as strongly determined by the exocyclic methylene lactone moiety, as by polarity factors.

Activity is most dramatically reduced when at least one hydroxyl group is present in the

molecule (3-epizaluzanin C, 7a-hydroxydehydrocostus lactone, micheliolide, and

pumilin). The successive replacement of double bonds with epoxide groups also caused a

significant loss of activity with the more polar diepoxides being less active than the

monoepoxide analogues. As contrasted by the activity of the 4P(15),10a(14)-diepoxide

and the 4a( 15), 10P( 14)-diepoxide, configurational and conformational influences seem

to exist but appear to be of lesser importance. Since the most lipophilic guaianolide

dehydrocostus lactone gives the highest antimycobacterial activity, it can be concluded

that activity is most strongly influenced by the polarity of a given molecule. Therefore,

within this structurally related group of sesquiterpene lactones, antimycobacterial activity

is most likely controlled by the transport through the outer lipid layer of mycobacteria.

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The root extracts of Inula helenium [Elecampane] (Asteraceae) and Rudbeckia

subtomentosa [Sweet Coneflower] (Asteraceae) were chemically investigated for their

active constituents. Chromatographic fractions of root extracts of I. helenium, which

exhibited significant activities against M. tuberculosis, provided the known

eudesmanolides alantolactone, isoalantolactone, and llaH , 13-dihydroisoalantolactone,

which had been previously isolated from the roots of /. helenium. Active fractions from

R. subtomentosa gave the known alloalantolactone and 3-oxoalloalantolactone. The

structures of the above eudesmanolides were established by spectroscopic methods

including ID and 2D NMR techniques as well as spectral comparison of previously

reported data. Peracid epoxidation of alantolactone and isoalantolactone gave the

derivatives a-epoxyalantolactone and a-epoxyisoalantolactone, respectively. Oxidation

of alantolactone with OSO 4 provided 11,13 dihydroxyalantolactone.

All natural and semisynthetic compounds were tested for biological activity

against M tuberculosis and demonstrated MICs ranging from 8 to >128 pg/ml. The

sesquiterpene lactones alantolactone, isoalantolactone, 11a, 13-dihydroisoalantolactone,

alloalantolactone, and 3-oxoalloalantolactone gave MICs of 32, 32, >128, 32, and 128

pg/ml, respectively. Oxidative modifications by epoxidation of alantolactone produced

5,6a-epoxyalantolactone, with a MIC of 8 jig/ml, a value significantly lower than

observed for alantolactone with an MIC of 32 jig/ml. In contrast, 4,15a-

epoxyisoalantolactone gave an MIC of 32 pg/ml. This value, which is the same as the

starting isoalantolactone, leads us to suggests that the 4(15) double bond in

isoalantolactone may be less important for biological activity, which could only be

confirmed with further modifications. Oxidation of alantolactone to 11, 13-

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dihvdroxyalantolactone resulted in complete loss of biological activity with an MIC of

>128 fig/ml. This strongly supports the necessity of the presence of the a, (3-unsaturated

lactone group, which is quite likely acting as an alkylation moiety. Further support for

the necessity of the a-methylene-y-lactone group is expressed in the high activity of

isoalantolactone (32 pg/ml), when compared to the nonactive 1 l(l3)-dihydroderivative

(>128 fig/ml).

Methanol extracts of the fruits of Meha volkensii provided two new and one

known triterpenoids, which were shown to be triterpenes of the euphane (20 R) series.

The new 12(3-hydroxykulactone and 6P-hydroxykulactone were shown to be hydroxyl

derivatives of kuiactone. The structures of 12P-hydroxykulactone and 6p-

hydroxykulactone were elucidated by ID and 2D NMR (l3C, lH, 'H-'H COSY, HMQC,

HMBC, and NOESY spectra) and FABMS studies. Also isolated was kulonate, which

had been previously obtained from Melia azedarach together with kuiactone. All three

compounds were evaluated for their inhibitory activity against M tuberculosis and were

shown to have potent MICs ranging from 4 pg/ml to 16 fig/m 1. 12P-Hydroxykuiactone

and kulonate showed MICs of 16 pg/ml, while 6P-hydroxykulactone had an MIC of 4

pg/ml suggesting that the presence of a hydroxyl group at C-6 in 6P-hydroxykulactone

significantly enhances biological activity.

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From: Norman R. Farnsworth To: Charles. L. Cantrell Date: Tuesday, February 24, 1998 4:34 PM Subject: Re: need permission to use published material within my dissertation

Sorry to be late in answering your previous messages. I have been ill at home for three weeks with severe sciatica and am still suffering but am trying to get through the hundred or so e-mails that have accumulated. By means of this message you have permission to utilize all of the data in your Phytomedicine article that will appear in Volume 5, issue 1, in your Ph.D. dissertation. If a signed letter is necessary, please let me know and I will type one up and send it. Apologies for the delay.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA

Charles Lowell Cantrell was bom on July 15, 1971 in Baton Rouge, Louisiana,

the son of Robert and Deborah Cantrell. He attended Oak Park Elementary in Lake

Charles, Louisiana, and Broadmoor Junior High School in Baton Rouge, Louisiana, to

complete his primary education in 1985. Then, he attended Walker High School in

Walker, Louisiana, and graduated in 1989.

His next three years were spent at Southeastern Louisiana University in

Hammond, Louisiana, pursuing a degree in Zoology. He transferred to Louisiana State

University in 1992 to complete his undergraduate education and graduated in 1994 with a

bachelor of science degree in Zoology and Physiology and a minor in Chemistry.

He began graduate school at Louisiana State University in Baton Rouge,

Louisiana, in the fall of 1994. He is currently a candidate for the degree of Doctor of

Philosophy in the Department of Chemistry, and his area of specialization is Organic

Chemistry. His research area, working under the direction of Dr. Nikolaus H. Fischer, is

in the field of Pharmacognosy with special emphasis on the isolation of plant natural

products possessing antituberculosis activity.

He is a member of the American Chemical Society, Phi Lambda Upsilon

(National Honorary Chemical Society), American Society of Pharmacognosy,

Phytochemical Society of North America, and the American Association for the

Advancement of Science.

173

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candidater Charles L. Cantrell

Major Field: Chemistry

Title of Dissertation: Antimycobacterial Natural Products from Higher Plants

Approved:

Maj Professor and Chai

Dean of the Graduate SctooT

EXAMINING COMMITTEE:

J . C | ca_X -JL _

Date of Bxamination:

18 March 1998

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