CHARACTERIZATION of BIOACTIVE PHYTOCHEMICALS from the STEM BARK of AFRICAN MAHOGANY Khaya Senegalensis (MELIACEAE) Huaping Zhang Clemson University, Zhahp@Hotmail.Com

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12-2008 CHARACTERIZATION OF BIOACTIVE PHYTOCHEMICALS FROM THE STEM BARK OF AFRICAN MAHOGANY Khaya senegalensis (MELIACEAE) Huaping Zhang Clemson University, [email protected]

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CHARACTERIZATION OF BIOACTIVE PHYTOCHEMICALS FROM THE STEM BARK OF AFRICAN MAHOGANY Khaya senegalensis (MELIACEAE)

A Dissertation Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Food Technology

by Huaping Zhang December 2008

Accetped by: Dr. Feng Chen, Committee Chair Dr. Alex Kitaygorodskiy Dr. R. Kenneth Marcus Dr. Xi Wang

i ABSTRACT

African mahogany Khaya senegalensis (Desr.) A. Juss (Meliaceae) is a large tree growing mainly in the sub-Saharan savannah forests from Senegal to Uganda. This plant is one of the most popular medicinal meliaceous plants in traditional African remedies, used as a bitter tonic, folk and popular medicine against malaria, fever, mucous diarrhea, and venereal diseases as well as an anthelmintic and a taeniacide remedy. Its extracts and chemical constituents have been the subject of extensive phytochemical and pharmacological investigations since 1960s. Anti-inflammatory, anti-bacterial, anthelmintic, and antiplasmodial activities of plant extracts have been reported.

Limonoids with anti-feeding, feeding deterrent and growth inhibitory properties, antifungal, and anti-sickling activity, as well as dimeric flavonoids with immunostimulating activity have been isolated from different parts of this plant. However, none of extracts or pure chemicals has been screened for biological activity such as antioxidant activity and anti-proliferative capacity against human cancer cell lines. Therefore, the main object of this study was to screen bioactive ingredients with anticancer and antioxidant activities from the plant through purification, isolation, structural elucidation, and bioassays.

Eleven natural products were isolated from the methanolic extract of stem bark of

Khaya senegalensis after extraction and purification, especially through crystallization and modern column chromatographic techniques using normal phase and reverse phase silica gel columns, Sephadex LH-20, MCI CHP20P, prep-HPLC, etc . Their structures were determined to be two ring D-seco limonoids 3α, 7 α-dideacetylkhivorin (1) and 1α,

3α, 7 α-trideacetylkhivorin ( 2), mexicanoloid limonoid khayanone ( 3), five khayanolide

ii limonoids 1-O-deacetylkhayanolide B (4), khayanolide B (5), khayanolide E ( 6), 1-O- deacetylkhayanolide E ( 7), and novel 6-dehydroxykhayanolide E ( 8), three lignans (-)- lyoniresinol ( 9), (-)-lyoniresin-9-yl-β-D-xylopyranoside ( 10 ), and (-)-lyoniresin-4'-yl-β-

D-glucopyranoside (11 ), respectively, through various spectroscopic methods including

IR, EI-MS (HREI-MS), LC-ESI-MS (accurate ESI-MS), extensive 1D and 2D NMR (1H ,

13 C, DEPT, 1H-1H COSY, HMQC or HSQC, HMBC, NOESY), and X-ray diffraction experiments.

The structures and stereochemistry of 1, 2, 3, 4, 6, and 8 were confirmed through X- ray crystallography. Based on the X-ray diffraction analyses and NMR data, two reported khayanolides 1α-acetoxy-2β,3 α,6,8 α,14 β-pentahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7- oate 12 and 1α,2 β,3 α,6,8 α,14 β-hexahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 13 are, in fact, 1-O-acetylkhayanolide B 4 and khayanolide B 5, respectively, and two reported phragmalins methyl 1α-acetoxy-6,8 α,14 β,30 β-tetrahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]- tricyclomeliac-7-oate 14 and methyl 1α,6,8 α,14 β,30 β-pentahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]- tricyclomeliac-7-oate 15 are, in fact, khayanolide E 6 and 1-O-deacetylkhayanolide E 7, respectively.

In the anti-proliferative bioassay, 1 and 2 showed significant growth inhibitory activities against MCF-7, SiHa, and Caco-2 tumor cells with IC 50 values in the range of

35-69 g/ml, while other compounds did not show anticancer activity even at high concentration 200 g/ml; 9, 10 , 11 exhibited strong antioxidant activities comparable to that of BHT, regarding the DPPH free scavenging activity.

iii

23 23 22 22 F O 20 MeO O O 20 18 21 7 18 12 12 21 17 17 11 H 11 OR 19 C O HO O 30 13 19 H 13 D 9 OH 14 16 6 14 16 2 1 9 8 A 10 O O 8 15 O 3 B O 15 H 4 5 10 1 1 R=Ac 5 4 30 7 OH 3 HO 6 2 R=H 2 28 3 28 23 H 29 29 22 O O 2' 7' 9' MeO 20 MeO 1' O 18 12 21 3' OH 7 17 8' 11 H 7 OR R4 13 O 1 19 4' 6' 8 R2O 6 OH 14 16 5' 9 1 9 OMe 5 8 O 2 10 15 6 4 1 30 O New compound 8 OR 28 1 MeO 5 3 OMe 29 4 2 4 R 1=Ac R 2=OH R 3=H R 4=OH OH R3 3 5 R 1=H R 2=OH R 3=H R 4=OH

R2 6 R 1=Ac R 2=R 3=O R 4=OH 9 R 1=H R 2=H

7 R 1=H R 2=R 3=O R 4=OH 10 R 2=H R 1=xyl 23 8 R 1=H R 2=R 3=O R 4=H 11 R 1=H R 2=glc 22 O O 20 18 21 12 MeO MeO 17 O O 11 7 O HO OH O H OH 13 HO 19 14 16 9 6 10 8 15 O O OR1 OH OH 5 1 OR 4 29 30 1 OH OH 2 28 3 H 12 R =Ac 14 R =Ac 1 O 1 R =H OH 13 R 1=H 15 1

iv DEDICATION

This dissertation is dedicated to my parents, Baocheng Zhang and Juxiang Xie and my parents in law, Zhenglun Chen and Fengying He, my beautiful wife Qing Chen, my lovely son Bowen Zhang. They not only shared every step of my study and research progress, but also are always the source of strength, support and encouragement.

v ACKNOWLEDGEMENTS

At first, I would like to thank my advisor, Dr. Feng Chen, for his guidance and patience of my graduate study in Clemson University. His high expectation, encouragement, knowledge, support and mentorship are greatly appreciated.

I am grateful to other committee members, Dr. Xi Wang (Department of Genetics and Biochemistry), Dr. R. Kenneth Marcus (Department of Chemistry), and Dr. Alex

Kitaygorodskiy (Department of Chemistry). Their professional knowledge initiates my passion in the theory of separation science, instrumental analysis and bioassays.

My special thanks go to Dr. Don VanDerveer (Department of Chemistry) for his much great help to do the X-ray diffraction analysis of the isolates, to Dr. Michael J.

Wargovich (Department of Cell and Molecular Pharmacology, Hollings Cancer Center,

Charleston) for the NIH grant, to Ms. Barbara Ramirez (Director of the Writing Center at

Clemson University) for her help in the preparation of this manuscript.

I am grateful to my colleagues Mr. Xiaohu Fan, Miss. Yen-Hui Chen, Ms. Juanjuan

Yin, the visiting scholar Dr. Xiaowen Wang, Dr. Huarong Tong, for their constructive suggestions on the experiments and help on data analyses.

I also appreciate Mrs. Kimberly Collins and Mr. James Findley for their generous administrative help. The help from inter-loan library is especially appreciated. I thank all other faculty and staff of the Department of Food Science and Human Nutrition for their friendship. In addition, I would express my gratitude to Dr. Liangwei Qu, Dr. Ping He, Dr.

Caoxi Luo, Dr. Xiaofei Jiang, Dr. Changfeng Wu, Dr. Limei Jin, Dr. Cao Li and Dr. Feng

Xu for their help in my daily life.

vi TABLE OF CONTENTS

Page

TITLE PAGE...... i

ABSTRACT...... ii

DEDICATION...... v

ACKNOWLEDGEMENTS...... vi

LIST OF TABLES...... ix

LIST OF FIGURES ...... x

CHAPTER

1. INTRODUCTION ...... 1

1.1 Botany of Khaya genus...... 1 1.2 Botany and distribution of Khaya senegalensis ...... 1 1.3 Medicinal usage of Khaya senegaleneis ...... 2 1.4 Khayanolides from Khaya senegalensis ...... 3 1.5 Purpose of our study ...... 4 1.6 References cited...... 6

2. PURIFICATION AND ISOLATION...... 11

2.1 General experimental procedures ...... 11 2.2 Plant material ...... 11 2.3 Extraction and partition...... 11 2.4 Purification and isolation ...... 12

3. STRUCTURAL DETERMINATION OF THE ISOLATES ...... 15

3.1 Introduction...... 15 3.2 General instruments and apparatus ...... 18 3.3 Structure identification of 3 α, 7 α-dideacetylkhivorin 1...... 18 3.4 Structure identification of 1 α, 3 α, 7 α-trideacetylkhivorin 2 ...... 33 3.5 Structure identification of khayanone 3...... 37 3.6 Structure identification of 1-O-acetylkhayanolide B 4...... 44

vii Table of Contents (Continued)

Page

3.7 Structure identification of khayanolide B 5 ...... 58 3.8 Structure identification of khayanolide E 6 ...... 61 3.9 Structure identification of khayanolide E 7 ...... 72 3.10 Structure elucidation of 6-dehydroxykhayanolide E 8 ...... 84 3.11 Structure identification of compounds 9-11 ...... 103 3.12 References cited ...... 116

4. BIOASSAY OF THE ISOLATES...... 120

4.1 Introduction...... 120 4.2 Experiments and methods ...... 121 4.3 Anti-tumor assay results ...... 123 4.4 Anti-oxidant assay results ...... 124 4.5 References cited ...... 127

5. SUMMARY...... 128

APPENDIX...... 130

viii LIST OF TABLES

Table Page

3-1 Crystal data and structure refinement of 1...... 31

3-2 Hydrogen-bond geometry (Å, °)...... 32

3-3 Crystal data and structure refinement of 2...... 35

3-4 Crystal data and structure refinement of 3...... 43

3-5 NMR data of 4 and those reported in the literature ...... 46

3-6 Crystal data and structure refinement of 4...... 57

3-7 NMR data of 5 and those reported in the literature ...... 60

3-8 NMR data of 6 and those reported in the literature ...... 63

3-9 Crystal data and structure refinement of 6...... 70

3-10 NMR data of 7 and those reported in the literature ...... 75

3-11 NMR data of 8 (δ in ppm) measured in CDCl 3 ...... 86

3-12 HR-EI-MS data of 8 ...... 91

3-13 Crystal data and structure refinement of 8...... 102

ix LIST OF FIGURES

Figure Page

2-1 Flow chart of the purification and isolation...... 14

3-1 Structures of limonoids 1-8, lignans 9-11 and reported 12 -15 ...... 17

3-2 The process of planner structure determination of 1 ...... 21

3-3 Accurate ESI-MS spectrum of 1 (3,7-dideacetylkhivorin)...... 22

3-4 1H NMR spectrum of 1 (3,7-dideacetylkhivorin)...... 23

3-5 13 C NMR spectrum of 1 (3,7-dideacetylkhivorin)...... 24

3-6 DEPT135 ° spectrum of 1 (3,7-dideactylkhivorin) ...... 25

3-7 DEPT90 ° spectrum of 1 (3,7-dideactylkhivorin) ...... 26

3-8 HMQC spectrum of 1 (3,7-dideacetylkhivorin) ...... 27

3-9 1H-1H COSY spectrum of 1 (3,7-dideacetylkhivorin)...... 28

3-10 HMBC spectrum of 1 (3,7-dideacetylkhivorin) ...... 29

3-11 Crystal structure of 1 (3, 7-dideacetylkhivorin) ...... 30

3-12 Crystal structure of 2 (1, 3, 7-trideacetylkhivorin)...... 34

3-13 Chemical structures of 1-3 and related compounds in literature...... 36

3-14 Accurate ESI-MS spectrum of 3 (khayanone)...... 39

3-15 1H NMR spectrum of 3 (khayanone)...... 40

3-16 13C NMR spectrum of 3 (khayanone)...... 41

3-17 Crystal structure of 3 (khayanone) ...... 42

3-18 Accurate ESI-MS spectrum of 4 (1-O-acetylkhayanolide B)...... 47

3-19 1H NMR spectrum of 4 (1-O-acetylkhayanolide B)...... 48

x List of Figures (Continued)

Page

3-20 13C NMR spectrum of 4 (1-O-acetylkhayanolide B)...... 49

3-21 DEPT135 ° spectrum of 4 (1-O-acetylkhayanolide B)...... 50

3-22 DEPT90 ° spectrum of 4 (1-O-acetylkhayanolide B)...... 51

3-23 HMQC spectrum of 4 (1-O-acetylkhayanolide B) ...... 52

3-24 1H-1H COSY spectrum of 4 (1-O-acetylkhayanolide B)...... 53

3-25 HMBC spectrum of 4 (1-O-acetylkhayanolide B) ...... 54

3-26 Structural revision of 12 -15 to 4-7 ...... 55

3-27 Crystal structure of 4 (1-O-acetylkhayanolide B) ...... 56

3-28 Accurate ESI-MS spectrum of 5 (khayanolide B)...... 59

3-29 LC-ESI-MS (negative ion mode) spectrum of 6 (khayanolide E)...... 64

3-30 LC-ESI-MS (positive ion mode) spectrum of 6 (khayanolide E)...... 65

3-31 1H NMR spectrum of 6 (khayanolide E) ...... 66

3-32 13C NMR spectrum of 6 (khayanolide E) ...... 67

3-33 DEPT135 ° spectrum of 6 (khayanolide E)...... 68

3-34 Crystal structure of 6 (khayanolide E)...... 69

3-35 Biogenetic pathways of B, D-seco limonoids ...... 71

3-36 Accurate ESI-MS spectrum of 7 (1-O-deacetylkhayanolide E) ...... 76

3-37 1H NMR spectrum of 7 (1-O-acetylkhayanolide E) ...... 77

3-38 13C NMR spectrum of 7 (1-O-acetylkhayanolide E)...... 78

3-39 DEPT135 ° spectrum of 7 (1-O-acetylkhayanolide E)...... 79

xi List of Figures (Continued)

Page

3-40 DEPT90 ° spectrum of 7 (1-O-acetylkhayanolide E)...... 80

3-41 HMQC spectrum of 7 (1-O-acetylkhayanolide E) ...... 81

3-42 1H-1H COSY spectrum of 7 (1-O-deacetylkhayanolide E) ...... 82

3-43 HMBC spectrum of 7 (1-O-deacetylkhayanolide E)...... 83

3-44 IR spectrum of 8 (6-dehydroxykhayanolide E) ...... 87

3-45 ESI-MS (positive ion mode) of 8 (6-dehydroxykhayanolide E) ...... 88

3-46 ESI-MS (negative ion mode) of 8 (6-dehydroxykhayanolide E) ...... 89

3-47 EI-MS spectrum of 8 (6-dehydroxykhayanolide E) ...... 90

3-48 1H NMR spectrum of 8 (6-dehydroxykhayanolide E)...... 92

3-49 13 C NMR spectrum of 8 (6-dehydroxykhayanolide E)...... 93

3-50 DEPT135 ° spectrum of 8 (6-dehydroxykhayanolide E)...... 94

3-51 DEPT90 ° spectrum of 8 (6-dehydroxykhayanolide E)...... 95

3-52 HSQC spectrum of 8 (6-dehydroxykhayanolide E) ...... 96

3-53 1H-1H COSY spectrum of 8 (6-dehydroxykhayanolide E)...... 97

3-54 HMBC spectrum of 8 (6-dehydroxykhayanolide E) ...... 98

3-55 NOESY spectrum of 8 (6-dehydroxykhayanolide E)...... 99

3-56 Structural elucidation of 8 (6-hydroxykhayanolide E) ...... 100

3-57 Crystal structure of 8 (6-dehydroxykhayanolide E) ...... 101

3-58 Accurate ESI-MS spectrum of 9 (lyoniresinol)...... 106

3-59 1H NMR spectrum of 9 (lyoniresinol) ...... 107

xii List of Figures (Continued)

Page

3-60 13 C NMR spectrum of 9(lyoniresinol)...... 108

3-61 DEPT135 ° spectrum of 9 (lyoniresinol)...... 109

3-62 DEPT90 ° spectrum of 9 (lyoniresinol)...... 110

3-63 HMQC spectrum of 9 (lyoniresinol) ...... 111

3-64 1H-1H COSY spectrum of 9 (lyoniresinol)...... 112

3-65 HMBC spectrum of 9 (lyoniresinol)...... 113

3-66 Accurate ESI-MS spectrum of 10 ...... 114

3-67 Accurate ESI-MS spectrum of 11 ...... 115

3-68 Cell viability (%) with different concentrations of 1 and 3...... 125

3-49 Dose-response curves of antiradical capacity of 9-11 and BHT ...... 126

xiii CHAPTER 1 INTRODUCTION

1.1 Botany of Khaya genus

The Meliaceae, the mahogany family, comprised of 51 genera and approximately

1400 species, is found primarily in pantropical locations. Many large trees in this family are well-known for their high quality timber, especially those in the genera --- Agliaia ,

Aphanamixis , Azadirachta , Carapa , Cedrela, Chukrasis , Dysoxylum , Entandrophragma ,

Guarea , Khaya , Melia , Soymida , Swietenia , and Trichilia (Banerji & Nigam, 1983). The most common commercial genus of this family in African is Khaya (African mahogany), which is composed of the 5 species: K. anthotheca , K. grandifoliola , K. ivorensis , K. nyasica, and K. senegalensis , found all over the world . All the species of the Khaya genus are well-known for their high quality hard wood, which is resistant to insect and fungal attack (Adesida et al ., 1971). In addition, the Khaya genus is closely related to the South

American genus Swietenia , the original source of mahogany.

1.2 Botany and distribution of Khaya senegalensis

The most distinct species, K. senegalensis , is also called “heavy African mahogany” because it is darker and heavier than other commercial mahoganies (weight when dried:

K. anthotheca 540 kg/m 3, K. grandifoliola 720 kg/m 3, K. ivorensis 530 kg/m 3, K. nyasica

590 kg/m 3, and K. senegalensis 800 kg/m 3). It is a large evergreen tree, reaching a height of approximately 15-24 m and a diameter of approximately 1.0-3.0 m, characterized by a dense evergreen crown of dark shining foliage, pinnate leaves, and round capsules. It grows primarily in the deciduous savannah forests, especially in the countries of Nigeria,

Senegal, Sudan, Uganda, and Zaire. Its dark and heavy timber, which is said to provide

1 the best surface-finishing of all the African mahoganies, is a popular timber for lorry bodies, construction work, and decking in boats, in addition to its traditional uses for furniture (Styles & White, 1991).

1.3 Medicinal usage of Khaya senegaleneis

Khaya senegalensis is one of the most popular medicinal meliaceous plants used in traditional African remedies. The decoction of its stem bark is commonly used as a bitter tonic in folk and popular medicines for malaria, fever, mucous diarrhea, and venereal diseases as well as for an anthelmintic and a taeniacide remedy (Iwu, 1993; Olayinka et al ., 1992). The stem bark extracts and the chemical constituent profile have been the subject of extensive phytochemical and pharmacological investigations since the 1960s

(Adesida et al ., 1971; Mulholland et al ., 2000; Narender et al ., 2007). These plant extracts have been reported to exhibit anti-inflammatory effects (Lompo et al ., 1998) as well as anti-bacterial (Koné et al ., 2004), anthelmintic (Ademola et al ., 2004), anti-tumor, anti-oxidant (Androulakis et al ., 2006), and anti-plasmodial activities (El-Tahir et al .,

1998). Two dimeric flavonoids, as well as 2,6-dihydroxy-p-benzoquinone, β-sistosterol, and 3-O-glucose-β-sistosterol, catechin, tannins, saponins, polysaccharides, and coumarins (Kayser & Abreu, 2001), with immunostimulating activity have been isolated from the extracts of its stem bark. Approximately 45 limonoids (Adesida et al ., 1971;

Olmo et al ., 1996; Olmo et al ., 1997, Govindachari& Kumari 1998; Khalid et al ., 1998;

Govindachari et al ., 1999; Mulholland et al ., 2000; Abdelgaleil et al ., 2003; Tchimene et al ., 2006) isolated from the different parts (leaf, seed, and stem bark) of this plant constitute the bitter principles of the plant extracts. Furthermore, some limonoids

2 exhibited feeding deterrent and growth inhibitory properties (El-Aswad et al ., 2003), and anti-feeding (Abdelgaleil & Nakatani, 2003), anti-fungal (Govindachari et al. , 1998;

Abdelgaleil et al ., 2004), and anti-sickling activities (Fall et al ., 1999).

1.4 Khayanolides from Khaya senegalensis

The nomenclature limonoid is derived from the compound limonin, one of the primary bitter components in citrus fruits. The structural determination of limonin in

1960 marked the beginning of limonoid (tetranortriterpenoid) chemistry (Arigoni et al .,

1960). These highly oxygenated, modified tetranortriterpenoids are commonly found in plants of the families Meliaceae, Rutaceae, Cneoraceae and Simaroubaceae of the order

Rutals. Approximately 1300 limonoids, exhibiting more than 35 different carbon frameworks created through ring fission, re-cyclization, reopening, re-closure, and skeletal rearrangements, have been observed over the past five decades, with new structural types continuing to appear (Rogers et al ., 1998; Tchuendem et al ., 1998; Zhang et al ., 2003; Yuan et al ., 2005; Fang et al ., 2008). Of these four families, Meliaceae is of particular interest because of the abundance and structural diversity of the limonoids present in its plant members (Connolly 1983; Taylor, 1984; Champagne et al ., 1992;

Mulholland et al ., 2000).

Specifically, African mahogany Khaya senegalenis (Meliaceae) is a rich source of limonoids, exemplifying a wide variety of structural types (Adesida et al ., 1971;

Mulholland et al ., 2000; Abdelgaleil et al ., 2003). Khayanolide limonoid, first isolated from K. senegalensis in 1996, results from a cleavage between C-1, C-2 and a linkage between C-1, C-30 of phragmalin (Olmo et al ., 1996). Past research has characterized the

3 following 11 khayanolide limonoids in two plants of the Khaya genus: 1-O- deacetylkhayanolide E from K. grandifoliola (Zhang et al ., 2008) and khyanolide A, B, C

(Abdelgaleil et al ., 2001), khayanolide D, E (Nakatani et al ., 2002), 1-O- acetylkhayanolide A (Nakatani et al ., 2001), 1-O-acetylkhayanolide B (Abdelgaleil et al .,

2000), 1 α-acetoxy-2β,3 α,6,8 α,14 β-pentahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate

(Olmo et al ., 1996), 1 α,2 β,3 α,6,8 α,14 β-hexahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7- oate and 1 α-acetoxy-3β,6,8 α-trihydroxy-2α–methoxy-2β,14 β–epoxy-[4.2.1 10,30 .1 1,4 ]- tricyclomeliac-7-oate (Olmo et al ., 1997) from K. senegalensis . However, the configurations of oxygenated C-6 in these reported khayanolides were not confirmed, except for that in khayanolide A, which was determined through X-ray crystallography and CD study (Abdelgaleil et al ., 2001).

1.5 Purpose of our study

Although various pure compounds, including the 45 limonoids, have been identified from the plant, none has been screened for antioxidant activity or anti-proliferative activity against different human cancer cell lines. A preliminary study (Androulakis et al .,

2006) indicated that the methanolic extract of the stem bark of K. senegalensis exhibited antioxidant activities and anti-proliferative, anti-inflammatory and pro-apoptotic effects on HT-29, HCT-15, and HCA-7 cells, suggesting that this popular herbal medicine has potential as a natural chemopreventive agent for colorectal cancer.

Based on these results, the report here screened bioactive ingredients in the plant guided by anticancer and antioxidant bioassays, investigated the structures of these remaining khayanolides fully, and provided solid scientific evidence for the traditional

4 medicinal use of the stem bark of Khaya senegalensis . This work was divided into the following three stages:

(1) The extraction, purification and isolation of the stem bark of Khaya senegalensis from the Republic of Guinea (West Africa) using a series of modern column chromatographic techniques (normal phase and reverse phase C-18 silica gel, Sephadex

LH-20, MCI gel CHP20P, HPLC-DAD/ELSD, and preparative HPLC-DAD);

(2) The structural elucidation of the isolates based on spectroscopic methods including IR, EI-MS (HREI-MS), LC-ESI-MS (accurate ESI-MS) (positive ion model and negative ion model), extensive 1D and 2D NMR (1H, 13 C, DEPT, 1H-1H COSY,

HMQC or HSQC, HMBC, and NOESY), and X-ray diffraction experiments. The configuration of oxygenated C-6 in khayanolides was determined through X-ray crystallography, the preferred arbiter to confirm the structure and stereochemistry of organic compounds. The different C-6 configurations in B,D-seco limonoids found in the

African mahogany Khaya genus and the original mahogany genus Swietenia were analyzed, the results suggesting a significant chemotaxonomy between the two genera.

(3) Anti-tumor assay against different cell lines (MCF-7, SiHa, and Caco-2 tumor cells) and DPPH antioxidant bioassay of the extracts, fractions, and purified single entities. The relationship between the structure and the activity was also investigated.

5 1.6 References cited .

Abdelgaleil SAM, Iwagawa T, Doe M, Nakatani M 2004 . Antifungal limonoids from the

fruits of Khaya senegalensis . Fitoterapia 75 : 566-572

Abdelgaleil SAM, Nakatani M 2003 . Antifeeding activity of limonoids from Khaya

senegalensis (Meliaceae). J Appl Ent 127: 236-239.

Abdelgaleil SAM, Okamura H, Iwagawa T, Doe M, Nakatani M 2000 . Novel rings B,D-

seco limonoids from the stem bark of Khaya senegalensis . Heterocycles 53: 2233-

2240.

Abdelgaleil SAM, Okamura H, Iwagawa T, Sato A, Miyahara I, Doe M, Nakatani M

2001 . Khayanolides, rearranged phragmalin limonoid antifeedants from Khaya

senegalensis . Tetrahedron 57: 119–126.

Ademola IO, Fagbemi BO, Idowe SO 2004 . Evaluation of the athelmintic activity of

Khaya senegalensis extract against gastrointestinal nematodes of sheep: in vitro and

in vivo studies. Vet Parasitol 122: 151-164.

Adesida GA, Adesogan EK, Okorie DA, Taylor DAH, Styles BT 1971 . The limonoid

chemistry of the genus Khaya (Meliaceae). Phytochemsitry 10: 1845-1853.

Androulakis XM, Muga SJ, Chen F, Koita Y, Toure B, Wargovich MJ 2006 .

Chemopreventive effects of Khaya senegalensis bark extract on human colorectal

cancer. Anticancer Res 26: 2397-2406.

6 Arigoni D, Barton DHR, Corey EJ, Jeger O, Caglioti L, Dev S, Ferrini PG, Glazier ER,

Melera A, Pradhan SK, Schaffner K, Sternhell S, Templeton JF, Tobinaga S 1960 .

The constitution of limonin. Experientia 16: 41-49.

Banerji B, Nigam SK 1983 . Wood constituents of Meliaceae. Fitoterapia 55: 3-36.

Champagne DE, Koul O, Isman MB, Scudder GGE, Towers GHN 1992 . Biological

activity of limonoids from the Rutales. Phytochemistry 31: 377-394.

Connolly JD 1983 . Chemistry of the limonoids of the Meliaceae and Cneoraceae. Annual

proceedings of the Phytochemical Society of Europe . 22: 175-213.

El-Aswad AF, Abdelgaleil SAM, Nakatani M 2003 . Feeding deterent and growth inhitory

properties of limonoids from Khaya senegalensis against the cotton leafworm,

Spodoptera littoralis . Pest Manage Sci 60: 199-203.

El-Tahir A, Ibrahim AM, Satti GMH, Theander TG, Kharazmi A, Khalid SA 1998 . The

potential antileishmanial activity of some Sudanese medicinal plants. Phytother Res

12: 576-579.

Fang X, Di YT, He HP, Liu HY, Zhang Z, Ren YL, Gao ZL, Gao S, Hao XJ 2008 .

Cipadonoid A, a novel limonoid with an unprecedented skeleton, from Cipadessa

cinerasecns . Org. Lett . 10: 1905-1907.

Fall AB, Vanhaelen-Fastré R, Vanhaelen M, Lo I, Toppet M, Ferster A, Fondu P 1999 . In

vitro antisickling activity of a rearranged limonoid isolated from Khaya senegalensis .

Planta Medica 65: 209-212.

Govindachari TR, Kumari GNK 1998 . Tetranortriterpenoids from Khaya senegalensis .

Phytochemistry 47: 1423-1425.

Govindachari TR, Suresh G, Banumathy B, Masilamani S, Gopalakrishnan G, Kumari

GNK 1999 . Antifungal activity of some B, D-seco limonoids from two meliaceous

plants. J Chem Ecol 25: 923-933.

Iwu M 1993 Hankbook of African medicinal plants, Pharmacognostical profile of

selected medicinal plants . CRC Press Inc. pp. 196-197.

Kayser O, Abreu PM 2001 . Antileishmanial and immunostimulating activities of two

dimeric proanthocyanidins from Khaya senegalensis . Pharm Biol 39: 284-288.

Khalid SA, Friedrichsen GM, Kharazmi A, Theander TG, Olsen CE, Christensen SB

1998 . Limonoids from Khaya senegalensis . Phytochemistry 49: 1769-1772.

Koné WM, Atindehou KK, Terreaux C, Hostettmann K, Traoré D, Dosso M 2004 .

Traditional medicine in North Côte-d’Ivoire: screening of 50 medicinal plants for

antibacterial activity. J Ethnopharmacol 93: 43-49

Lompo M, Nikiema JB, Guissou IP, Moes AJ, Fontaine J 1998 . The topical anti-

inflammatory effect of chloroform extract from Khaya senegalensis stem barks.

Phytother Res 12: 448-450.

Mulholland DA, Parel B, Coombes PH 2000 . The chemistry of the Meliaceae and

Ptaeroxylaceae of southern and eastern Africa and Madagascar. Curr Org Chem 4:

1011-1054.

Nakatani M, Abdelgaleil SAM, Kassem SMI, Takezaki K, Okamura H, Iwagawa T, Doe

M 2001 . Antifeedant rings B and D opened limonoids from Khaya senegalensis . J Na.

Prod . 64: 1261–1265.

Nakatani M, Abdelgaleil SAM, Kassem SMI, Takezaki K, Okamura H, Iwagawa T, Doe

M 2002 . Three new modified limonoids from Khaya senegalensis . J Nat Prod . 65:

1219–1221.

Narender T, Khaliq T, Shweta, Reddy KP, Sharma RK 2007 . Occurance, biosynthesis,

biological activity and NMR spectroscopy of D and B, D ring seco-limonoids of

Meliaceae family. Nat Prod Commu 2: 203-221.

Olayinka AO, Onoruvwe O, Loti TY 1992 . Cardiovascular effects in rodents of the

methanolic extract of the stem bark of Khaya senegalensis A. Juss. Phytother Res 6:

282-284.

Olmo, LRV, Silva MF das GF da, Fo ER, Vieira PC, Fernandes JB, Marsaioli AJ,

Pinheiro AL, Vilela EF 1996 . Rearranged limonoids from Khaya senegalensis .

Phytochemistry 42: 831-837.

Olmo LRV, Silva MF das GF da, Fo. ER, Vieira PC, Fernandes JB, Pinheiro AL, Vilela

EF 1997 . Limonoids from leaves of Khaya senegalensis . Phytochemistry 44: 1157-

1161.

Rogers LL, Zeng L, McLaughlin JL 1998 . Volkensinin: A New Limonoid from Melia

volkensii . Tetrahedron Lett . 39: 4623-4626.

Styles BT, White F 1991 . Flora of tropical East Africa: Meliaceae. Royal Botanic

Gardens, Kew publisher.

Taylor DAH 1984 . The chemistry of the limonoids from Meliaceae. In: Herz, W.,

Grisebach, H., Kirby, G.W. (Eds.), Progress in the Chemistry of Organic Natural

Products, vol. 45. Springer-Verlag, New York, pp. 1–102.

Tchimene MK, Ngamga D, Tane P, Stemer O, Connolly JD 2006 . 3,7-diacetyl-6-

hydroxykhivorin, a new limonoid from Khaya senegalensis (Meliaceae) Bull Chem

Soc Ethiop . 20: 69-73.

Tchuendem MHK, Ayafor F, Connolly JD, Sterner O 1998 . Khayalactone, a novel

limonoid from Khaya grandifoliola . Tetrahedron Lett . 39: 719-722.

Yuan XH, Li BG, Zhou M, Qi HY, Zhang GL 2005 . Cipadesins A-C: novel

tetranortriterpenoids from Cipadessa cinerascens . Org Lett . 7: 5051-5053.

Zhang H, Odeku OA, Wang XN, Yue JM 2008 . Limonoids from the stem bark of Khaya

grandifoliola . Phytochemistry 69: 271–275.

Zhang HP, Wu SH, Shen YM, Ma YB, Wu DG, Qi SH, Luo XD 2003 . A

pentanortriterpenoid with a novel carbon skeleton and a new pregnane from Trichilia

connaroides . Can J Chem . 81: 253-257.

10 CHAPTER 2 PURIFICATION AND ISOLATION

2.1 General experimental procedures

All chemicals used for extraction and separation were analytical grade (Fisher

Scientific, USA) and were used directly without further purification. The normal phase and reverse phase C-18 TLC plates used were purchased from Fisher Scientific. The column material reverse phase gel MCI CHP20P (high porous polymer, 37-75 m) was purchased from Mitsubishi Chemical Corporation. The RP C-18 gel (reverse phase C-18,

40-63 m) was purchased from Acros Corporation. The Sephadex LH-20 was purchased from Amersham Biosciences Corporation. The normal phase silica gel (200-300 mesh) was purhcased from Qingdao Haiyang Chemical Corporation. The TLC zones (normal phase silica gel and reverse phase C-18) were visualized by exposure to 10% alcohol sulfuric acid. Analytical HPLC-PDA (Shimadzu) was used to detect the purity of the isolates.

2.2 Plant material

The stem bark of Khaya senegalensis was collected from the martime plains near

Conakry, Republic of Guinea (West Africa), in November 2005 under the supervision of

Dr. Youssouf Koita, Ministere de Sante Publique, Departement Sante Communautaire,

Republic of Guinea. A voucher specimen (number 018/INSP/2005) has been deposited at the Ministry of Public Health, Republic of Guinea.

2.3 Extraction and partition

Air dried stem bark (495 g) of K. senegalensis was grounded using a mechanic machine and sieved through a 100 mesh sifter. Then 160 g of the plant powder was

11 extracted using 5 L MeOH for 24 h in a Soxhlet extractor, with a total of 3 batches needed to process the 495 g of bark. The pooled MeOH extract was filtered through cheese cloth, with the clean solution being condensed through a rotary evaporator in vacuum to remove the solvents. The 60 g extracts obtained were suspended in 300 ml water and then partitioned by 400 ml CHCl 3, EtOAC, and n-BuOH three times to achieve

CHCl 3 phase fraction (4.8 g), EtOAC phase fraction (15.2 g), n-BuOH phase fraction

(20.4 g) and the left H 2O phase fraction (6.8 g). All the 4 fractions were bioassayed using anti-tumor and antioxidant tests.

2.4 Purification and isolation

The concentrated CHCl 3 extract was subjected to column chromatography over silica gel (particle size 32-63 M), and the elution was conducted in an increasing polarity solvent system using n-hexane, 20% acetone in hexane, 50% acetone in hexane, and acetone, respectively. The fractions eluted by 50% acetone in hexane were pooled and subjected to column chromatography over reverse phase C 18 silica gel (particle size

40-63 m) and eluted with H 2O: MeOH (1:3). The fractions eluted resulted in compounds

1 (12 mg), 2 (11 mg) and colorless crystals (35 mg). The X-ray diffraction analysis indicated that the crystal was a mixture of two compounds having a ratio 1:1 that were difficult to be separated through normal phase column chromatography. As a result, the mixture was subjected to semi-preparative-HPLC using a reverse phase C-18 column eluted by gradient CH 3CN:MeOH:H 2O (15:15:70, 18:18:64, 20:20:60, 25:25:50,

30:30:40 and MeOH). Compound 6 (12 mg) was obtained from the CH 3CN:MeOH:H 2O

20:20:60 fractions while 8 (12 mg) was obtained from the MeOH fractions.

12 The concentrated EtOAc fraction (15 g) was separated using a silica gel (32-63 M) column eluted with chloroform/acetone with an increasing gradient of acetone (30:1 to pure acetone) to obtain six fractions (F1-F6). The F5 (3.5 g) was further separated into five fractions using a silica gel column (CHCl 3/MeOH 50:1 to 4:1). Repeated chromatography over reverse phase C-18 silica gel (40-63 m) and Sephadex LH-20 afforded compound 3 (13 mg) from the sub-fractions 3, 4 (20 mg) from the sub-fraction 4.

The fraction 4 (3.3 g) was purified using a silica gel column (CHCl3/EtOAc 20:1 to 2:1), giving five sub-fractions. Repeated column chromatography afforded compounds 5 (18 mg) and 7 (7 mg) from the sub-fraction 4.

The concentrated n-butanol fraction (20.4 g) was subjected to a MCI CHP20P (300 g, 32-63 m) column eluted with with an increasing gradient of water/ethanol (water, 5:1,

3:1 to pure ethanol) to obtain four fractions (F1-F4). The F3 (5 g) was further separated into five sub-fractions using a silica gel column with gradient elutes (CHCl 3/MeOH/H 2O

20:1:0.1, 10:1:0.1, 4:1:0.1). Repeated chromatography over reverse phase C-18 (40-63

m) eluted with MeOH and H 2O and Sephadex LH-20 eluted with MeOH afforded compounds 9 (33 mg), 10 (29 mg) and 11 (40 mg).

Suitable block-shaped crystals for further X-ray diffraction of 1, 2, 3 (in CHCl 3 and

MeOH), 4 (in CHCl 3), 6 and 8 (in CHCl 3 and MeOH) were obtained through optimization of the solvent condition for re-crystallization.

The flow chart of the procedures, beginning with the raw material, the stem bark of

Khaya senegalensis , and ending with the pure compounds 1-11 , is shown in the following

Figure 2-1.

495 g dried, powdered stem bark raw material

Grounded

Extracted by MeOH

MeOH extracts (60 g)

Suspended in water

Partitioned in CHCl 3, EtOAC and n-BuOH

CHCl 3 extract EtOAc extract BuOH extract H2O extract (4.8 g) (15.2 g) (20.4 g) (6.8 g)

1 12 mg 4 20 mg 9 33 mg 2 11 mg 5 18 mg 10 29 mg 3 13 mg 6 11 mg 11 40 mg 8 12 mg 7 7 mg

23 23 23 22 22 22 7' F O O OMeO 2' 9' 20 MeO MeO 20 1' O 20 18 O 3' OH 18 21 18 12 21 8' 12 7 12 21 7 17 17 17 11 11 H 11 H OR R4 O 7 1 OR 19 O HO 19 13 8 C 13 19 O R2O 4' 6' 30 H 13 OH 14 16 5' 14 D 6 9 OH 16 6 9 16 14 1 9 O OMe 2 1 9 5 10 8 2 8 O O 15 6 A 10 O 8 15 O 4 3 B 15 H 1 30 4 5 10 1 O 4 30 OR 5 7 OH 28 1 MeO 5 3 OMe HO 3 2 29 6 28 R3 2 4 H OH 28 R2 3 29 29 O 4 R =Ac R =OH R =H R =OH 9 R =H R =H 1 R=Ac 1 2 3 4 1 2 3 5 R =H R =OH R =H R =OH 2 R=H 1 2 3 4 10 R 2=H R 1=xyl 6 R =Ac R =R =O R =OH 1 2 3 4 11 R 1=H R 2=glc 7 R 1=H R 2=R 3=O R 4=OH 8 R =H R =R =O R =H 1 2 3 4

Figure 2-1 Flow chart of the purification and isolation

14 CHAPTER 3 STRUCTURAL DETERMINATION OF THE ISOLATES

3.1 Introduction

The structures and the stereochemistry of the 11 isolated natural products (1-11 )

(Figure 3-1) were characterized through various spectroscopic methods including IR, MS

(EI-MS, HREI-MS, LC-ESI-MS, accurate ESI-MS), NMR (1D and 2D NMR experiments 1H, 13 C, DEPT 90 ° and 135 °, 1H-1H COSY, HMQC or HSQC, HMBC, and

NOESY), and single crystal X-ray diffraction analyses. Among the isolates, 8 compounds

(1-8) belong to limonoids (tetranortriterpenoids) and 3 compounds (9-11 ) are lignans.

Compound 8 is a novel khayanolides while the findings from the research reported here indicates the first isolated compounds, 2, 7, 9, 10 , 11 , from the title plant.

The limonoids were elucidated as 3α, 7 α-dideacetylkhivorin ( 1), 1 α, 3 α, 7 α- trideacetylkhivorin ( 2), khayanone ( 3), 1-O-acetylkhayanolide B ( 4), khayanolide B ( 5), khayanolide E ( 6), 1-O-deacetylkhayanolide E ( 7), and 6-dehydroxylkhayanolide E ( 8).

These teternortriterpenoids are classified as three different carbon-frame types: (I) khivorin limonoids with ring D-seco ( 1 and 2), (II) mexicanolide limonoid with rings B,

D-seco and ring B recycles ( 3), and (III) khayanolide limonoids with a cleavage of C-1,

C-2 and a link of C-1, C-30 of rearranged phragmalin ( 4-8).

The structures and stereochemistry of 1, 2, 3, 4, 6, and 8 were confirmed through X- ray crystallography. This study is the first report of khayanone 3 as a mexicanolide with a

6 S configuration as determined through X-ray diffraction analyses. The absolute stereochemistry of 1-O-acetylkhayanolide B 4 was determined because of the presence of the chlorines of CHCl 3 molecules in the crystals.

15 The results obtained here confirmed the structures of khayanolides 4-7 reported in

2000-2003 by the Nakatani group and have necessitated the structure revision of the four limonoids reported in 1996-1997 by the Silvo group, isolated from the same part (stem bark) of the same species ( K. senegalensis ). Two khayanolides 1α-acetoxy-

2β,3 α,6,8 α,14 β-pentahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 12 and

1α,2 β,3 α,6,8 α,14 β-hexahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 13 are, in fact, 1-O- acetylkhayanolide B 4 and khayanolide B 5, respectively, and two phragmalins methyl

1α-acetoxy-6,8 α,14 β,30 β-tetrahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]-tricyclomeliac-7-oate 14 and methyl 1α,6,8 α,14 β,30 β-pentahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]-tricyclomeliac-7-oate 15 are, in fact, khayanolide E 6 and 1-O-deacetylkhayanolide E 7, respectively ( Figure 3-1).

All the C-6 configurations in khayanolides 4, 5, 6, and 7 were determined to be an S.

Based on the results from this study and on the biogenetic pathway view, the methyl 6- hydroxyangolensate in African mahogany K. senegalensis should have a C-6 S configuration while methyl 6-hydroxyangolensate in genuine mahogany Swietenia species should have a C-6 R configuration. This configuration, different from the oxygenated C-6 in B, D-seco limonoids, also suggests a different chemotaxonomy between the African mahogany Khaya genus and the genuine mahogany Swietenia genus.

The three lignans were identified as (-)-lyoniresinol ( 9), (-) lyoniresin-9-yl-β-D- xylopyranoside ( 10), and (-)-lyoniresin-4'-yl-β-D-glucopyranoside (11) (Figure 3-1).

16 23 23 22 22 F O 20 MeO O O 20 18 21 7 18 12 12 21 17 17 11 H 11 OR 19 C O HO O 30 13 19 H 13 D 9 OH 14 16 6 14 16 2 1 9 8 A 10 O O 8 15 O 3 B O 15 H 4 5 10 1 1 R=Ac 4 30 5 7 HO OH 2 R=H 3 2 6 28 3 28 23 H 29 29 22 O

O 2' 7' 9' MeO 20 MeO 1' O 18 12 21 3' OH 7 17 8' 11 H 7 OR R4 13 O 1 19 4' 6' 8 R2O 6 OH 14 16 5' 9 1 9 OMe 5 8 O 2 10 15 6 4 1 30 O New compound 8 OR 28 1 MeO 5 3 OMe 29 4 2 4 R 1=Ac R 2=OH R 3=H R 4=OH R3 5 R =H R =OH R =H R =OH OH 3 1 2 3 4 R2 6 R 1=Ac R 2=R 3=O R 4=OH 9 R 1=H R 2=H

Figure 3-1 Structures of limonoids 1-8, lignans 9-11 and the reported 12 -15

17 3.2 General instruments and apparatus

The optical rotation spectrum was obtained using a Perkin-Elmer-341 polarimeter.

The IR spectrum was recorded using a Nicolet-Magna-750-FTIR spectrometer with KBr pellets. The NMR spectra were measured using a Varian Mercury plus 300, a Bruker-

AV-300, a Bruker-DRX-400, or a Bruker-AV-500 instrument with 7-15 mg samples dissolved in 0.6 ml CDCl 3, CD 3OD, or CDCl 3+CD 3OD solvents with TMS serving as an internal reference. The mass spectra were conducted using a Finnigan MAT-95 mass spectrometer for EI-MS (HR-EI-MS) and LCQ-Deca or Waters Q-TOF micro TM mass spectrometer for LC-ESI-MS and accurate ESI-MS. The X-ray diffraction experiments were conducted using a Rigaku AFC8S diffractometer with a Mercury CCD detector or a

Bruker Smart Apex CCD.

3.3 Structure identification of 3 α, 7 α-dideacetylkhivorin 1

Compound 1 is soluble in CHCl 3 but not in MeOH. Its molecular formula was determined to be C28 H38O8 (molecular weight 502) based on accurate ESI-MS ( Figure 3-

3) and NMR data ( Figures 3-4 to 3-10 ). As a result, the degree of unsaturation is 10.

Its 1-D 1H NMR spectrum (Figure 3-4) indicates the presence of 6 singlet methyls

δ 2.10, 1.23, 1.03, 1.03, 1.01 and 0.90. Twenty-eight carbon signals were observed in the

13 C NMR spectrum (Broad Band Decoupling) ( Figure 3-5). The multiplicities of these carbons determined through DEPT 135 ° (Figure 3-6) and DEPT 90 ° (Figure 3-7) resulted in 6 CH 3, 4 CH 2, 10 CH, and 8 C, including two ester carbonyls ( δ 169.6, 168.3), a β-substituted furan ( δ 142.9, 141.1, 120.8, 110.0), four oxygenated methines ( δ 78.6,

75.9, 74.4, 70.3) and one oxygenated carbon ( δ 69.9). Based on the molecular formula

18 and the DEPT spectra, two hydroxyl groups must be present in a molecule of 1. The signals of the β-substituted furan ring in the 1H NMR spectrum occurred at δ 7.42 (1H, s,

H-23), 7.41 (1H, s, H-21), 6.34 (1H, s, H-22) and the corresponding carbon signals at

142.94 (C-23, d), 141.07 (C-21, d), 109.97 (C-22, d), respectively, in the HMQC spectrum ( Figure 3-8). The structural fragments of 1 was deduced as they are shown in

Figure 3-2 from the structures of the furan ring, the singlet of methyl groups, and the combination of coupling constants in the 1H NMR spectrum, chemical shift values in the

1H and 13 C NMR spectra, 2-D 1H-1H COSY ( Figure 3-9), HMQC and HMBC ( Figure 3-

10 ) spectra.

Finally, the structure and the relative stereochemistry of 1 were confirmed through single crystal X-ray diffraction experiment ( Figure 3-11 and Table 3-1). As a result, compound 1 was identified to be 3 α, 7 α-dideacetylkhivorin ( Figure 3-1).

X-ray crystallography confirmed that 1 contains four six-membered rings A—D, one three-membered ring E (C14/C15/O5), and one furan ring F linked to atom C17 of ring D through a C—C bond in an equatorial position, known as a D-seco type limonoid.

The ring junctions A/B, B/C and C/D are all trans , while D/E is cis . The six-membered rings A—D adopt chair, chair, twist boat and half-chair conformations, while rings E and

F are fairly planar moieties. The bond lengths and angles are within normal ranges, fairly close to their expected values, and the data are comparable with the corresponding values in those of a similar D-seco limonoid gedunin (Toscano et al. , 1996). The strong classical intra-molecular hydrogen bond, O3—H3···O1 and inter-molecular hydrogen-bonding interactions O4—H4···O3 connect the molecules to form a network ( Table 3-2).

19 Previous results reported that 3 α, 7 α-dideactylkhivorin 1 was originally isolated from the seeds of Khaya nyasica in Tanzania (Adesida et al. , 1971) and was also found to be present in the fresh seeds of Khaya senegalensis (Govindachari & Kumari, 1998) in

India and in the stem bark of Khaya ivorensis in the Democratic Republic of Congo

(Abdelgaleil et al. , 2005). Since there is only partial proton data for this compound presented in the literature, the 1H and 13 C NMR data are unambiguously assigned based on 2D NMR experiments as the following:

1 3α,7 α-dideacetylkhivorin (1): Colorless block-shaped crystals, C28 H38O8, H-

NMR (500 M Hz, in CDCl 3) δ: 7.42 (1H, s, H-23), 7.41 (1H, s, H-21), 6.34 (1H, s, H-22),

6.00 (1H, s, H-17), 4.85 (1H, s, H-1), 3.88 (1H, s, H-15), 3.54 (1H, brs, H-7), 3.41 (1H, brs, H-3), 2.81 (1H, m, H-9), 2.39 (1H, dd, J =11.0, 7.0 Hz, H-5), 2.29 (1H, m, H-2), 2.10

(3H, s, OAc), 1.97 (1H, m, H-2), 1.83 (1H, dd, J = 16.0, 11 Hz, H-6), 1.65 (1H, m, H-12),

1.62 (1H, m, H-11), 1.36 (1H, m, H-6), 1.32 (1H, m, H-11), 1.24 (1H, m, H-12), 1.23 (3H, s, H 3-18), 1.03 (3H, s, H 3-29), 1.03 (3H, s, H 3-30), 1.01 (3H, s, H 3-19), 0.90 (3H, s, H 3-

13 28); C NMR (125 M Hz, in CDCl 3 ) δ: 74.42 (C-1), 28.13 (C-2), 75.94 (C-3), 37.39 (C-

4), 34.97 (C-5), 26.58 (C-6), 70.31 (C-7), 43.41 (C-8), 35.20 (C-9), 41.09 (C-10), 14.56

(C-11), 26.55 (C-12), 38.36 (C-13), 69.91 (C-14), 57.21 (C-15), 168.31 (C-16), 78.61 (C-

17), 17.67 (C-18), 16.60 (C-19), 120.76 (C-20), 141.07 (C-21), 109.97 (C-22), 142.94 (C-

23), 22.12 (C-28), 28.38 (C-29), 18.63 (C-30), 169.59 (OAc), 21.56 (OAc). Accurate

ESI-MS m/z : 503.2484 [M+H] +, 1005.4966 [2M+H] +, 1027.4792 [2M+Na] +, 443.2285

+ + + [M-HOAc+H] , 425.2092 [M-HOAc-H2O+H] , 407.2061 [M-HOAc-2H 2O+H] .

20 HMQC showed the information which protons are attached to which carbons:

H 7.42 6.34 H 142.94 78.61 74.42 57.27 70.31 75.94 109.97 O H O H O H O H O H O 120.76 141.07 6.00 4.85 3.88 3.54 3.41 H 7.41

21.56 28.38 18.63 14.56 17.67 22.12 35.20 34.97 28.13 26.58 26.55 16.60 H H H H H H H H H H O H O H H H H H H H H H H H H H H 2.81 2.39 2.29 1.97 2.10 1.83 1.36 1.65 1.24 1.62 1.32 1.23 1.03 1.01 0.90

1H-1H COSY and HMQC exhibited the following fragments:

O 4.85 1.65 1.24 2.29 H H O H 74.42 26.58 1.62 H H 34.97 70.31 26.55 1.97 H 28.13 H H 1.32 H 14.56 75.94 2.39 H H 3.54 3.41 H 2.81 H 35.20 O 1.83 1.36

HMBC showed the following fragments including the quaternary carbons:

169.59 6.34 2.10 1.03 1.23 H O 1.01 109.97 O CH 18.63 CH 17.67 O 37.39 34.97 H3C 16.60 3 3 141.07 CH3 21.56 35.20 69.91 H 6.00 H H O 26.55 78.61 120.76 H 75.94 H 74.42 H H H 7.41 35.20 O O 78.61 O 22.12 28.38 H 43.41 38.36 H C 41.09 38.36 168.31 3 CH3 34.97 70.31 69.99 O 0.90 1.03 O O 57.21 H O H H 3.88

The planner structure of 1 was deduced as that on the O O left, after combining the above moieties and checking O O the NMR data including chemical shift values, O O coupling constants in 1-D NMR and the correlations in HO OH 2D NMR COSY and HMBC spectra for confirmation.

Figure 3-2 The process of planner structure determination of 1

Figure 3-3 Accurate ESI-MS spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-4 1H NMR spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-5 13C NMR spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-6 DEPT135 ° spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-7 DEPT90 ° spectrum of 1 (3,7-dideacetylkhivorin)

26 Figure 3-8 HMQC spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-9 1H-1H COSY spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-10 HMBC spectrum of 1 (3,7-dideacetylkhivorin)

Figure 3-11 Crystal structure of 1 (3, 7-dideacetylkhivorin)

30 Table 3-1 Crystal data and structure refinement of 1

Crystal description Colorless block

Empirical formula C28 H38 O8 Molecular weight 502.58 Temperature (K) 298(2) Wavelength (Å) 0.71073

Crystal system, space group Orthorhombic, P212121 Unit cell dimensions (Å) a = 10.907 (2), b = 14.200 (3), c = 17.391 (4) Volume (Å 3) 2693.5 (9) Number of molecules per unit cell, Z 4 Calculated density (Mg/m 3) 1.239 Absorption coefficient ( ) (mm -1) 0.09 Diffraction radiation type Mo Ka F(000) 1080 Crystal size 0.48 mm × 0.46 mm × 0.29 mm θ range for data collection ( °) 2.20 < θ < 25.14 Limiting indices -12 ≤ h ≤ 13, -16 ≤ k ≤ 16, -20 ≤ l ≤ 20

Reflections collected / unique 23488 / 2485 [ Rint = 0.0300] Completeness to θ = 25.14 99.8 % Absorption correction REQAB (multi-scan) Max. and min. transmission 0.9705 and 0.9355 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2711 / 0 / 333 Goodness-of-fit on F2 1.065

Final R indices [ I > 2 σ ( I)] R1 = 0.0407, ωR2 = 0.1045

R indices (all data) R1 = 0.0458, ωR2 = 0.1085 Largest diff. peak and hole (e Å -3) 0.15 and -0.18

Table 3-2 hydrogen-bond geometry (Å, °) of compounds 1-3

Compound D-H⋅⋅⋅A D⋅⋅⋅A H⋅⋅⋅A D⋅⋅⋅A D-H⋅⋅⋅A

O3-H3 ⋅⋅⋅O1 0.83 2.10 2.810 (2) 143

3, 7-dideactyl O4-H4 ⋅⋅⋅O3 i 0.83 1.93 2.758 (2) 171 khivorin 1 symmetry code: i x + 0.5, -y + 0.5, -z

O1-H1A ⋅⋅⋅O7 ii 0.83 2.24 3.015 (3) 155

1, 3, 7-trideactyl O4-H4 ⋅⋅⋅O3 iii 0.83 1.98 2.800 (2) 171 khivorin 2 symmetry code: ii x + 1, y, z; iii x – 0.5, -y + 1.5, -z + 1

O3-H3 ⋅⋅⋅O6 iv 0.83 1.877 2.695 168

O6-H6 ⋅⋅⋅O10 iv 0.83 1.915 2.740 172.35

O10-H10B ⋅⋅⋅O4 v 0.946 2.002 2.882 53.87

O10-H10A ⋅⋅⋅O11 0.903 2.720 2.902 92.43 Khayanone 3 O10-H10B ⋅⋅⋅O11 0.946 2.654 2.902 96.50

O11-H10D ⋅⋅⋅O10 0.72 2.351 2.902 157.69

O10-H10A ⋅⋅⋅O7 vi 0.903 2.179 3.082 178.28

Symmetry codes: iv 1.5-x, 0.5+y, 0.25-z; v 1.5-x, 0.5+y; vi x, y, -z

32 3.4 Structure identification of 1α, 3α, 7α-trideacetylkhivorin 2

Compound 2 (Figure 3-1) was soluble in CHCl 3 but not in MeOH. Colorless block- shaped crystals suitable for X-ray diffraction analysis were obtained in both solvents. Its structure and relative stereochemistry were determined unambiguously through single crystal X-ray diffraction experiments ( Figure 3-12 and Table 3-3). As a result, compound 2 was identified as 1α, 3α, 7α-trideacetylkhivorin. The compound was reported as a synthetic derivative through basic hydrolysis of khivorin (Bevan et al ., 1963) and recently isolated as a natural product from Khaya ivorensis (Abdelgaleil et al ., 2005).

Similar to compound 1, 2 contains four six-membered rings A–D, one three- membered ring E, and one furan ring F linked to ring D through a C—C bond in an equatorial position. The ring junctions A/B, B/C and C/D are all trans , while D/E is cis .

The six-membered rings A–D adopt chair, chair, twist boat and half-chair conformations, while rings E and F are essentially planar. The five-membered furan ring is disordered by a 180° rotation between the C17—C20 bond. All the bond lengths and angles are close to their expected values, and the data are comparable with the corresponding values of gedunin (Toscano et al ., 1996) ( Figure 3-13) and of 1,3-dideacetylkhivorin 1. Compound

2 exhibits strong classical intermolecular O1—H1A···O7, O4—H4···O3 hydrogen bond interactions linking the molecules to a stabilized network ( Table 3-2). Compounds 1 and

2 are typical D-seco khivorin limonoids ( Figure 3-13), which can be further transformed to B,D-seco limonoids, such as methyl angosenlate, commonly regarded as a precursor for mexicanolide limonoid (Mulholland et al ., 2000).

Figure 3-12 Crystal structure of 2 (1, 3, 7-trideacetylkhivorin)

34 Table 3-3 Crystal data and structure refinement of 2

Crystal description Colorless block

Empirical formula C26H36 O 7 Molecular weight 460.55 Temperature (K) 153 (2) Wavelength (Å) 0.71073

Crystal system, space group Orthorhombic, P212121 Unit cell dimensions (Å) a = 9.856 (2), b = 14.492 (3), c = 15.714 (3) Volume (Å 3) 2244.5 (8) Number of molecules per unit cell, Z 4 Calculated density (Mg/m 3) 1.363 Absorption coefficient ( ) (mm -1) 0.10 Diffraction radiation type Mo Ka F(000) 992 Crystal size 0.41 mm × 0.41 mm × 0.31 mm θ range for data collection ( °) 3.30 < θ < 25.10 Limiting indices -8 ≤ h ≤ 11, -17 ≤ k ≤ 17, -18 ≤ l ≤ 18

Reflections collected / unique 16840 / 2485 [ Rint = 0.037] Completeness to θ = 25.14 99.8 % Absorption correction REQAB (multi-scan) Max. and min. transmission 0.961 and 0.970 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2485 / 0 / 306 Goodness-of-fit on F2 1.071

Final R indices [ I > 2 σ ( I)] R1 = 0.0366, ωR2 = 0.0912

R indices (all data) R1 = 0.0387, ωR2 = 0.0944 Largest diff. peak and hole (e Å -3) 0.19 and -0.16

35 23

22 O O 20 18 OH

11 OR 17 19 30 13 O 9 H O 1 10 O havanensin H O 15 3 5 1 R=Ac HO OH H 7 2 R=H HO OH H O 28 O 29 O

O O O H O H O O methyl angosenlate H O OAc gedunin COOMe H 23 O 22 MeO O O R 20 MeO O 18 R OH 17 O 12 21 13 6 H C R D H 11 17 H 13 O 6 S O H OH 14 16 R HO 19 9 O B O S 8 15 O H A 6R-hydroxymexicanolide 10 5 1 30 H S 4 3 3 (khayanone) 2 O O 28 H MeO O 29 O O OH 17 MeO 13 O O 6 H R R H R OH 17 O O 13 6 H OH O H O O H swietenine O H 6R,8-dihydroxycarapin O O Figure 3-13 Chemical structures of 1-3 and related compounds in literature

36 3.5 Structure identification of khayanone 3

Compound 3 (Figure 3-1) was obtained as colorless block-shaped crystals in CHCl 3 and CH3OH solvents. Its molecular formula was determined to be C27 H35 O9 (molecular weight 502) based on accurate ESI-MS ( Figure 3-14) and NMR data ( Figures 3-15 and

3-16). Its NMR data are in good agreement with those reported for khayanone (Nakatani et al ., 2001), isolated from the stem bark of the same plant species, K. senegalensis , and elucidated through NMR and CD spectral analyses.

Single crystal X-ray diffraction experiments were conducted to determine the structure and the stereochemistry (Figure 3-17), specifically the configuration of oxygenated C-6. X-ray crystallography confirmed the structure of khayanone reported by the Nakatani group. This structure (Figure 3-1) is similar to that of mexicanolide with the absolute configurations of C-13 R and C-17 R (Sanni et al ., 1987). Refinement of the

Flack parameter failed to determine the absolute configuration of 3 because of the absence of heavy atoms. On the basis of the similarity to the biogenetic pathway of mexicanolide limonoids in nature, compound 3 has been assigned the name of 6S, 8α- dihydroxy-14,15-dihydrocarapin. The S configuration of C6 is different from those from

X-ray diffraction analysis of swietenine (C-6 R) originally isolated from southern

American mahogany Swietenia macrophylla (Solomon et al ., 2003) or 6 R, 8 α- dihydroxycarapin isolated from African mahogany Khaya anthotheca (Tchimene et al .,

2005) (Figure 3-13). This finding appears to be the first report of a 6 S configuration of mexicanolide limonoid revealed through X-ray diffraction analyses.

37 The six-membered rings A and B exist in boat conformations, the rings C and D in chair conformations, and the furan ring is planar. The crystal structure is stabilized by classic intermolecular hydrogen bonds (O3-H3 ⋅⋅⋅O6 and O6-H6 ⋅⋅⋅O10) and C-H⋅⋅⋅O interactions ( Table 3-2). In addition, It should be pointed out that there are two independent water molecules of crystallization, one (O10) is 100% occupied and the second (O11) is 12.5% occupied. In total, 9 water molecules are present in one crystal unit cell.

1 Khayanone 3 (6 S, 8 α-dihydroxy-14,15-dihydrocarapin): H NMR (in CDCl 3 and trace amount of CD 3OD, 300 MHz, ppm) δ 7.37 (1H, s, H-21), 7.36 (1H, s, H-23), 6.29

(1H, brs, H-22), 5.52 (1H, s, H-17), 4.34 (1H, s, H-6), 3.75 (3H, s, OMe), 3.03 (1H, d, J =

9.6 Hz, H-2), 2.81 (1H, d, J = 15 Hz, H-30 β), 2.70 (1H, d, J =19.2 Hz, H-15 α), 2.67 (1H, brs, H-5), 2.61 (1H, dd, J = 19.2, 7.8 Hz, H-15 β), 2.29 (1H, dd, J = 15, 9.6 Hz, H-30 α),

1.80 (1H, dd, J = 13.5, 5.0 Hz, H-9), 1.75 (1H, m, H-11 β), 1.66 (1H, m, H-12 α), 1.60

(1H, m, H-14), 1.29 (3H, s, H 3-19), 1.20 (3H, s, H 3-29), 1.15 (3H, s, H 3-28), 1.08 (1H,

13 brdt, J = 12.5, 2.4, H-11 α), 1.06 (1H, m, H-12), 0.90 (3H, s, H 3-18); C NMR (in CDCl 3 and trace amount of CD 3OD, 75 MHz, ppm) δ 213.33 (C-1), 54.43 (C-2), 214.61 (C-3),

50.31 (C-4), 46.04 (C-5), 70.64 (C-6), 175.81 (C-7), 72.81 (C-8), 61.32 (C-9), 50.32 (C-

10), 22.71 (C-11), 35.04 (C-12), 35.39 (C-13), 51.04 (C-14), 26.73 (C-15), 171.68 (C-16),

77.29 (C-17), 23.71 (C-18), 23.82 (C-19), 120.97 (C-20), 141.13 (C-21), 109.91 (C-22),

143.22 (C-23), 23.95 (C-28), 23.81 (C-29), 38.90 (C-30), 63.07 (OMe). Accurate ESI-MS

+ + + m/z : 503.2001 [M+H] , 1005.4114 [2M+H] , 485.1951 [M-H2O+H] , 467.1695 [M-

+ + 2H 2O+H] , 407.1953 [M-2H 2O-HCOOCH 3+H] .

Figure 3-14 Accurate ESI-MS spectrum of 3 (khayanone)

Figure 3-15 1H NMR spectrum of 3 (khayanone)

Figure 3-16 13C NMR spectrum of 3 (khayanone)

Figure 3-17 Crystal structure of 3 (khayanone)

42 Table 3-4 Crystal data and structure refinement of 3

Crystal description Colorless plate

Empirical formula C27 H34 O 9·1.125 H 2O Molecular weight 522.81 Temperature (K) 163(2) Wavelength (Å) 0.71073

Crystal system, space group Tetragonal, P4 1212 Unit cell dimensions (Å) a = 13.2315(19), c = 29.118(6) Volume (Å 3) 5097.8 (15) Number of molecules per unit cell, Z 8 Calculated density (Mg/m 3) 1.362 Absorption coefficient ( ) (mm -1) 0.104 Diffraction radiation type Mo Ka F(000) 2234 Crystal size 0.65 mm × 0.38 mm × 0.29 mm θ range for data collection ( °) 2.08 < θ < 25.14 Limiting indices -15 ≤ h ≤ 15, -15 ≤ k ≤ 12, -34 ≤ l ≤ 30

Reflections collected / unique 30465 / 4552 [ Rint = 0.0300] Completeness to θ = 25.14 99.8 % Absorption correction REQAB (multi-scan) Max. and min. transmission 0.9705 and 0.9355 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4552 / 0 / 350 Goodness-of-fit on F2 1.138

Final R indices [ I > 2 σ ( I)] R1 = 0.0375, ωR2 = 0.0906

R indices (all data) R1 = 0.0409, ωR2 = 0.0921 Largest diff. peak and hole (e Å -3) 0.221 and -0.201

43 3.6 Structure identification of 1-O-acetylkhayanolide B 4

Compound 4 (Figure 3-1) is not soluble either in CHCl 3 or in CH 3OH, but is soluble in a mixture of CHCl 3 and MeOH solvents. Its molecular formula was determined to be C29H36O11 (molecular weight 560) based on accurate ESI-MS (m/z 561.2185

[M+H] +, 1121.4210 [2M+H] +) (Figure 3-18) and NMR data.

Its 1D (Figures 3-19 to 3-22) and 2D NMR data ( Figures 3-23 to 3-25) measured in

CDCl 3 and trace CD 3OD are in good agreement with those of 1-O-acetylkhayanolide B obtained in CDCl 3 and trace CD 3OD (Abdelgaleil et al ., 2000) and 1α-acetoxy-

2β,3 α,6,8 α,14 β-pentahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 12 (Figure 3-1) tested in CDCl 3 and trace DMSO-D6 (Olmo et al ., 1996), both isolated from the stem bark of the same species, K. senegalensis . All (Tables 3-5) indicated similar chemical shift values, coupling constants, H-C long-range correlations and coupling patterns. However, the structures of 1-O-acetylkhayanolide B and 12 described in literature were found to differ in planar structures (i.e., an ether linkage between C-2 and C-14 in 1-O- acetylkhayanolide B vs. two independent hydroxyl groups at C-2 and C-14 in 12 ) and in stereochemistry (i.e., a β orientation of C-3 hydroxyl group and an assumed S configuration of C-6 in 1-O-acetylkhayanolide B vs. an α orientation of C-3 hydroxyl and an unresolved configuration of C-6 in 12 ). This discrepancy and the unconfirmed configuration of C-6 prompted the use of CHCl 3 solvent to obtain colorless block-shaped crystals suitable for X-ray diffraction. As a result, this research conducted an in-depth 3D structure study of 4 using single crystal X-ray diffraction analysis to determine its structure and stereochemistry.

44 The crystal structure of 4 with its atomic labeling shown in Figure 3-27 confirms the structure of 1-O-acetylkhayanolide B reported by the Nakatani group (Abdelgaleil et al .,

2000). The crystal data are listed in Table 3-6. The X-ray crystallography, however, did not support 2,14-dihydroxyl groups in 12 as described by the Silva group, as perhaps they did not conduct a mass spectroscopy (Olmo et al ., 1996). In addition, stereochemistry analysis obtained through the X-ray crystallography revealed that the five-membered- rings A1 (C-1, 29, 4, 5, and 10), B1 (C-1, 30, 8, 9, and 10) and B2 (C-2, 30, 8, 14 and O) have half-chair conformations and the furan ring E is planar while the six-membered ring

A2 (C-1, 30, 2, 3, 4 and 29) has a distorted chair conformation and rings C (C-8, 9, 10, 11,

12, 13, and 14) and D (C-17, 13, 14, 15, 16, and O) exhibit a chair conformation. The spatial proximity of 3.37 Å. between H-3 ( α orientation) and H-5 ( β orientation) results in the correlation observed between H-3 and H-5 in NOESY although they are on different sides of the plane. The 33.2º dihedral angle of H (2)-C (2)-C (3)-H (3) accounts for the coupling constant value JH-2,H-3 = 6.5 Hz. Although the JH-2,H-3 value and NOE correlation between H-3 and H-5 are the same, the conclusions from these results reported here differ from those of the Silva group (Olmo et al ., 1996) concerning a boat conformation of the six-membered ring A2 (C-1, 2, 3, 4, 29, 30) and an H-3 β orientation in 12 .

Furthermore, the absolute configuration of C-6 in 4 was confirmed as an S on the basis of the anomalous scattering of the chlorines of the CHCl 3 molecules in the crystal.

The Flack parameter (Flack, 1983) was used to confirm that the correct absolute configuration is the one presented in Figure 3-1. Based on these results, it was concluded that khayanolide 12 is, in fact, 1-O-acetylkhayanolide B 4 ( Figure 3-26).

45 Table 3-5 NMR data of 4 and those reported in the literature

Num. 4a 4a, # 12 b H C H C H C 1 91.2 91.4 91.1 2 4.43 (dd, 6.5, 9.5) 72.1 4.52 (dd, 6.7, 9.7) 72.0 4.45 (dd, 6.7, 9.5) 72.0 3 3.39 (d, 6.5) 78.2 3.48 (d, 6.7) 78.1 3.39 (brd, 6.7) 78.1 4 43.7 44.1 44.1 5 2.99 (d, 8.0) 39.1 3.02 (d, 7.4) 39.4 2.93 (d, 7.0) 39.1 6 4.10 (d, 8.0) 71.1 4.22 (d, 7.4) 71.6 4.15 (d, 7.0) 71.5 7 175.3 175.2 175.1 8 86.6 86.3 86.8 9 2.17 (d, 7.0) 55.2 2.22 (d, 7.6) 55.8 2.16 (brd, 7.0) 55.9 10 61.0 61.2 60.9 11 α 1.78 (m) 16.3 1.85 (m) 16.4 ------16.4 β 1.56 (m) 1.77 (m) ------12 α 0.90 (m) 26.1 0.99 (m) 26.1 0.81 (m) 26.0 β 1.78 (m) 1.84 (m) ------13 37.6 37.7 37.5 14 81.5 81.6 81.5 15 α 2.97 (d, 17.5) 31.9 3.05 (d, 18.8) 31.9 3.04 (d, 18.7) 32. 0 β 2.68 (d, 17.5) 2.75 (d, 18.8) 2.69 (d, 18.7) 16 171.6 170.9 170.3 17 5.58 (s) 81.1 5.60 (s) 80.9 5.50 (s) 80.5 18 1.02 (s) 14.4 1.10 (s) 14.4 1.05 (s) 14.4 19 1.20 (s) 18.0 1.29 (s) 18.0 1.21 (s) 18.1 20 120.5 120.6 120.6 21 7.39 (s) 141.0 7.45 (brd, 1.7) 141.0 7.33 (m) 140.9 22 6.34 (s) 110.0 6.40 (brd, 1.7) 110.0 6.33 (m) 110.1 23 7.33 (s) 142.7 7.40 (t, 1.7) 142.7 7.37 (m) 142.6 28 0.99 (s) 19.0 1.11 (s) 19.2 1.03 (s) 19.3 29 1.75 (d, 12.0) 41.3 1.83 (d, 12.2) 41.2 2.23 (d, 12.2) 41.1 2.21 (d, 12.0) 2.30 (d, 12.2) 1.74 (d, 12.2) 30 3.10 (d, 9.5) 58.5 3.18 (d, 9.7) 58.8 3.15 (d, 9.5) 58.7 OMe 3.70 (s) 52.2 52.5 3.69 (s) 52.2 OCOMe 1.97 (s) 171.0 3.77 (s) 170.7 1.97 (s) 170.4 21.7 2.03 (s) 21.7 22.0 Note: δ in ppm relative to the internal TMS, multiplicities (s, single; d, double; t, triplet; m, multiplet; br, broad; and coupling constants ( J inHz) are given in brackets. Assignments are based on extensive 2D NMR data including COSY, HMQC and HMBC. # data are from previous literature. “------” data are absent. The assignments of H-2, 30 of 12 from references were exchanged for comparison with a underline marker. a in

b CDCl 3 and trace CD 3OD; in CDCl 3 and trace DMSO-D6.

Figure 3-18 Accurate ESI-MS spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-19 1H NMR spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-20 13C NMR spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-21 DEPT135 ° spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-22 DEPT90 ° spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-23 HMQC spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-24 1H-1H COSY spectrum of 4 (1-O-acetylkhayanolide B)

Figure 3-25 HMBC spectrum of 4 (1-O-acetylkhayanolide B)

54 22 O O MeO 20 O 18 12 21 MeO 7 17 11 O H O O HO 19 13 OH 6 OH 14 16 HO

5 9 O 10 8 15 revised to be O 4 OH 1 30 O 28 OAc OAc 29 OH 2 3 4 OH O OH 12 O MeO O MeO O H HO O OH O OH HO O revised to be O OH O OH OH OH 23 5 22 OH O OH 13 O MeO 20 18 O 21 12 MeO 17 H O 11 HO O 7 13 O OH H OH HO 19 14 16 O 9 revised to be 6 10 8 O OAc OH 15 O 5 1 OAc 4 29 30 OH 2 28 3 H O 6 O O 14 O MeO O MeO H O HO O O H OH OH HO 6 O revised to be O OH OH O OH OH H O 7 O 15 Figure 3-26 Structural revision of 12 -15 to 4-7

Figure 3-27 Crystal structure of 4 (1-O-acetylkhayanolide B)

56 Table 3-6 Crystal data and structure refinement of 4

Crystal description Colorless block

Empirical formula C29 H36 O 11 ·0.79CHCl 3 Molecular weight 654.70 Temperature (K) 298 (2) Wavelength (Å) 0.71073

Crystal system, space group Monoclinic, P2 1 Unit cell dimensions (Å) a = 8.2283 (16), b = 11.057(2) c = 17.704(4), β = 99.68(3)º Volume (Å 3) 1587.8(5) Number of molecules per unit cell, Z 2 Calculated density (Mg/m 3) 1.369 Absorption coefficient ( ) (mm -1) 0.293 Diffraction radiation type Mo Ka F(000) 687 Crystal size 0.50 mm × 0.24 mm × 0.12 mm θ range for data collection ( °) 2.94 < θ < 25.05 Limiting indices -9 ≤ h ≤ 9, -12 ≤ k ≤ 13, -21 ≤ l ≤ 21

Reflections collected / unique 16840 / 2485 [ Rint = 0.037] Completeness to θ = 25.05 99.3 % Absorption correction REQAB (multi-scan) Max. and min. transmission 0.9657 and 0.8673 Data / restraints / parameters 5305 / 1 / 406 Goodness-of-fit on F2 1.096

Final R indices [ I > 2 σ ( I)] R1 = 0.0513, ωR2 = 0.1430

R indices (all data) R1 = 0.0539, ωR2 = 0.1447 Absolute structure parameter 0.04 (12) Largest diff. peak and hole (e Å -3) 0.445 and -0.351

57 3.7 Structure identification of khayanolide B 5

Compound 5 ( Figure 3-1) was found to have the molecular formula of C27H34O10

(molecular weight 518) as determined through ESI-MS (m/z 519.2224 [M+H] +, 541.2013

[M+Na] +) (Figure 3-28) and NMR experiments. Its NMR data (Tables 3-7) are similar to those of 1-O-khayanolide B 4 except for the absence of the acetyl group at C-1. The low- frequency shift of δ 84.0 (C-1) in 5 and δ 91.2 (C-1) in 4 suggests 5 is khayanolide B, a conclusion supported by the 2D NMR experiments. In fact, the NMR data of 5 are in good agreement with those reported for khayanolide B (Abdelgaleil et al ., 2001) and khayanolide 1α,2 β,3 α,6,8 α,14 β-hexahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 13

(Olmo et al., 1996) ( Figure 3-1), both isolated from the stem bark of the same species, K. senegalensis . Since the only difference between 13 and 12 is a substitute group change at

C-1 and khayanolide 12 was found to be the same as 4 through X-ray crystallography analysis, it was concluded based on spectral correlation that khayanolide 13 is, in fact, khayanolide B 5. In addition, the biogenetic pathway indicates the configuration of oxygenated C-6 of 2 is an S, the same as that in 4. {Note: (1) The Nakatani group

(Abdelgaleil et al ., 2001) reported the MS data of khayanolide as HRFABMS m/z

+ 519.2079 [M+H] , calcd for C27H35O10 519.2231. The Silvo group reported (Olmo et al.,

+ 1996) the following EI-MS m/z (rel. int.) data: 518 [M-H2O] (73), 380 (100), 95 (80).

Based on these data, it is concluded that peak 518 is the molecular ion peak. (2) 1-O- acetyllkhayanolide and corresponding 1-O-deacetyl khayanolide co-occurred as a pair frequently in Khaya senegalensis , for example as khayanolide A, 1-O-khayanolide A, 1-

O-acetylkhayanolide B, khayanolide B (Abdelgaleil et al ., 2003).}

Figure 3-28 Accurate ESI-MS spectrum of 5 (khayanolide B)

59 Table 3-7 NMR data of 5 and those reported in literatures

Num. 5 a 5a, # 13 b H C H C H C 1 84.0 84.3 83.5 2 4.26 (dd, 6.5,9.5) 72.3 4.50 (dd ,9.5, 8.6) 72.2 4.45 (dd, 9.5, 6.7) 72.3 3 3.20 (d, 6.5) 78.6 3.41 (d, 6.8) 78.5 3.42 (brd, 6.7) 78.2 4 42.7 42.7 42.7 5 3.00 (d, 7.8) 40.9 3.05 (d, 7.3) 40.9 2.88 (d, 6.8) 40.8 6 4.12 (d, 7.8) 71.3 4.21 (d, 7.3) 71.6 4.03 (dd, 6.8, 3.6) 71.4 7 175.3 175.4 175.1 8 86.9 87.0 86.6 9 2.20 (d, 7.6) 55.5 2.09 (d, 8.1) 56.1 2.03 (brd, 9.0) 55.3 10 59.3 59.4 59.2 11 α 1.85 (m) 16.3 1.86 m 16.5 1.60 m 16.4 β 1.75 (m) 1.74 m 1.74 m 12 α 0.91 (m) 26.1 0.97 m 26.0 0.62 (brd, 12.0) 25.9 β 1.81 (m) 1.85 m 1.78 m 13 37.6 37.7 37.1 14 81.5 81.5 80.7 15 α 3.04 (d, 17.5) 32.0 3.14 (d, 18.8) 32.0 2.86 (d, 12.0) 32.3 β 2.67 (d, 17.5) 2.78 (d, 18.8) 2.63 (d, 12.0) 16 171.5 171.4 170.5 17 5.55 (s) 81.1 5.61 (s) 81.0 5.61 (s) 81.0 18 1.02 (s) 14.4 1.10 (s) 14.4 0.96 (s) 14.5 19 1.24 (s) 17.8 1.21 (s) 17.7 1.07 (s) 18.6 20 120.5 120.7 121.3 21 7.41 (s) 141.0 7.45 (brs, 0.7) 140.9 7.58 (m) 141.2 22 6.34 (s) 110.0 6.41 (dd, 1.7, 0.7) 110.0 6.45 (m) 110.5 23 7.35 (s) 142.7 7.40 (t, 1.7) 142.6 7.62 (m) 143.2 28 0.95 (s) 19.1 1.09 (s) 19.2 0.95 (s) 19.7

29 pro-R 1.70 (d, 12.0) 44.5 1.88 (d, 12.2) 44.6 1.68 (d, 12.0) 45.3 Pro-S 2.13 (d, 12.0) 1.37 (d, 12.2) 1.16 (d, 12.0) 30 2.88 (d, 9.5) 63.2 2.60 (d, 9.5) 63.3 2.48 (d, 9.2) 63.1 OMe 3.62 (s) 52.2 3.77 52.1 3.57 (s) 51.4 Note: # data reported in the literature. δ in ppm relative to the internal TMS, multiplicities (s, single; d, double; t, triplet; m, multiplet; br, broad; and coupling constants ( J inHz) are given in brackets. a Assignments are based on extensive 2D NMR data including COSY, HMQC and HMBC. in CDCl 3 and b trace CD 3OD; in CDCl 3 and trace DMSO-D6. The proton signals of hydroxyls in 13 were also observed as δ 5.41 (d, 3.6 C-6, OH), 4.95 (brs), 4.50 (brs), 3.70 (d, 9.0, C-3 OH) reported in the literature.

60 3.8 Structure identification of khayanolide E 6

Compound 6 (Figure 3-1) is not soluble in MeOH but is soluble in the mixed solvents of CHCl 3 and MeOH. Its molecular formula was determined to be C29H34O11

(molecular weight 558) based on LC-ESI-MS (negative ion mode m/z 603.7

[M+HCOOH-H] +, 1115.6 [2M-H] + and positive ion mode m/z 559.3 [M+H] +, 581.3

[M+Na] +, 1117.5 [2M+H] +, 1139.4 [2M+Na] +) ( Figures 3-29 and 3-30 ) and NMR data

(Figures 3-31 to 3-33).

Its NMR data (Table 3-8) are in good agreement with those of khayanolide E

(Nakatani et al ., 2002) and phragmalin derivative methyl 1α-acetoxy-6,8 α,14 β,30 β- tetrahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]-tricyclomeliac-7-oate 14 (Figure 3-1) (Olmo et al .,

1997), both isolated from the stem bark of the same plant species, K. senegalensis .

However, the following differences between khayanolide E and 14 have been reported in past research: Khayanolide E exhibits an ether linkage between C-2 and C-14 and a proposed 6 S configuration while 14 exhibits two independent hydroxyl groups at C-14 and C-30 and an unresolved C-6 configuration; furthermore, khayanolide E is a khayanolide limonoid while 14 is a phragmalin limonoid because of the locations of C-2 and C-30. In addition, khayanolide E and 14 have essentially identical spectroscopic properties. These structural differences resulted in an in-depth 3D structure study of 6 using single crystal X-ray diffraction analysis. The block-shaped crystals suitable for X- ray diffraction experiments were obtained from the mixture solvents of CHCl 3 and

MeOH with slow solvent evaporation at room temperature. The crystal data of 6 are listed in Table 3-9.

61 The crystal structure of 6 with its atomic labeling shown in Figure 3-34 confirmed the structure of khayanolide E reported by the Nakatani group (Nakatani et al ., 2002), indicating that khyanolide 14 is the same as khayanolide E 6.

The reasons that the Silvo group (Olmo et al ., 1997) concluded phragmalin 14 , not khayanolide 6, were perhaps based on the following three factors: (1) they did not observe the molecular ion peak in the EI-MS spectrum report as EI-MS m/z (rel. int.): 446

+ [M-(CH 2=CO+HCOCOOMe)] , (100), 168 (15) (Olmo et al ., 1997). (2) they did not consider other possibilities besides phragmalin as being compatible with the 2D NMR data. It was taken for granted at that paper phragmalin limonoid was an intermediate from mexicanolide to khayanolide based on the view of biogenetic pathway (Olmo et al ., 1996;

Olmo et al ., 1997), leading to the proposed structure of phragmalin; (3) they did not take into account the important HMBC correlations between H-30 (1H, δ 3.28, d, J = 10.5) and C-9 ( δ 55.1, CH), and between H-2 (1H, δ 4.34, d, J = 10.5) and C-4 ( δ 51.4, C), observed in both the experiments reported here and those of the Nakatani group. These three factors proved critical in determining the structural similarities and differences between khayanolides and phragmalins.

Although phragmalin may be an intermediate in the transformation from mexicanolide to khayanolide ( Figure 3-35) in nature on the basis of the biogenetic pathway, until recently, no other phragmalins have been isolated and reported from the

Khaya genus except the incorrect 14 and the following 15 . Perhaps the concentration of phragmalin in the plants is too low or it is easily transformed to khayanolide with specific enzymes.

62 Table 3-8 NMR data of 6 and those reported in literatures

Num. 6a 6# a 14 c H C H C H C 1 90.8 91.4 90.1 2 4.34 (d, 10.5) 74.3 4.38 (d, 10.5) 74.0 4.40 (d, 10.4) 74.1 3 205.8 204.8 205.4 4 51.4 52.3 50.5 5 2.87 (brd,4.8) 40.9 3.02 (d, 4.9) 42.1 3.07 (d, 8.4) 40.0 6 4.32 (d, 4.8) 70.7 4.34 (d, 4.9) 71.7 4.13 (8.4,5.0) 69.6 7 174.4 174.9 173.4 8 86.8 87.7 86.6 9 2.20 (d, 9.3) 55.1 2.36 (brd,9.5) 57.6 2.30 (brd 8.4) 54.5 10 61.6 62.1 60.9 11 α 1.86 (m) 16.4 1.92 (m) 16.7 1.64-1.72 (m) 16.4 β 1.53 (m) 1.54 (m) 1.90 (m) 12 α 0.83 (m) 26.0 0.94 (m) 26.9 0.74 (brd,10.8) 26.4 β 1.63 (m) 1.71 (m) 1.64-1.72 (m) 13 37.5 37.5 37.0 14 83.5 83.3 83.4 15 α 3.06 (d, 18.6) 32.7 3.08 (d, 18.8) 32.5 2.96 (d, 18.8) 33.0 β 2.58 (d, 18.6) 2.64 (d, 18.8) 2.72 (d, 18.8) 16 171.6 170.0 169.7 17 5.44 (s) 80.9 5.52 (s) 79.9 5.26 (s) 80.0 18 1.03 (s) 14.2 1.10 (s) 14.2 1.03 (s) 14.7 19 1.34 (s) 18.6 1.41 (s) 20.1 1.33 (s) 18.0 20 120.4 120.6 120.7 21 7.34 (s) 141.1 7.40 (brs) 141.1 7.68 (s) 141.7 22 6.30 (s) 110.0 6.36 (d, 1.5) 110.1 6.47 (s) 110.5 23 7.33 (s) 142.9 7.39 (t, 1.7) 142.9 7.67 (s) 143.5 28 1.27 (s) 15.3 1.26 (s) 15.9 0.92 (s) 15.1 29 2.08 (d, 12.6) 40.8 2.12 (d, 12.7) 40.7 2.04 (d, 10.0) 40.7 2.75 (d, 12.6) 2.78 (d, 12.7) 2.55 (d, 10.0) 30 3.28 (d, 10.5) 58.5 3.37 (d, 10.5) 59.7 3.27 (d, 10.5) 58.1 OMe 3.69 (s) 52.2 3.75 (s) 52.5 3.61 (s) 51.8 OCOMe 2.01 (s) 171.2 2.07 (s) - 2.02 (s) 170.2 21.6 - 22.0 Note: # data reported in the literature. δ in ppm relative to the internal TMS, multiplicities (s, single; d, double; t, triplet; m, multiplet; br, broad; and coupling constants ( J inHz) are given in brackets. “-“ data are absent in the reference. The assignments of H-2, 30 of 14 from the reference were exchanged for

a c comparison with a underline marker. in CDCl 3 and trace CD 3OD; in DMSO-D6. The proton signals of hydroxyls in 14 were also observed as δ 2.86 (d, 9.6, C-3 OH), 2.54 (d, 7.2, C-6 OH).

Figure 3-29 ESI-MS (negative ion mode) spectrum of 6 (khayanolide E)

Figure 3-30 ESI-MS (positive ion mode) spectrum of 6 (khayanolide E)

Figure 3-31 1H NMR spectrum of 6 (khayanolide E)

Figure 3-32 13C NMR spectrum of 6 (khayanolide E)

Figure 3-33 DEPT135 ° spectrum of 6 (khayanolide E)

Figure 3-34 Crystal structure of 6 (khayanolide E)

69 Table 3-9 Crystal data and structure refinement of 6

Crystal description Colorless block

Empirical formula C29 H34 O11 Molecular weight 558.56 Temperature (K) 168 (2) Wavelength (Å) 0.71073

Crystal system, space group Monoclinic, P2 1 Unit cell dimensions (Å) a = 12.9096 (18), b = 8.0852 (17), c = 14.1008 (19), β = 116.925 (7)º Volume (Å 3) 1312.3 (5) Number of molecules per unit cell, Z 2 Calculated density (Mg/m 3) 1.414 Absorption coefficient ( ) (mm-1) 0.11 Diffraction radiation type Mo Ka F(000) 595 Crystal size 0.53 mm × 0.29 mm × 0.24 mm θ range for data collection ( °) 3.2 < θ < 25.1 Limiting indices -15 ≤ h ≤ 15, -9 ≤ k ≤ 9, -16 ≤ l ≤ 15

Reflections collected / unique 10799 / 4493 [ Rint = 0.043] Completeness to θ = 25.05 99.3 % Absorption correction REQAB (multi-scan) Max. and min. transmission 0.9657 and 0.8673 Data / restraints / parameters 4493 / 1 / 368 Goodness-of-fit on F2 1.050

Final R indices [ I > 2 σ ( I)] R1 = 0.0391, ωR2 = 0.1009

R indices (all data) R1 = 0. 0406, ωR2 = 0.1019 Absolute structure parameter 0.1 (8) Largest diff. peak and hole (e Å -3) 0.26 and -0.34

70 24 27

20 25

HO HO HO H Euphane (H-20 S) Tirucallane (H-20 R)

HO HO OH H HO H H

HO O O Four carbons C24, C25 C C26, C27 D are lost HO O tetranortriterpenoid O A B O O

HO OH H limonoid O

O O D- seco O

O 8 14 1 O B- seco H 30 O 6 H O O H recyclise COOCH HO OH 3 H

O O O

COOCH3 O COOCH O COOCH3 O OH 3 H OH

8 O O O 8 8 1 30 O O OH 1 1 29 30 30 29 OH 2 2 H 2 H O O 29 O Figure 3-35 Biogenetic pathways of B,D-seco limonoids

71 3.9 Structure identification of 1-O-deacetylkhayanolide E 7

Compound 7 is soluble in MeOH but not in CHCl 3. Its molecular formula was determined to be C27H32O10 (molecular weight 516) based on accurate ESI-MS (positive ion model m/z 517.1382 [M+H] +, 539.1089 [M+Na]+, 1033.2683 [2M+H] +, 1055.2358

[2M+Na] +) ( Figure 3-36) and NMR data.

Its 1D NMR ( Figures 3-37 to 3-40 ) and 2D NMR ( Figures 3-41 to 3-43) are similar to those of khayanolide E 7 except for the absence of the acetyl group at C-1. The low- frequency shift of δ 84.0 (C-1) in 7 and δ 90.8 (C-1) in 6 suggests 7 is 1-O-deacetyl khayanolide E, a conclusion supported by the 2D NMR experiments. In fact, the NMR data of 7 are in good agreement with those reported for 1-O-deacetyl khayanolide E isolated from K. grandfolia (Zhang, et al., 2008) and phragmalin derivative

1α,6,8 α,14 β,30 β-pentahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]-tricyclomeliac-7-oate 15 (Figure 3-

1), isolated from the stem bark of the same species, K. senegalensis (Olmo et al ., 1997).

Since the only difference between 15 and 14 is a substitute group change at C-1 and phragmalin 14 was found to be the same as 6 through X-ray crystallography analysis, it was concluded based on spectral correlation that phragmalin 15 is, in fact, 1-O-deacetyl khayanolide E 7.

In addition, the biogenetic pathway indicates the configuration of oxygenated C-6 of

7 is an S, the same as that of 6. Since different solvents were used in the research for these compounds, the NMR data of 7 assigned through 2D NMR experiments are listed for comparison in Tables 3-10 . It seems there is no significant difference in 13 C chemical shift value change (< 1 ppm) among those measured in CDCl 3, or DMSO-d6, or C 5D5N

72 1 and in the H chemical shift value change (< 0.2 ppm) between CDCl 3 and DMSO-d6

1 while differences are from the H NMR data (> 0.5-1 ppm) between CDCl 3 and C 5D5N.

The reasons that led the Silvo group (Olmo et al ., 1996; Olmo et al ., 1997) to conclude a phragmalin 15 instead of the khayanolide 7, are perhaps ascribed to the following factors similar to those concerning 14 and 6 : (1) wrong assignment of the observed molecular ion in the EI-MS spectrum reported as EI-MS m/z (rel. int.): 516 [M-

+ H2O] (73), 378 (100) (Olmo et al ., 1997). The 516 ion peak was, in fact, the molecular ion peak, just like those EI-MS m/z 516 [M] + (86), 378 (100) elucidated by the Jianmin

Yue group (Zhang et al ., 2008); (2) they did not consider other possibilities besides phragmalin as being compatible with the 2D NMR data; (3) they did not take into account the important HMBC correlations between H-30 and C-9, and between H-2 and C-4 observed in both the experiments reported here and those of the Jieming Yue group

(Zhang et al ., 2008). These correlations proved critical in determining the structural similarities and differences between khayanolides and phragmalins.

Overall, X-ray crystallography results of the structure reported here confirm the structure and the stereochemistry 1-O-deacetylkhayanolide B 4 and khayanolide E 6 reported by the Nakatani group, revealing that the two reported khayanolides 12 and 13 are, in fact, 1-O-acetylkhayanolide B 4 and khayanolide B 5, respectively, and that the two reported phragmalin derivatives 14 and 15 are, in fact, khayanolide E 6 and 1-O- deacetylkhayanolide E 7, respectively. These incorrect structures 12 -15 were cited by other research groups in addition to the Nakatani group. (Fall et al ., 1999; Mulholland et al ., 2000; Ferreira et al ., 2005; Narender et al ., 2007).

73 All the C-6 configurations in these khayanolides were determined to be S, the same as that of khayanolide A. In contrast, all the oxygenated C-6 configurations in the mexicanolide limonoids from the original mahogany Swietenia genus were reported to be

R, the same as that of swietenine isolated from Swietenia macrophylla and confirmed to have a 6 R configuration through X-ray crystallography (McPhail et al ., 1964). Past research accepted that methyl 6-hydroxyangolensate is a common precursor (Connolly et al ., 1964; Taylor, 1984) of further derived C-6 oxygenated limonoids such as mexicanolides, phragmalins, and khayanolides. However, the C-6 configuration of the methyl 6-hydroxyangolensate was not fully clarified in the past because its stereochemistry is unable to be determined through routine spectra. Considering that the configuration of oxygenated C-6 does not change during its transformation from methyl

6-hydroxyangolensate to mexicanolides, phragmalins, and khayanolides, it is reasonable to assume the methyl 6-hydroxyangolensate from the K. senegalensis (Adesida et al.,

1967) should have a 6 S configuration while methyl 6-hydroxyangolensate (Saad et al .,

2003) from the Swietenia species should have a 6 R configuration. The methyl 6 S- hydroxyangolensate from K. senegalensis was proposed by Taylor forty years ago, though it was not verified at that time (Adesida et al ., 1967; Adesogan et al ., 1968). The results reported here not only confirm the C-6 S configuration assignment of the khayanolides but also indirectly support the structure of methyl 6 S-hydroxyangolensate deduced by the Taylor group. The configuration difference of oxygenated C-6 in B, D- seco limonoids imply a significant chemotaxonomy between the African mahogany

Khaya genus and the genuine mahogany Swietenia genus.

74 Table 3-10 NMR data of 7 and those reported in the literature

Num. 7a 7b, # 15 c H C H C H C 1 84.0 84.8 83.6 2 4.24 (d, 10.4) 74.8 4.83 (d, 10.4) 75.8 4.37 (d, 10.3) 74.5 3 206.7 207.3 206.5 4 49.4 51.8 49.3 5 3.08 (d, 8.7) 42.2 4.09 (brs) 43.1 3.25 (d, 8.0) 41.9 6 4.25 (d, 8.7) 70.2 4.90 (brs) 72.7 4.22 (dd,8.0, 5.0) 70.0 7 174.2 175.9 173.6 8 87.1 88.5 86.8 9 2.28 (d, 9.3) 55.3 3.07 (d, 8.7) 56.8 2.28 (brd, 10.0) 54.6 10 59.5 59.4 59.3 11 α 2.04 (m) 16.1 2.16 17.1 1.77-1.83 (m) 16.2 β 1.81 (m) 1.92 1.90 (m) 12 α 0.91 (m) 26.4 2.22 26.6 0.85 (brd, 9.0) 26.3 β 1.81 (m) 1.08 (brd, 11.2) 1.77-1.83 (m) 13 37.4 38.4 36.9 14 83.4 84.3 83.0 15 α 3.06 (d, 18.8) 33.7 3.83 (d, 18.6) 34.2 3.10 (d, 18.6) 33.1 β 2.69 (d, 18.8) 3.36 (d, 18.6) 2.69 (d, 18.6) 16 172.0 170.4 169.9 17 5.43 (s) 81.2 5.85 (s) 80.7 5.40 (s) 80.2 18 1.03 (s) 13.9 1.39 (s) 14.6 1.11 (s) 14.6 19 1.24 (s) 17.2 1.94 (s) 21.0 1.33 (s) 18.3 20 120.8 121.9 120.7 21 7.41 (d, 1.7) 141.2 7.65 (s) 141.7 7.59 (m) 141.5 22 6.35 (d, 1.7) 110.0 6.55 (brs) 110.9 6.47 (m) 110.4 23 7.39 (d, 1.7) 142.8 7.64 (s) 143.4 7.56 (m) 143.3 28 0.91 (s) 14.4 1.72 (s) 16.2 0.99 (s) 15.3 29 1.72 (d, 12.0) 44.0 2.17 (s) 46.5 1.73 (d, 12.6) 44.3 2.03 (d, 12.0) 2.67 (s, 12.2) 2.11 (d, 12.6) 30 2.73 (d, 10.4) 63.2 3.49 (d, 10.4) 65.1 2.88 (d, 10.3 ) 62.7 OMe 3.62 (s) 51.3 3.73 (s) 52.2 3.66 (s) 51.6 Note: # data reported in the literature. δ in ppm relative to the internal TMS, multiplicities (s, single; d, double; t, triplet; m, multiplet; br, broad; and coupling constants ( J inHz) are given in brackets. The assignments of H-2, 30 of 15 from references were exchanged for comparison with a underline marker. a in

b c # CDCl 3 and trace CD 3OD; in C 5D5N; in DMSO-D6. reported in the literatures. The proton signals of hydroxyls in 15 c were also observed at δ 5.44 (d, 5.0), 4.97 (brs), 4.60 (s). The proton signals of hydroxyls in 7b,# were at δ 6.99 (brs, C-1 OH), 7.57 (brs, C-6 OH), 7.36 (brs, C-8 OH).

Figure 3-36 Accurate ESI-MS spectrum of 7 (1-O-deacetylkhayanolide E)

Figure 3-37 1H NMR spectrum of 7 (1-O-acetylkhayanolide E)

Figure 3-38 13C NMR spectrum of 7 (1-O-acetylkhayanolide E)

Figure 3-39 DEPT135 ° spectrum of 7 (1-O-acetylkhayanolide E)

Figure 3-40 DEPT90 ° spectrum of 7 (1-O-acetylkhayanolide E)

80 50

100

ppm (t1

7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm (t2)

Figure 3-41 HMQC spectrum of 7 (1-O-acetylkhayanolide E)

81 1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50 ppm (t1

4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 ppm (t2)

Figure 3-42 1H-1H COSY spectrum of 7 (1-O-deacetylkhayanolide E)

Figure 3-43 HMBC spectrum of 7 (1-O-deacetylkhayanolide E)

83 3.10 Structure elucidation of 6-dehydroxykhayanolide E 8

Compound 8 was soluble in CHCl 3 but not in MeOH. Its molecular formula was determined to be C29H34O10 (molecular weight 542) through LC-ESI-MS (positive ion mode Figure 3-45 and negative ion mode Figure 3-46), EI-MS (Figure 3-47), high resolution EI-MS ( Table 3-12 ), and NMR data ( Table 3-11 ). As a result, the degree of unsaturation is 13.

The strong IR absorption peaks (3431, 1736, 1703 cm -1) (Figure 3-44) indicate the presence of hydroxyl and ester carbonyl groups. The 1H NMR spectrum ( Figure 3-48) showed the presence of six singlet methyls δ 3.67 (-OMe), 2.02 (-OAc), 1.26, 1.10 and

0.99. Twenty-nine carbon signals were observed in the 13 C NMR spectrum ( Figure 3-49).

Their multiplicities of these carbons determined through DEPT 135 ° ( Figure 3-50 ) and

DEPT 90 ° ( Figure 3-51) resulted in 5 CH 3, 5 CH 2, 8 CH, and 11 C, including one ketone carbonyl ( δ 204.1), three ester carbonyls ( δ 172.5, 170.1, 169.7), a β-substituted furan ( δ

142.8, 141.2, 120.4, 110.1), three oxygenated carbons ( δ 90.9, 87.8, 83.5), two oxygenated methines ( δ 80.1, 74.5), and one oxygenated methyl ( δ 52.1). Based on the molecular formula and the DEPT spectra, only one hydroxyl group was present in the structure. The signals of the β-substituted furan ring in the 1H NMR spectrum occurred at

δ 7.40 (1H, s, H-23), 7.38 (1H, s, H-21), 6.36 (1H, s, H-22) and the corresponding carbon signals at 142.8 (C-23, CH), 141.2 (C-21, CH), 110.2 (C-22, CH), respectively, in the

HMQC spectrum ( Figure 3-52).

Based on the starting point of the furan ring and the combination of the coupling constants in 1H NMR spectrum; chemical shift values in proton and carbon spectra; and

84 the 1 H-1H COSY ( Figure 3-53), HMBC ( Figure 3-54), and NOESY ( Figure 3-55) correlations, the structure of 8 could be deduced as Figure 3-1, and the connection of these structural fragments are shown in Figure 3-56.

In fact, the NMR data of 8 are similar to those of khayanolide E 6, except for the presence of the C-6 methine ( 1H NMR: 1H, δ 4.32, d, J = 4.8 Hz; 13 C NMR: δ 70.7 CH) in 6 and the methylene assigned to C-6 ( 1H NMR 1H, δ 2.46, dd, J = 11.0, 17.0 Hz and

13 1H, δ 2.31, dd, J = 4.0, 17.0 Hz; C NMR: δ 34.0, CH 2) in 8, suggesting 8 was a 6- dehydroxy derivative of 6. This conclusion was supported through COSY, HMQC,

HMBC, NOESY experiments and finally confirmed through the single-crystal X-ray diffraction (Figure 3-57 and Table 3-13 ). The full assignments of the NMR data of 6- dehydroxylkhayanolide E 8 are listed in Table 3-11.

Similar to those of 1-O-acetylkhayanolide B 4 and khayanolide E 6, the five- membered-rings A 1 (C-1, 29, 4, 5, 10), B 1 (C-1, 30, 8, 9 10) and B 2 (C-2, 30, 8, 14 and O) of compound 8 have half-chair conformations, the furan ring E is planar, and the six- membered rings C, D exist in a chair conformation.

For 6-dehydroxykhayanolide E 8, colorless block-shaped crystals were obtained

20 -1 from CHCl 3 and MeOH solvents, [ α]D 26 (0.14, CHCl 3); IR νmax ( KBr) cm : 3430 (br),

2923, 1736 (s), 1703 (s), 1465, 1383, 1242 (s), 1167, 1014, 875; ESI-MS m/z (positive ion mode) 543.2 [M+H] +, 1085.5 [2M+H] +, 1107.5 [2M+Na] +; (negative ion mode) 587.4

[M+HCOOH-H] +, 1083.4 [2M-H] +, EI-MS m/z (rel. int.): 542 [M] + (65), 446 (48), 404

(100), 386 (19), 358 (37), 344 (49), 312 (13), 238 (53), 215 (37), 182 (18), 125 (21);

+ HREI-MS m/z 542.2151 [M] (calcd. for C 29 H34 O10 , 542.2152).

85 Table 3-11 NMR data of 8 (δ in ppm) measured in CDCl 3

Num. H C COSY HMBC (H to C) NOESY 1 90.9 C 2 4.42 (d, 10.5) 74.5 CH H-30 C-1, 3, 4 H-15 β, 30 3 204.0 C 4 51.8 C 5 3.47 (dd, 11.0, 4.0) 35.3 CH H2-6 C-4, 6, 7, 9, H-12 β, 6 6 2.46 (dd,11.0, 17.0) 34.0 CH 2 H-5, H-6 C-4, 5, 7, 10 H-5, 6, 28 2.31 (dd, 4.0, 11.0) H-5, H-6 C-4, 5, 7, 10 H-6 7 172.5 C 8 87.8 C 9 2.27 (brd, 9.5) 55.8 CH H2-11 C-5, 10, 11, 12, 19 H-6, 11, 19 10 59.4 C 11 α 1.98 (m) 16.3 CH 2 H-9, 11 β, 12 C-12, 13 H-11 β,12 β 1.52 (m) H-9, 11 α, 12 C-12, 13 H-11, 12

12 α 1.04 (m) 25.9 CH 2 H-11, 12 β C-11 H-11,12 β β 1.85 (m) H-11, 12 α C-11, 13 H-11, 12 13 37.7 C 14 83.5 C 15 α 3.14 (d, 18.5) 32.7 CH 2 H-15 α C-8, 16 H-15 β, 18 β 2.66 (d, 18.5) H-15 β C-8, 13, 16 H-15 16 169.7 C 17 5.55 (s) 80.1 CH C-12, 13, 18, 20, 21, 22 H-12 β, 22

18 1.10 (s) 14.3 CH 3 C-12, 13, 14, 18 19 1.26 (s) 18.5 CH 3 C-1, 5, 9, 10 20 120.5 C 21 7.39 (s) 141.2 CH C-20, 22, 23 H-17, 18, 22, 23 22 6.35 (s) 110.1 CH C-20, 21, 23 H-17, 18 23 7.37 (s) 142.8 CH C-20, 21, 22 H--21, 22 28 0.99 (s) 15.0 CH 3 C-3, 4, 5, 29 H-15 β 29 1.93 (d, 13.5) 40.6 CH 2 H-29 C-1, 3, 4, 10 3.14 (d, 13.5) H-29 C-1, 3, 5, 10 30 3.36 (d, 10.5) 60.0 CH C-1, 2, 3, 8, 9, 10 H-2, 15 β OMe 3.67 (s) 52.0 CH 3 C-7 OCOCH 3 2.02 (s) 170.1 C OCOCH3 21.9 CH 3 C-170.1

Note: 1H NMR 500 MHz and 13 C 125 MHZ. The important HMBC correlations between H-2 and C-4, between H-30 and C-9 are marked in bold. Multiplicities (s, single; d, double; t, triplet; m, multiplet; br, broad) and coupling constants ( J in Hz) are given in brackets.

Figure 3-44 IR spectrum of 8 (6-dehydroxylkhayanolide E)

Figure 3-45 ESI-MS (positive ion mode) of 8 (6-dehydroxykhayanolide E)

Figure 3-46 ESI-MS (negative ion mode) of 8 (6-dehydroxykhayanolide E)

Figure 3-47 EI-MS spectrum of 8 (6-dehydroxykhayanolide E)

90 Table 3-12 HR-EI-MS data of compound 8 (6-dehydroxykhayanolide E)

Mass Intensity %RA %RIC Delta (mmu) R+D Composition 71.08617 36755 20.62 0.26 -0.1 0.5 C5 H11 73.02961 20087 11.27 0.14 -0.7 1.5 C3 H5 O2 77.03990 12536 7.03 0.09 -0.8 4.5 C6 H5 79.05588 16098 9.03 0.11 -1.1 3.5 C6 H7 81.06986 21084 11.83 0.15 0.6 2.5 C6 H9 83.08552 23506 13.19 0.17 0.6 1.5 C6 H11 85.10142 30202 16.95 0.21 0.3 0.5 C6 H13 91.05428 28278 15.87 0.20 0.5 4.5 C7 H7 93.07042 15599 8.75 0.11 0.0 3.5 C7 H9 95.04981 18448 10.35 0.13 -0.1 3.5 C6 H7 O 95.08591 13818 7.75 0.10 0.2 2.5 C7 H11 97.10145 25500 14.31 0.18 0.3 1.5 C7 H13 105.0649 27352 15.35 0.19 111.9883 13676 7.67 0.10 119.0854 18733 10.51 0.13 0.7 4.5 C9 H11 121.0653 20657 11.59 0.15 0.0 4.5 C8 H9 O 123.0804 12607 7.07 0.09 0.5 3.5 C8 H11 O 125.0602 37752 21.18 0.27 0.0 3.5 C7 H9 O2 131.0855 16668 9.35 0.12 0.6 5.5 C10 H11 133.0646 15528 8.71 0.11 0.7 5.5 C9 H9 O 135.0806 19659 11.03 0.14 0.4 4.5 C9 H11 O 147.0804 20372 11.43 0.14 0.6 5.5 C10 H11 O 159.0801 15172 8.51 0.11 0.8 6.5 C11 H11 O 161.0962 12607 7.07 0.09 0.5 5.5 C11 H13 O 169.1007 13320 7.47 0.09 1.0 7.5 C13 H13 177.0916 17024 9.55 0.12 0.0 5.5 C11 H13 O2 182.0954 32054 17.99 0.23 -1.1 4.0 C10 H14 O3 188.0843 14246 7.99 0.10 -0.6 7.0 C12 H12 O2 207.1050 15884 8.91 0.11 -2.9 5.5 C12 H15 O3 208.1109 31911 17.91 0.22 -1.0 5.0 C12 H16 O3 214.1006 54492 30.58 0.38 -1.2 8.0 C14 H14 O2 215.1062 66387 37.25 0.47 1.0 7.5 C14 H15 O2 216.1134 22366 12.55 0.16 1.6 7.0 C14 H16 O2 220.1106 17522 9.83 0.12 -0.7 6.0 C13 H16 O3 237.1163 20585 11.55 238.1206 94666 53.12 0.67 0.0 5.0 C13 H18 O4 253.1219 16953 9.51 0.12 0.9 9.5 C17 H17 O2 271.1330 15599 8.75 0.11 0.4 8.5 C17 H19 O3 295.1336 12607 7.07 0.09 -0.2 10.5 C19 H19 O3 312.1364 22580 12.67 0.16 -0.2 10.0 C19 H20 O4 326.1500 13035 7.31 0.09 1.8 10.0 C20 H22 O4 327.1584 20016 11.23 0.14 1.2 9.5 C20 H23 O4 344.1633 86902 48.76 0.61 -0.9 9.0 C20 H24 O5 345.1591 20799 11.67 0.15 358.1775 66316 37.21 0.47 0.6 9.0 C21 H26O5 359.1811 17024 9.55 0.12 -1.1 17.5 C28 H23 372.1582 23150 12.99 0.16 -0.9 10.0 C21 H24 O6 386.1724 34689 19.46 0.24 0.5 10.0 C22 H26 O6 387.1798 25429 14.27 0.18 0.9 9.5 C22 H27 O6 404.1838 178221 100 1.25 -0.3 9.0 C22 H28 O7 405.1870 37254 20.90 0.26 -1.5 17.5 C29 H25 O2 446.1951 85620 48.04 0.60 -1.0 10.0 C24 H30 O8 447.1989 18448 10.35 0.13 482.1907 14317 8.03 0.10 542.2151 115181 64.63 0.81 0.1 13.0 C29 H34 O10 543.2199 31056 17.43 0.22 628.9650 13676 7.67 0.10 Mode: EI + VE + LMR; R+D: -2.0 > 60.0

Figure 3-48 1H NMR spectrum of 8 (6-dehydroxykhayanolide E)

Figure 3-49 13 C NMR spectrum of 8 (6-dehydroxykhayanolide E)

Figure 3-50 DEPT135 ° spectrum of 8 (6-dehydroxykhayanolide E)

Figure 3-51 DEPT90 ° spectrum of 8 (6-dehydroxykhayanolide E)

Figure 3-52 HSQC spectrum of 8 (6-dehydroxykhayanolide E)

96 1.00

1.50

2.00

2.50

3.00

3.50

4.00

ppm4.50 (t1

4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 ppm (t2)

97 Figure 3-53 1H-1H COSY spectrum of 8 (6-dehydroxylkhayanolide E)

100

150

200 ppm (t1

7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm (t2)

98 Figure 3-54 HMBC spectrum of 8 (6-dehydroxylkhayanolide E)

99 Figure 3-55 NOESY spectrum of 8 (6-dehydroxylkhayanolide E)

HMQC and DEPT H

H 22 23 H H CH H H H H O 3 15 17 2 5 30 H 29 20 O O H 21 O H

19 28 18 CH CH CH CH3 H H H H 3 3 3 6 9 11 12 H H H

H-H COSY H H H H H H H H H 9 11 H 12 15 H H 29 H 2 30 6 5 H

HMBC

19 18 CH 28 CH 3 CH3 CH 3 CH3 3 5 H 12 5 O O O H 10 H 4 O H 13 17 3 O O 17 O H H H 7 O 9 29 H 14

CH3 10 H O O O O 7 22 H 23 1 O 7 H 29 H O 9 H O 3 8 6 20 4 H 2 30 4 H 21 H 3 O H H 5 17 H H 5 H O O

23 O 22 O 20 O NOESY OMe chemical shift 12 21 plannar structure O O OMe 11 OH J value 7 17 O 19 OH 16 O 6 10 9 14 O 8 15 O 5 1 30 OAc O 28 4 29 OAc 2 O 3 O

100 Figure 3-56 Structural elucidation of 8 (6-hydroxykhayanolide E)

101 Figure 3-57 Crystal structure of 8 (6-dehydroxykhayanolide E)

Table 3-13 Crystal data and structure refinement of 8

Crystal description Colorless block

Empirical formula C29 H34 O10 Molecular weight 542.56 Temperature (K) 293 (2) Wavelength (Å) 0.71073

Crystal system, space group Monoclinic, P2 1 Unit cell dimensions (Å) a = 12.9044 (18), b = 8.0118 (12), c = 14.224 (2), β = 116.582 (2)º Volume (Å 3) 1315.1 (3) Number of molecules per unit cell, Z 2 Calculated density (Mg/m 3) 1.370 Absorption coefficient ( ) (mm -1) 0.10 Diffraction radiation type Mo Ka F(000) 576 Crystal size 0.42 mm × 0.36 mm × 0.13 mm θ range for data collection ( °) 1.6 < θ < 26.0 Limiting indices -15 ≤ h ≤ 15, -9 ≤ k ≤ 9, -14 ≤ l ≤ 17

Reflections collected / unique 7213 / 2753 [ Rint = 0.082] Completeness to θ = 25.05 99.3 % Absorption correction phi and omega scans Max. and min. transmission 1.000 and 0.789 Data / restraints / parameters 2753 / 1 / 359 Goodness-of-fit on F2 1.078

Final R indices [ I > 2 σ ( I)] R1 = 0.0630, ωR2 = 0.1578

R indices (all data) R1 = 0. 0706, ωR2 = 0.1617 Largest diff. peak and hole (e Å -3) 0.25 and -0.26

102

3.11 Structure identification of compounds 9-11

The molecular formula of 9 was determined to be C22 H28 O8 through accurate ESI-

MS ( Figure 3-58) and NMR spectra ( Figures 3-59 to 3-65). Its structure was identified as (-)-lyoniresinol because the NMR data were in good agreement with those previously reported (Dada et al ., 1989; Hanawa et al ., 1997).

The molecular formula of 10 was determined to be C27H36 O12 through accurate ESI-

MS ( Figure 3-66) and NMR data. Its structure was characterized as (-) lyoniresin-9-yl-β-

D-xylopyranoside because the NMR data were consistent with those previously reported in the literature (Miyamura et al ., 1983; Smite et al ., 1995). Furthermore, the downfield shift of C-9 from δ 63.9 in 9 to δ 70.9 in 10 , the large coupling constant of H-1′′ ( δ 4.20,

1H, d, J = 7.5 Hz), and the HMBC correlations of H-9 with C-1′′ and H-1′′ with C-9 indicated a β-xylose attachment at the C-9 position.

The molecular formula of 11 was determined to be C28H38O13 through accurate ESI-

MS (Figure 3-67) and NMR data. Its structure was shown to be (-) lyoniresin-4'-yl-β-D- glucopyranoside because these data were in good accord with those previously reported

(Buske et al ., 2001). The large coupling constant of the anomeric proton ( δ 4.87, 1H, d, J

= 8.0 Hz, H-1′′), the HMBC correlation between H-1′′ and C-4′ ( δ 138.3), and the downfield shift values of C-3′ from δ 148.4 in 9 to δ 152.9 in 11 and C-5′ from δ 147.4 in

9 to δ 152.3 in 11 confirmed a β-glucose attachment at the C-9 position. All the 1H and

13 NMR data of 9-11 were unambiguously assigned on the basis of the DEPT (90 and 135),

1H-1H COSY, HMQC and HMBC experiments.

103 This isolation of these three compounds is the first report of these lignans in Africa mahogany Khaya species.

(-) lyoniresinol (9): C 22 H28 O8, molecular weight 420, colorless needles from CHCl 3

1 and MeOH solvents, H NMR (500 MHz, in CD 3OD) δ 6.39 (2H, s, H-2 and H-6), 4.33

(1H, d, J = 6.0 Hz, H-7), 1.98 (1H, m, H-8), 3.45 (2H, m, H 2-9), 3.75 (6H, s, H 3-3-OMe and H 3-5-OMe), 6.62 (1H, s, H-2′), 2.73 (1H, dd, J = 15.0, 5.0, H-7′a), 2.60 (1H, dd, J =

15.0, 11.0, H-7′b); 1.62 (1H, m, H-8′), 3.62 (1H, dd, J = 11.0, 5.0, H-9′a), 3.51 (1H, dd, J

13 = 11.0, 6.5, H-9′b), 3.40 (3H, s, H 3-3′-OMe), 3.88 (3H, s, H3-5′-OMe); C NMR (125

MHz, in CD 3OD) δ 139.0 (s, C-1), 106.6 (d, C-2), 148.5 (s, C-3), 134.2 (s, C-4), 148.5 (s,

C-5), 106.6 (s, C-6), 42.0 (d, C-7), 48.7 (d, C-8), 63.9 (t, C-9), 56.5 (q, 3, 5 OMe), 129.9

(s, C-1′), 107.5 (d, C-2′), 148.4 (s, C-3′), 138.6 (s, C-4′), 147.4 (s, C-5′), 126.0 (s, C-6′),

33.3 (t, C-7′), 40.6 (d, C-8′), 66.5 (t, C-9′), 59.8 (q, 3 ′-OMe), 56.3 (q, 5 ′-OMe); accurate

ESI-MS (positive ion mode): m/z [M+Na]+ 443.1450, [2M+Na] + 863.3193.

lyoniresin-9-yl-βββ-D-xylopyranoside (10): C27H36 O12 , molecular weight 552,

1 amorphous, H NMR (300 MHz, in CD 3OD) δ 6.40 (2H, s, H-2 and H-6), 4.30 (1H, d, J

= 6.0 Hz, H-7), 1.99 (1H, m, H-8), 3.52 (1H, m, H-9a), 3.92 (1H, m, H-9b), 3.74 (6H, s,

H3-3-OMe and H 3-5-OMe), 6.60 (1H, s, H-2′), 2.71 (1H, dd, J = 15.0, 5.0, H-7′a), 2.62

(1H, dd, J = 15.0, 11.2, H-7′b); 1.69 (1H, m, H-8′), 3.63 (1H, dd, J = 11.0, 4.6, H-9′a),

3.51 (1H, dd, J = 11.0, 6.5, H-9′b), 3.37 (3H, s, H 3-3′-OMe), 3.86 (3H, s, H3-5′-OMe),

4.20 (1H, d, J = 7.5 Hz, H-1′′), 3.20 (1H, m, H-2′′), 3.30 (1H, m, H-3′′), 3.48 (1H, m, H-

4′′), 3.16 (1H, dd, J = 11.5, 10.2 Hz, H-6′′a), 3.82 (1H, dd, J = 11.5, 5.5 Hz, H-6′′b); 13 C

104 NMR (75 MHz, in CD 3OD) δ 139.0 (s, C-1), 106.5 (d, C-2), 148.6 (s, C-3), 134.1 (s, C-4),

148.6 (s, C-5), 106.5 (s, C-6), 42.6 (d, C-7), 46.1 (d, C-8), 70.9 (C-9), 56.8 (q, 3,5-OMe),

129.9 (s, C-1′), 107.7 (d, C-2′), 148.3 (s, C-3′), 138.5 (s, C-4′), 147.3 (s, C-5′), 126.1 (s,

C-6′), 33.7 (t, C-7′), 40.7 (d, C-8′), 65.7 (t, C-9′), 60.1 (q, 3 ′-OMe), 56.5 (q, 5 ′-OMe),

103.8 (d, C-1′′), 74.7 (d, C-2′′), 77.5 (d, C-3′′), 71.2 (d, C-4′′), 66.7 (t, C-5′′). Accurate

ESI-MS (positive ion mode) m/z 575.2018 [M+Na]+ .

lyoniresin-4'-yl-βββ-D-glucopyranoside (3): C28H38O13 , molecular weight 582,

1 amorphous, H NMR (300 MHz, in CD 3OD) δ 6.38 (2H, s, H-2 and H-6), 4.27 (1H, d, J

= 5.7 Hz, H-7), 1.96 (1H, m, H-8), 3.45 (2H, m, H 2-9), 3.78 (6H, s, H 3-3-OMe and H 3-5-

OMe), 6.69 (1H, s, H-2′), 2.74 (1H, dd, J = 15.0, 5.0, H-7′a), 2.60 (1H, dd, J = 15.0, 11.0,

H-7′b); 1.67 (1H, m, H-8′), 3.60 (1H, dd, J = 11.0, 5.0, H-9′a), 3.50 (1H, dd, J = 11.0, 6.5,

H-9′b), 3.40 (3H, s, H 3-3′-OMe), 3.88 (3H, s, H3-5′-OMe), 4.87 (1H, d, J = 8.0 Hz, H-1′′),

3.42 (1H, m, H-2′′), 3.37 (1H, m, H-3′′), 3.39 (1H, m, H-4′′), 3.15 (1H, m, H-5′′), 3.63

(1H, dd, J = 11.0, 6.0 Hz, H-6′′a), 3.72 (1H, dd, J = 11.0, 2.2 Hz, H-6′′b); 13 C NMR (75

MHz, in CD 3OD) δ 139.1 (s, C-1), 106.5 (d, C-2), 148.8 (s, C-3), 134.3 (s, C-4), 148.8 (s,

C-5), 106.5 (s, C-6), 42.5 (d, C-7), 48.5 (d, C-8), 62.8 (t, C-9), 56.5 (q, 3, 5 OMe), 134.8

(s, C-1′), 108.7 (d, C-2′), 152.9 (s, C-3′), 138.3 (s, C-4′), 152.3 (s, C-5′), 126.7 (s, C-6′),

33.7 (t, C-7′), 39.9 (d, C-8′), 65.9 (t, C-9′), 56.5 (q, 3 ′-OMe), 61.0 (q, 5 ′-OMe); 103.7 (d,

C-1′′), 75.6 (d, C-2′′), 77.5 (d, C-3′′), 71.0 (d, C-4′′), 78.0 (d, C-5′′), 62.1 (d, C-6′′).

Accurate ESI-MS (positive ion mode): m/z [M+Na]+ 605.2111.

105

Figure 3-58 Accurate ESI-MS spectrum of 9 ( lyoniresinol)

106

Figure 3-59 1H NMR spectrum of 9 (lyoniresinol)

107

Figure 3-60 13 C NMR spectrum of 9 (lyoniresinol)

108

Figure 3-61 DEPT135 ° spectrum of 9 (lyoniresinol)

109

Figure 3-62 DEPT90 ° spectrum of 9 (lyoniresinol)

110

Figure 3-63 HMQC spectrum of 9 (lyoniresinol)

111

Figure 3-64 1H-1H COSY spectrum of 9 (lyoniresinol)

112

Figure 3-65 HMBC spectrum of 9 (lyoniresinol)

113

Figure 3-66 Accurate ESI-MS spectrum of 10

114

Figure 3-67 Accurate ESI-MS spectrum of 11

115 3.12 References cited

Abdelgaleil SAM, Hashinaga F, Nakatani M 2005 . Antifungal activity of limonoids from

Khaya ivorensis . Pest Manag Sci 61: 186–190.

Abdelgaleil SAM, Okamura H, Iwagawa T, Doe M, Nakatani M 2000 . Novel rings B,D-

seco limonoids from the stem bark of Khaya senegalensis . Heterocycles 53: 2233-

2240

Abdelgaleil SAM, Okamura H, Iwagawa T, Sato A, Miyahara I, Doe M, Nakatani M

2001 . Khayanolides, rearranged phragmalin limonoid antifeedants from Khaya

senegalensis . Tetrahedron 57: 119–126.

Adesida GA, Adesogan EK, Okorie DA, Taylor DAH, Styles BT 1971 . The limonoid

chemistry of the genus Khaya (Meliaceae). Phytochemsitry 10: 1845–1853.

Adesida GA., Adesogan EK, Taylor DAH 1967 . Extractives from Khaya senegalensis A.

Juss. Chem Commu 790-791.

Adesogan EK, Taylor DAH 1968 . Extractives from Khaya senegalensis A. Juss. J Chem

Bevan CWL, Powell JW, Taylor DAH 1963 . West African timbers. Part V1. Petroleum

extracts from species of the genera Khaya, Guarea, Carapa, and Cedrela. J Chem

Soc 980–982.

116 Buske A, Schmidt J, Porzel A, Adam G 2001 . Alkaloidal, megastigmane and lignan

glucosides from Antidesma membranaceum (Euphorbiaceae). Eur J Org Chem 3537-

3543

Chantrapromma K, Sawean N, Fun HK, Chantropromma S, Rahmanb AA 2004 . 6-

hydroxymexicanolide. Acta Cryst E 60: o312-314.

Connolly JD, Henderson R, McCrindle R, Overton KH 1964 . The constitution of

swietenine, a novel tetranortriterpenoid. Tetrahedron Lett. 37: 2593-2597.

Dada G, Corbani A, Manitto P, Speranza G, Lunazzi L 1989 . Lignan glycosides from the

heartwood of European oak Quercus petraea . J Nat Prod . 52: 1327-1330.

Fall AB, Vanhaelen-Fastre R, Vanhaelen M, Lo I, Toppet M, Ferster A, Fondu P 1999 . In

vitro antisickling activity of a rearranged limonoid isolated from Khaya senegalensis

Planta Medica 65: 209-212.

Ferreira ICP, Cortez DAG, Silva MF das GF da, Rodrigues Fo E, Vieira PC, Fernandes

JB 2005 . Acylperoxylated and seco -mexicanolides from stems of Khaya anthotheca .

J Nat Prod 68: 413-416.

Flack HD, 1983 . On enantiomorph-polarity estimation. Acta Cryst A 39: 876-881.

Govindachari TR, Kumari GNK 1998 . Tetranortriterpenoids from Khaya senegalensis .

Phytochemistry 47: 1423-1425.

Hanawa F, Shiro M, Hayashi Y 1997 . Heartwood constituents of Betula maximowicziana .

Phytochemistry 45: 589-595.

117

McPhail AT, Sim GA 1964 . The constitution and absolute stereochemistry of swietenine.

Tetrahedron Lett 37: 2599-2601.

Miyamura M, Nohara T, Tomimatsu T, Nishioka I 1983. Seven aromatic compounds

from bark of Cinnamomum cassia. Phytochemistry 22: 215-218.

Mulholland DA, Parel B, Coombes PH 2000 . The chemistry of Meliaceae and

Ptaeroxylaceae of southern and eastern Africa and Madagascar. Curr Org Chem 4:

1011–1054.

Nakatani M, Abdelgaleil SAM, Kurawaki J, Okamura H, Iwagawa T, Doe M 2001 .

Antifeedant ring B and D opened limonoids from Khaya senegalensis J Nat Prod 64:

2161-1265.

Narender T, Khaliq T, Shweta, Reddy KP, Sharma RK 2007 . Occurrence, biosynthesis,

biological activity and NMR spectroscopy of D and B, D rings seco -limonoids of

Meliaceae family. Nat Prod Commu 2: 203-221.

Nakatani M, Abdelgaleil SAM, Kassem SMI, Takezaki K, Okamura H, Iwagawa T, Doe

M 2002 . Three new modified limonoids from Khaya senegalensis . J Nat Prod 65:

1219–1221.

Olmo LRV, Silva MFdasGFda, Rodrigues Fo E, Vieira PC, Fernandes JB, Marsaioli AJ,

Pinheiro AL, Vilela EF 1996 . Rearranged limonoids from Khaya senegalensis .

Phytochemistry 42: 831-837.

118 Olmo LRV, Silva MFdasGFda, Rodrigues Fo E., Vieira PC, Fernandes JB, Marsaioli AJ,

Pinheiro AL, Vilela EF 1996 . Rearranged limonoids from Khaya senegalensis .

Phytochemistry 42: 831-837.

Saad MMG., Iwagawa T, Doe M, Nakatani M 2003 . Swietenialides, novel ring D opened

phragmalin limonoid orthoesters from Swietenia mahogani JACQ . Tetrahedron 59:

8027-8033.

Sanni SB, Behim H, Beurskens PT 1987 . Structure and absolute configuration of Cedrela

odorata substance B (Mexicanolide). Acta Cryst C 43: 1400-1403.

Smite E, Pan H, Lundgren LN 1995 . Lignan glycosides from inner bark of Betula

pendula. Phytochemistry 40: 341-343.

Solomon KA, Malathi R, Rajan SS, Narasimhanb S 2003 . Swietenine. Acta Cryst E59 :

o1519-1521.

Tchimene MK, Tane P, Ngamga D, Connolly JD, Farrugia LJ 2005 . Four

tetranortripenoids from the stem bark of Khay anthotheca. Phytochemistry 66: 1088-

1093.

Toscano RA, Mata R, Calderon J, Segura R 1996 . Genudin, a D-seco limonoid. J Chem

Crystallogr 26: 707–711.

Zhang H, Odeku OA, Wang XN, Yue JM 2008 . Limonoids from the stem bark of Khaya

grandifoliola . Phytochemistry 69: 271–275.

119 Chapter 4 BIOASSAY OF THE ISOLATES

4.1 Introduction

All 11 isolates were tested in vitro for their anti-oxidant activities using a DPPH bioassay and anti-tumor activities (Figure 4-1) against different tumor cells. (-)- lyoniresinol 9, (-) lyoniresin-9-yl-β-D-xylopyranoside 10, and (-)-lyoniresin-4'-yl-β-D- glucopyranoside 11 (Figure 3-1) exhibited potent antioxidant activity comparable to the standard BHT of the DPPH radical scavenging test ( Figure 4-2) while others did not. It appears the glucose moiety attached to the aromatic ring phenol hydroxyl group enhanced the anti-oxidant ability.

Among the 11 isolates, two D-seco limonoids, 3α, 7 α-dideacetylkhivorin 1 and 1α,

3α, 7 α-trideacetylkhivorin 2 (Figure 3-1), showed significant growth inhibitory activities against MCF-7, Caco-2 and SiHa cancer cell lines with IC 50 values in the range of 35-69

g/ml ( Figure 4-1), while other compounds showed no activity even at the high concentration of 200 g/ml.

These findings are the first report of the anti-tumor and anti-oxidant activities of these natural chemicals. The research results obtained here provide solid scientific evidence for the traditional use of the stem bark of Khaya senegalensis as a folk remedy or tonic.

120 4.2 Experiments and methods

Anti-proliferative activities of isolates against different cancer cells

Sodium pyruvate, sterile cell culture penicillin, streptomycin, Rosewell Park

Memorial Institute 1640 (RPMI 1640), medium sodium bicarbonate, and trypsin EDTA solution were purchased from Sigma Chemical Co. (St. Louis, MO). Tissue culture plates were purchased from Costar (Cambridge, MA). Cosmic calf serum and fetal bovine serum were purchased from Hyclone (Logan, Utah).

Three cancer cell lines, MCF-7 (human breast cancer), Caco-2 (human colon cancer), and SiHa (cervical cancer) were purchased from American Type Culture

Collection (ATCC) (Rockville, MD). MCF-7 and SiHa were cultured in RPMI-1640 with

L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 unit/mL), streptomycin

(0.1 mg/mL), 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate and 10% cosmic calf serum. Caco-2 cells were grown in RPMI medium with 20% fetal bovine

o serum instead of 10% cosmic calf serum. All cells were incubated at 37 C with 5% CO 2 at 90-100% relative humidity. Medium renewal was conducted 2-3 times per week, and cells were sub-cultured when they reached approximately 80-90% confluence.

Prior to chemical treatment, 10 4 cells/well (100 µL) were seeded into a 96-well tissue culture plate and allowed to attach for 24 hours, then treated with defined concentrations of the tested chemicals in DMSO to obtain final concentrations of 200,

100, 50, 20 and 2 ppm. The DMSO concentration was kept at 2% per well. The negative control was the cells treated with DMSO only. Gossypol, an NIH-patented therapeutic agent for human cancer patients (Flack et al., 2000), was used as a positive control. After

121 an incubated period of 24 hours, cell proliferation was determined using the CellTiter 96 ® aqueous nonradioactivity cell proliferation assay according to the manufacturer’s recommendations (Promega, Madison, WI) and recorded on a Bio-Tek microplate reader at 490 nm. The 50% inhibition concentration (IC 50 ) was specified as the chemical concentration causing 50% inhibition of cell growth. Triplicates were conducted for each concentration of the tested chemicals. All the experiments were done at least three times on different days.

DPPH free radical-scavenging activity of the isolates

The antioxidant potency of the isolates were measured using the 2, 2-diphenyl-1- picrylhydrazyl radical (DPPH) free radical scavenging activity method of Chung et al .

(Chung, et al . 2002) with minor modifications.

A 400 L MeOH solution of the candidate samples was added to 400 L of a 250

M DPPH solution dissolved in methanol, to reach a final concentration of 125 M

DPPH. The mixture was shaken vigorously and incubated at room temperature in the darkness for 30 min. Absorbance of the resulting solutions was measured at 517 nm using the Spectronic @ 20 GENESYS TM spectrometer (Fisher Scientific Co. Fairlawn, NJ, USA) and using a 400 µL DPPH˙ solution plus a 400 µL methanol as the blank control and

BHT (butylated hrdroxytoluene) as the antioxidant standard. The discoloration of the

DPPH˙ solution indicated the presence of antioxidant chemicals, and lower absorbance of the reaction mixture indicated higher free radical scavenging activity.

The scavenging activity of free radical was determined using the equation following:

122 absorbance of sample at 517 nm Scavenging Effect (%)= (1 − ) × 100 absorbance of control at 517 nm

Statistical analysis

Data were reported as the mean ± standard deviation. All statistical analysis was conducted using the SAS V9.1 software for Windows (SAS Institute Inc., Cary, NC,

USA). Differences among all sample means were determined by analysis of variance

(one–way analysis of variance, ANOVA), p < 0.05.

4.3 Anti-tumor assay results

Compounds 1 and 2 with the positive control gossypol were tested in vitro for their antiproliferative activites against the three cancer cell lines MCF-7, Caco-2 and

SiHa. The results indicated that the growth of MCF-7, Caco-2 and SiHa cells was significantly inhibited by various concentrations of 1 and 2 in a dose-dependent manner

(Figure 4-1). The highest tested concentration of 200 g/ml inhibited cell growth approximately 66% for MCF-7, 70% for Caco-2 and 61% for SiHa. The IC 50 values of 1 were 69, 35, and 54 g/ml for MCF-7, Caco-2 and SiHa, respectively. The positive control gossypol resulted in IC 50 values of 24, 17, and 14 g/ml, for the corresponding cell lines of MCF-7, Caco-2 and SiHa, respectively. In contrast, compounds 3-11 were weaker inhibitors of cell growth than 1, 2, and gossypol, showing less than 10% inhibition of cell growth for MCF-7 and SiHa, and less than 18% inhibition for Caco-2, even at a concentration of 200 g/ml. The antiproliferative bioactivity of the compounds

1 and 2 may be due to their 14,15 β–epoxide moiety. Limonoids including such a functional group (for example, gedunin inhibited the growth of Caco-2 with an IC 50 value

123 of 8 g/ml; see Uddin et al ., 2007) were reported having a wide range of anti-feedant, anti-fungal, and anti-bacterial activities (Balunasa et al ., 2006; Champagne et al ., 1992;

Roy et al ., 2006). These results appear to be the first report of anti-tumor activity of limonoids isolated from K. senegalensis although the molecular mechanism for how 1, 2 or the reported gedunin (Uddin et al ., 2007) inhibit cancer cell growth is not known.

These results indicate that the anticancer agents 1 and 2 merit further investigation.

4.4 Anti-oxidant assay results

Figure 4-2 and Table 4-1 represent the DPPH free radical scavenging activity of the isolates 9-11 and BHT at different concentrations ( g/ml). The results reported here are the first description of the anti-oxidant capacity of these lignans, although they were previously isolated from difference natural sources before (see Chapter 3). The lignans 9-

11 exhibited strong antioxidant activities at the concentration of 100 g/ml. Among those isolates, the strongest antioxidant is compound 11 , which exhibited 88% DPPH free radical scavenging activities, comparable to those of BHT; In addition, compound 11 exhibited significantly higher antioxidant activity than 9 and 10 , which showed approximately 80%. Since both 10 and 11 have the same aglycone 9, it was concluded that the glucose moiety attached to C-4′ in the aromatic ring in 11 enhanced the antioxidant capacity, while the xylose moiety attached to C-9 in the side chain of 10 did not significantly affect the anti-oxidant ability. Although antioxidant activities of lignans are regarded into be correlated to anti-cancer activities (Saleem et al ., 2005), the bioassay results of compounds 1-2 and 9-11 reported here didn’t support there is a direct relationship between anti-oxidant activity and anti-cancer activity of the isolates.

124 MCF-7

120

100

60 1 (IC50 = 69 g/ml) 40 3 Cell Viability (%) Viability Cell 20

0 0 2 20 50 100 200 Concentration ( µg/ml)

Caco2 120

100

60 1(IC50 = 35 g/ml) 40 3 Cell Viability (%) 20

0 0 2 20 50 100 200 Concentration ( g/ml)

SiHa

120

100

60 1 (IC50 = 54 g/ml) 40

Cell Viability (%) 20 3

0 0 2 20 50 100 200 Concentration ( g/ml)

Figure 4-1 Cell viability (%) with different concentrations of compounds 1 and 3

125 100

60 9 10 40 11 BHT 20

Free radical scavengingFree activity (%) 0 1 10 100 concentration ( g/ml)

Conc.( g/ml) 100 10 1

9 80.37±0.64 c 24.09±0.36 c 3.56±0.73 c

10 79.89±0.48 c 21.44±0.66 b 3.56±0.55 c

11 88.04±0.65 b 42.99±1.00 a 9.35±0.46 b

BHT 92.28±0.398 a 42.26±1.25 a 11.71±0.43 a

Note: Each value (DPPH free radical scavenging activity) is expressed as mean ± SD (n = 3). Values within a column followed by a different letter represent a significant difference at p < 0.05.

Figure 4-2 Dose-response curves of antiradical capacity of 9-11 and BHT

126 4.5 References cited

Champagne DE, Koul O, Isman MB, Scudder GGE, Towers GHN 1992 . Biological

activity of limonoids from the Rutales. Phytochemistry 31: 377-394

Balunasa MJ, Jonesa WP, Chin YB, Mia QW, Farnswortha NR, Soejartoa DD, Cordella

GA, Swansona SM, Pezzutoa JM, Chai HB, Kinghorn AD 2005 . Relationships

between inhibitory activity against a cancer cell line panel, profiles of plants collected,

and compound classes isolated in an anticancer drug discovery project . Chemistry &

Biodiversity 3: 897-915..

Chung YC, Chang CT, Chao WW, Lin CF, Chou ST 2002 . Antioxidative activity and

safety of the 50% ethanolic extract from red bean fermented by Bacillus subtilis IMR-

NK1. J Agric Food Chem 50: 2454-2458.

Flack MR, Knazek R, Reidenberg M 2000 . Gossypol for the treatment of cancer. US

patent 6114397.

Roy A, Saraf S 2006 . Limonoids: overview of significant bioactive triterpenes distributed

in plants kingdom. Biol Pharm Bull 29: 191-201.

Saleem M, Kim HJ, Shai M, Lee YS 2005. An update on bioactive plant lignans. Nat

Prod Rep 22: 696-716.

Uddin SJ, Nahar L, Shilpi JA, Shoeb M, Borkowaki T, Gibbons S, Middleton M, Byres

M, Sarker SD 2007 . Gedunin, a limonoid from Xylocarpus granatum , inhibits the

growth of Caco-2 colon cancer cell line in vitro . Phytother Res 21: 757-761.

127 CHAPTER 5 SUMMARY

Guided by the anti-tumor and anti-oxidant bioassays, 11 compounds were isolated and screened from the stem bark of medicinal plant African mahogany Khaya senegalensis (Meliaceae) by modern column chromatography and crystallization. Their structures ( Figure 3-1) were identified and elucidated as 8 limonoids: 3α, 7α- dideacetylkhivorin 1, 1 α, 3 α, 7 α-trideacetylkhivorin 2, khayanone 3, 1-O- acetylkhayanolide B 4, khayanolide B 5, khayanolide E 6, 1-O-deacetylkhayanolide E 7,

6-dehydroxylkhayanolide E 8, and 3 lignans: (-)-lyoniresinol 9, (-) lyoniresin-9-yl-β-D- xylopyranoside 10, (-)-lyoniresin-4'-yl-β-D-glucopyranoside 11 based on the spectroscopic determination through IR, MS (EI-MS, HR-EI-MS, LC-ESI-MS, accurate

ESI-MS), 1D and 2D NMR ( 1H, 13 C, DEPT 90 ° and 135 °, 1H-1H COSY, HMQC or

HSQC, HMBC, and NOESY), and single crystal X-ray diffraction analysis.

The structures and the stereochemistry of 1, 2, 3, 4, 6, and the new compound 8 were confirmed by the single crystal X-ray diffraction experiments. Four reported limonoids 12 -15 ( Figure 3-1) in the literatures were revised to be 4, 5, 6, and 7, respectively, based on our obtained data. The main reasons led to the incorrect deduction in the literatures were discussed.

Ring D-seco limonoids 1 and 2 showed significant growth inhibitory activities against MCF-7, Caco-2 and SiHa cell lines with IC 50 values in the range of 35-69 g/ml.

The DPPH assay demonstrated lignans 9, 10 , and 11 were strong anti-oxidants comparable to BHT. The relationship between structure and activity were discussed.

128 Our research work has provided scientific proof of the traditional medicinal use of the stem bark of K. senegalensis , which can be a new candidate for screening novel anticancer agents. In addition, we appreciate financial support from South Carolina

Nutrition Research Consortium and a grant from National Institutes of Health,

R21CA107138.

129 APPENDIX

Publications and manuscripts

1. Zhang HP, VanDerveer D, Chen F, Wang X, Wargovich MJ 2007 . 3,7-

dideacetylkhivorin, Acta Cryst E 63: o4162.

2. Zhang HP, VanDerveer D, Wang X, Chen F, Wargovich MJ 2007 . 1,3,7-

trideacetylkhivorin, Acta Cryst E 63: o4599.

3. Zhang HP, VanDerveer D, Wang X, Chen F, Androulakis XM, Wargovich MJ

2007 . 6S,8a-dihydroxy-14,15-dihydrocarapin (Khayanone) from the Stem Bark of

Khaya senegalensis (Meliaceae): isolation and its crystal structure. J Chem

Crystallogr 37:463–467.

4. Zhang HP, Wang X, Chen F, Androulakis XM, Wargovich MJ 2007 . Anticancer

activity of limonoid from Khaya senegalensis . Phytother Res 21: 731-734.

5. Zhang HP, Chen F, Want X, Wu DG, Chen Q 2007 . Complete assignments of 1H

and 13C NMR data for rings A,B-seco limonoids from the seed of Aphanamixis

polystachya . Magn Reson Chem 45: 189–192.

6. Zhang HP, Tan JJ, VanDerveer D, Wang, X, Wargovich MJ, Chen F (submitted

in June 12 and accepted on Dec. 1 2008) Khayanolides from African mahogany

Khaya senegalensis (Meliaceae). Phytochemistry .

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