สารออกฤทธิ์ทางชีวภาพจากเปลาเงิน หัสคืน และกระเจาะ นางสาวจิราภรณ ทองตัน วิทยานิพนธนเี้ ปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบณ ั ฑิต สาขาวิชาเภสัชเคมีและผลิตภัณฑธรรมชาติ คณะเภสัชศาสตร จุฬาลงกรณมหาวิทยาลัย ปการศึกษา 2546 ISBN : 974-17-5114-1 ลิขสิทธิ์ของจุฬาลงกรณมหาวิทยาลัย BIOACTIVE COMPOUNDS FROM CROTON KONGENSIS, CROTON BIRMANICUS AND MILLETTIA KANGENSIS Miss Jiraporn Thongtan A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmaceutical Chemistry and Natural Products Faculty of Pharmaceutical Sciences Chulalongkorn University Academic Year 2003 ISBN : 974-17-5114 -1 Thesis Title Bioactive Compounds from Croton kongensis, Croton birmanicus and Millettia kangensis By Miss Jiraporn Thongtan Field of Study Pharmaceutical Chemistry and Natural Products Thesis Advisor Associate Professor Nijsiri Ruangrungsi, Ph.D. Thesis Co-Advisor Mr. Prasat Kittakoop, Ph.D Accepted by the Faculty of Pharmaceutical Sciences, Chulalongkorn University in Partial Fulfillment of the Requirements for the Doctor’s Degree …………………………Dean of the Faculty of Pharmaceutical Sciences (Associate Professor Boonyong Tantisira, Ph.D.) THESIS COMMITTEE …………………………………………………………………... Chairman (Associate Professor Ekarin Saifah, Ph.D.) ………………………………………….………………….. Thesis Advisor (Associate Professor Nijsiri Ruangrungsi, Ph.D.) ………………………………………….……………… Thesis Co-Advisor (Mr. Prasat Kittakoop, Ph.D.) ………………………………………………………..................... Member (Associate Professor Somyote Sutthivaiyakit, Ph.D.) ……………………………………………………………………..Member (Associate Professor Sumphan Wongseripipatana, Ph.D.) จิราภรณ ทองตัน: สารออกฤทธิ์ทางชีวภาพจากเปลาเงิน หัสคืน และ กระเจาะ (BIOACTIVE COMPOUNDS FROM CROTON KONGENSIS, CROTON BIRMANICUS AND MILLETTIA KANGENSIS) อาจารยที่ปรึกษา: รศ. ดร. นิจศิริ เรืองรังษี, อาจารยทปี่ รึกษารวม : ดร. ประสาท กิตตะคุปต, 161 หนา. ISBN: 974-17-5114-1 จากการศึกษาสารออกฤทธิ์ทางชีวภาพของเปลาเงิน หัสคืน และ กระเจาะ สามารถแยกสาร ในกลุมไดเทอรปน อยด 4 ชนิด ฟลาโวนอยด 7 ชนิด และอัลคาลอยด 1 ชนิด การพิสจู นโครงสราง ของสารทั้งหมดที่แยกไดอาศัยการวิเคราะหเชิงสเปคตรัมของ UV, IR, MS และ NMR รวมกับการ เปรียบเทียบขอมูลกับสารที่ทราบโครงสรางแลว พบวาสารที่แยกไดจากเปลาเงินประกอบดวยสาร ใหมที่มีโครงสรางในกลุม 8,9-secokaurane 2 ชนิดคือ คือ ent-8,9-seco-7α,11β-diacetoxy kaura-8(14),16-dien-9,15-dione, ent-8,9-seco-8,14-epoxy-7α-hydroxy-11β-acetoxy-16kauren-9,15-dione, สารที่เคยมีรายงานแลว 1 ชนิดคือ ent-8,9-seco-7α-hydroxy-11-acetoxy และสารที่เคยมีรายงานแลวในกลุม kaurane 1 ชนิด คือ ent7β-hydroxy-15-oxokaur-16-en-18-yl acetate สารที่แยกไดจากหัสคืนประกอบ ดวยสารที่เคยมี รายงานแลวในกลุม glutarimide alkaloid 1 ชนิดคือ julocrotine สารที่แยกไดจากกระเจาะ ประกอบดวยสารใหมในกลุม furanoflavonoids 2 ชนิด คือ 3-methoxy-6-hydroxy-[4˝,5˝:8,7]furanoflavone, 2,5,8-trimethoxy-[4˝,5˝:6,7]-furanoflavanone, pyranoflavonoid 1 ชนิด คือ 3,6-dimethoxy-2˝-dimethyl-[5˝,6˝:8,7]-pyranoflavone และ coumestan 1 ชนิด คือ 4΄hydroxy,5,6,7-trimethoxycoumestan สารที่เคยมีรายงานแลว 2 ชนิดคือ karanjin และ 3,6dimethoxy-[4˝,5˝:8,7]-furanoflavone และสารที่พบครั้งแรกจากธรรมชาติอีก 1 ชนิด คือ 5,8dimethoxy-[4˝,5˝:7,6]-furanoflavone สารที่แยกไดทั้งหมด 12 ชนิดถูกนําไปทดสอบฤทธิ์ทาง ชีวภาพไดแกฤทธิ์ตา นวัณโรค ฤทธิ์ตานมาลาเรีย และฤทธิ์ความเปนพิษตอเซลล พบวา ent-8,9kaura-8(14),16-dien-9,15-dione ent-8,9-seco-8,14-epoxy-7αseco-7α,11β-diacetoxykaura-8(14),16-dien-9,15-dione, hydroxy-11β-acetoxy-16-kauren-9,15-dione, ent-8,9-seco-7α-hydroxy-11-acetoxykaura -8(14),16-dien-9,15-dione และ ent-7β-hydroxy-15-oxokaur-16-en-18-yl acetate จากเปลา เงินมีฤทธิ์ตา นวัณโรค ฤทธิ์ตานมาลาเรีย และฤทธิ์ความเปนพิษตอเซลล ขณะที่ julocrotine จาก หัสคืนและ 5,8-dimethoxy-[4˝,5˝:7,6]-furanoflavone จากกระเจาะมีฤทธิ์ตานวัณโรค ภาควิชา เภสัชเวท ลายมือชื่อนิสิต………………………………………... สาขาวิชา เภสัชเคมีและผลิตภัณฑธรรมชาติ ลายมือชื่ออาจารยที่ปรึกษา………………………........ ปการศึกษา 2546 ลายมือชือ่ อาจารยที่ปรึกษารวม...................................... # # 4276953033 MAJOR: PHARMACEUTICAL CHEMISTRY AND NATURAL PRODUCTS KEY WORDS: 8,9-SECOKAURANE/KAURANE/ALKALOID/FLAVONOID/COUMESTAN/ANTI MALARIAL/ANTIMYCOBACTERIAL/CYTOTOXICITY/CROTON KONGENSIS/CROTON BIRMANICUS/MILLETTIA KANGENSIS JIRAPORN THONGTAN: BIOACTIVE COMPOUNDS FROM CROTON KONGENSIS, CROTON BIRMANICUS AND MILLETTIA KANGENSIS THESIS ADVISOR: ASSOC. PROF. NIJSIRI RUANGRUNGSI, Ph.D., THESIS CO-ADVISOR: MR. PRASAT KITTAKOOP Ph.D., 161 pp. ISBN: 974-17-5114-1 Chemical investigation of Croton kongensis, Croton birmanicus and Millettia kangensis, led to the isolation of four diterpenoids, seven flavonoids and an alkaloid. The structure determination of these compounds was accomplished by spectroscopic analyses (UV, IR, MS and NMR) and by comparison with previously reported data of known compounds. C. kongensis provided two new 8,9-secokauranes identified as ent-8,9-seco7α,11β-diacetoxykaura-8(14),16-dien-9,15-dione and ent-8,9-seco-8,14-epoxy-7α-hydroxy11β-acetoxy-16-kauren-9,15-dione, and also gave two known diterpenes, ent-8,9-seco-7αhydroxy-11-acetoxykaura-8(14),16-dien-9,15-dione and ent-7β-hydroxy-15-oxokaur-16-en18-yl acetate. C. birmanicus was isolated to yield a known glutarimide alkaloid, julocrotine. Isolation of a crude extract of M. kangensis afforded two new furanoflavonoids identified as 3-methoxy-6-hydroxy-[4˝,5˝:8,7]-furanoflavone and 2,5,8-trimethoxy-[4˝,5˝:6,7]-furanoflava none, a new pyranoflavonoid, 3,6-dimethoxy-2˝-dimethyl-[5˝,6˝:8,7]-pyranoflavone, a new coumestan (4΄-hydroxy,5,6,7-trimethoxycoumestan), a new natural product (5,8-dimethoxy[4˝,5˝:7,6]-furanoflavone), together with two known compounds, karanjin and 3,6-dimethoxy[4˝,5˝:8,7]-furanoflavone. The isolated compounds were evaluated for their biological activities, including antimycobacterial, antimalarial, and cytotoxic activities. ent-8,9-Seco7α,11β-diacetoxykaura-8(14),16-dien-9,15-dione, ent-8,9-seco-8,14-epoxy-7α-hydroxy-11βacetoxy-16-kauren-9,15-dione, 9,15-dione and ent-8,9-seco-7α-hydroxy-11-acetoxykaura-8(14),16-dien- ent-7β-hydroxy-15-oxokaur-16-en-18-yl from C. kongensis exhibited antimycobacterial, antimalarial, and cytotoxicity activities, while julocrotine from C. birmanicus and 5,8-dimethoxy-[4˝,5˝:7,6]-furanoflavone from M. kangensis showed mild antimycobacterial activity. Field of Study Pharmaceutical Chemistry and Natuaral Products Acadamic year 2003 Student’s signature…………………………... Advisor’s signature…………………………... Co-Advisor’s signature………………………. ACKNOWLEDGEMENTS I am grateful to the following persons who encouraged and supported my research fulfillment: Associate Professor Dr. Nijsiri Ruangrungsi, my thesis advisor, for his encouragement and intensive supervision through out this work. Dr. Prasat Kittakoop, my thesis co-advisor, National Center for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, for his excellent advice, valuable guidance and hearty encouragement through out my research study. I am grateful to the Thailand Graduated Institute of Science and Technology (TGIST), for a student grant. I would like to thank the Biodiversity Research and Training Program (BRT) for financial support. My special thank to Professor Dr. Yodhathai Thebtaranonth, researchers and all member of Bioresource Research Unit (BRU), BIOTEC, NSTDA, Thailand Science Park, are gratefully acknowledged for providing necessary research facilities, including UV, IR, HPLC, MS and NMR. . I would like to also thank Associate Professor Dr. Palangpon Kongsaeree and Dr. Teinthong Thongpanchang, Department of Chemistry, Faculty of Sciences, Mahidol University, Thailand, for providing the X-ray diffraction, optical rotation and MS measurements. I would like to thank the thesis committee for their constructive suggestions and critical review of this thesis. I am also grateful to my friends at the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for their friendship, kind support and encouragement through out the period. Finally, I wish to thank Mr. Nattarooj Kasemkosin and my family for their kind understanding, love, support and encouragement. CONTENTS Page ABSTRACT (Thai)………………………………………………………….. iv ABSTRACT (English)………………………………………………………. v ACKNOWLEDGEMENTS…………………………………………………. vi CONTENTS…………………………………………………………………. vii LIST OF TABLES…………………………………………………………... xiii LIST OF FIGURES…………………………………………………………. xiv LIST OF SCHEMES………………………………………………………… xix LIST OF ABBREVIATIONS AND SYMBOLS…………………………… xx CHAPTER I INTRODUCTION…………………………………………………….. 1 II HISTORICAL………………………………………………………… 12 1. 2. Classification and Bioactivities of Diterpenes from Some Croton Species………………………………………………….. 12 1.1 Casbane Diterpenes………………………………………. 12 1.2 Cembrane Diterpenes……………………………………... 13 1.3 Clerodane Diterpenes…………………………………….. 14 1.4 Cleistanthane Diterpenes…………………………………. 18 1.5 Kaurane Diterpenes………………………………………. 18 1.6 Labdane Diterpenes………………………………………. 19 1.7 Pimarane Diterpenes……………………………………… 19 1.8 Halimane Diterpenes……………………………………... 20 Miscellaneous…………………………………………………… 20 viii CONTENTS (continued) Page 3. III Classification and Biological Activities of Flavonoids from Millettia Species………………………………………………… 21 3.1 Flavanones and Isoflavanones……………………………. 21 3.2 Flavans and Isoflavans……………………………………. 24 3.3 Flavones and Isoflavones………………………………… 25 3.4 Chalcones…………………………………………………. 31 3.5 Rotenoids………………………………………………….. 33 3.6 Coumarins………………………………………………... 35 3.7 Quinones………………………………………………….. 35 4. Biosynthetic Relationship of Diterpenoids in Croton spp………. 35 5. Biosynthetic Relationship of Flavonoids in Millettia spp………. 36 EXPERIMENTAL…………………………………………………….. 36 1. Source of Material………………………………………………. 38 2. General Techniques……………………………………………... 38 2.1 Thin-Layer Chromatography……………………………... 38 2.2 Column Chromatography…………………………………. 39 2.3 2.2.1 Vacuum Liquid Column Chromatography…………. 39 2.2.2 Flash Column Chromatography…………………….. 39 2.2.3 Gel Filtration Chromatography……………………... 39 2.2.4 High Performance Liquid Chromatography………... 40 Spectroscopy……………………………………………… 40 2.3.1 Infrared (IR) Absorption Spectra…………………… 40 2.3.2 Ultraviolet (UV) Absorption Spectra……………….. 40 ix CONTENTS (continued) Page 2.3.3 Mass Spectra………………………………………... 40 2.3.4 Proton and Carbon-13 Nuclear Magnetic Resonance 2.4 (1H-NMR and 13C-NMR) Spectra………………………… 41 Physical Properties………………………………………... 41 2.4.1 Optical Rotations……………………………………. 41 2.4.2 X-ray Crystallography………………………………. 41 2.5 3. Solvents…………………………………………………… 41 Extraction and Isolation………………………………………… 42 3.1 42 Extraction and Isolation of Compounds from Croton kongensis………………………………………….. 3.1.1 Extraction…………………………………………… 42 3.1.2 Isolation of Compounds from CH2Cl2 extract………. 42 3.1.3 Isolation of Compounds CK 01 amd CK 03……… 42 3.1.4 Isolation of Compounds CK 02…………...………… 43 3.1.3 Isolation of Compounds CK 04…………...………… 43 3.2 Extraction and Isolation of Compounds from Croton birmanicus……………………………………….. 44 3.2.1 Extraction…………………………………………… 44 3.2.2 Isolation of Compounds from CH2Cl2 extract………. 44 3.2.3 Isolation of Compound CB 01……………………… 3.3 44 Extraction and Isolation of Compounds from Millettia kangensis………………………………………... 45 x CONTENTS (continued) Page 3.3.1 Extraction and Isolation of Compounds from the Leaves of Millettia kangensis………………………. 45 3.3.1.1 Extraction……………………….................... 45 3.3.1.2 Isolation of Compounds from CHCl3 extract.. 45 3.3.1.3 Isolation of Compounds MK 02 and MK 07.. 46 3.3.2 Extraction and Isolation of Compounds from the 4. Twigs of Millettia kangensis……………………….. 47 3.3.2.1 Extraction………………………................... 47 3.2.2.2 Isolation of Compounds from CH2Cl2 extract 46 3.2.2.3 Isolation of Compound MK 01…………… 47 3.2.2.4 Isolation of Compound MK 03…………… 47 3.2.2.5 Isolation of Compound MK 04…………… 48 3.2.2.6 Isolation of Compound MK 05…………… 48 3.2.2.7 Isolation of Compound MK 06…………… 48 Physical and Spectral Data of Isolated Compounds………….. 49 4.1 Compound CK 01……………………………………… 49 4.2 Compound CK 02……………………………………… 50 4.3 Compound CK 03……………………………………… 50 4.4 Compound CK 04……………………………………… 50 4.5 Compound CB 01……………………………………… 51 4.6 Compound MK 01……………………………………... 51 4.7 Compound MK 02……………………………………... 51 4.8 Compound MK 03……………………………………... 52 xi CONTENTS (continued) Page 5. IV 4.9 Compound MK 04……………………………………... 52 4.10 Compound MK 05……………………………………... 53 4.11 Compound MK 06……………………………………... 53 4.12 Compound MK 07……………………………………... 53 Biological Activities…………………………………………... 54 5.1 Antimycobacterial Activity……………………………. 54 5.2 Antimalarial Activity ………………………………….. 54 5.3 Cytotoxic Activity……………………………………... 54 RESULTS AND DISSCUSSION……………………………………... 55 1. 2. Structure Elucidation of Compounds Isolated from Croton kongensis…………….....................................………….. 56 1.1 Structure Elucidation of Compound CK 01……………. 56 1.2 Structure Elucidation of Compound CK 02……………. 60 1.3 Structure Elucidation of Compound CK 03……………. 64 1.4 Structure Elucidation of Compound CK 04……………. 67 Structure Elucidation of Compounds Isolated from Croton birmanicus……………………………………………….. 71 2.1 3. Structure Elucidation of Compound CB 01………......... 71 Structure Elucidation of Compounds Isolated from Millettia kangensis………………………………………………. 75 3.1 Structure Elucidation of Compound MK 01…………… 75 3.2 Structure Elucidation of Compound MK 02…………… 78 3.3 Structure Elucidation of Compound MK 03…………... 81 xii CONTENTS (continued) page 4. V 3.4 Structure Elucidation of Compound MK 04…………. 84 3.5 Structure Elucidation of Compound MK 05…………. 87 3.6 Structure Elucidation of Compound MK 06…………… 90 3.7 Structure Elucidation of Compound MK 07…………… 93 Biological Activities……………………………………………. 95 4.1 Bioactive Compounds from Croton kongensis……..…. 95 4.2 Bioactive Compounds from Croton birmanicus……….. 95 4.3 Bioactive Compounds from Millettia kangensis.............. 95 CONCLUSION……………………………………………………… 97 REFERENCES……………………………………………………………… 98 APPENDIX……………………………………………………….………… 107 VITA……………………………………………………….……………… 161 xiii LIST OF TABLES Table Page 1 Antimycobacterial and antimalarial activities of the crude extract….… 2 The 1H and 13 55 C-NMR Spectral Data of ent-8,9-seco-7α-Hydroxy-11- acetoxykaura-8(14),16-dien-9,15-dione and Compound CK 01 in CDCl3………………………………………………………………….. 59 3 The 1H and 13C-NMR Spectral Data of Compound CK 02 in CDCl3…. 63 4 The 1H and 13C-NMR Spectral Data of Compound CK 03 in CDCl3…. 66 5 The 1H and 13 C-NMR Spectral Data of ent-7β-Hydroxy-15-oxokaur- 16-en-18-eyl acetate and Compound CK 04 in CDCl3……………….. 6 The 1H and 13C-NMR Spectral Data of Julocrotine and Compound CB 01 in CDCl3…………………………………………………………… 7 70 1 74 H and 13C-NMR Spectral Data of Karanjin and Compound MK 01 in CDCl3………………………………………………………………….. 77 8 The 1H and 13C-NMR Data of Compound MK 02 in DMSO-d6……… 80 9 The 1H and 13 C-NMR Spectral Data of 3,6-Dimethoxy-[4˝,5˝:8,7]- furanoflavone and Compound MK 03…………………………………. 10 The 1H and 13 C-NMR Spectral Data of Compound MK 04 in CDCl3…………..……………………………………………………… 11 The 1H and 13 The 1H and 13 C-NMR Spectral Data The 1H and 13 C-NMR Spectral Data of Compound 92 MK 07 in DMSO-d6………………………………………………………………. 14 89 of Compound MK 06 in DMSO-d6………………………….…………………………………… 13 86 C-NMR Spectral Data of Compound MK 05 in CDCl3…………..……………………………………………………… 12 83 94 Biological Activities of Compounds from C. kongensis, C. birmanicus and M. kangensis……………………… 96 xiv LIST OF FIGURES Figure Page 1 Croton kongensis……………………………………………………. 9 2 Croton birmanicus…………………………………………………... 10 3 Millettia kangensis…………………………………………………... 11 4 UV Spectrum of Compound CK 01 (chloroform)….………………. 108 5 IR Spectrum of Compound CK 01 (neat)…………………………… 108 6 MS Spectrum of Compound CK 01…………………………………. 109 7 1 109 8 13 9 1 H- NMR Spectrum (CDCl3) of Compound CK 01………………… C-NMR Spectrum (CDCl3) of Compound CK 01………………… 110 H-1H COSY Spectrum (CDCl3) of Compound CK 01…………….. 110 10 NOESY Spectrum (CDCl3) of Compound CK 01…………….…….. 111 11 HMQC Spectrum (CDCl3) of Compound CK 01…………………… 111 12 HMBC Spectrum (CDCl3) of Compound CK 01…………………… 112 13 UV Spectrum of Compound CK 02 (chloroform)..………………… 112 14 IR Spectrum of Compound CK 02 (neat)..…………………………. 113 15 MS Spectrum of Compound CK 02………………………………… 113 16 1 114 17 13 18 1 H- NMR Spectrum (CDCl3) of Compound CK 02………………… C-NMR Spectrum (CDCl3) of Compound CK 02………………… 114 H-1H COSY Spectrum of Compound CK 02……………………… 115 19 NOESY Spectrum of Compound CK 02…………...……………..… 115 20 HMQC Spectrum (CDCl3) of Compound CK 02………………...…. 116 21 HMBC Spectrum (CDCl3) of Compound CK 02…………………… 116 22 UV Spectrum of Compound CK 03 (chloroform)…………………... 117 xv LIST OF FIGURES (continued) Figure Page 23 IR Spectrum of Compound CK 03 (neat)…………………………… 117 24 MS Spectrum of Compound CK 03…………………………………. 118 25 1 118 26 13 27 1 H- NMR Spectrum (CDCl3) of Compound CK 03………………… C-NMR Spectrum (CDCl3) of Compound CK 03………………… 119 H-1H COSY Spectrum (CDCl3) of Compound CK 03…………….. 119 28 NOESY Spectrum (CDCl3) of Compound CK 03…………..……… 120 29 HMQC Spectrum (CDCl3) of Compound CK 03………………….... 120 30 HMBC Spectrum (CDCl3) of Compound CK 03………………….... 121 31 UV Spectrum of Compound CK 04 (chloroform)…………………... 121 32 IR Spectrum of Compound CK 04 (neat).………………………….. 122 33 MS Spectrum of Compound CK 04…………………………………. 122 34 1 123 35 13 36 1 H- NMR Spectrum (CDCl3) of Compound CK 04………………… C-NMR Spectrum (CDCl3) of Compound CK 04………………… 123 H-1H COSY Spectrum (CDCl3) of Compound CK 04…………….. 124 37 NOESY Spectrum (CDCl3) of Compound CK 04…………..……… 124 38 HMQC Spectrum (CDCl3) of Compound CK 04…………………… 125 39 HMBC Spectrum (CDCl3) of Compound CK 04…………………… 125 40 UV Spectrum of Compound CB 01 (chloroform)……………….….. 126 41 IR Spectrum of Compound CB 01 (film)..………………………….. 126 42 MS Spectrum of Compound CB 01………………………………… 127 43 1 127 44 13 H- NMR Spectrum (CDCl3) of Compound CB 01………………… C-NMR Spectrum (CDCl3) of Compound CB 01………………… 128 xvi LIST OF FIGURES (continued) Figure Page 45 1 H-1H COSY Spectrum (CDCl3) of Compound CB 01……………... 128 46 NOESY Spectrum (CDCl3) of Compound CB 01………………….. 129 47 HMQC Spectrum (CDCl3) of Compound CB 01…………………… 129 48 HMBC Spectrum (CDCl3) of Compound CB 01…………………… 130 49 UV Spectrum of Compound MK 01 (methanol)…………………… 130 50 IR Spectrum of Compound MK 01 (film)…………………………... 131 51 MS Spectrum of Compound MK 01………………………………… 131 52 1 132 53 13 54 1 H- NMR Spectrum (CDCl3) of Compound MK 01………………... C-NMR Spectrum (CDCl3) of Compound MK 01……………….. 132 H-1H COSY Spectrum (CDCl3) of Compound MK 01……………. 133 55 NOESY Spectrum (CDCl3) of Compound MK 01……………….… 133 56 HMQC Spectrum (CDCl3) of Compound MK 01…………………... 134 57 HMBC Spectrum (CDCl3) of Compound MK 01…………………... 134 58 UV Spectrum of Compound MK 02 (methanol)…………………… 135 59 IR Spectrum of Compound MK 02 (film).………………………….. 135 60 MS Spectrum of Compound MK 02………………………………… 136 61 1 136 62 13 63 1 H- NMR Spectrum (DMSO-d6) of Compound MK 02……………. C-NMR Spectrum (DMSO-d6) of Compound MK 02……………. 137 H-1H COSY Spectrum (DMSO-d6) of Compound MK 02………… 137 64 NOESY Spectrum (DMSO-d6) of Compound MK 02………...……. 138 65 HMQC Spectrum (DMSO-d6) of Compound MK 02………………. 138 66 HMBC Spectrum (DMSO-d6) of Compound MK 02……………….. 139 67 UV Spectrum of Compound MK 03 (methanol)………….………… 139 xvii LIST OF FIGURES (continued) Figure Page 68 IR Spectrum of Compound MK 03 (film).…………………………. 140 69 MS Spectrum of Compound MK 03………………………………… 140 70 1 141 71 13 72 1 H- NMR Spectrum (CDCl3) of Compound MK 03………………... C-NMR Spectrum (CDCl3) of Compound MK 03……………….. 141 H-1H COSY Spectrum (CDCl3) of Compound MK 03……………. 142 73 NOESY Spectrum (CDCl3) of Compound MK 03…………………. 142 74 HMQC Spectrum (CDCl3) of Compound MK 03………………….. 143 75 HMBC Spectrum (CDCl3) of Compound MK 03…………………... 143 76 UV Spectrum of Compound MK 04 (chloroform)…………………. 144 77 IR Spectrum of Compound MK 04 (film).…………………………. 144 78 MS Spectrum of Compound MK 04………………………………… 145 79 1 145 80 13 81 1 H- NMR Spectrum (CDCl3) of Compound MK 04………………... C-NMR Spectrum (CDCl3) of Compound MK 04……………….. 146 H-1H COSY Spectrum (CDCl3) of Compound MK 04………….… 146 82 NOESY Spectrum (CDCl3) of Compound MK 04…………………. 147 83 HMQC Spectrum (CDCl3) of Compound MK 04………………..…. 147 84 HMBC Spectrum (CDCl3) of Compound MK 04………………...… 148 85 UV Spectrum of Compound MK 05 (chloroform)…………………. 148 86 1 149 87 13 88 1 H- NMR Spectrum (CDCl3) of Compound MK 05……………….. C-NMR Spectrum (CDCl3) of Compound MK 05……………….. 149 H-1H COSY Spectrum (CDCl3) of Compound MK 05…….……… 150 89 NOESY Spectrum (CDCl3) of Compound MK 05…………………. 150 90 HMQC Spectrum (CDCl3) of Compound MK 05…………………... 151 xviii LIST OF FIGURES (continued) Figure Page 91 HMBC Spectrum (CDCl3) of Compound MK 05…………………... 151 92 UV Spectrum of Compound MK 06 (chloroform)….……………… 152 93 IR Spectrum of Compound MK 06 (film)…..…………………….… 152 94 MS Spectrum of Compound MK 06………………………………… 153 95 1 153 96 13 97 H- NMR Spectrum (DMSO-d6) of Compound MK 06……………. C-NMR Spectrum (DMSO-d6) of Compound MK 06……………. 154 1 H-1H COSY Spectrum (DMSO-d6) of Compound MK 06………… 154 98 1 H-1H NOESY Spectrum (DMSO-d6) of Compound MK 06………. 155 99 HMQC Spectrum (DMSO-d6) of Compound MK 06………………. 155 100 HMBC Spectrum (DMSO-d6) of Compound MK 06………………. 156 101 UV Spectrum of Compound MK 07 (chloroform)…………………. 156 102 IR Spectrum of Compound MK 07 (film)….………………………. 157 103 MS Spectrum of Compound MK 07………………………………... 157 104 1 158 105 13 106 1 H- NMR Spectrum (DMSO-d6) of Compound MK 07……………. C-NMR Spectrum (DMSO-d6) of Compound MK 07……………. 158 H-1H COSY Spectrum (DMSO-d6) of Compound MK 07………… 159 107 NOESY Spectrum (DMSO-d6) of Compound MK 07……………… 159 108 HMQC Spectrum (DMSO-d6) of Compound MK 07………………. 160 109 HMBC Spectrum (DMSO-d6) of Compound MK 07………………. 160 xix LIST OF SCHEMES Scheme Page 1 Biosynthetic relationship of diterpenes in Croton spp……………. 36 2 Currently proposed interrelationship between flavonoid monomer... 37 3 Seperation of CH2Cl2 extract from the leaves of Croton kongensis.. 43 4 Seperation of CH2Cl2 extract from the roots of Croton birmanicus.. 45 5 Seperation of CHCl3 extract from the leaves of Millettia kangensis. 46 6 Seperation of CH2Cl2 extract from the twigs of Millettia kangensis. 49 LIST OF ABBREVIATIONS AND SYMBOLS α = Alpha [α]30D = Specific rotation at 30˚ and sodium D line (589 nm) β = Beta br d = Broad doublet (for NMR spectra) br t = Broad triplet (for NMR spectra) br s = Broad singlet (for NMR spectra) calcd = Calculated CDCl3 = Deuterated chloroform CHCl3 = Chloroform CH2Cl2 = Dichloromethane cm = Centimeter cm-1 = Reciprocal centimeter (unit of wave number) 13 = Carbon-13 Nuclear Magnetic Resonance d = Doublet (for NMR spectra) dd = Doublet of doublets (for NMR spectra) ddd = Doublet of doublet of doublets (for NMR spectra) DEPT = Distortionless Enhancement by Polarization Transfer DMSO-d6 = Deuterated dimethyl sulfoxide δ = Chemical shift ESIMS = Electrospray Ionization Mass Spectrometry ESITOFMS = Electrospray Ionization Time of Flight Mass Spectrometry EtOAc = Ethyl acetate g = Gram 1 = Proton Nuclear Magnetic Resonance C NMR H NMR xxi LIST OF ABBREVIATIONS AND SYMBOLS (continued) HMBC = 1 H-Detected Heteronuclear Multiple Bond Coherence HMQC = 1 H-Detected Heteronuclear Multiple Quantum Coherence HPLC = High Perfornance Liquid Chromatography HRESIMS = High Resolution Electrospray Ionization Mass Spectrometry Hz = Hertz IR = Infrared Spectrum J = Coupling constant Kg = Kilogram L = Liter λmax = Wavelength at maximal absorption ε = Molar absorptivity m = Multiplet (for NMR spectra) MeOH = Methanol mg = Milligram [M+H]+ = Protonated molecular ion MHz = Megahertz mL = Milliliter MW = Molecular weight m/z = Mass to charge ratio MS = Mass Spectrometry NMR = Nuclear Magnetic Resonance Spectroscopy NOESY = Nuclear Overhauser Effect Spectroscopy o = Ortho xxii LIST OF ABBREVIATIONS AND SYMBOLS (continued) p = Para νmax = Wave number at maximal absorption s = Singlet (for NMR spectra) t = Triplet (for NMR spectra) TLC = Thin Layer Chromatography UV = Ultraviolet UV-VIS = Ultraviolet and Visible Spectrophotometry CHAPTER I INTRODUCTION The genus Croton belongs to the family of Euphorbiaceae. It is distributed throughout Thailand and over all tropical countries. This genus consists of about 750 species. They are either trees or shrubs, occasionally rheophytic (one introduced species and annual herb), densely or sparsely clothed with stellate hairs or shinning scales, occasionally subglabrous. Leaves alternate or pseudo-verticillate, petiolate, subentire or crenate or dentate or occasionally lobed, penninerved or sometimes palminerved at the base, biglandular at junction of petiole and lamina; stipules minute or shortly filiform, sometimes obsolete. Flowers are mostly monoecious. Inflorescenses terminal, racemose, androgynaecious. The female flowers sometimes reduced to 1 basal long-pedicelled flower. Male flower: sepals mostly 5, free, imbricate or valvate; petals 5, free, often lanate at the apex; disk-glands small, opposite the sepals; stamens 5-30, mostly lanate at the base, inflexd at the apex in bud; pistillode absence. Female flower: sepals much as in male; petals mostly or vestigial; ovary 3-locular; styles variously divided into 2 or 4 linear or thickened branches or occasionally shortly flabellate. Capsule tricoccous, smooth or shortly muricate; seeds ovoid or ellipsoid, smooth, occasionally sparely stellate-lepidote (Shaw, 1980; Shaw, 1981). According to Smitinand (2001), the species of genus Croton found in Thailand are as follow (Smitinand, 2001). Croton acutifolius Esser จิมิจิยา Chi-mi-chi-ya, เปลา Plao, เปลาแพะ Plao pae, มะดอไก Madokai (Northern). C. argyratus Blume เปลา Plao (Prachuap Khiri Khun); เปลาเงิน Plao ngoen (Nong Khai). C. birmanicus Müll.Arg. = C. tiglium L. C. bonplandianus Daillon เปลาทุง Plao thung (General). 2 Croton cascarilloides Raeusch. เปลาเงิน Plao ngoen (Songkhla); เปลาน้ําเงิน Plao nam ngoen (Prachuap Khiri Khun). C. caudatus Geiseler กระดอหดใบขน Krado hot bai khon (Chanthaburi); โคคลาน Kho khlan (Nakon Ratchasima); ปริก Prik (Trang); โคคลานใบ ขน Kho khlan bai khon (General)กูเราะปริยะ Ku-ro-pri-ya (Malay-Narathiwat). C. columnaris Airy Shaw เปลาคํา Plao khum (Sukhothai). C. crassifolius Geiseler ปงคี Pang khi, พังคี Phang khi (Chiang Mai). C. cumingii Müll.Arg. = C. crassifolius Geiseler C. delpyi Gagnep. เปลา Plao, เปลานอย Plao noi, นมน้ําเขียว Nom nam khiao (Southeastern). C. griffithii Hook.f. จิก Chik, เปลา Plao (Peninsular). C. hirtus L.Her. เปลาลมลุก Plao lom luk (Peninsular). C. hutchinsonianus Hosseus เปลา Plao, เปลาแพะ Plao phae, เปลาเลือด Plao lueat, แมลาเลือด Mae la lueat, เหมือนฮอน Mueat hon (Northern). C. kerii Airy Shaw เปลา Plao (General). C. kongensis Gagnep. เปลาเงิน Plao ngoen, เปลานอย Plao noi (Northeastern); เปลาน้ําเงิน Plao nam ngoen (Eastern); เสปอตุ Se-po-tu (Karen-Chiang Mai). C. krabas Gagnep. ทรายขาว Sai khon (Northern); พริกนา Prik na (Central); ฝายน้ํา Fai num (Eastern). C. lachnocarpus Benth. ขี้อน Khi on (Southwestern). 3 Croton longissimus Gagnep. เปลานอย Plao noi {Lampang). C. mekongensis Gagnep. เปลาน้ําเงิน Plao num ngeon, พริกนา Prik na (Northern). C. oblongifolius Roxb .= C. roxburghii N.P. balakar. C. poilanei Gagnep. เปลาใหญ Plao yai (Southeastern). C. pierri Gagnep. = C. cascarilloides Raeusch. C. robustus Kurz เปลาเลือด Plao lueat (Lampang). C. rottleri Geiseler Gagnep. = C. cascarilloides Raeusch C. roxburghii N.P.Balakr. ควะวู Khwa-wu (Karen-Kanchanaburi), เปลาใหญ Plao yai (Central). C. santisukii Airy Shaw เปลาสันติสุข Plao santisuk (Southwestern). C. sepalinus Airy Shaw เปลาเงิน Plao ngoen (Peninsular). C. siamensis Craib = C. robustus Kurz C. stellatopilosus OHba เปลานอย Plao noi (Prachin Buri, Prachuap Khiri Khan) (Southeastern). C. thorelii Gagnep. เปลาตะวัน Plao tawan (Southeastern). C. tiglium L. บะกั้งB a kang (Phare), หัสคืน Has sa Khuen (Northern), สลอด Salot, หมากยอง Mak-yong (Shan-Mae Hong Son). C. tomentosus Müll.Arg. = C. cascarilloides Raeusch C. trachycaulis Airy Shaw กวาวะ Kwa wa, ขี้อน Khi on (Prachuap Khiri Khun). C. wallichii Müll.Arg. เปลา Plao, เปลานา Ploa na (General). Croton kongensis Gagnep. is an indigeneous plant, commonly known in Thai as ˝Plao Ngeon˝ or ˝Plao Noi˝, is frequently used in folk medicine. The leaves of C. kongensis are used in Indo-China for various stomach disorders including ulcers, and a decoction is externally applied for furuncles and impetigo. This plant is deciduous 4 shrub (1.8-3 M height), basal diameter 10-15 mm, bark thin, smooth, brown; male inflo-erect, sepals with scales medially brown, sides greyish petals, filaments pale light green, anthers pale light yellow; blade dull dark green above, silvery-greyish undemeath, small leaves, and creamy white flowers (van Valkenburg and Bunyapraphatsara, 2001). Croton birmanicus Müll.Arg. is exotic plant (Burma) known in Thai as ˝Has sa Khuen˝, is similar to Croton tiglium. This plant is shrub or small tree, 3-6 m high. Leaf simple, alternate, ovate, 4-7 cm wide, 7-10 cm long, brownish green. Inflorescence in axillary raceme, monoecious, pisillate flowers at base, staminate flowers upward. Fruit schizocarp, 3-lobed, 1-3 seeded (Saralamp, Chuakul, Temsiririrkkul et al., 1996). The plants in genus Millettia belongs to Family Leguminosae, subfamily Papilionoideae. These plants are trees or climbing shrubs, leaves odd-pinnate. Flower showy, in auxiliary racemes, often fascicled, simple or paniculate and terminal. Calyx campanulate; teeth short. Petals white or pink; standard ovate or orbicular; wing oblong. Stamens monodelphous or diadelphous, filaments filiform; anthers uniform. Ovary sessile, linear, few-ovuled; style filiform, incurved, glabrous, stigma capitate. Pod linear or oblong, coriaceous or woody, flattened or thick. Seeds lenticular or rainform (Chopra, Badhwar and Ghosh, 1965). Latin descriptions of Millettia kangensis Craib, according to Craib (1927), are as follow: Millettia kangensis Craib; species floribus inter maiores cum foliis iuvenilibus orientibus, vexillo basi calloso extra sericeo, ovario pubescente distinguenda. Arbor circa I0 m. alta (ex Kerr); ramuli iuventute densius breviter crispatim fulvo-pubescents, mox glabri, cortice brunneo vel cinereo-brunneo obtecti, lenticellis numeriosis prominaentibus. Folia 7-9 foliolata, petiole circa 4 cm, longo incluso circa I7 cm, longa, et rhachi subteretibus vel hoc superne late canaliculato indumento ei ramulorum invenilium simili obtectis; stipulae lineares, circa 3 mm longae; folila opposite, oblonga, oblongooblanceolata vel terminali obovato, apice breviter subito 5 acuminata, ad 8 cm longa et 4.2 cm lata, chartacea, supra primo sericea, mox adpresse pubescentia, subtus breviter molliter pubescentia, nervis lateralibus utrinque 8-IO supra conspicuis subtus prominulis, reticulatione gracili sub oculo armato subtus conspicus, petiolulo circa 3 mm longo suffulta, terminali a lateralibus fere 2 cm distante, dtipellis filiformibus pubescentibus circa 3.5 mm longis. Paniculae partials in paniculas terminals paucifoliatas vel efoliatas adflores e ramusculis lateralibus ad 3 cm, longis racemosim orti; bracteae angustae, circa 4 mm longae, deciduae; bracteolae binae, ad pedicelli apicem positae, circa 3 mm longae, angustae, deciduae; rhachis, ramuli, et pediceli densius fulvo-tomentelli vel etiam parce pubescentes; pedicelli ad I cm longi, breviter pubescentes. Calyx extra pubescens, ad 6.5 cm longus; lobi postici approximate, breves, laterals et anticus deltoidei, acuti, I.5 mm longi 2 mm lati. Vexillum oblongum, basi cordatulum, bicallosum, I.5 cm longum, 0.8 cm latum, dorso sericeum, ungui 3 mm longo suffultum; alae I4 mm longae, 4 mm latae, basi auriculatae, apicem versus angustatae, obtusae vel rotundatae, extra apicem versus sparse sericeae, ungui 5 mm longo suffultae; carnae petala basi auriculata, I2 mm longa, 4.5 mm lata, extra apice sericea, ungui 5 mm longo suffulta. Stamina monadelpha, vexillari basi tantum ab aliis libero. Ovarium I cm altum, subsessile, sericeum, stylobasi sericeo apicem versus glabro, ovules 7 (Craib, 1927) . According to Smitinand (2001), the species of genus Millettia found in Thailand are as follow (Smitinand, 2001). Millettia atropurpurea Wall. = Collerya atropurpurea (Wall.) Schott M. brandisiana Kurz กระพี้จั่น Kra phi chan, จั่น Chan, พี้จั่น Phi Chan (General); ปจั่น Pi Chan (Northern). M. caerulea Baker. ปวเปาะเดาะ Pua-po-do (Karen Mae Hong Son); ผักเยี่ยววัว Phak yiao wua (Nakhonsawan, Northern); หางไหลแดง Hang Lai daeng (Kanchanaburi). M. decipiens Prain ปารี Pa ri (Malay-Narathiwat). 6 Millettia extensa Benth. กาวเครือ Kao khruea, กวาวเครือ Kwao khruea (Chiang Mai); ตานครบ Tan krop (Lampang). M. glaucescens Kurz ยะดา Ya-daa (Malay-Narathiwat); หยีน้ํา Yi nam (Peninsular). M. kangensis Craib กระเจาะ Kra cho, ขะเจาะ Kha cho, ขะเจาะ น้ํา Kha cho nam (Chiang Mai). M. kityana Craib เครือขาวเย็น Khruea khao yen, ลางเย็น Lang yen, ฮางเย็น Hang yen (Northern). M. latifolia Dunn ขะเจาะ Kha cho (General). M. leucantha Kurz กะเซาะ Kaso (Central); กระเจาะ Kra cho, var. leucantha ขะเจาะ Kha cho (Northern); กระพี้เขาควาย Kra phi khao khwai (Prachuap Khiri Khan); ขะแมบ Kha maep, คําแมบ Kham maep (Chiang Mai). M. leucantha Kurz กระเจาะ Kra cho, ขะเจาะ Kha cho var. buteoides (Gagnep.) P.K. Loc (Lampang); กระทอน Kra thon, (M. buteoides Gagnep. var. (Phetchabun Phitsanulok); ไมกระทงน้ําผัก siamensis Craib, M. pendula Mai kra tong nam phak (Loei); Benth.) Sa thon (Saraburi); สาธร Sa thon (Ubon สะทอน Ratchathani). M. macrostachya Collett & Hemsl. ขะเจาะน้ํา Kha cho nam (Chiang Mai). var. macrostachya M. macrostachya Collett & Hemsl. ขะเจาะหลวง Kha cho luang, ขะเจาะใหญ 7 var. tecta Craib Millettia pachycarpa Benth. Kha cho yai (Narathiwat). เกถะ Ke-tha (Karen-Chiang Mai); เครือ ไหล Khruea lai (Chiang Mai). M. peguensis Ali ตอหิ To-hi (Karen-Kanchanaburi). (M. ovalifolia Kurz) M. pulcha Benth. Kurz จันพอ Chan pho (Northern). M. racemosa (Roxb.) Benth. = Endosamara racemosa (Roxb.)R. Geesink M. sericea (Vent.) Benth. จะไนโคะ Cha-nai-kho, ปาตู Paa-tu (Malay-Narathiwat); นอเราะ No-ro (Malay-Yala, Pattani); ยิมแมเกาะ Yimmae-ko (Malay-Yala); ออยสามสวน Oi sam suan (Nong Khai). M. thorelii Gagnep. = Derris thorelii Craib M. utilis Dunn สะทอนน้ําผัก Sathon nam phak (Loei). M. xylocarpa Miq. กะเจาะ Ka cho, ขะเจาะ Kha cho (General); คะแมด Kha maet (Chiang Mai); จักจั่น Chakkachan (Loei); พีพ ้ ง Phi phong (Phrae); ยะดา Ya-da (Malay-Yala); ไยยี Yai-yi (Karen-Mae Hong Son); สาธร Sa thon, หยีน้ํา Yi nam (Pattani-Yala). Several phytochemical studies on many species of Croton and Millettia have been reported but none on Croton kongensis, Croton birmanicus, and Millettia kangensis were found. 8 Our preliminary activity screening showed that a crude CH2Cl2 extract from the leaves of Croton kongensis exhibited antimalarial at IC50 0.9 µg/mL and antimycobacterial at MIC 12.5 µg/mL activity. A crude CH2Cl2 extract from the root of Croton birmanicus showed antimycobacterial activity at MIC 100 µg/mL. These crude CHCl3 extract from the leaves and CH2Cl2 extract from the twigs of Millettia kangensis exhibited antimycobacterial activity at MIC 100 µg/mL. Therefore, these plant extracts were selected for phytochemical investigation. Aims of this research work are as follows: 1. Isolation and purification of compounds from the leaves of Croton kongensis Gagnep., the roots of Croton birmanicus Müll.Arg., and the leaves and twigs of Millettia kangensis Craib. 2. Determination of chemical structures of isolated compounds. 3. Evaluation of biological activities of isolated compounds. 9 A B Figure 1 Croton kongensis Gagnep. A) Whole plants, B) Leaves and inflorescence 10 A B Figure 2 Croton birmanicus Müll.Arg. A) Whole plant, B) Leaves 11 B A D C Figure 3 Millettia kangensis Craib A) Whole plant, B) Leaves, C) Whole plant with inflorescene, D) Inflorescene CHAPTER II HISTORICAL The genus Croton belongs to the family Euphorbiaceae, distributed throughout Thailand, and several species have been used as ingredients in traditional medicine. Croton plants are used in folk medicine for antiinflammatory (Bettolo and Scarpati, 1979; Cai, Chen and Phillipson, 1993; Kubo, Asaka and Shibata, 1991; Mazzanti, Bolle, Matinoli et al., 1987), antibacterial (Chen, Cai and Phillipson, 1994), antimicrobial (Peres, Monache, Cruz et al., 1997), gastric ulcer (Craveiro, Andrade, Matos et al., 1980; Roengsumran, Petsom, Kuptiyanuwat et al., 2001), wound healing (Cai et al., 1993; Cai, Evans, Roberts et al., 1991; Milo, Risco, Vila et al., 2002; Pieters, De Bruyne, Mei et al., 1992), cancer (Cai et al., 1993; Cai et al., 1991; Milo et al., 2002), antitumor (Boonyarathanakornkit, Che, Fong et al., 1987; Ferrigni, Puynum, Anderson et al., 1982), dysentery (Milo et al., 2002), purgative (Asuzu, Gray and Waterman, 1988; Mazzanti et al., 1987), bronchitis, fever, malaria (Vigor, Fabre, Fouraste et al., 2002), nervous disturbances (Batatinha, de Souza-Spinosa and Bernardi, 1995), narcotic (Vigor, Fabre, Fouraste et al., 2001), aphrodisiac (Moulis and Fouraste, 1992), antidiabetic (Itokawa, Ichihara, Kojima et al., 1989; Kubo et al., 1991), antilipotropic (Itokawa et al., 1989), hypertension (Puebla, Lopez, Guerrero et al., 2003), syphilis (Babili, Moulis, Bon et al., 1998), hypoglycaemia (Maciel, Pinto, Arruda et al., 2000), and rheumatism (Cai et al., 1991). In addition, these plants showed cytotoxicity, and insecticidal activity (Smitt and Hogberg, 2002). Croton species contain a number of diterpenes. Typical diterpenes in Croton spp. are casbanes, cembranes, clerodanes, cleistanthanes, kauranes, labdanes, pimaranes, and halimanes. In addition, Croton species also produce phorbols, polysaccharides, flavonoids, lignans, benzofurans, sesquiterpenes, polyphenols, and alkaloids. 1. Classification and Bioactivities of Diterpenes from some Croton species 1.1 Casbane Diterpenes 13 In 1990, Moura and co-workers isolated a new macrocyclic diterpene [1] from the stems of Croton nepetaefolius (Moura, Monte and Filho, 1990). H OH H H OH H O [1] 1.2 Cembrane Diterpenes In 1998, Nareeboon isolated a new cembrane diterpene, namely 1isopropyl-4,8-dimethylcyclotetradeca-1,4,8-triol-2E,6Z,11E-triene-12-carboxylic acid [2], and two new diterpenes, 2β,3β-dihydroxy-labda-8(17),12(13),14(15)triene [3] and 2β,3β,11-trihydroxy-16-norlabd-8(17),12(13)-dien-14-one [4], from leaves of Croton joufra (Nareeboon, 1998). OH OH COOH [2] C. oblongifolius, a Thai medicinal plant, was found as a source of neocrotocembranal [5]. The compound 5 inhibited platelet aggregation induced by thrombin (IC50 = 47.21 µg/mL), and showed cytotoxicity against P-388 cells in vitro (IC50 = 6.48 µg/mL) (Roengsumran, Singtothong, Pudhom et al., 1999). CHO [5] 14 1.3 Clerodane Diterpenes Croton species is a rich source of clerodane diterpenes and nor-clerodane diterpenes. In 1972, 11-dehydro-hardwickiciic acid [6] was isolated from the stems bark of Croton oblongifolius by Aiyar and Seshadri (Aiyar and Seshadri, 1972). C. californicus Muell. Arg., an herbaceous shrub indigenous to the Sonoran Desert, Arizona, U.S.A., was found to posses an antimalarial (-)-hardwickiic acid [7] (Luzbetak, Torrance, Hoffmann et al., 1978). C. aromaticus L. is widely distributed in Sri Lunka, and used in ethnomedical preparations and in traditional agriculture. The air dried roots of this plant provided obtained a bioactive compound, (-)-hardwickiic acid [7], which showed insecticidal activity against Apis craccivora (Bandara, Wimalasiri and Bandara, 1987). O O H H COOH COOH [6] [7] Stems of C. sublyratus were found to posses plaunolide [8] and plaunol B [9], which exhibited antipeptic ulcer activity (Takahashi, Kurabayashi, Kiyazawa et al., 1983). Leaves and barks of C. haumanianus are used in folk medicine against gastric ulcer and antihypertensive, and used as an antiepileptic drug. Chemical investigation of the petroleum ether extract of C. haumanianus led to the isolation of crotocorylifuran [10] and crotohaumanoxide (Tchissambou, Chiaroni, Riche et al., 1990). 15 O O H H O O H H O O OH O O O O [8] H [9] O H O H . O CO2CH3 CO2CH3 [10] Chiromodine [11] and its monoacrtyl derivative [12] were isolated from the East Afican medicinal plant, Croton megalocarpus (Weckert, Hummer, Mensah et al., 1992). O O H O OCH3 RO [11] : R = H [12] : R = CH3CO In 1992, MenSah, I. A. et al. isolated chiromodine [13] and epoxychiromodine [14] from the bark of C. megalocarpus (Mensah, Achenbach, Thoithi et al., 1992). O O O H O O H OCH3 OCH3 HO OH [13] O O [14] 16 Croton cajucara Benth is a Brazilian medicinal plant, commonly called Sacaca, its cortices are known for their antidiabetic and antilipotropic properties. In 1989, Itokawa et al. isolated nor-clerodane diterpenes, trans-crotonon [15] and dehydrocrotonin [16] (Itokawa et al., 1989). O O H H O O O H O O H O H H [15] [16] In 1997, Farias et al. studied activities of trans-dehydrocrotonin [16], which was isolated from the bark of C. cajucara. Compound 16 demonstrated a significant hypoglycemic activity in alloxan-induced diabetic rats but not in normal rat, at oral dose of 25 and 50 mg/kg body weight (Farias, Rao, Viana et al., 1997). Compound 16 also showed antiulcerogenic activity on human promyelocytic leukaemia cells (Freire, Melo, Aoyama et al., 2002). C. sonderianus Muell. Arg. is used in folk medicine as a remedy for gastric disturbances. Antimicrobacterial terpenes, sonderianin [17], hardwickic acid [7], 12-hydroxyhardwickic acid [18], and sonderianial [19], were isolated from C. sonderianus (McChesney and Silveira, 1989). In 1994, Silveira. and McChesney. isolated 6α-hydroxyannonence [20], 6α,7β-dihydroxyannonence [21], and 6α,7β-diacetoxyannonence [22], from the roots of C. sonderianus (Silveira and McChesney, 1994). 17 O O OH O R H3CO HO O [17] : R = O O [18] [19] : R = αH, βOH O H R3 R1 R2 [20] : R1 = CH3, R2 = αOH, R3 = H [21] : R1 = CH3, R2 = αOH, R3 = OH [22] : R1 = CH3, R2 = αOAc, R3 = OAc In 1992, Moulis and Fouraste isolated crovalin [23], a clerodane diterpene, from the stem bark of Croton levatii Guill. (Moulis and Fouraste, 1992). O O H O H O H CO2CH3 [23] 1.4 Cleistanthane Diterpenes In 1999, Siriwat isolated 3,4-seco-cleistantha-4(18),13(17),15-trien-3-ioc acid [24] from stem barks of C. oblongifolius Roxb. (Siriwat, 1999). 18 HOOC [24] 1.5 Kaurane Diterpenes Croton lacciferus Linn. is a medicinally important plant commonly found in Sri Lanka and South India. The roots of C. lacciferus furnished three ent-kauranoids, 16α-H-ent-kauran-17-oic acid [25], ent-15β,16-epoxykauran-17-ol [26], and entkauran-15-en-3β,17-diol [27]. In addition, compounds 26 and 27 showed moderate insecticidal activity against Apis craccivora at a dose of 5 ppm per insect against 61% and 62% mortarity, respectively (Bandara, Wimalasiri and Macleod, 1988). CO2H H CH2OH CH2OH O HO [25] [26] [27] In 1998, Pattamadilok, isolated a kaurane diterpene, ent-kaur-16-en-19-oic [28], from the stem barks of C. oblonifolius, a Thai medicinal plant (Pattamadilok, 1998). HOOC [28] The kaurane diterpene, (-)-ent-kaur-16-en-19-oic acid [28], showed significant Na+, K+-ATPase inhibitory effect (IC50 = 2.2x10-5 M) (Ngamrojnanich, Sirimongkon, Roengsumran et al., 2003). 19 The leaves of Croton tonkinensis were previously found to have an inhibitory effect on malarial parasites, and yilded an ent-kaurane diterpenoid ent-7βhydroxy-15oxokaur-16-en-18-yl [29]. A novel ent-kaurane diterpenoid, ent-1αacetoxy-7β,14α-dihydroxy-kaur-16-en-15-on [30], has been isolated from this plant (Minh, Ngoc, Quang et al., 2003). OAc AcO H OH H O OH H [29] O OH [30] 1.6 Labdane Diterpenes In 2001, Sutthivaiyakit et al. isolated a new labdane diterpene, 2α,3αdihydroxy-labda-8,12,14-triene [31], from a Thai medicinal plant, C. joufra (Sutthivaiyakit, Nareeboon, Ruangrangsi et al., 2001). HO HO H [31] 1.7 Pimarane Diterpenes From the CHCl3 extract of leaves of C. joufra, 3β-hydroxy-19-O-acetylpimara-8,15-diene-7-one [32], was isolated (Sutthivaiyakit et al., 2001). OH O OCOCH3 [32] 20 1.8 Halimane Diterpenes Non-specific strong cytotoxic compounds, crotohalimaneic acid [33] and crotohalimoneic acid [34], were isolated from Croton oblongifolius (Roengsumran, Pornpakakul, Muangsin et al., 2004). O O O COOH COOH [33] [34] 2. Miscellaneous A cytotoxic alkaloid taspine [35] was isolated from South American Dragon’s blood (Croton spp.) (Pieters et al., 1992). H3CO N O O O O OCH3 [35] Julocrotin [36], a glutarimide alkaloid, was isolated from C. humilis (Stuart, McNeill, Kutney et al., 1973) and C. membranaceus (Aboagye, Sam, Massiot et al., 2000; Stuart et al., 1973). O N HN O [36] O 21 C. kongensis is an indigenous plant, and distributes in the North of Thailand. This plant is a shrub tree, and used as folk medicine. C. birmanicus is an exotic plant, similar to Croton tiglium. C. birmanicus is taller than C. tiglium. Several chemical studies on the Croton spp. have been reported but none on C. kongensis and C. birmanicus. The genus Millettia belongs to the family Leguminosae, these plants are used in traditional medicine as a laxative, a blood purifier, a dewormer, an analgesic, a diarrhoea (Irvine, 1961), an anti-plasmodial (Yenesew, Derese, Midiwo et al., 2003), an anthelmintic, and a purgative (Perrett, Whitfield, Sanderson et al., 1995). The Millettia spp. exhibits insecticidal (Gupta, Bhattacharyya, Mitra et al., 1983; Hooker, 1973; Singhal, Baruan, Sharma et al., 1983; Singhal, Sharma, Baruan et al., 1982), pesticidal (Gupta et al., 1983; Singhal et al., 1982), fish poison (Dagne and Bekele, 1990; Singhal et al., 1982), molluscicidal, and cercaricidal activities (Perrett et al., 1995). Previous chemical studies of genus Millettia have shown that they are a rich source of flavonoids and isoflavonoids (Hooker, 1973; Mahmoud and Waterman, 1985). Typical metabolites of Mellettia are flavanones, isoflavanones, flavanes, isoflavanes, flavones, isoflavones, chalcones, rotenoids, coumarins, and quinones. 3. Classification and Biological Activities of Flavonoids from Millettia Species. 3.1. Flavanones and Isoflavanones. From 1974 to 1980, Millettia ovalifolia had been intensively studied for chemical constituents, which led to the isolation of several flavanones, isoflavanones, flavones, isoflavones and chalcones. The flavanones millitenins A [37] and B [38], the chromenoflavanones, ovalichromenes A [39] and B [40], the prenylated flavanones, 7hydroxy-6,8-di-C-prenylflavanone [41] and 7-hydroxy-8-di-C-prenylflavanone [42], were isolated from this plant (Gupta and Krishnamurti, 1976; Islam, Gupta and Krishnamurti, 1980; Khan and Zaman, 1974). 22 O O O H3CO O O O H3CO O H3CO O O [37] [38] O O O O R O [39] R = OCH3 [40] R = H R4 R3 HO O R5 R2 R1 O [41] R1 = R4 = R5 = H, R2 = R3 = [42] R1 = R2 = R4 = R5 = H, R3 = In 1980, Millettia pachycarpa was found to possess a prenylated dihydroflavonol [43] (Singhal, Sharma, Thyagarajan et al., 1980). OH O O H OH O [43] In 1984, Baruah’s group isolated a dihydroflavanol, (2S)-3,7,4΄-trihydroxy8,3΄,5΄-triprenylflavanone [44], two flavanones, (-)-sophoranone [45] and its 5hydroxy derivative [46], and four pterocarpans from M. pulchra (Baruah, Baruah, Sharma et al., 1984). 23 OR2 R2O O R3 R1 O [44] R1 = R2 = R3 = H [45] R1 = OH, R2 = R3 = H [46] R1 = H, R2 = R3 = OH In 1989, Millettia ferruginea was chemically explored, and a pyranoflavanone 4΄-hydroxyisolonchocarpin [47], eight isoflavones, a chalcone, and a pterocarpene, were isolated from this plant (Dagne, Bekele and Waterman, 1989). OH O O O [47] Cytotoxic isoflavanones, pervilleanons [48] and its 3΄-O-demethyl derivative [49], were isolated from M. pervilleana (Galeffi, Rasoanaivo, Federici et al., 1997). R1O O OR1 OR2 O OCH3 [48] R1 = H, R2 = CH3 [49] R1 = R2 = H Sritularak’s group could isolate 6-methoxy-[2˝,3˝:7,8]-furanoflavanone [50] and 2,5-dimethoxy-4-hydroxy-[2˝,3˝:7,8]-furanoflavan (Sritularak, Likhitwitayawuid, Conrad et al., 2002a). from M. erythrocalyx 24 O O H3CO O [50] 3.2 Flavans and Isoflavans In 1989, Kumar, Krupadanam and Srimannarayana isolated three isoflavans, 3R(+)-millinol [51], 3R(+)-millinol-B [52], and 3R(+)-cyclomillinol [53], from a stem bark of Millettia racemosa (Kumar, Krupadanam and Srimannarayana, 1989). In 1994, Rao and Krupadanam isolated compounds 51, 52, 53, 3R(+)-isomillinol-B [54], 3R(-) vestitol [55], and 3R(-)-laxifloran [56], from M. racemosa. Compounds 53 and 55 showed significant antibacterial activity against Staphylococcus aureus and Escherichia coli (Rao and Krupadanam, 1994). Two prenylated isoflavans, neomillinol [57] and millinolol [58], were also isolated from M. racemosa (Rao, Prashant and Krupadanam, 1996). R3 O O O O OR1 OH OR2 OH [51] R1 = R2 = R3 = H [52] R1 = CH3, R2 = R3 = H [54] R1 = R3 = H, R2 = CH3 R1 O O [53] R3 O R2 OR2 O OH R3 OR4 [55] R1 = R4 = H, R2 = CH3, R3 = OCH3 [56] R1 = R2 = R3 = H, R4 = CH3 R1 OH [57] R1 = H, R2 = CH3 [58] R1 = CH3, R2 = H In 2002, Sritularak et al isolated 2,5-dimethoxy-4-hydroxy-[2˝,3˝:7,8]furanoflavan [59] from M. erythrocalyx (Sritularak et al., 2002a). 25 O O OCH3 H OH OCH3 [59] 3.3 Flavones and Isoflavones. In 1974, Millettia ovalifolia was isolated, and two flavones, milletenin C [60] and ovalifolin A [61], were obtained (Khan and Zaman, 1974). O O H3CO O O O O H3CO O O [60] [61] M. auriculata was isolated to yield auriculasin [62] and isoauriculasin [63] (Minhaj, Khan, Kapoor et al., 1976). Raju and Srimannarayana isolated aurmillone [64] from the seeds of M. auriculata (Raju and Srimannarayana, 1978), while Gupta’s group isolated an isoflavone isoaurmillone [65] from the pods (Gupta et al., 1983). Three new prenylated flavonones, 2΄-deoxyisoauriculatin [66], 2΄-O-methyliso auriculatin [67], and auriculatin [68], were isolated from M. auriculata (Rao, Prasad and Ganapaty, 1992). R1 O O R2 OH O OR3 [63] R1 = , R2 = OH, R3 = H, [64] R1 = , R2 = OH, R3 = 26 R2 HO O R1 OH O O [62] R1 = H, R2 = OCH3 [65] R1 = OCH3, R2 = H O O O O OH O R O O O [66] R = H O [68] [67] R = OCH3 Isolation of the aerial part of Millettia pachycarpa Benth. gave a new prenylated isoflavone, 5,7,4΄-trihydroxy-6,3΄-diprenylaisoflavone [69] (Singhal et al., 1980). In 1981, M. pachycarpa was chemically explored, and four new prenylated 5hydroxyisoflavones with 3,3 dimethyl-3-hydroxylpropyl group [70] and its isomers [71], [72] and [73] were obtained (Singhal, Sharma, Madhusudanan et al., 1981). In 1983, a new prenylated isoflavone, 5,7,3΄-trihydroxy-4΄-methoxy-6,8-diprenylated isoflavone [74] was isolated from M. pachycarpa (Singhal et al., 1983). HO HO O O OH OH [69] O OH OH O [74] OCH3 27 OH O O O O R1 R2 OH O R1 R2 O OCH3 O R3 [70] R1 = H, R2 = OCH3 [72] R1 =R3 = H, R2 = OCH3 [71] R1 = OCH3, R2 = H [73] R1 = OH, R2 =H, R3 = OCH3 In 1984, Millettia pulchara was chemically investigated to afford two new ptercarpans and two new prenylated isoflavones 5,7,2΄,4΄-tetrahydroxy-6,3΄diprenylisoflavone, together with its derivatives 75 and 76 (Baruah et al., 1984). HO O OH R O OH [75] R = OH [76] R = OCH3 M. hemsleyana, collected from the South of Thailand, was isolated by Mahmound and Waterman. The stem bark of M. hemsleyana has yielded a methylenedioxyflavone, 3΄,4΄-methylenedioxy-7-methoxyflavone [77] and three chalcones (Mahmoud and Waterman, 1985). O O H3CO O O [77] The barks and seed pods of M. ferruginea (Hochst.) Bak.subsp. ferruginea and darassana, endemic to Ethiopia, provided two new isoflavones [78,79], a new chalcone, a new flavanone and a new pterocarpene (Dagne et al., 1989). In 1990, 28 O-geranylated [80] and O-prenylated flavonoids [81] were isolated from Millettia ferruginea (Dagne, Bekele, Noguchi et al., 1990). Dagne et al. also isolated three novel C-prenylated isoflavonoids [82-84] from the seeds of M. ferruginea (Dagne et al., 1990). O O R3 R2 O R1 R4 R5 [78] R1 = R2 = OCH3, R3R4 = -OCH2O-, R5 = H [79] R1 = R2 = H, R3 = OH, R4 R5 = -OCH2OHO O O O O O O OCH3 [80] HO O [81] H3CO R2 O OCH3 O R1 O O O OCH3 OCH3 [82] R1 = H, R2 = OCH3 [84] [83] R1 = OCH3, R2 = H 3-Hydroxy-4΄-methoxyflavone [85] was isolated from the flowers of M. zechiana by Parvez and Ogbeide (Parvez and Ogbeide, 1989). OCH3 O OH O [85] 29 Isolation of seed pods of Millettia dura yielded four new isoflavones, durallone [86], 6-demethyldurallone [87], predurallone [88] and isoerythrinin-A 4΄(3-methylbuty-2-enyl)ether [89] (Yanesew, Midiwo and Waterman, 1996). O O R2 R1 O R3 [86] R1 = OCH3, R2 = R3 = CH3 [87] R1 = R2 = H, R3 = HO O O O O OCH3 H3CO O O OCH3 [88] O [89] A root bark of M. griffoniana was chemically investigated, and five new isoflavonoids [89-93], a new courmarin, and a new rotenoid, were isolated (Yanesew et al., 1996; Yankep, Fomun, Bisrat et al., 1998; Yankep, Mbafor, Fomun et al., 2001; Yankep, Njamen, Fotsing et al., 2003). O O O O O R1 R3 O O O [92] R2 [89] R1R2 = -OCH2O-, R3 = H [90] R1 = H, R2 = OCH3, R3 = CH2OH [91] R1 = R2 = OH, R3 = CH3 O O HO [93] O OCH3 Conrauinones A, B, C and D [94-97] were isolated from the stems bark of M. conraui (Fuendjiep, Nkengfack, Fomun et al., 1998a;b). 30 O O O OCH3 OH O H3CO OCH3 O O O O [95] O O [94] O HO R O O [96] R = OCH3 [97] R = H In 1998, Kamperdick et al. isolated a new furanoflavone [98] and a new pyranoisoflavonoid [99] from the leaves of Millettia ichyochtona (Kamperdick, Phuong, Sung et al., 1998). O O OCH3 O OCH3 OCH3 [98] O O H3CO OCH3 O [99] From the stem bark of M. usaramensis subsp. usaramensis, a new isoflavone norisojamicin [100] and anti-plasmodial compound [101] were isolated (Yanesew, Midiwo and Waterman, 1998; Yenesew et al., 2003). O O O O OCH3 O O [100] O OH O [101] OCH3 OCH3 In 1999, a new flavonol laurentiol [102] was isolated from the heart wood of M. laurentii (Kammaing, Free, Nkengfack et al., 1999). 31 OCH3 OH HO O OCH3 OH O [102] The novel pyranoisoflavones 103 and 105 were isolated from Millettia thonningii (Olivares, Lwande, Monache et al., 1982). O O O O R1 OCH3 O OCH3 O OR2 OCH3 [105] [103] R1 = OH, R2 = [104] R1 = H, R2 = H M. erythrocalyx afforded the new flavones, millettocalyxins A-C [106-108], and pongol methyl ether [109] (Sritularak, Likhitwitayawuid, Conrad et al., 2002b). R3 R1 O R O O O O OCH3 R2 O O [106] R1 = H, R2 = R3 = OCH3 [108] R = OCH3 [107] R1 = [109] R = H O , R2 = OCH3, R3 = H 3.4 Chalcones Chalcone derivatives, ovalitenins A-D [110-113], were isolated from the seeds of Millettia ovalifolia (Gupta and Krishnamurti, 1977; Islam et al., 1980). A new chalcone monoethoxychalcone [114] was obtained from Millettia pachcarpa (Singhal et al., 1983). 32 O OCH3 O OCH3 O O [110] [111] O O O O O O O O OCH3 O [113] [112] OCH3 O O [114] Two novel chalcones, dihydromillelltenone methyl ether [115] and dihydroisomilletenone methyl ether [116] (Mahmoud and Waterman, 1985), were isolated from the stem bark of M. hemsleyana, while a novel 4΄-Ogeranylisoliquiritigenin [117] (Yankep, Fomun and Dagne, 1997) and a known chalcone [118] were isolated from bark and seed pods of M. ferruginea (Dagne et al., 1989). Compound 117 was also found from the extract of M. griffoniana. O O O O H3CO OCH3 H3CO H3CO OCH3 OCH3 O O [115] [116] OH O OH OH [117] OH O O [118] 33 A new α-hydroxydihydrochalcone [119] was isolated from the stem bark of Mllettia usaramensis subsp. usaramensis (Yanesew et al., 1998), while a new chalcone [120] was isolated from M. erythrocalyx (Sritularak et al., 2002a). O O OCH3 O H3CO OH OH OCH3 O O [119] [120] In 2003, Phrutivorapongkul et al isolated anti-Herpes Simplex Virus (HSV) compounds [121 and 122], and cytotoxic compounds [123 and 124] from the stem barks of Millettia leucantha (Phrutivorapongkul, Lipipun, Ruangrungsi et al., 2003). R1 R5 R4 R3 R2 O [121] R1R2 -OCH2O-, R3 = H, R4= R5 = OCH3 [122] R1R2 -OCH2O-, R3 = R4 = R5 = OCH3 O H3CO OCH3 O R1 R2 O [123] R1 = H, R2 = OCH3 [124] R1 = OCH3, R2 = H 3.5 Rotenoids The roots of Millettia pachycarpa furnished a new rotenone, cis-12ahydroxyrot-2-enoic acid [125], and three known compounds [126-128]. A new rotenone griffonianone [129] was isolated from the root barks of M. griffoniana (Singhal et al., 1982; Yankep et al., 2001). 34 H O O O HO H O H O O O R R OCH3 OCH3 OCH3 OCH3 [125] R = OH [126] R = H [127] R =H [128] R = OH O O O HO OH O OCH3 [129] OCH3 The stem bark of M. usaramensis subsp. usaramensis provided four new rotenones, (+)-12a-epimillettosin [130], (+)-usararotenoid-A [131], (+)-12- dihydrousararotenoid-A [132], and (+)-usararotenoid-B [133], and an anti-plasmodial rotenoid usararotenoid C [134] (Yanesew et al., 1998; Yenesew et al., 2003). O H O O O O O OH O O H O OH O O O O [130] [131] H3CO O O H O OH O [135] O O O O OCH3 H H3CO O OH OH O O H O OH O O O O [132] [134] 35 3.6 Coumarins The seeds of Millettia thonningii have yielded the novel 3-phenylcoumarins, thonningine A [136] and thonningine B [137] (Khalid and Waterman, 1983). A new 3phenylcoumarin [138] was isolated from M. griffoniana (Yankep et al., 1998). O O O H3CO R1 R4 R3 OH O O O H3CO OCH3 OH R2 O [138] [136] R1R2 = -OCH2O-, R3 = R4 = H [137] R1= H, R2 = OCH3, R3 = R4 = H 3.7 Quinones A new isoflavan-quinone laurentiquinone [139] and its isomer [140] were isolated from the heartwood of M. laurentii (Kammaing et al., 1999). An antiinflammatory quinone [141] along with two known quinones 142 and 143 were isolated from the stem bark of M. versicolor (Fotsing, Yankep, Njamen et al., 2003). OCH3 HO O O R1 R1 O O R1 O O OCH3 O R2 [139] R1 = OCH3, R2 = H [140] R1 = H, R2 = OCH3 [141] R1 = H, R2 = OCH3 [142] R1 = R2 = H [143] R1 = OCH3, R2= H 4. Biosynthetic Relationship of Diterpenoids in Croton spp. The diterpenes possess twenty carbon atoms in their molecules. They are biogenetically derived from geranylgeranyl pyrophosphate (GGPP). The diterpene skeleton is the fascinating variation encountered in their core structure, these compounds could be classified into several types, such as mono-, bi-, tri-, tetra-, and pentacyclic diterpenes. The typical diterpenes in Croton spp. are casbane, cembrane, 36 clerodane, cleistanthane, kaurane, labdane, pimarane, and halimane. The relationship of diterpenes is displayed in scheme 1. In addition, the biosynthetic is also proposed (Devon and Scott, 1972). OPP Cembrane Geranylgeranyl pyrophosphate rearrangement Pimarane Labdane Halimane rearrangement Cleistanthane Stachane Clerodane O Kaurane Seco-kaurane Scheme 1 Biosynthetic relationship of diterpenes in Croton spp. 5.Biosynthetic Relationship of Flavonoids in Millettia spp. Flavonoids possess fifteen carbon atoms in their basic skeleton, which are derived from shikimate and acetate-malonate pathway. The typical flavonoids in Millettia spp. are flavanones, isoflavanones, flavanes, isoflavanes, flavones, isoflavones, chalcones, rotenoids, coumarins, and quinines. The relationship of flavonoids is displayed in scheme 2. (Markham, 1982). 37 OH Acetate-malonate pathway Shikimate pathway HOOC 3 2 3' HO 4' 2' OH Lignin OH 4 1 OH β Cinnamyl alcohols HO 5 O H 6 α 5' 6' OH O OH O (-)-Flavanone Chalcone OH HO HO 7 8 6 2 5 4 9 3 6' OH OH 5' 3' H 2' 9 10 5 OH (+)-Dihydroflavonol O 6' 4 6 OH 5' 2 H OH 4' O O OH O 8 HO HO OH O Dihydrochalcone Aurone OH O 4' CH O OH HO 3' 2' O OH O Flavone OH Isoflavone 5 4 HO O 6 7 6a 2 8 11a OH O 11 10 OH Pterocarpan (OH) OH OH HO O H HO O HO OH OH (+)-Catechin O OH Flavonol + O O OH OH (-)-Epicatechin HO O HO OH OH OH OH O O Rotenoid (OH) HO O OH OH H O OH Anthocyanidin OH O OH Coumestan Scheme 2 Currently proposed interrelationships between flavonoid monomer CHAPTER III EXPERIMENTAL 1. Source of Material The leaves of Croton kongensis Gagnep. and the roots of Croton birmanicus Müll.Arg. were collected from Maetang District, Chiangmai Province, Northern Thailand, in November 2001. The voucher specimens of C. kongensis (No. NR 1291951) and C. birmanicus (No. NR 2291951) have been deposited at the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. The leaves and twigs of Millettia kangensis Craib were collected from Maerim District, Chiangmai Province, Northern Thailand, in January 2002. The voucher specimen of M. kangensis (No. NR 3291951) has been deposited at the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. 2. General Techniques 2.1 Thin-Layer Chromatography (TLC) Technique : One dimension, ascending. Adsorbent : Silica gel 60 F254 precoated on aluminium plate (E. Merck). Layer thickness : 0.2 mm Plate size : 2 x 5.0 and 5 x 5 cm Detection : 1. Under ultraviolet light at wavelengths of 254 and 365 nm. 2. Dyeing reagents. 2.1 Anisaldehyde-H2SO4 reagent. (0.5% ethanolic solution of anisaldehyde with 5% sulphuric acid). Stained TLC plates give specific color spots with this reagent after heating at 80-100˚ C for 2-3 minutes. 39 2.2 Column Chromatography 2.2.1 Vacuum Liquid Column Chromatography Adsorbent : a. Silica gel 60 (No. 7734) particle size 0.0630.200 nm (70-230 mesh ASTM) (E. Merck). b. Silica gel 60 (No. 9385) particle size 0.0400.063 nm (70-230 mesh ASTM) (E. Merck). Packing method : Dry packing method. Sample loading : A sample was dissolved in a small amount of organic solvent, mixed with a small quantity of adsorbent, triturated, dried and placed on the top of column 2.2.2 Flash Column Chromatography Adsorbent : a. Silica gel 60 (No. 7734) particle size 0.0630.200 nm (70-230 mesh ASTM) (E. Merck). b. Silica gel 60 (No. 9385) particle size 0.0400.063 nm (70-230 mesh ASTM) (E. Merck). Packing method : Slurry method. Sample loading : A portion of sample was dissolved in a small amount of organic solvent and added to a small quantity of silica gel 60 with particle size 0.0630.200 nm, air dried and added onto the top of this column, for further elution. 2.2.3 Gel Filtration Chromatography Gel filter : Sephadex LH 20 (Phamacia). Packing method : Gel filter was suspended in the eluent and left standing to swell for 24 hours prior to use. It was then poured into the column and allowed to set tightly. Sample loading : The sample was dissolved in a small volume of 40 eluent and applied on top of the column. 2.2.4 High Performance Liquid Chromatography High pressure pump : Waters 600 Detector : Waters 996 Photodiode array detector. Column : 1. LiChroCart 250-10 HPLC-Cartridge 2. PrepNova-Pak cartridge 40x100mm, 6µm 60˚A 2.3 Spectroscopy 2.3.1 Infrared (IR) Absorption Spectra IR spectra (KBr disc and neat film) were obtained on a Bruker vector 22 spectrophotometer (National Center for Genetic Engineering and Biotechnology, BIOTEC, NSTDA, Thailand Science Park, Pathumthani, Thailand). 2.3.2 Ultraviolet (UV) Absorption Spectra UV (in methanol and chloroform) spectra were recorded on a Cary 1E UV-visible spectrophotometer UVIDEC-650 (National Center for Genetic Engineering and Biotechnology, BIOTEC, NSTDA, Thailand Science Park, Pathumthani, Thailand). 2.3.3 Mass Spectra Electrospray ionization mass spectra (ESIMS) were measured on a mass spectrometer LCT (LCMS) Micromass (National Center for Genetic Engineering and Biotechnology, BIOTEC, NSTDA, Thailand Science Park), and LCMS spectra were recorded on BRUKER mass spectrometer (Department of Chemistry, Faculty of Sciences, Mahidol University, Bangkok, Thailand). 41 Proton and Carbon-13 Nuclear Magnetic Resonance (1H-NMR and 2.3.4 13 1 C- NMR) Spectra H NMR (500 MHz) and 13C-NMR (125 MHz) spectra were obtained on a BRUKER AV500D spectrometer, and in some experiments the spectra were obtained on a BRUKER DRX400 spectrometer (National Center for Genetic Engineering and Biotechnology, BIOTEC, NSTDA, Thailand Science Park, Pathumthani, Thailand). 2.4 Physical Properties 2.4.1 Optical Rotations Optical Rotations were recorded in methanol and chloroform with sodium D line (589 nm) on a JASCO DIP-370 digital polarimeter (Department of Chemistry, Faculty of Sciences, Mahidol University, Bangkok, Thailand). 2.4.2 X-ray Crystallography X-ray Crystallographic data were measured at room temperature on a Bruker Nonius kappa CCD diffractometer (Department of Chemistry, Faculty of Sciences, Mahidol University, Bangkok, Thailand). 2.5 Solvents Column chromatography : All solvents are of commercial grade and are redistilled prior to use. HPLC : All solvents are HPLC grade. NMR : All deuterated solvents are NMR grade. 42 3. Extraction and Isolation 3.1 Extraction and Isolation of Compounds from Croton kongensis 3.1.1 Extraction The dried powder leaves of C. kongensis (1 kg) were macerated in CH2Cl2 (2x4L). The extracts were filtered and evaporated under reduced pressure, to give green gummy crude extract (11.11 g). 3.1.2 Isolation of Compounds from CH2Cl2 Extract The CH2Cl2 extract was dissolved in a lot of volumn of MeOH, filtered by filter paper, and then applied on the top of column. The fraction was isolated by gel filtration chromatography (on Sephadex LH-20), MeOH as eluent. Nine fractions (80 mL each) were collected. Fraction 5 was repeatedly chromatographed on a Sephadex LH-20 and a preperative HPLC (reversed-phase C18 column), to yield compounds CK01 and CK 02. Fraction 4 was subjected to Sephadex LH-20 chromatography, MeOH as eluent, to furnish compound CK04. Fraction 2 was re-chromatographed on Sephadex LH-20 and silica gel chromatography, furnishing compound CK02. Detail of isolation of the CH2Cl2 extract of C. kongensis are demonstrated in Scheme 3. 3.1.3 Isolation of Compounds CK 01 and CK 03 Fraction 5 was subjected to Sephadex LH-20 using MeOH as eluent, to afford nine fractions (50 mL per fraction). Fraction 7 was repeatedly chromatographed on a Sephadex LH-20 with MeOH, yielding twelve fractions. Fraction 5 was rechromatographed on Sephadex LH-20 also using MeOH as eluent, to give twelve fractions. Isolation of fraction 8 by reverse phase C18 HPLC with 60:40 acetonitrile and water gave compounds CK 01 (20 mg) and CK 03 (9 mg). 43 3.1.4 Isolation of Compound CK 02 Fraction 2 was re-chromatographed on a Sephadex LH-20, MeOH as eluent. Fraction 9 was subjected to column chromatography using silica gel 60 (No. 9385) as adsorbent, 5:95 of ethyl acetate and hexane as mobile phase to furnish compound CK 02 (12 mg). 3.1.5 Isolation of Compound CK 04 Fraction 4 was subject on Sephadex LH-20, MeOH as eluent gave nine fractions (50 mL per fraction). Fraction 7 was repeatedly chromatographed on a Sephadex LH-20 with MeOH, yielding eight fractions. Purification fraction 4 by Sephadex LH-20 (MeOH as mobile phase) furnished compound CK 04 (4 mg). The CH2Cl2 extract from leaves of Croton kongensis (11.11 g) Sephadex LH-20 (MeOH as eluent) Fr.1 Fr 3 Fr 6 Fr 4 Fr 2 Fr 7 Fr 8 Fr 9 Fr 5 Sephadex LH-20 (MeOH as eluent) Sephadex LH-20 (MeOH as eluent) Fr 4-1,…, Fr 4-7,…, Fr 4-9 Sephadex LH-20 (MeOH as eluent) Sephadex LH-20 (MeOH as eluent) Fr 5-1, …, Fr 5-7, …, Fr 5-9 Sephadex LH-20 (MeOH as eluent) Fr 4.7-1, …, Fr 4.7-5, …, Fr 4.7-8 Fr 2-1,…, Fr 2-9, …, Fr 2-13 Si CC. 5:95 EtOAc: Hexane Sephadex LH-20 (MeOH as eluent) Fr 5.7-1, …, Fr 5.7-5, …, Fr 5.7-12 Sephadex LH-20 (MeOH as eluent) Fr 4.7.5-1, …, Fr 4.7.5-4, …, Fr 4.7.5-7 Fr 5.7.5-1, …, Fr 5.7.5-8, …, Fr 5.7.5-12 Fr 2.9-1, …, Fr 2.9-(29-42), …, Fr 2.9-72 Preperative HPLC Reversed-phase C18 column 60:40 MeCN:H2O Compound CK 04 (4 mg) Compound CK 02 (12 mg) Compound CK 01 (20 mg) Compound CK 03 (9 mg) Scheme 3 Separation of a CH2Cl2 extract from the leaves of Croton kongensis 44 3.2 Extraction and Isolation of Compounds from Croton birmanicus 3.2.1 Extraction The dried roots of C. birmanicus (2 kg) were milled and macerated in CH2Cl2 (2x5L) and MeOH (2x5L). The extract was filtered and evaporated to dryness to give crude extract gummy (17.5 g for CH2Cl2 extract and 112.0 g for MeOH extract). 3.2.2 Isolation Compounds from CH2Cl2 Extract The CH2Cl2 extract was dissolved in CH2Cl2:MeOH (20:80), filtered, and then applied to Sephadex LH-20 (MeOH as eluent), to give eleven frations (100 mL each). Fraction 4 was re-chromatographed on Sephadex LH-20 (MeOH as eluent), furnishing compound CB 01 as shown in Scheme 4. 3.2.3 Isolation of Compound CB 01 Fractions 8-10 were combined and further isolatated by Sephadex LH-20, 20:80 of MeOH and CH2Cl2 as eluent to yield eleven fractions. Fraction 6 was separated by Sephadex LH-20 with 20:80 of MeOH and CH2Cl2 as mobile phase, yielding eight fractions. Fraction 6-8 were combined and re-chromatographed on Sephadex LH-20 (30:70 of MeOH and CH2Cl2 as eluent), gave fourty fractions (10 mL per fraction). Fraction 32-34 were purified by silica gel 60 (No. 7734) as adsorbent, 1% MeOH/CH2Cl2 as mobile phase to furnish compound CB 01 (13.8 mg). 45 The CH2Cl2 extract from the roots of Croton birmanicus (17.5 g) Sephadex LH-20 (20:80 CH2Cl2:MeOH as eluent) Fr 1 Fr 2 Fr 3 Fr 4 Fr 5 Fr 6 Fr 7 Fr 8 Fr 9 Fr 10 Fr 11 Sephadex LH-20 (20:80 CH2Cl2:MeOH as eluent) Fr 8910-1, …., Fr 8910-6, …, Fr 8910-11 Sephadex LH-20 (20:80 CH2Cl2:MeOH as eluent) Fr 89106-1,…, Fr 89106-(6-8),…, Fr 89106-10 Sephadex LH-20 (30:70 CH2Cl2:MeOH as eluent) Fr 8910668-1, …, Fr 8910668-6, …, Fr 8910668-8 Sephadex LH-20 (20:80 CH2Cl2:MeOH as eluent) Fr 89106686-1,…, Fr 89106686-(32-34), … Fr 89106686-40 Si CC 1% MeOH:CH2Cl2 Compound CB 01 (13.8 mg) Scheme 4 Separation of a CH2Cl2 extract from the roots of Croton birmanicus 3.3 Extraction and Isolation of Compounds from Millettia kangensis 3.3.1 Extraction and Isolation of Compounds from the Leaves of Millettia kangensis 3.3.1.1 Extraction The dried powder leaves (4.3 kg) of M. kangensis was extracted with CHCl3 (2x15L), filtered and evaporated, yielding deep green gum (52 g). 3.3.1.2 Isolation of compounds from CHCl3 extract The CHCl3 extract (52 g) was dissolved in a small volume of CHCl3, triturated with silica gel 60 (No. 7734), and dried under vacuum. It was separated by vacuum liquid column chromatography using a sintered glass filter 46 column of silica gel 60 (No. 7734). Fractions were collected (250 ml). Elution was performed in a polarity gradient manner with mixtures of hexane and ethyl acetate (100:0 to 0:100). Sixty fractions were collected, and combined similar fractions by TLC, yielding nine fractions. Fraction 8 was isolated, yielding compounds MK 02 and 07. 3.3.1.3 Isolation of compounds MK 02 and MK 07 Fraction 8 was subjected to Sephadex LH-20 (1:10 CHCl3:MeOH as eluent), yielding six fractions (50 mL per fraction). Fraction 2 was rechromatographed on Sephadex LH-20 (1:10 CHCl3:MeOH as eluent), which gave fifteen fractions (25 mL per fraction). Fractions 7-11 were isolated, yielding compounds MK 02 (5.4 mg) and 07 (.3 mg). The CHCl3 extract of Millettia kangensis leaves (52 g) vacuum liquid chromatography 100:0 0:100 Hexane:EtOAc Fr 1 Fr 2 Fr 3 Fr 4 Fr 5 Fr 6 Fr 7 Fr 8 Sephadex LH-20 1:10 CHCl3:MeOH Fr 9 Fr 8-1, …, Fr 8-2, …, Fr 8-6 Sephadex LH-20 1:10 CHCl3:MeOH Fr 82-1, …, Fr 82-(7-11), …, Fr 82-15 Si CC 1:100 MeOH : CHCl3 Fr 82711-1, …, Fr 82711-(19-40), Fr 82711-(41-53), … ,Fr 82711-(144) Compound MK 02 (5.4 mg) Compound MK 07 (4.3 mg) Scheme 5 Separation of a CHCl3 extract from the leaves of Millettia kangensis 47 3.3.2 Extraction and Isolation of Compounds from the Twigs of Millettia kangensis 3.3.2.1 Extraction The dried twigs (0.8 Kg) of M. kangensis were macerated in CH2Cl2 (2x3L). The extracts were filtered and evaporated under reduced pressure, to yield a deep green gum (11.4 g). 3.3.2.2 Isolation of compounds of CH2Cl2 extract The CH2Cl2 extract was fractionated by Sephadex LH-20 (MeOH as eluent), yielding eight fractions (each 100 mL). Recrytalization of fraction 5 afforded compound MK 06. Fraction 6 was isolated by Sephadex LH-20 and HPLC, yielding compounds MK 03 and MK 04. Fraction 7 was refractionated with Sephadex LH-20 and HPLC to give compounds MK 03 and MK 06. Fraction 8 was isolated with Sephadex LH-20 and HPLC, yielding compounds MK 03, MK 05 and MK 01. 3.3.2.3 Isolation of compound MK 01 Fraction 8 was fractionated by Sephadex LH-20 (MeOH as eluent), yielding eight fractions (50 mL per fraction). Fraction 4 was isolated by RP-C18 HPLC with 40:60 MeCN:H2O, to yield compounds MK 01 (1.7 mg), MK 03 (2.6 mg), and MK 06 (6.8 mg). 3.3.2.4 Isolation of compound MK 03 Fraction 7 was isolated by Sephadex LH-20 (MeOH as eluent), obtaining as nine fractions (50 mL per fraction). Fraction 7 was purified by RP-C18 HPLC (35:65 MeCN:H2O) to yield three fractions, while fraction 1 gave compound MK 03 (12.2 mg). Fraction 2 was isolated by RP-C18 HPLC (40:60 MeCN:H2O), furnished compounds MK 03 (1.3 mg) and MK 05 (6.4 mg). 48 Fraction 8 was fractionated by Sephadex LH-20 (MeOH as eluent), yielding eight fractions (50 mL per fraction). Fraction 4 was isolated by RP-C18 HPLC with 40:60 MeCN:H2O, to yield compounds MK 01 (1.7 mg), MK 06 (6.8 mg), and MK 03 (2.6 mg). 3.3.2.5 Isolation of compound MK 04 Fraction 6 was isolated by Sephadex LH-20 (MeOH as eluent), yielding nine fractions (30 mL per fraction). Fraction 5 was purified by RP-C18 HPLC with 25:75 MeOH:H2O, to yield compounds MK 04 (12.2 mg) and MK 03 (26.4). 3.3.2.5 Isolation of compound MK 05 Fraction 7 was isolated by Sephadex LH-20 (MeOH as eluent), obtaining as nine fractions (50 mL per fraction). Fraction 7 was purified by RP-C18 HPLC (35:65 MeCN:H2O) to yield three fractions,while fraction 1 gave compound MK 03 (12.2 mg). Fraction 2 was isolated by RP-C18 HPLC (40:60 MeCN:H2O), which furnished compounds MK 05 (6.4 mg) and MK 03 (1.3 mg). 3.3.2.5 Isolation of compound MK 06 Fraction 5 was recrystallized from MeOH, yielding compound MK 05 (260.1 mg). Fraction 8 was fractionated by Sephadex LH-20 (MeOH as eluent), yielding eight fractions (50 mL per fraction). Fraction 4 was isolated by RP-C18 HPLC with 40:60 MeCN:H2O, to yield compounds MK 06 (6.8 mg), MK 01 (1.7 mg), and MK 03 (2.6 mg). 49 The CH2Cl2 extract from the twigs of Millettia kangensis (11.4 g) Sephadex LH-20 MeOH as eluent Fr 2 Fr 1 Fr 3 Fr 4 Fr 5 Fr 6 Fr 7 Fr 8 Sephadex LH-20 MeOH as eluent recrystallize from MeOH Compound MK 06 (260.1 mg) Sephadex LH-20 MeOH as eluent Fr 7-1, …, Fr 7-7, …, Fr 7-9 HPLC 35:65 MeCN:H2O Sephadex LH-20 MeOH as eluent Fr 6-1, …, Fr 6-5, …, Fr 6-9 Fr 8-1, …, Fr 8-4, …, Fr 8-8 HPLC 40:60 MeCN:H2O Fr 77-1, Fr 77-2, Fr 77-3 HPLC 25:75 MeCN:H2O Compound MK 03 5.4 mg Compound MK 03 (26.4 mg) HPLC 40:60 MeCN:H2O Compound MK 03 (2.6 mg) Compound MK 06 (6.8 mg) Compound MK 04 (12.2 mg) Compound MK 03 (1.3 mg) Compound MK 05 (6.4 mg) Compound MK 01 (1.7 mg) Scheme 6 Separation of CH2Cl2 extract from the twigs of Millettia kangensis 4. Physical and Spectral Data of Isolated Compounds 4.1 Compound CK 01 Compound CK 01 was obtained as colourless oil, soluble in CHCl3 (20.0 mg, 2.0 x 10-3% based on dried weight of leaves). UV : λmax nm (log ε), in CHCl3; 239.2 (4.46), see Figure 4 IR : νmax cm-1, neat (CHCl3); 3444, 1647, see Figure 5 EITOFMS : m/z; 375.2412 [M+H]+, see Figure 6 [α]30D : -54.4˚ (c 0.45, CHCl3) 1 : δ ppm, 500 MHz, in CDCl3, see Table 2, and Figure 7 H NMR 13 C NMR : δ ppm, 125 MHz, in CDCl3, see Table 2, and Figure 8 50 4.2 Compound CK 02 Compound CK 02 was obtained as brown oil, soluble in CHCl3 (12.0 mg, 1.2 x -3 10 % based on dried weight of leaves). UV : λmax nm (log ε), in CHCl3; 241.4 (4.07) , see Figure 13 IR : νmax cm-1, neat (CHCl3); 2928, 1743, 1704, 1653, 1624, 1370, 1231, 1022, 932, see Figure 14 EITOFMS : m/z ; 439.2089 [M+Na]+ , see Figure 15 [α]30D : -147.6˚ (c 0.575, CHCl3) 1 : δ ppm, 500 MHz, in CDCl3, see Table 3, and Figure 16 H NMR 13 C NMR : δ ppm, 125 MHz, in CDCl3, see Table 3, and Figure 17 4.3 Compound CK 03 Compound CK 02 was obtained as brown oil, soluble in CHCl3 (9.0 mg, 9.0 x 10-4 % based on dried weight of leaves). UV : λmax nm (log ε), in CHCl3; 233.2 (3.48) nm, see Figure 22 IR : νmax cm-1, neat (CHCl3); 3445, 1748, 1733, 1697, 1684, 1646, 1636, 1558, 1540, 1374, 1219, see Figure 23 EITOFMS : m/z ; 413.1969 [M+Na]+ , see Figure 24 [α]30D : -16.7˚ (c 0.45, CHCl3) 1 : δ ppm, 500 MHz, in CDCl3, see Table 4, and Figure 25 H NMR 13 C NMR : δ ppm, 125 MHz, in CDCl3, see Table 4, and Figure 26 4.4 Compound CK 04 Compound CK 04 was obtained as colourless oil, soluble in CHCl3 (4.0 mg, 4.0 x 10-4% based on dried weight of leaves). UV : λmax nm (log ε), in CHCl3; 242.8 (3.68) , see Figure 31 IR : νmax cm-1, neat (CHCl3); 3443, 1635 , see Figure 32 51 EITOFMS : m/z ; 383 [M+Na]+ , see Figure 33 [α]30D : -4.0˚ (c 1.0, CHCl3) 1 : δ ppm, 500 MHz, in CDCl3, see Table 5, and Figure 34 H NMR 13 C NMR : δ ppm, 125 MHz, in CDCl3, see Table 5, and Figure 35 4.5 Compound CB 01 Compound CK 05 was obtained as brown oil, soluble in CHCl3 (13.8 mg, 6.9 x 10-4% based on dried weight of roots). UV : λmax nm (log ε), in CHCl3; 214 (2.47) , see Figure 40 IR : νmax cm-1, film; 350, 3063, 2931, 2856, 1726 and 1682 , see Figure 41 ESIMS : m/z ; 339.2 [M+Na]+ , see Figure 42 [α]30D : -20.6˚ (c 0.825, CHCl3) 1 : δ ppm, 400 MHz, in CDCl3, see Table 6, and Figure 43 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 6, and Figure 44 4.6 Compound MK 01 Compound MK 01 was obtained as colourless crystal, soluble in CHCl3 (1.7 mg, 1.7 x 10-4% based on dried weight of twigs). : λmax nm (log ε), in MeOH; 203 (3.16), 217 (3.25), 260 (3.13) and UV 303 (2.95), see Figure 49 IR : νmax cm-1, film; 3450, 2927,1637,1624 and 1458, see Figure 50 EITOFMS : m/z ; 293.0818 [M+H]+, see Figure 51 1 : δ ppm, 400 MHz, in CDCl3, see Table 7, and Figure 52 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 7, and Figure 53 4.7 Compound MK 02 Compound MK 02 was obtained as colourless plates, soluble in DMSO (5.4 mg, 1.3 x 10-4% based on dried weight of leaves). 52 : λmax nm (log ε), in MeOH; 280 (3.02) and 308 (3.17), UV see Figure 58 : νmax cm-1, film; 3265, 1593, 15661, 1483, 1466, 1396, 1358, 1316, IR 1229 and 1136, see Figure 59 ESIMS : m/z ; 331.3 [M+Na]+, see Figure 60 1 : δ ppm, 400 MHz, in DMSO-d6, see Table 8, and Figure 61 H NMR 13 C NMR : δ ppm, 100 MHz, in DMSO-d6, see Table 8, and Figure 62 4.8 Compound MK 03 Compound MK03 was obtained as colourless plates, soluble in CHCl3 (35.7 mg, 3.6 x 10-3% based on dried weight of twigs). : λmax nm (log ε), in MeOH; 203 (2.26), 206 (2.61) and 306 (2.48), UV see Figure 67 IR : νmax cm-1, film; 2918, 2849, 1753 and 1473 cm-1, see Figure 68 EITOFMS : m/z ; 345.48 [M+Na]+ , see Figure 69 1 : δ ppm, 400 MHz, in CDCl3, see Table 9, and Figure 70 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 9, and Figure 71 4.9 Compound MK 04 Compound MK04 was obtained as colourless plates, soluble in CHCl3 (12.2 mg, 1.2 x 10-3% based on dried weight of twigs). UV : λmax nm (log ε), in CHCl3; 282 (2.77), 312 (2.31) and 348 (2.04), see Figure 76 IR : νmax cm-1, film; 2919, 2845, 1735, 1473, 1463 and 1372, see Figure 77 EITOFMS : m/z ; 323.0919 [M+H]+, see Figure 78 1 : δ ppm, 400 MHz, in CDCl3, see Table 10, and Figure 79 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 10, and Figure 80 53 4.10 Compound MK 05 Compound MK05 was afforded as colourless crystals, soluble in CHCl3 (6.4 mg, 6.4 x 10-4% based on dried weight of twigs). : λmax nm (log ε), in CHCl3; 203 (2.11), 271 (2.62), 309 (2.05), UV see Figure 85 [α]30D -12.4˚ (c 0.50, CHCl3) 1 : δ ppm, 400 MHz, in CDCl3, see Table 11, and Figure 86 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 11, and Figure 87 4.11Compound MK 06 Compound MK06 was obtained as colourless needles, soluble in CHCl3 (266.9 mg, 2.7 x 10-2% based on dried weight of leaves). : λmax nm (log ε), in CHCl3; 214 (2.56), 246 (2.93), 280 (2.75) and UV 340 (2.71), see Figure 92 IR : νmax cm-1, film; 2923 and1615, see Figure 93 EITOFMS : m/z ; 387.1204 [M+Na]+ , see Figure 94 1 : δ ppm, 400 MHz, in CDCl3, see Table 12, and Figure 95 H NMR 13 C NMR : δ ppm, 100 MHz, in CDCl3, see Table 12, and Figure 96 4.12 Compound MK 07 Compound MK07 was displayed as colourless plates, soluble in DMSO (4.3 mg, 1.0 x 10-4% based on dried weight of leaves). UV : λmax nm (log ε), in CHCl3; 280 (2.55), 302 (2.84), 343 (2.94) and 358 (3.16) , see Figure 101 IR : νmax cm-1, film; 3374, 1731, 1621 and 1469, see Figure 102 EITOFMS : m/z ; 365.2 [M+Na]+ , see Figure 103 1 : δ ppm, 500 MHz, in CDCl3, see Table 13, and Figure 104 H NMR 54 13 : δ ppm, 125 MHz, in CDCl3, see Table 13, and Figure 105 C NMR 5. Biological Activities 5.1 Antimycobacterial Activity The antimycobacterial activity was assessed against Mycobacterium tuberculosis H37Ra using the Microplate Alamar Blue Assay (MABA). The standard drugs, isoniazid and kanamycin sulfate, used as reference compounds for the antimycobacterial assay, showed MIC values of 0.040-0.090 and 2.0-5.0 µg/mL, respectively, in the test systems (Collins and Franzblau, 1997). 5.2 Antimalarial Activity The antimalarial activity was evaluated against the parasite Plasmodium falciparum (K1, multidrug-resistant strain), which was cultured continuously according to the method of Trager and Jensen (Trager and Jansen, 1976). Quantitative assessment of antimalarial activity in vitro was determined by the microculture radioisotope technique based upon the method described by Desjardins, et al. The inhibitory concentration (IC50) represents the concentration which causes 50% reduction in parasite growth as indicated by the in vitro uptake of [3H]-hypoxanthine by P. falciparum. An IC50 value of 1 ng/mL was observed for the standard compound, artemisinin, in the same test system (Desjardins, Canfield, Haynes et al., 1979). 5.3 Cytotoxic Activity Cytotoxicity was determined by employing the colorimetric method described by Skehan and co-workers. The reference compound, ellipticine, exhibited activity toward Vero, KB and BC cell lines with the IC50 ranges of 0.2-0.3 µg/mL (Skehan, Storeng, Scudiero et al., 1990). CHAPTER IV RESULTS AND DISSCUSSION Preliminary bioactivity screening revealed that Croton kongensis, Croton birmanicus, and Millettia kangensis exhibited antimycobacterial and antimalarial activities. These results of bioactivities are summarized in Table 1. Table 1 Antimycobacterial and antimalarial activities of the crude extract Crude extract Antimycobacterial activity Antimalarial activity MIC (µg/mL) IC50 (µg/mL) The CH2CL2 leaves extract 12.50 0.90 The MeOH leaves extract 100.00 5.79 100.00 inactive The CH2Cl2 leaves extract 100.00 inactive The CH2Cl2 twigs extract 100.00 inactive C. kongensis C. birmanicus The CH2Cl2 roots extract M. kangensis The CH2Cl2 leaves extract of C. kongensis were isolated, yielding three 8,9secokauranes (CK 01-03) and a kaurane (CK 04). Compounds CK 01 and CK 02 are new compounds. The CH2Cl2 roots extract of C. birmanicus was isolated, furnishing a glutarimide alkaloid (CB 01). The CH2Cl2 leaves and twigs extracts from M. kangensis afforded five furanoflavonoids (MK 01-05), a pyranoflavonoid (MK 06), and a coumestan (MK 07). Compounds MK 03, 05, 06 and 07 are new compounds, while compound MK 04 is a new natural product. Structure elucidation of these compounds was performed by interpretion of their UV, IR, NMR, MS, and X-ray crystallographic data, and by comparison with previous reports. In addition, their antimycobacterial, antimalarial, and cytotoxic activities are displayed in Table 14. Although there have been various classes of diterpenoids isolated from genus Croton, the presence of 8,9-secokauranes has never been before reported. The 8,9secokauranes have been reported from two liverwort species and from several species 56 in the higher plant genus Rabdisia (Family Lamiaceae). This is the first report on the presence of 8,9-secokauranes in the plant genus Croton. Additionally, the presence of coumestan, the rare derivative of isoflavonoid, has never been before reported from genus Millettia. Coumestan, erosnin, has been reported from Pachyrrhizus erosus in 1977. This is the first report of coumestan skeleton in the plant genus Millettia. 1. Structure Elucidation of Compounds Isolated from Croton kongensis 1.1 Structure Elucidation of Compound CK 01 A compound CK 01 was obtained as colourless oil. CK 01 possessed a molecular formula C22H30O5, as revealed by the ESITOFMS spectrum, showing a prominent peak at m/z 375.2412 [M+H]+(Figure 6). The IR spectrum displayed OH stretching at ν 3444 cm-1, and C=O stretching at ν 1647 cm-1(Figure 5). The UV absorption showed λmax at 243 nm (Figure 4). The 1H-NMR spectrum (CDCl3) (Figure 7) of compound CK01 showed signals of four methyl singlets at δH 0.88 (3H), 1.05 (3H), 1.17 (3H), and 2.0 (3H), two singlet signals of exocyclic methylene at δH 5.33 (1H) and 5.95 (1H), a broad singlet signal of olefinic proton at δH 7.28 (1H), two doublet of doublet signals of δH 4.72 (1H, J = 11.9, 4.6 Hz) and δH 5.25 (1H, J = 5.4, 1.2 Hz), a broad singlet signal of methine proton at δH 3.6 (1H), doublet of quartet and doublet of doublet of doublet signals of methylene protons at δH 2.34 (1H, J = 14.7, 2.7 Hz) and δH 2.95 (1H, J = 14.7, 5.0, 1.2 Hz), and four methylene protons at δH 1.95-1.15. The 13 C-NMR spectrum (Figure 8) of compound CK01 revealed 22 signals, while DEPT135 spectrum (Figure 8) revealed four methyl carbons, six methylene carbons, five methine, and seven quaternary carbons. A carbonyl carbon and a methyl group were resonanced at δC 172.0 and 22.0, a characteristic of an acetate group. The downfield shift at δH 5.25 (H-11) and the HMBC (Figure 12) spectrum demonstrated that correlation from H-11 to δC 172.0 (C-1΄), 31.0 (C-12), and 213 (C-9), establishing the first substructure of CK 01 as shown below. 57 O 2, 1' H3C H H H 1 H-13C HMBC 12 11 O 9 O The 1H-1H COSY spectrum (Figure 9) of compound CK01 displayed cross peaks from H-2 (δH 1.50) to H-1 (δH 1.60) and δH H-3 (1.15), from H-5 (δH 1.80) to H6 (δH 1.92), while the HMBC spectrum revealed correlations from CH3-20 (δH 1.05) to C-10 (δC 34.0), from H-1 (δH 1.26) to C-10 (δC 34.0), from H-1 (δH 1.60) to C-2 (δC 17.7), from H-2 (δH 1.5) to δC C-4 (54.0), from H-3 (δH 1.51) to C-4 (δC 54.0), from CH3-19 (δH 1.17) to C-4 (δC 54.0), from H-5 (δH 1.8) to C-7 (δC 64.0), C-4 (δH 54.0), C-10 (δC 34.0), from H-5 (δH 1.8) to C-7 (δC 64), C-4 (δC 54), and C-10 (δC 34), from H-6 methylene (δH 1.95 and 1.52) to C-5 (δC 41.0), C-10 (δC 34), and C-7 (δC 64), and from H-7 (δH 4.72) to C-14 (δC 160), C-8 (δC 148.5), and C-15 (δC 194.5). Based on these spectral data, the second substructure was created as shown. H H H H H H 14 9 1 2 3 15 8 5 4 O 6 H 1 H-13C HMBC O 7 H HH A typical of exocyclic methylene was found in CK 01, exhibiting two singlet resonances at δH 5.33 (1H, s) and δH 5.95 (1H, s). The HMBC revealed the correlation from H-17 (δH 5.33 and δH 5.95) to C-16 (δC 148), and C-15 (δC 194.5), from H-12 (δH 2.35 and 2.95) to C-14; from H-13 (δH 3.6) to C-8 (δC 148.5); and from H-12 to C-11. These spectral data established the third partial structure of CK 01. H H 12 13 H 11 H H 16 14 8 H 17 H 15 1 H-13C HMBC O Combination of the first, second and the third fragments well assembled a gross structure of CK 01. Therefore compound CK 01 was identified as ent-8,9-seco7α-hydroxy-11-acetoxykaura-8(14),16-dien-9,15-dione, which is a known compound 58 previously isolated from a New Zealand liverwort, Lepidolaena taylorii (Perry, Burgess, Baek et al. 1999). 2' O 1' O 12 11 9 1 10 17 16 14 O 7 15 8 O 4 OH 18 19 CK 01 exhibited negative optical rotation ([α]30D -54.4˚, c 0.45, CHCl3) similar to ent-8,9-seco-7α-hydroxy-11-acetoxykaura-8(14),16-dien-9,15-dione, and therefore compound CK 01 possessed the same stereochemistry as that of ent-8,9-seco-7αhydroxy-11-acetoxykaura-8(14),16-dien-9,15-dione. Proton and carbon of compound CK 01 were completely assigned by analyses of 1H-1H COSY (Figure 9), NOESY (Figure 10), HMQC (Figure 11) and HMBC (Figure 12) spectral data as shown in Table 2. 2' O 1' O 9 1 10 H 12 11 17 16 14 O 7 15 8 4 H 18 O OH 19 Compound CK 01 59 Table 2: The 1H and 13 C-NMR Spectral Data of ent-8,9-seco-7α-Hydroxy-11- acetoxykaura-8(14),16-dien-9,15-dione and Compound CK 01 in CDCl3 Position ent-8,9-seco-7α-hydroxy-11-aceto Compound CK 01 xykaura-8(14),16-dien-9,15-dione (Perry et al., 1999). 1 δH (ppm), J (Hz) δC (ppm) δH (ppm), J (Hz) δC (ppm) No report 31.8 1.26 (1H, m) 31.6 1.60 (1H, m) 2 No report 17.9 1.50 (1H, m) 17.7 1.65 (1H, m) 3 No report 41.5 1.15 (1H, m) 41.4 1.51 (1H, m) 4 - 34.3 - 34.0 5 1.73 (dd, 6, 2) 40.6 1.8 (1H, br d, 6.1) 41.0 6 (S) 1.90 (ddd, 13, 6, 5) 32.4 1.95 (1H, br d, 1.0) 31.0 6 (R) 1.45 7 4.71 (dd, 12, 4) 63.8 4.72 (1H, dd, 11.9, 4.6) 64.0 8 - 148.5 - 148.5 9 - 212.2 - 213.0 10 - 54.7 - 54.0 11 5.23 (dd, 5, 1) 77.7 5.25 (1H, dd, 5.5, 1.2) 76.0 12 (R) 2.91 (ddd, 14, 5, 2) 37.1 2.95 (1H, ddd, 14.7, 5.0,1.2) 37.0 12 (S) 2.32 (ddd, 15, 6, 3) 13 3.57 (br m) 41.0 3.60 (1H, br s) 41.0 14 7.25 ( br d, 3) 159.1 7.28 (1H, d, 2.8) 160.0 15 - 194.7 - 194.5 148.2 - 148.0 113.0 5.33 (1H, s) 112.0 16 17 (E) 5.24 (br s) (Z) 5.88 (br s) 1.52 (1H, br d, 1.6) 2.34 (1H, dq, 14.7, 2.7) 5.95 (1H, s) 18 (ax) 0.95 (s) 34.1 0.88 (3H, s) 34.0 19 (eq) 1.03 (s) 22.2 1.17 (3H, s) 20.7 20 1.01 (s) 18.3 1.05 (3H, s) 18.0 1΄ - 169.1 - 172.0 2΄ - 20.8 2.0 (3H, s) 22.0 60 1.2 Structure Elucidation of Compound CK 02 A compound CK 02 was obtained as brown oil. A molecular formula of C24H32O6, [m/z 439.2089 [M+Na]+, calculated for 439.2097] was obtained from the ESITOFMS (Figure 15). The IR absorption (Figure 14) showed bands of C=O stretching at ν 1743 and 1704 cm-1, and the UV spectrum (figure 13) displayed λmax at 214 nm. The optical rotation of CK02 was negative, [α]30D -147.6˚ (c 0.575, CHCl3). The 1H-NMR spectrum (CDCl3) (Figure 16) of compound CK02 displayed signals of five methyl singlets at δH 1.14 (3H, s), 0.97 (3H, s), 1.06 (3H, s), 2.05 (3H, s) and 2.02 (3H, s), three methine doublet of doublets at δH 2.12 (1H, dd, J = 7.2, 0.2 Hz), δH 5.55 (1H, dd, J = 12.2, 4.5 Hz), δ H 5.28 (1H, dd, J = 5.5, 1.3), a broad singlet methine at δH 3.61 (1H, br s), two methylene doublet of doublet of doublets at δH 2.36 (1H, ddd, J = 14.6, 5.4, 2.8 Hz) and δH 2.95 (1H, ddd, J = 14.6, 5.0, 1.3 Hz), two olefinic broad singlets at δH 5.31 (1H, br s) and δH 5.94 (1H, br s), and five methylene multiplets at δH 1.26-0.98. The 13 C NMR spectrum (Figure 17) of compound CK02 revealed 24 signals, and the DEPT135 (Figure 17) spectral data revealed the presence of five methyl carbons, six methylene carbons, five methine carbons, and eight quaternary carbons. Two acetate groups were found in CK 02, having two singlet resonances of carbonyl at C-1΄ (δC 169.0) and C-1˝ (δC 169.7), two singlet resonances of methyl groups at C2΄ (δC 20.6) and C-2˝ (δC 20.9). In addition, the HMBC spectral data (Figure 21) demonstrated the correlation from H-11 (δH 5.28) to C-1΄ (δC 169.0), and from H-7 (δH 5.55) to C-1˝ (δC 169.7), placing an acetate at C-11 and C-7, respectively. The 1H-1H COSY spectrum (Figure 18) of compound CK 02 revealed correlations from H-1(δH 1.26) to H-3 (δH 1.59), from H-2 (δH 1.48) to H-3 (δH 1.59), from H-5 (δH 1.81) to H-6 (δH 1.98), and from H-7 (δH 5.55) to H-6 (δH 1.45 and 1.98). The HMBC spectrum of CK 02 demonstrated correlations from H-5 to C-9 (δC 212.1), from H-7 to C-8 (δC 145), C-14 (δC 159.3), C-1˝( δC 169.7), C-6 (δC 32.3), and C-15 (δC 193.5), and from H-6 to C-8 (δC 145.0), C-7 (δC 66.3). Therefore, the first substructure of CK 02 was assembled as shown 61 2' O CH3 1' H O 11 H 9 1 2 10 3 5 1 H-1H COSY H 14 13 15 O 6 H 8 7 O 1 H-13C HMBC O H 1" H3C O 2" The 1H-NMR spectrum of CK 02 displayed two broad singlet signals at δH 5.31 and δH 5.94, attributable to an exocyclic methylene. The HMBC correlations from H-7 (δH 5.55) to C-8, C-14, C15 and C-1΄, from H-11 (δH 5.28 ) to C-9 and C-11, from H12 (δH 2.36 and 2.95) to C-9, C-13, C-14 and C-16, from H-14 (δH 7.23) to C-15 and C-16, and from H-17 (δH 5.31 and 5.94) to C-13 and C-16. The construction of the second partial structure was by analyses of the above spectral data.. O CH3 H O H 12 11 9 14 O 7 H 17 13 16 15 8 1 H-13C HMBC O O H3C O The HMBC spectrum displayed correlations from CH3-18 (δH 1.14) to C-4 (δC 34.2), C-3 (δC 41.4) and C-5 (δC 40.4), from CH3-19 (δH 0.97) to C-4 (δC 34.2), C-3 (δC 41.4) and C-5 (δC 40.4), from CH3-20 (δH 1.06) to C-1 (δC 32.3), C-5 (δC 40.4), C9 (δC 212.1), and C-10 (δC 54.6), and from H-5 (δH 1.81) to C-4 (δC 34.2), and C-5 (δC 40.4). Therefore, the third partial structure is created. 20 1 10 2 3 4 5 19 18 9 O 1 H-13C HMBC 6 H On the basis of these spectral data compound CK 02 was assigned as an acetate derivative of the known ent-8,9-seco-7α-hydroxy-11-acetoxykaura-8(14),16-dien- 62 9,15-dione and identified as ent-8,9-seco-7α,11β-diacetoxykaura-8(14),16-dien-9,15dione (compound CK 01). Proton and carbon of compound CK 02 were completely assigned by analyses of 1H-1H COSY (Figure 18), NOESY (Figure 19), HMQC (Figure 20) and HMBC (Figure 21) spectral data as shown in Table 2. 2' O 1' O 9 1 10 17 16 14 O 7 4 15 8 O O H 18 H 12 11 1" 19 2" O Compound CK 02 Compound CK 02 exhibited a negative rotation similar to that of compound CK 01, so it is therefore reasonable to assume that the absolute configuration of compound CK 02 is the same as that of compound CK 01. Moreover, the coupling constants at H-7 (δH 5.55, dd, 12.2 and 4.5 Hz of CK 02; δH 4.71, dd, 12 and 4 Hz of CK 01) and H-11 (δH 5.28, dd, 5.5 and 1.3 Hz of CK 02; δH 5.23, dd, 5 and 1 Hz of CK 01) of compound CK 02 were also relatively close to those of CK01. 63 Table 3: The 1H and 13C-NMR Spectral Data of Compound CK 02 in CDCl3 Position δH (ppm), J (Hz) δC (ppm) 1 1.26 (1H, m) 32.3 1.57 (1H, m) 2 1.48 (1H, m) 17.8 1.60 (1H, m) 3 1.51 (1H, m) 41.4 1.59 (1H, m) 4 - 34.2 5 1.81 (1H, dd, 6.1, 1.2) 40.4 6 1.45 (1H, m) 32.3 1.98 (1H, m) 7 5.55 (1H, dd, 12.2, 4.5) 66.3 8 - 145.0 9 - 212.1 10 - 54.6 11 5.28 (1H, dd, 5.5, 1.3) 77.6 12 (α) 2.36 (1H, ddd, 14.6, 5.4, 2.8) 31.7 (β) 2.95 (1H, ddd, 14.6, 5.0, 1.3) 13 3.61 (1H, br s) 41.1 14 7.23 (1H, d, 2.7) 159.3 15 - 193.5 16 - 147.8 17 (E) 5.31 (1H, br s) 112.9 (Z) 5.94 (1H, br s) 18 1.14 (3H, s) 33.8 19 0.97 (3H, s) 22.0 20 1.06 (3H, s) 18.2 1΄ - 169.0 2΄ 2.08 (3H, s) 20.6 1˝ - 169.7 2˝ 2.02 (3H, s) 20.9 64 1.3 Structure Elucidation of Compound CK 03 A compound CK 03 was obtained as brown oil. A molecular formula of C22H30O6 [m/z 413.1969 [M + Na]+, calculated for [C22H30O6 + Na]+, 413.1940] was obtained from the ESITOFMS spectrum (Figure 24). The IR absorption (Figure 23) showed bands of OH-strecthing at ν 3445 cm-1, C=O stretching at ν 1748 and 1733 cm-1, and the UV spectrum (Figure 22) displayed λmax at 233 nm. The optical rotation of CK 03 was negative, [α]30D -16.7˚ (c 0.45, CHCl3). The 1H and 13 C NMR (CDCl3) spectra of CK 03 (Figures 25 and 26) resembled those of the 8,9-secokauranes CK 01 and CK 02, but an olefinic proton signal (δH 7.23) in CK 01 and CK 02 were replaced by an oxygenated methine signal (δH 3.84) in CK 03. These 1H and 13C NMR spectral data, together with the evidence from the ESITOFMS spectrum, indicated that compound CK 03 is an oxidized form of CK 01 in which the double bond C-8/C-14 (δC 64.7/60.9) is epoxidized. The HMBC spectrum (Figure 30) helped place an acetate ester at C-11 (δC 77.6), showing the correlations from H-11(δH 5.39) to C-1′ (δC 168.8) and from the singlet methyl (at δH 2.08, H-2′) to C-1′. Based upon these spectral data, compound CK 03 was identified as ent-8,9-seco-8,14-epoxy-7α-hydroxy-11β-acetoxy-16-kauren-9,15-dione. The protons and carbons in CK 03 were completely assigned by analysis of its 2D NMR spectra (Table 4)(Figures 25, 26, 27, 28, 29 and 30). 2' O 1' O H 11 O 14 O 1 8 7 O H-13C HMBC H OH The absolute stereochemistry of compound CK 03 was assumed to be the same as that of compounds CK 01 and CK 02 due to the similarity of negative rotations observed. The orientation of the epoxide in compound CK 03 was evident from the NOESY spectrum (Figure 28) whereupon H-13 (δH 3.28) showed a more intense cross peak with H-12α (δH 2.27) than with H-12β (δH 2.94), while the H-14 (δH 3.84) epoxy proton exhibited a cross peak with H-12β (δH 2.94). These spectral data implied 65 that H-14 in compound CK 03 is β, thus the oxirane ring is α-oriented. The stereochemistry of the 8,14-epoxide ring in 8,9-secokauranes has been oriented in the same manner as that previously reported (Perry, Burgess and Tangney 1996). O 2' 1' O 1 10 4 H 12 11 20 9 14 O 16 13 O 15 8 5 7 H OH 18 19 CK 03 O 17 66 Table 4: The 1H and 13C-NMR Spectral Data of Compound CK 03 in CDCl3 δH (ppm), J (Hz) Position 1 1.34 (1H, m) δC (ppm) 32.0 1.66 (1H, m) 2 1.52 (1H, m) 17.7 1.63 (1H, m) 3 1.51 (1H, m) 41.5 1.56 (1H, m) 4 - 34.5 5 2.12 (1H, dd, 7.2, 0.8) 39.0 6 1.20 (1H, m) 34.2 1.95 (1H, m) 7 4.73 (1H, dd, 11.8, 3.4) 61.8 8 - 64.7 9 - 211.6 10 - 54.3 11 5.39 (1H, dd, 6.1, 1.4) 77.6 12 (α) 2.27 (1H, ddd, 15.1, 6.1, 3.6) 30.5 (β) 2.94 (1H, ddd, 15.1, 4.6, 1.6) 13 3.28 (1H, br t, 1.5) 38.7 14 3.84 (1H, s) 60.9 15 - 195.8 16 - 146.8 17 (E) 5.36 (1H, d, 1.6) 118.2 (Z) 6.01 (1H, br s) 18 1.12 (3H, s) 33.9 19 1.00 (3H, s) 21.6 20 1.08 (3H, s) 18.1 1΄ - 168.8 2΄ 2.08 (3H, s) 20.6 67 1.4 Structure Elucidation of Compound CK 04 Compound CK 04 was obtained as colorless oil. It was assigned the molecular C22H32O4 by ESITOFMS [m/z 383 [M + Na]+(Figure 33). The IR adsorption bands (Figure 32) showed OH-stretching at ν 3443 cm-1, and C=O stretching at ν 1635 cm-1. The UV spectrum (Figure 31) of CK 04 showed λmax at 230 nm, and the optical rotation of CK 03 was negative, [α]30D -4.0˚ (c 1.0, CHCl3). The 1H-NMR spectrum (CDCl3) (Figure 34) of compound CK 04 displayed signals of three methyl singlets at δH 0.84 (3H, s), 1.15 (3H, s), and 2.11 (3H, s), five methylene multiplets at δH 2.08 (2H, m), δH 1.56 (2H, m), 1.50 (2H, m), 1.65 (2H, m), and δH 1.72 (2H, m) , a methylene doublet of doublet of triplet at 1.92 (2.6, 6.2, 13.3), a methylene triplet of doublet and doublet of triplet at δH 1.81 (1H, dt, 3.2, 12.8) and δH 0.75 (1H, td, 3.2, 13.1), a methylene singlet at δH 5.3 (1H, s) and δH 3.88 (1H, s), a methylene doublet at δH 3.67 (1H, d, 11.1) and δH 3.88 (1H, d, 11.2), a methylene singlets at δH 5.3 (1H, s) and δH 6.0 (1H, s), a methine doublet of doublet at δH 4.05 (1H, dd, 11.8, 4.4), a methine doublet at δH 1.25 (1H, d, 8.5), two methine broad singlets at δH 1.30 (1H, br s) and δH 3.12 (1H, br s), and a methine doublet of doublet at 4.05 (1H, dd, 11.8, 4.4). The 13 C-NMR spectral data (Figure 35) revealed three methyl carbons at δC 17.6 (19-CH3), δC 18.2 (20-CH3), and δC 21.2 (2΄-CH3), nine methylene carbons at δC 17.4 (C-2), δC 18.0 (C-11), δC 27.7 (C-6), δC 28.0 (C-14), δC 32.8 (C-12), δC 35.5 (C-3), δC 39.0 (C-1), δC 72.4 (C-18), and δC 115.0 (C-17), four methine carbons at δC 37.6 (C- 13), δC 46.3 (C-5), δC 71.0 (C-7), and δC 51.8 (C-9), and six quaternary carbons at δC 36.4 (C-4), δC 39.7 (C-10), δC 58.4 (C-8), and δC 149.2 (C-16), δC 171.3 (C-1΄) and δC 209.8 (C-15). The 1H-NMR spectral data (Figure 34) exhibited resonances of an acetate group at δH 2.10 (3H, s), an oxygenated methine proton at δH 4.05 (1H, dd, 11.8, 4.4), and a methylene proton bearing oxygen at δH 3.67 (1H, d, 11.1) and δH 3.88 (1H, d, 11.2). The 1H-1H COSY spectrum (Figure 36) demonstrated for the cross peak of methylene proton from δH 0.75 (H-1) to δH 1.52 and 1.65 (H-2), from δH 1.52 (H-2) to δH 1.45 (H-3), and from δH 4.05 (H-7) to δH 1.72 (H-6). The HMBC spectrum (Figure 68 39) showed correlations from δH 0.84 (18-CH3) to δC 36.4 (C-4), from δH 3.67 and δH 3.88 (H-18) to δC 171.3 (C-1΄), from δH 2.11 (2΄-CH3) to δC 171.3 (C-1΄), from δH 1.25 (H-5) to δC 39.0 (C-1) and δC 51.8 (C-9), from δH 1.15 (20-CH3) to δC 39.7 (C-10), and from δH 1.72 (H-6) to δC 71.0 (C-7) and δC 58.4 (C-8). Based on these spectral data the first substructure of compound CK04 is propossed as shown below. 9 2 3 1 4 O H-1H COSY 7 5 H 1 8 10 6 H OH 1 H-13C HMBC O The 1H-1H COSY spectrum (Figure 36) of compound CK 04 displayed a correlation between δH 3.12 (H-13) to δH 2.08 (H-14), while the HMBC spectrum of compound CK04 showed the correlations from δH 4.05 (H-7) to δC 209.8 (C-15 ), from δH 2.08 (H-14) to δC 58.4 (C-8), δC 209.8 (C-15), δC 149.2 (C-16), and from δH 5.3 and δH 6.0 (H-17) to δC 149.2 (C-16), δC 37.6 (C-13), δC 209.8 (C-15) and δC 58.4 (C-8), therefore the second fragment of compound CK 04 is assembled as shown. 12 11 17 13 16 9 1 H-13C HMBC 14 8 7 15 OH O Combination of the first and the second fragments established a gross structure of CK 04. Therefore compound CK 04 was identified as ent-7β-hydroxy-15-oxokaur16-en-18-eyl acetate, which is a known compound previously isolated from a Vietnamese folk medicine, Croton tonkinensis (Son, Giang and Taylor 2000). 11 20 2 3 '2 10 O 17 16 14 9 8 15 7 4 6 1' O 1 13 12 19 OH O Compound CK 04 69 Compound CK 04 exhibited negative optical rotation ([α]30D -4.0˚, c 1.0, CHCl3) similar to ent-7β-hydroxy-15-oxokaur-16-en-18-eyl acetate, and therefore compound CK 04 possibly possessed the same stereochemistry as that of ent-7βhydroxy-15-oxokaur-16-en-18-eyl acetate (Son et al. 2000). Proton and carbon of compound CK 04 were completely assigned by analyses of 1H-1H COSY (Figure 36), NOESY (Figure 37), HMQC (Figure 38) and HMBC (Figure 39) spectral data as shown in Table 5. 12 20 2 O 2' 1' 3 13 14 9 1 10 H 4 H O 11 6 8 17 16 15 7 O OH 19 18 Compound CK 04 70 Table 5: The 1H and 13C-NMR Spectral Data of ent-7β-Hydroxy-15-oxokaur-16en-18-eyl acetate (Son et al. 2000) and Compound CK 04 in CDCl3 Position ent-7β-Hydroxy-15-oxokaur-16-en- Compound CK 04 18-eyl acetate (CDCl3, 400MHz) (CDCl3, 500 MHz) (Son et al. 2000) δH (ppm), J (Hz) 1 α 1.79 (ddd, 3.5, 3.5, 13.0) 38.9 β 0.74 (tdq, 13.0, 4.0, 0.9) 2 α 1.65 (m) α 1.81 (1H, dt, 3.2, 12.8) δC (ppm) 39.0 β 0.75(1H, td, 3.2, 13.1) 17.5 β 1.50 (m) 3 δH (ppm), J (Hz) δC (ppm) 1.52 (1H, m) 17.4 1.49 (1H, m) α 1.38 (br d, 4.2) 35.4 β 1.35 (m) 1.45 (1H, m) 35.4 1.35 (1H, m) 4 - 36.4 - 36.4 5 1.28 (dd, 12.6, 1.8) 46.3 1.30 (1H, d, 12.5) 46.3 6 α 1.45 (q, 12.0) 27.7 1.72 (2H, m) 27.7 β 1.70 (ddd, 12.0, 4.4, 1.6) 7 4.05 (dd, 4.4, 12.0) 70.8 4.05 (1H, dd, 11.8, 4.4) 71.0 8 - 58.3 - 58.4 9 1.23 (br d, 8.5) 51.8 1.25 (1H, d, 8.5) 51.8 10 - 39.6 - 39.7 11 α 1.70 (m) 18.0 1.65 (1H, m) 18.0 β 1.47 (m) 12 1.50 (1H, m) α 1.96 (tdd, 13.0, 6.2, 2.7) 32.8 1.98 (2H, ddt, 2.6, 6.2, 13.3) 32.8 β 1.70 (m) 13 3.10 (m) 37.6 3.12 (1H, br s) 37.6 14 2.07 (br d) 27.9 2.08 (2H, m) 27.8 15 - 209.7 - 209.8 16 - 149.2 - 149.2 17 5.29 (t, 1.1) 115.0 5.3 (1H, s) 115.0 5.97 (t, 1.1) 18 6.0 (1H, s) 3.66 (d, 10.8) 72.3 3.87 (d, 10.8) 3.67 (1H, d, 11.1) 72.4 3.88 (1H, d, 11.2) 19 0.84 (s) 17.5 0.84 (3H, s) 17.6 20 1.14 (d, 0.9) 18.2 1.15 (3H, s) 18.2 1΄ 2΄ 2.10 (s) 171.2 21.1 2.11 (3H, s) 171.3 21.2 71 2. Structure Elucidation of Compounds Isolated from Croton birmanicus 2.1 Structure Elucidation of Compound CB 01 Compound CB 01 was obtained as brown oil. A molecular formula of C18H24N2O3 m/z 339.2 [M+Na]+ was obtained from the ESIMS spectrum (Figure 42). The IR spectrum (Figure 41) showed bands of NH stretching at ν 3350 cm-1, CH3 stretching at ν 3063 cm-1, CH2 stretching at ν 2931 cm-1, C=O stretching at ν 1726 and 1682 cm-1, and the UV spectrum (Figure 40) displayed λmax at 214 nm. The optical rotation of CB 01 was negative, [α]30D -20.6˚ (c 0.825, CHCl3). The 1H NMR (CDCl3) spectral data (Figure 43) of CB 01 displayed the signals of five non- equivalent methylene protons at δH 2.77 (H-14, t, 7.7), δH 3.94 (H-13, m), δH 2.66 (H-4, dd, 5.2), δH 2.45 and δH 1.62 (H-5, m), and δH 1.41 and δH 1.62 (H-10, m), two methine protons at δH 4.41 (H-6, m), and δH 2.14 (H-9, m), five aromatic protons at δH 7.14 (H-16 and H-20, m), δH 7.22 (H-17 and H-19, m), and δH 7.15 (H-18, m), and an exchangeable NH at δH 6.20 (H-7, br d, 5.0). The 13 C NMR and DEPT135 spectral data (Figure 44) of CB 01 revealed 16 signals of two methyl carbons at δC 11.9 (C-11) and δC 17.4 (C-12); five methylene carbons at δC 24.6 (C-5), δC 27.4 (C-10), δC 31.8 (C-4), δC 34.1 (C-14), and δC 41.8 (C13), two methine carbons at δC 51.4 (C-6) and δC 43.1 (C-9); six aromatic carbons at δC 126.7 (C-18), δC 128.6 (2xC, C-17 and C-19), δC 129.1 (2xC, C-16 and C-20) and δC 138.3 (C-15); and three carbonyl carbons at δC 171.1 (C-3), δC 172.0 (C-1), and δC 177.0 (C-8). The 1H-1H COSY spectrum (Figure 46) of CB 01 displayed cross peak from δH 2.45 to δH 1.62, from δH 2.74 to δH 2.66, and from δH 1.62 to δH 1.43, while the HMQC spectrum (Figure 47) of CB 01 showed correlations from 2.45 and 1.62 (H-4) to 24.6 (C-4), from δH 2.74 and δH 2.66 (H-5) to 31.8 (C-5), and from δH 1.62 and δH 1.43 (H-10) to 27.4 (C-10). A typical resonance of 1-substituted benzene ring in CB 01 could be observed, exhibiting three multiplet resonances at δH 7.14 (2H, m), δH 7.15 (1H, m), and δH 7.22 72 (2H, m). The 13C and HMQC spectral data indicated signals of 1-substituted benzene ring at δC 129.1 (2xC), 128.6 (2xC), 126.7 (1xC) and 138.0 (1xC) assignable to C-16 (C-20), C-17 (C-19), C-18 and C-15, respectively. The 1H-1H COSY spectrum of CB 01 displayed cross peak from δH 3.98 (H-14) to δH 2.77 (H-13), while the 1H-13C correlation of HMBC(Figure 48) demonstrated the correlations from H-16 (H-20) to C-18 and C-14, from H-17 (H-19) to C-16 (C-20) and C-15, from H-14 to C-13, C-15 and C-16 (C-20), and from H-13 to C-15. Based on these spectral data, a monosubstituted aromatic ring in CB 01 is constructed as shown below. 17 1 16 18 H-1H COSY 15 19 14 20 1 H-13C HMBC 13 The 1H-1H COSY spectrum of CB 01 revealed the cross peaks from δH 2.74 and 2.66 (H-5) to δH 2.45 and 1.62 (H-4), from δH 4.41 (H-6) to δH 2.74 and 2.66 (H5) and δH 6.2 (H-7), from δH 1.62 and 1.43 (H-10) to δH 1.10 (12-CH3), and from δH 2.15 (H-9) to δH 1.10 (11-CH3). The HMBC spectrum of CB 01 showed the correlations from H-4 to δC 171.1 (C-3), from H-6 to δC 172.0 (C-1), from H-9 to δC 177 (C-8), and from H-10 to δC 11.9 (C-11), from 11-CH3 to δC 27.4 (C-10) and δC 43.1 (C-9), and from 12-CH3 to δC 43.1 (C-9) and δC 27.4 (C-10). The 1H-1H COSY spectrum of CB 01 revealed the correlation of NH proton and H-6, and the downfield resonance of H-6 suggested the presence of amide linkage in CB 01. Therefore the second partial structure of compound CB 01 is assembled as shown. 12 O N2 1 3 O 4 H N 6 5 7 8 O 9 10 11 1 H-1H COSY 1 H-13C HMBC The HMBC spectrum of CB 01 assisted in placing the connection between the first partial and the second partial structure, showing the correlation from H-13 to C-1 and C-3. 73 O 13 1 N 1 H-13C HMBC N 3 O O Based on these spectral data, the gross structure of compound CB 01 was identified as julocrotine, a glutarimide alkaloid, which was previously isolated from Julocroton montevidensis Klotzsch (Nakano, Djerassi, Corral et al. 1961), Croton humilis (Stuart, McNeill, Kutney et al. 1973), and Croton membranaceus (Aboagye, Sam, Massiot et al. 2000). Proton and carbon of compound CB 01 were completely assigned by analyses of 1H-1H COSY (Figure 45), NOESY (Figure 46), HMQC (Figure 47) and HMBC (Figure 48) spectral data as shown in Table 6. 17 18 16 15 19 20 12 O 13 2 N 14 3 O H N 1 6 4 5 7 8 9 O Compound CB 01 10 11 74 Table 6: The 1H and 13C-NMR Spectral Data of Julocrotine (Aboagye et al. 2000) and Compound CB 01 in CDCl3 Position Julocrotoine (300 MHz, CDCl3) CB 01 (400 MHz, CDCl3) (Aboagye et al. 2000) δH (ppm), J (Hz) δC (ppm) δH (ppm), J (Hz) δC (ppm) 1 - 171.7 - 172.0 2 - - - - 3 - 170.9 - 171.1 4 2.72 (m) 31.6 2.74 (1H, m) 31.8 2.66 (1H, m) 5 2.51 (m) 24.3 1.71 (m) 2.45 (1H, m) 24.6 1.62 (1H, m) 6 4.52 (dd) 51.0 4.41(1H, td, 7.8, 5.4) 51.4 7 (NH) 6.38 (br s) - 6.20 (1H, br d, 5.0) - 8 - 176.6 - 177.0 9 2.23 42.8 2.15 (1H, q, 6.7) 43.1 10 1.48 (m) 27.1 1.43 (1H, m) 27.4 1.71 (m) 1.62 (1H, m) 11 0.95 (dd) 11.7 0.87 (3H, t, 8.5) 11.9 12 1.19 (d) 17.1 1.10 (3H, d, 6.9) 17.4 13 4.01 (m) 41.4 3.94 (2H, m) 41.8 14 2.82 (t) 33.6 2.77 (2H, t, 7.8) 34.1 15 - 138.0 - 138.0 16 7.21 (m) 128.8 7.14 (1H, m) 129.1 17 7.29 (m) 128.3 7.22 (1H, m) 128.6 18 7.29 (m) 126.5 7.15 (1H, m) 126.7 19 7.29 (m) 128.3 7.22 (1H, m) 128.6 20 7.21 (m) 128.8 7.14 (1H, m) 129.1 75 3. Structure Elucidation of Compounds Isolated from Millettia kangensis 3.1 Structure Elucidation of MK 01 The compound MK 01 was obtained as colourless crystal. The molecular formular C18H12O4 was determined by ESITOFMS (Figure 51), showing [M+H]+ peak at m/z 293.0818 (calc. for C18H13O4 293.0814). The IR spectrum (Figure 50) of MK 01 revealed the presence of a conjugated carbonyl stretching at ν1637, and aromatic ring stretching at ν 1624 and 1458 cm-1. The UV spectrum (Figure 49) demonstrated the absorption at λmax at 303, 260, 217, 203 nm. The 1H-NMR (CDCl3) spectral data of MK 01 (Figure 52) displayed a characteristic of a1-substituted aromatic ring, showing a doublet of doublet signal at δH 8.16 (2H, dd, J = 8.0, 2.1 Hz), and multiplet signal at δ H 7.56 (3H, m). The 13C and HMQC spectrum (Figures 53 and 56) of MK 01 showed signals of mono-substituted aromatic ring at δC 130.6 (C-1΄), 128.0 (C-2΄ and C-6΄), 128.2 (C-3΄ and C-5΄), and 130.2 (C-4΄). The HMBC spectrum (Figure 57) of MK 01 further revealed the correlation from H-2΄ to C-3΄, C-4΄, C-1΄, and C-2. Based upon these spectral data, the first substructure was constructed. H H 2' 3' H 4' 2 1 H-13C HMBC 1' Typical signals of an anellated furan ring on the 1H NMR spectrum of MK 01, showing two doublets at δH 7.19 (1H, d, 2.2 Hz) and at δH 7.76 (1H, d, 2.2 Hz). The coupling constant of 8.8 Hz for δH 8.21 (1H, d, 8.8 Hz) and δ 7.56 (1H, d, 8.8 Hz) indicated ortho coupling between H-5 and H-6, while the HMQC spectrum showed the attachment of H-5 (δH 8.81) to C-5 δC (121.5), and H-6 (δH 7.56) to C-6 (δC 109.57). The HMBC spectrum of MK 01 exhibited correlations from H-6 to C-8, C10, and C-5, from H-5 to C-4, and from 3-OCH3 protons to C-3. The assignment of the second partial structure of MK 01 was established as shown. 76 H H 2" 3" O O 1 H-13C HMBC H 6 OCH3 5 H O Combination of the two fragments as described earlier led to the construction of a gross structure of MK 01. Finally, the structure of MK 01 was confirmed by X-ray crystallography. Compound MK 01 was therefore identified as 3-hydroxy-[4˝,5˝:8,7]furanoflavone (Karanjin), which is a known agent previously isolated from Millettia leucantha (Phrutivorapongkul, Lipipun, Ruangrungsi et al. 2003). Proton and carbon of compound MK 01 were completely assigned by analyses of 1H-1H COSY (Figure 54), NOESY (Figure 55), HMQC (Figure 56) and HMBC (Figure 57) spectral data as shown in Table 7. 2" O 3' 3" 2' 8 1' 9 O 7 2 4' 5' 6' 6 5 10 4 3 OCH3 O MK 01 The structure of compound MK 01 is confirmed by single-crystal X-ray diffraction analysis. ORTEP PLOT of MK 01 77 Table 7: 1H and 13C-NMR Spectral Data of Karanjin and Compound MK 01 Position Karanjin (CDCl3, 400 Hz) Compound MK 01 (CDCl3, 400 Hz) (Phrutivorapongkul et al., 2003) δH (ppm), J (Hz) δC (ppm) δH (ppm), J (Hz) δC (ppm) 2 - 154.9 - 153.4 3 - 141.8 - 141.3 4 - 175.3 - 174.8 5 8.21 (1H, d, 8.8) 121.8 8.21 (1H, d, 8.8) 121.5 6 7.56 (1H, m) 110.0 7.56 (1H, m) 109.6 7 - 158.2 - 157.5 8 - 117.0 - 116.7 9 - 150.0 - 148.3 10 - 119.7 - 118.3 1΄ - 131.1 - 130.6 2΄ 8.15 (1H, m) 128.4 8.16 (1H, dd, 8.0, 2.1) 128.0 3΄ 7.56 (1H, m) 128.7 7.56 (1H, m) 128.2 4΄ 7.56 (1H, m) 130.7 7.56 (1H, m) 130.2 5΄ 7.56 (1H, m) 128.7 7.56 (1H, m) 128.2 6΄ 8.15 (1H, m) 128.4 8.11 (1H, dd, 8.0, 2.1) 128.0 2˝ 7.77 (1H, d, 2.4) 145.6 7.76 (1H, d, 2.2) 145.3 3˝ 7.19 (1H, dd, J = 2.4, 104.2 7.19 (1H, d, 2.2) 103.8 60.3 3.93 (3H, s) 59.8 1.2) 3-OCH3 3.93 (3H, s) 78 3.2 Structure Elucidation of MK 02 Compound MK 02 was obtained as colourless plate. The ESIMS (Figure 60) exhibited a peak of [M+Na]+ at m/z 331.3, calculated for C18H12O5. The UV absorptions (Figure 58) bands appeared at λmax 308 and 208 nm. The IR spectrum (Figure 59) showed conjugated carbonyl stretching at ν 1592.5 cm-1 and OH stretching at ν 3265 cm-1. The 1H-NMR spectrum (DMSO-d6) (Figure 61) exhibited typical signals of 1substituted benzene ring showing a multiplet signal at δH 7.59 (3H, m) and a doublet of doublet signal at δH 8.11 (2H, dd, J = 1.85, 7.98 Hz). The 13C and HMQC spectral data indicated signals of 1-substituted benzene ring at δC 128.20 (2xC), 128.80 (2xC), 130.72 (1xC) and 130.68 (1xC) assignable to C-2΄ (C-6΄), C-3΄(C-5΄), C-4΄ and C-1΄, respectively. The 1H-1H COSY (Figure 63) showed a correlation between δ H 7.59 (H4΄, H-6΄) and 8.11 (H-2΄, H-5΄), while the 1H-13C correlation of HMBC (Figure 66) demonstrated the correlations from H-2΄ to C-1΄, C-4΄ and C-2, and from H-3΄ to C-2΄ and C-4΄. Based on these spectral data, a mono-substituted aromatic ring in MK 02 was constructed as shown below. H H 3' 2 H 1 H-1H COSY 4' 2' 1' 5' 6' 1 H-13C HMBC The singlet signal at δH 7.33 was assigned to H-5, and the HMBC spectrum showed the correlations from H-5 to C-7 (δC 147.5 ppm), C-10 (δC 120.0 ppm) and C=O (δC 173.6 ppm). Chemical shifts of 13C-NMR spectrum (Figure 62) of MK 02 at δC 140.65 (C-6), 147.5 (C-7), 147.2 (C-2˝), 143.3 (C-9), 153.9 (C-2) and 140.7 (C-3) indicated that these carbons were oxygenated double bonds. Based on these spectral data, the second partial structure of MK 02 was created as shown. 79 O 7 O 6 O 2 1 3 5 H-13C HMBC OCH3 O H The 1H-NMR spectrum of MK 02 showed the characteristic of anellated furan ring with the signals at δH 8.19 (H-2˝, d, J = 2.17 Hz) and δH 7.44 (H-3˝, d, J= 2.09 Hz). The HMQC spectrum (Figure 65) revealed the attachment of furan protons to its corresponding carbons at δC 147.2 (C-2˝) and δC 104.9 (C-3˝). The 1H-1H COSY spectrum showed the correlation between H-3˝ and H-2˝, while the HMBC spectrum showed the correlations from H-2˝ to C-3˝, C-7 and C-8, and from H-3˝ to C-7 and C8. The third fragment of MK 02 is shown below. H 2" O H 3" 8 7 1 H-13C HMBC O H The NOESY spectrum (Figure 64) of MK 02 demonstrated the correlations between 6-OH and H-5, and between 3-OCH3 and H-6΄. The HMBC spectrum exhibited the correlation from 3-OCH3 protons to C-3. O O 1 2 6' 3 HO 6 5 H O H OCH3 H-13C HMBC NOESY On the basis of these spectral data, a gross structure of MK 02 was established as shown below. Compound MK 02 was therefore identified as 3-methoxy-6-hydroxy[4˝,5˝:8,7]-furanoflavone. MK 02 is an oxidized form of MK 01, and it is a new compound. Assignment of protons and carbons of MK 02 is in Table 8. 80 3' 2" 3" O 2' 9 O 4' 2 5' 6' 3 HO 10 6 4 5 OCH3 O MK 02 Table 8: The 1H and 13C-NMR Spectral Data of Compound MK 02 in DMSO-d6 a Position δH (ppm), J (Hz) δC (ppm) 2 - 153.9 3 - 141.7a 4 - 173.6 5 7.33 (1H, s) 102.6 6 - 140.6a 7 - 147.5 8 - 118.8 9 - 143.2a 10 - 120.0 1΄ - 130.6 2΄ 8.11 (1H, dd, 7.98, 2.14) 128.2 3΄ 7.58 (1H, m) 128.8 4΄ 7.59 (1H, m) 130.7 5΄ 7.58 (1H, m) 128.8 6΄ 8.11 (1H, dd, 7.98, 2.14) 128.2 2˝ 8.19 (1H, d, 2.17) 147.2 3˝ 7.45 (1H, d, 2.09) 104.9 3-OCH3 3.83 (3H, s) 59.7 6-OH 10.80 (1H, br s) - Assignments may be exchangeable. 81 3.3 Structure Elucidation of MK 03 Compound MK 03 was obtained as colourless plate. The molecular formular was determined as C19H14O5 by ESITOFMS (Figure 69), observing for [M+Na]+ at m/z 345.48. The IR spectrum (Figure 68) of MK 03 revealed the presence of a conjugated carbonyl at ν 1753 cm-1, and the UV spectrum (Figure 67) exhibited absorptions at λmax 203, 206 and 306 nm. The 1H-NMR (CDCl3) spectrum (Figure 70) of MK 03 showed signals at δH 3.92 (3H, s), 4.10 (3H, s), 7.18 (1H, d, J = 2.3 Hz), 7.54 (3H, m), 7.55 (1H, s), 7.75 (1H, d, J = 2.0 Hz) and 8.13 (2H, dd, J = 7.9, 1.8 Hz) ppm. The 13C-NMR spectrum (Figure 71) of MK 03 demonstrated signals at δC 56.6, 60.3, 100.1, 104.9, 118.9, 126.8, 128.5, 128.8, 130.7, 131.2, 141.7, 145.2, 146.1, 148.3, 154.8 and 175, while the DEPT135 spectrum(Figure 71) revealed the presence of eight methine, two methyl, and nine quaternary carbons. Evidence from IR spectrum and 13C NMR data suggested that compound MK 03 is a flavonoid. The pattern with doublet of doublet at δH 8.13 (2H, dd, J = 7.9, 1.8 Hz) and multiplet at δH 7.54 (3H, m) were typical signals of a mono-substituted aromatic ring. The 13 C and HMQC spectra (Figures 71 and 74) indicated signals of 1-substituted benzene ring at δC 128.5 (2xC), 128.8 (2xC), 130.7 (1xC), and 131.2 (1xC) assignable to C-2΄ (or C-6΄), C-3΄ (or C-5΄), C-4΄, and C-1΄, respectively. The HMBC spectrum (Figure 75) of MK 03 revealed the correlation from H-2΄ (or H-6΄) to C-1΄, C-3΄ (or C-5΄), C-4΄, and C-2. These spectral data assisted in the construction of the first partial structure of MK 03 as shown. H H 2' 3' H 4' 1 H-13C HMBC 1' 2 The 1H NMR spectrum of MK 03 further exhibited two doublet signals at δH 7.18 (1H, d, 2.3 Hz) and δH 7.55 (1H, d, 2.0 Hz), while the 1H-1H COSY spectrum (Figure 72) displayed the correlation between these two protons (H-2˝ and H-3˝). The 82 HMBC spectrum of MK 03 showed the correlation from H-2˝ to C-7 (δC 148.3), C-8 (δC 118.9), and C-3˝ (δC 104.9), and from H-3˝ to C-7, C-8, and C-2˝. The above spectral data led to the construction of the second partial structure of MK 03 as shown. H H 2" 1 H-1H COSY 3" O 8 1 H-13C HMBC 7 The 1H NMR spectrum of MK 03 displayed two methoxy singlets at δH 3.92 and δH 4.10, while the 1H-13C correlation of HMBC spectrum showed the correlation from 3-OCH3 protons to C-3 and from 6-OCH3 protons to C-6, establishing the attachment of 3-OCH3 and 6-OCH3. A singlet methine signal at δH 7.55 (1H, s) on aromatic ring A was assignable to H-5 by the HMBC correlations from H-5 to C-7 (δC 148.3), C-9 (δC 145.2), and C-4 (δC 175.0). Chemical shifts of 13C-NMR spectrum of MK 03 at δC 154.8 (C-2), 141.7 (C-3), 175 (C-4), 145.2 (C-6), 148.3 (C-7), 145.2 (C9), and 146.1 (C-2˝) indicated that these carbons were oxygenated double bonds. The NOESY spectrum (Figure 73) of MK 03 demonstrated the correlations between 6OCH3 and H-5. Analysis of these spectral data assisted in the construction of the third substructure of MK 03 as shown. O 2 4 3 1 H3CO 6 5 H O OCH3 H-13C HMBC NOESY Combination of the three fragments mentioned above led to the assignment of a gross structure of MK 03 (Table 9). Therefore compound MK 03 was identified as 3,6-dimethoxy-[4˝,5˝:8,7]-furanoflavone, which is a known substance previously isolated from M. ichthyochtona (Kamperdick 1998). Protons and carbons of MK 06 were assigned as shown in Table 9. 83 3' 2" 3" O 2' O 4' 2 5' 6' 3 H3CO OCH3 5 O MK 03 Table 9: The 1H and 13 C-NMR Spectral Data of 3,6-Dimethoxy-[4˝,5˝:8,7]- furanoflavone and Compound MK 03 Position 3,6-Dimethoxy-[4˝,5˝:8,7]-furano Compound MK 03 flavone (CDCl3, 300 MHz) (CDCl3, 400 MHz) (Kamperdick 1998) δH (ppm), J (Hz) δC (ppm) δH (ppm), J (Hz) δC (ppm) 2 - 154.6 - 154.8 3 - 141.5 - 141.7 4 - 174.8 - 175.0 5 7.56 (1H, s) 99.8 7.55 (1H, s) 100.1 6 - 144.1 - 145.2 7 - 148.1 - 148.3 8 - 118.7 - 118.9 9 - 144.9 - 145.2 10 - 120.4 - 126.8 1΄ - 131.0 - 131.2 2΄ 8.14 (1H, dd, 7.9, 1.2) 128.3 8.13 (1H, dd, 7.9, 1.8) 128.5 3΄ 7.56 (1H, m) 128.6 7.54 (1H, m) 128.8 4΄ 7.56 (1H, m) 130.5 7.54 (1H, m) 130.7 5΄ 7.56 (1H, m) 128.6 7.54 (1H, m) 128.8 6΄ 8.14 (1H, dd, 7.9, 1.2) 128.6 8.13 (1H, dd, 7.9, 1.8) 128.5 2˝ 7.77 (1H, d, 1.9) 145.9 7.75 (1H, d, 2.0) 146.1 3˝ 7.18 (1H, d, 1.9) 104.7 7.18 (1H, d, 2.3) 104.9 3-OCH3 3.93 (3H, s) 60.2 3.92 (3H, s) 60.3 6-OCH3 4.11 (3H, s) 56.5 4.10 (3H, s) 56.6 84 3.4 Structure Elucidation of MK 04 The compound MK 04 was obtained as colourless plate. A molecular formula of C19H14O5 for MK04 was deduced from the ESITOFMS spectrum (Figure 78), [M+Na]+ observed at m/z = 345.0742. The IR spectrum (Figure 77) of MK 04 revealed the presence of a conjugated carbonyl at ν 1735 cm-1, and the UV spectrum (Figure 76) exhibited absorptions at λmax 282, 312 and 348 nm. The 1H-NMR (CDCl3) spectrum (Figure 79) of MK 04 prominently exhibited signals of a methoxyl group at δH 4.10 and 4.26, a singlet signal of aromatic proton at δH 6.74 (1H, H-3), multiplet signals of five aromatic protons at δH 7.53 (2H, H-3΄ and H-5΄), 7.54 (1H, H-4΄) and 7.98 (2H, H-2΄ and H-6΄), two doublet signals typically for furan ring at δH 7.04 (1H, d, J = 2.2 Hz) and 7.65 (1H, d, J = 2.3 Hz) as H-3˝ and H-2˝. Analysis of 13C-NMR and DEPT135 spectral (Figure 80) data of MK 04 revealed the presence of nine quaternary, eight methine, and two methyl carbons. Compound MK 04 possessed 1-substituted benzene ring, as revealed by analysis of its spectral data. The 1H-1H COSY spectrum (Figure 81) exhibited the correlation of H-2΄ (or H-6΄) and H-3΄ (or H-5΄), while the 1H-13C HMQC spectrum (Figure 83) showed the attachment between H and C, e.g. H-2΄ (H-6΄) to C-2΄ (C-6΄), and H-3΄ (H-5΄) to C-3΄ (C-5΄). The long range 1H-13C signals on the HMBC spectrum (Figure 84) were observed from H-3΄ to C-4΄, C-2΄ and C-1΄, and from H-2΄ to C-4΄ and C-1΄. These spectral data assisted in the construction of the first partial structure of MK 04 as shown. H 3' H 2' H 4' 1 H -13C HMBC 1' The downfield shift and coupling constant of 2 Hz for H-2˝ and H-3˝ are typical to an anellated furan ring. The HMBC correlations were seen from H-2˝ to C- 85 3˝, C-7 and C-8, and from H-3˝ to C-2˝ and C-7, leading to the assignment of furan ring in MK 04. 8 O H 1 H -13C HMBC 7 2" 6 3" H Two downfield singlet methoxy signals at δH 4.10 (5-OCH3) and 4.26 (8OCH3) demonstrated the HMBC correlations to carbons at respective δC 147.1 (C-5) and 131.8 (C-8), indicating that the methoxy groups situated at C-5 and C-8. These spectral data implied the presence of the third fragment in MK 04 as shown. OCH3 8 1 H-13C HMBC 5 OCH3 A singlet signal at δH 6.75 (H-3) correlated to C-2, C-1΄ and C-9, as observed on the HMBC spectrum of MK 04. The NOESY spectrum (Figure 82) of MK 04 demonstrated the correlations between 5-OCH3 and H-3˝. The carbonyl carbon of MK 04 exhibited a signal at δC 178.7 which is a characteristic of a flavonoid skeleton. Combination of all partial structures mentioned earlier led to the assemble a gross structure for MK 04. OCH3 B O O A C 4 OCH3 O 2 1 3 H H -13C HMBC NOESY 86 Based upon these spectral data, compound MK 04 is a new natural product, and identified as 5,6-dimethoxy-[4˝, 5˝;6,7]-furanoflavone. Protons and carbons of MK 04 were assigned as shown in Table 10. 3' OCH3 O 8 7 9 4' 2' O 1' 2 5' 6' 2" 3" 6 5 10 3 4 OCH3 O MK 04 Table 10: The 1H and 13C-NMR Spretral Data of Compound MK 04 in CDCl3 Position δH (ppm), J (Hz) δC (ppm) 2 - 161.7 3 6.77 (1H, s) 107.4 4 - 178.7 5 - 147.6 6 - 119.8 7 - 149.5 8 - 131.7 9 - 147.1 10 - 114.0 1΄ - 130.5 2΄ 7.98 (1H, m) 126.3 3΄ 7.53 (1H, m) 129.2 4΄ 7.53 (1H, m) 131.7 5΄ 7.53 (1H, m) 129.2 6΄ 7.98 (1H, m) 126.3 2˝ 7.65 (1H, d, 2.3) 145.8 3˝ 7.04 (1H, d, 2.2) 105.4 5-OCH3 4.10 (3H, s) 62.6 8-OCH3 4.26 (3H, s) 61.8 87 3.5 Structure Elucidation of MK 05 A compound MK 05 was obtained as colourless crystal. The UV spectrum (Figure 85) showed λmax at 203, 271 and 309 nm. The 1H and 13 C NMR (CDCl3) spectral (Figures 86 and 87) of MK 05 revealed that compound MK 05 was flavonoid derivative. A typical flavonoid carbonyl carbon was observed at δC 189.0. The characteristics of 1-phenyl substitution were also observed in MK 05, showing δH at 7.64 (br d, J = 7.3 Hz, H-2΄or H-6΄), 7.47 (m, H-3΄ or H-5΄) and 7.41 (m, H-4΄) with δC at 125.5 (2xC, (C-2΄ or C-6΄)), 128.2 (2xC, (C-3΄ or C-5΄)), and 128.4 (1xC, (C-4΄)). Interestingly, there was a low field methoxy group (δH 3.05) and nonequivalent methylene protons at δH 3.00 (1H, d, J = 16.1 Hz) and δH 3.08 (1H, d, J = 16.1 Hz). The HMBC (Figure 91) well established the substructure shown below, correlations were observed from H-3΄ (or H-5΄) to C-1΄, H-2΄ (or H-6΄) to C-2; H-3 to C-2, C-4, and C-1΄, and 2-OCH3 protons to C-2. O 1 2 3 O H OCH3 H-13C HMBC H The other partial structure of MK 05 was constructed as shown. The furan on the aromatic ring showed typical resonances at δH 6.96 (d, J = 2.3 Hz) and 7.55 (d, J = 2.3 Hz) with 13 C resonances at respective δC 105.3 and 143.9. The NOESY spectrum (Figure 89) of MK 05 showed the correlation between 5-OCH3 protons and H-3˝, placing the position 5-OCH3 and the furan on the aromatic ring as shown. The HMBC spectrum showed the correlation from 8-OCH3 protons to a carbon with δC at 130.0 (C-8), and this upfield resonance of C-8 was from the shielding effect of the adjacent oxygen atoms at C-7 and C-9, readily confirming the presence of oxygenated C-7, C-8, and C-9 in MK 05. 88 OCH3 8 O 9 O 1 H-13C HMBC 7 2" 6 3" 10 5 OCH3 NOESY A gross structure of MK 05 was assembled by combination of the two partial structures, leading to a flavanone structure uniquely decorated with a methoxy group at C-2. On the basis of these spectral data, compound MK 05 was identified as a new compound, 2,5,8-trimethoxy-[4˝, 5˝:6, 7]-furanoflavanone. Proton and carbon of compound CK 04 were completely assigned by analyses of 1H-1H COSY (Figure 88), NOESY (Figure 89), HMQC (Figure 90) and HMBC (Figure 91) spectral data as shown in Table 11. 3' 2' OCH3 O 7 9 6 10 O 2 2" 1' OCH3 3" 5 4 3 OCH3 O MK 05 4' 5' 89 Table 11: The 1H and 13C-NMR Spectral Data of Compound MK 05 in CDCl3 Position δH (ppm), J (Hz) δC (ppm) 2 - 104.5 3 3.00 (1H, d, J = 16.1) 51.1 3.08 (1H, d, J = 16.1) 4 - 189.0 5 - 149.2 6 - 130.0 7 - 151.6 8 - 115.6 9 - 146.9 10 - 111.0 1΄ - 138.4 2΄ 7.67 (1H, br d, J = 7.3) 125.5 3΄ 7.47 (1H, m) 128.3 4΄ 7.41 (1H, m) 128.4 5΄ 7.47 (1H, m) 128.3 6΄ 7.67 (1H, br d, J = 7.3) 125.5 2˝ 7.55 (1H, d, 2.3) 143.9 3˝ 6.92 (1H, d, 2.3) 105.3 2-OCH3 3.05 (3H, s) 50.4 5-OCH3 4.09 (3H, s) 61.2 6-OCH3 4.06 (3H, s) 61.1 90 3.6 Structure Elucidation of Compound MK 06 Compound MK 06 was obtained as a colorless needle. The molecular formula was determined as C22H20O5 by ESITOFMS [M+Na]+ (Figure 94) for m/z 387.1204 (387.1240). The IR bands (Figure 93) showed C-O stretching of conjugated carbonyl at ν 1615 cm-1, and C-H stretching of CH3 at ν 2923 cm-1. The UV spectrum (Figure 92) showed λmax at 350, 341, and 285 nm. The 1H-NMR spectrum (Figure 95) (DMSO-d6) of MK 06 exhibited typical signals of 1-substituted aromatic ring, showing a doublet of doublet for H-2΄ (H-6΄) at δH 8.04 (2H, dd, 7.9, 1.8), a multiplet for H-3΄ (H-5΄) at δ H 7.58 (2H, m), and a multiplet for H-4΄ at δ H 7.58 (1H, m). The 13C-NMR and HMQC spectral (Figures 96 and 99) data also confirmed the presence 1-substituted benzene ring, showing signals at δC 130.7 (1xC), 128.1 (2xC), 128.8 (2xC), and 130.3 (1xC) assignable to C-4΄, C-2΄ (C-6΄), C-3΄ (C-5΄), and C-1΄, respectively. The 1H-1H COSY (Figure 97) showed a correlation between H-2΄ (or H-4΄) and H-3΄ (or H-5΄), while the HMBC spectrum (Figure 100) showed correlation from H-2΄ to C-2, and from H-5΄ to C-1΄ Based on these spectral data, the first partial structure of compound MK 06 was created as shown. H H 2' 3' 1 4' 1' 2 H-1H COSY 5' 6' 1 H-13C HMBC The 1H-NMR spectrum further revealed a methyl singlet at δH 1.46 (6H, s) deduced to geminal methyl groups (H-4˝ and H-5˝) and two doublet signals at δH 5.95 (1H, d, J = 10.0 Hz) and δH 6.90 (1H, d, J = 10.0 Hz). The HMBC spectrum displayed correlations from two methyl groups (H-4˝ and H-5˝) to C-2˝ and C-3˝, from H-2˝ to C-3˝ and C-8, and from H-1˝ to C-3˝, C-7 and C-9, leading to the assignment of an anellated pyrano ring attactched at C-7 and C-8. The HMBC spectral data showed correlations from H-5 to carbons at δC 145.7 (C-7), δ C 146.6 (C-9), and δC 172.9 (C4), from δH 3.88 (3H, s) to δC 146.8 (C-6), and from δH 3.81 (3H, s) to δC 140.4 (C-3). 91 The NOESY spectrum (Figure 98) of MK 06 showed the correlation between 6-OCH3 protons and H-5. On the basis of these spectral data, the second partial structure was assigned as shown. H 2" 4" 5" 3" O H3CO 7 1" 8 H O 4 6 5 H 1 H-13C HMBC 3 1 OCH3 O H-1H COSY NOESY Combination of the first and second fragment led to the construction of a gross structure of MK 06. Compound MK 06 was therefore identified as 3,6dimethoxy-2˝-dimethyl-[5˝,6˝:8,7]-pyranoflavone. 4" "5 3' 2" 3" 1" O 8 2' 9 O 4' 1' 2 5' 7 6' 3 H3CO 6 10 5 4 OCH3 O MK 06 The spectrum of compound MK 06 is confirmed by single-crystal X-ray diffraction analysis. Protons and carbons of MK 06 were assigned as shown in Table 12. ORTEP PLOT of MK 06 92 Table 12: The 1H and 13C-NMR Spectral Data of Compound MK 06 in DMSO-d6 Position δH (ppm), J (Hz) δC (ppm) 2 - 153.9 3 - 140.4 4 - 172.9 5 7.34 (1H, s) 103.9 6 - 146.8 7 - 145.7 8 - 109.9 9 - 146.6 10 - 116.7 1΄ - 130.3 2΄ 8.04 (1H, dd, J = 7.9, 1.7) 128.1 3΄ 7.58 (1H, m) 128.8 4΄ 7.57 (1H, m) 130.7 5΄ 7.58 (1H, m) 128.8 6΄ 7.57 (1H,m) 128.1 1˝ 6.90 (1H, d, J = 10.0) 114.7 2˝ 5.95 (1H, d, J = 10.0) 131.3 3˝ - 78.2 2xCH3 1.46 (6H, s) 27.6 3-OCH3 3.81 (3H, s) 59.7 6-OCH3 3.88 (3H, s) 55.8 93 3.7 Structure Elucidation of MK 07 The compound MK07 was obtained as colourless plate. The ESIMS (Figure 103) suggested molecular formular of MK 07 as C18H14O7, showing a peak of [M+Na]+ at m/z 365.2. The IR bands (Figure 102) revealed signals of OH stretching at ν 3374 cm-1, α,β conjugated lactone ring at ν 1731 cm-1, and aromatic moiety at ν 1621 and 1469 cm-1. The UV spectrum (Figure 101) showed λmax at 208, 302 and 358 nm. The 1H-NMR spectrum (DMSO-d6) of MK 07 (Figure 104) showed three signals on an aromatic ring at δH 7.82 (1H, d, 8.60), 6.92 (1H, dd, 8.59, 2.06) and 6.87(1H, d, 2.05), a typical for an ABX aromatic apin system. The 1H-1H COSY spectrum (Figure 106) showed connectivities between H-2΄ and H-3΄, and between H-3΄ and H-5΄. The 13 C-NMR spectrum (Figure 105) displayed a C-4΄ hydroxyl carbon at δC 160.3. The upfield carbonyl carbon at δC 161.4 ppm suggested the presence of conjugated cyclic ester, and the HMBC (Figure 109) correlations could be well observed from H-2΄ to C-4΄, and from H-3΄ to C-1΄. Based on spectral data substructure of MK 07 was created as shown. O O 1 H-1H COSY H 2 2' O 6' H 3' 1' 5' 4' 1 H-13C HMBC OH H The HMQC (Figure 108) data revealed that three methoxyl protons located at δH 3.78, 3.88 and 3.89 ppm, belong to δC 56.3, 61.4 and 63.2. The 1H-13C HMBC spectrum showed correlations from δH 3.88(5-OCH3) to δC 147.1 (C-5), δH 3.78 (6OCH3) to δC 140.7 (C-6), and δH 3.89 (7-OCH3) to δC 156.6 (C-5). The HMBC spectrum also showed the correlation from H-8 to C-10, C-9, and C-6. Thus, the combination of HMBC and HMQC spectral data demonstrated that three methoxyl groups and H-8 were located on ring A. 94 H 7 6 H3CO 1 H-13C HMBC 8 H3CO A 5 OCH3 Based on these spectral data, MK 07 was identified as 4΄hydroxy,5,6,7-trimethoxycoumestan, this is the fisrt report of a coumestan skeleton from Millettia spp. Proton and carbon of compound MK 07 were completely assigned by analyses of 1H-1H COSY (Figure 106), NOESY (Figure 107), HMQC (Figure 108) and HMBC (Figure 109) spectral data as shown in Table 13. 8 H3CO O O 2 7 2' H3CO 6 3' 5 4' OCH3 O 5' OH MK 07 Table 13: The 1H and 13C-NMR Spectral Data of Compound MK 07 in DMSO-d6 Position δH (ppm), J (Hz) δC (ppm) 2 - 161.4 3 - 102.7 4 - 156.7 5 - 147.1 6 - 140.7 7 - 153.6 8 7.32 (1H, s) 93.3 9 - 151.8 10 - 110.4 1΄ - 104.2 2΄ 7.82 (1H, d, 8.60) 123.3 3΄ 6.92 (1H, dd, 8.59, 2.06) 114.2 4΄ - 160.3 5΄ 6.87 (1H, d, 2.05) 103.0 6΄ - 155.1 5-OCH3 3.88 (3H, s) 61.4 6-OCH3 3.78 (3H, s) 56.8 7-OCH3 3.89 (3H, s) 63.2 95 4. Biological Activities The results of biological activities including antimycobacterial, antimalarial, and cytotoxicity are shown in Table 14. 4.1 Bioactive Compounds from Croton kongensis Compounds CK 01, 02, 03 and 04 exhibited significant antimalarial (Plasmodium falciparum K1), antimycobacterial (Mycobacterium tuberculosis H37Ra) and cytotoxicity (KB cell, BC cell and NCI-H187) activities. These results are demonstrated in Table 14. 4.2 Bioactive Compound from Croton birmanicus Compound CB 01 has displayed mild antimycobacterial activity against Mycobacterium tuberculosis, and this result is in Table 14. CB 01 did not exhibit cytotoxicity. 4.3 Bioactive Compounds from Millettia kangensis Compounds MK 01, 02, 03, 05, 06, and 07 did not possess antimycobacterial and antimalarial activities (Table 4), while compound MK 04 exhibit only mild antimycobacterial activity. All isolated compounds had no cytotoxicity. 96 Table 14 Biological Activities of Compounds from C. kongensis, C. birmanicus and M. kangensis Antimalariala Antimycobacterialb activity activity IC50 (µg/mL) MIC (µg/mL) CK 01 2.1 CK 02 Compound a b c d e Cytotoxicity IC50 (µg/mL)* Vero cell KB cellc BC celld NCI-H187e 6.25 0.90 1.25 1.13 0.32 2.8 25.0 3.16 13.84 inactive 1.10 CK 03 2.7 6.25 0.99 3.39 2.16 0.42 CK 04 1.3 6.25 N.D. N.D. inactive inactive CB 01 inactive 100 >50 inactive inactive inactive MK 01 N.D. N.D. N.D. N.D. N.D. N.D. MK 02 N.D. N.D. N.D. N.D. N.D. N.D. MK 03 inactive inactive >50 inactive inactive inactive MK 04 inactive 200 >50 inactive inactive inactive MK 05 N.D. N.D. N.D. N.D. N.D. N.D. MK 06 inactive inactive >50 inactive inactive inactive MK 07 N.D. N.D. N.D. N.D. N.D. N.D. Antimalarial activity against Plasmodium falciparum, K 1 multi-drug resistant strain. Antimycobacterial activity against Mycobacterium tuberculosis H37Ra. KB cell, Human epidermoid carcinoma cell lines of nasopharynx. BC cell, Human breast cancer cell lines. NCI-H187, Human small cell lung cancer cell lines. N.D.; Not determined. * IC50 (µg/mL) > 20; inactive >10-20; weakly active 5-10; moderately active < 5; strongly active CHAPTER V CONCLUSION Two new 8,9 secokauranes namely, ent-8,9-seco-7α,11β-diacetoxykaura8(14),16-dien-9,15-dione (CK 02) and ent-8,9-seco-8,14-epoxy-7α-hydroxy-11βacetoxy-16-kauren-9,15-dione (CK 03), were isolated from Croton kongensis leaves along with two known compounds ent-8,9-seco-7α-hydroxy-11-acetoxykaura-8 (14),16-dien-9,15-dione (CK 01), and compound ent-7β-hydroxy-15-oxokaur-16-en18-yl acetate (CK 04). A known glutarimide alkaloid, julocrotine (CB 01), was isolated from the roots of Croton birmanicus. Two new furanoflavoniods as 3methoxy-6-hydroxy-[4˝,5˝:8,7]-furanoflavone (MK 02) and 2,5,8-trimethoxy- [4˝,5˝:6,7]-furanoflavanone (MK 05), a novel pyranoflavonoid, 3,6-dimethoxy-2˝dimethyl-[5˝,6˝:8,7]-pyrano flavone (MK 06), a new coumestan, 4΄-hydroxy,5,6,7trimethoxycoumestan (MK 07), together with a new natural product (synthetically known) 5,8-dimethoxy-[4˝,5˝:7,6]-furanoflavone (MK 04), and two known compounds, karanjin (MK 01) and 3,6-dimethoxy-[4˝,5˝:8,7]-furanoflavone (MK 03), were isolated from the leaves and twigs of Millettia kangensis. Ent-8,9-seco-7α,11βdiacetoxykaura-8(14),16-dien-9,15-dione (CK 02), ent-8,9-seco-8,14-epoxy-7α- hydroxy-11β-acetoxy-16-kauren-9,15-dione (CK 03), ent-8,9-seco-7α-hydroxy-11acetoxykaura-8(14),16-dien-9,15-dione (CK 01), ent-7β-hydroxy-15-oxokaur-16-en18-yl acetate (CK 04) showed significant antimalarial activity against Plasmodium falciparum (at IC50 2.8, 2.7, 2.1, and 1.3 µg/mL, respectively), antimycobacterial activity against Mycobacterium tuberculosis H37Ra (at MIC 25.0, 6.25, 6.25, and 6.25 µg/mL, respectively). In addition, CK 01, CK 02 and CK 03 exhibited strongly active toward Vero and NCI-H187 cell lines, and compounds CK 01 and CK 03 showed strongly active against BC cell line. Julocrotine (CB 01) from the roots of C. birmanicus showed mild antimycobacterial against Mycobacterium tuberculosis H37Ra at MIC 100 µg/mL. 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Prod., 66(9). APPENDIX 108 2' O 1' O H 12 11 17 20 9 1 10 16 14 O 7 15 8 4 H 18 O OH 19 Figure 4 UV Spectrum of Compound CK 01 (chloroform) Figure 5 IR Spectrum of Compound CK 01 (neat) 109 Figure 6 MS Spectrum of Compound CK 01 CH3OH residue 2' O 1' O H 12 11 17 20 9 1 10 O 7 19 15 8 4 O 20 OH H 18 2′ 16 14 19 14 17 1,2 5 2,3 13 6 17 11 7 13 12 12 Figure 7 1H- NMR Spectrum (CDCl3) of Compound CK 01 18 110 7 11 17 14 9 10 13 16,8 1′ 4,18 1,6 3,5 2′ 2,20 12 19 15 Figure 8 13C NMR and DEPT Spectra of (CDCl3) Compound CK 01 CH3OH residue 19, 20 2′ 1 1,2 5 2,3 18 6 3 12 14 17 17 11 13 12 7 2' O 1' O H 12 11 17 20 9 1 10 16 14 O 7 15 8 4 H 18 O OH 19 Figure 9 1H-1H COSY Spectrum (CDCl3) of Compound CK 01 111 CH3OH residue 19, 20 2′ 1 1,2 5 2,3 18 6 3 14 17 17 11 13 12 12 7 Figure 10 NOESY Spectrum (CDCl3) of Compound CK 01 19, 20 CH3OH residue 2′ 1 17 14 17 11 7 1,2 5 2,3 6 18 3 13 12 12 1,6 2,20 19, 2′ 12 18 3,5 10 13 7 11 17 2' O 1' 16,8 14 O H 12 11 17 20 1′ 9 1 15 9 10 16 14 O 7 15 8 4 H 18 O OH 19 Figure 11 HMQC Spectrum (CDCl3) of Compound CK 01 112 CH3OH residue 2' O O H 12 11 9 10 15 O 8 7 O 4 OH H 18 17 14 16 14 17 11 1 1,2 5 2,3 6 18 3 17 20 1 19, 20 2′ 1' 13 12 12 7 19 1,6 19, 2′ 2,20 12 18 10 13 3,5 7 11 17 16,8 14 1′ 15 9 Figure 12 HMBC Spectrum (CDCl3) of Compound CK 01 2' O 1' O 9 1 10 17 16 14 O 7 4 15 8 O O H 18 H 12 11 1" 19 2" O Figure 13 UV Spectrum of Compound CK 02 (chloroform) 113 2' O 1' O 9 1 10 17 16 14 O 7 4 15 8 O O H 18 H 12 11 1" 19 2" O Figure 14 IR Spectrum of Compound CK 02 (neat) Figure 15 MS Spectrum of Compound CK 02 114 18 2' O 1' O 9 1 17 10 15 O 4 2″ 8 6 O 20 1 19 6 5 O H 1" 19 2" O 12 14 2,3 1,2,3 16 14 7 18 2′ H 12 11 17 12 17 7 11 13 Figure 16 1H- NMR Spectrum (CDCl3) of Compound CK 02 3,5,13 4,18 1,6 2′ 7 14 9 1′,1″ 15 17 8 16 11 2,20 19 10 12 Figure 17 13C NMR and DEPT Spectra (CDCl3) of Compound CK 02 115 20 2′,2″ 18 14 17 17 7 11 2,3,6 1,2,3 1 19 13 6 12 12 5 2' O 1' O 9 1 10 17 16 14 O 7 4 15 8 O O H 18 H 12 11 1" 19 2" O Figure 18 1H-1H COSY Spectrum (CDCl3) of Compound CK 02 14 17 17 7 11 20 18 2′,2″ 2,3,6 1,2,3 1 19 6 13 12 12 5 Figure 19 NOESY Spectrum (CDCl3) of Compound CK 02 116 18 2′,2″ 17 17 14 20 2,3,6 1,2,3 1 6 19 5 7 11 12 12 13 2,20 12 19 2′ 1,6 4,18 3,5,13 10 7 11 2' O 1' O 17 9 1 10 17 O 4 18 O 1" 19 14 15 8 O H 16 16 14 7 8 H 12 11 2" O Figure 20 HMQC Spectrum (CDCl3) of Compound CK 02 2′,2″ 2,3,6 17 17 14 7 11 13 18 1,2,3 1 19 6 12 12 5 2,20 1,612 19 2′ 4,18 3,5,13 10 7 11 17 8 14 20 16 1′,1″ 15 9 Figure 21 HMBC Spectrum (CDCl3) of Compound CK 02 117 O 2' 1' O H 12 11 20 9 14 13 16 15 1 10 4 17 O O 8 5 7 H O OH 18 19 Figure 22 UV Spectrum of Compound CK 03 (chloroform) Figure 23 IR Spectrum of Compound CK 03 (neat) 118 Figure 24 MS Spectrum of Compound CK 03 2' O 1' O 9 1 10 17 O 7 18 16 14 4 15 8 O 20 2′ O H 18 H 12 11 1,2 1" 19 2" 12 5 17 17 19 2,3 O 6 1 6 14 11 7 13 12 Figure 25 1H- NMR Spectrum (CDCl3) of Compound CK 03 119 O 2' 1' H 12 O 13 16 11 20 9 14 15 1 10 4 17 O O 8 5 7 H O OH 18 19 5,13 4,6,18 2,20 3 1 14 11 15 1′ 19 12 2′ 8 17 9 7 16 10 Figure 26 13C NMR and DEPT Spectra (CDCl3) of Compound CK 03 18 20 17 17 11 14 7 2′ 1,2 2,3 19 5 1312 12 6 6 1 Figure 27 1H-1H COSY Spectrum (CDCl3) of Compound CK 03 120 18 20 2′ 17 17 11 14 7 1,2 2,3 19 5 13 12 12 6 O 6 1 2' 1' H 12 O 17 13 16 11 20 9 14 O 15 1 O 10 8 O 5 4 7 H OH 18 19 Figure 28 NOESY Spectrum (CDCl3) of Compound CK 03 14 17 17 2′ 2,3 5 6 12 16 11 7 18 20 1,2 12 13 19 2,20 2′ 19 1 12 4,6,18 5,13 3 10 14 7 8 O 2' 1' O 11 H 12 11 20 9 14 13 16 O 15 1 10 4 O 8 5 7 H OH 18 19 17 Figure 29 HMQC Spectrum (CDCl3) of Compound CK 03 O 17 121 O 2' 1' O 13 16 10 O 8 5 7 H OH 18 19 2,20 19,2′ 1,12 5,13 3 10 14 7 8 17 O 15 1 4 H 12 11 20 9 14 14 17 O 17 2′ 5 11 13 12 7 1,2 18 20 2,3 19 12 6 1 6 4,6,18 11 17 16 1′ 15 9 Figure 30 HMBC Spectrum (CDCl3) of Compound CK 03 12 20 2 O 2' 1' 3 13 14 9 1 10 8 H 4 H O 11 6 17 16 15 7 O OH 19 18 Figure 31 UV Spectrum of Compound CK 04 (chloroform) 122 12 20 2 O 2' 1' 3 13 14 9 1 10 8 H 4 H O 11 6 17 16 15 7 O OH 19 18 Figure 32 IR Spectrum of Compound CK 04 (neat) Figure 33 MS Spectrum of Compound CK 04 123 12 20 2 O 2' 1' 3 13 14 9 1 10 4 6 17 16 8 H H O 11 2′ 15 20 7 O OH 19 18 19 6,11 17 17 18 7 9 14 3,5 12 2,3,11 1 1 18 13 Figure 34 1H- NMR Spectrum (CDCl3) of Compound CK 04 19 17 9 7 18 13 12 5 1 3 6,14 2,11,19,20 2′ 4 15 1′ 16 8 10 Figure 35 13C-NMR Spectrum (CDCl3) of Compound CK 04 124 2′ 17 17 6,11 3,59 14 2,3,11 12 1 1 18 18 13 7 20 19 12 20 2 O 2' 1' 3 11 9 1 10 8 H 4 H O 13 14 6 17 16 15 7 O OH 19 18 Figure 36 1H-1H COSY Spectrum (CDCl3) of Compound CK 04 17 17 18 18 7 13 20 19 2′ 6,11 3,59 14 2,3,11 12 1 1 Figure 37 NOESY Spectrum (CDCl3) of Compound CK 04 125 17 17 6,11 2019 2′ 3,5 9 142 3 11 12 1 1 18 18 13 7 2 11 19 20 19 6 14 2′ 13 31 12 5 9 718 12 17 20 2 O 2' 1' 3 13 14 9 1 10 8 H 4 H O 11 6 17 16 15 7 O OH 19 18 Figure 38 HMQC Spectrum (CDCl3) of Compound CK 04 17 17 18 18 7 13 6,11 2019 2′ 3,5 9 14 2,3,11 12 1 1 2,11,19,20 19 2′ 6,14 12 13 3,4 1 10 5 9 8 718 17 16 1′ 15 Figure 39 HMBC Spectrum (CDCl3) of Compound CK 04 126 17 18 16 13 15 19 20 12 O 2 N 14 3 O H N 1 6 4 7 5 8 9 11 10 O Figure 40 UV Spectrum of Compound CB 01 (chloroform) 17 18 16 19 20 12 O 13 15 2 N 14 3 O H N 1 6 4 5 7 8 O Figure 41 IR Spectrum of Compound CB 01 (film) 9 10 11 127 Figure 42 MS Spectrum of Compound CB 01 17 18 16 19 17,18,19 20 16,20 13 15 2 N 14 3 O H N 1 6 4 7 5 8 9 10 11 11 O 5,10 13 7 12 12 O 6 9 10 4 5 Figure 43 1H- NMR Spectrum (CDCl3) of Compound CB 01 128 16,20 17,19 16,20 17,19 18 1,3 8 83 1 4 14 5 12 11 13 410 9 14 6 13 105 12 11 9 6 18 15 15 Figure 44 13C NMR and DEPT Spectra (CDCl3)of Compound CB 01 17,18,19 12 16,20 4 5,10 5 9 10 7 11 13 6 17 18 16 13 15 19 20 12 O 2 N 14 3 O H N 1 6 4 5 7 8 9 10 11 O Figure 45 1H-1H COSY Spectrum (CDCl3) of Compound CB 01 129 16,20 17,18,19 12 11 4 5,10 5 13 6 7 9 10 17 18 16 19 20 12 O 13 15 2 N 14 3 O H N 1 6 4 7 5 8 10 9 11 O Figure 46 NOESY Spectrum (CDCl3) of Compound CB 01 17,18,19 4 16,20 5 12 11 5,10 10 9 7 6 13 11 12 4 10 5 14 13 96 17,19 18 16,20 15 1,3 8 Figure 47 HMQC Spectrum (CDCl3) of Compound CB 01 130 12 2" 17 18 16 20 3" 13 7 14 6 O 2' 8O 9 O 15 19 3' 2 N 1 17,18,19 4' 16,20 11 4 5,10 10 5 9 12 O H2 N 6 4 3 10 5 5 4 7 3 1' 8 5' 9 6' 10 OOCH3 11 13 7 6 O 11 12 5 4 10 13 14 96 17,19 18 16,20 15 1,3 8 Figure 48 HMBC Spectrum (CDCl3) of Compound CB 01 2" O 3' 3" 2' 8 1' 9 O 7 2 4' 5' 6' 6 5 10 4 3 OCH3 O Figure 49 UV Spectrum of Compound MK 01 (methanol) 131 2" O 3' 3" 2' 8 1' 9 O 7 5' 6' 6 5 10 4 O Figure 50 IR Spectrum of Compound MK 01 (film) Figure 51 MS Spectrum of Compound MK 01 2 4' 3 OCH3 132 2" O 7 2' 8 1' 9 O 3-OCH 3 6 6, 3′,4′,5′ 5 10 2 2″ 5' 3 4 OCH3 O 3″ 4' 6' O 2" 2′,6′ 5 3' 3" 3' 3" 2' 8 1' 9 O 7 2 4' 5' 6' 6 5 10 4 3 OCH3 O Figure 52 1H- NMR Spectrum (CDCl3) of Compound MK 01 2′ 3′,5′ 1′,6′ 2″ 5 6 3″ 3-OCH3 4 7 2 9 3 10 8 Figure 53 13C NMR and DEPT Spectra (CDCl3) of Compound MK 01 133 3-OCH3 6, 3′,4′,5′ 2′,6′ 5 3″ 2″ 2" O 3' 3" 2' 8 1' 9 O 7 2 4' 5' 6' 6 5 10 4 3 OCH3 O 3-OCH3 3″ 6, 3′,4′,5′ 2′,6′ 2″ 5 Figure 54 1H-1H COSY Spectrum (CDCl3) of Compound MK 01 3-OCH3 6, 3′,4′,5′ 2′,6′ 5 2″ 3″ 3-OCH3 3″ 6, 3′,4′,5′ 2′,6′ 2″ 5 Figure 55 NOESY Spectrum (CDCl3) of Compound MK 01 134 3-OCH3 6, 3′,4′,5′ 2′,6′ 5 2″ 3″ 3-OCH3 6 2′,3′,5′ 6′ 2" 3″ O 5 3' 3" 2' 8 1' 9 O 7 5' 6' 6 5 2″ 2 4' 10 4 3 OCH3 O Figure 56 HMQC Spectrum (CDCl3) of Compound MK 01 3-OCH3 6, 3′,4′,5′ 2′,6′ 5 2″ 3″ 3-OCH3 6 3″ 8 10 5 2′,3′,5′ 3 1,6′ 2″ 9 2 7 4 Figure 57 HMBC Spectrum (CDCl3) of Compound MK 01 135 3' 2" 3" O 2' 9 O 4' 2 5' 6' 3 HO 10 6 4 5 OCH3 O Figure 58 UV Spectrum of Compound MK 02 (methanol) Figure 59 IR Spectrum of Compound MK 02 (film) 136 Figure 60 MS Spectrum of Compound MK 02 3' 2" 3" O 2' 9 O 4' 2 5' 6' 3 3′,4′,5′ 2′,6′ 6-OH 2″ HO 10 6 4 5 3-OCH3 5 3″ Figure 61 1H- NMR Spectrum (DMSO-d6) of Compound MK 02 O OCH3 137 3′,4′,5′ 2′,6′ 5 7,2″ 1′ 10 8 3 6 9 2 4 3-OCH3 3″ Figure 62 13C NMR and DEPT Spectra (DMSO-d6) of Compound MK 02 3′,4′,5′ 3' 2" 3" O 2' O 9 4' 2 2′,6′ 5' 5 3-OCH3 6' 3 HO 10 6 OCH3 4 5 O 6-OH 2″ 3″ 3-OCH3 3″ 5 3′,4′,5′ 2′,6′ 2″ 6-OH Figure 63 1H-1H COSY Spectrum (DMSO-d6) f Compound MK 02 138 3′,4′,5′ 5 2′,6′ 6-OH 3-OCH3 3″ 2″ 3-OCH3 5 3″ 3′,4′,5′ 3' 2" 2′,6′ 2″ 3" O 2' O 9 4' 2 5' 6' 3 HO 10 6 4 5 OCH3 O 6-OH Figure 64 NOESY Spectrum (DMSO-d6) of Compound MK 02 2′,6′ 3′,4′,5′ 5 3-OCH3 3″ 2″ 3-OCH3 2′,6′ 3′,4′,5′ 3″ 5 2″ Figure 65 HMQC Spectrum (DMSO-d6) of Compound MK 02 139 2′,6′ 3′,4′,5′ 5 3-OCH3 3″ 6-OH 2″ 3-OCH3 3' 2" O 5 2' 9 O 4' 2 5' 6' 3 3″ HO 10 6 OCH3 4 5 8 2′,6′ 3′,4′,5′ 3" O 10 1′ 6 3 9 7,2″ 2 4 Figure 66 HMBC Spectrum (DMSO-d6) of Compound MK 02 3' 2" 3" O 2' O 4' 2 5' 6' 3 H3CO OCH3 5 O Figure 67 UV Spectrum of Compound MK 03 (methanol) 140 3' 2" 3" O 2' O 4' 2 5' 6' 3 H3CO OCH3 5 O Figure 68 IR Spectrum of Compound MK 03 (film) Figure 69 MS Spectrum of Compound MK 03 141 3' 2" 3" 2' O O 4' 2 5' 6' 3 H3CO OCH3 5 O 6-OCH3 3′,4′,5′ 2′,6′ 2″ 3-OCH3 3″ Figure 70 1H- NMR Spectrum (CDCl3) of Compound MK 03 3′,5′ 2′,6′ 2″ 4 6 2 79 3 6-OCH3 3″ 4′ 5 10 3-OCH3 8 Figure 71 13C NMR and DEPT Spectra (CDCl3) of Compound MK 03 142 2′,6′ 3′,4′,5′ 2″ 6-OCH3 3-OCH3 3″ 3-OCH3 6-OCH3 3' 2" O 3″ 3′,4′,5′ 3" 2' O 4' 2 5' 6' 2″ 2′,6′ 3 H3CO OCH3 5 O Figure 72 1H-1H COSY Spectrum (CDCl3) of Compound MK 03 6-OCH3 3′,4′,5′ 2″ 3-OCH3 3″ 3-OCH3 6-OCH3 3″ 3′,4′,5′ 2″ Figure 73 NOESY Spectrum (CDCl3) of Compound MK 03 143 3′,4′,5′ 2′,6′ 2″ 6-OCH3 3-OCH3 3″ 6-OCH3 3-OCH3 3″ 3' 2" 2′,3′,5′,6′ 4′ 3" O 2' O 2″ 4' 2 5' 6' 3 H3CO OCH3 5 O Figure 74 HMQC Spectrum (CDCl3) of Compound MK 03 6-OCH3 3-OCH3 3′,4′,5′ 2′,6′ 2″ 3″ 6-OCH3 3-OCH3 3″ 2′,3′,5′,6′ 4′ 2″ 5 8 10 6, 9 3 7 2 4 Figure 75 HMBC Spectrum (CDCl3) of Compound MK 03 144 3' OCH3 O 7 9 O 10 4 2 2" 3" 6 5 OCH3 O Figure 76 UV Spectrum of Compound MK 04 (chloroform) Figure 77 IR Spectrum of Compound MK 04 (film) 3 2' 4' 1' 5' 145 Figure 78 MS Spectrum of Compound MK 04 3' OCH3 O 7 9 O 10 4 2 2" 3" 6 5 OCH3 O 8-OCH3 5-OCH3 3′,4′,5′ 3 2″ 2′,6′ 3″ Figure 79 1H- NMR Spectrum (CDCl3) of Compound MK 04 3 2' 4' 1' 5' 146 3' OCH3 O 9 O 10 4 7 2 2' 4' 1' 5' 2" 3" 6 5 3 OCH3 O 3′,5′ 2′,6′ 2″ 5 4 2 7 3″ 4′ 3 5-OCH3 8-OCH 3 1′ 9 6 8 10 Figure 80 13C NMR and DEPT Spectra (CDCl3) of Compound MK 04 3′,4′,5′ 2′,6′ 2″ 8-OCH3 5-OCH3 3 3″ 5-OCH3 8-OCH3 3 3″ 3′,4′,5′ 2″ 2′,6′ Figure 81 1H-1H COSY Spectrum (CDCl3) of Compound MK 04 147 3′,4′,5′ 2′,6′ 2″ 8-OCH3 5-OCH3 3 3″ 5-OCH3 8-OCH3 3' OCH3 3 3″ O 7 9 O 10 4 2 2' 4' 1' 5' 2" 3′,4′,5′ 2″ 3" 6 5 3 OCH3 O 2′,6′ Figure 82 NOESY Spectrum (CDCl3) of Compound MK 04 8-OCH3 3′,4′,5′ 2″ 2′,6′ 3″ 5-OCH3 3 5-OCH3 8-OCH3 3″ 3 2′,6′ 3′,5′ 5,9 4′ 2″ 7 10 8 6, 1′ 2 4 Figure 83 HMQC Spectrum (CDCl3) of Compound MK 04 148 3′,4′,5′ 2″ 2′,6′ 8-OCH3 5-OCH3 3 3″ 5-OCH3 8-OCH3 3' OCH3 O 7 9 O 10 4 2 2' 4' 1' 5' 2" 3" 6 5 3″ 3 2′,6′ 4′ 3′,5′ 3 OCH3 O 10 8 6, 1′ 2″ 5,9 7 2 4 Figure 84 HMBC Spectrum (CDCl3) of Compound MK 04 3' 2' OCH3 O O 7 OCH3 2'' 4 3'' 5' 2 9 4' 3 5 OCH3 O Figure 85 UV Spectrum of Compound MK 05 (chloroform) 149 3' 2' OCH3 O O 7 9 OCH3 2'' 4 3'' 4' 5' 2 5-OCH3 3 5 OCH3 O 2′,6′ 2″ 8-OCH3 2-OCH3 3′,5′ 4′ 3″ Figure 86 1H- NMR Spectrum (CDCl3) of Compound MK 05 3′,5′ 2′,6′ 3″ 2″ 7 59 4 1′ 6 8 10 2 8-OCH3 3 2-OCH3 5-OCH3 Figure 87 13C NMR and DEPT Spectra (CDCl3) of Compound MK 05 150 2′,6′ 2″ 3′,5′ 5-OCH3 4′ 3″ 8-OCH3 2-OCH3 3' 2' OCH3 O O 7 9 OCH3 2'' 4 3'' 4' 5' 2 3 5 OCH3 O Figure 88 1H-1H COSY Spectrum (CDCl3) of Compound MK 05 2′,6′ 3′,5′ 2″ 4′ 3″ 5-OCH3 8-OCH3 2-OCH3 Figure 89 NOESY Spectrum (CDCl3) of Compound MK 05 151 5-OCH3 2′,6′ 3′,5′ 2″ 2-OCH3 8-OCH3 4′ 3″ 3 2-OCH3 8-OCH3 5-OCH3 2 3″ 2′,6′ 3′,5′ 2″ 5 10 8 1′ 9 3' 6 2' OCH3 O O 7 OCH3 2'' 7 4 3'' 5' 2 9 4' 3 5 OCH3 O 4 Figure 90 HMQC Spectrum (CDCl3) of Compound MK 05 2′,6′ 3′,5′ 2″ 4′ 3″ 5-OCH3 8-OCH3 2-OCH3 3 2-OCH3 8-OCH3 5-OCH3 2 10 8 2′,6′ 3′,5′ 6 1′ 2″ 9 5 7 3″ 4 Figure 91 HMBC Spectrum (CDCl3) of Compound MK 05 152 4" 3' 2" 3" 2' 1" 5" 8 O 9 O 4' 1' 5' 2 7 6' 3 H3CO 6 10 5 4 OCH3 O Figure 92 UV Spectrum of Compound MK 06 (chloroform) Figure 93 IR Spectrum of Compound MK 06 (film) 153 Figure 94 MS Spectrum of Compound MK 06 4" 3' 2" 3" 2' 1" 5" 8 O 9 O 4' 1' 5' 2 7 6' 3 H3CO 6 10 5 4 OCH3 O 3-OCH3 6-OCH3 3′,4′,5′ 2′,6′ 4″-CH3 5″-CH3 5 1″ 2″ Figure 95 1H- NMR Spectrum (DMSO-d6) of Compound MK 06 154 4" 3' 2" 3" 2' 1" 5" 8 O 9 O 4' 1' 5' 2 7 6' 3 H3CO 3′,5′ 6 10 4 5 OCH3 O 2′,6′ 4″ 5″ 6-OCH3 4′ 6,9 1′,2″ 2 73 4 10 3-OCH3 1″ 8 3″ 5 Figure 96 13C-NMR Spectrum (DMSO-d6) of Compound MK 06 6-OCH3 3′,4′,5′ 2′,6′ 5 1″ 3-OCH3 4″-CH3 2″ 5″-CH3 4″-CH3 5″-CH3 3-OCH3 6-OCH3 2″ 1″ 5 3′,4′,5′ 2′,6′ Figure 97 1H-1H COSY Spectrum (DMSO-d6) of Compound MK 06 155 3-OCH3 6-OCH3 3′,4′,5′ 4″-CH3 5 2′,6′ 1″ 5″-CH3 2″ 4″-CH3 5″-CH3 3-OCH3 6-OCH3 2″ 4" 3' 2" 3" 2' 1" 5" 1″ 8 O 5 9 O 4' 1' 5' 2 7 6' 3 H3CO 2′,6′ 3′,4′,5′ 6 10 5 4 OCH3 O Figure 98 NOESY Spectrum (DMSO-d6) of Compound MK 06 6-OCH3 3′,4′,5′ 2′,6′ 3-OCH3 4″-CH3 5 1″ 5″-CH3 2″ 4″ 5″ 6-OCH3 3-OCH3 3″ 2′,6′ 3′,5′1′,4′,2″ 5 1″ 8 10 7 6,9 3 2 4 Figure 99 HMQC Spectrum (DMSO-d6) of Compound MK 06 156 4" 3' 2" 3" 2' 1" 5" 8 O 9 4' 1' O 5' 2 7 6' 3 H3CO 6 10 5 OCH3 4 O 6-OCH3 3′,4′,5′ 3-OCH3 4″-CH3 5 2′,6′ 5″-CH3 1″ 2″ 4″ 5″ 6-OCH3 3-OCH3 3″ 2′,6′ 3′,5′1′,4′,2″ 5 1″ 8 10 6,9 7 3 2 4 Figure 100 HMBC Spectrum (DMSO-d6) of Compound MK 06 8 H3CO O H3CO O 2 7 1' 6 5 2' 3' 4 4' OCH3 O 5' OH Figure 101 UV Spectrum of Compound MK 07 (chloroform) 157 8 H3CO O H3CO O 2 7 1' 6 5 2' 3' 4 4' OCH3 O 5' OH Figure 102 IR Spectrum of Compound MK 07 (film) Figure 103 MS Spectrum of Compound MK 07 158 8 H3CO O 7 H3CO 6-OCH3 O 2 1' 6 5 5-OCH3 2' 3' 4 4' OCH3 O 7-OCH3 OH 5' 8 2′ 3′ 5′ 4′-OH Figure 104 1H- NMR Spectrum (DMSO-d6) of Compound MK 07 2′ 3′ 2 7 6′ 4 9 4′ 5′ 8 6-OCH3 5-OCH3 1′ 5 6 7-OCH3 10 3 Figure 105 13C NMR and DEPT Spectra (DMSO-d6) of Compound MK 07 159 5-OCH3 7-OCH3 3′ 2′ 8 6-OCH3 5′ Figure 106 1H-1H COSY Spectrum (DMSO-d6) of Compound MK 07 5-OCH3 8 3′ 2′ 5′ 6-OCH3 7-OCH3 4′-OH 8 H3CO O 7 H3CO O 2 1' 6 5 2' 3' 4 4' OCH3 O 5' OH Figure 107 NOESY Spectrum (DMSO-d6) of Compound MK 07 160 6-OCH3 5-OCH3 7-OCH3 8 3′ 2′ 5′ 6-OCH3 5-OCH3 7-OCH3 8 5′ 3′ 2′ 8 H3CO O 7 H3CO O 2 1' 6 5 2' 3' 4 4' OCH3 O 5' OH Figure 108 HMQC Spectrum (DMSO-d6) of Compound MK 07 5-OCH3 6-OCH3 7-OCH3 8 3′ 2′ 5′ 6-OCH3 5-OCH3 7-OCH3 8 5′1′ 3 10 3′ 2′ 9 6 76′ 5 4 4′ 2 Figure 109 HMBC Spectrum (DMSO-d6) of Compound MK 07 VITA Miss Jiraporn Thongtan was born on December 3, 1965 in Bangkok, Thailand. She received her Bachelor’s degree of Science in Chemistry from Ramkhamhaeng University in 1992, and Master’s degree of Science in Applied Chemistry from Ramkhamhaeng University in 1997. She received a student grant from theThailand Graduated Institute of Science and Technology (TGIST). Publication Thongtan, J., Kittakoop, P., Ruangrugsi, N., Saenboonrueng, J., and Thebtaranonth, Y. 2003. New antimycobacterial and antimalarial 8,9secokaurane diterpenes from Croton kongensis. J. Nat. Prod., 66(6): 868-870. Poster Presentations 1. Thongtan, J., Kittakoop, P., Ruangrugsi, N., and Saenboonrueng, J. Antimycobacterial and antimalarial principle from Croton kongensis. NRCT-JSPS CORE UNIVERSITY SYSTEM: The sixth NRCT-JSPS Joint Seminar in Pharmaceutical Sciences; Drug Development Through Biopharmaceutical Sciences. December 2-4, 2003, Bangkok, Thailand. 2. Thongtan, J., Kittakoop, P., Ruangrugsi, N., and Saenboonrueng, J. Antimycobacterial and antimalarial compounds from Croton kongensis and Croton birmanicus.The 20th Annual Research Meeting Pharmaceutical Sciences, December 1, 2003, Bangkok, Thailand. in