Summary of Chemistry doctoral thesis: Study on chemical constituents, biological activities of conifer species Dacrycarpus imbricatus and Fokienia hodginsii

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The objective of the thesis: Study on the chemical constituents of the species Dacrycarpus imbricatus (Blume) de Laub, Fokienia hodginsii (Dunn) Henry a. et Thomas; investigation in to their biological activities.

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Nội dung Text: Summary of Chemistry doctoral thesis: Study on chemical constituents, biological activities of conifer species Dacrycarpus imbricatus and Fokienia hodginsii

MINISTRY OF EDUCATION AND VIETNAM ACADEMY TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- DANG VIET HAU STUDY ON CHEMICAL CONSTITUENTS, BIOLOGICAL ACTIVITIES OF CONIFER SPECIES: DACRYCARPUS IMBRICATUS AND FOKIENIA HODGINSII Major: Organic Chemistry Code: 62 44 01 14 SUMMARY OF CHEMISTRY THE DOCTORAL THESIS HA NOI - 2017
Thesis was completed at: Institute of Chemistry Vietnam Academy of Science & Technology Advisers: 1. Assoc. Prof. Dr. Trinh Thi Thuy Institute of Chemistry- Vietnam Academy of Science & Technology 2. Dr. Tran Van Loc Institute of Chemistry- Vietnam Academy of Science & Technology 1st Reviewer: .............................................................................. 2nd Reviewer: ............................................................................. 3rd Reviewer: ............................................................................... The dissertation will be defended at: Graduate University Science and Technology - Vietnam Academy of Science & Technology- No. 18 Hoang Quoc Viet - Cau Giay - Hanoi. At............hour.............day............month............ 2017 Thesis can be found in: -The library of Graduate University Science And Technology -National Library of Vietnam
INTRODUCTION 1. The urgency of the thesis Thanks for industrial development, human life is increasingly improving. However, the downside of this development is environmental catastrophe. Moreover, abuse of chemicals and medicines plant protection in agriculture and food industry, together with the resistance of the virus species, the bacteria, the variation of this species do encounter evidence of human diseases such as cancer, HIV/AIDS, heart disease, diabetes, SARS, or recent viral fewers. To deal with the challenges of constant evolution of life, scientists must continuously invent new medicines providing selected effects, effective, and affordable to both human patients and the conservation of rare plant and animal species. According to scientists, studying the nature and mimic-nature are the best the way for human being to exist and adapt to their living environments. One of the approaches is researching new structures with potential activity that be able to be developed into medicines for humans, livestock, and crops from natural compounds. Over millions of years of evolution, these natural compounds have mounted up a flexible compatibility and relatively suitability with living organisms; especially, they also contain less toxic and perform morefriendly to environment. Located in the tropical monsoon climate, Vietnam has extremely diverse vegetation and abundant with approximately 13,000 species , of which 309 branches and 4,000 species can be adopted to make drugs. As a result, these are invaluable natural resources of the country. Although there are many types of herbs that can be used to make medicines for people, livestock and crops, their economic performance is still limited because the exploit and usage are still based on folk experience. Based on this scientific background, we chose the project "Study on chemical constituents, biological activities of conifer species Dacrycarpus imbricatus and Fokienia hodginsii". 2. The objective of the thesis - Study on the chemical constituents of the species Dacrycarpus imbricatus (Blume) de Laub, Fokienia hodginsii (Dunn) Henry a. et Thomas. - Investigation in to their biological activities 3. The main content of the thesis 1
- Purification and isolation of compounds from crude extracts of these species is techniques as column chromatography, flash column chromatography, etc. - Determine structure of the pure compounds by the combination of spectral methods such as IR, MS, 1D-2D-NMR, NMR, etc. - Some isolated compounds were tested biological activities in order to discover potential compounds. Chapter 1: OVERVIEW 1.1. Characteristics of plant and study of the genenus Dacrycarpus, family Podocarpaceae 1.1.1. Characteristics of plant genenus Dacrycarpus 1.1.1.1. Characteristics of plant species Dacrycarpus imbricatus 1.1.1.2. Characteristics of plant species Dacrycarpus dacrydioides 1.1.1.3. Characteristics of plant species Dacrycarpus vieillardii 1.1.2. Used in traditional medicine 1.1.3. The chemical constituents and biological activity of genera Dacrycarpus 1.1.3.1. Diterpene compounds 1.1.3.2. Flavonoids glucoside compounds 1.2. Characteristics of plants and study of the genus Fokienia. Henry & H.H. Thomas, family (Cupressaceae) 1.2.1. Family Cupressaceae (Cupressaceae) 1.2.2. Characteristics of plant of the genenus Fokienia. 1.2.3. Characteristics, distribution of species Fokienia hodginsii (Dunn) Henry a. H.H & Thomas 1.2.4. Use and application of traditional medicine 1.2.5. The chemical constituents and biological activity of species Fokienia hodginsii 1.2.6. Chemistry and biological activities of diterpene compounds Chapter 2: EXPERIMENT This chaper describes the collection, determination, extraction, isolation, spectral data, and bioactivity assays of isolated compounds from these plant materials. 2
2.1. Materials, chemicals and equipment 2.2. Research method 2.3. Extraction and isolation of the compounds from two researched trees 2.3.1. Dacrycarpus imbricatus Figure 2.1. Schematic diagram showing steps for the isolation of pure compounds from the bark of Dacrycarpus imbricatus 3
Figure 2.2. Schematic diagram showing steps for the isolation of pure compounds from the wood of Dacrycarpus imbricatus 4
Figure 2.3. Schematic diagram showing steps for the isolation of pure compounds from the twig and leaves of Dacrycarpus imbricatus  Spectral data of isolated compounds ● Compound 86 (β-sitosterol) colorless solid, Rf = 0.34 (n- hexane:DCM = 1:3). ● Compound 89 (catechin) red oil, Rf = 0.2 (DCM:MeOH:H2O = 4:1:0.1), [α]25D = +14o (MeOH, c 0.1), molecular formula: C15H14O6 1 H-NMR (CD3OD, 500MHz), δ (ppm), J (Hz): 6.86 (1H, d, J = 2 Hz, H-2’); 6.78 (1H, dd, J = 2, 6.5 Hz, H-6’); 6,74 (1H, dd, J = 2, 2 Hz, H-5’); 5
5.95 (1H, d, J = 2.5 Hz, H-8); 5.88 (1H, d, J = 2.5 Hz, H-6); 4.59 (1H, d, J = 8 Hz, H-2); 4.01 (1H, ddd, J = 5.5, 5, 5.5 Hz, H-3); 2.86 (1H, dd, J = 5.5, 5.5 Hz, H-4a); 2.53 (1H, dd, J = 8, 8 Hz, H-4b). 13 C-NMR (125MHz, CD3OD), δ (ppm): 157.8 (C-5); 157.5 (C-7); 156.9 (C-9); 146.2 (C-4’); 146.21 (C-3’); 132.2 (C-1); 120.0 (C-6’); 116.1 (C-2’); 115.2 (C-5’); 100.8 (C-10); 96.3 (C-6); 95.5 (C-8); 82.8 (C-2); 68.8 (C-3); 28.46 (C-4). ● Compound 87 (ponasterone A) white powder, Rf = 0.3 (DCM:MeOH = 4.5:0.5), tonc = 259-260oC, HR-ESI-MS m/z = 487,3029 [M+Na]+ (C27H44O6Na), m/z = 465.3209 [M+H]+ (C27H45O6), molecular formula C27H44O6 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, d, J = 2 Hz, H-7); 3.97 (1H, d, J = 2 Hz, H-3); 3.86 (1H, ddd, J = 4, 4.5, 3 Hz, H-2ax); 3.36 (1H, H-22); 3.17 (1H, m, H-9); 2.38 (1H, m, H-5); 2.14 (1H, m, H- 12ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.9 (1H, H-16α); 1.8 (1H, H- 12eq); 1.78 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H-4eq); 1.75 (1H, H- 24a); 1.72 (1H, H-20); 1.60 (1H, H-23a); 1.65 (1H, H-11ax); 1.65 (1H, H- 4ax); 1.53 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H-24b); 1.43 (1H, H- 1ax); 1.30 (1H, H-23b); 1.20 (3H, s, H-27); 0.98 (3H, s, H-26); 0.97 (3H, s, H-19); 0.94 (3H, H-21); 0.91 (3H, s, H-18). 13 C-NMR (125MHz, CD3OD), δ (ppm): 206.40 (C-6); 167.94 (C-8); 122.14 (C-7); 85.23 (C-14); 77.96 (C-20); 77.84 (C-22); 68.70 (C-2); 68.51 (C-3); 51.78 (C-5); 50.47 (C-17); 49.62 (C-13); 39.26 (C-10); 37.65 (C-23); 37.37 (C-1); 35.10 (C-9); 32.86 (C-4); 32.52 (C-12); 31.75 (C-15); 30.47 (C- 24); 29.21 (C-25); 24.40 (C-19); 23.40 (C-27); 22.75 (C-26); 21.50 (C-11); 21.50 (C-16); 20.99 (C-21); 18.32 (C-18). ● Compound 88 (20-hydroxyecdysone) colorless solid, Rf = 0.3 (DCM:MeOH = 4:1), [α]25D = 58o (MeOH, c 0.1), tonc = 244-246oC HR-ESI-MS m/z = 503.2972 [M+Na]+ (C27H44O7Na), m/z = 481.3151 [M+H]+ (C27H45O7), molecular formula C27H44O7. 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, J = 2 Hz, H- 7); 3.97 (1H, d, J = 1.5 Hz, H-3); 3.86 (1H, ddd, J = 3.5, 5, 3.5 Hz, H-2ax); 3.36 (1H, H-22); 3.17 (1H, t, J = 9Hz, H-9); 2.38 (1H, dd, J = 9.5; 5 Hz, H- 5); 2.16 (1H, m, H-12 ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.98 (1H, H- 16α); 1.8 (1H, H-12eq); 1.79 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H- 4eq); 1.75 (1H, H-24a); 1.72 (1H, H-20); 1.67 (1H, H-23a); 1.65 (1H, H- 11ax); 1.65 (1H, H-4ax); 1.59 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H- 24b); 1.43 (1H, H-1ax); 1.31 (1H, H-23b); 1.22 (3H, s, H-27); 1.22 (3H, s, H-26); 1.21 (3H, s, H-19); 0.98 (3H, H-21); 0.91 (3H, s, H-18). 6
13 C-NMR (125MHz, CD3OD), δ (ppm): 206.36 (C-6); 167.93 (C-8); 122.12 (C-7); 85.20 (C-14); 78.39 (C-20); 77.88 (C-22); 71.27 (C-25); 68.68 (C-2); 68.49 (C-3); 51.75 (C-5); 50.51 (C-17); 49.50 (C-13); 42.36 (C-24); 39.25 (C-10); 37.36 (C-1); 35.07 (C-9); 32.82 (C-4); 32.50 (C-12); 31.77 (C-15); 29.68 (C-26); 28.99 (C-27); 27.32 (C-23); 24.40 (C-19); 21.49 (C-16); 21.06 (C-21); 18.04 (C-18). ● Compound 84 (lambertic acid) white powder, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α]25D = +58o (MeOH, c 0.1), tonc = 129-130oC, molecular formula C20H28O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.98 (1H, d, J = 8.3 Hz, H-12); 6.52 (1H, d, J = 8.5 Hz, H-11); 3.12-3.14 (1H, m, H-15); 2.94 (1H, dd, J = 14.1, 6.3 Hz, H-7α); 2.74-2.76 (1H, m, H-7β); 2.26-2.28 (1H, m, H-6α); 2.01(1H, dd, J = 13.8 Hz; H-1α); 1.86 (1H, d, J = 13.4 Hz; H- 3α); 1.71-1.73 (1H, m, H-2α); 1.67-1.69 (1H, m, H-1β); 1.62-1.64 (1H, m, H-2β); 1.42 (1H, d, J = 13.4 Hz; H-5); 1.38 (1H, d, J = 13.4 Hz; H-6β); 1.34 (3H, d, J = 7 Hz, H-16); 1.33 (3H, d, J = 7 Hz, H-17); 1.18 (3H, s, H- 20); 1.06 (3H, s, H-18); 1.00 (1H, dd, J = 13, 4.3 Hz; H-3β). 13 C-NMR (125MHz, CD3OD), δ (ppm): 181,64 (C-19); 153,38 (C- 12); 147,37 (C-9); 133,61 (C-8); 127,30 (C-14); 127,17 (C-13); 112,58 (C- 11); 54,41 (C-5); 44,87 (C-4); 40,91 (C-6); 39,45 (C-10); 38,83 (C-1); 32,49 (C-3); 29,29 (C-15); 27,73 (C-18); 23,69 (C-20); 23,16 (C-16); 23,12 (C-17); 22,63 (C-7); 21,20 (C-2). ● Compound 79 (cassipouryl hexadecanoat, new compound) colorless oil, Rf = 0.5 (n-hexane:EtOAc = 5:0.2), m/z: 555.5126 [M+Na]+ (C36H68NaO2), molecular formula C36H68O2 . 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.33 (1H, t, J = 7.0 Hz, H-14); 4.58 (2H, d, J = 7.0 Hz; H-15); 2.29 (2H, t, J = 7.5 Hz, H-2’); 2.00 (1H, m, H-12a); 1.69 (3H, br s, H-20); 1.60 (2H, m, H-11); 1.58-1.60 (2H, m, H-3’); 1,52 (1H, m, H-5); 1.38 (1H, m, H-6); 1.36 (2H, m, H-9); 1.30 (2H, m, H-8); 1.28 (2H, m, H-7); 1.25 (2H, m, H-3); 1.25 (2H, m, H-4); 1.25 (1H, m, H-10a); 1.25 (26H, m, H-4’- H-13’); 1.25 (2H, s, H-14’); 1.25 (2H, br s, H-15’); 1.14 (2H, m, H-2); 1.08 (1H, m, H-10b); 1.08 (1H, m, H- 12b); 0.87 (3H, t, J = 7 Hz; H-16’); 0.86 (3H, s, H-16); 0.85 (3H, s, H-17); 0.845 (3H, d, J = 7; H-18); 0.838 (3H, d, J = 7 Hz; H-19). 13 C-NMR (125MHz, CDCl3), δ (ppm): 173.93 (C-1’); 142.58 (C-13); 118.21 (C-14); 61.19 (C-15); 39.86 (C-12); 39.38 (C-2); 37.44 (C-10); 37.37 (C-4); 37.30 (C-8); 36.64 (C-1); 34.42 (C-2’); 32.80 (C-6); 32.68 (C-9); 31.93 (C-14’); 29.70-29.16 (C4’-C13’); 27.98 (C-5); 25.04 (C-11); 24.80 (C-7); 24.80 (C-3’); 24.47 (C-3); 22.69 (C-16); 22.62 (C-17); 22.71 (C-15’); 19.74 (C-18); 19.71 (C-19); 16.36 (C-20); 14.10 (C-16’). 7
● Compound 85, (+)-spathulenol: colorless oil, Rf = 0.25 (n- hexane:EtOAc = 5:0.5), [α]25D = 5° (CHCl3, c 0.1) ESI-MS m/z: 221.3 [M+H]+, molecular formula C15H24O 1 H-NMR (500 MHz, CDCl3), δ (ppm), J (Hz): 4.69 (1H, s, H-14a); 4.66 (1H, s, H-14b); 2.42 (1H, dd, J = 13.0, 6.0 Hz, H-4a); 2.20 (1H, m, H- 6); 2.06 (1H, d, J = 13.0 Hz, H-4b); 1.77 (1H, m, H-8a); 1.55 (1H, m, H-8b); 1.32 (1H, d, J = 10.5 Hz, H-10); 1.28 (3H, s, H-15); 1.06 (3H, s, H-12); 1.03 (3H, s, H-13); 0.71 (1H, ddd, J = 11.0, 9.5, 6.0 Hz, H-2); 0.47 (1H, dd, J = 11.0, 9.5 Hz, H-1). 13 C-NMR (125MHz, CDCl3), δ (ppm): 153.46 (C-5); 106.26 (C-14); 80.99 (C-9); 54.35 (C-10); 53.41 (C-6); 41.75 (C-8); 38.87 (C-4); 29.92 (C-1); 28.66 (C-12); 27.50 (C-2); 26.72 (C-7); 26.08 (C-15); 24.79 (C-3); 20.27 (C-11); 16.34 (C-13). ● Compound cassipourol: colorless oil, Rf = 0.5 (n-hexane:DCM = 3:1.5), [α]25D = + 10.9° (CHCl3, c 0.1), molecular formula C20H38O 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.41 (1H, qt, J = 1.5, 7.0 Hz, H-14); 4.15 (2H, d, J = 7.0 Hz; H-15); 1.99 (1H, m, H-12a); 1.67 (3H, br s, H-20); 1.52 (1H, m, H-5); 1.51 (2H, m, H-11); 1.40 (1H, m, H-9); 1.38 (1H, m, H-10a), 1.38 (1H, m, H-6); 1.34 (2H, m, H-8); 1.29 (1H, m, H-10b); 1.28 (1H, m, H-2α); 1.28 (2H, m, H-7); 1.26 (2H, m, H-3); 1.25 (2H, m, H-4); 1.09 (1H, m, H-12b); 1.04 (1H, m, H-2β); 0.86 (3H, s, H-16); 0.853 (3H, d, J = 7, H-18); 0.85 (3H, s, H-17); 0.846 (3H, d, J = 7 Hz, H-19). 13 C-NMR (125MHz, CDCl3), δ (ppm): 140.30 (C-13); 123.11(C-14); 59.43 (C-15); 39.87 (C-12); 39.38 (C-2); 37.44 (C-10); 37.37 (C-8); 37.37 (C- 4); 36.67 (C-1); 32.70 (C-9); 32.70 (C-6); 27.98 (C-5); 25.14 (C-11); 24.79 (C-3); 24.47 (C-7); 22.71 (C-16); 22.62 (C-17); 19.75 (C-18); 19.71 (C-19); 16.17 (C-20). ● DIH03 the mixture of trans-communic acid (compound 81) and (compound 82) cis–communic acid, Yellowish oil, molecular formula C20H30O2 1 H-NMR compound 81 (500MHz, CDCl3), δ (ppm), J (Hz): 6.32 (1H, dd, J = 17.0, 10.5 Hz, H-14); 5.41 (1H, t, J = 6.0 Hz, H-12); 5.04 (1H, d, J = 17.0 Hz, H-15a); 4.88 (1H, d, J = 10.5 Hz, H-15b); 4.84 (1H, s, H-17a); 4.47 (1H, s, H-17b); 1.75 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65 (3H, s, H-20). 13 C-NMR compound 81 (125MHz, CDCl3), δ (ppm): 183.90 (C-19); 147.93 (C-8); 141.61 (C-12); 133.90 (C-14); 133.45 (C-13); 109.91 (C- 15); 107.66 (C-17); 56.43 (C-5); 56.29 (C-9); 44.21 (C-4); 40.37 (C-10); 39.27 (C-6); 38.49 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 23.31 (C-11); 19.93 (C-2); 12.83 (C-20); 11.84 (C-16). 8
1 H-NMR compound 82 (500MHz, CDCl3), δ (ppm), J (Hz): 6.78 (1H, dd, J = 17.5, 11.0 Hz, H-14); 5.31 (1H, t, J = 6.0 Hz, H-12); 5.18 (1H, d, J = 17.5 Hz, H-15a); 5.08 (1H, d, J = 11.0 Hz, H-15b); 4.84 (1H, s, H-17a); 4.49 (3H, s, H-17b); 1.77 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65 (3H, s, H-20). 13 C-NMR compound 82 (125MHz, CDCl3), δ (ppm): 183.90 (C-19); 147.93 (C-8); 133.86 (C-14); 131.67 (C-12); 131.56 (C-13); 113.25 (C- 15); 107.80 (C-17); 56.70 (C-9); 56.43 (C-5); 44.21 (C-4); 40.43 (C-10); 39.27 (C-6); 38.52 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 22.28 (C-11); 19.93 (C-2); 19.72 (C-16); 12.83 (C-20). ● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n- hexane:DCM = 1:3). ● Compound 83 (pimaric acid): colorless solid, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α]20D = + 87.3o (CHCl3, c 0.4), tonc = 217-219oC ESI-MS m/z: 303.6 [M+H]+, molecular formula C20H30O2 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.77 (1H, dd, J = 17.0, 10.5 Hz, H-15); 5.22 (1H, s, H-14); 4.90 (1H, dd, J = 17.0, 1.5 Hz, H- 16a); 4.88 (1H, dd, J = 10.5, 1.5 Hz, H-16b); 1.21 (3H, s, H-19); 1.04 (3H, s, H-17); 0.84 (3H, s, H-20). 13 C-NMR (125MHz, CDCl3), δ (ppm): 184.92 (C-18); 148.91 (C- 15); 136.63 (C-8); 129.13 (C-14); 110.16 (C-16); 50.58 (C-9); 48.84 (C-5); 47.28 (C-40); 38.30 (C-1); 37.74 (C-10); 37.40 (C-13); 37.05 (C-3); 35.48 (C-7); 34.46 (C-12); 26.04 (C-17); 24.91 (C-6); 18.57 (C-11); 18.16 (C-2); 16.78 (C-19); 15.22 (C-20). ● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid): colorless oil, Rf = 0.2 (n-hexane:EtOAc = 2:1), molecular formula C20H32O4 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.93 (1H, dd, J = 11, 11 Hz, H-14); 5.34 (1H, d, J = 17 Hz, H-15); 5.21 (1H, dd, J = 10.5, 10.5 Hz, H-15); 4.85 (1H, s, H-17a); 4.44 (1H, s, H-17b); 3.48 (1H, d, J = 10.5 Hz, H-12); 2.4 (1H, m, H-7); 2.18 (1H, d, J = 13 Hz, H-3); 2.03 (1H, m, H- 9); 1.97 (1H, m, H-7); 1.85 (1H, m, H-2α); 1.81-2.0 (2H, m, H-6); 1.79 (1H, m, H-1); 1.6 (1H, m, H-11); 1.53 (1H, m, H-2β); 1.4 (1H, m, H-5); 1.4 (1H, m, H-11); 1.33 (3H, s, H-16); 1.24 (3H, s, H-18); 1.15 (1H, m, H- 1); 1.07 (1H, m, H-3); 0.59 (3H, s, H-20). 13 C-NMR (125MHz, CDCl3), δ (ppm): 183.04 (C-19); 148.36 (C- 19); 140.88 (C-8); 114.57 (C-15); 106.71 (C-17); 75.98 (C-13); 75.84 (C- 12); 56.24 (C-5); 51.65 (C-9); 44.21 (C-4); 40.13 (C-10); 39.03 (C-1); 38.70 (C-7); 37.97 (C-3); 29.02 (C-16); 26.06 (C-6); 24.54 (C-18); 19.87 (C-2); 12.95 (C-20). 9
2.3.2. Fokienia hodginsii Figure 2.4. Schematic diagram showing steps for the isolation of pure compounds from the twig and leaves of Fokienia hodginsii ● Compound 81 (trans-communic acid) colorless solid, Rf = 0.5 (n- hexane: EtOAc = 4:1), [α]25D = +38o (MeOH, c 0.1), tonc = 130-132oC, molecular formula C20H30O2 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.34 (1H, dd, J = 17.5, 17 Hz, H-14); 5.40 (1H, t, J = 6.5 Hz, H-12); 5.04 (1H, d, J = 17.5 Hz, H- 15a); 4.88 (1H, d, J = 9.5 Hz; H-15b); 4.85 (1H, br s, H-17a); 4.47 (1H, br s, H-17b); 1.75 (3H, s, H-16); 1.22 (3H, s, H-18); 0.70 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 181.20 (C-19); 149.55 (C-8); 142.85 (C-14); 134.89 (C-12); 134.55 (C-13); 110.15 (C-15); 107.95 (C-17); 10
57.90 (C-9); 57.43 (C-5); 45.14 (C-4); 41.39 (C-10); 40.62 (C-1); 39.65 (C-3); 39.34 (C-7); 29.62 (C-18); 27.31 (C-6); 24.32 (C-11); 21.20 (C-2); 13.43 (C- 20); 12.02 (C-16). ● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n- hexane:DCM = 1:3). ● Compound 92 (totarolone): colorless solid, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α]25D = +101.5o (MeOH, c 0.1), tonc = 188-189oC, molecular formula C20H28O2. 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.95 (1H, d, J = 8.5 Hz, H-11); 6.57 (1H, d, J = 8.5 Hz, H-12); 3.26 (1H, s, H-15); 1.34 (3H, d, J = 3.5 Hz, H-16); 1.35 (3H, d, J = 3.5 Hz, H-17); 1.28 (3H, s, H-18); 1.16 (3H, s, H-19); 1.14 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 220.56 (C-3); 155.12 (C- 13); 140.11 (C-9); 134.93 (C-8); 132.05 (C-14); 124.81 (C-11); 115.58 (C- 12); 51.52 (C-5); 48.32 (C-4); 39.62 (C-1); 38.39 (C-10); 35.79 (C-2); 30.14 (C-7); 28.85 (C-15); 27.30 (C-18); 25.07 (C-20); 21,68 (C-6); 21,47 (C-19); 20,58 (C-16); 20,51 (C-17). ● Compound 90 (15-nor-labda-8(17),12E-diene-14-carboxaldehyde- 19-oic acid): colorless oil, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular formula C19H28O3 1 H-NMR (500MHz, CD3OD): δ (ppm), J (Hz): 9.31 (1H, d, J = 3 Hz; H- 15); 6.56 (1H, ddd, J = 3, 3, 6 Hz; H-12); 4.86 (1H, s, H-17a); 4.44 (1H, s, H- 17b); 1.76 (3H, d, J = 1.1 Hz, H-16); 1.24 (3H, s, H-18); 0.71 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 197.07 (C-14); 181.13 (C-19); 158.69 (C-12); 149.57 (C-8); 140.01 (C-13); 108.03 (C-17); 57.25 (C-5); 57.15 (C-9); 45.15 (C-4); 41.38 (C-10); 40.55 (C-1); 39.42 (C-3); 39.26 (C-7); 29.54 (C-18); 27.24 (C-6); 25.73 (C-11); 21.14 (C-2); 13.37 (C-20); 9.29 (C-16). ● Compound 91 (13-oxo-15,16-dinor-labda-8(17),11E-diene-19-oic acid): colorless oil, Rf = 0.57 (n-hexane:EtOAc = 4:1), [α]25D = +20.5o (MeOH, c 0.1), molecular formula C18H26O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 7.01 (1H, dd, J = 10.5, 10 Hz, H-11); 6.10 (1H, d, J = 16 Hz, H-12); 4.85 (1H, H-17a); 4.45 (1H, s, H-17b); 2.6 (1H, dd, J = 10.5 Hz, H-9); 2.47 (1H, m, H-3α); 2.29 (3H, s, H- 16); 2.18 (1H, m, H-7α); 2.04 (1H, m, H-3β); 1.98 (2H, m, H-6); 1.89 (1H, m, H-2α); 1.48 (1H, m, H-1α); 1.45 (1H, m, H-2β); 1.33 (1H, m, H-5); 1.24 (3H, s, H-18); 1.09 (1H, m, H-1β); 1.07 (1H, m, H-7β); 0.86 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 200.88 (C-13); 180.12 (C- 19); 149.89 (C-8); 148.67 (C-11); 134.58 (C-12); 108.87 (C-17); 61.35 (C- 9); 56.38 (C-5); 45.08 (C-4); 42.13 (C-1); 40.95 (C-10); 39.31 (C-3); 38.24 11
(C-7); 29.42 (C-18); 27.11 (C-14); 26.33 (C-6); 20.86 (C-2); 14.14 (C-20). ● Compound 93 (3β-hydroxytotarol): colorless oil, Rf = 0.55 (n- hexane:EtOAc = 4:1), [α]25D = +29o (MeOH, c 0.1), molecular formula C20H30O2 1 H-NMR (500MHz, CD3OD),δ (ppm), J (Hz): 6.92 (1H, d, J = 8.5 Hz, H-11); 6.53 (1H, d, J = 8.5 Hz, H-12); 4.1 (1H, d, J = 7 Hz, H-3); 3,24 (1H, dd, J = 5, 5 Hz, H-15); 2.97 (1H, dd, J = 6; 6 Hz, H-7α); 2.71 (1H, dd, J = 4, 7.5 Hz, H-7β); 2.30 (1H, m, H-1α); 2.03 (1H, s, H-6α); 19.6 (2H, m, H-2); 1.82 (1H, m, H-6β); 1.42 (1H, m, H-1β); 1.35 (3H, H-17); 1.32 (3H, H-16); 1.29 (1H, m, H-5); 1.17 (3H, s, H-18); 1.07 (3H, s, H-19); 0.88 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 155.32 (C-13); 142.29 (C-9); 134.89 (C-8); 132.07 (C-14); 123.78 (C-11); 115.26 (C-12); 79.58 (C-3); 50.93 (C-5); 39.87 (C-4); 39.27 (C-1); 38.56 (C-10); 30.29 (C-7); 28.96 (C-2); 28.76 (C-19); 25.61 (C-20); 20.56 (C-16&C-17); 20.43 (C-6); 16.08 (C-18). ● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid) colorless solid, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular formula C20H32O4 ● Compound 94 [(9S)-drummondol]: colorless oil, Rf = 0.5 (n- hexane:EtOAc = 4:1), molecular formula C13H20O4 1 H-NMR (500MHz, CD3OD) δ (ppm), J (Hz): 6.19 (1H, dd, J = 16.5, 5.5 Hz, H-8); 6.05 (1H, d, J = 16.5 Hz, H-7); 4.40 (1H, dq, J = 5.2, 6.5 Hz, H-9); 3.93 (1H, dd, J = 8, 2.5 Hz, H-11α); 3.67 (1H, d, J = 8 Hz, H-11β); 2.68 (1H, dd, J = 18, 2.5 Hz; H-2α); 2.37 (1H, dd, J = 18, 2.5 Hz, H-2β); 2.80 (1H, d, J = 17.5 Hz, H-4α); 2.45 (1H, dd, J = 17.5, 2.5 Hz; H-4β); 1.30 (3H, d, J = 6.5 Hz, H-10); 1.20 (3H, s, H-13); 0.98 (3H, s, H-12). 13 C-NMR (125MHz, CD3OD), δ (ppm): 211.23 (C-3); 140.68 (C-8); 125.66 (C-7); 87.45 (C-5); 82.34 (C-6); 78.39 (C-11); 68.87 (C-9); 53.94 (C-4); 53.23 (C-2); 48.49 (C-1); 23.97 (C-10); 19.17 (C-13); 15.65 (C-12). ● Compound 95 (vomifoliol): colorless oil, Rf = 0.5 (n- hexane:EtOAc = 4:1), molecular formula C13H20O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.90 (1H, d, J = 1 Hz, H-4); 5.83 (1H, d, J = 15.5 Hz, H-8); 5.79 (1H, d, J = 15.5 Hz, H-7); 4.34 (1H, dq, J = 6.5, 4 Hz, H-9); 2.54 (1H, d, J = 17 Hz, H-2α); 2.18 (1H, d, J = 17 Hz, H-2β); 1.95 (3H, d, J = 1 Hz, H-13); 1.26 (3H, d, J = 7 Hz, H-10); 1.06 (3H, s, H-12); 1.03 (3H, s, H-11). 13 C-NMR (125MHz, CD3OD), δ (ppm): 201.34 (C-3); 167.53 (C-5); 136.91 (C-8); 130.08 (C-7); 127.10 (C-4); 79.97 (C-6); 68.72 (C-9); 50.72 (C-2); 42.43 (C-1); 24.48 (C-11); 23.81 (C-10); 23.47 (C-12); 19.59 (C-13). 12
Chapter 3: RESULTS AND DISCUSSION 3.1. Determination the chemical structure of isolated compounds from Dacrycarpus imbricatus 13 Compounds have been isolated and determined the structure from Dacrycarpus imbricatus, including: 3 steroid 86, 87 and 88; 1 flavonoid 89; 1 sugar saccaroza (GB2), 1 secquiterpene 85; 7 diterpene, 3 compound diterpene labdane 80; 81 and 82, 2 compound diterpene abietane 83, 84 and 2 diterpene monocycle 78 and 79. ● Compound 78 and 79: Cassipourol and cassipouryl hexadecanoate (new compound) Compounds 78 and 79 were obtained as colorless semi-liquid compounds. The molecular formula of 78 was established as C20H38O from its HR-ESI-MS in combination with 13C NMR, and DEPT spectroscopic data. The 13C NMR and DEPT spectra indicated the presence of 20 13
carbons in this compound including 5xCH3, 9xCH2, 4xCH, 2xCq. These spectra also revealed the existence of the only one double bond (C 140.3, 123.1) in the structure suggesting compound 78 must be a monocyclic diterpene. The lH NMR showed signals of an olefinic proton as a quartet triplets at  5.41 (J = 1.5, 7.0 Hz); two oxymethylene protons as a doublet at H 4.15 (2H, J = 7.0 Hz). Remaining proton signals in the lH NMR spectrum were extremely overlapped, thus the chemical shift assignments were determined by HSQC experiment. Namely, three methyl singlets at H 1.67, 0.86, 0.85 correlated with the carbon signals at C 16.2, 22.6, 22.7, respectively in the HSQC spectra. In addtion, it could be assigned for two methyl doublets at H 0.846; 0.853/C19.7, 19.8; eight methylenes and three methines between  1.04 and  1.99 (Table 1). A comparison of the 1H and 13C NMR data of 78 with those of cassipourol indicated that they were identical [45] A comparison of the 1H and 13C NMR data of 79 with those of 78 indicated that they were almost identical except for the presence of signals for an aliphatic long-chain acid derivative. This group was determined through the apperance of one carbonyl ester (C 173.9), fourteen methylenes (between C 29.2 and 29.7 ppm) and one methyl group (C 14.1) beside the 20 carbons of cassipourol in the 13C NMR spectrum of 79 (Table 3.1). Similarly, it could be seen not only the resonances of 78 but also the resonances of methyl triplet at H 0.87 (J = 7 Hz), strong broad singlet at H ca 1.25 and a triplet at H 2.29 (J = 7.5 Hz) in the 1H NMR of 79. The singlet at  1.25 correlated to the signal of methylene carbon (at C ca. 29 ppm), while triplet at H 2.29 (J = 7.5 Hz) connected to a shifted downfield methylene at C 34.4 in the HSQC spectrum. The HR-ESI-MS exhibited molecular ion peak at m/z 555.5126 [M+Na]+ (calcd for C36H68NaO2, 555.5117) establishing a molecular formula of C36H68O2 to compound 79 and the fragment at m/z 318.2965 (C20H39NaO required 318.2899) indicating the presence of a cassipouryl moiety. These data allowed to identify aliphatic chain as a hexadecanoic acid derivative. The esterification of this acid with cassipourol on C-15 was suggested by the chemical shift of C-15 at C 61.19 (shifted downfield,  +1.76 ppm due to este RCOO group). Furthermore, it was also confirmed by the key HMBC correlations H-14/C-12, C-15; H-15/C-14, C-13, C-1’ (C=O); H- 2’/C-1’, C-3’, C-4’ and H-3’/C-2’, C-4’. 14
4 18 3 5 H 14 8 10 2 6 9 13 O 1' O 1 7 11 16 17 19 15 2' 3' 4' (CH2)11CH3 HMBC (H/C ) and NOESY ( ) Figure 3.4. Selected HMBC and NOESY correlations of compounds 79 The configurations of the chiral centers at C-5, C-6 and three methyl groups at C-16, C-17, and C-18 of 79 were similar to those of 78 due to the same phyto origin. This finding was deduced from their almost identical 13C NMR values and coupling constants. In addition, it was also supported by the correlations of Me-16 with H-6 and the Me-17, Me-18 with methylene group H2-7 in the NOESY spectrum. The trans (E stereochemistry) of the double bond C-13 and C-14 was determined by the correlation of Me-20 with oxymethylene protons H2-15 (no correlation between Me-20 with H-14). Consequently, the structure of 79 was elucidated as cassipouryl hexadecanoate, a new cassipourol dervative. Table 3.1.: 13C- and 1H NMR data for compounds 78 and 79 (125/500 MHz, CDCl3, ppm)a C 78 79 C H C H HMBC (HC) 1 36.7 - 36.6 - - 2 39.4 1.28 m, 1.04 m 39.4 1.14 m, 2H C-3; C-4; C-16; C-17 3 24.8 1,26 m 24.5 1.25 m C-2 4 37.4 1,25 m 37.4d* 1.25 m C-5; C-18 5 28.0 1.52 m 28.0 1.52 m C-3; 6 32.7 1.38 m 32.8 1.38 m C-4; C-7; C-18 7 24.5 1.28 m 24.8 1.28 m C-6; C-8, 8 37.4 1.34 m 37.3d* 1.30* m C-7; C-19 9 32.7 1.40 m 32.7 1.36 m C-8 10 37.4 1.38 m, 1.29 m 37.4 1.25 m, 1.08m C-8; C-9; C-19 11 25.1 1.51 m 25.0 1.60 m C-10; C-12 12 39.9 1.99 m, 1.09 m 39.9 2.00 m, 1.08 m C-20; C-11; C-10; C-14; C-13 13 140.3 - 142.6 - - 14 123.1 5.41 qt (1.5. 118.2 5.33 t (7.0) C-20; C-12; C-15 (w) 7.0) 15 59.4 4.15 d (7.0) 61.2 4.58 d (7.0) C-14; C-13; C-1’ (C=O); 16 22.7b 0.86 s 22.7e 0.86 s* C-1; C-2; C-17 17 22.6b 0.85 s 22.6e 0.85 s* C-1; C-2; C-16; 18 19.8c 0.853b d (7) 19.7f 0.845 d (7) C-5 15
19 19.7c 0.846 d (7) 19.7f 0.838 d (7) C-8; C-9 20 16.2 1.67 br s 16.4 1.69 br s C-12; C-14 C-1’ - - 173.9 - - 2’ - - 34.4 2.29 t (7.5) C-1’, C-3’, C-4’ 3’ - - 24.8 1.58-1.60 m C-2’, C-4’ 4’-13’ - - 29.7 - 1.25 m, 26 H * 29.2 14’ - - 31.9 1.25 s * 15’ - - 22.7 1.25* br s * 16’ - - 14.1 0.87 t (7) * a Assignments made on the basis of HSQC and HMBC spectroscopic data and in comparison with the literature values [45] b-h values having the same superscript in the respective columns may be interchanged. * Overlap signals (of C1’-C16’= hexadecanoate). Figure 3.5. HR-ESI-MS spectrum of compound 79 16
Figure 3.6. 1H-NMR spectrum of compound 79 (CDCl3, 500MHz) Figure 3.7. 13C-NMR spectrum of compound 79 (CDCl3, 125 MHz) 17
Figure 3.8. HSQC spectrum of compound 79 Figure 3.8. HSQC spectrum of compound 79 18