Triterpenoids from Erythrophleum fordii. LI Nan, YU Fang, YU Shi-Shan*. (
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union ...
Acta Botanica Sinica 植 物 学 报
2004, 46 (3): 371－374
http://www.chineseplantscience.com
Triterpenoids from Erythrophleum fordii LI Nan, YU Fang, YU Shi-Shan* (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China)
Abstract: A phytochemical investigation of the bark of Erythrophleum fordii Oliv. furnished five compounds. One is a new triterpenoid, namely 20S, 24S-epoxy-23S, 25-dihydroxy-dammarane-3-one (1), and four are known, identified to be 20S , 25-epoxy-24R -hydroxydammarane-3-one (2), 20S, 24S -epoxydammarane-3β, 25-diol (3), betulinic acid (4), morolic acid acetate (5). All the known compounds were isolated from the species for the first time. The structure of compound 1 was elucidated on the basis of spectroscopic analyses. Key words: Erythrophleum fordii ; triterpenoid; cytotoxic activities; KB cells; A2780 cells Erythrophleum fordii Oliv. is used as Chinese traditional medicine with the main actions: invigoration and promoting blood circulation. To date, no previous chemical and pharmacological investigations have been reported on it. In the course of screening for cytotoxic compounds, the 95% ethanol extract of the dry barks of E. fordii was partitioned into EtOAc and H2O to afford an EtOAc-soluble portion, which showed cytotoxic activities against KB cells with the IC50 0.08 µg/mL, A2780 cells with the IC50 3.1 µg/mL. Phytochemical investigation on the EtOAc fraction led to the isolation and identification of five compounds, one is new, named 20S, 24S-epoxy-23S, 25-dihydroxydammarane-3-one (1), and four are known (2－5), identified to be 20S, 25-epoxy-24R-hydroxydammarane-3-one (2), 20S, 24S-epoxydammarane-3β,25-diol (3), betulinic acid (4), morolic acid acetate (5). We report herein the isolation and structure elucidation of the new compound. The structures of the known compounds (2－5) were identified by the comparison of their 1H-NMR and 13C-NMR data with those reported in literatures (Mochammad et al.,1980; Antonio et al., 1981; Peter and Stephen, 1985; Carl et al., 1993). Bioactive tests exhibited that all the compounds had no cytotoxic activity against KB cells and A2780 cells.
1 Results and Discussion Compound 1 was obtained as colorless crystal, mp 202－ 。 203 ℃, ［α］１８ Ｄ +45 (c 1.00, CHCl3). Libermann-Burchard reaction was positive. The HR-FABMS gave a molecular ion at m/z 475.382 4 ([M+H]+) corresponding to the molecular formula C30H50O4. The IR spectrum of compound 1
indicated the presence of hydroxyl groups (3 408, 1 072 cm－1 ) and a carbonyl group (1 707 cm－1). The 1H-NMR spectrum (in CDCl3) of compound 1 showed eight methyl group signals at δ 1.38 (3H, s), 1.36 (3H, s),1.28 (3H, s), 1.08 (3H, s), 1.04 (3H, s), 1.00 (3H, s), 0.94 (3H, s), and 0.89 (3H, s). The 13C-NMR spectrum of compound 1 and DEPT experiments confirmed that the molecular contained 30 carbons, including eight methyl groups, nine methylenes, six methines (two oxygenated methines), and seven quaternary carbons. Compound 1 was assigned as a triterpenoid which showed a close structural similarity to cabraleone (20S, 24S-epoxy-25-hydroxy-dammarane-3-one) ( Peter and Stephen, 1985), except for additional signal of an oxo-carbon (δ 74.22) and differences in the chemical shifts around C-22 (Table 1). The EI-MS presented ion peaks at m/z 159, 141, 123, presumably derived from the side chain, which could thus include a hydroxylated tetrahydrofuryl isopropanol. The proton at δＨ 3.44 (H-24) resonate as doublets with coupling constant (J = 2.5 Hz), indicating the hydroxyl group at C-23 with a trans-proton. The structure of compound 1 was also supported by HMBC (Fig.1). The stereochemistry of C-20 should be S configuration, since the C-20 epimer (R configuration) of compounds with the tetrahydrofuran ring has been shown to be quite unstable and be converted easily to the corresponding tetrahydrofuran derivative. ( Masahiro et al., 1968 ) . Information on the additional stereochemistry of compound 1 was obtained by the use of selective NOE irradiation. In this experiment, irradiation at δＨ 3.44 (H-24) not enhance the signal at δＨ 1.38 (CH3-21), while irradiation
Received 27 Mar. 2003 Accepted 19 Sept. 2003 Supported by the National Natural Science Foundation of China (20272084) and the National Key Basic Research and Development Plan Program (G1998-051120). * Author for correspondence. Tel: +86 (0)10 63165324; Fax: +86 (0)10 63017757; E-mail: .
３７２
Fig.1. The selected HMBC correlations for compound 1, indicated by arrows from 1H to 13C.
at δＨ 4.43 (H-23) gave NOE on CH3-26 ( δＨ 1.36) and CH3-27 ( δＨ 1.36). On the basis of above data, the structure of compound 1 was established as 20S, 24S-epoxy-23S, 25dihydroxy-dammarane-3-one.
2 Experimental 2.1 General experimental procedures Melting points were determined on an XT-4 micromelting point apparatus and uncorrected. Optical rotations were obtained using a Perkin-Elmer 241 polarimeter. IR spectra were recorded using KBr disks on a Perkin-Elmer 683 FT infrared spectrometer. The NMR spectra were taken with TMS as internal standard on an Inova 500 FT-NMR spectrometer. Mass spectra were measured on VG ZAB-2F spectrometer. Silica GF254 for thin-layer chromatography analysis and silica gel (160－200 mesh) for column chromatography were produced by Qingdao Marine Chemical Company, Qingdao, China. Solvents and chemicals were analytical grade and purchased from Beijing Chemical Company, Beijing, China. 2.2 Plant materials Barks of Erythrophleum fordii Oliv. were collected from Guangxi Province of China, and identified by Prof. Liu ShouYang (Guangxi College of Chinese Traditional Medicine, Guangxi Province, China) in August 2000. Barks were harvested and air-dried at room temperature in darkness. A voucher specimen was deposited in Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. 2.3 Extraction and isolation The dried and chopped barks of E. fordii (15 kg) were extracted three times with 95% hot EtOH .The combined EtOH extract was evaporated under reduced pressure to yield a dark brown syrup ( 2 910 g ), which was partitioned
Ａｃｔａ Ｂｏｔａｎｉｃａ Ｓｉｎｉｃａ 植物学报 Ｖｏｌ．４６ Ｎｏ．３ ２００４
between EtOAc and H2O. The EtOAc-soluble layer (400 g) was directly chromatographed over silica gel by eluting with a gradient of CH2Cl2-MeOH (20:1 to 1:1) to afford twelve fractions. The fraction 3 (47.7 g) was chromatographed over silica gel with gradient of Petro ether-EtoAc (9:1 to 1:1) to yield compounds 1 (12 mg), 2 (10 mg), 3 (22 mg), 4 (47 mg), 5 (381 mg). 2.4 Identification 20S, 24S-epoxy-23S, 25-dihydroxy-dammarane-3-one (1) Colorless crystal; mp 202－203 ℃; [α]18D +45°(c 1.00, CHCl3); Libermann-Burchard reaction positive; IR (KBr) vmax 3 408, 1 707, 1 072 cm－1; EI-MS m/z 438 [M－2H2O]+ (2), 205 (23), 159 (18), 141 (50), 123 (100), 107 (44); HR-FABMS m/z [M+H]+ 475.382 4 (calcd. for C30H51O4, 475.378 7). 1H-NMR (500 MHz, CDCl3) δ 4.43 (1H, br, H-23), 3.44 (1H, d, J=2.5 Hz, H-24), 2.43－2.50 (2H, m, H-2), 1.40－1.50 (1H, m, H-17), 1.38, 1.36, 1.28, 1.08, 1.04, 1.00, 0.94, 0.89 (each 3H, s, 21, 26, 27, 28, 29, 19, 18, 30-Me); 13C-NMR (125 MHz, CDCl3), see Table 1. 20S, 25-epoxy-24R-hydroxy-dammarane-3-one (2) Colorless crystal (EtOAc); mp 188－189 ℃; [α]18D +37°(c 0.80, CHCl3); Libermann-Burchard reaction positive; IR (KBr) vmax 3 485, 1 685, 1 082, 1 020 cm－1; EI-MS m/z 440 [M－H2O]+ (2), 205 (20), 143 (42), 125 (100), 107 (50); HRFABMS m/z [M+H]+ 459.386 2 (calcd. for C30H51O3, 459.383 8). 1 H-NMR (500 MHz, CDCl3) δ 3.38 (1H, dd, J = 9, 6.5 Hz), 2.39－2.53 (2H, m), 1.21, 1.20, 1.18, 1.08, 1.04, 1.02, 1.00, 0.94, 0.86 (each 3H, s); 13C-NMR (125 MHz, CDCl3), see Table 1. 20S, 24S-epoxy-dammarane-3β, 25-diol (3) Colorless crystal (CH3OH); mp 158－159 ℃; [α]18D +24°(c 0.85, CHCl3); Libermann-Burchard reaction positive; IR (KBr) vmax 3 438, 1 045 cm－1; EI-MS m/z 424 (2) [M－H2O]+, 143 (100), 125 (50); 1H-NMR (500 MHz, CDCl3) δ 3.64 (1H, dd, J=15, 5 Hz, H-24), 3.20 (1H, dd, J=11.5, 4.5 Hz, αH-3), 1.19, 1.15, 1.11, 0.98, 0.98, 0.88, 0.85, 0.78 (each 3H, s, 27, 26, 21, 18, 28, 30, 19, 29-Me); 13C-NMR (125 MHz, CDCl3), see Table 1. Betulinic acid (4) Colorless crystal (CH3OH); mp 252－ 253 ℃; [α]18D －36°(c 1.00, CHCl3); Libermann-Burchard reaction positive; IR (KBr) vmax 3 454, 3 076, 1 689, 1 643, 1 043, 1 032, 984 cm－1; EI-MS m/z 456 (12) [M]+, 207 (50), 189 (100). 1H-NMR (500 MHz, C5D5N) δ 4.76 (2H, s, H-30), 3.44 (1H, dd, J = 9, 7.5 Hz, αH-3), 1.78 (3H, s, Me-29), 1.21, 1.05, 1.04, 0.99, 0.81 (each 3H, s); 13C-NMR (125 MHz, C5D5N), see Table 1. Morolic acid acetate (5) Colorless crystal (CH3OH), mp 263－265 ℃; [α]18D +20°(c 0.99, CHCl3); LibermannBurchard reaction positive; IR (KBr) vmax 3 446,1 734, 1 695 cm－1; EI-MS m/z 453 (2) [M－COOH]+, 203 (40), 189(100);
３７３
LI Nan et al.: Triterpenoids from Erythrophleum fordii Table 1
13
C-NMR (in CDCl3) data for compounds 1－5 and cabraleone
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 39.92 34.08 218.06 47.40 55.30 19.63 34.57 40.29 50.16 36.84 22.32 25.85 42.98 49.99 31.50 27.08 50.66 15.17 16.09 86.73 27.95 44.03 74.22 85.87 72.09 24.06 29.27
28 29 30 Spectra determined in 2 (CH2), or 3 (CH3).
(2) (2) (0) (0) (1) (2) (2) (0) (1) (0) (2) (2) (1) (0) (2) (2) (1) (3) (3) (0) (3) (2) (1) (1) (0) (3) (3)
26.73 (3) 20.97 (3) 16.31 (3) CDCl3; data reported
Cabraleone 39.96 (2) 34.12 (2) 217.96 (0) 47.42 (0) 55.41 (1) 19.71 (2) 34.66 (2) 40.35 (0) 50.23 (1) 36.91 (0) 23.36 (2) 25.84 (2) 43.35 (1) 50.04 (0) 31.46 (2) 27.80 (2) 49.84 (1) 15.23 (3) 16.08 (3) 86.40 (0) 24.11 (3) 34.91 (2) 27.05 (2) 86.50 (1) 70.27 (0) 26.40 (3) 26.78 (3) 27.15 (3) 21.02 (3) 16.34 (3) in ppm. Carbon types
2 39.90 34.13 18.24 47.41 55.34 19.67 34.55 40.30 50.16 36.84 22.18 25.41 42.02 50.27 30.18 27.66 51.55 15.13 16.06 74.56 21.85 31.85 25.11 75.58 76.10 26.72 25.50
3 39.06 27.42 78.95 38.97 55.86 18.28 35.29 40.38 50.83 37.15 21.80 25.84 42.85 50.03 31.44 27.42 49.84 15.48 16.23 86.54 24.05 34.77 26.36 86.30 70.25 27.14 27.81
4 39.3 28.3 78.2 39.6 56.0 18.8 34.9 41.2 51.0 37.6 21.2 26.2 38.7 42.9 31.3 32.9 56.7 47.8 49.8 151.4 30.3 37.6 28.7 16.4 16.5 16.5 14.9
5 38.6 23.7 81.0 37.8 55.6 18.1 34.5 40.7 51.1 37.1 20.9 26.0 41.4 42.6 29.4 33.5 47.9 136.7 133.3 32.1 33.4 33.3 27.9 16.5 16.0 16.7 14.9
29.95 27.99 178.9 181.1 20.98 15.35 19.5 30.3 16.50 16.40 110.0 29.1 determined by a DEPT experiment and reported as 0 (quaternary), 1 (CH),
H-NMR (500 MHz, CDCl3) δ 5.18 (1H, s, H-19), 4.48 (1H, dd, J=11, 6 Hz, H-3), 2.05 (3H, s, -OCOCH3), 1.00, 0.99, 0.98, 0.89, 0.85, 0.84, 0.79, 0.77 (each 3H, s); 13C-NMR (125 MHz, CDCl3), see Table 1.
1
department of pharmacology in our institute, for cytotoxicity testing. References: Antonio G G, Braulio M F, Pedro G, Melchor G H, Angel G R.
3
Bioassays
Cytotoxicity against human cancer cell lines for EtOAcsoluble portion was measured in a five-day MTT test at Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, using A2780 human epithelial carcinoma cells and KB human epidermoid cancer cells (Dominic et al., 1988). Cells were placed in a 96-well plate. The cells were dissolved in 0.2 mL of DMSO, and the absorbance at 540 nm was measured in a microplate reader. Acknowledgements: We are grateful to the department of medicinal analysis of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for performing EI-MS, NMR and IR, and thank the
1981.
13
C NMR spectra of olean-18-ene derivatives.
Phytochemistry, 20:1919－1921. Carl A E, Anthony C W, Mabry B, Rosalind Y W. 1993. Ergostanoids from Petunia inflata. Phytochemistry, 33:471－ 477. Dominic A S, Robert H S, Keneth D P. 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res, 48:4827－4833. Masahiro N, Nobutoshi T, Sachiko I, Osamu T. 1968. Triterpenes of Betula platyphylla. Tetrahedron Lett, 40:4239－4240. Mochammad S, Kazuo Y, Ryoh K, Osamv T. 1980. 13C nuclear magnetic resonance of lupane-type triterpenes, lupeol, betulin and betulinic acid. Chem Pharm Bull, 28:1006－1008.
３７４ Peter G W, Stephen A. 1985. Dammarane triterpenes from the stem bark of Commiphora dulzielii. Phytochemistry, 24:2925－
Ａｃｔａ Ｂｏｔａｎｉｃａ Ｓｉｎｉｃａ 植物学报 Ｖｏｌ．４６ Ｎｏ．３ ２００４ 2928.
(Managing editor: WANG Wei)
