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Enmein type diterpenoids from Isodon japonica
Phytochemistry 58 (2001) 543–546 www.elsevier.com/locate/phytochem
Enmein type diterpenoids from Isodon japonica Baolin Lia,*, Xianhua Tianb a
Department of Chemistry, Shaanxi Normal University, Xi’an 710062, People’s Republic of China b Life Science College, Shaanxi Normal University, Xi’an 710062, People’s Republic of China Received 8 December 2000; received in revised form 19 April 2001
Abstract Two enmein type diterpenoids, taibaijaponicains A and B, were isolated from the ethanol extract of the leaves and branches of Isodon japonica. Their structures are designated as 6b,11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7olide and 3b-acetoxy-6b,11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7-olide, respectively, on the basis of detailed spectroscopic analyses. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Isodon japonica; Labiatae; Taibaijaponicains A and B; Enmein type diterpenoids
1. Introduction Enmein was first isolated from Isodon japonica in 1958. Enmein type diterpenoids are bitter constituents of the plants of genus Isodon (Labiatae). They have varied biological activities, i.e. anti-bacterial, anti-inflammatory, anti-tumor, etc. (Cheng et al., 1987). Previous studies on I. japonica led to the isolation of more than 16 diterpenoids (Fujita et al., 1973; Li et al., 1982; Liu et al., 1982; Zhao et al., 1984; Wang et al., 1987; Zhao and Li., 1987; Meng et al., 1989; Xu et al., 1996). A further study of the minor diterpenoid constituents of this species was aimed at finding substances with even higher biological activities. We have isolated two new enmein type diterpenoids, taibaijaponicains A (1) and B (2), together with six known diterpenoids, norhendosin A, rabdophyllin G, dihydroenmein, nodosin, enmein, rabdosin A, and two other known compounds, b-sitosterol and oleanolic acid. In this paper, we report the isolation and structure elucidation of the new diterpenoids by means of spectroscopic methods including 1D and 2D NMR spectroscopy.
2. Results and discussion Taibaijaponicain A (1) was obtained as an amorphous powder. Its molecular formula was determined as * Corresponding author. Tel.: +86-29-5307788; fax: +86-295307774. E-mail address: [email protected] (B. Li).
C21H30O7 by positive HR–FABMS ([M+1]+ m/z 395.4644, calc. 395.4666) and FABMS ([M+Li]+ m/z 401, [M+Na]+ m/z 417). The 1H, 13C and DEPT NMR spectra of 1 (see Tables 1 and 2) showed signals for two methyl groups, one methoxy group, six methylene groups, seven methine groups, three quaternary carbons, one ketonic carbon and one ester carbonyl carbon. Taibaijaponicain A (1) contains a five-membered ketone group [max 1765 cm 1, c 212.3(s)], -lactone [max 1720 cm 1, H 4.77(1H, dd, J=11.2, 3.9 Hz), c 171.2(s)], and a hemiacetal group [H 5.27(1H, s), c 102.6(d)]. In addition, the IR and 1H NMR spectra also revealed that the compound possessed two hydroxyl groups [max 3358 cm 1, H 5.27 (1H, s), 4.30 (1H, ddd, J=10.6, 8.8, 3.4 Hz)]. Considering the known structures of diterpenoids from the genus Isodon, these data suggest that 1 was an enmein type diterpenoid of pentacyclic 6,7-seco-ent-kaurane type, with one methoxy group and two hydroxyl groups. Compound 1 did not show an absorption maximum band above 220 nm in the UV spectrum, which indicated the absence of a five-membered ketone conjugated with the exo-methylene group. The 13C NMR spectroscopic data of 1 were very similar to those of nodosin (3) (Takeda et al., 1994) except for one more methoxyl group and one less C–C double bond. Comparison of their 13C NMR spectral data indicated that the difference between 1 and 3 was only in the ring D. This meant that two hydroxyl groups were attached at C-6 and C11, and a methoxyl group was in ring D in compound 1. In the 1H NMR spectrum of 1, besides an AB peak at 3.92, 3.73 (each 1H, AB, d, J=9.2 Hz, H-20a, 20b),
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00245-X
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Table 1 1 H NMR and principal HMBC correlations of compounds 1 and 2 in acetone-d6a 1
2
Proton H, mult, intgr, (J, Hz)
HMBC (carbon)
1b 2a 2b 3a 3b 5b 6a 9a 11b 12a 12b 13b 14a 14b 16b 17a 17b 18 19 20a 20b OCH3 OAc
(2), 3, 9, (10), 20 4.85, dd, 1H, (10.8, 3.6) (2), 3, (10), 20 (1), (3), 4, 9, 10 2.12, m, 1H (1), (3), 4, 10 (1), (3), 4, 9, 10 2.34, m, 1H (1), (3), 4 1, (2), (4), 19 4.79, dd, 1H, (3.5, 3.6) (2), (4), 19, OAc(170.6 ppm) 1, (2), (4), 19 – – (4), (6), 9, (10), 20 2.73, s, 1H (4), (6), 9, 10 4, (5), 10, 20 5.26, s, 1H (5), 10, 20 1, (8), (10), (11), 12, 14, 15 2.13, bs d, 1H, (10.4) (8), (10), (11), 12 (9), 10, (12) 4.31, ddd, 1H, (10.4, 8.3, 3.1) (9), 10, (12) (11), (13), 14 2.41, m, 1H (11), (13), 14 9, (11), (13) 1.44, overlap, 1H n.o.b 9, 11, (12), (14), 17 2.80, m, 1H 11, (12), (14), 16 (8), (13) 1.82, m, 1H n.o. (8), (13) 1.50, m, 1H (8), (13) 11, 12, (13), 14, MeO 1.47, m, 1H (13), 14, 17 13, (16), MeO 3.65, 3.54, AB, dd, each 1H, (10.2, 4.3) 13, (16), MeO 13, (16), MeO 13, (16), MeO 3, (4), 5, 19 1.01, s, 3H (4), 5, 19 3, (4), 5, 18 0.98, s, 3H 3, (4), 5, 18 1, 9, (10), 11 3.93, 3.71, AB, d, each 1H, (9.1) 1, 6, 9, (10) 9, (10) 6, 9, (10) 17 3.32, s, 3H 17 – 2.07, s, 3H
a b
4.77, dd, 1H, (11.2, 3.9) 1.82, m, 1H 1.78, m, 1H 1.48, m, 1H 1.38, m, 1H 2.75, s, 1H 5.27, s, 1H 2.14, bs d, 1H, (10.6) 4.30, ddd, 1H, (10.6, 8.8, 3.4) 2.40, m, 1H 1.45, overlap, 1H 2.79, m, 1H 1.81, overlap, 1H 1.48, overlap, 1H 1.45, m, 1H 3.64, 3.56, AB, dd, each 1H, (10.4, 4.5) 1.00, s, 3H 0.97, s, 3H 3.92, 3.73, AB, d, each 1H, (9.2) 3.30, s, 3H –
dH, mult, intgr, (J, Hz)
HMBC (carbon)
Two-bond correlations are indicated in parentheses. n.o. Indicates no clear correlations with this proton.
there was an AB peak at 3.64, 3.56 (each 1H, AB, dd, J=10.4, 4.5 Hz, H-17a, 17b). In the HMBC spectrum of 1 (see Table 1), the signals at 3.64 and 3.56 correlated with the signals at 31.3 (d, C-13), 58.8 (d, C-16) and 56.4 (q, OCH3); the signal at 2.79 (1H, m, H-13b) showed a correlation with the signals at 69.3 ppm (t, C-17), 32.6 ppm (t, C-12) and 35.0 ppm (t, C-14). These results suggested that the methoxyl group should be located at the C-17 position. In enmein type diterpenoids, the ring A is in a chair conformation, the rings B1 and D are in envelope conformations and ring B2 is in a boat conformation. Ring C can adopt either a chair or boat conformation depending on the substituents present in ring C (Xu, 1985). In compound 1, the coupling constant between H-5b ( 2.75, s) and H-6 ( 5.27, s) was zero, which indicated that its dihedral angle approximated 90 , consistent with a borientation of the hydroxyl group at C-6 of 1. Ring C of 1 appears to be in a boat conformation judging from that the coupling constant of H-11 ( 4.30, ddd, J=10.6, 8.8, 3.4 Hz) with H-9a ( 2.14, bs d, J=10.6 Hz) and H-12a( 2.40, m), H-12b ( 1.45, overlap). For a chair conformation of the ring C, the dihedral angles between H-9a and H-11, H-11 and H-12a or H-12b should approximate 60 , and all of their coupling constants should be less than 4 Hz. This fact suggests that the hydroxyl group at C-11 of 1 was in the a-orientation. These results were further verified by a NOESY
Table 2 13 C NMR spectral data for compounds 1 and 2 in acetone-d6 Carbon
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe AcO
77.3, 24.2, 37.4, 32.1, 54.3, 102.6, 171.2, 57.7, 53.1, 50.9, 63.3, 32.6, 31.3, 35.0, 212.3, 58.8, 69.3, 33.1, 23.5, 73.9, 56.4,
2 d t t s d d s s d s d t d t s d t q q t q
77.2, 32.1, 71.6, 35.7, 54.2, 102.5, 171.1, 57.5, 53.0, 51.5, 63.4, 32.5, 31.4, 35.2, 212.4, 58.9, 69.4, 32.8, 22.4, 73.7, 56.2, 170.6, 20.9,
d t d s d d s s d s d t d t s d t q q t q s q
experiment, where correlations are shown in Fig. 1. In addition, in the NOESY spectrum of 1, the signal at 1.45 (1H, m, H-16) exhibited a NOE correlation with both the signals at 2.79 (1H, m, H-13b) and 1.81 (1H,
B. Li, X. Tian / Phytochemistry 58 (2001) 543–546
overlap, H-14a). This indicated that C-17 was in the aorientation. Therefore, taibaijaponicain A (1) was 6b, 11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-secoent-kaur-15-one-1,7-olide. Taibaijaponicain B (2) was obtained as an amorphous powder. Its molecular formula was determined as C23H32O9 by FABMS ([M+Li]+ m/z 459, [M+Na]+ m/z 475). The IR, 1H, 13C and DEPT NMR data of 2 (see Tables 1 and 2) were very similar to those of taibaijaponicain A (1) except for the presence of one acetoxyl group in 2 [max 1742 cm 1, H 2.07 (3H, s), c 170.6 (s), 20.9(q)]. The 13C NMR spectral data of 2 differed from those of 1 only at C-2, C-3 and C-4. The downfield shift of C-3 from 37.4 ppm in 1 to 71.6 ppm in 2 indicated that the acetoxyl group at C-3 in 2 had replaced the H in 1. These assignments were further confirmed by the HMBC spectrum of 2 (see Table 1). The relative configuration of the acetoxyl group at C3 of 2 was established on the basis of the coupling constants of H-3 ( 4.79, 1H, dd, J=3.5, 3.6 Hz) with H-2a ( 2.12, 1H, m) and H-2b ( 2.34, 1H, m). The coupling constants suggested that the dihedral angles between H-3 and H-2a, and H-2b approximated 60 , thus the H-3 was in the a-orientation and the acetoxyl group was in the b-orientation in compound 2. On the basis of the above evidence, the structure of taibaijaponicain B (2) was determined to be 3b-acetoxy-6b,11a-dihydroxy-16a-methoxymethyl6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7-olide.
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l2) at room temperature for 7 days. After removal of the solvent in vacuo, the residue was partitioned in H2O and extracted with petroleum ether (4 l3) and EtOAc (4 l3), respectively. The EtOAc extract (130 g) was subjected to CC on silica gel (3 kg, 200–300 mesh), eluting with CHCl3 and increasing proportions of Me2CO (from CHCl3 100% to Me2CO 100%). Fractions were combined by monitoring with TLC. All components were further purified by column chromatography and preparative TLC on silica gel to give bsitosterol (1.13 g, 0.016%), oleanolic acid (2.6 g, 0.037%), norhendosin A (273 mg, 0.0039%), rabdophyllin G (67 mg, 0.00096%), dihydroenmein (315 mg, 0.0045%), nodosin (3, 110 mg, 0.0016%), enmein (384 mg, 0.0055%), taibaijaponicain A (1, 236 mg, 0.0034%), taibaijaponicain B (2, 89 mg, 0.0013%), rabdosin A (95 mg, 0.0014%), orderly. 3.4. Taibaijaponicain A (1) An amorphous powder; [a]17 104.7 (c 0.49, aceD KBr 1 tone); IR max cm : 3358, 2925, 2800, 1765, 1720, 1504, 1461, 1264, 1126, 1072 and 1050; FABMS m/z: 401 [M+Li]+, 417 [M+Na]+; HR–FABMS m/z 395.4644 [M+1]+ for C21H30O7 (calc. 395.4666); 1H NMR spectral data (400 MHz) and principal HMBC correlations are listed in Table 1; DEPT, 13C NMR spectral data are listed in Table 2. Most of the NOESY correlations are shown in Fig. 1.
3. Experimental
3.5. Taibaijaponicain B (2)
3.1. General
An amorphous powder; [a]17 98.6 (c 0.5, acetone); D KBr 1 IR max cm : 3360, 2921, 1766, 1742, 1720, 1463, 1370,
FAB mass spectra were recorded on a ZAB-HS Instrument (data system: MASPEC II); HR–FAB mass spectrum was recorded in an Autospec 3000 instrument. IR spectra were recorded in KBr pellets on a Nicolet AVATAR 360 FT–IR spectrometer. The optical rotations were measured with a JASCO-20C polarimeter. NMR spectra were recorded on a Bruker AM-400 Instrument. The chemical shift values are given in ppm using TMS as the internal standard. 3.2. Plant material The plant material of I. japonica was collected in Taibai Mountain, Shaanxi Province, PR China, in August 1997. A voucher specimen (SNU 97-08-03, Li) was deposited in the Herbarium of Department of Biology, Shaanxi Normal University. 3.3. Extraction and isolation The dried powdered leaves and tender branches of I. japonica (7.0 kg) were extracted with 95% EtOH (18
Fig. 1. Major NOE correlations in 1.
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1264, 1052 and 908; FABMS m/z: 459 [M+Li]+, 475 [M+Na]+; 1H NMR spectral data (400 MHz) and principal HMBC correlations are listed in Table 1; DEPT, 13C NMR spectral data are listed in Table 2. Acknowledgements We are grateful for the financial support from the Natural Science Foundation of Shaanxi Province, PR China. We would like to thank Professor Xuan Tian, Department of Chemistry, Lanzhou University (PR China), for helpful discussions. References Cheng, P.Y., Guo, Y.W., Xu, M.J., 1987. Pharmaceutical prospects of Rabdosia species. Zhong Yao Tong Bao 12, 707. Fujita, E., Taoka, M., Nagao, Y., Fujita, T., 1973. Terpenoids. XXV. J. Chem. Soc., Perkin Trans. 1, 16, 1760.
Li, J.C., Liu, C.J., An, X.Z., Wang, M.T., Zhao, T.Z., Yu, S.Z., Zhao, G.S., Chen, R.F., 1982. Study on the antitumor constituent of Rabdosia japonica (burm. f) Hara. I. Structure of rabdosins A and B. Yao Xue Xue Bao 17, 682. Liu, C.J., Li, J.C., An, X.Z., Cheng, R.M., Chen, F.Z., Xu, Y.L., Wang, D.Z., 1982. Studies on the antitumor constituent of Rabdosia japonica. II. Structure of rabdosin C. Yao Xue Xue Bao 17, 750. Meng, X.J., Wang, Q.G., Chen, Y.Z., 1989. Diterpenoids from Rabdosia japonica. Phytochemistry 28 (4), 1163. Takeda, Y., Matsumoto, T., Otsuka, H., 1994. Longirabdolide C, a new diterpenoid from Rabdosia longituba. J Nat. Prod. 57 (5), 650. Wang, M.T., Zhao, T.Z., Li, J.C., Liu, C.J., An, X.Z., 1987. Structures of rabdosinate and rabdosinatol. Acta Chim. Sin. 45 (9), 871. Xu, F.M., Hu, H.P., Wang, Z.Q., 1996. Studies on diterpenoid constituents in Rabdosia japonica (Burm. f.) Hara. Zhong Guo Zhong Yao Za Zhi 21 (11), 678. Xu, G.Y., 1985. 1H NMR of Rabdosia diterpenoids. Acta Chim. Sin. 43, 35. Zhao, Q.C., Li, C.S., 1987. Chemical contituents of Rabdosia japonica var. glaucocalyx. Zhong Yao Tong Bao 12 (5), 294. Zhao, Q.Z., Chao, J.H., Wang, H.Q., Sun, H.D., 1984. Chemical constituent in Rabdosia japonica. Zhong Cao Yao 15 (2), 49.
Enmein type diterpenoids from Isodon japonica Baolin Lia,*, Xianhua Tianb a
Department of Chemistry, Shaanxi Normal University, Xi’an 710062, People’s Republic of China b Life Science College, Shaanxi Normal University, Xi’an 710062, People’s Republic of China Received 8 December 2000; received in revised form 19 April 2001
Abstract Two enmein type diterpenoids, taibaijaponicains A and B, were isolated from the ethanol extract of the leaves and branches of Isodon japonica. Their structures are designated as 6b,11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7olide and 3b-acetoxy-6b,11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7-olide, respectively, on the basis of detailed spectroscopic analyses. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Isodon japonica; Labiatae; Taibaijaponicains A and B; Enmein type diterpenoids
1. Introduction Enmein was first isolated from Isodon japonica in 1958. Enmein type diterpenoids are bitter constituents of the plants of genus Isodon (Labiatae). They have varied biological activities, i.e. anti-bacterial, anti-inflammatory, anti-tumor, etc. (Cheng et al., 1987). Previous studies on I. japonica led to the isolation of more than 16 diterpenoids (Fujita et al., 1973; Li et al., 1982; Liu et al., 1982; Zhao et al., 1984; Wang et al., 1987; Zhao and Li., 1987; Meng et al., 1989; Xu et al., 1996). A further study of the minor diterpenoid constituents of this species was aimed at finding substances with even higher biological activities. We have isolated two new enmein type diterpenoids, taibaijaponicains A (1) and B (2), together with six known diterpenoids, norhendosin A, rabdophyllin G, dihydroenmein, nodosin, enmein, rabdosin A, and two other known compounds, b-sitosterol and oleanolic acid. In this paper, we report the isolation and structure elucidation of the new diterpenoids by means of spectroscopic methods including 1D and 2D NMR spectroscopy.
2. Results and discussion Taibaijaponicain A (1) was obtained as an amorphous powder. Its molecular formula was determined as * Corresponding author. Tel.: +86-29-5307788; fax: +86-295307774. E-mail address: [email protected] (B. Li).
C21H30O7 by positive HR–FABMS ([M+1]+ m/z 395.4644, calc. 395.4666) and FABMS ([M+Li]+ m/z 401, [M+Na]+ m/z 417). The 1H, 13C and DEPT NMR spectra of 1 (see Tables 1 and 2) showed signals for two methyl groups, one methoxy group, six methylene groups, seven methine groups, three quaternary carbons, one ketonic carbon and one ester carbonyl carbon. Taibaijaponicain A (1) contains a five-membered ketone group [max 1765 cm 1, c 212.3(s)], -lactone [max 1720 cm 1, H 4.77(1H, dd, J=11.2, 3.9 Hz), c 171.2(s)], and a hemiacetal group [H 5.27(1H, s), c 102.6(d)]. In addition, the IR and 1H NMR spectra also revealed that the compound possessed two hydroxyl groups [max 3358 cm 1, H 5.27 (1H, s), 4.30 (1H, ddd, J=10.6, 8.8, 3.4 Hz)]. Considering the known structures of diterpenoids from the genus Isodon, these data suggest that 1 was an enmein type diterpenoid of pentacyclic 6,7-seco-ent-kaurane type, with one methoxy group and two hydroxyl groups. Compound 1 did not show an absorption maximum band above 220 nm in the UV spectrum, which indicated the absence of a five-membered ketone conjugated with the exo-methylene group. The 13C NMR spectroscopic data of 1 were very similar to those of nodosin (3) (Takeda et al., 1994) except for one more methoxyl group and one less C–C double bond. Comparison of their 13C NMR spectral data indicated that the difference between 1 and 3 was only in the ring D. This meant that two hydroxyl groups were attached at C-6 and C11, and a methoxyl group was in ring D in compound 1. In the 1H NMR spectrum of 1, besides an AB peak at 3.92, 3.73 (each 1H, AB, d, J=9.2 Hz, H-20a, 20b),
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00245-X
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Table 1 1 H NMR and principal HMBC correlations of compounds 1 and 2 in acetone-d6a 1
2
Proton H, mult, intgr, (J, Hz)
HMBC (carbon)
1b 2a 2b 3a 3b 5b 6a 9a 11b 12a 12b 13b 14a 14b 16b 17a 17b 18 19 20a 20b OCH3 OAc
(2), 3, 9, (10), 20 4.85, dd, 1H, (10.8, 3.6) (2), 3, (10), 20 (1), (3), 4, 9, 10 2.12, m, 1H (1), (3), 4, 10 (1), (3), 4, 9, 10 2.34, m, 1H (1), (3), 4 1, (2), (4), 19 4.79, dd, 1H, (3.5, 3.6) (2), (4), 19, OAc(170.6 ppm) 1, (2), (4), 19 – – (4), (6), 9, (10), 20 2.73, s, 1H (4), (6), 9, 10 4, (5), 10, 20 5.26, s, 1H (5), 10, 20 1, (8), (10), (11), 12, 14, 15 2.13, bs d, 1H, (10.4) (8), (10), (11), 12 (9), 10, (12) 4.31, ddd, 1H, (10.4, 8.3, 3.1) (9), 10, (12) (11), (13), 14 2.41, m, 1H (11), (13), 14 9, (11), (13) 1.44, overlap, 1H n.o.b 9, 11, (12), (14), 17 2.80, m, 1H 11, (12), (14), 16 (8), (13) 1.82, m, 1H n.o. (8), (13) 1.50, m, 1H (8), (13) 11, 12, (13), 14, MeO 1.47, m, 1H (13), 14, 17 13, (16), MeO 3.65, 3.54, AB, dd, each 1H, (10.2, 4.3) 13, (16), MeO 13, (16), MeO 13, (16), MeO 3, (4), 5, 19 1.01, s, 3H (4), 5, 19 3, (4), 5, 18 0.98, s, 3H 3, (4), 5, 18 1, 9, (10), 11 3.93, 3.71, AB, d, each 1H, (9.1) 1, 6, 9, (10) 9, (10) 6, 9, (10) 17 3.32, s, 3H 17 – 2.07, s, 3H
a b
4.77, dd, 1H, (11.2, 3.9) 1.82, m, 1H 1.78, m, 1H 1.48, m, 1H 1.38, m, 1H 2.75, s, 1H 5.27, s, 1H 2.14, bs d, 1H, (10.6) 4.30, ddd, 1H, (10.6, 8.8, 3.4) 2.40, m, 1H 1.45, overlap, 1H 2.79, m, 1H 1.81, overlap, 1H 1.48, overlap, 1H 1.45, m, 1H 3.64, 3.56, AB, dd, each 1H, (10.4, 4.5) 1.00, s, 3H 0.97, s, 3H 3.92, 3.73, AB, d, each 1H, (9.2) 3.30, s, 3H –
dH, mult, intgr, (J, Hz)
HMBC (carbon)
Two-bond correlations are indicated in parentheses. n.o. Indicates no clear correlations with this proton.
there was an AB peak at 3.64, 3.56 (each 1H, AB, dd, J=10.4, 4.5 Hz, H-17a, 17b). In the HMBC spectrum of 1 (see Table 1), the signals at 3.64 and 3.56 correlated with the signals at 31.3 (d, C-13), 58.8 (d, C-16) and 56.4 (q, OCH3); the signal at 2.79 (1H, m, H-13b) showed a correlation with the signals at 69.3 ppm (t, C-17), 32.6 ppm (t, C-12) and 35.0 ppm (t, C-14). These results suggested that the methoxyl group should be located at the C-17 position. In enmein type diterpenoids, the ring A is in a chair conformation, the rings B1 and D are in envelope conformations and ring B2 is in a boat conformation. Ring C can adopt either a chair or boat conformation depending on the substituents present in ring C (Xu, 1985). In compound 1, the coupling constant between H-5b ( 2.75, s) and H-6 ( 5.27, s) was zero, which indicated that its dihedral angle approximated 90 , consistent with a borientation of the hydroxyl group at C-6 of 1. Ring C of 1 appears to be in a boat conformation judging from that the coupling constant of H-11 ( 4.30, ddd, J=10.6, 8.8, 3.4 Hz) with H-9a ( 2.14, bs d, J=10.6 Hz) and H-12a( 2.40, m), H-12b ( 1.45, overlap). For a chair conformation of the ring C, the dihedral angles between H-9a and H-11, H-11 and H-12a or H-12b should approximate 60 , and all of their coupling constants should be less than 4 Hz. This fact suggests that the hydroxyl group at C-11 of 1 was in the a-orientation. These results were further verified by a NOESY
Table 2 13 C NMR spectral data for compounds 1 and 2 in acetone-d6 Carbon
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe AcO
77.3, 24.2, 37.4, 32.1, 54.3, 102.6, 171.2, 57.7, 53.1, 50.9, 63.3, 32.6, 31.3, 35.0, 212.3, 58.8, 69.3, 33.1, 23.5, 73.9, 56.4,
2 d t t s d d s s d s d t d t s d t q q t q
77.2, 32.1, 71.6, 35.7, 54.2, 102.5, 171.1, 57.5, 53.0, 51.5, 63.4, 32.5, 31.4, 35.2, 212.4, 58.9, 69.4, 32.8, 22.4, 73.7, 56.2, 170.6, 20.9,
d t d s d d s s d s d t d t s d t q q t q s q
experiment, where correlations are shown in Fig. 1. In addition, in the NOESY spectrum of 1, the signal at 1.45 (1H, m, H-16) exhibited a NOE correlation with both the signals at 2.79 (1H, m, H-13b) and 1.81 (1H,
B. Li, X. Tian / Phytochemistry 58 (2001) 543–546
overlap, H-14a). This indicated that C-17 was in the aorientation. Therefore, taibaijaponicain A (1) was 6b, 11a-dihydroxy-16a-methoxymethyl-6,20-epoxy-6,7-secoent-kaur-15-one-1,7-olide. Taibaijaponicain B (2) was obtained as an amorphous powder. Its molecular formula was determined as C23H32O9 by FABMS ([M+Li]+ m/z 459, [M+Na]+ m/z 475). The IR, 1H, 13C and DEPT NMR data of 2 (see Tables 1 and 2) were very similar to those of taibaijaponicain A (1) except for the presence of one acetoxyl group in 2 [max 1742 cm 1, H 2.07 (3H, s), c 170.6 (s), 20.9(q)]. The 13C NMR spectral data of 2 differed from those of 1 only at C-2, C-3 and C-4. The downfield shift of C-3 from 37.4 ppm in 1 to 71.6 ppm in 2 indicated that the acetoxyl group at C-3 in 2 had replaced the H in 1. These assignments were further confirmed by the HMBC spectrum of 2 (see Table 1). The relative configuration of the acetoxyl group at C3 of 2 was established on the basis of the coupling constants of H-3 ( 4.79, 1H, dd, J=3.5, 3.6 Hz) with H-2a ( 2.12, 1H, m) and H-2b ( 2.34, 1H, m). The coupling constants suggested that the dihedral angles between H-3 and H-2a, and H-2b approximated 60 , thus the H-3 was in the a-orientation and the acetoxyl group was in the b-orientation in compound 2. On the basis of the above evidence, the structure of taibaijaponicain B (2) was determined to be 3b-acetoxy-6b,11a-dihydroxy-16a-methoxymethyl6,20-epoxy-6,7-seco-ent-kaur-15-one-1,7-olide.
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l2) at room temperature for 7 days. After removal of the solvent in vacuo, the residue was partitioned in H2O and extracted with petroleum ether (4 l3) and EtOAc (4 l3), respectively. The EtOAc extract (130 g) was subjected to CC on silica gel (3 kg, 200–300 mesh), eluting with CHCl3 and increasing proportions of Me2CO (from CHCl3 100% to Me2CO 100%). Fractions were combined by monitoring with TLC. All components were further purified by column chromatography and preparative TLC on silica gel to give bsitosterol (1.13 g, 0.016%), oleanolic acid (2.6 g, 0.037%), norhendosin A (273 mg, 0.0039%), rabdophyllin G (67 mg, 0.00096%), dihydroenmein (315 mg, 0.0045%), nodosin (3, 110 mg, 0.0016%), enmein (384 mg, 0.0055%), taibaijaponicain A (1, 236 mg, 0.0034%), taibaijaponicain B (2, 89 mg, 0.0013%), rabdosin A (95 mg, 0.0014%), orderly. 3.4. Taibaijaponicain A (1) An amorphous powder; [a]17 104.7 (c 0.49, aceD KBr 1 tone); IR max cm : 3358, 2925, 2800, 1765, 1720, 1504, 1461, 1264, 1126, 1072 and 1050; FABMS m/z: 401 [M+Li]+, 417 [M+Na]+; HR–FABMS m/z 395.4644 [M+1]+ for C21H30O7 (calc. 395.4666); 1H NMR spectral data (400 MHz) and principal HMBC correlations are listed in Table 1; DEPT, 13C NMR spectral data are listed in Table 2. Most of the NOESY correlations are shown in Fig. 1.
3. Experimental
3.5. Taibaijaponicain B (2)
3.1. General
An amorphous powder; [a]17 98.6 (c 0.5, acetone); D KBr 1 IR max cm : 3360, 2921, 1766, 1742, 1720, 1463, 1370,
FAB mass spectra were recorded on a ZAB-HS Instrument (data system: MASPEC II); HR–FAB mass spectrum was recorded in an Autospec 3000 instrument. IR spectra were recorded in KBr pellets on a Nicolet AVATAR 360 FT–IR spectrometer. The optical rotations were measured with a JASCO-20C polarimeter. NMR spectra were recorded on a Bruker AM-400 Instrument. The chemical shift values are given in ppm using TMS as the internal standard. 3.2. Plant material The plant material of I. japonica was collected in Taibai Mountain, Shaanxi Province, PR China, in August 1997. A voucher specimen (SNU 97-08-03, Li) was deposited in the Herbarium of Department of Biology, Shaanxi Normal University. 3.3. Extraction and isolation The dried powdered leaves and tender branches of I. japonica (7.0 kg) were extracted with 95% EtOH (18
Fig. 1. Major NOE correlations in 1.
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1264, 1052 and 908; FABMS m/z: 459 [M+Li]+, 475 [M+Na]+; 1H NMR spectral data (400 MHz) and principal HMBC correlations are listed in Table 1; DEPT, 13C NMR spectral data are listed in Table 2. Acknowledgements We are grateful for the financial support from the Natural Science Foundation of Shaanxi Province, PR China. We would like to thank Professor Xuan Tian, Department of Chemistry, Lanzhou University (PR China), for helpful discussions. References Cheng, P.Y., Guo, Y.W., Xu, M.J., 1987. Pharmaceutical prospects of Rabdosia species. Zhong Yao Tong Bao 12, 707. Fujita, E., Taoka, M., Nagao, Y., Fujita, T., 1973. Terpenoids. XXV. J. Chem. Soc., Perkin Trans. 1, 16, 1760.
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