行政院國家科學委員會專題研究計畫 期中精簡報告
台灣產膨大羽珊瑚、紫羽珊瑚及笙珊瑚癌細胞毒殺成分之研
究(2/3)
計畫類別： 個別型計畫
計畫編號：
NSC91-2320-B-110-010-執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日
執行單位： 國立中山大學海洋資源學系(所)
計畫主持人： 杜昌益
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中
華
民
國 92 年 5 月 6 日
Abstr act:
Three new cytotoxic prostanoids, claviridenone E-G (1−3) and three new cytotoxic steroids,
stoloniferone E-G (4−6) were isolated from the methylene chloride solubles of the Formosan soft coral
Clavularia viridis Quoy and Gaimard. A cytotoxic cembranoid, claviolide (7) was isolated from the methylene chloride solubles of the Formosan soft coralClavularia violacea Quoy and Gaimard. The structures were elucidated by 1D and 2D NMR spectral analysis and their cytotoxicity against selected cancer cells was measured in vitro.
O COOMe AcO 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 O COOMe AcO 1 2 3 OHO OH H H H 4 OHO OH H H H O H OH OHO OH H H H OH O H 5 6 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 28 O COOMe AcO CH3 O CH3 CH3 AcO AcO O
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Compound 1 was shown to have a molecular formula of C23H32O5 as indicated by HREIMS and NMR
data. The IR spectrum of 1 showed absorption due to acetate ester (1735, 1235 cm-1) and α,β-unsaturated cyclopentenone (1705 cm-1) functionalities. The presence of a cross-conjugated system in 1, corresponding to that of the clavulones,4 was demonstrated by UV absorption at 226 (log ε 3.88) and 290 (log ε 4.04) nm. The 13C NMR and DEPT spectrum exhibited 23 carbon resonances which were attributable to two methyls (δ 21.4 q and 14.1 q), one methoxyl (δ 51.6 q), one ketone carbonyl (δ 193.9 s), two ester carbonyls (δ 173.6 s and 169.4 s), eight sp3 methylene (δ 33.3 t, 23.9 t, 32.7 t, 35.7 t, 27.5 t, 29.1 t, 31.6 t, 22.6 t), seven sp2 methines (δ 146.5 d, 125.4 d, 131.4 d, 134.8 d, 157.5 d, 121.3 d, 135.3 d), one sp2 quaternary carbon (δ 134.0 s), one sp3 quaternary carbon (δ 85.6 s) (Table 1). The 1H NMR spectrum of 1 disclosed five olefinic protons in the cross-conjugated system at δ 6.22 (1H, dt, J = 7.2, 15.0 Hz, H-5), 6.41 (1H, d, J = 6.0 Hz, H-10), 6.54 (1H, dd, J = 11.7, 15.0 Hz, H-6), 6.92 (1H, d, J = 11.7 Hz, H-7), 7.48 (1H, d, J = 6.0 Hz, H-11); two olefinic protons on carbon-carbon double bond at δ 5.17 m and 5.50 m; and a terminal methyl at δ 0.88 (3H, t, J = 6.9 Hz, H3-20). The analysis of 1H−1H COSY spectrum revealed a sequence of the correlations starting from a
doublet at δH 6.92 (1H, d, J = 11.7 Hz, H-7) and carried through to a triplet at δH 2.35 (2H, t, J = 7.5 Hz,
H-2), indicating the partial structure of H-7 through H-2 on the α-side chain shown as a bold line in Figure 1. The connectivity from H-13 to H-20 on the ω-side chain was indicated by the correlations in the 1H−1H COSY spectrum starting from two doublet of doublets at δH 2.70 (1H, dd, J = 14.4, 8.1 Hz,
H-13) and 2.96 (1H, dd, J = 14.4, 7.2 Hz, H-13) and ending with the methyl protons at δH 0.88 (3H, t, J
= 6.9 Hz, H-20). These spectroscopic findings showed 1 to have a structure similar to that of clavulone II,4 except for C-4 (CH2 in 1; CHOAc in clavulone II) on the α side chain. Assignments between the 1
H and 13C NMR signals were made based on HSQC correlations. The data from the HMBC spectrum fully supported the assigned structure and key HMBC correlations are shown in Figure 1.
The molecular formula of compound 2 was assigned as C23H32O5 by HREIMS and NMR data.
The 1H and 13C NMR spectra of 2 were very similar to those of 1 except for the 1H coupling constant between the two olefinic protons at H-5 and H-6 (10.8 Hz in 2; 15.0 Hz in 1) and the 13C chemical shifts at C-4 (δC 27.3 in 2; 32.7 in 1) and C-7 (δC 125.6 in 2; 131.4 in 1). Compound 2 was thus
assigned as a 5Z isomer of 1 based on the comparison of 1H and 13C NMR data with those of clavulone II.4 NOESY correlations from H-5 to H-6 and from H-4 to H-7 confirmed this assignment. The assignments of the 1H and 13C NMR signals were accomplished by COSY, HSQC, HMBC, and NOESY experiments.
The molecular formula of compound 3 was shown to be C23H32O5 by HREIMS and NMR data.
The 1H and 13C NMR spectra of 3 were also very similar to those of 1 except for the 1H chemical shift values at H-6 (δH 7.61 in 3; 6.54 in 1) and H-7 (δH 6.54 in 3; 6.92 in 1) and the 13C chemical shifts at
C-7 (δC 134.7 in 3; 131.4 in 1). Compound 3 was thus assigned as a 7Z isomer of 1 based on the
comparison of 1H and 13C NMR data with those of clavulone II.4 The assignments the 1H and 13C NMR signals were confirmed by COSY, HSQC, HMBC, and NOESY experiments.
Compound 4 had a molecular formula of C28H44O3 as indicated by HREIMS. 13
DEPT spectrum of 4 exhibited the presence of six methyls, six sp3 methylenes, nine sp3 methines, three sp2 methines, two sp3 quaternary carbons and two sp2 quaternary carbons. The IR spectrum of 4 showed absorption due to an α,β-unsaturated ketone (1676 cm-1). The presence of a conjugated enone system in 4 was also indicated by UV absorptions at 222 nm (log ε 3.79) and 280 (log ε 4.01) nm as well as 1H NMR [δ 6.18 (1H, d, J = 9.6 Hz), 6.19 (1H, d, J = 6.0 Hz), 6.99 (1H, dd, J= 9.6, 6.0 Hz)] and 13C NMR [δ 118.9 (CH), 126.7 (CH), 140.8 (CH), 157.8 (C)] spectra (Table 2). IR absorption at 3300 cm-1 and NMR signals at δH 4.58 (1H, brs) and 4.04 (1H, dt, J = 3.7, 10.5 Hz) as well as at δC
73.6 (CH) and 66.9 (CH) indicated the presence of two secondary hydroxyl groups. The spectral data of 4 exhibited some similarity to those of yonarasterol E,5 except for the presence of a trisubstituted double bond and lacking of the epoxide. All C−H correlations of 4 were detected in the HSQC experiment. The 1H−1H COSY spectrum exhibited partial structures a, b, and c (Figure 2). In the HMBC spectrum, partial structure a could be connected to b through two quaternary carbons (C-5 and C-10) and H3-19 (Figure 2). Partial structure b could be connected to c through the remaining
quaternary carbons (C-13) and H3-18. Based on these findings, the gross structure of 4 was concluded
as in Figure 2. The NOESY correlations (Figure 3) observed between H-11 and H-8, H-11 and H3-18,
H-11 and H3-19, H-4 and H-6, H-9 and H-14, H3-18 and H-8, H3-19 and H-8, H3-18 and H-20, H3-21
and H-12β, H-9 and H-12α indicated the relative configurations for each ring junction and chiral center. Stereochemistry at C-20 and C-24 was determined by comparison of 13C NMR data with those of yonarasterol E and stoniferone-c.5-6
HREIMS and 13C NMR data revealed 5 to have a molecular formula of C28H46O5. 13
C and 1H NMR data (Table 2) showed some similarity to 4, except for the presence of two additional hydroxyls and absence of the trisubstituted double bond. The location the hydroxyls on C-2 and C-4 was made based on 1H-1H COSY correlations from H-2 to H-3; H-3 to H-4; and HMBC correlations (Figure 2) from H-2 to C-1, C-3, C-4; H-3 to C-1, C-2, C-4, C-5; H-19 to C-1, C-5, C-9, C-10. The NOESY correlations (Figure 3) observed between H-4 and H3-19, H-4 and H-6, H-11 and H-8, H-11 and H3-18,
H-11 and H3-19, H-9 and H-14, H3-18 and H-8, H3-19 and H-8, H3-18 and H-20, H3-21 and
H-12β, H-9 and H-12α indicated the relative configurations for each ring junction and chiral center. The molecular formula of compound 6 was assigned as C28H44O5 by HREIMS and NMR data.
The 1H and 13C NMR spectra of 6 were very similar to those of 5 except for NMR signals due to the side chain. Stereochemistry at C-20 was determined by comparison of 13C NMR data with those of stoniferone-a.6
Compound 7 was isolated as a colorless oil, [α]25D –33.8° (c 0.05, CHCl3). HREIMS, 13C NMR,
and DEPT spectra established the molecular formula of 7 as C24H32O6. The IR spectrum of 7 indicated
the presence of the functionalities of ester group(s) (νmax 1730, 1240 cm-1) and α-methylene-γ-lactone
(νmax 1760, 1660 cm-1). The presence of the α-methylene-γ-lactone system in 7 was also demonstrated
by UV absorption at 210 (log ε 4.12) nm and signals at δ 5.57 (H-16a) and 6.27 (H-16b) in the 1H NMR
spectrum. The 1H NMR spectrum of 7 also showed signals for three olefinic protons at δ 5.02 (H-3), 5.09 (H-11), and 5.15 (H-7) ppm; three oxymethine protons either bearing three acetates or in the
γ-lactone group at δ 4.84 (H-2), 5.57 (H-6), and 5.71 (H-10); three olefinic methyl groups at δ 1.69 (H3-19), 1.73 (H3-20), and 1.83 (H3-18); and two methyl groups in acetate esters at δ 2.01 and 2.03.
The 1H−1H COSY spectrum exhibited correlations from H-13 to H-3, H-5 to H-6, H-9 to H-11. 1H-1H long-range correlations were also observed between H-1 to H2-16, H-3 to H3-18, and H-7 to H3-19, and
H-11 to H3-20. These spectroscopic findings and the nine degrees of unsaturations indicated that 7 was
a 14-membered cembrane-type diterpene skeleton with an α-methylene-γ-lactone. After assignments between all the C-H bondings were made based on HSQC experiment, the plane structure was determined by HMBC analysis. The correlations according to HMBC are shown in Figure 4. The stereochemistry for the three trisubstituted olefins of 7 was determined by NOESY analysis. The NOESY correlations between H-3 and H-5, H-7 and H-9, and H-11 and H-13 disclosed the all E
configurations for the three trisubstituted olefins. The chemical shift values at δC 19.9, 16.3, and 15.5
(for C-18, C-19, and C-20 respectively) also supported the all-E configurations.7 The relative configurations at C-1 and C-2 were determined by the coupling constant observed for H-1 and H-2 proton signals and NOESY correlations (Figure 5) between H-1 and H-3 and H-2 and H-13. The relative configurations of the remaining two chiral centers at C-6 and C-10 were deduced from the following NOE analysis. NOESY correlations (Figure 5) between H-2 and H-18, H-18 and H-7 and between H-18 and H-11 indicated that these protons (H-2, H-7, H-11, and H-18) were oriented to the same side, while NOESY correlations between H-1 and H-3, H-1 and H-20 demonstrated that these protons (H-1, H-3, and H-20) were oriented to the opposite face of the molecule. According to the relationships of these protons, the relative configurations at C-6 and C-10 were determined by NOESY correlations (Figure 5) between H-6 and H-3, H-6 and H-19, H-19 and H-10, and H-10 and H-20.
The cytotoxicity of compounds 1−7 is shown in Table 3. Compounds 2 and 4 exhibited potent
cytotoxicity against P-388, HT-29, and A549 cells. Compound 3 showed showed potent cytotoxicty against A549 cells.
