National Sun Yat-sen University Institutional Repository:Item 987654321/36936

行政院國家科學委員會專題研究計畫 成果報告

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(3/3)

中 華 民 國 93 年 8 月 20 日

行政院國家科學委員會補助專題研究計畫成果報告

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(3/3)

計畫類別：個別型計畫

計畫編號：

NSC92-2320-B-110-005

執行期間：

92 年 8 月 1 日至 93 年 7 月 31 日

計畫主持人：

杜 昌 益

成果報告類型：完整報告

處理方式：立即公開查詢

執行單位：

國立中山大學 海洋資源學系

中 華 民 國 93 年 8 月 1 日

中文摘要

台灣產膨大羽珊瑚、紫羽珊瑚及笙珊瑚分出之化合物如下：

Tubipolide (A-G) (1-7), tubiporone (8), claviridenone E-G (9−11), stoloniferone E-G (12−14),

及 claviolide (15)。

英文摘要

Eight new sesquiterpenes, tubipolide (A-G) (1-7), tubiporone (8) (novel carbon skeleton), and a known sesquiterpene, spirotubipolide have been isolated from the Formosan stolonifer

Tubipora musica Linnaeus. Three new cytotoxic prostanoids, claviridenone E-G (9−11) and three

new cytotoxic steroids, stoloniferone E-G (12−14) were isolated from the methylene chloride

solubles of the Formosan soft coral Clavularia viridis Quoy and Gaimard. A cytotoxic cembranoid, claviolide (15) was isolated from the methylene chloride solubles of the Formosan soft coral Clavularia 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.

keywords:

tubiporone, claviridenone E-G, stoloniferone E-G, claviolide.

內容

Tubipolide A (1) was shown to have molecular formula C17H20O4 as indicated by HREIMS and NMR data. Its UV absorption at λmax (log ε) 262 nm (3.08) exhibited the presence of a conjugated homoannular diene system, which was further confirmed by 1H NMR [δ 5.66 (H-1), 6.02 (H-2,) 6.00 (H-3)] and 13C NMR (δ 134.4, 123.8, 121.1, 136.7). The 1H NMR and 13C NMR spectra also showed signals due to an acetoxymethyl (δH 2.12, 4.66, δC 20.9, 66.1, 170.8), a methyl on a quaternary carbon (δH 0.98, δC 24.6, 36.5), two methylenes [δH 2.11 (H-6β), 2.85 (H-6α), 2. 47 (H-9β), 1.25 (H-9α), δC 27.1, 45.4].

The presence of a α-methyl-α,β-unsaturated-γ-lactone moiety was indicated by the following spectral data. IR: 1746 cm-1; UV: 226 (4.26) nm; 1H NMR: 1.82 (H-13), 4.66 (H-8), 13C NMR: 8.3 (C-13), 78.4 (C-8), 119.6 (C-11), 160.1 (C-7), 175.0 (C-12). These spectral data closely resembled those of the known α-methyl-α,β-unsaturated-γ-lactone.5 COSY correlations from H-5 to H2-6, H-8 to H2-9, as well as HMBC correlations from H2-6 to C-7 and C-11, from H2-9 to C-10, C-1, and C-14 confirmed the connection of ring A and ring C through C-6 and C-9. The relative stereochemistry of 1 was established by 2D NOESY experiment as shown in Figure 2.

The NOESY correlations between the angular methyl (H3-14) and the H-5 signal indicated the cis A/B ring juction. H-8 was assigned as β based on NOEs between H3-14 and H-9α; H-9β and H-8. Tubipolide B (2) had a molecular fomula of C15H18O2 (HREIMS and NMR). The UV and IR spectra indicated the presence of conjugated homoannular diene and α,β-unsaturated-γ-lactone moieties. The 13C and 1H NMR spectra of 2 were quite similar to those of 1, except for the acetoxymethyl was replaced by a methyl group (δH 1.86, δC 22.4) in 2. HMBC correlations between H3-15 and C-3, C-4 as well as COSY correlation between H-15 and H-3 confirmed this assignment. The relative stereochemistry of 2 was established by 2D NOESY experiment. The NOESY correlations between the angular methyl (H3-14) and the H-5 signal indicated the cis A/B ring junction. H-8 was assigned as β based on NOEs between H-14 and H-9α; H-9β and H-8.

HREIMS and NMR data revealed tubipolide C (3) to have a molecular formula of C17H20O5. The presence of a α-methyl-α,β-unsaturated-γ-hydroxy-γ-lactone moiety was indicated by the following spectral data; IR: 3504, 1747cm-1; UV: 236 (4.28) nm; 1H NMR: 1.79 (H-13), 13C NMR: 8.3 (C-13), 103.2 (C-8), 121.3 (C-11), 158.9 (C-7), 172.5 (C-12). The 13C and 1H NMR spectra of 3 resembled those of 1 except a hydroxy group replaced the proton at C-8 (δC 103.2) in

3. The assignment of hydroxy group at C-8 was confirmed by HMBC correlations between H-9

to C-8, H-6 to C-4, C-5, C-7, C-8, C-10; OH-8 and C-7, C-8, C-9. The relative stereochemistry at C-5 and C-10 of 3 was determined by NOESY correlations between H-5 and H3-14, H-6α, H3-15. The hydroxy group at C-8 was assigned as β based on the chemical shift of H3-14 was not influced by a deshielded effect of the hydroxyl.

Tubipolide D (4) had a molecular formula of C15H18O3 as indicated by HREIMS and NMR data. The presence of a α-methyl-α,β-unsaturated-γ-hydroxy-γ-lactone moiety was indicated by the following spectral data; IR: 3461, 1743 cm-1; UV: 235 (4.29) nm; 1H NMR: 1.85 (H-13), 13C NMR: 8.3 (C-13), 103.0 (C-8), 121.2 (C-11), 159.0 (C-7), 172.0 (C-12). The 13C and 1H NMR spectra of 4 exhibited close similarity to 3 except the acetoxyl methyl group at C-4 was replaced by a methyl (δH 1.87, δC 22.2). HMBC correlation between H3-15 and C-5, C-3, C-4 confirmed this assignment. The relative stereochemistry at C-5 and C-10 of 4 was determined by NOESY correlations between H-5 and H3-14, H-6α.

Tubipolide E (5) showed a higher molecular weight by 16 mass units than 4. The presence of a α-methyl-α,β-unsaturated-γ-hydroxy-γ-lactone moiety was indicated by the following spectral data; IR: 3556, 1746 cm-1; UV: 242 (4.28) nm; 1H NMR: 1.87 (H-13), 13C NMR: 8.3 (C-13), 102.9 (C-8), 122.1 (C-11), 159.7 (C-7), 171.9 (C-12). The 13C and 1H NMR data of 5 were similar to those of 4 except the presence of an enol ether linkage between C-2 (δC 139.9, δH 6.14) and C-3 (δC 139.6, δH 6.24). HMBC correlations from H-2 to C-1, C-3, and C-10; from H-1 to C-2 and C-5; from H3-15 to C-3, C-4, and C-5 confirmed the assignment of a oxepine ring. The

cis A/B ring junction was determined by NOESY correlation between H3-14 and H-5. Tubipolide F (6) had a molecular formula of C15H16O3 as determined by HREIMS. The presence of a α-methyl-α,β-unsaturated-γ-lactone moiety was indicated by the following spectral data; IR: 1739 cm-1; UV: 246 (4.24) nm; 1H NMR: 1.91 (H-13), 13C NMR: 8.5 (C-13), 149.0 (C-8), 120.6 (C-11), 148.0 (C-7), 171.3 (C-12). The structure of 6 was determined by comparing

the 1H NMR spectra with those of 5. Vinylic protons at δ 6.25 (H-3), 6.06 (H-2) and at 4.47 (H-1) indicated the same ring A structure. The lack of the lactonic proton as well as of the isolated methylene protons at C-9 and presence of an olefinic singlet at δ 5.65 (H-9) indicated a double bond at ∆8,9. HMBC correlations between H-9 and C-7, C-5, C-14; H3-14 and C-1, C-5, C-9, C-10; H2-6 and C-7, C-8; H-5 and C-10, C-9 confirmed this assignment.

The molecular formula of tubipolide G (7) was determined as C17H18O4 from HRFABMS. The 13C and 1H NMR data of 7 were analogous to those of 1 except the methylene (C-9) and a lactonic methine (C-8) were replaced by an olefin at ∆8,9 (δH 5.54, δC 112.8, 147.4). HMBC correlations between H2-9 and C-5, C-7, C-8, C-10, C-14; H3-14 and C-5, C-9, C-10; H2-6 and C-4, C-5, C-8, C-10, C-11 confirmed the position of the double bond (∆8,9). The relative stereochemistry at C-5 and C-10 of 7 was determined by NOESY correlations between H3-14 and H-5.

Tubiporone (8), which was obtained as a colorless oil, [α]25D +3.4° (c 0.03, CHCl3). Analysis of HRFABMS revealed a molecular formula of C17H22O6. Its IR spectrum (KBr) suggested the presence of an ester carbonyl (1746 cm–1) and a ketone carbonyl (1724 cm-1) group. The presence of a conjugated homoannular diene system in 1 was indicated by UV absorption at λmax (log ε) 262 nm (3.26) and 1H NMR [δ 5.50 (H-1), 5.89 (H-2), 5.95 (H-3). The 1H and 13C NMR spectra also showed signals due to an acetoxymethyl (δH 2.12, 4.58 , 4.78 , δC 21.0, 66.3, 171.0), a methyl on a quaternary carbon (δH 1.14, δC 25.7, 42.4), a methine (δH 2.58, δC 40.3), two methylenes [δH 1.68 (H-6β), 2.49 (H-6α), 2.56 (H-9β), 2.78 (H-9α); δC 38.2, 49.3], and a ketone [δC 207.7]. The COSY correlation (H-5 to H2-6) and HMBC correlations (H3-14 to C-1, C-5, C-9, and C-10; H-5 to C-3, C-4, C-6, C-7, and C-15; H2-9 to C-1, C-5, C-7, C-8, C-10, and C-14; H2-6 to C-4, C-5, C-7, C-8, and C-11) confirmed the relationships of the protons and carbons on the rings A and B. The presence of a 3-hydroxy-2-propanone side chain was indicated by the following spectral data; 1H NMR δ 2.40 (H-13), 4.55 (H-11), 3.62 (OH-11); 13C NMR 28.0 (C-13), 78.7 (C-11), 206.2 (C-12). HMBC correlations from H-13 to C-11 and C-12; H-11 to C-7 and C-8; H2-6 to C-4, C-5, C-7, C-8, and C-11; H2-9 to C-1, C-5, C-7, C-8, C-10, and C-14; H-5 to C-3, C-4, C-6, C-7, and C-15 confirmed the attachment of the 3-hydroxy-2-propanone side chain at C-7. The relative stereochemistry at C-5, C-7, C-10 was established by NOESY correlations from H-5 to H3-14, H-6α, and H-11; and from H-11 to H-6α, Η−9α, and Η−13.

Compound 9 was shown to have a molecular formula of C23H32O5 as indicated by HREIMS and NMR data. The IR spectrum of 9 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, 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). The 1H NMR spectrum of 9 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 1

H−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 9 to have a structure similar to that of clavulone II, except for C-4 (CH2 in 9; CHOAc in clavulone II) on the α side chain. Assignments between the 1H and 13C NMR signals were made based on HSQC correlations.

The molecular formula of compound 10 was assigned as C23H32O5 by HREIMS and NMR data. The 1H and 13C NMR spectra of 10 were very similar to those of 9 except for the 1H coupling constant between the two olefinic protons at H-5 and H-6 (10.8 Hz in 10; 15.0 Hz in 9) and the 13C chemical shifts at C-4 (δC 27.3 in 10; 32.7 in 9) and C-7 (δC 125.6 in 10; 131.4 in 9). Compound 10 was thus assigned as a 5Z isomer of 9 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 11 was shown to be C23H32O5 by HREIMS and NMR data. The 1H and 13C NMR spectra of 11 were also very similar to those of 9 except for the 1

H chemical shift values at H-6 (δH 7.61 in 11; 6.54 in 9) and H-7 (δH 6.54 in 11; 6.92 in 1) and the 13C chemical shifts at C-7 (δC 134.7 in 11; 131.4 in 9). Compound 11 was thus assigned as a 7Z isomer of 9 based on the comparison of 1H and 13C NMR data with those of clavulone II. The assignments the 1H and 13C NMR signals were confirmed by COSY, HSQC, HMBC, and NOESY experiments.

Compound 12 had a molecular formula of C28H44O3 as indicated by HREIMS. 13C NMR and DEPT spectrum of 12 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 12 showed absorption due to an α,β-unsaturated ketone (1676 cm-1). The presence of a conjugated enone system in 12 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 12 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 12 were detected in the HSQC experiment. The 1H−1H COSY

spectrum exhibited partial structures a, b, and c. In the HMBC spectrum, partial structure a could be connected to b through two quaternary carbons (C-5 and C-10) and H3-19. 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 12 was concluded as in Figure 2. The NOESY correlations 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.

HREIMS and 13C NMR data revealed 13 to have a molecular formula of C28H46O5. 13C and 1H NMR data showed some similarity to 12, 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 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 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 14 was assigned as C28H44O5 by HREIMS and NMR data. The 1H and 13C NMR spectra of 14 were very similar to those of 13 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.

Compound 15 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 15 as C24H32O6. The IR spectrum of 15 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 15 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 15 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 15 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 15 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 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 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 between H-6 and H-3, H-6 and H-19, H-19 and H-10, and H-10 and H-20.

The cis stereochemistry between the angular C-5 and C-10 positions for 1−8 was different

from the trans form of eudesmanolides found in higher plants. Tubipolide B (2), tubipolide G (7), and spirotubipolide exhibited cytotoxicity against P-388 cell line with ED50 of 3.69, 4.01, and 3.24 µg/mL, espectively. Compounds 10 and 12 exhibited potent cytotoxicity against P-388, HT-29, and A549 cells. Compound 11 showed showed potent cytotoxicty against A549 cells.

O H O AcO O H O O H O AcO O H O O H O AcO H O 1 2 3 ₄ 5 7 ₈ OH OH 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 O O H O O O OH O H O O OH H AcO 11 13

O COOMe AcO 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 9 10 OHO OH H H H 12 OHO OH H H _H O H OH 13 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 O _COOMe AcO OHO OH H H H OH O H 14 CH₃ O CH₃ CH₃ AcO AcO O 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 11