New Cytotoxic Constituents from the Formosan Soft Corals Clavularia viridis
and Clavularia violacea
Chang-Yih Duh,*,†Ali Ali H. El-Gamal,†,‡ Chih-Ju Chu,†Shang-Kwei Wang,§and Chang-Feng Dai⊥ Department of Marine Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, Department of Microbiology, Kaohsiung Medical University, Kaohsiung, Taiwan, and Institute of Oceanography, National Taiwan University, Taipei, Taiwan, Republic of China
Received April 19, 2002
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. A cytotoxic cembranoid, claviolide (7), was isolated from the methylene chloride solubles of the Formosan soft coral Clavularia violacea. The structures were elucidated by 1D and 2D NMR spectral analysis, and their cytotoxicity against selected cancer cells was measured in vitro.
The genus Clavularia has afforded many types of bio-active prostanoids, terpenoids, and steroids.1As part of our search for bioactive substances from marine organisms, the Formosan soft corals Clavularia viridis Quoy and Gaimard (class Anthozoa, subclass Octocorallia, order Stolonifera) as well as C. violacea Quoy and Gaimard were studied because their CH2Cl2extracts showed significant cytotox-icity against A549 (human lung adenocarcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lym-phocytic leukemia) cell cultures as determined by standard procedures.2,3Bioassay-guided fractionations resulted in the isolation of three new cytotoxic prostanoids, claviride-none E-G (1-3), and three new cytotoxic steroids, stolonif-erone E-G (4-6), from C. viridis as well as a new cytotoxic cembranoid, claviolide (7), from C. violacea.
Results and Discussion
Compound 1 was shown to have a molecular formula of C23H32O5as indicated by HREIMS and NMR data. The IR spectrum of 1 showed absorption due to acetate ester (1735, 1235 cm-1) and R,β-unsaturated cyclopentenone (1705 cm-1) functionalities. The presence of a cross-conjugated system in 1, corresponding to that of the clavulones,4was demonstrated by UV absorption at 226 (log 3.88) and 290 (log 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 and 169.4 s), eight sp3methylene (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 sp2methines (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), and 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 a carbon-carbon double bond at δ 5.17 and 5.50 m; and a terminal methyl at δ 0.88 (3H, t, J ) 6.9 Hz, H3-20). The analysis of the 1H-1H COSY spectrum (Figure 1) revealed a sequence of
the correlations starting from a doublet at δH6.92 (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 R-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 the1H-1H COSY spectrum starting from two doublets of doublets at δH2.70 (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 δH0.88 (3H, t, J ) 6.9 Hz, H-20). These spectroscopic findings showed 1 to have a structure similar to that of clavulone II,4except for C-4 (CH
2in 1; CHOAc * To whom correspondence should be addressed. Tel: 886-7-525-2000,
ext. 5036. Fax: 886-7-525-5020. E-mail: yihduh@mail.nsysu.edu.tw. †National Sun Yat-sen University.
‡On leave from Faculty of Pharmacy, Mansoura University, Egypt. §Kaohsiung Medical University.
⊥National Taiwan University.
10.1021/np0201873 CCC: $22.00 © 2002 American Chemical Society and American Society of Pharmacognosy Published on Web 09/17/2002
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in clavulone II) on the R-side chain. Assignments between the 1H and13C NMR signals were made on the basis of 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 C23H32O5by HREIMS and NMR data. The1H and13C 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 (δC27.3 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 on the basis of the comparison of1H and 13C NMR data with those of clavulone II.4NOESY correla-tions 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 C23H32O5by HREIMS and NMR data. The1H and13C NMR spectra of 3 were also very similar to those of 1 except for the1H chemical shift values at H-6 (δ
H7.61 in 3; 6.54 in
1) and H-7 (δH6.54 in 3; 6.92 in 1) and the13C chemical shifts at C-7 (δC134.7 in 3; 131.4 in 1). Compound 3 was thus assigned as a 7Z isomer of 1 on the basis of the comparison of1H and13C NMR data with those of clavulone II.4The assignments of the1H and13C NMR signals were confirmed by COSY, HSQC, HMBC, and NOESY experi-ments.
Compound 4 had a molecular formula of C28H44O3 as indicated by HREIMS.13C NMR and DEPT spectra of 4
exhibited the presence of six methyls, six sp3methylenes, nine sp3methines, three sp2methines, two sp3quaternary carbons, and two sp2quaternary carbons. The IR spectrum of 4 showed absorption due to an R,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 3.79) and 280 (log 4.01) nm as well as1H 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)] and13C NMR [δ 118.9 (CH), 126.7 (CH), 140.8 (CH), 157.8 (C)] spectra (Table 2). IR absorption at 3300 cm-1and NMR signals at δH4.58 (1H, br s) and 4.04 (1H, dt, J ) 3.7, 10.5 Hz) as well as at δC73.6 (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 the epoxide. All C-H correlations of 4 were detected in the HSQC experi-ment. The1H-1H COSY spectrum exhibited partial struc-tures 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. On the basis of 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, and H-9 and H-12R indicated the relative configurations for each ring junction and chiral center. Stereochemistry at C-20 and C-24 was determined by comparison of13C 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. 13C and 1H NMR data (Table 2) showed some similarity to 4, except for the presence of two additional hydroxyls and the absence of the trisubstituted double bond. The location of the hy-droxyls on C-2 and C-4 was made on the basis of1H-1H COSY correlations from H-2 to H-3 and 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; and H-19 to C-1, C-5, C-9, C-10. Table 1. 1H and13C NMR Spectral Data of 1-3 in CDCl
3 1 2 3 position 13Ca 1Hb 13Ca 1Hb 13Ca 1Hb 1 173.6 s 173.7 s 174.6 s 2 33.3 t 2.35 (t, 7.5) 33.4 t 2.34 m 33.5 t 2.36 (t, 7.5) 3 23.9 t 1.81 (t, 8.1) 24.5 t 1.63 (ddd, 13.5, 8.0, 7.5) 24.1 t 1.81 (t, 7.2) 32.7 t 2.33 m 27.3 t 2.36 m 32.5 t 2.29 (dd, 14.1, 6.9) 5 146.5 d 6.22 (dt, 15.0, 7.2) 146.5 d 6.04 (dt, 10.8, 8.1) 145.6 d 6.11 (dt, 15.2, 7.2) 6 125.4 d 6.54 (dd, 15.0, 11.7) 123.2 d 6.54 (dd, 12.3, 10.8) 126.7 d 7.61 (dd, 15.2, 11.4) 7 131.4 d 6.92 (d, 11.7) 125.6 d 7.25 (d, 12.6) 134.7 d 6.54 (d, 11.4) 8 134.0 s 135.5 s 133.3 s 9 193.9 s 194.0 s 194.4 s 10 134.8 d 6.41 (d, 6.0) 135.3 d 6.43 (d, 6.0) 136.9 d 6.36 (d, 6.3) 11 157.5 d 7.48 (d, 6.0) 157.8 d 7.48 (d, 6.0) 155.8 d 7.51 (d, 6.3) 85.6 s 85.5 s 85.7 s 13 35.7 t 2.96 (dd, 14.4, 7.2) 35.6 t 2.97 (dd, 14.0, 6.6) 35.6 t 2.88 (dd, 14.5, 7.5) 2.70 (dd, 14.4, 8.1) 2.70 (dd, 14.0, 8.4) 2.65 (dd, 14.5, 7.5) 14 121.3 d 5.17 m 121.3 d 5.17 m 120.6 d 5.23 m 15 135.3 d 5.50 m 134.9 d 5.51 m 135.4 d 5.53 m 16 27.5 t 1.97 (dd, 6.9, 5.1) 27.5 t 1.95 m 27.5 t 1.95 m 17 29.1 t 1.29 m 29.7 t 1.28 m 29.1 t 1.29 m 18 31.6 t 1.30 m 31.6 t 1.26 m 31.6 t 1.27 m 19 22.6 t 1.32 m 22.6 t 1.31 m 22.6 t 1.31 m 20 14.1 q 0.88 (t, 6.9) 14.1 q 0.88 (t, 6.9) 14.1 q 0.88 (t, 6.6) OCH3 51.6 q 3.68 s 51.7 q 3.69 s 51.6 q 3.68 s CH3CO 169.4 s 169.4 s 169.8 s CH3CO 21.4 q 2.04 s 21.4 q 2.04 s 21.4 q 1.99 s
aMultiplicities of resonances were deduced by DEPT experiments.bMultiplicities and J (Hz) values are presented in parentheses.
Figure 1. 1H-1H COSY and key HMBC correlations of 1.
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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, and H-9 and H-12R indicated the relative configurations for each ring junction and chiral center.
The molecular formula of compound 6 was assigned as C28H44O5by HREIMS and NMR data. The1H and13C 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 of13C NMR data with those of stoniferone-a.6
Compound 7 was isolated as a colorless oil, [R]25
D-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 functional-ities of ester group(s) (νmax1730, 1240 cm-1) and R-meth-ylene-γ-lactone (νmax1760, 1660 cm-1). The presence of the
R-methylene-γ-lactone system in 7 was also demonstrated by UV absorption at 210 (log 4.12) nm and signals at δ 5.57 (H-16a) and 6.27 (H-16b) in the 1H NMR spectrum. The1H 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 ac-etates 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 Table 2. 1H and13C NMR Spectral Data of 4-6 in CDCl
3 4 5 6 position 13Ca 1Hb 13Ca 1Hb 13Ca 1Hb 1 212.4 (C) 212.5 (C) 212.8 (C) 2 126.7 (CH) 6.18 (d, 9.6) 78.6 (CH) 7.02 (dd, 8.1, 1.2) 78.5 (CH) 7.01 (dd, 8.0, 1.5) 3 140.8 (CH) 6.99 (dd, 9.6, 6.0) 126.5 (CH) 6.70 (dd, 8.1, 6.3) 126.4 (CH) 6.70 (dd, 8.0, 6.5) 4 118.9 (CH) 6.19 (d, 6.0) 141.8 (CH) 4.63 (dd, 6.3, 1.2) 141.7 (CH) 4.62 (dd, 6.5, 1.5) 5 157.8 (C) 84.0 (C) 83.9 (C) 6 73.6 (CH) 4.58 br s 67.0 (CH) 4.08 m 66.9 (CH) 4.07 m 7 40.3 (CH2) 1.28 m 34.9 (CH2) 1.69 m 34.8 (CH2) 2.01 m 8 29.7 (CH) 2.08 m 27.8 (CH) 1.85 m 27.7 (CH) 9 58.1 (CH) 1.40 m 50.1 (CH) 2.00 (t, 9.9) 49.9 (CH) 2.00 (t, 10.5) 10 55.4 (C) 49.3 (C) 49.2 (C) 11 66.9 (CH) 4.04 m 67.1 (CH) 4.10 m 67.0 (CH) 4.10 m 12 49.5 (CH2) 2.39 (dd, 9.6, 6.0) 48.6 (CH2) 2.37 (dd, 12.6, 4.8) 48.5 (CH2) 2.37 (dd, 12.0, 4.5) 1.23 m 1.31 m 1.30 m 13 42.8 (C) 42.8 (C) 42.7 (C) 14 54.8 (CH) 1.12 m 54.4 (CH) 1.29 m 54.3 (CH) 15 24.5 (CH2) 1.13 m 24.4 (CH2) 1.12 m 24.3 (CH2) 1.57 m 1.63 m 16 28.2 (CH2) 1.28 m 28.2 (CH2) 1.32 m 28.1 (CH2) 1.87 m 1.88 m 17 55.9 (CH) 1.13 m 56.1 (CH) 1.24 m 56.0 (CH) 18 13.0 (CH3) 0.79 s 13.2 (CH3) 0.75 s 13.1 (CH3) 0.75 s 19 18.7 (CH3) 1.71 s 20.2 (CH3) 1.42 s 20.1 (CH3) 1.41 s 20 36.1 (CH) 1.38 m 36.3 (CH) 1.37 m 35.7 (CH) 21 19.9 (CH3) 0.96 (d, 6.3) 18.8 (CH3) 0.97 (d, 6.6) 18.5 (CH3) 0.98 (d, 6.6) 22 33.5 (CH2) 0.95 m 33.6 (CH2) 0.96 m 34.5 (CH2) 1.40 m 1.40 m 23 30.6 (CH2) 1.39 m 30.7 (CH2) 0.97 m 31.0 (CH2) 1.38 m 24 39.0 (CH) 1.20 m 39.1 (CH) 1.22 m 156.6 (C) 25 31.4 (CH) 1.55 m 32.0 (CH) 1.57 m 33.8 (CH) 26 20.5 (CH3) 0.86 (d, 6.6) 20.6 (CH3) 0.86 (d, 6.9) 22.1 (CH3) 1.03 (d, 6.5) 27 17.6 (CH3) 0.78 (d, 6.6) 17.7 (CH3) 0.79 (d, 6.7) 22.0 (CH3) 1.04 (d, 6.5) 28 15.4 (CH3) 0.79 (d, 6.3) 15.5 (CH3) 0.80 (d, 6.7) 106.0 (CH2) 4.66 br s 4.72 br s
aMultiplicities of resonances were deduced by DEPT experiments.bMultiplicities and J (Hz) values are presented in parentheses.
Figure 2. 1H-1H COSY and key HMBC correlations of 4 and 5.
Figure 3. Selective NOESY correlations of 4 and 5.
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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, and H-9 to H-11.1H-1H long-range correlations were also observed from H-1 to H2-16, H-3 to H3-18, 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 R-methylene-γ-lactone. After assignments between all the C-H bondings were made on the basis of HSQC experi-ment, the planar structure was determined by HMBC analysis. The correlations according to HMBC are shown in Figure 4. The stereochemistry for the three trisubsti-tuted 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.7The relative configurations at C-1 and C-2 were determined by the coupling constant observed for the 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 cor-relations (Figure 5) between H-2 and H-18, H-18 and H-7, and H-18 and H-11 indicated that these protons (H-2, H-7, H-11, and H-18) were oriented on the same side, while NOESY correlations between H-1 and H-3 and H-1 and H-20 demonstrated that these protons (H-1, H-3, and H-20)
were oriented on the opposite face of the molecule. Accord-ing 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 excep-tionally potent cytotoxicty against A549 cells.
Experimental Section
General Experimental Procedures. Optical rotations were determined on a JASCO DIP-181 polarimeter. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer, and IR spectra were recorded on a Hitachi 26-30 spectropho-tometer. The NMR spectra were recorded on a Bruker Avance
300 NMR spectrometer at 300 MHz for1H and 75 MHz for
13C, respectively, in CDCl
3using TMS as internal standard.
EIMS spectra were obtained with a JEOL JMS-SX/SX 102A mass spectrometer at 70 eV. Si gel 60 (Merck, 230-400 mesh) was used for column chromatography; precoated Si gel plates
(Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC
analysis.
Animal Material. The soft coral C. viridis was collected at Green Island, off Taiwan, in May 2001, at a depth of 1-2 m and was stored for 1 month in a freezer until extraction. A voucher specimen, NSUGN-052, was deposited in the Depart-ment of Marine Resources, National Sun Yat-sen University, Taiwan.
The soft coral C. violacea was collected at Green Island, off Taiwan, in October 2000, at a depth of 5-6 m and was stored for 1 week in a freezer until extraction. A voucher specimen, NSUGN-033, was deposited in the Department of Marine Resources, National Sun Yat-sen University, Taiwan.
Extraction and Isolation. The bodies of the soft coral C.
viridis were freeze-dried to give 1.60 kg of a solid, which was
extracted with CH2Cl2(4.0 L× 3). After removal of solvent in
vacuo, the residue (70 g) was chromatographed over Si gel 60 using n-hexane and n-hexane-EtOAc mixtures of increasing polarity. Elution by n-hexane-EtOAc (8:2) afforded fractions containing compounds 1-3. Elution by n-hexane-EtOAc (6: 4) afforded fractions containing compounds 4-6. Compounds 1-3 were further purified by Si gel column chromatography, by eluting with n-hexane-acetone (11:1). Compounds 4-6 were further purified by Si gel column chromatography by
eluting with CH2Cl2-EtOAc (7:3) and C18HPLC column by
using MeOH-H2O (85:15) as solvent system.
The bodies of the soft coral C. violacea were freeze-dried to
give 240 g of a solid, which was extracted with CH2Cl2(2.0 L
× 3). After removal of solvent in vacuo, the residue (20 g) was
chromatographed over Si gel 60 using CH2Cl2and CH2Cl2
-acetone mixtures of increasing polarity. Elution by CH2Cl2
afforded a fraction containing compound 7. Compound 7 were further purified by Si gel column chromatography by eluting with n-hexane-EtOAc (1:1).
Claviridenone E (1): oil (25 mg); [R]25
D +8.6° (c 0.30,
CHCl3); UV (MeOH) λmaxnm (log ) 226 (3.88), 290 (4.04); IR
(KBr) νmax1735, 1705, 1235 cm-1;1H and13C NMR, see Table
1; EIMS m/z 388 [M]+(1), 345 (2), 331 (4), 257 (3), 231 (10),
201 (12), 173 (18), 146 (28), 131 (42), 109 (36), 55 (100);
HREIMS m/z 388.2246 (calcd for C23H32O5, 388.2251).
Claviridenone F (2): amorphous solid (17 mg); [R]25
D+6.7°
(c 0.31, CHCl3); IR (KBr) νmax 1734, 1708, 1240 cm-1; UV
(MeOH) λmaxnm (log ) 224 (3.89), 288 (4.06);1H and13C NMR,
see Table 1; EIMS m/z 388 [M]+(1), 345 (2), 287 (2), 235 (8),
203 (9), 147 (15), 129 (26), 103 (26), 55 (100); HREIMS m/z
388.2248 (calcd for C23H32O5, 388.2251).
Claviridenone G (3): oil (7 mg); [R]25
D +5.4° (c 0.10,
CHCl3); UV (MeOH) λmaxnm (log ) 223 (3.89), 286 (4.06); IR
(KBr) νmax1738, 1710, 1230 cm-1;1H and13C NMR, see Table
1; EIMS m/z 388 [M]+(1), 346 (3), 329 (5), 235 (18), 203 (34),
109 (20), 55 (100); HREIMS m/z 388.2244 (calcd for C23H32O5,
388.2251).
Figure 4. 1H-1H COSY and key HMBC correlations of 7.
Figure 5. Selective NOESY correlations of 7. Table 3. Cytotoxicityaof 1-7 cell lines ED50(µg/mL) compound A549 HT-29 P-388 1 4.1× 10-1 1.02 1.1× 10-1 2 5.0× 10-3 5.1× 10-2 5.2× 10-7 3 5.1× 10-2 1.22 2.6× 10-1 4 3.2× 10-4 9.1× 10-3 1.2× 10-4 5 3.69 6.46 2.36 6 3.58 5.86 2.12 7 4.91 8.4× 10-1 3.8× 10-1
aFor significant activity of pure compounds, an ED
50of e4.0
µg/mL is required.
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Stoloniferone E (4): amorphous solid (6 mg); [R]25
D+10.0°
(c 0.05, CHCl3); UV (MeOH) λmax nm (log ) 222 (3.79), 280
(4.01); IR (KBr) νmax3300, 1676 cm-1;1H and13C NMR, see
Table 2; EIMS m/z 428 [M]+(1), 410 (2), 362 (1), 340 (2), 283
(2), 255 (5), 221 (5), 150 (100), 55 (86); HREIMS m/z 428.3299
(calcd for C28H44O3, 428.3292).
Stoloniferone F (5): amorphous solid (4 mg); [R]25
D-30.6°
(c 0.11, CHCl3); UV (MeOH) λmaxnm (log ) 208 (3.68); IR (KBr)
νmax 3460, 1680 cm-1;1H and13C NMR, see Table 2; EIMS
m/z 462 [M]+(1), 270 (1), 256 (1), 221 (1), 192 (3), 176 (5), 154
(45), 137 (100), 107 (66); HREIMS m/z 462.3342 (calcd for C28H46O5, 462.3347).
Stoloniferone G (6): amorphous solid (3 mg); [R]25
D-21.7°
(c 0.12, CHCl3); IR (KBr) νmax3510, 1678 cm-1;1H and 13C
NMR, see Table 2; EIMS m/z 460 [M]+ (1), 268 (1), 254 (1),
220 (1), 190 (4), 175 (6), 152 (40), 137 (100), 107 (76); HREIMS
m/z 460.3186 (calcd for C28H44O5, 460.3190).
Claviolide (7): oil (80 mg); [R]25
D -33.8° (c 0.05, CHCl3);
UV (MeOH) λmaxnm (log ) 210 (4.12); IR (KBr) νmax1760, 1730,
1660, 1240 cm-1;1H NMR δ 1.69 (3H, br s, H-19), 1.73 (3H, br s, H-20), 1.83 (3H, br s, H-18), 1.86 (2H, m, H-14), 2.01 (3H, s, OCOCH3), 2.03 (3H, s, OCOCH3), 2.09 (1H, m, H-13β), 2.16 (1H, m, H-5β), 2.18 (1H, m, H-9β), 2.31 (1H, m, H-13R), 2.40 (1H, m, H-1), 2.60 (1H, m, H-5R), 2.62 (1H, m, H-9R), 4.84 (1H, dd, J ) 4.0, 9.0 Hz, H-2), 5.02 (1H, br d, J ) 9.0 Hz, H-3), 5.09 (1H, br d, J ) 8.5 Hz, H-11), 5.15 (1H, br d, J ) 9.5 Hz, H-7), 5.57 (1H, m, H-6), 5.57 (1H, d, J ) 2.0 Hz, H-16), 5.71 (1H, m, H-10), 6.27 (1H, d, J ) 2.6 Hz, H-16);13C NMR δ 15.5 (q, C-20), 16.3 (q, C-19), 19.9 (q, C-18), 21.3 (q, 2× COCH3), 32.6 (t, C-14), 36.0 (t, C-13), 42.3 (t, C-5), 43.0 (d, C-1), 44.6 (t, C-9), 67.4 (d, C-10), 69.1 (d, C-6), 78.9 (d, C-2), 122.3 (t, C-16), 124.2 (d, C-3), 125.1 (d, C-11), 126.8 (d, C-7), 137.7 (s, C-8), 139.1 (s, C-15), 139.9 (s, C-4), 141.0 (s, C-12), 170.0 (s, C-17), 170.3 (s, 2× COCH3); EIMS m/z 416 [M]+(1), 356 (8), 306 (10), 296 (22), 153 (96), 135 (100); HEIMS m/z 416.2195 (calcd for C24H32O6, 416.2199).
Cytotoxicity Testing. P-388 cells were kindly supplied by Prof. J. M. Pezzuto, Department of Medicinal Chemistry and
Pharmacognosy, University of Illinois at Chicago; A549 and HT-29 were purchased from the American Type Culture Collection. Cytotoxic assays were carried out according to the
procedure described previously.3
Acknowledgment. We thank Prof. J. M. Pezzuto, Depart-ment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, for the provision of P-388 cell lines. This work was supported by grants from the National Science Council of Taiwan awarded to C.-Y.D. References and Notes
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