Soft corals belonging to genus Sinularia (Alcyoniidae) have been well recognized as a rich source of structurally unique and biologically active metabolites.1) During the course of our screening of bioactive metabolites from marine organisms,2—10) we have reported the isolation of b-caryophyllene-based sesquiterpenoids and diterpenoids (xe-niaphyllanes) from the genus Sinularia.9—11) Some of these metabolites showed selective in vitro cytotoxicity.9) In addi-tion to gibberosins A—H,11)a continuous chemical investiga-tion on the chemical constituents of the soft coral Sinularia
gibberosa TIXIER-DURIVAULT (Alcyoniidae) has again led to the isolation of seven new xeniaphyllanes, gibberosins G—M (1—7). We will describe herein the isolation, structure eluci-dation, and biological activity of these compounds.
The minced bodies of S. gibberosa were extracted exhaus-tively with EtOAc. The combined extract was concentrated under reduced pressure, and the residue was fractionated by open column chromatography on Si gel. The terpenoid-con-taining fractions (recognized by 1H-NMR measurement in CDCl3) were selected for further purification, using a column of the Sephadex LH-20 and the normal phase HPLC to
af-ford terpenoids 1—7. All isolated compounds were obtained as colorless oil.
Gibberosin G (1) was found to possess a molecular for-mula C20H30O4 from the quasimolecular ion peak appearing at m/z 357.2040 [MNa]in the HR-ESI-MS, correspond-ing to six degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxy (3421 cm1) and keto-car-bonyl (1684 cm1) functionalities. The 13C-NMR spectrum of 1 showed signals of twenty carbons (Table 1) among which eleven have very similar chemical shifts to those of the bicyclic structure (C-1 to C-11) of the known b-caryophyllene type metabolites,11)suggesting that 1 could be a b-caryophyllene-related diterpenoid. This could be further confirmed by the very similar 1H-NMR data (Table 2) of 1 with those of these known metabolites. The gross structure of 1 was successively established by the assistance of 1H–1H COSY and HMBC correlations as shown in Fig. 1. There-fore, the C-12 position of the keto-carbonyl in the molecule was interpreted by the HMBC correlations observed from H-13 (d 6.52, d, J15.5 Hz), and H3-18 (d 1.32, s), to C-12 (d 203.2, C). The hydroxy groups were also determined to be located at C-15 and C-16 due to the HMBC correlations ob-served from H-14 (d 6.87, d, J15.5 Hz), H3-17 (d 1.32, s), and H2-16 (d 3.55, dd, J11.0, 11.0 Hz), to C-15 (d 73.7, C). The NOE correlations (Fig. 2) observed by H3-20 with H-3b (d 2.09, m), H-3b with H-2b (d 1.57, m), H-2b with H3-18 (d 1.32, s), H3-18 with H-9 (d 2.72, q, J10.0 Hz), and that displayed by H-5 (d 2.89, dd, J10.0, 3.5 Hz) with H-1, but not with H-9, indicated the 1S*, 4S*, 5S*, 9R*, and 11S* configurations in 1. The double bond between C-13 and C-14 was determined to have a trans geometry based on the cou-pling constant (J15.5 Hz) between 13 (d 6.52, d) and H-14 (d 6.87, d). Further NOE analysis revealed that 1 pos-sessed the same configurations at C-1, C-4, C-5, C-9, and C-11 as those of the known metabolite (Fig. 1).11) Based on the above results, the structure of 1 was established as (1S*,4S*,5S*,9R*,11S*,13E)-15,16-dihydroxy-4,5-epoxy-xeniaphylla-8(19),13-dien-12-one.
Gibberosin H (2) had a molecular formula C22H32O5as es-tablished from its HR-ESI-MS (m/z 399.2148, [MNa]).
Xeniaphyllane-Derived Terpenoids from the Formosan Soft Coral
Sinularia gibberosa
Shin-Pin CHEN,aJui-Hsin SU,aAtallah Fouad AHMED,a,bChang-Feng DAI,cYang-Chang WU,dand
Jyh-Horng SHEU*,a,e
aDepartment of Marine Biotechnology and Resources, National Sun Yat-sen University; eAsian Pacific Ocean Research
Center, National Sun Yat-sen University; Kaohsiung 804, Taiwan, R.O.C.: bDepartment of Pharmacognosy, Faculty of
Pharmacy, Mansoura University; Mansoura 35516, Egypt: cInstitute of Oceanography, National Taiwan University; Taipei
112, Taiwan, R.O.C.: and dGraduate Institute of Natural Products, Kaohsiung Medical University; Kaohsiung 807,
Taiwan, R.O.C. Received May 10, 2007; accepted July 12, 2007
New xeniaphyllane-derived metabolites (1—7) were isolated from the EtOAc extract of the Formosan soft coral Sinularia gibberosa. The structures and relative configurations of these compounds were elucidated on the basis of extensive spectroscopic analysis (including 2D NMR) and by comparison of their spectral data with those of related compounds. In vitro cytotoxic evaluation of the above metabolites towards a limited panel of cancer cell lines is also described.
Key words xeniaphyllane-derived; soft coral; Sinularia gibberosa
© 2007 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: [email protected]
Table 1. 13C-NMR Spectral Data of Compounds 1—7 C # 1a) 2b) 3a) 4b) 5a) 6b) 7a) 1 45.2 (CH)c) 45.3 (CH) 44.9 (CH) 48.5 (CH) 48.4 (CH) 48.6 (CH) 48.6 (CH) 2 28.1 (CH2) 28.2 (CH2) 27.6 (CH2) 30.2 (CH2) 30.1 (CH2) 30.0 (CH2) 29.9 (CH2) 3 38.5 (CH2) 38.6 (CH2) 38.7 (CH2) 39.5 (CH2) 39.5 (CH2) 39.5 (CH2) 39.4 (CH2) 4 59.6 (C) 59.6 (C) 59.6 (C) 135.2 (C) 135.2 (C) 135.1 (C) 135.0 (C) 5 63.6 (CH) 63.6 (CH) 63.9 (CH) 124.7 (CH) 124.6 (CH) 124.8 (CH) 124.7 (CH) 6 30.0 (CH2) 30.0 (CH2) 30.2 (CH2) 28.1 (CH2) 28.1 (CH2) 28.3 (CH2) 28.2 (CH2) 7 29.6 (CH2) 29.7 (CH2) 29.4 (CH2) 34.7 (CH2) 34.6 (CH2) 34.6 (CH2) 34.5 (CH2) 8 150.6 (C) 150.7 (C) 151.3 (C) 153.6 (C) 153.6 (C) 153.5 (C) 153.4 (C) 9 47.3 (CH) 47.3 (CH) 48.0 (CH) 47.2 (CH) 47.1 (CH) 47.2 (CH) 47.1 (CH) 10 35.1 (CH2) 35.2 (CH2) 34.8 (CH2) 35.6 (CH2) 35.5 (CH2) 35.6 (CH2) 35.4 (CH2) 11 47.7 (C) 47.8 (C) 40.2 (C) 47.1 (C) 47.0 (C) 47.8 (C) 47.4 (C) 12 203.2 (C) 203.1 (C) 78.7 (CH) 203.9 (C) 203.9 (C) 213.6 (C) 213.2 (C) 13 123.3 (CH) 123.1 (CH) 27.8 (CH2) 123.7 (CH) 120.4 (CH) 36.6 (CH) 30.6 (CH2) 14 149.9 (CH) 148.8 (CH) 125.9 (CH) 149.6 (CH) 153.1 (CH) 116.5 (CH) 36.8 (CH2) 15 73.7 (C) 72.6 (C) 132.4 (C) 73.8 (C) 71.2 (C) 134.9 (C) 207.4 (C) 16 69.2 (CH2) 70.5 (CH2) 62.9 (CH2) 69.4 (CH2) 29.5 (CH3) 18.2 (CH3) 17 23.9 (CH3) 24.7 (CH3) 21.5 (CH3) 23.9 (CH3) 29.5 (CH3) 25.8 (CH3) 30.0 (CH3) 18 17.0 (CH3) 17.1 (CH3) 15.7 (CH3) 17.9 (CH3) 17.9 (CH3) 17.8 (CH3) 17.8 (CH3) 19 114.1 (CH2) 114.1 (CH2) 113.6 (CH2) 112.9 (CH2) 112.8 (CH2) 113.0 (CH2) 112.9 (CH2) 20 16.8 (CH3) 16.9 (CH3) 17.1 (CH3) 16.4 (CH3) 16.3 (CH3) 16.4 (CH3) 16.3 (CH3) OAc 171.1 (C) 170.6 (C) 20.9 (CH3) 20.9 (CH3) OAc 171.0 (C) 21.0 (CH3)
a) Spectra recorded at 125 MHz in CDCl3. b) 75 MHz in CDCl3at 25 °C. c) Deduced by distortionless enhancement by polarization transfer (DEPT).
Table 2. 1H-NMR Spectral Data of Compounds 1—7
H # 1a) 2b) 3a) 4b) 5a) 6b) 7a) 1 2.47 dd 2.47 dd 1.96 dd 2.32 dd 2.32 dd 2.32 dd 2.32 dd (10.0, 10.0)c) (9.6, 9.6) (10.0, 10.0) (9.6, 9.6) (10.0, 10.0) (9.6, 9.6) (9.5, 9.5) 2 a 1.87 m 1.87 m 1.63 m 1.74 m 1.74 m 1.69 m 1.69 m b 1.57 m 1.57 m 1.45 m 1.63 m 1.62 m 1.57 m 1.59 m 3 a 1.10 m 1.09 m 0.92 ddd 2.03 m 2.03 m 2.02 m 2.03 m (13.0, 13.0, 5.0) b 2.09 m 2.10 m 2.08 m 2.13 m 2.12 m 2.08 m 2.10 m 5 2.89 dd 2.86 dd 2.82 dd 5.31 m 5.33 dd 5.31 m 5.35 dd (10.0, 3.5) (10.5, 3.6) (10.0, 3.5) (9.5, 9.0) (9.5, 9.5) 6 a 2.24 m 2.23 m 2.25 m 2.00 m 2.00 m 1.97 m 2.00 m b 1.34 m 1.34 m 1.33 m 2.35 m 2.35 m 2.35 m 2.35 m 7 a 2.28 m 2.28 m 2.31 m 2.18 m 2.17 m 2.16 m 2.17 m b 2.12 m 2.12 m 2.16 m 1.99 m 2.00 m 1.99 m 2.02 m 9 2.72 q 2.70 q 2.63 q 2.40 q 2.40 q 2.37 q 2.38 q (10.0) (9.6) (9.0) (9.0) (9.0) (9.0) (10.5) 10 a 2.16 m 2.16 m 1.59 m 2.22 m 2.22 m 2.26 m 2.27 t (10.5) b 1.86 m 1.87 m 1.86 t 1.79 m 1.81 m 1.82 m 1.82 m (10.0) 12 4.75 dd (9.5, 4.0) 13 6.52 d 6.50 d 2.19 m 6.88 d 6.97 d 3.12 m 2.56 m (15.5) (15.3) (15.6) (15.0) 14 6.87 d 6.87 d 5.31 dd 6.56 d 6.43 d 5.31 m 2.71 m (15.5) (15.3) (7.5, 7.0) (15.6) (15.0) 16 3.55 dd 4.07 dd 4.49 d (12.0) 3.56 dd 1.39 s 1.62 s (11.0, 11.0) (9.0, 9.0) 4.61 d (12.0) (8.1, 8.1) 17 1.32 s 1.34 s 1.73 s 1.32 s 1.39 s 1.75 s 2.21 s 18 1.32 s 1.30 s 1.09 s 1.30 s 1.28 s 1.27 s 1.29 s 19 4.91 s 4.90 s 4.88 s 4.86 s 4.86 s 4.85 s 4.86 s 5.00 s 4.99 s 5.00 s 4.93 s 4.93 s 4.94 s 4.96 s 20 1.21 s 1.20 s 1.18 s 1.63 s 1.63 s 1.62 s 1.62 s OAc 2.08 s 2.05 s OAc 2.07 s
The IR spectrum indicated the presence of hydroxy (3443 cm1), ester carbonyl (1734 cm1), and keto-carbonyl (1685 cm1) moieties. It was found that the 1H- and 13 C-NMR spectral data of 2 (Tables 1, 2) were very similar to those of 1, however, the NMR chemical shifts for H2-16 and C-16 of 2 (d 4.07, 2H, dd, J9.0, 9.0 Hz; d 70.5) were found to be shifted to a lower field, in comparison with the analo-gous data of 1 (d 3.55, 2H, dd, J11.0, 11.0 Hz; d 69.2), suggesting that the hydroxy group of 1 was replaced by an acetoxy group in 2. The above observations revealed that 2 is simply the 16-O-acetyl derivative of 1.
Gibberosin I (3) exhibited a quasimolecular ion peak in the HR-ESI-MS at m/z 427.2461 [MNa] and showed NMR spectroscopic data (Tables 1, 2) consistent with a mo-lecular formula C24H36O5. Comparison of the
1
H- and 13 C-NMR data of compound 3 with those of 1 and 2 revealed that
3 has the same ring structure but with a different side chain.
The HMBC correlations observed from H3-18 to C-1, C-10, C-11, and an oxymethine carbon at d 78.7, assigned the latter oxygenated carbon to be C-12. This finding and the upfield chemical shift of C-11 (d 40.2), relative to those of metabo-lites 1 (d 47.7) and 2 (d 47.8), indicated the attachment of one acetoxy group at C-12. Furthermore, the protons of the oxymethylene group in 3 (d 4.49 and 4.61, each d,
J12.0 Hz) were found to exhibit HMBC correlations with
the carbonyl carbon of the other acetoxy group and an olefinic carbon (d 132.4, C, C-15), while the latter carbon was found to be correlated with the proton of an olefinic methyl (d 1.73, 3H, s, H3-17). Therefore, the trisubstituted double bond was positioned between C-14 and C-15 where an acetoxymethyl and a methyl should be located at C-15. The 1H–1H COSY correlations found from H-12 (d 4.75, dd, J9.5, 4.0 Hz) to H2-13 (d 2.19 m) and from H2-13 to the olefinic proton at d 5.31 (dd, J7.5, 7.0 Hz, H-14) further supported the C-14/C-15 position of the double bond. This
double bond was determined to have a cis geometry on the basis of the NOE interaction found between H-14 and H3-17. Furthermore, the analysis of the NOESY spectrum of 3 re-vealed the same relative configurations at C-1, C-4, C-5, C-9, and C-11 as in 1 and 2. Therefore, compound 3 was estab-lished as (1S*,4S*,5S*,9R*,11S*,14Z)-12,16-diacetoxy-4,5-epoxyxeniaphylla-8(19),14-dien.
Gibberosin J (4) exhibited a quasimolecular ion peak at
m/z 341.2092 [MNa]in the HR-ESI-MS, appropriate for a molecular formula C20H30O3 and six degrees of unsatura-tion. The 13C-NMR spectrum of 4 also revealed the presence of 20 carbon signals, characteristic for a xeniaphyllane-de-rived terpenoids (Table 1). Through extensive NMR experi-ments (1H-, 13C-NMR, COSY, HMQC, and HMBC), the structure of 4 was found to be close to that of 1 except that a trisubstituted epoxide at C-4/C-5 (dH2.89, 1H, dd, J10.0, 3.5 Hz, H-5; dC59.6, C, C-4; dC63.6, CH, C-5) in 1 was re-placed by a trisubstituted double bond (dH5.31, 1H, m, H-5; dC135.2, C, C-4; dC124.7, CH, C-5) in 4. The molecular framework of 4 was further established by the 1H–1H COSY and HMBC correlations as illustrated in Fig. 1. The relative configurations at C-1, C-9, and C-11 in 4 were found to be the same as those of 1—3 on the basis of the NOE cor-relations (Fig. 2). The E geometry of the 4,5-endocyclic double bond in 4 was indicated by the lack of NOE cor-relation between the olefinic methyl protons (d 1.63, s) attached at C-4 and H-5 (d 5.31, m) and the upfield shift of C-20 (d 20 ppm). Compound 4 was thus identi-fied as (1S*,9R*,11S*,4E,13E)-15,16-dihydroxyxeniaphylla-4,8(19),13-trien-12-one.
On the basis of its HR-ESI-MS (m/z 325.2146, [M Na]), the molecular formula of gibberosin K (5) was es-tablished as C20H30O2. The IR spectrum indicated the pres-ence of hydroxy (3566 cm1) and carbonyl (1684 cm1) func-tionalities. The 1H- and 13C-NMR data were very similar to those of compound 4. By comparison of the NMR spectral data of compound 4 with those of compound 5, it was found that a hydroxy-bearing methylene (dH 3.56, dd, J8.1, 8.1 Hz; dC69.4) at C-15 in 4 was replaced by a methyl in 5. Compound 5 was thus identified as (1S*,9R*,11S*,4E,13E)-15-hydroxyxeniaphylla-4,8(19),13-trien-12-one.
Gibberosin L (6) was found to have a molecular formula
Fig. 1. Selective 1H–1H COSY and HMBC Correlations of 1, 4, 6, and 7
C20H30O from the HR-ESI-MS (m/z 309.2192 [MNa]) and showed an IR absorption band of carbonyl (1717 cm1) functionality. The 13C-NMR data (Table 1) of 6 were found to be very similar to those of compound 5 from C-1 to C-11 and C-18 to C-20. The side-chain structure was elucidated by the 1H–1H COSY and HMBC correlations of 6 as shown in Fig. 1. Moreover, the NOE correlations for protons of the bi-cycle moiety were found to be the same as those of 5 and thus led to the determination of the 1S*, 9R*, and 11S* con-figurations of this compound. Also, the lack of NOE correla-tion between the H3-20 and H-5 and the upfield shift of C-20 (d 16.4) confirmed the E geometry of the 4,5-endocyclic double bond. The structure of 6 was then established as (1S*,9R*,11S*,4E)-xeniaphylla-4,8(19),14-trien-12-one.
A molecular formula C19H28O2for gibberosin M (7) was established by the HR-ESI-MS (m/z 311.1986 [MNa]) and NMR data (Tables 1, 2). The 13C-NMR spectrum exhib-ited nineteen carbon signals, including those of three methyls (d 30.0, 17.8, 16.3, each CH3), two keto-carbonyls (d 213.2, 207.4, each C), one exomethylene (d 153.4, C, and 112.9, CH2), one trisubstituted double bond (d 135.0, C, and 124.7, CH), and two ring-junctured methines (d 48.6, 47.1, CH) which identified compound 7 as a norxeniaphyllane. The 13 C-NMR spectroscopic data of compound 7 were found to be nearly identical to those of compounds 5 and 6 from C-1 to C-12 and C-18 to C-20, revealing the same bicyclic structure of 7 as those of 5 and 6. However, one methyl displayed a 3H singlet at d 2.21 in the 1
H-NMR spectrum and showed an HMBC correlation to one of the keto-carbonyl carbons (d 207.4, C), indicating the presence of a terminal acetyl group in the side chain of 7 instead of two methyls at C-15 in 5 and
6. This was further supported by the 1H–1H COSY and HMBC correlations as illustrated in Fig. 1. These findings to-gether with the analysis of NOE correlations for 7 deter-mined gibberosin M to be (1S*,9R*,11S*,4E)-16-norxenia-phylla-4,8(19)-dien-12,15-dione.
Cytotoxicity of metabolites 1—7 toward a limited panel of cancer cell lines was evaluated. Compounds 5 and 6 have been shown to exhibit moderate activity against A-549 (human lung carcinoma), Hep G2 (human hepatocellular car-cinoma), and MDA-MB-231 (human breast carcinoma) cell lines with IC50’s of 7.1, 12.4, and 5.5mg/ml, IC50’s of 11.3, 14.5, and 7.7mg/ml, respectively. Other metabolites were found to be inactive against the growth of the above three cancer cell lines (20.0 mg/ml).
Experimental
Optical rotations were measured on a Jasco DIP-1000 digital polarimeter. IR spectra were recorded on a Jasco FT-5300 infrared spectrophotometer. NMR spectra were recorded on a Bruker AVANCE DPX300 FT-NMR at 300 MHz for 1H and 75 MHz for 13C or on a Varian Unity INOVA 500
FT-NMR at 500 MHz for 1H and 125 MHz for 13C, in CDCl
3using TMS as an
internal standard. Low-resolution mass data and HR-MS data were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230—400 mesh) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography (HPLC) was performed on a Hitachi L-7100 apparatus equipped with a Bischoff refractive index detector and the Merck Hibar Si-60 column (250 mm21 mm, 7 mm).
Animal Material The soft coral Sinularia gibberosa was collected by hand using scuba equipment off the coast of northeastern Taiwan in May 2004, at a depth of 15—20 m, and was stored in a freezer until extraction. A voucher specimen was deposited in the Department of Marine
Biotechnol-ogy and Resources, National Sun Yat-sen University (voucher no. SC-20040621-5).
Extraction and Isolation The bodies of S. gibberosa (1.3 kg fresh weight) were minced and extracted exhaustively with EtOAc, and the extract was concentrated under reduced pressure to give a dark brown viscous residue (15.4 g). The residue was fractionated by open column chromatogra-phy on silica gel using an n-hexane and n-hexane–EtOAc mixture of increas-ing polarity to yield 32 fractions. Fraction 6, eluted with n-hexane/EtOAc (8 : 1), was subjected to Sephadex LH-20 column using acetone and fol-lowed by normal phase HPLC eluted with n-hexane/acetone (7 : 1), to afford compounds 3 (2.1 mg), and 6 (14.6 mg). Fraction 15, eluted with hexane/EtOAc (5 : 1), was further purified by silica gel column using n-hexane/EtOAc (5 : 1) as eluent to afford compound 7 (5.8 mg). Fraction 20, eluted with n-hexane/EtOAc (3 : 1), was further purified by normal phase HPLC using n-hexane/acetone (5 : 1 to 3 : 1) to give compound 5 (3.8 mg). Fraction 22, eluted with n-hexane/EtOAc (1 : 1), was further purified by nor-mal phase HPLC using n-hexane/acetone (2 : 1), to give compounds 1 (3.0 mg), 2 (1.2 mg), and 4 (2.4 mg).
Gibberosin G (1): Colorless oil; [a]D
2524.6° (c0.3, CHCl
3); IR (neat)
nmax 3421, 2924, 1684 cm1; 1H- and 13C-NMR data, see Tables 1 and 2;
ESI-MS m/z 357 ([MNa]); HR-ESI-MS m/z 357.2040 [MNa](Calcd for C20H30O4Na, 357.2042).
Gibberosin H (2): Colorless oil; [a]D
2520.0° (c0.24, CHCl
3); IR (neat)
nmax3443, 2966, 1734, 1685 cm1; 1H- and 13C-NMR data, see Tables 1 and
2; ESI-MS m/z 399 ([MNa]); HR-ESI-MS m/z 399.2148 [MNa] (Calcd for C22H32O5Na, 399.2147).
Gibberosin I (3): Colorless oil; [a]D
2575.0° (c0.3, CHCl
3); IR (neat)
nmax2926, 1734 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESI-MS
m/z 427 ([MNa]); HR-ESI-MS m/z 427.2461 [MNa] (Calcd for C24H36O5Na, 427.2460).
Gibberosin J (4): Colorless oil; [a]D
2535.0° (c0.16, CHCl
3); IR (neat)
nmax 3420, 2929, 1684 cm1; 1H- and 13C-NMR data, see Tables 1 and 2;
ESI-MS m/z 341 ([MNa]); HR-ESI-MS m/z 341.2092 [MNa](Calcd for C20H30O3Na, 341.2093).
Gibberosin K (5): Colorless oil; [a]D
2520.0° (c0.6, CHCl
3); IR (neat)
nmax 3566, 2926, 1684 cm1; 1H- and 13C-NMR data, see Tables 1 and 2;
ESI-MS m/z 325 ([MNa]); HR-ESI-MS m/z 325.2146 [MNa](Calcd for C20H30O2Na, 325.2143).
Gibberosin L (6): Colorless oil; [a]D
2514.4° (c1.5, CHCl
3); IR (neat)
nmax2934, 1717 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESI-MS
m/z 309 ([MNa]); HR-ESI-MS m/z 309.2192 [MNa] (Calcd for C20H30ONa, 309.2194).
Gibberosin M (7): Colorless oil; [a]D
2513.8° (c0.6, CHCl
3); IR (neat)
nmax2945, 1717 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESI-MS
m/z 311 ([MNa]); HR-ESI-MS m/z 311.1986 [MNa] (Calcd for C19H28O2Na, 311.1987).
Cytotoxicity Testing Compounds were assayed for cytotoxicity against A-549, Hep G2, and MDA-MB-231 cancer cells using the MTT [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method.12,13) Freshly
trypsinized cell suspensions were seeded into a 96-well microtiter plate at densities of 5000—10000 cells per well and then the test compounds were added from DMSO-diluted stock solutions. After 3 d in culture, attached cells were incubated with MTT (0.5 mg/ml, 1 h) and subsequently dissolved in DMSO. The absorbency at 550 nm was then measured using a microplate reader. The IC50is the concentration of agent that reduced cell growth by
50% under the experimental conditions.
Acknowledgments Financial support was provided by the Ministry of Education (C030313) and the National Science Council of Taiwan (NSC 95-2113-M-110-011-MY3) awarded to J.-H. Sheu.
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