A Cytotoxic Lobane Diterpene from the Formosan Soft Coral Sinularia
inelegans
Min-Chi Chai,†Shang-Kwei Wang,‡Chang-Feng Dai,§and Chang-Yih Duh*,†
Department of Marine Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, Department of Microbiology, Kaohsiung Medical College, Kaohsiung, Taiwan, and Institute of Oceanography, National Taiwan University, Taipei, Taiwan, Republic of China
Received October 29, 1999
A new cytotoxic lobane diterpene, ineleganene (1), was isolated from the Formosan soft coral Sinularia inelegans. The structure of compound 1 was determined by 1D and 2D spectral analysis.
In the search for bioactive substances from marine organisms, the soft coral Sinularia inelegans Tixier-Durivault (family Alcyoniidae) was studied because the hexane extracts showed significant cytotoxicity in A549 (human lung adenocarcinoma) and P-388 (mouse lympho-cytic leukemia) cell cultures as determined by standard procedures.1,2Bioassay-guided fractionations resulted in the isolation of a new cytotoxic lobane diterpene, inele-ganene (1).
The hexane-soluble fraction of S. inelegans was chro-matographed over Si gel to give a colorless oil, [R]25
D+9.6° (c 0.1, CHCl3). HRFABMS and the DEPT spectrum of 1 established the molecular formula to be C20H32. Thus, 5 degrees of unsaturation were determined for 1.1H and13C NMR spectral data (Table 1) showed that the structure of 1 contained four olefin groups, including a vinyl (δC150.4, d, C-8; 109.8, t, C-9; δH 5.82, dd, J ) 17.7, 10.2 Hz, H-8; 4.88 d, J ) 10.2 Hz, 4.91 d, J ) 17.7 Hz, H2-9), an isopropenyl group (δC 147.8, s, C-10; 112.1, t, C-11, 24.7, q, C-12; δH4.59, br s, 4.82, br s, H2-11; 1.71, s, H3-12), a methyl-bearing trisubstituted double bond (δC 131.6, s, C-13; 124.3, d, C-15; 25.6, q, C-14; δH5.13, m, H-15; 1.69, s, H3-14), and an isopentenyl group (δC107.1, t, C-19; 154.5, s, C-18; 17.7, q, C-20; 34.9, t, C-17; 27.3, t, C-16; δH4.74 br s, 4.79 br s, H2-19; 1.62, s, H3-20; 2.13, m, H2-16; 2.08, m, H2-17). These facts, in combination with the molecular formula, suggested the occurrence of one ring. The presence of the vinyl and isopropenyl groups together with a tertiary methyl group (δC16.6, q, C-7; δH1.01, s, H3-7) is reminis-cent of a 3-isopropenyl-4-methyl-4-vinylcyclohexane-1-yl moiety that is reported in lobane-type diterpenoids,3-5and this partial structure was confirmed by COSY, HMQC, and HMBC spectra. Further, the trisubstituted olefin and the isopentenyl groups were connected by HMBC and NOESY
correlations to form a 1,5-dimethyl-1(E),5-hexadiene-1-yl side chain, which was attached at the R (equatorial) position of C-4. Compound 1 exhibited cytotoxicity against A549 and P-388 cell lines with GI50values of 3.63 and 0.20 µg/mL, respectively.
Experimental Section
General Experimental Procedures. Optical rotation was determined on a JASCO DIP-181 polarimeter. The IR spectrum was recorded on a Hitachi 26-30 spectrophotom-eter.1H and13C NMR spectra were recorded with a Bruker AMX 400 NMR spectrometer at 400 and 100.6 MHz, respectively, in CDCl3using TMS as internal standard. The HMBC experiment was obtained using HMBC (optimized fornJ
C-H) 8 Hz) pulse sequences with a pulse gradient. 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.
* To whom correspondence should be addressed. Tel.: 886-7-525-2000, ext. 5036. Fax: 886-7-525-5020.
†National Sun Yat-sen University. ‡Kaohsiung Medical College. §National Taiwan University.
Table 1. NMR Data of 1a
position δH; mult. Jb δC; mult.c HMBC COSY NOESY
1 40.4; s 2 2.01; dd; 12.1, 5.0 52.9; d 3, 6, 7, 10, 11, 12 11 3R 1.51; m 33.4; t 4, 5, 13 4 7 β 1.58; m 4 1.92; m 44.5; d 6, 14 3 5 2.14; m 26.8; t 6 6 1.46; m 39.8; t 1, 2, 4, 7 5 8 7 1.01; s 16.6; q 2, 6, 8 3R, 9, 11, 12, 14 8 5.82; dd; 17.7,10.2 150.4; d 6, 7 9 6, 9 9 4.88; d, 10.2 109.8; t 1, 8 8 7, 8 4.91; d; 17.7 10 147.8; s 11 4.59; br s 112.1; t 2, 12 12 2, 3, 7 4.82; br s 12 1.71; s 24.7; q 2, 10, 11 11 7 13 131.6; s 14 1.69; s 25.6; q 13, 15 3R, 15 15 5.13; m 124.3; d 13 16 14, 16 16 2.13; m 27.3; t 15 15, 17 3R, 15 17 2.08; m 34.9; t 16, 18, 19, 20 16 19 18 154.5; s 19 4.74; br s 107.1; t 17, 18 17 4.79; br s 20 1.62; s 17.7; q 18 aSpectra recorded in CDCl 3. bJ values in Hz. cMultiplicity
deduced by DEPT and indicated by usual symbols.
843 J. Nat. Prod. 2000, 63, 843-844
10.1021/np990539e CCC: $19.00 © 2000 American Chemical Society and American Society of Pharmacognosy Published on Web 04/29/2000
Downloaded by NATIONAL TAIWAN UNIV on November 2, 2009 | http://pubs.acs.org
Animal Material. The soft coral S. inelegans was collected at Green Island off Taiwan in September 1997, at a depth of 12 m and was stored for 1 day in a freezer until extraction. A voucher specimen NSUGN-1024 (identi-fied by Prof. Chang-Feng Dai) was deposited in the Depart-ment of Marine Resources, National Sun Yat-sen Univer-sity, Taiwan.
Extraction and Isolation. S. inelegans specimens (wet wt 2.1 kg) were freeze-dried to give 980 g of a solid, which was extracted with CH2Cl2 (2 L × 3). After removal of solvent in vacuo, the residue (40 g) was partitioned between CH2Cl2and H2O. The CH2Cl2-soluble fraction was further partitioned between n-hexane and 10% aqueous MeOH. The n-hexane-soluble fraction was chromatographed over Si gel 60 using n-hexane and n-hexanes-EtOAc mixtures (the final ratio, 1:3) of increasing polarity. Elution by n-hexane afforded fractions containing 1. Compound 1 was finally purified by Sephadex LH-20 chromatography using n-heptane as eluting solvent.
Ineleganene (1): colorless oil (7 mg); [R]25
D+9.6° (c 0.10, CHCl3); IR (KBr) νmax3090, 2925, 1637 cm-1;1H and 13C NMR, see Table 1; EIMS m/z 272 [M]+(0.3), 258 (0.3), 244 (0.2), 230 (1), 216 (0.4), 147 (11), 133 (15), 121 (19), 41 (100); HREIMS m/z 272.2496 (calcd for C20H32, 272.2498).
Cytotoxicity Testing. P-388 cells were supplied by Prof. J. M. Pezzuto, University of Illinois at Chicago; A549 and HT-29 were purchased from the American Type Culture Collection. Cytotoxicity asssays were carried out according to the procedure described previously.6
Acknowledgment. We thank Prof. J. M. Pezzuto, Program
for Collaborative Research in Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, for the provision of P-388 line. This work was supported by a grant from the National Science Council of Taiwan (NSC-89-2314-B-110-002) awarded to C.-Y.D.
References and Notes
(1) Geran, R. I.; Greenberg, N. H.; MacDonald, M. M.; A. M. Schumacher, A. M.; Abbott, B. J. Cancer Chemother. Rep. 1972, 3, 1-91. (2) Hou, R.-S.; Duh, C.-Y.; Chiang, M. Y.; Lin, C.-N. J. Nat. Prod. 1995,
58, 1126-1130.
(3) Edrada, R. A.; Proksh, P.; Wray, V.; Witte, L.; van Ofwegen, L. J. Nat. Prod. 1998, 61, 358-361.
(4) Raju, B. L.; Subbaraju, G. V.; Rao, C. B.; Trimurtulu, G. J. Nat. Prod. 1993, 56, 961-966.
(5) Dunlop, W. W.; Wells, R. J. J. Aust. J. Chem. 1979, 32, 1345-1351. (6) Duh, C.-Y.; Wang, S.-K.; Weng, Y.-L.; Chiang, M. Y.; Dai, C.-F. J.
Nat. Prod. 1999, 62, 1518-1521. NP990539E
844 Journal of Natural Products, 2000, Vol. 63, No. 6 Notes
Downloaded by NATIONAL TAIWAN UNIV on November 2, 2009 | http://pubs.acs.org
