Polyoxygenated Sterols from the Formosan Soft Coral Sinularia gibberosa Atallah F. Ahmed, †,‡ Ya-Ting Hsieh, † Zhi-Hong Wen, † Yang-Chang Wu, § and Jyh-Horng Sheu* ,† Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura UniVersity, Mansoura 35516, Egypt, and Graduate Institute of Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China ReceiVed April 4, 2006 Chemical investigation on the dichloromethane-soluble fraction from the EtOH extract of Sinularia gibberosa Tixier- Durivault has led to the isolation of three new polyoxygenated sterols, gibberoketosterols B (1) and C (2) and gibberoepoxysterol (3), along with two known steroids, gibberoketosterol (4) and 24-methylenecholest-5-en-3-ol (5). These cholestane-type sterols possessing a 22,24(28)-conjugated diene (1 and 2) in the side chain or a 5R,6R-epoxide in the B-ring (3) were isolated for the first time from marine sources. Compound 4 showed significant inhibition against the up-regulation of the pro-inflammatory iNOS and COX-2 proteins of LPS-stimulated RAW264.7 macrophage cells, while 1 was found to be inactive. The cytotoxicity of 1-4 toward a limited panel of cancer cell lines is also reported. Marine organisms, including octocorals (Coelenterata), have been well-recognized as a rich source of 3-hydroxy sterols and their polyoxygenated analogues. 1,2 Worldwide chemical investigations on the steroidal contents of soft corals of the genus Sinularia have afforded various polyoxygenated steroids as derivatives of 24- methyl- and 24-methylenecholestan-3-ol 3-12 and their glyco- sides. 11,12 Some of these compounds have been shown to exhibit in Vitro cytotoxic activities toward several cancer cell lines. 7,8 Our current chemical investigation on S. gibberosa has again led to the isolation of three new polyoxygenated sterols, gibberoketosterols B(1) 13 and C (2) and gibberoepoxysterol (3), along with the known steroids gibberoketosterol (4) 8,13 and 24-methylenecholest-5-en-3- ol (5). 8 The structures of 1-3 were determined utilizing extensive spectroscopic analyses, including 2D NMR ( 1 H- 1 H COSY, HMQC, HMBC, and NOESY) spectroscopy, and by comparison of their NMR data with those of related compounds. The ability of 1 and 4 to inhibit up-regulation of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells has been evaluated. The cytotoxic activity of metabolites 1-4 against HepG2 (human liver carcinoma), MCF7, MDA-MB-23 (human breast carcinoma), and A-549 (human lung carcinoma) cells also is reported herein. Results and Discussion The sliced bodies of the soft coral S. gibberosa were homog- enized exhaustively with EtOH. The concentrated EtOH extract was partitioned between CH 2 Cl 2 and water. The combined CH 2 Cl 2 - soluble fraction was concentrated under reduced pressure, and the residue was repeatedly chromatographed to yield sterols 1-5 (see Experimental Section). The physical and spectral data of 4 were found to be in full agreement with those previously reported for gibberoketosterol isolated from the same organism. 8 Gibberoketosterol B (1) was isolated as a white powder. Its molecular formula was established as C 28 H 44 O 4 by HRESIMS (467.3138 m/z, [M + Na] + ) and NMR data (Tables 1 and 2), implying seven degrees of unsaturation. The UV spectrum of 1 showed an absorption maximum at 232 nm (log ǫ 4.06) due to a conjugated diene moiety in the molecule. The strong absorptions at ν max 3422 and 1714 cm -1 in the IR spectrum suggested the existence of hydroxy and ketone functionalities. Three hydroxyls in the molecule were estimated from the ion peaks appearing at m/z 426 (M - H 2 O) + , 408 (M - 2H 2 O) + , and 390 (M - 3H 2 O) + in the EIMS spectrum. Two of these hydroxyls exhibited the D 2 O- exchangeable proton signals in the 1 H NMR spectrum of 1 (in CDCl 3 , see Table 2, and in C 5 D 6 N, see Experimental Section). The 13 C NMR spectral data of 1 (Table 1), measured in CDCl 3 , indicated the presence of 28 carbon atoms of an oxosteroid attributable to five methyl, eight methylene (including one olefinic), 10 methine (including two olefinic and two oxygenated), and five quaternary carbons (including one olefinic, one oxygenated, and one ketonic). Moreover, the 1 H NMR spectral data of 1 (Table 2), particularly those of the two hydroxyl-bearing methines, one exomethylene, and five methyls, revealed that 1 was a derivative of 4. 8 The only difference was the appearance of the proton signals at δ 5.94 (1H, d, J ) 15.7 Hz) and 5.56 ppm (1H, dd, J ) 15.7, 8.8 Hz) due to a trans 1,2-disubstituted double bond in the side chain of 1. The 1 H- 1 H COSY correlations observed between the olefinic proton H-22 (δ 5.56) and H-20 (δ 2.15, m) and between H-20 and H 3 -21 (δ 1.05, d, J ) 6.6 Hz) indicated the C-22 and C-23 position of the double bond. This was further supported by the 1 H- 1 H COSY correlation between H-22 and H-23 (δ 5.94) and the HMBC correlations found between the exomethylene protons H 2 -28 (δ 4.83 * To whom correspondence should be addressed. Tel: 886-7-5252000, ext. 5030. Fax: 886-7-5255020. E-mail: sheu@mail.nsysu.edu.tw. † National Sun Yat-sen University. ‡ Mansoura University. § Kaohsiung Medical University. 1275 J. Nat. Prod. 2006, 69, 1275-1279 10.1021/np0601509 CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 08/17/2006