Anti-Inflammatory Polyoxygenated Steroids from the Soft Coral Sinularia sp. Jui-Hsin Su, 1;2 Ching-Li Lo, 1 Yi Lu, 1 Zhi-Hong Wen, 1 Chiung-Yao Huang, 1 Chang-Feng Dai, 3 and Jyh-Horng Sheu 1;2 1 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan 2 Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan 3 Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan Received June 11, 2008; E-mail: sheu@mail.nsysu.edu.tw Four new polyhydroxylated steroids (1–4) were isolated from a Formosan soft coral Sinularia sp. The structures of these metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analyses. Among these metabolites, 3 and 4 are rarely found marine steroids with a C-9/C-10 double bond. Compounds 1 and 2 have shown significant inhibition of the accumulation of the pro-inflammatory COX-2 protein of LPS-stimulated RAW264.7 macro- phage cells at 10 mM. Previous chemical investigations on the Formosan soft cor- als of the genus Sinularia have afforded several polyoxygenat- ed steroids. 1–5 Recently, we have investigated the chemical constituents of a Taiwanese soft coral Sinularia sp. and have isolated six new sesquiterpenoids. 6,7 Our continuing study on the chemical content of this soft coral also has resulted in the isolation of four new polyhydroxylated steroids (1–4) (Chart 1). The structures of the new metabolites were deter- mined on the basis of extensive spectroscopic analysis, includ- ing 2D NMR ( 1 H– 1 H COSY, HMQC, HMBC, and NOESY) spectroscopy. Furthermore, at a concentration of 10 mM both compounds 1 and 2 demonstrated an ability to inhibit the ac- cumulation of two pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. The soft coral Sinularia sp. was kept at 20  C immediately after collection. The frozen animals were extracted exhaustive- ly with EtOH, and then the concentrated EtOH extract was par- titioned between EtOAc and H 2 O. The combined EtOAc-solu- ble fraction was concentrated under reduced pressure and the residue was repeatedly chromatographed to yield metabolites 1–4. The HR-ESI-MS of 1 revealed a pseudomolecular ion peak at m=z 439.3554 [M + Na] þ , corresponding to the molecular formula C 28 H 48 O 2 (calcd for C 28 H 48 O 2 Na m=z 439.3552). The IR spectrum displayed absorption bands at 3437 and 1655 cm 1 for hydroxy group and carbon–carbon double- bond, respectively. The 1 H NMR spectrum (Table 1) revealed six methyl signals [ 1.04 (s), 0.92 (d, J ¼ 7:0 Hz), 0.86 (d, J ¼ 7:0 Hz), 0.79 (d, J ¼ 7:0 Hz), 0.77 (d, J ¼ 7:0 Hz), and 0.68 (s)], two oxymethine signals [ 3.99 (m) and 3.85 (s)], and an olefinic proton signal at  5.60 (d, J ¼ 5:5 Hz). In the 13 C NMR spectrum (Table 2), compound 1 showed 28 carbon resonances, with multiplicities determined by DEPT 90 and 135 experiments. The olefinic carbon signals appearing at  C HO H H OH 3 1 HO H H 1 3 5 7 9 10 11 13 14 18 19 17 OH 20 21 22 25 26 27 24 28 4 HO H H H OH 2 HO H H H OH Chart 1. 1616 Bull. Chem. Soc. Jpn. Vol. 81, No. 12, 1616–1620 (2008) Ó 2008 The Chemical Society of Japan Published on the web December 12, 2008; doi:10.1246/bcsj.81.1616