Cytotoxic Pregnane Steroids from the Formosan Soft Coral Stereonephthya crystalliana Shang-Kwei Wang, † Chang-Feng Dai, § and Chang-Yih Duh* ,⊥ Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung, Taiwan, Republic of China, Department of Microbiology, Kaohsiung Medical UniVersity, Kaohsiung, Taiwan, Republic of China, and Institute of Oceanography, National Taiwan UniVersity, Taipei, Taiwan, Republic of China ReceiVed October 1, 2005 Nine new steroids, stereonsteroids A-I(1-9), were isolated from the methylene chloride solubles of the Formosan soft coral Stereonephthya crystalliana Ku ¨kenthal. The structures were elucidated by extensive spectroscopic analysis, and their cytotoxicity against selected cancer cells was measured in vitro. The family Nephtheidae has afforded bioactive terpenes and steroids. 1 As part of our search for bioactive substances from marine organisms, the Formosan soft coral Stereonephthya crystalliana Ku ¨kenthal (family Nephtheidae) was studied because the CH 2 Cl 2 extract showed significant cytotoxicity to A549 (human lung adenocarcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cell cultures as determined by standard procedures. 2,3 Bioassay-guided fractionations resulted in the isolation of nine new steroids, stereonsteroids A-I(1-9). Results and Discussion Compound 1 was assigned a molecular formula of C 21 H 34 O 2 as shown by HREIMS, indicating 5 degrees of unsaturation. 13 C NMR and DEPT spectra of 1 exhibited the presence of one methyl, 10 methylene sp 3 C atoms, six methine sp 3 C atoms, one methine sp 2 C atom, two sp 3 quaternary carbons, and one methylene sp 2 C atom, indicating 1 was tetracyclic. The 1 H and 13 C (including DEPT and HSQC) NMR spectra (Tables 1 and 2) implied the presence of a tertiary methyl (δ H 0.64 s; δ C 13.3 q), a terminal vinyl group (δ H 4.97 d, J ) 16.2 Hz, 4.98 d, J ) 10.5 Hz; δ C 139.9 d, 114.5 t), an oxygenated methine (δ H 3.65 m; δ C 71.1 d), and an oxygenated methylene (δ H 3.81 d, J ) 11.4 Hz, 3.95 d, J ) 11.4 Hz; δ C 61.0 t). The foregoing spectral data and a literature survey provided evidence that 1 has a 3-ol pregnane skeleton, 4 with an oxygenated methylene group. This methylene group was assigned to C-19, on the basis of the absence of a methyl singlet (δ 0.80) assignable to the C-19 angular methyl and the presence of an AB doublet at δ 3.81 (J ) 11.4 Hz) and 3.95 (J ) 11.4 Hz). HMBC correlations between H 2 -19 and C-10, C-9, C-1, and C-5 confirmed this assignment. The relative stereochemistry of 1 was established by NOESY experiment. The NOESY correlations observed from H-20 to H 3 -18, from H-14 to H-17/H-9, from H 2 -19 to H-8/H-2, and from H-5 to H-3/H-9/H-1R indicated the relative configurations for each ring junction and chiral center. On the basis of these findings, the structure of 1 was established as pregna-20-diene- 3,19-diol. 4 Compound 2 had a molecular formula of C 23 H 34 O 3 as determined by HREIMS, indicating 6 degrees of unsaturation. The 1 H and 13 C NMR (including DEPT) spectra suggested the presence of a tertiary methyl (δ H 0.53 s; δ C 12.8 q), a terminal vinyl group (δ H 4.96 d, J ) 17.1 Hz, 4.97 d, J ) 10.5 Hz; δ C 139.5 d, 114.8 t), a secondary acetoxyl (δ H 4.72 m, 2.00 s; δ C 72.8 d, 170.8 s), and an aldehyde (δ H 10.03 s; δ C 208.3 s). Comparison of 1 H and 13 C NMR spectra data with those of 1 and a literature survey suggested that 2 has a 3-O-acetoxyl pregnane skeleton, with an aldehyde group. This aldehyde was assigned to C-19, on the basis of the absence of a methyl singlet (δ 0.80) assignable to the C-19 angular methyl. HMBC correlations between H-19 and C-10, C-9, C-1, and C-5 helped ascertain this assignment. The relative stereochemistry of 2 was deduced from a NOESY experiment. Therefore, the structure of 1 can be formulated as pregna-20-dien-3-acetoxyl-19-al. 4 The molecular formula of 3 proved to be C 29 H 46 O 7 from HRFABMS, DEPT, and 13 C NMR data. The seven degrees of unsaturation inherent in the molecular formula of 3 could be accounted for by only one carbon-carbon double bond and one ester carbonyl group. Hence, 3 possessed five rings. The 1 H NMR spectral data of 3 in CDCl 3 were similar to those of 1, except that there were additional signals at δ 4.0-5.8 and at δ 2.15, suggesting the presence of an acetylated sugar moiety in the molecule. The * 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. † Kaohsiung Medical University. § National Taiwan University. ⊥ National Sun Yat-sen University. 103 J. Nat. Prod. 2006, 69, 103-106 10.1021/np050384c CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 01/10/2006 Downloaded by NATIONAL TAIWAN UNIV on November 4, 2009 | http://pubs.acs.org Publication Date (Web): January 10, 2006 | doi: 10.1021/np050384c