Crassocolides A-F, Cembranoids with a trans-Fused Lactone from the Soft Coral Sarcophyton crassocaule Ho-Cheng Huang, †,‡ Atallah F. Ahmed, †,§ Jui-Hsin Su, † Chih-Hua Chao, † Yang-Chang Wu, | Michael Y. Chiang, ⊥ and Jyh-Horng Sheu* ,† Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China, Department of Chemical and Materials Engineering, Cheng Shiu UniVersity, Kaohsiung 833, Taiwan, Republic of China, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura UniVersity, Mansoura 35516, Egypt, Graduate Institute of Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, and Department of Chemistry, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China ReceiVed April 25, 2006 Six new polyoxygenated cembrane-based diterpenoids possessing a trans-fused R-methylene-γ-lactone, crassocolides A-F(1-6), have been isolated along with the known compound 7 from the ethyl acetate extract of a Taiwanese soft coral, Sarcophyton crassocaule. The structures of 1-6 have been established by detailed spectroscopic analysis, including 2D NMR ( 1 H- 1 H COSY, HMQC, HMBC, and NOESY) spectroscopy, while their absolute configurations were determined using a modified Mosher’s method for 1. The structure of 5 was further proven by X-ray diffraction analysis. The full assignment of NMR data of 7 is reported herein for the first time. The cytotoxicity of crassocolides 1-4, 6, and 7 against a limited panel of cancer cells was also determined. Marine terpenoids are of considerable interest due to their unique structures and wide range of biological activities. 1 The macrocyclic cembrane-type compounds have been found to be the main terpenoidal constituents in marine coelenterates. 1 Cembranoids possessing a γ-lactone 2-17 have been mostly isolated from octoc- orals (Alcyonaceae) belonging to the genera Sinularia, 2-5 Eunicea, 6-12 and Lobophytum. 13-17 Some of these metabolites have been shown to exhibit cytotoxic activity against the growth of various cancer cell lines. 2,6-8,14,15 In our continuing search for bioactive metabolites from soft corals of Taiwanese waters, 18-22 we have chemically investigated Sarcophyton crassocaule Moser and have succeeded in the isolation of six new polyoxygenated trans-fused cembrano- lides, crassocolides A-F(1-6), along with the known metabolite 7 23 from the EtOAc extract of the organism. The structures of 1-6 have been established by extensive spectroscopic analysis, including 2D NMR ( 1 H- 1 H COSY, HMQC, HMBC, and NOESY) spec- troscopy. Their absolute configurations were confirmed on the basis of the absolute structure of 1, determined by a modified Mosher’s method, and the absolute structure of 7, determined previously by X-ray crystallography. 24 The relative structure of 5 was further proven by X-ray diffraction analysis. The full 1 H and 13 C NMR data of 7 are reported herein for the first time. Cytotoxicity of metabolites 1-4, 6, and 7 against Hep G2 (human liver carcinoma), MCF-7, MDA-MB-231 (human breast carcinoma), and A-549 (human lung carcinoma) is also discussed. Results and Discussion The EtOAc extract of the frozen animal was fractionated by silica gel column chromatography, and the eluted fractions were subjected to further separation and purification utilizing normal-phase HPLC to yield cembranolides 1-7 (see Experimental Section). All new metabolites were isolated as colorless oils (except 5) and showed positive optical rotations. Spectroscopic data revealed that all of these metabolites are cembranoids containing R-methylene-γ- lactones. The HRFABMS (m/z 393.2275 [M + H] + ) of the most polar metabolite, crassocolide A (1), established the molecular formula C 22 H 32 O 6 , requiring seven degrees of unsaturation. The IR spectrum of 1 revealed the presence of hydroxy (ν max 3440 cm -1 ), R-meth- ylene-γ-lactone (ν max 1753 and 1653 cm -1 ), 2-17 and ester (ν max 1723 cm -1 ) moieties. The ion peaks at m/z 357 [M - 2H 2 O + H] + , 297 [M - 2H 2 O - AcOH + H] + in the FABMS indicated the presence of two hydroxyls and one acetoxyl in 1. The latter was indicated from the 1 H NMR data at δ 2.01 (3H, s) and the 13 C NMR data at δ C 20.8 (CH 3 ) and 169.5 (qC). The 22 carbon signals appearing in the 13 C NMR spectrum of 1 (Table 1) were identified by DEPT to belong to four methyls, six methylenes (including one olefinic carbon), six methines (including two vinylic CH), and six quaternary carbons. These data suggested 1 was a diterpenoid containing an acetoxy function. The NMR signals observed at δ C 170.0 (qC), 139.0 (qC), 122.6 (CH 2 ), 80.9 (CH), and 36.1 (CH) and δ H 6.27, 5.70 (each, 1H, d, J ) 2.5 Hz), 4.64 (1H, br s), and 3.31 (1H, m) revealed the presence of the R-methylene-γ-lactonic group as compared to those of similar metabolites. 2-17 Two trisubstituted double bonds were also designated from the NMR signals at δ C 136.7 (qC), 130.4 (qC), 127.4 (CH), and 125.4 (CH) and at δ H 5.29 (1H, dd, J ) 7.0, 4.5 Hz) and 5.09 (1H, dd, J ) 7.0, 5.0 Hz). The three 3H singlets appearing in the 1 H NMR at δ 1.74, 1.68, and 1.12 were assigned to two olefinic methyls and one methyl bound to a quaternary oxycarbon, respectively. The remaining one * Corresponding author. Tel: 886-7-5252000, ext. 5030. Fax: 886-7- 5255020. E-mail: sheu@mail.nsysu.edu.tw. † Department of Marine Biotechnology and Resources, National Sun Yat- sen University. ‡ Cheng Shiu University. § Mansoura University. | Kaohsiung Medical University. ⊥ Department of Chemistry, National Sun Yat-sen University. 1554 J. Nat. Prod. 2006, 69, 1554-1559 10.1021/np060182w CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 10/19/2006