Regular ArticleSoft corals are well known to be a rich source of terpene metabolites, especially diterpenes of the cembranoid series, 1,2) which are naturally occurring diterpenens containing a 14-carbon ring. Many of them are of great interest as they exhibit a wide range of biological activities including ichthyotoxic, anti-cancer, anti-inflammatory, anti-human immunodeficiency virus (HIV), and anti-microbial activities. [3][4][5][6][7][8][9] In the continuation of our ongoing research for bioactive marine natural products, we have investigated the n-hexane extract of the Lobophytum crassum (von Marenzeller, 1886) which has exhibited a significantly inhibitory effect on nitric oxide (NO) production against lipopolysaccharide (LPS)-induced Raw 264.7. Chemical investigation revealed that the nhexane-soluble material in this soft coral included four new (1-4) and fifteen known (5-19) cembranoids (Fig. 1). In this paper, we report on the isolation and structure elucidation of four new cembranoids 1-4 and also the determination of absolute stereochemistry of the new 1 and 2 and the known cembranoids 5 and 6. Furthermore, the inhibitory effect of some isolates was evaluated on NO production against Raw264.7.The frozen L. crassum collected in Okinawa was completely extracted in n-hexane. The extract was partitioned between n-hexane and aqueous methanol. The n-hexane soluble-material was subjected to silica gel flash column chromatography and recycling HPLC, followed by the reversed phase (RP) HPLC to afford four new cembranoids 1-4 and fifteen known cembranoids 5-19.The molecular formula of 1 was determined to be C-NMR spectra of 1 showed signals assignable to three olefinic methyls, one acetylmethyl, three trisubstituted double bonds, one exomethylene, two oxymethines, one aliphatic methine, five methylenes, and two ester carbonyls. Six of the eight degrees of unsaturation in 1 were attributed to four double bonds and two ester carbonyls (Tables 1, 2). The remaining two degrees of unsaturation were attributed to the bicyclic ring system of 1. Interpretation of correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) spectra, in combination with heteronuclear multiple bond correlation (HMBC) experiments, led to the planar structure of 1 (Fig. 2) easily merged these three spin systems and the presence of an a-methylene-l-lactone adjacent to the 14-membered ring. Thus, the planar structure of 1 was depicted in Fig. 2. The E-geometry of the D 3 and D 7 was determined by the nuclear Overhauser effect (NOE) correlations between H-3/H-5 and H-7/H-9 (d H 2.00), and the Zgeometry of the D 12 was determined by the strong NOE between H-13 and H-20. The cis-fused a-methylene-l-lactone and the 14-membered ring was deduced from the NOE correlation between H-1 and H-2. In addition, NOE correlations were observed between H-3 and H-2/H-5/H-7, H-7 and H-9/H11, H-13 and H-1/H-17/H-20. These data revealed the conformation of the 14-membered ring and the relative configuration of C-11. Thus, the relative stereochemist...
