Sarcocrassocolides M–O, Bioactive Cembranoids from the Dongsha Atoll Soft Coral Sarcophyton crassocaule

Three new cembranoids, sarcocrassocolides M–O (1–3), have been isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 1–3 were shown to exhibit moderate cytotoxicity toward a limited panel of cancer cell lines and display significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein.


Results and Discussion
The HRESIMS (m/z 429.1892 [M + Na] + ) of sarcrocrassocolide M (1) established the molecular formula C 22 H 30 O 7 , appropriate for eight degrees of unsaturation, and the IR spectrum revealed the presence of lactonic carbonyl (1757 cm −1 ) group. The 13 C NMR and DEPT (Table 1) spectroscopic data showed signals of three methyls (including one acetate methyl), five sp 3 methylenes, two sp 2 methylenes, five sp 3 methines (including four oxymethines), one sp 2 methines, one sp 3 and five sp 2 quaternary carbons (including two ester carbonyls). The NMR signals (Table 1) observed at δ C 169.1 (C), 139.3 (C), 121.6 (CH 2 ), 81.4 (CH), and 37.4 (CH), and δ H 6.28, 5.63 (each, 1H, d, J = 2.0 Hz), 4.61 (1H, t, J = 2.5 Hz), and 3.07 (1H, dt, J = 11.5, 2.5 Hz) showed the presence of an α-methylene-γlactonic group by comparing with similar NMR data of known cembranoids with the same five-membered lactone ring [22,23]. Signals resonating at δ C 60.1 (C), 60.0 (CH) and δ H 2.53 (1H, dd, J = 7.0, 4.0 Hz) revealed the presence of a trisubstituted epoxide. One trisubstituted and one 1,1-disubstituted double bond were also identified from NMR signals appearing at δ C 129.4 (C), 128.1 (CH), and δ H 5.46 (1H, dd, J = 7.0, 5.5 Hz), and at δ C 113.5 (CH 2 ), 146.6 (C), δ H 5.16 and 5.12 (1H, s, each), respectively. In the 1 H-1 H COSY spectrum, it was possible to identify three different structural units, which were assembled with the assistance of an HMBC experiment. Key HMBC correlations of H 3 -18 to C-3, C-4 and C-5; H 2 -19 to C-7, C-8 and C-9; H 3 -20 to C-11, C-12 and C-13 and H 2 -17 to C-1, C-15 and C-16 permitted the establishment of the carbon skeleton ( Figure 1). Furthermore, the acetoxy group positioned at C-13 was confirmed from the HMBC correlations of the methyl protons of an acetate (δ H 2.02) to the ester carbonyl carbon at δ C 169.2 and the oxymethine signal at 77.4 (C-13, CH). The 13 C NMR signals at δ C 87.1 (CH) and HRESIMS showed the presence of a hydroperoxy group at a methine carbon C-7 [9]. On the basis of the above analysis, the planar structure of 1 was established unambiguously. The relative structure of 1 was elucidated by the analysis of NOE correlations, as shown in Figure    Compound 2 possessed the same molecular formula (C 22 H 30 O 7 ) as that of 1, as revealed from HRESIMS. Furthermore, it was found that the NMR spectroscopic data of 2 (Table 1) were similar to those of 1. Analysis of the 2D NMR ( 1 H-1 H COSY, HMQC, and HMBC) correlations revealed that compound 2 possesses the same planar structure as that of 1. From the NOESY spectrum, it was found that H-7 (δ 4.38) showed a weak NOE interaction with H 3 -20 (1.78), but not with H-11 (δ 5.41), revealing the α-orientation of H-7. Further analysis of other NOE interactions revealed that 2 possessed the same relative configurations at C-1, C-3, C-4, C-13 and C-14, as those of 1 (Figure 2). Therefore, 2 was found to be the C-7 epimer of 1.     (4), sinularolide E (5), and 13-acetoxysarcocrassolide (6) (Chart 2), which were isolated by our previous study [24,25].  Table 2) showed that all compounds 1-3 were found to exhibit cytotoxicity against all or part of the above carcinoma cell lines. In this assay, the in vitro anti-inflammatory effects of compounds 1-3 were also tested. The inhibition of LPS-induced up-regulation of pro-inflammatory proteins, iNOS and COX-2 in RAW264.7 macrophage cells was measured by immunoblot analysis (Figure 4). At a concentration of 10 μM, compounds 1-3 were found to significantly reduce the levels of iNOS protein to 4.2 ± 1.6%, 52.9 ± 12.8%, and 22.7 ± 2.8%, respectively, relative to the control cells stimulated with LPS only. At the same concentration metabolites 2 and 3 did not show activity in inhibiting the expression of the pro-inflammatory COX-2 expression with LPS treatment, but compound 1 could reduce the expression of COX-2 to 62.8 ± 22.4%. Thus, compounds 1-3 might be useful anti-inflammatory agents, while 1 is a promising anti-inflammatory lead compound. Compound 1 could inhibit the expression of both iNOS and COX-2 which might be arisen from the presence of β-hydroperoxy group at C-7 by comparison with compound 2. . Relative intensity of the lipopolysaccharide (LPS) alone stimulated group was taken as 100%. * Significantly different from LPS alone stimulated group (*P < 0.05). a stimulated with LPS; b stimulated with LPS in the presence of 1-3 (10 μM).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter. Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. NMR spectra were recorded on a Varian 400MR FT-NMR (or Varian Unity INOVA500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1 H and 100 MHz (or 125 MHz) for 13 C in CDCl 3 . LRMS and HRMS were obtained by ESI on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230-400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography was performed on a Hitachi L-7100 HPLC apparatus with a Merck Hibar Si-60 column (250 × 21 mm, 7 μm) and on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm).

Animal Material
S. crassocaule (specimen No. 20070402) was collected by hand using scuba off the coast of Dongsha, Taiwan, in April 2007, at a depth of 5-10 m, and stored in a freezer until extraction. A voucher sample was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

In Vitro Anti-Inflammatory Assay
Macrophage (RAW264.7) cells were purchased from ATCC. In vitro anti-inflammatory activities of compounds 1-3 were measured by examining the inhibition of lipopolysaccharide (LPS) induced upregulation of iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in macrophages cells using western blotting analysis [27].

Conclusions
Our investigation demonstrated that the soft coral, S. crassocaule, could be a good source of bioactive substances. The isolated compounds 1-3, in particular 1, are potentially anti-inflammatory and may become lead compounds in the future drug development. Also, it is noteworthy to mention that cembranoids 1-3 possessing an α-methylene-γ-lactonic group with a rarely found 1,1-disubstituted double bond at C-19/C-8 and containing a hydroperoxy group at C-7, were discovered for the first time from corals of this species.