New Casbane Diterpenoids from a South China Sea Soft Coral, Sinularia sp

Six new casbane diterpenoids, named as sinularcasbanes A–F (1–6), along with six known analogues 7–12, were isolated from a South China Sea soft coral, Sinularia sp. The structures of the new compounds were elucidated by extensive spectroscopic analysis and by comparison with data reported in the literature. All compounds were evaluated for their cytotoxicity against selected cancer cell lines and the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages.


Introduction
The casbanes are a small group of diterpenoids strictly related to the cembrane skeleton, and characterized by the presence of a dimethyl-cyclopropyl moiety fused to the 14-membered ring. To date, approximately 33 casbane diterpenoids, in the majority of which the two rings forming the OPEN ACCESS macrocyclic structure are cis-fused, have been isolated and described mainly from some plants of the family Euphorbiaceae [1][2][3][4][5][6][7][8][9][10][11][12][13], as well as from two species of soft coral belonging to the genus Sinularia (S. microclavata and S. depressa) [14,15]. The biological potential of casbane diterpenoids has not been explored extensively, however some members of this group have been proven to display moderate cytotoxicity and antimicrobial activity [14,15].
Soft corals belonging to the genus Sinularia distribute widely in the tropical reef, and have been shown to be a plentiful source of diterpenoids, possessing a range of biological activities [16,17]. Our recent study of bioactive natural products from a Hainan soft coral, Sinularia sp., resulted in the isolation of six new casbane diterpenoids 1-6, along with six known analogues 7-12 ( Figure 1). The compounds isolated were evaluated for their cytotoxicity against selected cancer cell lines and their inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ). Herein we report the purification, structural elucidation, and bioactivity of these compounds.
Sinularcasbane C (3) has a molecular formula of C 20 H 32 O 2 as determined by HRESIMS data (m/z 327.2267 [M + Na] + ), requiring five degrees of unsaturation. The analysis of 1D and 2D NMR spectroscopic data revealed that the gross structure of 3 was closely related to 10-hydroxydepressin (8) [15], with the exception that C-5 was linked to a hydroxyl group instead of a ketone in the latter.
The NMR spectroscopic data of sinularcasbane D (4) (Tables 1 and 2) indicated that its structure was closely related to that of 3. The only difference was found to be the dehydroxylation of C-5 in 4 compared to 3, as evident from the HMBC correlation from H 3 -18 (δ H 1.64, s) to a methylene carbon C-5 (δ C 39.4), instead of a hydroxy-bearing methine carbon (δ C 79.3) as in 3. The molecular formula of 4, C 20 H 32 O as established by HRESIMS (m/z 311.2322 [M + Na] + ), was 16 amu less than that of 3 and further supported this structure assignment. The relative stereochemistry of 4 was in agreement with that of 3 based on the similar NMR and NOE data. Thus, compound 4 was defined as a C-5 dehydroxylated analogue of 3.
Compounds 1-12 were tested for their cytotoxicity against a panel of tumor cell lines including SGC7901 (human gastric carcinoma), A549 (human lung epithelial carcinoma), MCF7 (human breast carcinoma), HCT116 (human colonic carcinoma), B16 (mouse melanoma) and P815 (mouse lymphoblast-like mastocytoma). However, they showed no cytotoxic activity against all cell lines at 50 μM. In order to detect the anti-inflammatory property of these compounds, the test for inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ) was performed. The bioassay results revealed compounds 2 and 5 showed moderate inhibition with an inhibitory concentration 50% (IC 50 ) of 8.3 and 5.4 μM, respectively, whereas the other compounds showed weak activity (IC 50 > 10 μM). In addition, all compounds were weakly cytotoxic toward mouse PEMΦ (IC 50 > 10 μM).

General Experimental Procedures
Optical rotations were measured on a PoLAAR 3005 digital polarimeter. IR spectra were determined on a Bruker Equinox 55 spectrometer. 1 H and 13 C NMR and 2D NMR were recorded on a Bruker Avance 500 MHz NMR spectrometer using TMS as an internal standard. δ values were expressed in parts per million (ppm), and J values were reported in Hertz (Hz). HRESIMS data were obtained from a Bruker micrO TOF-QII instrument. Silica gel (200-300 mesh) for column chromatography and GF 254 silica gel for TLC was provided by Qingdao Marine Chemistry Co. Ltd. Reversed-phase HPLC chromatography was performed on an Agilent 1100 series instrument using a VWD G1314A detector at 210 nm and a YMC-Pack C 18 (10 μm, 250 × 10 mm) column.

Animal Material
The soft coral Sinularia sp. was collected off the coast of Ximao island, Hainan Province, China, in November 2011, at a depth of 8 m and was frozen immediately after collection. The specimen was identified by Xiu-Bao Li (South China Sea Institute of Oceanology, CAS, Guangzhou, China). A voucher specimen (HS201101) is deposited at the Institute of Natural Drugs Development, Wenzhou Medical College, China.
One-way analysis of variance was applied for all statistical analysis by independent experiments, and data were represented as means ± standard error of the measurement. Individual values were compared by t-test and a p-value <0.01 to evaluate the significance.

Conclusions
Sinularcasbanes A-F (1-6) together with six known casbane diterpenoids 7-12 were isolated from the EtOAc extract of the South China Sea soft coral Sinularia sp. All of the compounds exhibited no cytotoxic activity against SGC7901, A549, MCF7, HCT116, B16, and P815 tumor cells at 50 μM in the in vitro cytotoxicity assay. Sinularcasbanes B (2) and E (5) were found to show moderate inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ) with an IC 50 of 8.3 and 5.4 μM, respectively. NO plays an important role in the inflammatory process, therefore, sinularcasbanes B (2) and E (5) could be promising lead compounds for anti-inflammatory agents. Other studies should be performed to elucidate the mechanism by which these compounds inhibit the production of NO. A literature survey revealed that sinularcasbanes E (5) and F (6) are the first members of the casbane family with a carbon-carbon single bond between carbons C3 and C4. The present work provided a group of new casbane diterpenoids with which to enrich the chemical diversity of Sinularia corals.