Cembranoids from the Dongsha Atoll Soft Coral Lobophytum crassum

Chemical investigation of the Dongsha Atoll soft coral Lobophytum crassum has afforded four new cembranoids, crassumols A–C (1–3) and 13-acetoxysarcophytoxide (4). The structures of these isolated compounds were elucidated by extensive NMR and HRESIMS experiments. The cytotoxicity and anti-HCMV (Human cytomegalovirus) activities of 1–4 were evaluated in vitro. Compound 4 exhibited cytotoxicity against A-549 (human lung carcinoma) cell line with an ED50 of 3.6 μg/mL.


Introduction
Soft corals belonging to the genus Lobophytum (Alcyoniidae) have been well recognized as a rich source of secondary metabolites endowed with a range of structural diversity and various biological activities . Previous bioassay results of some cembraniods and their analogues have demonstrated remarkable pharmacological potential such as in vitro cytotoxicity against various cancer cell lines [2][3][4][5][6][7][8][9], anti-inflammatory properties [10][11][12], antimicrobial activities [10], and HIV-inhibitory activity [13]. In view of their scientific significance and potential usage, we have investigated the Dongsha Atoll soft coral Lobophytum crassum (Von Marenzeller, 1886) ( Figure 1) for the bioactive substances. Four

Results and Discussion
Specimens of L. crassum were frozen immediately after collection. Conventional extraction procedures were used, and the acetone extract was exhaustively partitioned between EtOAc and H 2 O to afford the EtOAc-soluble fraction, which was evaporated under vacuum to yield a dark brown gum (20 g). The resultant concentrated residue was subsequently subjected to fractionation with column chromatography and high-performance liquid chromatography, leading to the isolation of four new cembranoids, crassumols A-C (1-3) and 13-acetoxysarcophytoxide (4) (Figure 2).
It is possible that compound 2 arise from the precursor with a 7,8-epoxide. Transannular opening of the 7,8-epoxide by an 11-hydroxyl would give rise to an oxepane ring in compounds 2 as shown in Figure 9. The major metabolite, (+)-sarcophytoxide (5), should be the precursor for compounds 2-4. Therefore, the absolute configuration at C-2 for compounds 2-4 should be S.

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on a JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. The NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13 C or on a Varian Unity INOVA 500 FT-NMR spectrometer at 500 MHz for 1 H and 125 MHz for 13 C, respectively. Chemical shifts are expressed in δ (ppm) referring to the solvent peaks δ H 7.27 and δ C 77.0 for CDCl 3 , respectively, and coupling constants are expressed in Hz. ESI-MS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 230-400 mesh) and LiChroprep RP-18 (Merck, 40-63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) and precoated RP-18 F 254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phase column (Merck, Hibar LiChrospher RP-18e, 5 μm, 250 × 25 mm).

Biological Material
The soft coral L. crassum was collected by hand using SCUBA along the coast reefs offshore from the Dongsha Atoll in April 2007, at a depth of 6 m, and was stored in a freezer for 14 months until extraction. Identification was kindly verified by Prof. Chang-Feng Dai, Institute of Oceanography, National Taiwan University, Taiwan. A voucher specimen (TS-11) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

Extraction and Isolation
The frozen soft coral (2.2 kg) was chopped into small pieces and extracted exhaustively by maceration with fresh acetone for 24 h at room temperature. The quantity of solvent used for each extraction (2 L) was at least three times the amount of the soft coral material used. The acetone extracts were filtered and concentrated under vacuum to yield a brownish oily residue, which was subsequently partitioned between EtOAc and H 2 O. The resulting EtOAc-soluble residue (20.1 g) was subjected to silica gel 60 column chromatography (Si 60 CC) using hexane-EtOAc and EtOAc-MeOH of increasing polarity for elution to give roughly 40 fractions on the basis of the 1 H NMR spectroscopic data and TLC analyses.

Cytotoxicity Assay
Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [11,22,23]. P-388 cell line was kindly supplied by J. M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection.

Anti-HCMV Assay
To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity was expressed as IC 50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [24].