New Diterpenoids from Soft Coral Sarcophyton ehrenbergi

Continuing chemical investigation on the acetone extracts of the soft coral Sarcophyton ehrenbergi collected off the coast of San-hsian-tai, Taitong County, Taiwan led to the isolation of two new diterpenoids, ehrenbergol C and acetyl ehrenberoxide B (1 and 2). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses. Moreover, in vitro tests show that compounds 1 and 2 displayed antiviral activity towards human cytomegalovirus, with EC50 of 20 and 8.0 µg/mL, respectively.

Fifteen cembranoids were previously reported from the Taiwanese soft coral Sarcophyton ehrenbergi [19][20][21]. Continuing chemical investigation of the soft coral S. ehrenbergi ( Figure 1) collected at San-Hsian-Tai (Taitong County, Taiwan) resulted in the isolation of two new diterpenoids, designated as ehrenbergol C and acetyl ehrenberoxide B (1 and 2) ( Figure 2). Herein, we describe the purification, structure elucidation, cytotoxicity and antiviral evaluation of these metabolites.
All these data allowed us to identify compound 1 having the same planar framework as lobocrasol isolated from soft coral Lobophytum crassum [22]. With the gross structure of 1 in hand, the relative stereochemistry of compound 1 was deduced from NOESY correlations and Chem3D Ultra 9.0 ( Figure 4). The Z geometry of the Δ 11 double bond was established by the NOESY correlation observed between H-11 and H-10b and between H-10a and H-13b. NOESY correlations between H-7/H 3 -19, H-7/H 3 -18, and H 3 -18/H-3 indicated that these protons are on the same face of the ring system, thereby establishing the relative configuration of 1. The relative stereochemistry of C-7 and C-8 were different from lobocrasol; however, the absolute structure was not determined due to the limited amount of the sample.  Compound 2 analyzed for C 22 H 36 O 4 from HRESIMS and 13 C NMR spectroscopic data (Table 2), corresponding to five degrees of unsaturation. The IR spectrum of 2 at 3445 cm −1 demonstrated a broad absorption band diagnostic of hydroxy group. The presence of one oxygenated methine [δ H 4.05 (t, 1H, J = 3.2 Hz) and δ C 75.3 (C-7)] and an oxygenated quaternary carbon [δ C 80.4 (C-12)] implied that an oxygen bridge is probably present between C-7 and C-12, which was supported by the HMBC correlations from H-7 to C-12. The NMR spectroscopic data ( ; δ C 151.0 (C, C-1), 117.9 (CH, C-2), 123.5 (CH, C-3), and 132.6 (C, C-4)]. The above functionalities suggest that metabolite 2 must consist of a 14-membered ring diterpenoid incorporating an oxepane ring, a hydroxy, an acetoxy and a conjugated diene. Correlations deduced from extensive analyses of the 1 H-1 H COSY correlations of 2 in C 6 D 6 enabled initially the establishment of five partial structures. The structural fragments were subsequently interconnected by the HMBC correlations ( Figure 3). Two oxygen bearing carbons at δ C 87.0 (C) and 78.3 (CH) were ascribable to C-8 and C-11 on the basis of the HMBC correlations from Me-19 to C-7, C-8, and C-9 and from Me-20 to C-11, C-12, and C-13. The attachment of isopropyl to C-1 was established on the grounds of HMBC correlations from Me-16/Me-17 to C-15 and C-1. The positions of the conjugated double bonds at C-1/C-2 and C-3/C-4 were confirmed by the HMBC cross-peaks from Me-18 to C-3, C-4, and C-5, as well as a COSY correlation between H-2 and H-3. The planar structure of compound 2 was thus elucidated. The relative configuration and the detailed 1 H NMR spectroscopic data assignments of 2 were determined mainly by the assistance of the NOESY experiment ( Figure 5 Figure 5. Accordingly, the structure of 2 was determined as (7R*,8S*,11S*,12R*,1Z,3E)-8-acetoxy-11-hydroxy-7, 12-epoxycembra-1(2),3-diene.   a Spectra were measured in C 6 D 6 (400 MHz); b Spectra were measured in C 6 D 6 (100 MHz).
The cytotoxicities of compounds 1 and 2 against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells are shown in Table 3. Compounds 1 and 2 were also examined for antiviral activity against human cytomegalovirus (HCMV) using a human embryonic lung (HEL) and displayed antiviral activity against human cytomegalovirus, with EC 50s of 20 and 8.0 µg/mL, respectively.

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. UV and IR spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13 C. 1 H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δ H 7.27 for CHCl 3 or δ H 7.15 for C 6 D 6 , and coupling constants are expressed in Hertz (Hz). 13 C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δ C 77.0 for CDCl 3 or δ C 128.0 for C 6 D 6 . MS were recorded by 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-phased column (Merck, Hibar LiChrospher RP-18e, 5 µm, 250 × 25 mm).

Biological Material
The soft coral S. ehrenbergi was collected by SCUBA at San-Hsian-Tai, Taitong County, Taiwan, in July 2009 at a depth of 6 m and stored in a freezer until extraction. The voucher specimen (ST-13) was identified by Professor Chang-Feng Dai, National Taiwan University and deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

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 [24][25][26]. The provision of the P-388 cell line was supported 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. To measure the cytotoxic activities of tested compounds, five concentrations with three replications were performed on each cell line. Mithramycin was used as a positive control.

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 with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 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 [26][27][28].

Conclusions
This investigation of Taiwanese soft coral S. ehrenbergi collected has led to the isolation of two new ehrenbergol C and acetyl ehrenberoxide B (1 and 2). The carbon framework of 1 was identical to a cytotoxic diterpene, lobocrasol isolated from soft coral Lobophytum crassum. However, the stereochemistry of C-7 and C-8 of 1 were different from lobocrasol. Compounds 1 and 2 were not cytotoxic towards P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells. However, compounds 1 and 2 displayed antiviral activity towards human cytomegalovirus, with IC 50 of 20 and 8.0 μg/mL, respectively.