Cytotoxic and Antibacterial Cembranoids from a South China Sea Soft Coral, Lobophytum sp

Chemical examination of a South China Sea soft coral Lobophytum sp. led to the isolation of three new α-methylene-γ-lactone-containing cembranoids, (1R*,3R*,4R*,14R*,7E,11E)-3,4-epoxycembra-7,11,15(17)-trien-16,14-olide (1), (1R*,7S*,14S*,3E,11E)-7-hydroperoxycembra-3,8(19),11,15(17)-tetraen-16,14-olide (2), and (1R*,7S*,14S*,3E,11E)-18-acetoxy-7-hydroperoxycembra-3,8(19),11,15(17)-tetraen-16,14-olide (3), along with eleven known analogues 4–14. The structures of the new compounds were elucidated through extensive spectroscopic analysis, including 1D and 2D NMR data. Compounds 1–3 exhibited moderate cytotoxic activity against the selected tumor cell lines. Moreover, 2 and 3 were found to be moderate inhibitors against the bacteria S. aureus and S. pneumoniae.

Compound 1 was obtained as a colorless oil.  (Tables 1 and 2). IR absorptions at 1759 and 1660 cm −1 suggested the presence of an α-methylene-γ-lactone group [3,21]. This assumption was further supported by the 1 H NMR signals at δ H 6.26 (1H, d, J = 2.4 Hz, H-17a), 5.52 (1H, d, J = 2.4 Hz, H-17b), and 13 C NMR signals at δ C 169.9 (C, C-16), 138.3 (C, C-15), 120.8 (CH 2 , C-17), 77.1 (CH, C-14), and 42.6 (CH, C-1). Four olefinic carbon signals at δ C 135.1 (C, C-8), 130.1 (CH, C-11), 129.6 (C, C-12), and 122.3 (CH, C-7), and two olefinic proton signals at δ H 5.11 (1H, br d, J = 9.0 Hz, H-11) and 4.95 (1H, dd, J = 7.2, 1.8 Hz, H-7) were attributed to two trisubstituted double bonds. In addition, a trisubstituted epoxide was observed from carbon signals at δ C 62.3 (CH, C-3) and 60.8 (C, C-4), as well as an oxymethine proton at δ H 2.58 (1H, dd, J = 10.2, 1.2 Hz, H-3). Six degrees of unsaturation, accounted for by the functional groups from seven in the molecule, suggested the remaining of a cyclic structure in 1. By interpretation of 1 H-1 H COSY correlations, three partial structures extending from H 2 -13 to H-3, from H 2 -5 to H-7, and from H 2 -9 to H-11 were established. Moreover, the connectivities of these partial structures were established by HMBC correlations and revealed a 14-membered cembrane-type diterpenoid skeleton for 1 ( Figure 2). The location of the epoxide at C-3 and C-4 was indicated by the HMBC correlations observed from H 3 -18 to C-3, C-4, and C-5, while the locations of two double bonds at C-7/C-8 and C-11/C-12 were disclosed by the HMBC correlations from H 3 -19 to C-7, C-8, and C-9, and from H 3 -20 to C-11, C-12, and C-13. Thus, the gross structure of 1 was determined to be the same as isolobophytolide (8) [21], possessing an α-methylene-γ-lactone ring fused to a 14-membered ring at C-1 and C-14. However, the diagnostic upfield shift of C-2 (−6.3 ppm) due to the presence of the γ-gauche effect was observed in 1, compared to 8, suggesting the lactone ring at C-1 and C-14 was cis-fused. On the other hand, according to the general empirical rule that all cembrane diterpenes of known absolute configuration at C-1 reported from the order Alcyonacea belong to the α series [22], the configurations at C-1 and C-14 in 1 were assigned to be consistent with those of the co-occurring analogue lobophytolide A (14) [22]. The geometry of two double bonds at C-7/C-8 and C-11/C-12, and stereogenic centers at C-3 and C-4 were in agreement with those of isolobophytolide (8) on the basis of the similar NMR data. The stereochemistry of 1 as assigned above was further confirmed by the NOESY correlations ( Figure 3     (1) Compound 2 was also obtained as a colorless oil. The molecular formula of 2 was determined to be C 20  In addition, the presence of a hydroperoxyl group was disclosed by the NMR data at δ H 4.35 (1H, dd, J = 7.8, 4.2 Hz, H-7), 7.78 (1H, br s, OOH) and δ C 84.5 (CH, C-7), in association with the fact that two additional oxygen atoms remained in the molecule according to the HRESIMS data. All these data suggested that 2 possessed a cembrane skeleton with functionalities of an α-methylene-γ-lactone, two methyl-bearing trisubstituted double bonds, an exocyclic double bond, and a secondary hydroperoxyl group. Further interpretation of 1 H-1 H COSY and HMBC correlations established the gross structure of 2 as shown in Figure 2. The E geometry of two double bonds at C-3/C-4 and C-11/C-12, and the trans junction of the α-methylene-γ-lactone ring in 2 was determined based on the similar NMR data in comparison with those of the co-occurring analogue (1R*,14S*,3E,7E,11E)-cembra-3,7,11,15 (17)-tetraen-16,14-olide (4) [21]. The configuration at C-7 was assigned by comparison of the 13 C NMR chemical shift of C-7 with that of decaryiol D, a cembrane diterpene containing the same partial structure extending from C-5 to C-13 [25]. In decaryiol D, the absolute configuration at C-7 was determined as R unambiguously through structural transformation. While in compound 2, significant upfield shift of C-7 (−6.7 ppm) was observed, compared to decaryiol D, allowing the assignment of C-7 S* in 2. This assumption of stereochemistry for 2 was consistent with NOESY correlations (Figure 3 Compounds 1-3 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), and B16 (mouse melanoma). The bioassay results showed that compounds 1-3 possess moderate cytotoxicity against the selected tumor cell lines (Table 3). In addition, all compounds were evaluated for the antimicrobial activity against Staphylococcus aureus, S. pneumoniae, Pseudomonas aerugonisa, Saccharomyces cerevisiae, and Aspergillus fumigatus. The antibiotic assay revealed that compounds 2 and 3 exhibited moderate inhibition against Staphylococcus aureus and S. pneumoniae with the inhibitory rates around 90% at 20 μg/mL, but the other compounds were weak inhibitors against the two bacterial strains. All compounds showed weak effects against the microorganisms Pseudomonas aerugonisa, Saccharomyces cerevisiae, and Aspergillus fumigatus.

General Experimental Procedures
Optical rotations were determined with a PoLAAR 3005 digital polarimeter. IR spectra were obtained on a Bruker Equinox 55 spectrometer. 1 H and 13 C NMR and 2D NMR were recorded on a Bruker Avance 600 MHz NMR spectrometer using TMS as an internal standard. Chemical shifts (δ) were expressed in parts per million (ppm), and coupling constants (J) were reported in Hertz (Hz). HRESIMS data were recorded by a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Silica gel (200-300 mesh) for column chromatography and GF 254 silica gel for TLC was provided by Qingdao Marine Chemistry Co., Ltd. High-performance liquid chromatography (HPLC) chromatography was carried out using an Agilent 1100 series instrument equipped with a VWD G1314A detector at 210 nm and a YMC-Pack C 18 (10 μm, 250 × 10 mm) column.

Animal Material
The soft coral Lobophytum sp. was collected from the inner coral reef at a depth of 8 m in Sanya Bay, Hainan Island of China, in November 2011, and the fresh samples were frozen immediately after collection. The specimen was identified by Dr. Xiu-Bao Li (South China Sea Institute of Oceanology, CAS, Guangzhou, China). A voucher specimen (HS201105) is deposited at the Institute of Natural Drugs Development, Wenzhou Medical College, China.

Cytotoxicity Assay
The cytotoxic properties of the isolated compounds were tested in vitro using tumor cell lines including SGC7901 (human gastric carcinoma), A549 (human lung epithelial carcinoma), MCF7 (human breast carcinoma), HCT116 (human colonic carcinoma), and B16 (mouse melanoma) tumor cells by a modification of the MTT colorimetric method according to a previously described procedure [26,27]. The cell lines were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, CAS.

Antibiotic Assay
Antimicrobial bioassays were conducted in triplicate according to the method recommended by the National Center for Clinical Laboratory Standards (NCCLS) [28]. The bacterial strains Staphylococcus aureus, S. pneumoniae, and Pseudomonas aerugonisa were grown on Mueller-Hinton agar. The yeast, Saccharomyces cerevisiae, was grown on Sabouraud dextrose agar, and the fungus, Aspergillus fumigatus, was grown on potato dextrose agar. Targeted microbes (3-4 colonies) were prepared from broth culture (bacteria: 37 °C for 24 h; fungus: 28 °C for 48 h), and the final spore suspensions of bacteria (in MHB medium), yeast (in SDB medium), and fungus (in PDB medium) were 10 6 and 10 5 cells/mL and 10 4 mycelial fragments/mL, respectively. Testing compounds (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test microorganisms were added to each well (200 μL) (antimicrobial peptide AMP, streptomycin, and fluconazole were used as positive controls). After incubation, the absorbance at 595 nm was measured with a microplate reader (TECANT), and the inhibition rate was calculated and plotted versus test concentrations.