Norcembranoidal Diterpenes from the Cultured-Type Octocoral Sinularia numerosa

A known norcembranoidal diterpene, 5-episinuleptolide (1), along with a new analogue, 4α-hydroxy-5-episinuleptolide (2), were isolated from a cultured-type soft coral Sinularia numerosa. The structures of 1 and 2 were elucidated on the basis of spectroscopic methods and by comparison of the data with those of the related metabolites. Cytotoxicity of metabolites 1 and 2 against a panel of tumor cells is also described. Compound 2 exhibited moderate cytotoxicity toward CCRF-CEM cells with an IC50 value 4.21 μg/mL. Preliminary SAR (structure activity relationship) information was obtained from these two compounds.

4α-Hydroxy-5-episinuleptolide (2) was obtained as a white powder, [α] 20 D − 15 (c 0.9, CHCl3). The molecular formula C19H24O7 was determined (8 unsaturations) based on the HRESIMS (C19H24O7 + Na, m/z 387.14147, calculated 387.14142). From the 13 C NMR spectra (Table 1), 2 was found to possess an ester group (δC 169.2, C-19) and two ketone carbonyls (δC 211.5, C-6; 208.7, C-3). Two additional unsaturated functionalities were indicated by 13    The relative configuration of 2 observed in the NOESY spectrum corroborated the MM2 force field calculations which suggested the most stable conformation as shown in Figure 2 [16]. In the NOESY experiment for 2, it was found that one of the methylene protons at C-14 (δH 3.64) exhibited a correlation with H-1, but not with H-13, and therefore it was assigned as H-14β, and the other C-14 proton (δH 2.41) as H-14α. H-13 showed correlations with H-11 and H-14α, but not with H-1, and H-10 showed a correlation with H-11, as well as the lack of coupling was detected between H-10 and H-11, indicating the dihedral angle between H-10 and H-11 is approximately 90° and the configurations of chiral carbons C-10 and C-11 were assigned as S*-and R*-forms, respectively. One proton of C-9 methylene (δH 2.52) correlated with H-10 and H3-18, but not with H-11, and H3-18 showed a correlation with H-5, indicating that Me-18 and H-5 were β-oriented. A correlation was detected between OH-4 and H-11, indicating that the hydroxy group at C-4 should be α-oriented by modeling analysis. From the above evidences, the relative configurations of the chiral carbons of 2 were assumed to be 1R*, 4S*, 5S*, 8R*, 10S* and 11R*.  Table 2. CCRF-CEM cell line was more sensitive to the cytotoxic effects of 2. Compound 2 (4α-hydroxy-5-episinuleptolide) exhibited modest cytotoxicity toward CCRF-CEM cells, and was more potent than 1 (5-episinuleptolide), showing that the presence of a hydroxy substituent at C-4α position would enhance the activity.

Animal Material
Specimens of the cultured octocoral Sinularia numerosa were collected by hand in a 0.6 ton cultivation tank with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan, in 30 July 2014 and stored in freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-14009) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Molecular Mechasnics Calculations
Implementation of the MM2 force field [16] in CHEM3D PRO software from CambridgeSoft Corporation (ver. 9.0, Cambridge, MA, USA) was used to calculate molecular models.

MTT Antiproliferative Assay
CCRF-CEM (human acute lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia), K-562 (human chronic myelogenous leukemia), U-937 (human histiocytic lymphoma), DLD-1 (human colorectal adenocarcinoma), LNCaP (human prostatic carcinoma) and MCF7 (human breast adenocarcinoma) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded at 4 × 10 4 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds were dissolved in dimethyl sulfoxide (less than 0.02%) and made immediately of 1.25, 2.5, 5, 10 and 20 μg/μL prior to the experiments. After treatment for 72 h, the cytotoxicity of the tested compounds was determined using a MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128). The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD570 − OD620) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos labtec Instrument, Salzburg, Austria) to calculate the concentration that caused 50% inhibition (IC50), i.e., the cell concentration at which the light absorbance value of the experiment group was half that of the control group. These results were expressed a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments [17][18][19].

Conclusions
Octocorals have been well recognized as an important source of potential medicinal-use agents. However, because of the octocorals are claimed to be threatened species and most of the compounds from octocorals are difficult to obtain by chemical methods at this stage, bioactive substances from cultured-type marine invertebrates will play an important role in this field. Our further studies on a cultured soft coral Sinularia numerosa for the extraction of additional natural substances, have led to the isolation of a new norcembranoidal diterpene, 4α-hydroxy-5-episinuleptolide (2), and this compound was found to exhibit modest cytotoxicity against CCRF-CEM tumor cells. This study suggested that 4α-hydroxy-5-episinuleptolide (2) is worthy of further biomedical investigation.