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New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides

A novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E–G (2–4), were isolated by cytotoxicity-guided fractionation from soft corals Nephthea chabroli and Paralemnalia thyrsoides. The structures of the new compounds were determined by extensive analysis of spectroscopic data.


Results and Discussion
The molecular formula of chabrolin A (1) was determined as C 20 H 32 O from its HR-FAB-MS, 13 C NMR, and DEPT spectroscopic data. The 13 C NMR ( Figure S2) and DEPT spectrum of 1 exhibited the presence of four methyl, seven sp 3 methylene, three sp 3 methine, two sp 2 methine, two sp 3 quaternary, and two sp 2 quaternary carbons. The presence of two trisubstituted olefins in 1 was shown by the NMR data [δ H 5.59 t (J = 8.0 Hz), 5.13 t (J = 7.2 Hz); δ C 144. 5 (Table 1) pointed to a 1,2-disubstituted cyclopropane ring in 1. The 1 H NMR spectrum ( Figure S1) displayed signals for four tertiary methyl groups, (δ H 0.52, 0.64, 1.62, 1.69). The presence of the oxygen bearing sp 3 methine (δ C 74.1 CH) was shown in the 13 C NMR spectrum. By interpretation of 1 H-1 H COSY correlations (Figure 2 and Figure S3), it was possible to establish four partial structures of contiguous proton systems extending from H-1 to H-5, from H-7 to H-8, from H-10 to H-12, and from H-14 to H-16. HMBC correlations ( Figure S5) of (a) CH 3 -18 to C-5, C-6, C-7, and C-16, (b) H 2 -19 to C-8, C-9, C-10, C-13, and C-14, (c) H-8 to C-10, (d) CH 3 -20 to C-12, C-13, C-14, and C-19 connected four partial structures concluding the planar structure of 1, as shown in Figure 2. The above functionalities revealed that chabrolin A (1) possesses a novel diterpene tricyclic skeleton. The relative configuration of all chiral centers in 1 was deduced from a NOESY experiment ( Figure S6). Assuming the β-orientation of H 3

Animal Material
The octocoral N. chabroli was collected by hand using scuba at Green Islang, Taitong County, Taiwan, in August 2015, at a depth of 6 m. A voucher specimen (GN-100) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
The octocoral P. thyrsoides was collected by hand using scuba at San-Hsian-Tai, Taitong County, Taiwan, in July 2008, at a depth of 6 m. A voucher specimen (SST-07) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Extraction and Separation
The frozen soft coral was chopped into small pieces and extracted with acetone in a percolator at room temperature. The acetone extract of N. chabroli was concentrated to a brown gum, which was partitioned with EtOAc and H 2 O. The EtOAc-soluble residue (50 g) was subjected to Si 60 CC using n-hexane-EtOAc mixtures of increasing polarity for elution. Fraction 10, eluted with n-hexane-EtOAc (1:10), was purified by reverse-phase HPLC (MeOH-H 2 O, 85:15) to afford 1 (2.9 mg).
The frozen soft coral was chopped into small pieces and extracted with acetone in a percolator at room temperature. The acetone extract of P. thyrsoides was concentrated to a brown gum, which was partitioned with EtOAc and H 2 O. The EtOAc-soluble residue (10 g) was subjected to Si 60 CC using n-hexane-EtOAc mixtures of increasing polarity for elution. Fraction 8, eluted with n-hexane-EtOAc  Table 1; 13 C NMR data, see

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 [20,21]. 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 the 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 a 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 [22].

Conclusions
Cytotoxicity-guided fractionation of the ethyl acetate extract of the soft corals N. chablroli and P. thyrsoides led to the isolation of a novel diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton) as well as three new sesquiterpenoids. Compounds 1-4 displayed cytotoxicity against P-388, with ED 50 values of 3.18, 2.59, 3.31, and 2.49 µg/mL, respectively. However, Compounds 1-4 were not cytotoxic to A549 and HT-29 cell lines and did not show anti-HCMV activity. A plausible biosynthetic pathway of 1 is proposed in Scheme 1.

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 [20,21]. 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 the 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 IC50 (50% inhibitory concentration), or a 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 [22].