A new briarane-type diterpenoid from the South China Sea Gorgonian Dichotella gemmacea

One new briarane-type diterpenoid, dichotellide V (1), along with four known analogues, gemmacolide N (2), dichotellide J (3), junceelin A (4) and junceellolide A (5), was isolated from the South China Sea gorgonian Dichotella gemmacea. All of the isolated compounds (1–5) were established by comprehensive analysis of the spectral data, especially 1D and 2D NMR (HMQC and HMBC) spectra. The cytotoxic activities of these compounds were evaluated.


Introduction
Briarane-type diterpenoids are a group of highly oxidised secondary metabolites reported from marine organisms, particularly from octocorals (Blunt et al. 2011). These kinds of diterpenoids are characterised by a bicycle [8.4.0] ring system fused by a glactone group, with high oxidisation and esterification by all kinds of acyls, i.e. acetyl and isovalerate (Grote et al. 2006;Su et al. 2007;Sun et al. 2011;Lei et al. 2014). Since the first briarane-type diterpenoid, briarane A, was isolated from the West Indian gorgonian Briareum asbestinum in 1977 (Burks et al. 1977), more than 500 briarane family members have been discovered (Sung et al. 2002(Sung et al. , 2005(Sung et al. , 2008. Until now, these kinds of molecules have been continuously reported in an increasing number due to the structural complexity and interesting biological activities, such as cytotoxicity (Hoshino et al. 2005;Ishiyama et al. 2008), anti-inflammatory (Shin et al. 1989), antivirus (Subrahmanyam et al. 1998), insecticide (El Sayed et al. 1997, immunomodulation (Hamann et al. 1996) and antifouling (Qi et al. 2006). In the course of our ongoing screening for biologically active secondary metabolites from marine sources, we made a collection of the gorgonian Dichotella gemmacea off the coast of Meishan Island, Hainan province of China. Early investigation on this species resulted in the isolation and characterisation of a series of new briarane diterpenoids (Li, La, Li, et al. 2011;Li, La, Sun, 2011, 2012Sun et al. 2011Sun et al. , 2013, fatty acids (Wang et al. 2011) and steroidal glycosides . Our continuing studies of the EtOAc-soluble fraction from the EtOH extract of D. gemmacea have now led to the isolation of one new briarane-type diterpenoid, dichotellide V (1), along with four known analogues, gemmacolide N (2) (Li, La, Li, et al. 2011;Li, La, Sun, 2011), dichotellide J (3) , junceelin A (4) (Garcia et al. 1999) and junceellolide A (5) (Shin et al. 1989). The structures of these diterpenoids were determined by extensive spectroscopic analysis ( 1 H and 13 C NMR, DEPT, HSQC, HMBC, NOESY and HR-ESI-MS), aided by the comparison with reported data of related derivatives. Herein we report the isolation, structure elucidation and biological activities of these compounds.
The 1 H and 13 C NMR spectra of 1 showed great similarity to those of dichotellide J(3), except that three acetate groups at C-12, C-13 and C-16 in 3 were replaced by three isovalerate groups in 1, respectively. This assignment was confirmed by the HMBC correlations of H-12/H-13/H-16 to the corresponding carbonyl group (d C 172.1, 171.7,171.9) of the isovalerate group, respectively. Accordingly, the established planar structure of 1 was assigned and further supported by the 1 H-1 H COSY and HMBC spectra ( Figure S1). The relative configuration of 1 at the chiral centres was proved to be the same as that of 3 by a NOESY experiment ( Figure S2), showing a b configuration of H-7, H-12, H-13, H-14, Me-15, H-17 and CH 2 -20, and an a configuration of H-2, H-9, H-10 and Me-18. The geometry of the D 3 double bond was assigned as (Z) based on the proton coupling constant between H-3 and H-4 (J ¼ 10.0 Hz), while that of D 5 double bond was determined as (E) due to the NOESY correlation between H-6 and H 2 -16 ( Figure S2). Thus, the relative configuration of 1 was determined as 1R*, 2R*, 3Z, 5E, 7S*, 8R*, 9S*, 10S*, 11R*, 12R*, 13R*, 14R* and 17R* (Figure 1).
The four known compounds gemmacolide N (2), dichotellide J (3), junceelin A (4) and junceellolide A (5) were also isolated and identified by using their NMR, MS data and comparing their experimental data with those reported in the literature.
All the isolated briarane-type diterpenoids were tested for their cytotoxicity against six human tumour cell lines (H1975, U937, BGC823, MCF-7, A549 and Hela), using the CCK8 method (Wanka et al. 2013). However, none of the compounds exhibited cytotoxic effects on the tested cancer cell lines (IC 50 . 100 mM). Previous studies found that the number and the location of isovaleryl group substitution may increase and decrease the cytotoxicity, respectively (Li, La, Li, et al. 2011;Li, La, Sun, 2011;Li et al. 2013). For example, according to the results of Li et al. (2013), gemmacolide AF which has isovaleryl group at C-12 and acetyl group at C-13 showed cytotoxic activities against A549 and MG63 cells, but gemmacolide AG which has acetyl group at both C-12 and C-13 did not show any cytotoxic activities. They presented that the 12-Oisovalerate may show the positive contribution to the activity. Comparing the results of dichtellide V with these analogues, differentially functionalised group at C-12 and C-13 might be one of the factors to show cytotoxicity. This observation may encourage further investigations on briaranes and the clear structure -activity relationships.

Animal material
The gorgonian D. gemmacea were collected from Meishan Island, Hainan province of China in April 2009 (7 -10 m depth) and identified by Professor Hui Huang, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (No. M090405) has been deposited in the Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO 2 Water Jacketed Incubator with 5% CO 2 in air at 378C. Cell viability assay was determined by the CCK8 (Dojindo, Japan) assay. Cells were seeded at a density of 400-800 cells/well in 384-well plates and treated with various concentrations of compounds or solvent control. After 72 h incubation, CCK8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were plotted to determine the IC 50 values using Prism 5.0 (Graphpad Software Inc., USA).

Conclusions
The investigation of bioactive natural products from the EtOH extract of D. gemmacea has led to the isolation of one new briarane-type diterpenoid, dichotellide V (1), along with four known analogues, gemmacolide N (2), dichotellide J (3), junceelin A (4) and junceellolide A (5). All compounds were tested in vitro for cytotoxic activity against six human tumour cell lines by the CCK8 (DOjinDo, Kumamoto, Japan) method, However, none of the compounds exhibited cytotoxic effects on the tested cancer cell lines (IC 50 (100 mM).

Supplementary material
Supplementary material relating to this article is available online, alongside Tables S1 -S2 and Figures S1 -S7.