Norcembranoidal Diterpenes from a Formosan Soft Coral Sinularia sp.

Two norcembranoidal diterpenes, 5-episinuleptolide acetate (1) and scabrolide D (2), were isolated from a Formosan octocoral identified as Sinularia sp. The structures of norcembranoids 1 and 2 were established by spectroscopic methods and by comparison of the spectral data with those of known analogues and 1 was proven to be a new natural product. Norcembranoid 1 was found to exhibit cytotoxicity toward a panel of tumor cells.


Introduction
The search for bioactive natural products from marine organisms has been remarkably successful [1,2] and octocorals belonging to the genus Sinularia have proven to be rich sources of bioactive terpenoid analogues [3]. Among these terpenoid metabolites, the C 19 -norcembranoid diterpene derivatives played an important role [4]. In continuation of our search for new natural substances from the marine invertebrates collected off the waters of Taiwan at the intersection of the Kuroshio current and the South China Sea surface current, we have further isolated two norcembranoidal diterpenes, 5-episinuleptolide acetate (1) and scabrolide D (2), from an octocoral identified as Sinularia sp. (Figure 1). In this paper, we describe the isolation, structure determination and cytotoxicity of norcembranoids 1 and 2.

Results and Discussion
The molecular formula for norcembranoidal diterpene 1 was determined to be C 21 3 ), an ester group ( C 167.7, C-19) and two ketone carbonyls ( C 205.3, C-3; 214.5, C-6). Two additional unsaturated functionalities were indicated by 13 C resonances at δ C 127.6 (C-12), 147.2 (CH-13), 147.0 (C-15) and 110.4 (CH2-16), suggesting the presence of a trisubstituted olefin and an exocyclic carbon-carbon double bond. From the 1 H-1 H COSY spectrum of 1 (Table 1 and   The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 offered by computer modeling (Figure 3), in which the close contacts of atoms in space calculated were consistent with the NOESY correlations [5]. Most naturally occurring cembrane-type natural products from soft coral belonging to the order Alcyonacea have the H-1 in the -orientation [6].   By comparison of the spectral data with those of a known semisynthetic diterpene, compound 1 was elucidated unambiguously to be 5-episinuleptolide acetate [7]. To the best of our knowledge, norcembranoidal diterpene 1 was isolated for the first time from a natural source.
Norcembranoidial diterpene 2 was identified as scabrolide D, which had been isolated from a Formosan octocoral Sinularia scabra. Its spectral data were in full agreement with those previously reported [8].

General Procedures
Optical rotation values were measured with a Jasco-P1010 digital polarimeter. Infrared spectra were obtained on a Varian Diglab FTS 1000 FT-IR spectrophotometer. NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C in CDCl 3 at 25 °C. Proton chemical shifts were referenced to the residual CHCl 3 signal ( H 7.26 ppm). 13 C-NMR spectra were referenced to the center peak of CDCl 3 at  C 77.1 ppm. ESIMS and HRESIMS data were recorded on Bruker APEX II mass spectrometer. Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. HPLC was performed using a system comprised of a Hitachi L-7100 pump, a Hitachi L-7455 photodiode array detector and a Rheodyne 7725 injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 m) was used for HPLC.

Animal Material
Specimens of the soft coral Sinularia sp. were collected by hand using scuba equipment off the coast of Sansiantai, Taitung County, Taiwan in October 13, 2011, and stored in a freezer (−20 °C) until extraction. This organism was identified by comparison with previous descriptions [9]. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
The freeze-dried and mince material of Sinularia sp. (wet weight 1.30 kg, dry weight 328 g) was extracted with ethyl acetate (EtOAc) at 25 °C (2 L × 10). The EtOAc extract left after removal of the solvent (11.4 g) was separated by silica gel and eluted using n-hexane/EtOAc in a stepwise fashion from 100:1 to pure EtOAc to yield 13 fractions A-M. Fraction H was separated by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and EtOAc (9:4) as the mobile phase to afford compounds 1 (29.5 mg) and 2 (4.4 mg). (1) 13 C-NMR spectral data were found to be in full agreement with those of reported previously [8].

Molecular Mechanics Calculations
Implementation of the MM2 force field [5] in CHEM3D PRO software from CambridgeSoft Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to calculate molecular models.

Conclusions
Cembrane-and norcembrane-type natural products are major constituents of the extracts of Sinularia spp. octocorals distributed in the waters off Taiwan [8,. Our continuing studies on the chemical constituents of a wild-type soft coral Sinularia sp. has led to the isolation of a new natural product, 5-episinuleptolide acetate (1), which was found to exhibit modest cytotoxicity against K562, MOLT-4, HTC-116, DLD-1, T-47D and MDA-MB-231 tumor cells. This study suggested that 5-episinuleptolide acetate (1) is worthy of further biomedical investigation. The study material Sinularia sp. has begun to be transplanted to culturing tanks with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium, Taiwan for the extraction of additional natural products in order to establish a stable supply of bioactive material.