Oxygenated Cembranoids from the Soft Coral Sinularia flexibilis

Chemical examination of the Taiwanese soft coral Sinularia flexibilis led to the isolation of five cembrane-based diterpenoids 1–5, including two new metabolites, 11-acetylsinuflexolide (1) and 11-acetyldihydrosinuflexolide (2). The structures of the new metabolites were determined based on extensive spectroscopic analysis, particularly mass spectrometry and 2D NMR (1H–1H COSY, HMQC, HMBC, and NOESY) spectroscopy. Metabolites 1, 3 and 4 exhibited moderate to weak cytotoxicity to human tumor cell lines, HeLa, HEp-2, MCF-7 and MDA-MB-231.


Results and Discussion
Frozen samples of Sinularia flexibilis were extracted with EtOAc. The dry EtOAc extracts were fractionated by silica gel gravity column chromatography, and the eluted fractions were further purified by HPLC to yield cembranoids 1-5.
The HR-ESI-MS (m/z 417.2250 [M + Na] + ) of 11-acetylsinuflexolide (1) established the molecular formula C 22 H 34 O 6 , appropriate for six degrees of unsaturation. Inspection of the 13 C-NMR and DEPT spectroscopic data (Table 1) (Figures S1 and S2) showed signals of four methyls (including one acetate methyl), seven sp 3 methylenes, one sp 2 methylene, three sp 3 methines (including two oxymethines), one sp 2 methine, two sp 3 and four sp 2 quaternary carbons (including two ester carbonyls). The 13 C NMR signals appearing at δ C 166.6 (C), 140.4 (C), 125.5 (CH 2 ), 84.5 (CH), 36.7 (CH), and 29.3 (CH 2 ) were assigned to an α-exomethylenic-δ-lactone ring functionality by comparing the very similar NMR data of the cembranoids with the same six-membered lactone ring [23,24]. Resonances in the 13 C NMR spectrum of 1 at δ C 170.6 (C) supported the presence of one additional ester group ( Table 1). The ester was identified as acetate by the presence of one methyl resonance in the 1 H NMR spectrum at δ H 2.11 (3H, s) ( Table 1). Furthermore, carbon signals of three methyls (δ C 16.1, 25.4 and 25.5), one trisubstituted double bond (δ C 135.1, C; 127.2, CH), two oxygen-bearing methines (δ C 84.5 and 77.5), and two oxygenated quaternary carbons (δ C 74.8 and 73.7) were also determined. The 1 H-NMR spectral data revealed the presence of two olefinic methylene protons (δ 6.43, d, J = 2.0 Hz and 5.63, d, J = 2.0 Hz) and one olefinic methine proton (δ 5.26, dd, J = 7.5, 7.5 Hz). Furthermore, two oxygenated methines (δ 4.79, dd, J = 6.5, 2.5 Hz and 4.05, d, J = 11.5 Hz) were also designated from the 1 H NMR signals. By interpretation of 1 H-1 H COSY correlations ( Figure S5), it was possible to establish three partial structures from H-1 to H-3, from H 2 -5 to H-7, from H 2 -9 to H-11, and from H 2 -13 to H-1 through H 2 -14 ( Figure 3). These data, together with the HMBC correlations ( Figure 3) ( Figure S4) from H 2 -5 to C-3 and C-4, H 2 -9 to C-7 and C-8, and H 2 -13 to C-11 and C-12 established the connectivity within the 14-membered ring. Three methyl groups attached at C-4, C-8 and C-12 were confirmed by the HMBC correlations from H 3 -18 to C-3, C-4 and C-5, H 3 -19 to C-7, C-8 and C-9, H 3 -20 to C-11, C-12 and C-13. A 1,1-disubstituted double bond attached at C-15 was confirmed by the HMBC correlations from H 2 -17 to C-1, C-15 and C-16. Moreover, one acetoxy group positioned at C-11 was confirmed from the HMBC correlations of H-11 (δ 4.79) and protons of an acetate methyl (δ 2.11) to the ester carbonyl carbon at δ 170.6 (C). The E-configuration of one double bond at C-7/C-8 was assigned based on the 13 C NMR chemical shifts at C-19 (δ C 16.1). Thus, 1 was revealed as a cembranoid possessing an α-exomethylenic-δ-lactone ring, based on the above analysis. Furthermore, the relative stereochemistry of 1 was mostly confirmed to be the same as that of the known metabolite sinuflexolide (3) by comparison of the proton chemical shifts and coupling constants [24]. Further comparison of the 1 H and 13 C NMR data of 1 with those of 3, showed that 1 contains an extra acetyl group relative to 3. The chemical shift of H-11 in 3 (δ H 3.47, dd, J = 6.4, 2.4 Hz) was shifted downfield (δ H 4.79, dd, J = 6.5, 2.5 Hz) in 1, suggesting that 1 is the 11-acetyl derivative of 3. This was further supported by acetylation of 3 with acetic anhydride in pyridine to yield 1. Thus, compound 1 was established as the 11-acetyl derivative of 3.
11-acetyldihydrosinuflexolide (2) obtained as a white powder. The HRESIMS (m/z 419.2411, [M + Na] + ) and NMR data of 2 indicated the molecular formula, C 22 H 36 O 6 . Both the 1 H and 13 C NMR signals of 2 were found to be very closely related to those of compound 1, suggesting the same skeleton. Further comparison of NMR data of 2 with those of 1 (Table 1) (Figures S1-S10), revealed that the two exomethylene proton signals (δ H 6.43 and 5.63) in 1 was replaced by a methyl proton signal (δ H 1.35 d, J = 7.0 Hz) in 2. This was confirmed by the HMBC correlations ( Figure 3) from H 3 -17 to C-1, C-15 and C-16. The relative stereochemistry of all stereocenters except C-15 of 2 was determined to be the same as that of 1 by comparison of the proton shifts and coupling constants. The methyl group at C-15 was assigned the β-configuration primarily due to the NOE correlation between H 3 -17 and H-1. Furthermore, comparison of the NMR data between 2 and 5 confirmed both compounds have the same relative stereochemistry at C-15 [22]. Thus, the structure of 2 was established.  (Table 2).

General Procedures
Optical rotation values were measured using a Jasco P-1010 digital polarimeter. IR spectra were recorded on a Varian Digilab FTS 1000 Fourier transform infrared spectrophotometer. NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR (or Varian Unity INOVA 500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1 H-NMR and 100 MHz (or 125 MHz) for 13 C-NMR, respectively, in CDCl 3 . ESIMS and HRESIMS data were recorded with a Bruker APEX II mass spectrometer. Gravity column chomatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). Thin layer chomatography (TLC) was carried out on precoated Kieselgel 60 F254 (0.2 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 (Tokyo, Japan) and a Rheodyne 7725 injection (Cotati, CA, USA) port. A preparative normal phase column (Hibar 250 × 21.2 mm, Supelco, silica gel 60, 5 μm, Bellefonte, PA, USA) was used for HPLC.

Animal Material
The marine soft coral Sinularia flexibilis (Quoy and Gaimard, 1833) was collected by scuba divers at a depth of around 10 m off the coast of Pingtung County, Taiwan, in July 2012, and the sample was frozen immediately after collection. A voucher sample was deposited at the National Museum of Marine Biology and Aquarium, Taiwan (specimen No. 2012-0709-10).