Cembrane Derivatives from the Soft Corals, Sinularia gaweli and Sinularia flexibilis

A new norcembranoidal diterpene, 1-epi-sinulanorcembranolide A (1), and a new cembranoidal diterpene, flexibilin D (2), were isolated from the soft corals, Sinularia gaweli and Sinularia flexibilis, respectively. The structures of new metabolites 1 and 2 were elucidated by spectroscopic methods, and compound 2 was found to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 proteins of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In addition, S. flexibilis yielded a known cembrane, 5-dehydrosinulariolide (3); the structure, including its absolute stereochemistry, was further confirmed by single-crystal X-ray diffraction analysis.


Introduction
Octocorals belonging to the genus Sinularia, which are distributed widely in the tropical and subtropical Indo-Pacific Ocean, have been proven to be rich sources of bioactive terpenoid analogues [1][2][3][4]. In ongoing studies on the chemical constituents from marine invertebrates collected off the waters of Taiwan, the intersection of the Kuroshio current and the South China Sea surface current, the octocorals Sinularia gaweli and Sinularia flexibilis (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Alcyoniidae) were studied, as their organic extract was found to show meaning signals in NMR studies. In previous studies, a series of interesting secondary metabolites were isolated from the octocorals, S. gaweli [5][6][7] and S. flexibilis [8][9][10][11][12][13][14][15][16][17][18], collected in the waters of Taiwan. We further isolated a new norcembranoidal diterpene, 1-epi-sinulanorcembranolide A (1), from S. gaweli and a new cembranoidal diterpene, flexibilin D (2), along with a known cembrane, 5-dehydrosinulariolide (3) [10,13,[19][20][21][22][23], from S. flexibilis (Figure 1). In this paper, we describe the isolation, structure determination and bioactivity of terpenoids 1-3.   The IR spectrum of 1 showed strong bands at 3448, 1769, 1701 and 1687 cm -1 , consistent with the presence of hydroxy, ester, ketone and α,β-unsaturated ketone groups. The 13 C NMR and DEPT spectra of 1 showed that this compound had 19 carbons (Table 1), including two methyls, four sp 3 methylenes, five sp 3 methines, an sp 3 quaternary carbon, an sp 2 methylene and six sp 2 quaternary carbons. From the 13 C NMR spectrum (Table 1), 1 was found to possess two keto carbonyls (δ C 208.7, C-3; 198.0, C-6) and an ester group (δ C 174.7, C-19). Two additional unsaturated functionalities were indicated by 13 C resonances at δ C 147.1 (C-15), 145.6 (C-8), 129.9 (C-7) and 112.3 (CH 2 -17), suggesting the presence of a tetrasubstituted olefin and an exocyclic carbon-carbon double bond. Thus, from the above data, five degrees of unsaturation were accounted for, and 1 was identified as a tetracyclic compound.    The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1, ascertained using molecular mechanics calculations (MM2) [24]. In the NOESY experiment of 1 (Figure 3), one of the C-14 methylene protons (δ H 2.11) exhibited correlations with H-1 and H-13, but not with H-5, indicated that these protons were situated on the same face and were assigned as α protons, since H-5 is a β-substituent at C-5. H-11 showed correlations with H-10 and H-13, as well as the lack of coupling detected between H-10 and H-11, indicating that the dihedral angle between H-10 and H-11 is approximately 90° and that the lactone moiety and 11-hydroxy group were β-oriented by molecular modeling analysis. From the above evidence, the relative configurations of the chiral carbons of 1 were assumed to be 1S*, 5R*, 10S*, 11R*, 12R* and 13R*. By comparison of the spectral data with those of a known norcembranoidal diterpene, sinulanorcembranolide A (4) (Figure 1), which was also isolated from S. gaweli [7], 1 was found to be the 1-epi-compound of 4 and should be named as 1-epi-sinulanorcembranolide A. To the best of our knowledge, compounds 1 and 4 are the only two norcembranoidal diterpenes with carbon-carbon linkages between C-5/13 and C-7/12. A plausible biosynthetic pathway for the compounds of this type was proposed in our previous study [7]. On the basis of the overall unsaturation data, 2 was concluded to be a diterpenoid molecule possessing two rings. The 1 H NMR spectrum of 2 showed the presence of three methyl groups: two singlets at δ H 1.35 and 1.31, representing the methyl groups on oxygenated quaternary carbons, and a vinyl methyl at δ H 1.66.    The relative stereochemistry of 2 was elucidated by the analysis of NOE correlations, as shown in Figure 5. In the NOESY experiment of 2, H-13 was found to be correlated with H 3 -20, but not with H-1; this demonstrated that H-1, H-13 and Me-20 were α-, β-and β-oriented, respectively. Additionally, correlations between H-9 and H-13, and the absence of correlation between H-9/H 3 -19, reflected the E geometry of the double bond at C-8/9. The 4-hydroxy proton was found to be correlated with one of the C-11 methylene protons (δ H 1.69), indicating that the 4-hydroxy group was α-oriented. Based on the above findings, the structure of 2 was elucidated, and the chiral carbons of 2 were assigned as 1R*, 4R*, 12R* and 13S*. The known cembrane, 5-dehydrosinulariolide (=11-dehydrosinulariolide) (3) [10,13,[19][20][21][22][23], was first isolated from an Indonesian soft coral, Sinularia flexibilis [19], and its structure was elucidated by spectroscopic and chemical methods and confirmed by single-crystal X-ray diffraction analysis [13,20]. In this study, the absolute stereochemistry of 3 was established by single-crystal X-ray diffraction analysis for the first time ( Figure 6). The configurations for all chiral carbons of 3 were assigned as 1R, 4R, 12S and 13S. The proton and carbon chemical shifts for 3 were reassigned by detailed analyses of 1D and 2D NMR spectra (Table 3).  The in vitro anti-inflammatory effects of compounds 1-3 were tested. In this assay, the upregulation of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins expression of LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells was evaluated using immunoblot analysis. At a concentration of 20 μM, compound 2 was found to significantly reduce the levels of iNOS and COX-2 to 19.27 ± 2.72 and 30.08% ± 9.07%, respectively, relative to the control cells stimulated with LPS only (Figure 7). Levels of β-actin protein (internal control) demonstrated no significant difference among concentrations of 10, 20 and 50 μM of compound 2 or compared with LPS only. Thus, compound 2 might be promising as an anti-inflammatory agent, as it does not exhibit cytotoxicity to RAW264.7 macrophage cells.  Relative intensity of the lipopolysaccharide (LPS)-alone stimulated group was taken as 100%. Under the same experimental condition, CAPE (caffeic acid phenylethyl ester, 10 μM), reduces the levels of the iNOS and COX-2 to 2.8 ± 4.6 and 66.7% ± 9.6%, respectively. * Significantly different from LPS-alone stimulated group (p < 0.05).

Sinularia gaweli
Specimens of the octocoral, S. gaweli, were collected by hand using scuba equipment off the coast of Sansiantai, Taitung County, Taiwan in October, 2011, and stored in a freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-11007) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Sinularia flexibilis
Specimens of the octocoral, S. flexibilis, were collected by hand using scuba equipment off the coast of Southern Taiwan in July, 2011, and stored in a freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-11005) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Sinularia gaweli
Specimens of the soft coral, S. gaweli (wet weight 1.30 kg, dry weight 328 g), were extracted with ethyl acetate (EtOAc). 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-pure EtOAc to yield 13 fractions, A-M. Fraction I was separated by NP-HPLC, using a mixture of n-hexane and acetone (5:1) to yield 8 subfractions, 1-8.

Sinularia flexibilis
Sliced bodies of the soft coral, S. flexibilis (wet weight 3.0 kg, dry weight 950 g), were extracted with ethyl acetate (EtOAc). The EtOAc extract was separated by silica gel and eluted using n-hexane/EtOAc in a stepwise fashion from pure n-hexane-100:1-pure EtOAc to yield 11 fractions, A-K. Fraction H was repurified by NP-HPLC using a mixture of n-hexane and acetone (7:1, flow rate: 2.0 mL/min) as the mobile phase to afford 3 (58.7 mg, t R = 240 m). Fraction J was chromatographed on silica gel and eluted using n-hexane/EtOAc (stepwise, 2:1-1:1) to afford subfractions 1-10. Fraction J7 was separated by NP-HPLC using a mixture of n-hexane and acetone (2:1, flow rate: 2.0 mL/min) as the mobile phase to afford 2 (3.1 mg, t R = 85 m).

Molecular Mechanics Calculations
Implementation of the MM2 force field [24] in CHEM3D PRO software from CambridgeSoft Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to calculate molecular models.

In Vitro Anti-Inflammatory Assay
The macrophage (RAW264.7) cell line was purchased from ATCC. In vitro anti-inflammatory activity of compounds 1 and 2 was measured by examining the inhibition of lipopolysaccharide (LPS)-induced upregulation of iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in macrophage cells using Western blotting analysis [27][28][29].

Conclusions
In the present study, a new norcembranoidal diterpene, 1-epi-sinulanorcembranolide A (1), and a new cembranoidal diterpene, flexibilin D (2), were isolated from the soft corals, Sinularia gaweli and Sinularia flexibilis, respectively. In addition, a known cembrane, 5-dehydrosinulariolide (=11-dehydrosinulariolide) (3), which was found to show antitumor activities toward CAL-27 (human oral adenosquamous carcinoma) [30] and A2058 (human Caucasian metastatic melanoma) [31] cells and displayed neuroprotection in an in vitro Parkinson's model [32], was also obtained from S. flexibilis. The structure, including its absolute stereochemistry of 3 was further confirmed by single-crystal X-ray diffraction analysis for the first time in this study. Moreover, in the anti-inflammatory test, compound 2 was found to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 proteins of the LPS-stimulated RAW264.7 macrophage cells. Because octocorals are claimed to be endangered species and based on the potential medicinal use, the soft coral S. flexibilis has begun to be transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for exhibition and the extraction of additional natural products to establish a stable supply of bioactive material.