Terpenoids from the Octocoral Sinularia gaweli

Two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembrane diterpenoids, (–)-leptodiol acetate (3) and sinulacembranolide A (4) were isolated from the octocoral Sinularia gaweli and compounds 2–4 are new isolates. The structures of new terpenoids 2–4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 marcophage cells.


Results and Discussion
A known eudesmane sesquiterpenoid, verticillatol (1), was obtained in this study. This compound had been obtained from a Vietnam plant Litsea verticullata [6]. The NMR data and rotation value of 1 were identical to those of verticillatol described previously.
5α-Acetoxy-4(14)-eudesmene-1β-ol (2) was isolated as a colorless oil and the molecular formula for this compound was determined to be C17H28O3 (four units of unsaturation) using HRESIMS (C17H28O3 + Na, m/z 303.19293, calculated 303.19307). Comparison of the 13 C NMR and distortionless enhancement by polarization transfer (DEPT) data with the molecular formula indicated that there was an exchangeable proton, which required the presence of a hydroxy group. This deduction was supported by a broad absorption in the IR spectrum at 3465 cm -1 . The IR spectrum also showed a strong band at 1735 cm -1 , consistent with the presence of an ester group. From the 1 H and 13 C NMR spectra (Table 1), 2 was found to possess an acetoxy group (δH 2.03, 3H, s; δC 168.9, C; 21.8, CH3). An additional unsaturated functionality was indicated by 13 C resonances at δC 111.6 (CH2-14) and 145.1 (C-4), suggesting the presence of an exocyclic carbon-carbon double bond. Thus, the proposed skeleton of 2 was suggested to be a bicyclosesquiterpenoid.  From the 1 H-1 H COSY and HMBC spectrum of 2 (Table 1), permitted elucidation of the main eudesmane carbon skeleton. The relative configuration of 2 was elucidated by means of a NOESY (nuclear overhauser effect spectroscopy) experiment ( Figure 2). The NOEs of H-9α to H-1 and H-7α revealed the hydroxy group at C-1 and isopropyl group at C-7 to be β-oriented. The NOEs between H3-15/H-2β, H3-15/H-6β, and H3-15/H-8β assigned the methyl group at C-10 as β. 23 D +108 (c 0.8, MeOH)) [7] (In this reference, the alphabetical orders for C-14 and C-15 in this compound should be exchanged from the IUPAC recommendation), suggested that the acetoxy group at C-5 should be α-oriented. Since the absolute configuration of 4(15)-eudesmene-1β, 5α-diol had been determined by modified Mosher's method [7], we were able to assign the absolute configurations of all the chiral centers of 2 as 1R, 5S, 7R, 10S. Based on the above findings, the structure of 2 was, therefore, determined to be 5α-acetoxy-4(14)-eudesmene-1β-ol. The spectral ( 1 H, 13 C NMR and IR) data of 3 were in full agreement with those of a known cembrane analogue, leptodiol acetate, which was isolated from a Panama gorgonian coral identified as Leptogorgia sp. [8]. However, the optical rotation value of 3 ( The molecular formula for cembranoid 4 (sinulacembranolide A) was determined to be C25H30O10 (11 units of unsaturation) using HRESIMS (C25H30O10 + Na, m/z 513.17338, calcd. 513.17312). Absorption for hydroxy and carbonyl groups at 3481, 1756, and 1721 cm -1 were observed in the IR spectrum. The 13 C NMR and DEPT spectra of 4 (Table 2) showed the presence of 25 carbon signals assigned to 5 × CH3 (one methoxy group, two from acetyl groups), 4 × CH2 (one olefinic), 6 × CH (two olefinics and three oxymethines) and 10 quaternary carbons (four carbonyls and five olefinics). 1 H and 13 C NMR data of 4 ( Table 2) were similar with those of 3, particularly the chemical shifts for 11,12-epoxy group in 3 (δH 4.10, 1H, br s, H-11; δC 63.0, CH-11; 59.0, C-12) were replaced by a carbon-carbon double bond (δH 6.16, 1H, d, J = 1.2 Hz, H-11; δC 154.0, CH-11; 129.8, C-12) for molecule 4. Connectivity information obtained from 2D NMR, including 1 H-1 H COSY and HMBC experiments, unambiguously determined the planar structure of 4 ( Table 2). The relative configuration of 4 was elucidated from NOESY correlations (Figure 3). In the NOESY experiment for 4, it was found that one of the methylene protons at C-2 (δH 2.82) exhibited a correlation with H-1, but not with H-13 and, therefore, it was assigned as H-2α, and the other C-2 proton (δH 3.57) as H-2β. H-13 showed correlations with H-2β and H-11, but not with H-1, and H-11 showed a correlation with H-10, as well as a small coupling (J = 1.2 Hz) detected between H-10 and H-11, indicating the dihedral angle between H-10 and H-11 is approximately 90° and the geometry of the C-11/12 carbon-carbon double bond was Z form and C-10 possessing an relative configuration R*-form. H3-19 correlated with H-10 and H-11, but not with H-7, indicating that Me-19 and H-7 were β-and α-oriented at C-8 and C-7, respectively. From the above evidence, the relative configuration of chiral carbons of 4 were assumed to be 1S*, 7R*, 8S*, 10R*, and 13S*.
To the best of our knowledge, the C-7, C-8 vicinal diol-based cembranoid analogues are rarely found [9]. In previous studies on S. gaweli had afforded an interesting norcembranoid, sinulanorcembranolide A [3], and the biosynthetic pathway for sinulanorcembranolide A was proposed in a later study [10]. This observation is in agreement with the observation that cembrane diterpenoids from Alcyonacea have opposite configuration at C-1 compared to those obtained from Gorgonacea [11].    In the in vitro anti-inflammatory activity test, the upregulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins expression of LPS (lipopolysaccharide) -stimulated RAW264.7 macrophage cells was evaluated using immunoblot analysis. At a concentration of 10 μM, compound 4 was found to significantly reduce the levels of iNOS to 8.55% ± 3.32%, relative to the control cells stimulated with LPS only (Figure 4). Thus, compound 4 might be promising as an anti-inflammatory agent, as this compound did not exhibit cytotoxicity to RAW264.7 macrophage cells.

Animal Material
Specimens of the octocoral Sinularia gaweli (Verseveldt, 1978) were collected by hand using self-contained underwater breathing apparatus (SCUBA) equipment off the coast of Sansiantai, Taitung county, Taiwan on 7 May 2013, and stored in a freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-13031) was deposited in the National Museum of Marine Biology & Aquarium, Taiwan.

In Vitro Anti-Inflammatory Assay
Murine macrophage (RAW264.7) cell line was purchased from ATCC. In vitro anti-inflammatory activity of compounds 1-4 was measured by examining the inhibition of lipopolysaccharid (LPS)-induced up-regulation of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in macrophage cells using Western blotting analysis [12][13][14]. Briefly, inflammation in macrophages was induced by incubating them for 16 h in a medium containing only LPS (10 ng/mL) without compounds. For anti-inflammatory activity assay, compounds 1-4 and dexamethasone (10 μM) were added the cells 10 min before LPS challenge. The cells then underwent Western blot analysis. The immunoreactivity data are calculated with respect to the average optical density of the corresponding LPS-stimulated group. For statistical analysis, the data were analyzed by a one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls post hoc test for multiple comparisons. A significant difference was defined as a p value of <0.05.

Conclusions
Our studies on Sinularia gaweli for the extraction of additional natural substances, have led to the isolation of two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembranoids, (-)-leptodiol acetate (3) and sinulacembranolide A (4) and compounds 2-4 are new isolates. Terpenoid 4 is potentially anti-inflammatory and may become a lead compound in marine anti-inflammatory drug development. The octocoral Sinularia gaweli will be transplanted to culturing tanks located in the National Museum of Marine Biology & Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material.