Cembrane diterpenoids and their cyclized derivatives are the most abundant secondary metabolites of soft corals and gorgonians [1,2,3]. There is a wide range of structural complexity within this series. These cembranes represent the main chemical defense tools of animals against their natural predators such as other corals, and fishes, as well as the settlement of microorganisms [4,5]. In addition, cembranes also exhibit a wide range of biological activities including anti-inflammatory [6,7,8], and antitumor properties [9,10].

Genus Sinularia is one of the most widely distributed soft corals. It constitutes a dominant portion of the biomass in the tropical reef environment. Sinularia elaborates a rich harvest of secondary metabolites, including sesquiterpenes, diterpenoids, polyhydroxylated steroids, and polyamine compounds [11,12,13,14]. These metabolites were recently shown to possess a range of biological activities [15]. Cembranes are the most frequent secondary metabolites isolated from various Sinularia species [16,17,18].

Nuclear factor-kappa B (NF-κB) is a protein complex that controls the transcription of DNA. NF-κB plays a key role in regulating the immune response to infection. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development [19]. Our recent investigation of bioactive natural products from a Hainan soft coral, Sinularia sp., has led to the isolation of five new cembranes (1–5), along with nine other known diterpenoids (6–14) (Figure 1). In this paper, we report the isolation and structural elucidation of these diterpenoids and their activities as inhibitors of NF-κB.

Compound 1 was isolated as a colorless oil. The HRESIMS of 1 established its molecular formula as C₂₁H₃₈O₆, indicating three unsaturations. Resonances due to olefinic carbons (δ_C 133.9, 128.7), and one carboxyl (δ_C 178.9) in the ¹³C NMR spectrum accounted for two double-bond equivalents, indicating that 1 was a monocyclic compound (Table 1). Signals for a vinyl methyl at δ 1.68 (3H, s), one methoxy group at δ 3.68 (3H, s), a methyl doublet at δ 1.17 (3H, d, J = 7.0 Hz), and two tertiary oxygenated methyl groups at δ 1.15 (3H, s), and 1.08 (3H, s) were observed in the ¹H NMR spectrum (Table 2). Carbon signals at δ 70.6, 71.0, 75.3, and 75.7, and two proton signals at δ 3.52, and 3.65 indicated the presence of two secondary and two tertiary hydroxyl groups. A signal at δ 5.43 attributed to an olefinic proton, together with a methyl carbon signal at δ 17.0, indicated the E configuration for this double bond. These data suggested that 1 was a rearranged cembrane. Key HMBC correlations from H₃-20 to C-13, C-12, and C-11; H-11 to C-12, C-10, and C-20; H₃-19 to C-7, C-8, and C-9; H-7 to C-9, C-6, and C-19; H₃-18 to C-5, C-4, and C-3; and H-13 to C-14, and C-1 established the 14-membered ring structure of 1 (Figure 2). The isopropyl acid group was found based on the HMBC correlations observed from H₃-21 to C-1, C-15, and C-16; H-15 to C-1, C-2, C-21, and C-16. Furthermore, the methoxyl substituent was shown to be connected to position C-16 on the basis of the HMBC correlation between the oxymethyl protons (δ_H 3.68) and the carbonyl carbon (δ_C 178.9). The NMR spectra of compound 1 were almost identical with those of sinuflexibilin [20] with the exception that the exo-methylene proton resonances of the latter were replaced by a methyl doublet. The stereochemistry of 1 was determined on the basis of the chemical shift and NOESY spectrum (Figure 3). NOE correlations from H-3 to H-1, H-11, H₃-18 and from H-11 to H-20 indicated that all four hydroxy groups in 1 were β-oriented and H-1, H-3, H-11, H₃-18, and CH₃-20 were α-oriented with respect to this ring.

Compound 2 was isolated as a colorless oil. The HRESIMS of 2 established its molecular formula as C₂₀H₃₄O₅, indicating four unsaturations. The ¹H and ¹³C NMR spectra of 2 were similar to those of capillolide [21], with the exception that the exo-methylene proton resonances of the latter were replaced by a methyl doublet at δ 1.28 (3H, d, J = 7.5 Hz) coupled to a signal at δ 2.92. The relative stereochemistry of 2 was deduced mainly by NOESY and by comparison with that found for capillolide. Both H-3 and H-11 showed NOEs with H-1, which further correlated with H-15. Moreover, H₃-18 shared mutual NOE enhancement with H-3 and H-15. These observations indicated that all H-1, H-3, H-11, H₃-18, and H-15 were α-oriented with respect to this ring. Furthermore, it was found that the H₃-20 did not exhibit NOE response with H-11, indicating the β-configuration.

Compound 3 was isolated as a colorless oil. The HRESIMS of 3 established its molecular formula as C₂₁H₃₄O₄, indicating five unsaturations. The ¹H and ¹³C NMR spectra of 3 were similar to those of pseudoplexauric acid methyl ester except for the downfield shifts of C-3 (62.8→71.7), C-4 (60.7→75.2), and C-18 (16.9→23.4) (Table 1) [22], which indicated that two hydroxylated carbons replaced the 3,4-epoxy carbons of the known analogue. This was also supported by the molecular weight of 3, which was 18 amu higher than that of pseudoplexauric acid methyl ester, as indicated by the HRESIMS data. In the NOESY spectrum of 3, H-3 showed NOE with H-1, but not with H-18, justifying the assigned relative stereochemistry at C-1, C-3, and C-4.

Compound 4 was isolated as a colorless oil. The HRESIMS of 4 established its molecular formula as C₂₀H₃₂O₃, indicating five unsaturations. The ¹H and ¹³C NMR spectra of 4 were similar to those of 14-deoxycrassin [22,23], with the exception that the exo-methylene proton resonances of the latter were replaced by a methyl doublet at δ 1.32 (3H, d, J = 7.0 Hz) coupled to a signal at δ 2.09, which was confirmed by the HMBC experiment. Analyses of NOESY and NMR data revealed that the relative stereochemistry of 4 was the same as 14-deoxycrassin. The relative configuration of the secondary methyl group at C-15 was assigned to be on the same side as the H-1 proton of the δ-lactone ring by comparison of ¹H NMR spectral data with those of dihydrosinularin [δ 1.35 (3H, d, J = 7 Hz, H₃-17), 2.2 (1H, m, H-15)] and its 15-epimer [δ 1.21 (3H, H₃-17), 2.80 (1H, m, H-15)] [20,24].

Compound 5 was isolated as a colorless oil. The HRESIMS of 5 established its molecular formula as C₂₂H₂₈O₈, indicating nine unsaturations. The ¹H and ¹³C NMR spectra of 5 showed great similarity to those of 3α-ethoxyfuranocembrane [25] except that the ethoxyl was replaced by a methoxyl at C-3, and the acetoxy group was replaced by a hydroxy at C-11. The determination of the structure of 5 was further supported by detailed analysis of its 2D NMR data (Figure 2). The relative configuration of 5 was deduced by a NOESY experiment (Figure 3) and by comparison with those of 3α-ethoxyfuranocembranoid [25]. One proton attaching at C-2 and resonating at δ_H 2.48 was found to show NOE interactions with H-1 and was assigned arbitrarily as H-2α. The isopropenyl group located at C-1 should be β-oriented. NOE correlations between H-5 and H-7, H-7 and H-19 revealed the α-orientation of Me-19, and the Z configuration of the double bond Δ⁶⁽⁷⁾. The NOE interaction between H₃-19 and H₃-22 indicated the α-orientation of C-3. The trans-arrangement of H-10, and H-11 was implied by the coupling constant J (10, 11) ≈ 0 Hz. The NOE interaction between H-11 and H-13 indicated the Z configuration of the double bond at Δ¹²⁽¹³⁾ [25]. The 3α-ethoxyfuranocembranoid is an artifact created during isolation by reaction of the solvent ethanol with the natural product danielid [25]. However the danielid and its analogues have not been isolated in our investigation from Sinularia sp. In this context, whether compound 5 is created from danielid or its analogues remains to be established.

The identities of compounds 6–14 were established by comparison of their spectral data with those of the known compounds reported. They are capilloloid (6) [21], sethukarailin (7) [26], sinuladiterpenes I (8) [27], sinulaflexiolides H (9) [28], flexibilisin A (10) [29], (1R,13S,12S,9S,8R,5S,4R)-9-acetoxy-5,8:12,13-diepoxycembr-15(17)-en-16,4-olide (11) [21], 11-epi-sinulariolide acetate (12) [30], flexibilide (13) [21], mandapamate (14) [31].

Compounds 1–14 were evaluated for inhibition of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay. The results showed that only 13 exhibited a potent effect with IC₅₀ value of 5.30 μg/mL, while other compounds showed only marginal effects.

The investigation of bioactive natural products from a Hainan soft coral, Sinularia sp., has led to the isolation of five new cembranes, sinuflexibilins A–E (1–5), along with nine other known diterpenoids (6–14). Compound 13 exhibited significant inhibition activity of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay with an IC₅₀ of 5.30 μg/mL.