Our previous chemical examination of soft corals of the genus Sinularia led to the isolation and identification of various oxygenated cembrane-type metabolites [1–5]. Some of these metabolites exhibit anti-inflammatory activity [2–4] and/or cytotoxicity to the growth of some cancer cell lines [1]. Our ongoing research to discover bioactive metabolites from the soft coral Sinularia triangular (Tixier-Durivault, 1970; family Alcyoniidae) (Figure 1) led to the isolation of two new cembrane-based diterpenoids, triangulenes A (1) and B (2), along with three known metabolites, sinularin (3) [6], dihydrosinularin (4) [6], and (−)14-deoxycrassin (5) [7]. The structures of 1 and 2 were established by extensive spectroscopic analysis, including 2D NMR spectroscopy. The cytotoxicity of 1–5 to the human tumor cell lines CCRF-CEM (T-cell acute lymphoblastic leukemia) and DLD-1 (colon adenocarcinoma) was studied, and the ability of 1–5 to inhibit the expression of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in lipopolysaccharide (LPS)–stimulated RAW264.7 macrophage cells was also evaluated.

Frozen samples of S. triangular 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 (Figure 2).

The HRESIMS spectrum of triangulene A (1) contained a molecular ion peak consistent with the molecular formula C₂₀H₃₂O₂, indicating the molecule has five double-bond equivalent. A UV absorption maxima at 240 nm (logɛ = 4.0) was attributed to double bond conjugation. The IR spectrum of 1 revealed the presence of a carbonyl functionality (ν_max = 1703 cm⁻¹). The ¹³C NMR data of 1 showed the presence of 20 carbons (Table 1): five methyls, six sp³ methylenes, three sp³ methines (including an oxygenated carbon at δ 62.7), two sp² methines, and four quaternary carbons (including an oxygenated carbon at δ 61.5, two olefinic carbons with resonances at δ 148.3 and δ 129.3, and a keto-carbonyl at δ 209.8). The ¹H NMR data revealed the presence of two olefinic methine protons as doublets at δ 6.15 and δ 6.06. A proton signal at δ 2.71 (1H, dd, J = 8.4, 4.0 Hz) that correlated with a carbon signal at δ 62.7 in the HMQC spectrum of 1 was attributed to the proton of a trisubstituted epoxide. The gross planar structure of 1 was determined by detailed analysis of its 1D and 2D NMR spectra. From the ¹H–¹H COSY correlations (Figure 3), it was possible to establish five partial structures of consecutive proton spin systems extending from H-2 to H-3; H-8 to H₃-19; H₂-9 to H-11; H₂-13 to H₂-14; and H-15 to H₃-16 and H₃-17. The following key HMBC correlations permitted connection of the carbon skeleton: H-2 to C-1, C-14, and C-15; H-3 to C-5; H-5 to C-4 and C-6 (carbonyl carbon); H-7 to C-6, C-8, and C-9; H-13 to C-11 and C-12; H₃-16 and H₃-17 to C-1 and C-15; H₃-18 to C-3, C-4, and C-5; H₃-19 to C-7, C-8, and C-9; and H₃-20 to C-11, C-12, and C-13. Thus, 1 was found to possess a tetrasubstituted diene at C-1/C-2 and C-3/C-4, a ketone group at C-6, and a trisubstituted epoxide at C-11/C-12. The above results indicate that 1 possessed the same molecular framework as known cembranoids 6 and 7 (Figure 4), which were isolated previously from octocorals Eunicea tourniforti [8] and Eunicea sp. [9], respectively.

The relative configuration of 1 was determined from NOE correlations observed in the NOESY spectrum (Figure 5). The NOE correlations between H-2 and methyl protons H₃-16 and H₃-18 and between H-3 and H₂-5 indicated E configurations for the double bonds at C-1/C-2 and C-3/C-4. In addition, one proton of C-10 methylene (δ 1.92) was found to exhibit correlations with H-11 (δ 2.71, dd, J = 8.4, 4.0 Hz) and H₃-19 (δ 0.93, d, J = 6.8 Hz), indicating that these protons were situated on the same face; they were assigned as α protons, as C-20 methyl was β-oriented at C-12, which were verified by the absence of correlation between H-11 and H₃-20. Furthermore, H₃-20 correlated with protons of C-10 (δ 1.92 and 1.14) and C-14 (δ 2.37 and 2.28) methylenes, respectively. Consideration of molecular models found that H₃-20 was reasonably close to H₂-10 and H₂-14 when H₃-20 was β-oriented. Based on the above findings, the structure of 1, including its relative configuration was established, and the chiral centers for 1 were assigned as 8S*, 11S*, and 12S*. Furthermore, the chemical shifts of 1 were shifted downfield at C-7 (Δδ_C +1.7 ppm) and C-8 (Δδ_C +2.5 ppm) and upfield at C-19 (Δδ_C −0.8 ppm) relative to the corresponding chemical shifts of 7. On the basis of the above findings, we determined the relative structure of 1, which was determined to be the C-8 epimer of 7.

Triangulene B (2) had the same molecular formula (C₂₀H₃₂O₂) as 1, as indicated by HRESIMS and NMR spectra (Table 1). Comparison of the ¹H and ¹³C NMR data of 2 with those of 1 revealed that the two compounds possessed similar structures. The trisubstituted double bonds at C-1/C-2 and C-3/C-4 of 2 had Z geometries, as indicated by NOE interactions (Figure 5) between H-3 (δ 6.29) and H₃-18 (δ 1.93) and between H-2 (δ 6.19) and H-5 (δ 3.90). After determining the structure of 2, we discovered that its planar structure has been obtained previously as diterpenoid 8 from the octocoral Eunicea sp. [8]. Furthermore, we found that the NMR data for 2 were similar to those of 8, except that C-7 and C-8 of 2 were shifted markedly downfield (Δδ_C +3.9 ppm and Δδ_C +3.6 ppm, respectively) relative to the corresponding carbons of 8. Further analysis of other NOE interactions revealed that 1 and 2 possessed the same relative configurations at C-8, C-11, and C-12. Thus, the structure of 2 was established unambiguously.

Study of the cytotoxicity of diterpenoids 1–5 to human tumor cell lines CCRF-CEM and DLD-1 showed that 3 and 5 moderately inhibited the growth of the tested cell lines (the ED₅₀ values were 26.0 and 37.1 μM for 3 and 29.8 and 32.2 μM for 5 for CCRF-CEM and DLD-1, respectively). The in vitro anti-inflammatory effects of 1–5 were also tested. The inhibition of LPS-stimulated upregulation of the pro-inflammatory proteins iNOS and COX-2 in RAW264.7 macrophage cells was measured by immunoblot analysis. At a concentration of 10 μm, 3–5 reduced the levels of the iNOS protein to 1.2 ± 0.3%, 5.1 ± 1.6%, and 0.9 ± 0.7%, respectively, of the levels in control cells stimulated with LPS alone (set at 100%). At the same concentration, 4 and 5 markedly reduced the levels of COX-2 to 24.9 ± 7.4% and 5.9 ± 1.0%, respectively, relative to controls (Figure 6).