In the investigation of secondary metabolites from marine invertebrates, several terpenoid metabolites have been isolated from cultured octocorals Erythropodium [1], Klyxum simplex [2,3,4], Sinularia flexibilis [5], Sarcophyton trocheliophorum [6], Briareum excavatum [7,8,9,10,11,12,13,14,15] and Briareum sp. [16]. Some of these metabolites have been found to possess several kinds of biological activities, such as cytotoxic [2,4,5,8,16] and anti-inflammatory activities [3,4,11,12,13,14]. The current chemical investigation of cultured octocoral Lobophytum crassum (Figure 1) led to the discovery of three new cembranoids, culobophylins A–C (1–3), and two known compounds lobophylin B (4), and lobophylin A (5) [17]. The structures of 1–5 were established by detailed spectroscopic analysis, including extensive examination of 2D NMR (¹H–¹H COSY, HMQC and HMBC) correlations. The cytotoxicity of metabolites 1–5 against human promyelocytic leukemia (HL60), human breast carcinoma (MDA-MB-231) and human colon adenocarcinoma (HCT-116 and DLD-1) cell lines 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.

The EtOAc extract of the freeze-dried specimen was fractionated by silica gel column chromatography and the eluted fractions were further separated utilizing normal phase HPLC to yield metabolites 1–5 (Chart 1).

The new metabolite culobophylin A (1) had a molecular formula of C₂₀H₃₀O₃, which was determined by HRESIMS and NMR spectroscopic data. The IR spectrum of 1 showed absorption bands at 3458 and 1694 cm⁻¹, suggesting the presence of hydroxy and carbonyl groups. The ¹³C NMR data of 1 showed the presence of 20 carbons (Table 1): three methyls, six sp³ methylenes, one sp² methylene, three sp³ methines (including two oxygenated carbons at δ 76.6 and 75.6), two sp² methines, and one sp³ quaternary carbon. The remaining three signals appearing in the lower field region of the spectrum are due to the quaternary carbons of three olefinic carbons (δ 148.2, 132.9 and 131.8) and one aldehyde carbonyl (δ 194.7). From the ¹H NMR (Table 1) spectrum of 1, the presence of one aldehyde proton resonating as a singlet at δ_H 9.56 was observed. Moreover, the ¹H NMR data revealed the presence of two olefinic methylene protons (δ 6.33, J = 1.5 Hz; 6.14, d, J = 1.5 Hz) and two olefinic methine protons (δ 5.18, dd, J = 5.0, 5.0 Hz; 4.84, d, J = 7.5 Hz). Furthermore, two oxygenated methines (δ 4.66, ddd, J = 11.0, 6.0, 5.0 Hz; 3.96, dd, J =9.5, 4.0 Hz) and three methyls (δ 1.61, s; 1.56, s; 1.10, s) were also designated from the ¹H NMR signals. The planar structure and all of the ¹H and ¹³C chemical shifts of 1 were elucidated by 2D NMR spectroscopic analysis, in particular ¹H–¹H COSY and HMBC experiments (Figure 2). From the ¹H–¹H COSY correlations (Figure 2), it was possible to establish three partial structures of consecutive proton spin systems extending from H₂-5 to H-7; H-9 to H-11; H₂-13 to H-3. The following key HMBC correlations permitted connection of the carbon skeleton: H₂-5 to C-3 and C-4; H₂-13 to C-11 and C-12; H₂-16 to C-1, C-15 and C-17; H-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 three double bonds at C-7/C-8, C-11/C-12 and C-15/C-16 and an aldehyde group at C-15. Furthermore, the HMBC cross-peak from H-14 to C-3 suggested that C-3 and C-14 were linked through an oxygen to form a tetrahydrofuran ring. The relative configuration of 1 elucidated mainly from the NOESY spectrum was compatible with that of 1 offered by using the MM2 force field calculations which suggested the most stable conformations as shown in Figure 3. In the NOESY spectrum, both H₃-18 and H-14 showed NOEs with H-1 but not with H-3. Thus, assuming the β-orientation of H-1, H₃-18 and H-14 should be positioned on the β face. Moreover, H-3 should be positioned on the α face. Also, the NOE correlations of H₃-19 with H₂-6 but not with H-7 and H₃-20, with H-10a (δ 2.33) but not with H-11, indicated the E configuration of the double bonds between C-7/C-8 and C-11/C-12. Furthermore, the relative stereochemistry of 1 was mostly confirmed to be the same as that of 4 by comparison of the proton chemical shifts and coupling constants [17]. On the basis of the above findings and other detailed NOE correlations, the structure of 1 was established unambiguously.

Culobophylin B (2) was isolated as a colorless oil with the molecular formula C₂₀H₃₂O₃, which possesses five degrees of unsaturation, as indicated by HRESIMS (m/z 343.2251, [M + Na]⁺) and NMR spectroscopic data (Table 1). In addition, ¹H and ¹³C NMR spectroscopic data (Table 1) of 2 showed the structural unit of a 3,14-oxa-bridged tetrahydrofuran. By comparison of the NMR data of 2 with that of 4, it was found that the ¹H and ¹³C NMR data of 2 were very similar to those of 4 [17]. However, the ¹H and ¹³C NMR spectroscopic data revealed that the signals corresponding to one 1,1-disubstituted carbon–carbon double bond in 4 were not present and were replaced by one 1,1-disubstituted epoxide in 2 (δ_H 2.51, 1H, d, J = 4.5 Hz and δ_H 2.43, 1H, d, J = 5.0 Hz; δ_C 54.2, C and δ_C 50.8 CH₂) (Table 1). ¹H–¹H COSY and HMBC (Figure 2) further revealed that 2 possesses one 1,1-disubstituted epoxide at C-15. On the basis of the above observations, and with the assistance of additional 2D NMR (¹H–¹H COSY and HMBC) correlations, it was possible to establish the planar structure of 2, as illustrated in Figure 2. The relative stereochemistries of all stereocenters except C-15 of 2 were confirmed to be the same as those of 1 and 4 by comparison of the proton shifts, coupling constants, and NOE correlations of 2 with those of 1 and 4.

Culobophylin C (3) was obtained as a colorless oil and showed a [M + Na]⁺ ion peak in the HRESIMS spectrum corresponding to the molecular formula C₂₀H₃₀O₃, the same as that of 2. IR absorptions were observed at 3425 cm⁻¹, suggesting the presence of a hydroxy group in 3. The ¹³C NMR spectrum of 3 showed twenty signals accounting for three methyls, five sp³ methylenes, two sp² methylenes, five sp³ methines , one sp² methine and four quaternary carbons (including one oxygenated carbon at δ 74.4 and three olefinic carbons with resonances at δ 146.5, 144.4 and 132.5). The ¹H NMR data revealed the presence of four olefinic methylene protons (δ 5.28, 5.12, 4.83 and 4.73, each a singlet). Two proton signals at δ 3.46 ddd (1H, 8.5, 2.5, 2.0) and 3.10 (1H, d, J = 2.0 Hz) correlated with two carbon signals at δ 54.5 and 61.6 and in the HMQC spectrum of 3 were attributed to the proton of one 1,2-disubstituted epoxide. The planar structure and all of the ¹H and ¹³C chemical shifts of 3 were elucidated by 2D NMR spectroscopic analysis, in particular ¹H–¹H COSY and HMBC experiments (Figure 2). Thus, 3 was found to possess three double bonds at C-8/C-19, C-11/C-12 and C-15/C-16, one hydroxy group at C-4, one 1,2-disubstituted epoxide at C-6/C-7, and an oxa-bridged ether linkage at C-3/C-14. The relative configurations of the five chiral centers at C-3, C-4, C-6, C-7 and C-14 in 3 were elucidated by detailed NOE analysis, as shown in Figure 4. In these experiments, it was found that H₃-18 showed NOE interactions with H-14 and H-7. Thus, assuming the β-orientation of H₃-18, H-7 and H-14 should be positioned on the β face. The NOE correlation observed between H-14 and H-1 also reflected the β-orientation of H-1. Furthermore, the NOESY spectrum showed NOE interaction of H₃-20 with H-10, but not with H-11, revealing the E geometry of the C-11/C-12 double bond. On the basis of these results and other detailed NOE correlations, the structure of 3 was established unambiguously.

The cytotoxicities of compounds 1–5 against HL60, MDA-MB-231, DLD-1 and HCT-116 cancer cells are shown in Table 2. The results show that compound 1, the most potent of compounds 1–5, exhibited cytotoxicity against the HL60, MDA-MB-231, DLD-1 and HCT-116 cancer cell lines with IC₅₀s of 3.0, 16.8, 4.6 and 16.3 μg/mL, respectively. Furthermore, compound 2 exhibited moderate to weak cytotoxic activity against HL60, DLD-1 and HCT-116 cancer cell lines (the IC₅₀ values were 6.8, 16.2 and 16.7 μg/mL for HL60, DLD-1 and HCT-116, respectively). The other tested compounds were not cytotoxic (IC₅₀ > 20 μg/mL) toward the above four cancer cell lines. The in vitro anti-inflammatory effects of 1–5 were also tested. Furthermore, the anti-inflammatory activity of 1–5 against the accumulation of pro-inflammatory iNOS and COX-2 proteins in RAW264.7 macrophage cells stimulated with LPS was evaluated using immunoblot analysis. At a concentration of 10 µM, compounds 1–5 did not inhibit COX-2 and iNOS proteins expression relative to the control cells stimulated with LPS only (Figure 5).

In previous reports, several 3,14-ether linkage-related cembranoids were identified from the marine soft corals Sinularia gibberosa [23,24], Sarcophyton infundibuliforme [25] and Lobophytum sp. [17]. Among these compounds, only one (3,14-epoxy-1(E),7(E),11(E)-cembratrien-4,15-diol) has been found to possess moderate cytotoxicity toward three cancer cells (A-549, HT-29 and P-388) [24]. In the present study, only compound 1 exhibited significant cytotoxicity against the growth of HL60 and DLD-1 cancer cell lines. According to the structures of 1–5, it seems that the aldehyde group in compound 1 is critical for the cytotoxic activity of metabolites 1–5. It is worth noting that metabolite 2 is rarely found in cembranoids possessing an isopropyl moiety with an epoxide group [26].