Marine organisms, which have developed unique metabolic and physiological capabilities to ensure survival in extreme ocean habitats, offer the potential to produce new bioactive constituents that would not be observed from terrestrial organisms [1]. Soft corals belonging to the genus Sarcophyton (Alcyoniidae) have been well recognized as a rich source of terpenoids [1]. These constituents, mainly macrocyclic cembrane-type diterpenoids and their derivatives, represent important chemical defense substances for the animals against their natural predators [2]. Cembranoids have been previously reported to exhibit a range of biological activities including antitumor [3,4,5,6,7,8,9], ichthyotoxic [10], anti-inflammatory [11], neuroprotective [12], antibacterial [13], antiangiogenic [14], antimetastatic [14], anti-osteoporotic [15], and cytotoxic [16,17,18] properties. Among them, sarcophine (4) was reported to have antimetastatic activity [14].

Twelve cembranoids were previously reported from the soft coral Sarcophyton ehrenbergi [1,19]. The samples for our previous studies on the secondary metabolites of the soft coral S. ehrenbergi were all collected at Dongsha Atoll [19,20]. Chemical investigation of the Taiwanese soft coral S. ehrenbergi (Figure 1) collected at San-Hsian-Tai (Taitong County) has afforded three new cembranoids, designated as (+)-12-ethoxycarbonyl-11Z-sarcophine (1), ehrenbergol A and B (2 and 3) (Figure 2). Herein, we describe the purification, structure elucidation, cytotoxicity and antiviral evaluation of these metabolites in detail.

The HRESIMS of 1 exhibited a pseudomolecular ion peak at m/z 397.1993 [M + Na]⁺, consistent with the molecular formula of C₂₂H₃₀O₅, requiring eight degrees of unsaturation. IR absorption at 1754 cm⁻¹ and NMR signals (Table 1) at δ_C 174.4 (qC, C-16), 160.7 (qC, C-1), 124.2 (qC, C-15), 78.2 (CH, C-2), and 8.6 (CH₃, C-17); δ_H 5.57 (1H, dd, J = 10.0, 2.0 Hz, H-2) and 1.90 (3H, s, H₃-17) were indicative of an α,β-unsaturated γ-lactone functionality by comparison with those of similar metabolites, such as the corresponding data of sarcophine (4) [6]. The IR absorption bands at 1705 cm⁻¹ and NMR signals at δ_H 6.80 (1H, dd J = 10.4, 4.8 Hz, H-11), 4.23 (2H, m, H₂-21), 1.32 (1H, t, J = 7.2 Hz, H-22); δ_C 166.9 (qC, C-20), 131.1 (qC, C-12), and 142.0 (CH, C-11) indicated the presence of α,β-unsaturated ethyl ester [20]. In addition, a trisubstituted epoxide was present in 1 from its ¹H NMR signals at δ_H 2.56 (1H, br d, J = 6.4 Hz, H-7) and ¹³C NMR signals at δ_C 61.3 (qC, C-8) and 62.4 (CH, C-7). Moreover, the ¹³C NMR signals at δ_C 121.1 (CH, C-3), and 144.5 (qC, C-4) were assigned a trisubstituted double bond. The above functionalities account for seven of the eight degrees of unsaturation, suggesting a tricyclic structure in 1.

By interpretation of ¹H-¹H COSY correlations, it was possible to establish three partial structures of consecutive proton systems extending from H-2 to H-3, from H₂-5 to H-7 through H₂-6, from H₂-9 to H-11 through H₂-10, and from H₂-13 to H₂-14. Subsequently, the connectivities of these partial structures were further established by the HMBC correlations (Figure 3). HMBC correlations observed from H₃-19 to C-7, C-8, and C-9 indicated the position of the epoxide at C-7 and C-8. Moreover, the HMBC correlations from H-11 to C-9, C-10, C-12, C-13, and C-20 and from H₂-21 to C-20 as well as COSY correlation between H₂-21 and H₃-22, led the assignment of the ethoxycarbonyl at C-12. The locations of the double bond at C-3/C-4 was clarified by analysis of the HMBC correlations from Me-18 to C-3, C-4, and C-5. The molecular framework of 1 was further established by other HMBC correlations between H₂-14 to C-1, C-2, C-15 and H₃-17 to C-1, C-15, C-16.

The relative configuration of 1 assigned by a NOESY spectrum was compatible with that suggested by computer modeling, in which the close contacts of atoms calculated in space were consistent with the NOESY correlations (Figure 4). The presence of a NOESY cross peak between the vinylic H-11 and H₂-9 (δ_H 2.22) suggested the E geometry for the C-11/C-12 double bond, which was also identified by the chemical shift of H-11 at δ_H 6.80 [19,21]. The geometry of the trisubstituted olefin at C-3/C-4 was assigned as E based on the higher field chemical shift of the olefinic methyl signal for C-18 (δ_C 15.3). Furthermore, the crucial NOE correlations between H-2/Me-18, Me-18/H-6b (δ_H 1.72), Me-19/H-6b, Me-19/H-9b (δ_H 2.22), H-7/H-6a (δ_H 1.92), and H-7/H-9b, H-7/H-5a (δ_H 2.39), and H-3/H-5a demonstrated the 2S*, 7S*, and 8S* configurations as depicted in Figure 4. A careful analysis of all the NMR spectroscopic data (COSY, HSQC, HMBC, and NOESY) confirmed that 1 is actually the 12-ethoxycarbonyl derivative of (+)-11Z-sarcophine [19,22]. All of the NMR spectroscopic data of 1 were consistent with the structure shown as (+)-12-ethoxycarbonyl-11Z-sarcophine.

Ehrenbergol A (2) was assigned a molecular formula of C₂₁H₃₂O₄, according to its HRESIMS and NMR spectroscopic data (Table 2). The IR absorptions of 2 at 1715 cm⁻¹ revealed the presence of an α,β-unsaturated methyl ester functionality, which was confirmed by its NMR spectroscopic data [δ_H 6.89 (1H, dd, J = 10.4, 6.8 Hz, H-11) and 3.42 (3H, s, H₃-21); δ_C 167.6 (qC, C-20), 133.9 (qC, C-12), 141.1 (CH, C-11), and 51.2 (CH₃, COOMe)]. The NMR spectroscopic data also indicated that 2 possesses a trisubstituted epoxide [δ_H 2.67 (1H, dd, J = 10.8, 2.8 Hz, H-7); δ_C 60.6 (qC, C-8) and 62.1 (CH, C-7)], and two trisubstituted olefins [δ_H 6.53 (1H, d, J = 11.0 Hz, H-2) and 6.08 (1H, d, J = 11.0 Hz, H-3); δ_C 147.1 (qC, C-1), 118.4 (CH, C-2), 123.8 (CH, C-3), and 135.8 (qC, C-4)]. The above functionalities account for five of the six degrees of unsaturation, suggesting that 2 must consist of a 14-membered ring diterpenoid skeleton.

The structure of 2 was established through COSY and HMBC experiments (Figure 3). Crucial HMBC correlations from Me-18 to C-3, C-4, and C-5, from Me-19 to C-7, C-8, and C-9, from H-11 to C-10, C-12, C-13, and C-20, and from Me-16/Me-17 to C-15 and C-1 confirmed the connectivity among these partial structures (Figure 3). The position of the methoxycarbonyl at C-12 was established by the HMBC correlations from H-11 and H-13 to C-20 and from H₃-21 to C-20. A COSY experiment established a correlation between the two vinylic protons at δ_H 6.53 (H-2) and 6.08 (H-3). These results allowed the assignment of the planar structure of 2 as shown.

The configurations of all double bonds were determined from a NOESY experiment on 2. The crucial NOE correlations (Figure 5) between H-2 (δ_H 6.53)/Me-16 (δ_H 1.33), H-2/Me-18 (δ_H 1.44), and H-3 (δ_H 6.08)/H-5a (δ_H 2.12) indicated that the geometries of the conjugated diene at C-1/C-2 and C-3/C-4 were both E. The large coupling constant (J_2,3 = 11.0 Hz) further suggested the s-trans geometry of the conjugated double bonds [20,21]. The presence of a NOESY cross peak between the vinylic H-11 and H₂-9 made it possible to identify the configuration of the olefin at C-11/C-12 as the E geometry, which was also confirmed by the chemical shift of H-11 at δ_H 6.89 [20]. Moreover, the crucial NOESY correlations between Me-19/H-6b (δ_H 1.16), Me-19/H-9b (δ_H 1.94), H-7/H-6a (δ_H 1.97), H-7/H-9b (δ_H 1.95), and H-7/H-5a (δ_H 2.12) demonstrated the configurations of C-7 and C-8 as 7S∗ and 8S*, respectively. On the basis of the aforementioned observations and other detailed NOESY correlations (Figure 4), the structure of ehrenbergol A (2) was established.

The positive HRESIMS spectrum of ehrenbergol B (3) exhibited a pseudo molecular ion peak at m/z 387.2509 [M + Na]⁺, consistent with the molecular formula of C₂₂H₃₆O₄, implying five degrees of unsaturation. The presence of two oxygenated methine [δ_H 3.40 (d, 1H, J = 7.6 Hz) and δ_C 85.1 (C-7); δ_H 3.32 (dd, 1H, J = 11.2, 2.0 Hz) and δ_C 80.1 (C-11)] implied that an ether linkage is present between C-7 and C-11, which was confirmed by the HMBC correlations from H-7 to C-11, and from H-11 to C-7. The NMR spectroscopic data (Table 3) indicated that 3 possesses a conjugated diene [δ_H 5.89 (1H, d, J = 5.0 Hz) and 5.99 (1H, d, J = 5.0 Hz); δ_C 150.2 (qC, C-1), 119.6 (CH, C-2), 122.3 (CH, C-3), and 137.1 (qC, C-4)] and an acetoxy [δ_C 169.2 (qC), 21.9 (CH₃) and δ_H 1.67 (3H, s)];. The above functionalities account for three of the five degrees of unsaturation, implying that 3 is a cembranoid characterized by the presence of an ether linkage between C-7 and C-11.

The final assembly of 3 was determined by the information from COSY and HMBC experiments. The ¹H-¹³C long-range correlations as determined from the HMBC spectrum allowed the connectivity of the structural fragments around each methyl group to be deduced (Figure 3). The crucial NOESY correlations (Figure 6) proved that the geometries of the conjugated diene at C-1/C-2 and C-3/C-4 were both E. The coupling constant (J_2,3 = 5.0 Hz) further suggested the s-cis geometry of the above functionality [20,21]. The key NOESY correlations between H-3/H-14a (δ_H 2.22), H-14a/H-11, H-11/H-7, H-11/H-9a (δ_H 1.73), H-11/H-10a (δ_H 1.56), H-7/H-9b, H-7/H-3, H-7/H-5 (δ_H 2.16), Me-19/H-9b (δ_H 2.83), Me-20/H-10b (δ_H 1.39), and Me-20/H-13b (δ_H 1.80) suggested that H-7, and H-11 are on the same face (β), whereas Me-19 and Me-20 are oriented toward the other face (α), as shown in a computer generated 3D drawing. The above findings indicated the 7R*, 8S*, 11R*, and 12S* configurations as depicted in Figure 6. Therefore, the structure of 3 was elucidated as (7R*,8S*,11R*,12S*,1Z,3E)-8,12-dihydroxy-7,11-epoxycembra-1(2),3-diene 8-acetate.

The cytotoxicities of metabolites 1–3 against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells are shown in Table 4. Metabolites 1–3 were also examined for antiviral activity against human cytomegalovirus (HCMV) using a human embryonic lung (HEL) and displayed antiviral activity against human cytomegalovirus, with IC_50s of 60, 46, and 5.0 μg/mL, respectively.

The first investigation of soft coral S. ehrenbergi collected at San-Hsian-Tai (Taitong County, Taiwan) has led to the isolation of three new cembranoids, (+)-12-ethoxycarbonyl-11Z-sarcophine (1) as well as ehrenbergol A and B (2 and 3). Metabolites 1–3 were not cytotoxic towards P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells. However, metabolites 1–3 displayed antiviral activity towards human cytomegalovirus, with IC₅₀s of 60, 46, and 5.0 μg/mL, respectively. Ehrenbergol B is the first cembranoid from Taiwanese soft corals to show potent anti-HCMV activity.