In our continuing research on chemical constituents of marine invertebrates collected in Taiwan waters, a series of interesting and complex briarane-type diterpenoid derivatives (3,8-cyclized cembranoids), have been isolated from the octocorals belonging to the genus Briareum [1–21], Ellisella [18,22–25], and Junceella [16,26–38], and the compounds of this type were proven to possess various interesting bioactivities [39–41]. Because of its interesting and potential chemical constituents and a series of new briarane metabolites, including briaexcavatins I-Z [16–20] and excavatoids A-D [21] (Figure 1), the octocoral B. excavatum was transplanted to the National Museum of Marine Biology & Aquarium (NMMBA), Taiwan. We report herein the isolation, structure determination, and bioactivity of two new briaranes, excavatoids E (1) and F (2) (Figure 2), resulting from further studies on the chemical constituents of cultured B. excavatum. The structures of compounds 1 and 2 were established by extensive spectroscopic methods and these two metabolites have displayed moderate inhibitory effects on elastase release by human neutrophils.

Excavatoid E (1) was obtained as a white powder and the molecular formula of 1 was determined to be C₂₈H₃₈O₉ by analysis of 13C- and 1H-NMR data, in conjunction with DEPT results (Table 1); this conclusion was further confirmed by HRESIMS with m/z 541.2415 (calcd. for C₂₈H₃₈O₉Na, 541.2413). This showed that 1 contained 10 degrees of unsaturation. Comparison of the ¹H and DEPT data with the molecular formula indicated that there must be an exchangeable proton, requiring the presence of a hydroxy group, and this deduction was supported by a broad band in the IR spectrum at 3,463 cm⁻¹. The IR absorptions of 1 also showed the presence of α,β-unsaturated γ-lactone (1,743 cm⁻¹) and ester (1,740 cm⁻¹) groups. From the ¹³C-NMR spectrum (Table 1), compound 1 was found to possess an n-butyryloxy group (δ_C 13.6, q; 18.0, t; 35.9, t; 172.3, s); two acetoxy groups (δ_C 21.2, q; 20.9, q; 170.4, s; 169.1, s), a γ-lactone moiety (δ_C 173.9, s, C-19), a tetrasubstituted olefin (δ_C 160.8, s, C-8; 127.6, s, C-17), and two disubstituted olefins (δ_C 141.9, s, C-5; 123.9, d, CH-6; 130.5, s, C-11; 123.4, d, CH-12). Thus, from the above data, seven degrees of unsaturation were accounted for, and compound 1 must be tricyclic.

Moreover, a methyl singlet (δ_H 1.36, 3H, s, H₃-15), three vinyl methyls (δ_H 2.01, 3H, s, H₃-18; 1.85, 3H, s, H₃-16; 1.64, 3H, s, H₃-20), two pairs of methylene protons (δ_H 3.46, 1H, dd, J = 15.6, 6.0 Hz; 2.04, 1H, d, J = 15.6 Hz, H₂-4; 2.57, 1H, br d, J = 18.4 Hz; 2.15, 1H, m, H₂-13), an aliphatic methine proton (δ_H 3.25, 1H, br s, H-10), five oxymethine protons (δ_H 6.07, 1H, d, J = 8.8 Hz, H-7; 5.45, 1H, d, J = 2.4 Hz, H-2; 5.08, 1H, dd, J = 6.0, 6.0 Hz, H-14; 4.98, 1H, br s, H-9; 4.98, 1H, dd, J = 6.0, 2.4 Hz, H-3), two olefin protons (δ_H 5.58, 1H, br s, H-12; 5.21, 1H, d, J = 8.8 Hz, H-6), and two acetyl methyls (δ_H 2.12, 3H, s; 1.99, 3H, s) were observed in the ¹H-NMR spectrum of 1. An n-butyryl group (δ_H 0.94, 3H, t, J = 7.6 Hz; 1.61, 2H, m; 2.22, 2H, t, J = 7.6 Hz) was also observed in the ¹H-NMR spectrum and further confirmed by the ¹H-¹H COSY correlations and coupling constant analysis.

The gross structure of 1 was elucidated with the assistance of 2D NMR studies. From the ¹H-¹H COSY experiment of 1 (Table 2), it was possible to establish the separate spin systems that map out the proton sequences from H-2/3/4, H-6/7, and H-9/10. These data, together with the HMBC correlations between H-2/C-1, −3, −4; H-3/C-1; H₂-4/C-3, −5, −6; H-6/C-4; H-7/C-5, −6, −8; H-9/C-1, −8; and H-10/C-9, established the connectivity from C-1 to C-10 in the 10-membered ring (Table 2). The vinyl methyl groups attached at C-5 and C-11 were confirmed by the HMBC correlations between H₃-16/C-4, −5, −6; and H₃-20/C-10, −11, −12, and further supported by the allylic couplings between H-6/H₃-16 and H-12/H₃-20, respectively. The vinyl methyl group attached at C-17 was also established by the HMBC correlations between H₃-18 and C-8, −17, −19. The methylcyclohexene ring, which is fused to the 10-membered ring at C-1 and C-10, was elucidated by the ¹H-¹H COSY correlations between H-12/H₂-13/H-14 and by the HMBC correlations between H-2/C-14; H-10/C-11; H-14/C-1, −2; and H₃-20/C-10. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H-2/C-15; H-14/C-15; and H₃-15/C-1, −2, −10, −14. In addition, the HMBC correlations also revealed that two acetates should attach at C-2 and C-14, respectively. The remaining n-butyryloxy and hydroxy groups were positioned at C-3 and C-9 as indicated by analysis of ¹H-¹H COSY correlations and characteristic NMR signals analysis, although no HMBC correlation was observed between H-3 and the n-butyrate carbonyl.

Based on previous studies, all naturally occurring briarane-type diterpenoids have the C-15 methyl group trans to H-10, and these two groups are assigned as β- and α-oriented, respectively, as shown in most briarane derivatives [42–44]. The relative stereochemistry of 1 was established from a NOESY experiment (Figure 3), in which the NOE correlations of H-10 with H-3 and H-9; and H-3 with H-2, indicated that these protons are situated on the same face and were assigned as α protons since the C-15 methyl is the β-substituent at C-1. H-14 was found to exhibit a correlation with H₃-15 but not with H-10, revealing the β-orientation of this proton. One of the C-4 methylene protons (δ_H 3.46) exhibited a correlation with H₃-15 and was assigned as H-4β, while the other was denoted as H-4α (δ_H 2.04). A correlation observed between H-4β and H-7, reflected the β-orientation of H-7. The NOESY spectrum showed correlations of H-6/H₃-16 and H-12/H₃-20, revealing the Z geometry of C-5/6 and C-11/12 double bonds in 1.

The molecular formula of excavatoid F (2) was determined as C₂₈H₃₈O₁₂ by its HRESIMS (m/z 589.2257, calcd. for C₂₈H₃₈O₁₂Na, 589.2261). The IR spectrum showed bands at 3,487, 1,779, and 1,737 cm⁻¹, consistent with the presence in 2 of hydroxy, γ-lactone, and ester groups. From the ¹³C-NMR data of 2 (Table 1), a trisubstituted olefin was deduced from the signals of two carbons at δ_C 146.2 (s, C-5) and 117.6 (d, CH-6). A methyl-containing tetrasubstituted epoxy group was confirmed from the signals of two quaternary oxygenated carbons at δ_C 70.6 (s, C-8) and 64.5 (s, C-17), and from the chemical shifts of a tertiary methyl group (δ_H 1.73, 3H, s, H₃-18; δ_C 10.3, q, CH₃-18) (Table 1). Moreover, five carbonyl resonances appeared at δ_C 170.9 (s, C-19), 170.6, 170.5, 169.5, and 168.1 (4 × s, ester carbonyls), confirming the presence of a γ-lactone and four esters in 2. All the esters were identified as acetates by the presence of methyl resonances in the ¹H-NMR spectrum at δ_H 2.19, 2.07, 2.05, and 2.00 (each 3H × s).

The planar structure of 2 was determined mainly by 2D NMR studies. The coupling information in the ¹H-¹H COSY spectrum of 2 enabled identification of the proton sequences H-2/3/4, H-4/6 (by allylic coupling), H-6/7, H-6/H₃-16 (by allylic coupling), H-9/10, and H-12/13/14 (Table 2). These data, together with the correlations observed in an HMBC experiment of 2 (Table 2), the molecular framework of 2 could be further established. The HMBC correlations also indicated that the acetoxy groups should attach at C-2, −9, −12, and C-14. Thus, the remaining hydroxy group has to be positioned at C-11, as indicated by characteristic NMR signal analysis. The relative stereochemistry of 2 was elucidated from the NOE interactions observed in a NOESY experiment (Figure 4). In the NOESY spectrum of 2, correlations were observed between H-10 with H-2, −9, −12, and H₃-20, indicating that these protons should be positioned on the α face in 2 and Me-20 was positioned on the equatorial direction in the methylcyclohexane ring of 2. One proton attaching at C-3 (δ_H 2.65) was found to exhibit a correlation with H₃-15 and was assigned as H-3β proton. H-7 showed a correlation with H-3β, confirming the β-orientation for this proton. Furthermore, H₃-18 was found to show correlations with H-9, H₃-20, and H-7, and from molecular models, was found to be reasonably closed to H-9, H₃-20, and H-7; therefore, H₃-18 should be placed on the β face in the γ-lactone ring of 2. The Z-configuration of the C-5/C-6 double bond was elucidated by an interaction between H-6 (δ_H 5.25) and H₃-16 (δ_H 2.02). On the basis of the above results, the structure of 2, including the relative configuration, was elucidated.

In previous study, several diterpenoid derivatives of potential medical interest were isolated from a cultured gorgonian coral Erythropodium caribaeorum [45]. Because all corals are claimed to be threatened species, we wanted to maintain and culture these interesting specimens as sources of new and interesting natural products in our continuing search for novel substances from marine organisms originally collected in Taiwan waters, in the hope of identifying extracts that exhibit bioactivity. Briaranes 1 and 2 displayed moderate inhibitory effects on elastase release by neutrophils, and briarane 1 exhibited weak inhibitory effects on superoxide anion generation by human neutrophils at 10 μg/mL, respectively (Table 3). Furthermore, these two compounds were not cytotoxic toward the CCRF-CEM (human T-cell acute lymphoblastic leukemia) and DLD-1 (human colon adenocarcinoma) cells (ED₅₀ > 40 μg/mL). The possible bioactivity for these two compounds will be further studied if we can obtain enough material from the cultured B. excavatum in the future.