In our research on the chemical constituents of the marine invertebrates collected in Taiwan waters, a series of briarane-type diterpenoid derivatives had been isolated from various octocorals belonging to the genus Briareum (family Briareidae), Ellisella, and Junceella (family Ellisellidae), and the compounds of this type were proven to possess various interesting bioactivities [1–3]. Recently, our further chemical examination of Briareum excavatum has resulted in the isolation of two new highly oxidized briarane-type diterpenoids, excavatoids O (1) and P (2) (Scheme 1). The structures of compounds 1 and 2 were established by spectroscopic methods.

Excavatoid O (1) was obtained as a white powder and had a molecular formula C₃₀H₄₂O₁₃, as determined by HRESIMS (C₃₀H₄₂O₁₃ + Na, m/z found 633.2519; calculated 633.2523) indicating 10 degrees of unsaturation. The presence of hydroxy, lactone, and ester groups in 1 were evidenced by the IR absorptions at 3512, 1793, and 1741 cm⁻¹. It was found that the ¹H and ¹³C spectra of 1 in CDCl₃ revealed mostly broad peaks when measured at 25 °C. In order to make more reliable assignments of NMR signals of the stabilized conformers, the ¹H and ¹³C NMR spectra of 1 were measured at 0 °C in CDCl₃. In the ¹³C spectrum of 1, five ester carbonyl resonances appeared at δ_C 173.6, 170.8, 170.1, 169.5, and 169.3 (5 × s) (Table 1). In the above carbonyl carbons, three were identified as acetate carbonyls by the presence of three methyl resonances in the ¹H NMR spectrum at δ_H 2.18, 2.12, and 1.96 (each 3H × s) and one was identified as n-butyrate carbonyl by the presence of seven contiguous protons at δ_H 0.95 (3H, t, J = 7.2 Hz), 1.64 (2H, m), and 2.23 (2H, m) (Table 1). On the basis of the unsaturation data overall, 1 was concluded to be a briarane diterpenoid molecule possessing five rings. A tetra-substituted epoxide containing a methyl substituent was elucidated from the signals of two oxygenated quaternary carbons at δ_C 72.6 (s, C-8) and 63.3 (s, C-17); and further confirmed by the proton signal of a methyl singlet at δ_H 1.57 (3H, s, H₃-18). In addition, a tri-substituted epoxide containing a methyl substituent was deduced from the signals of an oxymethine (δ_H 3.11, 1H, d, J = 8.8 Hz, H-6; δ_C 63.0, d, C-6), a quaternary oxygen-bearing carbon (δ_C 62.1, s, C-5), and a methyl singlet at δ_H 1.35 (3H, s, H₃-16).

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/H-3/H₂-4, H-6/H-7, and H-9/H-10. These data, together with the HMBC correlations between H-2/C-1, -4; H-3/C-4; H₂-4/C-3, -5, -6; H-7/C-5, -6; H-9/C-1, -7, -8, -10; and H-10/C-1, -2, established the connectivity from C-1 to C-10 in the 10-membered ring (Table 2). The methyl group at C-5 was confirmed by the HMBC correlations between H₃-16/C-4, -5, -6. The methylcyclohexane 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-10/H-11/H-12/H₂-13/H-14 and H-11/H₃-20 and by the HMBC correlations between H-2/C-14; H-9/C-11; H-10/C-11, -12, -14; H-11/C-10, -20; H₂-13/C-1; H-14/C-1, -2; and H₃-20/C-10, -11, -12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H-2/C-15; and H₃-15/C-1, -2, -10, -14. In addition, the HMBC correlations also revealed that three acetates should be attached at C-2, C-9, and C-14, respectively. The remaining n-butyrate ester and hydroxy groups were positioned at C-3 and C-12 as indicated by analysis of ¹H-¹H COSY correlations and characteristic NMR signals analysis (δ_H 5.13, 1H, br s, H-3; δ_C 69.9, d, C-3; δ_H 3.96, 1H, br s, H-12; δ_C 69.3, d, C-12). These data, together with the HMBC correlations between H-7/C-17, -19 and H₃-18/C-8, -17, -19, were used to establish the molecular framework of 1.

Based on previous studies, all naturally occurring briarane-type diterpenoids have the C-15 methyl group as trans to H-10, and these two groups are assigned as β - and α-oriented, respectively, as shown in most briarane derivatives [1–3]. The relative stereochemistry of 1 was established from a NOESY experiment (Figure 1). In the NOESY experiment of 1, the correlations of H-10 with H-2, H-3, H-6, H-9, and H-11; and H-11 correlated with H-12, 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, revealing the β -orientation of this proton. The correlations between H₃-16 and H-3, H-6, reflected the α-orientation of H₃-16. H-7 correlated with H₃-15, indicating this proton should be β -oriented. Furthermore, H₃-18 showed a correlation with H-9. By detailed analysis of molecular models, H₃-18 was found to be reasonably close to H-9 when H₃-18 was placed on the β face in the γ-lactone moiety. Based on the above findings, the structure of 1 was elucidated unambiguously.

The molecular formula of excavatoid P (2) was determined as C₃₀H₄₃ClO₁₄ by its HRESIMS (m/z 685.2235, calculated for C₃₀H₄₃ClO₁₄ + Na, 685.2239). The IR spectrum showed bands at 3472, 1784, and 1734 cm⁻¹, consistent with the presence of hydroxy, γ-lactone, and ester groups in 2. From the ¹³C NMR data of 2 (Table 1), five carbonyl resonances appeared at δ_C 173.9, 172.0, 170.4, 170.3, and 170.0 (5 × s), confirming the presence of a γ-lactone and four esters in 2; three acetyl methyls (δ_H 2.43, 2.17, 2.03, each 3H × s) and an n-butyryl group (δ_H 0.98, 3H, t, J = 7.6 Hz; 1.66, 2H, m; 2.33, 2H, t, J = 7.6 Hz) were also observed. According to the above data, briarane 2 was found to be a tetracyclic compound with a γ-lactone, as no other unsaturated functional group could be found.

¹H NMR coupling information in the ¹H-¹H COSY spectrum of 2 enabled identification of the C-2/-3/-4, C-6/-7, C-9/-10/-11/-12/-13/-14, and C-11/-20 units (Table 2), which were assembled with the assistance of an HMBC experiment (Table 2). The HMBC correlations between protons and quaternary carbons, such as H-2, H-3, H-10, H-11, H₃-15/C-1; H-3, H-4, H-6, H-7, H₃-16, OH-5/C-5; H-6, H-9, H-10, H₃-18/C-8; H-9, H₃-18/C-17; and H-7, H₃-18/C-19, permitted elucidation of the carbon skeleton. A methyl at C-5 was established by the HMBC correlations between H₃-16/C-4, -5, -6 and H-4, H-6, OH-5/C-16. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H₃-15/C-1, -2, -10, -14; and H-10/C-15. The acetate esters at C-2 and C-9 were established by correlations between H-2 (δ_H 4.62), H-9 (δ_H 5.36) and the acetate carbonyls observed in the HMBC spectrum of 2. The n-butyrate ester positioned at C-4 was confirmed from the HMBC correlation between H-4 (δ_H 5.82) and the carbonyl carbon (δ_C 173.9) of n-butyrate ester. Thus, the remaining acetoxy group was positioned at C-14 as indicated by analysis of ¹H-¹H COSY correlations and characteristic NMR signals (δ_H 4.84, 1H, br s, H-14; δ_C 80.8, d, C-14). The presence of hydroxy groups at C-3 and C-12 was deduced from the ¹H-¹H COSY correlations between the hydroxy protons (δ_H 3.18, OH-3 and δ_H 2.17, OH-12) and H-3 (δ_H 5.07) and H-12 (δ_H 4.13), respectively. The C-5 hydroxy group was also confirmed by the HMBC correlations between the hydroxy proton (δ_H 2.36, OH-5) and C-4, -5, -16. Thus, the remaining chlorine atom in 2 should be attached C-6 by the ¹H-¹H COSY correlation between H-6 (δ_H 4.29) and H-7 (δ_H 5.26) and further supported by the HMBC correlations between H-6/C-4, -5, -7, -8, -16 and H-7, H₃-16/C-6.

The relative configuration of 2 was elucidated from the interactions observed in a NOESY experiment and from vicinal proton coupling constant analysis. In the NOESY experiment of 2 (Figure 2), the correlations of H-10 with H-3, H-11, and H-12, indicated that these protons were situated on the same face and were assigned as α protons since the C-15 and C-20 methyls are β -substituents at C-1 and C-11, respectively. H-2 exhibited an interaction with H-3, and no coupling was found between H-2 and H-3, indicating that the dihedral angle between H-2/H-3 is approximately 90° and the acetoxy group at C-2 should be β -oriented. H-14 was found to exhibit a response with H₃-15, showing that H-14 has a β -orientation. H-9 was found to show responses with H-11, H₃-18, and H₃-20. From modeling analysis, H-9 was found to be close to H-11, H₃-18, and H₃-20 when H-9 was α-oriented. Moreover, H₃-16 exhibited correlations with H-3 and H-6, and no coupling constant was detected between H-6 and H-7, suggesting the α-orientation of H₃-16 and H-6; and β-orientation of H-7. H-7 exhibited a correlation with H-4, and no coupling was found between H-3 and H-4, indicating that the dihedral angle between H-3 and H-4 is also approximately 90° and the n-butyrate ester group at C-4 was α-oriented. On the basis of the above results, the structure of 2 was elucidated. To the best of our knowledge, briarane 2 is the first briarane which possesses a 6β -chlorine atom.

In the biological activity testing, briaranes 1 and 2 displayed 16.9 and 16.1% inhibitory effects on elastase release by human neutrophils at 10 μg/mL, resepectively [4].