Among the diterpenoids isolated from octocorals, the briarane-type metabolites (3,8-cyclized cembranes) are a major group of compounds [1,2,3]. The compounds of this type were suggested to be of marine origin and the octocorals belonging to the genus Briareum have been proven to be the most important source of briarane-type compounds [4,5,6,7]. In previous studies, a series of interesting terpenoid derivatives, including briarane [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35], cembrane [36] and carotenoid [37], had been isolated from the octocorals belonging to the genus Briareum that were distributed in the waters off Taiwan, at the intersection point of the Kuroshio current and the South China Sea surface current. In a continuation of our search for new substances from the Formosan marine invertebrates, the chemical constituents of a specimen octocoral identified as Briareum sp. (Briareidae) were studied. A fraction of its organic extract (fraction H, see Experimental Section) displayed inhibitory effects on the generation of superoxide anion (inhibition rate 36.8%) and the release of elastase (inhibition rate 90.3%) at a concentration of 10 μg/mL. We further isolated two new briarane-type diterpenoids, briarenolides, F (1) and G (2) (Figure 1), from the octocoral Briareum sp. In this paper, we report the isolation, structure determination and bioactivity of briaranes 1 and 2.

Briarenolide F (1) was isolated as a white powder. The molecular formula of 1 was established as C₂₈H₄₀O₁₂ (nine degrees of unsaturation) from a sodium adduct at m/z 591 in the ESIMS spectrum and further supported by HRESIMS (C₂₈H₄₀O₁₂Na, m/z 591.2420, alculated 591.2417). The IR spectrum of 1 showed bands at 3498, 1789 and 1743 cm⁻¹, consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. The ¹³C NMR and DEPT spectra of 1 showed that this compound had 28 carbons (Table 1), including seven methyls, four sp³ methylenes, eight sp³ methines, three sp³ quaternary carbons, an sp² methine and five sp² quaternary carbons. From the ¹H and ¹³C NMR spectra (Table 1), 1 was found to possess two acetoxy groups (δ_H 1.99, 2.01, each 3H × s; δ_C 170.6, 2 × qC; 21.3, 2 × CH₃), an n-butyrate group (δ_H 0.94, 3H, t, J = 7.2 Hz; 1.63, 2H, sext, J = 7.2 Hz; 2.27, 2H, t, J = 7.2 Hz; δ_C 13.7, CH₃; 18.4, CH₂; 36.3, CH₂; 173.1, qC), a γ-lactone moiety (δ_C 171.0, qC-19) and a trisubstituted olefin (δ_H 5.65, 1H, br d, J = 13.6 Hz, H-4; δ_C 130.3, CH-4; 128.8, qC-5). The presence of a tetrasubstituted epoxide containing a methyl substituent was established from the signals of two quaternary oxygenated carbons at δ_C 68.8 (qC-8) and 58.4 (qC-17) and further confirmed by the proton signal of a methyl singlet at δ_H 1.49 (3H, s, H₃-18). Thus, from the above NMR data, five degrees of unsaturation were accounted for and 1 was identified as a tetracyclic compound.

¹H–¹H couplings in the COSY spectrum of 1 enabled identification of the C-2/-3/-4, C-6/-7, C-10/-11/-12/-13/-14, C-4/-16 (by allylic coupling) and C-11/-20 units (Table 1), which were assembled with the assistance of an HMBC experiment. The HMBC correlations between protons and quaternary carbons of 1, such as H-2, H-9, H-10, H-11, H₃-15/C-1; H-3β, H-6, H-7, H₃-16/C-5; H-6, H-9, H-10, H₃-18/C-8; H-9, H₃-18/C-17; and H₃-18/C-19, permitted the elucidation of the carbon skeleton (Table 1). The vinyl methyl at C-5 was confirmed by the allylic coupling between H-4/H₃-16 in the ¹H–¹H COSY spectrum and by the HMBC correlations between H₃-16/C-4, -5, -6 and H-6/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; H-2/C-15; and H-10/C-15. In addition, the carbon signal at δ_C 173.1 (qC) was correlated with the signal of the methylene protons at δ_H 2.27 in the HMBC spectrum and was consequently assigned as the carbon atom of the n-butyrate carbonyl. Additionally, the n-butyrate positioned at C-12 was confirmed by the connectivity between H-12 (δ_H 4.98) and the carbonyl carbon (δ_C 173.1, qC) of the n-butyrate. Furthermore, an acetate ester at C-2 was established by a correlation between H-2 (δ_H 5.22) and the acetate carbonyl (δ_C 170.6, qC) observed in the HMBC spectrum of 1. The presence of a hydroxy group at C-9 was deduced from the ¹H–¹H COSY correlation between a hydroxy proton (δ_H 2.95) and H-9 (δ_H 4.59). The presence of a hydroperoxy group in 1 was supported by a hydroperoxy proton signal at δ_H 8.71 as a broad signlet [22,32,38]. Due to absence of HMBC correlations for H-14 (δ_H 4.88) and the hydroperoxy proton (δ_H 8.71), the positions for the remaining acetoxy and hydroperoxy groups could not be determined by this method. By comparison the ¹H and ¹³C NMR data of C-14 oxymethine for 1 (δ_H 4.88; δ_C 74.9) with those of a known briarane analogue, excavatolide F (3) (δ_H 4.94; δ_C 74.1) (Figure 2) [10], which possesses a similar cyclohexane moiety as that of 1, the remaining acetoxy group in 1 was placed at C-14. Thus, the hydroperoxy group is positioned at C-6, an oxymethine at δ_C 84.9 (CH), by analysis of the ¹H–¹H COSY correlations and characteristic NMR signal analysis.

In all naturally-occurring briaranes, H-10 is trans to the C-15 methyl group, and these two groups are assigned as α- and β-oriented in most briarane derivatives [4,5,6,7]. The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment and was found to be compatible with that of 1 offered by computer modeling (Figure 3) [39] and that obtained from vicinal proton coupling constant analysis. In the NOESY experiment of 1, the correlations of H-10 with H-2, H-9, H-11 and H-12, but not with H₃-15 and H₃-20, indicated that these protons (H-2, H-9, H-10, H-11 and H-12) were situated on the same face, and these were assigned as α protons, since the C-15 and C-20 methyls are β-substituents at C-1 and C-11, respectively. H-14 was found to exhibit an interaction with H₃-15, but not with H-10, revealing the β-orientation of this proton. The configuration at C-9 is worthy of comment. H-9 was found to exhibit correlations with H-10, H-11, H₃-18 and H₃-20. From a consideration of molecular models, H-9 was found to be reasonably close to H-10, H-11, H₃-18 and H₃-20, while it was placed on the α face in 1. The C-16 vinyl methyl showed correlations with H-4 and H-6, demonstrating the Z configuration of Δ^4,5 and the hydroperoxy group at C-6 was α-oriented. The cis relationship between H-6 and H-7 was established by a correlation between H-6 and H-7 and a small coupling constant (J = 2.4 Hz) between these two protons. Moreover, an acetyl methyl (δ_H 2.01) exhibited correlations with H-12 and H-2, further supporting an acetoxy group was positioned on the α-position at C-14 in 1. Based on the above findings, the configurations of all chiral carbons of 1 were assigned as 1S*, 2S*, 6S*, 7S*, 8R*, 9S*, 10S*, 11R*, 12S*, 14S*, 17R*, and the structure of 1 was established unambiguously. To the best of our knowledge, briarane derivatives possessing a hydroperoxy group are rarely found [22,32,38] and briarenolide F (1) is the first briarane derivative possessing a 6-hydroperoxy group. A double bond positioned at C-4(5) in briarane-type metabolites is also rarely found [31,40,41,42].

Briarenolide G (2) was isolated as a white powder whose HRESIMS showed a molecular ion at m/z 397.1989 implying that 2 had the molecular formula C₂₂H₃₀O₅ (C₂₂H₃₀O₅Na, calculated 397.1991). The IR spectrum revealed absorptions for hydroxy (3397 cm⁻¹) and ester carbonyl (1757 and 1734 cm⁻¹) groups. The ¹H NMR data (Table 2) showed resonances due to an acetyl methyl (δ_H 2.03, 3H, s), three vinyl methyls (δ_H 1.78, 3H, br s, H₃-16; 1.87, 3H, d, J = 1.6 Hz, H₃-18; 1.63, 3H, d, J = 0.8 Hz, H₃-20), a quaternary methyl (δ_H 0.77, 3H, s, H₃-15), two olefinic protons (δ_H 5.29, 1H, br s, H-6; 5.12, 1H, m, H-12) and an oxymethine signal (δ_H 4.78, 1H, br s, H-14). The ¹³C NMR and DEPT spectra of 2 (Table 2) revealed the presence of a tetrasubstituted (δ_C 160.8, qC-8; 125.1, qC-17) and two trisubstituted (δ_C 144.4, qC-5; 124.6, CH-6; 136.3, qC-11; 117.8, CH-12) carbon-carbon double bonds, a hemiketal carbon (δ_C 106.7, qC-7), an acetate carbonyl (δ_C 170.9, qC), an α,β-unsaturated-γ-lactone carbonyl (δ_C 171.1, qC-19), a tetrasubstituted carbon atom bearing a carbon substituent (δ_C 39.1, qC-1) and an oxymethine (δ_C 77.2, CH-14).

From the ¹H–¹H COSY experiment of 2 (Table 2), it was possible to establish the separate spin systems that map out the proton sequences from H₂-2/H₂-3/H₂-4 and H₂-9/H-10. These data, together with the HMBC correlations between H₂-2/C-1, -3, -10; H₂-3/C-2; H-4β/C-3; H-6/C-4, -7; and H₂-9/C-7, -8, -10, established the connectivity from C-1 to C-10 in the ten-membered ring (Table 2). The vinyl methyl at C-5 was confirmed by the HMBC correlations between H₃-16/C-4, -5, -6; H-4β/C-16; and H-6/C-16, and further supported by the allylic coupling between H-6 and H₃-16. The methylcyclohexene ring, which is fused to the ten-membered ring at C-1 and C-10, was elucidated by the ¹H–¹H COSY correlations between H-12/H₂-13/H-14 and H-12/H₃-20 (by allylic coupling) and by the HMBC correlations between H₂-2/C-14, H₂-9/C-11 and H₃-20/C-10, -11, -12. The ring junction C-15 methyl group was positioned at C-1 from the HMBC correlations between H₃-15/C-1, -2, -10, -14. In addition, the acetate ester at C-14 was established by a correlation between H-14 (δ_H 4.78) and the acetate carbonyl observed in the HMBC spectrum of 2. The presence of a hydroxy group at C-7 was deduced from the HMBC correlations between the hydroxy proton (δ_H 3.35, 1H, s, OH-7) and C-6, C-7, and C-8. The C-7 hydroxy group was concluded to be a part of hemiketal constellation on the basis of a characteristic carbon signal at δ_C 106.7 (a quaternary hemiketal carbon, qC-7). These data, together with the HMBC correlations between H₃-18/C-8, -17, -19, were used to establish the molecular framework of 2.

NOESY measurements were carried out in order to deduce the relative stereochemical features of 2 (Figure 4). Thus, H₃-15 gave a correlation with H-14, but not with H-10, indicating that H₃-15 and H-14 are located on the same face (assigned as the β-face) and that H-10 lies on the opposite side, α-face. The NOESY spectrum showed correlations between H-6/H₃-16 and H-12/H₃-20, revealing the Z geometry of the C-5/6 and C-11/12 double bonds in 2. Due to the absence of NOESY correlations for the C-7 hydroxy group, the configuration at that chiral center could not be determined by this method. By comparison of the ¹³C NMR chemical shifts of C-6 (δ_C 124.6), C-7 (δ_C 106.7) and C-8 (δ_C 160.8) for 2 with those of an unnamed known 7β-hydroxybriarane analogue 4 (δ_C 124.8, C-6; 106.2, C-7, 160.1, C-8), which was obtained from a Caribbean octocoral Briareum polyanthes [43] (Figure 5), we deduced that the C-7 hydroxy group was β-oriented and the configuration of all the chiral carbons in 2 were assigned as 1R*, 7S*, 10S*, 14S*.

The in vitro anti-inflammatory effects of briaranes 1 and 2 were tested. Briarenolide F (1) was found to display a significant inhibitory effect on the generation of superoxide anion by human neutrophils (Table 3).

Briarane-type natural products (3,8-cyclized cembranoid) were found in various marine organisms, particularly with the octocorals belonging to the genus Briareum (family Briareidae) [4,5,6,7]. It is interesting to note that the briarane-type natural products are major constituents of the extracts of octocorals Briareum spp. distributed in the tropical and subtropical Indo-Pacific Ocean. In the past 35 years, over 500 briarane analogues have been obtained and the number is still increasing based on their structural complexity and interesting bioactivities. It is worth noting that only three hydroperoxybriarane analogues have been isolated to date [22,32,38] and that briarenolide F (1) is the first 6-hydroperoxybriarane. 7-Hydroxybriarane derivatives are also rarely found [43,50,51,52]; the new briarane, briarenolide G (2) was the first 7-hydroxybriarane derivative isolated from the octocorals collected off the waters of Taiwan. The study material Briareum sp. has begun to be transplanted in tanks for the extraction of natural products in order to establish a stable supply of bioactive material.