The briarane diterpenoids [1] continue to attract the attention of natural product chemists because of their structural complexity and interesting biological activities, such as anti-inflammatory [2], antiviral [3], cytotoxic [4,5,6], antifouling [7,8], immuno-modulatory [9], insecticidal [10] and reversal of multidrug resistance [11]. The structures of these diterpenoids are characterized by a highly oxygenated bicyclo[8.4.0]tetradecane skeleton that is frequently attached with a γ-lactone moiety. Since the first structural elucidation of briarein A isolated from Briareum asbestinum in 1977 [12], more than 450 briarane-type diterpenoids have been reported from Octocorallia, including Gorgonacea, Pennatulacea, Alcyonacea and Stolonifera [13,14,15]. In serial studies of the Taiwanese gorgonian corals, many new briaranes have been isolated, including juncenolides A–G from Junceella juncea [16,17,18], frajunolides A–K from J. fragilis [19,20] and briaviodiol A from B. violacea [21].

In this paper, we report the investigation of Taiwanese soft coral Briareum violacea that provided ten new briarane-type diterpenoids, briaviolides A–J (1–10) (Figure 1), along with six known analogues, solenolides A and D, excavatolide A, briaexcavatolide I, 4β-acetoxy-9-deacetystylatulide lactone and 9-deacetylstylatulide lactone. The structures of new compounds were established by spectroscopic and chemical methods. Among them, the structure of 1 was further confirmed by single-crystal X-ray analysis. The in vitro anti-inflammatory activities of new compounds (1–10) and new derivative 11 were also tested for their inhibition of elastase release and superoxide-anion generation from human neutrophils.

Briaviolide A (1) was isolated as colorless prisms. Its ESIMS revealed two isotopic [M + Na] ⁺ and [M + Na + 2]⁺ peaks for pseudo-molecular ions at m/z 521 and 523 (3:1) and HRESIMS at m/z 521.1554 [M + Na]⁺, indicating a molecular formula C₂₄H₃₁O₉Cl, which contains one chlorine atom and accounts for nine degrees of unsaturation. The IR spectrum of 1 showed absorption frequencies at 3524, 1767 and 1737 cm⁻¹, indicating the presence of hydroxyl, γ-lactone and carbonyl ester functionalities, respectively. The ¹H- and ¹³C-NMR spectroscopic data (Table 1 and Table 2) exhibited signals of two acetate methyl singlets at δ_H 2.09 (δ_C 21.1) and 2.17 (δ_C 22.1), with corresponding carbonyl signals at δ_C 170.3, 170.2, respectively. The carbonyls showed respective HMBC connectivity (Figure 2) with two methine doublets at δ_H 6.09 and 5.30, revealing the positions of each acetate group (C-2 and C-9, respectively). The carbonyl signal of a γ-lactone (δ_C 174.9) was connected with a secondary methyl doublet at δ_H 1.17 (δ_C 6.3) by the HMBC (H-19/C-18) and COSY (H-19/H-17) correlations. The connection between C-17 and C-8 (δ_C 83.9) was elucidated by the HMBC correlation from H-19 to C-8. The hydroxyl proton (δ_H 3.52) of C-8 was correlated with the adjacent lactonide carbon C-7 and C-9 by virtue of the HMBC correlations from 8-OH to C-7 and C-9. These correlations along with the H-6/H-7 (COSY) correlation fixed the position of the lactone ring. The signals of an exocyclic double bond were observed at δ_H 6.24 (br s) and 5.95 (d, J = 2.1 Hz) (δ_C 118.7) and were conjugated with an endocyclic double bond at δ_H 5.63 (dd, J = 11.4, 9.0 Hz; δ_C 131.0), 5.90 (d, J = 11.4 Hz; δ_C 128.0) by related HMBC and COSY correlations. The correlations of H-6/H-7 (COSY) and H-16/C-6 (HMBC) thus connected the conjugated double bond with the γ-lactone ring. The signals of a pair of methine protons at δ_H 3.23 (H-13) and δ_H 2.99 (H-14) were assigned for an epoxide ring. This moiety was neighbored on a quaternary carbon (δ_C 40.6) and a hydroxylated carbon (δ_C 70.5; δ_H 3.71) by the HMBC correlation of H-12/C-13 and H-14/C-1. The COSY correlations of H-12/H-11/H-20 and H-9/H-10 indicated the presence of two proton sequences. These two sequences were connected by the HMBC correlations of H-20/C-10 and H-9/C-11. By deducing the unsaturation of two acetates, conjugated diene, a γ-lactone ring and the epoxide ring, the remaining two degrees of unsaturation strongly suggested that Compound 1 possesses two additional rings belong to briarane-type diterpenoids.

A literature survey revealed that the ¹H- and ¹³C-NMR spectroscopic data of 1 showed similarity with those of briaexcavatolide I [22]. The only difference between them was 1 having two acetyl groups, while there are three in briaexcavatolide I. Acetylation of 1 gave a triacetyl product that was identical to briaexcavatolide I after comparing with their ¹H and ¹³C NMR data. Benzoylation of 1 yielded a monobenzoyl derivative 11 that confirmed the secondary hydroxyl group at C-12.

The relative configuration of 1 was determined by NOESY (Figure 3a) and X-ray diffraction analysis (Figure 3b). Naturally occurring briaranes have β-face of Me-15 and α-orientation of H-10. The NOESY correlations between H-2/H-16, H-2/H-10, H-10/H-12, H-12/H-11, H-11/H-9 and H-9/Me-19 required that all of these groups were in α-face, and correlations of H-6/H-7, H-7/H-17, Me-15/H-14 and H-14/H-13 indicated β-disposition for these groups. The correlations between H-16/H-2 and H-2/H-10 suggested that the conjugated diene had a s-cis geometry and forced the ten-membered ring to adopt a boat-like conformation with C-1 and C-5 at the bow and stern positions. An X-ray crystallographic analysis established the complete structure and stereochemistry of 1 as shown by the Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram stereo-drawing in Figure 3b. The negative optical rotation value of 1 was similar to that of briaexcavatolide I [23] in direction and magnitude, suggesting that 1 and briaexcavatolide I had 1S,10S-configurations in the ring junction. Thus, the structure of briaviolide A (1) was determined as (1S,2S,3Z,6S,7R,8R,9S,10S,11R,12R,13S,14R,17R)-6-chloro-13,14-epoxy-2,9-diacetoxy-8,12-dihydroxybriaran-3(4),5(16)-dien-18,7-olide.

Briaviolide B (2) was isolated as a colorless gum, having a molecular formula of C₂₇H₃₅O₁₀Cl as deduced from the high-resolution ESIMS. Similar to those of 1, Compound 2 showed IR bands at 3453, 1783, 1738 and 1732 cm⁻¹, indicating hydroxyl, γ-lactone and ester carbonyl functionalities, respectively. Comparisons of its ¹H- and ¹³C-NMR data (Table 1 and Table 2) with those of 1 revealed strong resemblance in all signals, except that the C-12 and H-12 signals in the NMR spectra of 2 were shifted downfield to δ_C 73.7 and δ_H 4.62 (d, J = 4.4 Hz), respectively, suggesting that Compound 2 had an ester group at position C-12. This ester group was revealed to be a propionyloxy group (δ_H 2.38, m; δ_H 1.15, t, J = 8.0 Hz). The structure of 2 was further supported by COSY, HSQC and HMBC experiments. The NOESY cross-peaks of 2 and 1 were quite similar, suggesting that they have the same relative configuration. Thus, briaviolide B (2) was established to be a 12-propionyloxy derivative of 1.

The HRESIMS and ¹³C-NMR data (Table 2) of Compound 3 suggested a molecular formula of C₂₈H₃₆O₁₂ that contains eleven degrees of unsaturation. It was found that the ¹H-, ¹³C-NMR and IR spectroscopic data of 3 were very similar to those of Compound 1, except for the signals of two more acetyl groups, including two methyl singlets, δ_H 2.07 (δ_C 21.0), δ_H 2.19 (δ_C 21.9), and the respective two carbonyls, δ_C 169.6, δ_C 170.3. The HMBC correlations (Figure 2) of H-6 (δ_H 5.75, d, J = 10.0 Hz)/δ_C 169.6 and H-12 (δ_H 4.63, d, J = 5.0 Hz)/δ_C 170.3 suggested that two acetoxyl group were attached at C-6 and C-12. Compound 3 was the first example of a briarane-type diterpenoid that contains an ester group at C-6. The configuration of Compound 3 was determined by using NOESY correlations (Figure 4) and comparing the data with those of 1. The NOESY correlations of Me-15/Me-20, H-14; H-13/H-14 and H-7/H-17 indicated that all these atoms were β-oriented and the correlations of H-10/H-2, H-9, H-12; H-12/Me-19 suggested that all of these atoms were α-oriented. In addition, the NOESY correlations between OH-8 (δ_H 3.48, s) and H-6, H-10, Me-19 confirmed the α-orientation of H-6 and OH-8. On the basis of the above observations, the structure of briaviolide C (3) was assigned as the 2,6,9,12-tetraacetyl derivative of 1, having 1S, 2S, 6R, 9S, 10S, 11R, 12R, 13S and 14R configurations.

A pseudo-molecular ion peak at m/z 537.1506 [M + Na]⁺ in the HRESIMS suggested that briaviolide D (4) had the molecular formula of C₂₄H₃₁O₁₀Cl with nine degrees of unsaturation. The presence of a chlorine atom was supported by an isotope peak at m/z 539 in the LRESI spectrum, having one third of the intensity relative to m/z 537. The NMR spectroscopic data (Table 1 and Table 2) and IR spectrum revealed that Compound 4 possessed an 8-hydroxybriarane structure similar to that of Compound 1, except for one additional hydroxyl group at C-11 (δ_C 77.0). This finding was confirmed from ¹³C-NMR data and HMBC correlations between H-10/C-11, H-12/C-11 and Me-20/C-11. The NOESY correlations of H-10/Me-11, H-12 required that the methyl group at C-11 be α-oriented. The configurations of other chiral centers are similar to Compound 1 as ascertained by NOESY experiments (Figure 4). Thus, the structure of briaviolide D (4) was established as (1S,2S,6S,7R,8R,9S,10S,11R,12S,13S,14R,17R)-6-chloro-13,14-epoxy-2,9-diacetoxy-8,11,12-trihydroxybriaran-3(4),5(16)-dien-18,7-olide.

The HRESIMS data of 5 agreed with the molecular formula C₂₉H₃₉O₁₁Cl containing a chlorine atom and 10 degrees of unsaturation. The IR bands at 3429, 1779 and 1738 cm⁻¹ suggested the presence of hydroxyl group, ester groups and a γ-lactone moiety. A detailed inspection of ¹H and ¹³C-NMR spectroscopic data (Table 1 and Table 2) of 5 indicated the presence of an 8-hydroxybriarane skeleton with two epoxides, two acetate esters and an isovalerate ester group. It was found that the spectroscopic data of Compound 5 were very similar to those of brianolide [23]. However, cross comparisons of ¹H- and ¹³C-NMR spectra showed that the 12-acetoxyl signals in brianolide were replaced by an isovalerate ester group (δ_H 2.20, m; 2.10, m; 0.96 d, 6H, J = 6.3 Hz); (δ_C 171.9, 43.2, 25.7, 22.3 × 2) in Compound 5. The configurations of 5 was deduced by the NOESY analysis (Figure 4) and was based on the X-ray analysis of brianolide [24]. It was observed that the introduction of the 3,4-epoxide ring on the skeleton does not affect the boat-like conformation of the macro-ring, as revealed by the same NOESY interactions between H-16/H-2 and H-2/H-10 as those of Compound 1. Thus, the structure of briaviolide E (5) was established as (1R,2R,3R,4R,6S,7R,8R,9S,10S,11R,12R,13S,14R,17R)-6-chloro-3(4),13(14),-diepoxy-2,9-diacetoxy-12-isovaleryloxy-8-hydroxybriaran-5(16)-dien-18,7-olide.

The HRESIMS of briaviolide F (6) showed a pseudo-molecular ion peak at m/z 593.2125 [M + Na]⁺, consistent with a molecular formula of C₂₈H₃₉O₁₀Cl (Δ = 10). Cross comparison of ¹H- and ¹³C-NMR spectroscopic data of 6 (Table 1 and Table 2) with those of 5 revealed that the differences were the substituents at C-2 and configurations at C-12. Comparison of the proton chemical shift of H-2 with that of milolide D [23] (Figure 5) could confirm the presence of a hydroxyl group at C-2 (δ_C 74.0) in 6. Signals of an acetyl group (δ_H 2.20; δ_C 169.8, 22.0) located at C-9 (δ_C 68.7) and one hexanoate group (δ_H 2.28, 1.61, 1.31, 1.31, 0.90; δ_C 173.1, 34.0, 24.8, 31.4, 22.4, 14.0) located at C-12 (δ_C 69.7) were determined by their HMBC correlations (Figure 2). The NOESY correlations (Figure 4) of H-10/H-2 and H-9, H-9/Me-19 suggested that H-2, H-9, H-10 and Me-19 were placed in α-orientation. Correlations of H-7/H-6 and H-17, H-12/H-13 and Me-20, H-14/H-13 and Me-15 agreed with β-orientations for all these groups. Thus, Compound 6 was established as a 2β-hydroxyl-12α-hexanoyl derivative of 5.

Briaviolide G (7) had the molecular formula C₂₆H₃₃O₁₁Cl, as determined by HRESIMS analysis. It was found that the ¹H-, ¹³C-NMR (Table 1 and Table 2) and IR spectroscopic data were very similar to those of Compound 6. However, the hexanoate group at C-12 in 6 was replaced by an acetyl group at C-2 in 7. This finding was supported by the HMBC correlations between H-2 (δ_H 5.23, d, J = 9.5 Hz)/carbonyl carbon (δ_C 170.0) and H-12 (δ_H 4.70, dd, J = 5.0, 2.5 Hz)/carbonyl carbon (δ_C 169.4). Comparing the ¹H- and ¹³C-NMR data of 7 with those of solenolide D [25] and briaexcavatolide E [22] indicated their resemblance, except for the chemical shifts around C-12 and C-13. Based on Kobayashi’s and Williams’s study [26], the configuration of 12-OH could be assigned (Figure 5). By comparison of the coupling constant of H-12/H-13 and the chemical shift of C-20, as well as the NOESY correlation between H-12 and Me-20 (δ_H 1.06), the acetyl group at C-12 was assigned to be α-face. The other NOSEY correlations (Figure 4) also indicated that Compound 7 had identical configurations as those of 6. Therefore, 7 was assigned a 2β-acetoxyl-12α-acetoxyl derivative of Compound 6.

Briaviolide H (8) was obtained as an amorphous gum. The molecular formula of 8 was determined as C₂₄H₃₄O₉ by its HRESIMS. The IR spectrum showed absorption bands due to hydroxyl group (3440 cm⁻¹), γ-lactone (1732 cm⁻¹) and ester carbonyl (1712 cm⁻¹). The ¹H- and ¹³C-NMR spectra of 8 in CDCl₃ gave broad signals, while those in acetone-d₆ were well resolved. Characteristic resonances due to three methyl protons (δ_H 1.02, d, J = 7.2 Hz; 1.08, s; 1.85, s; each 3H) and two acetyl protons (δ_H 1.90, s, 6H) were observed in the ¹H-NMR spectrum (Table 1). Signals of six oxygenated carbons (δ_C 74.8, 79.9, 83.6, 75.7, 69.4, 75.7), two acetyl carbons (δ_C 168.6 and 169.0) and a γ-lactone carbonyl carbon (δ_C 175.1) were observed in the ¹³C-NMR spectrum (Table 2). The HMBC correlations of H-2 (δ_H 5.34, d, J = 9.2 Hz)/carbonyl carbon (δ_C 168.6) and H-14 (δ_H 4.68, br s)/carbonyl carbon (δ_C 169.0) helped to locate two acetoxyl groups at C-2 and C-14. The above data suggested that Compound 8 is a highly oxygenated 8-hydroxybriarane-type diterpenoid. The ¹H-NMR spectrum of 8 was similar to that of frajunolide A [19], except that the chemical shift of H-12 (δ_H 4.13, dd, J = 10.8, 5.2 Hz; in CDCl₃) was located upfield as compared with frajunolide A (δ_H 5.35). The last remaining oxygen can only be accounted for by a hydroxyl group attached on C-9, because the chemical shift of H-9 (δ_H 4.68, br s) was also shifted upfield in comparison with frajunolide A (δ_H 5.37). The above data combined with HMBC correlations (Figure 2) revealed that 9- and 12-acetyl groups in frajunolide A were replaced by hydroxyl groups in 8. The NOESY correlations of H-2/H-10, H-10/H-9 and Me-19 in 8 suggested that the configurations of them were α-oriented. On the other hand, the correlations of H-6/H-7, H-7/H-17, H-14/Me-15 agreed with a β-configuration of H-7, H-14, Me-15 and H-17. The large coupling constant (J_6,7 = 9.6 Hz) confirmed the anti-parallel arrangement of H-6 and H-7 and the β-orientation of H-7 [19]. It was concluded that briaviolide H (8) has the structure of (1S,2S,6Z,7S,8R,9S,10S,12S,14S,17R)-2,14-diacetoxy-12-hydroxy-8,9-dihydroxybriaran-5(6)-dien-18,7-olide.

The molecular formula C₂₄H₃₄O₁₀ was assigned to Compound 9 from its HRESIMS and ¹³C NMR data (Table 2). The spectroscopic values of 9 suggested a briarane structure similar to that of 8 with one additional hydroperoxy group at C-12 (δ_C 83.8). The configuration of Compound 9 was further determined by the NOESY experiments (Figure 4), and the correlations revealed that 9 possessed the same relative configurations as those of 8. Thus, briaviolide I (9) was assigned as 12-hydroperoxyl derivative of Compound 8.

Briaviolide J (10) had the molecular formula C₂₆H₃₆O₁₀, as determined by its HRESIMS and DEPT ¹³C NMR data. The IR absorptions of 10 were found at 3426, 1732 and 1675 cm⁻¹, which indicated the presence of hydroxyl, a γ-lactone and ester groups. The ¹H- and ¹³C-NMR data (Table 1 and Table 2) revealed that 10 was an 8-hydroxybriarane-type diterpenoid and was structurally similar to 8 and 9. Comparisons of their NMR and MS data showed that the only difference between 8 and 10 was the presence of an acetate group at C-12 in 10. Acetylation of 8 afforded a product identical to Compound 10. Thus, it was concluded that 10 is 12-acetoxyl derivative of Compound 8.

In addition, six known briaranes, solenolides A and D [25], excavatolide A [26], briaexcavatolide I [22], 4β-acetoxy-9-deacetystylatulide lactone and 9-deacetylstylatulide lactone [27], were identified. The new isolated briaranes 1–10 and derivative 11 were evaluated for anti-inflammatory activities on superoxide-anion generation and elastase release by human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP)/Cytochalasin B (CB). The inhibition percentages of these compounds at the concentration of 10 μg/mL are summarized in Table 3. The bioassay data showed that Compounds 5 and 9 have moderate activities on both of superoxide-anion generation and elastase release, while Compound 11 has selective activity on the inhibition of elastase release.

Sixteen briarane diterpenoids, including ten new compounds briaviolides, A–J (1–10), were successfully isolated from the Taiwanese soft coral, Briareum violacea, and their structures determined. The inhibitory effects of the isolates and new derivative 11 on superoxide-anion generation and elastase release by human neutrophils in response to fMLP/CB were evaluated. Compounds 5 and 9 showed moderate anti-inflammatory activities at a concentration of 10 μg/mL. Compound 11, derived from Compound 1, showed better inhibition of elastase release than that of 1. Further comparison of the activities of those compounds may suggest that β-orientation and the chain length of ester groups at C-12 are important for the anti-inflammatory activities in briarane-type diterpenoids.