New Briarane Diterpenoids from Taiwanese Soft Coral Briareum violacea

Ten new briarane diterpenoids, briaviolides A–J (1–10), together with six known briaranes, solenolides A and D, excavatolide A, briaexcavatolide I, 4β-acetoxy-9-deacetystylatulide lactone and 9-deacetylstylatulide lactone, were isolated from the Taiwanese soft coral, Briareum violacea. Their structures were determined on the basis of spectroscopic data (1H- and 13C-NMR, 1H–1H COSY, HSQC, HMBC and NOESY), HR-MS and chemical methods. The absolute configuration of briaviolide A (1) was determined by X-ray crystallographic analysis. Compounds 5, 9 and derivative 11 showed moderate inhibitory activities on superoxide-anion generation and elastase release by human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine/Cytochalasin B (fMLP/CB).

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.

Results and Discussion
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 C 24 H 31 O 9 Cl, 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      Briaviolide B (2) was isolated as a colorless gum, having a molecular formula of C 27 H 35 O 10 Cl 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 1 H-and 13 C-NMR data (Tables 1 and 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 13 C-NMR data ( Table 2) of Compound 3 suggested a molecular formula of C 28 H 36 O 12 that contains eleven degrees of unsaturation. It was found that the 1 H-, 13 C-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)   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 C 24 H 31 O 10 Cl 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 (Tables 1 and 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 13 C-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 of briaviolide F (6) showed a pseudo-molecular ion peak at m/z 593.2125 [M + Na] + , consistent with a molecular formula of C 28   Briaviolide G (7) had the molecular formula C 26 H 33 O 11 Cl, as determined by HRESIMS analysis. It was found that the 1 H-, 13 C-NMR (Tables 1 and 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 1 H-and 13 C-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.
The molecular formula C 24 H 34 O 10 was assigned to Compound 9 from its HRESIMS and 13 C 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 C 26 H 36 O 10 , as determined by its HRESIMS and DEPT 13 C 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 1 H-and 13 C-NMR data (Tables 1 and 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. 65.05 ± 6.12 52.45 ± 6.34 a At a concentration of 10 μg/mL for each compound. Results are presented as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control value; b Positive control.

Animal Material
The gorgonian, Briareum violacea (Quoy and Gaimard), was collected in Pingtong County of southern Taiwan by scuba diving at a depth of 15 m, in May 2007. The fresh gorgonian was immediately frozen after collection and kept at −20 °C until being processed. A voucher specimen was deposited in the School of Pharmacy, College of Medicine, National Taiwan University.

Conclusions
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.