Anti-Inflammatory Cembranoids from a Formosa Soft Coral Sarcophyton cherbonnieri

The present investigation on chemical constituents of the soft coral Sarcophyton cherbonnieri resulted in the isolation of seven new cembranoids, cherbonolides F–L (1–7). The chemical structures of 1–7 were determined by spectroscopic methods, including infrared, one- and two-dimensional (1D and 2D) NMR (COSY, HSQC, HMBC, and NOESY), MS experiments, and a chemical reduction of hydroperoxide by triphenylphosphine. The anti-inflammatory activities of 1–7 against neutrophil proinflammatory responses were evaluated by measuring their inhibitory ability toward N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced superoxide anion generation and elastase release in primary human neutrophils. The results showed that all isolates exhibited moderate activities, while cherbonolide G (2) and cherbonolide H (3) displayed a more active effect than others on the inhibition of elastase release (48.2% ± 6.2%) and superoxide anion generation (44.5% ± 4.6%) at 30 µM, respectively.

Many studies have revealed that soft corals of the genus Sarcophyton are important sources of various types of natural products, some of them with notable bioactivies [25][26][27][28]. Our previous chemical study on Sarcophyton cherbonnieri led to the isolation of six new cembranoids cherbonolides A-E and one biscembranoid bischerbolide peroxide, along with a known compound, isosarcophine [24]. In continuation of our effort on discovery of new and bioactive compounds from marine animals, we further explored the chemical constituents of S. cherbonnieri. This investigation again led to the discovery of new cembranoids, cherbonolides F-L (1-7). The structures of 1-7 ( Figure 1) were determined by spectroscopic analysis, including two-dimensional (2D) NMR experiments and a chemical reaction. Compounds 2 and 4 were elucidated as cembranoids possessing an allylic peroxy group. Cembranoids of isosarcophine-type have been reported frequently [24,[27][28][29][30].
The screening of the in vitro anti-inflammatory activities through the inhibition of superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced primary human neutrophils was also performed in order to unveil the anti-inflammatory ability of these compounds. We report herein the isolation, structure determination, and bioactivity of the new metabolites 1-7.
Furthermore, analysis of nuclear Overhauser effect (NOE) correlations was applied to establish the relative configuration of 1, as shown in Figure 3. It was revealed that H-2 showed NOE correlation with H 3 -18, but not with H-3; therefore, assuming the β-orientation of H-2, H 3 -18 should be located on the β face. Moreover, H 3 -18 exhibited NOE correlation with H-7, but not with H-6, revealing the β-orientation of H-7 and the α-orientation of H-6. Both H-6 and H-7 exhibited NOE interactions with H 3 -19, thus established the β-orientation of H 3 -19 as shown in Figure 3. One methylene proton at C-13 exhibited NOE correlation with H-2 and was characterized as H-13β (δ H 0.99, m), while the other proton was assigned as H-13α (δ H 1.49, m). NOE correlations of H-13β with H-11 and H-13α with H 3 -20 reflected the β-orientation of H-11 and the α-orientation of H 3 -20. The E geometries of the trisubstituted C-3/C-4 and C-6/C-7 double bonds were also assigned from the NOE correlations of H 3 -18 (δ H 1.35, s) with H-2, but not with H-3, as well as the large coupling constant J = 16.0 Hz between H-6 and H-7, and the observed more shielded signal of C-18 (δ C 16.7). According to the above observations, the relative configuration of this compound was established. As 1 was isolated together with the previous reported compounds isosarcophine and cherbonolides A−E [24] from the same organism, it should possess the same (2S,8S,11R,12R)-configuration from the shared biosynthetic pathway.     The molecular formula of cherbonolide G (2) was found to be C 20 H 28 O 5 by analysis of HR-ESI-MS (m/z calculated 371.1829; found 371.1830, [M + Na] + ), revealing that 2 possesses an additional oxygen atom to that of 1. Moreover, both 1 and 2 showed the very similar 1 H-and 13 C-NMR data ( Table 1), except that the chemical shift of C-8 was shifted from δ C 71.7 of 1 to δ C 83.7 of 2. The very similar COSY, HMBC (Figure 2), and NOE ( Figure 3) correlations of 1 and 2 also revealed the very close structures for both compounds. However, the hydroxy group of 1 at C-8 was replaced by a hydroperoxy group in 2, with a broad singlet appearing at δ H 6.72 and the downfield shift of C-8. Accordingly, the molecular skeleton and the (2S,8S,11R,12R)-configuration of 2 were determined.
Cherbonolide H (3) has the same molecular formula as that of 1, as determined by HR-ESI-MS experiment. Moreover, most of the 1 H-1 H COSY and HMBC correlations (Figure 2) of 3 were similar to those of isosarcophine except for the presence of a hydroxyl at C-9 leading to the shift of CH-9 to lower field (δ C 76.2; δ H 3.68), and the shift of C-6/C-7 double bond of 1 to C-7/C-8 double bond of 3. Analysis of NOE correlations (Figure 3) showed that the β-oriented H-2 exhibited NOE interactions with both H 3 -18 and H-13β, but not with H-3, assigning the E-geometry of the trisubstituted C-3/C-4 double bond. These results, along with the found NOE correlations (Figure 3) of H-13α/ H 3 -20, H 3 -20/H-9, led to the assignment of the α-orientation of H-9.
Cherbonolide I (4) was found to contain one additional oxygen atom than 3, according to HR-ESI-MS experiment. These two compounds also showed very similar 1 H-1 H COSY and HMBC correlations, revealing the identical molecular framework of both compounds. NMR data of 3 and 4 were similar (Table 1), except for those of CH-9, suggesting that 4 is possibly the C-9 hydroperoxy derivative of 3. By analysis of NOE correlations (Figure 3), the E geometries of both C-3/C-4 and C-7/C-8 double bonds of 4 and the (2S,9R,11R,12R)-configuration were also established. Reduction of 4 by triphenylphosphine yielded 3, further confirming the structure of 4.
Cherbonolide J (5) was given as a colorless oil with a molecular formula C 20 H 30 O 5 on the basis of HR-ESI-MS data (m/z calculated for C 20 H 30 O 5 Na 373.1986; found 373.1984), revealing six degrees of unsaturation. The IR absorptions at 3443 and 1748 cm −1 were due to hydroxy and ester carbonyl groups, respectively. The 13 C-and 1 H-NMR spectroscopic data (Tables 1 and 3) of 5 measured at C 6 D 6 were very close to a known compound sarcophyolide E [29], and the 2D NMR (COSY, HSQC, and HMBC) correlation analysis revealed that both compounds had the same molecular framework (Figure 4). Detailed analysis of the NOE correlations ( Figure 5) showed that both compounds possessed the same relative configuration. However, the [α] D 25 values in CHCl 3 (−6 for 5 and +4.4 for sarcophyolide E) were close but with different signs, suggesting that 5 is the enantiomer of this known compound. The absolute configurations of 5 and sarcophyolide E were deduced by comparison of the circular dichroism (CD) spectroscopic data. As shown in Figure 6, the negative Cotton effect at 247 nm and positive effect at 228 nm for 5 in comparison with the positive and negative Cotton effects at 252 and 226 nm for sarcophyolide E [29], respectively, confirmed that 5 is the newly found enantiomer of sarcophyolide E.   Cherbonolide K (6) is a colorless oil which was shown to have the molecular formula C 20 H 28 O 4 by HR-ESI-MS experiment, appropriate for seven degrees of unsaturation. The infrared (IR) spectrum of 6 showed peaks of hydroxy and estercarbonyl groups at 3444 and 1763 cm −1 , respectively. 13 C-NMR data (Table 1) with signals at δ C 151.2 (C), 147.2 (C), 116.2 (CH), 72.7 (C), 123.6 (C), 169.5 (C), 9.1 (CH 3 ), and 29.9 (CH 3 ) and 1 H NMR data (Table 3) with signals at δ H 5.50 (s, 1H), 1.95 (s, 3H), and 1.41 (s, 3H) were attributed to the cembranoidal α-methyl-α,β-unsaturated-γ lactone ring with a conjugated 2,3-double bond that further connected with the methyl and hydroxyl substituted C-4. The above results were supported by HMBC correlations of 6 ( Figure 4) from H-3 (δ H 5.50) to C-1 (δ C 151.2), C-2 (δ C 147.2), and C-4 (δ C 72.7), H 3 -17 (δ H 1.95) to C-1, C-15 (δ C 123.6), and C-16 (δ C 169.5), and H 3 -18 (δ H 1.41) to C-3 (δ C 116.2) and C-4 (δ C 72.6). The remainder of the structure from C-5 to C-14 was found to be identical to isosarcophine [24]. Thus, the planar structure of 6 was established. Furthermore, the NOE correlation analysis shown in Figure 5 revealed the α-orientations of 4-OH and 12-CH 3 , β-orientation of H-11, (Z)-2,3-double bond, and (E)-7,8-double bond. An isomer of 6, cherbonolide L (7), was also subsequently isolated. The metabolite 7 had nearly the same NMR data as 6 except for CH 2 -5 and CH 2 -6. Thus, it can be assumed that 7 is the C-4 epimer of 6. Analysis of the 2D NMR correlations of 7 (Figures 4 and 6) further supported this assumption.
For the screening of bioactivities, the anti-inflammation activities of 1-7 toward inhibition of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion (O 2 •-) and release of elastase in primary human neutrophils were measured. The results (Table 4) showed that, although none of the isolates exhibited strong inhibitory activities in the assay, 2 and 3 were found to display notable ability to inhibit the elastase release (48.2% ± 6.2%) and superoxide anion generation (44.5% ± 4.6%) at 30 µM, respectively. In comparison with (+)-isosarcophine, cherbonolides A-E, and bischerbonolide peroide discovered previously from S. cherbonnieri [24], it was found that, although 2 and 3 exhibited weaker activities than bischerbonolide peroxide, they displayed comparable activities to those of cherbonolides A and C. In general, allylic oxidation at the 7,8-double bond of (+)-isosarcophine might be able to produce derivatives with stronger bioactivities.

Animal Materials
The marine organism S. cherbonnieri was collected and preserved as described previously [24].

Reduction of Cherbonolide I (4)
The solution of compound 4 (1.4 mg) in diethyl ether (5.0 mL) was added to an excess amount of triphenylphosphine (1.3 mg), and the mixture was stirred at room temperature for 4 h. The solvent of the solution was evaporated under reduced pressure to afford a residue, which was purified by silica gel column chromatography using n-hexane-acetone (3:1) as an eluent to yield 3 (1.0 mg, 75%). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki.

Statistical Analysis
Data were displayed as the mean ± SD, and comparisons were performed by one-way ANOVA with Dunnett analysis. All results were obtained from eight biological replicates. A probability value of 0.05 or less was considered to be significant. The Prism software (Version 5.0, GraphPad Software, San Diego, CA, USA) was used for the statistical analysis.

Conclusions
Our present examination of the chemical constituents of the soft coral S. cherbonnieri led to the discovery of new cembranoid compounds 1-7. All compounds were found to possess anti-inflammatory activity by exhibiting inhibitory effects on the generation of superoxide anion and elastase release in fMLF/CB-induced primary human neutrophils, and cherbonolides G and H (2 and 3) were found to be the most active in the inhibition of elastase release and superoxide anion generation, respectively.
As the marine environment is an important source of bioactive substances, and due to the high chemical diversity and specimen diversity of the Sarcophyton genus [27,28,34,35], it can be expected that new natural products and activities from soft corals of this genus can be continuously discovered in the future.

Conflicts of Interest:
The authors declare no conflict of interest.