Bioactive Capnosanes and Cembranes from the Soft Coral Klyxum flaccidum

Two new capnosane-based diterpenoids, flaccidenol A (1) and 7-epi-pavidolide D (2), two new cembranoids, flaccidodioxide (3) and flaccidodiol (4), and three known compounds 5 to 7 were characterized from the marine soft coral Klyxum flaccidum, collected off the coast of the island of Pratas. The structures of the new compounds were determined by extensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and spectroscopic data comparison with related structures. The rare capnosane diterpenoids were isolated herein from the genus Klyxum for the first time. The cytotoxicity of compounds 1 to 7 against the proliferation of a limited panel of cancer cell lines was assayed. The isolated diterpenoids also exhibited anti-inflammatory activity through suppression of superoxide anion generation and elastase release in the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-stimulated human neutrophils. Furthermore, 1 and 7 also exhibited cytotoxicity toward the tested cancer cells, and 7 could effectively inhibit elastase release. It is worth noting that the biological activities of 7 are reported for the first time in this paper.


Results and Discussion
The ethyl acetate (EtOAc) extract of K. flaccidum was initially fractionated over a silica gel column, and the eluted fractions displaying terpenoidal methyl, olefinic, and oxymethine proton signals in the 1 H NMR spectra were selected for further purification. One of these fractions was purified by repeated column chromatography to yield diterpenoids 1 to 7 (Figure 1), the structures were established on the basis of spectroscopic analyses (Supplementary Materials, Figures S1 to S28). Flaccidenol A (1) was isolated as a colorless oil and has a molecular formula of C 20 H 34 O 4 with four unsaturations, based on the sodium adduct ion peak [M + Na] + obtained by positive high-resolution electrospray ionization mass spectroscopy (HRESIMS). The infrared (IR) spectrum revealed the presence of hydroxy and olefinic functionalities (ν max 3391 and 1647 cm −1 ). This was evidenced from the carbon signals of a trisubstituted and one 1,1-disubstituted carbon-carbon double bonds [δ C 148.1 (C), 146.6 (C), 122.3 (CH), and 117.4 (CH 2 )] and three sp 3 oxygenated carbons [δ C 90.7 (CH), 82.8 (C), and 74.5 (C)] (Table 1), respectively, out of the 20 carbon signals in 13 C NMR spectrum of 1. Moreover, the remaining two unsaturations indicated that compound 1 was a bicyclic diterpenoid. Similar to cembranoids [32], the 1 Table 2). The analysis of correlation spectroscopy (COSY) of 1 indicated four partial structures bearing consecutive proton systems ( Figure 2). One of the partial structures included two ring-juncture methines [δ H /δ C 2.89/51.0 (CH) and 2.63/50.5 (CH)] and were positioned at C-3 and C-7 from the analysis of heteronuclear multiple-bond correlation (HMBC) spectra ( Figure 2), similar to those of 3,7-cyclized cembranoid (capnosane) diterpenoids [15,17,37,38]. Key HMBC correlations observed from H 3 -18 to C-3 and C-5; H 3 -19 to C-7 and C-9; H 2 -20 to C-11 and C-13; H-15 to C-2 were used to connect the four COSY correlated partial structures and to further establish the 3,7-linkage in the capnosane molecular skeleton. Moreover, the NMR signals at δ H 7.64 (1H, br s) and δ C 90.7 (CH) pointed out the presence of an allylic hydroperoxy group [39][40][41]. Finally, the detailed HMBC correlation analysis confirmed the hydroperoxyl, the two hydroxyls, the exomethylene, and the trisubstituted double bond to be at C-11, C-4, C-8, C-12, and C-1/C-2, respectively, and consequently the planar structure of 1 as shown in Figure 2. The relative configurations of 1 at C-3, C-4, C-7, C-8, and C-11 were proposed from the analysis of nuclear Overhauser effect spectroscopy (NOESY) correlations in combination with molecular modeling using molecular mechanical parameters (MM2 force field) calculations (  The molecular formula of compound 2 was found to be C 20 H 34 O 2 as deduced from HRESIMS and 13 C NMR data, appropriate for four degrees of unsaturation. The IR spectrum showed the presence of the hydroxy group (ν max 3391 cm −1 ) and olefinic functionality (ν max 1656 cm −1 ). The NMR spectroscopic data were found to be similar to those of pavidolide D [37], while detailed analyses of 2D NMR correlations (COSY and HMBC) revealed that both two compounds possessed the same molecular skeleton (Figure 2). On careful comparison of the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2, respectively) of 2 with those of pavidolide D, it was found that the ring-juncture protons H-3 (δ H 2.37, dd, J = 9.6, 7.8 Hz) and H-7 (δ H 2.65, ddd, J = 9.6, 7.8, 6.6 Hz) showed differentiable NMR data with the corresponding H-3 (δ H 2.55, t, J =10.3 Hz) and H-7 (δ H 1.98, m) of the known compound. Further NOE correlations analysis of 2, confirmed compound 2 to be a new capnosane diterpenoid that should be the 7-epimer of pavidolide D. The assignment of the cis ring juncture protons H-3 and H-7 in 2 was further supported by the similar upfield chemical shift C-7 in 2 (δ C 54.3) with that of trocheliophol F (δ C 54.2) [17] relative to those of the related capnosenes with trans fused rings (δ C ≥ 55.5) [16,17,37]. Furthermore, the E-geometry of C-11/C-12 double bond was determined from the NOE correlations observed between H 3 -20 and H-3 and between H-11 and H 3 -19, and from the chemical shift of C-20 (δ C < 20 ppm) [42].   (Tables 1 and 2), with those of a known metabolite gibberosene C [43], revealed that both compounds possessed the same 3(4),11(12)-diepoxyl groups in the cembrane ring-structure. The hydroxyl group at C-13 of gibbrosene C was acetylated, and the methylene group of C-14 was converted to acetoxyl-bearing methine, as support by the downfield chemical shifts of H-13 (δ H 5.49) and H-14 (δ H 5.89) of 3, and the three-bond correlations of both protons to two acetate carbonyl carbons unveiled from HMBC spectrum of 3. The planar structure of 3 was further confirmed by the detailed analyses of 1 H-1 H COSY and HMBC spectra (Figure 2). These results, along with extensive analysis of NOE correlations of 3 (Figure 3), determined the structure of 3 unambiguously.
Flaccidodiol (4) was isolated as a gum. HRESIMS of 4 exhibited a sodiated molecular ion peak at m/z 329.2448, corresponding to the molecular formula C 20 H 34 O 2 . IR absorptions at 3457 cm −1 indicated the presence of a hydroxyl group in 4. The NMR data (Tables 1 and 2) assigned the presence of an isopropyl group and three methyls, including two olefinic ones and one attaching to an oxygenated sp 3 carbon atom. It was found that the proton and carbon chemical shifts of 4 were very similar to those of a known dihydroxycembrane, sarcophytol T [34]. Thus, both compounds should be diastereomers. Furthermore, the proton signal of 1-OH exhibited NOE correlation with H-3, while H 3 -18 showed NOE correlation with H-2, revealing the position of both 1-OH and 4-OH to be in the same face of 4. Thus, the relative configuration for 4 was established. Cytotoxicities of metabolites 1 to 7 against the growth of human lung adenocarcinoma (A549), human colorectal adenocarcinoma (DLD-1), and mouse lymphocytic leukemia (P388D1) cell lines were screened. Compounds 1, 2, and 7 exhibited inhibitory activity against the growth of the tested cancer cells ( Table 3). The hydroperoxyl (as in 1) seem to potentiate the cytotoxic effect of the diterpenoid molecules. However, the diepoxide compound 3 could selectively inhibit the growth of P388D1 cancer cells relative to the inactive monoepoxide derivative (6). These compounds also exhibited the potent anti-inflammatory activity by suppressing O 2 −• generation and elastase release in the fMLF/CB-stimulated human neutrophils (Table 4). Compound 7 could effectively inhibit elastase release (IC 50 = 7.22 ± 0.85 µM), reducing the level of elastase release to 59.66 ± 0.83% at a concentration of 10 µM relative to the control group.

Animal Material
The soft coral Klyxum flaccidum Tixier-Durivault was collected along the coast of Pratas island, Taiwan, and stored until extraction as described before [32]. The organism was identified by one of the co-authors (C.-F.D.). A voucher sample (No. LI6) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

In Vitro Anti-Inflammatory Assay
Human neutrophils were obtained from blood by dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis and then incubated as previously described [46]. Neutrophils

Conclusions
Further chemical investigation on Formosan soft coral Klyxum flaccidum has led to the isolation and characterization of two new capnosanoids, flaccidenols A (1) and B (2); two new cembranoids, flaccidodioxide (3) and flaccidodiol (4); along with three known cembranoids. This is the first study to discover the rarely found capnosane diterpenoids from genus Klyxum. Diterpenoids 1, 2, and 7 exhibited significant cytotoxicity against a limited panel of cancer cell lines. The hydroperoxy group might be required to potentiate the cytotoxic effect, as shown by the capnosanoid 1. Moreover, 14-O-acetylsarcophytol B (7) could inhibit the elastase release in the fMLF/CB-stimulated human neutrophils effectively. This is the first time to disclose bioactivity for this known compound, which might be considered as an anti-inflammatory candidate.