Glaucumolides A and B, Biscembranoids with New Structural Type from a Cultured Soft Coral Sarcophyton glaucum

Glaucumolides A (1) and B (2), novel biscembranes composed of an unprecedented α,β-unsaturated ε-lactone, along with the known metabolites ximaolide A (3) and isosarcophytonolide D (4), were isolated from the cultured soft coral Sarcophyton glaucum. The structures of the new metabolites were determined by extensive spectroscopic analyses. Compounds 1 and 2 were shown to exhibit cytotoxicity against a limited panel of cancer cell lines. In anti-inflammation assay, compounds 1 and 2 displayed strong inhibition of superoxide anion generation and elastase release in human neutrophils stimulated by fMLP/CB. Furthermore, both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase protein, and compounds 1−3 were found to effectively reduce the expression of cyclooxygenase-2 protein, in lipopolysaccharide-stimulated RAW264.7 macrophage cells.

and 2, possessing a γ -and an ε -lactone rings, exhibited inhibition against the growth of human cancer cell lines, promyelocytic leukemia (HL-60), leukemic lymphoblasts (CCRF-CEM), acute T lymphoblastic leukaemia (MOLT-4), and erythroleukemia (K-562), as well as anti-inflammatory activities by significantly reducing the superoxide anion generation and elastase release in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB), and the expression of iNOS and COX-2 proteins in LPS-challenged RAW264.7 macrophage cells. In contrast, without the presence of the mentioned lactone rings, 3 only displayed weaker inhibition on COX-2 accumulation in the same macrophage cells.

Results
Glaucumolide A (1), [α ] 25 D − 207 (c 0.007, CHCl 3 ), was isolated as an amorphous solid. Its molecular formula, C 42 H 58 O 8 , was established by HRESIMS (713.4022 m/z, [M + Na] + ), implying 14 degrees of unsaturation. The presence of the hydroxyl group was suggested by an absorption band at 3499 cm -1 in the IR spectrum. The 13 C NMR spectroscopic data of 1 (Table 1) showed the presence of 42 carbon atoms, including eight methyls, 11 methylenes, 11 methines, and 12 quaternary carbons. Its NMR spectrum showed the signals of four vinyl methyls (δ H 2.12, s; 1.81, s; 1.65, s; 1.62, s; δc 20.2, 19.6, 17.9, 16.0),     The IR spectrum also suggested the presence of hydroxyl group in 2 (ν max 3434 cm -1 ). The 13 C NMR spectroscopic data (Table 1) of 2 were found to be resembled to those of 1. Detailed analysis of 1D and 2D NMR spectra of 2 revealed the similar gross structure as that of 1. However, it was found that H-12 (δ 5.98, s) showed significant NOE interaction with H 3 -19 (δ 1.89, s), and the signal of the C-19 in 2 was remarkably downfield-shifted (δ 19.6 in 1, 24.8 in 2), indicating a Z geometry of Scientific RepoRts | 5:15624 | DOi: 10.1038/srep15624 Δ 11 (12) in 2, in contrast to the 11E double bond in 1. These results and other NMR data including NOE correlations, established the structure of compound 2 to be the 11Z isomer of 1.
The absolute configurations of 1 and 2 were further confirmed by comparison of the CD (circular dichroism) spectroscopic data with structurally related compound. As shown in Fig. 5, the CD spectrum of 2 showed a broad negative Cotton effect at 258 nm (Δε = −7.1) due to enone n-π * transition absorption while the intense positive cotton effect at 209 nm (Δε = +27.4) resulted from the π -π * transition of the two isolated Δ 22 (23) and Δ 34(35) double bonds, very similar to that of bislatumlide C 10 . Thus, the absolute configurations for rings B and C should be the same for both bislatumlide C and 2. One the basis of above results and the fact that both 1 and 2 were isolated from the same organism, the structures and absolute configurations of both 1 and 2 were found to possess the 1S,2R,3S,4S,5S,21S,26R,27S.
A plausible biosynthetic pathway, involving a key Diels-Alder reaction, was postulated for the biosynthesis of compounds 1 and 2 in Figure 6. It is obvious that 4 is one of the two precursors of 1. Another proposed precursor should be the yet to be discovered compound 6.
[4+2] Endo cycloaddition of the dienophile 4 and diene 6 occurs between 1,2-Z double bond of 4 and ∆ 21(34) and ∆ 35(36) conjugated double bonds diene of 6. Although the possible involvement of a biosynthetic Diels-Alder reaction to afford biscembranoid has been mentioned frequently, no any other bicembranoid was found to be formed by using ε -lactone cembrane 6 or related cembranoidal ε -lactone as the diene precursor. In addition, almost all the previous cembrane dimers exhibited the reactive dienophile double bond at positions C-1 and C-14 (according to the numbering assigned to compound 4), whereas the dienophile double bond in 4 was located at C-1 and C-2. The Diels-Alder addition which arises from supra-supra transition state explains the trans stereochemistry of H-2 and lactone as well as the cis geometry of lactone and H-21. Analogously, the formation of biscembrane 2 by Diels-Alder cyclization of the 11Z isomer of 4, sarcophytonolide A (5) 6,9 , with compound 6 could be hypothesized.
The cytotoxicity of compounds 1-4 against four human cancer cell lines, HL-60, CCRF-CEM, MOLT-4, and K-562 was investigated. The results (Table 2) demonstrated that compound 2 exhibited significant cytotoxicity against HL-60 and CCRF-CEM cancer cell lines with ED 50 values of 3.8 ± 0.9 and 5.3 ± 1.4 μg/mL, respectively. Also, compound 1 exhibited cytotoxicity against the above two cell lines with ED 50 values of 6.6 ± 1.2 and 7.4 ± 1.5 μg/mL, respectively. Further, compounds 1 and 2 displayed weaker activity against MOLT-4 and K-562 cell lines (ED 50 11.0-19.2 μg/mL). In contrast, compound 3 was inactive toward all the tested cell lines. Perhaps the enhanced cytotoxicity of compounds 1 and 2 relative to 3 is owing to the presence of a α ,β -unsaturated ε -lactone ring.
The anti-inflammatory activities of compounds 1-4 on neutrophil pro-inflammatory responses were evaluated by measuring their ability in suppressing fMLP/CB-induced superoxide anion (O 2 − • )  generation and elastase release in human neutrophils, and the results were shown in Table 3. From the results, 1 and 2 showed strong inhibitions (88.42 ± 3.97 and 91.75 ± 3.08%, respectively.) toward superoxide anion generation at 10 μ M. Both of them also exhibited potent inhibitory activity against elastase release, with 88.94 ± 6.96 and 103.25 ± 1.89% inhibitions in the same fMLP/CB-stimulated cells at the same concentration. The IC 50 values of 1 and 2 in inhibiting the superoxide generation and elastase release were also measured. Although compound 4 did not exhibit strong activity in inhibiting superoxide anion generation, it was shown to display significant inhibitory activity in elastase release. The in vitro anti-inflammatory activity of compounds 1-3 was also studied. In this assay, the up-regulation of the proinflammatory iNOS and COX-2 proteins of LPS-stimulated RAW264.7 macrophage cells was evaluated using immunoblot analysis. The results (Fig. 7) showed that at concentrations of 5, 10, and 20 μM, compound 1 was found to significantly reduce the levels of iNOS and COX-2 to 59.4 ± 9.0 and 66.5 ± 4.4%; 31.3 ± 6.5 and 78.3 ± 5.0%; and −2.6 ± 2.7 and −0.5 ± 3.2%, respectively. At concentrations of 10 and 20 μM, compound 2 was found to significantly reduce the levels of iNOS and COX-2 to 75.9 ± 3.5 and 64.3 ± 6.9%; and 43.4 ± 5.0 and 6.0 ± 3.6%, respectively. Moreover, at 20 μM, 3 also reduced the level of COX-2 expression to 22.0 ± 6.5% in macrophage cells with LPS treatment. As they did not exhibit cytotoxicity to RAW264.7 cells, they might be promising anti-inflammatory agents. Also, 2 possessing promising cytotoxicity, could become a candidate for future anticancer drug development.

Discussion
Compounds 1 and 2 are structurally novel as they belong to a new type of biscembranoids using the not yet isolated ε -lactonecembrane 6 as the first time discovered diene precursor for the biosynthesis of biscembranoids by Diels-Alder reaction. Metabolites 1 and 2, with the presence of a α ,β -unsaturated ε -lactone ring, were shown to exhibit cytotoxicity against a limited panel of HL-60, CCRF-CEM, MOLT-4 and K-562 cancer cell lines. Compounds 1 and 2 also exhibited potent anti-inflammatory activity in inhibiting the superoxide generation and elastase release in fMLP/CB-induced human neutrophils. Furthermore, both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase protein, and compounds 1-3 were found to effectively reduce the expression of cyclooxygenase-2 protein, in lipopolysaccharide-stimulated RAW264.7 macrophage cells.

Conclusion
The unusual structural framework with α ,β -unsaturated ε -lactone system were reported here for glaucumolides A and B (1 and 2), along with a known biscembranolide ximaolide A (3), and isosarcophytonolide D (4) from the cultured soft coral S. glaucum. From the results of biological activities, it appears that compounds 1 and 2 might be useful for future biomedical applications. The discovery of glaucumolides with a novel carbon scaffold provides additional evidence that cultured soft corals might be a promising source of structurally novel bioactive natural products which could be used for further pharmacological investigation.

General Experimental Procedures. Optical rotations were measured on a Horiba High Sensitivity
Polarimeter SEPA-300. Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. CD spectra were recorded on a JASCO J-815 CD spectrophotometer. NMR spectra were recorded on a Varian 400MR FT-NMR instrument at 400 MHz for 1 H and 100 MHz for 13  Animal Material. The cultured soft coral Sarcophyton glaucum used in this study was originally collected from the wild and cultured for five years in an 80-ton cultivation tank (height 1.6 m) located in the National Museum of Marine Biology and Aquarium, Taiwan. The tank was a semiclosed recirculating aquaculture system and did not require deliberate feeding. To the best of our knowledge, this is the   Cytotoxicity Testing. Cell lines were purchased from the American Type Culture Collection (ATCC).
Cytotoxicity assays of compounds 1-4 were performed using the Alamar Blue assay 18,19 . To measure the cytotoxicity activities of tested compounds, three concentrations in DMSO with three replications were performed on each cell line. 5-Fluorouracil and DMSO were used as positive and negative controls, respectively in this assay.
Preparation of Human Neutrophils. Human neutrophils obtained from peripheral blood of healthy adult volunteers (20-30 years old) were enriched using a standard method of dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis 20,21 . Purified neutrophils were resuspended in a Ca 2+ -free HBSS buffer (pH 7.4) at 4 °C prior to use.
Statistical Analysis. Results are expressed as the mean ± SEM, and comparisons were made using Student's t-test. A probability value of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.
In vitro anti-inflammatory activities of compounds 1-3 were measured by examining the inhibition of lipopolysaccharide (LPS) induced upregulation of iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in macrophages cells using Western blotting analysis 25 . For statistical analysis, all of the data were analyzed by a one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls post hoc test for multiple comparisons. A significant difference was defined as a p value of < 0.05.