New Verticillene Diterpenoids, Eudesmane Sesquiterpenoids, and Hydroperoxysteroids from the Further Chemical Investigation of a Taiwanese Soft Coral Cespitularia sp.

An investigation of the chemical composition of a Formosan soft coral Cespitularia sp. led to the discovery of one new verticillene-type diterpenoid, cespitulactam M (1); one new eudesmane sesquiterpenoid, cespilamide F (2); and three new hydroperoxysteroids (3–5) along with twelve known analogous metabolites (6–17). In addition, one new derivative, cespitulactam M-6,2′-diacetate (1a), was prepared from compound 1. The structures were determined by detailed spectroscopic analyses, particularly HRESIMS and NMR techniques. Moreover, the in vitro cytotoxicity, anti-inflammatory, and antibacterial activity of 1–17 and 1a were evaluated.

With the planar structure of 1 determined, the relative stereochemistry of the three stereogenic centers, 1R*, 6S*, and 10R*, of 1, was assigned via the analysis of the NOESY spectrum ( Figure 3) , revealing the α-orientation of proton H-6. On the basis of the NOESY spectral analysis and MM2 force field analysis, the relative structure of cespitulactam M (1) was determined. The absolute configuration of 1 was suggested as 1R, 6S, and 10R by the proposed biosynthetic pathway as an intermediate from cespitularin C to cespitulamide C [7].
Furthermore, upon acetylation, compound 1 afforded the diacetate, cespitulactam M-6, 2'-diacetate (1a), which exhibited two additional three-proton acetyl singlets at δH 2.01 and δH 2.04. Through the comparison of 1 H and 13 C NMR spectra of 1 and 1a, the deshielding of H2-2′ from δH 3.85 to 4.31 and 4.12 suggested the location of an acetyl group at C-2′, while the deshielding of H-6 from δH 4.35 to 5.29 also indicated the location of an acetyl group at C-6. In addition, the HRESIMS of 1a revealed a molecular ion at m/z 466.2565, [M + Na] + (calculated for C22H33NO3Na, 466.2564).   Cespilamide F (2) was obtained as a white powder. The HRESIMS (m/z 300.1572 [M + Na] + , calculated for C16H23NO3Na, 300.1570) of 2 established the molecular formula C16H23NO3, appropriate for six degrees of unsaturation. Its IR spectrum also revealed the presence of amide (1693 cm −1 ) and hydroxy (3418 cm −1 ) groups. The 13 C NMR and HSQC spectroscopic data (Tables 1 and 2) illustrated signals of three methyls (including one methoxyl group appearing at δH 3.09 and δC 49.6 ppm), four sp 3 methylenes, one sp 2 methylene, two sp 3 methines, two sp 3 , and four sp 2 quaternary carbons (including one carbonyl carbon appearing at δC 174.3). The above data accounted for three of the six degrees of unsaturation, indicating a tricyclic structure for 2. From the COSY spectrum measured in CDCl3, we established two proton sequences from H2-1 to H2-3 and H-5 to H2-6. The key HMBC correlations of H2-6 to C-7 and C-8; H-9 to C-7, C-8, and C-10; H2-13 to C-7, C-11, and C-12; H3-14 to C-1, C-5, C-9, and C-10; H3-15 to C-3 and C-5; and H3-16 to C-8 permitted the connection of the carbon skeleton (supplementary materials, S12-S20). Based on the above analysis, the planar structure of 2 was established ( Figure 2).
The relative configuration of 3 was elucidated on the basis of the observed key NOE correlations (Figure 3). In particular, the stereo center of C-7 was the most significant result in this compound; thus, it was compared with the reported compounds 7β-hydroperoxycholesterol and its stereoisomer 7α-hydroperoxycholesterol [19]. In terms of the absolute configuration of the side chain, the 1D NMR spectra of compound 3 were compared with those of gorgosterol [21] and 7-oxogorgosterol [22] from previous research. It turned out that the chemical shifts of compound 3 were similar to that of 7-oxogorgosterol as 20R, 22R, 23R, and 24R. Thus, the absolute configuration of 7β-hydroperoxygorgosterol (3) was proposed.
7α-Hydroperoxygorgosterol (4) was isolated as a white powder. The HRESIMS exhibited a [M + Na] + ion peak at 481.3652 m/z, establishing a molecular formula of C 30 H 50 O 3 . By 2D NMR spectroscopy data, including HSQC, COSY, and HMBC (supplementary materials, S29-S36), compound 4 was displayed to possess the same molecular framework as that of 3. The molecular formula of 3 and 4 indicates that 4 is an isomer of 3. On the basis of the above references and its NOE correlations, compound 4 was revealed to be the C-7 epimer of 3, namely 7α-hydroperoxygorgosterol (4).
The HRESIMS of 7β-hydroperoxycampesterol (5) showed that it possesses the molecular formula C 28 H 48 O 3 (m/z 455.3495 [M + Na] + ). The IR spectrum of 5 showed the absorption of a hydroxy group (3383 cm −1 ). Comparison of the 1 H and 13 C NMR spectroscopic data of compound 5 with known compound 7α-hydroperoxycampesterol (6) suggested that the planar structure of 5 was the same as 7α-hydroperoxycampesterol [18].
Owing to the NMR data of 5 and the reported compound 7α-hydroperoxycampesterol (6), this suggests that 5 is an isomer of 6. Compound 5 was also compared with the known compounds 7β-hydroperoxycholesterol and 7α-hydroperoxycholesterol in order to define the stereo center C-7 [19]. In terms of the chiral center C-24 on the side chain, the 1D NMR data were compared with those of (24R)-campesterol and (24S)-methylcholesterol [23,24]. Based on the previous literature and NOE correlations, the absolute configuration of 5 was proposed to be 3S, 7R, and 24S.
Compounds 1-16 and 1a were also evaluated for their cytotoxicity to human lung adenocarcinoma (A549), human hepatocellular carcinoma (HepG2), and human breast adenocarcinoma (MDA-MB-231) cancer cell lines by using the Almar Blue assay [25,26]. The results showed that only 7β-hydroperoxycampesterol (5) exhibited cytotoxicity (IC 50 = 15.40, 18.74 µg/mL) toward the cell lines MDA-MB-231 and A549, compared with the positive control, doxorubicin (IC 50 0.30, 0.15 µg/mL), respectively, while others did not exhibit cytotoxicity within 20 µg/mL. Furthermore, the antibacterial activities of 3-7, 9, and 12 were tested against the growth of a limited panel of bacteria strains, including Bacillus subtilis, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis, Salmonella typhimurium, Serratia marcescens, Shigella sonnei, Staphylococcus aureus, and Yersinia enterocolitica. As a result, compound 5 exhibited antibacterial activities against S. enteritidis (inhibition zone: 6.0 mm) and K. pneumoniae (inhibition zone: 5.0 mm), and compound 9 showed an inhibition zone of 9.0 mm against K. pneumoniae at the dosage of 25 µg/disk by the disc diffusion method, compared with the positive control, ampicillin, against S. enteritidis (inhibition zone: 10.0 mm) and K. pneumoniae (inhibition zone: 5.0 mm) at the same dosage of 25 µg/disk, while others did not show activities to these bacteria strains.

Animal Material
The soft coral, Cespitularia sp., was collected by hand using SCUBA from the coast of Green Island, Taiwan, in June 2007, at a depth of 10-15 m and stored in a -20 • C freezer until extraction. The soft coral was identified by Professor Chang-Feng Dai, Institute of Oceanography, National Taiwan University. A voucher sample was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

In Vitro Antibacterial Assay
The antibacterial assay of compounds 1-17 and 1a was evaluated against B. subtilis The experiment for measuring cytokine was tested by enzyme-link immunosorbent assay (ELISA) from the previously reported method [28,29]. The DCs were manipulated with lipopolysaccharide (LPS, 100 ng/mL) from Escherichia coli 055:B5 and the following treatment with the isolated compounds for 24 h. The optical density of the production of TNF-α was measured at 450 nm using the ELISA reader.
3.6.2. Measurement of Nitric Oxide (NO) Production by DCs DC cells were seeded in 24-well plates at a density of 1 × 10 6 cells/mL. DCs were treated with each compound for 1 h and then stimulated with 100 ng/mL LPS for 24 h. The nitrite concentration in the medium was measured as an indicator of NO production through the Griess reaction. Briefly, 100 µL of cell culture supernatant was reacted with 100 µL of Griess reagent (1:1 mixture of 2% sulfanilamide and 0.2% N-(1naphthyl)ethylenediamine dihydrochloride in water) in 96-well plate at room temperature for 10 min, and absorbance at 540 nm was recorded using sandwich ELISA assays [28,29].

Statistical Analysis
The results are expressed as the mean ± SEM, and comparisons were made using one-way ANOVA by Tukey's post hoc test (GraphPad Prism 5.0, GraphPad Software, San Diego, CA, USA). A probability value of 0.05 or less was considered significant. The software Sigma Plot was used for the statistical analysis.