Steroids from the Soft Coral Sinularia crassa

One new sterol, crassarosterol A (1), and four new steroidal glycosides, crassarosterosides A–D (2–5) were isolated from the Formosan soft coral Sinularia crassa. The absolute configuration of 1 was determined using the Mosher’s method. The absolute configurations for the sugar moieties of 2–5 were determined by HPLC analysis on the o-tolylthiocarbamates derived from the liberated sugar after acid hydrolysis. Compounds 2 and 4 could significantly inhibit the expression of pro-inflammatory iNOS protein at 10 µM. In contrast, 1–3 were found to stimulate the expression of COX-2 protein at this concentration. Steroids 1 and 4 also showed cytotoxicity toward the selected human liver cancer cells.


Introduction
Soft corals have proven to be a rich source of terpenoids [1]. We previously have isolated a series of bioactive cembrane- [2][3][4] and norcembrane- [5][6][7][8] diterpenoids from the Formosan soft corals of the genus Sinularia. Previous chemical investigations on the soft coral Sinularia crassa have led to the isolation of structurally unique steroids and cembranoids, from which some have been shown to exhibit anti-inflammatory [9,10] and 5α-reductase inhibitory activities [11], respectively. Our continuing chemical investigation on S. crassa has led to the isolation of one new sterol, crassarosterol A (1), and four new steroidal glycosides, crassarosterosides A-D (2-5) (Figure 1). The structures of 1-5 have been established by extensive spectroscopic analysis, including 2D NMR ( 1 H-1 H COSY, HMQC, HMBC, and NOESY), chemical methods, and HPLC. The effect of 1-4 on the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells and the cytotoxicity of compounds 1-5 against a panel of cancer cell lines including human liver carcinoma (HepG2 and HepG3), human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A-549) were evaluated in order to discover bioactive natural products.

Results and Discussion
The HRESIMS of crassarosterol A (1) exhibited a pseudomolecular ion peak at m/z 453.3342 [M + Na] + , consistent with a molecular formula of C 28 H 46 O 3 and requiring six degrees of unsaturation. The 13 C NMR and DEPT spectroscopic data (Table 1) displayed 28 carbon signals, including five methyls, nine methylenes, ten methines, and four quaternary carbons. The IR spectrum revealed the presence of hydroxy groups (3389 cm -1 ). The carbon resonances at δ 141.2 (C), 121.3 (CH), 156.9 (C), and 106.3 (CH 2 ) suggested the presence of two double bonds. The above data coupled with the characteristic 1 H NMR signals for methyl groups at δ 0.92 (3H, s), 1.18 (3H, s), 1.04 (3H, d, J = 6.8 Hz), and 1.03 (6H, d, J = 7.2 Hz) and signals for olefinic protons at δ H 5.41 (1H, d, J = 5.6 Hz), 4.76 (1H, s), and 4.70 (1H, s) ( Table 2) suggested 1 to be a member of the 24-methylenecholesterol class [12,13]. The 1 H− 1 H COSY correlations allowed the assignment of four separated spin systems ( Figure 2). The presence of an sp 2 methylene substituent at C-24 was further confirmed by the HMBC correlations from H 2 -28 to C-23, C-24, and C-25 ( Figure 2). Likewise, the steroidal nucleus was confirmed by the HMBC correlations from H 3 -18 to C-12, C-13, C-14, and C-17 and H 3 -19 to C-1, C-5, C-9, and C- 10 [14]. Analysis of the 1 H NMR data of esters 1a and 1b resulted in the determination of a 3S configuration ( Figure 3).   Analysis of the HRESIMS and 13 C NMR spectroscopic data of crassarosteroside A (2) suggested a molecular formula of C 36 H 58 O 8 . The IR spectrum of 2 showed the presence of hydroxy (3461 cm −1 ) and carbonyl (1741 cm −1 ) groups. The latter was identified as an acetoxy group according to the carbon resonances at δ 170.9 (C) and 21.2 (CH 3 ) ( Table 1) (Table 2) [12,15,16]. The acetoxy group attached at C-2' of the α-fucose residue was evidenced from the downfield chemical shift of H-2' (δ 5.07), which was confirmed by the HMBC correlations from this proton to the acetate carbonyl carbon. The HMBC correlation from H-1' to C-3 revealed that the α-fucose residue was attached at C-3 of the steroidal aglycone. The absolute configuration of the sugar moiety in 2 was determined by reversed phase HPLC analysis of its o-tolylthiocarbamate [17]. The liberated fucose from acid hydrolysis of 2 was treated with L-cysteine methyl ester followed by reaction with o-tolylisothiocyanate to afford the corresponding o-tolylthiocarbamate derivative. The retention time of the liberated sugar derivative by HPLC analysis was found to be consistent with that of a standard L-fucose derivative.
Crassarosteroside B (3) gave the same molecular formula as that of 2 based on the analysis of the HRESIMS and 13 C NMR spectroscopic data ( Table 1). The NMR spectroscopic data of 3 were similar to those of 2, with some exceptions for those of the sugar residue. An HMBC correlation from the anomeric proton at δ 5.12 (H-1') to the carbon signal at δ 77.8 (C-3) connected the fucose residue to C-3 of the steroidal aglycone. The downfield proton chemical shift at δ 4.87 (1H, dd, J = 10.5, 4.0 Hz) was ascribed to the presence of an acetoxy group at C-3' ( Table 2). The detailed 2D NMR analysis confirmed the above elucidation. Likewise, the L-fucose residue was deduced according to RP HPLC analysis of the corresponding o-tolylthiocarbamate as described above. Crassarosteroside C (4) was assigned the same molecular formula as those of 2 and 3. A comparison of NMR spectroscopic data of 4 with those of 2 and 3 revealed that an acetoxy group should be located at C-4' of the fucose residue (Tables 1 and 2). This was evidenced by the 1 H NMR shift of H-4' at 5.20 (1H, d, J = 2.8 Hz). In the same manner, RP HPLC anslysis of the corresponding o-tolylthiocarbamate derived from the hydrolyte of 4 allowed the determination of the L-fucose moiety.
The HRESIMS and 13 C NMR spectroscopic data of crassarosteroside D (5) established a molecular formula of C 37 H 60 O 7 . The presence of an acetoxy group was evidenced by the 1 H NMR signal at δ 2.17 (3H, s) ( Table 1) and 13 C NMR signals at δ 171.3 (C) and 20.8 (CH 3 ) ( Table 2) as well as the IR absorption band at 1737 cm −1 . The NMR spectroscopic data for the sugar moiety of 5 were quite similar to those of 4, suggesting that they shared the same 4'-O-acetylfucose residue. Except for the sugar moiety, the remaining 29 carbon signals as well as the characteristic methyl signals at δ 1. and C-24. This rare steroidal side chain is the same as that of sarcophytosterol isolated previously by us from the soft coral Lobophytum sarcophytoides [18]. The NMR spectroscopic data for the aglycone moiety of 5 are almost the same as those of sarcophytosterol, except for some minor variations in 1 H and 13 C chemical shifts at C-2, C-3, and C-4 between both compounds. This is due to the attachment of the sugar residue at C-3 of the steroidal aglycone. Similarly, HPLC analysis of the relevant o-tolylthiocarbamate derived from the hydrolysis of 5 suggested the presence of L-fucose.   showed cytotoxicity toward HepG2 and HepG3 cell lines with IC 50 values of 17.6 and 18.9 µM, respectively. The other compounds were found to be inactive (IC 50 > 20 μM) toward the above cancer cell lines after 72 h exposure. The effect of steroids 1-4 on the expression of pro-inflammatory iNOS and COX-2 proteins in RAW264.7 macrophage cells stimulated with LPS was also evaluated using immunoblot analysis. At a concentration of 10 µM (Figure 4), steroid 2 was found to significantly reduce the level of iNOS protein to 12.9 ± 4.3% and 4 could reduce the iNOS espression to 50.1 ± 6.3%. However, these compounds could not effectively reduce the level of COX-2. On the contrary, 1-3 were found to stimulate the expression of COX-2.

General Experimental Procedures
The melting point was determined using a Fisher-Johns melting point apparatus. Optical rotations were determined with a JASCO P1020 digital polarimeter. IR spectrum was obtained on a JASCO FT/IR-4100 spectrophotometer. The NMR spectra were recorded on a Varian 400 MR NMR or Varian Unity INOVA 500 FT-NMR instrument at 400 or 500 MHz for 1 H (referenced to TMS, δ H 0.00 ppm for CDCl 3 ) and 100 or 125 MHz for 13 C (referenced to δ C 77.0 for CDCl 3 ). ESIMS and HRESIMS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, 230−400 mesh) and LiChroprep RP-18 (Merck, 40-63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) and precoated RP-18 F 254S plates (Merck, 1.05560) were used for TLC analyses. High-performance liquid chromatography (HPLC) was performed on a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 210 nm and a semi-preparative reversed-phase column (Merck, Hibar Purospher RP-18e, 5 μm, 250 × 10 mm).

Animal Material
The soft coral Sinularia crassa was collected by hand using scuba off the coast of Sansiantai, Taitung county, Taiwan, in July 2008, at a depth of 10 m, and was stored in a freezer. This soft coral was identified by one of the authors (C.-F.D.). A voucher specimen (specimen no. SST-03) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Preparation of (S)-and (R)-MTPA Esters of 1
To a solution of 1 (1.0 mg) in pyridine (0.4 mL) was added (R)-MTPA chloride (25 μL), and the mixture was allowed to stand for 3 h at room temperature. The reaction was quenched by the addition of 1.0 mL of H 2 O, and the mixture was subsequently extracted with EtOAc (3 × 1.0 mL). The EtOAc layers were combined, dried over anhydrous MgSO 4 , and evaporated. The residue was subjected to short silica gel column chromatography using n-hexane−EtOAc

Determination of Sugar Configuration
Authentic samples of D-fucose and L-cysteine methyl ester hydrochloride (each 0.5 mg) were dissolved in pyridine (0.1 mL) and heated at 60 °C for 1 h. To this mixture was added o-tolylisothiocyanate (0.5 mg in 0.1 mL pyridine) and heated at 60 °C for additional 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC (Mightysil RP-18 GP column; 4.6 × 250 nm; 25% CH 3 CN in 50 mM H 3 PO 4 ; 0.8 mL/min; 35 °C) and detected at 250 nm to give the retention time of the o-tolylthiocarbamate of sugar. The retention of the o-tolylthiocarbamate derived from L-fucose, L-cysteine methyl ester, and o-tolylisothiocyanate was obtained by the same manner.
A solution of the glycoside (0.4 mg for each) in 0.6 M HCl/dioxane (1:1 v/v, 0.2 mL) was heated at 80 °C for 24 h. After cooling, the solution was neutralized with Amberlite IRA-400, and the resin was removed by filtration. The filtrate was extracted with EtOAc. The aqueous layer was dried in vacuo and the afforded residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester (0.5 mg), followed by heating at 60 °C for 1 h. A 0.1 mL solution of o-tolylisothiocyanate (0.5 mg) in pyridine was added to the mixture, which was heated at 60 °C for additional 1 h, to yield the corresponding o-tolylthiocarbamate derivative. Reverse phase HPLC analysis of the o-tolylthiocarbamate derivatives derived from the hydrolytes of the glycosides 2-5 showed peaks at 28.2, 28.0, 28.1, and 27.9 min, respectively, while the t R values for standard L-fucose and D-fucose derivatives were observed at 28.0 and 25.4 min, respectively, suggesting the presence of a L-fucose residue in 2-5.

Cytotoxicity Testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Compounds were assayed for cytotoxicity against human liver carcinoma (HepG2 and HepG3), human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A-549) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [19]. Freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 5000-10000 cells per well with tested compounds added from DMSO-diluted stock. After 3 days in culture, attached cells were incubated with MTT (0.5 mg/mL, 1 h) and subsequently dissolved in DMSO. The absorbency at 550 nm was then measured using a microplate reader. The IC 50 is the concentration of agent that reduced cell growth by 50% under the experimental conditions.

In Vitro Anti-Inflammatory Assay
Macrophage (RAW264.7) cell line was purchased from ATCC. In vitro anti-inflammatory activities of tested compounds were measured by examining the inhibition of lipopolysaccharide (LPS) induced upregulation of iNOS and COX-2 proteins in macrophage cells using western blotting analysis [20,21].

Conclusions
Prior investigation of the genus Sinularia reported on several steroidal glycosides, however, all of these were found to possess a 24-methylene substituted side chain [22,23]. 5 is the first example of steroidal glycoside with a 23,24-dimethyl substitited side chain from soft coral of the genus Sinularia. Our biological data reveal that 2′-O-acetyl-L-fucose functionality plays an important role toward the inhibition of the pro-inflammatory iNOS. Steroidal glycoside 2 could be a useful anti-inflammatory agent, while steroids 1 and 4 have shown inhibitory activity toward the selected human liver cancer cells.