4-Methylenesterols from a Sponge Theonella swinhoei

Three new 4-methylenesterols, theonellasterol K (1), acetyltheonellasterol (2) and acetyldehydroconicasterol (3), along with two known sterols, theonellasterol (4) and theonellasterone (5), were isolated from the sponge Theonella swinhoei. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. Compound 1 exhibited significant cytotoxic activity against HCT-116, K562 and Molt 4 cancer cell lines.


Results and Discussion
The EtOAc extract of the freeze-dried specimen was fractionated by silica gel column chromatography and the eluted fractions were further separated utilizing normal phase HPLC to yield metabolites 1-5. The new compounds were given the trivial names theonellasterol K (1), acetyltheonellasterol (2) and acetyldehydroconicasterol (3). The known compounds were identified as theonellasterol (4) and theonellasterone (5). Theonellasterol K (1) was obtained as a white amorphous solid. The HRESIMS spectrum of 1 exhibited a pseudomolecular ion peak at m/z 481.3659 [M + Na] + , which established a molecular formula of C 30 H 50 O 3 , implying six degrees of unsaturation. IR absorptions were observed at 3362 and 3251 cm −1 , suggesting the presence of hydroxy groups in 1. The structure of this compound was deduced from its 13 C NMR and DEPT spectroscopic data (Table 1), which showed that the compound has 30 carbons, including six methyls, ten sp 3 methylenes, one sp 2 methylene, seven sp 3 methines (including one oxymethine), one sp 2 methine, three sp 3 quaternary carbons and two sp 2 quaternary carbons. From the 1 H NMR spectrum of 1, resonances of one olefinic methine proton (δ 5.68, d, J = 5.5 Hz), two olefinic methylene protons (δ 5.18 and 4.74, each s) and one oxygenated methine (δ 4.01, dd, J = 11.0, 5.0 Hz) were observed. Moreover, the 1 H NMR spectrum revealed the presence of one hydroperoxy proton resonating as a broad singlet at δ H 6.79. The planar structure and all of the 1 H and 13 C chemical shifts of 1 were elucidated by 2D NMR spectroscopic analysis, in particular 1 H-1 H COSY and HMBC experiments ( Figure 2). From the 1 H-1 H COSY correlations, it was found that one ring-juncture methine proton H-5 (δ 1.97) and one oxymethine proton H-3 (δ 4.01) exhibited allylic correlations with the exomethylene protons at C-30 (δ H 4.74 and 5.18). In addition, 1 H-1 H COSY spectral analysis established five partial structures of consecutive proton spin systems ( Figure 2). Further analysis of the HMBC correlations was employed successfully to establish the gross structure of 1 ( Figure 2). Thus, 1 was found to possess two double bonds at C-7/C-8 and C-4/C-30, one hydroxy group at C-3 and one hydroperoxy group at C-14. The relative configuration of 1, elucidated mainly from the NOESY spectrum, was compatible with that of 1 ascertained using molecular mechanics calculations (MM2), which suggested the most stable conformations, as shown in Figure 3. In the NOESY spectrum of 1, the NOE correlations between one of the methylene protons at C-11 (δ H 1.47) and both methyls (H 3 -18 and H 3 -19); H 3 -18 and H-20 as well as between H 3 -18, H 3 -19 and H-20 indicated that these protons adapt a β-orientation. H-5 was found to interact with H-3, but not with H 3 -19, revealing the β orientation of the hydroxy group at C-3. Moreover, the α-orientation of 14-OOH was further confirmed by the lower field chemical shift of H-17 (δ H 1.85, m). Furthermore, the chemical shifts of the side chain from C-20 to C-29 in 1 were nearly identical to those of 4 and 5. Thus, the structure of steroid 1 was established. After determining the structure of 1, we discovered that its molecular framework has been obtained as a known 4-methylenesterol conicasterol H, which was isolated previously from sponge Theonella swinhoei [18]. Acetyltheonellasterol (2) was isolated as a white powder with the molecular formula C 32 H 52 O 2 , which possesses seven degrees of unsaturation, as indicated by HRESIMS (m/z 491.3862, [M + Na] + ) and NMR spectroscopic data (Table 1). By comparison of the NMR data of 2 with those of 4, it was found that the 1 H and 13 C NMR data of 2 were very similar to those of 4, with the difference that 2 contains one more acetyl group relative to 4. The chemical shift of H-3 in 4 (δ H 4.01) was shifted downfield (δ H 5.15) in 2, suggesting that 2 is the 3-acetyl derivative of 4. We observed further that acetylation of 4 gave a product which was found to be identical to 2 by comparison of the physical and spectroscopic data. Thus, compound 2 was established as the 3-acetyl derivative of 4.
The new metabolite acetyldehydroconicasterol (3) was obtained as a white powder and possessed the molecular formula C 31 H 48 O 2 , as established from the HRESIMS and NMR data, implying eight degrees of unsaturation. Both the 1 H and 13 C NMR signals of 3 were found to be very closely related to those of compound 2, suggesting a very similar steroidal skeleton. By comparison of the NMR data of 3 with those of 2 (Table 1)

General Experimental Procedures
Optical rotation values were measured with a Jasco P-1010 digital polarimeter. IR spectra were recorded on a Varian Digilab FTS 1000 Fourier transform infrared spectrophotometer. The NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR (or Varian Unity INOVA 500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1 H-NMR and 100 MHz (or 125 MHz) for 13 C-NMR, respectively, in CDCl 3 . ESIMS were obtained with a Bruker APEX II mass spectrometer. Gravity column chromatography was performed on silica gel (230-400 mesh, Merck). TLC was carried out on precoated Kieselgel 60 F254 (0.2 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. High-performance liquid chromatography (HPLC) was performed using a system comprised of a Hitachi L-7100 pump and a Rheodyne 7725 injection port. A preparative normal phase column (250 × 21.2 mm, 5 μm) was used for HPLC.

Animal Material
The specimen of Theonella swinhoei was collected by scuba divers at a depth of 15-20 m from coral reefs off the coast of Pingtung, Taiwan. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan (specimen no. 2011SP-2). Taxonomic identification was performed by Prof. Wen-Been Chang of the National Museum of Marine Biology & Aquarium, Pingtung, Taiwan.

Molecular Mechanics Calculations
Implementation of the MM2 force filed in Chem3D Pro software [24] was used to calculate the molecular models.