New Cytotoxic Oxygenated Sterols from the Marine Bryozoan Cryptosula pallasiana

Six new sterols (1-6), together with seven known sterols (7-13), were isolated from the CCl4 extract of the marine bryozoan Cryptosula pallasiana, four (3-6) of which have already been reported as synthetic sterols. This is the first time that these compounds (3-6) are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC50 values from 14.73 to 22.11 µg/mL except for compounds 12 and 13.


Results and Discussion
The CCl 4 extract (12.9 g) of the marine bryozoan C. pallasiana was fractionated by Sephadex LH-20 chromatography to afford three major fractions (Frs. A-C). Fr. A (5.66 g) was further subjected to column chromatography (CC) over reversed-phase silica gel column (RP-18) and normal silica gel column, respectively, and then further purified by reverse semi-preparation HPLC to yield compounds 1-13 ( Figure 1).  (Table 1).
An extensive examination of 1 H NMR and 13 C NMR spectra data, to draw assistance from the data of 1 H-1 H COSY, HSQC and HMBC spectra, allowed the establishment of a sterol skeleton with a 5(6)-double bond (δ C 140.9 and 121.8), which was consistent with the literature [12]. The 1 H NMR spectrum showed five methyl resonance signals at δ H 0.69 (3H, s), 0.91 (3H, d, J = 6.6 Hz), 1.00 (3H, s), and 1.25 (6H, s), which were ascribed to the methyl groups 18, 21, 19 and 26/27, respectively. The resonances at δ H 3.52 (1H, m) and 5.35 (1H, t, J = 2.9 Hz) assigned for H-3 and the olefinic proton H-6, respectively, were indicative for Δ 5 mon-hydroxylated steroidal nucleus, which was confirmed by HMBC correlations from H-6 to C-4 (δ C 42.4), C-7 (δ C 32.0), C-8 (δ C 32.0) and C-10 (δ C 36.6) ( In the NOESY experiment, both H-3 and H-6 correlated with H-4α (δ H 2.28, m), indicating the β-orientation of the hydroxyl group on C-3 ( Figure 2), which was confirmed by the chemical shift of C-3 (δ C 71.9 > 70.0) [13]. After mapping all of the signals for each moiety by careful inspection of the 1D and 2D NMR spectra, compound 1 was unambiguously assigned as (23E)-25-methoxy-cholesta-5,23-dien-3β-ol. Sterol 1, not reported previously in the literature, is a new sterol with trans-double bonds between C-23 and C-24, together with a methoxy group at C-25 in the side chain.  13 C NMR spectra of 3 with those of 2 revealed that they shared the same 3β-hydroxy, 7β-methoxy Δ 5 -steroid nucleus but differed in the side chain. Compound 3 was finally assigned as 7β-methoxy-cholest-5-en-3β-ol due to the missing trans-double bonds between C-22 and C-23 in the 13 C NMR spectrum by comparison with 2. Compound 3 has been reported as a synthetic sterol with effective inhibition of cholesterol acyltransferase (ACAT) [9] and is reported here as a natural product for the first time.
Compound 4 was obtained as a white amorphous powder and was positive to Liebermann-Burchard test. The HR-ESI-MS spectrum showed the molecular ion peak at m/z 407.2928 [M + Na] + (C 26 H 40 O 2 Na, calculated for 407.2926) and EI-MS spectrum showed the molecular ion peak at m/z 384 [M] + corresponding to the molecular formula C 26 H 40 O 2 , with the help of NMR data. Compound 4 was assumed to have the same typical nucleus of 3β-hydroxy Δ 5 -steroid by comparing the 1D NMR data with those of 1, but differed in the side chain. The protons of trans-olefinic bonds in the side chain appeared at δ H 6.07 (1H, d, J = 15.5 Hz) and 6.78 (1H, m). The HSQC spectral data indicated that the proton H-24 (δ H 6.07) was connected to the carbon at δ C 132.8 (C-24) and H-23 (δ H 6.78) was connected to the carbon at δ C 147.6 (C-23), while the protons H 2 -22 (δ H 2.34, 1.98) were connected to the carbon at δ C 39.5 (C-22). The correlation of H-23 with H 2 -22 and H-24 in the 1 H-1 H COSY spectrum indicated that the double bonds were in C-23 and C-24. This was also supported by the key cross-peaks H-24 with C-22 and H-23 with C-22 in the HMBC experiment. Similarly, the downfield singlet methyl protons at δ H 2.25 (H 3 -26) exhibited HMBC correlations with C-25 (δ C 198.7) and C-24, and upfield methyl proton signals at δ H 0.95 (H 3 -21) with C-17 (δ C 55.9), C-20 (δ C 36.0) and C-22, suggesting the remnant connectivities of the side chain in compound 4. Accordingly, 4 was finally assigned as (23E)-3β-hydroxy-27-norcholesta-5,23-dien-25-one, which had been obtained by synthesis [10], but was reported as a natural product for the first time.
Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cells in vitro, using a MTT assay method. The results of their cytotoxicity are shown in Table  3. Although 12 and 13 did not show any apparent cytotoxicity, sterols 1-4, 7, 10 and 11 displayed moderate cytotoxicity to HL-60 cells with IC 50 values of 17.91, 21.30, 22.11, 15.05, 18.28, 15.12 and 14.73 g/mL, respectively. It appears that the cytotoxicity against HL-60 human myeloid leukemia cells of these sterols has a correlation with their structure. The present chemical study of the marine bryozoan C. pallasiana resulted in the isolation and characterization of six new sterols (1-6) and seven known sterols (7-13), four (3-6) of which have already been reported as synthetic sterols [9][10][11]. This is the first time that they (3-6) are reported as natural sterols. The structures of these new compounds are notable for the following viewpoints of natural product chemistry. Sterol 1, not reported previously in the literature, is characterized by an oxygenated methoxy group at C-25 in the side chain. In the nucleus of 2 and 3, the 7β methoxy group is a rare feature and first encountered among natural sterols. Compound 4 is specific in carbonylation at C-25 accompanied by a loss of a methyl group in the side chain. Sterols 5 and 6 are stereoisomeric with the C-24 with hydroxyl group in the side chain, which are reported as a natural source for the first time.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 343 polarimeter. 1D and 2D NMR spectra experiments were measured in CDCl 3 on a Bruker AVANCE-500 spectrometer, with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm and coupling constants in Hz. EI-MS spectra were obtained on a MAT212 mass spectrometer; ESI-MS and HR-ESI-MS spectra were taken on a Micromass Quattro mass spectrometer. Separation and purification were performed by CC on silica gel H (1040 m, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Pharmacia Inc., New Jersey, USA), reversed-phase Si gel (Lichroprep RP-18, 40-63 m, Merck Inc., Darmstadt, Germany). HPLC was carried out on a Dionex P680 liquid chromatograph equipped with a UV 170 UV/Vis detector at 206 nm using a YMC-Pack R﹠D ODS-A column (250  20 mm i.d., 5 m, YMC, Kyoto, Japan) for semi-preparation and a Thermo ODS-2 column (250  4.6 mm i.d., 5 m, Thermo Hypersi-Keystone Inc., Bellefonte, U.S.A.) for analysis. TLC detection was achieved by spraying the silica gel plates (Qingdao Marine Chemical Inc., Qingdao, China) with 20% H 2 SO 4 followed by heating.

Animal Material
The