Marine bryozoans are well known producers of bioactive secondary metabolites and important marine drug sources due to their remarkable antineoplastic activity [1]. Bryostatins isolated from the marine bryozoans Bugula neritina are a well known example [2]. Other bioactive secondary metabolities from marine bryozoans include alkaloids, sterols, as well as heteratom-containing compounds, which showed remarkable activities on tumor cell lines, such as murine lymphocytic leukemia P388, human myeloid leukemia HL-60, human leukemia U937, human hepatocellular liver carcinoma HepG2, etc. [3–6]. In our previous studies focused on B. neritina, a new antineoplastic macrolide, bryostatin 19, two ceramides and four cerebrosides, as well as a series of sterols were isolated from this bryozoan [5–8]. In the course of our ongoing investigations toward the isolation of biologically active secondary metabolites from marine bryozoans, Cryptosula pallasiana was investigated, another genus of marine bryozoans, collected from the coast of Huang Island in Qingdao City, Shandong Province of China. Herein, we report the isolation and structure identification of six new sterols (1–6) and seven known sterols (7–13), four (3–6) of which have already been reported as synthetic sterols [9–11]. This is the first time that these compounds (3–6) are reported as natural sterols. In addition, the cytotoxicity of the oxygenated sterols 1–4, 7 and 10–13 against HL-60 human myeloid leukemia cell line is also described.

The CCl₄ 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).

Compound 1 was obtained as a white amorphous powder and was positive to Liebermann-Burchard test. The positive ion mode HR-ESI-MS spectrum showed a pseudomolecular ion peak at m/z 437.3398 [M + Na]⁺ (C₂₈H₄₆O₂Na, calculated for 437.3396), which, together with the molecular ion peak at m/z 414 [M]⁺ in the positive ion mode EI-MS, enabled the determination of the molecular formula C₂₈H₄₆O₂, with the help of NMR data (Table 1).

An extensive examination of ¹H NMR and ¹³C NMR spectra data, to draw assistance from the data of ¹H-¹H 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 ¹H 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 Δ⁵ 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) (Figure 2), as well as ¹H-¹H COSY correlations from H-3 to H₂-2 and H₂-4. The partial structure of C-17 side chain in compound 1 was established based on EI-MS, 1D and 2D NMR data. The double bond of C-23 (δ_C 128.8) and C-24 (δ_C 136.8) was confirmed by the cross peaks of H-23 (δ_H 5.50, m) to C-22 (δ_C 39.3), C-24 and C-25 (δ_C 75.0); H-24 (δ_H 5.38, d, J = 15.8 Hz) to C-22 and C-23 in the HMBC spectrum, and was determined to be trans-disubstituted due to the large coupling constant (J_23,24 = 15.8 Hz). The methoxy group (δ_H 3.15 (3H, s), δ_C 50.4) at C-25 position was confirmed by the HMBC spectrum data (Figure 2) and fragment ion at m/z 382 [M − OCH₃ − H]⁺ in the EI-MS spectrum of 1. Based on the above analysis, the plane structure of 1 was determined (Figure 1).

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.

Compound 2 was isolated as an isomer of 1, due to the same formula of C₂₈H₄₆O₂ from EI-MS (m/z 414 [M]⁺) and HR-ESI-MS (m/z 437.3394 [M + Na]⁺ (C₂₈H₄₆O₂Na, calculated for 437.3396)), with the help of 1D NMR spectral data. Fragment at m/z 382 [M − OCH₃ − H]⁺ due to loss of a methoxy group, as well as the two ion fragments at m/z 271 [M − C₈H₁₅ − OCH₃ − H]⁺ and 253 [M − C₈H₁₅ − OCH₃ − H − H₂O]⁺, suggested the presence of the mono-hydroxylated and mono-methoxylated steroid with a mono-unsaturated side chain. The presence of a strong peak in the EI-MS at m/z 111 confirmed the presence of the C₈H₁₅ side chain. The partial structure of a Δ²² mono-unsaturated side chain was established from the HMBC spectrum of 2 (Figure 2), which showed correlations of H₃-27/C-25, H₃-26/C-25, H₃-26/C-24, H-23/C-24, H-23/C-20, H-22/C-23, H-22/C-20, H₃-21/C-17, H₃-21/C-20 and H₃-21/C-22. The position of the methoxy group was assigned as C-7 (δ_C 74.1) due to a strong broad singlet at δ_H 3.35 (3H, s) correlated to C-7 in the HMBC spectrum, and the partial structure of the steroidal nucleus was confirmed by the observation of the HMBC cross peaks from H₂-4 to C-3, C-5 and C-6; H-6 to C-4, C-8 and C-10; H₃-18 to C-13, C-14 and C-17, and H₃-19 to C-1 and C-10 (Figure 2). The configuration of the double bond between C-22 and C-23 was determined to be trans-disubstituted due to the large coupling constant (J_22,23 = 15.2 Hz) between H-22 and H-23. The β-orientation of the hydroxyl group on C-3 was deduced also from the NOESY correlations of H-3/H-4α and H-4α/H-6, and the β-orientation of the methoxy group on C-7 was deduced by observation of correlations of H-7α/H-6 and H-7α/H-14 in the NOESY experiment (Figure 2). Accordingly, compound 2 can be defined as (22E)-7β-methoxy-cholesta-5,22-dien-3β-ol.

Compound 3 was isolated as a white amorphous powder and was also positive to the Libermann-Burchard test. Its molecular formula was established as C₂₈H₄₈O₂ by observation of the molecular ion peak at m/z 439.3550 [M + Na]⁺ (C₂₈H₄₈O₂Na, calculated for 439.3552) in the HR-ESI-MS spectrum and ion peak at m/z 416 [M]⁺ in the EI-MS spectrum. Comparison of the ¹H NMR and ¹³C NMR spectra of 3 with those of 2 revealed that they shared the same 3β-hydroxy, 7β-methoxy Δ⁵-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 ¹³C 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]⁺ (C₂₆H₄₀O₂Na, calculated for 407.2926) and EI-MS spectrum showed the molecular ion peak at m/z 384 [M]⁺ corresponding to the molecular formula C₂₆H₄₀O₂, with the help of NMR data. Compound 4 was assumed to have the same typical nucleus of 3β-hydroxy Δ⁵-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 H₂-22 (δ_H 2.34, 1.98) were connected to the carbon at δ_C 39.5 (C-22). The correlation of H-23 with H₂-22 and H-24 in the ¹H-¹H 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 (H₃-26) exhibited HMBC correlations with C-25 (δ_C 198.7) and C-24, and upfield methyl proton signals at δ_H 0.95 (H₃-21) 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.

Another two stereoisomeric sterols, 5 and 6, were isolated as white amorphous powder, and were analyzed to share the same molecular formula of C₂₇H₄₄O₂ using EI-MS (m/z 400 [M]⁺) and HR-ESI-MS data. The ¹H NMR and ¹³C NMR data of 5 and 6 agreed with those of cholesta-5,25-diene-3β,24ξ-diol from red alga Galaxaura marginata [14]. Although, the configuration at C-24 was hard to determine due to the small difference in the chemical shift of C-24 in the ¹³C NMR spectrum between S and R epimers (Table 2), compound 5 was finally assigned as 24(R)-cholesta-5,25-diene-3β,24-diol due to no correlation between H-24 and H-20β in the NOESY experiment, whereas, the presence of H-24 correlated with H-20β in 6 confirmed the correct configuration of C-24 in 5. Furthermore, the results were confirmed by the ¹H NMR data of 5 and 6, consistent with the same synthetic sterols reported earlier [11]. Accordingly, 6 was assigned to be 24(S)-cholesta-5,25-diene-3β,24-diol. 5 and 6 as stereoisomeric sterols were isolated from a natural origin for the first time.

Comparing their MS and NMR data with those reported in the literature, the known sterols were identified as (23Z)-cholesta-5,23-diene-3β,25-diol (7) [15], cholest-5-ene-3β,7β-diol (8) [16], cholest-5-ene-3β,7α-diol (9) [16], (22E)-3β-hydroxy-24-norcholesta-5,22-dien-7-one (10) [16], (22E)-3β-hydroxycholesta-5,22-dien-7-one (11) [16], 3β-hydroxycholest-5-en-7-one (12) [16] and (4E,22E)-12β-hydroxy-24-norcholesta-1,4,22-trien-3-one (13) [17], respectively. The cis-double bonds between C-23 and C-24, together with an oxygenated hydroxyl group at C-25 in the side chain of 7 is scarce in natural sterols. Sterols 8–12 were previously isolated from two marine sponges Cliona copiosa [16] and Stelodoryx chlorophylla [18], and sterols 8–9 were also isolated from a soft coral Dendronephthya gigantean [19] and a marine bryozoan Biflustra grandicella [20]. Compound 13 was a highly functionalized C₂₆ steroid Δ^1,4-dien-3-one with 12β oxygen function, which is a rare structural feature among sterols and has been isolated from soft coral Gersemia rubiformis [17]. However, all of the known sterols were isolated for the first time from this species.

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 IC₅₀ values of 17.91, 21.30, 22.11, 15.05, 18.28, 15.12 and 14.73 μg/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 [9–11]. 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.