Sterols from Thai Marine Sponge Petrosia (Strongylophora) sp. and Their Cytotoxicity

Eight new sterols (1–5 and 11–13), together with eight known compounds (6–10 and 14–16) were isolated from marine sponge Petrosia sp. The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of some compounds against a panel of human cancer cell lines is also reported.

Compound 4 was obtained as an amorphous powder, and its molecular formula of C31H54O5 was established by the ESI-TOF MS at m/z 529.3858 [M + Na] + (calcd for C31H54NaO5, 529.3864). After comparing the 1 H and 13 C NMR spectral data with that of compound 1 (Tables 1 and 2), the proton and hydroxyl groups at C-3 in 1 were replaced by two methoxy groups in 4, based on the 1 H signals of two singlets at δ 3.16 and 3.07 and the 13 C signal of ketal at C-3 (δ 100.7). Therefore, compound 4 was established as 26,27-cyclo-24,27-dimethylcholestan-3,3-dimethoxy-7α,12β,20β-triol (or 7αhydroxyaragusterolketal I).
Compound 4 was obtained as an amorphous powder, and its molecular formula of C 31 H 54 O 5 was established by the ESI-TOF MS at m/z 529.3858 [M + Na] + (calcd for C 31 H 54 NaO 5 , 529.3864). After comparing the 1 H and 13 C NMR spectral data with that of compound 1 (Tables 1 and 2), the proton and hydroxyl groups at C-3 in 1 were replaced by two methoxy groups in 4, based on the 1 H signals of two singlets at δ 3.16 and 3.07 and the 13 C signal of ketal at C-3 (δ 100.7). Therefore, compound 4 was established as 26,27-cyclo-24,27-dimethylcholestan-3,3-dimethoxy-7α,12β,20β-triol (or 7α-hydroxyaragusterolketal I).
The molecular formula of compound 11 was determined as C 31 H 54 O 4 by APCI-TOF MS. The 1 H and 13 C NMR spectra of 11 (Tables 2 and 3, Figures S15 and S16) were similar to those of compound 4, suggesting a 12β-hydroxy group and dimethyl ketal at C-3. The main differences were that the position of the β-hydroxy group at C-7 in 4 was now at C-16 (δ H 4.33/δ C 74.2) and the hydroxy-substituted quaternary carbon at C-20 (δ C 74. 4)  spectrum. In addition, the relative stereochemistry of C-20-C-29 side chain at C-17 of compound 11 was confirmed by the similarity of the 1 H and 13 C chemical shifts to those of known petrosterol (19) [25] and 7-oxopetrosterol (10) [4]. The NOESY correlation between H-17 and H-12 further established the orientation of C-17 side chain as β-orientation. Thus, the structure of 11 was elucidated as 26,27-cyclo-24,27-dimethylcholestan-3,3-dimethoxy-12β,16α-diol. The IR spectrum showed characteristic absorption bands of a hydroxyl group at 3518 cm −1 and a carbonyl group at 1713 cm −1 . The 1 H and 13 C NMR spectra of the rings A-D of 12 (Tables 2 and 3, Figures S17 and S18) were similar to those ofxestokerol B (8) [22], suggesting the presence of the carbonyl group at C-3 (δ 210.3), α-OH at C-7 (δ 66.3), and β-OH at C-12 (δ 78.0). The main difference was the signals due to the side chain (C-20-C-27) at C-17, which was assigned by the analysis of 1 H-1 H COSY and HMBC correlations. The 1 H-1 H COSY spectrum showed the cross peak between the olefinic H-23 (δ 5.94) and methylene H-24 (δ 1.99 and 2.03), which in turn coupled to a methine H-25 (δ 1.66) of the isopropyl group and showed the HMBC correlations from CH 3 -21 to C-17 (δ 64.1); 20-OH to C-20 (δ 73.9), C-21 (δ 31.6), and C-17 (δ 64.1); and both H-22 and H-23 to C-20 (δ 73.9). All of these suggested that the side chain of 12 was 1-hydroxy-1,5-dimethyl-2-hexenyl unit. The trans geometry of the double bond at C-22 and C-23 was established from the large coupling constants of 15.5 Hz. In addition, the orientation of the hydroxy group at C-20 could be established as β-OH from the cross peak between H-22 and CH 3 -18, H-17 and CH 3 -21 in NOESY analysis (Figure 3). Thus, compound 12 was identified as 7α,12β,20β-trihydroxycholesta-22E-en-3-one.

C NMR and [α] D ) with literature values.
Although the 3-dimethyl ketal functionality of compounds 4 and 5 were assumed to have been artificially formed during the isolation and purification procedures, there have been some examples that the aragusteroketals A (16) [19] and B [24] possessing the 3-dimethyl ketal functionality have also been isolated from a marine sponge of Xestospongia sp. As an additional proof, the xestokerol B (8) and the aragusterol A (15) with carbonyl group at C-3 were subjected to conditions similar to those during the process of the isolation and purification for one month. No change in the thin layer chromatography (TLC) analyses was observed, suggesting that all isolated dimethyl ketal derivatives are naturally occurring.
In a previous study, sterols with a cyclopropane ring were reported to possess cytotoxicity toward various cancer cell lines [19,20,23]. In our study, several compounds (1-4, 6-8, 10, and 12-16) were evaluated for their cytotoxicity using a panel of human cancer cell lines, including MOLT-3 (acute lymphoblastic leukemia), HepG2 (hepatocarcinoma), A549 (human lung cancer), HuCCA-1 (human chlolangiocarcinoma), HeLa (cervical carcinoma), and MDA-MB-231 (hormone-independent breast cancer) as well as a normal cell line, MRC-5 (normal human embryonic lung fibroblasts). As shown in Table 4, all of the tested compounds, except for sterol 15, exhibited weak to moderate cytotoxicity, with the IC 50 values in the range of 11.23-103.5 µM. The most potent, compound 15, was cytotoxic, with the IC 50 values of 7.10 and 6.11 µM against HepG-2 and HeLa cell lines, respectively, while exhibiting moderate cytotoxicity with the IC 50 values of 12.84, 37.93, 37.58, and 18.01 µM against the other four cancer cell lines, MOLT-3, A549, HuCCA-1, and MDA-MB-231, respectively. It was noted that all of the tested compounds exhibited weaker cytotoxic activity than the positive control (etoposide or doxorubicin) and were noncytotoxic towards a normal cell line (MRC-5), with IC 50 values greater than 37.68 µM.

Animal Material
The sponge, Petrosia sp., was collected from the Similan Islands in the Andaman Sea, Phang Nga, Thailand in February 2011, by hand via scuba diving. It was identified by Dr. Sumaitt Putchakarn, Institute of Marine Science, Burapha University, Bangsaen, Chonburi, Thailand. A voucher specimen (No. CRI 589) was deposited at the Laboratory of Natural Products, Chulabhorn Research Institute.

Extraction and Isolation
The sponge (wet weight ca. 6.4 kg) was extracted with MeOH and concentrated under reduced pressure. The MeOH extract was partitioned between EtOAc and water. The EtOAc-soluble portion Compound 11. White amorphous powder; 1 H and 13 C NMR data (see Tables 2 and 3); APCI-TOF

Conclusions
The chemical investigation of Thai marine sponge Petrosia sp. led to the isolation of eight new (1-5 and 11-13) and eight known (6-10 and 14-16) sterols. Their structures were established by the basis of spectroscopic method. Some compounds (1-4, 6-8, 10, and 12-16) were evaluated for their cytotoxicity using a panel of human cancer cell lines. The most potent, compound 15, was cytotoxic, with the IC 50 values of 7.10 and 6.11 µM against HepG-2 and HeLa cell lines, respectively.