Polyhydroxylated Steroids from the Bamboo Coral Isis hippuris

In previous studies on the secondary metabolites of the Taiwanese octocoral Isis hippuris, specimens have always been collected at Green Island. In the course of our studies on bioactive compounds from marine organisms, the acetone-solubles of the Taiwanese octocoral I. hippuris collected at Orchid Island have led to the isolation of five new polyoxygenated steroids: hipposterone M–O (1–3), hipposterol G (4) and hippuristeroketal A (5). The structures of these compounds were determined on the basis of their spectroscopic and physical data. The anti-HCMV (human cytomegalovirus) activity of 1–5 and their cytotoxicity against selected cell lines were evaluated. Compound 2 exhibited inhibitory activity against HCMV, with an EC50 value of 6.0 μg/mL.


OPEN ACCESS
Some of the second types were reported to show cytotoxicity or a reversal of multidrug resistance activity [10]. The samples for previous studies on the secondary metabolites of Taiwanese octocoral I. hippuris were all collected at Green Island [5][6][7]12,14,15]. In our continued study of the bioactive metabolites from marine organism, the Taiwanese octocoral I. hippuris (Figure 1) collected at Orchid Island was selected for study since its acetone extract exhibited antiviral activity against HCMV. Bioactivity-guided fractionation resulted in the isolation of five polyoxygenated steroids: hipposterone M-O (1-3), hipposterol G (4), hippuristeroketal A (5) (Figure 2). We describe herein the isolation, structure elucidation, and biological activity of these compounds.
The positive HRESIMS of hipposterone O (3) established a molecular formula of C 35 H 54 O 10 . NMR data (Tables 1 and 2) of 3 showed the presence of a ketone (δ C 211.5), three ester cabonyls, two oxygenated sp 3 methines, two oxygenated sp 3 methylene, three oxygenated sp 3 quaternary carbons, two secondary methyls, three tertiary methyls, six non-oxygenated sp 3 methines, eight non-oxygenated sp 3 methylenes, and two non-oxygenated sp 3 quaternary carbons. By comparison of NMR spectroscopic data (Tables 1 and 2) of 3 with those of hippuristerone J [14], the primary acetoxy group at C-21 was shift to C-18 on the basis of HMBC correlations ( Figure 3) from H 2 -18 [δ H 4.23 (1H, d, J = 11.6 Hz) and 4.30 (1H, d, J = 11.6 Hz)] to C-12, C-13, C-14, C-17, and carbonyl carbon of 18-OAc. The stereochemistry of the side chain moiety was determined by comparison of the 1 H and 13 C NMR spectral data with those of hippuristerones J and K previously isolated from I. hippuris [14].    Hipposterol G (4) was isolated as a white powder, and its molecular formula, C 35 H 56 O 9 , was determined by HRESIMS. Its IR spectrum revealed the functionalities of hydroxyl (ν max 3471 cm −1 ) and ester carbonyl (ν max 1734 cm −1 ). NMR data (Tables 1 and 2) of 4 indicated the presence of three ester cabonyls, three oxygenated sp 3 methines, an oxygenated sp 3 methylene, three oxygenated sp 3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp 3 methines, eight non-oxygenated sp 3 methylenes, and two non-oxygenated sp 3 quaternary carbons. NMR data (Tables 1 and 2) of 4 were similar to those of hippuristerone G [16] with the absence of the ketone carbon signal at δ C 211.6 ppm and the presence of signal at δ H 3.60 ppm NOE correlation H-3/H-5 and chemical shift values for C-1-C-7 nuclei. This is in agreement with the results reported for 5α-cholestan-3β-ol, which allowed us to propose a β orientation of OH group at C-3 ( Figure 4). The stereochemistry of the side chain moiety was determined by comparison of the 1 H and 13 C NMR spectral data with those of hippuristerone A. The molecular formula of hippuristeroketal A (5) was found to be C 35 H 58 O 9 , as deduced from HRESIMS data. Its IR spectrum revealed the absorptions for hydroxyl (ν max 3471 cm −1 ) and ester carbonyl (ν max 1731 cm −1 ) groups. NMR data (Tables 1 and 2) of 5 indicated the presence of a ketal (δ C 100.7), two ester cabonyls, two oxygenated sp 3 methines, an oxygenated sp 3 methylene, three oxygenated sp 3 quaternary carbons, two secondary methyls, four tertiary methyls, six non-oxygenated sp 3 methines, eight non-oxygenated sp 3 methylenes, and two non-oxygenated sp 3 quaternary carbons. By comparison of the NMR spectroscopic data (Tables 1 and 2) of 5 resembled those of hippuristerone F [14] with the absence of ketone carbon at δ C 211.6 and the presence of two methoxyl signals [δ H 3.12 (3H, s), 3.02 (3H, s) and δ C 47.6 (CH 3 ), 47.5 (CH 3 )] in the molecule. The HMBC correlations (Figure 3) of the methoxyl protons with C-3 [δ C 100.7 (qC)], suggesting that C-3 was substituted by two methoxy groups. The stereochemistry of the side chain moiety was determined by comparison of the 1 H and 13 C NMR spectral data with those of hippuristerones F, H, and I previously isolated from I. hippuris [16]. Compound 5 was not an artifact because 1 H NMR signals for the dimethylketal were observed before MeOH treatment.
Metabolites 1-5 were not cytotoxic against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells with IC 50 values greater than 50 μg/mL. The anti-HCMV activity and cytotoxicity against of selected cell lines of 1-5 were evaluated. Compound 2 exhibited inhibitory activity against HCMV, with an EC 50 values of 6.0 μg/mL.

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13 C or on a Varian Unity INOVA 500 FT-NMR spectrometer at 500 MHz for 1 H and 125 MHz for 13 C, respectively. 1 H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peaks δ H 7.27 and 7.15 for CDCl 3 and C 6 D 6 , respectively, and coupling constants are expressed in Hz. 13 C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peaks δ C 77.0 and 128.0 for CDCl 3 and C 6 D 6 , respectively. ESI-MS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 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) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phase column (Merck, Hibar LiChrospher RP-18e, 5 μm, 250 × 25 mm).

Biological Material
The octocoral I. hippuris was collected by hand using scuba at Orchid Island, 70 km off the southeastern coast of Taiwan, in August 2008 at a depth of 9 m and stored in a freezer until extraction. The voucher specimen (LY-19) was identified by Prof. Chang-Feng Dai, National Taiwan University and deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

Extraction and Isolation
A specimen of octocoral I. hippuris (4.0 kg, wet weight) was minced and exhaustively extracted with acetone (3 × 3 L) at room temperature. The combined acetone extracts was then partitioned between H 2 O and EtOAc. The resulting EtOAc extract (25.6 g) was subjected to gravity silica gel 60 column chromatography (Si 60 CC) using n-hexane-EtOAc and EtOAc-MeOH of increasing polarity, to give 44 fractions.

Cytotoxicity Assay
Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [19,20]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection.

Anti-HCMV Assay
To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity was expressed as IC 50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [21].