Bioactive Pregnane Steroids from a South China Sea Gorgonian Carijoa sp.

A new pregnane steroid, 1, and three known analogues 2–4, have been isolated from a gorgonian Carijoa sp. collected from the South China Sea. The planar structure and relative configuration of 1 were elucidated from comprehensive spectroscopic data. Its absolute configuration was determined by application of the modified Mosher method. Compounds 1, 3 and 4 exhibited cytotoxicity against the human hepatoma cell line Bel-7402, with IC50 values of 9.33, 11.02 and 18.68 µM, respectively. Additionally, compound 1 exhibited promising antibacterial activity against Pseudomona puido, with a MIC value of 31 nM, which is approximately 5-fold more potent than ciprofloxacin (MIC = 156 nM).


Results and Discussion
Compound 1 was obtained as pale yellow oil.Its molecular formula was determined as C 21 H 28 O 2 by HRESIMS, suggesting eight degrees of unsaturation.Like 2, the 1 H-NMR spectrum of 1 (Table 1) showed the typical signals in the low-field region of a dienone system (H-1, H-2 and H-4) along with vinyl group (H-21a, H-21b and H-20) signals.The most obvious differences were the presence of one signal at δ H 4.10 (m H-15) in 1 instead of the corresponding signal δ H 5.10 (m, H-15) in 2, and the disappearance of the methyl group signal at δ H 2.05 (s, COCH 3 ) in 1.In the 13 C-NMR spectra, the methine carbon signal of C-15 was shifted upfield (δ C 69.0 in 1 vs. δ C 73.7 in 2) and the carbonyl carbon signal at δ C 170.5, together with the acetyl methyl carbon signal at δ C 21.6, had disappeared in 1, which was the result of a hydroxy substituent at C-15 in 1, instead of the acetoxy group in 2. Furthermore, the position of the hydroxy group was confirmed as C-15 on the basis of the 1 H-1 H COSY correlation between H-15 (δ H 4.10 m) and the hydroxyl proton (δ H 4.45 d J = 4.0).The contiguous sequence of correlations from H-6 to H-12, and from H-8 to H-21 in the 1 H-1 H COSY spectrum (Figure 2) and the HMBC correlations (Figure 2) between H-1/C-3 and C-5, H-4/C-6 and C-10, H-21/C17, as well as from H-18 to C-12, C-13, C-14 and C-17, and from H-19 to C-1, C-5, C-9 and C-10 (Table 1) indicated that 1 has a 3-one pregnane skeleton similar to that of 2, but with a hydroxy group at C-15.Treatment of 2 with NaOH in the presence of ethanol afforded 1 as the major product, which further confirmed the structure of 1.Because the relative configurations of 2 have been previously established, the chemical conversion from 2 allowed the determination of β-orientation of hydroxy group at C-15 in 1, and the NOESY correlations between H-14/H-15, H-15/H-16α and H-16α/H-17, as well as H-18/H-16β, H-16β/H-20 and H-20/H-18, also confirmed the β-stereochemistry of the hydroxy group at C-15.This configuration was also confirmed by the correlations between H-19/H-8 and H-8/H-18 in the NOESY spectrum (Figure 3).The absolute configuration of 1 was established by the modified Mosher method [16].Treatment of 1 with (S)-(+)-α-and (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) gave the corresponding (R)-and (S)-MTPA esters 1r and 1s, respectively.The 1 H-NMR signals of the two MTPA esters were assigned on the basis of their 1 H-1 H COSY spectra.The Δδ H(S-R) values were then calculated (Figure 4).Following the literature [16], the results indicated that the absolute configuration of C-15 was R. Therefore the absolute configurations at C-8, C-9, C-10, C-13, C-14 and C-17 in 1 were assigned as R, S, S, R, S and R, respectively.On the basis of the above evidence, the chemical conversion from 2 to 1 allowed the determination of the 8R,9S,10S,13R,14S,15R,17R configurations for 2 as the first report of its absolute configuration.Compound 2 differed from 1 only in the acetate group at C-15.Actually we used EtOAc for their extraction and isolation, but even though 1 was dissolved in EtOAc and stirred at 40 °C for one week, 2 was not detected in solution, therefore, it seems very unlikely that 2 is an artifact of the isolation procedure.Furthermore, 2 has also been isolated and confirmed as a new natural product from an Indopacific octocoral Carijoa sp.[13].
The antibacterial activity of compounds 1-4 was further evaluated in vitro against a panel of pathogenic bacteria [17], including Bacillus cereus, Tetragenococcus halophilus, Staphylococcus albus, Staphylococcus aureus, Escherichia coli, Pseudomonas putida, Nocardia brasiliensis, and Vibrio parahaemolyticus (Table 2).Compounds 1 and 2 showed a broad spectrum of antibacterial activity.Compound 1 showed significant antibacterial activity against S. aureus, S. albus, E. coli, V. parahaemolyticus and N. brasiliensis, with MIC values of 0.063, 1.00, 1.00, 4.00, and 0.500 μM, respectively, and exhibited promising inhibitory activity against P. putida, with an MIC value of 31 nM, which was approximately 5-fold more potent than that of ciprofloxacin (MIC = 156 nM).Compound 2 inhibited seven pathogenic bacteria, but not S. aureus, and was especially active against T. halophilus, with a MIC value of 312 nM.Additionally, Compound 3 showed antibacterial activity against B. cereus, S. aureus and T. halophilus with MIC values of 2.50, 0.156, and 1.25 μM, respectively.

General Procedures
Optical rotations were measured on a JASCO P-2000 digital polarimeter at room temperature.IR spectra were recorded on a Nicolet 6700 spectrometer.UV spectra were measured on a Nicolet Evolution 300 spectrophotometer.ESI-MS and HR-ESI-MS were recorded on a Q-Tof Premier LC mass spectrometer.NMR spectra were recorded on an AVANCE III 400 (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR) spectrometer and a JEOL Eclips-600 spectrometer.Chemical shifts (δ) were reported in ppm relative to an internal TMS standard, and coupling constant (J) was reported in Hz.HPLC separation was performed in an Agilent 1200 semi-preparative HPLC system coupled with variable wavelength detector.A XDB-C 18 preparative HPLC column (250 × 9.4 mm, 5 μm) was used.Analysis HPLC (Agilent 1200 HPLC system coupled with diode array detector) with XDB-C 18 HPLC column (150 × 4.6 mm, 5 μm) was used.All solvents used were of analytical grade (Shanghai Chemical Plant, Shanghai, China).Silica gel (200-300 mesh; Qingdao Marine Chemical Group Co., Qingdao, China), octadecylsilyl (ODS) silica gel (45-60 mm; Merck KGaA, Darmstadt, Germany), and Sephadex LH-20 (Amersham Biosciences Inc., Piscataway, NJ, USA) were used for column chromatography.Precoated silica gel GF254 plates (Yantai Zifu Chemical Group Co., Yantai, China) were used for TLC analysis.

Animal Materials
Gorgonian Carijoa sp.(1.1 kg, wet weight) was collected off the coral reef of Weizhou Island in the South China Sea, China, in April 2011, and was identified by Dr. Xiu-Bao Li, South China Sea Institute of Oceanology, Chinese Academy of Science.

Extraction and Isolation
The frozen specimen was extracted with 95% EtOH (3 × 2000 mL) three times at room temperature for one week, and the solvent was evaporated in vacuo.The residue was partitioned in H 2 O (500 mL) and extracted with EtOAc three times (3 × 1,000 mL) at room temperature.The EtOAc extract was concentrated in vacuo to afford 8 g of EtOAc residue, which was subjected to column chromatography (CC) on silica gel, using petroleum ether (b.p. 60-90 °C)-EtOAc (from 20:1 to 0:10) as eluent.By combining the fractions according to TLC (GF 254 ) monitoring, six fractions (Fr.1−Fr.6) were obtained.Fr. 2 (900 mg) was fractionated over silica gel CC eluted with petroleum ether−EtOAc gradients (from 25:1 to 3:1) to afford three sub-fractions (Fr.

Hydrolysis of Compound 2
Compound 2 (20 mg) was dissolved in ethanol (2.0 mL), then NaOH (40 mg) was added, and the solution was allowed to stir at room temperature for 12 h.After that the mixture was neutralized with excess hydrochloric acid, water (10 mL) was added, and the solution was then extracted with EtOAc (10 mL × 3).The organic solvent was removed with a high-vacuum pump and the crude mixture was subjected to preparative HPLC to obtain 1 (15 mg).