Bioactive Polyoxygenated Steroids from the South China Sea Soft Coral, Sarcophyton sp

Seven new polyoxygenated steroids (1–7) were isolated together with seven known analogues (8–14) from the South China Sea soft coral, Sarcophyton sp. The structures of the new compounds were identified on the basis of extensive spectroscopic analysis and comparison with reported data. All the steroids are characterized with 3β,5α,6β-hydroxy moiety, displaying carbon skeletons of cholestane, ergostane, gorgostane and 23,24-dimethyl cholestane. In the in vitro bioassay, metabolites exhibited different levels of antimicrobial activity against bacterial species Escherichia coli and Bacillus megaterium, and fungal species Microbotryum violaceum and Septoria tritici. No inhibition was detected towards microalga Chlorella fusca. Preliminary structure-activity analysis suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane moiety at the side chain may also contribute to the bioactivity.


Introduction
Soft corals are thought to produce various bioactive metabolites that chemically defend themselves from attack [1][2][3]. The soft coral Sarcophyton species (order Alcyonacea, family Alcyoniidae) are prolific in the South China Sea and are dominant in many coral reef areas [4]. Chemical research on the animals of this genus has established that it is a rich resource of steroids, diterpenes and tetraterpenes [5,6].
In the course of our ongoing screening for bioactive secondary metabolites from marine sources [7][8][9][10][11], we have made a collection of Sarcophyton sp. off Weizhou Island, Guangxi Province, China. Chemical investigation on the Et 2 O-soluble fraction of the acetone extract from Sarcophyton sp. resulted in the isolation of fourteen steroids (1-14, Figure 1) with 3β,5β,6β-hydroxy moiety. The sterols can be re-sorted into four clusters, due to their different carbon skeletons, namely cholesterol-, ergosterol-, gorgosterol-and 23,24-dimethyl cholesterol-types, displaying an excellent example of chemical diversity. The isolates were tested for in vitro antimicrobial activity against bacteria, fungi and a microalga. Here, we describe the isolation, structural elucidation and bioactivity of these new metabolites.

Results and Discussion
Freshly collected specimens of Sarcophyton sp. were kept at −20 °C before extraction. The acetone extract of the soft coral was partitioned between Et 2 O and H 2 O. Purification of the Et 2 O extract, by repeated column chromatography on silica and Sephadex LH-20 and followed by reversed-phase semi-preparative HPLC, yielded the pure compounds shown in 1-14 ( Figure 1).
Compound 1 was isolated as an optically active, white amorphous solid. The molecular formula was established as C 29 H 48 O 5 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) from the pseudo-molecular ion at m/z 499.3397 [M + Na] + , indicating six degrees of unsaturation. The infrared (IR) absorption at 3408 cm −1 and 1712 cm −1 showed the presence of hydroxyl and carbonyl groups in the molecule. This observation was in agreement with the signals in the 13 C nuclear magnetic resonance (NMR) and distortionless enhancement by polarization transfer (DEPT0 spectra ( Table 1) for 3 sp 2 carbon atoms (1 × OC=O, CH=C) at a lower field and 26 sp 3 carbon atoms at a higher field (1 × OC, 3 × OCH, 2 × C, 5 × CH, 9 × CH 2 , 6 × CH 3 ), accounting for two degrees of unsaturation. The remaining degrees of unsaturation were attributed to the presence of four rings in the molecule.
Furthermore, compound 4 was also obtained as an optically active, amorphous powder. Its formula C 29 (Tables 1 and 3). The double bond was readily assigned as ∆ 17(20) , due to the observation of the vinyl methyl singulate for H 3 -21 (δ 1.71, s) and its HMBC correlation with C-17, C-20 and C-22, and the HMBC correlation from H 3 -18 to C-17 provided further evidence. Meanwhile, the secondary alcohol at C-11 had to be assigned to C-15, due to the proton sequence from H-9 to H 2 -12 and from H-14 to H 2 -16, as established by the 1 H-1 H COSY experiment. The location of 15-OH was confirmed by the HMBC correlation from H-15 to both C-13 and C-17. The diagnostic interactions between H 3 -18 and H-15 in the NOESY spectrum indicated a β configuration of the proton. The R configuration at both C-23 and C-24 were suggested to be the same, biogenetically, as those in sarcosterol, 25-hydroxy sarcosterol and peridinosterol, produced by the soft corals, S. glaucum [35] and Sinularia mayi [36] and the symbiotic dinoflagellate [37], respectively. Biosynthetic studies suggested the same C-23 stereochemistry in sarcosterol and peridinosterol, due to the symbiotic relationship of soft coral and dinoflagellate [36]. The structure of compound 5 was tentatively determined to be (23R,24R)-23,24-dimethylcholesta-17(20)-en-3β,5α,6β-triol.
Compounds 6, 7 and 12-14 belong to the cluster of gorgosteroids, which are characterized as having a cyclopropane ring in the side chain. Compound 6 was isolated as an optically active, white amorphous solid. The molecular formula was established as C 32 H 54 O 6 by HR-ESI-MS. The NMR data of 6 were almost identical to those of 12, displaying characteristic proton signals for the cyclopropane ring at the very high field (δ H −0.13~0.46) (  , H-12β and 1.17, m, H-12α; δ C 46.5) in 12 was replaced by a oxygenated methine group (δ C 75.0, CH; δ H 3.95, d, J = 3.0 Hz, 1H) in 6. This hydroxyl group was assigned at C-12, due to HMBC correlation from H 3 -18 to H-12 and the proton connectivity of H-9/H-11/H-12. The β configuration of H-12 was deduced from its small coupling constant with H-11 and its nuclear Overhauser effect (NOE) effect with H 3 -18. The structure of 6 was thus elucidated as 11α-acetoxy-gorgostane-3β,5α,6β,12α-tetraol. Compound 7 was isolated as an optically active, white amorphous solid. The compound had the same formula (C 32 H 54 O 6 ) as 6 on the basis of HR-ESI-MS. Inspection of NMR spectra for 7 revealed great similarity with those of 6 concerning the signals in rings A, C, D and the side chain. Analysis of the 1 H-1 H COSY spectrum for the proton sequence H-9/H-11/H-12 and the HMBC correlation from H 3 -18 to C-12 led the assignment of 12-OAc and 11-OH in 7 instead of 11-OAc and 12-OH in 6. The configuration at chiral centers remains intact due to the analysis of related proton coupling constants and the NOE effects, establishing the structure of 7 as 12α-acetoxy-gorgostane-3β,5α,6β,11α-tetraol.
The isolation of an array of sterols demonstrates an excellent example of chemical diversity, displaying carbon skeletons of cholestane, ergostane, gorgostane and 23,24-dimethyl cholestane. The co-occurrence of these metabolites supported the biosynthesis proposal of side chains in dinosterol, peridinosterol and gorgosterol [32]. The methylation of the side chain of 1 may yield the side chain of 2, which further would be methylated to the side chain of the dinosterol derivative 4 and the peridinosterol analogues, 5 and 6. The side chain of 4 was then methylated to the side chain of sterols belonging to the gorgosterol family (6, 7, 12-14).
All isolated compounds, except 5, were tested in an agar diffusion assay for their antibacterial, antifungal and algicidal properties ( Table 4). All the metabolites exhibited antibacterial activity against the Gram-negative bacterium, Escherichia coli, the Gram-positive bacterium, Bacillus megaterium, and antifungal activity against the fungi, Microbotryum violaceum and Septoria tritici. However, 1, 2, 6, 7 and 8 exhibited considerable growth inhibitory activity with regard to bacteria and/or fungi species. This suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane side chain seem to also contribute to this bioactivity. None of the sterols showed inhibition of green alga Chlorella fusca.

General Experimental Procedures
Commercial silica gel (200-300 mesh, 10-40 mm; Yantai, China) and Sephadex LH-20 (Pharmacia) were used for column chromatography. Precoated silica gel plates (Yantai, GF 254 plate, 10-40 mm) were used for analytical thin-layer chromatography (TLC). Spots were visualized by heating Si gel plates sprayed with 10% H 2 SO 4 in EtOH. Semipreparative HPLC was carried out on an Agilent 1100 liquid chromatography equipped with a refractive index detector using a Zorbax 300 SB-C18 column (25 cm × 9.4 mm i.d.). Melting points were determined on an XT5-XMT apparatus. Optical rotations were measured with a Perkin-Elmer 341 polarimeter. NMR spectra were recorded in CHCl 3 on a Bruker DRX 400 or 500 spectrometer, and the 2D NMR spectra were obtained using standard pulse sequences. Chemical shifts are reported in parts per million (δ), with use of the residual CHCl 3 signal (δ H 7.26 ppm) as an internal standard for 1 H NMR and CDCl 3 (δ C 77.0 ppm) for 13 C NMR; Coupling constants (J) are reported in Hz. 1 H NMR and 13 C NMR assignments were complemented by heteronuclear single quantum correlation (HSQC), HMBC, 1 H-1 H COSY and nuclear Overhauser effect spectroscopy (NOESY) experiments. The following abbreviations were used to describe spin multiplicity: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br s = broad singlet; dd = doublet of doublets; ov = overlapped signals. HR-ESI-MS were recorded on a Micromass Quattro mass spectrometer. Optical rotations were measured in CHCl 3 with an Autopol IV polarimeter at the sodium D line (590 nm). Infrared spectra were recorded in thin polymer films on a Nexus 470 FTIR spectrophotometer (Nicolet, USA); peaks are reported in cm −1 .

Material and Methods
The soft coral Sarcophyton sp. was collected by hand using SCUBA off the coast of Weizhou Island, Guangxi Province of China, in October 2008, at a depth of 20 m and identified by Dr. Xiu-Bao Li, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (No. S-7-1) was deposited at the Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai, China.

Conclusions
Chemical investigation of the soft coral Sarcophyton sp. from the South China Sea led to the isolation and structural elucidation of fourteen polyoxygenated steroids (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) with 3β,5α,6β-hydroxy moiety, demonstrating an excellent example of chemical diversity. These compounds displayed different levels of antibacterial and antifungal bioactivities in the in vitro bioassay. Preliminary structure-activity analysis suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane moiety at the side chain may also contribute to the bioactivity. The interesting discovery may encourage further investigations on the sterols, the antibacterial and antifungal activity, and the structure-activity relationship.