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Sinularosides A and B, Bioactive 9,11-Secosteroidal Glycosides from the South China Sea Soft Coral Sinularia humilis Ofwegen Peng Sun,† Li-Yuan Meng,† Hua Tang, Bao-Shu Liu, Ling Li, Yanghua Yi, and Wen Zhang* Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People's Republic of China. S Supporting Information *
ABSTRACT: Two new 9,11-secosteroidal glycosides, namely, sinularosides A and B (1, 2), together with the known pregnene glycoside 3β-(β-xylopyranosyloxy)-5α-pregna-20-ene (3), were isolated from the South China Sea soft coral Sinularia humilis Ofwegen. The structures of these compounds were elucidated by a combination of detailed spectroscopic analyses, chemical methods, and comparison with reported data. This is the first report of 9,11-secosteroidal glycosides from a soft coral and from nature. In in vitro bioassays, the new compounds exhibited potent antimicrobial activities and showed no growth inhibition activity against the tumor cells HepG2 and Caco-2.
S
ecosteroids from marine sources are highly oxidized metabolites with one of the rings of the steroid tetracyclic skeleton oxidatively cleaved. The large family of metabolites can be divided into six principal groups, 5,6-, 8,9-, 8,14- 9,10-, 9,11-, and 13,17-secosteroids, on the basis of the ring-cleavage location.1 The largest group of this family, 9,11-secosteroids, are characterized by a cleaved C ring.1,2 The first example of 9,11-secosteroids was isolated as (22R,23R,24R)-22,23-methylene-23,24-dimethyl-9-oxo-9,11-secocholest-5-ene-3β,11-diol from the gorgonian Pseudopterogorgia americana in 1972.3 Since then, an increasing number of 9,11-secosteroids have been found mainly from soft corals,1,4−6 gorgonians,1 and sponges1 and individually from an ascidian, Aplidium conicum,7 and a mollusk, Trimusculus costatus.8 These metabolites were reported to exhibit diverse biological activities, e.g., cytocoxic, antiinflammatory, antimicrobial, and ichthyotoxic effects.1 In the course of our ongoing search for bioactive metabolites from marine sources,9,10 we collected Sinularia humilis Ofwegen from the South China Sea. Chemical studies on the animals of this genus have resulted in the isolation and characterization of polyhydroxylated sterols, cembrane diterpenoids, sesquiterpenoids, and glycolipids.2 Our investigation of the Et2O-soluble fraction from the acetone extract of S. humilis Ofwegen has now led to the isolation of two new 9,11-secosteroid glycosides, namely, sinularosides A and B (1, 2), as well as the known compound 3β-(β-xylopyranosyloxy)-5α-pregna-20-ene (3).11 Structures of the new compounds were elucidated by a combination of chemical methods and detailed analysis of their spectroscopic data, aided by the comparison with data of related derivatives. This is the first report of 9,11-secosteroid glycosides from a soft coral and from nature as well. We report herein on the isolation, structure elucidation, and bioactivity of these compounds. © 2012 American Chemical Society and American Society of Pharmacognosy
Freshly collected specimens of S. humilis Ofwegen were immediately frozen and stored at −20 °C before extraction. The Et2O-soluble portion of the acetone extract was subjected to repeated column chromatography on silica gel, Sephadex LH-20, and RP-HPLC to afford three pure steroid glycosides (1−3). The structure of the known compound 3 was determined as 3β-(β-xylopyranosyloxy)-5α-pregna-20-ene, a pregnene glycoside isolated previously from the soft coral Stereonephthya crystalliana. Its structure was determined by extensive spectroscopic analysis combined with careful comparisons with the reported data.11 Sinularoside A (1) was isolated as an optically active, colorless, amorphous solid. The molecular formula of 1 was established as C34H56O7 from the pseudomolecular ion at m/z 599.3929 [M + Na]+ in the HRESIMS spectrum, indicating seven degrees of double-bond equivalents. The IR spectrum showed the presence of hydroxy (3387 cm−1), ketone (1711 cm−1), and alkene (1637 cm−1) functionalities. This observation was in agreement with the signals in the 13C and DEPT spectra (Table 1) for five sp2 carbon atoms at lower field (1 × Received: July 7, 2012 Published: September 4, 2012 1656
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inant O-bearing methine protons resonated between δ 3.5 and 4.5 (5H) in conjunction with the presence of related secondary alcohol carbons (δC 67.1, 69.8, 71.5, 73.1), and an obvious acetal carbon (δC 98.9) suggested the presence of a sugar moiety in the molecule. The presence of a monosaccharide was further confirmed by the observation of the ESIMS ion for a fragment at m/z 437 [M + Na − 162]+, accounting for an additional one double-bond equivalent. The remaining doublebond equivalents were due to the presence of three rings in the aglycone of 1. The complete assignment of the sugar unit was accomplished by detailed analysis of NMR spectra. Starting from the anomeric proton (δH 5.48, d, J = 4.5 Hz, H-1′), analysis of the COSY spectrum readily established the proton sequence from H-1′ to H3-6′, as shown in Figure 1. The obvious HMBC
Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1 and 2a 1b position 1α 1β 2α 2β 3 4α 4β 5 6 7α 7β 8 9 10 11a 11b 12a 12b 13 14 15α 15β 16α 16β 17 18 19 20 21 22a 22b 23a 23b 24 25 26 27 28a 28b 1′ 2′
δC, type 31.7, CH2 29.0, CH2 76.9, CH 37.5, CH2
140.2, C 121.4, CH 31.8, CH2 42.2, CH 215.0, C 48.2, C 57.9, CH2 41.5, CH2 45.6, C 41.7, CH 25.7, CH2 23.6, CH2 49.6, 17.3, 22.7, 34.3, 19.3, 34.2,
CH CH3 CH3 CH CH3 CH2
31.8, CH2 156.4, 33.7, 21.7, 21.9, 106.5,
C CH CH3 CH3 CH2
98.9, CH 69.8, CH
3′
71.5, CH
4′ 5′ 6′ OAc
73.1, CH 67.1, CH 16.9, CH3
2c δH (J in Hz)
2.25, ov 2.03, ov 2.23, ov 1.90, ov 3.78, m 2.78, dd (9.5, 3.0) 2.50, ov 5.51, d (3.0) 2.13, ov 2.48, ov 3.23, dt (11.5, 6.0)
4.12, 4.17, 1.87, 2.02,
m m ov ov
2.88, 1.30, 1.83, 1.73, 1.40, 1.79, 0.86, 1.38, 1.67, 1.12, 1.40, 1.73, 2.15, 2.41,
m ov ov ov ov ov s s ov d (6.5) ov ov ov ov
2.35, ov 1.13, d (7.0) 1.14, d (7.0) 4.92, s 4.94, s 5.48, d (4.5) 4.67, dd (9.0, 4.5) 4.56, dd (9.0, 3.0) 4.28, br s 4.45, q (6.5) 1.63, d (6.5)
δC, type 31.1, CH2 28.7, CH2 77.5, CH 37.2, CH2
139.6, C 122.1, CH 32.8, CH2 43.4, CH 217.1, C 48.5, C 59.3, CH2 40.3, CH2 45.5, C 41.8, CH 25.3, CH2 24.3, CH2 49.2, 17.3, 22.8, 34.1, 19.3, 34.1,
CH CH3 CH3 CH CH3 CH2
31.7, CH2 156.6, 33.8, 21.8, 21.9, 106.1,
C CH CH3 CH3 CH2
97.2, CH 69.4, CH 70.0, CH 73.0, 65.3, 16.2, 171.3, 20.8,
CH CH CH3 C CH3
δH (J in Hz) 1.88, ov 1.52, ov 1.60, ov 1.39, ov 3.47, m 2.49, dd (9.6, 3.0) 2.26, ov 5.50, d (5.4) 2.04, ov 2.43, ov 3.03, dt (12.0, 7.2)
3.68, 3.84, 1.34, 1.67,
m m ov ov
2.62, 1.33, 1.70, 1.57, 1.32, 1.67, 0.69, 1.39, 1.44, 0.99, 1.13, 1.54, 1.89, 2.09,
m ov ov ov ov ov s s ov d (6.6) ov ov ov ov
Figure 1. Key HMBC and COSY correlations for 1 and 2.
correlation from H-1′ to C-5′ led to the formation of a pyranose. The relative configuration of the sugar moiety was determined by the analysis of the 1H−1H coupling constants in combination with a NOESY experiment (Figure 2). The
2.22, ov 1.02, d (7.0) 1.03, d (7.0) 4.66, s 4.73, s 5.03, d (4.2) 3.76, dd (9.8, 4.2) 3.94, dd (9.8, 3.3) 5.21, d (3.3) 4.12, q (6.6) 1.14, d (6.6)
Figure 2. Key NOESY correlations for 1 and 2.
distinct NOE effect between H-3′ and H-5′ indicated a 1,3diaxial relationship of the two protons. An axial orientation for H-2′ and an equatorial orientation of H-4′ were then deduced from the large coupling constant between H-2′ and H-3′ (JH2′,H3′ = 9.0 Hz) and the small coupling constant between H3′ and H-4′ (JH3′,H4′ = 3.0 Hz), respectively. Consequently, the small coupling constant between H-1′ and H-2′ (JH1′,H2′ = 4.5 Hz) suggested an equatorial orientation of the anomeric proton. The sugar subunit was assigned as α-fucopyranose by comparing its 13C NMR chemical shifts with the reported data.12 Its absolute configuration was identified to be Lfucopyranose by HPLC analysis of the thiocarbamoylthiazolidine derivatives of the sugar present in the acid hydrolysate of 1 and those of authentic D- and L-fucose.13 The additional 1H and 13C signals for the molecule were almost identical to those of 3β,11-dihydroxy-24-methylene9,11-secocholest-5-en-9-one, a 9,11-secosterol analogue previously isolated from the soft coral Sinularia sp.5,14 and S. leptoclados.4 The observation of the downfield shifted NMR signals for H-3/C-3 in 1 (δH 3.78, δC 76.9) with respect to
2.17, s
δ in ppm, assignments made by DEPT, COSY, HSQC, HMBC, and NOESY. bIn pyridine-d5, at 500 MHz for 1H and 125 MHz for 13C NMR experiments. cIn CDCl3, at 600 MHz for 1H and 150 MHz for 13 C NMR experiments. a
OC, 2 × CC) and 29 sp3 carbon atoms at higher field (6 × OCH, 1 × OCH2, 2 × C, 5 × CH, 9 × CH2, 6 × CH3), accounting for three double-bond equivalents. The predom1657
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those of the secosteroid (δH 3.48, δC 71.0)14 suggested the connectivity of the aglycone to the sugar moiety. The diagnostic HMBC correlation from the anomeric proton to C-3 further confirmed the linkage between the two subunits. The relative configurations at C-3, C-8, C-10, C-13, C-14, C-17, and C-20 in 1 were proven to be the same as those of the known analogue,5 showing the same NOE correlation patterns for H3-19 with H-4β and H-8, H-7α with H-14, H3-18 with H20, and H3-21 with H-17 (Figure 2). The structure of 1 was finally determined as 3β-O-(α-L-fucopyranosyl)-11-hydroxy-24methylene-9,11-secocholest-5-en-9-one. Sinularoside B (2) was isolated as an optically active, colorless, amorphous solid. The HRESIMS of 2 established the molecular formula C36H58O from the presence of the pseudomolecular ion at m/z 641.4026 [M + Na]+. The IR spectrum of 2 closely resembled that of 1, showing similar functionalities in the molecule. Analysis of the 1H and 13C NMR spectra of 2 also revealed similarities to 1 (Table 1), except for the presence of an additional acetyl group (δH 2.17, s; δC 171.3, C). The location of the acetoxy group at C-4′ was shown by the downfield shift of the respective proton signal at C-4′ from δ 3.79 in 1 (in CDCl3, Table S1) to δ 5.21 in 2, which was confirmed by the HMBC correlation between H-4′ and the ester carbonyl atom. The complete assignment of the acetylated 6′-deoxyhexose unit was accomplished by COSY, HMBC, and NOESY experiments and further confirmed by comparison of its 13C NMR chemical shifts with those of reported data.15 The structure of 2 was therefore determined as 3β-O-(4′-acetyl-α-L-fucopyranosyl)-11-hydroxy-24-methylene9,11-secocholest-5-en-9-one. The antimicrobial and cytotoxic activities of compounds 1 and 2 were evaluated. Both compounds exhibited antifungal activity against Microbotryum violaceum and Septoria tritici, antibacterial activity against the Gram-positive bacterium Bacillus megaterium, and moderate inhibitory activity against the microalga Chlorella f usca. Neither of the compounds were active toward the Gram-negative bacterium Escherichia coli (Table 2). Compounds 1 and 2 showed no activity against liver hepatocellular cells (HepG2) and human epithelial colorectal adenocarcinoma cells (Caco-2).
from the Red Sea sponge Erylus lendenfeldi.19 All of these possess an 8,9-secosteroid aglycone. This is the first report of 9,11-secosteroid glycosides from a soft coral and from nature as well. Interestingly, in contrast to the considerable activity displayed by their aglycone (IC50 = 9.1, 9.1, 9.9, and 4.5 μg/mL for tumor cells HepG2, A-549, MDA-MB-231, and MCF-7, respectively),6 the glycosides 1 and 2 did not show tumor cell growth inhibition toward the tested cells. This fact suggests that 3-O-glycosidation decreases the tumor cell growth inhibition activity of the compounds. On the other hand, these compounds showed antimicrobial activity against the tested fungi, the Gram-positive bacterium, and the microalga. The interesting structures and bioactivity may encourage further investigation of this family of metabolites.
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Table 2. Agar Diffusion Assays of Compounds 1 and 2 for Antimicrobial Activitya 1 2 penicillin streptomycin ketoconazole acetone
M. violaceum
S. tritici
E. coli
B. megaterium
C. f usca
17 17 \ \ 15 0
14 15 \ \ 14 0
0 0 20 25 \ 0
30 30 28 30 \ 0
12 14 \ \ \ 0
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured in CHCl3 on an Autopol-IV polarimeter at the sodium D line (590 nm). UV absorption spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer; peak wavelengths are reported in nm. IR spectra were recorded in thin polymer films on a Nexus-470 FT-IR spectrophotometer (Nicolet, USA); peaks are reported in cm−1. The NMR spectra were recorded at 300 K on Bruker DRX 400, 500, and Avance 600 spectrometers. Chemical shifts are reported in parts per million (δ), with use of the residual CDCl3 signal (δH 7.26 ppm; δC 77.0 ppm) and pyridine-d6 signal (δH 8.74, 7.58, 7.22; δC 150.3, 135.9, 123.9) as internal standards for 1H and 13C NMR and coupling constants (J) in Hz; assignments were supported by COSY, HSQC, HMBC, and NOESY experiments. The mass spectra and highresolution mass spectra were performed on a Q-TOF Micro mass spectrometer in m/z, resolution 5000; an isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound. Semipreparative HPLC was performed on an Agilent-1100 system equipped with a refractive-index detector using a YMC-Pack-ODS-A column (250 × 10 mm, 5 μm). Commercial silica gel (200−300 and 400−500 mesh; Yantai, China) was used for column chromatography. Precoated SiO2 plates (HSGF-254; Yantai, China) were used for analytical TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde H2SO4 reagent. Animal Material. The soft coral Sinularia humilis Ofwegen was collected from the South China Sea (N 21°05′, E 109°05′) in July 2008, at a depth of 16 m, and authenticated by Dr. Xiu-Bao Li (The South China Sea Institute of Oceanology, Chinese Academy of Sciences). A voucher specimen (ZS-8) was deposited at the Second Military Medical University. Extraction and Isolation. The frozen animals (2.6 kg, wet weight) were cut into small pieces and extracted ultrasonically with acetone (6 × 1.5 L) at room temperature. The combined extracts of S. humilis Ofwegen were partitioned between Et2O and H2O. The Et2O extract was concentrated under vacuum to give a dark residue (14.6 g). The Et2O extract was fractionated by column chromatography (CC) on silica (0:100 → 100:0 acetone/petroleum ether) followed by CC on Sephadex LH-20 (CH2Cl2/MeOH, 1:1) and repeated normal-phase CC on silica (CHCl3/MeOH, 7:3) to afford the steroid-containing fraction. This fraction was subjected to semipreparative reversed-phase HPLC (5 μm, 250 × 10 mm; 50% ACN/H2O; 2.5 mL/min) to give 1 (2.8 mg, tR 10.3 min), 2 (5.6 mg, tR 9.2 min), and 3 (1.3 mg, tR 14.5 min). Sinularoside A (1): colorless, amorphous solid; [α]25 D −51 (c 0.06, CHCl3); IR (film) νmax 3387, 2955, 2924, 2869, 1711, 1670, 1637, 1456, 1074, 1034 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 599 [M + Na]+, 437 [M + Na − 162]+; ESIMS m/z 611 [M + Cl]−; HRESIMS m/z 599.3929 [M + Na]+ (calcd for C34H56O7Na, 599.3924). Sinularoside B (2): colorless, amorphous solid; [α]25 D −58.3 (c 0.18, CHCl3); IR (film) νmax 3349, 2956, 2927, 2860, 1738, 1716, 1671 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 641 [M
a
0.05 mg of the test or control substances dissolved in acetone was applied to a filter disk and sprayed with the respective test organism. Radii of the zones of inhibition are given in mm. “\” not tested.
As an important group of marine secondary metabolites, steroid glycosides are widely distributed in echinoderms, sponges, soft corals, and algae.16 Although a large number of secosteroids have been discovered from marine invertebrates,1 the secosteroid glycosides are rare.16 To date, only three secosteroid glycosides have been reported: sarasinoside M from the Indonesian sponge Melophlus sarasinorum,17 sarasinoside A4 from the Australian sponge M. sarasinorum,18 and eryloside L 1658
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+ Na]+, 437 [M + Na − 204]+; ESIMS m/z 653 [M + Cl]−; HRESIMS m/z 641.4026 [M + Na]+ (calcd for C36H58O8Na, 641.4029). Acid Hydrolysis and Absolute Configuration Determination of Monosaccharide for Sinularoside A (1). The glycoside 1 (0.8 mg) was dissolved in 2 M aqueous CF3COOH (1.0 mL) at 120 °C for 2 h. The mixture was evaporated to dryness, and the residue was partitioned between CH2Cl2 and H2O. The aqueous phase was concentrated to furnish a monosaccharide residue. After drying under vacuum, the residue was dissolved in 0.4 mL of pyridine containing 2 mg of L-cysteine methyl ester hydrochloride and heated at 60 °C for 1 h. Phenyl isothiocyanate (2 μL) was then added, and the mixture was heated at 60 °C for 1 h. The reaction mixture was analyzed by reversed-phase HPLC, which was performed on an Agilent 1200 HPLC system (Agilent Technologies Inc., USA) equipped with a photodiode array detector and a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm) at 35 °C with isocratic elution of 25% CH3CN in 50 mM H3PO4 solution for 40 min and subsequent washing of the column with 90% CH3CN at a flow rate of 0.8 mL/min. The injection volume was 4 μL, and peaks were detected at 250 nm. The reaction conditions for authentic L- and D-fucose were the same as described above. The absolute configuration of the monosaccharide in 1 was confirmed to be L-fucose by comparison of the retention time of the thiocarbamoylthiazolidine derivative of the acid hydrolysate of 1 with those of standard samples: L-fucose (29.2 min) and D-fucose (26.4 min), respectively. Agar Diffusion Test for Biological Activity. Compounds 1 and 2 were dissolved in acetone at 1.0 mg/mL; 50 μL of the solution (0.05 mg) was pipetted onto a sterile filter disk (Schleicher & Schuell, 9 mm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organisms.20 The test organisms were the bacteria Escherichia coli and Bacillus megaterium (both grown on NB medium), the fungi Microbotryum violaceum and Septoria tritici (both grown on MPY medium), and the microalga Chlorella f usca (grown on CP medium). Ketoconazole (0.05 mg), penicillin (0.05 mg), and streptomycin (0.05 mg) were used as positive controls. These microorganisms were chosen because they (a) are nonpathogenic and (b) had in the past proved to be accurate initial test organisms for antibacterial and antifungal activities. Commencing at the outer edge of the filter disk, the radius of the zone of inhibition was measured in mm. Cytotoxicity Assay. Compounds 1 and 2 were evaluated for cytotoxicity against liver hepatocellular cells (HepG2) and human epithelial colorectal adenocarcinoma cells (Caco-2), using a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. Adriamycin was used as positive control, IC50 = 2.7 μM.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
HRESIMS and NMR spectra for compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 86 21 81871257. Fax: 86 21 81871257. E-mail:
[email protected]. Author Contributions †
These two authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research work was financially supported by Natural Science Foundation of China (Nos. 41076082, 81172979). 1659
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