2016 Volume 64 Issue 10 Pages 1523-1527
Eight highly hydroxylated steroids (1−8), including three new compounds as sodium salts of (24S)-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24-nonaol 6-sulfate (1), (24E)-5α-cholest-24-ene-26-yde-3β,6α,8,14,15α-pentaol 15-sulfate (2), and 5α-cholest-3β,6α,8,14,15α,24,25,26-octaol 15-sulfate (3), were isolated and elucidated from the methanol extract of the Vietnamese starfish Archaster typicus. The structure elucidation was done by spectroscopic methods including one and two dimensional (1D-, 2D-)NMR and Fourier transform ion cyclotron resonance (FT-ICR)-MS. The isolated compounds can be used as chemical markers for taxonomic identification of the starfish A. typicus.
The phylum Echinodermata is divided into five classes: Crinoidea (sea lilies and feather stars), Holoturoidea (sea cucumbers or holothurians), Echinoidea (sea urchins), Asteroidea (starfish or sea stars), and Ophiuroidea (brittle stars). Of which, starfish are benthic organisms widely found in all oceans. Starfish have attracted organic chemists, biochemists, and pharmacologists as a fascinating source of bioactive secondary metabolites especially polyhydroxylated steroids and steroidal glycosides.1,2) Polyhydroxylated steroids from starfish comprise four to nine hydroxy groups in steroidal nucleus and side chains. It is of special interest that all the groups were found only in limited positions. The majority of polyhydroxylated steroids possess a 3β,6α(or β),8,15α(or β),16β-pentahydroxycholestane skeleton, sometimes with additional OH groups at positions 4β,5α,7α(or β), and occasionally at 14α. The side chains of these compounds are very diverse, but (25S)-26-hydroxy structure is a common feature; the less common side chain is hydroxylated at C-24 with (S)-configuration. Polyhydroxylated steroids are also found in sulfated forms, with the sulfate group located at position 3β, 6α, 15α or 24.1–3)
The starfish Archaster typicus is abundantly found in Vietnamese seas. Previous studies on this species yielded nine unique polyhydroxylated steroids in the 1980s.4–6) Recently, several polyhydroxylated steroids of this type7,8) and asterosaponins9,10) were reported from this species. In continuation of our recent investigations on Vietnamese starfish,11) this paper deals with the isolation and structure elucidation of eight highly hydroxylated steroids (1–8, Fig. 1), including three new compounds 1–3, from A. typicus.
Using various chromatographic separations, eight highly hydroxylated steroids were isolated from a methanol extract of the Vietnamese starfish A. typicus. The known compounds were elucidated as 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol 6-sulfate (4),4) (24E)-5α-cholest-24-ene-3β,6α,8,14,15α,26-hexaol 15-sulfate (5),4) sodium 5α-cholest-25(27)-ene-3β,6α,8,14,15α,24,26-heptaol 15-O-sulfate (6),7) 27-nor-5α-cholestane-3β,4β,5,6α,8,14,15α,24α-octaol (7),4) and (24R)-27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24-nonanol (8)4) by detailed analysis of the spectroscopic data including one and two dimensional (1D-, 2D-)NMR and MS and comparison with those reported in the literatures.
Compound 1 was isolated as a colorless powder. Its molecular formula was determined as C27H47NaO12S by Fourier transform ion cyclotron resonance (FT-ICR)-MS at m/z 641.25834 [M+Na]+. The NMR features indicated a polyhydroxylated steroid with the presence of six oxymethine groups [δC 68.4 (C-3), 72.4 (C-4), 81.1 (C-6), 73.1 (C-7), 69.7 (C-15), and 78.1 (C-24)/δH 4.02 (1H, ddd, J=4.0, 5.0, 12.0 Hz, H-3), 4.07 (1H, d, J=4.0 Hz, H-4), 4.84 (1H, d, J=9.5 Hz, H-6), 4.39 (1H, d, J=9.5 Hz, H-7), 4.42 (1H, dd, J=5.0, 10.0 Hz, H-15), and 3.23 (1H, m, H-24)] and three quaternary oxygenated carbons [δC 78.7 (C-5), 80.1 (C-8), and 84.1 (C-14)]. In addition, the signals of two tert-methyl [δC 16.8 (C-18) and 17.4 (C-19)/δH 1.17 (H-18) and 1.40 (H-19), each 3H, s] and three sec-methyl [δC 19.0 (C-21), 17.5 (C-26), and 19.5 (C-27)/δH 0.89 (H-21), 0.91 (H-26), and 0.93 (H-27), each 3H, d, J=7.0 Hz] groups were also observed. The 1H- and 13C-NMR data of 1 were similar to those of 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol 6-sulfate (4),4) except for an additional presence of a methyl group. Detailed comparison of the 13C-NMR data of 1 (Table 1) with those of 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol 6-sulfate (4)4) confirmed the positions of all hydroxy groups on the steroid nucleus, which was also supported by heteronuclear multiple bond correlation (HMBC) and 1H–1H correlation spectroscopy (COSY, Fig. 2). The carbon signal for C-6 of 1 was strongly shifted downfield at δC 81.1 relative to that of 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol4) at δC 70.8 confirming the common location of a sulfate group at C-6.8) Positions of the last OH group at C-24 and the additional methyl group C-27 were assigned by COSY correlations of H2-23/H-24/H-25/H3-26 and HMBC cross-peaks of H-26/H-27 with C-24 and C-25 (Fig. 2). The 1H- and 13C-NMR data for the steroid nucleus of 1 were essentially identical to those of sodium cholest-25(27)-ene-3β,4β,5α,6α,7β,8,14,15α,24,26-decanol 6-sulphate8) indicating the same configurations for the steroid nucleus of these two compounds. This was also confirmed by a nuclear Overhauser effect spectroscopy (NOESY). The NOESY correlations of H-6 (δH 4.84) with H-19 (δH 1.40) and H-15 (δH 4.42) with H-18 (δH 1.17) indicated β-orientation for both H-6 and H-15 (Fig. 3). Proton H-4 (δH 4.07) showed no NOESY correlations with H-6 (δH 4.84) and/or H-19 (δH 1.40) suggesting for α-orientation of H-4. The small coupling constant between H-3 and H-4 (J3–4=4.0 Hz) and a large coupling constant between H-2 and H-3 (J2–3=12.0 Hz) indicated equatorial- and axial-orientations of H-4 and H-3, respectively. Moreover, the 13C-NMR chemical shifts at C-3 (δC 68.4) and C-4 (δC 72.4) of 1 were completely identical to those of sodium cholest-25(27)-ene-3β,4β,5α,6α,7β,8,14,15α,24,26-decanol 6-sulphate8) further confirming the α-orientation for both H-3 and H-4. The α-orientation of H-7 was assigned by a spatial proximity of H-7 (δH 4.39) with H-9 (δH 2.35). In addition, the common 24S-configuration of 1 was suggested by the coexistence of 1, 4, 7, and 8 in A. typicus and by analogy with all the reported steroids from starfish.1,2) Consequently, the structure of 1 was elucidated as sodium (24S)-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24-nonaol 6-sulfate.
| Position | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| δCa,b) | δHa,c) mult. (J in Hz) | δCa,b) | δHa,c) mult. (J in Hz) | δCb,d) | δHc,d) mult. (J in Hz) | |
| 1 | 33.0 | 1.39 m/1.62 m | 39.8 | 1.01 m/1.76 m | 38.6 | 1.01 m/1.75 m |
| 2 | 26.6 | 1.62 m/1.79 m | 31.4 | 1.50 m/1.76 m | 30.8 | 1.85 m/2.05 m |
| 3 | 68.4 | 4.02 ddd (4.0, 5.0, 12.0) | 72.1 | 3.50 m | 70.7 | 3.90 m |
| 4 | 72.4 | 4.07 d (4.0) | 32.3 | 1.23 m/2.19 m | 32.0 | 1.75 m/2.95 br d (12.0) |
| 5 | 78.7 | — | 53.2 | 1.06 m | 52.1 | 1.50 m |
| 6 | 81.1 | 4.84 d (9.5) | 67.6 | 3.66 dt (4.5, 11.0) | 66.4 | 4.33 dt (4.0, 10.5) |
| 7 | 73.1 | 4.39 d (9.5) | 44.6 | 1.72 m | 43.9 | 2.52 ddd (2.5, 11.0, 13.0) |
| 2.31 dd (4.0, 13.0) | 3.37 dd (3.5, 13.0) | |||||
| 8 | 80.1 | — | 79.3 | — | 78.3 | — |
| 9 | 41.0 | 2.35 dd (3.5, 12.5) | 48.8 | 1.67 m | 47.6 | 2.03 m |
| 10 | 40.8 | — | 37.6 | — | 36.7 | — |
| 11 | 18.2 | 1.30 m/1.72 m | 19.1 | 1.47 m/1.67 m | 18.2 | 1.55 m/1.19 m |
| 12 | 35.6 | 1.62 m/1.78 m | 34.9 | 1.59 m/1.81 m | 34.0 | 1.70 m/2.15 m |
| 13 | 49.9 | — | 48.1 | — | 46.8 | — |
| 14 | 84.1 | — | 85.4 | — | 84.5 | — |
| 15 | 69.7 | 4.42 dd (5.0, 10.0) | 77.7 | 4.96 dd (4.5, 11.0) | 76.2 | 5.77 m |
| 16 | 37.0 | 1.72 m/1.86 m | 37.3 | 2.00 m/2.13 m | 36.5 | 2.45 m/2.75 m |
| 17 | 51.4 | 1.95 m | 51.3 | 2.10 m | 50.9/50.8 | 2.45 m |
| 18 | 16.8 | 1.17 s | 17.4 | 1.19 s | 16.8 | 1.36 s |
| 19 | 17.4 | 1.40 s | 14.3 | 1.05 s | 13.9 | 1.29 s |
| 20 | 36.5 | 1.40 m | 36.1 | 1.47 m | 35.5/34.9 | 1.50 m |
| 21 | 19.0 | 0.89 d (7.0) | 18.6 | 0.94 d (7.0) | 18.4 | 0.94 d (7.0) |
| 22 | 33.6 | 1.00 m/1.64 m | 35.5 | 1.28 m/1.62 m | 33.5 | 1.06 m/2.21 m |
| 33.1 | 1.59 m/1.80 m | |||||
| 23 | 31.8 | 1.25 m/1.59 m | 26.8 | 2.35 m/2.45 m | 28.3 | 1.66 m/2.28 m |
| 27.9 | 1.92 m/2.01 m | |||||
| 24 | 78.1 | 3.23 m | 157.6 | 6.64 dt (1.0, 7.5) | 76.1 | 4.08 m |
| 75.3 | 4.04 m | |||||
| 25 | 34.5 | 1.64 m | 140.3 | — | 74.63 | — |
| 26 | 17.5 | 0.91 d (7.0) | 197.3 | 9.37 s | 68.22 | 4.08 m |
| 4.25 dd (4.5, 11.0) | ||||||
| 27 | 19.5 | 0.93 d (7.0) | 9.0 | 1.74 s | 19.2/19.1 | 1.59 s |
a) Recorded in CD3OD. b) 125 MHz. c) 500 MHz. d) Recorded in pyridine-d5. All assignments were done by HSQC, HMBC, 1H–1H COSY, and NOESY experiments.
) and HMBC (
) Correlations of 1–3
The FT-ICR-MS of 2 revealed a quasi-molecular ion peak at m/z 589.24230 [M+Na]+ confirming the molecular of 2 as C27H43NaO9S. Its NMR features are also typical for a polyhydroxylated steroid with presence of three oxymethine groups and two oxygenated quaternary carbons (Table 1). The 1H- and 13C-NMR data of 2 were similar to those of (24E)-5α-cholest-24-ene-3β,6α,8,14,15α,26-hexaol 15-sulfate (5),4) except for the presence of an aldehyde group [δC 197.3 (C-26)/δH 9.37 (1H, s, H-26)] in 2 instead of an oxymethylene group in 5. Detailed comparison of the 13C-NMR data of 2 with those of 54) and combination with COSY and HMBC experiments (Fig. 2) clearly confirmed the planar structure of 2. The relative configurations of 2 were assigned by the coexistence of 2 and 4 in A. typicus, good agreement of the 1H- and 13C-NMR data for the steroid nucleus of 2 with those of (23E)-27-nor-25-oxo-5α-cholest-23-ene-3β,6α,8,14,15α-pentaol 15-O-sulfate sodium salt,7) and by NOESY experiment. The proton signal of H-3 at δH 3.50 (1H, m, J1/2=11.0 Hz) is indicative for its α-orientation12) (versus t, J=2.5 or 3.0 Hz for β-orientation of H-3 without a large J value attributable to a diaxial coupling13,14)). This was further confirmed by the 13C-NMR chemical shift for C-3 of 2 at δC 72.17,12,15) and a NOESY correlation of H-3 (δH 3.50) with H-5 (δH 1.06). The β-orientation for both H-6 and H-15 was assigned by NOESY correlations of H-6 (δH 3.66) with H-19 (δH 1.05) and H-15 (δH 4.96) with H-18 (δH 1.19). The aldehyde proton H-27 (δH 9.37) showed a NOESY correlation with H-24 (δH 6.64) confirming the E-configuration of the double bond C-24/C-25 (Fig. 3). Thus, compound 2 was identified as sodium (24E)-5α-cholest-24-ene-26-yde-3β,6α,8,14,15α-pentaol 15-sulfate.
The 1H- and 13C-NMR data of 3 were similar to those of 2 (Table 1), 5,4) and 6,7) exept for difference in signals of the side chains. The FT-ICR-MS of 3 revealed a quasi-molecular ion peak at m/z 625.26342 [M+Na]+ corresponding to a molecular of C27H47NaO11S with the presence of three OH groups in the side chain. Detailed analysis of heteronuclear single quantum coherence (HSQC) and HMBC experiments confirmed positions of the three OH groups at C-24, C-25, and C-26 (Fig. 2). However, the 13C-NMR spectrum revealed two sets of signals with a ratio of 1 : 1 at C-17 (δC 50.9/50.8), C-20 (δC 35.5/34.9), C-22 (δC 33.5/33.1), C-23 (δC 28.3/27.9), C-24 (δC 76.1/75.3), and C-27 (δC 19.2/19.1) indicating for the presence of an inseparable mixture (attempts to separate these isomers by normal and reversed-phase HPLC were unsuccessful) of C-24 and/or C-25 epimers in 3. From all above evidence, the structure sodium 5α-cholest-3β,6α,8,14,15α,24,25,26-octaol 15-sulfate was elucidated for 3.
In summary, eight highly hydroxylated steroids 1–8, including three new compounds 1–3, were isolated and structure elucidated from the Vietnamese starfish A. typicus. The isolated compounds possess 3β,6α,8,14,15α-pentaol (compounds 2, 3, 5, 6), 3β,4β,5,6α,8,14,15α-heptaol (compound 7), and 3β,4β,5,6α,7β,8,14,15α-octaol (compounds 1, 4, 8) steroidal skeletons. To date, these three oxygenated patterns were only found in the species A. typicus.4–8) Thus, these kinds of polyhydroxylated steroids can be used as chemical markers for taxonomic identification of this starfish.
Optical rotations were determined using a Jasco P-2000 polarimeter. IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer. High resolution mass spectra were recorded on a Varian 910 FT-ICR mass spectrometer. The NMR spectra were recorded on a Bruker AVANCE III HD 500 spectrometer with tetramethylsilane (TMS) as an internal standard. Medium pressure liquid chromatography (MPLC) was carried out on a Biotage–Isolera One system. TLC was performed on Kieselgel 60 F254 (1.05715; Merck) or RP-18 F254s plates. Spots were visualized by spraying with 10% aqueous H2SO4 solution, followed by heating for 3–5 min. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck), YMC*GEL resins (ODS-A, 12 nm S-150 µm, YMC Co., Ltd.), Sephadex LH-20 (Sigma), and Diaion HP-20 (Supelco).
Biological MaterialThe samples of starfish A. typicus MÜLLER & TROSCHEL, 1840 were collected in Haiphong, Vietnam, in April 2014 and identified by Professor Do Cong Thung (Institute of Marine Resources and Environment, VAST). Voucher specimens (No: ĐAB-AT-04-2014) were deposited at the Institute of Marine Biochemistry, VAST, Vietnam.
Extraction and IsolationDried samples of A. typicus (5.0 kg) were powdered and extracted three times with MeOH (24 h each) at room temperature. The resulted solutions were filtered, combined and concentrated in vacuum to obtain crude MeOH residue (450 g). This was suspended in water and partioned in turn with n-hexane and CH2Cl2 to give the corresponding extracts: n-hexane (H, 160 g), CH2Cl2 (C, 180 g), and water layer (W). The water layer was passed through a Diaion HP-20 CC eluting with increasing concentration of MeOH in water (0, 25, 75, and 100%) to obtain three fractions, W1–W3, after removal of the fraction eluted with water. Fraction W1 (52 g, 100% MeOH) was crudely separated on Silica gel MPLC eluting with CH2Cl2–MeOH (20 : 1→1 : 1, v/v) to obtain five subfractions, W1A–W1E. Subfraction W1B (6.5 g) was further separated into six smaller fractions W1B1–W1B6 by YMC CC using eluent of MeOH–H2O (2 : 1, v/v). Fraction W1B2 (410 mg) was further separated by Silca gel CC eluted with EtOAc–MeOH–H2O (6 : 1 : 0.1, v/v), followed by Sephadex LH-20 CC with MeOH–H2O (1 : 1, v/v) to give compound 5 (5.3 mg). Compound 4 (5.0 mg) was purified from fraction W1B3 (700 mg) after subjecting it on Silica gel CC eluted with CH2Cl2–MeOH–H2O (4 : 1 : 0.1, v/v), followed by YMC CC with MeOH–H2O (1.5 : 1, v/v). Purification of fraction W1B4 (74 mg) by Silica gel CC using eluent of CH2Cl2–MeOH–H2O (5 : 1 : 0.1, v/v) to obtain compound 1 (3.7 mg). Fraction W1B5 (500 mg) was purified by Silica gel CC eluting with CH2Cl2–MeOH–H2O (5 : 1 : 0.1, v/v) to furnish compound 7 (15.0 mg). Compound 8 (6.0 mg) was crystalized in MeOH from fraction W1B6 (340 mg). Fraction W1D (20 g) was further separated by RP-18 MPLC eluting with MeOH–H2O (1 : 1, v/v) to give seven subfractions, W1D1–W1D7. Subfraction W1D2 (477 mg) was separated into two smaller fractions, W1D2A (10 mg) and W1D2B (247 mg), using Sephadex LH-20 CC with MeOH–H2O (1 : 1, v/v). Fraction W1D2B was further separated on Silica gel CC using eluent of EtOAc–MeOH–H2O (3 : 1 : 0.1, v/v), followed by YMC CC with acetone–H2O (1 : 3, v/v) to furnish compound 3 (4.0 mg). Finally, fraction W1D4 (848 mg) afforded compounds 2 (1.8 mg) and 6 (5.0 mg) after subjecting it on Silica gel CC eluted with EtOAc–MeOH–H2O (4 : 1 : 0.1, v/v).
Sodium (24S)-5α-Cholestane-3β,4β,5,6α,7β,8,14,15α,24-nonaol 6-Sulfate (1)Colorless powder; [α]D25 +50° (c=0.05, CHCl3); IR (KBr) νmax: 3437, 2945, 2868, 1643, 1561, 1259, 1061, and 969 cm−1; FT-ICR-MS m/z 641.25834 [M+Na]+ (Calcd for C27H47Na2O12S+, 641.25726); 1H- and 13C-NMR data, see Table 1.
Sodium (24E)-5α-Cholest-24-ene-26-yde-3β,6α,8,14,15α-pentaol 15-Sulfate (2)Colorless powder; [α]D25 +41° (c=0.05, MeOH); IR (KBr) νmax: 3451, 2925, 2875, 1635, 1570, 1373, 1232, 1049, and 611 cm−1; FT-ICR-MS m/z 589.24230 [M+Na]+ (Calcd for C27H43Na2O9S+, 589.24177); 1H- and 13C-NMR data, see Table 1.
Sodium 5α-Cholest-3β,6α,8,14,15α,24,25,26-octaol 15-Sulfate (3)Colorless powder; [α]D25 +25° (c=0.05, MeOH); IR (KBr) νmax: 3484, 2954, 2869, 1649, 1221, and 1064 cm−1; FT-ICR-MS m/z 625.26342 [M+Na]+ (Calcd for C27H47Na2O11S+, 625.26290); 1H- and 13C-NMR data, see Table 1.
This study was financially supported by a Grant from Vietnam Academy of Science and Technology (codes: VAST.TÐ.ÐAB.03/13–15 and VAST.TÐ.DLB.03/16–18). The authors are grateful to the Institute of Chemistry, VAST for measurement of the NMR and mass spectra.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials, 1D- and 2D-NMR spectra for the new compounds 1–3.
