1
␣
,3

,5

-Trihydroxy-24-methylenecholestan-6-one:
a novel steroid from a soft coral Sinularia gibberosa
Atallah F. Ahmed
a,b, Chang-Feng Dai
c, Yao-Haur Kuo
d, Jyh-Horng Sheu
a,∗aDepartment of Marine Resources, National Sun Yat-Sen University, Kaohsiung 804, Taiwan, ROC bDepartment of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
cInstitute of Oceanography, National Taiwan University, Taipei 106, Taiwan, ROC dNational Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC Received 4 December 2002; received in revised form 19 February 2003; accepted 5 March 2003
Abstract
A novel steroid, 1␣,3,5-trihydroxy-24-methylenecholestan-6-one (gibberoketosterol) (1), along with four known steroids, was isolated from the lipophilic extracts of a Taiwanese soft coral Sinularia gibberosa. The structure of the new metabolite was determined on the basis of extensive spectral analyses and chemical reaction. The relative stereochemistry of gibberoketosterol was established by the NOESY experiments and analysis of the pyridine-induced deshielding effect of the axial hydroxy groups. Gibberoketosterol is the first example of 1␣,3,5-trihydroxy-6-oxosteroids isolated from natural sources and was found to exhibit a moderate cytotoxicity against the growth of Hepa59T/VGH cancer cells.
© 2003 Elsevier Science Inc. All rights reserved.
Keywords: Sinularia gibberosa; 1␣,3,5-Trihydroxy-24-methylenecholestan-6-one; Gibberoketosterol; Soft coral; Cytotoxicity
1. Introduction
Marine organisms, including soft corals, have been well-recognized as a natural source of 3-hydroxy sterols and their oxygenated analogues[1]. Previous chemical in-vestigations on the steroidal contents of the soft coral species belonging to genus Sinularia, have led to the isolation and identification of varieties of polyoxygenated steroids[2–8]. Some of these compounds have been shown to exhibit cy-totoxic activity against the growth of the various cancer cell lines[6]. In our current chemical investigation on Sin-ularia gibberosa, we have succeeded in isolating a new oxygenated steroid along with four known steroids (Fig. 1) from the organic extracts. This paper deals with the isolation and structure elucidation of the new metabolite. The new steroid, 1␣,3,5-trihydroxy-24-methylenecholestan-6-one (gibberoketosterol) (1), is the first example of natural 1␣,3,5-trihydroxy-6-oxosteroids. The structure of 1 was deduced by a series of 2D-NMR experiments (1H-1H COSY, HMQC, HMBC, and NOESY) and by careful
analy-∗Corresponding author. Tel.:+886-7-5252000x5030;
fax:+886-7-5255020.
E-mail address: [email protected] (J.-H. Sheu).
sis of the pyridine-induced deshielding shifts effect exerted by axial hydroxy groups of this metabolite. Cytotoxicity of metabolites 1, 4, and 5 against Hepa59T/VGH (human liver carcinoma) cancer cells also is reported.
2. Experimental
2.1. General methods
Melting points were determined using a Fisher–Johns melting point apparatus. Optical rotations were measured on a Jasco DIP-1000 digital polarimeter. IR spectra were recorded on a Jasco FT-5300 infrared spectrophotometer. EIMS was obtained with a VG Quattro GC/MS spectrome-ter. HRMS spectra were recorded on a Finnigan MAT-95XL mass spectrometer. The NMR spectra were recorded on a Bruker AVANCE DPX300 FT-NMR at 300 MHz for1H and 75 MHz for13C or on a Varian Unity INOVA 500 FT-NMR at 500 MHz for 1H and 125 MHz for 13C, respectively, in CDCl3using TMS as internal standard, unless otherwise
in-dicated. Si gel (Merck; 230–400 mesh) was used for column chromatography. Precoated Si gel plates (Merck, Kieselgel 60 F-254; 0.2 mm) were used for analytical TLC.
0039-128X/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0039-128X(03)00036-9
Fig. 1. Steroids isolated from Sinularia gibberosa.
2.2. Organism
The soft coral S. gibberosa Tixier-Durivault (0.6 kg, wet weight) was collected by hand via SCUBA along the coast of Kenting, Taiwan, in December 2000, at a depth of 10–15 m, and stored in a freezer until extraction. A voucher sample was deposited at the Department of Marine Resources, Sun Yat-Sen University (specimen no. NHSC 002).
2.3. Extraction and isolation
The organism was exhaustively extracted with EtOH. The EtOH extract was filtered and concentrated under reduced pressure and the aqueous residue was parti-tioned with n-hexane, followed by dichloromethane to provide n-hexane fraction (4 g) and dichloromethane frac-tion (11 g). The n-hexane fracfrac-tion was chromatographed on Si gel 60 using acetone–n-hexane (stepwise, 0–100% acetone) to yield 20 fractions. Fraction 6 eluted with 5% acetone was further purified by normal phase MPLC using n-hexane–dichloromethane gradient (3:2 to 0:1) to yield 3 (10.0 mg). Fraction 14 eluted with 20% ace-tone was further purified by normal phase HPLC using dichloromethane–MeOH (98:2) to yield 2 (3.0 mg). The dichloromethane fraction was fractionated by normal phase MPLC and elution was performed with n-hexane–EtOAc (stepwise, 0–100% EtOAc) to yields 36 fractions. Frac-tion 26 eluted with 25% EtOAc was further purified by
normal phase HPLC using n-hexane–EtOAc (2:1) to af-ford 4 (34 mg). Fraction 30 eluted with 40% EtOAc was further chromatographed by normal phase HPLC using dichloromethane–MeOH (99:1 to 95:5, gradient) to af-ford 1 (18 mg). Fraction 36 eluted with EtOAc yielded
5 (17 mg) after being purified by recrystallization from
EtOAc.
2.3.1. Gibberoketosterol (1)
White solid: mp, 140–141◦C (EtOAc); [α]25D, −0.7◦ (c 0.38, CHCl3). IR (neat)νmax(cm−1): 3570 (br), 3015, 2936,
2872, 1707, 1219. 1H NMR (500 MHz, CDCl3) and 13C
NMR (125 MHz, CDCl3), seeTable 1.1H NMR (300 MHz,
pyridine-d5)δH: 4.89 (1H, dd,J = 14.0, 4.0 Hz, H-1), 4.87
(1H, s, H-28), 4.85 (1H, s, H-28), 4.48 (1H, brs, H-3), 1.39 (3H, s, 19-Me), 1.06 (6H, d,J = 6.6 Hz, 26-Me, 27-Me), 0.96 (3H, d,J = 6.6 Hz, 21-Me), 0.66 (3H, s, 18-Me).13C NMR (75 MHz, pyridine-d5) δC: 211.5 (s, C-6), 156.7 (s, C-24), 106.7 (t, C-28), 85.1 (s, C-5), 69.8 (d, C-1), 68.3 (d, C-3), 56.7 (d, C-14), 56.3 (d, C-17), 49.8 (s, C-10), 43.7 (d, C-9), 42.7 (t, C-7), 42.3 (s, C-13), 40.2 (t, C-12), 39.2 (t, C-2), 38.2 (t, C-4), 37.6 (d, C-8), 36.0 (d, C-20), 35.0 (t, C-22), 34.1 (d, C-25), 31.4 (t, C-23), 28.1 (t, C-16), 24.4 (t, C-15), 24.2 (t, C-11), 22.2 (q, C-27), 22.0 (q, C-26), 18.8 (q, C-21), 14.6 (q, C-19), 12.2 (q, C-18).1H NMR (300 MHz, DMSO-d6)δH: 5.02 (1H, s, OH), 4.69 (1H, s, H-28), 4.62 (1H, s, H-28), 4.56 (1H, d, J = 5.0 Hz, OH), 4.43 (1H, d, J = 6.8 Hz, OH), 3.98 (2H, m, H-1, H-3), 3.98 (1H,
Table 1
1H and13C NMR data for compound 1
C/H δH(ppm)a 13Cb 1 4.25 dd (13.0, 4.0)c 70.7 (d)d 2␣ 1.96 td (13.5, 3.0) 37.5 (t) 2 2.09 m 3 4.20 brs 67.7 (d) 4␣ 2.34 dd (14.5, 3.5) 37.3 (t) 4 1.62 brd (14.5) 5 83.7 (s) 6 211.5 (s) 7␣ 2.24 t (14.5) 41.2 (d) 7 2.43 dd (14.5, 5) 8 1.79 dd (12.5, 5.0) 37.4 (d) 9 1.99 dd (11.0, 5.0) 43.3 (d) 10 49.1 (s) 11␣ 2.20 m 23.6 (t) 11 1.47 brdd (12.5, 3.0) 12␣ 1.21 dd (14.5, 3.5) 39.7 (t) 12 2.02 dt (13.0, 4.0) 13 42.4 (s) 14 1.25 m 56.8 (d) 15␣, 1.54 m 24.2 (t) 16␣ 1.30 td (12.0, 5.0) 27.7 (t) 16 1.86 dd (11.0, 5.0) 17 1.16 t (9.5) 56.0 (d) 18 0.66 s 11.9 (q) 19 0.97 s 13.4 (q) 20 1.42 m 35.6 (d) 21 0.94 d (7.0) 18.5 (q) 22␣ 1.54 m 34.5 (t) 22 1.14 m 23␣ 2.10 brd (11.0) 30.9 (t) 23 1.89 m 24 156.7 (s) 25 2.21 m 33.8 (d) 26 1.02 d (7.0) 21.8 (d) 27 1.03 d (7.0) 22.0 (q) 28 4.72 s, 4.65 s 106.0 (t) 5-OH 4.50 s aSpectra recorded at 500 MHz in CDCl3 at 25◦C. bSpectra recorded at 125 MHz in CDCl3 at 25◦C. cJ values (in Hz) in parentheses.
dMultiplicity deduced by DEPT and indicated by usual symbols. The values are in ppm downfield from TMS.
m, H-1), 0.97 (3H, d, J = 6.6 Hz, 27-Me), 0.96 (3H, d, J = 6.6 Hz, 26-Me), 0.90 (3H, d, J = 6.4 Hz, 21-Me), 0.81 (3H, s, 19-Me), 0.59 (3H, s,18-Me). 13C NMR (75 MHz, DMSO-d6) δC: 210.8 (s, C-6), 156.0 (s, C-24), 106.7 (t, C-28), 83.6 (s, C-5), 72.5 (d, C-1), 66.7 (d, C-3), 55.9 (d, C-17), 55.8 (d, C-14), 48.4 (s, C-10), 42.4 (s, C-13), 42.1 (d, C-9), 41.7 (t, C-7), 39.4 (t, C-12), 37.7 (t, C-2), 37.1 (d, C-8), 36.6 (t, C-4), 35.2 (d, C-20), 34.3 (t, C-22), 33.2 (d, C-25), 30.6 (t, C-23), 27.6 (t, C-16), 24.0 (t, C-15), 23.3 (t, C-11), 22.0 (q, C-27), 21.8 (q, C-26), 18.6 (q, C-21), 13.9 (q, C-19), 11.9 (q, C-18). HREIMS (m/z): 446.3385 [M]+. C28H46O4requires 446.3398. EIMS (70 eV) (m/z): 447 [0.2,
(M+H)+], 446 [0.2, (M)+], 428 [0.2, (M–H2O)+], 410 [0.2,
(M–2H2O)+], 400 [0.4], 382 [1.0], 347 [2.3], 316 [3.5], 298
[3.6], 69 [100].
2.3.2. Acetylation of 1
A solution of gibberoketosterol (1) (13 mg, 0.032 mM) in pyridine (0.25 ml) was added with Ac2O (0.25 ml) and
the mixture was stirred at RT for 36 h. After evaporation of excess reagent, the residue was separated by column chro-matography on silica gel to give 1,3-diacetyl derivative 6 (CH2Cl2–MeOH= 98:2, 11 mg, 0.022 mmol, 69%), as fine
needles. 2.3.3. 1,3-Diacetate (6) The mp: 71–72◦C; [α]27D −33.5◦(c 0.5, CHCl3). IR (neat) νmax(cm−1): 3479 (br), 3030, 2926, 2874, 1732, 1710, 1215. 1H NMR (300 MHz, CDCl 3) δH: 5.51 (1H, dd,J = 9.3, 6.6 Hz, H-1), 5.20 (1H, brs, H-3), 4.72 (1H, s, H-28), 4.66 (1H, s, H-28), 2.45 (1H, dd,J = 14.8, 4.5 Hz, H-7b), 2.26 (1H, m, H-7␣), 2.24 (2H, m, H-4␣, H-25), 2.09 (3H, s, 3-OAc), 2.07 (3H, m, H-11␣, H-12, H-23␣), 2.06 (1H, m, H-2), 2.04 (3H, s, 1-OAc), 2.00 (1H, m, H-2␣), 1.99 (1H, m, H-23), 1.91 (1H, dd, J = 11.1, 3.7 Hz, H-9), 1.85 (1H, m, H-16), 1.78 (2H, brd, J = 14.8 Hz, H-4, H-8), 1.60 (2H, m, H2-15), 1.55 (1H, m, H-22␣), 1.52 (1H, m, H-11), 1.39 (1H, m, H-20), 1.29 (1H, m, H-16␣), 1.26 (1H, m, H-14), 1.22 (1H, m, H-12␣), 1.15 (1H, dd, J = 11.1, 3.4 Hz, H-17), 1.15 (1H, m, H-22), 1.03 (6H, d, J = 6.3 Hz, 26-Me, 27-Me), 0.96 (3H, s, 21-Me), 0.83 (3H, s, 19-Me), 0.67 (3H, s, 18-Me).13C NMR (75 MHz, CDCl3) δC: 211.5 (s, C-6), 171 (s, CO of 3-acetate), 170.1 (s, CO of 1-acetate), 156.7 (s, C-24), 106.1 (t, C-28), 81.8 (s, C-5), 72.6 (d, C-1), 68.8 (d, C-3), 56.9 (d, C-14), 56.1 (d, C-17), 48.7 (s, C-10), 43.5 (d, C-9), 42.4 (s, C-13), 41.4 (t, C-7), 39.7 (t, C-12), 37.2 (d, C-8), 35.7 (d, C-20), 35.0 (t, C-4), 34.6 (t, C-22), 33.9 (d, C-25), 31.0 (t, C-23), 31.0 (t, C-2), 27.8 (t, C-16), 24.3 (t, C-15), 23.1 (t, C-11), 22.0 (q, C-27), 21.9 (q, C-26), 21.5 (q, CH3of 1-acetate), 21.4 (q, CH3of 3-acetate), 18.6 (q, C-21), 12.9 (q, C-19), 12.0 (q, C-18). 2.4. Cytotoxicity assay
Hepa59T/VGH cells were purchased from the Ameri-can Type Culture Collection (ATCC). Cytotoxicity assay of the test compounds 1, 4, and 5 was performed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) colorimetric method[9,10].
3. Results and discussion
Gibberoketosterol (1) was isolated as translucent thin flakes, mp 140–141◦C. Its HREIMS exhibited a molecular ion peak at m/z 446.3385, corresponding to the molecular formula C28H46O4 and six units of unsaturation. The IR
spectrum showed absorption bands of hydroxy (3570 cm−1, broad) and carbonyl (1707 cm−1) groups. EIMS of 1 ex-hibited peaks at m/z 428 [M–H2O]+and 410 [M–2H2O]+,
suggesting the presence of at least two hydroxy groups in 1. The13C NMR spectrum displayed 28 signals, which were
Fig. 2.1H-1H COSY and key HMBC correlations for 1.
identified by the assistance of a DEPT spectrum as 5 methyl, 10 methylene, 8 methine, and 5 quaternary carbons (Table 1). Above data suggested that 1 is a hydroxylated steroid. The presence of a ketone and one 1,1-disubstituted double bond could be deduced from signals appearing at δ 211.5 (s), 106.0 (t), and 156.7 (s). Three signals appearing atδ 67.7 (d), 70.7 (d), and 83.7 (s) were due to carbons bonded to an oxygen. Comparison of the13C NMR spectral data of 1 (in DMSO-d6,) with those of the known 1,3,5-trihydroxylated
sterone (7) suggested that 1 could be an isomer of 7[5]. In the 1H NMR spectrum of 1 (in CDCl3; see Table 1), the
signals appearing atδ 4.25 (dd, J = 13.0, 4.0 Hz) and 4.20 (1H, brs) were assigned to H-1 and H-3, respectively, by the assistance of1H-1H COSY and HMBC spectra (Fig. 2). An additional signal atδ 4.50 (1H, s) was deduced to be the resonating peak of a tertiary hydroxy group attached to a quaternary carbon (δ 83.7). A 1,1-disubstituted double bond was further confirmed by1H NMR spectrum which showed signals at δ 4.72 (1H, s) and 4.65 (1H, s). 1H-1H COSY and HMBC spectral data further established three partial structures of sequential proton sets and long-range1H/13C correlations, respectively (Fig. 2). On the basis of above observations, the planar structure of 1 was established as 1,3,5-trihydroxy-24-methylenecholestan-6-one.
The relative stereochemistry of 1 was determined on the basis of the NOESY experiment for 1 (Fig. 3), and the ob-served pyridine-induced solvent shifts[11]. In the NOESY spectrum of 1, the axially oriented H-1 (δ 4.25, dd, J = 13.0,
Fig. 3. Observed NOESY correlations for 1 (R= H; solid arrows) and 6 (R = Ac; dashed and solid arrows). Table 2
13C NMR data for ring A and B carbons of 1 and 7
C 1a 7b 1 72.5 (d)c 73.2 (d) 2 37.7 (t) 41.3 (t) 3 66.7 (d) 61.6 (d) 4 36.6 (t) 39.0 (t) 5 83.6 (s) 81.6 (s) 6 210.8 (s) 209.9 (s) 7 41.7 (t) 36.0 (t) 8 37.1 (d) 36.4 (d) 9 42.1 (d) 39.3 (d) 10 48.4 (s) 44.3 (s)
aSpectra recorded at 75 MHz in DMSO-d6 at 25◦C.
bSpectra recorded at 100 MHz in DMSO-d6 at 25◦C (see ref.[5]).
cMultiplicity deduced by DEPT and indicated by usual symbols. The values are in ppm downfield from TMS.
4.0 Hz) exhibited a significant NOE with H3-19, suggesting
the␣-orientation of C-1 hydroxy group. The -configuration of C-3 hydroxy group was deduced owing to the lack of NOE correlations between H-3 and H-2, which showed NOE interaction with H-1. Careful investigation on the NOESY spectrum of the diacetate 6, obtained from the acetylation of
1, revealed significant NOE correlations between H-3 and
both of H-2␣ and H-4␣, and between H-4␣ and H-9, and fur-ther confirmed the above results. Furfur-thermore, the chemical shifts of carbons 2–5, 7, and 9–10 of 1 showed significant differences in comparison with the corresponding chemical shifts of 1␣,3,5␣-trihydroxy-24-methylenecholestan-6-one (7) [5](Table 2). Thus, 1 should be the 5-epimer of 7. On the basis of the above results, the configuration of 1 could be established as shown inFig. 2, reflecting the␣-equatorial orientation of 1-OH, and the-axial orientations of 3-OH and 5-OH. Moreover, the large pyridine-induced downfield shifts (δ = δCDCl3− δC6D5N; seeTable 3) experienced
upon H-1 (δ = −0.64 ppm) could be achieved only when both 3-OH and 5-OH are axially oriented on the same face of H-1[11]. Finally, the␣-downward orientation of ring A re-sulted from the-orientation of 5-OH was further interpreted by the NOE interactions, observed between H-4␣ (δ 2.34, dd,J = 14.5, 3.5 Hz) and both H-9 (δ 1.99, dd, J = 11.0, 5.0 Hz) and H-7␣ (δ 2.24, t, J = 14.5 Hz). Therefore, the
Table 3
Selected1H NMR chemical shifts of compounds 1 and 7 in CDCl3 and pyridine-d5
H δH in CDCl3 δHin C5 D5N 1a 7b 1c 7b H-1 4.25 dd (13.0, 4.0)d 4.00 brs 4.89 dd (14.0, 4.0) 4.19 brs H-3 4.25 brs 4.33 m 4.48 brs 5.4–5.6 m 18-Me 0.66 3H, s 0.69 3H, s 0.66 3H, s 0.63 3H, s 19-Me 0.97 3H, s 0.74 3H, s 1.39 3H, s 0.88 3H, s aSpectra recorded at 500 MHz at 25◦C.
bSpectra recorded at 400 MHz at 25◦C (see ref.[5]). cSpectra recorded at 300 MHz at 25◦C.
dJ values (in Hz) in parentheses.
structure of gibberoketosterol (1) could be established unam-biguously as 1 ␣,3,5-trihydroxy-24-methylenecholestan-6-one.
The known steroids 2, 3, 4, and 5 were also isolated from the lipophilic fractions of S. gibberosa and identified by comparison of their physical characters (mp and [α]D) and
spectral data with those of 3 -hydroxy-24-methylenecholest-5-en-7-one [12,13], 24-methylenecholesterol [14], preg-nenolone[6], and numersterol[4], respectively.
Although the ketosteroid 2 was previously isolated from two sponge species [12,13] and a higher plant [15], it is worthwhile to mention that this is the first time of isolating this metabolite from a coral. In addition, steroids con-taining 1␣,3,5-trihydroxy-6-oxo functionality have not been found before. Thus, gibberoketosterol (1) represents the first example of the natural 1 ␣,3,5-trihydroxy-6-oxosteroids.
The cytotoxicity of metabolites 1, 4, and 5 against the growth of Hepa59T/VGH cancer cells was evaluated and the results showed that compounds 1, 4, and 5 possess moderate cytotoxicity against this cell line with ED50’s 10.0, 9.3, and
6.8g/ml, respectively.
Acknowledgments
This work was supported by a grant from the National Science Council of the Republic of China (Contract No. NSC-89-2323-B-110-002) awarded to J.-H. Sheu.
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