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Sinugrandisterols A–D, trihydroxysteroids from the soft coral Sinularia grandilobata
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Sinugrandisterols A–D, trihydroxysteroids from the soft coral Sinularia grandilobata Atallah F. Ahmed a,b , Shu-Hui Tai a , Yang-Chang Wu c , Jyh-Horng Sheu a,d,∗ a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt c Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Asian Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan b
a r t i c l e
i n f o
Article history:
a b s t r a c t Four new trihydroxysteroids, sinugrandisterols A–D (1–4), have been isolated from the
Received 20 October 2006
CH2 Cl2 -soluble fraction of the EtOH extract of Sinularia grandilobata. The structures of these
Received in revised form
metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR)
22 December 2006
analysis. The cytotoxicity of 1–4 toward a limited panel of cancer cell lines is also reported.
Accepted 8 January 2007
© 2007 Elsevier Inc. All rights reserved.
Published on line 16 January 2007 Keywords: Trihydroxysteroids Sinularia grandilobata Cytotoxicity
1.
Introduction
Marine organisms, including octocorals (Coelenterata), have been shown to be a rich source of a variety of polyoxygenated steroids [1–12]. A few of these sterols have been found to possess an 5,6-epoxide [3–5]. Chemical investigations on the steroidal constituents of soft corals of the genus Sinularia have afforded various polyhydroxylated steroids as derivatives of 24-methyl- and 24-methylene-cholestane-3-ol [1,2,6–13], and their glycosides [12,13]. Some of these compounds exhibited in vitro cytotoxic activity toward various cancer cell lines [2] and potent inhibition for histamine release from the mast cells [8]. Our current chemical investigation on Sinularia grandilobata Verseveldt (Alcyonacea) has also led to the isolation of four new trihydroxylated sterols, sinu-
grandisterols A–D (1–4). The structures of these metabolites were identified as 24-methylenecholest-5-en-1␣,3,7-triol (1), 24-methylenecholesta-5,22E-dien-1␣,3,7-triol (2), 5,6epoxy-24-methylenecholesta -1␣,3,7-triol (3), and 5,6epoxy-24-methylenecholesta-22E-en-1␣,3,7-triol (4), on the basis of the spectroscopic (IR, MS, and 1D and 2D NMR) analysis. The NOESY correlations, NMR data comparison with related steroids, pyridine-induced solvent shift, and acetylation reaction were also utilized to confirm the number, positions, and orientations of the hydroxy substituents on rings A and B of these new steroids. The cytotoxic activity of metabolites 1–4 against HepG2 (human liver carcinoma), MCF7, MDA-MB-23 (human breast carcinomas), and A-549 (human lung carcinoma) cells also is reported herein.
∗ Corresponding author at: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan. Tel.: +886 7 5252000x5030; fax: +886 7 5255020. E-mail address: [email protected] (J.-H. Sheu). 0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2007.01.001
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s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
2.
Experimental
2.1.
General procedures
2.2.
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 Hitachi I-2001 infrared spectrophotometer. NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR or on a Bruker AVANCE 500 FT-NMR at 500 MHz for 1 H and 125 MHz for 13 C or Bruker AVANCE 300 FT-NMR at 300 MHz for 1 H and 75 MHz for 13 C, in CDCl , unless otherwise stated. Low-resolution mass 3 spectral data were obtained by EI or ESI with a VG QUATTRO GC/MS spectrometer. HRMS were recorded by ESI FT-MS on a BRUKER APEX II mass spectrometer. Silica gel (Merck, 230–400 mesh) was used for open column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. Isolation by HPLC was performed by Shimadzu SPD-10A instrument equipped with a normal phase column (Hibar Lichrosorb Si-60, 7 m, 250 mm × 25 mm) or a reversed phase column (Hibar Purospher RP-18e, 5 m, 250 mm × 10 mm).
Animal material
The soft coral S. grandilobata was collected by hand using SCUBA off the coast of Kenting, Taiwan, in June 2004, at depths of 15–20 m and stored in a freezer until extraction. A voucher sample (SC-62) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
2.3.
Extraction and isolation procedure
The frozen bodies of S. grandilobata (0.9 kg, wet weight) were sliced and exhaustively extracted with EtOH (1 L × 5). The EtOH extract was filtered and concentrated under reduced pressure. The residue was partitioned between CH2 Cl2 and H2 O to afford the CH2 Cl2 fraction (30 g). The dichloromethane fraction was chromatographed by Si gel by CC and eluted with EtOAc in n-hexane in (0–100%, gradient) then with MeOH in EtOAc (5–50%, gradient) to yield 17 fractions. The eluted fractions were monitored by 1 H NMR spectroscopy. Fraction 12 (0.2 g), eluted by EtOAc–n-hexane (1:1), was further purified primarily by Si gel CC using acetone–EtOAc–n-hexane (1:1:5) to give sub-
Table 1 – 1 H NMR data for sterols 1–4 H#
1a
2a
1 2␣ 2 3 4␣ 4 6 7 8 9 11␣ 11 12␣ 12 14 15␣ 15 16␣ 16 17 18 19 20 21 22
3.88 br s 2.07 m 1.75 m 4.03 m 2.40 dd (12.0, 4.0)c 2.31 dd (12.0, 12.0) 5.48 s 3.90 m 1.45 m 1.66 m 1.50 m 1.47 m 1.18 m 2.03 br d (14.0)c 1.22 m 1.85 m 1.43 m 1.90 m 1.31 m 1.14 m 0.70 3H, s 1.06 3H, s 1.42 m 0.95 3H, d (6.3) 1.16 m 1.54 m 1.88 m 2.08 m 2.23 septet (6.6) 1.03 3H, d (6.6) 1.03 3H, d (6.6) 4.66 s; 4.72 s
3.88 br s 2.09 m 1.77 m 4.03 m 2.43 dd (12.9, 4.5) 2.31 dd (12.9, 12.9) 5.50 s 3.90 m 1.43 m 1.68 m 1.52 m 1.49 m 1.22 m 2.00 br d (12.6) 1.37 m 1.77 m 1.43 m 1.73 m 1.33 m 1.19 m 0.73 3H, s 1.07 3H, s 2.15 m 1.07 3H, d (6.3) 5.58 dd (15.8, 8.1)
23 25 26 27 28 a b c
Spectra recorded at 300 MHz. 500 MHz in CDCl3 at 25 ◦ C. The J values are in Hz in parentheses.
5.95 d (15.8) 2.55 septet (6.9) 1.07 3H, d (6.9) 1.08 3H, d (6.9) 4.83 s; 4.85 s
3b 3.92 br s 2.08 m 1.74 ddd (13.0, 10.5, 2.0) 4.23 m 1.66 dd (13.5, 5.0) 2.02 dd (13.5, 10.5) 3.18 s 3.57 dd (8.5, 8.5) 1.43 m 1.63 m 1.44 m 1.33 m 1.10 m 1.96 m 1.15 m 1.96 m 1.37 m 1.89 m 1.29 m 1.08 m 0.66 3H, s 1.01 3H, s 1.40 m 0.94 3H, d (6.0) 1.14 m 1.53 m 1.87 ddd (15.0, 10.0, 5.0) 2.09 m 2.21 septet (7.0) 1.03 3H, d (7.0) 1.03 3H, d (7.0) 4.65 s; 4.71 s
4a 3.92 br s 2.09 dd (14.0, 5.0) 1.74 br dd (14.0, 14.0) 4.23 m 1.66 m 2.02 m 3.18 s 3.57 d (8.1) 1.44 m 1.66 m 1.44 m 1.33 m 1.16 m 1.94 m 1.20 m 1.90 m 1.25 m 1.67 m 1.30 m 1.15 m 0.69 3H, s 1.02 3H, s 2.18 m 1.03 3H, d (6.3) 5.58 dd (15.8, 8.7) 5.94 d (15.6) 2.55 septet (6.6) 1.07 3H, d (6.9) 1.08 3H, d (6.9) 4.83 s; 4.85 s
370
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
fractions A and B. Subfraction A was re-chromatographed by RP-18 HPLC using MeOH–H2 O (9:1) to afford subfractions A1 and A2. Subfraction A1 was purified by normal phase HPLC using CH2 Cl2 –MeOH (25:10) to yield 2 (1.8 mg). Subfraction A2 was also purified by normal phase HPLC using acetone–nhexane (1:2) to afford 1 (12.9 mg). Subfraction B was also re-chromatographed by RP-18 HPLC using MeOH–H2 O (9:1) to give crude steroid fractions B1 and B2, which were purified separately by normal phase HPLC using acetone–n-hexane (2:3) to afford 4 (0.9 mg) and 3 (2.7 mg), respectively.
2.3.1.
Sinugrandisterol A (1)
White powder; mp 140–142 ◦ C; Rf = 0.56 (Si gel, acetone– hexane, 1:1); [␣]D 25 –8 (c 1.2, CHCl3 ); IR (neat) max 3355, 2959, 2930, 2866, 1644, 1464, 1379, 1261 and 1055 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; 1 H NMR data (300 MHz, C5 D5 N) ı 6.26 (1H, br s, 3-OH), 6.02 (1H, br s, 1-OH), 5.91 (1H, s, H-6), 5.61 (1H, br s, 7-OH), 4.83 (1H, s, H28), 4.81 (1H, s, H-28), 4.76 (1H, m, H-3), 4.20 (1H, m, H-7), 4.14 (1H, br s, H-1), 2.78 (1H, m, H-4), 2.76 (1H, m, H-4), 2.60 (1H, br d, J = 11.4 Hz, H-2), 2.37 (1H, ddd, J = 11.7, 11.7, 3.0 Hz, H-9), 2.25 (2H, m, H-15 and H-23), 2.20 (1H, m, H-25), 2.15 (1H, br dd, J = 11.4, 11.4 Hz, H-2), 1.97 (1H, m, H-23), 1.95 (1H, m, H-12), 1.89 (1H, m, H-16), 1.81 (1H, m, H-11), 1.77 (2H, m, H-8 and H-15), 1.61 (1H, m, H-22), 1.46 (2H, m, H-11 and H-20), 1.36 (1H, m, H-14), 1.25 (1H, m, H-16), 1.18 (1H, m, H-22), 1.17 (1H, m, H-12),
1.10 (3H, s, H3 -19), 1.09 (1H, m, H-17), 1.03 (6H, d, J = 6.6 Hz, H3 26 and H3 -27), 0.95 (3H, d, J = 6.3 Hz, H3 -21), 0.74 (3H, s, H3 -18); 13 C NMR data (75 MHz, C D N) ı 157.0 (qC, C-24), 141.0 (qC, C5 5 5), 130.7 (CH, C-6), 106.9 (CH2 , C-28), 73.3 (CH, C-7), 72.8 (CH, C-1), 66.2 (CH, C-3), 57.3 (CH, C-14), 56.1 (CH, C-17), 43.5 (qC, C-13), 43.0 (CH2 , C-4), 42.3 (qC, C-10), 41.4 (CH, C-8), 40.6 (CH2 , C-2), 40.4 (CH, C-9), 40.2 (CH2 , C-12), 36.3 (CH, C-20), 35.4 (CH2 , C-22), 34.4 (CH, C-25), 31.6 (CH2 , C-23), 29.3 (CH2 , C-16), 27.7 (CH2 , C-15), 22.3 and 22.4 (each CH3 , C-26 and C-27), 21.1 (CH2 , C-11), 19.7 (CH3 , C-19), 19.3 (CH3 , C-21), 12.5 (CH3 , C-18); EIMS m/z 430 (1.2, [M]+ ), 412 (23.9, [M − H2 O]+ ), 394 (3.1, [M − 2H2 O]+ ), 376 (0.6, [M − 3H2 O]+ ), 213 (3.6), 173 (9.9), 161 (11.9), 145 (13.4), 131 (14.9), 121 (15.9), 105 (27.7); HRESIMS m/z 453.3342 [M + Na]+ (calcd for C28 H46 O3 Na, 453.3344).
2.3.2.
2.3.3.
Table 2 – 13 C NMR data for sterols 1–4 C# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a b c
1a 72.6 (CH)c 38.3 (CH2 ) 66.0 (CH) 40.9 (CH2 ) 140.0 (qC) 129.0 (CH) 73.0 (CH) 40.8 (CH) 39.5 (CH) 41.4 (qC) 20.2 (CH2 ) 39.3 (CH2 ) 43.0 (qC) 55.8 (CH) 26.6 (CH2 ) 28.5 (CH2 ) 55.3 (CH) 11.8 (CH3 ) 19.2 (CH3 ) 35.7 (CH) 18.8 (CH3 ) 34.7 (CH2 ) 31.0 (CH2 ) 156.8 (qC) 33.8 (CH) 21.9 (CH3 ) 22.0 (CH3 ) 106.0 (CH2 )
2a 72.7 (CH) 38.4 (CH2 ) 66.1 (CH) 40.9 (CH2 ) 140.0 (qC) 129.1 (CH) 73.1 (CH) 40.8 (CH) 39.6 (CH) 41.5 (qC) 20.2 (CH2 ) 39.2 (CH2 ) 43.0 (qC) 55.8 (CH) 26.6 (CH2 ) 28.6 (CH2 ) 55.3 (CH) 12.3 (CH3 ) 19.2 (CH3 ) 40.2 (CH) 20.6 (CH3 ) 135.8 (CH) 129.3 (CH) 153.0 (qC) 29.4 (CH) 22.1 (CH3 ) 22.4 (CH3 ) 109.7 (CH2 )
3b 72.5 (CH) 38.0 (CH2 ) 63.9 (CH) 41.2 (CH2 ) 66.2 (qC) 67.8 (CH) 74.6 (CH) 37.7 (CH) 39.9 (CH) 38.9 (qC) 21.1 (CH2 ) 39.4 (CH2 ) 43.1 (qC) 55.3 (CH) 27.3 (CH2 ) 28.5 (CH2 ) 55.1 (CH) 11.7 (CH3 ) 16.5 (CH3 ) 35.6 (CH) 18.8 (CH3 ) 34.6 (CH2 ) 31.0 (CH2 ) 156.8 (qC) 33.8 (CH) 21.8 (CH3 ) 22.0 (CH3 ) 106.0 (CH2 )
4a 72.5 (CH) 38.0 (CH2 ) 63.9 (CH) 41.2 (CH2 ) 66.2 (qC) 67.8 (CH) 74.6 (CH) 37.7 (CH) 39.9 (CH) 39.0 (qC) 21.1 (CH2 ) 39.3 (CH2 ) 43.1 (qC) 55.3 (CH) 27.3 (CH2 ) 28.6 (CH2 ) 55.1 (CH) 12.0 (CH3 ) 16.5 (CH3 ) 40.2 (CH) 20.7 (CH3 ) 135.8 (CH2 ) 129.3 (CH2 ) 153.0 (qC) 29.3 (CH) 22.1 (CH3 ) 22.4 (CH3 ) 109.7 (CH2 )
Spectra recorded at 75 MHz. 125 MHz in CDCl3 at 25 ◦ C. Attached protons were determined by DEPT experiments.
Sinugrandisterol B (2)
White powder; mp 108–110 ◦ C; Rf = 0.53 (Si gel, acetone– hexane, 1:1); [␣]D 25 −6 (c 1.0, CHCl3 ); IR (neat) max 3356, 2959, 2930, 2870, 1653, 1458, 1373, 1125 and 1053 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; EIMS m/z 428 (1.0, [M]+ ), 410 (5.7, [M − H2 O]+ ), 395 (3.5, [M − H2 O − Me]+ ), 392 (1.2, [M − 2H2 O]+ ), 314 (11.7), 303 (41.1), 285 (7.3), 249 (11.2), 173 (11.3), 145 (13.0), 133 (15.0), 123 (51.2), 105 (40.8); ESIMS m/z 451 [M + Na]+ ; HRESIMS m/z 451.3191 [M + Na]+ (calcd for C28 H44 O3 Na, 451.3188).
Sinugrandisterol C (3)
White powder; mp 222–224 ◦ C; Rf = 0.47 (Si gel, acetone– hexane, 1:1); [␣]D 25 + 46 (c 1.1, CHCl3 ); IR (neat) max 3361, 2959, 2930, 2866, 1647, 1458, 1375,1161 and 1059 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; 1 H NMR data (300 MHz, C5 D5 N) ı 6.58 (1H, d, J = 3.9 Hz, 1-OH), 6.42 (1H, br s, 3-OH), 6.16 (1H, d, J = 8.0 Hz, 7-OH), 4.97 (1H, m, H-3), 4.82 and 4.80 (each 1H, s, H2 -28), 4.18 (1H, br s, H-1), 4.00 (1H, br dd, J = 8.0, 8.0 Hz, H-7), 3.61 (1H, s, H-6), 2.64 (1H, dd, J = 12.0, 12.0 Hz, H-4), 2.62 (1H, m, H-2), 2.10 (1H, m, H-4), 2.40 (1H, m, H-15), 2.39 (1H, ddd, J = 11.4, 11.4, 5.0 Hz, H-9), 2.23 (1H, septet, J = 6.6 Hz, H-25), 2.12 (1H, m, H-23), 2.08 (1H, m, H-2), 1.99 (1H, m, H-23), 1.95 (1H, m, H-8), 1.89 (1H, m, H-12), 1.78 (1H, m, H16), 1.71 (1H, m, H-15), 1.56 (2H, m, H-11 and H-22), 1.46 (1H, m, H-11), 1.31 (1H, m, H-20), 1.29 (1H, m, H-14), 1.26 (1H, m, H-16), 1.15 (1H, m, H-22), 1.03 (1H, m, H-17), 1.02 (6H, d, J = 6.6 Hz, H3 26 and H3 -27), 1.01 (1H, m, H-12), 1.23 (3H, s, H3 -19), 0.92 (3H, d, J = 6.6 Hz, H3 -21), 0.64 (3H, s, H3 -18); 13 C NMR data (75 MHz, C5 D5 N) ı 157.0 (qC, C-24), 107.0 (CH2 , C-28), 75.0 (CH, C-7), 72.5 (CH, C-1), 70.2 (CH, C-6), 66.7 (qC, C-5), 64.1 (CH, C-3), 56.7 (CH, C-14), 55.8 (CH, C-17), 43.7 (CH2 , C-4), 43.5 (qC, C-13), 40.8 (CH, C-9), 40.3 (CH2 , C-12), 40.1 (qC, C-10), 39.8 (CH2 , C-2), 38.0 (CH, C-8), 36.3 (CH, C-20), 35.3 (CH2 , C-22), 34.3 (CH, C-25), 31.6 (CH2 , C-23), 29.3 (CH2 , C-16), 28.4 (CH2 , C-15), 22.4 and 22.3 (each CH3 , C-26 and C-27), 22.2 (CH2 , C-11), 19.4 (CH3 , C-21), 17.6 (CH3 , C19), 12.3 (CH3 , C-18); ESIMS m/z 469 [M + Na]+ ; HRESIMS m/z 469.3291 [M + Na]+ (calcd for C28 H46 O4 Na, 469.3294).
2.3.4.
Acetylation of 3
A solution of sinugrandisterol C (3) (1.1 mg, 0.0025 mmol) in pyridine (0.2 mL) was mixed with Ac2 O (0.2 mL) and the mixture was stirred at RT for 24 h. After evaporation of excess reagent, the residue was purified by normal phase HPLC, using Acetone–n-hexane = 1:3, to give a triacetyl derivative 3a
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
(1.4 mg, 0.0024 mmol, 96% yield). 3a: colorless gum; [␣]D 25 + 21 (c 0.7, CHCl3 ); IR (neat) max 2959, 2925, 2860, 1738, 1640, 1373 and 1238 cm−1 ; 1 H NMR (500 MHz, CDCl3 , selected data) ı 5.13 (1H, br s, H-1), 5.06 (1H, m, H-3), 4.82 (1H, d, J = 9.5 Hz, H-7), 4.71 (1H, H-28), 4.64 (1H, H-28), 3.22 (1H, s, H-6), 2.21 (1H, septet, J = 7.0 Hz, H-25), 2.12, 2.09, and 2.02 (each 3H, s, 3 OAc), 1.80 (1H, m, H-8), 1.11 (3H, s, H3 -19), 1.03 and 1.01 (each 3H, d, J = 7.0 Hz, H3 -26 and H3 -27), 0.91 (3H, d, J = 7.0 Hz, H3 -21), 0.65 (3H, s, H3 -18); ESIMS m/z 595 [M + Na]+ .
2.3.5.
Sinugrandisterol D (4)
White powder; mp 205–207 ◦ C; Rf = 0.44 (Si gel, acetone– hexane, 1:1); [␣]D 25 + 17 (c 0.4, CHCl3 ); IR (neat) max 3362, 2959, 2928, 2868, 1653, 1458, 1375 and 1055 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; ESIMS m/z 467 [M + Na]+ ; HRESIMS m/z 467.3135 [M + Na]+ (calcd for C28 H44 O4 Na, 467.3137).
2.4.
Cytotoxicity testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of the test compounds 1–4 were performed using the MTT [3-(4,5-dimethylthiazole-
371
2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [14,15].
3.
Results and discussion
The sliced bodies of the soft corals S. grandilobata were exhaustively extracted with EtOH. The EtOH extract was concentrated and the residue was partitioned between CH2 Cl2 and H2 O. The combined CH2 Cl2 –soluble fraction was concentrated and the residue was chromatographed on Si gel CC followed by normal and reversed phase HPLC to yield sterols 1–4 (see Section 2). Sinugrandisterol A (1) was obtained as a white powder and possessed a molecular formula C28 H46 O3 as determined by HRESIMS (453.3342 m/z, [M + Na]+ ), implying six degrees of unsaturation. The IR absorptions at max 3355 and 1644 cm−1 suggested the presence of hydroxy and olefinic functionalities. Three hydroxyls in the molecule were estimated from the ion peaks appearing at m/z 412 (M − H2 O)+ , 394 (M − 2H2 O)+ , and 376 (M − 3H2 O)+ in the EIMS spectrum, and from the NMR signals resonating (in CDCl3 , Tables 2 and 1) at ıC 73.0, 72.6, and 66.0 (each CH) and ıH 3.90 (1H, m), 3.88 (1H, br s), and 4.03 (1H, m), respectively. Measurement of the 1 H NMR spec-
Fig. 1 – 1 H–1 H COSY and HMBC correlations for 1 (C5 D5 N), 2 (C5 D1 N), and 3 (CDCl3 ).
372
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
Fig. 1 – (Continued ).
trum of 1 in C5 D5 N also exhibited three D2 O–exchangeable OH signals at ı 6.26, 6.02, and 5.61 (each 1H, br s). Furthermore, an exomethylene group and a trisubstituted double bond (ıC 156.8, qC, 140.0, qC, 129.0, CH and 106.0, CH2 ; ıH 5.48, 4.72 and 4.66, each 1H, s) were also indicated in the molecule of 1. Moreover, the 1 H NMR spectrum exhibited two singlets at ı 1.06 and 0.70 (each 3H, s) and two doublets at ı 1.03 (6H, d, J = 6.6 Hz) and 0.95 (3H, d, J = 6.3 Hz) attributable to two tertiary and three secondary methyls. These findings together with the 28 carbon signals appearing in the 13 C NMR spectrum (Table 2) suggested 1 as a trihydroxylated methylenecholestene. By the analysis of 1 H–1 H COSY and HMBC correlations (in C5 D5 N) the planar structure of compound 1 was unambiguously established as illustrated in Fig. 1, where the positions of three hydroxy groups and the two double bonds were determined to be at C-1, C-3, C-7, C-5/C-6, and C-24/C-28, respectively. The NOESY correlations, NMR data comparison, and pyridine-induced solvent shift were successively employed to establish the stereochemistry at C-1, C-3, and C-7. The hydroxy groups at C-1 and C-3 were determined to have the ␣ and  orientations, respectively, on the basis of strong NOE interactions of H3 -19 (ı 1.06, s) with H-1 (ı 3.88, br s), H3 -19 with H-4 (ı 2.31, dd, J = 12.0, 12.0 Hz), and H-4␣ (ı 2.40, dd, J = 12.0, 4.0 Hz) with H-3 (ı 4.03, m) (Fig. 2). The ␣ orientation of 1-OH was further confirmed by measuring the 1 H NMR spectrum of 1 in
Fig. 2 – Key NOESY correlations for 1–3.
373
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
C5 D5 N (see Section 2) where large pyridine-induced downfield shifts (ı = ıCDCl3 − ıC5 D5 N) [2,16] were observed for H-3 (ı = −0.73 ppm) and H-9 (ı = −0.71 ppm) due to the 1,3diaxial deshielding effect exerted by 1-OH in pyridine [16]. The significant NOE correlations displayed by H-7 (ı 3.90, m) with both H-9 (ı 1.66, m) and H-14 (ı 1.22, m), but not with H-8, revealed the  orientation of the 7-OH. This was supported by the quite similar chemical shifts of H-7 (ı 3.90, m), C-7 (ı 73.0, CH), and C-8 (ı 40.8, CH) of 1 with those of isodecortinol (5) [17] (ı 3.83, 73.0 and 40.8, respectively), measured in CDCl3 . Furthermore, the pyridine-induced downfield shift for the  oriented H-8 (ı = −0.32 ppm) due to the vicinal deshielding effect [16] by 7-OH in pyridine provided an additional evidence for the -orientation of the hydroxy function at C-7. On the basis of above findings, the structure of sinugrandisterol A (1) was unambiguously established as 24-methylenecholesta-5en-1␣,3,7-triol. Sinugrandisterol B (2) was also obtained as a white powder and exhibited a quasimolecular ion peak at m/z 451.3191 (M + Na)+ in the HRESIMS, appropriate for a molecular formula of C28 H44 O3 . It was found that the IR, MS, and 13 C NMR spectral data of 2 (Table 2) were very similar to those of 1, except the replacement of the carbon signals of an ethylene moiety (ı 34.7 and 31.0, each CH2 ) in 1 by a 1,2-disubstituted double bond (ı 135.8 and 129.3, each CH) in 2. This was also evidenced by the two olefinic proton signals appearing at ı 5.95 (d, J = 15.8 Hz) and 5.58, dd, J = 15.8, 8.1 Hz) in the 1 H NMR spectrum of 2. The 1 H–1 H COSY correlations (Fig. 1) observed between the latter proton and H-20 (ı 2.15, m), and between H-20 and H3 21 (ı 1.07, d, J = 6.3 Hz) indicated the C-22 and C-23 position of the double bond. This was further supported by the 1 H–1 H COSY correlation between H-22 and H-23 (ı 5.95) and by the HMBC correlations (Fig. 1) found from the exomethylene protons H2 -28 (ı 4.85 and 4.83, each 1H, s) to C-23 (ı 129.3, CH). The very similar NMR data (Tables 2 and 1) and NOESY correlations (Fig. 2) observed for protons within rings A-D in both 1 and 2 reflected the ␣, , and  orientations of the hydroxyls at C-1, C3, and C-7 in 2, respectively. Moreover, the coupling constant (J = 15.8 Hz) between H-22 and H-23, and the NOE interactions of H-23 with both H-20 and H-28 (ı 4.85, s), and H-22 with H-25 (ı 2.55, septet, J = 6.9 Hz) confirmed the E geometry of the 1,2disubstituted double bond in the side chain. These findings and other 2D NMR correlations (Figs. 1 and 2) established the structure of sinugrandisterol B (2) as 24-methylenecholesta5,22E-dien-1␣,3,7-triol.
Sinugrandisterol (3) was found to be more polar than 2 (see Section 2) and had a molecular formula C28 H46 O4 as determined by its HRESIMS (469.3291 m/z, [M + Na]+ ) and NMR data (Tables 1 and 2, and Section 2). The IR spectrum (max 3361 cm−1 ) indicated the presence of hydroxy functionality in 3. The presence of three hydroxy groups was supported by the acetylation of 3 to yield 3a, which exhibited three 3H singlet of acetate methyls in the 1 H NMR spectrum (ı 2.12, 2.09, and 2.02). Comparison of NMR data of compound 3 with those of 1 (Tables 1 and 2) indicated that 3 is another 24-methylenecholestane-triol. As in case of 1, measuring the 1 H NMR spectrum of 3 in C D N (see Section 2) showed three 5 5 D2 O-exchangeable hydoxyl protons at ı 6.58 (1H, d, J = 3.5 Hz, 1-OH), 6.42 (1H, br s, 3-OH), and 6.16 (1H, d, J = 8.0 Hz, 7-OH). Thus, the remaining fourth oxygen atom in the molecular formula of 3 was suggested to be incorporated in an epoxide (ıC 66.2, qC and 67.8, CH; ıH 3.18, 1H, s), which arised from the epoxidation of the trisubstituted double bond (ıC 140.0, qC and 129.0, CH; ıH 5.48, 1H, s) of 1. The C-5/C-6 location of this epoxide was proven by the HMBC correlations (Fig. 1) from the epoxymethine proton (ı 3.18, 1H, s) to C-7 (ı 74.6, CH), C-8 (ı 37.7, CH), C-10 (ı 38.9, CH), and the quaternary oxycarbon of the epoxide (ı 66.2, qC), while the latter carbon was found to be correlated with H2 -4 (ı 1.66, dd, J = 13.5, 5.0 Hz and 2.02, dd, J = 13.5, 10.5 Hz) and H3 -19 (ı 1.01, s). Also, the three hydroxyls were determined to be situated at C-1, C-3, and C-7 on the basis of correlations displayed in the 1 H–1 H COSY and HMBC spectra of 3 (Fig. 1). The similarity of the splitting patterns of H-1 and H-3 in compound 3 with those in 1 and 2 revealed the ␣ and  orientations of the hydroxy groups at C-1 and C-3, respectively. The ␣ orientation of 1-OH was further confirmed by the NOE interaction displayed by the  oriented H3 -19 with H-1. The large pyridine-induced downfield shift induced at H-3 (ı = −0.74 ppm) was due to the 1,3-diaxial deshielding effect exerted by 1-OH in pyridine, as observed in the case of 1, further confirming the ␣ and  orientations of 1-OH and 3-OH, respectively. Moreover, the  orientation of 7-OH was concluded from the splitting pattern and J value (d, ∼8.0 Hz) of H-7 with H-8 in CDCl3 (Table 2) and in C5 D5 N (after addition of D2 O), or in CDCl3 after acetylation (see Section 2). This was further supported by the NOE interactions observed for H-7 with the ␣ oriented H-9 (ı 1.63, m) and H-14 (ı 1.15, m), but not with the  oriented H-8 (ı 1.43, m). Furthermore, the C5/C6–epoxide was thought to possess the  orientated oxygen on the basis of NOE correlations (Fig. 2) displayed by the
Table 3 – Selected NMR data for sterols 3, 6 [4] and 7 [5] #
4 5 6 7 8 a b c
3 (CDCl3 )a
6 (CDCl3 )b
ıH
ıC
3.18 s 3.57 dd (8.5, 8.5) 1.43 m
41.2 66.2 67.8 74.6 37.7
3 (C5 D5 N)a
ıH
ıC
3.14 d (1.2) 3.51 m 1.43 m
41.7 67.3 67.5 74.7 38.3
Spectra recorded at 300 MHz for 1 H and 75 MHz for 13 C. 500 MHz for 1 H and 125 MHz for 13 C (Ref. [4]). 400 MHz for 1 H and 100 MHz for 13 C (Ref. [5]).
7 (C5 D5 N)c
ıH
ıC
3.61 s 4.00 br dd (8.0, 8.0) 1.95 m
43.7 66.7 70.2 75.0 38.0
ıH
ıC
3.47 s 3.79 dd (8.0, 7.6) 1.87 m
43.3 66.8 69.1 74.6 38.1
374
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
references
Table 4 – Cytotoxicity (IC50 g/mL) of sterols 1–4 Hep G2 1 2 3 4 Doxorubicin a b
9.1 6.8 –b –a 0.40
MCF-7 –a 19.3 18.9 –a 0.13
MDA-MB-231 9.2 –b –b –a 0.25
A549 17.2 16.9 18.9 –a 0.2
Compound is considered inactive when IC50 > 20 g/mL. Not tested.
epoxide proton H-6 (ı 3.18, s) with H-7 (ı 3.57, dd, J = 8.5, 8.5 Hz) and H-4␣ (ı 1.66, dd, J = 13.5, 5.0 Hz). Comparison of the NMR data of the C-4–C-8 moiety in rings A and B of 3 with those of the known marine sterols 6 [4] and 7 [5] (Table 3) showed close similarity and further confirmed the 5, 6 orientation of the epoxy oxygen as well as the  orientation of the hydroxyl at C-7. On the basis of above results, together with the detailed analysis of 2D NMR spectra of 3 (Figs. 1 and 2), the structure of sinugrandisterol C (3) was unambiguously established as 5,6-epoxy-24-methylenecholestane-1␣,3,7triol. The most polar compound, sinugrandisterol D (4), was determined to have a molecular formula of C28 H44 O4 by the quasimolecular ion peak at m/z 467.3135 (M + Na)+ in the HRESIMS and NMR data (Tables 2 and 1). Its IR spectrum (max 3362 cm−1 ) also revealed the presence of hydroxy group in the molecule. Moreover, it was found that the NMR data of A–D rings (C-1 to C-19 and H-1 to H3 -19) of 4 were quite similar to those of 3, indicating the same substitution and stereochemistry at C-1, C-3, C-5, C-6, and C-7. Also, the NMR data of the side chain (C-20 to C-28 and H-20 to H2 -28) of 4 were found to be almost the same as those of 2. On the basis of above findings, together with the analysis of the NOE correlations of 4, the structure of sinugrandisterol D (4) was determined as 5,6-epoxy-24-methylenecholest-22E-en-1␣,3,7triol. The cytotoxicity of compounds 1–4 against the growth of cancer cell lines of Hep G2, MCF-7, MDA-MB-231, and A-549 were also studied. The results (Table 4) showed that the 5,6 sterols 1 and 2 showed moderate cytotoxicity against the growth of Hep G2 and MDA-MB-231 cells (IC50 6.8–9.2 g/mL). Compounds 2 and 3 exhibited weak activity against A549 and MCF-7cancer cells (IC50 16.9 and 19.3 g/mL), while 1 was inactive against MCF-7 (IC50 > 20 g/mL) and showed only weakly cytotoxic activity against A549. Compound 4 showed no cytotoxicity against the tested cell lines.
Acknowledgments Financial support was provided by Ministry of Education (C030313) and National Science Council of Taiwan (NSC 952323-B-110-002) awarded to J.-H. Sheu.
[1] Blunt JW, Copp BR, Munro MHG, Northcote PT, Prinsep MR. Marine natural products. Nat Prod Rep 2006;23:26-L78, and previous reports in this series. [2] Ahmed AF, Dai CF, Kuo YH, Sheu JH. 1␣,3,5-Trihydroxy-24-methylenecholestan-6-one: a novel steroid from a soft coral Sinularia gibberosa. Steroids 2003;68:377–81. [3] Tanaka J, Trianto A, Musman M, Issa HH, Ohtani II, Ichiba T, et al. New polyoxygenated steroids exhibiting reversal of multidrug resistance from the gorgonian Isis hippuris. Tetrahedron 2002;58:6259–66. ¨ ¨ [4] Koljak R, Lopp A, Pehk T, Varvas K, Mu¨ urisepp AM, Jarving I, et al. New cytotoxic sterols from the soft coral Gersemia fruticosa. Tetrahedron 1998;54:179–86. [5] Rueda A, Zub´ıa E, Ortega MJ, Salva´ J. Structure and cytotoxicity of new polyhydroxylated sterols from the Caribbean gorgonian Plexaurella grisea. Steroids 2001;66:897–904. [6] D’Auria MV, Minale L, Riccio R. Polyoxygenated steroids of marine origin. Chem Rev 1993;93:1839–95. [7] Jin P, Deng Z, Pei Y, Fu H, Li J, Ofwegen LV, et al. Polyhydroxylated steroids from the soft coral Sinularia dissecta. Steroids 2005;70:487–93. [8] Shoji N, Umeyama A, Takei M, Arihara S. Potent inhibitors of histamine release: polyhydroxylated sterols from the Okinawan soft coral Sinularia abrupta. J Pharm Sci 1994;83:761–2. [9] Li R, Huang Z, Long K. Two polyhydroxylated steroids from the Chinese soft coral Sinularia microclavata. Steroids 1992;57:3–6. [10] Su J, Yu X, Zeng L, Mak TCW. Novel polyhydroxylated sterols from the soft coral Sinularia numerose. J Nat Prod 1989;52:934–40. [11] Jagodzinska BM, Trimmer JS, Fenical W, Djerassi CJ. Sterols in marine invertebrates. 51. Isolation and structure elucidation of C-18 functionalized sterols from the soft coral Sinularia dissecta. J Org Chem 1985;50:2988–92. [12] Rao CB, Satyanarayana C, Rao DS, Rao DV, Fahy E, Faulkner DJ. Metabolites of the soft coral Sinularia ovispiculata from the Indian ocean. J Nat Prod 1993;56:2003–7. [13] Tillekeratne LMV, Liyanage GK, Ratnasooriya WD, Ksebati MB, Schimtz FJ. A new spermatostatic glycoside from the soft coral Sinularia crispa. J Nat Prod 1989;52:1143–5. [14] Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetraxolium assay. Cancer Res 1988;48:589–601. [15] Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other cell lines. Cancer Res 1988;48:4827–33. [16] Demarco PV, Farkas E, Doddrell D, Mylari BL, Wenkert E. Pyridine-induced solvent shifts in the nuclear magnetic resonance spectra of hydroxylic compounds. J Am Chem Soc 1968;90:5480–6. [17] Ahmed VU, Aliya R, Perveen S, Shameel M. Sterols from marine green alga Codium decorticatum. Phytochemistry 1993;33:1189–92.
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journal homepage: www.elsevier.com/locate/steroids
Sinugrandisterols A–D, trihydroxysteroids from the soft coral Sinularia grandilobata Atallah F. Ahmed a,b , Shu-Hui Tai a , Yang-Chang Wu c , Jyh-Horng Sheu a,d,∗ a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt c Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Asian Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan b
a r t i c l e
i n f o
Article history:
a b s t r a c t Four new trihydroxysteroids, sinugrandisterols A–D (1–4), have been isolated from the
Received 20 October 2006
CH2 Cl2 -soluble fraction of the EtOH extract of Sinularia grandilobata. The structures of these
Received in revised form
metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR)
22 December 2006
analysis. The cytotoxicity of 1–4 toward a limited panel of cancer cell lines is also reported.
Accepted 8 January 2007
© 2007 Elsevier Inc. All rights reserved.
Published on line 16 January 2007 Keywords: Trihydroxysteroids Sinularia grandilobata Cytotoxicity
1.
Introduction
Marine organisms, including octocorals (Coelenterata), have been shown to be a rich source of a variety of polyoxygenated steroids [1–12]. A few of these sterols have been found to possess an 5,6-epoxide [3–5]. Chemical investigations on the steroidal constituents of soft corals of the genus Sinularia have afforded various polyhydroxylated steroids as derivatives of 24-methyl- and 24-methylene-cholestane-3-ol [1,2,6–13], and their glycosides [12,13]. Some of these compounds exhibited in vitro cytotoxic activity toward various cancer cell lines [2] and potent inhibition for histamine release from the mast cells [8]. Our current chemical investigation on Sinularia grandilobata Verseveldt (Alcyonacea) has also led to the isolation of four new trihydroxylated sterols, sinu-
grandisterols A–D (1–4). The structures of these metabolites were identified as 24-methylenecholest-5-en-1␣,3,7-triol (1), 24-methylenecholesta-5,22E-dien-1␣,3,7-triol (2), 5,6epoxy-24-methylenecholesta -1␣,3,7-triol (3), and 5,6epoxy-24-methylenecholesta-22E-en-1␣,3,7-triol (4), on the basis of the spectroscopic (IR, MS, and 1D and 2D NMR) analysis. The NOESY correlations, NMR data comparison with related steroids, pyridine-induced solvent shift, and acetylation reaction were also utilized to confirm the number, positions, and orientations of the hydroxy substituents on rings A and B of these new steroids. The cytotoxic activity of metabolites 1–4 against HepG2 (human liver carcinoma), MCF7, MDA-MB-23 (human breast carcinomas), and A-549 (human lung carcinoma) cells also is reported herein.
∗ Corresponding author at: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan. Tel.: +886 7 5252000x5030; fax: +886 7 5255020. E-mail address: [email protected] (J.-H. Sheu). 0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2007.01.001
369
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
2.
Experimental
2.1.
General procedures
2.2.
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 Hitachi I-2001 infrared spectrophotometer. NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR or on a Bruker AVANCE 500 FT-NMR at 500 MHz for 1 H and 125 MHz for 13 C or Bruker AVANCE 300 FT-NMR at 300 MHz for 1 H and 75 MHz for 13 C, in CDCl , unless otherwise stated. Low-resolution mass 3 spectral data were obtained by EI or ESI with a VG QUATTRO GC/MS spectrometer. HRMS were recorded by ESI FT-MS on a BRUKER APEX II mass spectrometer. Silica gel (Merck, 230–400 mesh) was used for open column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. Isolation by HPLC was performed by Shimadzu SPD-10A instrument equipped with a normal phase column (Hibar Lichrosorb Si-60, 7 m, 250 mm × 25 mm) or a reversed phase column (Hibar Purospher RP-18e, 5 m, 250 mm × 10 mm).
Animal material
The soft coral S. grandilobata was collected by hand using SCUBA off the coast of Kenting, Taiwan, in June 2004, at depths of 15–20 m and stored in a freezer until extraction. A voucher sample (SC-62) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
2.3.
Extraction and isolation procedure
The frozen bodies of S. grandilobata (0.9 kg, wet weight) were sliced and exhaustively extracted with EtOH (1 L × 5). The EtOH extract was filtered and concentrated under reduced pressure. The residue was partitioned between CH2 Cl2 and H2 O to afford the CH2 Cl2 fraction (30 g). The dichloromethane fraction was chromatographed by Si gel by CC and eluted with EtOAc in n-hexane in (0–100%, gradient) then with MeOH in EtOAc (5–50%, gradient) to yield 17 fractions. The eluted fractions were monitored by 1 H NMR spectroscopy. Fraction 12 (0.2 g), eluted by EtOAc–n-hexane (1:1), was further purified primarily by Si gel CC using acetone–EtOAc–n-hexane (1:1:5) to give sub-
Table 1 – 1 H NMR data for sterols 1–4 H#
1a
2a
1 2␣ 2 3 4␣ 4 6 7 8 9 11␣ 11 12␣ 12 14 15␣ 15 16␣ 16 17 18 19 20 21 22
3.88 br s 2.07 m 1.75 m 4.03 m 2.40 dd (12.0, 4.0)c 2.31 dd (12.0, 12.0) 5.48 s 3.90 m 1.45 m 1.66 m 1.50 m 1.47 m 1.18 m 2.03 br d (14.0)c 1.22 m 1.85 m 1.43 m 1.90 m 1.31 m 1.14 m 0.70 3H, s 1.06 3H, s 1.42 m 0.95 3H, d (6.3) 1.16 m 1.54 m 1.88 m 2.08 m 2.23 septet (6.6) 1.03 3H, d (6.6) 1.03 3H, d (6.6) 4.66 s; 4.72 s
3.88 br s 2.09 m 1.77 m 4.03 m 2.43 dd (12.9, 4.5) 2.31 dd (12.9, 12.9) 5.50 s 3.90 m 1.43 m 1.68 m 1.52 m 1.49 m 1.22 m 2.00 br d (12.6) 1.37 m 1.77 m 1.43 m 1.73 m 1.33 m 1.19 m 0.73 3H, s 1.07 3H, s 2.15 m 1.07 3H, d (6.3) 5.58 dd (15.8, 8.1)
23 25 26 27 28 a b c
Spectra recorded at 300 MHz. 500 MHz in CDCl3 at 25 ◦ C. The J values are in Hz in parentheses.
5.95 d (15.8) 2.55 septet (6.9) 1.07 3H, d (6.9) 1.08 3H, d (6.9) 4.83 s; 4.85 s
3b 3.92 br s 2.08 m 1.74 ddd (13.0, 10.5, 2.0) 4.23 m 1.66 dd (13.5, 5.0) 2.02 dd (13.5, 10.5) 3.18 s 3.57 dd (8.5, 8.5) 1.43 m 1.63 m 1.44 m 1.33 m 1.10 m 1.96 m 1.15 m 1.96 m 1.37 m 1.89 m 1.29 m 1.08 m 0.66 3H, s 1.01 3H, s 1.40 m 0.94 3H, d (6.0) 1.14 m 1.53 m 1.87 ddd (15.0, 10.0, 5.0) 2.09 m 2.21 septet (7.0) 1.03 3H, d (7.0) 1.03 3H, d (7.0) 4.65 s; 4.71 s
4a 3.92 br s 2.09 dd (14.0, 5.0) 1.74 br dd (14.0, 14.0) 4.23 m 1.66 m 2.02 m 3.18 s 3.57 d (8.1) 1.44 m 1.66 m 1.44 m 1.33 m 1.16 m 1.94 m 1.20 m 1.90 m 1.25 m 1.67 m 1.30 m 1.15 m 0.69 3H, s 1.02 3H, s 2.18 m 1.03 3H, d (6.3) 5.58 dd (15.8, 8.7) 5.94 d (15.6) 2.55 septet (6.6) 1.07 3H, d (6.9) 1.08 3H, d (6.9) 4.83 s; 4.85 s
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s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
fractions A and B. Subfraction A was re-chromatographed by RP-18 HPLC using MeOH–H2 O (9:1) to afford subfractions A1 and A2. Subfraction A1 was purified by normal phase HPLC using CH2 Cl2 –MeOH (25:10) to yield 2 (1.8 mg). Subfraction A2 was also purified by normal phase HPLC using acetone–nhexane (1:2) to afford 1 (12.9 mg). Subfraction B was also re-chromatographed by RP-18 HPLC using MeOH–H2 O (9:1) to give crude steroid fractions B1 and B2, which were purified separately by normal phase HPLC using acetone–n-hexane (2:3) to afford 4 (0.9 mg) and 3 (2.7 mg), respectively.
2.3.1.
Sinugrandisterol A (1)
White powder; mp 140–142 ◦ C; Rf = 0.56 (Si gel, acetone– hexane, 1:1); [␣]D 25 –8 (c 1.2, CHCl3 ); IR (neat) max 3355, 2959, 2930, 2866, 1644, 1464, 1379, 1261 and 1055 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; 1 H NMR data (300 MHz, C5 D5 N) ı 6.26 (1H, br s, 3-OH), 6.02 (1H, br s, 1-OH), 5.91 (1H, s, H-6), 5.61 (1H, br s, 7-OH), 4.83 (1H, s, H28), 4.81 (1H, s, H-28), 4.76 (1H, m, H-3), 4.20 (1H, m, H-7), 4.14 (1H, br s, H-1), 2.78 (1H, m, H-4), 2.76 (1H, m, H-4), 2.60 (1H, br d, J = 11.4 Hz, H-2), 2.37 (1H, ddd, J = 11.7, 11.7, 3.0 Hz, H-9), 2.25 (2H, m, H-15 and H-23), 2.20 (1H, m, H-25), 2.15 (1H, br dd, J = 11.4, 11.4 Hz, H-2), 1.97 (1H, m, H-23), 1.95 (1H, m, H-12), 1.89 (1H, m, H-16), 1.81 (1H, m, H-11), 1.77 (2H, m, H-8 and H-15), 1.61 (1H, m, H-22), 1.46 (2H, m, H-11 and H-20), 1.36 (1H, m, H-14), 1.25 (1H, m, H-16), 1.18 (1H, m, H-22), 1.17 (1H, m, H-12),
1.10 (3H, s, H3 -19), 1.09 (1H, m, H-17), 1.03 (6H, d, J = 6.6 Hz, H3 26 and H3 -27), 0.95 (3H, d, J = 6.3 Hz, H3 -21), 0.74 (3H, s, H3 -18); 13 C NMR data (75 MHz, C D N) ı 157.0 (qC, C-24), 141.0 (qC, C5 5 5), 130.7 (CH, C-6), 106.9 (CH2 , C-28), 73.3 (CH, C-7), 72.8 (CH, C-1), 66.2 (CH, C-3), 57.3 (CH, C-14), 56.1 (CH, C-17), 43.5 (qC, C-13), 43.0 (CH2 , C-4), 42.3 (qC, C-10), 41.4 (CH, C-8), 40.6 (CH2 , C-2), 40.4 (CH, C-9), 40.2 (CH2 , C-12), 36.3 (CH, C-20), 35.4 (CH2 , C-22), 34.4 (CH, C-25), 31.6 (CH2 , C-23), 29.3 (CH2 , C-16), 27.7 (CH2 , C-15), 22.3 and 22.4 (each CH3 , C-26 and C-27), 21.1 (CH2 , C-11), 19.7 (CH3 , C-19), 19.3 (CH3 , C-21), 12.5 (CH3 , C-18); EIMS m/z 430 (1.2, [M]+ ), 412 (23.9, [M − H2 O]+ ), 394 (3.1, [M − 2H2 O]+ ), 376 (0.6, [M − 3H2 O]+ ), 213 (3.6), 173 (9.9), 161 (11.9), 145 (13.4), 131 (14.9), 121 (15.9), 105 (27.7); HRESIMS m/z 453.3342 [M + Na]+ (calcd for C28 H46 O3 Na, 453.3344).
2.3.2.
2.3.3.
Table 2 – 13 C NMR data for sterols 1–4 C# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a b c
1a 72.6 (CH)c 38.3 (CH2 ) 66.0 (CH) 40.9 (CH2 ) 140.0 (qC) 129.0 (CH) 73.0 (CH) 40.8 (CH) 39.5 (CH) 41.4 (qC) 20.2 (CH2 ) 39.3 (CH2 ) 43.0 (qC) 55.8 (CH) 26.6 (CH2 ) 28.5 (CH2 ) 55.3 (CH) 11.8 (CH3 ) 19.2 (CH3 ) 35.7 (CH) 18.8 (CH3 ) 34.7 (CH2 ) 31.0 (CH2 ) 156.8 (qC) 33.8 (CH) 21.9 (CH3 ) 22.0 (CH3 ) 106.0 (CH2 )
2a 72.7 (CH) 38.4 (CH2 ) 66.1 (CH) 40.9 (CH2 ) 140.0 (qC) 129.1 (CH) 73.1 (CH) 40.8 (CH) 39.6 (CH) 41.5 (qC) 20.2 (CH2 ) 39.2 (CH2 ) 43.0 (qC) 55.8 (CH) 26.6 (CH2 ) 28.6 (CH2 ) 55.3 (CH) 12.3 (CH3 ) 19.2 (CH3 ) 40.2 (CH) 20.6 (CH3 ) 135.8 (CH) 129.3 (CH) 153.0 (qC) 29.4 (CH) 22.1 (CH3 ) 22.4 (CH3 ) 109.7 (CH2 )
3b 72.5 (CH) 38.0 (CH2 ) 63.9 (CH) 41.2 (CH2 ) 66.2 (qC) 67.8 (CH) 74.6 (CH) 37.7 (CH) 39.9 (CH) 38.9 (qC) 21.1 (CH2 ) 39.4 (CH2 ) 43.1 (qC) 55.3 (CH) 27.3 (CH2 ) 28.5 (CH2 ) 55.1 (CH) 11.7 (CH3 ) 16.5 (CH3 ) 35.6 (CH) 18.8 (CH3 ) 34.6 (CH2 ) 31.0 (CH2 ) 156.8 (qC) 33.8 (CH) 21.8 (CH3 ) 22.0 (CH3 ) 106.0 (CH2 )
4a 72.5 (CH) 38.0 (CH2 ) 63.9 (CH) 41.2 (CH2 ) 66.2 (qC) 67.8 (CH) 74.6 (CH) 37.7 (CH) 39.9 (CH) 39.0 (qC) 21.1 (CH2 ) 39.3 (CH2 ) 43.1 (qC) 55.3 (CH) 27.3 (CH2 ) 28.6 (CH2 ) 55.1 (CH) 12.0 (CH3 ) 16.5 (CH3 ) 40.2 (CH) 20.7 (CH3 ) 135.8 (CH2 ) 129.3 (CH2 ) 153.0 (qC) 29.3 (CH) 22.1 (CH3 ) 22.4 (CH3 ) 109.7 (CH2 )
Spectra recorded at 75 MHz. 125 MHz in CDCl3 at 25 ◦ C. Attached protons were determined by DEPT experiments.
Sinugrandisterol B (2)
White powder; mp 108–110 ◦ C; Rf = 0.53 (Si gel, acetone– hexane, 1:1); [␣]D 25 −6 (c 1.0, CHCl3 ); IR (neat) max 3356, 2959, 2930, 2870, 1653, 1458, 1373, 1125 and 1053 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; EIMS m/z 428 (1.0, [M]+ ), 410 (5.7, [M − H2 O]+ ), 395 (3.5, [M − H2 O − Me]+ ), 392 (1.2, [M − 2H2 O]+ ), 314 (11.7), 303 (41.1), 285 (7.3), 249 (11.2), 173 (11.3), 145 (13.0), 133 (15.0), 123 (51.2), 105 (40.8); ESIMS m/z 451 [M + Na]+ ; HRESIMS m/z 451.3191 [M + Na]+ (calcd for C28 H44 O3 Na, 451.3188).
Sinugrandisterol C (3)
White powder; mp 222–224 ◦ C; Rf = 0.47 (Si gel, acetone– hexane, 1:1); [␣]D 25 + 46 (c 1.1, CHCl3 ); IR (neat) max 3361, 2959, 2930, 2866, 1647, 1458, 1375,1161 and 1059 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; 1 H NMR data (300 MHz, C5 D5 N) ı 6.58 (1H, d, J = 3.9 Hz, 1-OH), 6.42 (1H, br s, 3-OH), 6.16 (1H, d, J = 8.0 Hz, 7-OH), 4.97 (1H, m, H-3), 4.82 and 4.80 (each 1H, s, H2 -28), 4.18 (1H, br s, H-1), 4.00 (1H, br dd, J = 8.0, 8.0 Hz, H-7), 3.61 (1H, s, H-6), 2.64 (1H, dd, J = 12.0, 12.0 Hz, H-4), 2.62 (1H, m, H-2), 2.10 (1H, m, H-4), 2.40 (1H, m, H-15), 2.39 (1H, ddd, J = 11.4, 11.4, 5.0 Hz, H-9), 2.23 (1H, septet, J = 6.6 Hz, H-25), 2.12 (1H, m, H-23), 2.08 (1H, m, H-2), 1.99 (1H, m, H-23), 1.95 (1H, m, H-8), 1.89 (1H, m, H-12), 1.78 (1H, m, H16), 1.71 (1H, m, H-15), 1.56 (2H, m, H-11 and H-22), 1.46 (1H, m, H-11), 1.31 (1H, m, H-20), 1.29 (1H, m, H-14), 1.26 (1H, m, H-16), 1.15 (1H, m, H-22), 1.03 (1H, m, H-17), 1.02 (6H, d, J = 6.6 Hz, H3 26 and H3 -27), 1.01 (1H, m, H-12), 1.23 (3H, s, H3 -19), 0.92 (3H, d, J = 6.6 Hz, H3 -21), 0.64 (3H, s, H3 -18); 13 C NMR data (75 MHz, C5 D5 N) ı 157.0 (qC, C-24), 107.0 (CH2 , C-28), 75.0 (CH, C-7), 72.5 (CH, C-1), 70.2 (CH, C-6), 66.7 (qC, C-5), 64.1 (CH, C-3), 56.7 (CH, C-14), 55.8 (CH, C-17), 43.7 (CH2 , C-4), 43.5 (qC, C-13), 40.8 (CH, C-9), 40.3 (CH2 , C-12), 40.1 (qC, C-10), 39.8 (CH2 , C-2), 38.0 (CH, C-8), 36.3 (CH, C-20), 35.3 (CH2 , C-22), 34.3 (CH, C-25), 31.6 (CH2 , C-23), 29.3 (CH2 , C-16), 28.4 (CH2 , C-15), 22.4 and 22.3 (each CH3 , C-26 and C-27), 22.2 (CH2 , C-11), 19.4 (CH3 , C-21), 17.6 (CH3 , C19), 12.3 (CH3 , C-18); ESIMS m/z 469 [M + Na]+ ; HRESIMS m/z 469.3291 [M + Na]+ (calcd for C28 H46 O4 Na, 469.3294).
2.3.4.
Acetylation of 3
A solution of sinugrandisterol C (3) (1.1 mg, 0.0025 mmol) in pyridine (0.2 mL) was mixed with Ac2 O (0.2 mL) and the mixture was stirred at RT for 24 h. After evaporation of excess reagent, the residue was purified by normal phase HPLC, using Acetone–n-hexane = 1:3, to give a triacetyl derivative 3a
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
(1.4 mg, 0.0024 mmol, 96% yield). 3a: colorless gum; [␣]D 25 + 21 (c 0.7, CHCl3 ); IR (neat) max 2959, 2925, 2860, 1738, 1640, 1373 and 1238 cm−1 ; 1 H NMR (500 MHz, CDCl3 , selected data) ı 5.13 (1H, br s, H-1), 5.06 (1H, m, H-3), 4.82 (1H, d, J = 9.5 Hz, H-7), 4.71 (1H, H-28), 4.64 (1H, H-28), 3.22 (1H, s, H-6), 2.21 (1H, septet, J = 7.0 Hz, H-25), 2.12, 2.09, and 2.02 (each 3H, s, 3 OAc), 1.80 (1H, m, H-8), 1.11 (3H, s, H3 -19), 1.03 and 1.01 (each 3H, d, J = 7.0 Hz, H3 -26 and H3 -27), 0.91 (3H, d, J = 7.0 Hz, H3 -21), 0.65 (3H, s, H3 -18); ESIMS m/z 595 [M + Na]+ .
2.3.5.
Sinugrandisterol D (4)
White powder; mp 205–207 ◦ C; Rf = 0.44 (Si gel, acetone– hexane, 1:1); [␣]D 25 + 17 (c 0.4, CHCl3 ); IR (neat) max 3362, 2959, 2928, 2868, 1653, 1458, 1375 and 1055 cm−1 ; 1 H and 13 C NMR data (CDCl3 ), see Tables 1 and 2, respectively; ESIMS m/z 467 [M + Na]+ ; HRESIMS m/z 467.3135 [M + Na]+ (calcd for C28 H44 O4 Na, 467.3137).
2.4.
Cytotoxicity testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of the test compounds 1–4 were performed using the MTT [3-(4,5-dimethylthiazole-
371
2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [14,15].
3.
Results and discussion
The sliced bodies of the soft corals S. grandilobata were exhaustively extracted with EtOH. The EtOH extract was concentrated and the residue was partitioned between CH2 Cl2 and H2 O. The combined CH2 Cl2 –soluble fraction was concentrated and the residue was chromatographed on Si gel CC followed by normal and reversed phase HPLC to yield sterols 1–4 (see Section 2). Sinugrandisterol A (1) was obtained as a white powder and possessed a molecular formula C28 H46 O3 as determined by HRESIMS (453.3342 m/z, [M + Na]+ ), implying six degrees of unsaturation. The IR absorptions at max 3355 and 1644 cm−1 suggested the presence of hydroxy and olefinic functionalities. Three hydroxyls in the molecule were estimated from the ion peaks appearing at m/z 412 (M − H2 O)+ , 394 (M − 2H2 O)+ , and 376 (M − 3H2 O)+ in the EIMS spectrum, and from the NMR signals resonating (in CDCl3 , Tables 2 and 1) at ıC 73.0, 72.6, and 66.0 (each CH) and ıH 3.90 (1H, m), 3.88 (1H, br s), and 4.03 (1H, m), respectively. Measurement of the 1 H NMR spec-
Fig. 1 – 1 H–1 H COSY and HMBC correlations for 1 (C5 D5 N), 2 (C5 D1 N), and 3 (CDCl3 ).
372
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
Fig. 1 – (Continued ).
trum of 1 in C5 D5 N also exhibited three D2 O–exchangeable OH signals at ı 6.26, 6.02, and 5.61 (each 1H, br s). Furthermore, an exomethylene group and a trisubstituted double bond (ıC 156.8, qC, 140.0, qC, 129.0, CH and 106.0, CH2 ; ıH 5.48, 4.72 and 4.66, each 1H, s) were also indicated in the molecule of 1. Moreover, the 1 H NMR spectrum exhibited two singlets at ı 1.06 and 0.70 (each 3H, s) and two doublets at ı 1.03 (6H, d, J = 6.6 Hz) and 0.95 (3H, d, J = 6.3 Hz) attributable to two tertiary and three secondary methyls. These findings together with the 28 carbon signals appearing in the 13 C NMR spectrum (Table 2) suggested 1 as a trihydroxylated methylenecholestene. By the analysis of 1 H–1 H COSY and HMBC correlations (in C5 D5 N) the planar structure of compound 1 was unambiguously established as illustrated in Fig. 1, where the positions of three hydroxy groups and the two double bonds were determined to be at C-1, C-3, C-7, C-5/C-6, and C-24/C-28, respectively. The NOESY correlations, NMR data comparison, and pyridine-induced solvent shift were successively employed to establish the stereochemistry at C-1, C-3, and C-7. The hydroxy groups at C-1 and C-3 were determined to have the ␣ and  orientations, respectively, on the basis of strong NOE interactions of H3 -19 (ı 1.06, s) with H-1 (ı 3.88, br s), H3 -19 with H-4 (ı 2.31, dd, J = 12.0, 12.0 Hz), and H-4␣ (ı 2.40, dd, J = 12.0, 4.0 Hz) with H-3 (ı 4.03, m) (Fig. 2). The ␣ orientation of 1-OH was further confirmed by measuring the 1 H NMR spectrum of 1 in
Fig. 2 – Key NOESY correlations for 1–3.
373
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
C5 D5 N (see Section 2) where large pyridine-induced downfield shifts (ı = ıCDCl3 − ıC5 D5 N) [2,16] were observed for H-3 (ı = −0.73 ppm) and H-9 (ı = −0.71 ppm) due to the 1,3diaxial deshielding effect exerted by 1-OH in pyridine [16]. The significant NOE correlations displayed by H-7 (ı 3.90, m) with both H-9 (ı 1.66, m) and H-14 (ı 1.22, m), but not with H-8, revealed the  orientation of the 7-OH. This was supported by the quite similar chemical shifts of H-7 (ı 3.90, m), C-7 (ı 73.0, CH), and C-8 (ı 40.8, CH) of 1 with those of isodecortinol (5) [17] (ı 3.83, 73.0 and 40.8, respectively), measured in CDCl3 . Furthermore, the pyridine-induced downfield shift for the  oriented H-8 (ı = −0.32 ppm) due to the vicinal deshielding effect [16] by 7-OH in pyridine provided an additional evidence for the -orientation of the hydroxy function at C-7. On the basis of above findings, the structure of sinugrandisterol A (1) was unambiguously established as 24-methylenecholesta-5en-1␣,3,7-triol. Sinugrandisterol B (2) was also obtained as a white powder and exhibited a quasimolecular ion peak at m/z 451.3191 (M + Na)+ in the HRESIMS, appropriate for a molecular formula of C28 H44 O3 . It was found that the IR, MS, and 13 C NMR spectral data of 2 (Table 2) were very similar to those of 1, except the replacement of the carbon signals of an ethylene moiety (ı 34.7 and 31.0, each CH2 ) in 1 by a 1,2-disubstituted double bond (ı 135.8 and 129.3, each CH) in 2. This was also evidenced by the two olefinic proton signals appearing at ı 5.95 (d, J = 15.8 Hz) and 5.58, dd, J = 15.8, 8.1 Hz) in the 1 H NMR spectrum of 2. The 1 H–1 H COSY correlations (Fig. 1) observed between the latter proton and H-20 (ı 2.15, m), and between H-20 and H3 21 (ı 1.07, d, J = 6.3 Hz) indicated the C-22 and C-23 position of the double bond. This was further supported by the 1 H–1 H COSY correlation between H-22 and H-23 (ı 5.95) and by the HMBC correlations (Fig. 1) found from the exomethylene protons H2 -28 (ı 4.85 and 4.83, each 1H, s) to C-23 (ı 129.3, CH). The very similar NMR data (Tables 2 and 1) and NOESY correlations (Fig. 2) observed for protons within rings A-D in both 1 and 2 reflected the ␣, , and  orientations of the hydroxyls at C-1, C3, and C-7 in 2, respectively. Moreover, the coupling constant (J = 15.8 Hz) between H-22 and H-23, and the NOE interactions of H-23 with both H-20 and H-28 (ı 4.85, s), and H-22 with H-25 (ı 2.55, septet, J = 6.9 Hz) confirmed the E geometry of the 1,2disubstituted double bond in the side chain. These findings and other 2D NMR correlations (Figs. 1 and 2) established the structure of sinugrandisterol B (2) as 24-methylenecholesta5,22E-dien-1␣,3,7-triol.
Sinugrandisterol (3) was found to be more polar than 2 (see Section 2) and had a molecular formula C28 H46 O4 as determined by its HRESIMS (469.3291 m/z, [M + Na]+ ) and NMR data (Tables 1 and 2, and Section 2). The IR spectrum (max 3361 cm−1 ) indicated the presence of hydroxy functionality in 3. The presence of three hydroxy groups was supported by the acetylation of 3 to yield 3a, which exhibited three 3H singlet of acetate methyls in the 1 H NMR spectrum (ı 2.12, 2.09, and 2.02). Comparison of NMR data of compound 3 with those of 1 (Tables 1 and 2) indicated that 3 is another 24-methylenecholestane-triol. As in case of 1, measuring the 1 H NMR spectrum of 3 in C D N (see Section 2) showed three 5 5 D2 O-exchangeable hydoxyl protons at ı 6.58 (1H, d, J = 3.5 Hz, 1-OH), 6.42 (1H, br s, 3-OH), and 6.16 (1H, d, J = 8.0 Hz, 7-OH). Thus, the remaining fourth oxygen atom in the molecular formula of 3 was suggested to be incorporated in an epoxide (ıC 66.2, qC and 67.8, CH; ıH 3.18, 1H, s), which arised from the epoxidation of the trisubstituted double bond (ıC 140.0, qC and 129.0, CH; ıH 5.48, 1H, s) of 1. The C-5/C-6 location of this epoxide was proven by the HMBC correlations (Fig. 1) from the epoxymethine proton (ı 3.18, 1H, s) to C-7 (ı 74.6, CH), C-8 (ı 37.7, CH), C-10 (ı 38.9, CH), and the quaternary oxycarbon of the epoxide (ı 66.2, qC), while the latter carbon was found to be correlated with H2 -4 (ı 1.66, dd, J = 13.5, 5.0 Hz and 2.02, dd, J = 13.5, 10.5 Hz) and H3 -19 (ı 1.01, s). Also, the three hydroxyls were determined to be situated at C-1, C-3, and C-7 on the basis of correlations displayed in the 1 H–1 H COSY and HMBC spectra of 3 (Fig. 1). The similarity of the splitting patterns of H-1 and H-3 in compound 3 with those in 1 and 2 revealed the ␣ and  orientations of the hydroxy groups at C-1 and C-3, respectively. The ␣ orientation of 1-OH was further confirmed by the NOE interaction displayed by the  oriented H3 -19 with H-1. The large pyridine-induced downfield shift induced at H-3 (ı = −0.74 ppm) was due to the 1,3-diaxial deshielding effect exerted by 1-OH in pyridine, as observed in the case of 1, further confirming the ␣ and  orientations of 1-OH and 3-OH, respectively. Moreover, the  orientation of 7-OH was concluded from the splitting pattern and J value (d, ∼8.0 Hz) of H-7 with H-8 in CDCl3 (Table 2) and in C5 D5 N (after addition of D2 O), or in CDCl3 after acetylation (see Section 2). This was further supported by the NOE interactions observed for H-7 with the ␣ oriented H-9 (ı 1.63, m) and H-14 (ı 1.15, m), but not with the  oriented H-8 (ı 1.43, m). Furthermore, the C5/C6–epoxide was thought to possess the  orientated oxygen on the basis of NOE correlations (Fig. 2) displayed by the
Table 3 – Selected NMR data for sterols 3, 6 [4] and 7 [5] #
4 5 6 7 8 a b c
3 (CDCl3 )a
6 (CDCl3 )b
ıH
ıC
3.18 s 3.57 dd (8.5, 8.5) 1.43 m
41.2 66.2 67.8 74.6 37.7
3 (C5 D5 N)a
ıH
ıC
3.14 d (1.2) 3.51 m 1.43 m
41.7 67.3 67.5 74.7 38.3
Spectra recorded at 300 MHz for 1 H and 75 MHz for 13 C. 500 MHz for 1 H and 125 MHz for 13 C (Ref. [4]). 400 MHz for 1 H and 100 MHz for 13 C (Ref. [5]).
7 (C5 D5 N)c
ıH
ıC
3.61 s 4.00 br dd (8.0, 8.0) 1.95 m
43.7 66.7 70.2 75.0 38.0
ıH
ıC
3.47 s 3.79 dd (8.0, 7.6) 1.87 m
43.3 66.8 69.1 74.6 38.1
374
s t e r o i d s 7 2 ( 2 0 0 7 ) 368–374
references
Table 4 – Cytotoxicity (IC50 g/mL) of sterols 1–4 Hep G2 1 2 3 4 Doxorubicin a b
9.1 6.8 –b –a 0.40
MCF-7 –a 19.3 18.9 –a 0.13
MDA-MB-231 9.2 –b –b –a 0.25
A549 17.2 16.9 18.9 –a 0.2
Compound is considered inactive when IC50 > 20 g/mL. Not tested.
epoxide proton H-6 (ı 3.18, s) with H-7 (ı 3.57, dd, J = 8.5, 8.5 Hz) and H-4␣ (ı 1.66, dd, J = 13.5, 5.0 Hz). Comparison of the NMR data of the C-4–C-8 moiety in rings A and B of 3 with those of the known marine sterols 6 [4] and 7 [5] (Table 3) showed close similarity and further confirmed the 5, 6 orientation of the epoxy oxygen as well as the  orientation of the hydroxyl at C-7. On the basis of above results, together with the detailed analysis of 2D NMR spectra of 3 (Figs. 1 and 2), the structure of sinugrandisterol C (3) was unambiguously established as 5,6-epoxy-24-methylenecholestane-1␣,3,7triol. The most polar compound, sinugrandisterol D (4), was determined to have a molecular formula of C28 H44 O4 by the quasimolecular ion peak at m/z 467.3135 (M + Na)+ in the HRESIMS and NMR data (Tables 2 and 1). Its IR spectrum (max 3362 cm−1 ) also revealed the presence of hydroxy group in the molecule. Moreover, it was found that the NMR data of A–D rings (C-1 to C-19 and H-1 to H3 -19) of 4 were quite similar to those of 3, indicating the same substitution and stereochemistry at C-1, C-3, C-5, C-6, and C-7. Also, the NMR data of the side chain (C-20 to C-28 and H-20 to H2 -28) of 4 were found to be almost the same as those of 2. On the basis of above findings, together with the analysis of the NOE correlations of 4, the structure of sinugrandisterol D (4) was determined as 5,6-epoxy-24-methylenecholest-22E-en-1␣,3,7triol. The cytotoxicity of compounds 1–4 against the growth of cancer cell lines of Hep G2, MCF-7, MDA-MB-231, and A-549 were also studied. The results (Table 4) showed that the 5,6 sterols 1 and 2 showed moderate cytotoxicity against the growth of Hep G2 and MDA-MB-231 cells (IC50 6.8–9.2 g/mL). Compounds 2 and 3 exhibited weak activity against A549 and MCF-7cancer cells (IC50 16.9 and 19.3 g/mL), while 1 was inactive against MCF-7 (IC50 > 20 g/mL) and showed only weakly cytotoxic activity against A549. Compound 4 showed no cytotoxicity against the tested cell lines.
Acknowledgments Financial support was provided by Ministry of Education (C030313) and National Science Council of Taiwan (NSC 952323-B-110-002) awarded to J.-H. Sheu.
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