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Journal of Chemistry, Vol. 45 (Special issue), P. 141 - 144, 2007 STUDY ON CHEMISTRY OF THE SPONGE PETROSIA NIRICANS LIVING IN VIETNAMESE SEA Received 15 October 2007 Tran Thu Huong1 , Nguyen Tuan Anh1 , Tran Thuong Quang1 , Chu Nhat Huy1 , Tran Thi Minh1, Chau Van Minh2, Phan Van Kiem 2 1 Faculty of Chemical Technology, Hanoi University of Technology 2 Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology summary Batilol (1), 5,8-epidioxycholest-6-en-3-ol (2), and cholesterol (3) were isolated from chloroform extract of the sponge Petrosia nigricans collected in Vietnam. Their structures were determined on the basic of the physicochemical and spectroscopic data. This is the first report of compounds 1, 2 and 3 from this species. Keywords: Petrosia nigricans, Batilol, Glycerol 1-O-octadecyl ether, 5,8-Epidioxycholest-6-en3-ol, Cholesterol. I - INTRODUCTION Highly functionalized steroids featuring biogenetically unprecedented structures have been found in a vast array of marine organisms, particularly sponges. It has been hypothesized that the sponge’s effect chemical modification of their dietary precursors in order to produce the diverse variation in steroidal content obtained from these sources [1]. Many steroids exhibit potent pharmacological properties, including cytotoxic [2] and ichthyotoxic [3] effects. As part of our current investigations on bioactive compounds from marine organisms in Vietnam, we report here the isolation and structural elucidation of batilol (1), 5,8epidioxycholest-6-en-3-ol (2), and cholesterol (3) from the sponge Petrosia nigricans. II - EXPERIMENTAL spectrum was obtained using an AGILENT 1200 LC-MSD Trap spectrometer. The 1H-NMR (500MHz) and 13C-NMR (125 MHz) spectra were recorded on Bruker AM500 FT-NMR spectrometer. Chemical shifts are referenced to using tetramethylsilan (TMS) as an internal standard. Column chromatography (CC) was performed on silica gel 230 - 400 mesh (0.040 0.063 mm, Merck) or YMC RP-18 resins (30 50 µm, FuJisilisa Chemical Ltd.). Thin layer chromatography (TLC) was performed on DCAlufolien 60 F254 (Merck 1.05715) or RP18 F254s (Merck) plates. The cytotoxic activity was evaluated by methods of Vanden et al [4] and Likhiwitayawuid et al [5]. The cancer cell lines are KB (Human epidemoid carcinoma), FL (Fibril sarcoma of Uterus) and Hep-G2 (Human Hepatoma Carcinoma). 2. Animal material 1. General experimental procedures The Electronspray Ionization (ESI) mass The specimens of Petrosia nigricans were collected at 30 - 45 m depth in Truong Sa 141 archipelago, Khanh Hoa province, Vietnam during May, 2007 and deep frozen until used. The scientific name was identified by Dr. Do Cong Thung, Institute of Marine Resources and Environment, Vietnamese Academy of Science and Technology, Vietnam. small pieces and extracted three times with hot MeOH (50oC for 3 hours each time) and then concentrated under reduced pressure to give 50 g MeOH extract. This extract was suspended in water and partitioned in turn with chloroform and n-buthanol. From the chloroform extract (30 g), three compounds: 1 (100 mg, white crystals), 2 (50 mg, white crystals), 3 (150 mg, crystals), was isolated by combination of normal and reversed phase chromatography methods. 3. Extraction and isolation Fresh frozen sample of the sponge Petrosia nigricans (10 kg) was well grinded, cut into 21 25 HO 3 18 OH 20 17 11 2 1 1 1' O 9 18' CH2(CH2)16CH3 O 3 1 14 8 10 5 HO 27 13 19 O 2 21 20 22 25 26 18 17 27 19 1 8 3 5 HO 6 7 3 Figure 1: The structures of 1 - 3 Table 1: NMR spectral data of 1 C 1 2 3 1’ 2’ 3’ 4’-15’ 16’ 17’ 18’ 2-OH 3-OH a a,b C 72.55 70.44 64.35 71.89 29.37 - 29.71 26.11 29.37 - 29.71 31.94 22.70 14.12 - Recorded in CDCl3, b125 MHz, c 500 MHz. 142 a,c mult. (J in Hz) 3.65 dd. (3.0. 11.0); 3.72 dd. (5.0. 11.0) 3.86 m 3.52 m 3.46 m 1.58 m 1.26 br s 1.26 br s 1.26 br s 1.26 br s 0.88 t (7.0) 2.60 d (5.0) 2.17 t (7.0) H 26 Table 2: NMR spectral data of 2 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 a,b C 34.71 30.11 66.44 36.94 82.16 135.40 130.77 79.46 51.09 36.95 20.62 39.44 44.74 51.59 a,c H mult. (J in Hz) 1.94/1.99 1.54/1.83 (m) 3.96 (m) 1.95/2.11 6.24 (d. 8.5) 6.51 (d. 8.5) 1.52 1.00 (m)/1.59 1.22 1.58 C 15 16 17 18 19 20 21 22 23 24 25 26 27 a,b C 23.42 28.23 56.44 12.62 18.58 35.22 18.16 35.95 23.80 39.44 27.98 22.53 22.79 a,c H mult. (J in Hz) 1.21/1.52 1.45 1.20 0.80 (s) 0.88 (s) 1.60 0.90 (d. 7.0) 1.70 (m) 1.32 (m) 1.24 1.42 0.86 (d. 7.0) 0.87 (d. 7.0) a Recorded in CDCl3, b125 MHz, c500 MHz. Batilol (1): White crystals; Positive ESI-MS: m/z 345.0 [M+H]+ and 367.0 [M+Na]+, (C21H44O3, M = 344); 1H-NMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3): see: Table 1. 5,8-Epidioxycholest-6-en-3-ol (2): White crystals; Positive ESI-MS: m/z 417.0 [M+H]+ and 439.0 [M+Na]+, (C27H44O3, M = 416); 1H-NMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3): see table 2. Cholesterol (3): White crystals, mp. 148.5oC, Positive ESI-MS: m/z 369.1 [MH2O+H]+ (C27H46O, M = 386); 1H-NMR (500 MHz, CDCl3) : 3.52 (1H, m, H-3), 5.35 (1H, br d, J = 5.0 Hz, H-6), 1.01 (3H, s, H-19), 0.92 (3H, s, H-21), 0.87 (3H, d, J = 7.0 Hz, H-27), 0.86 (3H, d, J = 7.0 Hz, H-26), and 0.68 (3H, s, H-18); 13C-NMR (125 MHz, CDCl3) : 37.2 (t, C-1), 31.6 (t, C-2), 71.8 (d, C-3), 42.2 (t, C-4), 140.7 (s, C-5), 121.7 (d, C-6), 31.8 (t, C-7), 31.8 (d, C-8), 50.1 (d, C-9), 36.5 (s, C-10), 21.1 (t, C11), 39.8 (t, C-12), 42.3 (s, C-13), 56.7 (d, C14), 24.3 (t, C-15), 28.3 (t, C-16), 56.1 (d, C17), 11.9 (q, C-18), 19.4 (q, C-19), 35.7 (d, C20), 18.7 (q, C-21), 36.1 (t, C-22), 23.8 (t, C23), 39.5 (t, C-24), 28.0 (d, C-25), 22.5 (q, C-26) and 22.8 (q, C-27). III - RESULTS AND DISCUSSION Compound 1 was isolated as white crystals. Its molecular formula, C21H44O3, was determined by positive ESI-MS (at m/z 345.0 [M+H]+ and 367.0 [M+Na]+) and NMR spectral data. The 1H-NMR spectrum of 1 showed typical signals of oxymethine and oxymethylene groups at 3.86 (1H, m), 3.72 (1H, dd, J = 5.0, 11.0 Hz), 3.65 (1H, dd, J = 3.0, 11.0 Hz), 3.52 (2H, m) and 3.46 (2H, m); protons of hydroxyl groups at 2.60 (1H, d, J = 5.0 Hz) and 2.17 (1H, t, J = 7.0 Hz); one methyl group at 0.88 (3H, t, J = 7.0 Hz) and methylene protons of a saturated long chain at 1.58 (2H, m) and 1.26 (30H, br s). The 13C-NMR spectrum of 1 exhibited typical signals of an unbranched long aliphatic chain at 31.94 (CH2), 29.71 (CH2), 29.67 (CH2), 29.62 (CH2), 29.60 (CH2), 29.47 (CH2), 29.37 (CH2), 26.10 (CH2), 22.70 (CH2) and 14.12 (CH3). The presence of an asymmetric mono-substituted glycerol group 143 CH2OCH2CH(OH)CH2OH was identified by carbon signals at 72.54 (CH2, C-1), 70.45 (CH, C-2), 64.33 (CH2, C-3) and 71.89 (CH2, C-1’) [9]. As the optical rotation of 1 was similar as that of (S)-chimyl alcohol, and as all other chiral glyceride-1-ethers known to date are (S) configured, the configuration of 3 at C-2 was proposed to be (S) [5, 6]. From all the above analysis, 1 was identified as glycerol mono-alkyl ether. The good agreement of the NMR spectral data of 1 with literature values [5] together with the quasi ion peaks at m/z 345.0 [M+H]+ and 367.0 [M+Na]+ (corresponding to the molecular formula of C21H44O3, M = 344) observed on the positive ESI mass spectrum led to the conclusion that the chemical structure of 1 is glycerol 1-Ooctadecyl ether (batilol). Compound 2 was obtained as white crystals. The positive ESI-MS (at m/z: 417.0 [M+H]+ and 439.0 [M+Na]+) and NMR spectral data indicated its molecular formula of C27H44O3. The 1H-NMR spectrum of 2 showed five methyl signals typical for a steroid structure at 0.80 (3H, s, H-18), 0.88 (3H, s, H-19), 0.90 (3H, d, J = 7.0 Hz, H-21), 0.86 (3H, d, J = 7.0 Hz, H-26), and 0.87 (3H, d, J = 7.0 Hz, H-27). In addition, an oxymethine group and a disubstituted double bond was identified by signals at 3.96 (1H, m, H-3) and 6.24 (1H, d, J = 8.5 Hz, H-6) 6.51 (1H, d, J = 8.5 Hz, H-7), respectively. The 13C-NMR spectrum of 2 showed 27 sterol-like carbon signals, including 4 quaternary, 8 methine, 10 methylene, and 5 methyl carbons. Of which one di-substituted double bond was confirmed at 135.40 (CH, C6)/130.77 (CH, C-7); two oxygen bearing quaternary carbons at 82.16 (C, C-5) and 79.46 (C, C-8); and one oxymethine carbon at 66.44 (CH, C-3). The large carbon chemical shifts at C-5 and C-8 suggested the presence of a peroxide bridge at these positions. From all the above analysis and the good agreement of the NMR spectral data of 2 with the literature values [7, 8], the chemical structure of this compound was established as 144 5,8-Epidioxycholest-6-en-3-ol. By the direct comparisons of the NMR spectral data and physicochemical properties of 3 with published literatures, compound 3 were identified as cholesterol [9]. All isolated compounds were tested for their cytotoxic activity. The results showed that compound 2 exhibited strongly cytotoxic effect against all three tested cell line with the IC50 values of 2.0 µg/ml (KB), 3.93 µg/ml (FL) and 2.4 µg/ml (Hep-G2). The other compounds were negative for all tested cell lines. Acknowledgment: The authors gratefully thank to the receipt of a grant from the Flemish Interuniversity Council for University Development cooperation (VLIR UOS) which enabled them to carry out this work. REFERENCES 1. W. A. Gallimore, M. Kelly, and P. J. Scheuer. 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