Chemical Constituents And Bioactivities Of Three Marine Sponges From Paracel Islands

The South China Sea is a rich resource of invertebrates possessing structurally unique secondary metabolites with various bioactivities such as antitumor, anti-virus, antibiosis, anti-inflammatory, anti-allergy, and immunomodulation bioactivities. As part of ongoing research on chemical constituents and bioactivities of marine organisms, we collected 31 species marine sponges from Paracel Islands of the South China Sea for antitumor screening, and 11 of them exhibited significant antitumor activity in vitro. In order to discover new bioactive compounds or leading compounds, we successfully investigated the three sponges, Phyllospongia foliascens, Phakellia fusca and Theonella swinhoei. A total of 66 compounds were obtained by chromatography techniques and 63 of them, including 17 steroids, 12 terpenoids, 11 cyclopeptides, 17 alkaloids, 5 miazines and 1 benzoic acid, were identified by spectroscopic methods. Among these compounds, 12 of them were proved to be new compounds. Some of sesterterpenes, cyclopeptides and steroids were cytotoxic against cancer cell lines P388, BEL-7402, SPC-A-1, HAC and Colo-26 in vitro.The acetone extract of marine sponge P. foliascens collected from Woody Island and Rocky Island exhibited antitumor activity against cancer cell lines HAC (IC50 < 1μg/mL) and Colo-26 (IC50 < 1μg/mL). 22 compounds (A1~A22) were isolated and purified from this sponge by solvent extraction and chromatography methods including VLC, LPLC, MPLC, HPLC on silica gel, ODS C-18 and Sephadex LH-20. Theses compounds, including 11 steroids and 11 sesterterpenes, were identified by 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC (HSQC), HMBC, NOESY (ROESY) and MS as:β-sitosterol (A1), cholesta-5,7-dien-3β-ol (A2), ergosta-5,7,24(28)-trien-3β-ol (A3), ergosta-5,7-dien-3β-ol (A4), (24E)-stigmasta-5,7,24(28)-trien-3β-ol (A5), stigmasta-5,7-dien-3β-ol (A6), ergosterol (A7), phylloketal (A8), phyllohemiketal A (A9), phyllofenone A (A10), phyllofolactone B (A11), 20,24α-dimethyl-scalaran-12α- ol-25,24-lactone (A12, phyllofolactone M), 24-oxo-24-homoscalar-16,25(26)-dien- 12α-ol (A13, phyllofenone D), phyllofenone B (A14), 20,24-dimethyl-24-oxo-25- norscalar-16-en-12α,18β-diol (A15, phyllofenone E), 24β-methyl-12-oxoscalaran- 16β-ol-25,24-lactone (A16, phyllofolactone L), phyllofolactone A (A17), phyllofolactone C (A18), (24E)-5α,6α-epoxystigmasta-7,24(28)-dien-3β-ol (A19), 5α,6α-epoxystigmasta-7,22-dien-3β-ol (A20), 5α,6α-epoxycholest-7,22-dien-3β-ol (A21), and 5α,6α-epoxycholest-7-en-3β-ol (A22). The five compounds A12, A13, A15, A16 and A19 were new compounds, ten steroids A1~A7, A20~A22 were isolated from this genus for the first time. Compounds A11 and A12 were stereoisomers at CH3-26 configuration, and were separated by HPLC and distinguished by 13C-NMR data. Compounds A13 and A14 were the only two phyllofolactones possessed a rareα,β-unsaturated ketone ring E from the South China Sea sponge. In the antitumor assays, compounds A10, A13, A14, A15, A16 and A17 exhibited cytotoxicity against cancer cell line P388 with IC50 values of 15.3, 6.5, 11.9, 30.2, 25.6 and 10.5μg/mL, respectively; compounds A13 and A14 showed cytotoxicity against cancer cell line BEL-7402 with IC50 values of 47.8 and 32.9μg/mL, respectively; compounds A10 and A15 exhibited cytotoxicity against cancer cell line SPC-A-1 with IC50 values of 23.6 and 29.3μg/mL, respectively; compounds A11, A12 and A15 showed cytotoxicity against cancer cell line HAC with IC50 values of 46.7, 32.5 and 45.1μg/mL, respectively; compounds A10, A13, A14 and A15 exhibited cytotoxicity against cancer cell line Colo-26 with IC50 values of 23.5, 11.9, 28.1, 26.9 and 42.7μg/mL, respectively.The ethanol extract of marine sponge P. fusca collected from Woody Island and Seven connected islets showed antitumor activity against cancer cell lines HAC (IC50 6.4μg/mL) and Colo-26 (IC50 < 1μg/mL). 27 compounds (B1~B27) were isolated and purified from this sponge by solvent extraction and chromatography methods including VLC, LPLC, MPLC, HPLC on silica gel, ODS C-18 and Sephadex LH-20. Theses compounds including 10 cyclopeptides and 17 alkaloids were identified by 1H-NMR, 13C-NMR, 1H-1H COSY (MQF-COSY), HMQC (HSQC), HMBC, TOCSY, HMQC-TOCSY, NOESY (ROESY), MS and X-Ray as: cyclo(-Pro1-Phe-Gly-Pro2- Thr- Leu-Trp-) (phakellistatin 13, B1), cyclo(-Pro1-Trp-Val-Pro2-Leu1-Thr-Pro3-Leu2-) (hymenamide H, B2), cyclo(-Pro1-Pro2-Tyr-Val-Pro3-Leu-Ile1-Ile2-) (hymenistatin 1, B3), cyclo(-Pro1-Pro2-Tyr-Val-Pro3-Leu1-Ile-Leu2-) (hymenamide G, B4), cyclo(-Pro1- Trp-Val-Pro2-Leu-Ile1-Pro3-Ile2-) (B5), cyclo(-Pro1-Trp-Ile-Pro2-Leu1-Thr-Pro3-Leu2-) (B6), cyclo(-Pro1-Tyr-Pro2-Ile1-Phe-Pro3-Ile2-) (B7), cyclo(-Pro1-Phe-Gly-Pro2-OMe- Glu-Leu-Trp-) (B8), cyclo(-Pro-Tyr1-Asp-Phe-Trp-Lys-Val-Tyr2-) (hymenamide J, B9), cyclo(-Pro-Phe-Asp-Ser-Lys-Ala-Val-Thr-Tyr-) (B10), aldisin (B11), 2-bromoaldisin (B12), 5-bromopyrrole-2-carboxamide (B13), 4,5-dibromopyrrole- 2-carboxamide (B14), 5-bromopyrrole-2-carboxylic acid (B15), dibromophakellin (B16), dibromoisophakellin (B17), monobromophakellin (B18), (Z)- debromohymenialdisine (B19), (Z)-hymenialdisine (B20), (Z)-debromoaxionhdantion (B21), (Z)-axionhdantion (B22), manzacidin C (B23), manzacidin A (B24), manzacidin B (B25), N-methylmanzacidin C (B26), and taurodispacamide A (B27). MALDI-TOF/TOF de novo sequence analysis of all cyclopeptides B1~B10 confirmed the NMR structure determination, and the absolute configuration of compound B1 was confirmed by CuKαX-Ray single crystal diffraction analysis. The five cyclopeptides B5, B6, B7, B8 and B10 were new compounds; compounds B2, B3, B4, B9, B23~B27 were isolated from this sponge for the first time. Compounds B9 and B10 were the hydrophilic cyclopeptides isolated from the n-Butanol fraction of genus Phakellia for the first time. Compound B10 appeared as two conformers in DMSO-d6, and the NMR data indicated a cis configuration for Pro in the major conformer and a trans configuration for Pro in the minor conformer. In the antitumor assays, compounds B3, B6 and B10 exhibited cytotoxicity against cancer cell line P388 with IC50 values of 8.3, 7.8 and 5.6μg/mL, respectively; compounds B1, B6 and B10 showed cytotoxicity against cancer cell line BEL-7402 with IC50 values of 12.7, 30.4 and 14.8μg/mL, respectively; compound B1 exhibited cytotoxicity against cancer cell line SPC-A-1 with an IC50 value of 11.6μg/mL; compounds B1, B3, B9 and B10 showed cytotoxicity against cancer cell line Colo-26 with IC50 values of 23.5, 11.9, 28.1, 26.9 and 42.7μg/mL, respectively.The ethanol extract of marine sponge T. swinhoei collected from Woody Island and Seven connected islets exhibited antitumor activity against cancer cell lines HAC (IC50 < 3μg/mL) and Colo-26 (IC50 < 1μg/mL). 14 compounds (C1~C14) were isolated and purified from this sponge by solvent extraction and chromatography methods including VLC, LPLC, MPLC, HPLC on silica gel, ODS C-18 and Sephadex LH-20. These compounds, including 1 triterpene, 6 steroids, 1 cyclopeptide, 5 miazines and 1 benzoic acid, were identified by 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC (HSQC), HMBC, NOESY (ROESY) and MS as: 32,35-anhydrobacteriohopanetetrol (C1), 7α-hydroxylconicasterol (C2), conicasterol (C3), theonellasterol (C4), 7α,14α-dihydroxylconicasterol (C5), 9α-hydroxyl-15- oxoconicasterol (C6), 3β,8β-dihydroxyl-4-methylene-B-norergosta-6-aldyhyde (C7), orbiculamide A (C8), thymine (C9), thymidine (C10), uracil (C11), thymidine-5'-carboxylic acid methyl ester (C12), thymidine-5'-carboxylic acid butyl ester (C13), and benzoic acid (C14). Compounds C6 and C7 were new 4-methylene steroids with 7-OH or 8-OH and B-nor framework; compounds C1, C12, C13 and C14 were isolated from this genus for the first time. In the antitumor assays, compounds C7 and C8 exhibited cytotoxicity against cancer cell line P388 with IC50 values of 40.3 and 0.65μg/mL, respectively; compound C8 showed cytotoxicity against cancer cell line BEL-7402 with an IC50 value of 1.7μg/mL; compounds C6, C7 and C8 exhibited cytotoxicity against cancer cell line SPC-A-1 with IC50 values of 27.9, 33.1 and 19.3μg/mL, respectively; compounds C5 and C8 showed cytotoxicity against cancer cell line Colo-26 with IC50 values of 24.7 and 2.3μg/mL, respectively.This dissertation describes the details of isolation and structure elucidation of new and bioactive compounds from three marine sponges collected from Paracel Islands of South China Sea by bioassay-guided separation. The results reveal the biodiversity and chemistry diversity of marine organism of Paracel Islands of the South China Sea, which is proved to be a potential resource of anticancer agents.