【作者】 张红军；

【导师】 易杨华； 林厚文；

【作者基本信息】 第二军医大学 ， 药物化学， 2009， 博士

【摘要】 我国南海海洋生物中含有许多结构新颖且具有抗肿瘤、抗病毒、抗菌、抗炎、抗过敏和免疫调节等不同生物活性的次生代谢产物,它们是寻找高效低毒药物或先导化合物的新来源。本文作者对我国南海西沙海域的海洋生物资源进行了考察,对采集到的31种海绵样本的93个提取部位进行了体外活性筛选,发现有11种海绵的提取物具有显著的体外抗肿瘤活性。通过多种现代色谱分离纯化技术,从其中的3种海绵Phyllospongia foliascens、Phakellia fusca和Theonella swinhoei中共分离得到了66个单体化合物;采用多种波谱方法鉴定了其中63个化合物的结构,包括:17个甾体类化合物、12个萜类化合物、11个环肽类化合物、17个生物碱类化合物、5个嘧啶类化合物和1个苯甲酸,其中新化合物12个。在体外药理活性筛选中,部分二倍半萜、环肽和甾体类化合物显示出细胞毒活性。采自永兴岛和石岛的杯叶海绵P. foliascens的丙酮提取物显示对小鼠肝癌腹水瘤HAC细胞和小鼠结肠癌Colo-26细胞的IC50值均小于1μg/mL。运用溶剂分步萃取以及减压柱色谱、低压柱色谱、中压柱色谱、凝胶柱色谱和高效液相制备等多种色谱分离方法,从其石油醚和二氯甲烷萃取部位分离鉴定了共22个化合物A1～A22,包括11个甾体类化合物和11个二倍半萜类化合物。运用1H-NMR、13C-NMR、1H-1H COSY、HMQC (HSQC)、HMBC、NOESY (ROESY)和MS等波谱技术,结合文献,确定了它们的结构分别为: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), 5α,6α-epoxycholest-7- en-3β-ol (A22);其中,4个二倍半萜类化合物A12、A13、A15、A16和1个甾体类化合物A19为新化合物;10个甾体类化合物A1～A7, A20～A22为首次从该属海绵中分离得到的化合物。化合物A11和A12是通过HPLC分离获得的CH3-26立体异构的phyllofolactones类二倍半萜,它们的构型是通过碳谱的化学位移确定的。化合物A13和A14是目前仅从我国南海海绵中获得的2个环E为含α,β-不饱酮的五元碳环结构的phyllofolactones类二倍半萜成分。体外抗肿瘤活性筛选中,化合物A10, A13, A14, A15, A16, A17对小鼠白血病P388细胞显示有细胞毒活性,IC50值分别为15.3, 6.5, 11.9, 30.2, 25.6, 10.5μg/mL;化合物A13, A14对人肝癌BEL-7402细胞显示有细胞毒活性,IC50值分别为47.8, 32.9μg/mL;化合物A10, A15对人肺癌SPC-A-1细胞显示有细胞毒活性,IC50值分别为23.6, 29.3μg/mL;化合物A11, A12, A15对小鼠肝癌腹水瘤HAC细胞显示有细胞毒活性,IC50值分别为46.7, 32.5, 45.1μg/mL;化合物A10, A13, A14, A15对小鼠结肠癌Colo-26细胞显示有细胞毒活性,IC50值分别为23.5, 11.9, 28.1, 26.9, 42.7μg/mL。采自永兴岛和七连屿附近的棕色扁海绵P. fusca的乙醇提取物对小鼠结肠癌Colo-26细胞的IC50值小于1μg/mL,对小鼠肝癌腹水瘤HAC细胞的IC50值为6.4μg/mL。运用溶剂分步萃取以及多种色谱分离方法,从其二氯甲烷和正丁醇萃取部位分离鉴定了共27个化合物B1～B27,包括10个环肽类化合物和17个生物碱类化合物。运用1H-NMR、13C-NMR、1H-1H COSY (MQF-COSY)、HMQC (HSQC)、HMBC、TOCSY、HMQC-TOCSY、NOESY (ROESY)、MS和X-Ray等波谱技术,结合文献,确定它们的结构分别为: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), taurodispacamide A (B27)。所有环肽化合物B1～B10都通过MALDI TOF/TOF从头测序方法获得了氨基酸残基连接信息,确证了NMR的结构鉴定。其中,5个环肽类化合物B5, B6, B7, B8, B10为新化合物;化合物B2～B4, B9, B23～B27为首次从该种海绵中获得。化合物B9和B10为首次从Phakellia属海绵正丁醇萃取部位获得的水溶性环肽。化合物B1的绝对构型通过CuKαX-Ray单晶衍射实验得到了确认。化合物B10在DMSO-d6中显示具有两个构象存在,通过脯氨酸残基碳化学位移Δδβγ值和NOESY相关信号证实在其主要构象和次要构象中脯酰胺肽键分别为反式和顺式构型。体外抗肿瘤活性筛选中,化合物B3, B6, B10对小鼠白血病P388细胞显示有细胞毒活性,IC50值分别为8.3, 7.8, 5.6μg/mL;化合物B1, B6, B10对人肝癌BEL-7402细胞显示有细胞毒活性,IC50值分别为12.7, 30.4, 14.8μg/mL;化合物B1对人肺癌SPC-A-1细胞显示有细胞毒活性,IC50值为11.6μg/mL;化合物B1, B3, B9, B10对小鼠结肠癌Colo-26细胞显示有细胞毒活性,IC50值分别为23.5, 11.9, 28.1, 26.9, 42.7μg/mL。本文所获得的环肽和生物碱类成分的其它的生物活性值得进一步研究。采自永兴岛和七连屿附近的隋氏蒂壳海绵T. swinhoei的乙醇提取物对小鼠肝癌腹水瘤HAC细胞的IC50值小于3μg/mL,对小鼠结肠癌Colo-26细胞的IC50值小于1μg/mL。运用溶剂分步萃取以及减压柱色谱、低压柱色谱、中压柱色谱、凝胶柱色谱和高效液相制备等多种色谱分离方法,从其二氯甲烷和正丁醇萃取部位分离鉴定了共14个化合物C1～C14,包括1个三萜类化合物、6个甾体类化合物、1个环肽类化合物、5个嘧啶类化合物以及1个苯甲酸。运用1H-NMR、13C-NMR、1H-1H COSY、HMQC (HSQC)、HMBC、NOESY (ROESY)和MS等波谱技术,结合文献,确定它们的结构分别为: 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 methylester (C12), thymidine-5′-carboxylic acid butyl ester (C13), benzoic acid (C14)。其中,化合物C6和C7为首次发现具有7-OH或8-OH以及降B环结构的4-methylene甾体类新化合物;化合物C1, C12, C13, C14为首次从该种海绵中获得。体外抗肿瘤活性筛选中,化合物C7, C8对小鼠白血病P388细胞显示有细胞毒活性,IC50值分别为40.3, 0.65μg/mL;化合物C8对人肝癌BEL-7402细胞显示有细胞毒活性,IC50值为1.7μg/mL;化合物C6, C7, C8对人肺癌SPC-A-1细胞显示有细胞毒活性,IC50值分别为27.9, 33.1, 19.3μg/mL;化合物C5, C8对小鼠结肠癌Colo-26细胞显示有细胞毒活性,IC50值分别为24.7, 2.3μg/mL。本课题采用活性筛选追踪的方法,运用多种现代色谱分离技术,对采自中国西沙海域的海绵提取物的体外活性和化学成分进行了研究并有新的活性成分的发现。特别是对天然产物中数量相对较少的二倍半萜和环肽类化学成分进行了系统的分离、结构测定和活性研究,为寻找海洋生物活性成分积累了新的研究资料。从杯叶海绵P. foliascens中得到的二倍半萜成分、从棕色扁海绵P. fusca中得到的生物碱和环肽类成分以及从隋氏蒂壳海绵T. swinhoei中获得的甾体和环肽类化合物在结构上既具有各自生物种属的化学特征,同时也体现出西沙海绵的独特性。研究结果同时也揭示了我国南海海洋生物具有的生物和化学多样性,显示出了南海海洋生物资源具有的研究潜力,为进一步开发和利用我国丰富的海洋生物资料提供了科学依据。更多还原

【Abstract】 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.更多还原