【作者】 齐君；

【导师】 王长云；

【作者基本信息】 中国海洋大学 ， 药物化学， 2013， 博士

【摘要】 由于陆生环境日益恶化，陆生资源日益匮乏，严重威胁人类生命健康的疑难病症频频发生，对新药的需求与日俱增。因此，人们把寻求新药的希望寄托在海洋这个特殊的生态系统。高压、高盐、低温、缺氧等特殊的海洋生态环境造就了海洋生物的特殊性和多样性，赋予了海洋天然产物结构的多样性和新颖性。结构多样的海洋天然产物也具有独特、显著的生物活性。其中，相当一部分海洋天然产物具有抗菌活性。研究表明海洋无脊椎动物及其共附生微生物是丰富的抗菌资源。本研究以三株海洋来源真菌及四种短指软珊瑚为研究对象，以抗菌活性筛选为指导，进行次级代谢产物研究，以期获得具有抗菌活性的化合物，从而为海洋活性天然产物以及海洋药物先导化合物的筛选和发现提供基础资料。本研究对3株海洋来源的真菌，青霉属真菌(Penicillium sp.)，茎点属真菌(Phoma sp.)，黄蓝状菌(Talaromyces sp.)和4种同时、同地采集的短指软珊瑚(Sinularia sp.)为研究对象，综合运用正、反相硅胶柱层析，SephadexLH-20凝胶柱层析及半制备HPLC等分离手段，NMR、MS、IR、UV、CD等波谱学技术，并结合化学方法，分离并鉴定了110个化合物，其中新化合物21个(1–3,6–8,11–14,36–38,40,50,66,80,81和91)。涉及9种结构类型，包括：异香豆素及苯并呋喃类化合物21个，色原酮类化合物10个，联苯醚类化合物6个，甾体化合物40个，萜类化合物11个，芳香族化合物7个，含氮化合物12个，醌类化合物2个以及内酯类化合物1个。从真菌Penicillium sp.中发现了仅在C-4位有取代基，而C-3位没有取代基的罕见的异香豆素类化合物(1,1A-1和1A-2)，运用Mosher法及CD量化计算的方法确定了化合物的绝对构型；从该菌中还发现含脂肪侧链的异香豆素类化合物(2–5)，通过与相似化合物比较CD谱图，结合Mosher法，确定了其绝对构型。从短指软珊瑚Sinularia sp.中首次分离获得了芳香化的amphilectane-type二萜化合物(80和81)。对分离获得的部分化合物进行了抗菌活性评价，此外还通过细胞毒、抗藤壶幼虫附着、斑马鱼胚胎毒性和卤虫致死等生物活性模型，对分离获得的单体化合物进行了生物活性评价，发现具有抗菌、细胞毒活性的化合物25个；具有抗藤壶幼虫附着、卤虫致死以及斑马鱼胚胎毒活性的化合物24个。Amphilectane二萜类化合物对Escherichia coli， Staphylococcus albus以及Vibrio anguillarum的抑制活性与阳性药环丙沙星相当，其中化合物80对S. albus的MIC为0.156μM，强于阳性药(MIC0.312μM)的活性。甾体类化合物15，18，21和22对鳗弧菌(Vibrio anguillarum)显示了强的抗菌活性，其MIC分别为0.39,0.78,0.78和0.39μM。甾体四环系统中的酮羰基、环氧以及过氧环基团是甾体类化合物抗菌活性的有效官能团。联苯醚类化合物显示了较强的抗菌活性，其中化合物39的抗菌活性明显高于化合物36和38的活性，表明苯环上甲氧基取代和糖基化作用会导致抗菌活性的减弱。除此之外，异香豆素类化合物2，4，5和7对斑马鱼胚胎的24h卵凝结的EC50值分别为6.42，5.68，8.87和7.13μg/mL，对斑马鱼胚胎72h致死IC50值分别为4.07，3.95，6.48和4.15μg/mL。构效关系研究发现，异香豆素类化合物C-3位脂肪侧链的存在和C-4位羟甲基的乙酰化增强了化合物的斑马鱼胚胎毒活性，C-7位被羟基取代后活性明显降低。研究结果表明，异香豆素及苯并呋喃类化合物、9,11-开环甾体、发生电子迁移的甾体类化合物以及联苯醚类化合物显示了多种生物学活性。对相同时间采自相同地域的四种短指软珊瑚Sinularia sp.1(GX-WZ-27-49)，Sinularia sp.2(GX-WZ-32-47)，Sinularia sp.3(GX-WZ-28-33)，Sinularia sp.4(GX-WZ-46)中的次级代谢产物进行了比较研究。结果表明，相同时间采自相同地域的同属不同种的短指软珊瑚的次级代谢产物结构类型存在差异，包括9,11-开环甾体、amphilectane二萜、孕甾、过氧化倍半萜等多种结构类型。本发现对该属短指软珊瑚的化学分类学研究具有一定的借鉴意义。综上所述，本研究通过抗菌活性筛选和化学筛选，从三株海洋来源真菌和四种短指软珊瑚中分离获得了结构类型丰富多样的海洋天然产物，运用Mosher法及ECD法确定了化合物的绝对构型。抗菌活性构效关系初步表明共轭酮羰基、环氧及过氧环基团是甾体类化合物的有效官能团。Amphilectane二萜的芳香化可以增强其抗菌活性。生物活性筛选评价表明，分离获得的化合物具有综合的化学生态学效应。本研究发现了海洋来源真菌和短指软珊瑚中的次级代谢产物具有丰富多样的结构类型和显著的抗菌活性，为海洋抗菌药物先导化合物的开发应用研究提供了化合物基础。更多还原

【Abstract】 Since terrestrial environment is deteriorating, terrestrial resources areincreasingly scarce, and the incurable diseases, a serious threat to human life andhealth, occur frequently. The demand for new drugs is increasing. Therefore, the hopeof seeking new drugs was put on the marine ecology environment. Marine ecologyenvironment which have a high pressure, high salt, hypothermia, hypoxia and otherspecial environmental characteristics may create abundant biodiversity which can beendowed with unique chemical diversity and distinct bioactivities. Among them, avariety of marine natural products exhibited antibacterial activities. Marineinvertebrates and microorganisms from coral reef ecosystem, rich in antibacterialsecondary metabolites, have become important bioresources for screening anddiscovery of lead compounds and marine drugs. In this study, the chemical diversityand antibacterial activity of secondary metabolites of three marine-derived fungi andfour soft corals Sinularia sp. have been investigated. These researches providedfoundations for the screening and discovery of bioactive marine natural products andlead compounds of marine drugs.In this study, under the guidance of the chemical and bioactive screening, threemarine-derived fungi including Penicillium sp., Phoma sp., Talaromyces sp. and foursoft corals Sinularia sp. collected from the same area at the same time wereinvestigated for their bioactive secondary metabolites. The compounds were isolatedby column chromatography on silica gel, Sephadex LH-20and preparative reversedphase HPLC, and identified by spectroscopy of NMR, MS, IR, UV, CD as well aschemical methods. From these organisms,110compounds were obtained, including 21new compounds (1–3,6–8,11–14,36–38,40,50,66,80,81and91). More than9structural types were found, including21isocoumarins and benzofurans,10chromones,6diphenyl ethers,40steroids,11terpenoids,7aromatic compounds,12nitrogen compounds,2quinone derivatives and1lactone. Compounds1,1A-1and1A-2were isolated from the sponge-derived fungus, Penicillium sp., which representa rare naturally occurring isocoumarin derivative with4-substitution, but nosubstituent at the3-position. The relative and absolute configurations of1weredetermined by quantum chemical calculation of its ECD spectrum combined with themodified Mosher’s method. The isocoumarins (2–5) with side chain at C-3, were alsoobtained from the Penicillium sp. fungus. The absolute configurations of compounds2–5were assigned by the modified Mosher’s method together with comparison of CDspectra. The new aromatic amphilectane-type diterpenes (80and81) were isolatedfrom Sinularia sp. for the first time.The isolated compounds were evaluated their antibacterial activity and otheractivities by various of screening modles including antifouling, ichthyotoxic, lethalityand so on. A variety of bioactive compounds had been found, including25antibacterial and cytotoxic activity compounds and24antifouling, lethality activity,and ichthyotoxic activity compounds. Amphilectane-type diterpenes showed stronginhibitory activity against Escherichia coli, Staphylococcus albus and Vibrioanguillarum. Compound80displayed strong inhibitory activity against S. albus withan MIC value of0.156μM, which was stronger than that of the positive controlciprofloxacin (MIC0.312μM). Steroids15,18,21and22exhibited inhibitory activityagainst Vibrio anguillarum with MIC values of0.39,0.78,0.78and0.39μM,respectively, which were stronger than or equivalent to that of the positive controlciprofloxacin. The results of antibacterial activity suggested that the ketone carbonylgroups, epoxy rings and peroxy groups in steroids played apparent role on theantibacterial activity. The diphenyl ethers exhibited antibacterial activity. Compound39displayed better potent antibacterial activity than compounds36and38, with thesubstituted methoxy and hexose residue. These results indicated that the antibacterialactivity might be weakened due to the substituent of methoxy and hexose residue. And the isocoumarins2,4,5and7displayed24h coagulated eggs of zebrafish at theconcentrate of6.42,5.68,8.87and7.13μg/mL,72h death of zebrafish at theconcentrate of4.07,3.95,6.48and4.15μg/mL, respectively. The study ofstructure-activity relationships about isocoumarins showed that the presence of3-substituted side chain and the acetylation of hydroxymethyl at C-4increased thezebrafish toxicity. The substituted hydroxyl at C-7probably weakened thezebrafish toxicity. The bioactive results indicated that isocoumarins, benzofurans,9,11-secosterols,13(14→8)abeo-8-ergostane and diphenyl ethers exhibited a varietyof biological activities.The secondary metabolites of four soft corals Sinularia sp.1(GX-WZ-27-49),Sinularia sp.2(GX-WZ-32-47), Sinularia sp.3(GX-WZ-28-33), Sinularia sp.4(GX-WZ-46) collected from the same area at the same time were compared andanalyzed. It indicated that the different species in Sinularia genus collected from thesame area at the same time shared similarities and differences. The secondarymetabolites included many structural types, such as9,11-secosterols,amphilectane-type diterpenes, pregnane, cyclic sesquiterpene peroxides, and so on. Itsupplied foundation for further investigation for the chemotaxonomic significance ofthe Sinularia genus.In conclusion, in present study under the guidance of the chemical and bioactivescreening, bioassay-guided fractionation of three marine-derived fungi and four softcorals Sinularia sp. led to the isolation of a variety of bioactive natural products. Theabsolute configurations of isocoumarins were assigned by the modified Mosher’smethod and TDDFT ECD calculation together with comparison of CD spectra. Theresults of antibacterial activity suggested that the ketone carbonyl groups, epoxy ringsand peroxy groups in steroids played apparent role on the antibacterial activity. Thearomatization of amphilectane-type diterpenes increased the antibacterial activity. Theresults of bioactive evaluation indicated that the isolated compounds exhibited avariety of biological activities, and some compounds have been found to possessecological activity and pharmacological activities at the same time. The studyindicated that the secondary metabolites of marine-derived fungi and soft corals display chemical diversity and pronounced antibacterial activity. It providedfoundations for the discovery and exploration of antibacterial marine natural products.更多还原