【作者】 杜林；

【导师】 顾谦群；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 特殊的生态环境孕育了特殊的生物多样性，而特殊的生物多样性往往孕育着特殊的化学多样性和生物活性的多样性。为了寻找骨架新颖且具有良好治疗作用的新药先导化合物，本论文以不同生源环境分离得到的海洋和陆生真菌作为研究对象，开展活性次级代谢产物的结构和生物活性研究。研究主要内容包括：活性菌株的分离筛选；代谢产物的分离纯化及化学结构的鉴定和构型的确定；新化合物生合成途径的探索；单体化合物生物活性的初步评价；活性化合物作用靶点和活性机制的初步探讨。从采集自不同纬度（山东、福建、广东、海南）的24个潮间带海泥、红树林根泥和火山口沉积物样品中共分离丝状真菌390株，采用海虾生物致死法和tsFt210细胞镜检细胞毒活性筛选模型，结合TLC及HPLC化学筛选，得到20株活性菌，从中选取火山口沉积物来源的桔青霉Penicillium citrinum HGY1-5和红树林根部海泥来源的灰绿曲霉Aspergillus glaucus HB1-19，以及其他来源的三株真菌—深海海泥来源的青霉Penicillium sp．F23-2和青霉Penicillium sp．F1，潮间带海泥来源的木霉Trichoderma sp．f-13—作为本论文的研究对象。对5株目标活性菌株发酵产物运用萃取，薄层色谱，正相、反相硅胶柱色谱，LH-20凝胶柱色谱，反相高压液相等化学分离纯化手段，从桔青霉P．citrinumHGY1-5的代谢产物中分离得到47个单体化合物（1-47）；从灰绿曲霉A．glaucusHB1-19的代谢产物中分离得到了34个单体化合物（42，43，48-79）；从青霉Penicillium sp．F23-2的代谢产物中分离得到11个单体化合物（80-90）；从青霉Penicillium sp．F1的代谢产物中分离得到了10个单体化合物（91-100）；从木霉Trichoderma sp．f-13的代谢产物中分离得到了11个单体化合物（46，101-110）；另外，以桔青霉P．citrinum HGY1-5对甾体进行生物转化得到三个单体化合物（111-113）；对桔霉素二聚体17的化学转化得到一个人工产物（114），共114个化合物。继而，利用理化性质和波谱学方法（IR，UV，MS，NMR，CD，X-ray）结合化学反应的方法（Mosher法，对溴苯甲酰酯化等）阐明了全部1 14个化合物的化学结构（Fig．1），结构类型按生合成来源分类包括：甾体类化合物21个（1-16，40，41，111-113），萜类化合物6个（85-90），聚酮类化合物56个（17-39，42-45，48-75，114），生物碱类化合物19个（76-84，91-100），sorbicillin衍生物11个（46，101-110），脑苷酯类化合物1个（47）。其中发现新化合物63个，包括13个具有罕见的bicyclo[4．4．1]A／B环的C25甾体化合物（1，3-13，16），2个新骨架的桔霉素三聚体（19，20），13个结构新颖的桔霉素二聚体（17，18，21-24，26-28，32-34，114），2个含有新颖的naphtho[1，2，3-de]chromene-2，7-dione骨架的灰绿霉素类聚酮化合物（48，49），2个含有新颖的spiro[5．5]undecane螺环骨架的灰绿霉素类聚酮化合物（50，51），14个蒽型的或萘型的芳香聚酮化合物（52-54，56，57，59-61，63-68），2个meleagrin型生物碱（80，81，其中80是一个新颖的meleagrin型生物碱与conidiogenone型二萜链接形成的复合化合物），2个roquefortine型二酮哌嗪生物碱（83，84），6个罕见的conidiogenone型二萜（85-90，目前仅两例报道），5个brevicompanine型二酮哌嗪生物碱（91-93，96，97），2个sorbicillin衍生物（101，107）。在研究中，我们首次采用稳定同位素标记，化学结构转化和甾体微生物转化的方法确定了C25甾体（1-16）骨架的生合成来源和各侧链类型化合物的亲缘关系；我们对桔霉素（36）人工聚合和降解反应机制进行了探索研究；采用X-ray单晶衍射方法确定了新骨架化合物灰绿霉素A（48）的结构，采用同位素标记结合NMR的方法确定了螺环新骨架化合物灰绿霉素C（50）的结构，并进一步采用在线手性HPLC-CD和计算机量子化学CD模拟相结合的方法，确定了灰绿霉素C外消旋混合物的本质及两个对映异构体的绝对构型；采用NOESY和CD相结合的方法首次确定了conidiogenone型二萜（85-90）的绝对构型；采用NOESY和氨基酸手性HPLC分析相结合的方法确定了brevicompanine型二酮哌嗪生物碱（91-93，96，97）的绝对构型。为了寻找化合物的治疗作用，我们采用抗肿瘤和神经保护两种不同的策略对单体化合物的生物活性进行评价。首先，采用MTT法、SRB法以及流式细胞术结合形态学检测的方法，对分离获得的单体化合物的抗肿瘤活性进行了初步评价，从中筛选出了20个活性化合物(细胞毒IC₅₀＜10μM)（17-22，24，46，48，49，61，80-82，86-88，90，102，114），进一步采用ELESA和Western blot等方法对活性化合物的分子靶点和作用机制进行初步探索。其中：1．新骨架化合物灰绿霉素A（48）对A-549和HL-60细胞的IC₅₀分别为0．13和0．28μM；该化合物在10μM时对蛋白激酶具有抑制作用，抑制率分别为Src（40．5％），KDR（43．4％）；在该浓度下灰绿霉素A（48）还可以明显的抑制拓扑异构酶Ⅱ的活性；初步的实验表明灰绿霉素A（48）对小鼠S₁₈₀肉瘤模型具有体内抗肿瘤活性；目前对灰绿霉素A（48）的临床前药效学评价正在深入进行当中。2．桔霉素聚合物17-20和114在1-10μM浓度下可以明显的诱导HL-60细胞发生凋亡，其作用具有时间依赖性和浓度依赖性；它们在10μM浓度下可以效应性的激活凋亡相关蛋白Caspase 3和8以及PARP，是一类新颖的肿瘤细胞凋亡诱导剂。3．Meleagrin生物碱82在10μM浓度下可以将HL-60细胞周期阻滞在G₂／M期，推测其作用机制可能与其9-OCH₃类似物oxaline一样，通过抑制微管蛋白聚合发挥作用；而生物碱与二萜链接型新骨架化合物meleagrin B（80）在10μM和5μM浓度下可以明显的诱导HL-60细胞发生凋亡，推测二萜片断的引入可能使其作用靶点发生了改变。4．Sorbicillin衍生物（46，101-110）对HL-60细胞的细胞毒测试表明，山梨酰基侧链2，3-双键还原会使相应的化合物细胞毒活性下降2-10倍，说明此类化合物侧链共轭系统与其抗肿瘤活性密切相关。对于100μM浓度下没有表现出明显的细胞毒活性的化合物，采用LPS诱导的BV2小胶质细胞炎症拮抗模型或G-蛋白偶联受体12（GPR12）激活模型评测其潜在的神经保护活性，首次发现10个新活性化合物（1，2，8，9，12，91，92，94，96，97）。其中：1．Brevicompanine型二酮哌嗪生物碱92和97在0．1μg／ml～80μg／ml浓度范围内对LPS诱导的BV2小胶质细胞炎症模型具有明显的抑制活性，这是首次发现此类二酮哌嗪生物碱的抗炎活性。2．C25甾体化合物1，2，8，9和12在10μM浓度下可以明显的激活GPR12，促进cAMP的释放。这是首次发现C25甾体化合物潜在的治疗活性。本论文从5株真菌次级代谢产物中共分离鉴定了63个新化合物，发现了五种新颖的化合物骨架类型，确定了其绝对构型，并对C25甾体，桔霉素衍生物和灰绿霉素类聚酮等新骨架化合物的生合成进行了系统的研究，丰富了天然产物的化学结构类型和生合成内容。经活性评价，共筛选出30个活性化合物（包括24个新化合物），其中发现了一个新骨架的抗肿瘤先导化合物灰绿霉素A（48），该化合物具有新颖的naphtho[1，2，3-de]chromene-2，7-dione骨架，体外细胞和分子水平有效，体内初步评价有效，是一个新颖的多靶点作用的抗肿瘤先导化合物；发现了两类新骨架的肿瘤凋亡诱导剂桔霉素聚合物17-20和生物碱与二萜链接型化合物meleagrin B（80），为新药研究提供了重要的先导结构。共分离筛选获得20株抗肿瘤活性菌株，发现了三株能产生系列新骨架抗肿瘤活性化合物的真菌灰绿曲霉A．glaucus HB 1-1 9，桔青霉P．citrinum HGY1-5和青霉Penicillium sp．F23-2，为开发真菌抗肿瘤药物提供了重要的药用资源活性菌株。更多还原

【Abstract】 Special environment may create specific biodiversity which can be endowed withunique chemical diversity and distinct bioactivities.In order to look for new leadcompounds with novel skeletons and potent bioactivities,a study was carried out toinvestigate the bioactive compounds derived from marine and terrestrial fungi.Studies include isolation and screening of bioactive fungal strains,purification andstructural elucidation of the secondary metabolites,exploring the biosyntheticpathways of the novel compounds,preliminary evaluation for bioactivities of purecompounds and the potential mechanisms and targets of the active compounds.390 fungal strains were isolated from 24 samples,including near shore sediment,sediment around the mangrove roots,and volcano ash,collected at distinct latitudes（Shandong,Fujian,Guandong,and Hainan）.Using brine shrimp lethality assay andtsFT210 cell line as bioactive screen model,together with applying the TLC andHPLC chemical screen assays,20 fungal strains were detected the cytotoxic activity.Among the bioactive strains,a volcano-ash derived fungus Penicillium citrinumHGY1-5 and a mangrove root sediment derived fungus Aspergillus glaucus HB1-19were picked out as the aimed strains.Two deep ocean sediment derived fungiPenicillium sp.F23-2 and Penicillium sp.F1,and an inshore sediment derived fungusTrichoderma sp.f-13 from other sources were also chosen. In all,114 compounds were isolated and purified,from the bioactive extracts of the5 aimed strains,by using solvent extraction,silica gel column,Sephadex LH20,PHPLC and etc.From fungus P.citrinum HGY1-5,47 compounds （1-47） wereisolated;from A.glaucus HB1-19,34 compounds （42,43,48-79） were isolated;fromPenicillium sp.F23-2,11 compounds （80-90） were isolated;from Penicillium sp.F1,10 compounds （91-100） were isolated;from Trichoderma sp.f-13,11 compounds （46,101-110） were isolated.Additionally,3 compounds （111-113） were obtained bybiotransformation of steroids using P.citrinum HGY1-5 and another compound （114）was prepared by chemical transformation of the citrinin dimmer 17.By means ofphysico-chemical properties and spectral analysis （IR,UV,MS,NMR,X-ray,etc.）,structures of all the 114 pure compounds were respectively determined.Among them there are 21 steroids （1-16,40,41,111-113）,6 diterpenes （85-90）,56polyketides （17-39,42-45,48-75,114）,19 alkaloids （76-84,91-100）,11 sorbicillinderivatives （46,101-110）,and one cerebroside （47）.63 compounds are new,including13 C25 steroids （1,3-13,16） with rare bicyclo[4.4.1] A/B rings,2 citrinin trimers （19,20） with novel skeletons,13 novel citrinin dimmers （17,18,21-24,26-28,32-34,114）,2 aspergiolide polyketides （48,49） with novel naphtho[1,2,3-de]chromene-2,7-dioneskeltons,2 aspergiolide polyketides （50,51） with novel spiro[5.5]undecane skeletons,14 anthracene or naphthalene polyketides （52-54,56,57,59-61,63-68）,2 meleagrinalkaloids（80,81,80 is a novel complex compound with a meleagrin alkaloid moietyand a conidiogenone diterpene moiety）,2 roquefortine diketopiperazine alkaloids （83,84）,6 conidiogenone diterpenes （85-90）,5 brevicompanine diketopiperazine alkaloids（91-93,96,97）,and 2 sorbicillin derivatives （101,107）.In the study,we firstinvestegated the biosynthetic origin of the C25 steroids and the chemical relationshipsof the distict side chains by the combination of the stable isotope labeling experiments,chemical transformation,and microbial transformation of several steroids;weexplored the polymerization and degradation mechanism of citrinin （36）.We firstdetermined the structure of the novle compound aspergiolide A （48） using X-raycrystallographic analysis.We first elucidated the constitution of a novel spirocyclicaromatic polyketide aspergiolide C （50） by a combination of spectroscopic methodsand isotope-labeling experiments.Aspergiolide C is a racemic mixture,its twoenantiomers were resolved by HPLC on a chiral phase and their absoluteconfigurations were assigned online,right from the peaks in the chromatogram,by acombination of HPLC-CD and quantum chemical CD calculations.We first established the absolute configurations of the conidiogenone diterpenes （85-90） by thecombination of NOESY and CD spectra.We established the absolute configurationsof the brevicompanine diketopiperazine alkaloids （91-93,96,97） by the combinationof the NOESY spectrum and chiral HPLC analysis for amino acids.In order to look for the potential curing effects of the compounds isolated,twodifferent strategies,antitumor and neuro-protection were applied.The antitumoractivities against several cancer cell lines of these compounds were assayed by MTT,SRB and flow cytometry methods.Among them 20 compounds （17-22,24,46,48,49,61,80-82,86-88,90,102,114） with significant bioactivities (cytotoxic,IC₅₀＜10μM)were found.The molecular targets or mechanisms of the active compounds werefurther explored using the methods such as ELESA and Western blot.1.The novelcompound aspergiolide A （48） showed potent cytotoxic activities against the A-549and HL-60 cell lines with the IC₅₀ values at 0.13 and 0.28μM.At 10μM,aspergiolideA （48） inhibited the protein kinases Src and KDR with the inhibitory rates at 40.5%and 43.4%,respectively.At the same concentration,it could also inhibitedtoposiomerose II potently.Aspergiolide A （48） also showed in vivo antitumor activitieson the S₁₈₀-sarcoma mice model.2.Citrinin trimers and dimers 17-20 and 114 couldinduce HL-60 cell apoptosis at 1-10μM.The activities were confirmed by the effectsof these compounds to activate caspases 3 and 8 and also PARP at 10μM.3.Meleagrin alkaloid 82 could block HL-60 cells through G₂/M phase at 10μM.Itindicted that this compound may inhibit tubulin polymerization as its 9-OCH₃ analogoxaline.The novel complex compound meleagrin B （80） could induce HL-60 cellapoptosis at 5 and 10μM.It indicated that the introduction of the diterpene moietyinto the structure may alter the the target of the alkaloid.4.The cytotoxic assay ofsorbicillin dericatives （46,101-110） showed both monomers and dimers withfull-unsaturated side chains all showed higher cytotoxity than the correspondingdihydro analogs.The potential neuro-protection activities of those without obvious cytotoxicactivities at 100μM were evaluated using the lipopolysaccharide （LPS）-inducedinflammation inhibition assay in BV2 microglial cell line or the cAMP generationassay in GPR12-CHO and WT-CHO cells.Ten compounds （1,2,8,9,12,91,92,94,96,97） were active,including:1.Brevicompanine diketopiperazine alkaloids 92 and97 showed potential anti-inflammatory effects on LPS-induced inflammatory model in BV2 microglial cells and Raw264.7 macrophages at 0.1μg/ml～80μg/ml.This isthe first report of the anti-inflammatory effect of such diketopiperazine alkaloids.2.C25 steroids 1,2,8,9 and 12 could induce the production of cAMP by activatingGPR12.This is the first report of the therapeutical effect of the C25 steroids.In this study,63 new compounds were isolated and identified from 5 aimed fungalstrains.Five novel skeleton types were discovered.We established their absoluteconfigurations and systematically studied the biosynthesis of some novle skeletoncompounds including C25 steroids,citrinin derivatives and aspergiolide polyketides.It enriched the chemical structure types and biosynthetic contents of natural products.Furthermore,30 active compounds were discovered （including 24 new compounds）including an antitumor lead compound aspergiolide A （48） with novelnaphtho[1,2,3-de]chromene-2,7-dione skeleton.This compound is active both in vitroand in vivo.It is a novel molecular-targeted antitumor lead compound which hasentered pre clinical study phase.We also discovered novel apopotosis inducers citrninderivatives 17-20 and meleagrin B （80）.It provided important lead structures for drugdiscovery.Twenty antitumor strains were screened from 390 fungal strains isolatedfrom 24 marine or terrestrial samples.Three important fungal strains,Aspergillusglaucus HB 1-19,Penicillium citrinum HGY1-5,and Penicillium sp.F23-2,which canproduce novel antitumor compounds,were discovered.It provided importantbioactive fungal strains for drug discovery.更多还原