Secondary Metabolites Of Five Flamentous Fungal Strains: Structures And Bioactivities

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.