Discovery And Bioactivity Of Staurosporine Derivatives From Marine Actinomycete Streptomyces Sp.A68

Staurosporines are a class of compounds of potential values to the development of medicine due to strong cytotoxic activity and protein kinase inhibitory activity.So far,most indolocarbazole products are obtained from actinomycetes,myxomycetes,cyanobacteria and marine invertebrates,among which marine actinomycete,as a staurosporine metabolites producer,has been proven to be of a better research value.In this research,one marine strain Streptomyces sp.A68,which was isolated from marine sediment samples from Dongtou County,Zhejiang Province,was selected for further comprehensive study.Based on the one strain-many compounds strategy(OSMAC),several methods were used in this research to explore the diversification of indolocarbazole metabolites of the strain A68,such as screening of culture medium,chemical ion co-culture,screening of resistant mutant strains,etc.And,the cytotoxic and kinase inhibitory activities of all these isolated compounds were evaluated respectively at last.The experiment was conducted using methods of separation and purification such as Sephadex LH-20 column chromatography,silica gel column chromatography,and high performance liquid chromatography.Modern spectroscopic techniques such as LC-MS,IR,UV and NMR were used for identification.One staurosporines analogue staurosporine(1)and one indole derivative tryptophol(2)were isolated from the liquid culture extracts of the strain A68.Based on the OSMAC strategy,the abundance of fermentation products of the strain was compared before and after changing the solid substrate and culture condition,and the rice medium was screened for large-scale fermentation as a better medium.The method of adding calcium fluoride in rice medium was used to ferment and culture the strain A68,where the secondary metabolites were stimulated by chemical ions to obviously change its metabolites.The components with obvious changes were identified according to HPLC analysis.The method of chemical mutagenesis was used to induce the strain A68 taking diethyl sulfate as the mutagen,and a total of six mutants DES-M1-M6 were screened out.Antibiotic treatment-mediacted ribosomal mutation was used to improve the abundance of indolocarbazole metabolites.Also,Rifampicin,Gentamicin,Streptomycin,Norfloxacin and Erythromycin were treated as mutegens respectively,and a total of 13 mutants,the R-M1,G-M1-M3,G-M5-M9,N-M1-M2 and E-M1-M2,were screened out,among which,the Rifampicin-resistant strain R-M1 with the most abundant metabolites was identified as an object strain for further study.Twenty-two compounds,eight new compounds included,were isolated and identified from the fermentation of the strain A68.The eight new compounds include one sesquiterpene derivative(4),one quinazolinone derivative 6-(indol-10-ylcabonyl-quinazolone)(5),four bisphenol A analogues(9R,22R)-bisphenol A bis(9,22-hydroxy-10,23-anthranilicacid-propyl)ether(6),(9R)-bisphenol A(9-hydroxy-10-anthranilicacid-propyl)ether(7),(9R)-bisphenol A(9-hydroxy-10-anthranilicacid-propyl)(22,23-dihy droxy-propyl)ether(8),(9R)-bisphenol A(9-hydroxy-10-anthranilicacid-propyl)(22-hydroxyl-23-methoxy-propyl)ether(9)and two holyrine A derivatives,3'-epi-N-acetyl-holyrine A(11)and 3'-N-acetyl-holyrine A(13).The strain A68 was cultured in the rice solid medium containing calcium fluoride,and two components with obvious changes were isolated and purified,followed by being identified as the indole oxazoles derivatives SF2583B(25)and SF2583A(26).The dominant mutant R-M1 was cultured in the rice solid medium,and six indolocarbazole derivatives were isolated from its metabolites,including three new compounds,namely 2',3'-epi-streptocarbazoles C(27),7-methyl-k252c(29)and 3'N-formyl-holyrine A(30).Finally,the cytotoxic and kinase inhibitory activities of these compounds were evaluated respectively,showing that compound 1(staurosporine)exhibits the most potent inhibitory activities and 3'-epi-N-acetyl-holyrine A(11)shows remarkable anti-PC3 cells activity and PKCa,ROCK ? inhibitory activities with IC50 values of 0.81,0.17,0.21?M respectively.Compound 6 as a bisphenol A derivate,also has a good protein kinase inhibitory activity,with IC50 values of 1.32 and 3.24?M against PKCa and ROCK ?respectively.