Studies On The Optimization Of Staurosporine Producing Strains And Synthesis Of Staurosporine Derivatives

The unique structure and good biological activity of indolecarbazole alkaloids draw attention and rouse interest of organic chemists and pharmaceutical scientists, in which staurosporine (ST) is the representative for its strong protein kinase C (PKC) inhibition. In this dissertation, a series of research work were based on ST, including the optimization of ST producing strains, isolation and identification of ST, and the synthsis of halo-derivatives of ST. First work includes screening ST producing strains, breeding high-yielding strains, metabolic regulation, secondary metabolites of mixed culture and staurosporine analogs from the high-yield mutant strain; In addition, after the scale fermentation of producing strain, we established the methods for the extraction and purification of ST, and ST derivatives were also synthesized to find new protein kinase C inhibitors of good activity and low toxicity.The actinomycetes preserved in our laboratory were seperated from the mud of Jiaozhou Bay, Qingdao and from the salt pond sedimen of Qingdao Dongfeng salt. After an extensive screening, we got three streptomyces to produce ST, i.e S. fradiae 007, S. arenae Z4007, and S. rubrolavendulae THW-12. However, the ST output of is low, only about 1μg/ml. In order to improve the fermentation titer of ST producing strains, ultraviolet and nitrosoguanidine (NTG) were used as mutagens in treating S. fradiae 007, S. arenae Z4007 and S. rubrolavendulae THW-12. As a result, three mutant strains with high yield of ST, i.e. S. fradiae M297, S. fradiae M301 and S. fradiae M315, were obtained from S. fradiae 007, in which the titer of M315 was 9.06μg·mL-1, nine times as that of the original 007 (1.02μg·mL-1). The genetic stability of high-producing strains was confirmed by experiments.In order to further improve the fermentation titer, we also carried out the work of metabolic regulation and mixed culture. We hoped that the new secondary metabolic pathways were induced by the addition of metabolic precursors, enzyme inhibitors, and competitive growth factors. Preliminary studies of five common methylation inhibitors (D-methionine, D, L-ethionine, S-adenosyl-L-homocysteine, sinefungin) showed that after the addition of sinefungin in the culture, THW-12 could produce demethylstaurosporine with the lower yield. Mixed culture was investigated on the influence of secondary metabolites by the use of Penicillium citrinum WC29-5, an epiphytic fungus with the mangrove plant, Aegiceras corniculata, and S. Fradiae 007. Ten compounds (1-10) were isolated and identified from the mix culture. The absolute configuration of compounds 6 was determined for the first time, and its cytotoxic activity was evaluated for the first time with IC50 values of 0.9 and 0.8μM on A549 and HL-60 cell lines, respectively. The results showed that secondary metabolites of mixed culture had great changes with that of the pure culture and the further research need to be carried out.After optimization of fermentation conditions, M315 was cultured in a 250L- large scale. Ten indolocarbazole alkaloids (11-20) including ST were isolated and identified. Among them, compounds 11-13 were new compounds with different substituents at postion 4’N, in which compoud 11 is a nitrile group, compoud 12 is a tryptophan moiety, and compoud 13 is a carbonoamino group.In order to obtain the halogenated derivatives of PKC412 and UCN-01, two anticancer drugs in clinical trial, we have designed and synthesized 45 new ST analogues belonged to three types substituted at the positions of C-7, N-4’ and aromatic ring. And "me-too" or "me-better" drugs were also expected. The antitumor activities were evaluated in cellular and molecular level by the MTT or SRB assays. Compounds 116, 117, 123, 194, 207, 222 showed significant cytotoxic activity against A549 and HL-60 cell lines with IC50 values of 0.8、0.5、5.1、1.0、0.6、0.4 and 1.1、1.4、4.9、1.1、0.9、0.9μM, respectively. Compouds 123, 124, 126, 204, showed significant inhibitory effect on PKCβ2 with IC50 values of 22.3,14.4, 37.0, 24.5 nM, respectively.In summary, we have got three staurosporine producing strains, and the fermentation titers were improved by the compound mutation method; effects on secondary metabolites were also investigated by adding metabolic precursor, methyltransferase in the culture, and growth competition between microbial species. Ten compounds were isolated and identified from the mixed culture broth, one of which displayed obvious cytotoxic activity. Besides, ten indole carbazole alkaloids including ST were obtained from the scale fermentation of mutant strain, 3 of which were new compounds. Forty-five new ST analogues belonge to three types also had been synthesized, some of which showed significant cytotoxicity on HL-60 and A549 cells and PKCβ2 inhibition.