Synthesis Of Uracil Derivatives And Establish The Determination Method Of Thymidylate Synthase Inhibitors Activity

Thymidylate synthase(TS) as a folate-dependent enzyme, catalyzes the reductive methylaton of dUMP by CH2H4folate to produce dTMP, which is subsequently metabolised to thymidine triphosphate (dTTP), exclusively for incorporation into DNA during synthesis and repair. TS plays a very important role in cell proliferation. The tumor cell proliferation and growth were suppressed by restaining the activity of TS. In recent years, TS becomes a hot target in cancer chemotherapy. Inhibitor of TS is an important direction of anticancer research. Consequently, achievements of this research are as follows:1Expression and purification of TS protein:The laboratory recombinant strains TS-pET-28b(+)/Rosseta(DE3) was used to express TS protein, TS molecular weight was36.0kDa, the yield was about42.1%of total protein content, best expression condition were:37℃,8h,IPTG concentration of1.5mM. The recombinant human TS with6*His-tag was purified by Ni-NTA affinity chromatography, which purity was up to90%.2Synthesis of mTHF:The optimized reaction condition was explored. From the point of purity, the optimum reaction conditions were60℃,1.5h, Na2S2O41.5g. The purity was up to66.1%.3Synthesis of compound A:This compound has two parts, one is uracil derivatives; another is quinazoline. According to pertinent literature, designed the route of synthesis as follows:inhibitor A was synthesized form m-toluidine through salt formation, oximation, hydrolysis, oxadative, cyclization and bromination reaction. The synthesized compounds were confirmed by melting point and1HNMR.4Activity determination of TS protein and inhibitor:By the method of ultraviolet spectrophotometer, the enzyme activity determined was0.0137U, and specific activity was0.11U/mg. The inhibitory concentration50%(IC50) of Tomudex and LY231514to refolding TS was respectively6.5×10-6M、7.4×10-5M. Intermediates6-9IC50was respectively:3.45×10-2M,4.62×10-4M,7.84×10-5M、4.62×10-5M.