Inhibition Assay: The inhibitory activity of the compound against human dUTPase was determined by measuring the production of [5-3H]deoxyuridine monophosphate from [5-3H]deoxyuridine triphosphate.
Inhibition Assay: The inhibitory activity of these compounds of the present invention against human dUTPase was determined by measuring the production of [5-3H]deoxyuridine monophosphate (hereinafter, referred to as [5-3H]dUMP) from [5-3H]deoxyuridine triphosphate (hereinafter, referred to as [5-3H]dUTP) according to a method shown below.Specifically, 0.2 mL in total of a solution containing 0.02 mL of 1 uM dUTP (including 588 Bq/mL [5-3H]dUTP), 0.05 mL of a 0.2 M Tris buffer solution (pH 7.4), 0.05 mL of 16 mM magnesium chloride, 0.02 mL of 20 mM 2-mercaptoethanol, 0.02 mL of a 1% aqueous solution of fetal bovine serum-derived albumin, 0.02 mL of varying concentrations of test compound solutions or pure water as a control, and 0.02 mL of a solution of human dUTPase, which is expressed in E. coli and then purified, was incubated at 37 C. for 15 minutes. After the incubation, the solution was heated at 100 C. for 1 minute to terminate the reaction, followed by centrifugation at 15000 rpm for 2 minutes.