Reaction #829479

Reaction equation

potassium phosphate

mercaptoethanol

dihydrofolic acid

NADPH

→

tetrahydrofolic acid

Conditions

Workup

1. 1
   workup.ADDITIONA solution of purified DHFR was added to a solution
2. 2
   workup.ADDITIONwas added to the mixture
3. 3
   Otherprepared above
4. 4
   OtherThe mixtures thus prepared
5. 5
   Othera reaction

Procedure

A solution of purified DHFR was added to a solution comprising a potassium phosphate buffer solution (pH: 7.5, 75 mM), mercaptoethanol (7.5 mM) and NADPH (0.25 mM) to make up to a total amount of 0.57 mi. Each of the serial dilutions of the compound according to the present invention in an amount of 0.015 ml was added to the mixture prepared above. The mixtures thus prepared were preliminarily heated at 37° C. for 5 minutes, followed by the addition of 0.015 ml of a 25 μM solution of dihydrofolic acid. The obtained mixtures were kept at 37° C. for 5 minutes to conduct a reaction. The resulting mixtures were each examined for the decrease in the absorbency at 339 nm per minute. The amount of tetrahydrofolic acid formed was determined on the basis of the decrease and compared with that of a control solution (containing none of the compounds according to the present invention) to calculate the 50% DHFR-inhibitory concentration (IC50). The results are given in Tables 1-A and 1-B.

Source