Folate Analogues. 30. Synthesis and Biological Evaluation of N¹⁰-Propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolic Acid and Related Compounds

The 5,6,7,8-tetrahydro derivative (1) of the powerful thymidylate synthase inhibitor N¹⁰-propargyl-5,8-dideazafolic acid (PDDF) has been synthesized and evaluated for its antifolate activity. A convenient method for the preparation of the key intermediate 2-amino-6-(bromomethyl)-4-hydroxy-5,6,7,8-tetrahydroquinazoline (18) is described. Two closely related analogues of 1 were also synthesized and evaluated for their antifolate activity and thymidylate synthase inhibition. N¹⁰-Propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (1) and N¹⁰-methyl and N¹⁰-hydrogen analogues 2 and 3 were weaker inhibitors of Lactobacillus casei thymidylate synthase compared to PDDF. N¹⁰-Methyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (2) exhibited the most potent antifolate activity against L. casei (IC₅₀= 2.8 nM) and Streptococcus faecium (IC50 = 0.57 nM). In intact and permeabilized murine leukemia LI210 cells, the replacement of the quinazoline moiety with its tetrahydro derivative resulted in a marked decrease in potency and a loss of the contribution of the propargyl substituent to enzyme inhibition, indicating an altered binding mode to thymidylate synthase.