Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10-CF₃CO-DDACTHF

Structurally-related, but non-polyglutamylatable, derivatives of 10-CF ₃CO-DDACTHF (1), which incorporate l-glutamine (2) and l-isoglutamine (3) in place of l-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR Tfase. While the l-glutamate α-carboxamide derivative 3 was much less effective as a rhGAR Tfase inhibitor (K_i = 4.8 μM) and inactive in cellular functional assays, the γ-carboxamide derivative 2 was found to be a potent and selective rhGAR Tfase inhibitor (K_i = 0.056 μM) being only 4-fold less potent than 1 (K _i = 0.015 μM). Moreover, 2 was effective in cellular functional assays exhibiting purine sensitive cytotoxic activity (IC₅₀ = 300 nM, CCRF-CEM) only 20-fold less potent than 1 (IC₅₀ = 16 nM), consistent with inhibition of de novo purine biosynthesis via selective inhibition of GAR Tfase. Like 1, 2 is transported into the cell by the reduced folate carrier. Unlike 1, the functional activity of 2 is not dependent upon FPGS polyglutamylation.