Metabolic inhibition by a new antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methyl-pyrido[2,3-d]pyrimidine (BW3O1U), an effective inhibitor of human lymphoid and dihydrofolate reductase-overproducing mouse cell lines

Abstract

The human lymphoblastoid cell line, WIL-2, and a mouse cell line, 3T6R400, that overproduces a mutant dihydrofolate reductase (DHFR) having greater than 100-fold increased resistance to methotrexate (MTX) inhibition, were used to compare the inhibitory properties of a novel lipid-soluble antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methyl-pyrido[2,3-d]pyrimidine (BW301U), with the 2,4-arylpyrimidine, 2,4-diamino-5-(3,4'-dichlorophenyl)-6-methylpyrimidine (DDMP) and with MTX. These studies demonstrated that, like DDMP, BW301U rapidly entered cells and inhibited the incorporation of dUrd into DNA. Drug association with cells was temperature-independent and apparently did not require active transport. BW301U inhibited cell growth by 50% at 0.025 microM, whereas MTX caused equivalent inhibition at 0.045 microM. Inhibition of DNA synthesis produced by 90 min of exposure to BW301U was completely reversed within 2 hr after washing cells and suspending them in drug-free medium. In contrast, inhibition of DNA synthesis in MTX-treated cells was not reversed by simply removing the antifolate, but required the addition of calcium leukovorin or thymidine to the drug-free medium in order to facilitate complete reversal of DNA synthesis. Finally, like DDMP, BW301U was approximately 1000 times more effective than MTX in inhibiting dUrd incorporation into the DNA of a DHFR gene-amplified cell line of mouse 3T6 cells.