Nucleoside Transport Inhibitors: Structure−Activity Relationships for Pyrimido[5,4-d]pyrimidine Derivatives That Potentiate Pemetrexed Cytotoxicity in the Presence of α1-Acid Glycoprotein

Membrane transport of nucleosides or nucleobases is mediated by transporters including the equilibrative nucleoside transporters (ENTs), and resistance to antitumor antimetabolite drugs may arise via salvage of exogenous purine or pyrimidine nucleosides or nucleobases by ENT transporters. The therapeutic utility of dipyridamole (3), a potent ENT inhibitor, is compromised by binding to the serum protein α1-acid glycoprotein (AGP). Derivatives and prodrugs of the ENT inhibitor 4,8-bis[(3,4-dimethoxybenzyl)amino]-2,6-bis[(2-hydroxypropyl)amino]pyrimido[5,4-d]pyrimidine (6, NU3108) are described, with improved in vivo pharmacokinetic properties and reduced AGP binding relative to dipyridamole. The mono- and diglycine carbamate derivatives were at least as potent as 6 and showed no reduction in potency by AGP. In a [3H]thymidine incorporation assay, employing COR-L23 cells, the diastereoisomers of 6 (IC50 = 26 nM) exhibited activity comparable with 3 (IC50 = 15 nM). The monophenyl carbamate and mono-4-methoxyphenyl carbamate exhibited the best ENT-inhibitory activity in the COR-L23 assay (IC50 = 8 and 4 nM, respectively). All of the new prodrugs were also highly effective at reversing thymidine/hypoxanthine rescue from pemetrexed cytotoxicity in the COR-L23 cell line.