Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity in vitro, but its clinical activity is compromised by binding to the plasma protein α 1 -acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the in vivo activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 in vitro and in vivo. Plasma NU3153 concentrations commensurate with rescue inhibition in vitro were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity in vivo. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (≥90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved in vivo using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver.

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 α(1)-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 [(3)H]thymidine incorporation assay, employing COR-L23 cells, the diastereoisomers of 6 (IC(50) = 26 nM) exhibited activity comparable with 3 (IC(50) = 15 nM). The monophenyl carbamate and mono-4-methoxyphenyl carbamate exhibited the best ENT-inhibitory activity in the COR-L23 assay (IC(50) = 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.

<div>Abstract<p>Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity <i>in vitro</i>, but its clinical activity is compromised by binding to the plasma protein α₁-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the <i>in vivo</i> activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 <i>in vitro</i> and <i>in vivo</i>. Plasma NU3153 concentrations commensurate with rescue inhibition <i>in vitro</i> were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity <i>in vivo</i>. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (≥90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved <i>in vivo</i> using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver. [Mol Cancer Ther 2009;8(7):1828–37]</p></div>

Supplementary Fig. S2 from Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity

Supplementary Fig. S1 from Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity