Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced α 1-acid glycoprotein binding

Abstract

Summary Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein α 1 -acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 µM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a K i value for inhibition of thymidine uptake of 0.1 µM compared to 0.28 µM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml -1). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC 90 by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 µM NU3076 and 1 µM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors.

Resistance-Modifying Agents. 11. Pyrimido[5,4-d]pyrimidine Modulators of Antitumor Drug Activity. Synthesis and Structure−Activity Relationships for Nucleoside Transport Inhibition and Binding to α1-Acid Glycoprotein

“…Accordingly, a number of compounds with potency comparable to that of DP, but only limited susceptibility to the effects of AGP, have been identified. Compounds 40 , 55 , 67 , and 73 have been shown to prevent thymidine rescue from the cytotoxic effects of the thymidylate synthase inhibitor nolatrexed in vitro, with 40 proving at least equipotent with DP in these resistance modulation experiments . Compounds 37 , 40 , and 41 have also been the subject of further detailed biological evaluation, which has shown that they can overcome the ability of thymidine and hypoxanthine to prevent the growth inhibitory activity of the new antifolate drug pemetrexed (MTA) .…”

“…Compounds 40, 55, 67, and 73 have been shown to prevent thymidine rescue from the cytotoxic effects of the thymidylate synthase inhibitor nolatrexed in vitro, with 40 proving at least equipotent with DP in these resistance modulation experiments. 44 Compounds 37, 40, and 41 have also been the subject of further detailed biological evaluation, which has shown that they can overcome the ability of thymidine and hypoxanthine to prevent the growth inhibitory activity of the new antifolate drug pemetrexed (MTA). 1 Their ability to reverse thymidine and hypoxanthine rescue was directly related to their NT inhibitory potency (Figure 1).…”

Recent Progress in Pyrimido[5,4-d]pyrimidine Chemistry

“…The studies aimed at improved activity or applicability compared to Dipy, for instance, in cancer-therapeutic use of analog RA25 (61a) (13WOP37129). Regarding nucleoside transport inhibition, remarkable effects have been found with ring homolog 113b and open-chain dialkylamino analog 226 (07JME3906, 13MI1), subst.-benzylamino compounds 124a, 208, and 227 (01MI1, 04JME4905), 208 derivative 209 and prodrug 210 (09MI1, 11JME1847), and alkoxy compounds, such as 189a and 228 (99MI2). Protected Dipy analog 128 was coupled through the amino group to give fluorescein conjugate 129 as a novel equilibrative nucleoside transport probe (11BCC1221).…”

Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity

“…We used a modified rapid mixing technique, combined with an inhibitor-stop method, to determine thymidine uptake into L1210 murine leukemia cells, as described previously (14,17 3 H]thymidine in the extracellular space that was carried through the oil layer.…”

“…As part of a program to develop potent pyrimidopyrimidine analogues of dipyridamole with reduced AGP binding, we have optimized our initial lead compound, NU3076, a pyrimidopyrimidine that has comparable potency to dipyridamole without being affected by AGP (14) . Several analogues that inhibit nucleoside transport even in the presence of physiologic concentrations of AGP were identified (15).…”