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

Abstract

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.

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

“…Notwithstanding solubility problems with diaryl carbamates ( 13 , 15 , 17 , 19 ), it was evident that the monoaryl carbamates ( 12 , 16 , 18 ) retained NT-inhibitory activity at least comparable with 6 , 20 , and 21 , whereas the corresponding diaryl carbamates ( 13 , 17 , 19 ) were weakly active ( 13 ) or inactive ( 17 , 19 ) where evaluation was possible (Figure A). Crucially, the desired lack of significant AGP binding in vitro for NU3108 ( 6 ) was also observed for the monoglycine carbamate ( 20 ) and the monoaryl carbamates 12 , 16 , and 18 , with no reduction in potency being evident in the presence of physiologically relevant concentrations (5 mg/mL) of AGP (Figure B) . By contrast, the NT-inhibitory activity of 3 was reduced by approximately 66% under these conditions.…”

“…While the 3 H-TdR transport assay served as a useful initial screen, a 3 H-TdR incorporation assay, employing the COR-L23 human lung carcinoma tumor cell line, was used for subsequent studies with selected compounds, as this assay provides a more representative value for NT inhibition . As expected, the parent pyrimidopyrimidine 6 (IC 50 = 26 ± 5 nM) exhibited activity comparable with 3 (IC 50 = 15 ± 4 nM), with 6 and the homochiral derivative 11 proving equipotent.…”

The rationality for using prodrug approach in drug discovery programs for new xenobiotics: opportunities and challenges

“…However, it appeared that the prodrug did not have very strong and sustained in vivo inhibition which is required to drastically reduce the viral load. It was suggested that the prodrug should possess a much higher potency (at least twofold greater) to have an equivalent effect to that of the pyrimidopyrimidine derivative of dipyridamole (Thomas et al 2009). …”

Synthesis, In Vitro Cytotoxicity and Bleomycin‐Dependent DNA Damage Evaluation of Some Heterocyclic‐Fused Pyrimidinone Derivatives

“…Isoxazolo[5,4‐ d ]pyrimidinones and pyrazolo[3,4‐ d ]pyrimidinones are potent anticancer agents . Pyrimido[4,5‐ d ]pyrimidinones display significant antitumor, anti‐inflammatory, and antimicrobial activities, prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity and the inhibition of cellular proliferation in breast cancer . According to the above‐mentioned findings and in the persistence of our strategies for the synthesis of novel bioactive heterocycles, we report here the synthesis of some heterocycles fused with pyrimidinone ring with the objective of evaluating their cytotoxicity and bleomycin‐dependent DNA damage activities.…”