Studies on enantioselectivity of chiral 4-acetylamino-6-alkyloxy-2-alkylthiopyrimidines acting as antagonists of the human A3 adenosine receptor

Three A3adenosine receptor (AR) antagonists (1-3) selected from 4-acylamino-6-alkyloxy-2-alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or thioether side chains. These compounds gave us the chance to evaluate whether their higher lipophilicity, reduced conformational freedom and chirality might improve the potency towards the A3AR. Racemic mixtures of 1-3 were resolved using chiral HPLC methods and the absolute configurations of the enantiomers were assigned by chiroptical spectroscopy and density functional theory calculations. We measured the affinity for human A1, A2A, A2Band A3ARs of the racemic mixtures and the pure enantiomers of 1-3 by radioligand competition binding experiments. Cell-based assays of the most potent enantiomers confirmed their A3AR antagonist profiles. Our research led to the identification of (S)-1 with high potency (0.5 nM) and selectivity as an A3AR antagonist. Moreover we built a docking-model useful to design new pyrimidine derivatives.