Adenosine 5'-O-(1-boranotriphosphate) derivatives as novel P2Y(1) receptor agonists

Abstract

P2-receptors (P2-Rs) represent important targets for novel drug development. Most ATP analogues proposed as potential drug candidates have shortcomings such as limited receptor-selectivity and limited stability that justify the search for new P2-R agonists. Therefore, a novel series of nucleotides based on the adenosine 5'-O-(1-boranotriphosphate) (ATP-alpha-B) scaffold was developed and tested as P2Y(1)-R agonists. An efficient four-step one-pot synthesis of several ATP-alpha-B analogues from the corresponding nucleosides was developed, as well as a facile method for the separation of the diastereoisomers (A and B isomers) of the chiral products. The potency of the new analogues as P2Y(1)-R agonists was evaluated by the agonist-induced Ca2+ release of HEK 293 cells stably transfected with rat-brain P2Y(1)-R. ATP-alpha-B A isomer was equipotent with ATP (EC50 = 2 x 10(-7) M). However, 2-MeS- and 2-Cl- substitutions on ATP-alpha-B (A isomer) increased the potency of the agonist up to 100-fold, with EC50 values of 4.5 x 10(-9) and 3.6 x 10(-9) M, compared to that of the ATP-alpha-B (A isomer). Diastereoisomers A of all ATP-alpha-B analogues were more potent in inducing Ca2+ release than the corresponding B counterparts, with a 20-fold difference for 2-MeS-ATP-alpha-B analogues. The chemical stability of the new P2Y(1)-R agonists was evaluated by 31P NMR under physiological and gastric-juice pH values at 37 degrees C, with rates of hydrolysis of 2-MeS-ATP-alpha-B of 1.38 x 10(-7) s-1 (t1/2 of 1395 h) and 3.24 x 10(-5) s-1 (t1/2 = 5.9 h), respectively. The enzymatic stability of the new analogues toward spleen NTPDase was evaluated. Most of the new analogues were poor substrates for the NTPDase, with ATP-alpha-B (A isomer) hydrolysis being 5% of the hydrolysis rate of ATP. Diastereoisomers A and B exhibited different stability, with A isomers being significantly more stable, up to 9-fold. Furthermore, A isomers that are potent P2Y(1)-R agonists barely interact with NTPDase, thus exhibiting protein selectivity. Therefore, on the basis of our findings, the new, highly water-soluble, P2Y(1)-R agonists may be considered as potentially promising drug candidates.