Adenosine 5‘-O-(1-Boranotriphosphate) Derivatives as Novel P2Y1 Receptor Agonists

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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-α-B) scaffold was developed and tested as P2Y1-R agonists. An efficient four-step one-pot synthesis of several ATP-α-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 P2Y1-R agonists was evaluated by the agonist-induced Ca2+ release of HEK 293 cells stably transfected with rat-brain P2Y1-R. ATP-α-B A isomer was equipotent with ATP (EC50 = 2 × 10-7 M). However, 2-MeS− and 2-Cl− substitutions on ATP-α-B (A isomer) increased the potency of the agonist up to 100-fold, with EC50 values of 4.5 × 10-9 and 3.6 × 10-9 M, compared to that of the ATP-α-B (A isomer). Diastereoisomers A of all ATP-α-B analogues were more potent in inducing Ca2+ release than the corresponding B counterparts, with a 20-fold difference for 2-MeS-ATP-α-B analogues. The chemical stability of the new P2Y1-R agonists was evaluated by 31P NMR under physiological and gastric-juice pH values at 37 °C, with rates of hydrolysis of 2-MeS-ATP-α-B of 1.38 × 10-7 s-1 (t1/2 of 1395 h) and 3.24 × 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-α-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 P2Y1-R agonists barely interact with NTPDase, thus exhibiting protein selectivity. Therefore, on the basis of our findings, the new, highly water-soluble, P2Y1-R agonists may be considered as potentially promising drug candidates.