P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel P2Y-R ligands, 2-thioether 5'-O-phosphorothioate adenosine derivatives (2-RS-ATP-alpha-S), were synthesized as potential insulin secretagogues. An efficient synthesis of these nucleotides and a facile method for separation of the chiral products are described. The enzymatic stability of the compounds toward pig pancreas type I ATPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)adenosine (2-hexylthio-ATP-alpha-S) isomers by ATPDase was 28% of that of ATP. Some 2-thioether 5'-(monophosphorothioate)adenosine derivatives (2-RS-AMP-S) exerted an inhibitory effect on ATPDase. The apparent affinity of the compounds to P2Y(1)-R was determined by measurement of P2Y-R-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K(0.5) values in the nanomolar range. 2-RS-AMP-S derivatives were full agonists, although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nanomolar concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective.

Activation of P2Y receptors on pancreatic β-cells by extracellular ATP bring about amplification of glucose-induced insulin secretion, which has been shown to reduce hyperglycemia in vivo. A new P2 receptor ligand, 2-benzylthio-ATP-α-S, was synthesized based on a combination of modifications of the ATP skeleton, as a potential insulin secretagogue. The two diastereoisomers of this ligand were separated and are designated A and B. The effects of these compounds on insulin secretion and vascular resistance in the rat isolated and perfused pancreas were evaluated in the presence of a slightly stimulating glucose concentration (8.3 mM) and were compared with ATP-α-S and ATP. Both isomers of 2-benzylthio-ATP-α-S (0.015-1.5 µM) induced a concentration-dependent increase in glucose-induced insulin release. The potency of isomer A was not significantly different from that of isomer B, and both were approximately 100-fold more potent than ATP. ATP-α-S induced a similar pattern of insulin response; however, it was only approximately 10-fold more potent than ATP. These compounds also induced vascular effects: ATP-α-S induced a vasodilatation and was transiently vasoconstrictor only at a high concentration, whereas the C2-substituted derivative constantly induced a vasoconstriction. The chemical stability of these ligands was evaluated under physiological conditions and gastric juice pH. Hydrolysis of 2-benzylthio-ATP-α-S has been studied both in pH 7.4 and pH 1.4 at 37°C using 31 P nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography. This compound exhibited high chemical stability with respect to hydrolysis of the glycosidic bond and desulfurization of the phosphorothioate moiety. Hydrolysis of the phospho ester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37°C), was slow, with a half-life of 264 h. Moreover, even at gastric juice conditions (pH 1.4, 37°C), hydrolysis of the terminal phosphate was the only detectable reaction, with half-life of 17.5 h. In conclusion, both isomers of 2-benzylthio-ATP-α-S are soluble in water and highly chemically stable. These compounds are highly potent and effective insulin secretagogues; however, they increase pancreatic vascular resistance. Drug Dev. Res. 53:33-43, 2001.