Antiplatelet Activity, P2Y(1) and P2Y(12) Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate

BACKGROUND: Diadenosine tetraphosphate (Ap(4)A), a constituent of platelet dense granules, and its P(1),P(4)-dithio and/or P(2),P(3)-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y(1) and P2Y(12). The most active of those analogs, diadenosine 5′,5″″-P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. OBJECTIVE: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y(1) and P2Y(12) receptor antagonism, and their metabolism in human plasma. METHODS: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y(1)-mediated changes in cytosolic Ca(2+), P2Y(12)-mediated changes in VASP phosphorylation, and metabolism in human plasma. RESULTS: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y(12) receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P(1)- and P(4)-phosphorothioate groups, the S(P)S(P) diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R(P)R(P) diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S(P)R(P) diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P(2),P(3)-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S(P)S(P) and the R(P)R(P) diastereomers. CONCLUSIONS: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y(1) and P2Y(12) receptors.