Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.