Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-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 P2Y1 and P2Y12 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, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-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 SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors

Antiplatelet activity, P2Y₁ and P2Y₁₂ inhibition, and metabolism in plasma of stereoisomers of diadenosine 5',5'″-P¹ ,P⁴-dithio-P²,P³-chloromethylenetetraphosphate.

Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5',5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma.We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma.The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-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 SPSP and the RPRP diastereomers.The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5\u27) tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents

Stability and metabolism of the four diastereomers of compound 1 in human plasma at 37 °C.

<p>t_1/2, half-life of first order elimination. For other abbreviations, see Figure 1.</p

Platelet-related properties of the four diastereomers of compound <b>1</b>.

a<p>VASP, vasodilator-stimulated phosphoprotein.</p>b<p>All comparisons <i>vs.</i> diastereomer <b>1.1</b>.</p>c<p>p<0.05.</p>d<p>p<0.01.</p>e<p>p<0.001.</p

³¹P NMR Spectra (¹H decoupled) of the diastereomers of compound 1 and their racemic mixture.

<p>In the left panel are the P¹,P⁴ regions, and in the right panel are the P²,P³ regions of the spectra. The traces are, from bottom to top: racemic mixture (RM), diastereomer <b>1.1</b>, <b>1.2</b>, <b>1.3</b>, and <b>1.4</b>.</p

Effects of compound 1 diastereomers on platelet aggregation and platelet ADP receptor signaling.

<p>Inhibition by the diastereomers of compound <b>1</b> of: A, 3 µM ADP-stimulated platelet aggregation; B, P2Y₁₂ mediated ADP-induced decrease of VASP phosphorylation; and C, P2Y₁-mediated ADP-induced intraplatelet Ca²⁺ level increase. VASP, vasodilator-stimulated phosphoprotein. For other abbreviation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094780#pone-0094780-g001" target="_blank">Figure 1</a>.</p

Reversed-phase HPLC separation of the diastereomers of compound 1.

<p>Panel A: chromatogram of the racemic mix of the diastereomers of compound <b>1</b> analyzed by reversed-phase HPLC using a XBridge C18 column, 3.5 µm, 4.6×150 mm (Waters Inc., Waltham, Mass. USA), linear gradient from 1 to 7% methanol in 20 mM potassium phosphate buffer, pH 7, 1 ml/min, detection by UV at 260 nm; Panel B: chromatograms of isolated individual diastereomers (<b>1.1</b>–<b>1.4</b>) of compound <b>1</b> analyzed using the same column and flow rate, but with isocratic elution with 7% methanol in 20 mM potassium phosphate buffer, pH 7.</p