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.
Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America; Hematology Division, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan.
Hung
H
Chang
GLSynthesis Inc., Worcester, Massachusetts, United States of America.
Ivan B
IB
Yanachkov
GLSynthesis Inc., Worcester, Massachusetts, United States of America.
Edward J
EJ
Dix
GLSynthesis Inc., Worcester, Massachusetts, United States of America.
Milka
M
Yanachkova
Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Youfu
Y
Li
Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Marc R
MR
Barnard
GLSynthesis Inc., Worcester, Massachusetts, United States of America.
George E
GE
Wright
Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America; Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
Alan D
AD
Michelson
Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America; Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
Andrew L
AL
Frelinger
D015226:Dinucleoside Phosphates / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects*; D010979:Platelet Function Tests; D058921:Purinergic P2Y Receptor Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology*; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*; D013237:Stereoisomerism
D015226:Dinucleoside Phosphates; D058921:Purinergic P2Y Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; C473681:diadenosine 5',5'''-(P(1),P(4)-dithio-P(2),P(3)-chloromethylene)tetraphosphate