Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y 1 while uridine containing dinucleotides have some level of agonist response on P2Y 2 and P2Y 6 . The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.
ADP is the cognate agonist of the P2Y 1 , P2Y 12 , and P2Y 13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y 1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the G i -linked P2Y 12 and P2Y 13 receptors, a chimeric G protein, G␣ q/i , was coexpressed to confer a capacity of these G i -linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC 50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC 50 ϭ 1.2 Ϯ 0.2 nM) agonist at the P2Y 1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y 12 receptor and very low activity at the P2Y 13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y 12 and P2Y 13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y 1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y 1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y 12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.The G protein-coupled P2Y receptor family is comprised of at least eight different human receptors that are activated by nucleoside diphosphates, nucleoside triphosphates, or nucleotide sugars to regulate a broad range of physiological responses including neurotransmission, muscle contraction, ion secretion, and platelet aggregation (Dubyak and ElMoatassim, 1993;Harden et al., 1998;Ralevic and Burnstock, 1998). A complex set of ectoenzymes that metabolize extracellular nucleotides has complicated the study of these receptors Zimmermann, 2000). This difficulty is exacerbated by the lack of selective agonists or antagonists for most of the P2Y receptors.
Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.
Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an ADP-induced platelet aggregation assay.
The selective N-alkylation to the intermediates such as (V) is the key step in the synthesis of the title compounds. -(ENKVIST, E.; RAIDARU, G.; URI*, A.; PATEL, R.; REDICK, C.; BOYER, J. L.; SUBBI, J.; TAMMISTE, I.; Nucleosides, Nucleotides
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