The P2Y 6 receptor is a cytoprotective G protein-coupled receptor (GPCR) activated by UDP (EC 50 , 0.30 μM). We compared and combined modifications to enhance P2Y 6 receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as α,β-methylene and extension of the terminal phosphate group into γ-esters of UTP analogues. The conformationally constrained (S)-methanocarba UDP is a full agonist (EC 50 0.042 μM). 4-Methoxyimino modification of pyrimidine enhanced P2Y 6 , preserved P2Y 2 and P2Y 4 , and abolished P2Y 14 receptor potency, in the appropriate nucleotide. N 4 -Benzyloxy-CDP (15, MRS2964) and N 4 -methoxy-Cp 3 U (23, MRS2957) were potent, selective P2Y 6 receptor agonists (EC 50 0.026 μM and 0.012 μM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-γ-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y 6 receptor potency, but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N 4 -methoxy-cytidine 5′-triphospho-γ-[1]glucose were active (EC 50 of 2.47 μM and 0.18 μM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y 6 receptor agonists may be enhanced by modest structural changes.

P2Y2 and P2Y4 receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We varied structurally phosphate and uracil moieties in analogues of pyrimidine nucleoside 5′-triphosphates and 5′-tetraphosphate esters. P2Y4 receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N4-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5′-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N4-(Phenylpropoxy)-CTP 16 (MRS4062), Up4-[1]3′-deoxy-3′-fluoroglucose 34 (MRS2927) and N4-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y4 over P2Y2 and P2Y6 receptors (EC50 values 23, 62 and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up4 activated P2Y2 but not P2Y4 receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y2 and P2Y4 receptor models indicated greater steric tolerance of N4-phenylpropoxy group at P2Y4. Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y4 receptor-selective agonists.

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