Novel analogues of the P2 receptor antagonist pyridoxal-5′-phosphate 6-azophenyl-2′,5′-disulfonate (2) were synthesized and studied as antagonists in functional assays at recombinant rat P2X 1 , P2X 2 , and P2X 3 receptors expressed in Xenopus oocytes (ion flux stimulation) and at turkey erythrocyte P2Y 1 receptors (phospholipase C activation). Selected compounds were also evaluated as antagonists of ion flux and the opening of a large pore at the recombinant human P2X 7 receptor. Modifications were made in the 4-aldehyde and 5′-phosphate groups of the pyridoxal moiety: i.e. a CH 2 OH group at the 4-position in pyridoxine was either condensed as a cyclic phosphate or phosphorylated separately to form a bisphosphate, which reduced potency at P2 receptors. 5-Methylphosphonate substitution, anticipated to increase stability to hydrolysis, preserved P2 receptor potency. At the 6-position, halo, carboxylate, sulfonate, and phosphonate variations made on the phenylazo ring modulated potency at P2 receptors. The p-carboxyphenylazo analogue, 4, of phosphate 2 displayed an IC 50 value of 9 nM at recombinant P2X 1 receptors and was 1300-, 16-, and >10000-fold selective for P2X 1 versus P2X 2 , P2X 3 , and P2Y 1 subtypes, respectively. The corresponding 5-methylphosphonate was equipotent at P2X 1 receptors. The 5-methylphosphonate analogue containing a 6-[3,5-bis(methylphosphonate)]-phenylazo moiety, 9, had IC 50 values of 11 and 25 nM at recombinant P2X 1 and P2X 3 receptors, respectively. The analogue containing a phenylazo 4-phosphonate group, 11, was also very potent at both P2X 1 and P2X 3 receptors. However, the corresponding 2,5-disulfonate analogue, 10, was 28-fold selective for P2X 1 versus P2X 3 receptors. None of the analogues were more potent at P2X 7 and P2Y 1 receptors than 2, which acted in the micromolar range at these two subtypes.

P2Y(1) receptors are activated by ADP and occur on endothelial cells, smooth muscle, epithelial cells, lungs, pancreas, platelets, and in the central nervous system. With the aid of molecular modeling, we have designed nucleotide analogues that act as selective antagonists at this subtype. The present study has tested the hypothesis that acyclic modifications of the ribose ring, proven highly successful for nucleoside antiviral agents such as gancyclovir, are generalizable to P2Y receptor ligands. Specifically, the binding site of the P2Y(1) receptor was found to be sufficiently accommodating to allow the substitution of the ribose group with acyclic aliphatic and aromatic chains attached to the 9-position of adenine. Three groups of adenine derivatives having diverse side-chain structures, each containing two symmetrical phosphate or phosphonate groups, were prepared. Biological activity was demonstrated by the ability of the acyclic derivatives to act as agonists or antagonists in the stimulation of phospholipase C in turkey erythrocyte membranes. An acyclic N(6)-methyladenine derivative, 2-[2-(6-methylamino-purin-9-yl)-ethyl]-propane-1, 3-bisoxy(diammoniumphosphate) (10), containing an isopentyl bisphosphate moiety, was a full antagonist at the P2Y(1) receptor with an IC(50) value of 1.60 micro¿. The corresponding 2-Cl derivative (11) was even more potent with an IC(50) value of 0.84 microM. Homologation of the ethylene group at the 9-position to 3-5 methylene units or inclusion of cis- or trans-olefinic groups greatly reduced antagonist potency at the P2Y(1) receptor. Analogues containing a diethanolamine amide group and an aryl di(methylphosphonate) were both less potent than 10 as antagonists, with IC(50) values of 14 and 16 microM, respectively, and no agonist activity was observed for these analogues. Thus, the ribose moiety is clearly not essential for recognition by the turkey P2Y(1) receptor, although a cyclic structure appears to be important for receptor activation, and the acyclic approach to the design of P2 receptor antagonists is valid.