Structure-activity relationships of pyridoxal phosphate derivatives as potent and selective antagonists of P2X(1) receptors

Novel analogues of the P2 receptor antagonist pyridoxal-5'-phosphate 6-azop hcnyl-2',5'-disulfonate (2) were synthesized and studied as antagonists in functional assays at recombinant rat P2X(1), P2X(2), and P2X(3) receptors e xpressed in Xenopus oocytes lion flux stimulation) and at turkey erythrocyt e P2Y(1) receptors (phospholipase C activation). Selected compounds were al so 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-ald ehyde and 5'-phosphate groups of the pyridoxal moiety: i.e. a CH2OH group a t the 4-position in pyridoxine was either condensed as a cyclic phosphate o r phosphorylated separately to form a bisphosphate, which reduced potency a t P2 receptors. 5-Methylphosphonate substitution, anticipated to increase s tability to hydrolysis, preserved P2 receptor potency. At the g-position, h alo, carboxylate, sulfonate, and phosphonate variations made on the phenyla zo ring modulated potency at P2 receptors. The p-carboxyphenylazo analogue, 4, of phosphate 2 displayed an IC50 value of 9 nM at recombinant P2X(1) re ceptors and was 1300-, 16-, and >10000-fold selective for P2X(1) versus P2X (2), P2X(3), and P2Y(1) subtypes, respectively. The corresponding Ei-methyl phosphonate was equipotent at P2X(1) receptors. The 5-methylphosphonate ana logue containing a 6-[3,5-bis(methylphosphonate)]phenylazo moiety, 9, had I C50 values of 11 and 25 nM at recombinant P2X(1) and P2X(3) receptors, resp ectively. The analogue containing a phenylazo 4-phosphonate group, 11, was also very potent at both P2X(1) and P2X(3) receptors. However, the correspo nding 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 subt ypes.