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.

1 We have compared the antagonist activity of trinitrophenyl-ATP (TNP-ATP) and diinosine pentaphosphate (Ip 5 I) on recombinant P2X receptors expressed in Xenopus oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia. 2 Slowly-desensitizing responses to a,b-methylene ATP (a,b-meATP) recorded from oocytes expressing P2X 2/3 receptors were inhibited by TNP-ATP at sub-micromolar concentrations. However, Ip 5 I at concentrations up to 30 mM was without e ect. 3 Nodose ganglion neurones responded to a,b-meATP with slowly-desensitizing inward currents. These were inhibited by TNP-ATP (IC 50 , 20 nM), but not by Ip 5 I at concentrations up to 30 mM. 4 In DRG neurones that responded to ATP with a rapidly-desensitizing inward current, the response was inhibited by TNP-ATP with an IC 50 of 0.8 nM. These responses were also inhibited by Ip 5 I with an IC 50 of 0.1 mM. Both antagonists are known to inhibit homomeric P2X 3 receptors. 5 Some DRG neurones responded to a,b-meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 mM Ip 5 I, the sustained component remained una ected. 6 In conclusion, Ip 5 I is a potent antagonist at homomeric P2X 3 receptors but not at heteromeric P2X 2/3 receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.