Pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (7a,
PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X₃ receptor antagonists as potential
antinociceptive agents. Based on the results of our previous report, all strong anionic groups in
PPADS including
phosphate and sulfonate groups were changed to
carboxylic acids or deleted. The unstable azo (-NN-) linkage of 7a was transformed to more stable
carbon-
carbon,
ether or
amide linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 = 400 ∼ 700 nM) at the hP2X₃ receptor in the two-
electrode voltage clamp (TEVC) assay system on the Xenopus oocytes. Introduction of bulky aromatic groups at the
carbon linker, as in compounds 13 h-n, dramatically improved the selectivity profiles of hP2X₃ when compared with mP2X₁ and hP2X₇ receptors. Among the substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and potency at the hP2X₃ receptor. In searching for effective substituents at the 4- and 3-positions, we found that compound 36j, with 4-carboxaldehyde, 3-propenoic
acid and 2-(m-phenoxy)benzyl groups, was the most potent and selective hP2X₃ receptor antagonist with an IC50 of 60 nM at hP2X₃ and marginal antagonistic activities of 10 μM at mP2X₁ and hP2X₇. Furthermore, using an ex-vivo assay system, we found that compound 36j potently inhibited
pain signaling in the rat dorsal horn with 20 μM 36j displaying 65% inhibition while 20 μM
pregabalin, a clinically available
drug, showed only 31% inhibition.