Structure-activity relationships and optimization of 3,5-dichloropyridine derivatives as novel P2X ₇ receptor antagonists

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X ₇ receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R ₂) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X ₇ antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R ₂ position optimized antagonistic activity. In the EtBr uptake assay in hP2X ₇-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC ₅₀ values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC ₅₀ = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X ₇ receptor targeted anti-inflammatory agents.