Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists

Antagonists
for the ATP-gated ion channel receptor P2X1 have potential
as antithrombotics and for treating hyperactive bladder and inflammation.
In this study, salicylanilide derivatives were synthesized based on
a screening hit. P2X1 antagonistic potency was assessed in 1321N1
astrocytoma cells stably transfected with the human P2X1 receptor
by measuring inhibition of the ATP-induced calcium influx. Structure–activity
relationships were analyzed, and selectivity versus other P2X receptor
subtypes was assessed. The most potent compounds, N-[3,5-bis­(trifluoromethyl)­phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 μM) and N-[3,5-bis­(trifluoromethyl)­phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 μM), displayed >500-fold
selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4
and P2X7 receptors, and inhibited collagen-induced platelet aggregation.
They behaved as negative allosteric modulators, and molecular modeling
studies suggested an extracellular binding site. Besides selective
P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor
inhibition were discovered. These compounds represent the first potent,
non-acidic, allosteric P2X1 receptor antagonists reported to date.