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

Abstract

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.

Update of P2X receptor properties and their pharmacology: IUPHAR Review 30

“…Only a few P2X1 receptor-selective antagonists have been developed ( Figure 2 ). Salicylamide derivatives with high potency and selectivity were recently described ( Tian et al, 2020 ), representing small, uncharged molecules, which act as negative allosteric modulators (NAMs). Some of them, for example, PSB-2014 ( 16b ), only partly inhibited hP2X1 receptors.…”

Recommended tool compounds and drugs for blocking P2X and P2Y receptors

“…2). Structurally related potent P2X3 receptor antagonists include AF-353 (12) and AF-906 ( 13), all of which are highly potent and perorally bioavailable. The more lipophilic antagonist 12 is even brain-permeant.…”

“…Due to its polyanionic character, it is well soluble in water, but its selectivity is limited since it also blocks the P2X3 receptor at somewhat higher concentration. The first relatively potent, selective P2X1 receptor antagonists have recently been described, salicylamide derivatives 5 and 6 [ 12 ]. These compounds act as allosteric inhibitors, and their binding site was proposed by docking studies to be located in the extracellular domain (see Fig.…”

“…2 ). To provide a structural hypothesis for the recently described series of allosteric P2X1 receptor antagonists, a homology model was generated based on the crystal structures of the P2X7 receptor [ 12 ]. The putative binding mode of PSB-2001 ( 5 ) and its close analogs partly overlaps with the allosteric binding site identified by the crystal structures of the antagonist-bound P2X7 receptor.…”

“…The aniline moiety of PSB-2001 is proposed to be embedded forming hydrophobic interactions with F92, F291, F293, F308, and V310, and the salicylate moiety is likely surrounded by another set of hydrophobic residues, F106 and W164 (see Fig. 2 ) [ 12 ].…”