Synthesis and biological evaluation of carboxamide and quinoline derivatives as P2X7R antagonists

Abstract

P2X7 receptor (P2X7R) has a key role in different pathological conditions, importantly overexpressed and activated in cancers. We explored the structure activity relationship (SAR) of three novel pyrazines, quinoline-carboxamide and oxadiazole series. Their selective inhibitory potency in Ca2+ mobilization assay using h-P2X7R-MCF-7 cells improved with phenyl ring substitutions (–OCF3, –CF3, and –CH3) in carboxamide and oxadiazole derivatives, respectively. However, highly electronegative fluoro, chloro, and iodo substitutions enhanced affinity. 1e, 2f, 2e, 1d, 2 g and 3e were most potent and selective toward h-P2X7R (IC50 values 0.457, 0.566, 0.624, 0.682, 0.813 and 0.890 µM, respectively) and were inactive at h-P2X4R, h-P2X2R, r-P2Y6R, h-P2Y2R, t-P2Y1R expressed in MCF-7 and 1321N1 astrocytoma cells. Cell viability (MTT assay at 100 µM, cell line) for 3e was 62% (HEK-293T), 70% (1321N1 astrocytoma) and 85% (MCF-7). >75% cell viability was noted for 2 g and >80% for 2e and 1d in all non-transfected cell lines. Anti-proliferative effects, compared to control (Bz-ATP), of selective antagonists (10 µM) were 3e (11%) 1d, (19%) 1e, (70%, P = 0.005) and 2f, (24%), indicating involvement of P2X7R. Apoptotic cell death by flow cytometry showed 1e to be most promising, with 35% cell death (PI positive cells), followed by 2e (25%), 2f (20%), and 1d (19%), compared to control. Fluorescence microscopic analysis of apoptotic changes in P2X7R-transfected cell lines was established. 1e and 2f at 1X and 2X IC50 increased cellular shrinkage, nuclear condensation and PI/DAPI fluorescence. In-silico antagonist modeling predicted ligand receptor interactions, and all compounds obeyed Lipinski rules. These results suggest that pyrazine, quinoline-carboxamide and oxadiazole derivatives could be moderately potent P2X7R antagonists for in vivo studies and anti-cancer drug development.