Structure−Activity Relationship and Solubility Studies of N1‐Substituted Quinoxaline‐2,3‐diones as Kainate Receptor Antagonists

Abstract

Kainate receptors are a class of ionotropic glutamate receptors that respond to the excitatory neurotransmitter glutamate in the central nervous system and play an important role in the development of neurodegenerative disorders and the regulation of synaptic function. In the current study, we investigated the structure‐ activity relationship of the series of quinoxaline‐2,3‐diones substituted at N1, 6, and 7 positions, as ligands of kainate homomeric receptors GluK1‐3 and GluK5. Pharmacological characterization showed that all derivatives obtained exhibited micromolar affinity at GluK3 receptors with Ki values in the range 0.1–4.4 μM range. The antagonistic properties of the selected analogues: N‐(7‐fluoro‐6‐iodo‐2,3‐dioxo‐3,4‐dihydroquinoxalin‐1(2H)‐yl)‐3‐sulfamoylbenzamide, N‐(7‐(1H‐imidazol‐1‐yl)‐6‐iodo‐2,3‐dioxo‐3,4‐dihydroquinoxalin‐1(2H)‐yl)‐3‐sulfamoylbenzamide and N‐(7‐(1H‐imidazol‐1‐yl)‐2,3‐dioxo‐6‐(phenylethynyl)‐3,4‐dihydroquinoxalin‐1(2H)‐yl)‐3‐sulfamoylbenzamide at GluK3 receptors, were confirmed by an intracellular calcium imaging assay. To correlate in vitro affinity data with structural features of the synthesized compounds and to understand the impact of the substituent in N1 position on ability to form additional protein‐ligand interactions, molecular modeling and docking studies were carried out. Experimental solubility studies using UV spectroscopy detection have shown that 7‐imidazolyl‐6‐iodo analogues with a sulfamoylbenzamide moiety at the N1 position are the best soluble compounds in the series, with molar solubility in TRISS buffer at pH 9 more than 3‐fold higher compared to NBQX, a known AMPA/kainate antagonist.

Small‐molecule positive allosteric modulation of homomeric kainate receptors GluK1‐3: development of screening assays and insight into GluK3 structure

“…We are currently using the assays to screen a range of positive allosteric modulators which will be reported elsewhere. In addition, the assays have been used to determine IC 50 values for new antagonists at GluK3 [52,53] and GluK1 [53].…”

Crystal structure of the GluK1 ligand-binding domain with kainate and the full-spanning positive allosteric modulator BPAM538

“…Grip Tite TM Human Embryonic Kidney (GT HEK293) (Invitrogen) cells were grown as previously described (Chałupnik et al 2022, 2023). For testing of PAM potentiation of agonist activity at GluK1( Q ) b and GluK2( VCQ ) a , a Ca 2+ -sensitive fluorescence-based assay were used.…”