N-(7-(1H-Imidazol-1-yl)-2,3-dioxo-6-(trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)-yl)benzamide, a New Kainate Receptor Selective Antagonist and Analgesic: Synthesis, X-ray Crystallography, Structure–Affinity Relationships, and in Vitro and in Vivo Pharmacology

Abstract

Selective pharmacological tool compounds are invaluable for understanding the functions of the various ionotropic glutamate receptor subtypes. For the kainate receptors, these compounds are few. Here we have synthesized nine novel quinoxaline-2,3-diones with substitutions in the 7-position to investigate the structure–activity relationship at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Compound 11 exhibited the highest binding affinity across GluK1–3 while having selectivity toward kainate vs AMPA receptors. Compound 11 potently inhibited glutamate evoked currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with K b values of 65 and 39 nM, respectively. The binding mode of 11 in the ligand binding domain of GluK1 was investigated by X-ray crystallography, revealing that 11 stabilizes the receptor in an open conformation, consistent with its demonstrated antagonism. Furthermore, 11 was tested for analgesic effects in the mouse tail flick test where it significantly increased tail flick latency at doses where 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo­[f]-quinoxaline-7-sulfonamide (NBQX) was ineffective.

Kainate Receptor Antagonists: Recent Advances and Therapeutic Perspective

“…In a mouse tail flick test for acute pain, 15 was shown to have an analgesic effect and was more effective than NBQX, suggesting that, contrary to an earlier hypothesis [75], the analgesic effects observed for early generation quinoxalinediones could also be mediated by kainate receptors. Furthermore, 15 did not show an adverse effect on motor coordination in the rotarod test as previously observed for NBQX, and it was found to cause an increase in locomotor activity [95].…”

“…SAR studies for the recently reported series of N1-substituted analogues confirmed that, also for compounds without a pyrrole ring in position 7 (and possessing a fluorine or nitro group instead), the N1-benzamide moiety was optimal for binding to the GluK1-3 recombinant receptors (11)(12)(13)(14) and sufficient to achieve an affinity profile similar to LU97175 (12), while compounds with smaller or non-aromatic substituents in position N1 showed a significantly lower KAR affinity (Table 2) [92]. Attempts to bioisosterically replace the pyrrolyl moiety in the structure of LU97175 resulted in another series of derivatives, among which a 7-imidazole analogue (15) exhibited the highest binding affinity across the recombinant receptors GluK1-3 and the 73-fold binding selectivity for GluK3 over the AMPA subtype GluA2 (Table 2) [95]. In a mouse tail flick test for acute pain, 15 was shown to have an analgesic effect and was more effective than NBQX, suggesting that, contrary to an earlier hypothesis [75], the analgesic effects observed for early generation quinoxalinediones could also be mediated by kainate receptors.…”

Discovery of the First Highly Selective Antagonist of the GluK3 Kainate Receptor Subtype

“…The predominant number of compounds was successfully docked to GluK1- and GluK3-LBD, with the top-ranking docking poses adopting a similar position inside both binding sites ( Supplementary Materials, Figure S3 ). The same characteristic pattern of interactions between the quinoxaline-2,3-dione scaffold and the D1 lobe of the receptor was observed for all ligands anchored, as in the case of other analogues in this chemical group, which were bound to the KAR or AMPAR subtypes [ 32 , 33 , 36 ]. A crucial role in these interactions is played by Arg523/525, Pro516/518, and Thr518/520 ( Figure 3 and Figure 4 , numbering of amino acids for GluK1 and GluK3 sequences, respectively).…”

“…Recently, we have published the results of studies on the new series of quinoxaline-2,3-dione derivatives and their pharmacological characterization at native and recombinant KA and AMPA receptors [ 32 , 33 ]. Among others, we found that aromatic amido substituents at the N1 position of the quinoxalinedione core were essential for binding to the KAR subunits GluK1 and GluK3, while a phenylethynyl moiety at the 6-position was beneficial for GluK3/GluK1 selectivity.…”

Regulation and dysregulation of neuronal circuits by KARs

“…We are in general lacking highly selective antagonists targeting the most abundant KAR isoform GluK2/GluK5, albeit UBP310 was also found to block synaptic receptors comprising GluK2/GluK5 KARs in vitro and in vivo (Pinheiro et al, 2013). Recently however quinoxaline-2,3-dione analogues have been synthetized and shown to have a good pharmacological selectivity at GluK3 over GluK1 and AMPARs (Møllerud et al, 2019). This may offer possibilities in the future to better understand the physiological role of the elusive GluK3 subunit.…”