Structure-activity relationships of alkyl- and alkoxy-substituted 1,4-dihydroquinoxaline-2,3-diones: potent and systemically active antagonists for the glycine site of the NMDA receptor.

We report on a series of alkyl- and alkoxy-substituted 1,4-dihydroquinoxaline-2,3-diones (QXs), prepared as a continuation of our structure-activity relationship (SAR) study of QXs as antagonists for the glycine site of the N-methyl-D-aspartate (NMDA) receptor. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) in rat brain cortical membranes. In general, methyl is a good replacement for chloro or bromo in the 6-position, and alkoxy-substituted QXs have lower potencies than alkyl- or halogen-substituted QXs. Ethyl-substituted QXs are generally less potent than methyl-substituted QXs, especially in the 6-position of 5,6,7-trisubstituted QXs. Fusion of a ring system at the 6,7-positions results in QXs with low potency. Several methyl-substituted QXs are potent glycine site antagonists that have surprisingly high in vivo activity in the maximal electroshock (MES) test in mice. Among these, 7-chloro-6-methyl-5-nitro QX (14g) (IC50 = 5 nM) and 7-bromo-6-methyl-5-nitro QX (14f) (IC50 = 9 nM) are comparable in potency to 6,7-dichloro-5-nitro QX (2) (ACEA 1021) as glycine site antagonists. QX 14g has an ED50 value of 1.2 mg/kg iv in the mouse MES assay. Interestingly, alkyl QXs with log P values of 0.5 or less tend to be more bioavailable than QXs with higher log P values. QX 14g has 440-fold selectivity for NMDA vs alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, as determined electrophysiologically under steady-state conditions in oocytes expressing rat cerebral cortex poly(A)+ RNA. Overall, 14g was found to have the best combination of in vitro and in vivo potency of all the compounds tested in this and previous studies on the QX series.

Pharmacology of ACEA-1416: a potent systemically active NMDA receptor glycine site antagonist.

Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1, 4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2, 3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites (Kb = 7.9 nM in oocytes, Kb = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower (Kb = 3.5 microM in oocytes, Kb = 1.6 microM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites (Kb > 5.9 microM) and metabotropic glutamate receptors (Kb > 57 microM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at approximately 2 min and the biological half-time of protection was approximately 60 min. The ED50 measured at peak efficacy was approximately 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.

Synthesis and structure-activity relationships of substituted 1,4-dihydroquinoxaline-2,3-diones: antagonists of N-methyl-D-aspartate (NMDA) receptor glycine sites and non-NMDA glutamate receptors.

A series of mono-, di-, tri-, and tetrasubstituted 1,4-dihydroquinoxaline-2,3-diones (QXs) were synthesized and evaluated as antagonists at N-methyl-D-aspartate (NMDA)/glycine sites and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-preferring non-NMDA receptors. Antagonist potencies were measured by electrical assays in Xenopus oocytes expressing rat whole brain poly(A)+ RNA. Trisubstituted QXs 17a (ACEA 1021), 17b (ACEA 1031), 24a, and 27, containing a nitro group in the 5 position and halogen in the 6 and 7 positions, displayed high potency (Kb approximately 6-8 nM) at the glycine site, moderate potency at non-NMDA receptors (Kb = 0.9-1.5 microM), and the highest (120-250-fold) selectivity in favor of glycine site antagonism over non-NMDA receptors. Tetrasubstituted QXs 17d,e were more than 100-fold weaker glycine site antagonists than the corresponding trisubstituted QXs with F being better tolerated than Cl as a substituent at the 8 position. Di- and monosubstituted QXs showed progressively weaker antagonism compared to trisubstituted analogues. For example, removal of the 5-nitro group of 17a results in a approximately 100-fold decrease in potency (10a,b,z), while removal of both halogens from 17a results in a approximately 3000-fold decrease in potency (10v). In terms of steady-state inhibition, most QX substitution patterns favor antagonism at NMDA/glycine sites over antagonism at non-NMDA receptors. Among the QXs tested, only 17i was slightly selective for non-NMDA receptors.