licostinel and 5-7-dichlorokynurenic-acid

A series of novel di- and trisubstituted 1,4-dihydroquinoxaline-2,3-diones (QXs) related to licostinel (Acea 1021) was synthesized and evaluated as antagonists for the glycine site of the N-methyl-D-asparate (NMDA) receptor. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [(3)H]-5,7-dichlorokynurenic acid ([(3)H]DCKA) in rat brain cortical membranes. Structure-activity relationship studies indicate that a cyano group is a good replacement for the nitro group in the 5-position of licostinel while 5-carboxy, 5-ester, 5-ketone and 5-amide derivatives showed reduced potency. 5,6-Cyclized analogues of licostinel also showed significantly reduced potency. Among the trisubstituted QXs investigated, 5-cyano-6,7-dichloro QX and 5-cyano-7-chloro-6-methyl QX are the most potent with IC(50) values of 32 nM and 26 nM, respectively.

Topics: Animals; Binding Sites; Cell Membrane; Cerebral Cortex; Glycine; Inhibitory Concentration 50; Kynurenic Acid; Quinoxalines; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship

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.

Topics: Animals; Anticonvulsants; Binding Sites; Binding, Competitive; Cerebral Cortex; Electrophysiology; Excitatory Amino Acid Antagonists; Glycine; Kynurenic Acid; Magnetic Resonance Spectroscopy; Mice; Molecular Structure; Oocytes; Quinoxalines; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Structure-Activity Relationship; Xenopus

The effects of five glycine site antagonists were comparatively examined on maximal and plateau currents evoked by 200 microM N-methyl-D-aspartate (NMDA) in the presence of 1 microM glycine in cultured cerebrocortical cells of the rat using whole-cell patch-clamp technique. 5,7-Dichlorokynurenic acid, ACEA-1021 (5-nitro-6,7-dichloro-quinoxalinedione), L-695,902 (methyl 7-chloro-4-hydroxy 2(1H)-quinolone-3-carboxylate), LY-294,619 (5,7-dichloro-3-(4-hydroxphenyl)-4-hydroxyquinolin-2(1H)-one ) and RPR-104,632 (2-(3-bromo-benzyl)-6,8-dichloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-3-carboxylic acid) caused concentration-dependent inhibition of NMDA-activated currents. However, antagonists showed different relative efficacies to block peak currents and plateau currents, characterised by the following IC50 ratios: L-695,902: 0.98; RPR-104,632: 1.06; ACEA-1021: 1.69; LY-294,619: 1.71; and 5,7-dichlorokynurenic acid: 3.42. Our findings indicate a heterogeneity of glycine site antagonists in affecting NMDA receptor desensitisation, and suggest potential differences in their pharmacologies.

Topics: Animals; Benzothiadiazines; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Glycine; Kynurenic Acid; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Quinolones; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

N-methyl-D-aspartate (NMDA) receptor antagonists show therapeutic potential as neuroprotectants, analgesics, and anticonvulsants. In this context, we used electrical recording techniques to study the in vitro pharmacology of two novel quinoxalinediones, i.e., ACEA-1021 and ACEA-1031 (5-nitro-6,7- dichloro- and 5-nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxalinedione, respectively). Assays with NMDA receptors expressed by rat brain poly(A)+ RNA in Xenopus oocytes and with NMDA receptors in cultured rat cortical neurons indicated that ACEA-1021 and ACEA-1031 are potent competitive antagonists at NMDA receptor glycine sites. Apparent dissociation constants (Kb values) for ACEA-1021 and ACEA-1031 ranged between 6 and 8 nM for oocyte assays and between 5 and 7 nM for neuronal assays. Cloned NMDA receptors expressed in oocytes showed up to 50-fold variation in sensitivity, depending upon subunit composition. For example, using fixed agonist concentrations (10 microM glycine and 100 microM glutamate) IC50 values for ACEA-1021 with four binary combinations were as follows: NMDA receptor (NR)1A/2A, 29 nM; NR1A/2B, 300 nM; NR1A/2C, 120 nM; NR1A/2D, 1500 nM. Measurement of EC50 for glycine and calculation of Kb for the inhibitors indicated that differences in IC50 values are due to subunit-dependent variations in glycine affinity (EC50 ranged between approximately 0.1 and 1 microM) combined with variations in affinity of the antagonists themselves (Kb of approximately 2-13 nM). In addition to the strong antagonism of NMDA receptors, ACEA-1021 and ACEA-1031 were also moderately potent competitive inhibitors of non-NMDA receptors activated either by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or by kainate. Antagonist affinities were similar whether measured with receptors expressed by rat brain poly(A)+ RNA in oocytes (Kb of 1-2 microM) or with cultured neurons (Kb of 1.5-3.3 microM). Our results suggest that the in vivo neuro-protective actions of ACEA-1021 and ACEA-1031 are predominantly due to inhibition at NMDA receptor glycine sites, although additional inhibition at non-NMDA receptors may play an ancillary role.

Topics: Animals; Binding Sites; Brain; Cells, Cultured; Cloning, Molecular; DNA, Complementary; Electrophysiology; Female; Glycine; Kynurenic Acid; Neurons; Oocytes; Quinoxalines; Rats; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; RNA, Complementary; RNA, Messenger; Transcription, Genetic; Xenopus laevis