In vtro parmacology of ACEA-1021 and ACEA-1031: Systemically ative qinoxalinediones with high affinity and selectivity for N-methyl-D-aspartate receptor glycine sites

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 (K_b 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 μM glycine and 100 μM 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 ∼0.1 and 1 μM) combined with variations in affinity of the antagonists themselves (K_b of ∼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 α-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 (K_b of 1-2 μM) or with cultured neurons (K_b of 1.5-3.3 μM). Our results suggest that the in vivo neuroprotective 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.