Synthesis of 1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3,4-f]- quinoxaline-2,3-dione and related quinoxalinediones: characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (and N-methyl-D-aspartate) receptor and anticonvulsant activity.

Four related series of substituted quinoxalinediones containing angular fused-piperidine rings have been synthesized as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists with potential as neuroprotective agents, primarily for acute therapy immediately following a stroke. The compounds were tested for their affinity to the AMPA, kainate, and strychnine-insensitive glycine receptor sites. In AMPA binding, the most potent compound was 27a (PNQX, IC50 = 63 nM), with affinity comparable to the literature standard 1 (NBQX, IC50 = 52 nM). Other 6-nitro analogs from the 9-aza series had comparable affinity at the AMPA receptor, as did 6-nitro-8-aza derivatives such as 13a (iPNQX, IC50 = 290 nM). The receptor binding profile of 27a differed from that of 1 in that 27a possessed significant affinity at the glycine site of the N-methyl-D-aspartate (NMDA) receptor, whereas 1 was essentially inactive. Three compounds, 26c, 26d, and 26e, demonstrated moderate selectivity for kainate relative to AMPA receptors. Selected analogs reported herein as well as in the literature were superimposed to generate an AMPA pharmacophore model, and 6-substituted compounds from the PNQX and iPNQX series were combined and analyzed via quantitative structure-activity relationship techniques. Compounds with high affinity at non-NMDA receptors were further characterized in functional assays in neuronal cell culture and in a cortical wedge preparation. Both 1 and 27a showed comparable effectiveness in an AMPA- and kainate-induced excitoxicity assay. Both inhibited AMPA-induced depolarizations in the cortical wedge. However, 27a also inhibited spontaneous epileptiform discharges in the cortical wedge (reversed by glycine), while 1 was ineffective. The combination of AMPA and NMDA antagonist activity may contribute to the 30-fold difference in potency between 27a and 1 in the maximal electroshock convulsant assay in mice. The significant in vivo potency of 27a suggests that it has potential clinical utility.

Synthesis and pharmacological evaluation of 4a-phenanthrenamine derivatives acting at the phencyclidine binding site of the N-methyl-D-aspartate receptor complex.

A novel series of octahydrophenanthrenamines and their heterocyclic analogues have been synthesized as potential noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor complex. The compounds were evaluated for their affinity at the phencyclidine (PCP) binding site by determining their ability to displace [3H]TCP from crude rat brain synaptic membranes. A wide range of affinities were observed, with the most potent analogs possessing IC50's equivalent to that of the reference agent MK-801 (3, dizocilpine). NMDA antagonist activity was demonstrated by prevention of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Selected compounds were also studied in vivo to determine their ability to prevent the lethal effects of systemically injected NMDA in the mouse. In general, the SAR of the phenanthrenamine series may be summarized as follows: (a) for the amino group at C4a, NHMe > NH2 > NHEt >> NC5H10; (b) for the B-ring substitution, X = CH2 > S > O; (c) unsaturation of the C ring decreases receptor affinity; (d) cis-ring fusion between the B and C rings is desirable; (e) 6-hydroxy or 6-methoxy substitution of the phenanthrenamine system identified an additional hydrogen bonding interaction that substantially increased receptor affinity; (f) spiro analogues (such as 55, IC50 = 3400 nM), which altered the point of attachment of the C ring, caused a substantial reduction in PCP-site affinity. Molecules from this series were useful for refining a pharmacophore model consistent with previous models of the PCP site. In this model, the (R)-(+)-phenanthrenamine 13 superimposes closely onto MK-801 (3), and the angular 4a-amino group is believed to hydrogen bond with a putative receptor site atom. In the phenanthrenamine and thiaphenanthrenamine series, the (R)-(+)-enantiomers (9, 13, and 44) are more potent by approximately 5-10-fold than their corresponding (S)-(-)-enantiomers with respect to their affinity for the PCP site, their ability to prevent accumulation of [45Ca2+] in cultured neuronal cells, and their protection against the lethal effects of NMDA in mice. In general, there was no separation between the dose that prevented NMDA lethality and the dose that produced ataxia in mice, except in the case of the thiaphenanthrenamines 41 and 43. We have not yet obtained evidence that this small separation in activity offers a therapeutic advantage in the treatment of cerebral ischemia or other neurodegenerative disorders.

Exploration of N-phosphonoalkyl-, N-phosphonoalkenyl-, and N-(phosphonoalkyl)phenyl-spaced alpha-amino acids as competitive N-methyl-D-aspartic acid antagonists.

A series of N-substituted alpha-amino acids containing terminal phosphonic acid groups has been synthesized as potential N-methyl-D-aspartate (NMDA) receptor antagonists. NMDA receptor affinity was determined by displacement of a known ligand ([3H]CPP) from crude rat brain synaptic membranes; an antagonist action was demonstrated by the inhibition of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Receptor affinity was significantly correlated with antagonist activity (Figure 1). Moderate affinity (IC50 = 1-2 microM) was retained for analogues (31 and 32, Table I; and 59 and 66, Table II) with reduced flexibility in their phosphonate side chains and is consistent with entropy playing a role in determining receptor affinity. Modeling studies suggest a folded conformation that brings the distal phosphonic acid group into close proximity with the alpha-carboxylate is required for binding. Each of the active analogues possess entropy-limiting features (double bonds, phenyl rings) in their side chains that allows the superposition of their key NH2, alpha-COOH, and distal PO3H2 groups with those of known competitive antagonists. Affinity decreased for analogues with alpha-carbon substitution, presumably because the alpha-substituent inhibits the folding of these structures into a bioactive conformation and occupies receptor-excluded volume. A complete description of the NMDA antagonist pharmacophore model is provided in a companion paper.