7-[9-Fluorenylmethoxycarbonyl(methyl)amino]heptanoic acid

The specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid (3H-APH), a potent N-methyl-D-aspartate (NMDA) antagonist, to extensively washed, previously frozen crude mitochondrial fractions of rat brain is described. Binding was optimal at physiological pH and temperature and, in Triscitrate buffer, attained equilibrium within 60 minutes. Scatchard analysis of the equilibrium data for forebrain revealed a single, non-interacting population of binding sites (BMapp = 15 picomoles/mg protein; KDapp = 3.6 uM; Hill coefficient = 0.92, r = 0.99; N = 5). Specific binding of the ligand was readily reversible by unlabeled APH and was absent in peripheral tissues including heart, lung, kidney, liver, spleen and striate muscle and in heat treated brain sonicates. An 8-fold variation in the amount of ligand bound to brain membranes prepared from different regions was observed with binding being greatest in the hippocampal formation and least in the midbrain. Kainic acid, NMDA and aspartic acid exhibited negligible affinity for the [3H]-APH site; in contrast, quisqualic acid, ibotenic acid, glutamatic acid, homocysteic acid and 2-amino-4-phosphono butyric acid were moderately potent displacers. The results indicate that [3H]-APH labels a quisqualate preferring site in vitro. Unlike the receptor labeled by [3H]-glutamate however, [3H]-APH binding was attenuated in the presence of chloride ions suggesting that this ligand may label a subpopulation of excitatory amino acid receptors.

It is shown that (a) peripheral injections of quinolinic acid cause neuronal excitation with a latency much less than that of convulsions due to quinolinic acid and (b) peripherally injected 2-amino-7-phosphono-heptanoic acid (2APH) does antagonise neuronal excitation due to quinolinic acid applied locally by microiontophoresis. It is concluded that the previously reported failure of 2APH to prevent quinolinic acid seizures is a reflection of different modes of action of quinolinic acid in causing neuronal excitation and convulsions, and does not contradict the suggestion that quinolinic acid acts at N-methyl-D-aspartate (NMDA) receptors in the brain.

A selection of biphenyl-analogues of 2-amino-7-phosphonoheptanoic acid (AP7), N-methyl-D-aspartate (NMDA) receptor antagonists with high affinity in vivo efficacy. The lead compound SDZ EAB 515 was found to inhibit L-phenylalanine uptake by the large neutral amino acid carrier in vitro and in vivo; active transport may thus confer a good bioavailability to this class of compounds. CNS effects were demonstrated by significant changes in 2-deoxyglucose-uptake in various brain regions at doses from 1 to 10 mg/kg i.p. With the most active agent, SDZ 220-581, full protection against maximal electroshock seizures (MES) was obtained at oral doses of 10 mg/kg in rats and in mice. The compound had a fast onset (< or = 1 hr) and a long duration (> or = 24 hr) of action. Motor-debilitating effects (impairment of rotarod performance) occurred at doses about 10 times higher than those required for protection against MES. Neuroprotective activity was demonstrated by the ability of the compounds to reduce the extent of quinolinic acid-induced striatal lesions in rats, in the dose range of 3-15 mg/kg (i.p.) or 10-50 mg/kg (p.o.). In the middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats, the test compounds reduced the infarct size by 40-50% when given i.v. before or by 20-30% when given i.v. 1 hr after MCAO. SDZ 220-581 provided 20-30% protection at > or = 2 x 10 mg/kg p.o. This compound also showed analgesic activity at low oral doses in a model of neuropathic pain, although higher doses were required in model of mechanical inflammatory hyperalgesia. Unexpectedly, SDZ 220-581 at low s.c. doses counteracted the antiparkinsonian effects of L-DOPA in MPTP-treated marmosets. (Sub)chronic administration of SDZ 220-581 did not reduce its ability to protect against quinolinic acid neurotoxicity, and no upregulation of NMDA receptors was detected using a [3H]CGP-39653 binding assay. In conclusion, from a series of biphenyl-AP7-derivatives, SDZ 220-581 is clearly the most active compound in vivo. Its pharmacological profile with a good, long-lasting oral activity might open up novel therapeutic applications for competitive NMDA receptor antagonists.