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1 The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the Nmethyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2 Radioligand binding experiments demonstrated that CGP 37849 potently (K1 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-( ±)-342-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nm, and was 4, 5 and 7 fold more potent than the antagonists ((±)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3 CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4 In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CAI pyramidal cell firing rate. In slices bathed in medium containing low Mg2+ levels, concentrations of CGP 37849 up to 10pM suppressed burst firing evoked in CAl neurones by stimulation of Schaffer collateral-commissural fibres without affecting the magnitude of the initial population spike; CGP 39551 exerted the same effect but was weaker. In vivo, oral administration to rats of either CGP 37849 or CGP 39551 selectively blocked firing in hippocampal neurones induced by ionophoreticallyapplied NMDA, without affecting the responses to quisqualate or kainate. al., 1988). Many studies have demonstrated that activation of the NMDA receptor is involved in the generation of epileptiform activity and in hypoxic-ischaemic neuronal damage, and conversely that NMDA receptor antagonists are anticonvulsant and cerebroprotective in animal models of epilepsy and stroke (see reviews by Meldrum, 1985;Rothman & Olney, 1986;Choi, 1988;Patel et al., 1988;Iversen et al., 1989;Albers et al., 1989 (Meldrum, 1985;Rothman & Olney, 1986;Albers et al., 1989;.Antagonism of NMDA receptor mechanisms potentially may be achieved by a number of different approaches. In addition to the transmitter recognition site, the NMDA receptor complex comprises an allosteric regulatory site and a channel binding domain (Foster & Fagg, 1987b), and possibly also sites defined by the actions of Zn2", polyamines, tricyclic antidepressants, ifenprodil and CGP 31358 (see Lodge, 1989;Baud et al., 1989). At present, however, the two most well characterized sites are (1) the transmitter recognition site, at which substances such as ...

Anticonvulsant properties of CGP 37849 and CGP 39551, two novel phosphono-amino acids which are competitive NMDA receptor antagonists, were examined in rodents. At optimal pretreatment times CGP 37849 suppressed electroshock-induced seizures in mice and rats with ED50s ranging from 8 to 22 mg/kg after oral administration, and 0.4 to 2.4 mg/kg after i.v. and i.p. injection. Relative to CGP 37849, CGP 39551 was more potent after p.o. (ED50 3.7-8.1 mg/kg), and less potent after i.v. or i.p. treatment (ED50 2.7-8.7 mg/kg). Following oral treatment, the duration of action of CGP 37849 was about 8 h, while CGP 39551 still showed good activity after 24 h (ED50 8.7 mg/kg, mouse; 21 mg/kg, rat). Both compounds were anticonvulsant at doses below those at which overt behavioural side effects were apparent. CGP 39551 delayed the development of kindling in rats at doses of 10 mg/kg p.o. and above, and showed weak anticonvulsant activity against pentylenetetrazol-evoked seizures. CGP 37849 and CGP 39551 are the first competitive NMDA antagonists to show oral anti-convulsant properties in a therapeutically-useful dose-range, and hence are interesting candidates for novel antiepileptic therapy in man.

Activation of N-methyl-D-aspartate (NMD)A)receptors is essential for synaptic plasticity in the central nervous system and contributes to neuronal death under various pathological conditions. Although several subunits have been cloned, the structure of NMDA receptors in situ is unresolved. By using a photoreactive antagonist with nanomolar affnity to the NMDA-binding site, three types of receptors were differentiated by their pattern of photoaffinity-labeled METHODS The photoaffinity ligand 125I-labeled CGP 55802A (specific radioactivity 1150 Ci/mmol; 1 Ci = 37 GBq) was synthesized as described elsewhere (R.H., H.A., and C.A., unpublished work). For radioligand binding, rat whole-brain membranes were washed with 100 mM Tris acetate, pH 8.0/0.03% (wt/vol) Triton X-100 and the same buffer without detergent. Aliquots (250 ug ofprotein per assay) were incubated with 1.0 nM '25I-labeled CGP 55802A for 30 min at room temperature in 100 mM Tris acetate, pH 8.0, with the level of nonspecific binding (18% of total binding) being determined in the presence of 2 mM NMDA or 1 mM glutamate. Rapid filtration through GF/C filters (Whatman) was followed by washing twice with 4 ml of ice-cold incubation buffer. For photoaffinity labeling (50 pg of protein per assay) the 30-min incubation was followed by irradiation with UV light (355 nm; 36-W TLD lamp, Philips) for 5 min at a distance of 3 cm. After being washed with 100 mM Tris acetate, pH 8.0, at 40C by centrifugation, the samples were subjected to 6.5% SDS/ PAGE followed by autoradiography (Kodak X-Omat AR film for 2-5 days) and densitometric analysis of the radiotraces (Shimadzu, Kyoto, CS-930). The pattern of photolabeled proteins was the same when Triton X-100 was omitted from the membrane preparation.For autoradiography, cryostat sections, kept in 100 mM Tris acetate, pH 8.0, for 40 min at room temperature, were incubated with 1 nM 125I-labeled CGP 55802A as described above for membranes. For reversible binding the sections were subsequently washed for 20 min in ice-cold incubation buffer (three buffer changes). In the case of photoaffinity labeling sections were exposed to 355-nm light for 5 min, followed by washing for 10 min at room temperature in incubation buffer containing 1 mM glutamate (three buffer changes). The sections were air-dried before exposure to film.Electrophysiological studies were done on transverse hippocampal slices (400 ,um) prepared from male Tif:RAI f(SPF) Abbreviations: NMDA, N-methyl-D-aspartate; Pn, postnatal day n. 8434

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. block, slow activation and inactivation kinetics, and the requirement for glycine as a co-agonist in channel gating (1-6). They play a crucial role in brain development, synaptic plasticity and the pathophysiology of major neurological disorders (7-11). NMDA receptors are assembled from the NR1 subunit in combination with at least one type of NR2 subunit (NR2A-2D) (12-15), giving rise to receptor subtypes with distinct electrophysiological and pharmacological properties (2, 16 -21).Inhibition of NMDA receptors by glycine site antagonists is of major interest for the therapeutic intervention in pathological central nervous system states like stroke, head injury, and epilepsy. At doses evoking remarkable anticonvulsant and neuroprotective effects, glycine site antagonists are less prone to psychotomimetic actions and exhibit less severe motoric and cognitive side effects than glutamate site antagonists (22). Further improvements are expected from the targeting of the glycine site of NMDA receptor subtypes.The NR1 subunit harbors main determinants of the glycine binding site in recombinant receptors as well as in situ. Recombinant homomeric NR1 receptors bind glycine agonists and antagonists (19,23,24), and various amino acid residues involved in high affinity binding have been identified by mutational analysis of the NR1 subunit (25-28). Similarly, in native receptors, the novel glycine antagonist and photoaffinity ligand [ 3 H]CGP 61594 labels exclusively the NR1 subunit of NMDA receptors in brain membranes.2 In contrast, the glutamate binding pocket is formed by the NR2 subunits (29). Interestingly, the properties of the glycine site are influenced by the type of NR2 subunit that is assembled with the NR1 subunit. Glycine displays an up to 10-fold lower potency at NR1/2A receptors than at NR1/2B, NR1/2C, or NR1/2D receptors, and a similar difference in affinity has also been observed for the glycine site agonists D-serine, 1-aminocyclobutanecarboxylic acid, D-alanine, 414 (16,19,21,[30][31][32][33]. However, glycine site antagonists (e.g. L-701,324, L-689,560, L-695,902, CKA) did not clearly differentiate between recombinant NMDA receptor subtypes (23,34). Mutational analysis of the NR1 subunit suggested structural differences in the binding domains of glycine site agonists and antagonists (25)(26)(27)(28). Some of the mutated residues affected the affinity for