piperazine-2,3-dicarboxylic acid (PBPD) is a moderate affinity, competitive N-methyl-D-aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using L-[ 3 H]-glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. 2 Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures -fluorenone or phenanthrene groups -retained or enhanced activity. 3 Relative to PBPD, the phenanthrene derivative (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) displayed a 30-to 78-fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16-fold (NR2B) to 94-fold (NR2C) increase in affinity over PBPD. 4 Replacement of the biphenyl group of PBPD with a 9-oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 5 2 0 -Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3-to 5-fold at NR2A-, NR2B-and NR2D-containing receptors, but had little effect on NR2C-containing receptors. In contrast, 4 0 -fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. 6 The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D-preferring antagonist yet reported and thus may be useful in defining NR2C/ NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity.

Long-term potentiation (LTP) and long-term depression (LTD) are persistent modifications of synaptic strength that have been implicated in learning, memory, and neuronal development. Despite their opposing effects, both forms of plasticity can be triggered by the activation of NMDA receptors. One mechanism proposed for this bidirectional response is that the specific patterns of afferent stimulation producing LTP and LTD activate to different degrees a uniform receptor population. A second possibility is that these patterns activate separate receptor subpopulations composed of different NMDA receptor (NR) subunits. To test this hypothesis we examined the inhibition of LTP and LTD by a series of competitive NMDA receptor antagonists that varied in their affinities for NR2A/B and NR2C/D subunits. The potency for the inhibition of LTP compared with inhibition of LTD varied widely among the agents. Antagonists with higher affinity for NR2A/B subunits relative to NRC/D subunits showed more potent inhibition of LTP than of LTD. D-3-(2-carboxypiperazine-4-yl)-1-propenyl-1-phosphonic acid, which binds to NR2A/B with very high affinity relative to NR2C/D, showed an approximately 1000-fold higher potency for LTP than for LTD. These results show that distinct subpopulations of NMDA receptors characterized by different NR2 subunits contribute to the induction mechanisms of potentiation and depression.

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The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist (2R,3S)-(1-biphenylyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD, 16b) displays an unusual selectivity with improved relative affinity for NR2C and NR2D vs NR2A and NR2B. Analogues of 16b bearing aroyl or aryl substituents attached to the N(1) position of piperazine-2,3-dicarboxylic acid have been synthesized to probe the structural requirements for NR2C/NR2D selectivity. A phenanthrenyl-2-carbonyl analogue, 16e, had >60-fold higher affinity for NR2C and NR2D and showed 3-5-fold selectivity for NR2C/NR2D vs NR2A/NR2B. The phenanthrenyl-3-carbonyl analogue (16f) was less potent but more selective, having 5- and 7-fold selectivity for NR2D vs NR2A and NR2B, respectively. Thus, antagonists bearing bulky hydrophobic residues have a different NR2 subunit selectivity than that of typical antagonists.

1 The presynaptic depressant action of L-2-amino-4-phosphonobutyrate (L-AP4) on the monosynaptic excitation of neonatal rat motoneurones has been differentiated from the similar effects produced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), (1S,3S)-ACPD and (2S,3S,4S)-u-(carboxycyclopropyl)glycine (L-CCG-I), and from the postsynaptic motoneuronal depolarization produced by (lS,3R)-ACPD, by the actions of two new antagonists, a-methyl-L-AP4 (MAP4) and a-methyl-L-CCG-I (MCCG). Such

1 We investigated the agonist and antagonist activities of 22 new phenylglycine and phenylalanine derivatives for metabotropic glutamate receptors (mGluRs) by examining their effects on the signal transduction of mGluR1, mGluR2 and mGluR6 subtypes expressed in Chinese hamster ovary cells. This analysis revealed several structural characteristics that govern receptor subtype specificity of the agonist and antagonist activities of phenylglycine derivatives.2 Hydroxyphenylglycine derivatives possessed either an agonist activity on mGluR,/mGluR6 or an antagonist activity on mGluR,.3 Carboxyphenylglycine derivatives showed an agonist activity on mGluR2 but an antagonist activity on mGluR1. 4 a-Methylation or a-ethylation of the carboxyphenylglycine derivatives converts the agonist property for mGluR2 to an antagonist property, thus producing antagonists at both mGluRj and mGluR2.5 Structurally-corresponding phenylalanine derivatives showed little or no agonist or antagonist activity on any subtypes of the receptors. 6 This investigation demonstrates that the nature and positions of side chains and ring substituents incorporated into the phenylglycine structure are critical in determining the agonist and antagonist activities of members of this group of compounds on different subtypes of the mGluR family. 7 We also tested two ax-methyl derivatives of mGluR agonists. (2S, 1'S, 2'S)-2-(2-Carboxycyclopropyl)glycine (L-CCG-I) is a potent agonist for mGluR2 but a-methylation of this compound changes its activity to that of an mGluR2-selective antagonist. In contrast, a-methylation of L-2-amino-4-phosphonobutyrate (L-AP4) results in retention of an agonist activity on mGluR6. Thus, a-methylation produces different effects, depending on the chemical structures of lead compounds and/or on the subtype of mGluR tested.

An understanding of the physiological and pathological roles of metabotropic glutamate receptors (mGluRs) is currently hampered by the lack of selective antagonists. Standard extracellular recording techniques were used to investigate the activity of recently reported mGluR antagonists on agonist‐induced depressions of synaptic transmission in the lateral perforant path of hippocampal slices obtained from 12–16 day‐old rats. The group III specific mGluR agonist, (S)‐2‐amino‐4‐phosphonobutanoate (L‐AP4) depressed basal synaptic transmission in a reversible and dose‐dependent manner. The mean (±s.e.mean) depression obtained with 100 μm L‐AP4 (the maximum concentration tested) was 74 ± 3% and the IC50 value was 3 ± 1 μm (n = 5). The selective group II mGluR agonists, (1S,3S)‐1‐aminocyclopentane‐1,3‐dicarboxylate ((1S,3S)‐ACPD) and (2S,1′R,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) also depressed basal synaptic transmission in a reversible and dose‐dependent manner. The mean depression obtained with 200 μm (1S,3S)‐ACPD was 83 ± 8% and the IC50 value was 12 ± 3 μm (n = 5). The mean depression obtained with 1 μm DCG‐IV was 73 ± 7% and the IC50 value was 88 ± 15 nM (n = 4). Synaptic depressions induced by the actions of 20 μm (1S,3S)‐ACPD and 10 μm L‐AP4 were antagonized by the mGluR antagonists, (+)‐α‐methyl‐4‐carboxyphenylglycine ((+)‐MCPG), (S)‐2‐methyl‐2‐amino‐4‐phosphonobutanoate (MAP4), (2S, 1′S,2′S)‐2‐methyl‐2‐(2′‐carboxycyclopropyl)glycine (MCCG), (RS)‐α‐methyl‐4‐tetrazolylphenylglycine (MTPG), (RS)‐α‐methyl‐4‐sulphonophenylglycine (MSPG) and (RS)‐α‐methyl‐4‐phosphonophenylglycine (MPPG) (all tested at 500 μm). (+)‐MCPG was a weak antagonist of both L‐AP4 and (1S,3S)‐ACPD‐induced depressions. MCCG was selective towards (1S,3S)‐ACPD, but analysis of its effects were complicated by apparent partial agonist activity. MAP4 showed good selectivity for L‐AP4‐induced effects. The most effective antagonist tested against 10 μm L‐AP4 was MPPG (mean reversal 90 ± 3%; n = 4). In contrast, the most effective antagonist tested against 20 μm (1S,3S)‐ACPD induced depressions was MTPG (mean reversal 64 ± 4%; n = 4). Both antagonists produced parallel shifts in agonist dose‐response curves. Schild analysis yielded estimated KD values of 11.7 μm and 27.5 μm, respectively. Neither antagonist had any effect on basal transmission or on depressions induced by the adenosine receptor agonist, 2‐chloroadenosine (500 nM; n = 3). We conclude that both group II and group III mGluRs can mediate synaptic depressions induced by mGluR agonists in the lateral perforant path. The mGluR antagonists MTPG, MPPG and MAP4 should be useful in determining the roles of group II and III mGluRs in the central nervous system.

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