Structure-activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid

Abstract

(2S*,3R*)-1-(biphenyl-4-carbonyl)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-[3H]-glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. 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. 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. Replacement of the biphenyl group of PBPD with a 9-oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 2'-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'-fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. 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. British Journal of Pharmacology (2004) 141, 508-516. doi:10.1038/sj.bjp.0705644

Figure 1

Structures of PBPD derivatives and reference compounds. PBPD derivatives were synthesized and tested as inhibitors of L-[³H]-glutamate binding to NMDA receptors in rat brain sections. Compounds that displayed less than 50% inhibition at 100 μM are listed as ‘Inactive Compounds'. The reference compound (R)-AA has the structure of (R)-AP5 in which the phosphonate group is replaced by a carboxy group. (R)-CPP has the structure of (R)-CPPene in which the double bond is reduced.

Figure 2

Representative autoradiographs showing compound inhibition of L-[³H]-glutamate binding to NMDA receptors. Autoradiographs are horizontal sections of adult rat brain in (a) absence of inhibitors, total binding, or (b) in the presence of 2 μM PPDA, (c) 6 μM CPP or (d) 500 μM NMDA, nonspecific binding. PPDA preferentially inhibits binding in the medial thalamus (MT) compared to the septum (Sp), striatum (Str), cerebral cortex (Ctx), hippocampus (HC), olfactory bulb (OB) and the cerebellum (CBLM).

Figure 3

A representative recording of antagonist blockade of NMDA receptor-mediated responses in Xenopus oocytes. NR1/NR2C RNA-injected oocytes were voltage clamped at −60 mV and inward currents were evoked by bath application of 10 μM L-glutamate plus 10 μM glycine (indicated by the heavy bar). Increasing concentrations of bath applied antagonist (UBP131) reduced the inward currents.

Figure 4

NMDA receptor subunit selectivity of PBPD compared to other competitive antagonists. Each compound was evaluated for the ability to block agonist-induced responses of NR1a/NR2 NMDA receptors expressed in Xenopus oocytes. IC₅₀ values were generated from dose–response curves and the corresponding K_i value is shown above. To compare the subunit selectivity profiles of drugs with various affinities, K_i values were normalized to the drug's NR1/NR2A affinity. The selectivities of PBPD and the novel antagonist PEAQX are shown in comparison with the well-established NMDA receptor antagonists (R)-AA, (R)-CPP, (R)-AP5 and (R)-CPPene. Data for (R)-AP5, (R)-CPPene and PBPD are from Buller & Monaghan (1997) and are presented for comparison. Absolute values and statistical differences are listed in Table 2.

Figure 5

NMDA receptor subunit selectivity of PBPD derivatives. Each compound was evaluated for the ability to block responses to 10 μM L-glutamate plus 10 μM glycine at each of the NR1a/NR2 NMDA receptors expressed in Xenopus oocytes. To compare the subunit selectivity profiles of drugs with various affinities, K_i values were normalized to the drug's NR1/NR2A affinity. Absolute values and statistical differences are listed in Table 2.