Antagonists selective for NMDA receptors containing the NR2B subunit.

In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.

Functional characterization of CP-465,022, a selective, noncompetitive AMPA receptor antagonist.

The hypothesis that aberrant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity contributes to epileptogenesis and neurodegeneration has prompted the search for AMPA receptor antagonists as potential therapeutics to treat these conditions. We describe the functional characterization of a novel quinazolin-4-one AMPA receptor antagonist, 3-(2-chloro-phenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one (CP-465,022). This compound inhibits AMPA receptor-mediated currents in rat cortical neurons with an IC(50) of 25 nM. Inhibition is noncompetitive with agonist concentration and is not use- or voltage-dependent. CP-465,022 is selective for AMPA over kainate and N-methyl-D-aspartate receptors. However, the compound is found to be equipotent for AMPA receptors composed of different AMPA receptor subunit combinations. This is indicated by the finding that CP-465,022 is equivalently potent for inhibition of AMPA receptor-mediated responses in different types of neurons that express different AMPA receptor subunits. Thus, CP-465,022 provides a new tool to investigate the role of AMPA receptors in physiological and pathophysiological processes.

CP-101,606, an NR2B subunit selective NMDA receptor antagonist, inhibits NMDA and injury induced c-fos expression and cortical spreading depression in rodents.

(1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. This compound was used to investigate the role of NR2B containing receptors in three responses to NMDA receptor activation in vivo. In mouse, CP-101,606 completely inhibited increases in fos-like immunoreactivity in dentate gyrus caused by a subconvulsant intraperitoneal dose of NMDA. In rat, the compound completely blocked cortical c-fos mRNA induction following focal injury in parietal cortex and the initiation and propagation of electrically induced cortical spreading depression. Inhibition of these responses by CP-101,606 indicates that c-fos induction and cortical spreading depression are dependent on activation of NMDA receptors containing the NR2B subunit. Since NMDA receptor dependent c-fos induction and cortical spreading depression may contribute to neuron loss after focal CNS injury, inhibition of these responses by CP-101,606 may contribute to the neuroprotective efficacy of the compound.

Separation of alpha 1 adrenergic and N-methyl-D-aspartate antagonist activity in a series of ifenprodil compounds.

Ifenprodil (1) represents a new class of N-methyl-D-aspartate (NMDA) antagonist. This drug also possesses potent activity at several other brain receptors (most notably alpha 1 adrenergic receptors). We have prepared the enantiomers and diastereomers of ifenprodil along with a series of partial structures in order to explore the basic structure activity relations within this class of compounds. From this study, it is clear that alpha 1 adrenergic and NMDA receptor activities may be separated by selection of the threo relative stereochemistry. Examination of the optical isomers of threo-ifenprodil (2) reveals that no further improvement in receptor selectivity is gained from either antipode. Individual removal of most of the structural fragments from the ifenprodil molecule generally results in less active compounds although fluorinated derivative 9 with threo relative stereochemistry is somewhat more potent and substantially more selective for the NMDA receptor. Finally a minimum structure for activity in this series (14) has been identified. This stripped-down version of ifenprodil possesses nearly equivalent affinity for the NMDA receptor with no selectivity over alpha 1 adrenergic receptors.

Quinazolin-4-one alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists: structure-activity relationship of the C-2 side chain tether.

A series of 6-fluoro-3-(2-chlorophenyl)quinazolin-4-ones has been prepared, which contains a 2-fluorophenyl ring attached to C-2 by a variety of two-atom tethers. These compounds were used to probe the structure-activity relationship (SAR) for AMPA receptor inhibition. The relative potencies of the new compounds ranged from 11 nM to greater than 10 microM. The differential activity of the compounds was rationalized on the basis of alterations of the 2-fluorophenyl positioning (planar and radial) relative to the quinazolin-4-one ring based on computational methods. From this effort, new AMPA receptor antagonists, containing the methylamino tether group, have been identified.

A unified approach to systematic isosteric substitution for acidic groups and application to NMDA antagonists related to 2-amino-7-phosphonoheptanoate.

A systematic approach to the replacement of acidic groups with potential bioisosteres is described. The strategy involves simple nucleophilic displacement of a common alkyl halide precursor with a variety of mercaptoazoles and related molecules. The mercaptoazoles and their oxidized derivatives (sulfinyl- and sulfonylazoles) represent a series of possible surrogates for acidic groups which span a pKa range from about 4.5-11.5. This simple strategy was extended to include 2-hydroxy- or 2-aminothiophenyl groups which function as relatively nonacidic isosteres for a phosphonic acid. By replacing the phosphonic acid of 2-amino-7-phosphonoheptanoate (AP-7) with these groups, we have synthesized novel N-methyl-d-aspartate (NMDA) antagonists.

(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol: a potent new neuroprotectant which blocks N-methyl-D-aspartate responses.

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (20, CP-101,606) has been identified as a potent and selective N-methyl-D-aspartate (NMDA) antagonist through a structure activity relation (SAR) program based on ifenprodil, a known antihypertensive agent with NMDA antagonist activity. Sites on the threo-ifenprodil skeleton explored in this report include the pendent methyl group (H, methyl, and ethyl nearly equipotent; propyl much weaker), the spacer group connecting the C-4 phenyl group to the piperidine ring (an alternating potency pattern with 0 and 2 carbon atoms yielding the greatest potency), and simple phenyl substitution (little effect). While potent NMDA antagonists were obtained with a two atom spacer, this arrangement also increased alpha 1 adrenergic affinity. Introduction of a hydroxyl group into the C-4 position on these piperidine ring resulted in substantial reduction in alpha 1 adrenergic affinity. The combination of these observations was instrumental in the discovery of 20. This compound potently protects cultured hippocampal neurons from glutamate toxicity (IC50 = 10 nM) while possessing little of the undesired alpha 1 adrenergic affinity (IC50 approximately 20 microM) of ifenprodil. Furthermore, 20 appears to lack the psychomotor stimulant effects of nonselective competitive and channel-blocking NMDA antagonists. Thus, 20 shows great promise as a neuroprotective agent and may lack the side effects of compounds currently in clinical trials.

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol.

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.

Neuroprotective effects of the N-methyl-D-aspartate receptor antagonists ifenprodil and SL-82,0715 on hippocampal cells in culture.

The N-methyl-D-aspartate (NMDA) antagonists ifenprodil and SL-82,0715 were examined for neuroprotective efficacy against glutamate toxicity of hippocampal neurons in culture. Hippocampal cells were grown on 96-well culture plates for 2 to 3 weeks and then exposed for a 15-min period to glutamate or NMDA. Neurodegeneration was quantified 24 hr after the excitotoxin exposure, by measuring the activity of lactate dehydrogenase leaked into the culture medium by the damaged cells. Glutamate induced a concentration-dependent increase in lactate dehydrogenase that reached 3-fold the activity of control cultures. The NMDA antagonists MK-801 and AP-7 blocked this neurotoxicity when added either during or after the glutamate exposure. Ifenprodil and SL-82,0715 blocked the neurotoxicity only when added during the excitotoxin exposure. Ifenprodil was 3 times more potent than SL-82,0715 in blocking glutamate or NMDA-induced neurotoxicity. Glycine did not reverse the neuroprotective effects of these antagonists. The neuroprotective effect of ifenprodil or SL-82,0715 did not appear to result from actions at alpha-1 adrenergic or sigma receptor sites because the alpha-1 adrenergic antagonist prazosin and the sigma ligands haloperidol, 3-(3-hydroxyphenyl)-N-propylpiperidine) and 1,3-di-o-tolylguanidine) showed no neuroprotective activity. We conclude that ifenprodil and SL-82,0715 protect cultured hippocampal neurons from excitotoxic damage by antagonizing NMDA receptors.

Methaqualone derivatives are potent noncompetitive AMPA receptor antagonists.

Quinazolin-4-one derivatives of methaqualone substituted at C-2 define a new class of noncompetitive antagonists at AMPA receptors.

Characterization of the binding site for a novel class of noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonists.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is an ionotropic glutamate receptor that mediates fast excitatory synaptic transmission throughout the central nervous system. In addition to the glutamate binding site, allosteric modulatory sites on the receptor are inferred from the ability of synthetic compounds to affect channel function without interaction with the glutamate binding site. We have identified a novel class of potent, noncompetitive AMPA receptor antagonists typified by CP-465, 022 and CP-526,427. The latter compound was radiolabeled and used to elucidate the pharmacology of one allosteric modulatory site. [(3)H]CP-526,427 labels a single binding site in rat forebrain membranes with a K(d) value of 3.3 nM and a B(max) of 7.0 pmol/mg of protein. The [(3)H]CP-526,427 binding site does not seem to interact directly with the glutamate binding site but overlaps with that for another class of AMPA receptor antagonists, the 2,3-benzodiazepines. This binding site is distinct from that for the antagonist Evans blue and for several classes of compounds that modulate AMPA receptor desensitization. These results indicate the existence of at least two physically distinct allosteric sites on the AMPA receptor through which channel activity or desensitization is modulated.

Atropisomeric quinazolin-4-one derivatives are potent noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists.

Piriqualone (1) was found to be an antagonist of AMPA receptors. Structure activity optimization was conducted on each of the three rings in 1 to afford a series of potent and selective antagonists. The sterically crowded environment surrounding the N-3 aryl group provided sufficient thermal stability for atropisomers to be isolated. Separation of these atropisomers resulted in the identification of (+)-38 (CP-465,022), a compound that binds to the AMPA receptor with high affinity (IC50 = 36 nM) and displays potent anticonvulsant activity.