Search

In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.

A series of compounds based on the mGluR5-selective ligand 2-methyl-6-(phenylethynyl)pyridine (MPEP) were designed and synthesized. The compounds were found to be either structural analogues of MPEP, substituted monomers, or dimeric analogues. All compounds retained mGluR5 selectivity with only weak or no activity at other mGluRs or iGluRs. The substituted analogue, 1,3-bis(pyridin-2-ylethynyl)benzene (19), is a potent negative modulator at mGluR5, whereas all other compounds lost potency relative to MPEP and showed that activity is highly dependent on the position of the nitrogen atom in the pyridine moieties. A homology modeling and ligand docking study was used to understand the binding mode and the observed selectivity of compound 19.

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. These receptors play an important role for the development and function of the nervous system, and are essential in learning and memory. However, iGluRs are also implicated in or have causal roles for several brain disorders, e.g. epilepsy, Alzheimer's disease, Parkinson's disease and schizophrenia. Their involvement in neurological diseases has stimulated widespread interest in their structure and function. Since the first publication in 1998 of the structure of a recombinant soluble protein comprising the ligand-binding domain of GluA2 extensive studies have afforded numerous crystal structures of wildtype and mutant proteins including different ligands. The structural information obtained combined with functional data have led to models for receptor activation and desensitization by agonists, inhibition by antagonists and block of desensitization by positive allosteric modulators. Furthermore, the structural and functional studies have formed a powerful platform for the design of new selective compounds.

The design and synthesis of new N1-substituted 3-carboxy- and 3-phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high-affinity interaction with these receptors was performed. We identified two highly potent and selective competitive NMDA receptor antagonists, (5S,alphaR)-1 and (5S,alphaR)-4, which exhibit good in vitro neuroprotective activity and in vivo anticonvulsant activity by i.p. administration, suggesting that these molecules may have potential use as therapeutic agents.

More than 50 structures have been reported on the ligand-binding core of the ionotropic glutamate receptor iGluR2 that belongs to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid-type of receptors. In contrast, the ligand-binding core of the kainic acid-type receptor iGluR5 has only been crystallized with three different ligands. Hence, additional structures of iGluR5 are needed to broaden the understanding of the ligand-binding properties of iGluR5, and the conformational changes leading to channel opening and closing. Here, we present two structures of the ligand-binding core of iGluR5; one as a complex with the partial agonist (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid (domoic acid) and one as a complex with the antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid ((S)-ATPO). In agreement with the partial agonist activity of domoic acid, the ligand-binding core of the iGluR5 complex is stabilized by domoic acid in a conformation that is 11° more open than the conformation observed in the full agonist (S)-glutamic acid complex. This is primarily caused by the 5-carboxy-1-methyl-hexa-1,3-dienyl moiety of domoic acid and residues Val685-Thr690 of iGluR5. An even larger domain opening of 28° is introduced upon binding of the antagonist (S)-ATPO. It appears that the span of domain opening is much larger in the ligand-binding core of iGluR5 (30°) compared with what has been observed in iGluR2 (19°). Similarly, much larger variation in the distances between transmembrane linker residues in the two protomers comprising the dimer is observed in iGluR5 as compared with iGluR2.

The erythro- and threo-amino-(3′-hydroxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids, stereoisomers of tricholomic acid, were synthesized along with the corresponding higher homologues erythro- and threo-amino-(3′-carboxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids. The target compounds were prepared via the 1,3-dipolar cycloaddition of a suitable nitrile oxide to (±)-2-tert-butoxycarbonylamino-3-buten-1-ol. Such a strategy allowed the synthesis of the two stereoisomeric amino acids in comparable amounts. The pharmacological activity of these compounds was investigated at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) by means of receptor binding assays to rat cortical membranes, electrophysiological tests and second messenger assays at cloned receptors expressed in CHO cells. Their pharmacological profiles were compared to those of l -glutamate and of the previously described selective NMDA receptor antagonists 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acids in order to highlight the effect of increasing/reducing the distance between the amino acid moiety and the distal acid group, which represent the two pharmacophoric entities.

The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant neuroprotective effect when tested in an oxygen glucose deprivation (OGD) cell culture test. The same compounds were preliminarily assayed using Xenopus oocytes expressing cloned rat NMDA receptors containing the NR1 subunit in combination with either NR2A, NR2B, NR2C, or NR2D subunit. In this assay, all three derivatives showed high antagonist potency with preference for the NR2A and NR2B subtypes, with derivative (-)-4 behaving as the most potent antagonist. The biological data are discussed on the basis of homology models reported in the literature for NMDA receptors and mGluRs.

The second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids reported herein proceeds via Strecker reaction of chiral ketimines, obtained from condensation of racemic 2-ethoxycarbonylmethylcyclopentanone and commercially available (S)- and (R)-1-phenylethylamine, respectively. In the key stereodifferentiating step, the cyanide addition leads to mixtures of diastereomeric alpha-amino nitrile-esters, the composition of which is independent of the reaction temperature and the type of the solvent, respectively. Hydrolysis of the alpha-amino nitrile-esters with concentrated H(2)SO(4) yielded diastereomeric mixtures of secondary alpha-amino amido-esters, which after separation were hydrogenolyzed and hydrolyzed each to the enantiomeric trans-1-amino-2-carboxymethylcyclopentanecarboxylic acids. Their configuration was completely established by NMR methods, CD spectra, and X-ray analysis of the trans-1S,2R-configured secondary alpha-amino amido-ester. In receptor binding assays and functional tests, trans-1S,2R-1-amino-2-carboxymethylcyclopentanecarboxylic acid hydrochloride was found to behave as a selective mGluR(2)-antagonist without relevant binding properties at iGluRs.

The two diastereomeric pairs of acidic amino acids 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid (8A/8B) and 4-(2-amino-2-carboxyethyl)-5,5-dimethyl-4,5-dihydroisoxazole-3-carboxylic acid (10A/10B) were prepared via a strategy based on a 1,3-dipolar cycloaddition. The four amino acids were tested at ionotropic and metabotropic glutamate receptors. None of the compounds was active, neither as agonists nor as antagonists, at 1 mM on metabotropic receptors (mGluR1, -2, -4, and -5 expressed in CHO cell lines). Conversely, the pair of stereoisomers 8A/8B showed a remarkable affinity, antagonist potency, and selectivity for NMDA receptors, when tested on ionotropic glutamate receptors. The affinity of 8A proved to be 5 times higher than that of diastereomer 8B (K(i) values 0.21 and 0.96 microM, respectively). Furthermore, compounds 8A and 8B exhibited a noteworthy anticonvulsant activity in in vivo tests on DBA/2 mice. Derivative 10A was inactive at all ionotropic glutamate receptors, whereas its stereoisomer 10B displayed a seizable binding to both NMDA and AMPA receptors.

In this study, we have determined and compared the pharmacological profiles of ibotenic acid and its isothiazole analogue thioibotenic acid at native rat ionotropic glutamate (iGlu) receptors and at recombinant rat metabotropic glutamate (mGlu) receptors expressed in mammalian cell lines. Thioibotenic acid has a distinct pharmacological profile at group III mGlu receptors compared with the closely structurally related ibotenic acid; the former is a potent (low μm) agonist, whereas the latter is inactive. By comparing the conformational energy profiles of ibotenic and thioibotenic acid with the conformations preferred by the ligands upon docking to mGlu1 and models of the other mGlu subtypes, we propose that unlike other subtypes, group III mGlu receptor binding sites require a ligand conformation at an energy level which is prohibitively expensive for ibotenic acid, but not for thioibotenic acid. These studies demonstrate how subtle differences in chemical structures can result in profound differences in pharmacological activity.

A series of 2-arylureidobenzoic acids (AUBAs) was prepared by a short and effective synthesis, and the pharmacological activity at glutamate receptors was evaluated in vitro and in vivo. The compounds showed noncompetitive antagonistic activity at the kainate receptor subtype GluR5. The most potent compounds showed more than 50-fold selectivity for GluR5 compared to GluR6 and the AMPA receptor subtypes GluR1-4. The structure-activity relationships for the AUBAs showed distinct structural requirements for the substituents on the two aromatic ring systems. Only para-substituents were tolerated on the benzoic acid moiety (ring A), whereas ring B tolerated a variety of substituents, but with a preference for lipophilic substituents. The most potent compounds had a 4-chloro substituent on ring A and 3-chlorobenzene (6b), 2-naphthalene (8h), or 2-indole (8k) as ring B and had IC(50) values of 1.3, 1.2, and 1.2 microM, respectively, in a functional GluR5 assay. Compound 6c (IC(50) = 4.8 microM at GluR5) showed activity in the in vivo ATPA rigidity test, indicating that 6c has better pharmacokinetic properties than 8h, which was inactive in this test. The AUBAs are the first example of a series of noncompetitive GluR5-selective antagonists and may prove to be important pharmacological tools and leads in the search for therapeutic glutamatergic agents.

On the basis of structural studies, chloro-homoibotenic acid (Cl-HIBO) was designed and synthesized. Cl-HIBO was characterized in binding and electrophysiology experiments on native and cloned subtypes of GluRs. Electrophysiological selectivities ranged from 275 to 1600 for GluR1/2 over GluR3/4. The potent AMPA receptor activity was strongly desensitizing and the neurotoxicity similar to AMPA. Thus, Cl-HIBO is the most subtype selective agonist reported to date on GluR1/2, and offers a new standard for selectively studying subtypes of AMPA receptors.

Stereomeric 3-carboxy-Δ 2 -isoxazoline–cyclopentane amino acids, which represent restricted conformations of glutamic acid, have been prepared through a strategy based on the 1,3-dipolar cycloaddition of ethoxycarbonylformonitrile oxide to a suitably protected 1-amino-cyclopent-2-enecarboxylic acid. These amino acids, assayed at iGluRs and mGluRs, proved to be inactive. The biological data have been accounted for through the comparison of their conformational profile with that of a 3-carboxy-Δ 2 -isoxazolinyl proline (CIP-A) and (±)-1-aminocyclopentane-1,3-dicarboxylic acid.

The design and synthesis of conformationally restricted analogues of alpha-amino acids is an often used strategy in medicinal chemistry research. Here we present the rational design, synthesis, and pharmacological evaluation of 2-azanorbornane-3-exo,5-endo-dicarboxylic acid (1), a novel conformationally restricted (S)-glutamic acid (Glu) analogue intended as a mimic of the folded Glu conformation. The synthesis of 1 was completed in its racemic form in eight steps from commercially available starting materials. As a key step, the first facially selective hydroboration of a 5-methylidene[2.2.1]bicyclic intermediate was investigated. In this transformation, the catalytic methodology of Wilkinson's/catechol borane proved superior to stoichiometric borane or dialkyl borane reagents, in terms of higher diastereomeric excess and chemical yield. To our surprise (+/-)-1 did not show affinity in binding studies on native 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) (IC(50)300 microM, [(3)H]AMPA) or kainic acid (IC(50)160 microM, [(3)H]kainic acid) receptors nor in binding studies on the cloned iGluR5,6 subtypes (IC(50)300 microM, [(3)H]kainic acid).

The synthesis and pharmacology of two potential glutamic acid receptor ligands are described. Preparation of the bicyclic 3-hydroxy-Δ 2 -isoxazoline-cyclopentane derivatives (±)- 7 and (±)- 8 was accomplished via 1,3-dipolar cycloaddition of bromonitrile oxide to suitably protected 1-amino-cyclopent-3-enecarboxylic acids. Their structure was established using a combination of 1 H NMR spectroscopy and molecular mechanics calculations carried out on the intermediate cycloadducts (±)- 11 and (±)- 12 . Amino acid derivatives (±)- 7 and (±)- 8 were assayed at ionotropic and metabotropic glutamic acid receptor subtypes and their activity compared with that of trans -ACPD and cis -ACPD. The results show that the replacement of the ω-carboxylic group of the model compounds with the 3-hydroxy-Δ 2 -isoxazoline moiety abolishes or reduces drastically the activity at the metabotropic glutamate receptors. Conversely, on passing from cis -ACPD to derivative (±)- 8 , the agonist activity at NMDA receptors is almost unaffected.

In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, determined using a [(3)H]AMPA binding assay and an electrophysiological model, respectively. The affinities and agonist activities obtained for (R)-TDPA (IC(50)=0.265 microM and EC(50)=6.6 microM, respectively) and (S)-TDPA (IC(50)=0.065 microM and EC(50)=20 microM, respectively) revealed a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific and subtype-selective agonist activity was observed for (S)-TDPA at group I metabotropic Glu (mGlu) receptors (EC(50)=13 microM at mGlu(5) and EC(50)=95 microM at mGlu(1)).

Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA) analogs, and the synthesis, stereochemistry, and enantiopharmacology of 3-methyl-AA (4a-d), 4-methyl-AA (5a-d), 5-methyl-AA (6a-d), and (E)-Delta(4)-5-methyl-AA (7a and 7b) are reported. The compounds were resolved using chiral HPLC and the configurational assignments of the enantiomers were based on X-ray crystallographic analyses, chemical correlation, and CD spectral analyses. The effects of the individual stereoisomers at ionotropic and metabotropic (S)-Glu receptors (iGluRs and mGluRs) were characterized. Compounds with S-configuration at the alpha-carbon generally showed mGluR2 agonist activity of similar or slightly lower potencies than (S)-AA [e.g., EC(50) = 76 microM for (2S,4S)-4-methyl-AA (5a) as compared to EC(50) = 35 microM for (S)-AA]. The position of the methyl substituent had a profound effect on the observed pharmacology, whereas the absolute stereochemistry at the methylated carbon atom had a very limited effect on pharmacology. Structure-activity relationships at iGluRs in the rat cortical wedge preparation showed a complex pattern, some compounds being NMDA receptor agonists [e.g., EC(50) =110 microM for (2S,5RS)-5-methyl-AA (6a,b)] and some compounds showing NMDA receptor antagonist effects [e.g., IC(50) = 300 microM for (2R,4S)-4-methyl-AA (5d)]. The two unsaturated analogs (S)- (7a) and (R)-(E)-Delta(4)-5-methyl-AA (7b) turned out to be a weak AMPA receptor agonist and a weak mixed NMDA/AMPA receptor antagonist, respectively.

A series of analogues of the glutamate receptor ligands (S)-2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) and AMOA were synthesized in which the 3-hydroxyisoxazole moiety was exchanged for a 3-hydroxypyrazole moiety. This exchange enables further substitution at the additional nitrogen atom in the heterocyclic core. Several of the analogues have activity at AMPA receptors equipotent to the antagonist ATPO, demonstrating that additional substitution can be accommodated in the antagonist binding site. Modelling studies offer an explanation for the pharmacological pattern observed for the compounds and suggest that this scaffold may be developed further to obtain subtype selective antagonists.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.

The glutamate receptor system is implicated in the development and maintenance of epileptic seizures, and animal studies have disclosed potent anticonvulsant activity of a number of inhibitors of AMPA and/or kainate (KA) receptor activity. These results make such inhibitors potential future antiepileptic drugs. Different series of compounds with inhibitory activity towards AMPA receptors have been developed. Most of these inhibitors are structurally derived from AMPA, quinoxalinedione or 2,3-benzodiazepine. In contrast, only a limited number of inhibitors of KA receptor activity have been developed, most of which contain quinoxalinedione or decahydroisoquinoline skeletons. In spite of promising anticonvulsant activity in various animal model studies, no AMPA/KA receptor inhibitors are in clinical use against epilepsy today. Based on molecular biology studies, AMPA and KA receptors are at present divided into four and five subtypes, respectively, and attempts to develop subtype selective compounds have been initiated. Future studies and development of such compounds will indicate whether AMPA/KA receptor inhibition is a feasible therapeutic strategy for the treatment of epilepsy.

Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5- tert -butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2 S ,4 R )-4-methylglutamic acid ((2 S ,4 R )-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropic glutamic acid receptors. The ( S )-enantiomers of E -4-(2,2-dimethylpropylidene)glutamic acid (( S )- 1 ) and E -4-(3,3-dimethylbutylidene)glutamic acid (( S )- 2 ) were shown to be selective and high affinity GluR5 ligands, with K i values of 0.024 and 0.39 μM, respectively, compared to K i values at GluR2 of 3.0 and 2.0 μM, respectively. Their affinities in the [ 3 H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC 50 >100 μM).

We have previously reported the synthesis and pharmacological characterization of analogues of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 1a), in which the methyl group was replaced by a phenyl group (APPA, 1b) or heteroaryl groups. While 2b and its 3-pyridyl analogue 2-amino-3-[3-hydroxy-5-(3-pyridyl)-4-isoxazolyl]propionic acid (3-Py-AMPA, 3) show very low affinity for AMPA receptors, introduction of heteroaryl substituents containing heteroatom in the 2-position provides potent AMPA receptor agonists. We here report the synthesis and pharmacology of 2-amino-3-(3-hydroxy-5-pyrazinyl-4-isoxazolyl)propionic acid (7) (IC50 = 1.2 microM), which is weaker as an AMPA agonist than AMPA (IC50 = 0.040 microM; EC50 = 3.5 microM) but comparable in potency with 2-Py-AMPA (4) (IC50 = 0.57 microM; EC50 = 7.4 microM), as determined in radioligand binding and electrophysiological experiments, respectively. The AMPA analogues 8a-c, containing 2-, 3-, or 4-methoxyphenyl substituents, respectively, and the corresponding hydroxyphenyl analogues, 9a-c, were also synthesized and evaluated pharmacologically. With the exception of 2-amino-3-[3-hydroxy-5-(2-hydroxyphenyl)-4-isoxazolyl]propionic acid (9a), which is a very weak AMPA agonist (IC50 = 45 microM; EC50 = 324 microM), none of these compounds showed detectable effect at AMPA receptors.

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC50 = 0.025 μM), low affinity in kainic acid binding (IC50 = 3.6 μM), and potent AMPA receptor agonist activity on cortical neurons (EC50 = 0.25 μM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC50 = 0.039 μM), but was found to be a relatively weak AMPA receptor agonist (EC50 = 12 μM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC50 = 220 μM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu2 receptor subtype (KB = 148 μM). Chirality 13:523–532, 2001. © 2001 Wiley-Liss, Inc.

We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2), on the other hand, was shown to be a selective AMPA receptor antagonist (IC(50) = 73 microM), more potent in electrophysiological experiments than AMOA (IC(50) = 320 microM). The isomeric analogue of 2, compound 5, did not show AMPA antagonist effects, but was a weak NMDA receptor antagonist (IC(50) = 540 microM). Finally, compound 3, which is an isomer of ACPA, turned out to be a very weak NMDA antagonist, and an AMPA receptor agonist approximately 1000 times weaker than ACPA. None of the compounds showed agonist or antagonist effects at metabotropic EAA receptors.

We have previously shown that the higher homologue of (S)-glutamic acid [(S)-Glu], (S)-alpha-aminoadipic acid [(S)-alpha-AA] is selectively recognized by the mGlu(2) and mGlu(6) subtypes of the family of metabotropic glutamic acid (mGlu) receptors. Furthermore, a number of analogues of (S)-alpha-AA, in which the terminal carboxyl group has been replaced by various bioisosteric groups, such as phosphonic acid or 3-isoxazolol groups, have been shown to interact selectively with different subtypes of mGlu receptors. In this paper we report the synthesis of the 3-pyrazolone bioisosteres of alpha-AA, compounds (RS)-2-amino-4-(1,2-dihydro-5-methyl-3-oxo-3H-pyrazol-4-yl)butyric acid (1) and (RS)-2-amino-4-(1,2-dihydro-1,5-dimethyl-3-oxo-3H-pyrazol-4-yl)butyric acid (2). At a number of steps in the reaction sequences used, the reactions took unexpected courses and provided products which could not be transformed into the target compounds, and attempts to synthesize the 2,5-dimethyl isomer of 2, compound 3, failed. An X-ray crystallographic analysis of the intermediate 1,2-dihydro-4-(2-hydroxyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) confirmed the expected regioselectivity of the reaction between methylhydrazine and alpha-acetylbutyrolactone (4). Neither 1 nor 2 showed significant effects at the different types of ionotropic glutamic acid receptors or at mGlu(1a) (group I), mGlu(2) (group II), and mGlu(4a) and mGlu(6) (group III) receptors, representing the three indicated groups of mGlu receptors.

( RS )-2-Amino-3-(5- tert -butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of ( RS )-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring ( S )-glutamic acid (( S )-Glu) receptors. We report here the separation of (+)- and (−)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and >99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of ( S )-(+)-ATPA and ( R )-(−)-ATPA. ( S )- and ( R )-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6+ KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, ( S )-ATPA showed AMPA receptor agonist effects (EC 50 =23 μM) approximately twice as potent as those of ATPA. ( R )-ATPA antagonized depolarizations induced by AMPA ( K i =253 μM) and by ( S )-ATPA ( K i =376 μM), and ( R )-ATPA antagonized the biphasic depolarizing effects induced by kainic acid ( K i =301 μM and 1115 μM). At cloned AMPA receptors, ( S )-ATPA showed agonist effects at GluR3 and GluR4 with EC 50 values of approximately 8 μM and at GluR1 (EC 50 =22 μM), producing maximal steady state currents only 5.4–33% of those evoked by kainic acid. ( R )-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with K i values of 33–75 μM. ( S )-ATPA produced potent agonist effects at GluR5 (EC 50 =0.48 μM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, ( S )-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by ( S )-ATPA and kainic acid were not significantly different. ( R )-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither ( S )- nor ( R )-ATPA showed significant effects at GluR6. ( S )-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6+KA2 receptors, whereas ( R )-ATPA was inactive. Thus, ( S )- and ( R )-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA ( S )-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.

(RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is a potent and selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, has previously been shown to desensitize AMPA receptors to a much lower degree than AMPA itself. We now report the synthesis of [3H]ACPA (32.5 Ci/mmol), the neurochemical and pharmacological characterization of [3H]ACPA binding, and a comparison of the distribution of [3H]ACPA, [3H]AMPA, and [3H](S)-5-fluorowillardiine binding sites in rat brain. Under equilibrium conditions, [3H]ACPA was shown to bind to a single population of receptor sites on rat brain membranes. [3H]ACPA was shown to bind with single and similar affinities (15-45 nM) to cloned AMPA receptor subunits (GluR1-4), expressed in insect cells, whereas a K(D) value of 330 nM was determined for the binding of [3H]ACPA to cloned kainic acid preferring GluR5 subunits. Whereas Bmax and K(D) values for [3H]ACPA binding, determined using filtration techniques, were different from such obtained in centrifugation assays, Bmax and K(D) values as well as association and dissociation constants were not significantly affected by the addition of the chaotropic agent KSCN. K(D) values, determined under equilibrium conditions, were, however, markedly different from K(D) values derived from kinetic data. Furthermore, the results of analyses of these kinetic data were consistent with the existence of two different populations of [3H]ACPA binding sites. The pharmacology of [3H]ACPA binding sites was characterized using a series of AMPA receptor agonists and antagonists. Whereas addition of KSCN had little effect on the affinities of AMPA receptor agonists for [3H]ACPA binding, this chaotropic agent reduced the affinities of AMPA receptor antagonists structurally related to AMPA. Based on these and previously reported data, the AMPA receptor agonists, ACPA, AMPA and (S)-5-fluorowillardiine, seem to bind to and activate AMPA receptors in a nonidentical fashion, and these three agonists together may be useful tools for studies of AMPA receptor mechanisms.

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.

Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (Ki=16 μm) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 μm), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 μm, respectively). Responses to MeGlu were unaffected by CTZ (100 μm), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 μg ml−1). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 μm). The remaining component was further reduced by ATPO with a Ki of 36 μm, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors. British Journal of Pharmacology (1998) 125, 1517–1528; doi:10.1038/sj.bjp.0702236

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.

2,3-Dihydropyrazolo[3,2-b]oxazoles were used as intermediates in a new method for preparation of N1-methyl-3-hydroxypyrazoles. Synthesis of this bicyclic system was achieved either by alkylation of 3-hydroxypyrazole with 1,2-dibromoethane or, with better yields, by cyclization of 1-tosyl-2-(2-hydroxyethyl)pyrazol-3-ones via a nitrogen to oxygen transfer of the tosyl group. Alkylation with methyl trifluoromethanesulfonate followed by dihydrooxazole ring-opening with sodium iodide, led to the 1-methyl-2-(2-iodoethyl)pyrazoles. Removal of the iodoethyl chain on N2 to give the target 3-hydroxypyrazoles was achieved either via a cyanation and then a decyanoethylation reaction or via an elimination of hydrogen iodide, followed by an iodine-based oxidation of the resulting vinylic derivative. Using the latter method, 1-methyl-3-hydroxypyrazoles were obtained in 58–73% yields from the corresponding 2,3-dihydropyrazolo[3,2-b]oxazoles.

The activity of the (R, S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist, (R,S) -2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO), at recombinant ionotropic glutamate receptors (GluRs) was evaluated using electrophysiological techniques. Responses at homo- or heterooligomeric AMPA-preferring GluRs expressed in human embryonic kidney (HEK) 293 cells (GluR1-flip) or Xenopus laevis oocytes (GluR1-4-flop or GluR1-flop + GluR2) were potently inhibited by ATPO with apparent dissociation constants (Kb values) ranging from 3.9 to 26 microM. A Schild analysis for kainate (KA)-activated GluR1 receptors showed ATPO to have a KB of 8.2 microM and a slope of unity, indicating competitive inhibition. The antagonism by ATPO at GluR1 was of similar magnitude at holding potentials between -100 mV and +20 mV. In contrast, ATPO (300 microM), does not inhibit responses to kainate at homomeric GluR6 or heterooligomeric GluR6/KA2 expressed in HEK 293 cells but activated GluR5 and GluR5/KA2 expressed in X. laevis oocytes. ATPO produced15% inhibition at the maximal concentration (300 microM) of current responses through NR1A + NR2B receptors expressed in X. laevis oocytes. Thus, ATPO shows a unique pharmacological profile, being an antagonist at GluR1-4 and a weak partial agonist at GluR5 and GluR5/KA2.

Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist for the pharmacological characterization of mGlu6 (Trends Pharmacol. Sci. Suppl. 1997, 37-39), and we here report the resolution, configurational assignment, and pharmacology of (S)- (6) and (R)- (7) Homo-AMPA. Using the "Ugi four-component condensation", 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10) was converted into the separable diastereomeric derivatives of 6 and 7, compounds 12 and 11, respectively. Deprotection of 12, in one or two steps, gave extensively racemized 6, which was converted in low yield into 6 (99.0% ee) through several crystallizations. 6 (99.7% ee) and 7 (99.9% ee) were finally obtained by preparative chiral HPLC. The configurational assignments of 6 and 7 were based on 1H NMR spectroscopic studies on 12 and 11, respectively, and circular dichroism studies on 6 and 7. Values of optical rotations using different solvents and the chiral HPLC elution order of 6 and 7 supported the results of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3 microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM).

In this study we have determined the pharmacology of a series of 1-aminocyclopentane-1,3-dicarboxylic acid (1,3-ACPD) analogues at cloned metabotropic glutamic acid (mGlu) receptors. The new analogues comprise the four possible stereoisomers of 1-amino-1-carboxycyclopentane-3-acetic acid (1,3-homo-ACPD) and the racemic mixture of (1RS,2RS)-1-amino-1-carboxycyclopentane-2-acetic acid (1RS,2RS-homo-ACPD), (1RS,2RS)-Homo-ACPD was shown to be a competitive mGlu2 receptor antagonist with a KB of 391 microM. (1S,3R)-Homo-ACPD and (1R,3R)-homo-ACPD were both shown to be mGlu2 receptor agonists with EC50 values of 122 and 105 microM, respectively. Compared to (S)-Glu both compounds displayed partial agonism with intrinsic activities of 79% and 47%, respectively. (1S,3S)-Homo-ACPD was also found to be a partial mGlu2 receptor agonist with an intrinsic activity of 27% compared to (S)-Glu. None of the compounds tested showed any activity at mGlu1a or mGlu4a receptors. These homo-ACPD's show a higher degree of subtype selectivity than the parent compound (1SR,3RS)-ACPD. In addition none of the compounds demonstrated any activity at ionotropic Glu receptors.

We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation using N-BOC protected ATAA and (R)- and (S)-phenylethylamine. Enantiomeric purities (ee > 98%) of (R)- and (S)-ATAA were determined using the Crownpak CR(-) and CR(+) columns, respectively. The absolute configuration of (R)-ATAA was established by an X-ray crystallographic analysis of the (R)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation (Ki > 1,000 microM).

Summary We have previously proposed the existence of a lipophilic cavity of the 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor recognition site capable of accommodating alkyl substituents of limited size in the 5-position of the isoxazole ring. In order to indirectly elucidate the approximate extent of this proposed cavity we have synthesized and pharmacologically characterized a number of AMPA analogues. For most of these AMPA analogues, a positive correlation between AMPA receptor affinity and agonist effect was observed. The only exception was demethyl-AMPA ( 8a ), which showed relatively high AMPA receptor affinity (IC 50 = 0.27 μM) but remarkably weak agonist potency (EC 50 = 900 μM). Whereas the ethyl analogue of AMPA (Et-AMPA) (IC 50 = 0.030 μM; EC 50 = 2.3 μM) has previously been shown to be slightly more potent than AMPA (IC 50 = 0.040 μM; EC 50 = 3.5 μM), substitutions of a propyl or a butyl group for the methyl group of AMPA to give 8b (IC 50 = 0.090 μM; EC 50 = 5.0 μM) or 8f (IC 50 = 1.0 μM; EC 50 = 32 μM), respectively, result in progressive loss of the AMPA agonist effect. Analogues containing larger groups, such as isopentyl ( 8e ), 1-propylbutyl ( 8g ), 2,2-dimethylpropyl ( 8h ), or benzyl ( 14 ) groups, were very weak or totally inactive as AMPA receptor ligands.

(RS)-2-Amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid (APPA), which is an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), shows the characteristics of a partial AMPA receptor agonist. Since (S)-APPA is a full AMPA agonist and (R)-APPA a competitive antagonist, the partial agonism observed for APPA, which is a 1:1 mixture of (S)- and (R)-APPA, is only apparent. These observations have prompted comparative pharmacological studies of different molar ratios of a series of AMPA and N-methyl-D-aspartic acid (NMDA) agonists and respective competitive antagonists, and of these agonists in the presence of fixed concentrations of antagonist. Using the rat cortical wedge preparation, the latter series of experiments showed the expected rightward parallel shifts of the dose-response curves. The former type of experiments, on the other hand, produced dose-response curves at different levels of maximal response, depending on the molar ratios of agonist and antagonist used. This phenomenon, which is in agreement with the theory for competitive receptor interaction, has been termed functional partial agonism, a new pharmacological concept of potential therapeutic utility. These results were obtained using AMPA, the AMPA agonist (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), the competitive AMPA antagonists (RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid (AMOA) and 6-nitro-7-sulfamoylbenzo[f] quinoxalin-2,3-dione (NBQX), NMDA, and the competitive NMDA antagonist (RS)-3-(2-carboxy-4-piperazinyl)propyl-1-phosphonic acid (CPP).

A series of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) analogues were evaluated for activity at homo-oligomeric glutamate1-flop (Glu1-flop) receptors expressed in Xenopus oocytes, using the two-electrode voltage clamp technique. (RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) (EC50, 2.4 microM), a homologue of AMPA having a carboxyl group as the terminal acidic functionality, was five times more potent than AMPA (EC50, 12 microM) and 20 times more potent than kainate (EC50, 46 microM). (RS)-2-Amino-3(3-hydroxy-5-trifluoromethyl-4-isoxazolyl)propionic acid (Tri-F-AMPA), in which an electronegative trifluoromethyl group is substituted for the methyl group on the isoxazole ring in the AMPA structure, was three times more potent than AMPA, whereas (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA), a bicyclic analogue of AMPA with highly restricted conformational flexibility was 10 times less potent than AMPA. The limiting slope of log-log plots of Glu1-flop receptor currents versus low agonist concentrations had a value of 1.7 for ACPA and kainate compared to 1.5 for Tri-F-AMPA and 1.3 for 5-HPCA and AMPA. The amplitude of responses evoked by near saturating concentrations of the agonists varied more than 7-fold. The sequence of efficacy was ACPA = kainate > Tri-F-AMPA > AMPA > 5-HPCA. Moreover, when saturating concentrations of Tri-F-AMPA and kainate were co-applied, the response was significantly greater than when each of the agonists was applied separately. The potency of the antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) (estimated KB, approximately 200 nM), to block currents mediated by Glu1-flop receptors was similar for all of the agonists tested in this study. These results indicate that relatively minor changes in the molecular structure of AMPA are associated with marked effects on potency and efficacy. In particular, it is suggested that the acidity of the terminal group plays a major role in determining the degree of receptor activation in the steady state.

(R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy)-5-methyl-4-isoxazolyl]N-tert-butyl-2- [N-[(S)-1-phenylethyl]benzamido]-acetamide (16 and 17, respectively) were synthesized and separated chromatographically. The absolute stereochemistry of 16 was confirmed by an X-ray analysis. Deprotection of these intermediates did, however, provide (R)- (8) and (S)- (9) AMAA, respectively, in extensively racemized forms. N-BOC-protected (R,S)-AMAA (21) was successfully resolved via diastereomeric salt formation using cinchonidine. The stereochemical purity and stability of 8 and 9 obtained via this resolution were determined using chiral HPLC. (R)-AMAA (8) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly. In electrophysiological studies using rat brain tissue, 8 (EC50 = 7.3 +/- 0.3 microM) was 1 order of magnitude more potent than 9 (EC50 = 75 +/- 9 microM) as an NMDA receptor agonist.

The (R)- and (S)-forms of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) were synthesized, using (S)-BOC-phenylalanine as a chiral auxiliary and their absolute stereochemistry correlated with that of (R)-Br-HIBO. The enantiomeric excesses for (R)-HIBO (1) (> 99.5%) and (S)-HIBO (2) (99.5%) were determined using chiral HPLC. Whereas compounds 1 and 2 were equipotent inhibitors of the binding of [3H]glutamic acid in the presence of calcium chloride, 2 showed AMPA agonist activity and 1 very weak NMDA agonist activity.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 μM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.

Abnormalities in the excitatory glutamate neurotransmitter system appear to be a prominent factor in Alzheimer's disease (AD). Whereas hypoactivity of this system is observed in some areas of Alzheimer brains, hyperactivity may play a role in the degenerative processes in other brain areas. This apparently paradoxical situation makes therapeutic intervention in the glutamatergic system in AD difficult and demands the development of unique therapeutic approaches. The involvement of the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) subtype of glutamate receptors in AD brain dysfunctions seems to be important, and compounds showing different modulatory activity at AMPA receptors are discussed in relation to therapeutic possibilities. Compounds enhancing excitatory activity at AMPA receptors may have beneficial effects on the learning and memory deficits observed in AD, whereas agents showing antagonistic or partial agonist profiles may block or delay the progressive neurodegeneration, which is a key phenomenon in AD. In vitro experiments with compounds capable of enhancing AMPA receptor activity have been performed. Such compounds without excitatory activity on their own may not show the excitotoxic properties characteristic of glutamate agonists. Another possibility for therapeutic intervention is the use of a partial agonist. The concept of "functional partial agonism" at the AMPA receptors is described with a specific example.

Direct functionalization of the 3-oxygenated isothiazole heteroaromatic parental system has not yet been reported in the literature. Here, we report the first regioselective lithiation of the 5-position of 3-(benzyloxy)isothiazole (4) using LDA in diethyl ether. The versatility of the methodology was explored by quenching with a variety of electrophiles to give the desired products 7a,b,d-g in 54-68% yield. Only benzoylation aiming at the synthesis of 7c was unsuccessful. Furthermore, a highly convergent synthesis of thioibotenic acid (1), the sulfur analogue of the neurotoxic natural product ibotenic acid, was carried out.

Three acidic amino acids were synthesized using the non-NMDA antagonist, (RS-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA), as a lead structure. (RS)-3-(carboxymethoxy)-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (1) was found to be a selective NMDA antagonist, whereas (RS)-2-amino-3-[3-(carboxymethoxy)-5-trifluoromethyl-4-isoxazolyl]propionic acid (2) was shown to be a non-NMDA antagonist.

The D-enantiomer of bromohomoibotenic acid (Br-HIBO) was inactive in electrophysiological experiments when administered alone, but enhanced depolarizations evoked by L-Br-HIBO or quisqualate when co-administered with these agonists. In addition, quisqualate induced a long-lasting (> 120 min) sensitization of cortical wedge neurons to D-Br-HIBO. This latter effect of D-Br-HIBO was similar to, but significantly more potent and selective, than the earlier observed quisqualate-induced sensitization of cortical neurones to depolarization by (S)-2-amino-4-phosphonobutyric acid (L-AP4).

The enantiomers of AMPA and its diethyl derivative were prepared by an enzymatic resolution using α-chymotrypsin in solution. Electrophysiological studies in vivo and in vitro showed the excitatory effects of AMPA to reside mainly in the L-enantiomer in agreement with the IC50 values of 0.02 μM and 76 μM observed for the L- and D-enantiomer, respectively, in [3H]AMPA binding assays.

The isoxazole amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) (1), which is a highly selective agonist at the AMPA subtype of excitatory amino acid (EAA) receptors, has been used as a lead for the development of two novel EAA receptor antagonists. One of the compounds, 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA, 7), was synthesized via O-alkylation by ethyl chloroacetate of the amino acid protected AMPA derivative 4. The other compound, 2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-+ ++oxoisoxazolin -4-yl]propionic acid (AMNH, 14) was synthesized with use of 4-(chloromethyl)-3-methoxy-5-methylisoxazole (8) as the starting material. The intermediate 4-(chloromethyl)-2-(3-methoxy-5-methylisoxazol-4-yl)methyl-5-me thylisoxazolin- 3-one (11) was converted into the acetamidomalonate (12), which was stepwise deprotected to give 14. Compounds 7 and 14 were stable in aqueous solution at pH values close to physiological pH. Neither 7 nor 14 showed detectable affinities for the receptor, ion channel, or modulatory sites of the N-methyl-D-aspartic acid (NMDA) receptor complex. Quantitative receptor autoradiographic and conventional binding techniques were used to study the affinities of 7 and 14 for non-NMDA receptor sites. Both compounds were inhibitors of the binding of [3H]AMPA (IC50 = 90 and 29 microM, respectively). Compounds 14 and 7 were both very weak inhibitors of the high-affinity binding of radioactive kainic acid [( 3H]KAIN). Compound 14, but not 7, was, however, shown to be an inhibitor of low-affinity [3H]KAIN binding (IC50 = 40 microM) as determined in the presence of 100 mM calcium chloride. In the rat cortical slice preparation, 7 was shown to antagonize excitation induced by 1 with some selectivity, whereas 14 proved to be a rather selective antagonist of KAIN-induced excitation. Both antagonists showed very weak effects on the excitatory effects of NMDA. Compound 7 was a poor antagonist of excitation by quisqualic acid (2), whereas 14 did not affect excitation by this nonselective AMPA receptor agonist. On cat spinal neurones, both 7 and 14 reduced excitations by 1 and KAIN, but, again, the excitatory effects of 2 were much less sensitive. Compound 14 and, in particular, 7 effectively protected rat striatal neurones against the neurotoxic effects of KAIN, whereas the toxic effects of 1 were reduced only by 7. Neither antagonist showed protection against the cell damage caused by intrastriatal injection of the NMDA agonist quinolinic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

The novel acidic amino acids 6a-c, 7, and 8 have been synthesized via 1,3-dipolar cycloadditions, using nitrile oxides and alkynes. The prepared compounds are heterocyclic analogues of glutamic acid with differing chain lengths. One of these compounds, (RS)-2-amino-3-(3-carboxy-5-methyl-4- isoxazolyl)propionic acid (ACPA, 8), was shown in [3H]AMPA binding studies to be more active than AMPA itself (IC50 = 20 nM compared to IC50 = 79 nM for AMPA). No affinity for NMDA receptors (NMDA-sensitive [3H]glutamic acid binding) was found, and only weak affinity in [3H]kainic acid binding (IC50 = 6.3 microM) was detected. The excitatory activity in rat cortical wedge also showed that ACPA was more potent than AMPA (EC50 = 1.0 microM compared to EC50 = 3.5 microM for AMPA). The depolarizing effect of ACPA could be fully antagonized by the selective non-NMDA antagonist 6-cyano-7-nitro-quinoxazoline-2,3-dione (CNQX), but was unaffected by the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (AP5).