Analogues of 3-hydroxyisoxazole-containing glutamate receptor ligands based on the 3-hydroxypyrazole-moiety: design, synthesis and pharmacological characterization.

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.

Design, synthesis and in vitro pharmacology of GluK1 and GluK3 antagonists. Studies towards the design of subtype-selective antagonists through 2-carboxyethyl-phenylalanines with substituents interacting with non-conserved residues in the GluK binding sites.

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.

Pharmacological characterization and modeling of the binding sites of novel 1,3-bis(pyridinylethynyl)benzenes as metabotropic glutamate receptor 5-selective negative allosteric modulators.

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.

Synthesis and pharmacological characterization at glutamate receptors of the four enantiopure isomers of tricholomic acid.

The two enantiomeric pairs of erythro- and threo-amino-(3'-hydroxy-4',5'-dihydro-isoxazol-5'-yl)-acetic acids were synthesized via the 1,3-dipolar cycloaddition of bromonitrile oxide to ( R)- or ( S)-3-( tert-butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine. The pharmacological profiles of the studied amino acids reflect the relationship between the activity/selectivity and the stereochemistry of the two stereogenic centers: while the (2 S,5' S) stereoisomer is an agonist at the AMPA and KA receptors, its (2 R,5' R) enantiomer interacts selectively with the NMDA receptors; the (2 S,5' R) stereoisomer is the only one capable to activate the mGluRs.

Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites.

Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric arylpropylsulfonamides were derived from known modulators and target the cyclothiazide-binding site at the AMPA receptors. The three stereoisomers--R,R, meso, and S,S--of the two constructed dimers were prepared, and in vitro testing showed the R,R forms to be the most potent stereoisomers. The biarylpropylsulfonamides have dramatically increased potencies, more than three orders of magnitude higher than the corresponding monomers. Dimer (R,R)-2a was cocrystallized with the GluR2-S1S2J construct, and an X-ray crystallographic analysis showed (R,R)-2a to bridge two identical binding pockets on two neighboring GluR2 subunits. Thus, this is biostructural evidence of a homomeric dimer bridging two identical receptor-binding sites.

Synthesis and pharmacological evaluation of novel conformationally constrained homologues of glutamic acid.

Twelve novel conformationally constrained homologues of glutamic acid have been synthesized and pharmacologically characterized at ionotropic glutamate receptors (iGluRs). Synthesis of the target compounds involved 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles. The structure to the compounds has been assigned by (1)H NMR and, in the case of derivatives (+/-)-4a, (+/-)-4b, (+/-)-5a, and (+/-)-5b, by means of an X-ray crystallographic analysis carried out on intermediate (+/-)-12a. The synthesized amino acids were found to be without affinity (K(i)/IC(50)>100microM) for iGluRs with the exception of compounds (+/-)-4b and (+/-)-5b, which showed a modest affinity for NMDA receptors (K(i)=34 and 13microM, respectively). The results indicate that the increased conformational constraints introduced by the cyclopropane ring and the spiro-attached proline ring are both detrimental to the pharmacological activity.

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Synthesis, binding affinity at glutamic acid receptors, neuroprotective effects, and molecular modeling investigation of novel dihydroisoxazole amino acids.

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.

Synthesis of novel N1-substituted bicyclic pyrazole amino acids and evaluation of their interaction with glutamate receptors.

N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors. Some of them turned out to be selective for the NMDA receptors, where they behaved as weak antagonists. The biological activity is mainly due to the interaction with the glutamate binding site, and not with the glycine co-agonist site.

The AMPA receptor binding site: focus on agonists and competitive antagonists.

It is generally agreed that (S)-glutamic acid (Glu) receptors are involved in the development of a number of diseases in the central nervous system (CNS), and ligands that interact with these receptors are of significant interest. Selective ligands are indispensable as tools for the elucidation of the physiological role of AMPA receptors and as leads for the development of therapeutic agents. Over the last decade a wide variety of such ligands have been developed and studies on the structure-activity relationships of these compounds have contributed to our understanding of the mechanisms involved in AMPA receptor activation and blockade. Series of selective agonists using the 3-isoxazolol amino acid ibotenic acid (2) as a lead compound have been designed and developed. Other heterocycles, such as the uracil moiety of willardiine (6), have also proved to be highly effective bioisosteres for the distal carboxyl group of Glu. For a number of reasons, the development of competitive antagonists with therapeutic potential has been hampered for example due to the limited solubility of key heterocyclic compounds structurally unrelated to Glu. However, some problems have been overcome, and series of water-soluble, potent and selective quinoxalinediones, indenoimidazones and isatine oximes have now been developed. At the turn of the millennium the crystal structure of GluR2 co-crystallized with different AMPA receptor ligands became available, opening a new era in the design of AMPA receptor ligands on a rational basis.

Bioisosteric modifications of 2-arylureidobenzoic acids: selective noncompetitive antagonists for the homomeric kainate receptor subtype GluR5.

2-Arylureidobenzoic acids (AUBAs) have recently been presented as the first series of selective noncompetitive GluR5 antagonists. In this paper we have modified the acidic moiety of the AUBAs by introducing different acidic and neutral groups, and similarly, we have replaced the urea linker of the AUBAs with other structurally related linkers. Replacing the acid with neutral substituents led to inactive compounds in all instances, showing that an acidic moiety is necessary for activity. Replacing the carboxylic moiety in 2a with a sulfonic acid (5c) or a tetrazole ring (5d) improved the potency at GluR5 receptors (compounds 5c and 5d showed IC(50) values of 1.5 and 2.0 muM, respectively, compared to compound 2a with IC(50) = 4.8 muM). Compound 5c did not show improved in vivo activity in the ATPA rigidity test compared to 2a, whereas compound 5d was 4 times more potent than 2a. All compounds wherein the urea linker had been replaced showed lower or no activity. The results described extend the knowledge of structure-activity relationships for the AUBAs, and compound 5d may prove to be a good candidate for studying GluR5 receptors in vitro and in vivo.

2-arylureidobenzoic acids: selective noncompetitive antagonists for the homomeric kainate receptor subtype GluR5.

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.

Selective antagonists at group I metabotropic glutamate receptors: synthesis and molecular pharmacology of 4-aryl-3-isoxazolol amino acids.

Homologation of (S)-glutamic acid (Glu, 1) and Glu analogues has previously provided ligands with activity at metabotropic Glu receptors (mGluRs). The homologue of ibotenic acid (7), 2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid (HIBO, 8), and the 4-phenyl derivative of 8, compound 9a, are both antagonists at group I mGluRs. Here we report the synthesis and molecular pharmacology of HIBO analogues 9b-h containing different 4-aryl substituents. All of these compounds possess antagonist activity at group I mGluRs but are inactive at group II and III mGluRs.

Design, synthesis, and pharmacology of a highly subtype-selective GluR1/2 agonist, (RS)-2-amino-3-(4-chloro-3-hydroxy-5-isoxazolyl)propionic acid (Cl-HIBO).

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.

Design, synthesis, and pharmacological characterization of novel, potent NMDA receptor antagonists.

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.

Synthesis and anticonvulsant activity of novel bicyclic acidic amino acids.

Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them behaved as antagonists at mGluR1,5 and as agonists at mGluR2. Furthermore, whereas (+/-)-6 was inactive at all ionotropic glutamate receptors, (+/-)-7 displayed a quite potent antagonism at the NMDA receptors. In the in vivo tests on DBA/2 mice, the compounds displayed an anticonvulsant activity. The interesting pharmacological profile of (+/-)-7 qualifies it as a lead of novel neuroprotective agents.

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

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.

Analogues of homoibotenic acid show potent and selective activity following sensitisation by quisqualic acid.

Quisqualic acid induces sensitisation of neurones to depolarisation by analogues of 2-amino-4-phosphonobutyric acid (AP4), phenylglycine, and homoibotenic acid (HIBO). Thus, after administration of quisqualate these analogues become active at concentrations at which they are otherwise inactive. The mechanisms behind quisqualate-induced sensitisation are poorly understood and have not previously been quantified properly. In this study, we have tested the activity of a number of 4-alkyl- and 4-aryl-substituted analogues of HIBO as regards quisqualate-sensitisation, and present a method for quantifying the sensitisation induced by quisqualate at cortical neurones. These analogues are generally more potent and selective than (S)-AP4 or its homologue (S)-AP5 following quisqualate-sensitisation. Furthermore, we found a statistically significant correlation between the ligands' ability to inhibit CaCl(2)-dependent (S)-[(3)H]glutamate uptake into rat cortical synaptosomes, and their potency following quisqualate-induced depolarisation. This demonstrates the involvement of a transport system in the mechanism underlying the quisqualate-effect.

Ibotenic acid and thioibotenic acid: a remarkable difference in activity at group III metabotropic glutamate 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 microm) 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.

Cognitive enhancing effects of an AMPA receptor positive modulator on place learning in mice.

This study presents an in vivo investigation of the arylpropylsulfonamide α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor positive modulator (R,R)-N,N-(2,20-[biphenyl-4-40-diyl]bis[propane-2,1-diyl])dimethanesulfonamide (PIMSD). The pharmacokinetics of the drug were examined in male C57BL/6J mice and the drug concentration in blood plasma determined after subcutaneous injection of 1mg/kg b.w. This analysis revealed a rapid increase of the plasma concentration, peaking within 30min after administration with a T(1/2) of approximately 30min and a peak plasma concentration of about 2µM. Analysis of brain tissue homogenates also indicated blood-brain barrier permeability of the compound. Cognitive enhancing effects of the drug were then studied on place learning in male C57BL/6J mice in a water maze. In order to elucidate the potential positive effects of PIMSD on spatial learning the muscarinergic antagonist scopolamine was utilized, which is known to impair spatial learning ability. The mice were divided into four groups and subjected to two sequential subcutaneous injections administered 25min prior to behavioural testing: (1) vehicle/vehicle; (2) PIMSD/vehicle; (3) scopolamine/vehicle; (4) PIMSD/scopolamine. PIMSD at a dose of 3mg/kg b.w. was able to partially reverse the impairment given by 0.5mg/kg b.w. scopolamine. These results suggest that arylpropylsulfonamides such as PIMSD may have a therapeutic use in the enhancement of cognitive function and support the hypothesis that AMPA receptor potentiation is one mechanism that can be targeted for diseases of cognitive impairment.