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Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β2-adrenergic receptor (β2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased β2/β1AR-selectivity, and/or substantially slower β2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β

We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound ...

Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-iPr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible determinants of the GluR4i/GluR1i selectivity. In a mutagenesis study, doubly mutating M686V/I687A in GluR1i in combination with either D399S or E683A increased both the potency and the maximal response of 2-Bn-Tet-AMPA at this receptor to levels similar to those elicited by the agonist at GluR4i. The dependence of the novel selectivity profile of 2-Bn-Tet-AMPA upon residues located outside of the orthosteric site underlines the potential for developing GluR subtype selective ligands by designing compounds with substituents that protrude beyond the (S)-Glu binding pocket.

G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1-3). Herein, we present the first structure-activity relationship study for the 2-arylindole antagonist 3, comprising the design, synthesis, and pharmacological evaluation of a focused library of 3-substituted 2-arylindoles. In a FRET-based inositol monophosphate (IP1) assay we identified compounds 7, 13e, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date.

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 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.

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.

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.

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).

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.

Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clinical trials to treat addiction. We evaluated a novel GABA-AT inactivator (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115, compound 1) and observed that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. By use of in vivo microdialysis techniques in freely moving rats and microPET imaging techniques, 1 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at (1)/(300) to (1)/(600) the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose (1)/(300) that of vigabatrin. Electroretinographic (ERG) responses in rats treated with 1, at doses 20-40 times higher than those needed to treat addiction in rats, exhibited reductions in ERG responses, which were less than the reductions observed in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, 1 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects.

Gamma-hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report the design and synthesis of the first (125)I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.

A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl glycine (NHP5G) derivatives are selectively recognized by N-methyl- d-aspartic acid (NMDA) receptors and that the ( R)-enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual subtypes among the NMDA receptors, providing new pharmacological tools. For example, 4-propyl NHP5G is an antagonist at the NR1/NR2A subtype but an agonist at the NR1/NR2D subtype. Molecular docking studies indicate that the substituent protrudes into a region that may be further exploited to improve subtype selectivity, thereby opening up a design strategy for ligands which can differentiate individual NMDA receptor subtypes.

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.

The natural product kainic acid is used as template for the rational design of a novel conformationally restricted (S)-glutamic acid (Glu) analogue, (1R,4S,5R,6S)-3-azabicyclo[3.3.0]octane-4,6-dicarboxylic acid (1a). The target structure 1a was synthesized from commercially available (S)-pyroglutaminol, in an enantioselective fashion, in 14 steps. Pharmacological characterization of 1a at human glutamate transporter subtypes 1, 2, and 3 yielded K(i) values of 127, 52, and 46 microM, respectively. Furthermore, binding studies at native ionotropic Glu (iGlu) receptors revealed low affinity for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-preferring iGlu receptors (IC(50) > 100 microM), whereas affinities for the KAIN-preferring iGlu receptors and the N-methyl-d-aspartate (NMDA)-preferring group of iGlu receptors were in the low micromolar range (IC(50) = 14 and 2.9 microM, respectively). At metabotropic Glu receptors (mGluR), EC(50) values for 1a were >1000 microM for mGluR1 and 4, representing group I and III, respectively, and approximately 1000 microM (agonist) for mGluR2, representing group II.

We have previously described (RS)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (4-AHCP) as a highly effective agonist at non-N-methyl-d-aspartate (non-NMDA) glutamate (Glu) receptors in vivo, which is more potent than (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) but inactive at NMDA receptors. However, 4-AHCP was found to be much weaker than AMPA as an inhibitor of [(3)H]AMPA binding and to have limited effect in a [(3)H]kainic acid binding assay using rat cortical membranes. To shed light on the mechanism(s) underlying this quite enigmatic pharmacological profile of 4-AHCP, we have now developed a synthesis of (S)-4-AHCP (6) and (R)-4-AHCP (7). At cloned metabotropic Glu receptors mGluR1alpha (group I), mGluR2 (group II), and mGluR4a (group III), neither 6 nor 7 showed significant agonist or antagonist effects. The stereoisomer 6, but not 7, activated cloned AMPA receptor subunits GluR1o, GluR3o, and GluR4o with EC(50) values in the range 4.5-15 microM and the coexpressed kainate-preferring subunits GluR6 + KA2 (EC(50) = 6.4 microM). Compound 6, but not 7, proved to be a very potent agonist (EC(50) = 0.13 microM) at the kainate-preferring GluR5 subunit, equipotent with (S)-2-amino-3-(5-tert-butyl-3-hydroxyisothiazol-4-yl)propionic acid [(S)-Thio-ATPA, 4] and almost 4 times more potent than (S)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionic acid [(S)-ATPA, 3]. Compound 6 thus represents a new structural class of GluR5 agonists. Molecular modeling and docking to a crystal structure of the extracellular binding domain of the AMPA subunit GluR2 has enabled identification of the probable active conformation and binding mode of 6. We are able to rationalize the observed selectivities by comparing the docking of 4 and 6 to subtype constructs, i.e., a crystal structure of the extracellular binding domain of GluR2 and a homology model of GluR5.

Homologation of analogues of the central excitatory neurotransmitter glutamic acid (Glu), in which the distal carboxy group has been bioisosterically replaced by acidic heterocyclic units, has previously provided subtype selective ligands for metabotropic Glu receptors (mGluRs). The (S)-form of the 1,2,5-thiadiazol-3-ol Glu analogue, 2-amino-3-(4-hydroxy[1,2,5]thiadiazol-3-yl)propionic acid (TDPA, 6), is an 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, which in addition stereospecifically activates group I mGluRs. We have now synthesized the (S)- and (R)-forms of 2-amino-4-(4-hydroxy[1,2,5]thiadiazol-3-yl)butyric acid (homo-TDPA, 7) and shown that whereas neither enantiomer interacts with AMPA receptors, (S)- and (R)-7 appear to be selective and equipotent agonists at group II mGluRs as represented by the mGluR2 subtype. The activities of (S)- and (R)-7 are rationalized by conformational analysis, comparison with the potent and specific group II mGluR agonist (-)-LY379268 [(-)-12], and docking to a homology model of mGluR2.

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.

(RS)-2-Amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (Bu-HIBO, 6) has previously been shown to be an agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors and an inhibitor of CaCl2-dependent [3H]-(S)-glutamic acid binding (J. Med. Chem. 1992, 35, 3512-3519). To elucidate the pharmacological significance of this latter binding affinity, which is also shown by quisqualic acid (3) but not by AMPA, we have now resolved Bu-HIBO via diastereomeric salt formation using the diprotected Bu-HIBO derivative 11 and the enantiomers of 1-phenylethylamine (PEA). The absolute stereochemistry of (S)-Bu-HIBO (7) (ee = 99.0%) and (R)-Bu-HIBO (8) (ee > 99.6%) were established by an X-ray crystallographic analysis of compound 15, a salt of (R)-PEA, and diprotected 8. Circular dichroism spectra of 7 and 8 were recorded. Whereas 7 (IC50 = 0.64 microM) and 8 (IC50 = 0.57 microM) were equipotent as inhibitors of CaCl2-dependent [3H]-(S)-glutamic acid binding, neither enantiomer showed significant affinity for the synaptosomal (S)-glutamic acid uptake system(s). AMPA receptor affinity (IC50 = 0.48 microM) and agonism (EC50 = 17 microM) were shown to reside exclusively in the S-enantiomer, 7. Compounds 7 and 8 did not interact detectably with kainic acid or N-methyl-D-aspartic acid (NMDA) receptor sites. Neither 7 nor 8 affected the function of the metabotropic (S)-glutamic acid receptors mGlu2 and mGlu4a, expressed in CHO cells. Compound 8 was shown also to be inactive at mGlu1 alpha, whereas 7 was determined to be a moderately potent antagonist at mGlu1 alpha (Ki = 110 microM) and mGlu5a (Ki = 97 microM). Using the rat cortical wedge preparation, the AMPA receptor agonist effect of 7 was markedly potentiated by coadministration of 8 at 21 degrees C, but not at 2-4 degrees C. These observations together indicate that the potentiation of the AMPA receptor agonism of 7 by 8 is not mediated by metabotropic (S)-glutamate receptors but rather by the CaCl2-dependent (S)-glutamic acid binding system, which shows the characteristics of a transport mechanism. After intravenous administration in mice, 7 (ED50 = 44 mumol/kg) was slightly more potent than AMPA (1) (ED50 = 55 mumol/kg) and twice as potent as Bu-HIBO (6) (ED50 = 94 mumol/kg) as a convulsant, whereas 8 was inactive. After subcutaneous administration in mice, Bu-HIBO (ED50 = 110 mumol/kg) was twice as potent as AMPA (ED50 = 220 mumol/kg) as a convulsant. Since 7 and Bu-HIBO (EC50 = 37 microM) are much weaker than AMPA (EC50 = 3.5 microM) as AMPA receptor agonists in vitro, the presence of a butyl group in the molecules of Bu-HIBO and 7 seems to facilitate the penetration of these compounds through the blood-brain barrier.