Probing α4ßδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4ß1δ/α4ß3δ receptor agonist on tonic and phasic inhibition in rat brain.

In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5ß2,3γ2S and ρ1 GABAARs and partial agonism at α6ß2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4ß1δ and α4ß3δ GABAARs was contrasted by its negligible activity at the α4ß2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 µm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6ß2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4ß2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4ßδ subtypes in the cells. Finally, whereas 100 µm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4ßδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ß2- and ß3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4ßδ GABAARs.

Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres.

The ionotropic GABAA receptors (GABAARs) are widely distributed in the central nervous system where they play essential roles in numerous physiological and pathological processes. A high degree of structural heterogeneity of the GABAAR has been revealed and extensive effort has been made to develop selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology models, has provided more insight into the molecular basis for binding. Similar binding modes are proposed for the heterocyclic GABA analogues covered in this review by use of ligand-receptor docking into the receptor homology model and the presented structure-activity relationships. A network of interactions between the analogues and the binding pocket is leaving no room for substituents and underline the limited space in the GABAAR orthosteric binding site when in the agonist conformation.

Selective GABA transporter inhibitors tiagabine and EF1502 exhibit mechanistic differences in their ability to modulate the ataxia and anticonvulsant action of the extrasynaptic GABA(A) receptor agonist gaboxadol.

Modulation of the extracellular levels of GABA via inhibition of the synaptic GABA transporter GAT1 by the clinically effective and selective GAT1 inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid; Gabitril] has proven to be an effective treatment strategy for focal seizures. Even though less is known about the therapeutic potential of other GABA transport inhibitors, previous investigations have demonstrated that N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502), which, like tiagabine, is inactive on GABA(A) receptors, inhibits both GAT1 and the extrasynaptic GABA and betaine transporter BGT1, and exerts a synergistic anticonvulsant effect when tested in combination with tiagabine. In the present study, the anticonvulsant activity and motor impairment associated with systemic administration of gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), which, at the doses used in this study (i.e., 1-5 mg/kg) selectively activates extrasynaptic α4-containing GABA(A) receptors, was determined alone and in combination with either tiagabine or EF1502 using Frings audiogenic seizure-susceptible and CF1 mice. EF1502, when administered in combination with gaboxadol, resulted in reduced anticonvulsant efficacy and Rotarod impairment associated with gaboxadol. In contrast, tiagabine, when administered in combination with gaboxadol, did not modify the anticonvulsant action of gaboxadol or reverse its Rotarod impairment. Taken together, these results highlight the mechanistic differences between tiagabine and EF1502 and support a functional role for BGT1 and extrasynaptic GABA(A) receptors.

Subtype selective kainic acid receptor agonists: discovery and approaches to rational design.

(S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system, activating the plethora of glutamate receptors (GluRs). In broad lines, the GluRs are divided into two major classes: the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (mGluRs). Within the iGluRs, five subtypes (KA1, KA2, iGluR5-7) show high affinity and express full agonist activity upon binding of the naturally occurring amino acid kainic acid (KA). Thus these receptors have been named the KA receptors. This review describes all-to our knowledge-published KA receptor agonists. In total, over 100 compounds are described by means of chemical structure and available pharmacological data. With this perspective review, it is our intention to ignite and stimulate inspiration for future design and synthesis of novel subtype selective KA receptor agonists.

Synaptic and extrasynaptic GABA transporters as targets for anti-epileptic drugs.

Inhibition of the GABA transporter subtype GAT1 by the clinically available anti-epileptic drug tiagabine has proven to be an effective strategy for the treatment of some patients with partial seizures. In 2005, the investigational drug EF1502 was described as possessing activity at both GAT1 and BGT-1. When combined with the GAT1 selective inhibitor tiagabine, EF1502 was found to possess a synergistic anti-convulsant action in the Frings audiogenic seizure-susceptible mouse model of reflex epilepsy. This effect was subsequently attributed to inhibition of BGT-1. In this study, the anti-convulsant effect of the GAT2/3 inhibitor SNAP-5114 was assessed in the Frings audiogenic seizure-susceptible mouse alone, and in combination with tiagabine and EF1502. The results showed that SNAP-5114 produced a synergistic anti-convulsant effect in combination with EF1502 but not when used in combination with tiagabine. These findings support anatomical evidence that GAT2/3 are most likely located at the synapse in close proximity to GAT1; whereas BGT-1 is located some distance away from the synapse and GAT1 and GAT2/3. Lastly, EF1502 and tiagabine were evaluated alone, and in combination, in the corneal kindled mouse model of partial epilepsy. The results of this evaluation provide further evidence in support of a role for BGT-1 in the control of seizure activity. In addition, they suggest that the combined inhibition of GAT1 and BGT-1 may afford some advantage over inhibiting either transporter alone.

Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction.

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.

Inhibition of the betaine-GABA transporter (mGAT2/BGT-1) modulates spontaneous electrographic bursting in the medial entorhinal cortex (mEC).

Disruptions in GABAergic neurotransmission have been implicated in numerous CNS disorders, including epilepsy and neuropathic pain. Selective inhibition of neuronal and glial GABA transporter subtypes may offer unique therapeutic options for regaining balance between inhibitory and excitatory systems. The ability of two GABA transport inhibitors to modulate inhibitory tone via inhibition of mGAT1 (tiagabine) or mGAT2/BGT-1 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), also known as EF1502) was evaluated using an in vitro model of spontaneous interictal-like bursting (SB). SBs were recorded extracellularly in combined mEC-HC horizontal brain slices (400 microm; 31+/-1 degrees C) obtained from KA-treated rats. Slice recordings demonstrated that EF1502 exhibited a concentration-dependent reduction in SB frequency. EF1502 significantly reduced SB rate to 32% of control at the 30 microM concentration, while reducing the area and duration of SB activity to 60% and 46% of control, respectively, at the 10 microM concentration. In contrast, the GAT1 selective inhibitor tiagabine (3, 10, and 30 microM) was unable to significantly reduce the frequency of SB activity in the mEC, despite significantly reducing both the duration (51% of control) and area (58% of control) of the SB at concentrations as low as 3 microM. The ability of EF1502, but not tiagabine, to inhibit SBs in the mEC suggests that this in vitro model of pharmacoresistant SB activity is useful to differentiate between novel anticonvulsants with similar mechanisms of action and suggests a therapeutic potential for non-GAT1 transport inhibitors.

4-aryl-5-(4-piperidyl)-3-isoxazolol GABAA antagonists: synthesis, pharmacology, and structure-activity relationships.

A series of 4-aryl-5-(4-piperidyl)-3-isoxazolol GABAA antagonists have been synthesized and pharmacologically characterized. The meta-phenyl-substituted compounds 9k and 9m and the para-phenoxy-substituted compound 9l all display high affinities (Ki=10-70 nM) and antagonist potencies in the low nanomolar range (Ki=9-10 nM). These potencies are significantly higher than those of previously reported 4-PIOL antagonists and considerably higher than that of the standard GABAA antagonist SR 95531.

5-Substituted imidazole-4-acetic acid analogues: synthesis, modeling, and pharmacological characterization of a series of novel gamma-aminobutyric acid(C) receptor agonists.

A series of ring-substituted analogues of imidazole-4-acetic acid (IAA, 4), a partial agonist at both GABAA and GABAC receptors (GABA = gamma-aminobutyric acid), have been synthesized. The synthesized compounds 8a-l have been evaluated as ligands for the alpha1beta2gamma2S GABAA receptors and the rho1 GABAC receptors using the FLIPR membrane potential (FMP) assay and by electrophysiology techniques. None of the tested compounds displayed activity at the GABAA receptors at concentrations up to 1000 microM. However, the 5-Me, 5-Ph, 5-p-Me-Ph, and 5-p-F-Ph IAA analogues, 8a,c,f,g, displayed full agonist activities at the rho1 receptors in the FMP assay (EC50 in the range 22-420 microM). Ligand-protein docking identified the Thr129 in the alpha1 subunit and the corresponding Ser168 residue in rho1 as determinants of the selectivity displayed by the 5-substituted IAA analogues. The fact that GABA, 4, and 8a displayed decreased agonist potencies at a rho1Ser168Thr mutant compared to the WT rho1 receptor strongly supported this hypothesis. However, in contrast to GABA and 4, which exhibited increased agonist potencies at a alpha1(Thr129Ser)beta2gamma2 mutant compared to WT GABAA receptor, the data obtained for 8a at the WT and mutant receptors were nonconclusive.

A tetrazolyl-substituted subtype-selective AMPA receptor agonist.

Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.

Astrocytic GABA Transporters: Pharmacological Properties and Targets for Antiepileptic Drugs.

Inactivation of GABA-mediated neurotransmission is achieved by high-affinity transporters located at both GABAergic neurons and the surrounding astrocytes. Early studies of the pharmacological properties of neuronal and glial GABA transporters suggested that different types of transporters might be expressed in the two cell types, and such a scenario was confirmed by the cloning of four distinctly different GABA transporters from a number of different species. These GABA-transport entities have been extensively characterized using a large number of GABA analogues of restricted conformation, and several of these compounds have been shown to exhibit pronounced anticonvulsant activity in a variety of animal seizure models. As proof of concept of the validity of this drug development approach, one GABA-transport inhibitor, tiagabine, has been developed as a clinically active antiepileptic drug. This review provides a detailed account of efforts to design new subtype-selective GABA-transport inhibitors aiming at identifying novel antiepileptic drug candidates.

Potent 4-arylalkyl-substituted 3-isothiazolol GABA(A) competitive/noncompetitive antagonists: synthesis and pharmacology.

The GABA(A) agonists muscimol (1), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, gaboxadol, 3), and the partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 6a) and their respective 3-isothiazolol analogues thiomuscimol (2), thio-THIP (4), and thio-4-PIOL (7a) are ligands at the GABA(A) orthosteric (recognition) site. The structure-activity relationships (SARs) between these structures are key elements of a 3D-pharmacophore model for GABA(A) agonists and competitive antagonists [Frølund, B.; Jørgensen, A. T.; Tagmose, L.; Stensbøl, T. B.; Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.; Krogsgaard-Larsen, P.; Liljefors, T. J. Med. Chem. 2002, 45, 2454-2468]. Prompted by this model, we now report the synthesis and SAR of a series of analogues of 7a, in which the 4-position of the 3-isothiazolol was substituted by alkyl or bulky aromatic groups such as naphthylmethyl and diphenylalkyl groups (7b-h). The compounds have been pharmacologically characterized using receptor binding assays and two-electrode voltage-clamped Xenopus oocytes expressing alpha1beta3gamma2S- and alpha4beta3delta-containing receptors. The compounds show SARs comparable with those of 6b-h but are generally 5-15 times more potent. The 2-naphthylmethyl, the 1-bromo-2-naphthylmethyl, and the 3,3-diphenylpropyl analogues, compounds 7e, 7f, and 7h, respectively, show affinity in the low-nanomolar range (K(i) 2-10 nM). Interestingly, 7e and 7h exhibited a mixed antagonist profile consisting of a noncompetitive component in the picomolar range and a competitive component at concentrations above 1 nM. This unique profile was shown not to be due to either use dependence or kinetic effects. This antagonist profile of 7e and 7h was particularly pronounced at alpha4beta3delta-containing GABA(A) receptors, which showed three- and 10-fold selectivity for 7h and 6h, respectively.

A novel selective gamma-aminobutyric acid transport inhibitor demonstrates a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS.

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. This transport system is an important therapeutical target in epileptic disorders, but potentially also in other neurological disorders. Thus, selective intervention in GABA uptake has been the subject of extensive research for several decades. In a series of lipophilic diaromatic derivatives of (RS)-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO), N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) turned out to be an equipotent inhibitor at the mouse transporters GAT1 and GAT2 (BGT-1) but inactive at GAT3 and GAT4. This novel pharmacological profile among GABA uptake inhibitors prompted a thorough investigation of the in vivo properties of this compound. These investigations have for the first time demonstrated a functional role for GABA transporter subtype GAT2/BGT-1, which points to the therapeutic relevance of inhibiting this transporter subtype. An overview of the development and characterisation of EF1502 is presented here.

Potent 4-aryl- or 4-arylalkyl-substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling.

We have previously described a series of competitive GABA(A) antagonists derived from the low-efficacy partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 4). The 2-naphthylmethyl analogue, 4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol (5), provided affinity for the GABA(A) receptor site higher than that of the standard GABA(A) receptor antagonist, SR 95531 (3). Molecular modeling studies of these compounds exposed a cavity at the receptor recognition site capable of accommodating aromatic groups of substantial size in the 4-position in the 3-isoxazolol ring. Here we present a series of analogues of 5, with various substituents in different positions in the naphthyl ring system (6a-k), and compounds with aromatic substituents directly attached to the 4-position of the 3-isoxazolol ring (7l-n). The compounds have been pharmacologically characterized using receptor-binding assays and electrophysiological whole-cell patch-clamp techniques. All of the tested compounds show affinity for the GABA(A) receptor site. While the 5-, 7-, and 8-bromo analogues, 6b-d, showed receptor affinities (K(i) = 45, 109, and 80 nM, respectively) comparable with that of 5 (K(i) = 49 nM), the 1-bromo analogue, 6a, provided the highest receptor affinity of the series (K(i) = 10 nM). Introduction of a series of different substituents in the 1-position in the 2-naphthyl ring system led to compounds, 6e-k, with retained high affinity for the GABA(A) receptor (K(i) = 16-250 nM). Introduction of a phenyl ring directly into the 4-position on the 3-isoxazolol ring gave a 41-fold increase in affinity relative to that of 4-PIOL. In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine, 6a showing antagonist potency (IC(50) = 42 nM) markedly higher than that of 3 (IC(50) = 240 nM). Molecular modeling studies, based on the compounds described, emphasized the importance of the distal ring in 5 for receptor affinity and the considerable dimensions of the proposed receptor cavity. Furthermore, the phenyl rings in 7l and in 6k were shown to represent highly favorable positions for an aromatic ring in previously unexplored receptor regions in terms of a pharmacophore model.

Convergent synthesis and pharmacology of substituted tetrazolyl-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid analogues.

The synthesis and pharmacological characterization of 1- and 2-alkyltetrazolyl analogues of (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA), a highly potent and selective agonist at AMPA receptors, are presented. A shorter and more convergent synthetic route than previously described, employing a new method for introducing the amino acid moiety, was developed for these derivatives. The 2-substituted isomers were selective agonists, and their activity correlated inversely with the size of the substituent. Structural explanations of the structure-activity relationship are provided.

GABAA receptor partial agonists and antagonists: structure, binding mode, and pharmacology.

A high degree of structural heterogeneity of the GABAA receptors (GABAARs) has been revealed and is reflected in multiple receptor subtypes. The subunit composition of GABAAR subtypes is believed to determine their localization relative to the synapses and adapt their functional properties to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown for only a few orthosteric ligands. Still, these examples show that it is indeed possible to obtain orthosteric subtype selectivity and they serve as models for further development in the orthosteric GABAAR ligand area. This review presents the very few existing structural classes of orthosteric GABAAR antagonists and describes the development of potent antagonists from partial agonists originally derived from the potent GABAAR agonist muscimol. In this process, several heterocyclic aromatic systems have been used in combination with structural models in order to map the orthosteric binding site and to reveal structural details to be used for obtaining potency and subtype selectivity. The challenges connected to functional characterization of orthosteric GABAAR partial agonists and antagonists, especially with regard to GABAAR stoichiometry and alternative binding sites are discussed. GABAAR antagonists have been essential in defining the tonic current but both remaining issues concerning the GABAARs involved and the therapeutic possibilities of modulating tonic inhibition underline the need for GABAAR antagonists with improved selectivity.

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.

GABA(A) receptor ligands and their therapeutic potentials.

The GABA(A) receptor system is implicated in a number of neurological diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP or isoguvacine. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds such as the GABA(A)agonists imidazole-4-acetic acid and THIP, showing highly subunit-dependent potency and maximal response. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effect. Only little information about direct acting GABA(A) receptor agonists in clinical studies is available. Results from clinical studies on the effect of the GABA(A) agonist THIP on human sleep pattern shows that the functional consequences of a direct acting agonist are different from those seen after administration of GABA(A) receptor modulators.

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.