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The multimodal antidepressant vortioxetine is thought to mediate its pharmacological effects via 5-HT

Brexpiprazole (OPC-34712, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one) is a novel drug candidate in clinical development for psychiatric disorders with high affinity for serotonin, dopamine, and noradrenaline receptors. In particular, it bound with high affinity (Ki1 nM) to human serotonin 1A (h5-HT1A)-, h5-HT2A-, long form of human D2 (hD2L)-, hα1B-, and hα2C-adrenergic receptors. It displayed partial agonism at h5-HT1A and hD2 receptors in cloned receptor systems and potent antagonism of h5-HT2A receptors and hα1B/2C-adrenoceptors. Brexpiprazole also had affinity (Ki5 nM) for hD3-, h5-HT2B-, h5-HT7-, hα1A-, and hα1D-adrenergic receptors, moderate affinity for hH1 (Ki = 19 nM), and low affinity for hM1 receptors (Ki1000 nM). Brexpiprazole potently bound to rat 5-HT2A and D2 receptors in vivo, and ex vivo binding studies further confirmed high 5-HT1A receptor binding potency. Brexpiprazole inhibited DOI (2,5-dimethoxy-4-iodoamphetamine)-induced head twitches in rats, suggestive of 5-HT2A antagonism. Furthermore, in vivo D2 partial agonist activity of brexpiprazole was confirmed by its inhibitory effect on reserpine-induced DOPA accumulation in rats. In rat microdialysis studies, brexpiprazole slightly reduced extracellular dopamine in nucleus accumbens but not in prefrontal cortex, whereas moderate increases of the dopamine metabolites, homovanillic acid and DOPAC (3,4-dihydroxy-phenyl-acetic acid), in these areas also suggested in vivo D2 partial agonist activity. In particular, based on a lower intrinsic activity at D2 receptors and higher binding affinities for 5-HT1A/2A receptors than aripiprazole, brexpiprazole would have a favorable antipsychotic potential without D2 receptor agonist- and antagonist-related adverse effects. In conclusion, brexpiprazole is a serotonin-dopamine activity modulator with a unique pharmacology, which may offer novel treatment options across a broad spectrum of central nervous system disorders.

International audience; The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6–8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a–d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low–mid nanomolar range.

The present study aimed at investigating the theory that free (unbound) active site concentrations are the best predictors of target binding of compounds blocking the serotonin transporter (Sert) in the central nervous system (CNS). Thirteen serotonin reuptake inhibitors were evaluated for their Sert-binding affinities in vitro and in vivo in rats together with their unbound fractions in plasma and brain. Cortical Sert occupancy was used in vivo to acquire EC₅₀-estimates from total plasma, free plasma, whole brain, and free brain concentrations after acute drug administration. The in vitro-in vivo Sert occupancy analyses showed that the best correlation was achieved when unbound brain concentrations were employed. Unbound brain concentrations also provided a better correlation when compared with unbound plasma concentrations, which could be related to lack of equilibrium between plasma and brain at time of measurements or involvement of active brain efflux processes. In addition, brain-free fractions were shown to be directly correlated to the lipophilicity of the compounds. These data emphasize the use and impact of applying free fraction data in assessment of pharmacological in vitro-in vivo correlations and demonstrates its use to validate in vivo Sert occupancy as pharmacodynamic marker for serotonin reuptake inhibitors in rats.

The synthesis and structure-activity relationship of a novel series of compounds with combined effects on 5-HT(3A) and 5-HT(1A) receptors and on the serotonin (5-HT) transporter (SERT) are described. Compound 5m (Lu AA21004) was the lead compound, displaying high affinity for recombinant human 5-HT(1A) (K(i) = 15 nM), 5-HT(1B) (K(i) = 33 nM), 5-HT(3A) (K(i) = 3.7 nM), 5-HT(7) (K(i) = 19 nM), and noradrenergic β(1) (K(i) = 46 nM) receptors, and SERT (K(i) = 1.6 nM). Compound 5m displayed antagonistic properties at 5-HT(3A) and 5-HT(7) receptors, partial agonist properties at 5-HT(1B) receptors, agonistic properties at 5-HT(1A) receptors, and potent inhibition of SERT. In conscious rats, 5m significantly increased extracellular 5-HT levels in the brain after acute and 3 days of treatment. Following the 3-day treatment (5 or 10 (mg/kg)/day) SERT occupancies were only 43% and 57%, respectively. These characteristics indicate that 5m is a novel multimodal serotonergic compound, and 5m is currently in clinical development for major depressive disorder.

The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogues is presented that addresses the influence of substitutions at the 4- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most potent analogue 1o displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacological tool. The excitatory amino acid transporters (EAATs) belong to the solute carrier family 1 (SLC1), and carry out the concentra- tive uptake of (S)-glutamate (Glu) (and aspartate (Asp)) from the glutamatergic synaptic cleft. 1-3 Five EAAT subtypes have been identified to date, in humans termed EAAT1-EAAT5. For historical reasons the nomenclature differs in rodents: the orthologues of human EAAT1, EAAT2 and EAAT3 subtypes are termed GLAST, GLT-1 and EAAC-1, respectively, whereas the nomenclature for EAAT4,5 is conserved across the species. The EAATs display differential localization and overall distribution patterns in the adult rodent brain. Whereas GLAST (EAAT1) and GLT-1 (EAAT2) are astroglial transporters, EAAC-1 (EAAT3) and EAAT4 are predominantly found in neurons. 4 GLT-1 (EAAT2) is the predominantly expressed subtype, found throughout the brain and spinal cord, and has been shown to be responsible for >90% of the Glu uptake in the adult brain. 4-6 GLAST (EAAT1) is found at the highest levels in the cerebellum with lower levels in the cortex and spinal cord, 7 and EAAC-1 (EAAT3) is expressed in high densities at postsynaptic terminals in the whole brain, particu- larly in the hippocampus, cerebellum, and basal ganglia. 4 EAAT4 is highly enriched in the Purkinje cells of the cerebel- lum, but the transporter has also been detected in the rat fore- and midbrain albeit in dramatically lower levels. 4,5,8,9 Finally, EAAT5 is found exclusively in the retina. 4,5,10 In agreement with the distribution and densities of the CNS subtypes, EAAT2 knockout mice have displayed pronounced loss of hippocampal neurons, seizures, and 50% mortality at 6 weeks of age. In contrast, knockout of GLAST (EAAT1), EAAC-1 (EAAT3), or EAAT4 in mice have been reported not to cause significant abnormalities in anatomy or in CNS-related phenotypes. 4,6 However, such studies may not be fully conclusive when it comes to elucidation of the physiological importance of the specific transporter subtype, as the absence of physiologic and phenotypic effects in the knockout mice may also be ascribed to compensatory mechanisms. Hence, we believe that valuable information about the physiological functions and thus thera- peutic potentials of a specific EAAT subtype may be addressed in a more subtle way by studying the actions of a selective ligand. 4

( S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system (CNS) activating the plethora of ionotropic Glu receptors (iGluRs) and metabotropic Glu receptors (mGluRs). In this paper, we present a chemo-enzymatic strategy for the enantioselective synthesis of five new Glu analogues 2a- f ( 2d is exempt) holding a functionalized substituent in the 4-position. Nine Glu analogues 2a- j are characterized pharmacologically at native 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), kainic acid (KA), and N-methyl- d-aspartic acid (NMDA) receptors in rat synaptosomes as well as in binding assays at cloned rat iGluR5-7 subtypes. A detailed in silico study address as to why 2h is a high-affinity ligand at iGluR5-7 ( K i = 3.81, 123, 57.3 nM, respectively), while 2e is only a high affinity ligand at iGluR5 ( K i = 42.8 nM). Furthermore, a small series of commercially available iGluR ligands are characterized in iGluR5-7 binding.

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.

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)).

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). 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 (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.

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.

Brexpiprazole (OPC-34712, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one) is a novel serotonin-dopamine activity modulator with partial agonist activity at serotonin 1A (5-HT1A) and D2/3 receptors, combined with potent antagonist effects on 5-HT2A, α1B-, and α2C-adrenergic receptors. Brexpiprazole inhibited conditioned avoidance response (ED50 = 6.0 mg/kg), apomorphine- or d-amphetamine-induced hyperactivity (ED50 = 2.3 and 0.90, respectively), and apomorphine-induced stereotypy (ED50 = 2.9) in rats at clinically relevant D2 receptor occupancies. Brexpiprazole also potently inhibited apomorphine-induced eye blinking in monkeys. The results suggest that brexpiprazole has antipsychotic potential. Brexpiprazole induced catalepsy (ED50 = 20) well above clinically relevant D2 receptor occupancies, suggesting a low risk for extrapyramidal side effects. Subchronic treatment with phencyclidine (PCP) induced cognitive impairment in both novel object recognition (NOR) and attentional set-shifting (ID-ED) tests in rats. Brexpiprazole reversed the PCP-induced cognitive impairment in the NOR test at 1.0 and 3.0 mg/kg, and in the ID-ED test at 1.0 mg/kg. However, aripiprazole (10 mg/kg) was ineffective in both tests, despite achieving relevant D2 occupancies. In the NOR test, the 5-HT1A agonist buspirone and the 5-HT2A antagonist M100907 [(R)-(2,3-dimethoxyphenyl)[1-(4-fluorophenethyl)piperidin-4-yl]methanol] partially but significantly reversed PCP-induced impairment. Furthermore, the effect of brexpiprazole was reversed by cotreatment with the 5-HT1A antagonist WAY100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate). The results indicate that brexpiprazole has antipsychotic-like activity and robust efficacy in relevant models of cognitive impairment associated with schizophrenia. The effects of brexpiprazole in the cognitive tests are superior to those of aripiprazole. We propose that the pharmacologic profile of brexpiprazole be based on its balanced effects on 5-HT1A, D2, and 5-HT2A receptors, with possible modulating activity through additional monoamine 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.

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.

( 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.

Piperidinyl-3-phosphinic acid 2, piperidinyl-3-methylphosphinic acid 3 and N-(4,4-diphenyl-3-butenyl)-piperidinyl-3-phosphinic acid 4 have been synthesized as bioisosteres of the corresponding amino carboxylic acids, which are potent and specific GABA-uptake inhibitors. The novel amino phosphinic acids were tested for their GABA-uptake inhibitory activity and 2 and 4 were identified as the first phosphinic acid based GABA-uptake inhibitors. The methylphosphinic acid 3 was found to be inactive.

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.

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 AMPA receptor agonist Thio-AMPA, the 3-isothiazolol analogue of AMPA was converted into the selective NMDA antagonist, 2, in which a 3-isothiazolone unit is a bioisosteric analogue of the peptide bond of the NMDA antagonist, γ-(R)-Glu-Gly. The isomeric 3-oxygenated isothiazole amino acid, 3, and the corresponding isothiazole phosphono amino acid 4 were also synthesized, and were shown to be selective AMPA receptor antagonists. Compound 1, in which the peptide bond of γ-(R)-Glu-Gly is replaced by an ester group, was synthesized and shown to be unstable in the test buffer system.

Potassium channels containing the KCNQ2 subunit play an important role in the regulation of neuronal excitability and therefore have been implicated in epilepsy. This study describes the expression of KCNQ2 subunit immunoreactivity in the basolateral amygdala in two rat models of temporal lobe epilepsy, (1) amygdala kindling and (2) spontaneously epileptic rats after status epilepticus induced by hippocampal electrical stimulation. KCNQ2 subunit immunoreactivity was assessed with a commercial antibody raised against a C-terminal part of the KCNQ2 protein. We show that KCNQ2 subunit immunoreactivity is upregulated in the basolateral amygdala in both models and that generalized seizures are required to induce this upregulation. We hypothesize that the upregulation of potassium channels containing the KCNQ2 subunit might represent a mechanism to counteract seizures in experimental temporal lobe epilepsy.

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b , of ( RS )-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and ( RS )-2-amino-3-(5- tert -butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies ( K b = 38–161 μM) similar to that of the AMPA receptor antagonist ( RS )-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) ( K b = 43–76 μM). Furthermore, the AMOA analogue, 1a , blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 ( K b = 19 μM). Unlike the iGluR antagonist ( S )-2-amino-3-[5- tert -butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [( S )-ATPO], the corresponding tetrazolyl analogue, 1b , lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure–activity relationships.

We have previously used homologation of (S)-glutamic acid (Glu) and Glu analogs as an approach to the design of selective ligands for different subtypes of Glu receptors. (RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is an isoxazole homolog of Glu, is a very potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) subgroup of Glu receptors and a moderately potent ligand for the kainic acid (KA) subgroup of Glu receptors. The enantiomers of ACPA were previously obtained by chiral HPLC resolution. Prompted by pharmacological interest in ACPA, we have now prepared the (S)- and (R)-enantiomers of ACPA by stereocontrolled syntheses using (1R,2R,5R)- and (1S,2S,5S)-2-hydroxy-3-pinanone, respectively, as chiral auxiliaries. Furthermore, the 5-ethyl analog of ACPA, Ethyl-ACPA, was synthesized, and (S)- and (R)-Ethyl-ACPA were also prepared using this method. The absolute configurations of (S)- and (R)-ACPA were established by X-ray crystallographic analysis of a protected (1S,2S,5S)-2-hydroxy-3-pinanone imine derivative of (R)-ACPA. The absolute stereochemistry of (S)- and (R)-Ethyl-ACPA was assigned on the basis of a comparison of their properties with those of the enantiomers of ACPA, employing elution order on chiral HPLC columns, as well as circular dichroism (CD) spectroscopy in combination with time-dependent density functional theory. The structural and electronic basis for the Cotton effect observed for such analogs is examined. The lower homolog of ACPA, (RS)-2-amino-2-(3-carboxy-5-methyl-4-isoxazolyl)acetic acid (1), which is a Glu analog, was also synthesized. Affinities and neuroexcitatory effects were determined using rat brain membranes and cortical wedges, respectively, at native AMPA, KA, and N-methyl-D-aspartic acid (NMDA) receptors. The molecular pharmacology of (S)- and (R)-ACPA and (S)- and (R)-Ethyl-ACPA was evaluated at homomeric cloned subtypes of AMPA receptors (iGluR1o,3o,4o) and of KA receptors (iGluR5,6), expressed in Xenopus laevis oocytes. The cloned receptors mGluR1alpha, mGluR2, and mGluR4a, expressed in CHO cell lines, were used to study the effects of the five compounds at metabotropic Glu receptors. In accordance with ligand-receptor complexes known from X-ray crystallography, the conformationally restricted Glu analog 1 was inactive at all Glu receptors studied, and the R-forms of ACPA and Ethyl-ACPA were very weak or inactive at these receptors. At AMPA receptor subtypes, (S)-ACPA and (S)-Ethyl-ACPA showed equally potent agonist effects at iGluR1o and iGluR3o, whereas (S)-Ethyl-ACPA was 6-fold more potent than (S)-ACPA at iGluR4o. (S)-ACPA and (S)-Ethyl-ACPA were approximately an order of magnitude less potent at iGluR5 than at AMPA receptor subtypes, and neither compound showed detectable effects at iGluR6. The binding mode of (S)-Ethyl-ACPA at iGluR2 was examined by docking to the (S)-ACPA-iGluR2 complex.

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-(5-tert-butyl-3-hydroxy-4-isothiazolyl)propionic acid (thio-ATPA), a 3-isothiazolol analogue of (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), has previously been shown to be a relatively weak AMPA receptor agonist at native (S)-glutamic acid ((S)-Glu) receptors (EC(50)=14 microM), comparable in potency with ATPA (EC(50)=34 microM). Recent findings, that (S)-ATPA is a potent (EC(50)=0.48 microM) and selective agonist at homomerically expressed ionotropic GluR5, prompted us to resolve thio-ATPA using chiral chromatography and pharmacologically characterize the two enantiomers at native as well as cloned ionotropic glutamate receptors. The enantiomers, (S)- and (R)-thio-ATPA, were obtained in high enantiomeric excess, and their absolute stereochemistry established by an X-ray crystallographic analysis. Electrophysiologically, the two enantiomers were evaluated in the rat cortical wedge preparation, and the S-enantiomer was found to be an AMPA receptor agonist (EC(50)=8.7 microM) twice as potent as the racemate, whereas the R-enantiomer was devoid of activity. In accordance with this, (S)-thio-ATPA proved to be an agonist at homomerically expressed recombinant AMPA receptors (GluR1o, GluR3o, and GluR4o) with EC(50) values of 5, 32 and 20 microM, respectively, producing maximal steady state currents of 78--168% of those maximally evoked by kainic acid, and 120-1600% of those maximally evoked by (S)-ATPA. At homomerically expressed GluR5, (S)-thio-ATPA was found to be a potent agonist (EC(50)=0.10 microM), thus being approximately five times more potent than (S)-ATPA. (R)-Thio-ATPA induced saturating currents with an estimated EC(50) value of 10 microM, most likely due to a contamination with (S)-thio-ATPA. At heteromerically expressed GluR6+KA2 receptors, (S)-thio-ATPA showed relatively weak agonistic properties (EC(50)=4.9 microM). Thus, (S)-thio-ATPA has been shown to be a very potent agonist at GluR5, and may be a valuable tool for the investigation of desensitization properties of AMPA receptors.

We have previously shown that the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA, 2), binds to AMPA receptors in a manner different from that of AMPA (1) itself and that 2, in contrast to 1, also binds to kainic acid receptor sites. To elucidate the structural requirements for selective activation of the site/conformation of AMPA receptors recognized by 2, a number of isosteric analogues of 2 have now been synthesized and pharmacologically characterized. The compound 2-amino-3-(5-carboxy-3-methoxy-4-isoxazolyl)propionic acid (3a) (IC(50) = 0.11 microM; EC(50) = 1.2 microM), which is a regioisostere of 2 with a methoxy group substituted for the methyl group, was approximately equipotent with 2 (IC(50) = 0.020 microM; EC(50) = 1.0 microM) as an inhibitor of [(3)H]AMPA binding and as an AMPA agonist, respectively, whereas the corresponding 3-ethoxy analogue 3b (IC(50) = 1.0 microM; EC(50) = 4.8 microM) was slightly weaker. The analogues 3c-e, containing C3 alkoxy groups, were an order of magnitude weaker than 3b, whereas the additional steric bulk of the alkoxy groups of 3f-i or the presence of an acidic hydroxyl group at the 3-position of the isoxazole ring of 3j prevented interaction with AMPA receptor sites. The 2-amino-3-(2-alkyl-5-carboxy-3-oxo-4-isoxazolyl)propionic acids 4a,b, i, which are regioisosteric analogues of 3a,b,i, showed negligible interaction with AMPA recognition sites. Similarly, replacement of the carboxyl group of 3b by isosteric tetrazolyl or 1,2,4-triazolyl groups to give 5 and 6, respectively, or conversion of 3b into analogue 7, in which the diaminosquaric acid group has been bioisosterically substituted for the alpha-aminocarboxylic acid unit, provided compounds completely devoid of effect at AMPA receptors. In contrast to the parent compound ACPA (2) (IC(50) = 6.3 microM), none of the analogues described showed detectable inhibitory effect on [(3)H]kainic acid receptor binding.

The pharmacological properties of [ 3 H]ATPA (( RS )-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert -butyl analogue of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [ 3 H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors ( K d ∼13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [ 3 H]ATPA binding with potencies similar to those obtained for competition of [ 3 H]kainate binding to GluR5. Saturable, high affinity [ 3 H]ATPA binding ( K d ∼4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [ 3 H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [ 3 H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

The pharmacology of (2S,4R)-4-methylglutamic acid, (2S,4S)-4-methylglutamic acid and (S)- and (R)-4-methyleneglutamic acids (obtained in high chemical and enantiomeric purity from racemic 4-methyleneglutamic acid by chiral HPLC using a Crownpak CR(+) column), was examined in binding experiments using rat brain ionotropic glutamate receptors, and in functional assays using cloned metabotropic glutamate (mGlu) receptors. As a notable result of these studies, (2S,4R)-4-methylglutamic acid and (2S,4S)-4-methylglutamic acid were shown to be selective for kainic acid receptors and mGlu receptors (subtypes 1alpha and 2), respectively, whereas (S)-4-methyleneglutamic acid showed high but rather non-selective affinity for the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid, NMDA and mGlu receptors (subtypes 1alpha and 2). Although none of the compounds were specific for any of the receptor subtypes, the results demonstrate that each of these structurally related compounds has a distinct pharmacological profile.

A series of acyclic and heterocyclic analogues of carbacholine (1) was synthesized using N-methylcarbacholine (MCC, 2), N,N-dimethylcarbacholine (DMCC, 3), and the corresponding tertiary amine (4) as leads. Whereas nicotinic acetylcholine receptor affinity was determined using [3H]nicotine as the radioactive ligand, [3H]oxotremorine-M ([3H]Oxo-M) and [3H]quinuclidinyl benzilate ([3H]QNB), in some cases supplemented with [3H]pirenzepine ([3H]PZ), were used as radioligands for muscarinic acetyicholine receptors on rat brain membranes. On the basis of receptor binding data, nicotinic/muscarinic (N/M) selectivity factors were determined, and muscarinic receptor efficacy (M agonist index) and M1 selectivity (M2/M1 index) estimated. In most cases, quaternized analogues showed higher affinity than the corresponding tertiary amines for muscarinic and, in particular, nicotinic receptor sites. Among the new compounds, N,N-diethylcarbacholine (9e) (IC50 = 0.046 microM), (S)-1-methyl-2-(N,N- dimethyl-aminocarbonyloxymethyl)pyrrolidine (17k) (IC50 = 0.068 microM), and the corresponding quaternized analogue, 18k (IC50 = 0.018 microM) showed the highest nicotinic receptor affinity. The tertiary amine, 17k showed much higher nicotinic receptor affinity than the acyclic analogue, 4 (IC50 = 5.7 microM), and the N/M selectivity factor determined for 17k (150) is an order of magnitude lower than that of nicotine (1400). THe N/M selectivity factors for MCC (2) and DMCC (3), previously reported to be highly selective nicotinic receptor ligands, were shown to be 6.5 and 60, respectively, the latter value being comparable with that of 18k (89).

We have previously shown that (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA, 2) is a functional partial agonist at the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) subtype of excitatory amino acid receptors, reflecting that (S)-APPA is a full agonist and (R)-APPA a competitive antagonist at AMPA receptors. We have now synthesized and pharmacologically characterized (RS)-2-amino-3-[3-hydroxy-5-(2-fluorophenyl)isoxazol-4-yl]propionic acid (2-F-APPA, 5a), 3-F-APPA (5b), 4-F-APPA (5c), (S)-4-F-APPA (6), (R)-4-F-APPA (7), and the fully and partially, respectively, saturated APPA (2) analogues, (RS)-2-amino-3-(hydroxy-5-cyclohexylisoxazol-4-yl)propionic acid (5d) and compound 5e containing a 1-cyclohexenyl ring. The absolute stereo-chemistry of 6 and 7 was established on the basis of comparative circular dichroism studies on 6, 7, and (S)- and (R)-APPA. 4-F-APPA (5c), (S-4-F-APPA (6), 5d, and 5e were shown to selectively inhibit [3H]AMPA binding and to activate AMPA receptors. Whereas (S)-4-F-APPA (6) showed full AMPA receptor agonism, (R)-4-F-APPA (7) was an AMPA receptor antagonist. Co-administration of (S)- and (R)-4-F-APPA to the rat cortical wedge preparation produced functional partial AMPA receptor agonism. Semi empirical calculations showed that the magnitude of the torsional angle of the bond connecting the two rings in the series of nonannulated bicyclic AMPA analogues appears to be of importance for the potency and efficacy of these compounds.

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.

Using ibotenic acid (2) as a lead, two series of 3-isoxazolol amino acid ligands for (S)-glutamic acid (Glu, 1) receptors have been developed. Whereas analogues of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [AMPA, (RS)-3] interact selectively with ionotropic Glu receptors (iGluRs), the few analogues of (RS)-2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid [HIBO, (RS)-4] so far known typically interact with iGluRs as well as metabotropic Glu receptors (mGluRs). We here report the synthesis and pharmacology of a series of 4-substituted analogues of HIBO. The hexyl analogue 9 was shown to be an antagonist at group I mGluRs. The effects of 9 were shown to reside exclusively in (S)-9 (K(b) = 30 microM at mGlu(1) and K(b) = 61 microM at mGlu(5)). The lower homologue of 9, compound 8, showed comparable effects at mGluRs, but 8 also was a weak agonist at the AMPA subtype of iGluRs. Like 9, the higher homologue, compound 10, did not interact with iGluRs, but 10 selectively antagonized mGlu(1) (K(b) = 160 microM) showing very weak antagonist effect at mGlu(5) (K(b) = 990 microM). The phenyl analogue 11 turned out to be an AMPA agonist and an antagonist at mGlu(1) and mGlu(5), and these effects were shown to originate in (S)-11 (EC(50) = 395 microM, K(b) = 86 and 90 microM, respectively). Compound 9, administered icv, but not sc, was shown to protect mice against convulsions induced by N-methyl-D-aspartic acid (NMDA). Compounds 9 and 11 were resolved using chiral HPLC, and the configurational assignments of the enantiomers were based on X-ray crystallographic analyses.