RESUMEN
In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompetitive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones possess marked anticonvulsant properties and high affinity for the noncompetitive binding site of the AMPAR complex. In this paper, we report the synthesis and pharmacological characterization of a full set of 2,3-benzodiazepin-4-ones in order to better define the structure-activity relationship (SAR) of this class of compounds. Binding assays and functional tests were performed to evaluate the antagonistic activity at the AMPARs. Through these results we have identified a potent AMPAR antagonist, 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (5c). This compound noncompetitively inhibited AMPAR-mediated toxicity in primary mouse hippocampal cultures with an IC(50) of 1.6muM and blocked kainate-induced calcium influx in rat cerebellar granule cells with an IC(50) of 6.4muM. Thus, 5c has the in vitro potential as therapeutic drug in the treatment of various neurological disorders.
Asunto(s)
Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacología , Fármacos Neuroprotectores/farmacología , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Animales , Benzodiazepinas/síntesis química , Benzodiazepinonas/química , Calcio/metabolismo , Células Cultivadas , Hipocampo/citología , Concentración 50 Inhibidora , Ácido Kaínico/toxicidad , Ligandos , Ratones , Estructura Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Isoxazoles/química , Propionatos/química , Receptores AMPA/agonistas , Tetrazoles/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Compuestos de Anilina , Animales , Sitios de Unión , Línea Celular , Femenino , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Isoxazoles/farmacología , Modelos Moleculares , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Propionatos/farmacología , Ratas , Receptores AMPA/genética , Receptores AMPA/fisiología , Homología de Secuencia de Aminoácido , Estereoisomerismo , Relación Estructura-Actividad , Tetrazoles/farmacología , Termodinámica , Xantenos , Xenopus , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
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.
Asunto(s)
Receptores AMPA/agonistas , Tetrazoles/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/metabolismo , Línea Celular , Cristalografía por Rayos X , Técnicas In Vitro , Insectos , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Ensayo de Unión Radioligante , Ratas , Receptores AMPA/química , Receptores AMPA/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Sinaptosomas/metabolismo , Tetrazoles/química , Tetrazoles/farmacología , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
This paper describes the synthesis of racemic 3,5-dihydro-5-methyl-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (+/-)-5, attempted stereoselective synthesis of its enantiomers, chiral HPLC resolution of the racemate, and assignment of the absolute configuration. Enantiomer (5S)-(-)-5 is provided with an in vivo anticonvulsant activity 8 times higher than its enantiomer (5R)-(+)-5. This result is confirmed in the in vitro test by the ability to inhibit the kainate-induced increase of the [Ca(2+)](i) in a primary culture of rat cerebellar granule cells which express alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. Binding affinity of compound (+/-)-5 at the AMPA and N-methyl-d-aspartic acid (NMDA) receptors was also evaluated.
Asunto(s)
Anticonvulsivantes/síntesis química , Benzodiazepinas/síntesis química , Benzodiazepinonas/síntesis química , Receptores AMPA/antagonistas & inhibidores , Animales , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/química , Benzodiazepinonas/farmacología , Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Técnicas In Vitro , Ácido Kaínico/farmacología , Ratones , Ratones Endogámicos DBA , Modelos Moleculares , Conformación Molecular , Prosencéfalo/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/tratamiento farmacológico , EstereoisomerismoRESUMEN
A series of 3-(alkylcarbamoyl)-2-aryl-1,2-dihydro-6,7-(methylenedioxy)-3H-quinazolin-4-ones, compounds 3-6, were synthesized, and screened as anticonvulsant agents in DBA/2 mice against sound-induced seizure (Table). The new compounds were found to display anticonvulsant properties inferior to those of the known dehydro congeners 1 and 2. The binding affinities of 1-6 at the AMPA and NMDA receptors were also evaluated.