RESUMEN
A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.
Asunto(s)
Azetidinas/farmacología , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Memoria/efectos de los fármacos , Azetidinas/síntesis química , Azetidinas/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The structure-activity relationships of 2-(piperidin-3-yl)-1H-benzimidazoles, 2-morpholine and 2-thiomorpholin-2-yl-1H-benzimidazoles are described. In the lead optimization process, the pK(a) and/or logP of benzimidazole analogs were reduced either by attachment of polar substituents to the piperidine nitrogen or incorporation of heteroatoms into the piperidine heterocycle. Compounds 9a and 9b in the morpholine series and 10g in the thiomorpholine series demonstrated improved selectivity and CNS profiles compared to lead compound 2 and these are potential candidates for evaluation as sedative hypnotics.
Asunto(s)
Bencimidazoles/síntesis química , Antagonistas de los Receptores Histamínicos H1/farmacología , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Bencimidazoles/farmacología , Sistema Nervioso Central/efectos de los fármacos , Diseño de Fármacos , Canal de Potasio ERG1 , Electrofisiología/métodos , Canales de Potasio Éter-A-Go-Go/química , Humanos , Hipnóticos y Sedantes/farmacología , Concentración 50 Inhibidora , Cinética , Microsomas Hepáticos/efectos de los fármacos , Modelos Químicos , Morfolinas/química , Nitrógeno/química , Piperidinas/química , Receptores Histamínicos H1/química , Relación Estructura-ActividadRESUMEN
A series of 2-(3-aminopiperidine)-benzimidazoles were identified as selective H(1)-antihistamines for evaluation as potential sedative hypnotics. Representative compounds showed improved hERG selectivity over a previously identified 2-aminobenzimidazole series. While hERG activity could be modulated via manipulation of the benzimidazole N1 substituent, this approach led to a reduction in CNS exposure for the more selective compounds. One example, 9q, retained a suitable selectivity profile with CNS exposure equivalent to known centrally active H(1)-antihistamines.
Asunto(s)
Bencimidazoles/química , Sistema Nervioso Central/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/química , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Bencimidazoles/síntesis química , Bencimidazoles/uso terapéutico , Descubrimiento de Drogas , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Antagonistas de los Receptores Histamínicos H1/síntesis química , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Humanos , Relación Estructura-ActividadRESUMEN
A series of indene analogs of the H(1)-antihistamine (-)-R-dimethindene was evaluated for selectivity in the search for potentially improved sedative-hypnotics. Variation of the 6-substitutent in the indene core in combination with a pendant electron rich heterocycle led to the identification of several potent H(1)-antihistamines with desirable selectivity over CYP enzymes, the M(1) muscarinic receptor and the hERG channel. These compounds were candidates for further ADME profiling and in vivo evaluation.
Asunto(s)
Antagonistas de los Receptores Histamínicos/uso terapéutico , Indenos/uso terapéutico , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacología , Humanos , Indenos/química , Indenos/farmacologíaRESUMEN
Analogs of the known H(1)-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/química , Indenos/química , Piridazinas/química , Receptores Histamínicos H1/química , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Animales , Citocromo P-450 CYP2D6/metabolismo , Dimetindeno/química , Electroencefalografía , Antagonistas de los Receptores Histamínicos H1/farmacocinética , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Humanos , Indenos/farmacocinética , Indenos/uso terapéutico , Microsomas Hepáticos/metabolismo , Modelos Animales , Piridazinas/farmacocinética , Piridazinas/uso terapéutico , Ratas , Receptores Histamínicos H1/metabolismo , Relación Estructura-ActividadRESUMEN
SAR of lead benzothiophene H(1)-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H(1)-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacocinética , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Tiofenos/farmacología , Tiofenos/farmacocinética , Antagonistas de los Receptores Histamínicos H1/química , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Humanos , Receptores Histamínicos H1/metabolismo , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/uso terapéuticoRESUMEN
The benzimidazole core of the selective non-brain-penetrating H(1)-antihistamine mizolastine was used to identify a series of brain-penetrating H(1)-antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H(1)-antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.
Asunto(s)
Bencimidazoles/antagonistas & inhibidores , Bencimidazoles/química , Encéfalo/efectos de los fármacos , Química Farmacéutica/métodos , Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/farmacología , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Animales , Diseño de Fármacos , Canal de Potasio ERG1 , Electroencefalografía/métodos , Electromiografía/métodos , Canales de Potasio Éter-A-Go-Go/química , Humanos , Modelos Químicos , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.
Asunto(s)
Acetamidas/farmacología , Antagonistas del Receptor de Adenosina A2 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Pirimidinas/farmacología , Acetamidas/síntesis química , Acetamidas/química , Antagonistas del Receptor de Adenosina A1 , Animales , Evaluación Preclínica de Medicamentos , Canales de Potasio Éter-A-Go-Go/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Ratas Wistar , Especificidad de la Especie , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.
Asunto(s)
Diseño de Fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Memoria/efectos de los fármacos , Pirazoles/síntesis química , Pirazoles/farmacología , Animales , Técnicas de Química Sintética , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformación Molecular , Permeabilidad , Pirazoles/química , Pirazoles/metabolismo , RatasRESUMEN
A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.
Asunto(s)
Memoria/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores de Fosfodiesterasa/químicaRESUMEN
The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.
Asunto(s)
Piridazinas/química , Piridazinas/farmacocinética , Receptores de Somatostatina/antagonistas & inhibidores , Receptores de Somatostatina/metabolismo , Somatostatina/antagonistas & inhibidores , Aminoácidos/química , Animales , Calcio/metabolismo , Agonistas de los Canales de Calcio/química , Agonistas de los Canales de Calcio/farmacocinética , Canales de Calcio/metabolismo , Células Cultivadas , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Neuronas/efectos de los fármacos , Ratas , Receptores de Somatostatina/químicaRESUMEN
The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.0(2.6)]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 microM, respectively, while EAAT1 activity was inhibited by 50% at 100 microM (IC50 values determined in oocytes were 1.3 microM (EAAT2), 52.5 microM (EAAT3) and 125.9 microM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]d-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited d-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes.
Asunto(s)
Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Heptanos/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inhibidores de la Captación de Neurotransmisores/farmacología , Sistema de Transporte de Aminoácidos X-AG/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Perros , Relación Dosis-Respuesta a Droga , Heptanos/química , Compuestos Heterocíclicos con 3 Anillos/química , Humanos , Inhibidores de la Captación de Neurotransmisores/química , RatasRESUMEN
In the present article, we report on a strategy to improve the physical properties of a series of small molecule human adenosine 2A (hA2A) antagonists. One of the aromatic rings typical of this series of antagonists is replaced with a series of aliphatic groups, with the aim of disrupting crystal packing of the molecule to lower the melting point and in turn to improve the solubility. Herein, we describe the SAR of a new series of water-soluble 2,4,6-trisubstituted pyrimidines where R1 is an aromatic heterocycle, R2 is a short-chain alkyl amide, and the typical R3 aromatic heterocyclic substituent is replaced with an aliphatic amino substituent. This approach significantly enhanced aqueous solubility and lowered the log P of the system to provide molecules without significant hERG or CYP liabilities and robust in vivo efficacy.
Asunto(s)
Acetamidas/uso terapéutico , Antagonistas del Receptor de Adenosina A2 , Pirimidinas/uso terapéutico , Acetamidas/síntesis química , Antagonistas del Receptor de Adenosina A1 , Animales , Conducta Animal/efectos de los fármacos , Catalepsia/inducido químicamente , Catalepsia/tratamiento farmacológico , Sinergismo Farmacológico , Haloperidol , Humanos , Pirimidinas/síntesis química , Ratas , Rotación , Solubilidad , Relación Estructura-ActividadRESUMEN
Analogues of the known H(1)-antihistamine R-dimethindene were profiled as potential agents for the treatment of insomnia. Several highly selective compounds were efficacious in rodent sleep models. On the basis of overall profile, indene 1d and benzothiophene 2a had pharmacokinetic properties suitable for evaluation in night time dosing. Compound 2a did not show an in vivo cardiovascular effect from weak hERG channel inhibition.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Animales , Encéfalo/metabolismo , Dimetindeno/metabolismo , Dimetindeno/farmacocinética , Dimetindeno/farmacología , Dimetindeno/uso terapéutico , Electroencefalografía/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Antagonistas de los Receptores Histamínicos H1/metabolismo , Antagonistas de los Receptores Histamínicos H1/farmacocinética , Humanos , Ratas , Receptores Muscarínicos/metabolismo , Sueño/efectos de los fármacos , Especificidad por SustratoRESUMEN
4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinson's disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.
Asunto(s)
Antagonistas del Receptor de Adenosina A2 , Enfermedad de Parkinson/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Catalepsia/inducido químicamente , Catalepsia/tratamiento farmacológico , Modelos Animales de Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Haloperidol , Humanos , Ligandos , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA(A) receptors from rat brain and acts as a positive allosteric modulator of GABA(A) receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA(A) receptor alpha subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA(A) receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA(A) receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for alpha1 subunit-containing receptors over GABA(A) receptors containing alpha2, alpha3, or alpha5 subunits. The EC(50) values were 2.6, 24, 60, and 77 nM for alpha1beta2gamma2, alpha2beta2gamma2, alpha3beta3gamma2, and alpha5beta2gamma2 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and >100 times more potent than zaleplon. Moreover, indiplon, up to 1 microM, did not potentiate GABA(A) receptors composed of alpha4beta2gamma2 and alpha6beta2gamma2 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.
Asunto(s)
Benzodiazepinas/farmacología , Hipnóticos y Sedantes/farmacología , Subunidades de Proteína/metabolismo , Receptores de GABA-A/metabolismo , Proteínas Recombinantes/metabolismo , Tiofenos/farmacología , Regulación Alostérica , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Unión Competitiva , Línea Celular , Clonación Molecular , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , RatasRESUMEN
The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.
Asunto(s)
Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Urea/análogos & derivados , Animales , Estructura Molecular , Pirrolidinas/química , Ratas , Receptores de la Hormona Hipofisaria/metabolismo , Relación Estructura-Actividad , Urea/síntesis química , Urea/química , Urea/farmacologíaRESUMEN
Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.
Asunto(s)
Inhibidores Enzimáticos/toxicidad , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/metabolismo , Heptanos/toxicidad , Compuestos Heterocíclicos con 3 Anillos/toxicidad , Hipocampo/efectos de los fármacos , Homeostasis/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG/fisiología , Animales , Animales Recién Nacidos , Ácido Aspártico/farmacología , Western Blotting/métodos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Transportador 2 de Aminoácidos Excitadores/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/citología , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Ratas , Factores de Tiempo , Tritio/metabolismoRESUMEN
In this study, we describe the pharmacological characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT2, the predominant glutamate transporter in forebrain regions. The rank order of potency determined for the inhibition of human EAAT2 was N(4)-[4-(2-bromo-4,5-difluorophenoxy)phenyl]-L-asparagine (WAY-213613) (IC(50) = 85 +/- 5 nM) > N(4)-(2'-methyl-1,1'-biphenyl-4-yl)-L-asparagine (WAY-213394) (IC(50) = 145 +/- 22 nM) = N(4)-[7-(trifluoromethyl)-9H-fluoren-2-yl]-L-asparagine (WAY-212922) (IC(50) = 157 +/- 11 nM) = 3-{[(4'-chloro-2-methyl-1,1'-biphenyl-4-yl)carbonyl]amino}-L-alanine (WAY-211686) (IC(50) = 190 +/- 10 nM). WAY-213613 was the most selective of the compounds examined, with IC(50) values for inhibition of EAAT1 and EAAT3 of 5 and 3.8 microM, respectively, corresponding to a 59- and 45-fold selectivity toward EAAT2. An identical rank order of potency [WAY-213613 (35 +/- 7 nM) > WAY-213394 (92 +/- 13 nM) = WAY-212922 (95 +/- 8 nM) = WAY-211686 (101 +/- 20 nM)] was observed for the inhibition of glutamate uptake in rat cortical synaptosomes, consistent with the predominant contribution of EAAT2 to this activity. Kinetic studies with each of the compounds in synaptosomes revealed a competitive mechanism of inhibition. All compounds were determined to be nonsubstrates by evaluating both the stimulation of currents in EAAT2-injected oocytes and the heteroexchange of d-[(3)H]aspartate from cortical synaptosomes. WAY-213613 represents the most potent and selective inhibitor of EAAT2 identified to date. Taken in combination with its selectivity over ionotropic and metabotropic glutamate receptors, this compound represents a potential tool for the further elucidation of EAAT2 function.
Asunto(s)
Ácido Aspártico/análogos & derivados , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Propionatos/farmacología , Animales , Ácido Aspártico/farmacología , Células CHO , Línea Celular , Cricetinae , Humanos , Modelos Moleculares , Propionatos/química , Ratas , Sinaptosomas/efectos de los fármacos , Sinaptosomas/fisiología , Xenopus laevisRESUMEN
Clinically used benzodiazepine and nonbenzodiazepine sedative-hypnotic agents for the treatment of insomnia produce their therapeutic effects through allosteric enhancement of the effects of the inhibitory neurotransmitter GABA at the GABA(A) receptor. Indiplon is a novel pyrazolopyrimidine sedative-hypnotic agent, currently in development for insomnia. Using radioligand binding studies, indiplon inhibited the binding of [(3)H]Ro 15-1788 (flumazenil) to rat cerebellar and cerebral cortex membranes with high affinity (K(i) values of 0.55 and 0.45 nM, respectively). [(3)H]Indiplon binding to rat cerebellar and cerebral cortex membranes was reversible and of high affinity, with K(D) values of 1.01 and 0.45 nM, respectively, with a pharmacological specificity consistent with preferential labeling of GABA(A) receptors containing alpha1 subunits. In "GABA shift" experiments and in measurements of GABA-induced chloride conductance in rat cortical neurons in culture, indiplon behaved as an efficacious potentiator of GABA(A) receptor function. In both the radioligand binding and electrophysiological experiments, indiplon had a higher affinity than zolpidem or zaleplon. These in vitro properties are consistent with the in vivo properties of indiplon as an effective sedative-hypnotic acting through allosteric potentiation of the GABA(A) receptor.