RESUMEN
A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Piridonas/síntesis química , Piridonas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Masculino , Microsomas Hepáticos/enzimología , Estructura Molecular , Piridazinas/química , Piridazinas/farmacología , Piridonas/química , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.
Asunto(s)
Benzamidas/síntesis química , Benzamidas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Pironas/síntesis química , Pironas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Benzamidas/química , Modelos Animales de Enfermedad , Perros , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Estructura Molecular , Piridonas , Pironas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38alpha. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.
Asunto(s)
Antiinflamatorios/química , Inhibidores de Proteínas Quinasas/química , Piridonas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Piridonas/síntesis química , Piridonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Unusual amino acids such as beta-methoxytyrosine (beta-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among beta-MeOTyr isomers was poor. The best separation of beta-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.
Asunto(s)
Aminoácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
Colyophilization with methyl-beta-cyclodextrin activates subtilisin Carlsberg by more than 200-fold in organic solvents, though this is a short-lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 degrees C) decreases exponentially with a t(1/2) of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the cross-linked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.
Asunto(s)
Subtilisina/química , Estabilidad de Enzimas , Liofilización , Semivida , Preservación Biológica , Solventes , Factores de TiempoRESUMEN
All 4 diastereomeric possibilities for the 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid (Dhtda) residue, found in the cyclic depsipeptide natural products papuamides A-D and mirabamides A-D, were stereoselectively synthesized using a Z-selective Wittig reaction of both enantiomers of 2,4-dimethylhex-2-enyl-triphenylphosphonium bromide with all four diastereoisomers of ethyl-3-formyl-2-methyl-1,4-dioxaspiro[4,4]nonane-2-carboxylate. To elucidate the configuration of Dhtda, the 1H- and 13C-NMR spectra of the synthetic isomers were compared to those of the natural residue. On the basis of that comparison, it is suggested that the likely configuration of the diastereomer present in Dhtda residue is either (2R,3S,8S) or (2S,3R,8S) in the papuamides and mirabimides.
RESUMEN
In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.