RESUMEN

Dual-parameter measurements of refractive index and methane concentration based on electromagnetic Fano resonance are proposed. Two independent Fano resonances can be produced through electric dipole and toroidal dipole resonance in an all-dielectric metasurface separately. The linear relationship between the spectral peak-shifts and the parameters to be measured will be obtained directly. The refractive index (RI) sensitivity and gas sensitivity are 1305.6 nm/refractive index unit (RIU), -0.295 nm/% for one resonance peak (dip1), and 456.6 nm/RIU, -0.61 nm/% for another resonance peak (dip2). Such a metasurface has simpler structure and higher sensitivity, which is beneficial for environmental gas monitoring or multi-parameter measurements.

RESUMEN

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Asunto(s)

Conducto Inguinal/anatomía & histología , Trasplante de Islotes Pancreáticos , Modelos Biológicos , Grasa Subcutánea/trasplante , Animales , Glucemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/terapia , Supervivencia de Injerto , Humanos , Islotes Pancreáticos/patología , Ratones Endogámicos C57BL , Perfusión , Supervivencia Tisular

RESUMEN

5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 µmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

Asunto(s)

5'-Nucleotidasa/genética , Cordyceps/química , Proteínas Fúngicas/genética , Genoma Fúngico , 5'-Nucleotidasa/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cordyceps/clasificación , Cordyceps/enzimología , Desoxiadenosinas/biosíntesis , Proteínas Fúngicas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Filogenia , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura

RESUMEN

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

Asunto(s)

Trasplante de Islotes Pancreáticos/métodos , Epiplón/fisiopatología , Animales , Masculino , Ratones

RESUMEN

INTRODUCTION: Tyrosine kinase 2 (Tyk2) is a non-receptor tyrosine-protein kinase, an enzyme that in humans is encoded by the TYK2 gene. Tyk2, together with three other family subtypes, namely, Jak1, Jak2, and Jak3, belong to the JAK family. Before 2014, far more publications and patents appeared in public domain attributing to the development of selective Jak2 and Jak3 inhibitors than those for selective Tyk2 and Jak1 inhibitors. AREAS COVERED: This review sought to give an overview of patents related to small molecule selective Tyk2 inhibitors published from 2015 to 2018. The article also covers clinical activities of small molecule selective Tyk2 inhibitors in recent years. EXPERT OPINION: As a key component of the JAK-STAT signaling pathway, Tyk2 regulates INFα, IL12, and IL23. Selective inhibition of Tyk2 can provide pharmacological benefits in the treatment of many diseases such as psoriasis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), cancer, and diabetes. The selectivity against other Jak family subtypes (such as Jak2) is crucial in order to minimize the potential side effects and to maximize the desired pharmacological effects. In this context, this review of recent selective Tyk2 inhibitor patents may prove valid, interesting, and promising within the therapeutic paradigm.

Asunto(s)

Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , Animales , Humanos , Quinasas Janus/metabolismo , Patentes como Asunto , Inhibidores de Proteínas Quinasas/efectos adversos , Factores de Transcripción STAT/metabolismo , Transducción de Señal , TYK2 Quinasa/genética

RESUMEN

Caterpillar fungus is a well-known fungal Chinese medicine. To reveal molecular changes during early and late stages of adenosine biosynthesis, transcriptome analysis was performed with the anamorph strain of caterpillar fungus. A total of 2,764 differentially expressed genes (DEGs) were identified (p ≤ 0.05, |log2 Ratio| ≥ 1), of which 1,737 were up-regulated and 1,027 were down-regulated. Gene expression profiling on 4-10 d revealed a distinct shift in expression of the purine metabolism pathway. Differential expression of 17 selected DEGs which involved in purine metabolism (map00230) were validated by qPCR, and the expression trends were consistent with the RNA-Seq results. Subsequently, the predicted adenosine biosynthesis pathway combined with qPCR and gene expression data of RNA-Seq indicated that the increased adenosine accumulation is a result of down-regulation of ndk, ADK, and APRT genes combined with up-regulation of AK gene. This study will be valuable for understanding the molecular mechanisms of the adenosine biosynthesis in caterpillar fungus.

Asunto(s)

Adenosina/biosíntesis , Hypocreales/genética , Hypocreales/metabolismo , Transcriptoma/genética , Perfilación de la Expresión Génica , Medicina Tradicional China , Redes y Vías Metabólicas/genética

RESUMEN

PURPOSE: To evaluate the effect of tear malate dehydrogenase 2 on monitoring ocular surface injury in mild dry eye (DE) disease. METHODS: A total of 15 DE patients (30 eyes) with mild subjective symptoms but no ocular surface fluorescein staining signs were enrolled in this study (DE group). The control group was 15 healthy age- and sex-matched volunteers (30 eyes). All subjects were asked to fill out a DE symptoms questionnaire and take different tests including tear MDH and MDH2 activities evaluation, tear breakup time (TBUT), Schirmer I, and slit-lamp examination of the ocular surface. We investigated different changes in tear MDH and MDH2 activities in the DE group and control group, discussed the association between tear MDH2 activity and DE symptoms, and the relationship between tear MDH2 activity and diagnostic tests (Schirmer I and TBUT). We also analyzed the changes in tear MDH2 activities after the treatment with artificial tears. RESULTS: Tear MDH activities in the DE group and control group were 288 ± 102 U/L and 259 ± 112 U/L, respectively, and this difference was not statistically significant (P > 0.05). The tear MDH2 activities in DE group were significantly increased compared with control group. Tear MDH2 was significantly and negatively correlated with the Schirmer's value (r = -0.733, P < 0.01) and the TBUT value (r = -0.841, P < 0.01). MDH2 also had a significant positive correlation with soreness symptoms (r = 0.687, P < 0.01). Treatment with artificial tears relieved or eliminated all discomfort symptoms, together with a considerable decrease in MDH2 activities (P < 0.01), but no significant changes in the Schirmer and the TBUT tests were observed. CONCLUSION: Tear MDH2 activity can indicate ocular surface injury in mild DE patients and may be used to monitor the response to therapy.

Asunto(s)

Síndromes de Ojo Seco/enzimología , Malato Deshidrogenasa/análisis , Encuestas y Cuestionarios , Lágrimas/enzimología , Adulto , Biomarcadores/análisis , Estudios de Casos y Controles , Síndromes de Ojo Seco/tratamiento farmacológico , Femenino , Fluoresceína , Humanos , Masculino , Persona de Mediana Edad , Soluciones Oftálmicas/administración & dosificación , Lámpara de Hendidura

RESUMEN

We have explored a series of spirocyclic piperidine amide derivatives (5) as tryptase inhibitors. Thus, 4 (JNJ-27390467) was identified as a potent, selective tryptase inhibitor with oral efficacy in two animal models of airway inflammation (sheep and guinea pig asthma models). An X-ray co-crystal structure of 4 x tryptase revealed a hydrophobic pocket in the enzyme's active site, which is induced by the phenylethynyl group and is comprised of amino acid residues from two different monomers of the tetrameric protein.

Asunto(s)

Asma/tratamiento farmacológico , Hipersensibilidad Respiratoria/tratamiento farmacológico , Inhibidores de Serina Proteinasa/farmacología , Compuestos de Espiro/síntesis química , Compuestos de Espiro/farmacología , Triptasas/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Inhibidores Enzimáticos del Citocromo P-450 , Modelos Animales de Enfermedad , Perros , Cobayas , Humanos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Estructura Molecular , Ratas , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacocinética , Ovinos , Espectrometría de Masa por Ionización de Electrospray , Compuestos de Espiro/farmacocinética , Tripsina/metabolismo , Triptasas/metabolismo

Asunto(s)

Plaquetas/efectos de los fármacos , Nucleósidos/síntesis química , Antagonistas del Receptor Purinérgico P2 , Tetrazoles/síntesis química , Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Humanos , Nucleósidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Transducción de Señal , Tetrazoles/farmacología

RESUMEN

We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.

Asunto(s)

Antagonistas de los Receptores de Hormonas Antidiuréticas , Benzazepinas/química , Compuestos de Espiro/química , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/química , Antihipertensivos/metabolismo , Antihipertensivos/farmacocinética , Benzazepinas/administración & dosificación , Benzazepinas/farmacocinética , Benzazepinas/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratas , Ratas Long-Evans , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/fisiología , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Vasopresinas/metabolismo

RESUMEN

Novel bis(indolyl)maleimide pyridinophanes 3, 9a, 9b, 10a, 10b, and 11 were prepared by cobalt-mediated [2+2+2] cycloaddition of an appropriate alpha,omega-diyne with an N,N-dialkylcyanamide. These macrocyclic heterophanes were found to be potent, selective inhibitors of glycogen synthase kinase-3beta. An X-ray structure of a co-crystal of GSK-3beta and 3 (IC(50)=3nM) depicts the hydrogen bonding and hydrophobic interactions in the ATP-binding pocket of this serine/threonine protein kinase.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Piridinas/farmacología , Adenosina Trifosfato/metabolismo , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Interacciones Hidrofóbicas e Hidrofílicas , Maleimidas/química , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Piridinas/química , Relación Estructura-Actividad

RESUMEN

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

Asunto(s)

Indazoles/síntesis química , Indoles/síntesis química , Maleimidas/síntesis química , Proteína Quinasa C/antagonistas & inhibidores , Animales , Línea Celular , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Cobayas , Humanos , Indazoles/farmacología , Indazoles/toxicidad , Indoles/farmacología , Indoles/toxicidad , Interleucina-8/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Síndrome de QT Prolongado/inducido químicamente , Maleimidas/farmacología , Maleimidas/toxicidad , Modelos Moleculares , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Proteína Quinasa C/química , Proteína Quinasa C beta , Relación Estructura-Actividad

RESUMEN

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.

Asunto(s)

Anticoagulantes/síntesis química , Fibrinolíticos/síntesis química , Cetonas/síntesis química , Oligopéptidos/síntesis química , Tiazoles/síntesis química , Trombina/antagonistas & inhibidores , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Electrocardiografía/efectos de los fármacos , Fibrinolíticos/química , Fibrinolíticos/farmacología , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Hipotensión/inducido químicamente , Técnicas In Vitro , Cetonas/química , Cetonas/farmacología , Modelos Moleculares , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Conejos , Estereoisomerismo , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Trombina/química , Tripsina/química , Trombosis de la Vena/prevención & control

RESUMEN

We investigated the formation of macrocycles from alpha,omega-diynes in cobalt-mediated co-cyclotrimerization reactions. Long-chain alpha,omega-diynes underwent metal-mediated [2 + 2 + 2] cycloadditions with nitriles, cyanamides, or isocyanates in the presence of CpCo(CO)2 (Cp = cyclopentadienide) to yield pyridine-containing macrocycles, i.e., meta- and para-pyridinophanes, such as 5m/5p, 35m/35p, and 41m/41p. The regioselectivity of these reactions was affected by the length and type of linker unit between the alkyne groups, as well as by certain stereoelectronic factors. An analogous alpha,omega-cyano-alkyne, 28, combined with an alkyne to yield two isomeric meta-pyridinophanes, such as 5m and 29m, and an ortho cycloadduct (benzannulation product), such as 29o. We developed a reaction protocol for these cobalt-based [2 + 2 + 2] cycloadditions that involves markedly improved conditions such that this process offers a convenient, flexible synthetic approach to macrocyclic pyridine-containing compounds. For example, diyne 6 reacted with p-tolunitrile in 1,4-dioxane to give 7p and 7m (7:1 ratio) in 87% yield at a moderate temperature of ca. 100 degrees C in 24 h without photoirradiation or syringe-pump addition. Isocyanates were also effective reactants, as exemplified by the formation of 44p almost exclusively (44p:44m > 50:1) in 64% yield from diyne 8 and 2-phenylethylisocyanate. By using this improved protocol we were able to co-cyclotrimerize long-chain alpha,omega-diynes with alkynes in certain cases to demonstrate a successful macrocyclic variant of the Vollhardt reaction. For instance, diyne 6 reacted with dipropylacetylene to give paracyclophane 57p and benzannulene 57o (2:1 ratio) in 29% yield.

RESUMEN

CpCo(CO)2-mediated cyclotrimerisation of bis-alkynes and cyanamides provides multisubstituted 2-aminopyridines, including macrocyclic products, such as 22 (50% yield).

Asunto(s)

Alquinos/síntesis química , Cobalto/química , Cianamida/síntesis química , Compuestos Organometálicos/química , Alquinos/química , Cianamida/química , Ciclización , Estructura Molecular , Compuestos Organometálicos/síntesis química

RESUMEN

A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.

Asunto(s)

Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Inhibidores de Proteínas Quinasas/química , Línea Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Maleimidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

Two approaches were developed to synthesize the novel 7-azaindolyl-heteroarylmaleimides. The first approach was based upon the palladium-catalyzed Suzuki cross-coupling or Stille cross-coupling of 2-chloro-maleimide 5 with various arylboronic acids or arylstannanes. The second approach was based upon the condensation of ethyl 7-azaindolyl-3-glyoxylate 12 with various acetamides. The hydroxypropyl-substituted 7-azaindolylmaleimide template was first used to screen different heteroaryls attached to the maleimide. Replacement of hydroxypropyl with different chain lengths and different functional groups were studied next. Many compounds synthesized were demonstrated to have high potency at GSK-3beta, good GS activity in HEK293 cells and good to excellent metabolic stability in human liver microsomes. Three representative compounds (21, 33, and 34) were demonstrated to have good selectivity against a panel of 80 kinase assays. Among them, compound 33 exhibited very weak inhibitions at the other 79 kinase assays, and behaved as a highly selective GSK-3beta inhibitor.

Asunto(s)

Compuestos Aza/síntesis química , Compuestos Aza/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Maleimidas/farmacología , Animales , Compuestos Aza/química , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Maleimidas/síntesis química , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Conejos , Especificidad por Sustrato

RESUMEN

Protease activated receptor-1 (PAR-1) is a key mediator of the cellular actions of alpha-thrombin. Thus, antagonism of this unique G-protein coupled receptor with a small molecule represents a means of selectively inhibiting thrombin's cellular actions without inhibiting its proteolytic activity. RWJ-58259 (alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)- amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide) is a potent and selective inhibitor of PAR-1 identified as part of a synthetic chemistry program based upon a de novo design approach. RWJ-58259 inhibited thrombin-induced platelet aggregation in human platelets with an IC50 of 0.37 microM without inhibiting thrombin's proteolytic activity or aggregation induced by other agonists. RWJ-58259 was not effective in guinea pig models of thrombosis. This reflected the presence of a second thrombin-sensitive receptor system in guinea pigs (PAR-3/4) and the selectivity of RWJ-58259 for PAR-1. However, RWJ-58259 was effective in a non-human primate model of thrombosis. Because human platelets have a PAR expression profile similar to the non-human primate, PAR-1 antagonism has the potential to be antithrombotic in humans. RWJ-58259 also inhibited thrombin-induced intracellular calcium signaling and proliferation in rat vascular smooth muscle cells. Perivascular application of RWJ-58259 in vivo significantly inhibited arterial injury-induced stenosis in a rat model of balloon angioplasty. These preclinical results suggest a potential clinical utility of RWJ-58259 for treatment of thrombotic disorders and vascular injury associated with acute coronary interventions and atherosclerosis. Given the potential role of PAR-1 in thrombin's actions in other cell types and disease states, RWJ-58259 provides a means for assessing additional clinical utilities of PAR-1 antagonism in disease conditions such as inflammation, cancer and neurodegeneration.

Asunto(s)

Indazoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Receptor PAR-1/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacología , Animales , Disponibilidad Biológica , Semivida , Humanos , Indazoles/química , Indazoles/farmacocinética , Músculo Liso Vascular/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Urea/química , Urea/farmacocinética

RESUMEN

[reaction: see text] Cobalt-mediated [2 + 2 + 2] cycloaddition of alpha,omega-diynes and isocyanates provides a direct approach to macrocyclic 2-oxopyridinophanes. This macrocyclization process, which proceeded most efficiently with aliphatic isocyanates, was conveniently performed at a moderate temperature (85 degrees C) without irradiation or syringe-pump addition.