RESUMO
This article describes the discovery of aryl hydroxy pyrimidinones and the medicinal chemistry efforts to optimize this chemotype for potent APJ agonism. APJ is a G-protein coupled receptor whose natural agonist peptide, apelin, displays hemodynamic improvement in the cardiac function of heart failure patients. A high throughput screen was undertaken to identify small molecule hits that could be optimized to mimic the apelin in vitro response. A potent and low molecular weight aryl hydroxy pyrimidinone analog 30 was identified through optimization of an HTS hit and medicinal chemistry efforts to improve its properties.
Assuntos
Receptores de Apelina/agonistas , Pirimidinonas/farmacologia , Descoberta de Drogas , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Pirimidinonas/síntese química , Relação Estrutura-AtividadeRESUMO
Monoacylglycerol acyltransferase 2 (MGAT2) is a membrane-bound lipid acyltransferase that catalyzes the formation of diacylglycerol using monoacylglycerol and fatty acyl CoA as substrates. MGAT2 is important for intestinal lipid absorption and is an emerging target for the treatment of metabolic diseases. In the current study, we identified and characterized four classes of novel MGAT2 inhibitors. We established both steady state and kinetic binding assay protocols using a novel radioligand, [(3)H]compound A. Diverse chemotypes of MGAT2 inhibitors were found to compete binding of [(3)H]compound A to MGAT2, indicating the broad utility of [(3)H]compound A for testing various classes of MGAT2 inhibitors. In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as Kon, Koff, and T1/2 were systematically defined. Of particular value, the residence times of inhibitors on MGAT2 enzyme were derived. We believe that the identification of novel classes of MGAT2 inhibitors and the detailed kinetic characterization provide valuable information for the identification of superior candidates for in vivo animal and clinical studies. The current work using a chemical probe to define inhibitory kinetics can be broadly applied to other membrane-bound acyltransferases.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Humanos , Ligantes , Camundongos , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante/métodos , Ratos , Proteínas Recombinantes/metabolismoRESUMO
To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes.
Assuntos
Bioensaio/métodos , Diglicerídeos/biossíntese , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Camundongos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Obesidade/genética , Ácido Palmítico/metabolismoRESUMO
A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.
Assuntos
Pelo Animal/metabolismo , Inibidores Enzimáticos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Doenças das Glândulas Sebáceas/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Mesocricetus , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ratos Sprague-Dawley , Doenças das Glândulas Sebáceas/induzido quimicamente , Estearoil-CoA Dessaturase/metabolismoRESUMO
MGAT2 inhibition is a potential therapeutic approach for the treatment of metabolic disorders. High-throughput screening of the BMS internal compound collection identified the aryl dihydropyridinone compound 1 (hMGAT2 IC50 = 175 nM) as a hit. Compound 1 had moderate potency against human MGAT2, was inactive vs mouse MGAT2 and had poor microsomal metabolic stability. A novel chemistry route was developed to synthesize aryl dihydropyridinone analogs to explore structure-activity relationship around this hit, leading to the discovery of potent and selective MGAT2 inhibitors 21f, 21s, and 28e that are stable to liver microsomal metabolism. After triaging out 21f due to its inferior in vivo potency, pharmacokinetics, and structure-based liabilities and tetrazole 28e due to its inferior channel liability profile, 21s (BMS-963272) was selected as the clinical candidate following demonstration of on-target weight loss efficacy in the diet-induced obese mouse model and an acceptable safety and tolerability profile in multiple preclinical species.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Doenças Metabólicas/tratamento farmacológico , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Relação Estrutura-AtividadeRESUMO
The APJ receptor and its endogenous peptidic ligand apelin have been implicated as important modulators of cardiovascular function, and APJ receptor agonists may be beneficial in the treatment of heart failure. In this article, we describe the discovery of a series of biphenyl acid derivatives as potent APJ receptor agonists. Following the identification of initial high-throughput screen lead 2, successive optimization led to the discovery of lead compound 15a. Compound 15a demonstrated comparable in vitro potency to apelin-13, the endogenous peptidic ligand for the APJ receptor. In vivo, compound 15a demonstrated a dose-dependent improvement in the cardiac output in male Sprague Dawley rats with no significant changes in either mean arterial blood pressure or heart rate, consistent with the hemodynamic profile of apelin-13 in an acute pressure volume loop model.
Assuntos
Receptores de Apelina/agonistas , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Receptores de Apelina/química , Receptores de Apelina/metabolismo , Compostos de Bifenilo/química , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
A novel series of dihydro- and tetrahydrotriazolopyridazine-1,3-dione-based amino acid derivatives were identified as very potent motilin receptor agonists. Incorporating one additional phenylethyl glycinamide subunit to 1 (EC(50) = 660 nM) was found to improve in vitro potency approximately 3000-fold, resulting in compound 10 (EC(50) = 0.22 nM). The more potent enantiomer 11A has an EC(50) of 0.047 nM in the motilin receptor functional assay and a K(i) of 0.7 nM in the binding assay. In addition, compound 11A was shown to have a significantly reduced tendency to cause receptor desensitization as compared with the motilin receptor agonist ABT-229.
Assuntos
Motilina/agonistas , Piridazinas/síntese química , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores de Neuropeptídeos/agonistas , Triazóis/síntese química , Sinalização do Cálcio/efeitos dos fármacos , Células HeLa , Humanos , Piridazinas/química , Piridazinas/farmacologia , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Neuropeptídeos/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologiaRESUMO
Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.
Assuntos
Fibrinolíticos/síntese química , Compostos de Fenilureia/síntese química , Antagonistas do Receptor Purinérgico P2Y/síntese química , Piridinas/síntese química , Ureia/análogos & derivados , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/tratamento farmacológico , Tempo de Sangramento , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Células HEK293 , Humanos , Masculino , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/química , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Piridinas/química , Piridinas/farmacologia , Ratos , Relação Estrutura-Atividade , Trombose/sangue , Trombose/tratamento farmacológico , Ureia/síntese química , Ureia/química , Ureia/farmacologiaRESUMO
Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.