RESUMO
We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.
Assuntos
Imidazóis/farmacologia , Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Imidazóis/síntese química , Imidazóis/química , Janus Quinase 1/metabolismo , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-AtividadeRESUMO
The discovery of the activating mutation V617F in the JH2 domain of Jak2 and the modulation of oncogenic Stat3 by Jak2 inhibitors have spurred a great interest in the inhibition of the Jak2/Stat pathway in oncology. In this Letter, we communicate the discovery of novel inhibitors of the Jak2/Stat5 axis, the N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives. The rationale, synthesis and biological evaluation of these derivatives are reported. Two lead analogs from this series, 6 and 9, displayed prolonged residence time on Jak2, at enzymatic level. Although 6 and 9 exhibited moderate selectivity in a selected kinase panel, we chose to test these inhibitors in vivo as a consequence to their long residence time. However, extended inhibition of Jak2 due to the long residence time, in the form of inhibiting phosphorylation of downstream Stat5, was not recapitulated in an in vivo setting.
Assuntos
Descoberta de Drogas , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Animais , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Conformação Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Wistar , Fator de Transcrição STAT5/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de TempoRESUMO
Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Nitrilas/síntese química , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Piridinas/química , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Nitrilas/química , Nitrilas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Thiazol-2-yl amine was identified as an isosteric replacement for pyrazol-3-yl amine during our efforts to identify potent and selective JAK2 inhibitors. The rationale, synthesis and biological evaluation of several analogs is reported, along with the in vivo evaluation of the lead compounds.
Assuntos
Aminas/síntese química , Antineoplásicos/síntese química , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Pirazóis/química , Tiazóis/química , Aminas/química , Aminas/farmacocinética , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Descoberta de Drogas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Nus , Microssomos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.
Assuntos
Fármacos Anti-HIV/química , Inibidores Enzimáticos/química , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Tiocarbamatos/química , Triazóis/química , Sequência de Aminoácidos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , Dados de Sequência Molecular , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiocarbamatos/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.
Assuntos
Indóis/uso terapêutico , Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Linhagem Celular Tumoral , Desenho de Fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Indóis/síntese química , Indóis/farmacocinética , Camundongos Nus , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The design, synthesis and biological evaluation of a series of pyrazol-3-ylamino pyrazines as potent and selective JAK2 kinase inhibitors is reported, along with the pharmacokinetic and pharmacodynamic properties of lead compounds.
Assuntos
Descoberta de Drogas , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Pirazóis/farmacologia , Animais , Cães , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazinas/síntese química , Pirazinas/química , Pirazóis/síntese química , Pirazóis/química , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The bisamide class of kinase inhibitors was identified as being active against CSF-1R. The synthesis and SAR of pyridyl and thiazolyl bisamides are reported, along with the pharmacokinetic properties and in vivo activity of selected examples.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Tiazóis/farmacologia , Células 3T3 , Amidas/química , Amidas/farmacocinética , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Feminino , Humanos , Camundongos , Camundongos Nus , Ratos , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Tiazóis/química , Tiazóis/farmacocinéticaRESUMO
Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays â¼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).
Assuntos
Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Through high throughput screening of various libraries, substituted styryl naphthalene 6 was identified as an HCMV protease inhibitor. Optimization of various regions of the lead molecule using parallel synthesis resulted in 1,6-substituted naphthalenes 19d-i. These compounds displayed good potency and were selective over elastase, trypsin, and chymotrypsin. The optimization approach on lead compound 6 in three different regions of the molecule using parallel solution-phase synthesis and the corresponding SAR are discussed in detail.
Assuntos
2-Naftilamina/síntese química , Citomegalovirus/química , Naftalenos/síntese química , Inibidores de Proteases/síntese química , Serina Endopeptidases/química , Sulfonamidas/síntese química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Bases de Dados Factuais , Naftalenos/química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Structure based design, synthesis, and biological evaluation of a novel series of 1-methyl-1H-imidazole, as potent Jak2 inhibitors to modulate the Jak/STAT pathway, are described. Using the C-ring fragment from our first clinical candidate AZD1480 (24), optimization of the series led to the discovery of compound 19a, a potent, orally bioavailable Jak2 inhibitor. Compound 19a displayed a high level of cellular activity in hematopoietic cell lines harboring the V617F mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a demonstrated significant tumor growth inhibition in a UKE-1 xenograft model within a well-tolerated dose range.
Assuntos
Antineoplásicos/síntese química , Imidazóis/síntese química , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Animais , Antineoplásicos/farmacologia , Cães , Descoberta de Drogas , Humanos , Imidazóis/farmacologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are a heterogeneous but related group of hematological malignancies characterized by clonal expansion of one or more myeloid lineages. The discovery of the Jak2 V617F gain of function mutation highlighted Jak2 as a potential therapeutic target in the MPNs. Herein, we disclose the discovery of a series of pyrazol-3-yl pyrimidin-4-amines and the identification of 9e (AZD1480) as a potent Jak2 inhibitor. 9e inhibits signaling and proliferation of Jak2 V617F cell lines in vitro, demonstrates in vivo efficacy in a TEL-Jak2 model, has excellent physical properties and preclinical pharmacokinetics, and is currently being evaluated in Phase I clinical trials.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Pirazóis/síntese química , Pirimidinas/síntese química , Fatores de Transcrição STAT/fisiologia , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Feminino , Humanos , Técnicas In Vitro , Janus Quinase 2/química , Camundongos , Camundongos Nus , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/metabolismoRESUMO
The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.
Assuntos
Aminopiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Janus Quinase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazóis/farmacologia , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/metabolismo , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Fenótipo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.
Assuntos
Citomegalovirus/enzimologia , Serina Endopeptidases/química , Soluções Tampão , Catálise , Citomegalovirus/genética , Dimerização , Ditiotreitol/química , Ácido Edético/química , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Humanos , Cinética , Morfolinas/química , Mutagênese Sítio-Dirigida , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Cloreto de Sódio/química , Especificidade por Substrato/genética , Sulfatos/química , Propriedades de Superfície , TitulometriaRESUMO
The synthesis and biological activity of pyrimidotetrazin-6-ones against HCMV protease is described. The mechanism of action for these inhibitors is the oxidation of several cysteine residues to generate cross-linked enzyme.