RESUMO
Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Assuntos
Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridonas/síntese química , Quinolinas/síntese química , Triazóis/síntese química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridonas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologiaRESUMO
mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.
Assuntos
Inibidores de Proteínas Quinases/química , Piridazinas/química , Piridinas/química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Benzimidazóis/química , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Imidazóis/química , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Estrutura Terciária de Proteína , Piridazinas/síntese química , Piridazinas/farmacocinética , Piridinas/síntese química , Piridinas/farmacocinética , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.
Assuntos
Naftiridinas/metabolismo , Piridazinas/metabolismo , Tiazóis/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Glutationa/química , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Microssomos Hepáticos/metabolismo , Conformação Molecular , Naftiridinas/química , Naftiridinas/farmacocinética , Naftiridinas/toxicidade , Ligação Proteica , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/toxicidade , Ratos , Fatores de Risco , Espectrometria de Massas por Ionização por Electrospray , Tiazóis/química , Tiazóis/toxicidadeRESUMO
Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.
Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/fisiologia , Neovascularização Fisiológica/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Sobrevivência Celular , Humanos , Camundongos , Camundongos Nus , Fosforilação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.
Assuntos
Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridazinas/síntese química , Triazóis/síntese química , Animais , Cristalografia por Raios X , Fator de Crescimento de Hepatócito/fisiologia , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/farmacologia , Ratos , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.
Assuntos
Antineoplásicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Naftalenos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/sangue , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure-activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure-activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.
Assuntos
Antineoplásicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Naftalenos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/sangue , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
1,2,3,4-Tetrahydroisoquinolines and 3,4-dihydroisoquinoline-1(2H)-ones were identified as potent and selective inhibitors of KDR. The discovery, synthesis, and structure-activity relationships of these novel inhibitors are reported. In vitro metabolism and pharmacokinetic profiles of the most interesting compounds are discussed.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/isolamento & purificação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Química Farmacêutica/métodos , Desenho de Fármacos , Endotélio Vascular/citologia , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Masculino , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Amplificação de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Necrose , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridonas/síntese química , Piridonas/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Animais , Antineoplásicos/farmacocinética , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Humanos , Camundongos , Modelos Moleculares , Piridonas/farmacocinética , Relação Estrutura-Atividade , Triazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Assuntos
Descoberta de Drogas , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Quinolinas/farmacologia , Triazóis/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/química , Quinolinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Triazóis/química , Triazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In acute myelogenous leukemia (AML), the FLT3 receptor tyrosine kinase (RTK) is highly expressed with 30% of patients expressing a mutated, constitutively active form of this protein. To inhibit this receptor, VX-322 was developed and found to be very potent against both the FLT3 and c-KIT RTKs with enzyme K(i) values of <1 nM and a cellular IC(50) between 1 and 5 nM. It was efficacious in a FLT3-ITD dependent myeloproliferative mouse model, doubling survival compared to other FLT3 inhibitors, with 25% of the mice cured. Upon treatment of primary AML patient blast cells, the dual inhibition of FLT3 and c-KIT was superior to inhibitors targeting a single RTK. Thus, this compound may represent an improved pharmacologic and selectivity profile that could be effective in the treatment of AML.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Triazóis/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Soro , Células Tumorais CultivadasRESUMO
A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Administração Oral , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Disponibilidade Biológica , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacocinética , Imidazóis/farmacologia , Concentração Inibidora 50 , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Especificidade por SubstratoRESUMO
A high-throughput screen of our compound archive revealed a novel class of dual FMS-like tyrosine kinase 3 (FLT3)/c-KIT inhibitors. With the help of molecular modeling, this class was rapidly optimized for both potency against FLT3 and FLT3/c-KIT and excellent potency in cell-based assays, leading to dose-dependent cell death in acute myelogenous leukemia (AML) patient blast samples. Ultimately, the AML patient blast data defined the preferred target profile as we designed and evaluated a set of FLT3 selective and FLT3/c-KIT dual molecules. Further optimization for pharmacokinetic properties resulted in the selection of the dual FLT3/c-KIT inhibitor, N(3)-(4-(trans-4-morpholinocyclohexyl)phenyl)-1-(pyridin-2-yl)-1H-1,2,4-triazole-3,5-diamine, VX-322 (compound 37), to move forward to preclinical evaluation.