RESUMO
PURPOSE: Aurora kinases are required for orderly progression of cells through mitosis, and inhibition of these kinases by siRNA or small molecule inhibitors results in cell death. We previously reported the synthesis of SCH 1473759, a novel sub-nanomolar Aurora A/B inhibitor. METHODS: We utilized SCH 1473759 and a panel of tumor cell lines and xenograft models to gain knowledge about optimal dosing schedule and chemotherapeutic combinations for Aurora A/B inhibitors. RESULTS: SCH 1473759 was active against a large panel of tumor cell lines from different tissue origin and genetic backgrounds. Asynchronous cells required 24-h exposure to SCH 1473759 for maximal induction of >4 N DNA content and inhibition of cell growth. However, following taxane- or KSP inhibitor-induced mitotic arrest, less than 4-h exposure induced >4 N DNA content. This finding correlated with the ability of SCH 1473759 to accelerate exit from mitosis in response to taxane- and KSP inhibitor-induced arrest. We tested various dosing schedules in vivo and demonstrated SCH 1473759 dose- and schedule-dependent anti-tumor activity in four human tumor xenograft models. Further, the efficacy was enhanced in combination with taxanes and found to be most efficacious when SCH 1473759 was dosed 12-h post-taxane treatment. CONCLUSIONS: SCH 1473759 demonstrated potent mechanism-based activity, and activity was shown to be enhanced in combination with taxanes and KSP inhibitors. This information may be useful for optimizing the clinical efficacy of Aurora inhibitors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imidazóis/farmacologia , Cinesinas/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Aurora Quinase A , Aurora Quinases , Linhagem Celular Tumoral , Esquema de Medicação , Feminino , Humanos , Imidazóis/administração & dosagem , Masculino , Camundongos , Camundongos Nus , Neoplasias/patologia , Pirazinas/administração & dosagem , Taxoides/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Óxidos N-Cíclicos , Quinases Ciclina-Dependentes/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Histonas/metabolismo , Humanos , Immunoblotting , Indolizinas , Camundongos , Camundongos Nus , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/química , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/química , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Aurora kinases are cell cycle regulated serine/threonine kinases that have been linked to cancer. Compound 1 was identified as a potent Aurora inhibitor but lacked oral bioavailability. Optimization of 1 led to the discovery of a series of fluoroamine and deuterated analogues, exemplified by compound 25, with an improved pharmacokinetic profile. We found that blocking oxidative metabolism at the benzylic position and decreasing the basicity of the amine are important to obtaining compounds with good biological profiles and oral bioavailability.
Assuntos
Antineoplásicos/síntese química , Flúor , Imidazóis/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/síntese química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Aurora Quinases , Disponibilidade Biológica , Linhagem Celular Tumoral , Deutério , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Histonas/metabolismo , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Macaca fascicularis , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Pirazinas/farmacocinética , Pirazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Benzopiranos/administração & dosagem , Benzopiranos/farmacologia , Cinesinas/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Tiadiazóis/administração & dosagem , Tiadiazóis/farmacologia , Administração Oral , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HCT116 , Haplorrinos , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Ratos , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
We report a series of potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors. Optimization of the solvent accessible 8-position led to improvements in both oral bioavailability and off-target kinase inhibition. Compound 25 demonstrates anti-tumor activity in an A2780 ovarian tumor xenograft model.
Assuntos
Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/farmacologia , Administração Oral , Animais , Aurora Quinases , Disponibilidade Biológica , Linhagem Celular Tumoral , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacocinética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/química , Pirazinas/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 µM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.
RESUMO
Several fast high performance liquid chromatography/atmospheric pressure ionization/tandem mass spectrometric (HPLC-API/MS/MS) methods were evaluated for the simultaneous determination of cladribine and clofarabine in mouse plasma samples. The chemical separation for analytes under reversed-phase conditions were achieved by using either ultra-performance liquid chromatography (UPLC) or micro-column HPLC coupled to either a quadrupole linear ion trap mass spectrometer (QTrap MS) or a triple quadrupole mass spectrometer. Atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) interfaces in the positive mode were employed prior to mass spectrometric detection. The effects of various dopant solvents on the APPI sensitivities of analytes and the internal standard were investigated. The matrix ionization suppression potential for the test compounds in plasma samples on fast HPLC-MS/MS methods was examined by a post-column infusion technique. In this work, these proposed approaches were successfully employed to determine the concentrations of cladribine and clofarabine in mouse plasma in the low ng/ml region. The mouse plasma levels of all analytes obtained by these fast HPLC-MS/MS methods were compared and found to be well correlated in terms of analytical accuracy.