RESUMO
The synthesis and structure-activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded compounds with improved enzymatic and cellular potency.
Assuntos
Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Quinases/metabolismo , Antineoplásicos/síntese química , Benzodiazepinonas/síntese química , Linhagem Celular Tumoral/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Inibidores Enzimáticos/síntese química , Células HeLa , Humanos , Modelos Químicos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
A series of isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones were synthesized as potent inhibitors against Pim-1 and Pim-2 kinases. The structure-activity-relationship studies started from a high-throughput screening hit and was guided by molecular modeling of inhibitors in the active site of Pim-1 kinase. Installing a hydroxyl group on the benzene ring of the core has the potential to form a key hydrogen bond interaction to the hinge region of the binding pocket and thus resulted in the most potent inhibitor, 19, with K(i) values at 2.5 and 43.5 nM against Pim-1 and Pim-2, respectively. Compound 19 also exhibited an activity profile with a high degree of kinase selectivity.
Assuntos
Isoxazóis/síntese química , Isoxazóis/farmacologia , Modelos Moleculares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinolinas/síntese química , Quinolinas/farmacologia , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Isoxazóis/química , Conformação Molecular , Estrutura Molecular , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Based on the crystallographic analysis of a urea-checkpoint kinase 1 (Chk1) complex and molecular modeling, a class of macrocyclic Chk1 inhibitors were designed and their biological activities were evaluated. An efficient synthetic methodology for macrocyclic ureas was developed with Grubbs metathesis macrocyclization as the key step. The structure-activity relationship studies demonstrated that the macrocyclization retains full Chk1 inhibition activity and that the 4-position of the phenyl ring can tolerate a wide variety of substituents. These novel Chk1 inhibitors exhibit excellent selectivity over a panel of more than 70 kinases. Compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal Chk1 inhibitors, which showed little or no single-agent activity but significantly potentiate the cytotoxicities of the DNA-damaging antitumor agents doxorubicin and camptothecin. These novel Chk1 inhibitors abrogate the doxorubicin-induced G2 and camptothecin-induced S checkpoint arrests, confirming that their potent biological activities are mechanism-based through Chk1 inhibition.
Assuntos
Antineoplásicos/síntese química , Compostos Macrocíclicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/química , Ureia/análogos & derivados , Ureia/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Dano ao DNA , Doxorrubicina/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologiaRESUMO
A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray crystallography, medicinal chemistry efforts led to the identification of compound 46d, with potent enzymatic activity against Chk1 kinase. While maintaining a low cytotoxicity of its own, compound 46d exhibited a strong ability to abrogate G2 arrest and increased the cytotoxicity of camptothecin by 19-fold against SW620 cells. Pharmacokinetic studies revealed that it had a moderate bioavailabilty of 20% in mice. Two important binding interactions between compound 46b and Chk1 kinase, revealed by X-ray cocrystal structure, were hydrogen bonds between the hinge region and the amide bond of the core structure and a hydrogen bond between the methoxy group and Lys38 of the protein.
Assuntos
Antineoplásicos/síntese química , Azepinas/síntese química , Benzodiazepinonas/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Azepinas/química , Azepinas/farmacologia , Benzodiazepinonas/química , Benzodiazepinonas/farmacologia , Disponibilidade Biológica , Camptotecina/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Doxorrubicina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.
Assuntos
Alcinos/síntese química , Antineoplásicos/síntese química , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Indenos/síntese química , Pirazóis/síntese química , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tiofenos/síntese química , Alcinos/efeitos adversos , Alcinos/farmacologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Ligação Competitiva , Linhagem Celular , Canal de Potássio ERG1 , Edema/induzido quimicamente , Edema/tratamento farmacológico , Estradiol , Canais de Potássio Éter-A-Go-Go/fisiologia , Feminino , Humanos , Indenos/efeitos adversos , Indenos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Técnicas de Patch-Clamp , Ligação Proteica , Pirazóis/efeitos adversos , Pirazóis/metabolismo , Pirazóis/farmacologia , Ensaio Radioligante , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/metabolismo , Tiofenos/farmacologia , Doenças Uterinas/induzido quimicamente , Doenças Uterinas/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5-7, 10, 13, 14, 19-21, 25, 27, and 31-34. These compounds significantly sensitize tumor cells to the DNA-damaging antitumor agent doxorubicin in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and camptothecin-induced S checkpoints, indicating that the potent biological activities of these compounds are mechanism-based through Chk1 inhibition. Kinome profiling analysis of a representative macrocyclic urea 25 against a panel of 120 kinases indicates that these novel macrocyclic ureas are highly selective Chk1 inhibitors. Preliminary PK studies of 1a and b suggest that the 14-member macrocyclic inhibitors may possess better PK properties than their 15-member counterparts. An improved synthesis of 2 and 20 by using 2-(trimethylsilyl)ethoxycarbonyl (Teoc) to protect the amino group not only readily provided the desired compounds in pure form but also facilitated the scale up of potent compounds for various biological studies.
Assuntos
Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases , Ureia/síntese química , Ureia/farmacocinética , Animais , Catálise , Quinase 1 do Ponto de Checagem , Células HeLa , Humanos , Compostos Macrocíclicos/farmacologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/farmacocinética , Proteínas Quinases/fisiologia , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
A variety of macrocyclic urea compounds were prepared as potent Chk1 inhibitors by modifying the C5 position of the benzene ring of the macrocyclic urea with ether moieties, aliphatic carbon chains, amide and halides. Enzymatic activity less than 20nM was observed in 29 of 40 compounds. Compounds 14, 46d, and 48j provided the best overall results in the cellular assays as they abrogated doxorubicin-induced cell cycle arrest (IC(50)=3.31, 3.08, and 3.13microM) and enhanced doxorubicin cytotoxicity (IC(50)=0.54, 1.27, and 0.96microM) while displaying no single agent activity, respectively.
Assuntos
Compostos Macrocíclicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Ureia/síntese química , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Células HeLa , Humanos , Compostos Macrocíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus preserving their genomic integrity. The protein kinases checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and mitogen-activated protein kinase-activated protein kinase 2 (MK2) have all been shown to be involved in cell cycle checkpoint control. Recently, cell cycle checkpoint abrogation has been proposed as one way to sensitize cancer cells to DNA-damaging agents due to the expected induction of mitotic catastrophe. Due to their overlapping substrate spectra and redundant functions, it is still not clear which kinase is mainly responsible for the cell cycle arrests conferred by clinically relevant chemotherapeutics. Thus, the issue remains about which kinase is the most therapeutically relevant target and, more importantly, whether multiple kinases might need to be targeted to achieve the best efficacy in light of recent studies showing superior efficacy for pan-receptor tyrosine kinase inhibitors. To clarify this issue, we investigated the roles of the three kinases in response to different genotoxic stresses through small interfering RNA-mediated specific target knockdowns. Our result showed that only the down-regulation of Chk1, but not of Chk2 or MK2, abrogated camptothecin- or 5-fluorouracil-induced S-phase arrest or doxorubicin-induced G(2)-phase arrest. This was followed by mitotic catastrophe and apoptosis. Moreover, double inhibition of Chk1 and Chk2 failed to achieve better efficacy than Chk1 inhibition alone; surprisingly, inhibition of MK2, in addition to Chk1 suppression, partially reversed the checkpoint abrogation and negated mitotic catastrophe. We further showed that this is due to the fact that in MK2-deficient cells, Cdc25A protein, which is critically required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other checkpoint kinases in addition to Chk1 would be nonproductive.
Assuntos
Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Doxorrubicina/farmacologia , Feminino , Fluoruracila/farmacologia , Células HeLa , Histonas/efeitos dos fármacos , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias/tratamento farmacológico , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno , Células Tumorais Cultivadas , Fosfatases cdc25/efeitos dos fármacos , Fosfatases cdc25/metabolismoRESUMO
Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Dano ao DNA , Regulação para Baixo , Fluoruracila/toxicidade , Proteínas Quinases/fisiologia , Apoptose/fisiologia , Caspase 3 , Caspase 8 , Caspases/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , DNA/análise , DNA/efeitos dos fármacos , Replicação do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Células HeLa , Humanos , Fosforilação , Poli(ADP-Ribose) Polimerases/fisiologia , Proteínas Quinases/genética , RNA Interferente Pequeno/genéticaRESUMO
The discovery of 1-(5-chloro-2-alkoxyphenyl)-3-(5-cyanopyrazin-2-yl)ureas as a new class of potent (IC(50) values of 3-10 nM) and selective inhibitors of Chk1 kinase was described. One of these compounds (2e) potentiates HeLa cells by over 22-fold against doxorubicin in an antiproliferation assay, and SW620 cells against camptothecin by 20-fold in an antiproliferation assay and 14-fold in a soft agar assay. Flow cytometry (FACS) analysis confirmed that 2e abrogated G2 checkpoint arrest of H1299 cells caused by doxorubicin and S checkpoint arrest caused by camptothecin.
Assuntos
Antineoplásicos/síntese química , Nitrilas/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
Mammalian Chk1 is an essential kinase for embryonic development and plays an important role in the cellular response to DNA damage. However, it remains unclear whether inhibition of Chk1 induces apoptosis in somatic cells. The uncertainty has become a critical issue for rationale design of Chk1 mechanism-based anticancer drugs. Here we show that Chk1 small interfering RNA (siRNA) effectively eliminates Chk1 protein expression without altering the cell cycle profile or inducing apoptosis in various human cancer cell lines under normal conditions. In the presence of DNA-damaging agents, however, Chk1 siRNA alone is sufficient to abrogate the DNA damage-induced G(2) checkpoint and significantly enhance apoptosis. Cell cycle kinetic profiles show that abrogation of G(2) arrest is mediated through shortening of the checkpoint. We also demonstrate that Chk1 siRNA enhances DNA damage-induced apoptosis in p53-deficient cancer cell lines and augments the growth inhibition conferred by DNA-damaging agents. These findings imply that Chk1 inhibitors will have low cytotoxicity on their own and can enhance the efficacy of DNA-damaging drugs.
Assuntos
Apoptose , Proteínas Quinases/fisiologia , Western Blotting , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Fase G2 , Células HeLa , Humanos , Cinética , Proteínas Quinases/biossíntese , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with subnanomolar to low single-digit nanomolar K(i) values and exhibit excellent selectivity against a panel of diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b (PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells with submicromolar EC(50) values. For example, compound 14j inhibited the growth of K562 cells with an EC(50) value of 1.7 muM and showed K(i) values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3, respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based. The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes, good permeability, modest protein binding, and no CYP inhibition below 20 muM concentration.
Assuntos
Antineoplásicos/síntese química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirimidinas/síntese química , Pirimidinonas/síntese química , Tiofenos/síntese química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Humanos , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteínas Proto-Oncogênicas c-pim-1/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinonas/farmacocinética , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Tiofenos/farmacocinética , Tiofenos/farmacologia , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ciclina B/metabolismo , Terapia Genética/métodos , Neoplasias/terapia , Proteínas Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Inibidores da Topoisomerase , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Ciclina B1 , DNA Topoisomerases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Feminino , Células HeLa , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Mitose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Quinases/genética , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report the synthesis and biological evaluation of 5-substituted 1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted kinase inhibitors. Initial efforts focused on the development of selective KDR inhibitors, while later strategies involved the improvement of potency toward multiple kinase targets. Thus, several compounds were identified as potent KDR, Flt1, Flt3, and c-Kit inhibitors.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Pirazóis/síntese químicaRESUMO
A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5''-ethyl-pyridin-2''-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/farmacologia , Quinase 1 do Ponto de Checagem , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Inibidores de Proteínas Quinases/química , Pirazóis/químicaRESUMO
A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (<10nM) against CHK-1. X-ray co-crystal structures of bound inhibitors demonstrated that two sub-series of this class of compounds, exemplified by 21 and 41, exhibit distinctive hydrogen bonding patterns in the specificity pocket of the active site. Two compounds, 41 and 43, were capable of potentiating doxorubicin and camptothecin, both DNA-damaging agents, in cell proliferation assays (MTS and soft agar assays) and abrogating G2/M checkpoint in a mechanism-based FACS assay.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Células HeLa , Humanos , Ligação de Hidrogênio , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Especificidade por SubstratoRESUMO
An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.
Assuntos
Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Nitrilas/química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.
Assuntos
Antineoplásicos/farmacocinética , Química Farmacêutica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/química , Animais , Antineoplásicos/química , Células CACO-2 , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desenho de Fármacos , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Camundongos , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Quinases/metabolismo , RatosRESUMO
A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.
Assuntos
Hidrogênio/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Administração Oral , Animais , Quinase 1 do Ponto de Checagem , Cianetos/química , Indenos/química , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Pirazóis/administração & dosagem , Pirazóis/síntese química , Ratos , Relação Estrutura-AtividadeRESUMO
Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R4, and to a lesser extent, R2, and R5 of the phenyl ring, and made a variety of N-aryl-N'-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds. Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest (IC50=1.7 microM) and enhanced doxorubicin cytotoxicity (IC50=0.44 microM) while displaying no single agent activity.