RESUMO
Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.
Assuntos
Cetonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Síndrome de Werner/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Cetonas/síntese química , Cetonas/química , Micro-Ondas , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Síndrome de Werner/tratamento farmacológico , Síndrome de Werner/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The fluorinated pyrimidine family of nucleosides continues to represent major current chemotherapeutic agents for treating solid tumors. We herein report their phosphate prodrugs, ProTides, as promising new derivatives, which partially bypass the dependence of the current drugs on active transport and nucleoside kinase-mediated activation. They are also resistant to metabolic deactivation by phosphorolytic enzymes. We report 39 ProTides of the fluorinated pyrimidine FUDR with variation in the aryl, ester, and amino acid regions. Notably, only certain ProTide motifs are successful in delivering the nucleoside monophosphate into intact cells. We also find that the ProTides retain activity in mycoplasma infected cells, unlike FUDR. Data suggest these compounds to be worthy of further progression.
Assuntos
Antineoplásicos/síntese química , Floxuridina/análogos & derivados , Floxuridina/síntese química , Compostos Organofosforados/síntese química , Pró-Fármacos/síntese química , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Transportador Equilibrativo 1 de Nucleosídeo/genética , Floxuridina/farmacologia , Humanos , Mycoplasma hyorhinis/enzimologia , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Pirimidina Fosforilases/metabolismo , Relação Estrutura-AtividadeRESUMO
A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.
Assuntos
Antineoplásicos/farmacologia , Floxuridina/análogos & derivados , Floxuridina/farmacologia , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Timidina Quinase/fisiologia , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Floxuridina/metabolismo , Humanos , Leucemia L1210 , Camundongos , Fosforilação , Pró-Fármacos/metabolismo , Timidina Quinase/antagonistas & inibidores , Timidina Fosforilase/fisiologia , Uridina Fosforilase/fisiologiaRESUMO
BACKGROUND: The pyrazolyl ketone motif of RO3201195, which exhibits good oral bioavailability and high selectivity for p38 MAPK over other kinases, is a key pharmacophore that could find application in the treatment of Werner syndrome. RESULTS AND DISCUSSION: Microwave irradiation promotes Knoevenagel condensation of a ß-ketonitrile and formamidine, to give ß-aminovinyl ketones, and their subsequent cyclocondensation with a subset of hydrazines to provide rapid access to a 24-membered library of pyrazolyl ketones. The library was evaluated in human hTERT-immortalized HCA2 dermal cells and Werner syndrome cells. CONCLUSION: Four compounds display comparable, if not slightly improved, potency over RO3201195.
Assuntos
Micro-Ondas , Inibidores de Proteínas Quinases/síntese química , Pirazóis/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Síndrome de Werner/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Werner syndrome provides a convincing model for aspects of the normal ageing phenotype and may provide a suitable model for therapeutic interventions designed to combat the ageing process. Cultured primary fibroblast cells from Werner syndrome patients provide a powerful model system to study the link between replicative senescence in vitro and in vivo pathophysiology. Genome instability, together with an increased pro-oxidant state, and frequent replication fork stalling, all provide plausible triggers for intracellular stress in Werner syndrome cells, and implicates p38 MAPK signaling in their shortened replicative lifespan. A number of different p38 MAPK inhibitor chemotypes have been prepared rapidly and efficiently using microwave heating techniques for biological study in Werner syndrome cells, including SB203580, VX-745, RO3201195, UR-13756 and BIRB 796, and their selectivity and potency evaluated in this cellular context. Werner syndrome fibroblasts treated with a p38 MAPK inhibitor reveal an unexpected reversal of the accelerated ageing phenotype. Thus the study of p38 inhibition and its effect upon Werner pathophysiology is likely to provide new revelations into the biological mechanisms operating in cellular senescence and human ageing in the future.
RESUMO
5-Aminopyrazol-4-yl ketones are prepared rapidly and efficiently using microwave dielectric heating from beta-ketonitriles by treatment with N,N'-diphenylformamidine followed by heterocyclocondensation by irradiation with a hydrazine. The inhibitory activity of RO3201195 prepared by this methodology was confirmed in hTERT-immortalized HCA2 and WS dermal fibroblasts at 200nM concentration, both by ELISA and immunoblot assay, and displays excellent kinase selectivity for p38alpha MAPK over the related stress-activated kinase JNK.
Assuntos
Inibidores Enzimáticos/síntese química , Cetonas/química , Micro-Ondas , Pirazóis/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , Química Farmacêutica/métodos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , MAP Quinase Quinase 4/metabolismo , Modelos Químicos , Naftalenos/química , Nitrilas/química , Isoformas de Proteínas , Pirazóis/química , Pirazóis/farmacologiaRESUMO
A benzopyranopyridine inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2) is prepared rapidly and efficiently in one step using microwave dielectric heating, whereas a substrate-selective p38 MAPK inhibitor was prepared using conventional heating techniques. The former had MK2 inhibitory activity above 2.5 microM concentration, whereas the latter showed no MK2 inhibition at 10 microM. However, rather than rescuing the reduced cellular growth rate and aged morphology of hTERT-immortalised WS dermal fibroblasts, both induce a state resembling stress-induced cellular senescence, suggesting that these inhibitors may have limited therapeutic use.