ABSTRACT
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.
Subject(s)
Ketones/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Small Molecule Libraries/pharmacology , Werner Syndrome/pathology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/pathology , Humans , Ketones/chemical synthesis , Ketones/chemistry , Microwaves , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Werner Syndrome/drug therapy , Werner Syndrome/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Floxuridine/analogs & derivatives , Floxuridine/pharmacology , Organophosphorus Compounds/pharmacology , Prodrugs/pharmacology , Thymidine Kinase/physiology , Animals , Cell Line, Tumor , Enzyme Activation , Floxuridine/metabolism , Humans , Leukemia L1210 , Mice , Phosphorylation , Prodrugs/metabolism , Thymidine Kinase/antagonists & inhibitors , Thymidine Phosphorylase/physiology , Uridine Phosphorylase/physiologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Ketones/chemistry , Microwaves , Pyrazoles/chemical synthesis , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Line , Chemistry, Pharmaceutical/methods , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , MAP Kinase Kinase 4/metabolism , Models, Chemical , Naphthalenes/chemistry , Nitriles/chemistry , Protein Isoforms , Pyrazoles/chemistry , Pyrazoles/pharmacologyABSTRACT
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.
