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
Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.
ABSTRACT
BACKGROUND: The ATP-competitive p38α MAPK inhibitor VX-745 exhibits an exquisite kinase selectivity profile, is effective in blocking p38 stress signaling in Werner syndrome dermal fibroblasts, has efficacy in clinical trials and may have therapeutic value against Werner syndrome. Previous synthetic routes, however, have only resulted in milligram quantities suitable for cell-based studies, whereas gram quantities would be required for in vivo use. RESULTS & DISCUSSION: Microwave irradiation using a stop-flow monomodal microwave reactor has been found to facilitate scale-up of the synthesis of VX-745. Ullmann-type C-S bond formation using thiophenol, chloropyridazine, copper(I) catalyst and diol ligand proceeds rapidly and efficiently in this apparatus for elaboration to the pyrimido[1,6-b]pyridazinone core of VX-745 on gram scale and with good overall yield. CONCLUSION: This method delivers the p38 inhibitor VX-745 in sufficient quantities for preclinical studies to rescue the aging phenotype in Werner syndrome.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Pyridazines/pharmacology , Pyrimidines/pharmacology , Werner Syndrome/drug therapy , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Humans , Magnetic Resonance Spectroscopy , Protein Kinase Inhibitors/therapeutic use , Pyridazines/therapeutic use , Pyrimidines/therapeutic use , Spectrometry, Mass, Electrospray IonizationABSTRACT
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.
Subject(s)
Microwaves , Protein Kinase Inhibitors/chemical synthesis , Pyrazoles/chemical synthesis , Small Molecule Libraries/chemical synthesis , p38 Mitogen-Activated Protein Kinases , Cell Line , Cell Line, Tumor , Humans , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Small Molecule Libraries/pharmacology , Werner Syndrome/pathology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Microwave irradiation promotes the rapid and efficient reaction of a thiophenol and aryl or heteroaryl halide using a copper or palladium catalyst and a range of ligands, depending upon substrate. Of particular utility is the use of copper(I) iodide (5 mol %) and trans-cyclohexane-1,2-diol as ligand under basic conditions and microwave irradiation to give the corresponding sulfide in high yield. This method for C-S bond formation is applied in the four-step synthesis of the clinical candidate VX-745 in 38% overall yield. The inhibitory activity of VX-745 against p38alpha MAPK is confirmed in Werner syndrome dermal fibroblasts at 1.0 microM concentration by immunoblot assay.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Pyridazines/chemical synthesis , Pyrimidines/chemical synthesis , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Protein Kinase Inhibitors/pharmacology , Pyridazines/pharmacology , Pyrimidines/pharmacology , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, InfraredABSTRACT
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.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Werner Syndrome/enzymology , Cell Line , Humans , Molecular Structure , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Substrate SpecificityABSTRACT
The p38 mitogen-activated protein kinase inhibitor VX-745 is prepared rapidly and efficiently in a four-step sequence using a combination of conductive heating and microwave-mediated steps. Its inhibitory activity was confirmed in hTERT immortalized HCA2 and WS dermal fibroblasts at 0.5-1.0 microM concentration by ELISA and immunoblot assay, and displays excellent kinase selectivity over the related stress-activated kinase JNK.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Pyridazines/chemical synthesis , Pyrimidines/chemical synthesis , Werner Syndrome/enzymology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Pyridazines/pharmacology , Pyrimidines/pharmacology , Werner Syndrome/pathologyABSTRACT
Microwave irradiation of substituted hydrazines and beta-ketoesters gives 5-aminopyrazoles in excellent yield, which can be transformed to the corresponding N-carbonyl derivatives by treatment with an isocyanate or chloroformate. Derivatization of 4-nitronaphth-1-ol using predominantly microwave heating methods and reaction with an N-pyrazole carbamate provides a rapid route to the N-pyrazole urea BIRB 796 in high purity, as a potent and selective inhibitor of p38alpha mitogen-activated protein kinase for the study of accelerated ageing in Werner syndrome cells.