ABSTRACT
A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.
Subject(s)
Cicatrix/prevention & control , Drug Discovery , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/chemistry , Pyridines/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Skin/drug effects , Animals , Models, Molecular , Phosphorylation , Rats , Receptor, Transforming Growth Factor-beta Type IABSTRACT
We explored the D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Francisella tularensis, a highly infectious gram-negative pathogen that has raised concern as a potential bioweapon, as a target for the development of novel chemotherapeutics. F. tularensis KdsD was expressed in Escherichia coli from a synthetic gene, purified, and characterized. A group of hydroxamates designed to be mimics of the putative enediol intermediate in the enzyme's catalytic mechanism were prepared and tested as inhibitors of F. tularensis KdsD. The best inhibitor, which has an IC(50) of 7 µM, is the most potent KdsD inhibitor reported to date.
Subject(s)
Alcohols/pharmacology , Aldose-Ketose Isomerases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Francisella tularensis/drug effects , Alcohols/chemical synthesis , Alcohols/chemistry , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Alkenes/chemical synthesis , Alkenes/chemistry , Alkenes/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Francisella tularensis/enzymology , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Inhibitory Concentration 50 , Molecular StructureABSTRACT
Inhibition of the cell cycle kinase, cyclin-dependent kinase-4 (Cdk4), is expected to provide an effective method for the treatment of proliferative diseases such as cancer. The pyrido[2,3-d]pyrimidin-7-one template has been identified previously as a privileged structure for the inhibition of ATP-dependent kinases, and good potency against Cdks has been reported for representative examples. Obtaining selectivity for individual Cdk enzymes, particularly Cdk4, has been challenging. Here, we report that the introduction of a methyl substituent at the C-5 position of the pyrido[2,3-d]pyrimidin-7-one template is sufficient to confer excellent selectivity for Cdk4 vs other Cdks and representative tyrosine kinases. Further optimization led to the identification of highly potent and selective inhibitors of Cdk4 that exhibit potent antiproliferative activity against human tumor cells in vitro. The most selective Cdk4 inhibitors were evaluated for antitumor activity against MDA-MB-435 human breast carcinoma xenografts in mice.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases/chemistry , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Models, Molecular , Proto-Oncogene Proteins/chemistry , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Stereoisomerism , Transplantation, HeterologousABSTRACT
A pharmacological approach to inhibition of cyclin-dependent kinases 4 and 6 (Cdk4/6) using highly selective small molecule inhibitors has the potential to provide novel cancer therapies for clinical use. Achieving high levels of selectivity for Cdk4/6, versus other ATP-dependent kinases, presents a significant challenge. The pyrido[2,3-d]pyrimidin-7-one template provides an effective platform for the inhibition of a broad cross-section of kinases, including Cdks. It is now demonstrated that the modification of pyrido[2,3-d]pyrimidin-7-ones to include a 2-aminopyridine side chain at the C2-position provides inhibitors with exquisite selectivity for Cdk4/6 in vitro. This selectivity profile is recapitulated in cells where the most selective inhibitors create a G(1) block at concentrations up to 100-fold the IC(50) for cell proliferation. On the basis of its selectivity profile and pharmacokinetic profile, compound 43 (PD 0332991) was identified as a drug candidate for the treatment of cancer.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinases/antagonists & inhibitors , Piperazines/chemical synthesis , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , G1 Phase/drug effects , Humans , Male , Piperazines/chemistry , Piperazines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thymidine/metabolismABSTRACT
PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 micromol/L) and Cdk6 (IC50, 0.016 micromol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridines/pharmacology , Xenograft Model Antitumor Assays , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Cyclin-Dependent Kinases/metabolism , Disease Models, Animal , Down-Regulation/drug effects , G1 Phase/drug effects , Humans , Mice , Molecular Structure , Neoplasm Transplantation/pathology , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation/drug effects , Piperazines/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/metabolism , Pyridines/chemistry , Retinoblastoma Protein/metabolism , Substrate SpecificityABSTRACT
An array of pharmaceutical compounds and impurities were used to investigate the applicability of atmospheric pressure ionization mass spectrometry (MS) to routinely detect coeluting impurities in HPLC (i.e. peak purity). Four drugs were individually tested against their related impurity set using a straightforward HPLC-MS peak purity strategy. For the investigated set, which represents 24 unique drug-impurity permutations, 75% of the coeluting impurities were detected at levels <1.0%, including one-third at 0.1% (%, w/w). Factors that affect the applicability of this peak purity approach are also discussed.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drug Contamination , Mass Spectrometry/methods , Aminosalicylic Acids/isolation & purification , Atmospheric Pressure , Digitoxin/isolation & purification , Digoxin/isolation & purification , Sensitivity and SpecificityABSTRACT
A simple, sensitive, and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method in the negative-ion electrospray ionization (ESI(-)) mode was validated for the quantitation of bromate (BrO(3)(-)) in potato snacks. Ground snack specimens ( approximately 0.5 g/sample) are spiked with Br(18)O(3)(-), stable-isotope labeled bromate internal standard (IS), and vortexed with a mixture of distilled/deionized water (dd water) and heptane. Subsequently, the specimens are centrifuged, and a small portion of the aqueous extract is isolated, diluted with dd water (1:4), and analyzed by HPLC-MS/MS. The methodology has a quantitation range of 10-1000 ppb, an accuracy of 1.5-7.5%, and a precision of 5.2-13.4% across the concentration range.
Subject(s)
Bromates/analysis , Chromatography, High Pressure Liquid/methods , Food Additives/analysis , Solanum tuberosum/chemistry , Tandem Mass Spectrometry/methods , Food AnalysisABSTRACT
The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.