ABSTRACT
A series of novel, potent 4-aminothienopyridine B-Raf kinase inhibitors was designed and synthesized using knowledge-based design. Compounds 5f, and 6k exhibited not only excellent potency in both enzyme assay (IC(50)=5.1, 16.6 nM) and cellular assay (IC(50)=0.2, 0.2 µM), but also had an outstanding selectivity profile against other kinases.
Subject(s)
Aminopyridines/chemistry , Phenylurea Compounds/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Thienopyridines/chemistry , Aminopyridines/chemical synthesis , Aminopyridines/metabolism , Drug Design , Drug Evaluation, Preclinical , Phenylurea Compounds/chemical synthesis , Phenylurea Compounds/metabolism , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity Relationship , Thienopyridines/chemical synthesis , Thienopyridines/metabolismABSTRACT
A potent series of inhibitors against the B-Raf(V600E) kinase have been developed that show excellent activity in cellular assays and good oral bioavailability in rats. The key structural features of the series are an arylsulfonamide headgroup, a thiazole core, and a fluorine ortho to the sulfonamide nitrogen.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sulfonamides/chemistry , Administration, Oral , Amino Acid Substitution , Animals , Binding Sites , Catalytic Domain , Fluorine/chemistry , Microsomes, Liver/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Structure-Activity RelationshipABSTRACT
Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.
Subject(s)
Alkynes/metabolism , Aniline Compounds/metabolism , ErbB Receptors/metabolism , Models, Molecular , Pyrimidines/metabolism , Animals , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Female , Isatin/analogs & derivatives , Isatin/metabolism , Mass Spectrometry , Mice , Mice, SCID , Molecular Structure , Pyrimidines/toxicity , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhibitors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent pharmacokinetic properties.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Imides/chemistry , Serine/chemistry , Threonine/chemistry , ortho-Aminobenzoates/chemistry , Animals , Aryl Hydrocarbon Hydroxylases/chemistry , Crystallography, X-Ray , Cytochrome P-450 CYP2C9 , Drug Design , Glycine/chemistry , Glycogen Phosphorylase/metabolism , Humans , Inhibitory Concentration 50 , Liver/enzymology , Models, Chemical , RatsABSTRACT
Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Carboxylic Acids/chemistry , Chemistry, Pharmaceutical/methods , Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Imides/pharmacology , ortho-Aminobenzoates/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/chemistry , Crystallography, X-Ray , Cytochrome P-450 CYP2C9 , Dogs , Drug Design , Glycine/chemistry , Glycogen Phosphorylase/metabolism , Humans , Imides/chemistry , Inhibitory Concentration 50 , Liver/enzymology , Molecular Conformation , Rats , ortho-Aminobenzoates/chemistryABSTRACT
Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC(50) values less than 1 microM against human tumor cells in vitro.
Subject(s)
Antineoplastic Agents/pharmacology , Chemistry, Pharmaceutical/methods , ErbB Receptors/chemistry , Pyrimidines/chemistry , Receptor, ErbB-2/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Lapatinib , Models, Chemical , Molecular Conformation , Quinazolines/pharmacologyABSTRACT
Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compounds containing the reference aniline.
Subject(s)
Aniline Compounds/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyrimidines/chemical synthesis , Receptor, ErbB-2/antagonists & inhibitors , Administration, Oral , Aniline Compounds/administration & dosage , Aniline Compounds/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Evaluation, Preclinical/methods , ErbB Receptors/metabolism , Growth Inhibitors/administration & dosage , Growth Inhibitors/chemical synthesis , Growth Inhibitors/pharmacology , Humans , Mice , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Receptor, ErbB-2/metabolismABSTRACT
Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Hypoglycemic Agents/chemical synthesis , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/pharmacology , Animals , Blood Glucose/analysis , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Disease Models, Animal , Hypoglycemic Agents/blood , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Mice , Molecular Conformation , Molecular Structure , Structure-Activity Relationship , Urea/pharmacology , ortho-Aminobenzoates/blood , ortho-Aminobenzoates/chemistryABSTRACT
The optimization of imidazo[1,2-a]pyridine inhibitors as potent and selective inhibitors of IGF-1R is presented. Further optimization of oral exposure in mice is also discussed. Detailed selectivity, in vitro activity, and in vivo PK profiles of an optimized compound is also highlighted.
Subject(s)
Chemistry, Pharmaceutical/methods , Pyridines/chemistry , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/chemistry , Administration, Oral , Aniline Compounds/chemistry , Animals , Cell Line, Tumor , Crystallography, X-Ray , Drug Design , Humans , Inhibitory Concentration 50 , Mice , Models, Chemical , Molecular Conformation , Pyridines/chemical synthesis , Pyridines/pharmacology , Receptor, Insulin/metabolismABSTRACT
A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.
Subject(s)
Antineoplastic Agents/chemistry , ErbB Receptors/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrrolidines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Administration, Oral , Animals , Mice , Pharmacokinetics , Pyrimidines/chemical synthesis , Pyrrolidines/chemical synthesis , Structure-Activity RelationshipABSTRACT
The synthesis of a 7-azaindole series of novel, potent B-Raf kinase inhibitors using knowledge-based design was carried out. Compound 6h exhibits not only excellent potency in both the enzyme assay (IC(50)=2.5 nM) and the cellular assay (IC(50)=63 nM), but also has an outstanding selectivity profile against other kinases.
Subject(s)
Chemistry, Pharmaceutical/methods , Indoles/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Drug Design , Humans , Inhibitory Concentration 50 , Models, Biological , Models, Chemical , Molecular Structure , Phosphotransferases/metabolism , Protein Kinase Inhibitors/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected molecules, including compound 15h, were found to be potent in enzymatic and cellular assays while also demonstrating exposure in the mouse from an oral dose.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Pyrimidines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Cell Line , Mice , Protein Binding , Pyrimidines/chemistry , Structure-Activity RelationshipABSTRACT
GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.
Subject(s)
Enzyme Inhibitors/chemistry , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , Quinazolines/chemistry , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Humans , Kinetics , Lapatinib , Models, Molecular , Molecular Sequence Data , Oncogene Proteins v-erbB/antagonists & inhibitors , Protein Conformation , Protein Structure, Secondary , Quinazolines/metabolism , Quinazolines/pharmacology , Substrate SpecificityABSTRACT
Glycogen synthase kinase 3 regulates glycogen synthase, the rate-determining enzyme for glycogen synthesis. Liver and muscle glycogen synthesis is defective in type 2 diabetics, resulting in elevated plasma glucose levels. Inhibition of GSK-3 could potentially be an effective method to control plasma glucose levels in type 2 diabetics. Structure-activity studies on a N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine series have led to the identification of potent and selective compounds with good cellular efficacy. Molecular modeling studies have given insights into the mode of binding of these inhibitors. Since the initial leads were also potent inhibitors of CDK-2/CDK-4, an extensive SAR was performed at various positions of the pyrazolo[1,5-b]pyridazin core to afford potent GSK-3 inhibitors that were highly selective over CDK-2. In addition, these inhibitors also exhibited very good cell efficacy and functional response. A representative example was shown to have good oral exposure levels, extending their utility in an in vivo setting. These inhibitors provide a viable lead series in the discovery of new therapies for the treatment of type 2 diabetes.
Subject(s)
Amines/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Pyridazines/chemistry , Pyridazines/pharmacology , Animals , Binding Sites , Cell Line , Enzyme Inhibitors/chemical synthesis , Glycogen Synthase Kinase 3/metabolism , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Protein Structure, Tertiary , Pyridazines/chemical synthesis , Rats , Structure-Activity RelationshipABSTRACT
Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.
ABSTRACT
Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAF(V600E) and the YUSIT1 BRAF(V600K) melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1(K59del)) or NRAS mutation (NRAS(Q61K) and/or NRAS(A146T)) with and without MEK1(P387S) in the BRAF(V600E) background and NRAS(Q61K) in the BRAF(V600K) background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRAS(Q61K) or NRAS(A146T) in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1(K59del), but not MEK1(P387S), decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27(kip1) protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Apoptosis/drug effects , Cell Growth Processes/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Imidazoles/administration & dosage , Imidazoles/pharmacology , Indoles/administration & dosage , Indoles/pharmacology , MAP Kinase Kinase 1/antagonists & inhibitors , Melanoma/enzymology , Oximes/administration & dosage , Oximes/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , VemurafenibABSTRACT
Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appended to this ring was favored. Selected molecules in the series demonstrated some activity against human tumor cell lines.
Subject(s)
Alkynes/chemistry , ErbB Receptors/antagonists & inhibitors , Pyrimidines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Models, Molecular , Molecular Structure , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity RelationshipABSTRACT
The identification of the kinase or kinases targeted by protein kinase inhibitors is a critical challenge in validating their use as therapeutic agents or molecular probes. Here, to address this problem, we describe a chemical genomics strategy that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets. By using this approach, we have identified two cyclin-dependent kinases, Cdk1 and Pho85, as the targets of the inhibitor GW400426 in Saccharomyces cerevisiae. We demonstrate that simultaneous inhibition of Cdk1 and Pho85, and not inhibition of either kinase alone, by GW400426 controls the expression of specific transcripts involved in polarized cell growth, thus revealing a cellular process that is uniquely sensitive to the multiplex inhibition of these two kinases. Our results suggest that the cellular responses induced by multiplex protein kinase inhibitors may be an emergent property that cannot be understood fully by considering only the sum of individual inhibitor-kinase interactions.
Subject(s)
CDC2 Protein Kinase/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Gene Expression Profiling , Protein Kinase Inhibitors/pharmacology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Genomics , Oligonucleotide Array Sequence AnalysisABSTRACT
A novel series of [1-(1H-benzimidazol-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl] arylhydrazones was synthesized and shown to potently inhibit glycogen synthase kinase-3 (GSK-3). In light of detailed structure-activity relationships and structural knowledge of the GSK-3 binding pocket, a benzimidazole substituent was incorporated onto the pyrazolopyrimidine core resulting in improved potency over previous analogs. More importantly, these derivatives show low nanomolar efficacy for stimulating glycogen synthesis in vitro and therefore may be useful in the treatment of type 2 diabetes mellitus.
Subject(s)
Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Hydrazones/pharmacology , Animals , Cell Line , Dogs , Enzyme Inhibitors/chemistry , Hydrazones/chemistry , Models, MolecularABSTRACT
A series of [1-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]arylhydrazones were discovered as novel inhibitors glycogen synthase kinase-3 (GSK-3). Based on initial modeling a detailed SAR was constructed. Modification of the interior binding aryl ring (Ar(1)) determined this to be a tight binding region with little room for modification. As predicted from the model, a large variety of modifications could be incorporated into the hydrazone aryl ring. This work led to GSK-3 inhibitors in the low nano-molar range.