ABSTRACT
Rapid clinical progress of hepatitis C virus (HCV) replication inhibitors, including these selecting for resistance in the NS5A region (NS5A inhibitors), promises to revolutionize HCV treatment. Herein, we describe our explorations of diverse spiropyrrolidine motifs in novel NS5A inhibitors and a proposed interaction model. We discovered that the 1,4-dioxa-7-azaspiro[4.4]nonane motif in inhibitor 41H (GSK2236805) supported high potency against genotypes 1a and 1b as well as in genotype 1b L31V and Y93H mutants. Consistent with this, 41H potently suppressed HCV RNA in the 20-day RNA reduction assay. Pharmacokinetic and safety data supported further progression of 41H to the clinic.
Subject(s)
Antiviral Agents/pharmacology , Genotype , Hepacivirus/drug effects , Pyrrolidines/pharmacology , Spiro Compounds/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Drug Design , Hepacivirus/genetics , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , Mutation , Pyrrolidines/chemistry , Pyrrolidines/pharmacokinetics , RNA, Viral/blood , Spiro Compounds/chemistry , Spiro Compounds/pharmacokineticsABSTRACT
Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients. We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure-activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Animals , Antiviral Agents/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Hepacivirus/physiology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Rats , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
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
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
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
We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced further as potential tool compounds for in vivo validation studies.
Subject(s)
Benzamides/chemistry , Benzamides/pharmacology , Benzothiazoles/chemistry , Kv1.3 Potassium Channel/antagonists & inhibitors , Amides , Animals , Benzothiazoles/pharmacology , Cell Line , Humans , Pancreatitis-Associated Proteins , Patch-Clamp Techniques , Rats , Structure-Activity RelationshipABSTRACT
We report the synthesis and in vitro activity of a series of novel N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several compounds, including compound 8b, showed similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions.
Subject(s)
Benzamides/chemistry , Benzothiazoles/chemistry , Kv1.3 Potassium Channel/antagonists & inhibitors , Amides , Animals , Benzamides/pharmacology , Benzothiazoles/pharmacology , Cell Line , Humans , Pancreatitis-Associated Proteins , Patch-Clamp Techniques , Structure-Activity Relationship , Sulfonamides , Urea/analogs & derivativesABSTRACT
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
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
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
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
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
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
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
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
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
Synthetic modifications on a 6-furanylquinazoline scaffold to optimize the dual ErbB-1/ErbB-2 tyrosine kinase inhibition afforded consistent SAR whereby a 4-(3-fluorobenzyloxy)-3-haloanilino provided the best enzyme potency and cellular selectivity. Changes made to the 6-furanyl group had little impact on the enzyme activity, but appeared to dramatically affect the cellular efficacy. The discovery of lapatinib emerged from this work.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Furans/chemistry , Furans/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Cell Line, Tumor , ErbB Receptors/genetics , ErbB Receptors/metabolism , Furans/chemical synthesis , Humans , Inhibitory Concentration 50 , Lapatinib , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Quinazolines/chemical synthesis , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Structure-Activity RelationshipABSTRACT
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.
