ABSTRACT
Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Purines/chemistry , Purines/pharmacology , src-Family Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Growth Inhibitors/chemistry , Growth Inhibitors/pharmacology , Humans , K562 Cells , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-abl/physiology , RatsABSTRACT
c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Enzyme Inhibitors/pharmacology , Interleukin-8/metabolism , Neoplasms/metabolism , Neovascularization, Pathologic/prevention & control , Vascular Endothelial Growth Factor A/metabolism , src-Family Kinases/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Animals , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Movement , DNA Primers , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoprecipitation , Mice , Mice, Inbred C3H , Neoplasms/blood supply , Phosphorylation , RNA, Small InterferingABSTRACT
In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Lung Neoplasms/drug therapy , Organophosphorus Compounds/pharmacology , Phosphines/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Administration, Oral , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mice, SCID , Molecular Conformation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Organophosphorus Compounds/administration & dosage , Organophosphorus Compounds/chemistry , Phosphines/pharmacology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Rats , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity RelationshipABSTRACT
Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.
Subject(s)
Drug Resistance, Neoplasm/genetics , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Crizotinib , Humans , Lung Neoplasms , Mutation , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.
Subject(s)
Fusion Proteins, bcr-abl/antagonists & inhibitors , Imidazoles/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Pyridazines/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Growth Processes/drug effects , Cell Line, Tumor , Crystallography, X-Ray , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Imidazoles/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, SCID , Models, Molecular , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/chemistry , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Pyridazines/chemistry , Signal Transduction/drug effectsABSTRACT
The deregulated, oncogenic tyrosine kinase Bcr-Abl causes chronic myeloid leukemia (CML). Imatinib mesylate (Gleevec, STI571), a Bcr-Abl kinase inhibitor, selectively inhibits proliferation and promotes apoptosis of CML cells. Despite the success of imatinib mesylate in the treatment of CML, resistance is observed, particularly in advanced disease. The most common imatinib mesylate resistance mechanism involves Bcr-Abl kinase domain mutations that impart varying degrees of drug insensitivity. AP23464, a potent adenosine 5'-triphosphate (ATP)-based inhibitor of Src and Abl kinases, displays antiproliferative activity against a human CML cell line and Bcr-Abl-transduced Ba/F3 cells (IC(50) = 14 nM; imatinib mesylate IC(50) = 350 nM). AP23464 ablates Bcr-Abl tyrosine phosphorylation, blocks cell cycle progression, and promotes apoptosis of Bcr-Abl-expressing cells. Biochemical assays with purified glutathione S transferase (GST)-Abl kinase domain confirmed that AP23464 directly inhibits Abl activity. Importantly, the low nanomolar cellular and biochemical inhibitory properties of AP23464 extend to frequently observed imatinib mesylate-resistant Bcr-Abl mutants, including nucleotide binding P-loop mutants Q252H, Y253F, E255K, C-terminal loop mutant M351T, and activation loop mutant H396P. AP23464 was ineffective against mutant T315I, an imatinib mesylate contact residue. The potency of AP23464 against imatinib mesylate-refractory Bcr-Abl and its distinct binding mode relative to imatinib mesylate warrant further investigation of AP23464 for the treatment of CML.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Enzyme Inhibitors/pharmacology , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Mutation/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphate/chemistry , Amino Acids/genetics , Amino Acids/metabolism , Apoptosis/drug effects , Benzamides , Cell Cycle/drug effects , Cell Division/drug effects , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/chemistry , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , HL-60 Cells , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Milk Proteins/metabolism , Models, Molecular , Nuclear Proteins/metabolism , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Piperazines/chemistry , Piperazines/pharmacology , Protein Structure, Tertiary , Pyridones/chemistry , Pyridones/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , STAT5 Transcription Factor , Trans-Activators/metabolismABSTRACT
Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Drug Design , Osteoporosis/drug therapy , Pyrimidines/chemical synthesis , src-Family Kinases/antagonists & inhibitors , Animals , Bone Diseases/drug therapy , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Models, Molecular , Purines/chemical synthesis , Purines/pharmacology , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.
Subject(s)
Bone Diseases/drug therapy , Purines/chemical synthesis , src-Family Kinases/antagonists & inhibitors , Adenosine Triphosphate/analogs & derivatives , Animals , Drug Delivery Systems , Drug Design , Durapatite/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Purines/pharmacology , Structure-Activity RelationshipABSTRACT
The design of bone-targeted pyrido[2,3-d]pyrimidin-7-ones as Src tyrosine kinase inhibitors is described. Leveraging SAR from known compounds and using structure-based methods, we were able to rapidly incorporate bone binding components, which maintained, and even increased potency against the target enzyme. Compound 4 displayed a high affinity for hydroxyapatite, a major constituent of bone, and demonstrated antiresoprtive activity in our cell-based assay.