ABSTRACT
IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and clogP. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Animals , Female , Humans , Indoles/pharmacokinetics , Interleukin-1 Receptor-Associated Kinases/immunology , Lipopolysaccharides/immunology , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Kinase Inhibitors/pharmacokinetics , Quinolines/pharmacokinetics , Signal Transduction/drug effectsABSTRACT
Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.
Subject(s)
Osteogenesis/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , src-Family Kinases/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Differentiation , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/cytology , Osteoclasts/drug effectsABSTRACT
The 7-alkene-3-quinolinecarbonitrile 20, a potent inhibitor of Src enzymatic and cellular activity with IC(50) values of 2.1 and 58 nM, respectively, had comparable efficacy to bosutinib in a colon tumor xenograft study.
Subject(s)
Antineoplastic Agents/chemistry , Protein Kinase Inhibitors/chemistry , Quinolines/chemistry , src-Family Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Humans , Mice , Mice, Nude , Microsomes/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Xenograft Model Antitumor Assays , src-Family Kinases/metabolismABSTRACT
Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).
Subject(s)
Isoenzymes/antagonists & inhibitors , Nitriles/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Humans , Interleukin-2/biosynthesis , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Nitriles/chemistry , Protein Kinase C-theta , Protein Kinase Inhibitors/chemistry , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
PKCtheta is a serine/threonine kinase involved in the regulation of IL2 production in T cells. It has recently become an attractive therapeutic target for a variety of immunological disorders. We describe the optimization of the enzymatic and cellular potency of a series of 5-vinyl-3-pyridinecarbonitrile inhibitors of PKCtheta. A binding model was developed that explains much of the SAR observed for this series, including the enzymatic potency observed for 19. An analysis of functional potency against various physiochemical parameters suggests that cellular potency is correlated with LogD(7.4), but not with cLogP, PAMPA permeability, or TPSA.
Subject(s)
Isoenzymes/antagonists & inhibitors , Nitriles/chemical synthesis , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Vinyl Compounds/chemical synthesis , Animals , Computer Simulation , Inhibitory Concentration 50 , Isoenzymes/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred C57BL , Models, Molecular , Nitriles/chemistry , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Vinyl Compounds/chemistry , Vinyl Compounds/pharmacologyABSTRACT
A series of 5-vinyl phenyl sulfonamide-3-pyridinecarbonitriles were prepared and evaluated as PKCtheta inhibitors. Optimization resulted in the identification of compound 15 with an IC(50) value 0.44 nM for the inhibition of PKCtheta with 150-fold selectivity over PKCdelta.
Subject(s)
Isoenzymes/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Pyridines/pharmacology , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Drug Design , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyridines/chemistry , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
A series of 5-vinyl-3-pyridinecarbonitriles were synthesized and evaluated as PKCtheta inhibitors. The systematic optimization of 4-[(4-methyl-1H-indol-5-yl)amino]-5-[(E)-2-phenylvinyl]-3-pyridinecarbonitrile 3 resulted in the identification of compound 23e as a potent PKCtheta inhibitor with good selectivity over PKCdelta.
Subject(s)
Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Animals , Humans , Isoenzymes/metabolism , Microsomes/metabolism , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606) and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transient middle cerebral artery occlusion (tMCAO)] and permanent [permanent middle cerebral artery occlusion (pMCAO)] focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3-30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.
Subject(s)
Aniline Compounds/therapeutic use , Brain Ischemia/drug therapy , Neuroprotective Agents/therapeutic use , Nitriles/therapeutic use , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Quinolines/therapeutic use , src-Family Kinases/antagonists & inhibitors , Aniline Compounds/administration & dosage , Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Animals , Blood Platelets/drug effects , Blood Platelets/enzymology , Brain/drug effects , Brain/enzymology , Brain/metabolism , Brain Ischemia/enzymology , Capillary Permeability , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Flow Cytometry , Humans , Injections, Intravenous , Male , Molecular Structure , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacokinetics , Neuropsychological Tests , Nitriles/administration & dosage , Nitriles/chemistry , Nitriles/pharmacokinetics , Piperazines/administration & dosage , Piperazines/chemistry , Piperazines/pharmacokinetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Quinolines/administration & dosage , Quinolines/chemistry , Quinolines/pharmacokinetics , Rats , Rats, Wistar , Time FactorsABSTRACT
Members of the protein kinase C (PKC) family of serine/threonine kinases have been targeted for drug discovery for over 20 years, initially focusing on inhibitors of the classic PKCs, which include the alpha, beta and gamma isoforms. Recently, inhibition of the activity of the novel PKC isoforms, namely Theta, delta, epsilon and eta has become a focus of research. PKCTheta, first identified in 1992, is a key enzyme in the regulation of T cell activation and survival. While T cells play critical roles in initiating and controlling the immune response, inappropriate or extended stimulation of T cells is responsible for many chronic inflammatory diseases. Mice with an engineered deficiency in PKCTheta have reduced incidence and severity of several inflammatory disorders including asthma, arthritis, inflammatory bowel disease, multiple sclerosis, and allograft rejection. This review summarizes the efforts directed towards the design of small molecule PKCTheta inhibitors as potential therapeutic agents.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Isoenzymes/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Humans , Isoenzymes/chemistry , Mice , Peptides/chemistry , Peptides/pharmacology , Protein Kinase C-theta , Small Molecule Libraries , Structure-Activity RelationshipABSTRACT
We earlier reported that 3-pyridinecarbonitriiles with a 4-methylindolyl-5-amino group at C-4 and a phenyl group at C-5 were inhibitors of PKCtheta. Keeping the group at C-4 of the pyridine core constant, we varied the water solubilizing group on the phenyl ring at C-5 and then replaced the C-5 phenyl ring with several monocyclic heteroaryl rings, including furan, thiophene and pyridine. Analog 6e with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-2-furyl group C-5 had an IC50 value of 4.5 nM for the inhibition of PKCtheta.
Subject(s)
Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Nitriles/chemistry , Nitriles/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Animals , Inhibitory Concentration 50 , Isoenzymes/genetics , Mice , Mice, Knockout , Protein Kinase C/genetics , Protein Kinase C-theta , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.
Subject(s)
Aminopyridines/chemistry , Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Aminopyridines/chemical synthesis , Aminopyridines/pharmacology , Animals , Benzofurans/chemical synthesis , Benzofurans/chemistry , Benzofurans/pharmacology , Half-Life , Humans , Interleukin-2/metabolism , Isoenzymes/metabolism , Mice , Microsomes, Liver/metabolism , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Rats , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
A series of 4-anilino-7-pyridyl-3-quinolinecarbonitriles was prepared as Src kinase inhibitors. A systematic SAR study of substitutions on both the pyridine ring and the 3-quinolinecarbonitrile core established the requirements for optimal activity. The lead compound, 17, showed potent activity in both the Src enzyme assay and cell assays, and demonstrated in vivo anti-tumor activity in a xenograft model.
Subject(s)
Aminoquinolines/chemistry , Aniline Compounds/chemistry , Antineoplastic Agents/chemistry , Protein Kinase Inhibitors/chemistry , Quinolines/chemistry , src-Family Kinases/antagonists & inhibitors , Aminoquinolines/chemical synthesis , Aminoquinolines/pharmacology , Aniline Compounds/chemical synthesis , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Humans , Mice , Mice, Nude , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Rats , Structure-Activity Relationship , Xenograft Model Antitumor Assays , src-Family Kinases/metabolismABSTRACT
The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCtheta inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC(50) value of 7.4nM for the inhibition of PKCtheta.
Subject(s)
Indoles/chemistry , Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Animals , Indoles/chemical synthesis , Indoles/pharmacology , Isoenzymes/metabolism , Mice , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Structure-Activity RelationshipABSTRACT
A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCtheta were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC(50) value of 18nM for the inhibition of PKCtheta.
Subject(s)
Isoenzymes/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Adenosine Triphosphate/chemistry , Animals , Anti-Inflammatory Agents/pharmacology , Chemistry, Pharmaceutical/methods , Drug Design , Humans , Inhibitory Concentration 50 , Isoenzymes/chemistry , Mice , Models, Chemical , Molecular Structure , Protein Isoforms , Protein Kinase C/chemistry , Protein Kinase C-theta , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Structure-Activity RelationshipABSTRACT
Thieno[2,3-b]pyridine-5-carbonitrile 16 with a 4-methyl-5-indolylamine at C-4 and a 5-methoxy-2-(dimethylamino)-methylphenyl group at C-2 had an IC(50) value of 16 nM for the inhibition of PKCtheta. While moderate inhibition of PKCdelta was also observed (IC(50)=130 nM), 16 had IC(50) values of greater than 5 microM against Lyn and other members of the Src kinase family.
Subject(s)
Chemistry, Pharmaceutical/methods , Indoles/chemical synthesis , Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Pyridines/chemistry , Drug Design , Enzyme Inhibitors/pharmacology , Humans , Indoles/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C-theta , Structure-Activity Relationship , src-Family Kinases/antagonists & inhibitorsABSTRACT
Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.
Subject(s)
Antineoplastic Agents/chemical synthesis , Nitriles/chemical synthesis , Quinolines/chemical synthesis , Receptor, IGF Type 1/antagonists & inhibitors , Humans , Nitriles/pharmacology , Quinolines/pharmacology , Structure-Activity RelationshipABSTRACT
In 2000, Wyeth reported that the 3-quinolinecarbonitrile ring system was a template for EGFR inhibitors. It soon became apparent that the group at C-4 of this core was responsible for kinase selectivity. A 4-(2,4-dichloro-5-methoxyanilino) substituent provided potent inhibitors of Src, a non-receptor tyrosine kinase that plays a key role in cell signaling. One compound from this series, SKI-606, bosutinib, is currently in clinical trials for the treatment of cancer.
Subject(s)
Quinolines/chemistry , Quinolines/pharmacology , src-Family Kinases/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Humans , Neoplasms/drug therapy , Structure-Activity RelationshipABSTRACT
A series of 2-alkenyl thieno[2,3-b]pyridine inhibitors of PKCtheta were synthesized as potential inflammatory modulators. This series led to the discovery of 2-alkenyl amides, which are exceptionally potent and selective inhibitors of PKCtheta. Compound 8 has an IC(50) of 3.8nM against PKCtheta and shows excellent selectivity over a variety of PKC isoforms.
Subject(s)
Isoenzymes/antagonists & inhibitors , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Thiophenes/chemical synthesis , Thiophenes/pharmacology , Animals , Combinatorial Chemistry Techniques , Mice , Models, Chemical , Molecular Structure , Nitriles/chemistry , Protein Kinase C-theta , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Thiophenes/chemistryABSTRACT
The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCtheta. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC(50) value of 7.5nM for the inhibition of PKCtheta.