ABSTRACT
PURPOSE: Bruton's tyrosine kinase (BTK) is a key component of B cell receptor (BCR) signaling, and as such a critical regulator of cell proliferation and survival. Aberrant BCR signaling is important in the pathogenesis of various B cell malignancies and autoimmune disorders. Here, we describe the development of a novel positron emission tomography (PET) tracer for imaging BTK expression and/or occupancy by small molecule therapeutics. METHODS: Radiochemistry was carried out by reacting the precursor with [18F]fluoride on a GE FX-FN TracerLab synthesis module to produce [18F]BTK-1 with a 6% decay-corrected radiochemical yield, 100 ± 6 GBq/µmol molar activity, and a radiochemical purity of 99%. Following intravenous administration of [18F]BTK-1 (3.63 ± 0.59 MBq, 0.084 ± 0.05 µg), 60-min dynamic images were acquired in two xenograft models: REC-1, an efficacious mantle cell lymphoma model, and U87MG, a non-efficacious glioblastoma model. Subsequent studies included vehicle, pretreatment (10 min prior to tracer injection), and displacement (30 min post-tracer injection) studies with different reversible BTK inhibitors to examine BTK binding. Human radiation dosimetry was estimated based on PET imaging in healthy rats. RESULTS: Uptake of [18F]BTK-1 was significantly higher in BTK expressing REC-1 tumors than non-BTK expressing U87MG tumors. Administration of BTK inhibitors prior to tracer administration blocked [18F]BTK-1 binding in the REC-1 tumor model consistent with [18F]BTK-1 binding to BTK. The predicted effective dose in humans was 0.0199 ± 0.0007 mSv/MBq. CONCLUSION: [18F]BTK-1 is a promising PET tracer for imaging of BTK, which could provide valuable information for patient selection, drug dose determination, and improving our understanding of BTK biology in humans.
Subject(s)
Fluorides , Protein Kinase Inhibitors , Humans , Animals , Rats , Adult , Agammaglobulinaemia Tyrosine Kinase/chemistry , Agammaglobulinaemia Tyrosine Kinase/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Receptors, Antigen, B-Cell , Positron-Emission TomographyABSTRACT
Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 µM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 µM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 µM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by â¼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.
Subject(s)
Arthritis, Experimental/prevention & control , Cyclohexylamines/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Leukocytes/drug effects , Leukocytes/immunology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Synovitis/prevention & control , Adaptor Proteins, Signal Transducing/metabolism , Animals , Arthritis, Experimental/complications , Arthritis, Experimental/pathology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Basophils/drug effects , Basophils/immunology , Biocatalysis/drug effects , Blood/drug effects , Blood/immunology , Blood/metabolism , Cell Degranulation/drug effects , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclohexylamines/administration & dosage , Cyclohexylamines/pharmacokinetics , Cyclohexylamines/therapeutic use , Disease Models, Animal , Edema/complications , Edema/pathology , Edema/prevention & control , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Foot/pathology , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Leukocytes/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Molecular Structure , Phosphorylation/drug effects , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Protein-Tyrosine Kinases/drug effects , Protein-Tyrosine Kinases/metabolism , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Rats, Inbred LewABSTRACT
Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.
Subject(s)
Anticoagulants/chemical synthesis , Anticoagulants/pharmacology , Benzamides/chemical synthesis , Benzamides/pharmacology , Drug Discovery/methods , Factor Xa Inhibitors , Pyridines/chemical synthesis , Pyridines/pharmacology , Administration, Oral , Animals , Anticoagulants/administration & dosage , Benzamides/administration & dosage , Catalytic Domain/drug effects , Cell Line , Dogs , Dose-Response Relationship, Drug , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Factor Xa/metabolism , Humans , Macaca fascicularis , Pyridines/administration & dosage , Rabbits , RatsABSTRACT
Anthranilamide-based benzamidine compound 4 and its N-substituted analogs were designed and examined as factor Xa inhibitors using substituted benzamidines as unconventional S4 binding element. A group of N,N-dialkylbenzamidines (11, 17 and 24) have been discovered as potent factor Xa inhibitors with strong anticoagulant activity and promising oral PK profiles.
Subject(s)
Anticoagulants/administration & dosage , Anticoagulants/chemical synthesis , Benzamidines/administration & dosage , Benzamidines/chemical synthesis , Factor Xa Inhibitors , ortho-Aminobenzoates/administration & dosage , ortho-Aminobenzoates/chemical synthesis , Administration, Oral , Animals , Anticoagulants/pharmacokinetics , Benzamidines/pharmacokinetics , Biological Availability , Dogs , Factor Xa/pharmacokinetics , Humans , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , ortho-Aminobenzoates/pharmacokineticsABSTRACT
Drug-induced QT prolongation arising from drugs' blocking of hERG channel activity presents significant challenges in drug development. Many, but not all, of our benzamidine-containing factor Xa inhibitors were found to have high hERG binding propensity. However, incorporation of a carboxylic acid group into these benzamidine molecules generally leads to hERG inactive compounds regardless where the carboxyl group is tethered within the molecules. The inhibitory effect of a carboxylic acid group on hERG binding has also been observed in many series of diverse structural scaffolds (including non-amidines). These findings suggest that the negatively charged carboxylate group causes unfavorable interaction within hERG channel binding cavity by electrostatic interaction.
Subject(s)
Benzamidines/metabolism , Carboxylic Acids/metabolism , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Factor Xa Inhibitors , HumansABSTRACT
A class of N,N-dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines has been discovered as potent factor Xa inhibitors with desirable in vitro and in vivo anticoagulant activity, but with low oral bioavailability. The 5-chloroindole and 6-chlorobenzo[b]thiophene groups are optimal as the factor Xa S1 binding elements. The strategy of incorporating a side chain on the piperazine nucleus to enhance binding affinity has been examined.
Subject(s)
Benzamidines/pharmacology , Factor Xa Inhibitors , Serine Proteinase Inhibitors/pharmacology , Benzamidines/chemistry , Benzamidines/pharmacokinetics , Biological Availability , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacokineticsABSTRACT
Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors. Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly potent in vitro, and show improved activity in human plasma-based thrombin generation assay.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Factor Xa Inhibitors , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/pharmacology , Administration, Oral , Animals , Biological Availability , Drug Design , Drug Evaluation, Preclinical , Humans , Molecular Structure , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thrombosis/drug therapy , Thrombosis/prevention & controlABSTRACT
Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure-activity relationship studies around 2 have led to a series of potent factor Xa inhibitors which are highly active in the human plasma-based thrombin generation assay with 2XTG values less than 1 microM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein thrombosis model, and also exhibits good oral bioavailability and a long half life in rats.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Factor Xa Inhibitors , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/pharmacology , Administration, Oral , Animals , Biological Availability , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Design , Humans , Molecular Structure , Rabbits , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thrombosis/drug therapyABSTRACT
A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)< or =2 nM) with improved in vitro anticoagulant activity (2xTG< or =1 microM) and respectable pharmacokinetic properties have been discovered.
Subject(s)
Amides/chemistry , Antithrombin III/chemistry , Factor Xa Inhibitors , Pyrazoles/chemistry , Amides/metabolism , Amides/pharmacology , Animals , Antithrombin III/metabolism , Antithrombin III/pharmacology , Humans , Protein Binding , Pyrazoles/metabolism , Pyrazoles/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Using N,N-dialkylated benzamidines as the novel P4 motifs, we have designed and synthesized a class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent and selective fXa inhibitors with significantly improved hydrophilicity and in vitro anticoagulant activity. These benzamidine-P4 fXa inhibitors have displayed excellent oral bioavailability and long half-life.
Subject(s)
Amides/chemical synthesis , Antithrombin III/chemical synthesis , Benzamidines/antagonists & inhibitors , Pyrazoles/chemical synthesis , Administration, Oral , Amides/administration & dosage , Amides/metabolism , Animals , Antithrombin III/administration & dosage , Antithrombin III/metabolism , Benzamidines/metabolism , Biological Availability , Drug Design , Humans , Pyrazoles/administration & dosage , Pyrazoles/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
OBJECTIVE: In this study we test the hypothesis that blood/plasma-based prothrombinase assays, rather than inhibition of purified factor Xa (fXa), are predictive of in vivo antithrombotic activity. METHODS AND RESULTS: Six fXa inhibitors with equivalent nanomolar Ki were studied in thrombin generation assays using human plasma/blood and endogenous macromolecular substrate. In all assays, benzamidine inhibitors were more potent (100 to 800 nmol/L) than the aminoisoquinolines (5 to 58 micromol/L) or neutral inhibitors (3 to 10 micromol/L). A similar rank order of compound inhibition was also seen in purified prothrombinase assays as well as in a rabbit model of deep vein thrombosis. CONCLUSIONS: Assays using prothrombinase with protein substrates are better predictors of in vivo efficacy than fXa Ki using amidolytic substrates.
Subject(s)
Benzamidines/pharmacology , Enzyme Inhibitors/pharmacology , Factor Xa Inhibitors , Fibrinolytic Agents/pharmacology , Isoquinolines/pharmacology , Prothrombin/metabolism , Thromboplastin/antagonists & inhibitors , Animals , Binding Sites/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Inhibitors/classification , Fibrinolytic Agents/classification , Humans , Male , Molecular Structure , Sensitivity and Specificity , Substrate Specificity , Thrombin/biosynthesis , Venous Thrombosis/prevention & controlABSTRACT
A series of benzoxazinone derivatives was designed and synthesized as factor Xa inhibitors. We demonstrated that the naphthyl moiety in the aniline-based compounds 1 and 2 can be replaced with benzene-fused heterobicycles and biaryls to give factor Xa inhibitors with improved trypsin selectivity. The P4 modifications lead to monoamidines which are moderately active. The benzoxazinones 41-45 are potent against factor Xa, retain the improved trypsin selectivity of the corresponding aniline-based compounds, and show strong antithrombotic effect dose responsively.
Subject(s)
Factor Xa Inhibitors , Oxazines/chemical synthesis , Oxazines/pharmacology , Aniline Compounds/chemical synthesis , Aniline Compounds/pharmacology , Animals , Binding, Competitive/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Design , In Vitro Techniques , Indicators and Reagents , Models, Molecular , Molecular Conformation , Rabbits , Structure-Activity Relationship , Thrombin/metabolism , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/pharmacologyABSTRACT
Based on DuPont Pharmaceuticals' monobenzamidine lead structure SN429, we have designed the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as a novel series of non-basic factor Xa inhibitors. We have discovered that the displacement of the benzamidine moiety with substituted 2-naphthyl structures not only results in highly potent factor Xa inhibitors, but also significantly increases their enzyme specificity and oral bioavailability.
Subject(s)
Factor Xa Inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Amides/chemistry , Animals , Drug Design , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Rats , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Several investigations of rigid alpha-keto cyclopropane cleavage by O-stannyl ketyls are summarized herein. Tricyclo[3.3.0.0(2,8)]octan-3-one ring systems were treated with nBu(3)SnH, which produced different ring-cleavage products depending on the location and type of substituent present. An examination of both radical-stabilizing substituents and stereoelectronic factors was initiated to understand what factors bias bond cleavage in a configurationally restricted alpha-ketocyclopropane via O-stannyl ketyls. A preference for cleavage of the cyclopropane bond with the best orbital overlap with the ketyl radical sp(2)-orbital even in the presence of radical stabilizing groups is indicated by these results. An O-stannyl ketyl ring scission-cyclization resulted in the novel synthesis of either a linear or an angular triquinane skeleton depending on the length and location an alkene tether on the tricyclo[3.3.0.0(2,8)]octan-3-one precursor.