ABSTRACT
Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction.
Subject(s)
Muscarinic Antagonists/chemistry , Quaternary Ammonium Compounds/chemistry , Receptors, Muscarinic/chemistry , Tyrosine/chemistry , Urea/analogs & derivatives , Animals , Bronchi/drug effects , Mice , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/metabolism , Structure-Activity Relationship , Urea/chemical synthesis , Urea/pharmacologyABSTRACT
Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic characteristics suitable for inhaled delivery.
Subject(s)
Biphenyl Compounds/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Muscarinic Antagonists/chemistry , Pulmonary Disease, Chronic Obstructive/drug therapy , Receptors, Muscarinic/chemistry , Administration, Inhalation , Animals , Biphenyl Compounds/chemical synthesis , Biphenyl Compounds/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Drug Discovery , Humans , Mice , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/pharmacokinetics , Rats , Receptors, Muscarinic/metabolism , Structure-Activity RelationshipABSTRACT
Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M(3) muscarinic acetylcholine receptor antagonists such as 14 (pA(2)=11.0) possessing good sub-type selectivity for M(3) over M(2). The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described.
Subject(s)
Amines/chemical synthesis , Chemistry, Pharmaceutical/methods , Receptor, Muscarinic M3/antagonists & inhibitors , Amides/chemistry , Amines/pharmacology , Asthma/drug therapy , Drug Design , Electrons , Humans , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Molecular Structure , Pulmonary Disease, Chronic Obstructive/drug therapy , Receptor, Muscarinic M3/chemistry , Structure-Activity RelationshipABSTRACT
SAR exploration of multiple regions of a tyrosine urea template led to the identification of very potent muscarinic acetylcholine receptor antagonists such as 10b with good subtype selectivity for M(3) over M(1). The structure-activity relationships (SAR) and optimization of the tyrosine urea series are described.
Subject(s)
Chemistry, Pharmaceutical/methods , Muscarinic Antagonists/chemical synthesis , Receptors, Muscarinic/chemistry , Tyrosine/chemistry , Urea/chemistry , Asthma/drug therapy , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Structure , Muscarinic Antagonists/pharmacology , Salts/chemistry , Structure-Activity RelationshipABSTRACT
A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M3 over M2 and M1 exhibited excellent inhibitory activity and long duration of action in a bronchoconstriction in vivo model in mice via intranasal administration. The novel inhaled mAChR antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease.
Subject(s)
Bronchoconstriction/drug effects , Bronchodilator Agents/pharmacology , Piperazines/pharmacology , Receptors, Muscarinic/drug effects , Administration, Intranasal , Animals , Bronchial Provocation Tests , Bronchoconstrictor Agents/pharmacology , Bronchodilator Agents/chemical synthesis , Bronchodilator Agents/chemistry , Disease Models, Animal , Drug Evaluation, Preclinical , Methacholine Chloride/pharmacology , Mice , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.
Subject(s)
Muscarinic Antagonists/pharmacology , Phenylurea Compounds/pharmacology , Quaternary Ammonium Compounds/pharmacology , Tyrosine/analogs & derivatives , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bronchoconstriction/drug effects , Drug Evaluation, Preclinical/methods , Guinea Pigs , Mice , Rats , Tyrosine/pharmacologyABSTRACT
In the course of our research program to develop novel muscarinic receptor antagonists for the treatment of COPD, new tropane carbamate derivatives were identified as potent anti-muscarinic agents. The synthesis, structure-activity relationships and pharmacological evaluation that led to the identification of compound 5o, are described.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Carbamates/chemical synthesis , Muscarinic Antagonists , Receptors, Muscarinic/drug effects , Tropanes/chemical synthesis , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bronchodilator Agents/chemical synthesis , Bronchodilator Agents/chemistry , Bronchodilator Agents/pharmacology , Carbamates/chemistry , Carbamates/pharmacology , Drug Evaluation, Preclinical , Inhibitory Concentration 50 , Mice , Molecular Structure , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/pharmacology , Pulmonary Disease, Chronic Obstructive/therapy , Tropanes/chemistry , Tropanes/pharmacologyABSTRACT
High-throughput screening of the corporate compound collection led to the discovery of a novel series of N-substituted-5-aryl-oxazolidinones as potent human CCR8 antagonists. The synthesis, structure-activity relationships, and optimization of the series that led to the identification of SB-649701 (1a), are described.
Subject(s)
Oxazolidinones/chemical synthesis , Oxazolidinones/pharmacology , Receptors, Chemokine/antagonists & inhibitors , Animals , CHO Cells , Chemotaxis, Leukocyte/drug effects , Computer Simulation , Cricetinae , Cricetulus , Drug Evaluation, Preclinical , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , Indicators and Reagents , Myotonin-Protein Kinase , Protein Serine-Threonine Kinases/drug effects , Receptors, CCR8 , Structure-Activity Relationship , Th2 Cells/drug effectsABSTRACT
The fluorescent styryl dyes FM1-43 and FM2-10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular-independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1-43 and FM2-10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1-43 and FM2-10 bound with moderate affinity (23-220 nM) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1-M5). In functional studies in vitro, FM1-43 and FM2-10 inhibited electrical field stimulation (EFS) and acetylcholine-induced cholinergic contractions of guinea-pig tracheal strips (IC50: FM1-43, 0.4 +/- 0.1; FM2-10, 1.6 +/- 0.1 microM; concentration of antagonist producing a 2-fold leftward shift in the acetylcholine concentration-response curve (Kb): FM1-43, 0.3 +/- 0.1; FM2-10, 15.8 +/- 10.1 microM). Neither compound inhibited EFS-evoked, non-adrenergic non-cholinergic nerve-mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole-mount preparations with 5 microM FM1-43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.
Subject(s)
Fluorescent Dyes/metabolism , Muscarinic Antagonists/metabolism , Pyridinium Compounds/metabolism , Quaternary Ammonium Compounds/metabolism , Receptors, Muscarinic/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Fluorescent Dyes/pharmacology , Guinea Pigs , Male , Muscarinic Antagonists/pharmacology , Muscle Contraction , Muscle, Smooth/drug effects , Pyridinium Compounds/pharmacology , Quaternary Ammonium Compounds/pharmacology , Receptors, Muscarinic/drug effects , Receptors, Muscarinic/genetics , Staining and Labeling/methods , Trachea/drug effects , TransfectionABSTRACT
We have previously identified a novel complex between the platelet-derived growth factor (PDGF)beta receptor and the sphingosine 1-phosphate receptor-1 (S1P1). The complex permits the utilization of active G-protein subunits (made available by constitutively active S1P1 receptor) by the PDGFbeta receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S1P1 receptor is predicted to reduce signal transduction from PDGFbeta receptor tyrosine kinase by blocking the constitutive activity of the G-protein coupled receptor. SB649146 is a novel inverse agonist of the S1P1 receptor. First, SB649146 displaced the S1P1 receptor agonist dihydrosphingosine 1-phosphate from membranes expressing the recombinant S1P1 receptor. Second, SB649146 reduced basal recombinant S1P1 receptor-induced GTPgammaS binding and S1P-induced GTPgammaS binding in membranes. Third, SB649146 blocked the S1P-induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S1P1 receptor. We now report that inverse agonism of the S1P1 receptor with SB649146 reduced the endocytosis of the PDGFbeta receptor-S1P1 receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse-agonist reduces growth factor-induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649146 also suggest that the constitutively active endogenous S1P1 receptor enhances PDGF-induced cell migration.
Subject(s)
Cell Movement/physiology , Platelet-Derived Growth Factor/pharmacology , Receptor, Platelet-Derived Growth Factor beta/physiology , Receptors, Lysosphingolipid/agonists , Animals , Cell Line , Cell Movement/drug effects , Cells, Cultured , Flavonoids/pharmacology , Guinea Pigs , Humans , Kidney , MAP Kinase Signaling System/drug effects , PC12 Cells , Rats , Receptors, Lysosphingolipid/physiology , Transfection , Tyrphostins/pharmacologyABSTRACT
Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.
Subject(s)
Bronchial Hyperreactivity/immunology , Chemokines, CC/physiology , Neurons/physiology , Receptor, Muscarinic M2/physiology , Receptors, Chemokine/antagonists & inhibitors , Animals , Bronchial Hyperreactivity/physiopathology , Bronchoalveolar Lavage Fluid , Chemokine CCL11 , Chemokines, CC/analysis , Disease Models, Animal , Female , Guinea Pigs , Ovalbumin/immunology , Parasympathetic Nervous System/immunology , Receptors, CCR3 , Receptors, Chemokine/physiology , Receptors, Interleukin-4/analysisABSTRACT
The phospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via transmembrane receptors S1P 1-5 and LPA 1-3, respectively. Both have been implicated in inflammatory responses. S1P and LPA receptor profiles on neutrophils of patients with pneumonia compared with healthy subjects were determined by PCR and Western blotting. Chemotaxis studies were performed to assess functional differences. S1P or LPA receptors were immunoprecipitated from neutrophils to assess receptor heterodimerization with CXCR1, an IL-8 receptor, by Western blotting. Receptors S1P 1, 4, and 5 and LPA 2 were expressed on neutrophils from both subject groups, but S1P 3 and LPA 1 receptor expression was mainly confined to neutrophils of patients with pneumonia. Chemotaxis of neutrophils from patients with pneumonia compared with control subjects was significantly increased in response to S1P and LPA. Pretreatment with S1P or LPA reduced IL-8-induced neutrophil chemotaxis and transcriptional expression of the CXCR1 receptor. Receptors S1P 3 and 4 and LPA 1 formed constitutive heterodimers with CXCR1. LPA treatment reduced the amount of LPA 1/CXCR1 heterodimer. Therefore, profiles of S1P and LPA receptors differ between neutrophils of patients with pneumonia and control subjects, with consequences for neutrophil function.
Subject(s)
Lysophospholipids/metabolism , Neutrophils/metabolism , Pneumonia/pathology , Receptors, Lysophosphatidic Acid/metabolism , Receptors, Lysophospholipid/metabolism , Sphingosine/analogs & derivatives , Adult , Aged , Aged, 80 and over , Case-Control Studies , Chemotaxis/drug effects , Dimerization , Female , Gene Expression , Humans , Lysophospholipids/pharmacology , Male , Middle Aged , Neutrophils/drug effects , Neutrophils/pathology , Pneumonia/metabolism , Protein Isoforms , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/metabolism , Receptors, Lysophosphatidic Acid/genetics , Reference Values , Sphingosine/metabolism , Sphingosine/pharmacologyABSTRACT
Abundant data from animal models and humans support the hypothesis that changes at the level of parasympathetic neuronal control of airway smooth muscle result in increased bronchoconstriction in response to vagal stimulation, leading to airway hyperresponsiveness. Neuronal inhibitory M2 muscarinic acetylcholine receptors on parasympathetic nerves are responsible for limiting acetylcholine release from these nerves. In humans with asthma, and after pulmonary inflammatory events in experimental animals, these receptors are dysfunctional, which results in airway hyperresponsiveness. Although it is unknown what mechanisms underlie airway hyperresponsiveness in chronic obstructive pulmonary disease, loss of parasympathetic control of airway smooth muscle is thought to be a contributing mechanism. As such, anticholinergic therapy is used extensively and with a high degree of success in the treatment of this condition. The future for inhaled anticholinergic compounds for the treatment of chronic obstructive pulmonary disease appears to rest in their combination with other agents, such as beta2 agonists and phosphodiesterase-4 inhibitors. Nonselective anticholinergic agents might be the best choice, because M2 muscarinic receptors on airway smooth muscle inhibit the generation and accumulation of cyclic adenosine monophosphate. Adequate concurrent blockade of M3 muscarinic receptors would be expected to counteract the enhanced acetylcholine release that would result from blockade of neuronal inhibitory M2 muscarinic receptors.
Subject(s)
Cholinergic Antagonists/therapeutic use , Lung/innervation , Parasympathetic Nervous System/physiopathology , Pulmonary Disease, Chronic Obstructive/physiopathology , Receptors, Muscarinic/physiology , Administration, Inhalation , Bronchial Hyperreactivity/physiopathology , Bronchodilator Agents/therapeutic use , Drug Therapy, Combination , Humans , Parasympathetic Nervous System/physiology , Pulmonary Disease, Chronic Obstructive/drug therapyABSTRACT
The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN-gamma and TNF-alpha, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF-alpha stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN-gamma stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF-alpha-induced CXCL10 expression in hASM was dependent on NFkappaB activation, and a salicylanilide NFkappaB inhibitor blocked the CXCL10 expression. In contrast, IFN-gamma stimulation resulted in transient NFkappaB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFkappaB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.