ABSTRACT
Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Purines/chemistry , Purines/pharmacology , Agammaglobulinaemia Tyrosine Kinase , Animals , Autoimmune Diseases/drug therapy , Autoimmune Diseases/enzymology , B-Lymphocytes/drug effects , Crystallography, X-Ray , Humans , Mice , Models, Molecular , Passive Cutaneous Anaphylaxis/drug effects , Protein-Tyrosine Kinases/metabolism , RatsABSTRACT
Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/toxicity , Esters/chemistry , Mice , Molecular Structure , Piperazine , Piperazines/chemistry , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in inflammation by catalyzing the release of arachidonic acid, a substrate for lipoxygenase and cyclooxygenase enzymes, from membrane phospholipids. In the present study we examined the role of cPLA(2) in inflammatory responses through the use of a specific inhibitor of the enzyme, cPLA(2), arachidonyl trifluoromethyl ketone (AACOCF3). Interestingly, we observed that AACOCF3 is an inhibitor of chronic but not acute inflammatory responses. Specifically, AACOCF3 inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice. Additionally, oral treatment of ovalbumin-sensitized/ovalbumin-challenged BALB/c mice with 20 mg/kg AACOCF3 prevented the development of airway hyper-responsiveness in a model of asthma. Furthermore, AACOCF3 decreased cellular recruitment in the airway lumen and airway inflammation after the ovalbumin challenge. Taken together, these results suggest that a potent and specific chemical inhibitor of cPLA(2) may be useful for the treatment of chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.
Subject(s)
Arachidonic Acids/administration & dosage , Cytosol/enzymology , Drug Delivery Systems/methods , Edema/enzymology , Edema/prevention & control , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Allergens/adverse effects , Animals , Asthma/drug therapy , Asthma/enzymology , Asthma/pathology , Cell Movement/drug effects , Chronic Disease , Cytosol/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Female , Group IV Phospholipases A2 , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/prevention & control , Leukocytes/pathology , Male , Mice , Mice, Inbred BALB C , Phospholipases A2ABSTRACT
Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.
Subject(s)
Genes, Reporter/genetics , Receptor, Adenosine A2A/genetics , Transfection/methods , Adenosine A2 Receptor Agonists , Adenosine-5'-(N-ethylcarboxamide)/pharmacology , Animals , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Genes, Reporter/drug effects , Humans , Receptor, Adenosine A2A/metabolism , beta-Galactosidase/metabolismABSTRACT
The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn's disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-alpha in vivo.