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
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.