ABSTRACT
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease linked to oxidative stress, which is associated with significant morbidity. The NADPH oxidase complex (NOX) produces reactive oxygen species (ROS) that are among the key markers for determining RA's pathophysiology. Therefore, understanding ROS-regulated molecular pathways and their interaction is necessary for developing novel therapeutic approaches for RA. Here, by combining mouse genetics and biochemistry with clinical tissue analysis, we reveal that in vivo Rubicon interacts with the p22phox subunit of NOX, which is necessary for increased ROS-mediated RA pathogenesis. Furthermore, we developed a series of new aryl propanamide derivatives consisting of tetrahydroindazole and thiadiazole as p22phox inhibitors and selected 2-(tetrahydroindazolyl)phenoxy-N-(thiadiazolyl)propanamide 2 (TIPTP, M.W. 437.44), which showed considerably improved potency, reaching an IC50 value up to 100-fold lower than an inhibitor that we previously synthesized reported N8 peptide-mimetic small molecule (blocking p22phox-Rubicon interaction). Notably, TIPTP treatment showed significant therapeutic effects a mouse model for RA. Furthermore, TIPTP had anti-inflammatory effects ex vivo in monocytes from healthy individuals and synovial fluid cells from RA patients. These findings may have clinical applications for the development of TIPTP as a small molecule inhibitor of the p22phox-Rubicon axis for the treatment of ROS-driven diseases such as RA.
Subject(s)
Arthritis, Rheumatoid/metabolism , Autophagy-Related Proteins/metabolism , Indazoles/administration & dosage , Indazoles/chemical synthesis , NADPH Oxidases/metabolism , Thiadiazoles/administration & dosage , Thiadiazoles/chemical synthesis , Aged , Animals , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/genetics , Autophagy-Related Proteins/genetics , Disease Models, Animal , Female , HEK293 Cells , Humans , Indazoles/chemistry , Indazoles/pharmacology , Inhibitory Concentration 50 , Mice , Middle Aged , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/genetics , Oxidative Stress , Protein Binding/drug effects , RAW 264.7 Cells , Reactive Oxygen Species/metabolism , Synovial Fluid/cytology , Synovial Fluid/drug effects , Synovial Fluid/metabolism , Thiadiazoles/chemistry , Thiadiazoles/pharmacologyABSTRACT
This article describes the synthesis and biological evaluation of a chemical library of mibefradil analogues to investigate the effect of structural modification on in vitro stability. The construction of the dihydrobenzopyran structure in mibefradil derivatives 2 was achieved through two efficient approaches based on a diastereoselective intermolecular Reformatsky reaction and an intramolecular carbonyl-ene cyclization. In particular, the second strategy through the intramolecular carbonyl-ene reaction led to the formation of a key intermediate 3 in a short and highly stereoselective way, which has allowed for practical and convenient preparation of analogues 2. Using this protocol, we could obtain 22 new mibefradil analogues 2, which were biologically tested for in vitro efficacies against T-type calcium channels and metabolic stabilities. Among the synthesized compounds, we found that analogue 2aa containing a dihydrobenzopyran ring and a secondary amine linker showed high % remaining activities of the tested CYP enzymes retaining the excellent T-type calcium channel blocking activity. These findings indicated that the structural modification of 1 was effective for improving in vitro stability, i.e., reducing CYP inhibition and metabolic degradation.
Subject(s)
Chemistry, Organic/methods , Mibefradil/analogs & derivatives , Mibefradil/chemical synthesis , Aldehydes/chemical synthesis , Aldehydes/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/metabolism , Crystallography, X-Ray , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Drug Stability , HEK293 Cells , Humans , Mibefradil/chemistry , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular ConformationABSTRACT
We have synthesized and biologically evaluated 1,4-diazepane derivatives as T-type calcium channel blockers. In this study, we discovered compound 4s, a potential T-type calcium channel blocker with good selectivity over hERG and N-type calcium channels. In addition, it exhibited favorable pharmacokinetic characteristics for further investigation of T-type calcium channel related diseases.
