Discovery of novel imidazo[4,5-b]pyridine derivatives as noncovalent reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) has emerged as a validated therapeutic target for B-cell malignancies and autoimmune disorders, and covalent BTK inhibitors have demonstrated remarkable clinical efficacy. However, because of concerns regarding their off-target effects and safety profiles, there is research interest in developing noncovalent, reversible inhibitors. Herein, we report the design, synthesis, and biochemical evaluation of novel imidazo[4,5-b]pyridine derivatives as noncovalent BTK inhibitors. Through a concise three-step synthetic route, we prepared 16 structurally diverse analogs (6a-p) bearing various substituents on the imidazole scaffold. The results of biochemical evaluation showed that 6b, 6o, and 6p are the most potent inhibitors, with IC₅₀ values of 1.14, 1.54, and 2.46 μM, respectively. The results of structure-activity relationship studies revealed that 2,4-dihydroxyphenyl substitution on the A-ring and extended functionalities (morpholinomethyl or 4-acetamido group) on the B-ring significantly enhanced the inhibition of BTK. The results of molecular docking studies elucidated key binding interactions and showed that the hydroxyl groups form hydrogen bonds with the Thr474 gatekeeper residue and the Met477 hinge region residue, whereas B-ring substituents extend toward the DFG motif. The preliminary kinase selectivity profiling of 6b against 39 kinases demonstrated promising selectivity, with significant inhibition observed primarily for BTK and c-Src. Although these compounds show moderate potency compared with clinically approved BTK inhibitors, their noncovalent, reversible nature offers potential advantages with regard to selectivity, toxicity, and pharmacological effects. Moreover, these compounds represent valuable starting points for further optimization toward developing novel therapeutics for B-cell malignancies and autoimmune disorders with potentially improved safety profiles for long-term treatment.