Design, synthesis, and biological evaluation of novel 3-oxo-2,3-dihydropyridazine derivatives as interleukin-2-inducible T-cell kinase (ITK) inhibitors

Abstract

This study reports the design, synthesis, and biological evaluation of a novel series of 3-oxo-2,3-dihydropyridazine derivatives, representing a previously unexplored scaffold for selective inhibition of interleukin-2-inducible T-cell kinase (ITK), with potential application in T-cell leukemia treatment. Cytotoxicity was assessed across a panel of ITK-expressing leukemia cell lines (Jurkat, CCRF-CEM), Bruton's tyrosine kinase (BTK)-positive lines (Ramos, K562), ITK/BTK-null cancer cells, and non-cancerous fibroblasts to determine therapeutic selectivity. Compound 9 emerged as the lead candidate, showing selective ITK inhibition in biochemical kinase assays (half-maximal inhibitory concentration, IC₅₀ = 0.87 µM) with no measurable BTK inhibition, moderate cytotoxicity in Jurkat cells (cellular IC₅₀ = 37.61 µM), and did not show measurable cytotoxicity in fibroblasts (IC₅₀ > 50 µM). In contrast, 22 exhibited greater potency in both kinase [IC₅₀ (ITK) = 0.19 µM] and cytotoxicity assay [IC₅₀ (Jurkat) = 11.17 µM], but showed partial BTK inhibition, indicating reduced selectivity. Structure–activity relationship analysis indicated that the 3,5-difluorophenyl and furan-2-ylmethyl groups in 22 contributed to potency, while the 3-fluorophenyl group in 9 was associated with improved selectivity. Importantly, western blot analysis confirmed that 9 reduced phosphorylation of ITK (Tyr551/Tyr511) and downstream extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204) in phytohemagglutinin-stimulated Jurkat cells, supporting on-target inhibition of ITK signaling. These results position 9 as a selective ITK inhibitor with a favorable therapeutic index, establishing a foundation for further optimization and preclinical development.