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.

Aragen Life Sciences Ltd Medicinal Chemistry Laboratory Division Hyderabad 500076 India

Department of Chemistry School of Science GITAM University Hyderabad 502102 Telangana India 91 9849869933

Institute of Molecular and Translational Medicine Czech Advanced Technologies and Research Institute Palacký University Olomouc Křížkovského 511 8 Olomouc 77900 Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital Olomouc Hněvotínská 1333 5 Olomouc 77900 Czech Republic 420 585 632 111

School of Chemistry and Physics College of Agriculture Engineering and Science Westville Campus University of KwaZulu Natal P Bag 10 54001 Durban 4000 South Africa 14093652520

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