Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are two important members of the Tec family with crucial roles in immune system function. Deregulation in ITK and BTK activity is linked to several hematological malignancies, making them key targets for cancer immunotherapy. In this study, we synthesized a series of azaspirooxindolinone derivatives and evaluated their cytotoxic activity against ITK/BTK-negative and positive cancer cell lines, followed by enzymatic inhibition studies to assess the ITK/BTK kinase selectivity of two hit compounds. Several compounds demonstrated selective cytotoxicity against ITK- or BTK-expressing cells. Compound 3d exhibited high cytotoxicity in ITK-positive Jurkat (IC50 = 3.58 µM) and BTK-positive Ramos (IC50 = 3.06 µM) cells, while compound 3j showed strong cytotoxicity in Ramos (IC50 = 1.38 µM) and Jurkat (IC50 = 4.16 µM) cells. Compounds 3a and 3e were selectively cytotoxic in Jurkat cells (IC50 = 9.36 µM and 10.85 µM, respectively), while compounds 3f and 3g were highly cytotoxic in Ramos cells (IC50 = 1.82 µM and 1.42 µM, respectively). None of the active compounds exhibited cytotoxicity in non-cancer cell lines (IC50 > 50 µM), demonstrating their selectivity for malignant cells. Enzyme inhibition assay showed that 3d is a selective ITK inhibitor (IC50 = 0.91 µM) with no detectable BTK inhibition, aligning with its strong activity in ITK-positive cells. In contrast, compound 3j did not inhibit ITK or BTK enzymatically, suggesting an alternative mechanism of action. These findings highlight 3d as a promising ITK inhibitor and warrant further investigation to elucidate its mechanism of action.

Aragen Lifesciences Pvt Ltd Medicinal Chemistry Laboratory Division Survey No 125 and 126 IDA Mallapur Hyderabad 500076 India

Aragen Lifesciences Pvt Ltd Medicinal Chemistry Laboratory Division Survey No 125 and 126 IDA Mallapur Hyderabad 500076 India; Department of Chemistry School of Science GITAM University Hyderabad 502102 Telangana India

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

Institute of Molecular and Translational Medicine Czech Advanced Technologies and Research Institute Palacký University Olomouc Křížkovského 511 8 779 00 Olomouc Czech Republic; Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital Olomouc Hněvotínská 1333 5 77900 Olomouc Czech Republic

NATCO Research Center Sanath Nagar Industrial Area Sanath Nagar Hyderabad 500018 Telangana India

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