Identification of a novel Azaspirooxindolinone-based PROTAC for selective BTK degradation and enhanced anticancer activity

Bruton's Tyrosine Kinase (Btk) is a key driver of hematological malignancies, autoimmune disorders, and neuroinflammation, making it an attractive therapeutic target. Proteolysis targeting chimeras (PROTACs) offer a novel strategy for Btk degradation via the E3 ubiquitin Ligase pathway. Here, we evaluated nine azaspirooxindolinone-based PROTAC derivatives for their cytotoxicity and BTK-targeting activity. Several compounds exhibited potent cytotoxicity against BTK-high RAMOS lymphoma cells without affecting non-cancer fibroblasts or normal T/B-cell lymphocytes. Among them, PROTAC 25 emerged as the most effective degraded, achieving a Dmax of 72.84 % and DC50 of 0.27 μM in a proteasome-dependent manner. Although PROTAC 25 was cytotoxic to IL-2-inducible T cell Kinase (Itk)-positive cells, Itk protein levels remained unaffected. Furthermore, kinase assays revealed that PROTAC 25 inhibited Btk kinase activity (IC₅₀ = 0.44 μM) with moderate selectivity over Itk (IC₅₀ = 2.16 μM). Notably, PROTAC 25 suppressed BTK-mediated downstream signaling in RAMOS cells, as evidenced by reduced phosphorylation of Btk and its downstream effector, p38 MAPK. These findings highlight PROTAC 25 as a promising Btk degrader with therapeutic potential and underscore the value of azaspirooxindolinone-based PROTACs in targeting BTK-driven diseases.