Optimization of Azaspirooxindolinone-based PROTACs targeting BTK: Synthesis, kinase profiling, and degradation analysis

Proteolysis-targeting chimeras (PROTACs) are a promising therapeutic modality for targeted protein degradation. Building upon our previous work on azaspirooxindolinone-based scaffolds, we synthesized a new series of BTK-targeting PROTACs using thalidomide as the CRBN-recruiting ligand. Among these, the lead compound — an azaspirooxindolinone-thalidomide conjugate with a two-methylene linker — exhibited the most potent anti-proliferative activity against BTK-high RAMOS cells (IC 50 = 4.19 ± 0.84 μM), with minimal cytotoxicity in non-cancerous fibroblasts. Molecular docking confirmed strong binding to both wild-type BTK (−10.1 kcal/mol) and the C481S mutant (−11.5 kcal/mol), and biochemical validation confirmed BTK inhibition and dose-dependent degradation. A 24 h treatment resulted in a DC 50 of 3.35 μM and Dmax of ~96% in RAMOS cells. These findings support the further development of this optimized PROTAC as a candidate BTK degrader for treating B-cell malignancies.