Biotinylated Diaryl Isoxazole Derivatives with Enhanced Janus Kinase 1 Inhibitory, Antiproliferative, and Apoptotic...

Isoxazole derivatives have emerged as promising scaffolds in anticancer drug discovery, yet their therapeutic potential remains to be fully harnessed. Here, we report on the synthesis and biological evaluation of two novel biotin-conjugated isoxazole derivatives (C160 and C161) related to the parent compound EB38. Across a panel of liver, prostate, and breast cancer cell lines, both derivatives exhibited significantly enhanced antiproliferative and pro-apoptotic activities compared to EB38, with consistently lower IC₅₀ values in 2D cultures and superior efficacy in 3D spheroid models. Mechanistic studies revealed that C160 and C161 robustly inhibit phosphorylation of JAK1 and STAT1, thereby suppressing JAK/STAT signaling and inducing apoptotic marker cleavage (PARP, Caspase-3, Caspase-7). Direct target engagement was further validated by CETSA and DARTS assays, which demonstrated thermal and proteolytic stabilization of endogenous JAK1 in the presence of C160. Molecular docking and HADDOCK simulations supported JAK1 as a direct cellular target, with binding affinities comparable to the clinically approved inhibitor Ruxolitinib. Importantly, zebrafish embryotoxicity assays confirmed that these derivatives do not elicit general cytotoxicity, even at doses substantially exceeding those effective in vitro. Collectively, our findings establish biotinylated isoxazole derivatives as potent JAK1-targeting anticancer agents with strong antiproliferative and pro-apoptotic activity, supporting their further development as promising candidates for targeted cancer therapy.