Structure-based optimization identified novel furyl-containing 2,4-diarylaminopyrimidine analogues as ALK/ROS1 dual...

Aiming to develop ALK/ROS1 dual inhibitors overcoming ceritinib-resistant G1202R mutant, a dedicated structure-guided modification campaign was conducted based on ALK co-crystal structures. Twenty eight diarylaminopyrimidine (DAAP) analogues possessing furan or tetrahydrofuran group were designed and synthesized, among which compound 16 bearing (dimethylamino)methyl)furan-2-yl)methyl)thio fragment was identified. Compound 16 exhibited significant cytotoxicity on ALK-positive Karpas299 and H2228 cells with IC₅₀ values of 20 nM and 110 nM. Meanwhile, compound 16 turned out as the most potent entity superior to ceritinib with IC₅₀ values of 2.8, 2.6, 3.8 and 2.3 nM against ALK^WT, ALK^L1196M, ALK^G1202R and ROS1^WT, respectively. Subsequently, western blot assay showed that compound 16 significantly suppressed ALK and its downstream protein expression in a dose-dependent manner. Alternatively, the Hoechst 33258 and AO/EB staining assays illustrated that compound 16 could induce H2228 cell apoptosis. Ultimately, the binding models of compound 16 with ALK^WT, ALK^G1202R as well as ROS1 clearly presented the essential interactions within the active site. Together, compound 16 was validated as a promising ALK/ROS1 dual inhibitor for ALK^G1202R mutation correlated tumors.