Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1<i>H</i>-benzo[<i>d</i>]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the “Clamp” Strategy

Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1<i>H</i>-benzo[<i>d</i>]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that <b>16b</b> was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAF^V600E with IC₅₀ values of 28, 3, and 3 nM, respectively. <b>16b</b> possessed an excellent selectivity over other 312 human-related kinases at 1 μM. In vitro, <b>16b</b> showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC₅₀ values of 0.011, 0.079, and 0.096 μM, respectively. CoIP experiments demonstrated that <b>16b</b> could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, <b>16b</b> demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that <b>16b</b> may be a promising MEK/RAF complex inhibitor and worthy of further development.