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

Herein,\nwe described the rational drug design and synthesis of\na series of 5-amino-4-fluoro-1<i>H</i>-benzo[<i>d</i>]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases.\nThe detailed screening cascades revealed that <b>16b</b> was\na preferred compound, which might act like a “clamp”\nto stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1,\nBRAF, and BRAF<sup>V600E</sup> with IC<sub>50</sub> values of 28,\n3, and 3 nM, respectively. <b>16b</b> possessed an excellent\nselectivity over other 312 human-related kinases at 1 μM. In\nvitro, <b>16b</b> showed potent antiproliferative activities\nagainst MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D\nKRAS) cells with IC<sub>50</sub> values of 0.011, 0.079, and 0.096\nμM, respectively. CoIP experiments demonstrated that <b>16b</b> could induce MEK/RAF complex formation. Most importantly, in the\nC26 syngeneic colorectal and HCT116 mice xenograft tumor models, <b>16b</b> demonstrated tumor growth inhibition of 70 and 93%, respectively,\nsuggesting that <b>16b</b> may be a promising MEK/RAF complex\ninhibitor and worthy of further development.