Structure-based design and synthesis of KX-01 analogs as potent antitumor agents targeting the tubulin colchicine binding site.

Our preliminary studies indicated that KX-01 inhibits tubulin polymerization in a reversible and concentration-dependent manner, resulting in dramatically low toxicity across various solid and liquid tumor types. However, KX-01 has not yet been approved as an anticancer agent due to its insufficient efficacy, and research on its derivatives remains limited. To improve its antitumor activity and investigate the structure-activity relationships (SARs), sixty-seven KX-01 analogs were designed and synthesized based on the KX-01-tubulin cocrystal structure. Among them, compound 8h exhibited the most potent antiproliferative activity, with IC50 values of 3.5 ± 0.6, 2.4 ± 0.2, 15.7 ± 3.1, 22.1 ± 1.9, and 7.3 ± 1.1 nM against HCT116, HeLa, A2780S, A2780T, and HT29 cells, respectively, indicating its potential to overcome multidrug resistance. Replacing the endocyclic nitrogen atom in the pyridine ring of KX-01 with an exocyclic fluorine atom directly results in the loss of Src inhibitory activity. The cocrystal of 8h-tubulin complex revealed that it simultaneously occupies the colchicine site in β-tubulin and a cavity in α-tubulin. In the HT29 xenograft model, orally administered 8h (5 mg/kg, once daily) showed marginally superior in vivo antitumor efficacy than KX-01.