Abstract: Microtubule-targeting drugs play a significant role in the treatment of metastatic and drug-resistant cancers. However, the emergence of resistance to microtubule-targeting drugs in clinical settings necessitates the development of molecules with novel modes of action and mechanisms to overcome drug resistance. The multi-target drug molecule strategy is an approach that has been developed in recent years to address complex diseases and drug resistance issues. A series of compounds based on tirbanibulin with increased Fsp3 have been prepared, which have been identified with ¹H NMR, ¹³C NMR and MS. The antiproliferative activities have been evaluated against HeLa and HepG2 with MTT method. The effect on microtubule and cell cycle distribution of compound 22 has been evaluated with immunofluorescence and flow cytometry. IC₅₀ of compound 22 against HeLa and HepG2 were 45 and 51 nmol·L^-1. And it destroys microfilament and arrests cell cycle at G2/M. The SAR show that pyridine moiety in tirbanibulin cannot be replaced with 2-imidazolidinone. Compound 22 was identified as a lead in the further research.