A series of 1-(3,5-dimethoxyphenyl)azetidine-2-ones were synthesised to evaluate their antiproliferative activity in MCF-7 breast cancer cells and HT-29 chemoresistant colon cancer cells. The 1,4-diarylazetidin-2-ones were designed by replacing the characteristic 3,4,5-trimethoxyphenyl Ring A of the antimitotic stilbene combretastatin CA-4 with a 3,5-dimethoxyphenyl substituent at N-1, together with phenyl, hydroxyl, and phenoxy substituents at C-3 of the four-membered ring. A panel of 12 novel compounds was synthesized and evaluated in estrogen receptor (ER)- and progesterone receptor (PR)-positive MCF-7 breast cancer cells followed with the more potent compounds further evaluated in HT-29 chemoresistant colon cancer cells. Cytotoxicity was determined by LDH assay. The structures of the 1-(3,5-dimethoxyphenyl)azetidine-2-ones 12i, 12k, 12o, 12p together with the 1-(3,5-dimethoxyphenyl)azetidine-2-one 12s were determined by X-ray crystallography. The trans configuration of the C-3 and C-4 substituents of the β-lactam ring was confirmed for compounds 12k and 12u. Molecular modelling and molecular dynamics studies examined the molecular interactions of the compounds with the colchicine binding site of tubulin. The 1-(3,5-Dimethoxyphenyl)-4-(4-ethoxyphenyl)-3-hydroxyazetidin-2-one 12l was identified as the most potent antiproliferative compound in the series (with an IC₅₀ value of 10 nM in MCF-7 breast cancer cells and 3 nM in HT-29 colon cancer cells) and with greater potency than CA-4 in the chemoresistant HT-29 cells. Computational docking studies predicted binding conformations for 12l and the related series of compounds in the colchicine binding site of tubulin and rationalised the impact of the 3,5-dimethoxyphenyl substituent at N-1 of the azetidine-2-one on activity. These findings indicate that the novel 1-(3,5-dimethoxyphenyl)-2-azetidinone 12l is a suitable candidate for further investigation as a potential antiproliferative microtubule-targeting agent for breast and chemoresistant colon cancers.