Cyclic bridged analogues of Combretastatin A-4: Design, synthesis and biological evaluation of 3-chloroazetidin-2-ones in breast cancer cells

Antimitotic drugs that target tubulin are among the most widely used chemotherapeutic agents; however, the development of multidrug resistance has limited their clinical activity. We have previously investigated the antiproliferative activity of the 1,4-diarylazetidin-2-ones (β-lactam) scaffold in breast cancer cell lines. We now report the synthesis and biological properties of a series of novel 3-chloro-β-lactams (2-azetidinone) and 3,3-dichloro-β-lactams, which are structurally related to Combretastatin CA-4. These compounds were evaluated as potential tubulin polymerization inhibitors and for their antiproliferative effects in breast cancer cells. A number of the compounds showed potent activity breast cancer cells e.g. compound 13 (3-chloro-4-(3-hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) with an IC₅₀ values of 17 nM and 32 nM in MCF-7 and MDA-MB-231 breast cancer cells respectively and displayed comparable cellular effects to those of Combretastatin A-4. Compound 13 displayed minimal cytotoxicity against non-tumorigenic HEK-293T cells and inhibited the in vitro polymerisation of tubulin with significant G₂/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 13 caused mitotic catastrophe by targeting tubulin. In addition, compound 13 promoted apoptosis by regulating the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2 and Mcl-1. Molecular docking was used to explore the potential molecular interactions between novel 3-chloro-β-lactams and residues in the colchicine binding active site cavity of b-tubulin. Collectively, these results suggest that 3-chloro-2-azetidinones such as compound 13 could be a promising lead compound for further clinical anti-cancer drug development.