A series of twenty-five 2-azitidinone (β-lactam) derivatives were synthesized and evaluated for anti-cancer properties against breast cancer, MCF-7 and MDA-MB-231. These β-lactam derivatives depicted significant cytotoxicity in cancer cell lines but not in normal human mammary epithelial cells, MEpiC. Interestingly, derivatives of 2-bromo ethyl acrylonitrile (19w) exhibited - potent anti-proliferative activity with IC50, 5.79 ± 0.01 μM in MCF-7 and 6.86 ± 0.009 μM in MDA-MB-231. In addition, an increased expression of pro-apoptotic genes (p53, Bax, Bid) as well as decreased mRNA expression of cyclins D1, E and Cdk 2, 6 along with cell cycle arrest at G1 phase was observed. 19w treatment has shown higher percentage of Annexin-positive cells indicating induction of apoptosis. Further, docking studies confirmed an interaction between 19w and ATP-binding catalytic site of AKT1. Mechanistically, 19w depicted dose-dependent decrease in phosphorylation of AKT and GSK-3β and significant decrease in AKT kinase activity. In conclusion, β-lactam derivative 19w is a potential anti-breast cancer therapeutic candidate targeting cell survival pathway (AKT/GSK3β).