Simple SummaryTo date, breast cancer remains a leading cause of cancer-related deaths among women. The triple-negative breast cancer (TNBC) subtype is the most aggressive and invasive form, and it frequently develops resistance to chemotherapy. Hormonal therapy can also be ineffective against TNBC. Herein, we synthesized two new chromene compounds and tested their activity against hormone-responsive and TNBC cells. We found that both compounds specifically inhibited the proliferation of TNBC cells with little or no effect on hormone-responsive cells. We found that our compounds disrupted the polymerization of microtubules within the cytoskeleton, which are required for proper cell division. Consequently, the TNBC cells underwent permanent growth arrest and cell death. Moreover, the chromene compounds efficiently blocked TNBC migration and inhibited the formation of blood vessels; these are two events crucial for cancer metastasis. Our findings highlight the therapeutic potential of these compounds against TNBC.AbstractBreast cancer continues to be the leading cause of cancer-related deaths among women worldwide. The most aggressive type of breast cancer is triple-negative breast cancer (TNBC). Indeed, not only does TNBC not respond well to several chemotherapeutic agents, but it also frequently develops resistance to various anti-cancer drugs, including taxane mitotic inhibitors. This necessitates the search for newer, more efficacious drugs. In this study, we synthesized two novel chromene derivatives (C1andC2) and tested their efficacy against a battery of luminal type A and TNBC cell lines. Our results show thatC1andC2significantly and specifically inhibited TNBC cell viability but had no effect on the luminal A cell type. In addition, these novel compounds induced mitotic arrest, cell multinucleation leading to senescence, and apoptotic cell death through the activation of the extrinsic pathway. We also showed that the underlying mechanisms for these actions ofC1andC2involved inhibition of microtubule polymerization and disruption of the F-actin cytoskeleton. Furthermore, both compounds significantly attenuated migration of TNBC cells and inhibited angiogenesis in vitro. Finally, we performed anin silicoanalysis, which revealed that these novel variants bind to the colchicine binding site in β-tubulin. Taken together, our data highlight the potential chemotherapeutic properties of two novel chromene compounds against TNBC.