Nitrogen mustards, widely used as chemotherapeutics, have limited safety and eﬃcacy. Mitochondria lack a functional nucleotide excision repair mechanism to repair DNA adducts and are sensitive to alkylating agents. Importantly, cancer cells have higher intrinsic mitochondrial membrane potential (Δψmt) than normal cells. Therefore, selectively targeting nitrogen mustards to cancer cell mitochondria based on Δψmt could overcome those limitations. Herein, we describe the design, synthesis, and evaluation of Mito-Chlor, a triphenylphosphonium derivative of the nitrogen mustard chlorambucil. We show that Mito-Chlor localizes to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death, resulting in a 80-fold enhancement of cell kill in a panel of breast and pancreatic cancer cell lines that are insensitive to the parent drug. Signiﬁcantly, Mito-Chlor
delayed tumor progression in a mouse xenograft model of human pancreatic cancer. This is a ﬁrst example of repurposing chlorambucil, a drug not used in breast and pancreatic cancer treatment, as a novel drug candidate for these diseases.