To develop novel GLS1 inhibitors as effective therapeutic agents for triple-negative breast cancer (TNBC), 25 derivatives were synthesized from the natural inhibitor withangulatin A (IC₅₀ = 18.2 μM). Bioassay optimization identified a novel and selective GLS1 inhibitor 7 (IC₅₀ = 1.08 μM). In MDA-MB-231 cells, 7 diminished cellular glutamate levels by blocking glutaminolysis pathway, further triggering the generation of reactive oxygen species to induce caspase-dependent apoptosis. Molecular docking indicated that 7 interacted with a new reacting site of allosteric binding pocket by forming various interactions in GLS1. The intraperitoneal administration of 7 at a dose of 50 mg/kg exhibited remarkable therapeutic effects and no apparent toxicity in the MDA-MB-231 xenograft model, indicating its potential as a novel GLS1 inhibitor for treatment of TNBC.