Discovery and in vitro and in vivo activity evaluation of novel baicalein phosphonium salt derivatives targeting mitochondrial MTHFD2 as anti-colon cancer agents.

Mitochondria are critical for the malignant proliferation of tumor cells and their adaptation to harsh microenvironments. Using baicalein as the substrate, we designed and synthesized a series of novel mitochondrial-targeting baicalein phosphonium salt derivatives, and evaluated their in vitro antiproliferative activities against four tumor cell lines (A549, HCT116, SW620, CT26) and the normal HEK293 cell line. Compound 2c exhibited potent activity against the colon cancer cell line HCT116 with an IC₅₀ of 5.17 ± 0.48 μM and a selectivity index (SI) of ≈3.42, outperforming the positive control doxorubicin (DOX, SI ≈ 2.19). HPLC analysis confirmed the accumulation of 2c in tumor mitochondria, while in silico studies suggested its binding to MTHFD2, a mitochondrially localized protein upregulated in cancer cells. Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) verified that 2c directly binds to MTHFD2 intracellularly, and enzymatic assays demonstrated an IC₅₀ of 0.066 ± 0.014 μM against MTHFD2. Furthermore, 2c remarkably increased intracellular reactive oxygen species (ROS) levels, induced mitochondrial membrane potential depolarization, arrested the cell cycle at G0/G1 phase, and promoted apoptosis. In a mouse colon cancer graft model, compound 2c achieved a 49.70% tumor inhibition rate at a dose of 20 mg/kg, with no obvious abnormalities observed in major organs. In conclusion, 2c is an effective mitochondrial MTHFD2 inhibitor with potential to develop into a potent anti-colon cancer drug.