Development of novel Copper(II)-hydrazone complexes for targeting the inhibition of human osteosarcoma growth

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults and remains associated with poor clinical outcomes due to recurrence, metastasis, and resistance to conventional chemotherapy. In recent years, copper-based compounds have emerged as promising anticancer agents because of their unique redox properties and ability to target multiple cellular pathways.

In this work, we synthesized and characterized two hydrazone ligands, (E)-N'-(2-hydroxybenzylidene)furan-2-carbohydrazide (L1) and (E)-N'-(2-hydroxybenzylidene)-4-methoxybenzohydrazide (L2), together with their corresponding Cu(II) complexes, CuL1 and Cu₂L2₂. The compounds were characterized by elemental analysis, spectroscopic techniques, high-resolution mass spectrometry, and single-crystal X-ray diffraction. The biological activity of the complexes was evaluated in human MG-63 osteosarcoma cells using the MTT assay. Reactive oxygen species (ROS) generation was assessed using dihydrorhodamine 123 (DHR123) after 24 h of treatment to gain insight into their mechanism of action. Their activity was further evaluated in 3D MG-63 osteosarcoma spheroids using a resazurin-based viability assay, while anti-migratory effects were assessed using the spheroid spreading assay.

Biological evaluation demonstrated that coordination to Cu(II) converted the inactive hydrazone ligands into highly potent anticancer agents. Both Cu(II) complexes exhibited potent cytotoxic activity in 2D cell cultures, with IC₅₀ values in the low micromolar range and significantly higher activity than cisplatin, whereas the free ligands showed no significant antiproliferative effect. Furthermore, treatment with CuL1 and Cu₂L2₂ induced a rapid increase in intracellular ROS, suggesting that oxidative stress contributes to their mechanism of action. The antitumor potential of the complexes was further assessed using multicellular tumor spheroids, where both compounds reduced spheroid viability, altered spheroid morphology, and significantly inhibited spheroid-derived cell migration.

Among the compounds studied, the binuclear complex Cu₂L2₂ displayed the highest antiproliferative activity in both 2D and 3D models. Overall, our findings demonstrate that coordination of hydrazone ligands to Cu(II) markedly enhances their biological activity and identify CuL1 and particularly the binuclear complex Cu₂L2₂ as promising candidates for the development of new copper-based therapeutics against osteosarcoma.