Evaluation of Pyrazolone-Based Hydrazones as Potential Therapeutic Agents Against Glioblastoma

Background: Glioblastoma (GBM) is the most aggressive subtype of malignant glioma. Current therapeutic options remain limited, highlighting the need for the development of novel compounds capable of improving the efficacy of standard treatments. This study aimed to evaluate the biological activity of a series of pyrazolone-based hydra-zone compounds (TPP) against GBM. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with the 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylethan-1-one, and tested on two human GBM cell lines, T98 and U87. The cytotoxic effects were evaluated via MTT assay. The most active compound was further investigated at IC₅₀ and IC₇₅ concentra-tions, to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔѰm) changes. Additional assays included colony formation, cell cycle analysis, and evaluation of DNA damage and apoptosis markers. Results: TPP25 exhibited the highest activity with IC₅₀ values of 11 and 13 µM on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization within 24 h, along with a significant reduction in colony formation. Cell cycle analysis revealed sub-G0 accumulation, consistent with increased cell death, confirmed by propidium iodide uptake. TPP25 also increased phosphorylated ɣ-H2A.X and cleaved Caspase-3 expression. Conclusions: TPP25 emerges as a promising candidate for GBM treatment, demonstrating significant cytotoxic activity.