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 hydrazone compounds (TPPs) in vitro GBM cell lines. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with 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 IC₂₅ concentrations to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨ_m) 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 11.01 μM (95% CI: 10.42 to 11.64) and 13.12 μM (95% CI: 10.23 to 16.87) on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization, along with a significant reduction in colony formation. Cell cycle analysis revealed accumulation in the sub-G0 phase, consistent with increased cell death, supported by propidium iodide uptake. Furthermore, the results suggest the involvement of an apoptotic-like mechanism as supported by Annexin V positivity, γ-H2AX upregulation and transient caspase-3 activation. Conclusions: TPP25 demonstrates significant in vitro cytotoxicity, likely driven by a pro-apoptotic mechanism. This profile positions it as a potential lead compound for further preclinical evaluation, supporting its future transition into in vivo GBM models.