{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T05:18:47Z","timestamp":1787721527677,"version":"build-2784847793"},"reference-count":24,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"EU","award":["2022488T5S"],"award-info":[{"award-number":["2022488T5S"]}]},{"name":"EU","award":["2022KC3X9L"],"award-info":[{"award-number":["2022KC3X9L"]}]},{"award":["2022488T5S"],"award-info":[{"award-number":["2022488T5S"]}],"id":[{"id":"https:\/\/ror.org\/019w4f821","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2022KC3X9L"],"award-info":[{"award-number":["2022KC3X9L"]}],"id":[{"id":"https:\/\/ror.org\/019w4f821","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>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 (\u0394\u03a8m) 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 \u03bcM (95% CI: 10.42 to 11.64) and 13.12 \u03bcM (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, \u03b3-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.<\/jats:p>","DOI":"10.3390\/ph19091335","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T09:20:02Z","timestamp":1787563202000},"page":"1335","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of Pyrazolone-Based Hydrazones as Potential Therapeutic Agents Against Glioblastoma"],"prefix":"10.3390","volume":"19","author":[{"given":"Giorgio","family":"Cameli","sequence":"first","affiliation":[{"name":"Immunopathology and Molecular Medicine Unit, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7885-0003","authenticated-orcid":false,"given":"Alessia","family":"Piergentili","sequence":"additional","affiliation":[{"name":"Chemistry Interdisciplinary Project (ChIP) Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1216-1368","authenticated-orcid":false,"given":"Eleonora","family":"Spinozzi","sequence":"additional","affiliation":[{"name":"Chemistry Interdisciplinary Project (ChIP) Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2765-4587","authenticated-orcid":false,"given":"Alessia","family":"Tombesi","sequence":"additional","affiliation":[{"name":"Chemistry Interdisciplinary Project (ChIP) Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4760-1204","authenticated-orcid":false,"given":"Riccardo","family":"Petrelli","sequence":"additional","affiliation":[{"name":"Chemistry Interdisciplinary Project (ChIP) Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8155-7211","authenticated-orcid":false,"given":"Loredana","family":"Cappellacci","sequence":"additional","affiliation":[{"name":"Chemistry Interdisciplinary Project (ChIP) Research Center, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4614-5649","authenticated-orcid":false,"given":"Maria Beatrice","family":"Morelli","sequence":"additional","affiliation":[{"name":"Immunopathology and Molecular Medicine Unit, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Becker, A.P., Sells, B.E., Haque, S.J., and Chakravarti, A. (2021). Tumor Heterogeneity in Glioblastomas: From Light Microscopy to Molecular Pathology. Cancers, 13.","DOI":"10.3390\/cancers13040761"},{"key":"ref_2","first-page":"139","article-title":"The Central Brain Tumor Registry of the United States Histopathological Grouping Scheme Provides Clinically Relevant Brain and Other Central Nervous System Categories for Cancer Registry Data","volume":"49","author":"Ostrom","year":"2022","journal-title":"J. Regist. Manag."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Obrador, E., Moreno-Murciano, P., Oriol-Caballo, M., L\u00f3pez-Blanch, R., Pineda, B., Guti\u00e9rrez-Arroyo, J.L., Loras, A., Gonzalez-Bonet, L.G., Martinez-Cadenas, C., and Estrela, J.M. (2024). Glioblastoma Therapy: Past, Present and Future. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25052529"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kr\u00f3likowska, K., B\u0142aszczak, K., \u0141awicki, S., Zajkowska, M., and Gudowska-Sawczuk, M. (2025). Glioblastoma\u2014A Contemporary Overview of Epidemiology, Classification, Pathogenesis, Diagnosis, and Treatment: A Review Article. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms262412162"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/S1366-0071(97)80033-2","article-title":"The Early History of Non-Opioid Analgesics","volume":"1","author":"Brune","year":"1997","journal-title":"Acute Pain"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"133044","DOI":"10.1016\/j.molstruc.2022.133044","article-title":"A Critical Review on Recent Trends on Pharmacological Applications of Pyrazolone Endowed Derivatives","volume":"1262","author":"Mustafa","year":"2022","journal-title":"J. Mol. Struct."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/j.cdc.2018.11.004","article-title":"Green Synthesis of Coumarin-Pyrazolone Hybrids: In Vitro Anticancer and Anti-Inflammatory Activities and Their Computational Study on COX-2 Enzyme","volume":"17\u201318","author":"Kulkarni","year":"2018","journal-title":"Chem. Data Collect."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"138634","DOI":"10.1016\/j.molstruc.2024.138634","article-title":"Synthesis, Anti-Inflammatory, Antibacterial, and Antioxidant Evaluation of Novel Pyrazole-Linked Hydrazone Derivatives","volume":"1312","author":"Kamat","year":"2024","journal-title":"J. Mol. Struct."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1108","DOI":"10.2174\/138955707782331722","article-title":"Privileged Structures: A Useful Concept for the Rational Design of New Lead Drug Candidates","volume":"7","author":"Duarte","year":"2007","journal-title":"Mini-Rev. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13561","DOI":"10.1021\/acs.inorgchem.2c02201","article-title":"Zinc(II) Complex with Pyrazolone-Based Hydrazones Is Strongly Effective against Trypanosoma Brucei Which Causes African Sleeping Sickness","volume":"61","author":"Marchetti","year":"2022","journal-title":"Inorg. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"144667","DOI":"10.1016\/j.molstruc.2025.144667","article-title":"Synthesis, Computational and Biological Evaluation of Imidazole-Pyrazole Hydrazones as Antitubercular Agents","volume":"1353","author":"Dhurjad","year":"2026","journal-title":"J. Mol. Struct."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kozmenko, Y.V., Khisamov, M.M., Revtovich, S.V., Korolev, S.P., Sherman, D.K., Spiridonov, V.V., Kalnina, L.B., Valuev-Elliston, V.T., Gottikh, M.B., and Kochetkov, S.N. (2025). Anti-HIV and Antimicrobial Activity of 7-Hydrazino-8-Hydroxyquinoline-Based Aromatic Hydrazones. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26178402"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14123","DOI":"10.1021\/acs.inorgchem.8b01935","article-title":"Ligand Design for N,O- or N,N-Pyrazolone-Based Hydrazones Ruthenium(II)-Arene Complexes and Investigation of Their Anticancer Activity","volume":"57","author":"Pettinari","year":"2018","journal-title":"Inorg. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"139523","DOI":"10.1016\/j.molstruc.2024.139523","article-title":"Synthesis, Anticancer and Antioxidant Activities of Novel Heterocyclic Phenolic Hydrazone Based Derivatives: Investigation of DFT Calculation, Molecular Docking and Drug-Likeness Studies","volume":"1319","author":"Xing","year":"2025","journal-title":"J. Mol. Struct."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Natorska-Chomicka, D., Gawro\u0144ska-Grzywacz, M., Patrejko, P., Herbet, M., Pi\u0105tkowska-Chmiel, I., Iwan, M., Dudka, J., and Popio\u0142ek, \u0141. (2026). Novel Experimental Therapeutic Approaches in Glioma\u2014New Hydrazide\u2013Hydrazones as Chemical Agents Sensitizing Glioblastoma Cell Line to Radiotherapy. Int. J. Mol. Sci., 27.","DOI":"10.3390\/ijms27125459"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7441","DOI":"10.1021\/acs.jmedchem.4c03150","article-title":"New Arylpiperazines as Potent and Selective Dopamine D4 Receptor Ligands Potentially Useful to Treat Glioblastoma","volume":"68","author":"Matteucci","year":"2025","journal-title":"J. Med. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Nov\u00e1s, M., and Matos, M.J. (2025). The Role of Trifluoromethyl and Trifluoromethoxy Groups in Medicinal Chemistry: Implications for Drug Design. Molecules, 30.","DOI":"10.3390\/molecules30143009"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"102437","DOI":"10.1016\/j.xpro.2023.102437","article-title":"Synthesis of fluorinated triphenylphosphonium analogs that improve cancer cell selectivity and in vivo detection","volume":"4","author":"Keyes","year":"2023","journal-title":"STAR Protoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"105670","DOI":"10.1016\/j.isci.2022.105670","article-title":"Fluorinated triphenylphosphonium analogs improve cell selectivity and in vivo detection of mito-metformin","volume":"25","author":"AbuEid","year":"2022","journal-title":"iScience"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"9357","DOI":"10.1074\/jbc.273.16.9357","article-title":"Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis","volume":"273","author":"Janicke","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1093\/neuonc\/nov315","article-title":"An intrinsic DFF40\/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: A common trait in human glioblastoma cells","volume":"18","author":"Velasco","year":"2016","journal-title":"Neuro Oncol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1458","DOI":"10.1038\/nprot.2006.238","article-title":"Analysis of apoptosis by propidium iodide staining and flow cytometry","volume":"1","author":"Riccardi","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"14165","DOI":"10.1039\/D2DT02430F","article-title":"Role of Hydrazone Substituents in Determining the Nuclearity and Antibacterial Activity of Zn(II) Complexes with Pyrazolone-Based Hydrazones","volume":"51","author":"Marchetti","year":"2022","journal-title":"Dalton Trans."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Aguzzi, C., Perinelli, D.R., Cespi, M., Zeppa, L., Mazzara, E., Maggi, F., Petrelli, R., Bonacucina, G., and Nabissi, M. (2023). Encapsulation of hemp (Cannabis sativa L.) essential oils into nanoemulsions for potential therapeutic applications: Assessment of cytotoxicological profiles. Molecules, 28.","DOI":"10.20944\/preprints202307.1840.v1"}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/9\/1335\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T04:21:55Z","timestamp":1787718115000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/9\/1335"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,24]]},"references-count":24,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ph19091335"],"URL":"https:\/\/doi.org\/10.3390\/ph19091335","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,24]]}}}