{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T06:10:24Z","timestamp":1785391824234,"version":"3.55.0"},"reference-count":41,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T00:00:00Z","timestamp":1769472000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003336","name":"Bulgarian National Science Fund","doi-asserted-by":"crossref","award":["KP-06-H61\/1"],"award-info":[{"award-number":["KP-06-H61\/1"]}],"id":[{"id":"10.13039\/501100003336","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: The development of small-molecule agents that selectively target DNA replication remains a central strategy in anticancer drug discovery. In this study, we report the biological characterization of a novel 6-nitro-benzodioxin-naphthalimide (NI) derivative (compound 5a), evaluated as a potential DNA-targeted anticancer lead. Methods\/Results: The antiproliferative activity of 5a was assessed in a small panel of human lung carcinoma cell models (A549, H1299) and a non-malignant control (MRC-5), revealing pronounced cytotoxic effects in tumor cells, accompanied by favorable selectivity indices. Mechanistic investigations demonstrated that treatment with 5a results in strong inhibition of DNA synthesis, as evidenced by a marked reduction in EdU incorporation and a robust induction of the DNA damage marker \u03b3H2AX. These effects were associated with cell-cycle perturbations characterized by accumulation in G1 and G2\/M phases, followed by activation of apoptotic pathways. Importantly, clonogenic survival assays confirmed that even transient exposure to 5a leads to a sustained loss of proliferative capacity, indicating irreversible long-term cellular damage. These results support a replication stress-driven mechanism of action for compound 5a, consistent with interference in DNA-associated processes during S phase. Conclusions: While the precise molecular initiating event remains to be elucidated, the observed biological profile positions 5a as a promising DNA-targeted lead structure with potential for further pharmaceutical optimization. These findings provide a solid foundation for the continued development of naphthalimide-based compounds as anticancer agents within a pharmaceutically relevant framework.<\/jats:p>","DOI":"10.3390\/pharmaceutics18020167","type":"journal-article","created":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T15:41:56Z","timestamp":1769528516000},"page":"167","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Benzodioxin-Annulated Naphthalimides as Potent DNA Replication Stress Inducers with Dual p53-Dependent and Independent Antitumor Activity"],"prefix":"10.3390","volume":"18","author":[{"given":"Zlatina","family":"Vlahova","sequence":"first","affiliation":[{"name":"Institute of Molecular Biology \u201cAkad. Roumen Tsanev\u201d, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6071-7949","authenticated-orcid":false,"given":"Lazar","family":"Lazarov","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology \u201cAkad. Roumen Tsanev\u201d, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0766-3934","authenticated-orcid":false,"given":"Maria","family":"Petrova","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology \u201cAkad. Roumen Tsanev\u201d, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0573-5685","authenticated-orcid":false,"given":"Shazie","family":"Yusein-Myashkova","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology \u201cAkad. Roumen Tsanev\u201d, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1427-7004","authenticated-orcid":false,"given":"Monika","family":"Mutovska","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry and Pharmacy, Sofia University \u201cSt. Kliment Ohridski\u201d, 1 J. Bourchier Blvd, 1164 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7830-1538","authenticated-orcid":false,"given":"Stanimir","family":"Stoyanov","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry and Pharmacy, Sofia University \u201cSt. Kliment Ohridski\u201d, 1 J. Bourchier Blvd, 1164 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6838-750X","authenticated-orcid":false,"given":"Yulian","family":"Zagranyarski","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry and Pharmacy, Sofia University \u201cSt. Kliment Ohridski\u201d, 1 J. Bourchier Blvd, 1164 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0116-0079","authenticated-orcid":false,"given":"Iva","family":"Ugrinova","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology \u201cAkad. Roumen Tsanev\u201d, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgaria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"105677","DOI":"10.1016\/j.bioorg.2022.105677","article-title":"Recent Developments on 1,8-Naphthalimide Moiety as Potential Target for Anticancer Agents","volume":"121","author":"Tandon","year":"2022","journal-title":"Bioorg. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Bhat, A.A., Gacem, A., Qasim, M.T., Almulla, N., Muzammil, K., and Shalabi, M.G. (2025). 1,8-Naphthalimide-Based DNA Intercalators and Anticancer Agents: A Systematic Review. Mol. Divers., 1\u201322.","DOI":"10.1007\/s11030-025-11251-1"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1601","DOI":"10.1039\/c2cs35467e","article-title":"Recent Advances in the Development of 1,8-Naphthalimide Based DNA Targeting Binders, Anticancer and Fluorescent Cellular Imaging Agents","volume":"42","author":"Banerjee","year":"2013","journal-title":"Chem. Soc. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Yakkala, P.A., Penumallu, N.R., Shafi, S., and Kamal, A. (2023). Prospects of Topoisomerase Inhibitors as Promising Anti-Cancer Agents. Pharmaceuticals, 16.","DOI":"10.3390\/ph16101456"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4657","DOI":"10.1039\/D5MD00205B","article-title":"Naphthalimide-Organometallic Hybrids as Multi-Targeted Anticancer and Luminescent Cellular Imaging Agents","volume":"16","author":"Magri","year":"2025","journal-title":"RSC Med. Chem."},{"key":"ref_6","first-page":"100285","article-title":"Naphthalimide-Polyamine Conjugates: A Promising Avenue for Targeted Anticancer Therapy","volume":"15","author":"Tian","year":"2025","journal-title":"Eur. J. Med. Chem. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Rykowski, S., Gurda-Wo\u017ana, D., Orlicka-P\u0142ocka, M., Fedoruk-Wyszomirska, A., Giel-Pietraszuk, M., Wyszko, E., Kowalczyk, A., St\u0105czek, P., Bak, A., and Kiliszek, A. (2021). Design, Synthesis, and Evaluation of Novel 3-Carboranyl-1,8-Naphthalimide Derivatives as Potential Anticancer Agents. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22052772"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1039\/D3PM00060E","article-title":"Insight into the Liposomal Encapsulation of Mono and Bis-Naphthalimides","volume":"1","author":"Dauda","year":"2024","journal-title":"RSC Pharm."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"15330338231225860","DOI":"10.1177\/15330338231225861","article-title":"Research Progress on Structure-Activity Relationship of 1,8-Naphthalimide DNA Chimeras Against Tumor","volume":"23","author":"Zhang","year":"2024","journal-title":"Technol. Cancer Res. Treat."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4225","DOI":"10.1039\/c0cc00078g","article-title":"Core-Substituted Naphthalenediimides","volume":"46","author":"Sakai","year":"2010","journal-title":"Chem. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ruan, W., Xie, Z., Wang, Y., Xia, L., Guo, Y., and Qiao, D. (2024). An Overview of Naphthylimide as Specific Scaffold for New Drug Discovery. Molecules, 29.","DOI":"10.3390\/molecules29194529"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1007\/BF00873971","article-title":"Phase II Study of Amonafide in Gastric Adenocarcinoma. An Illinois Cancer Center Trial","volume":"12","author":"Mullane","year":"1994","journal-title":"Investig. New Drugs"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/BF00877243","article-title":"Phase I Study of Mitonafide in Solid Tumors","volume":"10","author":"Llombart","year":"1992","journal-title":"Investig. New Drugs"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1007\/BF00180820","article-title":"Phase II Study of Mitonafide in Non-Small Cell Lung Cancer (NSCLC)","volume":"14","author":"Casado","year":"1996","journal-title":"Investig. New Drugs"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1007\/BF00210795","article-title":"Phase II Study of Mitonafide in Advanced and Relapsed Colorectal Cancer","volume":"14","author":"Abad","year":"1996","journal-title":"Investig. New Drugs"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lee, C.-C., Chang, C.-H., Huang, Y.-C., and Shih, T.-L. (2024). Novel 1,8-Naphthalimide Derivatives Inhibit Growth and Induce Apoptosis in Human Glioblastoma. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252111593"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Xu, Z., Zhou, Y., Wang, J., Mao, L., Li, W., and Xu, G. (2021). The Synthesis and Antitumor Activity of 1,8-Naphthalimide Derivatives Linked 1,2,3-Triazole. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.662432"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6287","DOI":"10.1039\/D5OB00657K","article-title":"1,8-Naphthalimide Derivatives as Small Molecules with Multi-Applications in Chemistry and Biology","volume":"23","author":"Sanaullah","year":"2025","journal-title":"Org. Biomol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1658","DOI":"10.1038\/s12276-022-00863-4","article-title":"DNA Damage Response Revisited: The P53 Family and Its Regulators Provide Endless Cancer Therapy Opportunities","volume":"54","author":"Abuetabh","year":"2022","journal-title":"Exp. Mol. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1038\/s41388-024-03219-6","article-title":"Breathing New Insights into the Role of Mutant P53 in Lung Cancer","volume":"44","author":"Chen","year":"2025","journal-title":"Oncogene"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1595","DOI":"10.1016\/j.jmb.2017.03.030","article-title":"Mutant P53 in Cancer: Accumulation, Gain-of-Function, and Therapy","volume":"429","author":"Yue","year":"2017","journal-title":"J. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Hern\u00e1ndez-Aceituno, J., Acosta, L., and Pi\u00f1eiro, J.D. (2015). Application of Time Dependent Probabilistic Collision State Checkers in Highly Dynamic Environments. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0119930"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"13978","DOI":"10.1038\/s41598-020-70597-8","article-title":"A Novel Naphthalimide That Selectively Targets Breast Cancer via the Arylhydrocarbon Receptor Pathway","volume":"10","author":"Gilbert","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"14399","DOI":"10.1021\/acsomega.1c01271","article-title":"Novel Mitochondria-Targeting and Naphthalimide-Based Fluorescent Probe for Detecting HClO in Living Cells","volume":"6","author":"Xu","year":"2021","journal-title":"ACS Omega"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2171028","DOI":"10.1080\/14756366.2023.2171028","article-title":"Carboranyl-1,8-Naphthalimide Intercalators Induce Lysosomal Membrane Permeabilization and Ferroptosis in Cancer Cell Lines","volume":"38","author":"Rykowski","year":"2023","journal-title":"J. Enzym. Inhib. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Wynne, C., and Elmes, R.B.P. (2024). Utilising a 1,8-Naphthalimide Probe for the Ratiometric Fluorescent Visualisation of Caspase-3. Front. Chem., 12.","DOI":"10.3389\/fchem.2024.1418378"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wang, J., Jia, X., Zhang, Y., Gao, Y., Zhang, L., Meng, C., Wang, Z., and Jiao, Y. (2025). Naphthalimide-Based Fluorescent Probe for Portable and Rapid Response to \u03b3-Glutamyl Transpeptidase. Molecules, 30.","DOI":"10.3390\/molecules30153174"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"16085","DOI":"10.1039\/D1SC04030H","article-title":"A Naphthalimide-Based Peptide Conjugate for Concurrent Imaging and Apoptosis Induction in Cancer Cells by Utilizing Endogenous Hydrogen Sulfide","volume":"12","author":"Singh","year":"2021","journal-title":"Chem. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2699","DOI":"10.1021\/acs.chemrev.3c00778","article-title":"Theranostic Fluorescent Probes","volume":"124","author":"Sharma","year":"2024","journal-title":"Chem. Rev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/0022-1759(83)90303-4","article-title":"Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays","volume":"65","author":"Mosmann","year":"1983","journal-title":"J. Immunol. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An Open-Source Platform for Biological-Image Analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2315","DOI":"10.1038\/nprot.2006.339","article-title":"Clonogenic Assay of Cells in Vitro","volume":"1","author":"Franken","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Mutovska, M., Anastasova, D., Simeonova, N., Zagranyarska, I., Vlahova, Z., Ugrinova, I., Stoyanov, S., and Zagranyarski, Y. (2024). One Pot Synthesis of New Powerful Building Blocks in 1,8-Naphthalimide Chemistry. Molbank, 2024.","DOI":"10.3390\/M1914"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"111701","DOI":"10.1016\/j.dyepig.2023.111701","article-title":"One-Pot Synthesis of Fused-Rings Heterocyclic Systems Based on Symmetrically Benzofuran Annulated 1,8-Naphthalimides","volume":"220","author":"Mutovska","year":"2023","journal-title":"Dye. Pigment."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Anastasova, D., Mutovska, M., Simeonova, N., Zagranyarska, I., Vlahova, Z., Stoyanov, S., and Zagranyarski, Y. (2024). A Gram Scale Synthesis of 3,4-Dihalogen Substituted 1,8-Naphthalimides. Molbank, 2024.","DOI":"10.3390\/M1918"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1002\/tcr.201600134","article-title":"1,8-Naphthalimide: A Potent DNA Intercalator and Target for Cancer Therapy","volume":"17","author":"Tandon","year":"2017","journal-title":"Chem. Rec."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7170","DOI":"10.1039\/D3CS00581J","article-title":"\u03c0-Electronic Ion Pairs: Building Blocks for Supramolecular Nanoarchitectonics via i\u03c0\u2013i\u03c0 Interactions","volume":"52","author":"Haketa","year":"2023","journal-title":"Chem. Soc. Rev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1093\/nar\/gki317","article-title":"Influence of the\u2014Interaction on the Hydrogen Bonding Capacity of Stacked DNA\/RNA Bases","volume":"33","author":"Mignon","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2589","DOI":"10.1021\/jm100025u","article-title":"A New Class of Naphthalimide-Based Antitumor Agents That Inhibit Topoisomerase II and Induce Lysosomal Membrane Permeabilization and Apoptosis","volume":"53","author":"Chen","year":"2010","journal-title":"J. Med. Chem."},{"key":"ref_40","first-page":"632","article-title":"Topoisomerase II Poisons Inhibit Vertebrate DNA Replication through Distinct Mechanisms","volume":"41","author":"Mehta","year":"2022","journal-title":"EMBO J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"639","DOI":"10.21873\/cdp.10266","article-title":"The Role of \u0393H2AX in Replication Stress-Induced Carcinogenesis: Possible Links and Recent Developments","volume":"3","author":"Fragkos","year":"2023","journal-title":"Cancer Diagn. Progn."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/2\/167\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T05:24:17Z","timestamp":1769664257000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/2\/167"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,27]]},"references-count":41,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["pharmaceutics18020167"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics18020167","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,27]]}}}