{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T10:23:32Z","timestamp":1775816612855,"version":"3.50.1"},"reference-count":32,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,4]],"date-time":"2026-01-04T00:00:00Z","timestamp":1767484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NextGeneration EUMUR PNRR Extended Partnership initiative on Emerging Infectious Diseases","award":["PE00000007"],"award-info":[{"award-number":["PE00000007"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Introduction: Despite the impressive progress carried out in the field of biomedical sciences in recent decades, the incidence of emerging and neglected lethal viral infections mainly belonging to the Coronaviridae, Filoviridae, Arenaviridae, Bunyaviridae, and Paramyxoviridae families has considerably impaired human health. The worldwide vaccination campaign at the end of the 1970s determined the eradication of smallpox. However, the growing number of cases of diseases linked to orthopoxvirus diseases, such as the recent epidemic of monkeypox zoonosis in various countries around the world, has increased the need for knowledge of these viral pathogens. To date, there is no specific treatement for Monkeypox virus (MPXV) infection. However, several antiviral drugs used to treat Smallpox and other viral infections could also be beneficial for Monkeypox disease. In this study we report the design and synthesis of new, variously substituted benzimidazole derivatives and the evaluation of their cytotoxicity and antiviral activity against representatives of the Orthopoxvirus genus, Vaccinia Virus (VV), closely related to variola virus and MPXV. Methods: A combination of cell-based assays and experimental techniques was used to investigate the cytotoxicity, antiviral activity, and mechanisms of action of the most interesting compound. Results: In our study, new, variously substituted benzimidazoles showed interesting EC50 values against vaccinia and MPXV and a cytotoxic profile in the high micromolar range. Conclusions: Our work shows that the new tested benzimidazole derivatives possess appealing activity and selectivity, accompanied by low cytotoxicity. These results set a valid foundation with which to identify potent and selective anti-Poxvirus agents.<\/jats:p>","DOI":"10.3390\/v18010071","type":"journal-article","created":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T15:28:57Z","timestamp":1767626937000},"page":"71","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Novel 2-Aryl-1H-Benzimidazole Derivatives and Their Aza-Analogues as Promising Anti-Poxvirus Agents"],"prefix":"10.3390","volume":"18","author":[{"given":"Valeria","family":"Manca","sequence":"first","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laura","family":"Locci","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3332-3845","authenticated-orcid":false,"given":"Roberta","family":"Ibba","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Pharmacy, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-9719-3512","authenticated-orcid":false,"given":"Laura","family":"Sanna","sequence":"additional","affiliation":[{"name":"Department of Chemical, Physical, Mathematical and Natural Sciences, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9457-7737","authenticated-orcid":false,"given":"Ilenia","family":"Lupinu","sequence":"additional","affiliation":[{"name":"Department of Chemical, Physical, Mathematical and Natural Sciences, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sandra","family":"Piras","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Pharmacy, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2791-9178","authenticated-orcid":false,"given":"Gabriele","family":"Murineddu","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Pharmacy, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1971-3989","authenticated-orcid":false,"given":"Gabriele","family":"Serreli","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of General Pathology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roberta","family":"Loddo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rebecca","family":"Piras","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-2124-6217","authenticated-orcid":false,"given":"Luca","family":"Virdis","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michela","family":"Isola","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Human Anatomy, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vanessa","family":"Palmas","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3999-4263","authenticated-orcid":false,"given":"Giuseppina","family":"Sanna","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Section of Microbiology and Virology, University of Cagliari, 09042 Monserrato, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonio","family":"Carta","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Pharmacy, University of Sassari, 07100 Sassari, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bunge, E.M., Hoet, B., Chen, L., Lienert, F., Weidenthaler, H., Baer, L.R., and Steffen, R. (2022). The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl. Trop. Dis., 16.","DOI":"10.1371\/journal.pntd.0010141"},{"key":"ref_2","unstructured":"WHO (2025, December 20). Available online: https:\/\/www.who.int\/."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e0009222","DOI":"10.1128\/cmr.00092-22","article-title":"Monkeypox Virus Infections in Humans","volume":"35","author":"Elsayed","year":"2022","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_4","unstructured":"(2025, April 29). WHO. Available online: https:\/\/www.who.int\/publications\/m\/item\/multi-country-outbreak-of-mpox--external-situation-report--51---29-april-2025."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"297","DOI":"10.18433\/jpps33138","article-title":"Monkeypox: Virology, Pathophysiology, Clinical Characteristics, Epidemiology, Vaccines, Diagnosis, and Treatments","volume":"25","author":"Soheili","year":"2022","journal-title":"J. Pharm. Pharm. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"100023","DOI":"10.1016\/j.mgmed.2025.100023","article-title":"The re-emergence of Mpox: A global public health threat amplified by mass gatherings","volume":"4","author":"Mweene","year":"2025","journal-title":"Mass Gather. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"102940","DOI":"10.1016\/j.jiph.2025.102940","article-title":"Mpox infection: A state-of-the-art overview of epidemiological, molecular, and clinical aspects following the 2024 public health emergency","volume":"18","author":"Zamani","year":"2025","journal-title":"J. Infect. Public Health"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"Eade7646","DOI":"10.1126\/scitranslmed.ade7646","article-title":"In vitro and in vivo efficacy of tecovirimat against a recently emerged 2022 monkeypox virus isolate","volume":"14","author":"Warner","year":"2022","journal-title":"Sci. Transl. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e04745-22","DOI":"10.1128\/spectrum.04745-22","article-title":"Identification of In Vitro Inhibitors of Monkeypox Replication","volume":"11","author":"Chiem","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"232","DOI":"10.15585\/mmwr.mm7209a4","article-title":"Interim Clinical Treatment Considerations for Severe Manifestations of Mpox\u2014United States, February 2023","volume":"72","author":"Rao","year":"2023","journal-title":"MMWR Morb. Mortal. Wkly Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"(2025). The PALM007 Writing Group. Tecovirimat for Clade I MPXV Infection in the Democratic Republic of Congo. N. Engl. J. Med., 392, 1484\u20131496.","DOI":"10.1056\/NEJMoa2412439"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e126","DOI":"10.1016\/S1473-3099(25)00014-3","article-title":"Antiviral activity of tecovirimat against monkeypox virus clades 1a, 1b, 2a, and 2b","volume":"25","author":"Postal","year":"2025","journal-title":"Lancet Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"199615","DOI":"10.1016\/j.virusres.2025.199615","article-title":"Tecovirimat is active against various MPXV strains, while cidofovir, brincidofovir, trifluridine, and gemcitabine have no detectable MPXV-specific antiviral activity","volume":"360","author":"Kato","year":"2025","journal-title":"Virus Res."},{"key":"ref_14","unstructured":"(2025, December 20). Mpox Antiviral Clinical Trial Advances as WHO Extends Global Emergency. Available online: https:\/\/globalbiodefense.com\/2025\/07\/17\/mpox-nv387-drug-trial-who-emergency-2025\/."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Marinescu, M. (2023). Benzimidazole-Triazole Hybrids as Antimicrobial and Antiviral Agents: A Systematic Review. Antibiotics, 12.","DOI":"10.20944\/preprints202307.0093.v1"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2937","DOI":"10.1016\/j.bmc.2010.02.037","article-title":"Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives","volume":"18","author":"Tonelli","year":"2010","journal-title":"Bioorganic Med. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ibba, R., Carta, A., Madeddu, S., Caria, P., Serreli, G., Piras, S., Sestito, S., Loddo, R., and Sanna, G. (2021). Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative. Viruses, 13.","DOI":"10.3390\/v13010058"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1055\/s-2007-965878","article-title":"A Simple and Efficient One-Pot Synthesis of 2-Substituted Benzimidazoles","volume":"4","author":"Bahrami","year":"2007","journal-title":"Synthesis"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1128","DOI":"10.1080\/00397910701863657","article-title":"New One-Pot Procedure for the Synthesis of 2-Substituted Benzimidazoles","volume":"38","author":"Sharghi","year":"2008","journal-title":"Synth. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/0166-0934(88)90134-6","article-title":"Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds","volume":"20","author":"Pauwels","year":"1988","journal-title":"J. Virol. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/j.ejmech.2017.12.083","article-title":"Quinoxaline derivatives as new inhibitors of coxsackievirus B5","volume":"145","author":"Carta","year":"2018","journal-title":"Eur. J. Med. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"113558","DOI":"10.1016\/j.fct.2022.113558","article-title":"Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation","volume":"172","author":"Casula","year":"2023","journal-title":"Food Chem. Toxicol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1084\/jem.113.4.657","article-title":"Spectrum and characteristics of the virus inhibitory action of 2- (\u03b1-hydroxybenzyl)-benzimidazole","volume":"113","author":"Eggers","year":"1961","journal-title":"J. Exp. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4893","DOI":"10.1016\/j.bmc.2014.06.043","article-title":"Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents","volume":"22","author":"Tonelli","year":"2014","journal-title":"Bioorganic Med. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1070\/RCR4832","article-title":"Benzimidazole nucleosides: Anti-viral and antitumour activities, methods of synthesis","volume":"87","author":"Kharitonova","year":"2018","journal-title":"Russ. Chem. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2186","DOI":"10.1128\/AAC.47.7.2186-2192.2003","article-title":"In Vitro Activities of Benzimidazole d- and l-Ribonucleosides against Herpesviruses","volume":"47","author":"Williams","year":"2003","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_27","unstructured":"Dahlin, J.L., Devanarayan, V., Foley, T.L., Glicksman, M., Gorshkov, K., Grotegut, S., Hall, M.D., Hoare, S., Inglese, J., and Iversen, P.W. (2004). Assay Guidance Manual, Eli Lilly & Company and the National Center for Advancing Translational Sciences."},{"key":"ref_28","unstructured":"Sittampalam, G., Gal-Edd, N., Arkin, M., Auld, D., and Austin, C. (2025, December 20). Analyzing Kinetic Binding Data, Assay Guidance, Available online: https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK569501\/."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ibba, R., Riu, F., Delogu, I., Lupinu, I., Carboni, G., Loddo, R., Piras, S., and Carta, A. (2022). Benzimidazole-2-Phenyl-Carboxamides as Dual-Target Inhibitors of BVDV Entry and Replication. Viruses, 14.","DOI":"10.3390\/v14061300"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"8562","DOI":"10.1021\/acs.jcim.4c01110","article-title":"AI Promoted Virtual Screening, Structure-Based Hit Optimization, and Synthesis of Novel COVID-19 S-RBD Domain Inhibitors","volume":"64","author":"Gkekas","year":"2024","journal-title":"J. Chem. Inf. Model."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"113211","DOI":"10.1016\/j.ejmech.2021.113211","article-title":"Design, synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles as inhibitors of ebola virus infection","volume":"214","author":"Ranjan","year":"2021","journal-title":"Eur. J. Med. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4721","DOI":"10.1021\/jm060269e","article-title":"Benzimidazole derivatives bearing substituted biphenyls as hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors: Structure-activity relationship studies and identification of a potent and highly selective inhibitor JTK-109","volume":"49","author":"Hirashima","year":"2006","journal-title":"J. Med. Chem."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/71\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T15:31:44Z","timestamp":1767627104000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/71"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,4]]},"references-count":32,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["v18010071"],"URL":"https:\/\/doi.org\/10.3390\/v18010071","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,4]]}}}