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.

In recent years, the emergence of drug-resistant pathogens and the limitations of current therapies have highlighted the need for innovative strategies to combat emerging viral infections. Natural compounds, derived from plants, are playing an increasingly significant role in the research of novel and effective therapies. Boswellia sacra, a frankincense-producing tree widely distributed in Yemen and Oman, and Nerium oleander, a common ornamental and medicinal plant, are examples of plants with well-documented antimicrobial properties. Their extracts have demonstrated good activity against a wide range of infections, which is attributed to the anti-inflammatory and immunomodulatory compounds they contain. Based on these findings, we assessed, in vitro, the broad-spectrum antiviral activity of combined extracts obtained from Boswellia sacra and Nerium oleander. The extract mixture NOBS7(1) was found to be active against the respiratory virus RSV, Herpesvirus simplex type 1, and Coxsackievirus 5. Furthermore, a combination of cell-based assays was performed to provide additional insights into their potential mechanism of action.