## 3 Results and Discussion
### Biological Activity
#### 3.2.2 Antiviral Activity  
Due to the ability of viruses to mutate and develop drug resistance, the search for new antiviral substances remains relevant. To assess the antiviral potential of phosphonate betulin derivatives, various types of viruses were selected. Bovine enteroviruses (BEV) belong to the _Picornaviridae_ family, and they are very stable in various environmental conditions due to their resistance to pH, temperature, and salinity fluctuations and exposure to disinfectants. Therefore, this virus has been utilized as model virus to test the virucidal activity of different compounds by Polish Standard PN-EN 14675 [58]. This virus is an environmental pollutant. BEV has a wide spectrum of hosts, including humans; however, it mainly poses a threat to cattle populations worldwide. In neonates and young animals, under certain conditions, it can cause severe and even fatal respiratory, digestive, and reproductive disorders. Among humans, it is assumed that every third person is exposed to this virus regardless of where they live (whether in urban or rural areas in close contact with domestic animals), but no association has been observed between BEV and any clinical disorder in humans [59]. Vesicular stomatitis virus (VSV) is a member of the genus _Vesiculovirus_ of the family _Rhabolovidae_. It causes vesicular lesions in the oral mucosa, tongue, lips, nostrils, hooves, and nipples of infected animals. The diseases it causes are limited to its natural hosts such as horses, cattle, and pigs. In humans, the infection is mild and asymptomatic [60]. The herpes simplex viruses HHV-1 and HHV-2 are members of the human _Herpes-viridae_ family. In 2016, it was found that worldwide among people aged 49 and under, approximately 491 million people live with HHV type 2 infection and 3752 million with HHV type 1 infection [61]. The symptoms of infection can range from mild to severe (from lesions to skin or mucous membranes of the mouth, lips, nose, genitals, or eyes to herpetic encephalitis) [62]. Among the adenovirus (AdV), there are serotypes responsible for many different clinical symptoms and syndromes, such as conjunctivitis, otitis media, rhinitis, keratitis, pharyngitis, and gastroenteritis. Often, infections caused by adenovirus are asymptomatic, which facilitates and increases the spread of the virus among the human population. HAdV5 belongs to endemic serotypes and mainly infects children [2]. Virucidal activity assays against BEV, VSV, HHV-1, and HAdV-5 were performed using the A549 cell line. The tested compounds were incubated with viruses for 2 h at room temperature with a non-toxic (CC\({}_{20}\)) concentration of each compound and then the \begin{table}
\begin{tabular}{c c} \hline \hline
**Compound** & **CC\({}_{20}\) [\(\mu\)M]** \\ \hline
**3** & 4.72 \\
**4** & 8.1 \\
**5** & 1.0 \\
**6** & 48.6 \\
**7a** & 33.4 \\
**7b** & 26.7 \\
**8a** & 60.22 \\
**8b** & 38.2 \\
**9** & 25.3 \\
**Betulin 1** & 28.22 a ** \\
**Acyclovir** & 196.08 a ** \\
**Ribavirin** & 409.84 a ** \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Cytotoxicity of compounds **3–9** and reference substances on human lung cancer cell line A549. mixtures were titrated on the A549 cell line. Simultaneously, the same amount of virus was incubated with culture medium as a control. After 48 h incubation (VSV and HHV-1) or 72 h for BEV and HAdV-5 virus-mediated cell death (cytopathic effect, CPE) was assessed using a microscope. All derivatives showed no virucidal activity against the tested viruses. Comparing the viral titers of controls and samples incubated with the tested betulin derivatives, the differences were found to be no more than one logarithm [42]. Effect on viral replication  
The effect of the tested compounds on virus replication was assessed depending on the time of their addition to the cell cultures. The tested compounds did not show a protective effect on cells A549 treated with the virus (Mode I) and did not affect the adsorption and penetration of the tested viruses (no change in virus titer--Mode II).