## 3 Results and Discussion
### Biological Activity
#### 3.2.2 Antiviral Activity
The results obtained are shown in Figures 2 and 3. Figure 3: Mode II: the activity of the studied compounds at the virus entry step of replication at a dose of 100 TCID\({}_{50}\) (with a median of three repetitions). Figure 2: Mode I: effect of the studied compounds on the sensitivity of the cells to infection with VSV, HHV-1, BEV, and HAdV-5, at a dose of 100 TCID\({}_{50}\) (with a median of three repetitions). Mode III assay infection of cells with a virus is followed by incubation with compounds and allows us to assess whether the active compounds affect the stage of DNA synthesis, virion assembly, and maturation. The results (Figure 4) show that derivative **3** reduced the EBCO and the HADV-5 titers by 0.5 log TCID\({}_{50}\) and 1.5 logTCID\({}_{50}\), respectively, compared to the virus control group. The reduction in virus infectivity for compound **3** corresponds to a percent reduction close to 90%. Compound **8a** reduced the EBCO titer by more than 2 log TCID\({}_{50}\), indicating a 99% reduction in virus infectivity. All tested compounds were inactive against VSV and HHV-1 viruses. Among all the tested compounds, only **3** (EC\({}_{50}\)--1.73 \(\mu\)M; SI--8.7) and **8a** (EC\({}_{50}\)--13.59 \(\mu\)M; SI--9.4) showed activity against the BEV virus. Additionally, derivative **3** had an effect on virus HAdV-5 and the EC\({}_{50}\) calculated for this compound was 2.59 \(\mu\)M (SI--5.8). The studied compounds have shown activity at the stage of DNA and protein synthesis, virion assembly, and maturation. This result is consistent with the data from the literature on the effects of natural compounds which inhibited HAdV-5 replication in the post-adsorption phase [2]. Compound **8a** likely interferes with the replication machinery involved in the synthesis of positive-sense RNA in BEV. It may inhibit key steps in the viral life cycle that are essential for the production of new virus particles. This disruption hampers the ability of BEV to replicate and spread. Compound **3** likely interferes with the replication processes associated with the synthesis of double-stranded DNA in HAdV-5. The absence of an envelope in both viruses indicates that the compound's action is more focused on intracellular processes rather than targeting an envelope structure. In summary, the compounds may act by specifically targeting the replication mechanisms of these viruses, disrupting key steps in the viral life cycle associated with their respective genomic and structural characteristics. Analyzing the structure of the tested derivatives, it can be seen that both betulin **1** and its 30-diethylphosphophonic derivative **4** showed no activity against the tested viruses. The obtained results indicate that the introduction of a phosphonate group to the isopropyl substituent of the betulin molecule has a positive effect on the virucidal activity. However, the position of this group is crucial; the activity is ensured by the vinyl position (29-substituted derivative **3**) while the allyl isomer (30-substituted derivative **4**) remains inactive. Conversion of compound **3** to the carboxylic derivative **6** resulted in inactivity. Similarly, esterification of the C3 hydroxyl group with dimethylglutaric anhydride created  
Figure 4: Mode III: effect of the studied compounds on reduction of the titer of tested viruses in the post-adsorption phase. Each compound was tested in triplicate. the inactive derivative **8b**. The introduction of a dimethylsucccinyl group at the C3 position with a shorter carbon chain (compound **8a**) provided a derivative active against BEV.