## 3 Results and Discussion
### In Silico Study
#### 3.3.1 Assessment of ADMET Properties and Drug Likeness of the Screened Molecules
The tested derivatives may have an inhibitory effect on P-glycoprotein, while only compound **3** can be both its substrate and inhibitor. P-gp transport protein participates in the protection of cells against the effects of potentially harmful factors; however, the therapeutic effect of orally taken drugs that are Pgp substrates is also reduced. By reducing its activity, Pgp inhibitors may increase the bioavailability of other drugs. This causes the concentration of these drugs to increase to toxic levels, resulting in the occurrence of many side effects. Knowledge of this type of relationship requires caution and monitoring of the applied therapy [64]. Viral diseases often attack patients with weakened immunity, who are already receiving treatment for other ailments. Therefore, with the applied pharmacotherapy, one can expect not only side effects from the drug used, but also the possibility of drug-drug interactions. Mutual drug interactions also depend on the activity of cytochrome P450 enzymes responsible for the metabolism of a given drug (mainly the CYP3A4 isoenzyme) [65]. All tested compounds may be substrates of the CYP3A4 isoenzyme; however, their inhibitory effect on CYP3A4 and other isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6) is not expected. The blood-brain barrier (BBB) not easily penetrated by tested compounds. The calculated logBB values range from \(-\)0.083 for compound **9** to \(-\)1.373 for the derivative **7b** (log BB \(<-\)1 are poorly distributed to the brain, logBB \(>\) 0.3 indicates readily cross the blood-brain barrier). The predicted total clearance (expressed as log (mL/min/kg) defines the volume of plasma that is completely cleared of drug per unit of time by the organ that eliminates the drug from the body. Knowledge of this parameter is essential for determining the size of the maintenance dose of the drug. The highest value was calculated for betulin **1**, the values for the other compounds are in the range \(-\)0.199 to \(-\)0.556. The toxicity of the tested compounds was determined based on the Ames test, inhibition of the potassium channels (hERG; human ether-a-go-go gene), calculation of the maximum human-tolerated dose (MRTD)) and oral rat acute toxicity (LD\({}_{50}\)--the amount of compound administered at a time that causes death to 50% of a group of test animals). For all tested compounds, the result of the Ames test, which evaluated the mutagenic potential of compounds using bacteria, was negative, which suggests a lack of carcinogenic activity. These compounds are also not hERG I and II inhibitors, so they should not be the cause of the development of acquired long QT syndrome, which leads to lethal ventricular arrhythmias. The maximum recommended tolerated dose (MRTD) of the test compound less than or equal to 0.477 log (mg/kg/day) is considered low and high above 0.477 log (mg/kg/day). For the compounds tested, the calculated values range from -0.433 to 0.533. The MRTD determines the size of the maximum recommended starting dose for pharmaceuticals in phase I clinical trials. Lethal dose values (LD\({}_{50}\)) are a standard measure of acute toxicity. For compounds **1**, **2-9**, the LD\({}_{50}\) ranged from 2.348 to 3.267 (mol/kg). Drug-induced liver damage is a major safety concern for drugs. pkCSM classifies a compound as hepatotoxic if it has shown at least one pathological or physiological event strongly associated with disruption of normal liver function [45]. Compounds **3-9** showed no hepatotoxicity. For compounds **3** and **8a**, which showed activity in in vitro studies, and for the starting betulin **1**, physicochemical parameters helpful in assessing bioavailability were determined (Table 7) and drug similarity was analyzed [46]. Considering two in vitro active semi-synthetic betulin derivatives, **3** and **8a**, and the initial betulin **1**, it can be seen that the introduction of new functional groups resulted in an increase in molar mass (MW; g/mol) from 442.72 (for betulin **1**) to the value of 578.80 and 720.91, respectively.