## 2 Results and Discussion
### Molecular Docking  
The investigated pyrazolo[4,3-e][1,2,4]triazine sulfonamides showed no antiproliferative activity against tumor cell lines in in vitro tests. However, our previous pharmacological studies showed that all chiral sulphonamides **8a-j** exhibited significant inhibitory activity on mushroom tyrosinase and jack bean urease [20]. Therefore, the biological activity of **8a-j** prompted us to investigate in silico the mechanism of action at the molecular level of the investigated pirazolotriazine sulfonamides by the molecular docking method. All chiral sulphonamides **8a-j** exhibited significant inhibitory activity on mushroom tyrosinase, with IC\({}_{50}\) values in the range of 27.9-40.17 uM, comparable to the activity of koijc acid (IC\({}_{50}\) = 16.69 uM), which was used as a reference compound in the test [20]. The most active compounds **8j** and **8b** showed inhibition of tyrosinase at IC\({}_{50}\) of 27.9 and 30.76 uM levels, respectively. Moreover, compounds **8a-j** were tested for their inhibitory effects on jack bean urease, exhibiting inhibitory activity that changed from an IC\({}_{50}\) of 0.037 uM for **8a** to 0.084 uM for **8b**, better than the activity of reference thiourea with an IC\({}_{50}\) value of 20.7 uM [20]. Tyrosinase is the enzyme which is responsible for the synthesis of melanin, a ubiquitous pigment in living organisms. In the crystalline state, _Agaricus bisprous_ mushroom tyrosinase occurs as tetramer H\({}_{2}\)L\({}_{2}\) subunits in complex with its inhibitor tropolone, forming a pre-Michaelis complex with the enzyme and a binuclear copper binding site in H subunit (PDB ID: 2Y9X) [47]. The results of the molecular docking of the most active in vitro testing of **8j** and **8b** to the binding site of tyrosinase are presented in Figure 7. The ligands **8j** and **8b** bound to the active site with the values of scoring function ChemPLP of 62.70 and 63.50, respectively. These values were significantly better than the value of 50.14 obtained for the re-docked molecule of tropolone, which indicates a greater affinity of **8j** and **8b** for the enzyme than that observed for tropolone. The molecule of **8j** bound to the binding site of tyrosinase by intermolecular hydrogen bonds N2... C (PHE264A) and C1... O (SER282A), with the distances between interacting atoms being 3.152 and 2.921 A, respectively. Figure 6: The proposed metabolic pathways of compounds **8m**, **8i** and **8j**. Moreover, short contacts between the hydroxyl group of (_R_)-(-)-leucinol substituent and Cu\({}^{2+}\) ions with distances of 2.493 and 3.474 A were observed. The molecule of **8b** interacted with the active site of tyrosinase through the O22... C (HIS263A; 3.001 A) hydrogen bond and, similar to **8j**, short contacts to the Cu\({}^{2+}\) ions with distances of 2.598 and 2.475 A were observed. Urease is a nickel-containing enzyme. Catalyzing the hydrolysis of urea and its inhibitors plays an important role in the therapy of human and plant disorders [20]. For the docking study, the crystal structure of a jack bean urease complex with acetohydroxamic acid, 1,2-ethanediol and two Ni\({}^{2+}\) ions in the binding site was used (PDB ID: 4H9M) [48]. Compounds **8a** and **8i**, being the most active in the test of the inhibitory effect on urease, bound to the active site of this enzyme with ChemPLP values of 77.60 and 66.72, respectively. It should be noted that the redocking of the acetohydroxamic acid molecule gave a relatively  
Figure 7: A view of the interaction of (**a**) **8j** and (**b**) **8b** with amino acids of the binding site in mushroom tyrosinase. smaller ChemPLP value of 39.08, which indicates that the molecules of **8a** and **8i** fit better to the active site than the molecule of acetohydroxamic acid. The molecule of **8a** bound to the active site of urease via O22... O (ASP494A), N1... N (ARG439A), C14... O (ALA636A) and C15... O (ALA636A) hydrogen bonds, with distances between the interacting atoms of 2.980, 2.871, 2.973 and 3.095 A, respectively.