3.3.4. Molecular Docking

The results of the molecular docking of the enantiomers of compounds **3a**–**d** to the four sites of albumin given by AutoDock Vina are presented in Table 2, while the results of AutoDock are presented in Table 3.

**Table 2.** The results of the docking study were made using AutoDock Vina expressed as the binding affinity of the studied compounds to the four targeted sites of HSA expressed as a variation of Gibbs free energy (ΔG kcal/mol).



**Table 3.** The results of the docking study were made using AutoDock expressed as the binding affinity of the studied compounds to the four targeted sites of HSA expressed as a variation of Gibbs free energy (ΔG kcal/moL) and the clustering analysis of the poses.

%C: percent of conformations in the same 2Å RMSD cluster of atom coordinates.

The analysis of the results obtained after the docking of the ligands at the four albumin sites using AutoDock Vina shows a major difference in interactions, depending on the size of the heterocycle from the structure of the compounds and less influenced by the type of the isomer.

The compounds from the present series that have the highest affinity for albumin are tetrahydroquinoline derivative **3c** and tetrahydroisoquinoline derivative **3d**. Compounds **3a** and **3b** (pyrrolidine and piperidine, respectively) have, for the four studied sites, a lower affinity than compounds **3c** and **3d**.

Taking into account the affinity for the four sites, compounds exhibit the highest affinity for site 3. The affinity for the site Sudlow 2 is lower than for site 3, while the affinity for the cleft site is lower than for Sudlow 2. From the present series of compounds, they exhibit for Sudlow 1, the lowest affinity, compared to the other three sites.

The results of the docking study using AutoDock for cross-validation of the results given by AutoDock Vina share the same pattern, the affinity of the compounds for albumin being influenced by the size of the heterocycle from the structure of the compounds and less by the type of enantiomer. The most reproducible conformations of the compounds according to the RMSD of the atom coordinates are the ones for compounds **3c** and **3d**. For them, the percentage of conformations from their total conformations generated are found in the same cluster with the top binding conformation is higher than the percentage for **3a** and **3b**. Considering this observation, it can be concluded that compounds **3c** and **3d** have a more repetitive binding in the studied sites compared to compounds **3a** and **3b**, confirming this reproducibility through the repeatability of the conformations found in approximately the same area.

The depiction of the interaction between the ligands and albumin was presented for both enantiomers of a compound, chosen from the present series as the best binding pair of enantiomers on a specific site (Sudlow 2, site 3 and cleft). No depiction was made for any ligand in Sudlow 1 site because the molecular docking study performed on both software indicated that the respective site has a marginal role in the binding of the compounds from the present series.

The Sudlow 2 site, being mainly hydrophobic, comprised of Leu460, Val456, Leu457, Leu453, Leu387, Val433, Leu430 and Val426, easily fits the lipophilic moieties of compounds **3a**–**d**. Both enantiomers of compounds **3c** and **3d** are involved in a *π-π* stacking with Tyr411, while the amidic oxygen acts like a hydrogen bond acceptor from the phenol of Tyr411. The ketone of ligands can interact with the sidechain of Asn391 as a hydrogen bond acceptor and with the positively charged sidechain of Arg410 via an ion–dipole interaction.

Visual analysis of the binding poses of enantiomers of **3c** indicates that there are some differences in the binding mode of the two isomers (Figure 5), but mainly the difference between them is minor.

**Figure 5.** The best binding conformation of enantiomers of compound **3c R** (**left**) and **S** (**right**) in the Sudlow 2 site of albumin. Carbon atoms of **3c** are depicted in magenta.

The binding of **3d** enantiomers in site 3 of albumin is depicted in Figure 6. In both cases, the positively charged sidechain of Arg186 interacts with the benzene ring of tetrahydroisoquinoline fragment through a *π*-cation interaction. The pair Tyr161-Tyr138 are involved in a double *π-π* stacking with one of the benzenes of **3d**, for enantiomer **3dR** is expected to appear two supplementary interactions: one of the ketones with the peptide bridge Tyr138-Leu139 and a hydrogen bond between Tyr161 as a donor and the nitrogen atom of **3d** as acceptor.

**Figure 6. 3d in site 3.** The best binding conformation of enantiomers of compound **3d R** (**left**) and S (**right**) in site 3 of albumin. Carbon atoms of **3d** are depicted in magenta.

The binding of **3d** enantiomers in the cleft of albumin is depicted in Figure 7**.** Both enantiomers are involved with the terminal benzene ring in a π-π stacking interaction with Tyr452. The ketone of **3d** is expected to interact with the sidechain of Asn429 via a hydrogen bond as an acceptor. The amidic oxygen of **3d** is expected to interact with the positively charged sidechain of Lys190 in the case of enantiomer R, while in the case of S enantiomer is expected to interact with the amide bridge between Val455-Val456. The tetrahydroisoquinoline fragment of **3d** of both enantiomers is predicted to interact with some hydrophobic residues, such as Leu463 or Pro421.

**Figure 7.** The best binding conformation of enantiomers of compound **3d R** (**left**) and S (**right**) in the cleft site of albumin. Carbon atoms of **3d** are depicted in magenta.
