3.3.5. Molecular Dynamics

The stability of the albumin complexes with the best binding ligands in the molecular docking study performed with AutoDock vina with the variation of the Gibbs free energy in the first half of energies was evaluated during 100 ns of molecular dynamics simulation. According to the results of the molecular docking study, all the potential complexes of the studied compounds in site 3 of albumin were simulated, and the complexes of compounds **3c** and **3d** in the site Sudlow 2 and in the cleft site. None of the complexes were simulated with ligands docked into the Sudlow 1 site due to their low affinity to the respective site.

The stability of the protein–ligand simulated systems in the molecular dynamics study was expressed by calculating the average root-mean-square deviation (RMSD) of the backbone of the protein, the average root-mean-square deviation (RMSD) of the heavy atoms of ligands, the radius of gyration (RG) of the protein and the hydrogen bonds between the ligand and the protein.

An overview of the results of the molecular dynamics study is presented in Table 4 as the RMSD of the backbone of the protein, in Table 5 as the RMSD of the heavy atoms of ligands, Table 6 as RG of the protein, and in Table 7 the evolution of the hydrogen bonds between protein and ligands.

**Table 4.** The root mean square deviation of the backbone of the protein from the systems evaluated in the molecular dynamics study (nm).



**Table 5.** The root mean square deviation of the heavy atoms of the ligands from the systems evaluated in the molecular dynamics study (nm).


**Table 5.** *Cont.*



**Table 6.** The radius of gyration of the protein from the systems evaluated in the molecular dynamics study (nm).



**Table 7.** The average number of hydrogen bonds between the ligand and the protein in the systems evaluated in the molecular dynamics study (no/ns).



It can be seen that compounds **3a** and **3b** (pyrrolidine and piperidine derivatives) gives complexes with albumin, which are less stable than those of compounds **3c** and **3d** (tetrahydroquinoline and tetrahydroisoquinoline hybrids). The increase of the nitrogen ring with a supplementary benzene ring leads to better stabilization of the albumin. On average, taking into account the data available for the complexes resulting from the binding of ligands into site 3, RMSD of the backbone of the protein is higher for complexes with compounds **3a** and **3b** than those with **3c** and **3d** (0.33 nm vs. 0.31 nm). The same trend is identified for the average RMSD of the ligands (0.56 nm vs. 0.44 nm) and the average RG of the protein (0.56 nm vs 0.44 nm). The highest stabilization of the backbone of the protein, expressed as the lowest RMSD of the backbone, was identified as **3cR** into site 3 and cleft, **3dR** into Sudlow 2 and cleft and **3dS** in Sudlow 2.

The changes in the position of the ligand expressed as RMSD of the heavy atoms of the ligand indicate that most of the predicted complexes are stable. Some exceptions were identified, such as compound **3bS** in site 3, **3cR** in cleft, **3dR** in Sudlow 2 and site 3 and **3dS** in site 3, which move significantly from their initial position.

Evaluating the hydrogen bonding between the ligand and the protein **3cR** and **3dS** are the ones that interact more via this type of bond than the other compounds.

Overall, the resumed data indicates that there is no obvious connection between the type of enantiomer of each compound and the parameters evaluated for the resulting complexes to express their stability. The stability of the complexes is influenced simultaneously by the type of nitrogen ring and the type of enantiomer.

Detailed information regarding the evolution of the stability of the complexes of compounds **3c** and **3d** in the Sudlow site 2 are presented in Table S1, of all **3a**–**d** compounds in site 3 of albumin in tables Tables S2–S4 for compounds **3c** and **3d** in the cleft site of albumin.

The data obtained after simulation of the complexes of compounds **3c** and **3d** into the Sudlow 2 site indicates that both compounds gave complexes with albumin with similar RMSD to the apo form of the protein. The complexes of **3d** (both enantiomers) lead to the best stabilization of the protein in terms of the RMSD of the protein backbone. Into the specified site, **3cR, 3cS** and **3dS** have the lowest movement, compared to **3dR,** which has a significant change in position during the simulation.

**3cR** and **3dS** are supposed to have significantly more hydrogen bonds compared to the other enantiomer. Again, this observation confirms the previous observation that the interaction between each enantiomer and protein is influenced by the type of nitrogen ring and the type of enantiomer.

When docked into site 3 of albumin, compounds **3aS** and **3cR** gave the most stable complexes in the present series. Significant instability of the complexes given by the other compounds was identified as follows: **3bS** leaves site 3 after approximately 20 ns, and **3cR** suffers a significant change of position at approximately 35 ns, resulting in continuous changes in the coordinates of the atoms of the backbone of albumin, **3cS** gave a stable complex until 85 ns of simulation, while both enantiomers of **3d** won't reach a convergence, having a continuous movement into the site 3 of albumin.

The complex of **3dR** in site 3 of albumin is the most stable from the current series. A high degree of stability expressed in terms of RMSD of the protein backbone was identified too, but it didn't reach a convergence point during the simulation and the RMSD of the protein backbone was found to slowly and continuously increase until the simulation ended. Anyhow, both R enantiomers gave more stable complexes than S enantiomers when docked into the cleft of albumin. **3cS** moved less into the cleft than **3cR**, but the RMSD of the backbone was similar to the apo form and even decreased during simulation, compared to **3cS**. **3dS** exhibited a significant change in position at approximately 70 ns, affecting a little the RMSD of the backbone of the protein at that time, increasing the RMSD of the protein backbone from complex over the apo form.

#### **4. Conclusions**

In conclusion, we have obtained four novel hybrid compounds combining a ketoprofen skeleton and an *N*-containing hetero ring. The newly discovered molecules have been thoroughly characterized and were subjected to a comprehensive mass spectral analysis. According to the *in vitro* and *in silico* experiments, the hybrid compounds have considerable *HPSA* and *in vitro* anti-inflammatory action as measured by *IAD*. Despite their lipophilic character, compounds **3b**-**d** have *HPSA* values comparable to quercetin. To neutralize damaging radicals in the cell membrane, lipophilic antioxidants are required. *In vitro*, anti-inflammatory activity was evaluated by *IAD*, as well as by molecular docking and molecular dynamics. Ligand–albumin interactions were demonstrated for both enantiomers of compound **3a**-**d**, which were chosen from the current series as the best binding pair of enantiomers at a given site (Sudlow 2, site 3, and Cleft). The highest *in vitro* anti-inflammatory efficacy is shown by hybrids **3c** and **3d**, which stabilize the albumin macromolecule by forming ligand–albumin complexes with Sudlow 2, site 3, and cleft. This interaction is responsible for preventing albumin denaturation during inflammatory processes. The stability of the albumin macromolecule is due to the fact that hybrids **3c** and **3d** participate in *π-π* arrangement with Tyr411 (Sudlow 2), with the pair Tyr161-Tyr138 (site 3), and Tyr452 (cleft). Furthermore, H-bonds are generated with the amide, ketone, and oxygen with the polar amino acid residues implicated in the Sudlow 2, site 3, and cleft structures. The in-silico studies completely confirm our *in vitro* experimental results for anti-inflammatory effects. All of this demonstrates that the hybrid compounds we synthesized inherit ketoprofen's anti-inflammatory capabilities, making them excellent candidates for future medications.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pr11061837/s1, Figure S1: 1H-NMR spectrum of compound **3a**; Figure S2: 1H-NMR spectrum of compound **3b**; Figure S3: 1H-NMR spectrum of compound **3c**; Figure S4: 1H-NMR spectrum of compound **3d**; Figure S5: 13C-NMR spectrum of compound **3a**; Figure S6: 13C-NMR spectrum of compound **3b**; Figure S7: 13C-NMR spectrum of compound **3c**; Figure S8: 13C-NMR spectrum of compound **3d**; Figure S9: UV spectrum of compound **3a**; Figure S10: UV spectrum of compound **3b**; Figure S11: UV spectrum of compound **3c**; Figure S12: UV spectrum of compound **3d**; Figure S13: ESI-HRMS of compound **3a**; Figure S14: Mass spectrum of **3a** obtained by positive ion ESI-MS/MS; Figure S15: ESI-HRMS of compound **3b**; Figure S16: Mass spectrum of **3b** obtained by positive ion ESI-MS/MS; Figure S17: ESI-HRMS of compound **3c**; Figure S18: Mass spectrum of **3c** obtained by positive ion ESI-MS/MS; Figure S19: ESI-HRMS of compound **3d**; Figure S20: Mass spectrum of **3d** obtained by positive ion ESI-MS/MS; Table S1: RMSD of protein backbone, RMSD of ligands and RG of protein in the molecular dynamics study when ligands **3c** and **3d** were docked into the Sudlow 2 site; Table S2: RMSD of protein backbone, RMSD of ligands and RG of protein in the molecular dynamics study when ligands **3a** and **3b** were docked into the site 3 of albumin; Table S3: RMSD of protein backbone, RMSD of ligands and RG of protein in the molecular dynamics study when ligands **3c** and **3d** were docked into the site 3 of albumin; Table S4: RMSD of protein backbone, RMSD of ligands and RG of protein in the molecular dynamics study when ligands **3c** and **3d** were docked into the cleft of albumin.

**Author Contributions:** Conceptualization, S.M. and D.B.; methodology, I.I.; software, G.M., S.M., S.O. and O.O.; validation, S.M., I.I. and D.B.; formal analysis, N.B., S.M., G.M., D.B., S.O. and P.N.; investigation, I.I.; resources, I.I. and O.O; data curation, S.M.; writing—original draft preparation, S.M., D.B. and G.M.; writing—review and editing, S.M. and D.B.; visualization, G.M.; supervision, I.I., S.O. and O.O; project administration, S.M.; funding acquisition, I.I. and O.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Scientific Research Fund of the University of Plovdiv, grant number ΦΠ23-XΦ-005.

**Data Availability Statement:** The data presented in this study are available in this article and supporting Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.
