## 3 Results
### MD Simulation and Stability
These findings could indicate that the **4d** molecule may have superior binding characteristics and could be a more stable therapeutic candidate when compared to Aspirin in terms of its interaction with the BSA protein. The RMSF quantifies the average shifts of the BSA protein during its interaction with each ligand. The average RMSF values for the **4d_BSA** and Aspirin_BSA complexes are both 0.16 nm, indicating that **4d** and Aspirin binding to the BSA protein results in similar levels of flexibility or stability in the active site region. This suggests that both ligands exert comparable effects on the protein's conformational dynamics, potentially reflecting analogous interaction modes or binding efficiencies. Furthermore, the low RMSF values emphasize the stability of these complexes, implying that the ligands maintain consistent interactions with the active site throughout the simulation. The Rg also offers a valuable and reliable approach for evaluating the stability of the BSA protein's folding or conformational changes when it is bound to a ligand. This metric provides insightful data on the compactness and structural integrity of the protein-ligand complex over the entire 300 ns MD simulation, making it an effective tool for assessing the dynamic stability of the interaction. Initially, the average Rg values for the **4d_BSA** and Aspirin_BSA complexes were 2.75 nm and 2.72 nm, respectively. This data clearly showed a slight discrepancy between the Rg values of each protein complex, indicating that both structures remained rigid and exhibited higher compactness throughout the 300 ns MD simulation. The SASA measures the surface area of a protein that is accessible to the solvent, providing insight into the extent to which the protein is stabilized by interactions with the solvent. The data in Table 6 reveal that the accessible surface areas for both complexes are almost identical. Specifically, the average SASA values for the **4d_BSA** and Aspirin_BSA complexes were 294.60 nm\({}^{2}\) and 292.99 nm\({}^{2}\), respectively. This indicates that both ligands, **4d** and Aspirin, interact with the BSA protein in a similar manner, with comparable solvent exposure, suggesting similar levels of stability and solvent interaction within the complexes. \begin{table}
\begin{tabular}{c c c} \hline  & **4d\_BSA Complex** & **Aspirin\_BSA Complex** \\ \hline RMSD (nm) & 0.180 \(\pm\) 0.01 & 0.324 \(\pm\) 0.02 \\ RMSF (nm) & 0.161 \(\pm\) 0.01 & 0.169 \(\pm\) 0.01 \\ Rg (nm) & 2.757 \(\pm\) 0.03 & 2.725 \(\pm\) 0.02 \\ SASA (nm\({}^{2}\)) & 294.606 \(\pm\) 1.25 & 292.994 \(\pm\) 1.10 \\ \hline \end{tabular}
\end{table} 
Table 6: The average values of RMSD, RMSF, Rg and SASA for BSA–ligand complexes over the 300 ns simulation period. All the data obtained demonstrate that the synthesized molecules are capable of effectively inhibiting the biological function of the BSA protein. These findings suggest that the results of this study hold significant potential for the development of new and effective anti-inflammatory drugs. Figure 6 illustrates the biomolecular stability of each system over 300 ns of simulation. Dynamic cross-correlation matrices were utilized to investigate the interconnected movements of structural configurations, providing insights into the stability of the BSA protein upon interaction with the reference ligand (Aspirin) and the synthesized compound (**4d**) derived from molecular dynamics simulations. These cross-correlation maps highlighted residual changes in the protein structure. Figure 7 depicts the movement of residues, both positive and negative, throughout the 300 ns simulation. The color scale represents the correlation levels, with blue indicating a positive correlation and red signifying a negative correlation. A positive correlation implies that residues move in the same direction, whereas a negative correlation indicates movement in opposite directions. The analysis of the dynamic cross-correlation matrix maps for both systems showed that their correlated motions were quite similar.