2.1.2. Molecular Modeling

Molecular modeling is a powerful tool employed by theoretical chemistry for quantitative predictions on guest–host interaction. The molecular docking analysis was performed using the Autodock 4.2.6 software together with the AutoDockTools [41]. The software applies a semi-empirical free energy force field and grid-based docking to assess conformations during docking process. The force field includes six pair-wise assessments (*V*) and an estimate of the conformational entropy lost upon binding (∆*Sconf*):

$$
\Delta G = \left(V\_{bound}^{L-L} - V\_{unbound}^{L-L}\right) + \left(V\_{bound}^{T-T} - V\_{unbound}^{T-T}\right) + \left(V\_{bound}^{T-L} - V\_{unbound}^{T-L} + \Delta S\_{conf}\right) \tag{1}
$$

where *L* makes mention of the "ligand" and *T* refers to the "target" in a ligand–target docking calculation. Each of the pair-wise energetic terms includes evaluations for dispersion/repulsion, hydrogen bonding, electrostatics and desolvation [42]. Following the redocking analysis, the root-mean-square deviation (RMSD) values lower than 0.4 Å have been calculated, suggesting the robustness and repeatability of the docking analysis.

The binding free energy value calculated for RSP/RM-β-CD inclusion complex (1:1) was −3.26 kcal/mol. Figure 3 presents the theoretical RSP/RM-β-CD inclusion complex, as rendered in the PyMOL [43] and Discovery Studio molecular visualization systems, simulated in a 1:1 molar ratio.

Analyzing the 3D images of the RSP/RM-β-CD (1:1) interaction, we noticed the presence of two Pi–sigma interactions, between pyrimidin-4-one cycle and the hydrogen (hydrogen methyl group) of a glucopyranose (2.28 and 2.73 Å). Two non-classical hydrogen bonds occur between the nitrogen group from the 4,5-dihydro-isoxazole heterocycle and the hydrogen from position 4 of a carbohydrate moiety. Four non-classical hydrogen bonds occur between the 1,2-oxazole heterocycle and the carbohydrate moiety hydrogen, with lengths of 2.3 Å approximately.

**Figure 3.** Molecular docking for 1:1 RSP/RM-β-CD inclusion complex. Figures (**a**,**b**) show the inclusion complex from the secondary face of RM-β-CD's cavity. The RSP molecule is represented in green and blue spheres while RM-β-CD is represented in red sticks (**a**); RSP is presented in sticks colored by element, RM-β-CD is presented in spheres colored by element (**b**); Image (**c**) show contacts between RSP and RM-β-CD, RSP is colored by element, while CD is presented in balls and sticks.
