2.1.3. MM-GBSA

To measure the strength of binding, the gmx\_MMPBSA library was utilized. Figure 7 shows the values of the energy components of MM-GBSA, and their standard deviations. The binding is mostly due to the van der Waals interaction (an average of −54.8 Kcal/Mol), followed by electrostatic interactions (an average of −25.07 Kcal/Mol) and a total binding energy of −40.38 Kcal/Mol. The amino acid contribution to the binding was measured via the decomposition of MM-GBSA, to know which amino acids are contributing most to the interaction (Figure 8). Eight amino acids showed a contribution to the binding, with values of less than −1 Kcal/Mol. L838, V846, K866, L887, V897, V914, C1043, and F1045 showed binding contributions of −1.07 Kcal/Mol, −1.12 Kcal/Mol, −1.49 Kcal/Mol, −1.37 Kcal/Mol, −1.27 Kcal/Mol, −1.11 Kcal/Mol, −3.21 Kcal/Mol, and −1.80 Kcal/Mol, respectively.

**Figure 7.** Different energy components obtained from the MM-GBSA analysis. Bars represent the standard deviation values.

**Figure 8.** Free binding energy decomposition of amino acids around 10 Å of the **7**-VEGFR-2 complex.

To know the numbers and types of interaction, the trajectory was clustered, and for each cluster, a representative frame was obtained that was used with the PLIP webserver. Table 1 shows the number and types of interactions for each frame. The predominant interaction is the hydrophobic interaction in all of the representative frames that support

the value of the van der Waals component in MM-GBSA analysis. In addition, PLIP outputs the .pse file that shows the 3D interaction pattern for each representative frame (Figure 9).

**Table 1.** Variation of interactions between compound **7** and VEGFR-2, as obtained from the PLIP webserver for the representative frame of each cluster.


**Figure 9.** *Cont*.

**Figure 9.** (**C1**–**C3**) Three-dimensional interaction between compound **7**-VEGFR-2 complex in each of the representative frames for each cluster. Amino acids are shown as blue sticks. Compound **7** is shown as orange sticks. Grey dashed lines: hydrophobic interaction. Blue solid lines: hydrogen bonds.

2.1.4. Density Functional Theory (DFT) Molecular Structure Optimization

The nucleophilic attack of *N*-(4-(hydrazinecarbonyl)phenyl)benzamide to 5-methoxy-1*H*-indole-3-carbaldehyde results in a Schiff base formation through the imine bond (C11- N13). The optimized structure of the formed Schiff base compound is represented in Figure 10. As shown in Figure 10, the imine bond length was found to be 1.28577 Å, while the (C9C11N13) and (C11N13C14) angles were found to be 118.21◦ and 129.37◦, respectively.

**Figure 10.** The optimized molecular structure of the selected compound at B3LYB/6-311++G(d,p).

Quantum Chemistry Calculations

The quantum chemistry calculations have been employed as a successful tool in the discovery of active compounds targeting various diseases such as prostate cancer [44], inflammation [45], and malignant glioblastoma [46]. The quantum chemistry calculations were conducted using the Gaussian(R) 09 program at the B3LYP level, together with the 6-311++G(d,p) basis set and the density functional theory (DFT) approach. As depicted in Figure 11, the electronic density of the highest occupied molecular orbital (HOMO) is localized on the heteroaromatic ring system, while in the lowest unoccupied molecular orbital (LUMO), the electronic density is located over the central linker and phenylbenzamide moieties. Frontier molecular orbital (FMO) theory suggested that HOMO serves as a donor, and LUMO serves as an acceptor for electrons. Both HOMO and LUMO have important roles in electronic investigations, and are essential to modern molecular biology and biochemistry when using quantum chemical calculations. A molecule is thought to be softer and more chemically reactive when its energy gap is small. A molecule is assumed to have greater chemical hardness and to be more stable when it has a large energy gap. The FMO gives very significant evidence for the stability, utilizing the difference in the energy (Egap) of the frontier orbitals. Chemical quantum parameters are related to the inhibition efficiency of compound **7**, such as the chemical potential (μ), global hardness (η), maximal charge acceptance (ΔNmax), and energy change (ΔE); global softness (σ), electronegativity (χ), electrophilicity index (ω), ionization potential (IP), and electron affinity (EA) were calculated according to the equations of Koopmans' theory (Table 2) (the equations were detailed in Supporting Data).

**Figure 11.** Energy gap (Egap), frontier molecular orbitals; HOMO and LUMO at the ground state at B3LYB/6-311++G(d,p).



Global quantum parameters, as well as the dipole moment (Dm) and the total ground state energy (TE), are calculated and summarized in Table 2. The results refer to the ability of comp. **7** to act as an inhibitor against VEGFR-2. For a system in equilibrium, the product of the density of states and probability distribution function gives the number of occupied states per unit volume for a given energy. This number is frequently used to study a variety of physical properties of materials. The total density of state analysis has been calculated and analyzed. The results confirmed the small energy gap of the compound under investigation, as depicted in Figure 12, which confirmed the reactivity of compound **7**. When the Egap of the border orbitals reduces, the inhibitor's efficiency increases [47].

**Figure 12.** The total density of state analysis for the compound under investigation at B3LYB/6- 311++G(d,p).

The Electron Density Maps

The reactivity strength of compound **7** can be predicted using DFT calculations based on the electron density of the donor atoms. The total electron density (TED) map, in Figure 13; represents the whole molecule's electron density. The red regions refer to the high electronegativity chemical sites, which are the O atoms of two carbonyls and methoxy groups in the investigated compound. Such active sites aid with electrophilic attack by amino acids (Cys917 and Glu833). In addition, the yellow-colored regions refer to atoms having a moderate electronegativity and that may form hydrophobic interactions, while the blue zones point to the most favorable positive regions, which accept electrons from the donor atoms of amino acids [48]. The electropositive regions are concentrated over the N-H groups. Such findings explain the nucleophilic attack of amino acids (Cys917 and Glu833) onto the NH groups of the 1*H*-indol and amide moieties, respectively. Furthermore, the possibility of hydrophobic interactions by the 1*H*-indol moiety, the central phenyl group, and the terminal hydrophobic phenyl group was supported by the yellow zones at these functional groups. The electrostatic surface potential (ESP) reveals the inhibition orientation of the molecule on the electrophilic amino acids (Figure 13), which is in the same orientation as the carbonyl and methoxy groups.

**Figure 13.** (**A**)**:** Total electron density (TED) and (**B**): electrostatic potential (ESP) maps of the selected compound (Ball and line form) at the 6-311G++(d,p) basis set. (**C**): ESP maps of the compound (stick form).
