*3.3. Docking Study*

Molecular docking studies have the potential to identify novel drug-like molecules that display high binding affinity for the target protein, and they can facilitate the understanding of biological activity data with the purpose of designing new compounds with improved activity. Hence, we extended our study to explore the conformational space and binding orientation of the synthesized pyrimidine derivatives.

The binding conformations of these compounds were explored by MOE-Dock module implemented in the MOE program [53]. The optimized conformers obtained from the Gaussian program were docked to the active site of urease. The docking results indicated that all the conformers were well accommodated inside the active site and were stabilized by various hydrophilic, hydrophobic and van der Waals interactions. The residues involved in these interactions were Arg439, Ala440, His492, Asp494, His593, His594, Asp633 Ala636 and Met637. Compound **3h** (IC50 = 6 ± 0.3 μM) showed strong inhibitory potential against urease as compared to **3b** (inactive) due to methyl substitution at the *para* position of the pyridine ring. Compound **3h** showed good interactions with the residues of the active site by coordinating with the bi-nickel center, via its carbonyl group at the pyrimidine ring and anchoring itself in a way that permitted strong interaction of the carbonyl group with the two nickel ions. Additionally, the two carbonyl groups on the pyrimidine ring acted as hydrogen bond acceptors and mediated hydrogen bond interaction with His492 (2.3 Å) and Arg439 (2.04 Å) of the binding pocket (Figure 2b).

**Figure 2.** Depiction of the docking results of low energy conformer: (**a**) **3a**, (**b**) **3h**, (**c**) **3d**, (**d**) **3j**, (**e**) **3m**, (**f**) **3f**, (**g**) **3p**, and (**h**) **5**. The key residues are presented as sticks models and nickel atoms are shown as green circles.

Compound **3a** (IC50 = 9 ± 2 μM), another active compound of the series, established various potential interactions with the active site of urease, as well as with nickel ions. The oxygen atom of the morpholine ring was involved in the chelation process with the nickel center in the catalytic site. This compound was further stabilized by two typical hydrogen bonds with His492 (2.8 Å) and Arg609 (3.0 Å). Apart from hydrogen bond interactions, a tetramine of the compound participated in the salt bridge interaction with the negatively charged Asp494 residue (Figure 2a). Compounds **3d** and **3l** (IC50 = 8 ± 0.3 μM) had almost similar structures, biological activities and patterns of binding interactions with the active site residues. The only difference was the interaction with metal ions. The lone pair of electrons for sulfur (C = S) of **3l** mediated strong interaction with the two nickel ions and hydrogen bond interaction with Arg609 (2.4 Å) and Met637 (2.8 Å), while in case of **3d**, the carbonyl group on the pyrimidine ring was involved in the interaction with the nickel ions as depicted in Figure 2c.

Compounds **3j** (IC50 =10 ± 0.9 μM) and **3k** (IC50 =11 ± 1 μM), which showed good biological activity, presented similar types of interactions. The additional effectiveness of **3j** compared to **3k** was due to the absence of a methyl group at the *meta* position of the benzene ring, which allowed the compound to establish close contacts with the active site residues (Figure 2d). The ring nitrogen of the pyridine is involved in two productive hydrogen bond interaction with His492 and Met637 at 2.54 and 3.4 Å, respectively. Moreover, the C = O of pyrimidine rind mediated a potential hydrogen bond interaction with the NH of His593 at 2.59 Å, while the compound was further stabilized through hydrophobic interactions with Ala440, Cme592, and His594.

The compounds with electron-withdrawing substitution, especially halogens, showed noteworthy inhibitory potential. For **3m**, **3n**, and **3o**, the presence of halogen atoms with their respective position on the benzene ring affected the binding pattern and orientation of the compound within the pocket (Figure 2e). Compounds **3f** (*o*-chloro phenyl) and **3o** (*o*-iodo phenyl) with IC50 = 10 ± 0.6 μM showed two hydrogen bond interactions with His492 and Ala440 (Figure 2f), while 3m and 3n were stabilized by a single hydrogen bond with His492. These compounds also displayed hydrophobic interaction with Ala440, His593 and Met637. Compounds **3c** (IC50 = 26 ± 1μM) and **3p** (IC50 = 22 ± 0.8 μM), both with moderate biological activity, established two hydrogen bond interactions with His492 and Ala440. However, only one interaction with the metallocenter was observed (Figure 2g). The compounds with low biological activity, as compared to the standard and other pyrimidine derivatives such as **3i** (IC50= 66 ± 2.4 μM) and **5** (IC50= 42 ± 2.3 μM), established single hydrogen bond interaction with the active site residue Arg439 while compound mostly established by hydrophobic interaction with the active site residues (Figure 2h). However, no interaction with nickel was observed for **3i**.

Indeed, the urease inhibition capacity of the synthesized pyrimidine derivatives is attributed to the mutual contribution of the distinct substitutions they bear. However, interaction with the metallocenter and hydrophilic interaction with Ala440, His492, His593, and Met637 are found to be crucial for the activity of these compounds.

### *3.4. Density Functional Theory (DFT)*

The physicochemical properties and frontier molecular orbitals (FMOs) of the new enaminone compounds play a crucial role in enhancing bioactivity. Khon-Sham's DFT approach with the B3LYP method was used for geometry optimization [54]. The electron-donating and -withdrawing ability of a compound can be explained by its HOMO and LUMO. The higher the energy value of HOMO, the greater the electron-contributing ability of the compound. The energy difference between HOMO and LUMO is an established parameter to measure the electron conductivity or degree of intermolecular charge transfer, which also affects bioactivity [55]. In this study, to examine the urease inhibition capacity of the pyrimidine derivatives, we randomly selected compounds for comparison with DFT results.

The results of HOMO-LUMO energies were plotted against the biological activity of the pyrimidine derivatives (Table 2). Good correlation was observed between biological activity and the energies of the LUMO orbitals. Compounds **3h**, **3k**, **3f**, and **3o** showed significant LUMO energy of −1.89, −2.24, −1.96 and −2.26 eV, respectively.


**Table 2.** Theoretical results of pyrimidine derivatives.

The visualization of the HOMO-LUMO orbitals of **3h** reflects the localization of FMO (frontier molecular orbital) (Figure 3). The negative and positive phases of orbitals are shown in green and red, respectively. For **3h**, HOMO is localized on the pyrimidine ring, distal pyridine moiety and carbonyl group, whereas LUMO is on the pi-bond adjacent to the pyrimidine ring. In contrast, for **3o**, electrons are delocalized mainly on the benzene ring with iodine group as a substituent. The influence of LUMO energy on the inhibitory activity of the compounds might be due to the presence of halogen substitution and the pyridine ring. The halogen substitution at *ortho* and *para* positions made a grea<sup>t</sup> contribution to the urease inhibition capacity of the derivatives. Notably, neither HOMO nor LUMO were located on the methyl group, which would explain why this group contributed the least to binding with the protein.

The energy gap for the most active compound **3h** of the series was −4.35. A similar energy di fference was observed for **3k**, **3f**, and **3o**. The smaller the di fference in HOMO-LUMO energies, the greater the chemical reactivity. For any potential interaction, electron transfer from high-lying HOMO to low-lying LUMO is always energetically favorable. Given this consideration, **3h**, **3k**, **3f**, and **3o** possess good activity, which correlated well with urease inhibitory activity.

**Figure 3.** The optimized geometries and surfaces of the HOMO (Highest occupied molecular orbital)-LUMO (Lowest unoccupied molecular orbital) of **3h**, **3k** and **3o** obtained at the B3LYP/6-31G (d, p) level.

Molecular electrostatic potentials (MEP) were run for compounds **3h**, **3k**, and **3o** at B3LYP/6-31G (d, p), providing information about the sites reactive towards nucleophilic and electrophilic attack, together with hydrogen-bond interactions. The potential of electrostatic interaction at the surface is shown by blue, red and green, representing the sites for positive, negative and no electrostatic potential, respectively (Figure 4). Moreover, the positive and negative regions of the maps were responsible for the nucleophilic and electrophilic reactivity of the compounds.

**Figure 4.** The molecular electrostatic potential (MEP) of **3h**, **3k,** and **3o**.
