Synthesis of piperazine-based benzimidazole derivatives as potent urease inhibitors and molecular docking studies

Abstract

The development of new bioactive compounds is important for progress in therapeutic research. In the present study, we describe the multistep synthetic approach to develop a library of novel benzimidazole analogs incorporating piperazine rings in order to increase their biological activity. In order to synthesize the desired benzimidazole analogs, the synthesis started with the easily accessible precursors between aniline and chloroacetyl chloride. It proceeded via a series of reactions, such as condensation, cyclization, and N-alkylation. TLC optimized each step, and spectroscopic methods such as CHN, IR, EIMS, ¹H-NMR, and ¹³C-NMR were used to characterize the final products. The urease inhibitory activity of the synthesized compounds was evaluated. It was discovered that almost all compounds were quite effective, even more potent (IC₅₀ = 0.15-12.17 µM) than the standard thiourea (IC₅₀ = 23.11 ± 0.21 µM). The structure-activity relationship (SAR) is also established, which displayed that compound 9 L (IC₅₀ = 0.15 ± 0.09 µM) with -NO₂ substitutions at meta position play a major role in urease inhibition and figure out as the most potent analog of the library. These results were further verified by molecular docking analysis, which indicated favorable binding energies and interactions of the compounds with the urease active site. This study not only depicts the importance of multistep synthesis but also the structure-based modification approach to produce new pharmacophores for therapeutic applications.

Keywords: Benzimidazole; Docking study; Piperazine; Urease inhibition.

Fig. 1

Representative urease inhibitors based on benzimidazole and piperazine (compounds A-F) and currently designed urease inhibitors 9a-n.

Scheme 1

Synthesis of benzimidazole derivatives 9a-n.

Scheme 2

NMR interpretation for compound 9 m (units for showed numbers are ppm).

Fig. 2

SAR of compounds 9a, l, m, h-k.

Fig. 3

SAR of compounds 9b-g and 9n.

Fig. 4

Lineweaver–Burk plot obtained through the kinetics study of the compound 9 L on urease enzyme.

Fig. 5

Illustration of superimposed docked orientation of all compounds over the urease active site in front (a) and 90 degree angle rotation (b). As an example, the orientation of compound 9a within the urease active site in front (c) and 90 degree angle rotation (d).

Fig. 6

Detailed depiction of the two different orientation of the most potent compounds 9 m (a, b) and 9 L (c, d) over the JB urease active site.

Fig. 7

RMSD representation of compound 9 L, urease complexed with compound 9 L, and urease backbone complexed with thiourea over a 100 ns MD simulation period (in yellow, red and green, respectively).

Fig. 8

(a) Representation of RMSF for the urease backbone when bound to thiourea (in green) and compound 9 L (in red). (b) Mapping of ligand binding sites over a 100 ns MD simulation period, highlighting α-helical and β-strand regions with light red and blue backgrounds, respectively.

Fig. 9

2D depiction of ligand-residue interactions of the MD simulation of urease-compound 9 L complex.

Fig. 10

Monitoring the distance between Ala440 and Ile599 urease residues while complexed with thiourea (green) and compound 9 L (yellow) throughout the entire MD simulation duration.