Mahmood J Jawad¹, Ali H Ijam¹, Jalal H Mohammed², Fatima Razzaq Mohammed³, Samir M Awad^{1, 4}, Mohamed F El-Shehry⁵, Ahmed A El-Rashedy^{6, 7}

Abstract

Purpose: To assess the metronidazole-resistant Helicobacter pylori properties of licorice extract and new metronidazole derivatives from 2-thiouracil moiety using in vitro and in silico models. Methods: In vitro anti-Helicobacter pylori activity of licorice extract and new compounds 4a, 4b, and 7 were evaluated against clinical isolates of Helicobacter pylori using the disk diffusion method with concentrations of 8, 16, and 32 μg/disk for each sample, using metronidazole as the standard. The docking studies were carried out using Auto Dock 4.2 on Helicobacter pylori urease enzyme with protein data bank (PDB) ID code 1E9Z as Helicobacter pylori inhibitor. Results: All synthesized compounds and licorice extract demonstrated significantly greater inhibitory activity against metronidazole-resistant H. pylori compared to metronidazole alone (p < 0.01 at 32 μg/disk). Compound 4a exhibited the highest potency, with a mean zone of inhibition of 28.5 ± 1.2 mm at 32 μg/disk, outperforming both metronidazole (12.3 ± 0.8 mm) and other derivatives. A synergistic combination of compound 4a and licorice extract resulted in a ~35 % increase in zone of inhibition compared to 4a alone. Docking studies confirmed that compound 4a had the strongest binding affinity to the urease active site (calculated binding energy: -9.8 kcal/mol), forming stable hydrogen bonds with key residues, which correlates with its superior efficacy. Conclusion: Compound 4a is promising lead compound with efficacy against resistant H. pylori. Furthermore, licorice extract acts as a potent synergistic agent, significantly enhancing the activity of 4a. Compound 4a-licorice combination represents a viable and novel therapeutic strategy to overcome metronidazole-resistant H. pylori infection and warrants further in vivo investigation.