Acid‐Stable Nitrofuran Triple Therapy With Rabeprazole and Amoxicillin Targeting Helicobacter pylori Infection

ABSTRACT Helicobacter pylori is an emerging antibiotic‐resistant and carcinogenic gastric pathogen. We developed nitrofuran derivatives with anti‐ H. pylori activity and found the analog CLI‐19‐009 (2‐nitro‐6,7‐dihydrobenzofuran‐4‐one N‐(cinnamoyl)hydrazone) inhibited H. pylori growth at 0.25 mg/L. Notably, CLI‐19‐009 contains a non‐conjugated hydrazone side chain, which may influence nitroreductive activation compared with classical nitrofurans and retain activity under acidic conditions. CLI‐19‐009 exhibited an additive effect with clinical antibiotics and proton pump inhibitors, cleared H. pylori on gastric epithelial cells, reduced bacterial motility, and decreased vacuolation. CLI‐19‐009 also showed low cytotoxicity (IC 50 >64 mg/L) and significantly improved survival of H. pylori ‐infected wax moth larvae compared with untreated controls ( p = 0.0002). Host‐cell RNA sequencing revealed that CLI‐19‐009 treatment downregulated inflammatory genes (IL1B, IL18, IL23A, MAPK1, NFKB1, TNF, and IRAK1) during host–pathogen interactions. Long‐term exposure generated CLI‐19‐009‐resistant clones harboring a single‐nucleotide polymorphism in rdxA in both lineages, demonstrated using whole‐genome sequencing; this finding suggests alterations in nitroreductive activation. CLI‐19‐009 showed in vitro synergy with rabeprazole and additive activity with amoxicillin, and these combinations reduced gastric inflammation and colonization compared with untreated mice when tested in a murine infection model ( p < 0.0001). These findings support further development of CLI‐19‐009 as an anti‐ H. pylori therapeutic alternative to clarithromycin‐based regimens.