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

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 (IC₅₀>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.