Broad‐Spectrum Antimicrobial and Antibiofilm Activities of Halogenated Anilines Against Uropathogenic <scp> <i>Escherichia coli</i> </scp> and <scp>ESKAPE</scp> Pathogens

Uropathogenic Escherichia coli (UPEC) is one of the leading causes of nosocomial infections and urinary tract infections (UTIs), capable of inducing a spectrum of conditions ranging from acute bladder cystitis to chronic pyelonephritis. The virulence arsenal of UPEC includes factors, such as curli fimbriae, hemolysin, motility elements and metallophores. Moreover, UPEC can form biofilm-like communities and quiescent intracellular reservoirs within host tissues, contributing to recurrent and persistent infections. This study investigates the antibiofilm and antimicrobial activities of two halogen-substituted aniline derivatives, 4-bromo-3-chloroaniline (4B3CA) and 3,5-dibromoaniline (3,5-DBA), against UPEC. The compounds demonstrated minimum inhibitory concentrations (MICs) of 200 μg/mL for 4B3CA and 100 μg/mL for 3,5-DBA, with both exhibiting a biofilm inhibition IC50 value of 10 μg/mL. Additionally, these derivatives showed antibiofilm activity against ESKAPE pathogens. Treatment with 4B3CA and 3,5-DBA led to significant downregulation of UPEC virulence- and biofilm-related genes, including those involved in curli production, fimbrial adhesion, motility, iron acquisition, quiescent colony formation, and stress response. Interestingly, a mild upregulation of hlyA, csrA and uvrY was noted, alongside a marked downregulation of the adenylate cyclase genes cyaA and crp. These findings suggest that inhibition of adenylate cyclase activity may be a primary mode of action, leading to both antimicrobial and antibiofilm effects. The presence of halogen atoms in these compounds appears to enhance their binding affinity to adenylate cyclase through stabilising halogen bond interactions. Furthermore, 3D-QSAR analysis indicates that electrostatic favourability at the third and fourth positions of the aniline ring is critical for bioactivity. Finally, in silico ADMET profiling and cytotoxicity assessments using Caenorhabditis elegans suggest that these aniline derivatives hold promise as therapeutic candidates, warranting further investigation.