Abstract
The increasing prevalence of multidrug-resistant bacteria presents a major public health
challenge, necessitating novel antimicrobial strategies. This study evaluates the antimicrobial
activity and mechanisms of action of three synthetic heterocyclic compounds: Compound A
(2-amino-4-phenylpyrimido[1,2-a]benzimidazole), Compound B (2-imino-4-phenyl-2H-1,3-
thiazino[3,2-a]benzimidazole), and Compound C (2-imino-4-phenyl-pyrimido[1,2-
a]benzoxazole). Their antibacterial effects were assessed against Escherichia coli (Gramnegative),
Staphylococcus aureus (Gram-positive), and Mycobacterium smegmatis (acid-fast)
using microbiological and mechanistic assays. All three compounds exhibited an MIC of
15.625 μg/mL, with species-specific MBC variations. E. coli displayed a higher MBC for
Compound B (62.5 μg/mL), while S. aureus and M. smegmatis had uniform MBCs of 31.25
μg/mL across all compounds. Mechanistic studies revealed that Compound B significantly
increased bacterial membrane permeability (57.0% in S. aureus, 43.6% in E. coli), suggesting
a membrane-destabilizing effect. Compound C exhibited the strongest efflux pump inhibition
in E. coli (50.3% ethidium bromide efflux reduction at 32 μg/mL), comparable to verapamil
(52.1% inhibition). Molecular docking studies predicted strong interactions between
Compound C and multiple efflux-associated proteins, particularly in 8TTE (-8.3 kcal/mol) and
8QKK (-8.0 kcal/mol), supporting its potential as an efflux pump inhibitor. Compound A also
exhibited high-affinity binding (-8.1 kcal/mol in 8TTE), reinforcing its inhibitory potential. In
contrast, Compound B displayed moderate docking scores (-7.5 to -7.9 kcal/mol), with stable
interactions in 4C48 involving key hydrophobic residues such as PHE727 and TRP809.
However, experimental validation of direct target binding is necessary. Synergy analysis
revealed that Compound B and Compound A exhibited strong synergy (FIC = 0.4) against S.
aureus, while the triple combination (A+B+C) produced additive effects (FIC = 1.4) rather
than enhanced synergy. Additionally, a non-linear fluorescence response was observed in M.
smegmatis, suggesting potential fluorescence quenching or altered permeability dynamics at
high concentrations. These findings highlight the potential of these compounds in antimicrobial
development, particularly for targeting efflux-mediated resistance and bacterial membrane
integrity. However, cytotoxicity testing was not conducted in this study, and future work will
be necessary to evaluate the safety profile of these compounds in mammalian systems. Further
studies are required to assess their cytotoxicity, antibiotic potentiation effects, and
pharmacokinetics to determine their clinical applicability.