Background: The rapid evolution of antibiotic resistance poses a serious threat to public health. The development of heterocyclic benzothiazole derivatives, as efficient and potential agents, has been the focus of antibacterial drug discovery.

Objective: Present study attempts to evaluate the antibacterial activity and mechanism of action of novel 2-(4’- aminophenyl) benzothiazole derivatives.

Methods: Antibacterial activity of novel benzothiazole derivatives was evaluated by agar disc diffusion method against a panel of susceptible Gram-positive and Gram-negative strains. The mechanism of action was explored by bactericidal kinetics, membrane depolarization, fluorescent assisted cell cytometry and DNA cleavage studies.

Results: Our findings revealed that compounds A07a and A07b turned out to be the most potent analogues having minimum inhibitory concentration values in the range of 3.91-31.2 µg/ml against Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa and Escherichia coli. The new benzothiazole derivatives displayed different modes of action as elucidated by the studies on intact bacterial cells and plasmid DNA. The structure activity relationship studies showed prominent activity of compound A07a containing oxime moiety on carbonyl carbon along with less bulky electron releasing and lipophillic group (methoxy and chloro) in phenyl ring at C2 position of 2-(4’-aminophenyl) benzothiazole ring system.

Conclusion: The potent antibacterial activity of compounds (A07a and A07b) was mediated by membrane perturbing and intracellular mode of actions. These results further validate the use of these derivatives in the treatment of microbial diseases and provide scope for further research.