We designed and synthesized a series of pyrazole-tagged alkanoyl (1–15) and oxoalkyl (16–31) derivatives of ciprofloxacin (CP) by modifying the nitrogen atom of the C-7 piperazinyl ring. Next, we evaluated their antibacterial activity, bactericidal kinetic, cytotoxicity, antibiofilm properties, and molecular interactions with bacterial topoisomerases. These compounds showed strong antibacterial activity against standard and clinical Gram-positive and Gram-negative pathogens, with several derivatives (5, 8, 19, 22, 23) with MIC values of 0.4–0.5 µg/mL being as effective as CP, and compounds 12 and 22 proving even more potent against S. aureus strains (MICs values of 0.25–0.4 µg/mL). Cytotoxicity tests on human HaCaT cells confirmed their low toxicity (IC50 ranged from 25 to >100 µM). Analogs 12 and 22 demonstrated both bactericidal activity and strong biofilm-inhibitory effects in most studies. They inhibited the formation of biofilms by the most susceptible S. aureus, S. epidermidis and S. pasteuri isolates at concentrations corresponding to the MIC or MBC. Notably, compound 12 was significantly more potent than CP at eradicating mature S. aureus biofilms, reducing biofilm mass two to three times more effectively than CP, and achieving biofilm survival rates of 18–57 %. Molecular docking studies confirmed that both derivatives bind to bacterial topoisomerases in a similar manner to CP, with differences in substituent orientation influencing their interactions. Overall, conjugate 12 emerges as a promising lead due to its strong antibacterial and antibiofilm properties, making it a potential candidate for further development.
