Synthesis and Evaluation of Ciprofloxacin-Nitroxide Conjugates as Anti-Biofilm Agents

Abstract

As bacterial biofilms are often refractory to conventional antimicrobials, the need for alternative and/or novel strategies for the treatment of biofilm related infections has become of paramount importance. Herein, we report the synthesis of novel hybrid molecules comprised of two different hindered nitroxides linked to the piperazinyl secondary amine of ciprofloxacin via a tertiary amine linker achieved utilising reductive amination. The corresponding methoxyamine derivatives were prepared alongside their radical-containing counterparts as controls. Subsequent biological evaluation of the hybrid compounds on preformed P. aeruginosa flow cell biofilms divulged significant dispersal and eradication abilities for ciprofloxacin-nitroxide hybrid compound 10 (up to 95% eradication of mature biofilms at 40 μM). Importantly, these hybrids represent the first dual-action antimicrobial-nitroxide agents, which harness the dispersal properties of the nitroxide moiety to circumvent the well-known resistance of biofilms to treatment with antimicrobial agents.

Keywords: antibiotic; biofilm; ciprofloxacin; nitroxide; radical.

Figure 1

NO donor prodrug cephalosporin-3′-diazeniumdiolate 1.

Figure 2

Structure of nitric oxide and the general structure of a nitroxide.

Figure 3

Ciprofloxacin 2.

Figure 4

TEMPOL 3 and alcohol derivative 4.

Scheme 1

Synthetic route to ciprofloxacin-nitroxide hybrid 10 and its corresponding methoxyamine derivative 11. Reagent and conditions: (a) (i) AcOH, MeOH 60 °C, 2 h. (ii) NaBH₃CN, 50 °C, 24 h; (b) 2 M NaOH, MeOH, 50 °C, 5 h.

Scheme 2

Synthetic route to ciprofloxacin-nitroxide hybrid 16 and its corresponding methoxyamine derivative 17. Reagent and conditions: (a) (i) AcOH, MeOH 60 °C, 2 h. (ii) NaBH₃CN, 50 °C, 24 h; (b) 2 M NaOH, MeOH, 50 °C, 5 h.

Figure 5

Confocal laser scanning microscopy images of 2 day old pre-formed P. aeruginosa PA14 biofilms grown in a flow cell at 37 °C, treated with 20 µM of (b) 10; (c) 11; (d) 16; (e) 17 for 24 h and then visualized with SYTO-9 (stains live cells green) and propidium iodide (stains dead cells red, merged colors appear as yellow to red). Panel (a) shows an untreated P. aeruginosa PA14 biofilms after 3 days. The scale bars represent 40 µm in length for images (a–c,e), and 50 μm for image (d). Each panel also shows the xy, yz and xz dimensions.

Figure 6

Confocal laser scanning microscopy images of 2 day old pre-formed P. aeruginosa PA14 biofilms grown in a flow cell at 37 °C, treated with 40 µM of (b) 10; (c) 11; (d) 16; (e) 17 for 24 h and then visualized with SYTO-9 (stains live cells green) and propidium iodide (stains dead cells red, merged colors appear as yellow to red). Panel (a) shows an untreated P. aeruginosa PA14 biofilms after 3 days. The scale bars represent 50 µm in length for images (a–d). Each panel also shows the xy, yz and xz dimensions.

Figure 7

Flow cell effluent harvested from preformed P. aeruginosa PA14 biofilms treated with 40 µM of compounds 10, 11, 16, and 17 after 24 h. Bacteria were plated for enumeration.