Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents

Abstract

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC₅₀ values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

Figure 1

Chemical structures of Ciprofloxacin, Norfloxacin, and Vosaroxin.

Figure 2

Antimicrobial (A) and antitumoral (B) structure–activity relationships of 4-quinolones.

Scheme 1. Synthetic Strategy for the Synthesis of Compounds 1–3a,b

Reagents and conditions: (i) 4-fluoro-1-nitro-2-(trifluoromethyl)benzene, DMSO, 120 °C, 1 h; (ii) CH₃OH, p-TsOH, 70 °C, 22 h; (iii) 4-fluoro-1-nitro-2-(trifluoromethyl)benzene, CH₃CN, 80 °C, overnight.

Scheme 2. Synthetic Strategy for the Synthesis of Final Compounds 6a–6d and 7a–7d

Reagents and conditions: (i) 1-aminoethanol or 1-aminopropan-3-ol, CH₃CN, 60 °C, overnight; (ii) CH₃SO₂Cl, TEA, dry CH₂Cl₂, 0 °C, then room temperature, 1 h; (iii) 2a and 2b, CH₃CN, reflux, overnight; (iv) aqueous NaOH 2 M, reflux, 24 h.

Figure 3

NO detection by a Griess test. Effects of 80 μM solution of 1a, 1b, and 7a–7d on NO production after 1 h of white light irradiation (mean ± SD of three independent experiments). Nitrite concentration was determined by comparing the test samples’ absorbance values to a standard curve generated by serial dilution of 50 μM NaNO₂.

Figure 4

IC₅₀ values obtained following 72 h treatment with Ciprofloxacin (C) and its derivatives in the MTT assay (mean ± ES 4/5 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 vs Cip).

Figure 5

IC₅₀ values obtained following 72 h treatment with Norfloxacin (N) and its derivatives in the MTT assay (mean ± ES 4/5 independent experiments; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs Nor).

Figure 6

IC₅₀ values obtained following 24 h treatment with compound 7b or 7c with 1 h irradiation and 48 h incubation in the drug-free medium and MTT assay (mean ± ES 3/4 independent experiments. *p < 0.05 vs white light).

Figure 7

IC₅₀ values obtained following 72 h treatment with DOX and the MTT assay (mean ± ES 4/5 independent experiments; ***p < 0.001 vs MCF7).

Figure 8

Summary of the most potent compounds synthesized in this work with their IC₅₀ values on the tested cancer cell lines.

Figure 9

Photodynamic treatment of P. aeruginosa PAO1 (a) and S. aureus ATCC 6538P (b). Bacteria were incubated in the dark with or without 7c (10 μM) for 10 min and then irradiated under blue light (20 J/cm²). Dark controls were not irradiated. Viable cells are expressed as CFU/mL. Values represent the mean of at least three independent experiments, *p < 0.05.

Figure 10

3D superposition of the best-docked pose for 1a bound to the Topo IIα (A) and binding site interactions (B). RMSD superposing on the receptor (C) and starting structure (D).