Pharmacological Characterization of 7-(4-(Piperazin-1-Yl)) Ciprofloxacin Derivatives

Pharmacological characterization of 7-(4-(piperazin-1-yl)) ciprofloxacin derivatives: antibacterial activity, cellular accumulation, susceptibility to efflux transporters, and intracellular activity

Béatrice Marquez, Vincent Pourcelle, Coralie M. Vallet,Marie-Paule Mingeot-Leclercq, Paul M. Tulkens, Jacqueline Marchand-Bruynaert, Françoise Van Bambeke

Supplemental material

S1. Synthesis and characterization of ciprofloxacin derivatives

a) 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-acetyl-piperazin-1-yl)-quinolone-3-carboxylic acid (1)

To a solution of 1-cyclopropyl-6-fluoro-1,4-dihydro-7-piperazin-1-yl-4-oxo quinoline-3-carboxylic acid hydrochloride (ciprofloxacin, 50 mg, 0.13 mmol) in 1,4-dioxane (2 mL) and NaOH 2M (0.5 mL) at 0°C, acetyl chloride (12 mg, 0.15 mmol) was added dropwise. The reaction mixture was stirred at room temperature for two hours and HCl 1M (1 mL) was added to reach pH 1. The precipitate was extracted with DCM and the organic phase dried on MgSO4.After solvent evaporation the collected solid was recrystallized from THF at -20°C to give 1 as a bright yellow powder (21 mg, 0.05 mmol) with 43 % yield.

Mp : 270-272°C.

1H NMR (300MHz, CDCl3)  (ppm): 1.23 (m, 2H, H-11), 1.42 (m, 2H, H-10), 2.19 (s, 3H, H-16), 3.31(m, 2H, H-14), 3.38 (m, 2H, H-14), 3.56 (m, 1H, H-9 ), 3.74 (m, 2H, H-13), 3.89 (m, 2H, H-13), 7.39 (d, 1H, 4JH-F = 7.0 Hz, H-8), 8.07 (d, 1H, 3JH-F = 12.9 Hz, H-5), 8.8 (s, 1H, H-2).

13C NMR (125MHz, CDCl3)  (ppm):177.4 (d, 4JC-F =2.5 Hz, C-4), 169.5 (C-15), 167.2 (C-12), 154.0 (d, 1JC-F = 250.0 Hz, C-6), 147.9 (C-2), 145.8 (d, 2JC-F = 10.6 Hz, C-7), 139.4 (C-8a), 120.6 (d, 3JC-F = 7.7 Hz, C-4a), 112.9 (d, 2JC-F = 22.5 Hz, C-5), 108.6 (C-3), 105.5 (d, 3JC-F = 4.2 Hz, C-8), 50.6 (C-13), 49.8 (C-13), 46.5 (C-14), 41.4 (C-14), 35.7 (C-9), 21.7 (C-16), 8.7 (C-10,11).

19F NMR (282.41MHz, CDCl3 )  (ppm): -121.1.

MS (APCI) m/z 374.2 [M + H+, 100%].

max (10-4M, CHCl3): 283nm (max= 10430), 322 (3230), 334 (3290).

b) 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-benzoyl-piperazin-1-yl)-quinolone-3-carboxylic acid (2)

To a solution of ciprofloxacin (50 mg, 0.14 mmol) in 1,4-dioxane (2 mL) and NaOH 2M (0.5 mL) at 0°C, benzoyl chloride (31 mg, 0.22 mmol) was added dropwise. The reaction mixture was stirred at room temperature for two hours and HCl 1M (1 mL) was added to reach pH 1. The precipitate was extracted with DCM and the organic phase dried on MgSO4.After solvent evaporation the collected solid was recrystallized from MeOH:iPr2O (1:2) at -20°C to give 2 (62 mg, 0.14 mmol) with a quantitative yield.

Mp: 317°C (decomposition).

1H NMR (300MHz, CDCl3 : CD3OD (10% v/v))  (ppm): 1.19 (m, 2H, H-11), 1.37 (m, 2H, H-10), 3.31(m, 4H, H-14), 3.52(m, 1H, H-9 ), 3.67(m, 2H, H-13), 3.95(m, 2H, H-13), 7.36(d, 1H, 4JH-F = 7.0 Hz, H-8), 7.40(m, 5H, H-17,18,19), 7.98(d, 1H, 3JH-F = 12.9 Hz, H-5), 8.73(s, 1H, H-2).

13C NMR (125MHz, CDCl3 : MeOD (10% v/v))  (ppm):177.1 (C-4), 171.1 (C-15), 167.6 (C-12), 153.7 (d, 1JC-F = 249.9 Hz, C-6), 147.8 (C-2), 145.4 (d, 2JC-F = 10.3 Hz, C-7), 139.1 (C-8a), 134.7(C-16), 130.3 (C-19), 128.7(C-18), 127.0(C-17), 120.3 (d, 3JC-F = 7.8 Hz, C-4a), 112.4 (d, 2JC-F = 23.3 Hz, C-5), 107.6 (C-3), 105.4 (d, 3JC-F = 2.6 Hz, C-8), 50.3 (C-13), 41.9 (C-14), 47.6 (C-13), 35.5 (C-9), 8.1 (C-10,11).

19F NMR (282.41MHz, CDCl3 : CD3OD (10% v/v))  (ppm): -121.1.

MS (APCI) m/z 436.2 [M + H+, 100%].

max (10-4M, CHCl3): 280nm (max=82414), 318 (18322), 334 (17154).

c) 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-ethyl-piperazin-1-yl)-quinolone-3-carboxylic acid (enrofloxacin) (3)

To a solution of ciprofloxacin (100 mg, 0.27 mmol) in CH3CN:H2O (1:1, 10 mL) with diisopropylamine (DIEA, 157µL, 0.9 mmol) at 0°C, bromoethane (61µL, 0.82 mmol) and KI (0.1%mol) were added under argon atmosphere. The reaction mixture was stirred at room temperature for 16 hours and the solvent was evaporated under reduced pressure. The reaction residue was dissolved in 40 mL of DCM and washed under vigorous stirring with 3 mL of aqueous solution adjusted to pH 6.5. The organic layer was dried on MgSO4 and evaporated under reduced pressure to obtain 3 (68 mg, 0.19 mmol) as a white solid with 70% yield.

Mp: 222.4°C.

1H NMR (300MHz, CDCl3)  (ppm): 1.17 (t, 3H, 3JH-H = 7.2 Hz, H-16), 1.21 (m, 2H, H-11), 1.40 (m, 2H, H-10), 2.55 (q, 2H, 3JH-H = 7.2 Hz, H-15), 2.72 (m, 4H, H-14), 3.40 (m, 4H, H-13), 3.52(m, 1H, H-9 ), 7.36(d, 1H, 4JH-F = 7.0 Hz, H-8), 7.40(d, 1H, 4JH-F = 7.1 Hz, H-8), 8.04(d, 1H, 3JH-F = 13.1 Hz, H-5), 8.78(s, 1H, H-2).

19F NMR (282.41MHz, CDCl3)  (ppm): -120.7.

MS (APCI) m/z 360.2 [M + H+, 56%].

max (10-4M, CHCl3): 284nm (max=37787), 326 (12765), 334 (12804).

d) 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-benzyl-piperazin-1-yl)-quinolone-3-carboxylic acid (4).

To a solution of ciprofloxacin (100 mg, 0.27 mmol) in CH3CN:H2O (1:1, 10 mL) with DIEA (157µL, 0.9 mmol) at 0°C, benzyl bromide (49µL, 0.41 mmol) and KI (cat) were added under argon atmosphere. The reaction mixture was stirred at room temperature for 16 hours. After evaporation of the solvent under reduced pressure and lyophilisation of the aqueous phase, the reaction residue was dissolved in 10 mL of DCM and washed under vigorous stirring with 2 mL of saturated NaHCO3 solution for one hour. The organic layer was dried on MgSO4 and evaporated under reduced pressure. The collected solid was dissolved in DCM, precipitated in Et2O and chromatographied on flash silica with DCM:MeOH:iPrOH (5:0.8:0.1) as eluant to obtain 4 (50 mg, 0.12 mmol) as a yellow solid with 44% yield.

Mp: 242.4°C;

1H NMR (300MHz, CDCl3 : CD3OD (10% v/v))  (ppm): 1.19 (m, 2H, H-11), 1.37 (m, 2H, H-10), 2.7 (m, 4H, H-14), 3.37 (m, 4H, H-13), 3.52(m, 1H, H-9 ), 3.62(2H, H-15), 7.28-7.36 (m, 6H, H-8, 17, 18, 19), 7.97(d, 1H, 3JH-F = 13.1 Hz, H-5), 8.73(s, 1H, H-2).

13C NMR (75MHz, CDCl3)  (ppm):177.1 (C-4), 167.1 (C-12), 153.7 (d, 1JC-F = 250.0 Hz, C-6), 147.4 (C-2), 146.0 (d, 2JC-F = 10.3 Hz, C-7), 139.1 (C-8a), 137.7(C-16), 129.2 (C-17), 128.4 (C-18), 127.3 (C-19), 119.7 (C-4a), 112.3 (d, 2JC-F = 23.4 Hz, C-5), 108.1 (C-3), 104.7 (d, 3JC-F = 3.3 Hz, C-8), 62.9 (C-15), 52.7 (C-14), 49.9 (C-13), 49.8 (C-13), 35.3 (C-9), 8.2 (C-10,11).

19F NMR (282.41MHz, CDCl3 : CD3OD (10% v/v))  (ppm): -120.6.

MS (APCI) m/z 442.5 [M + H+, 100%].

HRMS (ESI) calcd for C24H25N3O3F [M + H+]: 422.18799, found 422.18844.

max (10-4M, CHCl3): 279nm (max= 11901), 285 (13106), 326 (4117), 334 (4123)

Figure S2. HPLC chromatograms and UV spectra of ciprofloxacin and 4. Upper panel: (A) Chromatogram of standards in PBS: ciprofloxacin (CIP) + 4 (50/50, each molecule at 50 µg/mL); (B and C) chromatograms of cellular samples: cells incubated for 2h with ciprofloxacin (B) or 4 (C) (at 20 µg/mL) were collected in PBS, centrifuged and 50 µl of supernatant were injected. *: mobile phase peak. The inset shows at higher magnification the small peak appearing at 16 minutes. This peak is also present in standard (A), and may therefore not correspond to a metabolite produced in cells. Lower panel: UV spectra for the three peaks detected in HPLC, confirming the identity of compounds.