Pharmaceutical Research

, Volume 31, Issue 5, pp 1290–1301 | Cite as

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 BambekeEmail author
Research Paper



To evaluate pharmacological properties (antibacterial activity; accumulation in phagocytic cells; activity against intracellular bacteria; susceptibility to fluoroquinolone efflux transporters) of ciprofloxacin derivatives modified at C-7 of the piperazine ring.


N-acetyl- (1), N-benzoyl- (2), N-ethyl- (3), and N-benzyl- (4) ciprofloxacin were synthesized. MICs against Escherichia coli and Staphylococcus aureus were determined following CLSI guidelines. Cellular accumulation, subcellular distribution, and intracellular activity (towards S. aureus and Listeria monocytogenes) were determined in J774 mouse macrophages. Efflux in bacteria (NorA [S. aureus], Lde [L. monocytogenes]) and in macrophages (Mrp4) was assessed using the corresponding inhibitors reserpine and gemfibrozil, respectively.


All derivatives were active, though less than ciprofloxacin. 2 and 3 accumulated 2–3 fold more than ciprofloxacin in mouse macrophages but remained substrates for efflux by Mrp4. 4 was insensitive to NorA and Lde, accumulated approx 50-fold more than ciprofloxacin in macrophages, was barely affected by Mrp4, localized in the soluble fraction of cells, and was equipotent to ciprofloxacin against intracellular bacteria.


Benzyl substitution at C7 markedly affects the pharmacological profile of ciprofloxacin with respect to recognition by efflux transporters and cellular accumulation. N-benzyl-ciprofloxacin may serve as basis for designing molecules with higher intrinsic activity while remaining poorly susceptible to efflux.


antibacterial activity drug accumulation fluoroquinolones lipophilicity Mrp4 



ATP-Binding Cassette


American Type Culture Collection


Breast Cancer resistance Protein


Clinical and laboratory Standards Institute




Diisopropyl ethyl amine


Minimal Inhibitory Concentration


Multidrug-related Resistance Protein (human)


Multidrug-related resistance protein (murine)



Béatrice Marquez and Vincent Pourcelle contributed equally to this study. We thank S. Devouge, D. Timmerman, M. C. Cambier, C. Misson, N. Couwenbergh and M. Vergauwen for skilful technical assistance. We are grateful to P.C. Appelbaum (Hershey Medical Center, Hershey, PA), B. Ba (Université Victor Segalen Bordeaux 2, Bordeaux, France), P. Courvalin (Institut Pasteur, Paris, France), Y. Glupczynski (cliniques universitaires de l’UCL à Mont-Godinne, Yvoir, Belgium), and J.M. Pagès (Université de la Méditerranée, Marseille, France) for the kind gift of bacterial strains. B.M. was postdoctoral fellow of a FIRST-post doc programme of the Region Wallonne, C.M.V. was recipient of a doctoral grant from the Fonds pour la Recherche dans l’Industrie et l’Agriculture (FRIA) and F.V.B. is Maître de recherches of the Belgian Fonds pour la Recherche Scientifique (FRS-FNRS). This work was supported by the Région Wallonne, the Belgian Fonds pour la Recherche Scientifique Médicale (FRSM; grants 3.4.597.06 and 3.4.583.08) and the Belgian Federal Science Policy Office (Research project P6/19 [research action P6]).

Supplementary material

11095_2013_1250_MOESM1_ESM.doc (290 kb)
ESM 1 (DOC 290 kb)


  1. 1.
    Van Bambeke F, Michot JM, Van Eldere J, Tulkens PM. Quinolones in 2005: an update. Clin Microbiol Infect. 2005;11(4):256–80.PubMedCrossRefGoogle Scholar
  2. 2.
    Michot JM, Van Bambeke F, Mingeot-Leclercq MP, Tulkens PM. Active efflux of ciprofloxacin from J774 macrophages through an MRP-like transporter. Antimicrob Agents Chemother. 2004;48(7):2673–82.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Seral C, Carryn S, Tulkens PM, Van Bambeke F. Influence of P-glycoprotein and MRP efflux pump inhibitors on the intracellular activity of azithromycin and ciprofloxacin in macrophages infected by Listeria monocytogenes or Staphylococcus aureus. J Antimicrob Chemother. 2003;51(5):1167–73.PubMedCrossRefGoogle Scholar
  4. 4.
    Marquez B, Van Bambeke F. ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions. Curr Drug Targets. 2011;12(5):600–20.PubMedCrossRefGoogle Scholar
  5. 5.
    Alvarez AI, Perez M, Prieto JG, Molina AJ, Real R, Merino G. Fluoroquinolone efflux mediated by ABC transporters. J Pharm Sci. 2008;97(9):3483–93.PubMedCrossRefGoogle Scholar
  6. 6.
    Merino G, Alvarez AI, Pulido MM, Molina AJ, Schinkel AH, Prieto JG. Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion. Drug Metab Dispos. 2006;34(4):690–5.PubMedCrossRefGoogle Scholar
  7. 7.
    Marquez B, Caceres NE, Mingeot-Leclercq MP, Tulkens PM, Van Bambeke F. Identification of the efflux transporter of the fluoroquinolone antibiotic ciprofloxacin in murine macrophages: studies with ciprofloxacin-resistant cells. Antimicrob Agents Chemother. 2009;53(6):2410–6.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Marquez B, Ameye G, Vallet CM, Tulkens PM, Poirel HA, Van Bambeke F. Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin. PLoS One. 2011;6(12):e28368.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Haslam IS, Wright JA, O’Reilly DA, Sherlock DJ, Coleman T, Simmons NL. Intestinal ciprofloxacin efflux; the role of breast cancer resistance protein, BCRP (ABCG2). Drug Metab Dispos. 2011;39(12):2321–8.PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Michot JM, Heremans MF, Caceres NE, Mingeot-Leclercq MP, Tulkens PM, Van Bambeke F. Cellular accumulation and activity of quinolones in ciprofloxacin-resistant J774 macrophages. Antimicrob Agents Chemother. 2006;50(5):1689–95.PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Lismond A, Tulkens PM, Mingeot-Leclercq MP, Courvalin P, Van Bambeke F. Cooperation between prokaryotic (Lde) and eukaryotic (MRP) efflux transporters in J774 macrophages infected with Listeria monocytogenes: studies with ciprofloxacin and moxifloxacin. Antimicrob Agents Chemother. 2008;52(9):3040–6.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Vallet CM, Marquez B, Nhiri N, Anantharajah A, Mingeot-Leclercq MP, Tulkens PM, et al. Modulation of the expression of ABC transporters in murine (J774) macrophages exposed to large concentrations of the fluoroquinolone antibiotic moxifloxacin. Toxicology. 2011;290(2–3):178–86.PubMedCrossRefGoogle Scholar
  13. 13.
    Beyer R, Pestova E, Millichap JJ, Stosor V, Noskin GA, Peterson LR. A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrob Agents Chemother. 2000;44(3):798–801.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Letafat B, Emami S, Mohammadhosseini N, Faramarzi MA, Samadi N, Shafiee A, et al. Synthesis and antibacterial activity of new N-[2-(thiophen-3-yl)ethyl] piperazinyl quinolones. Chem Pharm Bull (Tokyo). 2007;55(6):894–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Pieroni M, Dimovska M, Brincat JP, Sabatini S, Carosati E, Massari S, et al. From 6-aminoquinolone antibacterials to 6-amino-7-thiopyranopyridinylquinolone ethyl esters as inhibitors of Staphylococcus aureus multidrug efflux pumps. J Med Chem. 2010;53(11):4466–80.PubMedCrossRefGoogle Scholar
  16. 16.
    Martinez M, McDermott P, Walker R. Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals. Vet J. 2006;172(1):10–28.PubMedCrossRefGoogle Scholar
  17. 17.
    Cormier R, Burda WN, Harrington L, Edlinger J, Kodigepalli KM, Thomas J, et al. Studies on the antimicrobial properties of N-acylated ciprofloxacins. Bioorg Med Chem Lett. 2012;22(20):6513–20.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wang S, Jia XD, Liu ML, Lu Y, Guo HY. Synthesis, antimycobacterial and antibacterial activity of ciprofloxacin derivatives containing a N-substituted benzyl moiety. Bioorg Med Chem Lett. 2012;22(18):5971–5.PubMedCrossRefGoogle Scholar
  19. 19.
    Lemaire S, Tulkens PM, Van Bambeke F. Contrasting effects of acidic pH on the extracellular and intracellular activities of the anti-gram-positive fluoroquinolones moxifloxacin and delafloxacin against Staphylococcus aureus. Antimicrob Agents Chemother. 2011;55(2):649–58.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 2012;22th informational supplement (MS100-S22).Google Scholar
  21. 21.
    Seral C, Van Bambeke F, Tulkens PM. Quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 macrophages. Antimicrob Agents Chemother. 2003;47(7):2283–92.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Al Dgither S, Alvi SN, Hammami MM. Development and validation of an HPLC method for the determination of gatifloxacin stability in human plasma. J Pharm Biomed Anal. 2006;41(1):251–5.PubMedCrossRefGoogle Scholar
  23. 23.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75.PubMedGoogle Scholar
  24. 24.
    Michot JM, Seral C, Van Bambeke F, Mingeot-Leclercq MP, Tulkens PM. Influence of efflux transporters on the accumulation and efflux of four quinolones (ciprofloxacin, levofloxacin, garenoxacin, and moxifloxacin) in J774 macrophages. Antimicrob Agents Chemother. 2005;49(6):2429–37.PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Vassault A. Lactate dehydrogenase. In: Bergemeyer HU, editors. Methods in enzymatic analysis. VHC Publishers, Veinheim, Federal Republic of Germany; 1987. p. 118–126.Google Scholar
  26. 26.
    Renard C, Vanderhaeghe HJ, Claes PJ, Zenebergh A, Tulkens PM. Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages. Antimicrob Agents Chemother. 1987;31(3):410–6.PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Aubert-Tulkens G, Van Hoof F, Tulkens P. Gentamicin-induced lysosomal phospholipidosis in cultured rat fibroblasts. Quantitative ultrastructural and biochemical study. Lab Invest. 1979;40(4):481–91.PubMedGoogle Scholar
  28. 28.
    Petersen U, Grohe K, Kuehle E, Zeiler H-J, Metzger KG. Quinolonecarboxylic acids and antibacterial agents containing these compounds. 17-12-1985;US19840576595 19840203.Google Scholar
  29. 29.
    Montero MT, Freixas J, Hernandez-Borrell J. Expression of the partition coefficients of a homologous series of 6-fluoroquinolones. Int J Pharm. 1997;149(2):161–70.CrossRefGoogle Scholar
  30. 30.
    Kerns EH, Di L. Lipophilicity methods. In: Kerns EH, Di L, editors. Drug-like properties: concepts, structure design and methods. San Diego, CA: Academic Press, Elsevier; 2008. p. 260–70.CrossRefGoogle Scholar
  31. 31.
    Alovero F, Nieto M, Mazzieri MR, Then R, Manzo RH. Mode of action of sulfanilyl fluoroquinolones. Antimicrob Agents Chemother. 1998;42(6):1495–8.PubMedCentralPubMedGoogle Scholar
  32. 32.
    Takenouchi T, Tabata F, Iwata Y, Hanzawa H, Sugawara M, Ohya S. Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother. 1996;40(8):1835–42.PubMedCentralPubMedGoogle Scholar
  33. 33.
    Piddock LJ, Jin YF, Griggs DJ. Effect of hydrophobicity and molecular mass on the accumulation of fluoroquinolones by Staphylococcus aureus. J Antimicrob Chemother. 2001;47(3):261–70.PubMedCrossRefGoogle Scholar
  34. 34.
    Godreuil S, Galimand M, Gerbaud G, Jacquet C, Courvalin P. Efflux pump Lde is associated with fluoroquinolone resistance in Listeria monocytogenes. Antimicrob Agents Chemother. 2003;47(2):704–8.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Neyfakh AA, Borsch CM, Kaatz GW. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrob Agents Chemother. 1993;37(1):128–9.PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Nikaido H, Pages JM. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev. 2011;36(2):340–63.PubMedCentralPubMedGoogle Scholar
  37. 37.
    Vallet CM, Marquez B, Ngabirano E, Lemaire S, Mingeot-Leclercq MP, Tulkens PM, et al. Cellular accumulation of fluoroquinolones is not predictive of their intracellular activity: studies with gemifloxacin, moxifloxacin and ciprofloxacin in a pharmacokinetic/pharmacodynamic model of uninfected and infected macrophages. Int J Antimicrob Agents. 2011;38(3):249–56.PubMedGoogle Scholar
  38. 38.
    Nikaido H, Thanassi DG. Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples. Antimicrob Agents Chemother. 1993;37(7):1393–9.PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Yu X, Zipp GL, Davidson III GW. The effect of temperature and pH on the solubility of quinolone compounds: estimation of heat of fusion. Pharm Res. 1994;11(4):522–7.PubMedCrossRefGoogle Scholar
  40. 40.
    Vazquez JL, Berlanga M, Merino S, Domenech O, Vinas M, Montero MT, et al. Determination by fluorimetric titration of the ionization constants of ciprofloxacin in solution and in the presence of liposomes. Photochem Photobiol. 2001;73(1):14–9.PubMedCrossRefGoogle Scholar
  41. 41.
    Lizondo M, Pons M, Gallardo M, Estelrich J. Physicochemical properties of enrofloxacin. J Pharm Biomed Anal. 1997;15(12):1845–9.PubMedCrossRefGoogle Scholar
  42. 42.
    Viswanadhan VN, Ghose AK, Revankar GR, Robins RK. Atomic physicochemical parameters for three dimensional structure directed quantitative structure-activity relationships. 4. Additional parameters for hydrophobic and dispersive interactions and their application for an automated superposition of certain naturally occurring nucleoside antibiotics. J Chem Inf Comput Sci. 1989;29(3):163–72.CrossRefGoogle Scholar
  43. 43.
    Klopman G, Li JY, Wang S, Dimayuga M. Computer automated log P calculations based on an extended group contribution approach. J Chem Inf Comput Sci. 1994;34(4):752–81.CrossRefGoogle Scholar
  44. 44.
    Ba BB, Arpin C, Vidaillac C, Chausse A, Saux MC, Quentin C. Activity of gatifloxacin in an in vitro pharmacokinetic-pharmacodynamic model against Staphylococcus aureus strains either susceptible to ciprofloxacin or exhibiting various levels and mechanisms of ciprofloxacin resistance. Antimicrob Agents Chemother. 2006;50(6):1931–6.PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Ghisalberti D, Masi M, Pages JM, Chevalier J. Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes. Biochem Biophys Res Commun. 2005;328(4):1113–8.PubMedCrossRefGoogle Scholar
  46. 46.
    Furet YX, Deshusses J, Pechere JC. Transport of pefloxacin across the bacterial cytoplasmic membrane in quinolone-susceptible Staphylococcus aureus. Antimicrob Agents Chemother. 1992;36(11):2506–11.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Béatrice Marquez
    • 1
    • 3
  • Vincent Pourcelle
    • 2
  • Coralie M. Vallet
    • 1
  • Marie-Paule Mingeot-Leclercq
    • 1
  • Paul M. Tulkens
    • 1
  • Jacqueline Marchand-Bruynaert
    • 2
  • Françoise Van Bambeke
    • 1
    Email author
  1. 1.Pharmacologie Cellulaire et Moléculaire, Louvain Drug Research InstituteUniversité catholique de LouvainBrusselsBelgium
  2. 2.Laboratoire de Chimie Médicinale, Institute of Condensed Matter and NanosciencesUniversité catholique de LouvainLouvain-la-NeuveBelgium
  3. 3.European CommissionBrusselsBelgium

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