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Chemical Physics Letters 442 (2007) 281–284

Structure of levofloxacin in hydrophilic and hydrophobicmedia: Relationship to its antibacterial properties

Alexandrine Lambert, Jean-Bernard Regnouf-de-Vains, Manuel F. Ruiz-Lopez *

SRSMC, Nancy-University, CNRS, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France

Received 3 May 2007; in final form 11 May 2007Available online 25 May 2007

Abstract

The relative stability of zwitterionic and uncharged neutral forms of the fluoroquinolone antibiotic levofloxacin has been investigatedusing quantum mechanical calculations at the B3LYP/6-31+G* level. Solvent effects have been incorporated using continuum and dis-crete-continuum models. The zwitterion is slightly more stable than the uncharged form in water where hydrogen bonds are shown toplay a key role. Energy transfer from water to an apolar medium has been computed as an attempt to estimate the energy involved incrossing a lipid bilayer. The obtained value (�2.9 kcal/mol) is consistent with the intermediate lipophilicity experimentally measured forthis drug.� 2007 Elsevier B.V. All rights reserved.

1. Introduction

Levofloxacin (Scheme 1) is one of the most prominentmembers of the quinolone class of antibiotics and is usedto treat a wide variety of bacterial infections. It is a fluoro-quinolone [1] a particular type of quinolones that combinethe presence of a fluor atom in position 6 and a piperazinesubstituent in position 7. As the other quinolones, levoflox-acin acts by inhibiting two fundamental enzymes involvedin cell division processes, Topoisomerase II, or DNA-gyr-ase, and Topoisomerase IV [1–6].

The carboxylic and piperazine amino functional groupsin fluoroquinolones may be charged in aqueous solutiondepending on pH. The acid-base properties of some deriv-atives were analyzed by Takacs-Novak et al. [7] usingpotentiometry, UV and NMR spectroscopy. These authorswere able to obtain microconstants and to quantify theconcentration ratio of charged over neutral forms as afunction of pH. They showed that the zwitterionic formof the molecules predominates at physiological pH thoughthe concentration of the uncharged neutral form is not neg-

0009-2614/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.cplett.2007.05.077

* Corresponding author. Fax.: +33 3 83 68 4372.E-mail address: manuel.ruiz@cbt.uhp-nancy.fr (M.F. Ruiz-Lopez).

ligible and that it may considerably vary from one deriva-tive to another. The electrophoretic behavior and pKa

values of some quinolones have been investigated by someauthors [8–10] who led to similar conclusions. It is there-fore generally admitted that fluoroquinolones in waterexhibits a zwitterionic structure at pH close to seven.

Nevertheless, the role played by the uncharged neutralform of fluoroquinolones in their mechanism of actionhas been put forward [11]. In fact, since fluoroquinolonetargets are within the cell cytoplasm, the molecule needsto cross the cytoplasmic membrane, as well as the outermembrane in the case of Gram-negative bacteria. Trans-port across the outer membrane may occur through porinproteins but diffusion across the lipid bilayer seems unlikelyto occur for charged forms of the molecule. Thus, thoughthe abundance of the uncharged neutral molecule in thephysiological media is minor, this species is probablyinvolved in reaching the cytoplasm.

Therefore, predicting theoretically the ionization state offluoroquinolone antibiotics in a particular media and pH isa relevant question. Quite surprisingly, this topic hasreceived very little attention. A recent theoretical study[12] devoted to aqueous solutions of norfloxacin has sug-gested that, though the zwitterionic form is substantially

Fig. 1. Lowest energy geometries of the levofloxacin molecule: uncharged(left) and zwitterionic form (right).

N

OHO

CH3

O

N

NH3C

F

O

Scheme 1.

282 A. Lambert et al. / Chemical Physics Letters 442 (2007) 281–284

stabilized by the solvent, the neutral form remains themajor species. This result is in apparent contradiction withexperimental data. To account for medium effects, theauthors used both, a continuum and a discrete-continuummodel, considering one or two explicit water molecules inthe later case. The continuum model usually provides areliable evaluation of solvation free energies, providedappropriate atomic radii parameters are used. Theseparameters are not unambiguously defined for ionic sys-tems and may be a source of error in the study of solvatedzwitterions. Moreover, explicit hydrogen bonds may repre-sent a fundamental contribution to stabilize zwitterions inwater [13,14] and computations using a continuum model(or a discrete-continuum model with a small number of sol-vent molecules) may lead to wrong conclusions.

The objective of the present work was to examine therole of the medium on the structure of levofloxacin. Wefocus on the following three points: (1) what is the relativestability of the uncharged form vs the zwitterionic form inmedia of variable dielectric permittivity, (2) what is the roleof hydrogen bonds in water, and (3) what is the energyrequirement to transfer levofloxacin from aqueous solutionto a hydrophobic environment.

2. Calculations

As a compromise between accuracy and computationalcost, quantum mechanical computations were carried outat the B3LYP/6-31+G* level. However, some MP2 andB3LYP computations were also done using more extendedbasis set for control purposes. The solvent was modeledeither by a continuum or a discrete-continuum approach.The continuum method developed in our group [15] hasbeen employed using dielectric constants from 2.0 (apolarmedia) to 78.4 (liquid water). The method assumes a mul-ticentric multipole moment development of the solventreaction field. Non-electrostatic solvation terms were com-puted using the PCM method [16] to analyze energy trans-fer of levofloxacin from water to cyclohexane. They wereassumed to be equal for uncharged and zwitterionic forms.In discrete-continuum calculations, a complex formed bylevofloxacin plus five explicit water molecules was consid-ered (see the discussion in the next section). Full geometryoptimization was carried out for levofloxacin or levofloxa-cin–water clusters in gas phase. Afterwards, single-pointenergy calculations were done in the continuum. Geometryoptimization in solution is expected to change a little the

results reported below but not to modify the main conclu-sions concerning the relative stabilities of the species. Zero-point energy and thermal contributions to the free energywere computed for the levofloxacin molecule in gas phaseusing the ideal gas approach; these values were assumedto be valid in solution. Calculations were done using theGAUSSIAN 03 program [17].

3. Results

As a preliminary study, we analyzed the most favorableconformation of the uncharged and zwitterionic neutralforms of levofloxacin in gas phase. Several conformationswere studied for the piperazine and carboxyl groups butonly the most stable ones (Fig. 1) will be considered below.

Computed energies in gas phase show the unchargedspecies to be much more stable than the zwitterion:DE = 78.9 kcal/mol, DG = 76.4 kcal/mol. Values do notchange much at higher computational levels (DE =80.8 kcal/mol and 82.7 kcal/mol at B3YP/6-311+G(2d,2p) and MP2/6-31+G* levels, respectively). In Fig. 2, werepresent the free energy difference as a function of thesolvent dielectric constant. In water, the difference is dras-tically reduced to DG = 17.0 kcal/mol. This strong effect ismainly connected to dipole moment values for theuncharged and zwitterionic systems which are 11.0D and41.5D in gas phase, respectively (14.4D and 51.9D inwater). Accordingly, the electrostatic free energy of solva-tion is much larger for the zwitterion (�70.0 kcal/mol) thanfor the uncharged species (�10.6 kcal/mol).

As shown, the continuum calculations still predict theuncharged form to be more stable in water. This resultmatches that reported for norfloxacin using a similar com-putational level [12] but is in contrast with experimentaldata. Clearly, the continuum model is not accurate enoughand according to previous works [13,14] one must take intoaccount the role of explicit hydrogen bonds in aqueoussolution. Preliminary calculations with one or two watermolecules did not show main changes with respect to thecontinuum model results, as also reported in the case ofnorfloxacin [12]. In fact, this number is clearly too low.The zwitterion contains two ionic sites. Solvation of theprotonated amine with one water molecule seems reason-able. Solvation of the carboxylate group is slightly moreintricate. Experimental and theoretical work for different

10

20

30

40

50

60

70

80

0 20 40 60 80

)lom/lack(

G

Fig. 2. Effect of the dielectric constant of the medium on the relativestability of the zwitterion (Z) vs. uncharged (U) form of the levofloxacinmolecule, DG = GZ � GU.

Table 1Contributions to the relative stabilization of zwitterionic (Z) vs uncharged(U) forms of levofloxacin in water solution

U Z Z � U

DG in gas phase +76.4Solvation energy1st shell �36.6 �71.6 �35.0Bulk �14.7 �56.8 �42.1DG in solution �0.7

Energy values in kcal/mol.

A. Lambert et al. / Chemical Physics Letters 442 (2007) 281–284 283

systems suggests a solvation of the carboxylate –COO�

group by roughly 3–5 water molecules (see the discussionin Ref. [18] and references cited therein). As a compromisewe have considered four water molecules. Thus, the totalnumber of water molecules considered in our explicit solva-tion model for the zwitterion is five, and for simplicity, wekeep the same number for the uncharged form. Increasingfurther this number would require a statistical treatmentsuch as the one made in Refs. [13,14]; it would probablyamplify a little the trend reported below.

The optimized geometries of the gas phase complexesare shown in Fig. 3 (optimization of the uncharged struc-ture was not completely achieved because of converge dif-ficulties; it was stopped when the energy changed by lessthan 10�5 atomic units during several cycles). Afterwards,the complexes where placed in a dielectric continuum withthe dielectric constant of water. A summary of results ispresented in Table 1. As shown, the zwitterion form isnow predicted to be slightly more stable than theuncharged form. The predicted [zwitterion]/[uncharged]concentration ratio at neutral pH and T = 298 K is 3.2,

Fig. 3. Geometries of the levofloxacin molecule interacting with five wa

in nice agreement with experimental data that rangebetween 1.4 and 9.5 for related fluoroquinolones [7].

Transfer to the lipid bilayer is obviously connected withthe hydrophilicity/hydrophobicity ratio of a drug. Thisproperty is usually estimated in terms of experimentalwater/n-octanol partition coefficients and this has encour-aged theoretical developments to predict such coefficientsusing continuum solvation approaches [19,20]. Experimen-tal data for fluoroquinolones have been reported [21–24]but difficulties for interpreting the values at a given pHhave been discussed on the basis of possible ionizationstates and interactions between them. It has been estab-lished, however, that levofloxacin possesses an intermediatelipophilic character, whereas other fluoroquinolones arehydrophilic (e.g. norfloxacin) or, on the contrary, exhibita strong lipophilicity (e.g. pefloxacin) [21]. Water/cyclohex-ane partition coefficients have also been employed as a con-venient property to estimate the lipophilicity of molecules[25,26] and we have preferred to consider these solventsin our study.

Since our calculations above show that levofloxacinexists mainly as a zwitterion in water and as an unchargedspecies in apolar media, the phase transfer energy mayroughly be written as

DGlevofloxacinwater!apolar solvent ¼ DGZ!U

water þ DGUwater!apolar solvent;

where

DGZ!Uwater ¼ GU

water � GZwater

stands for the energy necessary to transform the zwitterion(Z) into the uncharged neutral form (U) in water and

DGUwater!apolar solvent ¼ GU

apolar solvent � GUwater

ter molecules: uncharged form (left) and zwitterionic form (right).

Table 2Solvation energy contributions (continuum model) in cyclohexane andwater of the uncharged form of levofloxacin

Electrostatic Non-electrostatic Total

Cyclohexane �1.1 �2.3 �3.4Water �5.1 5.3 0.2Difference 4.0 �7.6 �3.6

Values in kcal/mol.

284 A. Lambert et al. / Chemical Physics Letters 442 (2007) 281–284

represents the energy necessary to transfer the unchargedspecies from water to the apolar solvent. The first termhas been computed above (0.7 kcal/mol). Computation ofthe second using the continuum model leads toDGU

water!apolar solvent ¼ �3:6 kcal=mol which, as shown inTable 2, results from the larger contribution (in absolutevalue) of the non-electrostatic terms. The transfer of levo-floxacin from the aqueous to the organic phase is thereforepredicted to be slightly exothermic (DGlevofloxacin

water!apolar solvent ¼�2:9 kcal=mol) in agreement with the experimentally deter-mined lipophilicity for this molecule [21].

In summary, our computations confirm both, the pre-dominance of the zwitterionic form of levofloxacin in waterand the lipophilicity character of this fluoroquinolone anti-biotic. The solvation models employed here can be usefulto classify novel fluoroquinolone compounds before theyare synthetized. Though the reported energies cannot beexpected to be quantitative owing to crude approximationsin the solvation models, our conclusions should be helpfulto undertake further theoretical calculations using moresophisticated approaches.

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