Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Triprotic site-specific acid–base equilibria and related properties of
fluoroquinolone antibacterials
Aura Rusu a , Gergő Tóth b , Levente Szőcs b , József Kökösi b , Márta Kraszni b ,
Árpád Gyéresi a , Béla Noszál b,∗
a
Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy, 540139 Targu Mures, 38 Gh. Marinescu, Romania
Department of Pharmaceutical Chemistry, Semmelweis University, Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences,
Budapest H-1092, Hőgyes Endre u. 9, Hungary
b
a r t i c l e
i n f o
Article history:
Received 11 January 2012
Received in revised form 27 February 2012
Accepted 28 February 2012
Available online 7 March 2012
Keywords:
NMR
Microspeciation
Fluoroquinolone
pKa
Protonation constant
a b s t r a c t
The complete macro- and microequilibrium analyses of six fluoroquinolone drugs – ciprofloxacin,
enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin – are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the
protonation macro- and microconstant values are reported. The macroconstants were determined by 1 H
NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive
methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full 1 H, 13 C and 15 N NMR assignments, NMR-pH
profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies
show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1 and N-4
piperazine nitrogens and concentration of the uncharged microspecies is way below the values published
earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and
microconstant values allow the pre-planned method development in techniques such as capillary zone
electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including
the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states
of protonation.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Medicinal quinolones are synthetic antibacterial agents, introduced into the therapy in the early 1960s, when nalidixic acid was
discovered for the treatment of urinary tract infections. A major
therapeutic progress occurred with the insertion of a fluorine
atom into the 6th position of the quinolone cycle, resulting in
fluoroquinolone compounds (FQs) with broad antimicrobial activity, including Gram-negative, Gram-positive, anaerobe and highly
resistant strains [1]. The addition of an extra ring or ring system,
containing basic nitrogen to the C-7 position further improves the
antibacterial activity and also, the pharmacokinetic properties. A
number of subsequent structural and formulatory modifications
have improved their therapeutic properties [2–5]. FQs poison the
catalytic activity of the bacterial topoisomerase by a sterically
∗ Corresponding author at: Department of Pharmaceutical Chemistry, Semmelweis University, Budapest H-1092, Hőgyes Endre u. 9, Hungary.
Tel.: +36 06 1 217 0891; fax: +36 06 1 217 0891.
E-mail address: [email protected] (B. Noszál).
0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2012.02.024
effective interaction with the single-stranded DNA gyrase complex
[6]. We studied 6 FQs, the structures of which are shown in Fig. 1.
It has long been known that the acid–base properties of drugs
influence their pharmacokinetic behavior. In particular, the activity
of FQs is reported to be pH-dependent, the binding of the bacterial
DNA gyrase enzyme is therefore assumed to be dependent on the
protonation states of FQs [7–9]. It is of crucial importance therefore
to know the exact values of the protonation constants of FQs. The
protonation of the compounds can be characterized in terms of
protonation macroconstants and microconstants.
Macroscopic equilibrium constants (expressed either as dissociation constants pKa or protonation constants log K) of multiprotic
molecules depict the acid–base properties of the compound as
a whole. These are useful parameters to characterize large drug
molecules [10]. The overall charge of the compound is primarily
related to the macroscopic protonation constant, whereas the moieties and site-specific charges can be determined from the microscopic protonation constants. Latters are also primarily related to
the interaction with the biological target. Microconstants describe
the proton binding ability of the individual functional groups and
are useful in calculating the pH-dependent concentrations of the
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
51
Fig. 1. The chemical structures and numbering of the six FQs studied.
different protonation isomers [11]. Protonation constants are also
important to establish quantitative structure–activity relationships
(QSAR) to design drugs of improved properties, and to develop
selective analytical methods (e.g. capillary electrophoresis).
Unfortunately, however, the reported acid–base properties of
FQs are highly controversial in the values of protonation constants
and even in the number of basic sites. The reported conflicting
values of norfloxacin are presented in Table 1.
The main objective of our research was either to identify
all the basic sites in the FQs, or to determine the protonation
macro- and microconstants, that play an important role in metal
complexation and DNA gyrase binding of FQs [16]. We used 1 H
NMR-pH titration for the determination of FQ macroconstants, and
combined spectroscopic-deductive method for the determination
of microconstants. To determine the protonation sites of FQs we
synthesized and used model compounds that contain reduced
number of basic sites.
Several important molecules have earlier been characterized by
NMR-pH titration [17–19] due to the numerous advantages of the
method: a number of compounds can be monitored simultaneously
in one single solution and the accuracy of the log K determination
is nowadays superior of the pH-potentiometric titrations, especially near the pH extrema, where potentiometric titrations are
no longer applicable. Using in tube indicator molecules and in situ
pH monitoring, 1 H NMR spectra can be acquired for single samples under circumstances of constant analyte concentration, ionic
strength and gradually varied pHs. The protonation of one basic
center can be followed selectively in many NMR-pH titrations [20].
In our FQ molecules all NMR nuclei are multiply influenced, their
chemical shifts reflect to some extent the protonation of every basic
center. Thus, to determine the site-specific protonation we also
introduced model molecules of reduced number of basic centers.
Model compounds are adequate to mimic the minor protonation
isomer, and the macroconstants of the auxiliary compound can
then be introduced into the microscopic protonation scheme of the
parent compound. It has been shown [21,22] that the electronic
effects of a carboxylic group and methyl ester; and those of an
aromatic hydroxyl group and its phenol ether are virtually identical on the adjoining moieties. In conclusion the knowledge of the
protonation macroconstants of the parent and model compounds
can be an adequate set of data to calculate of all the protonation
microconstants.
Although FQs have extensively been studied, a limited amount
of microconstants is available only. Here we report a complete set
of macro- and microconstants of six FQs, the data of which can
be further utilized to interpret and improve biological activity and
bioavailability during the development process of novel effective
antibacterial FQs.
2. Materials and methods
2.1. Materials
The FQs were purchased as follows: ciprofloxacin hydrochloride (CIP) from Ranbaxy, ofloxacin (OFL), norfloxacin (NOR) from
Smruthi Organics Ltd., pefloxacin mesylate (PEF) from Laropharm
Romania, moxifloxacin hydrochloride (MOX) from Bayer Schering
Pharma, enrofloxacin (ENR) and 1(2-fluorophenyl)piperazine
monohydrochloride from Sigma–Aldrich. Other substances:
dichloroacetic acid (Fluka), deuterium oxide (99.9%) and deuterium oxide (99.9%) containing 1% (w/w) 3-trimethylsilylpropane
sulfonic acid (DSS) (Sigma–Aldrich), dimethyl sulfoxide-d6 and
thionyl chloride (Merck). All other reagents were of analytical
grade. The deionized water was prepared with a Milli-Q Direct 8
Millipore system.
2.2. Synthesis of 7-chloro-6-fluoro-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (model compound B)
Synthesis of 7-chloro-6-fluoro-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid was performed by a literature method
[23]. Its chemical structure was checked by HRMS, 1 H NMR, 13 C
NMR and 15 N NMR techniques (see supplementary data).
2.3. Synthesis of N-acetyl-ciprofloxacin (model compound D)
of
7-(4-acetylpiperazin-1-yl)-1-cyclopropyl-6Synthesis
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid
was
performed by a literature method [24]. Its chemical structure
was checked by HRMS, 1 H NMR, 13 C NMR and 15 N NMR techniques
(see supplementary data).
52
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
Table 1
The reported protonation constants of NOR.
log K1
log K2
log K3
log K4
Technique
References
8.51
8.45
9.25
10.56
6.22
6.25
6.9
8.60
–
5.00
0.6
6.10
–
–
–
3.11
Potentiometry
CE
NMR
Potentiometry
[12]
[13]
[14]
[15]
2.4. Synthesis of CIP, ENR, MOX, NOR, OFL and PEF methyl esters
Methyl ester derivatives were prepared by a modified procedure of the synthesis of NOR-methyl ester [14]. First we prepared
the appropriate acyl chloride derivatives. In this procedure we used
only thionyl-chloride in excess for 60 min at 60 ◦ C. Then the solvent
was evaporated with benzene. In the last step we added methanol
to the mixture and the mixture was stirred for 24 h at room temperature and finally it was evaporated. CIP-methyl ester was used as
model compound C in latter experiment. The structure of the synthesized chloride salts were checked by HRMS, 1 H NMR and 13 C
NMR (see supplementary data).
2.5. NMR-pH titrations and other NMR experiments
All NMR measurements were carried out on a Varian VNMRS
spectrometer (599.9 MHz for 1 H, 150.9 MHz for 13 C and 60.8 for
15 N) with a dual 5 mm inverse-detection gradient (IDPFG) probehead. Standard pulse sequences and parameters were used to
obtain 1D 1 H, 13 C, NOE, COSY, HSQC, HMBC and NOESY spectra.
Assignments were made in DMSO-d6 solution (0.05–0.1 M).
Spectra were referenced to internal TMS. The 15 N NMR chemical shifts were measured by 1 H–15 N HMBC. The spectra were
referenced to external CH3 NO2 (0.00 ppm), chemical shifts were
obtained to a precision of 0.2 ppm.
The NMR-pH titrations were performed at 25.0 ± 0.1 ◦ C at a constant ionic strength (I = 1 mol/dm3 , auxiliary electrolyte: KCl) in 5%
D2 O–95% H2 O mixtures. This deuterium concentration proved to
be enough for the spectrometer lock system. Using such a small
amount of D2 O, the shift of pH-scale is within the deviation limit
(0.02 pH unit), according to the Gross–Butler–Purlee theory [25,26].
The spectra were referenced to internal DSS that is known to be
a pH independent NMR standard between pH 2 and 12 and was
successfully applied in strong acidic solutions as well [27,28]. For
the titration 1–3 mM solutions of FQs and their related compounds
were used.
For pH measurement a combined glass electrode (Metrohm
6.0234.110) was used in the pH range 2–12. The pH data are
pH meter readings based upon NIST primary standards: 0.05 M
potassium tetraoxalate (pH = 1.68), 0.05 M potassium hydrogen phthalate (pH = 4.01), 0.025 M KH2 PO4 + 0.025 M Na2 HPO4
(pH = 6.87) and 0.01 M borax (pH = 9.18) [29]. In solutions of pH ≤ 2
the pH was determined using dichloroacetic acid (1 mM concentration) as an in tube pH-indicator to avoid the glass electrode acid
error.
For the evaluation of protonation macroconstants Opium program and Origin 8.0 were used.
2.6. Potentiometric titration
The potentiometric titrations were carried out on a GLP conform computerized titration instrument (GLpKa) (Sirius Analitycal
Instrument) using combination Ag–AgCl pH electrode. The titrations were carried out under N2 atmosphere at constant (I = 0.15 M
KCl) ionic strength and at standard temperature 25.0 ± 0.1 ◦ C. The
electrode was calibrated with “four-parameter”TM calibration procedure that is based on alkalimetric titration of 0.5 M HCl [30].
RefinementProTM computer software was used for the calculation
of the protonation macroconstants. Bjerrum difference curve was
generated from the titration data. First approximate logs K values
were estimated at half-integer points of the difference curve and
this “seed” value was refined by a nonlinear least squares procedure.
2.7. UV-pH titration
UV-pH titrations were carried out on a Jasco V-550 diodearray spectrometer with 10 mm cuvettes at 25.0 ◦ C at 1 M
ionic strength. The solution concentrations during the titrations
were held constant, 0.075 mM for 1(2-fluorophenyl)piperazine
monohydrochloride and 0.025 mM for 7-chloro-6-fluoro-1-ethyl4-oxo-1,4-dihydro-quinoline-3-carboxylic acid. 10% methanol was
used to dissolve each compound. In case of such solutions a glass
electrode was calibrated with aqueous buffers and then these values were corrected with an appropriate value [31]. Absorption
spectra were recorded between 210 and 400 nm, and for the evaluation of protonation constants a wavelength was chosen to achieve
as much difference between the acidic and basic solutions as possible. Absorbance–pH datasets were fitted with Statistica 6.0.
2.8. HRMS analysis
The high resolution accurate masses were determined with
an Agilent 6230 time-of-flight mass spectrometer. Samples were
introduced by the Agilent 1260 Infinity LC system, the mass
spectrometer was operated in conjunction with a Jet Stream electrospray ion source in positive ion mode. Reference masses of
m/z 121.050873 and 922.009798 were used to calibrate the mass
axis during analysis. Mass spectra were processed using Agilent
MassHunter B.02.00 software.
3. Results and discussion
3.1. NMR analysis of the parent compounds
The proton assignment of the spectra was straightforward in
most cases. The proton chemical shifts of FQs (except for MOX) with
coupling constants are summarized in Table 2. Since the chemical
structure of MOX is different from the other FQs studied, assignments of this compound are shown in Table 3.
In the aromatic region 3 signs occur, except for OFL and MOX,
which have substitutions in C-8 position. The H-2 proton is of the
largest chemical shift. The chemical shift of this nucleus and H-5
depend on the substituent in N-1 position; if this substituent is a
cyclopropyl ring the H-2 chemical shift is reduced, while the H-5
chemical shift is increased. This phenomenon can be explained by
the orientation and the magnetic anisotropy cone of cyclopropyl
ring. The H-5 and H-8 signs are doublets because of the H–F coupling. The 3 J(H–F) and the 4 J(H–F) are approximately 13 and 7 Hz,
respectively, in all compounds.
The piperazine protons can be assigned on the basis of integral
values and only the H-6 and H-2 protons have cross peaks with the
aromatic H-8 proton in the NOESY experiment. Furthermore, the
1 H NMR data and HSQC correlations unambiguously indicate the
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
53
Table 2
The 1 H NMR data (chemical shifts in ppm, multiplicities with coupling constants in Hz and relative intensities) of CIP, ENR, NOR, OFL and PEF (tr. = trans-methylene proton,
cys. = cys-methylene proton, br. = broad, ax. = axial, eq. = equatorial).
CIP
ENR
NOR
OFL
PEF
2
5
8
2 , 6
8.69 s 1H
7.96 d(12.98) 1H
7.58 d(7.16) 1H
3.56 m 4H
8.66 s 1H
7.90 d(12.97) 1H
7.56 d(7.06) 1H
3.32 m 4H
8.94 s 1H
7.90 d(13.00) 1H
7.15 d(7.15) 1H
3.23 m 4H
8.96 s 1H
7.58 d(12.42) 1H
–
3.29 m 4H
3 , 5
12
13
14
15
16
3.31 m 4H
3.86 m 1H
1.33 m 2H (tr.)
1.20 m 2H (cys.)
–
–
2.58 m 4H
3.82 m 1H
1.31 m 2H (tr.)
1.18 m 2H (cys.)
2.41 q(7.1) 2H
1.05 t(7.1) 3H
2.89 m 4H
4.59 q(7.01) 1H
1.42 t(7.01) 3H
–
–
–
2.43 br. 4H
4.92 m 1H
4.58 dd(1.61, 11.40) 2H
1.45 d(7.01) 3H
2.23 s 3H
–
8.98 s 1H
7.97 d(12.91) 1H
7.28 d(7.15) 1H
3.89 br. 2H ax.
3.55 br. 2H eq.
3.30 br. 4H
4.62 q(7.00) 2H
1.43 t(7.00) 3H
2.32 s 3H
2.89 s 3H
mesylate
chemical shifts of both types (2/6 and 3/5) of the methylene moieties of the piperazine ring. In some cases the piperazine methylene
protons appear as broad peaks in DMSO-d6 or in D2 O, because of
ring motions. This fact depends on the protonation state of the
piperazine rings. In acidic media (pH < 4) the H-3 /H-5 protons
appear as broad signals in FQs. In the PEF all piperazine protons
have a broad signal and in the case of H-6 /H-2 protons the axial
and equatorial protons give separate peaks.
The proton assignment of pyrrolidino-piperidine ring (MOX) is
more complex. In the literature no complete MOX NMR assignment
was found. The pyrrolidino-piperidine protons can be assigned on
the basis of multiplicity, coupling constants and integral values
in 1 H spectra, 19 F–13 C coupling pattern in carbon spectra, and by
COSY, HSQC, HMBC, NOESY as well as 1D NOE experiments with
different mixing time. The anchor point of the assignment was that
O CH3 and the axial H-7 and the protons in the same side of the
ring are close in space, in agreement with the NOESY spectra of
500 ms mixing time.
The proton assignments of the cyclopropyl moiety in CIP, ENR
and MOX were in accordance with earlier literature data [32,33].
In the cyclopropyl ring the cys- and trans-methylene protons give
separate signals. The H-2 protons and the cys-methylene protons
are in correlation in NOESY experiment; the distance between the
trans-methylene proton and H-2 protons is larger. In the case of
MOX all the cyclopropyl protons give a separate signal, because the
O CH3 group inhibits the rotation of the cyclopropyl ring.
The 13 C NMR assignment of FQs was deduced from the twodimensional 1 H–13 C HSQC and HMBC experiments. Assignment of
carbons was supported by the values of 13 C–19 F coupling constants.
The carbon NMR assignments of FQs (except for MOX) with 13 C–19 F
coupling constants are presented in Supplementary Table S1. The
C–F coupling effect can also be observed at the piperazine C-2 and
C-6 atoms. The value of this 4 J(C–F) is about 5 Hz.
In all cases the 15 N chemical shifts were determined by means
of the 1 H–15 N HMBC spectra. The main difference of the 15 N NMR
spectra are in the chemical shift of outer piperazine nitrogen (N-4
or N-8 ) that can be explained by the hybridization state of nitrogen. The 15 N NMR assignments of FQs (except for MOX) are in
Supplementary Table S2.
3.2. Identification of the protonation sites in FQs
In order to determine the correct protonation sites of FQs
we used not only the parent molecules but also, some model
compounds, such as 1(2-fluorophenyl)piperazine monohydrochloride A, 7-chloro-6-fluoro-1-ethyl-4-oxo-1,4-dihydro-quinoline-3carboxylic acid B, CIP-methyl ester C and N4 -acetyl CIP D. These
model compounds helped to identify the protonation sites of the
appropriate moiety in the FQs. The structures of the model compounds are shown in Fig. 2.
In order to characterize the acid–base chemistry of FQs, we first
titrated CIP, and its two derivatives that contain a reduced number of basic sites (carboxymethyl-CIP and N4 -acetyl-CIP (model
compound C and D in Fig. 2)) by pH-potentiometry method. The
advantage of potentiometric titration is that the titration steps
are proportional to the number of protons bound/released. This
Table 3
1
H NMR, 13 C NMR and 15 N NMR data (chemical shifts in ppm, multiplicities with coupling constants in Hz, relative intensities and
parentheses) of MOX (tr. = trans-methylene proton, cys. = cys-methylene proton, ax. = axial, eq. = equatorial).
atom
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14 tr.
14 cys.
15 tr.
15 cys.
–
8.68 s 1H
–
–
7.68 d(13.21) 1H
–
–
–
–
–
–
3.60 s 3H
4.15 m 1H
1.03 m 1H
0.87 m 1H
1.19 m 1H
1.11 m 1H
H
13
C
–
150.31
106.31
175.90
106.44 (24.1)
152.37 (249.67)
136.53 (10.41)
140.21 (7.28)
134.45
117.20 (8.70)
165.73
61.75
40.51
9.56
8.30
15
N
−223.27
–
–
–
–
–
–
–
–
–
–
–
–
–
atom
1
1
2
3
3
4
4
5
5
6
7
7
8
9
9
3.88 m 1H
–
3.18 m 1H
2.91 m 1H
1.79 m 1H
1.74 m 1H
1.74 m 1H
1.68 m 1H
2.64 m 1H
3.57 dd 1H
4.05 dd(5.43, 12.13) 1H
–
3.75 dd(5.11, 10.58) 1H
3.85 dd 1H
ax.
eq.
ax.
eq.
ax.
eq.
ax.
eq.
ax.
eq.
H
19
F–13 C coupling constants in Hz in
13
C
54.30
–
41.40
20.31
17.34
36.34
54.39 (10.28)
–
51.73 (9.07)
15
N
–
−316.19
–
–
–
–
–
–
–
–
–
−335.38
–
–
54
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
Fig. 2. The constitutional formulas of the model compounds.
method is also appropriate to determine how many basic centers
are in a molecule in the 2–12 reliable range of pH-potentiometry.
In the case of CIP the potentiometric titration curve shows two
proton-association processes and the concomitant two basic centers and two protonation macroconstants (log K1 = 8.54 ± 0.01,
log K2 = 6.18 ± 0.01 (I = 0.15 M)). The molecules with reduced number of basic sites have one basic center and one protonation
constant only: log K = 8.48 ± 0.01 (I = 0.15 M) for carboxymethyl-CIP
and log K = 6.30 ± 0.01 (I = 0.15 M) for N-acetyl-CIP, clearly indicating that FQs in the pH range 2–12 have two protonating sites: the
carboxylate and the more basic piperazine N4 nitrogen.
We then investigated whether the other two nitrogens
(quinolone N1 and piperazine N1 ) are able to protonate below pH
2. 1 H NMR-pH titration is a proper method to solve this problem.
In our investigated compounds, however, all observed protons are
multiply influenced by the protonation of every basic site. Their
chemical shifts are composite ones. We used therefore two model
compounds that contain one of the expected protonation sites only.
The 1(2-fluorophenyl)piperazine monohydrochloride (model compound A in Fig. 2) contains the N-1 nitrogen in the piperazine ring
and the 7-chloro-6-fluoro-1-ethyl-4-oxo-1,4-dihydro-quinoline3-carboxylic acid (model compound B in Fig. 2) contains the N-1
nitrogen in the quinolone ring as a possible basic center. We
analyzed both model compounds by UV-pH titration and model
compound A by NMR-pH titration. Model compound B could not
be dissolved in the concentration range of the NMR-pH titration
technique. The UV-pH and the NMR-pH titration curves of model
compound A are shown in Supplementary Fig. S1. The absorbance
of model compound B did not change during UV-pH titration in
acidic media between pH 0–4. Consequently, only one macroconstant could be determined for this compound.
These investigations show that model compound A bears two
basic centers; the N-1 in piperazine ring protonates in very acidic
media only. The protonation macroconstant values from NMRpH titration: log K1 = 8.99 ± 0.02 (I = 1 M) and log K2 = 0.20 ± 0.04
(I = 1 M). Our results are different from those of Lin et al. [13] presented for the phenyl-piperazine compound. Model compound B
has one basic center: the carboxylate group only. The log K for this
compound from UV-pH titration is 6.08 ± 0.05 (I = 1 M).
In conclusion, FQs possess a total of three basic centers: the
two nitrogens in the piperazine ring and the carboxylate group.
The piperazine N1 nitrogen protonates in acidic media in a wellseparated manner from the other two basic centers. Thus the
protonation of FQs can be depicted in terms of three macroconstants and four microconstants, as shown in Fig. 3.
3.3. Determination of the macroconstants of parent FQs and their
derivatives
Evaluation of the protonation constants from 1 H NMR-pH titration curves was based on the principle that non-exchanging NMR
nuclei near the basic site sense different electronic environments
upon protonation. All carbon-bound protons could be observed.
Since protonation processes are fast on the NMR time scale, the
observed chemical shift of a nucleus can be expressed as a weighted
average of chemical shifts of the protonated and unprotonated
form. Weighting factors are the mole fractions.
ıobs. = ıL− L− + ıHL HL + ıH2 L+ H2 L+
=
ıL− + ıHL K1 [H+ ] + ıH2 L+ K1 K2 [H+ ]
1 + K1 [H+ ] + K1 K2 [H+ ]
2
2
(1)
where ıobs. is the observed chemical shift, L− , HL, and H2 L+ are the
non-protonated, mono- and diprotonated macrospecies, respectively, ıL− , ıHL , ıH2 L+ are the chemical shifts of the species in
subscript.
The N-1 nitrogen protonates in very acidic media where the
glass electrode function is distorted by the well-known acidic error
and the limiting chemical shift of the completely protonated form
is beyond reach at the applied, 1 M ionic strength. In order to
avoid the uncertainty of the glass electrode at pH ≤ 2 we used
dichloroacetic acid as an acidic NMR pH indicator. The pH of the
solution was determined from the actual chemical shift of the indicator molecule, using Eq. (2):
pH = log Kind + log
− ıHind
ıobs
ind
ıind − ıobs
ind
(2)
where log Kind is the protonation constant of the indicator (1.14
for dichloroacetic acid), ıind and ıHind are the chemical shift of
the species in subscript (ıind = 6.050 ppm, ıHind = 6.345 ppm for
dichloroacetic acid,) ıobs
is the observed chemical shift [28,34].
ind
At this acidic pH equation (1) is not applicable to determine
log K3 , because the chemical shift of the fully protonated form
cannot be measured at the applied ionic strength. Therefore, the
Perrin–Fabian method was used to determine the most acidic
macroconstants (log K3 ) [35].
ıobs.
=
L
ıH2 L+ (ıobs
− ıind ) + 10 log K ıHL (ıHind − ıobs
)
ind
ind
10 log K (ıHind − ıobs
) + (ıobs
− ıind )
ind
ind
(3)
where log K = log Kind − logKL .
Perrin and Fabian showed that relative protonation constants
can be determined more precisely and accurately in a multicomponent NMR-pH titration than log K values themselves [35]. Orgován
and Noszál [34] validated this method earlier and used for the quantitation of very high basicities where the limiting chemical shift
could not be reached either.
The protonation macroconstants of all the parent molecules are
shown in Table 4 and their derivatives in Table 5.
As an example Fig. 4 shows the NMR-pH profiles of ENR in the
aromatic region. H-8 proton shows a so-called “wrong way” shift
upfield. This upfield shift can be explained by pH-dependent conformational change of the piperazine ring.
The stepwise macroconstants show that the N-1 nitrogen protonation takes place in highly separated manner, as indicated by
the log K3 values of the parent molecules and log K2 values of the
methyl ester derivatives. The largest log K3 value belongs to MOX
that contains pyrrolidino-piperidine ring while the smallest log K3
value belongs to OFL due to the oxazine ring. The log K2 values of
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
55
Fig. 3. The micro- and macroprotonation schemes of the FQs examined. Microconstants with superscript C, N4 and N1 belong to the carboxylate, piperazine N4 nitrogen
and N1 nitrogen, respectively, and K1 , K2 , K3 are stepwise macroconstants. The superscripts on the microconstant indicate the protonating site, while the subscript (if any)
stand for the site already protonated.
Table 4
Protonation macroconstants of the parent molecules.
log K1
log K2
log K3
CIP
ENR
MOX
NOR
OFL
PEF
8.61 ± 0.01
6.30 ± 0.02
−0.21 ± 0.10
7.90 ± 0.02
6.22 ± 0.02
−0.19 ± 0.09
9.35 ± 0.01
6.31 ± 0.01
−0.08 ± 0.07
8.55 ± 0.01
6.20 ± 0.03
−0.20 ± 0.07
8.21 ± 0.01
6.13 ± 0.02
−0.43 ± 0.14
7.53 ± 0.01
6.22 ± 0.02
−0.23 ± 0.10
Table 5
The protonation macroconstants of the carboxymethylated compounds (1 = ciprofloxacin methyl ester, 2 = enrofloxacin methyl ester, 3 = moxifloxacin methyl ester,
4 = norfloxacin methyl ester, 5 = ofloxacin methyl ester, 6 = pefloxacin methyl ester).
log K1
log K2
1
2
3
4
5
6
8.57 ± 0.01
−0.23 ± 0.09
7.78 ± 0.01
−0.16 ± 0.05
9.33 ± 0.01
−0.09 ± 0.06
8.50 ± 0.01
−0.21 ± 0.06
8.14 ± 0.01
−0.38 ± 0.10
7.38 ± 0.01
−0.24 ± 0.07
the parent compounds are similar with a maximum difference of
0.18 log K units. The log K1 values depend on the number of substituents at the N-4 nitrogen, having relatively large values for
secondary amino groups (NOR, CIP, MOX) and modest basicities
for tertiary amino sites (OFL, ENR, PEF). This trend is known to be
an effect of the different hydration states of the protonated forms
of secondary and tertiary amines.
3.4. The complete microspeciation of FQs
Protonation microconstants can be determined by spectroscopy methods, provided that the concentrations of the related
microspecies are commensurable. Earlier the carboxylate protonation microconstants of FQs were determined by UV-pH titration.
Unfortunately the difference between the basicity of piperazine
N-4 and carboxyl groups is too large for reliable microconstant
determinations. For example, differences between the MOX macroconstants exceed three log K units. Thus, the deductive method for
the determination of FQ microconstants is more appropriate. In this
investigation the protonation of the methyl ester derivatives mimic
the protonation of N4 , when the carboxylate is in COOH status.
Thus, so the log K1 values of the esterified compounds are equal to
the log kCN4 microconstants of FQs. In accordance with the protonation scheme in Fig. 3, the following relationships between the
micro- and macroconstants can be formulated:
K1 = kN4 + kC
ˇ2 = K1 K2 = k
(4)
N4
N4
C
C
kN4
= k kC
(5)
Due to the large differences between the log K3 and log K2
N1 = log K equality can be set.
macroconstants, the log kN4
C
3
For further insight into the inter-moiety effects, the pairinteractivity parameter can be defined. The pair-interactivity
parameter quantifies how much the protonation of a basic center
reduces the basicity of the other basic site.
E N4 -C =
kN4
kCN4
=
kC
(6)
C
kN4
C
log E N4 -C = log kN4 − log kCN4 = log kC − log kN4
Fig. 4. NMR-pH titration curves of ENR in the aromatic region (H2, H5, H8 = chemical
shifts of aromatic protons).
(7)
All the 24 determined microconstants and 6 interactivity parameters are shown in Table 6.
C and log kC as well as the
It can be seen that the values of log kN4
interactivity parameters differ from the previous literature values
56
A. Rusu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 50–57
Table 6
Protonation microconstants and interactivity parameters of FQs.
N4 log k
log kC
log kCN4
C
log kN4
N1
log kN4
C
log E
4. Conclusions
CIP
ENR
MOX
NOR
OFL
PEF
8.61
6.34
8.57
6.30
−0.21
0.04
7.89
6.34
7.78
6.23
−0.19
0.11
9.35
6.33
9.33
6.31
−0.08
0.02
8.55
6.25
8.50
6.20
−0.20
0.05
8.21
6.20
8.14
6.14
−0.43
0.06
7.50
6.37
7.38
6.25
−0.23
0.12
[12]. These data show that only the deductive method is appropriate to determine the protonation microconstants of FQ in the minor
protonation pathway.
Comparing our analogous microconstants of the investigated
FQs, the carboxylate microconstants are similar. These values show
that the FQs have much weaker acid character than other aromatic carboxylic acid (for example benzoic acid log K = 4.19 [36]).
This property can be explained with the intramolecular H-bond
between the 3-carboxyl and the 4-carbonyl groups, which stabilise
the protonated form of the carboxyl group. The greatest differences
can be observed in the protonation of the piperazine N4 . Substitution in the N-4 position decreases the basicity of the compounds
and the interpretation of this process is similar to those described
for macroconstants. The interactivity parameters between the carboxylate and piperazine N4 are low due to the relative large
distance between these groups.
Using our microconstants the pH-dependent distribution diagrams of the 30 microspecies for the six FQs could be calculated.
As examples the pH-dependent distribution diagram of ENR and
MOX are shown in Fig. 5 and in Supplementary Fig. S2, respectively. The diagrams show that all compounds exist mainly in
zwitterionic form at the pH of the blood. It can be seen that the concentration of the neutral microspecies at this pH are much below
thus appeared in the previous literature data were shown. The
neutral microspecies play a role in the transport mechanism and
the zwitterionic ones in the binding of DNA gyrase. The distribution diagrams also show that the zwitterionic form fades away at
acidic pH, while the cationic ones becomes predominant, which
explains the pH-dependent activity of FQs [37]. In acidic media, at
the pH of the stomach the monoprotonated form is overwhelmingly dominant in all molecules but our microconstants show the
dicationic ones become also significant upon further lowering the
pH.
Fig. 5. The representative pH-dependent distribution of ENR microspecies.
We identified the protonation sites of FQs using the wellestablished therapeutic drugs and their model derivatives. Our
results show that the FQs have three protonation sites: the 3carboxylate group, the N-4 and the N-1 nitrogen in the piperazine
ring. The N-1 nitrogen protonates only in highly acidic media. By 1 H
NMR-pH titrations we determined all the macroconstants, and by
combination of NMR-pH titrations and a deductive method all the
microconstants for the studied FQs. The pH-dependent distribution
diagram of each FQ was calculated.
The site-specific acid base properties of FQs as distinctive
properties significantly influence their pharmacokinetic behavior
(absorption, distribution) and also, their protein/DNA-gyrase binding. On the basis of the determined macro- and microconstants
capillary zone electrophoresis separation can be developed and the
species-specific physicochemical properties can be interpreted and
improved for further FQ drug candidates.
Acknowledgements
This work was supported by OTKA T 73804 Grant and an Erasmus scholarship for Aura Rusu funded by the European Union. The
authors are grateful to Bayer Schering Pharma AG for providing
the moxifloxacin hydrochloride and to Laropharm, Romania, for
providing pefloxacin mesylate.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jpba.2012.02.024.
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