Protonation Equilibria of Quinolone Antibacterials
KRISZTINATAKACS-NovAK', BELANos~AL*,ISTVAN HERMECZ~~,
GEZA KERESZTURI~,
BENJAMIN PODANYln, AND GYORGY
SaSZ*
Received April 7, 1989, from the 'Institute for Pharmaceutical Chemistty, Semmelweis Medical University, Budapest, H-7092 Htlgyes Endre 1.9.,
the *Department of Inorganic and Analytical Chemistty, L. Elltvds University, Budapest, H-7443 Pf. 723, the "hinoin Pharmaceutical Works,
Budapest, H-7325Pf. 7 70, and the Alnstitute for Drug Research, Budapest, H-7325 Pf 82, Hungary.
Accepted for publication
January 5,1990.
Abstract 0 The aci&base properties of seven antibacterial 7-piperazinyl fluoroquinolone derivatives were studied by potentiometry and UV
and NMR spectroscopy. These molecules contain two proton-binding
sites of similar basiaty, namely, the piperazine amino and the carboxylate groups, as proven by 'H NMR spectroscopy. The basicities are
quantitated at the molecular level in terms of macroconstants, and also
at the submolecular level in terms of microconstants.The microconstants
are then used to calculate the concentration of the positive, zwitterionic,
neutral, and negatively charged species (microspeciation).The zwitterionic forms always predominate over their neutral protonation isomers,
but the zwitteri0nic:neutral concentration ratio is considerably different
for the examined fluoroquinolone derivatives.
The quinolone class of orally active antibacterials is widely
used in the treatment of urinary tract infections. The third
generation members of antibacterial quinolone derivatives
(2, 3, 4, and 5; see structures) are of broader spectrum and
greater activity than the earlier agents of this group, such as
nalidixic acid (12) and oxolinic acid (U.14
Several fluoroquinolone derivatives have recently been
synthesized and the mechanism of their action was extensively studied. These agents proved to be specific inhibitors of
the subunit-A of the bacterial topoisomerase DNA gyrase,
which controls the shape of DNA.4 Structure-activity relationships for fluoroquinoloneshave been studied,3.- characterizing qualitatively the influence of systematic structural
modifications on biological activity.6.6 Results of QSAR investigations have also appeared.3.7" There are, however, few
worksgJ0 reporting biologically important physicochemical
parameters, such as PK,and partition coefficient. These data
are important for a thorough understanding of absorption,
transport, and receptor binding of these drugs at the molecular level.
In the present work, we investigated the acid-base properties of seven amphoteric fluoroquinolone antibacterials, of
which norfloxacin (2) and pefloxacin (3) have been introduced
into human therapy (seestructures, next page) and two others
(4,5) are under clinical trial. We used potentiometric titration
and spectrophotometry for the determination of protonation
constants and 'H NMR spectroscopy to identify the proton
binding sites. On this basis, the acid-base properties are
depicted here a t the molecular level in terms of macroconstants and also at the submolecular level in terms of microconstants. Macroconstants quantitate the overall basicity of
the molecule, but they cannot be assigned to individual proton
binding sites." Microconstants describe the proton binding
ability of the individual functional groups and are useful in
calculating the pH-dependent concentrations of the different
protonation isomers (microspeciationL11-12NoszAl introduced
microspeciation for the characterization of polypeptides in
biological media.12
The microspeciation of drug molecules is also very impor0022-3549/90/1700- 7 023$01.W O
0 7990, American Pharmaceutical Association
H
n
F
H
- JH+N
A
F
CHfl-N-
H
F
CH3-N3-
H
H3Ch
HN
F
F
N-
u
HNDF
F
F
C H ~ - N ~ H3Ch
F
HN-N-
F
CHJ-S-ND0
H
-
H
F
H
H
H
-
tant since the affector-receptor association and, thus, the
biological action need both the drug molecule and the receptor
surface to be present in the appropriate complementary forms
of their microspecies.
Theoretical Section
Molecules with two proton-binding sites, such as fluoroquinolones (2-81, exist in four microscopic protonation forms in
solution. Two of the microspecies are protonation isomers.
The scheme of the interconversion between the four microspeJournal of Pharmaceutical Sciences I 1023
Vol. 79, No. 77, November 1990
The four microconstants can be expressed with microspecies concentrations as follows:
0
R = H , norfloxacin
R = CH3, pefloxacin
cies, as well as the relevant macro- and microconstants are
shown in Scheme I.
The pertinent equilibrium constants, expressed by means of
macro- and microspecies concentrations, are as follows:
[PHI
K ‘-[P-]
[H’]
= P1
(3)
where K, and K2are stepwise macroconstants, and p1and p2
are cumulative macroconstants.
(7)
where the superscript on k denotes the functional group
protonating in a given process, the subscript (if any) denotes
the already protonated group, and A and C refer to the amino
and carboxylate groups, respectively.
Relations between the micro- and macroconstants (PI, pz)
have been reported?
Macroconstants were determined using potentiometric titrations followed by a standard evaluation method,14 whereas
microconstants were determined by means of potentiometry
plus spectrophotometry, as described below.
3H
C
C H3- N
N
U
I
bH5
r+
pc-1
Scheme CProtonation scheme of pefloxacin.
1024 I Journal of Pharmaceutical Sciences
Vol. 79, No. 11, November 1990
K1=
Pi
-
PH
K2
PH?’
Experimental Section
Potentiometry-For the potentiometric measurements, a Radelkis
OP-21111 digital pH-meter (precision of display, 1 mV), a Radelkis
OP-930automatic burette (precision of reading, 0.001 cm’), and a
Radelkis OP-0808P combination electrode were used. Aliquots (50
cm3)of 5 x
M quinolone and 0.02 M HC1 solutions were titrated
by 1M NaOH under a N, atmosphere. All measurements were made
at 0.2 M ionic strength, using NaCl as the auxiliary electrolyte. The
temperature was kept a t 25.0 50.1”C, using a MLW-UP ultrathermostat. The electrode was calibrated with standard buffer solutions
(Merck) in the pH 2-11 range.
The complex products were calculated using a program written for
a Texas Instruments SR-52 calculator. The uncertainty of the constants varies between 0.03 and 0.05 log units, and the number of
parallel measurements is 19.
Spectrophotometry-We used combined potentiometry and spectrophotometry for the determination of the k’ microconstant values.
This method is baaed on the fact that the quinolone spectrum is
independent of the protonation state of the piperazine moiety, but it
is heavily influenced by that of carboxylate. Thus, the degree of
protonation at the carboxylate group can be selectively monitored by
spectrophotometry. Sharp isosbestic points can be found on the
pH-dependent electron spectra (see spectrum of pefloxacin in Figure
1).
Two aliquots of -5 x
M fluoroquinolone solutions were
prepared in either 0.001 M HCl or 0.001 M NaOH, with a total ionic
strength of 0.2 M. By mixing the acidic and basic stock solutions, five
solutions of different pH were obtained, and their spectra were
recorded on a Specord UV-VIS Zeim, PMG 11 spectrophotometer in
the wavelength range of 200 to 400 nm.
The fraction of protonation on the carboxylate group a t a given pH
(%oo-cpH))
can be calculated from spectrophotometric absorbance
data as follows:
where A,c ) and A,cooH, are experimental absorbance values at
H) is
extremely%asic and acidic pH values, respectively, and ern-(
the extent of protonation at the pH where the abeorbance is
Using relationships between),,,-~,,
and microspecies concentrations, as well as the equilibrium constants in eqs 1-7, the microconstant kC can be expressed a8 follows:
WOO-(pH) =
-
“;I + “11
“81 + “61 + “!I + “3
kC [H+l + pZ[H+12
1 + Ki[H+] + pZ[H+12
(11)
We calculated the average of log RC values from data obtained at
five different pH values. The accuracy of the constants varies between
0.07 and 0.14 log units and the number of parallel measurements is
35.Using the determined log kC value, the other microconstants were
calculated according to eqs 8 and 9.
Nuclear Magnetic Resonance Spectroscopy-Two -5 x lo-’ M
solutions of 3 and 4 were prepared in either 0.01 M DCl or 0.01 M
NaOD. The ‘H NMR spectra were recorded on a Bruker AC-250 N M R
spectrometer.
MaterialeThe quinolone derivatives were synthesized at Chinoin Pharmaceutical Works and used without further purification.
All reagents were of analytical grade.
Results and Discussion
(10)
300
350
A
Figure l-The pH dependence of UV spectra of pefloxacin. Key: (1)
0.001 M NaOH; (2) pH 7.0; (3)pH 6.7; (4) pH 6.4; (5) pH 6.1; (6) pH 5.8;
(7) 0.001 M HCI.
Macroeonstants-Seven of the quinolone derivatives (2-8)
contain carboxylate and the piperazine proton binding sites.
That protonation occurs at N; over other apparently basic
sites is proven by ‘H NMR measurements (Scheme II) and
supported by the following considerations: (i) the electronattracting effect of the aromatic ring diminishes the basicity
of the piperazine N; atom; and (ii) the quinolone ring nitrogen
does not have appreciable basicity in aqueous solution. The
greatest change in the chemical shifts was measured at the
N;-CH3 moiety, due to the protonation of the N; atom,
whereas the carboxyl deprotonation caused considerable
change at the C2-H chemical shift. This is in perfect
agreement with the fact that N;-acetylnorfloxacin (9) has
only one proton binding group (carboxylate), since the molecule loses amine basicity due to the acetylation of N;.
The first and second proton binding steps in the potentiometric titration curves of amphoteric fluoroquinolones are
overlapping owing to the comparable basicity of the carboxylate and piperazine proton binding sites. The complex products (& &) obtained from potentiometry are summarized in
Table I. On the basis of PK,data of monofunctional derivatives (9: PK, = 6.53; 11: pKa-+,= 8.481, it is evident that
the logK , anflog K, macroconstants mainly reflect the amine
and carboxylate basicity, respectively.
Structural differences in the fluoroquinolone derivatives
account for the following differences in macroconstants. The
log K,values of the four secondary amine type derivatives (2,
5, 6, 8) are greater than those of the tertiary amines (3, 7).
This is supported by literature data for relevant secondary
amines and tertiary amines [piperazine (flu,
= 9.71)16 and
N-CH3piperazine CPK,, = 8.98);16 piperidine (Nu
=
ll.l!W7 and N-CH3-piperidine
(flu
= 10.08)171. The
above trend is due to the different hydration states of the
protonated forms of secondary and tertiary amines, as proven
by theoretical calculations for a series of simple amines.18The
more water molecules involved in the hydrate sphere of the
protonated amine, the greater is the stabilization.
Journal of Pharmaceutical Sciences I 1025
Vol. 79, No. 11, November 1990
n
H*
8,723
7
OH'
3,031
C H 3 1,4681
3,704
I
C H 3 1,524
n
C H 3 2,907
C H 3 2,963
Scheme (I-The ' t i NMR chemical shlft data of (A) pefloxaan and (6)amifbxacin in basic and acidic solutions (Sppm).
T i b k I-Protonatlon Macroconslants of Oulnolomr 2-1 2
Compound
Number
log 82
log 81 = log K,
14.73
13.82
12.99
14.27
14.87
13.47
14.37
8.51.
7.80
7.57
8.78
9.33
8.13
8.39
-
8.48'
log 82 - log 81 =
log K,
lsoelectronlc
Point
~~
2
3
4
5
6
7
8
9
10
11
12
d
Norfloxadn
Pefloxacin
Amifloxacin
Lomefloxacin
8F-Norfloxacin
8F-Pefloxadn
8-Desfluorolomefloxacin
N-Acetylnorfloxadnb
Norfloxacin ethyleste?
Quinolonecarboxylic acid'
Nalidixic acidb
= 8.7 and pKpK,PK, = 6
2(ref 19).
=
-
-
6.51'
6.13',"
was determined using spectrophotometry.
Rotonation at this site is of no biological significance since it
occurs a t extremely low pH.
Microconstants-Microconstant values are given in Table
Il (the number of decimal places is proportional to the
accuracy of the data). The following conclusions can be drawn
from the microconstant values.
1. Microconstants belonging to the same proton binding
site (log k A and log ke;or log kC and log kz) are significantly
different values. This means that despite the great number of
intervening atoms between the two binding sites, the protonation a t one site significantly decreases the basicity of the
other site (log k A > log ke; log kc > log &:I.
2. The above observation can be quantitated as a measure
of the interaction between the two sites when protonation
Table ICProtonatlon Mlcrocon8tants oi Fluoroqulnolonei 2-8
Compound
Norfloxacin (2)
Pefloxadn (3)
Amifbxacln (4)
Lomefloxacin (5)
IF-Norfloxacin (6)
8F-Pefloxacin (7)
8-Desfluoro-lomefloxadn (8)
~
1Q2@
I Joumcrl of Phamtxwtca
'
ISciences
Vd. 79, No. 11, November 1990
-
-
6.2 (ref 10). Derivatives having only one protonatlon site. pK-,
The log K, values of our fluoroquinolone series show little
diversity. These compounds are weaker acids than either the
aromatic carboxylic acids or the aliphatic p-ketocarboxylic
acids. The weaker acidic character of these molecules may be
due to intramolecular H-bond formation stabilizing the protonated form of the carboxylate group (see structure below).
Some related compounds show similar acidity [nalidixic
acid (12) PK, = 6.13; 3-COOH-4-oxoquinolone (10) pKa =
6-53].
The differences between the log K, values of 6-F-7piperazinyl derivatives (2, 3, 8) are negligible (Table I). On
the other hand, compounds containing a second F atom in
position 8 have >0.5 log K, units greater acidity owing to the
additional negative inductive effect of the fluorine atom. The
decreased carboxylate basicity of amifloxacin (4) is obviously
substituent at the quinolone N, atom.
due to the -NH-CH,
7.37
6.91
6.50
7.14
7.44
6.73
7.19
6.22.
6.02
5.42
5.49
5.55
5.33
5.98
6.53'
log@
loge
log@
m6
8.5
7.2
6.7
5.8
6.0
6.1
5.7
7.0
7.6
7.1
7.2
8.3
8.8
7.7
7.4
6.3
6.1
5.7
5.7
5.7
5.6
6.0
~~~~~~
7.7
7.3
8.6
9.2
7.9
8.4
occurs at one of them. This interactivity parameter is considerable in all molecules, but decreases in the following
order: 6: 3.1, 5: 2.6, 7: 2.2, 4: 1.5, 8: 1.4, 2: 1.3, 3: 1.0.
3. Comparing the analogous microconstants of the seven
fluoroquinolones,the greatest differences occur in the entries
referring to piperazine protonation. The basicity of molecules
containing a secondary amine group is greater than the
basicity of tertiary amine derivatives. This is true for both the
log kA and log k$ values (see microconstants of 6 versus 7, 2
versus 3, 2 versus 4). The interpretation is the same as
described for macroconstants.
Methyl substitution ortho to the Nk atom decreases the
basicity to a small extent, presumably due to steric hindrance
(see corresponding log kA or log k$ values of 2 versus 8 and 6
versus 5). The basicity decreasing effect of 8-fluorosubstitution relative to monofluoro derivatives is considerable in those microconstants where the other proton binding
site has already been protonated: log k$ and log kz (6 versus
2, 7 versus 3, 5 versus 8).
The relative concentration for all microspecies in solution
can be calculated with the following equations:
l,oo
0,90
0'80
0,70
0,60
4
0'50
0,40
0.30
1
*[:=
"[O
+
=
+ P2[H'I2
(13)
kA[H']
1 + KI[H'] + P2[H'I2
(14)
1 + K1[Hf]
0,20
0,lO
0
kc[H']
*[; =
1 + K1[Hf] + P2[H'I2
"[:
P2[H'I2
=
1 + K1[Hf] + &[H'I2
1
(15)
(16)
The distribution diagram for the pefloxacin microspecies is
shown in Figure 2.
Analogous to pefloxacin, all compounds exist mainly in
zwitterionic forms between pH 3 and 11. The relative concentrations of microspecies at three biologically important pH
values are summarized in Table 111. The positively charged
form ([I) is present in 99.9% at pH 1, while the other
microspecies are minor components. At pH 7.4, all microspecies occur in commensurable concentrations. Considerable
differences exist in the ratio of protonation isomers of different
fluoroquinolones. The following order of the ratio of zwitterionic:nonionic forms can be set up: 8: 9.5; 2: 7.6; 3: 3.5; 5 =
6: 2.2; 7: 1.5; and 4: 1.4.
Fluoroquinolonemicrospeciation can be further utilized for
a deeper understanding of structure-activity relationships
and interpretation of bioavailability. For example, microspe-
3
2
4
5
6
7
9
8
1011
12
13
14
PH
Figure 2-The
microspeciation diagram of pefloxacin.
ciation may explain the differences in the biological absorption of fluoroquinolones; that is, at blood pH, the neutral form
concentration of compounds is different (34.0%for 8-fluoropefloxacin, while 25.3% for amifloxacin and only 10.2% for
norfloxacin).
Izumi et a1.20 have recently interpreted the poor binding of
pipedimic acid to human and rat albumin by the existence of
a zwitterionic form at neutral pH. As shown above, the
concentration of zwitterionic microspecies substantially varies within the examined series. Using microspeciation, the
different microforms a t physiological pH can be taken into
account in the interpretation of the protein binding of these
drugs. The fluoroquinolone microspeciation can also be used
for the determination of species-specific physicochemical
properties. For example, the octanol-water partition coefficient is a generally accepted descriptor of hydrophobicity in
QSAR. However, the true partition coefficient can only be
thoroughly understood if the four differently charged microspecies concentrations are known.
The antibacterial activity of piperazinyl-substituted fluoroquinolones was shown to be pH-dependent.21 A decrease in
Table ill-Percentage Dlstrlbutlon of Fiuoroqulnolone Microspecies at Phyalologicaily Important pH Values
~
2
3
4
5
6
7
8
1.9 x
1.5x
1.0x
5.4x
1.3 x
3.4 x
4.3 x
10-11
lo-'
10-11
lo-''
10-11
5.4 x 10-4
7.4 x
2.2 x
2.2 x 10-3
2.0 x
2.8 x
9.5 x 10-4
7.1x 10-5
2.1 x
1.6 x
1.0 x 10-3
8.9 x
1.9 x
1.0 x
99.9
99.9
99.9
99.9
99.9
99.9
99.9
6.7
27.7
40.1
3.9
1.1
15.5
8.9
77.9
53.9
34.1
65.6
67.5
50.4
80.0
10.2
15.2
25.3
29.3
30.1
34.0
8.3
~~~~~~
~
5.7
2.9
0.6
1.2
1.3
0.7
3.3
75.5
94.1
96.4
62.4
31.9
88.1
80.3
21.8
4.6
2.1
26.0
47.1
7.2
18.0
~
2.9
1.4
1.5
11.6
21.1
4.8
1.9
4.1 X lo-'
6.2X
9.4 x 10-4
1.2x lo-'
2.4x lo-*
2.6 x
1.9 x
Journal of Pharmaceutical Sciences I 1027
Vol. 79, No. 7 7 , November 1990
pH below 5.5 for norfloxacin22 and 5.0 for amifloxacinm
progressively decreases their activity. This phenomenon is
obviously related to the poor penetrating or receptor binding
ability of microspecies [:, which predominates at low pH.
References and Notes
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10.
11.
12.
13.
14.
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1028 I Journal of Pharmaceutical Sciences
Vol. 79, No. 11, November 1990
~~~~