Design, synthesis, antibacterial activity and physicochemical parameters of novel N-4-piperazinyl derivatives of norfloxacin

Article abstract of DOI:10.1016/j.bmc.2009.04.027

We report herein the synthesis of some N-Mannich bases in addition to different N-4 substituents of norfloxacin. The antibacterial activities of the newly synthesized compounds were evaluated and correlated with their physicochemical properties. Results r

Full text of DOI:10.1016/j.bmc.2009.04.027

Bioorganic & Medicinal Chemistry 17 (2009) 3879–3886
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, antibacterial activity and physicochemical parameters
of novel N-4-piperazinyl derivatives of norfloxacin
b
c
Gamal El-Din A. A. Abuo-Rahma a, , Hatem. A. Sarhan , Gamal F. M. Gad
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt
b Department of Pharmaceutics, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt
c Department of Microbiology, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the synthesis of some N-Mannich bases in addition to different N-4 substituents of nor-
floxacin. The antibacterial activities of the newly synthesized compounds were evaluated and correlated
with their physicochemical properties. Results revealed that some of the tested compounds exhibited
better inhibitory activities than the reference antibiotic norfloxacin against Pseudomonas aeruginosa,
Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus strains. Correlation results showed that
there is no single physicochemical parameter that can determine the effect of N-4 piperazinyl group
on the activity of these fluoroquinolones, where lipophilicity, molecular mass and electronic factors
may influence the activity.
Received 1 March 2009
Revised 13 April 2009
Accepted 14 April 2009
Available online 19 April 2009
Keywords:
Norfloxacin
Antibacterial
Mannich bases
Physicochemical properties
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Several N-4 piperazinyl derivatives of fluoroquinolones were
introduced and demonstrated various biological activities like ben-
Fluoroquinolone antibacterial agents represent a fast growing
group of antibiotics; various derivatives were synthesized and
tested for their antimicrobial activities. The new generations of flu-
oroquinolones achieved significant improvement in potency, spec-
trum and physicochemical properties.1 The SAR studies revealed
that, the fluorine atom and the 1-alkyl, 1,4-dihydro-4-oxo-quino-
line-3-carboxylic acid skeleton of fluoroquinolones is responsible
for potency represented in binding with type-II topoisomerase en-
zymes, DNA gyrase and topoisomerase IV.2 In addition, it is be-
lieved that the 6-fluoro and 7-piperazinyl groups are responsible
for the broad spectrum and antipseudomonal activities of fluoro-
quinolones.2 Moreover, it is clear that chemical modifications at
C-7 is suitable for controlling of the pharmacokinetic properties
and hence changes in the cell permeability of these antibiotics.3
The third generation fluoroquinolones as lomefloxacin and flerox-
acin exhibited longer duration of action, while sparfloxacin and
tosufloxacin revealed higher potency against Gram-positive cocci
and anaerobic bacteria than the earlier members4 The more re-
cently released fluoroquinolone, levofloxacin, possesses a broad-
spectrum antibacterial activity similar to that of earlier quinolones.
However, it has a greater activity against Gram-positive and atyp-
ical organisms.5
zenesulfonamide fluoroquinolones as broad spectrum antibacteri-
als6 and 7-{4-[2-(hydroxyiminoethyl]-1-piperazinyl derivatives
which demonstrated excellent activities against renal cancer.7
Also, isatine Mannich bases of gatifloxacin were emerged as potent
anticancer agents.8 In addition, research groups introduced re-
cently N-4 piperazinyl aralkyl,9 Mannich bases10 or cephalospo-
rin11 fluoroquinolone derivatives effective as potential
antimycobacterial agents. Other norfloxacin Mannich bases were
synthesized and showed anti-HIV, antifungal in addition to anti-
bacterial activities.12
N-Mannich bases have been used as potentially useful prodrug
candidates for imides, amides, amines, hydantoin and urea deriva-
tives.13 It is believed that the N-Mannich base functional group can
increase the lipophilicity of the parent amines at physiological pH
values by decreasing their protonation resulting in enhancement of
absorption through bio-membranes.14 The lipophilicity of fluoro-
quinolones can influence their ability to cross bacterial mem-
branes. Consequently, partition coefficient could be an important
parameter affecting the biological activity.15 Furthermore, it is
clear that the neutral species of fluoroquinolones are more lipo-
philic than zwitterionic form. So, factors that can affect N-4 proton-
ation, like steric and electronic effects or charge density, can also
affect lipophilicity.16 Moreover, it was proposed that lipophilicity
is a very important factor in the quinolones intestinal absorption17
that is responsible for the poor relationship between in vivo and
in vitro activity of fluoroquinolones.
* Corresponding author. Tel.: +20 103069431; fax: +20 862369075.
E-mail address: gamalaburahma@yahoo.com (G.E.-D.A.A. Abuo-Rahma).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.027

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G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
Herein, we report the synthesis of two series of norfloxacin
ference in melting point of this compound with that reported in lit-
erature (229–230 °C).1 Also, the 1H NMR spectrum of compound 3a
in DMSO-d6 showed the two –CH2– groups of the two hydroxy-
ethyl moieties and the piperazine moiety appeared as a multiplet
from 3.0 to 4.1 ppm. The 2 OH absorption appeared at d 5.3 ppm.
Elemental analysis data confirmed the N-quaternary derivative
3a. Heating at reflux of norfloxacin 1 with 3-chloro-5,6-diphenyl-
1,2,4-triazine using triethylamine as a base in acetonitrile afforded
the derivative 3c. On the other hand, preparation of the cyclopentyl
derivative 3b required stirring of norfloxacin 1 with cyclopentyl
bromide using sodium bicarbonate as a base and potassium iodide
as a catalyst in DMF at room temperature.
derivatives. The first series describes synthesis of bis(2-hydroxy-
ethyl)piperazin-1-ium bromide derivative with expected full posi-
tive charge at pH 7; 5,6-diphenyl 1,2,4-triazinyl and cyclopentyl N-
4 piperazinyl substituents in addition to the N-formyl derivatives,
the known active metabolite18 of norfloxacin. The second series de-
scribes synthesis of various N-4 piperazinyl Mannich bases as pro-
drugs of norfloxacin. The designed N-4 piperazinyl substituents
were chosen with expected variable lipophilicities, steric, elec-
tronic and molecular masses. Also, partitioning behavior of these
derivatives between octanol and buffer solutions (pH 7.4) is a mat-
ter of interest to be correlated with their antibacterial activities. It
seems reasonable to calculate the N-4 charge density, due to its
importance in determining the N-4 protonation, consequently, cor-
relation between partition coefficient, molecular mass and N-4
charge density with log MIC was carried out. The methods of syn-
thesis of the designed compounds are summarized in Schemes 1
and 2.
Mannich bases of norfloxacin 4a–f were prepared by heating at
reflux of norfloxacin 1 and the respective secondary amine, imide
with excess formaldehyde in ethanol. The yield of the formed Man-
nich bases range from 49.5% to 76%. The purity of the synthesized
compounds was checked by thin layer chromatography and ele-
mental analysis. The structural formulae of the products were as-
signed by spectral data. The 1H NMR spectra were identified by
their chemical shifts, multiplicities and coupling constants. In gen-
eral, 1H NMR spectra showed the characteristic chemical shifts for
the fluoroquinolone nucleus. It showed the absorption characteris-
tic for the –N–CH2CH3 as triplet–quartet; the piperazine 8 protons
appeared either as a multiplet or two multiplets in the range of
2.9–4.3 ppm, the doublet signal at a range of 6.8–7.25 ppm with
JH–F of about 6.6–7.5 Hz is characteristic for H-8; the doublet signal
at a range of 7.8–8.1 ppm with JH–F of about 12.0–14 Hz is charac-
teristic for H-5 and the singlet signal characteristic for H-2 at about
8.63–8.95 ppm. The 1H NMR spectrum of the N-formyl derivative 2
showed the characteristic signal for –N–CHO group as a singlet at
8.1 ppm. The 1H NMR of the Mannich bases 4a–f showed the char-
acteristic singlet (2H) for the –N–CH2–N– at 4.7–4.8 ppm in CDCl3
or 5.2–5.3 in DMSO-d6.
2. Results and discussion
2.1. Synthesis
Although the N-formyl derivative 2 was prepared previously1 in
50% yield using formic acid without spectroscopic specifications, it
was also recently prepared19 in DMF using an independent reac-
tion without spectroscopic data. It is prepared in this investigation
using a procedure reported for synthesis of other N-formyl20 by
refluxing norfloxacin 1 and formamide in glacial acetic acid in boil-
ing steam bath. It was difficult to react the formed N-formyl group
with amines or hydrazides. Heating at reflux of norfloxacin 1 with
four equivalents of bromoethanol in acetonitril afforded N-4 bis(2-
hydroxyethyl)piperazin-1-ium bromide derivative 3a in high yield.
The isolated compound exhibited a melting point over 300 °C.
Hence, the possibility of identification of the product as the tertiary
analogue, the N-4-hydroxyethyl derivative, is excluded where it is
reported in the literature with a variable melting point due to dif-
It is noteworthy noting that the 1H NMR spectrum of the Man-
nich base 4c in CDCl3 showed absorptions corresponding to two
tautomeric forms in the 3-methyl-pyrazole-5(4H)-one moiety. This
result is consistent with a previous finding about tautomerism in
OH
F
F
O
O
O
N
N
H
N
N
COOH
COOH
HCONH2
N
N
Br
HO
H3CH2C
3 a
H3CH2C
2
OH
F
Br
4
O
CH3CN
HN
N
COOH
N
H3CH2C
Br
1
TEA
CH3CN
N
KI
NaHCO3
N
Ph
F
Cl
Ph
N
O
F
N
N
Ph
Ph
N
N
O
COOH
N
N
N
N
COOH
H3CH2C
N
H3CH2C
3b
3 c
Scheme 1. Synthesis of compounds 2 and 3a–c.

G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
3881
F
F
R1
O
O
HN
HN
N
N
N
R2
COOH
R1
N
COOH
HCHO
N
R2
N
H3CH2C
H3CH2C
1
4a-f
b
a
c
d
f
e
4
O
N
O
O
O
N
N
N
R1 & R2
N
O
N
O
N
H3C
N
O
O
Scheme 2. Synthesis of the Mannich bases 4a–f.
pyrazolones.21 This may be attributed to the presence of both the
CH (A) and the NH-(B) isomers. The –N–CH2–N– group appeared
as two absorptions at d 4.69 and 4.36 ppm owing to the different
chemical environment corresponding to the two isomers. Other
absorption corresponding to the 4-CH2 moiety in pyrazolone tauto-
mer (A) multiplet at 3.38–3.50 ppm. In addition, a singlet charac-
teristic for the @CH group for the isomer (B) appears at 4.2 ppm.
The prevalence of the two tautomers is in the ratio of about 40%
of the tautomer (A) and 60% of tautomer (B). The possibility of
the presence of the –OH tautomer (C) is excluded due to the ab-
sence of both a downfield aromatic proton absorption correspond-
ing to the @CH and the enolic OH.
pneumonia than norfloxacin, but compounds 3a, 4c and 4e showed
good comparable activities. The N-formyl 2 and the Mannich base
4a showed excellent inhibition against S. aureus (MICs < 1). It is
noteworthy noting that the triazinyl derivative 3c and the Mannich
base 4f characterized by their relatively higher molecular weight
(550.28 and 528.53, respectively) exhibited no pronounced activi-
ties against all the tested strains.
2.3. Partition coefficient
The experimental partition coefficient between octanol and
phosphate buffer pH 7.4 expressed in (log D) of the newly synthe-
sized fluoroquinolones, 2, 3a, 3c, 4a–d, 4f in addition to the parent
norfloxacin ranged from 0.158 to ꢀ0.90 (Table 3). The results of the
calculated partition coefficient values (log P) are listed along with
the experimental partition coefficients for comparison in Table 3.
To determine the relationship between the calculated and experi-
mental partition coefficient values, a simple regression was per-
H3C
H3C
HN
H3C
H
H
H
N
N
H
N
N
N
O
O
OH
CH2
CH2
R
CH2
formed. There was found
a weak correlation between the
calculated and the experimental values (r2 = 0.29) Figure 1. This
weak correlation may be attributed to the zwitterionic nature of
the fluoroquinolones at the physiological pH which is close to
the isoelectric point (pH 7.0) that is not considered in the calcu-
lated log P. It is assumed that the partitioning of quinolones was
generally consistent with the knowledge that the neutral species
of like compounds is more lipophilic than the zwitterionic species.
The ratio of the zwitterions to the neutral species is a very impor-
tant feature to describe the lipophilicity of fluoroquinolones22
(Scheme 3).
R
R
(A)
(C)
(B)
R = norfloxacin
2.2. Antibacterial screening
The synthesized compounds 2, 3a–c, 4a–f in addition to the ref-
erence norfloxacin were tested for their in vitro antibacterial activ-
ity against P. aeruginosa, Escherichia coli, Klebsiella pneumonia and
Staphylococcus aureus strains. The diameter of the zones of inhibi-
tion (mm) are given in Table 1, their minimum inhibitory concen-
trations (MICs) are given in Table 2. From the obtained data, it is
obvious that, the pyrazolone Mannich base 4c exhibited good inhi-
bition activity against all the tested Gram-positive and Gram-neg-
ative organisms which is more pronounced than that exhibited by
the reference, norfloxacin. The succinimide Mannich base 4d and
the N-formyl analogue 2 revealed a better activity than norfloxacin
in all the tested strains except K. pneumonia, where the inhibition
is weak. Other compounds showed variable inhibition activities
against the used strains. In comparison, the N-formyl 2, the pyraz-
olone 4c and succinimide Mannich base 4d gave antipseudomonal
activities comparable to norfloxacin. The N-formyl 2 and the qua-
ternary derivative 3a, in addition to the Mannich bases 4c and 4d
showed better activity against E. coli than norfloxacin that is excel-
lent in case of the Mannich bases 4c and 4d (MICs < 1). None of the
newly synthesized compounds exhibited better activity against K.
2.4. Correlation between partition coefficient and log MIC
The parabolic relationship between the antibacterial activities
(log MIC) and both log D or the calculated log P has been fitted.
The following general regression equation was therefore applied.
Y ¼ a log X2 þ b log X þ C
where Y = log MIC; log X = log D or calcd log P; a, b and c are
constants.
The parabolic curve-fittings between log MIC and both the cal-
culated log P or the experimental log D values in all the tested
microorganisms are described. The correlation between calculated
log P and log MIC in case of P. aeruginosa showed strong positive
correlation (r2 = 0.94). (Fig. 2). On the other hand, this correlation
is weaker in case of K. pneumonia, E. coli and S. aureus (r2 = 0.85,
0.67, 0.62, respectively), Table 4.
The correlation between log D and log MIC is weaker than in
case of calculated log P where r2 = 0.54, 0.43, 0.38 and 0.25 for E.

3882
G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
Table 1
The diameter of the zone of inhibition (mm)
Compd
Norf.
Organisms
Diameter of inhibition zone (mm)
64 ( g/mL)
4 (
lg/mL)
16 (
lg/mL)
l
128 (
l
g/mL)
512 (lg/mL)
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
2
0
0
0
5
4
5
0
15
6
8
20
19
12
20
3
2
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
2
0
7
2
2
0
8
7
2
0
8
13
4
0
15
13
8
11
12
3a
3b
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
0
4
0
0
0
7
4
0
0
9
5
0
0
12
11
14
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
0
0
0
0
0
0
2
4
0
3
3
7
0
8
8
15
3c
4a
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
5
0
0
0
7
3
0
0
7
3
0
0
8
15
30
20
10
4b
4c
4d
4e
4f
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
0
3
0
0
0
6
3
0
0
7
8
0
15
10
20
3
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
8
0
0
3
8
2
0
3
10
3
7
10
6
18
10
11
3
1
3
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
8
0
9
0
15
4
13
0
20
12
15
0
22
13
27
5
31
22
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
0
0
0
0
0
0
4
0
10
0
8
12
9
10
0
0
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
Staphylococcus aureus
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
0
10
13
9
15
coli, K. pneumonia, S. aureus and P. aeruginosa strains, respectively,
Table 4. These non-significant correlation results may be explained
by the literature results22 which revealed that lipophilicity of the
molecule and hence penetration into bacterial cell is not the only
factor which can affect its activity. Indeed, other factors like the
affinity of the compounds for their target DNA Gyrase and Topoiso-
merase IV can affect their MIC.23 Furthermore, the heterogeneous
nature and the difference in the steric effect of the tested N-4-pip-
erazinyl substituent may play an important role in the accommo-
dation of these molecules on the active site of the enzyme.23 The
weaker correlation between log MIC and log D may be due to the
zwitterionic nature of fluoroquinolone analogues at the physiolog-
ical pH. Hence, protonation on the N-4 piperazinyl can influence
their lipophilicity. It is reported that the electronic factors can play
a pronounced effect on the protonation of N-4 piperazinyl group.23
Consequently, this may affect the state of the molecule to be either
in the neutral or zwitterionic form (Scheme 3). Furthermore, lipo-
philicity and so permeability into bacterial cells is a function of the
electronic effects. According to these findings, it is important to
calculate the residual charge on the piperazinyl N-4 of the synthe-
sized compounds and norfloxacin. The calculated charges for the
tested compounds are listed in Table 3. The parabolic curve-fittings
between the charge density and log D or log P are described. A
weak correlation between log D or log P and charge density
(r2 = 0.28 and 0.34, respectively) was found. These results revealed
that the N-4 charge density is not the only factor determining the
N-4 protonation and hence partition coefficient. Other factors, like
steric effect could possibly affect N-4 protonation. These findings
are consistent with a previous study about the hydrophobicity of
quinolones that revealed no good correlation between MIC with
hydrophobicity24 Similarly, another study revealed weak correla-
tion between MIC and cell accumulation.15,25
2.5. Correlation between molecular mass and log MIC
It is reported15,26 that the molecular mass and bulkiness of the
substituent at C-7 position hinder penetration of quinolones into
Gram-negative organisms through the porine channel, although
hydrophobic molecules appear to enter via the lipopolysaccharide
or across the lipid bilayer. Conversely, Gram-positive bacteria do

G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
3883
Table 2
these molecules to enzymes, there is no single physicochemical
parameter that can definitely be identified as an affluent on the
biological effect.
Minimum inhibitory concentration (MIC) of the tested active compounds and the
reference norfloxacin
Compd
Minimum inhibitory concentration (lg/mL)
P. aeruginosa
E. coli
K. pneumonia
S. aureus
4. Experimental
4.1. Chemistry
Norfl.
2
9
6
14
11
4
64
>512
128
4
8
64
<1
128
11
64
<1
128
10
128
10
13
>512
128
14
8
3a
3b
3c
4a
4b
4c
4d
4e
4f
>512
>512
>512
16
128
9
All the chemicals and solvents used were purchased from com-
mercial sources and were of the highest pure form. Melting points
(uncorrected) were determined on a Stuart electrothermal melting
point apparatus. 1H NMR spectra were run on GEMINI-200 NMR
spectrometer (200 MHz) and on MERCURY-300BB NMR spectrom-
eter (300 MHz) and also on Varian Em-360L NMR spectrometer
(60 MHz) using tetramethylsilan (TMS) as internal standard.
Chemical shifts are expressed in d ppm, the coupling constants J
are expressed in hertz. Mass spectra were performed on JEOL
JMS600 or HP mass spectrometers. Elemental microanalyses were
performed on a Perkin Elmer 2400 CHN Elemental analyzer or on a
Hereaus Vario EL apparatus. The reactions follow up and checking
the purity of the compounds were made by TLC (Kieselgel 60 F254
precoated plates, E. Merck, Dermastadt, Germany), the spots were
visualized by exposure to UV lamp at kmax 254 nm. Yields are of
purified product and were not optimized. Norfloxacin was pur-
chased from the (Medical Union Pharmaceuticals, Abu-sultan,
Ismailia, Egypt). 3-Chloro-5,6-diphenyl-1,2,4-triazine was pre-
pared as reported.28 3-Methyl-1H-pyrazol-5(4H)-one was pre-
pared as reported.29
<1
5
64
128
<1
128
128
128
9
64
4
>512
128
not possess an outer membrane, and so lack outer membrane pro-
teins and lipopolysaccharide. Hence accumulation of Gram-posi-
tive bacteria is thought to take place by simple diffusion across
the cytoplasmic membranes and not affected by increasing the
bulkiness of the N-7 substituents.27 Parabolic curve-fitting be-
tween molecular mass and log MIC for the tested compounds (Ta-
ble 4) resulted in a weak correlation (r2 = 0.19, 0.53 and 0.62) for
the Gram-negative organisms K. pneumonia, P. aeruginosa and
E. coli, respectively. Conversely, this correlation is stronger in case
of the Gram-positive S. aureus (r2 = 0.67) Table 4. These results are
consistent with a previous QSAR study about quinolones showing
that absorption is increased by higher lipophilicity up to a limit
due to steric hindrance.16
4.1.1. 1-Ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-1,4-dihydro-
4-oxoquinoline-3-carboxylic acid (2)
3. Conclusion
Norfloxacin (3.19 g, 0.01 mol) was heated with formamide
(50 mL) over a steam bath for 2 h. While standing in the cold over-
night, needle crystals were formed, which were filtered off. Recrys-
tallization with methanol afforded the pure product 2 as pale
A group of N-Mannich bases, as prodrugs of norfloxacin in addi-
tion to their related N-4-substituents; N-formyl, bis(2-hydroxy-
ethyl)piperazin-1-ium bromide, N-triazinyl and N-cyclopentyl
derivatives were prepared and identified by spectroscopic and ele-
mental methods. Some of the prepared compounds exhibited bet-
ter inhibitory activities than the reference antibiotic norfloxacin
against the tested microorganisms. The N-formyl 2 and the Man-
nich base 4d showed better activity than norfloxacin against
P. aeurginosa. The Mannich bases 4c and 4d exhibited a superior
yellow crystals, mp 292–293 °C in 73% yield; IR (KBr) cmꢀ1
:
3435, 3035, 2925, 1691, 1665, 1608, 1250, 1190, 802, 745; 1H
NMR spectrum (DMSO-d6) 400 MHz 1.39 (t, 3H, J = 7 Hz, –CH2CH3);
3.20–3.60 (m, 8H, piperazine 8H); 4.53 (q, 2H, J = 7 Hz, –CH2CH3);
7.14 (d, 1H, JH–F = 6.9 Hz, H-8); 7.80 (d, 1H, JH–F = 12.9 Hz, H-5);
8.10 (s, 1H, –CHO); 8.88 (s, 1H, H-2). FAB-MS m/z MH+ 348.19
(4.00%). Anal. Calcd for C17H18FN3O4 (347.13): Calcd: C, 58.78; H,
5.22; N, 12.10. Found: C, 58.59; H, 5.21; N, 12.14.
activity against E. coli (MICs < 1
the Mannich base 4a exhibited a superior activity against S. aureus
(MICs < 1 g/mL). In addition, the Mannich base 4c exhibited a pro-
lg/mL), while the N-formyl 2 and
l
4.1.2. 4-(3-Carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-
7-yl)-1,1-bis(2- hydroxyethyl)piperazin-1-ium bromide (3a)
A mixture of norfloxacin (0.79 g, 0.0025 mol) and bromoethanol
(1.24 g, 0.01 mol) in acetonitril (50 mL) was heated at reflux over-
night. The precipitated solid was filtered off, washed with hot
nounced broad spectrum activity against all the tested Gram-posi-
tive and Gram-negative microorganisms. Results revealed that
there is no significant correlation between log MIC and log P, log D
and molecular mass. Hence, assuming the similarity in affinity of
Table 3
Log p, log D, molecular mass, N-4 charge and log MIC for compounds 1, 2, 3a–c and 4a–f
No.
Correlation parameters
Mol. mass
Log MIC
Log P
Log D
N-4 Charge
P. aeruginosa
E. Coli
K. pneumonia
S. aureus
1
2
1.49
0.55
1.90
3.99
3.11
2.31
3.74
1.16
0.43
2.60
5.09
ꢀ0.41
0.087
0.158
ND
ꢀ0.072
ꢀ0.043
ꢀ0.043
ꢀ0.85
ꢀ0.80
ND
319.13
347.13
487.11
387.20
550.21
418.20
449.19
429.18
430.17
478.17
528.18
ꢀ0.209
0.417
0.739
0.95
0.78
2.71
2.71
2.71
1.20
2.11
0.95
0.70
1.81
2.11
1.45
1.04
0.60
1.81
2.71
2.11
0.60
0.00
0.00
2.11
2.11
0.00
2.11
1.00
1.11
2.71
2.11
1.15
0.90
2.11
0.95
1.81
1.81
0.00
2.11
1.04
1.81
0.00
2.11
1.00
0.60
2.71
2.11
3a
3b
3c
4a
4b
4c
4d
4e
4f
ꢀ0.143
0.188
ꢀ0.163
ꢀ0.127
ꢀ0.152
ꢀ0.149
ꢀ0.149
ꢀ0.145
ꢀ0.90

3884
G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
3.27 (m, 8H, piperazine 8H); 4.05 (m, cyclopentyl H-1); 4.47 (q,
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
2H, J = 7.4 Hz, N–CH2CH3); 7.05 (d, 1H, JH–F = 6.6 Hz, H-8); 7.80 (d,
1H, JH–F = 12.0 Hz, H-5); 8.85 (s, 1H, H-2); 15.25 (s, 1H, COOH).
EI-MS m/z MÅ+ 387.00 (28.30%). Anal. Calcd for C21H26FN3O3ꢂ
0.25H2O (387.20): C, 64.35; H, 6.81; N, 10.72. Found: C, 64.37; H,
6.66; N, 10.57.
y = -0.1282x - 0.1749
R2 = 0.2939
4.1.4. 1-Ethyl-6-fluoro-1,4-dihydro-7-(4-(5,6-diphenyl-1,2,4-
triazin-3-yl)piperazin-1-yl)-4-oxoquinoline-3-carboxylic acid (3c)
A mixture of norfloxacin (3.19 g, 0.01 mol), 3-chloro-5,6-diphe-
nyl-1,2,4-triazine (2.5 g, 0.01 mol) and TEA (1.2 g, 0.02 mol) in ace-
tonitril (50 mL) was heated at reflux overnight. The reaction
mixture was evaporated to dryness. To the residue was added
30 mL water, the precipitated solid was filtered off, washed with
ethanol. Crystallization from methanol/chloroform afforded 3.0 g
0
1
2
3
4
5
6
0.2
calc log p
Figure 1. Straight-line correlation between calculated log P and log D.
of yellow powder, mp > 300 °C; 54.55% yield; IR (KBr) cmꢀ1
:
3405, 3030, 2905, 1720, 1611, 1240, 1194, 796, 744; 1H NMR
200 MHz (CDCl3): d 1.63 (t, 3H, J = 7 Hz, –CH2CH3); 3.51 (br s, 4H,
piperazine 4H); 4.20–4.40 (m, 6H, piperazine 4H + N–CH2CH3);
6.93 (d, 1H, JH–F = 6.6 Hz, -H-8); 7.29–7.57 (m, 10H, Aromatic
10H); 8.10 (d, 1H, JH–F = 12.8 Hz, H-5); 8.70 (s, 1H, H-2); 15.09
(br s, 1H, –COOH). FAB-MS m/z MH+ 551.02 (4.30%) Anal. Calcd
for C31H27FN6O3ꢂ0.5H2O (550.21): C, 66.54; H, 5.04; N, 15.02.
Found: C, 66.55; H, 5.23; N, 15.08.
methanol and dried to afford 0.97 g of brownish yellow crystals,
mp > 300 °C, % yield = 79.88; IR (KBr) cmꢀ1: 3385, 3000, 2925,
1690, 1615, 1263, 1194, 797, 739; 1H NMR spectrum (DMSO-d6)
200 MHz 1.42 (t, 3H, J = 7 Hz, –CH2CH3); 3.0–4.1 (m, 16H, pipera-
zine 8H and –N–CH2CH2OH 8H); 4.60 (q, 2H, J = 7 Hz, –CH2CH3);
5.31 (br s, 2H, OH); 7.24 (d, 1H, JH–F = 7.2 Hz, -H-8); 7.89 (d, 1H,
JH–F = 14.2 Hz, -H-5); 8.92 (s, 2H, H-2), 15.23 (br s, 1H, -COOH).
EI-MS m/z: MꢀHBr 406.00 (3.00%), 364.30 (78%), 320.20 (100%).
Anal. Calcd for C20H27BrFN3O5 (487.11): Calcd: C, 49.19; H, 5.57;
N, 8.60. Found: C, 49.41; H, 5.41; N; 9.46.
4.1.5. General procedure for preparation of the Mannich bases
(4a–g)
To an equimolar mixture of norfloxacin and the respective
amine or imide in ethanol, 1 mL of formaline (37%) was added.
The reaction mixture was heated at reflux overnight, cooled. The
precipitated solid was filtered off under vacuum. Crystallization
from the respective solvent afforded the Mannich bases 4a–f in
40–76% yields, respectively.
4.1.3. 7-(4-Cyclopentylpiperazin-1-yl)-1-ethyl-6-fluoro-1,4-dihy
dro-4-oxoquinoline-3-carboxylic acid (3b)
A mixture of norfloxacin (0.5 g, 1.57 mmol), sodium bicarbonate
(0.13 g, 1.65 mmol), potassium iodide (0.05 g, 0.5 mmol) and
cyclopentyl bromide (0.278 g, 1.86 mmol) in 40 mL DMF was stir-
red at room temperature for 10 h. The reaction mixture was poured
into 20 mL water, extracted with chloroform (2 ꢁ 75) mL. The chlo-
roform extract was collected, dried over anhydrous sodium sulfate.
The dried solution was evaporated to dryness, crystallized from
ethanol/chloroform affording 0.42 g white powder, mp: 260–
262 °C, 70% yield; IR (KBr) cmꢀ1: 3445, 3025, 2845, 1724, 1610,
1247, 1208, 798, 746; 1H NMR 200 MHz (DMSO-d6): d 1.34 (t,
3H, J = 7 Hz, –CH2CH3); 2.42–2.66 (m, 8H, -cyclopentyl 8H); 3.1–
4.1.5.1. 1-Ethyl-6-fluoro-1,4-dihydro-7-(4-(morpholinomethyl)-
piperazin-1-yl)-4-oxoquinoline-3-carboxylic acid (4a).
Com-
pound 4a was prepared using the above general procedure in 63%
yield, crystallization solvent: DMF; mp = 287–288 °C; IR (KBr) cmꢀ1
:
3410, 3075, 2910, 1712, 1615, 1251, 1194, 818, 744; 1H NMR
300 MHz (DMSO-d6) d 1.42 (t, 3H, J = 6.9 Hz, –CH2CH3); 3.13–3.53
(m, 16H, piperazine 8H and morpholine 8H); 4.63(q, 2H, J = 6.9 Hz,
O
F
COOH
N
N
CH2CH3
N
R
Neutral form
O
N
O
F
COOH
F
COO
N
N
N
N
H
CH2CH3
CH2CH3
R
N
R
O
N
F
COO
N
N
H
CH2CH3
R
Zwitterion form
Scheme 3. The possible protonation states of norfloxacin derivatives.

G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
3885
dure in 49.5% yield; crystallization solvent: methanol/chloroform;
mp = 266–267 °C (dec.); IR (KBr) cmꢀ1: 3430, 3025, 2840, 1756,
1689, 1616, 1251, 1206, 797, 742; 1H NMR spectrum (CDCl3)
200 MHz, 1.60 (t, 3H, J = 6.9 Hz, –N–CH2CH3); 2.85 (s, 4H, –pyrroli-
din H); 2.9 (4H, piperazine H); 3.35 (m, 4H, – piperazine H); 4.35
(q, 2H, J = 7.20 Hz, N–CH2CH3); 4.65 (s, 2H, –N–CH2N–); 6.80 (d,
1H, JH–F = 7.20 Hz, -H-8); 8.05 (d, 1H, JH–F = 13.8 Hz, H-5); 8.65 (s,
1H, H-2); 15.10 (br s, 1H, –COOH). FAB-MS m/z MH+ 431.25
(3.00%) Anal. Calcd for C21H23FN4O5 (430.17): C, 58.60; H, 5.39;
N, 13.02. Found: C, 58.60; H, 5.40; N, 12.98.
Ps. aeur.
y = -0.387x4 + 1.7188x3 - 3.4619x2 + 6.9185x - 3.2724
R2 = 0.9487
6
5
4
3
2
1
0
4.1.5.5. 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-{4-[(1,3-diox-
oisoindolin-2-yl)methyl]piperazin-1-yl}quinoline-3-carboxylic
acid (4e). Compound 4e was prepared as white powder using
0
0.5
1
1.5
2
2.5
3
the above general procedure in 52.70% yield; crystallization sol-
vent: methanol/chloroform; mp = 256–257 °C (dec.); IR (KBr) cm
ꢀ1: 3450, 3030, 2881, 1761, 1702, 1611, 1245, 1203, 797, 732; 1H
NMR spectrum (CDCl3) 200 MHz, 1.57 (t, 3H, J = 6.9 Hz, –CH2CH3);
2.93 (br s, 4H, -piperazine H); 3.35 (br s, 4H, -piperazine H); 4.31
(q, 2H, J = 7.2 Hz, N–CH2CH3); 4.71 (s, 2H, –N–CH2N–); 6.81(d, 1H,
JH–F = 6.6 Hz, -H-8); 7.75–7.9 (m, 4H, benzene H); 7.97 (d, 1H, JH–
F = 12.9 Hz, H-5); 8.63 (s, 1H, H-2). EI-MS m/z MꢀH+ 477.00
(1.40%). Anal. Calcd for C25H23FN4O5 (478.17): C, 62.76; H, 4.85;
N, 11.71. Found: C, 62.34; H, 4.84; N, 11.70.
Calc. log P
Figure 2. Correlation between calc. log P and log MIC.
Table 4
Correlation coefficient (r2) between log MIC of the tested strains and log P, log D,
molecular mass and N-4-charge
Microorganism
Correlation parameters
Molecular mass
Log P
Log D
N-4-Charge
4.1.5.6. Preparation of 7-[4-(1,3-dioxo-2,3-dihydro-1H-2-phe-
nalenyl) piperazino]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-3-quin-
olinecarboxylic acid (4f). Compound 4f was prepared as white
P. aeurginosa
E. coli
K. pneumonia
S. aureus
0.948
0.67
0.842
0.616
0.246
0.539
0.42
0.529
0.626
0.197
0.677
0.580
0.155
0.582
0.249
0.378
powder using the above general procedure in 40% yield; crystalliza-
tion solvent: DMF; mp = 287–288 °C (dec.); IR (KBr) cmꢀ1: 3420,
3030, 2819, 1797, 1688, 1616, 1240, 1194, 796, 770, 691; 1H NMR
spectrum (DMSO-d6) 60 MHz, 1.60 (t, 3H, J = Hz, –CH2CH3); 2.9–4.0
(m, 8H, -piperazine H); 4.9 (q, 2H, J = Hz, N–CH2CH3); 5.3 (s,
2H, –N–CH2N–); 8.3–9.5 (m, 8H, 6H of benzene; H-5- and H-2 of
the fluoroquinolone ring); 13.2 (br s, 1H, –COOH, exchangeable with
D2O). EI-MS m/z MꢀH+ 527.00 (2.60%). Anal. Calcd for C29H25FN4O5
(528.18): C, 65.90; H, 4.77; N, 10.60. Found: C, 65.65; H, 4.77; N,
11.00.
N–CH2CH3);5.31(brs,2H, –N–CH2N–);7.25(d, 1H, JH–F = 7.5 Hz, H-8);
7.94 (d, 1H, JH–F = 13.2 Hz, H-5); 8.95 (s, 1H, H-2). 15.23 (br s, 1H,
COOH). Anal. Calcd for C21H27FN5O4 (418.20): C, 60.27; H, 6.50; N,
13.39. Found: C, 60.20; H, 6.09; N, 13.48.
4.1.5.2. 7-{4-[(1H-Benzo[d]imidazol-1-yl)methyl]piperazin-1-
yl}-1-ethyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (4b). Compound 4b was prepared using the above general
procedure in 76% yield; crystallization solvent: methanol/chloro-
form; mp = 262–264 °C (dec.); IR (KBr) cmꢀ1: 3395, 3030, 2880,
1725, 1609, 1249, 1197, 796, 744; 1H NMR 200 MHz (CDCl3) d
1.58 (t, 3H, J = 7.0 Hz, –CH2CH3); 2.90 (m, 4H, piperazine H);
3.34 (m, 4H, piperazine H) 4.34 (q, 2H, J = 7.0 Hz, –N–CH2CH3);
4.74 (s, 2H, –N–CH2N–); 6.83 (d, 1H, JH–F = 6.6 Hz, H-8); 7.57–
8.00 (m, 6H, benzimidazol 5H and H-5); 8.65 (s, 1H, H-2), 15.10
(br s, 1H, COOH). Anal. Calcd for C24H24FN5O3 (449.19): C,
64.13; H, 5.38; N, 15.58. Found: C, 64.22; H, 5.22; N, 15.88.
4.2. Antimicrobial screening30,31
Compounds 2, 3a–c and 4a–f were screened for their in vitro
antibacterial activity against P. aeruginosa, E. coli, K. pneumonia
(as Gram-negative bacteria) and S. aureus (as Gram-positive
organism).
Bacterial strains were supplied from department of Microbiol-
ogy, Faculty of Pharmacy, El-Minia University. Suspension of each
microorganism was prepared to 106 colony forming units (CFU/
mL). The tested compounds, as well as norfloxacin (Sigma, USA),
were dissolved in DMF to an initial concentration of 10 mg/mL
and dilutions of the test compounds were prepared at concentra-
4.1.5.3. 1-Ethyl-6-fluoro-1,4-dihydro-7-{4-[(4,5-dihydro-3-
methy-5-oxopyrazol-1-yl)methyl]piperazin-1-yl}-4-oxoquino-
line-3-carboxylic acid (4c). Compound 4c was prepared using
the above general procedure in 59% yield; crystallization solvent:
methanol/chloroform; mp = 255–257 (dec.); IR (KBr) cmꢀ1: 3445,
3030, 2995, 1785, 1714, 1610, 1250, 1207, 800, 757; 1H NMR spec-
trum (CDCl3) 300 MHz, d 1.59 (t, 3H, J = 7.2 Hz, –CH2CH3); 2.80 (s,
3H, –CH3 of the pyrazolone); 2.92 (m, 4H, piperazine H); 3.35 (m,
4H, piperazine H) 3.43 (m, 0.8H, –CH2 of the pyrazolone); 4.2 (s,
1.2H, @CH of the pyrazolone); 4.34 (q, 2H, J = 7.2 Hz, –N–CH2CH3);
4.37 (s, 1.2H, –N–CH2N–); 4.70 (0.8H, –N–CH2N–); 7.18 (d, 1H, JH–
F = 7.0 Hz, -H-8); 7.88 (d, 1H, JH–F = 13.6 Hz, -H-5); 8.94 (s, 1H, H-2).
15.37 (br s, 1H, –COOH). FAB-MS m/z MH++Na 454.30 (2.60%) Anal.
Calcd for C21H24FN5O4 (429.18): C, 58.73; H, 5.63; N, 16.31. Found:
C, 58.70; H, 5.68; N, 16.32.
tions 512, 128, 64, 16, 4 lg/mL.
4.2.1. Preparation of the inoculated media32
All strains were cultured on Muller-Hinton agar medium which
was supplied from Oxoid Chemical Co. UK, and prepared according
to the instructions of the manufacturers. The media were molten
on a water bath, inoculated with 0.5 mL of the culture of the spe-
cific microorganism and poured into sterile Petri dishes to form a
layer of about 3–4 mm thickness. The layer was allowed to cool
and harden. With the aid of cork-porer, cups of about 10 mm diam-
eter were done.
4.2.2. Agar diffusion technique31
Different concentrations of the tested compounds in DMF were
placed separately in cups in the agar medium. All plates were
4.1.5.4. 7-{4-[(2,5-Dioxopyrrolidin-1-yl)methyl]piperazin-1-yl}-
1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(4d). Compound 4d was prepared using the above general proce-

3886
G. A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 17 (2009) 3879–3886
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after 24 h. The minimum inhibitory concentration (MIC) was de-
fined to be the intercept of the grave of logarithm concentrations
versus diameter of the inhibition zones.
4.3. Determination of partition coefficient23,33
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The experimental partition coefficient (D) between n-octanol
and phosphate buffer was determined by a slight modification of
the method earlier described by Vazquez et al.23 Briefly, 100
lL
of a stock solution (0.2 mg/mL) was diluted with 1.9 mL of appro-
priate phosphate buffer solution (pH 7.4) and mixed with 2 mL of
octanol (the organic and aqueous phase was mutually saturated),
the vials were protected from light by wrapping in aluminum foil.
The two phases were vortexed for 3 min and agitated for 5 h in a
shaking water bath at 25 + 0.1 °C. After equilibration, the octanol
phase was removed with a Pasteur pipette and both phases were
assayed spectrophotometrically (k = 270–280 nm) to determine
drug concentration. The partition coefficient was calculated as
the ratio between molar concentration in n-octanol and aqueous
phase. The total concentration in both phases was measured by
spectrophotometry and the experimental partition coefficients
(D) were determined from:
16. Bermejo, B.; Merino, V.; Garrigues, T. M.; Pla Delfina, J. M.; Mulet, A.; Vizet, P.;
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Bermejo Sanz, M. Eur. J. Pharm. Biopharm. 2002, 53, 317.
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Sutherland, J. B. Appl. Microbiol. Biotechnol. 2001, 56, 474.
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29. Furniss, B. S.; Hannaford, A. J. O.; Smith, P. W. G.; Tatchell, A. R. Vogel’s
D ¼ Ci ꢀ Cw=Cw ꢂ Vw=Vo
where Ci and Cw represent the solute concentration in the aqueous
phase before and after distribution, respectively; Vw represents the
volume of the aqueous and Vo the volume of the organic phase. All
partition coefficient determinations were made in triplicate. Spec-
tronic Genesys, connected to an IBM computer loaded with the
Winspec application software (Milton Roy, USA) and Jenway 6505
(Jenway LTD., UK) ultraviolet–visible spectrophotometers with
matched 1 cm quartz cells were used throughout this study for all
measurements. The calcd log P was calculated by ACD/Labs (TM)
Software for MS Windows (TM). Copyright (C) 1993–1997 Ad-
vanced Chemistry Development, Inc. The calculation of charge den-
Textbook of Practical Organic Chemistry, 5th ed.; Longmann Scientific
Technical: UK, 1989. p 807.
&
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New York, 2000. p 114.
sity was carried out using CHEM
3D pro 10 program.
31. National Committee for Clinical Laboratory Standards (NCCLS) (2005):
Performance standards for antimicrobial susceptibility tests, Wayne, Pa.
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Tests. In Manual of Clinical Microbiology, 7th ed.; Muarry, P. R.; Baron, E. J.;
Yolken, R. C., Eds.; American Society for Microbiology: Washington, DC, USA,
1999, pp 1526–1543.
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