Mannich bases of 7-piperazinylquinolones and kojic acid derivatives: Synthesis, in vitro antibacterial activity and in silico study

Article abstract of DOI:10.1016/j.ejmech.2013.07.032

Novel Mannich bases of 7-piperazinylquinolones with kojic acid and chlorokojic acid were designed as new quinolone antibacterials. All compounds showed significant in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria. Particularly, chlorokojic derivative 2b was the most potent compound against Staphylococcus aureus and Pseudomonas aeruginosa (MIC values ≤0.19 μg/mL). Its activity was 4-8 times more than that of standard drug norfloxacin. The molecular docking study of compound 2b further supported the molecular basis of the designed compounds.

Full text of DOI:10.1016/j.ejmech.2013.07.032

European Journal of Medicinal Chemistry 68 (2013) 185e191
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Mannich bases of 7-piperazinylquinolones and kojic acid derivatives:
Synthesis, in vitro antibacterial activity and in silico study
,
b
c
d
Saeed Emami a , Ebrahim Ghafouri , Mohammad Ali Faramarzi , Nasrin Samadi ,
*
Hamid Irannejad a, Alireza Foroumadi e
a Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences,
Sari, Iran
b Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
c Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran
d Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Center, Tehran University of Medical Sciences,
Tehran, Iran
e Drug Design & Development Research Center, Tehran University of Medicinal Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Mannich bases of 7-piperazinylquinolones with kojic acid and chlorokojic acid were designed as
new quinolone antibacterials. All compounds showed significant in vitro antibacterial activity against
both Gram-positive and Gram-negative bacteria. Particularly, chlorokojic derivative 2b was the most
Received 23 May 2013
Received in revised form
15 July 2013
Accepted 18 July 2013
Available online 11 August 2013
potent compound against Staphylococcus aureus and Pseudomonas aeruginosa (MIC values ꢀ0.19
mg/mL).
Its activity was 4e8 times more than that of standard drug norfloxacin. The molecular docking study of
compound 2b further supported the molecular basis of the designed compounds.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Antibacterial activity
Quinolones
Kojic acid
Mannich bases
Docking study
1. Introduction
Quinolone antibacterial agents act by inhibiting two type II
bacterial topoisomerase enzymes, DNA gyrase and topoisomerase
Quinolones have emerged as a major class of chemotherapeutic
agents widely used to treat Gram-negative and Gram-positive
bacterial infections in both community and hospital settings [1].
Most of them currently in the market or under development are
generally characterized by a broad spectrum of antibacterial ac-
tivity, improved potency and excellent oral bioavailability [2].
However, their activity against clinically important Gram-positive
cocci including Staphylococci, Streptococci and Enterococci is
relatively moderate [3]. Moreover, the prevalence of newly
emerging virulence traits and drug resistance of the pathogens
toward the quinolones has become a serious problem [4]. Thus, the
design and development of new agents are of critical importance in
the field of antimicrobial chemotherapy.
IV [5]. Type II bacterial topoisomerase enzymes control the topol-
ogy and conformation of DNA during replication and transcription
processes [6]. DNA gyrase is responsible for introducing negative
supercoils into DNA in an ATP-dependent manner, while topo-
isomerase IV provides a potent decatenating activity and contrib-
uting to replication fork progression by relaxing positive supercoils
[7,8]. The inhibition of these essential enzymes by quinolones re-
sults in the disruption of DNA synthesis and, subsequently cell
death.
Structurally, the b-keto carboxylic acid part in the 1-alkyl-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid skeleton of quinolones
is essential for hydrogen bonding interaction with DNA bases in the
single stranded region of double helix of DNA created by the action
of the topoisomerase II enzymes [9]. It has been proposed that the
basic group at the C-7 position of quinolones is might interact with
the target enzyme [10e12]. In addition, structureeactivity rela-
tionship studies of quinolones have shown that the cell perme-
ability is dominantly controlled by C-7 side chains. Therefore, the
* Corresponding author. Tel.: þ98 151 3543082; fax: þ98 151 3543084.
E-mail address: sd_emami@yahoo.com (S. Emami).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.032

186
S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
O
O
type of substituents at the C-7 position has determinative effects on
their nature including antibacterial spectrum, potency, safety, and
pharmacokinetics properties [3]. In general, 5- and 6-membered
cyclic amines connected to the quinolone cycle by a CeN bond
have been proven to be the optimal substituents at the C-7 position
[13]. Numerous clinically important quinolones exemplified by
norfloxacin, ciprofloxacin, enoxacin, lomefloxacin and gatifloxacin
have piperazine substituent at C-7 position (Fig. 1). The previous
studies revealed that the C-7 position of quinolones is capable to
attach to the special bulky substituents [14e20].
Kojic acid is an antibiotic produced by some species of Asper-
gillus and Penicillium [21] and shows an inhibitory effect on the
growth of Escherichia coli and Staphylococcus aureus [22,23]. Also,
Wolf and Westveer have reported that chlorokojic acid (2-
chloromethyl-5-hydroxy-4H-pyran-4-one) have antimicrobial ac-
tivity [24]. Recently, Mannich bases of substituted piperazine and
chlorokojic acid (Fig. 2) have been described as antimicrobial
agents with significant activity against Gram-positive and Gram-
negative bacteria [25,26].
O
O
O
OH
R
OH
OH
N
Cl
HO
N
Cl
O
Kojic acid
Mannich bases of
chlorokojic acid
Chlorokojic acid
O
F
COOH
O
O
OH
N
X
N
N
Y
R1
R2
Designed compounds 2a-h
X=CH, COMe, N
R1 = cyclopropyl, ethyl
R
2 = H, Me
Y = OH, Cl
In our effort to find new N-substituted piperazinylquinolones,
we designed Mannich bases of 7-piperazinylquinolones with kojic
acid and chlorokojic acid (Fig. 2). Thus we report here, synthesis,
antibacterial activity evaluation and docking study of 7-[4-((3-
hydroxy-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]quinolone
derivatives 2aeh.
Fig. 2. Structures of kojic acid, chlorokojic acid and its Mannich bases with some
antimicrobial activity, and designed Mannich bases of 7-piperazinylquinolones with
kojic acid and chlorokojic acid as new antibacterial agents.
ring. The alcoholic hydroxyl group was appeared at 5.70 ppm as a
triplet due to the neighboring with methylene group. A doublet
signal at 4.32 ppm was assigned for methylene connected to the C-6
of pyran ring and hydroxyl group. The resonance of C-1 proton of
cyclopropyl was occurred at 3.79e3.82 ppm. Remaining protons
related to the methylene groups of piperazine and cyclopropyl rings
were appeared at the upfield of 3.34, 2.69 and 1.15e1.34 ppm,
respectively.
2. Chemistry
The title compounds 2aeh were synthesized starting from 7-
piperazinylquinolones 1aee and kojic acid derivatives as outlined
in Scheme 1. Kojic acid was converted to chlorokojic acid by using
thionyl chloride as reported in literature [27]. The Mannich bases
2aeh (Table 1) were prepared by the reaction of appropriate 7-
piperazinylquinolone with kojic acid or chlorokojic acid and
formalin in methanol at room temperature. The structures of target
compounds were confirmed by IR and NMR spectral data. For
example, the 1H NMR of compound 2a showed a singlet at
8.65 ppm which assigned for H-2 proton of quinolone ring. The H-5
and H-8 protons of quinolone ring displayed two doublets at 7.88
and 7.55 ppm with coupling constants of 13.6 and 7.6 Hz, respec-
tively. The spinespin splitting of H-5 and H-8 protons were due to
the presence of fluorine atom at C-6. The proton of pyran ring at C-5
was displayed at 6.34 ppm as a singlet peak. The broad singlet
signal at 9.08 ppm was related to the hydroxyl group at C-3 of pyran
3. Biology
The antibacterial activities of synthesized Mannich bases 2ae
h were evaluated by determination of their minimum inhibitory
concentrations (MICs) by using agar dilution method [28,29]. A
panel of Gram-positive (S. aureus ATCC 6538, Staphylococcus
epidermidis ATCC 12228, Bacillus subtilis ATCC 6633) and Gram-
negative (E. coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027,
Klebsiella pneumonia ATCC 10031) bacteria was used. The qui-
nolone antibacterial agent norfloxacin was used as reference
drug.
O
O
O
N
F
COOH
F
COOH
F
COOH
N
N
N
N
N
N
HN
HN
F
HN
CH3
CH3
Lomefloxacin (1c)
CH3
CH3
Norfloxacin (1a)
Enoxacin (1b)
O
N
O
N
F
COOH
F
COOH
N
N
OCH3
HN
HN
CH3
Ciprofloxacin (1d)
Gatifloxacin (1e)
Fig. 1. Structures of some 7-piperazinylquinolone antibacterial drugs.

S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
187
O
O
OH
SOCl2
OH
HO
Cl
Chlorokojic acid
O
O
Kojic acid
O
O
O
O
F
COOH
OH
F
COOH
O
O
Y
OH
N
X
N
N
X
N
N
Y
R1
HCHO
HN
R1
R2
2a-h
R2
Piperazinylquinolones 1a-e
X=CH, COMe, N
R1 = cyclopropyl, ethyl
R2 = H, Me
Y = OH, Cl
Scheme 1. Synthesis of designed compounds 2aeh.
4. Docking study
Augustin, Germany) was used for docking calculations. This pro-
gram considers ligand flexibility by changing the conformation of
the ligand in the active site while making the protein rigid. After
docking calculation, the best pose with the highest rank was
considered for visualization and further evaluation of the in-
teractions. 2D map of the ligand-active site complex was made by
LigPlot 1.4.4 [30].
Topoisomerase II DNAegyrase atomic coordinates in complex
with its bound inhibitor ciprofloxacin (PDB code: 2XCT) was
retrieved from Brookhaven Protein Data Bank. To set the initial
coordinates for the docking simulation, all water molecules, DNA
fragments and three subunits of enzyme were removed and
excluded from all calculations. The remaining subunit with its
Mn2þ cofactor was considered for the following calculations. Ligand
structure was sketched in HyperChem and optimized using semi-
empirical AM1 method in Gaussian98. The resulting minimized
conformation was then submitted to molecular docking simulation.
The lysine and arginine residues located in the binding site were
protonated and the side chain of acidic amino acids, aspartate and
glutamate, considered as carboxylate ion prior to any docking cal-
culations. Gasteiger charges were assigned to the ligand and whole
protein atoms. The binding site radius of the enzyme was set at any
residues 12 Ǻ distant from the X-ray crystallographic bound in-
hibitor (ciprofloxacin). LeadIT 2.0.2 (BiosolveIT GmbH, Sankt
5. Results and discussion
5.1. Antibacterial activity
The MIC values of compounds 2aeh in comparison with nor-
floxacin were presented in Table 2. At a glance, all compounds
showed significant antibacterial activity against both Gram-
positive and Gram-negative bacteria. Compounds 2b, 2c and 2d
with MIC values ꢀ0.78
against S. aureus. Compound 2b was the most potent compound
g/mL). Its activity was 4-fold
mg/mL exhibited potent inhibitory activity
against this strain (MIC ¼ 0.097
m
more than that of standard drug norfloxacin. Moreover, com-
pound 2b showed the highest growth inhibitory activity against
S. epidermidis, and B. subtilis, being as potent as norfloxacin. The
MIC values of test compounds against E. coli were in the range of
Table 1
Chemical structures of synthesized compounds 2aeh.
O
0.39e1.56
2aeh was less than norfloxacin. In the case of P. aeruginosa, com-
pound 2b with MIC value of 0.19 g/mL was the most potent
mg/mL. The susceptibility of E. coli toward compounds
F
COOH
O
O
m
OH
N
X
N
compounds, being 8 times more potent than norfloxacin. In addi-
tion, the activity of compounds 2a, 2c, 2d and 2f against
P. aeruginosa were comparable or superior to that of norfloxacin.
The MIC values of test compounds against K. pneumonia revealed
that compounds 2a, 2ceg showed relatively same inhibitory ac-
tivity while compound 2h was less active respect to the rest of
compounds. The activity of the most potent compound 2b
N
Y
R1
R2
Compound
X
Y
R1
R2
MW
Yield (%)
2a
2b
2c
2d
2e
2f
CH
CH
COMe
COMe
CH
OH
Cl
OH
Cl
OH
Cl
OH
OH
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Ethyl
Ethyl
Ethyl
Ethyl
H
H
CH3
CH3
H
H
CH3
H
485.46
503.91
529.51
547.96
473.45
491.90
505.47
474.44
83
77
76
82
78
20
44
90
(MIC ¼ 0.19
mg/mL) was comparable to that of norfloxacin against
the latter Gram-negative microorganism.
The comparison of the MIC values of kojic acid and chlorokojic
acid derivatives against different microorganisms revealed that in
the most cases chlorokojic acid derivatives were more active than
corresponding kojic acid analogues. The naphthyridine derivative
2h was less potent than its corresponding quinolone congener 2e.
CH
2g
2h
CF
N

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S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
mg/mL) of compounds 2aeh against different bacteria.
Table 2
The minimum inhibitory concentrations (MICs,
Compound
Staphylococcus aureus
Staphylococcus epidermidis
Bacillus subtilis
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
2a
2b
2c
2d
2e
2f
2g
2h
1.56
0.097
0.78
0.39
3.125
1.56
3.125
6.25
0.39
3.125
0.39
3.125
1.56
6.25
1.56
6.25
12.5
0.39
3.125
0.39
3.125
1.56
0.78
1.56
3.125
12.5
0.195
0.78
0.78
0.39
1.56
0.78
0.39
0.39
1.56
0.195
1.56
0.195
0.78
0.78
3.125
1.56
3.125
12.5
1.56
1.56
0.195
1.56
1.56
1.56
0.78
1.56
12.5
0.39
Norfloxacin
Thus, replacement of CH at 8-position with N decreased the anti-
bacterial activity against both Gram-positive and Gram-negative
bacteria. By comparing the antibacterial activity of cyclopropyl
derivative 2b and related N1-ethyl compound 2f, it is revealed that
the cyclopropyl is more favorable group at N1-position. The intro-
duction of the methoxy group at the C-8 position of quinolone and
methyl at the piperazine ring could not improve the antibacterial
activity (2c vs. 2a or 2d vs. 2b). Similarly, the comparison of com-
pounds 2e and 2g demonstrated that substitution of fluorine at C-8
and methyl at the 3-position of piperazine ring had no positive
effect on activity.
conjugate compounds showed significant antibacterial activity
against both Gram-positive and Gram-negative bacteria. Particu-
larly, compound 2b with MIC value of 0.097 mg/mL was the most
potent compound against S. aureus. Its activity was 4-fold more
5.2. Docking simulation
In order to evaluate the accuracy of our docking protocol, cip-
rofloxacin was docked into the topoisomerase II DNAegyrase
active site. The protein-inhibitor complex obtained from docking
simulation showed that the docked ciprofloxacin was almost
superimposed on the native co-crystallized one with RMSD being
0.862. The hydrogen bonds and interactions between the docked
ciprofloxacin and the amino acids were the same as those between
the native X-ray ciprofloxacin and the amino acids (Fig. 3A). The
docked ciprofloxacin forms coordination bond with Mn2þ ion by
the two oxygen atoms of carbonyl and carboxylate groups with
1.4 Ǻ and 2 Ǻ distance respectively while for the co-crystallized
ciprofloxacin, 2.1 Ǻ distance exists between the two oxygen
atoms of carbonyl and carboxylate groups and the Mn2þ ion.
Carboxylate group of the docked ciprofloxacin is hydrogen bonded
to the hydroxyl group of Ser1084 and the nitrogen atom of
piperazine ring forms hydrogen bonding with the side chain
carboxylate group of Glu477.
Compound 2b which is the most active compound exhibits very
similar mode of binding compared to the both co-crystallized and
docked ciprofloxacin. The carboxylate group of compound 2b is
hydrogen bonded to hydroxyl group of Ser1084 and NH2 group of
Ser1085. The 3-hydroxy group of pyrane-4-one ring makes
hydrogen bond with side chain NH2 group of Asn475. In addition,
the carbonyl oxygen atom of pyran-4-one is hydrogen bonded to
the amide nitrogen atom of Glu477. The carbonyl oxygen atom at
the 4-position of quinolone is coordinated to the Mn2þ with 1.6 Ǻ
distance (Fig. 3B). The interactions and distances can be better
visually inspected by the 2D map of compound 2b in the binding
site of topoisomerase II DNA gyrase in Fig. 4. Overall, compound 2b
can be docked in the binding site of enzyme in a similar manner to
ciprofloxacin with additional hydrogen bonds related to the 3-
hydroxy-pyran-4-one scaffold, supporting the molecular design of
compound 2b prototype.
6. Conclusion
Fig. 3. (A) Docked conformation of ciprofloxacin (ball and stick) and the co-crystallized
one (wire) in the active site of topoisomerase II DNAegyrase; (B) Binding mode of
compound 2b in the binding site of topoisomerase II DNAegyrase. For clarity only
amino acids within 8 Ǻ distant from the docked ligand are shown.
We designed Mannich bases of 7-piperazinylquinolones with
kojic acid and chlorokojic acid as new quinolone antibacterials. All

S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
189
acid according to the literature method [27]. The progress of re-
actions was checked by thin-layer chromatography (TLC) using pre-
coated silica gel 60 F254 plastic sheets. The UV lamp (254 nm) was
used for TLC visualization and detection of spots. Melting points
were determined in open glass capillaries using Bibby Stuart Sci-
entific SMP3 apparatus (Stuart Scientific, Stone, UK) and are un-
corrected. The IR spectra were recorded on a PerkinElmer FT-IR
spectrophotometer using KBr disks. The NMR spectra were recor-
ded using Bruker 400 or 500 spectrometers and chemical shifts are
expressed as
standard.
d (ppm) with tetramethylsilane (TMS) as internal
7.1.2. General procedure for the synthesis of Mannich bases 2aeh
To a mixture of 7-piperazinylquinolone 1aee (0.5 mmol) in
methanol (4 mL), 37% formalin (0.05 mL) and kojic acid or chlor-
okojic acid (0.5 mmol) were added, respectively. The mixture was
stirred at room temperature for 48e72 h. The resulting precipitated
solid was collected by filtration and washed with cold methanol.
The product was recrystallized from methanol to give pure com-
pounds 2aeh.
7.1.2.1. 1-cyclopropyl-6-fluoro-1,4-dihydro-7-[4-((3-hydroxy-6-
hydroxymethyl-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (2a). Yield 83%; mp 206e207 ꢁC; IR
)
(KBr, cmꢂ1 nmax: 3372 (OeH), 1724 (C]O), 1630 (C]O); 1H NMR
(400 MHz, DMSO-d6)
d
1.15e1.34 (m, 4H, cyclopropyl), 2.69 (br s,
4H, piperazine), 3.34 (br s, 4H, piperazine), 3.62 (s, 2H, CH2N),
3.79e3.82 (m, 1H, cyclopropyl), 4.32 (d, 2H, J ¼ 5.6 Hz, CH2-OH),
5.70 (t, 1H, J ¼ 6.0 Hz, CH2-OH), 6.34 (s, 1H, H5epyran), 7.55 (d, 1H,
JH,F ¼ 7.6 Hz, H8equinolone), 7.88 (d, 1H, JH,F ¼ 13.6 Hz, H5equi-
nolone), 8.65 (s, 1H, H2equinolone), 9.08 (br s, 1H, 3-OH pyran).
Anal. Calcd for C24H24FN3O7: C, 59.38; H, 4.98; N, 8.66. Found: C,
59.22; H, 5.05; N, 8.81.
7.1.2.2. 1-cyclopropyl-6-fluoro-1,4-dihydro-7-[4-((3-hydroxy-6-
chloromethyl-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (2b). Yield 77%; mp 209e210 ꢁC; IR
(KBr, cmꢂ1
)
nmax: 3421 (OeH), 1730 (C]O), 1650 (C]O); 1H
NMR (400 MHz, DMSO-d6)
d
1.15e1.34 (m, 4H, cyclopropyl), 2.71
(br s, 4H, piperazine), 3.34 (br s, 4H, piperazine), 3.68 (s, 2H,
CH2N), 3.80e3.83 (m, 1H, cyclopropyl), 4.68 (s, 2H, CH2Cl),
6.58 (s, 1H, H5epyran), 7.56 (d, 1H, JH,F ¼ 7.6 Hz, H8equinolone),
7.89 (d, 1H, JH,F ¼ 13.2 Hz, H5equinolone), 8.66 (s, 1H, H2equi-
nolone), 9.34 (br s, 1H, 3-OH pyran). Anal. Calcd for
C
24H23ClFN3O6: C, 57.20; H, 4.60; N, 8.34. Found: C, 57.14; H,
Fig. 4. 2D map of compound 2b in the binding site of topoisomerase II DNAegyrase.
Distances are in angstrom.
4.42; N, 8.55.
7.1.2.3. 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[3-methyl-
4-((3-hydroxy-6-hydroxymethyl-4-oxo-4H-pyran-2-yl)methyl)piper-
azin-1-yl]-4-oxoquinoline-3-carboxylic acid (2c). Yield 76%; mp
than that of standard drug norfloxacin. Moreover, compound 2b
showed potent activity against P. aeruginosa as important Gram-
190e191 ꢁC; IR (KBr, cmꢂ1
)
nmax: 3421 (OeH), 1732 (C]O), 1623
negative bacteria (MIC ¼ 0.19
m
g/mL). It was 8 times more
(C]O); 1H NMR (500 MHz, DMSO-d6)
d
0.9e1.12 (m, 4H, cyclo-
potent than norfloxacin. The molecular docking study of com-
pound 2b further supported the molecular basis of our design by
introducing 3-hydroxy-pyran-4-one scaffold into the7-pi
perazinylquinolones.
propyl), 1.12e1.3 (m, 3H, CH3epiperazine), 2.5e2.64 (m, 2H,
piperazine), 2.85e2.90 (m, 1H, piperazine), 2.92e3.03 (m, 1H,
piperazine), 3.66e3.69 (m, 1H, piperazine), 3.72 (s, 3H, OCH3),
3.80e3.86 (m,1H, piperazine), 4.15 (br s,1H, cyclopropyl), 4.31 (br s,
2H, CH2OH), 5.70 (br s,1H, CH2OH), 6.33 (s,1H, H5epyran), 7.2 (d, JH,
¼ 11.75 Hz, H5equinolone), 8.68 (s, 1H, H2equinolone), 9.09 (br
F
7. Experimental protocols
s, 1H, 3-OH pyran), 14.93 (br s, 1H, COOH). Anal. Calcd for
C26H28FN3O8: C, 58.97; H, 5.33; N, 7.94. Found: C, 59.11; H, 5.28;
7.1. Chemistry
N, 8.26.
7.1.1. General methods
All starting materials, reagents and solvents were purchased
from Merck Company. Chlorokojic acid was prepared from kojic
7.1.2.4. 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[3-methyl-
4-((3-hydroxy-6-chloromethyl-4-oxo-4H-pyran-2-yl)methyl)piper-
azin-1-yl]-4-oxoquinoline-3-carboxylic acid (2d). Yield 82%; mp

190
S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
186e187 ꢁC; IR (KBr, cmꢂ1
)
nmax: 3424 (OeH), 1723 (C]O), 1622
0.96e1.14 (m, 4H, cyclo-
7.2. Agar dilution method for MICs determination
(C]O); 1H NMR (500 MHz, DMSO-d6),
d
propyl), 1.17 (d, 3H, J ¼ 6.1 Hz, CH3epiperazine), 2.60e2.70 (m, 2H,
piperazine), 2.89 (d, 1H, J ¼ 11.4 Hz, piperazine), 2.98 (t, 1H,
piperazine), 3.16 (br s, 1H, piperazine), 3.77 (d, J ¼ 14.7 Hz, 1H,
piperazine), 3.87 (d, J ¼ 14.6 Hz, 1H, piperazine), 3.72 (s, 3H, OCH3),
4.15 (m, 1H, cyclopropyl), 4.68 (s, 2H, CH2Cl), 6.57 (s, 1H, H5epyran),
7.72 (d, 1H, JH,F ¼ 11.95 Hz, H5equinolone), 8.68 (s, 1H, H2equino-
lone), 9.3 (br s, 1H, 3-OH pyran), 14.96 (s, 1H, COOH). Anal. Calcd for
The antibacterial activity of compounds 2aeh were determined
against Gram-positive (S. aureus ATCC 6538, S. epidermidis ATCC
12228, B. subtilis ATCC 6633) and Gram-negative (E. coli ATCC 8739,
P. aeruginosa ATCC 9027, Klebsiella pneumoniae ATCC 10031) bac-
teria. The minimum inhibitory concentrations (MICs) of com-
pounds were determined by the conventional agar dilution method
[31]. For preparation of stock solution, the test compounds
(10.0 mg) were dissolved in DMSO (1 mL) and then diluted with
distilled water (9 mL). Further progressive two-fold serial dilution
with molten sterile MuellereHinton agar was performed to obtain
the required concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56,
C
26H27ClFN3O7: C, 56.99; H, 4.97; N, 7.67. Found: C, 56.70; H, 5.17;
N, 7.66.
7.1.2.5. 1-ethyl-6-fluoro-1,4-dihydro-7-[4-((3-hydroxy-6-
hydroxymethyl-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (2e). Yield 78%; mp 232e234 ꢁC; IR
0.78, 0.39, 0.19, 0.098, 0.049, and 0.025 mg/mL. The medium con-
taining the test compounds was dispensed into a sterile Petri-dish
and allowed to solidify. Petri-dishes were inoculated with 1e
5 ꢃ 104 CFU and incubated at 37 ꢁC for 18 h. The MIC was defined as
the lowest concentration of the test compound, which resulted in
no visible growth.
(KBr, cmꢂ1
)
nmax: 3430 (OeH), 1722 (C]O), 1630 (C]O); 1H NMR
(500 MHz, DMSO-d6),
d
1.40 (m, 3H, CH3), 2.67 (m, 4H, pipera-
zine), 3.62 (s, 2H, CH2N), 4.31 (m, 2H, CH2OH), 4.55e4.65 (m, 2H,
CH2CH3), 5.69 (br s, 1H, CH2OH), 6.34 (s, 1H, H5epyran), 7.16 (d,
1H, J ¼ 5.85 Hz, H8equinolone), 7.90 (d, JH,F ¼ 13.0 Hz, H5equi-
nolone), 8.95 (s, 1H, H2equinolone), 9.07 (br s, 1H, 3-OH pyran),
Acknowledgments
15.35 (s, 1H, COOH). 13C NMR (125 MHz, DMSO-d6)
d 14.20, 49.02,
49.39, 52.05, 53.37, 59.56, 105.89, 107.01, 108.93, 111.11 (d,
JC,F ¼ 22.7 Hz), 137.12, 143.75, 146.08, 146.26, 148.48, 152.9 (d,
JC,F ¼ 241 Hz), 166.08, 167.62, 173.57, 176.11. Anal. Calcd for
This work was supported by a grant from the Research Council
of Mazandaran University of Medical Sciences, Sari, Iran. A part of
this work was related to the Pharm.D thesis of Ebrahim Ghafouri
(Faculty of Pharmacy, Mazandaran University of Medical Sciences).
C
23H24FN3O7: C, 58.35; H, 5.11; N, 8.88. Found: C, 58.37; H, 5.02;
N, 8.49.
Appendix A. Supplementary data
7.1.2.6. 1-ethyl-6-fluoro-1,4-dihydro-7-[4-((3-hydroxy-6-
chloromethyl-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (2f). Yield 20%; mp 208e209 ꢁC;
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.07.032.
IR (KBr, cmꢂ1
)
nmax: 3430 (OeH), 1738 (C]O), 1652 (C]O), 1626
(C]O); 1H NMR (500 MHz, DMSO-d6),
d
1.41 (m, 3H, CH3), 2.7 (m,
References
4H, piperazine), 3.67 (s, 2H, CH2N), 4.58 (q, 2H, J ¼ 6.3 Hz, CH2CH3),
4.68 (s, 2H, CH2Cl), 6.58 (s, 1H, H5epyran), 7.17 (d, 1H, J ¼ 6.0 Hz,
H8equinolone), 7.91 (d, 1H, JH,F ¼ 13.1 Hz, H5equinolone), 8.95 (s,
1H, H2equinolone), 9.33 (br s, 1H, 3-OH pyran), 15.35 (s, 1H,
COOH). Anal. Calcd for C23H23ClFN3O6: C, 56.16; H, 4.71; N, 8.54.
Found: C, 56.42; H, 4.85; N, 8.43.
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192 ꢁC; IR (KBr, cmꢂ1
)
nmax: 3309 (OeH), 1726 (C]O), 1624 (C]O);
1.14 (d, 3H, CH3-piperazine), 1.42
1H NMR (500 MHz, DMSO-d6),
d
(m, 3H, CH3CH2), 2.59 (m, 3H, piperazine), 2.89 (m, 1H, piperazine),
3.00 (m, 1H, piperazine), 3.68 (d, J ¼ 14.2 Hz, 1H, piperazine), 3.80
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CH2CH3), 5.7 (br s, 1H, CH2OH), 6.33 (s, 1H, H5epyran), 7.82 (d, 1H,
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7.1.2.8. 1-ethyl-6-fluoro-1,4-dihydro-7-[4-((3-hydroxy-6-
hydroxymethyl-4-oxo-4H-pyran-2-yl)methyl)piperazin-1-yl]-4-oxo-
1,8-naphthyridine-3-carboxylic acid (2h). Yield 90%; mp 232e
234 ꢁC; IR (KBr, cmꢂ1
)
nmax: 3261 (OeH), 1711 (C]O), 1634 (C]O);
1.37 (t, 3H, J ¼ 7.0 Hz, CH3CH2), 2.63
1H NMR (400 MHz, DMSO-d6),
d
(t, 4H, J ¼ 4.4 Hz, piperazine), 3.59 (s, 2H, CH2N), 3.81 (t, 4H,
J ¼ 4.4 Hz, piperazine), 4.31 (d, 2H, J ¼ 6 Hz, CH2OH), 4.47 (q, 2H,
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pyran), 8.06 (d, 1H, JH,F ¼ 13.6 Hz, H5equinolone), 8.96 (s, 1H, H2e
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C
22H23FN4O7: C, 55.69; H, 4.89; N, 11.81. Found: C, 55.44; H, 5.02; N,
11.69.

S. Emami et al. / European Journal of Medicinal Chemistry 68 (2013) 185e191
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