Synthesis and in vitro activity of 1,2,4-triazole-ciprofloxacin hybrids against drug-susceptible and drug-resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2012.11.040

A series of novel 1,2,4-triazole-ciprofloxacin hybrids was designed, synthesised and evaluated in vitro against drug-susceptible and drug-resistant bacteria. A significant part of the compounds obtained showed antibacterial activity higher than the activity of ciprofloxacin, both towards Gram-positive and Gram-negative species. Despite relatively small number of synthesised derivatives, it was possible to observe important dependences between their structure and activity.

Full text of DOI:10.1016/j.ejmech.2012.11.040

European Journal of Medicinal Chemistry 60 (2013) 128e134
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and in vitro activity of 1,2,4-triazole-ciprofloxacin hybrids against
drug-susceptible and drug-resistant bacteria
,
a
b
b
c
Tomasz Plech a , Monika Wujec , Urszula Kosikowska , Anna Malm , Barbara Rajtar ,
*
Ma1gorzata Polz-Dacewicz c
Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Chodzki 4a, 20-093 Lublin, Poland
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University, Chodzki 1, 20-093 Lublin, Poland
Department of Virology, Faculty of Medicine, Medical University, Chodzki 1, 20-093 Lublin, Poland
a
ꢀ
b
ꢀ
c
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,2,4-triazole-ciprofloxacin hybrids was designed, synthesised and evaluated in vitro
against drug-susceptible and drug-resistant bacteria. A significant part of the compounds obtained
showed antibacterial activity higher than the activity of ciprofloxacin, both towards Gram-positive and
Gram-negative species. Despite relatively small number of synthesised derivatives, it was possible to
observe important dependences between their structure and activity.
Received 4 September 2012
Received in revised form
22 November 2012
Accepted 26 November 2012
Available online 4 December 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Fluoroquinolones
Mannich reaction
MRSA
Bactericidal effect
1. Introduction
[7,8]. An essential fault of fluoroquinolones is their restricted activity
towards Gram-positive bacteria, in particular Staphylococcus aureus
Ciprofloxacin (CPX) first patented in 1983 and approved for
clinical use in 1987, has ever since been the most frequently used
antibiotic from fluoroquinolone drug group [1]. CPX spectrum
covers both Gram-positive and Gram-negative bacteria, whereas the
activity towards Gram-positive bacteria is considerably weaker [2].
The reasons of the above mentioned differences in activity include:
(a) different affinity towards the enzymes constituting molecular
target, (b) differences in permeability of the drug to the inside of cells
of Gram-positive and Gram-negative bacteria [3]. Generally, the
antibacterial activity of fluoroquinolones is the result of inhibition of
DNA gyrase and topoisomerase IV e enzymes required for DNA
replication process [4]. In case of Gram-negative bacteria, fluo-
roquinolones show significantly stronger affinity to DNA gyrase than
to topoisomerase IV, whereas in Gram-positive bacteria the situation
is opposite [5]. There are also significant differences in transfer of the
antibiotics to bacterial cells. In case of Gram-positive bacteria fluo-
roquinolones permeate to the cell mainly through passive diffusion
[6], whereas they may be transferred to the inside of Gram-negative
bacteria through porin channels or directly through lipid bilayer
strain. The most common and the most rational method of modifi-
cation of fluoroquinolones structure is the introduction of new
substituents in C-7 position [9]. Earlier tests have proven that even
large substituents placed in this position do not have adverse effect
on drug’s permeability through bacterial membranes, and at the
same time they significantly modify the action strength and spec-
trum [2,10]. This led to obtaining, among other, hybrids containing
nitrothiophene [11], nitroimidazole [12], nitrofurane [13], 1,3,4-
thiadiazole [14,15], isatin [16], bromothiophene [17], acyl groups
[18] and other substituents in position C-7 [19e21]. However, it has
been frequently observed that chemical modifications resulting in
the increase of activity, as compared with Gram-positive bacteria,
led to significant decrease of activity, as compared with Gram-
negative bacteria [15,22].
The main purpose of our research was to design and to syn-
thesise derivatives of CPX showing considerable activity both
against Gram-negative and Gram-positive bacteria. In order to
achieve the intended result, we have decided to combine a mole-
cule of CPX with 1,2,4-triazole system. Our tests so far have shown
that disubstituted derivatives of 1,2,4-triazole demonstrate anti-
bacterial effect only on Gram-positive bacteria [23]. Preliminary
results (unpublished data) indicate also that the derivatives of the
kind do not show affinity to DNA gyrase and topoisomerase IV, i.e.
* Corresponding author. Tel.: þ48 081 532 05 19; fax: þ48 081 532 45 46.
E-mail address: tomasz.plech@umlub.pl (T. Plech).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.040

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
129
the main molecular targets for fluoroquinolone drugs. Therefore, it
is probable that the obtained CPX-triazole hybrids will combine the
mechanisms of action of fluoroquinolones and 1,2,4-triazoles.
Rationality of combination of the two structures is also spoken for
by the fact that replacement of hydrogen atom (NH group in the
1,2,4-triazole system) results in extension of the activity spectrum
on some Gram-negative bacteria [23]. Therefore, one may expect
that the compounds obtained by combination of CPX and 1,2,4-
triazole derivatives will strongly inhibit the growth of Gram-
positive bacteria, and at the same time maintain strong activity
towards the Gram-negative strains. Moreover, an important
problem related to the use of antibiotics is the growing number of
strains resistant to drugs. Therefore, it is recommended that newly
obtained substances with potential antibacterial effect should be
tested on drug-resistant strains (e.g. MRSA). The strain of this type
has been also used in our research.
in our earlier paper [23]. A series of new hybrids 3aer containing
CPX and 1,2,4-triazole derivatives were prepared by using the
Mannich reaction. The intermediates 2aer, ciprofloxacin and
formaldehyde (37%) in ethanolic solution were stirred at room
temperature for 12 h. The reaction yields ranged from 63 to 80%.
The structures of the novel compounds 3aer were determined
using 1H NMR, IR and elemental analyses.
2.2. Antibacterial activity
Antibacterial activity of compounds 3aer has been examined
with regard to drug-susceptible Gram-positive (S. aureus ATCC
25923, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis
ATCC 6633, Bacillus cereus ATCC 10876, Micrococcus luteus ATCC
10240) and Gram-negative bacteria (Escherichia coli ATCC 25922,
Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 9027)
(Table 1). Moreover, the research was also based on drug-resistant
strain of S. aureus (MRSA) (Table 2). The activity of synthesised
compounds has been compared with the activity of CPX, and in case
of MRSA strain e also with the activity of vancomycin.
2. Results and discussion
2.1. Chemistry
A significant part of the compounds obtained showed antibac-
terial activity higher than the activity of CPX, both towards Gram-
positive and Gram-negative bacteria. We have managed to
accomplish significant increase of activity towards the S. aureus
strain. Literature data show that poor antistaphylococcal effect of
CPX results from the weaker interaction with staphylococcal
enzymes [3]. However, in case of compounds 3aer, as many as 11
out of 18 derivatives acted more strongly than CPX. The highest
As it was shown in Scheme 1, the target compounds 3aer were
obtained in a three-step synthesis. Treatment of alkyl/aryl hydra-
zides with alkyl/aryl isothiocyanates gave the corresponding 1,4-
disubstituted thiosemicarbazides 1aer, which were subsequently
refluxed in 2% NaOH solution affording 1,2,4-triazole-5-thione
derivatives 2aer. All details concerning the synthesis of the 1,4-
disubstituted thiosemicarbazides and 1,2,4-triazoles can be found
O
O
(i)
+
R1
R2 NCS
NH
NH
R1
NH
R2
1a-r
NH NH2
S
(ii)
O
O
H
N
F
OH
S
N
2a-r
N
N
N
R1
R2
HN
(iii)
O
N
O
F
OH
R1
N
N
3a-r
N
R2
N
N
S
Scheme 1. Synthesis of compounds 3aer. Reagents and conditions: (i) EtOH, reflux, 5 min, (ii) 2% NaOH, reflux, (iii) EtOH, HCHO, rt., 12 h.

130
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
Table 1
Antimicrobial activity evaluation for compounds 3aer against drug-susceptible bacteria.
R1
R2
Minimum inhibitory concentrations (
m
M)
S. aureus
ATCC
25923
S. epidermidis B. subtilis
B. cereus
M. luteus
ATCC
10240
E. coli
ATCC
25922
P. mirabilis
ATCC
12453
P. aeruginosa
ATCC 9027
ATCC 12228
ATCC 6633 ATCC
10876
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-OHePh
3-OHePh
3-OHePh
3-OHePh
eH
4-OCH3ePh
4-BrePh
3-IePh
1.48
5.51
1.29
5.16
0.73
1.40
22.32
0.86
0.84
3.19
1.53
0.74
0.66
0.35
0.72
12.08
23.66
90.77
2.96
1.48
5.51
1.29
5.16
0.73
0.70
22.32
0.86
0.84
1.60
1.53
0.74
0.66
0.35
0.35
6.05
23.66
90.77
1.48
0.09
0.34
0.04
0.32
0.09
0.043
1.40
0.053
0.10
0.20
0.19
0.18
0.32
0.18
0.18
0.37
1.48
5.68
0.09
0.36
1.38
0.16
1.29
0.18
0.17
5.58
0.21
0.20
0.39
0.77
0.18
0.16
0.18
0.18
3.02
1.48
22.70
0.36
2.95
10.10
2.58
10.31
2.92
2.78
44.64
1.72
0.84
6.40
3.05
1.49
1.33
1.44
1.44
24.17
94.62
181.54
5.88
0.023
0.084
0.02
0.023
0.17
0.04
0.36
1.38
0.16
1.29
0.36
0.17
11.16
0.10
0.41
0.80
0.77
0.18
0.081
0.088
0.088
1.52
2.95
22.70
0.72
4-IePh
0.08
0.16
2,4-diFePh
2,4-diClePh
3,5-diClePh
eCH3
eC2H5
n-C4H9
n-C6H13
3-NO2ePh
3-IePh
2,4-diClePh
3,5-diClePh
3-IePh
0.022
0.043
0.70
0.022
0.043
0.70
0.053
0.051
0.098
0.19
0.023
0.041
0.022
0.044
0.18
0.053
0.051
0.098
0.047
0.023
0.041
0.044
0.022
0.093
0.36
3q
3r
eCH3
n-C3H7
3-IePh
3-IePh
0.74
2.83
0.045
0.71
0.024
Ciprofloxacin
Values in bold highlight activities that are higher (or equal) than that of ciprofloxacin.
activity, 8.5 times higher than of the reference antibiotic, was ob-
tained for 7-[4-{[4-(2,4-dichlorophenyl)-3-(3-hydroxyphenyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-
1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3n). M. luteus ATCC 10240 has also shown high sensitivity to
synthesised derivatives. In the group of 11 compounds with higher
activity than the activity of CPX, the strongest effect was achieved
by 3i compound (activity 7.5 times higher than in case of cipro-
floxacin). The modifications of CPX structure applied have proven
to be the least effective in case of B. subtilis ATCC 6633 strain. Only 5
derivatives have shown comparable (3a, 3i) or higher activity than
the reference drug (3c, 3f, 3h).
many life threatening infections [24,25]. One of the few drugs
effective in combating infections caused by MRSA is vancomycin
[25]. Therefore, CPX and vancomycin have been used by us as
reference drugs (Table 2). 3-Hydroxyphenyl derivatives (3leo)
showed particularly high anti-MRSA activity. 7-[4-{[4-(3,5-
Dichlorophenyl)-3-(3-hydroxyphenyl)-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3o) was 17 times
more active than CPX and nearly 8 times more active than vanco-
mycin. N4-Methyl (3h) and N4-ethyl (3i) derivatives were also
characterised by significant activity. On the grounds of MBC/MIC
ratio (Table 2) it has been stated that all these compounds (except
3c and 3f) showed bactericidal activity (MBC/MIC ꢀ 4), which is far
more advantageous than bacteriostatic effect [26].
Promising results of microbiological tests performed on drug-
susceptible strain of S. aureus made us check, whether 3aer
compounds are also effective against the methicillin-resistant
strain (MRSA). This type of staphylococcal strain is the cause of
For all chemicals applicable in the treatment of bacterial infec-
tions it is important that the compounds do not cause toxic effects.
Table 2
Lipophilicity and antimicrobial activity evaluation for compounds 3aer against MRSA strain.
R1
R2
LogPa
Staphylococcus aureus Microbank
MBC/MIC
14001 (MRSA)
MIC (
m
M)
MBC (mM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-ClePh
3-OHePh
3-OHePh
3-OHePh
3-OHePh
eH
4-OCH3ePh
4-BrePh
3-IePh
3.18
3.94
4.40
4.21
3.59
4.62
4.83
1.85
2.23
3.29
4.30
2.12
3.24
3.46
3.68
1.63
1.72
2.79
ꢁ0.70
0.74
2.76
0.64
2.58
0.73
0.70
22.32
0.42
0.41
1.60
1.53
0.36
0.66
0.35
0.74
5.51
5.16
5.16
0.73
44.64
44.64
0.86
0.84
1.60
1
2
8
2
1
64
2
2
2
1
2
2
1
2
4
2
1
1
4-IePh
2,4-diFePh
2,4-diClePh
3,5-diClePh
eCH3
eC2H5
n-C4H9
n-C6H13
3-NO2ePh
3-IePh
2,4-diClePh
3,5-diClePh
3-IePh
3.05
0.74
0.66
0.71
0.088
6.05
23.66
181.54
1.48
0.35
12.08
23.66
181.54
5.92
3q
3r
eCH3
n-C3H7
3-IePh
3-IePh
Ciprofloxacin
Vancomycin
4
0.68
2.70
Values in bold highlight activities that are higher than that of antibiotic(s).
a
LogP was calculated using Molinspiration Calculator (http://www.molinspiration.com/cgi-bin/properties).

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
131
Concentration which inhibits bacteria growth cannot disturb
proper functioning of normal human cells. Antibacterial
times increase of activity. In the group of 3-hydroxyphenyl deriv-
atives (3leo), the type and location of substituents attached to the
second phenyl group in position N-4 seems less important. All four
compounds (3leo) feature similar activity. Only in case of the MRSA
substances’ toxicity profile may be characterised by the ratio EC50
/
MIC [27]. EC50 is the concentration of the substance which inhibits
cells growth in 50% in proportion to the growth of control cells. The
bigger the difference between EC50 and MIC values is the lesser is
the risk of toxic effects. For two newly synthesised derivatives of
ciprofloxacin (3o and 3i), showing strong activity against MRSA
strain, there has been determined cytotoxicity (EC50) towards HEK-
293 (human embryonic kidney) cells. EC50 values for compounds
strain the 3,5 substitution is privileged (MIC ¼ 0.088
mM). On the
other hand, the presence of aromatic system in position N-4 is not
required. It is confirmed by the activity of N-4 alkyl derivatives
(3hek), where the compound 3i (ethyl) is even 7-times more active
than CPX. However, the longer the alkyl chain is, the activity
decreases significantly.
3o and 3i were 36.09 ꢂ 4.05
m
g/mL (52.95 ꢂ 5.94
m
M) and
While analysing the activity of compounds with para
substituted phenyl rings (3a, 3b, 3d) it is clear that the electron-
donating substituent (OCH3) wields more positive influence on
the antibacterial activity than the electron-withdrawing groups (Br,
I). What is more, it is possible to achieve an increase of activity if the
electron-withdrawing substituent from para position will be placed
in meta position. Further improvement of antibacterial effect is
possible through the use of 2,4-disubstituted phenyl ring in posi-
tion N-4.
To conclude, 1,2,4-triazole derivatives offer enormous possibil-
ities of modification of antibacterial activity of CPX. Through
appropriate adjustment of substituents in position C-3 and N-4, we
are able to obtain new compounds with strong action force. The
preliminary SAR observations we have made as regards antibacte-
rial activity of CPX-1,2,4-triazole hybrids will be soon used for
designing larger number of derivatives of the type.
23.13 ꢂ 1.19 g/mL (39.66 ꢂ 2.04
m
mM), respectively. In case of the
compound 3o the EC50 value was approximately 600 times higher
than the minimum concentration inhibiting the growth of MRSA
strain. Whereas for compound 3i the ratio EC50/MIC was close to
100. Therefore, it may be concluded that these derivatives have
positive cytotoxicity profile. Later on there will be determined
cytotoxicity levels for the other derivatives of ciprofloxacin.
The increase of activity of new derivatives of CPX (3aer)
towards Gram-positive bacteria may be caused by two reasons, at
least. The first one is related to the permeation process of drug
molecules across the bacterial cell wall. Gram-positive bacteria wall
is rich in peptidoglycan and is well permeable for hydrophobic
substances. Whereas, CPX has the nature of a zwitterion with two
ionisable groups (NH of piperazine, COOH) [15,28]. It may be the
reason of hindered permeability of drug molecules to the inside of
bacteria. The consequence of combination of CPX with 1,2,4-
triazoles (2aer) is the restriction of ionic character and the
increase of lipophilic properties of compounds 3aer (clogP values
are presented in Table 2). In the consequence, it may be expected
that transfer of these derivatives to the bacterial cells should be
facilitated. The other reason of the increased activity against Gram-
positive bacteria may be related to the presence of an additional
mechanism of antibacterial activity, resulting from the presence of
a 1,2,4-triazole fragment.
The literature data shows, that it is difficult to obtain the fluo-
roquinolone derivatives for which the increase of the antibacterial
activity against Gram-positive bacteria would not be related to the
decrease of activity towards Gram-negative bacteria [15,22]. The
most significant loss of activity is usually observed in case of
P. aeruginosa strain [15]. Its low sensitivity to CPX is usually related
to the existence of the very effective efflux system [29]. Therefore, it
should be emphasised that in most of the derivatives referred to
herein, there have been observed a significant increase of anti-
bacterial activity towards P. aeruginosa ATCC 9027. For instance,
3-hydroxyphenyl derivatives (3leo) acted up to 8 times stronger
than CPX.
3. Experimental
3.1. General comments
All reagents and solvents were purchased from Alfa Aesar (Ward
Hill, USA) and Merck Co. (Darmstadt, Germany). Melting points
were determined by using Fisher-Johns apparatus (Fisher Scientific,
Schwerte, Germany) and are uncorrected. The 1H NMR spectra were
recorded on a Bruker Avance spectrometer (Bruker BioSpin GmbH,
Germany) using TMS as an internal standard and CDCl3 as a solvent.
The IR spectra (KBr) were obtained on a PerkineElmer 1725X FTIR
spectrophotometer. Elemental analyses were performed on an AMZ
ꢀ
851 CHX analyser (PG, Gdansk, Poland) and the results were within
ꢂ0.4% of the theoretical values.
3.2. General procedure for the synthesis of ciprofloxacin derivatives
(3aer)
1 mmol of the respective 1,2,4-triazole derivative (2aer) was
dissolved (with heating) in 40 mL of anhydrous ethanol and then
equimolar amount of ciprofloxacin and formaldehyde solution
(37%) were added. The obtained suspension was stirred at room
temperature for 12 h. The precipitate formed was filtered off, dried,
and crystallised from ethanol to give compounds 3aer. The inter-
mediates 1aer and 2aer were prepared according to the literature
methods [23].
2.3. Structureeactivity relationships (SAR) observations
Despite relatively small number of synthesised derivatives, it is
still possible to observe significant dependences between their
structure and activity. Synthesised compounds differ from each
other by the type of substituents in position C-3 and N-4 of the
1,2,4-triazole ring. From the microbiological point of view, the key
factor is the presence of phenyl ring in position C-3. Derivatives
with alkyl substituent (3q, 3r) or having no substituent in position
C-3 (3p), showed low activity, especially towards Gram-positive
bacteria. Additionally, the more the alkyl fragment was extended,
the activity decreased more and more. Beside the presence of
phenyl ring (in position C-3), an extremely important factor is the
electronic nature of substituent attached to this ring. The presence
of electron-donating hydroxyl group is advantageous. It is clearly
visible in the example of compounds 3g and 3o, in case of which the
replacement of chlorine atom with hydroxyl group causes even 60-
3.2.1. 7-[4-{[3-(3-Chlorophenyl)-4-(4-methoxyphenyl)-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3a)
Yield: 72%, m.p. 228e230 ꢃC, 1H NMR (CDCl3): 1.14e1.24 (m, 2H,
cyclopropyl), 1.33e1.46 (m, 2H, cyclopropyl), 3.14e3.23 (m, 4H,
piperazine), 3.38e3.45 (m, 4H, piperazine), 3.56e3.61 (m, 1H,
cyclopropyl), 3.74 (s, 3H, OCH3), 5.39 (s, 2H, CH2 of linker), 7.08e
8.01 (m, 10H, AreH), 8.76 (s, 1H, CH), 14.90 (s, 1H, COOH). IR (KBr,
n
, cmꢁ1): 3435 (OH), 3064 (CHarom.), 2921, 2806 (CHaliph.), 1723,
1643 (C]O), 1587 (C]N), 1326 (C]S). Elemental analysis for

132
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
C33H30ClFN6O4S (661.14). Calculated: C 59.95, H 4.57, N 12.71.
Found: C 59.80, H 4.71, N 12.78.
analysis for C32H26Cl3FN6O3S (700.01). Calculated: C 54.91, H 3.74, N
12.01. Found: C 55.03, H 3.76, N 12.08.
3.2.2. 7-[4-{[4-(4-Bromophenyl)-3-(3-chlorophenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3b)
3.2.7. 7-[4-{[3-(3-Chlorophenyl)-4-(3,5-dichlorophenyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-
1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3g)
Yield: 67%, m.p. 256e258 ꢃC, 1H NMR (CDCl3): 1.13e1.25 (m, 2H,
cyclopropyl), 1.34e1.44 (m, 2H, cyclopropyl), 3.13e3.21 (m, 4H,
piperazine), 3.34e3.42 (m, 4H, piperazine), 3.52e3.60 (m, 1H,
cyclopropyl), 5.35 (s, 2H, CH2 of linker), 6.98e8.08 (m, 10H, AreH),
Yield: 70%, m.p. 249e251 ꢃC, 1H NMR (CDCl3): 1.20e1.30 (m, 2H,
cyclopropyl), 1.38e1.48 (m, 2H, cyclopropyl), 3.16e3.24 (m, 4H,
piperazine), 3.39e3.45 (m, 4H, piperazine), 3.55e3.64 (m, 1H,
cyclopropyl), 5.39 (s, 2H, CH2 of linker), 7.08e7.59 (m, 8H, AreH),
8.05 (d, 1H, AreH, J ¼ 13.07 Hz), 8.81 (s, 1H, CH), 14.82 (s, 1H,
8.75 (s, 1H, CH), 14.87 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3513 (OH),
3092 (CHarom.), 2963, 2854 (CHaliph.), 1734, 1639 (C]O), 1570 (C]
N), 1332 (C]S). Elemental analysis for C32H27BrClFN6O3S (710.02).
Calculated: C 54.13, H 3.83, N 11.84. Found: C 54.02, H 3.89, N 11.76.
COOH). IR (KBr, n
, cmꢁ1): 3444 (OH), 3069 (CHarom.), 2945, 2821
(CHaliph.), 1736, 1628 (C]O), 1577 (C]N), 1326 (C]S). Elemental
analysis for C32H26Cl3FN6O3S (700.01). Calculated: C 54.91, H 3.74, N
12.01. Found: C 54.83, H 3.83, N 12.11.
3.2.3. 7-[4-{[3-(3-Chlorophenyl)-4-(3-iodophenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3c)
3.2.8. 7-[4-{[3-(3-Chlorophenyl)-4-methyl-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3h)
Yield: 75%, m.p. 220e221 ꢃC, 1H NMR (CDCl3): 1.19e1.28 (m, 2H,
cyclopropyl), 1.39e1.47 (m, 2H, cyclopropyl), 3.16e3.25 (m, 4H,
piperazine), 3.39e3.47 (m, 4H, piperazine), 3.56e3.65 (m, 1H,
cyclopropyl), 5.39 (s, 2H, CH2 of linker), 7.08e8.04 (m, 10H, AreH),
Yield: 65%, m.p. 142e144 ꢃC, 1H NMR (CDCl3): 1.15e1.22 (m, 2H,
cyclopropyl), 1.35e1.42 (m, 2H, cyclopropyl), 3.06e3.13 (m, 4H,
piperazine), 3.32e3.38 (m, 4H, piperazine), 3.50e3.57 (m, 1H,
cyclopropyl), 3.69 (s, 3H, CH3), 5.29 (s, 2H, CH2 of linker), 7.25e7.70
(m, 5H, AreH), 7.99 (d, 1H, AreH, J ¼ 13.15 Hz), 8.75 (s, 1H, CH),
8.78 (s, 1H, CH), 14.85 (s, 1H, COOH). IR (KBr,
3041 (CHarom.), 2964, 2889, 2782 (CHaliph.), 1725, 1634 (C]O), 1575
(C]N), 1329 (C]S). Elemental analysis for 32H27ClFIN6O3S
n
, cmꢁ1): 3487 (OH),
C
(757.02). Calculated: C 50.77, H 3.59, N 11.10. Found: C 50.85, H 3.43,
N 11.17.
15.06 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3507 (OH), 3039 (CHarom.),
2983, 2871, 2802 (CHaliph.), 1724, 1635 (C]O), 1584 (C]N), 1320
(C]S). Elemental analysis for C27H26ClFN6O3S (569.05). Calculated:
C 56.99, H 4.61, N 14.77. Found: C 57.11, H 4.57, N 14.82.
3.2.4. 7-[4-{[3-(3-Chlorophenyl)-4-(4-iodophenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3d)
3.2.9. 7-[4-{[3-(3-Chlorophenyl)-4-ethyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (3i)
Yield: 77%, m.p. 258e260 ꢃC, 1H NMR (CDCl3): 1.15e1.23 (m, 2H,
cyclopropyl), 1.34e1.43 (m, 2H, cyclopropyl), 3.13e3.20 (m, 4H,
piperazine), 3.34e3.43 (m, 4H, piperazine), 3.55 (br. s, 1H, cyclo-
propyl), 5.34 (s, 2H, CH2 of linker), 7.00e8.01 (m, 10H, AreH), 8.74
Yield: 63%, m.p. 138e140 ꢃC, 1H NMR (CDCl3): 1.14e1.22 (m, 2H,
cyclopropyl), 1.32e1.42 (m, 5H, 2H-cyclopropyl þ CH3), 3.04e3.12
(m, 4H, piperazine), 3.31e3.40 (m, 4H, piperazine), 3.48e3.59 (m,
1H, cyclopropyl), 4.18 (q, 2H, CH2, J ¼ 7.21 Hz), 5.27 (s, 2H, CH2 of
linker), 7.23e7.65 (m, 5H, AreH), 7.97 (d, 1H, AreH, J ¼ 13.12 Hz),
(s, 1H, CH), 14.79 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3505 (OH), 3036
(CHarom.), 2945, 2830 (CHaliph.), 1730, 1633 (C]O), 1582 (C]N),
1323 (C]S). Elemental analysis for C32H27ClFIN6O3S (757.02).
Calculated: C 50.77, H 3.59, N 11.10. Found: C 50.89, H 3.48, N 11.11.
8.74 (s, 1H, CH), 14.96 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3468 (OH),
3100 (CHarom.), 2935, 2857 (CHaliph.), 1727,1641 (C]O),1579 (C]N),
1322 (C]S). Elemental analysis for C28H28ClFN6O3S (583.08).
Calculated: C 57.68, H 4.84, N 14.41. Found: C 57.70, H 4.99, N 14.28.
3.2.5. 7-[4-{[3-(3-Chlorophenyl)-4-(2,4-difluorophenyl)-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3e)
3.2.10. 7-[4-{[3-(3-Chlorophenyl)-4-(n-butyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(3j)
Yield: 66%, m.p. 210e212 ꢃC, 1H NMR (CDCl3): 1.18e1.27 (m, 2H,
cyclopropyl), 1.39e1.49 (m, 2H, cyclopropyl), 3.16e3.25 (m, 4H,
piperazine), 3.39e3.47 (m, 4H, piperazine), 3.55e3.64 (m, 1H,
cyclopropyl), 5.39 (s, 2H, CH2 of linker), 6.99e7.57 (m, 8H, AreH),
8.00 (d, 1H, AreH, J ¼ 13.20 Hz), 8.76 (s, 1H, CH), 14.90 (s, 1H,
Yield: 74%, m.p. 116e118 ꢃC, 1H NMR (CDCl3): 0.88 (t, 3H, CH3,
J ¼ 7.43 Hz), 1.14e1.41 (m, 6H, 4H-cyclopropyl þ CH2), 1.61e1.78 (m,
2H, CH2), 3.04e3.12 (m, 4H, piperazine), 3.31e3.38 (m, 4H, piper-
azine), 3.49e3.59 (m, 1H, cyclopropyl), 4.11 (t, 2H, CH2, J ¼ 7.81 Hz),
5.27 (s, 2H, CH2 of linker), 7.26e7.66 (m, 5H, AreH), 7.96 (d, 1H, Are
COOH). IR (KBr, n
, cmꢁ1): 3457 (OH), 3084 (CHarom.), 2885, 2799
(CHaliph.), 1721, 1631 (C]O), 1578 (C]N), 1340 (C]S). Elemental
analysis for C32H26ClF3N6O3S (667.10). Calculated: C 57.61, H 3.93, N
12.60. Found: C 57.76, H 4.07, N 12.65.
H, J ¼ 13.10 Hz), 8.73 (s, 1H, CH), 14.98 (s, 1H, COOH). IR (KBr,
n,
cmꢁ1): 3476 (OH), 3073 (CHarom.), 2963, 2893 (CHaliph.), 1729, 1626
3.2.6. 7-[4-{[3-(3-Chlorophenyl)-4-(2,4-dichlorophenyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-
1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3f)
(C]O), 1577 (C]N), 1334 (C]S). Elemental analysis for
C30H32ClFN6O3S (611.13). Calculated: C 58.96, H 5.28, N 13.75.
Found: C 58.85, H 5.25, N 13.79.
Yield: 80%, m.p. 239e240 ꢃC, 1H NMR (CDCl3): 1.20e1.26 (m, 2H,
cyclopropyl), 1.39e1.47 (m, 2H, cyclopropyl), 3.15e3.25 (m, 4H,
piperazine), 3.37e3.45 (m, 4H, piperazine), 3.60 (br. s, 1H, cyclo-
propyl), 5.40 (s, 2H, CH2 of linker), 7.13e7.64 (m, 8H, AreH), 8.01 (d,
1H, AreH, J ¼ 13.19 Hz), 8.79 (s,1H, CH),14.84 (s,1H, COOH). IR (KBr,
3.2.11. 7-[4-{[3-(3-Chlorophenyl)-4-(n-hexyl)-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-cyclopropyl-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3k)
Yield: 75%, m.p. 152e154 ꢃC, 1H NMR (CDCl3): 0.74 (t, 3H, CH3,
J ¼ 6.96 Hz), 1.02e1.64 (m, 12H, 4H-cyclopropyl þ 4ꢄ CH2), 3.06e
3.12 (m, 4H, piperazine), 3.30e3.38 (m, 4H, piperazine), 3.45e
3.52 (m, 1H, cyclopropyl), 4.02 (t, 2H, CH2, J ¼ 7.75 Hz), 5.34 (s,
n
, cmꢁ1): 3486 (OH), 3096, 3036 (CHarom.), 2955, 2848, 2803 (CHa-
liph.), 1728, 1637 (C]O), 1584 (C]N), 1317 (C]S). Elemental

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
133
2H, CH2 of linker), 7.08e7.61 (m, 5H, AreH), 8.02 (d, 1H, AreH,
piperazine), 3.32e3.42 (m, 4H, piperazine), 3.54 (br. s, 1H, cyclo-
propyl), 5.27 (s, 2H, CH2 of linker), 7.31e8.09 (m, 6H, AreH), 8.21 (s,
J ¼ 13.15 Hz), 8.74 (s, 1H, CH), 14.87 (s, 1H, COOH). IR (KBr,
n,
cmꢁ1): 3484 (OH), 3066 (CHarom.), 2927, 2834, 2780 (CHaliph.), 1730,
1H, CH of triazole), 8.79 (s, 1H, CH), 14.65 (s, 1H, COOH). IR (KBr, n,
1629 (C]O), 1572 (C]N), 1317 (C]S). Elemental analysis for
cmꢁ1): 3487 (OH), 3072 (CHarom.), 2956, 2834, 2787 (CHaliph.), 1732,
C
32H36ClFN6O3S (639.18). Calculated: C 60.13, H 5.68, N 13.15.
1641 (C]O), 1582 (C]N), 1320 (C]S). Elemental analysis for
Found: C 60.02, H 5.75, N 13.19.
C26H24FIN6O3S (646.48). Calculated: C 48.30, H 3.74, N 13.00.
Found: C 48.39, H 3.65, N 13.11.
3.2.12. 1-Cyclopropyl-6-fluoro-7-[4-{[3-(3-hydroxyphenyl)-4-(3-
nitrophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}
piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3l)
Yield: 72%, m.p. 168e170 ꢃC, 1H NMR (CDCl3): 1.22e1.33 (m, 2H,
cyclopropyl), 1.43e1.54 (m, 2H, cyclopropyl), 3.13e3.20 (m, 4H,
piperazine), 3.31e3.40 (m, 4H, piperazine), 3.56e3.62 (m, 1H,
cyclopropyl), 5.40 (s, 2H, CH2 of linker), 5.65 (s, 1H, AreOH), 7.05e
3.2.17. 1-Cyclopropyl-6-fluoro-7-[4-{[4-(3-iodophenyl)-3-methyl-
5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3q)
Yield: 73%, m.p. 268e270 ꢃC, 1H NMR (CDCl3): 1.15e1.28 (m, 2H,
cyclopropyl), 1.35e1.43 (m, 2H, cyclopropyl), 2.20 (s, 3H, CH3),
3.03e3.13 (m, 4H, piperazine), 3.30e3.39 (m, 4H, piperazine),
3.48e3.59 (m, 1H, cyclopropyl), 5.22 (s, 2H, CH2 of linker), 7.22e
8.05 (m, 6H, AreH), 8.77 (s, 1H, CH), 14.76 (s, 1H, COOH). IR (KBr,
7.94 (m, 10H, AreH), 8.72 (s, 1H, CH), 14.68 (s, 1H, COOH). IR (KBr, n,
cmꢁ1): 3504 (OH), 3068 (CHarom.), 2904, 2810 (CHaliph.), 1732, 1633
(C]O), 1574 (C]N), 1322 (C]S). Elemental analysis for
n
, cmꢁ1): 3483 (OH), 3071 (CHarom.), 2932, 2858 (CHaliph.), 1725,
C
32H28FN7O6S (657.67). Calculated: C 58.44, H 4.29, N 14.91. Found:
1624 (C]O), 1579 (C]N), 1317 (C]S). Elemental analysis for
C27H26FIN6O3S (660.50). Calculated: C 49.10, H 3.97, N 12.72. Found:
C 58.56, H 4.13, N 14.82.
C 49.11, H 4.08, N 12.59.
3.2.13. 1-Cyclopropyl-6-fluoro-7-[4-{[3-(3-hydroxyphenyl)-4-(3-
iodophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}
piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3m)
Yield: 67%, m.p. 194e196 ꢃC, 1H NMR (CDCl3): 1.29 (br. s, 2H,
cyclopropyl), 1.62 (br. s, 2H, cyclopropyl), 3.17e3.28 (m, 4H, pipera-
zine), 3.35e3.41 (m, 4H, piperazine), 3.57e3.61 (m, 1H, cyclopropyl),
5.35 (s, 2H, CH2 of linker), 5.70 (s,1H, AreOH), 6.94e7.86 (m,10H, Are
3.2.18. 1-Cyclopropyl-6-fluoro-7-[4-{[4-(3-iodophenyl)-3-(n-
propyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}
piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3r)
Yield: 69%, m.p. 260e262 ꢃC, 1H NMR (CDCl3): 0.93 (t, 3H, CH3,
J ¼ 7.50 Hz),1.15e1.29 (m, 4H, 2H-cyclopropyl þ CH2), 1.35e1.44 (m,
2H, cyclopropyl), 2.46 (t, 2H, CH2, J ¼ 7.68 Hz), 3.10 (br. s, 4H,
piperazine), 3.36 (br. s, 4H, piperazine), 3.55 (br. s, 1H, cyclopropyl),
5.25 (s, 2H, CH2 of linker), 7.20e8.05 (m, 6H, AreH), 8.77 (s, 1H, CH),
H), 8.71 (s, 1H, CH), 14.84 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3531, 3444
(OH), 3056 (CHarom.), 2881, 2779 (CHaliph.), 1719, 1631 (C]O), 1580
(C]N), 1334 (C]S). Elemental analysis for C32H28FIN6O4S (738.57).
Calculated: C 52.04, H 3.82, N 11.38. Found: C 52.11, H 3.76, N 11.46.
14.87 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3452 (OH), 3104 (CHarom.),
2874, 2807 (CHaliph.), 1735, 1632 (C]O), 1570 (C]N), 1334 (C]S).
Elemental analysis for C29H30FIN6O3S (688.55). Calculated: C 50.59,
H 4.39, N 12.21. Found: C 50.52, H 4.43, N 12.32.
3.2.14. 7-[4-{[4-(2,4-Dichlorophenyl)-3-(3-hydroxyphenyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(3n)
3.3. Antimicrobial activity evaluation
Yield: 69%, m.p. 240e242 ꢃC, 1H NMR (CDCl3): 1.19e1.31 (m, 2H,
cyclopropyl), 1.41e1.52 (m, 2H, cyclopropyl), 3.11e3.22 (m, 4H,
piperazine), 3.31e3.39 (m, 4H, piperazine), 3.59 (br. s, 1H, cyclo-
propyl), 5.36 (s, 2H, CH2 of linker), 5.66 (s, 1H, AreOH), 7.06e7.78
(m, 8H, AreH), 8.05 (d, 1H, AreH, J ¼ 13.15 Hz), 8.75 (s, 1H, CH),
The antimicrobial activity of the compounds was screened on the
Gram-negative bacteria strains (E. coli ATCC 25922, P. mirabilis ATCC
12453, P. aeruginosa ATCC 9027) and on the Gram-positive bacteria
(S. aureus ATCC 25923, S. aureus Microbank 14001, S. epidermidis
ATCC 12228, B. subtilis ATCC 6633, B. cereus ATCC 10876, M. luteus
ATCC 10240). All stock solutions of the tested compounds were dis-
solved in dimethyl sulfoxide. Microbial suspensions were prepared in
sterile saline (0.85% NaCl) with an optical density of 0.5 McFarland
standard - 150 ꢄ 106 CFU/mL (CFU e colony forming units). The
medium with dimethyl sulfoxide at the final concentration and
without the tested compounds served as a negative control.
Preliminary antibacterial potency of compounds 3aer was screened
using the agar dilution method on the basis of the growth inhibition
on the Mueller-Hinton agar to which the tested compounds at
14.96 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3540, 3464 (OH), 3032
(CHarom.), 2911, 2803 (CHaliph.), 1726, 1628 (C]O), 1577 (C]N), 1325
(C]S). Elemental analysis for C32H27Cl2FN6O4S (681.56). Calcu-
lated: C 56.39, H 3.99, N 12.33. Found: C 56.45, H 4.08, N 12.45.
3.2.15. 7-[4-{[4-(3,5-Dichlorophenyl)-3-(3-hydroxyphenyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(3o)
Yield: 63%, m.p. 254e256 ꢃC, 1H NMR (CDCl3): 1.15e1.26 (m, 2H,
cyclopropyl), 1.36e1.48 (m, 2H, cyclopropyl), 3.16e3.25 (m, 4H,
piperazine), 3.32e3.40 (m, 4H, piperazine), 3.49e3.56 (m, 1H,
cyclopropyl), 5.39 (s, 2H, CH2 of linker), 5.71 (s, 1H, AreOH), 6.95e
7.82 (m, 8H, AreH), 8.00 (d,1H, AreH, J ¼ 13.21 Hz), 8.72 (s, 1H, CH),
concentrations 1000 mg/mL were added. Then the antibacterial
activity of the compounds with inhibitory effect was determined by
broth microdilution technique. The activity was expressed as the
minimal concentration of the compound that inhibits the growth of
the bacteria (MIC). The MBC (minimal bactericidal concentration),
defined as the lowest concentration of each compound that resulted
in >99.9% reduction in CFU of the initial inoculum, was also deter-
mined. In order to take into consideration the molar mass of
respective derivatives, MIC and MBC values were presented in the
14.83 (s, 1H, COOH). IR (KBr, n
, cmꢁ1): 3513 (OH), 3080 (CHarom.),
2967, 2824 (CHaliph.), 1725, 1624 (C]O), 1571 (C]N), 1334 (C]S).
Elemental analysis for C32H27Cl2FN6O4S (681.56). Calculated: C
56.39, H 3.99, N 12.33. Found: C 56.30, H 4.11, N 12.21.
form of molar concentrations (mM).
3.2.16. 1-Cyclopropyl-6-fluoro-7-[4-{[4-(3-iodophenyl)-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (3p)
3.4. Cytotoxicity assay
Yield: 70%, m.p. 257e259 ꢃC, 1H NMR (CDCl3): 1.17e1.30 (m, 2H,
cyclopropyl), 1.34e1.46 (m, 2H, cyclopropyl), 3.07e3.15 (m, 4H,
HEK-293 (human embryonic kidney) cells were obtained from
the American Type Culture Collection (ATCC CRL-1573) and were

134
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 128e134
antibacterials:
(1989) 3886e3894.
a cooperative drug-DNA binding model, Biochemistry 28
grown in Eagle’s Minimal Essential Medium (MEM; Sigma) sup-
plemented with 10% foetal bovine serum (FBS; Sigma). 100 U/mL of
[11] A. Foroumadi, S. Mansouri, Z. Kiani, A. Rahmani, Synthesis and in vitro anti-
bacterial evaluation of N-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-yl] piper-
azinyl quinolones, Eur. J. Med. Chem. 38 (2003) 851e854.
[12] A. Foroumadi, F. Soltani, M.H. Moshafi, R. Ashraf-Askari, Synthesis and in vitro
antibacterial activity of some N-(5-aryl-1,3,4-thiadiazole-2-yl)piperazinyl
quinolone derivatives, Il Farmaco 58 (2003) 1023e1028.
[13] A. Foroumadi, R. Ashraf-Askari, M.H. Moshafi, S. Emami, A. Zeynali, Synthesis
and in vitro antibacterial activity of N-[5-(5-nitro-2-furyl)-1,3,4-thiadiazole-2-
yl]piperazinyl quinolone derivatives, Pharmazie 58 (2003) 432e433.
[14] A. Foroumadi, S. Mansouri, S. Emami, J. Mirzaei, M. Sorkhi, N. Saeid-Adeli,
A. Shafiee, Synthesis and antibacterial activity of nitroaryl thiadiazole-
levofloxacin hybrids, Arch. Pharm. Chem. Life Sci. 339 (2006) 621e624.
[15] A. Foroumadi, S. Emami, A. Hassanzadeh, M. Rajaee, K. Sokhanvar,
M.H. Moshafi, A. Shafiee, Synthesis and antibacterial activity of N-(5-
benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-
yl)piperazinyl quinolone derivatives, Bioorg. Med. Chem. Lett. 15 (2005)
4488e4492.
[16] L.S. Feng, M.L. Liu, S. Zhang, Y. Chai, B. Wang, Y.-B. Zhang, K. Lv, Y. Guan,
H.Y. Guo, Ch. L. Xiao, Synthesis and in vitro antimycobacterial activity of 8-
OCH3 ciprofloxacin methylene and ethylene isatin derivatives, Eur. J. Med.
Chem. 46 (2011) 341e348.
[17] A. Foroumadi, S. Emami, M. Mehni, M.H. Moshafi, A. Shafiee, Synthesis and
antibacterial activity of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and N-[(2-
5-bromothiophen-2-yl)-2-oximinoethyl]derivatives of piperazinyl quino-
lones, Bioorg. Med. Chem. Lett. 15 (2005) 4536e4539.
[18] M.G. Rabbani, M.R. Islam, M. Ahmad, A.M.L. Hossion, Synthesis of some NH-
Derivatives of ciprofloxacin as antibacterial and antifungal agents,
Bangladesh J. Pharmacol. 6 (2011) 8e13.
[19] R. Cormier, W.N. Burda, L. Harrington, J. Edlinger, K.M. Kodigepalli, J. Thomas,
R. Kapolka, G. Roma, B.E. Anderson, E. Turos, L.N. Shaw, Studies on the anti-
microbial properties of N-acylated ciprofloxacins, Bioorg. Med. Chem. Lett. 22
(2012) 6513e6520.
penicillin and 100 mg/mL of streptomycin were added to the media.
The cell cultures were incubated at 37 ꢃC in a humidified atmo-
sphere with 5% CO2.
The investigated compounds were dissolved in dimethyl sulf-
oxide (50 mg/mL) and then diluted in cell culture media supple-
mented with 2% FBS. HEK-293 cells were placed into 96-well plastic
plates (Nunc, Roskilde, Denmark) at a cell density of 3 ꢄ 105 cells per
well. After 24 h of incubation at 37 ꢃC, the media were removed and
cells treated with the derivatives, diluted in media at final concen-
trations of 2e500 m
g/mL. Cell cultures were incubated at 37 ꢃC for
48 h. The cytotoxicity was estimated using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) that is cleaved into
a coloured formazan product by metabolically active cells, according
to the assay described by Takenouchi and Munekata [30]. The
quantity of the formazan product was measured in an automatic
plate reader. From the obtained results the EC50 (concentration of
the substance which inhibits cells growth in 50% in proportion to the
growth of control cells) values were calculated. The results were
given as mean ꢂ SD of three independent experiments.
Acknowledgements
The project was supported by the Research Grant for Young
Scientists.
[20] J.C. McPherson III, R. Runner, T.B. Buxton, J.F. Hartmann, D. Farcasiu,
}
I. Bereczki, E. Roth, S. Tollas, E. Ostorházi, F. Rozgonyi, P. Herczegh, Synthesis
of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone anti-
bacterials, Eur. J. Med. Chem. 47 (2012) 615e618.
References
[21] S. Wang, X.-D. Jia, M.-L. Liu, Y. Lu, H.-Y. Guo, Synthesis, antimycobacterial and
antibacterial activity of ciprofloxacin derivatives containing a N-substituted
benzyl moiety, Bioorg. Med. Chem. Lett. 22 (2012) 5971e5975.
[22] A. Foroumadi, S. Ghodsi, S. Emami, S. Najjari, N. Samadi, M.H. Faramarzi,
L. Beikmohammadi, F.H. Shirazi, A. Shafiee, Synthesis and antibacterial activity
of new fluoroquinolones containing a substituted N-(phenethyl)piperazine
moiety, Bioorg. Med. Chem. Lett. 16 (2006) 3499e3503.
[23] T. Plech, M. Wujec, A. Siwek, U. Kosikowska, A. Malm, Synthesis and antimi-
crobial activity of thiosemicarbazides, s-triazoles and their Mannich bases
bearing 3-chlorophenyl moiety, Eur. J. Med. Chem. 46 (2011) 241e248.
[24] T.D. Tran, T.H. Do, N.C. Tran, T.D. Ngo, T.N.P. Huynh, C.D. Tran, K.M. Thai,
Synthesis and anti methicillin resistant Staphylococcus aureus activity of
substituted chalcones alone and in combination with non-beta-lactam anti-
biotics, Bioorg. Med. Chem. Lett. 22 (2012) 4555e4560.
[25] T. Welte, M.W. Pletz, Antimicrobial treatment of nosocomial methicillin-
resistant Staphylococcus aureus (MRSA) pneumonia: current and future
options, Int. J. Antimicrob. Agents 36 (2010) 391e400.
[26] G.A. Pankey, L.D. Sabath, Clinical relevance of bacteriostatic versus bactericidal
mechanisms of action in the treatment of Gram-positive bacterial infections,
Clin. Infect. Dis. 38 (2004) 864e870.
[27] F. Kautzky, A. Hartinger, L.D. Köhler, H.J. Vogt, In vitro cytotoxicity of anti-
microbial agents to human keratinocytes, J. Eur. Acad. Dermatol. Venereol. 6
(1996) 159e166.
[28] M.J. Nieto, F.L. Alovero, R.H. Manzo, M.R. Mazzieri, Benzenesulfonamide
analogs of fluoroquinolones. Antibacterial activity and QSAR studies, Eur. J.
Med. Chem. 40 (2005) 361e369.
[29] H. Nikaido, Antibiotic resistance caused by Gram-negative multidrug efflux
pump, Clin. Infect. Dis. 27 (1998) 23e41.
[30] T. Takenouchi, E. Munekata, Amyloid beta-peptide-induced inhibition of MTT
reduction in PC12h and C1300 neuroblastoma cells: effect of nitroprusside,
Peptides 19 (1998) 365e372.
[1] J.T. Cross Jr., Fluoroquinolones, Semin. Pediatr. Infect. Dis. 12 (2001) 211e223.
[2] S. Emami, A. Shafiee, A. Foroumadi, Structural features of new quinolones and
relationship to antibacterial activity against Gram-positive bacteria, Mini Rev.
Med. Chem. 6 (2006) 375e386.
[3] M. Berlanga, M.T. Montero, J. Hernandez-Borrell, M. Vinas, Influence of the cell
wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and
Gram-positive bacteria, Int. J. Antimicrob. Agents 23 (2004) 627e630.
[4] P.S. Charifson, A.L. Grillot, T.H. Grossman, J.D. Parsons, M. Badia, S. Bellon,
D.D. Deininger, J.E. Drumm, C.H. Gross, A. LeTiran, Y.S. Liao, N. Mani,
D.P. Nicolau, E. Perola, S. Ronkin, D. Shannon, L.L. Swenson, Q. Tang,
P.R. Tessier, S.K. Tian, M. Trudeau, T.S. Wang, Y.Y. Wei, H. Zhang, D. Stamos,
Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topo-
isomerase IV possessing potent antibacterial activity: intelligent design and
evolution through the judicious use of structure-guided design and structure-
activity relationships, J. Med. Chem. 51 (2008) 5243e5263.
[5] F. Alovero, X.-S. Pan, J. Morris, R. Manzo, L. Fisher, Engineering the specificity
of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of
ciprofloxacin change its primary target in Streptococcus pneumoniae from
topoisomerase IV to gyrase, Antimicrob. Agents Chemother. 44 (2000) 320e
325.
[6] H. Nikaido, D.G. Thanassi, Penetration of lipophilic agents with multiple
protonation sites into bacterial cells: tetracyclines and fluoroquinolones as
examples, Antimicrob. Agents Chemother. 37 (1993) 1393e1399.
[7] J.S. Chapman, N.H. Georgopapadakou, Routes of quinolone permeation in
Escherichia coli, Antimicrob. Agents Chemother. 32 (1988) 438e442.
[8] A.J.H. Marshall, L.J.V. Piddock, Interaction of divalent cations, quinolones and
bacteria, J. Antimicrob. Chemother. 34 (1994) 465e483.
[9] T.D. Gootz, K.E. Brighty, Fluoroquinolone antibacterials: SAR, mechanism of
action, resistance, and clinical aspects, Med. Res. Rev. 16 (1996) 433e486.
[10] L.L. Shen, L.A. Mitscher, P.N. Sharma, T.J. O’Donnell, D.W.T. Chu, C.S. Cooper,
T. Rosen, A.G. Pernet, Mechanism of inhibition of DNA gyrase by quinolone