Novel N-4-Piperazinyl Ciprofloxacin-Ester Hybrids: Synthesis, Biological Evaluation, and Molecular Docking Studies

Article abstract of DOI:10.1134/S1070363220080265

Abstract: A series of novel N-4-piperazinylciprofloxacin-ester hybrids has been synthesized and the structures confirmed by¹H and ¹³C NMR, FT-IRspectral data, and elemental analysis. The products have been tested in vitro for their antibacterial activity againstsix bacterial strains (MRSA, Staphylococcusepidermidis, Bacillussubtilis, Escherichia coli,Salmonella enterica, and Klebsiella pneumoniae) and have demonstrated goodantibacterial activity with MIC values range 6.25–200 μg/mL. Antifungal andcytotoxic activities of the products have been tested against Candida kefyr and human leukemia K562 cell line,respectively. All compounds inhibit growth of K562 cells more efficiently thanthe parent ciprofoxacin in a dose- and duration-dependent way. Molecular dockingstudies performed for the compound 3i indicatesthat similarly to ciprofloxacin it can act as an inhibitor of S. aureus DNA gyrase.

Full text of DOI:10.1134/S1070363220080265

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 8, pp. 1558–1565. © Pleiades Publishing, Ltd., 2020.
Novel N-4-Piperazinyl Ciprofloxacin-Ester Hybrids: Synthesis,
Biological Evaluation, and Molecular Docking Studies
A. Shahbazia, H. Mostafavia,*, G. Zarrinib, and M. Mahdavib
a Department of Organic Chemistry and Biochemistry, University of Tabriz, Tabriz, 5166614766 Iran
b Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz, 5166614766 Iran
*e-mail: hmostafavi@tabrizu.ac.ir
Received May 29, 2020; revised July 30, 2020; accepted August 6, 2020
Abstract—A series of novel N-4-piperazinyl ciprofloxacin-ester hybrids has been synthesized and the structures
confirmed by 1H and 13C NMR, FT-IR spectral data, and elemental analysis. The products have been tested in vitro
for their antibacterial activity against six bacterial strains (MRSA, Staphylococcus epidermidis, Bacillus subtilis,
Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae) and have demonstrated good antibacterial
activity with MIC values range 6.25–200 μg/mL. Antifungal and cytotoxic activities of the products have been
tested against Candida kefyr and human leukemia K562 cell line, respectively. All compounds inhibit growth of
K562 cells more efficiently than the parent ciprofoxacin in a dose- and duration-dependent way. Molecular dok-
king studies performed for the compound 3i indicates that similarly to ciprofloxacin it can act as an inhibitor of
S. aureus DNA gyrase.
Keywords: antibacterial, anticancer, molecular docking, quinolone, ciprofloxacin
DOI: 10.1134/S1070363220080265
INTRODUCTION
ciprofloxacin and evaluated their biological properties.
Various types of aromatic, heteroaromatic and mono-
terpene alcohols that demonstrated antibacterial or
anticancer properties [8–12] have been used in the
synthesis. Also the target compounds were tested for
anticancer activity against human leukemia K562 cell
line. Docking studies have been performed for compound
3i for determining its possible bindings modes with
S. aureus DNA gyrase.
Ciprofloxacin is a second-generation fluoroquinolone
antibiotic that is widely used for treatment of a variety
of infections. The widespread use and misuse of
fluoroquinolones have resulted in development of
bacterial resistance to those which made it necessary
to produce novel drugs with the broad-spectrum of
antibiotic activity, high potency and few side effects [1].
Modification of the structures of existing fluoroquinolone
agents is a fruitful strategy to overcome the resistance
effect and increase their potency [2].
RESULTS AND DISCUSSION
Synthesis of the target compounds was carried out
by a two-step procedure depicted in Scheme 1. The
intermediate compounds 2a–2j were synthesized by
treatment of alcohols 1a–1j with chloroacetic acid in
the presence of 1,3-dicyclohexylcarbodiimide (DCC),
4-dimethylaminopyridine (DMAP) and CeCl3·7H2O.
Without a catalyst, by-products were formed. The
esterification method was first optimized for the reaction
of β-naphthol (1 mmol) with chloroacetic acid (1.2 mmol)
(Table 1, entries 1–14). The effect of three catalysts,
SnCl2, CeCl3·7H2O, and Montmorillonite K10, was
studied in the presence of DMAP (15 mol %) and DCC
(1.2 mmol) in DCM media. The best result was achieved
with CeCl3·7H2O. The following screening of reaction
NatureofsubstituentsinpositionC7 offluoroquinolones
has a considerable impact on their antibacterial potency,
spectral characteristics, solubility, pharmacokinetics,
bioavailability, safety, and the preferred target of those
[3, 4]. Physicochemical properties of fluoroquinolones,
such as charge, lipophilicity and molecular mass play a
significant role in their penetration into the bacterial cell
[5, 6]. Introduction of carboxymethyl in N4 position of the
piperazine ring of ciprofloxacin can lead to its increased
antibacterial activity [7].
Based on the above and in development of our ongoing
research program of creating drug agents with high
potency and broad-spectrum of antibiotic activity, we
have synthesized various esters of N-4-carboxymethyl
1558

NOVEL N-4-PIPERAZINYL CIPROFLOXACIN-ESTER HYBRIDS
Scheme 1. Synthesis of the target compounds 3a–3j.
1559
conditions indicated 20 mol % of CeCl3·7H2O and
20 mol % of DMAP in DCM solvent as optimum.
Comparison of antibacterial activity of compounds 3a
and 3e indicated that replacement of naphthol by phenol
reduced lipophilicity and antibacterial activity (except
for E. coli) of compound 3e. The product 3i had lower
lipophilicity compared to compounds 3d and 3f but
demonstrated higher antibacterial activity than the latter
ones. Replacing pyridin-2-ylmethyl with thiophen-2-
ylmethyl and 4-methoxybenzyl reduced the antibacterial
activity. Compounds 3g and 3h had the same molecular
weight and close ClogP values but antibacterial activity
of 3h was 2–8 times higher than 3g. Compound 3i
characterized by the lowest lipophilicity, in most cases
exhibited better or equal antibacterial activity with the
other products. The above indicated that lipophilicity
of the alkoxy(aryloxy) moiety did not play an essential
role in potency of the products.Antifungal activity of the
compounds was tested against Candida kefyr, and all of
those exhibited moderate antifungal activity.
In the second step, the reaction of 2a–2j with
ciprofloxacin in the presence of TEA as a base gave
the corresponding ciprofloxacin derivatives. The target
1
compounds were identified by FT-IR, H NMR, 13C
NMR, and elemental analysis.
Antimicrobial activity. According to the accumulated
antimicrobial activity data (Table 2) the synthesized
compounds exhibited moderate to good activities against
Gram-positive and Gram-negative bacteria. The pyridine
and menthol derivatives 3h and 3i were the most active
against Escherichia coli. Compound 3b demonstrated
the highest activity against Klebsiella pneumonia. All
synthesized compounds showed low activity against
K. pneumoniae and S. enterica. The products 3c and 3i
exhibited the best activity against MRSA, and compounds
3h and 3j were the most active against B. subtilis.
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SHAHBAZI et al.
Table 1. Optimization of synthesis of β-naphthol
Entry no.
1
2
3
4
5
6
7
8
9
10
11
12b
13
14
Catalyst (mol %)
SnCl2 (15%) + DMAP (15%) + DCC (1.2 mmol)
Solvent
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
THF
Time, h
12
Yielda, %
65
Montmorillonite k10 (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (15%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (10%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (20%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (25%) + DMAP (15%) + DCC (1.2 mmol)
CeCl3·7H2O (20%) + DMAP (10%) + DCC (1.2 mmol)
CeCl3·7H2O (20%) + DMAP (20%) + DCC (1.2 mmol)
CeCl3·7H2O (20%) + DMAP (25%) + DCC (1.2 mmol)
CeCl3·7H2O (20%) + DMAP (20%) + DCC (1.2 mmol)
DMAP (15%) + DCC (1.2 mmol)
12
12
12
12
12
12
12
12
12
12
12
12
12
24
68
74
70
63
51
67
71
76
76
75
78
78
78
61
DMF
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
15
a Isolated yields.
b Optimized condition.
Table 2. Minimum inhibitory concentrations of the products
MIC, μg/mL
E.coli K. pneumoniae S. enterica MRSA B. subtilis S. epidermidis
Comp.
no.
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
cpiro
Mw
ClogP
C.kefyr
25
25
50
50
50
50
50
25
25
25
100
50
100
100
200
200
200
200
200
100
100
100
1.56
50
50
25
25
25
25
515.54
583.62
535.57
485.53
465.48
509.53
527.64
527.64
480.50
555.61
385.82
2.31
2.64
2.01
1.27
1.13
1.61
3.99
3.97
0.13
2.97
1.86
100
100
25
100
100
12.5
12.5
25
200
200
200
200
200
100
100
200
3.123
12.5
50
100
50
100
50
12.5
50
50
50
50
50
50
6.25
25
6.25
1.56
50
50
50
50
50
25
25
25
25
50
5
1.56
6.25
0.39
Anticancer activity. Cytotoxicity of the products was
evaluated for antiproliferative activity against human
leukemia K562 cell line using the colorimetric MTT
assay. For each compound, various concentrations
(10–150 μM) were tested for 24, 48, and 72 h (Table 3).
Molecular docking. Molecular docking of compound
3i was carried out using MOE 2015.10 in order to
determine possible binding modes of the compound to
the S. aureus DNA gyrase. Compound 3i demonstrated
good antibacterial activity against most bacterial strains;
therefore, it was selected for molecular docking. The
co-crystallized ligand, ciprofloxacin, was re-docked into
active site of target protein for validating the docking
protocol. The root mean square deviation (RMSD) for
re-dock pose of the co-crystallized ligand was found
to be 0.7963 (<2.0 Å), which confirmed the docking
validation. Re-docking results showed that the docked
The results of the MTT assay indicated that all
compounds significantly inhibited growth of K562 cells
in a dose- and duration-dependent manner. Among the
newly synthesized compounds, 3g, 3h, and 3j derivatives
with the highest lipophilicity exhibited the best antitumor
activity in comparison with the other synthesized
compounds.
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NOVEL N-4-PIPERAZINYL CIPROFLOXACIN-ESTER HYBRIDS
1561
Table 3. Cytotoxicity against human leukemia K562 cell line
ciprofloxacin was almost superimposed on the native co-
crystallized ligand and exhibited similar interactions as
that of the original crystal structure. The best-ranked pose
of compound 3i was selected on the basis of docking score
(S). In vitro bioassays data and the molecular docking
results suggested that compound 3i was a potential
S. aureus DNA gyrase inhibitor. The best ranking pose
of compound 3i is shown in Fig. 1.
IC50, μM
Compound
24 h
>150
>150
98±7.0
>150
145±2.0
148±2.0
42±3.0
32±2.0
84±1.0
60±5.0
>150
48 h
150±5.0
105±3.0
81±4.0
>150
120±3.0
60±5.0
40±1.0
30±1.0
67±3.0
42±4.0
>150
72 h
90±2.0
70±5.0
18±2.0
50±2.0
40±5.0
56±4.0
30±3.0
20±2.0
46±4.0
17±3.0
>150
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
cipro
Doxa
The 2D ligand interactions of compound 3i in DNA
gyrase A demonstrated that two oxygen atoms of the
4-carbonyl and 3-carboxylate groups of quinolone ring
formed coordination bonds with Mn2+ with 2.60 and
2.40 Å distances, respectively. Quinolone ring formed π–π
stacking interactions with the aromatic ring of DG38 and
DG59. The methylene of piperazine and DC13 formed
CH-π contacts (3.66 Å).
1.6±0.1
0.83±0.05
a Doxorubicin (Dox) as a positive control.
0.54±0.02
EXPERIMENTAL
4-dimethylaminopyridine (DMAP) (20 mol %), and
CeCl3·7H2O (20 mol %) in dry dichloromethane
(4 mL) was ad. ded N,N′-dicyclohexylcarbodiimide (DCC)
(1.2 mmol). The mixture was stirred for 30 min at 0°C and
then for 12 h at room temperature. The suspension was
filtered and washed with dichloromethane. The organic
phase was washed with 2 M HCl (3 × 10 mL) and brine
(3 × 10 mL), dried over MgSO4, filtered and concentrated
on a rotary evaporator. The crude residue was used
without further purification in the next step.
All reagents were purchased from Fluka, Merck and
Sigma-Aldrich companies. Melting points (uncorrected)
were determined on an Electrothermal MEL-TEMP
apparatus model 9100. IR spectra were recorded on a
Shimadzu (model 8101M) FT-IR spectrophotometer
using KBr disks. 1H and 13C NMR spectra were measured
on a Bruker Spectrospin Avance 400 MHz spectrometer
in CDCl3 or DMSO-d6 at 25°C. Reactions progress was
monitored by TLC performed on Merck silica gel 60
F254 plates using a UV lamp for visualization of spots
at 254 and 365 nm. Elemental analysis was carried out
on a Heraeus CHN-O-rapid analyser.
Synthesis of compounds 3a–3j. A mixture of
ciprofloxacin.HCl (1 mmol) with TEA (3 mmol) was
stirred in DMSO (5 mL) at room temperature for 5 min.
A compound 2a–2j (1 mmol) was ad. ded to the solution
of ciprofloxacin. The reaction mixture was stored to its
completion (according to TLC). Distilled water (20 mL)
Synthesis of alkyl and aryl 2-chloroacetate deri-
vatives 2a–2j. To a stirred at 0°C mixture of an
alcohol 1a–1j (1mmol), chloroacetic acid (1.2 mmol),
(a)
(b)
Fig. 1. (a) 3D and (b) 2D docking of compound 3i with S. aureus DNA gyrase.
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SHAHBAZI et al.
7-{4-[2-(4-Allyl-2-methoxyphenoxy)-2-oxoethyl]-
piperazin-1-yl}-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (3c). White
solid, yield 79%, mp 190–191°C. IR spectrum, ν,
cm–1: 1770 (C=O, ester), 1724 (C=O, carboxylic acid),
was ad. ded and pH of the mixture was adjusted to 6 by
2 M HCl which resulted in precipitation of white or light
yellow solid. The suspension layer was extracted with
DCM (3 × 15 mL). The combined organic layers were
washed with brine (3 × 15 mL) and dried over MgSO4. The
organic layer was concentrated under reduced pressure
on a rotary evaporator. The product was washed with
distilled water (3 × 5 mL) and diethyl ether (3 × 5 mL).
The corresponding pure compounds 3a–3j were obtained
as white or light yellow solids.
1
1626 (C=O, quinolone). H NMR spectrum, δ, ppm:
1.17–1.24 m (2H, CH2, cyclopropyl), 1.35–1.43 m
(2H, CH2, cyclopropyl), 2.97 br. s (4H, 2CH2,
piperazine), 3.38 d (2H, J = 6.7 Hz, CH2=CH–
CH2), 3.44 t (4H, 2CH2, piperazine), 3.52–3.59 m
(1H, CH cyclopropyl), 3.64 s (2H, N–CH2–CO), 3.82 s
(3H, OCH3), 4.9–5.2 m (2H, CH2=CH–CH2), 5.8–6.1 m
(1H, CH2=CH–CH2), 6.6–6.9 m (2H, C6H3), 6.97 d (J =
7.9 Hz, 1H, C6H3), 7.38 d (J= 7.1 Hz, 1H, CH8 cipro), 8.0 d
(J = 12.9 Hz, 1H, CH5 cipro), 8.76 s (1H, CH2 cipro),
15.03 s (1H, COOH). 13C NMR spectrum, δC, ppm: 7.1,
34.2, 38.9, 48.4 d (J = 4.5 Hz), 51.2, 54.7, 57.5, 103.9 d
(J = 3 Hz), 106.9, 111.2 d (J = 23 Hz), 111.5, 115.2, 118.7 d
(J = 7.8 Hz), 119.6, 121.2, 135.8, 136.4, 137.9, 138.2,
144.7 d (J = 10 Hz), 146.3, 149.5, 152.6 d (J = 251 Hz),
165.9, 166.8, 175.9 d (J = 2.4 Hz). Found, %: C 64.83,
H 5.82, N 7.84. C29H30FN3O6. Calculated, %: C 65.04,
H 5.65, F 3.55, N 7.85, O 17.92. M 535.
1-Cyclopropyl-6-fluoro-7-{4-[2-(naphthalen-
2-yloxy)-2-oxoethyl]piperazin-1-yl}-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (3a). White solid,
yield 70%, mp 149–150°C. IR spectrum, ν, cm–1: 1768
(C=O, ester), 1726 (C=O, carboxylic acid), 1629 (C=O,
quinolone). 1H NMR spectrum, δ, ppm: 1.13–1.18 m (2H,
CH2, cyclopropyl), 1.25–1.40 m (2H, CH2, cyclopropyl),
3.02 br. s (4H, 2CH2, piperazine), 3.81 br. s (1H, CH
Cyclopropyl), 3.89 br. s (2H, N–CH2–CO), 7.33–7.39 d. d
(J = 8.8, 1.9 Hz, 1H, C10H7), 7.49–7.60 m (3H, C10H7 +
CH8 cipro), 7.72 s (1H, C10H7), 7.84–8.03 m (4H, C10H7 +
CH5 cipro), 8.64 s (1H, CH2 cipro), 15.20 s (1H, COOH).
13C NMR spectrum, δC, ppm: 7.6, 35.8, 48.9, 51.5, 57.5,
106.4, 106.7, 110.9 d (J = 23 Hz), 118.5, 118.7, 121.4,
125.9, 126.8, 127.5, 127.7, 129.4, 131, 133.3, 139, 144.9,
147.7, 147.9, 152.9 d (J = 249 Hz), 165.9, 168.4, 176.2.
Found, %: C 67.43, H 5.27, N 8.12. C29H26FN3O5.
Calculated, %: C 67.56, H 5.08, F 3.69, N 8.15, O 15.52.
M 515.
1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[2-oxo-2-
(thiophen-2-ylmethoxy)ethyl]piperazin-1-yl}-1,4-
dihydroquinoline-3-carboxylic acid (3d). White solid,
yield 69%, mp 179–180°C. IR spectrum, ν, cm–1: 1737
(C=O, ester), 1726 (C=O, carboxylic acid), 1626 (C=O,
quinolone). 1H NMR spectrum, δ, ppm: 1.17–1.23 m (2H,
CH2, cyclopropyl), 1.33–1.43 m (2H, CH2, cyclopropyl),
2.83 t (4H, 2CH2, piperazine), 3.36 s (2H, N–CH2–
CO), 3.40 t (4H, 2CH2, cyclopropyl), 3.57 m (1H, CH,
cyclopropyl), 5.35 s (2H, COOCH2), 7.00 d. d (J = 4.8,
3.7 Hz, 1H, CH4 thiophen), 7.12 d (J = 3.3 Hz, 1H, CH3
thiophen), 7.32–7.35 d (J = 5.2 Hz, 1H, CH5 thiophen),
7.35–7.38 d (J = 7.2 Hz, 1H, CH8 cipro), 7.92 d (J =
13 Hz, 1H, CH5 cipro), 8.71 s (1H, CH2 cipro), 15.02 s
(1H, COOH). 13C NMR spectrum, δC, ppm: 7.1, 34.2,
48.5 d (J = 4.8 Hz), 51.3, 57.8, 59.5, 103.8 d (J = 3 Hz),
106.8, 111.1 d (J = 23 Hz), 118.7, 125.8, 126, 127.4,
136.3, 137.9, 144.7 d (J = 10.4 Hz), 146.2, 152.4 d (J =
251 Hz), 165.8, 168.6, 175.8. Found, %: C 58.87, H 5.16,
N 8.57. C24H24FN3O5S. Calculated, %: C 59.37, H 4.98,
F 3.91, N 8.65, O 16.48, S 6.6. M 485.
1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[2-oxo-2-(2-
oxo-1,2-diphenylethoxy)ethyl]piperazin-1-yl}-1,4-
dihydroquinoline-3-carboxylic acid (3b). White solid,
yield 75%, mp 178–179°C. IR spectrum, ν, cm-1: 1727
(C=O, ester), 1698 (C=O, carboxylic acid), 1628 (C=O,
1
quinolone). H NMR spectrum, δ, ppm: 1.15–1.22 m
(2H, CH2, cyclopropyl), 1.35–1.42 m (2H, CH2,
cyclopropyl), 2.90 t (4H, 2CH2, piperazine), 3.40 q (4H,
2CH2, piperazine), 3.45–3.62 m (3H, N–CH2–CO + CH,
cyclopropyl), 6.92 s (1H, O–CH–CO), 7.34–7.50 m (8H,
C6H5 + CH8 cipro), 7.50–7.56 m (1H, C6H5), 7.90–8.01 m
(3H, C6H5 + CH5 cipro), 8.73 s (1H, CH2 cipro), 15.06 s
(1H, COOH). 13C NMR spectrum, δC, ppm: 7.1, 34.2,
48.5, 51.1, 57.4, 77, 103.9, 107, 111.3 d (J = 23 Hz),
118.8, 127.6, 127.7, 128.2, 128.5, 131.9, 132.6, 133.3,
138, 144.7 d (J = 9.4 Hz), 146.3, 152.6 d (J = 235 Hz),
165.9, 168.3, 176, 192.3. Found, %: C 68.06, H 5.36, N
7.33. C33H30FN3O6. Calculated, %: C 67.91, H 5.18, F
3.26, N 7.2, O 16.45. M 583.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-(2-oxo-2-
phenoxyethyl)piperazin-1-yl]-1,4-dihydroquinoline-
3-carboxylic acid (3e). White solid, yield 61%. mp
217–218°C. IR spectrum, ν, cm–1: 1766 (C=O, ester),
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NOVEL N-4-PIPERAZINYL CIPROFLOXACIN-ESTER HYBRIDS
1563
1725 (C=O, carboxylic acid), 1626 (C=O, quinolone).
1H NMR spectrum, δ, ppm: 1.18–1.21 m (2H, CH2,
cyclopropyl), 1.35–1.41 m (2H, CH2, cyclopropyl), 2.94 t
(4H, 2CH2, piperazine), 3.43 t (4H, 2CH2, piperazine),
3.56 m (1H, CH, cyclopropyl), 3.60 s (2H, N–CH2–CO),
7.08–7.15 m (2H, C6H5), 7.22–7.29 m (1H, C6H5), 7.35–
7.43 m (3H, C6H5 + CH8 cipro), 7.95 d (J = 13 Hz, 1H,
CH5 cipro), 8.72 s (1H, CH2 cipro), 15.02 s (1H, COOH).
13C NMR spectrum, δC, ppm: 7.1, 34.2, 48.6 d (J =
4.8 Hz), 51.3, 57.8, 103.8 d (J = 2.6 Hz), 106.9, 111.2 d
(J = 23 Hz), 118.7 d (J = 8 Hz), 120.2, 124.9, 128.3, 137.9,
144.7 d (J = 10.5 Hz), 146.2, 149.1, 152.5 d (J = 251 Hz),
165.8, 167.4, 175.9. Found, %: C 64.32, H 5.29, N 8.91.
C25H24FN3O5. Calculated, %: C 64.51, H 5.2, F 4.08, N
9.03, O 17.19. M 465.
(CH3)2CCH], 7.37 d (J = 7.1 Hz, 1H, CH8 cipro), 7.98 d
(J = 13 Hz, 1H, CH5 cipro), 8.73 s (1H, CH2 cipro), 15.02 s
(1H, COOH). 13C NMR spectrum, δC, ppm: 7.1, 16.6,
18.3, 24.3, 24.6, 28.4, 34.2, 34.4, 35.8, 48.6 d (J = 4.8),
51.4, 58, 62.3, 103.8, 107, 111.3 d (J = 24 Hz), 118.8 d
(J = 7.7 Hz), 123.4, 130.3, 138, 144.7 d (J = 10.4 Hz),
146.3, 152.5 d (J = 251 Hz), 165.9, 169.1, 176. Found,
%: C 65.89, H 7.41, N 7.68. C29H38FN3O5. Calculated,
%: C 66.01, H 7.26, F 3.6, N 7.96, O 15.16. M 527.
1-Cyclopropyl-6-fluoro-7-[4-(2-{[(1R,2S,5R)-
2-isopropyl-5-methylcyclohexyl]oxy}-2-oxoethyl)-
piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (3h). White solid, yield 77%, mp 218–
219°C. IR spectrum, ν, cm–1: 1737 (C=O, ester), 1628
(C=O, Quinolone). 1H NMR spectrum, δ, ppm: 0.78 d [3H,
J = 6.9 Hz, (CH3)2CH)], 0.81–0.88 m [1H, CH3CH(CH2)
CH2CH2], 0.88–0.94 m [6H, CH3CH + (CH3)2CH],
0.96–1.13 m (2H, CH–CH2–CH + CH–CH–CH2–CH2),
1.17–1.26 m (2H, CH2, cyclopropyl), 1.36–1.43 m
(3H, CH2, cyclopropyl, + O–CH–CH), 1.43–1.56 m
(1H, CH2–CH–CH2), 1.65–1.73 m (2H, CH–CH2–
CH2–CH), 1.79–1.90 m [1H, (CH3)2CH], 1.95–2.05 m
(1H, O–CH–CH2), 2.83 br. d (4H, 2CH2, piperazine),
3.30 d. d (J = 23 Hz, 16.5, 2H, N–CH2–CO) 3.41 br. s
(4H, 2CH2, piperazine), 3.57 br. s (1H, CH, cyclopropyl),
4.73–4.82 m (1H, O–CH–CH), 7.37 d (J = 7.1 Hz, 1H,
CH8 cipro), 7.97 d (J = 13 Hz, 1H, CH5 cipro), 8.74 s (1H,
CH2 cipro), 15.03 s (1H, COOH). 13C NMR spectrum, δC,
ppm: 7.1, 15.3, 19.6, 20.9, 22.4, 25.3, 30.3, 33.1, 34.2,
39.9, 45.9, 48.6 d (J = 4.7 Hz), 51.4, 58.2, 73.7, 103.8 d
(J = 3.2 Hz), 107, 111.3 d (J = 23.4 Hz), 118.8 d (J =
8.04 Hz), 138, 144.5 d (J = 10.3 Hz), 146.3, 152.5 d (J =
251 Hz), 165.9, 168.6, 176. Found, %: C 65.92, H 7.39,
N 7.92. C29H38FN3O5. Calculated, %: C 66.01, H 7.26,
F 3.6, N 7.96, O15.16. M 527.
1-Cyclopropyl-6-fluoro-7-{4-(2-[(4-methoxybenzyl)-
oxy)-2-oxoethyl]piperazin-1-yl}-4-oxo-1,4-di-
hydroquinoline-3-carboxylic acid (3f). White solid,
yield 67%, mp 179–180°C. IR spectrum, ν, cm–1: 1744
(C=O, ester), 1730 (C=O, carboxylic acid), 1628 (C=O,
quinolone). 1H NMR spectrum, δ, ppm: 1.18–1.22 m (2H,
CH2, cyclopropyl), 1.35–1.43 m (2H, CH2, cyclopropyl),
2.8–2.86 t (4H, 2CH2, piperazine), 3.34 s (2H, N–CH2–
CO), 3.37–3.41 t (4H, 2CH2, piperazine), 3.53–3.61 m
(1H, CH, cyclopropyl), 3.81 s (3H, OCH3), 5.12 s (2H,
COOCH2), 6.87–6.91d(J=8.5Hz, 2H, C6H4), 7.29–7.34d
(J = 8.5 Hz, 2H, C6H4), 7.36 d (J = 7.1 Hz, 1H, CH8
cipro), 7.96 d (J = 13 Hz, 1H, CH5 cipro), 8.73 s (1H,
CH2 cipro), 15.01 s (1H, COOH). 13C NMR spectrum,
δC, ppm: 7.1, 34.2, 48.6 d (J = 4.8 Hz), 51.4, 54.2, 57.9,
65.3, 103.8 d (J = 2.9 Hz), 107, 111.3 d (J = 23 Hz),
112.9, 118.8 d (J = 8.1 Hz), 129.25, 129.29, 138, 144.7 d
(J = 10.5 Hz), 146.3, 152.1 d (J = 251 Hz), 158.7,
165.9, 168.8, 176. Found, %: C 63.89, H 5.48, N 8.12.
C27H28FN3O6. Calculated, %: C 63.65, H 5.54, F 3.73,
N 8.25, O 18.84. M 509.
1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[2-oxo-2-
(pyridin-2-ylmethoxy)ethyl]piperazin-1-yl}-1,4-
dihydroquinoline-3-carboxylic acid (3i). White solid,
yield 76%, mp 190–191°C. IR spectrum, ν, cm–1: 1757
(C=O, ester), 1730 (C=O, carboxylic acid), 1628 (C=O,
1-Cyclopropyl-7-(4-{2-[(3,7-dimethyloct-6-en-1-yl)-
oxy]-2-oxoethyl}piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (3g). Light yellow
solid, yield 74%, mp 97–98°C. IR spectrum, ν, cm–1: 1739
(C=O, ester), 1627 (C=O, quinolone). 1H NMR spectrum,
δ, ppm: 0.93 d (3H, CH3–CH), 1.15–1.22 m (3H, CH2,
cyclopropyl + C=CH2–CH2–CH2), 1.25–1.85 m [12H,
CH2, cyclopropyl + (CH3)2CCHCH2CH2CH(CH3)CH2],
1.90–2.08 m (2H, CCHCH2), 2.81–2.86 t (4H, 2CH2,
piperazine), 3.32 s (2H, N–CH2–CO), 3.37–3.43 t (4H,
2CH2, piperazine), 3.52–3.61 m (1H, CH, cyclopropyl),
4.15–4.22 m (2H, COOCH2), 5.05–5.11 m [1H,
1
quinolone). H NMR spectrum, δ, ppm: 1.18–1.23 m
(2H, CH2, cyclopropyl), 1.36–1.43 m (2H, CH2,
cyclopropyl), 2.87 t (4H, 2CH2, piperazine), 3.40 t (4H,
2CH2, piperazine), 3.45 s (2H, N–CH2–CO), 3.52–3.59 m
(1H, CH, cyclopropyl), 5.30 s (1H, OCH2), 7.26 m (1H,
N–CH=CH), 7.37 d [J = 7.2 Hz, 2H, OCH2C(=N)CH +
CH8 cipro], 7.73 t. d (J = 7.69 Hz, 1.63, 1H, N–CH=CH–
CH), 7.97 d (J = 13 Hz, 1H, CH5 cipro), 8.61 d (J =
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 90 No. 8 2020

1564
SHAHBAZI et al.
4.3 Hz, 1H, N–CH–CH), 8.73 s (1H, CH2 cipro), 15.02 s
(1H,COOH).13CNMRspectrum,δC,ppm:7.1,34.2,48.4d
(J = 4.4 Hz), 51.3, 57.5, 66, 103.9, 107, 111.3 d (J =
23 Hz), 118.9 d (J = 7.5 Hz), 121, 122, 135.8, 138,
144.6 d (J = 10.5 Hz), 146.3, 148.5, 152.5 d (J = 251 Hz),
154, 165.8, 168.2, 176. Found, %: C 62.64, H 5.37, N
11.68. C25H25FN4O5. Calculated, %: C 62.49, H 5.24, F
3.95, N 11.66, O 16.65. M 480.
various concentrations (10–150 μM) of the synthesized
compounds for 24, 48, and 72 h. At the end of this
incubation, 10 μLof MTT (final concentration, 0.5 mg/mL
in PBS) was ad. ded to each well. The plates were
incubated in darkness for 4 h at 37°C, then the medium
of each well culture was carefully removed. Formazan
crystals produced by mitochondrial reductase from live
cells, were dissolved in 200 μL of DMSO. Absorbance
values were determined using a multi-well plate reader
(Elx 800 Microplate Reader, BioTEK) at 570 nm to
confirm the number of living cells. The IC50 value was
determined by plotting the percent of viability against
the dose of the compound ad. ded. The IC50 values of
target compounds were estimated by the MTT assay.
Doxorubicin (Dox) and untreated cells were used as
positive and negative controls, respectively. Data were
expressed as the average of three analyses ± standard
deviation.
7-{4-[2-(Benzhydryloxy)-2-oxoethyl]-
piperazin-1-yl}-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (3j). White solid,
yield 74%, mp 220–221°C. IR spectrum, ν, cm–1: 1744
(C=O, ester), 1726 (C=O, carboxylic acid), 1626 (C=O,
1
quinolone). H NMR spectrum, δ, ppm: 1.15–1.21 m
(2H, CH2, cyclopropyl), 1.34–1.39 m (2H, CH2,
cyclopropyl), 2.83 t (4H, 2CH2, piperazine), 3.79 t (4H,
2CH2, piperazine), 3.45 s (2H, N–CH2–CO), 3.50–3.56 m
(1H, CH, cyclopropyl), 6.96 s (1H, OCH), 7.28–7.37 m
(11H, 10CH, C6H5 + CH8 cipro), 7.98 d (J = 13 Hz, 1H,
CH5 cipro), 8.75 s (1H, CH2 cipro), 15.02 s (1H, COOH).
13C NMR spectrum, δC, ppm: 7.1, 34.2, 48.7 d (J =
4.7 Hz), 51.4, 58, 76.2, 103.9, 107, 111.4 d (J = 23 Hz),
118.8 d (J = 7.4 Hz), 126.1, 127, 127.5, 138, 138.8, 144.8 d
(J = 10.6 Hz), 146.4, 152.7 d (J = 251 Hz), 165.9, 168.1,
176.1. Found, %: C 69.07, H 5.61, N 7.45. C32H30FN3O5.
Calculated, %: C 69.18, H 5.44, F 3.42, N 7.56, O 14.4.
M 555.
Molecular docking analysis. The molecular
operating environment (MOE) 2015.10 software was used
for molecular docking calculations. The crystal structure
of gyrase DNAfrom S. aureus (PDB ID code 2XCT) was
obtained from RCSB Protein Data Bank (http://www.
rcsb.org/pdb/). The enzyme was subjected to protein
preparation using quick preparation method of MOE
2015.10 software. All parameters were kept as default
values. The 3D structure of compound 3i was constructed
by using ChemBio3D ultra 14.0 software. Ionization state
of the ligand at pH 7 was determined using the Protomers
panel in MOE software and the ionization state with the
highest population was selected. Compound 3i structure
was optimized to its min energy using MMFF94x force
field, and partial atomic charges were ad. ded in the MOE
software. The active sites of enzymes were identified
by the site finder application in MOE and defined as all
residues enclosed within 9 Å radius sphere centred by
the bound ligand. Docking calculations were done using
Triangle Matcher as the docking-placement function for
generating 1000 docking poses of each ligand. The best 30
poses were preselected based on the London dG scoring
function and further refined to produce the final ranking
by rescoring using the GBVI/WSA dG scoring function.
In vitro antimicrobial activity. All products were
assessed for their in vitro antibacterial activity against
three strains of Gram-positive bacteria: MRSA(S. aureus
ATCC 43300), Staphylococcus epidermidis (ATCC
12228), Bacillus subtilis (ATCC 6633), and three strains
of Gram-negative bacteria: Escherichia coli (ATCC
25922), Salmonella enterica (ATCC 14028), Klebsiella
pneumoniae (ATCC BAA-1705), and a fungus Candida
kefyr (ATCC 38296). Minimum inhibitory concentrations
(MICs) of the synthesized compound were determined
by the broth microdilution method in accordance with
the guidelines of the Clinical and Laboratory Standards
Institute (CLSI) [13].
MTT assay for cell viability. The anti-proliferative
effect of all synthesized compounds was measured on the
chronic myeloid leukemia cell lines (K562) employing
the MTT assay. The K562 cells were seeded in 96-well
microassay culture plates and grown in RPMI1640
supplemented with 10% fetal bovine serum (FBS),
1% penicillin and streptomycin in a 5% CO2 incubator
at 37°C for 24 h. The cells were then treated with
CONCLUSIONS
We have synthesized a series of novel N-4-piperazinyl
ciprofloxacin-ester hybrids. The structures of target
compounds have been characterized by spectral data.
Their antimicrobial and anticancer activity tests have
indicated good activities, especially against MRSA and
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 90 No. 8 2020

NOVEL N-4-PIPERAZINYL CIPROFLOXACIN-ESTER HYBRIDS
1565
and Rajak, H., J. Enzyme Inhib. Med. Chem., 2013,
vol. 28, p. 1.
https://doi.org/10.3109/14756366.2011.611943
Staphylococcus epidermidis. All products have shown
higher activity against gram-positive bacteria than gram-
negative bacteria. Compounds 3h and 3j are more potent
than the other products against B. subtilis. Compounds
3c and 3i demonstrate the highest antibacterial activity
against MRSA strain. Lipophilicity alone does not play
the major role in antibacterial potential of the synthesized
compounds. According to docking studies, compound 3i
and ciprofloxacin can form the coordination bond with
Mn2+ ion and π–π stacking interactions with DNAbases.
Cytotoxic activity of all products has been evaluated
against cell line K562, and their activity determined to
be higher than the parent ciprofloxacin.
6. Foroumadi, A., Ghodsi, S., Emami, S., Najjari, S.,
Samadi, N., Faramarzi, M.A., Beikmohammadi, L.,
Shirazi, F.H., and Shafiee, A., Bioorg. Med. Chem.
Lett., 2006, vol. 16, p. 3499.
https://doi.org/10.1016/j.bmcl.2006.03.103
7. Fardeau, S., Dassonville-Klimpt,A.,Audic, N., Sasaki,A.,
Pillon, M., Baudrin, E., Mullié, C., and Sonnet, P.,
Bioorg. Med. Chem., 2014, vol. 22, p. 4049.
https://doi.org/10.1016/j.bmc.2014.05.067
8. Sabbah, D.A., Saada, M., Khalaf, R.A., Bardaweel, S.,
Sweidan, K., Al-Qirim, T., Al-Zughier, A.,
Halim, H.A., and Sheikha, G.A., Bioorg. Med.
Chem. Lett., 2015, vol. 25, p. 3120.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the
Research Council of the University of Tabriz.
https://doi.org/10.1016/j.bmcl.2015.06.011
9. Marchese, A., Barbieri, R., Coppo, E., Orhan, I.E.,
Daglia, M., Nabavi, S. F., Izadi, M., Abdollahi, M.,
Nabavi, S.M., and Ajami, M., Crit. Rev. Microbiol.,
2017, vol. 43, p. 668.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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