New antiproliferative 7-(4-(N-substituted carbamoylmethyl)piperazin-1-yl) derivatives of ciprofloxacin induce cell cycle arrest at G2/M phase

Article abstract of DOI:10.1016/j.bmc.2016.07.070

New N-4-piperazinyl derivatives of ciprofloxacin 2a–g were prepared and tested for their cytotoxic activity. The primary in vitro one dose anticancer assay experienced promising cytotoxic activity against different cancer cell lines especially non-small c

Full text of DOI:10.1016/j.bmc.2016.07.070

Accepted Manuscript
New antiproliferative 7-(4-(N-substituted carbamoylmethyl) piperazin-1-yl de-
rivatives of ciprofloxacin induce cell cycle arrest at G2/M Phase
Hamada H.H. Mohammed, Amer Ali Abd El-Hafeez, Samar H. Abbas, El-
Shimaa M.N. Abdelhafez, Gamal El-Din A. Abuo-Rahma
PII:
S0968-0896(16)30593-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.070
BMC 13183
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 May 2016
28 July 2016
31 July 2016
Please cite this article as: Mohammed, H.H.H., El-Hafeez, A.A.A., Abbas, S.H., Abdelhafez, E.M.N., Abuo-Rahma,
G.E-D., New antiproliferative 7-(4-(N-substituted carbamoylmethyl) piperazin-1-yl derivatives of ciprofloxacin
induce cell cycle arrest at G2/M Phase, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.07.070
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New antiproliferative 7-(4-(N-substituted carbamoylmethyl) piperazin-1-yl derivatives
of ciprofloxacin induce cell cycle arrest at G2/M Phase
Hamada H. H. Mohammed1,§, Amer Ali Abd El-Hafeez2,§, Samar H. Abbas1, El-Shimaa M.
N. Abdelhafez1, Gamal El-Din A. Abuo-Rahma1*
1Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia 61519-
Egypt.
2Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National
Cancer Institute, Cairo University, Cairo, Egypt.
§: Both authors contributed equally to this work.
*The corresponding Author: e-mail: gamal.aborahma@mu.edu.eg, Mobile: +201003069431
Abstract
New N-4-piperazinyl derivatives of ciprofloxacin 2a-g were prepared and tested for their
cytotoxic activity. The primary in vitro one dose anticancer assay experienced promising
cytotoxic activity against different cancer cell lines especially non-small cell lung cancer.
Independently, compounds 2b, 2d, 2f and 2g showed anticancer activity against human non-
small cell lung cancer A549 cells (IC50 = 14.8, 24.8, 23.6 and 20.7 µM, respectively)
compared to the parent ciprofloxacin (IC50 >100 μM) and doxorubicin as a positive control
(IC50= 1 μM). The flow cytometric analysis for 2b showed dose dependent G2/M arrest in
A549 cells. Also, 2b increased the expression of p53 and p21 and decreased the expression of
cyclin B1 and Cdc2 proteins in A549 cells without any effect on the same proteins expression
in WI-38 cells. Specific inhibition of p53 by pifithrin-α reversed the G2/M phase arrest
induced by the 2b compound, suggesting contribution of p53 to increase. Taken together, 2b
induced G2/M phase arrest via p53/p21 dependent pathway. The results indicate that 2b can
be used as a lead compound for further development of new derivatives against non-small
cell lung cancer.
Keywords: Antiproliferative, Ciprofloxacin, Cytotoxicity, G2/M cell cycle arrest, p53/p21
pathway.

Introduction
Cancer is considered one of the most important causes of death globally.1 The
clinical use of chemotherapeutic agents in treatment of cancer is commonly associated with
severe side effects and lack of selectivity.2 As a result, there has been a growing interest for
development of new more selective anticancer agents with fewer side effects in different
parts of world by research groups.3,4 On the other hand, fluoroquinolones are synthetic
antimicrobial agents depicting broad spectrum and potent activity5,6 by targeting bacterial
DNA gyrase and topoisomerases.7,8 Besides their antibacterial activity, Fluoroquinolones
showed other biological activities such as antifungal9,10, antitubercular11,12, antitumor13,14
anti-HIV-1 integrase and anti- HCV-NS3 helicase.15
,
Quite recently, considerable attention has been paid to fluoroquinolones as
antiproliferative agents.16,17 DNA topoisomerases are essential enzymes that perform
important cellular functions involved in replication, recombination, and packaging and
unfolding of DNA in chromatin.16,18,19 Topoisomerase II (topo II) is the primary target of
fluoroquinolones as antitumor agents.20 Moreover, ciprofloxacin is a fluoroquinolone that
inhibits topoisomerase II in eukaryotic, including mammalian cells.21 It is worth to mention
that at a concentration exceeding 80 μg/ml ciprofloxacin was found to induce apoptosis,
while at concentration of 25 μg/ml it inhibits the proliferation of Jurkat cells without cell
death.22 Ciprofloxacin is also known for its anticancer properties by inducing G2 cell cycle
arrest and apoptosis of different cancer cells.23 Therefore, ciprofloxacin can be used as
adjuvant therapy for certain cancers.24
Several publications have appeared in recent years documenting the ability of
fluoroquinolones to induce apoptosis and cell cycle arrest in different cancer cell lines either
alone or in combination with other chemotherapeutic agents.25,26 The growth inhibitory effect
of ciprofloxacin22,23,26–28 and moxifloxacin26,29 is well established by inducing apoptosis
and cell cycle arrest in different cancer cell lines leading to cell death. Also, gatifloxacin is a
fourth-generation fluoroquinolone which induces S and G2-Phase cell cycle arrest in
pancreatic cancer cells via p21/p27/p53.30 It is clear in the literature that introduction of a
substituent on N4-piperazinyl moiety of ciprofloxacin dramatically alters the biological
profile of the parent quinolone from antibacterial to anticancer candidates and /or improves
the physicochemical properties.31–33 A series of N-4-piperazinyl-ciprofloxacin-chalcone
hybrids displayed significant anticancer activity against different cancer cell lines.16 Also,
another research study indicates that introduction of O-benzyl moiety on oxime group of N-

(2-oxyimino) piperazinyl quinolone series alters the biological activity of piperazinyl
quinolones from antibacterial to anticancer activity.34
Based on the previously mentioned findings, the aim of this research is to design and
synthesize new 7-(4-(N-substituted carbamoylmethyl) piperazin-1-yl derivatives of
ciprofloxacin for the purpose of improving the weak anticancer activity of ciprofloxacin.
In addition, the flow cytometric and western blotting analyses for compound 2b was carried
out to indicate its ability to arrest A549 cells in the G2/M phase via p53/p21 dependent
pathway.
2. Results and discussion
2.1 Chemistry
The target compounds and intermediates were prepared as outlined in scheme 1.The
acylated amine intermediates 1a-g were prepared as reported16,35 by reaction of the
appropriate amine with bromoacetyl bromide using potassium carbonate as a base in
dichloromethane. Alkylation of ciprofloxacin with 2-bromoacetamide derivatives 1a–g was
achieved in acetonitrile in the presence of trimethylamine (TEA) to afford the target
1
compounds 2a-g in a good yield.16,36 The prepared compounds were identified by IR, H-
13
1
NMR, C-NMR and HRMS. The H-NMR data for compounds 2a-g showed the known
characteristic pattern for the parent ciprofloxacin. In addition, the characteristic singlet of -N-
CH2- moiety of the amidic linker appeared at  3.24 ppm. Also, IR shows intense strong
sharp peak at 1680-1690 cm -1 which corresponding to C=O of the amidic linker.
Scheme 1: Synthesis of the target compounds 2a-g

2.2. Biological investigations
2. 2 .1. Antiproliferative activity
For screening of antiproliferative activity, compounds 2b, 2d and 2e were selected by the
National Cancer Institute (NCI) according to the protocol of the drug evaluation branch of the
NCI, Bethesda, USA for in vitro anticancer screening. Primary in vitro one dose anticancer
assay was performed in full NCI 60 cell lines derived from nine tumor subpanels, including
leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer cell lines.
The selected compounds were added at a single concentration (10ˉ5 M) and the culture was
incubated for 48 h. End point determinations were made with a protein binding dye
sulforhodamine B (SRB). Results for each compound were reported as a mean graph of the
percent growth of the treated cells when compared to the untreated control cells. The results
of NCI screening for the tested compounds are illustrated in Table 1.
Results showed that compound 2b revealed moderate cell growth inhibition activity
against fifteen cell lines; among them non-small cell lung cancer NCI-H322M, colon cancer
HT-29 CNS cancer U251, melanoma LOX IMVI, UACC-257, renal cancer ACHN, CAKI-
1and breast Cancer MDA-MB-23/ATCC, BT-549, T-47D. A complete cell death was
recorded for the non-small cell lung cancer NCI-H522cell line where the growth percent is -
113.73. Also, results showed significant cell growth inhibition activity against renal cancer
UO-31 cell line where the growth percent is 7.15. On the other hand, compound 2d exhibited
moderate cell growth inhibition against leukemia LOX IMVI and renal cancer A498 only and
it showed remarkable cell growth inhibition activity against non-small cell lung cancer NCI-
H322M, renal cancer 786-0 and breast cancer T-47D. In addition, compound 2e exhibited
remarkable cell growth inhibition activity against leukemia CCRF-CEM and non-small cell
lung cancer NCI-H522.
All the tested compounds showed very weak or no cytotoxicity against leukemia HL-60(TB),
MOLT-4, non-small cell lung cancer EKVX, NCI-H23, NCI-H460, colon cancer COLO 205,
HCC-2998, SW-620, CNS cancer SF-268, SF-295, SF-539, melanoma MALME-3M, MDA-
MB-435, SK-MEL-2, SK-MEL-28, ovarian cancer IGROV1, OVCAR-3, OVCAR-4,
OVCAR-5, NCI/ADR-RES, renal cancer SN12C, TK-10, prostate cancer DU-145 and breast
cancer MCF7, HS 578T, MDA-MB-468 cell lines.

Table 1: % Growth inhibition of the tested compounds 2b, 2d and 2e.
Panel/Cell Line
% Growth Inhibition
Panel/Cell Line
% Growth Inhibition
2b 2d 2e
2b
2d
2e
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
23.58
10.5
31.91 5.67
3.53
27.77 3.36
28.94
0
0
23.18
8.40
2.65
0
2.80
0
38.92 39.05 16.05
8.25
5.64
0
18.25 8.75
11.66 1.51
0
0
0
5.16
12.93 2.72
28.85 3.22
0
7.36
0
0
0
30.77 7.18
6.62
0
0
42.99 16.94 3.24
36.39 10.52 0.79
0
HOP-62
HOP-92
28.26 12.98 8.50
Ovarian Cancer
IGROV1
21.06 5.92
19.93 16.75
18.66
6.22
7.56
14.72
0
21.24 6.00
0
0
0
0
0
0
6.06
0
0
0
0
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0
0
0
0
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
5.51
7.04
47.19 26.39
12.05 6.32
113.7 12.88 24.43
3
0
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
SK-OV-3
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
36.59 17.03 5.77
29.87 20.91 15.24
0.11
14.16
26.94 9.38
17.00
42.14 0.04
17.14 6.61
2.63
0
0
0
0
0
1.21
0
0
31.41 37.65
0
0
46.77
32.79
0
0
0
0
KM12
25.29 11.85
9.89 4.00
13.94 nd
0
SW-620
CNS cancer
SF-268
SF-295
SF-539
0
0
2.78
10.54
0
8.82
6.72
0
0
0
0
92.85 17.87
12.14 2.32
13.06 5.31
16.92 12.33
Breast Cancer
MCF7
MDA-
5.73
39.75 13.79
0
0
0
SNB-19
MB231/ATCC
HS 578T
BT-549
SNB-75
U251
20.31 12.00
55.84 5.80
0
0
12.31 5.32
44.16 5.78
4.61
7.42
Prostate Cancer
PC-3
DU-31
T-47D
MDA-MB-468
40.88 24.51 9.38
14.04 6.39
19.92 11.63 14.49
0.90
0
0
0
*|nd: not detected

Independently, the inhibitory effect on growth caused by ciprofloxacin derivatives 2a-g on
human non-small cell lung cancer A549 and normal human lung fibroblast WI-38 cell lines
was evaluated using BrdU incorporation assay (Fig. 1A and B). A549 and WI-38 cells were
exposed to different concentrations (1, 5, 10, 15, 25, 50, 75 or 100 µM) from ciprofloxacin,
ciprofloxacin derivatives 2a-g or the reference doxorubicin for 24 h using DMSO as a
negative control. From the results in Table 2, it is obvious that 2b, 2d, 2f and 2g compounds
have cytotoxic activity against A549 cells (IC50 = 14.8-24.8 µM), while compounds 2a, 2c,
2e and ciprofloxacin have very weak or no anticancer activity (IC50 > 100 μM) in comparison
with doxorubicin as a positive control (IC50 = 1 μM). All the tested compounds have no
cytotoxic effect against WI-38 cells (IC50 >100) in comparison with doxorubicin (IC50 = 12.3
μM). The p-chlorophenyl derivative 2b (IC50 = 14.8 μM) was the most potent among all the
tested compounds against A549 cell line. From the results, it can be deduced that
ciprofloxacin derivatives 2b, 2d, 2f and 2g suppress cell proliferation in a dose-dependent
manner in A549 cells but not in WI-38 cells.

Fig. 1 Ciprofloxacin derivatives suppress cell proliferation in a dose-dependent manner in
A549 but not WI-38. (A) The effect of ciprofloxacin derivatives on the proliferation of A549
cells. (B) The effect of ciprofloxacin derivatives on the proliferation of W1-38 cells. The
results compared with the parent ciprofloxacin. DMSO served as a negative control.
Doxorubicin served as a positive control. Representative data of three independent
experiments are shown as means ± SE of three replicates.

Table 2. Cytotoxic activity of tested compounds against A549 and WI-38
In vitro Cytotoxicity IC50 (µM)
Compounds
R
A549
>100
14.8
>100
24.8
>100
23.6
20.7
>100
1
WI-38
>100
>100
>100
>100
>100
>100
>100
>100
12.3
2a
2b
2c
2d
2e
2f
Ph
4-Cl
2-OCH3-Ph
4-OCH3-Ph
3,4,5-tri-OCH3-Ph
4-CH3-Ph
Cyclohexy
2g
Ciprofloxacin
Doxorubicin
2. 2. 2. Cell cycle analysis
Regulation of cell growth is mainly controlled through cell cycle control mechanisms.37
Proliferation inhibition can be triggered by cell cycle arrest in cancer cells.38,39 During cell
cycle, the G2/M checkpoint is a potential target for cancer therapy. It prevents DNA-damaged
cells from entering mitosis and allows for the repair of DNA that was damaged in late S or G2
phases prior to mitosis.40 Induction of cell cycle arrest is a common mechanism proposed for
the cytotoxic effects of anticancer-drug containing quinolone ring.25-30
Accordingly, to explore the alteration in the cell cycle phases after treatment of A549 cells
with the most active cytotoxic ciprofloxacin derivative 2b, the cells were subjected to flow
cytometric analysis. The analysis indicates that A549 cells treated with 2b for 24 h
accumulated in the G2/M phase in a dose-dependent manner (22.6, 32.6 and 64%) compared
with untreated control (12.61%), doxorubicin (1 µM, 22.5%) and ciprofloxacin (100 µM,
16.8%) as shown in Fig. 2 (A and B). This result indicates that compound 2b induces cell
cycle arrest in the G2/M phase in A549 cells and this result is in a good agreement with
previous studies where quinolones have been shown to induce G2/M arrest in cancer cells.23,30

Fig. 2 Cell cycle distribution of A549 cells after treatment with 2b for 24 h. (A)
Representative data for the distribution of cells at each stage of the cell cycle determined by
flow cytometric analysis after incubation with control as 0 µM 2b, 100 µM ciprofloxacin, 1
µM doxorubicin or 5, 10, 15 and 25 µM 2b. (B) Percentage of cells in the sub G1, G1, S and
G2/M after 24 h incubation with control as 0 µM, 2b, 100 µM ciprofloxacin, 1 µM
doxorubicin or 5, 10, 15 and 25 µM 2b. The values are the means ± SEM of three different
experiments. ∗, P < 0.05 and ∗∗, P < 0.01 indicate a significant difference compared with
DMSO negative control treatment.

2.2.3. Compound 2b induced cell cycle arrest via p53/p21waf1 pathway in A549 cells.
The intracellular signaling was studied using western blotting analysis to understand the
molecular mechanisms by which 2b induces G2/M phase arrest (Fig. 3, A-E). p53, a tumor
suppressor protein, has been shown to play a key role in the regulation of cell cycle and cell
death in the cellular response to DNA damaging agents like quinolones.30 Activation of p53
by stress such as DNA damage may lead to arrest cell cycle at G2/M phase by activating its
target genes especially p21 or by inhibiting cyclins and CDKs directly.41-43 Since A549 cells
express wild type p53, we wished to check the possible alterations in expression of p53. The
data showed that 2b increased the expression of p53 protein in a dose-dependent manner
(Fig. 3, A and B). These data are in line with a previous report for quinolones.30 The p53-
target gene, p21 (waf-1/cip-1), is one of the major cyclin dependent kinase inhibitors which
has a broad spectrum of specificity in the cell cycle proteins and is able to arrest cell cycle at
G2/M checkpoint by inhibiting cyclin B1-Cdc2 complex.44 In the present study, expression of
p21have been increased in A549 cells after exposure to different concentrations of 2b in a
dose-dependent manner (Fig. 3, A and C). The obtained data is in a good correlation with
previous studies where quinolones have been shown to increase the expression level of p21 in
cancer cells.30 Cyclins and cyclin-dependent kinases (CDKs) determine the progress of cell
through cell cycle. On the other hand, the activity of CDKs is negatively regulated by a
variety of CDKs inhibitors, of which p21 have been shown to play a key role in transition
from G2 to M phase of cell cycle.44 Among the CDKs, Cdc2 is activated in association with
cyclin B1 in the G2/M phase progression.45 In this study, we found that cyclin B1 and Cdc2 is
down-regulated in A549 cells, after treating with different concentrations of 2b in a dose-
dependent manner (Fig. 3; A, D and E). Our results in agreement with the common
mechanism of quinolones like gatifloxacin which arrests cells in G2 phase via inactivation of
cdc2-cyclinB1 complex,30 suggesting that decreasing of Cdc2 and Cyclin B1 expression
might be the downstream molecular mechanism of p53/p21 pathway through which 2b
induced G2/M arrest.
We also tested the effect of 2b on the p53, p21, cyclin B1 and Cdc2 proteins expression in
WI-38 cells via western blotting (Fig. 4, A-E). The results indicated that 2b has no effect on
the monitored proteins which indicate the 2b selectivity toward the cancer cells. Based on the
mentioned results, it is clear that 2b induces cell cycle arrest in A549 cells via upregulation of
p53 and p21 and downregulation of cyclin B1 and Cdc2 proteins without any effect on WI-
38.

Fig. 3 The effect of 2b on the expression of p53 and the cell cycle regulator proteins in A549
cells. (A) Western blotting analysis showed that levels of p53 and p21 were increased while
levels of cyclin B1 and Cdc2 were decreased following treatment with 2b in a dose-

dependent manner in comparison with DMSO as a negative control and ciprofloxacin.
Doxorubicin used as a positive control: Lane 1, control; lane 2, ciprofloxacin (100 µM); lane
3, 1 μM doxorubicin; lane 4, 5 μM 2b; lane 5, 10 μM 2b; lane 6, 15 μM 2b; lane 7, 25 μM
2b. β - Actin was served as a loading control. (B-E) The relative protein expression levels of
p53, p21, cyclin B1 and Cdc2, compared to β - Actin were quantified by Image J. The values
are the means ± SEM of three different experiments. ∗, P < 0.05 and ∗∗, P < 0.01 indicate a
significant difference compared with DMSO negative control treatment.

Fig. 4 The effect of 2b on the expression of p53 and the cell cycle regulators proteins in
WI-38 cells. (A) Western blotting analysis showed that levels of p53, p21, cyclin B1 and
Cdc2 not change following treatment with 2b in comparison with DMSO as a negative

control and ciprofloxacin. Doxorubicin used as a positive control: Lane 1, control; lane 2,
ciprofloxacin (100 µM); lane 3, 1 μM doxorubicin; lane 4, 5 μM 2b ; lane 5, 10 μM 2b;
lane 6, 15 μM 2b; lane 7, 25 μM 2b. β - Actin was served as a loading control. (B-E) The
relative protein expression levels of p53, p21, cyclin B1 and Cdc2, compared to β - Actin
were quantified by Image J. The values are the means ± SEM of three different
experiments. ∗, P < 0.05 and ∗∗, P < 0.01 indicate a significant difference compared with
DMSO negative control treatment.
2.2.4. Association of p53 expression with 2b-induced cell cycle arrest
Tumor suppressor protein, p53, has been identified to play an important role in cell cycle
progression, and its induction after treatment with 2b compound has also observed. (Fig. 3 A
and B). Therefore, to confirm the role of p53 in 2b induced G2/M phase arrest, we used
selective p53 inhibitor pifithrin-α [PFT-α, 2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-
(4-methylphenyl) ethanone] to block p53 expression and performed cell cycle analysis in
A549 cells. PFT-α has been reported to inhibit p53 function temporally in vitro46 by a
reversible inhibition of p53-dependent transactivation of p53-responsive genes and to protect
cells against a variety of genotoxic agents.47 Our western blotting results showed reduction in
2b induced p53 and p21 expressions, post PFT- α treatment (Fig 5, A-C). Corresponding cell
cycle analysis showed that addition of PFT- α was able to reverse the cell cycle block
induced by the 2b compound (Fig 6, A-B). In particular, PFT- α treatment reduces G2/M
phase from 63.9% to 23.7% in 2b treated cells (Fig 6, A-B). Altogether, the above results
suggest that 2b compound induced G2/M phase cell cycle arrest is mediated by p53/p21
dependent pathway.

Fig. 5 Effect of PFT-α on 2b induced signaling in A549 cells. (A) Western blotting
analysis showed that levels of p53 and p21 expression after incubation with 25 μM 2b and
20 μM PFT-α for 24 h were significantly decreased: Lane 1, vehicle; lane 2b, 25 μM; lane
3, PFT-α 20 μM; lane 4, 2b (25 μM) with PFT-α (20 μM) (B-C) The relative protein
expression levels of p53 and p21 compared to β-Actin. β- Actin served as a loading control.
The values are the means ± SEM of three different experiments. ∗∗, P < 0.01 indicate a
significant difference compared with 2b (25 μM) treatment.

Fig. 6 Effect of 2b on the cell cycle analysis after p53 inhibition by PFT-α in A549 cells.
(A) Representative data are shown for the proportion of G2/M cells determined by flow
cytometric analysis post p53 inhibition by PFT- α (B) Proportion of cells in G2/M after p53
inhibition and 24 h incubation with 25 μM 2b. The values are the means ± SEM of three
different experiments. ∗∗, P < 0.01 indicate a significant difference compared with 2b (25
μM) treatment.

3. Conclusions
New N-4-piperazinyl derivatives of ciprofloxacin were prepared and their structural
formula had been identified by different spectroscopic techniques. The results obtained
indicate that compounds 2b, 2d, 2f and 2g experienced cytotoxic activity compared to the
parent ciprofloxacin and Doxorubicin as a reference. Also, compound 2b displayed
promising cytotoxic activity among all the tested compounds. From the above results, it is
obvious that the presence of p- substituent on the phenyl ring of phenylacetamide residue is
essential for cytotoxic activity where the p-chloro derivative 2b is the most active.
Meanwhile, the presence of one or more
electron donating group on the phenyl moiety
reduces and/ or eliminates the cytotoxic activity as in compounds 2d and 2e respectively. On
the other hand, replacement of the phenyl ring with cyclohexyl moiety fairly retains the
cytotoxic activity. Studying the mechanism of action of compound 2b was accomplished
using flow cytometric analysis. Compound 2b showed dose dependent G2/M cell cycle arrest
in A549 cell line. Also, 2b increased the expression of p53 and p21 and decreased the
expression of cyclin B1 and Cdc2 proteins in A549 cells without any effect on WI-38 cells.
Specific inhibition of p53 by pifithrin-α reduced the percentage of cells accumulated in G2/M
phase, suggesting contribution of p53 to G2/M phase arrest in A549 cells. Therefore,
compound 2b is considered a promising candidate that requires further investigation and
optimization for treatment of non-small cell lung cancer.
4. Experimental section
4.1. Chemistry
Reactions were monitored by TLC, using Merck 9385 pre-coated aluminum plate silica gel
(Kieselgel 60) 5 cm×20 cm plates with a layer thickness of 0.2 mm. The spots were detected
by exposure to UV-lamp at λ=254 nm. Melting points were determined on Stuart
electrothermal melting point apparatus and were uncorrected. IR spectra were recorded as
1
KBr disks on a Brukar Vector 22 IR spectrophotometer. NMR spectra (400 MHz for H, 100
MHz for 13C) were observed in DMSO-d6 on Bruker AM400 spectrometer with
tetramethylsilane as the internal standard. Chemical shifts () values are given in parts per
million (ppm) using DMSO-d6 as solvent and coupling constants are designated as (J) in Hz.
Splitting patterns are designated as follows: s, singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quartet; m, multiplet; brs, broad singlet. High-resolution mass spectra (HRMS)
were recorded on Thermo Scientific Q Exactive™ Orbitrap mass spectrometer and reported

as mass/charge (m/z) with percent relative abundance. The intermediates 1a-g were prepared
according to reported procedures.16, 35
4.1.1. General procedure for synthesis of compounds 2a-g
To a mixture of ciprofloxacin (0.66 g, 0.02 mole) and the respective 2-bromoacetamide
derivatives (0.02 mole) in acetonitrile (50 mL), TEA (0.04 mole) was added. The mixture
was heated under reflux for 6-8 h. solvent was removed under vacuum and the resulting solid
was recrystallized from acetonitrile.16
4.1.1.1.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(phenyl)amino)ethyl)piperazin-1-yl)-
1,4-dihydroquinoline-3-carboxylic acid 2a
Pale white crystal; 0.71g 85 yield; mp: 267-268 C ; 1H-NMR (400 MHz, DMSO-d6)  1.19-
1.20 (2H, m, cyclopropyl-H), 1.34-1.35 (2H, m, cyclopropyl-H), 2.79-2.81 (4H, m, piperazinyl-
H), 3.26 (2H, s, -N-CH2-CO), 3.43-3.45 (4H, m, piperazinyl-H), 3.81-3.83 (1H, m, cyclopropyl-
H), 7.08 (1H, t, J = 8 Hz, Ar-H), 7.30-7.34 (2H, dd, J = 8 Hz, Ar-H), 7.57 (1H, d, J = 8 Hz, H-
8), 7.65 (2H, d, J = 8 Hz, Ar-H), 7.88(1H, d, J = 12Hz, H-5), 8.65 (1H, s, H-2), 9.86 (1H, s, -
NH-CO) 15.10 (1H, brs, COOH); 13C-NMR (100 MHz, DMSO-d6)  8.06, 36.26, 49.81, 52.79,
61.87, 106.65, 107.34, 111.31, 119.07, 120.11, 123.97, 129.09, 138.98, 139.68, 145.55, 148.28,
152.33, 166.37, 168.55, 176.82 ; ESI-MS (m/z): calcd. For C25H24FN4O4 463.17871, found
463.17938 [M-H] -.
4.1.1.2.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(4-chlorophenyl)amino)ethyl)
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid 2b
1
Pale yellow crystal; 0.79 g, 88.26 yield; mp 237-238 C; H-NMR (400 MHz, DMSO-d6) 
1.19-1.20 (2H, m, cyclopropyl-H), 1.32-1.37 (2H, m, cyclopropyl-H), 2.79-2.81 (4H, m,
piperazinyl-H), 3.26 (2H, s, -N-CH2-CO ), 3.43-3.45 (4H, m, piperazinyl-H), 3.81-3.83 (1H, m,
cyclopropyl-H), 7.36 (2H, d, J = 8 Hz, Ar-H), 7.57 (1H, d, J = 8 Hz, H-8), 7.69 (2H, d, J = 8
Hz, Ar-H), 7.88 (1H, d, J = 16Hz , H-5), 8.65 (1H, s, H-2), 9.83 (1H, s, -NH-CO) 15.10 (1H,
13
brs, COOH); C-NMR (100 MHz, DMSO-d6)  8.03, 36.27, 49.83, 52.78, 61.87, 106.67,
107.38, 111.56, 119.11, 121.66, 127.57, 128.96, 138.04, 139.69, 145.64, 148.30, 152.23,
166.28, 168.75, 176.82; ESI-MS (m/z): calcd. For C25H23ClFN4O4 497.13973, found 497.14047
[M-H] -.

4.1.1.3.1-Cyclopropyl-6-fluoro-7-(4-(2-((2-methoxyphenyl) amino)-2-oxoethyl)piperazin-1-
yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 2c
Pale yellow crystal 0.79 g, 89 yield; mp 268-269 C; 1H-NMR (400 MHz, DMSO-d6)  1.19-
1.21 (2H, m, cyclopropyl-H), 1.34-1.36 (2H, m, cyclopropyl-H), 2.81-2.83 (4H, m, piperazinyl-
H), 3.27 (2H, s, -N-CH2-CO ), 3.44-3.46 (4H, m, piperazinyl-H), 3.84-3.86 (1H, m,
cyclopropyl-H), 3.90 (3H, s, OCH3), 6.94-6.95 (1H, m, Ar-H), 7.06-7.08 (2H, m, Ar-H), 7.61
(1H, d, J = 8 Hz, H-8), 7.90 (1H, d, J = 12Hz, H-5), 8.19 (1H, d, J = 8 Hz, Ar-H), 8.67 (1H, s,
H-2), 9.86 (1H, s, -NH-CO) 15.09 (1H, brs, COOH); 13C-NMR (100 MHz, DMSO-d6)  8.01,
36.29, 50.25, 52.85, 56.56, 61.76, 106.85, 107.39, 111.57, 119.24, 121.06,124.31, 127.62,
139.69, 145.53, 148.35, 152.23, 154.71, 166.32, 168.12, 176.81; ESI-MS (m/z): calcd. For
C26H26FN4O5 493.18927, found 493.18997 [M-H] -.
4.1.1.4.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(4-methoxyphenyl)amino)ethyl)
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid 2d
1
white crystal;0.73 g, 82 yield; mp 223-224 C; H-NMR (400 MHz, DMSO-d6)  1.18-1.20
(2H, m, cyclopropyl-H), 1.33-1.35 (2H, m, cyclopropyl-H), 2.78-2.80 (4H, m, piperazinyl-H),
3.23 (2H, s, -N-CH2-CO), 3.42-3.44 (4H, m, piperazinyl-H), 3.74 (3H, s, OCH3), 3.80-3.82
(1H, m, cyclopropyl-H), 6.89 (2H, d, J = 8 Hz, Ar-H), 7.55 (3H, d, J = 8. Hz, 2Ar-H & H-8),
7.89 (1H, d, J = 12 Hz, H-5), 8.66 (1H, s, H-2), 9.57 (1H, s, -NH-CO) 15.12 (1H, brs, COOH) ;
13C-NMR (100 MHz, DMSO-d6)  8.03, 36.29, 49.82, 52.82, 55.69, 61.87, 106.70, 107.30,
111.54, 114.29, 119.07, 121.66, 132.22, 139.67, 145.66, 148.33, 152.23, 154.71, 166.32,
168.04, 176.81; ESI-MS (m/z): calcd. For C26H26FN4O5 493.18927, found 493.18991 [M-H] -.
4.1.1.4.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(3,4,5-trimethoxyphenyl)amino)
ethyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid 2e
1
pale white crystal ; 0.81 g, 81.26 yield; mp 229-230 C; H-NMR (400 MHz, DMSO-d6) 
1.19-1.21 (2H, m, cyclopropyl-H), 1.34-1.35 (2H, m, cyclopropyl-H), 2.79-2.81 (4H, m,
piperazinyl-H), 3.21 (2H, s, -N-CH2-CO), 3.44-3.46 (4H, m, piperazinyl-H), 3.65 (3H, s,
OCH3), 3.78 (6H, s, OCH3), 3.81-3.83 (1H, m, cyclopropyl-H), 7.07 (2H, s, Ar-H), 7.58 (1H, d,
J = 8 Hz, H-8), 7.89 (1H, d, J = 16 Hz, H-5), 8.66 (1H, s, H-2), 9.60 (1H, s, -NH-CO), 15.10
13
(1H, brs, COOH); C-NMR (100 MHz, DMSO-d6)  8.03, 36.27, 49.82, 52.79, 56.43, 60.59,
62.00, 98.32, 106.67, 107.39, 111.58, 119.11, 134.52, 135.16, 139.70, 145.63, 148.32, 152.23,

153.25, 166.28, 168.40, 176.83,); ESI-MS (m/z): calcd. For C28H30FN4O7 553.2104, found
553.21130 [M-H] -.
4.1.1.6.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(p-tolylamino)ethyl)piperazin-1-yl)-
1,4-dihydroquinoline-3-carboxylic acid 2f
1
hite crystal ;0.78 g, 90 yield; mp 235-236 C; H-NMR (400 MHz, DMSO-d6)  1.17-121
(2H, m, cyclopropyl-H), 1.32-135 (2H, m, cyclopropyl-H), 2.27 (3H, s, CH3), 2.78-2.80 (4H, m,
piperazine-H), 3.24 (2H, s, -N-CH2-CO), 3.43-3.45 (4H, m, piperazine-H), 3.82-3.83 (1H, m,
cyclopropyl-H), 7.12 (2H, d, J = 8 Hz, Ar-H), 7.52 (2H, d, J = 8 Hz, Ar-H), 7.58 (1H, d, J = 8
Hz, H-8), 7.89 (1H, d, J = 16Hz, H-5), 8.66 (1H, s, H-2), 9.59 (1H, s, -NH-CO) 15.10 (1H, brs,
COOH); 13C-NMR (100 MHz, DMSO-d6)  8.03, 20.85, 36.27, 49.86, 52.81, 61.88, 106.70,
107.39, 111.57, 119.12, 120.10, 129.45, 132.87, 136.54, 139.70, 145.65, 148.31, 152.24,
166.29, 168.26, 176.84; ESI-MS (m/z): calcd. For C26H26FN4O4 477.19436, found 477.19473
[M-H] -.
4.1.1.7.1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-(cyclohexylamino)-2-oxoethyl)piperazin-1-yl)-
1,4-dihydroquinoline-3-carboxylic acid 2g
1
hite crystal ; 0.69 g, 61.61 yield; mp 259-260 C; H-NMR (400 MHz, DMSO-d6)  1.19-
1.35 (10H, m, cyclopropyl-4H & cyclohexyl-6H), 1.67-1.77 (4H, m , cyclohexyl-H), 2.70-2.72
(4H, m, piperazinyl-H), 3.03(2H, s, N-CH2-CO), 3.39-3.41 (4H, m, piperazinyl-H), 3.61-3.64
(1H, m, cyclohexyl-H), 3.83 (1H, m, cyclopropyl-H), 7.47-7.48 (1H, brs, NH-CO) , 7.57 (1H,
d, J = 8 Hz, H-8), 7.88 (1H, d, J = 12Hz, H-5), 8.66 (1H, s, H-2), 15.10 (1H, brs, COOH);
13C-NMR (100 MHz, DMSO-d6)  8.03, 24.97, 25.64, 32.82, 36.28, 47.68, 49.73, 52.78, 61.29,
106.70, 107.39, 111.58, 119.13, 139.71, 145.58, 148.34, 152.24, 166.33, 168.16, 176.86; ESI-
MS (m/z): calcd. For C25H30FN4O4 469.22566, found 469.22592 [M-H] -.
4.2. Biological investigations
Cell culture and reagents
Human lung adenocarcinoma (A549) and human lung fibroblast (WI-38) cell lines were
obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were
cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Co., LLC., St.
Louis, MO, USA) or Eagle's Minimum Essential Medium (EMEM, Sigma-Aldrich)
respectively, containing 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml penicillin,

and 100 μg/ml streptomycin (Life technologies) in a humidified atmosphere with 5% CO2 at
37°C. Ciprofloxacin derivatives were chemically synthesized (2a-g). Ciprofloxacin was taken
from (Pharco Pharmaceutical Company, Egypt). Doxorubicin and Pifithrin α were purchased
from Sigma-Aldrich Co., LLC. All chemicals used in this study were of the analytical or cell-
culture grade.
4.2.1. Proliferation assay
The methodology of the NCI anticancer screening has been described in detail elsewhere
(http://www.dtp.nci.nih.gov.) Briefly, the primary anticancer assay was performed at
approximately 60 human tumor cell lines panel derived from nine neoplastic diseases, in
accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute,
Bethesda. Tested compounds were added to the culture at a single concentration (10-5 M) and
the cultures were incubated for 48 h. End point determinations were made with a protein
binding dye, SRB. Results for each tested compound were reported as the percent of growth
of the treated cells when compared to the untreated control cells. The percentage growth was
evaluated spectrophotometrically versus controls not treated with test agents.
On the other hand, proliferation assay for A549 and WI-38 cells was quantified as BrdU
incorporation using a Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche Diagnostics,
Indianapolis, IN, USA). A549 and WI-38 cells were seeded as 5 × 103 cells/well and cultured
overnight in a 96-well plate. The cells were treated with 1, 5, 10, 15, 25, 50, 75 or 100 μM
ciprofloxacin derivatives 2a-g, ciprofloxacin, doxorubicin as a positive control, or DMSO as
a negative control for 24 h. BrdU was added to achieve a final concentration of 10 μM and
cultured for further two h. BrdU incorporation by the cells was quantified according to the
manufacturer’s instructions.
4.2.2. Cell cycle analysis
Cell cycle arrest of A549 cells induced by 2b was analyzed by FACS Calibur flow cytometer
(BD Biosciences, San Jose, CA, USA) using the BD PharmingenTM FITC-BrdU Flow Kit
(BD Biosciences), according to the manufacturer’s instructions. Briefly, A549 cells were
treated with 5, 10, 15 or 25 μM 2b, 100 μM ciprofloxacin, 1 μM doxorubicin or DMSO
(control) for 24 h. After harvesting, the cells were stained with FITC-labeled anti-BrdU
antibody and 7AAD for cell cycle analysis in PBS containing 1% BSA and 0.1% sodium

azide. Quantitative analysis of the FACS data was done by using Flowjo software (Flowjo,
Ashland, OR, USA)
4.2.3. Immunoblotting analysis
A549 cells were harvested, washed twice with chilled PBS, and lysed with ice-cold lysis
buffer containing 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 µg/ml
aprotinin, 5 µg/ml pefabloc SC (4-(2-Aminoethyl) benzenesulfonyl fluoride hidrochloride), a
protease inhibitor cocktail, 1 % phosphatase inhibitor cocktail and 50 mM Tris–HCl (pH 7.4)
after 24 h incubation with 2b, ciprofloxacin, doxorubicin, or DMSO (control). The cell
lysates were kept on ice for 30 min after gently vortex, and then centrifuged at 14,000g for 15
min at 4°C. The supernatants were loaded onto SDS-PAGE gel immediately after protein
extraction, or otherwise the supernatant was stored at −80°C until use. The protein
concentration was determined by Bradford assay (Bio-Rad Laboratories, ‎)ASC‎,AC‎,selucreH
-t‎ elcreocμ‎ 03‎ ,rroct‎ fcfrnHoHocl‎ neHrelc‎ rrc‎ .fuuclcoct‎ rc‎ r’e‎ rfccafurclelhH‎ ocHrlcuroccH
neot’r‎cf cell lysate were loaded onto a 15% SDS-PAGE gel. Proteins separated on a SDS-
PAGE gel were transferred onto a PVDF membrane. The PVDF membrane was incubated in
blocking buffer containing 3% non-fat milk powder, 1% bovine serum albumin (Sigma-
Aldrich), and 0.5% Tween-20 in PBS for 1 h. Subsequently, the PVDF membrane was
incubated with anti-p53 polyclonal antibody (pAb), anti-p21 monoclonal antibody (mAb,
clone no. EA10), anti-cyclin B1 pAb or anti-cdc2 mAb (clone no. POH1) (Cell Signaling
Technologies, Danvers, MA, USA) or anti- β-Actin mAb (clone no. 8H10D10, Cell
Signaling) for overnight, and followed by HRP-conjugated anti-rabbit IgG (Cell Signaling
Technologies) or HRP-conjugated anti-mouse IgG (Becton Dickinson Co, Durham, NC,
USA) for 1 h with agitation at room temperature. Finally the specific bindings to the each
primary antibody were detected on an X-ray film (Konica Minolta Medical Imaging, Wayne,
NJ, USA) with ECL prime immunodetection reagent (GE Healthcare, Little Chalfont, UK).
4.2.4. p53 inhibitor (pifithrin-α; PFT-α) experiments
PFT-α (10 µM) was added to the cells with ~50 confluency. After 24 h the cells were
incubated with 25 µM 2b and/or 10 µM PFT-α (total PFT-α concentration were therefore 20
µM) for 24h. DMSO was used as control. In order to confirm the inhibition of p53, whole
cell lysates were prepared and western blotting analyses were performed for p53 and p21
expressions as described above. Cell cycle analysis by flow cytometry was performed to

analyze the effect of PFT-α on 2b induced G2/M phase arrest as described above under cell
cycle analysis protocol.
4.2.5. Statistical analysis
Data are represented as means ± SD. Student’s t-test was performed to determine the
statistical significance between 2b and control. Statistical significance was defined as *P <
0.05 or **P<0.01. The IC50 of compounds was calculated by Graph Pad Prism 5 (Version 5.01,
GraphPad Software, San Diego, CA, USA). The data shown in the figures are representative
data for three independent experimental results.
Acknowledgment
The authors would like to thank the Development Therapeutics Program of the National
Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer activity. Also, the
authors gratefully appreciate the efforts of Dr. Safwat M. Rabee regarding HRMS analysis.
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Graphical abstract

Highlights
 New N-4 piperazinyl derivatives of ciprofloxacin
 Cytotoxic activity against different cancer cell lines especially non-small cell lung
cancer
 The flow cytometric analysis showed dose dependent G2/M arrest in A549 cells.
 Induce G2/M arrest via p53/p21 pathway.