Piperazine clubbed with 2-azetidinone derivatives suppresses proliferation, migration and induces apoptosis in human cervical cancer HeLa cells through oxidative stress mediated intrinsic mitochondrial pathway

Article abstract of DOI:10.1007/s10495-018-1439-x

Abstract: Piperazine scaffolds or 2-azetidinone pharmacophores have been reported to show anti-cancer activities and apoptosis induction in different types of cancer cells. However, the mechanistic studies involve in induction of apoptosis addressing thes

Full text of DOI:10.1007/s10495-018-1439-x

Apoptosis
https://doi.org/10.1007/s10495-018-1439-x
ORIGINAL PAPER
Piperazine clubbed with 2-azetidinone derivatives suppresses
proliferation, migration and induces apoptosis in human cervical
cancer HeLa cells through oxidative stress mediated intrinsic
mitochondrial pathway
Rashmin Khanam1 · Raj Kumar2 · Iram Iqbal Hejazi1 · Syed Shahabuddin3 · Ramovatar Meena2 · Vikrant Jayant2 ·
Prabhat Kumar2 · Abdul Roouf Bhat4 · Fareeda Athar1
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Piperazine scaffolds or 2-azetidinone pharmacophores have been reported to show anti-cancer activities and apoptosis
induction in different types of cancer cells. However, the mechanistic studies involve in induction of apoptosis addressing
these two moieties for human cervical cancer cells remain uncertain. The present study emphasizes on the anti-proliferating
properties and mechanism involved in induction of apoptosis for these structurally related azoles derivatives in HeLa can-
cer cells. 1-Phenylpiperazine clubbed with 2-azetidione derivatives (5a–5h) were synthesized, characterized using various
spectroscopic techniques and evaluated for their in-vitro anti-proliferative activities and induction of apoptosis. Further, we
also evaluated oxidative stress generated by these synthetic derivatives (5a–5h). Cell viability studies revealed that among
all, the compound N-(3-chloro-2-(3-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl) acetamide 5e remarkably
inhibited the growth of HeLa cells in a concentration dependent manner having IC50 value of 29.44 1.46 µg/ml. Morpho-
logical changes, colonies suppression and inhibition of migration clearly showed the antineoplasicity in HeLa cells treated
with 5e. Simultaneously, phosphatidylserine externalization, DNA fragmentation and cell-cycle arrest showed ongoing
apoptosis in the HeLa cancer cells induced by compound 5e in concentration dependent manner. Additionally, generation
of intracellular ROS along with the decrease in mitochondrial membrane potential supported that compound 5e caused
oxidative stress resulting in apoptosis through mitochondria mediated pathway. Elevation in the level of cytochrome c and
upregulation in expression of caspase-3 clearly indicated the involvement of the intrinsic pathway of programmed cell death.
In brief; compound 5e could serve as a promising lead for the development of an effective antitumor agent.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10495-018-1439-x) contains
supplementary material, which is available to authorized users.
* Abdul Roouf Bhat
abroouf@yahoo.co.in
* Fareeda Athar
fathar@jmi.ac.in
1
Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, New Delhi 110025, India
2
Radiation and Cancer Therapeutic Lab, School of Life
Sciences, Jawaharlal Nehru University, New Delhi 110067,
India
3
Research Centre for Nano-Materials and Energy Technology
(RCNMET), School of Science and Technology, Sunway
University, 47500 Selangor, Malaysia
4
Department of Chemistry, Sri Pratap College, Cluster
University, Srinagar 190001, India
Vol.:(0123456789)
1 3

Apoptosis
Graphical Abstract
Characteristic apoptotic morphological features
N
N
Wound
healing
assay
O
O
Morphological
changes
HN
N
Cl
Colonial
assay
AO-EB
fluorescent
staining assay
O2N
Compound 5e IC50 29.44 1.46 µg/ml
(HeLa Cancer cell)
DCFH-DA
assay
Cell cycle
assay
Western blot
DNA fragmentation
Mitochondrial membrane potential (ΔѰm)
Keywords Piperazines · 2-Azetidinones · Apoptosis · Anticancer activity · Oxidative stress
Abbreviations
MTT 3-(4,5-Dimethylthiazol-2-yl)-2.5-diphenyl-
tetrazolium bromide
regulating the apoptotic pathways leading to phagocytosis by
neighboring cells. There are mainly two pathways, extrinsic
death receptor pathway and mitochondrial (intrinsic) path-
way involved in the execution of apoptosis [4]. Although
inducing apoptosis through chemotherapy is one of the main
approaches used in cancer treatment, but unfortunately many
drugs used in chemotherapy suffer from various side effects
and attain resistance [5]. Thus, in order to improve the clini-
cal outcomes in cancer, new chemotherapeutics directing
towards apoptosis transduction signals must be discovered
[6].
DCFH-DA 2′,7′-Dichlorofluorescin diacetate
AO
Acridine organe
ROS
MMP
RPMI
Reactive oxygen species
Mitochondrial membrane potential
Roswell Park Memorial Institute medium
Introduction
Piperazine scaffolds are azole derivatives constituting
important structural framework for many drugs backbones
and displaying wide spectrum of biological activities like
anti-oxidation and anti-inflammation [7]. Piperazines are
of great significances since they act as specific ligands for
many biological targets and also possess an elastic binding
feature with common substrates [8]. Interacting with DNA
is another distinct characteristic shown by the piperazine
derivatives [9]. In addition to these, piperazines have
been shown to exhibit anticancer [10], antifungal [11],
antibacterial and antimalarial activities [12]. Piperazine
derivatives could substantially inhibit the proliferation
of various types of cancer such as colon, breast and leu-
kemia [13]. Recently, it has been found that piperazines
containing compounds are likely to be more active and
effective in treating cancer comparatively to the known
Cervical cancer is the second most commonly occurring can-
cer and also a third leading cause of deaths among women
worldwide [1]. There are several environmental elements
like carcinogens, viruses, chemicals, radiations including
germline mutations as one of the genetic factors that initiate
cancer and progressively leads to malignant cells, invasion
and metastasis. In-spite of much substantial progress been
made in the treatment of cervical cancer involving combina-
tional therapies of surgery, chemotherapy and radiotherapy,
the possibilities of remission, relapse and metastasis still exit
in comparison to other types of cancer with morality rate of
30% within 5 years of treatment [2].
Improper regulation of programmed cell death promi-
nently contributes to both cancer development and drug
responsiveness [3]. There are different gene families
1 3

Apoptosis
anticancer drugs such as taxane, sorafenib, cisplatin and
doxorubicin and also, they are broadly prescribed as anti-
cancer drugs [14]. Imatinib used for treating myelogenous
leukemia consist of piperazine nucleus, thereby increasing
its anticancer profile [15]. The anticancer activities of pip-
erazine derivatives are due to their potential in inhibiting
growth factors, enzymes and kinases possibly involved in
the cellular immortality and carcinogenesis such as focal
adhesion kinase (FAK) [16], insulin like growth factor-1
receptor (IGF-1R) [17] or to trigger oxidative stress caus-
ing mitochondrial impairment and apoptosis [18]. On the
other hand, azetidin-2-one derivatives exhibits an exten-
sive spectra of biological activities including antimicro-
bial, anti-tubercular, anti-cancer, anti-inflammatory, anti-
malarial, etc. [19]. Chemical reactivity of azetidin-2-one
ring which accounts for its enhanced biological profile is
contributed by the substituted nitrogen atom and phenyl
derivatives at C-4 position [20]. The anticancer activities
of 2-azetidinones derivatives are related to their ability
to inhibit proliferation either by inducing DNA damage
[21], or by activating. Adenosine monophosphate activated
protein kinase (AMPK) [19] and by bringing downstream
PI3K/AKT/GSK-3β (glycogen synthase kinase) signaling
pathways [20]. Inducing apoptosis directly is an impor-
tant characteristic feature of these azole derivatives which
enables them in removing cancer cells effectively, thereby
making them perfect candidate for cancer drug develop-
ment. The aim of this study is to evaluate the anti-prolif-
erative properties of piperazine/2-azetidinone derivatives
against human cervical cancer cells and also to evaluate
the effectiveness of these synthetic derivatives as apoptosis
inducers (Fig. 1).
Materials and methods
Chemistry
All the chemicals used in experiments were purchased
from Sigma-Aldrich, Merck (Germany) and were
employed without carrying any further purification. MEL-
temp apparatus was employed to determine the melting
points for all derivatives and results were uncorrected. For
assessing the purity of compounds, CHN Elemental ana-
lyzer (Vario EL-III) was used and values so obtained were
found to be within range of 0.3% of the observed val-
ues. IR spectra for all synthetic compounds were taken on
Bruker Tensor 37 FTIR spectrometer and wave numbers
were calculated in cm−1. NMR (1H NMR and 13C NMR)
spectra were obtained using Bruker Advance 300 MHz
spectrometer with DMSO-d6 as solvent. The chemical
shift (δ) values and coupling constant (J) were mentioned
in parts per million (ppm) and hertz respectively along
with reference residual peaks of CDCl3 (δ 7.24, 1HNMR;
δ 77.0, 13CNMR) or DMSO-d6 (δ 2.49, 1HNMR, δ 39.70,
13C NMR). ESI-MS spectra were recorded on AB-Sciex
2000.
General protocol for synthesis of piperazine clubbed
with 2-azetidione derivatives (5a–5h)
To the solution of N′-(4-substitutedbenzylidene)-2-(4-
phenylpiperazin-1-yl) acetohydrazide (4a–4h) (0.01 mol)
in dioxane (10–15 ml), chloroaetylchloride (0.01 mol)
and triethylamine (0.01 mol) was added carefully drop-
wise while maintaining the stirring at 0–5 °C. The reac-
tion mixture was allowed to reflux for 24 h. The reac-
tion mixture on completion was poured onto crushed ice
which precipitates, filtered, and dried. The crude product
so obtained was further purified through recrystallization
using mixture of solvents (DCM/methanol).
Molecular hybridization
Y
X
approach
HN
N
NR
O
N-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-2-(4-phenyl-
piperazin-1-yl)acetamide (5a) Yield: 64.6%; mp: 155–
157 °C; Anal Calcd for C21H23ClN4O2: C, 63.23; H,
5.81; N, 14.05%; found C, 63.21; H, 5.80; N, 14.03%.
IR (νmax cm−1): 3132, 3081 (Ar–H), 3093 (NH stretch),
1754 (2-azetidinone ring –C=O), 1624 (C=O of –CONH),
1595, 1505 (C=C aromatic), 1380 (C–N), 813 (C–Cl);
1HNMR (DMSO-d6) δ (ppm): 8.57 (s, 1H, CONH), 7.31
(s, 1H, phenyl ring), 7.20–7.16 (d, 2H, J = 6.6 Hz, aro-
matic protons), 6.84–6.80 (d, 2H, J = 6.6 Hz, aromatic
protons), 7.52–7.42 (m, 5H, phenylpiperazine), 6.32 (s,
2H, –NCH2), 5.32 (s, 1H, CH–Ar, 2-azetidione ring),
3.37–3.34 (q, 4H, –CH2–N–CH2, phenylpiperazine ring,
Azetidin-2-one scaffold
Piperazine scaffold
N
N
O
NH
Y
N
Piperazine-Azetidin-2one hydrid
O
X
Fig. 1 General structure of piperazine clubbed with azetidin-2-one
derivatives
1 3

Apoptosis
J = 6.7 Hz), 2.75–2.73 (t, 4H, –CH2–N–CH2, phenylpip-
erazine ring, J = 6.67 Hz), 2.45 (s, 1H, CH–Cl, 2-aetidi-
one ring); 13CNMR (DMSO-d6) δ (ppm) 163.5, 152.67,
145.45, 128.88, 128.3, 126.7, 122.2, 119.68, 116.11, 70.3,
59.1, 49.75, 45.31; ESI-MS m/z: [M + H]+ 399.89.
J=6.671 Hz, phenylpiperazine ring), 2.43 (s, 1H, –CH–Cl,
2-aetidione ring); 13CNMR (DMSO-d6) δ (ppm): 161.5,
151.67, 143.40, 129.78, 128.29, 126.64, 121.47, 118.78,
116.01, 71.13, 60.21, 49.65, 45.21; ESI-MS m/z: [M+H]+
429.91.
N - ( 3 - c h l o r o - 2 - ( 4 - c h l o r o p h e n y l ) - 4 - o x a z a e t i -
din-1-yl)-2-(4-phenylpiperazine-1-yl)acetamide (5b) Yield:
66.7%; mp: 169–171 °C; Anal Calcd for C21H22Cl2N4O2:
C, 58.21; H, 5.12; N, 12.93% found C, 58.21; H, 5.10; N,
12.93% IR (νmax cm−1): 3100, 3018 (Ar–H), 3086 (NH
stretch), 1752 (2-azetidinone ring C=O), 1604 (C=O of
–CONH), 1593, 1515 (C=C aromatic), 1387 (C–N), 780
(C–Cl); 1HNMR (DMSO-d6) δ (ppm): 8.53 (s, 1H, CONH),
7.18–7.14 (d, 2H, J=6.6 Hz), 6.82–6.78 (d, 2H, J=6.7 Hz),
7.51–7.41 (m, 4H, phenyl), 6.42 (s, 2H, –NCH2), 5.43 (s,
1H, CH–Ar, 2-azetidione ring), 3.35–3.33 (q, 4H, –CH2–N–
CH2, phenylpiperazine ring, J=6.67 Hz), 2.73–2.71 (t,
4H, –CH2–N–CH2, phenylpiperazine ring, J=6.8 Hz), 2.43
(s, 1H, –CH–Cl, 2-aetidione ring); 13CNMR (DMSO-d6)
δ (ppm), 167.67, 155.17, 146.17, 129.98, 127.44, 125.7,
121.2, 117.83, 115.57, 74.43, 56.91, 45.85, 43.11; ESI-MS
m/z: [M+H]+ 434.33.
N-(3-chloro-2-(3-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phe-
nylpiperazin-1-yl)acetamide (5e) Yield: 61.96%; mp:
163–165 °C; Anal Calcd for C21H22ClN5O4: C, 56.82; H,
5.00; N, 15.78%; found C, 56.80; H, 5.00; N, 15.76%. IR
(νmax cm−1): 3330, 3181 (Ar–H), 2993 (NH stretch), 1734
(C=O of 2-azetidinone ring), 1614 (C=O of –CONH),
1591, 1515 (C=C aromatic), 1383 (C–N), 793 (C–Cl);
1HNMR (DMSO-d6) δ (ppm): 8.85 (s, 1H, CONH), 8.05
(s, 1H), 7.45–7.30 (m, 2H), 7.28–7.20 (m, 5H, phenyl), 6.26
(s, 2H, –NCH2), 6.85–6.82 (d, 2H, J=6.7 Hz), 5.26 (s, 1H,
CH–Ar, 2-azetidione ring), 3.33–3.30 (q, 4H, –CH2–N–
CH2, J=5.12 Hz, phenylpiperazine ring), 2.66–2.64 (t, 4H,
–CH2–N–CH2, J=6.671 Hz, phenylpiperazine ring) 2.51
(s, 1H, –CH–Cl, 2-aetidione ring); 13CNMR (DMSO-d6)
δ (ppm): 167.62, 153.41, 141.36, 131.52, 130.99, 129.45,
128.82, 115.76, 110.95, 76.67, 62.91, 43.76, 41.91; ESI-MS
m/z: [M+H]+ 444.88.
N - ( 3 - c h l o r o - 2 - ( 2 - h y d r o x y p h e n y l ) - 4 - o x o a z e t i -
din-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide (5c) Yield:
54.16%; mp: 173–175 °C; Anal Calcd for C21H23ClN4O:
C, 60.79; H, 5.59; N, 13.50%; found C, 60.60; H, 5.57; N,
13.53%. IR (νmax cm−1): 3234, 3111 (Ar–H), 3102 (NH
stretch), 1766 (azetidinone ring –C=O), 1631 (C=O of –
CONH), 1605, 1515 (C=C aromatic), 1399 (C–N), 781
N-(3-chloro-2-(2-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phe-
nylpiperazin-1-yl)acetamide (5f) Yield: 57.86%; mp: 158–
161 °C; Anal Calcd for C21H22ClN5O4: C, 56.82; H, 5.00; N,
15.78% found C, 56.80; H, 5.00; N, 15.76%. IR (νmax cm−1):
3329, 3176 (Ar–H), 3112 (NH stretch), 1744 (C=O of 2-aze-
tidinone ring), 1621 (C=O of –CONH), 1592, 1505 (C=C
aromatic), 1380 (C–N), 813 (C–Cl); 1HNMR (DMSO-d6) δ
(ppm): 8.76 (s, 1H, CONH), 7.64–7.57 (m, 4H), 6.82–6.79
(d, 2H, J=6.8 Hz), 7.50–7.40 (m, 5H, phenyl), 6.31 (s, 2H,
–NCH2) 5.22 (s, 1H, CH–Ar, 2-azetidione ring), 3.37–3.34
(q, 4H, –CH2–N–CH2, J=6.8 Hz, phenylpiperazine ring),
2.73–2.71 (t, 4H, –CH2–N–CH2, J=6.67 Hz, phenylpipera-
zine ring), 2.43 (s, 1H, –CH–Cl, 2-aetidione ring); 13CNMR
(DMSO-d6) δ (ppm): 160.5, 150.67, 143.55, 129.79, 126.63,
125.67, 121.2, 117.78, 115.21, 70.54, 59.91, 49.75, 45.31;
ESI-MS m/z: [M+H]+ 444.88.
1
(C–Cl); HNMR (DMSO-d6) δ (ppm): 10.31 (s, 1H), 8.34
(s, 1H, CONH), 7.56–7.44 (m, 4H), 7.71–7.67 (m, 5H, phe-
nyl), 6.22 (s, 2H, –NCH2), 5.22 (s, 1H, CH–Ar, 2-azetidione
ring), 3.33–3.29 (q, 4H, –CH2–N–CH2, phenylpiperazine
ring, J=6.8 Hz), 2.75–2.73 (t, 4H, phenylpiperazine ring
–CH2–N–CH2, J=6.66 Hz), 2.42 (s, 1H, –CH–Cl, 2-aetidi-
one ring); 13CNMR (DMSO-d6) δ (ppm):165.37, 158.17,
155.15, 131.28, 129.27, 124.17, 120.12, 117.58, 114.91,
69.98, 61.1, 52.19, 43.21; ESI-MS m/z: [M+H]+ 415.89.
N - ( 3 - c h l o r o - 2 - ( 2 - m e t h o x y p h e n y l ) - 4 - o x o a z e t i -
din-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide (5d) Yield:
57.66%; mp: 181–179 °C; Anal Calcd for C22H25ClN4O3:
C, 61.61; H, 5.87; N, 13.06%; found C, 61.61; H, 5.83; N,
13.03%. IR (νmax cm−1): 3129, 3083 (Ar–H), 3091 (NH
stretch), 1756 (C=O of 2-azetidinone ring), 1616 (C=O of
–CONH), 1593, 1502 (C=C aromatic), 1382 (C–N), 815
(C–Cl); 1HNMR (DMSO-d6) δ (ppm): 9.66 (s, 1H, CONH),
8.33 (s, 3H, OCH3), 7.51–7.41 (m, 5H, phenyl), 7.21–7.17
(m, 4H), 6.30 (s, 2H, CH2), 5.31 (s, 1H, CH, –Ar, 2-aze-
tidionering), 3.36–3.33 (q, 4H, –CH2–N–CH2, J=6.81 Hz,
phenylpiperazine ring), 2.74–2.72 (t, 4H, –CH2–N–CH2,
N-(3-chloro-2-(4-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phe-
nylpiperazin-1-yl)acetamide (5g) Yield: 54.16%; mp: 186–
189 °C, Anal Calcd for C21H22ClN5O4: C, 56.82; H, 5.00;
N, 15.78%; found C, 56.80; H, 5.00; N, 15.76%. IR (νmax
cm−1): 3332, 3191 (Ar–H), 3100 (NH stretch), 1678 (C=O
of 2-azetidinone ring), 1601 (C=O of –CONH), 1587,
1
1554 (C=C aromatic), 1288 (C–N), 767 (C–Cl); HNMR
(DMSO-d6) δ (ppm): 9.03 (s, 1H, CONH), 7.60–7.56 (m,
2H), 6.74–6.70 (d, 2H, J=6.8 Hz), 7.43–7.33 (m, 5H, phe-
nyl), 6.45 (s, 2H, –NCH2), 5.72 (s, 1H, CH–Ar, 2-azetidi-
one ring), 3.77–3.74 (q, 4H, –CH2–N–CH2, J=6.67 Hz,
phenylpiperazine ring), 2.55–2.53 (t, 4H, –CH2–N–CH2
1 3

Apoptosis
J=5.0 Hz, phenylpiperazine ring), 2.66 (s, 1H, –CH–Cl,
2-aetidione ring); 13CNMR (DMSO-d6) δ (ppm): 162.68,
157.87, 143.58, 127.17, 122.73, 123.50, 120.96, 117.89,
114.71, 71.52, 52.81, 42.68, 40.71; ESI-MS m/z: [M+H]+
444.88.
Cell culture
Human cervical cancer cell (HeLa) line was brought from
National Center for Cell Science, Department of Biotechnol-
ogy, Pune, India. Cells were incubated at 37 °C with 5% CO2
in RPMI-1640 medium as monolayer supplemented with
10% (v/v) FBS, antibiotics (penicillin 100 U/ml, streptomy-
cin 10 μg/ml) and 1 mmol/l sodium pyruvate solution.
N - ( 3 - c h l o r o - 2 - ( 4 - a m i n o p h e n y l ) - 4 - o x o a z e t i -
din-1-yl)-2-(4-phenylpiperazin-1-yl)acteamide (5h) Yield:
69.26%; mp: 177–179 °C; Anal Calcd for C21H24ClN5O2:
C, 60.94; H, 5.84; N, 16.92%; found C, 60.91; H, 5.80; N,
16.93%. IR (νmax cm−1): 3131, 3085 (Ar–H), 3096 (NH
stretch), 1752 (C=O of 2-azetidinone ring), 1622 (C=O of
–CONH), 1592, 1515 (C=C aromatic), 1387 (C–N), 802
(C–Cl); 1HNMR (DMSO-d6) δ (ppm): 8.55 (s, 1H, CONH),
7.51–7.47 (d, 2H, J=6.7 Hz), 7.49–7.39 (m, 5H, phenyl),
7.23–7.19 (m, 2H), 6.31 (s, 2H, –NCH2) 5.32 (s, 1H, CH–
Ar, 2-azetidione ring), 5.01 (s, 2H, –NH2), 3.41–3.39 (q,
4H, –CH2–N–CH2, J=46.6 Hz, phenylpiperazine ring),
2.66–2.65 (t, 4H, –CH2–N–CH2, J=6.67 Hz, phenylpipera-
zine ring), 2.58 (s, 1H, –CH–Cl, 2-aetidione ring); 13CNMR
(DMSO-d6) δ (ppm): 167.34, 157.87, 140.91, 131.56,
130.99, 129.48, 128.83, 123.18, 123.10, 116.82, 113.517,
Cell proliferation (MTT assay)
To evaluate the proliferation of HeLa cells, MTT assay
was used. 96-well plate was used to seed cells having den-
sity of 5×103 cells/well and kept overnight for attachment.
Media was replaced and cells were exposed for 24 h to
different concentrations (12.5–100 μg/ml) of compounds
(5a–5h). 20 μl containing 5 mg/ml of MTT was poured
to each well 4 h before the incubation would complete.
The media was changed and 200 μl of DMSO was added
followed by incubation at room temperature for another
10 min. ELISA reader was used to observed absorbance
at 595 nm. The % of inhibition was calculated using fol-
lowing formula [22]:
Mean OD of untreated cells (control) − mean OD of treated cells
% Inhibition =
× 100
Mean OD of untreated cells (control)
74.83, 62.34, 46.95, 42.864, 40.62 ESI-MS m/z: [M+H]+
414.16.
IC50 values were calculated for compounds from the
curves by plotting % inhibition versus concentration of
compounds.
Pharmacology
Morphological changes
Cells in equal number 1 × 103 were seeded in 60 mm well
plates followed by treatment with various concentration of
compound (5e). Cell morphology was observed after 24 h
by using microscope (Nikon Eclipse Ti-S, Tokyo, Japan).
Antibodies and reagents
All chemicals including RPMI-1640, fetal bovine serum,
trypsin and antibodies were brought from GIBCO Grand
Island, New York, USA while reagents like bovine serum
albumin (BSA), ethidium bromide (EtBr), acridine orange
(AO), Triton X-100, and 3-(4,5-dimethylthiazol-2-yl)-2.5-di-
phenyl-tetrazolium bromide (MTT), JC-1 dye, propidium
iodide (PI), RNAse-A, proteinase-K were brought from
Sigma-Aldrich, St. Louis, MO, USA. Primary mice β-actin,
primary rabbit cytochrome-c, primary mice caspase-3 and
secondary antimice Ig-G-HRP and antirabbit Ig-G-HRP
antibodies were purchased from Santa Cruz Biotechnology,
Texas, USA. All other important chemicals used were of
molecular biology grade.
Clonogenic assay
1×103 cells/well were seeded in six well plates. After incu-
bating cells for 37 °C and replacing media twice in a week,
cells were washed twice thoroughly with PBS (phosphate
buffer saline) and were fixed for 10 min in a fixative solu-
tion. Cells were stained for 10 min using crystal violet solu-
tion and then colonies were observed through naked eyes.
Wound healing assay
1 × 103 cells/well was seeded equally in six well plates.
After 80% confluence of cells growth, a scratch was made
1 3

Apoptosis
using 200 µl pipette tip in each well plate and exposed
to various concentrations of compound (5e). The photo-
graphs of cell scratch were taken at 0 and 24 h by using
microscope (Nikon Eclipse Ti-S, Tokyo, Japan) and the
percentage of wound thickness was calculated as a ratio of
percentage of wound thickness after 24 h to initial wound
thickness.
10 min at around 500 rpm. The supernatant was discarded
and washing was done with cold PBS twice. 500 µl DNA
lysis buffer (20 mM EDTA, 10 mM Tris–HCl pH 8.0, 0.2%
Triton X-100 and 100 µg/ml proteinase-K) was used for lys-
ing the cells pellet at 37 °C for 1.5 h. To get the supernatant
containing DNA, the samples were subjected to centrifuga-
tion for 5 min at 6000×g. To equal volume of isopropanol,
the supernatant was added and 25 µl 4M NaCl making a final
concentration upto 100 mM and left for overnight incubation
at − 20 °C. Sample at room temperature was centrifuged
at 6000×g for 25 min. A solution of 50 µl double distilled
water and 2 µl RNase A (10 mg/ml) was used to dissolve
the pellets and left for incubation for 1 h at 37 °C. Spec-
trophotometer, NanoDrop (NanoDrop, Wilmington, USA)
was used to quantify the extracted DNA. 2% agarose gel
containing 1 μg/1 ml EtBr was used to electrophorese the
DNA samples. The gel was examined and ultraviolet gel
documentation system was used to take images.
Cell cycle distribution analysis
60 mm dish was used to seed HeLa cells, after which cells
were left for attachment followed by cells treatment with
compound 5e for 24 h. Harvesting and centrifugation of
cells at 400×g was done for 10 min. This step was fol-
lowed by discarding and fixation of pellets with 70% etha-
nol. Further washing of cells were done using PBS Cells
were then stained with 500 μl of PI solution and 25 µl of
RNAase and left for incubation in dark room for 30 min
at 37 °C. Fluorescence emission of PI-DNA complex to
quantified apoptotic cells was done using FACS Caliber
instrument (Becton Dickinson, Franklin Lakes, NJ, and
USA). Each sample taken for examination consists of
20,000 cells. The distribution pattern of DNA content was
expressed as subG1, G1, S, and G2/M phases.
Generation of intracellular reactive oxygen species (ROS)
Production of intracellular ROS (reactive oxygen species)
in HeLa cells was measured using 2′,7′-dichlorofluorescein-
diacetate (DCFHDA) dye. Cells with density of 5×105 cell/
well were seeded over cover-slip of six-plated well and kept
overnight for incubation for the attachment. Next day, cells
were exposed to fresh media having different concentrations
of compound (5e). Incubation of cells for 4 h at 37 °C was
done. Then cells were stained with 40 μM DCFHDA for
30 min. Cover slip was adhered on glass slide and fluores-
cence microscope (Nikon Eclipse Ti-E, Tokyo, Japan) was
used for observe the results..
Annexin V-FITC/propidium iodide (AV/PL) staining assay
Annexin-V-FITC/propidium iodide staining kit was
employed for analysis apoptosis. Six-well plate containing
cells were exposed to different concentrations of compound
5e for 24 h. Cells were harvested followed by washing using
cold PBS resuspended in 100 μl binding buffer and mixed
for about 30 min at room temperature containing annexin
V-FITC and PI. Cells were analyzed through flow cytometry.
Assessment of mitochondrial membrane potential
To access the loss of the mitochondrial membrane
potential, fluoroprobe 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimidazol-carbocyanine iodide (JC-1 dye) has
been used extensively. Six-well plate was used for growing
cells followed by exposure to various concentrations of com-
pound (5e). After treating cells for 4 h, washing was done
using PBS followed by staining cells with 2 µg/ml of JC-1
dye and left for incubation for 30 min in dark at 37 °C. Cells
were washed thoroughly with PBS (phosphate buffer saline)
and live imaging microscope was used for capturing images.
Fluorimeter was also used for measuring the mitochondrial
membrane potential which requires treatment of cells with
various concentration of compound 5e. Cells were incubated
at 37 °C for 4 h which were then stained for 20 min with the
mitochondrial-membrane-permeable dye JC-1 (10 μg/ml in
PBS) at 37 °C. Following this, harvesting and washing with
phosphate buffer saline was done and measurements at were
taken 530/590 nm.
Assessment of nuclear morphology of apoptotic cells
by acridine orange/ethidium bromide (AO/EtBr)
The AO/EtBr dual staining assay was used to evaluate the
apoptosis in HeLa cells. Cells were suspended in a mixture
containing AO/EtBr dual staining solution in 1:1 ratio (1 mg/
ml of AO in PBS+1 mg/ml of EtBr in PBS) for 2 min. To
visualize cells, fluorescence microscopy (Nikon ECLIPSE
Ti-E, Tokyo, Japan) was used. Cells were differentiated on
the basis of fluorescence emitted where live; apoptotic and
necrotic cells were seen as green, orange and red respec-
tively in merged image.
DNA fragmentation assay
After treating cells with compound 5e for 24 h, cells were
collected through trypsinization and centrifugation for
1 3

Apoptosis
Protein extraction and western blot analysis
after carrying out purification through recrystallization.
Ethyl-2-(4-phenylpiperazin-1-yl) acetate (2) and 2-(4-phe-
nylpiperazin-1-yl) acetohydrazide (3) were synthesized
using to reported protocol [23]. The reaction between ace-
tohydrazine derivatives (3) with various aromatic aldehydes
using ethanol as solvent to give Schiff base (4a–4h) was
done using reported procedure [24]. Synthesis of final com-
pounds (5a–5h) was carried out using Staudinger reaction
which involves cyclization between substituted acetic acid
acids or acid chlorides and imines in presence of base [25].
All synthesized compounds were obtained in yield of about
55–75% and were purified using Column chromatography
hexane/ethylacetate in ratio of (7:3) as eluting medium. All
the synthesized compounds were shown to in accordance
with the spectroscopic data obtained from FT-IR, 1HNMR,
13C NMR, ESI MS and CHN analysis (Figs. S.1, S.2, S.3
and S.4).
100 mm plate was used for seeding the cells in equal num-
ber. After 80% cells confluent, cells were exposed for 24 h to
various concentration of compound (5e). Cells were trypsi-
nized and washed with PBS. Radio immuno precipitation
assay (RIPA) buffer was used for extracting and estimating
protein and protein concentration respectively. The protein
was separated in 8% SDS-PAGE gel and was transferred
to nitrocellulose membrane. Obstruction of membrane was
done using 5% BSA in PBS. It was then probed with primary
antibody and incubated with a secondary antibody conju-
gated using horseradish peroxidase (HRP). ECL kit was used
to detect the bands and images were permanently captured in
X-ray films. Results were analyzed with respect to loading
control (β-actin).
Cytochrome c in cytosolic fraction
Pharmacology
4 °C was maintained to carry all steps. After treating with
compound 5e, cells were trypsinized and washed with PBS
and resuspended into 500 μl fractionation buffer [Buffer
HEPES (pH 7.4) 20 mM, KCI 10 mM, MgCl2 2 mM, EDTA
1 mM, EGTA 1 mM, DTT 1 mM protease inhibitors cock-
tail-50 μl/10 ml buffer] and left for incubation for 15 min
over ice. 1 ml syringe was used to pass cells suspension
through a 27 gauge needle 10–15 times and left on ice for
further 20 min and centrifuged at 720×g (3000 rpm) for
5 min Supernatant was collected and again centrifuged at
8000 rpm (10,000×g) for 5 min. Pellet was discarded and
supernatant was used to study cytochrome-c in cytosolic
fraction. Bradford dye was used to estimate protein concen-
tration in cytosolic fraction. The extracted protein was sepa-
rated in 8% SDS-PAGE gel for western blotting analysis.
In vitro inhibition of cell growth by N-(3-chloro-2-(3-nitr
ophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl)
acetamide (5e)
The anti-proliferative effect of newly synthesized
N-(3-chloro-2-(4-aryl)-4-oxoazetidin-1-yl)-2-(4-phenyl-
piperazin-1-yl) acetamide derivatives (5a–5h) on human
cervical cancer cell line (HeLa) has been evaluated by MTT
assay (Table 1). The cells were treated with final compounds
(5a–5h) along with standard anticancer drug 5-Fluroura-
cil in the concentration range of 12.5–100 μg/ml for 24 h.
Among all, compounds 5e showed the highest inhibition
towards HeLa cells with IC50 value 29.44 1.46 µg/ml in
comparison to the reference anticancer drug 5-Fluorouracil
which exhibited less toxicity towards HeLa cells with very
high IC50 value 112.16 0.02 µg/ml. Compound 5h also
showed moderate activity having IC50 value 61.69 0.08 µg/
ml, while other compounds of the series were found to be
inactive as they did not showed any activity upto 100 µg/
ml (Fig. S.5).
Data analysis
All the data was represented as a mean SD for three sets
of independent experiments and value of significant (p val-
ues) were evaluated using an unpaired student’s t test using
the GraphPad Prism version 7.0 software (San Diego, CA,
USA) and p values <0.05 were considered to be statistically
relevant.
Evaluation for drug likeness properties of synthesized
compounds
All the synthesized compounds were evaluated for some
molecular properties based on Lipinski’s rule of five which
are physically and pharmaceutically significant. All the syn-
thesized compounds exhibited optimum drug likeness prop-
erties without violating Lipinski’s rule of five (Table 2) [26].
Results
Chemistry
Synthesis of all compounds was carried out using simple
and feasible chemical reactions as shown in Scheme 1. The
initial reactant (1) used in the synthesis of key intermediate
hydrazide (3) was purchased from Sigma and was employed
1 3

Apoptosis
Scheme 1 Synthesis of pip-
erazine clubbed 2-azetidione
derivatives (5a–5h)
O
Cl
N
N
N
N
N
NH2NH2.H2O
C2H5OH
O
Dry Acetone, K2CO3
O
N
H
O
NH2
N
H
O
(1)
(2)
(3)
gl.acetic acid
Ar-CHO
N
Chloroacetylacetate
TEA
N
N
N
O
O
Ar
N
H
N
NHN CH-Ar
(5a-5h)
(4)
O
Cl
Ar =
OH
OCH3
Cl
5b
5c
5d
5a
NO2
NO2
NH2
5h
NO2
5g
5f
5e
Morphological changes of HeLa cells induced by N-(3-chlor
o-2-(3-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpipera-
zin-1-yl)acetamide (5e)
whereas, untreated cells (control) appeared to be healthy in
nature with normal shape and size (Fig. 2).
Decreased in number of HeLa cells in clonogenic
assay by N-(3-chloro-2-(3-nitrophenyl)-4-oxoazeti-
din-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide (5e)
Compound 5e induced morphological changes in HeLa cells
treated for 24 h in concentration dependent manner. The
treated cells showed various morphological changes such
as cell shrinkage, detachments and dead cells formation
Besides the effect of compound 5e on cell viability and mor-
phology, the number and size of cells were also evaluated
1 3

Apoptosis
Table 1 Anti-proliferative
activity of piperazine clubbed
2-azetidinone derivatives
(5a–5h)
Compound no.
Ar group
HeLa
IC50 (µg/ml) SD
> 100
5a
> 100
5b
Cl
N
> 100
5c
5d
5e
OH
N
O
Ar
> 100
OCH3
NO2
N
H
N
29.44 1.46
O
Cl
> 100
5f
NO2
> 100
5g
5h
NO2
61.69 0.08
NH2
5-Fluorouracil
112.16 0.02
Bold values represent the IC50 values of active compounds
No activity>100 µM. The values (in µg/ml) represent the mean SE of three independent experiments.
IC50 is the concentration required to inhibit 50% of the cell population
HeLa human cervical cancer cell line
Table 2 Molecular properties of
Compounds no. Mi logp TPSA No. of atoms MW
nON nOHNH Volume Nrotb
compounds
Desirable value
<5
<140
–
<500
<10
<5
–
<10
5a
5b
5c
5d
5e
5f
2.40
3.08
1.92
2.46
2.33
2.36
2.36
1.48
55.88 28
55.88 29
76.11 29
65.12 30
398.89
433.34
414.89
428.92
443.89
443.89
443.89
413.91
6
6
7
7
9
9
9
7
1
1
2
1
1
1
1
3
353.69
367.20
414.89
379.24
377.02
377.02
377.02
364.98
3
5
5
6
6
6
6
5
101.7
101.7
101.7
31
31
31
5g
5h
81.91 29
MW molecular weight, nviolations number of violations, natoms number of atoms, nON number of hydro-
gen bond acceptors, nOHNH number of hydrogen bond donors, Nrotb number of rotatable bonds, mi logp
logp value predicted by Molinspiration, TPSA topological polar surface area, MV molecular volume
using clonogenic assay. A cluster of approximately least 50
cells constitutes a colony. The clonogenic assay showed that
the number and size of colonies significantly decreased when
treated with compound 5e in a concentration dependent man-
ner in comparison to the untreated (control) cells which pro-
liferated to form colonies (Fig. 3).
Inhibition of migration of HeLa cells by N-(3-chloro-2-(3-n
itrophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl)
acetamide (5e)
In order to perform the in vitro cell migration assay wound
was created on a confluent monolayer of cultured HeLa cells
and exposed to different concentrations of 5e (i.e. 12.5, 25
1 3

Apoptosis
Fig. 2 Morphological changes such as detachments and dead cells were observed in HeLa cells treated for 24 h with compound 5e in concentra-
tion dependent manner. (Images were captured using Nikon ECLIPSE Ti-S microscope at ×10 magnification)
Fig. 3 Clonogenic assay (crystal
violet staining) (arrow repre-
senting decrease in number and
size of colonies in HeLa cells
treated for 24 h with compound
5e in concentration dependent
manner)
and 50 µg/ml). The result showed that after 24 h, the thick-
ness of wound gap decreased considerably for the untreated
cells (control) cells in comparison with treated cells. The
result showed that for treated cells the wound gap increased
due to inhibition of migration of cells by compound 5e in
concentration dependent manner (Fig. 4a). The percentage
of wound thickness was found to be increasing from 56.81%
(control) to 57.94% (solvent DMSO), 64.02% (12.5 µg/
ml), 76.66% (25 µg/ml) and 92.8% (50 µg/ml) respectively
(Fig. 4b), thus signifying the compound 5e effectively
1 3

Apoptosis
Fig. 4 a Wound-healing assay showed that 5e inhibited the migra-
tion of HeLa cells. The photographs of cell scratch were taken
using microscope (Nikon ECLIPSE Ti-S) at 0 and 24 h. b Relative
percentage of thickness of wound quantified using Graphpad prism
7. Data are presented as mean SD of three separate experiment
****p<0.0001, ***p<0.001 with respect to control
suppressed the migration of HeLa cells in a concentration
dependent manner.
of phosphatidylserine (PS) in cell membrane of HeLa
cells using annexin-V/propidium (AV/PI) dual staining
dye. The result showed increase in the total percentage
of apoptotic cells (early and late stage) and dead cells
from (control) 8.87–9.28% (DMSO), (17.95)% 12.5 μg/
ml, (34.14)% 25 μg/ml and (95.23)% 50 μg/ml concentra-
tions of compound 5e respectively (Fig. 5c). The signifi-
cant increase in total percentage of apoptotic cells (early
and late) from 7.87% (control) to 8.31% (DMSO), 16.67%
(12.5 μg/ml), 32.98% (25 μg/ml) and 94.68% (50 μg/ml) in
a concentration-dependent manner clearly showed that the
compound 5e induced apoptosis in HeLa cells (Fig. 5d).
Concomitantly, compound 5e induced apoptosis was fur-
ther shown by change in membrane integrity and mor-
phology of nucleus. AO/EtBr fluorescent staining assay
differentiates the live, apoptotic and dead cells. The results
showed that after 24 h, untreated cells (control) exhibited
nuclei with normal morphology having intact membrane,
uniform chromatin and appeared green in color, conversely
nucleus of the AO/EtBr stained cells treated with different
concentrations of compound 5e, showed all three (green,
N-(3-chloro-2-(3-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phe-
nylpiperazin-1-yl)acetamide (5e) induced G2/M cell cycle
arrest and apoptosis in HeLa cells
Flow cytometric analysis of PI-stained cells showed that
after 24 h treatment, the ratio of HeLa cells increased
in the G2/M phase (p < 0.05) from 11.51% (control) to
11.54% (DMSO), 13.06, 26.92 and 34.86% at 12.5, 25
and 50 µg/ml concentrations of compound 5e respec-
tively (Fig. 5a). Concurrently, a noteworthy reduction
(p < 0.05) in the population of HeLa cells in Sub-G1
phase was observed. These results clearly indicated that
compound 5e led to apoptosis by arresting G2/M phase
of cell cycle (Fig. 5b). To further evaluate apoptosis
induced by compound 5e, we analyzed the disturbances
in symmetry of plasma membrane, nuclear morphological
changes and internucleosomal DNA fragmentation. Flow
cytometry was employed to quantify the externalization
1 3

Apoptosis
Fig. 5 a Cell cycle analysis of HeLa cells treated with different con-
centrations of 5e for 24 h. The cell cycle distribution was performed
by using propidium iodide staining and analyzed by flow cytometry
BD FACS CALIBUR 4C. b Histogram of cell cycle distribution (%)
in HeLa cells treated with different concentrations of 5e for 24 h.
Values are expressed as means SD of three different experiments
with p<0.05. c Flow cytometry analysis of apoptosis induced by
compound 5e in HeLa cells treated in concentration dependent man-
ner using through annexin V/PI staining quadrants; lower left (AV/
PI-ve)-viable cells; upper right (AV/PI+ve)—late apoptosis; lower
right (AV+ve)—early apoptosis and upper left (PI+ve)—second-
ary necrotic cells. d Increase in the population of apoptotic cells
(early and late) with respect to different concentrations of compound
5e after 24 h in HeLa cells. e Compound 5e induced nuclear mor-
phological changes in HeLa cells treated in concentration dependent
manner related to apoptosis analyzed using EtBr/AO/staining. Green,
orange and red fluorescence corresponds to live, apoptotic and dead
cells respectively. Images were captured by fluorescence microscope
(Nikon ECLIPSE TiE) at ×10 magnification. f Effect of 5e compound
on HeLa cells by DNA ladder assay 24 h post treatment: 2% agarose
gel showing the cleavage of HeLa cells genomic DNA (control) intact
bands; (compound 5e, 12.5, 25, 50 µg/ml) forming DNA ladders
1 3

Apoptosis
(c)
(d)
Fig. 5 (continued)
orange and red) florescence representing definitive damage
of cell membrane (Fig. 5e). Further, DNA fragmentation
was evaluated using gel electrophoresis. DNA ladder assay
showed an intact DNA bands in untreated cells (control),
while treated cells showed deteriorated bands forming lad-
der pattern in concentration dependent manner (Fig. 5f).
Generation of intracellular ROS levels using DCFH-DA
assay by N-(3-chloro-2-(3-nitrophenyl)-4-oxoazeti-
din-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide (5e)
To explore whether ROS played a prominent role in
5e-induced apoptosis, we used a fluorescent probe DCFH-
DA to analyze the generation of cellular ROS in HeLa
cells. The intensity of green fluorescence corresponds to
the level of ROS produced in the cells. It was observed
1 3

Apoptosis
Fig. 5 (continued)
1 3

Apoptosis
Fig. 6 Effect of different concentrations of 5e on ROS level in HeLa cells after 6 h. Mark increased fluorescence with increasing concentration
of compound 5e. Images were captured by fluorescence microscope (Nikon ECLPSE Ti-E) at ×20 magnification
clearly that after 24 h, the compound 5e significantly
increased the intensity of fluorescence in a concentration-
dependent manner in HeLa cells, whereas control cells
showed only a weak and diffused green fluorescence
(Fig. 6).
from 100% (control) to 81.69, 77.84 and 73.09% at 12.5, 25
and 50 μg/ml concentrations of 5e respectively.
Release of cytochrome c and activation of caspase-3 using
western blotting by N-(3-chloro-2-(3-nitrophenyl)-4-ox-
oazetidin-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide (5e)
Loss of mitochondrial membrane potential (ΔѰm) using
JC-1 dye cell growth by by N-(3-chloro-2-(3-nitrophenyl)-
4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl)acetamide
(5e)
The depolarization of mitochondrial membrane potential
generally occurs before or in combination with release of
cytochrome c from mitochondria to cytosol, which is one
of the important processes that occur before apoptosis.
In this regard, we have measured the expression of cyto-
solic cytochrome c in HeLa cells after exposed to different
concentrations of compound 5e. The western blot results
showed elevation in the level of cytochrome c in the cytosol
fraction (Fig. 8a). The translocation of cytochrome c from
the mitochondria into cytosol ultimately results in apopto-
sis through activation of caspases. The results also showed
that the compound 5e significantly increased the activity
of caspase-3 in concentration dependent manner (Fig. 8a).
The change in the protein fold was analyzed using Image J
software (Fig. 8b). All these results collectively showed that
compound 5e induce apoptosis in HeLa cells by the gen-
eration of cellular ROS through the intrinsic mitochondrial
mediated apoptotic pathway (Fig. 9).
Mitochondrial membrane gets depolarized on loss of ΔѰm
damaging the mitochondrial functions and resulting in cell
death. Variations in mitochondrial membrane potential was
evaluated using JC-1 dye, as it forms red fluorescent aggre-
gates in mitochondria corresponding to high membrane
potential, whereas it forms green fluorescent monomers
predominantly in cytosolic form, representing a collapse of
membrane As shown in (Fig. 7a), after 24 h, cells treated
with different concentrations of compound 5e exhibited
strong green fluorescence in comparison to untreated (con-
trol) cells which displayed high red fluorescence. The results
from (Fig. 7b) showed the decrease in percentage of ΔѰm
1 3

Apoptosis
Red
Merge
Green
(a)
(b)
150
100
50
**
***
***
0
l
l
l
l
O
o
m
m
/
m
/
/
r
S
t
g
g
g
n
M
µ
µ
µ
o
5
5
0
D
.
C
2
5
2
1
Concentration of compound 5e
Fig. 7 a JC-1 staining representing the mitochondrial membrane
depolarization of HeLa cells exposed to different concentrations of 5e
after 8 h: image were captured by live imaging microscope (Nikon
ECLIPSE Ti) at ×20 magnification. b Effect of 5e on the loss of
mitochondrial membrane potential (ΔΨm) of HeLa cells using JC-1
dye: fluorescence was measured at 530/590 nm. Results are presented
as mean SD of triplicates **p<0.01, ***p<0.001, with respect to
control
modifications within structural framework. The substitu-
ents present on the phenyl ring on 2-azetidinone ring are
important in analyzing structure–activity relationship (SAR)
(Scheme 1). Compounds with unsubstituted phenyl ring (5a)
were found to be less active and effective towards HeLa
cancer cells in comparison with the substituted phenyl ring
(5b–5h). It was observed that phenyl ring substituted with
strong electron donating group (–NH2) (5h) at para posi-
tion and strong electron withdrawing group (–NO2) (5e) at
meta position respectively showed higher activity than the
standard drug (5-Fluorouracil) towards HeLa cancer cells
in concentration dependent treatment. Phenyl ring having
ortho substitution with either strong or weak electron with-
drawing or donating group showed no activity (5b–5d, 5f).
Among all, compound 5e show highest inhibitory effect with
IC50 value of 29.44 1.46 µg/ml on HeLa cells. The signifi-
cant anti-proliferative effect of compound 5e towards HeLa
Discussion
In the current study, a new series of N-phenylpiperazines
containing 2-azetidinones derivatives were synthesized,
characterized using high resolution spectroscopic tech-
niques such as FT-IR, 1HNMR, 13CNMR, ESI-MS and CHN
analysis and evaluated for their anti-proliferative effects on
(HeLa) human cervical cancer cell line using MTT assay
[27]. 5-Fluorouracil was taken as a standard drug for assay.
The IC50 values (μg/ml) for both synthesized compounds
(5a–5h) and standard drug (5-Fluorouracil) were repre-
sented in Table 1. The in-vitro cytotoxic studies show that
out of all, only two compounds 5e and 5h significantly inhib-
its the growth of human cervical cancer cell line (HeLa) in
a concentration dependent manner.
MTT results show that the anti-proliferating property
of the synthesized compounds is due to the variations and
1 3

Apoptosis
Cytochrome C
5
4
3
2
1
0
(b)
(a)
****
****
****
l
l
l
l
o
O
m
m
/
m
/
r
/
S
t
g
g
g
µ
n
M
µ
µ
o
5
5
0
5
D
.
C
2
2
1
Caspase 3
5
4
3
2
1
0
****
***
Caspase 3
***
β-actin
l
l
l
l
o
O
m
/
m
/
m
/
r
S
t
g
g
g
µ
n
M
µ
µ
o
5
.
5
0
5
D
C
2
2
1
Fig. 8 a HeLa cells exposed to different concentrations of 5e for
24 h then total protein and cytosolic protein extract were analyzed by
Western blotting for cytochrome c in cytosolic protein fraction and
caspase-3 in total cells protein extract. b Bar graph representing the
fold changes in the protein expression compared to loading control
β-actin. Data expressed as mean SEM of triplicates. ***p<0.001
and ****p<0.0001 significant vs. control
Fig. 9 Proposed mechanism
for induction of apoptosis by
N-(3-chloro-2-(3-nitrophenyl)-
4-oxoazetidin-1-yl)-2-(4-
phenylpiperazin-1-yl) acetamide
(5e) in human cervical cancer
cell lines (HeLa)
N
N
O
O
HN
N
Cl
Compound 5e
O2N
Cytosol
ROS
Mitochondrial permeability
Cytochrome C
Nucleus
Activates
Caspase-3
Cell cycle
arrest
Apoptosis
DNA damage
cancer cells encourage us to further exploit its effect at cel-
lular level, principally targeting the molecular mechanism
involve in induction of apoptosis.
Recent studies have described the cytotoxic effect of some
piperazine derivatives like β-elemene piperazine, 1,4-bis-(4-
(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine, chloroalkyl
1 3

Apoptosis
piperazine and 6-(4 substituted piperazine-1-yl)-9-(β-d-
ribofuranosyl) purine in several cancer cells via apoptosis
induction, although, these derivatives activated only extrinsic
pathway of apoptosis [28]. On the other hand, 2-azetidinones
derivatives used in the preparation of some of anticancer drugs
like paclitaxel (Taxol), docetaxel (Taxotere) and also exhibits
anti-proliferation by inducing apoptosis in many human cancer
cell lines such as breast, prostate and head and neck involving
cell cycle arrest and caspase activation, however the mecha-
nism remains unclear [29]. We herein demonstrate the pos-
sible mechanism of action of N-(3-chloro-2-(3-nitrophenyl)-
4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl) acetamide (5e)
compound (conjugate of piperazine/2-azetidinone) (Fig. 1) in
human cervical cancer cell (HeLa) via inducing apoptosis
through oxidative stress. Our investigations provide the better
understanding of the mechanism of action of compound 5e and
basis for the future development of drug for cervical cancer.
Apoptosis is prime target for many anticancer drugs
according to the various reports [30, 31]. In the present
study, the changes in cell morphology reveal that com-
pound 5e is cytotoxic and causes cell death in a concentra-
tion dependent manner (Fig. 2). Cells undergo apoptosis and
inhibit the proliferation within 24 h of treatment. This was
further shown through colony formation assay that displayed
reduction in number and size of colonies (Fig. 3) [32]. We
evaluated PS externalization, DNA fragmentation, ROS
production, variation in mitochondrial membrane potential,
translocation of cytochrome-c (cyt-c) and activation of cas-
pases 3 as mediators of apoptosis. Therefore, we suggest the
involvement of mitochondria mediated apoptotic pathway in
compound 5e induced HeLa cells (Fig. 9).
increased extensively in HeLa cells (Fig. 6). Increase of
ROS beyond a threshold damage DNA through activation
of mitochondrial-related apoptotic signaling and also lead
to mitochondrial dysfunction as shown by decrease in the
mitochondria membrane potential in HeLa cells treated with
compound 5e (Fig. 7a, b) [37]. Disruption of mitochondrial
membrane potential released cytochrome c which in turn,
activates executioners of caspases, ultimately leading to cell
death/DNA fragmentation (Fig. 5f) [38]. From western blot
analysis it is reveal that compound 5e enhance transloca-
tion of cytochrome c into cytosol leading to activation of
caspase-3 (Fig. 8a, b). Thus, compound 5e can be expected
as a potent inducer of morphological variations involving
apoptotic molecular events in the HeLa cancer cells related
with its cytotoxic potential.
Conclusion
Our results revealed that compound 5e (N-(3-chloro-2-(3-
nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl)
acetamide) inhibited the growth of HeLa cervical cancer
cells by bringing apoptosis through ROS-mediated mito-
chondrial pathway. However, further investigation of its
in vivo activity is required to explore its tumor reducing
potential.
Acknowledgements The research work has been funded by UGC (F
no-43-172/2014 (SR)).
Compliance with ethical standards
Evaluation of membrane blebbling and condensation of
chromatin through AO/EtBr staining demonstrated the mor-
phological changes significant to the apoptotic event (Fig. 5e)
[31]. It is evident that on increasing the concentration of com-
pound 5e from 12.5 to 50 µg/ml, the shift from early to the
late stage of apoptotic events occurred, suggesting that treating
HeLa cancer cells at higher concentrations of compound 5e
can induce necrosis in these cells (Fig. 5c, d) [33]. To further
support the occurrence of apoptosis, we carried out cell cycle
analysis in the HeLa cancer cells through PI staining [34]. On
analyzing the various cell cycle phases, increase in the popula-
tion of G2/M phase was observed with concomitant decrease
in sub-G1 phase. These investigations confirm that cell death
is initiated by apoptosis (Fig. 5a, b) [35]. Besides cell death
compound 5e was found to be effective in inhibiting the migra-
tion potential of the HeLa cells in vitro. Migration of cells is
an important factor in cancer as it plays a major role in tumor
progression and metastatic cascade. A substantial decrease of
HeLa cell migration was noticed in vitro at ½×IC50, IC50 and
2×IC50 concentrations of compound 5e (Fig. 4a, b) [36].
Mitochondria are the chief source of ROS regulating the
viability of cells. Our finding showed that ROS generation
Conflict of interest All authors declare no conflict of interest.
Ethical approval This article has no studies concerned with human
participants or animals.
References
1. Khazaei S, Ramachandran V, Abdul Hamid R, Esa NM, Etemad
A, Moradipoor S, Ismail P (2017) Flower extract of Allium atro-
violaceum triggered apoptosis, activated caspase-3 and down-reg-
ulated antiapoptotic Bcl-2 gene in HeLa cancer cell line. Biomed
Pharmacother 89:1216–1226
2. World Health Organization (2014) GLOBOCAN2012: estimated
cancer incidence, mortality and prevalence worldwide in 2012.
International agency for research on cancer, population fact sheets
(Islamic Republic of Iran)
3. Fulda S (2009) Tumor resistance to apoptosis. Int J Cancer
124:511–515
4. Thangam R, Sathuvan M, Poongodi A, Suresh V, Pazhanichamy
K, Sivasubramanian S, Kanipandian N, Ganesan N, Rengasamy R,
Thirumurugan R (2014) Activation of intrinsic apoptotic signaling
pathway in cancer cells by Cymbopogon citratus polysaccharide
fractions. Carbohydr Polym 107:138–150
1 3

Apoptosis
22. Dhivya R (2015) In virto antiprolifeative and apoptosis-inducing
properties of a mononuclear copper (II) complex with dppz ligand,
i = n two genotypically different breast cancer cell lines. Springer,
New York
5. Alshatwi AA, Periasamy VS, Athinarayanan J, Elango R (2016)
Synergistic anticancer activity of dietary tea polyphenols and
bleomycin hydrochloride in human cervical cancer cell: caspase-
dependent and independent apoptotic pathways. Chem Biol Inter-
act 247:1–10
23. Wani MY, Ahmad A, Shiekh RA, Al-Ghamdi KJ, Sobral AJ
(2015) Imidazole clubbed 1,3,4-oxadiazole derivatives as poten-
tial antifungal agents. Bioorg Med Chem 23:4172–4180
24. Unver Y, Sancak K, Celik F, Birinci E, Kucuk M, Soylu S, Burnaz
NA (2014) New thiophene-1,2,4-triazole-5(3)-ones: highly bioac-
tive thiosemicarbazides, structures of Schiff bases and triazole-
thiols. Eur J Med Chem 84:639–650
6. Ahmed W, Van Etten RA (2013) Signal transduction in the chronic
leukemias: implications for targeted therapies. Curr Hematol
Malig Rep 8:71–80
7. Asif M (2015) Piperazine and pyrazine containing molecules and
their diverse pharmacological activities. Int J Adv Sci Res 1:05–11
8. Berkheij M, van der Sluis L, Sewing C, den Boer DJ, Terpstra JW,
Hiemstra H, Bakker WII, van den Hoogenband A, van Maarse-
veen JH (2005) Synthesis of 2-substituted piperazines via direct
α-lithiation. Tetrahedron Lett 46:2369–2371
25. O’Boyle NM, Greene LM, Bergin O, Fichet JB, McCabe T, Lloyd
DG, Zisterer DM, Meegan MJ (2011) Synthesis, evaluation and
structural studies of antiproliferative tubulin-targeting azetidin-
2-ones. Bioorg Med Chem 19:2306–2325
9. Wilson WD, Barton HJ, Tanious FA, Kong S-B, Strekowski L
(1990) The interaction with DNA of unfused aromatic systems
containing terminal piperazino substituents: intercalation and
groove-binding. Biophys Chem 35:227–243
26. Morris GM (1998) Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function.
J Comput Chem 19:1639–1662
27. Cohen G, Sun X, Snowden R, Dinsdale D, Skilleter D (1992) Key
morphological features of apoptosis may occur in the absence of
internucleosomal DNA fragmentation. Biochem J 286:331–334
28. Yu Z, Wang R, Xu L, Xie S, Dong J, Jing Y (2011) β-Elemene
piperazine derivatives induce apoptosis in human leukemia cells
through downregulation of c-FLIP and generation of ROS. PLoS
ONE 6:e15843
10. Gillet R, Jeannesson P, Sefraoui H, Arnould-Guerin M-L, Kirki-
acharian S, Jardillier J-C, Pieri F (1997) Piperazine derivatives
of butyric acid as differentiating agents in human leukemic cells.
Cancer Chemother Pharmacol 41:252–255
11. Upadhayaya RS, Sinha N, Jain S, Kishore N, Chandra R, Arora SK
(2004) Optically active antifungal azoles: synthesis and antifun-
gal activity of (2R, 3S)-2-(2, 4-difluorophenyl)-3-(5-{2-[4-aryl-
piperazin-1-yl]-ethyl}-tetrazol-2-yl/1-yl)-1-[1, 2, 4]-triazol-1-yl-
butan-2-ol. Bioorg Med Chem 12:2225–2238
29. Geesala R, Gangasani JK, Budde M, Balasubramanian S, Vaidya
JR, Das A (2016) 2-Azetidinones: synthesis and biological eval-
uation as potential anti-breast cancer agents. Eur J Med Chem
124:544–558
12. Chaudhary P, Kumar R, Verma AK, Singh D, Yadav V, Chhillar
AK, Sharma G, Chandra R (2006) Synthesis and antimicrobial
activity of N-alkyl and N-aryl piperazine derivatives. Bioorg Med
Chem 14:1819–1826
30. Shyam H, Singh N, Kaushik S, Sharma R, Balapure AK (2017)
Centchroman induces redox-dependent apoptosis and cell-cycle
arrest in human endometrial cancer cells. Apoptosis 22:570–584
31. Aarts M, Sharpe R, Garcia-Murillas I, Gevensleben H, Hurd MS,
Shumway SD, Toniatti C, Ashworth A, Turner NC (2012) Forced
mitotic entry of S-phase cells as a therapeutic strategy induced by
inhibition of WEE1. Cancer Discov 2:524–539
13. Yoshida M, Maehara Y, Sugimachi K (1999) MST-16, a novel
bis-dioxopiperazine anticancer agent, ameliorates doxorubicin-
induced acute toxicity while maintaining antitumor efficacy. Clin
Cancer Res 5:4295–4300
14. Hatnapure GD, Keche AP, Rodge AH, Birajdar SS, Tale RH,
Kamble VM (2012) Synthesis and biological evaluation of novel
piperazine derivatives of flavone as potent anti-inflammatory and
antimicrobial agent. Bioorg Med Chem Lett 22:6385–6390
15. Shaquiquzzaman M, Verma G, Marella A, Akhter M, Akhtar W,
Khan MF, Tasneem S, Alam MM (2015) Piperazine scaffold: a
remarkable tool in generation of diverse pharmacological agents.
Eur J Med Chem 102:487–529
32. Kumar R, Tiku A (2018) Galangin induces cell death by modulat-
ing the expression of glyoxalase-1 and Nrf-2 in HeLa cells. Chem
Biol Interact 279:1–9
33. Tentner AR, Lee MJ, Ostheimer GJ, Samson LD, Lauffenburger
DA, Yaffe MB (2012) Combined experimental and computational
analysis of DNA damage signaling reveals context-dependent
roles for Erk in apoptosis and G1/S arrest after genotoxic stress.
Mol Syst Biol 8, 568
16. Sun J (2017) Discovery of a series of 1,3,4-oxadiazole-2(3H)-
thione derivatives containing piperazine skeleton as potential FAK
inhibitors. Bioorg Med Chem Lett 25:2593–2600
34. Gasparri F, Cappella P, Galvani A (2006) Multiparametric
cell cycle analysis by automated microscopy. J Biomol Screen
11:586–598
17. Guo FJ, Sun J, Gao LL, Wang XY, Zhang Y, Qian SS, Zhu HL
(2015) Discovery of phenylpiperazine derivatives as IGF-1R
inhibitor with potent antiproliferative properties in vitro. Bioorg
Med Chem Lett 25:1067–1071
35. Liu Y-J, Liang Z-H, Hong X-L, Li Z-Z, Yao J-H, Huang H-L
(2012) Synthesis, characterization, cytotoxicity, apoptotic induc-
ing activity, cellular uptake, interaction of DNA binding and anti-
oxidant activity studies of ruthenium (II) complexes. Inorg Chim
Acta 387:117–124
18. da Silva DD, Silva MJ, Moreira P, Martins MJ, Valente MJ, Car-
valho F, de Lourdes Bastos M, Carmo H (2016) In vitro hepa-
totoxicity of ‘Legal X’: the combination of 1 benzylpiperazine
(BZP) and 1 (m trifluoromethylphenyl) piperazine (TFMPP) trig-
gers oxidative stress, mitochondrial impairment and apoptosis.
Springer, Heidelberg
36. Khanam R, Ahmad K, Hejazi II, Siddique IA, Kumar V, Bhat
AR, Azam A, Athar F (2017) Inhibitory growth evaluation and
apoptosis induction in MCF-7 cancer cells by new 5-aryl-2-butyl-
thio-1,3,4-oxadiazole derivatives. Cancer Chemother Pharmacol
80:1027–1042
19. Mehta PD, Sengar NP, Pathak AK (2010) 2-Azetidinone–a new
profile of various pharmacological activities. Eur J Med Chem
45:5541–5560
37. Hejazi II, Khanam R, Mehdi SH, Bhat AR, Rizvi MMA, Islam
A, Thakur SC, Athar F (2017) New insights into the antioxidant
and apoptotic potential of Glycyrrhiza glabra L. during hydrogen
peroxide mediated oxidative stress: an in vitro and in silico evalu-
ation. Biomed Pharmacother 94:265–279
20. Arya N, Jagdale AY, Patil TA, Yeramwar SS, Holikatti SS,
Dwivedi J, Shishoo CJ, Jain KS (2014) The chemistry and bio-
logical potential of azetidin-2-ones. Eur J Med Chem 74:619–656
21. Smith DM, Kazi A, Smith L, Long TE, Heldreth B, Turos E, Dou
QP (2002) A novel beta-lactam antibiotic activates tumor cell
apoptotic program by inducing DNA damage. Mol Pharmacol
61:1348–1358
38. Simon HU, Haj-Yehia A, Levi-Schaffer F (2000) Role of reac-
tive oxygen species (ROS) in apoptosis induction. Apoptosis
5:415–418
1 3