Diarylidenecyclopentanone derivatives as potent anti-inflammatory and anticancer agents

Article abstract of DOI:10.1007/s00044-020-02578-5

Cancer is often associated with chronic inflammation. In order to develop potential anticancer and anti-inflammatory agents a series of 26 diarylidenecyclopentanones (DACPs) Ia–Iv, II, III, and IV were synthesized. Five of the synthesized DACPs are novel (Ih, Ij, Ik, Is, and Iv), derivative Iv was characterized using single-crystal X-ray diffraction study. All the synthesized derivatives were tested for their anti-inflammatory as well as cytotoxicity properties. Compound Is is found to have the highest anti-inflammatory activity (93.67%) by inhibiting PGE₂ (prostaglandin E₂) production. Three of the DACPs (Io, It, and Iu) were observed to have high cytotoxicity with IC₅₀ value of 8.73 ± 0.06 μM (Io), 12.55 ± 0.31 μM (It), and 11.47 ± 0.15 μM (Iu) against HeLa cells. Further staining and cell cycle analysis was done using these three DACPs to understand their mechanism of action. The G₀/G₁ phase was observed to be the longest one through which the cells undergo apoptosis.

Full text of DOI:10.1007/s00044-020-02578-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02578-5
ORIGINAL RESEARCH
Diarylidenecyclopentanone derivatives as potent anti-inflammatory
and anticancer agents
Nitesh Tamang1 Gayathri Ramamoorthy2 Mayank Joshi3 Angshuman Roy Choudury3 Siva Kumar B.1
●
●
●
●
●
Nageswara Rao Golakoti1 Mukesh Doble2
●
Received: 18 February 2020 / Accepted: 28 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Cancer is often associated with chronic inflammation. In order to develop potential anticancer and anti-inflammatory agents
a series of 26 diarylidenecyclopentanones (DACPs) Ia–Iv, II, III, and IV were synthesized. Five of the synthesized DACPs
are novel (Ih, Ij, Ik, Is, and Iv), derivative Iv was characterized using single-crystal X-ray diffraction study. All the
synthesized derivatives were tested for their anti-inflammatory as well as cytotoxicity properties. Compound Is is found to
have the highest anti-inflammatory activity (93.67%) by inhibiting PGE2 (prostaglandin E2) production. Three of the DACPs
(Io, It, and Iu) were observed to have high cytotoxicity with IC50 value of 8.73 0.06 µM (Io), 12.55 0.31 µM (It), and
11.47 0.15 µM (Iu) against HeLa cells. Further staining and cell cycle analysis was done using these three DACPs to
understand their mechanism of action. The G0/G1 phase was observed to be the longest one through which the cells undergo
apoptosis.
Abbreviations
NCCS
NCI
PBS
PGE2
PI
National Centre for Cell Science
National Cancer Institute
phosphate buffered saline
Prostaglandin E2
AO
Acridine orange
COX
cyclooxygenase
DACP
DMEM
ETBR
Diarylidenecyclopenatnone
Dulbecco's Modified Eagle Medium
Ethidium bromide
Propidium Iodide
mPGES-1 membrane-associated PGE synthase-1
MTT
3-(4,5- dimethylthiazole-2-yl)-2,5-diphenylte-
trazolium bromide
Introduction
Cancer is a chronic disease that claims millions of lives
every year and for the year 2018 itself, it was responsible
for 9.6 million deaths (WHO 2018). There are different
ways to treat cancer–surgery, radiation therapy, che-
motherapy, immunotherapy, targeted therapy, hormone
therapy, stem cell transplants, and precision medicine (NCI
2020). To treat solid tumors either surgery or radiation is
carried out. Chemotherapy is widely used since the drug
diffuses into the cancerous cells. Chemotherapy has to be
undergone (especially during the metastasis stage) when the
primary tumor cells have been transmitted to other parts of
the human body (Steeg 2006). Even though it has been
widely used for the treatment of cancer, these drugs come
with many side effects such as inflammation, immunosup-
pression, and digestive tract lining disorders. So, search for
better anticancer drugs with fewer side effects along with
high potency is required (Axelrad et al. 2016). Epidemio-
logical studies show the relation between mortality due to
These authors contributed equally: Nitesh Tamang, Gayathri
Ramamoorthy
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02578-5) contains supplementary
material, which is available to authorized users.
* Nageswara Rao Golakoti
gnageswararao@sssihl.edu.in
* Mukesh Doble
mukesh.doble0@gmail.com
1
Department of Chemistry, Sri Sathya Sai Institute of Higher
Learning, Puttaparthi, Andhra Pradesh, India
2
Department of Biotechnology, Indian Institute of Technology,
Chennai, Tamil Nadu, India
3
Department of Chemical Sciences, Indian Institute of Science
Education and Research (IISER), Mohali, Punjab, India

Medicinal Chemistry Research
cancer and chronic inflammation is 15–20% and inflam-
mation and cancer are connected by both intrinsic and
extrinsic pathways (Mantovani et al. 2008). A lot of triggers
such as microbial infections, autoimmune diseases,
inflammatory reactions, physical and chemical agents are
associated with chronic inflammation to dysregulate the
inflammatory response (Eiró and Vizoso 2012). When the
inflammatory response is failed or unresolved, chronic
inflammation plays a significant role in promotion, initia-
tion, malignant, metastasis of many human cancer tissues
(Lorusso and Rüegg 2008). Inflammatory enzymes like
cyclooxygenase (COX) and membrane-associated PGE
synthase-1 (mPGES-1) are important targets for anti-
inflammatory and anti-cancer strategy (Nakanishi et al.
2010; Ghosh et al. 2010). Prostaglandin E2 (PGE2) is
generated through the sequential action of COX and
mPGES-1 enzymatic reactions. Dysregulated function or
over-production of PGE2 has been associated with tumor
progression and inflammation (Ricciotti and FitzGerald
2011). Hence, to target inflammation is to significantly
reduce the incidence of human cancer.
Anti-inflammatory activities of the compounds were
estimated by measuring the decrease in PGE2 production
when treated with the compounds in inflammation induced
HeLa cells. Anticancer activities of the compounds were
studied by three different ways namely, by (1) measuring
the cytotoxicity by MTT assay; (2) observing the morpho-
logical changes in the cells after compound treatment. This
can tell if the cells die due to apoptosis (such as nuclear
fragmentation and chromatin condensation) or necrosis and
(3) cell cycle analysis—find percentage of the population in
various phases namely, G0/G1, S, and G2/M. This infor-
mation can be used to, monitor the effect of an anticancer
treatment.
Materials and methods
Synthesis
Chemicals used in the study were procured from Sigma-
Aldrich (Bangalore, India) and SD-fine (Maharashtra,
India).
Two different Schemes (1 and 2) were used for the
synthesis of DACPs. The compounds which we had diffi-
culty in synthesizing using Scheme 1 (base catalyzed) were
synthesized using Scheme 2 (acid catalyzed).
Mono-carbonyl analogs of Curcumin such as chalcones
and bis-chalcones have been synthesized and reported with
various biological activities (Fig. 1) such as anti-inflammatory
(Liang et al. 2008, 2009b; Wang et al. 2012; Wu et al. 2013;
Liu et al. 2014; Qian et al. 2015; Zhao et al. 2015), anticancer
(Liang et al. 2009a; Zhao et al. 2010; Wiji Prasetyaningrum
et al. 2018; Liu et al. 2018), antioxidant (Putri et al. 2018) and
antibacterial activities (Liang et al. 2008).
Base catalyzed reaction
In our studies, we have synthesized chalcones (Fig. 2a)
and reported their anti-bacterial activity (Kar et al. 2018). We
have also synthesized bis-chalcones (Fig. 2b) and reported
their anti-inflammatory activity (Kar et al. 2019) and anti-
malarial activity (Joshi et al. 2018). In our current study, we
have synthesized a class of diarylidenecyclopentanones
(DACPs), mono-carbonyl analogs of curcumin, and studied
their SAR for anti-inflammatory and cytotoxicity properties.
The DACPs (Ia–Il, II, III, and IV) were synthesized (Lee
et al. 2009) by slowly adding aq. NaOH (1.74 gm) to the
mixture of arylaldehyde (0.0171 mol) and cyclopentanone
(0.0085 mol) prepared earlier in ethanol–water (1:1) mix-
ture (30–50 ml) and kept in an ice bath. After the addition of
NaOH was completed the reaction mixture was allowed for
10 more minutes in the ice bath and thereafter it was
removed and stirred at room temperature till the completion
Fig. 1 Biological activities of
mono-carbonyl analogs of
curcumin with some of the most
active analogs

Medicinal Chemistry Research
(a)
(b)
Fig. 2 Molecular structure of:
a chalcones; b bis-chalcones
Scheme 1 Base catalyzed
synthesis of
diarylidenecyclopentanones
Scheme 2 Acid catalyzed
synthesis of
diarylidenecyclopentanones
of the reaction (monitored by TLC). After completion, the
mixture was added to ice-cold water and stirred followed by
filtration. The solid obtained was then dried and recrys-
tallized from chloroform–methanol mixture. In the case of
the compound Ij containing nitro group, the reaction mix-
ture was first purged with nitrogen and nitrogen atmosphere
was maintained throughout the reaction.
mixture was poured into ice-cold water and stirred followed
by filtration. The solid obtained was washed till the
washings are neutral, dried, and recrystallized from
chloroform–methanol mixture. DACPs (Im–Iv) were syn-
thesized using this method (Du et al. 2006). For compound
V the reaction contents were neutralized using aq. NaOH
before filtration.
Acid catalyzed reaction
Instrumentation
Arylaldehyde
(0.0171 mol)
and
cyclopentanone
The 1HNMR and 13CNMR spectra were obtained using
BRUKER 400 MHz with TMS as the internal standard and
CDCl3/DMSO-d6 as the solvent. AGILENT 6430 Triple Quad
LC/MS and Shimadzu 2010 plus were employed for recording
mass spectra. Thermo-Nicolet Avatar 370 spectrophotometer
(0.0085 mol)) were added into a round bottom flask con-
taining acetic acid saturated with HCl gas (15–30 ml). After
addition, the mixture was stirred till completion of the
reaction (checked by TLC). Upon completion, the reaction

Medicinal Chemistry Research
was used for recording FT-IR spectra. The spectra were
recorded between 400 and 4000 cm−1 using KBr pellets.
Shimadzu 2450 spectrophotometer was used for recording
UV–Vis spectra. The range of wavelength used was
200–600 nm.
concentration of the compounds for 48 h. Subsequently,
20 µl of MTT (5 mg/ml in phosphate buffered saline (PBS)
w/v) was added and plate was incubated for 4 h. After the
incubation, 200 µl of DMSO was added. The viable cells
have formed purple color formazan products and the
amount of purple formazan products were measured using
ELISA multimode plate reader (version 4.10.3005.1440) at
570 nm. The increasing concentration of the compound’s
absorbance were compared to untreated control absorbance.
The IC50 values of the compounds were analyzed by non-
linear regression using GraphPad software.
Single crystal X-ray diffraction data were collected on
Rigaku XtaLABmini X-ray diffractometer equipped with
Mercury CCD detector with graphite monochromatic Mo-
Kα radiation (λ = 0.71073 Å) at room temperature
(24.85 °C) using ω scans. The data were reduced using
CrysAlisPro 1.171.39.35c and the space group determina-
tion was done using Olex2 (Dolomanov et al. 2009). The
crystal structures were solved by using ShelXT (Sheldrick
2015) and were refined using ShelXL (Sheldrick 2008)
through Olex2 suite. All the hydrogen atoms were geome-
trically fixed and refined using the riding model. Absorption
correction was done by a multi-scan method. Data collec-
tion, crystal structure solution, and refinement details are
listed in the Supplementary section . All the packing and
interaction diagrams have been generated using Mercury
3.9.40 software (Macrae et al. 2008)
Cell morphology analysis
The apoptotic changes were evaluated by acridine orange/
ethidium bromide (AO/ETBR) staining. Briefly, the cells
were seeded in 96 well plate. After the cells reached 70%
confluence, cells were exposed to the compounds with a
concentration of 10 µM for 48 h. After 48 h incubation, the
supernatant from the plate was removed and cells were
washed with PBS. Subsequently, 2–3 drops of 1:1 ratio AO/
ETBR (10 µl/ml of ETBR and 0.1 mg/ml of AO) staining
solution were added. The stained plate was incubated at
37 °C for 15 min and the cell morphological changes were
visualized under fluorescent microscope. Images of both
live and dead cells were taken and finally, they were merged
using the Image J software system.
Biological activity
Cell culture and maintenance
The anti-cancer activity of the compounds was determined
in HeLa cells procured from National Centre for Cell Sci-
ence, Pune. The cervical cancer cell line was grown in
DMEM (Dulbecco’s Modified Eagle Medium) containing
essential antibiotics (1%) and Fetal bovine serum (10%)
procured from Sigma Aldrich, India. They were maintained
in a humidified atmosphere of 5% CO2 incubated at 37 °C.
Cell cycle analysis
The cell cycle analysis was done using Flow Cytometer.
The cells were seeded in 96 well cell culture dishes.
After the cells reached 70% confluence, the cells were
treated with compounds at 10 µM concentration for 48 h.
After the treatment, cells were harvested by trypsinization
and the cells were fixed with 70% ethanol (stored at −20 °C
for 24 h). The fixed cells were centrifuged (8000 rpm for
5 min) and washed again with PBS. The pellet was resus-
pended in 500 µl PBS, 5 µl of RNAs followed by adding
10 µl of propidium iodide (PI). The PI stained cells were
incubated at 37 °C for 30 min in dark condition. The cell
cycle analysis was performed using FACS Calibur software
and the results were compared with untreated control cells.
PGE2 assay
The cells were seeded in a 24 well plate. After the cells
reached 80% confluence, TNF alpha (50 ng/ml) was used to
induce inflammation. Then they were treated with 10 µM
compounds for 48 h and without compounds were taken as a
positive control Licofelone used as a positive control. The
PGE2 was measured by the Cayman PGE2 ELISA kit, the
absorbance was read at 410 nm (Enspire, PerkinElmer, USA).
The percentage inhibition of PGE2 production by the com-
pounds was calculated by normalizing the value obtained
from cells treated with TNFα alone (Puratchikody et al. 2019).
Results and discussion
Determination of cytotoxicity
Characterization
Cytotoxicity of the drugs was measured by MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide)
assay. Briefly, 104 cells were seeded into 96 well plate.
After 24 h incubation, the cells were treated with increasing
The synthesized compounds were purified by recrystalli-
zation and were characterized using UV–Vis, IR, 1H-NMR,
13C-NMR, and mass spectrometry. The spectral data for all
the compounds showed the expected λmax ~250 and 350 nm,

Medicinal Chemistry Research
Fig. 3 a Asymmetric unit of 2,5-bis((E)-2-chloro-3-hydroxy-4-meth-
oxybenzylidene)cyclopentanone (Iv); b H-bond in a layer of 2,5-Bis
((E)-2-chloro-3-hydroxy-4-methoxybenzylidene)cyclopentanone (Iv)
molecule; c molecular interaction of 2,5-Bis((E)-2-chloro-3-hydroxy-
4-methoxybenzylidene)cyclopentanone (Iv) in crystal packing
infrared bands, NMR signals and molecular ion peaks
confirmed their molecular structure.
The complete spectral data along with the assignments
are provided in the Supplementary section.
The broad band at 3200–3500 cm−1 in IR and signal at
around δ 10.00 ppm in 1H NMR spectra of Im, In, Ir, Is, It,
Iu, and Iv confirm the presence of aromatic –OH, which is
further confirmed by D2O exchange. The presence of
–OCH3 group in Ig, Ir, Is, It, and Iv was confirmed by the
observed C–O stretch at 1300 cm−1, 6H singlet at δ 3.8 ppm
in 1H NMR spectra and the signal at around δ 55.00 ppm in
13C NMR. The strong bands at around1500 and 1300 cm−1
in IR spectra confirm the presence of NO2 group in com-
pounds Io, Ip, Iq, and Ij. In all the compounds Ib, Ic, Id,
Ih, and Ij containing –Cl group, band at ~1100 cm−1 was
observed for the aromatic C–Cl stretch and [M]+, [M+2]+
peaks were observed in the mass spectra further confirming
the presence of –Cl group. Compound If contains the iso-
propyl group and is confirmed by signal at δ 1.27 ppm (6H,
doublet), δ 1.29 ppm (6H, doublet), δ 2.97 ppm (2H, septet)
X-ray diffraction analysis of single crystals of 2,5-bis((E)-2-
chloro-3-hydroxy-4-methoxybenzylidene) cyclopentanone
(Iv)
Crystal growth: The crystal growth of Iv was done by slow
evaporation growth technique at ambient temperature. The
purified compound was used for growing single crystals
using a chloroform/methanol mixture (1:1, v/v). Yellow
colored rod-shaped crystals were obtained.
Crystal structure description: The single crystal of Iv is
crystallized in monoclinic system with I2/a space group
(CCDC number is 1958556). The asymmetric unit contains
half molecule of the compound as shown in Fig. 3a.
Molecular interaction in crystal packing is shown in Fig. 3b,
c. Two molecules within a layer interacting by strong H-
bond [O1…H2-O2 (2.302 Å), which are arranged anti-
parallelly. We can see weak hydrogen bond and van der
Waals interaction between interlayer of Iv which are
arranged above, below and in the plane of molecule in 3D
packing (Fig. 3c). Table containing the detailed crystal-
lographic data of Iv is provided in the Supplementary
section.
1
in H NMR along with signals at δ 23.92 ppm and δ 34.23
ppm in 13C NMR. Compound Ii contains dimethyl amino
group which is confirmed by signals at δ 3.00 ppm (12H,
1
singlet) of H NMR along with δ 41 ppm in 13C NMR. In
the mass spectrum of compound IV [M + 2]+ peak of 9%
[RA] was observed confirming the presence of sulfur. The
mass spectra of all the compounds showed molecular ions
further supporting their structures.

Medicinal Chemistry Research
Table 1 In vitro anti-inflammatory testing results
Biological activity
Assigned No. % inhibition of
PGE2 production
Assigned No. % inhibition of
PGE2 production
Anti-inflammatory
Ia
Ib
Ic
Id
Ie
If
n.a.
In
58.64 2.1
50.91 7.7
n.a.
Twenty-six DACP derivatives were tested for their anti-
inflammatory activity and the results are tabulated in
Table 1. It was observed that compounds with hydroxyl
group showed good activity whereas the derivatives with
chloro substitution showed no activity. Interestingly the
activity of the compounds containing hydroxyl group varied
with changes in the position of the substitution (ortho, para)
and also with the presence of a different functional group.
Compound Is which contains methoxy at ortho position and
hydroxyl at para position showed the best activity among
all the 26 derivatives. Compounds Im (2-OH) and In (4-
OH) are positional isomers. From the observed data, it is
clear that In is 20 times more potent than Im showing the
necessity of the hydroxyl substitution at the para position.
The presence of methoxy group at ortho position where the
hydroxyl group is at para position enhances the activity (Is)
by twofold with respect to In. On the contrary, methoxy at
meta position decreases the activity of the compound It (3-
OMe-4-OH) when compared with In. Subsequently, when
both the meta positions are substituted with methoxy group
(Iu) activity becomes zero. Io (2NO2), Ie (-3CF3), and It
(3-OMe-4-OH) showed moderate activity. Among all the
compounds containing nitro group, substitutions at meta
and para positions were observed to be inactive. The
aforementioned inactivity of compounds with chloro sub-
stitution adds to the fact that when coupled with the –CF3
group at the meta position makes Ik inactive as compared to
the moderate activity observed in Ie. Compound III showed
a moderate activity among all the heteroaromatic derivatives
that were tested. Licofelone showing 97.36 2.4% inhibi-
tion is used as a positive control.
n.a.
Io
n.a.
Ip
n.a.
Iq
n.a.
48.36 1.2
n.a.
Ir
12.52 9.7
93.28 0.5
49.21 0.15
n.a.
Is
Ig
Ih
Ii
n.a.
It
n.a.
Iu
n.a.
Iv
n.a.
Ij
n.a.
II
n.a.
Ik
Il
n.a.
II
41.62 4.21
n.a.
n.a.
IV
Im
–
3.21 3.3
–
V
n.a.
Licofelone
97.36 2.4
n.a. no activity
Table 2 In vitro cytotoxicity testing results against HeLa cells
CODE % cell proliferation
(10 µM)
CODE % cell proliferation
(10 µM)
Ia
Ib
Ic
Id
Ie
If
91.27 4.00
90.57 6.66
81.70 4.76
90.24 5.89
82.05 6.99
71.50 4.14
84.83 2.19
81.52 1.01
69.44 1.38
85.74 4.28
76.83 0.06
78.19 0.20
86.26 2.28
–
In
Io
Ip
Iq
Ir
80.27 7.34
33.39 2.61
79.95 6.25
90.73 5.14
83.33 8.81
89.39 2.77
61.52 7.64
56.17 1.82
103.29 4.34
73.97 4.78
84.07 2.71
73.10 5.14
96.96 5.08
76.37 4.04
Is
Ig
Ih
Ii
It
Iu
Iv
II
Ij
Ik
Il
III
IV
V
Cytotoxicity
Im
–
5-flu
These DACP derivatives were tested for their cytotoxicity
effect against HeLa cells. The results are shown in Table 2.
The percentage inhibition values reveal that seven DACPs
showed significant inhibition at 10 µM in comparison to the
reference drug 5-fluorouracil (5-flu).
5-flu 5-fluorouracil
Bold font indicates active compounds
Three of the DACP derivatives showed good activity
with % cell proliferation of 33.39 2.61(Io), 61.52 7.64
(It), and 56.17 1.82 (Iu). It was observed that the presence
of electron donating hydroxyl group in the para position and
methoxyl group in the meta position, It (-3OMe-4OH) and
Iu (-4OH-3,5-MeO) had good activity compared to other
DCAP derivatives. Derivative with electron donating group
at ortho position Im (-2OH) has less activity than derivative
with electron accepting group at ortho position Io (-2NO2).
When the benzene ring is replaced with different
heteroaromatic rings we observed change in the activity. The
activity increased when replaced by furan moiety and thio-
phene moiety whereas decreased when it was replaced with
pyridine moiety as compared to the parent compound Ia.
The three compounds which exhibited the best activities
were tested further and their IC50 values were calculated. It
was observed that the compound Io was the most potent
anticancer compound with an IC50 value of 8.73 0.06 µM
and the reason may be attributed to the presence of nitro
group in the carbon skeleton. Nitro group undergoes an

Medicinal Chemistry Research
enzymatic reduction in a biological system producing
reactive species which can cause cell damage by oxidative
stress (Silva Lopes et al. 2015). The electron withdrawing
nitro group is crucial for inhibiting cancer cell growth and
has also been found in niclosamide and benzyl chloride
derivatives (Kauerova et al. 2016). The phenyl ring with
nitro moiety in ortho position is known to enhance the
anticancer effect of the curcumin related compounds
(Lima et al. 2016). It was also demonstrated that 2-
nitrobenzylidene derivatives of the indoline-3-one inhibit
HT-29, K562 and HepG2 with an IC50 in a range of 2.04,
2.33, and 2.24 µM, respectively (Jia et al. 2014). Compound
It (-3OMe-4OH) and Iu (-4OH-3,5-MeO) are the second
and the third potent compounds with IC50 values of 11.47
0.15 µM and 12.55 0.31 µM, respectively. The reference
drug 5-flu showed an IC50 of 15.02 µM indicating that the
three DACPs have very potent activity. Compound It
contains hydroxy and methoxy group at meta and para
positions respectively. Similarly, compound Iu also has a
methoxy group occupying the meta position and a hydroxy
at para position. Para position of curcumin analogs is an
important position where appropriate substitution may lead
to a drastic improvement in their biological activity (Pan
et al. 2016). Compound Iu is a more potent compound
because it has methoxy group occupying both the meta
positions compared to that of compound It whose only one
meta position is occupied by methoxy group. There are
reports about compounds containing hydroxy and methoxy
groups influencing the anticancer activity (IC50 2.54 μM)
against CEM/ADR5000 cells (Das and Manna 2016). Ear-
lier it was found that introducing methoxy substituent in
aromatic rings increases the antiproliferative effect against
HeLa within a range of 1 to 5 µM concentration (Tandon
et al. 2009).
Cell morphology
From the family of protease, caspases play a crucial role in
the apoptosis pathway. The biochemical and morphological
changes in the cells are due to apoptotic pathways. Cancer
generally develops upon the deregulation in the apoptosis
pathway (Pistritto et al. 2016). We have analyzed cell
morphology changes by staining with the acridine orange/
ethidium bromide method. The apoptosis associated chan-
ges in cells can be studied using this dual fluorescent
staining method. Acridine orange stains all the cells
whereas only the cells that have lost their cytoplasmic
membrane integrity will be stained by ethidium bromide
because of which live cells appear green. Bright green dots
in the nuclei are generally due to the nuclear fragmentation
and chromatin condensation which are observed in the early
apoptotic cells. The late apoptotic cells have condensed and
Fig. 4 AO/ETBR staining
images: a untreated HeLa cells,
b Io treated HeLa cells, c It
treated HeLa cells, d Iu treated
HeLa cells

Medicinal Chemistry Research
Fig. 5 Diarylidenecyclopentanone treated HeLa cells. a untreated HeLa cells, b Io treated cells, c It treated cells, d Iu treated cells
often fragmented nuclei and hence appear orange. Necrotic
cells also have orange nuclei but are displayed as normal
nuclear morphology (Ribble et al. 2005). The normal live
HeLa cells were seen as green dots in the absence of any
DACPs (untreated cells). In the DACP series, a drastic
decrease in the number of cells with the Io treatment was
observed (Fig. 4).
It and Iu treated cells also showed a decrease in the
number of cells but are lesser compared with Io. Overall,
we observed nuclear fragmentation and chromatin con-
densation for compounds treated cells in AO/ETBR staining
indicating them to be apoptosis inducers.
Fig. 6 The percentage of cells in different phases of the cell cycle is
plotted here for untreated and compound treated cells for DACPs
The cell cycle is controlled by various groups of cyclin
and its dependent protein kinases. For the progression of G1
(growth phase) Cdk4, Cdk6, and D-type cyclins (D1, D2,
and D3) complexes are necessary. To enter the S phase
(DNA synthesis) from the late G1 phase, Cdk2, and E-type
cyclins (E1 and E2) play a significant role whereas for the
progression of the S phase Cdk2 and A-type cyclin (A1 and
A2) complexes are required. The cells then enter into the G2
phase (growth phase). The G2/M transition and progression
Cell cycle analysis
FACS (Fluorescence-activated cell sorting) was employed to
study the cell cycle analysis and performed with cells incu-
bated with 10 µM of the compounds Io, It, and Iu for 48 h.
FACS image (Fig. 5 and 6) shows that the majority of the cells
are present in the G0/G1 phase in the untreated HeLa cells.

Medicinal Chemistry Research
Fig. 7 a untreated HeLa cells,
b Io treated cells at 50 µM
of M phase (mitosis) involve Cdk1 and B-type cyclin (B1
and B2) complexes (Shin et al. 2010). The Io treated cells
were highly accumulated in G0/G1 phase and exhibited
82.82% population in the G0/G1 phase. About 21.74 and
50.34% of cells were accumulated in subG0 and G0/G1
phases for the compound It. Hence, the compound It might
be downregulating the cyclinE, cyclinD1, Cdk2 protein
levels (Wang et al. 2015). Compound Iu treated cells were
vastly accumulated in the late subG0 phase and in the early
S phase. A significant portion of the cell population was
observed in S phase and this may indicate the inhibition of
mitosis and cancer cell proliferation (Luo et al. 2012). The
earlier reports with similar results indicated that the arrest of
the cell cycle at subG0 phase of HeLa aligned with Cdk1
downregulation (Khazaei et al. 2017).
The DACPs treated (compound Io, It, and Iu) cells were
highly accumulated in subG0 phase especially compound Iu
treated cells were vastly accumulated in the late subG0 phase.
The earlier reports have indicated that the arrest of the cell
cycle at subG0 phase of HeLa is due to Cdk1 downregulation
(Khazaei et al. 2017) leading to apoptosis in HeLa. Overall,
the cell culture studies show that the HeLa cells, when treated
with the DACP derivatives, show accumulation in the G0/G1
phase. This emphasizes that G0/G1 is the longest phase
through which the cells undergo apoptosis hence the treated
cells accumulate (Chen et al. 2017).
Fig. 8 The percentage of cells in different phases of the cell cycle is
plotted here for untreated and compound Io treated cells at 50 µM
property inhibiting PGE2 production and anticancer poten-
tial against HeLa cells. Compound Is with methoxy at ortho
and hydroxy at para position was observed to have the best
activity with 93.67% inhibition of PGE2 production
(10 µM) among all the DACPs. From the anti-inflammatory
studies, we conclude that the presence of the hydroxy group
is important for anti-inflammatory activity. The activity of
the hydroxy containing DACPs varied depending upon the
position of the substitution. The cytotoxicity studies showed
that the three compounds Io, It, and Iu to be the most active
among all the 26 DACPs. These three compounds
were chosen for further analysis. The anticancer potency of
the three compounds selected have their IC50 values in the
range of 8–13 µM. Cell cycle analysis was also carried out
to determine the arrested phase of the HeLa cells and was
concluded that the G0/G1 is the longest phase through which
the cells undergo apoptosis.
The compound Io at higher concentration (50 µM) has
drastically decreased the number of cells in all the cell cycle
phases when compared with the untreated control (Fig. 7
and 8).
Acknowledgements Authors dedicate the work to Founder Chancellor
Bhagawan Sri Sathya Sai Baba. The authors thank CRIF, SSSIHL for
providing the characterization facilities. We also thank Sri Swayam-
siddha Kar and Dr. Sai Manohar Chelli for their valuable inputs.
Nitesh Tamang thanks MOTA for the NFST fellowship (201819-
NFST-WES-02502) and Mayank Joshi thanks DST for Inspire fel-
lowship (IF160793).
Conclusion
In this study, 26 diarylidenecyclopentanones were synthe-
sized, characterized, and tested for their anti-inflammatory

Medicinal Chemistry Research
Compliance with ethical standards
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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