Regioselective synthesis and biological studies of novel 1-aryl-3, 5-bis (het) aryl pyrazole derivatives as potential antiproliferative agents

Article abstract of DOI:10.1007/s11010-016-2887-7

Pyrazole moiety represents an important category of heterocyclic compound in pharmaceutical and medicinal chemistry. The novel 1-aryl-3, 5-bis (het) aryl pyrazole derivatives were synthesized with complementary regioselectivity. The chemical structures we

Full text of DOI:10.1007/s11010-016-2887-7

Mol Cell Biochem
DOI 10.1007/s11010-016-2887-7
Regioselective synthesis and biological studies of novel 1-aryl-3,
5-bis (het) aryl pyrazole derivatives as potential antiproliferative
agents
1
2
•
Hanumappa Ananda1 Kothanahally S. Sharath Kumar Mayilaadumveettil Nishana
•
•
Mahesh Hegde1 Mrinal Srivastava2 Raghava Byregowda1 Bibha Choudhary3
Sathees C. Raghavan2 Kanchugarakoppal S. Rangappa1
•
•
•
•
•
Received: 3 August 2016 / Accepted: 10 November 2016
Ó Springer Science+Business Media New York 2016
Abstract Pyrazole moiety represents an important cate-
gory of heterocyclic compound in pharmaceutical and
medicinal chemistry. The novel 1-aryl-3, 5-bis (het) aryl
pyrazole derivatives were synthesized with complementary
regioselectivity. The chemical structures were confirmed
by IR, 1H NMR, 13C NMR, and mass spectral analysis. The
chemical entities were screened in various cancer cell lines
to assess their cell viability activity. Results showed that
the compound 3-(1-(4-bromophenyl)-5-phenyl-1H-pyra-
zol-3-yl) pyridine (5d) possessed maximum cytotoxic
effect against breast cancer and leukemic cells. The cyto-
toxicity was confirmed by live–dead cell assay and cell
cycle analysis. Mitochondrial membrane potential,
Annexin V-FITC staining, DNA fragmentation, Hoechst
staining, and western blot assays revealed the ability of
compound 5d to induce cell death by activating apoptosis
in cancer cells. Thus, the present study demonstrates that
compound 5d could be an attractive chemical entity for the
development of small molecule inhibitors for treatment of
leukemia and breast cancer.
Keywords Pyrazole Á Regioselectivity Á Dithioesters Á
Breast cancer Á MCF7 Á Apoptosis
Introduction
One of the major global challenges that the health system
faces today is to provide an effective treatment for cancer,
which significantly contributes to the global disease bur-
den. Cancer is a disease characterized by the uncontrolled
proliferation of cells which can invade and spread to
adjacent sites of the body. The most common cancers
include that of breast, prostate, lung, colon, and blood [1].
Each year, nearly 1.3 million women are diagnosed with
breast cancer [2]. Breast cancer is not a single disease
entity, but a summation of subclasses with varying clinical
outcome [3]. Although this is one of the cancers that can be
effectively treated in many patients, still there is a need for
effective small molecule inhibitors.
Among the cancers, leukemia is considered as one of the
most aggressive and life-threatening hematological malig-
nancies. Although leukemia is sensitive to Chemothera-
peutic agents, there are only few effective chemicals that
are used currently [4]. Hence, there is an increased need for
identification of novel chemical inhibitors that can be used
for cancer therapy.
Electronic supplementary material The online version of this
article (doi:10.1007/s11010-016-2887-7) contains supplementary
material, which is available to authorized users.
& Sathees C. Raghavan
sathees@biochem.iisc.ernet.in
The pyrazole moiety is one of the key building blocks in
several drugs, and it possesses a wide range of pharma-
cological activities [5–10]. Several commercial drugs such
as sildenafil [11], celecoxib [12], and Fipronil are highly
substituted pyrazole derivatives, and they have proved to
be good ligands for estrogen receptors. For the last two
decades, John A. Katzenellenbogen and co-workers have
been working on pyrazole analogues as estrogen receptor
antagonists [13]. Recently, we have been able to synthesize
& Kanchugarakoppal S. Rangappa
rangappaks@chemistry.uni-mysore.ac.in
1
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysuru 570006, India
2
Department of Biochemistry, Indian Institute of Science,
Bangalore 560012, India
3
Institute of Bioinformatics and Applied Biotechnology,
Electronics City, Bangalore 560 100, India
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Mol Cell Biochem
and identify compounds such as 5-amino pyrazole and
4-thiazolidinone derivatives which induce apoptosis
[14, 15]. The present study examines the effect of series of
novel aryl (het)pyrazole derivatives in inducing cytotoxi-
city in five human cell lines.
Biology
Chemicals and reagents
All the chemicals used in present study were purchased
from Sigma-Aldrich, USA, Sisco Research Laboratories
(SRL), India, and antibodies were purchased from Santa
Cruz Biotechnology, USA.
To continue our previous studies to develop new
chemotherapeutic agents [16, 17], we have synthesized
novel 1-aryl-3,5-bis(het)aryl pyrazoles 4a–e (Table 1)
via cyclocondensation of monothio-1,3-diketones with
different arylhydrazines [18]. Further, we have also
synthesized other regioisomers 5a–e (Table 2) directly
from active methylene ketones 1a–d via one-pot, three-
component reaction (Fig. 1). The structures of these
1-aryl-3,5-bis(het)aryl pyrazoles also resemble tamox-
ifen in which the ethylene bridge was replaced with
pyrazole ring. All the synthesized derivatives were
evaluated in vitro against cancer cell lines such as MCF7,
T47D, K562, and MOLT4, and transformed normal
human embryonic kidney cell line, HEK 293T, and the
mouse embryonic fibroblast cell line, MEF. All the above
results confirm that 3-(1-(4-bromophenyl)-5-phenyl-1H-
pyrazol-3-yl) pyridine (5d) (Fig. 2) could induce cell
death by activating apoptotic pathways in various cancer
cell lines.
Cell lines and culture
Human cell lines were procured from National Centre for
Cell Science, Pune, India and used for the study. The cells
used were human chronic myelogenous leukemia (CML)
cell line (K562), human T cell leukemia cell line
(MOLT4), human breast adenocarcinoma cell lines
(MCF7, T47D), human embryonic kidney cell line (HEK
293T), and mouse embryonic fibroblast cell line (MEF).
Further, cell lines were cultured and maintained in RPMI
1640 (Roswell Park Memorial Institute), MEM (Minimum
Essential Medium), and DMEM (Dulbecco’s Modified
Eagle’s Medium) containing 10% fetal bovine serum
(FBS), 100 U/ml of penicillin, and 100 lg of streptomycin/
mL at 37 °C in 98% humidity with 5% CO2.
MTT assay
Materials and methods
Cytotoxic effect of the compounds against the breast carci-
noma and leukemic cells was assessed using 3-(4,5-Dime-
Thin-Layer Chromatography (TLC) was used to monitor
the progress of the reaction, which was visualized under
UV light. Agilent–Varian NMR spectrometer was used to
record 1H NMR and 13C NMR spectra operating at 400 and
100 MHz, respectively, with tetramethylsilane as internal
standard. High-resolution mass spectrometer (ESI) was
used to record the mass spectra. IR spectra were recorded
in KBr on Shimadzu FT-IR model 8300 spectrophotome-
ter. Column chromatography was performed using 60:120
silica gel.
thyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
(MTT) assay [19–22]. Cells were seeded at a density of
0.5 9 105 cells/ml in 24-well plates, and the cells were
treated with compounds. Initially, all the compounds were
tested at 1, 10, 50, and 100 lM for 48 h, and IC50 values
were determined. Further, the potent compound 5d was
tested in different cancer cell lines at different concentra-
tions (5, 10, 20, and 40 lM) for MTT assay. Cells were
harvested post 48- and 72-h treatment, respectively, and
Table 1 Regioselective synthesis of 1,3,5-trisubstituted pyrazoles 4a–e
Entrya 2 (monothio-1,3-diketones)
Ar3(phenyl
hydrazines)
Products
Yield
(%)b
4a
3-(4-methoxyphenyl)-1-phenyl-3-
thioxopropan-1-one
(4-fluorophenyl)
hydrazine
1-(4-fluorophenyl)-3-(4-methoxyphenyl)-5-
phenyl-1H-pyrazole
86
4b
4c
3-(3-phenyl-3-thioxopropanoyl)benzonitrile
phenylhydrazine
phenylhydrazine
3-(1,3-diphenyl-1H-pyrazol-5-yl)benzonitrile
3-(1,3-diphenyl-1H-pyrazol-5-yl)pyridine
92
84
3-phenyl-1-(pyridin-3-yl)-3-thioxopropan-1-
one
4d
4e
a
3-phenyl-1-(pyridin-3-yl)-3-thioxopropan-1-
one
(4-bromophenyl)
hydrazine
3-(1-(4-bromophenyl)-3-phenyl-1H-pyrazol-5-
yl)pyridine
81
78
1-(3-methylthiophen-2-yl)-3-phenyl-3-
thioxopropan-1-one
phenylhydrazine
5-(3-methylthiophen-2-yl)-1,3-diphenyl-1H-
pyrazole
Reaction conditions: 2(1.0 mmol), arylhydrazine (1.1 mmol), in EtOH, reflux, 4 h
b
Isolated yield
123

Mol Cell Biochem
Table 2 Synthesis of regioisomeric 1, 3, 5-trisubstituted pyrazole derivatives 5a–e via one-pot, three-component reaction
Entrya 1 (Acetophe nones)
2 (Dithioesters)
Ar3(phenyl
hydrazines)
Products
Yield
(%)b
5a
Acetophenone
methyl
4-methoxybenzodithioate
(4-fluoro
phenyl)hydrazine
1-(4-fluorophenyl)-5-(4-methoxyphenyl)-3-
phenyl-1H-pyrazole
76
5b
5c
5d
3-acetyl benzonitrile
methyl benzodithioate
Phenyl hydrazine
Phenyl hydrazine
3-(1,5-diphenyl-1H-pyrazol-3-yl)benzonitrile 85
3-(1,5-diphenyl-1H-pyrazol-3-yl)pyridine 80
1-(pyridin-3-yl)ethanone methyl benzodithioate
1-(pyridin-3-yl)ethanone methyl benzodithioate
(4-bromo
phenyl)hydrazine
3-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol- 79
3-yl)pyridine
5e
1-(3-methylthiophen-2-
yl)ethanone
methyl benzodithioate
Phenyl hydrazine
3-(3-methylthiophen-2-yl)-1,5-diphenyl-1H- 74
pyrazole
a
Reaction conditions: Ketone 1(1.0 mmol), dithioester 2(1.2 mmol), NaH (2.0 mmol, 100%), MeI (1.2 mmol), Ar3NHNH2 (1.1 mmol), in DMF,
90 °C, 4–6 h
b
Isolated yield
Fig. 1 Regioselective synthesis of 1-aryl-3, 5-bis(het)aryl pyrazole derivatives. It was done via cyclocondensation of monothio-1,3-diketones
with different arylhydrazines and one-pot, three-component system
transferred to 96-well plates. MTT (0.5 mg/ml) was added
and incubated at 37 °C for 4 h; the resulting formazan
crystals were dissolved by adding solubilizing agent (10%
SDS and 50% dimethyl formamide) for 2 h, and readings
were acquired at 570 nm (Bio-Rad, USA). Since the com-
pounds were dissolved in DMSO, equivalent volume of
DMSO corresponding to the highest concentration alone
served as vehicle control.
reduction in cell number was determined. Each experiment
was repeated for a minimum of three independent times with
good agreement, and the values were plotted and presented.
Live–dead cell assay
MCF7 cells were seeded in 12-well plates (0.5 9 105 cells
per ml); the cells were treated with compound 5d (10, 20,
and 40 lM) and cultured for 48 h. The cells were then
trypsinized and harvested, washed with phosphate buffered
saline (PBS), and stained with ethidium homodimer-1
(5 lg/ml) and Calcein-AM (1 lg/ml) (Sigma-Aldrich,
USA) in dark for 20 min at RT. The reading for a mini-
mum of 10,000 cells was acquired in flow cytometer
(FACS Verse, BD Biosciences, USA). For live cells, an
excitation of 480 nm and emission of 527/532 nm, and for
Trypan blue assay
The cell viability following addition of the compound was
tested using Trypan blue assay as described previously
[23, 24]. Briefly, cells were treated with compound 5d (5, 10,
20, and 40 lM) for 48 and 72 h, and mixed with 0.4% trypan
blue. The viable cells were counted using hemocytometer, and
123

Mol Cell Biochem
Fig. 2 Spectral data of compound 5d. a HRMS, b 13C NMR, c 1H NMR
dead cells, an excitation of 528 nm and emission of
617 nm were used [25]. Results were analyzed in WinMDI
2.9 software and presented in dot plot. Quantification of
live and dead cells was plotted in bar diagram based on two
plates (0.75 9 105 cells/ml) and treated with different
concentrations of compound 5d (10, 20, and 40 lM) for
48 h. DMSO-treated cells served as vehicle control. Cells
were harvested, washed with PBS, and stained with JC-1
independent experiments.
dye
(5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimida-
zolo-carbocyanine iodide) (0.5 lg/ml) (Calbiochem, USA)
at 37 °C for 30 min. The samples were washed, resus-
pended in PBS, and acquired by FACS (FACS Verse, BD
Biosciences, USA) using Cell Quest Pro Software. A
minimum of 10,000 cells were measured, and 2,4-dinitro-
phenylhydrazine (2,4-DNP) was used as positive control.
Data were analyzed using WinMDI 2.9 software and
plotted.
Cell cycle analysis
Cell cycle analysis was performed as described earlier [26].
MCF7 and K562 cells (0.75 9 105 cells/ml) were treated
with compound 5d (5, 10, 20, and 40 lM) and cultured for
48 h. Cells were harvested and washed with PBS, treated
with RNase A (50 lg/ml) (Sigma-Aldrich, USA), stained
with propidium iodide (Sigma-Aldrich, USA), suspended in
PBS, and analyzed by FACS (FACS Verse, BD Biosciences,
USA) using Cell Quest Pro Software. Data analysis was
performed using Flowing Software (Version 2.5).
Annexin V- FITC staining
To identify apoptotic cells following treatment with com-
pound 5d in MCF7 cells, Annexin V-FITC and propidium
iodide staining were carried out [29]. Briefly, MCF7 cells
(0.5 9 105 cells per ml) were seeded in 12-well plates, and
cells were treated with compound 5d (20 and 40 lM,
48 h). Cells were harvested, washed, suspended in cell-
Mitochondrial membrane potential assay
JC-1 staining was performed as described previously
[27, 28]. MCF7 and K562 cells were seeded in 12-well
123

Mol Cell Biochem
Statistical analysis
binding buffer, and stained with Annexin V-FITC (0.2 mg/
ml) and propidium iodide (0.05 mg/ml) (Santacruz, USA).
Cells were then subjected to FACS analysis (FACS Verse,
BD Biosciences, USA) (10,000 cells were acquired). The
results were analyzed in Flowing Software (Version 2.5),
and data are presented as bar diagram with error bars based
on three independent experiments.
The values were expressed as mean SEM for samples,
and statistical analysis was performed using Graph Pad
Software 5.1. One-way ANOVA followed by Dunnett’s
test was performed for determining the significance. The
values were considered as statistically significant if the p-
value was equal to or less than 0.05 (0.05*, 0.005**,
0.0005***).
DNA fragmentation assay
To quantify the percentage of nuclei undergoing DNA
fragmentation upon treatment with compound 5d, DNA
fragmentation assay was performed [30]. Briefly, MCF7
cells were cultured in the presence of 5d (5, 10, 20, and
40 lM) for 48 h. Cells were harvested, and genomic DNA
was extracted. Cells were lysed using lysis buffer
(100 mM NaCl, 10 mM Tris (pH 8.0), 0.25% Triton
X-100, 1 mM EDTA, and 100 lg/ml Proteinase K) and
incubated at 55 °C for 5 h. Following RNase A (50 lg/
ml) treatment (37 °C, 1 h), samples were deproteinized
using phenol:chloroform, and DNA was precipitated in
the presence of 1/10th volume of 3 M sodium acetate (pH
5.2) and equal volume of isopropanol. DNA pellet was
washed with 70% ethanol, dried, and dissolved in TE
buffer. The DNA samples were electrophoresed on agar-
ose gel (2%), visualized following ethidium bromide
staining, and photographed. DMSO-treated cells were
used as control.
Results
Study of novel regioselectively synthesized
compounds on human cancer cell lines identified 5d
as potent anticancer molecule
Regioselectively synthesized novel pyrazole derivatives
(4a–e and 5a–e) were screened for their ability to induce
cytotoxicity in different cancer cell lines (MCF7, T47D,
K562, and MOLT4). MTT assay showed that treatment
with different concentrations of the compounds (1, 10, 50,
and 100 lM, 48 h) resulted in cell death at varying levels.
Among the compounds, 5d showed maximum inhibition on
cell viability, followed by 4d (Table 3), while other iso-
mers of pyrazole did not affect cell viability significantly
on tested cell lines. Further, 5d and 4d were screened on
MOLT4 cells where 5d showed significant cytotoxicity.
Interestingly, 5d did not inhibit the cell viability in normal
cell lines, HEK 293T and MEF. The IC50 of 5d was
Hoechst staining
To check the chromatin condensation upon 5d treatment in
MCF7 cells, Hoechst staining was carried out [31]. Briefly,
MCF7 cells were treated with 5d (20 and 40 lM, 48 h),
processed and stained with Hoechst dye (1 lM). Images
were captured using Carl Zeiss Axioscope A1 microscope
(Germany). Cells with condensed chromatin were counted
and presented (n [ 300 cells).
Table 3 Inhibition studies of pyrazole derivatives
Compound Cell lines (IC50 Value in lM)
No.
MCF-7 T47D K562 MOLT4 HEK 293T MEF
4a
78.1
99.8
[100
53.7
[100
53.7
65.9
90
[100 [100 N.T
[100 [100 N.T
[100 [100 N.T
N.T
N.T
N.T
N.T
N.T
N.T
N.T
N.T
[50
N.T
N.T
N.T
N.T
N.T
N.T
N.T
N.T
N.T
N.T
[100
N.T
N.T
4b
4c
4d
49
44.8 [100 N.T
61 [100 N.T
85.1 95.1
Western blot analysis
4e
5a
Western blotting was performed as described previously
[32]. MCF7 cells were treated with compound 5d (20 and
40 lM; 48 h); cells were harvested and proteins were
extracted using RIPA buffer. Resulting samples were
resolved on SDS-PAGE (10%), transferred to PVDF
membrane, and western blotting was performed using
various primary antibodies, such as activated PARP, acti-
vated Caspase 3, Smac/DIABLO, and GAPDH. The blot
was washed with PBST buffer, incubated with respective
secondary antibody, and visualized using chemilumines-
cence method.
5b
61.9 [100 N.T
[100 [100 N.T
5c
5d
25.4
77.9
36.1
[100 N.T
N.T
26.2 28
5e
N.T
N.T
Paclitaxel
0.0096 N.T
Antiproliferative properties of 1-Aryl-3, 5-bis (het) aryl Pyrazole
derivatives (4a–4e and 5a–5e) on MCF7, T47D, K562, MOLT4, HEK
293T, and MEF cells. IC50 values were based on MTT assay results at
48-h time point
N.T not tested
123

Mol Cell Biochem
determined to be 25.4, 36.1, 26.2, 28.0, [50.0,
and [100 lM in MCF7, T47D, K562, MOLT4, HEK
293T, and MEF cells, respectively (Table 3).
5d, a remarkable change in the cell cycle pattern was
observed during the cell cycle distribution, particularly in
the subG1 population following treatment 40 lM of 5d
(Fig. 5a, c). However, we observed fewer changes at lower
concentrations (5, 10, and 20 lM). Studies were also car-
ried out at time intervals of 24, 36, and 48 h after treating
with 30 lM of compound 5d in MCF7 cells. In both the
cases, notable accumulation of subG1 population was
observed suggesting that 5d promotes cell death through
apoptosis (Fig. 5e). The percentage of cell populations in
MCF7 and K562 at each phase of cell cycle is represented
in the bar diagram (Fig. 5 b, d, f).
Based on the above results, detailed cytotoxic studies
were carried out for compound 5d on both breast cancer
and leukemic cell lines using MTT and trypan blue assays
(48 and 72 h) (Fig. 3). Trypan blue assay showed reduced
number of live cells following treatment with compound
5d (5, 10, 20, and 40 lM) in MCF7, T47D, K562, and
MOLT4 cell lines. Besides, MTT assay results confirmed
the reduction in cell viability.
Live–dead cell assay on MCF7 cells treated
with compound 5d
Compound 5d treatment induced changes
in mitochondrial membrane potential
To examine the cell death induced by compound 5d on
MCF7 cells, live–dead cell assay was carried out using
calcein-AM and ethidium homodimer staining. Calcein
stains live cells, while ethidium homodimer stains only
dead cells by virtue of the broken cell membrane. A dose-
dependent (10, 20, and 40 lM for 48 h) increase in dead
cells and decrease in number of calcein-AM-stained cells
was observed following treatment with 5d in MCF7 cells,
indicating cell death following treatment with 5d (Fig. 4a,
b). These results confirmed the ability of compound 5d to
induce cell death in MCF7 cells.
To check the effect of compound 5d on mitochondrial
membrane potential (Dwm), the experiment was carried out
by staining with JC-1 following treatment with 5d on
MCF7 and K562 cells (10, 20, and 40 lM, 48 h). Changes
in the ratio of red to green fluorescence are an indication of
lower mitochondrial membrane potential. In healthy
mitochondria, JC-1 forms aggregates and gives red fluo-
rescence; on the other hand, in apoptotic or unhealthy/dead
cells with low mitochondrial membrane potential, JC-1
remains in the monomeric form which gives only green
fluorescence. We observed an increase in the number of
cells which are positive for green fluorescence after 48 h
treatment in both MCF7 and K562 cells (Fig. 6a, c), sug-
gesting the involvement of mitochondrial apoptotic
machinery in induction of cell death upon treatment with
compound 5d. The percentage of low and high mitochon-
drial cell populations in MCF7 and K562 is represented in
the bar diagram (Fig. 6 b, d).
5d treatment leads to accumulation of the cells
in the subG1 phase of the cell cycle
The effect of compound 5d (5, 10, 20, and 40 lM; 48 h) on
cell cycle progression in MCF7 and K562 cells was ana-
lyzed by flow cytometric analysis (Fig. 5). On addition of
MCF7
K562
293T
T47D
MOLT4
A
C
E
G
I
150
100
50
150
100
50
150
100
50
150
100
50
150
100
50
48 h
72 h
48 h
72 h
48 h
72 h
48 h
72 h
48 h
72 h
*
*
**
***
***
***
***
**
**
**
***
**
**
***
***
***
***
***
***
***
10 20 40
Concentration (µM)
0
0
0
0
0
C
5
10 20
40
C
5
10 20
40
40
C
5
10 20 40
C
5
C
5
10 20
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
B
D
F
H
J
150
100
150
100
48 h
72 h
48 h
72 h
150
100
150
100
150
100
50
48 h
48 h
72 h
48 h
72 h
72 h
*
**
*
*
***
***
**
**
***
***
*** ***
***
***
50
0
***
***
***
**
50
0
50
50
***
***
***
***
***
***
***
10 20 40
0
0
0
C
5
10 20 40
C
5
10 20 40
C
5
10 20 40
C
5
10 20 40
C
5
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
Fig. 3 Cytotoxicity effect of 5d on MCF7, K562, 293T, T47D, and
MOLT4 cells. a, c, e, g, i MTT assay showing effect of 5d (5, 10, 20,
and 40 lM; 48 and 72 h) on cell viability in different cells. Results are
represented as percentage of cell viability at different concentrations of
5d. b, d, f, h, j Trypan blue assay showing impact of 5d (5, 10, 20, and
40 lM at 48 and 72 h) treatment on cell viability. In all samples, ‘‘C’’
stands for DMSO-treated control. Each experiment was carried out for
a minimum of 3 times. Error bars are given as SEM. The values were
considered as statistically significant if the p-value was equal to or less
than 0.05 (0.05*, 0.005**, 0.0005***). (Color figure online)
123

Mol Cell Biochem
A
Control
10 µM
20 µM
40 µM
Calcein -AM
B
Live Cells
Dead Cells
100
50
0
C
10
20
40
Concentraꢀon (µM)
Fig. 4 Live–dead cell assay of 5d-treated MCF7 cells. a Dot plot
representing MCF7 cells treated with 5d (10, 20, and 40 lM). Live
cells are stained with Calcein-AM and dead cells are stained with
ethidium homodimer. b Bar diagram representing percentage positive
cells for Calcein-AM and ethidium homodimer
A
B
Control
5 µM
10 µM
20 µM
40 µM
G1
G2/M
S
100
SubG1
50
0
PI-A
PI-A
PI-A
PI-A
PI-A
C
5
10
20
40
Concentration (µM)
C
E
D
Control
5 µM
10µM
20 µM
40 µM
G1
G2/M
S
100
SubG1
50
0
PI-A
PI-A
24 h
PI-A
PI-A
PI-A
C
5
10
20
40
Concentration (µM)
F
Control
36 h
48 h
G1
G2/M
S
100
50
0
SubG1
C
24
36
48
PI-A
PI-A
PI-A
PI-A
Time (h)
Fig. 5 Effect of 5d treatment on cell cycle progression in MCF7 and
K562 cells. a, c, e Histograms representing cell cycle distribution of
DMSO-treated control cells and 5d-treated MCF7 and K562 cells (5,
10, 20, and 40 lM for 48 h and 24, 36, and 48 h at 30 lM),
respectively. b, d, f Bar diagram showing quantification of percent-
age of cell population in different phases of cell cycle shown in MCF7
and K562, respectively
123

Mol Cell Biochem
A
Control
2,.4 DNP
10 µM
20 µM
B
R1
R2
100
50
0
40 µM
C
10
20
40
2.4-DNP
Concentration (µM
)
C
Control
2,.4 DNP
10 µM
20 µM
D
R1
R2
100
50
0
40 µM
C
10
20
40
2.4 DNP
Concentration (µM)
Fig. 6 Evaluation of loss of mitochondrial membrane potential
following 5d treatment in MCF7 and K562 cells. a, c Density plot
representing JC-1-stained MCF7 and K562 cells at various concen-
trations of 5d treatment (10, 20, and 40 lM). 2, 4-DNP-treated MCF7
and K562 cells were used as a positive control. b, d Bar graph
showing percentage of red fluorescent cells against green fluorescent
cells, which indicates high and low MMP, respectively. (Color
figure online)
123

Mol Cell Biochem
Compound 5d induces apoptosis
MCF7 cells (20 and 40 lL for 48 h), which suggested
induction of apoptosis. Moreover, western blot results
confirmed the overexpression of Smac/DIABLO and the
cleavage of PARP1, and Caspase 3 in 5d-treated cases due
to the activation of apoptosis (Fig. 8a–c).
Translocation of membrane phosphatidylserine (PS) from
the inner side of the plasma membrane to the outer surface
is a hallmark of apoptosis. We studied induction of apop-
tosis in MCF7 cells by compound 5d using Annexin
V-FITC and propidium iodide double staining followed by
flow cytometry (Fig. 7a, b). We observed an increase in the
number of cells stained with Annexin V-FITC and pro-
pidium iodide in a dose-dependent manner upon treatment
with 5d (20 and 40 lM) in MCF7 cells compared to
DMSO-treated control cells, suggesting induction of
apoptosis.
Discussion
Identification of novel small molecule inhibitors for control
of proliferating neoplastic cells is needed in the field of
medicinal chemistry and pharmaceutical science. Pyrazole
heterocycle is considered as extremely versatile building
blocks in chemical biology (Fig. 9). In that route, we have
reported previously [18] an efficient protocol for the syn-
thesis of highly substituted 1-aryl-3,5-bis(het) aryl pyra-
zoles via cyclocondensation of arylhydrazines with either
1,3-bis(het)aryl monothio-b-diketones or 1,3-bis(het)aryl-
3-(methyl thio)-2-propenones; the substituents at the 3- and
5- positions of pyrazoles are complementary to each other
in terms of regioselectivity. By employing the same strat-
egy while continuing our research in the field of chemical
biology, novel 1,3,5-trisubstituted pyrazole derivatives 4a–
5d treatment results in DNA fragmentation,
chromatin condensation, and activation of apoptotic
markers
DNA fragmentation is a hallmark of activation of apopto-
sis. 5d-treated MCF7 cells (5, 10, 20, and 40 lL for 48 h)
showed prominent DNA ladder formation in a concentra-
tion-dependent manner. Besides, Hoechst staining con-
firmed significant chromatin condensation in 5d-treated
A
Control
20
Necrosis
M
40 M
B
50
40
30
20
10
0
Early apoptosis
Late apoptosis
C
20
40
Concentration (µM)
Fig. 7 Annexin V-FITC/PI staining of 5d-treated MCF7 cells.
a MCF7 cells were treated with 5d (20 and 40 lM) for 48 h and
processed for Annexin V-FITC/PI double staining. Lower left
quadrant shows cells which are negative for both Annexin V-FITC
and PI; lower right quadrant shows only Annexin V-positive cells,
which are in early stage of apoptosis; upper left quadrant shows only
PI-positive cells which are necrotic/dead cells; and upper right
quadrant shows both Annexin V- and PI-positive cells, which are
undergoing late apoptosis. b Bar graph shows the percentage of cell
population in different stages of apoptosis
123

Mol Cell Biochem
A
B
Control
20 µM
40 µM
M
C
5
10 20 40 (µM)
Bp 10000
1500
1000
800
600
400
200
Sample
details
No. of cells
counted
Condensed
cells
% of
condensed cells
C
C
20
40 (µM)
Control
692
943
354
86
12.4
Cleaved PARP
20 µM
40 µM
347
183
36.7
51.6
Activated Caspase 3
Smac/DIABLO
GAPDH
Fig. 8 Evaluation of mechanism of 5d-induced apoptosis. a Agarose
gel profiles showing DNA fragmentation. Following treatment with
5d (5, 10, 20, and 40 lL) in MCF7 cells, the chromosomal DNA was
extracted and used for gel electrophoresis. ‘‘M’’ is marker. b Hoechst
staining showing the chromatin-condensed nuclei in apoptotic cells
following treatment with compound 5d (20 and 40 lM) in MCF7.
c Western blotting studies showing expression levels of apoptosis-
related proteins upon 5d treatment (20 and 40 lM) in MCF7 cells.
The expression profile of PARP1, Caspase3, and Smac/DIABLO
proteins are shown after 5d exposure. GAPDH served as loading
control
e and 5a–e (Fig. 1) with complementary regioselectivity
were synthesized. Base-induced thioacylation of different
(het)aryl methyl ketones 1a–d with dithioesters 2a–c in the
presence of sodium hydride in DMF yielded the key
intermediate 1,3-substituted monothio-1,3-diketones 3a–d
in excellent yields. We have essentially focused on the
discovery of new heterocyclic small molecules which tar-
get kinases such as p38a MAP, Src, B-Raf, EGFR, and
VEGFR-2 relevant in cancer [33], and other proteins such
as CB1 receptor antagonists [34], Histone Deacetylase
Probes [35], Heat shock proteins [36], and bacterial dihy-
droorotate dehydrogenase (DHODase) [37] are also tar-
geted. In the similar manner, we have screened a few of the
derivatives (4a–e and 5a–e) which were synthesized from
different monothio 1,3-diketones bearing various sub-
stituents and tested in various cancer cell lines. Among
them, compound 5d was found to be enhanced, and its
synthesis is attained as shown in Fig. 1.
showed the reduction in cell viability in a dose-dependent
manner. Interestingly, its effect was less in noncancerous
cells, particularly in MEF cells. Evaluation of molecular
mechanism of action of compound 5d, in MCF7 and
K562, using flow cytometry revealed increase in dead cell
population in both cancer cells. Depolarization in mito-
chondrial membrane potential leading to apoptosis was
also evident when both the cancer cell lines were treated
with 5d.
Pyrazole moiety is suggested to affect the regulation of
gene expression and epigenetic signaling pathways in
cancer and numerous other diseases [38]. DNA fragmen-
tation, chromatin condensation, and activation of apoptotic
markers studies showed that compound 5d efficiently
inhibit the proliferation of tumor cells by inducing apop-
tosis (Figs. 7a, b, 8a–c). Previous studies have shown that
pyrazole selectively inhibits Hsp90 in small-cell lung
cancer [39] and estrogen receptor and osteoporosis inhi-
bitor in breast cancer [40]. Therefore, it is possible that
pyrazole heterocycle is an effective scaffold for improve-
ment of inhibitors against diverse targets.
Synthesized pyrazole derivatives are tested for their
ability to reduce cell viability in MCF7, T47D, MOLT4,
and K562 cancer cells. Cells treated with compound
123

Mol Cell Biochem
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Conclusion
Thus, in vitro studies with the compound 5d (1-aryl-3,
5-bis (het) aryl pyrazole) revealed that it has antiprolifer-
ative and apoptotic effects in leukemic and breast adeno-
carcinoma cells. Hence, compound 5d can be further
derivatized to improve the potency and can be developed as
a cancer therapeutic.
Acknowledgements The authors acknowledge to the CSIR Project,
Govt. of India for financial support to K.S.R. for the project vide No.
01 (2434)/10/EMR-II Dated 28.12.2010. SCR acknowledges the
financial assistance from IISc-DBT partnership programme. H.A.
acknowledges ICMR-SRF (No 45/54/2013-PHA/BMS, Dated:
08.12.2014) for research fellowship. Authors also thank Vashishta
from the IISc FACS facility, Bengaluru, and Central instrumentation
facility, Institute of excellence (IOE), University of Mysore, Mysuru.
15. Sharath Kumar KS, Hanumappa A, Vetrivel M, Hegde M, Girish
YR, Byregowda TR, Rao S, Raghavan SC, Rangappa KS (2015)
Antiproliferative and tumor inhibitory studies of 2,3 disubstituted
4-thiazolidinone derivatives. Bioorg Med Chem Lett
25:3616–3620. doi:10.1016/j.bmcl.2015.06.069
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