Synthesis and antiproliferative studies of curcumin pyrazole derivatives

Article abstract of DOI:10.1007/s00044-016-1628-5

A series of curcumin pyrazole derivatives (3a–e) were synthesized. The chemical structures were determined by ¹H and ¹³C NMR spectroscopic techniques and their purity was confirmed by LC–MS and melting point determination. The compou

Full text of DOI:10.1007/s00044-016-1628-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1628-5
ORIGINAL RESEARCH
Synthesis and antiproliferative studies of curcumin pyrazole derivatives
1 ●
2 ●
Honnalagere Ramesh Puneeth Hanumappa Ananda
2 ●
2 ●
Kothanahally S. Sharath Kumar Kanchugarakoppal S. Rangappa
Angatahally Chandrashekariah Sharada
1
Received: 25 January 2015 / Accepted: 27 June 2016
©
Springer Science+Business Media New York
●
●
●
Abstract A series of curcumin pyrazole derivatives (3a–e)
Keywords Leukemic cells Cytotoxicity Anticancer Cell
were synthesized. The chemical structures were determined
cycle
1
13
by H and C NMR spectroscopic techniques and their
purity was confirmed by LC–MS and melting point deter-
mination. The compounds were screened for anti-
cancer effects on different cancer cell lines by
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide) assay. The analogs demonstrated growth inhibi-
tory effect on MCF-7, HeLa, and K562 cell lines with
significant IC50 values. Compound 3b exhibited a high
degree of cytotoxicity against cancer cells and minimum
growth inhibitory effects against normal cells HEK293T
and hence further, cell cycle analysis and mitochondrial
membrane potential studies (JC-1 assay) were conducted by
utilizing flow cytometry against K562 cells. This compound
effectively arrested cell cycle progression at SubG1 phase
and cells exhibited decreased membrane potential in a
concentration-dependent manner with fluorescence shifting
from red to green. Our findings suggest that compound 3b
could be a promising anticancer agent since it effectively
inhibited cell proliferation and can be selected for further
in vitro and in vivo investigations.
Introduction
The fight against cancer is a global effort. Despite of sig-
nificant advances in diagnosis and treatment, cancer is still a
life threatening disease around the world. Recent investi-
gations have focused on naturally occurring chemopreven-
tive compounds possessing anticancer potential with
minimal toxicity. Most of the anticancer drugs approved
during 1981–2002 are natural products and their derivatives
(Newman et al., 2003).
Curcumin is a polyphenol compound obtained from the
rhizome (turmeric) of the plant Curcuma longa and is the
major active constituent in turmeric. Pharmacologically, cur-
cumin has been found to be safe even at very high doses in
human clinical trials (Aggarwal et al., 2003). It has been
widely used for various ailments due to its medicinal proper-
ties (Aggarwal et al., 2007). The anticancer potential of cur-
cumin has been demonstrated in various cell lines (Aggarwal
et al., 2006; Senft et al., 2010) and can induce apoptosis in a
variety of cell lines including GBC-SD, HT-29, JURKAT,
H520 (Liu et al., 2013; Wang et al., 2013; Gopal et al., 2014).
However, curcumin is not regarded as a potent ther-
apeutic molecule as potential utility of curcumin is restricted
because of its in vivo bioavailability. Curcumin decomposes
rapidly in neutral and basic conditions. In addition, curcu-
min extensively undergoes in vitro and in vivo Phase I and
Phase II metabolism (Wang et al., 1997; Pan et al., 1999;
Anand et al., 2007).
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-016-1628-5) contains supplementary material,
which is available to authorized users.
*
Angatahally Chandrashekariah Sharada
sharadaac@gmail.com
1
2
Department of Biochemistry, Yuvaraja’s College, University of
Mysore, Mysuru 570005 Karnataka, India
Multiple approaches are being hunted to overcome these
limitations. Synthetic chemical modifications of curcumin
Department of Studies in Chemistry, Manasagangotri, University
of Mysore, Mysuru 570006 Karnataka, India

Med Chem Res
structure have been studied and various curcumin analogs are
synthesized to improve the therapeutic profile of the natural
product. New analogs of curcumin exhibited antiangiogenic,
antitumor, and growth suppressive activities 30 times than
natural curcumin (Chandru and Sharada, 2007; Davis et al.,
compounds obey the Lipinski’s rules within four violation;
molecular weight below 500 Da, hydrogen bond donor (<5)
and acceptor (<10). QPlogPo/w (octanol/water partition
coefficient) for all the compounds is <5. Total solvent
accessible surface area (SASA), hydrophobic component of
the SASA (FOSA), and hydrophilic component of the SASA
(FISA) were analyzed for the compounds that were abiding
the ranges in Qikprop physiochemical properties (Data not
shown). Qualitative model for human oral absorption was
predicted, Curcumin showed high oral absorption com-
paratively against 3a, 3c, 3d, and 3e which showed low, and
3b showed medium oral absorption (Table 2). Polar nitrogen
and oxygen, and Van der Waals surface area (PSA) of
compounds were fulfilling the limit in physicochemical
calculation. All the ligands satisfy the values of partition
coefficient of octanol/gas (QPlogPoct), water/gas (QPlogPw)
and brain/blood (QPlogBB), skin permeability (QPlogKp),
aqueous solubility (QPlogS), and predicted for compounds
lie in the allowed solubility and permeability range (Data not
shown) (Fig. 2)
2008; Youssef et al., 2005; Ohori et al., 2006) and an analog
of curcumin showed potent growth inhibitory activities on
both prostate and breast cancer cell lines and was 50 times
more potent than curcumin (Fuchs et al., 2009). In this context,
we synthesized ethanone pyridine curcumin analogs and
cyclopropoxy curcumin analogs which exhibited in vivo
growth inhibitory and antiangiogenic effects against a mouse
tumor model (Chandru and Sharada, 2007; Chandru et al.,
2007, 2008) in our previous investigations.
In this line of continuation, in the present study, we have
synthesized and investigated the antiproliferative potential
of curcumin pyrazole derivatives against various cancer cell
lines.
Molecule docking has been successfully carried out to
design novel potential leads. A total of five validated
potential leads are suggested from the study among which
Results
Chemistry
3
b satisfy ADME drug criteria. ADME result suggest that
the oral administration potency might be medium in case of
b, but its availability to get into moderate when compared
Curcumin analogs (3a–3e) was synthesized by base cata-
lyzed cyclization of different phenyl hydrazines with cur-
cumin in the presence of ethanol under reflux condition
3
to range of 95 % standard drugs. Thus it validates that 3b
could be a potential cytotoxic agent.
(
Fig. 1). Both electron-donating substituent and electron
withdrawing substituent on phenyl hydrazines underwent
cyclization smoothly with curcumin to produce pyrazole
derivatives (Mayadevi et al., 2012; Kumar et al., 2012;
Pramod et al., 2012; Mishra et al., 2008). Proton and carbon
NMR spectral data are convinced with the proposed struc-
ture and physicochemical data of all the compounds are
listed in Table 1.
Biological activities
Cell growth suppression analysis by MTT assay
All compounds were evaluated for in vitro cytotoxicity
against three cancer cell lines, MCF-7, HeLa, and K562
cells by MTT assay. Paclitaxel drug was used as positive
control and curcumin was taken as a standard reference.
The results of cytotoxicity studies of curcumin analogs
(3a–3e) were recorded at different doses and at different
time intervals, and are presented as IC50 values, the mean
Bioavailability studies
ADME (absorption, distribution, metabolism, and excretion)
properties were calculated in Qikprop simulation. All the
Fig. 1 Synthesis of pyrazole
analogs of curcumin

Med Chem Res
Table 1 Curcumin pyrazole analogs, synthesis of curcumin pyrazole derivatives
a
b,c
Entry
1
2
3
Time (h)
13
Yield
(%)
91 (Mayadevi et al., 2012)
2
14
89 (Kumar et al., 2012)
3
15
75 (Pramod et al., 2012)
4
14
86
5
12
92 (Mishra et al., 2008)
a
3
Curcumin (1 equivalent), phenyl hydrazines (1.5 equivalent) and NaHCO (1.5 equivalent)
b
c
Isolated yield
Literature reported compound
growth inhibitory concentration and are summarized in
Table 3.
significant growth inhibitory effects against all the three
cancer cell lines. Compound 3b was tested for cytotoxicity
against HEK293T cells and was found to be nontoxic.
Among the cancer cells tested, K562 cells were more
sensitive to this compound and were selected for further
studies.
The data obtained by MTT assay showed that compound
3
a, 3b, and 3c have considerable inhibitory effects on the
growth of cancer cell lines after 48 and 72 h of treatment.
Compound 3b was found to be more effective and showed

Med Chem Res
The effect of curcumin analog 3b on cell cycle progression
After preliminary investigations of MTT assay, the effect of
compound 3b on cell cycle progression was examined by
fluorescence activated cell sorter. The concentrations were
selected based on growth inhibitory studies. K562 cells
were processed, stained with propidium iodide, and sub-
jected to flow cytometric analysis. The histogram of control
cells (DMSO-treated) showed a standard pattern of cell
cycle progression. Cell population in different cell cycle
phases of control was found to be 38.86, 30.64, 13.38, and
8.89 % in G1, S, SubG1, and G2 phases respectively. Cell
cycle progression was altered upon addition of the com-
pound 3b. The 10 and 20 µM concentrations of compound
3
b did not induce a significant effect on cell cycle pro-
gression. The cell distribution pattern in 40 µM, compound
b treated K562 cells was found to be 8.89, 13.15, 23.53,
3
and 39.64 % in G1, S, SubG1, and G2 phases, respectively.
In this concentration at 40 µM, compound 3b treated cells
were significantly arrested in the SubG1 phase showing
accumulation of cells.
JC-1 assay to detect changes in mitochondrial membrane
potential by flow cytometry
The cell death induced by compound 3b in K562 cells was
analyzed by JC-1 staining assay by flow cytometric analy-
sis. Changes in the mitochondrial membrane potential were
determined by red vs. green fluorescence where healthy
mitochondria gives out red fluorescence and apoptotic/dead
cells emits green fluorescence because of lack of mito-
chondrial membrane potential. Cells exposed to the 2,
4-DNP was used as positive control and DMSO-treated
cells served as a vehicle control. Minimum 10,000 events
were acquired and analyzed by using CellQuest Pro soft-
ware (BD, USA). Panels representing flow cytometric
density plots were used to determine the results, which are
depicted in Fig. 3. DMSO-treated cells showed 26.5 % of
green fluorescence whereas 2, 4-DNP-treated cells showed
6
5
8.92 % of green fluorescence. Compound 3b exhibited
3.60, 86.62, and 93.30 % of green fluorescence at 10, 20,
and 40 µM, respectively. Results revealed that compound
b induced a significant depolarization of mitochondrial
3
membrane potential at 20 and 40 µM that was indicated by
shifting of fluorescence from red to green. The effect was
increased linearly in a concentration-dependent manner in
leukemic (K562) cells.
Discussion
Cancer treatment is one of the many focused areas of interest
in the current research. Chemoprevention has evolved as a

Med Chem Res
A
Compound 3b
K562 CELL CYCLE ANALYSIS
Control
10 µM
20 µM
40 µM
B
Fig. 2 Effect of curcumin analog 3b (10, 20, and 40 µM, respectively)
on cell cycle progression in K562 cells. a Histogram obtained after
FACS analysis for K562 cells. b Bar graph showing the percentage of
cells in the SubG1, G1, S, and G2 phases of the cell cycle. The data
presented is derived from two independent experiments and error bars
are indicated
Table 3 Effect of of curcumin analogs (3a–3e) against three human
cancer cell lines in MTT assay expressed in IC50 values
et al., 2005; Thangapazham et al., 2006). Curcumin inhibits
various cell cycle phases in different cell lines (Liu et al.,
2007; Anand et al., 2008; Lee et al., 2009). Though curcumin
Compounds
Cell lines (IC50 values in µM)
fulfills the criteria of an ideal chemopreventive drug, various
efforts have been undergone to overcome the limitations of
curcumin and to improve its therapeutic efficacy. The devel-
opment of chemical modifications in the structure of curcumin
is one of the possible approaches to advance its anticancer
efficiency (Anand et al., 2008; Fuchs et al., 2009). On the
other hand, pyrazoles are the crucial bioactive moiety and
received considerable attention in the pharmacological
research with promising anticancer activities in various cancer
models (Balbi et al., 2011; Koca et al., 2013; Shamroukh
et al., 2014).
HeLa
MCF-7
K562
HEK293T
Curcumin
46.60
42.54
45.56
56.45
78.12
83.12
0.0061
37.84
52.88
34.99
43.60
>100
81.88
0.0053
28.86
36.99
25.55
42.35
85.21
75.12
0.0049
NT
3
3
3
3
3
a
b
c
NT
>1000
NT
d
e
NT
NT
Paclitaxel
NT
NT Not tested
In this perspective, a series of curcumin pyrazole deri-
vatives (3a–3e) were synthesized. These compounds were
assessed for bioavailability and screened for anticancer
activities. The efficiency of anticancer drugs can be assessed
by their ability to suppress the proliferation of tumor cells.
MTT assay is a reliable method to measure the proliferative
rate and cell death (Ferrari et al., 1990). Cytotoxicity of
curcumin pyrazoles (3a–3e) was investigated by using MTT
assay on HeLa, MCF-7, and K562 cell lines. All the com-
pounds in the series exhibited growth inhibitory effects on
all the three cancer cell lines. Compound 3b was found to
be more potent and was more effective than curcumin in
HeLa and K562 cell lines. Compound 3b was also found to
be nontoxic with negligible growth inhibitory effects
against normal cells HEK293T.
finer strategy to control malignancy with minimum secondary
effects than classical chemotherapy (Tsao et al., 2004;
Steward & Brown, 2013). But, chemotherapeutic and che-
mopreventive drugs usually lack multiple targeting ability on
cancer cells and are non-selective in their action and hence
toxic to normal healthy cells with undesirable side effects. An
ultimate drug should target cancer cells in multiple ways and
should possess minimal toxicity against normal cells. Many
natural products are screened for their antitumor potential
from the past few decades in this aspect (Devi et al., 1999;
Rocha et al., 2001; Yin et al., 2013). Curcumin, an active
component of turmeric inhibits malignant cells through mul-
tiple pathways and also ensures cytoprotection selectively
against normal cells due to its polyphenolic nature (Duvoix

Med Chem Res
A
R1
R1
R1
Negative Control
10 µM
20 µM
B 2
00
R1
R2
150
R1
100
R1
50
0
NC
10
20
40
2.4-DNP
Concentration (µM)
40 µM
2.4-DNP
Fig. 3 Effect of curcumin analog 3b (10, 20, and 40 µM, respectively)
on mitochondrial membrane potential by JC-1 staining assay. a Dot
plot representing JC-1 stained K562 cells at various concentrations of
compound 3b. b Bar graph showing percentage of red vs. green
fluorescing cells, which represents high and low mitochondrial mem-
brane potential. (R2 and R1 respectively)
K562 cells were more sensitive to compound 3b treat-
ment than other two cell lines screened and hence K562
cells were selected for further studies.
We utilized the flow cytometry to detect the cell cycle
progression. DMSO-treated K562 cells (control) underwent
a normal cell cycle with high amount of cell populations in
G1 phase whereas cells accumulated significantly at SubG1
phase (39.64 %) after treatment with compound 3b at 40
µM concentration for 48 h. Accumulation of cells in the
SubG1 phase is an early indicator of apoptosis (Fujii et al.,
potential collapses resulting in distribution of JC-1 reagent
throughout the cell in a monomeric state that fluoresces
green color. We employed JC-1 staining assay to investi-
gate the effect of compound 3b on the mitochondrial
membrane potential. The ratio of the red to green fluores-
cence in the compound treated with K562 cells sig-
nificantly declined at 48 h compared to the control in a
dose-dependent manner. Compound 3b is capable of dis-
rupting mitochondrial membrane potential significantly at
low concentrations (10, 20, and 40 µM) which was an
indication of apoptosis.
2
013). Our findings suggest that compound 3b induces cell
death, which resulted in increased cell population in the
SubG1 phase, which may lead to apoptosis, thereby
affecting its proliferation and was further confirmed by
mitochondrial membrane studies.
Anticancer compounds inducing apoptosis always show
a rapid collapse in the mitochondrial membrane potential.
Loss of mitochondrial membrane potential is one of the
early events in apoptotic cascade and it can be detected by
JC-1 staining, a sensitive assay (Ly et al., 2003). JC-1 is a
cationic dye that can selectively enter into mitochondria
where it aggregates and shows red fluorescence in normal
cells. In apoptotic cells, the mitochondrial membrane
From the studies we found that the compound 3b treat-
ment effectively inhibits the proliferation of K562 cells via
an induction of apoptosis. However, further experiments
need to be performed to investigate the specific markers of
apoptotic cells and further mechanism of action of com-
pound 3b in future in vitro studies.
Conclusion
A series of curcumin pyrazole derivatives were synthesized
and are evaluated for their cytotoxic potential. All the

Med Chem Res
curcumin pyrazole derivatives showed considerable growth
inhibitory activities in all the cell lines (HeLa, MCF-7, and
K562), whereas compound 3b exhibited significant activity.
The electron-donating capacity of chlorine atom could be
the key aspect for the better activity than other substituents.
Compound 3b was found to be selectively nontoxic against
normal cells and was more potent against cancer cells.
Experimental studies against K562 cells revealed that
compound 3b induced cell death by SubG1 phase arrest in
cell cycle and induced cell damage by causing depolariza-
tion of mitochondrial membrane potential. Thus from the
findings we can conclude that, the synthetic analog of
curcumin, compound 3b [4,4′-(1E,1′E)-2,2′-(1-(3-chlor-
ophenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl)bis(2-
methoxyphenol] could be a promising, inexpensive antic-
ancer drug effective at nontoxic doses. The structural
modification of compound 3b to improve its bioavailability
without affecting its activity is underway in our laboratory.
4,4′-(1E,1’E)-2,2′-(1-phenyl-1H-pyrazole-3,5-diyl)bis
(ethene-2,1-diyl)bis(2-methoxyphenol) (3a) Pale brown
solid (MeOH) compound (3a) was prepared from curcumin
(1) (1.35 mmol, 0.50 g) and phenylhydrazine hydrochloride
(2a) (2.03 mmol, 0.29 g) according to the general proce-
dure. The product obtained as a pale brown solid was
purified from methanol: 0.54 g (91 %); m.p. 89–91 °C
1
(Mayadevi et al., 2012; 88–89 °C); H NMR (400 MHz,
DMSO-d6): δ 3.75 (s, 3H, OCH ), 3.81 (s, 3H, OCH ),
3
3
6.72–6.76 (m, 3H), 6.90–7.04 (m, 5H), 7.11–7.19 (m, 3H),
7.41–7.45 (m, 1H), 7.49–7.56 (m, 4H, ArH), 9.12 (s, 1H,
1
3
OH), 9.23 (s, 1H, OH); C NMR (100 MHz, DMSO-d6):
δ 56.0 (OCH ), 56.1 (OCH ), 101.2 (ArC–H), 110.1 (2×
3
3
ArC–H), 111.0 (Pyrazole C=C), 112.6 (ArC–H), 116.0 ( 2×
ArC–H), 116.1 (C=C), 117.8 (ArC–H), 120.6 (ArC–H),
125.1 (C=C), 128.0 (ArC–H), 128.2 (ArC–H), 128.8
(ArC–H), 129.7 (2× Ar–C), 131.1 (C=C), 133.2 (C=C),
139.7 (ArC–N), 142.7 (Pyrazole C–N), 147.2 (ArC–OH),
1
1
47.8 (ArC–OH), 148.2 (ArC–OCH ), 148.3 (ArC–OCH ),
3 3
51.4 (Pyrazole C=N). LCMS (ESI) m/z [M + 1] 441.51;
+
Anal. calcd for C H N O : C, 73.62; H, 5.49; N, 6.36; O,
2
7 24 2 4
Experimental
Chemistry
14.53 Found: C, 73.65; H, 5.52; N, 6.34; O, 14.55.
4,4′-(1E,1’E)-2,2′-(1-(3-chlorophenyl)-1H-pyrazole-3,5-
diyl)bis(ethene-2,1-diyl)bis(2-methoxyphenol) (3b) Brown
solid this (MeOH) compound (3b) was prepared from cur-
cumin (1) (1.35 mmol, 0.50 g) and 3-chloro phenylhy-
drazine hydrochloride (2b) (2.03 mmol, 0.36 g) according to
the general procedure. The product obtained as a brown
All the chemicals were supplied from Merck, Aldrich, and
Fluka. The melting points were determined on capillary
tubes on a Büchi oil heated melting point apparatus and are
uncorrected. Reactions were monitored by TLC using pre-
solid was purified from methanol: 0.57 g (89 %); m.p.
coated sheets of silica gel G/UV-254 of 0.25 mm thickness
1
1
16–118 °C; H NMR (400 MHz, DMSO-d6): δ 3.79
1
(
Merck 60F254) using UV light for visualization. H and
C NMR spectra were recorded on a NMR spectrometer
(s, 3H, OCH ), 3.84 (s, 3H, OCH ), 6.78–6.85 (m, 3H),
3 3
1
3
6
.96–7.22 (m, 8H), 7.51–7.64 (m, 4H, ArH), 9.17 (s, 1H,
operating at 400 and 100 MHz, respectively, using the
residual solvent peaks as reference relative to SiMe . Mass
spectra were recorded using high resolution mass spectro-
meter. Infrared spectra were recorded on Shimadzu FT-IR
model 8300 spectrophotometer.
1
3
OH), 9.29 (s, 1H, OH). C NMR (100 MHz, DMSO-d6):
4
δ 56.06 (OCH ), 56.11 (OCH ), 101.8 (ArC–H), 110.1
3
3
(
(
ArC–H), 110.9 (Pyrazole C=C), 112.4 (ArC–H), 116.0
ArC–H), 116.1 (C=C), 117.5 (ArC–H), 120.7 (ArC–H),
120.9 (ArC–H), 123.5 (ArC–H), 124.7 (C=C), 127.8
(
(
ArC–H), 128.1 (Ar–C), 128.7 (Ar–C), 131.4 (C=C), 131.6
ArC–H), 133.7 (C=C), 134.0 (ArC–Cl), 140.9 (Pyrazole
General procedure for the synthesis of compounds (3a–3e)
C–N), 143.0 (ArC–N), 147.3 (ArC–OH), 147.9 (ArC–OH),
48.2 (ArC–OCH ), 148.3 (ArC–OCH ), 151.9 (Pyrazole
C=N). LCMS (ESI) m/z [M + 1] 475.97; Anal. calcd for
1
3
3
To a solution of curcumin (1 equivalent) in ethanol,
+
NaHCO (1.5 equivalent), and different phenylhydrazine
3
C H ClN O : C, 68.28; H, 4.88; Cl, 7.46; N, 5.90; O,
2
7
23
2 4
hydrochlorides (1.5 equivalent) were added. The resulting
mixture was refluxed for 12–15 h and the reaction was
monitored by TLC. After completion of the reaction, mix-
ture was evaporated under reduced pressure and the
resulting residue was dissolved with ethyl acetate and
washed with water followed by the brine solution. The
organic layer was dried over anhydrous sodium sulfate and
evaporated under vacuum to get the crude product, which
was purified by column chromatography using hexane:
ethyl acetate as an eluent.
1
3.48 Found: C, 68.32; H, 4.91; Cl, 7.49; N, 5.89; O, 13.51.
4,4′-(1E,1′E)-2,2′-(1-(2,4-dinitrophenyl)-1H-pyrazole-3,5-
diyl)bis(ethene-2,1-diyl)bis(2-methoxyphenol) (3c) Red
solid (MeOH) compound (3c) was prepared from curcumin
(1) (1.35 mmol, 0.50 g) and 2,4-dinitro phenylhydrazine
hydrochloride (2c) (2.03 mmol, 0.47 g) according to the
general procedure. The product obtained as a red solid was
purified from methanol. 0.54 g (75 %); m.p. 119–121 °C;
1
(Pramod et al., 2012, 118–119 °C); H NMR (400 MHz,

Med Chem Res
DMSO-d6): δ 3.78 (s, 3H, OCH ), 3.88 (s, 3H, OCH ),
(ArC–OCH ), 148.0 (ArC–OCH ), 148.9 (ArC–OCH ),
154.1 (Pyrazole C=N). LCMS (ESI) m/z [M + 1] 471.54
3
3
3
3
3
1
3
+
6
.65–6.82 (m, 4H), 6.91–7.48 (m, 10H).
C NMR
(
100 MHz, DMSO–d6): δ 56.2 (OCH ), 56.4 (OCH ),
Anal. calcd for C H N O : C, 71.47; H, 5.57; N, 5.95; O,
3
3
28 26 2 5
1
1
09.1 (ArC–H), 109.3 (ArC–H), 110.3 (Pyrazole C=C),
16.1 (C=C), 116.5 (ArC–H), 116.7 (ArC–H), 120.4
17.00 Found : C, 71.49; H, 5.60; N, 5.97; O, 17.02.
(
1
ArC–H), 122.7 (ArC–H), 122.9 (ArC–H), 123.3 (ArC–H),
24.1 (C=C), 127.9 (ArC–H), 130.1 (2× Ar–C), 130.9
Bioavailability studies
(
(
(
(
+
4
1
C=C), 131.5 (C=C), 136.6 (Pyrazole C–N), 139.8
Compound preparation and ADME Prediction
ArC–N), 142.6 (ArC–NO ), 146.5 (ArC–NO ), 147.5
2
2
ArC–OH), 147.8 (ArC–OH), 149.3 (ArC–OCH ), 149.6
QikProp, the prediction program used to calculate ADME
properties. Compounds were drawn using Maestro 2D
sketcher, each structure was launched in Ligprep for energy
minimization using the OPLS force field 2005 and geo-
metrically optimized. Lipinski’s values/ADME prediction
was executed by using QikProp v3.3. Qikprop modules
provide the ranges of molecular predicting properties for
comparing the properties of a particular molecule with those
of 95 % of known drugs (Lipinski et al., 2001).
3
ArC–OCH ), 152.5 (Pyrazole C=N). LCMS (ESI) m/z [M
3
+
1] 531.53; Anal. calcd for C H N O : C, 61.13; H,
2
7 22 4 8
.18; N, 10.56; O, 24.13 Found: C, 61.15; H, 4.21; N,
0.58; O, 24.12.
4
-(3,5-bis(4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl)
benzonitrile (3d) Yellowish brown solid (MeOH) com-
pound (3d) was prepared from curcumin (1) (1.35 mmol,
0
.50 g) and 4-cyano phenylhydrazine hydrochloride (2d)
(
2.03 mmol, 0.34 g) according to the general procedure. The
Biological studies
product obtained as a yellowish brown solid was purified
from methanol: 0.54 g (86 %); m.p. 119–121 °C; H NMR
1
Three different cancer cell lines, MCF-7, HeLa, and K562
cells, were selected for the preliminary anticancer screening
of curcumin pyrazole derivatives (3a–3e). MTT assay was
employed to assess the growth inhibitory potential of the
curcumin analogs (Sharath Kumar et al., 2015) with few
modifications. Further, analysis of progression of cell cycle
and mitochondrial membrane potential assay were per-
formed by flow cytometry to confirm the mechanism of
induction of cell death in compound 3b treated K562 cells
(
400 MHz, DMSO-d6): δ 3.80 (s, 3H, OCH ), 3.85 (s, 3H,
3
OCH ), 6.79–6.91 (m, 3H), 6.99–7.23 (m, 8H), 7.75–7.77
(
3
d, J = 8.4 Hz, 2H, ArH), 8.01–8.03 (d, J = 8.8 Hz, 2H,
1
3
ArH). C NMR (100 MHz, DMSO–d6): δ 56.0 (OCH3),
6.1 (OCH ), 102.7 (ArC–H), 109.8 (ArC–H), 110.1
5
3
(
(
ArC–H), 111.1 (ArC–H), 112.4 (Pyrazole C=C), 116.0
2× ArC–H), 116.1 (C=C), 117.3 (ArC–H), 118.9 (C≡N),
1
(
(
(
20.8 (ArC–H), 121.1 (ArC–H), 125.0 (C=C), 128.1
Ar–C), 128.6 (Ar–C), 132.1 (C=C), 134.1 (C=C), 134.2
2× ArC–H), 143.2 (Pyrazole C–N), 143.4 (ArC–N), 147.5
2× ArC–OH), 148.0 (ArC–OCH ), 148.3 (ArC–OCH ),
(Vinaya et al., 2011; Sharath Kumar et al., 2014).
3
3
Cell lines and culture
+
1
52.6 (Pyrazole C=N). LCMS (ESI) m/z [M + 1] 466.52;
Anal. calcd for C H N O : C, 72.24; H, 4.98; N, 9.03; O,
2
8 23 3 4
HeLa (Human cervical cancer cell line), MCF-7 (Human
breast cancer cell line), K562 (human myelogenous leuke-
mia cell line), and HEK293T (Human embryonic kidney
cell line) cells were purchased from National centre for cell
science, Pune, India. Cells were grown in RPM1
1640 supplemented with 10 % heat inactivated fetal bovine
serum, 100 U/ml of penicillin and 100 µg of streptomycin/
ml and incubated at 37 °C in a humidified atmosphere with
5 % CO2.
1
3.75 Found: C, 72.27; H, 4.99; N, 9.06; O, 13.78.
4
,4′-(1E,1′E)-2,2′-(1-(4-methoxyphenyl)-1H-pyrazole-3,5-
diyl)bis(ethene-2,1-diyl)bis(2-methoxyphenol) (3e) Dark
brown solid (MeOH) compound (3e) was prepared from
curcumin (1) (1.35 mmol, 0.50 g) and 4-methoxy phe-
nylhydrazine hydrochloride (2d) (2.03 mmol, 0.35 g)
according to the general procedure. The product obtained as
a dark brown solid and was purified from methanol: 0.58 g
1
(
92 %); m.p. 107–109 °C; (Mishra et al., 2008; 106 °C); H
NMR (400 MHz, DMSO-d6): δ 3.90 (s, 6H, OCH ), 3.91
MTT assay
3
(
(
s, 3H, OCH ), 6.80 (s, 1H), 6.98–7.02 (m, 4H), 7.08–7.19
3
m, 6H), 7.29–7.41 (m, 4H). C NMR (100 MHz, DMSO-
1
3
The cytotoxic effects of the synthesized compounds against
the cervical carcinoma, breast carcinoma, and leukemic
cells (5 × 10 cells) were assessed using 3-(4,5-dimethyl-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
Embryonic kidney cell lines were utilized to assess the
growth suppressive effects of the potent cytotoxic com-
pound in the series by MTT assay. The test compounds
d6): δ 55.4 (OCH ), 56.4 (OCH ), 56.8 (OCH ), 107.8
3
3
3
5
(2× ArC–H), 110.3 (ArC–H), 110.8 (Pyrazole C=C), 112.9
(2× ArC–H), 115.6 (2× ArC–H), 117.9 (C=C), 118.1
(ArC–H), 119.5 (ArC–H), 122.2 (ArC–H), 124.6 (C=C),
1
29.6 (2× Ar–C), 129.9 (C=C), 130.7 (C=C), 132.3 (Pyr-
azole C–N), 137.8 (ArC–N), 146.1 (2× ArC–OH), 146.8

Med Chem Res
were dissolved in DMSO and treated with different con-
centrations of synthesized compounds 3a–3e (10, 20, and
samples and are statistically analyzed. One-way ANOVA
followed by Dunnett test was used; in each case experi-
mental samples were compared with control and sig-
nificance was determined. p-value ≤ 0.05 was considered as
statistically significant.
4
0 µM, respectively). Cells in the control wells received the
same volume of medium containing DMSO. After 48 and
2 h treatment cells were harvested and incubated
7
with MTT (0.5 µg/ml) for 4 h at 37 °C in 96-well plate. The
blue MTT formazan precipitate formed in the viable cells is
solubilized by the addition of 70 µl DMSO. The suspension
is placed in microvibrator for 5 min and absorbance was
measured at 540 nm using multimode reader (Varioskan
Flash Multimode, Thermo scientific, USA). The experiment
was performed in triplicate and repeated at least for
three times.
Acknowledgments The authors express sincere gratitude to
Dr. S Nagendra, Managing Director and Chief executive officer,
Scintilla BIO-MARC pvt. Ltd, Bangalore, India for providing tech-
nical assistance regarding cell culture facilities for the work.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Cell cycle analysis using fluorescent activated cell sorter
analysis
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