Synthesis, antiproliferative activity, and molecular docking studies of curcumin analogues bearing pyrazole ring

Article abstract of DOI:10.1007/s00044-015-1457-y

Several curcumin analogues bearing pyrazole were synthesized and characterized by IR, NMR, and mass spectral data. There were four tested compounds among 11 synthesized compounds, which were evaluated for antiproliferative activity and showed significant activity in both one-dose and five-dose assays. The antiproliferative effects were tested on a panel of 60 cell lines, according to the National Cancer Institute screening protocol. The most active compounds among the series were 3,5-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-carboxamide (3k) which showed mean percent growth inhibition of 116.09 in one-dose assay at 10 μM, and GI₅₀ values were ranging between 0.0912 and 2.36 μM in five-dose assay. The best results were recorded on the leukaemia cell lines with value ranging from 0.0912 to 0.365 μM. All the tested compounds showed broad-spectrum antiproliferative activity over different cancer cell lines. When compared with the standard drug paclitaxel, the compound 3k showed superior activity on nearly 42 cell lines. The molecular docking study was performed to explore the binding interaction of these curcumin analogues with the active site of EGFR tyrosine kinase (EGFR-TK). The hydroxyl group of both phenyl rings was important for the rein-geminated hydrogen bonding by either side chain or backbone with the active site of EGFR-TK. Graphical Abstract: Four curcumin analogues were evaluated for their antiproliferative activity and showed promising results. The molecular docking studies showed that all the compounds (3a-k) were well accommodated in the EGFR tyrosine kinase.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-015-1457-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1457-y
ORIGINAL RESEARCH
Synthesis, antiproliferative activity, and molecular docking
studies of curcumin analogues bearing pyrazole ring
1
1
2
•
Mohamed Jawed Ahsan
2
•
Kavita Choudhary
3
•
Surender Singh Jadav
4
Sabina Yasmin
•
Md. Yousuf Ansari
•
Reddymasu Sreenivasulu
Received: 26 January 2015 / Accepted: 27 August 2015
Ó Springer Science+Business Media New York 2015
Abstract Several curcumin analogues bearing pyrazole
were synthesized and characterized by IR, NMR, and
mass spectral data. There were four tested compounds
among 11 synthesized compounds, which were evaluated
for antiproliferative activity and showed significant
activity in both one-dose and five-dose assays. The
antiproliferative effects were tested on a panel of 60 cell
lines, according to the National Cancer Institute screening
protocol. The most active compounds among the series
spectrum antiproliferative activity over different cancer
cell lines. When compared with the standard drug pacli-
taxel, the compound 3k showed superior activity on
nearly 42 cell lines. The molecular docking study was
performed to explore the binding interaction of these
curcumin analogues with the active site of EGFR tyrosine
kinase (EGFR-TK). The hydroxyl group of both phenyl
rings was important for the rein-geminated hydrogen
bonding by either side chain or backbone with the active
site of EGFR-TK.
were
3,5-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-
carboxamide (3k) which showed mean percent growth
inhibition of 116.09 in one-dose assay at 10 lM, and GI50
values were ranging between 0.0912 and 2.36 lM in five-
dose assay. The best results were recorded on the leu-
kaemia cell lines with value ranging from 0.0912 to
Graphical Abstract Four curcumin analogues were eval-
uated for their antiproliferative activity and showed
promising results. The molecular docking studies showed
that all the compounds (3a–k) were well accommodated in
the EGFR tyrosine kinase.
0
.365 lM. All the tested compounds showed broad-
&
&
Mohamed Jawed Ahsan
jawedpharma@gmail.com
Kavita Choudhary
kkchoudhary21@gmail.com
1
Department of Pharmaceutical Chemistry, Maharishi Arvind
College of Pharmacy, Ambabari, Jaipur, Rajasthan 302 039,
India
2
3
Department of Pharmaceutical Chemistry, Birla Institute of
Technology, Mesra, Ranchi, Jharkhand 835 215, India
Keywords Antiproliferative activity Á
Pharmacoinformatics Department, National Institute of
Pharmaceutical Education and Research (NIPER), Hajipur
Curcumin analogues Á EGFR tyrosine kinase Á
Molecular docking Á One-dose assay Á Five-dose assay
8
44 102, India
4
Department of Chemistry, Acharya Nagarjuna University,
Nagarjuna Nagar 522 510, India
123

Med Chem Res
Introduction
(Normanda et al., 2002). The involvement of the EGFR
family of tyrosine kinases in cancer proliferation suggests
that an inhibitor which blocks the tyrosine kinase activity
of the entire EGFR family could have significant thera-
peutic potential (Mendelsohn and Baselga, 2000). Gefi-
tinib, erlotinib, lapatinib, canertinib, PD 153035, AG 1295,
AG 1385, etc., are examples of EGFR tyrosine kinase
inhibitors (TKIs), a biological target for human cancer.
Although several EGFR-TKIs have been clinically vali-
dated for the treatment of cancer patients, the search for
new active molecules against EGFR-TK is still continuing.
Encouraged by our previous research and validated target
as EGFR-TK, we report herein the synthesis, antiprolifer-
ative, and molecular docking study of some curcumin
analogues containing pyrazole nucleus. In another study
also, it was reported that curcumin analogues have anti-
EGFR activity (Yadav et al., 2014).
An estimated 14.6 million cancer cases and 8.2 million
death tolls occurred globally in 2012 with nearly 8 %
increased deaths from 7.6 million, as reported in a previous
survey in 2008 (WHO World Cancer Report, 2014). The
chemotherapy of cancer is usually associated with various
side effects, and in the recent era, new drug pipelines are
drying up (Aydemir and Bilaloglu, 2003). Also the demand
for relatively more effective and safer agents for cancer
treatment has been a great surge. Researchers and phar-
maceutical companies around the world are ardently affi-
anced in the research and development to find a more
pleasant solution for the treatment of cancer.
It has been observed that natural products from plants
and their synthetic and semi-synthetic analogues are
expected to play a vital role in creating new and better
chemotherapeutic agents (Cragg and Newman, 2005;
Balaji et al., 2015; Carmona et al., 2003; Koepfli et al.,
1
947). Curcumin modulates numerous targets which
Results and discussion
Chemistry
include growth factors, growth factor receptor transcription
factors, cytokines, enzymes, and gene-regulating apoptosis
(
Pari et al., 2008). Medicinal chemists have exploited four
different sites for chemical modification of curcumin that
include aryl side chain, diketo group, double bonds, and
active methylene group and synthesized a number of cur-
cumin analogues with improved biological activity (Vyas
et al., 2013; Balaji et al., 2015). Earlier we have reported
curcumin analogues bearing pyrazole/pyrimidine rings, and
the results were significantly high with maximum mean
growth percent (GP) of -28.71 in one-dose assay and GI50
values ranging between 0.0079 and 1.86 lM in five-dose
assay and also showed inhibition of EGFR with IC50 of
The curcumin analogues (3a–k) described in the study
are given in Table 1, and the reaction sequence for the
Table 1 Physical constants of the curcumin analogues (3a–k)
O
O
Ar
NH2
N
N
N
N
O
O
O
O
HO
3a-j
OH
HO
3k
OH
Compound Ar
NSC
code
–
Yield Mp (°C)
(%)
77
Reported Found
OH
3
a
–
170–172
O
O
2
9.2 lM (Ahsan et al., 2013). Since curcumin itself is more
toxic to cancerous cells as compared to the normal human
cell, it can be concluded that their analogues would have
more specificity to the cancer cells (Kunwar et al., 2008).
In another study, curcumin showed autophagic and apop-
totic death of K562 cell line (leukaemia) (Jia et al., 2009).
Various other biological activities, viz. antiproliferative
–
71
215–217a 212–214c
3
3
b
c
H
2
N
777,944 78
–
–
160–162c
Cl
Cl
168–170c
3
d
–
74
–
–
72
62
105–106b 104–106c
207–209a 206–208
3e
N
(
Sharma et al., 2015; Ahsan et al., 2013; Liang et al.,
009), antibacterial (Lal et al., 2012; Sahu et al., 2012;
OH
3
f
2
–
65
–
120–122c
Zhichang et al., 2012), anti-HIV (Singh et al., 2010), anti-
inflammatory (Saja et al., 2007), antimalarial (Mishra
et al., 2008), and many more, have been reported for cur-
cumin analogues. These recent developments made cur-
cumin as an ideal lead compound for future drug discovery.
Epidermal growth factor receptor tyrosine kinase
3g
S
138–140c
130–132c
128–130c
3
3
h
i
777,945 82
76
–
–
–
O
3
H C
3j
777,,946 74
777947 84
–
O
204-206b 200–202c
3
k
–
(
EGFR-TK) is one among the attractive targets for human
a
b
c
Zhichang et al. (2012)
Sahu et al. (2012)
Balaji et al. (2015)
cancer. EGFR belongs to ErbB family of tyrosine kinase
proteins which includes four members: ErbB-1/HER1,
ErbB-2/Neu/HER2, ErbB-3/HER3, and ErbB-4/HER4
1
23

Med Chem Res
Conc. H SO4
2
ArCOOH
C H COOH
ArCOOC H5
2
+
2
5
NH NH .H O EtOH
2
2
2
O
Ar
N
N
ArCONHNH
2
O
O
2
a-j
HO
3a-j
OH
AcOH
O
O
O
O
HO
OH
1
O
NH2
N
N
AcOH
O
O
HO
OH
2
NH C(=O)NHNH2
3k
2
k
Scheme 1 Protocol for the synthesis of curcumin analogues (3a–k) bearing pyrazole ring
synthesis is summarized in Scheme 1. A mixture of 1,7-
bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione
Antiproliferative activity
(
curcumin) and aryl hydrazides/semicarbazide was
Four compounds were evaluated for their antiproliferative
activity in both one-dose and five-dose assays. The
antiproliferative screening data of the compounds are given
in Table 2. The most active compound of the series was 3k,
active on all the 60 cell lines with mean growth percent
inhibition (%GI) of 116.09, and was found to be highly
active on OVCAR-3 (ovarian cancer; %GI = 172.72), SK-
MEL-2, MDA-MB-435 (melanoma cancer; %GP =
152.65 and 145.96, respectively), and RMPI-8226 (leu-
kaemia; %GI = 143.96) cell lines. The compound 3j was
found to be the most active on COLO-205 (colon cancer;
%GI = 166.17), RXF-393 (renal cancer; %GI = 154.83),
RMPI-8226 (leukaemia; %GI = 127.35), and MDA-MB-
435 (melanoma cancer; %GI = 122.53) with mean %GI of
103.31. The compound 3h showed mean percent GI of
100.92 and was found to be highly active on COLO-205
(colon cancer; %GP = 164.28), RXF-393 (renal cancer;
%GI = 142.56), and RMPI-8226 (leukaemia; %GI =
134.22), while the compound 3c showed mean %GI of
82.39 and was found to be highly active on MDA-MB-435,
SK-MEL-5 (melanoma cancer; %G1 = 145.22 and 132.37,
respectively), RMPI-8226, and HL-60(TB) (leukaemia;
%GI = 116.05 and 112.43, respectively). The curcumin
analogues (3c, 3h, 3j, and 3k) showed broad-spectrum
antiproliferative activity against different cancer cell lines.
When compared with the standard drug paclitaxel, com-
pound 3c showed superior activity on 26 cell lines, com-
pound 3h showed superior activity on 37 cell lines,
compound 3j showed superior activity on 40 cell lines,
refluxed in glacial acetic acid (AcOH) to obtain the
curcumin analogues bearing pyrazole ring (3a–k). The
yields of the title compounds were ranging from 62 to
8
4 % after recrystallization with absolute ethanol. The
completion of reaction was monitored by TLC using
mobile phase, hexane/ethylacetate (6:4), and purity of
the compounds was checked by elemental analyses.
Both the analytical and spectral data (IR, NMR, and
MS) of the synthesized compounds were in full har-
mony with the proposed structures. The aryl hydrazides
(
(
2a–j) were synthesized as per the reported method
Yar et al., 2007). Aromatic acid (0.005 mol) and
absolute ethanol (20 ml) were refluxed in the presence
of 1 ml conc. H SO for 8–12 h, the reaction mixture
2
4
was cooled and neutralized, and aryl esters (liquid)
were separated by separating funnel. In the final step,
the aryl ester was refluxed with hydrazine hydrate in
ethanol for 12 h. The excess of solvent was removed
under reduced pressure, and then the reaction mixture
was poured into the crushed ice to obtain the aryl
hydrazides (2a–j). The compounds 3b–k were reported
earlier (Zhichang et al., 2012; Sahu et al., 2012; Balaji
et al., 2015). The compounds 3b and 3f reported earlier
1
13
were fully characterized by IR, H NMR, C NMR,
and mass spectral data, while rest of the reported
compounds were not fully characterized. Earlier we
have reported some curcumin analogues with anti-
malarial activity (Balaji et al., 2015).
123

Med Chem Res
Table 2 Sixty human tumour cell line anticancer screening data of curcumin analogues
Panel/cell line
3c
3h
3j
3k
GP
% GI
GP
% GI
GP
% GI
GP
% GI
Leukaemia
CCRF-CEM
HL-60(TB)
K562
0.63
99.37
112.43
87.76
0.17
99.83
109.93
98.31
-11.58
-15.62
-6.97
111.58
115.62
106.97
118.44
127.35
109.23
-22.86
-25.07
-23.04
-33.01
-43.96
-3.77
122.86
125.07
123.04
133.01
143.96
103.77
-12.43
2.24
-9.93
1.69
MOLT-4
5.80
94.20
1.08
98.92
-18.44
-27.35
-9.23
RPMI-8226
SR
-16.05
3.07
116.05
96.93
-34.22
4.44
134.22
95.56
Non-small cell lung cancer
A549/ATCC
HOP-62
12.41
20.28
-2.95
48.40
21.79
28.50
4.86
87.59
79.72
102.95
51.60
78.21
71.50
95.14
4.22
2.82
-0.11
13.65
1.94
9.62
4.69
–
95.78
97.18
100.11
86.35
98.06
90.38
95.31
3.98
5.20
96.02
94.80
110.72
86.99
92.04
89.30
95.63
106.80
3.16
-3.02
-42.76
11.92
96.84
103.02
142.76
88.08
HOP-92
-10.72
13.01
7.96
NCI-H226
NCI-H23
-4.00
-1.78
0.57
104.00
101.78
99.43
NCI-H322 M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
11.70
4.37
–
-6.80
-21.16
121.16
98.67
52.05
0.95
1.37
47.95
99.05
85.90
89.22
83.58
-64.28
17.20
1.23
164.28
82.80
98.77
93.89
90.61
95.37
-66.17
16.44
-0.56
5.59
166.17
83.56
100.56
94.41
93.67
95.86
-37.65
7.85
137.65
92.15
139.15
95.67
96.53
99.00
-39.15
4.33
HCT-15
14.10
10.78
16.42
6.11
KM12
9.39
6.33
3.47
SW-620
4.63
4.16
1.00
CNS cancer
SF-268
26.97
47.96
10.05
45.60
-2.93
16.81
73.03
52.04
89.95
54.40
102.93
83.19
9.64
8.28
90.36
91.72
6.45
6.61
93.55
93.39
-10.72
4.87
110.72
95.13
SF-295
SF-539
-23.14
7.03
123.14
92.97
-24.41
12.80
-16.03
0.80
124.41
87.20
-26.27
4.94
126.27
95.06
SNB-19
SNB-75
0.66
99.34
116.03
99.20
-40.88
-10.93
140.88
110.93
U251
-2.58
102.58
Melanoma
LOX IMVI
MALME-3M
M14
13.87
44.62
-1.11
-45.22
–
86.13
55.38
101.11
145.22
–
5.18
-0.49
0.24
94.82
100.49
99.76
110.77
–
4.50
3.93
94.50
96.07
1.48
-4.92
98.52
104.92
166.21
145.96
152.65
124.63
133.04
99.17
-15.10
-22.53
-34.06
-8.32
-13.30
0.12
115.10
122.53
134.06
108.32
113.30
99.88
-66.21
-45.96
-52.65
-24.63
-33.04
0.83
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
-10.77
–
19.47
-32.37
63.24
22.50
80.53
132.37
36.76
77.50
-4.66
-31.84
-5.67
1.59
104.66
131.84
105.67
98.41
-0.83
100.83
-47.19
147.19
33.96
7.65
66.04
92.35
63.51
10.09
70.91
78.08
11.42
1.55
88.58
98.45
12.78
1.05
87.22
98.95
1.18
-72.72
-6.24
-30.67
-6.69
7.68
98.82
172.72
106.24
130.67
106.69
92.32
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
36.49
79.91
29.09
21.92
7.27
92.73
-7.85
-18.77
-5.24
25.97
107.85
118.77
105.24
74.03
-18.69
-17.05
21.71
118.69
117.05
78.29
1
23

Med Chem Res
Table 2 continued
Panel/cell line
3c
3h
3j
3k
GP
% GI
GP
% GI
GP
% GI
GP
% GI
Renal cancer
7
86-0
19.50
80.50
92.51
88.46
82.09
84.66
86.40
NT
10.25
89.75
109.09
94.60
89.10
142.56
90.93
NT
2.92
97.08
114.82
92.36
-45.85
-24.40
-11.20
14.12
145.85
124.40
111.20
85.88
NT
A498
7.49
11.54
17.91
15.34
13.60
-9.09
5.40
-14.82
7.64
ACHN
CAKI-1
20.90
-42.56
9.07
20.44
-54.83
12.76
24.71
-1.75
79.56
RXF-393
SN 12C
154.83
87.24
NT
-5.22
4.79
105.22
95.21
122.11
TK-10
NT
NT
75.29
UO-31
0.56
99.44
9.97
90.03
101.75
-22.11
Prostate cancer
PC-3
4.59
95.41
80.11
3.01
96.99
82.75
-9.28
109.72
85.42
-23.46
123.46
97.89
DU-145
19.89
17.25
16.58
2.11
Breast cancer
MCF7
20.14
31.84
10.11
11.19
1.45
79.86
68.16
89.89
88.81
98.55
82.39
–
-0.78
15.19
9.84
100.78
84.81
90.16
107.75
104.29
99.08
–
-3.07
12.54
0.54
103.07
87.46
99.46
104.93
108.43
103.31
–
-16.39
3.70
116.39
96.30
111.69
107.56
114.35
116.09
–
MDA-MB-231/ATCC
HS 578T
T-47D
-11.69
-7.56
-14.35
-16.09
86.84
-7.75
-4.29
0.92
-4.93
-8.43
-3.31
92.14
MDA-MB-468
Mean
17.61
143.89
Range
85.99
NT not tested, GP growth percent, % GI percent growth inhibition
while the most active compound (3k) of the series regis-
tered superior activity on 47 cell lines. The comparison of
percent growth inhibition (%GI) expressed by compound
ranging between 0.201 and 40.5 lM. The best results were
recorded on the leukaemia cell lines with the value ranging
from 0.155 to 0.404 lM (Fig. 3). A number of three tested
cancer cell lines presented TGI value with [100 lM, the
best result being noted on the MDA-MB-435 (melanoma)
with GI50 value of 0.201 lM. Only in 38 cell lines, com-
pound 3j registered LC50 value with [100 lM. The com-
pound 3h showed GI50 ranging between 0.218 and
2.90 lM. The best results were recorded on the leukaemia
cell lines with value ranging from 0.233 to 0.464 lM
(Fig. 4). A number of five tested cancer cell lines presented
TGI value with [100 lM, the best result being noted on
the MDA-MB-435 (melanoma) with GI50 value of
0.233 lM. Only in 37 cell lines, compound 3h registered
LC50 value with [100 lM, while the compound 3c showed
GI50 ranging between 0.228 and 4.20 lM. The best results
were recorded on the leukaemia cell lines with value
ranging from 0.228 to 1.83 lM (Fig. 5). A number of two
tested cancer cell lines presented TGI value
with [100 lM, the best result being noted on the SR
(leukaemia) with GI50 value of 0.228 lM. Only in 30 cell
lines, compound 3c registered LC50 value with [100 lM.
The five-dose assay screening data of four compounds are
3
c, 3h, 3j, 3k, and paclitaxel against the 58 NCI human
cancer cell lines at 10 lM is shown in Fig. 1. The
antiproliferative activity data of paclitaxel were obtained
from NCI data warehouse index (http://dtp.nci.nih.gov/
dtpstandard/dwindex/index.jsp). The antiproliferative
activity of curcumin analogues reported here was found to
be comparatively less than that of the earlier reported
curcumin analogues in single-dose assay (Ahsan et al.,
2
013).
Since all these curcumin analogues (3c, 3h, 3j, and 3k)
were found to be active on all the 60 cancer cell lines in
one-dose assay and hence selected for five-dose assay. The
compound 3k showed GI50 ranging between 0.0912 and
2
.36 lM. The best results were recorded on the leukaemia
cell lines and the value ranging from 0.0912 to 0.365 lM
Fig. 2). A number of four tested cancer cell lines pre-
(
sented TGI value with [100 lM, the best results being
noted on the MDA-MB-435 (melanoma) with 0.346 lM
(
GI ). Only in 31 cell lines, compound 3k registered LC
50 50
value with [100 lM. The compound 3j showed GI50
123

Med Chem Res
%
GI of 3c
% GI of 3h
% GI of 3j
% GI of 3k
% GI of Paclitaxel
1
1
1
1
1
80
60
40
20
00
8
6
4
2
0
0
0
0
0
Fig. 1 Comparison of percent growth inhibition (% GI) expressed by compound 3c, 3h, 3j, 3k, and paclitaxel against the NCI human cancer cell
lines at 10 lM
Fig. 2 Five-dose anticancer screening of compound 3k on leukaemia
cell lines
Fig. 3 Five-dose anticancer screening of compound 3j on leukaemia
cell lines
given in Table 3. The curcumin analogues reported earlier
Molecular docking
had GI50 value between 0.004 and 3.39 lM (Ahsan et al.,
2
013), while the GI50 of the curcumin analogues reported
The EGFR tyrosine kinase (PDB: 2J5F) was downloaded
from PDB (http://www.rcsb.org/pdb/explore.do?structu
reId=2j5f), it contains the bound ligand, and it has the
covalent bond with the residue Cys797. As we reported
earlier, the active site of EGFR tyrosine kinase is defined
here lies between 0.0912 and 40.5 lM. The antiprolifera-
tive activity of the curcumin analogues in five-dose assay
reported earlier was found to be more potent than that of
the curcumin analogues reported here.
1
23

Med Chem Res
well as backbone H-bonding with the hydroxyl and meth-
oxy functional groups present on phenyl ring of curcumin
analogues (3a–k). In addition, residues Phe723, Lys745,
and Asp855 (motif 1) were responsible for hydrophobic as
well as p–p interactions with the phenyl portion of pyra-
zole nucleus and p-cationic interaction along with side
chain hydrogen bonds with rest of the residues, whereas the
last binding site containing residue Lys875 (motif 2) was
responsible for interactions with hydroxyl and methoxy
functional groups of another phenyl ring, homogenous to
that of first one, i.e. hydrophobic binding site. Apart from
all these, residues Arg841 and Asn842 contributed to
charged interactions with hydroxyl group phenyl ring
present on pyrazoline of compound 3f. The ligand 3c
showed hydrophobic interactions with residues Lys745,
Leu792, and Met793 and p–p cationic interactions (motif
Fig. 4 Five-dose anticancer screening of compound 3h on leukaemia
cell lines
1) with phenyl portion of pyrazole nucleus with residues
Phe723, while residue Lys875 was responsible for inter-
actions with the hydroxy and methoxy functional groups of
another phenyl ring similar to that of the hydrophobic
cavity (Fig. 7). The ligand 3h showed hydrophobic inter-
actions with residues Leu792 and Met793 and p–p cationic
interactions (motif 1) with phenyl portion of pyrazole
nucleus with residues Phe723, while residue Lys875 was
responsible interactions with hydroxyl and methoxy func-
tional groups of another phenyl ring similar to that of the
hydrophobic cavity (Fig. 8). The interaction of ligands 3j
(Fig. 9) and ligand 3k (Fig. 10) with the EGFR tyrosine
kinase showed interaction with residues Leu792, Met793,
Lys845, and Phe723. The compounds 3j and 3k had
interaction with more residues in the hydrophobic cavity of
EGFR tyrosine kinase active site and found to have max-
imum antiproliferative activity than compounds 3c and 3h.
The binding mode of the compounds reported earlier
showed hydrophobic interaction with more residues
Fig. 5 Five-dose anticancer screening of compound 3c on leukaemia
cell lines
(
leu718, Pro794, Met793, Ala743, Leu844, Val726, and
Cys797) than that of the present compounds (3c, 3h, 3j and
k) against the EGFR tyrosine kinase active site. The
3
by the presence of residues Phe723, Thr790, Lys745,
Gln791, Gly796, Leu792, Met793, Asp800, and Asp855,
which makes it hydrophobic binding site along with the
bound ligand 34-JAB (N-[4-(3-bromophenylamino)-
quinazoline-6-yl]-acrylamide) (Blair et al., 2007). It has
been perceived that compounds 3a–k were accountable for
reiterated ligand receptor interactions by getting accom-
modated in the active site (Fig. 6). As we previously
reported the important residues of EGFR tyrosine kinase
antiproliferative activity of some of the reported com-
pounds was found to be more significant than that of the
present compounds. The strong hydrophobic interaction
could be the reason of more antiproliferative activity of the
reported compounds (Ahsan et al., 2013).
Finally it has been concluded that all the compounds
(
3a–k) were well accommodated in binding site of 34-JAB
of the EGFR tyrosine kinase, and the hydroxy and methoxy
function groups of both phenyl rings were important for the
rein-geminated hydrogen bonding by either side chain or
backbone as discussed above. The binding energy through
extra-precision (XP) mode was calculated and is listed in
Table 4.
(
(
Ahsan et al., 2013) with various hypothetic binding motifs
hydrophobic cavity, motif 1, and motif 2), it also contains
the residues Met793 and Gln791 (hydrophobic cavity),
important for the continuous and almost all side chain as
123

Med Chem Res
Table 3 NCI in vitro testing results of compounds of the compounds 3c, 3h, 3j, and 3k at five-dose level in lM
S. no. Panel/cell line 3c (NSC 777,944) 3h (NSC 757,926) 3j (NSC 757,929)
3k (NSC 763,375)
TGI LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
1
.
Leukaemia
CCRF-CEM
HL-60(TB)
K262
1.64
1.58
[100 [100 0.333 [100 [100 0.304 [100 [100 0.295
7.62 [100 0.385 NT [100 0.335 NT [100 0.352
[100 0.434 [100 [100 0.404 [100 [100 0.350
[100 [100
NT [100
0.807 16.4
[100 [100
[100 [100
MOLT-4
1.64
1.83
11.9
6.33
[100 0.464 [100 [100 0.372 NT
[100 0.365
[100 0.293
[100 0.0912
RPMI-8226
SR
[100 0.411 2.24
[100 0.233 NT
[100 0.351 NT
[100 0.155 NT
NT
NT
[100
[100
0.228 3.60
2
.
Non-small cell lung cancer
A549/ATCC
EKVX
3.30
12.8
NT
58.4
NT
2.80
NT
1.66
NT
[100 1.37
14.0
NT
[100 0.491
11.3
NT
[100
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
HOP-62
2.08
3.97
3.51
2.35
3.03
2.03
6.92
22.2
23.2
7.95
14.1
20.4
30.3
NT
NT
NT
NT
NT
HOP-92
[100 NT
[100 2.66
[100 1.56
NT
0.609 4.88
[100 0.861
[100 1.38
[100 0.647
[100 1.07
[100 0.318
4.40
4.40
3.53
3.15
1.30
45.3
[100
[100
NT
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
9.88
6.59
10.1
[100 2.06
70.9 0.84
[100 1.29
38.9 0.37
8.52
4.69
10.5
13.3
38.9
1.68
[100 0.526 10.4
[100 0.323 1.49
44.2
0.595 4.32
[100 0.267 0.785 9.21
0.252
0.964 5.30
3
.
3.78
3.94
2.15
1.54
3.42
1.24
3.12
13.4
12.8
47.9
26.1
100
61.7
2.07
4.49
11.3
NT
0.433 2.09
9.19
NT
2.36
6.03
11.2
1.11
2.01
5.28
1.86
1.82
[100
[100
6.30
[100 2.90
[100 2.16
[100 1.56
26.8 0.314
[100 0.312
13.7 2.18
[100 0.492 6.76
[100 0.388 3.79
41.5
66.8
0.362 2.69
0.338 5.96
45.8
HT29
[100 1.66
4.50
[100 0.482 2.24
25.8
KM12
14.4
1.38
75.4
74.0
0.461 [100 [100 0.387 18.1
[100 0.351
[100 0.398
[100
[100
SW-620
0.684 10.9
[100 0.455 11.9
[100 0.476 8.42
4
.
CNS cancer
SF-268
2.65
3.13
1.24
2.80
1.99
2.94
13.6
12.4
6.07
12.1
10.3
11.6
51.0
57.9
32.1
50.2
41.3
45.0
0.834 NT
[100 0.439
[100 1.16
[100 0.279
[100 0.691
4.83
2.74
1.45
10.0
1.26
10.5
[100
NT
SF-295
2.01
6.48
91.6
1.64
6.28
SF-539
0.475 3.91
[100 0.365 2.21
NT
SNB-19
2.43
[100 [100 1.09
18.8
[100
3.55
[100
SNB-75
0.545 3.45
19.3
0.395 1.64
5.37
0.311
U251
1.21
11.2
[100 0.597 10.9
[100 0.526
5
.
Melanoma
LOX IMVI
MALME-3M
M14
1.69
1.31
1.50
NT
[100 0.609 9.02
40.8 0.644 4.98
[100 0.399 2.64
[100 0.467 3.15
[100 0.556 3.59
[100 0.493
[100 0.509
[100 0.283
2.75
2.28
1.03
[100
NT
10.1
15.2
63.0
0.218 0.587 54.6
0.516 2.51 24.4
1.21 15.1
0.532 3.17
2.68 11.5
0.883 4.76
0.328 1.32
5.21
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
0.449 10.7
44.2
91.3
44.7
42.1
49.0
37.1
0.201 0.593 17.1
0.360 0.934 NT
0.102
0.40
0.346 1.74
1.92
2.49
2.27
3.79
2.72
58.5
12.2
11.6
13.2
10.1
1.13
3.26
1.13
9.06
NT
5.66
[100
NT
[100 0.602 7.45
[100 0.339 1.52
9.78
0.609
[100 0.296
NT 1.53
[100 0.457
88.4
2.03
12.1
2.02
[100
[100
[100 40.5
6
.
3.17
2.67
2.02
3.66
15.4
7.72
34.9
18.2
[100 NT
[100 1.12
[100 1.11
NT
NT
NT
NT
NT
NT
NT
NT
OVCAR-3
OVCAR-4
OVCAR-5
5.27
6.39
80.0
[100 0.449 2.28
[100 0.695 5.44
[100 0.427
[100 0.57
2.19
5.03
[100
44.2
94.3
2.77
[100 2.61
[100 [100 1.76
[100 [100
1
23

Med Chem Res
Table 3 continued
S. no. Panel/cell line
3c (NSC 777,944)
3h (NSC 757,926)
3j (NSC 757,929)
3k (NSC 763,375)
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
OVCAR-8
2.80
2.9
8.14
18.0
4.35
[100 1.85
16.7
[100 0.86
13.1
[100 0.543
[100 0.435
22.8
4.44
1.93
[100
[100
31.3
NCI/ADR-RES
SK-OV-3
[100 0.792 10.9
11.0 0.69 2.64
[100 0.492 10.5
1.83
88.0
0.465 1.80
0.445 1.84
6.57
0.392
7
.
Renal cancer
86-0
7
2.55
2.71
1.03
2.97
NT
16.2
9.60
13.7
15.0
NT
[100 0.916 4.53
39.7
24.8
6.66
26.9
0.372
1.11
1.37
3.48
10.2
10.0
2.33
5.94
4.73
1.69
4.53
11.7
[100
43.0
NT
A498
3.43
53.2
45.9
NT
2.41
6.93
2.25
7.26
ACHN
0.419 29.2
[100 0.388 17.2
[100 0.452 15.2
[100 0.613 2.35
[100 0.635 19.3
[100 0.342
[100 0.368
CAKI-1
0.73
1.14
38.4
3.48
44.5
9.09
RXF-393
SN 12C
NT
0.562
2.35
4.20
1.39
13.0
14.7
10.1
[100 1.53
[100 0.585
[100
32.8
6.20
TK-10
47.1
3.64
2.58
54.9
24.5
1.91
6.66
34.3
21.7
1.30
0.37
UO-31
0.466 4.31
0.393 3.20
8
.
.
Prostate cancer
PC-3
2.66
3.03
15.5
13.9
[100 0.915 4.34
44.1 1.48 8.63
77.4
85.4
0.496 3.78
0.668 4.56
[100 0.439
2.31
1.63
[100
4.29
DU-145
27.6
0.482
9
Breast cancer
MCF7
2.43
2.84
3.23
1.83
1.92
2.64
16.0
19.6
[100 0.333 2.52
[100 1.45 20.9
82.3
0.303 1.57
35.3
0.314
1.45
12.8
2.52
1.58
3.68
1.52
7.50
MDA-MB-231/ATCC
HS 578T
BT-549
[100 0.575 22.6
[100 0.411 NT
[100 0.466 2.32
[100 0.737 3.95
[100 0.655
[100 0.259
[100 0.395
[100 0.507
[100
[100
5.77
[100 [100 0.428 NT
8.32
5.86
7.90
[100 0.758 3.93
T-47D
[100 1.33
76.0 1.17
5.17
4.58
[100
[100
MDA-MB-468
[100 0.37
2.00
63.1
0.298
NT not tested
Experimental
Chemistry
All the chemicals were supplied by Merck (Germany),
Konark Herbal (India), and S. D. Fine Chemicals (India).
Melting points were determined by open tube capillary
method and are uncorrected. IR spectra were obtained on a
1
Schimadzu 8201 PC, FT-IR spectrometer (KBr pellets). H
NMR spectra were recorded on a Bruker AC 400 MHz
spectrometer using TMS as internal standard in DMSO d6.
Mass spectra were recorded on a Bruker Esquire LCMS
using ESI, and elemental analyses were performed on
PerkinElmer 2400 Elemental Analyzer.
Fig. 6 Graphical illustration of binding modes of 12 proposed
curcumin analogues (3a–k) in the binding pocket of EGFR tyrosine
kinase. The ligands and interacting residues are shown in stick and
line, respectively
General method for the synthesis of 3,5-bis(4-
hydroxy-3-methylstyryl)-1H-pyrazole-1-yl-
(
aryl)methanone analogues (3a–j)
The docking score of the compounds 3a–k varies
between -5.549 and -7.569. The docking score of our
previously reported compounds varies between -5.935 and
1
,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-
dione (curcumin) (0.005 mol) and aryl hydrazides
0.005 mol) were refluxed in glacial acetic acid (AcOH)
for 12 h. The progress of reaction was monitored
-7.702. The docking score of the compound 3j was found
to be comparatively less than those of earlier reported
(
compounds (Ahsan et al., 2013).
123

Med Chem Res
Fig. 7 Two-dimensional
interaction of the ligand 3c with
the binding pocket of EGFR
tyrosine kinase
Fig. 8 Graphical illustration of
binding modes of ligand 3h in
the binding pocket of EGFR
tyrosine kinase
throughout by TLC plates (silica gel G) using mobile
phase, hexane/ethylacetate (6:4), and the spots were
identified by iodine vapours or UV light. The excess of
solvent was removed under reduced pressure, and then the
reaction mixture was then poured into the crushed ice. The
solid mass was filtered, washed, dried, and recrystallized
with ethanol furnished the title of 3,5-bis(4-hydroxy-3-
2-[3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-
caboxyloyloxy]benzoic acid (3a) Light yellow solid
(EtOH), mp 170–172 °C; yield 77 %; IR (KBr) vmax: 3402,
-
1
1
1785, 1765, 1569, 1334 cm
.
H NMR (400 MHz,
), 6.61 (1H, s, CH=C),
3
DMSO-d ): d 3.83 (6H, s, OCH
6
6.76 (2H, d, J = 11.4 Hz, CH and CH), 6.91 (2H, d,
J = 11.4 Hz, CH and CH), 6.94–7.02 (4H, m, ArH), 7.14
(2H, s, ArH), 7.43–7.45 (2H, d, J = 8.0 Hz, ArH),
7.92–7.94 (2H, d, J = 6.4 Hz, ArH), 9.15 (1H, s, ArOH),
methylstyryl)-1H-pyrazole-1-yl-(aryl)methanone
logues (3a–j).
ana-
1
23

Med Chem Res
Fig. 9 Graphical illustration of
binding modes of ligand 3j in
the binding pocket of EGFR
tyrosine kinase
Fig. 10 Graphical illustration
of binding modes of ligand 3k
in the binding pocket of EGFR
tyrosine kinase
Table 4 Docking and E model scores of the curcumin analogues
3a–k)
13
1
0.17 (2H, s, ArOH); C NMR (100 MHz, DMSO-d ): d
6
(
0
1
82.81 (C=O), 174.09 (C=O, benzoic acid), 148.46 (C
Ar
S. no
1
Compounds
Docking
score
E model
score
OCH ), 148.38 (2 9 C OCH ), 143.00 (2 9 C OH),
3
Ar
3
Ar
1
34.92 (Cpyr=N), 134.85 (2 9 ArC–CH), 134.66 (CpyrN),
0
0
1
30.31 (C3 ), 129.99 (2 9 C –CH), 129.39 (C5 Ar),
Ar
Reference/34-JAB
-8.288
-68.491
Ar
0
0
(
3a
3b
3c
3d
3e
3f
Blair et al., 2007)
129.19 (C6 Ar), 128.97 (C2 Ar), 128.86 (2 9 ArCH=CH),
128.48 (2 9 C6 ), 128.41 (2 9 C5 ), 128.35 (2 9 C1 ),
2
3
4
5
6
7
8
9
-7.569
-6.617
-6.999
-7.021
-6.712
-6.984
-5.845
-6.028
-6.351
-5.549
-6.619
-72.116
-73.957
-72.616
-80.274
-71.689
-77.823
-70.045
-71.984
-68.524
-72.366
-66.680
Ar
Ar
Ar
0
123 (C4 ), 92.64 (C ), 55.10 (2 9 OCH ); EIMS m/
Ar
pyr
3
?
z = 529.1 [M] . Anal. Calcd. for C H N O : C, 65.90;
2
9 24 2 8
H, 5.58; N, 5.30. Found: C, 65.92; H, 5.56; N, 5.32.
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(4-
aminophenyl)methanone (3b) Yellow solid (EtOH), mp
12–214 °C (found), 215–217 °C (reported) (Zhichang
et al., 2012); yield 71 %; IR (KBr) vmax: 3399, 3125, 1762,
3
3g
3h
3i
2
10
11
12
-1 1
1
562, 1344 cm . H NMR (400 MHz, DMSO-d ): d 3.81
6
3j
(6H, s, OCH ), 4.26 (2H, bs, NH ), 6.59 (1H, s, CH=C),
3 2
3k
6.74 (4H, d, J = 7.6 Hz, ArH), 6.90 (2H, d, J = 11.6 Hz,
123

Med Chem Res
CH and CH), 6.99 (2H, d, J = 16.8 Hz, CH and CH), 7.12
2H, s, ArH), 7.68–7.70 (2H, d, J = 8.4 Hz, ArH),
J = 11.8 Hz, CH and CH), 6.87–6.88 (4H, m, ArH), 7.12
1
(2H, s, ArH), 9.14 (2H, s, ArOH); C NMR (100 MHz,
3
(
0 0
7
1
1
1
.86–7.88 (2H, d, J = 8.4 Hz, ArH), 9.14 (1H, s, ArOH),
1
DMSO-d ): d 182.19 (C=O), 151.12 (C3 and C5 pyri),
6
Ar
3
0.25 (1H, s, ArOH); C NMR (100 MHz, DMSO-d ): d
146.35 (2 9 C OCH ), 145.78 (Cpyr=N), 144.82
Ar 3
0
(2 9 C OH), 141.12 (C1 pyri), 134.32 (2 9 ArC–CH),
Ar
6
82.81 (C=O), 154.61 (C NH2), 151.71 (2 9 C OCH ),
Ar
Ar
3
0
0
44.12 (2 9 C OH), 146.71 (Cpyr=N), 134.32 (2 9 ArC–
Ar
131.71 (C2
and C6 ), 128.97 (2 9 C1 ), 123.83
Ar Ar
Ar
0
0
0
0
and C6 pyri) 120.17
pyri
CH), 130.81 (C2 and C6 ), 128.91 (2 9 C1 ), 123.81
Ar
(2 9 ArCH=CH), 123.44 (C2
Ar
Ar
(
2 9 ArCH=CH), 120.11 (2 9 C6 ), 116.91 (2 9 C5 ),
Ar
(2 9 C6 ), 116.51 (2 9 C5 ), 112.24 (2 9 C1 ), 107.92
Ar Ar Ar
Ar
0
0
?
(Cpyr), 56.10 (2 9 OCH ); EIMS m/z = 469.4 [M] . Anal.
1
16.63 (C3
and C5 ), 112.21 (2 9 C1 ), 106.92
Ar
Ar
Ar
3
?
Cpyr), 56.12 (2 9 OCH ); EIMS m/z = 408.5 [M] . Anal.
(
Calcd. for C H N O : C, 69.07; H, 4.94; N, 8.95. Found:
27 23 3 5
3
Calcd. for C H N O : C, 69.55; H, 5.21; N, 8.69. Found:
28 25 3 5
C, 69.09; H, 4.92; N, 8.94.
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(2-
hydroxyphenyl)methanone (3f) Light Brown (EtOH), mp
C, 69.57; H, 5.19; N, 8.70.
3
3
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(4-
1
68–170 °C; yield 74 %; IR (KBr) vmax: 3402, 3398, 1752,
chlorophenyl)methanone (3c) Yellow solid (EtOH), mp
1
-1 1
1
557, 1347 cm . H NMR (400 MHz, DMSO-d ): d 3.81
60–162 °C; yield 78 %; IR (KBr) vmax: 3406, 1745, 1562,
6
-
1
337, 698 cm . H NMR (400 MHz, DMSO-d ): 3.83
1
(6H, s, OCH ), 6.59 (1H, s, CH=C), 6.74 (2H, d,
3
1
6
J = 11.8 Hz, CH and CH), 6.85 (2, d, J = 11.8 Hz, ArH),
(
6H, s, OCH ), 6.61 (1H, s, CH=C), 6.75 (2H, d,
3
6
.82–7.92 (9H, m, ArH), 7.14 (2H, s, ArH), 9.13 (1H, s,
1
J = 11.6 Hz, CH and CH), 6.91 (2H, d, J = 11.6 Hz, CH
3
ArOH), 10.88 (1H, s, ArOH), 11.76 (1H, s, ArOH);
C
and CH), 6.93-7.89 (10H, m, ArH), 9.14 (2H, s, OH), 9.89
1
3
NMR (100 MHz, DMSO-d ): d 165.21 (C=O), 161.12
(
1H, s, OH); C NMR (100 MHz, DMSO-d ): d 181.98
6
6
0
(C2 OH), 152.23 (2 9 C OCH ), 147.78 (Cpyr=N),
Ar
(
C=O), 147.51 (2 9 C OCH ), 144.02 (2 9 C OH),
3
Ar
3
Ar
Ar
0
1
47.11
(2 9 C OH),
Ar
136.71
0
(C4 Ar),
133.42
1
1
45.71 (Cpyr=N), 140.11 (C Cl), 134.32 (2 9 ArC–CH),
Ar
0
0
(2 9 ArCH=CH), 131.10 (C6 ), 128.99 (2 9 C1Ar),
Ar
31.71 (C2
and C6 ), 128.97 (2 9 C1 ), 123.83
Ar Ar
Ar
0
123.61 (2 9 ArCH=CH), 121.6 (C5 ), 119.9 (2 9 C6Ar),
Ar
(
2 9 ArCH=CH), 120.17 (2 9 C6 ), 116.51 (2 9 C5 ),
Ar
Ar
0
0
116.61 (2 9 C5 ), 112.21 (2 9 C2 ), 107.31 (Cpyr),
Ar Ar
1
16.53 (C3
and C5 ), 112.24 (2 9 C1 ), 107.92
Ar
Ar
Ar
?
6.31 (2 9 OCH ); EIMS m/z = 484.5 [M] . Anal. Calcd.
?
Cpyr), 56.10 (2 9 OCH ); EIMS m/z = 503.0 [M] ; Anal.
5
(
3
3
for C H N O : C, 69.41; H, 4.99; N, 5.78. Found: C,
28 24 3 6
Calcd. for C H ClN O : C, 66.87; H, 4.61; N, 5.57.
2
8
23
2 5
69.39; H, 4.96; N, 5.80 (Zhichang et al., 2012).
Found: C, 66.84; H, 4.63; N, 5.54 (Balaji et al., 2015).
3
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(2-
3
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(2-
thiophenyl)methanone (3g) Light yellow solid (EtOH),
mp 120–122 °C; yield 65 %; IR (KBr) vmax: 3396, 1735,
chlorophenyl)methanone (3d) Yellow solid (EtOH), mp
1
68–170 °C; yield 74 %; IR (KBr) vmax: 3412, 1751, 1569,
-1 1
1
-1 1
1563, 1337 cm . H NMR (400 MHz, DMSO-d
): d 3.83
6H, s, OCH ), 6.61 (1H, s, CH=C), 6.76 (2H, d,
6
329, 699 cm . H NMR (400 MHz, DMSO-d ): d 3.83
6
(
3
(
6H, s, OCH ), 6.61 (1H, s, CH=C), 6.76 (2H, d,
3
J = 12.8 Hz, CH and CH), 6.89 (2H, d, J = 12.8 Hz, CH
1
and CH), 6.92–7.88 (9H, m, ArH), 10.53 (2H, s, OH);
J = 12.1 Hz, CH and CH), 6.90 (2H, d, J = 12.1 Hz, CH
1
and CH), 6.94-8.17 (10H, m, ArH), 9.15 (2H, s, OH);
3
3
C
C
NMR (100 MHz, DMSO-d ): d 182.21 (C=O), 152.23
6
NMR (100 MHz, DMSO-d ): d 181.83 (C=O), 147.53
6
0
(2 9 C OCH ), 145.21 (C1 ), 147.11 (2 9 C OH),
Ar
3
Ar
Ar
(
2 9 C OCH ), 143.32 (2 9 C OH), 141.71 (Cpyr=N),
Ar 3 Ar
0
0
1
44.78 (Cpyr=N), 137.71 (C5 Ar), 135.11 (C3 Ar), 133.49
0
37.11 (C Cl), 136.01 (C4 Ar), 134.39 (2 9 ArC–CH),
Ar
1
1
0
(2 9 ArCH=CH), 128.98 (C4 ), 128.19 (2 9 C1Ar),
Ar
0
0
31.71 (C5 Ar), 129.41 (C3 ), 128.97 (2 9 C1 ), 127.11
Ar
Ar
1
23.69 (2 9 ArCH=CH), 120.91 (2 9 C6 ), 116.71
Ar
0
(
C5 Ar), 123.13 (2 9 ArCH=CH), 120.19 (2 9 C6Ar),
(2 9 C5 ), 112.21 (2 9 C2 ), 106.37 (Cpyr), 56.12
Ar Ar
1
5
16.21 (2 9 C5 ), 112.14 (2 9 C1 ), 107.92 (Cpyr),
Ar Ar
?
?
6.12 (2 9 OCH ); EIMS m/z = 503.0 [M] . Anal. Calcd.
(2 9 OCH
); EIMS m/z = 475.1 [M] . Anal. Calcd. for
3
3
C H N O S: C, 65.81; H, 4.65; N, 5.90. Found: C, 65.84;
26 22 2 5
for C H ClN O : C, 66.87; H, 4.61; N, 5.57. Found: C,
2 5
2
8
23
H, 4.65; N, 5.94 (Balaji et al., 2015).
6
6.85; H, 4.63; N, 5.55 (Balaji et al., 2015).
3
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(4-
3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(phe-
nyl)ethanone (3h) Yellow solid (EtOH), mp 138–140 °C;
yield 82 %; IR (KBr) vmax: 3401, 1751, 1565, 1338 cm .
pyridyl)methanone (3e) Yellow solid (EtOH), mp
-
1
1
2
1
04–106 °C (found), 105–106 °C (reported) (Sahu et al.,
1
012); yield 74 %; IR (KBr) vmax: 3401, 1753, 1567, 1543,
-
H NMR (400 MHz, DMSO-d ): d 3.66 (2H, s, CH ), 3.83
6
2
1 1
342 cm . H NMR (400 MHz, DMSO-d ): d 3.81 (6H, s,
(6H, s, OCH ), 6.61 (1H, s, CH=C), 6.76 (2H, d,
3
6
OCH ), 6.60 (1H, s, CH=C), 6.75 (2H, d, J = 11.8 Hz, CH
3
J = 11.8 Hz, CH and CH), 6.89 (2H, d, J = 11.8 Hz, CH
1
3
and CH), 6.82 (4H, d, J = 7.8 Hz, ArH), 6.87 (2H, d,
and CH), 6.94–7.81 (11H, m, ArH), 9.15 (2H, s, OH);
C
1
23

Med Chem Res
NMR (100 MHz, DMSO-d ): d 192.28 (C=O), 151.83
TLC plates (silica gel G) using mobile phase, hexane/
ethylacetate (6:4), and the spots were identified by iodine
vapours or UV light. The excess of solvent was removed
under reduced pressure, and then the reaction mixture was
then poured into the crushed ice. The solid mass was fil-
tered, washed, dried, and recrystallized with ethanol fur-
nished the title of 3,5-bis(4-hydroxy-3-methylstyryl)-1H-
pyrazole-1-carboxamide/carbothioamide analogues (3k).
6
(
2 9 C OCH ), 147.71 (C =N), 145.11 (2 9 C OH),
Ar 3 pyr Ar
0 0
35.91 (C1 Ar), 133.49 (2 9 ArCH=CH), 129.71 (C2 Ar
1
0
0
0
and C6 Ar), 129.31 (C3 and C5 Ar), 128.09 (2 9 C1Ar),
Ar
0
1
27.98 (C4 Ar), 123.68 (2 9 ArCH=CH), 120.19
(
2 9 C6 ), 116.65 (2 9 C5 ), 112.01 (2 9 C2 ), 107.65
Ar Ar Ar
(
Cpyr), 56.10 (2 9 OCH ), 35.56 (CH ); EIMS m/
3
2
?
z = 482.6 [M] . Anal. Calcd. for C H N O : C, 72.18;
2
9 26 2 5
H, 5.43; N, 5.81. Found: C, 72.14; H, 5.47; N, 5.79 (Balaji
et al., 2015).
3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-car-
boxamide (3k) Brown solid (EtOH), mp 200–202 °C
found), 200–202 °C (reported) (Sahu et al., 2012; Balaji
3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(phe-
(
noxyl)methanone (3i) Brown solid (EtOH), mp 130–
et al., 2015); yield 84 %; IR (KBr) vmax: 3407, 1745, 1561,
-1
132 °C; yield 76 %; IR (KBr) vmax: 3402, 1755, 1569,
-1 1
1334 cm . H NMR (400 MHz, DMSO-d ): d 3.79 (2H, s,
1
1
334 cm ; H NMR (400 MHz, DMSO-d ): d 3.81 (6H, s,
6
6
OCH ), 6.59 (1H, s, CH=C), 6.73 (2H, d, J = 12.1 Hz, CH
3
OCH ), 3.83 (6H, s, OCH ), 6.61 (1H, s, CH=C), 6.76 (2H,
2
3
and CH), 6.81 (4H, d, J = 12.1 Hz, CH and CH), 6.90–7.36
d, J = 11.8 Hz, CH and CH), 6.90 (2H, d, J = 11.8 Hz,
(4H, m, ArH), 7.12 (2H, s, ArH), 9.14 (2H, s, ArOH); d
CH and CH), 6.94–7.38 (10H, m, ArH), 9.15 (2H, s, OH);
1
181.21 (C=O), 148.32 (2 9 C OCH ), 147.81 (Cpyr=N),
Ar 3
3
C NMR (100 MHz, DMSO-d ): d 192.18 (C=O), 162.11
6
145.35 (2 9 C OH), 135.66 (CpyrN), 133.85 (2 9 ArCH=
Ar
0
(
(
C1 ), 151.83 (2 9 C OCH ), 146.71 (Cpyr=N), 144.11
Ar
Ar
3
CH), 128.81 (2 9 C1Ar), 123.86 (2 9 ArCH = CH),
0
2 9 C OH), 133.49 (2 9 ArCH=CH), 129.76 (C3 and
Ar
0
Ar
1
1
21.33 (2 9 C6 ), 117.33 (2 9 C5 ), 112.55 (2 9 C2 ),
Ar Ar Ar
0
C5 Ar), 123.69 (2 9 ArCH=CH), 121.32 (C4 Ar), 120.18
?
07.12 (Cpyr), 56.21 (2 9 OCH ); EIMS m/z = 407.1 [M] .
0 0
2 9 C6 ), 116.85 (2 9 C5 ), 114.33 (C2 and C6 Ar),
Ar Ar Ar
3
(
Anal. Calcd. for C H N O : C, 69.86; H, 5.20; N, 10.31.
22 21 3 5
1
12.61 (2 9 C2 ), 107.72 (C ), 72.56 (CH ), 56.12
Ar pyr 2
Found: C, 69.85; H, 5.24; N, 10.26.
?
(
2 9 OCH ); EIMS m/z = 499.1 [M] . Anal. Calcd. for
3
C H N O : C, 69.87; H, 5.26; N, 5.62. Found: C, 69.84;
29 26 2 6
Antiproliferative activity
H, 5.25; N, 5.64 (Balaji et al., 2015).
,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(4-
methoxyphenyl)methanone (3j) Brown solid (EtOH), mp
3
The antiproliferative activity of the was carried out at
National Cancer Institute (NCI US) on leukaemia, mela-
noma, lung, colon, CNS, ovarian, renal, prostate, and breast
cancers cell lines. There were nine panels of cell lines with
nearly 60 in number according to the standard screening
protocol, reported elsewhere (http://dtp.nci.nih.gov; Monks
et al., 1991; Boyd and Paull, 1995; Shoemaker, 2006;
Alley et al., 1988; Grever et al., 1992). All the curcumin
analogues were synthesized and the structure of the com-
pounds submitted online to the official site of NCI for
antiproliferative screening. Among 11 compounds, only
four compounds were selected for antiproliferative
screening. NCI have their own selection procedure of the
compounds for antiproliferative screening based on the
novelty of heterocyclic ring system, drug-like properties
utilizing the concept of privileged scaffolds, structure
based on computer-aided drug design, etc., while the
structures containing problematic linkage or functional
groups (e.g. nitro, nitroso, –N–N–, –N=N–, imine, semi-
carbazone, thioamides, thioureas) are avoided for suc-
cessful drug development (http://dtp.nci.nih.gov; Ahsan
et al., 2013). Growth percent (GP) is the percent growth in
the cell lines, while logGI50, logTGI, and logLC50 are the
logarithm molar concentration producing 50 % growth
inhibition (GI50), a total growth inhibition (TGI), and a
50 % cellular death (LC50), respectively.
1
28–130 °C; yield 74 %; IR (KBr) vmax: 3407, 1745, 1561,
-1 1
1
334 cm : H NMR (400 MHz, DMSO-d ): d 3.83 (9H,
6
s, OCH ), 6.61 (1H, s, CH=C), 6.76 (2H, d, J = 12.2 Hz,
3
CH and CH), 6.92 (2H, d, J = 12.2 Hz, CH and CH),
6
.92–7.92 (10H, m, ArH), 9.15 (1H, s, OH), 10.28 (1H, s,
1
3
OH). C NMR (100 MHz, DMSO-d ): d 166.5 (C=O),
1
6
0
65.1 (C4 Ar), 151.3 (2 9 C OCH ), 147.3 (Cpyr=N),
Ar
3
1
44.9 (2 9 C OH), 133.9 (2 9 ArCH=CH), 133.4 (Cpyr–
Ar
0
0
N), 130.9 (C2
and C6 ), 128.8 (2 9 C1 ), 123.6
Ar
Ar
Ar
0
(
(
2 9 ArCH=CH), 122.9 (C1 Ar), 120.1 (2 9 C6 ), 116.8
Ar
0
0
2 9 C5 ), 114.8 (C3
Ar
and C5 Ar), 112.0 (2 9 C2Ar),
Ar
1
07.7 (Cpyr), 56.2 (2 9 ArOCH ), 55.9 (ArOCH ); EIMS
3 3
?
m/z = 499.1 [M] . Anal. Calcd. for C H N O : C, 69.87;
2
9 26 2 6
H, 5.26; N, 5.62. Found: C, 69.85; H, 5.24; N, 5.65 (Balaji
et al., 2015).
Method for the synthesis of 3,5-bis(4-hydroxy-3-
methylstyryl)-1H-pyrazole-1-carboxamide (3k)
1
,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-
dione (curcumin) (0.005 mol) and semicarbazide (0.005
mol) were refluxed in glacial acetic acid (AcOH) for 12 h.
The progress of reaction was monitored throughout by
123

Med Chem Res
Molecular docking studies
Acknowledgments Antiproliferative data were provided by
National Cancer Institute (NCI US), Bethesda, MD, USA. We are
grateful for all help provided by Prof. Doug Smallwood and Mr.
Mohammed Nayel. The people holding the management of Maharishi
Arvind College of Pharmacy, Jaipur, Rajasthan, India, are acknowl-
edged for providing research facilities. We are also grateful to Dr.
Reddy Institute of Life Science, Hyderabad, Andhra Pradesh, India,
for providing spectral data of synthesized compounds.
Protein structure
The X-ray crystallographic structure of EGFR tyrosine
˚
kinase (PDB: 2J5F) with resolution 3.00 A, R value 0.194
(
observed), was obtained from protein data bank (www.
rcsb.org).
Compliance with ethical standards
Conflict of interest The authors confirm that this article content has
no conflicts of interest.
Protein preparation
The protein preparation wizard (Maestro, version 8.5,
Schrodinger, LLC, 2008) was employed to refine the pro-
tein structure. Bond orders were assigned, hydrogens were
added, and water molecules were deleted. Metals have not
been found in present protein. Heterostate for co-crystal-
lized ligand was generated using Epik, and hydrogen bond
of protein was allocated using Protassign. The energy
minimization of protein was exerted by Impref minimiza-
tion at pH 7.0 and OPLS2005 force field.
References
Ahsan MJ, Khalilullah H, Yasmin S, Jadav SS, Govindasamy J (2013)
Synthesis, characterisation, and in vitro anticancer activity of
curcumin analogues bearing pyrazole/pyrimidine ring targeting
EGFR tyrosine kinase. Bio Med Res Int 2013:239354. doi:10.
1
155/2013/239354
Alley MC, Scudiero DA, Monks PA, Hursey ML, Czerwinski MJ, Fine
DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR (1988)
Feasibility of drug screening with panels of human tumor cell lines
using a microculture tetrazolium assay. Cancer Res 48:589–601
Aydemir N, Bilaloglu R (2003) Genotoxicity of two anticancer drugs,
gemcitabine and topotecan, in mouse bone marrow in vivo.
Mutat Res 537:43–51
Receptor grid generation and molecular docking protocol
The receptor grid was generated around the reference/
bound ligand 34-JAB (N-4-[3-bromophenylamino)-quina-
zolin-6-yl]-acrylamide with default parameters using
GLIDE5.0 which was implemented in Schrodinger’s LLC
and was also employed for the automated flexible molec-
ular docking protocol (Blair et al., 2007). All the three-
dimensional structures of ligands were drawn using Mae-
stro, prepared by ligprep, and output was docked in the
EGFR tyrosine kinase active site. The extra-precision
docking (XP) was employed for the current docking sim-
ulation score.
Balaji SN, Ahsan MJ, Jadav SS, Trivedi V (2015) Molecular
modeling, synthesis and antimalarial potentials of curcumin
analogues containing heterocyclic ring. Arab J Chem. doi:10.
1016/j.arabjc.2015.04.011
Blair JA, Rauh D, Kung C, Yun CH, Fan Q, Rode H, Zhang C, Eck
MJ, Weiss WA, Shokat KM (2007) Structure-guided develop-
ment of affinity probes for tyrosine kinases using chemical
genetics. Nat Chem Bio 4:229–238
Boyd MR, Paull KD (1995) Some practical considerations and
applications of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev Res 34:91–109
Carmona D, Saez J, Granados H, Perez E, Blair S, Angulo A,
Figadere B (2003) Antiprotozoal 6-substituted-5,6-dihydro-al-
pha-pyrones from Raimondia cf. monoica. Nat Prod Res
1
7:275–280
Cragg GM, Newman DJ (2005) Biodiversity: a continuing source of
novel drug leads. Pure Appl Chem 77:7–24
Conclusion
Development therapeutic program NCI/NIH (2014). http://dtp.nci.nih.
gov. Accessed 13 Nov 2014
Grever MR, Schepartz SA, Chabner BA (1992) The National Cancer
Institute: cancer drug discovery and development program.
Semin Oncol 19:622–638
Jia Y, Li J, Qin ZH, Liang ZQ (2009) Autophagic and apoptotic
mechanisms of curcumin-induced death in K562 cells. J Asian
Nat Prod Res 11:918–928
Koepfli JB, Mead JF, Brockman JA (1947) An alkaloid with high
antimalarial activity from Dichroa Febrifuga1. J Am Chem Soc
A series of eleven curcumin analogues bearing pyrazole
nucleus were synthesized in satisfactory yield, and four
compounds were evaluated for their in vitro antiprolifera-
tive activity at single-dose and five-dose assays. The cur-
cumin analogues showed potential antiproliferative activity
on the various cancer cell lines. All the compounds (3a–
k) were well accommodated in binding site of 34-JAB of
the EGFR tyrosine kinase, and the hydroxyl and methoxy
function groups of both phenyl rings were important for the
rein-geminated hydrogen bonding by either side chain or
backbone. The curcumin analogues bearing pyrazole
nucleus discovered in the present studies may provide
invaluable therapeutic intervention in the treatment of
cancer and may provide lead for further discovery.
6
9:1837
Kunwar A, Barik A, Mishra B, Rathinasamy K, Pandey R,
Priyadarsini K (2008) Phytochemicals as modulators of neo-
plastic phenotypes. Biochem Biophy Act 1780:673–679
Lal J, Gupta SK, Thavaselvam D, Agrawal DD (2012) Design,
synthesis, synergistic antimicrobial activity and cytotoxicity of
4-aryl substituted 3,4-dihydropyrimidinones of curcumin. Bioorg
Med Chem 22:2872–2876
1
23

Med Chem Res
Liang G, Shao L, Wang Y, Zhao C, Chu Y, Xiao J, Zhao Y, Li X,
Yang S (2009) Exploration and synthesis of curcumin analogues
with improved structural stability both in vitro and in vivo as
cytotoxic agents. Bioorg Med Chem 17:2623–2631
Sharma R, Jadav SS, Yasmin S, Bhatia S, Khalilullah H, Ahsan MJ
(2015) Simple efficient and improved synthesis of Biginelli-type
compounds of curcumin as anticancer agents. Med Chem Res
24:236–244
Mendelsohn J, Baselga J (2000) The EGF receptor family as targets
for cancer therapy. Oncogene 19:6550–6565
Shoemaker RH (2006) The NCI60 human tumour cell line anticancer
drug screen. Nat Rev Cancer 6:813–823
Mishra S, Karmodiya K, Surolia N, Surolia A (2008) Synthesis and
exploration of novel curcumin analogues as anti-malarial agents.
Bioorg Med Chem 16:2894–2902
Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D,
Hose C, Langley J, Cronise P, Vaigro-Wolff A, Gray-Goodrich
M, Campbell H, Mayo J, Boyd M (1991) Feasibility of a high-
flux anticancer drug screening using a diverse panel of cultured
human tumor cell lines. J Natl Cancer Inst 83:757–766
Singh RK, Rai D, Yadav D, Bhargava A, Balzarini J, DeClercq E
(2010) Synthesis, antibacterial and antiviral properties of
curcumin bioconjugates bearing dipeptide, fatty acids and folic
acid. Eur J Med Chem 45:1078–1086
Vyas A, Dandawate P, Padhye S, Ahmad A, Sarkar F (2013)
Perspectives on new synthetic curcumin analogs and their
potential anticancer properties. Curr Pharm Des 19:2047–2069
WHO World Cancer Report 2014. Retrieved from http://www.
nydailynews.com/life-style/health/14-million-people-cancer-
2012-article-1.1545738. Retrieved on 12 Dec 2013
NCI Data Warehouse Index (2014). http://dtp.nci.nih.gov/
dtpstandard/dwindex/index.jsp. Accessed 13 Nov 2014
Normanda N, Maiello MR, Luca AD (2002) Epidermal growth factor
receptor tyrosine kinase inhibitors (EGFR-TKIs): Simple drugs
with a complex mechanism of action? J Cell Physiol 194:13–19
Pari L, Tewas D, Eckel J (2008) Role of curcumin in health and
disease. Arch Physiol Biochem 114:127–149
Sahu PK, Sahu PK, Gupta SK, Thavaselvam D, Agarwal DD (2012)
Synthesis and evaluation of antimicrobial activity of 4H-
pyrimido[2,1-b]benzothiazole, pyrazole and benzylidene deriva-
tives of curcumin. Eur J Med Chem 54:366–378
Yadav IS, Nandekar PP, Shrivastava S, Sangamwar A, Chaudhury A,
Agarwal SM (2014) Ensemble docking and molecular dynamics
identify knoevenagel curcumin derivatives with potent anti-
EGFR activity. Gene 539:82–90
Yar MS, Siddiqui AA, Ali A (2007) Synthesis and anti tuberculostatic
activity of novel 1,3,4-oxadiazole derivatives. J Chin Chem Soc
54:5–8
Zhichang L, Yinghong W, Yuanqin Z, Qinxiang X (2012) Synthesis
and antibacterial activities of N-substituted pyrazole curcumin
derivatives. Chin J Org Chem 32:1487–1492
Saja K, Babu MS, Karunagaran D, Sudhakaran PR (2007) Anti-
inflammatory effect of curcumin involves down regulation of MMP-
9
in blood mononuclear cells. Int Immunopharm 7:1659–1667
123