Some 1,3,5-trisubstituted pyrazoline derivatives targeting breast cancer: Design, synthesis, cytotoxic activity, EGFR inhibition and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2020.103780

Different 1,3,5-trisubstituted pyrazoline derivatives 2a-c, 3-c, 4a-f, 6a-c, 7a-f and 8a-d were prepared via condensation reaction of the appropriate chalcone 1a-c or 5a-c with various hydrazine derivatives. All compounds were screened for their cytotoxicity against breast MCF-7 cancer cell line and the normal fibroblasts WI-38. Thirteen compounds 2a, 3a, 3c, 4a-d, 6c, 7d, 7e, 8b, 8d and 8f revealed promising cytotoxicity against MCF-7 compared to the reference standard staurosporine and they were safe to the normal fibroblasts WI-38. In addition, compounds 3c, 6c, 7d, 8b and 8d elicited higher cytotoxicity than erlotinib and exhibited promising EGFR inhibitory activity at submicromolar level comparable to that of erlotinib except for compound 8b that may exert its cytotoxicity via another mechanism besides EGFR inhibition. Molecular docking of 3c, 6c, 7d, 8b and 8d in the active site of EGFR confirmed the obtained results.

Full text of DOI:10.1016/j.bioorg.2020.103780

Journal Pre-proofs
Some 1,3,5-Trisubstituted Pyrazoline Derivatives Targeting Breast Cancer:
Design, Synthesis, Cytotoxic activity, EGFR inhibition and Molecular dock-
ing
Riham F. George, Manal Kandeel, Dina Y. El-Ansary, Ahmed M. ElKerdawy
PII:
S0045-2068(19)32114-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103780
YBIOO 103780
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 December 2019
17 March 2020
19 March 2020
Please cite this article as: R.F. George, M. Kandeel, D.Y. El-Ansary, A.M. ElKerdawy, Some 1,3,5-Trisubstituted
Pyrazoline Derivatives Targeting Breast Cancer: Design, Synthesis, Cytotoxic activity, EGFR inhibition and
Molecular docking, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103780
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Some 1,3,5-Trisubstituted Pyrazoline Derivatives Targeting Breast Cancer: Design,
Synthesis, Cytotoxic activity, EGFR inhibition and Molecular docking
Riham F. Georgea*, Manal Kandeelb, Dina Y. El-Ansaryb Ahmed M. ElKerdawya,c
,
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
bPharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical
Industries, Future University in Egypt, Cairo, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, Newgiza,km 22
Cairo–Alexandria Desert Road, Cairo, Egypt.
Declarations of interest: none
Abstract
Different 1,3,5-trisubstituted pyrazoline derivatives 2a-c, 3-c, 4a-f, 6a-c, 7a-f and 8a-d
were prepared via condensation reaction of the appropriate chalcone 1a-c or 5a-c with
various hydrazine derivatives. All compounds were screened for their cytotoxicity against
breast MCF-7 cancer cell line and the normal fibroblasts WI-38. Thirteen compounds 2a,
3a, 3c, 4a-d, 6c, 7d, 7e, 8b, 8d and 8f revealed promising cytotoxicity against MCF-7
compared to the reference standard staurosporine and they were safe to the normal
fibroblasts WI-38. In addition, compounds 3c, 6c, 7d, 8b and 8d elicited higher
cytotoxicity than erlotinib and exhibited promising EGFR inhibitory activity at
submicromolar level comparable to that of erlotinib except for compound 8b that may
exert its cytotoxicity via another mechanism besides EGFR inhibition. Molecular
docking of 3c, 6c, 7d, 8b and 8d in the active site of EGFR confirmed the obtained
results.
Key words: Pyrazolines; Chalcones; Hydrazine; MCF7; EGFR
* Corresponding author: Riham F. George, e-mail: riham.eskandar@pharma.cu.edu.eg
1

1. Introduction
Breast cancer is one of the most common cancer types in both developed and
developing countries. Despite the presence of advanced diagnostic techniques for its early
detection, breast cancer occupies the first place in the detected new cancer cases in the
US annually (268,600 cases), in addition to its high death rate of 41,760 deaths annually
[1]. These records reflect the presence of a defect in the breast cancer treatment which
may result from the emergence of a resistance to the used therapies [1]. Therefore, there
is an urgent need for the discovery of more efficient anticancer agents acting on clinically
validated targets.
Epidermal Growth Factor Receptor (EGFR) is one of the tyrosine kinases (TKs) that
play a crucial role in the regulation of several cellular functions such as cell growth,
survival, proliferation, differentiation, and apoptosis [2]. However, in many cancer types
as hepatocellular carcinoma, breast cancer, non-small cell lung cancer, pancreatic cancer,
colorectal carcinoma, melanoma, and glioblastoma, there are significant resistance to the
used EGFR inhibitors [3]. Therefore, four generations of EGFR inhibitors have been
discovered to combat different types of cancer and to solve the problem of resistance
emerged from each precedent generation [4].
Pyrazoline is a privileged scaffold found in many bioactive agents with diverse biological
activities [5-10]. For example, compound I and the thiazolyl-pyrazoline derivative II
displayed potent cytotoxic activity against breast cancer cell line (MCF-7) with IC50 of
0.08 and 0.07 µM, respectively, through EGFR inhibition with IC50 of 0.07 and 0.06 µM,
respectively, whereas, the positive control erlotinib, in these studies, showed IC50 of 0.03
and 0.02 µM, respectively [10,11]. Moreover, the pyrazoline derivative III exhibited
promising cytotoxicity against MCF-7 with IC50 of 0.16 µM in comparison to sorafenib
(IC50 = 0.19 µM) which used as reference standard in this study [12]. Additionally, the
2,4-disubstituted thiazole-based pyrazoline derivative IV elicited equipotent cytotoxic
activity against breast cancer cell line (MCF-7) compared to cisplatin with IC50 of 7.50
µM [13] (Figure 1).
2

N
N
F
S
N
N
N
O
S
H2N
Cl
I
II
Cl
N
N
N
S
N
Cl
S
N
C
NH
NH
O
N
Cl
S
F
III
IV
F
F
Figure 1. Reported 1,3,5-trisubstituted-4,5-dihydro-1H-pyrazolines with cytotoxic
activity against breast cancer cell line (MCF-7) and/or EGFR inhibitory activity.
Inspired by these data, this work focuses on the synthesis of 1,3,5-trisubstituted-4,5-
dihydro-1H-pyrazolines with styryl and aryl moieties incorporated at positions 3 and/or 5
(General formulae A and B, Figure 2) in order to study the effect of this positional
isomerism on the cytotoxic activity. Furthermore, different substituents at the para
position of the aryl and styryl moieties will be used to investigate the impact of their
electronic effects on the obtained activity. Additionally, at the pyrazoline position 1,
various groups will be inserted as acetyl (compounds 2a-c, 6a-c), carboxamide
(compounds 3a-cand 7a-c) and its bioisostere carbothioamide (compounds 7d-f) to study
the effect of these structural modifications on the obtained activity. Moreover, attachment
of phenyl or p-methoxyphenyl moiety at position 1 of the pyrazoline ring was carried out
(compounds 4a-c, 8a-f) to study the influence of their lipophilicity and steric effect on
the obtained activity (Figure 2).
3

All the designed compounds will be screened for their cytotoxicity against breast
cancer cell line (MCF-7) as well as normal fibroblasts (WI-38) to ensure their safety to
normal cells and selectivity towards cancerous cells. Additionally, compounds eliciting
promising anticancer activity will be tested for their EGFR inhibitory activity to shed
light on their possible mode of action. Furthermore, molecular docking simulation of the
most active EGFR inhibitors in the receptor active site will be performed to validate the
obtained experimental results and to illustrate their binding pattern and binding
interactions with the key amino acids in the EGFR active site.
Y
X
X
N
N
N
N
R
R
A
B
X,Y= H,Cl.OCH3
R= Different aryl moieties, CONH2, CSNH2, COCH3
R
R
N
N
N
N
R
R1
X
R1
X
6a-c, 7a-f
2a-c, 3a-c
R= H, Cl, OCH3
R
R1= NH2, CH3
R
X= O, S
N
N
N
N
R
R1
8a-f
R1
4a-c
R= H, Cl, OCH3
R1= H, OCH3
Figure 2. Structure of the target 1,3,5-trisubstituted-4,5-dihydro-1H-pyrazolines
4

2. Results and discussion
2.1. Chemistry
The synthetic pathways used for preparation of the target compounds are illustrated in
Schemes 1 and 2. (1E, 4E)-1,5-diarylpenta-1,4-dien-3-ones (1a-c) were prepared as
reported via Claisen Schmidt reaction of the appropriate benzaldehyde and acetone in
ethanol under basic conditions [14-16]. Similarly, the condensation of cinnamaldehyde
and the appropriate acetophenones in ethanol in the presence of sodium hydroxide
afforded the chalcones 5a-c [17, 18]. Furthermore, the reaction of 1a-c or 5a-c with
hydrazine hydrate in glacial acetic acid afforded the reported 1-acetyl-5-aryl-3-styryl-4,5-
1
dihydro-1H-pyrazolines (2a-c) or their positional isomers 6a-c, respectively [19,20]. H
NMR spectra of 2a-c confirmed the cyclization due to the appearance of 3 doublet of
doublets at 2.89-3.05, 3.51-3.62 and 5.42-5.57 ppm, respectively assigned for HA, HM and
HX of the pyrazoline ring in addition to a singlet signal at 2.27-2.40 ppm attributed to
the acetyl methyl protons. Furthermore, two doublets for the two olefinic protons
appeared at 6.63-6.79 and 6.99-7.14 ppm, respectively with high coupling constant (J =
16.32-16.40 Hz) confirming E conformation of olefinic protons at the double bond. On
1
the other hand, H NMR spectrum of 6a-c presented a singlet signal at δ 2.43-2.45 ppm
corresponding to CH3 protons of the acetyl group in addition to two doublets of doublet
for HA, HM protons of pyrazoline ring at 3.11-3.16, 3.54-3.58, respectively. Furthermore,
a multiplet for HX of pyrazoline ring appeared at 5.29-5.35 ppm due to coupling with HA,
HM at C-4 of pyrazoline as well as olefinic proton at side chain. Consequently, this
olefinic proton appeared as doublet of doublet at 6.22-6.23 ppm due to coupling with Hx
of pyrazoline and the neighboring olefinic proton that appeared as a doublet at 6.61-6.63
ppm.
Moreover, reaction of 1a-c with semicarbazide hydrochloride in ethanol in the presence
of sodium hydroxide resulted in formation of the reported 1-carboxamide pyrazoline
derivative 3a [21] and the new congeners 3b and 3c. The reaction of 5a-c with either
semicarbazide hydrochloride or thiosemicarbazide adopting the same reaction conditions
resulted in compounds 7a-f. IR spectrum of compounds 3a-c showed NH2 bands at 3478-
3441, 3498-3398 cm-1 and C=O band at 1676-1660 cm-1. 13C NMR of 3b and 3c revealed
signals for CH2 and CH carbons of the pyrazoline ring at 42.0 and 59.4-59.5 ppm,
5

respectively along with C=O signal at 155.0-159.0 ppm, in addition to two signals at 55.3
and 55.4 ppm due to two OCH3 groups of compound 3c. IR spectrum of 7a-f showed
NH2 bands at 3495-3275, 3402-3197 cm-1, C=O band at 1681-1674 cm-1 for compounds
1
7a-c and C=S band at 1253-1246 cm-1 for compound 7d-f. H NMR of compounds 7a-f
showed, two doublets of doublet for HA, HM at 3.03-3.16, 3.49-3.61, respectively and a
multiplet for HX of pyrazoline ring at 5.10-5.26 ppm along with a doublet of doublet and
a doublet at 6.18-6.29 and 6.54-6.64 ppm, respectively attributed to the olefinic protons.
Moreover, an exchangeable singlet signal of NH2 protons appeared at 5.30-5.52 ppm
which disappeared upon deuteration.
Additionally, 1-phenyl-4,5-dihydro-1H-pyrazoline derivatives 4a-c or their analogs 8a-c
were obtained according to the reported procedure [14, 20, 22] via reaction of
phenylhydrazine with the chalcones 1a-c or 5a-c, respectively, in absolute ethanol in the
presence of glacial acetic acid. However, the reaction of p-methoxyphenylhydrazine
hydrochloride with either 1a-c or 5a was carried out in ethanol to give the corresponding
1-(4-methoxyphenyl)-pyrazoline analogs 4d-f or 8d, respectively. Whereas, using the
same conditions in case of reaction of 5b and 5c with p-methoxyphenylhydrazine
hydrochloride affording the open condensation products 8e and 8f instead of their
pyrazoline congeners. 1H NMR spectrum of 4a-f confirmed the pyrazoline ring formation
by the presence of three doublets of doublet for HA, HM and HX of the pyrazoline ring at
2.94-3.03, 3.65-3.86 and 5.18-5.26 ppm, respectively, and two doublets for two olefinic
proton at 6.46-6.54 ppm and 7.07-7.38 ppm. 13C NMR of 4d-f showed a signal referred to
CH2 and CH carbons of the pyrazoline ring at 42.2-45.4 and 61.0-64.8 ppm, respectively
1
along with the methoxy signal at 55.3-55.6 ppm. H NMR spectra of 8a-d exhibited two
doublets of doublet for HA, HM at 3.12-3.16, 3.63-3.67, respectively and a multiplet for
HX of pyrazoline ring at 4.80-5.16 ppm, in addition to a doublet of doublet and a doublet
1
at 6.15-6.36 and 6.58-6.92 ppm attributed to the olefinic protons. On the other hand, H
NMR spectra of 8e and 8f lacked the AMX pattern of pyrazoline protons and showed a
singlet signal at δ 5.32-6.95 ppm corresponding to NH proton and two doublets for four
olefinic protons at 6.75-6.85 ppm and 7.04-7.16 ppm negating the pyrazoline ring
formation and indicating that only the condensed products 8e and 8f were obtained.
6

CHO
R
R
R
O
(a)
2
acetone
O
app. benzaldehyde
1a-c
(b)
(d)
(c)
R
R
R
R
R
R
N
N
N
N
N
N
NH2
O
O
2a-c
3a-c
R1
1a, 2a, 3a: R=H
1b, 2b, 3b: R=Cl
1c, 2c, 3c: R=OCH3
4a-f
4a: R= H , R1= H
4b: R= Cl, R1= H
4c: R= OCH3, R1= H
4d: R= H, R1= OCH3
4e: R= Cl, R1= OCH3
4f: R= R1= OCH3
Reagents and conditions: (a) NaOH, absolute ethanol, RT, 1.5h; (b) Hydrazin hydrate, glacial acetic acid, 8h; (c)
Semicarbazide.HCl, NaOH, EtOH, 8h; (d)Phenyl hydrazine , glacial acetic acid, EtOH, 8h or p-methoxy phenyl hydrazine
HCl, EtOH, 8h.
Scheme 1. The synthetic pathway for compounds 2a-c, 3a-c and 4a-f
CHO
O
O
(a)
H3C
R
R
5a-c
R
app. acetophenone
Cinnamaldehyde
(d)
(b)
(c)
R
R1
R
N
N
N
N
X
O
N
N
H2N
NH
6a-c
N
7a-f
7a: R = H, X= O
7b: R= Cl, X= O
7c: R= OCH3, X= O
7d: R= H, X= S
7e: R= Cl, X= S
7f: R= OCH3, X= S
5a, 6a: R= H
5b, 6b: R= Cl
5c, 6c: R= OCH3
R1
R
8a-d
8e,f
8a: R = R1= H
8b: R= Cl, R1= H
8c: R= OCH3, R1 = H
8d: R= H, R1= OCH3
8e: R=Cl, R1= OCH3
8f: R= R1= OCH3
Reagents and conditions: (a) NaOH, EtOH, RT, 20h for 5a and 1h for 5b,c; (b) Hydrazine hydrate, glacial acetic acid 8h; (c) Semicarbazide HCl
or thiosemicarbazide, NaOH, EtOH, 8h; (d) Phenyl hydrazine, glacial acetic acid, EtOH, 8h or p-methoxy phenyl hydrazine HCl, EtOH, 8h.
Scheme 2. Synthetic pathways for compounds 6a-c, 7a-f and 8a-f.
7

2.2.
In vitro cytotoxic activity
It should be noted that although some of the targeted final compounds in the present
investigation such as 2a-c, 3a, 4a-c, 6a-c, 8a and 8c are previously reported, none of
them has been screened for potential cytotoxic activity. Therefore, they were synthesized
and screened for their cytotoxicity to clarify the SAR study. Thus, twenty-seven
synthesized compounds; 2a-c, 3a-c, 4a-f, 6a-c, 7a-f and 8a-f were evaluated for their in
vitro cytotoxicity against breast cancer cell line (MCF-7) in addition to normal fibroblast
cells (WI38) using MTT assay [23-25]. Staurosporine and erlotinib were used as
reference standards; staurosporine is a cell permeable indolocarbazole alkaloid isolated
from Streptomyces staurosporeus exhibiting an anticancer activity through a potent, non-
selective inhibition of protein kinases [26]. From the obtained results (Table 1), it can be
observed that the tested compounds revealed moderate to potent cytotoxicity against
breast cancer cell line (MCF-7) with IC50 ranged between 3.79 – 37.73 μM compared to
staurosporine and erlotinib (which showed IC50 = 10.61 and 4.74 μM, respectively).
Compounds 2a, 3a, 3c, 4a-d, 6c, 7d, 7e, 8b, 8d and 8f exhibited higher activity compared
to staurosporine, while compounds 3c, 6c, 7d, 8b and 8d elicited comparable activity to
erlotinib.
Furthermore, the following structure activity relationships could be concluded:
In the 1-acetylpyrazoline derivatives 2a-c and their positional isomers 6a-c, it was found
that the presence of styryl moiety at position 3 and phenyl ring at position 5 of the
pyrazoline ring 2a represented the highest activity (IC50 = 6.62 μM) followed by the 4-
methoxyphenyl derivative 2c and its 4-chloro analog 2b (IC50 = 17.47 and 29.19 μM,
respectively). On the other hand, changing the position of styryl moiety to position 5 of
the pyrazoline ring resulted in a decrease in the activity in case of the unsubstituted 6a
and 4-chloro derivatives 6b (IC50 = 22.01 and 31.61 μM, respectively) in comparison to
their analogs 2a and 2b. Whereas, the 4-methoxyphenyl derivative 6c revealed the
highest activity among these series with IC50 of 4.33 μM which was superior to both
sataurosporine and erlotinib.
Furthermore, the1-carboxamide pyrazoline derivative with 4-methoxyphenyl substituent
at position 5, compound 3c, displayed a significant activity (IC50 = 4.33 μM) followed by
8

the unsubstituted 3a and 4-chloro 3b congeners (IC50 = 10.59 and 16.87 μM,
respectively). The 5-styryl-1-carboxamide derivatives 7a and 7c showed a significant
decline in the cytotoxic activity (IC50 = 19.08 and 37.73 μM, respectively) compared to
their analogs 3a and 3c.The 4-chloro derivative 7b was nearly equipotent to 3b (IC50 =
15.05 and 16.87 μM). Moreover, replacing oxygen by sulfur to obtain the corresponding
carbothioamide analogs 7d-f resulted in a significant enhancement in the cytotoxic
activity (IC50 = 4.53, 7.18 and 22.40 μM, respectively) compared to their carboxamide
derivatives 7a-c which could be attributed to the increase in their lipophilicity.
Additionally, 1-phenyl/1-(4-methoxyphenyl)-3-styryl-4,5-dihydro-1H-pyrazolies 4a and
4c showed higher activity (IC50 = 6.61 and 6.14 μM, respectively) relative to their chloro
derivative 4b (IC50 = 10.20 μM). However, the presence of the styryl moiety at position 5
of the pyrazoline ring led to a decrease in the cytotoxic activity of the unsubstituted 8a
and 4-methoxyphenyl congener 8c (IC50 = 12.95 and 20.48 μM, respectively). The chloro
derivative 8b revealed the highest activity in this series comparable to 4a-c (IC50 = 3.93
μM). Finally, the 1-(4-methoxyphenyl)-5-phenyl-3-styrylpyrazoline derivative 4d
demonstrated a higher activity (IC50 = 6.66 μM) than its chloro 4e and methoxy 4f
analogs (IC50 = 18.06 and 19.51 μM, respectively). Moreover, the positional isomer 8d
elicited a superior activity (IC50 = 3.79 μM) in comparison to 4d concluding that
compound 8d was the most potent compound in comparison to both staurosporine and
erlotinib. Notably, the condensed open products 8e and 8f showed a higher activity
(IC50= 13.28 and 6.00 μM, respectively) in comparison to the cyclized pyrazoline analogs
4e and 4f (IC50 = 18.06 and 19.51 μM, respectively).
Regarding the safety of the tested compounds towards the normal cell line, compounds
eliciting higher cytotoxicity against MCF-7 than staurosporine and erlotinib (compounds
2a, 3a, 3c, 4a-d, 6c, 7d, 7e, 8b, 8d and 8f) were selected to be screened for their
cytotoxicity against normal fibroblasts (WI-38). The tested compounds were found to be
more selective to MCF-7 cancer cell line than to the normal cell line WI-38 as they
revealed cytotoxicity against normal fibroblasts WI-38 with IC50 of 11.24-54.22 μM and a
selectivity index of 1.5-11.3. Therefore, they can be considered to be safe against the
normal cell line.
9

2.3.
EGFR inhibitory activity
In order to investigate the mechanism of action of the most active compounds 3c, 6c, 7d,
8b and 8d that revealed the highest cytotoxicity against MCF-7 compared to erlotinib,
they were tested for their EGFR inhibition using erlotinib as a reference standard. The
results as IC50 (μM) are presented in Table 2.
From the obtained results, it can be noted that the EGFR inhibitory activity of the tested
compounds was consistent with their cytotoxicity except for compound 8b that exhibited
moderate EGFR inhibition (IC50 = 1.39 μM) and promising cytotoxicity (IC50= 3.93 μM).
Therefore, it may act via another mechanism in addition to the EGFR inhibition. On the
other hand, compounds 3c, 6c, 7d and 8d exhibited submicromolar inhibitory activity of
EGFR (IC50 = 0.33, 0.46, 0.34 and 0.33 μM, respectively) comparable to erlotinib (IC50 =
0.23 μM) suggesting that they may exert their cytotoxic activity via EGFR inhibition.
Table 1. Cytotoxicity of the tested compounds against MCF-7 and WI38 cell lines (IC50,
µM) and selectivity index (SI)
R
R
R
R
R
N
N
N
N
NH
N
R
N
N
N
N
R
R1
2a-c, 3a-c
X
R1
X
R1
8a-d
R1
8e,f
R1
4a-c
6a-c, 7a-f
Selectivity
index (SI)*
Cytotoxicity against (IC50, µM)
Compd. No.
R1
X
R
MCF-7
WI38
17.37±0.77
2.5
2a
H
CH3
O
6.92±0.24
NT**
NT**
25.39±1.13
NT**
NT**
2.3
NT**
3.6
3.0
2b
2c
3a
3b
3c
4a
4b
Cl
OCH3
H
Cl
OCH3
H
CH3
CH3
NH2
NH2
NH2
H
O
O
O
O
O
-
29.19±1.58
17.47±0.07
10.59±0.38
16.87±0.95
4.46±0.11
6.61±0.18
10.20±0.44
NT**
16.23±0.65
20.05±0.75
15.72±0.71
1.5
Cl
H
-
21.47±0.93
20.61±1.16
3.5
3.1
4c
4d
OCH3
H
H
OCH3
-
-
6.14±0.15
6.66±0.25
10

4e
4f
Cl
OCH3
H
Cl
OCH3
H
Cl
OCH3
H
Cl
OCH3
H
Cl
OCH3
H
Cl
OCH3
-
OCH3
OCH3
CH3
CH3
CH3
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
OCH3
OCH3
OCH3
-
-
-
18.06±0.73
19.51±1.23
22.01±0.93
31.61±1.19
4.33±0.06
19.082±0.83
15.051±0.71
37.73±1.42
4.53±0.28
7.18±0.37
22.40±1.52
12.95±0.82
3.93±0.07
20.48±1.35
3.79±0.04
13.28±0.61
6.01±0.22
10.61±0.62
4.74±0.14
NT**
NT**
NT**
NT**
NT**
NT**
NT**
11.3
NT**
NT**
NT**
8.6
6a
6b
6c
7a
7b
7c
7d
7e
7f
8a
8b
O
O
O
O
O
O
S
S
S
-
-
-
-
-
-
-
-
NT**
49.13±2.18
NT**
NT**
NT**
39.10±2.37
54.22±3.29
NT**
NT**
11.24±0.52
NT**
25.56±1.59
NT**
28.02±2.18
17.50±1.12
NT**
7.5
NT**
NT**
2.9
NT**
6.7
NT**
4.7
1.6
8c
8d
8e
8f
Staurosporine
Erlotinib
-
NT**
-
*SI = activity of the tested compounds (IC50) against normal cell line (WI-38)/ activity of the tested
compounds (IC50) against cancer cell line.
**NT: Not tested
2.4.
Molecular docking study
This study was performed for compounds 3c, 6c, 7d, 8b and 8d that were tested for their
EGFR inhibitory activity in order to validate the obtained results and investigate their
binding mode in the EGFR active site. Docking setup was first validated by self-docking
of the co-crystallized ligand (Erlotinib) in the vicinity of the binding site of the enzyme
with energy score (S) = −10.89 kcal/mol and RMSD of 1.47 Å and with the ability to
reproduce all the key interactions accomplished by the co-crystallized ligand with the key
amino acids in the binding site. H-bonding with Met769, through water mediated H-
bonding with Thr766 and cation-π interaction with Lys721 (Figures 1 and 2 in
supplementary materials).
The ability of the tested compounds to interact with the key amino acids in the binding
site rationalizes their good activity as indicated by their binding pattern and docking
score compared to that of Erlotinib (Figures 3-7 and table 2). The general binding pattern
of the tested compounds 3c, 6c, 7d and 8d is that they interact through hydrogen bonding
with the key amino acid Met769. They also interact through hydrophobic interaction by
11

their phenyl moiety with the hydrophobic side chains of the amino acids Val702, Ala719,
Met742 and Leu764.
Compound 3c, showing the best experimental EGFR inhibitory activity with IC50 of 0.33
µM, showed the highest predicted binding affinity with docking score of −11.16 kcal/mol.
Moreover, it showed a promising binding mode as it interacts through hydrogen bonding with the
key amino acid Met769 by its pyrazoline nitrogen at position 2 and with the amino acid Gln767
by its carboxamide substituent at position 1 of the pyrazoline ring. Furthermore, its 4-
methoxyphenyl moiety at position 5 of the pyrazoline ring enhances the binding affinity through
its hydrophobic interaction with the hydrophobic side chains of the amino acids Val702,
Ala719 and Leu820. On the other hand, although compound 8b showed a hydrophobic
interaction with the hydrophobic side chains of the amino acids Leu694, Val702, Met769
and Leu820 by its 4-chlorophenyl moiety at position 3 of the pyrazoline ring, its inability
to interact with the key amino acid Met769 in the active site of EGFR (Figure 6) results
in a low predicted docking score of −9.55 kcal/mol which is reflected inits moderate
experimental EGFR inhibitory activity (IC50= 1.39 µM). Thus, the obtained results
confirmed that the binding mode was consistent with the EGFR inhibitory activity of the
tested compounds.
Table 2: Docking energy scores (S) in kcal/mol of tested compounds, erlotinib, amino
acids involved in binding and their EGFR inhibitory activity (IC50µM)
Compound
Docking score
(kcal/mol)
−11.16
Amino acids involved in
binding
EGFR IC50 (µM)
3c
6c
7d
Met769 and Gln767
0.33
0.46
0.34
−10.86
−9.40
Met769 and Lys721
Met769, Gln767 and
Thr766
-
8b
8d
−9.55
−10.52
−10.89
1.39
0.33
0.23
Met769
Erlotinib
Met769 and Thr766
12

Figure 3. 2D diagram of compound 3c in the EGFR binding site.
Figure 4. 2D diagram of compound 6c in the EGFR binding site.
13

Figure 5. 2D diagram of compound 7d in the EGFR binding site.
Figure 6. 2D diagram of compound 8b in the EGFR binding site.
14

Figure 7. 2D diagram of compound 8d in the EGFR binding site.
3. Conclusion
Twenty seven 1,3,5-trisubstituted pyrazolines were prepared by condensation reaction of
the appropriate chalcone 1a-c or 5a-c with different hydrazine derivatives. Compounds
2a, 3a, 3c, 4a-d, 6c, 7d, 7e, 8b, 8d and 8f revealed higher activity than staurosporine and
they were safe to the normal fibroblasts. Moreover, compounds 3c, 6c, 7d, 8b and 8d
exerted comparable cytotoxicity and EGFR inhibition relative to erlotinib except
compound 8b which revealed moderate EGFR concluding that it may exert its
cytotoxicity via another mechanism in addition to EGFR inhibition. Molecular docking of
compounds 3c, 6c, 7d, 8b and 8d in the active site of EGFR validated the obtained
results. Therefore, 1-substituted-5-aryl-3-styryl-4,5-dihydro-1H-pyrazolines and their
positional isomers may represent interesting scaffolds for further optimization to obtain
more promising cytotoxic agents with EGFR inhibitory activity.
15

4. Experimental
4.1.
Chemistry
Melting points were detected using a Stuart SMP10 melting point apparatus. IR spectra
(KBr) were performed on a Shimadzu FT-IR 8400S spectrophotometer. NMR spectra
(CDCl3) were carried out on a Bruker Ascend 400/R (1H: 400 MHz, 13C: 100 MHz)
spectrometer. Mass spectra were measured on a GCMS-QP1000 EX spectrometer at 70
e.v. Elemental analyses were obtained utilizing FLASH 2000 CHNS/O analyzer,
Thermo Scientific. Compounds 1a-c [14-16], 2a-c [19], 3a [21], 4a-c [22], 5a-c [17,18],
6a-c, 8c [20] and 8a [14] were prepared according to the reported procedures and the
data for the reported target compounds are present in the supplementary materials.
4.1.1. General procedure for preparation of compounds 3a-c
To a mixture of semicarbazide HCl (2.0 mmole) and the appropriate chalcones (1a-c)
(2.0 mmole) in absolute ethanol (7 mL), sodium hydroxide (0.24 g, 6.0 mmole) was
added. The reaction mixture was heated under reflux for 12 h till the reaction completed.
Then, it was poured onto ice water with stirring, and the obtained precipitate was filtered,
washed with water, dried and crystallized from ethanol.
4.1.1.1.
(E)-5-(4-Chlorophenyl)-3-(4-chlorostyryl)-4,5-dihydro-1H-pyrazole-1-
carboxamide (3b)
Pale yellow crystals; yield (0.56 g, 93%); m.p. 139-140oC; IR ν (cm-1): 3450-3398 (NH2),
1
3000-3050 (C-H aromatic), 2924 (C-H aliphatic), 1670 (C=O), 1573-1404 (C=C); H
NMR (CDCl3, 400 MHz, δ, ppm): 1.79 (broad s, 2H, NH2 exchanged with D2O), 2.97
(dd, J = 5.20, 17.20 Hz, 1H, pyrazoline HA), 3.62 (dd, J = 11.60, 17.20 Hz, 1H,
pyrazoline HM), 5.43 (dd, J = 5.20, 11.60 Hz, 1H, pyrazoline HX), 6.65 (d, J = 16.40 Hz,
1H, olefinic H), 7.01 (d, J = 16.40 Hz, 1H, olefinic H), 7.16 (d, J = 8.40 Hz, 2H,
aromatic H),7.30 (d, J = 8.40 Hz, 2H, aromatic H), 7.33 (d, J = 8.80 Hz, 2H, aromatic
13
H), 7.38 (d, J = 8.40 Hz, 2H, aromatic H); C NMR (CDCl3, 100 MHz, δ, ppm): 41.6
(CH2 of pyrazoline), 59.5 (CH of pyrazoline), 121.0, 127.0, 128.1, 129.1, 129.2, 133.4,
134.2, 134.8, 135.4, 140.8, 152.6 (aromatic and olefinic carbons), 155.0 (C=O); Anal.
16

calcd. for C18H15Cl2N3O; C, 60.02; H, 4.20; N, 11.66; Found; C, 60.26; H, 4.37; N,
11.95.
4.1.1.2.
(E)-5-(4-Methoxyphenyl)-3-(4-methoxystyryl)-4,5-dihydro-1H-pyrazole-1-
carboxamide (3c)
Pale yellow crystals; yield (0.55 g, 91%); m.p. 215-216oC; IR ν (cm-1): 3498-3441 (NH2),
1
3008 (C-H aromatic), 2912 (C-H aliphatic), 1660 (C=O), 1554-1415 (C=C); H NMR
(CDCl3, 400 MHz, δ, ppm): 2.41 (broad s, 2H, NH2 exchanged with D2O), 3.02 (dd, J =
4.84, 17.16 Hz, 1H, pyrazoline HA), 3.60 (dd, J = 11.84, 17.24 Hz, 1H, pyrazoline HM),
3.79 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 5.43 (dd, J = 4.80, 11.80 Hz, 1H, pyrazoline
HX), 6.68 (d, J = 16.36 Hz, 1H, olefinic H), 6.86-6.96 (m, 5H, 4H aromatic H, 1H
olefinic H), 7.18 (d, J = 8.56 Hz, 2H, aromatic H),7.41 (d, J = 8.60 Hz, 2H, aromatic H);
13C NMR (CDCl3, 100 MHz, δ, ppm): 41.7 (CH2 of pyrazoline), 55.3 (OCH3), 55.4
(OCH3), 59.4 (CH of pyrazoline), 114.2, 114.3, 118.5, 126.6, 128.4, 128.6, 134.7, 136.2,
153.2, 155.1 (aromatic and olefinic carbons), 159.0 (C=O); MS m/z [%]: 351.12 [M+,
72.31], 307.27 [100]; Anal. calcd. for C20H21N3O3; C, 68.36; H, 6.02; N, 11.96; Found;
C, 68.59; H, 6.24; N, 12.20.
4.1.2. General procedure for preparation of compounds 4a-f
Phenyl hydrazine (0.24 g, 2.2 mmole) was added to the appropriate chalcones (1a-c) (2.0
mmole) in absolute ethanol (7 mL) and glacial acetic acid (1 mL). The reaction mixture
was heated under reflux for 12 h till the reaction completed. Then, it was poured onto ice
water with stirring; the obtained precipitate was filtered, washed with water, dried and
crystallized from ethanol to obtain compounds (4a-c). Compounds 4d-f were prepared
similarly using equimolar amounts of 4-methoxyphenylhydrazine HCl and the
appropriate chalcones (1a-c) (2.0 mmole) in absolute ethanol (7 mL).
4.1.2.1. (E)-1-(4-Methoxyphenyl)-5-phenyl-3-styryl-4,5-dihydro-1H-pyrazole (4d)
Yellow crystals; yield (0.50 g, 66%); m.p. 120-121℃; IR ν (cm-1): 3059-3001 (C-H
aromatic), 2931-2904 (C-H aliphatic), 1604-1454 (C=C); 1H NMR (CDCl3, 400 MHz, δ,
ppm): 3.03 (dd, J = 8.00, 16.80 Hz, 1H, pyrazoline HA), 3.68-3.747 (m, 4H, 1H,
17

pyrazoline HM, 3H, OCH3), 5.20 (dd, J = 8.00, 12.40 Hz, 1H, pyrazoline HX), 6.54 (d, J
= 16.40 Hz, 1H, olefinic H), 6.77 (d, J = 9.20 Hz, 2H, aromatic H), 6.98 (d, J = 8.80 Hz,
2H, aromatic H), 7.22-7.38 (m, 9H, 8H aromatic, 1H olefinic H), 7.46 (d, J = 7.20 Hz,
13
2H, aromatic H); C NMR (CDCl3, 100 MHz, δ, ppm): 45.4 (CH2 of pyrazoline), 55.5
(OCH3), 61.0 (CH of pyrazoline), 104.2, 114.5, 127.1, 127.2, 127.5, 127.9, 128.3, 128.4,
128.6, 128.7, 128.8, 129.4, 130.1, 134.4, 136.1, 160.0 (aromatic and olefinic carbons);
Anal. calcd. for C24H22N2O; C, 81.33; H, 6.26; N, 7.90; Found; C, 81.50; H, 6.53; N,
8.12.
4.1.2.2.
(E)-5-(4-Chlorophenyl)-3-(4-chlorostyryl)-1-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazole (4e)
Yellow crystals; yield (0.60 g, 86%); m.p. 150-151℃; IR ν (cm-1): 3043-3001 (C-H
aromatic), 2927-2904 (C-H aliphatic), 1508-1465 (C=C); 1H NMR (CDCl3, 400 MHz, δ,
ppm): 2.96 (dd, J = 7.44, 16.64 Hz, 1H, pyrazoline HA), 3.65-3.75 (m, 4H, 1H,
pyrazoline HM, 3H, OCH3), 5.18 (dd, J = 8.00, 10.88 Hz, 1H, pyrazoline HX), 6.47 (d, J
= 16.24 Hz, 1H, olefinic H), 6.78 (d, J = 8.88 Hz, 2H, aromatic H), 6.94 (d, J = 8.88 Hz,
2H, aromatic H), 7.18 (d, J = 16.16 Hz, 1H, olefinic H), 7.25 (d, J = 8.28 Hz, 2H,
aromatic H), 7.31-7.38 (m, 6H, aromatic H); 13C NMR (CDCl3, 100 MHz, δ, ppm): 42.2
(CH2 of pyrazoline), 55.6 (OCH3), 64.6 (CH of pyrazoline), 104.6, 114.3, 114.5, 114.9,
120.5, 126.7, 127.4, 127.6, 127.7, 128.8, 128.9, 129.0, 129.3, 129.7, 129.9, 132.5, 133.4,
133.5, 134.6, 135.5, 138.4, 140.8, 143.2, 150.4, 159.2 (aromatic and olefinic carbons);
MS m/z [%]: 423.53 [M+, 6.98], 78.12 [100]; Anal. calcd. for C24H20Cl2N2O; C, 68.09;
H, 4.76; N, 6.62; Found; C, 68.32; H, 4.98; N, 6.81.
4.1.2.3.
(E)-1,5-Bis(4-methoxyphenyl)-3-(4-methoxystyryl)-4,5-dihydro-1H-
pyrazole (4f)
Yellow crystals; yield (0.50 g, 71%); m.p.173-174℃; IR ν (cm-1): 3070-3001 (C-H
aromatic), 2931-2848 (C-H aliphatic), 1604-1465 (C=C); 1H NMR (CDCl3, 400 MHz, δ,
ppm): 2.98-3.01 (m, 1H, pyrazoline HA), 3.74-3.86 (m, 4H, 1H, pyrazoline HM, 9H, 3
OCH3), 5.18 (broad s, 1H, pyrazoline HX), 6.50 (d, J = 15.76 Hz, 1H, olefinic H), 6.77
(d, J = 8.52 Hz, 2H, aromatic H), 6.87-6.96 (m, 6H, aromatic H), 7.10 (d, J = 16.08 Hz,
18

1H, olefinic H), 7.24 (d, J = 8.48 Hz, 2H, aromatic H), 7.40 (d, J = 7.16 Hz, 2H,
aromatic H); 13C NMR (CDCl3, 100 MHz, δ, ppm): 42.5 (CH2 of pyrazoline), 55.3
(OCH3), 55.5 (OCH3), 55.6 (OCH3), 64.8 (CH of pyrazoline), 103.5, 114.0, 114.2, 114.3,
114.4, 119.7, 126.9, 127.2, 127.8, 128.1, 129.5, 130.0, 131.8, 134.6, 139.1, 144.6, 159.0,
159.6, 159.7, 159.9 (aromatic and olefinic carbons); Anal. calcd. for C26H26N2O3; C,
75.34; H, 6.32; N, 6.76; Found; C, 75.58; H, 6.59; N, 6.95.
4.1.3. General procedure for preparation of compounds 7a-f
To a mixture of semicarbazide HCl or thiosemicarbazide (2.0 mmole) and the appropriate
chalcones (5a-c) (2.0 mmole) in absolute ethanol (7 mL), sodium hydroxide (0.24 g, 6.0
mmole) was added. The reaction mixture was heated under reflux for 12 h till the reaction
completed. Then, it was poured onto ice water with stirring, and the obtained precipitate
was filtered, washed with water, dried and crystallized from ethanol.
4.1.3.1. (E)-3-Phenyl-5-styryl-4,5-dihydro-1H-pyrazole-1-carboxamide (7a)
Brown crystals; yield (0.50 g, 81%); m.p. 241-242oC; IR ν (cm-1): 3275-3205 (NH2),
3055-3024 (C-H aromatic), 2920-2850 (C-H aliphatic), 1670 (C=O), 1573-1442 (C=C);
1H NMR (CDCl3, 400 MHz, δ, ppm): 3.14 (dd, J = 5.00, 17.40 Hz, 1H, pyrazoline HA),
3.59 (dd, J = 11.60, 17.36 Hz, 1H, pyrazoline HM), 5.19-5.25 (m, 1H, pyrazoline HX),
5.52 (broad s, 2H, NH2 exchanged with D2O), 6.29 (dd, J = 6.80, 15.80 Hz, 1H, olefinic
H), 6.64 (d, J = 15.84 Hz, 1H, olefinic H), 7.21-7.31(m, 3H, aromatic H), 7.38-7.44 (m,
5H, aromatic H), 7.72-7.73 (m, 2H, aromatic H); 13C NMR (CDCl3, 100 MHz, δ, ppm):
40.0 (CH2 of pyrazoline), 58.3 (CHof pyrazoline), 126.4, 126.7, 127.8, 127.9, 128.5,
128.7, 130.1, 131.1, 131.4, 136.3, 152.3 (aromatic and olefinic carbons), 155.6 (C=O);
MS m/z [%]: 291.81 [M+, 55.76], 211.60 [100]; Anal. calcd. for C18H17N3O; C, 74.20; H,
5.88; N, 14.42; Found; C, 74.03; H, 6.09; N, 14.56.
4.1.3.2. (E)-3-(4-Chlorophenyl)-5-styryl-4,5-dihydro-1H-pyrazole-1-carboxamide (7b)
White crystals; yield (0.50 g, 83%); m.p. 250-251oC; IR ν (cm-1): 3325-3263 (NH2),
3051-3020 (C-H aromatic), 2931-2862 (C-H aliphatic), 1674 (C=O), 1589-1564 (C=C);
19

1H NMR (CDCl3, 400 MHz, δ, ppm): 3.12 (dd, J = 5.24, 17.40 Hz, 1H, pyrazoline HA),
3.58 (dd, J = 11.60, 17.40 Hz, 1H, pyrazoline HM), 5.20-5.26 (m, 1H, pyrazoline HX),
5.41 (broad s, 2H, NH2 exchanged with D2O), 6.28 (dd, J = 6.88, 15.84 Hz, 1H, olefinic
H), 6.63 (d, J = 15.84 Hz, 1H, olefinic H), 7.23 (t, J = 7.20 Hz, 1H, aromatic H), 7.31(d,
J = 7.60 Hz, 2H, aromatic H), 7.40 (t, J = 8.36 Hz, 4H, aromatic H), 7.65 (d, J = 8.56
13
Hz, 2H, aromatic H); C NMR (CDCl3, 100 MHz, δ, ppm): 39.9 (CH2 of pyrazoline),
58.5 (CH of pyrazoline), 126.7, 127.6, 127.7, 127.8, 128.5, 129.0, 130.0, 131.3, 136.0,
136.2, 151.1 (aromatic and olefinic carbons), 155.2 (C=O); MS m/z [%]: 327.18 [(M++
2), 3.04], 325.18 [M+, 10.79], 44.15 [100]; Anal. calcd. for C18H16ClN3O; C, 66.36; H,
4.95; N, 12.90; Found; C, 66.59; H, 5.12; N, 13.14.
4.1.3.3. (E)-3-(4-Methoxyphenyl)-5-styryl-4,5-dihydro-1H-pyrazole-1-carboxamide (7c)
Off white crystals; yield (0.50 g, 83%); m.p. 205-206oC; IR ν (cm-1): 3278-3197 (NH2),
3055-3028 (C-H aromatic), 2920-2846 (C-H aliphatic), 1681 (C=O), 1589-1516 (C=C);
1H NMR (CDCl3, 400 MHz, δ, ppm): 3.11 (dd, J = 5.12, 17.32 Hz, 1H, pyrazoline HA),
3.57 (dd, J = 11.52, 17.32 Hz, 1H, pyrazoline HM), 3.87 (s, 3H, OCH3), 5.17-5.23 (m,
1H, pyrazoline HX), 5.41 (broad s, 2H, NH2 exchanged with D2O), 6.29 (dd, J = 6.84,
15.84 Hz, 1H, olefinic H), 6.63 (d, J = 15.84 Hz, 1H, olefinic H), 6.95 (d, J = 8.72 Hz,
2H, aromatic H), 7.23 (t, J = 7.16 Hz, 1H, aromatic H), 7.31 (d, J = 7.28 Hz, 2H,
aromatic H), 7.39 (d, J = 7.36 Hz, 2H, aromatic H), 7.66 (d, J = 8.76 Hz, 2H, aromatic
H); 13C NMR (CDCl3, 100 MHz, δ, ppm): 40.1 (CH2 of pyrazoline), 55.4 (OCH3), 58.1
(CH of pyrazoline), 114.1, 124.1, 126.6, 127.7, 128.0, 128.1, 128.5, 131.0, 136.4, 152.0,
161.1 (aromatic and olefinic carbons), 155.5 (C=O); Anal. calcd. for C19H19N3O2; C,
71.01; H, 5.96; N, 13.08; Found; C, 71.28; H, 6.19; N, 13.23.
4.1.3.4. (E)-3-Phenyl-5-styryl-4,5-dihydro-1H-pyrazole-1-carbothioamide (7d)
Yellow crystals; yield (0.50 g, 77%); m.p. 240-241oC; IR ν (cm-1): 3495-3398 (NH2),
3059-3028 (C-H aromatic), 2920-2850 (C-H aliphatic), 1577-1446 (C=C), 1246 (C=S);
1H NMR (CDCl3, 400 MHz, δ, ppm): 3.16 (dd, J = 5.04, 17.36 Hz, 1H, pyrazoline HA),
3.61 (dd, J = 11.52, 17.36 Hz, 1H, pyrazoline HM), 5.21-5.24 (m, 1H, pyrazoline HX),
5.41 (broad s, 2H, NH2 exchanged with D2O), 6.29 (dd, J = 6.84, 15.84 Hz, 1H, olefinic
20

H), 6.64 (d, J = 15.84 Hz, 1H, olefinic H), 7.23 (t, J = 7.20 Hz, 1H, aromatic H), 7.31 (d,
J = 7.12 Hz, 2H, aromatic H),7.39 (d, J = 7.28 Hz, 2H, aromatic H), 7.44 (t, J = 7.24 Hz,
3H, aromatic H), 7.71-7.74 (m, 2H, aromatic H); 13C NMR (CDCl3, 100 MHz, δ, ppm):
40.1 (CH2 of pyrazoline), 58.4 (CH of pyrazoline), 126.4, 126.7, 127.7, 127.8, 128.5,
128.7, 130.2, 131.3, 136.3 (aromatic and olefinic carbons), 172.2 (C=S); MS m/z [%]:
307.25 [M+, 25.33], 115.84 [100]; Anal. calcd. for C18H17N3S; C, 70.33; H, 5.57; N,
13.67; Found; C, 70.49; H, 5.79; N, 13.51.
4.1.3.5. (E)-3-(4-Chlorophenyl)-5-styryl-4,5-dihydro-1H-pyrazole-1-carbothioamide (7e)
Brown crystals; yield (0.50 g, 79%); m.p. 200-201oC; IR ν (cm-1): 3471-3402 (NH2),
3059-3028 (C-H aromatic), 2927-2854 (C-H aliphatic), 1593-1554 (C=C), 1253 (C=S);
1H NMR (CDCl3, 400 MHz, δ, ppm): 3.03 (dd, J = 4.96, 17.32 Hz, 1H, pyrazoline HA),
3.49 (dd, J = 11.52, 17.28 Hz, 1H, pyrazoline HM), 5.10-5.13 (m, 1H, pyrazoline HX),
5.30 (broad s, 2H, NH2 exchanged with D2O), 6.18 (dd, J = 6.84, 15.84 Hz, 1H, olefinic
H), 6.54 (d, J = 15.76 Hz, 1H, olefinic H), 6.98-7.21 (m, 4H, aromatic H), 7.30 (t, J =
8.60 Hz, 3H, aromatic H), 7.56 (d, J = 8.52 Hz, 2H, aromatic H); 13C NMR (CDCl3, 100
MHz, δ, ppm): 45.2 (CH2 of pyrazoline), 65.2 (CH of pyrazoline), 116.8, 118.4, 126.7,
127.6, 127.8, 128.2, 128.5, 129.1, 129.5, 130.5. 135.2, 137.2 (aromatic and olefinic
carbons), 172.3 (C=S); Anal. calcd. for C18H16ClN3S; C, 63.24; H, 4.72; N, 12.29; Found;
C, 63.52; H, 4.95; N, 12.53.
4.1.3.6.
(E)-3-(4-Methoxyphenyl)-5-styryl-4,5-dihydro-1H-pyrazole-1-carbothioamide
(7f)
Brown crystals; yield (0.40 g, 63%); m.p. 205-206oC; IR ν (cm-1): 3460-3371 (NH2),
3059-3001 (C-H aromatic), 2927-2839 (C-H aliphatic), 1600-1419 (C=C), 1253 (C=S);
1H NMR (CDCl3, 400 MHz, δ, ppm): 3.12 (dd, J = 5.12, 17.28 Hz, 1H, pyrazoline HA),
3.58 (dd, J = 11.48, 17.28 Hz, 1H, pyrazoline HM), 3.87 (s, 3H, OCH3), 5.17-5.23 (m,
1H, pyrazoline HX), 5.36 (broad s, 2H, NH2 exchanged with D2O), 6.28 (dd, J = 6.88,
15.88 Hz, 1H, olefinic H), 6.63 (d, J = 15.84 Hz, 1H, olefinic H), 6.95 (d, J = 8.80 Hz,
2H, aromatic H), 7.23 (t, J = 7.20 Hz, 1H, aromatic H), 7.31 (d, J = 7.20 Hz, 2H,
aromatic H), 7.39 (d, J = 7.40 Hz, 2H, aromatic H), 7.66 (d, J = 8.76 Hz, 2H, aromatic
21

13
H); C NMR (CDCl3, 100 MHz, δ, ppm): 40.3 (CH2 of pyrazoline), 55.4 (OCH3), 58.5
(CH of pyrazoline), 114.2, 123.5, 126.7, 127.0, 128.0, 128.2, 128.5, 132.0, 136.1, 154.0,
156.1 (aromatic and olefinic carbons), 172.5 (C=S); Anal. calcd. for C19H19N3OS; C,
67.63; H, 5.68; N, 12.45; Found; C, 67.87; H, 5.80; N, 12.72.
4.1.4. General procedure for preparation of compounds 8a-f
Phenyl hydrazine (0.24 g, 2.2 mmole) was added to the appropriate chalcones (5a-c) (2.0
mmole) in absolute ethanol (7 mL) and glacial acetic acid (1 mL). The reaction mixture
was heated under reflux for 12 h till the reaction completed. Then, it was poured onto ice
water with stirring, the obtained precipitate was filtered, washed with water, dried and
crystallized from ethanol to obtain compounds (8a-c). Compounds 8d-f were prepared
similarly using equimolar amounts of 4-methoxyphenylhydrazine HCl and the
appropriate chalcones (5a-c) (2.0 mmole) in absolute ethanol (7 mL).
4.1.4.1.
(E)-3-(4-Chlorophenyl)-1-phenyl-5-styryl-4,5-dihydro-1H-pyrazole (8b)
Brown crystals; yield (0.50 g, 75%); m.p.100-101oC; IR ν (cm-1): 3059-3028 (C-H
aromatic), 2924-2854 (C-H aliphatic), 1597-1492 (C=C); 1H NMR (CDCl3, 400 MHz, δ,
ppm): 3.12 (dd, J = 6.96, 16.84 Hz, 1H, pyrazoline HA), 3.63 (dd, J = 12.00, 16.00 Hz,
1H, pyrazoline HM), 5.13-5.16 (m, 1H, pyrazoline HX), 6.15 (dd, J = 7.04, 16.00 Hz, 1H,
olefinic H), 6.92 (d, J = 16.32 Hz, 1H, olefinic H), 7.06-7.60 (m, 10H, aromatic H), 7.87
(d, J = 8.44 Hz, 2H, aromatic H), 7.94 (d, J = 8.40 Hz, 2H, aromatic H); 13C NMR
(CDCl3, 100 MHz, δ, ppm): 41.7 (CH2 of pyrazoline), 61.3 (CH of pyrazoline), 113.6,
120.1, 124.6, 125.5, 126.4, 126.6, 126.7, 127.1, 128.0, 128.1, 128.3, 128.5, 128.6, 128.8,
129.0, 129.2, 129.3, 130.1, 132.8, 146.9 (aromatic and olefinic carbons); Anal. calcd. for
C23H19ClN2; C, 76.98; H, 5.34; N, 7.81; Found; C, 76.79; H, 5.60; N, 8.05.
4.1.4.2. (E)-1-(4-Methoxyphenyl)-3-phenyl-5-styryl-4,5-dihydro-1H-pyrazole (8d)
o
Brown crystals; yield (0.40 g, 53%); m.p.105-106 C; IR ν (cm-1): 3055-3028 (C-H
aromatic), 2954-2835 (C-H aliphatic), 1512-1462 (C=C); 1H NMR (CDCl3, 400 MHz, δ,
ppm): 3.13 (dd, J = 8.40, 16.80 Hz, 1H, pyrazoline HA), 3.63 (dd, J = 11.60, 16.80 Hz,
1H, pyrazoline HM), 3.78 (s, 3H, OCH3), 4.80-4.87 (m, 1H, pyrazoline HX), 6.36 (dd, J =
7.60, 16.00 Hz, 1H, olefinic H), 6.67 (d, J = 16.00 Hz, 1H, olefinic H), 6.87 (t, J = 8.80
22

Hz, 3H, aromatic H), 7.03 (d, J = 9.20 Hz, 2H, aromatic H), 7.15-7.54 (m, 5H, aromatic
13
H), 7.74 (d, J = 7.20 Hz, 2H, aromatic H), 7.94 (d, J = 7.60 Hz, 2H, aromatic H); C
NMR (CDCl3, 100 MHz, δ, ppm): 43.6 (CH2 of pyrazoline), 55.6 (OCH3), 62.3 (CH of
pyrazoline), 100.6, 114.4, 114.6, 125.6, 126.4, 126.9, 127.3, 127.6, 128.4, 128.6, 128.8,
129.0, 129.2, 130.4, 130.9, 134.1, 135.9, 143.6, 150.7, 160.1 (aromatic and olefinic
carbons); MS m/z [%]: 354.30 [M+, 7.18], 77.11 [100]; Anal. calcd. for C24H22N2O; C,
81.33; H, 6.26; N, 7.90; Found; C, 81.19; H, 6.53; N, 8.14.
4.1.4.3.
(E)-1-((2E,4E)-1-(4-Chlorophenyl)-5-phenylpenta-2,4-dien-1-ylidene)-2-(4-
methoxyphenyl)hydrazine (8e)
Off white crystals; yield (0.50 g, 69%); m.p. 135-136oC; IR ν (cm-1): 3417 (NH), 3047-
1
3020 (C-H aromatic), 2916-2850 (C-H aliphatic), 1516-1442 (C=C); H NMR (CDCl3,
400 MHz, δ, ppm): 3.91 (s, 3H, OCH3), 6.85 (d, J= 16.28 Hz, 1H, olefinic H), 6.95 (s,
1H, NH exchanged with D2O), 7.06 (d, J = 8.42 Hz, 2H, aromatic H), 7.16 (d, J = 16.28
Hz, 1H, olefinic H), 7.30-7.45 (m, 9H, 2H olefinic H, 7H, aromatic H), 7.49 (d, J = 8.84
13
Hz, 2H, aromatic H), 7.87 (d, J = 8.52 Hz, 2H, aromatic H); C NMR (CDCl3, 100
MHz, δ, ppm): 55.6 (OCH3), 100.5, 114.4, 115.5, 126.7, 127.0, 127.1, 127.4, 128.4,
128.8, 128.9, 129.4, 129.8, 131.6, 132.4, 133.6, 136.4, 142.9, 150.5, 159.5 (aromatic and
olefinic carbons); Anal. calcd. for C24H21ClN2O; C, 74.12; H, 5.44; N, 7.20; Found; C,
74.19; H, 5.67; N, 7.33.
4.1.4.4.
(E)-1-(4-Methoxyphenyl)-2-((2E,4E)-1-(4-methoxyphenyl)-5-phenylpenta-2,4-
dien-1-ylidene)hydrazine (8f)
Brown crystals; yield (0.30 g, 42%); m.p.101-102oC; IR ν (cm-1): 3421 (NH), 3055-3001
1
(C-H aromatic), 2931-2835 (C-H aliphatic), 1600-1512 (C=C); H NMR (CDCl3, 400
MHz, δ, ppm): 3.76 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.75 (d, J = 16.80 Hz, 2H,
olefinic H), 6.81 (s, 1H, NH exchanged with D2O), 6.88 (d, J = 8.80 Hz, 2H, aromatic
H), 6.94 (d, J = 8.80 Hz, 2H, aromatic H), 7.04 (d, J = 16.40 Hz, 2H, olefinic H), 7.24-
7.34 (m, 5H, aromatic H), 7.38 (d, J = 8.80 Hz, 2H, aromatic H), 7.75 (d, J = 8.80 Hz,
13
2H, aromatic H); C NMR (CDCl3, 100 MHz, δ, ppm): 55.5 (OCH3), 55.6 (OCH3),
100.1, 113.8, 114.0, 114.2, 114.4, 115.7, 126.6, 127.1, 127.3, 127.6, 128.3, 128.8, 128.9,
23

129.1, 130.7, 131.8, 132.1, 132.7, 136.2, 141.5, 142.6, 144.0, 151.5, 159.5 (aromatic and
olefinic carbons); Anal. calcd. for C25H24N2O2; C, 78.10; H, 6.29; N, 7.29; Found; C,
78.23; H, 6.45; N, 7.50.
4.2.
In vitro Cytotoxicity activity
The cytotoxic activity screening was performed at confirmatory diagnostic unit,
VACSERA, Dokki, Cairo, Egypt. Culture medium was removed to a centrifuge tube, the
cell layer rinsed with 0.25% (w/v) 0.53 mM EDTA solution to remove all traces of serum
which contains Trypsin inhibitor, 2.0 to 3.0 mL of Trypsin EDTA solution was added to
flask and observe cells under an inverted microscope until cell layer is dispersed (usually
within 5 to 15 minutes). Followed by adding 6.0 to 8.0 mL of complete growth medium
and aspirate cells by gently pipetting, then the cell suspension is transferred to the
centrifuge tube with the medium and cells, centrifuge at approximately 125 X g for 5 to
10 minutes and the supernatant wad discarded. Moreover, the cell pellet was resuspended
in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture
vessels and incubate cultures at 37°C for 24 hrs. After treatment of cells with the serial
concentrations of the compound to be tested incubation was carried out for 48 h at 37ºC,
then the plates were examined under the inverted microscope and proceeded for the MTT
assay. The cultures were removed from incubator into laminar flow hood or other sterile
work area. Each vial of MTT [M-5655] reconstituted to be used with 3 ml of medium or
balanced salt solution without phenol red and serum. Add reconstituted MTT in an
amount equal to 10% of the culture medium volume, then the cultures were returned to
incubator for 2-4 hours depending on cell type and maximum cell density. (An incubation
period of 2 hours is generally adequate but may be lengthened for low cell densities or
cells with lower metabolic activity). Incubation times should be consistent when making
comparisons. After the incubation period, cultures were removed from incubator and the
resulting formazan crystals were dissolved by adding an amount of MTT Solubilization
Solution [M-8910] equal to the original culture medium volume. Furthermore, gentle
mixing in a gyratory shaker would enhance dissolution. Occasionally, especially in dense
cultures, pipetting up and down [trituration] may be required to completely dissolve the
MTT formazan crystals. Finally, the absorbance was measured spectrophotometrically at
24

a wavelength of 570 nm. The background absorbance of multiwell plates was measured
at 690 nm and subtracted from the 450 nm measurement. Tests performed in multiwell
plates can be read using the appropriate type of plate reader [23-25].
4.3.
EGFR inhibitory activity
It was tested at the confirmatory diagnostic unit, VACSERA, Dokki, Cairo, Egypt. The
master mixture (6 μL 5X Kinase Buffer + 1 μL ATP (500 μM) + 1 μL 50 X PTK
substrate + 17 μL water) was prepared then, 25 μL to every well was added. 5 μL of
Inhibitor solution of each well labeled as “Test Inhibitor” was added. However, for
the“Positive Control" and “Blank”, 5 μL of the same solution without inhibitor (Inhibitor
buffer) was added. 3 ml of 1X Kinase Buffer by mixing 600 μL of 5X Kinase Buffer with
2400 μL water was prepared. So, 3 ml of 1X Kinase Buffer became sufficient for 100
reactions. To the wells designated as "Blank", 20 μl of 1X Kinase Buffer was added.
EGFR enzyme on ice was thawed. Upon first thaw, briefly the tube containing enzyme
was spun to recover full content of the tube. The amount of EGFR required for the assay
and dilute enzyme to 1 ng/μL with 1X Kinase Buffer was calculated. Moreover, the
remaining undiluted enzyme in aliquots was stored at -80°C. The reaction was initiated
by adding 20 μL of diluted EGFR enzyme to the wells designated “Positive Control” and
"Test Inhibitor Control", after that it was incubated at 30°C for 40 minutes. After the 40
minutes reaction, 50 μL of Kinase-Glo Max reagent was added to each well and the plate
was covered with aluminum foil and incubated at room temperature for 15minutes.
Luminescence was measured using the microplate reader.
4.4.
Molecular docking study
All the molecular modeling studies were carried out using Molecular Operating
Environment (MOE, 2010.10) software. All minimizations were performed with MOE
until an RMSD gradient of 0.1 kcal∙mol−1Å−1 with MMFF94x force field and the partial
charges were automatically calculated. The X-ray crystallographic structure of Epidermal
Growth Factor Receptor (EGFR) co-crystallized with the 4-anilinoquinazoline derivative
(Erlotinib) (PDB ID: 1M17) was downloaded from the protein data bank [27, 28]. Water
molecules and ligands that are not involved in binding were removed. Next, the protein
25

was prepared for docking study using Protonate 3D protocol in MOE with default
options. The co-crystalized ligand (Erlotinib) was used to define the binding site for
docking. Triangle Matcher placement method and London dG scoring function were used
for docking. Docking setup was first validated by self-docking of the co-crystallized
ligand (Erlotinib) in the vicinity of the binding site of the enzyme with energy score (S) =
−10.89 kcal/mol and RMSD of 1.47 Å and with the ability to reproduce all the key
interactions accomplished by the co-crystallized ligand with the key amino acids in the
binding site. H-bonding with Met769, through water mediated H-bonding with Thr766
and cation-π interaction with Lys721 (Figures 1 and 2 in supplementary materials). The
validated setup was then used in predicting the ligands receptor interactions at the
binding site for the compounds of interest.
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29