Design, synthesis and biological evaluation of pyrazolyl-nitroimidazole derivatives as potential EGFR/HER-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.11.040

A series of novel pyrazole-nitroimidazole derivatives had been arranged and evaluated for their EGFR/HER-2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on four kinds of cancer cell lines (MCF-7, Hela, HepG2, B16-F10).

Full text of DOI:10.1016/j.bmcl.2015.11.040

Accepted Manuscript
Design, synthesis and biological evaluation of pyrazolyl-nitroimidazole deriv-
atives as potential EGFR/HER-2 kinase inhibitors
Xiang-Xiang Tao, Yong-Tao Duan, Long-Wang Chen, Dan-Jie Tang, Meng-Ru
Yang, Peng-Fei Wang, Chen Xu, Hai-Liang Zhu
PII:
S0960-894X(15)30248-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.11.040
BMCL 23296
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2015
31 October 2015
13 November 2015
Please cite this article as: Tao, X-X., Duan, Y-T., Chen, L-W., Tang, D-J., Yang, M-R., Wang, P-F., Xu, C., Zhu,
H-L., Design, synthesis and biological evaluation of pyrazolyl-nitroimidazole derivatives as potential EGFR/HER-2
kinase inhibitors, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.11.040
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Design, synthesis and biological evaluation of
pyrazolyl-nitroimidazole derivatives as potential
EGFR/HER-2 kinase inhibitors
Xiang-Xiang Tao†, Yong-Tao Duan†, Long-Wang Chen†, Dan-Jie Tang, Meng-Ru
Yang, Peng-Fei Wang, Chen Xu*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 163
Xianlin Road, Nanjing 210023, People’s Republic of China
1

Abstract
A series of novel pyrazole-nitroimidazole derivatives had been arranged and
evaluated for their EGFR/HER-2 tyrosine kinase inhibitory activity as well as their
antiproliferative properties on four kinds of cancer cell lines (MCF-7, Hela, HepG2,
B16-F10). The bioassay results showed most of the designed compounds exhibited
potential antiproliferation activity, with the IC50 values ranging from 0.13 μM to 128.06
μM in four tumor cell lines. Among them, compound 5c exhibited remarkable inhibitory
activity against EGFR/HER-2 tyrosine kinase with IC50 value of 0.26 μM / 0.51 μM
respectively, comparable to the positive control erlotinib (IC50 = 0.41 μM for HER-2 and
IC50 = 0.20 μM for EGFR) and lapatinib (IC50 = 0.54 μM for HER-2 and IC50 = 0.28 μM
for EGFR). Molecular modeling simulation studies were performed in order to predict the
biological activity of the proposed compounds and activity relationship (SAR) of these
pyrazole-nitroimidazole derivatives.
Keywords:
Pyrazole-nitroimidazole derivatives
EGFR/HER-2 inhibitors
Anticancer activity
Cytotoxicity
Molecular docking and QSAR
2

The epidermal growth factor receptor (EGFR) and the homologous HER-2/ErbB-2
kinases, which are members of the type 1 or ErbB/HER receptor tyrosine kinase (RTK)
family and closely implicated in cell proliferation, survival, adhesion, migration and
differentiation,1 have emerged as the most viable anticancer molecular targets in this
family of four members, the others being HER-3 and HER-4.2,3 The over expression or
activating mutation of EGFR and/or HER-2 have always been observed in breast, ovarian,
and non-small cell lung cancers4,5,6 making them attractive therapeutic targets in cancers
treatment.7 EGFR and HER-2 are the hottest targets in current research and their over
expression or abnormal activation often cause cell malignant transformation.8 Also they
have relationship with postoperative adverse, radiotherapy and chemotherapy resistance
and tumor angiogenesis.9 The blockade of EGFR and ErbB-2 has been clinically
validated as an attractive approach for cancer therapy.10,11
In previous study, metronidazole/secnidazole (Figure 1), kinds of nitroimidazole,
have been studied extensively for use as radiosensitizers due to their affinity for hypoxic
tumors and molecular markers of hypoxic regions in solid tumors.12 As we know,
2-Nitroimidazole derivatives, such as misonidazole and etanidazole, have been reported
to have potent radiosensitising ability.13,14 On the mechanism level, nitroimidazole
derivatives attracted considerable attention as they showed a tendency to penetrate and
accumulate in regions of tumors,15,16 and were capable of undergoing bioreduction to
yield electrophilic substances which can damage protein and nucleic acids. Our previous
investigations have provided evidence of the antitumour properties of nitroimidazole
ester derivatives and compound a (Figure 1) showed perfect antitumour activity with
IC50 value of 0.62 µM for EGFR and 2.15 µM for HER-2.17
3

Figure 1. Chemical structures of some reported compounds
Also, pyrazolines have various activities and have versatile use in medicinal
chemistry, it has been reported that anticancer compounds such as celecoxib, SC-5584,
which have 4,5-dihydropyrazoles, the core structure of numerous biologically active
compounds, always possess a wide range of bioactivities (anti-viral/anti-tumor,18,19,20
antibacterial,21,22 anti-inflammatory,23 analgesic,24 fungistatic,25 and anti-hyperglycemic
activity26). According to Richard Ducray et al. , 3 - alkoxy - 1H - pyrazolo [3, 4 - d]
pyrimidines analogues (3A - 1 - PP) (Figure 1) showed potent EGFR and HER-2
receptor tyrosine kinase inhibitory activity, with IC50 values superior to positive control
drug.27 Besides, a series of novel compounds (for example: compound b) (Figure 1)
containing the pyrazole motifs were reported as potent anticancer agents targeting EGFR
tyrosine kinase and some of them had demonstrated potent anticancer activity.28
4

So far few reports have been dedicated to design and synthesize any
anticancer/antitumor compound containing pyrazole and nitroimidazole. Herein, we
describe the synthesis and structure-activity relationship (SAR) of a series of pyrazole
derivatives containing nitroimidazole group, and present their in vitro antitumour activity
and enzyme inhibition activity. Docking simulations are performed using the X-ray
crystallographic structure of the EGFR in complex with an inhibitor to explore the
binding modes of these compounds at the active site.
A series of novel pyrazole-nitroimidazole derivatives (Table 1) were synthesized by
the routes outlined in Scheme 1. Compounds 3a - j were synthesized through the
reactions of 4-formylbenzoic acid with substituted acetophenone 2a - j in ethanol by
adding rapidly a stirred solution of NaOH in H2O at room temperature. Synthesized
compounds 3a - j were treated with hydrazine hydrate in CH3COOH which led to the
formation of compounds 4a - j. The synthesis of compounds 5a - j / 6a - j was performed
by reactions of the obtained compounds 4a - j in the presence of metronidazole /
secnidazole using EDC and DMAP as catalyst in CH2Cl2. The refined compounds were
finally obtained by subsequent purification with chromatography. All of the target
compounds gave satisfactory NMR data and analytical data, which in accordance with
their designed structures.
Table 1. Chemical structures of pyrazolyl-nitroimidazole derivatives
R1
H
R2
H
R1
OCH2CH3
OCH2CH3
Cl
R2
H
Compounds
Compounds
5a
6a
5b
6b
5f
H
CH3
H
CH3
H
6f
Br
Br
5g
6g
CH3
Cl
CH3
5

CF3
CF3
H
CH3
H
F
F
H
CH3
H
5c
6c
5d
6d
5e
6e
5h
6h
5i
OCH3
OCH3
NO2
I
CH3
H
I
CH3
H
6i
CH3
CH3
5j
6j
NO2
CH3
CH3
Scheme 1a.
aReagents and conditions: (i) Ethanol, NaOH / H2O=1:10, RT, 8h; (ii) CH3COOH, Hydrazine
Hydrate, reflux, 12h; (iii) CH2Cl2, EDC, DMAP, reflux,12 h.
Four cultured cancer cell lines: human hepatoma cells (HepG2), human breast
cancer cells (MCF-7), human cervical cancer cell line (Hela) as well as mouse melanoma
cells (B16-F10) were exploited in the anticancer assay. The test followed the MTT assay
and used erlotinib and lapatinib under identical conditions as control. The IC50 of the
compounds against these cancer cells were presented in Table 2. Results revealed that
6

some synthesized compounds exhibited potential antiproliferative activities, with the
most promising IC50 0.13 μM.
Table 2. In vitro anticancer activities (IC50, µM a) of all compounds against tumor cell lines
IC50
(µMa±
SD)
IC50
(µMa±
SD)
IC50
(µMa±
SD)
IC50
(µMa±
SD)
R1
R2
Compounds
Hela b
MCF-7 b
HepG2 b
B16-F10 b
H
H
CH3
H
48.82±0.17 58.21±1.67 42.21±4.12 62.73±3.11
45.21±1.18 49.92±0.89 43.81±1.91 68.24±2.16
5a
H
6a
Br
Br
2.82±0.08
5.56±0.13
3.82±0.27 13.09±0.07
9.02±0.12 10.76±0.20
5b
CH3
H
11.78±0.14 8.92±0.36
6b
CF3
CF3
OCH3
OCH3
NO2
NO2
OCH2CH3
OCH2CH3
Cl
0.13±0.05
0.42±0.02
0.96±0.13
2.18±0.08
0.33±0.16
2.21±0.08
0.19±0.08
5.87±0.14
5c
CH3
H
6c
27.87±0.17 30.20±0.26 15.81±0.15 36.21±0.52
35.13±1.15 27.98±0.56 25.21±0.79 40.58±0.62
5d
CH3
H
6d
0.15±0.10
3.78±0.27
1.13±0.07
3.33±0.56
1.75±0.23
0.38±0.12
5e
CH3
H
3.62±0.55 12.22±0.13
6e
31.35±0.41 20.62±0.56 19.28±0.40 38.91±1.17
47.65±1.23 40.62±1.72 38.82±0.89 46.56±1.12
5f
CH3
H
6f
4.13±0.07
2.27±0.11
3.31±0.12
4.82±0.25
5g
CH3
H
Cl
15.62±0.17 8.82±0.27 12.20±0.17 22.19±0.18
6g
F
2.45±0.11
7.42±0.51
7.53±0.21
1.67±0.05
5.21±0.21
4.85±0.17
1.89±0.03
4.19±0.10
5h
F
CH3
H
8.89±0.21 15.52±0.10
6.28±0.09 11.77±0.22
9.98±0.21 19.13±0.08
6h
I
5i
I
CH3
H
25.62±0.72 9.77±0.23
6i
5j
CH3
CH3
76.77±1.21 70.65±7.78 50.16±1.77
72.12±1.45 92.12±0.89 58.12±1.82
>100
>100
CH3
6j
0.20±0.06
0.12±0.02
0.10±0.01
0.12±0.05
0.27±0.06
0.17±0.08
0.21±0.03
Erlotinib
Lapatinib
0.47±0.03
aAntiproliferation activity was measured using the MTT assay. Values are the average of six independent experiments
run in triplicate. Variation was generally 5-10%.
bCancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University.
On the whole, these compounds exhibit stronger anticancer activities against Hela
and HepG2 cell lines. Against Hela, compounds 5e (IC50 = 0.15 µM), 6c (IC50 = 0.42 µM)
are comparable to erlotinib (IC50 = 0.20 µM). Then against B16-F10, compounds 5e (IC50
= 0.38 µM) are comparable to lapatinib (IC50 = 0.21 µM). Notably, compound 5c showed
broad-spectrum anticancer activity against all the four cell lines, with IC50 of 0.13 to 0.96
7

µM. These results suggested that compound 5c is more potent than other compounds
overall.
To enhance the antiproliferation activity against Hela or HepG2 cell lines,
electron-withdrawing groups tended to be preferable to electron-donating groups for
pyrazolyl-nitroimidazole derivatives. This conclusion is especially true when substituent
R2 comes to be constant and substituent R1 varies simultaneously, because the order of
electron-withdrawing potential is: trifluoromethyl > halogen > hydrogen > methyl and the
order of anticancer potential is: 5c > 5h > 5i > 5a > 5j.
To further illustrate the effect of substituent R2 on cell antiproliferation, we also
carried out the control comparison by maintaining substituent R1 unchanged. The results
indicated that when substituent R2 was hydrogen rather than methyl, anticancer activity
of the compound always reached a favorable level. On account of diverse nitroimidazoles
with alteration of substituent R2, the massive disparities in value of IC50 existed between
series 5 (5a - j) and series 6 (6a - j). Comparison of mean value and the most potent value
of IC50 of series 5 (5a - j) or series 6 (6a - j) against Hela and HepG2 cell lines implied
that substituent R2 determine the bioactivity of pyrazolyl-nitroimidazole derivatives to a
great extent.
To test whether the inhibition of cell growth of Hela was related to cell apoptosis,
Hela cell apoptosis induced by compound 5c was determined using flow cytometry. The
uptake of Annexin V-PE was significantly increased, and the uptake of normal cells was
significantly decreased in a density-dependent manner. Finally the percentage of total
apoptotic cells was markedly elevated in a dose-dependent manner from 24.22% to
80.9% at 48 h. As shown in Figure 2, we treated Hela cells with different doses of 5c and
found that the 0.13 μM-treated group displayed double apoptosis than control-treated
group. Meanwhile, we compare apoptosis of 0.26 μM-, 0.52μM-, and 1.04 μM-treated
groups with that of 0.13 μM-treated group, then drew the conclusion that the percentage
of apoptotic cell significantly increased after treatment with high doses of 5c.
8

Figure 2. Hela cells treated with 0.13, 0.26, 0.52 and 1.04 µM 5c for 48 hours were collected and
processed for analysis. The percentage of early apoptotic cells in the lower right quadrant (annexin
V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant
(annexin V-FITC positive/PI positive cells). Images were representative of three independent
experiments. Data were mean ± S.E.M. of three independent experiments. Values represent the mean,
n = 3. ***P < 0.001.
9

All compounds were evaluated for their abilities to inhibit the auto-phosphorylation
of EGFR and HER-2 kinases using a solid-phase activity-test kit. The inhibition constants
(IC50) of the compounds were summarized in Table 3. For the given compounds, it was
observed that the IC50 value for inhibition of EGFR kinase was higher than that observed
of HER-2 kinase. Obviously, they had the same trends. It was evident that there is also a
reasonable correlation between the EGFR and HER-2 inhibitory activities; thus, this was
not surprising in view of the high sequence homology of the catalytic domains of these
two kinases. As shown in Table 3, among the tested compounds, compound 5c showed
potent anticancer activity with IC50 of 0.26 and 0.51 μM against EGFR and HER-2
respectively, which were comparable to the positive control erlotinib (IC50 = 0.41 μM for
HER-2 and IC50 = 0.20 μM for EGFR) and lapatinib (IC50 = 0.54 μM for HER-2 and IC50
= 0.28 μM for EGFR). Other tested compounds displayed moderate inhibitory activities
with IC50 ranging from 0.60 to 25.58 μM. The results of EGFR and HER-2 kinases
inhibitory activity of the tested compounds correlated with the structural relationships
(SAR) of their inhibitory effects on the cell proliferation assay. This suggests that the
potent inhibitory effects of the synthetic compounds on the cell proliferation assay were
causally related to their kinase inhibitory activities.
Table 3. Inhibition activities of all compounds against EGFR and HER-2
IC50
(µMa±SD)
IC50
(µMa±SD)
Compounds
EGFR
HER-2
5a
6a
5b
6b
5c
6c
5d
6d
5e
6e
5f
9.58±0.24
12.24±1.13
4.13±0.20
5.96±0.82
0.26±0.03
6.67±0.11
8.54±0.55
21.31±0.52
0.60±0.02
1.45±0.06
15.16±2.86
14.04±1.12
17.00±10.77
3.45±0.25
8.27±1.01
0.51±0.06
5.62±0.33
17.25±1.63
12.05±6.33
2.06±0.09
3.53±0.08
14.93±1.02
10

6f
26.92±1.58
7.37±0.56
9.26±0.56
5.02±0.33
6.58±0.73
1.26±0.15
10.69±1.02
10.92±1.11
25.58±1.09
0.20±0.02
0.28±0.03
18.12±2.69
4.10±0.89
9.96±1.12
4.07±1.01
8.54±0.63
2.75±0.24
12.66±2.54
13.16±1.46
18.89±2.11
0.41±0.02
0.54±0.02
5g
6g
5h
6h
5i
6i
5j
6j
Erlotinib
Lapatinib
aEGFR/HER-2 inhibitory activity was measured using the MTT assay. Values are the average of six independent
experiments run in triplicate. Variation was generally 5-10%.
Six compounds selected on the basis of their antiproliferative activity were
evaluated for their toxicity against human kidney epithelial cell 293T with the median
cytotoxic concentration (CC50) data of tested compounds by the MTT assay. These
compounds were tested at multiple doses to assess the viability of 293T. As shown in
Table 4, six compounds were selected on the basis of their potent IC50 values against
EGFR/HER-2 tyrosine kinase and tumor cell lines. Judging from the median cytotoxic
concentration (CC50) data, it demonstrated that the compounds selected according to their
anticancer activity exhibit hypotoxicity in vitro against human kidney epithelial cell
293T.
Table 4. The median cytotoxic concentration (CC50) data of tested compounds
CC50, µMa
98.86
CC50, µMa
137.91
Compounds
Compounds
5g
5c
5e
5h
6c
78.83
168.27
6e
Erlotinib
117.52
92.60
104.76
Lapatinib
123.17
aThe cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50%
(CC50).
In order to gain better understanding on the potency of the 20 compounds and guide
further SAR studies, we proceeded to examine the interaction of these compounds with
11

EGFR (PDB code: 1M17) by molecular docking, which was performed by simulation of
the 20 compounds into the ATP binding site in EGFR. All docking runs were applied the
Lamarckian genetic algorithm of Auto-Dock 3.5. The obtained results were presented in
Figure 3 and Figure 4(b), which showed the optimal binding mode of compound 5c
interacting with the 1M17 protein. The amino acid residues which had interaction with
EGFR were labeled. In the binding mode, compound 5c was nicely bound to EGFR via
three charge interactions, one π-cation interaction, one hydrogen bond interaction and one
Van der Waals interaction. Lys721 formed one π-cation interaction with imidazole ring
and one charge interaction with oxygen atom of nitro group on imidazole ring. And more,
two charge interactions were found between Asp831, Glu738 and nitrogen atom of nitro
group on imidazole ring. Meanwhile, a Van der Waals interaction was formed between
oxygen atom of carbonyl group on pyrazole ring and Cys773. At last, one hydrogen bond
interaction displayed in presence of Lys692 and fluoride atom of trifluoromethyl group.
12

Figure 3. Molecular docking 3D model of the interaction between compound 5c and EGFR enzyme:
for clarity, only interacting residues are displayed.
Figure 4. (a) Molecular docking 2D model of the interaction between compound 6c and EGFR
enzyme: for clarity, only interacting residues are displayed. (b) Molecular docking 2D model of the
interaction between compound 5c and EGFR enzyme: for clarity, only interacting residues are
displayed.
In addition, the predicted binding interaction energy was used as the criterion for
ranking; the estimated interaction energies of other synthesized compounds were ranging
13

from -53.05 to -43.30 kcal/mol, as displayed in Figure 5 with histogram. The selected
compound of 5c had a best estimated binding free energy of -54.11 kcal/mol for EGFR.
These molecular docking results, along with the biological assay data, suggested that
compound 5c is a potential inhibitor of EGFR.
Figure 5. The histogram about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of all compounds
for EGFR
Besides, for further understanding the influence of substituent R2 on bioactivity of
compounds, we also compare molecular modeling result of compound 6c with that of
compound 5c. As shown in Figure 4(a), in the binding mode of compound 6c interacting
with the 1M17 protein, compound 6c was bound to EGFR via one charge interactions,
one π-cation interaction, and one hydrogen bond interaction. Obviously, interactions
between compounds 6c and 1M17 protein decreased, which in accordance with the
differences between compounds 5c and 6c in predicted binding interaction energy
presented in Figure 5.
In order to acquire a systematic SAR profile on 20 compounds as antitumor agents
and explore the more powerful EGFR inhibitors, 3D-QSAR model was built to choose
activity conformation of the designed molecular and reasonably evaluated the designed
14

molecules by using the corresponding pIC50 values which were converted from the
obtained IC50 (μM) values of EGFR inhibition and performed by built-in QSAR software
of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The way of this transformation was
derived from an online calculator developed from an Indian clinical chemistry lab
(http://www.sanjeevslab.org/tools-IC50.html). The training and test set was divided by the
random diverse molecules method of DS 3.5, in which the test set accounts for 25%
while the training set was set to 75%. The training set composed 15 agents and 5 agents
were consisted of the relative test set, which had been presented in Table 5. The success
of this model depended on docking study and the reliability of previous study about
activity data.
Table 5. The experimental and predicted inhibitory activity of all compounds by 3D-QSAR models
based upon active conformation achieved by molecular docking
EGFR
Compounds a
Residual error
Actual pIC50
Predicted pIC50
5.018
5.384
6.521
5.068
6.221
4.819
5.132
5.299
5.899
4.961
4.912
5.224
5.175
4.671
5.838
4.569
5.033
5.181
4.971
4.592
5.068
5.354
6.487
5.327
6.237
5.289
5.434
5.221
5.761
5.247
4.865
4.941
5.238
4.549
5.819
4.538
5.039
5.065
4.962
4.758
-0.050
0.030
0.034
-0.259
-0.016
-0.470
-0.302
0.078
0.138
-0.286
0.047
0.283
-0.063
0.122
0.019
0.031
-0.006
0.116
0.009
-0.166
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
aThe underlined for the test set, and the rest for training.
15

In default situation, the alignment conformation of each molecule that possessed the
lowest CDOCKER_INTERACTION_ENENGY among the ten docked poses. The
3D-QSAR model generated from DS 3.5, defined the critical regions (steric or
electrostatic) affecting the binding affinity. The graphical relationship of observed and
predicted values had been illustrated in Figure 6(c), in which the plot of the observed
IC50 versus the predicted values showed that this model could be used in prediction of
activity.
(a) & (b)
(c)
16

Figure 6. (a) Isosurface of the 3D-QSAR model coefficients on Van der Waals grids. The green
triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates
negative coefficients. (b) Isosurface of the 3D-QSAR model coefficients on electrostatic potential
grids. The blue triangle mesh represents positive electrostatic potential and the red area represents
negative electrostatic potential. (c) Using linear fitting curve to compare the predicted pIC50 value
with that of experiment.
A contour plot of the electrostatic field region favorable (in blue) or unfavorable (red)
for anticancer activity based on EGFR target were shown in Figure 6(b) while the energy
grids corresponding to the favorable (in green) or unfavorable (yellow) steric effects for
the EGFR affinity were shown in Figure 6(a). It was widely acceptable that a better
inhibitor based on the 3D-QSAR model should have strong Van der Waals attraction in
the green areas and a polar group in the blue electrostatic potential areas (which were
dominant close to the skeleton).
In summary, a series of pyrazole-nitroimidazole derivatives were synthesized and
their biological ability was evaluated for EGFR/HER-2 tyrosine kinase inhibition as well
as MTT assay in vitro. Results showed these compounds possessed potent
antiproliferative activity against EGFR/HER-2 tyrosine kinase with IC50 values in
microM range. Among them, the promising IC50 values of compound 5c against
EGFR/HER-2 tyrosine (0.26 μM / 0.51 μM) and human cervical cancer cell line (Hela)
(0.13 μM) indicated that compound 5c could be the potential EGFR/HER-2 tyrosine
kinase inhibitor and anti-tumor agent. Docking simulation was carried out to find the
probable binding models and poses by means of DS 3.5 (Discovery Studio 3.5, Accelrys,
Co. Ltd). QSAR models facilitated us to validate the reasonable design of EGFR/HER-2
inhibitors in the future.
17

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Figure Captions
Table 1. Chemical structures of pyrazole-nitroimidazole derivatives.
Table 2. In vitro anticancer activities (IC50, µM) of all compounds against tumor cell lines.
Table 3. Inhibition activities of all compounds against EGFR and HER-2.
Table 4. The median cytotoxic concentration (CC50) data of tested compounds.
Table 5. The experimental and predicted inhibitory activity of all compounds by 3D-QSAR models
based upon active conformation achieved by molecular docking
Figure 1. Chemical structures of some reported compounds.
Figure 2. Hela cells treated with 0.13, 0.26, 0.52 and 1.04 µM 5c for 48 hours were collected and
processed for analysis. The percentage of early apoptotic cells in the lower right quadrant (annexin
V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant
(annexin V-FITC positive/PI positive cells). Images were representative of three independent
experiments. Data were mean ± S.E.M. of three independent experiments. Values represent the mean,
n =4. ***P < 0.001.
Figure 3. Molecular docking 3D model of the interaction between compound 5c and EGFR enzyme:
for clarity, only interacting residues are displayed.
Figure 4. (a) Molecular docking 2D model of the interaction between compound 6c and EGFR
enzyme: for clarity, only interacting residues are displayed. (b) Molecular docking 2D model of the
interaction between compound 5c and EGFR enzyme: for clarity, only interacting residues are
displayed.
Figure 5. The histogram about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of all compounds
for EGFR
Figure 6. (a) Isosurface of the 3D-QSAR model coefficients on Van der Waals grids. The green
triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates
negative coefficients. (b) Isosurface of the 3D-QSAR model coefficients on electrostatic potential
19

grids. The blue triangle mesh represents positive electrostatic potential and the red area represents
negative electrostatic potential. (c) Using linear fitting curve to compare the predicted pIC50 value
with that of experiment.
Scheme 1a.
Graphical abstract
20