Synthesis, biological evaluation, and molecular docking studies of novel 2-styryl-5-nitroimidazole derivatives containing 1,4-benzodioxan moiety as FAK inhibitors with anticancer activity

Article abstract of DOI:10.1016/j.bmc.2014.04.005

A series of 2-styryl-5-nitroimidazole derivatives containing 1,4-benzodioxan moiety (3a-3r) has been designed, synthesized and their biological activities were also evaluated as potential antiproliferation and focal adhesion kinase (FAK) inhibitors. Among all the compounds, 3p showed the most potent activity in vitro which inhibited the growth of A549 with IC ₅₀ value of 3.11 μM and Hela with IC₅₀ value of 2.54 μM respectively. Compound 3p also exhibited significant FAK inhibitory activity (IC₅₀ = 0.45 μM). Docking simulation was performed for compound 3p into the FAK structure active site to determine the probable binding model.

Full text of DOI:10.1016/j.bmc.2014.04.005

Accepted Manuscript
Synthesis, biological evaluation, and molecular docking studies of novel 2-styr-
yl-5-nitroimidazole derivatives containing1,4-benzodioxan moiety as FAK in-
hibitors with anticancer activity
Yong-Tao Duan, Yong-Fang Yao, Wei Huang, Jigar A. Makawana, Shashikant
B. Teraiya, Nilesh j. Thumar, Dan-Jie Tang, Xiang-Xiang Tao, Zhong-Chang
Wang, Ai-Qin Jiang, Hai-Liang Zhu
PII:
S0968-0896(14)00249-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.005
BMC 11500
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 March 2014
2 April 2014
4 April 2014
Please cite this article as: Duan, Y-T., Yao, Y-F., Huang, W., Makawana, J.A., Teraiya, S.B., Thumar, N.j., Tang,
D-J., Tao, X-X., Wang, Z-C., Jiang, A-Q., Zhu, H-L., Synthesis, biological evaluation, and molecular docking
studies of novel 2-styryl-5-nitroimidazole derivatives containing1,4-benzodioxan moiety as FAK inhibitors with
anticancer activity, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.005
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Synthesis, biological evaluation, and molecular docking studies of
novel 2-styryl-5-nitroimidazole derivatives
containing1,4-benzodioxan moiety as FAK inhibitors with anticancer
activity
Yong-TaoDuan†, Yong-Fang Yao†, Wei Huang, Jigar A. Makawana, Shashikant
B. Teraiya, Nilesh j. Thumar, Dan-Jie Tang, Xiang-Xiang Tao, Zhong-Chang
Wang, Ai-Qin Jiang*,Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Medical school,
Nanjing University, Nanjing 210093, People’s Republic of China
*Corresponding author. Tel. & fax: +86-25-83592672; e-mail: zhuhl@nju.edu.cn
†These two authors equally contributed to this paper.
1

Abstract:
A
series
of
2-styryl-5-nitroimidazole
derivatives
containing1,4-benzodioxan moiety (3a-3r) has been designed, synthesized and their
biological activities were also evaluated as potential antiproliferation and focal
adhesion kinase (FAK) inhibitors. Among all the compounds, 3p showed the most
potent activity in vitro which inhibited the growth of A549 with IC50 value of 3.11 µM
and Hela with IC50 value of 2.54 µM respectively. Compound 3p also exhibited
significant FAK inhibitory activity (IC50 =0.45 µM). Docking simulation was
performed for compound 3p into the FAK structure active site to determine the
probable binding model.
Keywords:
2-styryl-5-nitroimidazole derivatives
Focal adhesion kinase
Structure–activity relationship
Molecular docking
2

1.Introduction：
Focal adhesion kinase (FAK) is a 125 kDanonreceptor tyrosinekinase that modulates
cell adhesion, migration, proliferation and survival in response to extracellular
signals.1-4 In addition to being a key player inregulating normal cellular activities such
as adhesion, migration and survival, FAK is also implicated in cancer cell invasion,
metastasis and survival.5-8Accordingly, FAK inhibition is considered as an effective
antineoplastic strategy by inducingapoptosis and sensitizing tumor cells to
chemotherapy. Different approaches to inhibit FAK with FAK antisense
oligonucleotides,9-10 dominant-negative C-terminal domain of FAK, FAK-CD or
FRNK or FAK siRNA11-12 caused decrease the cellular viability, growth inhibition or
apoptosis. Couple of FAK inhibitors with mono- and bicycic core structures have
been reported, some of them is in clinical phase 1 testing.13-17
In previous study, metronidazole is a kind of nitroimidazole moiety has been
studied extensively as radiosensitisers due to its affinity for hypoxic tumors and
molecular markers of hypoxic regions in solid tumours.18 On the mechanism level,
nitroimidazole derivatives have attracted considerable attention as they showed a
tendency to penetrate and accumulate in regions of tumors19 and can undergo
bioreduction to yield electrophilic substances which can damage protein and nucleic
acids. In 2012, Dim.et.cn reported a series of imidazole-based candidate FAK
inhibitors.20 In addition, to our knowledge, few reports available in literature which
described the synthesis and FAK inhibitory activity of compounds of modifications at
the 2-position of the nitroimidazole ring are published. It is apparent that nitro group
of metronidazole plays a key role in the metabolic activation.21 Metronidazole
derivatives substituted in the 2-methyl group may be promising for developing new,
potent and safe lead compounds.
Compounds containing 1,4-benzodioxan template also have received significant
attention in chemical, medicinal and pharmaceutical research as this structural
scaffold is found in a variety of drugs. For example, the mesylate salt of doxazosin
`isan effective drug for treatment of hypertension.22 The 6-position substituted
3

1,4-benzodioxan is known as a non-steroidal anti-inflammatory drug.23 Our research
group ever firstly reported that 1,4-benzodioxan moiety as potential anticancer agents
targeted FAK.24-26 In continuation to extend our research on antitumor compounds
with FAK inhibitory activity, herein we describe the synthesis and
structure–activityrelationships of a series of 2-styryl-5-nitroimidazole derivatives
containing 1,4-benzodioxan moiety as antitumor agents. Biological evaluation was
also carried out for screening potential FAK inhibitors of the synthesized compounds.
The apoptosis study was also carried out by flow cytometry to understand the
preliminary mechanism of synthesized compounds. Docking simulations were
performed to investigate the inhibitor interaction with FAK and explore the binding
mode of compounds at the active site.
2. Results and discussion
2.1 Chemistry
2,3-Dihydro-benzo[1,4]dioxine-5-carboxylic acid was prepared in three steps as
shown in Scheme 1. Firstly, 3,4-dihydroxybenzoic acid was catalyzed by concentrated
sulfuric acid in methanol to give methyl 3,4-dihydroxybenzoate. Secondly, treatment
of compound 1 with dibromoethane in acetone gave compound 2, which was then
saponified
(NaOH
(aq),
MeOH,
and
THF)
yielding
2,3-Dihydro-benzo[1,4]dioxine-5-carboxylic acid (compound 3). According to
method A, compounds 1a-1r were synthesized by the reaction of metronidazole with
different substituted benzaldehydes in DMSO by adding rapidly a stirred solution of
sodium methoxide in methanol at room temperature. Synthesized compounds 1a-1r
were treated with 4-methylbenzoyl chloride using triethylamineas catalyst in CH2Cl2
which led to the formation of compounds 2a-2r. The refined compounds were finally
obtained by subsequent purification using chromatography. A mixture of
2,3-Dihydro-benzo[1,4]dioxine-5-carboxylic acid, compounds 2a-2r and K2CO3 in
DMF was refluxed to provide the desired compounds 3a-3r. Meanwhile, the desired
products could be produced following another pathway depicted in method B.
Compound 5 was prepared according to the modified procedure of Kummerle et
al.27Active 2,3-Dihydro-benzo[1,4]dioxine-5-carboxylic acid and metronidazole were
4

dissolved in CH2Cl2 followed by dropwise addition triethylamine giving compound 6
with yield of 85%. The target compounds 3a-3r were synthesized by the reaction of
compound 6 with different substituted benzaldehydes in DMSO by adding rapidly a
stirred solution of sodium methoxide in methanol at room temperature. All
synthesized compounds gave satisfactory analytical and spectroscopic data which
were in full accordance with their depicted structures.
2.2. Biological activity
The inhibitory activity of the compounds is due to cell apoptosis or toxic effect, so
cytotoxicity test was performed before detecting biological activity. All synthesized
compounds were detected for their cytotoxicity on macrophages cells. The
pharmacological results of compounds were summarized in Table 1. According to the
data, it has been observed that most of the compounds were found to have low
toxicity.
2.2.1 Antiproliferative activity
All the synthesized compounds 3a-3r were screened for their in vitro antitumour
activities against two cultured cell lines (adeno carcinomic human alveolar basal
epithelial cells (A549) and human cervical cancer cell line (Hela) by CCK-8 method
as compared to the potent positive control staurosporine under identical conditions.
The results were mentioned in Table 1. According to Table 1, most of the synthesized
compounds exhibited greater anticancer activity against one or both of the cell lines
used. Among all, compounds 3p and 3q displayed the most potent anticancer
activities (IC50= 3.11, 2.54 and 5.01, 4.95 µM against A549, and Hela respectively) as
comparable to positive control staurosporine with IC50 of 3.05, 2.72 µM against A549
and Hela).
Meanwhile, a comparison of the substitution on benzene ring was demonstrated as
follows: for compounds 3a-3c, the potency order was found as meta>ortho>para in
–Cl substituted benzene ring while, the activity gradient was found as
para>meta>ortho in compounds 3d-3f containing –Br substituted benzene ring. The
potent inhibitory action order could be summarized as –OCH3< -CH3< -F/-Cl/-Br with
change in different substitution on benzene ring. Compound 3r with a bare phenyl
5

ring showed about 6-fold decreasing activity as compared to positive control.
However, compound 3r exhibited superior activity than compounds with
electron-donating groups on benzene ring and inferior than compounds with
electron-withdrawing groups.
From the above-mentioned analysis, it could be concluded that the compounds with
electron-withdrawing groups on phenyl ring were found to be the most favorable for
the antitumor activity.
2.2.2 Apoptosis analysis by Annexin V-PE fluorescence-activated cell sorting
(FACS)
To test whether the inhibition of cell growth of Hela was related to cell apoptosis,
Hela cell apoptosis induced by compound 3p 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 time-dependent manner. Finally the percentage
of early apoptotic cells was markedly elevated in a density-dependent manner from
10.60% to 4.66% at 48 h (Fig.1).
2.2.3 Invitro enzyme inhibition activity
To examine whether the compounds inhibit FAK, the FAK inhibitory potency of
compounds 3a-3r was examined and the results were summarized in Table 2. Most of
the tested compounds displayed potent FAK inhibitory activities. Among them,
compound 3p displayed the most efficient inhibitory activity with IC50 value of 0.45
µM as compared to reference drug staurosporine with IC50value of 0.50 µM. SAR
analysis result of FAK inhibitory activity of the tested compounds was consistent with
that of their anticancer activities. This consistency suggested that the potent anticancer
activities of synthesized compounds were likely related to their FAK inhibitory
activities.
2.3 Computational
2.3.1 Molecular docking
To gain better understanding on the potency of studied compounds and guide
further SAR study, we proceeded to examine the interaction of compound 3p with
FAK crystal structure (2ETM pdb). The molecular docking was performed by
6

inserting compound 3p into ATP binding site of FAK. All docking runs were applied
CDOCKER Dock protocol of Discovery Studio 3.5. The binding modes of compound
3p and FAK were depicted in Fig. 2. In the binding mode, compound 3p is potently
bound to the ATP binding site of FAK via hydrophobicinteractions and binding is
stabilized by two hydrogen bonds, one π–cation interaction and two charge
interactions. The end group of ARG426 was formed one hydrogen bond (angle
O···H–O
=
124.4°, distance
=
2.25 Å) with oxygen atom of
2,3-Dihydro-benzo[1,4]dioxine moiety. Meanwhile, another hydrogen bond (O···H–F
= 143.3°, distance = 2.31 Å) was formed between fluorine atom and LYS454. Also the
phenyl ring formed a π-cation interaction with LYS454. Besides, GLU430 and
GLU506 were respectively formed two charge interactions with two nitrogen
atoms.The docking calculation of all the compounds was also depicted in Table 2.
The CDocker Energy (energy of the ligand–receptor complexes) agreed with the FAK
inhibitory trend for all the synthesized compounds.
2.3.2 3D-QSAR model
In order to acquire a systematic SAR profile on 18 compounds as antitumor agents
and to explore the more powerful FAK inhibitors, 3D-QSAR model was built to
choose activity conformation of the designed molecular and reasonably evaluated the
designed molecules by using the corresponding pIC50 values which were converted
from the obtained IC50 (µM) values of FAK 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
dicinal chemistry lab (http://www.sanjeevslab.org/tools-IC50.html). The training and
test set was divided by the random diverse molecules method of DS3.5, in which the
test set accounted for 83% while the training set was set to 17%. The training set
composesd4 agents and 14 agents were consisted of the relative test set. The success
of this model depended on docking study and the reliability of previous study about
activities data.
In default situation, the alignment conformation of each molecule that possessed
the lowest CDOCKER_INTERACTION_ENENGY among the ten docked poses. The
7

3D-QSARmodel generated from DS 3.5, defined the critical regions (steric or
electrostatic) affecting the binding affinity. It was a PLS model that set up 230
independent variables (conventional R2 =0.84). The graphical relationship of observed
and predicted values had been illustrated in Fig. 3a, in which the plot of the observed
IC50 versus the predicted values showed that this model could be used in prediction of
activity.
A contour plot of the electrostatic field region favorable (in blue) or unfavorable
(red) for anticancer activity based on telomerase target was shown in Fig. 3b while the
energy grids corresponding to the favorable (in green) or unfavorable (yellow) steric
effects for the telomerase affinity were shown in Fig. 3c. 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). As shown in these two pictures, this
promising model would provide a guideline to design and optimize more effective
FAK inhibitors based on the a series of 2-styryl-5-nitroimidazole derivatives
containing 1,4-benzodioxan moiety and pave the way to further study in future.
3. Conclusion
In this paper, a series of 2-styryl-5-nitroimidazole derivatives containing
1,4-benzodioxan moiety has been synthesized and evaluated for their anti-cancer
activity. The bioactivity assay results showed that compound 3p exhibited the most
potent inhibitory activity for FAK with IC50 value of 0.45 µM as compared to the
positive control staurosporine as well as good activity against A549 with IC50 value of
3.11 µM and Hela with IC50 value of 2.54 µM better than the reference drug
staurosporine. The most potent inhibitor 3p was nicely bound into active site of FAK.
Also apoptosis assay results showed that compound 3p induced apoptosis of
stimulated Hela cells. Finally, QSAR models were built with previous activity data
and binding conformations to provide a reliable tool for reasonable design of FAK
inhibitors in the future.
4. Experiments
4.1 Chemistry
8

All chemicals and reagents used in the current study were of analytical grade. The
reactions were monitored by thin layer chromatography (TLC) on Merck precoated
silica GF254 plates. Melting points (uncorrected) were determined on a XT4 MP
1
apparatus (Taike Corp, Beijing, China). All the H NMR spectra were recorded on a
Bruker DPX 300 model Spectrometer in DMSO- d6 and chemical shifts were reported
in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid instrument and
were within 0.4% of the theoretical values. ESI-MS spectra were recorded on a
Mariner System 5304 Mass spectrometer.
4.2 General method of synthesis of compounds3a-3r
Protocatechuic acid (1 mmol) in methanol (30 mL) was treated with concentrated
sulfuric acid (0.5 mL) under 90ºC overnight. The sovlent was removed leaving oil
which was dissovled in ethyl acetate (20 mL) and extracted with water (40 mL). After
drying the organic layer with anhydrous Na2SO4 and evaporating the solvent under
reduced pressure a solid appeared. The solid was recyrstallized from ethanol to obtain
the compound 1. In the three-necked flask under a nitrogen atmosphere, compound
1(1 mmol) was dissolved in dry acetone (10 mL), and then added anhydrous
potassium carbonate (2 mmol). After that, the acetone solution containing
dibromomethane (1 mmol) was added dropwise then refluxed for 24 h. The reaction
solution was evaporated reduced pressure distillation. The appropriate amount of
water was added in the residue and extracted with ethyl acetate (3×40 mL). Combined
organic layer and dried with anhydrous magnesium sulfate. The solvent was removed
by reduced pressure steam to give compound 2. Compound 2 was then saponified
(NaOH (aq) : MeOH : THF = 1 : 1 : 1) at 75 ºC for two hours. Filtered and
recrystallization led to 2,3-Dihydro-benzo[1,4]dioxine-5-carboxylic acid (compound
3).
Method A: Reaction of metronidazole (12 mmol) with different substituted
benzaldehyde (16 mmol) in 6 mL DMSO by adding rapidly a stirred solution of
sodium methoxide (12.8 mmol) in methanol at room temperature resulted in the
formation of target compounds 1a-1r.To a stirred solution of compounds 1a-1r (7
mmol) in CH2Cl2 (15 mL), 4-methylbenzoyl chloride (7mmol) and triethylamine (10
9

mL) at room temperature were added and stirred for 5 hours. The precipitate was
purified by column chromatography using EtOAc/hexane (1:6) aseluent to give pure
compounds 2a-2r. Equimolar portions of the synthesized compounds 2a-2r (5mmol),
compound 3 (5mmol) and K2CO3 (5mmol) were dissolved in approximately 15 mL of
DMF. The reaction solution was allowed to stir overnightat room temperature. The
precipitate was purified by column chromatography using EtOAc/hexane (1:9) as
eluent to give the pure compounds 3a-3r.
Method B: SOCl2 (10ml) was added to a stirred solution of compound 3 (20mmol) in
anhydrous DMF (50 mL). The reaction solution was allowed to stir at room
temperature for approximately 4 h. Then, active compound 4 (15mmol) and
metronidazole (15mmol) were dissolved in CH2Cl2 followed by drop wise addition
triethylamine and compound 5 was obtained with yield of 85%. Compound 5
(10mmol), different substituted benzaldehydes (12mmol) and NaOH (15mmol) were
dissolved in DMSO (30 ml) at room temperature. The appropriate amount of water
was then added in the residue and filtered. The resulting solid was collected and
washed with cold water, dried and crystallized from anhydrous ethanol to get the
desired compounds. All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their depicted structures.
1.
(Z)-2-(2-(2-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate （3a）
yield 64.5%. m.p. 188 ~ 190 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.26 (s, 1H),
8.06 (d, J = 14.7 Hz, 1H), 7.99-7.79 (m, 1H), 7.54-7.47 (m, 2H), 7.43-7.35 (m, 2H),
7.09 (q, J = 7.9 Hz, 1H), 6.92 (q, J = 8.1 Hz, 1H), 6.52 (t, J = 7.9 Hz, 1H), 5.01 (t, J =
4.7 Hz, 2H), 4.58 (t, J = 4.7 Hz, 2H), 4.13 (s, 4H) ESI-MS: 456.09 [M+H]+.
Anal.Calcd for C22H18ClN3O6: C, H, N;
2. (Z)-2-(2-(3-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate （3b）
yield 64.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.25 （s,1H）,
8.01 (t, J = 7.1 Hz, 2H), 7.77-7.69 (m, 2H), 7.42 (t, J = 7.5 Hz, 1H), 7.17 (t, J = 7.6
10

Hz, 1H), 6.93 （d, J = 7.8 Hz, 1H）, 6.42 (d, J = 7.9 Hz, 2H), 5.06 (t, J = 4.4 Hz, 2H),
4.75 (t, J = 4.2 Hz, 2H), 4.17 (s, 4H), ESI-MS: 456.09 [M+H]+. Anal.Calcd for
C22H18ClN3O6: C, H, N;
3. (Z)-2-(2-(4-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate （3c）
yield 67.5%. m.p. 187 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.25 (s, 1H),
7.89 (s, 1H), 7.71 (d, J = 12. 3 Hz, 1H), 7.64-7.61 (m, 1H), 7.52 (d, J = 7.6 Hz, 1H),
7.42 (d, J = 6.8 Hz, 2H), 7.11 (d, J = 7.9 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.53-6.48
(m, 1H), 5.02 (t, J = 8.4 Hz, 2H), 4.57 (t, J = 8.2 Hz, 2H), 4.13 (s, 4H),ESI-MS:
456.09 [M+H]+. Anal.Calcd for C22H18ClN3O6: C, H, N;
4.
(Z)-2-(2-(2-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3d)
yield 61.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.26 (s, 1H),
7.74 (d, J = 4.5 Hz, 2H), 7.61 (d, J = 6.9 Hz, 2H), 7.47-7.32 (m, 2H), 7.13 (q, J = 4.4
Hz, 1H), 6.95 (q, J = 3.3 Hz, 1H), 6.55 (t, J = 5.9 Hz, 1H), 5.04 (t, J = 4.4 Hz, 2H),
4.59 (t, J = 4.3 Hz, 2H), 4.12 (s, 4H), ESI-MS: 502.04[M+H]+. Anal.Calcd for
C22H18BrN3O6: C, H, N;
5.
(Z)-2-(2-(3-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3e)
yield 63.5%. m.p. 186 ~ 187 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.23 (s, 1H),
7.77 (d, J = 4.4 Hz, 2H), 7.65 (d, J = 6.9 Hz, 1H), 7.50-7.35 (m, 3H), 7.16 (q, J = 4.4
Hz, 1H), 6.99 (q, J = 3.7 Hz, 1H), 6.55 (t, J = 5.9 Hz, 1H), 5.01 (t, J = 4.5 Hz, 2H),
4.56 (t, J = 4.3 Hz, 2H), 4.13 (s, 4H), ESI-MS: 502.04[M+H]+. Anal.Calcd for
C22H18BrN3O6: C, H, N;
6.
(Z)-2-(2-(4-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3f)
11

yield 62.5%. m.p. 187 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.25 (s, 1H),
7.74-7.67 (m, 3H), 7.60 (d, J = 9 Hz, 2H), 7.47 (d, J = 14.6 Hz, 1H), 7.10 (q, J = 4.0
Hz, 1H), 6.92 (q, J = 3.6 Hz, 1H), 6.52 (t, J = 7.9 Hz, 1H), 4.99 (t, J = 4.4 Hz, 2H),
4.56 (t, J = 4.5 Hz, 2H), 4.13 (s, 4H), ESI-MS: 502.04[M+H]+. Anal.Calcd for
C22H18BrN3O6: C, H, N;
7.
(Z)-2-(2-(2-fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3g)
yield 64.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.25 (s, 1H),
7.92 (t, J = 7.9 Hz, 1H), 7.83 (d, J = 6.9 Hz, 1H), 7.51-7.40 (m, 2H), 7.29-7.23 (m,
2H), 7.10 (q, J = 3.9 Hz, 1H), 6.90 (q, J = 4.0 Hz, 1H), 6.50 (t, J = 7.9 Hz, 1H), 4.99
(t, J = 4.7 Hz, 2H), 4.58 (t, J = 4.7 Hz, 2H), 4.13 (s, 4H), ESI-MS: 440.12[M+H]+.
Anal.Calcd for C22H18FN3O6: C, H, N;
8.
(Z)-2-(2-(3-fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3h)
yield 62.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.23 (s, 1H),
7.97 (d, J = 6.9 Hz, 1H), 7.86 (d, J = 6.9 Hz, 1H), 7.55-7.44 (m, 3H), 7.29 (d, J = 4.5
Hz, 1H), 7.14 (q, J = 3.3 Hz, 1H), 6.94 (q, J = 4.4 Hz, 1H), 6.56 (t, J = 7.9 Hz, 1H),
4.95 (t, J = 4.4 Hz, 2H), 4.55 (t, J = 4.3 Hz, 2H), 4.13 (s, 4H), ESI-MS:
440.12[M+H]+. Anal.Calcd for C22H18FN3O6: C, H, N;
9.
(Z)-2-(2-(4-fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3i)
yield 65.5%. m.p. 188 ~ 190 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.21 (s, 1H),
7.95 (t, J = 6.9 Hz, 1H), 7.85 (d, J = 6.9 Hz, 2H), 7.51 (d, J = 6.6 Hz, 2H), 7.29 (m,
1H), 7.14 (q, J = 3.3 Hz, 1H), 6.96 (q, J = 4.4 Hz, 1H), 6.54 (t, J = 7.7 Hz, 1H), 4.96
(t, J = 4.4 Hz, 2H), 4.58 (t, J = 4.4 Hz, 2H), 4.11 (s, 4H), ESI-MS: 440.12[M+H]+.
Anal.Calcd for C22H18FN3O6: C, H, N;
12

10.
(Z)-2-(2-(2-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3j)
yield 66.5%. m.p. 187~ 188 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.26 (s, 1H), 7.93
(t, J = 6.6 Hz, 1H), 7.83 (t, J = 6.6 Hz, 2H), 7.58 (d, J = 3.6 Hz, 1H), 7.33 (m, 2H),
7.16 (q, J = 3.6 Hz, 1H), 6.99 (q, J = 4.8 Hz, 1H), 6.57(t, J = 7.8 Hz, 1H), 4.95 (t, J =
4.4 Hz, 2H), 4.62 (t, J = 4.8 Hz, 2H), 4.12 (s, 4H), 2.32 (s, 3H),ESI-MS:
436.14[M+H]+. Anal.Calcd for C23H21N3O6: C, H, N;
11.
(Z)-2-(2-(3-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3k)
yield 62.5%. m.p. 188 ~ 190 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.24 (s, 1H),
7.90 (d, J = 6.9 Hz, 1H), 7.82 (t, J = 6.6 Hz, 2H), 7.56 (d, J = 3.6 Hz, 1H), 7.29 (m,
2H), 7.18 (q, J = 3.6 Hz, 1H), 6.94 (q, J = 4.4 Hz, 1H), 6.62 (t, J = 7.3 Hz, 1H), 4.98
(t, J = 4.8 Hz, 2H), 4.61 (t, J = 4.8 Hz, 2H), 4.13 (s, 4H), 2.25 (s, 3H), ESI-MS:
436.14[M+H]+. Anal.Calcd for C23H21N3O6: C, H, N;
12.
(Z)-2-(2-(4-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3l)
yield 68.5%. m.p. 189 ~ 190 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.27 (s, 1H),
7.85 (d, J = 12.9 Hz, 1H), 7.46-7.39 (m, 3H), 7.36-7.26 (m, 2H), 7.07 (q, J = 3.8 Hz,
1H), 6.90 (q, J = 3.3 Hz, 1H), 6.57 (t, J = 7.9 Hz, 1H), 4.87 (t, J = 7.7 Hz, 2H), 4.58 (t,
J = 4.6 Hz, 2H), 4.13 (s, 4H), 2.43 (s, 3H) ESI-MS: 436.14[M+H]+. Anal.Calcd for
C23H21N3O6: C, H, N;
13.
(Z)-2-(2-(2-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3m)
yield 66.5%. m.p. 188 ~ 190 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.21 （s, 1H）,
7.81 (d, J = 4.5 Hz, 1H), 7.41 (t, J = 6.3 Hz, 1H), 7.44-7.28 (m, 4H), 6.98 （t, J = 5.1
Hz, 2H）, 6.52 (t, J = 5.8 Hz,1H), 4.96 (t, J = 3.3, 2H),4.62 (t, J = 6.5 Hz, 2H), 4.16 (s,
4H), 3.76 (s, 3H), ESI-MS: 452.14 [M+H]+. Anal.Calcd for C23H21N3O7: C, H, N;
13

14.
(Z)-2-(2-(3-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3n)
yield 68.5%. m.p. 187 ~ 188 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.23 （s, 1H）,
7.71 (d, J = 14.5 Hz, 1H), 7.41 (d, J = 14.3 Hz, 1H), 7.34-7.25 (m, 3H), 7.10 (d, J =
7.8 Hz, 1H), 6.92 （t, J = 9.1 Hz, 2H）, 6.49 (t, J = 7.8 Hz, 1H), 4.99 (t, J = 4.3 Hz,
2H), 4.55 (t, J = 4.5 Hz, 2H), 4.11 (s, 4H), 3.77 (s, 3H), ESI-MS: 452.14 [M+H]+.
Anal.Calcd for C23H21N3O7: C, H, N;
15.
(Z)-2-(2-(4-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3o)
yield 65.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.18 （s, 1H）,
7.79 (d, J = 6.5 Hz, 2H), , 7.44-7.31 (m, 3H), 7.21 (d, J =4.8 Hz, 2H), 6.98 （t, J = 6.1
Hz, 2H）, 6.51 (t, J = 7.8 Hz, 2H), 4.97 (t, J = 4.4 Hz, 2H),4.57 (t, J = 4.5 Hz, 2H),
4.14 (s, 4H), 3.75 (s, 3H), ESI-MS: 452.14 [M+H]+. Anal.Calcd for C23H21N3O7: C, H,
N;
16.
(Z)-2-(2-(2-chloro-6-fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3p)
yield 67.5%. m.p. 187 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.27 (s, 1H),
7.85 (d, J = 12.5 Hz, 1H), 7.43-7.26 (m, 4H), 7.08-7.05 (m, 1H), 6.92-6.88 (m, 1H),
6.60-6.54 (m, 1H), 4.87 (t, J = 4.6 Hz, 2H), 4.58 (t, J = 4.6 Hz, 2H), 4.12 (s, 4H),
ESI-MS: 474.08 [M+H]+. Anal.Calcd for C22H17FClN3O6: C, H, N;
17.
(Z)-2-(2-(2-(naphthalen-1-yl)vinyl)-5-nitro-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3q)
yield 68.5%. m.p. 187 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.26 (s, 1H),
7.85 (d, J = 5.8 Hz, 1H) , 7.55-7.32 (m, 4H), 7.18-7.08 (m, 4H), 6.82 (t, J = 4.8 Hz,
2H), 6.65 (d, J = 3.9 Hz, 1H), 4.86 (t, J = 4.7 Hz, 2H), 4.57 (t, J = 4.6 Hz, 2H), 4.13 (s,
4H), ESI-MS: 472.14 [M+H]+. Anal.Calcd for C26H21N3O6: C, H, N;
14

18.
(Z)-2-(5-nitro-2-styryl-1H-imidazol-1-yl)ethyl
2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (3r)
yield 66.5%. m.p. 188 ~ 189 ℃；1H NMR (DMSO-d6, 300 MHz) δ: 8.25 (s, 1H),
7.88 (d, J = 5.5 Hz, 1H) , 7.45-7.25 (m, 4H), 7.08-7.05 (m, 3H), 6.92 (d, J = 4.4 Hz,
1H), 6.60 (d, J = 3.3 Hz, 1H), 4.89 (t, J = 4.4 Hz, 2H), 4.55 (t, J = 4.6 Hz, 2H), 4.13 (s,
4H), ESI-MS: 422.13 [M+H]+. Anal.Calcd for C22H18N3O6: C, H, N;
5. Bioassay conditions
5.1 FAK inhibitory assay
To evaluate the effect of the compounds on FAK assembly in vitro, varying
concentrations were pre-incubated with 10 µMFAK in glutamate buffer at 30 ℃and
then cooled to 0℃. After addition of GTP, the mixtures were transferred to 0℃
cuvettes in a recording spectrophotometer and warmed up to 30 ºC and the assembly
of FAK was observed turbid metrically. The IC50was defined as the compound
concentration that inhibited the extent ofassembly by 50% after 20 min incubation.
5.2 Cell proliferation assay
CCK-8 is much more convenient and helpful than MTT for analyzing cell
proliferation, because it can be reduced to soluble formazan by dehydrogenase in
mitochondria and has little toxicity to cells. Cell proliferation was determined using
CCK-8 dye (BeyotimeInst Biotech, China) according to manufacture′s instructions.
Briefly, 1-5 × 103 cells per well were seeded in a 96-well plate, grown at 37 ℃ for
12 h. Subsequently, cells were treated with the target compounds at increasing
concentrations in the presence of 10% FBS for 24 or 48 h. After 10 µL CCK-8 dye
was added to each well, cells were incubated at 37 ℃ for 1-2 h and Plates were read
in a Victor-V multilabel counter (Perkin-Elmer) using the default europium detection
protocol. Percent inhibition or IC50 values of compounds were calculated by
comparison with DMSO-treated control wells.
5.3 Flow cytometry
Cells (1.3 × 105 cells/mL) were cultured in the presence or not of novobiocin
analogues at 200 µM. Nvb at the same concentration served as reference inhibitor.
15

After treatment for 48 and 72 h, cells were washed and fixed in PBS/ ethanol (30/70).
For cytofluorometric examination, cells (104 cells/mL) were incubated for 30 min in
PBS/ Triton X100, 0.2% /EDTA 1 mM, and propidium iodide (PI) (50 µg/mL) in PBS
supplemented by RNase (0.5 mg/mL). The number of cells in the different phases of
the cell cycle was determined, and the percentage of apoptotic cells was quantified.
Analyses were performed with a FACS Calibur (Becton Dickinson, Le Pont de Claix,
France). Cell Quest software was used for data acquisition and analysis.
6. Virtual calculation
6.1 Experimental protocol of docking study
Molecular docking of compounds 3a-3r into the FAK catalytic subunit (PDB code:
2ETM) was carried out using the Auto-Dock software package (version3.5) as
implemented through the graphical user interface Auto-DockTools (ADT 1.4.6).
6.2 3D-QSAR
Ligand-based 3D-QSAR approach was performed by QSAR software of DS 3.5
(Discovery Studio 3.5, Accelrys, Co.Ltd). The training sets were composed of
inhibitors with the corresponding pIC50 values which were converted from the
obtained IC50 (µM), and test sets comprised compounds of data sets. All the definition
of the descriptors can be seen in the “Help” of DS 3.5 software and they were
calculated by QSAR protocol of DS 3.5. The alignment conformation of each
molecule was the one with lowest interaction energy in the docked results of
CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on
the cross-validated correlation coefficient, which qualifies the predictive abilityof the
models. Scrambled test (Y scrambling) was performed to investigate the risk of
chance correlations. The inhibitory potencies of compoundswererandomly reordered
for 30 times and subject to leave-one-out validation test, respectively. The models
were also validated by test sets, in which the compounds are not included in the
training sets. Usually, one can believe that the modeling is reliable, when the R2 for
test sets is larger than 0.6.
Acknowledgment
This work was supported by the project (No. BY2012136) from the Science &
16

Technology
Agency
of
Jiangsu
Province
and
by
the
projects
(Nos.CXY1213&CXY1222) from the Science
Lianyuangang City of Jiangsu Province.
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18

Legends
Figure 1.Compound 3p induced apoptosis in HELA cells with the density of 1.6, 8.0,
40.0, 200.0 µg/mL.HELA cells were treated with for 48 h.
Figure 2.Molecular docking modeling of compound 3p with tubulin: (2A) 2D Ligand
interaction diagram of compound 3p with PLK1. (2B) 3D model of the interaction
between compound 3p and the PLK1 binding site.
Figure 3.(3A) Using linear fitting curve to compare the predicted pIC50 value with
that of experiment.(3B) 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. (3C) 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.
Scheme 1.General method of the preparation of compounds 3a-3r. Reagents and
conditions: (I) methanol, concentrated sulfuric acid, 90℃, 8 h; (II) dibromoethane,
potassium carbonate, acetone, 70℃, 12 h; (III)NaOH (aq), MeOH, THF (85–95%);
Method A: (IV) Sodium methoxide, different aldehydes, DMSO, methanol, room
temperature, 4h; (V) 4-toluene sulfonyl chloride; CH2Cl2, TEA, room temperature , 5
h. (VI) 2,3-dihydro-1,4-benzodioxine-5-carboxylic acid,DMF, K2CO3, reflux,
overnight; Method B: (VII) SOCl2, DMF, reflux, 4 h; (VIII) CH2Cl2, TEA, 8 h. (IX)
NaOH, different aldehydes, DMSO, methanol, room temperature, overnight.
20

Table 1
In vitro anticancer activities (IC50, µM) of compounds 3a – 3r against human
tumorcell lines and cytotoxicity assay of the compounds on macrophages cells (CC50,
µM).
IC50
Compounds
CC50
A549
Hela
20.32
10.54
7.87
8.97
5.90
3a
18.34
12.27
13.32
14.43
20.40
19.23
15.44
17.89
10.43
6.45
3b
15.76
14.33
12.34
7.54
12.78
12.54
11.71
9.76
3c
3d
3e
3f
3g
8.21
7.10
9.09
9.90
3h
10.55
17.23
15.73
18.66
21.78
17.65
20.55
3.11
15.23
21.44
16.25
12.48
17.67
21.56
25.23
2.54
3i
3j
3k
7.67
3l
15.65
8.43
3m
3n
6.67
3o
35.53
26.55
11.43
-
3p
3q
3r
5.01
4.95
16.23
3.05
15.01
2.72
Staurosporine
21

Table 2
Fak inhibitory activity and docking calculation of compounds 3a-3r.
∆ Gb
∆ Gb
(-kcal/mol)
CDocker
Energy
43.21
IC50(µM)
IC50(µM)
(-kcal/mol)
CDocker
Energy
51.43
Compounds
Compounds
Tubulininhibitation
Tubulininhibitation
6.55
16.21
26.54
30.07
24.43
28.67
0.45
3a
3b
3c
3d
3e
3f
3k
3.42
58.34
31.45
3l
18.42
12.43
10.34
1.75
40.65
29.56
3m
46.11
33.43
3n
48.23
29.78
3o
60.31
68.71
3p
4.34
55.34
1.01
64.11
3g
3h
3i
3q
5.21
52.78
3r
Staurosporinea
20.56
0.50
38.65
8.01
50.02
-
23.54
35.32
3j
aUsed as a positive control
22

Figure 1.Compound 3p induced apoptosis in HELA cells with the density of 1.6, 8.0,
40.0, 200.0 µg/mL.HELA cells were treated with for 48 h.
PI
Annexin V-FITC
23

Figure 2.Molecular docking modeling of compound 3p with tubulin: (2A) 2D Ligand
interaction diagram of compound 3p with PLK1. (2B) 3D model of the interaction
between compound 3p and the PLK1 binding site.
2A
2B
24

Figure 3.(3A) Using linear fitting curve to compare the predicted pIC50 value with
that of experiment.(3B) 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. (3C) 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.
3A
R2 = 0.84
Pre
dica
ted
pIC
50
Actural pIC50
3B
3C
Scheme 1.General method of the preparation of compounds 3a-3r. Reagents and
25

conditions: (I) methanol, concentrated sulfuric acid, 90℃, 8 h; (II) dibromoethane,
potassium carbonate, acetone, 70℃, 12 h; (III)NaOH (aq), MeOH, THF (85–95%);
Method A: (IV) Sodium methoxide, different aldehydes, DMSO, methanol, room
temperature, 4h; (V) 4-toluene sulfonyl chloride; CH2Cl2, TEA, room temperature , 5
h. (VI) 2,3-dihydro-1,4-benzodioxine-5-carboxylic acid,DMF, K2CO3, reflux,
overnight; Method B: (VII) SOCl2, DMF, reflux, 4 h; (VIII) CH2Cl2, TEA, 8 h. (IX)
NaOH, different aldehydes, DMSO, methanol, room temperature, overnight.
COOCH3
COOH
OH
OH
HO
HO
COOCH3
O
O
O
O
I
II
III
O
OH
2
3
1
R1
HC
HC
Method A
R1
R1
HC
HC
O
HC
HC
O
N
N
C
O
O
O
O2N
N
N
HO
N
HO
N
H3C
S
O
N
N
VI
IV
V
O
O2N
O2N
1a-1r
O2N
2a-2r
3a-3r
R1
HC
HC
Method B
N
N
O
Cl
O
O
O
O
N
N
COOH
O
C
C
C
O
O
O
O2N
O
O
O2N
VIII
IX
VII
O
O
3a-3r
5
4
3a o
:
3b m
3c p
:
3d o
3e m
3f
p
3g o
:
3h m 3i p
-F;
3j o
-F; : -CH3;
R=
-Cl;
:
-Cl;
-Cl;
:
-Br;
:
-Br;
:
-Br;
-F;
:
:
3k m
: -CH3;
3l p
3m o
:
3n m
3o p
:
3p
3q
3r
:
-CH3;
-OCH3;
:
-OCH3;
-OCH3; : 2-F-6-Cl; : fused Ph ; : H
26

Synthesis, biological evaluation, and molecular docking studies of
novel 2-styryl-5-nitroimidazole derivatives containing
1,4-benzodioxan moiety as FAK inhibitors with anticancer activity
Yong-Tao Duan†, Yong-Fang Yao†, Shashikant B. Teraiya, Nilesh j. Thumar,
Dan-Jie Tang, Xiang-Xiang Tao, Ai-Qin Jiang*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
People’s Republic of China
A series of 2-styryl-5-nitroimidazole derivatives containing1,4-benzodioxan moiety
(3a-3r) has been designed, synthesized and their biological activities were also
evaluated as potential antiproliferation and focal adhesion kinase (FAK) inhibitors.
Among all the compounds, 3p showed the most potent activity in vitro which
inhibited the growth of A549 with IC50 value of 3.11 µM and Hela with IC50 value of
2.54 µM respectively. Compound 3p also exhibited significant FAK inhibitory
activity (IC50 =0.45 µM). Docking simulation was performed for compound 3p into
the FAK structure active site to determine the probable binding model.
27