Synthesis and biological evaluation of substituted N-(2-(1H-benzo[d]imidazol-2-yl)phenyl)cinnamides as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104191

A new series of N-(2-(1H-benzo[d]imidazol-2-yl)phenyl) cinnamides was prepared and evaluated for their in vitro cytotoxic activity using various cancer cell lines viz. A549 (human non-small cell lung cancer), MDA-MB-231 (human triple negative breast cancer), B16-F10 (mouse melanoma), BT-474 (human breast cancer), and 4 T1 (mouse triple negative breast cancer). In the series of tested compounds, 12h showed potent cytotoxic activity against non-small cell lung cancer cell line with IC₅₀ value of 0.29 ± 0.02 μM. The cytoxicity of most potent compound 12h was also tested on NRK-52E (normal rat kidney epithelial cell line) and showed less cytotoxicity compared to cancer cells. Tubulin polymerization assay indicated that the compound 12h was able to impede the cell division by inhibiting tubulin polymerization. Moreover, molecular docking study also suggested the binding of 12h at the colchicine-binding site of the tubulin protein. Cell cycle analysis revealed that the compound 12h arrests G2/M phase. In addition, 12h induced apoptosis in A549 cell lines was evaluated by various staining studies like acridine orange, DAPI, analysis of mitochondrial membrane potential, annexin V-FITC, and DCFDA assays.

Full text of DOI:10.1016/j.bioorg.2020.104191

BioorganicChemistry103(2020)104191
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of substituted N-(2-(1H-benzo[d]
imidazol-2-yl)phenyl)cinnamides as tubulin polymerization inhibitors
Kavitha Donthiboinaa, Pratibha Anchib, Sowmyasree Gurramb, Geeta Sai Mania,
⁎
⁎
⁎
Jaya Lakshmi Uppua, Chandraiah Godugub, , Nagula Shankaraiaha, , Ahmed Kamala,c,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
b Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
c School of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi 110062, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of N-(2-(1H-benzo[d]imidazol-2-yl)phenyl) cinnamides was prepared and evaluated for their in
vitro cytotoxic activity using various cancer cell lines viz. A549 (human non-small cell lung cancer), MDA-MB-
231 (human triple negative breast cancer), B16-F10 (mouse melanoma), BT-474 (human breast cancer), and
4 T1 (mouse triple negative breast cancer). In the series of tested compounds, 12h showed potent cytotoxic
Benzimidazole
Apoptosis
Cytotoxicity
Colchicine binding site
Flow cytometry
Reactive oxygen species
Cell migration
activity against non-small cell lung cancer cell line with IC50 value of 0.29
0.02 µM. The cytoxicity of most
potent compound 12h was also tested on NRK-52E (normal rat kidney epithelial cell line) and showed less
cytotoxicity compared to cancer cells. Tubulin polymerization assay indicated that the compound 12h was able
to impede the cell division by inhibiting tubulin polymerization. Moreover, molecular docking study also sug-
gested the binding of 12h at the colchicine-binding site of the tubulin protein. Cell cycle analysis revealed that
the compound 12h arrests G2/M phase. In addition, 12h induced apoptosis in A549 cell lines was evaluated by
various staining studies like acridine orange, DAPI, analysis of mitochondrial membrane potential, annexin V-
FITC, and DCFDA assays.
Molecular modeling
1. Introduction
broad spectrum of pharmacological activities such as antioxidant, anti-
inflammatory, antitumor, cytoprotective, antimicrobial and tyrosinase
Microtubules are crucial elements of cytoskeleton composed of α-β-
tubulin heterodimers [1]. They possess a significant role in modulating
cellular activities such as mitosis, preserving cell structure, cell sig-
naling and intracellular transport [2]. Dynamic instability of these fi-
laments contribute a decisive role in the spindle formation during mi-
tosis [3]. Hence, microtubules serve as effective target for various anti-
mitotic agents. Tubulin binding agents can be categorized based on the
binding site such as laulimalide, taxane/epothilone, vinca alkaloid and
colchicine sites [4]. Small molecules like colchicine (1), combretastatin
A-4 (2), nocodazole (3), E7010 (4) and HMN-241 (5, Fig. 1) interact
with the colchicine binding site of tubulin. These molecules disturb
microtubule formation by inhibiting tubulin assembly [5]. Over the
past decade, various molecules were designed to affect tubulin dy-
namics [6]. Most of the studies revealed that planar or biaryl config-
uration is the required feature for the molecules to bind at the colchi-
cine binding site [7].
inhibition [8]. Anti-mutagenesis property of cinnamic acid is due to its
ability to interact with cellular nucleophiles such as glutathione (GSH)
and cysteine. The presence of α,β-unsaturated carbonyl moiety is re-
sponsible for the cellular interactions [9]. Various reports proved the
antimitotic activity of cinnamides [10]. 2-Methyl cinnamide (6), cin-
namic mustard derivative of distamycin A (PNU-157911, 7, Fig. 1) are
the congeners of the cinnamic acid noted with anticancer activity [11].
Hence, cinnamic acid and its derivatives serve as fascinating moieties to
develop new molecules with anticancer property. On the other hand,
benzimidazole is a noteworthy and potent pharmacophore with various
activities like anti-inflammatory, antibacterial, antiviral, anti-
proliferative, H3 receptor-antagonistic, anti-hypertensive, anthelmintic,
antifungal, anti-HIV, and antioxidant [12]. Benzimidazole is also an
isostere of purine nucleoside, it can easily interact with biomolecules
like proteins in the living system [13]. Various natural and synthetic
benzimidazole derivatives are reported with remarkable anticancer
activity [14]. Nocodazole (3) and HOECHST 33,258 (8) with
Cinnamic acids are widely distributed as natural substances having
⁎ Corresponding authors at: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
(A. Kamal).
E-mail addresses: chandra.niperhyd@gov.in (C. Godugu), shankar@niperhyd.ac.in (N. Shankaraiah), ahmedkamal@iict.res.in (A. Kamal).
https://doi.org/10.1016/j.bioorg.2020.104191
Received 24 January 2020; Received in revised form 15 July 2020; Accepted 17 August 2020
Availableonline26August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
O
O
O
O
NH
O
H
N
S
O
O
O
HO
NH
O
O
O
N
O
2
1
3
O
H
O
S
N
O
O
O
NH2
N+
O-
HN
N
N
O
S
O
O
4
5
6
OH
OH
O
H
Cl
N
O
N
H
N
N
N
NH
N
N
Cl
8
7
distamycin A
N
Fig. 1. Representative bioactive molecules possessing anticancer activity by inhibiting tubulin.
benzimidazole moiety (Fig. 1) are well-known antineoplastic agents.
Over the past decade, rational drug design has given emphasis to
hybrids of different pharmacophoric units owing to their effect on
multiple targets and improved affinity when compared to individual
scaffolds [15]. In continuation of our interest to develop new anticancer
molecules by molecular hybridization approach [16], herein we at-
tempted to develop new hybrids of benzimidazole and cinnamic acid
(Fig. 2) resulted with N-(2-(1H-benzo[d]imidazol-2- yl)phenyl)cinna-
mides 12a–v and further screened for their in vitro cytotoxic activity.
initiated by the treatment of different substituted ortho-amino anilines
10a–e with isatoic anhydride (9) in the presence of acetic acid affords
the key intermediates 11a–e. The coupling of variously substituted
cinnamic acids 11f–k with 2-(1H-benzo[d]imidazol-2-yl)anilines 11a–e
by using EDCI and HOBt in DMF resulted the desired compounds 12a–v
in excellent yields. All the synthesized compounds were confirmed by
NMR spectroscopy and HRMS. In 1H NMR of 12a, the characteristic
amide proton was observed at δ 13.21 ppm and sharp singlet of NeH
was observed at δ 13.48 ppm. Remaining protons were observed in the
range of 8.90–6.94 ppm. In 13C NMR of 12a, the carbonyl carbon was
observed at δ 164.4 ppm, remaining signals appeared between δ 151.4
to 116.0 ppm. All the compounds 12a–v showed similar pattern of
signals in 1H as well as in 13C NMR spectra. HRMS of all the compounds
showed characteristic [M+H]+ peak equivalent to their molecular
formulae.
2. Results and discussion
2.1. Chemistry
Strategy for the synthesis of N-(2-(1H-benzo[d] imidazol-2-yl)
phenyl)cinnamides 12a–v depicted in Scheme 1. This synthesis was
2.2. Biological evaluation
2.2.1. Cytotoxic activity
Compounds 12a–v were screened for their in vitro cytotoxic po-
tential by dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay [17] against a panel of tumor cell lines viz. A549 (human
non-small cell lung cancer), MDA-MB-231 (human triple negative
breast cancer), B16-F10 (mouse melanoma), BT-474 (human breast
cancer), and 4 T1 (mouse triple negative breast cancer). Preliminary
screening was performed for the compounds at 30 µM concentration,
where the compounds exhibited > 50% inhibition of cell viability
(IC50) was further selected to generate dose response curve (DRC). The
obtained IC50 values were compared with standard nocodozole as
shown in Table 1. However, the safety of the active compound was also
evaluated in normal rat kidney epithelial cell line (NRK-52E). From the
Table 1, it can be inferred that around 30% of compounds i.e., 12b,
12d, 12h, 12j, 12 k, 12 l, 12n, 12p, 12u and 12v exhibited moderate
to potent cytotoxic activity, amongst which 12h showed most potent
cytotoxic activity with IC50 values ranging from 0.2 to 1.5 µM on dif-
ferent cancer cell lines.
Structure activity relationship (SAR) of this new series was ex-
emplified by the potency based on diversified substitution on the
phenyl ring of cinnamic acid as well as phenyl ring of benzimidazole
(Fig. 3). The results suggest that presence of electron donating group at
Fig. 2. Design of new N-(2-(1H-benzo[d]imidazol-2-yl)phenyl)cinnamides via
molecular hybridization approach.
2

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
O
R
H2N
R
N
AcOH, 90 oC,
1-3 h, 60-65%
O
N
H
H2N
N
H
9
O
H2N
10a
10b:
10c:
: R = H
11a e
-
COOH
R = CH3
R = Cl
EDCI, HOBt
DMF, rt, 5-6 h
80-85%
10d
10e
: R = F
: R = OMe
R1
11f-k
R
N
N
H
HN
O
R1
12a-v
12a: R, R1 = H
12l: R = 4-Me, :R1 = 3,4,5-tri OMe
12m: R = 4-Cl, R1 = H
12b: R = H, R1 = 2-OC2H5
12c: R = H, R1 = 4-Br
12n: R = 4-Cl, R1 = 2-OC2H5
12o: R = 4-Cl, R1 = 4-Br
12d: R = H, R1 = 2-Br,4-6-di OMe
12e: R = H, R1 = 4-Cl
12f: R = H, R1 = 3,4,5-tri OMe
12g: R = 4-Me, R1 = H
12h: R = 4-Me, R1 = 2-OC2H5
12i: R = 4-Me, R1 = 4-Br
12j: R = 4-Me, R1 = 2-Br, 4-6-di OMe
12k: R = 4-Me, R1 = 4-NO2
12p: R = 4-Cl, R1 = 2-Br,4-6-di OMe
12q: R = 4-Cl, R1 = 4-NO2
12r: R = 4-Cl, R1 = 3,4,5-tri OMe
12s: R = 4-F, R1 = 2-OC2H5
12t: R = 4-F, R1 = 3,4,5-tri OMe
12u: R = 4-OMe, R1 = 2-OC2H5
12v = 4-OMe, R1 = 4-Br
Scheme 1. Synthetic strategy for N-(2-(1H-benzo[d] imidazol-2- yl)phenyl)cinnamides 12a-v.
A and B rings are responsible for the activity. Compounds 12 s–t having
F group on the A ring are less potent and showing IC50 values > 30 μM.
Similarly, the presence of Cl group on the A ring also diminished the
activity (12n and 12p), apart from that 12 m, 12o, 12q having electron
with-drawing groups on both rings made less active on all the tested
cancer cell lines.
compounds 12f and 12r which have 3,4,5-tri OMe substitution on
phenyl ring of cinnamide diminished the activity, this might be due to
the steric hindrance.
Finally, the compound 12h was selected as the lead compound as it
has shown greater potency than standard nocodazole against the tested
tumor cell lines and was further evaluated its effect on normal cells
Compounds 12b and 12d showed moderate cytotoxicity against all
the tested cancer cell lines with IC50 values ranging from 3.4 to
21.7 μM, this is due to the presence of electron donating groups on B
ring, whereas 12a, 12c, and 12e having no substitution on A ring and
NRK-52E, the results showed less cytotoxicity (1.58
0.43 μM)
compared to cancer cells. With these encouraging results, we further
investigated the activity of 12h at cellular level on A549 cells.
electron deficient groups on
B ring leads to loss of activity.
2.2.2. Evaluation of cell morphology
Interestingly, 12j–l and 12u-v having methyl and methoxy substitution
respectively on A ring have shown significant activity. Furthermore,
12h having methyl group on A ring and 2-ethoxy on B ring was potent
with remarkable cytotoxicity against the cancer cell lines A549
Morphological examination of the cancer cell line A549 upon
treatment with the compound 12h was performed. Cells were treated
with 0.1, 0.25, 0.5 μM concentrations of compound 12h. Cells were
examined and images were captured using phase contrast microscope.
From Fig. 4, it is clearly observed that by increasing the concentration
of the compound 12h there was distinct loss of viable cells compared to
control with a significant morphological changes and cell detachment.
(0.29
(1.25
(0.49
0.02 μM), MDA-MB-231 (1.48
0.04 μM), BT-474 (0.42
0.020 μM), B16-F10
0.10 μM), and T1
4
0.14 μM). The conclusions from the SAR analysis revealed
based on IC50 values, it depicts that no substitution on the phenyl ring
of cinnamides 12a, 12 g, 12 m and 12 s resulted in loss of activity. In
addition, the presence of electron deficient groups (F, Cl, Br and NO2)
on at least one of the phenyl (benzimidazole or cinnamide) ring leads to
show lowering or loss of activity. Electron rich substitution on either of
the rings resulted in moderate to good activity, except for the
2.2.3. Acridine orange staining
While developing the new anti-neoplastic agents, apoptosis induc-
tion is considered as preferable property [18]. Hence, extent of apop-
tosis induced by the active compound 12h was evaluated by Acridine
orange (AO) staining. Acridine orange a fluorescent dye, stains nuclei
3

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
Table 1
In vitro cytotoxic (IC50 value in μM)a activity of the synthesized compounds 12a–v.
Compound
A549b
MDA-MB-231c
B16-F10d
BT-474e
4T1f
NRK-523g
12a
12b
12c
> 30
6.37
> 30
> 30
5.25
> 30
21.73
> 30
11.42
> 30
> 30
> 30
0.42
> 30
4.14
–
–
–
–
–
–
–
0.11
0.23
7.37
0.18
0.27
1.69
0.98
0.59
1.13
0.18
2.69
> 30
3.43
> 30
6.97
> 30
21.32
> 30
> 30
> 30
1.25
> 30
11.13
> 30
> 30
> 30
0.49
12d
12e
12f
> 30
> 30
> 30
0.29
> 30
> 30
> 30
1.48
12g
12h
12i
0.02
0.02
0.04
2.52
0.10
0.00
0.14
1.58
0.43
> 30
2.60
> 30
1.95
> 30
8.57
> 30
0.65
> 30
1.68
–
12j
0.19
0.09
0.17
0.12
0.97
1.48
0.68
–
12k
12l
4.78
4.32
20.09
24.59
> 30
2.08
4.18
14.58
23.85
> 30
14.44
> 30
12.47
> 30
> 30
> 30
> 30
1.53
0.16
7.90
–
18.02
> 30
7.25
0.26
25.75
> 30
2.18
3.73
0.41
0.99
2.75
1.09
1.54
0.50
2.81
–
12m
12n
12o
12p
12q
12r
> 30
6.02
–
0.58
2.06
0.11
1.76
1.27
–
> 30
8.98
> 30
7.94
> 30
13.37
> 30
> 30
> 30
> 30
1.87
> 30
26.45
> 30
> 30
> 30
> 30
1.71
–
1.63
–
> 30
> 30
> 30
> 30
1.99
> 30
> 30
> 30
> 30
0.51
–
–
12s
–
12t
-
12u
12v
Nocodazole
0.46
3.13
0.12
0.09
1.78
0.02
0.06
0.05
0.08
1.50
4.42
4.57
–
0.30
0.81
4.42
5.24
3.87
1.29
0.05
5.12
0.33
0.08
5.77
4.44
2.15
1.50
2.70
Nd
a
Concentration required for half maximal inhibition mean
SEM of three individual experiments performed in triplicate. bHuman non-small cell lung cancer cell
line. cHuman triple negative breast cancer cell line. dMouse melanoma cell line. eHuman breast cancer cell line. fMouse triple negative breast cancer cell line. gNormal
rat kidney epithelial cell line. Nd: Not determined.
electron donating substituents
increasing potency
R
N
A
N
H
HN
O
B
R1
electron rich substituents
increasing the potency
Fig. 3. SAR analysis for anticancer activity of compounds 12a–v.
green in color by diffusing through intact cell membrane. From the
Fig. 5, it can be implied that control cells exhibited normal morphology
whereas the compound 12h treated A549 cells have shown sign of early
apoptosis such as rounding of the cells (change in actual morphology of
cell) with lowest dose (0.1 µM). However, with increase in dose (0.25,
0.5 μM) cornering of nuclei (displacement of nuclei from the centre),
formation of micronuclei was observed and represented in Fig. 5.
Fig. 4. Phase contrast images of A549 cells after 24 h treatment with different
concentrations of compound 12 h and compound induced morphological
changes were observed at 200X magnification.
nuclei in a dose dependent manner where as control cells posses intact
nuclei.
2.2.4. DAPI staining
2.2.5. Measurement of reactive oxygen species by DCFDA staining
DAPI (4′,6-Diamidino-2-phenyl indole) is a fluorescent nuclear stain
that can bind A-T rich sequence of minor grove of DNA by which it can
detect nuclear morphological changes [19]. DAPI stains apoptotic cell
in bright color due to condensed nucleus, therefore DAPI staining was
performed to detect apoptosis induced by the compound 12h on the
cancer cell line A549. From Fig. 6, it was observed that compound 12h
treated cells were stained bright with fragmented, horse shoe shaped
Intrinsic apoptotic cascade initiates the raise in reactive oxygen
species (ROS) through oxidative damage of mitochondrial membrane,
collapse of membrane potential [20]. 2′,7′–Dichlorofluorescein diace-
tate (DCFDA), a fluorescent dye itself converts into non-fluorescent
compound by cellular esterases and further oxidized to highly fluor-
escent DCF by ROS generated within the cell. The extent of fluorescence
depends upon amount of ROS generated. Therefore, DCFDA assay was
4

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
mitochondrial membrane potential is the process that occur during
apoptosis [21]. Thus, effect of the compound 12h on mitochondrial
membrane potential was measured using lipophilic JC1 dye [22].
Normal polarized mitochondria possesses J aggregates and emits red to
orange colour fluorescence, whereas J monomers present in depolar-
ized mitochondria exhibits green colour fluorescence. From Fig. 8, it
was clear that there was a increase in depolarized cells (P2 population)
in a dose dependent manner, when A549 cells treated with compound
12h at 0.1, 0.25, 0.5 μM concentrations for 24h and stained with JC1
dye.
Control
0.1 μM
0.25 μM
0.5 μM
2.2.7. Scratch wound healing assay
Cancerous cells have the ability to multiply and migrate throughout
the body by intravasing into blood circulation, subsequently attaching
to a distant site and finally extravasing by making its own identity. This
invasion is currently considered as a main reason for death in cancer
patients termed by metastatic progression. Hence, to evaluate the
property of the compound 12h to inhibit metastasis or cell migration,
cell culture wound healing assay was performed on the A549 cell lines.
It can be observed from the Fig. 9, that the % wound closure was found
to be in dose dependent manner i.e., it was observed to be
Fig. 5. Non-small cell lung cancer cells were treated for 24 h with different
concentrations of compound 12 h and stained with Acridine Orange. Apoptotic
characteristics and less number of viable cells (highlighted with orange arrows)
were noticed at 200X magnification. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this
article.)
36.77
3.52, 11.44
4.16, 6.76
1.88, 4.64
0.34 for control,
0.05 µM, 0.1 µM, 0.25 µM concentrations of 12h.
2.2.8. Annexin V-FITC/Propidium iodide staining assay
Quantitative determination of the percentage of the cells under-
going apoptosis by the treatment with 12h Annexin V-FITC/PI dual
staining assay was performed. This study allows the detection of live
cells (Q1-LL; AV-/PI-), early apoptotic cells (Q2-LR; AV+/PI-), late
apoptotic cells (Q3-UR; AV+/PI+) and necrotic cells (Q4-UL; AV-/PI+).
A549 cells were treated with 0.1, 0.2 and 0.5 µM of compound 12h for
24h and stained with Annexin V-FITC/PI. From the Fig. 10, it was
observed that there was increase in the percentage of late apoptotic
cells [from 5.57% (control) to 27.37% (at 0.1 µM) 32.52% (at 0.25 µM)
and 37.05% (at 0.5 µM)] in a dose dependent manner, which suggest
that the compound 12h induced apoptosis in A549 cells.
Control
0.1 μM
2.2.9. Cell cycle analysis
Most of the anticancer agents elicit cytotoxic effect either by indu-
cing apoptosis or by inhibiting cell cycle progression [23]. To evaluate
the compound 12h induced cell cycle arrest, cell cycle analysis was
performed, where A549 cells were treated with the compound 12h at
0.1, 0.25, 0.5 μM concentrations for 24h and stained with propidium
iodide. From the Fig. 11, it was evident that compound 12h altering cell
cycle progression profoundly. Compared to DMSO treated control cells,
compound treated cells have shown more G2/M population (control:
19.5%, 0.25 μM: 29.0%, 0.5 μM: 50.3%) and less G0/G1 population
(control: 49.9%, 0.25 μM: 21.2%, 0.5 μM: 4.9%), these results indicated
that compound 12h alter the cell cycle by arresting progression at G2/
M phase.
0.5 μM
0.25 μM
Fig. 6. DAPI staining to evaluate the morphology of nucleus. A549 cells were
treated with 0.1, 0.25, 0.5 μM concentrations of compound 12 h for 24 h and
images were captured using fluorescence microscope. Appoptotic parameters
highlighted with yellow arrows. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
2.2.10. Effect of compound on tubulin polymerization
Tubulin is an essential element for the cell replication; in vitro re-
sults revealed that cytotoxic effect of the compound 12h and flow cy-
tometric analysis proved arrest of G2/M phase of the cell cycle. To
know the antimitotic activity of the compound 12h, tubulin poly-
merization assay was performed, as tubulin is the critical element in the
mitosis. The effect of compound 12h on tubulin polymerization in a cell
free in vitro tubulin assay using paclitaxel as standard at 3 μM con-
centration, in comparison with control groups illustrated in Fig. 12. The
compound 12h has shown inhibition of tubulin polymerization with
performed to evaluate the potency of the active compound 12h to in-
duce ROS. From the Fig. 7, it was observed that when the cancer cell
line A549 was treated with compound 12h, there was an increase in
generation of ROS (from increased fluorescence of 88.80 to 200.15 at
480/535 nm dose dependently) which was evident with the generated
fluorescence in comparison to control cells. This concludes that the
compound 12h was able to induce apoptosis by the generation of ROS.
IC50 value of 4.64
0.09 µM
2.2.6. Effect on mitochondrial membrane potential (ΔΨm)
Mitochondria are cellular sources of energy, plays crucial role in the
process of apoptotic events. Loss of mitochondrial membrane integrity
by the oxidative damage due to increased ROS and dissipation of
2.2.11. Molecular docking studies
Molecular docking simulation studies were performed to interpret
the mode of binding and type of interactions with active site of tubulin
5

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
0.1μM
Control
0.25 μM
0.5 μM
Fig. 7. DCFDA assay illustrating activity of compound 12h on generation of ROS at concentrations 0.1, 0.25 and 0.5 µM in A549 cancer cell line after 24 h treatment.
Fig. 8. Image representing ratio of J aggregates
(orange colour P1) to J monomers (P2 blue colour).
Fluorescence of JC-1 was analysed using Flow cyt-
ometer (BD FACSVerseTM
, USA). Here 10,000
events for each group was calculated for the ana-
lysis. (For interpretation of the references to color
in this figure legend, the reader is referred to the
web version of this article.)
(PDB ID: 3E22) [24] by using GLIDE (Grid based Ligand Docking with
Energetics) docking module of Schrödinger suite 2017–1. Glide has
been optimized for docking accuracy and also to dock prepared ligands
in a reasonable time with the active site of a receptor [29]. This study
explains that top ranked confirmation of compound 12h bind
effectively at the colchicine-binding site of the tubulin. From Fig. 13, it
can be noticed that the carbonyl oxygen of amide acts as hydrogen bond
acceptor in sequential hydrogen bonding with NH2 of Asn249
(d = 3.61 Å), Ala250 (d = 3.61 Å), Asp251 (d = 3.79 Å) of colchicine
binding site of tubulin, these interactions play a crucial role in binding.
6

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
Fig. 9. A) Images of wound closure observed after 0 and 24 hrs upon treatment with compound 12 h (at magnification 100X). B.Graph represents the calculated
percentage wound closure at various doses of 12 h. Data represented **p < 0.01 vs control. Data was analysed by one way ANOVA followed Tukeys multiple
comparision.
In addition, various hydrophobic interactions were noticed with active
site of amino acids, Val238, Cys241, Leu242, Leu248, Ala250, Leu252,
Leu255, Met259, Val315, Ala316, Ala317, Val318, Val351, Ala354, and
Ile378. These interactions stabilize the binding of potent compound
12h to the colchicine-binding site of tubulin. Based on these observa-
tions, it can be inferred that molecular docking simulation studies
proved the rationalization of tubulin inhibitory activity of most potent
compound 12h.
3. Conclusion
With an aim to develop new cytotoxic agents, a series of N-(2-(1H-
benzo[d]imidazol-2-yl)phenyl) cinnamides 12a–v was synthesized and
well characterized. Initially, these compounds were screened for the in
vitro cytotoxic activity using various cancer cell lines such as A549
(human non-small cell lung cancer), MDA-MB-231 (human triple ne-
gative breast cancer), B16-F10 (mouse melanoma), BT-474 (human
breast), and 4 T1 (mouse triple negative breast cancer). Compound 12h
Fig. 10. Annexin V-FITC/propidium iodide (PI)
dual staining assay. A549 cells were treated with
different concentrations compound 12 h, labelled
with Annexin V-FITC/PI, and observed for apop-
tosis using flow-cytometer. Cells in the lower left
quadrant (Q1-LL: AV-/PI-): live cells; lower right
quadrant (Q2-LR: AV+/PI-): early apoptotic cells;
upper right quadrant (Q3-UR: AV+/PI+): late
apoptotic cells and upper left quadrant (Q4-UL:
AV-/PI+): necrotic cells.
7

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
SUB G: 11.7
G0/G1:41.1
S: 3.7
SUB G: 6.4
G0/G1: 49.9
S: 4.8
G2/M: 17.6
G2/M: 19.5
SUB G: 13.1
G0/G1:21.2
S:4.7
SUB G: 13.1
G0/G1: 4.9
S: 4.4
G2/M:29.0
G2/M: 50.3
Fig. 11. Flow cytometric analysis of A549 cells by the treatment of compound 12 h. DNA histogram represents different stages of cell cycle i.e., SubG1, G0/G1, S, G2/
M. Cell cycle results of each phase of treated cells were compared with control cells.
expressed best cytotoxic potential against lung cancer cell line (A549)
with an IC50 value of 0.29 0.02 µM,. In vitro tubulin assay results
4. Experimental section
explained that compound 12h was able to interrupt mitosis by in-
hibiting tubulin polymerization and molecular docking study also
supported the tubulin binding property of compound 12h. Cell cycle
analysis resulted that compound 12h was able to arrest G2/M phase.
Additional studies such as acridine orange, DAPI staining, annexin V-
FITC/propidium iodide staining, analysis of mitochondrial membrane
potential and ROS assays proved compound 12h induced apoptosis.
Moreover, wound healing assay proved that compound 12h could in-
terfere migration of tumor cells. Finally, we believe that 12h could be
the lead compound for the development of chemical library that would
serve as a potential entity to treat cancer.
Commercially available starting materials, reagents and solvents
were used directly without any further purification. TLC was performed
on 0.25 mm silica gel 60-F254 plates. UV light was used to detect pro-
duct formation by visualizing spots. 1H and 13C spectra were recorded
on 500 MHz spectrometers using tetramethylsilane as internal standard.
Chemical shifts were reported in parts per million (ppm) downfield
from tetramethylsilane. Spin multiplicities were described as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet).
Coupling constants were reported in Hertz. HRMS analyses were ac-
quired on single quadruple and carried out using ESI technique 70 eV.
All molecular formulae were mentioned by adding extra proton to re-
present [M+H]+. Column chromatography was performed by using
60–120 mm silica-gel with hexane and ethyl acetate. Melting points
Fig. 12. Effect of compound 12 h on tubulin polymerization.
8

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
Fig. 13. Docking pose of the most active compound
12 h. (Yellow colour ball and stick) in the colchi-
cine-binding site of tubulin and the interacting
amino acids (PDB ID: 3E22). The red dashed lines
represent hydrogen bonds. (For interpretation of
the references to color in this figure legend, the
reader is referred to the web version of this article.)
were determined with an Electro thermal melting point apparatus and
were uncorrected.
J = 15.6 Hz, 1H). 13C NMR (125 MHz, DMSO-d6): δ 164.5, 151.4,
141.2, 139.0, 134.8, 131.2, 130.5, 129.5, 128.6, 127.8, 123.5, 123.3,
120.6, 116.0. HRMS (ESI): m/z calculated for [M+H]+ C22H18N3O:
340.1450 found 340.1453.
4.1. General procedure [25] to synthesize substituted 2-(1H-benzo[d]
imidazol-2-yl) aniline (10a–e)
4.2.2. (E)-N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(2-ethoxyphenyl)
acrylamide (12b)
To a mixture of isatoic anhydride (9, 1 mmol) and ortho-phenylene
diamine (10, 1 mmol) was added glacial acetic acid (2 mL) and allowed
to stir at 90 °C for 1–3h, reaction was monitored by TLC. After com-
pletion of the reaction, acetic acid was evaporated. Saturated solution
of NaHCO3 was used to quench the reaction mixture and extracted with
ethyl acetate (3x20 mL). The combined organic layers dried over an-
hydrous Na2SO4 filtered, concentrated and then purified by column
chromatography using ethyl acetate and hexane (15%) as eluent to
afford the corresponding 2-(1H-benzo[d]imidazol-2-yl)aniline 10a-e.
Off-white solid; Yield: 82%, 65 mg; M.p: 195–198 °C; FT-IR (cm−1):
3457.0, 3267.9, 3004.2, 2970.9, 2873.8, 1738.4, 1667.4, 1621.9,
1586.3, 1043.9; 1H NMR (500 MHz, DMSO-d6): δ 13.37 (s, 1H), 13.21
(s, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.98 (d,
J = 15.8 Hz, 1H), 7.87–7.76 (m, 2H), 7.61 (d, J = 7.3 Hz, 1H), 7.53 (t,
J = 7.6 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.36–7.25 (m, 3H), 7.13 (d,
J = 8.3 Hz, 1H), 7.05 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 15.8 Hz, 1H),
4.20 (q, J = 13.6, 6.7 Hz, 2H), 1.45 (t, J = 6.8 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6): δ 164.9, 157.9, 151.46, 142.6, 139.0, 136.9,
134.00, 131.9, 131.1, 129.7, 127.9, 124.0, 123.5, 123.4, 123.3, 122.7,
121.2, 120.6, 119.1, 115.9, 113.1, 112.0, 64.4, 15.2. HRMS (ESI): m/z
calculated for [M+H]+ C24H22N3O2 384.1712; found 384.1738.
4.2. General procedure [26] for the synthesis of substituted N-(2-(1H-benzo
[d]imidazol-2-yl)phenyl)cinnamamide (12a–v)
To a stirred solution of substituted cinnamic acids 11f-k (1 equiv.)
in dry DMF (5 mL), EDCI (1.05 equiv.), HOBt (1.05 equiv), were added
and stirred for 30 min at room temperature. Later, 2-(1H-benzo[d]
imidazol-2-yl)aniline 11a-e (1.05 equiv.) was added to the reaction
mixture and allowed to stir at room temperature until consumption of
starting material. The reaction mixture was then diluted with EtOAc
(20 mL), washed with cold H2O (2 times). The organic layer was dried
over Na2SO4 filtered and concentrated. The resulting residue was pur-
ified by column chromatography using EtOAc/Hexane to afford the
final compounds 12a-v.
4.2.3. (E)-N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(4-bromophenyl)
acrylamide (12c)
Off-white solid; Yield: 78%, 55 mg; M.p: 272–276 °C; FT-IR (cm−1):
3340.5, 3012.5, 2946.5, 1742.5, 1640, 1585, 1350, 950; 1H NMR
(500 MHz, DMSO-d6): δ 13.50 (s, 1H), 13.38 (s, 1H), 8.85 (d,
J = 7.6 Hz, 1H), 8.18 (s, 1H), 7.89–7.62 (m, 7H), 7.53 (s, 1H),
7.42–7.21 (m, 3H), 6.97 (d, J = 15.4 Hz, 1H). 13C NMR (125 MHz,
DMSO-d6): δ 164.3, 151.3, 140.2, 140.0, 138.8, 134.2, 132.4, 131.3,
131.1, 130.7, 130.6, 127.8, 124.2, 123.7, 123.6, 120.7, 116.1. HRMS
(ESI): m/z calculated for [M+H]+ C22H17N3BrO 418.0555; found
418.0585.
4.2.1. N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)cinnamamide (12a)
Off-white solid; Yield: 80%, 60 mg; M.p: 190–192 °C; FT-IR (cm−1):
3360.5, 3014.5, 2936.5, 1740.5, 1610, 1585, 1350, 1050; 1H NMR
(500 MHz, DMSO-d6): δ 13.47 (s, 1H), 13.21 (s, 1H), 8.86 (d,
J = 8.3 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 6.9 Hz, 2H),
7.75 (m, 2H), 7.57–7.41 (m, 5H), 7.3–7.24 (m, 3H), 6.93 (d,
4.2.4. (E)-N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(2-bromo-4,6-
dimethoxyphenyl)acrylamide (12d)
Off-white solid; Yield: 75%, 60 mg; M.p: 210–213 °C; FT-IR (cm−1):
3347.0, 3227.9, 3024.2, 2960.9, 2853.8, 1748.4, 1657.4, 1631.9,
9

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
1596.3, 1023.9; 1H NMR (500 MHz, DMSO-d6): δ 13.47 (s, 1H), 13.20
(s, 1H), 8.85 (d, J = 7.2 Hz, 1H), 8.21–8.12 (m, 1H), 8.02–7.82 (m,
2H), 7.74–7.49 (m, 4H), 7.38–7.23 (m, 3H), 6.96 (d, J = 15.2 Hz, 1H),
3.96 (s, 3H), 3.86 (s, 3H) ppm; 13C NMR (125 MHz, DMSO-d6): δ 168.0,
164.4, 151.8, 151.6, 151.3, 149.2, 141.9, 138.9, 138.9, 131.1, 128.3,
127.8, 127.6, 126.3, 125.8, 124.9, 124.4, 123.5, 120.8, 120.5, 119.6,
116.7, 116.5, 116.2, 116.1, 111.0, 110.7, 110.1, 56.6, 56.5. HRMS
(ESI): m/z calcd for [M+H]+ C24H21Br81N3O3 480.0766; found
480.0782.
NMR (500 MHz, DMSO): δ 13.61 (s, 1H), 12.73 (s, 1H), 8.50–8.30 (m,
2H), 8.29–8.15 (m, 3H), 7.99 (d, J = 8.3 Hz, 3H), 7.95–7.78 (m, 2H),
7.75–7.66 (m, 1H), 7.43 (t, J = 13.0 Hz, 1H), 6.75 (d, J = 16.0 Hz,
1H), 2.09 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 167.9, 164.2,
143.3, 143.0, 140.0, 138.7, 134.0, 132.4, 132.31, 130.6, 128.3, 127.7,
124.9, 124.2, 124.0, 123.7, 123.5, 120.6, 119.6, 116.1, 110.1, 21.8.
HRMS (ESI): m/z calculated for [M+H]+ C23H19Br81N3O 434.0691;
found 434.0692.
4.2.10. (E)-3-(2-Bromo-4,6-dimethoxyphenyl)-N-(2-(5-methyl-1H-benzo
[d]imidazol-2-yl)phenyl)acrylamide(12j)
4.2.5. (E)-N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(4-nitrophenyl)
acrylamide (12e)
Off-white solid; Yield: 70%, 50 mg; M.p: 268–279 °C; FT-IR (cm−1):
3265.7, 3006.5, 2970.7, 1738.8, 1669.5, 1590.7, 1433.7, 1260.2; 1H
NMR (500 MHz, DMSO-d6): δ 13.52 (bs, 1H), 13.05 (bs, 1H), 8.83 (t,
J = 9.4 Hz, 1H), 8.14 (d, J = 7.7 Hz, 1H), 7.93 (dd, J = 12.5, 6.7 Hz,
1H), 7.76–7.63 (m, 1H), 7.56 (s, 1H), 7.54–7.45 (m, 1H), 7.38 (s, 1H),
7.34–7.26 (m, 2H), 7.17–7.07 (m, 1H), 6.94 (d, J = 15.5 Hz, 1H), 3.96
(s, 3H), 3.85 (s, 3H), 2.44 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ
164.4, 151.6, 149.2, 149.2, 138.8, 138.8, 130.9, 127.6, 126.3, 126.1,
124.50, 124.4, 123.5, 120.7, 116.5, 116.2, 116.1, 116.0, 110.9, 110.6,
56.6, 56.4, 21.8. HRMS (ESI): m/z calcd for [M+H]+ C25H23BrN3O3
492.0923; found 492.0925.
Yellow solid; Yield: 79%, 58 mg; M.p: 283–285 °C; FT-IR (cm−1):
3457.0, 3016.5, 3004.2, 2970.9, 1738.4, 1668.7, 1590.4, 1349.5,
1052.4; 1H NMR (500 MHz, DMSO-d6): δ 13.60 (s, 1H), 13.23 (s, 1H),
8.85 (d, J = 7.7 Hz, 1H), 8.34–8.08 (m, 5H), 7.94 (s, 1H), 7.84 (d,
J = 15.8 Hz, 1H), 7.65–7.50 (m, 2H), 7.33 (s, 3H), 7.15 (d,
J = 15.7 Hz, 1H). 13C NMR (125 MHz, DMSO-d6): δ 163.6, 151.2,
148.3, 141.5, 138.8, 138.7, 131.2, 129.7, 127.8, 127.6, 124.5, 123.8,
122.7, 120.7, 119.5, 116.2, 112.0. HRMS (ESI): m/z calcd for [M+H]+
C22H17N4O3 385.1301; found 385.1320.
4.2.6. (E)-N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (12f)
White solid; Yield: 80%, 62 mg; M.p: 252–255 °C; FT-IR (cm−1):
3456.9, 3204.5, 3004.3, 2970.9, 1738.8, 1621.9, 1584.5, 1366.2,
1217.1, 1043.9; 1H NMR (500 MHz, DMSO-d6): δ 13.44 (s, 1H), 13.21
(s, 1H), 8.85 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.90 (d,
J = 6.9 Hz, 1H), 7.75–7.46 (m, 4H), 7.32 (s, 2H), 7.15 (s, 2H), 6.93 (d,
J = 15.8 Hz, 1H), 3.91 (s, 6H), 3.74 (s, 3H). 13C NMR (125 MHz,
DMSO-d6): δ 164.7, 153.6, 151.3, 142.6, 141.5, 139.6, 139.0, 134.0,
131.2, 130.5, 127.8, 124.0, 123.5, 122.83, 122.7, 120.7, 119.3, 119.0,
115.9, 112.1, 112.00, 106.2, 60.7, 56.5. HRMS (ESI): m/z calcd for [M
+H]+ C25H24N3O4 430.1767; found 430.1768.
4.2.11. (E)-N-(2-(5-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-
nitrophenyl)acrylamide (12 k)
Yellow solid; Yield: 78%, 52 mg; M.p: 211–214 °C; FT-IR (cm−1):
3457.0, 3016.6, 2970.8, 1738.6, 1600.8, 1523.6, 1347.5, 1229.4; 1H
NMR (500 MHz, DMSO-d6): δ 13.62 (d, J = 19.4 Hz, 1H), 13.08 (d,
J = 18.4 Hz, 1H), 8.85 (d, J = 7.5 Hz, 1H), 8.31 (s, 2H), 8.24–8.06 (m
3H), 7.98 (d, J = 8.2 Hz, 1H), 7.88–7.77 (m, 2H), 7.52 (d, J = 6.8 Hz,
1H), 7.39 (s, 1H), 7.31 (s, 1H), 7.14 (d, J = 14.2 Hz, 1H), 2.49 (s, 3H).
13C NMR (125 MHz, DMSO-d6): δ 167.5, 163.7, 148.5, 148.3, 141.7,
141.5, 141.2, 138.9, 138.6, 131.0, 129.7, 128.3, 127.6, 124.8, 124.5,
124.3, 124.1, 123.7, 120.7, 119.5, 116.3, 1010.1, 21.8. HRMS (ESI): m/
z calcd for [M+H]+ C23H19N4O3 399.1457; found 399.1455.
4.2.7. N-(2-(5-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)cinnamamide
(12 g)
Off-white solid; Yield: 75%, 50 mg; M.p: 248–250 °C; FT-IR (cm−1):
3357.0, 3026.5, 2970.9, 2960.5, 1738.4, 1669.5, 1542.3, 1317.7,
1126.4; 1H NMR (500 MHz, DMSO-d6): δ 13.51 (s, 1H), 13.05 (s, 1H),
8.86 (s, 1H), 8.15 (s, 1H), 7.99–7.16 (s, 4H), 7.63–7.36 (m, 5H),
7.34–7.08 (d, 2H), 6.93 (s, 1H), 2.50 (s, 3H). 13C NMR (125 MHz,
DMSO-d6): δ 164.6, 150.7, 141.2, 138.8, 134.9, 134.2, 133.5, 130.9,
130.5, 129.5, 128.6, 127.6, 125.4, 124.3, 123.5, 123.2, 120.6, 118.9,
116.2, 111.6, 22.2. HRMS (ESI): m/z calcd for [M+H]+ C23H20N3O
354.1606; found 354.1632.
4.2.12. (E)-N-(2-(5-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(3,4,5-
trimethoxyphenyl) acrylamide (12 l)
White solid; Yield: 73%, 53 mg; M.p: 256–259 °C; FT-IR (cm−1):
3436.5, 3106.8, 3014.3, 2960.5, 1738.6, 1621.9, 1584.5, 1366.2,
1227.1, 1053.9; 1H NMR (500 MHz, DMSO-d6): δ 13.49 (s, 1H), 13.06
(s, 1H), 8.84 (d, J = 7.4 Hz, 1H), 8.14 (d, J = 7.7 Hz, 1H), 7.72–7.65
(m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.39 (s, 1H), 7.28 (t, J = 7.4 Hz, 1H),
7.13 (s, 3H), 6.91 (d, J = 15.6 Hz, 1H), 3.91 (s, 6H), 3.73 (s, 3H), 2.48
(s, 3H). 13C NMR (126 MHz, DMSO): δ 164.7, 153.7, 150.9, 141.3,
139.7, 138.7, 134.2, 133.5, 132.0, 130.9, 130.6, 127.6, 125.4, 124.3,
123.3, 123.1, 122.9, 120.6, 118.8, 116.07, 111.6, 106.1, 60.7, 56.9,
21.8. HRMS (ESI): m/z calcd for [M+H]+ C26H26N3O4 444.1923;
found 444.1956.
4.2.8. (E)-3-(2-Ethoxyphenyl)-N-(2-(5-methyl-1H-benzo[d]imidazol-2-yl)
phenyl)acrylamide (12 h)
Off-white solid; Yield: 80%, 62 mg; M.p: 200–202 °C; FT-IR (cm−1):
3263.5, 2970.9, 2840.8, 1738.4, 1667.4, 1621.9, 1576.3, 1023.9; 1H
NMR (500 MHz, DMSO-d6): δ 13.37 (s, 1H), 13.21 (s, 1H), 8.88 (d,
J = 8.2 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 8.01–7.94 (m, 1H), 7.79 (d,
J = 7.4 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.62 (s, 1H), 7.50 (q,
J = 15.5, 7.9 Hz, 1H), 7.45–7.37 (m, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.18
– 7.10 (m, 2H), 7.07 – 7.03 (m, 1H), 6.98 (d, J = 15.8 Hz, 1H), 4.19 (q,
J = 12.1, 5.6 Hz, 2H), 2.51 (s, 3H), 1.45 (t, J = 6.8 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6): δ 165.0, 157.9, 150.8, 143.00, 140.7, 138.8,
136.8, 134.1, 133.4, 131.8, 131.0, 129.8, 127.6, 125.4, 124.3, 123.3,
121.2, 120.6, 118.7, 116.0, 113.1, 111.6, 64.3, 21.7, 15.2. HRMS (ESI):
m/z calcd for [M+H]+ C25H24N3O2 398.1869; found 398.1895.
4.2.13. N-(2-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)cinnamamide
(12 m)
Off-white solid; Yield: 78%, 55 mg; M.p: 244–246 °C; FT-IR (cm−1):
3456.9, 3204.5, 3004.3, 2970.9, 1738.8, 1621.9, 1584.5, 1366.2,
1217.1, 1043.9; 1H NMR (500 MHz, DMSO): δ 13.32 (s, 1H), 13.27 (s,
1H), 8.89 – 8.81 (m, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.91–7.97 (m, 1H),
7.84 (t, J = 8.0 Hz, 2H), 7.61 (dd, J = 8.7, 4.8 Hz, 1H), 7.54 (m, 1H),
7.52 – 7.45 (m, 3H), 7.40 (dd, J = 8.8, 2.2 Hz, 1H), 7.34–7.27 (m, 1H),
7.23 – 7.13 (m, 1H), 6.94 (d, J = 15.7 Hz, 1H). 13C NMR (125 MHz,
DMSO-d6): δ 164.3, 152.5, 143.3, 140.4, 138.9, 134.3, 132.3, 132.2,
131.5, 131.3, 130.8, 130.7, 130.6, 127.9, 124.0, 123.8, 123.6, 120.7,
115.7, 113.1. HRMS (ESI): m/z calcd for [M+H]+ C22H17ClN3O
374.1060; found 374.1084.
4.2.9. (E)-3-(4-Bromophenyl)-N-(2-(5-methyl-1H-benzo[d]imidazol-2-yl)
phenyl)acrylamide (12i)
Off-white solid; Yield: 70%, 50 mg; M.p: 213–215 °C; FT-IR (cm−1):
3247.0, 3016.5, 2970.7, 1738.8, 1669.5, 1542.3, 1375.7, 1229.4; 1H
10

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BioorganicChemistry103(2020)104191
4.2.14. (E)-N-(2-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-3-(2-
ethoxyphenyl)acrylamide (12n)
4.2.19. (E)-3-(2-Ethoxyphenyl)-N-(2-(5-fluoro-1H-benzo[d]imidazol-2-
yl)phenyl)acrylamide (12 s)
Off-white solid; Yield: 75%, 50 mg; M.p: 239–241 °C; FT-IR (cm−1):
3456.9, 3225.6, 3016.4, 2970.5, 1738.5, 1535.7, 1366.2, 1217.1,
1047.4; 1H NMR (500 MHz, DMSO-d6): δ 13.36 (s, 1H), 13.12 (s, 1H),
8.88 (d, J = 7.1 Hz, 1H), 8.14 (d, J = 7.7 Hz, 1H), 8.00 (t, J = 15.4 Hz,
1H), 7.94–7.78 (m, 2H), 7.64–7.49 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H),
7.38–7.26 (m, 2H), 7.13 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 6.6 Hz, 1H),
6.96 (d, J = 15.8 Hz, 1H), 4.19 (d, J = 6.6 Hz, 2H), 1.43 (t, J = 6.7 Hz,
3H). 13C NMR (125 MHz, DMSO-d6): δ 164.9, 157.7, 152.9, 152.5,
143.5, 141.4, 139.1, 136.7, 134.8, 132.8, 131.9, 131.5, 129.5, 128.0,
123.4, 123.2, 121.2, 120.7, 118.6, 115.6, 113.3, 113.1, 111.7, 64.3,
15.0. HRMS (ESI): m/z calcd for [M+H]+ C24H21ClN3O2 418.1322;
found 418.1325.
Off white solid; Yield: 70%, 52 mg; M.p: 190–193 °C; FT-IR (cm−1):
3456.9, 3204.5, 3004.3, 2970.9, 1738.8, 1621.9, 1584.5, 1366.2,
1217.1, 1043.9; 1H NMR (500 MHz, DMSO-d6): δ 13.35 (s, 1H), 13.17
(s, 1H), 8.88 (s, 1H), 8.19–7.76 (m, 3H), 7.66 (s, 2H), 7.25–7.57 (m,
3H), 7.21–6.91 (m, 3H), 6.53 (d, J = 15.0 Hz, 1H), 4.26–4.05 (m, 2H),
1.53–1.29 (m, 3H). 13C NMR (125 MHz, DMSO-d6): δ 164.9, 157.5,
143.3, 139.4, 136.8, 132.1, 131.9, 130.5 (d, J = 162.1 Hz), 129.1,
127.9, 127.1, 124.6, 123.3, 123.2 (d, J = 51.2 Hz), 121.2, 121.1,
120.6, 119.7, 119.4, 115.8, 113.0, 110.2, 64.4, 15.1. HRMS (ESI): m/z
calcd for [M+H]+ C24H21FN3O2 402.1618; found 402.1625.
4.2.20. (E)-N-(2-(5-Fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-3-(3,4,5-
trimethoxyphenyl) acrylamide (12 t)
White solid; Yield: 67%, 50 mg; M.p: 279–281 °C; FT-IR (cm−1):
3453.9, 3104.5, 2960.9, 1738.8, 1621.7, 1574.5, 1356.2, 1217.1,
1023.9; 1H NMR (500 MHz, DMSO-d6): δ 13.31 (s, 1H), 13.23 (s, 1H),
8.84 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.68 (d,
J = 15.7 Hz, 2H), 7.54 (t, J = 7.7 Hz, 1H), 7.34–7.17 (m, 3H), 7.14 (s,
2H), 6.93 (d, J = 15.7 Hz, 1H), 3.91 (s, 6H), 3.73 (s, 3H). 13C NMR
(125 MHz, DMSO-d6): δ 164.7, 153.6, 141.5, 139.8, 138.9, 131.3,
130.5, 127.8, 123.5, 122.7, 120.7, 115.9, 106.4, 60.6, 56.5. HRMS
(ESI): m/z calcd for [M+H]+ C25H23FN3O4 448.1673; found 448.1679.
4.2.15. (E)-3-(4-Bromophenyl)-N-(2-(5-chloro-1H-benzo[d]imidazol-2-
yl)phenyl)acrylamide (12o)
Off-white solid; Yield: 67%, 48 mg; M.p: 258–260 °C; FT-IR (cm−1):
3456.8, 3265.6, 3016.5, 2970.8, 1738.5, 1631.9, 1586.0, 1366.2,
1217.0, 1059.5; 1H NMR (500 MHz, DMSO-d6): δ 13.37 (s, 1H), 13.21
(s, 1H), 8.85 (d, J = 6.9 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.17–8.11
(m, 1H), 7.81 (s, 2H), 7.74–7.56 (m, 4H), 7.55 (t, J = 7.6 Hz, 1H),
7.40–7.27 (m, 2H), 7.00 (d, J = 15.7 Hz, 1H). 13C NMR (125 MHz,
DMSO-d6): δ 164.3, 152.5, 143.3, 140.4, 138.9, 134.3, 132.4, 132.3,
131.5, 131.3, 130.9, 130.7, 130.6, 127.9, 124.0, 123.8, 123.6, 120.8,
115.7, 113.2. HRMS (ESI): m/z calcd for [M+H]+ C22H16Br81ClN3O
454.0145; found 454.0146.
4.2.21. (E)-3-(2-Ethoxyphenyl)-N-(2-(5-methoxy-1H-benzo[d]imidazol-2-
yl)phenyl)acrylamide (12u)
White solid; Yield: 80%, 50 mg; M.p: 282–284 °C; FT-IR (cm−1):
3453.9, 3104.5, 2901.9, 1738.8, 1661.7, 1586.5, 1321.6, 1217.1,
1025.9; 1H NMR (500 MHz, DMSO-d6): δ 13.34 (s, 1H), 13.09 (s, 1H),
8.88 (s, 1H), 8.13 (s, 1H), 7.98 (, J = 15.0 Hz, 1H), 7.84 – 7.61 (m, 2H),
7.39 (m, 4H), 7.18 – 6.90 (m, 4H), 4.21 (s, 2H), 3.86 (s, 3H), 1.53 – 1.39
(m, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.9, 158.1, 157.7, 157.4,
138.9, 138.7, 137.5, 136.3, 134.9, 132.1, 130.7, 129.5, 127.2, 124.6,
123.4, 121.3, 120.6, 119.9, 115.9, 113.1, 112.2, 64.3, 55.9, 15.1.
HRMS (ESI): m/z calcd for [M+H]+ C25H24N3O3 414.1819; found
414.1814.
4.2.16. (E)-3-(2-Bromo-4,6-dimethoxyphenyl)-N-(2-(5-chloro-1H-benzo
[d]imidazol-2-yl)phenyl)acrylamide (12p)
Off-white solid; Yield: 65%, 45 mg; M.p: 190–191 °C; FT-IR (cm−1):
3457.0, 3016.3, 2970.8, 1738.7, 1591.3, 1433.3, 1216.9, 1023.5; 1H
NMR (500 MHz, DMSO-d6): δ 13.29 (s, 1H), 12.45 (s, 1H), 8.65 (m, 1H),
8.33–7.84 (m, 4H), 7.79–7.49 (m, 3H), 7.45–7.17 (m, 2H), 6.79 (m,
1H), 3.88 (s, 6H). 13C NMR (125 MHz, DMSO-d6): δ 168.0, 151.8,
149.2, 141.9, 139.1, 138.9, 131.6, 128.3, 128.0, 127.7, 126.0, 124.9,
120.9, 120.4, 119.6, 116.8, 116.1, 116.0, 115.7, 110.1, 110.0, 56.5.
HRMS (ESI): m/z calcd for [M+H]+ C24H20Br81ClN3O3 514.0356;
found 514.0364.
4.2.22. (E)-3-(4-Bromophenyl)-N-(2-(5-methoxy-1H-benzo[d]imidazol-2-
yl)phenyl)acrylamide (12v)
White solid; Yield: 82%, 55 mg; M.p: 285–286 °C; FT-IR (cm−1):
3456.9, 3124.5, 2901.9, 1738.8, 1631.7, 1566.5, 1331.6, 1227.1,
1025.9; 1H NMR (500 MHz, DMSO-d6): δ 13.48 (s, 1H), 13.12 (s, 1H),
8.85 (s, 1H), 8.13 (s, 1H), 7.81 (s, 2H), 7.71–7.65 (m, 3H), 7.56–7.41
(m, 2H), 7.31–7.27 (m, 2H), 7.08–6.95 (m, 2H), 3.86 (s, 3H).13C NMR
(125 MHz, DMSO-d6): δ 164.2, 140.0, 138.5, 138.0, 134.2, 132.41,
130.8, 130.8, 130.7, 130.7, 127.1, 124.2, 123.7, 123.6, 120.7, 120.1,
116.2, 112.4, 56.0. HRMS (ESI): m/z calcd for [M+H]+ C23H19BrN3O2
447.0582; found 447.0587.
4.2.17. (E)-N-(2-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-
nitrophenyl)acrylamide (12q)
Yellow solid; Yield: 67%, 47 mg; M.p: 218–220 °C; FT-IR (cm−1):
3454.7, 3016.5, 2970.9, 2920.0, 1738.8, 1694.7, 1621.9, 1584.5,
1366.2, 1206.5, 1092.5; 1H NMR (500 MHz, DMSO-d6): δ 13.22 (s, 2H),
8.30–8.57 (m, 1H), 8.28–8.14 (m, 3H), 7.99 (d, J = 7.7 Hz, 3H),
7.75–7.66 (m, 2H), 7.57–7.37 (m, 3H), 6.75 (d, J = 15.9 Hz, 1H). 13
C
NMR (125 MHz, DMSO-d6): δ 167.3, 148.5, 143.3, 141.7, 141.4, 130.7,
129.7, 128.2, 127.5, 124.8, 124.4, 124.2, 123.6, 119.5, 110.2. HRMS
(ESI): m/z calcd for [M+H]+ C22H16ClN4O3 419.0911; found
419.0918.
4.3. Biological procedures
4.3.1. Material and Methods:
4.2.18. (E)-N-(2-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (12r)
Human lung cancer cell line (A549) mouse breast cancer cell line
(4 T1), human breast cancer cell line (MDA-MB-231), mouse melanoma
cell line (B16-F10), human breast cancer cell line (BT-474), normal
kidney cell line (NRK-52E) were obtained from National Center for Cell
Sciences (NCCS, Pune, India). Cells were cultured in respective
(Dulbecco's Modified Eagle's medium) DMEM), RPMI (Roswell Park
Memorial Institute medium, Sigma Aldrich, St. Louis, USA) and media
was supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and
1% stabilized antibiotic, penicillin–streptomycin mixture (Gibco, USA).
Cells were maintained at 37 °C in a CO2 incubator with 5% CO2 and
90% relative humidity. Trypsin-EDTA required for cell culture was
obtained from (Gibco, USA). 4′, 6-Diamidino-2-phenylindole (DAPI),
fluorescent dyes acridine orange (AO) were from Sigma Aldrich, St.
White solid; Yield: 70%, 50 mg; M.p: 239–241 °C; FT-IR (cm−1):
3453.9, 3214.5, 3024.3, 2940.9, 1738.8, 1621.9, 1574.5, 1346.2,
1227.1, 1043.9; 1H NMR (500 MHz, DMSO-d6): δ 13.37 (s, 1H), 13.19
(s, 1H), 8.84 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.03–7.83
(m, 1H), 7.67 (d, J = 14.8 Hz, 1H),7.61 (s, 1H), 7.55 (t, J = 7.6 Hz,
1H), 7.38–7.27 (m, 2H), 7.14 (s, 2H), 6.93 (d, J = 15.7 Hz, 1H), 3.91 (s,
6H), 3.75 (d, J = 13.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): δ
164.7, 153.6, 152.8, 143.5, 141.5, 139.7, 138.8, 134.9, 132.8, 131.7,
130.6, 127.9, 127.2, 124.0, 123.5, 122.8, 120.8, 118.8, 115.7, 113.4,
111.7, 106.3, 60.6, 56.5. HRMS (ESI): m/z calcd for [M+H]+
C25H23ClN3O4 464.1377; found 464.1385.
11

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
Louis, USA, while Dichlorofluorescin diacetate (DCFDA) and JC-1 dyes
with compound 12 h at different concentrations (0.1, 0.25. 0.5 µM) for
24 h, and then cells were incubated with JC-1 stain (2.5 mg/mL) [28]
for 30 min. Post incubation, adhered cells were collected by trypsini-
zation, washed with PBS and cells were resuspended in a solution of
PBS. Then samples were analyzed for JC-1 fluorescence using flow-
cytometer (BD FACSVerseTM, USA).
were procured from Invitrogen, USA.
4.3.2. Anticancer activity
Cytotoxic activity of compounds 12a-v of the series was carried out
by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay. Cells were plated in a 96 well plate at a density of 5x103
cells per well, supplemented with 10% FBS and they were incubated for
24 h at 37 °C in 5% CO2 later they were treated with the compounds in
respective concentrations in the culture media with vehicle controls and
known standard (nocodozole). After 48 h of treatment, 100 µL of MTT
reagent (0.5 mg/mL) dissolved in serum free media was added to each
well and incubated for 4 h. Then the supernatant media was removed
carefully, washed with PBS. The formazan crystals formed were dis-
solved in 200 µL of DMSO and absorbance was taken at 570 nm in
multimode plate reader (Spectra Max M4, Molecular devices, US).
4.3.8. Scratch wound healing assay
To perform this assay, A549 cells (4 × 105/ well) were seeded in 24
well plates and grown until formation of monolayer. After formation of
monolayer, it was wounded with micro pipette tip carefully.
Monolayers were washed twice with PBS to remove all the non-ad-
herent cells. Then cells were treated with 0.05, 0.1 and 0.25 µM con-
centrations of compound 12 h and cells were allowed to migrate into
the wound area. Images of wounds from all the groups were taken at 0
and 24 h post wound induction. After 24 h post treatment, migrated
cells were observed and compared with that of the control cells (DMSO
treatment), the same was photographed under Carl Zeiss Phase contrast
Microscope at 10X objective lens and width was measured using the
Zenn software and percentage wound closure was calculated as mi-
gration of cancer cells towards the wound or gap’.
4.3.3. Cell morphology by phase contrast imaging
A549 cells were plated with density of 1x105 cells/mL in 24 well
culture plates and allowed to adhere by incubating at 37 °C in 5% CO2
atmosphere for 24 h. Later, cells were incubated with various con-
centrations of compound 12 h (0.1, 0.25 and 0.5 µM). After 24 h of
incubation with compound 12h, morphological changes and viability of
cells were observed and respective images were captured by using
phase contrast microscope (Nikon, Inc. Japan) at 100X magnification.
4.3.9. Annexin v FITC / propidium iodide binding assay
In order to determine the extent of apoptosis induced cell death
annexin V FITC and propidium iodide staining was performed by using
the Annexin V FITC apoptosis detection kit according to manufacturer’s
protocol (Sigma Aldrich). In brief, cells were seeded at a density of
1 × 106 cells per well in 12 well plates and allowed to adhere for
overnight. Cells were treated with different concentrations of 0.1, 0.25
and 0.5 µM compounds along with the vehicle control. After 24 h of
incubation, cells were harvested, washed with PBS and stained with
Annexin V FITC and propidium iodide. Early and late apoptotic cells
were quantified using flow cytometer (BD FACSVerseTM, USA) based
software (BD Biosciences).
4.3.4. Acridine orange (AO) staining
Apoptosis induced by the compound 12h was studied by AO
staining, for this, A549 cells were plated with density of 2x105 cells/mL
in 24 well plates. After allowing the cells to adhere to plates for over-
night, they were treated with different concentrations of compound
12h (0.1, 0.25 and 0.5 µM) and incubated at 37 °C in an atmosphere of
5% CO2 for 24 h. For AO staining, 1 mg/mL concentration was added
into wells to reach concentration of 10 µg/mL. After 10 min, cells were
washed thoroughly and visualized under fluorescence microscope
(Nikon, Inc. Japan) with excitation 488 nm and emission 550 nm wa-
velengths at 100X magnifications and representative images were
captured.
4.3.10. Flow cytometry for cell cycle analysis
To examine the effect of 12h on cell cycle, A549 cells were plated
with the density of 1x106/well in 12 well plates for 24 h and then cells
were treated with 0.1, 0.25 and 0.5 µM concentrations of compound
12h for 24 h, and then adhered cells were collected by trypsinisation,
washed with PBS (pH 7.4) and fixed. Following fixation, cells were
rewashed with PBS and stained with (450 µL) Propidium Iodide (PI) for
15 min in dark at room temperature. The stained samples were then
analysed by flow cytometry (BD FACSVerseTM, USA). 10,000 events/
group were considered for analysing results.
4.3.5. DAPI staining
Compound 12h induced nuclear changes were studied using DAPI
staining [27]. Here cells were seeded at the density of 2x105 cells/well
and allowed to adhere for overnight. After that cells were incubated
with compound 12h for 24 h. After incubation, cells were washed with
PBS and permealised for 5 min with 0.1% Tween 20, later cells were
fixed with 4% paraformaldehyde followed by staining with 1 mM DAPI.
Fluorescence microscope was used to observe control and treated cells
with wavelengths of excitation at 350 nm and emission at 460 nm using
DAPI filter at 100X magnifications and representative images were
captured.
4.3.11. Tubulin polymerization assay
To demonstrate the effect of 12 h on tubulin polymerization, fluor-
escence based cell free in vitro tubulin polymerization assay was per-
formed according to the manufacturer’s protocol (BK011, Cytoskeleton,
Inc.). The reaction mixture for the experiment contain Porcine brain
tissue (2 mg/mL) in 80 mM PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA,
1.0 mM GTP and glycerol, experiment was performed in the presence of
test compound 12h (final concentration of 1.25, 2.5, 5.0, 10.0 µM),
control and standard (paclitaxel) at 3 µM concentration. Polymerization
was monitored by time dependent increase in the fluorescence at 37 °C by
a fluorescence reporter incorporated into microtubules. Fluorescence
emission at 440 nm (excitation wavelength is 360 nm) was measured by
using a spectramax M4 multi-mode micro -plate detection System. The
compound concentration required to inhibit 50% of tubulin assembly in
comparison to control (IC50) was calculated.
4.3.6. Measurement of reactive oxygen species (ROS) generation by DCFDA
staining
A549 cells were plated at a density of 2x105 cells/mL in 24 well
plates and allowed to adhere for overnight. Later, cells were treated
with compound 12h at concentrations of 0.1, 0.25 and 0.5 µM of for
24 h. Then the media was replaced with culture medium containing
DCFDA dye (10 mM) and incubated for 30 min in dark. Fluorescence
spectroscopy (SpectraMax, Molecular devices) was used to detect the
intensity of this fluorescence from cells at an excitation and emission
wavelength of 480 and 535 nm, respectively and the corresponding
images were captured at 100X magnification.
4.3.12. Molecular docking simulation study
4.3.7. Measurement of mitochondrial membrane potential by JC-1 staining
A549 cells were cultured in 12 well plates at a density of 5x105
cells/mL and allowed to adhere over night. Next day, cells were treated
The 3D structure of compound 12h was built on Maestro Molecule
Builder of Schrödinger. The built molecule was optimized using
OPLS_2005 force field in LigPrep module of Schrödinger 2017–1, Glide
12

K. Donthiboina, et al.
BioorganicChemistry103(2020)104191
7.4 software [29]. Docking procedure was followed using the standard
protocol implemented in Maestro, 11.1 and the compound 12h was
docked against the colchicine binding site of α/β-tubulin interphase
(PDB ID: 3E22). The ligand–protein complex was analyzed for inter-
actions and the 3D pose of most active compound 12h was taken using
Schrödinger.
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