Design, synthesis and biological evaluation of novel imidazole-chalcone derivatives as potential anticancer agents and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104904

Novel imidazole-chalcone derivatives were designed and synthesized as tubulin polymerization inhibitors and anticancer agents. The antiproliferative activity of the imidazole-chalcone was assessed on some human cancer cell lines including A549 (adenocarci

Full text of DOI:10.1016/j.bioorg.2021.104904

Bioorganic Chemistry 112 (2021) 104904
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel imidazole-chalcone
derivatives as potential anticancer agents and tubulin
polymerization inhibitors
,
b
,
,
Sara Rahimzadeh Oskueia 1, Salimeh Mirzaeic 1, Mohammad Reza Jafari-Nikb,
Farzin Hadizadeha b, Farhad Eisvandd, Fatemeh Mosaffaa, Razieh Ghodsia
,
,b,*
a Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
b Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
c Department of Medicinal Chemistry, Faculty of Pharmacy, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
d Department of Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel imidazole-chalcone derivatives were designed and synthesized as tubulin polymerization inhibitors and
anticancer agents. The antiproliferative activity of the imidazole-chalcone was assessed on some human cancer
cell lines including A549 (adenocarcinoma human alveolar basal epithelial cells), MCF-7 (human breast cancer
cells), MCF-7/MX (mitoxantrone resistant human breast cancer cells), and HEPG2 (human hepatocellular car-
cinoma cells). Generally, the imidazole-chalcone derivatives exhibited more cytotoxicity on A549 cancer cells in
comparison to the other three cell lines, among them compounds 9j’ and 9g showed significant cytotoxicity with
Chalcone
Imidazole
Tubulin inhibitors
Anticancer activity
Molecular docking
Apoptosis
IC50 values ranging from 7.05 to 63.43 μM against all the four human cancer cells. The flow cytometry analysis of
A549 cancer cells treated with 9g and 9j’ displayed that these compounds induced cell cycle arrest at the G2/M
phase at low concentrations and increased the number of apoptotic cells (cells in subG1 phase) at higher con-
centrations. They have also inhibited tubulin polymerization similar to combretastatin A-4 (CA-4). Annexin V
binding staining assay in A549 cancer cells revealed that compound 9j’ induced apoptosis (early and late).
Finally, molecular docking studies of 9j’ into the colchicine-binding site of tubulin presented the probable in-
teractions of these compounds with tubulin.
1. Introduction
[7–17] There are three well known binding sites in tubulin including the
vinca domain, colchicine, and paclitaxel sites [18,19]. Numerous anti-
Cancer is a multifactorial disease and considered one of the most
serious illnesses worldwide. Although the biological pathways involved
in cancer became more clear over time, potent and effective anticancer
drugs are needed for the treatment of cancer. The existing anticancer
agents formulated from natural and synthetic products exert toxicity due
to the low site-specificity, which leads to healthy cell damage and drug
resistance [1–3].
mitotic agents are derived from both natural and synthetic sources.
Anti-mitotic agents such as taxanes and vinca alkaloids have been uti-
lized for the clinical treatment of different cancerous patients in the last
decades [4,19]. However, high toxicity, poor solubility, low oral
bioavailability render these agents less optimum for clinical treatment of
cancer [20] Therefore, scientists are encouraged to develop novel anti-
mitotic agents in order to overcome the mentioned drawbacks.
Microtubules, comprising
α
-β tubulin heterodimer, are important
Chalcones are essential pharmacophores of many natural products
such as curcumin, flavokawain, millepachine, and xanthohumol [21].
Chalcone-based derivatives have received notable attention because of
their wide ranges of biological activities, such as anti-oxidant, anti-
bacterial, anti-fungal, anti-proliferative [22–25], and antidiabetic ac-
tivities [26]. Over the last few years, many attempts have been done for
components of the eukaryotic cytoskeleton. They control several cellular
functions including motility regulation, cell signaling, secretion, cell
architecture in interphase [4–6]. Since microtubules have an important
role in the life cycle of the cell, they have been recognized as major
targets for the development of novel anticancer compounds recently
* Corresponding author.
1
These authors contributed equally in this study.
https://doi.org/10.1016/j.bioorg.2021.104904
Received 5 January 2021; Received in revised form 3 April 2021; Accepted 7 April 2021
Available online 20 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
the development of chalcone derivatives as tubulin polymerization in-
hibitors such as chalcone 1 and compounds A-D (Fig. 1) [15,23,27–36].
It is reported that chalcones may act as Michael acceptors and alkylate
numerous critical proteins of the tubulin-microtubule system [37],
thereby chalcone has been considered as an advantaged scaffold for the
design and development of new anti-cancer agents [31]. Some imidazole
derivatives also are reported as tubulin inhibitors such as compounds E-
G (Fig. 1) [22,38–41]. On the other hand, many studies demonstrated
that natural products possessing imidazole ring which are derived from
marine including eleutherobin, sarcodictyin A, sarcodictyin B (Fig. 1),
exhibit tubulin inhibition activity [42]. The biological importance and
relative shortage of these natural products have prompted several
groups to undertake the total chemical synthesis of these products and
their derivatives. They reported the structure-activity relationships
derived from these studies. The most noticeable result was the signifi-
(human hepatocellular carcinoma cells). Compounds that displayed the
most antiproliferative activity were examined for their effects on in-
duction of apoptosis and cell cycle arrest using flow cytometry, these
compounds were also evaluated to inhibit microtubule polymerization.
Besides, to describe the obtained results of biological assessments,
docking studies were performed.
2. Results and discussion:
2.1. Synthesis
Compounds 7a and 7b were synthesized using the procedures re-
ported before [44] As illustrated in Scheme 1, benzyl amine (1), dihy-
droxyacetone (2) and potassium thiocyanate (3) in a solution of acetic
acid and 1-butanol were stirred for 72 h to afford (1-benzyl-2-mercapto-
1H-imidazol-5-yl) methanol (4). Then methyl iodide or ethyl iodide was
added to a solution of compound 4 in methanol in the presence of an
aqueous solution of sodium hydroxide to produce compounds 5 and 6.
These compounds (5 and 6) were oxidized by magnesium dioxide
(MnO2) under reflux conditions with chloroform to afford compound 7.
Finally, Compounds 9a-9j’ were synthesized by applying Claisen-
Schmidt condensation between compound 7a or 7b and different ace-
tophenones (8a-8j) in the presence of the methanolic sodium hydroxide.
Oxidation of 9j with oxone in THF-water gave the oxidized methyl-
sulfonyl compound 9k. The structures of compounds were confirmed
by nuclear magnetic resonance (1HNMR, 13C NMR) and mass
spectrometry.
cant role of the α, β–unsaturated hetero-aromatic side chain for both
antiproliferative activities and tubulin binding. Moreover, the natural
imidazole ring displayed the optimal activity, whereas replacing the
imidazole ring with other aromatic rings such as pyridine, thiazole, or
oxazole led to a decrease in activity [43]. Therefore, according to these
findings, in this study new imidazole-chalcone derivatives are designed
and synthesized as tubulin inhibitors. The rationale for the design of the
imidazole-chalcone hybrids is represented in Fig. 1. The anti-
proliferative activity of synthesized compounds was evaluated against
four different cancer cell lines including A549 (adenocarcinoma human
alveolar basal epithelial cells), MCF-7 (human breast cancer cells), MCF-
7/MX (mitoxantrone resistant human breast cancer cells), and HEPG2
Fig. 1. Chemical structures of known tubulin inhibitors and our designed compounds.
2

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Scheme 1. Reagents and conditions.
2.2. Biological evaluation
membrane because of their increased lipophilicity or enhanced in-
teractions with residues in the active site of tubulin [45–50]. It is also
concluded that the higher cytotoxic activity of non-polar compounds
could be attributed to their higher lipophilicity leading to better
permeability to lipophilic cell membranes. Compounds 9h and 9h’ have
less cytotoxicity than compounds 9g and 9g’, despite having a phenyl
group at position 4 (C-4) of A-ring. This may be because of the steric
hindrance of the phenyl ring in the active site of tubulin.
2.2.1. In vitro anticancer activity
To evaluate the cytotoxic effects of these newly imidazole-chalcone
derivatives, MTT assay was performed against four cancer cell lines
including A549 (adenocarcinoma human alveolar basal epithelial cells),
MCF-7 (human breast cancer cells), MCF-7/MX (mitoxantrone resistant
human breast cancer cells), and HEPG2 (human hepatocellular carci-
noma cells) using combretastatin A-4 (CA-4) as the positive control. As
shown in Table 1, most of our compounds exhibited moderate to high
antiproliferative activity, with IC50 values in the micromolar range.
Generally, the imidazole-chalcone derivatives showed more cytotoxicity
on A549 cancer cells in comparison to the other three cell lines. HEPG2
cancer cells were not so sensitive to our compounds except 9j’. Among
the synthesized compounds, 9j’ and 9g displayed significant cytotox-
Compound 9a possessing bromine atom (Br) at position 4 (C-4) of A-
ring displayed strong cytotoxic activity against A549 cancer cells,
comparable to the most potent compound 9j’, which may be due to the
proper size and lipophilicity of the bromine (Br). Replacing Br with
chlorine and fluoride reduced the potency of the compound. Oxidizing
compound 9j increased the cytotoxicity of this compound on MCF-7
(compare the cytotoxicity of 9k with its parent 9j). Clog P (Calculated
logP) is an important factor in membrane permeability and hence
antiproliferative activity. Clog values of the compounds also were
determined [51]. These compounds had ClogP values in range of
2.48–4.01 (Table 1). As shown in Table 1, in general, there is not a
significant correlation between ClogP values and MTT data in our
compounds.
icity with IC50 values ranging from 7.05 to 63.43 μM against all four
human cancer cells. Compound 9j’ possessing three methoxy groups on
A-ring (Scheme 1) demonstrated stronger activity than the other com-
pounds, since trimethoxyphenyl moiety is an important pharmacophore
in some strong tubulin inhibitors such as CA-4, the increased activity of
9j’ is likely due to the extensive interactions of the trimethoxyphenyl
group with tubulin. Based on the IC50 values of the synthesized com-
pounds, compounds 9g and 9g’ possessing naphthalene group, showed
an acceptable potency with IC50 values of 11.7 µM and 13.96 µM on A-
549 cell line, and 14.4 µM and 24.07 µM on MCF7/MX cell line,
respectively. This may be due to their ability to penetrate the cell
2.2.2. Tubulin polymerization assay
Based on the acquired antiproliferative activities of imidazole-
chalcone hybrids on human cancer cells, 9j’ and 9g (the most potent
antiproliferative compounds) and 9j (which did not show significant
3

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Table 1
The in vitro antiproliferative activities (IC50 (µM)a) of compounds 9a-9j’ against human cancer cell lines.
Compound
R1
R2
R3
R4
A-549
MCF-7
HEPG 2
MCF-7 MX
Clog P
9a
9b
9c
9d
9e
H
H
H
H
H
Br
F
H
H
H
H
H
SCH3
SCH3
SCH3
SCH3
SCH3
8.84 ± 2.31
13.31 ± 2.12
>100
27.94 ± 3.41
17.53 ± 3.11
29.08 ± 3.17
>100
66.35 ± 3.76
>100
85.63 ± 4.89
22.14 ± 2.74
>100
3.44
3.19
3.31
2.77
3.55
Cl
51.71 ± 4.15
>100
N2O
87.02 ± 5.34
14.87 ± 3.66
>100
28.3 ± 3.55
>100
29.75 ± 2.44
9f
H
CH3
H
H
SCH3
SCH3
26.01 ± 3.44
11.7 ± 1.62
21.24 ± 2.53
14.85 ± 2.73
>100
32.89 ± 3.15
14.94 ± 1.91
3.04
3.41
9g
63.43 ± 5.33
9i
H
OCH3
OCH3
OCH3
Br
H
SCH3
>100
28.3 ± 2.78
>100
ND
ND
3.42
3.76
2.48
3.35
3.44
3.39
4.01
9j
OCH3
OCH3
H
OCH3
OCH3
H
SCH3
66.47 ± 4.85
ND
>100
>100
>100
>100
>100
>100
54.86 ± 2.94
>100
9k
9a’
9b’
9c’
9e’
SO2CH3
S-Ethyl
S-Ethyl
S-Ethyl
S-Ethyl
18.33 ± 3.15
14.6 ± 2.14
>100
78.9 ± 4.87
85.42 ± 5.76
>100
>100
H
F
H
17.4 ± 2.66
>100
H
Cl
H
76.13 ± 3.77
33.42 ± 2.94
H
H
17.89 ± 2.44
>100
9g’
9h’
H
H
S-Ethyl
S-Ethyl
13.96 ± 2.48
>100
>100
>100
>100
>100
24.07 ± 3.56
>100
3.59
3.88
H
9i’
H
OCH3
OCH3
H
S-Ethyl
S-Ethyl
>100
33.42 ± 3.46
9.88 ± 1.56
0.43 ± 0.14
ND
ND
3.71
3.86
3.61
9j’
OCH3
OCH3
7.05 ± 1.12
0.86 ± 0.23
21.97 ± 2.23
0.63 ± 0.17
20.2 ± 2.76
1.49 ± 0.47
CA4
a
Compound concentration required to inhibit the cells growth by 50%. ND; not determined.
antiproliferative activity) were assessed for in vitro inhibitory effects on
tubulin polymerization at 50 and 100 µM concentrations. In this eval-
uation, compounds paclitaxel, (a polymerization promoter), and CA-4,
(a polymerization suppressor), were used as the references. As shown
in Fig. 2, 9j’ at the concentrations of 50 and 100 µM inhibited tubulin
polymerization by 42.85% and 60.71% respectively, and 9g inhibited
tubulin polymerization by 25% and 55.71% at the concentrations of 50
and 100 µM, respectively. The tubulin inhibitory activity of Compound
9j was less than those of 9j’ and 9g which is consistent with their
antiproliferative activities as well. inhibited The results confirmed that
these compounds are able to inhibit tubulin polymerization in a dose-
A549 cells were evaluated by flow cytometry. Since cell cycle arrest
causes DNA fragmentation, the percentage of sub-G1 apoptotic cells in
cancer cells treated with compounds 9j’ and 9g, was measured. As
displayed in Figs. 3 and 4, treatment of A549 cells with 9j’ (10, 20, and
40 μM) for 48 h, induced cell cycle arrest at G2/M phase and increased
the percentage of cells from 14.11% in the control group to 34.9% at the
concentration of 10 µM, but at higher concentrations, the cells number
at sub-G1 phase increased, which indicating the cells were subjected to
apoptosis when treated by higher concentrations. when A549 cell line
was treated with 9g at various concentrations for 48 h, the percentages
of cells in the G2/M phase were 29.78% and 32.92% at concentrations of
10 µM and 20 µM (in comparison to 14.11% arrested cells in the control
group), at 40 µM concentration the percentage of cells in the subG1
phase was increased. These results further confirmed that 9j’ and 9g
possibly exerted their antiproliferative activity through cell cycle arrest
at the G2/M phase and finally induction of cellular apoptosis.
dependent manner and in a mode similar to that of CA-4 (5
μM).
These data revealed that one of the mechanisms of antiproliferative
activity of these compounds may be due to inhibition of tubulin
polymerization.
2.2.3. Cell cycle analysis using flow cytometry
Antimitotic drugs, by the destruction of the microtubular network,
induce cell cycle arrest at the G2/M phase. To investigate the mecha-
nism of cytotoxicity of compounds 9j’ and 9g, the effects of these
compounds at different concentrations on the cell cycle progression of
2.2.4. Apoptosis assay
Compound 9j’ was evaluated by Annexin V FITC/PI (AV/PI) dual
staining assay to characterize the mode of cell death induced by these
compounds. This assay can detect between live cells (Q4), early
4

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Neg.control
Paclitaxel
CA-4(5µM)
9j ' (100µM)
9j ' (50µM)
9g (50 µM
9g (100 µM)
9j 50 µM)
9j (100 µM
0
10
20
30
40
50
60
Time (min)
Fig. 2. Effects of compounds 9g, 9j’ and 9j on in vitro tubulin polymerization.
apoptotic cells (Q3), late apoptotic cells (Q2), and necrotic cells (Q1). In
this study A549 cells were treated with 9j’ for 24 h at 10, 20, and 40 µM
concentrations (Fig. 5). The results showed an accumulation of total
apoptotic cells (early and late apoptotic cells) from 3.1% (in untreated
control) to 11.54% (10 µM), 50.7% (20 µM), and 54.2% (40 µM),
respectively (Fig. 5). The findings indicated that 9j’ can induce cellular
apoptosis (in a dose-dependent manner) and these results are consistent
with its antiproliferative effect in the tested cell lines.
7.05 to 63.43 μM against all four human cancer cells. Compound 9j’
possessing three methoxy groups on A-ring exhibited potent anti-
proliferative activity against all cancer cells, which’s in agreement with
the potent tubulin inhibitors with trimethoxyphenyl pharmacophore
such as CA-4. The flow cytometry analysis of A549 cancer cells treated
with 9g and 9j’ showed that low concentrations of these compounds
induced cell cycle arrest at the G2/M phase, while at higher concen-
trations they increased the number of apoptotic cells (cells in subG1
phase). Annexin V-FITC/PI staining assay in A549 cancer cells treated
with 9j’ displayed that 9j’ induced cell apoptosis in a dose-dependent
manner. Besides, compounds 9j’ and 9g, inhibited tubulin polymeriza-
tion similar to CA-4. Finally, molecular docking studies of 9j’ into the
colchicine binding site of tubulin demonstrated the probable in-
teractions of this compound with tubulin.
2.3. Molecular modeling studies
To investigate the possible binding mode of the synthesized
imidazole-chalcone hybrids with tubulin, docking studies of these
compounds were performed at the colchicine binding site of the tubulin
crystal structure (PDB ID: 4O2B) using MOE 2015.10.
4. Experimental
All the synthetic compounds were well accommodated inside the
colchicine binding site of tubulin. The docking pose of 9j’, the most
potent compound in the series, in complex with tubulin was shown in
Figs. 6 and 7. Compound 9j’, has shown two hydrogen bond interactions
4.1. Chemistry
All needed reagents were bought from Merck and Acros. The prog-
ress of all reactions was monitored using thin-layer chromatography
(TLC) with 0.25 mm silica gel plates (60 GF-254- Merck & Co (Darm-
stadt, Germany)). Melting points were measured by a Thomas Hoover
capillary instrument. NMR spectra were acquired using a Bruker FT-300
MHz instrument. Compounds were solved in Chloroform-D or DMSO-D6
before acquiring NMR spectra. Coupling constant (J) values were
measured in hertz (Hz) and spin multiples are given as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet). The mass spectra were
acquired using a 3200 QTRAP LC/MS triple quadrupole mass spec-
trometer possessing an electrospray ionization (ESI) interface.
with the catalytically active residues Ser 178
dashed line in Fig. 6A) a cation-
Further, several hydrophobic interactions between the compound 9j’
α
and Ala 316β and (red
π
interaction with Asn 258β (Fig. 6A).
and other residues, such as Glu183
α
, Thr 224 , Lys 254β, Asn 101 , Val
α
α
351β, Lys 352β, and Leu 248β were observed (Fig. 6B). These various
hydrophobic interactions and hydrogen bonds formation of 9j’ with
tubulin can describe the inhibitory effect of this compound.
3. Conclusion
In the current study, a new series of imidazole-chalcone hybrids were
synthesized as tubulin inhibitors. The cytotoxic activity of the synthe-
sized compounds was evaluated against four cancer cell lines including
MCF7, A549, HepG2, and MCF7/MX by MTT assay. Generally, the
imidazole-chalcone derivatives displayed more cytotoxicity on A549
cancer cells in comparison to the other three cell lines, among them, 9j’
and 9g exhibited strong cytotoxic activity with IC50 values ranging from
4.2. General procedure for the synthesis of 3-(1-benzyl-2-(methylthio or
ethylthio)-1H-imidazol-4-yl)-1-phenylpropan-1-one (9a-9j’)
An aqueous solution of sodium hydroxide 2 N (2 ml) was added to 1-
benzyl-2-mercapto-1H-imidazole-5-carbaldehyde derivatives (7a or 7b,
5

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Fig. 3. Flow cytometry analysis of compound 9j’ in A549 cancer cells.
1 mmol) in methanol (5 ml), then the appropriate acetophenone (8a-8j,
1 mmol) was added to the mixture and was stirred at room temperature
for 24–48 h (monitored by TLC). Upon completion, HCl (2 N) was added
until a precipitate formed. The crude product was filtrated, washed with
cold ethanol and recrystallized in methanol.
(ppm)1.25–1.30 (t, 3H, CH3, J = 7.2 Hz), 3.29–3.36 (SCH2, J = 7.2 Hz),
5.57 (s, 2H, CH2), 7.13–7.16 (m, 2H, ArH), 7.31–7.42 (m, 3H, ArH),
7.53–7.58 (d, 1H, CH = CH, J = 15.3 Hz), 7.74–7.78 (d, 2H, 4-bromo-
phenyl, H2 &H6, J = 8.7 Hz), 7.89–7.94 (d, 1H, CH = CH, J = 15.3
Hz), 7.98–8.01 (d, 2H, 4-bromophenyl, H3 &H5, J = 8.7 Hz), 9.62 (s, 1H,
imidazole); 13CNMR (75 MHz-DMSO‑d6): δ (ppm), 15.04, 28.77, 48.31,
126.77, 127.19, 128.06, 128.51, 128.64, 129.49, 130.89, 132.00,
132.36, 135.76, 136.55, 145.59, 187.75; LC-MS [52]: 427.3 (M + H)+.
4.2.1. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
bromophenyl) prop-2-en-1-one (9a)
Yield: 62%; mp = 203–205 ◦C; 1HNMR (300 MHz-DMSO‑d6): δ
(ppm) 2.80 (s, 3H, SCH3), 5.55 (s, 2H, CH2), 7.15–7.18 (m, 2H, ArH),
7.31–7.44 (m, 3H, ArH), 7.53–7.58 (d, 1H, CH = CH, J = 15.3 Hz),
7.77–7.79 (d, 2H, 4-bromophenyl, H2 &H6, J = 8.7 Hz), 7.87–7.92 (d,
1H, CH = CH, J = 15.3 Hz), 7.97–8.00 (d, 2H, 4-bromophenyl, H3 &H5,
J = 8.7 Hz), 8.50 (s, 1H, imidazole); 13CNMR (75 MHz-DMSO‑d6): δ
(ppm) 16.34, 48.23, 122.381, 126.87, 128.06, 128.55, 128.60, 129.52,
130.88, 131.88, 132.38, 135.63, 136.58, 147.42; LC-MS [52]: 414.3 (M
+ H)+.
4.2.3. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
fluorophenyl) prop-2-en-1-one (9b)
Yield: 42%; mp = 183–185 ◦C; 1H NMR (300 MHz-DMSO‑d6): δ
(ppm) 2.64–2.65 (s, 3H, CH3, J = 4.5 Hz), 5.40 (s, 2H, CH2), 7.08–7.11
(d, 2H, ArH, J = 8.4 Hz), 7.28–7.37 (m, 3H, ArH), 7.39–7.42 (m, 2H,
ArH), 7.52–7.57 (d, 1H, HC = CH, J = 15.6 Hz), 7.64–7.70 (d, 1H, HC =
CH, J = 15.6 Hz), 8.05 (s, 1H, imidazole), 8.07–8.09 (d, 1H, ArH, J = 5.4
Hz), 8.10–8.12 (d, 1H, ArH, J = 5.7 Hz); 13CNMR (75 MHz-DMSO‑d6): δ
(ppm) 15.47, 47.36, 116.06 (CF), 116.34 (CF), 118.85, 126.44, 128.18,
129.40, 129.87, 131.54, 131.64, 134.26, 134.78, 136.81, 148.55; LC-MS
(ESI): 353.4 (M + H)+, 375.5 (M + Na)+.
4.2.2. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(4-bromophenyl)
prop-2-en-1-one (9a’)
Yield: 75%; mp = 153–155 ◦C; 1HNMR (300 MHz-DMSO‑d6): δ
6

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Fig. 4. Flow cytometry analysis of compound 9g in A549 cancer cells.
4.2.4. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(4-fluorophenyl)
prop-2-en-1-one (9b’)
4.2.6. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(4-chlorophenyl)
prop-2-en-1-one (9c’)
Yield: 41%; mp = 134–136 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 1.28–1.32 (t, 3H, CH3, J = 14.4 Hz), 3.14–3.21 (q, 2H, CH2, J =
7.2), 5.42 (s, 2H, CH2), 7.07–7.09 (d, 2H, ArH, J = 6.9 Hz), 7.28–7.41
(m, 5H, ArH), 7.51–7.56 (d, 1H, HC = CH, J = 15.6 Hz), 7.65–7.70 (d,
1H, HC = CH, J = 15.3 Hz), 8.06 (s, 1H, imidazole), 8.07–8.09 (d, 1H,
ArH, J = 5.4 Hz), 8.10–8.12 (d, 1H, ArH, J = 5.7 Hz); 15.53, 27.85,
47.38, 118.56, 126.56, 128.16, 129.39, 130.17, 130.58, 131.39, 134.55,
136.73, 136.90, 138.37, 147.53, 187.63; LC-MS(ESI): 367.4 (M + H)+.
Yield: 63%; mp = 148–150 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 1.27–1.32 (t, 3H, CH3, J = 7.2 Hz), 3.14–3.21 (q, 2H, CH2, J = 7.3
Hz), 5.42 (s, 2H, CH2), 7.06–7.09 (d, 2H, ArH, J = 7.2 Hz), 7.27–7.32 (t,
1H, ArH, J = 7.2 Hz), 7.36–7.41 (t, 2H, ArH, J = 7.2 Hz), 7.52–7.57 (d,
1H, HC = CH, J = 15.6 Hz), 7.59–7.62 (d, 2H, ArH, J = 8.7 Hz),
7.63–7.68 (d, 1H, HC = CH, J = 15.6 Hz), 8.00–8.03 (d, 2H, ArH, J = 8.7
Hz), 8.07 (s, 1H, imidazole); 13CNMR(75 MHz-DMSO‑d6): δ (ppm)
15.63, 27.58, 47.38, 118.56, 126.56, 128.16, 129.30, 130.17, 130.58,
131.39, 134.55, 136.73, 136.90, 138.37, 147.53, 187.63; LC-MS (ESI):
383.4 (M + H)+.
4.2.5. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
chlorophenyl) prop-2-en-1-one (9c)
Yield: 59%; mp = 183–1855 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 2.69 (s, 3H, CH3), 5.46–5.50 (s, 2H, CH2), 7.12–7.17 (t, 2H, ArH),
7.29–7.46 (m, 3H, ArH), 7.56–7.66 (t, 3H, ArH), 7.71–7.77 (d, 1H, HC =
CH, J = 15.6 Hz), 8.03–8.08 (m, 2H, ArH), 8.20 (s, 1H, imidazole); LC-
MS(ESI): 369.4 (M + H)+.
4.2.7. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
nitrophenyl)prop-2-en-1-one (9d)
Yield: 12%; mp = 165–167 ◦C; 1HNMR(300 MHz-DMSO‑d6): δ (ppm)
2.72 (s, 3H, SCH3), 5.48 (s, 2H, CH2), 7.12–7.14 (m, 2H, ArH), 7.30–7.43
(m, 3H, ArH), 7.57–7.62 (d, 1H, CH = CH, J = 15.3 Hz), 7.77–7.78 (d,
1H, CH = CH, J = 15.3 Hz), 8.21–8.34 (m, 5H, ArH); 13CNMR(75 MHz-
7

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Annexin V-FITC
(FL-1)
Fig. 5. Effects of 9j’ on the apoptosis of A549 cancer cells after 24 h using annexin V/PI double staining test by flow cytometry.
Fig. 6. Binding mode of 9j’ in colchicine binding site. A) Hydrogen bonding of 9j’ with colchicine binding site of tubulin (red dashed line). B) Hydrophobic in-
teractions of 9j’ with tubulin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
8

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
Fig. 7. The 2D representation of the interaction between compound 9j’ in the crystal structure of tubulin.
DMSO‑d6): δ (ppm) 15.78, 47.79, 129.14, 124.32, 126.78, 128.38,
129.47, 130.09, 130.35, 131.63, 131.95, 136.21, 142.68, 148.61,
150.25, 187.73; LC-MS (ESI): 380.4 (M + H)+.
7.2 Hz), 5.42 (s, 2H, CH2), 7.05–7.09 (m, 3H, ArH), 7.13–7.15 (m, 2H,
ArH), 7.23–7.31 (m, 2H, ArH), 7.35–7.40 (m, 2H, ArH), 7.45–7.55 (m,
2H, ArH), 7.63–7.68 (d, 1H, CH = CH, J = 15.3 Hz), 8.03–8.05 (m, 3H,
ArH); 13CNMR(75 MHz-DMSO‑d6): δ (ppm), 15.52, 27.64, 47.40,
117.74, 119.00, 120.40, 125.20, 126.55, 128.13, 129.37, 129.41,
130.80, 131.26, 132.89, 134.07, 136.94, 147.14, 155.52, 161.56,
187.27; LC-MS (ESI):441.6 (M + 1)+, 463.3 (M + Na)+.
4.2.8. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
phenoxyphenyl) prop-2-en-1-one (9e)
Yield: 44%; mp = 144–146 ◦C; 1HNMR(300 MHz-DMSO‑d6): δ (ppm)
2.52 (s, 3H, SCH3), 5.39 (s, 2H, CH2), 7.05–7.08 (m, 2H, ArH), 7.08–7.11
(m, 2H, ArH), 7.11–7.15 (m, 2H, ArH), 7.23–7.31(m, 2H, ArH),
7.35–7.40(m, 2H, ArH), 7.44–7.51(m, 2H ArH), 7.51–7.56 (d, 1H, CH =
CH, J = 15.3 Hz), 7.63–7.68 d, 1H, CH = CH, J = 15.3 Hz); 13CNMR(75
MHz-DMSO‑d6): δ (ppm) 15.05, 47.38, 117.74, 118.83, 120.39, 125.19,
126.63, 128.16, 129.38, 130.79, 131.25, 131.58, 132.91, 133.98,
136.81, 148.34, 155.53, 161.55, 187.24; LC-MS (ESI): 427.3 (M + Na)+.
4.2.10. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(p-tolyl)prop-
2-en-1-one (9f)
Yield: 79%; mp = 118–120 ◦C; 1HNMR(300 MHz-DMSO‑d6): δ (ppm)
2.39(s, 3H, Toluene) 2.76 (s, 3H, SCH3), 5.51 (s, 2H, CH2), 7.14–7.17
(m, 2H, H3 & H5, p-tolyl), 7.30–7.43 (m, 5H,ArH), 7.50–7.55 (d, 1H, CH
= CH, J = 15.3 Hz), 7.82–7.87 (d, 1H, CH = CH, J = 15.3 Hz), 7.93–7.96
(d, 2H, p-tolyl, H2&H6, J = 15.6 Hz), 8.40 (s, 1H, imidazole); 13CNMR
(75 MHz-DMSO‑d6): δ (ppm) 16.12, 21.67, 48.12, 126.79, 126.87,
127.13, 128.06, 128.55, 128.60, 129.52, 130.88, 131.88, 132.38,
135.63, 136.58, 147.42; LC-MS (ESI): 349.4 (M + H)+.
4.2.9. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(4-
phenoxyphenyl) prop-2-en-1-one (9e’)
Yield: 41%; mp = 127–129 ◦C; 1HNMR (300 MHz-DMSO‑d6): δ
(ppm)1.27–1.31 (t, 3H, CH3, J = 7.2 Hz), 3.13–3.20 (q, 2H, SCH2, J =
9

S. Rahimzadeh Oskuei et al.
Bioorganic Chemistry 112 (2021) 104904
4.2.11. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(naphthalen-
2-yl)prop-2-en-1-one (9g)
129.43, 131.25, 133.36, 133.57, 135.39, 142.43, 149.21, 153.09,
188.05; LC-MS(ESI): 425.4 (M + H)+, 447.4 (M + Na)+.
Yield: 54%; mp = 189–191 ◦C; 1HNMR(300 MHz-DMSO‑d6): δ (ppm)
2.51 (s, 3H, SCH3), 5.56 (s, 2H, CH2), 7.17–7.20 (m, 2H, ArH), 7.31–7.45
(m, 3H, ArH), 7.59–7.64 (d, 1H, CH = CH, J = 15.6 Hz), 7.66–7.73 (m,
2H, ArH), 8.01–8.14 (m, 5H, ArH) 8.79 (s, 1H, imidazole); 13CNMR(75
MHz-DMSO‑d6): δ (ppm) 16.21, 48.19, 122.381, 124.41, 126.83,
127.54, 128.23, 128.50, 129.01, 129.29, 129.52, 130.03, 130.81,
132.00, 132.69, 135.01, 135.56, 135.83, 147.74, 188.42; LC-MS (ESI):
385.5 (M + H)+.
4.2.17. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (9j’)
Yield: 61%; mp = 82–84 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ (ppm)
1.35–1.40 (t, 3H, CH3, J = 7.3 Hz), 3.17–3.24 (q, 2H, CH2, J = 7.3 Hz),
3.87 (s, 6H, OCH3), 3.92 (s, 3H, OCH3), 5.32 (s, 2H, CH2), 7.10–7.13 (d,
4H, ArH, J = 6.9 Hz), 7.18–7.24 (d, 1H, HC = CH, J = 15.3 Hz),
7.29–7.39 (m, 3H, ArH), 7.61–7.66 (d, 1H, HC = CH, J = 15.3 Hz), 7.73
(s, 1H, imidazole); 13C NMR (75 MHz, CDCl3): δ (ppm) 15.00, 27.91,
47.98, 56.36, 60.97, 105.83, 118.31, 126.24, 128.05, 129.10, 129.52,
131.08, 133.36, 133.62, 135.55, 142.43, 148.19, 153.10, 188.10; LC-MS
(ESI): 439.5 (M + H)+.
4.2.12. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(naphthalen-2-
yl)prop-2-en-1-one (9g’)
Yield: 57%; mp = 103–105 ◦C; 1HNMR (300 MHz-DMSO‑d6): δ
(ppm)1.28–1.32 (t, 3H, CH3, J = 7.2 Hz), 3.14–3.22 (s, 2H, SCH2, J =
7.2 Hz), 5.45 (s, 2H, CH2), 7.10–7.12(m, 2H, ArH), 7.28–7.33(d, 1H, CH
= CH, J = 15.6 Hz), (7.37–7.42 (m, 2H, ArH), 7.60–7.70 (m, 3H, ArH),
7.82–7.87 (, 1H, CH = CH, J = 15.6 Hz) , 7.99–8.03 (m, 2H, ArH),
8.099–8.12 (m, 2H, ArH); 13CNMR(75 MHz-DMSO‑d6): δ (ppm), 15.57,
27.64, 47.46, 119.115, 124.50, 126.56, 127.42, 128.17, 128.88, 129.05,
129.40, 129.65, 129.97, 130.32, 131.53, 132.72, 134.36, 135.43,
136.94, 147.38, 188.65; LC-MS (ESI): 399.4 (M + H)+.
4.3. Synthesis of (E)-methyl 1-benzyl-5-(3-oxo-3-(3,4,5-trimethoxy-
phenyl)prop-1-en-1-yl)-1H-imidazole-2-sulfinate (9k)
Compound 9j (300 mg, 0.7 mmol) was dissolved in THF (6 ml) and
1.5 g oxone in THF/water was added. The mixture was stirred at room
temperature for 3 h, after evaporation of THF, the residue was washed
with water and recrystallized in ethanol.
Yield: 87%; mp = 170–172 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 3.87 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 5.86 (s, 2H, CH2),
7.15–7.18 (d, 2H, ArH, J = 8.4 Hz), 7.29–7.32 (m, 1H, ArH), 7.33–7.36
(d, 2H, ArH, J = 6.9 Hz), 7.37–7.42 (m, 2H, ArH), 7.51–7.56 (d, 1H, HC
= CH, J = 15.3 Hz), 7.92–7.97 (d, 1H, HC = CH, J = 15.3 Hz); 13CNMR
(75 MHz-DMSO‑d6): δ (ppm), 43.55, 48.35, 56.69, 60.68, 106.71,
124.38, 126.64, 128.16, 128.35, 129.38, 131.62, 132.83, 133.75,
136.62, 142.76, 145.75, 153.40; LC-MS (ESI): 457.5 (M + H)+, 479.4
(M + Na)+.
4.2.13. (E)-1-([1,1′-biphenyl]-4-yl)-3-(1-benzyl-2-(ethylthio)-1H-
imidazol-4-yl)prop-2-en-1-one (9h’)
Yield: 51%; mp = 198–200 ◦C; 1HNMR (300 MHz-DMSO‑d6): δ
(ppm)1.26–1.31 (t, 3H, CH3, J = 7.2 Hz), 3.24–3.31 (q, 2H, SCH2, J =
7.2 Hz), 5.54 (s, 2H, CH2), 7.32–7.51 (m, 6H, ArH), 7.52–7.60 (d, 1H,
CH = CH, J = 15.6 Hz), 7.732–7.88 (m, 3H, ArH), 7.88–7.93 (d, 1H, CH
= CH, J = 15.6 Hz), 8.12–8.152 (d, 2H, J = 8.7 Hz), 8.262–8.42 (s, 1H,
imidazole); 13CNMR(75 MHz-DMSO‑d6): δ (ppm), 15.43, 28.47, 48.07,
126.72, 127.33, 127.45, 127.512, 128.42, 128.49, 129.37, 129.47,
129.572, 129.619, 131.944, 135.944, 135.54, 138.311, 145.103,
188.09; LC-MS (ESI): 425.3 (M + H)+.
4.4. Biological evaluation
4.4.1. Antiproliferative activity assay
4.2.14. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(4-
methoxyphenyl) prop-2-en-1-one (9i)
To evaluate the antiproliferative activity, several human cancer cell
lines, including A549 (adenocarcinoma human alveolar basal epithelial
cells), MCF-7 (human breast cancer cells), MCF-7/MX (mitoxantrone
resistant human breast cancer cells), and HEPG2 (human hepatocellular
carcinoma cells) were grown in RPMI complete culture medium and
then with a density of 5 × 103 cells/mL of the culture medium were
seeded into 96-well plates and incubated at 37 ◦C in a 5% CO2 incubator.
After 24 h, the culture medium was substituted with a medium having
positive control CA-4 as well as diverse concentrations of new synthe-
sized compounds and RPMI as the negative control. After incubation at
37 ◦C for 48 h, the cell viability was determined using the MTT assay.
The IC50 was defined as the compound concentration required to inhibit
cell proliferation by 50%. The data were analyzed with GraphPad Prism
[47].
Yield: 71%; mp = 152–154 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 2.67 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 5.27 (s, 2H, CH2),
6.90–6.93 (d, 2H, ArH, J = 8.7 Hz), 7.11–7.13 (d, 2H, ArH, J = 6.9 Hz),
7.26–7.38 (m, 4H, ArH), 7.56–7.62 (d, 1H, HC = CH, J = 15.3 Hz), 7.70
(s, 1H, imidazole), 7.85–7.88 (d, 2H, ArH, J = 8.7 Hz); 13C NMR (75
MHz, CDCl3): δ (ppm) 15.56, 47.89, 55.48, 113.79, 118.93, 126.37,
128.08, 128.54, 129.10, 130.59, 130.92, 131.39, 132.70, 135.46,
148.65, 163.37, 187.60; LC-MS(ESI): 365.4 (M + H)+, 387.4 (M + Na)+.
4.2.15. (E)-3-(1-benzyl-2-(ethylthio)-1H-imidazol-4-yl)-1-(4-
methoxyphenyl) prop-2-en-1-one (9i’)
Yield: 78%; mp = 143–145 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 1.35–1.39 (t, 3H, CH3, J = 7.3 Hz), 3.16–3.23 (q, 2H, CH2, J = 7.3
Hz), 3.86 (s, 3H, OCH3), 5.30 (s, 2H, CH2), 6.90–6.93 (d, 2H, ArH, J =
8.7 Hz), 7.09–7.12 (d, 2H, ArH, J = 7.2 Hz), 7.26–7.38 (m, 4H, ArH),
7.56–7.61 (d, 1H, HC = CH, J = 15.3 Hz), 7.71 (s, 1H, imidazole),
7.85–7.88 (d, 2H, ArH, J = 9 Hz); 13C NMR (75 MHz, CDCl3): δ (ppm)
15.03, 27.99, 47.91, 55.48, 113.79, 118.94, 126.32, 128.02, 128.68,
129.07, 130.59, 130.93, 131.22, 132.91, 135.66, 147.62, 163.37,
187.66; LC-MS(ESI): 379.4 (M + H)+.
4.4.2. Tubulin polymerization assay
Tubulin polymerization assay was done by using a commercial kit
(cytoskeleton, cat. #BK011P). In brief, tubulin was suspended in ice-
cold G-PEM buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM
GTP, 20% (v/v) glycerol) and added to wells on a 96-well plate con-
taining 9g, 9j and 9j’ (at 50 and 100 µM concentration), Paclitaxel at 3
µM concentration, CA4 at 5 µM concentration and control (no drug),
then Samples were mixed well, and tubulin polymerization was moni-
tored by excitation at 360 nm and emission at 420 nm for 60 min at 1
min intervals [7].
4.2.16. (E)-3-(1-benzyl-2-(methylthio)-1H-imidazol-4-yl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (9j)
Yield: 81%; mp = 171–173 ◦C; 1H NMR(300 MHz-DMSO‑d6): δ
(ppm) 2.67 (s, 3H, CH3), 3.86 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 5.28 (s,
2H, CH2), 7.11–7.13 (t, 3H, ArH, J = 6.6 Hz), 7.18–7.23 (d, 1H, HC =
CH, J = 15.6 Hz), 7.28–7.38 (m, 3H, ArH), 7.60–7.66 (d, 1H, HC = CH, J
= 15.6 Hz), 7.71 (s, 1H, imidazole); 13C NMR (75 MHz, CDCl3): δ (ppm)
15.45, 47.94, 56.35, 60.96, 105.82, 118.21, 126.27, 128.10, 129.13,
4.4.3. Cell cycle analysis using flow cytometry
A549 cells were seeded into 6-well plates (2.5 × 10 5 cells/well) for
24 h, then treated with different concentrations of compounds 9j’ and 9g
and vehicle alone (0.05% DMSO) and were incubated for 48 h. After the
cells were washed with PBS and fixed with 70% ethanol, then washed
10

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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
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Acknowledgment
We thank the research deputy of Mashhad university of medical
sciences for the financial support of this study (as part of the thesis of
Sara Rahimzadeh Oskuee).
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