New thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2019.111784

A new series of thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety were synthesized as diaryl-heterocylic analogs of combretastatin A-4 with anticancer activity. The cytotoxicity evaluation of synthesized compounds against cancer cell lin

Full text of DOI:10.1016/j.ejmech.2019.111784

Journal Pre-proof
New thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety as
anticancer agents
Mahsa Ansari, Mohammad Shokrzadeh, Saeed Karima, Shima Rajaei, Marjan Fallah,
Nasrin Ghassemi Barghi, Majid Ghasemian, Saeed Emami
PII:
S0223-5234(19)30936-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111784
EJMECH 111784
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 June 2019
Revised Date: 10 October 2019
Accepted Date: 10 October 2019
Please cite this article as: M. Ansari, M. Shokrzadeh, S. Karima, S. Rajaei, M. Fallah, N.G. Barghi,
M. Ghasemian, S. Emami, New thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety
as anticancer agents, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111784.
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Graphical Abstract

New thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety as anticancer
agents
a
Mahsa Ansari , Mohammad Shokrzadeh b, Saeed Karima c, Shima Rajaei c, Marjan Fallah b,
Nasrin Ghassemi barghi b, Majid Ghasemian c, Saeed Emami d,*
a
Pharmaceutical Sciences Research Center, Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
b Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical
Sciences, Sari, Iran
c Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical
Sciences (SBMU), Tehran, Iran
d Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A new series of thiazole-2(3H)-thiones containing 4-(3,4,5-trimethoxyphenyl) moiety were
synthesized as diaryl-heterocylic analogs of combretastatin A-4 with anticancer activity. The cytotoxicity
evaluation of synthesized compounds against cancer cell lines (A549, MCF-7 and SKOV3) revealed that
most of them had potent cytotoxic activity toward all tested cell lines (IC50s <10 µg/mL). Among them, 3-
(chlorobenzyl) derivatives 5c and 5d showed the best inhibitory effect on MCF-7 cells (IC50 values of
1.14 and 2.41 µg/mL, respectively). Furthermore, the ability of tubulin polymerization inhibition and
apoptosis induction were evaluated for the promising compounds 5c and 5d. Results suggested that these
compounds remarkably inhibit tubulin polymerization and induce apoptosis resulting in cell death. In
vitro studies revealed that these compounds had no significant cytotoxicity against normal cells at the
concentrations required for growth inhibition of cancer cells. In vitro biding assay and in silico docking
study also confirmed the binding of prototype compound to the colchicine binding site of tubulin.
Keywords: Anticancer agents; Thiazole-2(3H)-thione; Cytotoxic activity; 3,4,5-trimethoxyphenyl;
Tubulin polymerization
* Corresponding author. Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University
of Medical Sciences, Sari, Iran. E-mail: sdemami12@gmail.com; semami@mazums.ac.ir
1

1. Introduction
Nowadays, cancer after cardiovascular disease has become one of the most prominent factors of
death worldwide. Deaths due to cancer are globally about 17% of total human deaths [1]. The
lack of efficient and safe drugs is a driving force for continuous efforts to discovery of new
anticancer agents [2].
Microtubule/tubulin is a promising target for design and development of new anticancer agents.
Microtubules are major components of the cytoskeleton and are formed by tubulin
polymerization during interphase which have pivotal role in division of cells [3,4]. Hereupon,
diverse molecules inhibiting tubulin assembly and suppressing microtubule formation have been
synthesized and reported as anti-mitotic agents [5,6]. One of the most potent anti-mitotic agents
which soon gained the attention of medicinal chemists and cancer biologists is combretastatin A-
4 (CA-4, Fig. 1), a trimethoxyphenyl (TMP) containing stilbenoid that first was found in
Combretum caffrum [7]. CA-4 belongs to a group of tubulin binding agents which inhibits
intensely tubulin polymerization and disrupts microtubule dynamics by binding to the colchicine
binding site (CBS) in beta tubulin. Other well-known colchicine binding drugs containing TMP
moiety are AVE8062, and BCN-105P (Fig. 1) [8,9].
Figure 1. Structures of some potent anti-mitotic agents targeting CBS of tubulin.
2

SAR studies on CA-4 demonstrated that both TMP moiety and cis configuration in alkene bridge
which connects two aromatic rings are essential structural features and required for the activity
of this compound [10]. Since olefinic double bond in CA-4 is instable and undergoes geometrical
isomerization, considerable efforts have been made to modify the structure of CA-4, maximizing
its stability and bioavailability. Replacement of the olefinic bridge with diverse heterocyclic
rings such as thiophene, tetrazole, 1,2,3-triazole, 1,2,3-thiadiazole, 1,3-oxazole, and oxazolone
was the main modifications [11,12]. In particular, Nam et al. have reported two series of
combretoxazolones including 3,4-diaryloxazolones and 4,5-diaryloxazolones as potent antitumor
agents. The representative compound A (Fig. 2) with IC50 values of 1.1-4.3 nM showed potent
cytotoxic activity against B16, MCF-7, HCT116, A549, and PC-3 cell lines. This compound had
effective antitumor activity at the dose of 30 mg/kg/day in BDF1 mice bearing B16 murine
melanoma cells [13].
Figure 2. Structures of lead compounds (A and B) and newly designed compounds.
In continuation of our works on tubulin-targeting anticancer agents [14-17] we have recently,
designed and synthesized several 3-(TMP)-2(3H)-thiazole thiones B as new CA-4-modified
analogs. These compounds are combretoxazolone analogs in which both oxygen atoms of central
3

heterocyclic ring have been replaced with sulfur atoms [18]. This isosteric replacement could
affect the size and electronic character of central ring, resulting in possible alteration of binding
properties and increase of lipophilicity of the new designed molecules namely thiazole thiones B.
The cytotoxicity assay of compounds B toward T47D, MCF-7 and MDA-MB-231 cancer cell
lines revealed that 4-methyl derivative (R= 4-Me) with IC50 values of 11.8-19.7 µg/mL had the
highest activity against all tested cell lines. In conductance with our previous publications, 4-
(TMP)-thiazole-2(3H)-thione derivatives (Fig. 2) as new series of anticancer agents targeting
tubulin were designed and in these new series of compounds, the TMP moiety has been
displaced from position 3 to position 4 of thiazole-2(3H)-thione core structure.
2. Results and discussion
2.1. Chemistry
The synthetic route followed for the preparation of 4-(TMP)thiazole-2(3H)-thiones 5a-h was
illustrated in Scheme 1. Firstly, treatment of 3,4,5-trimethoxyacetophenone (1) with cupper (II)
bromide in refluxing chloroform-ethyl acetate afforded α-bromo-3,4,5-trimethoxyacetophenone
(2) [19]. In the next step, the in situ generated cabodithioate form the reaction of different
primary amines (3) with CS2 in the presence of potassium acetate, was condensed with
compound 2 to give 4-hydroxythiazolidine-2-thione intermediates 4a-e [20]. Subsequently, these
alcoholic compounds were refluxed with HCl 0.5% in methanol to yield pure target compounds
5a-f [21]. On the other hand, the appropriate N-aryldithiocarbamate salt (6) prepared according
to the literature method [21], was treated with α-bromo- compound 2 in absolute ethanol at room
temperature. After addition of hydroxylamine hydrochloride (as catalyst), the reaction mixture
was heated under microwave irradiation (T1= 150 ºC, t1= 15 min, t2= 30 min). In these cases, the
obtained products were characterized to be corresponding 4-(TMP)thiazole-2(3H)-thiones 5g,h.
4

S
S
Br
R
N
MeO
MeO
MeO
MeO
MeO
MeO
O
O
a, 86%
b, 65-77%
OH
4a: R= H
4b: R= Ph
4c: R= 2-Cl-Ph
4d R= 4-Cl-Ph
4e: R= 4-F-Ph
NH2
R
OMe
OMe
OMe
3
1
2
4a-e
H
N
S
HNEt3
d, 62-67%
R
c, 63-88%
S
6
S
S
R
R
N
S
N
S
MeO
MeO
MeO
MeO
5a: R= H
5b: R= Ph
5c: R= 2-Cl-Ph
5d R= 4-Cl-Ph
5e: R= 4-F-Ph
5f: R= 4-OH-Ph
OMe
OMe
5a-f
5g: R= 4-Cl
5h: R= 3,4-Cl2
Scheme 1. Synthesis of thiazolidine-2-thiones 4a-e and thiazole-2(3H)-thiones 5a-h. Reagents and conditions: (a)
CuBr2, CHCl3-EtOAc, reflux; (b) CS2, CH3COOK, MeOH, rt; (c) HCl 0.5%, MeOH, reflux; (d) absolute ethanol, rt,
and then HONH2.HCl, MW.
2.2. Biology
2.2.1. Cell viability assay
The cytotoxicity of all newly synthesized compounds 4a-e and 5a-h towards human cancer cell
lines A549 (lung), MCF-7 (breast) and SKOV3 (ovarian) was examined by employing the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [22]. Etoposide was
served as a positive control. The IC50 values of the tested compounds are reported in Table 1.
5

Table 1. Cytotoxicity (IC50 values, µg/mL) of compounds 4a-e and 5a-h against human cancer cell lines
(Note: the related IC50 values in µM are shown into parentheses below the values in µg/mL).
Compound
4a
A549
MCF-7
SKOV3
3.21 ± 0.41
(10.2)
8.23 ± 0.12
(26.1)
4.23 ± 0.14
(13.4)
5.02 ± 0.12
(12.8)
5.22 ± 0.17
(13.4)
5.38 ± 0.16
(13.8)
4b
6.19 ± 0.23
(14.6)
5.22 ± 0.10
(12.3)
5.13 ± 0.18
(12.1)
4
c
4.91 ± 0.14
(11.6)
7.97 ± 0.09
(18.8)
7.68 ± 0.28
(18.1)
4d
8.74 ± 0.22
(21.4)
4.92 ± 0.17
(12.0)
5.05 ± 0.22
(12.4)
4e
5.20 ± 0.24
(17.5)
8.22 ± 0.15
(27.7)
6.94 ± 0.13
(23.4)
5a
5b
4.52 ± 0.12
(12.1)
5.18 ± 0.22
(13.9)
6.52 ± 0.24
(17.5)
5.62 ± 0.23
(13.8)
1.14 ± 0.18
(2.8)
5.50 ± 0.18
(13.5)
5c
2.72 ± 0.15
(6.7)
2.41 ± 0.11
(5.9)
7.37 ± 0.08
(18.1)
5d
6.18 ± 0.16
(15.8)
3.91 ± 0.10
(10.0)
7.29 ± 0.16
(18.6)
5e
6.07 ± 0.22
(15.6)
8.58 ± 0.24
(22.0)
7.27 ± 0.18
(18.7)
5f
5g
6.04 ± 0.23
(15.4)
6.63 ± 0.08
(16.9)
6.95 ± 0.11
(17.7)
5.16 ± 0.10
(12.1)
4.11 ± 0.21
(9.6)
7.62 ± 0.25
(17.9)
5h
3.52 ± 0.23
(6.0)
3.37 ± 0.24
(5.7)
5.41 ± 0.06
(9.2)
Etoposide
The values are mean of three experiments.
Based on the observed results, the majority of compounds exhibited good cytotoxic activity,
displaying IC50 values in the range of 1.14-8.74 µg/mL. The reference drug etoposide showed
6

IC50 values between 3.37 and 5.41 µg/mL against tested cell lines. The highest inhibitions were
obtained with 3-(chlorobenzyl)thiazole thione derivatives 5c and 5d against MCF-7 cells,
displaying IC50 values of 1.41 and 2.41 µg/mL. The activity of 5c was better than that of
etoposide against MCF-7 cell line. Also, the 3-(4-chlorobenzyl)thiazole thione 5d was found to
be the most potent compound against A549 cell line (IC50 = 2.72 µg/mL). Nevertheless, the 3-
methyl derivative from 4-hydroxythiazolidine-2-thione series (compound 4a) was next potent
compound. Moreover, thiazolidine-2-thiones 4a-c, 4e along with thiazole-2(3H)-thione 5c were
found to be the most active derivatives against SKOV3 cells.
A survey on the IC50 values of benzyl analogs 4b-e and 5b-e against A549 and MCF-7 cells
revealed that the activity of thiazole-2(3H)-thiones is slightly better than that of their
corresponding intermediates 4-hydroxythiazolidine-2-thiones. In the case of SKOV3 cell line,
the differences between thiazole-2(3H)-thiones and 4-hydroxythiazolidine-2-thiones were not
significant, with the exception of 4-fluorophenyl derivatives 4e (IC50 = 5.05 µg/mL) and 5e (IC50
= 7.29 µg/mL). Furthermore, by comparing 3-(4-chlorobenzyl)thiazole thione 5d and 3-(4-
chlorophenyl)thiazole thione 5g, it can be concluded that the insertion of methylene between 4-
chlorophenyl moiety and thiazole-2(3H)-thione ring improves the potency towards A549 and
MCF-7 cancer cells.
As described in Introduction, firstly we have designed and synthesized 3,4-diarylthiazole-2(3H)-
thiones bearing a TMP moiety at 3-position [18]. Modification of substituents on 4-phenyl ring
resulted in 4-methyl derivative with promising activity against breast cancer cells (IC50s < 20
µg/mL), which should be improved using different strategy. Thus, we displaced the
pharmacophoric portion TMP from 3-position to 4-position of the central core thiazole-2(3H)-
thione to generate 4-(TMP)-thiazole-2(3H)-thiones. The obtained compounds in these series
showed significantly lower IC50 values especially against MCF-7 cells. For example, 4-(4-
chlorophenyl)- analog (7, Fig. 3) from 3-(TMP)thiazole thione series was inactive on MCF-7, its
reverse analog 5g had IC50 value of 6.63 µg/mL.
7

Figure 3. Structure-activity relationships of thiazole-2(3H)-thiones
Introduction of a methylene linker between 4-chlorophenyl and 4-(TMP)thiazole thione in
compound 5g resulted in compound 5d with improved potency (IC50= 2.41 µg/mL). Further
moving of chloro group from para to ortho position of benzyl moiety produced compound 5c
with highest activity against MCF-7 (Fig. 3). So, it proved that regional isomerization of 3-TMP
derivatives to 4-TMP and replacement of another aryl ring with benzyl moiety could
significantly increase the cytotoxic activity.
2.2.2. Effect of compounds 5c and 5d on normal cells L929 and red blood cells
Although all compounds showed pronounced cytotoxicity against cancer cell lines, but the
important issue that should be considered was selectivity of compounds. Therefore, the
representative compounds 5c and 5d were tested against normal cells L929 (mouse fibroblast
cell line) in vitro. As seen in Fig. 4, the viability of normal cells was not affected significantly at
low concentrations by these compounds. Even at high concentrations of 200 µg/mL, the percent
of viability was more than 50%. Accordingly, the IC50 values of compounds 5c and 5d are
determined to be > 200 µg/mL. Since the IC50 values of compounds 5c and 5d against cancer cell
lines were in the range of 1.14-7.37 µg/mL, thus they have acceptable selectivity against cancer
cells over normal cells. As investigated below, the possible mechanism of designed compounds
is inhibition of tubulin polymerization which has pivotal role in cell division. Thus, the increased
8

rate of cell division in cancer cells and significant divergent of metabolic pathways in cancer
cells make them more sensitive to anti-mitotic agents over the normal cells.
Some of the chemotherapeutic agents have potential toxicity due to hemolysis which results in
anemia [23]. Thus we studied the hemolytic activity of representative compounds 5c and 5d in
erythrocytes. Based on the results (Fig. 5), the hemolysis rate of the tested compounds was less
than 25% at the concentration of 10 µg/mL. Notably, compound 5d showed no significant
hemolytic activity at the concentrations of 1 and 5 µg/mL, compared to control. It should be
noted that the rates of hemolysis in negative control group (CT) and positive control (Triton X,
TX) were 6.2 and 100%, respectively.
Figure 4. Cytotoxic activity of compounds 5c and 5d toward normal cells L929 at the concentrations of 1, 10, 50
100 and 200 µg/mL in comparison with cisplatin (50 µg/mL). Each experiment was repeated three times and the
values were calculated as Mean ± SD. Significantly different from control (**P <0.01; ***P <0.001 ****P <
0.0001).
9

A
B
C
Figure 5. The rates of hemolysis induced by compounds 5c (A), 5d (B) and etoposide (C) in human erythrocytes at
the concentrations of 1, 5 and 10 µg/mL. The negative control (CT) consisted of erythrocytes treated with DMSO
and the positive control consisted of erythrocytes treated with Triton X-100 (TX, 2% v/v). Each experiment was
repeated three times and the values were calculated as Mean ± SD. Significantly different from CT (*P < 0.05;
****P < 0.0001). Significantly different from TX (####P < 0.0001).
2.2.3. Investigation of the genotoxicity of compounds 5c and 5d by comet assay
The comet assay is a simple and sensitive method based on single cell gel electrophoresis that
enables the detection of DNA damage in individual eukaryotic cells. In this method, the
electrophoresis of nucleic acids from a single cells immobilized in agarose layer, results in
migration of the DNA fragments in an electric field related to their mass, making a tail. The
10

percentage of fragmented DNA in the comet tail as well as the tail length and tail moment is
considered as parameters for evaluating potential genotoxicity of compounds [24,25].
Accordingly, the potential genotoxic effect of compounds 5c and 5d was explored by the
alkaline comet assay in normal cells L929. The compounds 5c and 5d were tested at the
concentration of 10 µg/mL and compared to etoposide (0.5 µg/mL) as positive control. As
parameters scored, we used tail length, % DNA in tail and tail moment, and the results were
presented in Fig. 6. Based on the results, etoposide (as reference drug) at lower concentration of
0.5 µg/mL showed significant levels of DNA damage in L929 cells. We observed that compound
5c at 10 µg/mL concentration didn’t induce a significant increase in DNA damage as compared
to the negative control group. On the other hand, compound 5d increased significantly (P < 0.01)
the level of DNA fragmentation in L929 cells as compared to the control group. However, the
level of DNA damage produced by compound 5d (10 µg/mL) was significantly less than that
caused by etoposide (0.5 µg/mL), indicating the lower genotoxicity of 5d.
Figure 6. Quantitative representation of the DNA damage evaluated by the comet assay in L929 cells as tail
parameters: (A) Tail length; (B) % DNA in tail and (C) tail moment. The cells were exposed to the compounds 5c
and 5d (10 µg/mL) and compared to etoposide (0.5 µg/mL) and negative control (untreated). Each graph has been
represented as Mean ± SEM. Significantly different from control (**P < 0.01; ***P < 0.001). Significantly different
from etoposide (####P < 0.0001).
11

2.2.4. Effect of compounds 5c and 5d on tubulin polymerization
To evaluate whether the novel synthesized 4-(TMP)-thiazole-2(3H)-thiones could target the
microtubule system also to elucidate the inhibitory effect of them on tubulin polymerization in
vitro, compounds 5c and 5d which showed better cytotoxic activities were chosen for further
evaluations. Colchicine was included as the reference drug and the polymerization was measured
according to the literature method [26, 27]. The obtained results indicated that these compounds
could inhibit tubulin polymerization at a concentration-dependent manner (Fig. 7). The IC50
values of compounds 5c and 5d were 5.14 and 9.97 µg/mL, respectively.
2.2.5. Colchicine-tubulin binding inhibition by compound 5c
Previous studies revealed that the TMP-derived tubulin inhibitors such as colchicine and
combretastatin A-4 can bind to the CBS in tubulin, resulting in microtubule depolymerization
[9]. In our study, the in vitro bioassay indicated that the prototype compounds 5c and 5d as
diaryl-heterocylic analogs of combretastatin A4 are also capable to inhibit tubulin
polymerization. In order to investigate the binding of ligand 5c to tubulin at the CBS, we used
the fluorescence assay of the colchicine-tubulin complex as reported previously [28-30]. In the
fluorescence assay, if a ligand has binding affinity to the CBS, colchicine molecule is displaced
by the ligand and the fluorescence intensity is reduced. Accordingly, we determined the changes
in the fluorescence of the colchicine-tubulin complex after exposure to compound 5c. Fig. 8
shows that the addition of compound 5c to the different mixtures of tubulin (0.25 µM) and
colchicine (0.1, 0.25, 0.5 or 1 µM) significantly reduces the relative fluorescence intensity
(F/F0). These findings indicate that compound 5c has specific affinity to the colchicine pocket
and prevents the binding of colchicine to tubulin.
12

Figure 7. Tubulin polymerization inhibitory activity of compounds 5c and 5d.
Figure 8. Inhibition of the binding of colchicine to tubulin by ligand 5c. The concentrations of colchicine (Col) are
presented in the figure.
13

2.2.6. Cell cycle analysis
The effect of compounds 5c and 5d on cell division in MCF-7 cell line was investigated by
determining the distribution of the cells in different phases of the cell cycle using flow
cytometric technique. The results show that compounds 5c and 5d significantly increased the
number of cells in the sub-G1 (Fig. 9). The percentage of cells in the sub-G1 after treatment with
5c and 5d increased from 0.66 to 14.74 and 17.28%, respectively. As the cells were treated with
5c and 5d, the percentage of cell in G1 phase reached from 44.45 to 78.02 and 54.57
respectively. Therefore, a significant increase in G1 phase cell with decrease percentage of cells
in G2/M phase, revealed that this compounds can arrest cell cycle in G1 and S phase.
Control
5c
5d
Sub-G1: 0.66
G1: 44.45
S: 38.2
Sub-G1: 14.74
G1: 78.02
S: 7.17
Sub-G1: 17.28
G1: 54.57
S: 22.43
G2/M: 16.69
G2/M: 0.07
G2/M: 5.72
Figure 9. Effects of compounds 5c and 5d on cell cycle in MCF-7 cells. The cells were treated with compounds 5c
and 5d (12µg/mL) for 48 h. The control group was untreated cells.
2.2.7. Annexin-V binding assay
Flow cytometry assay using propidium iodide (PI) and protein Annexin-V in SKOV3 cells was
carried out to evaluate the potential of 5c and 5d to induce apoptosis in cancer cells [31]. The
SKOV3 cells were treated with different concentrations (2 and 4 µg/mL) of compounds 5c and
5d for 48 h. Then the cells were stained with the Annexin-V-FITC apoptosis detection kit. As
shown in Fig. 10, quadrant plots have been divided into four regions labeled Q1–Q4
14

(Q1 = necrotic cells, Q2 = late-apoptotic cells, Q3 = viable cells, and Q4 = early-apoptotic cells).
The total percent of apoptotic cells are obtained by summation of Q2 and Q4. According to these
plots, compounds 5c and 5d relatively induce apoptosis in SKOV3 cells in a concentration-
dependent manner. Results demonstrated the rise in the percentage of apoptosis from 18.54 and
19.75 in 2 µg/mL to 27.41 and 21.69 in 4 µg/mL, respectively.
2.3. Molecular modeling for the tubulin-binding
Molecular docking studies were accomplished to further confirm the capability of the promising
compounds 5c and 5d for binding to the CBS of tubulin. AutoDock software (version 4.2) was
used for this purpose. The coordinates of the target co-crystallized with colchicine were extracted
from the Protein Data Bank (PDB ID: 4O2B). Colchicine was firstly re-docked into the pocket of
the tubulin to validate the docking protocol.
Docking poses shown in Fig. 11a and Fig. 11c indicate that the TMP scaffold in both 5c and 5d
was interred in the hydrophobic pocket of the CBSin the β-chain and surrounded by the residues
of amino acids such as βAsn249, βAsn258, βAla250, βLys254, βleu255, and βLys352, similar to
that of colchicine. 2D representation of compound 5c displayed two hydrogen bonding
interactions between the methoxy group and amino acid residues βAsn258 and βAla317. Besides
this, binding mode of 5c also established a pi-sigma interaction between the thiazole thione ring
and βLeu248 and two pi-alkyl interactions between 2-chlorophenyl moiety and amino acid
residues βLys352 and βAla316 in addition to some pi-sulfur and sulfur-x interactions with the
residues of βAla354 and βMet259, respectively. Compound 5d, showed one hydrogen bonding
interaction between the methoxy group and βAsn258. Furthermore, as can be seen in Fig. 11d,
several secondary interactions like van der Waal and polar interactions with the residues of
βLeu255, βVal315, βThr314, βAla317, βLys245 and βThr353 were involved. Further van der
Waal interaction between βLeu248 and central thiazole thione ring was a pi-sigma interaction.
The p-chlorophenyl group of 5d was perfectly cinctured by several alkyl and pi-alkyl
interactions with the hydrophobic residues of βAla354, βMet259, βLys352 and βAla316.
15

Figure 10. Flow cytometry of 5c- or 5d-treated SKOV3 cells at the doses of 2 and 4 µg/mL after 48 h; A: 5c (2
µg/mL), B: 5c (4 µg/mL), C: 5d (2 µg/mL), D: 5d (4 µg/mL).
16

A
B
C
D
Figure 11. 3D and 2D presentations for the binding modes of compounds 5c (A, C), and 5d (B, D) with tubulin
(PDB ID: 4O2B).
17

3. Conclusion
In the field of anticancer drug design and discovery, combretastatin-modified analogs have been
considered as attractive target molecules to find new anti-tubulin agents. The hit molecules
namely 3-(TMP)-2(3H)-thiazole thiones were a starting point for finding anti-tubulin agents
derived from combretastatin with 2(3H)-thiazole thione core structure. In this work, we
improved the anticancer activity of primary hits by displacing TMP moiety on the 2(3H)-thiazole
thione to introduce 4-(TMP)-2(3H)-thiazole thiones as new lead compounds. The cytotoxicity
assay against cancer cell lines (A549, MCF-7 and SKOV3) revealed that all compounds had
remarkable cytotoxic activity (IC50 values <10 µg/mL). Among them, the 3-(2-chlorobenzyl)-
and 3-(4-chlorobenzyl)- analogs (compounds 5c and 5d, respectively) with IC50 values of 1.14
and 2.41 µg/mL, exhibited the highest inhibitory activity against MCF-7 breast cancer cell line.
The in vitro tubulin polymerization assay proved that compounds 5c and 5d could interfere with
microtubule assembly with an IC50 <10 µg/mL. The in vitro colchicine-tubulin binding assay and
molecular modeling study indicated that these compounds bind at the CBS of the tubulin. The
representative compounds 5c and 5d had no significant effect on the viability of tested normal
cells (L929) at the range of concentrations required for the inhibition of cancer cell proliferation,
indicating the selective action and good safety profile of the designed compounds.
4. Experimental
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. The triethylammonium phenylcarbamodithioate salts 6 were prepared
according to the literature method [21].
4.1.1. Synthesis of compound 2
To a warm and well-stirred suspension of powdered cupper (II) bromide (10 mmol, 2.23 g) in
CHCl3-EtOAc (20 mL, 1:1), a solution of 1-(3,4,5-trimethoxyphenyl)ethan-1-one (1, 10 mmol,
18

2.10 g) in CHCl3 (10 mL) was added, and the reaction mixture was refluxed for 4 h. After
completion of the reaction, the reaction mixture was filtered to remove produced cupper (I)
bromide and the solution was evaporated using rotary evaporator under vacuum. Then the oily
product was crystallized from hexane-ether to afford pure compound 2. Yield: 86%; m.p.: 65-67
°C; 1H NMR (400 MHz, CDCl3) δ: 3.93 (s, 6H, 2×OMe), 3.94 (s, 3H, OMe), 4.41 (s, 2H, CH2),
13
7.24 (s, 2H, H-2 and H-6 Ar). C NMR (100 MHz, CDCl3) δ: 190.32, 153.25, 143.42, 128.99,
106.65, 61.11, 56.39, 30.62. EI-MS (m/z, %): 290 (M+2, 18), 288 (M+, 17), 196 (15), 195 (100),
181 (10), 137 (8).
4.1.2. General procedure for the preparation of compounds 4a-e
A mixture of methylamine (40% solution in water, 2 mmol) or benzylamine derivatives (3, 2
mmol) and potassium acetate (2 mmol, 196.3 mg) in methanol (3 mL) was stirred in an ice bath
following by addition of carbon disulfide (2 mmol, 152 mg, 0.12 mL). After 2h, compound 2 (1
mmol, 289 mg) was added portion wise during 30 min. After consumption of compound 2
(monitored by TLC), water (10 mL) was added and the mixture was kept in refrigerator
overnight to complete precipitation. The solid product was filtrated, washed with water and dried
to give alcoholic intermediates 4a-e.
4-Hydroxy-3-methyl-4-(3,4,5-trimethoxyphenyl)thiazolidine-2-thione (4a)
Yield: 72%; m.p.: 110-112 °C; IR (KBr, cm-1): 3352, 2833, 1656, 1595, 1505, 1437, 1332, 1129,
1066, 869, 678. 1H NMR (400 MHz, CDCl3) δ: 3.05 (s, 3H, NMe), 3.52 (d, 1H, J= 12.4 Hz, Ha),
3.73 (d, 1H, J= 12.4 Hz, Hb), 3.89 (s, 3H, OMe), 3.90 (s, 6H, 2×OMe), 6.70 (s, 2H, H-2 and H-
6). 13C NMR (100 MHz, DMSO-d6) δ: 195.32, 153.35, 137.97, 136.99, 103.23, 100.00, 60.46,
56.36, 42.93, 32.86. EI-MS (m/z, %): 315 (M+, 6), 297 (30), 282 (16), 242 (38), 210 (6), 195
(100), 181 (9), 167 (7), 152 (14), 137 (9), 122 (6), 104 (14), 73 (42).
3-Benzyl-4-hydroxy-4-(3,4,5-trimethoxyphenyl)thiazolidine-2-thione (4b)
Yield: 77%; m.p.: 150-152 °C; IR (KBr, cm-1): 3405, 3086, 2829, 1594, 1400, 1318, 1239, 1126,
997, 841, 702, 465. 1H NMR (400 MHz, CDCl3) δ: 3.34 (s, 1H, OH), 3.42 (d, 1H, J= 12.4 Hz,
19

Ha), 3.73 (s, 6H, 2×OMe), 3.77 (d, 1H, J= 12.4 Hz, Hb), 3.87 (s, 3H, OMe), 4.84 (d, 1H, J= 14.8
Hz, CH2 benzyl), 4.91 (d, 1H, J= 14.8 Hz, CH2 benzyl), 6.57 (s, 2H, H-2 and H-6), 7.14-7.19 (m,
2H, H-2′ and H-6′), 7.20-7.25 (m, 3H, H-3′, H-4′ and H-5′). 13C NMR (100 MHz, DMSO-d6) δ:
197.14, 152.76, 137.91, 137.51, 136.05, 128.58, 128.06, 126.99, 104.21, 100.22, 60.35, 56.11,
49.07, 42.77. EI-MS (m/z, %): 391 (M+< 1), 373 (100), 358 (13), 340 (66), 282 (16), 267 (6),
251 (78), 236 (16), 224 (30), 209 (20), 195 (18), 181 (10), 150 (9), 121 (6), 91 (93), 65 (13).
3-(2-Chlorobenzyl)-4-hydroxy-4-(3,4,5-trimethoxyphenyl)thiazolidine-2-thione (4c)
Yield: 65%; m.p.: 162-164 °C; IR (KBr, cm-1): 2965, 2926, 1593, 1509, 1461, 1443, 1343, 1126,
974, 889, 748. 1H NMR (400 MHz, CDCl3) δ: 3.44 (d, 1H, J= 12.4 Hz, Ha), 3.74 (s, 3H, OMe),
3.79 (d, 1H, J= 12.8 Hz, Hb), 3.96 (s, 6H, 2×OMe), 4.87 (s, 2H, CH2 benzyl), 6.61 (s, 2H, H-2
and H-6), 7.04 (d, 1H, J= 6.8 Hz, H-6′), 7.12-7.32 (m, 2H, H-4′ and H-5′), 7.35 (d, 1H, J= 6.4,
H-3′). 13C NMR (100 MHz, DMSO-d6) δ: 197.80, 152.72, 137.88, 135.67, 133.82, 131.44,
129.26, 129.11, 128.68, 126.95, 104.11, 100.08, 60.30, 56.17, 46.49, 42.71. EI-MS (m/z, %): 425
(M+< 1), 372 (4), 289 (11), 242 (14), 195 (79), 184 (21), 152 (7), 125 (100), 109 (5), 89 (19), 63
(8).
3-(4-Chlorobenzyl)-4-hydroxy-4-(3,4,5-trimethoxyphenyl)thiazolidine-2-thione (4d)
Yield: 69%; m.p.: 158-160 °C; IR (KBr, cm-1): 3254, 3014, 2941, 1597, 1509, 1444, 1416, 1399,
1
1233, 958, 665. H NMR (400 MHz, CDCl3) δ: 3.45 (d, 1H, J= 12.4 Hz, Ha), 3.52 (br s, 1H,
OH), 3.75 (s, 6H, 2×OMe), 3.81 (d, 1H, J= 12.4 Hz, Hb), 3.88 (s, 3H, OMe), 4.77 (d, 1H, J= 14.8
Hz, CH2 benzyl), 4.90 (d, 1H, J= 14.8 Hz, CH2 benzyl), 6.57 (s, 2H, H-2 and H-6), 7.12 (d, 2H,
J= 8.8 Hz, H-2′ and H-6′), 7.18 (d, 2H, J= 8.8 Hz, H-3′ and H-5′). 13C NMR (100 MHz, DMSO-
d6) δ: 197.13, 152.79, 137.93, 136.52, 135.99, 131.63, 129.92, 128.01, 104.21, 99.98, 60.33,
56.08, 48.29, 42.75. EI-MS (m/z, %): 425 (M+< 1), 242 (21), 195 (100), 183 (15), 167 (3), 152
(6), 137 (4), 125 (80), 109 (4), 89 (15), 77 (4), 63 (6).
3-(4-Fluorobenzyl)-4-hydroxy-4-(3,4,5-trimethoxyphenyl)thiazolidine-2-thione (4e)
Yield: 69%; m.p.: 146-148 °C; IR (KBr, cm-1): 340, 2967, 1601, 1509, 1439, 1318, 1167, 1125,
1
851. H NMR (400 MHz, CDCl3) δ: 3.44 (br s, 1H, OH), 3.45 (d, 1H, J= 12.4 Hz, Ha), 3.76 (s,
20

6H, 2×OMe), 3.80 (d, 1H, J= 12.8 Hz, Hb), 3.88 (s, 3H, OMe), 4.72 (s, 1H, CH2 benzyl), 4.84 (d,
1H, J= 2.8 Hz, CH2 benzyl), 6.59 (s, 2H, H-2 and H-6), 6.90 (t, 2H, J= 8.4 Hz, H-3′ and H-5′),
7.17 (dd, 2H, J= 8.4 and 5.6 Hz, H-2′ and H-6′). 13C NMR (100 MHz, CDCl3) δ: 198.06, 162.09
(d, JC,F= 245.4 Hz), 153.17, 138.50, 133.73, 132.88 (d, JC,F= 3.5 Hz), 130.40 (d, JC,F= 7.9 Hz),
115.05 (d, JC,F= 21.3 Hz), 103.61, 100.06, 60.92, 56.09, 48.64, 44.04. EI-MS (m/z, %): 409 (M+<
1), 391 (5), 358 (3), 292 (32), 242 (17), 210 (4), 195 (100), 183 (11), 167 (5), 152 (8), 124 (67),
109 (74), 83 (11), 66 (3).
4.1.3. General procedure for the preparation of compounds 5a-f
A solution of corresponding alcoholic compound 4 in methanol (3 mL) was treated with 0.5%
HCl in water (10 mL). The mixture was refluxed for 2-4 h to completing the reaction (checked
by TLC). Then the mixture was concentrated under reduced pressure, cooled to room
temperature and kept in refrigerator (4 °C) overnight. The precipitated solid was separated by
filtration, and washed with water to give pure final compounds 5a-f.
3-Methyl-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5a)
Yield: 88%; m.p.: 116-120 °C; IR (KBr, cm-1): 2932, 2833, 1584, 1500, 1307, 1241, 1127, 1182,
1
978, 835, 670. H NMR (400 MHz, CDCl3) δ: 3.61 (s, 3H, Me), 3.91 (s, 6H, 2×OMe), 3.93 (s,
3H, OMe), 6.51 (s, 1H, H-5 Thiazoline), 6.55 (s, 2H, H-2 and H-6). 13C NMR (100 MHz,
CDCl3) δ: 188.16, 153.62, 144.71, 139.42, 126.02, 108.23, 106.49, 60.98, 56.34, 36.28. EI-MS
(m/z, %): 297 (M+, 6), 256 (56), 224 (7), 192 (36), 160 (49), 128 (53), 96 (27), 64 (100).
3-Benzyl-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5b)
Yield: 73%; m.p.: 132-134 °C; IR (KBr, cm-1): 2934, 2835, 1580, 1500, 1409, 1364, 1236, 1192,
1
1124, 1030, 986, 831, 694. H NMR (500 MHz, CDCl3) δ: 3.58 (s, 6H, 2×OMe), 3.86 (s, 3H,
OMe), 5.38 (s, 2H, CH2 benzyl), 6.25 (s, 2H, H-2 and H-6), 6.51 (s, 1H, H-5 Thiazoline), 7.02
13
(d, 2H, J= 7.0 Hz, H-2′ and H-6′), 7.20-7.32 (m, 3H, H-3′, H-4′and H-5′). C NMR (100 MHz,
DMSO-d6) δ: 188.25, 153.11, 144.70, 138.85, 136.29, 128.92, 127.63, 126.83, 125.94, 110.32,
107.41, 60.52, 56.15, 50.97. EI-MS (m/z, %): 373 (M+, 100), 358 (83), 340 (38), 282 (10), 251
21

(49), 236 (10), 224 (19), 209 (14), 195 (5), 181 (6), 166 (4), 150 (6), 135 (3), 121 (4), 106 (3), 91
(59), 65 (8).
3-(2-Chlorobenzyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5c)
Yield: 63%; m.p.: 147-149 °C; IR (KBr, cm-1): 3336, 2965, 1678, 156, 1504, 1442, 1329, 1242,
1
1123, 1117, 751. H NMR (400 MHz, CDCl3) δ: 3.60 (s, 6H, 2×OMe), 3.84 (s, 3H, OMe), 5.45
(s, 2H, CH2 benzyl), 6.27 (s, 2H, H-2 and H-6), 6.60 (s, 1H, H-5 Thiazoline), 7.04 (d, 1H, J= 6.8
Hz, H-6′), 7.19-7.33 (m, 2H, H-4′ and H-5′), 7.35 (dd, 1H, J= 7.6 and 1.2 Hz, H-3′). 13C NMR
(100 MHz, DMSO-d6) δ: 188.12, 162.06, 153.13, 148.22, 144.50, 133.20, 132.71, 131.21,
130.98, 130.11, 127.95, 125.33, 122.25, 107.13, 60.46, 56.13, 49.03. EI-MS (m/z, %): 407 (M+,
59), 392 (7), 372 (97), 356 (5), 339 (6), 312 (7), 297 (23), 282 (27), 266 (7), 251 (100), 236 (47),
208 (24), 193 (17), 178 (17), 163 (7), 142 (8), 125 (94), 107 (14), 89 (22), 63 (12).
3-(4-Chlorobenzyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5d)
Yield: 71%; m.p.: 138-140 °C; IR (KBr, cm-1): 3096, 2931, 1584, 1496, 1338, 1239, 1187, 1126,
986, 557. 1H NMR (400 MHz, CDCl3) δ: 3.67 (s, 6H, 2×OMe), 3.90 (s, 3H, OMe), 5.38 (s, 2H,
CH2 benzyl), 6.26 (s, 2H, H-2 and H-6), 6.53 (s, 1H, H-5 Thiazoline), 6.98 (d, 2H, J= 8.4 Hz, H-
2′ and H-6′), 7.27 (d, 2H, J= 8.4 Hz, H-3′ and H-5′). 13C NMR (100 MHz, CDCl3) δ: 189.11,
153.23, 144.60, 139.34, 134.33, 133.49, 128.72, 128.43, 125.47, 108.74, 106.74, 60.96, 56.00,
50.60. EI-MS (m/z, %): 407 (M+, 100), 392 (8), 374 (31), 339 (9), 282 (16), 267 (3), 251 (61),
236 (20), 224 (22), 209 (15), 193 (2), 181 (8), 150 (7), 125 (54), 89 (10), 63 (4).
3-(4-Fluorobenzyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5e)
Yield: 78%; m.p.: 128-130 °C; IR (KBr, cm-1): 3101, 2932, 1606, 1585, 1509, 1497, 1363, 1223,
1
1162, 1128, 768. H NMR (400 MHz, CDCl3) δ: 3.67 (s, 6H, 2×OMe), 3.90 (s, 3H, OMe), 5.38
(s, 2H, CH2 benzyl), 6.28 (s, 2H, H-2 and H-6), 6.53 (s, 1H, H-5 Thiazoline), 6.94-7.12 (m, 4H,
F-Ph). 13C NMR (100 MHz, DMSO-d6) δ: 188.22, 161.73 (d, JC,F= 242.6 Hz), 153.17, 144.54,
138.93, 132.40, 129.17, 125.95, 115.67 (d, JC,F= 21.2 Hz), 107.49, 60.59, 56.26, 50.24, 47.65.
EI-MS (m/z, %): 391 (M+, 67), 376 (5), 358 (27), 282 (10), 266 (3), 251 (44), 224 (15), 209 (11),
195 (3), 181 (6), 166 (3), 150 (5), 124 (4), 109 (100), 83 (10), 63 (3).
22

3-(4-Hydroxybenzyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5f)
Yield: 73%; m.p.: 113-115 °C; IR (KBr, cm-1): 3336, 2931, 1597, 1531, 1446, 1410, 1239, 1121,
883, 668. 1H NMR (400 MHz, CDCl3) δ: 3.67 (s, 6H, 2×OMe), 3.89 (s, 3H, OMe), 5.34 (s, 2H,
CH2 benzyl), 6.29 (s, 2H, H-2 and H-6), 6.51 (s, 1H, H-5 Thiazoline), 6.73 (d, 2H, J= 8.4 Hz, H-
3′ and H-5′), 6.89 (d, 2H, J= 8.4 Hz, H-2′ and H-6′). 13C NMR (100 MHz, DMSO-d6) δ: 196.68,
156.53, 152.75, 137.90, 136.16, 129.68, 127.84, 114.78, 104.25, 102.21, 60.41, 56.10, 48.61,
42.80. EI-MS (m/z, %): 389 (M+< 1), 256 (3), 242 (11), 210 (17), 195 (75), 181 (2), 165 (6), 152
(6), 137 (6), 122 (4), 107 (100), 91 (2), 77 (19), 64 (4), 51 (5).
4.1.4. Synthesis of 3-(4-chlorophenyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5g)
A suspension of triethylammonium (4-chlorophenyl)carbamodithioate (1 mmol, 305 mg) and
compound 2 (1 mmol, 289 mg) in absolute ethanol (5 mL) was stirred at room temperature for 3
h. After complete consumption of the starting materials, hydroxylamine hydrochloride (3 mmol,
207 mg) was added and the reaction was continued under microwave irradiation for 30 min.
Then, water (10 mL) was added and the mixture was kept in refrigerator overnight. The
precipitate was separated by filtration, washed with water and recrystallized from ethanol to give
pure compound 5g. Yield: 62%; m.p.: 123-125 °C; IR (KBr, cm-1): 3003, 2932, 1606, 1580,
1
1483, 1321, 1122, 1083, 987, 710, 466. H NMR (250 MHz, CDCl3) δ: 3.66 (s, 6H, 2×OMe),
3.82 (s, 3H, OMe), 6.12 (s, 1H, H-5 Thiazoline), 6.27 (s, 2H, H-2 and H-6), 7.07 (d, 2H, J= 7.2
Hz, H-2′, and H-6′), 7.40 (d, 2H, J= 7.2 Hz, H-3′ and H-5′). 13C NMR (100 MHz, DMSO-d6) δ:
189.58, 149.80, 148.42, 144.34, 138.36, 132.26, 131.45, 130.14, 122.71, 113.25, 111.67, 109.88,
55.83. EI-MS (m/z, %): 395 (M+2, 13), 393 (M+, 37), 377 (31), 362 (13), 304 (40), 256 (18), 211
(8), 193 (17), 169 (17), 149 (13), 127 (31), 111 (40), 85 (66), 69 (82), 57 (100).
4.1.5. Synthesis of 3-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxyphenyl)thiazole-2(3H)-thione (5h)
A suspension of triethylammonium (3,4-dichlorophenyl)carbamodithioate (1 mmol, 339 mg) and
compound 2 (1 mmol, 289 mg) in absolute ethanol (5 mL) was stirred at rt for 3 h. After
23

complete consumption of the starting materials, the reaction mixture was refluxed for 48 h. Then,
water (10 mL) was added and the mixture was kept in refrigerator overnight. The precipitate was
separated by filtration, washed with water and recrystallized from ethanol to give pure compound
5h. Yield: 67%; m.p.: 180-182 °C; IR (KBr, cm-1): 2968, 2836, 1656, 1582, 1505, 1414, 1320,
1
1243, 1128, 1033, 992, 755. H NMR (400 MHz, CDCl3) δ: 3.71 (s, 6H, 2×OMe), 3.84 (s, 3H,
OMe), 6.29 (s, 2H, H-2 and H-6), 6.65 (s, 1H, H-5 Thiazoline), 7.07 (d, 1H, J= 8.4 Hz, H-6′),
7.42 (s, 1H, H-2′), 7.50 (d, 1H, J= 8.4 Hz, H-5′). 13C NMR (100 MHz, CDCl3) δ: 193.28, 153.16,
143.22, 138.90, 134.56, 132.56, 131.01, 130.50, 129.21, 126.80, 123.65, 106.31, 61.02, 56.34.
EI-MS (m/z, %): 427 (M+< 1), 281 (8), 207 (70), 195 (57), 181 (8), 161 (17), 146 (10), 135 (29),
119 (16), 105 (83), 91 (24), 77 (100), 64 (80), 51 (55).
4.2. Biological tests
4.2.1. Cell-based assays
4.2.1.1. Drug/sample solution preparation for cell-based assays
The stock solution of compounds and standard drugs was prepared in DMSO. In the cell viability
assay the stock solution was diluted to the desired concentrations in culture medium to reach the
final concentrations of 100, 50, 10, 5 and 1 µg/mL. The final concentration of DMSO in culture
medium was less than 1%, which had no effect on cell viability. In the hemolysis assay, an
aliquot (10 µL) of stock solution was added to diluted erythrocytes (190 µL), resulting final
concentrations of 1, 5 and 10 µg/mL. The selected concentrations for other tests were mentioned
in each corresponding section.
4.2.1.2. Cytotoxicity assay
Three human cancer cell lines (A549, MCF-7 and SKOV3) and normal cells L929 were
purchased from Pasture Institute, Tehran (Iran). The cytotoxic activities of thiazolidine-2-thiones
4a-e and thiazole-2(3H)-thiones 5a-h were evaluated in comparison with reference drug
24

etoposide by using MTT assay [22]. The detailed procedure can be followed in Supplementary
Material.
4.2.1.3. Hemolysis assay
The in vitro hemolytic activity of compound 5c and 5d was determined against human red blood
cells according to the reported method as described in Supplementary Material [32].
4.2.1.4. Cell cycle analysis
MCF-7 cells were cultured in 12-well plates with optimum density (25 ×104 cells/well). After 24
h, cells were treated with 12µg/mL of compounds 5c and 5d and incubated for 48 h. After
harvesting, cells were washed with cold PBS twice, suspended in 70% ethanol (for cell fixation)
at least for 3 h. Afterward, 5 µL of RNase A (10 mg/mL) was added and incubated at room
temperature for 1 h. Then, 10 µL of propidium iodide (PI, 50 µg/mL) was added. Finally,
percentages of cells in different phases of the cell cycle were determined using a FACS Calibur
flow cytometer. The cytographs were analyzed by Flowjo software.
4.2.1.5. Annexin V-FITC analysis for apoptosis
The apoptotic effect of compounds 5c and 5d was evaluated by Annexin-V FITC/PI assay [31].
SKOV3 cells were cultured in 6-well plates (1 × 106 cells/well) and incubated for 24 h. Then the
medium was removed and a complete medium containing 5c or 5d at 2 and 4 µg/mL
concentrations was added. After 48 h, the cells from the supernatant were harvested by
trypsinization, washed with PBS at 3000 rpm and stained with 5 mL of Annexin-V/FITC (Sigma
Chemical Co) in PBS at rt for 15 min and then with PI solution (Keygen Biotech, China) for
another additional 10 min. Flow cytometric analyses were implemented using a FACScan
(Becton Dickinson) appointed with a single 488 nm argon laser. The EXPO32 ADC analysis
software was used for calculating the percentage of apoptotic cells.
25

4.2.1.6. Alkaline comet assay
The possible effect of promising compounds 5c and 5d on DNA damage in normal cells L929
was investigated by alkaline single cell gel electrophoresis method with slightly modification
[24,25]. L929 Cells were seeded in 24 well plate (250000 cell/well). The cells were exposed with
5c and 5d at the concentration of 10 µg/mL for 1 h. Untreated cells and cell treated with
etoposide (0.5 µg/mL) were considered as negative and positive controls, respectively.
Afterwards, 10 µL of cell suspensions were mixed with 100 µL of low-melting-point agarose
(LMA). The mixture was expanded onto microscope slides which were pre-coated with 1.0%
normal-melting point agarose (NMA). After solidification of the agarose (4 °C, 10 min), slides
were lysed in cold lysis buffer including 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base, pH 10,
containing 10% DMSO and 1% TritonX-100 (4 °C, 1 h). Then, slides were submerged in
electrophoretic buffer including 1 mM Na2-EDTA, 300 mM NaOH, pH>13 for 40 min to open
DNA prior to electrophoresis. The electrophoresis was performed at 25 V and a current of 300
mA for 20 min. Then, the slides were washed with cold neutralization buffer solution three times
for 15 min. Subsequently, the slides were stained with 30 µL ethidium bromide and observed
with a fluorescence microscope. The most reliable factors, tail moment, tail length and tail DNA
percentage were measured using Comet Score software project (CASP) to assess DNA damage.
4.2.2. Tubulin extraction and tubulin polymerization assay
Ethics approval for the use of sheep brain for tubulin extraction was granted by the Ethics
Committee of the Shahid Beheshti University of Medical Sciences. Sheep fresh brains were
obtained from a local slaughterhouse and rapidly transported to the laboratory on ice in 0.9%
saline solution for further activities. Tubulin protein was extracted from the sheep brain by three
cycles of temperature-dependent polymerization/depolymerization as described in
Supplementary Material based on reported method [33]. Tubulin was re-suspended in a buffer
and incubated in the presence and absence of compounds 5c and 5d at the final concentrations of
0.5, 2, 10 and 20 µg/mL for 15 min on ice. For induction of tubulin polymerization, GTP (final
concentration 1 mM) was added at 37 °C and tubulin assembly was monitored turbidimertically
26

at 350 nm [26]. The concentration that inhibits tubulin polymerization by 50% (IC50) was
determined using area under the curve (AUC). The AUC of the untreated controls were
considered as 100% polymerization. The IC50 value for each compound was computed using
GraphPad Prism Software.
4.2.3. Fluorescence assay of colchicine-tubulin complex
The tubulin inhibitor 5c was subjected to binding assay using fluorescence technique according
to the reported method [34,35]. The purified tubulin (0.25 µM) was incubated at 37°C with the
different concentrations of colchicine (0.1, 0.25, 0.5 or 1 µM) for 1 h. The tubulin-colchicine
complexes were treated with compound 5c at the final concentration of 1 and 5 µM.
Fluorescence emission spectra for each experiment (n=3) were recorded at 430 nm following
excitation at 360 nm using Cytation 3, multi-mode imaging and plate Reader (BioTek, USA).
Fluorescent data was corrected by subtracting the background fluorescence intensity for
compound 5c at each concentration. “F0” and “F” are the fluorescence intensity (a.u) of the
[tubulin+colchicine] and [tubulin+colchicine+ligand 5c] mixtures, respectively.
4.3. Molecular docking studies
The binding mode of promising compounds 5c and 5d to the CBS of tubulin was investigated
using AutoDock software (version 4.2). The 3D structure of compounds was constructed and
optimized by ChemBioDraw Ultra16.0 and ChemBio3D Ultra 16.0 softwares. The β-subunit of
the target (chain D) was chosen and the rest was deleted. The final receptor pdbqt file was
prepared following AutoDock protocol [36]. A grid map with 40, 40, and 40 grid points (X, Y, Z
dimensions) and Centroid: X = 17.0192, Y = 65.9939, Z =43.3901 was selected. The pose with
the best scoring was selected and visualized for analyzing the involved interactions. The 3D, 2D
and pose of studied compounds 5c and 5d were taken using discovery studio 2016.
Acknowledgments
27

This study was a part of MA's Ph.D. thesis and supported by a grant (No. 831) from the Research
Council of Mazandaran University of Medical Sciences.
References
[1] WHO Factsheet, Sept 2018, https://www.who.int/news-room/fact-sheets/detail/cancer
[2] G. Bérubé, Expert Opin. Drug Discov. 14 (2019) 331–334.
[3] K.E. Arnst, S. Banerjee, H. Chen, S. Deng, et al., Med. Res. Rev. 39 (2019) 1398–1426.
[4] Y. Ilan, J. Cell Physiol. 234 (2019) 7923–7937.
[5] F. Naaz, M.R. Haider, S. Shafi, M.S. Yar, Eur. J. Med. Chem. 171 (2019) 310–331.
[6] K. Haider, S. Rahaman, M.S. Yar, A. Kamal, Expert Opin. Ther. Pat. 29 (2019) 623–641.
[7] L.M. Nainwal, M.M. Alam, M. Shaquiquzzaman, A. Marella, et al, Expert Opin. Ther. Pat.
(2019), doi: 10.1080/13543776.2019.1651841.
[8] T.J. Kim, M. Ravoori, C.N. Landen, A.A. Kamat, L.Y. Han, C. Lu. Cancer Res. 67 (2007)
9337–9345.
[9] C. Avendaño, J.C. Menéndez, Anticancer Drugs Targeting Tubulin and Microtubules, In:
Medicinal Chemistry of Anticancer Drugs (Second Edition), 2015, pp. 359–390.
[10] S. Arora, A.F. Gonzalez, K. Solanki. Int. J. Pharm. Sci. Rev. Res. 22 (2013) 168–174.
[11] Z.S. Seddigi, M.S. Malik, A.P. Saraswati, S.A. Ahmed, et al., Med. Chem. Comm. 8 (2017)
1592–1603.
[12] S.N.A. Bukhari, G.B. Kumar, H.M. Revankar, H.L. Qin, Bioorg. Chem. 72 (2017) 130–147.
[13] N.H. Nam, Y. Kim, Y.J. You, D.H. Hong, H.M. Kim, B.Z. Ahna, Bioorg. Med. Chem. Lett.
11 (2001) 3073–3076.
[14] H. Aryapour, G.H. Riazi, A. Foroumadi, S. Ahmadian, et al., Med. Chem. Res. 20 (2011)
503–510.
[15] H. Aryapour, G.H. Riazi, S. Ahmadian, A. Foroumadi, et al., Pharm. Biol. 50 (2012) 1551–
1560.
[16] H. Mirzaei, M. Shokrzadeh, M. Modanloo, A. Ziar, et al., Bioorg. Chem. 75 (2017) 86–98.
[17] H. Mirzaei, S. Emami, Eur. J. Med. Chem. 121 (2016) 610–639.
[18] M. Banimustafa, A. Kheirollahi, M. Safavi, S.K. Ardestani, et al., Eur. J. Med. Chem. 70
(2013) 692–702.
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