Synthesis, cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.10.030

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl- thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.

Full text of DOI:10.1016/j.bmc.2013.10.030

Bioorganic & Medicinal Chemistry 21 (2013) 7648–7654
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxic evaluation and molecular docking study
of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as
tubulin polymerization inhibitors
a
a
b
c
a
Marjan Salehi , Mohsen Amini , Seyed Nasser Ostad , Gholam Hossein Riazi , Amir Assadieskandar ,
c
d,
⇑
Bentolhoda Shafiei , Abbas Shafiee
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Rational Drug Use Research Center, Tehran University of Medical Sciences, Tehran, Iran
Institute of Biochemistry and Biophysics, University of Tehran, Tehran 13145-1384, Iran
b
c
d
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combre-
tastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell
lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The
biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity
against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring
resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted
derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was eval-
uated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry
Received 19 September 2013
Revised 21 October 2013
Accepted 22 October 2013
Available online 30 October 2013
Keywords:
2
-Alkylthio-4,5-diaryl-thiazole
Tubulin inhibitor
Cytotoxicity
2
analysis confirmed that the synthesized compounds led to cell cycle arrest at the G /M phase. Docking
simulation was performed to insert these compounds into the crystal structure of tubulin at the colchi-
cine binding site to determine a probable binding model.
Cell cycle analysis
Molecular docking
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
Microtubules are attractive molecular targets for anticancer
low aqueous solubility and the isomerization of its cis-double
bond into a more thermally stable, but inactive, trans-isomer.6
As CA-4 has a simple structure, with only two aromatic rings
linked by a double bond in the cis configuration, a wide number
of CA-4 analogues have been developed and synthesized to date.
Among these analogues, compounds in which the olefinic bond is
replaced with five-membered rings are promising targets. Imidaz-
therapeutics because microtubule polymerization dynamics can
greatly affect critical processes, such as cell signaling and mito-
sis. At the same time, polymerization dynamics can be affected
1
2
by natural products and synthetic small molecules. There are
7
8
9
10
three major binding sites on tubulin, the taxus, the vinca, and
the colchicine binding site.3 Taxanes are examples of compounds
that bind to the taxus binding site and stabilize the microtubule,
whereas the majority of compounds that bind to the vinca and
colchicine binding sites destabilize the microtubule and promote
depolymerization by inhibition of tubulin. The first colchicine
site inhibitor, colchicine (Fig. 1), has limited therapeutic applica-
tion because of its high cytotoxicity. Combretastatin A-4 (CA-4)
ole, triazole, thiazole, and pyrazoline are example of five-
membered heterocyclic bridges.
In continuation of our research on tubulin inhibitors,1
1,12
a ser-
ies
of
2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole
(Fig. 1) analogues of CA-4 were synthesized, and their cytotoxicity
and tubulin polymerization inhibitory activity were evaluated. The
binding mode of these compounds to tubulin was also studied by
molecular docking.
4
3
(Fig. 1), isolated from the South African willow tree Combretum
caffrom, strongly inhibits the polymerization of tubulin by binding
to the colchicine site. Although CA-4 expresses high levels of
in vitro activity, it does not show efficacy in vivo because of its
2
2
. Results and discussion
.1. Chemistry
5
2
-Alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles
were
⇑
Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
synthesized by the reaction of different
a-bromoketones with methyl
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.030

M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
7649
OH
R
H CO
O
3
H CO
3
N
H
CH3
S
N
H CO
3
SR'
H CO
3
OCH3
O
H CO
3
OCH3
H CO
3
OCH3
OCH3
OCH3
5a-h
Colchicine
CA-4
Figure 1. Structure of colchicine, combretastatin A-4, and 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles (5a–h).
and benzyl carbamodithioate 4a–b. In the first step,
a–d were prepared from bromination of 1-(2,3,4-trimethoxy-
phenyl)-2-(4-substitutedphenyl)-1-ethanones 2a–d in glacial acetic
a
-bromoketones
2.2. Biological study
3
To test the anticancer activity of the synthesized compounds,
the antiproliferative activity of all the derivatives against three
cancer cell lines, human colon adenocarcinoma (HT-29), human
breast adenocarcinoma (MCF-7), and human stomach adenocarci-
1
3
acid. These ethanones 2a–d were synthesized by reaction of
,2,3-trimethoxybenzene and appropriate phenyl acetic acid 1a–d
1
14
according to the literature. From two expected regio isomers,
-aryl-1-(2,3,4-trimethoxyphenyl)ethanone and 2-aryl-1-(3,4,5-tri-
2
noma (AGS), as well as a mouse embryonic fibroblast cell line,
NIH-3T3, was evaluated by an MTT colorimetric assay.1
7,18
CA-4
methoxyphenyl)ethanone one of the former was obtained. The struc-
ture of 2a–d were confirmed by NMR spectroscopy. In another
experiment, ammonium dithiocarbamate was prepared by passing
was used as a positive control. The cultured cells were treated with
several concentrations of test compounds for 48 h. The ability of
these analogues to inhibit cell growth is summarized in Table 1.
The results revealed that most of the tested compounds showed
moderate to potent cytotoxic activities. Structure-activity relation-
ship (SAR) studies showed that the nature of the benzylthio group
on the thiazole ring has a profound influence on the cytotoxicity of
the compounds. For example, compounds 5d and 5f, both of which
contain the 2-(benzylthio) group on thiazole rings, were active
1
5
ammonia through a solution of carbon disulfide in tetrahydrofuran.
In the next step, methyl and benzyl dithiocarbamate were obtained
from reaction of methyl and benzyl iodide with ammonium dithio-
carbamate. Finally, treatment of different
a-bromoketones with
twofold excess of 4a–b in methanol afforded the desired 4-(2,3,4-tri-
methoxyphenyl)-5-aryl-thiazole-2-thio substituted compounds
5
a–h (Scheme 1).
The CA-4 was synthesized as positive control according to the
(IC50 <20 lM) in all three cancer cell lines. In contrast, the
replacement of the 2-(benzylthio) group on the thiazole ring with
a 2-(methylthio) moiety, namely compounds 5c and 5e, led to a
1
6
literature. All of synthesized compounds were characterized by
1
13
H NMR, C NMR and CHN analysis.
R
R
R
OCH3
H CO
OCH3
3
(
a)
(b)
OCH3
OCH3
+
OCH3
OCH3
Br
HOOC
1
1
1
1
a, R=H
O
OCH3
O
OCH3
b, R=Cl
c, R=F
2a-d
3a-d
d, R=NO
2
(
c)
S
S
(
d)
CS + 2NH
2
3
-
+
H N
S NH
H N
SR'
2
4
2
(
e)
4
a, R'=Me
4
b, R'=Benzyl
R
S
N
SR'
H CO
OCH3
3
5a-h
OCH3
Scheme 1. Reagents and conditions: (a) H PO , (CF CO) O, 25 °C, 1 min; (b) Br , CH COOH, 25 °C, 2 h; (c) THF, 25 °C; (d) methanol, benzyl or methyl iodide, 25 °C; (e)
3 4 3 2 2 3
methanol, reflux, 24 h.

7
650
M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
Table 1
Structure and cytotoxicity of analogues of CA-4
R
S
SR'
N
H CO
OCH3
3
OCH3
0
Cytotoxicitya (IC50
AGS
27.2 ± 1.2
Compounds
R
R
, lM)
HT-29
MCF-7
NIH-3T3
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
H
Cl
Cl
F
F
NO
NO
CH
CH
CH
CH
CH
CH
CH
CH
3
2
3
2
3
2
3
2
77.9 ± 2.3
46.0 ± 1.7
>100
8.9 ± 0.7
80.3 ± 2.7
18.5 ± 0.9
45.3 ± 2.0
33.8 ± 1.5
1.58 ± 0.5
N.Db
22.1 ± 1.4
56.5 ± 1.8
95.7 ± 2.2
20.5 ± 0.8
42.1 ± 0.9
41.5 ± 1.0
>100
Ph
Ph
Ph
Ph
27.3 ± 0.6
38.5 ± 1.5
>100
N.Db
12.6 ± 0.9
N.Db
18.5 ± 1.1
15.8 ± 1.2
97.8 ± 2.6
18.1 ± 0.4
>100
g
h
2
2
N.Db
11 ± 1.3
0.67 ± 0.2
13.8 ± 0.9
0.064 ± 0.04
16.3 ± 1.1
0.097 ± 0.08
CA-4
a
Values are the mean ± SD. All the experiments were performed at least two times.
N.D, not determined.
b
reduction in the cytotoxic activity (IC50 >80
with para chloro substituted on the phenyl ring and the 2-(benzyl-
thio) group had the highest cytotoxic activity against the HT-29
cell line (IC50 = 8.9 lM). This compound also showed good activity
l
M). The compound 5d
the corresponding cytotoxic activities. The order of inhibition of
tubulin polymerization was CA-4 > 5d > 5f > 5h. The compound
with the 2-(benzylthio) group on the thiazole ring and the para
chloro substituted 5-phenyl ring was the most potent inhibitor of
tubulin polymerization. From these results, it seems that tubulin
is a possible target for the synthesized compounds.
against the MCF-7 and AGS cell lines. Moreover, the replacement of
chloro substituted with fluoro and nitro led to less active com-
pounds. In contrast, 5b with the 5-phenyl group and the 2-methyl-
thio substitution on the thiazole ring had little cytotoxic activity
against all the cell lines. The results suggest that removing the para
substitution from the 5-phenyl ring led to a decrease in the potency.
Generally, most of the tested compounds showed no cytotoxic activ-
ity or only moderate cytotoxic activity against the NIH-3T3 cell line.
Finally, the introduction of the 2-(benzylthio) group on the
thiazole ring seemed to play an important role in the cytotoxic
activity. Furthermore, the presence of the para substitution in the
3. Molecular modeling
Molecular modeling studies were performed to investigate the
binding ability of the synthesized compounds to the colchicine
binding site of a, b-tubulin (PDB: 1SA0). Docking studies revealed
that the 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole
compound, as well as CA-4, occupied the colchicine binding site
of a, b-tubulin mostly buried in the b subunit (Fig. 4). In these com-
5
-phenyl ring in the 2-(benzylthio) derivatives led to optimal
pounds, the oxygen atom of the 4-methoxy substituent formed a
hydrogen bond with Cysb241 in the b subunit, similar to that seen
in the X-ray complex of DAMA-colchicine with tubulin.
The 5-phenyl ring moiety lay in the hydrophobic pocket be-
tween Vala181, Metb259, and Lys352. Furthermore, the thiazole
effects against all the cancer cell lines. The same results were ob-
served when 5a and 5b and 5g and 5h were compared. However,
the cytotoxic effect of the synthesized compounds 5a–d was less
than that of the reference CA-4 in all the tested cell lines.
As the inhibition of tubulin polymerization has been implicated
ring interacted with Asnb258 in compounds with the 2-(benzyl-
thio) group, but replacements of this group with a 2-(thiomethyl
moiety) increased the distance between these two groups. For
example, the observed distance between thiazole and Asn258
/M phase cell cycle arrest in various cancer cell lines,1 the ef-
9
in G
2
fect of compound 5d on the cell cycle progression of the NIH-3T3
cells was determined by flow cytometry analysis (Fig. 2). The
0
0
NIH-3T3 cells were treated with compound 5d (25 and 50
for 24 h, and CA-4 (0.05 M) was used as a positive control. The
findings indicated that 5d results in weak accumulation of cells
in the G /M phase (37.64% compared to 35.70% for untreated cells)
in 25 M and in more evident accumulation (57.41%) at a higher
concentration of 50 M. These results demonstrated that the com-
pound 5d could arrest cells in the G /M phase.
After determination of the cytotoxic activity of the synthesized
compounds, the abilities of some derivatives in inhibiting the poly-
merization of tubulin were studied. For this purpose, the effect of
three potent compounds 5d, 5f, and 5h on the polymerization of
l
M)
was 3.05 ÅA for 5d, whereas this value was 4.85 ÅA for 5c. The in-
crease in the distance led to a lack of interaction between the thi-
azole ring and Asn258 in 5c. It is possible that this difference in the
distance between the two molecules is one of the factors leading to
the decreased antiproliferative activity observed for 5c and 5d. The
docking scores varied from ꢀ7.45 to ꢀ6.38 kcal/mol for com-
pounds with the 2-(benzylthio) group and were 1 kcal/mol lower
than the average 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-
methyl-thiazole score values.
l
2
l
l
2
4. Conclusions
purified tubulin was evaluated at a final concentration of 10 lM.
CA-4 was also evaluated by this assay as a positive control. Figure 3
shows that all three compounds decreased the polymerization
activity of the microtubule relative to the control and indicates
the rational relationship between the inhibition of tubulin and
In conclusion, the synthesis and biological evaluation of a series
of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles were
discussed. Most of the synthesized compounds showed moderate
to potent cytotoxic activity against the four cell lines. It seems that

M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
7651
Figure 2. Effect of compound 5d (25 and 50 lM) and CA-4 (0.05 lM) on the cell cycle of the NIH-3T3 cells.
the introduction of the 2-(benzylthio) group on the thiazole ring
played an important role in the cytotoxic activity and that the para
substitution in the 5-phenyl ring in the 2-(benzylthio) derivatives
led to optimal effects against all the cancer cell lines. The flow
cytometry analysis and microtubule polymerization assay con-
firmed that the synthesized compounds led to cell cycle arrest at
spectra were record on Bruker FT-500 MHz spectrometer. The
1
3
instrument was set as 125 MHz for acquiring C NMR spectra.
Coupling constant (J) values are presented in hertz (Hz) and spin
multiples are given as s (singlet), d (double), t (triple) and m (mul-
tiple). Elemental analyses were carried out with a Perkin Elmer
Model 240-C apparatus. Elemental analyses were within ± 0.4%
of theoretical values for C, H and N. Ethylenebis(oxyethylenenitril-
the G
The molecular modeling studies revealed that these compounds
could strongly bind to the colchicine binding site of , b-tubulin
2
/M phase by inhibitory effects against the microtubules.
0
o) tetraacetic acid (EGTA), guanosine-5 -triphosphate type II-S
0
a
(GTP), adenosine-5 -triphosphate (ATP), phenylmethylsulphonyl
through hydrogen bond interactions with Cysb241 and that the
thiazole ring also interacted with Asnb258 in compounds with
the 2-(benzylthio) group. The ability of some of the compounds
to inhibit tubulin polymerization shows that tubulin is a good tar-
get for diarylthiazole derivatives.
4
fluoride (PMSF), glycerol, MgSO were purchased from Sigma
(Deisenhofen, Germany). Piperazine-1,4-bis(2-ethanesulfonic acid)
(PIPES), dimethyl sulfoxide (DMSO) and all starting material for
synthesis were purchased from Merck (Darmstadt, Germany).
MTT was purchased from Carl Roth (Karlsruhe, Germany). MgSO
1 M) was added to both GTP and ATP 100 mM stock solutions,
as a ratio of 1:10 (v/v). All cell line obtained from Pasteur Institute
Tehran, Iran). The cells were cultured in RPMI-1640 medium (Sig-
ma) supplemented with 10% heat-inactivated fetal bovine serum
FBS; Gibco, USA), penicillin (100 U/ml), and (100 g/ml) strepto-
4
(
5
5
. Experimental section
.1. General
(
(
l
Melting points were taken on a Kofler hot-stage apparatus
Reichert, Vienna, Austria) and are uncorrected. The H NMR
mycin (Roche, Germany) at 37 °C in a humidified incubator with
1
(
5% CO . All of the test compounds, used in this study, were
2

7
652
M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
2
3
H
5
.75 (s, 3H, SCH
H, OCH ), 6.67 (d, J = 8.5 Hz, 1H, H
-Ar ), 7.21–7.23 (m, 5H, Ar
5.99 (OCH ), 60.76 (OCH ), 60.85 (OCH
-Ar ), 126.01 (C -Ar ), 127.62 (C -Ar ), 128.31 (C3,5-Ar
), 132.14 (C -Ar ), 133.57 (C -Ar ), 142.32 (C
46.87 (C -Ar ), 152.01 (C -Ar ), 154.11 (C
-thiazole). MS, m/z (%) 373 (M , 100), 267 (73), 207 (28), 171
14), 121 (17), 79 (13). Anal. Calcd for C19 : C, 61.10; H,
3
), 3.57 (s, 3H, OCH
3
), 3.81 (s, 3H, OCH
-Ar ), 7.05 (d, J = 8.5 Hz, 1H,
); C NMR (CDCl ) d: 16.68 (SCH ),
), 107.15 (C -Ar ), 121.83
3
). 3.88 (s,
3
3
4
1
3
2
4
5
3
3
3
3
3
5
4
(
(
C
C
1
4
6
4
4
5
5
), 128.52
2,6-Ar
5
1
5
3
4
5
-thiazole),
1
4
4
2
4
4
-thiazole), 163.33
+
(
(
C
2
3 2
H19NO S
5
.13; N, 3.75. Found: C, 61.22; H, 5.33; N, 3.54.
5
.2.1.2. 4-(2,3,4-Trimethoxyphenyl)-5-(phenyl)-2-(benzylthio)-
1
thiazole (5b).
Yield, 52%; mp 91–94 °C; H NMR (CDCl
), 3.82 (s, 3H, OCH ), 3.90 (s, 3H, OCH ), 4.49 (s,
), 6.70 (d, J = 8.5 Hz, 1H, H -Ar ), 7.05 (d, J = 8.5 Hz, 1H,
), 7.21–7.23 (m, 5H, Ar ), 7.27–7.32 (m, 3H, benzyl),
.41–7.48 (m, 2H, benzyl); C NMR (CDCl ) d: 38.93 (CH ), 56.01
), 107.19 (C -Ar ), 121.36
), 127.69 (C -Benzyl), 127.80 (C -Ar ),
), 128.54 (C2,6-Ar ), 128.66 (C2,6-Benzyl), 129.16
(C3,5-Benzyl), 131.90 (C -Ar ), 134.48 (C -Ar ), 136.36 (C -Benzyl),
42.20 (C -thiazole), 146.66 (C -Ar ), 152.01 (C -Ar ), 154.21
-thiazole), 161.26 (C -thiazole). MS, m/z (%) 449 (M , 11), 281
23), 252 (15), 207 (100), 96 (17), 73 (19). Anal. Calcd for C25
: C, 66.79; H, 5.16; N, 3.12. Found: C, 66.85; H, 5.29; N, 3.02.
3
) d:
3
2
H
.54 (s, 3H, OCH
H, SCH
-Ar
3
3
3
2
3
4
2
4
5
1
3
7
3
2
(
OCH
3
), 60.79 (OCH
3
), 60.88 (OCH
3
5
4
(
C
1
-Ar
4
), 126.07 (C -Ar
6
4
4
4
5
Figure 3. Effect of compounds 5d, 5f, 5h, and CA-4 on the polymerization of
tubulin. The results represent the mean of duplicate experiments for each
compound.
1
28.34 (C3,5-Ar
5
5
1
5
3
4
1
1
5
4
4
2
4
+
(
(
C
4
2
dissolved in DMSO (dimethyl sulfoxide) as 1 mM stock. For cell cul-
ture tests, DMSO was used at the final concentrations <0.1% (v/v).
DMSO alone in the mentioned concentration did not show any
cytotoxic effect.
H
23
3 2
NO S
5
(
.2.1.3.
methylthio)-thiazole (5c).
NMR (CDCl ) d: 2.74 (s, 3H, SCH
OCH ), 3.88 (s, 3H, OCH ), 6.68 (d, J = 8.5 Hz, 1H, H
J = 8.5 Hz, 1H, H -Ar ), 7.15 (d, J = 8.5 Hz, 2H, H3,5-Ar
J = 8.5 Hz, 2H, H2,6-Ar
OCH ), 60.91 (OCH ), 60.96 (OCH
Ar ), 125.89 (C -Ar ), 128.71 (C3,5-Ar
-Ar ), 132.12 (C -Ar ), 133.45 (C
47.33 (C -Ar ), 151.97 (C -Ar ), 154.64 (C
thiazole). MS, m/z (%) 409 (M +2, 33), 407 (M , 100), 301 (25),
81 (37), 266 (61), 207 (87), 73 (37). Anal. Calcd for C19
: C, 55.94; H, 4.45; N, 3.43. Found: C, 56.07; H, 4.56; N, 3.35.
4-(2,3,4-Trimethoxyphenyl)-5-(4-cholorophenyl)-2-
1
Yield, 59%; mp 150–151 °C.
), 3.61 (s, 3H, OCH ), 3.83 (s, 3H,
-Ar ), 7.03 (d,
), 7.21 (d,
) d: 16.62 (SCH ), 56.0
), 107.31 (C -Ar ), 121.54 (C
), 129.45 (C2,6-Ar ), 130.72
-Ar ), 142.52 (C -thiazole),
-thiazole), 163.65 (C
H
5
5
.2. Chemistry
3
3
3
3
3
3
4
.2.1. General procedure for the preparation of 5(a–h)
2
4
5
1
3
To a solution of ammonium dithiocarbamate (1 mmol) in meth-
5
); C NMR (CDCl
3
3
anol, methyl iodide (1.5 mmol) (or benzyl iodide) was added. The
reaction mixture was stirred at room temperature for 1 h. Then sol-
vent was evaporated under reduced pressure. Water (10 mL) was
added to the residue and extracted with dichloromethane. The or-
ganic phase was separated, dried by sodium sulfate and evaporated
under vacuum. The residue (1.5 mmol) was added to a solution of
(
3
3
3
5
4
1
-
4
6
4
5
5
(
C
1
5
3
4
4
5
5
1
4
4
2
4
+
4
2
-
+
2
H18ClNO3-
a-bromo-1,2-(p-substituted)diaryl-1-ethanone (1 mmol) in meth-
S
2
anol. The mixture was refluxed overnight. Then the reaction was
cooled to room temperature and the precipitated product was fil-
tered and recrystallized from ethanol to yield 2-alkylthio-4-
5
.2.1.4.
4-(2,3,4-Trimethoxyphenyl)-5-(4-cholorophenyl)-2-
1
(
benzylthio)-thiazole (5d).
NMR (CDCl ) d: 3.58 (s, 3H, OCH
H, OCH ), 4.49 (s, 2H, SCH ), 6.69 (d, J = 8.5 Hz, 1H, H
d, J = 8.5 Hz, 1H, H -Ar ), 7.13 (d, J = 8.5 Hz, 2H, H3,5-Ar
J = 8.5 Hz, 2H, H2,6-Ar ), 7.29-7.35 (m, 3H, benzyl), 7.43–7.46 (m,
Yield, 67%; mp 75–77 °C;
), 3.83 (s, 3H, OCH ), 3.90 (s,
-Ar ), 7.05
),7.21 (d,
H
(
2,3,4-trimethoxyphenyl)-5-aryl-thiazole derivatives.
3
3
3
3
(
3
2
3
4
5
.2.1.1. 4-(2,3,4-Trimethoxyphenyl)-5-(phenyl)-2-(methylthio)-
2
4
5
1
thiazole (5a).
3
Yield, 65%; mp 107–110 °C; H NMR (CDCl ) d:
5
Figure 4. Binding modes of DAMA-colchicine (A) and the comparison between the binding modes of 5c (magenta in B) and 5d (green in B).

M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
7653
2
H, benzyl); 13C NMR (CDCl
OCH ), 107.30 (C -Ar ), 121.49 (C
-benzyl), 128.65 (C2,6-benzyl), 128.71 (C3,5-Ar
3,5-benzyl), 129.48 (C2,6-Ar ), 130.64 (C -Ar ), 133.01 (C
-Ar ), 136.43 (C -benzyl), 142.20 (C
), 151.95 (C -Ar ), 154.23 (C
3
) d: 38.67 (CH
2
), 56.01 (OCH
-Ar ), 127.67
), 129.12
-Ar ),
-thiazole), 147.36
3
), 60.95
(C
151.80 (C
z (%) 494 (M , 11), 281 (34), 253 (21), 207 (100), 133 (12), 73
(23). Anal. Calcd for C25 : C, 60.71; H, 4.48; N, 5.66.
1
-Ar
5
), 142.61 (C
5
-thiazole), 146.58 (C
4
-Ar
4
), 148.96 (C
4 5
-Ar ),
(
(
(
3
5
4
1
-Ar ), 125.92 (C
4
6
4
2
-Ar ), 154.63 (C
4
4
-thiazole), 163.52 (C
2
-thiazole). MS, m/
+
C
C
4
5
5
1
5
4
5
22 2 5 2
H N O S
1
33.55 (C
-Ar
3
4
1
5
Found: C, 60.34; H, 4.73; N, 5.85.
(
C
4
4
2
4
4
-thiazole), 161.27 (C
2
-thia-
+
+
zole). MS, m/z (%) 485 (M +2, 3), 483 (M , 10), 281 (35), 207
5.3. Biology
(
100), 147 (15), 73 (22). Anal. Calcd for C25
3 2
H22ClNO S : C, 62.03;
H, 4.58; N, 2.89. Found: C, 62.25; H, 4.61; N, 2.77.
5.3.1. Growth inhibition assay
The response of three different carcinoma cell lines HT-29
(colon carcinoma cell), AGS (stomach carcinoma cell) and MCF7
(breast carcinoma cells) to synthesized compounds was evaluated
by the determination of cell survival using MTT assay. NIH-3T3
(mouse fibroblast cells) also used as normal cell line. CA-4 was ap-
plied as a positive control. Cells from different cell lines were cul-
tured in 96-well plates at the density of 8000–10,000 viable cells
per well and incubated for 48 h to allow cell attachment. Then cell
were treated with various concentrations of each compound
5
(
.2.1.5.
methylthio)-thiazole (5e).
NMR (CDCl ) d: 2.78 (s, 3H, SCH
OCH ). 3.91 (s, 3H, OCH
J = 8.5 Hz, 2H, H3,5-Ar
4-(2,3,4-Trimethoxyphenyl)-5-(4-flourophenyl)-2-
1
Yield, 43%; mp 98–101 °C;
H
3
3
), 3.61 (s, 3H, OCH
), 6.68 (d, J = 8.5 Hz, 1H, H
3
), 3.81 (s, 3H,
-Ar ), 6.95 (t,
), 7.19 (dd,
) d: 16.64
), 107.26 (C -Ar ),
-Ar ), 126.03 (C
), 133.93
), 152.16 (C -Ar ),
-thiazole), 161.67 (d, JCF = 242.0 -
3
3
3
4
5
2 4
), 7.05 (d, J = 8.5 Hz, 1H, H -Ar
13
J = 8.5 Hz, J = 5.5 Hz, 2H, H2,6-Ar
5
);
), 60.91 (OCH
), 121.58 (C
C NMR (CDCl
3
(
SCH
3
3
), 56.0 (OCH ), 60.76 (OCH
3
3
5
4
1
15.54 (d, JCF = 21.25 Hz,C3,5-Ar
5
1
4
6
-
Ar
4
), 128.31 (C
-Ar ), 142.22 (C
54.15 (C -thiazole), 160.18 (C
Hz,C -Ar ). MS, m/z (%) 391 (M , 100), 300 (14), 285 (76), 207
17), 189 (15), 139 (19). Anal. Calcd for C19 18FNO : C, 58.29;
H, 4.63; N, 3.58. Found: C, 58.35; H, 4.71; N, 3.35.
1
-Ar
5
), 130.01 (d, JCF = 8.75 Hz, C2,6-Ar
5
(0.001–100 lM). Cells were then incubated for another 24 or
(
C
3
4
5
-thiazole), 147.03 (C -Ar
4
4
2
4
48 h (depends to cell cycle of each cell line). At the end of this
period the response of cells to compounds were evaluated by
determining cell survival using 3-(4,5-dimethylthiazoyl-2-yl)2,5-
diphenyl tetrazolium bromide. For this purpose Cells were washed
1
4
2
+
4
5
(
H
3 2
S
in PBS, and 20 lL of MTT reagent (5 mg/ml) in phosphate buffered
serum (PBS) was added to each well and the cells were incubated
5
.2.1.6. 4-(2,3,4-Trimethoxyphenyl)-5-(4-flourophenyl)-2-(ben-
for another 4 h at 37 °C. Finally the supernatants were then aspi-
rated and dimethyl sulfoxide (100 lL) was added to each well.
1
18
zylthio)-thiazole (5f).
CDCl ) d: 3.56 (s, 3H, OCH
OCH ), 4.48 (s, 2H, SCH ), 6.69 (d, J = 8.5 Hz, 1H, H
J = 8.5 Hz, 2H, H3,5-Ar ), 7.04 (d, J = 8.5 Hz, 1H, H -Ar
J = 8.5 Hz, J = 5.5 Hz, 2H, H2,6-Ar ), 7.29–7.37 (m, 3H, Benzyl),
.43-7.46 (m, 2H, Benzyl); 13C NMR (CDCl
) d: 38.71 (CH ), 56.0
OCH ), 60.72 (OCH ), 60.90 (OCH ), 107.26 (C -Ar ), 115.54 (d,
CF = 21.25 Hz, C3,5-Ar ), 121.52 (C -Ar ), 125.98 (C -Ar ), 127.66
-benzyl), 128.19 (C -Ar ), 128.64 (C2,6-benzyl), 129.13
3,5-benzyl), 130.04 (d, JCF = 7.5 Hz, C2,6-Ar ), 133.26 (C -Ar ),
-benzyl), 142.39 (C -thiazole), 147.06 (C -Ar ), 151.97
), 154.16 (C -thiazole), 160.85 (C -thiazole), 162.20 (d,
CF = 246.0 Hz,C -Ar ). MS, m/z (%) 467 (M , 44), 281 (37), 253
18), 207 (100), 91 (18), 73 (20). Anal. Calcd for C25 22FNO : C,
4.22; H, 4.74; N, 3.00. Found: C, 64.25; H, 4.81; N, 2.98.
Yield, 74%; mp 81–82 °C; H NMR
), 3.82 (s, 3H, OCH ), 3.90 (s, 3H,
-Ar ), 6.93 (t,
),7.17 (dd,
(
3
3
3
The absorbance was determined by plate reader (Anthous 2020;
Austria) for each well at a test wavelength of 550 nm against a
standard reference solution at 690 nm. Each experiment was per-
formed in three replicates. The concentration required for 50%
inhibition of cell viability (IC50) were determined by a nonlinear
regression analysis and expressed in mean ± SD.
3
2
3
4
5
2
4
5
7
(
J
3
2
3
3
3
5
4
5
1
4
6
4
(
(
C
C
4
1
5
5.3.2. Cell cycle analysis
6
5
3
4
For flow cytometric analysis of DNA content, 10 NIH-3T3 cells
1
36.48 (C
-Ar
1
5
4
4
were treated with (25 and 50
centrifugation, the cell pellet was fixed in 75% ethanol at kept in
4 °C for 0.5 h. The cell pellet was resuspended in 500 L of PBS con-
taining 0.1% (v/v) Triton X-100, 10 g/mL propidium iodide (PI, Sig-
ma, St. Louis, MO), and 100 g/mL RNase A and incubated in 37 °C
lM) compound 5d for 24 h. After
(
C
2
4
4
2
+
J
(
6
4
5
l
H
S
3 2
l
l
for 0.5 h. Finally, the fluorescence cell was measured by FACS-Cal-
5
.2.1.7.
4-(2,3,4-Trimethoxyphenyl)-5-(4-nitrophenyl)-2-
ibur flow cytometer (BD Biosciences, San Jose, CA).
1
(
methylthio)-thiazole (5g).
NMR (CDCl ) d: 2.77 (s, 3H, SCH
OCH ). 3.91 (s, 3H, OCH ), 6.72 (d, J = 8.5 Hz, 1H, H
J = 8.5 Hz, 1H, H -Ar ), 7.36 (d, J = 8.5 Hz, 2H, H3,5-Ar
J = 8.5 Hz, 2H, H2,6-Ar
OCH ), 61.01 (OCH ), 61.11 (OCH
Ar ), 123.84 (C3,5-Ar ), 125.76 (C -Ar
-Ar ), 139.17 (C -Ar ), 142.55 (C
49.0 (C -Ar ), 151.82 (C
Yield, 81%; mp 161–162 °C;
), 3.61 (s, 3H, OCH ), 3.82 (s, 3H,
-Ar ), 7.08 (d,
), 8.10 (d,
) d: 16.55 (SCH ), 56.04
), 107.15 (C -Ar ), 121.18 (C
), 128.44 (C2,6-Ar ), 130.65
-thiazole), 146.48 (C -Ar ),
), 154.64 (C -thiazole), 165.83 (C
H
3
3
3
5.3.3. In vitro tubulin polymerization assay
Microtubule protein (MTP) was isolated from fresh sheep brain4
and then MAP-free tubulin was purified from the microtubule pro-
3
3
3
4
2
4
5
1
3
20
5
); C NMR (CDCl
3
3
tein by DEAE chromatography. The protein content was esti-
(
3
3
3
5
4
1
-
mated by Bradford method using BSA as a standard.21
4
5
6
4
5
For investigation of tubulin polymerization test compounds in
(
C
3
4
1
5
5
4
4
final concentration 10
lM were preincubated with purified tubulin
1
4
5
2
-Ar
4
+
4
2
-
(12 M) and PEM buffer (100 mM PIPES, pH 6.9, 1 mM MgSO
l
4
, and
thiazole). MS, m/z (%) 418 (M , 16), 281 (38), 253 (19), 207 (100),
1 mM EGTA) for 15 min and then cooled to 0 °C. After adding the
final 1 mM concentration of GTP, the assembly was initiated by
warming the solution from 0 to 37 °C and polymerization process
was monitored by observing the variations in absorbance at
350 nm. The results represent the mean of duplicate
7
3 (25). Anal. Calcd for C19
18 2 5 2
H N O S : C, 54.53; H, 4.34; N, 6.69.
Found: C, 54.75; H, 4.47; N, 6.48.
5
.2.1.8. 4-(2,3,4-Trimethoxyphenyl)-5-(4-nitrophenyl)-2-(ben-
1
22
zylthio)-thiazole (5h).
CDCl ) d: 3.58 (s, 3H, OCH
OCH ), 4.52 (s, 2H, SCH ), 6.75 (d, J = 8.5 Hz, 1H, H
J = 8.5 Hz, 1H, -Ar ), 7.29–7.38 (m, 5H, benzyl), 7.45 (d,
J = 8.5 Hz, 2H, H2,6-Ar ), 8.09 (d, J = 8.5 Hz, 2H, H3,5-Ar
); 13C NMR
CDCl ) d: 38.49 (CH ), 56.05 (OCH ), 61.02 (OCH ), 61.07 (OCH ),
07.59 (C -Ar ), 121.08 (C -Ar ), 123.82 (C3,5-Ar ), 125.78 (C
), 127.79 (C -benzyl), 128.50 (C2,6-benzyl), 128.70 (C2,6-Ar
-Ar ), 136.18 (C
Yield, 75%; mp 140–141 °C; H NMR
), 3.82 (s, 3H, OCH ), 3.92 (s, 3H,
-Ar ), 7.08 (d,
experiments.
(
3
3
3
3
2
3
4
5.4. Molecular docking
H
2
4
5
5
The X-ray crystal structure of the DAMA-colchicine–tubulin
complex (PDB code 1SA0) was used as the tubulin protein tem-
(
1
Ar
3
2
3
3
3
2
3
5
4
1
4
5
6
-
plate. The 3D structures of ligand molecules were built, opti-
mized and saved in Mol2 format with the aid of the hyperchem
8.0 program. These partial charges of Mol2 files were further
4
4
5
),
1
29.12 (C3,5-Benzyl), 131.40 (C
3
4
1
-benzyl), 139.11

7
654
M. Salehi et al. / Bioorg. Med. Chem. 21 (2013) 7648–7654
modified by using the ADT package (version 1.5.4) so that the
charges of the nonpolar hydrogens atoms assigned to the atom to
which the hydrogen is attached. The resulting files were saved as
pdbqt files. The box center was set to the center of co-crystalized
ligand, DAMA colchicines (x = 117.2187, y = 90.1800, z = 6.2898)
and a grid of 40 - 40 - 40 was used to ensure that the area probed
was adequate for the ligands to explore all possible binding modes.
Moreover, the number of generations and maximum number of en-
ergy evaluations was set to 150 and 2,500,000, respectively. To val-
idate the utility of method for docking ligand into the active site,
DAMA colchicine was built using the hyperchem8.0, energy mini-
mized and docked to the active site using the above parameter
and the best scored pose of DAMA colchcine compared with the
crystal structure. At the end of a docking job with multiple runs, re-
sults differing by less than 0.5 Å in positional root–mean–square
deviation (RMSD) were clustered together and the results of the
4. Aryapour, H.; Riazi, G. H.; Foroumadi, A.; Ahmadian, S.; Shafiee, A.; Karima, O.;
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most favorable free energy of binding were selected as the resul-
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Assadieskandar, A.; Amini, M.; Salehi, M.; Sadeghian, H.; Alimardani, M.;
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Acknowledgment
This research has been supported by Tehran University of Med-
ical Sciences & Health Services Grant No. 20276.
1
1
1
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.030.
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