Synthesis and biological evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones as combretastatin analogs

Article abstract of DOI:10.1016/j.ejmech.2013.10.046

A series of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5 were designed as new heterocyclic analogs of combretastatin A-4 (CA-4). Indeed, the olefinic core structure of CA-4 has been replaced by 2(3H)-thiazole thione. The general synthetic strategy to prepare compounds 5 was based on the cyclocondensation reaction between triethylammonium N-(trimethoxyphenyl)dithiocarbamate and appropriate phenacyl halide. The cytotoxic activity evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5 against human cancer cell lines T47D, MCF-7 and MDA-MB-231 demonstrated that 4-methyl analog 5f showed the highest activity against all cell lines. Compound 5f had no significant toxicity towards non-tumoral cells MRC-5 and its cytotoxicity was apparently selective for cancer cells. The results of bioassays showed that the representative compound 5f depolymerized tubulin, inhibited cell proliferation, and induced apoptosis in cancer cells.

Full text of DOI:10.1016/j.ejmech.2013.10.046

European Journal of Medicinal Chemistry 70 (2013) 692e702
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 3-(trimethoxyphenyl)-2(3H)-
thiazole thiones as combretastatin analogs
Mandana Banimustafa a, Asma Kheirollahi b, Maliheh Safavi c,
,
g,
*
Sussan Kabudanian Ardestani c, Hassan Aryapour d, Alireza Foroumadi e, Saeed Emami f
a Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
b Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
c Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran
d Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran
e Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran
f Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
g Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, 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 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5 were designed as new heterocyclic analogs of
combretastatin A-4 (CA-4). Indeed, the olefinic core structure of CA-4 has been replaced by 2(3H)-
thiazole thione. The general synthetic strategy to prepare compounds 5 was based on the cyclo-
condensation reaction between triethylammonium N-(trimethoxyphenyl)dithiocarbamate and appro-
priate phenacyl halide. The cytotoxic activity evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones
5 against human cancer cell lines T47D, MCF-7 and MDA-MB-231 demonstrated that 4-methyl analog 5f
showed the highest activity against all cell lines. Compound 5f had no significant toxicity towards non-
tumoral cells MRC-5 and its cytotoxicity was apparently selective for cancer cells. The results of bioassays
showed that the representative compound 5f depolymerized tubulin, inhibited cell proliferation, and
induced apoptosis in cancer cells.
Received 6 June 2013
Received in revised form
14 October 2013
Accepted 16 October 2013
Available online 25 October 2013
Keywords:
Anticancer activity
Cytotoxicity
Combretastatin analogs
2(3H)-Thiazole thiones
Thiazolidine-2-thione
Tubulin polymerization
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
[4]. This compound was found to be a potent antiproliferative agent
against a broad spectrum of cancer cell lines including multi drug-
Currently, cancer is one of the most serious problem threats
human health in the world [1]. Chemotherapy has still been an
important fundament for cancer treatment. Drugs that perturb
microtubule/tubulin dynamics are used widely in cancer chemo-
therapy [2]. Several binding sites including taxane, vinca alkaloids
and colchicine binding sites have been identified on tubulin. Anti-
mitotic agents with the capability of binding at the colchicine site of
tubulin have received much attention and some of them such as
combretastatin A-4 (CA-4) and its water-soluble prodrug CA-4P are
undergoing clinical trials as antitumor drugs [2,3].
resistant cells [5,6].
Structureeactivity relationship studies on CA-4 derivatives have
established that pharmacophore structure for binding to tubulin is
the cis-orientation of the two ethenyl-bridged aromatic rings which
one of them bearing 3,4,5-trimethoxy substituents [7e9]. The cis-
stilbene structure of CA-4 is unstable and during storage and
administration, the cis-configuration is isomerized to trans-form
results in decreasing of both antitubulin activity and cytotoxicity
[10,11]. Moreover, the double bond reduction of CA-4 leads com-
pound with moderately reduced activity [8,9,12]. Accordingly,
considerable efforts have been focused on the cis-restriction of
pharmacophoric backbone (Fig. 1), particularly by the replacement
of the double bond with heterocyclic rings [13,14]. For example, a
number of five-membered heterocyclic analogs of CA-4 such as
imidazoles, pyrazoles, thiazoles, triazoles, oxazolones and fur-
anones, have been reported as cis-restricted biologically active
congeners of CA-4 [13e15].
CA-4 is the most biologically significant member of cis-stilbenes
isolated from the bark of African willow tree Combretum caffrum
* Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy, Mazandaran University of Medical Sciences, Sari 48175-861, Iran. Tel.: þ98
151 3543082; fax: þ98 151 3543084.
E-mail address: sd_emami@yahoo.com (S. Emami).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.046

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
693
MeO
MeO
MeO
OMe
MeO
MeO
MeO
MeO
MeO
S
N
S
MeO
OH
R
R
(A)
(B)
(C)
2(3H)-Thiazole thiones
Combretastatin A-4
(CA-4)
Cyclic combretastatin
analogues
Fig. 1. (A) Structure of combretastatin A-4 (CA-4) as unstable cis-stilbenes undergoing to trans-isomerism; (B) cyclic combretastatin analogs locked as cis-oriented stilbenoids; (C) 3-
(trimethoxyphenyl)-2(3H)-thiazole thiones as new heterocyclic analogs of CA-4.
With the aim of developing new heterocyclic analogs of CA-4,
we designed a series of 3-(trimethoxyphenyl)-2(3H)-thiazole thi-
one derivatives in which the olefinic core structure of CA-4 has
been replaced by 2(3H)-thiazole thione (Fig. 1). Thus, we report
here the convenient synthesis and biological activity of 3-(trime-
thoxyphenyl)-2(3H)-thiazole thiones and their related compounds.
3a, a signal was observed at 3.79 ppm that belongs to the protons of
CH2. The chemical shift value of aromatic H-2 and H-6 signal
(8.05 ppm) was in accord to the protons ortho to the carbonyl
group. The 13C NMR data showed that the resonance related to the
carbonyl carbon of compound 3a was occurred at 197.96 ppm.
Cyclization of compound 3a to 5j resulted in disappearance of
carbonyl signal from this region of 13C NMR spectrum. These data
confirmed the acyclic form of intermediate 3a.
2. Chemistry
In the 1H NMR spectra of compound 4a, two characteristic dou-
blets at 3.62 and 3.80 ppmwith geminal coupling constants w12.3 Hz
indicated the diastereotopicnature of C-5 methylene protonsbecause
of neighboring with C-4 stereocenter in thiazolidine ring.
The synthesis of target compounds 5aej was carried out ac-
cording to Scheme 1. Firstly, 3,4,5-trimethoxyaniline (1a) was
treated with carbon disulfide in triethylamine, at room tempera-
ture to obtain N-(trimethoxyphenyl) dithiocarbamate salt 2. Com-
pound 2 was reacted with appropriate phenacyl halide to produce a
gummy adduct, which was dehydrated by refluxing in 0.5% HCl to
give 4-aryl-3-(3,4,5-trimethoxyphenyl)-2(3H)-thiazole thione 5.
Structurally, the intermediate adduct would be either acyclic S-
phenacyl dithiocarbamate 3 or cyclic 4-hydroxythiazolidine-2-
thione 4. In the case of the reaction of dithiocarbamate 2 and 4-
methoxylphenacyl bromide, the intermediate was isolated and
characterized as acyclic adduct 3a. Moreover, the reaction 4-
bromophenacyl bromide or 2,4-dichlorophenacyl chloride with
the dithiocarbamate salt 2 resulted in corresponding cyclic adducts
which were unambiguously purified and characterized as 4-
hydroxythiazolidine-2-thione 4a,b. It should be noted that com-
pounds 4a and 4b which have a chiral center at the C-4 of their
thiazolidine-2-thione scaffold are racemates. Due to the problem of
isolation and purification the rather labile intermediates 3 and 4,
the rest of final compounds 5 were obtained from refluxing of crude
mixture of 3 and 4 in 0.5% hydrochloric acid.
Furthermore, the IR, 1H NMR, 13C NMR and MS spectral data
were used for structural characterization of target compounds 5.
Representatively, the 1H NMR spectrum of compound 5b showed
six protons as a singlet at 3.56 ppm, which was related to the 3- and
5-methoxy groups of trimethoxyphenyl moiety. The protons of 4-
methoxy group were appeared at 3.69 ppm as a singlet peak. A
singlet at 6.57 ppm attributed to the H-5 of 2(3H)-thiazole thione.
The H-2 and H-6 aromatic protons of trimethoxyphenyl moiety
were appeared at 6.26 ppm upfield respect to the other aromatic
protons. The protons of 4-bromophenyl displayed two doublets at
6.86 and 7.27 ppm bearing coupling constant of 8.35 Hz. In the
decoupled 13C NMR of compound 5b, 13 signals was detected which
confirmed correct carbon skeleton of the compound. The mass
spectral data of compound 5b provided further evidence of its
correct structure. The molecular ion peak was observed at 437 and
439 as expected for m/z values of Mþ and [M þ 2].
3. Biology
For preparation of 3-(4-methoxyphenyl) and 3-(3,4-
dimethoxyphenyl) analogs of
5
(compounds 6a,b), we have
3.1. Cytotoxicity assay
attempted to synthesize corresponding dithiocarbamates by
following the general procedure which was used for compound 2.
Due to the low solubility of 4-methoxyaniline and 3,4-
dimethoxyaniline in triethylamine, these attempts were failed.
Luckily, compounds 6a,b were prepared by using an one-pot
sequential reaction (Scheme 2). Thus, treatment of 4-
methoxyaniline or 3,4-dimethoxyaniline with carbon disulfide in
DMF in the presence of K3PO4 afforded corresponding dithiocarba-
mate which was subsequently reacted with phenacyl bromide. The
crude product was refluxed in 0.5% HCl to give pure compound 6.
As described above, the intermediate compounds 3a, 4a and 4b
were isolated and unambiguously characterized by spectral data.
The IR spectrum of compound 3a showed a strong band at
1681 cmꢀ1 due to the carbonyl group. The NH absorption of com-
pound 3a was appeared at 3313 cmꢀ1. In the 1H NMR of compound
The in vitro cytotoxic activity of synthesized compounds against
three human cancer cell lines, including MCF-7, MDA-MB-231 and
T47D was determined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide) colorimetric assay [16]. MTT
assay is based on reduction of the tetrazolium salt to blue colored
formazan by mitochondrial dehydrogenases in viable cells. The
relative cell viability was expressed as the mean percentage of
viable cells comparing to control cells, and the inhibition percent-
ages of compounds were assessed by the following formula:
[(Abscontrol
ꢀ Abstreated cells)/Abscontrol cells] ꢁ 100. The IC50
cells
values (the concentration of compound required to decrease 50% of
cell viability) were calculated from the concentrationeresponse
curves by regression analysis. The IC50 values of the compounds
against different cancer cell lines are shown in Table 1.

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M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
OMe
OMe
OMe
MeO
MeO
MeO
MeO
S
MeO
MeO
S
a
b
N
H
O
S
N
H
S
NH2
2
HNEt3
1a
3
R
OMe
OMe
MeO
MeO
MeO
MeO
S
S
c
N
N
S
S
OH
R
R
5
_
( )-
+
4
R= H; 4-Br; 4-Cl, 2,4-Cl2; 4-F; 4-CH3; 4-Ph; 4-OH; 4-OCH3; 3,4-(OH)2
Scheme 1. Synthesis of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5. Reagents and conditions: (a) CS2, Et3N, rt; (b) appropriate phenacyl halide, acetone; (c) 0.5% HCl, reflux.
3.2. Tubulin polymerization assay
the apoptosis induction was evaluated by Annexin-V binding and 7-
AAD uptake test. Annexin V-PE was used to quantitatively deter-
mine the percentage of cells within a population that are under-
going apoptosis [18]. Using this technique, cells that are viable are
Annexin V-PE and 7-AAD negative; cells that are in early apoptosis
are Annexin V-PE positive and 7-AAD negative; and cells that are in
late apoptosis or necrosis are both Annexin V-PE and 7-AAD posi-
tive. The double stained cells were analyzed by flow cytometry.
To further characterize the effects of selected compound 5f on
microtubule polymerization, we examined microtubules assembly
incubated with different concentration of compound 5f using UVe
Visible spectrometer. The absorbances (l 350 nm) were recorded
for a period of 30 min and the results were compared to the DMSO-
treated control group to evaluate the relative degree of change in
optical density. The percent of inhibition was calculated as follow: %
inhibition ¼ (1 ꢀ A350 sample/A350 control) ꢁ 100.
4. Results and discussion
3.3. Acridine orange/ethidium bromide staining test
The IC50 values of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones
5aej in comparison with etoposide as standard drug are presented
in Table 1. Among the compounds 5aej, 4-methyl analog 5f showed
the highest activity against all cell lines with IC50 values 11.8e
The potential of compounds 4b and 5f to induce apoptosis in
T47D and MCF-7 cells was determined morphologically by acridine
orange/ethidium bromide double staining test. Using fluorescence
microscopy, cells can be distinguished as live cells (uniformly
stained green) and apoptotic cells that are stained orange because
of cell membrane destruction and the intercalation of ethidium
bromide between the nucleotide bases of DNA [17].
19.7
icant growth inhibitory activity against all tested cell lines (IC50
values < 50 g/mL).
mg/mL. Moreover, compounds 5c, 5e and 5h exhibited signif-
m
The comparison of IC50 values of 5f and 5h with those of
unsubstituted analog 5a revealed that the introduction of methyl or
hydroxyl group on 4-phenyl ring increases the cytotoxic activity.
The compounds 5bee containing halogen substituent showed
lower activities against T47D cells compared to unsubstituted
counterpart 5a. Thus, halogen substitution could not improve the
inhibitory activity against T47D cell line. While, the introduction of
4-fluoro- or 2,4-dichloro- substituents improved the cytotoxic ac-
tivity toward MCF-7 and MDA-MB-231 (compounds 5c and 5e vs.
5a). The O-methylation of 4-hydroxy derivative (compound 5j
compared to 5h) reduced the cytotoxic potential against all cell
lines. The observed activity with 4-hydroxy derivative 5h and 3,4-
dihydroxy analog 5i demonstrated that the insertion of second
hydroxy group could not improve the cytotoxic activity.
To investigate the impact of 3,4,5-trimethoxyphenyl function-
ality on cytotoxic activity, we synthesized and evaluated the mono-
methoxy and dimethoxy congeners of compound 5a (compounds
6a and 6b, respectively). Interestingly, these compounds showed
comparable or superior activity respect to the corresponding 3,4,5-
trimethoxyphenyl derivative 5a. These findings revealed that the
intrinsic cytotoxic activity of compounds significantly depends on
3,4-diaryl-2(3H)-thiazole thione backbone.
3.4. Flow cytometry analyses of the apoptotic cells with Annexin V-
PE and 7-aminoactinomycin D (7-AAD) double staining
The MCF-7 cells were treated with IC50 concentrations of the
selected compounds 4b, 5f and etoposide as reference drug. Then,
R
H3CO
NH2
1. CS2, K3PO4, DMF,
S
Phenacyl bromide
N
S
2. HCl 0.5%, reflux
R
OCH3
1b: R= H
6a: R= H
1c: R= MeO
6b: R= MeO
Scheme 2. One-pot sequential synthesis of 3-(4-methoxyphenyl)- and 3-(3,4-
dimethoxyphenyl)-2(3H)-thiazole thiones 6.

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
695
Table 1
Cytotoxic activity (IC50
,
m
g/mL) of compounds 3e6.
Structure
Compound
T47D
MCF-7
MDA-MB-231
OCH3
OCH3
H3CO
H3CO
S
3a
29.9 ꢂ 5.2
18.6 ꢂ 4.6
16.4 ꢂ 3.6
N
S
H
O
OMe
MeO
MeO
S
N
S
4a
16.3 ꢂ 3.3
16.5 ꢂ 1.0
15.1 ꢂ 2.7
OH
Br
OMe
MeO
MeO
S
N
S
4b
16.9 ꢂ 4.0
9.6 ꢂ 2.5
14.5 ꢂ 2.1
OH
Cl
Cl
OMe
OMe
MeO
MeO
S
5a
31.6 ꢂ 3.8
>100
52.0 ꢂ 14.1
N
N
S
MeO
MeO
S
S
S
S
S
5b
>100
>100
58.0 ꢂ 1.4
Br
OMe
OMe
OMe
MeO
MeO
N
N
N
S
S
S
5c
5d
5e
38.9 ꢂ 6.7
38.5 ꢂ 2.1
19.5 ꢂ 1.9
F
MeO
MeO
>100
>100
>100
Cl
MeO
MeO
43.4 ꢂ 8.8
23.6 ꢂ 3.2
49.0 ꢂ 5.2
(continued on next page)
Cl
Cl

696
M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
Table 1 (continued )
Compound
Structure
T47D
MCF-7
MDA-MB-231
OMe
MeO
MeO
S
S
N
5f
16.3 ꢂ 3.7
19.7 ꢂ 0.8
11.8 ꢂ 1.3
Me
OMe
MeO
MeO
S
S
N
5g
5h
5i
>100
45.3 ꢂ 10.6
35.1 ꢂ 3.1
>100
>100
Ph
OMe
OMe
MeO
MeO
S
S
N
N
26.4 ꢂ 6.2
22.9 ꢂ 7.2
HO
MeO
MeO
S
S
24.9 ꢂ 3.7
29.8 ꢂ 0.3
HO
OH
OMe
MeO
MeO
S
S
N
5j
>100
>100
53.8 ꢂ 8.4
MeO
MeO
MeO
S
S
N
N
6a
23.2 ꢂ 2.9
88.5 ꢂ 12.9
36.8 ꢂ 5.6
OMe
S
S
6b
33.1 ꢂ 8.2
17.6 ꢂ 1.5
19.1 ꢂ 1.4
Etoposide
8.2 ꢂ 1.2
9.4 ꢂ 3.2
7.2 ꢂ 1.6

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
697
Furthermore, the cytotoxic activity of acyclic and alcoholic in-
termediates (compounds 3a and 4a,b, respectively) were also
evaluated against test cell lines (Table 1). Surprisingly, by
comparing the IC50 values of acyclic analog 3a with those of het-
erocyclic congener 5j, it was demonstrated that acyclic form had
more cytotoxicity than compound 5j. It was worthy of note that the
alcoholic derivatives 4a and 4b showed more potent activity
respect to the corresponding dehydrated analogs 5b and 5e,
respectively. Alcoholic compound 4b was as potent as standard
drug etoposide against MCF-7 cells. Its activity against the latter cell
line was superior than all tested compounds.
The cytotoxic activity of promising compounds 4b and 5f were
also evaluated on non-tumoral cell line MRC-5. Results of MTT
assay showed that compounds 4b and 5f have less cytotoxic activity
against non-tumoral cells. The IC50 values of compounds 4b and 5f
on MRC-5 cells were 47.3 ꢂ 4.2
mg/mL and >100 mg/mL, respec-
tively. According to the results, the cytotoxicity of these compounds
was apparently selective for cancer cell lines T47D, MCF-7 and
MDA-MB-231. Particularly, compound 5f had no significant toxicity
towards non-tumoral cell line MRC-5 (Selectivity index > 5).
To verify whether the cytotoxic activity of the designed com-
pounds was correlated to tubulin inhibition, the inhibitory effect of
the most active compound 5f on the polymerization of purified
tubulin was evaluated. The effect of compound 5f on the microtu-
bules polymerization is shown in Fig. 2. The results showed that
compound 5f evidently inhibited the assembly of purified sheep
brain MTP in a concentration dependent manner (Fig. 2).
It is well documented that exposure to microtubule-targeting
agents leads to malformed mitotic spindles, mitotic arrest, and
apoptosis. Thus, we next examined the effects of selected com-
pounds 4b and 5f on the apoptosis of T47D and MCF-7 cells by
acridine orange/ethidium bromide double staining technique. T47D
and MCF-7 cells were treated with and without IC50 concentration
of compounds 4b and 5f for 24 h and stained with a mixture of
acridine orange and ethidium bromide. Analysis of the acridine
orange/ethidium bromide staining results revealed that the test
compounds 4b and 5f induced apoptosis in T47D and MCF-7 cell
lines. As shown in Fig. 3, the non-apoptotic control cells were
stained green and the apoptotic cells had orange particles in their
nuclei due to nuclear DNA fragmentation. The appearance of
chromatin condensation and nuclear fragmentation are evident in
this figure.
To further confirm of apoptosis induction by compounds 4b and
5f, the treated MCF-7 cells were subjected to Annexin V/7-AAD
double staining followed by flow cytometry analyses (Figs. 4 and
5). Annexin V/7-AAD flow cytometric analyses revealed that cells
undergo apoptosis after treatment with IC50 concentrations of
compounds 4b and 5f. As shown in Fig. 5, compounds 4b and 5f
induced 51.67% and 66.47% apoptosis in the cancer cells, respec-
tively. Therefore, it is evident that these compounds have strong
anti-proliferative activity.
Fig. 2. Inhibition of tubulin polymerization by compound 5f. (A) Kinetics of the in-
hibition of microtubule assembly in different concentrations of compound 5f. (B) Plot
of percent tubulin polymerization against log concentration (M) of compound 5f at
37 ꢃC.
against human cancer cell lines, including T47D, MCF-7 and
MDA-MB-231 demonstrated that 4-methyl analog 5f showed the
highest activity against all cell lines. The cytotoxicity of compound
5f was apparently selective for tested cancer cell lines and com-
pound 5f had no significant toxicity towards non-tumoral cell line
MRC-5. Besides 2(3H)-thiazole thiones 5, 4-hydroxy-3-(3,4,5-
trimethoxyphenyl)-4-(2,4-dichlorophenyl)thiazolidine-2-thione
(4b) which was separated and characterized as a stable interme-
diate showed better profile of antiproliferative activity against
tested cell lines. The representative compound 5f depolymerized
tubulin, inhibited cell proliferation, and induced apoptosis in can-
cer cells. These compounds can be considered as more stable an-
alogs of combretastatin A-4 for further biological evaluations.
5. Conclusion
In summary, we designed a series of 3-(trimethoxyphenyl)-
2(3H)-thiazole thiones 5 as new heterocyclic analogs of CA-4, in
which the olefinic core structure of CA-4 replaced by 2(3H)-thiazole
thione. The general synthetic strategy employed to prepare the 3-
(trimethoxyphenyl)-2(3H)-thiazole thiones 5 was based on the
cyclocondensation reaction between N-(trimethoxyphenyl)dithio-
carbamate and appropriate phenacyl halide. In some cases, the
intermediate adducts including acyclic S-phenacyl dithiocarbamate
3 or cyclic 4-hydroxythiazolidine-2-thione 4 were purified and
characterized as individual compounds. The cytotoxic activity
6. Experimental protocols
6.1. Chemistry
6.1.1. General methods
Triethylammonium
N-(3,4,5-trimethoxyphenyl)dithiocarba-
mate was prepared according to the literature method [19].
Required phenacyl halides including 2-bromo-40-hydrox-
yacetophenone and 2-bromo-40-chloroacetophenone were syn-
thesized by bromination of corresponding acetophenones using
CuBr2 in CHCl3eEtOAc [20]. Other phenacyl halides were
evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones
5

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M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
Fig. 3. Acridine orange/ethidium bromide double staining of T47D (A) and MCF-7 (B) cells with characteristic symptoms of apoptosis: (a) DMSO 1% as control; (b) etoposide as
positive control; (c) cells treated with IC50 concentration of compound 4b for 24 h; (d) cells treated with IC50 concentration of compound 5f for 24 h. White arrow indicates live cells
and dashed arrow indicates apoptotic cells. The images of cells were taken with a fluorescence microscope at 400ꢁ.
commercially available materials from Merck or Fluka companies.
The progress of reactions was checked by thin-layer chromatog-
raphy (TLC) using silica gel 60 F254 plastic sheets (Merck). The
ultra-violet light (254 nm) was used for TLC visualization. Yields are
based on isolated product and were not optimized. Melting points
were determined in open glass capillaries using Bibby Stuart Sci-
entific SMP3 apparatus (Stuart Scientific, Stone, UK) and are un-
corrected. The IR spectra were obtained on a PerkinElmer FT-IR
spectrophotometer using KBr disks. The NMR spectra were recor-
ded using a Bruker 500 spectrometer and chemical shifts are
in refrigerator overnight. The precipitated solid was collected by
filtration and washed with water. The product was recrystallized
from methanol or diethyl ether to give pure 3-(trimethoxyphenyl)-
2(3H)-thiazole thione 5.
6.1.4. General procedure for the synthesis of 3-(4-methoxyphenyl)-
and 3-(3,4-dimethoxyphenyl)-2(3H)-thiazole thiones 6
A
mixture of 4-methoxyaniline or 3,4-dimethoxyaniline
(1 mmol) and K3PO4 (215 mg, 1 mmol) in DMF (7.5 mL) was
cooled in ice bath, and then CS2 (380 mg, 5 mmol) was added. After
stirring for 20 min, 2-bromoacetophenone (199 mg, 1 mmol) was
added and the mixture was allowed to warm to room temperature
gradually. Water (20 mL) was added to the mixture and left in
refrigerator overnight. The precipitated solid was collected by
filtration and washed with water. The obtained crude product was
mixed with 0.5% HCl (20 mL) and methanol (4 mL), and the mixture
was refluxed for 45 min. After cooling up to room temperature, the
reaction mixture was left in refrigerator overnight. The precipitated
solid was collected by filtration and washed with water. The
product was recrystallized from methanol to give compound 6.
expressed as
d (ppm) with tetramethylsilane (TMS) as internal
standard. The mass spectra were obtained using a HP 5937 Mass
Selective Detector (Agilent technologies).
6.1.2. General procedure for the synthesis of intermediates S-
phenacyl dithiocarbamate 3 or 4-hydroxythiazolidine-2-thione 4
N-(Trimethoxyphenyl) dithiocarbamate salt 2 (1.5 mmol) was
dissolved in acetone (10 mL) and the solution was stirred in an ice
bath. An appropriate phenacyl halide (1.5 mmol) was added por-
tionwise over a 30 min period. After completion of the reaction
(consumption of phenacyl halide, 0.5e2 h), the minimum volume
of water to cause solution was added and the cold solution was
stirred for 15 min longer. After evaporation of acetone under
reduced pressure, a viscose oily residue was separated. Water
(5 mL) was added to the oily residue and the aqueous phase was
decanted. The crude product was crystallized from methanol or
diethyl ether.
6.1.5. Physicochemical and spectral data of synthesized compounds
6.1.5.1. 4-Methoxyphenacyl N-(3,4,5-trimethoxyphenyl)dithiocarba-
mate (3a). Yield 60%; mp 142e143 ꢃC; IR (KBr, cmꢀ1
)
nmax: 3313,
3076, 2938, 2838, 1681, 1599, 1508, 1454, 1312, 1261, 1177, 1127,
1015, 987, 835. 1H NMR (500 MHz, CDCl3)
d: 3.79 (s, 2H, CH2), 3.85
(s, 6H, 3-OCH3 and 5-OCH3), 3.87 (s, 3H, 4-OCH3), 3.91 (s, 3H, 40-
OCH3), 4.75 (br s, 1H, NH), 6.35 (s, 2H, H-2 and H-6), 7.00 (d, 2H,
J ¼ 8.82 Hz, H-30 and H-50), 8.05 (d, 2H, J ¼ 8.82 Hz, H-20 and H-60).
6.1.3. General procedure for the synthesis of 3-(trimethoxyphenyl)-
2(3H)-thiazole thiones 5
13C NMR (125 MHz, CDCl3)
d: 56.02, 56.49, 56.68, 61.36, 101.19,
N-(Trimethoxyphenyl) dithiocarbamate salt 2 (1.5 mmol) was
dissolved in acetone (10 mL) and the solution was stirred in an ice
bath. An appropriate phenacyl halide (1.5 mmol) was added por-
tionwise over a 30 min period. After completion of the reaction
(consumption of phenacyl halide, 0.5e2 h), the minimum volume
of water to cause solution was added and the cold solution was
stirred for 15 min longer. After evaporation of acetone under
reduced pressure, a viscose oily residue was separated. Water
(5 mL) was added to the oily residue and the aqueous phase was
decanted. The oily crude product was mixed with 0.5% HCl (20 mL)
and methanol (4 mL), and the mixture was refluxed for 45 min.
After cooling up to room temperature, the reaction mixture was left
107.19, 114.06, 114.51, 127.83, 131.59, 153.29, 153.71, 160.45, 164.74,
197.96. MS (m/z, %): 406 (Mþ, 5), 389 (100), 343 (30), 288 (16), 164
(18),135 (43). Anal. Calcd for C19H21NO5S2: C, 56.00; H, 5.19; N, 3.44.
Found: C, 56.23; H, 5.08; N, 3.50.
6.1.5.2. (ꢂ)-4-Hydroxy-3-(3,4,5-trimethoxyphenyl)-4-(4-
bromophenyl)thiazolidine-2-thione (4a). Yield 92%; mp 134e
135 ꢃC; IR (KBr, cmꢀ1
)
nmax: 3265, 2933, 1593, 1504, 1403, 1198,
1072, 990, 833, 820, 781, 631, 567. 1H NMR (500 MHz, CDCl3)
d: 3.57
(s, 6H, 3-OCH3 and 5-OCH3), 3.62 (d, 1H, J ¼ 12.25 Hz, H-5a Thia-
zolidine), 3.68 (s, 3H, 4-OCH3), 3.80 (d, 1H, J ¼ 12.28 Hz, H-5b
Thiazolidine), 6.23 (s, 2H, H-2 and H-6), 7.33 (s, 4H, BrPh), 7.53 (s,

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
699
Fig. 4. Histogram of flow cytometric analysis of MCF-7 cells treated with compounds 4b and 5f. Cells were stained with Annexin V/7-AAD and quantitated by flow cytometry. MCF-7
cells were treated with DMSO 1% (negative control) or with IC50 concentrations of etoposide (positive control) and compounds 4b and 5f. Fluorescence intensity of Annexin V-PE
was shifted to the right.

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M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
Fig. 5. Flow cytometric analysis of MCF-7 cells treated with compounds 4b and 5f. Cells were stained with Annexin V/7-AAD and quantitated by flow cytometry. Percentage of PE-
positive events (% apoptotic cells) was calculated.
1H, OH). 13C NMR (125 MHz, CDCl3/DMSO-d6)
d: 44.99, 56.37, 56.53,
6.1.5.5. 3-(3,4,5-Trimethoxyphenyl)-4-(4-bromophenyl)tꢀh1iazole-
2940, 1598, 1505, 1418, 1275, 1263, 1231, 1131, 955, 750. 1H NMR
61.04, 100.77, 107.32, 123.16, 128.38, 130.44, 131.58, 132.37, 133.85,
137.89, 140.51, 153.00, 197.77. MS (m/z, %): 455 (Mþ, 5), 423 (45),
408 (42), 395 (35), 243 (38),194 (64),183 (100), 155 (34), 89 (35), 76
(26), 50 (37). Anal. Calcd for C18H18BrNO4S2: C, 47.37; H, 3.98; N,
3.07. Found: C, 47.33; H, 4.22; N, 3.05.
2(3H)-thione (5b). Yield 73%; mp 239e240 ꢃC; IR (KBr, cm
) nmax:
(500 MHz, CDCl3) : 3.56 (s, 6H, 3-OCH3 and 5-OCH3), 3.69 (s, 3H, 4-
d
OCH3), 6.26 (s, 2H, H-2 and H-6), 6.57 (s, 1H, Thiazole-H), 6.86 (d,
2H, J ¼ 8.35 Hz, H-30 and H-50), 7.24 (d, 2H, J ¼ 8.35 Hz, H-20 and H-
60). 13C NMR (125 MHz, CDCl3/DMSO-d6)
d: 56.62, 61.19, 106.68,
6.1.5.3. (ꢂ)-4-Hydroxy-3-(3,4,5-trimethoxyphenyl)-4-(2,4-
dichlorophenyl)thiaꢀz1olidine-2-thione (4b). Yield 47%; mp 151e
109.87, 123.90, 129.86, 130.34, 131.99, 132.34, 132.96, 138.62, 143.98,
153.71. MS (m/z, %): 439 (M þ 2, 100), 437 (Mþ, 89), 424 (50), 392
(23), 194 (27), 183 (43), 89 (34). Anal. Calcd for C18H16BrNO3S2: C,
49.32; H, 3.68; N, 3.20. Found: C, 49.34; H, 3.55; N, 3.14.
152 ꢃC; IR (KBr, cm
) nmax: 3431, 2939, 1592, 1503, 1318, 1231, 1121,
1071, 947, 820. 1H NMR (500 MHz, CDCl3)
d: 3.43 (d, 1H,
J ¼ 12.37 Hz, H-5a Thiazolidine), 3.69 (s, 6H, 3-OCH3 and 5-OCH3),
3.75 (s, 3H, 4-OCH3), 4.24 (d, 1H, J ¼ 12.36 Hz, H-5b Thiazolidine),
6.61 (s, 2H, H-2 and H-6), 7.13 (dd, 1H, J ¼ 8.62 and 2.08 Hz, H-50),
7.31 (d, 1H, J ¼ 2.08 Hz, H-30), 7.78 (d, 1H, J ¼ 8.61 Hz, H-60). 13C NMR
6.1.5.6. 3-(3,4,5-Trimethoxyphenyl)-4-(4-fluorophenyl)tꢀh1iazole-
2(3H)-thione (5c). Yield 47%; mp 190e191 ꢃC; IR (KBr, cm
) nmax:
3060, 2932, 1601, 1505, 1459, 1415, 1271, 1226, 1136, 1004, 852, 729.
(125 MHz, CDCl3/DMSO-d6)
d
: 42.52, 56.46, 56.70, 61.13, 61.82,
1H NMR (500 MHz, CDCl3)
d
: 3.73 (s, 6H, 3-OCH3 and 5-OCH3), 3.87
99.37, 106.51, 198.62, 127.50, 127.77, 130.80, 133.43, 136.00, 138.03,
152.88. MS (m/z, %): 427 (32), 253 (10), 225 (93), 210 (73), 182 (61),
173 (100), 152 (65), 140 (19), 124 (18), 109 (43), 96 (29), 86 (22), 75
(22), 57 (14). Anal. Calcd for C18H17Cl2NO4S2: C, 48.43; H, 3.84; N,
3.14. Found: C, 48.71; H, 3.90; N, 2.95.
(s, 3H, 4-OCH3), 6.42 (s, 2H, H-2 and H-6), 6.64 (s, 1H, Thiazole-H),
6.95e6.99 (m, 2H, H-30 and H-50), 7.09e7.14 (m, 2H, H-20 and H-60).
13C NMR (125 MHz, CDCl3)
d: 56.73, 61.39, 106.80, 109.44, 116.14 (d,
JC,F ¼ 21.75 Hz), 127.29, 130.96 (d, JC,F ¼ 8.5 Hz), 133.12, 138.81,
144.31, 153.88, 163.39 (d, JC,F ¼ 249.38 Hz), 190.32. Anal. Calcd for
C18H16FNO3S2: C, 57.28; H, 4.27; N, 3.71. Found: C, 57.25; H, 4.32; N,
6.1.5.4. 3-(3,4,5-Trimethoxyphenyl)-4-phenyltꢀh1iazole-2(3H)-thione
3.70.
(5a). Yield 66%; mp 184e185 ꢃC; IR (KBr, cm
) nmax: 2934, 2827,
1600, 1505, 1450, 1228, 1126. 1H NMR (500 MHz, CDCl3)
d
: 3.71 (s,
6.1.5.7. 3-(3,4,5-Trimethoxyphenyl)-4-(4-chlorophenyl)tꢀh1iazole-
2928, 1597, 1505, 1274, 1262, 1231, 1133, 1092, 1050, 821. 1H NMR
6H, 3-OCH3 and 5-OCH3), 3.85 (s, 3H, 4-OCH3), 6.42 (s, 2H, H-2 and
H-6), 6.64 (s, 1H, Thiazole-H), 7.11 (d, 2H, J ¼ 7.0 Hz, H-20 and H-60),
7.25 (t, 2H, J ¼ 7.5 Hz, H-30 and H-50), 7.31 (t,1H, J ¼ 7.5 Hz, H-40). MS
(m/z, %): 359 (Mþ, 100), 344 (43), 312 (18), 258 (18), 135 (35), 105
(31), 77 (37), 43 (20). Anal. Calcd for C18H17NO3S2: C, 60.14; H, 4.77;
N, 3.90. Found: C, 60.32; H, 5.03; N, 3.77.
2(3H)-thione (5d). Yield 70%; mp 214e215 ꢃC; IR (KBr, cm
) nmax:
(500 MHz, CDCl3) : 3.76 (s, 6H, 3-OCH3 and 5-OCH3), 3.90 (s, 3H, 4-
d
OCH3), 6.43 (s, 2H, H-2 and H-6), 6.66 (s, 1H, Thiazole-H), 7.08 (d,
2H, J ¼ 8.22 Hz, H-30 and H-50), 7.27 (d, 2H, J ¼ 8.03 Hz, H-20 and H-
60). MS (m/z, %): 395 (M þ 2, 45), 393 (Mþ, 100), 378 (40), 346 (14),

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
701
292 (14), 181 (11), 168 (17), 152 (16), 136 (20), 123 (13), 109 (13), 89
(11), 81 (10), 66 (13), 57 (10). Anal. Calcd for C18H16ClNO3S2: C,
54.88; H, 4.09; N, 3.56. Found: C, 54.76; H, 3.88; N, 3.86.
Thiazole-H), 6.78 (d, 2H, J ¼ 8.81 Hz, H-30 and H-50), 7.04 (d, 2H,
J ¼ 8.81 Hz, H-20 and H-60). 13C NMR (125 MHz, CDCl3)
d: 55.71,
56.70, 61.38, 106.86, 108.49, 114.32, 123.49, 130.36, 133.44, 145.50,
153.79, 160.59. Anal. Calcd for C19H19NO4S2: C, 58.59; H, 4.92; N,
3.60. Found: C, 58.75; H, 5.11; N, 3.36.
6.1.5.8. 3-(3,4,5-Trimethoxyphenyl)-4-(2,4-dichlorophenyl)thiazole-
2(3H)-thione (5e). Yield 39%; mp 173e174 ꢃC; IR (KBr, cmꢀ1
)
nmax
3107, 2935, 1600, 1504, 1465, 1308, 1292, 1232, 1123, 1096. 1H NMR
(500 MHz, CDCl3) : 3.76 (s, 6H, 3-OCH3 and 5-OCH3), 3.85 (s, 3H, 4-
:
6.1.5.14. 3-(4-Methoxyphenyl)-4-phenylthiazole-2(3H)-thione (6a).
d
Yield 15%; mp 141e142 ꢃC; IR (KBr, cmꢀ1
)
nmax: 3029, 2837, 1609,
OCH3), 6.47 (s, 2H, H-2 and H-6), 6.67 (s, 1H, Thiazole-H), 7.11 (d,1H,
1514, 1284, 1257, 1171, 1052, 764, 728. 1H NMR (500 MHz, CDCl3)
d:
J ¼ 8.24 Hz, H-60), 7.19 (dd, 1H, J ¼ 8.24 and 1.92 Hz, H-50), 7.38 (d,
3.85 (s, 3H, OCH3), 6.66 (s, 1H, Thiazole-H), 6.93 (d, 2H, J ¼ 8.89 Hz,
H-3 and H-5), 7.12e7.15 (m, 4H, H-2, H-6, H-20 and H-60), 7.27 (t, 2H,
J ¼ 7.61 Hz, H-30 and H-50), 7.32 (t, 1H, J ¼ 7.35 Hz, H-40). Anal. Calcd
for C16H13NOS2: C, 64.18; H, 4.38; N, 4.68. Found: C, 64.10; H, 4.49;
N, 4.81.
1H, J ¼ 1.9 Hz, H-30). 13C NMR (125 MHz, CDCl3)
d: 56.66, 61.32,
106.16, 111.62, 127.58, 128.74, 130.12, 132.69, 133.30, 135.72, 137.21,
138.79, 140.57, 153.7. MS (m/z, %): 427 (Mþ, 88), 412 (29), 382 (12),
326 (13), 167 (18), 149 (31), 137 (30), 123 (44), 109 (54), 97 (85), 83
(86), 69 (93), 57 (100), 43 (85). Anal. Calcd for C18H15Cl2NO3S2: C,
50.47; H, 3.53; N, 3.27. Found: C, 50.61; H, 3.52; N, 3.44.
6.1.5.15. 3-(3,4-Dimethoxyphenyl)-4-phenylthiazole-2(3H)-thione
(6b). Yield 24%; mp 168e169 ꢃC; IR (KBr, cmꢀ1
)
nmax: 1598, 1516,
1442, 1295, 1244, 1220, 1079, 1052, 1023, 766, 732. 1H NMR
(500 MHz, CDCl3) 3.78 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.65 (s,
6.1.5.9. 3-(3,4,5-Trimethoxyphenyl)-4-(p-tolyl)thiazole-2(3H)-thione
(5f). Yield 75%; mp 164e165 ꢃC; IR (KBr, cmꢀ1
)
nmax: 1598, 1505,
d
1463, 1417, 1267, 1231, 1128, 717. 1H NMR (500 MHz, CDCl3)
d: 2.33
1H, Thiazole-H), 6.71 (d, 1H, J ¼ 2.01 Hz, H-2), 6.80 (dd, 1H, J ¼ 8.52
and 2.19 Hz, H-6), 6.87 (d, 1H, J ¼ 8.54 Hz, H-5), 7.13 (d, 2H,
J ¼ 7.61 Hz, H-20 and H-60), 7.25e7.35 (m, 3H, H-30 and H-40 and H-
50). Anal. Calcd for C17H15NO2S2: C, 61.98; H, 4.59; N, 4.25. Found: C,
61.92; H, 4.57; N, 4.00.
(s, 3H, CH3), 3.74 (s, 6H, 3-OCH3 and 5-OCH3), 3.88 (s, 3H, 4-OCH3),
6.43 (s, 2H, H-2 and H-6), 6.61 (s, 1H, Thiazole-H), 7.01 (d, 2H,
J ¼ 8.05 Hz, H-30 and H-50), 7.07 (d, 2H, J ¼ 7.94 Hz, H-20 and H-60).
13C NMR (125 MHz, CDCl3)
d: 21.65, 56.69, 61.36, 106.88, 108.87,
128.29,128.85, 129.19,129.58, 133.39, 139.80, 145.66, 153.74,190.25.
Anal. Calcd for C19H19NO3S2: C, 61.10; H, 5.13; N, 3.75. Found: C,
61.11; H, 5.01; N, 3.64.
6.2. Biological activity
6.2.1. Cell lines and cell culture
6.1.5.10. 3-(3,4,5-Trimethoxyphenyl)-4-(4-biphenyl)tꢀh1iazole-2(3H)-
The human cancer cell lines MCF-7, MDA-MB-231 and T47D
were obtained from Pasture Institute, Tehran (Iran). The cells were
thione (5g). Yield 33%; mp 209e210 ꢃC; IR (KBr, cm
) nmax: 1584,
1504, 1417, 1231, 1127, 1004, 753. 1H NMR (500 MHz, CDCl3)
d
: 3.65
cultured in RPMI 1640 containing 10% FBS, 1% L-Glutamine, and
(s, 6H, 3-OCH3 and 5-OCH3), 3.79 (s, 3H, 4-OCH3), 6.69 (s, 2H, H-2
and H-6), 7.23 (s,1H, Thiazole-H), 7.34e7.71 (m, 9H, Biphenyl). Anal.
Calcd for C24H21NO3S2: C, 66.18; H, 4.86; N, 3.22. Found: C, 66.20; H,
4.99; N, 3.07.
PenicillineStreptomycin, and then cells were incubated at 37 ꢃC in a
humidified atmosphere with 5% CO2.
6.2.2. Cytotoxicity assay
The in vitro cytotoxic activity of test compounds was determined
by MTT assay [16]. Briefly, cells in the log-phase of growth were
harvested by trypsinization, seeded in 96 well plates (Nunc,
Denmark) for 24 h. Then, the cells were treated with various con-
centrations of the compounds for 48 h. Etoposide and DMSO were
used as positive and negative controls, respectively. The final con-
centration of DMSO was less than 2%. After 48 h, the culture me-
6.1.5.11. 3-(3,4,5-Trimethoxyphenyl)-4-(4-hydroxyphenyl)thiazole-
2(3H)-thione (5h). Yield 53%; mp 254e255 ꢃC; IR (KBr, cmꢀ1
)
nmax
3400, 1602, 1505, 1464, 1278, 1233, 1128, 834. 1H NMR (500 MHz,
DMSO-d6) : 3.64 (s, 6H, 3-OCH3 and 5-OCH3), 3.66 (s, 3H, 4-OCH3),
:
d
6.58 (s, 2H, H-2 and H-6), 6.64 (d, 2H, J ¼ 8.55 Hz, H-30 and H-50),
7.03 (s, 1H, Thiazole-H), 7.05 (d, 2H, J ¼ 8.52 Hz, H-20 and H-60), 9.70
(s, 1H, OH). MS (m/z, %): 375 (Mþ, 100), 360 (76), 329 (92), 293 (26),
274 (21), 205 (20), 180 (29), 172 (28), 150 (68), 137 (31), 118 (65), 109
(36), 97 (36), 89 (48), 81 (34), 69 (40), 57 (40), 43 (36). Anal. Calcd
for C18H17NO4S2: C, 57.58; H, 4.56; N, 3.73. Found: C, 57.31; H, 4.48;
N, 3.85.
dium was removed, and cells were incubated with 200 mL of MTT
solution (0.5 mg/mL) for 4 h. Then, the supernatant was removed
and the formazan crystals were dissolved using DMSO. The absor-
bance was read at 492 nm with an ELISA plate reader (Exert 96, Asys
Hitch, Ec Austria) after 30 min.
6.1.5.12. 3-(3,4,5-Trimethoxyphenyl)-4-(3,4-dihydroxyphenyl)thꢀia1-
nmax: 3390, 3111, 1599, 1505, 1306, 1233, 1126, 1025. 1H NMR
6.2.3. Tubulin polymerization assay
zole-2(3H)-thione (5i). Yield 52%; mp 237e238 ꢃC; IR (KBr, cm
)
Sheep brain microtubule protein was isolated by two
cycles of polymerization-depolymerization in the PEM buffer
[100 mM PIPES, pH 6.9, 1 mM MgSO4 and 1 mM ethylene glycol
tetraacetic acid (EGTA)], according to the method described by
Sengupta et al. [21]. Tubulin was purified from the microtubule
protein by phosphocellulose chromatography [22]. The tubulin
solution was rapidly frozen as drops in liquid nitrogen and stored
at ꢀ70 ꢃC until used. Protein concentration was determined by
the method of Bradford with bovine serum albumin as the
standard [23]. The purity of tubulin was determined using
polyacrylamide gel electrophoresis, which was performed by the
Laemmli method [24].
(500 MHz, DMSO-d6) : 3.65 (s, 6H, 3-OCH3 and 5-OCH3), 3.66 (s,
d
3H, 4-OCH3), 6.50 (dd, 1H, J ¼ 8.18 and 2.01 Hz, H-60), 6.57 (s, 2H, H-
2 and H-6), 6.59 (d, 1H, J ¼ 8.18 Hz, H-50), 6.60 (d, 1H, J ¼ 2.01 Hz, H-
20), 6.98 (s,1H, Thiazole-H), 9.00 (s,1H, OH), 9.22 (s,1H, OH). MS (m/
z, %): 391 (Mþ, 100), 376 (51), 344 (23), 290 (17), 166 (34), 149 (35),
134 (68), 120 (35), 109 (46), 97 (45), 83 (40), 69 (48), 57 (54), 43
(43). Anal. Calcd for C18H17NO5S2: C, 55.23; H, 4.38; N, 3.58. Found:
C, 55.20; H, 4.46; N, 3.56.
6.1.5.13. 3-(3,4,5-Trimethoxyphenyl)-4-(4-methoxyphenyl)thiazole-
2(3H)-thione (5j). Yield 80%; mp 174e175 ꢃC; IR (KBr, cmꢀ1
)
nmax
1599, 1505, 1465, 1417, 1275, 1249, 1052, 833. 1H NMR (500 MHz,
CDCl3) : 3.74 (s, 6H, 3-OCH3 and 5-OCH3), 3.79 (s, 3H, 4-OCH3),
3.88 (s, 3H, 40-OCH3), 6.43 (s, 2H, H-2 and H-6), 6.58 (s, 1H,
:
The tubulin polymerization assay was carried out based on re-
ported method with some modifications [25]. Tubulin pellets were
thawed and centrifuged at 0 ꢃC to remove any aggregated or de-
natured tubulin. A drug/DMSO-tubulin pre-incubation, without
d

702
M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702
GTP, was performed for 15 min at 0 ꢃC in ice. After adding three
mL
Appendix A. Supplementary data
GTP (final concentration, 1 mM), the drug/DMSO-tubulin samples
were immediately placed in a Varian Cary 100 UV/visible spectro-
photometer, initiated polymerization by setting the temperature
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.046.
controller at 37 ꢃC. The absorbance (
l 350 nm) for a period of
30 min was recorded and the results were compared to the DMSO-
treated control cells to evaluate the relative degree of change in
optical density. DMSO was used at the final concentrations of <4%
(v/v).
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Acknowledgments
This work was supported by a grant from the Research Council
of Mazandaran University of Medical Sciences, Sari, Iran. A part of
this work was related to the Pharm. D thesis of Mandana Bani-
mustafa (Faculty of Pharmacy, Mazandaran University of Medical
Sciences).
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