Design, synthesis, cytotoxic evaluation and tubulin inhibitory activity of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazole derivatives

Article abstract of DOI:10.1016/j.bmc.2013.03.011

A new series of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazoles were synthesized and their cytotoxic activities in vitro against four different cell lines (HT-29, MCF-7, NIH-3T3, AGS) were evaluated. Compound 6g bearing 3,4,5-trimethoxyphenyl moiety on ring A and 4-methoxy substituent on ring B displayed potent cytotoxic activity against all cell lines. Flow cytometry analysis and microtubule polymerization assay confirmed that cytotoxic activities of this compound were related to inhibitory effect against microtubules polymerization. Molecular modeling studies revealed that compound 6g could strongly bind to the colchicine binding site of α,β-tubulin through hydrogen bond interactions with Thrα179 and Cysβ241.

Full text of DOI:10.1016/j.bmc.2013.03.011

Bioorganic & Medicinal Chemistry 21 (2013) 2703–2709
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, cytotoxic evaluation and tubulin inhibitory activity
of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazole
derivatives
Amir Assadieskandar a, Mohsen Amini a, Seyed Nasser Ostad b, Gholam Hossein Riazi c,
Tayebe Cheraghi-Shavi c, Bentolhoda Shafiei c, Abbas Shafiee d,
⇑
a Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b Department of Toxicology and Pharmacology, Faculty of Pharmacy and Rational Drug Use Research Center, Tehran University of Medical Sciences, Tehran, Iran
c Neuroorganic Laboratory, Institute of Biochemistry and Biophysics, University of Tehran, Tehran 13145-1384, Iran
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 new series of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazoles were synthesized and their
cytotoxic activities in vitro against four different cell lines (HT-29, MCF-7, NIH-3T3, AGS) were evaluated.
Compound 6g bearing 3,4,5-trimethoxyphenyl moiety on ring A and 4-methoxy substituent on ring B dis-
played potent cytotoxic activity against all cell lines. Flow cytometry analysis and microtubule polymer-
ization assay confirmed that cytotoxic activities of this compound were related to inhibitory effect
against microtubules polymerization. Molecular modeling studies revealed that compound 6g could
Received 13 February 2013
Revised 12 March 2013
Accepted 13 March 2013
Available online 21 March 2013
Keywords:
strongly bind to the colchicine binding site of
Thr 179 and Cysb241.
a,b-tubulin through hydrogen bond interactions with
Cytotoxicity activity
Cell cycle analysis
Tubulin polymerization
Tautomerization
a
Ó 2013 Elsevier Ltd. All rights reserved.
2-Alkylthio-1H-imidazole
1. Introduction
structure are important for cytotoxic and antitubulin activities
(Fig. 1).8,9 To retain the cis configuration, different diaryl-heterocy-
clic analogs of CA-4 such as furan, thiophene,10 pyrazole,11 imidaz-
ole,12 isoxazole,13 thiadiazole,14 triazole,15 tetrazole,16 and
thiazole,17 have been synthesized, and their activities have been
evaluated. The investigation of various substitutes on the aryl rings
has revealed that methoxy substitute significantly affected the po-
tency and tubulin activity in order that diaryl-heterocycles pos-
sessing 3,4,5-trimethoxyphenyl on ring A display low nanomolar
cytotoxicity and retain the potency and efficacy of CA-4 when 4-
methoxyphenyl (with or without one of the 3-hydroxy, 3-amino
and 3-fluoro substitutions) is positioned on ring B. However, the
activity of some promising compounds is not greatly different from
reference compound CA-4.
In light of the structural features of the above mentioned com-
pounds, some 4,5-diaryl-imidazole-2-thione analogues bearing a
3,4,5-trimethoxyphenyl moiety as one of the aryl rings, which
are identical to the A-ring of CA-4, were synthesized and the effect
of OCH3 substitutions on the other aryl moiety was studied. Cell
cycle analysis was conducted for the most potent compounds,
and their antitubulin activity was investigated. Furthermore, dock-
ing studies were performed to evaluate the binding mode of the ac-
tive compound at the colchicine binding site of tubulin proteins.
Microtubules play important role in many cellular processes
such as cell division, cell signaling, and intracellular transport,
and they influence the cell shape as well.1 Thus far, three different
microtubule small-molecule binding sites—vinca, taxane, and col-
chicine sites—have been characterized.2 The importance of vinca
alkaloids and taxanes in the treatment of human cancers has al-
ready been shown;3 furthermore, extensive research programs
have focused on introducing new anticancer drugs that inhibit
microtubule polymerization through colchicine binding sites.4,5
Combretastatin A-4 (CA-4) 1, which was isolated from the bark
of the South African tree Combretum caffrum,6 is a cytotoxic and
antitubulin agent that binds to tubulin at the colchicine binding
site.7 The structure of this compound has a 3,4,5-trimethoxy
substituted pattern, which resembles the trimethoxyaryl ring of
colchicine 2, that shows optimal cytotoxic activity.
Structure–activity relationship (SAR) studies showed that the
3,4,5-trimethoxy-substitution and cis configuration in the CA-4
⇑
Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.011

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A. Assadieskandar et al. / Bioorg. Med. Chem. 21 (2013) 2703–2709
tauto-isomerization in (4 or 5)-aryl-2-aroyl-(1H)-imidazole
O
H3CO
H3CO
H3CO
H3CO
derivatives. In the latter study, variable-temperature 1H NMR
spectroscopy was used to investigate the barrier of H–N
tauto-isomerization.20 In another study, Laufer et al., attributed
tauto-isomerization in 4,5-diaryl-2-(methylthio)-1H-imidazole to
the sterically restricted free rotation of the bulky residues like qui-
nolyl ring around its sigma bond with the imidazole core.21
A
N
H
CH3
B
H3CO
OCH3
OH
O
OCH3
OCH3
1
2
2.2. Biological evaluation
Figure 1. Structures of microtubule targeting agents, combretastatin A-4 1 and
colchicine 2.
The cytotoxicity of compounds 6a–g and 7a–g, was evaluated
against three human cancer cell lines—human colon carcinoma
cells HT-29, human breast adenocarcinoma cell line MCF-7, human
caucasian gastric adenocarcinoma cell line AGS—as well as fibro-
blast cell line NIH-3T3 by MTT assay. CA-4 was used as the positive
control. The cytotoxicity activities of the synthesized compounds
are summarized in Table 1. Generally, most tested compounds
showed moderate to potent cytotoxic activities. Among the syn-
thesized compounds, the 2-alkylthio-4-(3,4,5-trimethoxyphenyl)-
5-(4-methoxyphenyl)-1H-imidazole structure in compounds 6g
and 7g showed the most potent activity against all tested cell lines
2. Results and discussion
2.1. Synthesis
4,5-Diaryl-1H-imidazole-2(3H)-thiones were synthesized by
the reaction of different benzoins and ammonium thiocyanate.18
The cyanide ion-catalyzed condensation of 3,4-dimethoxy or
3,4,5-trimethoxy benzaldehyde is a convenient synthetic method
for the symmetrical benzoins 3a,b. On the other hand, for prepar-
ing of unsymmetrical benzoins, 3,4,5-trimethoxybenzaldehyde
was reacted with benzoyl chloride, potassium cyanide and aqueous
potassium hydroxide in the presence of tetra-butylammonium
hydrogen sulfate to obtain cyanohydrinbenzoate 4. In the second
step, the cyanohydrin was treated with different methoxy benzal-
dehydes to give related benzoin benzoate. Hydrolysis of resulted
compound in acetonitrile and sodium hydroxide under the atmo-
sphere of argon provided related unsymmetrical benzoins 3c–g19
(Scheme 1).
(IC50 = 0.06–0.75 lM). SAR studies showed that the nature of the
methoxy substituted group on the phenyl ring had a profound
influence on the cytotoxicity of these compounds, for example,
compounds 6a and 7a, both of which contain 3,4,5-trimethoxy
substituent in both rings, are inactive (IC50 >50 lM). Moreover,
removing 5-methoxy from both rings yielded to compounds 6b
and 7b, both of which showed enhanced cytotoxic activity
(IC50 = 15–36 lM). Clearly, the number of methoxy substitutions
in both A and B rings has important effect on the cytotoxicity of
Treatment of different benzoins with a 10-fold excess of ammo-
nium thiocyanate in n-butanol afforded the desired 4,5-diaryl-1H-
imidazole-2(3H)-thiones 5a–g. Finally, alkylation of 6a–g and 7a–g
using alkyl iodide in basic media afforded the title compounds
(Scheme 2).
this series of compounds.
In compounds 6c–g and 7c–g, 3,4,5-trimethoxy substitutes in
phenyl (ring A) were retained and the effect of methoxy groups
on the second phenyl (ring B) was evaluated. As shown in Table 1
and 4-methoxy substitutes in compounds 6g and 7g were
favorable and showed significant cytotoxic activity. Adding extra
methoxy groups at the meta- and ortho-positions (compounds
6c, 7c and 6d, 7d respectively) and replacement with methoxy
group at ortho-position of ring B (compounds 6f and 7f), led to a
decrease in potency. A comparison of the cytotoxic activities of
compounds 6c–g and 7c–g revealed that the effects of different
methoxy groups in the second phenyl (ring B) against HT-29 and
As shown in Figure 2, when the 1H NMR spectrum of 6g was col-
lected in DMSO-d6, two sets of signals in nearly 1:1 ratio were
detectable. This observation indicated a slow equilibrium conver-
sion between two imidazole tautomers A and B on the NMR time
scale. However, in CDCl3 the interconversion of the two isomers
is faster on the NMR time scale and consecutively the resonances
are averaging to single broad signals. Khalili et al. reported similar
OCH3
OCH3
O
O
O
O
H3CO
H3CO
H3CO
a
OCH3
H3CO
H3CO
H
H
+
+
OH
H3CO
OCH3
3a
OCH3
OCH3
OCH3
OCH3
O
O
H3CO
H3CO
H3CO
H3CO
a
H3CO
H3CO
H
H
OH
3b
O
H
O
O
Ph
O
OCH3
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
b
c
H
CN
OH
OCH3
OCH3
OCH3
4
3c-g
Scheme 1. Reagent and condition: (a) KCN, ethanol 50%, reflux; (b) PhCOCl, KCN, CH2Cl2/H2O, rt; (c) ArylCHO, NaOH, benzene, rt.

A. Assadieskandar et al. / Bioorg. Med. Chem. 21 (2013) 2703–2709
2705
OCH3
OCH3
H3CO
H3CO
R2
H
N
R3
R4
N
O
a
b
R1
R2
R1
R2
S
SR6
H3CO
H3CO
N
H
N
H
OH R5
R3
R3
R5
R5
R1
R4
R4
3a-g
6,7a-g
5a-g
Scheme 2. Reagents and conditions: (a) NH4SCN, n-butanol, reflux; (b) alkyl iodide, CH3OH, reflux.
Figure 2. (a) Tautomerization in compound 6g; (b) 1H NMR spectra of 6g in CDCl3 at room temperature; (c) 1H NMR spectra of 6g in DMSO-d6 at room temperature.
MCF-7 cell lines was para > meta, para > ortho, para >
ortho > ortho, meta. In NIH-3T3 and AGS, a similar pattern with
one replacement was observed in the following order para > meta,
para > ortho, para > ortho, meta > ortho. From these observations,
it has been concluded that only one methoxy group at the para po-
sition of ring B has beneficial effect on the cytotoxic activity, and
additional methoxy groups at the ortho and/or meta positions
are not favorable.
Finally, increasing the size of the thiol substituent from a
methyl to an ethyl group led to a decrease in potency. However,
there were two exceptions—compounds f and d—where methoxy
groups were substituted at ortho and ortho, para positions of ring
B, respectively. In these compounds, replacement of the
2-thiomethyl with the corresponding ethyl group led to increased
cytotoxic activity. Overall, the obtained results demonstrated that
the introduction of 3,4,5-trimethoxyphenyl in the 2-methylthio-
imidazole ring and the presence of 4-methoxy substituted in ring
B led to optimal effects against all cancer cell lines. Although the
activity of compound 6g is not greatly different from reference
compound CA-4, design and synthesis of new diaryl-heterocyclic
analogs would still merit consideration in order to increase the
richness of structural repertoire and optimizing the lead com-
pound’s property.
Considering the design strategy toward tubulin inhibition and
the similarity of the synthesized compounds and CA-4, flow
cytometry experiments were performed to determine the effect
of compound 6g on the cell cycle of NIH-3T3 cells, specifically, to
determine whether the synthesized compound could lead to cell
cycle arrest at the G2/M phase. NIH-3T3 cells were treated with
compound 6g (0.10 lM) for 24 h, and CA-4 (0.05 lM) was used
as a positive control. The percentages of G2/M population were
91.93% in NIH-3T3 cells that were exposed to compound 6g as
compared to 35.70% in the control group. These results demon-
strated that compound 6g could arrest cells in G2/M phase (Fig. 3).

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A. Assadieskandar et al. / Bioorg. Med. Chem. 21 (2013) 2703–2709
Table 1
In vitro cytotoxic activities of synthesized compounds 6a–g and 7a–g
OCH3
A
H3CO
N
R1
R2
SR6
N
H
B
R3
R5
R4
Compounds
R1
R2
R3
R4
R5
R6
Cytotoxicity (IC50, l
M)a
HT-29
MCF-7
NIH-3T3
AGS
6a
7a
6b
7b
6c
OCH3
OCH3
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
>50
>50
ndb
>50
>50
ndb
ndb
ndb
24.13 1.43
21.17 2.06
4.82 0.79
21.04 3.11
18.57 0.84
6.56 1.12
27.51 1.81
36.70 3.50
21.73 1.14
18.57 0.96
0.25 0.04
0.36 0.05
0.18 0.02
17.16 1.62
15.05 2.10
4.04 0.23
13.19 1.76
4.15 0.35
3.55 0.26
18.09 1.97
22.90 3.11
12.21 0.89
10.99 0.74
0.52 0.04
0.75 0.08
0.40 0.03
35.80 2.56
27.60 3.06
6.01 0.86
14.12 0.78
11.91 2.84
7.25 1.98
30.18 1.68
27.17 1.51
38.73 1.87
36.23 0.67
0.06 0.01
0.26 0.06
0.02 0.01
34.84 1.30
33.63 4.35
10.57 0.20
37.19 0.69
23.22 1.09
18.61 3.61
35.31 1.63
38.98 4.69
48.43 4.48
40.70 5.66
0.06 0.00
0.07 0.01
0.04 0.02
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
7c
6d
7d
6e
7e
6f
H
OCH3
OCH3
H
H
H
7f
6g
7g
CA-4
H
H
H
a
IC50, compound concentration required to inhibit cell proliferation by 50%.
nd: Not determined.
b
Figure 3. Effect of compound 6g (0.10 lM) and CA-4 (0.05 lM) on the cell cycle of NIH-3T3 cells.
To confirm whether the cytotoxic activities of the designed
compounds were related to tubulin interaction, the inhibitory ef-
fect of promising compounds 6g, 7g, 6c and compound 7c on the
polymerization of purified tubulin was evaluated. All compounds
were evaluated at final concentration of 5
lM. CA-4 was used as
a positive control (2 M). Figure 4 shows the rational relationship
l
between the tubulin inhibition and the corresponding cytotoxic
activities. The order of inhibition of tubulin polymerization was
CA-4 > 6g > 7g > 6c > 7c. This result suggested that tubulin is a po-
tential target for synthesized compounds.
To investigate the binding interaction of the synthesized com-
pounds to the colchicine binding site of
a,b-tubulin, a docking
study was performed on compound 6g. The results showed that
compound 6g occupied the colchicine binding site with and orien-
tation similar to that of crystallized colchicine fitted in a binding
cavity. The trimethoxyphenyl in ring A was positioned in the
hydrophobic pocket between Alab250, Leub242, Valb238, and
Figure 4. Effect of compounds 7c, 6c, 7g, 6g (5 lM) and CA-4 (2 lM) on tubulin
polymerization.

A. Assadieskandar et al. / Bioorg. Med. Chem. 21 (2013) 2703–2709
2707
was prepared according to a literature procedure and used as a po-
sitive control.22
4.1.1. 2-(Methylthio)-4,5-bis(3,4,5-trimethoxyphenyl)-1H-
imidazole (6a)
Yield, 76%; mp 90–92 °C; IR (KBr, cmÀ1):
m 3260, 1582, 1506,
1408, 1239, 1120, 1002, 840; 1H NMR (500 MHz, CDCl3): d 2.71
(s, 3H, SCH3), 3.76 (s, 12H, 3,5-OCH3), 3.85 (s, 6H, 4-OCH3), 6.76
(br s, 4H, H2,6-phenyl). Anal. Calcd for C22H26N2O6S: C, 59.18; H,
5.87; N, 6.27. Found: C, 59.34; H, 5.62; N, 6.41.
4.1.2. 2-(Ethylthio)-4,5-bis(3,4,5-trimethoxyphenyl)-1H-
imidazole (7a)
Yield, 74%; mp 80–82 °C ; IR (KBr, cmÀ1):
m 3272, 1588, 1511,
1419, 1229, 1122, 1004, 835; 1H NMR (500 MHz, CDCl3): d 1.40
(t, J = 7.5 Hz, 3H, SCH2CH3), 3.16 (q, J = 7.5 Hz, 2H, SCH2CH3), 3.76
(s, 12H, 3,5-OCH3), 3.85 (s, 6H, 4-OCH3), 6.76 (br s, 4H, H2,6-phe-
nyl). Anal. Calcd for C23H28N2O6S: C, 59.98; H, 6.13; N, 6.08. Found:
C, 59.74; H, 6.43; N, 6.37.
Figure 5. Superimposition of the assumed conformation of compound 6g on the
top of the X-ray structure of DAMA–colchicine (shown in pink).
4.1.3. 4,5-Bis(3,4-dimethoxyphenyl)-2-(methylthio)-1H-
imidazole (6b)
Yield, 79%; mp 89–91 °C ; IR (KBr, cmÀ1):
m 3236, 1506, 1465,
1255, 1132, 1029, 860, 763 ; 1H NMR (500 MHz, CDCl3): d 2.67
(s, 3H, SCH3), 3.76 (s, 6H, 3-OCH3), 3.89 (s, 6H, 4-OCH3), 6.83 (d,
J = 8.5 Hz, 2H, H5-phenyl), 7.05 (d, J = 8.5 Hz, 2H, H6-phenyl), 7.06
(s, 2H, H2-phenyl); 13C NMR (CDCl3): d 17.31, 55.72, 55.85,
111.06, 120.25, 124.98, 132.55, 140.61, 148.40, 148.74; MS, m/z
(%) 386 (M+, 100), 371 (16), 353 (15), 313 (16). Anal. Calcd for
Alab316, and the oxygen atom of the 4-methoxy substituent
formed a hydrogen bond with Cysb241 in b subunit which was al-
ways found in the interaction of colchicines and tubulin protein in
crystal form. In addition, the 4-methoxy group on ring B can inter-
act with the hydrophobic pocket Alaa180, Vala181, Thrb314,
Valb315, Asnb258, and Metb259 in which only one methoxy group
at the para-position was favorable to cytotoxic activity. Further-
more, the hydrogen atom of NH in the imidazole-2-thiomethyl ring
C20H22N2O4S: C, 62.16; H, 5.74; N, 7.25. Found: C, 62.32; H, 5.87;
N, 7.38.
can form a hydrogen bond with the CO of Thr
postulated that compound 6g would strongly bind to the colchi-
a179. These results
4.1.4. 4,5-Bis(3,4-dimethoxyphenyl)-2-(ethylthio)-1H-imidazole
(7b)
cine binding site between the
toxic activity (Fig. 5).
a and the b subunits and show cyto-
Yield, 76%; mp 77–80 °C; IR (KBr, cmÀ1):
m 3226, 1511, 1470,
1250, 1137, 1024, 860, 758; 1H NMR (500 MHz, CDCl3): d 1.39 (t,
J = 7.5 Hz, 3H, SCH2CH3), 3.13 (q, J = 7.5 Hz, 2H, SCH2CH3), 3.76 (s,
6H, 3-OCH3), 3.89 (s, 6H, 4-OCH3), 6.83 (d, J = 8.5 Hz, 2H, H5-phe-
nyl), 7.05 (d, J = 8.5 Hz, 2H, H6-phenyl), 7.06 (s, 2H, H2-phenyl).).
Anal. Calcd for C21H24N2O4S: C, 62.98; H, 6.04; N, 6.99. Found: C,
62.72; H, 6.27; N, 6.75.
3. Conclusions
In conclusion, the synthesis and biological evaluation of a series
of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazoles
was discussed. Most of these synthesized compounds showed
moderate to potent cytotoxic activity against four cell lines. The
SAR study demonstrated that 3,4,5-trimethoxyphenyl moiety on
ring A and 4-methoxy substituent on ring B had beneficial effect
on the cytotoxicity activity. The flow cytometry analysis and
microtubule polymerization assay confirmed that the synthesized
compound led to cell cycle arrest at the G2/M phase by inhibitory
effect against microtubules. Finally, molecular modeling studies
revealed that compound 6g could strongly bind to the colchicine
4.1.5. 5-(3,4-Dimethoxyphenyl)-2-(methylthio)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (6c)
Yield, 67%; mp 99–101 °C; IR (KBr, cmÀ1):
m 3286, 1582, 1521,
1408, 1250, 1127, 1024, 840, 768; 1H NMR (500 MHz, CDCl3): d
2.67 (s, 3H, SCH3), 3.73 (s, 6H, 3,5-OCH3-4-aryl), 3.77 (s, 3H,
3-OCH3-5-aryl), 3.85 (s, 3H, 4-OCH3-4-aryl), 3.89 (s, 3H, 4-OCH3-
5-aryl), 6.76 (br s, 2H, H2,6-4-phenyl), 6.84 (d, J = 8.5 Hz, 1H,
H5-5-phenyl), 7.01–7.10 (m, 2H, H2,6-5-phenyl). Anal. Calcd for
binding site of
a,b-tubulin through hydrogen bond interactions
C21H24N2O5S: C, 60.56; H, 5.81; N, 6.73. Found: C, 60.72; H, 5.68;
with Thr 179 and Cysb241.
a
N, 6.89.
4. Experimental section
4.1. Chemistry
4.1.6. 5-(3,4-Dimethoxyphenyl)-2-(ethylthio)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (7c)
Yield, 64%; mp 75–77 °C; IR (KBr, cmÀ1):
m 3267, 1582, 1506,
1413, 1255, 1132, 1029, 840, 762; 1H NMR (500 MHz, CDCl3): d
1.38 (t, J = 7.5 Hz, 3H, SCH2CH3), 3.12 (q, J = 7.5 Hz, 2H, SCH2CH3),
3.71 (s, 6H, 3,5-OCH3-4-aryl), 3.75 (br s, 3H, 3-OCH3-5-aryl), 3.84
(br s, 3H, 4-OCH3-4-aryl), 3.88 (br s, 3H, 4-OCH3-5-aryl), 6.76 (br
s, 2H, H2,6-4-phenyl), 6.83 (d, J = 8.5 Hz, 1H, H5-5-phenyl), 7.05
(m, 2H , H2,6-5-phenyl). 13C NMR (CDCl3): d 15.29, 29.52, 60.92,
104.86, 111.08, 120.54, 124.39, 127.84, 132.42, 137.35, 139.36,
148.68, 148.81, 153.14; MS, m/z (%) 430 (M+, 100), 415 (34), 400
(38), 367 (9), 343 (14), 313 (16). Anal. Calcd for C22H26N2O5S C,
61.38; H, 6.09; N, 6.51. Found: C, 61.56; H, 6.24; N, 6.76.
1H NMR spectra were recorded on a 500 MHz Bruker spectrom-
eter using CDCl3 or DMSO-d6 as solvent. 13C NMR spectra were re-
corded on a 125 MHz Bruker spectrometer using CDCl3 or DMSO-d6
as solvent. Chemical shifts (d) are reported in ppm relative to tet-
ramethylsilane (TMS) as internal standard. Infrared spectra were
acquired on a Nicolet Magna 550-FT spectrometer. IR spectra of
solids were recorded in KBr and the absorption band was given
in wave numbers
0.4 of the theoretical values for C, H and N. Combretastatin A-4
m
in cmÀ1. Elemental microanalyses were within

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4.1.7. 5-(2,4-Dimethoxyphenyl)-2-(methylthio)-4-(3,4,5-
(t, J = 7.5 Hz, 3H, SCH2CH3), 3.15 (q, J = 7.5 Hz, 2H, SCH2CH3), 3.72
(s, 6H, 3,5-OCH3-4-aryl), 3.85 (s, 3H, 4-OCH3-4-aryl), 3.89 (s, 3H,
2-OCH3-5-aryl), 6.83 (s, 2H, H2,6-4-phenyl), 6.90 (t, 1H, J = 8 Hz,
H5-5-phenyl), 7.00 (d, 1H, J = 8 Hz, H3-5-phenyl), 7.29 (t, 1H,
J = 8 Hz, H4-5-phenyl), 7.40 (d, 1H, J = 8 Hz, H6-5-phenyl); 13C
NMR (CDCl3): d 15.29, 29.52, 55.61, 55.93, 60.89, 104.82, 111.28,
118.89, 120.81, 125.51, 129.50, 129.65, 130.89, 137.11, 138.91,
152.99, 156.11; MS, m/z (%) 400 (M+, 100), 385 (29), 367 (7), 313
(7), 298 (7). Anal. Calcd for C21H24N2O4S: C, 62.98; H, 6.04; N,
6.99. Found: C, 62.63; H, 6.32; N, 6.67.
trimethoxyphenyl)-1H-imidazole (6d)
Yield, 65%; mp 82–84 °C; IR (KBr, cmÀ1):
m 3246, 1613, 1465,
1413, 1214, 1127, 1024, 835; 1H NMR (500 MHz, CDCl3): d 2.68
(s, 3H, SCH3), 3.73 (s, 6H, 3,5-OCH3-4-aryl), 3.83 (s, 3H, 4-OCH3-
5-aryl), 3.85 (s, 3H, 4-OCH3-4-aryl), 3.86 (s, 3H, 2-OCH3-5-aryl),
6.44 (dd, J = 8.5 Hz, J = 3 Hz, 1H, H5-5-phenyl), 6.55 (d, J = 3 Hz,
1H, H3-5-phenyl), 6.83 (s, 2H, H2,6-4-phenyl), 7.28 (d, 1H,
J = 8.5 Hz, H6-5-phenyl). Anal. Calcd for C21H24N2O5S: C, 60.56; H,
5.81; N, 6.73. Found: C, 60.84; H, 5.69; N, 6.60.
4.1.8. 5-(2,4-Dimethoxyphenyl)-2-(ethylthio)-4-(3,4,5-
4.1.13. 5-(4-Methoxyphenyl)-2-(methylthio)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (7d)
trimethoxyphenyl)-1H-imidazole (6g)
Yield, 60%; mp 74–76 °C; IR (KBr, cmÀ1):
m
3272, 1613, 1501,
Yield, 67%; mp 82–84 °C; IR (KBr, cmÀ1):
m 3262, 1588, 1495,
1413, 1214, 1116, 830; 1H NMR (500 MHz, CDCl3): d 1.39 (t,
J = 7.5 Hz, 3H, SCH2CH3), 3.10 (q, J = 7.5 Hz, 2H, SCH2CH3), 3.75 (s,
6H, 3,5-OCH3-4-aryl), 3.83 (s, 3H, 4-OCH3-5-aryl), 3.85 (s, 3H, 4-
OCH3-4-aryl), 3.88 (s, 3H, 2-OCH3-5-aryl), 6.45 (d, J = 8.5 Hz, 1H,
H5-5-phenyl), 6.56 (s, 1H, H3-5-phenyl), 6.86 (s, 2H, H2,6-4-phenyl),
7.28 (d, J = 8.5 Hz, 1H, H6-5-phenyl); 13C NMR (CDCl3): d 15.30,
29.54, 55.46, 55.59, 55.90, 60.87, 98.83, 104.62, 104.78, 111.98,
129.71, 130.19, 131.69, 136.82, 138.37, 152.92, 157.40, 160.83;
MS, m/z (%) 430 (M+, 100), 415 (39), 397 (14), 328 (16), 211 (21),
195 (19), 165(37). Anal. Calcd for C22H26N2O5S C, 61.38; H, 6.09;
N, 6.51. Found: C, 61.64; H, 6.23; N, 6.78.
1511, 1413, 1244, 1127, 830; 1H NMR (500 MHz, CDCl3): d 2.67
(s, 3H, SCH3), 3.72 (s, 6H, 3,5-OCH3-4-aryl), 3.83 (s, 3H, 4-OCH3-
5-aryl), 3.85 (s, 3H, 4-OCH3-4-aryl), 6.76 (br s, 2H, H2,6-4-phe-
nyl), 6.89 (d, 2H, J = 8 Hz, H3,5-5-phenyl), 7.38–7.53 (m, 2H,
H2,6-5-phenyl). 1H NMR (500 MHz, DMSO-d6): d 2.59 (s, 3H,
SCH3), 3.61 and 3.66 (two s, 6H, OCH3), 3.64 and 3.67 (two s,
3H, OCH3), 3.75 and 3.78 (two s, 3H, OCH3), 6.70 and 7.76
(two s, 2H, H2,6-4-phenyl), 6.90 and 7.00 (two d, J = 8.5 Hz, 2H,
H3,5-5-phenyl), 7.37 and 7.45 (two d, J = 8.5 Hz, 2H, H2,6-5-phe-
nyl), 12.40 (br s, 1H, NH); 13C NMR (CDCl3): d 17.11, 55.32,
55.95, 60.90, 104.58, 113.99, 128.86, 129.34, 129.51, 137.14,
140.73, 153.13, 159.18; MS, m/z (%) 386 (M+, 100), 371 (46),
353 (7), 313 (8), 135(7), 97 (12), 69 (16). Anal. Calcd for
4.1.9. 5-(2,3-Dimethoxyphenyl)-2-(methylthio)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (6e)
C20H22N2O4S: C, 62.16; H, 5.74; N, 7.25. Found: C, 62.36; H,
5.89; N, 7.53.
Yield, 63%; mp 77–79 °C; IR (KBr, cmÀ1):
m
3262, 1588, 1465,
1398, 1270, 1116, 999, 830, 830, 748; 1H NMR (500 MHz, CDCl3):
d 2.69 (s, 3H, SCH3), 3.76 (s, 6H, 3,5-OCH3-4-aryl), 3.79 (s, 3H, 3-
OCH3-5-aryl), 3.86 (s, 3H, 4-OCH3-4-aryl), 3.92 (s, 3H, 2-OCH3-5-
aryl), 6.85 (s, 2H, H2,6-4-phenyl), 6.86–6.89 (m, 1H, H4-5-phenyl),
6.93–7.02 (m, 2H, H5,6-5-phenyl); 13C NMR (CDCl3): d 17.02,
55.88, 56.00, 60.76, 60.90, 105.00, 111.83, 112.13, 122.06, 122.37,
124.26, 129.33, 137.67, 141.10, 146.10, 153.03; MS, m/z (%) 416
(M+, 100), 401 (51), 386 (20), 312 (15), 267 (17), 91 (16). Anal.
Calcd for C21H24N2O5S: C, 60.56; H, 5.81; N, 6.73. Found: C,
60.82; H, 5.63; N, 6.58.
4.1.14. 2-(Ethylthio)-5-(4-methoxyphenyl)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (7g)
Yield, 63%; mp 64–66 °C; IR (KBr, cmÀ1):
m 3246, 1588, 1495,
1509, 1403, 1255, 1116, 830; 1H NMR (500 MHz, CDCl3): d 1.39
(t, J = 7.5 Hz, 3H, SCH2CH3), 3.11 (q, J = 7.5 Hz, 2H, SCH2CH3), 3.72
(s, 6H, 3,5-OCH3-4-aryl), 3.83 (s, 3H, 4-OCH3-5-aryl), 3.86 (s, 3H,
4-OCH3-4-aryl), 6.76 (br s, 2H, H2,6-4-phenyl), 6.90 (d, 2H,
J = 8 Hz, H3,5-5-phenyl), 7.38–7.53 (m, 2H, H2,6-5-phenyl). Anal.
Calcd for C21H24N2O4S: C, 62.98; H, 6.04; N, 6.99. Found: C,
62.71; H, 6.27; N, 6.73.
4.1.10. 5-(2,3-Dimethoxyphenyl)-2-(ethylthio)-4-(3,4,5-
trimethoxyphenyl)-1H-imidazole (7e)
4.2. Cytotoxicity assay
Yield, 59%; mp 65–67 °C; IR (KBr, cmÀ1):
m 3262, 1583, 1470,
1396, 1276, 1122, 999, 835, 748; 1H NMR (500 MHz, CDCl3): d
1.39 (t, J = 7.5 Hz, 3H, SCH2CH3), 3.16 (q, J = 7.5 Hz, 2H, SCH2CH3),
3.75 (s, 6H, 3,5-OCH3-4-aryl), 3.77 (s, 3H, 3-OCH3-5-aryl), 3.85 (s,
The cytotoxic activity was measured using the MTT assay in
four different cell lines (HT-29, MCF-7, NIH-3T3 and AGS).23 Cells
from different cell lines were seeded in 96-well plates at the den-
sity of 8000–10,000 viable cells per well and incubated for 24 h. At-
tached cells were treated with various concentrations of
3H, 4-OCH3-4-aryl), 3.91 (s, 3H, 2-OCH3-5-aryl), 6.87 (s, 2H, H2,6
4-phenyl), 6.87–6.89 (m, 1H, H4-5-phenyl), 6.93–7.02 (m, 2H,
5,6-5-phenyl). Anal. Calcd for C22H26N2O5S C, 61.38; H, 6.09; N,
-
H
compounds 6, 7a–g (0.001–100
lM) and incubated for another
6.51. Found: C, 61.69; H, 6.27; N, 6.77.
48 h. In the following, 20 L of MTT (3-(4, 5-dimethylthiazol-2-
l
yl)-2,5-diphenyl tetrazolium bromide) solution (5 mg/mL) were
4.1.11. 5-(2-Methoxyphenyl)-2-(methylthio)-4-(3,4,5-
added to each well and incubated for additional 4 h. Finally,
trimethoxyphenyl)-1H-imidazole (6f)
100 lL dimethyl sulfoxide was added and absorbance of each well
Yield, 67%; mp 92–94 °C; IR (KBr, cmÀ1):
m
3264, 1588, 1511,
was measured by plate reader (Anthous 2020; Austria) at a test
wavelength of 550 nm against a standard reference solution at
690 nm. Two independent experiments in triplicate were per-
formed for determination of sensitivity to each compound. The
IC50 values were determined by a nonlinear regression analysis
and expressed in mean SD.
1413, 1244, 1127, 1004, 758; 1H NMR (500 MHz, CDCl3): d 2.69
(s, 3H, SCH3), 3.73 (s, 6H, 3,5-OCH3-4-aryl), 3.84 (s, 3H, 4-OCH3-
4-aryl), 3.90 (s, 3H, 2-OCH3-5-aryl), 6.82 (s, 2H, H2,6-4-phenyl),
6.89 (t, 1H, J = 8 Hz, H5-5-phenyl), 6.99 (d, 1H, J = 8 Hz, H3-5-phe-
nyl), 7.29 (t, J = 8 Hz, 1H, H4-5-phenyl), 7.39 (d, 1H, J = 8 Hz, H6-
5-phenyl). Anal. Calcd for C20H22N2O4S: C, 62.16; H, 5.74; N, 7.25.
Found: C, 62.31; H, 5.51; N, 7.62.
4.3. Cell cycle analysis
4.1.12. 2-(Ethylthio)-5-(2-methoxyphenyl)-4-(3,4,5-
For flow cytometric analysis of DNA content, 106 NIH-3T3 cells
trimethoxyphenyl)-1H-imidazole (7f)
were treated with (0.10
gation, 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 containing
lM) compound 6g for 24 h. After centrifu-
Yield, 62%; mp 72–74 °C; IR (KBr, cmÀ1):
m
3268, 1588, 1511,
1419, 1244, 1116, 1004, 758; 1H NMR (500 MHz, CDCl3): d 1.40
l

A. Assadieskandar et al. / Bioorg. Med. Chem. 21 (2013) 2703–2709
2709
0.1% (v/v) Triton X-100, 10
lg/mL propidium iodide (PI, Sigma, St.
References and notes
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0.5 h. Finally, the fluorescence cell was measured by FACS-Calibur
flow cytometer (BD Biosciences, San Jose, CA).
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Acknowledgments
This research has been supported by Tehran University of Med-
ical Sciences & Health Services Grant No. 15098, INSF (Iran Na-
tional Science Foundation), and INEF (Iranian National Elite
Foundation).
28. http://accelrys.com/products/discovery-studio.