Synthesis and biological evaluation of some 1,2-disubstituted benzimidazole derivatives as new potential anticancer agents

Article abstract of DOI:10.1002/ardp.201200452

The synthesis of some new 1-(2-aryl-2-oxoethyl)-2-[(morpholine-4-yl) thioxomethyl]benzimidazole derivatives and investigation of their anticancer activities were the aims of this work. 2-(Chloromethyl)benzimidazole compound was reacted with sulfur and morpholine via Willgerodt-Kindler reaction to give 2-[(morpholine-4-yl)thioxomethyl]benzimidazole. Then, the obtained compound was reacted with appropriate α-bromoacetophenone derivatives in the presence of potassium carbonate to give the final products. Structure elucidation of the final compounds was achieved by FT-IR, ¹H NMR spectroscopy and MS spectrometry. The anticancer activities of the final compounds were evaluated by MTT assay, BrdU method, and flow cytometric analysis on C6, MCF-7, and A549 tumor cells. Most of the synthesized compounds exhibited considerable selectivity against the MCF-7 and C6 cell lines. New 1-(2-aryl-2-oxoethyl)-2- [(morpholine-4-yl)thioxomethyl]benzimidazole derivatives were synthesized and investigated for their anticancer activities against C6, MCF-7, and A549 tumor cells. Most of the synthesized compounds exhibited considerable selectivity against the MCF-7 and C6 cell lines. Copyright

Full text of DOI:10.1002/ardp.201200452

Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
403
Full Paper
Synthesis and Biological Evaluation of Some 1,2-
Disubstituted Benzimidazole Derivatives as New Potential
Anticancer Agents
Leyla Yurttas¸1, S¸ eref Demirayak2, Gu¨ls¸en A. C¸ iftc¸i3, S¸ afak Ulusoylar Yıldırım4,
and Zafer A. Kaplancıklı1,5
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey
2
˙
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medipol University, sItanbul, Turkey
3 Department of Biochemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey
4 Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey
5 Department of Pharmaceutical Chemistry, Graduate School of Health Sciences, Anadolu University,
Eskisehir, Turkey
The synthesis of some new 1-(2-aryl-2-oxoethyl)-2-[(morpholine-4-yl)thioxomethyl]benzimidazole
derivatives and investigation of their anticancer activities were the aims of this work.
2-(Chloromethyl)benzimidazole compound was reacted with sulfur and morpholine via
Willgerodt–Kindler reaction to give 2-[(morpholine-4-yl)thioxomethyl]benzimidazole. Then, the
obtained compound was reacted with appropriate a-bromoacetophenone derivatives in the
presence of potassium carbonate to give the final products. Structure elucidation of the final
compounds was achieved by FT-IR, 1H NMR spectroscopy and MS spectrometry. The anticancer
activities of the final compounds were evaluated by MTT assay, BrdU method, and flow cytometric
analysis on C6, MCF-7, and A549 tumor cells. Most of the synthesized compounds exhibited
considerable selectivity against the MCF-7 and C6 cell lines.
Keywords: Anticancer activity / Benzimidazole / BrdU / Flow cytometric analysis / Thioamide
Received: November 30, 2012; Revised: January 21, 2013; Accepted: February 4, 2013
DOI 10.1002/ardp.201200452
Introduction
anti-helmintic, anti-HIV, antihistaminic, antiulcer, cardio-
tonic, and antihypertensive; they have become a key building
block for a variety of compounds that play crucial roles in the
function of a number of biologically important molecules [3,
4]. In addition, it has been extensively utilized as a drug
scaffold in medicinal chemistry and is an important phar-
macophore due to being a structural isostere of naturally
occurring nucleotides [5–8]. In recent years, benzimidazole
derivatives have attracted particular interest due to their
anticancer activity or as potential in vitro anticancer agents
[9, 10]. In particular, they have been explored as topoisomer-
ase I, PARP-1, kinase Chk2, Pgp and tyrosine kinases, metallo
and serine protease inhibitors [11, 12]. Benzimidazole as
‘‘lead’’ molecule binds with other heterocyclic compounds.
It acts by intercalation or blocks cell growth by inhibiting the
enzymes directly responsible for the formation of nucleic
acids. This inhibition is believed to prevent DNA transcrip-
tion, which ultimately leads to cell death, which explains the
use of these drugs to treat cancer [13, 14]. Nocodazole [15],
Cancer treatment often encompasses more than one approach,
and the strategy adopted depends largely on the nature of the
cancer and how far it has progressed. At the present time, the
main treatment strategies are still surgery, radiotherapy, and
chemotherapy [1]. In cancer chemotherapy, besides drugs in
use, there is much interest in the design of new anticancer
agents especially small molecules that bind to DNA with
sequence selectivity and noncovalent interactions [2].
Benzimidazoles are the privileged components of many
bioactive compounds. Because of their synthetic utility and
broad range of pharmacological activities such as antifungal,
Correspondence: Dr. Leyla Yurttas¸, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir,
Turkey.
E-mail: lyurttas@anadolu.edu.tr
Fax: þ902223350750
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L. Yurttas¸ et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
oncodazole, carbandazim [16], bendamustin [17], Hoechst-
33258 [18], Hoechst-33342 [19], and IMET 3393 [12] are some
of the benzimidazole carrying anticancer drugs which are in
clinic or preclinic stage.
in which intermediate product 1-(2-aryl-2-oxoethyl)-2-substi-
tuted benzimidazole derivatives exhibited higher anticancer
activity than final pyrazino[1,2-a]benzimidazole compounds,
we planned to synthesize 1-(2-aryl-2-oxoethyl)-2-[(morpholine-
4-yl)thioxomethyl]benzimidazole derivatives via Willgerodt–
Kindler reaction and to investigate their prospective anti-
cancer activities.
Additionally, in recent studies thioamide linkage has been
preferred to amide linkage in newer peptide drug design
studies because of the conformational changes. The slightly
changed electron distribution of the thioamide structure
with regard to the amide structure represents a conservative Results and discussion
modification of the peptide backbone, which has been
reported to improve the enzymatic stability and bioactivity
[20, 21]. On the other hand, it is known that gefitinib [22] is
one of the morpholine carrying anticancer drugs. There are
also a lot of studies about anticancer activity of benzimida-
zole and morpholine including compounds [11, 23, 24].
Motivated by the above observations and as an extension of
our previous works on pyrazino[1,2-a]benzimidazoles [25–27]
Chemistry
Target molecules (3–18) were synthesized in three steps as
shown in Scheme 1. In the first step, 2-(chloromethyl)benz-
imidazole (1) was synthesized via Phillips method. 1,2-
Diaminobenzene and chloroacetic acid were refluxed in
4 N HCl solution to obtain an irritant intermediate product.
In the second step, 1H-2-[(morpholine-4-yl)thioxomethyl]-
NH2
Cl
N
i
+
Cl
COOH
N
H
NH2
O
1
N
S
N
ii
HN
O
+
1
2
S8
+
N
H
2
O
N
N
+
iii
R
Br
N
S
O
O
R
3 : H
4 : 3-OCH3
5 : 3-Cl
6 : 3-F
7 : 3-NO2
8 : 4-CH3
9 : 4-OCH3
10 : 4-Br
R
_
3 18
11 : 4-Cl
12 : 4-F
13 : 2,4-diCH3
14 : 3,4-diOCH3
15 : 2,4-diCl
16 : 2,5-diCl
17 : 3,4-diCl
18 : 3,4-diF
R
Scheme 1. The synthetic protocol of the compounds (3–18). Reagents and conditions: (i) 4 N HCl, reflux; (ii) Et3N, DMF; (iii) K2CO3,
acetone.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
1,2-Disubstituted Benzimidazole Derivatives as Potential Anticancer Agents
405
Table 1. IC50 valuesa) (mg/mL) for compounds 3–18 in A549, C6,
MCF-7 cancer cell lines.
benzimidazole (2) was obtained from 2-(chloromethyl)benz-
imidazole (1) via Willgerodt–Kindler reaction. Basically, the
Willgerodt-Kindler reaction is the reaction of aryl, alkyl
ketones and secondary amines with sulfur to give thioamides
via oxidation and rearrangement [28]. In this study, unlike
classical procedures, we used benzylic structure (1) as a start-
ing material instead of aryl, alkyl ketone to obtain the thio-
amide structure [29]. 2-(Chloromethyl)benzimidazole (1),
sulfur, triethylamine and morpholine were used with a
relative ratio of 1:2.5:3:1.2 to obtain 1H-2-[(morpholine-4-
yl)thioxomethyl]benzimidazole (2) compound and the reac-
tion was realized with 63% yield. Finally, 1H-2-[(morpholine-4-
yl)thioxomethyl]benzimidazole (2) was reacted with appro-
priate a-bromoacetophenone derivatives via bimolecular
nucleophilic substitution (SN2) reaction to give the intended
1-(2-aryl-2-oxoethyl)-2-[(morpholine-4-yl)thioxomethyl]benz-
imidazole derivatives (3–18).
Compounds
A549
C6
MCF-7
3
4
5
6
7
8
9
10
11
12
146.67 ꢁ 2.4
>500
131.67 ꢁ 1.6
200 ꢁ 2.8
161.67 ꢁ 3.2
>500
106.67 ꢁ 2.7
303 ꢁ 3.4
98.3 ꢁ 2.5
440 ꢁ 2.9
475 ꢁ 4.3
17.3 ꢁ 2.8
13 ꢁ 3.7
270 ꢁ 3.2
353.33 ꢁ 1.8
330 ꢁ 2.2
58.33 ꢁ 2.5
27.7 ꢁ 2.7
230 ꢁ 4.5
15.66 ꢁ 1.8
>500
>500
48 ꢁ 2.2
24.33 ꢁ 1.2
55 ꢁ 2.3
9.33 ꢁ 4.5
>500
22.7 ꢁ 3.2
15.3 ꢁ 1.7
>500
13
14
15
16
17
18
303.33 ꢁ 3.3
108.33 ꢁ 4.6
>500
>500
140 ꢁ 2.8
13.8 ꢁ 3.6
420 ꢁ 3.2
>500
295 ꢁ 2.4
39.7 ꢁ 2.3
7.8 ꢁ 2.8
5.17 ꢁ 3.9
17.33 ꢁ 3.2
205 ꢁ 2.5
386.67 ꢁ 1.7
415 ꢁ 4.1
123.33
323.33 ꢁ 4.8
>500
90 ꢁ 1.8
Cisplatin
Doxorubicin
19 ꢁ 1.8
14.67
9.67
Structures of the obtained compounds were elucidated by
spectral data. In the IR spectra, significant stretching bands
belonging to –C¼N and –C¼C were observed in between
1612–1350 cmꢀ1 and bands belonging to –C–N and –C–O
were observed at about 1286–981 cmꢀ1. In the NMR spectra
of the final compounds, a singlet peak at about 6.20 ppm was
observed belonging to –CH2 of the oxoethyl moiety; a peak
about 195.73–191.94 ppm belonging to the –C¼O structure
and 186.38–184.97 ppm belonging to the –C¼S structure.
Peaks belonging to the morpholine ring were assigned as
four different triplets, commonly. Hydrogen and carbon
atoms close to the oxygen atom were observed at 4.30–
3.81 ppm and 66.93–66.04 ppm. Hydrogen and carbon atoms
close to the nitrogen atom were observed at 3.75–3.69 ppm
and 51.91–48.84 ppm in the NMR spectra. The other peaks
belonging to aromatic and aliphatic protons of variable side
chains were observed at the estimated areas. In the mass
spectra of the copmpounds, Mþ1 peaks agreed well with
the calculated molecular weight of the target compounds.
17.33 ꢁ 2.4
a)
Cytotoxicity of the compounds. Incubation for 24 h. IC50 is the
drug concentration required to inhibit 50% of the cell growth.
The values represent mean ꢁ SD of triplicate determinations.
The IC50 values of the compounds were determined for
A549 cell line in the range of 15.66–500 mg/mL. Compounds
8, 9, and 11, which were including methyl, methoxy, and
chloro substituents on the phenyl ring, had significant cyto-
toxic activity with IC50 values lower than 58.33 mg/mL.
Compound 11 possessed the lowest IC50 value, which was
15.66 mg/mL, whereas cisplatin and doxorubicin IC50 values
were 19 and 17.33 mg/mL against A549 cells, respectively.
Compounds 8, 9, 10, 11, and 14 exhibited remarkable cyto-
toxic activities against C6 cell line. Compound 11 showed the
highest cytotoxic activity with a IC50 value of 9.33 mg/mL
whereas cisplatin and doxorubicin had IC50 values of 14.67
and 9.67 mg/mL, respectively. MCF-7 cells were the most
susceptible cells to the compounds 8, 9, 10, 11, 14, and 18.
Cytotoxic properties of the 1-(2-aryl-2-oxoethyl)-2-[(morpho-
line-4-yl)thioxomethyl]benzimidazole derivatives (3–18) var-
ied according to the substituents on the phenyl ring. In
general, compounds 3, 5, 8, 9, 14, and 18 were the most
cytotoxic molecules. Compounds 3, 5, 8, 9, 10, 14, and 17
showed maximum activities against MCF-7 cells and mini-
mum activities against A549 cells. Compounds 6, 7, and 16
exhibited the highest activities against A549 cells, on the
other hand compounds 4, 11, and 15 displayed the highest
actitivity to C6 cells. Compound 10 failed to provide 50%
cytotoxicity on the three cell types even with the highest
concentration (500 mg/mL).
Biological results
Cytotoxicity of the compounds
Cytotoxical potentials of the compounds against tumor cells
were assessed by the colorimetric MTT assay. The MTT test is
based on the cleavage of the yellow tetrazolium salt to form a
soluble blue formazan product by mitochondrial enzymes.
The amount of formazan produced is directly proportional to
the number of living cells [30]. The cytotoxic activities of the
synthesized compounds (3–18) were compared against
positive controls by using A549 (human non-small cell lung
cancer), C6 (rat glioma), and MCF-7 (human breast carcinoma)
cell lines. Tested concentrations for compounds were in
between 3.9 and 500 mg/mL and for controls (doxorubicin
and cisplatin) were in between 0.98 and 500 mg/mL. The
corresponding IC50 values are listed in Table 1.
Synthesized compounds (3–18) exhibited the highest cyto-
toxic activities against MCF-7 cells, the lowest cytotoxical
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
activities against A549 cells. Compounds 8, 9, 10, 11, 14, and
18 destroyed 50% of the MCF-7 cells at doses up to 40 mg/mL.
All studied concentrations of compounds 6, 7, 12, and 13
showed unsufficient cytotoxical effect over C6 cells.
is based on a colorimetric measurement of the carcinogenic
DNA synthesis inhibition ratio [31]. This study was performed
for the highest cytotoxic eight compounds 3, 5, 8, 9, 10, 11, 14,
and 18, which were determined by the MTT test. A549, C6,
and MCF-7 cells were incubated with three different concen-
trations (IC50/2, IC50, and IC50 ꢂ 2) of the compounds for 24
and 48 h time periods. The tested compounds showed time-
and dose-dependent inhibitory activity on DNA synthesis of
the tumor cells. Cisplatin and doxorubicin were used as
positive controls.
With regard to the substituent effect in cytotoxic activities
of the studied compounds, 4-methyl, 4-methoxy, and 4-chloro
substituents had remarkable influence on the cytotoxic
activities and also 3-chloro, 4-bromo, 3,4-dimethoxy, 3,4-
difluoro substituents had attracted attention with compar-
able effects. In addition, compounds 15 and 16 which had
substitution on ortho position of the phenyl ring and com-
pound 7 which was bearing electron withdrawing substitu-
ent were both observed with low cytotoxic activities. The
compound 9 which was including a methoxy substituent
on para position of the phenyl ring exhibited remarkable
activity, but this was not observed for the meta methoxy
including derivative (compound 4). Besides compound 18
bearing 3,4-difluoro substituent had good cytotoxic activity,
it could not be determined for the compound 6 with 3-fluoro
substituent and compound 12 with 4-fluoro substituent.
Figure 1 shows the DNA % synthesis inhibitory activity of
the compounds 3, 5, 8, 9, 10, 11, 14, 18, and standard drugs on
A549 cells. Among all of the compounds, compound 11 was
determined as the most active compound. Compound 11 was
found to have 59.82 and 68.86% DNA synthesis inhibition at
15.66 and 23.49 mg/mL doses after 48 h of incubation
whereas cisplatin was found to have 59.23 and 62.26% inhi-
bition at 19 and 27.5 mg/mL doses, respectively. According to
these results, it was observed that compound 11 had more
antiproliferative activity on A549 cells than cisplatin.
Furthermore, it was determined that compounds 9, 14,
and 18 had lower antiproliferative activities and compounds
3, 5, and 8 were inactive on the A549 cell line.
DNA synthesis inhibition
Proliferation of the tumor cells (A549, C6, MCF-7) was
measured by incorporation of BrdU, a thymidine analog that
is incorporated into DNA during the S phase, and the method
The DNA % synthesis inhibitory activity of the compounds
3, 5, 8, 9, 10, 11, 14, 18, and standard drugs on C6 cells is seen in
Figure 1. DNA % synthesis inhibitory activity of the compounds 3, 5, 8, 9, 10, 11, 14, 18 and standard drugs on A549 cells. Mean percent
absorbance of the untreated control (assessed in the presence of DMSO used as a solvent and assumed as 0%), and three different
concentrations (a ¼ IC50/2, b ¼ IC50, c ¼ 2 ꢂ IC50) of test compounds and cisplatin are given. Data points represent means for three
independent experiments ꢁ SD of nine independent wells. p < 0.05.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
1,2-Disubstituted Benzimidazole Derivatives as Potential Anticancer Agents
407
Figure 2. DNA % synthesis inhibitory activity of the compounds 3, 5, 8, 9, 10, 11, 14, 18, and standard drugs on C-6 cells. Mean percent
absorbance of the untreated control (assessed in the presence of DMSO used as a solvent and assumed as 0%), and three different
concentrations (a ¼ IC50/2, b ¼ IC50, c ¼ 2 ꢂ IC50) of test compounds and cisplatin are given. Data points represent means for three
independent experiments ꢁ SD of nine independent wells. p < 0.05.
Fig. 2. DNA % inhibition was increased with the increasing
incubation period (24 and 48 h) for all of the compounds. This
increase can be clearly appreciated in particular for the com-
pound 18. Measurement of the inhibition for the compound
18 at lower dose (IC50/2) after 48 h incubation was seven times
higher than at 24 h of measurement and there was a twofold
increase at the other two doses (IC50, and IC50 ꢂ 2). It was
observed that compounds 8, 9, 11, 14, and 18 had the highest
inhibitory activities on C6 cells DNA synthesis. In fact, com-
pound 11 had higher activity than both cisplatin and doxor-
ubicin with values of 45.53 and 61.96% at a concentration of
4.67 mg/mL. Also compound 14 had 27.85 and 82.88% inhibi-
tory activity after 24 and 48 h incubation, at 8.67 mg/mL
whereas cisplatin had 43.84 and 68.70% inhibitions at
7.34 mg/mL. In addition to these, compounds 3 and 5 exhibited
moderate inhibitor activity on C6 cell line.
activity results, only compound 9 exceeded 50%. Time-
dependent increase of % inhibition was seen at most for
compounds 5 and 9. Among all of the tested compounds,
compound 9 was found to cause maximum inhibition of DNA
and compound 5 was found to cause minimum inhibition.
Compound 9 was observed to have an approximate inhibitory
activity with a value of 80.51% at 13 mg/mL concentration
whereas cisplatin had 76.54% inhibition at 11.7 mg/mL con-
centration after 48 h incubation.
In all evaluation of DNA synthesis inhibition results, A549
cells were the most resistant cells against all tested com-
pounds. Compounds had antiproliferative activity mostly
against the MCF-7 cell line. Among the tested compounds,
compound 11 had significant antiproliferative activity
against all three cell lines and also compounds 8 and 9
had high activity against C6 and MCF-7 cell lines. These three
compound (8, 9, and 11) have attracted attention with para
substituted phenyl moiety. It was also determined that 4-
methyl, 4-methoxy, and 4-chloro substituents support the
antiproliferative effect positively.
Figure 3 shows the DNA % synthesis inhibitory activity of
the compounds 3, 5, 8, 9, 10, 11, 14, 18, and standard drugs on
MCF-7 cells. Although it was not up to standard drugs, it was
seen that many tested compounds showed significant anti-
proliferative activity at IC50 concentrations, after 48 h incu-
bation period. At IC50 concentrations, DNA % inhibitions of
the compounds 8, 10, and 14 were below 48% while the
inhibitions of the other compounds were increased up to
80.51%. According to the 24 h incubation antiproliferative
Induction of apoptosis
Apoptosis is a regulated process of cell death that occurs
during embryonic development as well as maintenance of
tissue homeostasis. Annexin V labeled with FITC can identify
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L. Yurttas¸ et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
Figure 3. DNA % synthesis inhibitory activity of the compounds 3, 5, 8, 9, 10, 11, 14, 18, and standard drugs on MCF-7 cells. Mean percent
absorbance of the untreated control (assessed in the presence of DMSO used as a solvent and assumed as 0%), and three different
concentrations (a ¼ IC50/2, b ¼ IC50, c ¼ 2 ꢂ IC50) of test compounds and cisplatin are given. Data points represent means for three
independent experiments ꢁ SD of nine independent wells. p < 0.05.
and quantify apoptotic cells on a single-cell basis by flow
cytometry. Staining cells with propidium iodide and Annexin
V-FITC enables the distinction of live, apoptotic, dead and late
apoptotic or necrotic cells [32]. The percentages detected in
this test for each cell type, at different concentrations and
periods of time, provide information on the mechanism
involved in the cell death. With the purpose of detecting
In Fig. 5, it is seen the flow cytometric analysis diagram for
MCF-7 cell line. The populations of apoptotic cells induced by
compounds 8, 9, 11, and 14 were 22.1, 7.2, 9.1, and 6.8%,
respectively whereas cisplatin induced 34.5% apoptosis. The
results above demonstrated that the synthesized compounds,
in general, induced apoptosis of C6 and MCF-7 tumor cells.
the induced cellular death (necrosis or apoptosis), studies of Conclusion
flow cytometry were performed on compounds 8, 9, 11, 14,
and cisplatin in C6 and MCF-7 cells. These compounds were
incubated for 24 h at IC50 concentration and the results are
shown in Figs. 4 and 5. The four areas in the diagrams stand for
necrotic cells (Q1, positive for PI and negative for Annexin/FITC,
left square on the top), live cells (Q3, negative for annexin and
PI, left square at the bottom), late apoptotic or necrotic cells
(Q2, positive for annexin and PI, right square on the top) and
apoptotic cells (Q4, negative for PI and positive for annexin,
right square at the bottom), respectively.
The synthesis and anticancer activity of sixteen 1-(2-aryl-
2-oxoethyl)2-[(morpholine-4-yl)thioxomethyl]benzimidazole
derivatives (3–18) have been reported in this work. It was
determined that many synthesized compounds had consider-
able anticancer activity against the C6 and MCF-7 cell lines.
However compound 11 including 4-chlorophenyl substituent
was the most active compound against the A549 cell line.
Additionally, compound 11 possessed higher cytotoxic activity
than doxorubicin and cisplatin with a IC50 value of 9.33 mg/mL
against A549 cells and 15.66 mg/mL. According to the DNA
synthesis inhibition studies, compounds 8 and 9 inhibited
DNA synthesis on C6 and MCF-7 cells and compound 11 inhibited
DNA synthesis on all three cells and there was a time dependent
increase of inhibition ratios. Compounds 8, 9, and 14 were
determined that they affected C6 tumor cells by the apoptotic
pathway and compound 11 by the necrotic pathway. Also
compound 8 and 11 affected MCF-7 cells by the apoptotic way.
In Fig. 4, as for C6 cell line, compound 14 showed the
highest population of apoptotic cells (20.5%) of the tested
compounds which was 1.1-fold higher than for cisplatin.
Compound 8 and 9 produced a comparable population of
apoptotic cells with a percentage of 10.2 and 13.0%, respect-
ively according to cisplatin’s percentage of 17.8%. However
compound 11 provoked necrotic induction in C6 cells after
24 h treatment.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
1,2-Disubstituted Benzimidazole Derivatives as Potential Anticancer Agents
409
Figure 4. Flow cytometric analysis of the distribution of C6 cells treated with the selected compounds and cisplatin. Annexin V-PI analysis in
C6 cells, following 24 h of drug treatment of cisplatin and compounds 8, 9, 11, and 14 by Annexin-V-FITC method at IC50 concentrations,
which are 14.67, 48.0, 24.33, 9.33, and 17.33 mg/mL in the order indicated in figure. Each condition was analyzed in a histogram, which
displays two parameters, Annexin V-FITC and PI, as represented. The dual parametric dot plots show the necrotic cells in the upper left
quadrant, Q1 (Annexin V-negative/PI-positive; the late apoptotic cells in the upper right quadrant, Q2 (Annexin V-positive/PI-positive); the
viable cell population in the lower left quadrant, Q3 (Annexin V-negative/PI-negative) and the early apoptotic cells in the lower right quadrant,
Q4 (Annexin V-positive/PI-negative). Results are representative of one of three independent experiments. Percentages of Q1, Q2, Q3, and
Q4 are measured as 0.3, 12.8, 81.9, and 5.0% for cisplatin; 1.4, 5.6, 88.5, and 4.6% for compound 8; 1.1, 8.3, 85.9, and 4.7% for compound 9;
26.0, 3.3, 70.7, and 0% for compound 11; 1.1, 14.5, 78.4, and 6.0% for compound 14, respectively.
by Electrothermal 9100 digital melting point apparatus and
4-Methyl, 4-methoxy, 4-chloro, and 3,4-dimethoxy phenyl
were uncorrected. NMR data was recorded by Bruker 500 MHz
including derivatives have been identified as the most
spectrometer. Mþ1 peaks were determined by AB Sciex-3200
effective compounds for anticancer activity. Accordingly,
Q-TRAP LC/MS/MS system.
methoxy and chloro substituents at the para position of
the phenyl ring increased anticancer activity. In this study,
2-(Chloromethyl)benzimidazole (1)
1,2-Diaminobenzene (200 mmol, 21.6 g) and chloroacetic acid
it was seen once again that 1-(2-aryl-2-oxoethyl)-2-substituted
benzimidazole derivatives were anticancer compounds.
Substitution of the benzimidazole ring on second position
with thiomorpholinide moiety did not contribute a signifi-
cant increase in activity. In future studies, it is thought
to substitute 1-(2-aryl-2-oxoethyl)benzimidazole derivatives
with amide and/or chloro, methoxy including aryl moieties.
(300 mmol, 28.4 g) were refluxed in 300 mL 4 N HCl solution for
3–4 h. Then the reaction mixture was cooled to room tempera-
ture and neutralized with sodium bicarbonate. The precipitate
was filtered and washed with water to give pure product. Yield:
75%, m.p. 1628C (ref. 1658C) [33].
1H-2-[(Morpholine-4-yl)thioxomethyl]benzimidazole (2)
A mixture of 2-(chloromethyl)benzimidazole (120 mmol, 19.9 g),
sulfur (300 mmol, 9.6 g), and triethylamine (360 mmol,
50.50 mL) in 150 mL dimethylformamide was kept for 30 min,
morpholine (144 mmol, 12.56 mL) was added, and the reaction
mixture was stirred for 3 h at room temprature. The mixture was
then poured into water, the precipitate was filtered off and the
Experimental
Chemistry
All chemicals were purchased from Merck or Sigma–Aldrich
Chemical companies. All melting points (m.p.) were determined
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Figure 5. Flow cytometric analysis of the distribution of MCF-7 cells treated with the selected compounds and cisplatin. Annexin V-PI
analysis in C6 cells, following 24 h of drug treatment of cisplatin and compounds 8, 9, 11, and 14 by Annexin-V-FITC method at IC50
concentrations, which are 7.8, 17.3, 13, 15.3, and 13.8 mg/mL in the order indicated in figure. Each condition was analyzed in a histogram,
which displays two parameters, Annexin V-FITC and PI, as represented. The dual parametric dot plots show the necrotic cells in the upper left
quadrant, Q1 (Annexin V-negative/PI-positive; the late apoptotic cells in the upper right quadrant, Q2 (Annexin V-positive/PI-positive); the
viable cell population in the lower left quadrant, Q3 (Annexin V-negative/PI-negative) and the early apoptotic cells in the lower right quadrant,
Q4 (Annexin V-positive/PI-negative). Results are representative of one of three independent experiments. Percentages of Q1, Q2, Q3, and
Q4 are measured as 3.8, 20.7, 61.8, and 13.8% for cisplatin; 1.5, 8.5, 76.5, and 13.6% for compound 8; 1.1, 2.6, 91.6, and 4.6% for compound
9; 1.3, 3.5, 89.7, and 5.6% for compound 11; 0.9, 2.6, 92.3, and 4.2% for compound 14, respectively.
obtained compound was crystallized from ethanol. Yield:
63%, m.p. 2508C (ref. 248–2498C) [34]. IR (KBr, cmꢀ1): nmax
thioxomethyl]benzimidazole (3)
1-(2-Phenyl-2-oxoethyl)-2-[(morpholine-4-yl)-
3250–2623 (N–H), 1593–1446 (C¼C and C¼N), 1280–1114
(C–O, C–N and C¼S). 1H NMR (500 MHz, dimethyl sulfoxide
(DMSO)-d6, ppm): d 3.71 (t, J ¼ 4.74 Hz, 2H, N–CH2), 3.82 (t,
J ¼ 4.84 Hz, 2H, N–CH2), 4.24 (t, J ¼ 4.73 Hz, 2H, O–CH2), 4.38
(t, J ¼ 4.85 Hz, 2H, O–CH2), 7.25 (t, J ¼ 7.59 Hz, 1H, Ar–H), 7.31 (t,
J ¼ 7.55 Hz, 1H, Ar–H), 7.54 (d, J ¼ 7.95 Hz, 1H, Ar–H), 7.70 (d,
J ¼ 8.09 Hz, 1H, Ar–H), 12.96 (1H, s, NH). 13C NMR (125 MHz,
DMSO-d6, ppm): d 50.88, 53.64, 66.72, 67.42, 113.24, 121.18,
123.68, 125.09, 135.19, 143.49, 150.18, 186.55.
Yield: 78%, m.p. 1948C. IR (KBr, cmꢀ1): nmax 3057 (aromatic C–H),
2972 (aliphatic C–H), 1693 (C¼O), 1452 (C¼C, C¼N), 1277–1112
(C–O, C–N and C¼S), 761–744 (monosubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm):
d 3.70–3.72 (m, 4H,
N(CH2)2), 3.83 (t, J ¼ 4.69 Hz, 2H, O–CH2), 4.26 (t, J ¼ 4.76 Hz,
2H, O–CH2), 6.20 (s, 2H, CO–CH2), 7.29–7.35 (m, 2H, Ar–H), 7.62–
7.68 (m, 3H, Ar–H), 7.73–7.78 (m, 2H, Ar–H), 8.13 (d, J ¼ 7.16 Hz,
2H, Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 49.14, 51.52, 53.
89, 66.49, 66.89, 112.07, 121.02, 123.93, 124.70, 129.51, 130.29,
135.54, 135.59, 136.73, 142.22, 150.66, 186.31, 194.82. MS (EI) m/z:
366 (100%), 279 (69%), 251 (75%).
1-(2-Aryl-2-oxoethyl)-2-[(morpholine-4-yl)thioxomethyl]-
benzimidazole derivatives (3–18)
1H-2-[(Morpholine-4-yl)thioxomethyl]benzimidazole (2.02 mmol,
0.5 g), appropriate a-bromoacetophenone derivative (2.02 mmol)
and potassium carbonate (2.02 mmol, 0.28 g) were stirred for 5 h.
After the reaction was finished, the solvent was evaporated. The
residue was washed with water and filtered to obtain pure
product.
1-[2-(3-Methoxyphenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (4)
Yield 82%, m.p. 1708C. IR (KBr, cmꢀ1): nmax 3043 (aromatic C–H),
2980–2918 (aliphatic C–H), 1687 (C¼O), 1596–1433 (C¼C, C¼N),
1271–1031 (C–O, C–N and C¼S), 752 (1,3-disubstituted benzene).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
1,2-Disubstituted Benzimidazole Derivatives as Potential Anticancer Agents
411
1H NMR (500 MHz, DMSO-d6, ppm): d 3.69–3.71 (t, J ¼ 4.02 Hz,
4H, N(CH2)2), 3.82 (t, J ¼ 4.09 Hz, 2H, O–CH2), 3.86 (s, 3H, O–CH3),
4.26 (t, J ¼ 4.69 Hz, 2H, O–CH2), 6.17 (s, 2H, COCH2), 7.28–7.34 (m,
3H, Ar–H), 7.55 (t, J ¼ 7.94 Hz, 1H, Ar–H), 7.59 (s, 1H, Ar–H), 7.65
(d, J ¼ 7.90 Hz, 1H, Ar–H), 7.71–7.74 (m, 2H, Ar–H). 13C NMR
(125 MHz, DMSO-d6, ppm): d 49.15, 51.63, 53. 87, 56.33, 66.56,
66.90, 112.07, 114.09, 121.03, 121.36, 121.86, 123.94, 124.70,
131.54, 136.70, 136.87, 142.23, 150.68, 161.00, 186.29, 194.67.
MS (EI) m/z: 396 (100%), 309 (24%), 281 (23%), 237 (14%), 151 (29%),
108 (13%), 86 (15%).
1279–1109 (C–O, C–N and C¼S), 748 (1,4-disubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 2.43 (s, 3H, CH3), 3.69–3.72
(m, 4H, N(CH2)2), 3.83 (t, J ¼ 4.63 Hz, 2H, O–CH2), 4.26 (t,
J ¼ 4.69 Hz, 2H, O–CH2), 6.16 (s, 2H, COCH2), 7.29–7.34 (m, 2H,
Ar–H), 7.44 (d, J ¼ 8.04 Hz, 2H, Ar–H), 7.66 (d, J ¼ 8 Hz, 1H,
Ar–H), 7.73 (d, J ¼ 8 Hz, 1H, Ar–H), 8.03 (d, J ¼ 8.16 Hz, 2H,
Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 21.91, 49.13, 51.34,
53.89, 66.55, 66.88, 112.02, 121.02, 123.90, 124.68, 129.60, 130.82,
133.30, 136.74, 142.22, 146.30, 150.73, 186.34, 194.29. MS (EI) m/z:
380 (100%), 293 (16%), 265 (39%), 135 (70%), 91 (26%), 86 (14%).
1-[2-(3-Chlorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
1-[2-(4-Methoxyphenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (5)
thioxomethyl]benzimidazole (9)
Yield 83%, m.p. 2178C. IR (KBr, cmꢀ1): nmax 3057 (aromatic C–H),
2974–2912 (aliphatic C–H), 1693 (C¼O), 1453 (C¼C, C¼N), 1277–
1116 (C–O, C–N, and C¼S), 744 (1,3-disubstituted benzene).
Yield 85%, m.p. 1818C. IR (KBr, cmꢀ1): nmax 3053 (aromatic C–H
gerilim bandı), 2914–2860 (aliphatic C–H), 1682 (C¼O), 1602–
1454 (C¼C, C¼N), 1230–1030 (C–O, C–N, and C¼S), 750 (1,4-
disubstituted benzene). 1H NMR (500 MHz, DMSO-d6, ppm): d
3.69–3.71 (m, 5H, N(CH2)2, O–CH), 3.90 (s, 3H, O–CH3), 4.25 (m,
3H, O–CH2), 6.17 (s, 2H, COCH2), 7.15 (d, J ¼ 8.76 Hz, 2H, Ar–H),
7.28–7.34 (m, 2H, Ar–H), 7.65 (d, J ¼ 6.93 Hz, 1H, Ar–H), 7.73 (d,
J ¼ 7.73 Hz, 1H, Ar–H), 8.11 (d, J ¼ 8.75 Hz, 2H, Ar–H). 13C NMR
(125 MHz, DMSO-d6, ppm): d 49.11, 51.05, 53.90, 56.64, 66.55,
66.88, 111.98, 115.44, 120.99, 123.87, 124.66, 128.37, 131.94,
136.75, 142.21, 150.83, 165.46, 186.38, 193.02. MS (EI) m/z: 396
(100%), 309, (14%), 281 (30%), 151 (57%), 136 (11%), 121 (11%), 108
(21%), 86 (14%).
1H NMR (500 MHz, DMSO-d6, ppm):
d 3.70–3.74 (m, 4H,
N(CH2)2), 3.82 (t, J ¼ 4.62 Hz, 2H, O–CH2), 4.27 (t, J ¼ 4.72 Hz,
2H, O–CH2), 6.19 (s, 2H, COCH2), 7.32 (m, 2H, Ar–H), 7.67 (t,
J ¼ 7.71 Hz, 2H, Ar–H), 7.73 (d, J ¼ 7.78 Hz, 1H, Ar–H), 7.84 (d,
J ¼ 7.50 Hz, 1H, Ar–H), 8.07 (d, J ¼ 7.82 Hz, 1H, Ar–H), 8.18 (s, 1H,
Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 49.17, 51.71, 53. 86,
66.53, 66.90, 112.12, 121.03, 123.97, 124.73, 128.14, 129.26,
132.30, 135.19, 135.22, 136.67, 137.35, 142.23, 150.68, 185.00,
193.94. MS (EI) m/z: 399.5 (100%), 312.5 (23%), 284.5 (26%), 155
(39%) 86 (18%).
1-[2-(3-Fluorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
1-[2-(4-Bromophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (6)
thioxomethyl]benzimidazole (10)
Yield 87%, m.p. 2068C. IR (KBr, cmꢀ1): nmax 3063 (aromatic C–H),
2981–2862 (aliphatic C–H), 1693 (C¼O), 1591–1439 (C¼C, C¼N),
1275–1116 (C–O, C–N and C¼S), 740 (1,3-disubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.74 (m, 4H, N(CH2)2),
3.83 (t, J ¼ 4.75 Hz, 2H, O–CH2), 4.27 (t, J ¼ 4.65 Hz, 2H, O–CH2),
6.18 (s, 2H, COCH2), 7.29–7.35 (m, 2H, Ar–H), 7.62–7.74 (m, 4H,
Ar–H), 7.94–7.99 (m, 2H, Ar–H). 13C NMR (125 MHz, DMSO-d6,
ppm): d 49.16, 51.75, 53.87, 66.54, 66.89, 112.08, 115.70, 121.04,
123.97, 124.74, 125.75, 133.00, 135.01, 135.46, 136.67, 142.23,
149.50, 150.68, 185.00, 193.94. MS (EI) m/z: 384 (100%), 297 (33%),
269 (36%), 268 (15%), 237 (16%), 139 (48%), 86 (23%).
Yield 81%, m.p. 2248C. IR (KBr, cmꢀ1): nmax 3010 (aromatic C–H
gerilim bandı), 2900–2858 (aliphatic C–H), 1685 (C¼O), 1510
(C¼C, C¼N), 1274–1226 (C–O, C–N, and C¼S), 742 (1,4-disubsti-
tuted benzene). 1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.72
(m, 4H, N(CH2)2), 3.82 (t, J ¼ 4.62 Hz, 2H, O–CH2), 4.26 (t,
J ¼ 4.69 Hz, 2H, O–CH2), 6.16 (s, 2H, COCH2), 7.29–7.34 (m, 2H,
Ar–H), 7.66 (d, J ¼ 7.85 Hz, 1H, Ar–H), 7.73 (d, J ¼ 7.80 Hz, 1H,
Ar–H), 7.86 (d, J ¼ 8.45 Hz, 2H, Ar–H) 8.06 (d, J ¼ 8.50 Hz, 2H,
Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 49.16, 51.51, 53.88,
66.54, 66.88, 112.09, 121.03, 123.95, 124.72, 129.71, 131.52, 133.38,
134.57, 136.69, 142.22, 150.53, 186.23, 194.18. MS (EI) m/z: 445
(100%), 359 (21%), 331 (34%), 201 (63%), 120 (19%), 86 (19%).
1-[2-(3-Nitrophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
1-[2-(4-Chlorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (7)
Yield 80%, m.p. 2318C. IR (KBr, cmꢀ1): nmax 3086 (aromatic C–H),
2910–2866 (aliphatic C–H), 1697 (C¼O), 1529–1350 (C¼C, C¼N),
1276–1115 (C–O, C–N and C¼S), 742 (1,3-disubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.73 (m, 4H, N(CH2)2),
3.81 (t, J ¼ 4.91 Hz, 2H, OCH2), 4.25 (t, J ¼ 4.94 Hz, 2H, OCH2), 6.25
(s, 2H, COCH2), 7.29–7.32 (m, 2H, Ar–H), 7.66 (d, J ¼ 6.70 Hz, 1H,
Ar–H), 7.72 (d, J ¼ 7.20 Hz, 1H, Ar–H), 7.92 (t, J ¼ 8 Hz, 1H, Ar–H),
8.53–8.58 (m, 2H, Ar–H), 8.80 (s, 1H, Ar–H). 13C NMR (125 MHz,
DMSO-d6, ppm): d 49.21, 51.91, 53.88, 66.52, 66.93, 112.20, 121.06,
123.94, 124.02, 124.76, 129.61, 132.13, 135.78, 136.68, 142.25,
149.52, 150.44, 186.15, 193.94. MS (EI) m/z: 411 (100%), 324
(29%), 296 (20%), 250 (19%), 249 (13%), 130 (14%), 86 (26%).
thioxomethyl]benzimidazole (11)
Yield 89%, m.p. 2188C. IR (KBr, cmꢀ1): nmax 3020 (aromatic C–H),
2966–2856 (aliphatic C–H), 1685 (C¼O), 1589–1398 (C¼C, C¼N),
1275–1115 (C–O, C–N and C¼S), 742 (1,4-disubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 3.67–3.73 (m, 4H, N(CH2)2),
3.83 (t, J ¼ 4.69 Hz, 2H, O–CH2), 4.27 (t, J ¼ 4.76 Hz, 2H, O–CH2),
6.17 (s, 2H, COCH2), 7.29–7.35 (m, 2H, Ar–H), 7.66 (d, J ¼ 7.12 Hz,
1H, Ar–H), 7.71–7.74 (m, 3H, Ar–H), 8.15 (d, J ¼ 8.59 Hz, 2H,
Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 49.16, 51.54, 53.88,
66.54, 66.88, 112.09, 121.03, 123.96, 124.72, 130.41, 131.47, 134.24,
136.69, 140.53, 142.22, 150.54, 186.24, 193.95. MS (EI) m/z: 399.5
(100%), 312.5 (23%), 284.5 (41%), 155 (82%), 86 (20%).
1-[2-(4-Methylphenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (8)
1-[2-(4-Fluorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (12)
Yield 86%, m.p. 1828C. IR (KBr, cmꢀ1): nmax 3020 (aromatic C–H),
2976–2864 (aliphatic C–H), 1684 (C¼O), 1510–1454 (C¼C, C¼N),
Yield 86%, m.p. 2048C. IR (KBr, cmꢀ1): nmax 3047 (aromatic C–H),
2918–2856 (aliphatic C–H), 1697 (C¼O), 1595–1456 (C¼C, C¼N),
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1276–1105 (C–O, C–N, and C¼S), 837 (1,4-disubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.72 (m, 4H, N(CH2)2),
3.83 (t, J ¼ 4.63 Hz, 2H, O–CH2), 4.27 (t, J ¼ 4.70 Hz, 2H, O–CH2),
6.18 (s, 2H, COCH2), 7.29–7.35 (m, 2H, Ar–H), 7.47 (t, J ¼ 8.81 Hz,
2H, Ar–H), 7.67 (d, J ¼ 7.05 Hz, 1H, Ar–H), 7.74 (d, J ¼ 7.09 Hz,
1H, Ar–H), 8.23 (t, J ¼ 7.13 Hz, 2H, Ar–H). 13C NMR (125 MHz,
DMSO-d6, ppm): d 49.15, 51.46, 51.49, 66.55, 66.89, 112.06, 117.22,
121.03, 123.94, 124.71, 132.31, 132.73, 136.71, 142.22, 150.62,
168.13, 186.28, 193.46. MS (EI) m/z: 384 (94%), 297 (23%), 269
(48%), 139 (100%), 109 (10%), 95 (14%), 86 (20%).
C¼N), 1274–1027 (C–O, C–N and C¼S), 842–748 (1,2,5-trisubsti-
tuted benzene). 1H NMR (500 MHz, DMSO-d6, ppm): d 3.71 (t,
J ¼ 4.75 Hz, 2H, N–CH2), 3.78 (t, J ¼ 5 Hz, 2H, N–CH2), 3.81 (t,
J ¼ 4.5 Hz, 2H, O–CH2), 4.30 (t, J ¼ 4.75 Hz, 2H, O–CH2), 6.08
(s, 2H, COCH2), 7.31–7.38 (m, 2H, Ar–H, C6–H), 7.68–7.77
(m, 4H, Ar–H), 8.20 (s, 1H, Ar–H). 13C NMR (125 MHz, DMSO-d6,
ppm): d 48.92, 53.43, 53.54, 66.04, 66.45, 111.55, 120.33, 123.37,
124.10, 130.00, 130.17, 132.00, 133.16, 133.69, 135.70, 137.00,
138.10, 141.31, 149.40, 184.97, 194.31. MS (EI) m/z: 436 (100%),
402 (20%), 349 (44%), 321 (38%).
1-[2-(2,4-Dimethylphenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
1-[2-(3,4-Dichlorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (13)
thioxomethyl]benzimidazole (17)
Yield 84%, m.p. 1708C. IR (KBr, cmꢀ1): nmax 30536 (aromatic C–H),
2976–2856 (aliphatic C–H), 1693 (C¼O), 1612–1429 (C¼C, C¼N),
1275–985 (C–O, C–N and C¼S), 748 (1,2,4-trisubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 2.37 (s, 3H, CH3), 2.40 (s, 3H,
CH3), 3.71–3.75 (m, 4H, N(CH2)2), 3.86 (t, J ¼ 4.75 Hz, 2H, O–CH2),
4.28 (t, J ¼ 4.75 Hz, 2H, O–CH2), 6.05 (s, 2H, COCH2), 7.21 (s, 1H,
Ar–H), 7.27–7.35 (m, 3H, Ar–H), 7.73 (t, J ¼ 7.25 Hz, 2H, Ar–H), 8.07
(d, J ¼ 7.5 Hz, 1H, Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm):
d 21.49, 21.61, 48.82, 52.56, 53.60, 66.07, 66.47, 111.39, 120.28,
123.17, 123.96, 127.18, 130.22, 131.95, 133.23, 135.91, 139.12,
141.28, 143.55, 143.63, 185.40, 195.73. MS (EI) m/z: 394 (100%),
360 (12%), 307 (43%), 279 (31%), 264 (23%), 260 (10%), 246 (11%).
Yield 82%, m.p. 2218C. IR (KBr, cmꢀ1): nmax 3084 (aromatic C–H),
2904–2856 (aliphatic C–H), 1686 (C¼O), 1475–1379 (C¼C, C¼N),
1275–1028 (C–O, C–N, and C¼S), 741 (1,3,4-trisubstituted
benzene). 1H NMR (500 MHz, DMSO-d6, ppm):
d 3.71 (t,
J ¼ 4.75 Hz, 2H, N–CH2), 3.74 (t, J ¼ 4.75 Hz, 2H, N–CH2), 3.82
(t, J ¼ 4.5 Hz, 2H, O–CH2), 4.27 (t, J ¼ 4.75 Hz, 2H, O–CH2),
6.18 (s, 2H, COCH2), 7.29–7.34 (m, 2H, Ar–H), 7.66 (d, J ¼ 8 Hz,
1H, Ar–H), 7.73 (d, J ¼ 7.5 Hz, 1H, Ar–H), 7.92 (d, J ¼ 8.5 Hz, 1H,
Ar–H), 8.06 (d, J ¼ 8.5 Hz, 1H, Ar–H), 8.39 (s, 1H, Ar–H). 13C NMR
(125 MHz, DMSO-d6, ppm):
d 48.88, 51.38, 53.53, 66.12,
66.50, 111.44, 120.31, 123.25, 124.00, 128.73, 130.79, 131.84,
132.54, 134.86, 135.82, 137.55, 141.38, 149.58, 185.04,
192.11. MS (EI) m/z: 436 (100%), 349 (28%), 321 (30%), 191
(15%).
1-[2-(3,4-Dimethoxyphenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (14)
Yield 83%, m.p. 1598C. IR (KBr, cmꢀ1): nmax 3082 (aromatic C–H),
2935–2850 (aliphatic C–H), 1680 (C¼O), 1585–1425 (C¼C, C¼N),
1251–1018 (C–O, C–N, and C¼S), 746 (1,3,4-trisubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.72 (t, J ¼ 4.25 Hz, 4H,
N(CH2)2), 3.82–3.86 (m, 5H, O–CH2 ve O–CH3), 3.90 (s, 3H, O–CH3),
4.27 (t, J ¼ 4.75 Hz, 2H, O–CH2), 6.14 (s, 2H, COCH2), 7.18 (d,
J ¼ 8.5 Hz, 1H, Ar–H), 7.28–7.34 (m, 2H, Ar–H), 7.54 (s, 1H,
Ar–H), 7.63 (d, J ¼ 8.25 Hz, 1H, Ar–H), 7.73 (d, J ¼ 7.75 Hz, 1H,
Ar–H), 7.84 (dd, J ¼ 2, 8.5 Hz, 1H, Ar–H). 13C NMR (125 MHz, DMSO-
d6, ppm): d 48.84, 50.74, 53.56, 56.16, 56.40, 66.16, 66.52, 110.81,
111.29, 111.65, 120.29, 123.14, 123.65, 123.91, 135.89, 141.37,
149.25, 150.02, 154.46, 185.22, 191.94. MS (EI) m/z: 426 (100%),
339 (11%), 311 (30%), 260 (8%), 181 (9%), 151 (9%).
1-[2-(3,4-Difluorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (18)
Yield 81%, m.p. 2038C. IR (KBr, cmꢀ1): nmax 3049 (aromatic C–H),
2977–2850 (aliphatic C–H), 1689 (C¼O), 1610–1419 (C¼C, C¼N),
1286–1117 (C–O, C–N and C¼S), 739 (1,3,4-trisubstituted
benzene). 1H NMR (500 MHz, DMSO-d6, ppm): d 3.70–3.74
(m, 4H, N(CH2)2), 3.82 (t, J ¼ 4.5 Hz, 2H, O–CH2), 4.27 (t,
J ¼ 4.75 Hz, 2H, O–CH2), 6.17 (s, 2H, COCH2), 7.29–7.34 (m, 2H,
Ar–H), 7.63 (d, J ¼ 8 Hz, 1H, Ar–H), 7.70–7.75 (m, 2H, Ar–H, C7–H),
8.04 (brs, 1H, Ar–H), 8.24 (t, J ¼ 9.25 Hz, 1H, Ar–H). 13C NMR
(125 MHz, DMSO-d6, ppm): d 48.86, 51.28, 53.55, 66.13, 66.50,
111.38, 118.28, 118.42, 118.76, 118.90, 120.32, 123.24, 124.00,
126.76, 132.13, 135.82, 141.31, 149.40, 184.97, 194.31. MS (EI) m/z:
402 (100%), 368 (12%), 315 (54%), 287 (54%), 257 (11%).
1-[2-(2,4-Dichlorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (15)
Anticancer screening
Cell cultures
Yield 86%, m.p. 1908C. IR (KBr, cmꢀ1): nmax 3043 (aromatic C–H),
2908–2864 (aliphatic C–H), 1716 (C¼O), 1579–1411 (C¼C, C¼N),
1277–981 (C–O, C–N and C¼S), 800–739 (1,2,4-trisubstituted benzene).
1H NMR (500 MHz, DMSO-d6, ppm):d 3.70–3.72 (m, 2H, N–CH2), 3.77 (t,
2H, J ¼ 4.75 Hz, O–CH2), 3.81–3.82 (m, 2H, N–CH2), 4.30 (t,
J ¼ 4.75 Hz, 2H, O–CH2), 6.07 (s, 2H, COCH2), 7.30–7.37 (m, 2H,
Ar–H), 7.69–7.75 (m, 3H, Ar–H), 7.87 (s, 1H, Ar–H), 8.09 (d,
J ¼ 8.5 Hz, 1H, Ar–H). 13C NMR (125 MHz, DMSO-d6, ppm): d 48.94,
50.58, 53.36, 66.07, 66.47, 111.44, 120.36, 122.94, 123.35, 124.34,
128.29, 131.12, 132.16, 133.89, 135.70, 138.10, 141.31, 149.38,
184.97, 194.31. MS (EI) m/z: 435 (100%), 349 (20%), 321 (21%), 191 (9%).
A549 (human lung adenocarcinoma cells), C6 (rat glioma cells)
and MCF-7 (human breast cancer cells) cell lines were used in the
studies. The cells were incubated in 90% RPMI supplemented
with 10% fetal bovine serum (Gibco, UK). All media were supple-
mented with 100 IU/mL penicillin–streptomycin (Gibco, UK) and
cells were incubated at 378C in a humidified atmosphere of 95%
air and 5% CO2. L-Glutamine (2 mM), sodium pyruvate (1 mM),
and insulin (10 mM) were used for A549 and MCF-7 cells.
Exponentially growing cells were plated at 2 ꢂ 104 cells/mL into
96-well microtiter tissue culture plates (Nunc, Denmark) and
incubated for 24 h (the optimum cell number for cytotoxicity
assays was determined in preliminary experiments). Stock
solutions of compounds were prepared in dimethyl sulfoxide
(DMSO) and further dilutions were made with fresh culture
medium.
1-[2-(2,5-Dichlorophenyl)-2-oxoethyl]-2-[(morpholine-4-yl)-
thioxomethyl]benzimidazole (16)
Yield 88%, m.p. 2178C. IR (KBr, cmꢀ1): nmax 3086–3040 (aromatic
C–H), 2927–2860 (aliphatic C–H), 1703 (C¼O), 1460–1400 (C¼C,
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Arch. Pharm. Chem. Life Sci. 2013, 346, 403–414
1,2-Disubstituted Benzimidazole Derivatives as Potential Anticancer Agents
413
flow cytometer. The results were analyzed by using FCSExpress
software and represented as percentage of normal and apoptotic
cells at various stages [38]. The percentage of apoptotic cells was
calculated from the number of cells in sub-G1 phase, represent-
ing fragmented cell vesicles. The four areas in the diagrams stand
for necrotic cells (Q1, positive for PI and negative for annexin/
FITC, left square on the top), live cells (Q3, negative for annexin
and PI, left square at the bottom), late apoptotic or necrotic cells
(Q2, positive for annexin and PI, right square on the top) and
apoptotic cells (Q4, negative for PI and positive for annexin, right
square at the bottom), respectively. The experiment was repeated
three times.
MTT assay
The cytotoxic activities of the tested compounds were deter-
mined by cell proliferation analysis using standard 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
[35, 36]. A549, C6 and, MCF-7 cells were cultured in 96-well
flat-bottom plates at 378C for 24 h (2 ꢂ 104 cells per well). All
of the compounds were dissolved in DMSO individually and
added to culture wells at varying concentrations (3.9–500 mg/mL),
the highest final DMSO concentration was under 0.1%. After 24 h
drug incubation at 378C, 20 mL MTT solution (5 mg/mL MTT
powder in PBS) was added to each well. Then 3 h incubation
period was maintained in the same conditions. Purple formazan
was generated at the end of the process which is the reduction
product of the MTT agent by the mitochondrial dehydrogenase
enzyme of intact cells. Formazan crystals were dissolved in
100 mL DMSO and the absorbance was read by ELISA reader
(OD570nm). The percentage of viable cells was calculated based
on the medium control.
The authors present their thanks to Anadolu University BIBAM (Tu¨rkiye)
for anticancer test results and NMR spectra analyses.
The authors have declared no conflict of interest.
DNA synthesis inhibition assay
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