Synthesis of 2-substituted-N-[4-(1-methyl-4,5-diphenyl-1H-imidazole-2-yl) phenyl] acetamide derivatives and evaluation of theiranticancer activity

Article abstract of DOI:10.1016/j.ejmech.2010.04.015

In the present study 18 novel imidazole-(benz)azole and imidazoleepiperazine derivatives were synthesized in order to investigate their probable anticancer activity. The structures of the compounds were confirmed by IR,¹H NMR and EI-MS spectral data. Cytotoxicity (MTT),analysis of DNA synthesis and detection of apoptotic DNA assays were applied to determine anticancer activity of the compounds against colon (HT-29) and breast (MCF-7) carcinoma cell lines. Most of the compounds,showed greater activity against HT-29 cells than MCF-7 cells. Some of them indicated considerable cytotoxicity against both of the carcinogenic cell lines. However,their inhibitory activity on DNA synthesis was relatively poor. Anticancer activity screening results revealed that 11,12 and 13 were the most active compounds in the series. They exhibited significant cytotoxicity against both of the carcinogenic cell lines and caused DNA fragmentation of the HT-29 cells.

Full text of DOI:10.1016/j.ejmech.2010.04.015

European Journal of Medicinal Chemistry 45 (2010) 3320e3328
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 2-substituted-N-[4-(1-methyl-4,5-diphenyl-1H-imidazole-2-yl)
phenyl]acetamide derivatives and evaluation of their anticancer activity
,
b
b
ꢀ a
Yusuf Özkaya , Ilhan Is¸ ıkdag , Zerrin Incesu , Güls¸ en Akalın
_
_
*
a Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskis¸ehir, Turkey
b Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study 18 novel imidazole-(benz)azole and imidazoleepiperazine derivatives were
synthesized in order to investigate their probable anticancer activity. The structures of the compounds
were confirmed by IR, 1H NMR and EI-MS spectral data. Cytotoxicity (MTT), analysis of DNA synthesis and
detection of apoptotic DNA assays were applied to determine anticancer activity of the compounds
against colon (HT-29) and breast (MCF-7) carcinoma cell lines. Most of the compounds, showed greater
activity against HT-29 cells than MCF-7 cells. Some of them indicated considerable cytotoxicity against
both of the carcinogenic cell lines. However, their inhibitory activity on DNA synthesis was relatively
poor. Anticancer activity screening results revealed that 11, 12 and 13 were the most active compounds in
the series. They exhibited significant cytotoxicity against both of the carcinogenic cell lines and caused
DNA fragmentation of the HT-29 cells.
Received 25 January 2010
Received in revised form
11 April 2010
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Cancer
Imidazole
Azole, benzazole
Piperazine
HT-29
Ó 2010 Elsevier Masson SAS. All rights reserved.
MCF-7
1. Introduction
compounds which exhibit cytotoxicity against various types of
human cancer cell lines at micromolar concentrations [11]. Dacar-
DNA intercalators are the chemotherapeutics which exhibit
anticancer activity by inserting between the base pairs of the
double helix and causing a significant change of DNA conformation
[1]. They bind to DNA by non-covalent interactions and constitute
DNAeintercalator complex. The only recognized forces that main-
tain the stability of the DNAeintercalator complex, even more than
DNA alone, are van der Waals, hydrogen bonding, polarization and
hydrophobic forces [2e6]. The compounds which bear heteroatoms
such as nitrogen, sulphur and oxygen increase the strength of the
complex by forming hydrogen bonds with DNA. The force of
interaction between compound and DNA usually correlates with
the anticancer activity [7e9]. Besides, when one or more nitrogen
heteroatoms exist on the chemical structure, intercalating chro-
mophore possesses a polarized character and optimal interaction
occurs [6]. Based on such reasons the presence of heteroatoms in
the compounds plays an important role on exhibiting the anti-
cancer activity.
bazine [12], zoledronic acid [13], tipifarnib [14,15] and azathioprine
[16] are the imidazole ring bearing anticancer agents. In addition to
imidazole, some other azole and benzazole ring systems such as
benzoxazole, benzothiazole, triazole, tetrazole, thiadiazole and
thiazoline can be shown as pharmacophore groups which are
responsible for the anticancer activity [17e22]. Piperazine is
another nitrogenous moiety which often subjected to studies to
develop new chemotherapeutic agents. Anticancer agents, razox-
ane (ICRF-154), dexrazoxsane (ICRF-187) and MST-16, are the
compounds including this moiety [23,24].
Resistance improvement against the former anticancer agents
creates a research area in development of new anticancer agents.
Nevertheless, it is rather difficult to develop a new agent which can
selectively inhibit the proliferation of abnormal cells with least or
no effect on normal cells [25]. Therefore, cancer chemotherapy is
very important for medicinal chemists and the studies are still
being carried on to develop new chemotherapeutic agents that are
probable to indicate activity on various cancer types [26,27].
Prompted from the observations described above and the
chemotherapeutic value of the nitrogenous chemical scaffolds,
synthesis and anticancer activity evaluation of some novel imid-
azole-(benz)azole and imidazoleepiperazine derivatives were
examined in this study.
Imidazole is a nitrogen containing heterocyclic ring which
possesses biological and pharmaceutical importance. Thus, imid-
azole compounds have been an interesting source for researchers
for more than a century [10]. Lepidiline A and B are the imidazole
* Corresponding author. Tel.: þ90 2223350580/3772; fax: þ902223350750.
E-mail address: yozkay@anadolu.edu.tr (Y. Özkay).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.015

Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
3321
2. Results and discussion
aminophenyl)-4,5-diphenyl-1H-imidazole (5). 2-Chloro-N-[4-(1-
methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (6) was
prepared via acetylation of the 5 with chloroacetyl chloride. At final
step the 6 was reacted with appropriate thiol-(benz)azoles or cor-
responding piperazine derivatives to give 2-substituted-N-[4-(1-
methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide deriva-
tives (7e23).
Structures of the obtained compounds were elucidated by
spectral data. In the IR spectra, significant stretching bands
belonging to NeH, C]O, C]N, C]C and deformation bands
belonging to monosubstituted and 4-substitutedphenyl were
observed at expected regions. The entire aromatic and aliphatic
2.1. Chemistry
For the synthesis of the target compounds the reaction sequence
outlined in the Scheme 1 was followed. The reaction steps
including dimerisation of benzaldehyde to benzoin (1), oxidation of
the 1 to benzil (2), cyclisation of the 2 with 4-nitrobenzaldehyde to
2-(4-nitrophenyl)-4,5-diphenyl-1H-imidazole (3) and N-methyla-
tion of the
3 to 1-methyl-2-(4-nitrophenyl)-4,5-diphenyl-1H-
imidazole (4), were performed according to previous studies
[28e31]. Reduction of the 4 with Zn/HCl produced 1-methyl-2-(4-
Scheme 1. Synthesis of 2-substitued-N-[4-(1-methyl-4,5-diphenyl-1H-imidazole-2-yl) phenyl]acetamide derivatives (6e23) Reagents and conditions; a: NaCN, H2O/EtOH, reflux 1
h, b: (CH3COO)2Cu, NH4NO3, AcOH, reflux 2 h, c: 4-Nitrobenzaldehyde, CH3COONH4, AcOH, reflux 3 h d: 1) NaH/THF, R.T. 15 min. 2) CH3I reflux 3 h, e: Zn, EtOH/HCl, R.T. and then
reflux 1 h, f: TEA, ClCH2COCl, benzene, ice bath and then R.T. 1 h, g: Appropriate thiol-(benz)azole, K2CO3, acetone, reflux 12 h, h: Corresponding 1-substituted piperazine, K2CO3,
acetone, reflux 24 h.

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Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
proton peaks in the 500 MHz 1H NMR spectra were also recorded
at estimated areas. The mass spectra (EI-MS) of the compounds
showed [M þ 1] peaks, in agreement with their molecular
weight.
implicated to cytotoxicity test in order to determine the contribu-
tion of variable groups to activity.
Cytotoxic activity of the compounds against HT-29 and MCF-7
cell lines is presented in Tables 1 and 2. In the first group the 7, 10,
11, 12 and 13 displayed higher cytotoxic activity against both of the
cell lines than the 6. Cytotoxicity of the 15 was lower than that of
the 6. The other thiol-(benz)azole moiety bearing compounds 8, 9
and 14 showed approximate cytotoxic activity to the 6. These
results suggest that benzoxazole-2-thiol, benzothiazole-2-thiol,
1,2,4-triazole-5-thiol, 1,2,3,4-tetrazole-5-thiol and 1,2,4-thiadia-
zole-3-thiol moieties supply a substantial contribution to increase
of the cytotoxicity. On the other hand, determination of lower
cytotoxicity with the 15 than the 6 indicates that substitution of the
6 with 1-methyl-imidazole-2-thiol causes an activity decrease. The
observed findings also refer that 5-methyl-benzoxazole-2-thiol, 5-
chloro-benzoxazole-2-thiol and thiazoline-2-thiol substituted
compounds have no effect on the cytotoxicity.
2.2. Biochemistry
Anticancer activity screening of the 6e23 against HT-29 and
MCF-7 cell lines was performed at three steps. In the first step,
cytotoxicities of the compounds were determined by MTT method.
Afterwards, DNA synthesis inhibition was analyzed for the
compounds possessing significant cytotoxic activity. Finally,
apoptotic DNA fragmentation method was used for determination
of internucleosomal DNA ladders, which are potent inducers of
apoptosis, caused by compounds.
2.2.1. Cytotoxicity assay (MTT)
In the second group, most of the compounds exhibited lower
cytotoxicity against both of the cell lines than the 6 (Tables 1 and 2).
In the research for new anticancer agents, the most common
screening methods employ cytotoxicity tests against a panel of
cancer cell lines. These are high throughput screening assays,
revealing compounds with the highest cytotoxic activity [32]. MTT,
based on the ability of metabolically active cells to convert the pale
yellow MTT dye to a spectrophotometrically quantifiable blue for-
mazan product, is one of the most preferred cytotoxicity tests [33].
In the MTT test, HT-29 and MCF-7 cell lines were incubated with
In the 0.64e32
revealed greater cytotoxic activity than the 6. However, when
concentrations were raised to 80 g/mL, cytotoxic activity of the 6
mg/mL concentrations range, only the 19 and 23
m
increased significantly and seemed as higher than those of the 19
and 23. Cytotoxicity test results of the 16e23 demonstrate that
substitution of the 6 with 1-substituted piperazine derivatives
creates no important effect on the activity.
the various concentrations (0.64, 1.6, 6.4, 16, 32 and 80
mg/mL) of
When cytotoxic activity of the imidazole-(benz)azoles is
compared with that of the imidazoleepiperazines, it can be clearly
seen that the first group compounds display greater cytotoxicity.
The 7, 11, 12 and 13 are the most cytotoxic compounds in the series.
Moreover, cytotoxicities of the 11, 12 and 13 against HT-29 cells and
cytotoxicities of the 11 and 13 against MCF-7 cells are very close to
that of cisplatin. These results show that thiol-azole and thiol-
benzazole moieties have more contribution to increase cytotoxic
activity than piperazine ring. Such positive contribution to cyto-
toxicity can be explained by hydrogen bonding capacity of the first
group. The 7e15 bear a sulphur atom between N-[4-(4,5-diphenyl-
1H-imidazole-2-yl)phenyl]acetamide substructure and azole or
benzazole rings. On the other hand, the second group compounds
6e23. After the completion of incubation period (24 h), cytotoxic
activity of the compounds was examined and IC50 values were
calculated. Anticancer agent cisplatin was used as a positive control.
Compounds subjected to cytotoxicity test were designed to
contain two different pharmacophore groups which exist on the
chemical structures of some anticancer agents and are estimated to
be responsible for the anticancer activity. N-[4-(4,5-diphenyl-1H-
imidazole-2-yl)phenyl]acetamide substructure was fixed in all of
the compounds. This substructure was substituted from second
position of the acetamide moiety in order to classify the
compounds in two different groups. The first group was constituted
by using different azole and benzazole heterocycles and named
imidazole-(benz)azoles. The second group was imidazo-
leepiperazines and formed by substitution of corresponding
piperazine derivatives. The starting compound (6) was also
16e23 are constituted by
a direct linkage between same
substructure and piperazine ring. Hence, the imidazole-(benz)
Table 1
Cytotoxic activity of the compounds 6e23 against HT-29 cell line.
Compounds
Concentration (
0.64
m
g/mL)
1.6
IC50 (mg/mL)
6.4
16
32
80
6
7
8
9
7.2 ꢀ 1.8
24.8 ꢀ 2.1
11.9 ꢀ 2.2
11.0 ꢀ 1.4
9.8 ꢀ 1.7
14.4 ꢀ 2.2
41.0 ꢀ 2.3
17.4 ꢀ 4.3
15.5 ꢀ 2.1
15.3 ꢀ 3.3
40.8 ꢀ 2.9
49.8 ꢀ 2.3
39.9 ꢀ 2.5
30.4 ꢀ 2.7
23.8 ꢀ 2.6
27.4 ꢀ 1.2
13.9 ꢀ 2.0
8.0 ꢀ 1.8
15.1 ꢀ 3.0
44.9 ꢀ 2.5
29.4 ꢀ 1.5
25.6 ꢀ 2.7
36.8 ꢀ 3.2
43.1 ꢀ 3.4
60.6 ꢀ 2.8*
73.0 ꢀ 3.2*
31.0 ꢀ 1.3
30.7 ꢀ 2.9
28.8 ꢀ 2.6
17.2 ꢀ 2.8
12.2 ꢀ 2.9
46.5 ꢀ 2.2
26.8 ꢀ 5.4
27.7 ꢀ 2.7
17.8 ꢀ 3.2
41.5 ꢀ 4.6
71.3 ꢀ 1.7*
23.7 ꢀ 1.6
56.2 ꢀ 2.7*
42.5 ꢀ 1.0
37.7 ꢀ 2.3
47.2 ꢀ 1.8
59.1 ꢀ 1.2*
72.5 ꢀ 2.0*
83.3 ꢀ 1.4*
43.3 ꢀ 2.7
36.5 ꢀ 0.4
33.7 ꢀ 5.3
28.4 ꢀ 2.1
28.2 ꢀ 2.8
55.4 ꢀ 3.3*
37.0 ꢀ 3.2
30.6 ꢀ 2.1
21.8 ꢀ 1.3
52.9 ꢀ 3.2*
89.1 ꢀ 3.6*
53.2 ꢀ 3.3*
79.3 ꢀ 1.7*
61.9 ꢀ 2.7*
59.5 ꢀ 2.2*
66.0 ꢀ 2.3*
86.1 ꢀ 2.7*
84.0 ꢀ 3.6*
87.1 ꢀ 2.5*
59.4 ꢀ 1.5*
41.1 ꢀ 1.6
49.1 ꢀ 3.0
38.8 ꢀ 1.8
29.8 ꢀ 2.9
60.5 ꢀ 2.4*
40.0 ꢀ 3.1
42.3 ꢀ 1.1
29.9 ꢀ 2.5
55.6 ꢀ 1.7*
98.2 ꢀ 2.4*
76.1 ꢀ 3.4*
81.5 ꢀ 1.5*
79.6 ꢀ 2.0*
69.8 ꢀ 2.1*
82.7 ꢀ 2.5*
88.0 ꢀ 2.5*
89.0 ꢀ 2.8*
90.5 ꢀ 3.2*
75.2 ꢀ 2.7*
69.4 ꢀ 2.3*
53.1 ꢀ 2.4
51.2 ꢀ 2.4*
45.4 ꢀ 3.0
63.7 ꢀ 3.0*
54.5 ꢀ 1.2*
54.2 ꢀ 3.1*
51.2 ꢀ 2.6*
60.7 ꢀ 1.6*
99.3 ꢀ 1.3*
28.7
11
20.6
22.4
18
10.7
1.6
2.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Cisplatin
32.0 ꢀ 1.7
38.0 ꢀ 1.3
34.8 ꢀ 2.1
16.8 ꢀ 2.0
13.6 ꢀ 1.9
18.8 ꢀ 2.2
7.4 ꢀ 1.3
21
47.5
35.5
75
3.1 ꢀ 2.7
>80
17.3 ꢀ 1.9
15.5 ꢀ 0.8
15.4 ꢀ 2.6
5.2 ꢀ 1.6
26.0 ꢀ 0.5
26.0 ꢀ 2.6
26.4 ꢀ 1.3
10.1 ꢀ 2.5
16.4 ꢀ 2.8
48.1 ꢀ 2.1
8.6
65.5
62
77.3
12
11.3 ꢀ 2.2
41.3 ꢀ 1.4
1.7
Results are expressed as the mean % of MTT absorbance (ratio of absorbance in test compound treated and control cells). Data points represent means of three independent
experiments ꢀSD of twelve independent wells. p < 0.05. *Significantly different from control cells.

Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
3323
Table 2
Cytotoxic activity of the compounds 6e23 against MCF-7 cell line.
Compounds
Concentration (
m
g/mL)
1.6
IC50 (mg/mL)
0.64
6.4
16
32
80
6
7
8
9
3.4 ꢀ 0.7
18.7 ꢀ 1.4
2.4 ꢀ 0.9
1.1 ꢀ 1.1
0.9 ꢀ 0.6
33.1 ꢀ 2.2
21.9 ꢀ 1.6
35.1 ꢀ 1.3
10.3 ꢀ 1.5
10.3 ꢀ 1.1
1.1 ꢀ 0.9
4.7 ꢀ 0.4
1.2 ꢀ 1.1
12.1 ꢀ 1.0
7.2 ꢀ 0.9
9.4 ꢀ 2.2
1.0 ꢀ 0.9
4.8 ꢀ 2.1
38.4 ꢀ 0.7
4.3 ꢀ 1.4.
14.2 ꢀ 1.0
42.3 ꢀ 1.0
27.7 ꢀ 3.6
24.6 ꢀ 0.5
27.0 ꢀ 3.4
59.2 ꢀ 2.6*
55.9 ꢀ 0.9*
67.8 ꢀ 1.2*
18.6 ꢀ 1.8
27.2 ꢀ 1.3
28.6 ꢀ 1.6
16.4 ꢀ 1.8
11.1 ꢀ 1.2
37.7 ꢀ 1.5
23.8 ꢀ 1.3
21.8 ꢀ 3.4
29.0 ꢀ 1.6
33.4 ꢀ 1.0
67.1 ꢀ 3.1*
15.2 ꢀ 2.2
56.0 ꢀ 1.9*
40.0 ꢀ 1.8
40.3 ꢀ 1.5
50.3 ꢀ 1.8*
61.2 ꢀ 1.0*
63.7 ꢀ 1.6*
74.9 ꢀ 1.0*
41.8 ꢀ 1.2
32.4 ꢀ 2.7
30.1 ꢀ 1.9
32.8 ꢀ 3.2
19.4 ꢀ 1.8
59.6 ꢀ 0.8*
28.3 ꢀ 2.0
26.0 ꢀ 2.3
12.0 ꢀ 2.3
46.2 ꢀ 2.5
86.3 ꢀ 2.7*
42.8 ꢀ 2.0
71.5 ꢀ 1.3*
59.7 ꢀ 1.2*
57.7 ꢀ 0.4*
59.0 ꢀ 1.2*
89.2 ꢀ 0.8*
77.5 ꢀ 1.4*
83.4 ꢀ 1.9*
52.5 ꢀ 2.3*
43.4 ꢀ 1.8
46.2 ꢀ 3.4
38.6 ꢀ 1.3
25.8 ꢀ 2.4
63.6 ꢀ 1.8*
33.6 ꢀ 1.9
31.5 ꢀ 0.9
17.7 ꢀ 2.4
52.8 ꢀ 3.5*
98.5 ꢀ 2.2*
70.0 ꢀ 0.9*
76.3 ꢀ 2.4*
69.1 ꢀ 2.2*
65.1 ꢀ 2.1*
76.6 ꢀ 2.2*
89.3 ꢀ 1.9*
77.7 ꢀ 3.4*
85.2 ꢀ 2.8*
70.0 ꢀ 1.4*
64.0 ꢀ 3.3*
50.3 ꢀ 2.7*
46.3 ꢀ 2.3
34.7 ꢀ 2.9
72.5 ꢀ 3.7*
49.5 ꢀ 2.4
49.3 ꢀ 2.8
34.5 ꢀ 1.1
52.9 ꢀ 2.4*
99.4 ꢀ 1.6*
42.6
11.2
21.7
22.4
15.8
3.2
27.4 ꢀ 3.9
2.5 ꢀ 2.1
1.9 ꢀ 0.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Cisplatin
2.1 ꢀ 1.3
42.8 ꢀ 1.6
34.3 ꢀ 1.1
37.5 ꢀ 0.7
16.2 ꢀ 1.0
17.5 ꢀ 0.8
2.9 ꢀ 1.4
4.5
3.2
26.7
46.5
76.5
>80
>80
10.7
>80
>80
>80
21
14.6 ꢀ 0.9
1.9 ꢀ 0.8
21.3 ꢀ 2.5
13.3 ꢀ 2.0
19.3 ꢀ 1.6
27.3 ꢀ 3.2
11.8 ꢀ 1.9
44.7 ꢀ 1.9
2.6
Results are expressed as the mean percent of MTT absorbance (ratio of absorbance in test compound treated and control cells). Data points represent means of three
independent experiments ꢀSD of twelve independent wells. p < 0.05. *Significantly different from control cells.
azoles have more heteroatoms which may provide more capability
to interact with DNA by hydrogen bonds. Due to the correlation
between force of binding to DNA and anticancer activity, greater
cytotoxic activity with the first group can be related to the number
of heteroatoms.
Consequently, analysis of DNA synthesis indicates that the 7, 11,
12 and 13 have greater inhibitory effects on DNA synthesis of the
HT-29 cell line. However, DNA synthesis inhibitory activity of the
cisplatin against both of the cell lines is significantly greater than
that of the tested compounds. Furthermore, the results of DNA
synthesis analysis are not as significant as cytotoxicity test results.
These findings demonstrate that anticancer ability of the 7, 11, 12
and 13 is not related to their DNA synthesis inhibitory activity.
2.2.2. Analysis of DNA synthesis
This immunostaining procedure is based on measuring the
incorporation of bromodeoxyuridine (BrdU) into nuclear DNA in
place of thymidine during the S-phase of the cell cycle using
specific anti-BrdU antibodies [34]. Hence, such method provides
a colorimetric measurement for DNA synthesis inhibition ratio of
the carcinogenic cells.
DNA synthesis of the carcinogenic cell lines were analyzed for
the 7, 11, 12 and 13 which indicated significant cytotoxic activity
with MTT test. Cisplatin was used as a positive control. For 24 h and
48 h time periods, HT-29 and MCF-7 cells were incubated with three
different concentrations of the compounds that were determined
according to their IC50. Tested compounds showed time- and dose-
dependent inhibitory activity on DNA synthesis of both cell lines.
DNA synthesis inhibitory activity of the compounds on HT-29
cells is presented in Fig. 1. On the HT-29 cell line, applied concen-
trations of the 7 are approximate to those of the 11. When
compared the DNA synthesis inhibitory activity of these
compounds each other, it is clear that the 11 has higher activity
than the 7. After comparing inhibitory activity of the 11 with that of
2.2.3. Detection of apoptotic DNA fragmentation
This method was carried out for the most cytotoxic compounds
against HT-29 cell line hence this cell line was more sensitive to the
compounds. HT-29 cells were incubated with the compounds at
two different concentrations. IC50 and lower concentrations were
preferred for this purpose. When incubation period (24 h) was over,
DNA of the cells were isolated by using apoptotic DNA ladder kit
and monitored in UV gel visualization system. Activity of the
compounds on genomic DNA of the HT-29 cell line is given in Fig. 3.
As seen in Fig. 3, IC50 of the 11, 12 and 13 cause DNA fragmen-
tation (lanes 2, 3 and 4). However, when applied concentrations are
decreased DNA ladders disappear (lanes 6, 7 and 8). The 7 makes no
damage on DNA of the HT-29 cells at the applied concentrations.
These findings can be explained by different constituents of the
tested compounds. Benzoxazole ring is a fragment of the 7 and
includes two heteroatoms. On the other hand, the 11, 12 and 13
involve 1,2,4-triazole, 1,2,3,4-tetrazole and 1,2,4-thiadiazole rings
which bear more than two heteroatoms. Therefore, polarization
characters and hydrogen bonding capacities of the 11, 12 and 13 are
expected to be better than those of the 7. As a result, stronger
interaction between DNA and 11, 12 and 13, which probably cause
to DNA fragmentation, may occur.
the 12 at the concentrations of 3.2
mg/mL and 6.4 mg/mL, it is
determined that the 12 is more potent to inhibit the DNA synthesis
than the 11. The 13 is the most effective compound against HT-29
cell line because at the concentration of 1.6 mg/mL, DNA synthesis
inhibitory activity of the 13 is greater than that of the 12. All of these
comparisons enable to conclude the inhibitory activity ordering of
the compounds as 13 > 12>11 > 7.
3. Conclusion
DNA synthesis inhibitory activity of the compounds on MCF-7
cells is presented in Fig. 2. If the DNA synthesis inhibitory activity of
the compounds on MCF-7 cell line is compared each other by
following the same way used for HT-29 cells, it can be noticed that
the activity orderings of the compounds on HT-29 and MCF-7 cells
seem to be quite similar. However, such activity is more notable
against HT-29 cell line. This finding suggests that HT-29 cells are
more sensitive to the compounds than MCF-7 cells.
The preliminary in vitro anticancer investigation of novel imid-
azole-(benz)azole and imidazoleepiperazine derivatives has indi-
cated the anticancer potency of the tested compounds. In general,
imidazole-(benz)azole compounds exhibited greater anticancer
activity than imidazoleepiperazine derivatives. Especially the
compounds 11, 12 and 13 indicated significant anticancer activity
against colon carcinoma cell line. These three compounds showed

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Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
100,00
90,00
80,00
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
HT-29 Cell line
24 h
48 h
7a
7b
7c 11a 11b 11c 12a 12b 12c 13a 13b 13c Cpt-a Cpt-b Cpt-c
Compounds and Doses
Fig. 1. DNA synthesis inhibitory activity of the compounds 7, 11, 12 and 13 on HT-29 cells. Mean percent absorbance of the untreated (assessed in the presence of DMSO used as
a solvent and assumed as 0%), and three different concentrations (7a 11 g/mL; 7b 6.4 g/mL; 7c 3.2 g/mL; 11a 10 g/mL; 11b 6.4 g/mL; 11c 3.2 g/mL; 12a 4 g/mL; 12b 3.2 g/
mL; 12c 1.6 g/mL; 13a 2.7 g/mL; 13b 1.6 g/mL; 13c 0.64 g/mL; Cpt-a 3.2 g/mL; Cpt-b 1.7 g/mL; Cpt-c 0.64 g/mL) of test compounds and cisplatin were given. Data points
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
represent means for three independent experiments ꢀSD of nine independent wells. p < 0.05.
substantial cytotoxicity and caused DNA fragmentation of the HT-29
cells. However, they displayed poor inhibitory activity on DNA
synthesis. Such results refer that anticancer ability of the compounds
arises afterDNA synthesisof thecellshasbeencompleted.Theresults
implement this synthesis strategy can be replacement of an alter-
native heterocyclic ring system instead of 4,5-diphenylimidazole,
which is participated in all of the tested compounds.
also reveal that anticancer ability of the compounds is related to their
cytotoxic and apoptotic effects on HT-29 cell lines.
4. Materials and methods
In the present study, 1,2,4-triazole, 1,2,3,4-tetrazole and 1,2,4-
thiadiazole rings are the main heterocycles that are responsible for
the anticancer activity. Significant contribution to anticancer
activity with the presence of these ring systems have encouraged
us to make some modifications on basic structure of obtained
compounds to achieve more active derivatives in further studies.
For this purpose it is considered to synthesize some new
compounds that carry 1,2,4-triazole-5-thiol, 1,2,3,4-tetrazole-5-
thiol and 1,2,4-thiadiazole-3-thiol moieties and show structural
similarity to the compounds presented in this study. First choice to
4.1. Chemistry
Chemicals used in syntheses were obtained from Acros, Aldrich,
Alfa-Aesar, Fluka or Merck chemical companies. Melting points
(m.p.) of the target compounds were measured in Electrothermal
9001 Digital Melting Point Apparatus and uncorrected. IR and 1H
NMR spectra were recorded on Shimadzu, 8400 FTIR spectrometer
as KBr pellets and on Bruker UltraShield 500 MHz spectrometer in
DMSO-d6, respectively. EI-MS data were obtained on Quattromicro
Mass spectrometer.
100,00
MCF-7 Cell line
90,00
80,00
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
24 h
48 h
7a
7b
7c 11a 11b 11c 12a 12b 12c 13a 13b 13c Cpt-a Cpt-b Cpt-c
Compounds and Doses
Fig. 2. DNA synthesis inhibitory activity of the compounds 7, 11, 12 and 13 on MCF-7 cells. Mean percent absorbance of the untreated (assessed in the presence of DMSO used as
a solvent and assumed as 0%), and three different concentrations (7a 11.2 g/mL; 7b 6.4 g/mL; 7c 3.2 g/mL; 11a 4.2 g/mL; 11b 3.2 g/mL; 11c 1.6 g/mL; 12a 5 g/mL; 12b 3.2 g/
mL; 12c 1.6 g/mL; 13a 2.8 g/mL; 13b 1.6 g/mL; 13c 0.64 g/mL; Cpt-a 3.2 g/mL; Cpt-b 1.7 g/mL; Cpt-c 0.64 g/mL) of test compounds and cisplatin were given. Data points
represent means for three independent experiments ꢀSD of nine independent wells. p < 0.05.
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m

Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
3325
evaporated until dryness. The residue was washed with water and
recrystallized from ethanol to give the 7e15.
4.1.4.1. 2-Benzoxazole-2-yl-sulfanyl-N-[4-(1-methyl-4,5-diphenyl-
1H-imidazole-2-yl)phenyl]acetamide (7). Yield 78%. m.p. 232 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3349 (NeH), 1682 (C]O), 1599e1395 (C]C
and C]N), 837 (1,4-disubstituted benzene), 746e702 (mono-
substituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d
(ppm):
3.48 (3H, s, imidazole NeCH3), 4.46 (2H, s, COeCH2), 7.13e7.24
(3H, m, C6H5eH), 7.33e7.38 (2H, m, benzoxazole 3,4eH),
7.42e7.56 (7H, m, C6H5eH), 7.64e7.69 (2H, m, benzoxazole
2,5eH), 7.78 (4H, s, C6H4eH), 10.75 (H, s, NHeCO). EI-MS (m/z):
C
C
M þ 1: 517.
Fig. 3. Effects of the compounds 7, 11, 12 and 13 on genomic DNA of HT-29 cells. M:
DNA marker (10,000-50 bp); C: Control (DNA of HT-29 cell line); 1: DNA of HT-29 cell
line treated with 11 mg/mL of 7; 2: DNA of HT-29 cell line treated with 10 mg/mL of 11;
3: DNA of HT-29 cell line treated with 1.6 mg/mL of 12; 4: DNA of HT-29 cell line
treated with 2.7 mg/mL of 13; 5: DNA of HT-29 cell line treated with 6.4 mg/mL of 7; 6:
DNA of HT-29 cell line treated with 6.4 mg/mL of 11; 7: DNA of HT-29 cell line treated
with 1 mg/mL of 12; 8: DNA of HT-29 cell line treated with 1.6 mg/mL of 13.
4.1.4.2. 2-(5-Methyl-benzoxazole-2-yl)-sulfanyl-N-[4-(1-methyl-4,5-
diphenyl-1H-imidazole-2-yl)phenyl]acetamide (8). Yield 84%. m.p.
215 ꢁC. IR (KBr) nmaks(cmꢃ1): 3350 (NeH), 1676 (C]O), 1599e1395
(C]C and C]N), 837 (1,4-disubstituted benzene), 775e698
(monosubstituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d
(ppm): 2.40 (3H, s, benzoxazole eCH3), 3.48 (3H, s, imidazole
NeCH3), 4.43 (2H, s, COeCH2), 7.12e7.25 (4H, m, C6H5eH and
benzoxazole C4eH), 7.42e7.56 (9H, m, C6H5eH and benzoxazole
4.1.1. Synthesis of the starting materials (1e4)
C2,5eH), 7.77 (4H, s, C6H4e), 10.75 (H, s, NHeCO). EI-MS (m/z):
The starting materials; benzoin (1) (m.p. 135 ꢁC; lit. m.p. 134
[28]), benzil (2) (m.p. 94 ꢁC; lit. m.p. 94e95 ꢁC [29]), 2-(4-nitro-
phenyl)-4,5-diphenyl-1H-imidazole (3) (m.p. 240 ꢁC; lit. m.p.
242 ꢁC [30]) and 1-methyl-2-(4-nitrophenyl)-4,5-diphenyl-1H-
imidazole (4) (m.p. lit. 196 ꢁC; m.p. 198 ꢁC [31]), were synthesized
according to previous studies.
M þ 1: 531.
4.1.4.3. 2-(5-Chloro-benzoxazole-2-yl)-sulfanyl-N-[4-(1-methyl-4,5-
diphenyl-1H-imidazole-2-yl)phenyl]acetamide (9). Yield 73%. m.p.
217 ꢁC. IR (KBr) nmaks(cmꢃ1): 3349 (NeH), 1676 (C]O),
1599e1397 (C]C and C]N), 829 (1,4-disubstituted benzene),
775e698 (monosubstituted benzene). 1H NMR (500 MHz)
4.1.2. 1-Methyl-2-(4-aminophenyl)-4,5-diphenyl-1H-imidazole (5)
The compound 4 (26.625 g, 75 mmol) was dissolved in the
mixture of 200 mL EtOH and 200 mL HCl (25%). Zinc dust (7.3125 g,
1125 mmol) was divided into ten portions (0.73125 g ꢂ 10) and each
portion was added to the stirring solution in 15 min intervals. Once
the addition of the zinc was completed, reaction mixture was
refluxed for 1 h. Hot solution was allowed to cool down, poured into
ice water and then neutralized by using 10% NaOH solution. The
precipitate was extracted with chloroform (3 ꢂ100 mL). The extracts
were combined and filtered over anhydrous Na2SO4. Solvent was
evaporated and the white residue was recrystallized from ethanol to
give the 5. Yield 66%. m.p. 206 ꢁC; lit. m.p. 204 ꢁC [31].
(DMSO-d6) d(ppm): 3.49 (3H, s, imidazole NeCH3), 4.47 (2H, s,
COeCH2), 7.12e7.25 (3H, m, C6H5eH), 7.38e7.58 (8H, m,
C6H5eH and benzoxazole C4eH), 7.72 (2H, m, benzoxazole
C
2,5eH), 7.77 (4H, s, C6H4eH), 10.75 (H, s, NHeCO). EI-MS (m/z):
M þ 1: 551.
4.1.4.4. 2-Benzothiazole-2-yl-sulfanyl-N-[4-(1-methyl-4,5-diphenyl-
1H-imidazole-2-yl)phenyl]acetamide (10). Yield 87%. m.p. 226 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3339 (NeH), 1676 (C]O), 1599e1395 (C]C and
C]N), 839 (1,4-disubstituted benzene), 762e708 (mono-
substituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm): 3.48
(3H, s, imidazole NeCH3), 4.47 (2H, s, COeCH2), 7.12e7.24 (3H, m,
C6H5eH), 7.38e7.58 (9H, m, C6H5eH and benzothiazole C3,4eH),
7.77 (4H, s, C6H4eH), 7.86 (H, d, J ¼ 8.10 Hz, benzothiazole C5eH),
8.05 (H, d, J ¼ 7.97 Hz, benzothiazole C2eH),10.74 (H, s, NHeCO). EI-
MS (m/z): M þ 1: 533.
4.1.3. 2-Chloro-N-[4-(4,5-diphenyl-1H-imidazole-2-yl)phenyl]
acetamide (6)
The 5 (16.25 g, 50 mmol) was dissolved in 100 mL of benzene
and 8.5 mL (60 mmol) of triethylamine was added. This mixture
was allowed to stir on an ice bath. 4.8 mL (60 mmol) of chloroacetyl
chloride and 10 mL of benzene mixture was added drop by drop.
After completion of dropping, reaction mixture was stirred for 1 h
at room temperature (R.T.). Benzene was evaporated and brown
residue was recrystallized from ethanol to give the 6. Yield 78%. m.
p. 200 ꢁC. IR (KBr) nmaks (cmꢃ1): 3370 (NeH), 1682 (C]O),
1605e1400 (C]C and C]N), 841 (1,4-disubstituted benzene),
772e696 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
4.1.4.5. 2-(4-Methyl-4H-1,2,4-triazole-3-yl)-sulfanyl-N-[4-(1-
methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide
(11).
Yield 81%. m.p. 259 ꢁC. IR (KBr) nmaks(cmꢃ1): 3237 (NeH), 1694 (C]
O), 1605e1398 (C]C and C]N), 845 (1,4-disubstituted benzene),
775e696 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
d6) d(ppm): 3.48 (3H, s, imidazole NeCH3), 3.63 (3H, s, triazole
NeCH3), 4.12 (2H, s, COeCH2), 7.12e7.24 (3H, m, C6H5eH),
0
7.42e7.57 (7H, m, C6H5eH), 7.75 (4H, q, Ja,a ¼ 8.89 Hz, Jb,
b0
d6)
d(ppm): 3.48 (3H, s, imidazole NeCH3), 4.31 (2H, s, COeCH2),
¼ 8.92 Hz, C6H4eH), 8.60 (H, s, triazole C2eH), 10.60 (H, s,
7.13e7.25 (3H, m, C6H5eH), 7.43e7.57 (7H, m, C6H5eH), 7.78 (4H, s,
C6H4eH), 10.50 (H, s, NHeCO). EI-MS (m/z): M þ 1: 402.
NHeCO). EI-MS (m/z): M þ 1: 481.
4.1.4.6. 2-(1-Methyl-1H-1, 2, 3, 4-tetrazole-5-yl)-sulfanyl-N-[4-
(1-methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (12).
Yield 85%. m.p. 251 ꢁC. IR (KBr) nmaks(cmꢃ1): 3268 (NeH), 1692 (C]
O), 1607e1395 (C]C and C]N), 841 (1,4-disubstituted benzene),
783e702 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
4.1.4. General procedure for 2-substituted-sulfanyl-N-[4-(1-methyl-
4,5-diphenyl-1H-imidazole-2-yl)phenyl] acetamide derivatives
(7e15)
A mixture of the 6 (0.803 g, 2 mmol), appropriate (benz)azole-
thiol derivative (2 mmol) and K2CO3 (0.276 g, 2 mmol) in acetone
(30 mL) was refluxed for 12 h. After cooling, the solution was
d6)
d(ppm): 3.48 (3H, s, imidazole NeCH3), 4.02 (3H, s, tetrazole
NeCH3), 4.35 (2H, s, COeCH2), 7.12e7.24 (3H, m, C6H5eH),

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Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
0
0
7.42e7.56 (7H, m, C6H5eH), 7.76 (4H, q, Jaa ¼ 8.82 Hz, Jbb ¼ 8.78 Hz,
7.74 (2H, d, J ¼ 8.70 Hz, C6H4, C2,6eH), 7.82 (2H, d, J ¼ 8.69 Hz, C6H4,
3,5eH), 9.91 (H, br, NHeCO). EI-MS (m/z): M þ 1: 480.
C6H4eH), 10.60 (H, s, NHeCO). EI-MS (m/z): M þ 1: 482.
C
4.1.4.7. 2-(5-Methyl-1,2,4-thiadiazole-3-yl)-sulfanyl-N-[4-(1-methyl-
4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (13). Yield 82%.
m.p. 234 ꢁC. IR (KBr) nmaks(cmꢃ1): 3349 (NeH), 1676 (C]O),
1599e1391 (C]C and C]N), 839 (1,4-disubstituted benzene),
775e698 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
4.1.5.3. 2-[4-(2-Dimetylaminoethyl)piperazine-1-yl]-N-[4-(1-methyl-
4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (18). Yield 61%.
m.p. 130 ꢁC. IR (KBr) nmaks(cmꢃ1): 3236 (NeH), 1694 (C]O),
1603e1400 (C]C and C]N), 853 (1,4-disubstituted benzene),
781e700 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
d6)
d(ppm): 2.69 (3H, s, thiadiazole eCH3), 3.48 (3H, s, imidazole
d6) d(ppm): 2.14 (6H, s, eN(CH3)2), 2.33 (4H, m, eCH2CH2), 2.48 (4H,
NeCH3), 4.33 (2H, s, COeCH2), 7.12e7.24 (3H, m, C6H5eH),
7.42e7.56 (7H, m, C6H5eH), 7.76 (4H, s, C6H4eH), 10.59 (H, s,
NHeCO). EI-MS (m/z): M þ 1: 498.
br, piperazine C3,5eH), 2.54 (4H, br, piperazine C2,6eH), 3.15 (2H, s,
COeCH2), 3.48 (3H, s, imidazole NeCH3), 7.12e7.24 (3H, m,
C6H5eH), 7.42e7.57 (7H, m, C6H5eH), 7.74 (2H, d, J ¼ 8.55 Hz, C6H4,
C
2,6eH), 7.82 (2H, d, J ¼ 8.53 Hz, C6H4, C3,5eH), 9.91 (H, br, NHeCO).
4.1.4.8. 2-Thiazoline-2-yl-sulfanyl-N-[4-(1-methyl-4,5-diphenyl-1H-
imidazole-2-yl)phenyl]acetamide (14). Yield 76%. m.p. 215 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3339 (NeH), 1676 (C]O), 1599e1395 (C]C and
C]N), 839 (1,4-disubstituted benzene), 777e704 (mono-
EI-MS (m/z): M þ 1: 523.
4.1.5.4. 2-(4-Phenylpiperazine-1-yl)-N-[4-(1-methyl-4,5-diphenyl-
1H-imidazole-2-yl)phenyl]acetamide (19). Yield 79%. m.p. 227 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3281 (NeH), 1676 (C]O), 1597e1397 (C]C and
C]N), 856 (1,4-disubstituted benzene), 796e698 (mono-
substituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm):
3.46e3.52 (5H, s, imidazole NeCH3 and thiazoline C3e2H),
4.12e4.18 (4H, m, COeCH2 and thiazoline C2e2H), 7.12e7.24 (3H,
m, C6H5eH), 7.42e7.56 (7H, m, C6H5eH), 7.76 (4H, s, C6H4eH),10.59
(H, s, NHeCO). EI-MS (m/z): M þ 1: 485.
substituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm): 2.71
(4H, t, J ¼ 4.45 Hz and J ¼ 4.57 Hz, piperazine C2,6eH), 3.23 (4H, t,
J ¼ 4.27 Hz and J ¼ 4.76 Hz, piperazine C3,5eH), 3.25 (2H, s,
COeCH2), 3.48 (3H, s, imidazole NeCH3), 6.79 (H, t, J ¼ 7.23 Hz and
J ¼ 7.34 Hz, pNeC6H5, C4eH), 6.97 (2H, d, J ¼ 8.38 Hz, pNeC6H5,
4.1.4.9. 2-(1-Methyl-1H-imidazole-2-yl)-sulfanyl-N-[4-(1-methyl-
4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (15). Yield 79%.
m.p. 220 ꢁC. IR (KBr) nmaks(cmꢃ1): 3241 (NeH), 1682 (C]O),
1613e1402 (C]C and C]N), 849 (1,4-disubstituted benzene),
775e696 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
C2,6eH), 7.12e7.25 (5H, m, C6H5eH and pNeC6H5, C3,5eH),
7.42e7.57 (7H, m, C6H5eH), 7.75 (2H, d, J ¼ 8.37 Hz, C6H4, C2,6eH),
7.84 (2H, d, J ¼ 8.49 Hz, C6H4, C3,5eH), 10.00 (H, br, NHeCO). EI-MS
(m/z): M þ 1: 528.
d6)
d(ppm): 2.95 (3H, s, imidazole NeCH3), 3.48 (3H, s, 4,5-
diphenyl-imidazole NeCH3), 4.23 (2H, m, COeCH2), 6.96e7.14 (3H,
4.1.5.5. 2-(4-Benzylpiperazine-1-yl)-N-[4-(1-methyl-4,5-diphenyl-
1H-imidazole-2-yl)phenyl]acetamide (20). Yield 73%. m.p. 206 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3331 (NeH), 1688 (C]O), 1595e1400 (C]C and
C]N), 839 (1,4-disubstituted benzene), 781e696 (monosubstituted
0
m, C6H5eH), 7.38e7.54 (7H, m, C6H5eH), 7.75 (4H, q, Jaa ¼ 7.64 Hz,
0
Jbb ¼ 7.67, C6H4eH), 7.97 (H, d, J ¼ 8.04 Hz, imidazole C3eH), 8.22
(H, d, J ¼ 7.85 Hz imidazole C2eH),11.90 (H, s, NHeCO). EI-MS (m/z):
M þ 1: 480.
benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm): 2.47 (4H, br,
piperazine C2,6eH), 2.56 (4H, br, piperazine C3,5eH), 3.17 (2H, s,
COeCH2), 3.48 (3H, s, imidazole NeCH3), 3.50 (2H, s, C6H5eCH2),
7.13e7.25 (8H, m, C6H5eH and C6H5eCH2), 7.42e7.57 (7H, m,
C6H5eH), 7.74(2H, d,J¼ 8.70Hz,C6H4, C2,6eH), 7.81(2H, d, J¼ 8.73Hz,
C6H4, C3,5eH), 9.90 (H, br, NHeCO). EI-MS (m/z): M þ 1: 542.
4.1.5. General procedure for 2-(4-substitutedpiperazine-1-yl)-N-[4-
(1-methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide
derivatives (16e23)
A mixture of 6 (0.803 g, 2 mmol), corresponding 1-substituted
piperazine derivative (3 mmol) and K2CO3 (0.276 g, 2 mmol) in
acetone (30 mL) was refluxed for 24 h. After cooling, the solution
was evaporated until dryness. The oily residue was treated with
5 mL of ether. Solidified product was filtered, washed with water
and recrystallized from ethanol to give the 16e23.
4.1.5.6. 2-[4-(4-Methoxybenzyl)piperazine-1-yl]-N-[4-(1-methyl-
4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide (21). Yield 75%.
m.p. 192 ꢁC. IR (KBr) nmaks(cmꢃ1): 3378 (NeH), 1684 (C]O),
1599e1397 (C]C and C]N), 841 (1,4-disubstituted benzene),
785e706 (monosubstituted benzene). 1H NMR (500 MHz) (DMSO-
4.1.5.1. 2-(4-Methylpiperazine-1-yl)-N-[4-(1-methyl-4,5-diphenyl-1H-
imidazole-2-yl)phenyl]acetamide (16). Yield 69%. m.p. 122 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3241 (NeH), 1697 (C]O), 1603e1408 (C]C and
C]N), 847 (1,4-disubstituted benzene), 781e700 (monosubstituted
d6) d(ppm): 2.43 (4H, br, piperazine C2,6eH), 2.55 (4H, br, pipera-
zine C3,5eH), 3.17 (2H, s, COeCH2), 3.42 (2H, s, C6H5eCH2), 3.48 (3H,
s, imidazole NeCH3), 3.74 (3H, s, OeCH3), 6.89 (2H, d, J ¼ 8.58 Hz,
C6H4eOCH3 C3,5eH), 7.12e7.23 (5H, m, C6H5eH and eC6H4eOCH3
benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm): 2.18 (3H, s,
C
2,6eH), 7.42e7.57 (7H, m, C6H5eH), 7.74 (2H, d, J ¼ 8.64 Hz, C6H4,
piperazine NeCH3), 2.40 (4H, br, piperazine C3,5eH), 2.54 (4H, br,
piperazine C2,6eH), 3.16 (2H, s, COeCH2), 3.48 (3H, s, imidazole
NeCH3), 7.12e7.24 (3H, m, C6H5eH), 7.42e7.57 (7H, m, C6H5eH),
7.74 (2H, d, J ¼ 8.65 Hz, C6H4, C2,6eH), 7.82 (2H, d, J ¼ 8.61 Hz, C6H4,
C2,6eH), 7.81 (2H, d, J ¼ 8.67 Hz, C6H4, C3,5eH), 9.90 (H, br, NHeCO).
EI-MS (m/z): M þ 1: 572.
4.1.5.7. 2-[4-(4-Methylbenzyl)piperazine-1-yl]-N-[4-(1-methyl-4,5-
diphenyl-1H-imidazole-2-yl)phenyl]acetamide (22). Yield 71%. m.p.
198 ꢁC. IR (KBr) nmaks(cmꢃ1): 3241 (NeH), 1694 (C]O), 1605e1406
(C]C and C]N), 835 (1,4-disubstituted benzene), 781e698
C3,5eH), 9.91 (H, br, NHeCO). EI-MS (m/z): M þ 1: 466.
4.1.5.2. 2-(4-Ethylpiperazine-1-yl)-N-[4-(1-methyl-4,5-diphenyl-1H-
imidazole-2-yl)phenyl]acetamide (17). Yield 74%. m.p. 171 ꢁC. IR
(KBr) nmaks(cmꢃ1): 3295 (NeH), 1684 (C]O), 1593e1395 (C]C and
C]N), 839 (1,4-disubstituted benzene), 774e698 (monosubstituted
(monosubstituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d
(ppm): 2.28 (3H, s, eCH3), 2.44 (4H, br, piperazine C2,6eH), 2.55
(4H, br, piperazine C3,5eH), 3.16 (2H, s, COeCH2), 3.43 (2H, s,
C6H5eCH2), 3.48 (3H, s, imidazole NeCH3), 6.89 (2H, d, J ¼ 8.58 Hz,
benzene). 1H NMR (500 MHz) (DMSO-d6)
d(ppm): 1.01 (3H, t,
J ¼ 7.14 Hz and J ¼ 7.20 Hz, eCH2CH3), 2.34 (2H, q, J ¼ 7.17 Hz and
J ¼ 7.18 Hz, eCH2CH3), 2.44 (4H, br, piperazine C3,5eH), 2.56 (4H, br,
piperazine C2,6eH), 3.15 (2H, s, COeCH2), 3.48 (3H, s, imidazole
NeCH3), 7.12e7.24 (3H, m, C6H5eH), 7.42e7.57 (7H, m, C6H5eH),
C6H4eCH3 C3,5eH), 7.12e7.24 (7H, m, C6H5eH and eC6H4eCH3),
7.42e7.56 (7H, m, C6H5eH), 7.74 (2H, d, J ¼ 8.68 Hz, C6H4, C2,6eH),
7.81 (2H, d, J ¼ 8.68 Hz, C6H4, C3,5eH), 9.98 (H, br, NHeCO). EI-MS
(m/z): M þ 1: 556.

Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3320e3328
3327
4.1.5.8. 2-[4-(4-Chlorobenzyl)piperazine-1-yl]-N-[4-(1-methyl-4,5-
diphenyl-1H-imidazole-2-yl)phenyl]acetamide (23). Yield 82%. m.p.
196 ꢁC. IR (KBr) nmaks(cmꢃ1): 3331 (NeH), 1688 (C]O), 1593e1402
(C]C and C]N), 843 (1,4-disubstituted benzene), 787e698
experiments were repeated three times. For each concentration of
the compounds, triplicate wells were used.
4.2.4. Detection of apoptotic DNA ladder
(monosubstituted benzene). 1H NMR (500 MHz) (DMSO-d6)
d
HT-29 cells (1 ꢂ 106 cells/mL) were harvested into dishes and
incubated with various doses of the test compounds for 24 h. After
the incubation period, cells were washed in PBS, tripsynezed, put
(ppm): 2.46 (4H, br, piperazine C2,6eH), 2.56 (4H, br, piperazine
3,5eH), 3.17 (2H, s, COeCH2), 3.47 (3H, s, imidazole NeCH3), 3.49
C
(2H, s, C6H5eCH2), 7.12e7.24 (3H, m, C6H5eH), 7.34 (2H, d,
J ¼ 8.44 Hz, 4-CleC6H4, C2,6eH), 7.39 (2H, d, J ¼ 8.43 Hz, 4-CleC6H4,
into tubes and then mixed. Binding buffer (200
incubated at R.T. for 10 min then isopropanol (100
and mixed. The mixture was filtrated and centrifuged at 8000 rpm.
Washing buffer (500 l) was added and centrifuged again at the
same rpm. Filter tubes were solubilisated with elution buffer and
analyzed by 1% agarose gel electrophoresis (containing 500 g/mL
of ethidium bromide) at 50V for 120 min. Approximately 20 g of
m
l) was added and
ml) was added
C
C
3,5eH), 7.42e7.56 (7H, m, C6H5eH), 7.74 (2H, d, J ¼ 8.68 Hz, C6H4,
2,6eH), 7.81 (2H, d, J ¼ 8.71 Hz, C6H4, C3,5eH), 9.90 (H, br, NHeCO).
m
EI-MS (m/z): M þ 1: 576.
m
m
4.2. Biochemistry
DNA was loaded in each well, visualized under a UV light and
photographed (Fig. 3).
4.2.1. Cell cultures
MCF-7 cells were maintained in 90% Dulbecco’s modified Eagle’s
medium (DMEM) (Sigma, Deisenhofen, Germany), 1 mM sodium
4.2.5. Statistics
pyruvate (Sigma, Deisenhofen, Germany), 10
m
g/mL human insulin
The SPSS for Windows 11.5 computer program was used for
statistical analyses. Statistical comparison of the results obtained
from controls, groups, and time periods parameters were carried
out by the one-way analyses of variance (ANOVA) test and post hoc
analyses of group differences were performed by the Tukey test.
Results were expressed as mean ꢀ SD.
(Sigma, Deisenhofen, Germany) and 10% (v/v) of fetal bovine serum
(FBS) (Gibco, U.K.). HT-29 cells were cultured in 90% McCoy’s 5A
(Sigma, Deisenhofen, Germany) and 10% fetal bovine serum (FBS)
(Gibco, U.K.). All media were supplemented with penicillin/strep-
tomycin at 100 U/mL and cells were incubated at 37 ꢁC in a 5% CO2/
95% air humidified atmosphere.
Acknowledgement
4.2.2. MTT assay
A
tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
This work was financially supported by Anadolu University
Scientific Research Projects Commission. Project number:
070324.
nyltetrazolium bromide (MTT), was used as colorimetric
substrate for measuring cytotoxicity (MTT assay). The assay was
carried out according to previous study [35]. Briefly, HT-29 and
a
MCF-7 cells were cultured in 96 well plates and 0.64e80 mg/ml
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