Evaluation of Cytotoxic Activity of New Benzimidazole-Piperazine Hybrids Against Human MCF-7 and A549 Cancer Cells

Article abstract of DOI:10.1007/s11094-020-02119-9

A series of benzimidazole-piperazine hybrids (14 – 37) were designed, synthesized and evaluated for their cytotoxic activity against human lung (A549) and breast (MCF-7) cancer cell lines. Preliminary evaluation revealed that most of these hybrid molecules (i.e., 16 – 25) exhibited noteworthy and preferential antiproliferative effect against human lung cancer (A549) with IC₅₀ values of 2.8 – 7.8 μM. Among the synthesized molecules, compound 17 showed the most balanced cytotoxic effect against lung (A549) and breast (MCF-7) cancer cells with IC₅₀ values of 5.4 and 4.2 μM, respectively. To explore the mechanistic aspects fundamental to the observed activity, further biological studies of compounds 16, 17 and 22 were carried out. In addition, these compounds induced PARP-1 cleavage and caspase 7 activation, caused morphological changes such as bleb formation in the treated cells, and significantly increased the nuclear fragmentation. Taken all together, our findings indicate that cytotoxic activities of newly synthesized benzimidazole-piperazine hybrids are mediated through the apoptotic cell death induction. These benzimidazole derivatives have the potential for further development as anticancer agents.

Full text of DOI:10.1007/s11094-020-02119-9

DOI 10.1007/s11094-020-02119-9
Pharmaceutical Chemistry Journal, Vol. 53, No. 11, February, 2020 (Russian Original Vol. 53, No. 11, November, 2019)
EVALUATION OF CYTOTOXIC ACTIVITY OF NEW
BENZIMIDAZOLE-PIPERAZINE HYBRIDS AGAINST
HUMAN MCF-7 AND A549 CANCER CELLS
Aysun Özdemir,1,* Sümeyye Turanlý,2 Burcu Çalýþkan,2
Mustafa Arka,1 and Erden Banoglu2
Original article submitted April 1, 2019.
A series of benzimidazole-piperazine hybrids (14 – 37) were designed, synthesized and evaluated for their
cytotoxic activity against human lung (A549) and breast (MCF-7) cancer cell lines. Preliminary evaluation re-
vealed that most of these hybrid molecules (i.e., 16 – 25) exhibited noteworthy and preferential
antiproliferative effect against human lung cancer (A549) with IC50 values of 2.8 – 7.8 mM. Among the syn-
thesized molecules, compound 17 showed the most balanced cytotoxic effect against lung (A549) and breast
(MCF-7) cancer cells with IC50 values of 5.4 and 4.2 mM, respectively. To explore the mechanistic aspects
fundamental to the observed activity, further biological studies of compounds 16, 17 and 22 were carried out.
In addition, these compounds induced PARP-1 cleavage and caspase 7 activation, caused morphological
changes such as bleb formation in the treated cells, and significantly increased the nuclear fragmentation.
Taken all together, our findings indicate that cytotoxic activities of newly synthesized
benzimidazole-piperazine hybrids are mediated through the apoptotic cell death induction. These
benzimidazole derivatives have the potential for further development as anticancer agents.
Keywords: MCF-7, A549, benzimidazole, anticancer activity, apoptosis, cytotoxicity.
1. INTRODUCTION
ature precedence also reveals that benzimidazole nucleus has
been studied to develop tubulin polymerization inhibitors
[8, 9], topoisomerase inhibitors [10, 11] and sirtuin inhibitors
[12, 13] with potential antitumor activity.
Cancer is recognized as the second leading cause of
deaths worldwide following heart diseases, and the global
burden from cancer is anticipated to grow to about 22 million
new cancer cases and 13 million deaths by 2030 [1]. Thus, an
unending endeavour worldwide has been dedicated to the
discovery of new chemotypes as small-molecule cancer ther-
apeutics, which are prone to further development for inter-
vening with this complex disease. In this context, benzimi-
dazole ring is frequently encountered in contemporary me-
dicinal chemistry as a privileged substructure for developing
bioactive molecules including novel anticancer therapeutics
[2 – 5]. Bendamustine, the well-known benzimidazole deriv-
ative with a unique mechanism of action to activate apop-
tosis and inhibit mitotic checkpoints, has recently been ap-
proved by the FDA for the treatment of chronic lymphocytic
leukemia and non-Hodgkin’s lymphoma [6, 7]. Further liter-
Piperazine structure is another common structural motif
in anticancer agents, and a large number of compounds with
a piperazine fragments appeared in the literature as possess-
ing antiproliferative activity against various cancer cells
[14 – 17]. In particular, a series of piperazine analogs have
also been developed by Samjin Pharmaceutical to inhibit
microtubule synthesis, cell cycle progression, and angio-
genesis with promising antiproliferative activities against co-
lon, prostate, breast, lung and leukemia cell lines, as well as
effectively suppressing experimental tumors in small animal
models [18, 19]. Another recent study [20] showed that in-
corporation of an arylpiperazine moiety in purine nucleoside
analogs produced compounds with senescence-induced cell
death in liver cancer cells.
In the course of our longstanding commitment on devel-
oping bioactive heterocycles [5, 21 – 24], we have relied on
the aforementioned data for the design, synthesis and biolog-
ical evaluation of new benzimidazole derivatives amalgam-
ated with an aryl piperazine residue through a methylene
1
Department of Pharmacology, Faculty of Pharmacy, Gazi University,
06330 Ankara, Turkey.
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi Uni-
versity, 06330 Ankara, Turkey.
*
e-mail: aysunozdemir@gazi.edu.tr
1036
0091-150X/20/5311-1036 © 2020 Springer Science+Business Media, LLC

Evaluation of Cytotoxic Activity
1037
linker. In this context, we hereby report the straightforward
synthesis of these novel benzimidazole-piperazine hybrids,
which were evaluated for their antitumor activities against
human lung and breast cancer cell lines (Table 1).
pletion, the reaction mixture was diluted with water,
extracted with EtOAc, dried over anhydrous Na SO and
2
4
a
TABLE 1. IC Values (mM) of Compounds 14 – 37 for Human
2. EXPERIMENTAL
50
Lung (A549) and Breast (MCF-7) Cancer Cell Lines
2.1. Materials and Methods
All chemicals were purchased from Sigma Aldrich
Chemicals (Sigma Aldrich Corp., St. Louis, MO, USA) and
Merck Chemicals (Merck KGaA, Darmstadt, Germany). The
1H and 13C NMR spectra were recorded in CDCl or
3
DMSO-d on a Varian Mercury 400 MHz spectrometer using
6
tetramethylsilane as the internal standard. All chemical shifts
were recorded as d (ppm). All coupling constants are ex-
pressed in Hertz units. High resolution mass spectra data
(HRMS) were collected using Waters LCT Premier XE Mass
Spectrometer (high sensitivity orthogonal acceleration
time-of-flight instrument) operating in ESI (+) or ESI (-)
method, also coupled to an AQUITY Ultra Performance Liq-
uid Chromatography system (Waters Corporation) using a
UV detector monitoring at 254 nm. The purity of all final
compounds was >97%, according to the UPLC-MS method
using (A) water + 0.1% formic acid and (B) acetonit-
rile + 0.1% formic acid eluents at a flow rate of 0.3 mL/min
in Aquity BEH C18 column (2.1 ´ 100 mm, 1.7 mm). Mel-
ting points were determined with an SMP-II Digital Melting
Point Apparatus and remained uncorrected (Schorpp Geaete-
technik, Germany). The reactions were followed by LC-MS
or TLC on precoated Merck silica gel plates. The developed
plates were visualized using 254 nm or 365 nm UV expo-
sure. Flash chromatography was performed with a Combi-
flash Rf automated flash chromatography system with
RediSep columns (Teledyne-Isco, Lincoln, NE, USA) eluted
using dichloromethane : methanol solvent gradient. All mi-
crowave irradiation experiments were carried out in Biotage
Initiator+ microwave apparatus with Biotage sealed micro-
wave vials (Biotage, Uppsala, Sweden).
IC50 (mM)
Compd.
R1
R2
No
A549
>18
MCF-7
>18
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
2-Cl
2-Cl
2-F
3-F
10.3 ± 2.1
8.0 ± 0.3
5.4 ± 1.1
5.8 ± 1.1
6.5 ± 0.8
4.6 ± 0.6
3.6 ± 0.4
2.8 ± 0.4
4.5 ± 0.7
4.2 ± 0.5
7.8 ± 0.8
>18
9.1 ± 0.2
19.9 ± 1.8
4.2 ± 0.1
>18
2-Cl
4-F
2-Cl
4-CF3
3,4-diCl
3,5-diCl
2-F
2-Cl
2-Cl
>18
4-Cl
11.0 ± 0.4
9.2 ± 0.8
16.0 ± 7.5
13.4 ± 1.3
>18
4-Cl
3-F
4-Cl
4-F
4-Cl
4-CF3
3,4-diCl
3,5-diCl
2-F
4-Cl
4-Cl
>18
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
3,4,5-triOMe
3,4,5-triOMe
3,4,5-triOMe
3,4,5-triOMe
3,4,5-triOMe
3,4,5-triOMe
>18
3-F
>18
>18
4-F
7.4 ± 0.7
11.4 ± 0.8
9.4 ± 2.0
>18
12.0 ± 0.9
17.1 ± 0.7
>18
2.2. Chemistry
4-CF3
3,4-diCl
3,5-diCl
2-F
2-Hydroxymethylbenzimidazole was obtained by
Philips reaction of o-phenylenediamine with glycolic acid.
Acetylation of 2-hydroxymethylbenzimidazole gave com-
pound 1 (1H-benzo[d]imidazol-2-yl)methyl acetate) by anal-
ogy with reported procedures [42]. The structure of synthe-
>18
>18
>18
3-F
12.5 ± 2.1
>18
16.5 ± 0.8
>18
1
sized compounds was confirmed by means of H NMR, 13C
4-F
NMR, and high-resolution mass spectrometry (HRMS). The
synthesis of compound 2 – 37 is described below.
4-CF3
3,4-diCl
3,5-diCl
>18
13.2 ± 1.6
17.3 ± 7.5
>18
>18
General procedure for the synthesis of compounds
2 – 5. To an ice-cooled solution of NaH (60% in oil,
6.84 mmol, 1.2 eq) in 15 mL DMF, compound 1 (5.7 mmol,
1 eq) was added and stirred for 15 min. After addition of the
corresponding benzyl halide (5.98 mmol, 1.05 eq), the reac-
tion was stirred for 6 h at room temperature (RT). Upon com-
8.2 ± 0.8
a
IC values were calculated from the cell growth inhibition per-
50
centage obtained at four different concentrations; data are expressed
as mean ± SD.

1038
Aysun Özdemir et al.
Scheme 1. Synthesis of benzimidazole derivatives 14 – 37. Reagents and conditions: (a) benzyl halide derivatives, NaH, DMF; (b) LiOH,
MeOH:H O (1:1); (c) SOCl , CH Cl ; (d) piperazine. derivatives, Et N, DMF.
2
2
2
2
3
evaporated to dryness. The residue was triturated with hex-
ane : ethyl acetate, and the precipitate was filtered to give the
desired product.
mide with 1. Yield 74%; mp 93.0 – 95.0°C. 1H NMR
(400 MHz, CDCl ): d 1.94 (3H, s), 5.36 (2H, s), 5.42 (2H, s),
3
6.99 (2H, d, J = 8.8 Hz), 7.23 – 7.34 (5H, m), 7.84 (1H, m).
HRMS (m/z): [M+H]+ calcd for C H N O Cl: 315.0900;
General procedure for the synthesis of compounds
6 – 9. To a solution of compound 2 – 5 (4 mmol, 1 eq) in of
17 16
2
2
found: 315.0903.
methanol (25 mL) were added LiOH.H O (12 mmol, 3 eq)
2
(1-(2,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)me-
and water (7 mL) and the mixture was stirred at room tem-
perature for 3 h. After completion of the reaction, the reac-
tion mixture was diluted with water, acidified to pH 5 with
4N HCl, and the precipitate was filtered, washed with water,
and dried.
thyl acetate (4). Obtained by the reaction of 2,4-dichloro-
1
benzyl bromide with 1. Yield 83%; mp 116.1 – 118.0°C. H
NMR (400 MHz, CDCl ): d 1.90 (3H, s), 5.36 (2H, s), 5.48
3
(2H, s), 6.40 (1H, d, J = 8.0 Hz), 7.08 (1H, dd, J = 8.2,
2.0 Hz), 7.20 (1H, d, J = 8 Hz), 7.28 – 7.36 (2H, m), 7.48
(1H, d, J = 2.0 Hz), 7.86 (1H, d, J = 8 Hz). HRMS (m/z):
[M+H]+ calcd for C H Cl N O : 349.0511; found:
General procedure for the synthesis of alkyl chlorides
(10 – 13). To the solution of compound 6 – 9 (3 mmol, 1 eq)
in DCM (10 mL) was added SOCl (7.8 mmol, 2.6 eq) under
17 15
2
2
2
2
349.0497.
nitrogen atmosphere and the mixture was stirred at room
temperature for 2.5 h. After completion of the reaction, the
mixture was evaporated at reduced pressure and the residue
(1-(3,4,5-trimethoxybenzyl)-1H-benzo[d]imidazol-2-
yl)methyl acetate (5). Obtained by the reaction of
3,4,5-trimethoxybenzyl chloride with 1. Yield 86%; mp
was neutralized with NaHCO solution. The precipitate was
3
1
85.7 – 86.2°C. H NMR (400 MHz, CDCl ): d 1.98 (3H, s),
filtered under vacuum and dried.
3
General procedure for the synthesis of compounds
14 – 37 (Scheme 1). To the solution of compound 10 – 13
(0.5 mmol, 1 eq) in DMF (1.6 mL) were added appropriate
phenyl piperazine derivative (0.55 mmol, 1.1 eq) and
triethylamine (1.1 mmol, 2.2 eq) and the resulting mixture
was heated by microwave irradiation at 100°C for 15 min-
utes. The reaction mixture was cooled to RT, poured into wa-
ter, and the precipitate was filtered under vacuum. The crude
product was purified by flash chromatography using
DCM : MeOH (90 : 10) gradient.
3.72 (6H, s), 3.81 (3H, s), 5.36 (2H, s), 5.37 (2H, s), 6.26
(2H, s), 7.29 – 7.32 (3H, m), 7.82 – 7.84 (1H, m). HRMS
(m/z): [M+H]+ calcd for C H N O : 371.1607; found:
20 23
2
5
371.1605.
(1-(2-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)metha-
nol (6). Obtained by the hydrolysis reaction of 2. Yield 98%;
mp 143.1 – 143.4°C. HRMS (m/z): [M+H]+ calcd for
C H ClN O: 273.0795; found: 273.0799. CAS:
15 14
2
341021-39-4.
(1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)metha-
(1-(2-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl
acetate (2). Obtained by the reaction of 2-chlorobenzyl chlo-
ride with 1. Yield 87%; mp 109.1 – 110.5°C. 1H NMR
nol (7). Obtained by the hydrolysis reaction of 3. Yield 99%;
mp 128.1 – 130.0°C. HRMS (m/z): [M+H]+ calcd for
C H ClN O: 273.0795; found: 273. 0802. CAS:
(400 MHz, CDCl ): d 1.86 (3H, s), 5.36 (2H, s), 5.53 (2H, s),
15 14
2
3
7187-31-7.
6.47 (1H, d, J = 8.0 Hz), 7.10 (1H, t, J = 7.6 Hz), 7.23 – 7.35
(4H, m), 7.46 (1H, d, J = 8.0 Hz), 7.86 (1H, d, J = 7.6 Hz).
HRMS (m/z ): [M+H]+ calcd for C H N O Cl: 315.0900;
1-(2,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)me-
thanol (8). Obtained by the hydrolysis reaction of 4. Yield
83%; mp 144.4 – 145.8°C. HRMS (m/z): [M+H]+ calcd for
C H Cl N O: 307.0405; found: 307.0414. CAS:
17 16
2
2
found: 315.0894.
(1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl
acetate (3). Obtained by the reaction of 4-chlorolbenzyl bro-
15 13
2
2
500149-05-3.

Evaluation of Cytotoxic Activity
1039
(1-(3,4,5-trimethoxybenzyl)-1H-benzo[d]imidazol-2-
J
= 241.7 Hz). HRMS (m/z): [M+H]+ calcd for
C-F
yl)methanol (9). Obtained by the hydrolysis reaction of 5.
Yield 74%; 167.5 – 168.0°C. 1H NMR (400 MHz, CDCl ): d
C H ClFN : 435.1752; found: 435.1753.
25 25 4
1-(2-chlorobenzyl)-2-((4-(4-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (16). Prepared by the re-
action of 10 with 1-(4-fluorophenyl)piperazine. Yield 76%;
3
3.71 (6H, s), 3.80 (3H, s), 4.90 (2H, s), 5.40 (2H, s), 6.37
(2H, s), 7.24 – 7.28 (3H, m), 7.71 – 7.73 (1H, m). HRMS
(m/z): [M+H]+ calcd for C H N O : 329.1501; found:
1
mp 159.6 – 160.2°C. H NMR (400 MHz, CDCl ): d 2.62
3
18 21
2
4
(4H, bt), 2.87 (4H, bt), 3.82 (2H, s), 5.67 (2H, s), 6.53 (1H, d,
J = 8.0 Hz), 6.77 – 6.80 (2H, m), 6.93 (2H, t, J = 8.6 Hz),
7.07 (1H, t, J = 7.6 Hz), 7.20 (1H, t, J = 7.6 Hz), 7.27 – 7.33
(3H, m), 7.43 (1H, d, J = 8.0 Hz), 7.82 (1H, d, J = 7.6 Hz).
329.1491.
1-(2-chlorobenzyl)-2-(chloromethyl)-1H-benzo[d]imi-
dazole (10). Prepared by the reaction of 6 with SOCl . Yield
83%; mp 230°C (decomp.) HRMS (m/z): [M+H]+ calcd for
2
13C NMR (100 MHz, CDCl ): d 45.03, 49.90, 53.18 , 55.77,
C H Cl N :
291.0456;
found:
291.0446.
CAS:
15 13
2
2
3
1038339-61-5.
109.62, 115.47 (d, J = 22.1 Hz), 117.89 (d, J = 7.7 Hz),
C-F
C-F
1-(4-chlorobenzyl)-2-(chloromethyl)-1H-benzo[d]imi-
119.95, 122.46, 123.28, 126.86, 127.20, 128.72, 129.61,
dazole (11). Prepared by the reaction of 7 with SOCl . Yield
95%; mp 93.0 – 95.0°C. HRMS (m/z): [M+H]+ calcd for
132.2, 134.34, 136.11, 142.16, 147.81 (d, J = 2.3 Hz),
2
C-F
151.13, 157.20 (d, J = 237.7 Hz). HRMS (m/z): [M+H]+
C-F
C H Cl N : 291.0456; found: 291.0455. CAS: 7189-18-6.
calcd for C H ClFN : 435.1752; found: 435.1713.
15 13
2
2
25 25
4
1-(2,4-dichlorobenzyl)-2-(chloromethyl)-1H-benzo[d]-
1-(2-chlorobenzyl)-2-((4-(4-(trifluoromethyl)phenyl)p
iperazin-1-yl)methyl)-1H-benzo[d]imidazole (17). Pre-
pared by the reaction of 10 with 1-(4-(trifluoromethyl)-
phenyl)piperazine. Yield 47%; mp 178.9 – 180.0°C. 1H
imidazole (12). Prepared by the reaction of 8 with SOCl .
2
Yield 99%; mp 126.3 – 127.5°C. HRMS (m/z): [M+H]+ calcd
for C H Cl N : 325.0066; found: 325.0056. CAS:
15 12
3
2
7197-04-8.
NMR (400 MHz, CDCl ): d 2.61 (4H, t, J = 5.0 Hz), 3.02
3
1-(3,4,5-trimethoxybenzyl)-2-(chloromethyl)-1H-ben-
(4H, t, J = 5.0 Hz), 3.83 (2H, s), 5.67 (2H, s), 6.53 (1H, d,
J = 7.2 Hz), 6.82 (2H, d, J = 8.8 Hz), 7.06 (1H, td, J = 7.4,
1.1 Hz), 7.19 (1H, td, J = 7.8, 1.3 Hz), 7.26 – 7.34 (3H, m),
7.41 – 7.45 (3H, m), 7.82 (1H, d, J = 7.0 Hz). 13C NMR
zo[d]imidazole (13). Prepared by the reaction of 9 with
SOCl . Yield 61%; mp 118.3 – 120.1°C. 1H NMR
2
(400 MHz, CDCl ): d 3.74 (6H, s), 3.82 (3H, s), 4.85 (2H, s),
3
5.45 (2H, s), 6.34 (2H, s), 7.30 – 7.36 (3H, m), 7.80 – 7.84
(1H, m). HRMS (m/z): [M+H]+ calcd for C H ClN O :
(100 MHz, CDCl ): d 45.03, 47.69, 52.85, 55.74, 109.62,
3
114.56, 119.94, 120.57 (q, J = 32.7 Hz), 122.53, 124.69
18 20
2
3
C-F
347.1162; found: 347.1159.
(q, J = 267.6 Hz, CF ), 126.33 (q, J = 3.8 Hz), 126.79,
C-F
3
C-F
1-(2-chlorobenzyl)-2-((4-(2-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (14). Prepared by the re-
action of 10 with 1-(2-fluorophenyl)piperazine. Yield 79%;
127.19, 128.77, 129.62, 132.16, 134.29, 136.09, 142.06,
150.94, 153.10. HRMS (m/z): [M+H]+ calcd for
C H ClF N : 485.1720; found: 485.1723.
26 25
3
4
1
mp 168.7 – 169.5°C. H NMR (400 MHz, CDCl ): d 2.63
1-(2-chlorobenzyl)-2-((4-(3,4-dichlorophenyl)pipera-
zin-1-yl)methyl)-1H-benzo[d]imidazole (18). Prepared by
the reaction of 10 with 1-(3,4-dichlorophenyl)piperazine.
Yield 82%; mp 161.2 – 162.8°C. 1H NMR (400 MHz,
3
(4H, t, J = 4.8 Hz), 2.84 (4H, bt), 3.83 (2H, s), 5.67 (2H, s),
6.52 (1H, dd, J = 7.6, 1.2 Hz), 6.77 – 6.82 (1H, m),
6.83 – 6.93 (1H, m), 6.95 – 7.08 (3H, m), 7.18 – 7.26 (4H,
m), 7.43 (1H, dd, J = 8.0, 1.2 Hz), 7.80 – 7.82 (1H, m). 13C
NMR (100 MHz, CDCl ): d 45.02, 50.17 (d, J = 3.2 Hz),
CDCl ): d 2.59 (4H, t, J = 5.0 Hz), 2.90 (4H, t, J = 5.2 Hz),
3
3.82 (2H, s), 5.65 (2H, s), 6.51 (1H, dd, J = 7.6, 1.6 Hz), 6.64
(1H, dd, J = 8.8, 3.2 Hz), 6.85 (1H, d, J = 2.8 Hz), 7.06 (1H,
td, J = 8.0, 1.3 Hz), 7.18 – 7.33 (5H, m), 7.42 (1H, dd,
J = 7.8, 1.0 Hz), 7.81 – 7.83 (1H, m). 13C NMR (100 MHz,
3
C-F
53.23, 55.86, 109.54, 116.08 (d, J = 20.6 Hz), 118.87 (d,
C-F
J
= 2.6 Hz), 119.97 , 122.36, 122.46 (d, J = 8.4 Hz),
C-F
C-F
123.17, 124.35 (d, J = 3.9 Hz), 128.64, 129.57, 132.18,
C-F
134.45, 136.15, 139.96 (d, J = 8.3 Hz), 142.27, 151.31,
CDCl ): d 45.03, 48.39, 52.82, 55.69, 109.61, 115.38,
C-F
3
155.68 (d, J = 244.3 Hz). HRMS (m/z): [M+H]+ calcd for
117.24, 119.94, 122.24, 122.53, 123.36, 126.78, 127.21,
128.77, 129.62, 130.37, 132.16, 132.77, 134.29, 136.08,
142.03, 150.49, 150.91. HRMS (m/z): [M+H]+ calcd for
C H Cl N : 485.1067; found: 485.1067.
C-F
C H ClFN : 435.1752; found: 435.1754.
25 25
4
1-(2-chlorobenzyl)-2-((4-(3-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (15). Prepared by the re-
action of 10 with 1-(3-fluorophenyl)piperazine. Yield 64%;
25 24
3
4
1-(2-chlorobenzyl)-2-((4-(3,5-dichlorophenyl)piperazin-
1-yl)methyl)-1H-benzo[d]imidazole (19). Prepared by the
reaction of 10 with 1-(3,5-dichlorophenyl)piperazine. Yield
1
mp 135.7 – 136.8°C. H NMR (400 MHz, CDCl ): d 2.59
3
(4H, t, J = 5.0 Hz), 2.94 (4H, t, J = 5.0 Hz), 3.81 (2H, s),
5.66 (2H, s), 6.47 – 6.59 (4H, m), 7.04 – 7.32 (6H, m), 7.41
(1H, d, J = 8.0 Hz), 7.81 (1H, d, J = 8.0 Hz). 13C NMR
1
55%; mp 158.3 – 161.2°C. H NMR (400 MHz, DMSO): d
2.43 (4H, bt, J = 4.8 Hz), 2.86 (4H, bt, J = 4.8 Hz), 3.80 (2H,
s), 5.68 (2H, s), 6.50 (1H, dd J = 7.6, 1.2 Hz), 6.81 (3H, s),
7.15 – 7.27 (4H, m), 7.40 – 7.44 (1H, m), 7.50 (1H, dd,
(100 MHz, CDCl ): d 45.00, 48.35, 52.96, 55.82, 102.64 (d,
3
J
= 25.0 Hz), 105.86 (d, J = 21.1 Hz), 109.59, 111.17
C-F
C-F
J = 8.4, 1.2 Hz), 7.65 – 7.69 (1H, m). 13C NMR (DMSO-d ):
(d, J = 2.0 Hz), 119.97, 122.42, 123.25, 126.82, 127.18,
6
C-F
128.72, 129.61, 130.03 (d, J = 10.3 Hz), 132.16, 134.33,
d 44.66, 46.75, 52.12, 54.70, 110.18, 112.81, 116.92, 119.13,
C-F
136.13, 142.22, 151.09, 152.77 (d, J = 9.6 Hz), 163.78 (d,
121.75, 122.64, 126.98, 127.34, 128.68, 129.31, 131.33,
C-F

1040
Aysun Özdemir et al.
134.56, 135.04, 136.01, 141.83, 151.29, 152.36. HRMS
(m/z): [M+H]+ calcd for C H Cl N : 485.1067; found:
114.66, 119.98, 120.78 (q, J = 32.0 Hz), 122.44, 123.24,
C-F
124.69 (q, J = 269.6 Hz), 126.39 (q, J = 3.8 Hz),
25 24
3
4
C-F
C-F
485.1068.
127.77, 129.09, 133.68, 134.94, 135.90, 142.24, 150.61,
153.10. HRMS (m/z): [M+H]+ calcd for C H ClF N :
1-(4-chlorobenzyl)-2-((4-(2-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (20). Prepared by the re-
action of 11 with 1-(2-fluorophenyl)piperazine. Yield 63%;
26 25
3
4
485.1720; found: 485.1722.
1-(4-chlorobenzyl)-2-((4-(3,4-dichlorophenyl)piperazin-
1-yl)methyl)-1H-benzo[d]imidazole (24). Prepared by the
reaction of 11 with 1-(3,4-dichlorophenyl)piperazine. Yield
1
mp 191.6 – 192.1°C. H NMR (400 MHz, CDCl ): d 2.68
3
(4H, t, J = 4.8 Hz), 2.98 (4H, bt), 3.82 (2H, s), 5.56 (2H, s),
1
68%; mp 109.2 – 113.0°C. H NMR (400 MHz, CDCl ): d
6.85 – 6.94 (2H, m), 6.97 – 7.05 (4H, m), 7.22 – 7.30 (5H,
m), 7.78 – 7.80 (1H, m). 13C NMR (100 MHz, CDCl ): d
3
2.64 (4H, t, J = 5.0 Hz), 3.05 (4H, t, J = 5.0 Hz), 3.81 (2H,
s), 5.55 (2H, s), 6.69 (1H, dd, J = 8.8, 2.8 Hz), 6.90 (1H, d,
J = 2.8 Hz), 7.02 (2H, d, J = 8.0 Hz), 7.22 – 7.31 (6H, m),
3
46.80, 50.29 (d, J = 3.3 Hz, 53.30, 55.86, 109.62, 116.13
C-F
(d, J
20.5 Hz), 118.94 (d, J = 3.2 Hz), 119.97, 122.32,
C-F
C-F =
7.78 – 7.80 (1H, m). 13C NMR (100 MHz, CDCl ): d 46.80,
122.58 (d, J = 8.4 Hz), 123.10, 124.42 (d, J = 3.9 Hz),
3
C-F
C-F
48.56, 52.90, 55.72, 109.68, 115.46, 117.34, 119.95, 122.45,
123.25, 127.75, 129.07, 130.43, 132.78, 133.67, 134.91,
135.87, 142.15, 150.46, 150.55. HRMS (m/z): [M+H]+ calcd
for C H Cl N : 485.1067; found: 485.1045.
127.83, 129.03, 133.57, 135.11, 135.93, 139.45 (d,
= 8.4 Hz), 142.31, 150.91, 155.71 (d, J = 245.0 Hz).
J
C-F
C-F
HRMS (m/z): [M+H]+ calcd for C H ClFN : 435.1752;
25 25
4
found: 435.1753.
25 24
3
4
1-(4-chlorobenzyl)-2-((4-(3,5-dichlorophenyl)pipera-
zin-1-yl)methyl)-1H-benzo[d]imidazole (25). Prepared by
the reaction of 11 with 1-(3,5-dichlorophenyl)piperazine.
Yield 62%; mp 154.1 – 154.4°C. 1H NMR (400 MHz,
1-(4-chlorobenzyl)-2-((4-(3-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (21). Prepared by the re-
action of 11 with 1-(3-fluorophenyl)piperazine. Yield 66%;
1
mp 160.1 – 162.0°C. H NMR (400 MHz, CDCl ): d 2.64
3
CDCl ): d 2.63 (4H, t, J = 5.0 Hz), 3.07 (4H, t, J = 5.0 Hz),
(4H, t, J = 5.0 Hz), 3.09 (4H, t, J = 5.0 Hz), 3.80 (2H, s),
5.55 (2H, s), 6.50 – 6.56 (2H, m), 6.61 – 6.64 (1H, dd,
J = 8.2, 2.2 Hz), 7.03 (2H, d, J = 8.8 Hz), 7.14 – 7.30 (6H,
m), 7.79 (1H, dd, J = 7.0, 1.4 Hz). 13C NMR (100 MHz,
3
3.80 (2H, s), 5.54 (2H, s), 6.69 (2H, d, J = 2.0 Hz), 6.79 (1H,
t, J = 2.0 Hz), 7.01 (2H, d, J = 8.4 Hz), 7.21 – 7.31 (5H, m),
7.78 – 7.80 (1H, m). 13C NMR (100 MHz, CDCl ): d 46.80,
3
48.12, 52.84, 55.71, 109.70, 113.90, 119.08, 119.96, 122.47,
123.26, 127.73, 129.09, 133.70, 134.88, 135.43, 135.86,
142.14, 150.48, 152.41. HRMS (m/z): [M+H]+ calcd for
C H Cl N : 485.1067; found: 485.1069.
CDCl ):
d
46.79, 48.52, 53.05, 55.84, 102.74 (d,
3
J
= 25.0 Hz), 106.04 (d, J = 21.2 Hz), 109.67, 111.21
C-F
C-F
(d, J = 2.5 Hz), 119.96, 122.37, 123.16, 127.79, 129.05,
C-F
130.11 (d, J = 9.7 Hz), 133.64, 134.95, 135.90, 142.27,
25 24
3
4
C-F
1-(2,4-dichlorobenzyl)-2-((4-(2-fluorophenyl)pipera-
zin-1-yl)methyl)-1H-benzo[d]imidazole (26). Prepared by
the reaction of 12 with 1-(2-fluorophenyl)piperazine. Yield
150.72,
152.87
(d,
J
= 9.6 Hz),
163.80
(d,
C-F
J
= 241.7 Hz). HRMS (m/z): [M+H]+ calcd for
C-F
C H ClFN : 435.1752; found: 435.1753.
25 25
4
1
23%; mp 179.2 – 180.0°C. H NMR (400 MHz, CDCl ): d
1-(4-chlorobenzyl)-2-((4-(4-fluorophenyl)piperazin-1-
yl)methyl)-1H-benzo[d]imidazole (22). Prepared by the re-
action of 11 with 1-(4-fluorophenyl)piperazine. Yield 64%;
3
2.92 (4H, s), 3.06 (4H, s), 4.04 (2H, s), 5.73 (2H, s), 6.50
(1H, d, J = 8.4 Hz), 6.86 (1H, td, J = 8.8, 1.2 Hz),
6.92 – 7.11 (4H, m), 7.25 – 7.38 (3H, m), 7.48 (1H, d,
J = 2.4 Hz), 7.86 (1H, dd, J = 7.2, 1.6 Hz).13C NMR
1
mp 124.2 – 126.1°C. H NMR (400 MHz, CDCl ): d 2.65
3
(4H, bt, J = 4.0 Hz), 2.99 (4H, bt, J = 4.0 Hz), 3.80 (2H, s),
5.55 (2H, s), 6.80 – 6.83 (2H, m), 6.94 (2H, t, J = 8.6 Hz),
7.03 (2H, d, J = 8.4 Hz), 7.21 – 7.28 (5H, m), 7.78 (1H, d,
(100 MHz, CDCl ): d 45.21, 49.40, 53.09, 54.05, 110.01,
3
116.18 (d, J = 20.6 Hz), 119.11 (d, J = 2.2 Hz), 119.80,
C-F
C-F
J = 7.6 Hz). 13C NMR (100 MHz, CDCl ): d 46.78, 50.05,
123.06 (d, J = 7.6 Hz), 123.40, 124.20, 124.52 (d,
C-F
3
J
= 3.1Hz), 127.63, 127.95, 129.74, 132.37, 133.10,
53.24, 55.86, 109.64, 115.51 (d, J = 22.5 Hz), 117.92 (d,
C-F
C-F
134.39, 135.31, 139.30, 140.95, 150.20, 156.67 (d,
= 244.6 Hz). HRMS (m/z): [M+H]+ calcd for
J
= 7.7 Hz), 119.95, 122.33, 123.12, 127.82, 129.03,
C-F
J
133.59, 135.02, 135.93, 142.29, 147.76, 150.86, 157.25 (d,
= 237.9 Hz). HRMS (m/z): [M+H]+ calcd for
C-F
C H Cl FN : 469.1362; found: 469.1364.
J
25 24
2
4
C-F
1-(2,4-dichlorobenzyl)-2-((4-(3-fluorophenyl)pipera-
zin-1-yl)methyl)-1H-benzo[d]imidazole (27). Prepared by
the reaction of 12 with 1-(3-fluorophenyl)piperazine. Yield
C H ClFN : 435.1752; found: 435.1754.
25 25
4
1-(4-chlorobenzyl)-2-((4-(4-(trifluoromethyl)phenyl)p
iperazin-1-yl)methyl)-1H-benzo[d]imidazole (23). Pre-
pared by the reaction of 11 with 1-(4-(trifluoromethyl)phe-
1
56%; mp 127.0 – 127.5°C. H NMR (400 MHz, CDCl ): d
3
1
nyl)piperazine. Yield 68%; mp 147.2 – 148.4°C. H NMR
2.79 (4H, s), 3.10 (4H, s), 3.99 (2H, s), 5.71 (2H, s),
6.49 – 6.57 (3H, m), 6.60 – 6.63 (1H, m), 7.09 (1H, dd,
J = 8.4, 2.0 Hz), 7.15 – 7.21 (1H, m), 7.25 – 7.27 (1H, m),
7.32 – 7.40 (2H, m), 7.47 (1H, d, J = 2 Hz), 7.87 (1H, m).
(400 MHz, CDCl ): d 2.65 (4H, t, J = 5.0 Hz), 3.16 (4H, t,
3
J = 5.0 Hz), 3.81 (2H, s), 5.55 (2H, s), 6.87 (2H, d,
J = 8.6 Hz), 7.02 (2H, d, J = 9.6 Hz), 7.21 – 7.31 (5H, m),
7.46 (2H, d, J = 8.6 Hz), 7.78 – 7.80 (1H, m). 13C NMR
13C NMR (100 MHz, CDCl ): d 45.10, 48.14, 52.85, 54.37,
3
103.06 (d, J = 24.4 Hz), 106.48 (d, J = 20.6 Hz),
(100 MHz, CDCl ): d 46.82, 47.89, 52.96, 55.80, 109.70,
C-F
C-F
3

Evaluation of Cytotoxic Activity
1041
110.02, 111.46 (d, J = 3.0 Hz), 119.52, 123.60, 124.33,
(1H, t, J = 1.8 Hz), 7.10 (1H, dd, J = 8.4, 2.0 Hz), 7.28 (1H,
d, J = 7.2 Hz), 7.34 – 7.42 (2H, m), 7.48 (1H, d, J = 2.0 Hz),
C-F
127.66, 127.87, 129,74, 130.20 (d, J = 9.2 Hz), 132.11,
C-F
7.88 (1H, d, J = 7.2 Hz). 13C NMR (100 MHz, CDCl ): d
133.04, 134.48, 135.14, 140.09, 149.27, 152.34 (d,
3
J
= 9.9 Hz), 163.77 (d, J = 242.4 Hz). HRMS (m/z):
C-F
45.10, 47.83, 52.72, 54.33, 110.05, 114.12, 119.34, 119.42,
123.82, 124.51, 127.71, 127.80, 129.78, 131.98, 133.02,
134.58, 134.99, 135.49, 149.44, 152.18. HRMS (m/z):
[M+H]+ calcd for C H Cl N : 519.0677; found: 519.06792.
C-F
[M+H]+ calcd for C H Cl FN : 469.1362; found:
25 24
2
4
469.1349.
1-(2,4-dichlorobenzyl)-2-((4-(4-fluorophenyl)pipera-
zin-1-yl)methyl)-1H-benzo[d]imidazole (28). Prepared by
the reaction of 12 with 1-(4-fluorophenyl)piperazine. Yield
25 23
4
4
2-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-1-(3,4,5-
trimethoxybenzyl)-1H-benzo[d]imidazole (32). Prepared
by the reaction of 13 with 1-(2-fluorophenyl)piperazine.
Yield 51%; mp 136.7 – 137.2°C. 1H NMR (400 MHz,
CDCl3): d 2.70 (4H, bt), 3.04 (4H, bt), 3.70 (6H, s), 3.79
(3H, s), 3.84 (2H, s), 5.54 (2H, s), 6.27 (2H, s), 6.85 – 6.94
(2H, m), 6.97 – 7.05 (2H, m), 7.23 – 7.30 (3H, m),
1
37%; mp 158.3 – 159.2°C. H NMR (400 MHz, CDCl ): d
3
2.78 (4H, s), 2.99 (4H, s), 3.95 (2H, s), 5.68 (2H, s), 6.49
(1H, d, J = 8.4 Hz), 6.83 – 6.86 (2H, m), 6.93 – 6.97 (2H,
m), 7.08 (1H, dd, J = 8.4, 2.0 Hz), 7.24 – 7.27 (1H, m),
7.30 – 7.37 (2H, m), 7.47 (1H, d, J = 2.0 Hz), 7.85 (1H, d,
J = 7.6 Hz). 13C NMR (100 MHz, CDCl ): d 44.96, 49.85,
7.74 – 7.80 (1H, m). 13C NMR (100 MHz, CDCl ): d 47.44,
3
3
52.99, 54.75, 109.82, 115.63 (d, J = 21.3Hz), 118.37 (d,
C-F
50.35 (d, J = 3.2 Hz), 53.36, 55.85, 56.16, 60.86, 103.40,
C-F
J
= 6.8 Hz), 119.78, 123.22, 124.00, 127.61, 127.89,
C-F
109.73, 116.14 (d, J = 20.5 Hz), 118.90 (d, J = 2.6 Hz),
C-F
C-F
129.65, 132.50, 133.01, 134.29, 135.44, 141.01, 147.24,
149.75, 157.58 (d, J = 238.9 Hz). HRMS (m/z): [M+H]+
119.89, 122.21, 122.58 (d, J = 7.7 Hz), 123.00, 124.41 (d,
C-F
C-F
J
J
J
= 3.2 Hz), 132.22, 136.11, 137.51, 139.85 (d,
= 8.3 Hz), 142.28, 151.00, 153.68, 155.70 (d,
= 244.3 Hz). HRMS (m/z): [M+H]+ calcd for
C-F
C-F
C-F
calcd for C H Cl FN : 469.1362; found: 469.1360.
25 24
2
4
1-(2,4-dichlorobenzyl)-2-((4-(4-(trifluoromethyl)phe-
nyl)piperazin-1-yl)methyl)-1H-benzo[d]imidazole (29).
Prepared by the reaction of 12 with 1-(4-(trifluoromethyl)-
C H FN O : 491.2458; found: 491.2461.
28 32
4
3
2-((4-(3-fluorophenyl)piperazin-1-yl)methyl)-1-(3,4,5-
trimethoxybenzyl)-1H-benzo[d]imidazole (33). Prepared
by the reaction of 13 with 1-(3-fluorophenyl)piperazine.
Yield 56%; mp 123.3 – 124.1°C. 1H NMR (400 MHz,
1
phenyl)piperazine. Yield 37%; mp 97.5 – 98.9°C. H NMR
(400 MHz, CDCl ): d 2.77 (4H, bt), 3.16 (4H, bt), 4.00 (2H,
3
s), 5.71 (2H, s), 6.50 (1H, d, J = 8.8 Hz), 6.87 (2H, d,
J = 8.8 Hz), 7.09 (1H, dd, J = 8.0, 2.0 Hz), 7.26 – 7.28 (1H,
m), 7.33 – 7.40 (2H, m), 7.46 – 7.48 (3H, m), 7.88 (1H, d,
CDCl ): d 2.83 (4H, s), 3.23 (4H, s), 3.71 (6H, s), 3.80 (3H,
3
s), 3.98 (2H, s), 5.62 (2H, s), 6.27 (2H, s), 6.53 – 6.57 (2H,
J = 7.6 Hz). 13C NMR (100 MHz, CDCl ): d 45.09, 47.56,
3
m), 6.64 (1H, d, J = 7.2 Hz), 7.21 – 7.15 (1H, m), 7.31 – 7.37
(3H, m), 7.83 – 7.85 (1H, m). 13C NMR (100 MHz, CDCl ):
52.81, 54.43, 110.02, 114.89, 119.43, 121.12 (q,
3
J
= 32.8 Hz), 123.71, 124.41, 124.61 (q, J = 269 Hz),
C-F
C-F
d 47.71, 48.16, 52.92, 54.51, 56.22, 60.88, 103.00 (d,
126.43 (q, J = 3.8 Hz), 127.68, 127.85, 127.95, 129.97,
C-F
J
= 25.2 Hz), 103.42, 106.47 (d, J = 21.4 Hz), 110.19,
C-F
C-F
132.08, 133.01, 134.51, 135.08, 149.45, 152.84. HRMS
(m/z): [M+H]+ calcd for C H Cl F N : 519.1330; found:
111.41 (d, J = 2.3 Hz), 119.49, 123.14, 123.91, 130.22 (d,
C-F
26 24
2
3
4
J
= 9.9 Hz), 131.49, 135.54, 137.77, 140.64, 152.36 (d,
519.1321.
C-F
J
= 9.9 Hz), 153.82, 163.78 (d, J = 242.3 Hz). HRMS
1-(2,4-dichlorobenzyl)-2-((4-(3,4-dichlorophenyl)pipe-
razin-1-yl)methyl)-1H-benzo[d]imidazole (30). Prepared
by the reaction of 12 with 1-(3,4-dichlorophenyl)piperazine.
Yield 59%; mp 134.1 – 135.7°C. 1H NMR (400 MHz,
C-F
C-F
(m/z): [M+H]+ calcd for C H FN O : 491.2458; found:
28 32
4
3
491.2442.
2-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-1-(3,4,5-
trimethoxybenzyl)-1H-benzo[d]imidazole (34). Prepared
by the reaction of 13 with 1-(4-fluorophenyl)piperazine.
CDCl ): d 2.74 (4H, s), 3.02 (4H, s), 3.98 (2H, s), 5.69 (2H,
3
s), 6.49 (1H, d, J = 8.4 Hz), 6.68 (1H, dd, J = 8.4, 2.8 Hz),
6.89 (1H, d, J = 2.8 Hz), 7.09 (1H, dd, J = 8.4, 1.6 Hz),
7.25 – 7.28 (2H, m), 7.32 – 7.40 (2H, m), 7.47 (1H, d,
Yield 36%; 113.8 – 114.9°C. 1H NMR (400 MHz, CDCl ): d
3
2.94 (4H, bt), 3.20 (4H, bt), 3.70 (6H, s), 3.79 (3H, s), 4.04
(2H, s), 5.65 (2H, s), 6.27 (2H, s), 6.87 – 6.97 (4H, m),
7.34 – 7.36 (3H, m), 7.83 – 7.85 (1H, m). 13C NMR
J = 1.6 Hz), 7.87 (1H, m). 13C NMR (100 MHz, CDCl ): d
3
45.06, 48.27, 52.78, 54.46, 109.99, 115.67, 117.58, 119.46,
122.80, 123.66, 124.37, 127.68, 127.82. 129.73, 130.48,
132.13, 132.84, 133.01, 134.49, 135.12, 139.92, 149.53,
150.21. HRMS (m/z): [M+H]+ calcd for C H Cl N :
(DMSO-d ): d 46.92, 48.70, 52.60, 54.52, 55.83, 59.84,
6
104.27, 110.48, 115.11 (d, J = 21.8 Hz), 116.98 (d,
C-F
J
= 7.7 Hz), 118.91, 121.48, 122.26, 132.94, 135.73,
C-F
25 23
4
4
519.0677; found: 519.0676.
136.71, 141.87, 147.74 (d, J = 1.9 Hz), 151.06, 152.91,
C-F
155.89 (d, J = 234.0 Hz). HRMS (m/z): [M+H]+ calcd for
1-(2,4-dichlorobenzyl)-2-((4-(3,5-dichlorophenyl)pipe-
razin-1-yl)methyl)-1H-benzo[d]imidazole (31). Prepared
by the reaction of 12 with 1-(3,5-dichlorophenyl)piperazine.
Yield 55%; mp 157.8 – 159.4°C. 1H NMR (400 MHz,
C-F
C H FN O : 491.2458; found: 491.2460.
28 32
4
3
2-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)meth-
yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzo[d]imidazole (35).
Prepared by the reaction of 13 with 1-(4-(trifluorome-
thyl)phenyl)piperazine. Yield 40%; mp 161.3 – 162.5°C. 1H
CDCl ): d 2.74 (4H, s), 3.05 (4H, s), 4.00 (2H, s), 5.70 (2H,
3
s), 6.49 (1H, d, J = 8.4 Hz), 6.68 (2H, d, J = 1.8 Hz), 6.81

1042
Aysun Özdemir et al.
compound were observed in a phase contrast microscope,
and the images were captured with Leica DMIL and recorded
with Leica (DFC 420C) digital camera. Immunofluorescence
microscopy has also been used to evaluate the morphological
changes that may occur in the nuclei of cells. After the treat-
ment with compounds, the cells were washed 2 times with
500 mL cold phosphate buffered saline (PBS). Cells were
fixed with 2% paraformaldehyde for 15 min and then
permeabilized with 0.1% Triton X-100 for 5 min. After
washing the cells three times with PBS, they were incubated
for 5 min with 4¢,6-diamidino-2-phenylindole (DAPI). Cells
were examined in Leica DMIL inverted fluorescence micro-
scope. Fragmented nuclei were counted randomly and the
number of fragmented nuclei was expressed as percentage of
the total number of nuclei.
NMR (400 MHz, CDCl ): d 2.68 (4H, t, J = 5.2 Hz), 3.21
3
(4H, t, J = 5.0 Hz), 3.70 (6H, s), 3.79 (3H, s), 3.84 (2H, s),
5.54 (2H, s), 6.26 (2H, s), 6.88 (2H, d, J = 8.8 Hz),
7.26 – 7.31 (3H, m), 7.46 (2H, d, J = 8.8 Hz), 7.79 – 7.81
(1H, m). 13C NMR (100 MHz, CDCl ): d 47.43, 47.89,
3
52.96, 55.70, 56.18, 60.86, 103.37, 109.79, 114.63, 119.87,
120.78 (q, J = 32.7 Hz), 122.37, 123.18, 124.66 (q,
C-F
J
= 269.1 Hz), 126.36 (q, J = 3.6 Hz), 132.05, 136.04,
C-F
C-F
137.61, 142.09, 150.63, 153.07, 153.73. HRMS (m/z):
[M+H]+ calcd for C H F N O : 541.2427; found:
29 32
3
4
3
541.2433.
2-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-1-
(3,4,5-trimethoxybenzyl)-1H-benzo[d]imidazole (36). Pre-
pared by the reaction of 13 with 1-(3,4-dichlorophenyl)pipe-
razine. Yield 50%; mp 145.5 – 147.1°C. 1H NMR (400 MHz,
CDCl ): d 2.66 (4H, bt, J = 4.8 Hz), 3.09 (4H, bt,
Western blot. After the administration of compounds,
MCF-7 cells were homogenized in a homogenization buffer
(50 mM Tris-HCl pH 7.5, 400 mM NaCl, 2 mM EGTA,
1 mM EDTA, 1 mM DTT and protease inhibitors). Then, the
protein content of the samples was determined using the
Bradford method. Aliquots (25 mg) of protein samples were
loaded on 10% SDS PAGE gels. After gel electrophoresis,
the proteins were transferred to the nitrocellulose membrane
(350 mA, 1 hour). Then membranes were blocked with 5%
milk powder for 1 hour at room temperature. After blocking,
membranes were blotted overnight with PARP-1 (Santa
Cruz) and caspase 7 (Becton Dickinson) primer antibody.
Membranes were incubated with corresponding HRP-conju-
gated secondary antibodies for 2 hours after washing three
times with TBS-T (Tris-buffered saline, 0.1% Tween 20).
Membranes were then treated with chemiluminescence-spe-
cific solutes (ECL) and visualized. GAPDH was used as
loading control.
3
J = 4.8 Hz), 3.71 (6H, s), 3.79 (3H, s), 3.82 (2H, s), 5.53 (2H,
s), 6.26 (2H, s), 6.70 (1H, dd, J = 9.2, 3.2 Hz), 6.91 (1H, d,
J = 3.2 Hz), 7.24 – 7.30 (4H, m), 7.78 – 7.81 (1H, m). 13C
NMR (100 MHz, CDCl ): d 47.39, 48.62, 52.95, 55.80,
3
56.19, 60.89, 103.34, 109.75, 115.43, 117.29, 119.92,
122.30, 122.41, 123.11, 130.44, 132.12, 132.78, 136.11,
137.56, 142.26, 150.48, 150.77, 153.72. HRMS (m/z):
[M+H]+ calcd for C H Cl N O : 541.1773; found:
28 31
2
4
3
541.1776.
2-((4-(3,5-dichlorophenyl)piperazin-1-yl)methyl)-1-
(3,4,5-trimethoxybenzyl)-1H-benzo[d]imidazole (37). Pre-
pared by the reaction of 13 with 1-(3,5-dichlorophenyl)pipe-
razine. Yield 77%; mp 158.9 – 159.2°C. 1H NMR (400 MHz,
CDCl ): d 2.65 (4H, t, J = 5.0 Hz), 3.12 (4H, t, J = 5.0 Hz),
3
3.70 (6H, s), 3.79 (3H, s), 3.82 (2H, s), 5.52 (2H, s), 6.25
(2H, s), 6.70 (2H, d, J = 1.8 Hz), 6.79 (1H, t, J = 1.8 Hz),
7.25 – 7.31 (3H, m), 7.78 – 7.80 (1H, m). 13C NMR
(100 MHz, CDCl ): d 47.41, 48.15, 52.87, 55.68, 56.18,
Live cell Imaging. Cell images were captured using
JULI Cell Analyzer with a 4x magnification objective.
100.000 MCF-7 cells were seeded in 35 mm petri dishes
(Ibidi). On the next day, the cells were treated with 18 mM
compound 22. The images of the cells were taken every
15 min and the morphological changes were monitored for
48 h.
3
60.88, 103.33, 109.78, 113.88, 119.08, 119.89, 120.37,
123.19, 132.04, 135.43, 136.05, 137.59, 142.10, 150.59,
152.42, 153.73. HRMS (m/z): [M+H]+ calcd for
C H Cl N O : 541.1773; found: 541.1776.
28 31
2
4
3
2.3. Biology
2.4. Statistical Analysis
Cell Culture. MCF-7 and A549 cells were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% FBS (fetal bovine serum), 100 U/mL penicillin/strepto-
Statistical significance (P < 0.05) was assessed using one
way analysis of variance (ANOVA) followed by Tukey’s
tests when analyzing multiple groups. All values were ex-
pressed as the mean SEM.
mycin in an incubator with 5% CO at 37°C. When the cells
2
reached 60 – 70% confluence, the newly synthesized benz-
imidazole derivatives were applied to cells at increasing log-
arithmic concentrations for 48 hours. Benzimidazole deriva-
tives were dissolved in DMSO (0.2%), and DMSO alone was
administered to the cells as a control group.
3. RESULTS AND DISCUSSION
3.1. Chemistry
Cytotoxic Activity. The cytotoxic activity of the test
compounds was determined using MTT assay as described
previously [25].
Compounds 14 -– 37 were prepared following the reac-
tion sequence illustrated in Scheme 1 using the known gen-
eral procedures. Hence, 2-acetyloxymethylbenzimidazole
(1), which was obtained by acetylation of 2-hydroxymethyl-
Phase contrast and immunofluorescence microscopy.
Changes in cell morphology after 48 h administration of test

Evaluation of Cytotoxic Activity
1043
benzimidazole according to well-known methods[26], has
been treated with substituted benzyl halides in the presence
of a base to obtain N-benzylated derivatives 2 – 5. Hydroly-
sis of these intermediates (2 – 5) in aqueous alkali provided
N-benzyl-2-hydroxymethylbenzmidazoles (6 – 9), which
were further converted to alkyl chlorides 10 – 13 by reaction
with thionyl chloride. Finally, these intermediate alkyl chlo-
rides were treated with appropriate phenylpiperazine deriva-
tives to achieve target compounds 14 – 37. All compounds
were purified by automated flash chromatography and chec-
ked for purity by TLC and UPLC before being tested in bio-
logical assays (According to the peak area percentage of
UPLC analysis, the purity was >97%). The structures of syn-
1
thesized compounds were confirmed by means of H NMR,
13C NMR and high-resolution mass spectrometry (HRMS).
3.2. Biology
Cytotoxic activity. MTT assay was performed to evalu-
ate the cytotoxic effects of the synthesized benzimidazole-
piperazine hybrids (14 – 37) against selected human cancer
cell lines, namely human lung (A549) and breast (MCF-7)
cancer cell lines. The IC values, defined as the half maxi-
50
mal inhibitory concentration, are summarized in Table 1. The
results revealed that all the synthesized benzimidazole-pipe-
razine hybrids exhibited good to moderate antiproliferative
activity with IC values ranging from 2.8 to >18 mM.
50
Among the tested hybrids, compounds 17 – 5, 28 and 30
showed noteworthy cytotoxicity with IC values ranging
50
from 2.8 to 9.4 mM (Table 1).
The study of structure – activity relationships revealed
that most of compounds (18 -– 25, 28 – 30, 33, 35) in the test
series showed clear preference for A549 cancer cells but
were less efficient against MCF-7 cancer cells (Table 1). It is
noteworthy to observe that mono chloro substitution pattern
on the N-benzyl ring (either at 2- or 4-position, compounds
17 – 25) have significant impact on the activity against A549
Fig. 1. Phase contrast images of cancer cells after the incubation
with 18 mM of compounds 16, 17 and 22 for 48 h: (A) MCF-7 cells;
(B) A549 cells. Blubbing cells are indicated by arrows. Photographs
were taken at ´200 magnification; scale bar corresponds to 100 mm.
cells (IC = 2.8 – 7.8 mM, Table 1). Furthermore, the po-
50
tency gradually declined with bulky substituents on N-benzyl
ring such as 2,4-diCl (26 – 31) and 3,4,5-triOMe (32 – 37)
(IC = 7.4 – >18 mM, depending on the cell line).
50
Within the series, 4-Cl-benzyl analogs having differently
substituted phenyl attachment at N4 of the piperazine nu-
significantly decreased by 2-F (26), 3-F (27), 4-CF (29) and
3
3,5-diCl (31) phenyl ring substitutions with IC values be-
cleus (2-F, 3-F, 4-F, 4-CF , 3,4-diCl, 3,5-diCl in 20 – 25)
50
3
tween 11.4 and 18 mM, depending on the cell line. Intro-
showed the most potent cytotoxic activity for A549 cells
ducing bulkiness to N-benzyl (3,4,5-triOCH , 32–37) in the
(IC = 2.8 – 7.8 mM), while the activity against MCF-7 cells
3
50
same piperazine-phenyl series clearly is not tolerated well
for the cytotoxic potency for both A549 and MCF-7 cells
(Table 1). Thus, among this series of compounds, a chlorine
is preferred either at ortho or para position of the N-benzyl
ring along with para fluorine or trifluoromethyl in the
piperazine-phenyl part (i.e., 16, 17, 22 and 23), affording po-
tent anti-proliferative activity in tested tumor cells in the low
micromolar range. Based on the overall results acquired from
cytotoxicity screening, selected compounds with noteworthy
antiproliferative activity against A549 cells (16, 17 and 22
was less pronounced (IC = 9.2 – >18 mM for MCF-7). For
50
2-Cl-benzyl congeners with the same piperazine-phenyl sub-
stitutions (14 – 19), the potency was moderate for both cell
lines, however, para trifluoromethyl substitution on the
piperazine-phenyl ring improved the cytotoxic activity, and
compound 17 appeared to be equally efficient in both cancer
cells (IC = 5.4 mM for A549 and 4.2 mM for MCF-7). Next,
50
2,4-dichloro-N-benzyl congeners (26 – 31) were evaluated
for their anti-proliferative activity. As compared to the mono
chloro substituted-N-benzyl analogs, the cytotoxic potency

1044
Aysun Özdemir et al.
Fig. 2. Nuclear fragmentation of MCF-7 cells after the incubation with 18 mM of compounds 16, 17 and 22 for 48 h: (A) fluorescence micros-
copy images of MCF-7 cells; (B) histograms of percentage nuclear fragmentation in MCF-7 cells (* significant difference compared to control,
P < 0.05, n = 3).
with IC values 8.0, 5.4 and 2.8, respectively) were taken up
compounds, the number of viable cells was reduced in com-
parison to the vehicle control along with the distinctive mor-
phological features such as cells detachment from
substratum, cell shrinkage and bleb formation as a morpho-
logical marker of apoptotic cell death.
50
for detailed biological studies.
Morphological changes. To investigate if the selected
compounds cause morphological changes in the tested cell
lines leading to the loss of cell viability and induce apoptosis,
A549 and MCF-7 cells were treated with 16, 17 and 22 at
It is also well known that cells that are subject to
apoptosis demonstrate nuclear condensation and nuclear
fragmentation, which can be detected by staining with DAPI
and fluorescence microscopy. Therefore, MCF-7 and A549
18 mM for 48 h. After treatment, cells were monitored for
morphological changes with phase contrast microscope. As
can be seen from Fig. 1, following the treatment with these
Fig. 3. Nuclear fragmentation of A549 cells after the incubation with 18 mM of compounds 16, 17 and 22 for 48 h: (A) fluorescence micros-
copy images of A549 cells; (B) histograms of percentage nuclear fragmentation in A549 cells (* significant difference compared to control,
P < 0.05, n = 3).

Evaluation of Cytotoxic Activity
1045
Fig. 4. Benzimidazole-piperazine hybrids induce PARP-1 cleavage and caspase 7 activation in cancer cells: (A) representative immunoblot for
PARP-1 cleavage in MCF-7 cells after the incubation with 18 mm of compounds 16, 17, 22 at for 48 h; (B) representative immunoblot for
caspase 7 activation in MCF-7 cells after the incubation with 18 mm of compounds 16, 17, 22 for 48 h; (C) densitometric analysis of blots for
cleaved PARP-1; (D) densitometric analysis of blots for caspase 7 (* significant difference from control, P < 0.05; data present mean ± SEM
from 3 determinations; Cont = control cells; 16, 17, 22 refer to treatment for 48 h with 18 mM of test compound).
cells were treated with 16, 17 and 22 at increasing concentra-
tions for 48 hours and stained with DAPI. Nuclear fragmen-
tation was observed in both cancer cell lines after the treat-
ment of all selected compounds (Figs. 2A/3A). Cells that un-
derwent nuclear fragmentation were counted and calculated
as a percentage of the total cell number. As can be inferred
from Fig. 2B/3B, these benzimidazole-piperazine hybrids
significantly increased the number of cells with nuclear frag-
mentation compared to the control group in MCF-7 and
A549 cells in a concentration-dependent manner. Nuclear
fragmentation is a nuclear morphological change that is usu-
ally observed in apoptotic cell death, indicating that the
newly synthesized benzimidazole-piperazine hybrids might
induce apoptosis.
22 at 18 mM (Fig. 4B, D) suggesting that treatment of newly
synthesized compounds resulted in caspase 7 activation.
In concluding, our results suggest that it is possible to de-
velop better cytotoxic agents through introduction of proper
substituents on piperazine and N-benzyl ring while the
benzimidazole nucleus is open for further structural optimi-
zation. Meanwhile, the comparable positions of substituents
on the piperazine-phenyl ring and N-benzyl ring played a
key role in adjusting cytotoxicity of the derivatives. As a re-
sult, compound 17 might become a promising new lead com-
pound for further development of potential anticancer agents.
An ongoing comprehensive structural optimization and phar-
macological study, beyond the scope of this work, may pro-
vide additional evidence for these benzimidazole-piperazine
hybrids acting as potential anticancer agents in the future.
Effects of compounds 16, 17 and 22 on cell apoptosis.
PARP-1 cleavage in MCF-7 cells was also examined to con-
firm whether caspase activation was stimulated in com-
pound-induced apoptotic cell death. Our results showed that
while cleaved PARP-1 was not detectable at lower inhibitor
ACKNOWLEDGMENTS
This work was supported by the Scientific and Techno-
logical Research Council of Turkey (TUBITAK Grant No:
115S016).
concentrations (1 – 10 mM), all selected compounds led to
apoptotic cell death at 18 mM as demonstrated by the pres-
ence of cleaved PARP protein (Fig. 4A, C). It is known
MCF-7 cells are deficient in caspase-3 (27). Since it is
known caspase 7 and caspase 3 share same substrates and
caspase 7 binds to PARP-1 and improves its cleavage (28),
we determined the caspase 7 activation after the incubation
of benzimidazole-piperazine hybrids in MCF-7 cells. Our re-
sults revealed that there is a certain decrease in procaspase 7
levels after the treatment of cells with compound 16, 17 and
CONFLICT OF INTEREST
The authors state no conflict of interest.
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