Synthesis and evaluation of anticancer properties of novel benzimidazole ligand and their cobalt(II) and zinc(II) complexes against cancer cell lines A-2780 and DU-145

Article abstract of DOI:10.1016/j.ica.2019.118977

Eighteen new cobalt(II) or zinc(II) complexes of benzimidazole bearing 1-benzyl and 2-phenyl moieties were synthesized from the reaction of appropriate benzimidazole ligands and CoCl₂ or ZnCl₂. Their structural characterizations were

Full text of DOI:10.1016/j.ica.2019.118977

Accepted Manuscript
Research paper
Synthesis and evaluation of anticancer properties of novel benzimidazole ligand
and their cobalt(II) and zinc(II) complexes against cancer cell lines A-2780 and
DU-145
Ülkü Yılmaz, Suat Tekin, Nesrin Buğday, Kemal Yavuz, Hasan Kü ç ükbay,
Süleyman Sandal
PII:
S0020-1693(19)30765-0
https://doi.org/10.1016/j.ica.2019.118977
118977
DOI:
Article Number:
Reference:
ICA 118977
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 May 2019
21 June 2019
23 June 2019
Please cite this article as: U. Yılmaz, S. Tekin, N. Buğday, K. Yavuz, H. Kü ç ükbay, S. Sandal, Synthesis and
evaluation of anticancer properties of novel benzimidazole ligand and their cobalt(II) and zinc(II) complexes against
cancer cell lines A-2780 and DU-145, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.
2019.118977
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Synthesis and evaluation of anticancer properties of novel benzimidazole ligand and their
cobalt(II) and zinc(II) complexes against cancer cell lines A-2780 and DU-145
Ülkü Yılmaz1,2, Suat Tekin3, Nesrin Buğday1, Kemal Yavuz1, Hasan Küçükbay1*, Süleyman
Sandal2
1İnonu University, Art and Science Faculty, Department of Chemistry, Malatya, TURKEY
2Malatya Turgut Özal University, Battalgazi Vocational School, Battalgazi, Malatya,
TURKEY
2Department of Physiology, Faculty of Medicine, İnönü University, Malatya, Turkey
Abstract
Eighteen new cobalt(II) or zinc(II) complexes of benzimidazole bearing 1-benzyl and 2-phenyl
moieties were synthesized from the reaction of appropriate benzimidazole ligands and CoCl2
or ZnCl2. Their structural characterizations were done by IR, NMR (1H, 13C) and UV-VIS
spectrometers. Cytotoxic activities of eighteen new complexes and three benzimidazole ligands
were determined using A-2780 (human ovarian) and DU-145 (human prostate) cell lines.
Antitumor properties of all compounds were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cell viability assay for the tested benzimidazole
derivatives was performed and the LogIC50 values of the compounds were calculated after a
24-hour treatment. All tested benzimidazole derivatives showed higher or comparable
antitumor activity against A-2780 cell lines compared to the standard drug docetaxel with a
LogIC50 value of -0.81 µM (p<0.05). Eight of the examined compounds (1, 3, 5, 6, 7, 9, 10 and
13) showed high cytotoxic activity against A-2780 compared to the standard drug docetaxel.
While the LogIC50 of the docetaxel was -0.81 µM for A-2780 cells at 24 h, the IC50 values of
compounds 1, 3, 5, 6, 7, 9, 10 and 13 were -0.97, -1.30, -0.22, 0.13, -0.16, -0.73 and -0.53 µM,
1

respectively. Three of the compounds 1, 18 and V showed high cytotoxic activity against DU-
145 compared to docetaxel (p<0.05). While the LogIC50 of the docetaxel was -1.13 µM for DU-
145 cells at 24 h, the LogIC50 values of compounds 1, 18 and V were 0.84, -0.38 and -0.66 µM,
respectively.
Keywords: Benzimidazole complexes, cytotoxic, A-2780 cell lines, DU-145 cell lines.
*Correspondence: hasan.kucukbay@inonu.edu.tr(HK)
1. Introduction
Today, drug resistance is one of the most important problems in the treatment of all diseases.
Drug resistance also poses serious problems in cancer treatment. Cancer is the second leading
cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018. Globally,
about 1 in 6 deaths is due to cancer. A correct cancer diagnosis is essential for adequate and
effective treatment because every cancer type requires a specific treatment regimen that
encompasses one or more modalities such as surgery, radiotherapy, and chemotherapy[1].
Therefore, there is a serious need for the synthesis of novel, effective, inexpensive, and low-
side-effect drug candidates. Today, cisplatin (cis-diamminedichloroplatinum(II) is the most
widely used anticancer drug in the treatment of various types of cancer despite the undesirable
side effects and its high price. On the other hand, combination therapies with cisplatin and other
metal-containing drugs are thought to overcome drug resistance and reduce toxicity [2].
Therefore, much attention has focused on designing new type of efficient and inexpensive
anticancer agents with a broad range of anticancer activity with decreased side effects. In
connection with these purposes, several benzimidazole derivatives and their metal complexes
have been studied and obtained promising anticancer activity against several cell lines such as
A549 (Lung) [3], A2780 (ovarian) [4], MCF-7 (breast) [5][6][7], BEAS-2B (bronchial
epithelia) [5][3], HeLa (cervical-uterine) [6], PC3 (prostate) [4][5], DU-145 (prostate) [8],
2

HCT-15 (colon) [5], SMMC7721 (liver) [5], EC109 (esophagus) [9], U373 (glioblastoma) [5],
SiHa (cervical) [10], SGC7901 (gastric) and HT1080 (fibrosarcoma) [11], HEK-293T (kidney)
[12]. On the other hand, benzimidazoles, which are important biologically active compounds,
have a wide variety of biological activities [13].
In our previous studies we have also synthesized and published many biologically active
benzimidazole and benzimidazole metal complexes [14][15][16][17][18][19][3][20].
Metal complexes are generally more active than the corresponding ligands and there are some
reports related with this situation in the literature [21][22].
According to the literature knowledge and our previous experiences on the benzimidazole
derivatives, in this study it was aimed to synthesized new cobalt(II) and zinc(II) complexes
incorporating 1-benzyl, 2-phenyl substituted benzimidazole moieties (Scheme 1) in an attempt
to obtain new active compounds having cytotoxic activities against human cancer cell lines A-
2780 and DU-145.
2. Experimental
The starting materials and reagents used in the reactions were supplied commercially by Aldrich
or Merck Chemical Co. The solvents were dried by standard methods and freshly distilled prior
1
13
to use. H-NMR (400 MHz) and C-NMR (100 MHz) spectra were recorded using a Bruker
Avenced III 400 MHz Ultrashield high performance digital FT NMR spectrometer. Infrared
spectra were recorded with ATR equipment in the range 4000 - 200 cm-1 on a Perkin-Elmer
FT-IR spectrophotometer. Elemental analyses were performed by LECO CHNS-932 elemental
analyzer. UV-Vis spectra were measured on a Perkin-Elmer Lambda 35 spectrophotometer.
The melting points were recorded using an Electrothermal-9200 melting point apparatus and
are uncorrected. Benzimidazole complexes 1-18 were synthesized from benzimidazole ligands
(I-IX) with CoCl2.6H2O or ZnCl2 in EtOH. Benzimidazole ligands (I1 [23], II [24], III [24], IV
3

[24], VI [25], VII [25], VIII [26], IX [27] were prepared according to the literature procedures.
The benzimidazole ligand, (E)-2-phenyl-1-(4-styrylbenzyl)-1H-benzo[d]imidazole (V) was
synthesized for the first time in this work using the Mizoroki-Heck reaction. similar to the
literature method [28][29].
Y
1) Y= Cl, R= H, M= Zn
2) Y= Cl, R= H, M= Co
3) Y= Br, R= H, M= Zn
R
N
4) Y= Br, R= H, M= Co
5) Y= Me, R= H, M= Zn
6) Y= Me, R= H, M= Co
Y
N
Cl
7) Y= Ph, R= H, M= Zn
8) Y= Ph, R= H, M= Co
9) Y= Styryl, R= H, M= Zn
10) Y= Styryl, R= H, M= Co
11) Y= Cl, R= Cl, M= Zn
12) Y= Cl, R= Cl, M= Co
13) Y= Me, R= Cl, M= Zn
14) Y= Me, R= Cl, M= Co
15) Y= H, R= H, M= Zn
16) Y= H, R= H, M= Co
17) Y= H, R= Cl, M= Zn
18) Y= H, R= Cl, M= Co
M
N
N
MCl2
+
Cl
N
2
R
N
R
I) Y= Cl, R= H
II) Y= Br, R= H
III) Y= Me, R= H
IV) Y= Ph, R= H
V) Y= Styryl, R= H
VI) Y= Cl, R= Cl
VII) Y= Me, R= Cl
VIII) Y= H, R= H
IX) Y= H, R= Cl
Y
Scheme 1. Synthesis of novel benzimidazole metal complexes.
1) Synthesis of (E)-2-phenyl-1-(4-styrylbenzyl)-1H-benzo[d]imidazole (V)
A mixture of 1-(4-bromobenzyl)-2-phenylbenzimidazole (1.00 g, 2.75 mmol), styrene (0.31 g,
3.00 mmol), K2CO3 (0.83 g, 6.00 mmol), Pd(OAc)2 (% 2 mmol) and PPh3 (% 4 mmol) was
heated in DMF (3 mL)-H2O (3 mL) in a Schleck tube on a water bath for 6 h. The mixture was
than cooled and the product extracted with ethyl acetate (25 mL X 3). The extracts were dried
with anhydrous Na2SO4 and the solvent was removed under reduced pressure. The residue was
crystallized from ethanol to provide cream color crystals V 0.79 g (75%), mp: 171-172 °C. Anal
Calcd for C28H22N2 (386,5) (%): C 87.01, H 5.74, N 7.25. Found (%): C 86.90, H 5.65, N 7.32.
ν(C=N): 1614 cm-1, ν(C=C): 1511 cm-1. 1H-NMR (400 MHz, CDCl3) δ: 7.81 (d, 1H, Ar-H, J= 8 Hz
), 7.64-7.62 (m, 2H, Ar-H), 7.43-7.38 (m, 6H, Ar-H, CH=CH), 7.29-7.25 (m, 3H, Ar-H), 7.18-
7.16 (m, 4H, Ar-H), 7.02(d, 2H, Ar-H, J= 8 Hz), 7.01(s, 2H, Ar-H), 5.38 (s, -CH2-, 2H) ppm.
13C-NMR (100 MHz, DMSO-d6) δ: 153.7, 143.2, 137.4, 136.8, 136.7, 136.4, 130.6, 130.3,
4

129.5, 129.2, 129.1, 128.2(d), 127.3, 127.0, 126.9, 123.2, 122.7, 119.8, 111.6 (Ar-C and -
CH=CH-), 47.8 (-CH2-).
2) Synthesis of dichlorobis(1-(4-chlorobenzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (1)
A mixture of 1-(4-chlorobenzyl)-2-phenyl-1H-benzimidazole (I) (0.50 g, 1.57 mmol), ZnCl2
(0.11 g, 0.80 mmol) and EtOH (20 mL) was heated under reflux for 5h. The mixture was filtered
off while hot. The obtained cream color crude product was crystallized from EtOH/DMF (1:1).
Yield: 0.49 g, 80%, mp: 242-243 °C. Anal Calcd for C40H30Cl4N4Zn (773.9) (%): C 62.08, H
1
3.91, N 7.24. Found (%): C 61.82, H 3.84, N 7.15. ν(C=N): 1599 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.79-7.77 (m, 2H, Ar-H), 7.73-7.70 (m, 4H, Ar-H), 7.55-7.52 (m, 6H, Ar-H),
7.50-7.48 (m, 2H, Ar-H), 7.36 (d, 4H, Ar-H, J= 8 Hz), 7.29-7.26 (m, 4H, Ar-H), 7.01 (d, 4H,
Ar-H, J= 8 Hz), 5.59 (s, 4H, -CH2-). 13C-NMR (100 MHz, DMSO-d6) δ: 153.9, 142.9, 136.3,
136.1, 132.6, 130.4, 130.2, 129.6, 129.3, 129.2, 128.5, 123.4, 122.9, 119.8, 111.6 (Ar-C), 47.3
(-CH2-).
Similar to the procedure above, compounds 2-18 were synthesized from appropriate 1,2-
disubstituted benzimidazole and CoCl2.6H2O or ZnCl2.
3) Dichlorobis(1-(4-chlorobenzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (2)
Yield: 0.50 g, 83%, mp: 217-219 °C. Anal Calcd for C40H30Cl4N4Co (767.4) (%): C 62.60, H
3.94, N 7.30. Found (%): C 62.40, H 3.90, N 7.21. ν(C=N): 1603 cm-1.
4) Dichlorobis(1-(4-bromobenzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (3)
Yield: 0.46 g, 78%, mp: 290-292 °C. Anal Calcd for C40H30Br2Cl2N4Zn (862.8) (%): C 55.68,
1
H 3.50, N 6.49. Found (%): C 55.47, H 3.48, N 6.35. ν(C=N): 1595 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.79-7.77 (m, 2H, Ar-H), 7.72-7.70 (m, 4H, Ar-H), 7.55-7.53 (m, 6H, Ar-H), 7.49
(d, 6H, Ar-H, J= 8 Hz), 7.29-7.26 (m, 4H, Ar-H), 6.95 (d, 4H, Ar-H, J= 8 Hz), 5.56 (s, 4H, -
13
CH2-). C-NMR (100 MHz, DMSO-d6) δ: 153.8, 142.9, 136.8, 136.1, 132.2, 130.4, 130.2,
129.6, 129.3, 128.8, 123.4, 122.9, 121.1, 119.8, 111.6 (Ar-C), 47.4 (-CH2-).
5) Dichlorobis(1-(4-bromobenzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (4)
Yield: 0.44 g, 75%, mp: > 300 °C. Anal Calcd for C40H30Br2Cl2N4Co (856.4) (%): C 56.10, H
3.53, N 6.54. Found (%): C 56.01, H 3.42, N 6.44. ν(C=N): 1593 cm-1.
5

6) Dichlorobis(1-(4-methylbenzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (5)
Yield: 0.52 g, 85%, mp: 287-288 °C. Anal Calcd for C42H36Cl2N4Zn (733.1) (%): C 68.82, H
1
4.95, N 7.64. Found (%): C 69.03, H 5.09, N 7.60. ν(C=N): 1615 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.79-7.72 (m, 6H, Ar-H), 7.55-7.46 (m, 8H, Ar-H), 7.28-7.24 (m, 4H, Ar-H), 7.09
(d, 4H, Ar-H, J= 8 Hz), 6.89 (d, 4H, Ar-H, J= 8 Hz), 5.53 (s, 4H, -CH2-), 2.23 (s, 6H, CH3).
13C-NMR (100 MHz, DMSO-d6) δ: 153.9, 142.9, 137.2, 136.2, 134.2, 130.4, 129.8, 129.6,
129.3, 126.5, 123.3, 122.8, 119.7, 111.7 (Ar-C), 47.8 (-CH2-), 21.1 (CH3).
7) Dichlorobis(1-(4-methylbenzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (6)
Yield: 0.51 g, 84%, mp: 294-295 °C. Anal Calcd for C42H36Cl2N4Co (726.6) (%): C 69.43, H
4.99, N 7.71. Found (%): C 69.39, H 5.08, N 7.93. ν(C=N): 1615 cm-1.
8) Dichlorobis(1-(4-phenylbenzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (7)
Yield: 0.42 g, 70%, mp: 295-296 °C. Anal Calcd for C52H40Cl2N4Zn (857.2) (%): C 72.86, H
1
4.70, N 6.54. Found (%): C 72.70, H 4.62, N 6.50. ν(C=N): 1590 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.78-7.75 (m, 6H, Ar-H), 7.62-7.54 (m, 16H, Ar-H), 7.43 (t, 4H, Ar-H, J= 8 Hz),
7.35 (d, 2H, Ar-H, J= 8 Hz), 7.31-7.25 (m, 4H, Ar-H), 7.10 (d, 4H, Ar-H, J= 8 Hz), 5.64 (s,
4H, -CH2-). 13C-NMR (100 MHz, DMSO-d6) δ: 153.8, 143.0, 139.9, 139.8, 136.5, 136.2, 130.5,
130.4, 129.6, 129.4, 129.3, 128.0, 127.6, 127.2, 127.0, 123.3, 122.9, 119.8, 111.7 (Ar-C), 47.7
(-CH2-).
9) Dichlorobis(1-(4-phenylbenzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (8)
Yield: 0.43 g, 73%, mp: > 300 °C. Anal Calcd for C52H40Cl2N4Co (850.8) (%): C 73.41, H 4.74,
N 6.59. Found (%): C 73.20, H 4.60, N 6.48. ν(C=N): 1609 cm-1.
10) Dichlorobis(1-(4-styryllbenzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (9)
Yield: 0.39 g, 66%, mp: 247-248 °C. Anal Calcd for C56H44Cl2N4Zn (909.3) (%): C 73.97, H
4.88, N 6.16. Found (%): C 73.79, H 4.72, N 6.00. ν(C=N): 1611 cm-1, ν(C=C): 1514 cm-1. 1H-NMR
(400 MHz, DMSO-d6) δ: 7.77-7.74 (m, 6H, Ar-H), 7.56-7.52 (m, 16H, Ar-H and HC=CH),
7.37 (t, 4H, Ar-H, J= 6 Hz), 7.28-7.26 (m, 6H, Ar-H), 7.20 (s, 4H, Ar-H), 7.01 (d, 4H, Ar-H),
13
5.60 (s, -CH2-, 4H). C-NMR (100 MHz, DMSO-d6) δ: 153.8, 143.1, 137.4, 136.8, 136.7,
136.4, 130.6, 130.4, 129.6, 129.3, 129.2, 129.1, 128.2, 128.1, 127.3, 127.0, 126.9, 123.2, 122.8,
119.8, 111.6 (Ar-C and alkene-C), 47.8 (-CH2-).
6

11) Dichlorobis(1-(4-styryllbenzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (10)
Yield: 0.40 g, 68%, mp: 260-261 °C. Anal Calcd for C56H44Cl2N4Co (902.8) (%): C 74.50, H
4.91, N 6.21. Found (%): C 74.35, H 4.79, N 6.18. ν(C=N): 1595 cm-1, ν(C=C): 1515 cm-1.
12) Dichlorobis(1-(4-chlorobenzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)zinc(II) (11)
Yield: 0.47 g, 78%, mp: 276-277 °C. Anal Calcd for C40H28Cl6N4Zn (842.8) (%): C 57.01, H
1
3.35, N 6.65. Found (%): C 56.88, H 3.20, N 6.45. ν(C=N): 1651 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.78-7.72 (m, 6H, Ar-H), 7.61 (d, 4H, Ar-H, J= 8 Hz), 7.52-7.50 (m, 2H, Ar-H),
7.36 (d, 4H, Ar-H, J= 8 Hz), 7.29-7.27 (m, 4H, Ar-H), 7.02 (d, 4H, Ar-H, J= 8 Hz), 5.59 (s,
4H, -CH2-). 13C-NMR (100 MHz, DMSO-d6) δ: 152.6, 142.9, 136.3, 135.3, 132.6, 131.3, 129.4,
129.3, 129.2, 128.5, 123.5, 123.0, 119.9, 111.6 (Ar-C), 47.3 (-CH2-).
13) Dichlorobis(1-(4-chlorobenzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)cobalt(II) (12)
Yield: 0.48 g, 81%, mp: 290-291 °C. Anal Calcd for C40H28Cl6N4Co (836.3) (%): C 57.45, H
3.37, N 6.70. Found (%): C 57.30, H 3.40, N 6.62. ν(C=N): 1597 cm-1.
14) Dichlorobis(1-(4-methylbenzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)zinc(II) (13)
Yield: 0.52 g, 87%, mp: 270-271 °C. Anal Calcd for C42H34Cl4N4Zn (801.9) (%): C 62.91, H
1
4.27, N 6.99. Found (%): C 62.75, H 4.17, N 6.68. ν(C=N): 1659 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.82-7.77 (m, 6H, Ar-H), 7.64 (d, 4H, Ar-H, J= 8 Hz), 7.54-7.51 (m, 2H, Ar-H),
7.33-7.28 (m, 4H, Ar-H), 7.12 (d, 4H, Ar-H, J= 8 Hz), 6.92 (d, 4H, Ar-H, J= 8 Hz), 5.57 (s,
4H, -CH2-), 2.26 (s, 6H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ: 152.7, 142.8, 137.2, 136.3,
135.3, 134.1, 131.4, 129.8, 129.4, 129.3, 126.5, 123.5, 122.9,119.8, 111.8 (Ar-C), 47.8 (-CH2),
21.1 (CH3).
15) Dichlorobis(1-(4-methylbenzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)cobalt(II) (14)
Yield: 0.54 g, 90%, mp: 282-283 °C. Anal Calcd for C42H34Cl4N4Co (795.5) (%): C 63.41, H
4.31, N 7.04. Found (%): C 63.22, H 4.23, N 7.03. ν(C=N): 1602 cm-1.
16) Dichlorobis(1-benzyl)-2-phenyl-1H-benzimidazole-KN3)zinc(II) (15)
Yield: 0.52 g, 84%, mp: 238-239 °C. Anal Calcd for C40H32Cl2N4Zn (705.0) (%): C 68.15, H
1
4.58, N 7.95. Found (%): C 68.04, H 4.37, N 7.80. ν(C=N): 1604 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.84 (d, 2H, Ar-H, J= 8 Hz), 7.74-7.71 (m, 4H, Ar-H), 7.53-7.48 (m, 8H, Ar-H),
7

7.29-7.23 (m, 10H, Ar-H), 7.00 (d, 4H, Ar-H, J= 8 Hz), 5.57 (s, 4H, -CH2-). 13C-NMR (100
MHz, DMSO-d6) δ: 153.9, 137.2, 136.1, 130.5, 129.7, 129.3, 129.2, 128.0, 126.6, 123.4, 123.0,
119.7, 111.7 (Ar-C), 48.0 (-CH2-).
17) Dichlorobis(1-benzyl)-2-phenyl-1H-benzimidazole-KN3)cobalt(II) (16)
Yield: 0.50 g, 81%, mp: 278-279 °C. Anal Calcd for C40H32Cl2N4Co (698.6) (%): C 68.78, H
4.62, N 8.02. Found (%): C 68.70, H 4.54, N 7.93. ν(C=N): 1593 cm-1.
18) Dichlorobis(1-benzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)zinc(II) (17)
Yield: 0.47 g, 77%, mp: 238-239 °C. Anal Calcd for C40H30Cl4N4Zn (773.9) (%): C 62.08, H
1
3.91, N 7.24. Found (%): C 62.22, H 3.93, N 7.03. ν(C=N): 1601 cm-1. H-NMR (400 MHz,
DMSO-d6) δ: 7.81-7.79 (m, 2H, Ar-H), 7.75 (d, 4H, Ar-H, J= 8 Hz), 7.59 (d, 4H, Ar-H, J= 8
Hz), 7.52-7.50 (m, 2H, Ar-H), 7.31-7.23 (m, 10H, Ar-H), 6.99 ( d, 4H, Ar-H, J= 4 Hz), 5.59 (s,
4H, -CH2-). 13C-NMR (100 MHz, DMSO-d6) δ: 152.7, 142.7, 137.2, 136.2, 135.3, 131.4, 129.4,
129.3, 129.1, 128.0, 126.6, 123.5, 123.0, 119.8, 111.7 (Ar-C), 48.0 (-CH2-).
19) Dichlorobis(1-benzyl)-2-(4-chlorophenyl)-1H-benzimidazole-KN3)cobalt(II) (18)
Yield: 0.47 g, 77%, mp: 238-239 °C. Anal Calcd for C40H30Cl4N4Co (767.4) (%): C 62.60, H
3.94, N 7.30. Found (%): C 62.47, H 3.90, N 7.24. ν(C=N): 1592 cm-1.
4.5. Cell lines and culture conditions
The human cancer lines, ovarian cancer (A2780) and prostate cancer (DU-145) were used for
in vitro screening experiments. The cell lines were both purchased from the American Type
Culture Collection (ATCC). All cells were fed in 25 and 75 cm2 flasks with RPMI-1640
medium (containing 10% fetal bovine serum, 100 U/ mL penicillin and 0.1 mg/mL
streptomycin) in two days apart. In cells with a carbon dioxide (5% CO2) incubator (Panasonic,
Japan), the cells maintained at 37 °C and in a humid environment were separated from the flasks
using a solution of trypsin-EDTA (Sigma-Aldrich, USA) when confluent. The viability of the
8

cells was determined using 0.4% trypan blue and experiments were started when the viability
was above 90%.
4.5.1. MTT Assay
Antitumor activities of these substances were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay[30]. Cells were removed using a trypsin-EDTA
solution from flasks and counted by hemocytometer to determine cytotoxic effects. 15x103 cells
per well were plated in 96-well including 200 µL of RPMI-1640 medium. Cells were incubated
at 37 °C in a CO2 incubator for 24 hours to adhere to a 96-well plate base. When the incubation
ended, concentrations of 0.1, 1, 10 and 100 10 µM of the benzimidazole derivatives were added
to the wells in which the cells were contained. Incubation with cancer cells for 24 hours at 37
°C in a CO2 incubator was performed to determine the effects of different concentrations of
benzimidazole derivatives on cell viability for 24 hours. When the incubation was over, 0.5
mg/mL of MTT solution in sterile PBS was prepared and added to 96-well plates.
After MTT was added, the plates were incubated again for 3 hours. After this time, incubation
was stopped by adding dimethylsulphoxide (DMSO) to the wells, and the optical densities of
the cells in the plates were read on a spectrophotometer (Synergy HTX, USA) at a wavelength
of 550 nm[31]. Cell viability percentage was calculated by proportioning the absorbance values
obtained from benzimidazole ligands and their cobalt (II) and Zinc (II) complexes applied wells
to that of control group. MTT trials were performed ten times in triplicate on different days and
the inhibitory logarithmic 50 (LogIC50) values of the applied compounds were calculated based
on MTT results using the GraphPad prism 6 program on a computer.
Statical analysis
9

The IBM SPSS Statistics 24.0 (Windows) package program was used in the analysis.
Conformity to normal distribution was evaluated by Shapiro Wilk test. Intergroup comparisons
of quantitative variables were measured by Kruskal Wallis H test. When significant statistical
differences were determined between groups, multiple comparisons were made with Bonferroni
correction Mann Whitney U test. All P values <0.05 were considered statistically significant.
LogIC50 values of melatonin and agomelatine were calculated using Graphpad prism 6 program
on the computer based on the MTT results obtained from the experiments.
3. Results and discussion
3.1. Synthesis and characterization of the benzimidazole complexes
The cobalt(II) and zinc(II) complexes of the benzimidazole derivatives were prepared
from the reaction of the appropriate 1,2-disubstituted benzimidazole ligand and metal salt in
good yields between 66-90%. The complexes were crystallized in a DMF/EtOH mixture.
Benzimidazole ligand V was synthesized from the reaction of benzimidazole ligand II and
styrene through the Heck-Mizoroki cross-coupling reaction with a good yield. The structures
1
of Zn(II) benzimidazole complexes (1, 3, 5, 7, 9, 11, 13, 15 and 17) were elucidated by H
NMR, 13C NMR and IR spectrometric analyses. Due to paramagnetic properties of Co(II)
benzimidazole complexes (2, 4, 6, 8, 10, 12, 14, 16 and 18) we could not observe appropriate
NMR signals even more scans in diluted solvents. For this reason, the structures of Co(II)
benzimidazole complexes were elucidated by IR and UV-Vis spectrometric and elemental
analyses. While IR spectra of the compounds show the strong ν(C=N) bands in free
benzimidazoles at about 1600 cm-1 region, this bands shift slightly to higher wavelengths for
the corresponding Co(II) and Zn(II) complexes. The red shift indicates the coordination of a
tertiary nitrogen atom of the benzimidazole ligand to Co(II) and Zn(II) atoms. These types of
red shifts are also reported in the literature [32] [33]. We have also observed these types of red
10

shift in our previous studies [34][35]. IR spectra of compound V showed the ν(C=N) and ν(C=O)
stretching bands at 1614 and 1511 cm-1, respectively, and these stretching bands were observed
at 1611 and 1514 cm-1 for the corresponding Zn(II) complex and 1595 and 1515 cm-1 for the
corresponding Co(II) complex. The ν(C=N) and ν(C=O) stretching frequencies of some other
known benzimidazole ligands (I, III and IX) are reported as range of 1610 to 1635 cm-1 for
ν(C=N) and 1475 to 1495 cm-1 for ν(C=O) in the literature [24].
While the characteristic CH2 resonances for the benzyl group at position 1 of the
benzimidazole ligands were observed between 5.37-5.49 ppm [24][25], they resonated between
5.53-5.64 ppm for the Zn(II) complexes of the benzimidazole ligands (1, 3, 5, 7, 9, 11, 13, 15
and 17) . As expected, these benzylic methylene peaks shifted to downfield (Δδ≈ 0.15- to 0.16
ppm) for the Zn(II) complexes.
The general chemical shift to the downfield in both 1H and 13C NMR compared to the
free benzimidazole ligands reveal the formation of the expected Zn(II) benzimidazole
complexes.
13
The coordination to the Zn(II) ion for compound 9 shifts the C NMR signals of the
complex downfield from those of the free ligand V (Δδ≈ 0.1 ppm) for the carbon atom at
position 2 of the benzimidazole ring. The carbon resonances for the carbon atom at position 2
of the benzimidazole ligands of Zn(II) complexes (1, 3, 5, 7, 9, 11, 13, 15 and 17) were observed
between 152.6-153.9 ppm. All other aliphatic and aromatic protons and carbons were observed
in the expected regions. 1H and 13 C NMR, FTIR and UV-VIS spectra of all compounds can be
found in the supplementary information file provided with this manuscript.
The UV-Vis spectra of new benzimidazole complexes (1-18) and three benzimidazole
ligands (III, V and VII) were determined in the 200-800 nm region in DMSO. Compound III,
V and VII have absorption maxima at 323, 307, 267, 207; 332, 309, 297, 240 and 314, 310,
11

202, respectively, and these bands are attributed n- π* and π-π* transitions. In the complexes,
these peaks are observed nearly similar. The d-d bands for all cobalt(II) complexes (2, 4, 6, 8,
10, 12, 14, 16 and 18) were observed in the range of 670 (є= 110 M-1.cm-1) to 679 nm (є= 60
M-1.cm-1). All cobalt(II) complexes showed a single d-d band. Since zinc(II) has no unpaired
d-electrons, no absorption peak was observed in the visible region for these complexes.
3.2. Biological Activity
3.2.1. Cytotoxicity Studies
Cytotoxic activities of eighteen new complexes and three benzimidazole ligands were
determined using A-2780 (human ovarian) and DU-145 (human prostate) cell lines. In order to
obtain the cytotoxic properties of the newly synthesized cobalt(II) and zinc(II) benzimidazole
derivatives on A-2780 and DU-145 cells, the respective cell lines were incubated with
increasing concentrations (0-100 µg/mL) of the compounds for 48 h and then subjected to an
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A time-dependent
cell viability assay for the tested benzimidazole derivatives was performed and the LogIC50
values of the compounds were calculated after a 24 h treatment. LogIC50 values calculated for
the A-2780 and DU-145 cells based on the results of MTT assays for a 24-hour interaction of
benzimidazole derivatives are presented in Table 2.
Table 2 should be inserted here
As can be seen in Table 2, all tested benzimidazole derivatives showed higher or
comparable antitumor activity against A-2780 cell lines compared to the standard drug
docetaxel with a LogIC50 value of -0.81 µM. Among the tested 18 complexes, those that
incorporated zinc(II) metal and had chlorine, bromine or styryl at position 4 of the benzyl
12

substituent (1, 3, 4 and 9) showed higher cytotoxicity on A-2780 than the other compounds and
the standard drug docetaxel at 0.1µM. A nearly similar result was obtained at 1 µM
concentration for the A-2780 cell line. The cell viability results of A-2780 and DU-145 cells
after a 48-h treatment with the three benzimidazole ligands (III, V, VII) and eighteen
benzimidazole complexes of Zn(II) and Co(II) (1-18) are shown in Tables 3 and 4, respectively.
As shown in Table 3, all tested benzimidazole derivatives demonstrated anticancer activity
against the A-2780 cell line at 10 and 100 µM concentrations with P < 0.05, except compounds
12 and 16 at 10 µM concentration. Three of the compounds (1, 2 and 18) showed antitumor
activity against A-2780 cells at 0.1 µM concentration with P< 0.05. Thirteen of the tested
compounds also exhibited antitumor activity against A-2780 cells at 10 µM concentration with
P< 0.05. As can be seen in Tables 2 and 3, in accordance with literature information [21, 22],
the metal complexes in this study generally showed better anticancer activity than the
corresponding ligands on the A-2780 cell line. Three of the compounds 1, 18 and V showed
high cytotoxic activity against DU-145 compared to docetaxel as shown in Tables 2 and 4.
While the LogIC50 of the docetaxel was -1.13 µM for DU-145 cells at 24 h, the LogIC50 values
of compounds 1, 18 and V were 0.84, -0.38 and -0.66 µM, respectively. In contrast to literature
information [21, 22], anticancer activity against DU-145 cells was found more effective in
ligand V than in the corresponding metal complexes 9 and 10.
Tables 3 and 4 should be inserted here
4. Conclusions
Eighteen new benzimidazole Zn(II) and Co(II) metal complexes and three benzimidazole
ligands (V is new) were synthesized successfully and their anticancer activities were evaluated
on human cancer cell lines (A2780 and DU-145). Compounds 1, 3, 4 and 9 showed better
13

anticancer activity than the standard drug docetaxel at a concentration of 0.1 μM against the
A2780 cell line. On the other hand, only 1, 18 and V showed comparable anti-cancer activity
at 100 μM concentration against DU145. In conclusion, the result of this study is encouraging
us to continue our anticancer activity screening studies on benzimidazole derivatives.
Acknowledgements
We thank Inönü University, Turkey (BAPB-Grand No: 2014/33) for financial support.
Declaration of interest
The authors declare no conflicts of interest.
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Table 1. Electronic absorption spectral bands and magnetic moments of novel benzimidazole
ligands and their metal complexes.
Compounds Electronic absorption bands* λmax (nm)
Intraligand and charge transfer bands
275, 250, 249
d-d Bands
III
19

V
VII
1
278, 250, 248
312, 309, 282
331, 284, 251
282, 253, 242
275, 239, 233
274, 243, 234
342, 278, 242
279, 242, 233
279, 254, 233
289, 248, 239
261, 243, 234
364, 280, 246
282, 243, 230
361, 280, 252
356, 280, 252
386, 348, 333
279, 250, 231
278, 254, 242
278, 246, 234
356, 279, 247
2
671
671
679
670
678
679
678
679
678
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
* DMSO used as a solvent.
Table 2. LogIC50 (µM) concentrations calculated for A2780 and DU-145 cells in the
GraphPad Prizm 6 program of benzimidazole derivatives.
20

LogIC50 (µM) Concentration
Comp. no
A2780 cell DU-145 cell
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-0.97
1.25
-1.30
1.13
-0.22
0.13
-0.16
1.39
-0.73
-0.53
1.52
2.28
0.41
1.60
1.24
2.94
2.18
3.18
2.24
1.59
2.95
-0.81
0.84
2.93
1.83
4.42
2.15
1.82
1.06
12.60
1.90
2.03
5.59
2.97
6.44
2.92
2.32
1.66
1.97
-0.38
2.27
-0.66
0.27
-1.13
17
18
III
V
VII
Docetaxel (Ref. Drug)
Table 3. The cell viability results of A2780 cells after a 48-h treatment with three benzimidazole
21

ligands (III, V, VII) and eighteen benzimidazole complexes of Zn(II) and Co(II) (1-18).†
A2780 Cell viability (%)
Solvent
Comp. no
Control
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
100±6
(DMSO)
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
94±3
0.1 μM
21±3*
76±7*
43±7
42±9
98±15
76±10
67±7
96±8
52±10
60±5
87±5
97±6
91±4
90±5
95±4
94±2
91±6
76±7*
91±7
92±6
96±3
54±6*
1 μM
25±2
10 μM
21±4*
72±8*
10±2*
30±4*
4±0*
100 μM
4±0*
1
2
75±6*
19±6*
34±8*
27±3*
55±8*
57±6*
92±6
22±4*
4±0*
3
4
18±4*
4±0*
5
6
27±4*
6±0*
12±1*
5±0*
7
8
61±7*
10±1*
28±6*
50±5*
95±3
30±4*
6±0*
9
42±6*
38±6*
82±6*
95±3
10
11
12
13
14
15
16
17
18
III
V
22±9*
46±8*
64±11*
11±1*
44±4*
3±0*
64±9*
82±3
28±5*
58±6*
73±6*
89±4
90±7
89±8
67±5*
55±6*
59±4*
54±4*
44±6*
66±5*
0±0*
89±5
67±8*
63±7*
70±3*
61±6*
68±6*
10±1*
73±11*
80±4*
66±6*
80±7
VII
Docetaxel (Ref. Drug) 100±6
31±5*
* P < 0.05 0.05
† The changes in cell viability caused by benzimidazole ligands and benzimidazole
complexes are compared with the control data. Each data point is an average of 8 viability
measurements.
Table 4. The cell viability results of DU-145 cells after a 48-h treatment with three
22

benzimidazole ligands (III, V, VII) and eighteen benzimidazole complexes of
Zn(II) and Co(II) (1-18).‡
DU-145 Cell viability (%)
Solvent
Comp. no
Control
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
100±5
(DMSO)
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
92±2
0.1 μM
76±3*
78±8*
84±13
84±17
95±2
1 μM
63±10*
66±7*
82±9*
77±7*
94±4
10 μM
50±4*
68±6*
78±8*
84±7
100 μM
29±4*
55±9*
41±5*
64±6*
28±6*
52±6*
36±4*
90±5
1
2
3
4
5
69±5
6
95±4
90±4
54±8*
45±4*
94±5
7
94±3
70±7
8
94±5
93±2
9
97±3
84±7
75±8*
67±7*
76±7*
80±6
46±5*
50±8*
71±8*
72±8*
102±5
78±6
10
11
12
13
14
15
16
17
18
III
V
89±4
74±6*
79±8*
96±6
84±5
101±7
104±4
97±5
104±4
94±4
102±4
97±3*
75±8*
80±6*
73±8*
39±10*
92±5
99±5
79±8
56±8*
33±5*
42±7*
26±5*
55±6*
24±2*
31±4*
0±0*
78±6*
75±8*
51±5*
87±3
78±8*
75±6*
53±6
87±4
54±7*
80±5
40±5*
46±11*
28±4*
36±2*
33±4*
9±2*
VII
Docetaxel (Ref. Drug) 100±5
45±5*
* P < 0.05 0.05
‡ The changes in cell viability caused by benzimidazole ligands and benzimidazole
complexes are compared with the control data. Each data point is an average of 8 viability
measurements.
Highly active anticancer candidates against to A2780 cell lines were synthesized.
23

Anticancer properties against DU-145 cell lines of new complexes were tested.
New Zn (II) and Co (II) complexes of benzimidazole with anticancer activity.
Eighteen new cobalt (II) or zinc (II) complexes of benzimidazole bearing 1-benzyl and 2-
phenyl moieties were synthesized from the reaction of appropriate benzimidazole ligands and
13
CoCl2 or ZnCl2. Their structural characterizations were done by IR, NMR (1H, C) and UV-
VIS spectrometers. Cytotoxic activities of eighteen new complexes and three benzimidazole
ligands were determined using A-2780 (human ovarian) and DU-145 (human prostate) cell
lines. Antitumor properties of all compounds were determined by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Cell viability assay for the tested benzimidazole
derivatives was performed and the LogIC50 values of the compounds were calculated after a
24-hour treatment. All tested benzimidazole derivatives showed higher or comparable
antitumor activity against A-2780 cell lines compared to standard drug docetaxel with -0.81
µM LogIC50 value (p<0.05). Eight of the examined compounds (1, 3, 5, 6, 7, 9, 10 and 13)
showed high cytotoxic activity against A-2780 compared to standard drug docetaxel. While the
LogIC50 of the docetaxel was -0.81 µM for A-2780 cells at 24 h, the IC50 values of compounds
1, 3, 5, 6, 7, 9, 10 and 13 were -0.97, -1.30, -0.22, 0.13, -0.16, -0.73 and -0.53 µM, respectively.
Three of the compounds 1, 18 and V showed high cytotoxic activity against DU-145 compared
to docetaxel (p<0.05). While the LogIC50 of the docetaxel was -1.13 µM for DU-145 cells at
24 h, the LogIC50 values of compounds 1, 18 and V were 0.84, -0.38 and -0.66 µM, respectively.
24

Synthesis and evaluation of anticancer properties of novel benzimidazole ligands and
their cobalt (II) and Zinc (II) complexes against cancer cell lines A-2780 and DU-145.
Ülkü Yılmaz, Suat Tekin, Nesrin Buğday, Kemal Yavuz, Hasan Küçükbay*, Süleyman Sandal
25