Cytotoxic and antimicrobial potential of benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.202100076

New benzimidazole derivatives were synthesized and their structures were characterized by spectroscopic and microanalysis techniques. The cytotoxic properties of ten benzimidazole derivatives, five of which were synthesized in our previous studies, were d

Full text of DOI:10.1002/ardp.202100076

Received: 25 February 2021
Revised: 19 March 2021
Accepted: 25 March 2021
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DOI: 10.1002/ardp.202100076
F U L L P A P E R
Cytotoxic and antimicrobial potential of benzimidazole
derivatives
Hasan Küçükbay1
| Mustafa Uçkun2
| Elif Apohan2
| Özfer Yeşilada2
1Department of Chemistry, Faculty of Arts
and Sciences, İnönü University, Malatya,
Turkey
Abstract
New benzimidazole derivatives were synthesized and their structures were char-
acterized by spectroscopic and microanalysis techniques. The cytotoxic properties of
ten benzimidazole derivatives, five of which were synthesized in our previous studies,
were determined against the lung cancer cell line, A549, and the healthy lung epithelial
cell line, BEAS‐2B. Among the ten compounds tested, based on the 72‐h incubation
results, compound 12 was the most cytotoxic against the A549 cell line, whereas
against the BEAS‐2B cell line, it was as cytotoxic as cisplatin. The IC50 values of
compound 12 were 3.98 and 2.94 µg/ml for A549 and BEAS‐2B cells, respectively. The
cisplatin values were 6.75 and 2.75 µg/ml for A549 and BEAS‐2B cells, respectively.
Compounds 10, 8, 7, and 13 showed toxic effects against A549 cells, but were less toxic
against BEAS‐2B cells than cisplatin. The antimicrobial activity of these compounds
against pathogenic bacteria and yeasts was also evaluated based on their minimum
inhibitory concentration (MIC) values. The compounds, except 12 and 13, generally
showed higher antimicrobial activity against yeasts, compared with bacteria. Compound
12 showed better activity against Pseudomonas aeruginosa and Staphylococcus aureus
than against Escherichia coli. Compounds 7, 8, and 11 were the most effective ones
against the microorganisms, and yeasts were highly sensitive to these compounds with
MIC values of 25–100 µg/ml.
2Department of Biology, Faculty of Arts and
Sciences, İnönü University, Malatya, Turkey
Correspondence
Hasan Küçükbay, Department of Chemistry,
Faculty of Arts and Sciences, İnönü University,
44280 Malatya, Turkey.
Email: hasan.kucukbay@inonu.edu.tr
Elif Apohan, Department of Biology, Faculty
of Arts and Sciences, İnönü University, 44280
Malatya, Turkey.
Email: elif.apohan@inonu.edu.tr
K E Y W O R D S
A549 cells, antimicrobial activity, BEAS‐2B cells, benzimidazole derivatives, cytotoxicity
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1
INTRODUCTION
natural benzimidazole compound is N‐riosyldimethylbenzimidazole,
which is the ligand of the cobalt atom in vitamin B12.[4] The first
Benzimidazole, which has been studied with interest since the 1800s,
is found in the structure of many natural compounds and can be sold
commercially as medicine, continues to maintain its privileged place
among the heterocyclic compounds. Examples of benzimidazole
compounds sold as drugs in pharmacies are albendazole, omeprazole,
lansoprazole, pantoprazole, rabeprazole and tenatoprazole, etonita-
zine, galeterone, mavatrep, and dovitinib.[1–3] The most important
synthesis of benzimidazole was the preparation of 2,5 (or 2,6)‐
dimethylbenzimidazole by Hoebrecker by the reduction of 2‐nitro‐4‐
methylacetanilide with Sn/HCl in 1872.[5] Benzimidazole compounds,
which have attracted attention since their first synthesis, are being
studied with great interest today and continue to maintain their
privileged properties. According to the report of the World Health
Organization, cancer caused one of every six deaths in 2018 and
Dedicated to Professor Christian Bruneau.
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch Pharm. 2021;e2100076.
wileyonlinelibrary.com/journal/ardp
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KÜÇÜKBAY ET AL.
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became the second leading cause of death globally. According to the
same report, the most common cancers in men are lung, prostate,
colorectal, stomach, and liver cancer, whereas the most common in
women are breast, colorectal, lung, cervical, and thyroid cancer.
Globally, according to the 2020 report from the International Agency
for Research on Cancer, it is estimated that 1 in 5 people develop
cancer in their lifetime, and 1 in 8 men and 1 in 11 women die from
the disease.[6] Global aging population and socioeconomic risk fac-
tors, the ineffectiveness of the drugs used, and the proximity of
toxic values to therapeutic values are among the main factors that
trigger this increase. Lung cancer is the first cause of cancer deaths
worldwide, leading to about 1.6 million deaths per year.[7] Lung
cancer is comprised of small cell lung cancer (SCLC) and non‐SCLC
(NSCLC), among which NSCLC takes up approximately 85% of lung
cancer cases.[8] In recent years, significant increases in cancer cases
have made the development of anticancer drugs and the synthesis
of more active compounds among scientists' primary targets. In the
literature, there are notable studies on the anticancer properties of
1‐substituted benzimidazole and their 1,3‐disubstituted salts and
metal complexes such as cobalt and zinc.[9] As we observed sig-
nificant anticancer activities in some benzimidazole derivatives in
our recent studies,[10–12] we examined the anticancer properties of
other benzimidazole derivatives in this study. Benzimidazole is an
isostere of a purine‐based nucleic acid,[13] and benzimidazole de-
rivatives are an important class of heterocyclic compounds with
antimicrobial, antioxidant, anticancer, antiproliferative, and anti-
tumor activities.[14–20] Heterocyclic compounds containing het-
eroatoms such as nitrogen, sulfur, and oxygen have the ability to
make strong hydrogen bonding with DNA. The interaction strength
between such compounds and DNA is generally associated with
anticancer activity.[21,22] Benzimidazole containing two nitrogen
atoms is also an important pharmacophore in this respect. In
addition to the antitumor properties of benzimidazoles, the pre-
sence of a number of clinically approved benzimidazole derivatives
such as albendazole, mebendazole, tiabendazole, omeprazole, as-
temizole, enviradin, candesartan, and telmisartan raises the ex-
pectation that the new benzimidazoles will also exhibit potential
bioactivities.[23] As microorganisms develop resistance to anti-
biotics, new antimicrobial agents with antimicrobial activity are
needed. For this purpose, the antibacterial and antifungal activities
of these benzimidazole derivatives, against various bacteria and
yeasts, were also determined.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthesis pathway utilized to prepare the benzimidazole deri-
vatives 4, 5, and 7–11 is shown in Scheme 1. The 1‐substituted
benzimidazole compounds used as starting material were synthe-
sized by refluxing the appropriate benzimidazole with alkyl halide
containing silicon in the presence of KOH in ethyl alcohol using
Pozharskii and Simonov method.[24] Then, the 1‐substituted ben-
zimidazoles prepared were reacted with suitable alkyl halides for
1,3‐benzimidazole salts, and cobalt(II) chloride and zinc(II) chloride
for cobalt(II) and zinc(II) complexes, respectively. Compounds 1,[25]
2,[25] 3,[26] 6,[27] 12,[25] 13,[25] and 14[25] published in our previous
studies were synthesized again and used after their purity was
checked with the data in the literature. Although compounds 4
and 5 are synthesized from benzimidazole, they can be 5‐ or
6‐substituted due to their tautomers.[28] We have verified this si-
tuation in our previous studies with the single‐crystal X‐ray dif-
fraction method. Therefore, when naming these compounds, they
SCHEME 1 Synthesis pathways of the benzimidazole derivatives. DMF, dimethylformamide

KÜÇÜKBAY ET AL.
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were numbered as 5(6)‐substituted benzimidazoles.[29,30] When the
1H nuclear magnetic resonance (NMR) spectra of the compounds
are examined, the absence of the NH peak observed around 12 ppm
in dimethyl sulfoxide (DMSO)‐d6 of the compounds 4 and 5 in-
dicates that the first position of the benzimidazole is alkylated.
Although the proton at position 2 of the benzimidazole was ob-
served around 8.10 ppm, it shifted downfield by about
0.70–0.90 ppm due to the electron‐withdrawing chlorine and nitro
substituents in compounds 4 and 5. In compounds 7–11, which
converted to a salt structure by alkylation of the third position of
benzimidazole, the proton at position 2 shifted to the range of
9.48–9.87 ppm. These values show a downfield shift of about
0.7–1.5 ppm, compared with the corresponding 1‐substituted
benzimidazoles. The peak of carbon at position 2 of benzimida-
zole salts is generally reported in the range of 142 4 ppm in the
literature, and the values in our present study are in agreement
with the values in the literature.[31]
containing cyano group at the third position of benzimidazole moiety
has less effect on healthy lung cell lines (BEAS‐2B cell lines).
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2.2.2
Antimicrobial studies
The minimum inhibitory concentration (MIC) test was used to eval-
uate the antibacterial and antifungal activities of the compounds
against bacteria and yeasts, respectively. As shown in Table 3,
compound 1 had no antimicrobial activity against the microorgan-
isms. All the compounds showed low antibacterial activity against
Escherichia coli with MIC values equal or above 800 µg/ml. Therefore,
among the bacteria used, E. coli was the most resistant bacterium
against these compounds. Compounds 7, 8, and 11 were the most
effective compounds against E. coli. However, compound 12 was
detected as the most effective compound against Staphylococcus
aureus and Pseudomonas aeruginosa with MIC values of 100 and
200 µg/ml, respectively. Furthermore, the MIC value of compound 7
was also 200 µg/ml against S. aureus. Compounds 7, 8, and 11 were
the most effective compounds against the microorganisms, and
yeasts were highly sensitive to these compounds with the MIC va-
lues of 25–100 µg/ml. The MIC values of compound 7 for Candida
albicans and Candida tropicalis were in the range of 50–100 µg/ml.
However, compounds 8 and 11 with the MIC values of 25 µg/ml
showed the highest antifungal activity against these yeasts. When
the SAR of compounds 7, 8, 11, and 12 with the highest antimicrobial
activity are examined, it is seen that at position 5 of the benzimi-
dazole ring, electron‐releasing methyl or electron‐withdrawing
chlorine and nitro substituents contribute positively to anti-
microbial activity, compared with those without substituents (-
Table 3, compounds 6, 9, and 10). However, in benzimidazole metal
complexes, compound 12 without substituents at the fifth position of
the benzimidazole ring exhibited the highest antibacterial activity,
whereas its antifungal activity was found to be lower than those
without complexes. When the SAR of compounds 12, 13, and 14 was
examined, cobalt atom contributed more positively to antimicrobial
activity than the zinc atom.
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2.2
Pharmacology/biology
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2.2.1
Cytotoxicity studies
Lung adenocarcinoma (A549) and healthy lung bronchial epithelium
(BEAS2B) cells were incubated with increasing concentrations
(0–100 µg/ml) of the mentioned benzimidazole derivatives for 24, 48,
and 72 h with the aim of investigating the cytotoxic effects of the
compounds on these cells. After incubation periods (24, 48, and
72 h), cytotoxicity was evaluated colorimetrically by MTT assay.
Tables 1 and 2 show the IC50 (concentration required to inhibit tu-
mor cell proliferation by 50%) values of the compounds. Among the
10 compounds tested, according to the result of the 72‐h incubation,
whereas compound 12 is the most cytotoxic against A549 cell line, it
has as much cytotoxic effect as cisplatin on BEAS2B cell line. The
IC50 value of compound 12 was 3.98 µg/ml for A549 and 2.94 µg/ml
for BEAS2B. The IC50 value of cisplatin was 6.75 µg/ml for A549 and
2.75 µg/ml for BEAS2B. Compounds 10, 8, 7, and 13 showed a toxic
effect against A549, but they were less toxic than cisplatin against
BEAS‐2B. Abdel‐Mohsen et al.[32] developed benzimidazole con-
jugates with pyrimidine and explored their anticancer activities
against 12 carcinoma cell lines (KB, SK OV‐3, SF‐268, NCI H460,
RKOP27, HL60, U937, K562, G361, SK‐MEL‐28, GOTO, NB‐1).
When the structure–activity relationships (SAR) of the compounds
are examined, it is understood that benzimidazole–cobalt complexes
12 and 14 show higher cytotoxicity than other benzimidazole deri-
vatives (Table 1). This effect could possibly be due to the para-
magnetic property of cobalt. When the benzimidazole structures
showing high cytotoxicity in the second and third ranks are ex-
amined, it is seen that the alkyl group at the third position of ben-
zimidazole in compounds 9 and 10 contains oxygen atom and cyano
group, which can increase the nucleophilic property, respectively.
When Table 2 is examined, it is seen that among compounds 9, 10,
12, and 14 showing high cytotoxic activity, the alkyl structure
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3
CONCLUSION
Of the ten compounds tested, based on the 72‐h incubation results,
compound 12 was the most cytotoxic against the A549 cell line,
whereas in the BEAS2B cell line, it was as cytotoxic as cisplatin. The
IC50 value of compound 12 was 3.98 and 2.94 µg/ml for A549 and
BEAS2B, respectively. Cisplatin values were 6.75 and 2.75 µg/ml for
A549 and BEAS2B, respectively. The antimicrobial activity of the
compounds was investigated based on MIC values, and compounds 8
and 11 showed the highest antifungal activity against yeasts with an
MIC value of 25 µg/ml. Compound 12 showed better activity against
P. aeruginosa and S. aureus than it did against E. coli. All of the com-
pounds except compounds 12 and 13 generally showed higher an-
timicrobial activity against yeasts, compared with bacteria.

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TABLE 1 IC50 (µg/ml) values of benzimidazole derivatives
TABLE 2 IC50 (µg/ml) values of benzimidazole derivatives
against A549 cells
against BEAS‐2B cells
Time (h)
24
Time (h)
24
Compounds
1
48
72
Compounds
1
48
72
66.47
51.31
38.11
62.33
49.14
35.79
6
7
>100
30.83
>100
36.26
83.40
38.51
6
7
>100
86.35
61.36
61.16
57.89
63.34
74.13
8
65.85
35.49
28.49
8
53.46
43.73
9
57.84
79.03
37.09
37.39
26.22
24.35
9
37.96
>100
22.66
>100
28.55
87.63
10
10
11
12
>100
4.69
98.25
3.17
13.56
2.94
11
12
71.00
12.45
48.46
4.58
64.35
3.98
13
98.11
71.85
60.70
13
83.18
83.69
68.53
14
46.00
15.12
24.19
14
20.21
12.76
9.26
Cisplatin
4.72
2.79
2.75
Cisplatin
23.55
6.48
6.75

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TABLE 3 MIC values (µg/ml) of benzimidazole complexes
Compounds
1
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
Candida albicans
Candida tropicalis
–
–
–
–
–
6
7
1600
800
800
400
1600
200
400
50
400
100
8
800
400
400
25
25
9
1600
1600
400
800
400
100
800
100
800
10
1600
11
12
800
400
200
400
100
25
25
1600
400
400
13
1600
800
1600
800
800
(Continues)

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TABLE 3 (Continued)
Compounds
14
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
Candida albicans
Candida tropicalis
1600
800
800
400
800
Gentamicin
Fluconazole
0.78
0.39
3.12
–
–
–
–
–
0.39
0.39
Abbreviation: MIC, minimum inhibitory concentration.
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4
EXPERIMENTAL
Chemistry
4.1.3
Synthesis of 1‐trimethylsilylmethyl‐5(6)‐
chlorobenzimidazole (4)
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4.1
(Chloromethyl)trimethylsilane (0.92 ml, 6.55 mmol) was added to a
mixture of 5(6)‐chlorobenzimidazole (1.0 g, 6.55 mmol) and KOH
(0.37 g, 6.55 mmol) in EtOH (10 ml), and the mixture was heated
under reflux for 4 h. The mixture was then cooled, and potassium
chloride was filtered off and washed with a little EtOH. The solvent
was then removed from the filtrate in vacuum. The residue was
washed twice with water (20 ml) and crystallized from EtOH/DMF
(2:1). Yellow oily compound 1 was obtained in moderate yield
(1.04 g, 66%). M.p.: 231–233°C; υ(CN) = 1577 cm−1. Anal. found: C,
55.18; H, 6.24; N, 11.48%. Calculated for C11H15N2ClSi: C, 55.33;
H, 6.33; N, 11.73%. 1H NMR (400 MHz, DMSO) δ 8.80 (s, 1H,
NCHN), 7.96 (d, 1H, Ar–H, J = 8 Hz), 7.64 (d, 1H, Ar–H, J = 8 Hz),
3.97 (s, 2H, CH2Si), −0.08 (s, 9H, [(CH3)3Si]). 13C NMR (101 MHz,
DMSO) δ 145.3 (NCHN), 142.3, 133.4, 127.4, 122.2, 119.7, and
113.7 (Ar–C), 39.3 (CH2Si) and −0.7 [(CH3)3Si].
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4.1.1
General
The starting chemicals and solvents used in this study were commer-
cially supplied from Sigma‐Aldrich, Acros, Merck, and Tekkim compa-
nies. The human lung adenocarcinoma (A549) cancer cell line was
provided by Prof. Dr. Fikrettin Sahin (Yeditepe University, Department
of Genetics and Bioengineering, Istanbul/Turkey). All bacteria and
yeasts used in this study were obtained from stock cultures in the
Biotechnology Laboratory, Inonu University. Nuclear magnetic re-
sonance (1H NMR, 13C NMR) spectra (see the Supporting Information)
were recorded using a Bruker Advance III 400 MHz spectrometer in
DMSO‐d6. Chemical shifts are reported in parts per million (ppm) and
the coupling constants (J) are expressed in Hertz (Hz). Elemental
analyses were performed by LECO CHNS‐932 elemental analyzer.
Infrared spectra were recorded with ATR equipment in the range of
Compound 5 was synthesized according to the method used in
4000–650 cm−1 on
a
PerkinElmer Spectrum one FTIR spectro-
the synthesis of compound 4. M.p.: 128–129°C; υ(CN) = 1579 cm−1
Anal. found: C, 61.68; H, 5.50; N, 13.52%. Calculated for
16H17N3O2Si: C, 61.71; H, 5.50; N, 13.49%. 1H NMR (400
.
photometer. Melting points (mp) were measured in open capillary
tubes and were uncorrected, using a Gallenkamp MPD350.BM3.5
apparatus. Compounds 1,[25] 2,[25] 3,[26] 6,[27] 12,[25] 13,[25] and 14[25]
published in our previous studies were synthesized again and used
after their purity was checked with the data in the literature.
The InChI codes of the new compounds, together with some
biological activity data, are provided as Supporting Information.
C
MHz, DMSO) δ 8.83 (s, 1H, NCHN), 7.40 (s, 1H, Ar–H), 8.21 (d, 1H,
Ar–H, J = 8 Hz), 8.13 (d, 1H, Ar–H, J = 8.0 Hz), 7.54–7.28 (m, 5H,
Ar–H), 4.30 (s, 2H, CH2Si), 0.35 (s, 6H, [(CH3)2SiPh]). 13C NMR
(101 MHz, DMSO) δ 146.7 (NCHN), 144.9, 134.7, 133.9, 131.6,
128.0, 127.6, 122.9, 121.6, 115.1, 110.1 (Ar–C), 38.1 (CH2Si), −3.9
[(CH3)2SiPh].
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4.1.2
General procedure for the synthesis of
compounds 1–5[24]
4.1.4
General procedure for the synthesis of
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compounds 6–11
Appropriate alkyl halide (0.01 mol) was added to a mixture of ap-
propriate benzimidazole (0.01 mol) and KOH (0.01 mol) in EtOH
(10 ml), and the resulting mixture was refluxed for 4 h. The mixture
was then cooled, and potassium halide was filtered off and washed
with a little EtOH. The solvent was then removed from the filtrate in
vacuum. The residue was washed twice with water (20 ml) and
crystallized from EtOH/DMF (2:1) to yield desired compounds 1–5.
A mixture of an appropriate 1‐substituted benzimidazole (0.010 mol)
and an appropriate alkyl halide (0.011 mol) in dimethylformamide
(DMF) (5 ml) was heated under reflux for 3 h. The mixture was then
cooled and the volatiles were removed under vacuum. The residue
was crystallized from a DMF/ethanol mixture (1:1) to yield target
compounds 6–11.

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4.1.5
Synthesis of 5‐chloro‐3‐methyl‐1‐
148.1 (NCHN), 134.8, 134.4, 132.2, 131.4, 130.5, 128.4, 126.9, 126.6,
120.3, 114.5 (Ar–C), 113.9 (NC), 45.8 (CNCH2), 38.0 (CH2Si), 25.2
(CH2N), 14.1 (CH2CH2)CH2, −3.7 [(CH3)2SiPh].
[(trimethylsilyl)methyl]‐2,3‐dihydro‐1H‐benzo[d]‐
imidazolium iodide (7)
A mixture of 1‐trimethylsilylmethyl‐5(6)‐chlorobenzimidazole (1.1 g,
4.60 mmol) and iodomethane (0.30 cm3, 4.80 mmol) in DMF (5 ml)
was heated under reflux for 3 h. The mixture was then cooled and
the volatiles were removed under vacuum. The residue was crys-
tallized from a DMF/ethanol mixture (1:1). White crystals of the title
compound 7 (1.34 g, 76%) were obtained, m.p. 201–202°C; υ(CN) =
1559 cm−1. Anal. found: C, 37.34; H, 4.53; N, 7.05%. Calculated for
C12H18N2ClISi: C, 37.86; H, 4.77; N, 7.36%. 1H NMR (400 MHz,
DMSO) δ 9.49 (s, 1H, NCHN), 8.17 (s, 1H, Ar–H, 8.02 (d, 1H, Ar–H,
J = 8.0 Hz), 7.64 (d, 1H, Ar–H, J = 8.0 Hz), 4.11 (s, 2H, CH2Si), 3.96 (s,
3H, CH3), −0.00 (s, 9H, [(CH3)3Si]). 13C NMR (101 MHz, DMSO) δ
146.5 (NCHN), 144.9, 134.7, 133.0, 129.0, 117.8, 115.8 (Ar–C), 40.00
(CH2Si), 35.73 (CH3), −0.44 [(CH3)3Si].
1‐{[Dimethyl(phenyl)silyl]methyl}‐3‐methyl‐5‐nitro‐1H‐benzo[d]‐
imidazol‐3‐ium iodide (11)
M.p.: 169–170°C; υ(C═N) = 1630 cm−1. Anal. found: C, 62.48; H, 6.18; N,
12.76. Calculated for C17H20IN3O2Si: C, 62.55; H, 6.18; N, 12.87. 1H
NMR (400 MHz, DMSO) δ 9.74 (s, 1H, NCHN), 9.04 (s, 1H, Ar–H), 8.41
(d, 1H, Ar–H, J = 8 Hz), 8.13 (d, 1H, Ar–H, J = 8 Hz), 7.94 (d, 1H, Ar–H,
J = 8.0 Hz), 7.55–7.26 (m, 5H, Ar–H), 4.50 (s, 2H, CH2Si), 4.16 (s, 3H,
NCH3), 0.42 (s, 6H, [(CH3)2SiPh]. 13C NMR (101 MHz, DMSO) δ 147.8
(NCHN), 145.5, 135.3, 134.1, 131.5, 130.4, 128.5, 128.4, 120.9, 115.5,
110.9 (Ar–C), 38.6 (CH2Si), 34.5 (NCH3), −3.8 [(CH3)2SiPh].
|
4.1.6
General procedure for the synthesis of
Compounds 8, 9, 10, and 11 were synthesized according to the
method used in the synthesis of compound 7.
compounds 12–14[25]
A solution of an appropriate 1‐substituted benzimidazole (0.02 mol)
and cobalt or zinc(II) chloride (0.01 mol) in DMF (5 ml) was heated
under reflux for 2 h. The mixture was then cooled to room tempera-
ture, and then the solvent was removed from the filtrate in vacuo. The
resulting precipitate was then crystallized from EtOH/DMF (2:1).
3‐Ethyl‐5‐methyl‐1‐[(trimethylsilyl)methyl]‐1H‐benzo[d]imidazol‐3‐
ium iodide (8)
M.p.: 97–98°C; υ(CN) = 1576 cm−1. Anal. found: C, 67.92; H, 9.37; N,
11.44%. Calculated for C14H23N2ISi: C, 67.96; H, 9.37; N, 11.32%. 1H
NMR (400 MHz, DMSO) δ 9.48 (s, 1H, NCHN), 7.85 (d, 1H, Ar–H,
J = 8 Hz), 7.79 (s, 1H, Ar–H), 7.41 (d, 1H, Ar–H, J = 8 Hz), 4.41 (q, 2H,
CH2CH3, J = 8 Hz), 4.06 (s, 2H, CH2Si), 2.43 (s, 3H, CH3), 1.41 (t, 3H,
CH2CH3, J = 8 Hz), 0.00 (s, 9H, [(CH3)3Si]). 13C NMR (101 MHz, DMSO)
δ 142.3 (NCHN), 138.6, 134.3, 131.1, 130.1, 115.3, 115.0 (Ar–C), 44.1
(CH2CH3), 39.9 (CH2Si), 23.4 (CH3), 16.7 (CH2CH3), −0.50 [(CH3)3Si].
|
4.2
Biological assays
|
4.2.1
Cytotoxicity study
In this study, the human lung adenocarcinoma (A549) provided by Prof.
Dr. Fikrettin Sahin (Yeditepe University, Department of Genetics and
Bioengineering, Istanbul/Turkey) and healthy human lung bronchial
epithelium (BEAS‐2B) cancer cell lines were used. The cells were
maintained in Dulbecco's modified Eagle's medium growth medium
containing 10% fetal bovine serum and 1% penicillin/streptomycin at
37°C in 5% CO2. The cells were seeded at 5 × 103 cells/well in a 96‐well
plate. After 24 h, cells were exposed to the compounds (range con-
centration, 0–100 µg/ml) prepared in DMSO (in the final culture medium
was <0.1%) and the cells were incubated for 24, 48, and 72 h. The cells
were, then, treated with 20 µL of MTT for 4 h at 30°C to evaluate
cytotoxic activity of the analyzed compounds. The purple formazan
crystals formed by alive cells were solubilized in DMSO solution and
optical density was measured at 570 nm. For each concentration, 12
wells were used and IC50 values (µg/ml) were defined as the compound
concentrations that reduced the absorbance by 50% with respect to
control values. Cisplatin was also used as a control agent.
1‐{[Dimethyl(phenyl)silyl]methyl}‐3‐(2‐ethoxyethyl)‐1H‐benzo[d]‐
imidazol‐3‐ium chloride (9)
M.p.: 121–122°C; υ(C═N) = 1590 cm−1. Anal. found: C, 70.69; H, 8.01; N,
8.29%. Calculated for C20H27N2OSi: C, 70.75; H, 8.02; N, 8.25%. 1H
NMR (400 MHz, DMSO) δ 9.73 (s, 1H, NCHN), 8.07 (d, 1H, Ar–H,
J = 8 Hz), 7.88 (d, 1H, Ar–H, J = 8.0 Hz), 7.62 (d, 1H, Ar–H, J = 8.0 Hz),
7.56–7.50 (m, 6H, Ar‐H), 4.70 (q, 2H, CH2CH3, J = 4 Hz), 4.51 (s, 2H,
CH2Si), 3.78 (t, 2H, OCH2, J = 4 Hz), 3.43 (t, 2H, NCH2, J = 4 Hz), 1.03 (t,
3H, CH2CH3, J = 4 Hz), −0.39 (s, 6H, [(CH3)2PhSi]). 13C NMR (101 MHz,
DMSO) δ 141.9 (NCHN), 134.8, 134.1, 131.9, 131.4, 130.4, 128.4,
126.9, 126.5, 114.4, 114.1 (Ar–C), 66.1 (CH2CH3), 47.1(CH2Si), 37.9
(CH2CH2O), 34.2 (CH2CH2O), 15.6 (CH3), −4.0 [(CH3)2PhSi].
3‐(3‐Cyanopropyl)‐1‐{[dimethyl(phenyl)silyl]methyl}‐1H‐benzo[d]‐
imidazol‐3‐ium chloride (10)
M.p.: 108–109°C; υ(C≡N) = 2200 cm−1, υ(C═N) = 1615 cm−1. Anal. found:
C, 71.73; H, 7.22; N, 12.62%. Calculated for C20H24N3ClSi: C, 71.81; H,
7.23; N, 12.56%. 1H NMR (400 MHz, DMSO) δ 9.87 (s, 1H, NCHN),
8.11 (d, 1H, Ar–H, J = 8 Hz), 7.94 (d, 1H, Ar–H, J = 8.0 Hz), 7.67–7.54 (m,
4H, Ar–H), 7.53–7.33 (m, 3H, Ar–H), 4.61 (t, 2H, CH2CN, J = 6 Hz), 4.46
(s, 2H, CH2Si), 2.65 (t, 2H, CH2N, J = 8 Hz), 2.19 (t, 2H, CH2CH2CH2,
J = 6 Hz), −0.43 (s, 6H, [(CH3)2SiPh]). 13C NMR (101 MHz, DMSO) δ
|
4.2.2
Antimicrobial study
Bacteria E. coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa
ATCC 27853, and yeasts C. albicans and C. tropicalis were used to test

8 of 8
KÜÇÜKBAY ET AL.
|
the antimicrobial activity. These bacteria and yeasts are stock cul-
tures in the Biotechnology Laboratory, Inonu University.
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40, 393. https://doi.org/10.3906/kim-1510-15
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Ö. Yesilada, Y. Baran, J. Organomet. Chem. 2017, 828, 52. https://
doi.org/10.1016/j.jorganchem.2016.11.020
In this study, MIC values of the studied compounds were in-
vestigated to detect their antibacterial and antifungal activities, and
thus to determine the sensitivity of the microorganisms. All the
compounds used were prepared by dissolving them in DMSO, and
their serial dilutions were made in sterile 96‐well microplates. The
cultures of the microorganisms were prepared by growing bacteria
and yeasts in Mueller–Hinton agar and Sabouraud dextrose agar
media, respectively. Pure colonies from these cultures were used to
prepare the cell suspensions based on the McFarland standard. An
appropriate amount from these suspensions was inoculated into each
well. Sterility control and growth control wells were also used. The
plates were incubated at 37°C for 24 h for bacteria and 48 h for
fungi, respectively. The lowest concentration of each compound with
no growth was recorded as the MIC value. Gentamicin was used as a
positive control against bacteria and fluconazole for yeasts.
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ACKNOWLEDGMENT
[21] J. Akhtar, A. A. Khan, Z. Ali, R. Haider, M. S. Yar, Eur. J. Med. Chem.
2017, 125, 143. https://doi.org/10.1016/j.ejmech.2016.09.023
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https://doi.org/10.1016/j.ejmech.2010.11.007
The authors acknowledge İnönü University, Malatya, Turkey, for fi-
nancial support.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
[24] A. F. Pozharskii, A. M. Simonov, Russ. J. Gen. Chem. 1963, 33, 172.
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ORCID
Hasan Küçükbay
Mustafa Uçkun
http://orcid.org/0000-0002-7180-9486
https://orcid.org/0000-0001-5486-6684
[26] H. Küçükbay, N. Şireci, Ü. Yilmaz, S. Deniz, M. Akkurt, Z. Baktir,
O. Büyükgüngör, Turkish J. Chem. 2012, 36, 201. https://doi.org/10.
3906/kim-1109-5
Elif Apohan
https://orcid.org/0000-0002-9074-0525
https://orcid.org/0000-0003-0038-6575
Özfer Yeşilada
[27] Ü. Yılmaz, H. Küçükbay, N. Şireci, M. Akkurt, S. Günal, R. Durmaz,
M. N. Tahir, Appl. Organomet. Chem. 2011, 25, 366. https://doi.org/
10.1002/aoc.1772
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How to cite this article: H. Küçükbay, M. Uçkun, E. Apohan,
Ö. Yeşilada. Cytotoxic and antimicrobial potential of
benzimidazole derivatives. Arch. Pharm. 2021;e2100076.
https://doi.org/10.1002/ardp.202100076
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