Journal of the Indian Chemical Society 101 (2024) 101445

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Journal of the Indian Chemical Society

journal homepage: www.journals.elsevier.com/journal-of-the-indian-chemical-society

Cytotoxic activity properties of a benzimidazolium salt against lung, liver,

and colon cancer cell lines
Senem Akkoc a,b,*
a
Suleyman Demirel University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 32260, Isparta, Turkiye
b
Bahcesehir University, Faculty of Engineering and Natural Sciences, 34353, Istanbul, Turkiye

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, a known benzimidazolium salt was synthesized in two steps starting from N-phenyl-o-phenyl­
Benzimidazolium salt enediamine. This compound was tested in vitro for 72 h against different cancer cell lines (A549 - lung cancer,
Cancer HepG2 - liver cancer, DLD-1 - colon cancer) and to compare the activity of this compound with cisplatin, a widely
Cytotoxic activity
used chemotherapeutic agent. The evaluation of compound 4 as a potential anticancer agent against different
cancer types was achieved by examining the cytotoxic effects of this compound through IC50 values using the
MTT method. It was found that compound 4 showed different activity depending on the cell type and had a high
cytotoxic effect (IC50: 15.68 μM), especially against lung cancer cell line for 72 h incubation time.

1. Introduction analgesic [12], anticonvulsant [13], antidiabetic [13] and antifungal

[14]. In addition, there are active ingredients containing benzimidazole
Cancer, one of the leading causes of death worldwide, is caused by nuclei in the structure of some drugs with high therapeutic effects and
various abnormalities in the DNA sequence. Cancer can be broadly market value used in the clinic. Abemaciclib used in the treatment of
described as a condition characterized by the uncontrolled growth of early-stage breast cancer, and bendamustine used in the treatment of
abnormal cells that can invade and spread to other areas of the body. chronic lymphocytic leukemia, non-Hodgkin lymphoma, and multiple
The growth, spread, and metabolite production of cancer cells cause myeloma are among the active substances belonging to the group of
disorders in tissues. This leads to disruption of normal body functions. antineoplastic agents. Emedastine used in seasonal allergic conjuncti­
Cancer incidence and mortality are increasing rapidly worldwide vitis and bilastine used in symptomatic treatment of allergic rhino­
[1]. Cancer is one of the leading causes of death in both economically conjunctivitis and urticaria are antihistamine agents. Fig. 1 shows the
underdeveloped countries and economically developed countries. molecular structure of some of these active substances.
Smoking, poor eating habits, lack of physical activity, and advanced age In here, a known benzimidazolium salt was prepared and tested in
at first birth are just some of the factors known to increase the risk of different cancer cell lines including lung, liver and colon. The results
cancer [2]. demonstrated that this compound had cytotoxic effect in the lung
Ring systems containing at least one heteroatom other than (A549) cancer cell line with an IC50 value of 15.68 μM.
hydrogen and carbon atoms are called heterocyclic compounds. Benz­
imidazole is a heterocyclic compound formed by the fusion of the 2. Experimental SECTION
imidazole ring with the benzene ring at the 4,5 position. The benz­
imidazole core has a structure that shows important biological activity 2.1. Synthesis of N-phenylbenzimidazole
properties. Anticancer agents, which can also be called antiproliferative,
antitumor, or antineoplastic, are used in the treatment of various types A 20 % excess of N,N-dimethylformamide dimethylacetal (32.61
of cancer through different mechanisms. mmol) was added to N-phenyl-o-phenylenediamine (27.17 mmol)
Benzimidazole ring-containing compounds have antibacterial [3], (Fig. 2) [15]. The mixture was stirred at water bath temperature for 3 h,
antioxidant [4], anticancer [5,6], antiparasitic [7], antiviral [8], anti­ allowing MeOH and HNMe2 to evaporate. The residue was removed by
hypertensive [9], antihelminthic [10], anti-inflammatory [11], distillation in vacuo. The product was obtained with a 93 % yield.

* Suleyman Demirel University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 32260, Isparta, Turkiye.
E-mail address: [email protected].

https://doi.org/10.1016/j.jics.2024.101445
Received 30 September 2024; Accepted 22 October 2024
Available online 22 October 2024
0019-4522/© 2024 Indian Chemical Society. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar
technologies.
S. Akkoc Journal of the Indian Chemical Society 101 (2024) 101445

Fig. 1. Some active substances containing benzimidazole nucleus.

Table 1
IC50 results for 4 and cisplatin in human cancer cell lines.
Compounds IC50 (μM)

A549 HepG2 DLD-1

4 15.68 365.4 1497.0

Cisplatin 9.879 37.32 N.T.*

N.T.*: Not tested.

Fig. 2. The synthesis scheme of N-phenyl-o-phenylenediamine. glutamax. Cells, which were seeded at 5 × 103 cells/well density, were
exposed to compound 4 and cisplatin at 200, 100, 50, and 25 μM con­
centrations for 72 h [16,17]. Absorbance values at 590 nm were
measured on an Elisa reader.

3. Results and discussion

3.1. Cytotoxic activity studies

The cytotoxic effects of compound 4 tested in the study in different

cancer cell lines for 72 h were evaluated according to IC50 (half maximal
inhibitory concentration) values (Table 1). These values indicate the
concentration at which the compound can kill 50 % of the cells and are
Fig. 3. The synthesis scheme of a benzimidazolium salt 1H NMR (300 MHz,
an important parameter for measuring efficacy. Cisplatin was used as a
DMSO‑d6), δ: 10.26 (s, 1H, NCHN); 7.70–8.27 (m, 9H, Ar–H); 4.69 (t, 2H, J:
reference drug and the efficacies of these two agents were compared.
5.08 Hz, NCH2CH2OH); 3.94 (t, 2H, J: 4.93 Hz, NCH2CH2OH); 3.47 (s, 1H,
Compound 4 exhibited a moderate cytotoxicity with an IC50 value of
NCH2CH2OH). 13C NMR (75 MHz, DMSO‑d6), δ: 143.3 (NCHN); 113.9, 114.9,
125.7, 127.3, 127.8, 130.9, 131.9, 132, and 133.6 (Ar–C); 59.2 (NCH2CH2OH); 15.68 μM in the lung cancer cell line. This value indicates that the
50.4 (NCH2CH2OH) [15]. IR ν(CN) = 1554.14 cm− 1. compound has a significant effect against A549 cells. Cisplatin showed a
stronger cytotoxic effect with an IC50 value of 9.879 μM in the same cell
2.2. 1-Phenyl-3-(2-hydroxyethyl)benzimidazolium bromide, 4 line. This result reveals that cisplatin is more effective than compound 4
in A549 cell line. These results suggest that compound 4 may be a po­
This known compound was synthesized according to the literature tential drug candidate for the treatment of lung cancer, but its efficacy
[15]. 2-Bromoethanol (10.13 mmol) was added to a solution of N-phe­ remained slightly lower compared to the positive control.
nylbenzimidazole (10.10 mmol) in 5 mL of DMF and stirred at 60 ◦ C for The IC50 value of compound 4 in the liver cancer cell line was
18 h (Fig. 3). After cooling the reaction mixture to room temperature, determined as 365.4 μM. This is a very high value and expresses that
diethyl ether (15 mL) was added and the salt was precipitated, filtered, compound 4 has a low cytotoxic effect against HepG2 cells. In other
and dried under vacuum. Compound 4 was purified by crystallization in words, this result shows that the compound is ineffective in liver cancer
an ethyl alcohol-diethyl ether (1:2) mixture. The product was obtained cells. The IC50 value of cisplatin in the HepG2 cell line is 37.32 μM,
in a high yield of 89 %. which indicates that it is much more effective compared to compound 4.
These findings suggest that compound 4 may not have a specific target
against HepG2 cells due to its limited effect against liver cancer.
2.3. The cytotoxic activity studies The IC50 value of compound 4 in the DLD-1 cell line is a very high
value of 1497 μM. This indicates that the compound is almost inactive in
Lung (A549), liver (HepG2), and colon (DLD-1) cancer cell lines, colon cancer cells. Such a high IC50 value shows that compound 4 re­
which were purchased from American Type Culture Collection (ATCC, quires very high concentrations for a cytotoxic effect to occur in colon
USA) were cultured in DMEM supplemented with 10 % FBS and 1 % cancer cells.

2
S. Akkoc Journal of the Indian Chemical Society 101 (2024) 101445

relatively lower cytotoxicity in the HepG2 cell line. Cisplatin, on the

other hand, showed a significantly stronger effect in the HepG2 cell line.
The viability rate, which was 65.55 % at 25 μM, decreased to 13.77 % at
200 μM of cisplatin. These data reveal that cisplatin is much more
effective than compound 4 in HepG2 cells and suppresses the prolifer­
ation of cancer cells more strongly.
Compound 4 showed the lowest cytotoxic effect in the DLD-1 cell line
compared to other cell lines. Cell viability, which was 99.36 % at 25 μM,
decreased to only 88.59 % with increasing concentration up to 200 μM.
These results indicate that compound 4 has a very limited cytotoxic
effect on DLD-1 cells and exhibits a lower inhibition rate than other cell
lines.

4. Conclusion

N-phenylbenzimidazole was synthesized by heating N-phenyl­

orthophenylenediamine with N,N-dimethylformamide dimethylacetal.
As a result, a benzimidazolium salt with phenyl substituent was pre­
pared in two steps by alkylation of N-phenylbenzimidazole with 2-bro­
moethanol. The results of this study revealed that the cytotoxic
activity of compound 4 varied significantly depending on the cell type.
While compound 4 was relatively effective on lung cancer cells (A549),
its activity on liver (HepG2) and colon cancer (DLD-1) cells was found to
be limited for the concentrations studied.

Conflict of interest

The authors have no conflict of interest to declare.

Ethics committee approval

The author declares that the ethics committee approval is not

required for this study.

Declaration of competing interest

There is no declaration of any potential competing interests in this

study.

Acknowledgements

The author would like to thank the Suleyman Demirel University

Research Fund (TSG-2024-9516) in Turkiye for their financial support.

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