In vitro cytotoxicity in A549, Hepg2, MCF-7, and DLD-1 cancer cell lines and ADME/toxin analysis of a benzimidazole derivative

2.1 Synthesis of a N-Fenilbenzimidazol

N-Phenyl-o-phenylenediamine (5 g; 27.17 mmol) was mixed with a 20% excess of N,N-dimethylformamide dimethylacetal (3.89 g; 32.61 mmol). To enable the elimination of MeOH and HNMe₂, it was heated for three hours in a water bath. The remaining yellow oily residue was distilled off in vacuo.

2.2 Synthesis of 1-Phenyl-3-naphthalenomethylbenzimidazolium chloride, se-182

The palladium metal complex of this substance was published in the article, but the NMR information of this substance was not provided (Akkoç et al., 2014). In 5 milliliters of dimethyl ether (DMF), 1.15 gr of 1-chloromethylnaphthalene (6.52 mmol) was combined with 1.27 gr of N-phenylbenzimidazole (6.55 mmol) and stirred at 60°C for 18 hours. The salt was precipitated using 15 mL of diethyl ether when the reaction liquid cooled. The salt was subsequently filtered and vacuum dried. The se-182 product underwent crystallization purification with a 1:2 ratio of ethyl alcohol to diethyl ether (Fig. 1).

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Fig. 1.

Synthesis scheme of a benzimidazolium salt.

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Yield: 93%, m.p.: 220-221°C. IR ν_(CN)= 1595.98 cm^-1. ¹H NMR (300 MHz, CDCl₃), δ: 6.65 (s, 2H, NCH₂C₁₀H₇); 7.28-7.89 (m, 16H, Ar-H); 11.70 (s, 1H, 2-CH). ¹³C NMR (75.47 MHz, CDCl₃), δ: 49.6 (NCH₂C₁₀H₇); 122.8, 124.9, 125.4, 126.5, 127.5, 127.7, 128.1, 128.4, 129.1, 130.2, 130.7, 130.9, 131.3, 131.7, 133, and 133.9 (Ar-C); 143.4 (2-CH).

2.3 Diphenyltetrazolium bromide (MTT) test analysis of growth inhibition

The cytotoxicity of the benzimidazole derivative se-182 was assessed in four lines, i.e., HepG2, A549, MCF-7, and DLD-1 utilizing a typical MTT reduction test. The cell lines were obtained from the American Type Culture Collection (ATCC) in the United States and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% GlutaMAX. In contrast, Michigan Cancer Foundation-7 (MCF-7) cells were cultured in Roswell Park Memorial Institute (RPMI) medium instead of DMEM. The selection of cell culture medium was based on the distinct needs of each cell line to ensure maximum development and physiological relevance. The DMEM and RPMI media are extensively used, and each has been formulated to address specific cellular requirements. The literature indicates that DMEM has been used for the cultivation of HepG2, A549, and DLD-1 cells (Akkoc, 2024). Conversely, RPMI was used for the growth of MCF-7 cells. Consequently, these two media were used in cell culture in our study.

The synthesized compound (se-182) was dissolved in 0.5% dimethyl sulfoxide (DMSO) and administered to the cells. Similarly, the negative control cells were subjected to DMSO at the same ratio. Cisplatin was dissolved in phosphate-buffered saline. Cells sown at a density of 5 × 10³ cells/well were subjected to treatment with cisplatin and se-182 for 72 hours, at concentrations varying from 25 to 200 μM (Akkoc and Muhammed, 2024). The same conditions were used to cultivate cells that contained cisplatin (positive control drug). The treated cells’ old media were swapped out for new media after a 72-hr incubation period. Each well was filled with 5 mg/mL of MTT reagent in DMEM, and the cells were then incubated for a further 2-3 hours at 37°C. Following the treatment, each well received 200 μL of DMSO after the supernatants were properly removed. Using a multiwall plate reader, absorbance was measured at 590 nm. Drug sensitivity was reported as the drug dose needed to lower cell viability by 50% (IC₅₀).

2.4 ADME and toxicity (ADMET) properties

Utilizing the ChemBioDraw Ultra 14.0 program, the SMILES (Simplified Molecular Input Line Entry System) notation for the synthetic chemical se-182, C12=CC=CC=C1N(C3=CC=CC=C3)C=[N+]2CC4=CC=CC5=C4C=CC=C5.[Cl-], was produced. The ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) characteristics were computed using this molecular model. The SwissADME webpage (Daina et al., 2017) was used to compute ADME characteristics, whilst the Protox 3.0 webserver was employed to assess toxicity properties (Banerjee et al., 2018).

3.1 Cytotoxic activity studies

A compound (se-182) and cisplatin were evaluated for their cytotoxicity at 200, 100, 50, and 25 µM concentrations in tested cancer cell lines for 72 hours with an MTT assay. The findings on anticancer efficacy show that se-182 may markedly reduce the viability of all four cell types in a dose-dependent manner. The DLD-1 cell line demonstrated increased viability. The findings have been shown in Table 1 and Fig. 2.

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Fig. 2.

Cell viability assays for A549, HepG2, MCF-7, and DLD-1 cells. The graphs indicate the rate of viability for relevant cells after 72-hour exposure to different compounds (se-182 and cisplatin) at varying concentrations (25, 50, 100, 200 µM).

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The MTT test utilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to assess cell viability. This test employs mitochondrial dehydrogenase, found in metabolically active cells, to convert yellow MTT into a purple formazan product (Banerji et al., 2013). After treatment with various concentrations of se-182, HepG2 and A549 cells were the most affected cells compared to DLD-1 and MCF-7. Based on the statistical difference between the tested sample concentration and the positive control, cisplatin caused a 50% inhibition (IC₅₀) on the treated cell line’s viability. The viability of A549 cells treated with se-182 was higher than those treated with cisplatin. The se-182 (15.58 µM, p < 0.01) was more potent than cisplatin (37.32 µM) in affecting the viability of HepG2 cell line and exhibited a higher cytotoxic effect with a lower IC₅₀ value. Similarly, se-182 had a cytotoxic effect (65.89 µM, p < 0.01) against the DLD-1 cell line (Table 1). Leica Inverted microscope images of MCF-7 cells have been given below (Figs. 3-6).

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Fig. 3.

Zero-hour images of negative control cells and cells treated with different concentrations (200-25 µM) of compound se-182.

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Fig. 4.

Twenty-four-hour images of negative control cells and cells treated with different concentrations (200-25 µM) of compound se-182.

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Fig. 5.

Forty-eight-hour images of negative control cells and cells treated with different concentrations (200-25 µM) of compound se-182.

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Fig. 6.

Seventy-two-hour images of negative control cells and cells treated with different concentrations (200-25 µM) of compound se-182.

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3.2 ADME and toxicity (ADMET) properties

In pharmacology and pharmacokinetics, the acronym ADME stands for absorption, distribution, metabolism, and excretion. A medicine’s long-term effects on the body may be measured by looking at these four letters, which stand for descriptors. As a result, the drug development sector relies heavily on ADME parameter determination for novel compounds. Physicochemical, lipophilicity, solubility, pharmacokinetic, and drug-likeness characteristics make up these measures. The SwissADME web server was used to determine these markers (Table 2).

When assessing potential novel drugs using ADME metrics, drug-likeness characteristics are crucial. Companies are considering some guidelines for potential new drugs. Pharmacia (Egan), Bayer (Muegge), Pfizer (Lipinski), Amgen (Ghose), and GSK (Veber) are the firms and regulations in question. The most prominent of these rules is the rule of five proposed by Lipinski. Molecular weight, MLOGP, and the number of hydrogen bond acceptors and donors are among the physicochemical and lipophilicity properties of compounds that are considered in Lipinski’s rule of five (Lipinski et al., 2012). Substances must have a molecular weight value that is between 150 and 500 Da. The MLOGP number needs to be < 4.15. It requires < 5 hydrogen bond donor atoms and < 10 hydrogen bond acceptor atoms. In accordance with Lipinski’s Rule of Five, the synthesized molecule se-182 exhibited a molecular weight < 500, a hydrogen bond acceptor count < 10, a hydrogen bond donor count x 5, and an MLOGP of ∼ 4.15. It meets all requirements except for MLOGP.

Toxicity properties of compound se-182 were calculated using the Protox-II web server. The IC₅₀ values of toxic doses are typically expressed in mg/kg of body mass. The IC₅₀ is the exposure threshold at which 50% of the test subjects succumb. Toxicological categories are delineated using the extensively used Global Harmonized System (GHS) for the categorization and labeling of substances. The class was rated first to sixth, signifying was worst to best. When the toxicity properties of compound se-182 were examined in Table 2, it was observed that the IC₅₀ value was 1000 mg/kg, while the toxicity class was fourth.

The boiled-egg model is important for gastric absorption. In the model, substances with white patches are more easily absorbed than those without. The region in yellow represents the fraction of the total mass that is able to cross the blood-brain barrier (BBB). The yellow section of the model represents chemicals that have breached the BBB and may have therapeutic potential for disorders affecting the central nervous system (CNS). By comparing the synthesized molecule se-182 to the boiled egg model, we can see that it falls within the yellow zone, suggesting that it may have use in treating CNS problems.

Before a medicine is formulated for use in the pharmaceutical or clinical trial fields, two fundamental criteria are examined for each drug: HIA and CNS absorption (Majid et al., 2022). Substances must traverse the BBB to influence the CNS. Concurrently, drugs that are inert inside the CNS must be limited to prevent any detrimental effects on it. Consequently, the capacity to surmount this obstacle is vital (Hassan et al., 2022). Reduced BBB permeability and HIA in isolated flavones point to a low risk of detrimental effects on the CNS. Fig. 7 displays the EGG-BOILED curve. This method facilitates the evaluation of a drug’s capacity to traverse the BBB and its HIA. The methodology comprises two primary regions: the HIA and the BBB penetration area (yolk). If any component is inside the gray area, it indicates insufficient HIA or BBB penetration.

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Fig. 7.

Boiled-egg model. PGP: P-glycoprotein..

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