Yavuz BMC Chemistry(2024) 18:146
https://doi.org/10.1186/s13065-024-01241-z

BMC Chemistry

RESEARCH

Open Access

Synthesis of new two 1,2-disubstituted
benzimidazole compounds: their in vitro
anticancer and in silico molecular docking
studies
Sevtap Caglar Yavuz1*
Abstract
In this study, two new molecules were synthesized from the reaction of 2-methyl-1H-benzo[d]imidazole with aryl
halides in the presence of a strong base. The structures newly of synthesized 1,2-disubstituted benzimidazole
compounds were characterized using spectroscopic techniques (FT-IR, 1HNMR, 13CNMR) and chromatographic
technique (LC/MS). For discovering an effective anticancer drug, the developed heterocyclic compounds werescreened against three different human cancer cell lines (A549, DLD-1, and L929). The results demonstrated that of
IC50 values of compound 2a were higher as compared to cisplatin for the A549 and DLD-1 cell lines. The frontier
molecular orbital (FMO), and molecular electrostatic potential map (MEP) analyses were studied by using DFT(density functional theory) calculations at B3LYP/6-31G** level of theory. The molecular docking studies of thesynthesized compound with lung cancer protein, PDB ID: 1M17, and colon cancer antigen proteins, PDB ID: 2HQ6
were performed to compare with experimental and theoretical data. Compound 2a had shown the best binding
affinity with -6.6 kcal/mol. It was observed that the theoretical and experimental studies carried out supported
each other.
Keywords 1,2-disubstituted benzimidazole, DFT, Spartan 10, Anticancer activity, Cytotoxicity studies, ADMET,
Molecular docking

Introduction
Heterocycles are the basis of drug discovery and form thecore structure of approximately 80% of pharmaceuticals[1]. The benzimidazole nucleus, a heterocyclic compoundwith a broad spectrum of biological activity, is considereda biological pharmacophore [2]. There is a wide varietyof drugs available that have benzimidazoles as their core,*Correspondence:
Sevtap Caglar Yavuz
sevtap.yavuz@erzincan.edu.tr
1
Department of Medical Services and Technicians, Ilic Dursun Yildirim
Vocational School, Erzincan Binali Yildirim University, Erzincan, Türkiye

examples of some of the few blockbuster drugs such as
candesartan, omeprazole, and albendazole [3]. Benzimidazole derivatives also easily interact with the biopolymers of living systems, being structural isosteresof inherently consisting nucleotides [4], and are widelyused in the improving of recent curative agents against
many illnesses. Benzimidazole and its derivatives, whichhave been extensively researched in the pharmaceuticalfield, have versatile pharmacological properties that arevery useful in drug development. Many pharmacologicalactivities have been described for benzimidazole derivatives, anti-cancer, anti-tubercular, anti-acetylcholinesterase, anti-viral, anti-protozoal, analgesic, anti-malarial,© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Yavuz BMC Chemistry(2024) 18:146

anti-inflammatory, anti-fungal, and anti-histamine[5–11]. The different activities of benzimidazole and its
derivatives may result from different groups on the benzimidazole nucleus [12].
Cancer is one of the deadliest diseases in the world.
This disease, which begins with the abnormal and uncontrolled growth of cells, destroys body tissue and causes
metastasis [13, 14].
The death rate from cancer is set to almost double
in the coming years, according to an info issued by theWorld Health Organization (WHO) [15].
Biological therapies such as radiotherapy, chemotherapy, surgery, immunotherapy, hormone therapy, and genetherapy can be used alone or in combination in the treatment of cancer [16]. Due to the complex nature of cancer,
there are currently no drugs that can destroy only cancer cells in patients [17]. The majority of methods used
to treat many cancer diseases are toxic, and have caused
morbidity [18].
Therefore, there is a great need to develop new cancer
treatment approaches to target only cancer cells withoutharming normal cells and to discover new and powerful
chemotherapeutics with minimal side effects. Although
there are few anticancer drugs containing benzimidazole
core (nokodazole, carbendazim, bendamustine) (Fig. 1)
on the market, there are many studies in the literature on
the anticancer effectiveness of benzimidazoles.
The epidermal growth factor receptor (EGFR) is afamily of cellular transmembrane tyrosine kinases thatare overexpressed in many human tumors, includingcolorectal, prostate, ovarian, breast, and lung cancers.
Abnormal activation of EGFR is common in many typesof cancer and is associated with apoptosis, angiogenesis,
and metastasis [19].
Cyclophilins (Cyps) are a family of proteins that are
ubiquitous in organisms and have biological functions
such as promoting intracellular protein folding and participating in pathological processes such as inflammationand tumors. Cyps inhibit the proliferation and differentiation of cells and support apoptosis. Cyps are also known
to be involved in many pathological processes including

Page 2 of 12

viral infections, cardiovascular disease, inflammatory
responses, and cancer [20].
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase. FAK is involved in tumor growth and various
cell functions, including motility, proliferation, adhesion, metastasis, apoptosis, chemo-resistance, and angiogenesis, related to integrin signal transduction. In many
cancers, such as prostate, lung, and breast cancer, FAK
expression has been detected. Therefore, FAK is expected
to serve as a new potential target in tumor therapy [21].
The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling pathway
is the significant intracellular pathways regulating cell
growth, motility, metabolism and angiogenesis. Activating the PI3K/AKT/mTOR pathway helps improving oftumor and endurance to cancer treatments. In almost all
human cancers, such as breast cancer, colorectal cancer,
and haematological malignancies, the PI3K/AKT/mTOR
signalling pathway has been reported to be dysregulated.
The efficacy of PI3K inhibitors in inhibiting tumor progression is becoming increasingly clear [22].
Due to the information provided above, EGFR, Cyps,
FAK, and PI3K proteins for molecular docking were used
as a target.
In-silico drug design or computer-aided drug design ismade predictable by computer simulations and modeling of many important parameters related to moleculesthat can be drug candidates, using different sub-fields
of artificial intelligence. It is generally use high performance molecular dynamics simulation software (GROMACS, AMBER, and NAMD etc.) [23–27] known for
their extensive simulation capabilities and accuracy, andmolecular docking software (SeeSAR and Autodock etc.)[23, 28].
Benzimidazole is a heterocyclic ring that is frequently
used in drug development due to its different biologicaland pharmacological effects and is an important structure that plays a key role in new drug candidates. In thisstudy, two 1,2-disubstituted benzimidazole compounds
were designed and synthesized that may have new potential anticancer effects. The docking analysis were carried

Fig. 1 Chemical structure of some commercial benzimidazole-derived anticancer drugs

Yavuz BMC Chemistry(2024) 18:146

out using UCSF Chimera software with its AutoDock
Vina tool. The cytotoxic effects of the compounds 2a and2b on A549, DLD-1, and L929 cell lines were tested for
48 h.
With respect to the outcomes acquired compound (2a)had cytotoxic activities against human cancer cell lines(A549, DLD-1, and L929) with IC50 values of 111.70,
185.30, and 167.3 mM, respectively. The potential mechanism of action for these two compounds was exploredthrough molecular docking. In addition, DFT computations were achieved using the B3LYP/6-31G** basis set topredict the structural and electronic features of the synthesized novel benzimidazole derivatives. Thanks to the
FMO analysis of the compounds, an idea was obtained
about their chemical reactivity and stability. MEP mapsof the compounds were obtained in three dimensions andregions open to electrophilic and nucleophilic attacks
were identified.

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It was mixed 1 h at 25oC. Then, 3-chlorobenzyl chloride(0.003 mol) was slowly added to this solution, and thereaction mixture was stirred for 12 h at 80 oC. The aimed
final product was washed a few times with EtOH anddried under vacuum.
Color: Cream. IR: 1447 (C = N); 2916 and 3043 cm− 1(C-H). 1HNMR (400 MHz, DMSO-d6, 298 K), δ: 2.51(s, 3H, CH2NCCH3); 5.49 [s, 2H, NCH2C6H4(Cl)-3];
7.02–7.57 (m, 8H, Ar-H). 13CNMR (100 MHz, DMSOd6, 298 K), δ: 13.8 (CH2NCCH3); 46.1 [NCH2C6H4(Cl)-3];
152.6, 142.0, 139.6, 135.3, 133.9, 131.2, 128.1, 126.8,
125.7, 122.6, 118.6, 114.6, 110.5 (Ar-C and NCN). Elemental analysis for C15H13N2Cl (256.74 g/mol) %: Found
C: 70.05; H: 5.21; N: 10.82. Anal. Calc. C: 70.17; H: 5.10;
N: 10.91. LC/MS (m/z): Anal. Calcd. for C15H13N2Cl is
256.74; found is 257.
Synthesis procedure of compound 2b

The essential chemical substances, such as 2-methylbenzimidazole, 3-chlorobenzyl chloride, 2,3,5,6-tetramethylbenzyl chloride, ethyl alcohol (EtOH), potassiumhydroxide (KOH) were purchased from Sigma-Aldrich,
Merck, and Isolab. 1HNMR (400 MHz) spectra and13
CNMR (100 MHz) spectra were recorded on a Bruker
AM 400 MHz NMR spectrometer with DMSO-d6 as
the solvent. The FT-IR analysis was performed by FT-IR
spectrometer (Nicolet 6700, Thermo Scientific). LC-MS/
MS spectra were recorded using a Shimadzu 8040
LC-MS spectrophotometer. The compounds were prepared according to the literature [29, 30] (Scheme 1).
KOH is a prototypical strong base used in a variety oflaboratory and chemical synthesis applications due toits effectiveness and reliability in providing high concentrations of hydroxide ions. In this study, we used KOH,
which is a strong base. The reaction was conducted at 80o
C for 12 h.

2-methylbenzimidazole (0.5 g) was dissolved in EtOH
(4 ml), and KOH (0.004 mol) was added to this solution.
It was mixed 1 h at 25 oC. Then, 2,3,5,6-tetramethylbenzyl chloride (0.003 mol) was slowly added to this solution,and the reaction mixture became cloudy after a while.
The reaction mixture was stirred for 12 h at 80 oC. The
aimed final product was washed a few times with EtOH
and dried under vacuum.
Color: White. IR: 1436 (C = N); 2918 and 3054 cm− 1(C-H). 1HNMR (400 MHz, DMSO-d6, 298 K),
δ: 2.05, 2.19, and 2.37 [s, 15H, CH2NCCH3 andNCH2C6H(CH3)4]; 5.42 (s, 2H, NCH2C6H4(CH3)4);
6.76–7.49 (m, 5H, Ar-H). 13CNMR (100 MHz, DMSOd6, 298 K), δ:14.6, 15.8, and 20.6 [CH2NCCH3 andNCH2C6H(CH3)4]; 44.5 [NCH2C6H(CH3)4]; 152.8, 142.2,
135.6, 134.0, 132.1, 131.8, 121.9, 121.5, 118.6, 110.6 (ArC and NCN). Elemental analysis for C19H22N2 (278.40 g/mol) %: Found C: 82.08; H: 7.84; N: 10.04. Anal. Calc. C:
81.97; H: 7.97; N: 10.06. LC/MS (m/z): Anal. Calcd. for
C19H22N2 is 278.40; found is 279 (See supplementary filefor NMR, IR and LC/MS spectra of compounds).

Synthesis procedure of compound 2a

Cytotoxic activity studies

Materials and methods
Chemicals & equipments

2-methylbenzimidazole (0.5 g) was dissolved in EtOH
(4 ml), and KOH (0.004 mol) was added to this solution.

Cytotoxic activity analyses of 2a and 2b were carriedout by the process designated in the literature [31–34].

Scheme 1 Reaction representation of 2-methyl-1H-benzo[d]imidazole with aryl halides

Yavuz BMC Chemistry(2024) 18:146

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Colon (DLD-1; ATCC® CCL-221™), lung (A549; ATCC®
CCL-185™) cancer, cell lines were purchased from theAmerican Type Culture Collection (ATCC, USA). DLD1, A549, and L929 cells were cultured in Dulbecco’s
modified Eagle’s Medium-high-glucose (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX™. After the cells reached 90% density, they were
removed from the flask using trypsin-EDTA and counted.
The cell seeding was done at a density of 5 × 103 cells/wellinto sterile 96-well plates. After seeding, the plates wereincubated for 24 h. After the incubation period was completed, the medium in the wells was removed and fresh
medium was added instead. The DLD-1, A549, and L929
cells were exposed to the compounds (2a and 2b) at
300, 150, 75, 37.5 and 18.75 µM concentrations for 48 h.
After the cells were incubated for 48 h, the medium wasremoved. The MTT stock solution (50 µL, 5 mg/mL) was
added to the plate wells and incubated for an additional
2 h. After the 2-hour incubation period, the dye in themedium was carefully slowly removed and 200 µL DMSO
was added in its place. It was left to mix slowly for half anhour. Absorbance values were measured in the Epoch 2
Elisa plate reader device at 590 nm. IC50 values were calculated by GraphPad Prism Software 5.
Methods of calculation

The theoretical calculations of synthesized compounds
were performed using Spartan’10 software packages [35].
The geometry of the synthesized compounds has been
optimized by B3LYP method with 6-31G** [36] origin
using DFT technique. The docking analyses were accomplished using UCSF Chimera software with its AutoDock
Vina tool [37]. Some druggability, pharmacokinetics,
and toxicity analyses were estimated owing to online
web tools. The used web servers were ADMETlab [38],admetSAR [39], SwissADME [40], Pro Tox-II (Banerjee
and Ulker, 2022). Biovia Discovery Studio was used tovisualize and analyze the docking results. [41]. Crystal
data of the 1M17, 1MP8, 2HQ6, and 1E8X macromolecules are obtained from the RCSB Protein Data Bank
website (https://www.rcsb.org/ ).

Results and discussion
Spectral characterization of compounds 2a and 2b

Two new 1,2-disubstituted benzimidazole compounds
were synthesized in one step (Scheme 1). The obtained
Table 1 IC50 outcomes for compounds 2a and 2b in A549, DLD1, and L929 cell lines
Compounds
2a2bCisplatin

IC50 (µM)
A549
111.70 ± 6.22
176.80 ± 4.66
9.79 ± 0.91

DLD-1
185.30 ± 5.87
> 300
55.58 ± 4.05

L929
167.30 ± 4.79
> 300
7.41 ± 0.47

compounds were characterized by means of FT-IR spectroscopy, 1HNMR, 13CNMR, and elemental analysis. The
1
HNMR spectra of the compounds were run at 400 MHz
in DMSO-d6, and also 13CNMR spectra of the compounds were run at 100 MHz in DMSO-d6. In 1HNMR,
the signal related to a methyl group 2-position on benzimidazole ring resonated upfield at δ 2.51, and 2.39 ppm
as 3 H singlets for 2a and 2b, respectively. The 13CNMR
spectra were run in DMSO-d6 at 100 MHz. The imidazole-linked methyl carbon signals resonated at δ 13.76
ppm for 2a, δ 14.59 ppm for 2b. The aryl-linked methylcarbon signals resonated at δ 46.06 ppm for 2a, and at δ
21.45 ppm and 58.47 ppm for 2b. The IR spectrum output of the compounds demonstrated the stretching frequencies of the characteristic C = N peak at 1447.81, and1436.87 cm-1 for 2a and 2b, respectively. The stretchingfrequency bands of C–H were observed at 2916.93 and3043.48 cm− 1 for 2a, 2918.58 and 3054.74 cm− 1 for 2b.
Cytotoxic activity studies

The newly synthesized compounds 2a and 2b were
tested against three different cell lines as in vitro. Using
the MTT assay method, the compounds were appraisedfor their cytotoxicity at 18.75, 37.5, 75, 150, and 300 µM
in the A549, DLD-1, and L929 cell lines. In the study, apositive control drug (cisplatin) was used as a referencecompound for comparison under the same experimental
conditions. The cancer cell lines were exposed to synthesized compounds for a period of 48 h. The results are
given in Table 1.
It is known that colon cancer is a common type of cancer worldwide and can cause death if not diagnosed early.
Lung cancer, which occurs as a result of the uncontrolled
growth of abnormal cells, is another type of cancer thatis most common and causes the most deaths worldwide.
For this reason, many studies on lung and colon cancer
cell lines continue to be conducted in the literature tofind new drug candidates [42, 43]. For this reason, colonand lung cell lines were preferred as cancer cell lines
in this study. Mouse fibroblast cell line was used as thehealthy cell.
Compound 2a had cytotoxic effects with IC50 valuesof 111.70 ± 6.22, 185.30 ± 5.87, and 167.30 ± 4.79 µM for
A549, DLD-1, and L929, respectively. Compound 2b
had cytotoxic effects with IC50 values of 176.80 ± 4.66,> 300, and > 300 µM for A549, DLD-1, and L929, respectively. Although compound 2b was found to be inactivein DLD-1 and L929 cell lines, the same compound wasfound quite less active against A549 cell line. The specifically compound 2a inhibited the proliferation of A549
cancerous cells rather than DLD-1 cancer cells. Compounds 2a and 2b appeared to be much less effective
against the A549 and DLD-1 cell lines than the referencedrug.

Yavuz BMC Chemistry(2024) 18:146

While compound 2a has the chloro group on the benzyl ring at the 3-position, compound 2b contains substituents with the methyl group at the 2,3,5,6-positions.
Compound 2a showed better, albeit low, activity against
the cell lines studied than compound 2b.
The different substituents on the benzyl ring (electron
donating or withdrawing) may be responsible for thesedifferences.
The change in viability rates depending on concentration is given in Figs. 2, 3 and 4. Compound 2b was the
medicine candidate with the least efficient level. Even
when used at a high concentration of 300 µM, a cell
viability rate of around 40% was observed. Therefore,
compound 2b does not have a high toxic effect on A549,
DLD-1, and L929 cell lines.
Compound 2a, which has a chloride group, was themore potent compound than compound 2b and induced
significant cytotoxicity at 150 and 300 µM. Compound 2ashowed cytotoxicity, especially at a concentration of 300µM. The compound 2-methyl-3-(3-chlorobenzyl)benzimidazole 2a can inhibit DNA replication and transcription by binding or intercalating with DNA. This effect canstop cell division and lead to apoptosis in cancer cells.
The compounds (2a, 2b) inhibited the proliferation of
fast cell division lung cancerous cells rather than colonand fibrosarcoma cancerous cells. Inactive compound2b appears to be able to inhibit the growth of colon cells
only at the highest concentration 300 µM. It was foundthat compound 2a inhibited the growth of colon cells
more at 300 and 150 µM.
Computational studies

The FMOs are called the highest occupied molecularorbital (HOMO) and the lowest unoccupied molecular

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Fig. 2 Cell viability ratio of A549

Fig. 3 Cell viability ratio of DLD-1

orbital (LUMO). [44]. According to Fukui’s FMO theory, the chemical reactivity of the molecule in terms of
HOMO or LUMO electron density is interpreted andhelps us know how electric charges move within themolecule. While the HOMO molecular orbital symbolizes the highest occupied orbital filled by electrons, the
LUMO orbital is described as the lowest unfilled orbital

Fig. 4 Cell viability ratio of L929 (Noted: blue: 2a, orange: 2b, and gray: cisplatin)

Yavuz BMC Chemistry(2024) 18:146

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Table 2 Effect of solvents on the HOMO-LUMO energies forcompounds 2a and 2b
Mediumgas
water
ethanolgas
water
ethanolCompound
(2a)

(2b)

EHOMO(eV)-5.9043-6.1917-6.1877-5.6877-6.1075-6.0908

ELUMO(eV)-0.6482-0.5252-0.5271-0.1896-0.4631-0.4476ΔE(eV)
5.25615.66655,66065.49815.64445.6432

Fig. 5 HOMO and LUMO orbitals for compounds 2a and 2b[45]. FMOs are significant parameters in quantum chemistry that play a role in defining the global and local
properties of the molecule. The larger the gap between
HOMO-LUMO orbitals, the harder and the less reactive the molecule will be as electron flow will be less. On
the other hand, the smaller the HOMO-LUMO gap, thesofter the molecule and the more reactive it is. Parameters such as molecular chemical hardness, electronegativity, chemical potential, and softness were calculated using
HOMO and LUMO energies. Effect of different solvent
mediums on the HOMO-LUMO energies for compounds
2a and 2b were shown in Table 2, it is possible to see thatthe water environment had the highest energy range witha value of 5.6665 eV and 5.6444 eV, respectively. For thetitle compounds, especially in the aqueous environment,
the ΔE energy gaps were larger than in the gaseous environment and methanol environment. HOMO and LUMO
orbitals for title compounds were displayed in Fig. 5. For
compound 2a, EHOMO=-5.9043 eV, ELUMO=-0.6482 eV, andΔE = 5.2561 eV were found. The fact that the structure

Table 3 Global reactivity descriptors of the title molecules in thegaseous ambient
Parameters
EHOMO
ELUMO
Energy band gap (ΔE = ELUMO-EHOMO)
Ionization potential (I = -EHOMO)
Electron affinity (A = -ELUMO)
Electronegativity (χ =(I + A)/2)
Chemical hardness (η= (I-A)/2)
Chemical softness (σ = 1/2η)Chemical potential (µ = −(I + A)/2)
Electrophilicity index (ω = µ2/2η)Maximum charge transfer index (ΔNmax = -µ/η)

(2a)
(2b)
Values(eV)-5.9043-5.6877-0.6482-0.18965.25615.49815.90435.68770.64820.1896
3.2763
2,93872.62812.74910.19020.1819-3.2763-2,9387
2.04221.5707
1.2466
1.0689has high energy range, high hardness (η = 2.6281 eV),and low softness parameters (σ = 0.1902 eV) shows that
it is quite stable with low chemical activity and highkinetic stability. For compound 2b, EHOMO=-5.6877 eV,
ELUMO=-0.1896 eV, and ΔE = 5.4981 eV were found. Its
high hardness (η = 2.7491 eV) and low softness parameter (σ = 0.1819 eV) indicate that this compound has highchemical stability and low reactivity. The chemical potential values of compounds 2a and 2b were determined tobe -3.2763 eV and − 2.9387 eV, respectively and the electronegativity index values were 3.2763 eV and 2.9387 eV,
respectively. In gaseous environment the calculated
energy gap values are less than 5 eV, which stands forthat this compounds are a good bioactive material. Otherchemical reactivity parameters of the synthesized compounds were shown in Table 3.
In order to understand the charge distribution of thesynthesized compounds, the results obtained from thecalculations made with the B3LYP/6-31G** level werevisualized in three dimensions using the Spartan’10 program. Obtained resulting molecular electrostatic potential surface maps are given in Fig. 6.
Dissimilar values of the electrostatic potential on thesurface are given in distinct colors (blue to red). In MEPmaps, the red regions show nucleophilic regions, which
are atoms or groups of atoms rich in electron density,
while the blue regions show electrophilic regions, which
are atoms or groups of atoms that are poor in electron
density. The green color points out neutral electrostatic
potential [46]. In Fig. 6, both mono-chloro and tetramethyl substituted benzene have a high electron density
inside the aromatic ring (red color). This means that thearomatic C-H has become more basic.
Molecular docking

Molecular docking computations are an important calculation technique that estimates the binding affinity ofligands to receptor proteins in molecular pharmaceuticals

Yavuz BMC Chemistry(2024) 18:146

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ID: 1M17, PDB ID: 1MP8, PDB ID: 2HQ6 and PDB ID:
1E8X of molecule 2a were shown in Table 4. Molecule
2a constituted secondary interactions (van der Waals,
hydrogen bonding, π-anion, π-sigma, alkyl, and π-alkyl)
with the EGFR tyrosine kinase (1M17). On the other
hand same molecule constituted secondary interactions(van der Waals, π-cation, amide-π stacked, alkyl, andπ-alkyl) with the 2HQ6 protein which identified serologically as the cyclophilin CeCYP16-like domain of coloncancer. Hydrogen bonds describe principle stabilization
potencys in biomolecular structure. Van der Waals forces
are also very important in the formation of proteinligand complexes, and it is noted that these interactions
are important in determining the binding affinity of theligand to the protein. On the other hand, the hydrophobic interaction types such as π alkyl, π-sigma, and π-πstacked, types of electrostatic interactions as attractive
charge, pi-anion, pi-cation interactions assist increase
the interaction of the ligand in the binding pocket of thereceptor [51]. It can be seen in Figs. 8 and 10 that 1MP8
and 1E8X proteins also make secondary interactions withcompound 2a. Such bonds encountered in docking analyses are important for the structural unity of many biological molecules such as protein and DNA, as well as for
drug-receptor interactions. These interactions are shownin Figs. 7, 8, 9 and 10.
In silico pharmacokinetics/ADME-Tox studies
Fig. 6 MEP surfaces of compounds 2a and 2b[47]. Benzimidazole derivatives are compounds that cancontribute to designing efficient bioactive molecules forthe discovery and development of innovative drugs, particularly in the therapy of cancer [48]. Benzimidazole
derivatives have been often used for their anticancer
activity against different human cancer cell lines [49, 50].
Therefore, it was performed molecular docking of thecompound 2a with anticancer protein targets from coloncancer (PDB code: 2HQ6), tumor invasion and metastasis (PDB code: 1MP8), cancer cells proliferation andtumor growth (PDB code: 1E8X) and lung cancer (PDB
code: 1M17). Active sites of proteins were described asincluding residues at the centre of the active site of theprotein. A grid box size of 30 Å × 30 Å × 30 Å was used
for the grid generation in all docking studies. The molecules were optimized using the Spartan’10 package program for molecular docking calculation. It was created.pdb file of optimized structure. In the computations performed using these files, the biological activities of molecule 2a were compared with the proteins known in thestages of colon cancer and lung cancer. Good energy values of the docking results (-6.6 kcal/mol, -6.4 kcal/mol,-4.8 kcal/mol, and − 3.9 kcal/mol respectively) for PDB

Computer-aided approaches are able to potential toaccelerate drug discovery in terms of help minimize thecosts, and reduce the time [52]. Computer-aided drug
discovery includes in silico assessment, modification,
analysis and optimization of computational identification of potential drug targets [53]. According to Lipinski rule, the molecule should comply specific factors to
be improved as an orally drug [54]. According to Lipinski rule, these parameters are: molecular weight (shouldbe ≤ 500 Dalton), hydrogen bond acceptor (should benumber of HBAs ≤ 10), hydrogen bond donor (should benumber of HBDs ≤ 5), lipophilicity (should be clogP < 5)and number of rotatable bonds (should be < 10) [55].
The pharmacokinetics/ADME results demonstrated
that synthesized compounds comply with Lipinski rule.
These compounds displayed molecular weight less than
500 proposing that these compounds can be well moved
in the body. In 80% of the total drugs the molecularweight is below 450 daltons. When a compound meetsthese rules, it is more likely to have cell membrane permeability and be easily absorbed in the body [56, 57]. To
be an effective medicine, a substance must be soluble
in both water and oil. Medicines taken by mouth must
pass through the intestinal lining and penetrate the cell
membrane to reach the interior of a cell. The model compound for the cell membrane is octanol, known as log

Yavuz BMC Chemistry(2024) 18:146

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Table 4 Molecular docking results of the molecule 2a

Pow and this value stands for the octanol/water partition coefficient. Low lipophilicity (logP < 5) frequently
contributes to good oral absorption land high solubility.

Another importance of this coefficient is that it is used
as a descriptive in the calculations made for the bloodbrain barrier (BBB) in an aqueous environment. One ofFig. 7 Graphs of the protein-ligand interaction for the most steady complexes (PDB ID: 1M17)

Yavuz BMC Chemistry(2024) 18:146

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Fig. 8 Graphs of the protein-ligand interaction for the most steady complexes (PDB ID: 1MP8)

Fig. 9 Graphs of the protein-ligand interaction for the most steady complexes (PDB ID: 2HQ6)Table 5 Physicochemical properties predictions of the synthesized compounds 2a and 2b
Comp.
No.
2a
2b

Lipinski parameters
MWa
HBAb
256.73
1
278.391

HBDc
00

iLogPd2.62
3.08

Molar Ref.e
75.46
90.31

the most important features for drug candidate molecules is the ability to cross the BBB. It is especially essential for drugs used in central nervous system diseases topass through the BBB. It was predicted exhibit high gastrointestinal (GI) absorption and BBB ​​permeability of thesynthesized compounds. It cannot be concluded that thecompounds have exactly the desired pharmacokinetics
required for the development of an orally available drug.
Drug likeness evaluation for the compounds (2a, 2b)
is assessed on their in silico pharmacokinetics studies
depended on the Lipinski rule (Table 5).

Violations
00
anROTBf
2
2

TPSAg
17.82
17.82

BBBh
Yes
Yes

GI ABSi
High
High

Molecular weight. b Hydrogen Bond Acceptor. c
Hydrogen Bond Donor. d Partition Coefficient. e Molar
Refractivity. f Number of rotable bonds. g Topological
Polar Surface Area. h Blood Brain Barrier. i Gastrointestinal absorption.
Toxicity analysis was carried out using the Protox-II
online tool [58] which ensures knowledge about carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity
related to the compounds. It has acute toxicity estimation
findings such as toxicity class classification from 1 (toxic)
to 6 (non-toxic). It was estimated that the synthesizedYavuz BMC Chemistry(2024) 18:146

Page 10 of 12

Fig. 10 Graphs of the protein-ligand interaction for the most steady complexes (PDB ID: 1E8X)

Table 6 The toxicity computation of synthesized 2a and 2b by Pro-tox II web toolTox. class
Comp.
2a
2b

IV
IV

Organ toxicity (probability)
Hepatotoxicity
Carcinogenicity
Inactive
Inactive
Inactive
Inactive

compounds were classified as acute toxicity class 4(slightly toxic). The results recommend that compound
2a is non-carcinogenic and has no effect on immunotoxicity, cytotoxicity, and mutagenicity. On the other hand,
it shows that the 2b compound is active on mutagenicity(Table 6).

Conclusion
Two 1,2-disubstituted benzimidazole compounds weresynthesized, characterized, offered, and debated herein.
DFT analysis of the molecular structure, together with
the HOMO-LUMO orbitals, was carried out by using theSpartan’10 program software. Assessment of the anticancer activity of new compounds was performed and demonstrated moderate activity also the compounds showed
good inhibitory activity against molecular docking. The
pharmacokinetic properties of the synthesized molecules
were predicted using ADMET, and 2a molecule has no
toxic effect were kept as hepatotoxicity, carcinogenicity,
immunotoxicity, mutagenicity, and cytotoxicity. However, molecule 2b has only mutagenicity toxicity. The
ProTox-II program used to predict various toxicologicalproperties of compounds is not sufficient on its own andhas classified synthesized compounds as acute toxicity
class 4 (slightly toxic). In vitro studies also support thisprediction. In particular, compound 2b was found to be
toxic to two human cell lines (DLD-1 and L929).
In this study, the synthesized compounds 2a and 2b
were tested in vitro against different cancer cell lines. The

Immunotoxicity
Inactive
Inactive

Mutagenicity
Inactive
ActiveCytotoxicity
Inactive
Inactive

results showed that 2a, had slightly antiproliferative activity compared to cisplatin for the A549, DLD-1, and L929
cell lines while 2b was only found to be active against
A549 cell line. The molecular docking behaviour of compound 2a with lung cancer protein, PDB ID: 1M17 andcolon cancer antigen proteins, PDB ID: 2HQ6 and PDB
ID: 1E8X were compared with experimental and theoretically. The predicted binding energies were found tobe -6.6 kcal/mol, -4.8 kcal/mol, and -3.9 kcal/mol respectively. The docking results showed that compound 2a wasinhibited through secondary interactions. Compound 2acompared to compound 2b was softer and more reactive
because the HOMO-LUMO energy gap is smaller thancompound 2b. It could be said that compound 2a showed
better biological activity than compound 2b in the invitro analyses. It showed that substituents with electron
withdrawing capacity (-Cl), as in compound 2a, makethe molecule polar and show good activity. In general, it
could be said that compound 2a may exhibit a therapeutic potential for the prevention of lung cancer based on
the cytotoxic analyses obtained. It can be concluded thatthese compounds affect the inhibitory potential depending on whether the substituents have electron-withdrawing or electron-donating properties.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01241-z.

Yavuz BMC Chemistry(2024) 18:146

Supplementary Material 1
Acknowledgements
The author thank Erzincan Binali Yildirim University-Scientific Research Unit for
supplying under grant project number FBA-2023-865 and go to Senem Akkoc
for giving support for the cytotoxicity studies.
Author contributions
S.C.Y: Conceptualization, Methodology, Visualization, Investigation, Writing
original draft, review, and editing.
FundingErzincan Binali Yildirim University-Scientific Research Unit: FBA-2023-865.
Data availability
All data generated or analyzed during this study are included in thispublished article and its Supplementary information files. Analytical data
including copies of mass spectra, FT-IR, 1HNMR, and 13CNMR is available in the
Supplementary information.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 4 April 2024 / Accepted: 4 July 2024

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