Synthesis, antibacterial, and anti HepG2 cell line human hepatocyte carcinoma activity of some new potentially benzimidazole-5-(aryldiazenyl)thiazole derivatives

Article abstract of DOI:10.3390/molecules23123285

The paper describes the synthesis and biological evaluation of some new benzimidazole derivatives as potent clinical drugs that are useful in the treatment of some microbial infections and tumor inhibition. The starting compound 2-(bromomethyl)-1H-benzimidazole (1) was prepared, and hence underwent interesting functionalization reactions to afford several series of benzimidazole-5-(aryldiazenyl)thiazole derivatives: 3a–c, 7a–c, and 8a–c. The antibacterial activities of the synthesized compounds were evaluated by calculation of the inhibition zone diameter (mm) and the determination of minimum inhibitory concentration (μg/mL) against selected pathogenic bacteria Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria).Noticeable efficiency was found based on in vitro screening for their antioxidant activity and cytotoxicity effect against the human liver cancer cell line (HepG2) and human hepatocyte carcinoma cells at relatively high concentrations.

Full text of DOI:10.3390/molecules23123285

molecules
Article
Synthesis, Antibacterial, and Anti HepG2 Cell Line
Human Hepatocyte Carcinoma Activity of Some New
Potentially Benzimidazole-5-(Aryldiazenyl)Thiazole
Derivatives
Mohamed E. Khalifa 1,
*
, Adil A. Gobouri 1, Fahad M. Kabli 1, Tarik A. Altalhi 1,
Abdulraheem S. A. Almalki 1 and Mahmoud A. Mohamed 2
1
Department of Chemistry, faculty of Science, Taif University, Taif 21974, Saudi Arabia;
agobouri00@yahoo.com (A.A.G.); fmk_37@hotmail.com (F.M.K.); tmmba@windowslive.com (T.A.A.);
drasaalmalki1@gmail.com (A.S.A.A.)
Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza 12613, Egypt;
2
elcamel.mahmoud@gmail.com
*
Correspondence: mohamedezzat200@hotmail.com; Tel.: +966-569846966
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 16 November 2018; Accepted: 9 December 2018; Published: 11 December 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The paper describes the synthesis and biological evaluation of some new benzimidazole
derivatives as potent clinical drugs that are useful in the treatment of some microbial infections
and tumor inhibition. The starting compound 2-(bromomethyl)-1H-benzimidazole (
prepared, and hence underwent interesting functionalization reactions to afford several series
of benzimidazole-5-(aryldiazenyl)thiazole derivatives: 3a 7a , and 8a . The antibacterial
activities of the synthesized compounds were evaluated by calculation of the inhibition zone
diameter (mm) and the determination of minimum inhibitory concentration ( g/mL) against selected
1) was
–
c,
–
c
–c
µ
pathogenic bacteria Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative
bacteria).Noticeable efficiency was found based on in vitro screening for their antioxidant activity and
cytotoxicity effect against the human liver cancer cell line (HepG2) and human hepatocyte carcinoma
cells at relatively high concentrations.
Keywords: benzimidazoles; antimicrobial activity; antitumor; HepG2 cell line; pathogenic bacteria
1. Introduction
The promising therapeutic potential of benzimidazole derivatives could be traced
back to 1944and Woolley, who speculated that benzimidazole could act similar to purines
to elicit some biological responses [
1].
The optimization of substituents around the
benzimidazole nucleus resulted in many drugs with useful therapeutic activities such asmethyl
(5-benzoyl-1H-benzimidazol-2-yl)carbamate (mebendazole) as parasitic worm infestations,
(RS)-2-([3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methylsulfinyl)-1H-benzo[d]imidazole
(lansoprazole) and (RS)-6-(difluoromethoxy)-2-[(3,4-dimethoxypyridin-2-yl)methylsulfinyl]-1H-
benzo[d]imidazole (pantoprazole) as proton pump inhibitors; 1-[(4-fluorophenyl)methyl]-N-[1-[2-(4-
methoxyphenyl)ethyl]-4-piperidyl]benzoimidazol-2-amine (astemizole) as antihistaminic[
triazole-thiazol derivatives as analgesic ]; 2-[2-(4-chlorophenyl)-1,3-benzoxazol-5-
yl]propanoic acid (benoxaprofen) as anti-inflammatory [ ]; methyl [5-(propylthio)-1H-
benzoimidazol-2-yl]carbamate (albendazole) as antimicrobials [ 10]; 1-(isopropylsulfonyl)-6-(1-
phenylprop-1-en-1-yl)-1H-benzo[d]imidazol-2-amine (enviradine)
antitubercular 13 14]; 4-(1H-1,3-benzodiazol-2-yl)-1,3-thiazole(thiabendazole)
2];
[3–6
4–7
8–
as
antiviral
[11
,
12];
as
[
,
Molecules 2018, 23, 3285; doi:10.3390/molecules23123285
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3285
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antihelmenthic [15–18]; picoline-imidazoles as anticonvulsant [19
,20]; 4-[5-[bis(2-chloroethyl)amino]-
1-methylbenzimidazol-2-yl]butanoic acid (bendamustine) as anticancer [21
2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methanesulfinyl]-1H-benzimidazole (omeprazole) as
antiulcer [24 25]; angiotension-II receptor antagonists [26]; 2-ethoxy-1-([4-[2-(2H-1,2,3,4-tetrazol-
5-yl)phenyl]phenyl]methyl)-1H-1,3-benzodiazole-7-carboxylic acid (candesarten) as
antihypertensive [27]; alkynyl benzimidazoles as modulators of metabotropic glutamate
receptors [ 16 18 24 28 30]; and N-ribosyl-dimethyl benzimidazole as an axial ligand for
cobalt in vitamins [19 31 32].Aiming to prepare heterocyclic systems with some role in biological
–23]; 5-methoxy-
,
3, – , , –
,
,
applications, we report in the present work the synthesis of novel derivatives of benzimidazoles by
convenient methods. In vitro screening for their antimicrobial activities was determined against
selected pathogenic bacteria, namely Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli
(Gram-negative bacteria) using inhibition zone diameter (mm) and minimum inhibitory concentration
methodologies comparing with Kanamycin and Ampicillin, respectively, as standard antibacterial
agents. Their antioxidant and antitumor activities against the HepG2 human hepatocyte carcinoma
cell line were also evaluated.
2. Results
Benzimidazoles are hybrid heterocyclic compounds that are formed from fused benzene and
imidazole rings with broad industrial, biological, and clinical applications. The associated rich
literature with benzimidazole moieties prompted us to synthesize new functionalized benzimidazole
derivatives and study their potential as bioactive compounds, continuing our previous efforts related
to the synthesis of new pharmacological compounds [33–41].
2.1. Chemistry
Synthesis of the starting 2-(bromomethyl)-1H-benzimidazole (1) proceeded smoothly according
to the reported method by the reaction of o-phenylenediamine with bromoacetic acid in the presence
of 4 M hydrochloric acid, as shown in Scheme 1.
Scheme 1. Synthesis of 2-(bromomethyl)-1H-benzimidazole (1).
The IR spectrum of 2-(bromomethyl)-1H-benzimidazole (
3372–3025 cm−1 (NH, =CH aromatic, and –CH aliphatic). The 1H-NMR spectrum displayed a singlet
signal at 4.85 ppm due to the methylene protons (2H, CH2–Br), a singlet at 10.32 ppm due to the
NH–benzimidazole proton, and at 7.21–7.60 ppm due to the aromatic protons. The spectral analysis
1) revealed intense IR bands at
δ
δ
δ
was found to be in good agreement with the literature [42].
The direct condensation of 2-(bromomethyl)-1H-benzimidazole (
1) with 2-aminothiazole
was carried out to give the N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine (
2) [43],which
underwent diazo coupling with different aryl diazonium chorides, yielding the corresponding
N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)thiazol-2-amines (3a–c), as shown in Scheme 2.

Molecules 2018, 23, 3285
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N
S
HN
N
Br
N
N
S
H2N
+
N
H
N
H
2
1
NaOAc, EtOH
0-5 °C
Cl
Ar N N
N
HN
N
Ar
S
N N
N
H
3a-c
Ar
3a
3b
3c
p-NO2C6H4
p- CH3C6H4
p-CH3COC6H4
Scheme 2. Synthesis steps for the preparation of benzimidazole derivatives(3a–c).
The structures of the isolated compounds ( and 3a ) were secured by their spectral analysis as
2
–c
described in the experimental section, where a characteristic singlet signal at
C4-thiazole proton was displayed.
δ 6.90 ppm due to the
The reaction of thiourea with
the sulfur atom due to its high nucleophilicity to give thiouronium salts [44]. Whereas, conducting the
reaction under acidic medium (e.g., trifluoroacetic acid) afforded the alkylthiourea compounds [45 47].
Thus, the reaction of 2-(bromomethyl)-1H-benzimidazole ( ) with thiourea in acidic medium gave
the previously reported derivative 1-((1H-benzimidazol-2-yl)methyl)thiourea ( ) [45] through the
α-haloalkyl or α-halocarbonyl compounds often proceed through
–
1
4
suggested mechanism in Scheme 3. The structure of the obtained compound was secured by its
spectral analysis, and was in good agreement with the literature.
Scheme 3. Mechanism of formation of alkylthiourea benzimidazole derivative (4).
The thiourea derivative
at exploiting the reactivity of the thioureido moiety to build up the target compounds.
Thus, 1-((1H-benzimidazol-2-yl)methyl)thiourea ( ) was cyclized into the thiazole derivatives
2-(((1H-benzimidazol-2-yl)methyl)amino)thiazol-4(5H)-one ( ) and N-((1H-benzimidazol-2-yl)methyl)-
4-phenylthiazol-2-amine ( ), by treating with -halocarbonyl compounds (i.e., chloroacetyl chloride
and phenacyl chloride). The aminothiazolyl derivatives ( and ) were then allowed to couple
with different aromatic diazonium chloride compounds, affording the target arylazo compounds;
4 was then incorporated in a set of investigations aiming
4
5
6
α
5
6
2-(((1H-benzimidazol- 2-yl)methyl)imino)-5-(4-aryl)diazenyl)thiazolidin-4(5H)-one derivatives (7a
and N-((1H-benzimidazol-2-yl)methyl)-5-(4-aryl)diazenyl)-4-phenylthiazol-2-imine derivatives (8a
respectively, as shown in Scheme 4.
–
–
c
c
)
)

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Scheme 4. Synthesis of arylazo derivatives (7a–c) and (8a–c). The formation of compounds (7a–c and
8a–c) was elucidated by their spectral analyses.
2.2. Biological Impact of the Synthesized Compounds
2.2.1. Antibacterial activity
The antibacterial activities of novel synthesized compounds (100
µg/mL in DMSO), were
determined in vitro by using the disc diffusion method [48] against Staphylococcus aureus (S. aureus)
ATCC 25923 as Gram-positive bacteria and Escherichia coli (E. coli) ATCC 25922 as Gram-negative
bacteria compared with the standard drugs Kanamycin and Ampicillin, respectively. The results of the
assessments are plotted in Figure 1.
20
S. aureus
SD
E. coli
15
10
5
0
-5
The standard drugs and the synthesized dervativies
Figure 1. Antimicrobial activity of the tested benzimidazole derivatives vis-à-vis selected pathogenic
bacteria strains comparing with standard drugs.
2.2.2. Minimum Inhibitory Concentration (MIC)
The Minimum Inhibitory Concentration for the synthesized compounds was determined using a
spot-on-lawn assay [49]. The collected data are listed in Table 1. The lowest MIC value is the highest
antibacterial effect against the target bacterial strain.

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Table 1. The minimum inhibitory concentration (MIC) of effective tested compounds and positive reference standards.
MIC, µg/mL (Average ± SD)
Tested Strains
Reference Control
Tested Compounds
Ampicillin Kanamycin
3a
-
3b
-
24 ± 2.21
3c
7a
-
-
7b
-
-
7c
-
8a
8b
-
-
8c
E. coli
S. aureus
2.1 ± 0.09
-
14 ± 1.54
16 ± 1.05
12 ± 0.095
-
11.3 ±0.87
32 ± 3.95
-
27 ± 1.23
28 ± 1.3
21 ± 1.64
E. coli: Escherichia coli; S. aureus: Staphylococcus aureus, Values are mean ± SD, n = 3.

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The antibacterial study showed that some of the tested compounds exhibited variable
antimicrobial activities against both bacteria Gram-negative and Gram-positive bacteria at a
concentration of 100
µg/mL. The presence of substituted electron withdrawing groups such as nitro
and acetyl groups in compounds 3c
,
8a, and 8c showed activities against E. coli (Gram-negative
bacteria), while most of the synthesized compounds exhibited moderate activity against S. aureus
(Gram-positive bacteria).
Compound 8c showed the highest affect against tested bacterial strains compared with the rest of
the synthesized benzimidazoles under consideration due to the presence of an electron-withdrawing
group (COR group) on the phenyl ring. It should be noted that the thiazolone moiety resulted in a
dramatic reduction of the antibacterial efficiency (e.g., compounds 7a–c).
2.2.3. Antioxidant Potential of the Synthesized Compounds
DPPH Radical Scavenging Assay
Antioxidant capacities for the synthesized compounds were screened using a DPPH free radical
scavenging assay incorporating a metastable free radical that is capable of accepting hydrogen
radicals from antioxidants in solution. The method is based upon different mechanisms to provide
complementary insight into the antioxidant activity of the synthesized compounds [50]. The
reaction between DPPH and antioxidants can be monitored by the decrease in the absorbance of the
violet-colored free radicals. Inhibition percentages of DPPH radical by selected tested compounds were
plotted in Figure 2, and the measure of drug potency in terms of half maximal effective concentration
(EC50) values were plotted in Figure 3 (i.e., the compound concentration where 50% of its maximal
effect is observed). The high antioxidant and scavenging activities of the tested compounds may be
due to the resonance delocalization of the nitrogen lone pairs through the double bonds. This structure
may lead to radical formation at more than one site, especially on the benzene ring attached to the
electron-withdrawing groups (e.g., 3a and 3c: the highest DPPH value and the lowest EC50 value,
respectively). This conclusion is also supported by previously reported results [51].
DPPH Radical scavanging activity
100
20 ppm
50 ppm
80
100 ppm
60
40
20
0
-20
Trolox
3a
3b
3c
7a
7b
7c
8a
8b
8c
Synthesized benzimidazole derivatives
Figure 2. Inhibition percentage average results of DPPH radical by the tested synthesized
benzimidazoles based on three independent experiments.

Molecules 2018, 23, 3285
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Half maximal effective concentration (EC50)
700
600
500
400
300
200
100
0
3a
3b
3c
7a
7b
7c
8a
8b
8c
Synthesized benzimidazole derivatives
Figure 3. The average results of EC50 for the tested synthesized benzimidazoles based on three
independent experiments.
2.2.4. Evaluation of Cytotoxicity Effects of Benzimidazoles and its Derivatives Against the Human
Liver Cancer Cell Line (HepG2).
All of the compounds were screened in vitro for possible potential antitumor molecules against
human liver cancer cell line (HepG2) and showed variable anticancer activity. The Neutral Red (NR)
technique [52] was used to evaluate the cytotoxic effects of gradient concentrations (100
µg/mL to
500 g/mL) of the tested compounds on the human liver (HepG2) cell line and in terms of cell viability
µ
percentage, which measured the potency of the compound in inhibiting tumor growth (Figure 4).
100 µg/mL
250 µg/mL
500 µg/mL
100
80
60
40
20
0
3a
3b
3c
7a
7b
7c
8a
8b
8c
Synthesized benzimidazole derivatives
Figure 4. The viability % of human liver cancer (HepG2) cell line treated with the synthesized
benzimidazoles at 100 g/mL, 250 g/mL, and 500 g/mL based on three independent experiments.
µ
µ
µ

Molecules 2018, 23, 3285
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As shown in Figure 4, the tested compounds exhibited antitumor activities in different
concentrations, where increasing the concentration led to less percent viability. The benzimidazole
derivatives (3) and (8) in common showed higher activity than the other selected compounds that were
tested. The presence of the 4-phenyl function with or without attached electron-withdrawing groups
increased the antitumor activity due to their high antioxidant and free radical scavenging ability (e.g.,
3a and 8b). This also could be explained by the previously reported facts [51], and is supported by the
literature [53].
3. Materials and Methods
3.1. Chemical Reagents
All of the chemical reagents that were used were of analytical grade or chemically pure and
supplied by Sigma Aldrich Co., Darmstadt, Germany.
3.2. Micro and Spectroscopic Analysis
All of the corrected melting points are in degree centigrade and measured using a Stuart
SMP20 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). The infrared spectra were
recorded on a PerkinElmer Alpha platinum-ATR spectrometer; the 1H-NMR (300 MHz) and 13C-NMR
(75 MHz) spectra were recorded on a Varian Mercury VXR-300 spectrometer (Varian Inc., Palo Alto, CA,
USA) and the chemical shifts were related to that of the solvent DMSO-d6 using tetramethylsilane (TMS)
as an internal standard. MS spectra (HRMS) were acquired on TRACE GC Ultra gas chromatograph
mass spectrometry, coupled with a THERMO mass spectrometer detector ISQ Single Quadrupole
Mass Spectrometer (THERMO Scientific Corp., Waltham, MA, USA)and were obtained by electron
ionization (EI) at 70 eV, using a spectral range of m/z 50–1000.All of the microanalyses and spectral
analyses were performed by the Micro Analytical Centers of Taif University-Saudi Arabia (IR spectra,
HRMS), Cairo University (1H-NMR, 13C-NMR), and National Research Center-Egypt (Mass spectra).
3.3. Biological Analysis
All of the used kits for biochemical parameters were supplied by the Diagnostic Company,
Maidenhead, UK. All of the chemicals that were used were supplied by Gibco® Life Technologies,
Waltham, MA, USA. The biological tests were performed by the biotechnology unit, faculty of
Agriculture-Cairo University, Egypt. Human transformed cell lines from liver (HepG2) were obtained
from the Egyptian Company for Vaccine and Serum (VACSERA, Cairo, Egypt). The statistical package
for social science (SPSS) 1999 program version (IBM, Armonk, NY, USA) was used for all of the
analyses [54].
3.3.1. Antibacterial Evaluation
The disc diffusion procedure was used to evaluate the antibacterial activity of the synthesized
compounds [53]. Uninoculated agar medium was poured in sterilized Petridishes and left for
solidifying. One hundred milliliters of trypticase soy agar + 0.6% yeast extract were melted and
◦
cooled to 50 C, and then inoculated by the culture of the examined microorganism. Inoculated
◦
medium was poured over the previous layer and left to solidify at 4 C (surface layer should be
constant in volume and horizontally homogenous). Discs of Whatman No. 1 filter paper (6.0 mm in
diameter) were sterilized by autoclaving at 121 ◦C for 15 min. Each individual tested compound was
dissolved in 10% dimethyl sulfoxide (DEMSO) with a final concentration 100 mg/mL. An accurate
volume (15 µL) of each tested compound (100 mg/mL) was aseptically added to each disc and left to
dry. Each disc was aseptically placed on the middle of an agar plate surface (in triplicate) and left at
◦
4 C. Fifteen
µL of DEMSO (10%) was aseptically added to the disc and used as control. Then, 15
µL of
Kanamycin (30
µg/mL) was aseptically added to the disc and used as a positive standard reference for

Molecules 2018, 23, 3285
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Gram-positive strains, and Ampicillin (10
µg/mL) was used for Gram-negative strains. The inhibition
clear zones (mm) were recorded in all of the samples and equal zero in no-inhibition results.
3.3.2. Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of synthesized compounds was assayed by using a
spot-on-lawn assay [49]. Cells of laboratory strains (LAB strains) were grown in de Man, Rogosa &
◦
Sharpe MRS broth (Oxoid) supplemented with 0.5% CaCO3 at 30 C with shaking (120 strokes per min
(SPM)) to the early stationary phase, and the initial pH of the medium was adjusted to pH 6.0. Culture
fluid was obtained by removing the cells by centrifugation, and was filtered with a cellulose acetate
membrane filter (pore size 0.2
twofold with MRS broth (pH 5.0). Soft nutrient agar (20 mL) was solidified in a sterile dish (140 mm
by 100 mm) after the addition of 60 L of an overnight culture of Listeria monocytogenes SUB635. This
strain was used as an indicator in the assay, since a clearer inhibitory zone was obtained with this
organism than with B. subtilis. After 30 min of drying, 10 L of each diluted sample was spotted onto
µm; Advantec, Tokyo, Japan). The resulting sample was serially diluted
µ
µ
the plate. The resulting plates were incubated at 30 ◦C overnight, and the titer was defined as the
reciprocal of the highest dilution (2n) that resulted in the inhibition of the indicator lawn. Thus, the
arbitrary unit (AU) of antibacterial activity per milliliter was defined as 2n × 1000 µL/10 µL.
To examine the spectrum of activity of the purified bacteriocin against various bacterial strains,
the test described above was performed with soft agar containing cells of test strains grown in a
suitable medium to the early stationary phase. The abilities to inhibit a certain bacterial strain were
compared by using the highest dilution that resulted in a growth inhibition zone.
3.3.3. Assay of Cytotoxicity
Neutral Red (NR) assay [55] was used to evaluate the cytotoxicity effects of prepared compounds.
30.000 Cells/well were plated in 96-well plates in quadruplets for each treatment. 24 h later, cells were
washed with pre-warmed phosphate-buffered saline (PBS) and 125
concentration of each synthesized compound. Afterwards, cells were washed with pre-warmed PBS,
and 125
L of the NR reagent was added in each well. The plates were incubated at 37 ◦C with 5%
CO2, for a further 5 h. After incubation, plates were taken out, gently washed three times with PBS,
and 100 L of NR desorb solution was added per well. Plates were protected from light and shaken on
µL of media with an appropriate
µ
µ
a shaker at 60 rpm for 30 min, followed by keeping the plates still for 5 min. Plates were taken to a
96-well plate reader, and their absorbance was measured in each well at 540 nm. Each value is based
on means of the absorbance values acquired from the quadruplet of treatments and normalized to the
mean value of untreated control and expressed as % cell death according to the following equation
(Equation (1)):
Abs540 treated sample
Abs540 untreated sample
% Cell death =
× 100
(1)
The data is based on three independent experiments.
3.3.4. Antioxidant Potential
The tested compounds were analyzed for their antioxidant potential according to the reported
technique [56]. The stock solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was prepared by dissolving
◦
24 mg of DPPH in 100 mL of methyl alcohol (MeOH), and then stored at 20 C in the dark. The working
solution was obtained by diluting 10 mL of stock solution with 45 mL of MeOH, in order to obtain
an absorbance of 1.1
concentrations (20
±
0.1 units at 515 nm. A volume of 10
µ
L of different synthesized compounds’
µ
g/mL, 50 g/mL, and 100 g/mL) was added to 990 µ
µ
µ
L of 0.094 mM DPPH
freshly working solution up to total volume of one mL. Assays were continuously monitored at 515 nm
◦
over a one-hour period at 25 C. Changes in absorbance were minimal for all of the samples after
50 min. The antioxidant abilities were expressed as µM Trolox equivalents. Each sample was analyzed

Molecules 2018, 23, 3285
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in triplicate. The inhibition percentage of the DPPH radical by the samples was calculated according
to Equation (2):
[Ab − Aa]
% Inhibition =
× 100
(2)
Ab
where, Ab is the absorption of the blank sample (t = 0 min), and Aa is the absorption of the tested
compound or standard substance solution (t = 30 min). The EC50 value is defined as the concentration
of antioxidant in the reactive system that is necessary to decrease the initial DPPH concentration by
50%, and is calculated from the data obtained.
3.4. Synthesis and Structural Analysis
3.4.1. General Procedure for the Synthesis of 2-(Bromomethyl)-1H-Benzimidazole (1)
The 2-bromomethyl benzimidazole (1) was prepared according to the literature procedure [48,57,58],
where O-phenylenediamine (2.16 g, 20 mmol) was dissolved in 30–40 mL of four M of HCl by heating.
The bromoacetic acid (4.16 g, 30 mmol) was added thoroughly, and then, the reaction mixture was boiled
under reflux for five h. The mixture was neutralized with ammonia, and the solid product was filtered,
washed with water, and dried. The obtained product was recrystallized from acetone to give red crystals.
M.p.: 60–62 ◦C (59–61 ◦C [42]), yield: 3.6 g, 86%.
3.4.2. Synthesis of N-((1H-Benzimidazol-2-yl)Methyl)Thiazol-2-Amine (2)
A mixture of 2-(bromomethyl)-1H-benzimidazole
1 (2.11 g, 10 mmol), 2-aminothiazole (1 g,
10 mmol), and potassium iodide KI (1.66 g, 10 mmol) in ethanol (50 mL) was heated under reflux for
6 h. Afterwards, an aqueous solution of potassium hydroxide KOH (1.66 g, 10 mmol, in five mL of
H2O) was added and heating was continued for a further 2 h. The cold reaction mixture was poured
onto ice-cold water, and the precipitated solid product was filtered off, dried, and recrystallized from
ethanol to give reddish brown crystals of N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine (2). M.p.:
> 250 ◦C (398 ◦C [43]), yield: 1.6 g, 69%.
3.4.3. General Procedure for the Synthesis of the N-((1H-Benzimidazol-2-yl)Methyl)-5-
(Aryldiazenyl)Thiazol-2-Amine (3a–c)
The corresponding aryl diazonium chlorides were prepared by adding cold sodium nitrite solution
(0.69 g, 10 mmol in 10 mL of H2O) to a cold suspension of 10 mmol of different aromatic amines (namely
p-nitroaniline, 1.38 g; p-toluidine, 1.07 g; and p-aminoacetophenone, 1.35 g) in concentrated HCl (4–6 mL)
with stirring. To a cold solution of N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine 2 (2.3 g, 10 mmol) in
ethanol (40 mL) and sodium acetate (1.6 g, 20 mmol), a cold aqueous solution from the corresponding
◦
aromatic diazonium salts was added dropwise with stirring at 0–5 C for 2 h. The solid products obtained
were filtered off, washed with water followed by cold ethanol, dried, and recrystallized from ethanol to
give N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)thiazol-2-amine (3a–c).
N-((1H-benzimidazol-2-yl)methyl)-5-((4-nitrophenyl)diazenyl)thiazol-2-amine (3a). Brown solid, M.p.:
◦
>250 C, Yield: 2.4 g, 63%. IR: νmax = 3740–2850 (NH, =CH aromatic, –CH aliphatic), 1620 (C=N),
1451(CH2) and 1335, 1569 (NO2) cm−1. H-NMR:
δ
/ppm = 3.51 (s, 2H, CH2 aliphatic), 5.87 (s, 1H,
1
NH), 6.95–7.65 (m, 8H, Ar–H; s, 1H, C4–thiazole–H), 10.90 (s, 1H, NH–benzimidazole ring). 13C-NMR:
/ppm = 43.2, 115.2 (2C), 123.0 (2C), 123.8 (2C), 126.6 (2C), 134.7, 138.8 (2C), 140.7, 141.5, 147.2, 147.8,
163.2. MS (m/z, %): 379 (M+, 100.0). HRMS calc. for C17H13N7O2S: 379.0851, found: 379.0873.
δ
◦
N-((1H-benzimidazol-2-yl)methyl)-5-(p-tolyldiazenyl)thiazol-2-amine (3b). Brown solid, M.p.: >250 C,
Yield: 1.8 g, 54%. IR: νmax = 3740–2850 (NH=CH aromatic, –CH aliphatic), 1620 (C=N), 1453 (CH2),
1
and 1389 (CH3) cm−1. H-NMR:
δ
/ppm = 2.49 (s, 2H, CH2 aliphatic), 3.40 (s, 3H, CH3), 5.87 (s, 1H,
NH), 6.92–7.64 (m, 8H, Ar–H; s, 1H, C4–thiazole–H), 10.99 (s, 1H, NH–benzimidazole ring). 13C-NMR:

Molecules 2018, 23, 3285
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δ
/ppm = 21.3, 43.2, 115.2 (2C), 123.0 (2C), 125.6, 128.8 (2C), 129.0 (2C), 138.3, 138.8 (2C), 140.7, 141.5,
147.2, 163.2. MS (m/z, %): 348 (M+, 1.06). HRMS calc. forC18H16N6S: 348.1157, found: 348.1168.
1-(4-((2-(((1H-benzimidazol-2-yl)methyl)amino)thiazol-5-yl)diazenyl)phenyl)ethan-1-one (3c).Brown solid,
◦
M.p.: >250 C, Yield: 1.7 g, 47%. IR: νmax = 3740–2850 (NH, =CH aromatic, –CH aliphatic), 1667
(CO acetyl), 1619 (C=N), 1453 (CH2), and 1388 (CH3) cm−1
.
1H-NMR:
δ
/ppm = 2.44 (s, 2H, CH2
aliphatic), 3.43 (s, 3H, CH3), 5.87 (s, 1H, NH), 7.16–7.74 (m, 8H, Ar–H; s, 1H, C4–thiazole–H), 10.90
(s, 1H, NH-benzimidazole ring). 13C-NMR:
δ/ppm = 26.3, 43.3, 115.2 (2C), 123.0 (2C), 128.8 (4C),
133.1, 136.8, 138.8 (2C), 140.7, 141.5, 147.3, 163.2, 197.0. MS (m/z, %): 376 (M+, 100.0). HRMS calc. for
C19H16N6OS: 376.1106, found: 376.1118.
3.4.4. Synthesis of 1-((1H-Benzimidazol-2-yl)Methyl)Thiourea (4)
A mixture of 2-(bromomethyl)-1H-benzimidazole
1 (2.11 g, 10 mmol) and thiourea (1.14 g,
15 mmol) in ethanol (30 mL) containing catalytic amount of trifluoroacetic acid (0.02 mL), was well
stirred under reflux for 2 h. The reaction mixture was allowed to cool, and the solid product that was
formed was filtered off, washed with cold ethanol, dried, and recrystallized from ethanol to obtain a
brown solid; M.p.: 129–130 ◦C, Yield: 2 g, 99%. IR: νmax = 3740 (NH2), 3251–3020 (NH, =CH aromatic,
1
–CH aliphatic), 1601 (C=N) and 1485(CH2) cm−1. H-NMR:
δ
/ppm = 3.39 (d, 2H, J = 7.4 Hz, CH2
aliphatic), 5.87 (t, 1H, NH), 7.18–7.48 (m, 4H, Ar–H), 9.50 (s, 2H, NH2), 10.90 (s, 1H, NH–hydrazo),
11.31 (s, 1H, NH–benzimidazole ring). 13C-NMR:
δ/ppm = 44.7, 115.2 (2C), 123.0 (2C), 138.8 (2C),
141.5, 182.5. MS (m/z, %): 206 (M+, 100.0). HRMS calc. for C9H10N4S: 206.0626, found: 206.0649 [45].
3.4.5. Synthesis of (2-((1H-Benzimidazol-2-yl)Methyl)Amino)Thiazo-4(5H)-One (5)
To a well stirred solution of 1-((1H-benzimidazol-2-yl)methyl)thiourea
4 (2.06 g, 10 mmol) in
20 mL of ethanol containing 0.5 mL of triethylamine (TEA), a chloroacetylchloride (1.12 mL, 10 mmol)
was added over one h at room temperature. The reaction mixture was then heated, and stirring was
continued at 60 ◦C for five h. The mixture was allowed to cool, and the solid product formed was
collected, dried, and recrystallized from ethanol to yield orange crystals; M.p.: >250 ◦C (284 ◦C [59]),
Yield: 1.8 g, 75%.
3.4.6. Synthesis of N-((1H-Benzimidazol-2-yl)Methyl)-4-Phenylthiazol-2-Amine (6)
A mixture of 1-((1H-benzimidazol-2-yl)methyl)thiourea 4 (2.06 g, 10 mmol) in 20 mL of ethanol
containing a few drops of piperidine, and phenacylchloride (1.54 g, 10 mmol) was allowed to stir
under reflux for six h. The red dish brown solid product formed was collected, washed from methanol,
dried, and recrystallized from ethanol; M.p.: 219–220 ◦C, Yield: 2.7 g, 88%. IR: νmax = 3366–2950 (NH,
=CH aromatic, –CH aliphatic), 1629 (C=N), and 1444(CH2) cm−1
.
1H-NMR:
δ
/ppm = 2.49 (s, 2H,
CH2 aliphatic), 5.32 (s, 1H, NH), 7.30–7.64 (m, 9H, Ar–H), 7.90 (s, 1H, C–4 thiazole ring), 10.10 (s, 1H,
NH–benzimidazole ring). 13C-NMR:
δ/ppm = 43.3, 107.8, 115.2 (2C), 123.0 (2C), 127.5, 128.4 (2C), 134.8
(2C), 136.3, 138.8 (2C), 141.5, 158.2, 161.3. MS (m/z, %): 306 (M+, 18.4). HRMS calc. for C17H14N4S:
306.0939, found: 306.0960.
3.4.7. General Procedure for the Synthesis of the Arylazo Derivatives (7a–c) and (8a–c)
The corresponding aryl diazonium chlorideswere prepared by adding cold sodium nitrite
solution 0.69 g, 10 mmol in 15 mL of H2O) to a cold suspension of different aromatic
amines (10 mmol) in concentrated HCl (4–6 mL) with continuous stirring. To a cold solution
of (2-((1H-benzimidazol-2-yl)methyl)amino)thiazo-4(5H)-one
5 (2.46 g,10 mmol) and/or N-((1H-
benzimidazol-2-yl)methyl)-4-phenylthiazol-2-amine (3.06 g, 10 mmol) in ethanol (20 mL) and sodium
6
acetate (1.6 g, 20 mmol), a cold aqueous solution from the corresponding aromatic diazonium
salts were added dropwise with stirring at 0–5 ◦C for two h. The solid products obtained
were filtered off, washed with water followed by cold ethanol, dried, and recrystallized from

Molecules 2018, 23, 3285
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ethanol to give 2-(((1H-benzimidazol-2-yl)methyl)imino)-5-(2-aryldiazenyl)thiazolidin-4-one (7a
and N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)-4-phenylthiazol-2(3H)-imine (8a–c).
–c)
2-(((1H-benzimidaz◦ol-2-yl)methyl)amino)-5-((4-nitrophenyl)diazenyl)thiazol-4(5H)-one (7a). Dark brown
solid; M.p.: >250 C, Yield: 2.9 g, 73%. IR: νmax = 3366–2961 (NH, =CH aromatic, –CH aliphatic),
1
1635 (CO), 1585 (C=N), 1438 (CH2) and 1332, 1519 (NO2) cm−1. H-NMR:
δ
/ppm = 2.50 (s, 2H, CH2
aliphatic), 4.11 (s, 1H, NH), 7.18–7.89 (m, 8H, Ar–H), 9.70 (s, 1H, NH–benzimidazole ring). 13C-NMR:
δ
/ppm = 43.5, 74.6, 115.2 (2C), 120.5 (2C), 123.0 (2C), 124.1 (2C), 138.8 (2C), 141.5, 157.4, 157.0, 158.2,
173.3. MS (m/z, %): 395 (M+, 0.45). HRMS calc. for C17H13N7O3S: 395.0801, found: 395.0822.
2-(((1H-benzimidazol-2-yl)methyl)amino)-5-(p-tolyldiazenyl)thiazol-4(5H)-one (7b). Dark brown solid; M.p.:
◦
>250 C, Yield: 1.9 g, 53%. IR: νmax = 3627–2799 (NH, =CH aromatic, –CH aliphatic), 1654 (CO),
1595 (C=N), 1441 (CH2) and1408 (CH3) cm−1
.
1H-NMR:
δ
/ppm = 2.50 (s, 3H, CH3), 3.08(s, 2H,
CH2 aliphatic), 4.89 (s, 1H, NH), 7.21–8.48 (m, 8H, Ar–H) and 8.91 (s, 1H, NH–benzimidazole ring).
13C-NMR:
δ/ppm = 21.3, 43.7, 74.6, 115.2 (2C), 122.5 (2C), 123.0 (2C), 129.1 (2C), 138.8 (2C), 141.5,
147.7, 157.7, 158.2, 173.4. MS (m/z, %): 364 (M+, 0.18). HRMS calc. for C18H168N6OS: 364.1106,
found: 364.1131.
2-(((1H-benz◦imidazol-2-yl)methyl)amino)-5-((4-acetylphenyl)diazenyl)thiazol-4(5H)-one (7c). Brown solid;
M.p.: >250 C, Yield: 1.9 g, 49%. IR: νmax = 3627–2990 (NH, =CH aromatic, –CH aliphatic), 1660
1
(CO), 1591 (C=N), 1431(CH2) and 1402 (CH3) cm−1. H-NMR:
δ
/ppm = 2.49 (s, 3H, CH3), 3.32 (s, 2H,
CH2 aliphatic), 4.11 (s, 1H, NH), 7.16–7.98 (m, 8H, Ar–H) and 9.54 (s, 1H, NH–benzimidazole ring).
13C-NMR:
δ/ppm = 26.4, 43.7, 74.6, 115.2 (2C), 122.5 (2C), 123.0 (2C), 129.1 (2C), 133.6, 138.8 (2C), 141.5,
155.1, 158.2, 173.3, 197.0. MS (m/z, %): 393.11 (M+, 20.5). HRMS calc. for C19H15N6O2S: 392.1055,
found: 392.1071.
N-((1H-benzimidazol-2-yl)methyl)-5-((4-nitrophenyl)diazenyl)-4-phenylthiazol-2-amine (8a). Brown solid;
M.p.: 183–185 ◦C, Yield: 3.2 g, 72%. IR: νmax = 3366–2950 (NH, =CH aromatic, –CH aliphatic), 1629
1
(C=N), 1444 (CH2) and 1322, 1513 (NO2) cm−1. H-NMR:
δ
/ppm = 2.50 (s, 2H, CH2 aliphatic), 5.32
/ppm =
(s, 1H, NH), 7.52–8.31(m, 13H, Ar–H), 8.99 (s, 1H, NH–benzimidazole ring). 13C-NMR:
δ
44.2, 89.6, 115.2 (2C), 123.0 (2C), 123.8 (2C), 126.5 (2C), 127.9, 128.3 (2C), 128.6 (2C), 134.1, 138.8 (2C),
141.5, 147.8, 154.1, 158.2, 161.0. MS (m/z, %): 455 (M+, 24.9). HRMS calc. for C23H17N7O2S: 455.1164,
found: 455.1145.
N-((1H-benzimidazol-2-yl)methyl)-4-phenyl-5-(p-tolyldiazenyl)thiazol-2-amine (8b). Brown solid; M.p.:
◦
162–163 C, Yield 4 g, 95%. IR: νmax = 3385–2964 (NH, =CH aromatic, –CH aliphatic), 1636 (C=N),1475
(CH2), and 1407 (CH3) cm−1.1H-NMR:
δ
/ppm = 2.25 (s, 3H, CH3), 3.44 (s, 2H, CH2 aliphatic), 4.22
(s, 1H, NH), 7.09–7.57 (m, 13H, Ar–H), 10.14 (s, 1H, NH–benzimidazole ring). 13C-NMR:
δ
/ppm =
21.3, 44.2, 89.6, 115.2 (2C), 123.0 (2C), 127.8, 128.3 (2C), 128.6 (2C), 128.7 (2C), 129.0 (2C), 134.1, 138.4,
138.8 (2C), 141.5, 145.1, 158.2, 161.0. MS (m/z, %): 424 (M+, 1.36). HRMS calc. for C24H20N6S: 424.1470,
found: 424.1452.
1-(4-((2-(((1H-benzimidazol-2-yl)methyl)amino)-4-phenylthiazol-5-yl)diazenyl)phenyl)ethan-1-one (8c).Brown
solid; M.p.: 130–131 ◦C, Yield: 4.3 g, 96%. IR: νmax = 3295–2855 (NH, =CH aromatic, –CH aliphatic),
1656 (CO acetyl), 1588 (C=N), 1474 (CH2), and 1407 (CH3) cm−1
.
1H-NMR:
δ
/ppm = 2.54 (s, 3H,
CH3), 3.43 (s, 2H, CH2 aliphatic), 4.32 (s, 1H, NH), 7.31–8.43 (m, 13H, Ar–H) and 10.94 (s, 1H,
NH–benzimidazole ring). 13C-NMR:
δ/ppm = 26.4, 44.2, 89.6, 115.2 (2C), 123.0 (2C), 128.3 (2C), 128.6
(5C), 128.7 (2C), 134.1, 136.4, 138.8 (2C), 141.5, 152.2, 158.2, 161.0, 197.0. MS (m/z, %): 452 (M+, 0.23).
HRMS calc. for C25H20N6OS: 452.1419, found: 452.1423.

Molecules 2018, 23, 3285
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4. Conclusions
In the development of this work, the newly synthesized series of benzimidazole derivatives 3a
–c,
7a
–c
, and 8a derived from 2-(bromomethyl)-1H-benzimidazole were prepared in satisfactory yields
–c
and fully characterized. They exhibited in vitro variable antimicrobial activities when subjected to
the bacterial strains Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative
bacteria) compared with Kanamycin and Ampicillin as standard drugs, respectively. Their inhibition
effect against DPPH (the lowest EC50 values) compared to Trolox (EC50 = 55.13) was shown as well.
They were screened for their antitumor activities against human liver cancer cell line (HepG2) to
evaluate the cytotoxic effects of gradient concentrations (100–500
percentage and EC50 values. The tested compounds exhibited noticeable efficiency against the tumor
cell line under investigation at the higher concentration. The benzimidazole derivatives (3a ) and
8b) showed higher activity than the other selected compounds that were tested. Presence of electron
withdrawing groups increased the antitumor activities as discussed.
µg/mL) in terms of cell viability
,
c
(
Author Contributions: M.E.K., A.A.G., F.M.K., T.A.T. and A.S.A.M. conceived, designed the experiments,
performed the experiments, analyzed the data, contributed reagents/materials/analysis tools and wrote the paper.
M.A.M. designed, supervised and performed the biological experiments.
Funding: This work was funded by the King Abdulaziz City for Science and Technology (KACST)-Saudi Arabia
(project No. 1-17-01-022-0006). The Authors acknowledge support from the Scientific Research Unit of Taif
University, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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Sample Availability: Samples of the compounds 3–8 are available from the authors.
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