Proceedings

Chemical Synthesis and Hemi-Synthesis of Novel

Benzimidazoles Derivatives Using Microwave-
Assisted Process: Chemical Characterization,
Bioactivities and Molecular Docking †
AsmaâSameut 1,2, Sarah Zanndouche 1,3, Chaimaa Boumaza 1,2, Chaima Dikes 1,3
and Borhane Eddine Cherif Ziani 1,*
1 Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques CRAPC-Bouismail-Tipaza,
Algeria
2 Department of Biology, University of Sâad Dahleb-Bilda, Algeria

3 Department of Biology, University of Science and Technology Houari Boumediene-Algiers, Algeria

* Correspondence: [email protected] or [email protected]; Tel.: +213-673-954-968

† Presented at the 24th International Electronic Conference on Synthetic Organic Chemistry, 15 November–

15 December 2020; Available online: https://ecsoc-24.sciforum.net/.

Published: date

Abstract: Benzimidazoles derivatives represent a class of heterocyclic compounds that exhibit a

wide range of pharmaceutical properties. The present study aimed to investigate the in-vitro
antioxidant and antimicrobial activities of newly synthesized benzimidazoles derivatives.
Compound 1b, (2-(1H-1,3-benzodiazol-2-yl) phenol) was synthesized by reacting o-
phenylenediamine (OPA) with chemical salicylaldehyde, while compound 2b, (2-(2-[(1E)-2-
phenylethenyl]-1H-1,3-benzodiazole) and 3b, (2-[(1E)-2,6-dimethylhepta-1,5-dien-1-yl]-1H-1,3-
benzodiazole) were obtained through hemi-synthesis process of respectively the cinnamon
(cinnamaldehyde, 90.54%), and lemongrass (cis-citral, 43.9%) essential oils previously characterized
by GCMS. Compounds 4b, (2-phenyl-1H-benzimidazole) and 5b, (5-(1H-benzimidazol-2-
yl)benzene-1,2,3-triol) were synthesized with click chemistry method by reacting the OPA with
Benzoic acid and gallic acid directly in ethanol under microwave irradiation MW 400 MHz. The
structure/purity of the synthesized compounds was clarified by spectroscopy ATR-FTIR and NMR
1H. Compounds 1b–5b were screened for their antioxidant activity by using four complementary in-

vitro assays: DPPH scavenging activity, ferric ion reducing power, β-carotene bleaching inhibition,
and TBARS formation inhibition. All the tested compounds showed antioxidant potential with
different performances. Antimicrobial activity was investigated against ATCC strains (three Gram-
bacteria: Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa, one Gram+ bacteria:
Staphylococcus aureus, and one yeast stain Candida albicans) through the determination of MIC and
MBC by using the microdilution method and rapid colorimetric test of p-iodonitrotetrazolium
chloride (INT). Compound 5b exhibited the highest potential especially against S. aureus (MIC =
0.156 mg·mL−1) followed S. typhi and C. albicans (MIC = 0.3125 mg·mL−1), then E.coli and P. aeruginosa.
Compound 1b also showed a great potential against S. aureus and C. albicans (MIC ˂ 0.3125 mg·mL−1),
followed by E.coli and S. typhi (MIC = 0.3125 mg·mL−1), and P. aeruginosa (MIC = 0.625 mg·mL−1). A
further molecular docking was proceeded using AutoDock Vina software on S. aureus thymidylate
kinase TMK-protein to highlight the structure-activity relationship of the potent molecules.

Keywords: benzimidazoles; synthesis; hemi-synthesis; microwave-assisted; NMR 1H; antioxidant

activity; antimicrobial activity; in-silico docking

1. Introduction

Chem. Proc. 2020, 1, Firstpage-Lastpage; doi: FOR PEER REVIEW www.mdpi.com/journal/chemproc

Chem. Proc. 2020, 1, FOR PEER REVIEW 2

The search for novel antimicrobial compounds in clinical microbiology is prompted by the need
to counteract the growing number of infectious diseases caused by multidrug-resistant strains (MDR:
Multi-Drug-Resistant and TDR: Totally drug-resistant) [1]. Bacterial resistance has dramatically
reduced the effectiveness of the majority of treatments available today, and an increasing number of
diseases have become more difficult to treat. Hence, it’s a crucial point to develop new therapeutic
agents and broad-spectrum pharmaceutical probes for clinical trials [2]. The natural biomolecules of
vegetable origin due to their chemical diversity such as the phenolic acids and chiral monoterpene
aldehydes, offer unlimited possibilities for new drug discovery through organic synthesis patterns.
Those structurally assorted compounds may offer biological potentialities such as the antioxidant
[3,4] and the antimicrobial properties [5] slightly linked to their structure configuration. Moreover,
they are a good candidate for the hemi-synthesis of new bioactive agents targeting a particular
biological activity or protein functionality. Benzimidazoles are heterocyclic compounds that
represent with their derivatives an interesting class of molecules of great importance in medicinal
chemistry, due to the large diversity of biological properties that they may present (antibacterial,
antiviral, antioxidant, anticancer, anti-inflammatory…etc.) [6]. Recently, in-silico docking has made
great strides in predicting the molecular interactions that hold a protein and ligand in the binding
site stimulating the progress of new drugs development [7,8]. The docking of small molecules and
the virtual screening of candidate compounds have become an integral part in the biomedical field
and drug design. Several software have been developed to provide a procedure to predict the
interaction of small molecules with protein targets, and incorporate flexibility within docking
algorithms, such as: AutoDock and AutoDock vina programs.
In this study, we contributed to perform:
• A chemical synthesis and hemi-synthesis of new benzimidazole derivatives,
• A physicochemical characterization (purification and structural analysis of the synthesized
compounds by 1H NMR spectroscopy and FTIR,
• Evaluation of antioxidant and antimicrobial activities by in-vitro assays,
• Evaluation of Docking scores of the synthesized compounds on 4QGH protein of Staphylococcus
aureus Thymidylate kinase (TMK).

2. Methods

2.1. Plant Material and Extraction Procedure

Lemongrass (Cymbopogon citratus L.) plant was collected from Tipaza province, Algeria. While
cinnamon (Cinnamomum verum L.) bark strips were obtained from a local market of Tipaza downtown
city. The extraction of essential oils was performed by hydrodistillation method using a Clevenger
type apparatus [9]. 100 g of vegetal material was steamed in 500 mL of boiled water for three hours.
After evaporation, the EO was condensed and collected in a shaded bottle (vial) after elimination of
water.

2.2. Essential Oils Analysis

The components of the extracted essential oils were analyzed by gas chromatography/mass
spectrometry (GC/MS) using a Hewlett Packard-6890 system, equipped with HP-5MS capillary
column (30 m × 0.25 mm × 0.25 μm); directly coupled to a selective mass detector Hewllet Packard-
5973; Helium was used as the carrier gas (1 mL/min). The analysis was performed using the following
temperature program: 60 °C–300 °C to 3 °C/min; without division for 1.50 min; with a sample volume
of 2 µ L of essential oil solution. The injector and detector temperatures were set at 240 °C. The ion
source temperature was 180 °C, and mass spectra were obtained in EIMS at 70 eV electron energy.
Identification of compounds was based on a comparison of mass spectra of each peak with those
recorded in the MS library (NIST02 and Wiley7) and comparing retention indices and mass spectra
with literature data.

2.3. Synthesis Procedure

Chem. Proc. 2020, 1, FOR PEER REVIEW 3

2.3.1. Synthesis/Hemi Synthesis of Three Benzimidazole Aldehydes Derivatives

The amino derivative 1,2-phenylenediamine (OPA) is first diluted in ethanol (15 mL) to form an
initial solution of reagent 1, and then the chemical salicylaldehyde (1 mmol of corresponding
aldehyde, calculated on the basis of its corresponding molecular mass, approximately 0.5 mL) is
added to an ethanolic solution with few drops of HCl 0.05%. For Hemi synthesis, the initial solution
was mixed with EO 1 mmol of cinnamaldehyde (~90%) and citral (~44%) calculated on the basis of
their percentage in the EO under constant agitation at room temperature, and few drops of HCl 0.05%
was added (Figure 1). The reaction was left for reflux (1 h) under adapted microwave extractor 400
MHz. The crystals of compound 1b, 2b and 3b were gradually formed in the reaction medium. The
precipitates were filtered and washed with cold ethanol after the required time and then purified by
thin layer chromatography (TLC) and column chromatography.

Figure 1. Synthesis procedure and Chemical structure of synthesized molecules obtained by

Chemsketch software.

2.3.2. Synthesis of Benzimidazole Phenolic Acids Derivatives

The total chemical synthesis reaction is carried out by the condensation of OPA with the
appropriate phenolic acids (0.03 mol). The reaction is initiated under microwave irradiation
conditions at 400 MHz (Figure 1). Initially, the OPA (3.24 g, 0.03 mole) was dissolved in ethanol in
the presence of a few drops of NH4Cl (5%) in a glass recipient (microwave synthesis reactor) of 30 mL
at room temperature until completely dissolved to form solution 1; After, each phenolic acid (in molar
correspondence) was also dissolved in an ethanol (30 mL) in the reactor and heated to 140 °C under
10 bars of internal pressure for 5 min. Therefore, it was cooled to room temperature, thus solution 2
is formed; The two solutions are mixed and let to react under microwave irradiation at 400 MHz for
10 min to obtain a precipitate of 2-(4-phenyl substituted)-1H-benzimidazoles; After cooling at room
temperature, the precipitated product was washed with cold dichloromethane or hexane, dried to
room temperature, and recrystallized in ethanol.
Chem. Proc. 2020, 1, FOR PEER REVIEW 4

2.4. Fractionating/Purification
To determine the migration pattern of all the synthetized compounds, a thin-layer
chromatography (TLC) was performed on a thin plate of silica GF-254 with fluorescein developer
deposited on a support and visualized under UV at 254/360 nm. The simples were diluted in ethanol
and deposited on the bottom of the silica plate by spots. The plate was placed in a vessel containing
the migration solvent, allowing the solvent to run to the top edge of the plate. The migration solvent
was a mixture of hexane, dichloromethane and ethanol (2:6:2 v/v/v). After migration, the
chromatography plate is then read directly under UV light, the spots appear without having to resort
to a developer. Afterward, a column chromatography was performed to separate and purify the final
products of the hemi-synthesized molecules since the reaction may occur also on other aldehydes of
the EO mixture. The separation is carried out by gravity on silica particles of 70 to 200 nm where the
solvent flows by drip. The eluent (mobile phase) used here initially the dichloromethane/hexane
(50:50 v/v) which allows the elution of the non-polar fraction followed by ethanol and chloroform,
which separates the fraction strongly retained by silica. The benzimidazole molecules produced are
driven by the mobile phase, and they are recovered in 250 mL beakers to be dried under vacuum (45
°C).

2.5. Structural Analysis

The structural analysis was first monitored by infrared spectroscopy (ATR-FTIR) to determine
the functional groups of the purified molecules. The spectra are recorded in a few minutes without
any limitation concerning the size of the studied molecule. FTIR spectroscopy in the 4000e400 cm1
region was also used to measure trends and reaction patterns in real time, providing very specific
information on kinetics, the mechanism, the reaction path and the influence of variables on reaction
performance. A 1H NMR Analysis were then performed on a Bruker 400 spectrometer (Bruker,
Wissembourg, France, 400 MHz for 1H), in DMSO-d6 as solvent. Chemical shifts (δ) were reported
in ppm and coupling constants (J) in Hz and the internal standard was TMS. In a 5 mm diameter
NMR tube, 500 uL of sample (synthetic molecules or standard solution) is inserted and a capillary
tube containing a solution of TSP-d4 dosed at 2.1 mmol proton/L. This solution serves as a reference
for chemical displacements (6 ‘H = 0.00 ppm). Depending on the concentration of the samples, 64 to
128 accumulations were made over a spectral width of 3200 Hz. The use of Brüker’s Topspin 2.6 NMR
software was used for data processing. Structural drawing and naming attribution were monitored
by Chemketch software and NMR sectra was processed by MestRnova program. Brüker’s Topspin
CMC 2.6 was used for structure authentification.

2.6. Bioactivities Properties Evaluation

2.6.1. Antioxidant Activity

The antioxidant activity of the synthesized benzimidazoles was evaluated using four
complementary in-vitro tests: DPPH radical-scavenging, reducing power (RP), inhibition of β-
carotene bleaching/linoleate, and inhibition of lipid peroxidation in ovine brain cells homogenates
(TBARS). The molecules are dissolved in ethanol with a well-known volume to set an initial
concentration [C= 3 mg·mL−1] that is diluted at different concentrations until EC50 is determined
(Concentration providing 50% antioxidant activity or 0.5 absorbance in the reductive power;
Expressed in µ g/mL) [10]. DPPH radical-scavenging and RP activity was measured using an ELX800
microplate reader (Bio-Tek Instruments,Inc.; Winooski, VT, USA) at 515 and 690 nm respectively, and
calculated as a percentage of reagents discoloration. Inhibition of β-carotene bleaching was evaluated
by the neutralization levels of linoleate free radicals that avoids β-carotene bleaching. Lipid
peroxidation inhibition in ovine brain homogenates was evaluated by the decrease in thiobarbituric
acid reactive substances (TBARS); the color intensity of the malondialdehyde-thiobarbituric acid
(MDA-TBA) was measured by its absorbance at 532 nm. BHT and Trolox was used as positive
controls.
Chem. Proc. 2020, 1, FOR PEER REVIEW 5

2.6.2. Antimicrobial Activity

The synthesized compounds were tested for their antibacterial activity against three Gram-
bacterial strains: Escherichia coli (ATCC® 10145), Salmonella typhi (ATCC® 19430), and Pseudomonas
aeruginosa (GEP ATCC® 10145GFP™), one Gram- strain: Staphylococcus aureus (ATCC® 10832™) and
one yeast strain: Candida albicans (ATCC® 90819™). MIC and MBC values were determined by a
micro-dilution method and a rapid colorimetric test of p-iodonitrotetrazolium chloride (INT) [11].
Briefly, stock solutions of 100 mg·mL−1 were prepared for each compound (1b–5b) in DMSO, and 100
μL of each stock solution was diluted in 400 μL of MHB (Mueller Hinton broth) or TSB (Trypton Soya
broth) according to bacterial requirements (resulting in a solution of 20 mg·mL−1). Subsequently, 10
μL inoculum (1.5 108 CFU/mL) of fresh bacterial cultures were added to all wells containing tested
concentrations in the range of 20–0.156 mg·mL−1. The microplate is then incubated at 37 °C for 24 h.
The MIC of the sample was determined after addition of INT (0.2 mg·mL−1, 20 µ L) and incubation at
37 °C in the oven (Jouan, Berlin, Germany) for 30 min, where viable microorganisms reduced yellow
dye to pink. MIC was defined as the lowest concentration of molecules that prevented this change
and allowed complete inhibition of bacterial growth. To determine the minimal bactericidal
concentrations (MBC), each negative well and positive control culture (10 μL) were sub-cultured into
96-well micro-plates containing culture medium and further incubated at 37 °C for 24 h. Gentamicine
(10 Ug) and Ceftazidime (30 Ug) were used as positive controls for bacterial strains and Nystatine
was used for the yeast strain.

2.7. Molecular Docking Study

Molecular docking simulations were done using AutoDock Vina software on S. aureus
thymidylate kinase TMK-protein. A crystalized structure of TMK-protein (PDB: 4QGH) was selected
and obtained from the Protein Data Bank (http://www.rcsb.org/structure/4QGH). The protein was
prepared for molecular docking by removing ligand heteroatoms and water molecules, and by
addition of polar hydrogens on AutoDock tools 1.5.7 software (ADT, The Scripps Research Institute,
La Jolla, CA, USA). The ligand 1b and 5b, was prepared for molecular docking simulation by setting
the torsion tree and rotatable, nonrotatable bonds present in the ligand through AutoDock tools
software [12], The binding scores of the receptor proteins is identified by Biovia DS visualizer. The
molecular docking affinity of the receptors/ligand is validated basing on the obtained binding energy
(ΔG) and the predicted inhibition constant (Ki).

3. Results and Discussion

3.1. GCMS Profiles of Essential Oils of Cinnamon and Lemongrass

The GCMS profile of cinnamon EO showed variations in the chemical constituents. The GCMS
chromatogram showed that the major compound found throughout the cinnamon oil was
cinnamaldehyde at 90.54% (Table A1). Then the remaining compounds are minor elements present
in very small amounts. Other aldehydes such as benzaldehyde, hydrocinnamaldehyde and 4-
methoxycinnamaldehyde were also detected in very small amounts. However, the descending order
of the major compounds present in whole cinnamon oil is indicated as follows: -cinnamaldehyde
(90.54%) > coumarin (2.87%) > hydrocinnamaldehyde (0.92%). For lemongrass, the oil was dominated
by monoterpene hydrocarbons (Table A2). This monoterpenic fraction was characterized by a high
percentage of cis-citral (43.53%), neral (34.87 %) and β-Myrcene (4.55 %). Other aldehyde components
were identified such as the (R)-(+)-citronellal and trans-chrysanthemal in very low concentrations.
Chem. Proc. 2020, 1, FOR PEER REVIEW 6

3.2. Separation and Purification of Synthetic Products

Qualitative analysis of the fractions obtained by thin-layer chromatography enabled the
separation of the fractions and revealed a considerable number of constituents visualized under UV
light at 254–360 nm and the chromatographic profiles and column chromatography is used to
separate/fractionate products. (2-(1H-1,3-benzodiazol-2-yl) phenol) was synthesized by reacting o-
phenylenediamine (OPA) with chemical salicylaldehyde, while compound 2b, (2-[(1E)-2-
phenylethenyl]-1H-1,3-benzodiazole) and 3b, (2-[(1E)-2,6-dimethylhepta-1,5-dien-1-yl]-1H-1,3-
benzodiazole) were obtained through hemi-synthesis process of respectively the cinnamon
(cinnamaldehyde, 90.54 %), and lemongrass (citral, 43.9%) essential oils previously characterized by
GCMS. The reaction of OPA with benzoic and gallic acids gives the benzimidazolic compounds 2-
phenyl-1H-benzimidazole (4b) and 5-(1H-benzimidazol-2-yl)benzene-1,2,3-triol (5b) respectively. In
the synthesis reaction, the 1H-benzimidazole heterocycle substituted in position 2 was synthesized
by reacting the diamine group of the OPA with the aldehyde function (-COH) of the corresponding
aldehyde and the acid function (-COOH) of phenolic acid. These compounds are named by the
ChemSketch software.

3.3. Structural Analysis

The infrared analysis performed by ATR-FTIR spectroscopy informs on functional groups and
covalent bonding of the compound produced, it is based on the absorption of light by most of the
molecules in the infrared region of the electromagnetic spectrum and by converting this absorption
into molecular vibration. This absorption corresponds specifically to the bonds present in the
molecule (N–H), (C–H), (C=H), (C–H) stretching and (C–C–C) out of plane bending. The spectrum of
the most obtained compounds showed a significant absorbance rates estimated at about 90%
absorbance between region 3160 and 3469 cm−1 and 75% absorbance between region 1000 and 1500
cm−1, with a specific C-N stretching band at 1368 cm−1 of the benzimidazole ring. In the other hand,
the RMN1H analysis of the synthetized compounds reveals globally the presence of a 2H singlet at
8.1, 7.6 ppm corresponding to the two pyrrolytic protons. We also note the presence of a multiplet
between 7.16 and 7.08 ppm corresponding to the 4 aromatic protons. Also, two doublets are observed
at 7.7 and 7.6 ppm attributable to the four aromatic protons. The chemical shifts of the compounds
are represented as follow:
2-(1H-1,3-benzodiazol-2-yl) phenol (1b): 1H NMR, (400 MHz, DMSO-d6): δ 7.12 ppm (1H, ddd, J = 7.7,
7.5, 1.2 Hz), 7.23–7.33 ppm (2H, 7.28 (ddd, J = 8.1, 7.6, 1.6 Hz), 7.30 ppm (ddd, J = 8.3, 1.2, 0.4 Hz)),
7.41 ppm (1H, ddd, J = 7.7, 7.6, 1.2 Hz), 7.54 ppm (1H, ddd, J = 8.3, 7.6, 1.7 Hz), 7.72–7.79 ppm (2H,
7.75 (ddd, J = 7.7, 1.6, 0.5 Hz), 7.75 ppm (ddd, J = 7.7, 1.7, 0.4 Hz)), 7.93 ppm (1H, ddd, J = 8.1, 1.2, 0.5
Hz).
2-[(1E)-2-phenylethenyl]-1H-1,3-benzodiazole (2b): 1H NMR, (400 MHz, DMSO-d6): δ 7.05 ppm (1H, ddd,
J = 7.7, 7.6, 1.3 Hz), 7.10–7.22 ppm (2H, 7.17 (d, J = 14.0 Hz), 7.17 ppm (ddd, J = 8.1, 7.6, 1.4 Hz)), 7.26–
7.46 ppm (6H, 7.36 (dddd, J = 8.0, 7.6, 1.5, 1.5 Hz), 7.42 ppm (d, J = 14.0 Hz), 7.41 ppm (dddd, J = 8.1,
1.8, 1.5, 0.5 Hz), 7.31 ppm (tdd, J = 8.0, 1.6, 0.5 Hz)), 7.61 ppm (1H, ddd, J = 8.1, 1.3, 0.4 Hz), 7.86 ppm
(1H, ddd, J = 7.7, 1.4, 0.4 Hz).
2-[(1E)-2,6-dimethylhepta-1,5-dien-1-yl]-1H-1,3-benzodiazole (3b): 1H NMR, (400 MHz, DMSO-d6): δ
1.53–1.54 ppm (6H, 1.54 (s), 1.54 (s)), 1.76 ppm (3H, s), 2.06–2.14 ppm (4H, 2.12 (t, J = 7.4 Hz), 2.10 ppm
(td, J = 7.4, 7.2 Hz)), 5.26 ppm (1H, t, J = 7.2 Hz), 6.43 ppm (1H, s), 7.05 ppm (1H, ddd, J = 7.9, 7.6, 1.3
Hz), 7.21 ppm (1H, ddd, J = 8.1, 7.6, 1.5 Hz), 7.58 ppm (1H, ddd, J = 8.1, 1.3, 0.5 Hz), 7.71 ppm (1H,
ddd, J = 7.9, 1.5, 0.5 Hz).
2-phenyl-1H-benzimidazole (4b): 1H NMR, (400 MHz, DMSO-d6): δ 7.05 ppm (1H, ddd, J = 7.7, 7.6, 1.3
Hz), 7.12–7.22 ppm (2H, 7.17 (d, J = 14.0 Hz), 7.17 ppm (ddd, J = 8.1, 7.6, 1.4 Hz)), 7.26–7.46 ppm (6H,
7.36 (dddd, J = 8.0, 7.6, 1.5, 1.5 Hz), 7.42 ppm (d, J = 14.0 Hz), 7.41 ppm (dddd, J = 8.1, 1.8, 1.5, 0.5 Hz),
7.31 ppm (tdd, J = 8.0, 1.6, 0.5 Hz)), 7.61 ppm (1H, ddd, J = 8.1, 1.3, 0.4 Hz), 7.86 ppm (1H, ddd, J = 7.7,
1.4, 0.4 Hz).
Chem. Proc. 2020, 1, FOR PEER REVIEW 7

5-(1H-benzimidazol-2-yl)benzene-1,2,3-triol (5b): 1H NMR, (400 MHz, DMSO-d6): δ 7.06 ppm (1H, ddd,
J = 8.1, 6.8, 1.4 Hz), 7.17 ppm (2H, d, J = 2.4 Hz), 7.42 ppm (1H, ddd, J = 8.0, 6.8, 1.3 Hz), 7.64 ppm (1H,
ddd, J = 8.1, 1.3, 0.5 Hz), 7.87 ppm (1H, ddd, J = 8.0, 1.4, 0.5 Hz).

3.4. Bioactivities Properties

3.4.1. Antioxidant Activity

Due to the complexity of oxidation processes and the diverse nature of antioxidants, there is no
universal method by which antioxidant activity can be measured quantitatively in a precise manner.
Oftentimes, it is necessary to combine the responses of different and complementary tests to have an
indication on the antioxidant capacity of the sample to be tested [13]. In the present study, the new
benzimidazoles reported were screened for their antioxidant activity by using four in vitro assays:
DPPH free radicals scavenging, reducing power, β-carotene bleaching inhibition and TBARS
formation inhibition. The results are expressed in EC50values (µ g/mL) as summarized in Table 1. It
is well known that reactive oxygen species (ROS) which can be superoxide radicals, hydroxyl and
peroxyl…etc. are causes of oxidative stress associated with various chronical diseases and DNA
damages leading to carcinogenesis [14]. The six molecules (1b–5b) showed antioxidant activity with
different performances. Compounds 1b and 5b showed the highest activity with significant EC50s
<200 µ g/mL, for DPPH and β-carotene. Compound 3b showed the highest values for the DPPH test
and iron reducing power, respectively, while compound 2b gave relatively average results.
Compound 1b exhibited the highest potential closely similar to the synthetic antioxidant drug
“Trolox” used as standard of comparison. with EC 50 = 53 μg/mL in comparison with the Trolox
which represents the standard with an estimated value of 51 μg/mL for the DPPH test. The values
obtained for the RP test show that those compounds had a high reducing iron potential especially of
the compound 1b and 5b (54 and 96 µ g/mL). Compounds 2b and 3b gave also a good result (101 and
102 µ g/mL). The degree of discoloration of β-carotene is measured by spectrophotometry and used
as an estimation of antioxidant activity. Based on the results obtained, compound 5b appears to be
the best inhibitor of linoleic acid oxidation (EC50 = 94 µ g/mL), followed by 5b (132 µ g/mL) and 2b
(181 µ g/mL). The same observation was found with the antiperoxidal activity where the compounds
showed a good lipid peroxidation inhibitory activity either for molecule 5b (101 µ g/mL) and
compounds 1b/3b (EC50 = 134 µ g/mL). The antioxidant response in herein considered in relation to
chemical structure that determine the redox behavior of the synthesized molecules, it is found that
benzimidazole substituted in position 2 containing a free hydroxyl groups which are compounds 1b
and 5b have a significant antioxidant activity, including free radical trapping power, iron ion
reducing power and lipid peroxidation inhibiting capacity, which are very remarkable. Those are the
most promising benzimidazoles for the development of antioxidant drugs.

Table 1. Antioxidant activity.

Antioxidant Activity EC5O (µg/mL)

Synthetized Molecule
DPPH Test Ferric ion Reducing Power β-Carotene TBARS
1b 53 ± 1 54 ± 4 192 ± 7 134 ± 2
2b 139 ± 4 101 ± 7 181 ± 5 156 ± 52
3b 220 ± 15 102 ± 22 220 ± 11 134 ± 2
4b 767 ± 6 544 ± 4 872 ± 37 1554 ± 25
5b 78 ± 5 96 ± 8 94 ± 3 101 ± 7
BHT 23 ± 3 30 ± 6 48 ± 5 76 ± 1
Trolox 51 ± 4 44 ± 4 63 ± 2 84 ± 6

3.4.2. Antimicrobial Activity

The search for new antimicrobial compounds in clinical microbiology is driven by the need to
counteract the growing rate of infectious diseases caused by food borne and/or multi-drug resistant
Chem. Proc. 2020, 1, FOR PEER REVIEW 8

strains (MDR: Multi-Drug-Resistant and TDR: Totally drug-resistant). Currently, the bacterial
resistance is leading to a growing need for new and effective anti-infective materials to prevent and
delay infections associated with implants and devices. The antibacterial activity of the synthetized
molecules (1b–5b) has been tested against 4 bacterial ATCC strains and one yeast strain. The results
are expressed in MIC and MBC values (mg·mL−1) as represented in the Table 2. Results clearly
demonstrated different degrees of bacteria growth inhibition. Gram-positive bacteria S. aureus was
more sensitive to the tested molecules presenting MIC values ranging from 0.156 to 1.25 mg·mL−1
comparing with other strains. Compound 5b was likely the most active compound by presenting
MIC value similar to the standard antibiotic Ceftazidime (MIC = 156 mg·mL−1). According to the
chemical characterization, the molecules with hydroxyl groups were the most active (compound 1b
and 5b). These molecules can be qualified as bactericidal and fungicidal. However, they can be used
as antibiotics because of their ability to complex with soluble extracellular proteins and with bacterial
cell walls, often resulting in inactivation and loss of function [15]. The antimicrobial activities of
products containing hydroxyl groups may involve different modes of action, namely destabilization
and permeability of the cytoplasmic membrane and inhibition of enzymes by oxidized products,
possibly by reaction with sulfhydryl groups or by more non-specific interactions with proteins [16].

Table 2. Antibacterial activity.

P.
Synthetized E. coli S. aureus S. typhi C. albicans
aeruginosa
Molecule
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
1b 0.3125 2.5 ˂0.3125 1.25 0.625 5 0.3125 5 ˂0.3125 2.5
2b 0.3125 10 0.3125 5 1.25 >10 0.3125 5 0.625 2.5
3b 0.3125 5 0.3125 2.5 2.5 5 0.3125 5 0.3125 5
4b 2.5 2.5 1.25 1.25 >10 >10 2.5 >10 5 >10
5b 0.625 2.5 0.156 0.625 2.5 5 0.3125 5 0.3125 5
Antibiotics
Gentamicine
˂0.078 ˂0.078 0.156 ˂0.156 nt
10Ug
Ceftazidime
˂0.156 0.156 0.156 ˂0.156 nt
30Ug
Nystatine nt nt nt nt ˂0.078
nt: not tested.

3.5. Molecular Docking Results

The thymidylate kinase is a key enzyme that is involved in DNA replication and reparation
mechanisms in most bacterial strains. It catalyzes phosphorylation of deoxythymidine
monophosphate (dTMP) to deoxythymidine diphosphate (dTDP). However, it is necessary to interest
to this protein for the search for new molecules that avoid the existing resistance mechanisms, for
this thymidylate kinase as a new target of anti S. aureus drug [13]. The designed molecules 1b and 5b
could inhibit the ligand binding-induced receptor confirmed by a virtual molecular docking using
Autodock vina software. The docking scores of the binding affinity (ΔG) and inhibition constant (Ki)
are presented in Table 3. The molecular docking could confirm the possible binding patterns that
may occur with the synthesized compound against the thymidylate kinase protein. Autodock vina
scores makes clear that 1b and 5b molecules have a significant ΔG values of −8.3 (Ki = 0.812 µ m) and
−9.4 Kcal/Mol (ki = 0.127 µ m) with 4QGH binding sites respectively (Table 3). This interaction affinity
is due to the existence of potential H-bond donor and H-bond acceptor groups as well as the
hydrophobic interactions with the docked molecules. the N−H bonds of the NH amine and the
hydrogen atoms of the hydroxyl group OH may adopt different binding position with the active
pocket of the protein inducing the receptor inhibition being able to arrest the bacterial DNA
replication and reparation mechanisms.
Chem. Proc. 2020, 1, FOR PEER REVIEW 9

Table 3. Dicking scores of 1b and 5b on 4QGH.

Binding Energy, Inhibition Constant,

Protein Interacting Residue
ΔG (Kcal/Mol) ki (µm)
TMK (4QGH) 1b −8.3 0.812
5b −9.4 0.127

4. Conclusions
Overall, the current study is designed to develop new bioactive drugs. Thus, a set of five new
benzimidazoles derivatives were synthetized by reacting o-phenylenediamine with several
aldehydes and phenolic acids through chemical synthesis and hemi-synthesis using a quick
microwave-assisted processes. Hemi-synthesis products were purified using column
chromatography and the developed molecules were characterized by ATR-FTIR and NMR 1H
spectroscopy. All synthetized compounds were screened for their antioxidant and antimicrobial
activities by using several invitro assays. Among all the panel of the new benzimidazoles derivatives,
compound 2-(1H-1,3-benzodiazol-2-yl) phenol (1b) and 5-(1H-benzimidazol-2-yl) benzene-1,2,3-triol
(5b) showed significant potential. Hence, these compounds may serve as lead molecules to develop
antimicrobial and antioxidant drugs. Additionally, compound 1b and 5b [2-(1E)-2-phenylethenyl-
1H-1,3-benzodiazole] were docked with S. aureus thymidylate kinase TMK-protein (4QGH) using
AutoDock Vina software to highlight the structure-activity relationship of these molecules. The
results showed a great binding score. Further assays should be performed on cytotoxicity as well as
in-vivo experimentation to validate their possible introduction to the pharmaceutical trials.

Author Contributions: Conceptualization, methodology, software and validation, B.E.C.Z.; formal analysis and
investigation, all the authors; resources and data curation, all the authors; writing—original draft preparation,
A.S.; writing—review and editing, S.Z., C.D. and B.E.C.Z.; visualization, C.B.; supervision, B.E.C.Z. All authors
have read and agreed to the published version of the manuscript.”

Funding: This research received no external funding and is a part of a Master thesis research developed by the
four first authors under a research thematic of new bioactive molecules for pharmaceutical uses.

Acknowledgments: The authors are grateful to the scientific and technical research centre (CRAPC) of Tipaza-
Algeria- for technical support.

Conflicts of Interest: the authors declare that they have no conflicts of interest regarding this manuscript.

Appendix A

Table A1. GCMS composition of Cinnamomun verum L. bark strips.

Rt (min) Concentration % Compound

4.3 0.02 Tetrachloroethylene
8.6 0.04 γ-Terpinene
9.4 0.03 Camphene
10.6 0.25 Benzaldehyde
14.6 0.05 D-Limonene
23.1 0.05 1,2-Chromene
24.6 0.92 Hydrocinnamaldehyde
25.3 0.07 Phenyl 2-Propynyl Ether
34.9 90.54 Cinnamaldehyde
38.2 0.61 α-Cubebene
39.1 0.06 Oxirane
39.4 0.05 α-Copaene
39.8 0.07 (+)-Sativene
40.7 0.04 (-)-Isosativene
41.3 0.05 β-Thujene
Chem. Proc. 2020, 1, FOR PEER REVIEW 10

43.2 0.03 Benzenamine

43.8 2.87 Coumarin
45.0 0.21 Naphthalene
46.1 0.05 Amide Hydrocinnamique
46.5 0.66 α-Cadinene
47.9 0.90 δ-Cadinene
48.5 0.19 1H-3a,7-Methanoazulene
49.2 1.13 4-Methoxycinnamaldehyde
53.4 0.08 2,4-Hexadiene
55.9 0.36 γ-Cadinene

Table A2. GCMS composition of Cymbopogon citratus L.

Rt (Min) % Compound
4.6 0.02 Tridodecylamine
7.8 0.08 D-Limonene
12.2 4.55 β-Myrcene
14.6 0.1 α-Limonene
15.4 0.31 α-Pinene
16.1 0.33 β-Ocimene
16.4 0.06 Myrcenylacetat
19.3 0.05 Nortricyclene
19.8 0.4 Furan
20.3 1.52 L-Linalool
21.3 0.06 Fenchol
22.5 0.23 Cyclohexene
23.3 0.44 Trans-Chrysanthemal
23.6 0.35 (R)-(+)-Citronellal
24.5 0.81 Cyclopropene
25.8 1.29 7-Methyl-1-Nonyne
28.4 0.11 O-Mentha-1(7),8-Dien-3-Ol
30.5 34.87 Neral
32.8 43.88 Cis-Citral
33.7 0.32 Geranial
34.2 0.22 Geranyl Vinyl Ether
35.8 3.5 Geraniol
38.4 0.24 Nerol
39.5 3.37 Nerol Acetate
40.2 0.65 Geranic Acid
41.3 0.21 β-Caryophyllene
42.4 0.17 α-Bergamotene
50.1 0.06 Neryl Acetate
51.4 0.08 β-Citronellal
54.1 0.14 Trans-β-Farnesene
70.8 0.18 Farnesyl
72.5 0.06 Trans-Caryophyllene
75.3 0.11 Cyclopropane Carboxamide
76.7 0.2 α-Trans-Sequicyclogeraniol
78.3 0.31 Farnesol
79.6 0.14 3,7-Nonadien-2-Ol
80.0 0.07 Geranylacetone
Chem. Proc. 2020, 1, FOR PEER REVIEW 11

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