Syntheses of N-substituted benzimidazolone derivatives: DFT calculations, Hirshfeld surface analysis, molecular docking studies and antibacterial activities

Article abstract of DOI:10.1016/j.molstruc.2019.127174

New benzimidazolone derivatives (2–5) were synthesized and characterized using NMR and single crystal X-ray diffraction techniques. Along with the experimental data, the predicted spectral data were also obtained using density functional theory (DFT) at the B3LYP/6-31G(d,p) level of theory. In addition, the closest contacts between the active atoms of the compounds were identified through Hirshfeld surface analyses, molecular docking studies, and DFT calculations. The antibacterial activities of derivatives (2–5) against gram-positive and gram-negative microbial strains, such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were also evaluated, and the results obtained showed the antibacterial activities of derivatives (2–5) using a minimum inhibitory concentration (MIC) assay.

Full text of DOI:10.1016/j.molstruc.2019.127174

Journal Pre-proof
Syntheses of N-substituted benzimidazolone derivatives: DFT calculations, Hirshfeld
surface analysis, molecular docking studies and antibacterial activities
Asmaa Saber, Nada Kheira Sebbar, Yusuf Sert, Nabil Alzaqri, Tuncer Hökelek,
Lhoussaine El Ghayati, Ahmed Talbaoui, Joel T. Mague, Yassir Filali Baba, Martine
Urrutigoîty, El Mokhtar Essassi
PII:
S0022-2860(19)31283-9
DOI:
https://doi.org/10.1016/j.molstruc.2019.127174
MOLSTR 127174
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 July 2019
Revised Date: 28 September 2019
Accepted Date: 1 October 2019
Please cite this article as: A. Saber, N.K. Sebbar, Y. Sert, N. Alzaqri, T. Hökelek, L. El Ghayati,
A. Talbaoui, J.T. Mague, Y.F. Baba, M. Urrutigoîty, E.M. Essassi, Syntheses of N-substituted
benzimidazolone derivatives: DFT calculations, Hirshfeld surface analysis, molecular docking studies
and antibacterial activities, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127174.
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Syntheses of N-substituted benzimidazolone derivatives: DFT calculations,
Hirshfeld surface analysis, molecular docking studies and antibacterial activities
Asmaa Saber a, Nada Kheira Sebbara,b,*, Yusuf Sertc,*, Nabil Alzaqrid, Tuncer Hökeleke, Lhoussaine El
Ghayatia, Ahmed Talbaouif, Joel T. Magueg, Yassir Filali Babah, Martine Urrutigoîtyk, El Mokhtar
Essassia
aLaboratoire de Chimie Organique Hétérocyclique, Centre de Recherche des Sciences des Médicaments, Pôle de Compétences
Pharmacochimie, Mohammed V University in Rabat, Faculté des Sciences, Av. Ibn Battouta, BP 1014 Rabat, Morocco
bLaboratoire de Chimie Appliquée et Environnement, Equipe de Chimie Bioorganique Appliquée, Faculté des sciences,
Université Ibn Zohr, Agadir, Morocco
cBozok University Department of Physics Yozgat/Turkey
dDepartment of Chemistry, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
e Department of Physics, Hacettepe University, 06800 Beytepe, Ankara, Turkey
fLaboratoire de Biologie des Pathologies Humaines, Faculté des Sciences, Université Mohammed V, Rabat, Morocco,
gDepartment of Chemistry, Tulane University, New Orleans, LA 70118, USA
hLaboratoire de Chimie Organique Appliquée, Université Sidi Mohamed Ben Abdallah, Faculté des Sciences et Techniques, BP
2202 Fez, Morocco.
kLaboratoire de Catalyse, Chimie Fine et Polymères, Ecole Nationale Supérieure des Ingénieurs en Arts Chimiques et
Technologiques, 118 route de Narbonne, 31077 Toulouse Cedex 04, France.
Abstract
New benzimidazolone derivatives (2-5) were synthesized and characterized using NMR and
single crystal X-ray diffraction techniques. Along with the experimental data, the predicted
spectral data were also obtained using density functional theory (DFT) at the B3LYP/6-31G(d,p)
level of theory. In addition, the closest contacts between the active atoms of the compounds were
identified through Hirshfeld surface analyses, molecular docking studies, and DFT calculations.
The antibacterial activities of derivatives (2-5) against gram-positive and gram-negative
microbial strains, such as Staphylococcus aureus, Escherichia coli, and Pseudomonas
aeruginosa were also evaluated, and the results obtained showed the antibacterial activities of
derivatives (2-5) using a minimum inhibitory concentration (MIC) assay.
Keywords: benzimidazolone, crystal structure, Hirshfeld surface, molecular docking,
DFT, antibacterial activity.
_______________
Corresponding authors:
Dr. Nada Kheira Sebbar
Dr. Yusuf Sert
Tel: +212615011615; E-mail: snounousebbar@gmail.com, yusufsert1984@gmail.com

1. Introduction
In recent years, heterocyclic compounds have received considerable attention due to
their significant importance in pharmacological and agricultural fields [1–7]. Notably,
nitrogen heterocyclic compounds are known to exhibit excellent biological and
pharmaceutical activities [3-5]. In fact, benzimidazole derivatives have been extensively
studied in different areas of chemistry, including pharmaceutical and other chemical
industries [5-7]. The benzimidazole core has several active sites and provides a great
responsiveness, making it an excellent heterocyclic precursor in the syntheses of new
heterocyclic compounds. With respect to the biological applications of benzimidazolone
derivatives, these derivatives are found to possess potent antioxidant [8], antiparasitic [9],
anthelmintic [10], antiproliferative [11], anti-HIV [12], anticonvulsant [13], anti-
inflammatory [14], antihypertensive [15], antineoplastic [16] and anti-trichinellosis [17]
activities. In addition, they have been used as proteases, since proteases have been linked with
several disease states, including thrombosis, inflammation, bronchoconstriction, and tumor
growth and invasion [18]. The incorporation of the benzimidazolone nucleus is an important
synthetic strategy in studies of antimicrobial drug discovery. Due to the varied bioactivities
exhibited by the benzimidazoles, efforts have been made from time to time to generate
libraries of these compounds and screen them for potential biological activities [18-23].They
have also been reported to function as precursors for the syntheses of the related compounds,
such as N-ribosyl-dimethylbenzimidazole present in nature, which serves as an axial ligand
for cobalt in vitamin B12 [24], and possesses anti-diabetic [25] and anti-corrosion activities
[26-30]. Fig. 1 provides some examples of bioactive molecules possessing benzimidazole
moieties [31-33].
In continuation of our research work on the development of N-substituted benzimidazole
derivatives and evaluation of their potential pharmacological activities recently performed by
our team [16, 34], it seemed interesting to prepare molecules of original structures
incorporated with benzimidazole motifs and evaluate their activities. The synthesized
compounds (2-5) were characterized by single crystal X-ray diffraction and Nuclear Magnetic
Resonance (1H and 13C NMR) techniques. The structures were confirmed by predicting the
corresponding spectroscopic data and Z-matrix coordinates using DFT calculations at the
B3LYP/6-31G(d,p) level of theory and molecular docking studies. The compounds (2-5) were
further identified using Hirshfeld surface analyses, molecular docking studies and DFT
calculations. Moreover, the antibacterial activities of the compounds (2-5) were evaluated

against gram-positive and gram-negative bacteria such as Staphylococcus aureus, Escherichia
coli, and Pseudomonas aeruginosa.
2. Material and methods
2.1. Spectral data measurements
The spectroscopic characterizations of the synthesized compounds (2-5) were
achieved by recording NMR spectra, which were measured on a Bruker Avance DPX 300
instrument. The chemical shifts (δ) were expressed in ppm down field from TMS
[tetramethylsilane, Si(CH3)4], which has been assigned to a chemical shift of zero, TMS as an
internal reference. Thin layer chromatography (TLC) and column chromatography were
carried out on silica plates (Merck 60 F254) and silica gel (Merck 60, 230-400 mesh),
respectively. Melting points of the compounds (2-5) were measured in open capillaries and
were uncorrected.
2.2. Syntheses of compounds (2-5) by alkylation reactions under PTC conditions.
Initially, we prepared benzimidazolone 2 by condensation of o-phenylenediamine 1
with ethyl acetoacetate in xylene at reflux for 6 h (Step a). Thereafter, 1-(prop-1-en-2-yl)-3-
(prop-2-ynyl)-1H-benzimidazol-2(3H)-one 3 was prepared with a good yield (91%) via
alkylation reaction of compound 2 by propargyl bromide under phase transfer catalysis (PTC)
conditions using tetra-n-butylammonium bromide (TBAB) as a catalyst and potassium
hydroxide as a base in CH2Cl2 at room temperature (Step b) (Scheme 1). In this paper, we
present the synthesis of benzimidazolone 4 by means of N-3 deprotection (Step c) as
described by Weber et al, 1992 [35] and Mondieig et al, 2013 [19]. The action of benzyl
chloride on 4 in hot DMF, for 6 h in the presence of tetra n-butylammonium bromide (TBAB)
as a catalyst and K2CO3 as a base, led to production of alkylated product 5 in good yield
(76%) (Step d) (Scheme 1). The structures of the compounds (2-5) were determined by the
1
usual spectroscopic methods, such as H NMR, 13C NMR, and were further confirmed by X-
ray crystallographic studies.

Scheme 1. Syntheses of benzimidazole derivatives (2-5).
2.2.1. Experimental part:
* Procedure for the synthesis of 2: [36]
o-Phenylenediamine (1.0 g, 9 mmol) and ethyl acetoacetate (1.2 mL, 9 mmol) were
heated in xylene (10 mL) for 6 h. The mixture was set aside for the growth of colourless
crystals of N-isopropenyl benzimidazol-2-one (yield, 90%).
1-Isopropenyl-1H-1,3-benzimidazol-2(3H)-one: 2
Yield (%) = 90%; mp =392 K; 1H NMR (300 MHz, CDCl3): 22.24 (d, 3H, 4JH-H = 3 Hz,
4
4
CH3),5.25 (d, 1H, JH-H = 3 Hz, CH2), 5.40 (d, 1H, JH-H = 3 Hz, CH2), 7.05-7.14 (m, 4H,
CHarom),10.00 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): 154.44 (C=O), 137.84 (Cq), 130.27
(Cq), 128.27 (Cq), 121.93 (CHarom), 121.43 (CHarom), 113.93 (CH2), 109.79 (CHarom), 109.14
(CHarom), 20.22 (CH3).

* Procedure for the synthesis of 3:
Propargyl bromide (11.4 mmol) was added to a mixture of 1-isopropenyl-1H-1,3-
benzimidazol-2(3H)-one 2 (5.7 mmol), potassium hydroxide (5.7 mmol) and tetra-n-butyl
ammonium bromide (0.15 mmol) in CH2Cl2 (15 mL). Stirring was continued at room
temperature for 48 h. Salts were removed by filtration, and the filtrate was concentrated under
reduced pressure. The residue obtained was purified by recrystallization from ethanol to
obtain colourless crystals in 91% yield.
1-(prop-1-en-2-yl)-3-(prop-2-ynyl)-1H-benzimidazol-2(3H)-one: 3
1
4
Yield (%) = 91; mp =331K; H NMR (300 MHz, CDCl3): 2.22 (d, 3H, JH-H = 3 Hz,
CH3), 2.30 (t, 3H, 3JH-H = 3 Hz, CH), 4.69 (d, 2H, 4JH-H = 3 Hz, CH2C≡CH), 5.20 (d, 1H, 4JH-H
4
= 3 Hz, CH2), 5.35 (d, 1H, JH-H = 3 Hz, CH2), 7.10-7.30 (m, 4H, CHarom). 13C NMR
(75 MHz, CDCl3): 151.99 (C=O), 137.99 (Cq), 129.07 (Cq), 128.72 (Cq), 121.86 (CHarom),
121.78 (CHarom), 113.32 (CH2), 109.20 (CHarom), 108.57 (CHarom), 77.06(CH), 72.82 (C≡CH),
30.40 (CH2), 20.22 (CH3).
* Procedure for the synthesis of 4:
A solution of 1-(prop-1-en-2-yl)-3-(prop-2-ynyl)-1H-benzoimidazol-2(3H)-one 3 (7.0
mmol) in DMF (10 mL) was treated with a cold sulfuric acid solution (15 mL, 50%) for 12 h.
The precipitate obtained was filtered, washed several times with water and subsequently with
acetone, and dried to obtain 4, in 75% yield [34].
1-(prop-2-ynyl)-1H-benzoimidazol-2(3H)-one, 4
1
3
Yield (%) = 75; mp =399 K; H NMR (300 MHz, DMSO): 3.31 (t, 1H, JH-H = 3 Hz,
CH), 4.68 (d, 2H, 4JH-H = 3 Hz, CH2C≡CH), 7.04-7.21 (m, 4H, CHarom), 10.99 (s, 1H, NH).13C
NMR (75 MHz, DMSO):154.01 (C=O), 129.79 (Cq), 128.72 (Cq), 121.86 (CHarom), 121.17
(CHarom), 109.44 (CHarom), 108.75 (CHarom), 79.01 (CH), 74.99 (C≡CH), 29.85 (CH2).

* Procedure for the synthesis of 5: [37]
To a solution of 1-(prop-2-ynyl)-1H-benzoimidazol-2(3H)-one (3.42 mmol), benzyl
chloride (6.81 mmol), and potassium carbonate (6.42 mmol) in DMF (15 mL), a catalytic
amount of tetra- n-butylammonium bromide (0.37 mmol) was added. The mixture was stirred
for 24 h, the solid material was removed by filtration, and the solvent was evaporated under
vacuum. The solid product obtained was purified by recrystallization from ethanol to obtain
colourless crystals in 76% yield.
1-benzyl-3-(prop-2-ynyl)-benzimidazol-2-one, 5
1
3
Yield (%) = 76; mp =361 k; H NMR (300 MHz, CDCl3): 2.30 (t, 1H, JH-H = 3 Hz,
CH), 4.72 (d, 2H, 4JH-H = 3 Hz, CH2C≡CH), 5.07 (s, 2H, CH2), 6.87-7.32 (m, 9H, CHarom). 13C
NMR (75 MHz, DMSO): 153.65 (C=O), 136.17 (Cq), 129.38 (Cq), 128.82 (2CHarom), 128.57
(Cq), 127.80 (CHarom), 127.55(2CHarom), 121.83 (CHarom), 121.58 (CHarom), 108.54 (CHarom),
108.50 (CHarom), 77.16(CH), 72.81 (C≡CH), 45.10 (CH2-Ph), 30.67 (CH2).
2.3. Single crystal X-ray diffraction
2.3.1. Crystal structures of benzimidazole derivatives (2-5)
The molecular and crystal structures of the benzimidazole derivatives (2-5), which were
obtained by condensation, N-3 deprotection and alkylation reactions were clarified. It is
interesting to note that all compounds crystallize in the monoclinic system with the space
groups of P21/c (for compounds 3-5) and C 2/c (for compound 2). The crystallographic data
have been deposited in Cambridge Crystallographic Data Center (CCDC) with the deposition
numbers of CCDC NC2184 (for compound 2), CCDC 1941910 (for compound 3), CCDC
GK2546 (for compound 4) and CCDC 1879758 (for compound 5).
2.4. Theoretical details
Recently, Density Functional Theory (DFT) method has been demonstrated to have an
advantage in computations of organic or inorganic compounds because of its superiority over
other methods. This method is very crucial due to both cost of calculations and reliable results

for complex molecules in quantum chemistry; the foundation of this method was established
in the 1970s. From a literature survey, we can say that the DFT method is used in many fields
from dense matter physics and computational physics to computational chemistry and the
method is both popular and versatile. In this theorem an “s” function, usually Becke 3-Lee-
Yang-Parr (B3LYP) is preferred, which is probably the best one [38, 39]. In this study, for
computations by the B3LYP and 6-31G(d,p) methods, the basis set was selected and all
calculations were carried out employing the Gaussian 09W package program [40].
Furthermore, the Gauss View 9 interface program [41] was used for visualization of
properties of the computed quantities. Firstly, molecular optimizations of the compounds (2-
5) were carried out starting with the atom coordinates determined by the crystallographic
studies and other computations were made based on the optimized structures. Calculations of
the intermolecular interactions were performed using the Crystal Explorer 3.1 program [42];
this method is known as the Hirshfeld surface analysis. Also, in this study, Highest Occupied
Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) analysis of
the compounds (2-5) were performed and the distributions of the groups were obtained using
the Chemissian software [43]. Later, 1H and 13CNMR chemical shifts of the compounds (2-5)
were computed by using the GIAO-Gauge-Including Atomic Orbital approach with the
B3LYP functional and a 6-31G(d,p) basis set, and then compared with the experimental NMR
spectra in a DMSO solution. Finally, molecular docking studies between the compounds (2-5)
and topoisomerase II enzyme (PDB code : 1JIJ) were carried out employing the
AutoDockVina free software program [44].
3. Results and discussion
3.1. Molecular Structures
As can be seen in Fig. 3, the compounds (2-5) are out of plane (all structures have C1
symmetry) and the atom numbering schemes were those used in the structure determinations.
Each compound was optimized starting from the observed crystal structure using the B3LYP
method and 6-31G(d,p) basis set and the bond lengths and angles were determined. Important
optimized parameters and their experimental values are given in Table 2. The experimental
values were in the solid phase but the calculated values were in the gas phase, so some
differences may be seen as frequently occured.
To the best of our knowledge, no computational analysis of 3 appears in the literature;
however, compounds (2, 4 and 5) were examined as previously reported [36-37]. When we

focused on the benzimidazol-2-one group, which is common to all structures, the N1-C7, N2-
C7, and O1=C7 bond lengths were calculated as 1.390 Å, 1.409 Å and 1.219 Å (for 2), 1.404
Å, 1.395 Å and 1.221 Å (for 3), 1.400 Å, 1.391 Å and 1.220 Å (for 4) and 1.392 Å, 1.396 Å
and 1.225 Å (for 5), respectively, using the B3LYP method and 6-31G(d,p) basis set.
Regarding
1-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-6-methoxy-1H-benzimidazol-
2(3H)-one [45], the N1-C1, N2-C1, and O1-C1 (according to the given atom numbering
scheme) the bond lengths were calculated as 1.359 Å, 1.383 Å and 1.242 Å, respectively,
using the DFT/B3LYP method/function. In this study, the basis set, D95V++**, by El Bakri
et. al [45] was used.
In the present study, the N1-C7-O1, N2-C7-O1 and N1-C7-N2 bond angles were
computed as 127.0°, 128.0° and 105.0° (for 2), 128.0°, 126.2° and 105.7° (for 3), 127.1°,
128.1° and 104.8° (for 4) and 127.3°, 127.0° and 105.8° (for 5), respectively, employing the
DFT/B3LYP method and 6-31G(d,p) basis set. For 1-[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]-6-methoxy-1H-benzimidazol-2(3H)-one [45], the N1-C1-O1, N2-C1-O1 and N1-
C1-N2 bond angles were calculated as 127.82°, 125.65° and 106.53°, respectively, using the
DFT/B3LYP method and D95V++** basis set level. Furthermore, comparison of the selected
theoretical bond lengths and angles by the B3LYP method and 6-31G(d,p) basis set show that
there is an excellent agreement between them. The other optimized molecular structure
parameters can be seen and compared in Table 2.
3.2. Hirshfeld surface calculations
The Hirshfeld surface analyses of the compounds (2-5) were carried out by using Crystal
Explorer 3.1 program [41]. In these calculations, the experimental atom positions were used
to obtain the intermolecular contacts and the contributions of the inter-contacts related to the
Hirshfeld surfaces, which are presented in Figs. 4 (a-d) and Figs. S1-S3 (Supporting
Information a-d since Hirshfeld surface analyses have been reported previously for the
compounds 2, 4 and 5). For 3, Fig. 4 (a) represents the 3D Hirshfeld surface (Dnorm), and Figs.
4 (b-d) represent the 2D fingerprint plots. In the figures, the three colours are represented on
the surfaces; red, blue and white. The dark red spots indicate powerful hydrogen bond points
in the crystals, white spots indicate contacts near the van der Waals separation, and blue spots
indicate longer contacts [46-49]. As can be seen in Fig. 4 (a), the interacting distances for O-
H, H-H, and C-H bonds were obtained as 2.326 Å, 2.525 Å to 2.863 Å, and 2.958 Å to 3.659

Å, respectively. Finally, the contributions of the inter-contacts to the Hirshfeld surfaces are H-
H (52.2%), C-H/H-C (23.8%) and O-H/H-O (11%); the quantitative results of the Hirshfeld
surface analysis are shown in Figs. 4 (b-d).
3.3. HOMO-LUMO Analyses
Considering the fact that HOMO-LUMO energies are the key factors in defining
quantum chemical interactions, the importance of this analysis becomes clearer [50]. The
highest occupied molecular orbital (HOMO) energies, the lowest unoccupied molecular
orbital (LUMO) energies, and their related quantities and orbital distributions (percentage
distribution according to the related groups) of the compounds (2-5) were computed using the
DFT/B3LYP method and 6-31G(d,p) basis set. The contributions to HOMOs and LUMOs,
and HOMO and LUMO energy values and other related parameters of the compounds (2-5)
are tabulated in Tables 3 and 4, respectively, and the resulting orbital pictures are shown in
Fig. 5. In Table 3, the contributions to HOMOs and LUMOs are determined by employing the
Chemissian software [43] as shown. As can be seen, the group with the highest electron
density is the benzimidazol-2-one moiety for both HOMO and LUMO distributions, that is,
both HOMOs and LUMOs are mainly concentrated in this group. This indicates that the
electronic transitions and charge transfers are over this group. Additionally, the HOMO–
LUMO gaps of the compounds (2-5) were calculated as 5.401 eV, 5.426 eV, 5.500 eV and
5.381 eV, respectively. From these results, we can conclude that the sum of the electronic and
zero-point energies of compound 5 is relatively low (the most stable structure according to the
energy results) and its gap is small. Furthermore, by using HOMO and LUMO values, other
crucial parameters such as the ionization potential, electron affinity, chemical hardness,
electronic chemical potential, electrophilicity index and softness were also calculated (Table
3).
3.4. NMR Analyses
1
13
The theoretical H-NMR and C-NMR chemical shifts were calculated using CDCl3
solutions (for 2, 3 and 5) and d6-DMSO (for 4) by the DFT/B3LYP and 6-31G(d,p) basis set
method with the application of IEFPCM model and GIAO method. The experimental and
calculated proton 1H- and 13C-NMR chemical shifts are shown in Tables 5 and 6.

3.5. Molecular Docking Studies
For understanding the binding interactions between a ligand and its receptor,
molecular docking studies are very important especially for drug design. In this section,
molecular docking analyses were performed with the help of the AutoDockVina program
[44]. In the computations, both the ligands and receptor pdb (protein data bank) formats were
prepared using the Discover Studio Visualizer 4.0 (DSV 4.0) software [51] and pdb format
for the target receptor was obtained from the Protein Data Bank [52, 53]. For the analysis
between the ligands (2-5) and topoisomerase II enzyme-1 JIJ (receptor), the molecular
docking positions (grid positions) were taken as 38x58x124 Å3, 40x40x126 Å3, 40x40x126
Å3 and 44x52x126 Å3, respectively. According to the rotatable bonds of the ligands (2-5), ten
modes were superposed and their affinity energy values and root mean square (RMS) values
were calculated (Table 7). From Table 7, we can surmise that the best docking pose is
between 4 and 1JIJ receptor with a binding energy of -6.6 kcal/mol involving two hydrogen
bonds. Additionally, the inhibition constant (Ki) is 14.5266x10-6 M (for 4) derived from
Ki=exp(ꢀG/RT), where ꢀG, R and T are the binding free energy, gas constant (1.9872036 ×
10-3 kcal/mol) and room temperature (298.15 K), respectively. This docking model is shown
in Figs. 6 (a and b). Most of the hydrogen bonding interactions (Figure 6) is between the
GLU302 residue and H1 with a bond distance of 2.94 Å and between LYS305 residue and O1
atom with a 3.09 Å distance, additionally, there are π-π stacking interactions between the
PHE273-PHE306 residues and the centroid of the benzimidazole unit. For ligands (2, 3 and
5), the molecular docking surfaces around the ligands and 2D structures are given in Figs. S4-
S6 (Supporting Information).
Finally, the molecular docking results revealed that ligands (2, 3 and 5) may exhibit
some activities against the active site of topoisomerase II enzyme; from this point of view,
this study will be useful to take a step forward for future antibacterial studies.
4. Study of Antibacterial activity
4.1. Microorganism used and inoculum preparation
The microorganisms tested included the following bacteria: Staphylococcus aureus
(ATCC-25923), Escherichia coli (ATTC-25922), and Pseudomonas aeruginosa (ATCC-
27853). All pathogenic microorganisms isolated from patients were stored in the culture

collection of the Microbiology Department (Microthec Unity) at the National Institute of
Hygiene (Rabat, Morocco). They were maintained in a brain heart infusion (BHI) at -80 °C.
Prior to the experiment, the cultures were prepared by sub-culturing 1 mL of each culture
stock in 9 mL of BHI broth.
4.2. Disc diffusion method
The “agar disc diffusion method” was employed for the determination of the
antibacterial activities of the synthesized compounds (2-5). The principle of this technique is
to estimate the bacteriostatic activities of the antibacterial agents by measuring the growth
inhibition zones of the bacteria around the discs impregnated with the test samples placed on
the agar plates. It is mostly used as a preliminary step to further studies, because it provides
access to essential qualitative results. We have adapted a similar method described in some
studies [54, 55]. The test samples were firstly dissolved in DMSO (1%), which does not have
an adverse effect on microbial growth. Briefly, the test was performed in sterile petri plates
containing an agar medium. Sterile medium (30 mL) was poured into petri plates, and after
solidification, 100 µL [106 colony-forming units (cfu)/mL] of fresh cultures of different
bacterial species (one microorganism per petri plate) were swabbed on the plates. Sterile filter
paper discs (6 mm in diameter), impregnated with 6 µL of each sample at a concentration of
50 mg/mL, were placed on the surface of the agar. The petri plates were sealed with sterile
laboratory film to avoid evaporation of the test samples, and then incubated at 30 °C for 24 h.
Diameters of the inhibition zones were measured in millimetres (mm). In addition, the
antibacterial activities of the synthesized compounds (2-5) were compared with commercially
available antibiotics, such as chloramphenicol (Chlor) and ampicillin (Amp). 1% DMSO
alone was used as a negative control. The plates were then incubated at 37 °C for 24 h. After
incubation, the diameter of the zones of inhibition were measured in mm and recorded [56,
57]. The experiment was carried out in triplicate. Compounds (2-5) showed an average
antibacterial activity (Figure 7 and Table 8).
The results are presented in the form of antibiograms below:
Disc diffusion assays were employed to determine the MIC of the compounds (2-5).
The results of the antibacterial activity of the compounds (2-5) exhibit growth inhibition in
the three bacterial strains (Staphylococcus aureus, Escherichia coli, and Pseudomonas
aeruginosa). Compound 2 obtained by the condensation of o-phenylenediamine 1 with ethyl
acetoacetate gave an anti-bacterial activity of MIC = 12.5 ꢁg/mL for Staphylococcus aureus,

Pseudomonas aeruginosa, and MIC = 3.125 ꢁg/mL for Escherichia coli. Moreover, in order
to increase the inhibitory activity of 2 we alkylated it with propargyl bromide. Interestingly,
the presence of a prop-1-yne group in compounds (3, 4, and 5) showed a similar activity
against the two bacterial strains as that tested by 5, with an MIC of 12.5 ꢁg/mL for
Escherichia coli and Pseudomonas aeruginosa and MIC = 3.125 ꢁg/mL for Staphylococcus
aureus. However, compound 3 showed a better activity with an MIC of 6.25 ꢁg/mL for
Staphylococcus aureus, 25 ꢁg/mL for Pseudomonas aeruginosa, and 12.5 ꢁg/mL for
Escherichia coli. Indeed, the deprotection of N-3 of compound 3 also showed a better
activity, with an MIC of 12.5 ꢁg/mL for Staphylococcus aureus and Escherichia coli, and 25
ꢁg/mL for Pseudomonas aeruginosa. Notably that the derivatives functionalized with ester
groups and benzene rings possess the highest antibacterial activity (92% of the pathogenic
bacteria were sensitive to these compounds). Studies, such as, anti-inflammatory, anti-fungal,
antiparasitic, and anti-cancer tests should also be performed, as the literature reveals a lot of
interesting results on these subjects. Also, other bacteria should be tested to expand this
investigation.
5. Conclusions
The studies performed in this work show that benzimidazole constitutes an interesting core
present in several drugs and natural products Moreover, we report the synthesis of novel
benzimidazole derivatives 2-5. Their structures have been elucidated thanks to X-ray
crystallography and spectroscopic techniques. The theoretical approach used allows a relatively
1
good reproduction of X-ray geometrical parameters, spectral data, and H and 13C NMR
chemical shifts. Hirshfeld surface analysis was employed to confirm the existence of
intermolecular interactions in compounds 2-5. The experimental spectroscopic data were well
reproduced by using quantum chemical DFT and in silico based molecular docking studies. The
benzimidazolone derivatives 2-5 exhibited antibacterial activity and could act as potent
antibacterial compounds.
Acknowledgements
The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for participating in this work through research group no (RG- 1440-141). Authors
would like to thank Prof. Dr. Fatih Ucun for his helpful contribution and Gaussian Calculations
and JTM thanks Tulane University for support of the Tulane Crystallography Laboratory.

Abbreviations
Chlor-Chloramphenicol;
Amp-Ampicillin;
DMSO-dimethylsulfoxyde
MIC-Minimum Inhibitor Concentration
BHI-Brain heart infusion
HOMO-highest occupied molecular orbital
LUMO-lowest unoccupied molecular orbital
DSV 4.0-Discover Studio Visualizer 4.0
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Figure Captions
Figure 1. Examples of bioactive molecules derived from benzimidazoles.
Figure 2. ORTEP plots with the ellipsoids drawn at the 50% probability level of the compounds
(2-5). 2: 1-(prop-1-en-2-yl)-1H-benzoimidazol-2(3H)-one: [31]; 3: 1-(prop-1-en-2-yl)-3-(prop-
2-ynyl)-1H-benzoimidazol-2(3H)-one; 4: 1-(prop-2-ynyl)-1H-benzoimidazol-2(3H)-one and 5:
1-benzyl-3-(prop-2-yn-1-yl)-2,3-dihydro-1H-1,3-benzodiazol -3-one.
Figure 3. The optimized structures of benzodiazaol-2-one derivatives (2-5).
Figure 4. The 3D Hirshfeld surface (a) and 2D fingerprint histograms of 3.
Figure 5. HOMO and LUMO plots of 2-5 in B3LYP/6-31G(d,p).
Figure 6. The molecular docking results of 4 with 1JIJ protein, surfaces around ligand (a) and 2D
forms (b).
Figure 7. Antibacterial activities of the compounds (2-5) vis-a-vis bacteria tested
(Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) are summarized in the
figure below. Chlor, Chloramphenicol (30 µg/mL); Amp, Ampicillin (10 µg/mL); DMSO, dimethylsulfoxyde
(1 %).

Table 1. Experimental details
Compound 2 [36]
Compound 3
Crystal data
Compound 4
Compound 5
CCDC Deposition
number
NC2184
1941910
1879758
GK2546
Chemical formula
Mr
C10H10N2O
174.20
C13H12N2O
212.25
C10H8N2O
172.18
C17H14N2O
262.30
Crystal system, space
group
Monoclinic, C2/c
Monoclinic, P21/c
Monoclinic, P21/c
Monoclinic, P21/c
Temperature (K)
100
150
150
298
15.8724 (2),
6.0971 (1),
17.9313 (3)
90.961 (2)
1735.07 (5)
8
15.3197 (4),
9.4602 (2),
7.2933 (2)
92.467 (1)
1056.02 (5)
4
4.5116 (2), 17.9709 8.3567 (2), 9.2040
a, b, c (Å)
(7), 10.6508 (4)
(2), 17.7868 (4)
β (°)
V (Å3)
Z
94.405 (2)
860.99 (6)
4
94.559 (1)
1363.74 (5)
4
Radiation type
µ (mm−1)
Crystal size (mm)
Mo Kα
0.09
Cu Kα
0.69
Cu Kα
0.72
Cu Kα
0.64
0.35 × 0.30 × 0.18 0.28 × 0.12 × 0.05 0.37 × 0.09 × 0.02 0.23 × 0.20 × 0.19
Data collection
Bruker X8 APEXII Bruker D8
VENTURE
Bruker D8
Bruker D8
VENTURE
PHOTON 100
CMOS
VENTURE
PHOTON 100
CMOS
Diffractometer
PHOTON 100
CMOS
No. of measured,
independent and
observed [I> 2σ(I)]
reflections
2231
7854, 2053, 1838
6789, 1586, 1319
13551, 2778, 2433
Rint
0.023
0.030
0.053
0.032
Refinement
0.036, 0.090, 1.05
R[F2> 2σ(F2)], wR(F2), 0.039, 0.114, 0.98
0.041, 0.100, 1.13
0.038, 0.108, 1.05
S
No. of reflections
2506
2053
1586
2778
No. of parameters
ꢀρmax, ꢀρmin (e Å−3)
123
0.39, 0.21
183
0.24, −0.25
150
0.18, −0.22
238
0.15, −0.12

Table 2. The selected optimized structure parameters of the title compounds (2-5)
Bond angles (°)
Bond lenghts (Å)
Optimized bond
angles
X-Ray
DFT/B3LYP/
6-31 G(d,p)
Optimized
bond lenghts
X-Ray
DFT/B3LYP/
6-31 G(d,p)
Compound 2
C7—N1—C1
C4—C5—H5
C7—N1—H1
C1—N1—H1
C5—C6—N2
O1—C7—N2
O1—C7—N1
C6—N2—C8
C7—N2—C6
C9—C8—N2
N1—C1—C6
C1—C2—C3
C4—C3—C2
N2—C8—C10
C2—C1—N1
N1—C7—N2
C7—N2—C8
110.29 (7)
121.5
111.5
121.2
121.1
127.3
132.1
128.0
127.0
126.8
106.6
120.1
106.5
117.6
120.9
115.6
131.8
105.0
123.5
O1—C7
C3—H3
N1—C7
C4—C5
N1—C1
C4—H4
N1—H1
C5—C6
N2—C7
N2—C6
C8—C9
N2—C8
C8—C10
C1—C6
1.2338 (11)
0.9500
1.3663 (11)
1.3963 (13)
1.3868 (11)
0.9500
1.219
1.085
1.390
1.400
1.388
1.085
1.006
1.390
1.409
1.405
1.337
1.430
1.504
1.410
122.5 (11)
127.2 (11)
131.94 (8)
125.86 (8)
127.42 (8)
126.52 (7)
109.21 (7)
119.50 (8)
107.14 (7)
117.45 (9)
120.99 (9)
115.54 (8)
131.46 (8)
106.72 (7)
124.13 (7)
0.871 (9)
1.3827 (12)
1.3878 (11)
1.4016 (10)
1.3225 (14)
1.4319 (11)
1.4960 (13)
1.3999 (11)
Compound 3
C7—N1—C6
C7—N1—C8
C6—N1—C8
C7—N2—C1
C7—N2—C11
C1—N2—C11
C2—C1—N2
C2—C1—C6
N2—C1—C6
C5—C6—N1
C1—C6—N1
O1—C7—N2
O1—C7—N1
N2—C7—N1
C9—C8—N1
C9—C8—C10
N1—C8—C10
109.41(10)
123.86(10)
126.44(10)
110.51(10)
123.10(11)
126.22(11)
130.92(11)
122.11(11)
106.97(10)
132.25(11)
107.05(10)
126.53(11)
127.46(11)
106.01(10)
119.88(12)
124.03(12)
116.07(11)
109.5
123.5
126.9
110.5
121.6
127.7
131.4
121.6
106.9
132.0
107.2
126.2
128.0
105.7
120.1
124.3
115.6
N2—C11
O1—C7
N1—C7
N1—C6
N1—C8
N2—C7
N2—C1
C8—C9
C8—C10
C1—C2
C4—C5
C5—C6
C1—C6
C3—C4
C2—C3
1.4525(16)
1.2233(15)
1.3925(15)
1.4035(16)
1.4301(15)
1.3812(16)
1.3890(16)
1.3280(19)
1.4909(18)
1.3836(18)
1.3942(19)
1.3822(17)
1.3980(17)
1.3933(19)
1.3923(19)
1.450
1.221
1.404
1.405
1.430
1.395
1.391
1.336
1.504
1.389
1.401
1.389
1.409
1.398
1.400
Compound 4
C7—N1—C6
C7—N1—C8
C6—N1—C8
C7—N2—C1
C5—C6—N1
C1—C6—N1
C2—C1—N2
C2—C1—C6
N2—C1—C6
O1—C7—N2
O1—C7—N1
N2—C7—N1
109.64(12) 110.6
O1—C7
N1—C7
N1—C6
N1—C8
N2—C7
N2—C1
C1—C2
C1—C6
C2—C3
C2—H5
C3—C4
C3—H4
1.239(2)
1.382(2)
1.395(2)
1.455(2)
1.364(2)
1.390(2)
1.383(2)
1.391(2)
1.388(2)
0.951(19)
1.388(3)
0.97(2)
1.220
1.400
1.394
1.453
1.391
1.391
1.388
1.411
1.401
1.085
1.397
1.085
124.23(14) 121.6
126.11(13) 127.8
110.06(13) 111.3
131.43(15) 131.9
106.58(13) 106.9
131.56(15) 132.3
121.25(15) 121.3
107.19(13) 106.5
127.89(14) 128.1
125.60(14) 127.1
106.51(14) 104.8

N1—C8—C9
C10—C9—C8
C9—C10—H10
C1—C2—C3
C2—C3—C4
111.29(13) 113.8
176.99(18) 178.7
C4—C5
C4—H3
C5—C6
1.391(3)
0.94(2)
1.384(2)
1.401
1.085
1.389
177.6(15)
179.0
116.99(16) 117.6
121.76(17) 121.2
Compound 5
C7—N1—C6
C7—N1—C11
C6—N1—C11
C7—N2—C1
C7—N2—C8
C1—N2—C8
C2—C1—N2
C2—C1—C6
N2—C1—C6
O1—C7—N2
O1—C7—N1
N2—C7—N1
N2—C8—C9
N1—C11—C12
C5—C6—N1
C2—C3—C4
C3—C4—C5
110.18(9)
123.09(10) 122.2
126.62(10) 127.7
110.1
O1—C7
N1—C7
N1—C6
N1—C11
N2—C7
N2—C1
N2—C8
C1—C2
C1—C6
C2—C3
C2—H2
C3—C4
C4—C5
C9—C10
1.2163(15)
1.3823(16)
1.3869(15)
1.4632(15)
1.3775(16)
1.3875(15)
1.4525(16)
1.3737(17)
1.3985(16)
1.383(2)
1.225
1.392
1.394
1.458
1.396
1.394
1.453
1.388
1.411
1.401
1.084
1.398
1.401
1.207
110.34(9)
110.2
123.32(11) 122.0
126.34(11) 128.0
131.74(11) 131.8
121.46(11) 121.4
106.80(10) 106.8
126.95(12) 127.0
127.23(12) 127.3
105.82(10) 105.8
111.93(11) 113.8
113.05(10) 114.0
132.12(11) 131.8
121.16(13) 121.2
121.56(14) 121.3
0.959(17)
1.384(2)
1.388(2)
1.166(2)
Table 3. The contributions to HOMOs and LUMOs of compounds (2-5).
MOs
Compound 2
Benzoimidazol-2-one
Prop-1-en-2yl
93%
89%
7%
11%
HOMO
LUMO
Compound 3
MOs
Benzoimidazol-2-one
Prop-1-en-2yl
6%
14%
Compound 4
Prop-2-ynyl
93%
86%
1%
0%
HOMO
LUMO
MOs
Benzoimidazol-2-one
Prop-2-ynyl
98%
100%
2%
0%
HOMO
LUMO
Compound 5
Prop-2-ynyl
MOs
HOMO
LUMO
Benzoimidazol-2-one
benzyl
4%
89%
95%
11%
1%
0%

Table 4. HOMO and LUMO Energy Values and other related parameters of compounds (2-5).
Parameters (eV)
ELUMO (eV)
Compound 2
-0.221
Compound 3
-0.200
Compound 4
-0.177
Compound 5
-0.212
-5.622
-5.627
-5.677
-5.593
EHOMO (eV)
5.401
5.426
5.500
5.381
Energy band gap |EHOMO-ELUMO
|
Ionization potential (I = -EHOMO
Electron affinity (A = -ELUMO
)
5.622
0.221
5.627
0.200
5.677
0.177
5.593
0.212
)
2.700
0.185
2.922
2.713
0.184
2.914
2.750
0.182
2.927
2.690
0.186
2.903
Chemical hardness (h = (I-A)/2)
Chemical softness (z = 1/2h)
Electronegativity (χ = (I+A)/2)
Chemical potential (ꢀ= -(I+A)/2)
-2.922
1.581
-2.914
1.564
-2.927
1.558
-2.903
1.566
Electrophilicity index (w = ꢀ2/2h)
-840.681218
Sum of Electronic and zero-point Energies (a.u)
-571.661776
-687.073056
-570.418136
Table 5. The experimental and computed 13C NMR isotropic chemical shifts (with respect to TMS, all values
are in ppm) of compounds (2-5).
2
3
4
5
Atoms
C1
C2
C3
C4
C5
C6
C7
C8
δexp.
δcal.
Atoms
C1
C2
C3
C4
C5
C6
C7
C8
δexp.
δcal.
Atoms
C1
C2
C3
C4
C5
C6
C7
C8
δexp.
δcal.
Atoms
C1
C2
C3
C4
C5
C6
C7
C8
δexp.
δcal.
128.27 116.29
109.79 97.21
121.93 109.99
121.43 109.49
109.14 98.02
130.27 118.67
154.44 136.89
137.84 131.62
113.93 99.61
129.07 117.54
108.57 97.64
121.86 110.16
121.78 110.04
109.20 98.02
128.72 117.62
151.99 137.06
137.99 131.62
113.32 100.08
20.22
30.40
72.82
77.06
128.72 115.88
109.44 97.85
121.86 110.31
121.17 109.88
108.75 97.53
129.79 117.87
154.01 137.47
29.85
74.99
79.01
129.38 116.96
108.54 97.39
121.58 109.75
121.83 109.98
108.50 97.02
136.17 117.73
153.65 139.22
30.67
72.81
77.16
45.10
128.57 126.92
128.82 116.99
128.82 117.01
127.80 115.66
127.55 115.90
127.55 116.55
24.01
64.45
60.00
24.57
64.16
60.13
39.62
C9
C10
C9
C9
C10
C9
20.22
13.24
C10
C11
C12
C13
13.28
24.10
63.98
60.15
C10
C11
C12
C13
C14
C15
C16
C17

Table 6. The experimental and computed 1H NMR isotropic chemical shifts (with respect to TMS, all values are in
ppm) of compounds (2-5).
2
3
4
5
Atoms δexp. δcal.
Atoms
H2
H3
H4
H5
H9A
H9B
H10A
H10B
H10C
H11A
H11B
H13
δexp.
δcal.
Atoms
H1
H5
H4
H3
δexp.
δcal.
Atoms
H2
H3
H4
H5
H8A
H8B
H10
H11A
H11B
H13
H14
H15
H16
H17
δexp.
δcal.
H1
H2
H3
H4
6.75
7.23
7.39
7.41
7.45
7.55
7.49
7.48
7.49
5.72
5.48
2.05
2.02
3.59
4.37
5.36
2.23
7.00
7.55
7.51
7.51
7.38
4.42
5.24
2.29
7.51
7.43
7.39
7.34
4.42
5.45
2.23
4.41
5.81
8.03
7.72
7.65
7.71
7.82
7.04-
7.21
7.10-
7.30
6.87-
7.32
7.05
-
7.14
H5
5.35
5.20
2.22
2.22
2.22
4.69
4.69
2.30
H2
4.72
4.72
2.30
5.07
5.07
H9A
H9B
H10A
H10B
H10C
NH1
5.25 5.48
5.40 5.68
2.24 2.00
2.24 3.59
2.24 1.98
10.0
H8A
H8B
H10
NH2
4.68
4.68
3.31
10.99
6.87-
7.32
Table 7. AutoDockVina results of the binding affinity and RMSD values of different poses in 1JIJ
inhibitor of compounds 2-5.
Mode
2-1JIJ
rmsdl.b.
3-1JIJ
rmsdl.b.
Affinity
(kcal/mol)
-6.5
rmsdu.b.
Affinity
(kcal/mol)
-5.9
rmsdu.b.
1
2
3
4
5
6
7
8
9
0.000
20.458
36.621
20.140
1.731
10.553
20.467
20.070
40.735
2.531
0.000
21.739
37.555
21.310
2.873
12.161
21.663
21.413
41.840
3.338
0.000
36.198
36.572
37.662
35.963
19.720
11.303
36.933
36.729
1.528
0.000
37.853
38.498
39.435
37.762
21.405
13.549
38.758
38.493
3.927
-5.6
-5.3
-5.3
-5.3
-5.3
-5.2
-5.1
-5.1
-5.8
-5.6
-5.6
-5.5
-5.4
-5.4
-5.1
-5.0
10
-5.0
-4.9
4-1JIJ
5-1JIJ
Mode
Affinity
(kcal/mol)
-6.6
rmsdl.b.
rmsdu.b.
Affinity
(kcal/mol)
-6.5
rmsdl.b.
rmsdu.b.
1
2
3
4
5
6
7
8
9
0.000
23.334
1.816
20.775
20.805
37.308
30.494
19.363
20.042
21.277
0.000
24.717
2.125
22.032
22.065
38.393
31.430
20.805
21.409
22.643
0.000
3.613
41.713
2.130
0.000
5.677
43.692
3.961
-6.2
-5.4
-5.4
-5.4
-5.3
-5.2
-5.2
-5.1
-6.4
-5.4
-5.4
-5.2
-5.1
-5.1
-5.0
-4.9
2.707
5.852
23.257
21.777
23.218
23.357
12.323
24.770
23.109
25.255
25.933
15.645
10
-5.0
-4.8

Table 8. Antibacterial activity of the compounds (2-5) represented as Minimum Inhibitory
Concentration [MIC (µg/mL)]
Compounds
MIC (µg/mL)
Staphylococcus Escherichia Pseudomonas Staphylococcus
aureus
12.5
6.25
3.125
6.25
coli
3.125
12.5
6.25
12.5
6.25
aeruginosa
aureus
12.5
6.25
3.125
6.25
2
3
4
12.5
25
6.25
12.5
5
Chlor
12.5
6.25
12.5