1 H -Benzimidazole-5-carboxamidine derivatives: Design, synthesis, molecular docking, DFT and antimicrobial studies

Article abstract of DOI:10.1039/d0nj01899f

In this study, 15 new N-(cyclohexyl)-2-substituted-1H-benzimidazole-5-carboxamidine derivatives that could be new antimicrobial agents were synthesized and their antimicrobial activities were determined using the microdilution method. Some of the derivatives showed significant efficacy against MRSA and VREF with an MIC value of 8 μg mL-1 compared to reference drugs. Molecular docking studies of the compounds against PBP4 and active and allosteric regions of PBP2a were performed and estimated ADME profiles were calculated. The nitrogens of the amidine group of M7, one of the most effective antimicrobial compounds compared to reference drugs, formed two separate hydrogen bonds with ASP275 (1.77 ?) and ASP295 (1.83 ?) in the allosteric region of PBP2a. Geometric optimization parameters, MEP analysis, and HUMO and LUMO quantum parameters of M7 were calculated using DFT/B3LYP theory and the 6-311G(d,p) basis set and the results are displayed.

Full text of DOI:10.1039/d0nj01899f

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1H-Benzimidazole-5-carboxamidine derivatives:
design, synthesis, molecular docking, DFT and
antimicrobial studies†
Cite this: New J. Chem., 2020,
44, 21309
Meryem Erol, *a Ismail Celik,ab Ozlem Temiz-Arpaci,b Hakan Goker,b
Fatma Kaynak-Onurdagc and Suzan Oktenc
In this study, 15 new N-(cyclohexyl)-2-substituted-1H-benzimidazole-5-carboxamidine derivatives that
could be new antimicrobial agents were synthesized and their antimicrobial activities were determined
using the microdilution method. Some of the derivatives showed significant efficacy against MRSA and
VREF with an MIC value of 8 mg mLÀ1 compared to reference drugs. Molecular docking studies of the
compounds against PBP4 and active and allosteric regions of PBP2a were performed and estimated
ADME profiles were calculated. The nitrogens of the amidine group of M7, one of the most effective
antimicrobial compounds compared to reference drugs, formed two separate hydrogen bonds with
ASP275 (1.77 Å) and ASP295 (1.83 Å) in the allosteric region of PBP2a. Geometric optimization
parameters, MEP analysis, and HUMO and LUMO quantum parameters of M7 were calculated using
DFT/B3LYP theory and the 6-311G(d,p) basis set and the results are displayed.
Received 16th April 2020,
Accepted 18th November 2020
DOI: 10.1039/d0nj01899f
rsc.li/njc
and neurological diseases.4 Therefore, the discovery of new and
more effective antimicrobial drugs is very important and many
1. Introduction
Infectious diseases caused by bacteria and fungi are one of studies have been performed to design new agents.
the most important threats to public health. In recent years, The final stages of cell wall biosynthesis in bacteria are
there have been serious problems with resistance development carried out by penicillin-binding proteins (PBPs), which
and toxicity in the treatment of hospital infections caused by form cross-links in peptidoglycan chains. Since inhibition of
resistant Gram-positive bacteria and opportunistic fungal infec- these enzymes reduces the structural integrity of the cell wall,
tions caused by suppression of the immune system for various beta-lactam is the target of antibiotics. In methicillin-sensitive
reasons.1 In particular, methicillin-resistant Staphylococcus aur- S. aureus (MSSA), there are five PBPs, while a different PBP,
eus (MRSA) and vancomycin-resistant Enterococcus faecalis called PBP2 or PBP2a, is synthesized in MRSAs.5 Unlike
(VREF) infections have increased significantly due to the ran- other PBPs, PBP2/2a shows a low affinity for antibiotics in the
dom use of antibacterial drugs.2 The mortality rate in infectious b-lactam structure. Therefore, in the presence of b-lactam
diseases is the second most common cause of death due to group antibiotics, it is the only transpeptidase capable of
cardiovascular diseases and antibiotics are the most widely sustaining peptidoglycan synthesis by performing the function
used drugs in the world.3 Every year, 700 000 people die world- of high-affinity PBPs.6,7 PBPs encode genes called mecA and
wide due to bacterial infectious diseases that are resistant to mecC, and these genes are located on the SCCmec cassettes in
antibiotics only. In the prevention or treatment of such infec- the bacterial chromosome. The mechanism of resistance is the
tious diseases, it is estimated that if new remedies cannot be synthesis of a new penicillin-binding protein (PBP2a) encoded
developed, the number of people dying each year will reach by the mec-A gene.8–10 In E. faecalis, decreased sensitivity to
10 million people worldwide by 2050, and this figure will be b-lactam antibiotics is due to the expression of PBP4.11,12
much higher than the deaths caused by cancer, cardiovascular
The benzimidazole ring system has an important role to play
in many pharmacological activities such as antimicrobial,13–17
antiviral,18 anthelmintic,19 antihistamine,20 antiulcer,21
antihypertensive,22 anticancer,23 antioxidant,24 spasmolytic,25
anticonvulsant,26 anti-inflammatory,27 antiasthmatic,28 and
analgesic.29 Therefore, studies on these derivatives have been
increased in recent years. Research to date reveals that the
a Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Kayseri, Turkey. E-mail: eczacimeryem@gmail.com
b Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Ankara, Turkey
c Trakya University, Faculty of Pharmacy, Department of Pharmaceutical
Microbiology, Edirne, Turkey
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nj01899f benzimidazole ring system is mostly substituted from the 1st,
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in the presence of 6 mL of DMF. The reaction medium was
cooled, water was added and precipitated, the product was
crystallized from ethylacetate-n-hexane (50 : 50) and compound 1
was obtained.39,43,44 Absolute EtOH was treated with dry HCl
gas for about 40–45 minutes (Pinner reaction). Acid-ethanol
(6–7 mL) was added to compound 1 (10 mmol) until the
product dissolved and was left to stir at room temperature for
5–6 days. At the end of the period, it was diluted with dry
ether, filtered under vacuum, and dried at room temperature
(compound 2). The imidate esters (5 mmol) were left to stir for
2–3 days at room temperature with ammonia gas (6–7 mL)
passed through EtOH. At the end of the period, some of the
EtOH was evaporated, diluted with ether, allowed to be vacuum
filtered and dried (compound 3).45 Amidine (0.75–0.80 g) was
subjected to a reduction in ethanol with 40 mg of a 10% Pd–C
catalyst. The resulting product was obtained by filtering
through a Celite bed (compound 4).43,46,47 The relevant alde-
hyde derivatives (15 mmol) that provided the cyclization of the
benzimidazole ring were dissolved in 50 mL of EtOH. A solution
of 1.6 g Na2S2O5 in 10 mL of water was added slowly over the
aldehyde solution. The reaction mixture was shaken vigorously
and more EtOH was added. The mixture was kept in the
refrigerator for a few hours, and the precipitate was filtered
off and dried.48 The targeted result products were obtained by
reacting aldehyde salts and compound 4 in DMF for 6 hours at
100 1C.49,50 At the end of the period, the reaction medium was
precipitated with Na2CO3 solution and filtered. A portion of the
resulting crude product was purified by column chromatogra-
phy using a chloromethane : methanol : ammonia (10 : 1 : 0.1)
solvation mixture, while the other was crystallized from metha-
nol. In the last step, the HCl salts of the compounds were
obtained from HCl gas-passed EtOH.72 The intermediate pro-
ducts (compound 1, 2, 3, and 4) are not original.51 A list of the
synthesized compounds (Table S1, ESI†) and their physical and
spectral data are reported in the ESI.†
Fig. 1 Designed 1H-benzimidazole-5-carboxamidine derivatives.
2nd, and 5th positions.30,31 Groups in these positions are
generally thought to play a role in their impact and severity.
Since this ring system is a structural bioisostere of adenine and
guanine structures carrying purine nuclei, it is thought that
they can demonstrate their microbiological activities in this
way.32 In vivo and in vitro activity studies have shown that
aromatic amidine and diamidine group compounds have
strong activity against a large number of bacteria, amoeba,
protozoa, and viruses. The mechanisms of action of this group
of compounds are still not fully elucidated, possibly due to their
action by more than one mechanism of action. The intensely
emphasized mechanism is reported to be that aromatic
amidine and diamidine derivatives act by binding to the
minor cavity of adenine and thymine-rich DNA and inhibiting
one or more DNA-dependent enzymes or inhibiting direct
transcription.33–38 In new drug development studies, it is
known that new compounds with a stronger effect can be
found with the combination of different groups of pharmaco-
phores in the same molecule. In some studies, microbiological
activities of compounds carrying a benzimidazole ring and
amidine group against existing bacteria and their isolates were
investigated and some derivatives were found to be more
effective than standard drugs.4,39–42
2.2. Antimicrobial activity
In light of this information, it was aimed in this study to
synthesize some new mono cationic benzimidazole carboxami-
dine derivatives (Fig. 1), to clarify the structures of the com-
pounds using 1H-NMR, 13C-NMR, mass spectroscopy, and
elemental analysis methods and to investigate in vitro antimi-
crobial activities compared to clinical reference drugs. Estima-
tion of ADME profiles of all compounds, molecular docking
2.2.1. Microorganisms and standard drugs. Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococ-
cus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and
Candida albicans ATCC 10231 standard strains and clinical
isolates provided from Trakya University Health Center for
Medical Research and Practice (Hospital) were used in the
study. Ampicillin (Sigma), vancomycin (Sigma), ciprofloxacin
(Sigma), meropenem (Sigma), gentamycin (Sigma), and ampho-
tericin B (Sigma) were used as standard antimicrobial agents.
2.2.2. Microdilution method. Stock solutions of the test
compounds were prepared in DMSO (Merck). Mueller Hinton
Agar (MHA) (Merck), Mueller Hinton Broth (MHB) (Merck),
Sabouraud Dextrose Agar (SDA) (Merck), Sabouraud Liquid
Medium (SLM) (Merck), and RPMI-1640 medium (Sigma) with
L-glutamine buffered with 3-[N-morpholino]-propansulfonic
acid (MOPS) (Sigma) (pH: 7) were used in the study. Suscepti-
bility testing was performed according to the guidelines of the
¨
studies with Schrodinger, molecular mechanics generalized
born surface area (MM-GBSA) calculations, and molecular
reactivity analyses (HOMO–LUMO) were performed. Also, mole-
cular electrostatic potential (MEP) analysis and geometric opti-
mization of M7, which has one of the highest antimicrobial
activities in vitro, were performed.
2. Experimental
2.1. General procedures for the preparation of compounds
(M1–M15)
4-Chloro-3-nitrobenzonitrile (2 g, 10.95 mmol) and cyclohexy- Clinical and Laboratory Standards Institute (CLSI) M100-S2552
lamine (2.3 mL, 20 mmol) were reacted at 100 1C for 4–5 hours and M27-A3.53 100 mL of MHB and RPMI-1640 medium with
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L-glutamine (Sigma) buffered with MOPS (pH: 7) were added to is calculated with the formula. Calculations were made using the
58,59
¨
each well of the microplates for bacteria and fungi, respectively. Schrodinger Prime module.
The bacterial suspensions used for inoculation were prepared
at 105 CFU mLÀ1 by diluting fresh cultures at McFarland
2.5. DFT/B3LYP calculations
0.5 density. Suspensions of the yeast at McFarland density were
Quantum chemical calculations of the compounds were carried
diluted 1 : 100 and 1 : 20 respectively and 2.5 Â 103 CFU mLÀ1
were inoculated to the twofold-diluted solution of the com-
pounds. Stock solutions of the tested compounds and standard
drugs were diluted two-fold in the wells of the microplates so
the solution of the synthesized compounds was prepared at
512, 256, 128, 64, 32, 16, 8, and 4 mg mLÀ1, and standard drugs
were prepared at 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 mg mLÀ1
concentrations. All solvents and diluents, pure microorgan-
isms, and pure media were used in control wells. A 10 mL
microorganism inoculum was added to each well of the micro-
plates. Microplates including bacteria were incubated at 37 1C
for 16–20 h and microplates including fungi were incubated
at 35 1C for 24–48 h. After incubation, the lowest concentration
of the compounds that completely inhibits macroscopic growth
was determined and reported as the minimum inhibitory
concentration (MIC). All organisms were tested in triplicate in
each run of the experiments. Solvents, pure microorganisms,
and pure media were used as control wells.
out using the Gaussian 09 package program60 and DFT/B3LYP
theory and the 6-311G(d,p) basis set.61–68 HOMO–LUMO energies
of all compounds and other electronic parameters calculated from
these energies (ionization potential, electron affinity, electronega-
tivity, chemical hardness, chemical softness, chemical potential,
and electrophilic index, etc.) were calculated. Also, the molecular
electrostatic potential (MEP) and optimized geometrical structure
results of M7, which is one of the compounds showing the best
antimicrobial activity, were theoretically obtained. First, the M7
was optimized to find its minimum energy and its most stable
structure. The results are shown using the GaussView 6 program.69
2.6. Theoretical ADME predictions
One of the biggest problems of drug design is that compounds
with strong biological effects also do not have good pharmaco-
kinetic effects.70 Therefore, for the evaluation of the absorp-
tion, distribution, metabolism, and elimination profiles of the
synthesized compounds, physicochemical parameters such as
log P, PSA, nrotb, % human oral absorption, molecular weight,
and number of hydrogen bond donor–acceptor were calculated
using Schrodinger Maestro’s QikProp program.71
2.3. Molecular docking studies
The molecular docking study was carried out using the Maestro
11.5 program (Schrodinger Inc. USA)54 in 5 stages: protein
preparation, ligand preparation, grid box creation, displaying
docking, and docking results. The crystal structure of S. aureus
PBP2a in complex with Quinazolinone (PDB ID: 4CJN at 1.95 Å
resolution),55,56 and crystal structure of E. faecalis PBP4 in the
3. Results and discussion
3.1. Chemistry
ceftaroline-bound form (PDB ID: 6MKI at 2.98 Å resolution)11 M1–M15 was synthesized using the methods given in the
were imported from the protein data bank (https://www.rcsb. Experimental section and shown in Scheme 1. After the purity
org/). Ligands were minimized using ‘LigPrep’ and prepared controls of the synthesized compounds (TLC studies) and
with the OPLS3 force field with possible ionizations at pH = 7 Æ their melting points were determined, spectral analyses were
2. ‘Protein Preparation Wizard’ was used for the protein struc- performed to prove the structures. 1H-NMR, 13C-NMR, Mass
tures: preprocess, the removal of water and other molecules spectral analysis, and elemental analysis were found to prove
was minimized after optimizing using PROKA pH: 7.0. ‘Recep- the expected structures. First, the reaction product of 4-chloro-
tor Grid Generation’ was used for the binding site: for the 4CJN 3-nitrobenzonitrile and cyclohexylamine in the presence of
allosteric site x: 45.05, y: 44.42 and z: 7.36 coordinates, for the DMF was used to prepare 1. Subsequently, compound 2 was
4CJN active site x: À35.89, y: À12.57 and z: À23.65 coordinates formed by the Pinner reaction method. The resulting unstable
and for the 6MKI active site x: 35.24, y: 1.34 and z: 10.06 imidate esters were rapidly converted to compound 3 with
coordinates were created in 20 Â 20 Â 20 Å. Ligands and NH3/EtOH, followed by reduction of the nitro group on the
proteins prepared for the binding site determined using Pd/C catalyst with hydrogen gas to obtain compound 4, and
‘Ligand Docking’ were docked using Extra Precision (XP). then M1–15 was obtained in the presence of aldehyde salts
MM-GBSA values were calculated according to the obtained and DMF. HCl salts of the compounds were obtained from HCl
docking poses.57
gas-passed EtOH.
In the resulting compounds, aromatic protons were between
6.85–8.35 ppm and cyclohexyl protons were between 1.14–4.42
2.4. MM-GBSA free energy calculations
MM-GBSA is a popular approach used to estimate protein–ligand ppm (protons are not separated). Amidine group protons in
free energies. It allows us to examine whether key interactions are some spectra as a wide 4H singlet between 9.08 and 9.36 ppm,
established and stabilized during molecular dynamics simulation. and in some spectra as 2H and 2H singlet peaks, were observed,
The stability of the binding is determined by subtracting the but in some spectra could not be observed due to paramagnetic
hollow protein and single ligand energies from the total energy of shift. In mass spectral analysis of the compounds, the ion formed
the protein–ligand complex. DGbind = Ecomplex (minimized) À as a result of the breaking of the M + H ion or cyclohexyl group
[Eligand (unbound, minimized) + Ereceptor (unbound, minimized)] was monitored at 100% relative densities. Elemental analyses of
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the compounds showed an average activity against E. coli ATCC
25922 and multi-drug resistant E. coli (MIC: 16–256 mg mLÀ1),
while M8 exhibited the best activity against both strains
(MIC: 16 ug mLÀ1). Even if M8 did not show more activity than
reference drugs, it may be a lead compound to develop new
molecules against E.coli. The compounds generally had a
weak antimicrobial effect against Gram-negative P. aeruginosa
ATCC 27853 and P. aeruginosa isolate (MIC: 64–128 mg mLÀ1).
While half of the compounds against S. aureus ATCC 29123
showed good activity with MIC: 8 mg mLÀ1, they showed weaker
efficacy compared to reference drugs. The results of the M4,
M7, M8, and M12 derivatives against MRSA (MIC: 8 mg mLÀ1
)
are quite satisfactory compared to ampicillin and mero-
penem. The compounds showed a broad range of activity
against the Gram-positive bacterium E. faecalis ATCC 29212
(MIC: 8–512 mg mLÀ1) compared to the reference drugs. M7,
M8, and M12 derivatives against VREF showed better efficacy
than vancomycin and ampicillin with MIC: 8 mg mLÀ1. The
antifungal activities of the compounds against C. albicans were
in the range of 8 to 4512 mg mLÀ1 and much less effective than
amphotericin B.
When the microbiology findings were evaluated in general,
it was found that the tested derivatives showed more important
antimicrobial activity against Gram-positives than Gram-
negatives, and the best antibacterial activity was observed
against MRSA and VREF. Benzimidazole does not have a
positive effect on the activity of the presence of groups
(such as methyl, ethyl) that give electrons to the ring on
the phenyl ring at position 2; instead, it has been found to
increase antimicrobial activity by substituting it with electron
attracting groups such as chlorine, bromine, carboxy, and
benzyloxy (Table 1).
Scheme 1 Synthesis pathways of the target compounds.
the compounds were compared with theoretical calculations
to determine the purity of the compounds and the number
of other components. The results of the elemental analysis
and all spectral data were fully compatible with the molecules
presented.
3.2. In vitro antimicrobial activities
All synthesized compounds M1–M15 were tested for in vitro
antibacterial and antifungal activity against standard strains
and drug-resistant isolates. It was carried out according to
the broth microdilution technique described by CLSI. The
MIC of each compound was determined and compared with
ampicillin, gentamycin, vancomycin, meropenem, ciprofloxa-
cin, and amphotericin B. According to microbiological results,
Table 1 In vitro antimicrobial MIC values (mg mLÀ1) of the synthesized compounds (M1–M15) and reference drugs
Compound
A
B
C
D
E
F
G
H
I
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
Vancomycin
Ampicillin
Meropenem
Ciprofloxacin
Gentamicin
AmphotericinB
128
128
32
128
128
64
128
16
128
128
128
32
128
128
32
—
2
128
128
32
128
128
64
128
16
256
128
128
32
128
128
32
—
2
128
128
128
64
64
64
64
64
64
64
64
128
64
128
128
—
—
0.5
1
0.5
—
128
128
128
64
512
64
64
64
64
64
64
128
64
128
128
—
—
0.5
2
48
—
16
8
8
8
512
32
8
8
128
256
16
8
8
128
32
32
16
16
8
512
64
8
64
32
32
16
512
32
8
64
32
32
16
512
64
8
64
32
128
16
512
128
8
4512
128
128
64
64
16
128
128
—
—
—
—
—
0.5
8
8
8
256
256
32
8
16
128
64
o0.0625
48
48
0.5
2
128
128
64
8
64
128
64
o0.0625
2
—
2
—
—
128
128
64
8
64
128
64
48
48
—
o0.0625
2
o0.0625
o0.0625
0.25
—
o0.0625
o0.0625
0.25
—
o0.0625
0.5
1
2
—
—
—
—
A: E.coli ATCC 25922, B: E. coli isolate, C: P. aeruginosa ATCC 27853, D: P. aeruginosa isolate, E: S. aureus ATCC 29213, F: S. aureus isolate (MRSA), G:
E. faecalis ATCC 29212, H: E. faecalis isolate (VREF), I: C. albicans ATCC 10231. E. coli isolate is susceptible to the tested antimicrobial agents. S.
aureus isolate is a methicillin resistance (MRSA) isolate. Antibacterial drugs are not tested against fungi, while antifungal drugs are not tested
against bacteria. P. aeruginosa is naturally resistant to ampicillin. Gram (À) bacteria used in this study are resistant to vancomycin.
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3.3. Molecular docking
While the synthesized derivatives have a lower effect compared
to reference drugs against S. aureus and E. faecalis, we were
focused on the resistance proteins of these strains as they
exhibit higher activity against MRSA and VREF. As mentioned
in the introduction, it was concluded that the proteins act on
PBP2a (PDB: 4CJN) and PBP4 (PDB: 6MKI). Docking studies
suggest that the compounds may have been linked by binding
to the 4CJN allosteric site. The Glide emodel scores of the
docking studies performed in the 4CJN allosteric site generally
were calculated to be lower than the active site. One of the most
effective antimicrobial compounds, M7, formed 1.77 Å and
1.83 Å length hydrogen bonds between the two separate nitro-
gens of the amidine group and the ASP275 and ASP295 in the
4CJN allosteric site. M7 showed hydrophobic interactions with
GLY296, TYR297, LYS148, ASN146, GLU145, and ILE144. The
2D and 3D interaction diagram of M7 in the allosteric site of
PBP2a (PDB: 4CJN) is given in Fig. 2.
In the 6MKI binding site, M7 formed a single nitrogen of
the amidine group and two separate hydrogen bonds
between GLU624 and GLU635 with a length of 1.84 Å. It showed
hydrophobic interactions with THR622, GLY621, THR620,
VAL467, SER482, ASN484, THR665, and SER663. The reference
drug ampicillin formed hydrogen bonding with ASP666,
SER482, THR620 and THR622, and hydrophobic interactions
with SER663, THR665, GLY621, SER637, TYR605, VAL666 and
Fig. 3 2D and 3D interaction between M7 with PBP4 (6MKI) at the active
site.
TYR607 (see also ESI†). Fig. 3 shows 2D and 3D interactions of
the M7’s 6MKI active site.
The amidine group is required for hydrogen bond for-
mation. Hydrogen bonding with GLU624 in the 6MKI active
site is important, and the amidine group in the compound
framework formed hydrogen bonding with GLU624. Glide
emodel, MM-GBSA (dG Bind), and XP Gscore scores of all
compounds are given in Table 2. Accordingly, while there was
a good correlation between Glide emodel scores and antimi-
crobial effect, there was a weak relationship between MM-GBSA
(dG Bind) and XP Gscore scores.
3.4. Molecular reactivity analyses
Boundary orbitals in the molecule are named as HOMO and
LUMO. HOMO is the ‘Highest Occupied Molecular Orbital’ in a
molecule. LUMO, on the other hand, can be defined as the
‘Lowest Unoccupied Molecular Orbital’ in a molecule. These
orbitals describe intermolecular interactions. The energy dif-
ference between the HOMO and LUMO orbitals is a measure of
the chemical stability of the molecules and plays a major role in
determining the chemical and spectroscopic properties of the
molecules.73 Also, molecular properties such as ionization
potential (IP), electron affinity (EA), electronegativity (X),
chemical hardness (Z), chemical softness (S), chemical
potential (m), and electrophilic index (o) could be determined
using this energy range.74–76 The HOMO–LUMO energies are
given in Table 3 and other electronic properties are given
Table S2 (ESI†). Fig. 4 shows the regions where M7’s HOMO
Fig. 2 2D and 3D interaction between M7 with PBP2a (4CJN) at the
allosteric site.
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Table 2 Calculated Glide emodel, MM-GBSA (dG Bind), and XP Gscore values of the compounds and reference drugs
4CJN
6MKI
Allosteric site
Active site
Active site
Compound
Glide emodel MM-GBSA XP Gscore Glide emodel MM-GBSA XP Gscore Glide emodel MM-GBSA XP Gscore
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
À33.96
À29.91
À30.49
À35.28
À35.54
À32.09
À46.28
À37.34
À41.41
À39.69
À39.69
À43.39
À43.39
À30.26
À37.98
À75.60
À36.56
À31.87
À40.45
À47.25
À45.20
À33.80
À39.53
À38.83
À33.05
À41.28
À36.62
À29.65
À40.17
À40.17
À39.30
À39.30
À42.51
À38.63
À57.85
À27.08
À40.88
À44.19
À37.05
À3.78
À4.20
À2.26
À1.21
À4.40
À2.77
À3.22
À2.71
À3.00
À4.22
À4.22
À3.58
À3.58
À3.02
À3.90
À6.23
À3.58
À3.41
À4.55
À2.67
À37.20
À29.63
À23.96
À29.14
À28.13
À23.37
À32.89
À3.54
À28.53
À40.29
À29.50
À31.68
À33.19
À36.64
À41.49
À19.45
À19.27
À27.49
À38.15
À50.30
À47.39
À38.98
À31.40
À30.43
À32.61
À17.92
À32.54
À54.78
À4.02
À4.42
À5.63
0.19
À46.30
À49.52
À49.46
À52.06
À50.57
À44.039
À60.38
À55.45
À46.49
À55.83
À44.96
À55.40
À46.75
À46.76
À50.91
À70.73
À43.25
À77.75
À48.57
À46.66
À63.20
À65.37
À60.55
À61.33
À63.40
À51.10
À61.70
À56.67
À38.83
À41.43
À58.49
À62.24
À65.17
À59.89
À59.36
À95.36
À25.30
À68.84
À46.11
À52.33
À7.56
À7.83
À7.60
À7.33
À7.46
À4.39
À6.53
À4.28
À7.21
À8.44
À4.99
À4.97
À7.59
À7.38
À7.81
À6.77
À4.93
À2.08
À5.18
À5.21
0.02
À3.94
À3.87
À2.29
À4.78
À4.71
À3.06
À4.52
À4.20
À2.42
À4.63
À5.48
À3.71
À2.18
À6.47
À5.99
À29.67
À37.21
À36.85
À50.60
À25.21
À39.57
À32.86
À70.03
À32.24
28.64
Vancomycin
Ampicillin
Gentamycin
Meropenem
À37.23
Ciprofloxacin À32.61
À47.96
information about the formation of intramolecular hydrogen
bonds. Potential increases are listed as red o orange o yellow
o green o blue.77 While the red color is related to the regions
where the electrons are dense, the blue color represents the
electropositive points. Fig. 5 shows the MEP map of M7. When
the MEP map of M7 is examined, the electrophilic regions are
around the N atom and the nucleophilic regions are around the
C and H atoms. The hydrogen bonding of the amidine group of
M7 with ASP275, ASP295, GLU624, and GLU635 in the PBP4
and PBP2a binding sites shows that the molecular docking
results are consistent with MEP analysis.
Table 3 Calculated HOMO–LUMO energy values of M1–M15
Compound
HOMO
LUMO
DE
M1
M2
M3
M4
M5
M6
M7
M8
À0.20910
À0.20827
À0.20508
À0.21955
À0.21066
À0.18287
À0.21133
À0.20563
À0.21330
À0.21256
À0.20803
À0.19774
À0.20220
À0.20696
À0.21853
À0.04849
À0.04766
À0.04260
À0.05577
À0.05722
À0.02934
À0.03620
À0.06337
À0.07899
À0.06870
À0.04758
À0.04087
À0.04392
À0.05216
À0.05794
0.16061
0.16061
0.16248
0.16378
0.15344
0.15353
0.17513
0.14226
0.13431
0.14386
0.16045
0.15687
0.15828
0.1548
M9
M10
M11
M12
M13
M14
M15
3.6. Geometry optimization
The geometry of the molecule is of great importance in
the theoretical calculations of molecular energy and other
properties. Even small changes in molecular geometry affect
the energy of the molecule. Geometry optimization is the
optimization step in which the geometry of the molecular
system has the lowest energy corresponding to the base state
by changing the geometric parameters (bond length, bond
angles, and dihedral angle) of the structure. When the mole-
cules are most stable, they also correspond to atomic sequences
where their energy is minimal. The experimental geometric
structure of the compounds is unknown.78 In some recent
0.16059
and LUMO orbitals are localized. A moderate relationship
can be established between the HOMO–LUMO values of the
compounds and their antimicrobial effects against MRSA and
VREF. One of the most active compounds against both MRSA
and VREF, M7, has the highest quantum parameters with
HOMO = À0.21133, LUMO = À0.03620 and DE = 0.17513.
3.5. MEP analysis
MEP is a method that allows us to understand the molecular studies, experimental and computational studies of the molecular
polarity while correlating the electronegativity, charge, dipole geometry of compounds have been reported as comparisons.79,80
moment, and chemical reaction rate of a compound and also The numbered optimized geometrical structure of M7 is given
provides information about the net electrostatic effect created in Fig. 6.
by the total charge. An MEP energy map was used to identify
regions where the electron density of M7 is located and thus to
3.7. Theoretical ADME prediction
predict the reactive regions of electrophilic and nucleophilic Oral bioavailability studies of the compounds (absorption,
attacks. The importance of MEP is also that molecules show distribution, metabolism, and elimination) are essential to
both size and shape as well as positive, negative, and neutral eliminate compounds with unacceptable pharmacokinetic
electrostatic potential regions. It also provides important properties and for successful drug discovery studies. In silico
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Jorgensen’s three rules, and there should be no more than one
violation of drug-like compounds. Software data such as mole-
cular weight, log P, polar surface area, hydrogen donor number,
hydrogen acceptor number, rotatable bond number, and
volume are presented in Table S3 (ESI†) with violations of the
third and fifth rules. From the table, it can be seen that all
the compounds comply with these rules. This increases the
probability that the compounds are potential drug molecules.
4. Conclusions
In this study, a series of N-(cyclohexyl)-2-substituted-1H-
benzimidazole-5-carboxamidine derivatives, which we hope
may be a new antimicrobial agent, were designed and synthe-
sized and their antimicrobial activity was determined. M7, M8,
and M12 derivatives have been observed more effectively com-
pared to reference drugs against both MRSA and VREF, with an
MIC value of 8 mg mLÀ1. Derivatives show more antibacterial
activity against Gram-positive than Gram-negative; in particu-
lar, binding of groups attracting electrons to the phenyl ring
has been observed to significantly increase the antimicrobial
activity. For one of the most effective antimicrobial com-
pounds, M7, 2D/3D interactions were displayed in the 4CJN
active and allosteric site and 6MKI active site, and DFT calcula-
tions were performed to estimate its geometric structure and
electronic properties. The nitrogens of the amidine group made
two separate hydrogen bonds in both sites, and when the scores
of all compounds were examined, a good relationship was
found between Glide emodel scores and antimicrobial effect.
According to all these results, the synthesized compounds
showed promising antimicrobial activity.
Fig. 4 (a) HOMO and (b) LUMO plots of M7.
Conflicts of interest
There are no conflicts to declare.
Fig. 5 MEP map of M7.
Acknowledgements
ADME and toxicological screening systems can provide an
opportunity to predict performance in vivo. Estimates of ADME
properties of all synthesized compounds (M1–M15) were
performed with QikProp. This software evaluates drug candi-
dates’ ADME profiles according to Lipinski’s five rules and
NMR, mass spectra, and elemental analysis of the compounds
were performed by Ankara University Faculty of Pharmacy Central
Laboratory and Erciyes University Technology and Research Center
(TAUM). This work was supported by The Scientific and Technolo-
gical Research Council of Turkey (TUBITAK) Grant [315S333].
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