Synthesis, Molecular Docking, Molecular Dynamics, DFT and Antimicrobial Activity Studies of 5-substituted-2-(p-methylphenyl)benzoxazole Derivatives

Article abstract of DOI:10.1016/j.molstruc.2021.130151

In this study, new 2-(p-methylphenyl)-5-(2-substitutedacetamido)benzoxazole derivatives were synthesized, and antimicrobial activities on six bacteria and their twelve drug-resistant isolates and one fungus and its two drug-resistant isolates were investigated by microdilution method. B1 against Staphylococcus aureus isolate, and B1 and B2 against Escherichia coli isolate showed more potent antimicrobial activity with MIC value of 16 μg/mL than some of the reference drugs. The compounds' interactions on the DNA gyrase enzyme were evaluated by molecular docking and molecular dynamics simulations. Docked compounds have demonstrated superimposition in the DNA gyrase ATP binding site with similar protein-ligand interactions. With 50 ns duration molecular dynamics simulations, the average RMSD value of the DNA gyrase subunit B protein and B1, B2, B3, and B4 complexes were measured at about 0.15 nm. The ligands-bound DNA gyrase subunit B protein is a little less RMSF value and more stable than the apoprotein form between 45-49 residues in the active site amino acids region. Geometric optimization parameters, HOMO-LUMO orbital energies, and other electronic parameters derived from these energies, MEP, and NBO analysis were performed the DFT/B3LYP theory and 6-311G (d,p) basis set. The ΔE: LUMO-HOMO of the two most active compounds B1 and B2 are 4.2928 and 4.3219, respectively. The compounds' predicted ADME profiles were in line with Lipinski and other limiting rules.

Full text of DOI:10.1016/j.molstruc.2021.130151

Journal of Molecular Structure 1234 (2021) 130151
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, Molecular Docking, Molecular Dynamics, DFT and
Antimicrobial Activity Studies of
5-substituted-2-(p-methylphenyl)benzoxazole Derivatives
Meryem Erola,∗, Ismail Celika, Gulcan Kuyucuklub
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey
b Department of Medical Microbiology, Faculty of Medicine, Trakya University, Edirne, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, new 2-(p-methylphenyl)-5-(2-substitutedacetamido)benzoxazole derivatives were synthe-
sized, and antimicrobial activities on six bacteria and their twelve drug-resistant isolates and one fungus
and its two drug-resistant isolates were investigated by microdilution method. B1 against Staphylococcus
aureus isolate, and B1 and B2 against Escherichia coli isolate showed more potent antimicrobial activ-
ity with MIC value of 16 μg/mL than some of the reference drugs. The compounds’ interactions on the
DNA gyrase enzyme were evaluated by molecular docking and molecular dynamics simulations. Docked
compounds have demonstrated superimposition in the DNA gyrase ATP binding site with similar protein-
ligand interactions. With 50 ns duration molecular dynamics simulations, the average RMSD value of
the DNA gyrase subunit B protein and B1, B2, B3, and B4 complexes were measured at about 0.15 nm.
The ligands-bound DNA gyrase subunit B protein is a little less RMSF value and more stable than the
apoprotein form between 45-49 residues in the active site amino acids region. Geometric optimization
parameters, HOMO-LUMO orbital energies, and other electronic parameters derived from these energies,
MEP, and NBO analysis were performed the DFT/B3LYP theory and 6-311G (d,p) basis set. The ꢀE: LUMO-
HOMO of the two most active compounds B1 and B2 are 4.2928 and 4.3219, respectively. The compounds’
predicted ADME profiles were in line with Lipinski and other limiting rules.
Received 11 January 2021
Revised 7 February 2021
Accepted 9 February 2021
Available online 23 February 2021
Keywords:
Benzoxazole
antimicrobial activity
molecular docking
molecular dynamics
DFT
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
developed broad-spectrum antibiotics, the tendency of pharmaceu-
tical companies to develop drugs used in chronic diseases, and
Infectious diseases represent an increasing global public health
problem. Community-borne infections such as respiratory tract in-
fections, diarrhea, sepsis caused by resistant bacteria, catheter-
related infections, hospital infections observed in intensive care
units, skin, soft tissue infections, and pneumonia affect a severe
part of the human population cause high morbidity and mortal-
ity. Microorganisms develop many types of resistance as a result of
the increasing frequency of antibiotic use and the increase in the
number of antibiotics used [1,2]. Due to the increasing prevalence
of highly resistant Pseudomonas aeruginosa, methicillin-resistant
S. aureus (MRSA), vancomycin-resistant Enterococcus faecalis and
carbapenem-resistant Klebsiella pneumoniae, especially in hospital-
acquired infections, antibiotic resistance is a very serious threat to
public health today. Reasons such as rapid resistance against newly
high costs in the production process cause a significant decline in
antibiotic groups’ discovery and development [3-5].
Benzoxazole ring is an important heterocyclic ring system in
drug design due to its structural similarity to adenine and guanine
bases. It is thought that its antimicrobial activity can be achieved
by DNA gyrase enzyme inhibition, such as nucleic acid synthesis
inhibition or fluoroquinolone group antibiotics [6]. This approach
further increases the interest in benzoxazole derivatives. Benzox-
azole scaffold can participate in several energy-preferred interac-
tions with the receptors. Oxygen and nitrogen atoms in its struc-
ture can act as hydrogen bond acceptors. Due to its lipophilic
character, hydrophobic interactions with the receptor protein are
possible [7]. It is also open to chemical modifications, so many
derivatives can be created [8]. Among the benzoxazole deriva-
tive compounds, those substituted in the C2 and C5 positions
have been extensively studied so far and are among the strik-
ing structures in studies to develop effective antimicrobial com-
pounds [9,10]. In addition, 2,5-disubstituted benzoxazole deriva-
∗
Corresponding author at: Meryem Erol, Erciyes University, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Kayseri, Turkey.
E-mail address: eczacimeryem@gmail.com (M. Erol).
https://doi.org/10.1016/j.molstruc.2021.130151
0022-2860/© 2021 Elsevier B.V. All rights reserved.

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Fig. 1. Some marketed drugs containing benzoxazole ring
tives have been reported to have broad-spectrum pharmacological
effects such as anti-inflammatory [11], antiulcer [12], antihelmintic
[13], antiparkinsonian [14], analgesic [15], antiepileptic [16], anti-
malarial [17], and anticancer [18] and antialzheimer [19,20]. Ben-
zoxazole acts as the main active ingredient in some marketed
medicines, including calcimycin (antibiotic), prinaberel (estrogen
receptor agonist), pardoprunox (antiparkinsonian), flunoxaprofen,
benoxaprofen (NSAII), boxazomycin B (antibacterial) (Fig. 1).
2.1.1. General procedure for the synthesis of B1-B4
Firstly, 5 mmol of 2,4-diaminophenol dihydrochloride and 5
mmol of p-methyl benzoic acid were reflux at 25g polyphospho-
ric acid (PPA) catalyst at 160°C-190°C for about 3 h. At the end of
the reaction, the reaction content was poured onto ice, and the 10%
NaOH solution was added until the medium was alkaline. The pre-
cipitate was filtered, crystallized from ethanol-water, and dried. In
step 2, 5 mmol 5-amino-2-(p-methylphenyl)benzoxazole was dis-
solved in 100 ml diethyl ether, while 10 mmol NaHCO3 was dis-
solved in 50 ml distilled water. While mixing the ether and the
water phase in the magnetic stirrer in the ice bath, 5 mmol of 2-
chloroacetylchloride was added slowly, and stirring was continued
overnight. At the end of the reaction, the reaction medium was fil-
tered, crystallized from ethanol-water, and dried. Finally, 1 mmol
2-(p-methylphenyl)-5-(2-chloroacetamido)benzoxazole was reacted
with 1 mmol piperazine/piperidine derivatives and morpholine at
room temperature for 24 h in the presence of 1 mL triethylamine
(TEA) and 5 mL dimethylformamide (DMF). At the end of the re-
action, water was added to the medium. The compound precipi-
tated and then crystallized from ethanol-water or purified by col-
umn chromatography (mobile phase: ethyl acetate-n-hexane (1:1).
All compounds are original. Results of 1H-NMR and 13C-NMR spec-
tral data support the structure of the compounds.
Therefore,
in
this
study,
2-(p-methylphenyl)-5-(2-
substitutedacetamido)benzoxazole derivates were synthesized
to connect with the continuation of the above issues of our
previous studies, and their antimicrobial activities were evaluated
by the microdilution method. DNA gyrase creates the negative su-
perhelix required for chromosome replication and is an important
therapeutic target in designing effective antibacterial compounds.
Although this enzyme is not found in mammals, it is found in
bacteria. It is also a well-researched target that plays an essen-
tial role in bacterial DNA replication. For this reason, molecular
docking and molecular dynamics simulation were investigated to
determine benzoxazoles play a role in DNA gyrase inhibition (PDB
ID: 4KTN). Also, HOMO-LUMO energies, molecular electrostatic po-
tential (MEP) analysis, natural bond orbital (NBO) atomic charges,
and geometric parameters of molecules (bond length, bond angle,
dihedral angles) were calculated using the DFT/B3LYP theory and
6-311G (d,p) basis set. The predicted absorption, distribution,
metabolism, and excretion (ADME) profiles of the compounds
were in line with Lipinski and other limiting rules.
2-(p-methylphenyl)-5-(2-(4-methylpiperazine-1-
yl)acetamido)benzoxazole (B1): White powder, yield 62%, m.p:
205°C. 1H-NMR δ ppm (DMSO-d6): 2.17 (s, 3H, -CH3), 2.20 (s, 3H,
-CH3), 3.14 (s, 2H, -CH2-CONH-), 3.39 (s, 4H, piperazine (2)-CH2),
3.48 (s, 4H, piperazine (2)-CH2), 7.41 (d, 2H, Jo=7.6 Hz, phenyl
H-3’,5’), 7.58 (d, 2H, Jo= 8.4 Hz, phenyl H-2’,6’), 7.70 (d, H, Jo= 8.4
Hz, benzoxazole H-6), 8.07 (d, H, Jo=8.4 Hz, benzoxazole H-7), 8.15
(s, H, benzoxazole H-4), 9.88 (s, H, -NH). 13C-NMR δ ppm (DMSO-
d6): 21.63, 49.93, 53.33, 55.61, 62.25, 111.00, 114.69, 117.85, 124.14,
127.68, 130.37, 136.29, 142.62, 146.82, 153.33, 163.57, 168.72.
2-(p-methylphenyl)-5-(2-(4-acetylpiperazine-1-
2. Experimental details
2.1. Chemistry
The chemicals used in the synthesis and purification of the
compounds were obtained from Sigma-Aldrich, Acros Organics,
Fluka, and Riedel de Haen and were used without purification. The
melting points of the obtained compounds were determined us-
ing the capillary method on a Büchi B540 apparatus and given un-
corrected. Thin-layer chromatography (TLC) was used to monitor
the reactions’ progress and purity. SilicaGel HF254 chromatoplates
(0.3 mm) were used for TLC, and ethyl acetate: n-hexane (1:1) was
used as the mobile phase. Camag UV lamp with UV light at 254
and 366 nm wavelength was used to visualize the plates’ spots.
1H-NMR and 13C-NMR spectral analyses were performed with Var-
ian Mercury 400 High-Performance Digital FT-NMR Spectrometer,
and dimethylsulfoxide-d6 (DMSO-d6) was used as the solvent.
yl)acetamido)benzoxazole (B2): White powder, yield 55%, m.p:
211°C. 1H-NMR δ ppm (DMSO-d6): 1.99 (s, 3H, -CH3), 2.02 (s, 3H,
-COCH3), 3.20 (s, 2H, -CH2-CONH-), 3.38-3.44 (m, 4H, piperazine
(2)-CH2), 3.51-3.64 (m, 4H, piperazine (2)-CH2), 7.42 (d, 2H, Jo=8.0
Hz, phenyl H-3’,5’), 7.53 (d, 2H, Jo= 8.8 Hz, phenyl H-2’,6’), 7.91 (d,
H, Jo= 8.0 Hz, benzoxazole H-6), 8.09 (d, H, Jo=8.8 Hz, benzoxa-
zole H-7), 8.15 (s, H, benzoxazole H-4), 9.96 (s, H, -NH). 13C-NMR
δ ppm (DMSO-d6): 21.62, 47.53, 53.08, 55.82, 61.84, 110.77, 118.24,
124.13, 127.36, 127.67, 128.89, 130.34, 136.36, 142.59, 146.83,
163.56, 168.58, 169.43.
2-(p-methylphenyl)-5-(2-(piperazine-1-
yl)acetamido)benzoxazole (B3): White powder, yield 53%, m.p:
2

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
185°C. 1H-NMR δ ppm (DMSO-d6): 2.45 (s, 3H, -CH3), 2.77-2.89
(m, 8H, piperazine (4)-CH2), 3.10 (s, 2H, -CH2-CONH-), 7.44 (d, 2H,
Jo=8.4 Hz, phenyl H-3’,5’), 7.64 (d, 2H, Jo= 8.4 Hz, phenyl H-2’,6’),
7.88 (d, H, Jo= 8.4 Hz, benzoxazole H-6), 8.12 (d, H, Jo=8.4 Hz,
benzoxazole H-7), 8.18 (s, H, benzoxazole H-4), 9.87 (s, H, -NH).
13C-NMR δ ppm (DMSO-d6): 21.63, 46.72, 52.67, 61.83, 111.01,
113.11, 118.23, 124.12, 126.21, 130.38, 136.26, 137.29, 146.84, 155.19,
163.58, 168.61.
tested in triplicate in each run of the experiments. Solvents, pure
microorganisms, and pure media were used as control wells.
3. Computational and theoretical details
3.1. Molecular docking
The crystal structure of DNA gyrase ATP binding domain
of E. faecalis in complex with
a small molecule inhibitor
2-(p-methylphenyl)-5-(2-(morpholine-4-
((3S)-1-[2-(pyrido[2,3-b]pyrazin-7-ylsulfanyl)-9H-pyrimido[4,5-
b]indol-4-yl]pyrolidin-3-amine) was downloaded from the
RSCB protein database website with the code PDB ID: 4KTN
(https://www.rcsb.org/). The active site∗was created at coordinates
yl)acetamido)benzoxazole (B4): White powder, yield 57%, m.p:
190°C. 1H-NMR δ ppm (DMSO-d6): 2.40 (s, 3H, -CH3), 3.16 (s,
2H, -CH2-CONH-), 3.64-3.73 (m, 8H, piperazine (4)-CH2), 7.42 (d,
2H, Jo=8.0 Hz, phenyl H-3’,5’), 7.59 (d, 2H, Jo= 8.4 Hz, phenyl
H-2’,6’), 8.07 (d, H, Jo= 8.0 Hz, benzoxazole H-6), 8.10 (d, H,
Jo=8.8 Hz, benzoxazole H-7), 8.15 (s, H, benzoxazole H-4), 9.94 (s,
H, -NH). 13C-NMR δ ppm (DMSO-d6): 21.63, 33.40, 54.36, 62.69,
110.71, 118.22, 124.15, 127.22, 128.78, 130.36, 136.28, 142.16, 146.71,
146.82, 163.56, 169.08.
∗
3
˚
x: -9.852, y: -6.045, z: -15.499 and 20 20 20 A . The protein was
prepared in pdbqt file format using AutoDock Tools 1.5.6 program
[23]. Ligands were drawn with Chem3D 19.0 program, minimized,
saved in pdb format, and converted to pdbqt file format with
AutoDockTools 1.5.6 program. The molecular docking studies were
carried out with the latest AutoDock Vina. The results were dis-
played in 2D and 3D with Discovery Studio 2020 Client program
[24].
2.2. Antimicrobial evaluation
3.2. Molecular dynamics simulations
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC
27853, Staphylococcus aureus ATCC 29213, Enterococcus faecalis
ATCC 29212, Acinetobacter baumannii NTCC 13304, Klebsiella pneu-
moniae ATCC 700603, 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. Standard drugs of ampicillin (Sigma), vancomycin
(Mayne Pharma), cefotaxime (Sigma), ciprofloxacin (Sigma), gen-
tamicin (Sigma), meropenem (Sigma), fluconazole (Sigma), and
amphotericin B were used as standard antimicrobial agents. Stock
solutions of the test compounds were prepared in DMSO (Merck).
Ampicillin was prepared in phosphate buffer solution, and other
antibiotic solutions were prepared in sterile distilled water ac-
cording to the guidelines of CLSI M100-S28 and M27-A3 [21,22].
Mueller Hinton Agar (MHA) (Merck), Mueller Hinton Broth (MHB)
(Merck), Sabouraud Dextrose Agar (SDA) (Merck), Sabouraud Liq-
uid Medium (SLM) (Merck), and RPMI-1640 medium (Sigma) with
L-glutamine buffered pH:7 with 3-[N-morpholino]-propanesulfonic
acid (MOPS) (Sigma) were used for microbial cultures. Bacterial
isolates were subcultured in Mueller Hinton Agar (MHA) plates and
incubated overnight at 37°C, and C. albicans were subcultured in
Sabouraud Dextrose Agar (SDA) plates at 35°C for 24-48 h. Pure
colonies were transferred to MHB and SLM for bacteria and fungi,
respectively. They were incubated in the appropriate conditions
overnight. After incubation, the inoculation’s bacterial suspensions
were prepared at 105 CFU/mL by diluting fresh cultures at McFar-
land 0.5 density (108 CFU/mL). Yeast suspensions were also pre-
pared according to McFarland 0.5 density, and a working suspen-
sion was made by a 1:50 dilution followed by a 1:20 dilution of
the stock suspension (2.5 × 103 CFU/mL).
Molecular dynamics simulations were performed using the Gro-
macs 2020.4 version [25]. DNA gyrase subunit B protein and B1-
B4 ligands topology was created with pdb2gmx script and CgenFF
with Charmm36-Jul2020 force field, respectively [26]. The molec-
ular dynamics simulations were performed under periodic bound-
ary conditions with V-rescale and Berendsen’s coupling algorithms
and Newtonian leap-frog MD integrator. The solvating was formed
with a TIP3P water molecule, and seven 11 Na+ ions for neutraliza-
tion were added to the system. The total system energy was min-
imized by the protein-ligand canonical ensemble (amount of sub-
stance, N, pressure, P and temperature, T (NVT)) and isothermal-
isobaric (amount of substance, N, volume, V and equilibrium steps
temperature, T (NPT) were carried out 300 K and 1 atm for 100 ps.
Molecular dynamics of 50 ns duration were run. Root mean square
deviation (RMSD), root mean square fluctuation (RMSF), the radius
of gyration (Rg), intramolecular and intermolecular hydrogen bond
analyzes between DNA gyrase subunit B protein and B1, B2, B3,
and B4 ligands were measured. The data obtained from molecular
dynamics studies were graphed with the QtGrace tool.
3.3. DFT/B3LYP calculations
All theoretical calculations of B1-B4 were made using the
DFT/B3LYP method, 6-311G (d,p) basis set, and Gaussian 09 pack-
age program [27]. Theoretically, the geometric parameters of the
molecule (bond length, bond angle, and dihedral angles), HOMO-
LUMO orbital energies, and other electronic parameters obtained
from these energies, NBO analysis, and MEP analysis were per-
formed. The results were viewed with the GaussView 6.0 program
[28].
Susceptibility testing was performed with MHB for bacteria, and
RPMI-1640 medium with L-glutamine buffered pH:7 with 3-[N-
morpholino]-propanesulfonic acid (MOPS) for fungi. The solution
of the newly synthesized compounds and standard drugs were pre-
pared at 512, 256, 128, 64, 32, 16, 8, 4 μg/mL, and different stock
concentrations for every antimicrobial agent were prepared, re-
spectively by diluting the stock concentrations in a microdilution
tray with a multichannel pipette. After dilution, a 10 μl bacterial or
fungal inoculum was added to each well of the microdilution trays.
The trays were incubated at 37°C for bacteria and 35°C for fungi
in a humid chamber, and MIC endpoints were read after 24 h of
incubation. The compound’s lowest concentration that completely
inhibits macroscopic growth was determined, and minimum in-
hibitory concentrations (MICs) were reported. All organisms were
3.4. Theoretical ADME predictions
A drug candidate compound should have high pharmacological
activity, low toxicity profile, and ideal pharmacokinetics. Within
the scope of computer-aided drug development studies in recent
years, estimates of absorption, distribution, metabolism, and ab-
sorption (ADME) profiles of drug candidates can be made. In this
study, various pharmacokinetic parameter values such as log P,
TPSA, nrotb, molecular weight, and hydrogen bond donor-receptor
number of the compounds synthesized using the Molinspiration
software were calculated [29]. Drug-likeness scores were computed
using the Molsoft program [30].
3

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Scheme 1. General synthesis method of B1-B4
Table 1
4. Results and discussion
Minimal inhibitory concentration values (μg/mL) of the B1-B4
against Gram (+) bacterial strains compared to reference drugs
4.1. Chemistry
Compound
S. a.
S. a.∗
S.a.∗∗
E.f.
E.f.∗
E.f.∗∗
In this study, 4 original compounds were synthesized using
the method given in the literature (Scheme 1) [31-34]. When
the final products are examined, the p-methyl phenyl structure
in the 2nd position of the benzoxazole ring; in the 5th posi-
tion, there are morpholine, piperazine, N-methyl piperazine, N-
acetyl piperazine groups attached to the structure of 2-substituted
acetamido. In the synthesis of these derivatives, primarily 2,4-
diaminophenol dihydrochloride was reacted with p-methyl ben-
zoic acid at 160-190°C for 3-4 h under PPA catalysis (A1). Subse-
quently, the product obtained was treated with chloroacetylchlo-
ride, and amidifications of the benzoxazole structures from the 5th
position were carried out (A2). In the last step, the targeted com-
pounds were reached due to the reaction of 2-(p-methylphenyl)-
5-(2-chloroacetamido)benzoxazole with morpholine, piperazine, N-
methylpiperazine, N-acetyl piperazine in the presence of DMF and
TEA (B1-B4). The structure of the compounds was confirmed based
on spectral data. In 1H-NMR spectra of B1-B4, NH protons’ signal
appeared as single bands at 9.87-9.94 ppm. Also, aliphatic protons
were between 1.99-3.73 ppm and aromatic protons between 7.41-
8.18 ppm. The aromatic and carbonyl groups’ carbon peaks were
between 110.71-169.43 ppm, and the aliphatic carbon peaks were
between 21.62-62.69 ppm. All carbon signals appeared at their ex-
pected positions. Also, the compounds’ yields are fairly good, rang-
ing from 53% to 62%.
B1
32
64
64
64
0.5
0.5
0.25
0.5
2
16
64
64
64
˃16
2
32
64
64
128
˃16
2
16
64
64
64
2
32
32
32
32
˃16
32
˃8
˃4
˃8
-
32
32
32
32
˃16
32
˃ 8
˃4
˃8
-
B2
B3
B4
Ampicillin
Vancomycin
Gentamicin
Ciprofloxacin
Cefotaxime
Meropenem
2
˃16
˃16
˃16
-
˃16
˃16
8
4
2
4
-
-
-
S. a: S. aureus ATCC 29213, S.a∗: S. aureus isolate 1, S.a.∗∗: S. aureus
isolate 2, E. f.: E. faecalis ATCC 29212, E. f. ∗: E. faecalis isolate 1, E.
f.∗∗: E. faecalis isolate 2.
Staphylococcus aureus isolates are MRSA (resistant to all beta-
lactam antibiotics). It is also sensitive to vancomycin.
E. faecalis isolates are resistant to vancomycin (VRE). Also, they are
resistant to ampicillin and ciprofloxacin.
two isolates with MIC: 32 μg/mL they exhibited fairly close activity
to ampicillin.
While the compounds show weak activity against E. coli; B1
and B2 showed against E. coli isolates 1 and 2 a very close ac-
tivity to gentamicin and cefotaxime. Compounds showed activity
close to gentamicin against P. aeruginosa. The compounds exhibited
very poor activity on A. baumannii compared to reference drugs
and showed moderate activity to gentamicin with MIC: 32 μg/mL
against A. baumannii isolate 1. B1 showed activity against A. bau-
mannii isolate 2, comparable to gentamicin. Compounds showed
fairly weak activity against K. pneumoniae and both isolates.
The antifungal activities of the compounds against C. albicans
and two isolates were observed to be MIC: 64 μg/mL, and were
found to be less effective when compared with fluconazole and
amphotericin B.
4.2. Antimicrobial evaluation
The observed MIC values (μg/mL) of B1-B4 and reference drugs
were presented in Table 1, Table 2, and Table 3. The activity of
the compounds against S. aureus ranged from MIC: 32-64 μg/mL,
and their activity was lower when compared to reference drugs
(MIC: 0.5-2 μg/mL). B1 showed better activity against S. aureus
isolate 1 with MIC: 16 μg/mL compared to ampicillin, gentamicin,
ciprofloxacin, and cefotaxime, and also showed activity very close
to ampicillin, gentamicin and ciprofloxacin with MIC: 32 μg/mL
against S. aureus isolate 2. The compounds showed moderate activ-
ity against E. faecalis between MIC: 16-64 μg/mL, and on the other
Based on these results, benzoxazole derivatives’ antimicro-
bial activities against standard strains were found to be lower
compared to reference drugs in general. However, against drug-
resistant isolates antimicrobial activity is comparable or better
than reference drugs. The microbial susceptibility tendency listed
4

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Table 2
Minimal inhibitory concentration values (μg/mL) of the B1-B4 against Gram (-) bacterial strains compared to reference drugs.
Compound
E. c.
E. c.∗
E. c.∗∗
P. a.
P.a.∗
P.a.∗∗
A. b.
A. b.∗
A. b.∗∗
K. p.
K. p.∗
K. p.∗∗
B1
64
16
16
64
64
˃16
-
16
32
32
32
˃16
-
64
32
32
32
-
32
16
16
32
-
64
32
32
32
-
32
32
32
32
-
32
32
32
32
-
16
32
32
32
-
64
128
64
64
-
128
128
128
128
-
128
128
128
128
-
B2
128
128
128
8
B3
B4
Ampicillin
Vancomycin
Gentamicin
Ciprofloxacin
Cefotaxime
Meropenem
-
-
-
-
-
-
-
-
-
-
0.5
0.016
0.125
-
˃8
˃2
˃8
-
˃8
˃2
˃8
-
0.5
0.125
-
16
4
16
4
2
16
-
16
-
2
˃16
-
˃16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
˃ 8
˃ 8
2
˃8
8
E.c.: E.coli ATCC 25922, E.c.∗: E.coli isolate 1, E. c.∗∗: E.coli isolate 2, P.a.: P. aeruginosa ATCC 27853, P.a.∗: P. aeruginosa isolate
1, P.a.∗∗: P. aeruginosa isolate 2, A. b.: Acinetobacter baumannii NTCC 13304, A.b.∗: Acinetobacter baumannii isolate 1 A.b.∗∗
Acinetobacter baumannii isolate 2, K. p.: Klebsiella pneumoniae ATCC 700603, K. p.∗: Klebsiella pneumoniae isolate 1, K.p.∗∗
Klebsiella pneumoniae isolate 2.
:
:
E. coli isolate resistant to ciprofloxacin (Contains Broad Spectrum Beta-Lactamase Enzyme) P. aeruginosa isolates are resistant
to ciprofloxacin and gentamicin. K. pneumoniae isolates are resistant to gentamicin and carbapenem. A. baumannii isolates are
resistant to ciprofloxacin and gentamicin.
Table 3
4.4. Molecular Dynamics Simulations
Minimal inhibitory concentration values (μg/mL)
of the B1-B4 against fungal strains compared to
reference drugs.
The molecular dynamics simulations study was performed to
investigate the stability and alteration of the protein-ligand com-
plex structures obtained from AutoDock Vina molecular docking.
Molecular dynamics simulations of 50 ns duration were carried
out to analyze further the interactions of the complex structures
formed with the DNA gyrase subunit B protein (PDB ID: 4KTN) and
B1, B2, B3, and B4. The DNA gyrase subunit B protein was simu-
lated with apo form without ligand to validate the molecular dy-
namics system. RMSD measurement of the protein apo form and
holo form structures according to the protein backbone atoms was
measured. Fig. 3 shows that the apo form and holo form structure
showed an RMSD value below 0.23 nm. Throughout 50 ns, the av-
erage RMSD value of the DNA gyrase subunit B protein and B1, B2,
B3, and B4 complexes were measured at about 0.15 nm. Accord-
ingly, the B1, B2, B3, and B4 compound formed a structure stable
and consistent with the DNA gyrase subunit B protein.
Compound
C. a.
C. a.∗
C. a∗∗
B1
64
64
64
64
64
˃4
0.5
64
64
64
64
˃4
1
B2
64
B3
64
B4
64
Fluconazole
Amphotericin B
0.125
0.5
C.a.: C. albicans ATCC 10231, C.a.∗: C. albicans
isolate 1, C.a.∗∗: C. albicans isolate 2
are as follows: E. coli> S. aureus> E. faecalis> P. aeruginosa> A.
baumannii> K. pneumoniae> C. albicans. Those with substituents in
the 4th position of piperazine contributed positively to the activity
compared to those who did not. The methyl group affected the ac-
tivity more positively than the acetyl group, and the replacement
of the piperazine group with the morpholine group decreased the
activity, albeit partially.
The fluctuation of apoprotein and holoprotein structures was
calculated on a residue basis by selecting c-alpha. As shown in
Fig. 3, the RMSF value of the apo form and protein-ligand com-
plex structures were measured below about 0.25 nm. The ligands-
bound DNA gyrase subunit B protein is a little less fluctuating and
more stable than the apoprotein form between 45-49 residues in
the active site amino acids region.
4.3. Molecular docking study
Molecular docking is a method based on the adaptation of the
compounds designed for targets with known receptor structure to
the receptor spaces, their interactions with amino acids, and ex-
amining them from various angles [35]. DNA gyrase is a critical
therapeutic target in designing effective antibacterial compounds.
Although this enzyme is not found in eukaryotes such as mam-
mals, it is found in bacteria. It also plays a good role in bacte-
rial DNA replication and is a well-researched target. Therefore, it
is used to design compounds that are selectively effective against
bacteria [36]. One of the benzoxazoles’ mechanisms is inhibiting
the DNA gyrase enzyme, as mentioned in the introduction.
B1, B2, B3, and B4 respectively showed binding energy of -8.1
kcal/mol, -8.3 kcal/mol, -7.8 kcal/mol, -7.8 kcal/mol to the DNA gy-
rase enzyme. The binding energies and antimicrobial activity re-
sults were consistent with each other. The bonds formed by the
compounds with the active site were shown in 2D and 3D in Fig. 2.
It has been observed that the compounds interact with this en-
zyme with at least one hydrogen bond, van der Waals, pi-alkyl
interactions, pi-sigma interactions, amide-pi-stacked interactions.
B1, B2, and B3 formed hydrogen bond with GLN A: 85, while B4
formed hydrogen bonds with HIS A: 101. The compounds generally
exhibited similar interaction poses.
The radius of gyrations tells about protein stability, so Rg mea-
surement was performed to measure the compactness of apo form
and DNA gyrase subunit B - B1, B2, B3, and B4 protein-ligand com-
plex structures. As shown in Fig. 4, apo and holo form Rg values
for 50 ns formed a stable curve between 1.67 nm and 1.72 nm. No
degradation occurred in the compactness of the complex formed
by the protein with the ligand. According to RMSD, RMSF, and Rg
analysis BCAB and DNA gyrase subunit B protein form a stable and
stable interaction if we look at molecular dynamics simulations in
general.
The intramolecular H-bond number, which is a stability mea-
sure of the protein structure, was analyzed for holo form and apo
form. As shown in Fig. 4, the number of intramolecular H bonds of
the holo form and apo form has a similar intramolecular H bond
profile. B1, B2, B3, and B4 binding to DNA gyrase subunit B pro-
tein was not decreased protein stability. While the SOCS-2 apopro-
tein formed an average of 151.50 intramolecular H-bonds, the B1,
B2, B3, and B4 and DNA gyrase subunit B protein complex formed
an average of 152.64, 151.27, 149.40, and 150.42 intramolecular H
bonds throughout 50 ns, respectively. On the other hand, the H
bond analysis formed by the interaction of B1, B2, B3, and B4 with
5

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Fig. 2. The docked conformation of B1-B4 at DNA gyrase ATP binding site. H-bond, alkyl-π, and sigma-π interactions are represented by green, pink, and violet dotted lines,
respectively. The hydrogen bonding pocket is shown for clarity.
Fig. 3. Root-mean-square deviation (RMSD) and root mean square fluctuation (RMSF) analysis of the apo and holo form of DNA gyrase subunit B throughout 50 ns.
6

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Fig. 4. Radius of gyration (Rg) (left) and intramolecular H bonds number graph (right) of DNA gyrase subunit B protein apo and holo forms graph over 50 ns.
Fig. 5. Intermolecular H bonds number between DNA gyrase subunit B (PDB ID: 4KTN) - B1, B2, B3, and B4 ligands for 50 ns.
the DNA gyrase subunit B protein was performed. As shown in
Fig. 5, throughout 50 ns simulation, it was observed that B1, B2,
B3, and B4 generally formed an H-bond between 1 and 3 numbers.
appears that B2 has a high tendency to accept electrons, while B3
has more electron donation. The atomic orbital components of the
frontier molecular orbitals were shown in Fig. 6.
4.5. Molecular reactivity analyses
4.6. MEP analysis and Geometry Optimization
In all multi-electron molecular systems, the interaction with
other molecules is explained by the boundary molecular orbitals
called HOMO (highest occupied molecular orbital) and LUMO (low-
est unoccupied molecular orbital). The energy values obtained re-
sult from HOMO-LUMO (highest energy-filled molecular orbital -
lowest energy non-bonding empty molecule orbital). The energy
difference between these two orbitals can be used to explain the
chemical behavior of the molecule. The wider the HOMO-LUMO
range in a molecule, the more stable the molecule, while the
smaller the molecule, the more unstable it is. [37,38]. The com-
pounds’ stability order is B2>B4>B1>B3. By using HOMO and
LUMO boundary orbital energies, chemical properties such as ion-
ization potential, electron affinity, chemical potential, electronega-
tivity, chemical hardness, chemical softness, electrophilicity index
can be calculated. The ionization potential is the minimum en-
ergy required to remove an electron from the gas phase’s molecule.
Electron affinity is defined as the amount of energy that increases
when an electron is added to a gas phase molecule. Electroneg-
ativity refers to an atom’s force in a molecule to attract elec-
trons. Chemical hardness is a measure of charge transfer inhibition
within the molecule, and molecules with high chemical hardness
value cannot have little or no charge transfer within the molecule.
Calculated values were presented in Table 4. The HOMO energy of
the compounds is localized on the 2-(p-methylphenyl)benzoxazole
and the amide group, while the LUMO energy is localized only on
the 2-(p-methylphenyl)benzoxazole. This result indicates that the
substituted group does not affect the HOMO and LUMO energy. It
It is a type of analysis that deals with the electrostatic poten-
tial of a molecule, electronegativity, and partial charges on dif-
ferent atoms. MEP surface analysis study is a useful method for
understanding the ability to bond with biological molecules, in-
cluding charge-dipole, dipole-dipole, and quadrupole-dipole inter-
actions. MEP map shows the shape, size, dipole moment, elec-
trophilic and nucleophilic reactive regions of the molecule, and
a color-coding system is used to define MEP. The surface map is
determined by colors ranging from red to blue, starting with the
electron-rich part, towards the less rich regions [38,39]. In the
MEP map, the electrostatic potential increases in the order of red
<orange <yellow <green <blue. Red-colored parts represent neg-
ative electrostatic potential regions, while blue colored parts rep-
resent positive electrostatic potential regions. Regions containing
fewer electrons are shown in yellow; green-colored parts are the
regions where the electrostatic potential is zero. In the MEP map
of B1-B4, it has been observed that the red and yellow regions are
mainly concentrated on oxygen and nitrogen atoms, and the blue
and green regions are concentrated around carbon and hydrogen
atoms (Fig. 7).
In processes such as frequency and energy calculations in
molecules, and the determination of thermodynamic properties,
the molecule’s equilibrium geometry is determined first. The equi-
librium geometry of a molecule is the geometry in which the en-
ergy of that molecule is minimum, and the molecule is in the most
stable state in this state. The process of determining the equilib-
rium geometry of the molecule is called geometric optimization.
7

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Table 4
Calculated frontier molecular orbital parameters of B1-B4.
Compound
HOMO (eV)
LUMO (eV)
Gap ꢀE
ӀP (-HOMO)
EA (-LUMO)
η (IP − EA)/2
μ −(IP+EA)/2
S 1/2η
X (IP + EA)/2
ω (μ2 /2η)
B1
B2
B3
B4
-5.8265
-5.9173
-5.8202
-5.8597
-1.5336
-1.5954
-1.5322
-1.5564
4.2928
4.3219
4.2879
4.3032
5.8265
5.9173
5.8202
5.8597
1.5336
1.5954
1.5322
1.5564
2.1464
2.1609
2.1439
2.1516
-3.6800
-3.7564
-3.6762
-3.7081
0.2329
0.2313
0.2332
0.2323
3.7563
3.6762
3.7080
3.8867
1.5773
1.6324
1.5759
1.5976
Gap ꢀE: LUMO-HOMO, IP: Ionization potential, EA: Electron affinity, X: Electronegativity, η: Chemical hardness, S: chemical softness, μ: Chemical potential, ω:
Electrophilic index.
Fig. 6. HOMO and LUMO diagram of B1-B4
When the arrangement of the atoms that make up the molecules
change, the molecule’s energy also changes. The relationship be-
tween molecular conformation and energy is indicated by the po-
tential energy surface (PES). The general minimum represents the
lowest point of the PSI, and the local minimum represents the low-
est point in a region in the PES, and the minimums in the equi-
librium geometry of a molecule correspond to different conforma-
tions or structural isomers in the single-molecule case. The most
stable arrangement of all the atoms of the B1-B4 in space has been
determined (Fig. 7). The bond lengths (Ǻ), bond angles (°), and di-
hedral angles (°) of B1 and B2 were given in the Supplementary
Information section.
bution of C1 atom orbitals to the 1st σ bond is 49.65 percent, the
contribution of the C6 atom is 50.35 percent. Hybridization per-
centages of C1 atom orbitals within s, p, and d orbitals are 33.80,
64.16, and 0.04 percent, respectively. It is seen that the resulting
σ bonding hybridized orbital has sp1.73 character. Likewise, if the
2nd π bond is examined; the Contributions of C1 and C6 atoms
to this bond are 51.01 and 48.99 percent, respectively. Due to the
bond’s natural character, the s atomic orbital contribution is 0 per-
cent. The d orbital contribution in the π bond formed as a result of
p orbitals’ interaction is also seen as 0.04 percent and the character
of the bond-forming hybrid orbital is sp1.00. Table S8 shows the
second-order perturbation analysis based on NBO. The E(2) value
in the table is the stabilization energy obtained from the second-
order perturbation theory analysis. It expresses the average inten-
sity of the donor-acceptor interaction and forms the NBO analysis
basis.
4.7. NBO analysis
NBO analysis is a highly effective computational method to
explain and interpret intramolecular and intermolecular interac-
tions. It explains phenomena such as electron exchanges, hydro-
gen bonds, hyperconjugation, atomic hybridization, molecular hy-
bridization, and electron delocalization in a system. More specifi-
cally, NBOs are orthonormal clusters of localized "maximally filled"
orbitals that give the most accurate Lewis-like definition of elec-
tron density. That is, they are bond orbitals with maximum elec-
tron density. Lewis-type NBOs represent the localized natural
Lewis structure (NLS) of the wave function, while the remain-
ing non-Lewis-type NBOs describe the remaining delocalization ef-
fects. While explaining these interactions, charge transfers from
full molecular orbitals (donors) of the molecule to empty molec-
ular orbitals (acceptors) are taken as the basis.
Supplementary Information contains the NBO/NPA load values
of the compounds. It is seen that the C2, C4, C5, C8, C13, and C18
atoms have positive natural charge values. It has been determined
that this situation is due to the bonding of the relevant atoms with
oxygen and nitrogen atoms with high electronegativity. Among the
carbon atoms, the C18 atom has the highest positive natural charge
value. It was concluded that this was because the C18 atom was
the carbonyl group’s atom. Among the carbon atoms at the se-
lected computational levels, C3, C6, and C16 atoms have the high-
est negative natural charge values. These atoms have the highest
negative values
due to the inductive electron withdrawal from
the hydrogen atoms they are bound to. All oxygen atoms have a
negative charge, and all hydrogen atoms have a positive charge.
NBO analysis of B1-B4 is given in the Supplementary Informa-
tion section. It shows in detail the formation of hybrid molecu-
lar orbitals in these calculations and the atomic orbitals that con-
tribute to it. For example, considering the double bond between
C1=C6 atoms in the B1 molecule, while the total electronic contri-
4.8. In silico ADME prediction
Examination of physicochemical parameters gives a preliminary
idea for evaluating a drug in terms of the route of use. These
8

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Fig. 7. MEP plot (left) and optimized molecular structure (right) of B1-B4
Table 5
Calculated ADME parameters of B1-B4
Comp.
LogP ≤5
TPSA -
%ABS -
MW ≤500
nON ≤10
nOHNH ≤5
nviolations ≥1
nrotb -
Volume -
Drug-likeness score
B1
B2
B3
B4
3.54
2.90
2.94
3.50
61.61
78.78
70.39
67.60
87.74
81.82
84.71
85.67
364.45
392.46
350.42
351.41
6
7
6
6
1
1
2
1
0
0
0
0
4
4
4
4
340.10
359.08
323.16
319.74
0.78
0.81
0.60
0.52
MW: Molecular weight. TPSA: Topological polar surface area. %ABS: Percentage absorption. nrotb: Number of rotatable bonds. nON: Number of hydrogen
acceptors. nOHNH: Number of hydrogen donors. LogP: Log octanol/water partition coefficient
properties are important not only for oral use but also for top-
ical applications. Therefore, the determination of the synthesized
compounds’ physicochemical properties has gained importance in
terms of acting by predicting the ADME properties. Lipophilic, es-
timated volume, topological polar surface area, %ABS, molecular
weight, rotatable bond number, hydrogen-bonded receptor num-
ber, hydrogen bond donor count of all compounds were given in
Table 5. According to the five rules of Lipinski (molecular logP
≤ 5, molecular weight ≤ 500, number of hydrogen bond recep-
tors ≤ 10, and number of hydrogen bond donors ≤ 5), an ac-
tive oral drug must fully comply with this rule or violate at most
one of them [40]. This rule does not determine whether the com-
pounds are pharmacologically active, but compounds that follow
the rule are less likely to be eliminated during clinical trials, so
they have a higher chance of reaching the market. The absorption
percentage was calculated using % ABS=109-(0.345xTPSA) [41], and
the compounds showed a good absorption profile in the range of
81.82-87.74%. According to these results, it is seen that the result
molecules do not violate Lipinski’s five rules. B1 and B2, the most
effective antimicrobial compounds, have the highest drug-likeness
score (Table 5).
5. Conclusion
Synthesis, molecular docking, molecular dynamics, DFT, ADME
estimation, and antimicrobial activities of 2-(p-methylphenyl)-5-(2-
substitutedacetamido)benzoxazole derivatives have been reported.
B1 and B2 showed better antimicrobial activity against isolates
compared to reference drugs. As
a result of molecular dock-
ing studies performed with AutoDock Vina, B1, B2, B3, and B4
in the DNA gyrase ATP binding site showed -8.1 kcal/mol, -8.3
kcal/mol, -7.8 kcal/mol, -7.8 kcal/mol interaction energies, respec-
tively. Molecular dynamic simulation studies have shown that B1,
B2, B3, and B4 remain stable in the active binding site. B2, which
has the highest gap ꢀE = 4.3219 in DFT studies, showed high an-
timicrobial activity. Also, ADME profiles of compounds are suitable
for restrictive rules.
9

M. Erol, I. Celik and G. Kuyucuklu
Journal of Molecular Structure 1234 (2021) 130151
Author Statement
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draft; Ismail Celik: Writing draft, in silico studies, interpretation of
discussion, results; Gulcan Kuyucuklu: Biological measurements,
interpretation of discussion and results of biological measurements
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Declaration of competing interest
No potential conflict of interest was reported by the authors.
Acknowledgment
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