Virtual screening of some heterocyclic structures toward novel antibacterial agents

Article abstract of DOI:10.1007/s13738-017-1262-2

Infectious diseases and their treatment are among the most important issues in global health and economy. Moreover, increasing prevalence of antibiotic-resistant pathogenic bacteria necessitates the considerable need for discovering new drugs. Some heterocyclic structures with dihydropyridine (DHP) scaffold have been reported as antimicrobial agents. Herein, we report a structure-based virtual screening of a pool of 3,5-disubstituted DHPs and a post-analysis of virtual hits through in vitro antibacterial assessment. Four top-ranked DHP structures (6a–d) were found to interact with the relevant target active sites and exhibited superior stereoelectronic features within their enzyme inhibition. Selected compounds were synthesized and assessed for their antibacterial activity via microdilution method. Results of this study represented a significant application of multi-step virtual screening strategy in identifying privileged DHP structures as good starting points for further developments toward more potent antibacterial agents.

Full text of DOI:10.1007/s13738-017-1262-2

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-017-1262-2
ORIGINAL PAPER
Virtual screening of some heterocyclic structures toward novel
antibacterial agents
1
1
2
3
Zahra S. Hosseini · Mohammad Reza Housaindokht · Nima Razzaghi‑Asl · Ramin Miri
Received: 30 April 2017 / Accepted: 30 November 2017
©
Iranian Chemical Society 2017
Abstract
Infectious diseases and their treatment are among the most important issues in global health and economy. Moreover, increas-
ing prevalence of antibiotic-resistant pathogenic bacteria necessitates the considerable need for discovering new drugs. Some
heterocyclic structures with dihydropyridine (DHP) scaꢀold have been reported as antimicrobial agents. Herein, we report
a structure-based virtual screening of a pool of 3,5-disubstituted DHPs and a post-analysis of virtual hits through in vitro
antibacterial assessment. Four top-ranked DHP structures (6a–d) were found to interact with the relevant target active sites
and exhibited superior stereoelectronic features within their enzyme inhibition. Selected compounds were synthesized and
assessed for their antibacterial activity via microdilution method. Results of this study represented a signiꢁcant application
of multi-step virtual screening strategy in identifying privileged DHP structures as good starting points for further develop-
ments toward more potent antibacterial agents.
Keywords Antibiotic resistance · Dihydropyridine · Docking · Virtual screening · In vitro
Introduction
of bacteria are resistant to at least one of the most commonly
used drugs. Therefore, existing drugs can’t properly respond
to the control and treatment of many infectious diseases, and
hence, design and development of new and more eꢀective
antibacterial agents are an urgent requirement in human life
[2].
The remarkable role of bacterial infections in mortality has
become a big concern and human life has been always asso-
ciated with risks related to such infectious diseases. During
the past century, many prosperous eꢀorts have been per-
formed to control infectious diseases, but the prevention
and treatment of infectious diseases still remained a serious
problem in public health, leading to over 13 million deaths
each year [1]. Within antimicrobial therapy, antibiotic resist-
ance is one of the major challenges and is usually considered
to be a consequence of antibiotic misuse. Nowadays, many
Considerable advances in the field of computational
chemistry and protein crystallography facilitated most of
the traditional eꢀorts toward discovery of novel chemo-
therapeutic agents [3]. In silico or computer-aided drug
design is a rational drug discovery technique that serves
as a fundamental part of modern drug discovery projects.
In this regard, one of the most eꢂcient strategies is virtual
screening (VS) of chemical databases with the aim of ꢁnd-
ing novel bioactive compounds [4]. Virtual screening tries
to eꢂciently reduce the deꢁned chemical space to a much
smaller subset that can be synthesized and assessed through
biological targets with the aim of achieving potential lead/
drug candidates. It has been postulated that VS can reduce
costs and increase hit rates in a typical lead discovery path
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-017-1262-2) contains
supplementary material, which is available to authorized users.
*
Mohammad Reza Housaindokht
housain@um.ac.ir
1
2
3
Department of Chemistry, Faculty of Sciences, Ferdowsi
University of Mashhad, Mashhad 9177948974, Iran
[
5] and therefore may be an alternative approach to high-
throughput screening (HTS) [6].
Department of Medicinal Chemistry, School of Pharmacy,
Ardabil University of Medical Sciences, Ardabil, Iran
Structure-based virtual screening (SBVS) utilizes dock-
ing algorithms to explore the conformational space of
ligand–receptor complex [7]. The outputs are top-ranked
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
Vol.:(0
1
123456789)
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Journal of the Iranian Chemical Society
ligand conformations that represent the probable binding
modes in the active site of the macromolecular target. SBVS
methods require the information of the 3D structure of a tar-
get obtained from X-ray crystallography or nuclear magnetic
resonance spectroscopy. Such information can be retrieved
form a protein data bank (PDB; http://www.rcsb.org) or
homology modeled from a homologous protein possessing
close sequence identity and order [8].
substituted phenyl derivatives, while aryl or 5/6-membered
heteroaryl rings were substituted on the C4 position of the
DHP core. All of the 3D structures were subjected to geo-
metric optimization using semi-empirical PM3 method.
Structure‑based virtual screening (SBVS)
Several VS strategies have been successfully applied to
identify novel antibacterial compounds [9–11]. In the pre-
sent contribution, a pool of DHP compounds was subjected
to SBVS technique with the aim of ꢁnding new antibacterial
agents. Besides calcium channel blocking activity which is
the major biological eꢀect of DHP compounds, there are
several other eꢀects such as p-glycoprotein inhibition [12],
cyclooxygenase-2 inhibition [13], antituberculosis [14] and
anticonvulsant [15].
SBVS was carried out using automated ꢃexible ligand
module of the advanced program AutoDock 4.2 [19]. The
high-resolution crystal structures of selective therapeutic
targets from selected pathogenic bacteria E. coli (PDB
ID 3FV5) and S. aureus (PDB ID 1SFJ) were obtained
from the Brookhaven Protein Data Bank (RCSB) (http://
www.rcsb.org). Before docking the ligands into the mac-
romolecular active sites, the enzymes were processed
by removal of the substrate cofactor as well as cognate
ligands along with all crystallographic water molecules.
All the missing residues were reconstructed through the
“check for missing atoms” and “repair missing atoms”
module incorporated into the Misc module of the Auto-
Dock tools environment.
1,4-Dihydropyridine compounds with 3 and 5N-naphthyl
amide substituents exhibited higher inhibitory eꢀect on S.
aureus and E. coli with regard to methyl ester containing
derivatives [16]. It was also revealed that within N-naph-
thyl compounds, 4-chloro phenyl moiety in the C4 of DHP
ring produced compounds with higher activity against S.
aureus than ciproꢃoxacin [16]. In N-benzothiazolyl DHPs,
2D schematic representations of ligand–enzyme inter-
actions were all reproduced by Ligplot program [20].
The interaction/binding between DHPs and target
enzyme active site were modeled using Lamarckian
genetic algorithm (LGA). Fifty independent GA runs were
performed for each ligand under study. For Lamarckian
GA, 27,000 maximum generations, a gene mutation rate of
0.02, and a crossover rate of 0.8 were used. The grid maps
of docking studies were generated by AutoGrid program.
The size of grid was set in a way to include only the active
site but also considerable portions. For this purpose, the
3
3
,5-estric derivatives showed better activity proꢁle than
,5-diketone derivatives against S. aureus [17]. In a research
on 3,5-bis amide derivatives, it was observed that incor-
poration of N-(3,4-dichlorophenyl) and N-(2,4,5-tri chloro-
phenyl) rings into the nitrogen of amide group led to the
increase in antibacterial eꢀect against cloxacillin-resistant
S. aureus [18].
In the light of above explanations and with the aim of
developing DHP-based antibacterial compounds and also to
further extend the scope of DHP derivatives as privileged
biological structures, structures with nitrogen/sulfur hetero-
cycles within the amide substituents and also phenyl deriva-
tives on the C4 of the ring were considered for this study. We
report the identiꢁcation of a few antibacterial compounds.
For this purpose, multi-step VS of a collection of 3,5-bis-
N-aryl carbonylated-4-aryl-1,4-dihydropyridine structures
followed by synthesis, characterization and in vitro antibac-
terial assessment was performed.
3
x, y, z-coordinates of the grid were set to 60 × 60 × 60 Å
around the mass centers of the cognate ligands as the cata-
lytic site of enzyme (spacing 0.375 Å). Cluster analysis
was performed on the docked results using an RMSD tol-
erance of 2 Å.
Filtration criteria
A variety of ꢁltration criteria including estimated binding
aꢂnity within multiple target conformations, conformational
population of top-ranked cluster and chemical interactions
with the key residues of the macromolecular active sites
were considered to ꢁlter the candidates and hence pick up
the molecules that might hit the target successfully. It should
be notiꢁed that crystal structures from bacteria E. coli (PDB
ID 3FV5) and S. aureus (PDBID 1SFJ) were utilized to run
the SBVS project and primary hit candidates were selected
on the basis of predicted binding aꢂnities within these crys-
tallographic systems.
Experimental
Ligand dataset
A chemical collection including about 1000 diverse
3
,5-bis-carbamoylated 1,4-dihydropyridine structures
was designed and prepared electronically (Supplemen-
tary ꢁle 1). Chemical moieties incorporated into the car-
bamoyl groups of the DHP structures included phenyl or
1
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Journal of the Iranian Chemical Society
Chemistry
General procedure for the synthesis of substituted
,4‑dihydropyridines (6a–d)
1
All of the reagents for the synthesis were obtained from
Merck Chemical Company and used without further puri-
N-Aryl-3-oxobutanamide (3a and 3b) (2 mmol), correspond-
ing aldehyde (4a–c) (1 mmol) and ammonium acetate (5)
(2 mmol) were all mixed and reꢃuxed in 20 mL absolute eth-
anol for 24–48 h. The progress of the reaction was checked
by TLC and on completion of the reaction; the product was
ꢁltered, washed with small portions of cold ethanol and
then dried under vacuum to give the pure compounds 6a–d.
Physical and characteristic data of the ꢁnal compounds are
summarized in Table 1.
ꢁ
cation. Melting points were recorded by a Reichert–Jung
1
hot-stage microscope and were uncorrected. H NMR spec-
tra were determined on a Bruker FT-500 MHz spectrometer
in chloroform-d or DMSO-d depended on the solubility of
1
6
samples (Supplementary ꢁle 2). All the chemical shifts were
reported as (δ) values (ppm) against tetramethylsilane as an
internal standard. FT-IR (KBr) spectra were determined by
a Nicolet FT-IR Magna 550 spectrophotometer.
The analytical thin-layer chromatography (TLC) on pre-
coated silica gel 60 F254 aluminum plates (Merck, Ger-
many) was applied to check the progress of the reactions
and purity of the products. Preparative thin-layer chroma-
2,6‑Dimethyl‑3,5‑bis‑N‑(2‑benzothiazolyl)
carbamoyl‑4‑(3‑ꢀuorophenyl)‑1,4‑dihydropyridine
(6a)
tography was performed with prepared glass-backed plates
2
1
(
20 × 20 cm , 500 μ) using silica gel (Merk Kieselgel 60
Yellow precipitate; Yield 38%; H NMR (CDCl ): δ
3
HF254, Art. 7739).
(ppm) 11.96 (2H, s, NH-amide), 9.05 (1H, s, NH-DHP),
6
.91–7.98 (12H, m, ArH), 5.43 (1H, s, C4H-DHP), 2.30
−
1
(
6H, s, CH -DHP); IR (KBr) ν (cm ): 3415.3 (N–H, DHP),
3
General procedure for the synthesis
of N‑aryl‑3‑oxobutanamides (3a and 3b)
2913.2 (C–H, aliphatic), 1619.8 (C=O, amide), 1548.4
(C=C, alkene).
N-Aryl-3-oxobutanamides (3a and 3b) were synthesized
according to the modiꢁed method of Clemens via conden-
sation of 2,2,6-trimethyl-1,3-dioxine-4-one (1) and corre-
sponding arylamines (2a and 2b) [21].
2,6‑Dimethyl‑3,5‑bis‑N‑(2‑benzothiazolyl)
carbamoyl‑4‑(4‑chlorophenyl)‑1,4‑dihydropyridine
(6b)
1
Yellow precipitate; Yield 36%; H NMR (CDCl ): δ
3
(
ppm) 11.94 (2H, s, NH-amide), 9.05 (1H, brs, NH-DHP),
N‑(2‑Benzothiazolyl)‑3‑oxobutanamide (3a)
7.18–8.01 (12H, m, ArH), 5.40 (1H, s, C4H-DHP), 2.52
−
1
(
6H, s, CH -DHP); IR (KBr) ν (cm ): 3397.5 (N–H, DHP),
3
1
Pale yellow crystals; Yield 65%; m.p. 214–216 °C; H NMR
2965.1 (C–H, aliphatic), 1623.3 (C=O, amide), 1592.7
(C=C, alkene).
(
DMSO-d ) d (ppm) 11.65 (brs, 1H, NH-amide), 7.86 (d,
6
J = 7.6 Hz, 1H, C4′-H benzothiazole), 7.68 (d, J = 8.0 Hz,
1
H, C7-H benzothiazole), 7.35 (t, J = 8.4 Hz, 1H, C5′-H
2,6‑Dimethyl‑3,5‑bis‑N‑(4‑methyl‑2‑benzothiazolyl)
carbamoyl‑4‑(3‑bromophenyl)‑1,4‑dihydropyridine
(6c)
benzothiazole), 7.20 (t, J = 8.0 Hz, 1H, C6′-H benzothia-
zole), 3.73 (s, 2H, COCH CO), 2.23 (s, 3H, CH CO); IR
2
3
−
1
(
KBr) ν (cm ): 3178.7 (N–H, amide), 3067.5 (C–H, aro-
1
matic), 2917.3 and 2859.2 (C–H, aliphatic), 1719.4 (C=O,
ketone), 1661.3 (C=O, amide).
Yellow precipitate; Yield 45%; H NMR (CDCl ): δ (ppm)
3
11.60 (2H, brs, NH-amide), 6.98–7.58 (10H, m, ArH),
5
.86 (1H, s, NH-DHP), 5.19 (1H, s, C4H-DHP), 2.57 (6H,
s, CH -DHP), 2.35 (6H, s, CH -benzathiazole); IR (KBr)
3
3
−
1
N‑(4‑Methyl‑2‑benzothiazolyl)‑3‑oxobutanamide
ν (cm ): 3313.1 (N–H, DHP), 2923.2 (C–H, aliphatic),
(3b)
1655.4 (C=O, amide), 1529 (C=C, alkene).
Pale yellow precipitate; Yield 80%; m.p. 158–160 °C;
2,6‑Dimethyl‑3,5‑bis‑N‑(4‑methyl‑2‑benzothiazolyl)
carbamoyl‑4‑(4‑chlorophenyl)‑1,4‑dihydropyridine
(6d)
1
H NMR (DMSO-d ): δ (ppm) 12.48 (NH-amide, s, 1H),
6
7
.14–7.85 (ArH, m, 3H), 3.73 (COCH CO, s, 2H), 2.20
2
(
(
CH CO, s, 3H), 2.54 (C4′-CH -benzothiazol, s, 3H); IR
3 3
−
1
1
KBr) ν (cm ): 3167.5 (N–H, amide), 3054.91 (C–H, aro-
Yellow precipitate; Yield 36%; H NMR (CDCl ): δ (ppm)
3
matic), 2982.35 (C–H, aliphatic), 1724.34 (C=O, ketone),
9.85 (2H, brs, NH-amide), 7.07–7.54 (10H, m, ArH), 6.04
1
660.4 (C=O, amide).
(1H, brs, NH-DHP), 5.05 (1H, s, C4H-DHP), 2.52 (6H,
1
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Journal of the Iranian Chemical Society
Table 1 Physical and analytical data of synthesized DHP compounds 6a–d
Comp. code
Chemical structure
Mol. formula (mol. wt.)
Physical
mp (°C)
Yield (%)
38
appearance
6
a
H
N
C H FN O S (555.65)
Yellow
Decompose
29 22
5
2
H
H
N
S
N
S
N
O
O
F
N
H
6
b
C H ClN O S (572.1)
Yellow
Decompose
36
N
29 22
5
2 2
H
N
H
S
N
S
N
O
O
N
Cl
H
6
6
c
C H BrN O S 644.6
Yellow
Yellow
218–220
178–180
45
36
N
31 26
5
2 2
H
N
H
S
N
S
N
O
O
N
Br
H
d
C H ClN O S 600.15
31 26 5 2 2
N
H
N
H
N
S
S
N
O
O
N
Cl
s, CH -DHP), 2.26 (6H, s, CH -benzathiazole); IR (KBr)
rows for each case consisted of only culture media (Muel-
ler–Hinton broth), without bacterial suspension (microplate
1). The positive control rows consisted of culture media
3
3
−
1
ν (cm ): 3313.7 (N–H, DHP), 2921.2 (C–H, aliphatic),
1
658.7 (C=O, amide), 1592.8 (C=C, alkene).
(
Mueller–Hinton broth) and the bacterial suspension (micro-
Biological assessment
plate 12). The positive control rows lacked a test compound.
Ciproꢃoxacin was used as standard. MICs were recorded
after incubating the inoculated microplates at 37 °C for 24 h.
Antibacterial activities of DHP derivatives (6a–d) were
determined through a microdilution broth method according
to CLSI protocol. In this method, the 96-well microplate was
used and the stock solutions of each chemically synthesized
compound were prepared. At the next step, various tested
concentrations of compounds were prepared (1–512 µg/
mL) and loaded into deꢁned rows of 96-well microplates
in sterile microplates 2–11. 100 µL sterile Mueller–Hinton
broth was poured in sterile microplates 2–12 and after add-
ing an appropriate amount of stock solution to microplates
Results and discussion
Crystal structures selected in this study (PDB ID 3FV5)
and (PDB ID 1SFJ) are related to E. coli topoisomerase IV
and S. aureus 3-dehydroquinate dehydratase, respectively.
Topoisomerase IV is essential for unlinking or deactivating
DNA following DNA replication. Topoisomerase IV is also
responsible for relaxing positive supercoils. Topoisomerase
IV is the target for quinolone-based antibacterial compounds
that is able to overset the enzyme into a DNA damaging
agent [22].
2
, using broth, the volume was brought to 200 µL. Twofold
serial dilutions were carried out from microplates 2–11, and
excess broth (100 µL) was discarded from the microplate
8
1
1. 100 µL of standard inoculums (1.5 × 10 CFU/mL) was
added to microplates 2–12 in follow. The negative control
1
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Journal of the Iranian Chemical Society
3
-Dehydroquinate dehydratase is an enzyme which cata-
Table 2 AutoDock 4.2 validation results for bacterial holo PDB
structures (3FV5 and 1SFJ)
lyzes the conversion of 3-dehydroquinate into 3-dehydro-
shikimate in the third step of the shikimate pathway [23].
The shikimate pathway, an essential pathway in many plants,
bacteria and parasites [24], allows plants, fungi and bacteria
for biosynthesis of aromatic amino acids [23], so has become
important in development of herbicides and antimicrobial
agents [24].
PDB code Resolu-
tion (Å)
GA runs Maximum Popula-
RMSD
no. of
energy
evalua-
tions
tion in the from
optimum reference
cluster
(out of
structure
(Å)
1
00)
5
5
3
1
FV5
SFJ
1.80
2.40
50
50
25 × 10
25 × 10
48
50
0.61
1.18
Docking validation
A docking method was validated by re-docking of the co-
crystallographic ligands with the codes of DHK (1SFJ) and
SBVS
1
EU (3FV5). For the docked molecules, top-ranked binding
modes of DHK and 1EU were compared with the original
crystallographic poses (Table 2). On the basis of data, both
of the selected targets could be used in this study since the
RMSD values were found to be lower than 2 Å (0.61 and
Modern drug discovery techniques rely on the VS of chemi-
cal entities to identify hit/lead candidates [25]. Molecules
−
1
with binding energies lower than − 8 kcal mol were
removed from the study. On the basis of this ꢁltration, ꢁfty
molecules were selected. Obtained compounds (in silico
candidates) were subjected to a multi-ꢁltration protocol. The
ꢁltration criteria were chosen as conformational population
of top-ranked cluster and interaction with key residues of
the relevant targets.
1
.18 for 3FV5 and 1SFJ, respectively).
Another important factor is the number of ligand con-
formations in the top-ranked cluster. As it is obvious from
the data, all the top-ranked docking clusters were supported
by high conformational populations. Moreover, top clus-
ters showed good RMSDs with regard to crystallographic
ligands. In the next step, AutoDock parameters that were
used to validate the method (desired docking parameters in
the software) could be applied to VS of chemical structures.
To explain more, those binding energies that were sup-
ported with populated top-ranked clusters and superior
binding interactions passed the filter. On the basis of
this strategy, four molecules (6a–d) were picked up as
Fig. 1 Hierarchical view of
the multi-step VS procedure to
identify antibacterial DHPs
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Journal of the Iranian Chemical Society
Scheme 1 Synthetic route to
DHP derivatives: (i) xylene,
reꢃux, 1–4 h; (ii) EtOH, reꢃux,
2
4–48 h
hit compounds for synthesis and antibacterial assessment
Fig. 1).
and 6b 256 µg/mL) against S. aureus. Compound 6c was the
only compound that exhibited antibacterial activity against
E. coli (256 µg/mL).
(
Chemistry
Structure binding relationship of DHP derivatives
Selected DHP molecules (6a–d) were prepared through the
synthetic route depicted in Scheme 1. Molecular condensa-
tion of N-aryl-acetoacetamides (3a and 3b), aromatic alde-
hydes (4a–c) and ammonium acetate (5) in absolute ethanol
aꢀorded the desired 2,6-dimethyl-4-aryl/heteroaryl-3,5-bis-
N-(aryl/heteroaryl) carbamoyl-1,4 dihydropyridines (6a–d)
in fairly good yields (36–45%) (Table 1).
Binding residues of candidate targets (E. coli topoisomerase
IV 3FV5 and S. aureus 3-dehydroquinate dehydratase 1SFJ)
are summarized in Table 4. Compound 6a did not show any
hydrogen bonds with the residues of topoisomerase IV. This
feature was unique for compound 6a since other candidate
molecules exhibited H bonds in the active site of target. For
more clariꢁcation, estimated binding energies of diꢀerent
ligand–enzyme complexes are also summarized in Table 4.
The NH of the DHP ring participated in hydrogen bond
interactions with oxygen atom of aspartate group in Asp45
Biological evaluation
Compounds 6a, 6b, 6c and 6d were subjected to in vitro anti-
bacterial activity assessment against E. coli and S. aureus.
The utilized method was microdilution broth. Results are
summarized in Table 3.
As shown in Table 3, screened DHP derivatives exhib-
ited variable antibacterial activities against gram-positive
bacteria S. aureus and gram-negative bacteria E. coli. In
the whole series, the MIC (minimum inhibitory concentra-
tion) values of various tested chemical compounds ranged
between 64 and 256 µg/mL against S. aureus. Compound 6d
displayed the highest antibacterial activity against S. aureus
with MIC 64 µg/mL. The remaining compounds showed
medium (6c 128 µg/mL) or weak antibacterial eꢀects (6a
Table 3 In vitro antibacterial
Comp.
Minimum inhibitory
concentration (μg/
mL)
activities of DHP compounds
6
a–d in microdilution method
S. aureus
E. coli
6
6
6
6
a
b
c
256
256
128
64
–
–
256
–
d
1
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Journal of the Iranian Chemical Society
Table 4 Interaction energies and H-bond/hydrophobic contact characteristics of virtual screened hits within E. coli topoisomerase IV (3FV5)
and S. aureus 3-dehydroquinate dehydratase (1SFJ)
Comp. Target Estimated
No
Involved amino acid Involved
H-bond Involved residues (hydrophobic interaction)
ΔG (kcal/
of H
bonds
atom of
ligand
length
(Å)
b
mol)
6a
6b
6c
6d
6a
6b
3FV5
3FV5
− 8.65
− 8.72
0
2
1
2
1
3
–
–
–
Val165, Ser43, Val39, Asn42, Gln46, Pro75, Ile90,
Arg93, Asp45, Met74, Thr163, Asp69, Val67
Arg72….NH
O13
N6
N6
2.96
3.04
3.51
His79, Asp77, Pro75, Arg132, Met74, Asp69, Glu46,
2
Asp45….OD2
Asn42….OD1
Thr163, Gly73, Arg72, Ile78
3FV5 − 10
Asp45, Asn42, Met74, Ile90, 72, Gly73, Ile90, Ala86,
Leu89, Pro75, Thr163, Asn42, Glu136, Ala86,
3FV5
1SFJ
1SFJ
− 8.22
Arg72….NH
O13
N6
N6
2.92
3.09
3.04
Met74, Leu89, Asn42, Ile90, Ile116, Arg93, Arg132,
2
Asp45….OD2
Tyr77….OH
Glu46, Gly73, Thr163
− 10.11
− 10.23
Pro220, Gly224, Phe135, Gln221, Pro223, Glu136,
Lys72, Ala222
Tyr77….OH
Tyr77….OH
Gly75….O
N20
N14
N25
2.88
2.97
3.26
Arg37, Lys72, Pro220, Ala222, Phe135, Thr11, Gln221,
Pro223, Tyr77, Gly75
6
6
c
1SFJ
1SFJ
− 10.30
− 10.67
3
2
Tyr77….OH
Gln221….O
Gly75….O
N6
2.79
2.57
3.14
Lys72, Tyr77, Gln221, Gly75, Thr9, Ala222, Gln225,
Lys160, Arg37, His133, Arg70, Phe135, Pro223,
Glu136, Leu73
N20
N14
d
Glu136….OE2
N20
3.20
Gln221, Thr11, Arg37, Gly75, Lys72, Leu73, Asn134,
Tyr77….OH
N25
2.91
His133, Glu136, Phe135, Tyr77, Pro223
(
compounds 6b and 6d) and oxygen atom of amide carbonyl
above, the most potent compound 6d against S. aureus as
gram-positive bacteria showed signiꢁcant hydrophobic con-
tacts in the active site if target.
in the side chain of Asn42 (compound 6c).
Interacted residues of topoisomerase showed that varia-
tion of substituents in the C3 and C5 of DHP ring might sig-
niꢁcantly change the binding pattern of candidate molecules.
This could be obvious from the data of Table 4. Relatively
diꢀerent hydrophobic residues were found to make contacts
with DHP scaꢀolds. Analysis of data indicated that Met74
and Thr163 interacted with all of the DHP structures and
hence seemed important hydrophobic participants.
Conclusion
A multi-step VS methodology, chemical synthesis and
in vitro antibacterial assessment were applied to identify
potential antibacterial scaꢀolds with 3,5-bis-N-aryl car-
bamoylated-4-aryl-1,4-dihydropyridine structure. Results
showed that the most potent in vitro hit could potentiality
inhibit the growth of gram-positive bacteria S. aureus (MIC
64 μg/mL). On the basis of obtained results, 4-methyl ben-
zothiazole-3,5-bis-N-aryl carbamoylated-4-aryl-1,4-dihydro-
pyridine structure may provide the opportunity to further
development into more potent anti S. aureus agents. Results
of such studies may be important since there is an urgent
requirement to build up bioactive compounds that trigger
several targets within a pathogen system.
In the case of compound 6d, two nitrogen atoms of ben-
zothiazole ring participated in hydrogen bond interactions
with Tyr77 and Glu136 residues of S. aureus 3-dehydro-
quinate dehydratase target (1SFJ). Tyr77 was involved in
H-bond interaction with DHP nitrogen in compounds 6a and
6
c. Compound 6d made hydrophobic contact with Asn134
and such interaction could not be detected for other com-
pounds under study. When comparing compounds 6b (MIC
2
56 μg/mL) and 6c (MIC 128 μg/mL), it was seen that both
of the molecules made three hydrogen bonds with the active
site of the target, while more hydrophobic residues were
involved in binding to compound 6c. Lower antibacterial
eꢀects of compounds 6a and 6b might be in accordance with
less hydrophobic contacts through aromatic rings. Moreover,
it was revealed that the halogen substituent of compound 6d
did not participate in any loose contacts.
Funding Funding was provided by Ferdowsi University of Mashhad
(
Grand No. Research Project No. 3/32040 - 1393/07/19).
References
Results of the present study might be further exploited.
Candidate compounds against gram-negative bacteria must
have an acceptable polarity, while such rationalization is not
signiꢁcant for gram-positive bacteria. As it was explained
1
2
.
.
M.L. Cohen, Nature 406, 762 (2000)
H. Mahdavi, O. Rahmani, A.R. Shaverdi, J. Iran. Chem. Soc. 14,
37 (2017)
1
3

Journal of the Iranian Chemical Society
3
.
S. Mandal, M. Moudgil, S.K. Mandal, Eur. J. Pharmacol. 625, 90
14. N.C. Desai, A.R. Trivedi, H.C. Somani, K.A. Bhatt, Chem. Biol.
Drug Des. 86, 370 (2015)
(
2009)
4
5
.
.
D. Horvath, J. Med. Chem. 40, 2412 (1997)
15. R. Surendra Kumar, A. Idhayadhulla, A. Jamal Abdul Nasser, S.
Kavimani, S. Indumathy, Indian J. Pharm. Sci. 72(6), 719 (2010)
16. N. Rao, K. Lakshimi, Sci. Rev. Chem. Commun. 3(3), 141 (2013)
17. P.K. Mithlesh, P.K. Pareek, K. Ravikant, K.G. Ojha, Der. Pharma.
Chem. 3(2), 66 (2011)
Y. Biran, C.L. Masters, K.J. Barnham, A.I. Bush, P.A. Adlard, J.
Cell Mol. Med. 13, 61 (2009)
6
7
.
.
T. Tomašić, A. Kovač, G. Klebe, D. Blanot, S. Gobec, D. Kikelj,
L.P. Mašič, J. Mol. Model 18, 1063 (2012)
A. Srinivas Reddy, S. Priyadarshini Pati, P. Praveen Kumar, H.N.
Pradeep, G. Narahari Sastry, Curr. Protein Pept. Sci. 8, 329 (2007)
C.N. Cavasotto, Curr. Top. Med. Chem. 11, 1528 (2011)
H. Azizian, F. Nabati, A. Shariꢁ, F. Siavoshi, M. Mahdavi, M.
Amanlou, J. Mol. Model 18, 2917 (2012)
18. T. Akbarzadeh, A. Fallah Tafti, N. Samadi, A. Foroumadi, M.
Amanlou, M.A. Faramai, A. Shaꢁee, DARU 18(2), 118 (2010)
19. G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew,
D.S. Goodsell, A.J. Olson, J. Comput. Chem. 30, 2785 (2009)
20. A.C. Wallace, R.A. Laskowski, J.M. Thornton, Protein Eng. 8,
127 (1995)
8
9
.
.
1
0. P.J. Ballester, M. Mangold, N.I. Howard, R.L. Robinson, C. Abell,
J. Blumberger, J.B. Mitchell, J.R. Soc, Interface 9(77), 3196
21. R.J. Clemens, J.A. Hyatt, J. Org. Chem. 50, 2431 (1985)
22. J. Kato, H. Suzuki, H. Ikeda, J. Biol. Chem. 267, 25676 (1992)
23. K.M. Herrmann, Plant Physiol. 107, 7 (1995)
(
2012)
1
1
1
1. Z. Yu, L. Song, L. Tong, Y. Wang, W. Zheng, X. Gong, Wei Sheng
Wu Xue Bao 56(2), 219 (2016)
24. K.M. Herrmann, Plant Cell 7, 907 (1995)
2. M. Katoh, M. Nakajima, H. Yamazaki, T. Yokoi, Pharm. Res.
25. J. Jiaranaikulwanitch, C. Boonyarat, V.V. Fokin, O. Vajragupta,
Bioorg. Med. Chem. Lett. 20, 6572 (2010)
1
7(10), 1189 (2000)
3. I. Sabakhi, V. Topuzyan, Z. Hajimahdi, B. Daraei, H. Areꢁ, A.
Zarghi, Des. IJPR 14(4), 1087 (2015)
1
3