Design, synthesis, in-silico studies and biological screening of quinazolinone analogues as potential antibacterial agents against MRSA

Article abstract of DOI:10.1016/j.bmc.2019.05.012

Type or The emergence of resistance to antibiotic has developed a complicated situation in the treatment of bacterial infections. Considering the antimicrobial resistance phenomenon as one of the greatest challenge of medicinal chemists for search of better anti-bacterial agents, which have potential narrow spectrum activity with low development of resistance potential and low toxicity to host. Cross-linking of peptidoglycan is a key step catalyze by Penicillin binding protein (PBP) to maintain integrity of cell wall in bacterial cell. However, these Penicillin binding protein (PBP) has developed resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to acquisition of additional PBP2a. Various Quinazolinone analogues are reported in literature as potential anti-bacterial agents against MRSA. In present study new quinazolinone analogues has been designed, guided by molecular docking, In-silico and MM-GBSA study. Newly designed molecules have been synthesized by medicinal chemistry route and their characterization was done by using IR, NMR, & HR-MS techniques. Biological evaluation of synthesized compounds has been done on wild type Gram-negative (Escherichia coli), Gram-positive (Staphylococcus aureus) and resistant MRSA bacterial strains using Streptomycin, Kanamycin and Linezolid as standard drugs respectively. The in vitro evaluation results have shown that compound 5f is active with MIC value 15.625 μg/mL against S. aureus and with MIC value 31.25 μg/mL against MRSA.

Full text of DOI:10.1016/j.bmc.2019.05.012

Accepted Manuscript
Design, synthesis, in-silico studies and biological screening of Quinazolinone
analogues as potential antibacterial agents against MRSA
Shahnawaz I. Qureshi, Hemchandra K. Chaudhari
PII:
S0968-0896(19)30528-0
https://doi.org/10.1016/j.bmc.2019.05.012
BMC 14899
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 March 2019
6 May 2019
7 May 2019
Please cite this article as: Qureshi, S.I., Chaudhari, H.K., Design, synthesis, in-silico studies and biological screening
of Quinazolinone analogues as potential antibacterial agents against MRSA, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.05.012
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Design, synthesis, in-silico studies and
biological screening of Quinazolinone
analogues as potential antibacterial agents
against MRSA
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Shahnawaz I. Qureshi, Hemchandra K. Chaudhari*
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg,
Matunga (E), Mumbai, Maharashtra, India. 400019

Bioorganic & Medicinal Chemistry
Design, synthesis, in-silico studies and biological screening of Quinazolinone
analogues as potential antibacterial agents against MRSA
Shahnawaz I. Qureshi, Hemchandra K. Chaudhari*
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga (E), Mumbai, Maharashtra, India. 400019
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Type or The emergence of resistance to antibiotic has developed a complicated situation in the
treatment of bacterial infections. Considering the antimicrobial resistance phenomenon as one of
the greatest challenge of medicinal chemists for search of better anti-bacterial agents, which
have potential narrow spectrum activity with low development of resistance potential and low
toxicity to host. Cross-linking of peptidoglycan is a key step catalyze by Penicillin binding
protein (PBP) to maintain integrity of cell wall in bacterial cell. However, these Penicillin
binding protein (PBP) has developed resistance in methicillin-resistant Staphylococcus aureus
(MRSA) due to acquisition of additional PBP2a. Various Quinazolinone analogues are reported
in literature as potential anti-bacterial agents against MRSA. In present study new quinazolinone
analogues has been designed, guided by molecular docking, In-silico and MM-GBSA study.
Newly designed molecules have been synthesized by medicinal chemistry route and their
characterization was done by using IR, NMR, & HR-MS techniques. Biological evaluation of
synthesized compounds has been done on wild type Gram-negative (Escherichia coli), Gram-
positive (Staphylococcus aureus) and resistant MRSA bacterial strains using Streptomycin,
Kanamycin and Linezolid as standard drugs respectively. The in vitro evaluation results have
shown that compound 5f is active with MIC value 15.625 μg/mL against S. aureus and with
MIC value 31.25 μg/mL against MRSA.
Available online
Keywords:
Penicillin binding protein 2a
Antibacterial
Microbial resistance
Docking
MM-GBSA
2009 Elsevier Ltd. All rights reserved.
1. Introduction
to acquisition of additional penicillin binding protein (PBP),
designated PBP2a. 5
Antibiotics are among the most frequently prescribed
medications today although microbial resistance due to
evolutionary pressures and misuses threatens their continued
efficacy. Antimicrobial resistance is one of the most serious
health threats. Bacterial resistance to antibiotics is growing up
day by day in both communities as well as in hospitals.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of
the most widespread and virulent nosocomial pathogens.1
According to the report of Centers for diseases control and
prevention (CDC) of March 2019, it was estimated that nearly
1,19,000 noninvasive Methicillin Resistant Staphylococcus
aureus (MRSA) infection involving both healthcare and
community associated infection, causing more than 20,000
deaths in the year 2017.2 The microorganisms mainly involved in
the resistance process, were called the ESKAPE pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacterbaumanii, Pseudomonas aeruginosa,
and enterobacteriaceae). Among these organisms, Methicillin
Resistant Staphylococcus aureus (MRSA) alone accounts for
most of the death in US than of HIV/AIDS and tuberculosis
combined.3 During the past decades, β-lactam antibiotics were
the choice of drugs for S. aureus infections. However, these
agents become ineffective after the emergence of MRSA.4
MRSA resistance to β-lactam class of antibiotics developed due
Newer analogs of β-lactam, Cephalosporin antibiotics such as
ceftaroline and ceftobiprole are FDA (Food and drug
Administration) approved drugs to show anti-MRSA activities.
However, they are not orally bioavailable require intravenous
infusion after every 8-12 hrs.6 The MRSA resistant enzyme has a
closed active site regulated by allosteric mechanism hence, β-
lactam not able to inhibit MRSA.7 Researchers have shown that
ceftaroline has an ability to bind non-covalently to the allosteric
site at a distance of 60 Å apart from the active site. This binding
leads to conformational changes which results in the opening of
the active site, and then the another molecule of ceftaroline bind
to active site and cause inhibition.8 Resistance to this antibiotic
has already emerged by disruption of allosteric mechanism.9
Other antibiotics has been introduced such as oxazolidinones,
linezolid and tedizolid for MRSA strains, however resistance has
been already emerged for all these antibiotics.10, 11
There exists an urgent need of novel class of antibiotic for
MRSA infections. Researchers has recently reported a new class
of antibiotic 4-(3H)-Quinazolinones. The Quinazolinone class of
antibacterial is of particular importance; because they do not β-
lactam ring but able to inhibit PBP’s. This Quinazolinone class of
antibacterial has mechanism of action similar to ceftaroline, first
they bind non-covalently to allosteric site which then result in

opening of active site and then another molecule of
Quinazolinone bind to active site and cause cell wall inhibition.12
Allosteric site: Allosteric site was defined by removing co-
crystallized ligand and generating the grid box at the centroid of
the workspace ligand as selected in the receptor folder. The grid
was generated by applying van der waals radii of 1.0 Å with the
partial atomic charge less than 0.25 defaults. The ligands similar
in size to the workspace ligand were allowed to dock into the
active site. No constraints such as Positional, H-bonding or
Hydrophobic were defined.
Literature survey provides various highly active
Quinazolinone antibacterial showed several folds higher activity
towards the enzyme as compared to standard drugs (Table 1).13
An interest was created to study the structural features of these
compounds responsible for their high activity against MRSA
strains which may be helpful in designing new analogues.
2.1.3. Active site: No co-crystallized ligand is present at active
site. The grid box was generated around the active site for
docking by selecting following residues Pro401, Gly402, Ser403,
Lys406, Tyr446, Ser461, Ser462, Asp463, Asn464, Ile465,
Phe466, Tyr519, Gly520, Gln521, Ser598, Gly599, Thr600,
Ala601, Glu602, Leu603, Arg612, Gln613, Ile614, Ala642.15
In our paper, we are now reporting a modeling study for
design and development of Quinazolinone analogues effective
against MRSA strains. These studies can provide useful
information for the design of new analogues of Quinazolinone
antibacterial with improved activity. Our study includes Structure
based design approach docking study using Glide. A reported
crystal structure of S. aureus PBP2a in complex with
Quinazolinone (PDB ID-4CJN) were obtained from protein data
bank and performed docking study. Docking procedures were
used to identify the correct conformation of ligands in the
binding pocket of a protein and to predict the interaction between
the ligand and the protein. Docking is one method in which the
binding of ligand to a receptor can be explored. Furthermore,
Prime MM-GBSA of docked complex has been performed to
determine binding free energy. We have also performed
Adsorption Distribution, Metabolism and Excretion (ADME)
prediction studies. All these studies have been used to design
new molecules which then synthesized by appropriate synthetic
route and tested against bacterial strains for anti-bacterial
activity.
2.1.4. Ligand Docking
OPLS 2005 force field was used during docking. The receptor
grid generated previously was selected for the docking of ligands
prepared using Lig prep. Standard Precision (SP) feature was
used to perform flexible docking. The van der waals radii were
scaled to 0.80 default and default partial cutoff charge of 0.15 to
decrease the penalties for close contacts. No constraints were set
to defined ligand-receptor interactions. The structure output form
was set to pose viewer file so as to view the output of the
resulting docking studies from pose-viewer.
2.1.5. Viewing Docking Results
Pose-Viewer was used to see docking results. The H-bonding and
salt bridge interaction between ligands and receptor were
visualized using default settings to analyze the binding modes of
the ligands to receptor.
2. Materials and Methods
2.1 Docking studies
No constraints were set to defined ligand-receptor interactions.
Computer model score system that encompasses the grid score,
proprietary GLIDE score, and the internal energy strain were
used to rank the final ligand binding poses. The score function of
Glide, or Glide score, was used for binding affinity prediction
and ligand ranking.14
Glide module (version 7.1, Schrödinger, LLC, NY) were
used to perform the docking studies installed on Redhat Linux
workstation.14 Docking is a process by which the ligand or the
inhibitor binds to the receptor site of target protein and they fit
together in a 3D space. Docking studies involves the posing,
ranking or scoring. Posing involve determination of conformation
and orientation of ligands fit at the receptor site. While ranking or
scoring involve the estimation of binding free energy when
ligand bind to active site which then rank the ligands based on
their docking score. Docking study comprises of five steps ligand
preparation, protein preparation, receptor grid generation, actual
docking procedure and finally viewing the docking results using
the pose-viewer.
2.2. ADME predictions by QikProp
Almost half of proposed drug fail during the development
process, in spite of good efficacy and favorable toxicity profile
due to inappropriate pharmacokinetic profile. Therefore, now a
days medicinal chemist integrates ADME prediction into drug
design and lead optimization strategies. Absorption, Distribution,
Metabolism, and Excretion (ADME) prediction software
QikProp (version 4.3, Schrödinger, LLC, NY) is a quick,
accurate program designed by Professor William L. Jorgensen.
QikProp predicts pharmaceutically important properties of
organic molecules. In addition to predicting molecular properties,
QikProp also provides a range for comparing a particular
molecule’s properties with those of 95% of known drugs.
QikProp also flags 30 types of reactive functional groups that
may cause false positives in high-throughput screening (HTS)
assays.16
2.2.1 Ligand & Protein Preparation
As described previously all the ligands for docking studies
were prepared by using Lig prep module. Protein preparation
was performed by using Protein Preparation Wizard of Maestro
software. Crystal structure of S. aureus PBP2a in complex with
Quinazolinone (PDB ID-4CJN at 1.95 Å resolution) was taken
from protein data bank and then the following modification done
on protein structure such as add missing hydrogen, assign proper
bond orders, treat metal (i.e., delete bonds to metal and adjust the
formal charge on metal and neighboring atoms), and to delete
water molecules that are more than 5 Å from the heterogeneous
groups. The H bonds were optimized using sample orientations.
All the polar hydrogen was displayed. Finally, the protein
structure was minimized to the default Root Mean Square
Deviation (RMSD) value of 0.30. The protein structure was
minimized using OPLS 2005 force field. The protein structure
thus prepared is ready for docking.
2.3.
MM-GBSA free energy calculations
Molecular Mechanics-Generalized born surface area (MM-
GBSA) calculates binding free energies for molecules by
combining molecular mechanics calculations and continuum
(implicit) solvation models. Molecular mechanics estimates the
enthalpy contributions for protein-ligand interaction, whereas
implicit solvents models were used to estimate free energies of
solute solvent interaction.
2.1.2. Receptor Grid Generation
The docked poses were minimized using the local optimization
feature in Prime and also the ligand strain energies. Energies of

the ligand enzyme complexes were calculated using Prime MM-
GBSA technology with all the enzyme residues being held
frozen.17
The binding free energy ΔGbind was estimated by equation
below:
ΔGbind
=
Ecomplex(minimized)
-
[Eligand
(unbound,minimized) + Ereceptor (unbound, minimized) ]
Where ΔGbind is the calculated relative free energy that includes
both ligand and receptor strain energy. Ecomplex(minimized) is
the MM/GBSA energy of the minimized complex, and Eligand
(unbound, minimized) is the MM/GBSA energy of the ligand
after removing it from the complex and allowing it to relax.
Ereceptor (unbound, minimized) is the MM/GBSA energy of
protein after separating it from the ligand.18
3. Experimental
3.1 Chemicals
All solvents used were of laboratory grade and were obtained
from research gate. Anthranilic acid, triethyl ortho-acetate, 3-
nitro aniline, benzoic acid derivatives, benzaldehyde derivatives
were commercially available and were obtained from Avra Pvt
ltd, and SD fine chemicals.
3. 2. Instrumentation
Melting points were determined on Buchi B-545 melting point
apparatus. Compounds were routinely checked for purity in
Silica Gel Thin Layer Chromatography (TLC) plates and UV
lamp was used for visualization of spots. The IR spectra were
recorded in KBr pellets on “Perkin Elmer FTIR
Spectrophotometer” and “Shimadzu FTIR-Spectrophotometer”.
1H & 13C NMR spectra were recorded on 400 MHz NMR
spectrometer in CDCl3 using tetramethylsilane (TMS) as an
internal standard. Mass spectra were recorded on Agilent 5975
MSD.
Scheme 1 Synthesis route for designed Quinazolinone ligands.
3.4. Chemistry
3.4.1 General procedures for preparation of compounds (1a-e)
Carboxylic acid (1.5 eq.) is added to amine (1 eq.) and
trimethylamine (TEA) (3 eq.) in dichloromethane (DCM), then
1.5 eq. of thionyl chloride (SOCl2) is added dropwise at room
temperature. The mixture is stirred overnight at room
temperature. After completion of reaction as monitored by thin
layer chromatography (TLC), the recovery of the reaction
product is performed by evaporating the solvent under reduced
pressure. The resulting residue is then taken up in ethyl acetate
and washed first with 1N HCl and then with 1N NaOH. The
organic phase was dried over anhydrous sodium sulphate
(Na2SO4), and evaporated to dryness under vaccum.20
3.3. Antimicrobial Evaluation
Resazurin Microtitre (REMA) Assay, a colorimetric method for
determining the ability of drugs/compounds to inhibit viability of
bacteria cell is based on reduction of a dye resazurin. Viability of
bacterial cells is detected by change in color of the resazurin dye,
and the intensity of colour is directly proportional to number of
viable bacteria cell present in medium. Resazurin (7-hydroxy-3-
oxido-3H-phenoxazine 10-oxide) is a blue dye, it is non-
fluorescent in nature, viable bacterial cells irreversibly reduced
this dye to pink colored and highly fluorescent end product
resorufin. Non-viable cells rapidly loose metabolic capacity and
thus not able to generate a fluorescent signal. Resazurin dye is a
redox indicator used in cell viability assays for bacteria and
mammalian cells etc. Usually it is available commercially as the
sodium salt.19
N-(3-nitrophenyl) benzamide (1a). TLC carried out in Hexane:
Ethyl acetate (80:20) Rf= 0.37; Molecular formula: C13H10N2O3,
Nature: light yellow solid, Yield: 60%, M.P: 153-154°C (lit. MP:
152-153°C)21, IR: 3360cm-1 (N-H stretch), 1660cm-1 (C=O
amide), 1523cm-1 (C-NO2).
2-methyl-N-(3-nitrophenyl) benzamide (1b). TLC carried out in
Hexane: Ethyl acetate (80:20) Rf= 0.454; Molecular formula:
C14H12N2O3, Nature: light yellow solid, Yield: 65%, M.P: 140-
14°C, IR: 3287cm-1 (N-H stretch), 2925cm-1 (alkane C-H
stretch), 1654cm-1 (C=O amide), 1527cm-1 (C-NO2).
2-(4-methoxyphenyl)-N-(3-nitrophenyl) acetamide (1c). TLC
carried out in Hexane: Ethyl acetate (80:20) Rf= 0.5; Molecular
formula: C15H14N2O3, Nature: yellow solid, Yield: 70%, M.P:
162-163°C, IR: 3243cm-1 (N-H stretch), 1661cm-1 (C=O amide),
1524cm-1 (C-NO2).
3,4-dimethoxy-N-(3-nitrophenyl) benzamide (1d). TLC carried
out in Hexane: Ethyl acetate (80:20) Rf= 0.416; Molecular
formula: C15H14N2O5, Nature: light yellow solid, Yield: 60%,

M.P: 159-160°C, IR: 3322 cm-1 (N-H stretch), 1650 cm-1 (C=O
amide), 1510 cm-1 (C-NO2).
3.4.4. General procedures for preparation of compounds (4a-e):
Compound (1a-e) (1eq.) and compound (2a-e) (1eq.) were
suspended in 5 mL of glacial acetic acid. The suspension
dissolved completely upon heating. The reaction was refluxed for
15-30min. After completion of reaction (as monitored by TLC),
reaction mixture was allowed to cooled, and then 10 mL of water
was added. The resulting precipitate was then filtered and was
washed with water, cold ethanol, and hexanes to give the
product.25
N-(3-nitrophenyl) thiophene-2-carboxamide (1e). TLC carried
out in Hexane: Ethyl acetate (80:20) Rf= 0.4; Molecular formula:
C11H8N2O3S, Nature: light brown solid, Yield: 55%, M.P: 154-
155°C, IR: 3379 cm-1 (N-H stretch), 1658 cm-1 (C=O amide),
1527 cm-1 (C-NO2).
3.4.2 General procedures for preparation of compounds (2a-e):
N-(3-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)
benzamide
Dissolve 1eq. of nitro compound in 10mL of Ethanol, 0.5mL of
water and then iron powder (3eq.) and Calcium chloride (CaCl2)
(1eq.) were added to the reaction mixture. The resulting reaction
mixture was allowed to reflux at 60°C for 4-5 h. Progress of the
reaction was monitored by TLC. Spots on TLC plates were
visualized under UV lamp or by staining it with 0.2% ninhydrin
in ethanol solution and charring after elution. After completion,
the reaction mixture was filtered to remove the iron residues; the
recovery of the reaction product is performed by evaporating the
solvent under reduced pressure. The resulting residue is then
taken up in ethyl acetate. The organic extracts were washed with
H2O (3 × 10 mL), brine (2 × 10 mL), and dried over Na2SO4, the
organic phase was evaporated. Purification of the compound has
been performed by Silica column chromatography using Hexane:
Ethyl acetate (80:20) as eluting solvent.22
(4a): TLC carried out in Hexane: Ethyl acetate (60:40) Rf= 0.13;
cream color solid (70%); M.P: 248-249°C; IR: 3290cm-1 (N-H
stretch), 1658cm-1 (C=O amide); HRMS: m/z calcd for
C22H17N3O2: 355.39 [M+H]+; found: 356.1565.
2-methyl-N-(3-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)
benzamide (4b). TLC carried out in Hexane: Ethyl acetate
(60:40) Rf= 0.192; cream color solid (70%); M.P: 239-240oC; 1H
NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H),
7.81 – 7.72 (m, 2H), 7.65 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.0 Hz,
1H), 7.44 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 8.0 Hz, 1H), 7.21 (d, J =
7.4 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H),
6.81 (d, J = 7.7 Hz, 1H), 2.47 (s, 3H), 2.27 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 168.24 (s), 162.42 (s), 154.09 (s), 147.44
(s), 140.09 (s), 137.88 (s), 136.44 (s), 135.82 (s), 134.72 (s),
131.09 (s), 130.21 (s), 130.17 (s), 126.93 (s), 126.88 (s), 126.79
(s), 126.63 (s), 125.60 (s), 123.13 (s), 120.52 (s), 120.45 (s),
119.53 (s), 30.93 (s), 24.32 (s); IR: 3308 (N-H stretch), 2970
(alkane C-H stretch), 1661 (C=O amide) cm-1; HRMS: m/z calcd
for C23H19N3O2: 369.42 [M+H]+; found: 370.1740.
N-(3-aminophenyl) benzamide (2a). TLC carried out in Hexane:
Ethyl acetate (70:30) Rf= 0.136; Molecular formula: C13H12N2O,
Nature: light brown solid, Yield: 80%, M.P: 119-120°C (lit. MP:
124°C)23, IR: 3435cm-1 and 3346cm-1 (NH2 stretch), 1654cm-1
(C=O amide).
2-(4-methoxyphenyl)-N-(3-(2-methyl-4-oxoquinazolin-3(4H)-yl)
phenyl) acetamide (4c). TLC carried out in Hexane: Ethyl acetate
(50:50) Rf= 0.088; Nature: cream color solid (70%); M.P: 202-
203°C; IR: 3278cm-1 (N-H stretch), 2933 cm-1 (C-H stretch,
alkane) 1656cm-1 (C=O amide); HRMS: m/z calcd for
C24H21N3O3: 399.16 [M+H]+; found: 399.8000.
N-(3-aminophenyl)-2-methyl benzamide (2b). TLC carried out in
Hexane: Ethyl acetate (70:30) Rf= 0.16; Molecular formula:
C14H14N2O, Nature: light brown solid, Yield: 80%, M.P: 135-
136°C, IR: 3435cm-1 and 3350cm-1 (NH2 stretch), 2925cm-1
(alkane C-H stretch), 1644cm-1 (C=O amide).
N-(3-aminophenyl)-2-(4-methoxyphenyl) acetamide (2c).TLC
carried out in Hexane: Ethyl acetate (70:30) Rf= 0.12; Molecular
formula: C15H16N2O2, Nature: light brown solid, Yield: 75%,
M.P: 122-123oC, IR: 3435cm-1 and 3346cm-1 (NH2 stretch),
2926cm-1 (alkane C-H stretch), 1651cm-1 (C=O amide).
3,4-dimethoxy-N-(3-(2-methyl-4-oxoquinazolin-3(4H)-
yl)phenyl) benzamide (4d). TLC carried out in Hexane: Ethyl
acetate (50:50) Rf= 0.115; Nature: cream color solid (70%); M.P:
246-247°C; IR: 3268cm-1 (N-H stretch), 1655 cm-1 (C=O amide);
HRMS: m/z calcd for C24H21N3O4: 415.15 [M+H]+; found:
415.8000.
N-(3-aminophenyl)-3,4-dimethoxybenzamide (2d). TLC carried
out in Hexane: Ethyl acetate (70:30) Rf= 0.096; Molecular
formula: C15H16N2O3, Nature: brown solid, Yield: 70%, M.P:
131-132°C, IR: 3371 cm-1 and 3289 cm-1 (NH2 stretch), 2841 cm-
1 (alkane C-H stretch), 1649cm-1 (C=O amide).
N-(3-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)thiophene-2-
carboxamide (4e). TLC carried out in Hexane: Ethyl acetate
(60:40) Rf= 0.157; Nature: cream color solid (70%); M.P: 279-
280°C; IR: 3340 cm-1 (N-H stretch), 3078 cm-1 (alkane C-H
stretch), 1666 cm-1 (C=O amide); HRMS: m/z calcd for
C20H15N3O2S: 360.12 [M+H]+; found: 361.9000.
N-(3-aminophenyl) thiophene-2-carboxamide (2e). TLC carried
out in Hexane: Ethyl acetate (70:30) Rf= 0.12; Molecular
formula: C11H10N2OS, Nature: brown solid, Yield: 70%, M.P:
132°C, IR: 32296 cm-1 and 3194cm-1 (NH2 stretch), 1662cm-1
(C=O amide).
3.4.5. General procedures for preparation of compounds (5a-j):
Compound (4a-e) (1eq.) was suspended in 5 mL of glacial acetic
acid and dissolved upon heating, to which substituted
benzaldehyde (1eq.) was added. The reaction was then refluxed
for 24-48 h. Progress of the reaction monitored by TLC. After
completion of reaction, 10 mL of water was added to the cooled
reaction mixture. The resulting precipitate was filtered and
washed with water followed by cold ethanol and hexanes to give
the product. Purification of the products has been performed by
Silica column chromatography using Hexane: Ethyl acetate
(70:30) as eluting solvent.26
3.4.3. General procedures for preparation of compound (3): A
stirred mixture of anthranilic acid (1eq.) and triethyl ortho-acetate
(2eq.) was heated under reflux for 15-20 min. Progress of the
reaction was monitored by TLC. After completion of reaction (as
monitored by TLC), the reaction mixture was allowed to cooled
to 0 °C and the precipitate thus obtained was filtered off and
recrystallized from hexane to give white to yellow needles like
crystals.24
(E)-N-(3-(2-(4-fluorostyryl)-4-oxoquinazolin-3(4H)-
2-methyl-4H-benzo[d][1,3]oxazin-4-one (3).TLC carried out in
Hexane: Ethyl acetate (90:10) Rf= 0.481; Molecular formula:
C9H7NO2, Nature: light brown solid, Yield: 75%, M.P: 80°C, IR:
2970cm-1 (alkane C-H stretch), 1738cm-1 (lactone C=O),
yl)phenyl)benzamide (5a). TLC carried out in Hexane: Ethyl
acetate (60:40) Rf= 0.260; Nature: yellow solid, Yield: 60%,
M.P: 267-268°C, IR: 3345cm-1 (N-H stretch), 1664cm-1 (C=O
amide), 1602cm-1 (alkene C=C stretch); HRMS: m/z calcd for
C29H20FN3O2: 461.49 [M+H]+; found: 462.1791.

(E)-N-(3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-
2H); 13C NMR (101 MHz, CDCl3) δ: 169.86, 162.81, 158.85,
153.09, 151.33, 147.87, 140.27, 139.06, 136.99, 134.87, 131.39,
131.35, 130.46, 130.31, 129.76, 129.67, 127.47, 127.04, 126.71,
126.46, 123.29, 120.77, 120.56, 119.59, 119.22, 119.18, 115.98,
115.76, 114.29, 55.23, 43.57; IR: 3276cm-1 (N-H stretch),
3070cm-1 (alkene C=C-H stretch), 2929cm-1 (alkane C-H stretch),
1656cm-1 (C=O amide), 1601cm-1 (alkene C=C stretch), 808cm-1
(C-Cl stretch); HRMS: m/z calcd for C31H24ClN3O3: found:
[M+H]+: 521.7, [M+H]++:523.7.
yl)phenyl)benzamide (5b). TLC carried out in Hexane: Ethyl
acetate (60:40) Rf= 0.32; Nature: light yellow solid, Yield: 50%,
M.P: 290°C, 1H NMR (400 MHz, CDCl3) δ 9.48 (s, 1H), 8.21 (d,
J = 8.6 Hz, 1H), 7.87 (d, J = 14.1 Hz, 1H), 7.85 (d, J = 5.3 Hz,
4H), 7.73 (d, J = 5.1 Hz, 2H), 7.50 (d, J = 7.7 Hz, 2H), 7.43 (d, J
= 7.9 Hz, 3H), 7.36 (dd, J = 10.3, 7.2 Hz, 4H), 6.91 (d, J = 7.8
Hz, 1H), 6.52 (d, J = 15.5 Hz, 1H); IR: 3330cm-1 (N-H stretch),
2223cm-1 (CN stretch), 1658cm-1 (C=O amide), 1604cm-1 (alkene
C=C stretch), HRMS: m/z calcd for C30H20N4O2: 468.25 [M+H]+;
found: 469.2000.
(E)-N-(3-(2-(4-fluorostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
3,4-dimethoxybenzamide (5g). TLC carried out in Hexane: Ethyl
acetate (50:50) Rf= 0.260; cream color solid (50%); M.P: 239-
(E)-N-(3-(2-(4-fluorostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
2-methyl benzamide (5c). TLC carried out in Hexane: Ethyl
acetate (60:40) Rf= 0.348; cream color solid, Yield: 60%, M.P:
1
240°C; H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.29 (d, J =
7.5 Hz, 1H), 7.92 (d, J = 11.8 Hz, 1H), 7.89 (s, 1H), 7.78 (d, J =
5.0 Hz, 2H), 7.62 (s, 1H), 7.52 – 7.40 (m, 3H), 7.37 (t, J = 8.0
Hz, 1H), 7.29 (dd, J = 8.4, 5.4 Hz, 2H), 6.96 (t, J = 8.5 Hz, 2H),
6.75 (dd, J = 16.2, 7.9 Hz, 2H), 6.33 (d, J = 15.5 Hz, 1H), 3.85 (s,
3H), 3.85 (s, 3H); IR: 3465 cm-1 (N-H stretch), 3083 cm-1 (alkene
C=C-H stretch), 2938 cm-1 (alkane C-H stretch), 1676cm-1 (C=O
amide), 1601cm-1 (alkene C=C stretch), HRMS: m/z calcd for
C31H24FN3O4: 521.22 [M+H]+; found: 522.3000.
1
214-215°C, H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 7.9 Hz,
2H), 7.93 (d, J = 15.5 Hz, 1H), 7.79 (dd, J = 16.8, 7.6 Hz, 3H),
7.67 (d, J = 11.3 Hz, 1H), 7.45 (d, J = 7.6 Hz, 4H), 7.38 – 7.29
(m, 2H), 7.19 (d, J = 6.4 Hz, 1H), 7.13 (s, 1H), 6.99 (t, J = 8.5
Hz, 2H), 6.86 (s, 1H), 6.37 (d, J = 15.6 Hz, 1H), 2.49 (s, 3H); 13
C
NMR (101 MHz, CDCl3) δ 168.15 (s), 164.71 (s), 162.35 (s),
162.21 (s), 151.33 (s), 147.70 (s), 138.89 (s), 136.65 (s), 135.73
(s), 134.76 (s), 131.48 (s), 131.45 (s), 131.20 (s), 130.34 (s),
129.75 (s), 129.67 (s), 127.34 (s), 127.08 (s), 126.83 (s), 126.65
(s), 125.65 (s), 123.94 (s), 123.90 (s), 120.62 (s), 120.58 (s),
119.90 (s), 119.40 (s), 115.99 (s), 115.78 (s), 30.93 (s); IR:
3288cm-1 (N-H stretch), 2927cm-1 (alkane C-H stretch),
1656cm-1 (C=O amide), 1601cm-1 (alkene C=C stretch),
HRMS: m/z calcd for C30H22FN3O2: 475.0025 [M+H]+; found:
476.1943.
(E)-N-(3-(2-(4-chlorostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
3,4-dimethoxybenzamide (5h). TLC carried out in Hexane: Ethyl
acetate (50:50) Rf= 0.291; cream color solid, (45%); M.P: 251-
252°C, IR: 3346 cm-1 (N-H stretch), 2934 cm-1 (alkane C-H
stretch), 1661 cm-1 (C=O amide), 1603 cm-1 (alkene C=C
stretch), HRMS: m/z calcd for C31H24ClN3O4: 537.15 [M+H]+;
found: [M+H]+: 537.8, [M+H]++: 539.8.
(E)-N-(3-(2-(4-fluorostyryl)-4-oxoquinazolin-3(4H)-
(E)-N-(3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
2-methyl benzamide (5d). TLC carried out in Hexane: Ethyl
acetate (60:40) Rf= 0.442; light yellow, Yield: 65%, M.P: 245-
yl)phenyl)thiophene-2-carboxamide (5i). TLC carried out in
Hexane: Ethyl acetate (50:50) Rf= 0.368; Cream color solid
(60%); M.P: above 295°C, IR: 3325 cm-1 (N-H stretch), 3078 cm-
1
246°C; H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 7.4 Hz, 1H),
1
(alkene C=C-H stretch) 1658 cm-1 (C=O amide), 1604 cm-1
8.17 (s, 1H), 7.94 (d, J = 15.5 Hz, 1H), 7.77 (d, J = 7.8 Hz, 3H),
7.69 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.53 – 7.37 (m,
5H), 7.27 (s, 1H), 7.19 (d, J = 7.1 Hz, 1H), 7.14 (t, J = 7.1 Hz,
1H), 6.90 (d, J = 6.9 Hz, 1H), 6.56 (d, J = 15.5 Hz, 1H), 2.48 (s,
3H); IR: 3255cm-1 (N-H stretch), 2228cm-1 (CN stretch),
1665cm-1 (C=O amide), 1603cm-1 (alkene C=C stretch), HRMS:
m/z calcd for C31H22N4O2: 482.9018 [M+H]+; found: 482.9000.
(alkene C=C stretch), HRMS: m/z calcd for C27H18FN3O2S:
467.11 [M+H]+; found: 468.0000.
(E)-N-(3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-
yl)phenyl)thiophene-2-carboxamide (5j). TLC carried out in
Hexane: Ethyl acetate (50:50) Rf= 0.259; Light brown (55%);
M.P: above 295°C, IR: 3340cm-1 (N-H stretch), 3066cm-1 (alkene
C=C-H stretch) 2222cm-1 (CN stretch), 1666cm-1 (C=O amide);
HRMS: m/z calcd for C28H18N4O2: 474.12 [M+H]+; found:
474.8000.
(E)-N-(3-(2-(4-fluorostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
2-(4-methoxyphenyl) acetamide (5e). TLC carried out in Hexane:
Ethyl acetate (50:50) Rf= 0.25; brownish yellow solid (60%);
1
M.P: 236-23°C; H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 7.8
3.5. Testing of Antimicrobial activity
Hz, 1H), 8.20 (s, 1H), 7.90 (d, J = 15.5 Hz, 1H), 7.78 (d, J = 5.2
Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.56 – 7.44 (m, 1H), 7.44 –
7.34 (m, 2H), 7.29 – 7.16 (m, 6H), 6.91 (d, J = 7.3 Hz, 1H), 6.85
(d, J = 8.5 Hz, 2H), 6.35 (d, J = 15.5 Hz, 1H), 3.76 (s, 3H), 3.51
(s, 2H); 13C NMR (101 MHz, CDCl3) δ 169.82 (s), 162.70 (s),
151.18 (s), 147.80 (s), 140.16 (s), 138.88 (s), 136.97 (s), 135.53
(s), 134.88 (s), 133.62 (s), 130.49 (s), 130.33 (s), 129.04 (s),
129.01 (s), 128.75 (s), 127.50 (s), 127.05 (s), 126.81 (s), 126.37
(s), 123.37 (s), 123.35 (s), 120.74 (s), 120.62 (s), 120.54 (s),
120.02 (s), 119.58 (s), 119.21 (s), 114.35 (s), 114.32 (s), 55.24
(s), 43.62 (s); IR: 3276cm-1 (N-H stretch), 1646cm-1 (C=O
amide), 1689cm-1 (alkene C=C stretch); HRMS: m/z calcd for
C31H24FN3O3: 505.8025 [M+H]+; found: 505.8000.
The MIC of synthesized molecules was determined by micro
broth dilution method i.e. Resazurin microtitre assay method
(REMA). The following steps were carried out:
Preparation of Nutrient broth (200 ml):
Material required:
Nutrient broth…………………....3.25 g
Distilled water…………………...250 ml
Method
(E)-N-(3-(2-(4-chlorostyryl)-4-oxoquinazolin-3(4H)-yl)phenyl)-
2-(4-methoxyphenyl) acetamide (5f). TLC carried out in Hexane:
Ethyl acetate (50:50) Rf= 0.36; light yellow solid (45%); M.P:
243-244°C; 1H NMR (400 MHz, CDCl3) δ: 8.34 (s, 1H), 8.29 (d,
J = 8.1 Hz, 1H), 7.92 (d, J = 15.4 Hz, 1H), 7.78 (d, J = 8 Hz, 2H),
7.70 (d, J = 7.5 Hz, 1H), 7.54 – 7.43 (m, J = 4Hz, 1H), 7.38 (d, J
=8Hz, 2H), 7.28 (dd, J = 8.4, 5.3 Hz, 2H), 7.21 (d, J = 8.4 Hz,
2H), 6.96 (t, J = 8.5 Hz, 2H), 6.90 (d, J = 7.3 Hz, 1H), 6.84 (d, J
= 8.5 Hz, 2H), 6.30 (d, J = 15.5 Hz, 1H), 3.75 (s, 3H), 3.49 (s,
The nutrient broth was dissolved in 250 ml of water. The media
was then sterilized by moist heat sterilization method using
autoclave. The freshly prepared and sterilized media was used for
evaluation.
Preparation of bacterial cell culture: The strains of S. aureus
(wild type), E. coli (wild type) was obtained from institute itself,
while the strain of Methicillin Resistant Staphylococcus aureus
(MRSA) (ATCC 33592) was obtained from National facility of

biopharmaceuticals, G N Khalsa College. The cultures of
bacterial strains were prepared individually by inoculating in
o
nutrient broth and kept in incubator for 24h at temperature 37 C,
OD580 adjusted to 0.1 (approx. 107 cells/mL).
Preparation of drug and test compound stock solution: Stock
solution (5mg/ml) of the test compounds and standard drug were
prepared in DMSO in 2 mL of Eppendorf tube. In case of Wild
type strains Streptomycin and kanamycin is used as standard
drugs, while linezolid was used as standard drug in MRSA
strains. Dilution of stock solution: 100μL of stock solution of test
compound was taken into an Eppendorf tube and volume was
made up to 1mL with nutrient broth. From this 100 μL of
solution was serially diluted into each well of 96 microtitre plate
which previously contained 100μL of nutrient broth. Preparation
of 0.02% Resazurin solution: 20mg of Resazurin solution was
dissolved into 100mL of sterile water.
3.6. Assay protocol
100μl of nutrient broth was added into each well of the microtitre
plate.
100μL test compound was added into the 1st well of a particular
concentration and serially diluted (two-fold dilution) till 5th well
i.e. four dilutions.100μL of homogenized bacterial cell culture
suspension (105 cells per well) was added to all the wells (well
with or without drug). After this, the plate was labelled and it was
incubated at 37°C for 20-24 h in incubator. After 24 h, 30μL of
0.02 % Resazurin solution was added, plate was observed after
30 min. and MIC was determined by visual inspection of color
change from blue (inhibition) to pink (growth).27
Figure 2. 2D Interaction between most active reported molecule
A and amino acid present in Penicillin binding protein 2a (4CJN)
at allosteric site.
Figure 1. 2D Interaction between native ligand and amino acid
present in Penicillin binding protein 2a (4CJN) at allosteric site
Figure 3. 2D Interaction between least active reported molecule
B and amino acid present in Penicillin binding protein 2a (4CJN)
at allosteric site.

Figure 7. A) 2D and B) 3D Interaction between most docked
ligand 8 and amino acid present in target protein (4CJN) at active
site.
Figure 4. A) 2D and B) 3D Interaction between most active
reported molecule A and amino acid present in Penicillin binding
protein 2a (4CJN) at active site.
4. Results and Discussion
4. 1 Molecular Docking
4.1.1 Allosteric site
All molecules were docked using 4CJN target protein.
Validation of the docking was performed by removing the co-
crystallized ligand (Native ligand) from the allosteric site and
then re-docked to the same site, and the RMSD was found to be
0.014 Å (less than 2Å). To further validate the docking results,
we have incorporated two marketed active drug molecule such as
ceftaroline (active moiety of prodrug ceftarolinefosamil) and
ceftobiprole (active moiety of prodrug ceftobiprole medocaril) in
our docking study.8 Result of docking studies in form of docking
score is shown in Table 1.
The results showed that the marketed drug ceftaroline and
ceftobiprole has docking score of -5.558Kcal/mol and -4.64
Kcal/mol. The result indicates that most active reported molecule
A has the little lower binding affinity towards PBP2a as
compared to marketed drugs with docking score of -4.579
Kcal/mol. However, most active reported molecule A has higher
binding affinity than a native ligand (reported molecule). The
docking score of native ligand was found to be -3.581 kcal/mol.
Also, we observed that least active reported molecule B from
data set has lowest binding affinity towards PBP2a with docking
score of –1.199 Kcal/mol.
Figure 5. A) 2D and B) 3D Interaction between least active
reported molecule B and amino acid present in Penicillin binding
protein 2a (4CJN) at active site.
The binding pose of native ligand molecule (re-docked pose) is
shown in Figure 1. Several favorable interactions between ligand
and enzyme were observed; ligand shows two hydrogen bonding
interaction (pink dotted line) between oxygen of carboxylic acid
with ARG298.
The binding pose of most active reported molecule A is shown
in Figure 2. Several favorable interactions between ligand and
enzyme were observed; ligand shows three hydrogen bonding
interaction (pink dotted line) with receptor include nitrogen of
mesyl group with ASN146; Nitrogen Cyano grp with ARG293;
oxygen of Quinazolinone ring with TYR105.
The binding pose of least active reported molecule B is shown
in Figure 3. No favorable interactions between ligand and
enzyme were observed. This docking interaction with least active
molecule B indicate that electron withdrawing group might be
unfavorable. Therefore, the least active reported molecule B is
showing least docking score.
Figure 6. 2D Interaction between most docked ligand 8 and
amino acid present in target protein (4CJN) at allosteric site.
4.1.2 Active site
Similarly, to validate the docking results, we have included
two marketed active drug molecule such as ceftaroline and
ceftobiprole in our docking study. Result of docking studies in
form of docking score is shown in Table 1.

The results showed that the marketed drug ceftaroline and
ceftobiprole has docking score of -5.334Kcal/mol and -5.291
Kcal/mol at active site. The result indicates that most active
reported molecule A has the little lower binding affinity towards
PBP2a as compared to marketed drugs with docking score of -
4.834 Kcal/mol. However, most active reported molecule A has
little higher binding affinity than a native ligand. The docking
score of native ligand was found to be -4.795 kcal/mol. Also, we
observed that least active reported molecule B from data set has
lowest binding affinity towards PBP2a with docking score of –
1.603 Kcal/mol.
ceftobiprole medocaril. Overall, the percentage human oral
absorption for 77 reported molecules ranged from 38.37% to
100%.
All these pharmacokinetic parameters were found to be within
acceptable range defined for human use see in Table 2, thereby
indicating their potential as drug-like molecule.
4.1.4 Prime MM-GBSA calculations
The top ranked molecules were further subjected to Prime
MM-GBSA calculations (Table 1). At allosteric site, the most
active reported molecule A was found to have binding free
energy (dG bind) of -47.41kcal/mol, slightly lower than drug
ceftaroline which has highest binding free energy (dG bind) of -
50.36 kcal/mol, while dG bind of drug ceftobiprole was found to
be -42.13kcal/mol slightly lower than most active reported
molecule A. Whereas native ligand has lower binding free energy
compare to most active reported molecule A of -38.35kcal/mol
and least active reported molecule B has weaker binding free
energy of -22.42 kcal/mol. Similarly, at active site most active
reported molecule A was found to have binding free energy (dG
bind) of -43.43kcal/mol, slightly lower than drugs ceftaroline and
ceftobiprole which has highest binding free energy (dG bind) of -
54.89kcal/mol and -44.38kcal/mol respectively. Whereas native
ligand has lower binding free energy compare to most active
reported molecule A of -28.12kcal/mol and least active reported
molecule B has weaker binding free energy of -25.57kcal/mol.
Similar to the docking study, Prime MM-GBSA has been
validated by using known actives and inactive and also standard
drugs are incorporated in validation. This Prime MM-GBSA
calculation can be used to score new ligands in order to design
more potent analogues.
The binding pose of most active reported molecule A at active
site in 2D and 3D image is shown in Figure 4; Several favorable
interactions between ligand and enzyme was observed in 2D
interaction image, H-bond interaction (pink dotted line) between
nitrogen of mesyl group and TYR446, oxygen of quinazolinone
with LYS430 and nitrogen of cyano group with MET641. It also
shows π-cation interaction (red line) with LYS430. 3D
interaction image shows weak H-bond interaction (orange dotted
line) with various residues but no interaction with SER403
residue which is required for acylation in case of β-lactam
antibiotics. Hence it indicates that non-covalent interactions at
active site may be important for activity.
The binding pose of least active reported molecule B at active
in 2D and 3D interaction image is shown in Figure 5; 2D
interaction image shows only one π-cation interaction (red line)
with LYS597. 3D interaction image shows weak H-bond
interaction (orange dotted line). Here also no interaction with
SER403 residue. Similar to allosteric site, the least active
reported molecule B is not showing much interaction with the
amino acid residues. These may conclude that the reasons for low
activity of least active reported molecule might be due to less or
no interaction with residue at allosteric and active site.
4.1.5. Design of New molecules
Aforementioned, Structure based drug design studies helps in
prediction of the structural requirement for design of novel potent
4-(3H)-Quinazolinone antibacterial molecules. Docking studies
revealed that hydrogen-bonding interaction with the substitution
present at meta position of aromatic ring substituted at 3 position
of quinazolinone ring might be important for activity. Docking
studies also indicate that no interaction is observed for
quinazolinone with SER403 residue at active site, it may be the
non-covalent interactions at active site that may be responsible
for anti-bacterial activity. An important aspect for inhibitor
design will be to improve binding affinity by increasing the
number of noncovalent interactions between inhibitor and active
site.
The docking study concludes that quinazolinone molecule is
not showing any interaction with SER403 at active site, which
indicate that the anti-bacterial activity of molecules may be due
to non-covalent interactions. The most active reported molecule
A shows several favorable interactions with substitution present
at 3 position of quinazolinone at both allosteric and active site,
may be this position is important for modification to further
improve in activity. Docking study can be used to design new
more potent analogues.
4.1.3 In-silico pharmacokinetic prediction
QikProp software gives predicted information about molecular
weight, partition coefficient (logPo/w), stars, % oral absorption
values and Lipinski’s properties (Rule of Five) and apparent
Caco-2 cell permeability (QPPCaco) in nm/sec. To validate the
result of In-silico studies, we have incorporated two marketed
prodrugs such as ceftaroline fosamil and ceftobiprole medocaril
in our ADMET prediction study. We were studied 77 compounds
from that most active and least active reported molecules B along
with the prodrug ceftaroline fosamil and ceftobiprole medocaril
are mentioned in Table 2.
In order to design new inhibitor, it should provide larger
number of non-covalent stabilizing interactions. Alternatively,
non-β-lactam compounds that bind tightly to the active site
without the need for acylation with non-covalent interactions
may also provide highly effective inhibitor.28 The most active
reported molecule A was considered for modification to further
improve in activity. Literature indicated that direct substitution at
Quinazolinone ring is not allowed.13 However, modification is
allowed at meta position of aromatic ring substituted at 3 position
of quinazolinone and at para position of aromatic ring substituted
at 2 position of quinazolinone. The molecules containing amine
group at meta position of aromatic ring substituted at 3 position
of quinazolinone have good biological activity.13,29–31 Also the
molecules containing electron withdrawing group at para
For the 77 reported molecules, the partition coefficient
(logPo/w) was found to be in range between 2.3 to 5.1 resemble
with drugs ceftaroline fosamil and ceftobiprole medocaril for the
estimation of absorption and distribution of drug within the body.
Caco-2 cell permeability (QPPCaco), a key factor to
determine drug metabolism and its access to biological
membrane ranged in between0.568 to 1151.21nm/sec, from
which most active molecule shows an absorption value of 853.02
nm/sec, more than the active drugs ceftaroline fosamil and
positionof aromatic ring substituted at
2
position of
quinazolinone have good biological activity.13 Hence,
modification is done at R2 position of quinazolinone by adding
carbamoyl (-NHCO-) linker, followed by substitution with
electron withdrawing group such as F, Cl, CN at R1position of

Quinazolinone. New ligands mentioned in Table 3 have been
designed based upon the above mentioned structure-based and
ligand-based drug design studies.
band at 1661cm-1 corresponding to carbonyl (C=O) of amide, and
a band at 1524cm-1 corresponding to characteristic C-NO2
respectively. IR spectrum of compound (2c) shows a two
characteristic absorption bands at 3435cm-1 and 3346cm-1 for
NH2 stretch, and a band at 2926cm-1 corresponding to C-H
stretch of alkane, while absorption band at 1651cm-1
corresponding for carbonyl (C=O) of amide respectively. IR-
spectrum of compounds 3 shows an absorption bands at 2970cm-1
characteristic for C-H stretch of alkane, while a band at 1738cm-1
corresponding to carbonyl (C=O) of lactone. IR spectrum of
compound 4c shows an absorption band at 3278cm-1
corresponding to N-H stretch, and a band at 2933cm-1
corresponding to C-H stretch of alkane, while at 1656cm-1
corresponding to characteristic of carbonyl (C=O) amide. IR
spectrum of compound 5f shows an absorption band at 3278cm-1
corresponding to N-H stretch, and a bands at 3070cm-1 and
2929cm-1 corresponding for C-H stretch of alkene and alkane
4.1.6. Docking and MM-GBSA of designed molecules
All new designed molecules have been docked using same
target protein 4CJN. The docking study was carried out to
explore the interaction mechanism between inhibitors and the
receptor. The score function of Glide, gives highest binding
affinity prediction as well as molecule ranking. All molecules
along with their Glide docking score have been listed in Table 3.
The binding pose of most docked ligand 8 at allosteric site
shown in Figure 6. Several favorable interaction has been
observed in 2D interaction image; it shows π-π interaction (green
line) and π-cation (red line) of benzene ring with the residues
TYR297 and LYS273 respectively. It also shows H-bond
interaction (pink dotted line) between oxygen of methoxy group
with the residue LYS316.
respectively, and
a
band at 1656cm-1 corresponding to
characteristic of carbonyl (C=O) amide, Alkene C=C show a
band at 1601cm-1, while a band at 808cm-1 for C-Cl stretch.
The binding pose of most docked ligand 8 at active site in 2D
and 3D is shown in Figure 7. Several favorable interaction has
been observed in 2D interaction image; it shows two π-π
interaction (green line) between p-Cl phenyl and a substituted
phenyl attached at 2-position of Quinazolinone with the residues
TYR446. It also shows H-bond interaction (pink dotted line)
between oxygen of amide with the residue TYR446. Similar kind
of interaction is seen in 3D interaction image with some weak H-
bond interaction (orange dotted line) and aromatic H-bond
interaction (light blue dotted line). In 3D image H-bond is
indicated by yellow dotted line, while π-π interaction is indicated
by blue dotted line. Here also no interaction is observed with
SER403 residue at active site.
1
Proton H-NMR of compound 5f shows signal at δ: 8.34 (s,
1H,-CONH), 8.29 (d, J = 8.1 Hz, 1H, AR-H), 7.92 (d, J = 15.4
Hz, 1H, C=C-H), 7.78 (d, J = 8Hz, 2H, Ar-H attached next to
alkene), 7.70 (d, J = 7.5 Hz, 1H, Ar-H), 7.54 – 7.43 (m, J = 4Hz,
1H, Ar-H), 7.38 (d, J =8Hz, 2H, Ar-H attached next to Cl), 7.28
(dd, J = 8.4, 5.3 Hz, 2H, Ar-H), 7.21 (d, J = 8.4 Hz, 2H, Ar-H
attached next to NHCOCH2-), 6.96 (t, J = 8.5 Hz, 2H, Ar-H),
6.90 (d, J = 7.3 Hz, 1H, Ar-H), 6.84 (d, J = 8.4 Hz, 2H, Ar-H
attached next to O-CH3), 6.30 (d, J = 15.5 Hz, 1H, C=C-H), 3.75
(s, 3H, CH3), 3.49 (s, 2H, CH2).
Carbon 13C NMR of compound 5f shows 31 carbon signal see
in supporting section of which characteristic signal include: C=O
of amide at 169.86, C=O of quinazolinone at 162.81, aromatic
carbon attached to OCH3 at 158.85, carbon of alkene at 139.06,
aromatic carbon attached to Cl at 134.87, carbon of alkene at
114.29, carbon of OCH3 at 55.23, carbon of CH2 at 43.57.
Furthermore, all designed molecules were further subjected to
Prime MM-GBSA calculations using same target protein 4CJN
(Table 3). Ligand 8 has shown highest dG bind score of -52.723
and -54.628 at allosteric and active site respectively. Based on
the results of docking and MM-GBSA calculation and
pharmacokinetic prediction, the ligands that show good docking
and dG bind score with good pharmacokinetic predictions were
considered for synthesis and biological screening. The 10 best
ranked designed molecules include ligand no 1, 3, 5, 6, 7, 8, 9,
11, 12 and 15 have been selected for synthesis and biological
screening against microorganism.
The mass spectra of compounds containing Chlorine atoms (5f
and 5h) showed fragments corresponding to the typical chlorine
isotope (35Cl and 37Cl). Thus, the mass spectrum of 5f shows its
M+ and M+2 peaks at m/z 521.7 (66.67%) and m/z 523.7
(33.33%), respectively. (see supporting information for details)
4.3. Biological Evaluation
4.2 Synthesis of Designed Ligands
All synthesized compounds were evaluated for in-vitro anti-
bacterial activity. The MIC values of all synthesized
quinazolinone are as shown in Table 4. All of the synthesized
derivative shows good range of activities against S. aureus
(gram-positive) organism (see Figure supporting information).
However, all of these compounds show weak activity against E.
coli (gram-negative) organisms having Minimum inhibitory
concentration (MIC) ≥ 62.5 μg/mL. Among all the compounds
active against S. aureus, compound 5f show remarkable activity
with a MIC ≥ 15.625 μg/mL. Moreover, compound 5i show
moderate activity with a MIC ≥ 31.25 μg/mL. Compounds 5b,
5d, 5e, 5g, 5h and 5j show average activity against S. aureus
with MIC ≥ 62.5 μg/mlL. while rest of the compounds 5a and 5c
show very weak activity with MIC ≥ 125 μg/mL. Benzamide
substitution at aromatic ring of 3 position of quinazolinone with
4-fluoro styryl (compound 5a) at 2 position of quinazolinone
shows weak activity of MIC ≥ 125 μg/mL, while 4-cyano styryl
(compound 5b) substitution show little increase in activity with
MIC ≥ 62.5 μg/mL. Adding methyl at 2-position of benzamide
shows no improvement in activity with MIC ≥ 125 μg/mL and
MIC ≥ 62.5 μg/mL for 4-fluoro styryl (compound 5c) and 4-
All the designed compounds were synthesized by using
literature reported procedures (Scheme 1) see in experimental
section. N-(3-nitrophenyl)-benzamide (1a) and its derivatives
(1b-e) were synthesized from literature procedure with few
modifications includes using of 1.5 eq. of carboxylic acid instead
of 1 eq. of carboxylic acid indicated in procedure and keep the
reaction mixture for overnight.20 These nitro compounds were
then reduced to corresponding amine by using mixture of Fe,
CaCl2, water in Ethanol solvent to yield amine compounds (2a-e)
22
2-methyl-4H-benzo[d][1,3]oxazin-4-one (3) was synthesized
by reacting anthranilic acid with triethyl ortho-acetate and reflux
for 15-20min.24 Compound (2a-e) was then reacted with
compound (3) under acidic medium to yield compounds (4a-e).25
Compounds (5a-j) were synthesized by condensation of
compounds (4a-e) with substituted benzaldehyde under acidic
medium and then reflux for 24-48 h.26
Compounds (1-5) were characterized by elemental analysis
such as IR, 1H-NMR, 13C-NMR, MS (see supporting
information). For example, IR-spectrum of compound (1c) shows
an absorption bands at 3243cm-1 corresponding to N-H stretch, a

cyano styryl (compound 5d) substitution respectively. Adding
two methoxy group at 3 and 4 position of benzamide substituted
at aromatic ring of 3 position of quinazolinone show little
improvement in activity with both 4-fluoro styryl (compound 5g)
and 4-Chloro styryl (compound 5h) substitution at 2 position of
quinazolinone with an MIC ≥ 62.5 μg/mL.
Although replacing benzamide with thiophene-2-carboxamide
shows improvement in activity, 4-fluoro styryl (compound 5i)
substitution at 2 position of quinazolinone show moderate
activity with MIC ≥ 31.25 μg/mL, while 4-cyano styryl
(compound 5j) substitution at 2 position of quinazolinone shows
weak activity with MIC ≥ 62.5 μg/mL.
However, adding methylene linker in between amide and 4-
methoxy phenyl substituted at aromatic ring of 3 position of
quinazolinone shows no improvement in activity with 4-fluoro
styryl (compound 5e) substitution at 2 position of quinazolinone
with MIC ≥ 62.5 μg/mL, while 4-Chloro styryl (compound 5f)
substitution show good activity with MIC ≥ 15.625 μg/mL.
Moreover, all synthesized compounds were also tested against
Methicillin resistant staphylococcus aureus (MRSA) (ATCC).
Compound 5f and compound 5i shows good activity with MIC ≥
31.25 μg/mL, while rest of the compounds 5a, 5b, 5c, 5d, 5e, 5g,
5h, and 5j shows weak activity with MIC ≥ 62.5 μg/mL.
Table 1. Summary of Docking and MM-GBSA score at allosteric and active site. Docking sore MM-GBSA
Docking sore
MM-GBSA
Allosteric site
-4.579
Active site
-4.834
Allosteric site
-47.41
Active site
-43.43
Most active reported molecule A
Least active reported molecule B
Native ligand
Ceftaroline
Ceftobiprole
-1.199
-3.581
-5.558
-4.64
-1.603
-4.795
-5.334
-5.291
-22.42
-38.35
-50.36
-42.13
-25.57
-28.12
-54.89
-44.38
Table 2. Summary of QikProp studies.
Most active reported
Least active reported
Ceftaroline fosamil
Ceftobiprole medocaril
molecule A
molecule B
#stars
LogP o/w
QPPCaco
0
1
4.261
464
100
0
2
1.667
545
100
2
2
2.466
690
95
2.509
853.02
76.19
0
% Human oral absorption
Rule of five[a]
2
[a]value indicate violation in Lipinski’s rule of five.
Where,
#stars: A large number of stars suggest that a molecule is less drug-like than molecules with few stars.
Log P: Predicted octanol/water partition coefficient.
QPPCaco: Predicted apparent Caco-2 cell permeability in nm/sec. Caco-2 cells is a model for the gut-blood barrier. QikProp predictions are for non-active
transport.
Percent Human Oral Absorption: Predicted human oral absorption on 0 to 100% scale.
aRule of Five: Number of violations of Lipinski’s rule of five. The rules are: mol-MW < 500, logP o/w ≤ 5, donorHB ≤ 5, acceptHB ≤ 10. Compounds that satisfy
these rules are considered as druglike molecule.
Table 3. Designed molecules
Docking score
Allosteric Active
MM-GBSA (dG bind)
Ligand
no.
Rule of
five[a]
R
R1
LogP
Allosteric
site
Active
site
site
site
1
2
3
H
H
F
Cl
F
-3.538
-3.672
-49.752
-54.437
-44.573
-41.158
-35.76
-43.64
1
1
1
5.174
5.146
5.396
-3.594
-3.929
-2.849
-3.684
2-CH3

4
5
6
2-CH3
Cl
F
-3.584
-4.413
-4.066
-3.183
-3.886
-3.686
-54.646
-51.445
-59.734
-32.095
-40.979
-43.319
2
2
2
5.612
4.792
4.649
3,4-DiOCH3
3,4-DiOCH3
Cl
7
8
F
-3.964
-4.674
-3.865
-4.327
-50.221
-52.723
-51.977
-54.628
1
1
4.916
4.758
Cl
9
F
-3.618
-3.618
-3.632
-2.992
-52.99
-38.124
-36.021
1
0
5.012
4.924
10
Cl
-51.539
11
12
13
H
CN
CN
CN
-4.187
-4.261
-3.979
-3.464
-3.796
-3.898
-47.929
-50.275
-55.977
-42.367
-46.09
1
1
1
5.124
4.6
2-CH3
3,4-DiOCH3
-24.979
4.764
14
15
CN
CN
-3.834
-3.627
-4.086
-3.655
-51.64
-39.512
-41.733
0
1
4.747
5.862
-53.981
16
17
18
2-OH
2-OH
2-OH
F
-3.196
-3.149
-3.674
-3.067
-3.141
-3.737
-30.58
-33.598
-39.906
-24.168
-35.739
-44.249
1
0
1
6.118
4.843
6.584
Cl
CN
19
20
21
F
-4.02
-3.624
-4.157
-3.138
-2.645
-2.834
-54.948
-55.573
-50.637
-32.061
-37.253
-37.222
2
1
0
6.785
5.507
4.317
Cl
CN

22
23
24
F
-4.4
-3.546
-3.043
-4.258
-49.873
-47.381
-50.035
-31.571
-21.429
-40.381
0
0
1
4.564
3.217
6.465
Cl
-4.043
-4.579
CN
25
26
4-Cl
4-Cl
F
-3.727
-3.728
-3.553
-4.187
-47.751
-59.819
-32.372
-36.475
2
1
6.694
6.174
CN
Table 4 Antimicrobial activities of test compounds
Bacteria MIC (µg/mL)
Molecule no
R
R1
Gram-negative
Gram-positive
Staphylococcus
aureus
Methicillin- Resistant Staphylococcus aureus
Escherichia coli
(MRSA)
5a
5b
5c
5d
H
H
2-CH3
2-CH3
F
CN
F
62.5
62.5
62.5
62.5
125
62.5
125
62.5
62.5
62.5
62.5
CN
62.5
5e
5f
F
62.5
62.5
62.5
62.5
Cl
15.625
31.25
5g
5h
3,4-DiOCH3
3,4-DiOCH3
F
Cl
62.5
62.5
62.5
62.5
62.5
62.5
5i
F
62.5
31.25
31.25
5j
CN
62.5
7.8
62.5
7.8
62.5
-
Streptomycin
Standard drugs
Kanamycin
Linezolid
7.8
-
7.8
-
-
7.8

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The present research has been carried out to develop new
molecules by structure based drug design. Docking studies has
been carried out to explore the interaction between ligands and
the receptor. Docking study explained the importance of
hydrogen bonding interaction with the substitution present at
meta position of aromatic ring substituted at 3 position of
quinazolinone ring for activity. The most active reported
molecule A displays a H-bond interaction between amine of
mesyl group with residues of target protein and therefore, the
position has been selected for modification. Furthermore, all
designed molecules were further subjected to Prime MM-GBSA
calculations to determine binding free energy.
Based on the result of molecular modelling studies 26 new
ligands have been designed which were ranked as good ligand by
using docking interaction study and prime MM-GBSA
calculation and then in-silico pharmacokinetic properties of
designed molecules were determines by ADME prediction
software, Qikprop. These predictions help to identify potential
drug like molecule. Out of 26 designed ligands, 10 best ranked
ligands have been selected depend upon their docking, MM-
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synthesis and followed by biological screening of anti-bacterial
activity.
These 10 designed molecules have been synthesized by
appropriate conventional synthetic route see in experiment
section. After synthesis, these molecules were screened for
biological activity against three bacterial strains namely E. coli,
S. aureus, Methicillin resistant Staphylococcus aureus. All of
these ligands show weak activity against gram negative organism
E. coli, while shows moderate to good activity against gram
positive organism S. aureus of which ligand 5f has shown most
potent activity with MIC ≥ 15.625μg/mL against S. aureus.
These ligands were also tested against resistant strain of S.
aureus i.e. MRSA, the ligand 5f and 5i shows good activity with
MIC ≥ 31.25μg/mL. Thus, the molecular modelling studies assist
in design of new analogues that would further enhance the
biological activity.
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In present study, Structure based drug design has been
performed for Quinazolinone molecule with Penicillin binding
protein 2a (PBP2a). This study will help to understand how non
β-lactam able to inhibit cell wall inhibition similar to β-lactam.
This studies further help to identify the potential lead molecules
by virtual screening and optimization of the lead molecules.
Acknowledgments
SIQ and HKC thank to Merck India for providing financial
facility.
References and notes
Supplementary Material
Supplementary information file is provided
1. Boucher, H.W.; Talbot, G.H.; Bradley, J.S.; Edwards, J.E.;
Gilbert, D.; Rice, L.B.; Scheld, M.; Spellberg, B.; Bartlett, J. Clin.
Infect. Dis. 2009, 48, 1-12.
2. Staph infection can kill, Cent. Dis. Control Prev. 2019.
https://www.cdc.gov/vitalsigns/staph/pdf/vs-0305-staph-
infections-H.pdf (accessed April 25, 2019).
Click here to remove instruction text...

Highlights:
 Resistance in methicillin-resistant
Staphylococcus aureus (MRSA) due to
acquisition of additional PBP2a.
 CADD explore the structural features of
Quinazolinone as anti-MRSA agents.
 New molecules have been designed by
using docking using PBP2a target.
 Most compound were active against S.
aureus, While few show activity against
MRSA.
 The compound 5f shows good activity
against MRSA.