In-silico design, synthesis and evaluation of novel DNA-gyrase B inhibitors

Article abstract of DOI:10.1007/s00044-013-0518-3

2-Quinolones are an important class of compounds, isomeric to 4-quinolones. They may become promising candidates for exploiting more useful therapeutically active molecules. DNA-gyrase has drawn much attention as a selected target for finding potent anti-bacterial agents against multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and penicillin-resistant Streptococci pneumonia. The objective of the present study was to study the molecular docking simulations on 2-quinolone analogs as probable candidates for inhibiting DNA gyrase subunit-B of S. aureus. In the present study, docking simulations were carried out on the reported inhibitors of DNA-gyrase subunit A and B using docking software. Based on it, series of 2-quinolone analogs (compound 1-8) were designed, synthesized, characterized, and evaluated for their anti-bacterial activity against S. aureus and E. coli. Out of the eight test compounds, compound-2 showed good anti-bacterial activity against S. aureus and E. coli as compared with the rest of the other compounds. The rational approach to lead discovery has prompted a better insight into developing more specific 2-quinolones as potential antibacterial agents.

Full text of DOI:10.1007/s00044-013-0518-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0518-3
ORIGINAL RESEARCH
In-silico design, synthesis and evaluation of novel DNA-gyrase
B inhibitors
•
Umesh Shiroya Milan Patel
Received: 4 September 2012 / Accepted: 29 January 2013
Ó Springer Science+Business Media New York 2013
Abstract 2-Quinolones are an important class of com-
pounds, isomeric to 4-quinolones. They may become
promising candidates for exploiting more useful therapeu-
tically active molecules. DNA-gyrase has drawn much
attention as a selected target for finding potent anti-bacte-
rial agents against multi-drug resistant strains such as
methicillin-resistant Staphylococcus aureus, vancomycin-
resistant enterococci, and penicillin-resistant Streptococci
pneumonia. The objective of the present study was to study
the molecular docking simulations on 2-quinolone analogs
as probable candidates for inhibiting DNA gyrase subunit-
B of S. aureus. In the present study, docking simulations
were carried out on the reported inhibitors of DNA-gyrase
subunit A and B using docking software. Based on it, series
of 2-quinolone analogs (compound 1–8) were designed,
synthesized, characterized, and evaluated for their anti-
bacterial activity against S. aureus and E. coli. Out of the
eight test compounds, compound-2 showed good anti-
bacterial activity against S. aureus and E. coli as compared
with the rest of the other compounds. The rational
approach to lead discovery has prompted a better insight
into developing more specific 2-quinolones as potential
antibacterial agents.
Introduction
The increasing use of anti-bacterial agents such as beta-
lactams, macrolides, vancomycin, or quinolones has
resulted in the emergence of multi-drug resistant patho-
gens, especially gram-positive bacteria (Tanitame et al.,
2004a, b). Widespread emergence of bacterial resistance to
present drugs represents a serious problem in treatment of
bacterial infections. Modern approaches toward develop-
ment of new potential inhibitors are based on knowledge of
structure and function of proteins specific to bacteria. One
of them is DNA gyrase, a characteristic and essential
bacterial enzyme the inactivation of which leads to bacte-
rial death. For this reason, gyrases have been chosen as
targets for antibacterial agents (Gradisar et al., 2007).
DNA-gyrase has drawn much attention as a selected target
for finding potent anti-bacterial agents against multi-drug
resistant strains such as methicillin-resistant Staphylococ-
cus aureus (MRSA), vancomycin-resistant enterococci
(VRE), and penicillin-resistant S. pneumonia (PRSP).
DNA-gyrase, a typical type-II topoisomerase, has been
known to cause DNA replication, transcription, and
recombination. DNA-gyrase catalyzes the ATP-dependent
introduction of negative supercoils into bacterial DNA as
well as the decatenation and unknotting of DNA. The
DNA-gyrase enzyme consists of two subunits, A and B, of
molecular mass 97 and 90 kDa, respectively, with the
active enzyme being an A2B2 complex (Liu et al., 2009,
Brvar et al., 2010).
Keywords Antibacterial activity ꢀ Docking simulations ꢀ
DNA-gyrase ꢀ 2-quinolones
U. Shiroya (&)
17/Gopi Park Society, Hirawadi Road, Bapunagar,
Ahmedabad 382350, Gujarat, India
e-mail: u_shiroya@yahoo.com
The structure activity relationships of compounds based
on nalidixic acid have led to a large group of synthetic
antibacterial agents known collectively as quinolones. The
4-quinolones such as ciprofloxacin, ofloxacin, lomefloxa-
cin, enoxacin are established synthetic antibacterial agents
which act as DNA gyrase inhibitors (Mitscher et al., 1986).
U. Shiroya ꢀ M. Patel
Department of Pharmaceutical Chemistry, L.B.Rao Institute
of Pharmaceutical Education and Research, Bethak Road,
Khambhat 388620, Gujarat, India
123

Med Chem Res
In silico approaches
The benzopyrone ring in compounds of coumarins and
flavones is isosteric to quinolones and has shown DNA
gyrase inhibition (Gradisar et al., 2007). The 2-quinolones,
also called carbostyrils or 1-aza coumarins, are isosteric to
coumarins and isomeric to 4-quinolones and could become
a potential candidate for antibacterial activity (Thomas
et al., 2010). Reports are available on DNA gyrase inhi-
bition by quinolones, coumarins (chlorobiocin, novobio-
cin), and cyclothialidines (Wentland et al. 1988, Boehm
et al. 2000, Gray et al., 2003, Foroumadi et al., 2003,
Tanitame et al., 2005). Quinolones are known to inhibit the
DNA breakage–reunion cycle by binding to the subunit A
and by blocking the gyrase–DNA complex, whereas the
latter acts on subunit B. More recently, GyrB inhibitors
from various chemical classes have been reported includ-
ing indazole, pyrazole, benzimidazole, phenol, and ind-
olinone (Tanitame et al. 2004a, 2004b, Angehrn et al.,
2004, Liu et al., 2008, Brvar et al., 2010, Ronkin et al.,
2010).
One of the most important considerations in docking sim-
ulations is the selection of appropriate docking and scoring
algorithm. There are several algorithms and number of
scoring functions available today for docking. Roughly, the
scoring functions are categorized into knowledge based and
empirical categories. In the present study, an attempt was
made to work with Arguslab for the docking calculations.
The crystal structure of 24-kDa N-terminal fragment
of DNA-gyrase B complex with inhibitor, 4-methyl-5(3-
(Methylsulfanyl)-1H-pyrazol-5-yl)-2-thiophen-2-yl-1,3-thi-
azole (PDB code: 3G75, www.rcsb.org) was retrieved from
protein data bank in PDB format as starting point. All water
molecules from protein structure were deleted. Protein
structure consists of two chains A and B; among them
chain-B was deleted. In protein structure, missing bond
orders, hybridization states, charges, and angles were
assigned, and explicit hydrogen molecules were added
using the different parameters. The protein structure was
energy minimized by Line search method using geometry
optimization method. The Validation of docking algo-
rithms (Fig. 1) was performed by changing different
docking parameters until the lowest RMSD value was
In the present study, docking simulations were carried
out on the reported inhibitors of DNA-gyrase subunit A and
B using docking software. Based on it, series of 2-quino-
lone analogs (Compound 1 to 8) were designed, synthe-
sized, characterized, and evaluated for their anti-bacterial
activity against S. aureus and E. coli.
˚
obtained. By using the Grid resolution 0.8 A and
Lamarckian genetic algorithms dock engine, while other
parameters were set as default, the RMSD value of best
˚
pose of ligand was obtained as 1.62 A.
Materials and methods
All molecular docking simulations were carried out
using Arguslab Docker (ALD) version 4.0.1 installed
on Intel Pentium-4 machine. All the ligand structures
were constructed using Chemsketch software version-5.2
Chemistry
The chemical structure and three-dimensional (3D) struc-
tures were drawn using Structure Builder (Marvin-Sketch
version-5.2, ChemAxon). Docking studies were done
using the facilities of Molecular Modeling (Arguslab
4.0.1, Mark Thompson and Planaria Software LLC). The
melting point was determined using a melting point
apparatus (DBK Precision melting point apparatus) and
found to be uncorrected. The reactions were monitored
by thin-layer chromatography (TLC), and the Rf values
were determined using TLC plates with the solvent sys-
tem (Toluene : Methanol (4:2)). The kmax values for the
synthesized compounds were established by ultraviolet
(UV)–visible spectrometer (UV-1700 Pharmaspec, Shi-
madzu Corporation, Kyoto). The IR studies were done
with FT-IR (DRS-8400, Shimadzu Corporation, Japan).
The NMR study was carried out by the instrument NMR
using DMSO-d6 solvent (Bruker Avance-II 500 MHz
FT-NMR, TOPSPIN 1.3 Version), and the mass spectral
studies were done using LC–MS (MDS SCIEX API 2000
LC–MS/MS).
Fig. 1 Showing the validation of docking algorithm using protein
structure 3G75
123

Med Chem Res
installed on Intel Pentium-4 machine. The ligands were
saved in MDL-Mol file. The ligands were then imported in
Mervin-view software version-5.2 installed on Intel Pen-
tium-4 machine, and explicit hydrogen atoms were added
and converted to 3D conformations using Mervin-view
software. The ligands were then saved in a single MDL-
Mol file. The ligands were energy minimized by Line
search method using geometry optimization function in
Argus-lab, and then lower energy conformations were
selected for further studies. Different docking parameters
were taken as per validation of docking algorithms
(Table 1). All docking calculations were carried out using
grid based A-Score function with a Grid resolution of 0.8
protein residues. Secondly, the carboxylic acid group was
selected as the hydrogen bond donor to be attached to the
phenyl ring. A total of eight acid derivatives were designed
for molecular docking study. The docking studies (com-
pound-A) of 7-amino-4-methylquinolin-2(1H)-one (Fig. 2)
revealed the interaction of N-1 of 2-quinolone with Ser-55
and the hydrogen bonding between C-7 amino of molecule
and Thr-173. The docking studies (Fig. 3) of 4-((4-methyl-
2-oxo-1,2-dihydroquinolin-7yl)amino)benzoic acid with a
24-kDa fragment of DNA-gyrase B from S.aureus (Com-
pound-2) revealed the one hydrogen bonding between
carboxylic acid group of molecule and Ser-129 residue and
´
˚
A. The binding site on the receptor was defined as
extending in X, Y, and Z directions around dock molecule
´
˚
with binding site box size of around 15 A. Lamarckian
genetic dock algorithm with maximum of 1,000 genera-
tions was used through the calculations keeping all other
parameters as default. The lowest binding free energy pose
of conformation was displayed and saved in pdb format.
Then pdb file was imported into the Molegro virtual viewer
version 2010 2.0.0 for analyzing different interactions such
as hydrogen bonding, hydrophobicity, electrostatic inter-
actions, docking view etc.
Design of synthesized test compounds
and their docking
Molecular docking studies offer precise information for
studying the interaction between the ligand and the protein
residue and aids as a guide for the drug design. In the
present study, the key feature was focused on the optimi-
zation of the amino group attached to C-7 of 2-quinolone.
Initially, Molecular docking simulations were carried out
using 7-amino-4-methylquinolin-2(1H)-one as reported in
the Thomas et al.. Then side chain of 7-amino-4-methyl-
quinolin-2(1H)-one was extended and the substituted phe-
nyl group and heterocycles were chosen for their steric,
hydrophobic, and van der Waal interactions with the
Fig. 2 Showing the docking studies of 7-amino-4-methylquinolin-
2(1H)-one (Compound-A) revealed the interaction of N-1 of 2-quin-
olone with Ser-55 and the hydrogen bonding between C-7 amino of
molecule and Thr-173
Table 1 Showing the docking parameters used for the docking
simulations
Different docking parameters taken for docking simulation
˚
Grid resolution = 0.8 A
Cross over rate = 0.8
Genetic algorithms dock engine Elitism = 5
Calculation type = DOCK
Scoring function = A Score
Local search rate = 0.06
Mutation rate = 0.2
Convergence = 0.0015936
Binding site box
size = 15 9 15 9 15 A
Fig. 3 Showing the docking studies of 4-((4-methyl-2-oxo-1,2-dihy-
droquinolin-7yl)amino)benzoic acid (Compound-2) revealed the
hydrogen bonding between carboxylic acid group of molecule and
Ser-129, the hydrophobic interaction between substituted phenyl ring
and Ileu-175 residue and the hydrogen bonding between N-1 of
2-quinolone and Asn-54 residue
˚
Local search
convergence = 0.000159 A
Local search steps = 20
˚
Population size = 50
Maximum generations = 1,000
123

Med Chem Res
the hydrophobic interaction between substituted phenyl
ring and Ileu-175 residue and one hydrogen bonding
between N-1 of 2-quinolone and Asn-54 residue. Molecu-
lar docking was carried out for total eight derivatives of
2-quinolones; among them Compound-2 and Compound-7
showed better binding interactions within active site of
DNA-gyrase B subunit.
Synthesis of 7-amino-4-methylquinolin-2(1H)-one (com-
pound-A, Fig. 4) included 10.8 g (0.1 mol) of m-phenyl-
enediamine and 12.64 ml (0.1 mol) of ethylacetoacetate,
which were taken together in a 250 ml round bottomed flask.
The reaction mixture was refluxed at 150 °C for 18 h on an
oil bath. At the end of the reaction period, 200 ml of water
was added to the flask, and the contents were heated on a hot
plate to the boiling temperature of water. The mixture was
then filtered; the filtrate was chilled in refrigerator till the
precipitation appeared. The precipitate was retrieved, dried
in air, recrystallized from methanol, and was characterized.
Synthesis
The chemicals and reagents used in the project work were
of AR and LR grade, procured from s.d.fine Chem. Ltd.,
National Chemicals, Qualigens Fine Chemicals, Ranbaxy
fine chemicals limited, Shital Chemical Industries, and
Finar reagents. The chemicals were used without further
purification.
Synthesis of derivatives (Fig. 4)
A mixture of 5.77 g (0.166 mol) 7-amino-4-methyl-
quinolin-2(1H)-one, 0.026 mol of different halo-acids
(o-chlorobenzoic acid, p-chlorobenzoic acid, m-bromo-
benzoic acid, 2-chloropyridine-3-carboxylic acid, 4-chlor-
opyridine-3-carboxylic acid, 6-chloropyridine-3-carboxylic
acid, 3-chloropyrazine-2-carboxylic acid, and 5-chloropyr-
azine-2-carboxylic acid), 0.82 g of K2-CO3, and 0.2 g of
copper foil and 0.02 g tin powder was heated under gentle
reflux for 2–3 h in a 250 ml round bottomed flask. The
mixture was cooled, and to the semi solid mass, a mixture of
5 ml of concentrated hydrochloric acid and 5 ml of water
Based on the molecular docking simulations studies, it
was decided to synthesize 2-quinolone and its derivatives.
The present study utilizes Conrad-Limpach synthesis for
the preparation of 7-amino-4-methyl-2-quinolone (Furniss
and Hannaford, 2005). It was obtained by the reaction
of m-phenylene diamine with ethylacetoacetate to form
the amido-ketone by cyclization at high temperature. The
nature of substituted anilines, the temperature, and the
solvent used greatly influence the synthesis.
Fig. 4 Showing the general
scheme of synthesis for
substituted 2-quinolone analogs
Compound Code
Compound-1
Compound-2
Compound-3
-Ar
o-chlorobenzoic acid
p-chlorobenzoic acid
m-bromobenzoic acid
Compound-4
Compound-5
2-chloropyridine-3-carboxylic acid
4-chloropyridine-3-carboxylic acid
Compound-6
Compound-7
6-chloropyridine-3-carboxylic acid
5-chloropyrazine-2-carboxylic acid
Compound-8
3-chloropyrazine-2-carboxylic acid
123

Med Chem Res
˚
˚
were added. The resulting mixture was shaken vigorously,
filtered, and the residue was dissolved in 4 ml of 4 N NaOH.
Then, decolorizing carbon was added and boiled for
2–3 min and filtered the hot mixture. The mixture was
cooled, and the filtrate was acidified with conc. hydrochloric
acid and filtered. The residue was washed with about 100 ml
of water and dried at 100 °C.
cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2 amide
3,200–3,300, NH (b) amide 1,550–1,600, C=O (str) 1,630–
1,660, C=C (str) aromatic 1,400–1,450, CH (str) aromatic
2,850–3,000, CH (str) aromatic in methyl 1,250–1,300,
C=O (str) in carboxylic acid 1,700–1,750, OH (str) in
carboxylic acid 2,500–2,800, C–O (str) in carboxylic acid
1,200–1,300. MASS m/e ratio: 295.10 [M?1]?, 267.11
[M–CHO]?, 227.07 [M ? 1] [M–C4H5O]?, 122.03 [M?1]
[M–C10H10ON2]?, 107.05 [M?2] [M–C11H10O3]?. 1H-
NMR (500 MHz, DMSO-d6): 2.44 (s, 3H, CH3), 4.08 (s,
1H, Ar–NH), 6.34 (s, 1H, Ar–H), 6.89 (s, 1H, Ar–H), 7.14
(d, 1H, Ar–H), 7.21 (d, 1H, Ar–H), 7.73 (d, 2H, Ar–2H),
7.81 (d, 2H, Ar–2H), 8.05 (s, 1H, Ar–NH), 11.0 (s, 1H, Ar–
COOH). 13C-NMR (500 MHz, DMSO-d6): 19, 110, 111,
121, 122, 128, 131, 138, 148, 162, 169.
Spectral studies
Compound-A (7-amino-4-methylquinolin-2(1H)-one)
The yield was 75 %, the melting point 265–268 °C, and the
Rf value 0.60. Spectroscopic analysis showed IR (KBr)
˚
cm-1: NH2 (str) 3,300–3,500, NH (str) 2 amide 3,200–
3,300, NH (b) amide 1,550–1,600, C=O (str) 1,630–1,660,
C=C (str) aromatic 1,400–1,450, CH (str) aromatic in
methyl 1,250–1,300, CH (str) aromatic 2,850–3,000.
MASS m/e ratio: 175.08 [M?1]?, 147.08 [M–CHO]?,
107.05 [M–C4H5O]?, 93.00 [M–C4H6NO]?. 1H-NMR
(500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 6.05 (d, 1H,
Ar–H), 6.27 (s, 2H, Ar–NH2), 6.35 (s, 1H, Ar–H), 6.90
(s, 1H, Ar–H), 7.12 (d, 1H, Ar–H), 8.05 (s, 1H, Ar–NH).
13C-NMR (500 MHz, DMSO-d6): 19, 107, 109, 110, 121,
127, 138, 146, 148, 162.
Compound-3 3-((4-methyl-2-oxo-1,2-dihydroquinolin-
7yl)amino)benzoic acid
The yield was 80 %, the melting point 178–180 °C, and the
Rf value 0.52. Spectroscopic analysis showed IR (KBr)
˚
˚
cm-1: NH (str) 2 amine 3,150–3,300, NH (str) 2 amide
3,100–3,300, NH (b) amide 1,550–1,615, C=O (str) 1,630–
1,660, C=C (str) aromatic 1,400–1,450, CH (str) aromatic
2,850–3,000, CH (str) aromatic in methyl 1,250–1,300,
C=O (str) in carboxylic acid 1,700–1,750, OH (str) in
carboxylic acid 2,500–2,800, C–O (str) in carboxylic acid
1,200–1,300. MASS m/e ratio: 295.30 [M?1]?, 267.65
[M–CHO]?, 227.02 [M?1] [M–C4H5O]?, 122.32 [M?1]
[M–C10H10ON2]?, 107.15 [M?2] [M–C11H10O3]? 1H-
NMR (500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 4.01 (s,
1H, Ar–NH), 6.35 (s, 1H, Ar–H), 6.90 (s, 1H, Ar–H), 7.11
(d, 1H, Ar–H), 7.21 (d, 1H, Ar–H), 7.27 (s, 1H, Ar–H),
7.41 (t, 1H, Ar–H), 7.57 (d, 1H, Ar–H), 7.97 (d, 1H, Ar–H),
8.02 (s, 1H, Ar–NH), 11.05 (s, 1H, Ar–COOH). 13C-NMR
(500 MHz, DMSO-d6): 19, 110, 111, 117, 120, 121, 126,
128, 129, 134, 137, 138, 139, 148, 162, 166.
Compound-1 2-((4-methyl-2-oxo-1,2-dihydroquinolin-
7yl)amino)benzoic acid
The yield was 72 %, the melting point 140–143 °C, and the
Rf value 0.49. Spectroscopic analysis showed IR (KBr)
˚
˚
cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2 amide
3,200–3,300, NH (b) amide 1,550–1,600, C=O (str) 1,630–
1,660, C=C (str) aromatic 1,400–1,450, CH (str) aromatic
2,850–3,000, CH (str) aromatic in methyl 1,250–1,300,
C=O (str) in carboxylic acid 1,700–1,750, OH (str) in
carboxylic acid 2,500–2,800, C–O (str) in carboxylic acid
1,200–1,300. MASS m/e ratio: 295.10 [M?1]?, 267.11
[M–CHO]?, 227.07 [M?1] [M–C4H5O]?, 122.03 [M?1]
[M–C10H10ON2]?, 107.05 [M?2] [M–C11H10O3]?. 1H-
NMR (500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 4.05 (s,
1H, Ar–NH), 6.35 (s, 1H, Ar–H), 6.90 (s, 1H, Ar–H), 7.02
(t, 1H, Ar–H), 7.11 (d, 1H, Ar–H), 7.21 (d, 1H, Ar–H), 7.54
(t, 1H, Ar–H), 7.85 (d, 1H, Ar–H), 8.05 (s, 1H, Ar–NH),
8.42 (d, 1H, Ar–H), 11.0 (s, 1H, Ar-COOH). 13C-NMR
(500 MHz, DMSO-d6): 19, 110, 112, 113, 118, 121, 128,
131, 135, 138, 162, 169.
Compound-4 2-((4-methyl-2-oxo-1,2-dihydroquinolin-7-
yl)amino)nicotinic acid
The yield was 68 %, the melting point 155–158 °C, and the
Rf value 0.44. Spectroscopic analysis showed IR (KBr)
˚
˚
cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2 amide
3,200–3,300, NH (b) amide 1,550–1,600, C=O (str) 1,630–
1,660, C=C (str) aromatic 1,400–1,450, CH (str) aromatic
2,850–3,000, CH (str) aromatic in methyl 1,250–1,300,
C=O (str) in carboxylic acid 1,700–1,750, OH (str) in
carboxylic acid 2,500–2,800, C–O (str) in carboxylic acid
1,200–1,300. C=N (str) aromatic 1,450–1,515. MASS m/e
ratio: 296.30 [M?1]?, 267.13 [M–CHO]?, 227.06 [M?1]
[M–C4H5O]?, 159.05 [M?1] [M–C7H8ON2]?, 124.03
[M?1] [M–C10H10ON2]?, 107.15 [M?2] [M–C11H10O3]?
Compound-2 4-((4-methyl-2-oxo-1,2-dihydroquinolin-
7yl)amino)benzoic acid
The yield was 78 %, the melting point 194–197 °C, and the
Rf value 0.45. Spectroscopic analysis showed IR (KBr)
123

Med Chem Res
1H-NMR (500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 4.00
(s, 1H, Ar–NH), 6.38 (s, 1H, Ar–H), 6.95 (s, 1H, Ar–H),
7.14 (d, 1H, Ar–H), 7.21 (d, 1H, Ar–H), 7.25 (t, 1H, Ar–H),
7.96 (d, 1H, Ar–H), 8.02 (s, 1H, Ar–NH), 8.28 (d, 1H, Ar–
H), 11.04 (s, 1H, Ar–COOH). 13C-NMR (500 MHz,
DMSO-d6): 19, 106, 108, 110, 112, 113, 121, 127, 138,
139, 142, 148, 151, 161, 162, 169.
(s, 1H, Ar–NH), 8.17 (s, 1H, Ar–H), 11.0 (s, 1H, Ar–
COOH). 13C-NMR (500 MHz, DMSO-d6): 19, 106, 107,
110, 112, 120, 127, 137, 140, 142, 148, 150, 158, 161, 166.
Compound-7 5-((4-methyl-2-oxo-1,2-dihydroquinolin-7-
yl)amino)pyrazine-2 carboxylic acid
The yield was 71 %, the melting point 205–207 °C, and
the Rf value 0.50. Spectroscopic analysis showed IR
Compound-5 4-((4-methyl-2-oxo-1,2-dihydroquinolin-7-
yl)amino)nicotinic acid
˚
˚
(KBr) cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2
amide 3,200–3,300, NH (b) amide 1,550–1,600, C=O (str)
1,630–1,660, C=C (str) aromatic 1,400–1,450, CH (str)
aromatic 2,850–3,000, C–N (str) aromatic 1,080–1,200,
CH (str) aromatic in methyl 1,250–1,300, C=O (str) in
carboxylic acid 1,700–1,750, OH (str) in carboxylic acid
2,500–2,800, C–O (str) in carboxylic acid 1,200–1,300,
C=N (str) aromatic 1,500–1,550. MASS m/e ratio: 297.09
[M?1]?, 269.10 [M–CHO]?, 254.07 [M?1] [M–
C2H4O]?, 229.06 [M?1] [M–C4H5O]?, 125.04 [M?1]
[M–C10H10ON2]?, 106.05 [M?1] [M–C9H9O3N2]?. 1H-
NMR (500 MHz, DMSO-d6): 2.40 (s, 3H, CH3), 4.02 (s,
1H, Ar–NH), 6.38 (s, 1H, Ar–H), 6.91 (s, 1H, Ar–H),
7.10 (d, 1H, Ar–H), 7.24 (d, 1H, Ar–H), 8.36 (s, 1H, Ar–
H), 8.61 (s, 1H, Ar–H), 8.01 (s, 1H, Ar–NH), 11.13 (s,
1H, Ar–COOH). 13C-NMR (500 MHz, DMSO-d6): 19,
107, 108, 110, 120, 127, 133, 136, 138, 142, 146, 148,
160, 162, 165.
The yield was 73 %, the melting point 180–182 °C, and the
Rf value 0.54. Spectroscopic analysis showed IR (KBr)
˚
˚
cm-1: NH (str) 2 amine 3,100–3,350, NH (str) 2 amide
3,225–3,300, NH (b) amide 1,550–1,600, C=O (str)
1,630–1,660, C=C (str) aromatic 1,400–1,450, CH (str)
aromatic 2,850–3,000, CH (str) aromatic in methyl
1,250–1,300, C=O (str) in carboxylic acid 1,700–1,750,
OH (str) in carboxylic acid 2,500–2,800, C–O (str) in
carboxylic acid 1,200–1,340. C=N (str) aromatic
1,450–1,550. MASS m/e ratio: 296.29 [M?1]?, 280.06
[M–CH3]?, 267.10 [M–CHO]?, 227.16 [M?1] [M–
C4H5O]?, 159.28 [M?1] [M–C7H8ON2]?, 124.16 [M?1]
[M–C10H10ON2]?, 107.15 [M?2] [M–C11H10O3]? 1H-
NMR (500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 4.01 (s,
1H, Ar–NH), 6.34 (s, 1H, Ar–H), 6.92 (s, 1H, Ar–H), 7.05
(d, 1H, Ar–H), 7.12 (d, 1H, Ar–H), 7.20 (d, 1H, Ar–H),
8.02 (s, 1H, Ar–NH), 8.71 (d, 1H, Ar–H), 8.73 (s, 1H, Ar–
H), 11.0 (s, 1H, Ar–COOH). 13C-NMR (500 MHz, DMSO-
d6): 19, 106, 108, 110, 120, 127, 137, 141, 148, 151, 152,
157, 162, 169.
Compound-8 3-((4-methyl-2-oxo-1,2-dihydroquinolin-7-
yl)amino)pyrazine-2-carboxylic acid
The yield was 76 %, the melting point 210–213 °C, and the
Rf value 0.52. Spectroscopic analysis showed IR (KBr)
˚
˚
Compound-6 6-((4-methyl-2-oxo-1,2-dihydroquinolin-7-
yl)amino)nicotinic acid
cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2 amide
3,225–3,300, NH (b) amide 1,550–1,600, C=O (str)
1,630–1,660, C=C (str) aromatic 1,400–1,450, CH (str)
aromatic 2,850–3,000, C–N (str) aromatic 1,080–1,200,
CH (str) aromatic in methyl 1,250–1,300, C=O (str) in
carboxylic acid 1,700–1,750, OH (str) in carboxylic acid
2,500–2,800, C–O (str) in carboxylic acid 1,200–1,300,
C=N (str) aromatic 1,450–1,550. MASS m/e ratio:
297.28 [M?1]?, 269.10 [M–CHO]?, 254.07 [M?1]
[M–C2H4O]?, 229.06 [M?1] [M–C4H5O]?, 184.07
The yield was 74 %, the melting point 167–170 °C, and the
Rf value 0.46. Spectroscopic analysis showed IR (KBr)
˚
˚
cm-1: NH (str) 2 amine 3,100–3,300, NH (str) 2 amide
3,200–3,300, NH (b) amide 1,550–1,600, C=O (str)
1,630–1,660, C=C (str) aromatic 1,400–1,450, CH (str)
aromatic 2,850–3,000, CH (str) aromatic in methyl 1,250–
1,300, C=O (str) in carboxylic acid 1,700–1,750, OH (str)
in carboxylic acid 2,500–2,800, C–O (str) in carboxylic
acid 1,200–1,300. C=N (str) aromatic 1,475–1,550. MASS
m/e ratio: 296.32 [M?1]?, 267.10 [M?1] [M–CHO]?,
227.16 [M?1] [M–C4H5O]?, 183.07 [M?1] [M–
C5H6O3]?, 124.16 [M?1] [M–C10H10ON2]?, 107.15
[M?2] [M–C11H10O3]?.1H-NMR (500 MHz, DMSO-d6):
2.41 (s, 3H, CH3), 4.03 (s, 1H, Ar–NH), 6.35 (s, 1H, Ar–
H), 6.82 (d, 1H, Ar–H), 6.90 (s, 1H, Ar–H), 7.11 (d, 1H,
Ar–H), 7.21 (d, 1H, Ar–H), 7.72 (d, 1H, Ar–H), 8.01
[M?1]
[M–C5H6O3]?,
125.04
[M?1]
[M–
C10H10ON2]?, 106.05 [M?1] [M–C9H9O3N2]?. 1H-
NMR (500 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 4.01
(s, 1H, Ar–NH), 6.35 (s, 1H, Ar–H), 6.91 (s, 1H, Ar–
H), 7.11 (d, 1H, Ar–H), 7.25 (d, 1H, Ar–H), 7.84 (d,
1H, Ar–H), 8.02 (s, 1H, Ar–NH), 8.40 (d, 1H, Ar–H),
11.03 (s, 1H, Ar–COOH). 13C-NMR (500 MHz, DMSO-
d6): 19, 107, 108, 110, 121, 127, 136, 138, 142, 148,
157, 162, 168.
123

Med Chem Res
Anti-bacterial activity
compound (Compound-A) were synthesized. The purity of
test compounds was checked by melting point determina-
tion and thin layer chromatography, and structures were
established by IR, 1H-NMR, and mass spectral studies. The
yields of final test compounds (Table 2) were in the range
of 65–80 %. The kmax values of the synthesized compounds
(Table 2) were found to be in the range of 265–295 nm.
Antimicrobial activity was determined by the agar diffusion
method. All eight synthesized test compounds were tested
against two species of bacteria, namely, S. aureus (gram-
positive) and E. coli (gram-negative). Stock solutions of
synthesized test compounds and standard drug were pre-
pared in DMSO. Ciprofloxacin was used as standard at a
concentration of 1,500 lM. The test compounds were used
at a concentration of 1,500 lM. Nutrient agar medium
(peptic digest of animal tissue 5 gms/liter, beef extract 1.50
gms/liter, yeast extract 1.50 gms/liter, sodium chloride 5
gms/liter, and agar 15 gms/liter in distilled water at a pH
7.4 0.2 and 25 °C, sterilized by autoclave at 15 lb pres-
sure at 121 °C for 15 min) was used for the agar diffusion
method. The petri-dishes were thoroughly washed and
sterilized in a hot air oven at 160 °C for 1 h. The inoculum
was added to the medium, which was poured into sterile
petri-dishes for solidifying. Wells (bores) were made in the
medium using a sterile borer after solidification. To the
respective bores, 0.1 ml of the test and standard solutions
were added. A control bore containing only DMSO was
maintained in each plate. The petri-dishes were kept at
room temperature for 30 min for diffusion to take place,
then incubated at 37 °C for 24 h. The zone of inhibition was
observed and measured using a scale.
The substitution on the parent molecule decreased the kmax
.
Out of the eight test compounds synthesized and tested for
their antibacterial activity (Table 3), compound-2 showed
activity better than rest of the other test compounds against
E. coli and S. aureus. Further, from the docking simulation
studies, it was found that compound-7 showed good inter-
actions within active site of the target enzyme DNA gyrase.
However, in reality when tested against the organisms, it
did not generate any useful data, which might be due to
impurity profile or improper diffusion of test compounds
into the agar medium. Based on the in silico approaches,
the parent test compound 7-amino-4-methyl-2-quinolone
(Compound-A) and its derivatives (Compound 1 to 8) were
synthesized. All the test compounds were characterized by
TLC (Toluene: methanol 4:2), melting point determination,
1
UV, IR, H-NMR, and mass spectral data. The test com-
pounds were obtained in the yield of 65–80 %. The pres-
ence of a singlet peak at 6.27 (NH2) in 1H-NMR of
compound-A and its absence, with presence of a new sin-
glet peak at 4.05 (NH) and the presence of a new singlet
peak at 11.0 (COOH) confirmed the characterization of
compound-A and its derivatives, respectively. The mass
spectra of all the test compounds were characterized by the
presence of molecular ion peak. The UV absorption char-
acteristics of the test compounds were then determined by
their kmax values. Out of the eight test compounds syn-
thesized and tested for their antibacterial activity, com-
pound-2 showed activity better than rest of the other test
compounds against E. coli and S. aureus.
Result and discussion
The designing of a theoretical molecule before arriving at
the New Chemical Entities is a promising approach. This
can be achieved by the rational approach to new drug
discovery. In the present study, docking has generated an
insight into developing an ideal DNA gyrase inhibitor from
2-quinolone class. Thus, in the present study, an approach
of designing 2-quinolone derivatives by docking simula-
tions, followed by the synthesis, characterization, and
biological evaluation was carried out.
Conclusion
Molecular docking was carried out for total eight
derivatives of 2-quinolones. Among them, compound-2 and
compound-7 showed better binding interactions within
active site of DNA-gyrase B subunit. The starting mate-
rials for the synthesis of 7-amino-4-methyl-2-quinolone
were m-phenylene diamine and ethylacetoacetate. The
m-phenylene diamine was treated with b-ketoester (ethy-
lacetoacetate) at high temperature to give 7-amino-4-
methylquinolin-2(1H)-one (Compound-A), the parent
compound. It was purified by recrystallization from meth-
anol, which gave pale brown colored needle-shaped crys-
tals. The test compound was characterized by melting point
determination and thin layer chromatography. Further,
eight acid derivatives (Compound 1 to 8) of the parent
The rational approach to lead discovery has prompted a
better insight into developing more specific 2-quinolones
analogs as potential antibacterial agents. The data showing
docking have definitely generated useful parameters nee-
ded for an ideal bacterial DNA gyrase inhibitor. The
rational approach to lead discovery presents a direct cor-
relation between an ideal therapeutically built up molecule
and the synthesized test compound. However, the present
study revealed that this method was not solely responsible
for generating functional activities of test compounds in
reality. This approach has also paved the way for gener-
ating more useful 2-quinolone analogs in future studies as
bacterial DNA gyrase inhibitors.
123

Med Chem Res
Table 2 Showing the melting point, yield, Rf value, and kmax of synthesized test compounds
Compound code
Synthesized compounds
Molecular
formula
Molecular
weight
Melting
point (°C)
Yield (%)
75
Rf value
kmax
(nm)
Compound-A
C10H10N2O
174.20
294.30
265–268
0.60
357.0
Compound-1
Compound-2
C17H14N2O3
140–143
72
78
0.49
276.0
C17H14N2O3
294.30
194–197
0.45
265.5
Compound-3
Compound-4
Compound-5
C17H14N2O3
C16H13N3O3
C16H13N3O3
294.30
295.29
295.29
178–180
155–158
180–182
80
68
73
0.52
0.44
0.54
271.0
268.0
273.5
Compound-6
C16H13N3O3
295.29
167–170
74
0.46
287.0
Compound-7
C15H12N4O3
296.09
205–207
71
0.50
267.5
123

Med Chem Res
Table 2 continued
Compound code
Synthesized compounds
Molecular
formula
Molecular
weight
Melting
point (°C)
Yield (%)
76
Rf value
kmax
(nm)
Compound-8
C15H12N4O3
296.09
210–213
0.52
294.0
4-thiadiazole-2-yl] piperazinyl quinolones. Eur J Med Chem 38:
851–854
Table 3 Showing the antibacterial activity of the synthesized test
compounds
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Compound code
Zone of inhibition (mm)
S. aureus
E. coli
1
2
3
4
5
6
7
8
9
10
Compound-A
Compound-1
Compound-2
Compound-3
Compound-4
Compound-5
Compound-6
Compound-7
Compound-8
Ciprofloxacin
5.5
8.0
4.0
7.0
12.8
7.0
4.2
7.0
–
13.5
7.0
5.0
9.0
3.0
9.0
8.5
8.0
14.0
7.5
2-(1-(2-substituted-phenyl)-5-methyl
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Acknowledgments The authors are grateful to Mr. Milan N. Patel,
and Mr. Sagar S. Patel for their encouragement throughout this study
and helpful discussions. Thanks are also due to members of the
microbiology section for their support and members of CSMCRI,
Bhavnagar for spectral measurements.
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