Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent Antimicrobial Agent

Article abstract of DOI:10.1002/jhet.3431

A series of quinoxalinone derivatives were synthesized by the reaction of o-phenylenediamine with oxalic acid to yield 1, 4-dihydro quinoxaline-2, 3-dione (1) and then treated with thionyl chloride to yield 2, 3 dichloro quinoxaline (2). This was further reacted with hydrazine hydrate to produce 2, 3-dihydrazinyl quinoxaline (3). This was finally reacted with substituted aromatic aldehydes to produce 2,3-bis[2-(sustituted benzylidine) hydrazinyl] quinoxalines (4). These quinoxalinone derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy and MASS spectral data. All the synthesized compounds were evaluated for their antimicrobial activity. The results of the antimicrobial study revealed that compounds 4c, 4d, and 4i were active and exhibited better inhibitory activities as compared to standard drug ciprofloxacin. The results were further checked with protein legend interaction by using docking studies, and all the compounds exhibited good docking scores between ?8.72 and ?11.29?kcal/mol against dihydrofolate reductase protein fragment from Staphylococcus aureus (PDB ID-4XE6). Among all compound, 4c has shown maximum docking score and found in agreement to in vitro studies.

Full text of DOI:10.1002/jhet.3431

Month 2018
Design, Synthesis, and Characterization of Quinoxaline Derivatives as a
Potent Antimicrobial Agent
Dhansay Dewangan,a
Kartik Nakhate, Achal Mishra,
Alok Singh Thakur,c Harish Rajak,d Jaya Dwivedi,e
a
b
*
Swapnil Sharma,f and Sarvesh Paliwalf
aRungta College of Pharmaceutical Sciences and Research, Bhilai 490024, Chhattisgarh, India
bShri Shankaracharya Institute of Pharmaceutical Science, Junwani 490020, Chhattisgarh, India
cSri Rawatpura Sarkar Institute of Pharmacy, Kumhari 490042, Chhattisgarh, India
dDepartment of Pharmacy, Guru Ghasidas Central University, Bilaspur 495009, Chhattisgarh, India
eDepartment of Chemistry, Banasthali University, Banasthali 304022, Rajasthan, India
fDepartment of Pharmacy, Banasthali University, Banasthali 304022, Rajasthan, India
*E-mail: achal.mishra03@gmail.com
Received July 3, 2018
DOI 10.1002/jhet.3431
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A series of quinoxalinone derivatives were synthesized by the reaction of o-phenylenediamine with oxalic
acid to yield 1, 4-dihydro quinoxaline-2, 3-dione (1) and then treated with thionyl chloride to yield 2, 3
dichloro quinoxaline (2). This was further reacted with hydrazine hydrate to produce 2, 3-dihydrazinyl
quinoxaline (3). This was finally reacted with substituted aromatic aldehydes to produce 2,3-bis[2-(sustituted
benzylidine) hydrazinyl] quinoxalines (4). These quinoxalinone derivatives were characterized by infrared
spectroscopy and nuclear magnetic resonance spectroscopy and MASS spectral data. All the synthesized
compounds were evaluated for their antimicrobial activity. The results of the antimicrobial study revealed
that compounds 4c, 4d, and 4i were active and exhibited better inhibitory activities as compared to standard
drug ciprofloxacin. The results were further checked with protein legend interaction by using docking stud-
ies, and all the compounds exhibited good docking scores between À8.72 and À11.29 kcal/mol against
dihydrofolate reductase protein fragment from Staphylococcus aureus (PDB ID-4XE6). Among all com-
pound, 4c has shown maximum docking score and found in agreement to in vitro studies.
J. Heterocyclic Chem., 00, 00 (2018).
© 2018 Wiley Periodicals, Inc.

D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
INTRODUCTION
500MHz spectrometer using tetramethylsilane as an
internal reference, and the mass spectroscopy (MS) were
recorded on Aapi 3000 LC-MS.
Antimicrobial therapy nowadays is widely used for the
treatment and prevention of the most of the microbial
infections. Unfortunately, the long-term use and misuse
of these agents contribute to severe adverse effect and
development of resistance. The failure of recently
available antimicrobial therapies and development of
resistance, increased a load of adverse effects. It suggest
us to develop new therapeutic agents with minimal
adverse effect and also enhanced antimicrobial spectrum
which could decrease the acquaintance of resistance in
microorganisms [1–5].
The synthesized compounds 4(a–l) were examined for their
in vitro antibacterial activities against two gram-negative
bacteria [E. coli (MTCC 443) and P. aeruginosa (MTCC
424)] and two gram-positive bacteria [S. aureus (MTCC 96)
and S. pyogenes (MTCC 442)]. Zone of inhibition of all
derivatives was determined and compared with standard
ciprofloxacin which was used as reference drug. Finally,
biological studies were correlates with docking study.
General method of synthesis of 1, 4-dihydro quinoxaline-2,
3-dione (1). An aqueous solution of oxalic acid dihydrate
(0.238 mol, 15 g) and 4.5 mL of concentrated HCl (4N)
was heated to 100°C followed by the addition of
o-phenylenediamine (0.204 mol, 11 g) with continuous
stirring. The reaction mixture was refluxed at 100°C for
1 h. The precipitate was obtained after addition of
reaction mixture to the crushed ice and was filtered and
washed with water; compound was recrystallized from
the precipitate and was obtained after addition of reaction
mixture to the crushed ice [27].
Synthesis of 2, 3 dichloro quinoxaline (2). A mixture of
quinoxaline-2, 3-dione (0.1 mol, 8.1 g) and freshly
distilled thionyl chloride (SOCl2, 60 mL) was refluxed
with N,N-dimethylformamide (5 mL) for 2 h. Resulting
reaction mixture was left for 30 min. The reaction
mixture was slowly poured into ice water with stirring.
The resulting reddish solid was filtered and washed with
water. The precipitate was recrystallized from a mixture
of chloroform and n-hexane [28,29].
Echinomycin, levomycin, and actinoleutins antibiotics are
the drugs containing quinoxaline as basic nucleus and are
widely used in the clinical practice. Echinomycin inhibits
microbial RNA synthesis by intercalation in double-
stranded DNA through nucleotide sequence selection [6,7].
Quinoxaline derivatives are extensively spattered
bioactive class of heterocyclic compound and act as a
bioisostere of naphthalene, benzothiophene, and quinoline
which are known leads for many antibiotics [8–11].
Quinoxaline and its derivatives exhibited diverse
pharmacological properties like antibacterial [12–14],
antitubercular [15,16], antiviral [17], antifungal [18,19],
anticancer [20,21], antimalarial [22,23], and anti-
inflammatory [24]. Various reports have shown that
quinoxaline analogs were used as potential dihydrofolate
reductase (DHFR) inhibitors [25]. DHFR is a lead enzyme
in the biosynthesis of folic acid and nucleic acids through
catalysis of the 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate.
Moreover, it is actively involved in the production of purine
and pyrimidine base. Hence, DHFR has become a crucial
and established potential target in antimicrobial therapy [26].
In the present study, new quinoxaline derivatives were
synthesized, and their structures were confirmed by
physicochemical and spectroscopic data. The minimal
inhibitory concentration (MIC) of derivatives was
assessed on two gram-positive strains S. aureus and
S. pyogenes and two gram-negative Escherichia coli and
Pseudomonas aeruginosa by disk diffusion method using
ciprofloxacin as a standard drug. Furthermore, the
derivatives were subjected to docking study to understand
the protein–ligand interaction.
Synthesis of 2, 3-dihydrazinyl quinoxaline (3).
A
mixture of 2, 3 dichloro quinoxaline (4.6 g, 0.01 mol)
and hydrazine hydrate (0.64 g, 0.01 mol) was dissolved
in absolute ethanol and refluxed for 16 h on a water bath.
After completion of the reaction, the reaction mixture
was cooled and poured onto the crushed ice. The solid
product so obtained was separated and crystallized to
yield 2, 3-dihydrazinyl quinoxaline [30,31].
Synthesis of 2,3-bis(2-(sustituted benzylidine) hydrazinyl)
quinoxalines (4). Equimolar quantities of 2, 3-dihydrazinyl
quinoxaline and appropriate substituted aromatic aldehydes
were refluxed on a water bath for 10 h using absolute
ethanol as a solvent. After completion of reaction, the
mixture was poured onto crushed ice, and the solid product
so obtained was recrystallized to give 2,3-bis(2-(substituted
benzylidine) hydrazinyl) quinoxalines [32].
EXPERIMENTAL
An open capillary method was adopted to determine the
melting points, and the purity of compounds was checked
by thin-layer chromatography (TLC). Fourier transform
infrared (FTIR) spectra (KBR, cm-1) were recorded on
Perkins Elmer Infrared-283 FTIR, nuclear magnetic
resonance spectroscopy (1H-NMR) were on a Bruker
IN VITRO ANTIBACTERIAL SCREENING OF
SYNTHESIZED COMPOUNDS
The in vitro antimicrobial studies of all the synthesized
compounds were performed against the gram-positive
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent
Antimicrobial Agent
bacteria [S. aureus (MTCC 96) and S. pyogenes (MTCC
442)] and gram-negative bacteria [E. coli (MTCC 443)
and P. aeruginosa (MTCC 424)] by using the cup-plate/
disc diffusion method. Ciprofloxacin was used as
standard for antimicrobial studies. Nutrient agar (peptone
10 g, beef extract 10 g, agar 20 g, sodium chloride 5 g,
and purified water 1000 mL) was used as culture media
for the studies. The ingredients were dissolved in water,
and pH is adjusted to 7.2 to 7.4 by using dilute
acid/alkali. The resulting solution was autoclave at 120°C
for 20 min; 30–35 mL of nutrient agar was transferred to
the petridish; 1000 and 500 μg/disc concentrations of the
test compounds are prepared; and dimethylformamide
was used as vehicle. Aseptically nutrient agar plates were
prepared to get a thickness of 5–6 mm and allowed them
to solidify and invert the plate to prevent condensate
falling on the agar surface. The plates were dried at 37°C
just before inoculation. The standard inoculums were
inoculated in the plates prepared earlier aseptically by
dipping a sterile swab in the inoculums, removing the
excess of inoculums by pressing and rotating the swab
firmly against the sides of the culture tube above the level
of the liquid, and finally streaking the swab all over the
surface of sterile culture media. The sterilized discs for
the test drugs were placed in the Petri dishes aseptically.
Incubate the Petri dish at 37°C
0.2°C for about
18–24 h, after placing them in the refrigerator for 1 h to
facilitate uniform diffusion. The average zone diameter of
the plates was measured and recorded. All synthesized
compounds were tested for antibacterial activity [33].
Minimal inhibitory concentration of the test compounds
was performed for microorganisms used in the primary
screening by using the micro-dilution susceptibility
method in Muller–Hinton broth. Dimethyl sulphoxide
was used as a solvent to dissolve the standard antibiotic
and test compounds at 64 mg/mL concentration. The
Figure 1. Representation of the series of reaction for the synthesis of different quinoxaline derivatives.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent
Antimicrobial Agent
Table 2
Spectral data of synthesized quinoxaline derivatives.
Derivatives
IR (KBr cm-1)
1H-NMR δ (ppm) (DMSO-d6)
MS
4a
3112.22 (Ar-C-H str.), 1591.46 (Ar-C=C str.),
1232.60 (Ar-C-C str.), 1491.10 (C=N str.),
1231.10 (-C-N- str.), 1372.90 (C-F str.)
3088.68 (Ar-C-H str.), 1545.28 (C=C str.),
1639.71 (C=N str.), 1177.44 (Ar-C-C str.),
1126.44 (C-N str.), 722.78 (C-Cl str.)
6.94-7.65 (m 8H, Ar-H), 4.1 (s 2H, -hydrazine),
7.60–8.20 (d 4H, quinoxaline ring), 8.14
(s 2H, -imine)
7.14-7.85 (m 8H, Ar-H), 4.0 (s 2H, -hydrazine),
7.68-8.07 (d 4H, quinoxaline ring), 8.20
(s 2H, -imine)
7.11-7.88 (m 8H, Ar-H), 4.2 (s 2H, -hydrazine),
7.50-8.15 (d 4H, quinoxaline ring), 8.10
(s 2H, -imine)
7.30-7.75 (m 8H, Ar-H), 4.0 (s 2H, -hydrazine),
7.68-8.27 (d 4H, quinoxaline ring), 8.10
(s 2H, -imine)
6.65-7.75 (m 8H, Ar-H), 4.2 (s 2H, -hydrazine),
7.68-8.20 (d 4H, quinoxaline ring), 8.12
(s 2H, -imine), 3.45 (s 6H,-OCH3)
6.71-7.25 (m 6H, Ar-H), 4.1 (s 2H, -hydrazine),
7.58-8.28 (d 4H, quinoxaline ring), 8.15
(s 2H, -imine), 3.75 (s 12H,-OCH3)
6.65-7.31 (m 4H, Ar-H), 4.5 (s 2H, -hydrazine),
7.68-8.22 (d 4H, quinoxaline ring),
8.13 (s 2H, -imine), 3.73 (s 18H,-OCH3)
7.11-7.88 (m 8H, Ar-H), 4.0 (s 2H, -hydrazine),
7.68-8.04(d 4H, quinoxaline ring), 8.00
(s 2H, -imine)
7.30-7.60 (m 8H, Ar-H), 4.0 (s 2H, -hydrazine),
7.60-8.10 (d 4H, quinoxaline ring), 8.15
(s 2H, -imine)
6.70-7.45 (m 10H, Ar-H), 4.25 (s 2H, -hydrazine),
7.65-8.25 (d 4H, quinoxaline ring), 8.15
(s 2H, -imine)
401+
4b
4c
4d
4e
4f
434+
434+
434+
425+
485+
545+
456+
456+
365+
457+
3133.51 (Ar-C-H str.), 1522.43 (C=C str.),
1622.43 (C=N str.), 1116.44 (C-N str.),
1117.44 (Ar-C-C str.), 742.70 (C-Cl str.)
3077.89 (Ar-C-H str.), 1667.89 (Ar-C=C str.),
1257.78 (Ar-C-C str.), 1557.89 (C=N str.),
1180.56 (-C-N- str.), 752.70 (C-Cl str.)
2922.56 (Ar-C-H str.), 1633.56 (Ar-C=C str),
1111.67 (Ar-C-C str.), 1531.12 (C=N str.),
1223.87 (-C-N- str.), 1022.65 (C-O-C str. OCH3)
2880.45 (Ar-C-H str.), 1621.76 (Ar-C=C str.),
1118.45 (Ar-C-C str.), 1531.10 (C=N str.),
1266.45 (-C-N- str.), 1082.60 (C-O-C str. OCH3)
3134.78 (Ar-C-H str.), 1662.89 (Ar-C=C str.),
1136.89 (Ar-C-C str.), 1521.21 (C=N str.),
1254.76 (-C-N- str.), 1132.30 (C-O-C str. OCH3)
3068.45 (Ar-C-H str.), 1642.67 (Ar-C=C str.),
1115.78 (Ar-C-C str.), 1510.90 (C=N str.),
1220.43 (-C-N- str.), 1287.12 (NO2 str.)
2920.56 (Ar-C-H str.), 1633.56 (Ar-C=C str.),
1131.67 (Ar-C-C str.), 1541.12 (C=N str.),
1220.87 (-C-N- str.), 1237.12 (NO2 str.)
2912.12 (Ar-C-H str.), 1625.45 (Ar-C=C str.),
1225.60 (Ar-C-C str.), 1525.10 (C=N str.),
1165.10 (-C-N- str.)
4g
4h
4i
4j
4k
3123.20 (Ar-C-H str.), 1580.45 (Ar-C=C str.),
1240.60 (Ar-C-C str.), 1452.10 (C=N str.),
1225.10 (-C-N- str.), 1018.65 (C-O-C str. OCH3),
3443.89 (O-H str.)
3057.89 (Ar-C-H str.), 1647.89 (Ar-C=C str.),
1237.78 (Ar-C-C str.), 1556.89 (C=N str.),
1150.56 (-C-N- str.), 3393.89 (O-H str.)
6.70-7.70 (m 6H, Ar-H), 4.2 (s 2H, -hydrazine),
7.68-8.20 (d 4H, quinoxaline ring), 8.10
(s 2H, -imine), 3.75 (s 6H,-OCH3), 5.1 (s 2H,-OH)
4l
6.80-7.40 (m 6H, Ar-H), 4.10 (s 2H, -hydrazine),
7.68-8.20 (d 4H, quinoxaline ring), 8.12 (s 2H, -imine),
5.0 (s 2H,-OH)
397+
Table 3
Combustion analysis of quinoxaline derivatives.
Combustion analysis
Derivatives
Theoretical value
Observed values
C (66.97%) H (4.09%) Cl (9.26%) N (21.29%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
C (65.66%) H (4.01%) Cl (9.44%) N (20.88)
C (60.70%) H (3.70%) Cl (16.29%) N (19.31%)
C (60.70%) H (3.70%) Cl (16.29%) N (19.31)
C (60.70%) H (3.70%) Cl (16.29%) N (19.31)
C (67.59%) H (5.20%) O (7.50%) N (19.71%)
C (64.19%) H (5.39%) O (13.15%) N (17.27%)
C (61.53%) H (5.53%) O (17.56%) N (15.38%)
C (57.89%) H (3.53%) O (14.02%) N (24.55%)
C (72.11%) H (4.95%) N (22.94%)
C (59.49%) H (3.63%) Cl (16.61%) N (19.69%)
C (61.91%) H (3.63%) Cl (15.96%) N (19.31)
C (60.70%) H (3.70%) Cl (16.29%) N (18.92)
C (66.23%) H (5.30%) O (7.65%) N (19.31%)
C (65.48%) H (5.47%) O (13.15%) N (16.92%)
C (60.27%) H (5.63%) O (17.90%) N (15.07%)
C (59.04%) H (3.43%) O (13.73) N (25.04%)
C (73.41%) H (4.909%) N (23.24%)
4j
4k
4l
C (62.87%) H (4.84%) O (13.96%) N (18.33%)
C (66.32%) H (4.55%) O (8.03%) N (21.09%)
C (65.66%) H (4.01%) F (9.44%) N (20.88%)
C (63.87%) H (4.74%) O (14.23%) N (18.01%)
C (66.46%) H (4.53%) O (7.97%) N (20.66%)
C (65.47%) H (4.07%) F (9.31%) N (20.67%)
Journal of Heterocyclic Chemistry
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D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
solution was twofold diluted (64 to 0.5 mg/mL). A
prepared suspension of the standard microorganisms was
added to each dilution in a 1:1 ratio. Growth (or its lack)
of microorganisms was determined visually after
incubation for 24 h at 37°C. The lowest concentration at
which there was no visible growth (turbidity) was taken
as the MIC [34].
the way of the corresponding synergy of the test
compound with the potential target. From the protein data
bank, corresponding protein structure of DHFR from
S. aureus (PDB ID-4XE6) was obtained. Chem draw
ultra 10.0 was used for 3D molecule development, and
the structure was saved as.pdb format. Genetic Algorithm
dock and Argus dock (shape-based search algorithm)
were used for docking simulation. In the present study,
Argus dock was selected in docking engine to perform
the docking simulation, and for calculations, “Dock”
option was chosen as the calculation type. The protein–
ligand interaction was determined by using Pymol 1.3
software [36].
MOLECULAR DOCKING STUDY
Molecular docking study was performed using free
available docking software Argus Lab 4.0 [35] to observe
Table 4
Antibacterial activity of synthesized quinolaxine derivatives using plate-hole diffusion method.
Zone of inhibition (mm)
Gram-positive bacteria
S. aureus S. pyogenes
Gram-negative bacteria
P. aeruginosa
E. coli
Compounds
500 μg/mL
1000 μg/mL
500 μg/mL
1000 μg/mL
500 μg/mL
1000 μg/mL
500 μg/mL
1000 μg/mL
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
11
12
14
13
12
12
09
12
13
13
11
12
13
--
21
22
23
23
17
20
16
22
23
22
20
22
23
--
11
13
14
14
10
13
10
12
13
15
12
14
13
--
19
20
23
23
17
20
18
20
22
22
20
21
22
--
13
14
16
15
12
12
--
11
15
14
14
11
15
--
20
22
23
23
17
21
--
22
22
22
21
22
24
--
12
13
14
13
--
19
20
22
22
--
--
--
10
12
13
13
10
14
14
--
14
20
21
21
19
22
23
--
Ciprofloxacin
DMF (control)
Table 5
Antimicrobial activity of the synthesized compounds expressed as MIC (mg/mL).
Gram-positive bacteria Gram-negative bacteria
S. pyogenes
Compounds
S. aureus
E. coli
P. aeruginosa
1.97
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
1.10
1.09
0.97
1.01
2.87
2.97
3.03
2.14
1.04
2.89
1.87
1.97
0.97
1.28
1.12
0.92
1.03
3.28
3.31
3.43
1.98
1.06
2.91
2.12
2.08
0.89
1.89
1.37
0.93
0.96
3.17
2.97
3.23
1.87
1.10
2.76
2.28
1.83
0.93
1.84
0.98
1.02
3.14
3.48
3.17
2.73
1.07
3.01
1.98
1.79
0.96
Ciprofloxacin
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent
Antimicrobial Agent
RESULTS AND DISCUSSION
3-dihydrazinyl quinoxaline with different aromatic
aldehydes. Chemical structure, melting point, and other
physical data were mentioned in Table 1. Formation of
different 2,3-bis(2-(sustituted benzylidine) hydrazinyl)
quinoxalines derivatives was established by recording
The series of reactions are given in Scheme 1 of
Figure 1. Quinoxaline derivatives were synthesized by
the reaction of o-phenylenediamine with oxalic acid to
yield 1, 4-dihydro quinoxaline-2, 3-dione and then treated
with thionyl chloride to yield 2, 3 dichloro quinoxaline.
This was further reacted with hydrazine hydrate to
produce 2, 3-dihydrazinyl quinoxaline. This was finally
reacted with the substituted aromatic aldehyde to produce
1
there IR, H-NMR, and MS.
The characterization of structure was performed by the
FTIR, 1H-NMR, MS, and elemental analysis. The IR
spectra helped to confirm the structure by assuring the
presence of different functional molecule. The IR spectra
of compound III (intermediate) showed the appearance of
peak around 1482 and 1260 cm-1 and were suggested the
presence of C=N str. and C-N str. group, respectively, in
compound as closed heterocyclic ring (quinoxaline). The
support for quinoxaline heterocycle was made by the
appearance of IR around 3025 cm-1 for C-H str. which is
of fused aromatic ring. This interpretation was
complemented by the appearance of NMR multiplet peak
around δ 7.2–7.8 for Ar-C-H. Furthermore, the IR spectra
2,3-bis(2-(substituted
benzylidine)
hydrazinyl)
quinoxaline. The purity of the derivatives was confirmed
by TLC and melting point. Structure of these derivatives
was set up by determining infrared spectroscopy (IR),
1H-NMR, and MS. In addition to this, the synthesized
derivatives were screened for their antimicrobial activities.
The synthetic way used to produce different quinoxaline
derivatives is outlined in Scheme 1. In total, 12 different
quinoxaline derivatives were prepared by treating 2,
Table 6
Docking parameters used in Argus Lab 4.0.1.
Compound ID(s)
Constant term
vdW coefficient
H-bond coefficient neutral–neutral
Rotors coefficient
Hydrophobic coefficient
4m
4n
4o
4q
4q
4r
4s
4t
4u
4v
4w
4x
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
Grid parameters: spacing 0.375 Å and grid sizes 80X Å, 80Y Å, and 80Z Å.
Table 7
Properties of ligands on the basis of their molecular structure.
Molecular parameters*
Compound ID(s)
Strech
Bend
Strech–bend Torsion Non-1,4 VDW 1,4 VDW Dipole/dipole
Total energy (kcal/mol)
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.77
1.03
0.82
0.81
1.34
1.93
2.85
85.88
0.93
0.73
1.38
0.96
3.36
3.79
3.36
3.37
8.06
8.48
12.35
83.18
3.36
5.68
7.74
3.98
0.03
0.09
0.06
0.05
0.12
À20.06
À17.67
À20.18
À19.64
À17.70
À2.17
À2.76
À1.90
À2.98
À2.92
À2.03
À9.08
À10.57
À4.77
À2.92
À3.21
À6.45
À3.75
16.21
17.59
17.11
17.05
21.55
27.04
32.40
8.66
18.05
15.64
21.86
16.40
1.48
1.52
1.51
1.49
1.43
2.80
3.30
À0.55
1.46
1.38
1.00
2.50
À0.95
4.45
À029
0.22
12.78
28.97
41.10
À94.13
0.23
7.56
12.98
11.14
À0.04
À0.11
À237.30
0.09
0.86
À29.30
À18.64
À12.72
À12.51
À8.91
4j
4k
4l
0.05
À0.04
À0.04
*Data generated using ChemBioDraw 13 software after MM2 energy minimization.
Journal of Heterocyclic Chemistry
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D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
Table 8
Chemical properties of ligands
Compound ID(s)
Molecular formula
Molecular mass (g/mol)
CLogP values*
CMR
Gibbs energy: (kJ/mol)
4a
4b
4c
4d
4e
4f
4g
4h
4i
C22H16F2N6
C22H16Cl2N6
C22H16Cl2N6
C22H16Cl2N6
C24H22N6O2
C26H26N6O4
C28H30N6O6
C22H16N8O4
C22H16N8O4
C22H18N6
402
435
435
435
426
486
546
457
457
366
458
398
6.55
6.49
7.69
7.69
6.69
6.12
4.50
5.04
5.04
6.24
5.82
7.44
11.66
12.61
12.61
12.61
12.86
14.1
15.33
12.85
12.85
11.63
13.17
11.93
302.11
662.85
662.85
662.84
388.61
59.95
À268.71
--
--
4j
4k
4l
717.27
33.99
362.65
C24H22N6O4
C22H18N6O2
*Data generated using ChemBioDraw 13 software.
Figure 2. Interaction study and pose view of compound 4c with binding domain of 4XE6 protein-hydrogen binding of analog 4e with amino acids, amine
group of hydrazine, and ring nitrogen of quinoxaline interact with 46THR and 49SER residues and bond distance 2.3 Å and 2.9 Å. [Color figure can be
viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent
Antimicrobial Agent
Table 9
Docking results of ligands and standard drug against DHFR (4XE6).
Compound
ID (s)
Binding energy No. of hydrogen Bond length of
H-bond with
receptor residue
(kcal/mol)
bonds
H-bonds in Å
Enzyme’s binding site residue
4a
À9.74
1
2.77
28LEU
57ARG(beta strand), 50ILE(coil), 20LEU(coil),
28LEU(alpha helix), 54LEU(beta strand),
29LYS(alpha helix), 32LYS(alpha helix),
52LYS(alpha helix), 92PHE(coil), 55PRO(beta
strand), 46THR(alpha helix), 31VAL(alpha
helix)
4b
À10.21
À11.29
À11.17
À9.70
1
2
2
2
3
2.92
28LEU
57ARG(beta strand), 30HIS(alpha helix),
50ILE(coil), 20LEU(coil), 28LEU(alpha helix),
54LEU(beta strand), 40LEU(beta strand),
29LYS(alpha helix), 32LYS(alpha helix),
33LYS(alpha helix), 52LYS(alpha helix),
42MET(beta strand), 92PHE(coil), 55PRO(beta
strand), 331VAL(alpha helix)
7ALA(beta strand), 18ASN(coil), 19GLN(coil),
15GLY(coil), 93GLY(coil), 14ILE(coil),
50ILE(coil), 5LEU(beta strand), 20LEU(coil),
28LEU(alpha helix), 54LEU(beta strand),
92PHE(coil), 98PHE(alpha helix), 123PHE(beta
strand), 49SER(coil), 121THR(beta strand),
6VAL(beta strand), 31VAL(alpha helix)
57ARG(beta strand), 27ASP(alpha helix),
50ILE(coil), 20LEU(coil), 28LEU(alpha helix),
54LEU(beta strand), 29LYS(alpha helix),
32LYS(alpha helix), 45LYS(alpha helix),
92PHE(coil), 53PRO(beta strand), 55PRO(beta
strand), 46THR(alpha helix), 31VAL(alpha
helix)
57ARG(beta strand), 30HIS(alpha helix),
50ILE(coil), 20LEU(coil), 28LEU(alpha helix),
54LEU(beta strand), 29LYS(alpha helix),
32LYS(alpha helix), 33LYS(alpha helix),
52LYS(beta strand), 92 PHE (coil), 53PRO(beta
strand), 55PRO(beta strand), 49SER(coil),
46THR(alpha helix), 31VAL(alpha helix)
7ALA(beta strand), 57ARG(beta strand),
18ASN(coil), 27ASP(alpha helix), 19GLN(coil),
15GLY(coil), 14ILE(coil), 50ILE(coil),
113ILE(beta strand), 5LEU(beta strand),
20LEU(coil), 24LEU(coil), 28LEU(coil),
40LEU(beta strand), 54EU(beta strand),
32LYS(alpha helix), 16PHE(coil), 92PHE(coil),
35SER(coil), 49SER(coil), 46THR(alpha helix),
22TRP(coil), 6VAL(beta strand), 31VAL(alpha
helix)
4c
4d
4e
4f
2.30
2.90
46THR
49SER
2.60
2.80
46THR
49SER
2.30
2.80
52LYS
28LEU
À9.78
2.63
2.66
2.69
7ALA
20LEU
49SER
4g
À8.72
4
2.34
2.40
2.72
2.99
12ARG
10LEU
12ARG
12ARG
12ARG(coil), 9ASP(coil), 11GLN(coil),
114GLU(coil), 129GLU(coil), 8HIS(beta strand),
10LEU(coil), 123PHE(beta strand),
128PHE(coil), 124PRO(beta strand),
125PRO(beta strand), 127THR(beta strand),
126TYR(beta strand), 112VAL(beta strand)
18ASN(coil), 27ASP(alpha helix), 19GLN(coil),
23HIS(coil), 50ILE(coil), 20LEU(coil),
24LEU(coil), 28LEU(alpha helix), 92PHE(coil),
49SER(coil), 22TRP(coil), 31VAL(alpha helix)
7ALA(beta strand), 18ASN(coil), 19GLN(coil),
17GLU(coil), 15GLY(coil), 93GLY(coil),
94GLY(coil), 14ILE(coil), 50ILE(coil),
5LEU(beta strand), 20LEU(coil), 45LYS(alpha
helix), 16PHE(coil), 92PHE(coil), 98PHE(alpha
helix), 49SER(coil), 46THR(alpha helix),
121THR(beta strand), 6VAL(beta strand)
4k
4i
À10.05
À10.85
3
5
2.13
2.40
2.82
20LEU
22TRP
49SER
2.15
2.39
2.57
2.65
2.99
20LEU
18ASN
121THR
49SER
49SER
(Continues)
Journal of Heterocyclic Chemistry
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D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
Table 9
(Continued)
Compound
ID (s)
Binding energy No. of hydrogen Bond length of
H-bond with
receptor residue
(kcal/mol)
bonds
H-bonds in Å
Enzyme’s binding site residue
4j
À10.77
0
-
-
7ALA(beta strand), 27ASP(alpha helix),
93GLY(coil), 94GLY(coil), 23HIS(coil),
15ILE(coil), 50ILE(coil), 5LEU(beta strand),
20LEU(coil), 24LEU(coil), 28LEU(alpha helix),
54LEU(beta strand), 32LYS(alpha helix),
92PHE(coil), 98PHE(alpha helix), 22TRP(coil),
6VAL(beta strand), 31VAL(alpha helix)
7ALA(beta strand), 57ARG(beta strand),
18ASN(coil), 27ASP(alpha helix), 19GLN(coil),
51GLY(coil), 93GLY(coil), 14ILE(coil),
50ILE(coil), 5LEU(beta strand), 20LEU(coil),
28LEU(alpha helix), 40LEU(beta strand),
54LEU(beta strand), 32LYS(alpha helix), 92PHE
(coil), 35SER(coil), 49SER(coil), 46THR(coil),
22TRP(coil), 6VAL(beta strand), 31VAL(alpha
helix)
4k
À10.08
4
2.49
2.74
2.80
2.99
49SER
50ILE
7ALA
7ALA
4l
À10.60
À7.57
4
3
2.42
2.65
2.68
2.83
27ASP
27ASP
22TRP
24LEU
7ALA(beta strand), 27ASP(alpha helix),
23HIS(coil), 30HIS(alpha helix), 50ILE(coil),
5LEU(beta strand, 20LEU(coil), 24LEU(coil),
28LEU(coil), 40LEU(beta strand), 54LEU(beta
strand), 92PHE(coil), 25PRO(alpha helix),
111THR(beta strand), 22TRP(coil), 6VAL(beta
strand), 31VAL(alpha helix)
7ALA(beta strand), 18ASN(coil), 29GLN(coil),
95GLN(alpha helix), 15GLY(coil), 93GLY(coil),
94GLY(coil), 14ILE(coil), 50ILE(coil),
5LEU(beta strand), 20LEU(coil), 54LEU(beta
strand), 45LYS(alpha helix), 16PHE(coil),
92PHE(coil), 98PHE(alpha helix), 49SER(coil),
46THR(alpha helix), 121THR(beta strand),
6VAL(beta strand), 31VAL(alpha helix)
Ciprofloxacin
2.71
2.93
2.94
6VAL
7ALA
121THR
Grid parameters (Argus Lab.): spacing 0.375 Å and grid sizes 80X Å, 80Y Å, and 80Z Å.
containing a peak at 3424 cm-1 for primary and absorption
in the region 1120–1165 cm-1 for N-N str. confirm the
presence of attached hydrazine group. The hydrazine
group in also confirmed by the NMR absorption at δ 4.2
due to secondary amine (-NH-) and a doublet peak
around δ 2.2 for primary amine (-NH2) group. The
formation of compound 4a–l was assured due to the
manifestation of NMR peak for secondary amine at
around 4.3 and the absence of NMR peak for primary
amine due to its conversion to tertiary amine after the
attachment of benzal group with the formation of imine
bond (-N=CH-Ar). The attachment of substituted
aromatic ring in different derivatives (4a–l) was well
supported by NMR spectra as on the presence of cluster
of multiplet peaks around δ 7.4–8.6 for fused aromatic
ring of quinoxaline and δ 6.2–7.3 for substituted aromatic
as benzal. The presence of substituted ring was further
confirmed by the observation of IR peak around 3393
and 3443 cm-1 for -OH group in compounds 4l and 4k,
respectively, and 1022 and 1083 cm-1 C-O-C str.
(-OCH3) for compounds 4e and 4f, respectively. The
presence of these substituent groups, namely, -OH and
–OCH3 groups in synthesized derivatives, were cross
confirmed by NMR spectra around δ 5.00 and 3.45,
respectively. The mass spectra of quinoxaline derivative
(4a) showed a molecular ion peak at m/z 401 which is in
conformity with the molecular formula C22H16F2N6. In
the same way, spectral data of remaining derivatives are
given in Tables 2 and 3.
The synthesized compounds were tested for antibacterial
activity in vitro against microorganisms such as
S. pyogenes and S. aureus (gram-positive) and
P. aeruginosa and E. coli (gram-negative) using cup-
plate/disc diffusion method. Ciprofloxacin was used as
standard drug. The obtained results revealed that the
tested compounds indicated varying extent of activity
against the tested microorganisms Tables 4 and 5. In
the given results, compounds (4c, 4d, and 4i) were
highly active against both gram-positive and gram-
negative bacterial strain. The compounds 4c (23mm),
4d (23mm), and 4i (22mm) were significantly active
against E. coli and S. aureus when compared to
standard Ciprofloxacin (23 mm). Compounds 4e and 4f
showed no activity against P. aeruginosa, and
Journal of Heterocyclic Chemistry
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Design, Synthesis, and Characterization of Quinoxaline Derivatives as a Potent
Antimicrobial Agent
compound 4g was insensitive against E. coli. The rest of
estimated derivatives were exhibited good to moderate
activity against the selected strains of bacteria. The
MIC study was performed for synthesized compounds
for MIC determination micro-dilution susceptibility
method was used. The results were shown in Tables 5.
The gram-positive and gram-negative bacteria are
sensitive to most of the tested compounds, and some
compounds particularly 4c, 4d, and 4i have shown
significant activity against both type of microbial strains
than others. Antimicrobial activity of derivatives may
be attributed to the presence of electron withdrawing
groups. The presence of methoxy groups on the phenyl
side chain at the quinoxaline ring does not exhibit the
biological activity like standard ciprofloxacin.
The docking study gives an idea about an interaction
between the test compound and the potential target protein
as shown in Tables 6, 7, and 8. So to rationalize the
potency of the derivatives as an antimicrobial agent, all
synthesized derivatives were docked against DHFR
S. aureus (PBD ID-4XE6) obtained from protein data bank
(RCSB). It is well known that available docking software
has good success in generating active pose of the ligands
but that software are not generating docking scores in such
a good rate [37,38]. But in the current study, docking
scores seem to be in good agreement with the experimental
findings. The binding energy of drug-receptor complex and
standard drug are shown in Table 6. All synthesized
molecules interacted with the active sites of enzyme DHFR
from S. aureus (PBD ID-4XE6) through various bonds like
hydrogen, van der Waals, p-cation, p-anion, p-alkyl, p-
donor hydrogen, p-sulphur, carbon-hydrogen bond, and p-
sigma. Test compound showed the different modes of
binding with amino acids located at an active site of DHFR
(Fig. 2). The nitrogen atom of hydrazine group and
quinoxaline ring nitrogen heteroatom was forming the
hydrogen bond with amino groups of protein extracted as
PDB with specific bond distance. The ring nitrogen of
quinoxaline and the nitrogen atom of hydrazine group in
compound 4c (49 SER 2.9A°, 46 THR, 2.3A°) and the
nitrogen atom of quinoxaline of compound 4d (49 SER
2.8A°, 46THR 2.6A°) make the hydrogen bond with an
amino group of serine and threonine (Table 9). These
amino acids make a cascade to facilitate binding and
holding of the test compound in the active site of DHFR
protein. The binding energies of the all the derivatives
showed a good score of docking between À8.72 and
À11.29 kcal/mol and were better as compared to standard
drug ciprofloxacin. The compounds 4c (À11.29 kcal/mol),
4d (À11.17 kcal/mol), and 4i (À10.85 kcal/mol) represent
the promising docking score as compared with the standard
DHFR inhibitor ciprofloxacin (À7.57 kcal/mol) (Table 9).
The derivatives attributed the electron withdrawing group
having the better docking score than other substituents at
the same positions. Out of 21 amino acids of residue of
DHFR which is responsible for the formation of bonds
Figure 3. The figures A, B, C, represent 3D model of docking zone of ciprofloxacin, compounds 4c and 4d on the enzyme dihydrofolate reductase
(4XE6) in cartoon (1), solid surface (2), and transparent (3), respectively. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
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D. Dewangan, K. Nakhate, A. Mishra, A. S. Thakur, H. Rajak, J. Dwivedi, S. Sharma, and S. Paliwal Vol 000
with ciprofloxacin, 15 were found to be similar in
compounds like 4c [viz. 7ALA(beta strand), 18ASN(coil),
15GLY(coil), 93GLY(coil), 14ILE(coil), 50ILE(coil),
5LEU(beta strand), 20LEU(coil), 54LEU(beta strand),
considered that compounds 4c, 4d, and 4i exhibited
optimistic antimicrobial activity; furthermore, docking
studies suggested that compounds 4c and 4d were
interacted with protein more efficiently, and hence, these
derivatives could be further studied for their mechanisms
of action in depth and could be developed as effective
antimicrobial agents.
92PHE(coil),
121THR(beta
98PHE(alpha
strand), 6VAL(beta
helix),
49SER(coil),
strand), and
31VAL(alpha helix)] and 6 were found to be similar in
compounds like 4d [viz. 50ILE(coil), 20LEU(coil),
54LEU(beta strand45LYS(alpha helix), 92PHE(coil),
46THR(alpha helix), and 31VAL(alpha helix)]. The
compound 4c showed binding site alignment pattern
resemblance to that of ciprofloxacin with amino acid chain
of DHFR (4XE6) as shown in Figure 3.
Acknowledgments. The authors thank to the Management of
Shri Shankaracharya Institute of Pharmaceutical Science, Bhilai,
and Management of Rungta College of Pharmaceutical Sciences
and Research, Bhilai, for providing necessary infrastructural
facilities and IISER, Bhopal, for providing immense help in the
spectral analysis.
STRUCTURE ACTIVITY RELATIONSHIP
REFERENCES AND NOTES
From the results of in vivo antimicrobial activity of
newly prepared quinoxaline derivatives, the following
structural activity relationship was derived. The tested
derivatives 4c, 4d, and 4i were found to be promising
active against the tested strain of microbes. Almost all the
analogs showed good activity against all strains of bacteria
(Table 4 and 5). The antimicrobial activity of synthesized
compounds was compared to ciprofloxacin. The
structure–activity relationship study recommended that
electron withdrawing group substitutes were exhibiting
better activity against almost all the bacteria. Compounds
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substituents at the meta and para position of phenyl ring
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selected strains. Derivatives having an attachment of
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least effect on selected bacterial strains. Derivatives with
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used strains as compared to standard.
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Antimicrobial Agent
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in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet