Synthesis of Novel Substituted Phenyl-3-Hydrazinyl-Quinoxaline-2-Amine Derivatives: Evaluation of Antimicrobial Activity and Its Molecular Docking Studies

Article abstract of DOI:10.1002/jhet.3003

New series of quinoxaline derivatives (4a–4h) were synthesized by treating 2-chloro-3-hydrazinyl quinoxalin (3) with various anilines. Compound 3 was obtained from the 2,3-dichloroquinoxaline 2 which was prepared from 4-dihydroquinoxaline-2,3-dione (1). All synthesized compounds (4a–4h) were characterized by various spectral techniques, that is, IR, ¹H-NMR, mass spectroscopy, and elemental analysis and completion of reaction were confirmed by TLC. In vitro antimicrobial activity of synthesized compounds was evaluated using disc diffusion assay against gram-positive and gram-negative microbial strains, and then, the minimum inhibitory concentration and IC₅₀ values of compounds were also determined. The results of antimicrobial study revealed that compounds 4e, 4g, and 4a were active and exhibited better inhibitory activities as compared with standard drug amoxicillin. Docking studies were performed by using Argus lab, and all the compounds exhibited good docking scores between ?9.53 and ?7.94?kcal/mol against dihydrofolate reductase protein fragment from Staphylococcus aureus (PDB ID-4XE6). Among all compounds, 4e has shown the maximum docking score and found in agreement to in vitro studies.

Full text of DOI:10.1002/jhet.3003

Month 2017
Synthesis of Novel Substituted Phenyl-3-Hydrazinyl-Quinoxaline-2-
Amine Derivatives: Evaluation of Antimicrobial Activity and Its Molecular
Docking Studies
*
Sarvesh Paliwal, Swapnil Sharma, Jaya Dwivedi, and Achal Mishra
Department of Pharmacy, Banasthali Vidyapith University, Tonk, Niwai, Rajasthan, India
*
E-mail: achal. mishra03@gmail.com
Received February 3, 2017
DOI 10.1002/jhet.3003
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
New series of quinoxaline derivatives (4a–4h) were synthesized by treating 2-chloro-3-hydrazinyl
quinoxalin (3) with various anilines. Compound 3 was obtained from the 2,3-dichloroquinoxaline 2 which
was prepared from 4-dihydroquinoxaline-2,3-dione (1). All synthesized compounds (4a–4h) were character-
1
ized by various spectral techniques, that is, IR, H-NMR, mass spectroscopy, and elemental analysis and
completion of reaction were confirmed by TLC. In vitro antimicrobial activity of synthesized compounds
was evaluated using disc diffusion assay against gram-positive and gram-negative microbial strains, and
then, the minimum inhibitory concentration and IC50 values of compounds were also determined. The results
of antimicrobial study revealed that compounds 4e, 4g, and 4a were active and exhibited better inhibitory
activities as compared with standard drug amoxicillin. Docking studies were performed by using Argus
lab, and all the compounds exhibited good docking scores between À9.53 and À7.94 kcal/mol against
dihydrofolate reductase protein fragment from Staphylococcus aureus (PDB ID-4XE6). Among all com-
pounds, 4e has shown the maximum docking score and found in agreement to in vitro studies.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
benzothiophene, and quinoline which are known leads for
many antibiotics [6–9]. Quinoxaline and its derivatives
continuously attract researcher because of their wide
spectrum of biological properties like antibacterial
[10,11], antivirus [12], antifungal [13], antitubercular
[14], anticancer [15], and neuroprotective [16]. Some
quinoxaline-containing antibiotics such as echinomycin,
actinoleutin, and levomycin are among the most widely
prescribed antimicrobials. Echinomycin is derived
naturally from Streptomyces echinatus and is known to
inhibit RNA synthesis via nucleotide sequence-selective
intercalation [17]. It is also reported that echinomycin
selectively binds with DNA sequences by quinoxaline
chromophore [18]. Several studies have indicated that
quinoxaline analogs can be used as potential
dihydrofolate reductase (DHFR) inhibitors [19]. It is a
well-known fact that DHFR is a key enzyme in the folate
Worldwide, development of resistance is becoming a
serious threat in antimicrobial therapy and considered as
major public health concern. Antimicrobial agents are
widely used in the prevention and treatment of various
microbial infections. In addition, misuse and prolong use
of antimicrobials also contribute to severe adverse effects
that further aggravate the situation in recipient [1–3].
In view of successive failure of antimicrobial therapy
and increased burden of adverse effects, there is an
urgent need to develop new therapeutic agents with
enhanced antimicrobial activity with minimal or no
adverse effects which could reverse the acquired
resistance in microorganism [4,5].
Quinoxaline derivatives are biologically active class of
heterocyclic compounds. It is a bioisoster of naphthalene,
© 2017 Wiley Periodicals, Inc.

S. Paliwal, S. Sharma, J. Dwivedi, and A. Mishra
Vol 000
metabolism and is associated with biosynthesis of nucleic
acids through catalysis of the NADPH reduction of
7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Moreover,
in conjunction to thymidylate synthase, it is actively
involved in the production of purine and pyrimidine base.
Hence, DHFR has become a crucial and established
target in antimicrobial therapy [20].
Molecular docking studies help in identification of
unorthodox small molecular scaffold as well as give a
clear insight, in understanding the properties of any
compound such as binding energy, electron distribution,
hydrogen bond and donor acceptors, polarizability,
hydrophobicity, and protein ligand interaction with
selectivity/affinity for the target, drug likeness, and lead
determination [21].
Molecular docking study was performed using Argus
Lab 4.0 docking software [22] to find the mode of
corresponding interactions of test compound with the
target. Protein structure of dihydrofolate reductase from
Staphylococcus aureus (PDB ID-4XE6) was obtained
from the Protein Data Bank. For 3D molecule
development in .pdb format, ChemDraw Ultra 10.0 has
been used. In Argus Lab 4.0 program, options for
docking algorithm are genetic algorithm (GA) dock and
Argus dock (shape-based search algorithm). In the
present study, Argus dock was selected as docking engine
to perform the docking simulation, and for calculation,
“Dock” was chosen as the calculation type. The
protein–ligand interaction was determined by using
Pymol 1.3 software [23].
In line with this, the present study was designed to
synthesize some new derivatives of quinoxaline and
examine them for their antimicrobial activity in robust
disc diffusion model. In addition, docking studies have
also been carried out to understand the behavior of
synthesized compounds toward the possible therapeutic
target DHFR enzyme.
General
method
of
synthesis
of
1,4-dihydroquinoxaline-2,3-dione (1) [24].
Aqueous
solution of oxalic acid dihydrate (0.238 mol, 15 g) and
4.5 mL of con. HCl was heated to 100°C followed by the
addition of O-phenylenediamine (0.204 mol, 11 g) with
continuous stirring. Temperature was maintained 100°C
for 30 min. The precipitate was obtained after addition of
reaction mixture to the crushed ice, filtered, and washed
with water. The compound was recrystallized from the
precipitate that was obtained after addition of reaction
mixture to the crushed ice.
EXPERIMENTAL
Yield: 90% (gray color fine needle-shaped crystals), m.
The chemicals were purchased from Loba Chemicals
(India) and Aldrich Chemical Company (USA). All
recorded melting points were determined on a laboratory
melting point apparatus using open capillary method and
were uncorrected. Purity of compounds and progress of
reactions were checked by thin layer chromatography
using silica gel-G on microslide glass plates, and spots
were detected under iodine vapor. IR spectra were recorded
in Shimadzu FTIR-8400 spectrophotometer using KBr disc
and 1H NMR spectra in JEOL FT-NMR spectrometer
(300 MHz) using TMS as an internal standard. All
chemical shift values were recorded as δ (ppm). The mass
spectra were recorded on an Agilent 7890A GC with
Agilent 5975C MS. Detection of elements in synthesized
compounds was carried out on Carlo Erba EA 1108
elemental analyzer that was within 0.4% of the theoretical
values.
The synthesized compounds 4a–4h were examined for
their in vitro antibacterial activities against two
gram-positive and gram-negative bacterial strains, that
is, Bacillus subtilis (MTCC 10619), Staphylococcus
aureus (MTCC 3160), Escherichia coli (MTCC1652),
and Salmonella enteric (MTCC 733) using disc
diffusion method. All the bacterial strains were
procured from MTCC, Chandigadh, India. Minimum in-
hibitory concentration (MIC) and IC50 of each compound
were determined and compared with standard
amoxicillin.
p. À360–362°C; IR Vmax (KBr, in cmÀ1): 3042, 3226,
1
1650; H NMR (300 MHz, DMSO-d6, δ): 7.03–8.02 (4H,
m, Ar-C-H), 7.74 (1H, d, ÀNH of quinoxaline), FAB-MS
(m/z): 162 [M+]; Anal. Calcd for C8H16N2O2: C, 59.26;
H, 3.73; N, 17.28, O, 19.73. Found: C, 59.23; H, 3.71;
N, 17.27, O, 19.70.
Synthesis of 2,3-dichloroquinoxaline (2) [25]. A mixture
of quinoxaline-2,3-dione (0.1 mol, 8.1 g) and freshly
distilled phosphorous oxy trichloride (POCl3, 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.
Yield: 70% (brick red color powder), m.p. À150–152°C.
IR Vmax (KBr, in cmÀ1): 3017.2997, 730; 1H NMR
(300 MHz, DMSO-d6, δ): 7.43–7.72 (4H, m, Ar-C-H),
FAB-MS (m/z): 197 [M+], 199 [M + 2], 201 [M + 4]
(9:6:1); Anal. Calcd for C8H4Cl2N2: C, 48.28; H, 2.03; Cl,
35.62, N, 14.07. Found: C, 48.26; H, 2.04; Cl, 35.64, N,
14.10.
Synthesis of 2-chloro-3-hydrazinyl quinoxaline (3) [26].
The compound 2 (4.6 g, 0.01 mol) and hydrazine hydrate
(0.64 g, 0.01 mol) were dissolved in absolute ethanol and
refluxed for 16 h on water bath. After completion of
reaction, the reaction mixture was cooled and poured
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Synthesis of Phenyl-3-Hydrazinyl-Quinoxaline-2-Amine Derivatives
onto the crushed ice. The solid product so obtained was
separated and crystallized to yield compound 3.
3.37 (1H, s, ÀNH of 2˙ amine), 3.79 (3H, s, ÀOCH3);
FAB-MS (m/z): 281 [M+]; Anal. Calcd for C15H15N5O:
C, 64.04; H, 5.37; N, 24.90; O, 5.69. Found: C, 64.07;
H, 5.37; N, 24.88; O, 5.68.
Yield: 75% (yellow color fine powder), m.p. À294–
296°C. IR Vmax (KBr, in cmÀ1): 3017.2997, 1578, 3367;
1H NMR (300 MHz, DMSO-d6, δ): 7.63–7.78 (4H, m,
Ar-C-H), 3.84 (1H, s, ÀNH of hydrazine), 2.13 (2H, s,
ÀNH of hydrazine) FAB-MS (m/z): 194 [M+], 196
[M + 2] (3:1); Anal. Calcd for C8H4ClN4: C, 49.37; H,
3.63; Cl, 18.22, N, 28.79. Found: C, 49.34; H, 3.66; Cl,
18.23, N, 28.76.
N-(4-chlorophenyl)-3-hydrazinylquinoxalin-2-amine 4e.
Yield: 67% (light yellow fine crystals), m.p. À204–
206°C, IR Vmax (KBr, in cmÀ1): 3464, 3314, 2914, 1536;
1H NMR (300 MHz, DMSO-d6, δ): 7.22–7.66 (4H, m,
Ar-C-H), 7.67–7.79 (4H, m, ÀCH of quinoxaline), 3.76
(1H, s, ÀNH of hydrazine), 2.07 (2H, s, ÀNH of
hydrazine), 3.53 (1H, s, ÀNH of 2˙ amine); FAB-MS (m/
z): 285 [M+]; Anal. Calcd for C14H12ClN5: C, 58.85; H,
4.23; Cl, 12.41; 24.51. Found: C, 88.67; H, 4.27; Cl,
12.42; N, 24.53.
Synthesis
of
substituted
phenyl-3-hydrazinyl-quinoxaline-2-amine 4a–4h.
The
compound 3 (1.94 g, 0.01 mol) and the appropriate
commercially available substituted aniline were
suspended in ethanol (10 mL) and stirred under reflux
for 8 h. The contents were cooled at room temperature.
The precipitates of the compound were filtered off,
washed, and recrystallized with ethanol.
N-(4-bromophenyl)-3-hydrazinylquinoxalin-2-amine 4f.
Yield: 64% (yellow color fine power), m.p. À200–202°C,
IR Vmax (KBr, in cmÀ1): 3423, 3314, 2921, 1547; 1H
NMR (300 MHz, DMSO-d6, δ): 7.02–7.36 (4H, m, Ar-C-
H), 7.43–7.67 (4H, m, ÀCH of quinoxaline), 3.41 (1H, s,
ÀNH of hydrazine), 2.0 (2H, s, ÀNH of hydrazine), 3.27
(1H, s, ÀNH of 2˙ amine); FAB-MS (m/z): 329 [M+];
Anal. Calcd for C14H12BrN5: C, 50.93; H, 3.66; Br, 24.20;
N, 21.21. Found: C, 50.89; H, 3.69; Br, 24.24; N, 21.18.
3-Hydrazinyl-N-phenylquinoxalin-2-amine 4a.
Yield:
68% (brown color fine crystals), m.p. À198–200°C, IR
1
Vmax (KBr, in cmÀ1): 3229, 3298, 2727, 1587; H NMR
(300 MHz, DMSO-d6, δ): 6.18–7.51 (5H, m, Ar-C-H),
7.62–7.79 (4H, m, ÀCH of quinoxaline), 3.74 (1H, s,
ÀNH of hydrazine), 2.43 (2H, s, ÀNH of hydrazine),
3.43 (1H, s, ÀNH of 2˙ amine); FAB-MS (m/z): 251
[M+]; Anal. Calcd for C14H13N5: C, 66.92; H, 5.21; N,
27.87. Found: C, 66.88; H, 5.19; N, 27.88.
N-(4-flurophenyl)-3-hydrazinylquinoxalin-2-amine 4g.
Yield: 69% (light brown fine crystals), m.p.
À193–195°C, IR Vmax (KBr, in cmÀ1): 3378, 3327,
2876, 1587; 1H NMR (300 MHz, DMSO-d6, δ):
7.31–7.46 (4H, m, Ar-C-H), 7.63–7.78 (4H, m, ÀCH of
quinoxaline), 3.83 (1H, s, ÀNH of hydrazine), 1.87 (2H,
s, ÀNH of hydrazine), 3.68 (1H, s, ÀNH of 2˙ amine);
FAB-MS (m/z): 269 [M+]; Anal. Calcd for C14H12FN5:
C, 62.44; H, 4.49; F, 7.06; N, 26.01. Found: C, 62.47; H,
4.49; F, 7.07; N, 26.03.
N-(3,5-dimethoxyphenyl)-3-hydrazinylquinoxalin-2-amine
4b. Yield: 72% (faint gray power), m.p. À187–190°C, IR
1
Vmax (KBr, in cmÀ1): 3347, 3318, 2826, 1573, 1035; H
NMR (300 MHz, DMSO-d6, δ): 5.68–5.73 (3H, m, Ar-C-
H), 7.35–7.47 (4H, m, ÀCH of quinoxaline), 3.97 (1H, s,
ÀNH of hydrazine), 2.13 (2H, s, ÀNH of hydrazine),
3.37 (1H, s, ÀNH of 2˙ amine), 3.78 (6H, s, ÀOCH3);
FAB-MS (m/z): 311 [M+]; Anal. Calcd for C16H17N5O2:
C, 61.72; H, 5.50; N, 22.49; O, 10.28. Found: C, 60.76;
H, 5.47; N, 22.48; O, 10.27.
3-Hydrazinyl-N-(4-nitrophenyl)quinoxalin-2-amine 4h.
Yield: 73% (creamy white power), m.p. À189–191°C, IR
1
Vmax (KBr, in cmÀ1): 3394, 3317, 2891, 1594, 1356; H
NMR (300 MHz, DMSO-d6, δ): 6.81–8.16 (4H, m, Ar-C-
H), 7.64–7.81 (4H, m, ÀCH of quinoxaline), 3.92 (1H, s,
ÀNH of hydrazine), 1.91 (2H, s, ÀNH of hydrazine),
3.73 (1H, s, ÀNH of 2˙ amine); FAB-MS (m/z): 296
[M+]; Anal. Calcd for C14H12N6O2: C, 56.75; H, 4.09; N,
28.36; O, 10.80. Found: C, 56.78; H, 4.07; N, 28.38; O,
10.76.
3-Hydrazinyl-N-(3,4,5-trimethoxyphenyl)quinoxalin-2-
amine 4c. Yield: 64% (light gray long crystals), m.p.
À202–204°C, IR Vmax (KBr, in cmÀ1): 3437, 3273,
1
2914, 1557, 1175; H NMR (300 MHz, DMSO-d6, δ):
5.52–5.61 (2H, m, Ar-C-H), 7.65–7.79 (4H, m, ÀCH
of quinoxaline), 3.53 (1H, s, ÀNH of hydrazine), 2.21
(2H, s, ÀNH of hydrazine), 3.21 (1H, s, ÀNH of 2˙
amine), 3.81 (9H, s, ÀOCH3); FAB-MS (m/z): 341
[M+]; Anal. Calcd for C17H19N5O3: C, 59.81; H, 5.60;
N, 20.52; O, 14.06. Found: C, 59.83; H, 5.57; N,
22.54; O, 14.02.
BIOLOGICAL EVALUATION
Antimicrobial activity.
The in vitro antimicrobial
3-Hydrazinyl-N-(4-methoxyphenyl)quinoxalin-2-amine
4d. Yield: 72% (dark brown power), m.p. À192–194°C,
activity was performed using the disc diffusion method,
and the MIC and IC50 of each compound were
determined against B. subtilis MTCC 10619, S. aureus
MTCC 3160, E. coli MTCC 1652, and S. enteric MTCC
733. Amoxicillin was used as standard.
IR Vmax (KBr, in cmÀ1): 3449, 3287, 2907, 1573, 1121; 1H
NMR (300 MHz, DMSO-d6, δ): 7.02–7.51 (4H, m, Ar-C-
H), 7.67–7.79 (4H, m, ÀCH of quinoxalin), 3.41 (1H, t,
ÀNH of hydrazine), 2.21 (2H, d, ÀNH of hydrazine),
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S. Paliwal, S. Sharma, J. Dwivedi, and A. Mishra
Vol 000
Disc diffusion assay. The antimicrobial susceptibility of
compounds (4a–4h) was tested by disc diffusion method
[19,27]. The bacterial cultures (which has been adjusted to
0.5 McFarland standard) were used to lawn Muller Hinton
agar plates evenly using a sterile swab. In this method,
sterile paper discs (0.5 mm) impregnated with test
compounds (4a–4h) dissolved in DMSO at 200 μg/mL
concentration were used. These discs were placed on
bacterial lawn Mueller Hinton agar surface. Each
experimental plate contains of three discs: one positive
control, which is a standard antibiotic disc (amoxicillin
10 μg/mL), one negative control (DMSO 100%), and a
test compound-treated disc. The plates were incubated at
37°C for 24 h. After incubation, the plates were examined
for zone of inhibition. The inhibition zone was then
measured. The test was performed thrice to ensure
reliability.
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.
The concentrations afford 50% inhibition of bacteria
growth (IC50) that was computed from the dose–response
curves. MIC and IC50 values were studied for the same
bacterial strains in the disc diffusion assay. Amoxicillin
was used as positive control, and the culture medium with
no bacteria was used as negative control.
RESULT AND DISCUSSION
Chemistry. Quinoxaline derivatives were synthesized
according to Scheme 1. 2,3-Dichloroquinoxaline (2) [25]
was prepared by chlorination of 2,3-dihydroxyquinoxaline
(1) [24], which, in turn, prepared by the condensation of
the commercially available 1,2-diaminobenzene with
oxalic acid in aqueous hydrochloric acid. Treatment of (2)
with hydrazine hydrate yielded 3-hydrazino compound (3)
[26]; finally, compound (3) was refluxed with a solution of
substituted aniline in ethanol to afford substituted phenyl-
3-hydrazinyl-quinoxaline-2-amine 4a–4h.
Quinoxaline derivatives were synthesized according to
Scheme 1. 2,3-Dichloroquinoxaline (2) [25] was prepared
by chlorination of 2,3-dihydroxyquinoxaline (1) [24],
which, in turn, prepared by the condensation of the
commercially available 1,2-diaminobenzene with oxalic
Determination of minimum inhibitory concentration and
IC50.
The MIC values of compounds (4a–4h) were
determined by serial dilution method [19]. It is defined as
minimum concentration of the test compound which
prevents the visible growth of microorganism. For the
experiment, the selected microorganism was grown in
Mueller Hinton broth to 0.5 McFarland standard turbidity.
The test compounds were dissolved in DMSO and diluted
up to 500 μg/mL concentration for the test. Further serial
dilution was made in concentration range from 0.1 to
400 μg/mL in 10-mL sterile dilution tubes. A prepared
suspension of the standard microorganisms was added to
Scheme 1
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Synthesis of Phenyl-3-Hydrazinyl-Quinoxaline-2-Amine Derivatives
acid in aqueous hydrochloric acid. Treatment of (2) with
hydrazine hydrate yielded 3-hydrazino compound (3) [26];
finally, compound (3) was refluxed with a solution of
substituted aniline in ethanol to afford substituted phenyl-
3-hydrazinyl-quinoxaline-2-amine 4a–4h).
ÀOCH3 group and compound 4h confirmed by the
peak at 1356 cmÀ1 (sym. str. Ar-NO2) in the IR
spectrum.
Biological studies.
Results of antibacterial study
clearly revealed that most of the tested compounds
were active and caused significant inhibition of gram
positive and negative strains of bacteria. In particular,
compounds 4e and 4g, that is, chloro and fluro
derivatives of quinoxalines, exhibited most significant
activity among all derivatives (Tables 1 and 2). In
addition, among all synthesized quinoxaline derivatives,
compound 4h (with MIC range of 0.97 and 0.91 μg/
mL) had comparable activity to standard amoxicillin
against E. coli and S. enteric. Also, compound 4f
demonstrated good activity against E. coli with MIC
value 0.96. Similarly, compound 4d displayed good
activity against S. enteric and compound 4c against S.
aureus at MIC from 0.89 and 0.97 μg/mL. In
conclusion, N-phenyl (4a), chlorophenyl (4e), and
flurophenyl (4g) derivatives of quinoxaline exhibited the
best antimicrobial activity against both strains of
organism when compared with standard amoxicillin
(Tables 1 and 2).
Docking study. The docking study gives an idea about
interaction between test compound and target protein. In
the present study, test compounds were docked over
targeted protein dihydrofolate reductase from S. aureus
(PBD ID-4XE6). Test compound showed different modes
of binding with amino acids located at active site of
dihydrofolate reductase (Fig. 1).
In docking study, it was found that the nitrogen atom of
hydrazine group and nitrogen heteroatom of quinoxaline
ring in test compounds were available for the formation of
hydrogen bond with different amino groups of taken PBD
file with specific bond distance. Nitrogen atom of
quinoxaline ring and hydrazine group in compound 4a
(7ALA, 2.89 Å) and nitrogen atom of quinoxaline of
compound 4e (7ALA, 2.37 Å) formed hydrogen bond
with amino group of alanine. While in compound 4g,
All the synthesized compounds were characterized by
IR, 1H NMR, and mass spectra which were found
consistent to the assigned structure. The IR spectra of
all synthesized compounds showed some characteristic
peaks that indicated the presence of particular groups.
In the IR spectra of compound, the presence of
absorption band, in the IR spectrum, at 1650 and
3226 cmÀ1 assignable to C=O group and NH of
quinoxaline indicated the formation of compound 1
which was further confirmed by the appearance of
signal at 7.03–8.02 ppm due to the four aromatic
protons and at 7.74 ppm due to NH protons of
quinoxaline ring [24]. The disappearance of strong
absorption peak of C=O together with the presence of
absorption peak at 730 cmÀ1 due to C–Cl confirmed
the formation of compound 2 [25]. The formation of
compound
3
ascertained by the appearance of
absorption peak at 1578 and at 3367 cmÀ1 due to N–N
and N–H groups of hydrazine also by the other peaks
which appeared at 3.84 ppm for one proton and at
1
2.13 ppm for two protons of in H NMR spectra [26].
In addition, compound also showed [M + 2] and
[M + 4] peak in the mass spectrum. The compound 4a
displayed peak as compound 3 and some additional
peak at 6.18–7.51 ppm due to aromatic protons and a
singlet at 3.94 ppm assigned to N–H of secondary
amine. Similarly, the compounds 4b–4h exhibited the
peaks in the same region as of 4a; along with this, it
also showed some additional peaks around 3.78 ppm
(ÀOCH3) in compound; IR peak at 1035 cmÀ1 (sym.
str. C–O–C), 1175 cmÀ1 (sym. str. C–O–C), and
1121 cmÀ1 (sym. str. C–O–C) of C–OCH3 corroborated
the formation of 4b, 4c, and 4d, while compounds 4e
and 4f were ascertained by the absence of peak for
Table 1
Screening of antimicrobial activity of the synthesized compounds.
Compounds
Staphylococcus aureus
Bacillus subtilis
++
++
+
Escherichia coli
Salmonella Enteric
4a
4b
4c
4d
4e
4f
++
++
++
++
+
+
+
+
+
+
+++
++
+++
+++
+++
+
+++
+
++
+++
+++
+
++
++
++
++
+++
+++
++
++
+++
++
+++
4g
4h
Amoxeciline
Key to symbols: À inactive (inhibition zone <6 mm), slightly active = + (inhibition zone 7–9 mm), moderately active = ++ (inhibition zone 10–13 mm),
and highly active = +++ (inhibition zone >14 mm).
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S. Paliwal, S. Sharma, J. Dwivedi, and A. Mishra
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Table 2
Antimicrobial activity of the synthesized compounds expressed as MIC and IC50 (unit).
Gram-positive bacteria
Gram-negative bacteria
Staphylococcus
aureus
Staphylococcus
aureus
Bacillus
subtilis
Bacillus
subtilis
Escherichia
coli
Escherichia
coli
Salmonella
enteric
Salmonella
enteric
Compounds
MIC
IC50
0.44
0.56
0.98
0.61
0.43
0.48
0.46
0.49
0.49
MIC
0.93
1.97
2.73
1.25
0.84
1.13
0.97
1.10
0.87
IC50
0.46
0.98
1.36
0.62
0.42
0.56
0.48
0.55
0.43
MIC
0.97
0.82
0.89
0.97
0.92
0.96
0.89
0.97
0.95
IC50
0.48
0.41
0.44
0.48
0.46
0.48
0.44
0.48
0.47
MIC
0.87
0.82
1.87
1.08
0.82
1.09
0.83
0.91
0.89
IC50
4a
4b
4c
4d
4e
4f
4g
4h
0.89
1.12
1.97
1.23
0.87
0.97
0.93
0.98
0.98
0.43
0.41
0.93
0.54
0.41
0.54
0.41
0.45
0.44
Amoxicillin
Figure 1. Interaction study of compound 4e with binding domain of 4XE6 protein. Hydrogen binding of analog 4e with amino acids and amine group of
hydrazine interacts with 7ALA residue and bond distance 2.37 Å. [Color figure can be viewed at wileyonlinelibrary.com]
nitrogen atom of hydrazine group forms hydrogen bond
with amino group of aspartate (127 ASP, 2.73 Å) (Fig. 1).
These amino acids make a cascade to facilitate binding
and holding of the test compound in the active site of
dihydrofolate reductase protein.
respectively. Compounds 4e (À9.17 kcal/mol) and 4g
(À9.13 kcal/mol) containing the chloro group and fluro
group displayed better docking score than compound 4a
(À9.09 kcal/mol) which do not contain any substitution
on the same position.
All compounds showed good docking score (Table 3);
compounds 4e, 4g, and 4a exhibited the considerable
docking scores À9.53, À9.13, and À9.09 kcal/mol,
CONCLUSION
Some new quinoxalines were synthesized from
O-phenylenediamine, and all synthesized derivatives were
characterized by IR, 1H NMR, mass spectroscopy, and
elemental analysis. In addition, derivatives were examined
against two gram-positive and two gram-negative bacterial
strains. Of all the synthesized quinoxaline derivatives, 4e,
4g, and 4a have shown good antibacterial activity, while
other compounds had shown moderate activity against the
test organism which revealed that compound with the
electron-withdrawing group or unsubstituted group was
helpful in enhancing the antimicrobial activity.
Table 3
Molecular docking study (binding energy) data.
Compound no.
Docking score (kcal/mol)
4a
4b
4c
4d
4e
4f
À9.09
À7.94
À8.13
À8.11
À9.53
À8.95
À9.13
À8.71
4g
4h
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Synthesis of Phenyl-3-Hydrazinyl-Quinoxaline-2-Amine Derivatives
[10] Suresh, M.; Lavanya, P.; Sudhakar, D.; Vasu, K.; Rao, C. V. J
Chem Pharm Res 2010, 2, 497.
Synthesized compounds with electron-withdrawing group
exhibited better antimicrobial activity compared with
electron-donating group containing compound on the
phenyl group. In addition, molecular docking studies
suggested that compounds 4e, 4g, and 4a interacted with
protein more efficiently, and hence, these can be further
developed to improve their antimicrobial activity. This
effectiveness makes these substances attractive antibacterial
candidates. In vitro efficacy and safety of these compounds
are being planned for their further development.
[11] Tandon, V. K.; Yadav, D. B.; Maurya, H. K.; Chaturvedi, A.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet