Graph Theoretical Analysis, in Silico Modeling, Synthesis, Anti-Microbial and Anti-TB Evaluation of Novel Quinoxaline Derivatives

Article abstract of DOI:10.1055/s-0043-120198

Background We designed to synthesize a number of 2-(2-(substituted benzylidene) hydrazinyl)-N-(4-((3-(phenyl imino)-3,4-dihydro quinoxalin-2(1 H)-ylidene)amino) phenyl) acetamide S1-S13 with the hope to obtain more active and less toxic anti-microbial and anti-TB agents. Methods A series of novel quinoxaline Schiff bases S1-S13 were synthesized from o-phenylenediamine and oxalic acid by a multistep synthesis. In present work, we are introducing graph theoretical analysis to identify drug target. In the connection of graph theoretical analysis, we utilised KEGG database and Cytoscape software. All the title compounds were evaluated for their in-vitro anti-microbial activity by using agar well diffusion method at three different concentration levels (50, 100 and 150 μg/ml). The MIC of the compounds was also determined by agar streak dilution method. Results The identified study report through graph theoretical analysis were highlights that the key virulence factor for pathogenic mycobacteria is a eukaryotic-like serine/threonine protein kinase, termed PknG. All compounds were found to display significant activity against entire tested bacteria and fungi. In addition the synthesized scaffolds were screened for their in vitro antituberculosis (anti-TB) activity against Mycobacterium tuberculosis (Mtb) strain H ₃₇ Ra using standard drug Rifampicin. Conclusion A number of analogs found markedly potent anti-microbial and anti-TB activity. The relationship between the functional group variation and the biological activity of the evaluated compounds were well discussed. The observed study report was showing that the compound S6 (4-nitro substitution) exhibited most potent effective anti-microbial and anti-TB activity out of various tested compounds.

Full text of DOI:10.1055/s-0043-120198

Original Article
Thieme
Graph Theoretical Analysis, In Silico Modeling, Synthesis,
Anti-Microbial and Anti-TB Evaluation of Novel Quinoxaline
Derivatives
Authors
1
2
1
2
Govindaraj Saravanan , Theivendren Panneer Selvam , Veerachamy Alagarsamy , Selvaraj Kunjiappan ,
Shrinivas D. Joshi , Murugan Indhumathy , Pandurangan Dinesh Kumar
3
4
5
AbSTꢀAꢁT
Affiliations
1
2
3
Department of Pharmaceutical Chemistry, MNR College of
Background We designed to synthesize a number of
2-(2-(substituted benzylidene) hydrazinyl)-N-(4-((3-(phenyl
imino)-3,4-dihydro quinoxalin-2(1 H)-ylidene)amino) phenyl)
acetamide S1-S13 with the hope to obtain more active and less
toxic anti-microbial and anti-TB agents.
Pharmacy, Fasalwadi, Sangareddy-502294, Telangana, India
Department of Pharmaceutical Chemistry, Karavali College of
Pharmacy, Vamanjoor, Mangalore- 575028, Karnataka, India
Department of Pharmaceutical Chemistry, Sonia
Education Trust′s College of Pharmacy, Sangolli Rayanna
Nagar, Dharwad-580002, Karnataka, India
Methods A series of novel quinoxaline Schiff bases S1-S13
were synthesized from o-phenylenediamine and oxalic acid by
a multistep synthesis. In present work, we are introducing
graph theoretical analysis to identify drug target. In the con-
nection of graph theoretical analysis, we utilised KEGG data-
base and Cytoscape software. All the title compounds were
evaluated for their in-vitro anti-microbial activity by using agar
well diffusion method at three different concentration levels
(50, 100 and 150 µg/ml). The MIC of the compounds was also
determined by agar streak dilution method.
4
5
Department of Biotechnology, P.S.R Engineering College,
Sevalpatti, Sivakasi, Tamilnadu, India
Hindu College of Pharmacy, Amaravathi Road,
Guntur-522002, Andhra Pradesh, India
Key words
Graph theoretical analysis, Quinoxaline, Schiff base,
Anti-bacterial, Anti-fungal and Anti-tubercular
Results The identified study report through graph theoretical
analysis were highlights that the key virulence factor for patho-
genic mycobacteria is a eukaryotic-like serine/threonine protein
kinase, termed PknG. All compounds were found to display sig-
nificant activity against entire tested bacteria and fungi. In ad-
dition the synthesized scaffolds were screened for their in vitro
antituberculosis (anti-TB) activity against Mycobacterium tuber-
culosis (Mtb) strain H37Ra using standard drug Rifampicin.
Conclusion A number of analogs found markedly potent
anti-microbial and anti-TB activity. The relationship between
the functional group variation and the biological activity of the
evaluated compounds were well discussed. The observed study
report was showing that the compound S6 (4-nitro substitu-
tion) exhibited most potent effective anti-microbial and anti-TB
activity out of various tested compounds.
received 04.08.2017
accepted 17.09.2017
Bibliography
DOI https://doi.org/10.1055/s-0043-120198
Published online: 2017
Drug Res
©
Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Dr. T. Panneerselvam M. Pharm., Ph.D
Professor
Karavali College of Pharmacy
Vamanjoor , Mangalore
Karnataka, India
Tel.: + 91 75988224818; 9113656109
tpsphc@gmail.com
Introduction
threatening diseases. These bacterial parasites cause food poison-
ing, rheumatic fever and diarrhoea that affect millions of individu-
als in developing countries. More than 50 million people worldwide
are infected and up to 1,10,000 of these die every year. Amoxicil-
lin, Norfloxacin, Ciprofloxacin are the most common drugs used for
this bacterial infection but are associated with severe side effects.
The diverse parasitic bacteria such as Staphylococcus aureus,
Staphylococcus pyogenes, Salmonella typhimurium, and Escheri-
chia coli have significant impact on the mucosal health of humans.
Infection with S. aureus, S. pyogenes, S. typhimurium, and E. coli
may have resulted in massive destruction of host tissue and life
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
Toxicity and resistance to the drugs also play an important role in
the failure of treatment [1]. The rapid development of bacterial
strains resistant to anti-bacterial agents poses a significant threat
to global health [2]. In particular, attention has focused on the
Gram positive organism Staphylococcus aureus because many
strains of this organism are now resistant against clinically useful
antibiotics like Methicillin and Vancomycin [3]. The problem is fur-
ther compounded by the rapid emergence of multidrug resistant
organisms. For example, it look only a few years after the introduc-
tion of Linezolid [4], an oxazolidinone derivative, for clinical use be-
fore reports of resistant organisms and clinical failures emerged
impact ionization. All the IR spectra were recorded in KBr pellets
on a Jasco FT-IR 410 spectrometer. Elemental analyses were per-
formed on a Perkine Elmer model 2400C analyzer and were with-
in ± 0.4 % of the theoretical values.
Network topology analysis
Graph theoretical analysis [17–19] was performed by using Kyoto
Encyclopedia of Genes and Genomes database and network of pro-
tein interaction (hsa: 05152) in homo sapiens was in use to identi-
fy the influential proteins. The pathway was converted to a graph
with proteins as nodes and interactions as edges and it was repre-
sented in ▶Figs. 1 and ▶2. The network was carries 124 nodes and
167 edges, moreover the computed Degree Centrality, Between-
ness Centrality, Eigenvector Centrality, Radiality Centrality, Stress
Centrality and Closeness Centrality was used to identify the signif-
icant proteins from the network. Based on these measures with its
threshold values, the target PknG was identified and Mtb drug tar-
get, PknG has more attention than ESAT-6 because of its interac-
tion between 27 tuberculosis proteins and may help in the spread
of disease. Based on the report of graph theoretical analysis, it was
very clear proven that the PknG is a significant target to identify ac-
tive quinoxiline motifs.
[
5, 6]. In addition the statistics was indicates that millions of peo-
ple worldwide die annually from difficulty of TB. In coincidence with
the advent of the AIDS plague and the improved mobility of human
populations, has led to the appearance of frequent multidrug- re-
sistant Mycobacterium tuberculosis (Mtb). Given this situation,
there is an urgent need to discover and develop new anti-bacterial
agents. Fortunately, several promising heterocyclic compounds
have been identified. It was found that quinoxaline and its analogs
was one such important heterocyclic compound. A variety of chem-
ical modifications were carried out so far around the quinoxaline
nucleus. The quinoxalines have emerged as anti-microbial agents
of an immense interest because of their broad spectrum of in-vitro
activity and their in-vivo chemotherapeutic activity [7–14]. Schiff
bases have gained importance because of physiological and phar-
macological activities associated with them. Compounds contain-
ing azomethine group (–C=N–) in the structure are known as Schiff
bases, which are usually synthesized by the condensation of prima-
ry amines and active carbonyl groups. Schiff bases are well known
for their pharmacological properties as anti-bacterial, antifungal,
anti-cancer and anti-viral agents [15]. On the other hand, diverse
chemotherapeutic agents contain pharmacophore like phenolic
hydroxy, nitro, and chlorine substitutions, are reported to possess
anti-TB and anti-microbial activities [16]. Based on these findings,
and in continuation of our drug research program concerning syn-
thesis of new safer and more biologically active heterocyclic com-
pounds, it was of interest to synthesize a new series of quinoxaline
Schiff base derivatives with the hope to obtain more active and less
toxic anti-TB and anti-microbial agents.
Insilico modeling
Molecular docking was used to clarify the binding mode of the com-
pounds to provide straightforward information for further struc-
tural optimization. The “Sybyl-x 2.0, Tripos International, St. Louis,
MO, USA, 2012.”, was used to perform screening of docking based
on earlier reported method [20]. The crystal structure of Protein
kinase PknG from Mycobacterium tuberculosis in Complex with
Tetrahydrobenzothiophene (PDB ID 2PZI) was extracted from the
Brookhaven Protein Database (PDB http://www.rcsb.org/pdb). The
proteins were prepared for docking by adding polar hydrogen atom
with Gasteiger-Huckel charges and water molecules were removed.
The 3D structure of the ligands was generated by the SKETCH mod-
ule implemented in the SYBYL program (Tripos Inc., St. Louis, USA)
and its energy-minimized conformation was obtained with the help
of the Tripos force field using Gasteiger-Huckel charges and mo-
lecular docking was performed with Surflex-Dock program that is
interfaced with Sybyl-X 2.0. and other miscellaneous parameters
were assigned with the default values given by the software.
Experimental
Biological Methods
General
All the synthesized compounds were screened for anti-microbial
activities by agar well diffusion method. The anti-bacterial activity
of the compounds was evaluated against one Gram-positive bac-
teria (Bacillus subtilis ATCC 6633) and two Gram-negative bacteria
(Klebsiella pneumoniae ATCC 13883 & Pseudomonas aeruginosa
ATCC 27853). The anti-fungal activities of the synthesized com-
pounds were evaluated against three fungi (Trichoderma ATCC
26921, Aspergillus niger ATCC 9029 and Aspergillus flavus ATCC
10124). Bacterial strains were cultured over night at 37°C in Mul-
ler-Hinton broth and the yeast was cultured over night at 30°C in
nutrient agar medium for anti-bacterial and anti-fungal activity
tests. Ciprofloxacin and Ketoconozole were used as standard drugs
for anti-bacterial and anti-fungal activities respectively. Nutrient
agar medium and Sabouraud dextrose agar medium (Hi-Media Lab-
The chemicals and reagents used were obtained from various
chemical units Qualigens, E. Merck India Ltd., CDH, and SD Fine
Chem. These solvents used were of LR grade and purified before
their use. The Graph theoretical analysis was performed by using
KEGG database and Cytoscape software. The docking study was
performed using Sybyl-x 2.0, Tripos International, St. Louis, MO,
USA, 2012.” The silica gel G used for analytical chromatography
(
TLC) was obtained from E. Merck India Ltd. All the melting points
1
were taken in open glass capillary and are uncorrected. H-NMR
spectra were recorded at 500 MHz on Bruker Avance-500 NMR
spectrometer in CDCl using tetramethylsilane (TMS) as an inter-
nal standard. The chemical shifts are reported in ppm scale. Mass
spectra were obtained on a JEOL-SX-102 instrument using electron
3
Saravanan G et al. Potent Quinoxaline Derivatives Drug Res

▶
Fig. 1 The interaction of PknG pathway with nodes and edges.
oratories, India) was used as the medium for the study of anti-bac-
terial and anti-fungal activity respectively.
5 × 10– 5cfu/ml and applied to plates with serially diluted com-
pounds in dimethyl formamide to be tested and incubated at 37°C
for 24 and 48 h for bacteria and fungi, respectively. The MIC was
considered to be the lowest concentration of the test substance
exhibiting no visible growth of bacteria or fungi on the plate and
the observed MIC data was represented in ▶Taꢂle 2.
Paper disc diffusion technique
The sterilized (autoclaved at 120°C for 30min) medium (40–50°C)
was inoculated (1 ml/100 ml of medium) with the suspension
(
5×10– 5 cfu/ml) of the microorganism (matched to McFarland bar-
MABA Assay
ium sulphate standard) and poured into a petridish to give a depth
of 3–4 mm. The paper impregnated with the test compounds (50,
The synthesized scaffolds were screened for their in vitro anti-TB
activity against Mtb H37Ra and bioassay was performed using the
microplate Alamar blue assay (MABA) [23] and INH was chosen as
the reference compound. The indication of color change from blue
to pink and minimum inhibitory concentration (MIC) was calculat-
ed based on earlier reported method [24] and the observed MIC
data was represented in ▶Taꢂle 2.
1
00 and 150 µg/ml in dimethyl formamide) was placed on the so-
lidified medium. The plates were pre-incubated for 1h at room tem-
perature and incubated at 37 °C for 24 and 48 h for antibacterial
and antifungal activities, respectively. Ciprofloxacin (150 µg/ml/
disc) and Ketoconazole (150µg/ml/disc) were used as standard for
antibacterial and antifungal activities respectively [21]. All the ob-
served zone of inhibition is presented in ▶Taꢂle 1.
Synthetic methods
Minimum inhibitory concentration (MIC)
Synthesis of quinoxaline-2,3(1 H,4 H)-dione (1)
MIC of the compound was determined by agar streak dilution meth-
od [22]. Using dimethyl formamide a stock solution of the synthe-
sized compound were prepared in a concentration of 125µg/ml.
Graded quantities of the stock solution of test compounds were
incorporated in a specified quantity of molten sterile agar (nutri-
ent agar for antibacterial activity and Sabouraud’s dextrose agar
for antifungal activity) medium to get various concentrations of
serially diluted test compounds. A specified quantity of the medi-
um (40–50°C) containing the compound was poured into a petrid-
ish to give a depth of 3–4mm and allowed to solidify. Suspension
of the microorganism were prepared to contain approximately
An equimolar solution of o-phenylenediamine (1.08g, 0.01mol) and
oxalic acid (0.9g, 0.01mol) in 4N hydrochloric acid (90ml) was re-
fluxed in water bath for 4h and the reaction mixture was placed in
refrigerator overnight to produce quinoxaline-2,3(1H,4H)-dione 1.
The needle shaped solid obtained was filtered, washed and re-crys-
tallized by using ethanol and dried. Yield: 73%, m.p.: 291–294°C. IR
(KBr, cm– 1): 3351 & 3279 (NH), 3084 (Ar-CH), 1698 (C= O), 1616
1
(C=C). H NMR (CDCl , 500MHz) δ ppm: 7.23–7.89 (m, 4H, Ar-H),
3
8.34 (s, 1H, CONH of quinoxaline), 8.51 (s, 1H, CONH of quinoxa-
+
line). EI-MS m/z: 162 (M ). Anal. Cald for C H N O : C, 59.26; H,
8
6
2
2
3.73; N, 17.28. Found: C, 59.43; H, 3.72; N, 17.22.
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
▶
Fig. 2 The visualization of interaction of PknG using Cytoscape.
Synthesis of 3-(4-aminophenylimino)-3,4-dihydroquinoxa-
Synthesis of N-(3-(phenylimino)-3,4-dihydroquinoxa-
lin-2(1 H)-one (2)
lin-2(1 H)-ylidene)benzene-1,4-diamine (3)
To a solution of quinoxaline-2,3(1H,4H)-dione 1 (1.62g, 0.01mol)
in dimethyl formamide (30ml), p-amino aniline (1.62g, 0.015mol)
was added and the whole mixture was refluxed for 6h at 150 °C and
cooled overnight. The solid obtained was filtered, washed with
water, dried and recrystallised using ethanol. Yield: 80 %, m.p.:
A mixture of 3-(4-aminophenylimino)-3,4-dihydroquinoxa-
lin-2(1 H)-one 2 (2.52 g, 0.01 mol) and aniline (0.931 g; 0.01 mol)
in ethanol (20 ml) was refluxed for 5 h in a water bath. Latter the
mixture was cooled and poured in ice cold water. The precipitated
product 3 was filtered, dried and recrystallised using alcohol. Yield:
70 %, m.p.: 248–250 °C. IR (KBr, cm– 1): 3367 & 3243 (NH), 3071
1
81–183°C. IR (KBr, cm– 1): 3342 & 3256 (NH), 3053 (Ar-CH), 1678
1
1
(
C=O), 1612 (C=N), 1591 (C=C). H NMR (CDCl , 500MHz) δ ppm:
(Ar-CH), 1624 (C = N), 1609 (C = C). H NMR (CDCl , 500 MHz) δ
3
3
4
8
.13 (s, 2 H, NH ), 4.45 (s, 1 H, NH of quinoxaline), 7.02–7.97 (m,
H, Ar-H), 8.26 (s, 1H, CONH of quinoxaline). EI-MS m/z: 252 (M ).
ppm: 4.05 (s, 2 H, NH ), 4.28 (s, 1 H, NH of quinoxaline), 4.49 (s,
1H, NH of quinoxaline), 6.84–7.80 (m, 13H, Ar-H). EI-MS m/z: 327
2
2
+
+
Anal. Cald for C14H12N O: C, 66.65; H, 4.79; N, 22.21. Found: C,
(M ). Anal. Cald for C20H17N : C, 73.37; H, 5.23; N, 21.39. Found:
4
5
6
6.82; H, 4.80; N, 22.14.
C, 73.12; H, 5.25; N, 21.45.
Saravanan G et al. Potent Quinoxaline Derivatives Drug Res

▶
Taꢂle 1 Anti-microbial activity (zone of inhibition in mm) of title compounds S1-S13.
Micro-
ꢁonc.
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
ꢁon
Std
organism
(µg/ml)
bacillus
suꢂtilis
50
15
19
27
14
20
30
15
18
30
9
14
19
25
14
17
29
13
16
29
9
4
8
8
3
8
17
20
28
15
22
31
18
21
30
10
15
23
13
17
19
14
16
22
1
3
7
1
2
8
-
2
4
7
-
-
4
6
-
8
14
20
8
6
9
10
16
23
11
15
25
9
7
10
15
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
00
50
50
00
50
50
00
50
50
00
50
50
00
50
50
00
50
15
22
10
13
25
7
27
13
4
12
4
14
5
Kleꢂsiella
pneumoniae
1
1
8
7
2
7
-
3
7
-
11
24
6
8
8
29
30
21
18
22
13
3
11
2
15
5
17
5
Pseudomonas
aeruginosa
1
1
7
11
24
6
7
1
6
-
1
5
-
3
5
-
11
22
6
8
14
27
6
9
13
-
13
-
15
3
16
3
Trichoderma
1
1
14
21
11
17
18
12
15
21
13
19
10
15
18
11
15
20
5
10
16
7
5
2
5
-
2
4
-
3
4
-
9
5
11
18
8
5
16
-
14
-
14
7
10
3
10
5
Aspergillus
niger
1
1
4
13
15
8
3
2
6
-
2
5
-
3
4
-
11
14
8
5
14
16
9
6
15
2
15
2
9
11
5
Aspergillus
flavus
4
1
1
3
13
17
3
2
7
3
7
3
5
12
15
5
13
19
7
17
16
10
11
Con: Control (DMF), Std: Standard drug in 150 µg/ml (Ciprofloxacin for bacteria & Ketoconazole for fungi), -: No zone of inhibition observed
▶
Taꢂle 2 Minimum inhibitory concentration (MIC in µg/ml) of title compounds S1-S13.
Microorganism
bacillus suꢂtilis
S1
S2
S3
S4
S5
S6
S7
62.5
62.5
S8
62.5
125
S9
125
S10
S11
S12
15.62
7.81
S13
Std
7.81
3.9
7.81
7.81
7.81
7.81
31.25
62.5
15.62 31.25
15.62 62.5
7.81
7.81
15.62 31.25
15.62 31.25
31.25
31.25
Kleꢂsiella
125
pneumoniae
Pseudomonas
aeruginosa
15.62 31.25
62.5
31.25 62.5
31.25 125
15.62 125
125
125
31.25 62.5
31.25 62.5
31.25
62.5
7.81
Trichoderma
15.62 15.62
31.25 31.25
125
125
15.62 125
15.62 125
125
125
125
125
31.25
62.5
62.5
62.5
7.81
Aspergillus
niger
62.5
15.62 62.5
1.95
125
62.5
31.25 62.5
3.90 7.81
62.5
15.62
Aspergillus
flavus
15.62 15.62
62.5
7.81 125
125
125
15.62
31.25
7.81
7.81
Mycoꢂecterium
tuꢂerculosis
1.95
1.95
-
-
1.95
15.62
15.62
31.25
1.95
0.0092
-
: Inactive at tested concentrations
Synthesis of 2-chloro-N-(4-((3-(phenylimino)-3,4-dihydro-
quinoxalin-2(1 H)-ylidene) amino)phenyl)acetamide (4)
4
7
.21 (s, 1 H, NH of quinoxaline), 4.56 (s, 1 H, NH of quinoxaline),
.18–8.25 (m, 13 H, Ar-H), 8.34 (s, 1 H, CONH). EI-MS m/z: 405
1
(M+ 2), 403 (M ). Anal. Cald for C22H18ClN O: C, 65.43; H, 4.49; N,
+
N -(3-(Phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)ben-
5
zene-1,4-diamine 3 (3.27 g; 0.01 mol) was dissolved in glacial ace-
17.34. Found: C, 65.60; H, 4.47; N, 17.38.
tic acid (50 ml) containing of saturated solution of sodium acetate
Synthesis of 2-hydrazinyl-N-(4-((3-(phenylimino)-3,4-dihy-
(
50 ml). The mixture was warmed to dissolve the substance com-
droquinoxalin-2(1 H)-ylidene) amino)phenyl)acetamide (5)
pletely. The solution was cooled in ice bath with stirring. A solution
of chloroacetyl chloride (1.34 g; 0.012 mol) was added drop wise
to the above stirred solution so that the vigorous reaction did not
take place. After half an hour the product separated 4 is filtered,
washed with water, dried, and recrystallised using alcohol. Yield:
Equimolar quantity of 2-chloro-N-(4-((3-(phenylimino)-3,4-dihy-
droquinoxalin-2(1 H)-ylidene)amino)phenyl)acetamide 4 (4.03 g;
0.01 mol) and hydrazine hydrate (0.05 g; 0.01 mol) in ethanol
(30 ml) was refluxed for 10 h on a water bath. The resulting solu-
tion was cooled and poured in ice cold water with stirring. The prod-
uct 5 obtained was filtered, dried and recrystallised from ethanol.
Yield: 71 %, m.p.: 210–212 °C. IR (KBr, cm– 1): 3340 & 3268 (NH),
7
4 %, m.p.: 186–189 °C. IR (KBr, cm– 1): 3364 & 3282 (NH), 3092
(
7
Ar-CH), 2989 (CH -CH), 1682 (C = O), 1640 (C = N), 1613 (C = C),
2
1
68 (C-Cl). H NMR (CDCl , 500 MHz) δ ppm: 4.09 (s, 2 H, CH ),
3
2
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
2
-(2-(2-Chlorobenzylidene)hydrazinyl)-N-(4-((3-
3
040 (Ar-CH), 2975 (CH -CH), 1693 (C = O), 1632 (C = N), 1606
2
1
(phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S4)
(
(
C = C). H NMR (CDCl , 500 MHz) δ ppm: 2.03 (s, 2 H, NH ), 2.66
3 2
s, 1H, NH of CH NH), 3.95 (s, 2H, CH of CH NH), 4.38 (s, 1H, NH
2
2
2
of quinoxaline), 4.50 (s, 1 H, NH of quinoxaline), 7.31–8.04 (m,
Yield: 77 %, m.p.: 226–228 °C. IR (KBr, cm– 1): 3364 & 3267 (NH),
+
1
3 H, Ar-H), 8.12 (s, 1 H, CONH). EI-MS m/z: 399 (M ). Anal. Cald
3054 (Ar-CH), 2976 (CH -CH), 1679 (C = O), 1639 (C = N), 1590
2
1
for C22H21N O: C, 66.15; H, 5.30; N, 24.55. Found: C, 65.92; H,
(C = C), 785 (C-Cl). H NMR (CDCl , 500 MHz) δ ppm: 2.31 (s, 1 H,
7
3
5
.32; N, 24.63.
NH of CH NH), 3.97 (s, 2 H, CH of CH NH), 4.45 (s, 1 H, NH of qui-
2
2
2
noxaline), 4.64 (s, 1H, NH of quinoxaline), 6.82–7.95 (m, 17H, Ar-
H), 8.28 (s, 1 H, CONH), 8.39 (s, 1 H, CH of CH = N). EI-MS m/z: 523
General procedure for the synthesis of 2-(2-(substituted-
benzylidene)hydrazinyl)-N-(4-((3-(phenylimino)-3,4-dihyd-
roquinoxalin-2(1 H)-ylidene)amino)phenyl) acetamide
M+ 2), 521 (M ). Anal. Cald for C29H24ClN O: C, 66.73; H, 4.63; N,
+
(
7
1
8.78. Found: C, 66.95; H, 4.61; N, 18.72.
(
S1-S13)
2
-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-N-(4-((3-
Title compounds was synthesized by adding appropriate aromatic/
heterocyclic aldehydes (0.01mol) slowly with the well stirred mix-
ture of 2-hydrazinyl-N-(4-((3-(phenylimino)-3,4-dihydroquinoxa-
lin-2(1H)-ylidene)amino)phenyl)acetamide 5 (3.99g; 0.01mol) in
ethanol (50 ml), and glacial acetic acid (2–3 drops). The pH was
maintained to 5–6. The reaction mixture was then refluxed for a
period of 4–6 h, and kept aside for about 2 h. The products were
separated by filtration, washed, and vacuum dried. Finally the prod-
ucts were recrystallised using ethanol to get pure form.
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S5)
Yield: 74 %, m.p.: 257–260 °C. IR (KBr, cm– 1): 3345 & 3254 (NH),
3079 (Ar-CH), 2958 (CH -CH), 1681 (C = O), 1643 (C = N), 1629
(C = C). H NMR (CDCl , 500 MHz) δ ppm: 2.19 (s, 1 H, NH of
CH NH), 3.15 (s, 6 H, N(CH ) ), 3.62 (s, 2 H, CH of CH NH), 4.09
(s, 1 H, NH of quinoxaline), 4.36 (s, 1H, NH of quinoxaline), 7.03–
7.86 (m, 17H, Ar-H), 8.04 (s, 1H, CONH), 8.31 (s, 1H, CH of CH=N).
2
1
3
2
3 2
2
2
+
EI-MS m/z: 530 (M ). Anal. Cald for C31H30N O: C, 70.17; H, 5.70;
8
2
-(2-(3-Nitrobenzylidene)hydrazinyl)-N-(4-((3-
N, 21.12. Found: C, 69.94; H, 5.72; N, 21.18.
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S1)
Yield: 72 %, m.p.: 277–279 °C. IR (KBr, cm– 1): 3353 & 3289 (NH),
2-(2-(4-Nitrobenzylidene)hydrazinyl)-N-(4-((3-
(phenylimino)-3,4-dihydroquinoxalin-2(1H)-ylidene)
amino)phenyl)acetamide (S6)
3
095 (Ar-CH), 2953 (CH -CH), 1673 (C = O), 1621 (C = N), 1591
2
1
Yield: 73 %, m.p.: 221–224 °C. IR (KBr, cm– 1): 3336 & 3274 (NH),
(
(
C=C), 1534 & 1328 (NO ). H NMR (CDCl , 500MHz) δ ppm: 2.26
s, 1H, NH of CH NH), 3.91 (s, 2H, CH of CH NH), 4.20 (s, 1H, NH
2
3
3034 (Ar-CH), 2941 (CH -CH), 1675 (C = O), 1573 (C = N), 1549
2
2
2
2
1
of quinoxaline), 4.45 (s, 1 H, NH of quinoxaline), 6.90–7.72 (m,
(C=C), 1529 & 1310 (NO ). H NMR (CDCl , 500MHz) δ ppm: 3.02
2
3
1
7 H, Ar-H), 8.49 (s, 1H, CONH), 8.65 (s, 1H, CH of CH = N). EI-MS
(s, 1H, NH of CH NH), 3.69 (s, 2H, CH of CH NH), 4.25 (s, 1H, NH
2 2 2
+
m/z: 532 (M ). Anal. Cald for C29H24N O : C, 65.40; H, 4.54; N,
2
of quinoxaline), 4.41 (s, 1 H, NH of quinoxaline), 7.16–8.24 (m,
17 H, Ar-H), 8.43 (s, 1H, CONH), 8.78 (s, 1H, CH of CH = N). EI-MS
8
3
1.04. Found: C, 65.61; H, 4.53; N, 20.98.
+
m/z: 532 (M ). Anal. Cald for C29H24N O : C, 65.40; H, 4.54; N,
8
3
2
-(2-(2-Nitrobenzylidene)hydrazinyl)-N-(4-((3-
2
1.04. Found: C, 65.62; H, 4.55; N, 21.11.
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S2)
Yield: 75 %, m.p.: 244–246 °C. IR (KBr, cm– 1): 3348 & 3271 (NH),
2-(2-(Furan-2-ylmethylene)hydrazinyl)-N-(4-((3-
(phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S7)
3
069 (Ar-CH), 2983 (CH -CH), 1697 (C = O), 1645 (C = N), 1620
2
1
Yield: 70 %, m.p.: 190–194 °C. IR (KBr, cm– 1): 3336 & 3284 (NH),
(
(
C=C), 1546 & 1312 (NO ). H NMR (CDCl , 500MHz) δ ppm: 2.43
s, 1H, NH of CH NH), 3.56 (s, 2H, CH of CH NH), 4.42 (s, 1H, NH
2
3
3062 (Ar-CH), 2985 (CH -CH), 1690 (C = O), 1620 (C = N), 1598
2
2
2
2
1
of quinoxaline), 4.58 (s, 1 H, NH of quinoxaline), 7.24–8.19 (m,
(C = C), 1086 (C-O-C). H NMR (CDCl , 500 MHz) δ ppm: 2.04 (s,
3
1
7 H, Ar-H), 8.32 (s, 1H, CONH), 8.50 (s, 1H, CH of CH = N). EI-MS
1 H, NH of CH NH), 3.83 (s, 2H, CH of CH NH), 3.98 (s, 1H, NH of
quinoxaline), 4.31 (s, 1 H, NH of quinoxaline), 7.29–8.21 (m, 16H,
2
2
2
+
m/z: 532 (M ). Anal. Cald for C29H24N O : C, 65.40; H, 4.54; N,
8
3
2
1.04. Found: C, 65.19; H, 4.57; N, 21.09.
Ar-H), 8.36 (s, 1 H, CONH), 8.57 (s, 1 H, CH of CH = N). EI-MS m/z:
+
4
77 (M ). Anal. Cald for C27H23N O : C, 67.91; H, 4.85; N, 20.53.
7
2
2
-(2-(2-Hydroxybenzylidene)hydrazinyl)-N-(4-((3-
Found: C, 68.10; H, 4.83; N, 20.48.
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S3)
Yield: 70 %, m.p.: 230–234 °C. IR (KBr, cm– 1): 3531 (OH), 3335 &
2-(2-(3-Phenylallylidene)hydrazinyl)-N-(4-((3-
(phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S8)
3
242 (NH), 3088 (Ar-CH), 2960 (CH -CH), 1674 (C = O), 1616
2
1
Yield: 73 %, m.p.: 285–287 °C. IR (KBr, cm– 1): 3351 & 3247 (NH),
(
C=N), 1589 (C=C). H NMR (CDCl , 500MHz) δ ppm: 2.07 (s, 1H,
3
NH of CH NH), 3.70 (s, 2 H, CH of CH NH), 4.17 (s, 1 H, NH of qui-
noxaline), 4.49 (s, 1 H, NH of quinoxaline), 5.13 (s, 1H, OH), 7.06–
3074 (Ar-CH), 2963 (CH -CH), 1679 (C = O), 1648 (C = N), 1612
2
2
2
2
1
(C = C). H NMR (CDCl , 500 MHz) δ ppm: 2.35 (s, 1 H, NH of
3
8
.38 (m, 17H, Ar-H), 8.52 (s, 1H, CONH), 8.64 (s, 1H, CH of CH=N).
CH NH), 3.58 (s, 2H, CH of CH NH), 4.16 (s, 1H, NH of quinoxa-
line), 4.42 (s, 1 H, NH of quinoxaline), 5.43 (d, 1H, CH = CH), 6.29
(d, 1 H, CH = CH), 6.97–7.83 (m, 18 H, Ar-H), 8.13 (s, 1 H, CONH),
2
2
2
+
EI-MS m/z: 503 (M ). Anal. Cald for C29H25N O : C, 69.17; H, 5.00;
N, 19.47. Found: C, 68.93; H, 5.02; N, 19.54.
7
2
Saravanan G et al. Potent Quinoxaline Derivatives Drug Res

+
1
8
.40 (s, 1 H, CH of CH = N). EI-MS m/z: 513 (M ). Anal. Cald for
(C = C), 1068 (C-O-C). H NMR (CDCl , 500 MHz) δ ppm: 2.33 (s,
3
C31H27N O: C, 72.50; H, 5.30; N, 19.09. Found: C, 72.27; H, 5.31;
1H, NH of CH NH), 3.65 (s, 2H, CH of CH NH), 3.82 (s, 3H, OCH ),
7
2
2
2
3
N, 19.01.
4.20 (s, 1 H, NH of quinoxaline), 4.57 (s, 1 H, NH of quinoxaline),
.26–8.39 (m, 17 H, Ar-H), 8.51 (s, 1H, CONH), 8.78 (s, 1H, CH of
7
2
-(2-Benzylidenehydrazinyl)-N-(4-((3-(phenylimino)-3,4-
dihydroquinoxalin-2(1H)-ylidene)amino)phenyl)acetamide
S9)
Yield: 71 %, m.p.: 249–251 °C. IR (KBr, cm– 1): 3345 & 3239 (NH),
+
CH = N). EI-MS m/z: 517 (M ). Anal. Cald for C30H27N O : C, 69.62;
7
2
H, 5.26; N, 18.94. Found: C, 69.83; H, 5.24; N, 18.89.
(
Results and discussion
3
090 (Ar-CH), 2957 (CH -CH), 1684 (C = O), 1618 (C = N), 1581
2
1
(
C = C). H NMR (CDCl , 500 MHz) δ ppm: 2.18 (s, 1 H, NH of
3
Chemistry
CH NH), 3.95 (s, 2H, CH of CH NH), 4.33 (s, 1H, NH of quinoxa-
2
2
2
line), 4.56 (s, 1 H, NH of quinoxaline), 7.34–8.22 (m, 18 H, Ar-H),
A series of novel 2-(2-(substitutedbenzylidene)hydrazinyl)-N-(4-
((3-(phenylimino)-3,4-dihydro quinoxalin-2(1H)-ylidene)amino)
phenyl)acetamide S1-S13 were synthesized from o-phenylenedi-
amine and oxalic acid by a multistep synthesis. The synthetic path-
way by which compounds synthesized were illustrated in ▶Fig. 3.
Initially o-phenylenediamine was treated with oxalic acid to obtain
quinoxaline-2,3(1H,4H)-dione 1 by a ring closure reaction with re-
moval of two water molecule. Latter, obtained quinoxa-
line-2,3(1 H,4 H)-dione 1 undergone Schiff base reaction with p-
phenylenediamine and produced 3-(4-aminophenylimino)-3,4-di-
hydroquinoxalin-2(1 H)-one 2. In the succeeding step, compound
8
.40 (s, 1 H, CONH), 8.52 (s, 1 H, CH of CH = N). EI-MS m/z: 487
+
(
M ). Anal. Cald for C H N O: C, 71.44; H, 5.17; N, 20.11. Found:
2
9
25 7
C, 71.63; H, 5.16; N, 20.04.
2
-(2-(4-Methylbenzylidene)hydrazinyl)-N-(4-((3-
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S10)
Yield: 75 %, m.p.: 203–206 °C. IR (KBr, cm– 1): 3360 & 3257 (NH),
3
056 (Ar-CH), 2972 (CH -CH), 1672 (C = O), 1638 (C = N), 1604
2
1
(
C = C). H NMR (CDCl , 500 MHz) δ ppm: 2.21 (s, 1 H, NH of
3
1
CH NH), 2.78 (s, 3H, CH ), 3.79 (s, 2H, CH of CH NH), 4.14 (s, 1H,
2 was treated with aniline to produce N -(3-(phenylimino)-3,4-di-
2
3
2
2
NH of quinoxaline), 4.36 (s, 1H, NH of quinoxaline), 7.19–8.07 (m,
hydroquinoxalin-2(1 H)-ylidene)benzene-1,4-diamine 3 by Schiff
base reaction. In the next step, compound 3 was treated with chlo-
ro acetyl chloride to get a corresponding chloro acetyl derivative 4
[2-chloro-N-(4-((3-(phenylimino)-3,4-dihydroquinoxalin-2(1H)-
ylidene)amino)phenyl) acetamide] by chloro acetylation in pres-
ence of acetic acid and sodium acetate. In the next step compound
1
7 H, Ar-H), 8.25 (s, 1H, CONH), 8.37 (s, 1H, CH of CH = N). EI-MS
+
m/z: 501 (M ). Anal. Cald for C30H27N O: C, 71.84; H, 5.43; N,
7
1
9.55. Found: C, 71.60; H, 5.45; N, 19.61.
2
-(2-(4-Hydroxybenzylidene)hydrazinyl)-N-(4-((3-
(
phenylimino)-3,4-dihydro quinoxalin-2(1 H)-ylidene)
4
was treated with hydrazine hydrate to produce a corresponding
amino)phenyl)acetamide (S11)
2
-hydrazinyl acetamide derivative 5 [2-hydrazinyl-N-(4-((3-(phenyl
Yield: 79 %, m.p.: 198–201 °C. IR (KBr, cm– 1): 3590 (OH), 3357 &
imino)-3,4-dihydro quinoxalin-2(1H)-ylidene)amino)phenyl)aceta-
mide]. Finally, title compounds 2-(2-(substituted benzylidene)
hydrazinyl)-N-(4-((3-(phenylimino)-3,4-dihydro quinoxalin-2(1H)-
ylidene) amino)phenyl) acetamide S1-S13 were synthesized by
treating compound 5 with different aromatic/heterocyclic alde-
hydes by a Schiff base reaction according to the ▶Fig. 3. TLC was
performed throughout the reactions to optimize the reactions for
purity and completion.
3
280 (NH), 3076 (Ar-CH), 2973 (CH -CH), 1674 (C = O), 1585
2
1
(
C=N), 1550 (C=C). H NMR (CDCl , 500MHz) δ ppm: 2.29 (s, 1H,
3
NH of CH NH), 3.16 (s, 2 H, CH of CH NH), 4.02 (s, 1 H, NH of qui-
2
2
2
noxaline), 4.38 (s, 1 H, NH of quinoxaline), 5.34 (s, 1H, OH), 7.01–
8
.25 (m, 17H, Ar-H), 8.85 (s, 1H, CONH), 9.03 (s, 1H, CH of CH=N).
+
EI-MS m/z: 503 (M ). Anal. Cald for C29H25N O : C, 69.17; H, 5.00;
7
2
N, 19.47. Found: C, 69.39; H, 5.02; N, 19.50.
1
IR, H NMR, mass spectra, and elemental analyses of the syn-
2
-(2-(4-Chlorobenzylidene)hydrazinyl)-N-(4-((3-
thesized compounds are in accordance with the assigned struc-
tures. The IR spectra of all synthesized compounds showed some
characteristic peaks indicating the presence of particular groups.
Formation of the quinoxaline-2,3(1H,4H)-dione ring in compound
1 was confirmed by the presence of absorption peak at 3351 &3279
and 1698 cm– 1 in IR due to presence of NH and C = O stretching,
respectively. This is further supported by the appearance of two
singlet in NMR at δ 8.34 & 8.51ppm corresponds to CONH of qui-
noxaline ring. The formation of compounds 2 was confirmed by the
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
amino)phenyl)acetamide (S12)
Yield: 74 %, m.p.: 265–268 °C. IR (KBr, cm– 1): 3330 & 3265 (NH),
3
076 (Ar-CH), 2969 (CH -CH), 1692 (C = O), 1621 (C = N), 1586
2
1
(
C = C), 764 (C-Cl). H NMR (CDCl , 500 MHz) δ ppm: 2.09 (s, 1 H,
3
NH of CH NH), 3.84 (s, 2 H, CH of CH NH), 4.37 (s, 1 H, NH of qui-
noxaline), 4.43 (s, 1H, NH of quinoxaline), 6.95–8.01 (m, 17H, Ar-
H), 8.34 (s, 1 H, CONH), 8.66 (s, 1 H, CH of CH = N). EI-MS m/z: 523
2
2
2
M+ 2), 521 (M ). Anal. Cald for C29H24ClN O: C, 66.73; H, 4.63; N,
+
appearance of singlet at δ 4.13ppm for two protons in its H NMR
1
(
7
1
8.78. Found: C, 66.50; H, 4.64; N, 18.82.
spectra which might be assigned to NH group. Disappearance of
2
carbonyl group absorbance in IR spectrum around 1700cm– 1 and
2
-(2-(4-Methoxybenzylidene)hydrazinyl)-N-(4-((3-
1
appearance of two singlet in H NMR spectra at δ 4.05 and 4.49ppm
(
phenylimino)-3,4-dihydroquinoxalin-2(1 H)-ylidene)
for one proton each corresponds to NH of quinoxaline ring confirms
the formation of compound 3. Appearance of sharp absorbance
peak in IR spectra at 1682 and 768 cm– 1 due to presence of C = O
and C-Cl stretching, respectively confirms the formation of com-
amino)phenyl)acetamide (S13)
Yield: 76 %, m.p.: 293–295 °C. IR (KBr, cm– 1): 3356 & 3272 (NH),
3
097 (Ar-CH), 2980 (CH -CH), 1685 (C = O), 1640 (C = N), 1619
2
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
H
N
H
N
NH2 HO
O
O
O
O
N
4
N HCl
NH C H NH
2
+
2
6
4
NH2 HO
N
H
N
H
O
NH2
H
C H NH
6
5
2
N
N
N
O
H
N
N
N
ClCH COCl
2
Cl
N
H
N
H
CH COOH
3
CH COONa
3
N
H
NH2
1
0h
NH NH2 . H O
2
2
H
N
N
N
O
H
N
N
H
N
NH2
H
R
CHO
H
N
N
N
O
H
N
N
H
N
H
N
R
R
R
R
CH3
CH3
S1 =
S2 =
S5 =
N
S10 =
CH3
NO2
S6 =
NO2
S11 =
S12 =
S13 =
OH
Cl
O
O
2
N
S7 =
S8 =
S3 =
S4 =
C
C
H
H
OCH3
HO
Cl
S9 =
▶
Fig. 3 Synthetic scheme.
pound 4. This is further confirmed by appearance of singlet at δ
IR spectrum of title compound shows absorption bands at 3330–
1
3364 & 3239–3289 cm– 1, 3034–3097 cm– 1, 2941–2985 cm– 1
8
.34 ppm for one proton in its H NMR spectra which might be as-
,
signed to CONH group. Further presence of a two protons in NH2
of compound 5 was confirmed by the appearance of singlet at δ
1672–1697 cm– 1, 1573–1648 cm– 1, and weak band at 1549–
1629 cm– 1, which can be assignable to NH, Ar-H, CH -H, C = O,
2
1
2
.03 ppm. This is further supported by appearance of singlet in H
C=C, and C=N vibrations, respectively. IR spectrum of compounds
S1, S2 and S6 showed absorption band in the region of 1529–
1546 cm– 1 and 1310–1328 cm– 1 which may be assigned to NO2
NMR spectra at δ 2.66 and 3.95ppm for one and two protons, re-
spectively which might be assigned to NH and CH of CH NH. The
2
2
Saravanan G et al. Potent Quinoxaline Derivatives Drug Res

▶
Fig. 4 Docked view of all the compounds at the active site of the enzyme PDB ID: 2PZI.
▶
Taꢂle 3 Surflex Docking score (kcal/mol) of the derivatives.
ꢁ
ompound ꢁode
ꢁ Scorea
6.20
5.92
5.91
5.81
5.73
5.71
5.49
5.23
4.91
4.70
4.68
3.47
3.36
ꢁrash Scoreꢂ
− 2.04
− 5.39
− 3.13
− 1.56
− 3.82
− 1.16
− 3.52
− 2.83
− 1.84
− 2.12
− 3.82
− 2.50
− 0.84
Polar Scorec
2.15
D Scored
PMF Scoree
− 11.794
− 33.140
− 63.590
− 35.726
− 36.900
− 29.481
− 46.508
− 64.174
− 28.976
− 43.669
− 11.803
− 46.216
− 15.626
G Scoref
ꢁhem Scoreg
− 33.618
46.500
S6
− 145.421
− 194.872
− 195.043
− 129.754
− 204.548
− 138.943
− 202.452
− 194.606
− 165.176
− 197.668
− 164.413
− 137.175
− 104.621
− 251.859
− 332.882
− 340.197
− 185.072
− 309.049
− 225.376
− 316.260
− 309.830
− 252.999
− 300.765
− 266.513
− 214.545
− 149.543
S4
2.69
S12
S3
0.51
− 40.375
− 34.638
− 41.291
− 33.436
− 41.800
− 36.356
− 33.282
37.517
3.46
S11
S5
1.03
1.22
S10
S1
0.71
1.25
S7
0.83
S13
S8
0.71
0.91
− 35.859
− 35.192
− 26.310
S9
2.65
S2
1.35
aCScore (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor
and reports the output of total score; b Crash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are
favorable. Negative numbers indicate penetration; c Polar indicating the contribution of the polar interactions to the total score. The polar score may
be useful for excluding docking results that make no hydrogen bonds; d D-score for charge and van der Waals interactions between the protein and
the ligand; e PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of Mean Force, PMF); G-score
showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies; g Chem-score points for H-bonding, lipophilic contact,
and rotational entropy, along with an intercept term.
f
In silico screening
group. Compound S3 and S11 showed absorption bands at 3531–
590 cm– 1 due to presence of OH stretching. In addition presence
3
The PknG inhibition activity of 2-(2-(substituted benzylidene)
hydrazinyl)-N-(4-((3-(phenylimino)-3,4-dihydroquinoxalin-2(1H)-
ylidene)amino)phenyl) acetamide S1-S13 compounds docked with
active site of crystal structure of PknG. The molecular docking stud-
ies were performed on the crystal structure of Protein kinase PknG
from Mtb in Complex with Tetrahydrobenzothiophene using the
surflex-dock programme of sybyl-X 2.0 software. All the 13 inhibi-
tors were docked into the active site of enzyme as shown in
▶Fig. 4a and b. The predicted binding energies of the compounds
are listed in ▶Taꢂle 3. The docking study revealed that all the com-
pounds have showed very good docking score against Mtb. As de-
picted in the ▶Fig. 5a-c, compound S6 makes two hydrogen bond-
ing interactions at the active site of the enzyme (PDB ID: 2PZI), hy-
drogen atom present at hydrazineyl group makes an interaction
with carbonyl group of ILE292 (-NH ------ O=C-ILE292, 2.12Å), hy-
drogen atom present at the quinoxaline ring makes hydrogen
of chlorine in compounds S4 and S12 were confirmed by the ap-
pearance of absorption bands at 764–785cm-1 in IR spectrum and
appearance of M+ 2 peaks in mass spectrum. Compound S7 and S13
showed absorption bands at 1068–1086cm– 1 due to presence of
C-O-C vibrations. The proton magnetic resonance spectrums of
synthesized compounds were recorded in CDCl . The following con-
3
1
clusions can be derived by comparing the H NMR spectra of title
compounds:
▪
▪
▪
▪
▪
▪
▪
A singlet at δ 2.04–3.02 ppm for NH of CH NH,
2
A singlet at δ 3.16–3.97 ppm for CH of CH NH,
2
2
A singlet at δ 3.98–4.45 ppm for NH of quinoxaline,
A singlet at δ 4.31–4.64 ppm for NH of quinoxaline,
A multiplet at δ 6.82–8.39 ppm for Ar-CH,
A singlet at δ 8.04–8.85 ppm for CONH,
A singlet at δ 8.31–9.03 ppm for N = CH.
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
▶
Fig. 5 Docked view of compound S6 at the active site of the enzyme PDB: 2PZI.
▶
Fig. 6 Interaction of compound S4 at the binding site of the enzyme (PDB ID: 2PZI).
▶
Fig. 7 Interaction of compound S12 at the binding site of the enzyme (PDB ID: 2PZI).
bonding interaction with carbonyl group of GLY237 (NH-----
As depicted in ▶Fig. 7a-c, compound S12, makes four hydro-
gen bonding interactions at the active site of the enzyme (PDB ID:
2PZI), among those, two interactions were of nitrogen atom of hy-
drazineyl group with hydrogen of LYS181 (-N ------ H-LYS181,
2.68Å, 2.57Å), hydrogen atom of carboxamide group with carbon-
yl group of ASN281 (NH ------ O = C-ASN281, 2.42 Å) and the car-
bonyl group of the carboxamide has one hydrogen bonding with
hydrogen of LYS181 (C = O-----H-LYS181, 2.73 Å). ▶Fig. 8 repre-
sents the selected area of interest for comparative analysis.
-
O = C-GLY237, 2.06 Å).
As depicted in ▶Fig. 6a-c, compound S4, makes five hydrogen
bonding interactions at the active site of the enzyme (PDB ID: 2PZI),
among those, two interactions were of hydrogen atom of quinox-
aline ring with oxygen of GLU280 (-NH ------ O-GLU280, 2.41 Å,
2
.50 Å),
hydrogen atom of acetamide group makes an interaction with
carbonyl group of ILE292 (-NH ------ O = C-ILE292, 1.88 Å), hydro-
gen atom of hydrazineyl group makes hydrogen bonding interac-
tion with carbonyl group of GLU233 (NH ------ O = C-GLU233,
Antimicrobial activity
2
.21 Å) and remaining another hydrogen bonding interaction
All the synthesized compounds were evaluated for their in vitro an-
tibacterial and antifungal activity. A comparison of antimicrobial
activity of the synthesized compounds with that of standard drugs
raised from the nitrogen atom of hydrazineyl group with hydrogen
of VAL235 (N ----- H-VAL235).
Saravanan G et al. Potent Quinoxaline Derivatives Drug Res

H
N
N
N
O
H
N
N
H
N
H
N
R
ANTI-TB ACTIVITY
MOST FAVORABLE
ANTIMICROBIAL ACTIVITY
MOST FAVORABLE
UNFAVORABLE
R
R
NO2
NO2
S6
S6
FAVORABLE
HO S3
FAVORABLE
CH3
CH3
N
S5
S1
S1
NO2
NO2
S2
S2
O N
2
O N
2
S4
S4
Cl
Cl
CH3
Cl
Cl
S10
S12
S12
▶
Fig. 8 Selected area of interest for comparative analysis.
was effectively presented in ▶Taꢂle 2. All compounds were found
to exhibit significant activity against all the tested bacteria and
fungi. Compounds S1, S2, S4, S6, S10 and S12 showed excellent
antimicrobial properties against the entire microorganisms that
were included in the present evaluation. When compared to stand-
ard drug, compound S6 showed remarkable activity against Bacil-
lus subtilis ATCC 6633, Klebsiella pneumoniae ATCC 13883, Tricho-
derma ATCC 26921, and Aspergillus niger ATCC 9029. In addition
compound S6 showed equipotent activity against Pseudomonas
aeruginosa ATCC 27853, and Aspergillus flavus ATCC 10124. More-
over, compound S1 exhibited more activity against Klebsiella pneu-
moniae ATCC 13883 when compared with the reference drug Cip-
rofloxacin. Among the tested compounds against Bacillus subtilis
ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Trichoderma
ATCC 26921, and Aspergillus niger ATCC 9029 compound S1
showed equipotent activity like standard drug; whereas compound
S2 shown equipotent activity against Klebsiella pneumoniae ATCC
13883 and Aspergillus niger ATCC 9029. Compounds S3, S5, S11
and S13 showed moderate to good anti-microbial activity against
tested micro organism. Rest of compounds S7, S8 and S9 showed
weaker anti-microbial activity.
All the synthesized compounds were subjected to MIC (mini-
mum inhibitory concentration) studies against all microorganisms.
The MICs of Ciprofloxacin and Ketoconazole were determined in
parallel experiments in order to control the sensitivity of the test
organisms. MIC values of the compounds and the standards are
presented in ▶Taꢂle 2. As seen in ▶Taꢂle 2 While all compounds
showed lower activities than the standard against B. subtilis, com-
Saravanan G et al. Potent Quinoxaline Derivatives… Drug Res

Original Article
Thieme
pounds S1, S2 and S6 showed the same activity (MIC: 7.81µg/ml).
Against K. pneumoniae, all compounds exhibited lower activity
Conclusions
(
MIC: 7.81–125µg/ml) than standard (MIC: 3.9µg/ml). Similarly all
In summary, a series of novel quinoxaline Schiff bases (S1-S13) were
synthesized and evaluated for anti-microbial activities against var-
ious pathogenic microorganisms. IR, H-NMR, Mass spectroscopy
test compounds exhibited lower activity (MIC: 15.62–125µg/ml)
against P. aeruginosa than standard Ciprofloxacin (MIC: 7.81 µg/
ml). Compared to Ketoconazole (MIC: 7.81µg/ml) entire test com-
pounds exhibited lesser activity (MIC: 15.62–125 µg/ml) against
Trichoderma. Compound S6 showed the same activity (MIC:
1
and elemental analyses data of the synthesized compounds are in
accordance with the assigned structures. All compounds were
found to display significant activity against entire tested bacteria,
fungi and Mtb H37Ra. Towards entire tested bacteria, fungi & Mtb
H37Ra compounds S1, S2, S4, S6, S10 and S12 showed excellent
anti-microbial and anti-TB properties. SAR studies indicated that
nature of the substituent attached at 2-hydrazinyl acetamide is im-
portant for anti-microbial and anti-TB activities. The chemical
structure and anti-microbial and anti-TB activities are relationship
of the synthesized compounds revealed that the compounds hav-
ing electron withdrawing moiety exhibited better activity than
compounds having electron releasing moieties; whereas the un-
substituted derivative exhibited weaker activity. On correlating the
structures of the sample candidate within the same substituent,
para substituted derivatives showed potent activity than corre-
sponding ortho / meta substituted analogs. Out of thirteen tested
compounds, 2-(2-(4-nitrobenzylidene)hydrazinyl)-N-(4-((3-
(phenylimino)-3,4-dihydroquinoxalin-2(1H)-ylidene) amino)phe-
nyl)acetamide S6 exhibited most potent anti-bacterial, anti-fungal
and anti-TB activities compared to Ciprofloxacin and Ketoconazole.
As a result the compound S6 could therefore serve as a pilot mol-
ecule for further development as novel class of anti-microbial
agent.
1
5.62 µg/ml) as Ketoconazole against A. niger, while others dem-
onstrated lower activity (MIC: 31.25–125 µg/ml) than standard.
Compound S6 exhibited the same activity (MIC: 7.81µg/ml) against
A. flavus, while other compounds exhibited lower activity (MIC:
1
5.62–125 µg/ml) than standard Ketoconazole.
In vitro anti TB screening
Herein we report anti TB screening of title compounds 2-(2-(sub-
stituted benzylidene) hydrazinyl)-N-(4-((3-(phenylimino)-3,4-di-
hydroquinoxalin-2(1 H)- ylidene) amino) phenyl) acetamide S1-
S13. The anti-TB effect of these synthetic compounds on the
growth of Mtb H37Ra was recorded after 7 days of incubation at
3
0
7°C using by MABA method. There are six different concentrations
.97, 1.95, 3.90, 7.81, 15.62 and 31.25µg/ml were used. The data
of the anti-TB activity screening reveals that the compound S3, S5
was inactive in all concentrations against MTB H37Ra strain. In the
MABA screening, compounds S1, S2, S4, S6 and S12 were sensi-
tive at 1.95µg/ml concentration; compound S10 were sensitive at
3
.90µg/ml concentration; compounds S11 and S13 were sensitive
at 7.81 µg/ml concentration; compounds S7, and S8, were sensi-
tive at 15.62 µg/ml concentration and compound S9 were sensi-
tive at 31.25 µg/ml concentration. As like antimicrobial activity it
is interesting to highlight that compounds S1, S2, S4, S6 and S12
showed enhanced activity and these compounds were well
thought-out as a most effective scaffolds against microbial strains
and was found to indicate alike anti-TB potency.
Acknowledgement
The authors are thankful to the management of MNR College of
Pharmacy, Fasalwadi, Sangareddy, Telangana, India, for providing
infrastructure facilities to carry out this research work. The authors
gratefully acknowledge the Central Instrumentation Facility, IIT
Chennai, India for the spectral analysis of the compounds used in
this study. The authors also thank Kalasalingam University, Tamil-
nadu and VIT University, Vellore for providing insilico facilities to
carry out this research work.
Structure activity relationships (SAR)
Structure activity relationship (SAR) studies indicated that nature
of the substituent attached at 2-hydrazinyl acetamide is important
for anti-microbial and anti-TB activity ▶Fig. 8. In general it was
found that most potent anti-microbial and anti-TB activities was
exhibited compounds (S1, S2, S4, S6 and S12) possessing electron
withdrawing substituent like nitro, chlorine, and methyl group on
phenyl ring attached to 2-hydrazinyl acetamide. While other com-
pounds containing electron donating substituents such as meth-
oxy, hydroxyl and dimethylamino groups (S3 and S5) exhibited
moderate in-vitro anti-microbial activity and poor anti-TB activity.
The unsubstituted / heterocyclic derivative (S7, S8 and S9) showed
moderate to weaker activity. The chemical structure and anti-mi-
crobial and anti-TB activities are relationship of the synthesized
compounds revealed that the compounds having electron with-
drawing moiety exhibited better activity than compounds having
electron releasing moieties; whereas the unsubstituted derivative
exhibited weaker activity. On correlating the structures of the sam-
ple candidate within the same substituent, para substituted deriv-
atives (S6 and S12) showed potent activity than corresponding
ortho/meta substituted analogs (S1, S2 and S4).
Conflict of Interest
The authors have declared no conflict of interest.
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