Synthesis and antimicrobial activity of some new substituted quinoxalines

Article abstract of DOI:10.3390/molecules24224198

A number of new symmetrically and asymmetrically 2,3-disubstituted quinoxalines were synthesized through functionalization of 2,3-dichloroquinoxaline (2,3-DCQ) with a variety of sulfur and/or nitrogen nucleophiles. The structures of the obtained compounds were established based on their spectral data and elemental analysis. The antimicrobial activity for the prepared compounds was investigated against four bacterial species and two fungal strains. The symmetrically disubstituted quinoxalines 2, 3, 4, and 5 displayed the most significant antibacterial activity, while compounds 6a, 6b, and the pentacyclic compound 10 showed considerable antifungal activity. Furthermore, compounds 3f, 6b showed broad antimicrobial spectrum against most of the tested strains.

Full text of DOI:10.3390/molecules24224198

molecules
Article
Synthesis and Antimicrobial Activity of
Some New Substituted Quinoxalines
Mohamed A. El-Atawy 1,2,
*
, Ezzat A. Hamed 2, Mahjoba Alhadi 2 and Alaa Z. Omar 2
1
Chemistry Department, Faculty of Science, Taibah University, Yanbu 46423, Saudi Arabia
Chemistry Department, Faculty of Science, Alexandria University, P.O. 426 Ibrahemia,
Alexandria 21321, Egypt; ezzat.awad@alexu.edu.eg (E.A.H.); mahjoba.alhadi@yahoo.com (M.A.);
Alaazaki@alexu.edu.eg (A.Z.O.)
2
*
Correspondence: mohamed.elatawi@alexu.edu.eg; Tel.: +966-569-191-532
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 12 September 2019; Accepted: 12 November 2019; Published: 19 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A number of new symmetrically and asymmetrically 2,3-disubstituted quinoxalines were
synthesized through functionalization of 2,3-dichloroquinoxaline (2,3-DCQ) with a variety of sulfur
and/or nitrogen nucleophiles. The structures of the obtained compounds were established based on
their spectral data and elemental analysis. The antimicrobial activity for the prepared compounds was
investigated against four bacterial species and two fungal strains. The symmetrically disubstituted
quinoxalines
2, 3, 4, and 5 displayed the most significant antibacterial activity, while compounds
6a 6b, and the pentacyclic compound 10 showed considerable antifungal activity. Furthermore,
,
compounds 3f, 6b showed broad antimicrobial spectrum against most of the tested strains.
Keywords: quinoxalines; antimicrobial; thioether; thioglycolic acid and pentacyclic compounds
1. Introduction
The quinoxaline nucleus is one of the most important heterocyclic scaffolds. Quinoxalines exist
in several different biologically active compounds including some antibiotics such as levomycin,
actinoleutin and echinomycin which are known to be active against several transplantable tumors [
Beside that they have different applications in many fields such as agricultural [ ], fluorescent
materials [ ], dyes [ ], electroluminescent materials [ ], organic semiconductors [ ], organic light
emitting devices [ ], and medicinal chemistry. Various synthetic quinoxalines exhibit miscellaneous
therapeutic applications in medicinal research such as anticancer [ 10], anti-inflammatory [ 11],
antiviral [12 13], antidiabetic [14 15], antidepressant [16], anthelmintic [ ], antituberculosis [17],
antibacterial [18 19], and antiprotozoal [20 21]. Two clinical examples for drugs containing quinoxaline
1].
2
3
4
5
6
7
8–
9,
,
,
2
,
,
are illustrated in Figure 1. Namely, chlorosulfaquinoxaline [22] and R-(+)-XK469 [23]. The former
drug has immunosuppressive activity and potential antineoplastic activity [24] the second one is
an anticancer drug that has activity against solid tumors [25]. Furthermore, quinoxalines are used in
the agricultural field as herbicides, insecticides, and fungicides [2]. Thus, due to this diversity of useful
applications for quinoxalines, scientists pay much attention to the classical synthetic methods, their
modification, and for new methods for synthesizing quinoxalines to ensure the availability of more
functionalized quinoxalines.
Organosulfur compounds are well defined as organic molecules having one or more carbon-sulfur
bonds. These compounds constitute very diverse chemical structures and display several bioactive
properties as well as antimicrobial; the antimicrobial activity of some organosulfur compounds has
been reported against a wide spectrum of fungi, bacteria, and viruses [26]. Some of these organosulfur
compounds are also used in the treatment of various types of tumors caused by Helicobacter pylori
along with gastric ulcers. Moreover, existence of sulfur and nitrogen usually improve the efficiency
Molecules 2019, 24, 4198; doi:10.3390/molecules24224198
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4198
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of some compounds for the treatment of several types of fungal, bacterial, and different pathogen
causing diseases [27 29]. Recently, antimicrobial activity of trisubstituted quinoxaline derivatives has
–
been reported [19]. It was observed that 4-triflouromethylanilino, 4-hydroxyanilino or phenylthio
groups at positions 2 and/or 3 of quinoxaline ring are responsible for good to moderate antibacterial
activity. However, pipridino or morpholino groups at these positions reduced the antibacterial activity.
Furthermore, most of tested quinoxalines showed moderate to weak antifungal activity. Inspired and
motivated by these findings and by the importance of sulfur and nitrogen containing compounds,
we tried to improve the antimicrobial activity of quinoxaline by its functionalization with sulfur and/or
nitrogen. In order to identify new candidates that may be of value in designing new, selective, and
less toxic antimicrobial agents. Herein we report our results on functionalization of quinoxaline with
a variety of sulfur and nitrogen nucleophiles using classical methods and testing the products for their
biological activities.
Figure 1. Examples for drugs containing quinoxaline moiety.
2. Results and Discussion
2.1. Chemistry
Among the different methods for synthesizing symmetrically 2,3-disubstituted quinoxalines,
there are two common methods widely used. The first one includes cyclocondensation reaction
between symmetric alpha diketone such as benzil derivatives and o-phenylenediamine [30–32].
In general, this procedure needs use of a strong acid catalyst, high temperature, and long reaction
times. Moreover, the main drawback of this method is its scope, which is limited to the synthesis of
dialkyl or diaryl quinoxaline. The second method involves nucleophilic substitution reaction between
2,3-DCQ and nucleophiles [19]. This method has broader scope due to the availability of the starting
materials and the ease of their preparation. In the current work, a number of symmetrically 2,3-
disubstituted quinoxalines have been prepared through simple nucleophilic displacement on 2,3-DCQ
using either sulfur or nitrogen nucleophiles affording products 2a–e and 3a–g, respectively as shown
in Scheme 1. The yields of the reaction varied from good to excellent yield. The starting compound
1
was prepared by cyclocondensation reaction between o-phenylenediamine and oxalic acid in strong
acidic medium, to give quinoxaline-2,3-dione [33]. Which, on subsequent chlorination with thionyl
chloride or phosphorus oxychloride afforded the desired starting 2,3-DCQ 1. The S-aryl or NH-aryl
groups of the products 2a–e and 3a–g, respectively, bear a variety of substituents including halogens
(F, Br, and Cl), alkyl and alkoxy groups. Infrared spectrum of products 3a–g showed bands in the region
3200–3300 cm−1 that can be ascribed to the absorption characteristic for N-H stretching. Moreover,
proton NMR (DMSO-d6) of 3a–g revealed broad D2O-exchangeable singlets at
9.85, 8.84, and 8.87 ppm, respectively, due to the existence of NH protons. The infrared spectrum of
products 2e 3d–g, and
exhibited absorption bands at 2916–2999 cm−1 corresponding to the aliphatic
C-H stretching. The methyl groups of compounds 2e 3d 3e 3g, and 3f were displayed in both
proton and 13C-NMR spectrum. Accordingly, methyl protons of 2e 3d revealed peaks in 1H-NMR
2.42, 2.30 ppm, respectively. Additionally, 13C-NMR spectrum of 2e
at 3d exhibited signals at δ
δ 11.93, 9.63, 9.16, 8.92,
,
4
,
,
,
,
δ
,
21.28, 20.51 ppm respective to their methyl carbon. The methyl groups of 3e showed two singlets in
1H-NMR at 2.23 and 2.26 ppm and two signals in 13C-NMR at 18.89 and 19.72 ppm. However, the
methoxy groups of 3g were observed in 1H-NMR spectrum as more deshielded singlets at 3.76 and

Molecules 2019, 24, 4198
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1
3.81 ppm and in 13C-NMR spectrum as two signals at 55.78 and 55.40 ppm. H-NMR spectrum of 3f
exhibited a singlet at 3.76 ppm attributable to methoxy protons. Furthermore, methoxy carbon of 3f
appeared as a signal at 55.22 ppm in 13C-NMR. The formation of compound
4
has been confirmed by
the two singlets that appeared in 1H-NMR at 11.50, 4.17 ppm due to COOH and CH2, respectively.
Additionally, 13C-NMR of
showed two signals at 169.53 and 32.49 ppm corresponding to the carbons
of the carboxymethyl moiety (CH2COOH). Furthermore, Conformation of the symmetrical structures
of the products (2a–e 3a–g, and ) has been achieved mainly through NMR spectroscopic data. As it is
well known that symmetry-equivalent atoms must absorb at the same chemical shift. Accordingly,
due to the molecular symmetry of the products (2a–e 3a–g, and ) their quinoxaline moiety showed
4
,
4
,
4
up two peaks in proton NMR and two quaternary carbons in 13C-APT-NMR. As a prototype, the
13C-APT-NMR of compound 2a in DMSO-d6 revealed the presence of three quaternary carbons at
128.70, 140.37, and 153.73 ppm. The last two peaks (153.73 and 140.37 ppm) were assigned to the
quaternary carbons of the quinoxaline moiety of 2a, while the peak that appeared at 128.70 ppm
was referred to as the ipso quaternary carbon of the thiophenyl ring. Nevertheless, the different
environments for the protons or carbons of non-symmetrical structures (5, 6a,b, 7, 8, 9a–f, and 10) led
to appearance of four protons and four quaternary carbons corresponding to their quinoxaline moiety
as illustrated in Figure 2.
Scheme 1. Synthesis of sym. 2,3-disubstituted quinoxaline.
As an extension to this study, a variety of asymmetrically 2,3-disubstituted quinoxaline
containing sulfur and/or nitrogen substituents have been synthesized (Scheme 2). One of
the challenges of this work was how to find a simple and efficient method for the synthesis
of asym. disubstituted quinoxalines. In a previously reported method, vinylic substitution
reaction occurred between 1,1-dimethylsulfanyl-2-nitroethene and appropriate aniline derivative
to afford (N-(1-(methylthio)-2-nitrovinyl)aniline which on subsequent reductive cyclization gave
2-chloro-3-(methylthio)quinoxaline [34] as a precursor for transformation into asym. disubstituted
quinoxalines. However, a more convenient approach for the synthesis of these quinoxaline derivatives
is the substitution of one of the chlorines at the 2- or 3-position of 2,3-DCQ by nucleophiles. The reaction
was conducted at room temperature with continuous stirring to avoid occurrence of double substitution

Molecules 2019, 24, 4198
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on
acid, and hydrazine to afford compounds
chlorinated quinoxaline as analogue to several biologically active quinoxalines [35]. Reaction of either
6a with hydrazine or reaction of with thiophenol gave the same compound , which on subsequent
1
. Consequently, 2,3-DCQ was allowed to react with 4-chloroaniline, thiophenol, thiosalicylic
5,
6a 6b, and , respectively. These products contain
,
7
7
8
condensation with different aromatic aldehyde afforded hydrazones 9a–f. Furthermore, 2,3-DCQ was
reacted with benzimidazole-2-thiol as a binucleophile affording the pentacyclic compound 10. Thus,
we synthesized 19 new 2,3-disubstituted quinoxaline derivatives (2b–e 3a
;
; 3e; 3f; 4; 5; 6a,b; 8; 9a–f)
and seven previously reported compounds ( 2a 36]; 3b 3c 37]; 3d 3g, 7) using a simple and efficient
1;
[
;
[
;
synesthetic method with the goal of discovering their antimicrobial activity.
Figure 2. Quinoxaline moiety of 2a showed only two protons and two quaternary carbon in NMR. 6b
showed four different types of protons and four different quaternary carbons, which correspond to its
quinoxaline moiety.
Scheme 2. Synthesis of asym. 2,3-disubstituted quinoxaline.
Previously, hydrazones derived from quinoxaline have been reported for their antimicrobial and
anti-inflammatory effects [9,38–40]. That enhanced us to design quinoxaline derivatives 9a–f which
contain both thioether and arylhydrazones functionalities at 2- and 3-positions of quinoxaline ring for
testing their biological activity.
The newly prepared compounds 5–10 gave analytical and spectral data in agreement with
their structures. For example, compounds 5, 7, 8, and 9a–f showed the characteristic infrared
absorption bands for N-H stretching at 3300–3358 cm−1 in addition, proton NMR of those compounds
showed D2O-exchangeable broad singlets, which confirmed the existence of NH and/or NH2 groups.
Condensation reaction of
Those Schiff bases showed a new singlet at
Furthermore, the azomethine carbon appeared in carbon NMR at δ 138–145 ppm.
8
with six different aromatic aldehydes led to formation of Schiff bases 9a–f.
δ
8.46–8.72 ppm corresponding to azomethine proton.

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2.2. Biological Evaluation
In vitro antimicrobial screening of the synthesized compounds 2a–d
,
3a–g
,
4,
5,
6a,b
,
8, and 9a–f was
estimated against four bacterial species, namely Bacillus subtilis, Staphylococcus aureus as a Gram-positive
bacterium. Moreover, Escherichia coli and Proteus vulgaris as a Gram-negative bacterium. In addition,
the study also included two fungal strains, namely Candida albicans and Aspergillus flavus. Gentamycin
and Ketoconazole were used as antibacterial and antifungal reference drugs, respectively. Dimethyl
sulfoxide (DMSO) was used as solvent as well as negative reference. The activities of the tested
compounds are summarized in Table 1. The data obtained allowed the following observations and
conclusions: the negative reference, dimethyl sulfoxide, did not show any inhibition zone against all
the tested strains. The antibacterial positive reference, Gentamycin, showed an average inhibition
zone of 24.00, 26.00, 30.00, and 25.00 mm against the strains of Staphylococcus aureus; Bacillus subtilis;
Escherichia coli and Proteus vulgaris, respectively. The antifungal positive reference, Ketoconazole,
showed an average inhibition zone of 20.00 mm, 16.00 mm against the strains of, Candida albicans and
Aspergillus flavus, respectively. The tested compounds showed significant antibacterial activity against
the strains of Staphylococcus aureus, Bacillus subtilis, and Escherichia coli relative to the strain of Proteus
vulgaris. Furthermore, among all the tested compounds, only compounds 6a
compound 10 have considerable antifungal activity. Compounds 3f 6b showed broad antimicrobial
spectrum against most of the tested strains. The symmetrically disubstituted quinoxalines
and presented the most significant antibacterial activity. However, the asymmetrically substituted
quinoxalines , and 10 showed reduced antibacterial activity. The hydrazine revealed a slightly
, 6b and the pentacyclic
,
2, 3, 4,
5
8
,
9
8
lower antimicrobial activity than its corresponding hydrazones 9a–g.
Table 1. Antimicrobial activities of compounds 2–6 and 8–10.
Antibacterial Activity Zone of Inhibition (mm)
G+ bacteria
Antifungal Activity Zone of Inhibition (mm)
G− bacteria
Compounds
S. aureus
B. subtilis
E. coli
P. vulgaris
C. albicans
A. flavus
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
3g
4
5
6a
6b
8
9a
9b
9c
9d
9e
9f
10
26
22
25
28
13
19
35
16
26
30
37
27
22
22
29
15
22
17
14
20
19
13
13
-
30
19
20
44
12
28
41
30
24
29
29
38
37
35
31
9
18
13
12
11
10
15
26
-
18
25
27
34
14
39
37
32
27
30
28
12
37
24
35
9
25
18
22
14
19
17
24
-
15
39
31
11
24
23
15
27
16
28
16
15
24
19
23
11
23
20
21
12
18
11
15
-
17
16
13
11
19
18
18
11
18
17
17
18
18
25
22
7
10
11
10
11
19
11
34
20
-
14
12
15
5
14
9
16
19
11
14
15
15
12
17
11
9
9
11
15
7
5
8
27
16
-
Ketoconazole
Gentamycin
DMSO
24
0
26
0
30
0
25
0
0
0
S. aureus, Staphylococcus aureus (RCMB010010); B. subtilis, Bacillus subtilis (RCMB 015 (1) NRRL B-543); E. coli,
Escherichia coli (RCMB 010052) ATCC 25955; P. vulgaris, Proteus vulgaris RCMB 004 (1) ATCC 13315; C. albicans,
Candida albicans RCMB 005003 (1) ATCC 10231; A. flavus, Aspergillus flavus (RCMB 002002).

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The minimum inhibitory concentration (MIC) is the lowest concentration causing full inhibition of
the tested microorganism’s growth. MIC is a further test applied for selected compounds with
good antimicrobial activity. The MIC were determined via the double dilution technique [41].
Six concentrations were prepared for each selected compound (8, 16, 32, 64, 128, and 256
µ
g/mL).
◦
The bacteria were inoculated and incubated at 37 C for 24 h in nutrient broth medium; however,
the fungal strains were inoculated in malt extract broth and incubated for 48 h. MIC values of the tested
compounds for different microorganisms are given in Table 2. As summarized in Table 2, the pentacyclic
compound 10 exhibited the highest antifungal activity against both Candida albicans and Aspergillus
flavus with MIC 16
antibacterial activity against the Gram-negative bacterium Escherichia coli with MIC 8
Compounds 2d 3c , and 6a presented the highest antibacterial activity against the Gram-positive
bacterium Bacillus subtilis with MIC 16 g/mL. Besides that, many of the tested compounds presented
a good antibacterial activity comparable to Gentamycin (i.e., compounds 2a 2c 2d 3c 3f 3g , and 6b
6b, and 10 against Bacillus subtilis; compounds
µg/mL. On the other hand, the tested compounds 2d and 3c showed the highest
µg/mL. Moreover,
,
, 4
µ
,
,
,
,
,
, 4
against Staphylococcus aureus; compounds 2a
, 3d, 3f, 3g,
2c and 3d against Proteus vulgaris).
Table 2. The minimal inhibitory concentration (MIC: µg/mL) of the tested compounds.
Compounds
S. aureus
B. subtilis
E. coli
P. vulgaris C. albicans
A. flavus
2a
2b
2c
2d
3a
3b
3c
32
64
32
32
128
64
16
>256
64
64
64
64
8
>256
16
8
64
8
16
128
256
32
64
64
128
256
128
64
64
256
64
>256
128
>256
64
32
>256
256
32
16
64
64
3d
3e
3f
3g
4
5
6a
6b
10
256
64
32
32
32
256
64
32
256
-
32
32
128
32
32
16
32
16
32
32
16
128
32
16
>256
16
64
16
32
-
16
128
32
64
>256
32
32
32
32
-
>256
64
128
128
64
64
16
32
16
64
128
256
128
64
256
32
>256
16
32
Ketoconazole
Gentamycin
-
32
32
-
16
16
-
S. aureus, Staphylococcus aureus (RCMB010010); B. subtilis, Bacillus subtilis (RCMB 015 (1) NRRL B-543); E. coli,
Escherichia coli (RCMB 010052) ATCC 25955; P. vulgaris, Proteus vulgaris RCMB 004 (1) ATCC 13315; C. albicans,
Candida albicans RCMB 005003 (1) ATCC 10231; A. flavus, Aspergillus flavus (RCMB 002002).
3. Experimental
3.1. Instruments and Apparatus
Melting points were determined by MEL-TEMP II melting point apparatus in open glass
capillaries and were uncorrected. The IR spectra were recorded as potassium bromide (KBr) discs
on a Perkin-Elemer FT-IR (Fourier-Transform Infrared Spectroscopy), Faculty of Science, Alexandria
University. The NMR spectra were carried out at ambient temperature (~25 ◦C) on a (JEOL) 500 MHz
spectrophotometer using tetramethylsilane (TMS) as an internal standard, NMR Unit, Faculty of Science,
Mansoura University. Elemental analyses were analyzed at the Micro analytical Unit, Faculty of Science,
Cairo University. The biological evaluation was carried out in the Medical Mycology Laboratory of the
Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt.

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3.2. Agar Disk-Diffusion Method
The desired compounds were dissolved in DMSO (which has no inhibition activity) to obtain
concentrations of 250 ppm and soaked in filter paper disks of 5 mm. The test was performed on
medium potato dextrose agar (PDA) which contains an infusion of 200 g potatoes, 6 g dextrose, and
15 g agar. Uniform size filter paper disks (three disks per compound) were impregnated with equal
volume (10 µL) from the specific concentration of dissolved tested compounds and then carefully
placed on the incubated agar surface. After incubation for 36 h at 27 ◦C in the case of bacteria and for
48 h at 24 ◦C in the case of fungi, inhibition of the organisms (evidenced by a clear zone surrounding
each disk) was measured and used to calculate the mean of inhibition zones [42].
3.3. Determination of MIC
All the bacteria were incubated and activated at 37 ◦C for 24 h inoculation into nutrient broth and
the fungi were incubated in malt extract broth for 48 h. The compounds were dissolved in DMSO and
then diluted using cautiously adjusted Mueller–Hinton broth. Two-fold serial concentrations dilution
method (8, 16, 32, 64, 128, and 256 µg/mL) of some compounds were employed to determine the MIC
values. In each case, triplicate tests were performed, and the average was taken as the final reading.
The tubes were then inoculated with the test organisms, grown in their suitable broth at 37 ◦C for 24 h
for tested microorganisms (1
×
108 CFU/mL for bacteria and 1
×
106 CFU/mL of fungi), each 5 mL
received 0.1 mL of the above inoculum and was incubated at 37 ◦C for 24 h. The lowest concentration
showing no growth was taken as the minimum inhibitory concentration (MIC); shown in Table 2.
3.4. Synthesis of Quinoxaline-2,3-dione
o-Phenylenediamine (27.9 g, 0.25 mol), oxalic acid (32.5 g, 0.36 mol), and 4 N HCl (150 mL) were
refluxed for 4 h then cooled. The solid separated was filtered, washed, and used without further
purification, yield (86%), m.p. > 300 ◦C [lit. > 300 ◦C] [43], 1H-NMR (500 MHz, DMSO-d6, 300 K),
δ
11.91 (s, 2H, 2 NH, D2O exchangeable), 7.11 (dd, 2H, J = 6.1, 3.3 Hz, H6 and H7) and 7.07 (dd, 2H,
J = 6.0, 3.3 Hz, H5 and H8) ppm.
3.5. Synthesis of 2,3-DCQ (1)
Method A: Equimolar quantities of quinoxaline-2,3-dione (16.013 g, 0.1 mol) and phosphorus
oxychloride (15.333 g, 0.1 mol) were mixed and the resulting mixture was refluxed on a water bath
for 2 h. The reaction mixture was then cooled to room temperature and the resulting precipitate was
isolated by filtration, washed with water, then crystalized from rectified spirit as colorless crystals,
69% yield; m.p. 143–145 ◦C [lit. 148–150 ◦C] [44].
Method B: A mixture of quinoxaline-2,3-dione (3.08 g, 0.019 mol) in 1,2-dichloroethane (25 mL)
and thionyl chloride (4 mL, 0.055 mol) dissolved in few drops of dimethylformamide was heated
on water bath for 4 h. The reaction mixture was then cooled to room temperature and the resulting
precipitate was isolated by filtration, washed with water, then crystalized from rectified spirit as
colorless crystals, 82% yield; m.p. 143–145 ◦C. 1H-NMR (500 MHz: CDCl3),
δ 8.01 (dd, 2H, J = 6.4,
3.5 Hz, H6 and H7) and 7.79 (dd, 2H, J = 6.4, 3.4 Hz, H5 and H8) ppm.
3.6. Synthesis of 2,3-bis(arylthio)quinoxaline (2a–e)
In a typical experiment a solution of 2,3-DCQ
1 (1 g, 0.005 mol) and 0.015 mol of appropriate
thiol (namely, 4 mL thiophenol, 1.28 mL p- fluorothiophenol, 2.85 g p-bromothiophenol, 2.16 g
p-chlorothiophenol or 1.86 g p-methylthiophenol) and triethylamine (3.03 g, 0.03 mol) in 30 mL
methanol was refluxed for 2 h, monitored by TLC. It was then cooled by ice-water, filtered, and washed
with water and the separated solid was then crystalized from benzene-petroleum ether as crystals.

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3.6.1. 2,3-Dithiophenylquinoxaline (2a)
◦
Yellow crystals, 1.47 g (85%) yield; m.p. 112–115 C. IR (KBr), 3052 (sp2 = C-H), 1580 (C=N)
1
and 749, 682 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, CDCl3, 300 K),
δ
7.67–7.62 (m, 8H, Ar-H) and 7.46–7.48 (m, 6H, Ar-H) ppm. 13C-NMR (APT) (125 MHz, CDCl3,
300 K),
δ 153.73 (C), 140.37 (C), 135.08 (CH), 129.09 (CH), 128.70 (C), 128.32 (CH), 127.88 (CH) ppm.
C20H14N2S2 requires: C, 69.36%; H, 4.05%; N, 8.09%. Found: C, 69.14%; H, 4.27%; N, 8.31%.
3.6.2. 2,3-Di(thio-4-fluorophenyl)quinoxaline (2b)
Colourless crystals, 2.13 g (93%) yield; m.p. 147–150 ◦C. IR (KBr), 3037 (=C-H), 1586 (C=N) and
1
752, 636 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, CDCl3, 300 K),
δ 7.62
(m, 6H, Ar-H), 7.47 (dd, 2H, J = 6.3, 3.4 Hz, H8) and 7.16 (t, 4H, J = 8.6 Hz, Ar-H) ppm. 13C-NMR
1
(APT) (125 MHz, CDCl3, 300 K),
δ
163.53 (d, JC,F =250.3 Hz, CF), 153.41 (C), 140.35 (C), 137.49
(d, 3JC,F = 8.5 Hz, CH), 128.60 (CH), 127.87 (CH), 123.52 (d, 4JC,F = 2.5 Hz, C), 116.47 (d, 2JC,F = 22.2 Hz,
CH) ppm. C20H12N2S2 F2 requires: C, 52.63%; H, 2.63%; N, 6.14%. Found: C, 55.47%; H, 2.87%;
N, 6.27%.
3.6.3. 2,3-Di(thio-4-bromophenyl)quinoxaline (2c)
Colorless crystals, 2.3 g (94%) yield; m.p. 212-215 ◦C. IR (KBr), 3058 (sp2 = C-H), 1629 (C=N),
1
and 758, 622 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, CDCl3, 300 K),
0
0
0
δ
7.67 (0dd, 2H, J = 6.3, 3.5 Hz, H8), 7.58 (d, 4H, J = 8.4 Hz, H3 and H5 ), and 7.50 (m, 6H, H7, H2 ,
and H6 ) ppm. 13C-NMR (APT) (125 MHz: CDCl3), δ 152.99 (C), 140.40 (C), 136.59 (CH), 132.39 (CH),
128.84 (CH), 127.92 (CH), 127.66 (C), 123.87 (C) ppm.C20H12N2S2Br2 requires: C, 47.62%; H, 2.38%;
N, 5.56%. Found: C, 47.89%; H, 2.54%; N, 5.73%.
3.6.4. 2,3-Di(thio-4-chlorophenyl)quinoxaline (2d)
Pale yellow crystals, 1.87 g (90%) yield; m.p. 198–200 ◦C. IR (KBr), 3103 (sp2 = C-H), 1631 (C=N)
1
and 756, 625 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, CDCl3, 300 K),
0
δ
7.66 (dd, 2H, J = 3.5 Hz, H8), 70.56 (d, 4H, J = 8.5 Hz, H30 and H5 ), 7.49 (dd, 2H, J = 3.4 Hz, H7), and 7.43
(d, 4H, J = 8.5 Hz, H2 , and H6 ) ppm. 13C-NMR (APT) (125 MHz, CDCl3, 300 K),
δ 153.05 (C), 140.39
0
(C), 136.41 (CH), 135.57 (C), 129.36 (CH), 128.78 (CH), 127.90 (CH), 126.97 (C) ppm. C20H12N2S2Cl2
requires: C, 57.83%; H, 2.89%; N, 6.75%. Found: C, 57.61%; H, 3.11%; N, 6.89%.
3.6.5. 2,3-Di(thio-4-methylphenyl)quinoxaline (2e)
Colorless crystals, 1.59 g (85%) yield; m.p. 150 ◦C. IR (KBr), 3049 (sp2 = C-H), 2968 (sp3-C-H),
1596 (C=N), and 751, 592 (C-S, asymmetric and symmetric stretching) cm−1
.
1H-NMR (500 MHz,
0
0
CDCl3, 300 K), 7.65 (dd, 4H, J = 6.3, 3.5 Hz, H8), 7.53 (d, 4H, J = 8.1 Hz, H2 , and H6 ), 7.44 (dd, 2H,
J = 6.4, 3.4 Hz, H7), 7.27 (d, 4H, J = 8.0 Hz, H3’and H5’), and 2.42 (s, 6H, 2CH3) ppm. 13C-NMR (APT)
(125 MHz, CDCl3, 300 K), δ 154.01 (C), 140.32 (C), 139.26 (C), 135.13 (CH), 129.96 (CH), 128.19 (CH),
127.83 (CH), 124.95 (C), 21.28 (CH3) ppm. C22H18N2S2 requires: C, 70.5%; H, 4.8%; N, 7.4%. Found: C,
70.78%; H, 4.73%; N, 7.68%.
3.7. Synthesis of N2,N3-Diarylquinoxaline-2,3-diamine (3a–g)
In a typical experiment a solution of 2,3-DCQ 1 (0.5 g, 0.0025 mol) and 0.025 mol of appropriate
amine; (namely, 2-aminopyridine (2.35 g), aniline (2.33 g), m-chloroaniline (3.19 g), p-toludine (2.68 g),
3,4-dimethylaniline (3.03 g), p-anisidine (3.08 g) or 3,4-dimethoxyaniline (3.83 g)) and triethylamine
(0.5 g, 0.005 mol) in 20 mL acetonitrile was refluxed for 20 to 40 h with constant stirring, monitored
by TLC. The reaction mixture was then cooled by ice-water, filtered, washed with water to give
quinoxalines 3a–g as solid crystals which were purified by crystallization from alcohol.

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(Note: as an exception the reaction of 2-aminopyridine with 2,3-DCQ was carried out in
dimethylformamide instead of acetonitrile).
3.7.1. N2,N3-Di(pyridin-2-yl)quinoxaline-2,3-diamine (3a)
Yellow crystals, 0.59 g (75%) yield; m.p. > 250 ◦C. IR (KBr), 3372 (NH), 3107 (=C-H), and 1667
1
(C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K),
δ 11.96 (broad s, 1H, NH, D2O exchangeable),
11.91 (broad s, 2H, NH, D2O exchangeable), 7.38–7.26 (m, 2H, Ar-H), and 7.15–7.02 (m, 10H, Ar-H)
ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K), 132.82 (C), 129.03 (C), 125.60 (C), 124.63 (CH),
δ
123.78 (CH), 123.04 (CH), 123.01 (CH), 115.13 (CH), 114.22 (CH) ppm.C18H14N6 requires: C, 68.7%; H,
4.4%; N, 26.7%. Found: C, 68.99%; H, 4.63%; N, 26.43%.
3.7.2. N2,N3-Diphenylquinoxaline-2,3-diamine (3b)
◦
◦
Yellow crystals, 1.31 g (84%) yield; m.p. 228–230 C (Lit. 223 C) [45]. IR (KBr), 3311 (NH),
3075 (=C-H), and 1642 (C=N) cm−1. H-NMR (5000MHz, DMSO-d6, 300 K),
δ
9.63 (br. s, 2H, NH, D2O
1
0
0
exchangeable), 7.93 (d, 4H, J = 7.9 Hz, H2 , and H6 ), 7.55 (dd, 2H, J = 6.2, 3.4 Hz, H8), 7.41 (t, 4H, H3
and H5 ), 7.34 (dd, 2H, J = 6.2, 3.4 Hz, H7), and 7.10 (t, 2H, J = 7.4 Hz, H4 ) ppm. 13C-NMR (APT)
0
0
(125 MHz, DMSO-d6, 300 K),
δ 141.32 (C), 139.37 (C), 133.94 (C), 128.93 (CH), 125.66 (CH), 124.13 (CH),
123.79 (CH), 121.53 (CH) ppm.C20H16N4 requires: C, 76.92%; H, 5.13%; N, 17.95%. Found: C, 77.12%;
H, 5.29%; N, 18.24%.
3.7.3. N2,N3-Bis(3-chlorophenyl)quinoxaline-2,3-diamine (3c)
Yellow crystals, 0.75 g (81%) yield; m.p. 122-124 ◦C. IR (KBr), 3303 (NH), 3054 (=C-H), and 1575
1
0
(C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K),
δ 9.16 (s, 2H, NH, D2O exchangeable), 8.09 (t, 2H,
0
J = 2.0 Hz, H5 ), 7.81 (dd, 2H, J = 8.2, 1.5 Hz, H4 ), 7.59 (dd, 2H, J = 6.1, 3.4 Hz, H8), 7.44–7.38 (m, 4H, H7,
0
0
and H2 ), and 7.11 (dd, 2H, J = 7.9, 1.6 Hz, H6 ) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K),
δ
141.76 (C), 140.95 (C), 136.04 (C), 132.95 (C), 130.27 (CH), 125.81 (CH), 125.60 (CH), 122.03 (CH), 119.50
(CH), 118.52 (CH) ppm.C20H14N4S2Cl2 requires: C, 62.99%; H, 3.67%; N, 14.70%. Found: C, 62.84%; H,
3.75%; N, 14.81%.
3.7.4. N2,N3-Dip-tolylquinoxaline-2,3-diamine (3d)
Yellow crystals, 0.67 g (79%) yield; m.p. 145–147 ◦C (Lit. 147-149 ◦C) [46]. IR (KBr), 3388 (NH),
1
3026 (=C-H), 2916 (-C-H), and 1640 (C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K),
δ
8.92 (br. s,
0
0
2H, NH, D2O exchangeable), 7.76 (d, 4H, J = 8.4 Hz, H2 , and H6 ), 7.049 (dd, 2H, J = 6.1, 3.5 Hz, H8),
0
7.30 (dd, 2H, J = 6.0, 3.4 Hz, H7), 7.20 (d, 4H, J = 8.3 Hz, H3 , and H5 ), and 2.30 (s, 6H, 2CH3) ppm.
13C-NMR (APT) (125 MHz, DMSO-d6, 300 K),
δ 141.21 (C), 137.57 (C), 136.16 (C), 131.58 (C), 129.05
(CH), 125.25 (CH), 124.89 (CH), 120.77 (CH), 20.51 (CH3) ppm.C22H20N4 requires: C, 77.65%; H, 5.88%;
N, 16.47%. Found: C, 77.47%; H, 5.98%; N, 16.62%.
3.7.5. N2,N3-Bis(3,4-dimethylphenyl)quinoxaline-2,3-diamine (3e)
◦
Yellow crystals, 0.38 g (84%) yield; m.p. 258–261 C. IR (KBr), 3390 (NH), 3154 (=C-H), 2881
(sp3 -C-H), and 1634 (C=N) cm−1
.
1H-NMR (500 MHz, DMSO-d6, 300 K):
δ
9.85 (s, 2H, NH, D2O
exchangeable), 7.70–7.58 (m, 4H, Ar-H), 7.55 (dd, 2H, J = 6.0, 3.5 Hz, H8), 7.32 (dd, 2H, J = 6.0, 3.4 Hz,
H7), 7.19 (d, 2H, Ar-H), 2.26 (s, 6H, 2CH3), and 2.23 (s, 6H, 2CH3) ppm. 13C-NMR (APT) (125 MHz,
DMSO-d6, 300 K): δ 141.18 (C), 136.54 (C), 131.81 (C), 129.81 (CH), 127.80 (C), 126.17 (C), 125.34 (CH),
122.83 (CH), 119.21 (CH), 118, 72 (CH), 19.72 (CH3), 18.89 (CH3) ppm.C24H24N4 requires: C, 91.3%; H,
6.52%; N, 15.2%. Found: C, 78.45%; H, 6.71%; N, 15.47%.

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3.7.6. N2,N3-bis(4-methoxyphenyl)quinoxaline-2,3-diamine (3f)
Green crystals, 0.72 g (78%) yield; m.p. 116–118 ◦C. IR (KBr): 3387 (NH), 3070 (=C-H), 2949 (-C-H),
1
and 1656 (C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):
δ
8.84 (br. s, 2H, NH, D2O exchangeable),
0
0
7.77 (d, 4H, J = 9.0 Hz, H2 , and H6 ), 7.45 (dd, 2H, J = 6.2, 3.5 Hz, H8), 7.26 (dd, 2H, J = 6.0, 3.4 Hz,
0
0
H7), 6.98 (d, 4H, J = 9.0 Hz, H3 , and H5 ), and 3.76 (s, 6H, 2 OCH3) ppm. 13C-NMR (APT) (125 MHz,
DMSO-d6, 300 K): δ 155.06 (C), 141.33 (C), 136.21 (C), 133.11 (C), 125.12 (CH), 124.66 (CH), 122.48 (CH),
113.87 (CH), 55.22 (OCH3) ppm. C22H20N4O2 requires: C, 70.97%; H, 5.38%; N, 15.05%. Found: C,
70.73%; H, 5.47%; N, 14.89%.
3.7.7. N2,N3-Bis(3,4-dimethoxyphenyl)quinoxaline-2,3-diamine (3g)
Dark green crystals, 0.41 g (76%) yield; m.p. 208–210 ◦C (Lit. 214 ◦C). IR (KBr): 3372 (NH), 3059
1
(=C-H), 2936 (-C-H), and 1615 (C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):
δ
8.87 (br. s, 2H,
0
NH, D2O exchangeable), 7.66 (d, 2H, J = 2.4 Hz, H2 ), 7.49 (dd, 2H, J = 6.2, 3.5 Hz, H8), 7.38 (dd, 2H,
0
0
J = 8.6, 2.4 Hz, H6 ), 7.29 (dd, 2H, J = 6.1, 3.4 Hz, H7), 6.99 (d, 2H, J = 8.7 Hz, H5 ), 3.81 (s, 6H, 2 OCH3),
and 3.76 (s, 6H, 2 OCH3) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K): δ 148.49 (C), 144.56 (C),
141.18 (C), 136.09 (C), 133.63 (C), 125.21 (CH), 124.79 (CH), 112.61 (CH), 112.06 (CH), 106.05 (CH), 55.78
(OCH3), 55.40 (OCH3) ppm. C24H24N4O4 requires: C, 66.6%; H, 5.55%; N, 12.9%. Found: C, 66.87%; H,
5.62%; N, 12.78%. Spectral data of 3g are in accordance with those reported in the literature [47].
3.8. Synthesis and Characterization of 2,2’-(Quinoxaline-2,3-diylbis(sulfanediyl))diacetic acid (4)
A solution of 2,3-DCQ
1 (1 g, 0.005 mol) and a solution of thioglycolic acid (1.38 g, 0.015 mol) and
triethyl amine (3.03 g, 0.03 mol) in 30 mL methanol was refluxed for 4 h. The reaction was monitored
by TLC until 2,3-DCQ was totally consumed. The reaction mixture was then cooled by ice-water and
the separated solid was filtered then crystalized from a mixture of methanol and water.
Yellow crystals, 1.78 g (93%) yield; m.p. 227–229 ◦C. IR (KBr): 3426 (OH), 2999 (-C-H), 1705 (C=O),
1
1560 (C=N), and 759, 675 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz,
DMSO-d6, 300 K):
δ
7.83 (dd, 2H, J = 6.3, 3.4 Hz, H8), 7.67 (dd, 2H, J = 6.2, 3.4 Hz, H7), and 4.17 (s, 4H,
169.53 (CO), 152.60 (C), 138.97 (C), 128.94
2 CH2) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K):
δ
(CH), 127.16 (CH), 32.49 (CH2) ppm.C12H10N2S2O4 requires: C, 37.6%; H, 2.6%; N, 7.3%. Found: C,
37.75%; H, 2.47%; N: 7.49%.
3.9. Synthesis and Characterization of 3-Chloro-N-(4-chlorophenyl)quinoxalin-2-amine (5)
To a solution of p-chloroaniline (3.18 g, 0.025 mol) and triethylamine (0.5 g, 0.005 mol) in 5 mL
dimethylformamide, a solution of 2,3-DCQ
1 (0.5 g, 0.0025 mol) in 10 mL dimethylformamide was
added with constant stirring. After the completion of addition, the reaction mixture was refluxed
for 40 h, cooled by ice-water, filtered, washed with water, and the product formed was purified by
crystallization from hot ethanol. Yellow crystals, 0.55 g (76%) yield; m.p. 227–229 ◦C. IR (KBr): 3357
1
(NH), 3099 (=C-H), and 1676 (C=N) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):
δ 9.13 (br. s, 1H,
0
0
NH, D2O exchangeable), 7.92 (d, 2H, J = 8.9 Hz, H2 and H6 ), 7.56 (dd, 1H, J = 6.0, 3.5 Hz, Ar-H), 7.44
0
0
(d, 2H, J = 8.8 Hz, H3 , and H5 ), 7.41–7.30 (m, 2H, Ar-H), and 7.28–7.12 (m, 1H, Ar-H) ppm. 13C-NMR
(APT) (125 MHz, DMSO-d6, 300 K): 151.43(C), 140.57 (C), 138.97 (C), 138.63 (C), 135.96 (C), 128.51
δ
(CH), 128.36 (CH), 125.89 (C), 125.52 (CH), 121.91 (CH), 121.29 (CH), 115.08 (CH) ppm.C14H9N3Cl2
requires: C, 57.9%; H, 3.10%; N, 14.4%. Found: C, 58.13%; H, 3.29%; N, 14.67%.
3.10. Synthesis of 2-Chloro-3-(arylthio)quinoxaline (6a–b)
A solution of 2,3-DCQ 1 (1 g, 0.005 mol) and appropriate thiol (Namely, thiophenol or thiosalicylic
acid) (0.015 mol) and triethylamine (1.01 g, 0.01 mol) in 30 mL methanol was stirred for 4 h, monitored
by TLC. The reaction mixture was then cooled by ice-water, filtered, washed with water, and purified
by crystallization.

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3.10.1. 2-Chloro-3-(phenylthio)quinoxaline (6a)
◦
Colorless crystals (benzene-petroleum ether), 1.25 g (92%) yield; m.p. 145 C. IR (KBr): 3068
(=C-H), 1562 (C=N), and 753, 683 (C-S, asymmetric and symmetric stretching) cm−1. 1H-NMR (500 MHz,
CDCl3, 300 K):
δ
7.93–7.90 (m, 1H, Ar-H), 7.69–7.65 (m, 1H, Ar-H), 7.65–7.58 (m, 4H, Ar-H), and
155.30 (C), 144.32 (C), 141.21
7.51–7.46 (m, 3H, Ar-H) ppm. 13C-NMR (APT) (125 MHz, CDCl3, 300 K):
δ
(C), 139.68 (C), 135.56 (CH), 130.10 (CH), 129.62 (CH), 129.28 (CH), 129.15 (CH), 128.12 (C), 128.01 (CH),
127.98 (CH) ppm.C14H9N2SCl requires: C, 61.65%; H, 3.30%; N, 10.28%. Found: C, 61.92%; H, 3.19%;
N, 10.48%.
3.10.2. 2-(3-Chloroquinoxalin-2-ylthio)benzoic Acid (6b)
◦
Dark yellow crystals (methanol-water mixture), 1.4 g (92%) yield; m.p. > 250 C. IR (KBr):
3430 (OH), 3062 (=C-H), 1681 (C=O), 1586 (C=N), and 744, 646 (C-S, asymmetric and symmetric
1
stretching) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):
δ 8.01 (d, 1H, J = 6.4 Hz, Ar-H), 7.98–7.93
(m, 1H, Ar-H), 7.77 (dd, 1H, J = 6.3, 3.5 Hz, Ar-H), 7.70 (m, 1H, Ar-H), 7.61 (d, 1H, J = 7.9 Hz, Ar-H), 7.53
(m, 2H, Ar-H), and 7.33 (t, 1H, J = 7.9 Hz, Ar-H) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K):
δ
167.54 (CO), 153.65 (C), 139.95 (C), 138.84 (C), 135.69 (CH), 135.19 (C), 133.73 (CH), 133.65 (C), 133.19
(CH), 131.50 (CH), 130.55 (CH), 128.40 (C), 128.00 (CH), 125.90 (CH), 124.90 (CH) ppm.C15H9N2SO2Cl
requires: C, 56.8%; H, 2.8%; N, 8.8%. Found C, 56.97%; H, 3.13%; N, 8.61%.
3.11. Synthesis of 2-Chloro-3-hydrazinylquinoxaline (7)
A solution of 2,3-DCQ 1 (5 g, 0.025 mol) and hydrazine hydrate (2.7 g, 0.0055 mol) in 15 mL ethanol
was stirred for 24 h with constant stirring, the reaction being monitored by TLC. The reaction mixture
was then filtered and washed with water and the product was crystalized from benzene-petroleum
ether as dark orang crystals, 82% yield; m.p. > 250 ◦C [lit. >300 ◦C] the spectral data agreed with the
previously reported data [48].
3.12. Synthesis of 2-Hydrazinyl-3-(phenylthio)quinoxaline (8)
To a solution of thiophenol (7.00 g, 0.069 mol) and 0.138 mL of triethylamine in 20 mL methanol,
a solution of 2-chloro-3-hydrazinylquinoxaline
7 (4.5 g, 0.023 mol) in 20 mL methanol was added.
Then the reaction mixture was refluxed for 30 min, monitored by TLC, cooled by ice-water, filtered,
washed with water,◦and purified by crystallization from benzene. Orange crystals, 6.3 g (92%)
yield; m.p. 210–212 C. IR (KBr): 3300 (NH), 3237 (NH2), 3033 (=C-H), 1627 (C=N), and 750, 683
1
(C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K): δ 7.62–7.54
(m, 1H, Ar-H), 7.52–7.49 (m, 1H, Ar-H), 7.50–7.47 (m, 1H, Ar-H), 7.47–7.40 (m, 2H, Ar-H), 7.11 (t, 1H,
J = 8.3 Hz, Ar-H), 7.02 (d, 1H, J = 8.9 Hz, Ar-H), 6.92 (d, 1H, J = 9.0 Hz, Ar-H), 6.80 (t, 1H, J = 7.6 Hz,
Ar-H), 5.61 (br.s, 2H, NH2, D2O exchangeable), and 4.58 (br.s, 1H, NH, D2O exchangeable). C14H12N4S
requires: C, 62.67%; H, 4.48%; N, 20.90%. Found: C, 62.43%; H, 4.59%; N, 21.16%.
3.13. Synthesis of (2-(2-Arylidenehydrazinyl)-3-(phenylthio)quinoxaline (9a–f)
In a typical experiment a solution of 2-hydrazinyl-3-(phenylthio)quinoxaline
8 (0.5 g,
0.0018 mol) and appropriate aldehyde (0.0018 mol) (Namely, benzaldehyde, p-nitrobenzaldehyde,
m-nitrobenzaldehyde, p-methoxybenzaldehyde, p-chlorobenzaldehyde or p-hydroxybenzaldehyde) in
ethanol-DMF (20–2 mL) was refluxed for 6 h with constant stirring, the reaction being monitored by
TLC. The reaction mixture was then cooled by ice-water, filtrated, washed with water. Finally, purified
by crystallization from a mixture of methylene chloride-petroleum ether.
3.13.1. 2-(2-Benzylidenehydrazinyl)-3-(phenylthio)quinoxaline (9a)
Orange crystals, 0.5 g (90%) yield; m.p. 187–190 ◦C. IR (KBr): 3358 (NH), 3049 (=C-H), 1613 (C=N),
1
and 748, 627 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):

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δ
9.50 (br. s, 1H, NH, D2O exchangeable), 8.65 (s, 1H, CH), 7.82–7.86 (m, 2H, Ar-H), 7.67–7.60 (m, 2H,
Ar-H), 7.48–7.38 (m, 6H, Ar-H), 7.37 (d, 1H, J = 8.0 Hz, Ar-H), 7.34–7.26 (m, 1H, Ar-H), and 7.05–7.14
(m, 1H, Ar-H) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K):
158.12 (C), 156.52 (CH), 143.74
δ
(C), 135.47 (CH), 134.94 (C), 132.65 (C), 130.56 (C), 130.44(CH), 129.22 (CH), 129.07 (CH), 128.66 (CH),
128.61 (CH), 128.44 (CH), 128.41 (C), 126.80 (CH), 122.36 (CH), 115.18 (CH) ppm. C21H16N4S requires:
C, 70.79%; H, 4.49%; N, 15.73%. Found: C, 70.96%; H, 4.61%; N, 15.79%.
3.13.2. 2-(2-(4-Nitrobenzylidene)hydrazinyl)-3-(phenylthio)quinoxaline (9b)
Orange crystals (methylene chloride), 0.72 g (97%) yield; m.p. 200–202 ◦C. IR (KBr): 3344 (NH),
3064 (=C-H), 1599 (C=N), 1547, 1332 (NO2, asymmetric and symmetric stretching), and 746, 626
(C-S, asymmetric and symmetric stretching) cm−1 1H-NMR (500 MHz, DMSO-d6, 300 K):
. δ 8.90
(brs, 1H, NH, D2O exchangeable), 8.72 (s, 1H, CH), 8.30 (d, 2H, J = 8.6 Hz, Ar-H), 7.98 (d, 2H, J = 8.8 Hz,
Ar-H), 7.63 (m, 2H, Ar-H), 7.48–7.44 (m, 3H, Ar-H), 7.38 (d, 1H, J = 8.0 Hz, Ar-H), 7.31 (t, 1H, J = 7.4 Hz,
Ar-H), and 7.12 (d, 2H, J = 7.6 Hz, Ar-H) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6, 300 K):
δ 157.90
(C), 154.19 (CH), 147.98 (C), 144.51 (C), 141.22 (C), 135.44 (CH), 132.96 (C), 130.20 (C), 129.28 (CH),
129.11 (CH), 129.10 (C), 128.65 (CH), 128.49 (CH), 127.05 (CH), 123.82 (CH), 122.86 (CH), 115.51(CH)
ppm. C21H15N5SO2 requires: C, 62.84%; H, 3.74%; N, 17.46%. Found: C, 63.04%; H, 3.95%; N, 17.72%.
3.13.3. 2-(2-(3-Nitrobenzylidene)hydrazinyl)-3-(phenylthio)quinoxaline (9c)
Dark orange crystals (methanol-dimethylformamide), 0.71 g (96%) yield; m.p. 201–203 ◦C. IR (KBr):
3350 (NH), 3072 (=C-H), 1617 (C=N), 1522, 1342 (NO2, asymmetric and symmetric stretching), and 741,
1
654 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, DMSO-d6, 300 K):
δ
8.88
(brs, 1H, NH, D2O exchangeable), 8.70 (m, 2H, CH, and Ar-H), 8.22 (d, 1H, J = 7.6 Hz, Ar-H), 8.10
(d, 1H, J = 7.2 Hz, Ar-H), 7.63 (m, 2H, Ar-H), 7.57 (t, 1H, J = 7.2 Hz, Ar-H), 7.50–7.44 (m, 3H, Ar-H),
7.43–7.29 (m, 3H, Ar-H), and 7.16 (t, 1H, J = 4.7 Hz, Ar-H) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6,
300 K):
δ 157.92 (C), 154.36 (CH), 148.32 (C), 144.25 (C), 136.82 (C), 135.30 (CH), 134.36 (CH), 132.81 (C),
130.32 (C), 130.13 (CH), 129.26 (CH), 129.10 (CH), 128.56 (CH), 128.54 (C), 127.02 (CH), 124.53 (CH),
122.66 (CH), 122.42 (CH), 115.38 (CH) ppm. C21H15N5SO2 requires: C, 62.84%; H, 3.74%; N, 17.46%.
Found: C, 63.13%; H, 3.85%; N, 17.67%.
3.13.4. 2-(2-(4-Methoxybenzylidene)hydrazinyl)-3-(phenylthio)quinoxaline (9d)
◦
Dark orange crystals, 0.6 g (93%) yield; m.p. 193–195 C. IR (KBr): 3359 (NH), 3053 (=C-H),
1
2963 (-C-H), 1615 (C=N), and 744, 654 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR
(500 MHz, DMSO-d6, 300 K): δ 8.60 (s, 1H, CH), 7.90 (s, 1H, Ar-H), 7.68–7.59 (m, 3H, Ar-H), 7.52–7.47
(m, 2H, Ar-H), 7.42–7.46 (m, 3H, Ar-H), 7.43–7.38 (m, 1H, Ar-H), 7.34 (t, 1H, J = 9.8 Hz, Ar-H), 7.11
(m, 1H, Ar-H), 6.94 (m, 1H, Ar-H), and 3.85 (s, 3H, CH3) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6,
300 K):
δ 161.23 (C), 158.22 (C), 156.24 (CH), 143.30 (C), 135.46 (CH), 132.56 (C), 130.72 (CH), 130.13
(C), 129.18 (CH), 129.06 (CH), 128.70 (C), 128.37 (CH), 127.60 (C), 126.91 (CH), 122.15 (CH), 115.27
(CH), 114.14 (CH), 55.36 (OCH3) ppm.C22H18N4S O requires: C, 68.39%; H, 4.66%; N, 14.51%. Found:
C: 67.98; H: 4, 79; N: 14.32%.
3.13.5. 2-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(phenylthio)quinoxaline (9e)
Dark orange crystals, 0.6 g (91%) yield; m.p. 197–199 ◦C. IR (KBr): 3355 (NH), 3050 (=C-H), 1615
1
(C=N), and 747, 637 (C-S, asymmetric and symmetric stretching) cm−1. H-NMR (500 MHz, DMSO-d6,
300 K):
7.42–7.27 (m, 4H, Ar-H), and 7.16 (m, 1H, Ar-H) ppm. 13C-NMR (APT) (125 MHz, CDCl3, 300 K):
153.38 (C), 150.26 (CH), 138.96 (C), 130.50 (CH), 129.94 (C), 128.94 (C), 127.75 (C), 125.52 (C), 125.02
δ 8.65 (s, 1H, CH), 7.74–7.78 (m, 2H, Ar-H), 7.68–7.58 (m, 3H, Ar-H), 7.48–7.44 (m, 3H, Ar-H),
δ
(CH), 124.26 (CH), 124.13 (CH), 123.76 (CH), 123.66 (C), 123.53 (CH), 122.03 (CH), 117.50 (CH), 110.25
(CH) ppm.C21H15N4S Cl requires: C, 64.53%; H, 3.84%; N, 14.34%. Found: C: 64.39%; H: 3.97%;
N: 14.58%.

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3.13.6. 4-((2-(3-(Phenylthio)quinoxalin-2-yl)hydrazono)methyl)phenol (9f)
Orange crystals, 0.6 g (90%) yield; m.p. 203–205 ◦C. IR (KBr): 3339 (NH), 3256 (OH), 3057 (=C-H),
1598 (C=N), and 748, 659 (C-S, asymmetric and symmetric stretching) cm−1 1H-NMR (500 MHz,
DMSO-d6, 300 K): 11.01 (brs, 1H, OH, D2O exchangeable), 10.00 (brs, 1H, NH, D2O exchangeable),
.
δ
8.46 (s, 1H, CH), 7.87 (d, 2H, J = 8.0 Hz, Ar-H), 7.62–7.58 (m, 2H, Ar-H), 7.52 (d, 1H, J = 8.1 Hz, Ar-H),
7.50–7.45 (m, 3H, Ar-H), 7.28 (t, 1H, J = 7.4 Hz, Ar-H), 7.13 (d, 1H, J = 7.7 Hz, Ar-H), 7.00 (t, 1H,
J = 9.5 Hz, Ar-H), and 6.86 (d, 2H, J = 8.6 Hz, Ar-H) ppm. 13C-NMR (APT) (125 MHz, DMSO-d6,
300 K):
δ 159.83 (C), 158.23 (C), 156.52 (CH), 143.07 (C), 135.43 (CH), 132.48 (C), 130.76 (C), 130.29 (CH),
129.13 (CH), 129.01 (CH), 128.69 (C), 128.30 (CH), 126.85 (CH), 126.02 (C), 122.02 (CH), 115.47 (CH),
114.94 (CH) ppm. C21H16N4SO requires: C, 67.74%; H, 4.30%; N, 15.05%. Found: C, 67.93%; H, 4.43%;
N, 15.21%.
3.14. Synthesis of Benzimidazo[20,10:2,3]thiazolo[4,5-b]quinoxaline (10)
To a solution of 2-mercaptobenzimidazole (1.13 g, 0.0075 mol) and triethyl amine (1.51 g, 0.015 mol)
in 15 mL methanol, a solution of 2,3-DCQ 1 (0.5 g, 0.0025 mol) in 15 mL methanol was added with
constant stirring. Then the mixture was refluxed for 4 h, monitored by TLC. The reaction mixture was
then cooled by ice-water, filtered, washed with water, and crystalized from hot ethanol.
Colorless crystals, 0.65 g (94%) yield; m.p. 198–200 ◦C. IR (KBr): 3049 (=C-H) and 1565 (C=N) cm−1
1H-NMR (500 MHz, CDCl3, 300 K):
7.97 (dd, 1H, J = 8.1, 1.4 Hz, Ar-H), 7.85 (dd, 1H, J = 8.2, 1.4 Hz,
Ar-H), 7.69–7.57 (m, 4H, Ar-H), and 7.29 (dd, 2H, J= 6.0, 3.1 Hz, Ar-H) ppm. 13C-NMR (APT) (125 MHz,
CDCl3, 300 K): 153.29 (C), 152.81 (C), 152.33 (C), 145.82 (C), 145.28 (C), 138.57 (C), 137.76 (C), 129.52
.
δ
δ
(CH), 127.40 (CH), 127.21 (CH), 126.79 (CH), 122.94 (CH), 122.54 (CH), 118.80 (CH), 110.61 (CH) ppm.
C15H8N4S requires: C, 65.2%; H, 2.89%; N, 20.2%. Found: C, 64.89%; H, 3.04%; N, 20.52%.
13C-NMR and 1H-NMR spectrums are available in Supplementary Materials.
4. Conclusions
In summary, we described a simple and efficient synthetic method for 19 new quinoxalines with
evaluation of their antimicrobial activity. The synthesized compounds were fully characterized using
melting point, IR, 1H-NMR, 13C-APT-NMR, and elemental analysis. The tested compounds showed
reasonable antibacterial activity against the strains of Staphylococcus aureus; Bacillus subtilis; Escherichia
coli. Furthermore, among all the tested compounds, only 6a
considerable antifungal activity. The pentacyclic compound 10 exhibited the highest antifungal activity
against both Candida albicans and Aspergillus flavus having a MIC value of 16 g/mL. On the other hand,
compounds 2d and 3c showed the highest antibacterial activity against the Gram-negative bacterium
Escherichia coli having MIC value of 8 g/mL. The results indicated that the sulfur functionality on
, 6b and the pentacyclic compound 10 have
µ
µ
quinoxaline may enhance its antifungal activity, while functionalization of quinoxaline with aromatic
amines enhance its antibacterial activity. Further investigations of this aspect are in progress.
Supplementary Materials: Supplementary materials are available online.
Author Contributions: E.A.H., M.A.E.-A. and A.Z.O. designed the research; A.Z.O., M.A.E.-A. and M.A.
performed the experimental work and analyzed the data; E.A.H., M.A.E.-A. and A.Z.O. wrote the manuscript;
E.A.H., M.A.E.-A. and A.Z.O. revised the manuscript. All the authors discussed, edited, and approved the
final version.
Funding: This research received no external funding.
Conflicts of Interest: Authors declare no conflicts of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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