Design, synthesis, antimicrobial activity and molecular docking studies of some novel di-substituted sulfonylquinoxaline derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104164

2,3-Dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride 1 was prepared via reaction of o-phenylene diamine with oxalic acid followed by chlorosulfonation with excess chlorosulfonic acid. A series of new sulfonylquinoxaline derivatives 2–6 were obtained upon reacting compound 1 with different types of amines. 2,3-Dichloro-6-morpholinosulfonylquinoxaline derivative 6 was subjected to further chemical reactions to afford many derivatives of 6-morpholino 2,3-disubstitutedquinoxalines, thus reaction of compound 6 with different secondary amines yielded mono and di secondary aminoquinoxaline derivatives 7–10 depending on the reactivity difference of the two chlorine atoms. Hydrazinolysis of compound 7 furnished hydrazino quinoxaline derivatives 11a-c. Additionally triazolo and pyrazolyl quinoxaline derivatives 12–14 were obtained through the reaction of compound 11a with phenyl isothiocyanate, formylpyrazole and ethyl acetoacetate. All the synthesized compounds were screened for their antibacterial and antifungal activities. Compounds 7a, 9b, 10a, 10c, 10f and 11c showed good to moderate antimicrobial activity against the tested Gram-positive, Gram-negative bacteria and fungi with MIC values ranging from 2.44 to 180.14 μM. Their MBC values were also evaluated using the same tested microorganisms. Moreover, screening against multi-drug resistant strains revealed the promiscuity of these new derivatives, especially compound 7a that showed comparable antibacterial activity (MIC 4.91–9.82 μM) with Norfloxacin (MIC 2.44–9.80 μM). Furthermore, these compounds were evaluated as DNA Gyrase inhibitors and the obtained results were in the range of 15.69–23.72 μM. Immunomodulatory effect was also investigated and compounds 7a, 11c, 10f, 10c, 10a and 9b showed high immunostimulatory action with ratio (142.6 ± 0.4, 135.7 ± 0.5, 117.8 ± 0. 39, 112.5 ± 0. 83, 86.4 ± 0. 47, 72.8 ± 0. 77) respectively. Molecular docking studies of the promising derivatives into DNA Gyrase binding site proved the usefulness of hybridizing quinoxaline scaffold with SO₂ and morpholine moieties as a hopeful strategy in designing new DNA Gyrase binding molecules.

Full text of DOI:10.1016/j.bioorg.2020.104164

BioorganicChemistry104(2020)104164
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, antimicrobial activity and molecular docking studies of
some novel di-substituted sulfonylquinoxaline derivatives
⁎
⁎
Yousry A. Ammara, , Awatef A. Faragb, Abeer M. Alib, Ahmed Ragaba, , Ahmed A. Askarc,
⁎
Doaa M. Elsisib, Amany Belald,
a Department of Chemistry, Faculty of Science (Boys), Al-Azhar University, Nasr City, Cairo, Egypt
b Department of Chemistry, Faculty of Science (Girls), Al-Azhar University, Nasr City, Cairo, Egypt
c Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, Nasr City, Cairo, Egypt
d Medicinal Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
2,3-Dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride 1 was prepared via reaction of o-phenylene dia-
mine with oxalic acid followed by chlorosulfonation with excess chlorosulfonic acid. A series of new sulfo-
nylquinoxaline derivatives 2–6 were obtained upon reacting compound 1 with different types of amines. 2,3-
Dichloro-6-morpholinosulfonylquinoxaline derivative 6 was subjected to further chemical reactions to afford
many derivatives of 6-morpholino 2,3-disubstitutedquinoxalines, thus reaction of compound 6 with different
secondary amines yielded mono and di secondary aminoquinoxaline derivatives 7–10 depending on the re-
activity difference of the two chlorine atoms. Hydrazinolysis of compound 7 furnished hydrazino quinoxaline
derivatives 11a-c. Additionally triazolo and pyrazolyl quinoxaline derivatives 12–14 were obtained through the
reaction of compound 11a with phenyl isothiocyanate, formylpyrazole and ethyl acetoacetate. All the synthe-
sized compounds were screened for their antibacterial and antifungal activities. Compounds 7a, 9b, 10a, 10c,
10f and 11c showed good to moderate antimicrobial activity against the tested Gram-positive, Gram-negative
bacteria and fungi with MIC values ranging from 2.44 to 180.14 μM. Their MBC values were also evaluated using
the same tested microorganisms. Moreover, screening against multi-drug resistant strains revealed the pro-
miscuity of these new derivatives, especially compound 7a that showed comparable antibacterial activity (MIC
4.91–9.82 μM) with Norfloxacin (MIC 2.44–9.80 µM). Furthermore, these compounds were evaluated as DNA
Gyrase inhibitors and the obtained results were in the range of 15.69–23.72 µM. Immunomodulatory effect was
also investigated and compounds 7a, 11c, 10f, 10c, 10a and 9b showed high immunostimulatory action with
6-Morpholinosulfonylquinoxaline
Immunomodulatory effect
Antibacterial
Antifungal
MDRB
S. aureus DNA gyrase
Molecular docking
ratio (142.6
0.4, 135.7
0.5, 117.8
0. 39, 112.5
0. 83, 86.4
0. 47, 72.8
0. 77) respectively.
Molecular docking studies of the promising derivatives into DNA Gyrase binding site proved the usefulness of
hybridizing quinoxaline scaffold with SO2 and morpholine moieties as a hopeful strategy in designing new DNA
Gyrase binding molecules.
1. Introduction
development of new antibacterial agents to fight against microbial in-
fections. The demand for novel chemotherapeutic antibacterial agents
The upsurge and widespread of multi-drug resistant microorganisms
such as S. epidermidis [1,2], S. aureus [3,4], P. aeruginosa [5,6], E. coli
[7,8], E. faecium [9,10], S. apiospernum [11] had been reported as a
major threats to human health. Abscess of the brain is a dreadful
complication of E. coli infection [12]. Amoxicillin, Norfloxacin and
Ciprofloxacin are the most common drugs used for the treatment of E.
coli infections [13] but they are still associated with several side effects.
Toxicity and drug resistance are important factors that can lead to
treatment failure [14]. Therefore, there is an urgent need for the
represent an attractive strategy in the field of medicinal chemistry.
After many years of extensive studies on structural modification of
known antibacterial scaffolds, it is still difficult to deliver new leads,
therefore, the focus of such antibacterial research has moved to the
identification of novel chemical classes to target invading bacteria [15].
The study of quinoxaline derivatives has become of much interest in
recent years on account of their antibacterial, antiviral, anti-cancer,
anti-fungal, anti-helminthic and insecticidal [16–18] activities. The
quinoxaline ring has frequently been used as a component of various
⁎ Corresponding authors at: Medicinal Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Egypt (A. Belal).
E-mail addresses: yossry@azhar.edu.eg, yossry@yahoo.com (Y.A. Ammar), ahmed_ragab@azhar.edu.eg (A. Ragab), amany.mehani@pharm.bsu.edu.eg (A. Belal).
https://doi.org/10.1016/j.bioorg.2020.104164
Received 5 April 2020; Received in revised form 11 July 2020; Accepted 13 August 2020
Availableonline25August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
antibiotic molecules, such as echinomycin, levomycin, and actindeutin,
which inhibit the growth of Gram-positive bacteria and are active
against various transplantable tumors. Triostin C (I) is an antibiotic that
has quinoxaline core and exhibited activity against gram positive bac-
teria, its biological activity has been attributed to binding with the DNA
of susceptible cells through bifunctional intercalation of the quinoxa-
line moiety [19–22]. Furthermore, Sanna and co-workers reported that
quinoxaline derivatives bearing electron-withdrawing groups at the 6-
or 7-positions have antibacterial, antifungal, and anticancer activities
[23–26]. Hence, lipophilicity and the electronic properties of the sub-
stituents affect the biological activities of these compounds. The dis-
covery of sulfonamides as antibacterial agents was one of the most
fascinating area of chemotherapeutic agents and they were used suc-
cessfully in the treatment of a variety of bacterial infections. The sul-
fonamide group is considered as a pharmacophore which is present in
several biologically active molecules, in particular antimicrobial agents,
for example, sulfacetamide (II) [27–29].
compound 3 showed absorption bands at υ 3497, 2260, 1673 cm−1
related to NH, C^N and C]O groups respectively. 1H NMR spectrum
revealed the appearance of three singlet signals at δ 4.42, 11.92 and
11.94 ppm corresponding for CH2 and two NH protons in addition to
the aromatic protons which appeared in the region of 7.02–7.56 ppm.
13C NMR spectrum showed signals at δ 15.45 for methylene group and
three singlet signals at δ 143.99, 155.63 and 155.71 ppm corresponding
to carbonyl and -C]N groups as well as the aromatic carbons. Its mass
spectral data showed molecular ion peak at m/z = 381 (5%) which
agreed with the molecular mass of the compound (C17H11N5O4S) as
well as base peak was observed at m/z = 75. Also, compound 1 was
treated with theophylline in dimethylformamide under reflux to obtain
compound 4. The IR spectrum of compound 4 displayed the presence of
absorption bands at υ 3250, 1707&1662 cm−1 assignable to NH & 2 C]
O groups, respectively. 1H NMR spectrum displayed two singlet signals
at δ 3.22 and 3.43 ppm corresponding to two methyl protons that at-
tached to nitrogen of pyrimidine nucleus, multiplet signals at δ
7.02–7.42 ppm related to the four aromatic protons and another one
singlet signal at δ 9.96 ppm due to NH proton. 13C NMR spectrum
showed signals at δ 34.85 for two methyl groups and two singlet signals
at δ 155.71 and 155.78 ppm for carbonyl groups in addition to the
aromatic carbons between 113.21 and 143.70 ppm. Mass spectrum
displayed prominent molecular ion peak at m/z = 404 (16%) and base
peak at m/z = 180.
Also, numerous sulfonamide derivatives have been reported as
carbonic anhydrase inhibitors [30], anticancer [31–34], and anti-in-
flammatory agents [35]. Furthermore, recent reports suggested that
groups like morpholine, piperidine and piperazine can help in im-
proving pharmacological properties. Linezolid (III) is a synthetic anti-
biotic used for the treatment of infections caused by multi-resistant
bacteria as streptococcus and methicillin-resistant S. aureus (MRSA), by
inhibiting bacterial protein synthesis [36–40]. In a continuation of our
endeavors towards the development of potent and effective anti-
microbial agents [41–44] and especially anti-bacterial agents derived
from quinoxalines [45–49], this investigation deals with the rational
design of new molecular hybrids as potential antimicrobial agents.
These hybrids were designed to incorporate 6-morpholinosulfonylqui-
noxaline scaffold linked to various bioactive heterocyclic moieties at
position-2, 3 through different atom spacers, as shown in Fig. 1, the
reference drugs Triostin C (I), Sulfacetamide (II) and Linezolid (III)
were lunched through fragment based drug design to help us in the
design strategy of the new target compounds. The new compounds were
evaluated for their antibacterial activity to investigate the effect of such
structural modification on the biological effect.
The starting material 6-morpholinosulfonyl-2,3-dichloroquinoxa-
line (6)[51] was prepared in good yield through chlorination of com-
pound 5c with phosphorus oxychloride. The reactivity of di-
chloroquinoxaline derivative 6 towards some nitrogenous compounds
as mono nucleophile was discussed. Thus, the interaction of dichloro
derivative 6 with one mole of piperidine as a cyclic secondary amine in
acetonitrile furnished a sole product which was formulated as 2-or 3-
piperidino 6-morpholinosulfonylquinoxaline (7) or (8) respectively.
According to the effect of the sulfonyl group, the authors favor isomer 7
due to the 2-position is presumed to be preferentially substituted due to
the (-M) effect of the sulfonyl group in structure 7. This means that the
2-carbon will be more susceptible to nucleophilic attack and the reac-
tion proceed according to nucleophilic substitution through addition
elimination mechanism. The supporting evidence of compound 7a was
confirmed by microanalyses and spectral data. The 1H NMR spectra
indicated the presence of one multiplet and three triplets at δ 1.68,
2.91, 3.58, 3.62 ppm corresponding for piperidinyl and morpholinyl
protons besides to the aromatic protons. 13C NMR spectra showed sig-
nals at δ 24.14, 25.63, 46.44, 50.04 and 65.73 ppm for the piperidinyl
and morpholinyl carbons while the aromatic carbons of quinoxaline
moiety were observed from δ 128.09 to 154.04 ppm.
2. Results and discussion:
2.1. Chemistry
The synthetic strategies adopted for the synthesis of the inter-
mediates and target compounds are depicted in schemes 1-3. At first, o-
phenylenediamine was treated with oxalic acid in the presence of 4 N
HCl to afford 2,3-(1H,4H)-quinoxalinedione [25], subsequent treatment
with chlorosulfonic acid to produce 2,3-quinoxalinedione-6-sulfonyl
chloride (1) [50]. Interaction of the sulfonyl chloride derivative 1 with
m-anisidine as a primary aromatic amine led to the formation of N-(3-
methoxyphenyl)-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfona-
mide (2). Its IR spectrum showed absorption bands at υ 3228 and 1693
and 1388, 1145 cm−1 representing NH, C]O and SO2 groups respec-
tively. The 1H NMR spectrum showed one singlet signal at δ 3.65 ppm
corresponding to methoxy group together with the aromatic protons as
multiplet at δ 6.56–7.93 ppm and three exchangeable signals at δ 10.31,
12.06 and 12.12 ppm due to 3 NH protons. Its 13C NMR spectrum
showed signals at δ 55.44 for methoxy group of m-anisidine and two
singlet signals at δ 155.35 and 160.14 ppm for carbonyl and the carbon
attached to methoxy group in addition to the aromatic carbons between
δ 105.88–139.29 ppm. The mass spectral data showed molecular ion
peak at m/z = 347 (19%) which agreed with the molecular formula
(C15H13N3O5S) while its base peak was observed at m/z = 116.
Similarly, interaction of quinoxaline derivative 1 with some selected
bioactive heterocyclic secondary amines furnished the corresponding
sulfonamide derivatives 3–5. Structures of these compounds were elu-
cidated by elemental analysis and spectroscopic data. IR spectrum of
The interaction of the dichloro derivative 6 with a cyclic secondary
amines such as N-methylpiperazine and morpholine consumed one
mole and produced a single product in each case, which was named as
2-N-methylpiprazinyl and 2-morpholinyl derivatives 7b,c respectively
(Scheme 2), based on elemental analyses and spectral data in experi-
mental section. On the other hand, the interaction of compound 6 with
two moles of the cyclic secondary amines yielded symmetrical 2,3-
disubstitituted quinoxaline-6-morpholino-sulfonyl derivatives 9a-c. The
structure of compound 9b was proven by IR analysis, which displayed
absorption bands at υ 1603, 1354, 1156 cm−1 for C]N, SO2 groups. 1H
NMR spectrum demonstrated new signals for two N-methyl piperazine
moiety as well as morpholine protons at δ 2.22–3.62 ppm in addition to
signals due to aromatic protons between δ 7.60 and 7.86 ppm that
appeared as two doublet and one singlet signals. Moreover, 13C NMR
data showed signals at δ 44.75 ppm for two methyl carbons, δ 46.46,
46.87, 54.64 ppm for two piperazine rings, in addition to morpholine
carbons at δ 46.15, 65.75 ppm and aromatic carbons in the range of δ
124.11–140.17 ppm and two signals at 148.81, 149.30 for two C]N.
The structure of compound 9 was also, confirmed via the reaction of
compound 7 with another mole of the same secondary cyclic amines.
In order to obtain different cyclic secondary amines in the 2-and 3-
2

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Fig. 1. Design strategy of the new quinoxaline derivatives based on the reference drugs (Triostin C (I), Sulfacetamide (II) and Linezolid (III) through fragment based
drug design.
positions of quinoxaline to study the effect of type and position on the
biological activity, compounds 7a-c were reacted with another cyclic
secondary amines where unsymmetrical 2,3-diamines were obtained.
The structure elucidation of compounds 10a-f were performed from
elemental analyses and their spectroscopic data. IR spectra of com-
pound 10f showed absorption peaks at υ 1599, 1350, 1152 cm−1 for
C]N & SO2 moieties. 1H NMR spectrum displayed singlet signal at δ
2.24 ppm for methyl protons, triplet signals at δ 2.51, 3.56 ppm for
piperazine ring protons and triplet signals at δ 2.93, 3.47, 3.64 and
3.78 ppm for two morpholinyl moieties. Furthermore, 13C NMR analysis
demonstrated signals at δ 46.13 ppm for methyl carbon, δ 46.92,
54.54 ppm for piperazine moiety and δ 46.46, 47.49, 65.75 and
66.12 ppm for both morpholinyl carbons. Hydrazine hydrate was re-
acted with 7a-c under reflux condition in acetonitrile and underwent
hydrazinolysis to afford the corresponding 3-hydrazino quinoxaline
derivatives 11a-c. IR spectra of compound 11a as an example, which
displayed absorption bands at υ 3317, 3201 cm−1 for (NH2, NH), υ
1608, 1346 and 1161 cm−1 due to C]N beside SO2 respectively.1H
NMR data showed two new singlet signals exchangeable with D2O at δ
4.63, 8.34 ppm related to NH2 and NH protons respectively, in addition
to signals due to morpholine, piperidine and aromatic protons.
Moreover, 13C NMR spectra demonstrated signals at δ 24.93, 26.32 &
48.15 ppm for piperidine carbons, δ 46.42, 65.77 ppm for morpholine
heterocyclic compounds of potential biological activity [52]. Thus, it
was of our interest to study the reactivity of hydrazine derivative 11a
towards a variety of chemical reagents. Refluxing compound 11a with
phenyl isothiocyanate caused cyclization and the obtained product was
8-(morpholinosulfonyl)-N-phenyl-4-(piperidin-1-yl)-[1,2,4]triazolo
[4,3-a]quinoxalin-1-amine (12). The structure of the prepared com-
pounds was elucidated based on elemental analysis and spectral data.
IR displayed the absence of NH2 absorption band and the presence of
absorption peaks at υ 3248, 1612 & 1374, 1172 cm−1 for NH, C]N &
SO2 groups. 1H NMR data displayed signals at δ 1.18–1.22 ppm (as
multiplet), 3.07 ppm (as a triplet) for piperidine protons, δ 3.02 and
3.56 ppm as triplet signals due to morpholine protons. In addition to
aromatic protons in the range of δ 6.90–7.61 ppm and an exchangeable
signal at δ 10.70 ppm due to NH proton. Furthermore, condensation of
hydrazine quinoxaline derivative 11a with 1,3-diphenyl-1H-pyrazole-4-
carbaldehyde produced Schiff's base derivative 13. IR spectrum of
compound 13 displayed the presence of an absorption peaks at ν 3417,
1597 & 1350, 1165 cm−1 for NH, C]N, SO2 groups. 1H NMR analysis
demonstrated a new singlet signal for pyrazole proton at δ 8.26 ppm
and an exchangeable singlet signal at δ 9.04 ppm due to NH proton
together with signals due to morpholine, piperidine and aromatic pro-
tons. The mass spectra displayed a molecular ion peak at m/z = 622
(21%) corresponding to a molecular formula (C33H34N8O3S).
carbons in addition to, aromatic carbons in the range of
δ
Finally, the interaction of hydrazine derivative 11a with ethyl
acetoacetate afforded the pyrazolone derivative 14 through cyclization,
which was found in tautomer with its hydroxyl derivative (Scheme 3).
The IR spectrum demonstrated absorption bands at ν 3414, 1616 and
111.80–137.45 ppm and 152.71 ppm for the C]N group.
Hydrazine derivatives are versatile reagents and have been ex-
tensively used as synthetic starting materials for the synthesis of several
3

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Scheme 1. Synthesis of new sulfonylquinoxaline derivatives 2–6.
1342, 1156 for OH, C]N and SO2 groups. Also, 1H NMR spectra re-
vealed a new exchangeable singlet signal at δ 12.41 ppm due to OH
proton as well as singlet signal at δ 3.03 ppm for methyl group in
pyrazole core. Its mass spectral data showed a molecular ion peak at m/
z = 458 (21%) which agreed with the molecular mass of the compound
(C21H26N6O4S) while the base peak was observed at m/z = 105.
2.2. Biological activity evaluation
2.2.1. Antimicrobial activity evaluation
Anti-microbial activity of the newly synthesized quinoxaline deri-
vatives 2–13 was evaluated against three Gram-positive strains (B.
subtilis ATCC 6633, S. aureus ATCC 29213 and E. faecalis ATCC 29212),
three Gram-negative (P. aeruginosa ATCC 27853, E. coli ATCC 25922
Scheme 2. Synthesis of 2,3-disubstituted-6-(morpholinosulfonyl)quinoxaline derivatives 7a-11c.
4

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Scheme 3. Synthesis of 6-(morpholinosulfonyl)quinoxaline derivatives containing triazole or pyrazole 12–14.
Table 1
Antimicrobial activity screening of the synthesized compounds (In vitro).
Code
Table (1): In vitro Antimicrobial activity of the synthesized compoundswith mean diameter of inhibition zone (mm)
Gram-positive
Gram-negative
Fungi
B. subtilis
S. aureus
E. faecalis
E. Coli
P. aeruginosa
S. typhi
C. albicans
F. oxysporum
2
14
21
15
17
20
21
28
30
25
21
22
25
24
12
26
21
13
25
16
14
31
12
25
na
0. 31
0. 32
0. 12
0. 34
0. 58
0. 39
0.22
0.21
0. 24
0. 5
12
15
21
14
18
19
25
27
21
19
21
21
22
na
23
16
na
21
15
12
28
15
25
na
0. 78
13
11
14
17
13
17
30
31
18
22
19
23
21
14
24
24
14
22
17
na
30
na
22
na
0. 21
15
24
18
19
18
19
26
28
22
23
20
26
23
19
21
14
12
20
14
13
26
12
23
na
0. 24
0. 32
0. 52
0. 65
0. 14
0. 32
0.62
0.65
0. 18
0.81
0. 44
0. 37
0. 14
0. 33
0.14
0.4
17
15
14
18
17
21
17
22
19
12
18
23
21
12
20
na
na
18
na
na
21
na
20
na
0. 65
0. 65
0. 54
0. 53
0. 44
0. 24
0. 34
0. 14
0.2
14
14
13
12
13
24
28
27
20
19
15
19
23
na
22
na
15
24
11
10
26
na
21
na
0. 24
12
16
18
13
15
16
23
24
22
18
17
18
21
13.0
21
19
12
17
13
9
0. 25
0. 32
0. 45
0. 75
0. 65
0. 34
0.25
0.33
0. 14
0.56
0. 73
0. 58
0. 45
0.2
14
13
11
15
17
13
19
20
18
11
9
0. 37
0. 65
0. 35
0. 68
0. 75
0. 95
0.15
3
0. 42
0. 35
0. 65
0. 75
0. 32
0.19
0. 52
0. 45
0. 74
0. 85
0. 36
0. 34
0. 44
0. 34
0. 55
0. 23
0. 34
0. 92
0. 17
0. 33
0. 44
0. 14
0. 3
0. 96
0. 45
0. 85
0. 83
0. 21
0. 21
0. 46
0. 29
0. 16
0. 17
0. 2
4
5a
5b
5c
6
7a
0.89
0.79
7b
7c
0. 15
0. 12
0. 17
0. 27
0. 24
0. 5
0. 2
0.15
9a
0. 33
0. 21
0. 78
0.91
0. 66
0.11
0. 4
0. 87
0. 77
0. 25
0. 3
0. 62
0. 94
0. 78
9b
10a
10b
10c
10d
10e
10f
11a
11b
11c
13
S1
14
19
na
19
na
na
16
na
na
22
13
na
18
0. 13
0. 5
0. 61
0.2
0. 31
0.16
0. 64
0. 25
0.22
0. 53
0.36
0.2
0. 44
0.88
0.21
0.2
0. 29
0. 44
0.62
0.17
0. 42
0. 5
0.33
0. 75
0. 13
0.35
0. 74
0.51
0.65
0. 5
0.64
0. 16
0.22
0. 22
0. 96
0.12
0.42
0.17
0. 32
0.62
0. 67
0.24
0. 54
0.15
0. 61
25
17
na
22
0.33
0. 38
0.45
S2
0.2
0.32
*na: No activity, *S1 = Tetracycline, S2 = Amphotericin B
and S. typhi ATCC 6539), in addition to two fungal strains namely C.
albicans (ATCC 10231) and F. oxysporum (RCMB 008002). The anti-
microbial screening was determined by measuring inhibition zone
(mm) via conventional paper disk diffusion method [53,54], data re-
presented in Table 1, Tetracycline and the antifungal drug Amphoter-
icin B were used as refrence drugs.
The results of antimicrobial activity screening revealed that com-
pounds 6, 7a, 11c showed better antibacterial activity than
Tetracycline against the three tested Gram-positive strains. While
compounds 6, 10c showed better activity against B. subtilis and E.
faecalis strains, compounds 9b, 10d displayed good activity against E.
faecalis. On the other hand, compounds 7b, 9b and 10f showed
5

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
comparable activity against B. subtilis, and compounds 7c & 10f re-
vealed comparable activity towards E. faecalis. Moreover, compounds
7a, 11c exhibited promising antibacterial activity than tetracycline
against the three tested Gram-negative microorganisms. Compounds 3,
6, 9b showed better activity than tetracycline against E. coli, com-
pounds 5c, 9b, 10a displayed good activity against P. aeruginosa,
compounds 5c, 10a, 10c, 10f demonstrated good activity against S.
typhi. On the other hand, compounds 7c, 10a and 10c showed com-
parable activity against E. coli tested strain.
displayed better antibacterial activity on S. typhi (MIC of 40.18, 60.29,
33.76, and 14.12 μM) than Tetracycline (MIC of 33.63 μM). Compound
11c with bis-morpholine moiety as well as 3-hydrazinoquinaxoline
derivatives exhibited antifungal potential with (MIC of 19.79,
39.59 μM) on C. albicans and F. oxysporum strains, additionally, com-
pound 7a has displayed the second-best antifungal activity among the
tested compounds (MIC of 23.30, 46.63 μM) than the standard positive
control Amphotericin B (MIC of 16.81, 33.63 μM).
For further exploration of the most active compounds 7a, 9b, 10a,
10c, 10f and 11c, minimum bactericidal concentrations (MBC) were
determined using the conventional paper disk diffusion method
[53,54]. The obtained results are represented in Table 3 & Fig. S2,
bactericidal activity revealed that compounds 7a and 11c showed very
strong bactericidal activity on all tested Gram-positive, Gram-negative
and fungi strains compared to the standard positive control Tetra-
cycline and Amphotericin B as shown from MBC values in Table 3.
Interestingly, compounds 10a, 10c and 10f displayed better bacter-
icidal activity on B. subtilis with MBC values between 30.37 and
38.14 μM, while both 10c and 10f displayed good activity against E.
faecalis with MBC values 16.93, 39.99 μM compared to tetracycline
(MBC of 43.72, and 100.90 μM respectively).
Compounds 7a & 11c revealed observed antifungal activity which is
higher than that of the reference drug Amphotericin B against C. albi-
cans and F. oxysporum microorganisms. On the other hand, compounds
6, 10a, 10c showed better activity against F. oxysporum, while com-
pound 7b showed comparable activity to the reference standard
Amphotericin B against both fungal strains C. albicans and F. oxy-
sporum microorganisms.
2.2.2. Minimum Inhibitory/Bactericidal concentrations (MIC)/(MBC) and
SAR study
Depending on the antimicrobial screening results for all compounds,
previously represented in (Table 1), the minimum inhibitory con-
centrations (MIC) of the most active quinoxaline derivatives (7a, 9b,
10a, 10c, 10f, 11c) were determined using the conventional paper disk
diffusion method [53,54], data are represented in Table 2 & Fig. S1.
Compound 7a that contains piperidine and chlorine moieties as well as
quinoxaline sulphonyl morpholine surprisingly showed very strong
antibacterial potential on B. subtilis and E. faecalis (4.91, 2.44 μM) re-
spectively, compared to the standard positive control tetracycline
(MIC = 33.63 & 67.27 μM).
Compounds 7a, 10a and 10c exhibited better fungicidal activity on
C. albicans (MBC of 44.26, 61.02, and 50.86 μM) than the standard
positive control Amphotericin B (MIC of 37.26 μM), while compound
11c showed to be the most promising derivative against the tested two
fungal strains C. albicans, F. oxysporum with MBC values (31.66 and
67.30 μM) when compared to Amphotericin B (37.26 and 70.62 μM)
respectively.
While compound 11c with bis-morpholine moieties and 3-hy-
drazinoquinoxaline exhibited the most significant activity among all
derivatives against S. aureus with MIC value 14.12 μM compared to the
standard reference tetracycline (MIC = 67.27 μM). Interestingly,
compounds 9b, 10a, 10c and 10f also displayed better antibacterial
potential on B. subtilis (MIC of 32.84, 20.08, 16.95 and 16.88 μM) re-
spectively, than the standard positive control Tetracycline that ex-
hibited MIC value at 33.63 μM. Moreover, compounds 7a & 10a that
have piperidinyl moiety in position two of the quinoxaline sulfonamide
core showed better antibacterial potential on S. aureus (MIC: 19.67 &
60.29 μM) than Tetracycline (MIC of 67.27 μM), it is observed that the
presence of chlorine atom in position three gave more potent effect than
N-methyl piperazine.
2.2.3. Drug resistance study
For further screening of the best active derivatives 7a, 9b, 10a, 10c,
10f and 11c, they were evaluated against a panel of drug-resistant
Gram-positive bacterial strains S. aureus (ATCC 43300), S. aureus (ATCC
33591), multidrug-resistant Gram-negative E. coli (ATCC BAA-196) and
P. aeruginosa (ATCC BAA-2111) [55,56]. As shown in Table 4 & Fig.
S3A, B the selected compounds exhibited moderate to good potency
against all the tested multi-drug resistant bacterial (MDRB) strains with
MIC values of (4.91–32.84 µM), Fig. S3A. As for the tested strains S.
aureus, E. coli and P. aeruginosa, it was found that compound 7a has
showed a comparable antibacterial activity (MIC 4.91, 9.82, 4.91, and
9.82 µM) to Norfloxacin (MIC 3.91, 2.44, 4.91, and 9.81 µM), however,
Tetracycline as another positive control doesn’t give any results against
the tested resistant strains. Compound 7a displayed the best results
against (MDRB) compared with Norfloxacin, these may be due to the
presence of chlorine atom in position three in addition to piperidinyl
moiety in position two. The other five compounds also showed good
activity against MDRB, near to that of the standard commercial drug
Norfloxacin.
Compounds 9b, 10c, 10f & 11c displayed better antibacterial po-
tential on E. faecalis (MIC of 16.42, 8.46, 19.99 and 4.94 μM) than the
standard positive control Tetracycline (MIC of 67.27 μM). On the other
hand, compound 7a exhibited very strong antibacterial potential on E.
coli, P. aeruginosa and S. typhi (MIC of 19.67, 69.96 and 9.82 μM)
compared to Tetracycline (MIC of 16.81, 67.27 and 33.63 μM).
Furthermore, compounds 9b, 11c showed better antibacterial potential
on E. coli (MIC of 19.44, 19.79 μM) when compared to tetracycline
(MIC of 16.81 μM), whilst compounds 10a, 10c, 10f and 11c have
Next, we have evaluated the MBC for the most promising com-
pounds against MDRS as shown in Table 4, Fig. S3B. Notably, almost all
the tested derivatives 7a, 9b, 10a, 10c, 10f and 11c showed remarkable
Table 2
Minimal inhibitory concentrations (MIC) (μM) of the most active compounds.
Cpd. No.
Gram-positive
Gram-negative
Fungi
B. subtilis
S. aureus
E. faecalis
E. Coli
P. aeruginosa
S. typhi
C. albicans
F. oxysporum
7a
4.91
32.84
20.08
16.95
16.88
9.88
33.63
–
19.67
131.41
60.29
120.49
135.11
14.12
67.27
–
2.44
16.42
135.69
8.46
19.99
4.94
67.27
–
19.67
19.44
67.84
60.29
135.11
19.79
16.81
–
69.96
116.69
135.69
135.69
180.14
79.22
67.27
–
9.82
23.30
58.38
33.91
33.91
67.55
19.79
–
46.63
131.41
67.84
76.42
119.98
39.59
–
9b
65.70
40.186
60.29
33.76
14.12
33.63
–
10a
10c
10f
11c
S1
S2
16.81
33.63
*S1 = Tetracycline, S2 = Amphotericin B.
6

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Table 3
Minimum bactericidal concentrations (MBC) and minimum fungicidal concentration (µM) of the selected compounds against pathogenic microbes.
Cpd. No.
Gram-positive
Gram-negative
Fungi
B. subtilis
S. aureus
E. faecalis
E. coli
P. aeruginosa
ATCC27853
S. typhi
C. albicans
F. oxysporum
RCMB 008002
ATCC 6633
ATCC29213
ATCC 29212
ATCC 25922
ATCC 6539
ATCC 10231
7a
9.82
39.35
249.67
90.42
240.99
216.18
26.82
94.17
–
4.88
39.35
38.89
135.69
120.58
256.71
35.61
20.17
–
139.93
186.70
257.81
271.38
360.29
150.51
94.17
–
16.70
118.26
80.37
90.42
60.76
26.82
47.08
–
44.26
87.57
61.02
50.86
114.83
31.66
–
74.60
183.97
101.75
145.20
191.97
67.30
–
9b
52.54
38.14
33.91
30.37
16.80
43.72
–
32.84
271.38
16.93
39.99
9.38
10a
10c
10f
11c
Tetr.
Am. B
100.90
–
37.26
70.62
Table 4
Mean diameter of inhibition zone (mm), minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) (µM) of the selected compounds
against multidrug resistant bacteria (MDRB).
Code Mean diameter of inhibition zone (mm) and minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) (µM) of the most potent
synthesized compounds against MDRB.
S. aureus
S. aureus
E. coli
P. aeruginosa
ATCC 43300
ATCC 33591
ATCC BAA-196
ATCC BAA-2111
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
7a
25
15
19
17
18
22
–
0.16
0.4
4.91
32.84
13.56
16.95
16.88
11.25
–
9.82
65.70
25.77
32.19
33.76
22.51
–
23
22
17
21
15
25
–
0.22
0.5
9.82
8.19
16.95
12.04
19.19
4.94
–
19.65
15.57
33.91
24.09
34.54
8.391
–
26
19
22
21
20
23
–
0.66
0.11
0.15
0.23
0.44
0. 2
4.91
32.84
8.46
16.95
19.99
15.84
–
9.82
25
23
20
22
21
24
–
0. 24
0. 99
0. 66
0.56
9.82
13.14
19.27
9.63
16.88
13.18
–
19.65
24.95
34.69
14.45
33.76
25.04
–
9b
65.70
16.08
32.19
39.99
30.09
–
10a
10c
10f
11c
Tetr.
Nor.
0. 23
0.77
0.11
0.65
0. 35
0.49
0.91
0.12
0.33
0. 88
25
0.5
3.91
8.80
26
0.5
2.44
4.88
27
0.98
4.91
11.05
24
0.47
9.80
14.68
activities against MDRS used in this study. Where the promising com-
pounds exhibited MBC values range between 8.39 and 65.70 µM
compared with Norfloxacin (4.88–14.68 µM). Compound 7a showed
MBC value 9.82 µM against S. aureus (ATCC 43300), however, Nor-
floxacin MBC value was 8.80 µM. The bactericidal activity of com-
pounds 7a and 10a that having piperidinyl moiety in position two
showed MBC values (9.82, and 16.08 µM) respectively against E. coli
ATCC BAA-196 compared to 11.05 µM for Norfloxacin and this differ-
ence in activity for quinoxaline derivatives can be attributed to chloro
and/or N-methyl piperazine moieties. Finally, for P. aeruginosa ATCC
BAA-2111 compound 10c with bis-piperidinyl beside 6-morpholino-
sulfonylquinoxaline derivatives showed the best MIC and MBC values
9.63 and 14.45 µM against 9.80 and 14.68 µM assigned for the positive
control.
Ciprofloxacin was used as a standard reference and the results were
illustrated in (Table 5 and Fig. S4), IC50 values are expressed in (µM).
2-piperidinyl derivatives 7a, 10a and 2-morpholinyl derivative 11c
showed better activity (IC50 = 17.10, 15.69 & 17.69 µM) than the
standard drug, Ciprofloxacin (IC50 = 26.31 µM), while compounds 10f
and 9b exhibited DNA Gyrase inhibition at IC50 value equal to 18.85
and 19.21 µM respectively. The structure activity relationship showed
that the most promising quinoxaline derivative 10a that involving 2-
pipridinyl in position two of quinoxaline nucleus as well as 4-N-methyl
piperazinyl in position 3 in addition to 6-morpholinosulfonyl group in
quinoxaline moiety followed by the quinoxaline derivatives 7a and 11c
are more active than the other substituted analogs 9b, 10c & 10f.
Furthermore, it was found that two quinoxaline derivatives 10a and
10c that contain the same substituents [(4-methylpiperazin-1-yl) and
(2-piperidin-1-yl)] in position two and three to quinoxaline moiety
showed nearly 8 µM differ in activity and that difference in activity
illustrate that presence of piperidin-1-yl and methylpiperazin-1-yl in
position 2 and 3 is preferred and caused higher activity against DNA
gyrase. In the same way, quinoxaline derivative 10f in which piper-
idinyl in position 2 was replaced with morpholine showed decrease in
activity with nearly 3 µM. It is also observed that compounds 9b and
10f with 4-methylpiperazine moiety at position three revealed DNA
Gyrase inhibition (IC50 = 19.21, and 18.85 µM) respectively, and this
difference can be attributed to presence of morpholine core instead of
N-methyl piperazine in position 2 in the quinoxaline scaffold.
2.2.4. DNA Gyrase inhibition activity
Derivatives with promising activity 7a, 9b, 10a, 10c, 10f and 11c
were selected for screening as DNA gyrase inhibitors, as DNA Gyrase
represents an important target for antimicrobial candidates.
Table 5
S. aureus DNA gyrase inhibitory activity of the investigated compounds.
Compound
DNA gyrase Supercoiling inhibition
IC50 in µM
7a
17.10
19.21
15.69
23.72
18.85
17.69
26.31
1.16
1.22
1.63
1.13
1.9
9b
10a
2.2.5. Immunomodulatory activity for most active compounds
10c
The promising compounds depending on the previous results were
investigated in vitro to evaluate their immunomodulatory activity. The
neutrophils play a major role as a killer cell for many types of infections
[57]. The primary function of neutrophils is the intracellular killing of
10f
11c
1.55
1.64
Ciprofloxacin
7

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Table 6
removing the co-crystallized ligand and re-docking again into the
Gyrase binding site at RMDS value equal 1.44 (Supp. Data). The ob-
tained results revealed the promiscuity of the new quinoxalines to bind
into the DNA Gyrease enzyme. The binding free energy and types of
possible interactions in addition to, the interacting moiety of our
docked compounds are represented in Table S1.
Intracellular killing activities of the tested compounds.
Compound
Intracellular killing activity %
7a
142.6
72.8
0. 4
0.77
0. 47
0.83
0. 39
0. 5
9b
10a
10c
10f
11c
86.4
As shown from the obtained data quinoxaline core was able to form
arene-cation interactions with different amino acids in the DNA Gyrase
binding site as Lys 103 and Arg 76. Hybridizing quinoxaline scaffold
with SO2 and morpholine moieties proved to be a useful strategy in
building up new molecules with good ability of the binding into DNA
Gyrase binding site as morpholine and/or SO2 showed hydrogen
bonding interactions with Arg 76, Arg 136 and Gly 117. Further sub-
stitution with piperazine as compound 9b and NH2 group as compound
11c also extended the ability of these compounds to form hydrogen
bonding with Asp 73 and Arg 136 amino acids respectively.
112.5
117.8
135.7
microbes. The NBT reduction experiment was used to assess our se-
lected compounds [53–54,58], the obtained results (Table 6 and Fig.
S5) reflected the considerable effect of our compounds in killing ability
toward neutrophils. Intracellular killing activities are presented by
percentages (%). Compounds 7a, 9b, 10a, 10c, 10f and 11c showed
good potency as immunomodulatory agents, the highest im-
munostimulatory action was assigned to compounds 7a, 10c, 10f and
3. Conclusion
11c with activity range between 117.8
0. 39 and 142.6
0.4.
New twenty-four quinoxaline derivatives were designed and syn-
thesized, they were screened for their antimicrobial activity against six
bacterial strains and two fungal species. Six compounds (7a, 9b, 10a,
10c, 10f and 11c) revealed promising antibacterial activity with MIC
value range of 2.44–180.14 μM. Additionally, these compounds showed
good results against the tested multi-drug resistant strains, especially
compound 7a that showed comparable antibacterial activity (MIC 4-91-
9.82 µM) to that of Norfloxacin (MIC 2.44–9.80 µM). Furthermore,
compounds 7a, 10a & 11c exhibited potent DNA Gyrase inhibition
(IC50 = 17.10, 15.69, and 17.69 µM) higher than the standard drug
Ciprofloxacin (IC50 = 26.31 µM). Therefore, they can be considered as
a hit for further optimization to obtain more active antibacterial agents.
Molecular docking studies revealed that these compounds (7a, 9b, 10a,
10c, 10f and 11c) have binding mode and an affinity to DNA Gyrase
binding site comparable to that of Ciprofloxacin. Also, it has shown the
importance of hybridizing the quinoxaline scaffold with morpholine
and SO2 moieties for better binding with DNA Gyrase. Finally,
2.3. Molecular docking study
The DNA Gyrase enzyme is one of the topoisomerases classes (to-
poisomerase II), these enzymes are involved in winding and unwinding
of DNA during the process of replication and transcription. Gyrase en-
zyme affects the topological state of DNA, hence it is considered as an
important intracellular target for antibacterial agents as
a re-
presentative model for other DNA topoisomerases [59]. This fact en-
couraged us to investigate the binding mode, docking score energy and
the expected type of interactions between the hopeful new molecules
and binding site of the DNA Gyrase enzyme. Compounds 7a, 9b, 10a,
10c, 10f, 11c, that showed DNA Gyrase inhibition activity at a range
from 15.69 to 23.72 µM in addition to Ciprofloxacin as a reference drug
were docked into DNA Gyrase binding site, the obtained results are
represented in Table S1, Figs. 2A-5B and the other figures are provided
in the supplementary data files. The validation step was performed by
Fig. 2A. 2D for Ciprofloxacin docked into DNA Gyrase binding site.
8

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Fig. 2B. 3D for Ciprofloxacin docked into DNA Gyrase binding site.
compounds 7a, 10c, 10f and 11c showed to have a good im-
4.1. Chemistry
munomodulating activity.
4.1.1. 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride (1)
It was prepared according to previously reported method [50].
Yield: 88%; as White crystals from ethanol/DMF mixture; m.p.:
348–350 °C; IR: ν/cm−1: 3354, 3169 (NH), 3038 (CH-Ar), 2942, 2841
(CH-aliph), 1674 (C]O), 1387, 1161 (SO2); 1H NMR (400 MHz, DMSO)
δ/ppm: 7.02 (d, J = 8.3 Hz, 1H), 7.29 (dd, J = 8.3, 1.6 Hz, 1H), 7.42
(s, 1H), 11.91 (s, 1H, NH, exchangeable with D2O), 11.93 (s, 1H, NH,
exchangeable with D2O); 13C NMR (101 MHz, DMSO) δ/ppm: 113.21,
114.73, 121.07, 125.13, 126.05, 143.86, 155.67 (C]O), 155.75 (C]
O); Anal. Calcd. for C8H5ClN2O4S (260.65): C, 36.87; H, 1.93; N, 10.75;
Found: C, 36.83; H, 1.89; N, 10.71
4. Experimental
All solvents and reagents were freshly distilled and purified ac-
cording to standard procedures. All melting points are recorded on di-
gital Gallen Kamp MFB-595 instrument and are uncorrected. The IR
spectra (KBr) (cm−1) were detected on a Shimadzu 440 spectro-
photometer 1H NMR and 13C NMR spectra (δ, ppm) were performed at
the Main Chemical Warfare Laboratories, Chemical Warfare
Department, Ministry of Defense, Cairo, Egypt, on a Varian Gemini 500
(400 MHz and 101 MHz) spectrometer, deuterated dimethyl sulfoxide
(DMSO‑d6) was used as a solvent and TMS as an internal standard;
chemical shifts are expressed in δ ppm. The data were presented as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = tri-
plet, q = quartet, m = multiplet, br = broad, app = apparent), cou-
pling constant (s) in Hertz (Hz), and integration. Mass spectra were
recorded on Thermo Scientific ISQLT mass spectrometer at the Regional
Center for Mycology and Biotechnology, Al-Azhar University.
Elemental analyses were carried out at Micro Analytical Unit, Cairo
University.
4.1.2. N-(3-methoxyphenyl)-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-
sulfonamide (2)
2,3-dioxoquinoxaline derivatives 1 (10 mmol, 2.6 g) was dissolved
in dry dimethylformamide (20 mL), followed by addition of m-anisidine
(10 mmol, 1.23 g) and the resulting mixture was kept under stirring at
room temperature for 10 h. The reaction mixture was then poured into
water (200 mL) and the solid formed was recrystallized from DMF to
give desired product 2 as pale brown powder.
9

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Fig. 3A. 2D for compound 7a docked into DNA Gyrase binding site.
Yield: 75%; m.p.: 308–310 °C; IR: ν/cm−1: 3228 (NH), 3059(CH-
Ar), 2947, 2835 (CH-aliph.), 1693 (C]O), 1388, 1145 (SO2); 1H NMR
(400 MHz, DMSO) δ/ppm: 3.65 (s, 3H, –OCH3), 6.56 (d, J = 8.2,
1.8 Hz, 1H, Ar-H), 6.65 (d, J = 7.4 Hz, 1H, Ar-H), 7.08–7.10 (m, 1H,
Ar-H), 7.17 (d, J = 8.5 Hz, 1H, Ar-H), 7.44 (dd, J = 8.4, 1.7 Hz, 1H, Ar-
H), 7.54 (s, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 10.31, 12.06, 12.12 (3 s, 3H,
3NH exchangeable with D2O); 13C NMR (101 MHz, DMSO) δ/ppm:
55.44 (CH3), 105.88, 109.44, 112.18, 114.19, 115.97, 121.94, 126.26,
129.81, 130.46, 133.88, 139.29, 155.35 (2C]O), 160.14 (]CeO); MS
(m/z, %): 56(65%), 90 (42%), 109 (44%), 116 (100%), 121 (59%), 154
(45%), 161 (86%), 235 (40%), 262 (66%), 303 (49%), 325 (41%), 332
(56%), 346 (M+−1, 17%), 347 (M+, 19%), 348 (M++1, 17%), 349
(M++2, 10%); Anal. Calcd. for C15H13N3O5S (347.35): C, 51.87; H,
3.77; N, 12.10; Found: C, 51.85; H, 3.73; N, 12.06
4.1.3. 2-(1-((2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-6-yl)sulfonyl)-1H-
benzo[d]imida-zol-2-yl)acetonitrile (3)
A mixture of 2,3-dioxoquinoxaline derivatives 1 (10 mmol, 2.6 g)
and 2-(1H-benzo[d]imidazol-2-yl)acetonitrile (10 mmol, 1.57 g) in di-
oxane (20 mL), containing 3 drops of TEA, was heated under reflux for
1 h and then stirred at room temp. till completion of the reaction which
was monitored by TLC. The solid obtained after completion was
Fig. 3B. 3D for compound 7a docked into DNA Gyrase binding site.
10

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Fig. 4A. 2D for compound 10a docked into DNA Gyrase binding site.
filtered, washed with ethanol and recrystallized from DMF to give
compound 3 as pale brown powder.
O); MS (m/z, %): 45(40%), 65 (40%), 75 (100%), 89 (69%), 103 (64%),
116 (92%), 120 (51%), 264 (49%), 381 (M+, 5%); Anal. Calcd. for
C17H11N5O4S (381.37): C, 53.54; H, 2.91; N, 18.36; Found: C, 53.50; H,
2.85; N, 18.31.
Yield: 70%; m.p.:˃360 °C; IR: ν/cm−1: 3497 (NH), 3052(CH-Ar),
2943, 2840 (CH-aliph.), 2260 (C^N), 1673 (C]O), 1387, 1195 (SO2);
1H NMR (400 MHz, DMSO) δ/ppm: 4.42 (s, 2H, CH2), 7.02 (d,
J = 8.3 Hz, 2H, Ar-H), 7.22 (d, J = 6.0 Hz, 1H, Ar-H), 7.30 (d,
J = 8.3 Hz, 2H, Ar-H), 7.43 (s, 1H, Ar-H), 7.56 (d, J = 6.0 Hz, 1H, Ar-
H), 11.92, 11.94 (2 s, 2H, 2NH exchangeable with D2O); 13C NMR
(101 MHz, DMSO) δ/ppm: 15.45 (CH2), 113.20, 114.67, 117.16,
121.06, 122.06, 123.97, 125.08, 125.96, 127.20, 128.87, 131.96,
134.12, 138.04, 143.99 (C]N-imidazole), 155.63 (C]O), 155.71 (C]
4.1.4. 6-((1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)
sulfonyl)-1,4-di-hydroquino-xaline-2,3-dione (4)
2,3-dioxoquinoxaline derivatives 1 (10 mmol, 2.6 g) was dissolved
in dry dimethylformamide (20 mL), followed by addition of 1,3-di-
methyl-3,7-dihydro-1H-purine-2,6-dione (10 mmol, 1.8 g) and the re-
sulting mixture was heated under reflux for 1 h. The precipitate formed
Fig. 4B. 3D for compound 10a docked into DNA Gyrase binding site.
11

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
Fig. 5A. 2D for compound 11c docked into DNA Gyrase binding site.
while hot was filtered, washed with ethanol and recrystallized from
DMF to give compound 4 as white crystals.
(101 MHz, DMSO) δ/ppm: 34.85 (2(CH3)), 113.21, 114.79, 121.07(2C),
125.16, 126.12 (2C), 141.00, 143.70, 155.71 (2C = O), 155.78
(2C = O); MS (m/z, %): 102(44%), 122 (82%), 180 (100%), 323 (62%),
325 (49%), 339 (50%), 404 (M+, 16%); Anal. Calcd. for C15H12N6O6S
(404.36): C, 44.56; H, 2.99; N, 20.78; Found: C, 44.51; H, 2.81; N,
20.66.
Yield: 88%; m.p.: 320–322 °C; IR: ν/cm−1: 3250 (NH), 3025(CH-
Ar), 2943, 2823 (CH-aliph.), 1707, 1662 (C]O), 1381, 1174 (SO2); 1H
NMR (400 MHz, DMSO) δ/ppm: 3.22 (s, 3H, CH3), 3.43 (s, 3H, CH3),
7.02 (d, J = 8.3 Hz, 1H, Ar-H), 7.30 (dd, J = 8.3, 1.7 Hz, 1H, Ar-H),
7.42 (s, 2H, Ar-H), 9.96 (s, 2H, NH exchangeable with D2O); 13C NMR
Fig. 5B. 3D for compound 11c docked into DNA Gyrase binding site.
12

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
4.1.5. Synthesis of 6-(alk-1-ylsulfonyl)-1,4-dihydroquinoxaline-2,3-dione
(5a,b)
4.1.10. 3-Chloro-6-morpholinosulfonyl-2-(substitutedamine)quinoxaline
(7a-c)
A mixture of 2,3-dioxoquinoxaline derivatives 1 (10 mmol, 2.6 g)
and piperidine or 1-methyl piperazine (20 mmol) in dimethylforma-
mide (20 mL) was refluxed for 5 h. The solid obtained was precipitated
while hot, filtered, dried and recrystallized from DMF to give desired
product 5a,b.
A solution 2,3-dichloroquinoxaline derivatives 6 (10 mmol, 3.48 g)
and the requisite cyclic secondary amine (10 mmol) in 30 mL of acet-
onitrile was refluxed for 8 h. The reaction mixture was cooled and then
quenched onto crushed ice and stirred until the product precipitated
out. The precipitate was filtered, washed with water, dried and re-
crystallized from the proper solvent.
4.1.6. 6-(piperidin-1-ylsulfonyl)-1,4-dihydroquinoxaline-2,3-dione (5a)
As white crystals; yield: 73%; m.p.: 317–320 °C; IR: ν/cm−1:3142
(NH), 3040(CH-Ar), 2951, 2831 (CH-aliph.), 1684 (C]O), 1405, 1169
(SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.53 (m, 2H, CH2), 1.66 (m,
4H, C-(CH2)2), 2.98 (t, 4H, N(CH2)2), 7.03 (d, J = 8.3 Hz, 1H, Ar-H),
7.28 (dd, J = 8.3, 1.6 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 8.22, 11.91
(2 s, 2H, 2NH exchangeable with D2O); Anal. Calcd. for C13H15N3O4S
(309.34): C, 50.48; H, 4.89; N, 13.58; Found: C, 50.35; H, 4.76; N,
13.85.
4.1.11. 3-Chloro-6-morpholinosulfonyl-2-(piperidin-1-yl)quinoxaline (7a):
As yellow crystals from ethanol; yield: 75%; m.p.:148–150 °C; IR: ν/
cm−1: 3048 (CH-Ar), 2952, 2907, 2878, (CH-aliph.), 1559 (C]N),
1338, 1166 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.68 (m, 6H,
3(CH2)-pip), 2.91 (t, 4H, -N(CH2)2-morph), 3.58 (t, 4H, N(CH2)2-pip),
3.62(t, 4H, O(CH2)2), 7.88 (dd, J = 8.7, 2.0 Hz, 1H, Ar-H), 7.92 (d,
J = 8.7 Hz, 1H, Ar-H), 8.12 (s, 1H, Ar-H); 13C NMR (101 MHz, DMSO)
δ/ppm: 24.14 (CH2-pip), 25.63(C-(CH2)2-pip), 46.44 (N(CH2)2-morph),
50.04 (N(CH2)2-pip), 65.73 (O(CH2)2), 128.09, 128.14, 128.31, 132.06,
136.40, 142.54, 143.20 (CleC]N), 154.04 (NeC]N); Anal. Calcd. for
C
17H21ClN4O3S (396.89): C, 51.45; H, 5.33; N, 14.12; Found: C, 51.41;
4.1.7. 6-((4-methylpiperazin-1-yl)sulfonyl)-1,4-dihydroquinoxaline-2,3-
dione (5b)
H, 5.24; N, 14.03.
As light rose crystals; yield: 70%; m.p.: 290–292 °C; IR: ν/
cm−1:3182 (NH), 3047(CH-Ar), 2939, 2835 (CH-aliph.), 1708 (C]O),
1384, 1161(SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.54 (s, 3H,
eNeCH3), 2.88 (t, 4H, N4(CH2)2), 3.10 (t, 4H, N1(CH2)2), 7.31 (d,
J = 8.4 Hz, 1H, Ar-H), 7.44 (dd, J = 8.4, 1.9 Hz, 1H, Ar-H), 7.49 (s, 1H,
Ar-H), 12.07, 12.26 (2 s, 2H, 2NH exchangeable with D2O); Anal. Calcd.
for C13H16N4O4S (324.36): C, 48.14; H, 4.97; N, 17.27; Found: C, 48.06;
H, 4.93; N, 17.15
4.1.12. 3-Chloro-2-(4-methylpiperazin-1-yl)-6-
morpholinosulfonylquinoxaline (7b):
As shiny light brown crystals from acetonitrile/DMF; yield: 76%;
m.p.:208–210 °C; IR: ν/cm−1: 3054 (CH-Ar), 2901, 2864 (CH-aliph.),
1610 (C]N), 1345, 1164 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm:
2.24 (s, 3H, eNeCH3), 2.53 (t, 4H, N4(CH2)2-piperazine), 2.93 (t, 4H, N
(CH2)2-morph), 3.62 [(t, 8H, O(CH2)2 + N1(CH2)2-piperazine), 7.90
(dd, J = 8.8, 1.9 Hz, 1H, Ar-H), 7.94 (d, J = 8.7 Hz, 1H, Ar-H), 8.13 (s,
1H, Ar-H); 13C NMR (101 MHz, DMSO) δ/ppm: 46.44 [(CH3),
(N1(CH2)2-piperazine)], 48.60 (N(CH2)2-morph), 54.35 (N4(CH2)2-pi-
perazine), 65.73 (O(CH2)2), 128.12, 128.33, 128.38, 132.49, 136.61,
142.33, 143.07 (CleC]N), 153.68 (NeC]N); Anal. Calcd. for
4.1.8. 6-(morpholine-4-sulfonyl)-1,4-dihydroquinoxaline-2,3-dione (5c)
A
solution of 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl
chloride (1) (10 mmol, 2.6 g) in dioxane (20 mL), was treated with
(20 mmol, 1.74 mL) morpholine and the resulting mixture was kept
under stirring at room temperature for 2 h. The precipitate formed was
collected by filtration and recrystallized from ethanol/DMF mixture to
give 5c as white crystals.
C
17H22ClN5O3S (411.91): C, 49.57; H, 5.38; N, 17.00; Found: C, 49.51;
H, 5.34; N, 16.85.
4.1.13. 3-Chloro-2-morpholino-6-morpholinosulfonylquinoxaline (7c)
As yellow crystals from acetonitrile; yield: 60%; m.p.:163–165 °C;
IR: ν/cm−1: 3079(CH-Ar), 2964, 2920, 2898, 2859 (CH-aliph.), 1603
(C]N), 1348, 1156 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.92 (t,
4H, N(CH2)2), 2.98 (t, 4H, N(CH2)2), 3.64 (t, 4H, O(CH2)2), 3.80 (t, 4H,
O(CH2)2), 8.16 (dd, J = 8.9,1.9 Hz, 1H, Ar-H), 8.34 (d, J = 8.8 Hz, 1H,
Ar-H), 8.42 (s, 1H, Ar-H); Anal. Calcd. for C16H19ClN4O4S (398.86): C,
48.18; H, 4.80; N, 14.05; Found: C, 48.13; H, 4.76; N, 14.01.
Yield: 90%; m.p.:338–340 °C; IR: ν/cm−1:3253, 3148 (NH),
3069(CH-Ar), 2955, 2863 (CH-aliph.), 1678 (C]O), 1379, 1154 (SO2);
1H NMR (400 MHz, DMSO) δ/ppm: 2.84 (t, 4H, N(CH2)2), 3.62 (t, 4H, O
(CH2)2), 7.30 (d, J = 8.4 Hz, 1H, Ar-H), 7.41 (dd, J = 8.4, 1.9 Hz, 1H,
Ar-H), 7.47 (s, 1H, Ar-H), 12.05, 12.20 (2 s, 2H, 2NH exchangeable
with D2O), 13C NMR (101 MHz, DMSO) δ/ppm: 46.26 (-N(CH2)2),
65.71 (-O(CH2)2), 114.92, 116.12, 122.82, 126.56, 128.65, 130.23,
155.26, 155.63 (2C = O); MS (m/z, %):46(100%), 63 (86%), 73 (50%),
75 (90%), 76 (44%), 77 (50%), 129 (50%), 132 (41%), 309 (M+−2,
10%), 311 (M+, 13%), 312 (M++1, 24%), 314 (M++3, 6%); Anal.
Calcd. for C12H13N3O5S (311.31): C, 46.30; H, 4.21; N, 13.50; Found: C,
46.25; H, 4.15; N, 13.43
4.1.14. 2,3-sym.bis(sec-amino)-6-morpholinosulfonylquinoxaline (9a-c):
Method A:
A solution of compound 6 (10 mmol, 3.48 g) and cyclic secondary
amines (20 mmol) in 30 mL of acetonitrile was refluxed for 8 h. Then,
the solvent was evaporated under reduced pressure. The resulting
precipitate was filtered, dried and recrystallized from the proper sol-
vent.
4.1.9. 2,3-dichloro-6-(morpholinosulfonyl)quinoxaline (6) according
to reported method [51]
Method B:
As off-white needles from acetonitrile; yield: 85%;; m.p.:183–185
°C; IR: ν/cm−1: 3074(CH-Ar), 2962, 2918, 2856 (CH-aliph.), 1600 (C]
N), 1350, 1154 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.99 (t, 4H, N
(CH2)2), 3.62 (t, 4H, O(CH2)2), 8.15 (dd, J = 8.8, 2.0 Hz, 1H, Ar-H),
8.33 (d, J = 8.8 Hz, 1H, Ar-H), 8.39 (s, 1H, Ar-H); 13C NMR (101 MHz,
DMSO) δ/ppm: 46.36 (-N(CH2)2), 65.73 (-O(CH2)2), 128.55, 129.04,
130.24, 137.56, 139.78, 142.18, 147.35, 148.15 (2C-Cl); MS (m/z, %):
49 (53%), 63 (41%), 73 (44%), 74 (57%), 75 (63%), 78 (58%), 104
(69%), 116 (100%), 264 (67%), 265 (52%), 324 (55%), 345 (M+−3,
A mixture of compound 7a-c (10 mmol) and the same secondary
amines (10 mmol) in 30 mL of acetonitrile was refluxed for 6 h. After
the reaction time, the solvent was evaporated, and the resulting pre-
cipitate was washed with H2O, filtered, dried and recrystallized from
the proper solvent.
4.1.15. 6-morpholinosulfonyl-2,3-bispiperidinoquinoxaline (9a)
As pale yellow crystals from dioxane; yield: 70%; m.p.:173–175 °C;
IR: ν/cm−1: 3061(CH-Ar), 2965, 2921, 2859 (CH-aliph.), 1637 (C]N),
1348, 1162 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.52 (m, 6H,
3(CH2)-pip), 1.64 (m, 6H, 3(CH2)-pip), 2.95 (t, 4H, N(CH2)2-morph),
3.45 (t, 4H, N(CH2)2-pip), 3.54 (t, 4H, N(CH2)2-pip), 3.61 (t, 4H, O
13%), 348 (M+
,
10%), 350 (M++2, 4%); Anal. Calcd. for
C
12H11Cl2N3O3S (348.20): C, 41.39; H, 3.18; N, 12.07; Found: C, 41.35;
H, 3.11; N, 11.96.
13

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
(CH2)2), 7.55 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H), 7.71 (d, J = 8.5 Hz, 1H,
Ar-H), 7.81 (s, 1H, Ar-H), 13C NMR (101 MHz, DMSO) δ/ppm: 24.50
(2(CH2)-pip), 25.65 (2(CH2)2-pip), 25.70 (2(CH2)2-pip), 46.46 (N
(CH2)2-morph), 47.72 (N(CH2)2-pip), 47.81 (N(CH2)2-pip), 65.75 (O
(CH2)2), 123.77, 126.05, 126.85, 130.38, 136.54, 140.25, 149.11 (C]
N), 149.53 (N-C]N); Anal. Calcd. for C22H31N5O3S (445.58): C, 59.30;
H, 7.01; N, 15.72; Found: C, 59.23; H, 6.92; N, 15.59.
morph), 47.46 (N(CH2)2-morph), 47.86, 63.72, 65.74 (O(CH2)2), 66.20,
66.79 (O(CH2)2), 124.10, 126.21, 127.03, 130.73, 136.38, 140.38,
148.68 (C]N), 149.52 (C]N); Anal. Calcd. for C21H29N5O4S (447.55):
C, 56.36; H, 6.53; N, 15.65; Found: C, 56.32; H, 6.49; N, 15.59.
4.1.21. 2-(4-methylpiperazin-1-yl)-3-(piperidin-1-yl)-6-morpholinosulf-
onylquinoxaline (10c)
As off-white powder from acetonitrile; yield: 75%; m.p.:220–222 °C;
IR: ν/cm−1: 3054(CH-Ar), 2920, 2852 (CH-aliph.), 1595 (C]N), 1337,
1160 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.64 (m, 6H, 3(CH2)-
pip), 2.26 (s, 3H, CH3), 2.48 (t, 4H, N4(CH2)2-piperazine), 2.89 (t, 4H, N
(CH2)2-morph), 2.98 (t, 4H, N1(CH2)2-piperazine), 3.46 (t, 4H, N
(CH2)2-pip), 3.62 (t, 4H, O(CH2)2), 7.58 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H),
7.73 (d, J = 8.6 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H); MS (m/z, %): 42
(34%), 53 (32%), 63 (100%), 75 (58%), 88 (94%), 143 (61%), 460
(M+, 9%); Anal. Calcd. for C22H32N6O3S (460.60): C, 57.37; H, 7.00; N,
18.25; Found: C, 57.34; H, 6.98; N, 18.20.
4.1.16. 2,3-bis(4-methylpiperazine-1-yl)-6-morpholinosulfonylquinoxaline
(9b)
As pale yellow powder from acetonitrile; yield: 61%; m.p.:148–150
°C; IR: ν/cm−1: 3059(CH-Ar), 2906, 2853 (CH-aliph.), 1603 (C]N),
1354, 1156 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.22 (s, 6H,
2(CH3)), 2.48 (t, 8H, 2[N4(CH2)2-piperazine]), 2.89 (t, 4H, N(CH2)2-
morph), 3.48 (t, 4H, N1(CH2)2-piperazine), 3.55 (t, 4H, N1(CH2)2-piper-
azine), 3.62 (t, 4H, O(CH2)2), 7.60 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H), 7.75
(d, J = 8.6 Hz, 1H, Ar-H), 7.86 (s, 1H, Ar-H); 13C NMR (101 MHz,
DMSO) δ/ppm: 44.75 (2(CH3)), 46.15 (N(CH2)2-morph), 46.46
(N1(CH2)2-piperazine), 46.87 (N1(CH2)2-piperazine), 54.64 (2[N4(CH2)2-
piperazine]), 65.75 (O(CH2)2), 124.11, 126.28, 127.18, 130.94, 136.59,
140.17, 148.81 (C]N), 149.30 (C]N); Anal. Calcd. for C22H33N7O3S
(475.61): C, 55.56; H, 6.99; N, 20.62; Found: C, 55.52; H, 6.95; N, 20.78
4.1.22. 2-(4-methylpiperazin-1-yl)-3-morpholino-6-morpholinosulf-
onylquinoxaline (10d)
As off-white needles from acetonitrile; yield: 76%; m.p.:140–142 °C;
IR: ν/cm−1: 3048(CH-Ar), 2918, 2862 (CH-aliph.), 1610 (C]N), 1349,
1165 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.27 (s, 3H, CH3), 2.54
(t, 4H, N4(CH2)2-piperazine), 2.91 (t, 4H, N(CH2)2-morph), 3.51 (t, 4H,
N(CH2)2-morph), 3.64 (t, 8H, 2[O(CH2)2]), 3.80 (t, 4H, N1(CH2)2-pi-
perazine), 7.63 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H), 7.78 (d, J = 8.6 Hz, 1H,
Ar-H), 7.89 (s, 1H, Ar-H); 13C NMR (101 MHz, DMSO) δ/ppm: 43.56
(-N-CH3), 45.87, 46.43 (N1(CH2)2-piperazine), 46.66 (N(CH2)2-morph),
47.54 (N(CH2)2-morph), 54.37 (N4(CH2)2-piperazine), 65.73 (O
(CH2)2), 66.08 (O(CH2)2), 124.21, 126.31, 127.22, 131.10, 136.54,
140.19, 148.68 (C]N), 149.20 (C]N); Anal. Calcd. for C21H30N6O4S
(462.57): C, 54.53; H, 6.54; N, 18.17; Found: C, 54.46; H, 6.47; N,
18.11.
4.1.17. 2,3-bis(morpholino)-6-morpholinosulfonylquinoxaline (9c)
As yellow crystals from acetonitrile; yield: 70%; m.p.:198–200 °C;
IR: ν/cm−1: 3062(CH-Ar), 2947, 2873, 2856 (CH-aliph.), 1620 (C]N),
1344, 1161 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.87 (t, 4H, N
(CH2)2), 3.51 (t, 4H, N(CH2)2), 3.58 (t, 4H, N(CH2)2), 3.62 (t, 4H, O
(CH2)2), 3.76 (t, 8H, 2O(CH2)2), 7.64 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H),
7.78 (d, J = 8.6 Hz, 1H, Ar-H), 7.88 (s, 1H, Ar-H); MS (m/z, %): 119
(54%), 151 (100%), 214 (64%), 381 (43%), 447 (M+−2, 33%), 449
(M+, 31%); Anal. Calcd. for C20H27N5O5S (449.53): C, 53.44; H, 6.05;
N, 15.58; Found: C, 53.40; H, 5.97; N, 15.52
4.1.18. 2,3-unsym.bis(sec-amino)-6-morpholinosulfonylquinoxaline (10a-
f):
A mixture of compound 7a-c (10 mmol) and requisite cyclic sec-
ondary amine (10 mmol) in 30 mL of acetonitrile was refluxed for 6 h.
The reaction mixture was cooled and solid obtained after cooling was
filtered, washed with acetonitrile, dried and recrystallized from the
proper solvent.
4.1.23. 2-Mopholino-6-morpholinosulfonyl-3-piperidinoquinoxaline (10e)
As yellow crystals from acetonitrile; yield: 80%; m.p.:128–130 °C;
IR: ν/cm−1: 3048(CH-Ar), 2918, 2862 (CH-aliph.), 1610 (C]N), 1349,
1165 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.62 (m, 6H, 3(CH2)-
pip), 2.98 (t, 4H, N(CH2)2-morph), 3.45 (t, 4H, N(CH2)2-morph), 3.53(t,
4H, N(CH2)2-pip), 3.64 (t, 4H, O(CH2)2), 3.80 (t, 4H, O(CH2)2), 7.61
(dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 7.87 (s,
1H, Ar-H); 13C NMR (101 MHz, DMSO) δ/ppm: 24.48, 25.47, 25.70 (C-
(CH2)3-pip), 46.47 (N(CH2)2-morph), 47.40, 47.72 (N(CH2)2-morph),
47.81, 47.95 (N(CH2)2-pip), 65.75 (O(CH2)2), 66.24 (O(CH2)2), 123.94,
126.20, 127.13, 130.92, 136.79, 140.04, 149.17 (C]N), 149.22 (C]
N); Anal. Calcd. for C21H29N5O4S (447.55): C, 56.36; H, 6.53; N, 15.65;
Found: C, 56.30; H, 6.57; N, 15.71
4.1.19. 3-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)-6-morpholinosulf-
onylquinoxaline (10a):
As yellow crystals from ethanol/acetonitrile; yield: 80%;
m.p.:213–215 °C; IR: ν/cm−1: 3064(CH-Ar), 2957, 2894, 2854 (CH-
aliph.), 1617 (C]N), 1344, 1160 (SO2); 1H NMR (400 MHz, DMSO) δ/
ppm: 1.65 (m, 6H, 3(CH2)-pip), 2.24 (s, 3H, CH3), 2.50 (t, 4H,
N4(CH2)2-piperazine), 2.90 (t, 4H, N(CH2)2-morph), 3.50 (t, 4H,
N1(CH2)2-piperazine), 3.55 (t, 4H, N(CH2)2-pip), 3.64 (t, 4H, O(CH2)2),
7.60 (dd, J = 8.6, 2.1 Hz, 1H, Ar-H), 7.74 (d, J = 8.6 Hz, 1H, Ar-H),
7.85 (s, 1H, Ar-H); MS (m/z, %): 58 (32%), 73 (32%), 90 (34%), 117
(66%), 144 (100%), 159 (92%), 318 (28%), 460 (M+, 8%); Anal. Calcd.
for C22H32N6O3S (460.60): C, 57.37; H, 7.00; N, 18.25; Found: C, 57.33;
H, 6.94; N, 18.20.
4.1.24. 3-(4-Methylpiperazin-1-yl)-2-morpholino-6-morpholinosulf-
onylquinoxaline (10f)
As yellow crystals from ethanol; yield: 77%; m.p.:180–182 °C; IR: ν/
cm−1: 3042(CH-Ar), 2959, 2920, 2854 (CH-aliph.), 1599 (C]N), 1350,
1152 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 2.24 (s, 3H, -N-CH3),
2.51 (t, 4H, N4(CH2)2-piperazine), 2.93 (t, 4H, N(CH2)2-morph), 3.47 (t,
4H, N(CH2)2-morph), 3.56(t, 4H, N1(CH2)2-piperazine), 3.64 (t, 4H, O
(CH2)2), 3.78(t, 4H, O(CH2)2), 7.63 (dd, J = 8.5, 2.0 Hz, 1H, Ar-H),
7.81 (d, J = 8.6 Hz, 1H, Ar-H), 7.89 (s, 1H, Ar-H); 13C NMR (101 MHz,
DMSO) δ/ppm: 46.13 (-N-CH3), 46.46 (N(CH2)2-morph), 46.92
(N1(CH2)2-piperazine), 47.49 (N(CH2)2-morph), 54.54 (N4(CH2)2-pi-
perazine), 65.75 (O(CH2)2), 66.12 (O(CH2)2), 124.17, 126.32, 127.27,
131.10, 136.69, 140.12, 148.81 (C]N), 149.19 (C]N); Anal. Calcd.
for C21H30N6O4S (462.57): C, 54.53; H, 6.54; N, 18.17; Found: C, 54.48;
H, 6.50; N, 18.12
4.1.20. 3-morpholino-2-(piperidin-1-yl)-6-morpholinosulfonylquinoxaline
(10b)
As yellow crystals from acetonitrile; yield: 73%; m.p.:113–115 °C;
IR: ν/cm−1: 3035 (CH-Ar), 2988, 2958, 2894, 2853 (CH-aliph.), 1628
(C]N), 1344, 1161 (SO2); 1H NMR (400 MHz, DMSO) δ/ppm: 1.62 (m,
6H, 3(CH2)-pip), 2.87 (t, 4H, N(CH2)2-morph), 3.49 (t, 4H, N(CH2)2-
morph), 3.55 (t, 4H, O(CH2)2), 3.60 (t, 4H, N(CH2)2-pip), 3.77 (t, 4H, O
(CH2)2), 7.59 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 7.74 (d, J = 8.6 Hz, 1H,
Ar-H), 7.85 (s, 1H, Ar-H), 13C NMR (101 MHz, DMSO) δ/ppm: 24.47
((CH2) -pip), 25.50 (2(CH2)-pip), 43.28 (N(CH2)2-pip), 46.44 (N(CH2)2-
14

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
4.1.25. 3-Hydrazino-2-sec.amino-6-morpholinosulfonylquinoxalines (11a-
c):
J = 7.3 Hz, 2H, Ar-H), 7.47 (d, J = 7.5 Hz, 1H, Ar-H), 7.50 (s, 1H, Ar-
H), 7.61 (d, J = 7.9 Hz, 1H, Ar-H), 10.70 (s, 1H, NH, exchangeable with
D2O); Anal. Calcd. for C24H27N7O3S (493.59): C, 58.40; H, 5.51; N,
19.86; Found: C, 58.34; H, 5.45; N, 19.82.
To a mixture of compound 7a-c (10 mmol), hydrazine hydrate
(20 mmol), 25 mL of acetonitrile was added and refluxed for 6 h. After
the reaction time, the solvent was evaporated under reduced pressure,
and the resultant solid was washed with H2O and dried. The obtained
precipitate was recrystallized from the proper solvent.
4.1.30. 4-((3-(2-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-
2-(piperidin-1-yl)-6-morpholinosulfonylquinoxaline (13)
A mixture of 3-hydrazino quinoxaline derivatives 11a (10 mmol,
3.92 g) and 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (10 mmol,
2.48 g) was refluxed in acetonitrile (20 mL) in presence of few drops of
acetic acid for 8 h. The progress of reaction was monitored by TLC.
Upon completion of reaction, the reaction mixture was cooled. The
product precipitated out was filtered, washed with acetonitrile and
purified by recrystallization from acetonitrile to give compound 13 as
yellow crystals.
4.1.26. -Hydrazino-2-piperidino-6-morpholinosulfonylquinoxaline (11a)
As yellow crystals from acetonitrile; yield: 85%; m.p.: 280–282 °C;
IR: ν/cm−1: 3317, 3201 (NH2, NH), 3070(CH-Ar), 2931, 2854 (CH-
aliph.), 1608 (C]N), 1346, 1161 (SO2); 1H NMR (400 MHz, DMSO) δ/
ppm: 1.55–1.71 (m, 6H, 3(CH2)-pip), 2.86 (t, 4H, N(CH2)2-morph),
3.64(t, 4H, N(CH2)2-pip), 3.88 (t, 4H, O(CH2)2), 4.63 (s, 2H, NH2 ex-
changeable with D2O), 7.10 (d, J = 8.2 Hz, 1H, Ar-H), 7.35 (s, 1H, Ar-
H), 7.72 (d, J = 8.8 Hz, 1H, Ar-H), 8.34 (s, 1H, NH exchangeable with
D2O); 13C NMR (101 MHz, DMSO) δ/ppm: 24.93 ((CH2) -pip), 26.32
(2(CH2) -pip), 46.42 (N(CH2)2-morph), 48.15 (N(CH2)2-pip), 65.77 (O
(CH2)2), 111.80, 120.37, 124.22, 127.64, 130.99, 131.88, 137.45,
152.71(C]N); Anal. Calcd. for C17H24N6O3S (392.48): C, 52.03; H,
6.16; N, 21.41; Found: C, 51.97; H, 6.11; N, 21.38
Yield: 57%; mp: 223–225 °C; IR: ν/cm−1: 3417 (NH), 3047 (CH-Ar),
2931, 2850 (CH-aliph), 1597 (C]N), 1350, 1165 (SO2); 1H NMR
(400 MHz, DMSO) δ/ppm: 1.69 (m, 2H, CH2-pip), 1.74–1.81 (m, 4H, C-
(CH2)2-pip), 2.93 (t, 4H, N(CH2)2-morph), 3.27 (t, 4H, N(CH2)2-pip),
3.63 (t, 4H, O(CH2)2-morph),7.29 (s, 2H, Ar-H), 7.43–7.45 (m, 3H, Ar-
H), 7.50 (t, J = 6.4 Hz, 2H, Ar-H), 7.59 (t, J = 5.0 Hz, 2H, Ar-H), 7.64
(s, 1H), 7.69 (s, 2H, Ar-H), 7.93 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H, Ar-H),
8.26 (s, 1H, Ar-H), 9.04 (s, 1H, NH exchangeable with D2O); 13C NMR
(101 MHz, DMSO) δ/ppm: 24.58 (CH2-pip), 26.46 ((CH2)2-pip), 46.36
(N(CH2)2-morph), 57.27 (N(CH2)2-pip), 65.76 (O(CH2)2-morph),
116.00, 118.82, 119.43, 122.95, 123.66, 126.37, 126.66, 126.74,
126.92, 127.31, 127.81, 129.01, 129.20, 129.40, 130.04, 130.30,
130.75, 131.56, 140.83, 141.32, 146.99(C]N), 147.06(C]N),
148.89(C]N), 150.78 (C]N); MS (m/z, %): 45 (100%), 64 (62%), 69
(44%), 80 (48%), 93 (55%), 130 (86%), 301 (43%), 622 (M+, 13%);
Anal. Calcd. For C33H34N8O3S (622.75): C, 63.65; H, 5.50; N, 17.99;
Found: C, 63.61; H, 5.48; N, 17.94.
4.1.27. 3-Hydrazino-2-(4-methylpiperazine)-6-
morpholinosulfonylquinoxaline (11b)
As pale yellow crystals from acetonitrile; yield: 65%; m.p.: 265–267
°C; IR: ν/cm−1: 3338, 3228 (NH2, NH), 3068(CH-Ar), 2912, 2864 (CH-
aliph.), 1601 (C]N), 1353, 1158 (SO2); 1H NMR (400 MHz, DMSO) δ/
ppm: 2.22 (s, 3H, -N-CH3), 2.49 (t, 4H, N4(CH2)2-piperazine), 2.93 (t,
4H, N(CH2)2), 3.04 (t, 4H, N1(CH2)2-piperazine), 3.63 (t, 4H, O(CH2)2),
4.39 (s, 2H, NH2 exchangeable with D2O), 5.70 (s, 1H, NH exchange-
able with D2O), 7.73 (d, 2H, Ar-H), 8.27 (s, 1H, Ar-H); MS (m/z, %): 41
(59%), 55 (100%), 69 (53%), 83 (85%), 136 (82%), 143 (51%), 194
(52%), 245 (57%), 258 (60%), 262 (53%), 387 (54%), 406 (M+−1,
28%); Anal. Calcd. for C17H25N7O3S (407.49): C, 50.11; H, 6.18; N,
24.06; Found: C, 50.05H, 6.14; N, 23.95
4.1.31. 3-(3-Methyl-2,4-dihydro-5H-pyrazol-5-one-1-yl)-6-
morpholinosulfonyl-2-piperidinoquino xaline (14)
A solution of 3-hydrazino quinoxaline derivatives 11a (10 mmol,
3.92 g) and ethyl acetoacetate (10 mmol, 1.3 mL) was heated on a
boiling water bath in a fume cupboard for 15 min. with occasional
stirring. The heavy syrup was allowed to cool, and 15–20 mL of ether
was added and stirred the mixture vigorously to get crystalline the
desired product. The precipitate was filtered, washed thoroughly with
ether and then recrystallized from ethanol / dioxane to get yellow
crystals of (14).
4.1.28. 3-Hydrazino-2-(morpholino)-6-morpholinosulfonylquinoxaline
(11c)
As yellow crystals from acetonitrile; yield: 72%; m.p.: 273–275 °C;
IR: ν/cm−1: 3331, 3211 (NH2, NH), 3052(CH-Ar), 2969, 2901, 2857
(CH-aliph.), 1594 (C]N), 1340, 1150 (SO2); 1H NMR (400 MHz,
DMSO) δ/ppm: 2.93 (t, 4H, N(CH2)2), 3.06 (t, 4H, N(CH2)2), 3.64 (t,
4H, O(CH2)2), 3.80 (t, 4H, O(CH2)2), 4.41 (s, 2H, NH2 exchangeable
with D2O), 5.73 (s, 1H, NH exchangeable with D2O), 7.76 (d, 2H, Ar-H),
8.29 (s, 1H, Ar-H); 13C NMR (101 MHz, DMSO) δ/ppm: 46.36 (N
(CH2)2), 47.38 (N(CH2)2), 65.76 (O(CH2)2), 66.67 (O(CH2)2), 126.41,
126.60, 126.99, 128.04, 140.90, 147.30, 148.93(C]N); MS (m/z, %):
137 (50%), 149 (44%), 171 (67%), 178 (100%), 183 (93%), 216 (49%),
223 (59%), 248 (66%), 178 (100%), 258 (45%), 269 (58%), 283 (55%),
296 (83%), 324 (93%), 359 (62%), 368 (53%), 369 (60%), 374 (43%),
394 (M+, 21%); Anal. Calcd. for C16H22N6O4S (394.45): C, 48.72; H,
5.62; N, 21.31; Found: C, 48.65; H, 5.57; N, 21.26.
Yield: 48%; m.p.: 290–292 °C; IR: ν/cm−1: 3414 (OH), 3070 (CH-
Ar), 2924, 2854 (CH-aliph), 1616 (C]N), 1342, 1156 (SO2); 1H NMR
(400 MHz, DMSO) δ/ppm: 1.69 (m, 6H, 3(CH2)-pip), 2.93 (t, 4H, N
(CH2)2-morph), 3.03 (s, 3H, CH3), 3.63 (t, 8H, [O(CH2)2] + [N(CH2)2-
pip]), 7.58 (d, J = 8.6 Hz, 1H, Ar-H), 7.71 (s, 1H, CH), 7.81 (dd,
J = 8.6,1.8 Hz, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 12.41 (s, 1H, OH ex-
changeable with D2O); 13C NMR (101 MHz, DMSO) δ/ppm: 14.17
(CH3), 23.63 (CH2-pip), 25.37 ((CH2)2-pip), 45.40 (N(CH2)2-morph),
64.80 (N(CH2)2-pip), 65.76 (O(CH2)2-morph), 115.05, 122.72, 125.41,
126.37, 127.69, 132.72, 139.88, 146.04, 147.95, 149.05,
151.31(]CeOH); MS (m/z, %): 43 (92%), 105 (100%), 135 (83%), 178
(53%), 202 (47%), 350 (43%), 439 (41%), 458 (M+, 21%), 461 (M+
+3, 48%); Anal. Calcd. for C21H26N6O4S (458.54): C, 55.01; H, 5.72; N,
18.33; Found: C, 54.96; H, 5.68; N, 18.30.
4.1.29. 8-(morpholinosulfonyl)-N-phenyl-4-(piperidin-1-yl)-[1,2,4]
triazolo[4,3-a]quinoxalin-1-amine (12)
To a mixture of 3-hydrazino quinoxaline derivatives 11a (10 mmol,
3.92 g) dissolved in (20 mL) of acetonitrile and few drops of TEA,
(10 mmol, 1.35 mL) of phenyl isothiocyanate was added drop wise with
stirring. The mixture was refluxed for 6 h, then cooled and the resulting
solid was filtered, dried and recrystallized from dioxane to give white
powder of product 12.
4.2. Biological activity
4.2.1. Antimicrobial activity
Yield: 65%; m.p.: 185 °C (deco.); IR: ν/cm−1: 3248 (NH), 3045 (CH-
Ar), 2978, 2939, 2881 (CH-aliph), 1612 (C]N), 1374, 1172 (SO2); 1H
NMR (400 MHz, DMSO) δ/ppm: 1.18–1.20 (m, 6H, 3(CH2)-pip), 3.02 (t,
4H, N(CH2)2-morph), 3.07 (t, 4H, N(CH2)2-pip), 3.56 (t, 4H, O(CH2)2),
6.90 – 6.94 (m, 1H, Ar-H), 7.27 – 7.31 (m, 2H, Ar-H), 7.39 (d,
Gram-positive (Bacillus subtilis ATCC 6633, Staphylococcus aureus
ATCC 29213 and Enterococcus faecalis ATCC 29212), Gram-negative
(Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and
Salmonella typhi ATCC 6539), Candida albicans (ATCC 10231) and
Fusarium oxysporum (RCMB 008002) were used in our In vitro
15

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
antimicrobial activity evaluations at the bacteriology laboratory,
Botany and Microbiology Department, Faculty of Science, Al-Azhar
University, Cairo, Egypt. The antimicrobial potential of newly synthe-
sized organic compounds was investigated towards the tested micro-
organisms and expressed as the diameter of the inhibition zones ac-
cording to the agar plate diffusion method [55]. Briefly, 100 µL of the
test bacteria/fungi were grown in 10 mL of fresh media until they
reached a count of approximately 108 cells/mL for bacteria or 105 cells/
mL for fungi. One mL of each sample (at 0.5 mg/mL) was added to each
well (10 mm diameter holes cut in the agar gel). The plates were in-
cubated for 24 h at 37 °C (for bacteria and yeast) and for 72 h at 27 °C
(for filamentous fungi), each test was repeated three times. After in-
cubation, the microorganism's growth was observed. Tetracycline was
used as standard antibacterial drugs while Amphotericin B was used as
standard antifungal drug. The resulting inhibition zone diameters were
measured in millimeters and used as criterion for the antimicrobial
activity. Solvent controls (DMSO) were included in every experiment as
negative control. DMSO was used for dissolving the tested compounds
and showed no inhibition zones, confirming that it has no influence on
growth of the tested microorganisms.
4.3. Molecular docking study
PDB code: 4DUH was obtained from Protein Data Bank. 4DUH re-
presents the crystal structure for a complex between a small molecule
and DNA gyrase of E. coli, 24 kDa domain [68]. Refinement process was
achieved by removal of water chains, then protonation step and energy
minimization were done using Merck Molecular force field (MMff 94x),
finally detecting the binding site was attained by Molecular Operating
Environment docking tool 10.2008 (MOE). The selected compounds
were drawn as 2D by Chem Draw, saved as mol files, retrieved by MOE,
protonated and subjected to energy minimization then saved as mdb
file to be ready for docking simulation. First, we started with verifica-
tion step by re-docking the co-crystallized ligand into DNA Gyrase
binding site at RMDS value equal 1.44, the docking score energy is
−15.77 Kcal/mol. 2D and 3D figures for the verification process are
provided in the supplementary data file. After that the saved mdb file
for the selected compounds was used for docking simulation into DNA
Gyrase binding site, the obtained data are represented in Table S1 and
Figs. 2A-5B, in addition to the other figures that are supplied in sup-
plementary data files.
4.2.2. Evaluating the minimal inhibition concentration (MIC)
Declaration of Competing Interest
A conventional technique termed paper disk diffusion was used to
investigate the MIC of the active compounds [56,60–62] through em-
ploying a 12.7 mm diameter filter paper (Whatman, Germany). Bacteria
were grown in a media of nutrient agar, while fungi and yeasts were
The authors declared that there is no conflict of interest.
Appendix A. Supplementary material
́
grown in a media of Sabourauds agar. The synthesized compounds were
dissolved and loaded on paper disks with different concentrations.
Loading the drying disks over the agar plates' surface inoculated with
the selected microorganisms was carried out, then growth inhibition
was tested when incubated (at 37 °C for a day) for the bacterial strains
and yeasts and fungi (at 27 °C for three days); Also, MIC confirmed by
using the broth micro-dilution procedure described in the Clinical and
Laboratory Standards Institute (CLSI) guidelines [63]. Where repeating
of every experiment for three-times was performed for reproducibility.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104164.
References
[1] N. Nayak, T.C. Nag, G. Satpathy, S.B. Ray, Ultrastructural analysis of slime positive
& slime negative Staphylococcus epidermidis isolates in infectious keratitis, Ind. J.
Med. Res. 125 (2007) 767–771.
[2] A. Masunari, L.C. Tavares, A new class of nifuroxazide analogues: Synthesis of 5-
nitrothiophene derivatives with antimicrobial activity against multidrug-resistant
Staphylococcus aureus, Bioorg. Med. Chem. 15 (12) (2007) 4229–4236, https://doi.
org/10.1016/j.bmc.2007.03.068.
4.2.3. Multidrug resistant bacteria (MDRB) assay
The test microorganisms that were used in the present study are
multidrug resistant Gram-positive Strains, (Staphylococcus aureus
ATCC 43300 and Staphylococcus aureus ATCC 33591), multidrug re-
sistant Gram-negative (Escherichia coli ATCC BAA-196 and
Pseudomonas aeruginosa ATCC BAA-2111), they were used to define
the antimicrobial activity of the newly synthesized organic compounds.
Antimicrobial screening and MICs were performed by the same method
mention before [55–56,64–65] except we used both Norfloxacin, and
Tetracycline as the positive control.
[3] G.W. Kaatz, F. McAleese, S.M. Seo, Multidrug resistance in Staphylococcus aureus
due to overexpression of a novel multidrug and toxin extrusion (MATE) transport
protein, Antimicrob. Agents and Chem. 49 (5) (2005) 1857–1864, https://doi.org/
10.1128/aac.49.5.1857-1864.2005.
[4] J. Strahilevitz, Q.C. Truong-Bolduc, D.C. Hooper, DX-619, a Novel Des-Fluoro(6)
Quinolone Manifesting Low Frequency of Selection of Resistant Staphylococcus
aureus Mutants: Quinolone Resistance beyond Modification of Type II
Topoisomerases, Antimicrob. Agents and Chem. 49 (12) (2005) 5051–5057,
https://doi.org/10.1128/aac.49.12.5051-5057.2005.
[5] L.B. Jensen, S. Baloda, M. Boye, F.M. Aarestrup, Antimicrobial resistance among
Pseudomonas spp. and the Bacillus cereus group isolated from Danish agricultural
soil, Environ. Int. 26 (2001) 581–587, https://doi.org/10.1016/s0160-4120(01)
00045-9.
4.2.4. DNA gyrase
[6] K.K.Y. Wong, R.E.W. Hancock, Insertion Mutagenesis and Membrane Topology
Model of the Pseudomonas aeruginosa Outer Membrane Protein OprM, J.
Bacteriology 182 (9) (2000) 2402–2410, https://doi.org/10.1128/jb.182.9.2402-
2410.2000.
The In vitro enzyme inhibition determination for the most active
compounds, were carried out in the confirmatory diagnostic unit,
Vacsera, Egypt. The evaluation performed through profiling of the
tested compounds against S. aureus DNA gyrase according to the re-
ported method [66].
[7] D. Edoh, B. Alomatu, Comparison of antibiotic resistance patterns between la-
boratories in Accra East, Ghana, African J. Sci. Tech. 8 (1) (2007) 1-7. https://
www.ajol.info/index.php/ajst/article/view/155912/145540.
[8] J.I. Lundin, D.A. Dargatz, B.A. Wagner, J.E. Lombard, A.E. Hill, S.R. Ladely,
P.J. Fedorka-Cray, Antimicrobial Drug Resistance of Fecal Escherichia coli and
Salmonella spp, Isolates United States Dairy Cows, Foodborne Pathogens Disease 5
(1) (2008) 7–19, https://doi.org/10.1089/fpd.2007.0018.
4.2.5. Studying the activity of the intracellular killing
The activity of the intracellular killing was studied through a nitro-
blue tetrazolium (NBT) reduction method through the developed
Baehner and Nathan technique [67]. In HBSS, the isolated neutrophils
and the tested compound were incubated at 37 °C (for half an hour)
followed by extracting the blue formazon (reduced dye) through pyr-
idine and the spectrophotometric measurements were carried out at
515 nm. A negative control sample (which includes all the reagents
without the neutrophil suspension) was used for comparative studies.
Absorbance variation between the negative control and the cell cultures
which actively phagocyte latex particles express the index of the neu-
trophils' intracellular killing activity.
[9] J.H. Kang, M.S. Lee, Characterization of a bacteriocin produced by Enterococcus
faecium GM-1 isolated from an infant, J. Applied Micro. 98 (5) (2005) 1169–1176,
https://doi.org/10.1111/j.1365-2672.2005.02556.x.
[10] S. Kumar, S. Kohlhoff, G. Valencia, M.R. Hammerschlag, R. Sharma, Treatment of
vancomycin-resistant Enterococcus faecium ventriculitis in a neonate, Inter. J.
Antimicro. Agents 29 (6) (2007) 740–741, https://doi.org/10.1016/j.ijantimicag.
2006.11.025.
[11] D. Goldblatt, A.J. Thrasher, Chronic granulomatous disease. Immunodeficiency
review, Clin. Exp. Immunol. 122 (1) (2000) 1–9, https://doi.org/10.1046/j.1365-
2249.2000.01314.x.
[12] J. Sonntag, D. Kaczmarek, G. Brinkmann, G. Kammler, H.H. Hellwege,
Complicating neonatal Escherichia coli meningitis, Z. Geburtshilfe Neonatol. 208
16

Y.A. Ammar, et al.
(1) (2004) 32–35, https://doi.org/10.1055/s-2004-815521.
BioorganicChemistry104(2020)104164
dihydroquinolines as potent inhibitors of Mycobacterium tuberculosis, Eur. J. Med.
Chem. 164 (2019) 171–178, https://doi.org/10.1016/j.ejmech.2018.12.043.
[37] V. Kumar, S. Patel, R. Jain, New structural classes of antituberculosis agents, Med.
Res. Rev. 38 (2) (2017) 684–740, https://doi.org/10.1002/med.21454.
[38] G. Fernandes, C. Man Chin, J. Dos Santos, Advances in Drug Discovery of New
Antitubercular Multidrug-Resistant Compounds, Pharmaceuticals 10 (4) (2017) 51,
https://doi.org/10.3390/ph10020051.
[13] A.S. Puerto, J.G. Fernández, J.de D.L. del Castillo, M.J.S. Pino, G.P. Angulo, In vitro
activity of β-lactam and non–β-lactam antibiotics in extended-spectrum β-lacta-
mase–producing clinical isolates of Escherichia coli, Diag. Micro. Inf. Dis. 54 (2)
(2006) 135–139, https://doi.org/10.1016/j.diagmicrobio.2005.08.018.
[14] C.M. Nolan, E.G. Chalhub, D.G. Nash, T. Yamauchi, Treatment of Bacterial
Meningitis with Intravenous Amoxicillin, Antimicro. Agents Chem. 16 (2) (1979)
171–175, https://doi.org/10.1128/aac.16.2.171.
[39] R. Norrby, Linezolid-a review of the first oxazolidinone, Expert Opin.
Pharmacother. 2 (2) (2001) 293–302, https://doi.org/10.1517/14656566.2.2.293.
[40] J. Dotis, E. Iosifidis, M. Ioannidou, E. Roilides, Use of linezolid in pediatrics: a
critical review, Inter. J. Infec. Dise. 14 (8) (2010) 638–648, https://doi.org/10.
1016/j.ijid.2009. 10.002.
[15] A. Kamal, M.N.A. Khan, K.S. Reddy, K. Rohini, G.N. Sastry, B. Sateesh, B. Sridhar,
Synthesis, structure analysis, and antibacterial activity of some novel 10-substituted
2-(4-piperidyl/phenyl)-5,5-dioxo[1,2,4]triazolo[1,5-b][1,2,4]benzothiadiazine de-
rivatives, Bioorg. Med. Chem. Let. 17 (19) (2007) 5400–5405, https://doi.org/10.
1016/j.bmcl.2007.07.043.
[41] Y.A. Ammar, A.A. Farag, A.M. Ali, S.A. Hessein, A.A. Askar, E.A. Fayed, D.M. Elsisi,
A. Ragab, Antimicrobial evaluation of thiadiazino and thiazolo quinoxaline hybrids
as potential DNA gyrase inhibitors; design, synthesis, characterization and mor-
phological studies, Bioorg. Chem. 99 (2020) 103841, , https://doi.org/10.1016/j.
bioorg.2020.103841.
[16] G. W. H. Cheeseman, R.F. Cookson, in: A.Weissberge, E.C. Taylor (Eds.), The
Chemistry of Heterocyclic Compounds: A Series Of Monographs Chapter XXXVII
Pyrazoloquinoxalines, J Wiley and Sons, New York, 35 (1979) 1-27, 35-38.
[17] K. T. Potts, A. R. Katritzky, Comprehensive heterocyclic chemistry: the structure,
reactions, synthesis and uses of heterocyclic compounds, pergamon press, New
York, 3(1984) 157, 197.
[42] H.F. Rizk, M.A. El-Borai, A. Ragab, S.A. Ibrahim, Design, synthesis, biological
evaluation and molecular docking study based on novel fused pyrazolothiazole
scaffold, J. Iran. Chem. Soc. (2020), https://doi.org/10.1007/s13738-020-01944-9.
[43] M.A.M.S. El-Sharief, S.Y. Abbas, M.A. Zahran, Y.A. Mohamed, A. Ragab,
Y.A. Ammar, New 1,3-diaryl-5-thioxo-imidazolidin-2,4-dione derivatives:
Synthesis, reactions and evaluation of antibacterial and antifungal activities,
Zeitschrift Für Naturforschung B 71 (8) (2016) 875–881, https://doi.org/10.1515/
znb-2016-0054.
[18] E.A. Fayed, Y.A. Ammar, A. Ragab, N.A. Gohar, A.B.M. Mehany, A.M. Farrag, In
vitro cytotoxic activity of thiazole-indenoquinoxaline hybrids as apoptotic agents,
design, synthesis, physicochemical and pharmacokinetic studies, Bioorg. Chem. 100
(2020) 103951, , https://doi.org/10.1016/j.bioorg.2020.103951.
[19] T. Korzybski, Z. Kowszyk-Gindifer, W. Kurylowicz, Antibiotics: origin, nature and
properties, Pergamon Press, 1967, p. 1.
[20] H. Ishikawa, T. Sugiyama, K. Kurita, A. Yokoyama, Synthesis and antimicrobial
activity of 2,3-bis(bromomethyl)quinoxaline derivatives, Bioorg. Chem. 41–42
(2012) 1–5, https://doi.org/10.1016/j.bioorg.2011.12.002.
[44] A.S. Hassan, A.A. Askar, A.M. Naglah, A.A. Almehizia, A. Ragab, Discovery of New
Schiff Bases Tethered Pyrazole Moiety: Design, Synthesis, Biological Evaluation,
and Molecular Docking Study as Dual Targeting DHFR/DNA Gyrase Inhibitors with
Immunomodulatory Activity, Mol. 25 (11) (2020) 2593, https://doi.org/10.3390/
molecules25112593.
[21] Y.B. Kim, Y.H. Kim, J.Y. Park, S.K. Kim, Synthesis and Biological Activity of New
Quinoxaline Antibiotics of Echinomycin Analogues, Chem. Inform. 35 (19) (2004),
https://doi.org/10.1002/chin.200419208.
[45] Y.A. Ammar, M.A.M.Sh. El-Sharief, M.M. Ghorab, Y.A. Mohamed, A. Ragab,
S.Y. Abbas, New Imidazolidineiminothione, Imidazolidin-2-one and
Imidazoquinoxaline Derivatives: Synthesis and Evaluation of Antibacterial and
Antifungal Activities, Cur. Org. Syn. 13 (3) (2016) 466–475, https://doi.org/10.
2174/1570179412666150817221.
[22] M.A.Z. El-Attar, R.Y. Elbayaa, O.G. Shaaban, N.S. Habib, A.E. Abdel Wahab,
I.A. Abdelwahab, S.A.M. El-Hawash, Design, synthesis, antibacterial evaluation and
molecular docking studies of some new quinoxaline derivatives targeting dihyr-
opteroate synthase enzyme, Bioorg. Chem. 76 (2018) 437–448, https://doi.org/10.
1016/j.bioorg.2017.12.017.
[46] H.-A.S. Abbas, A.R. Al-Marhabi, S.I. Eissa, Y.A. Ammar, Molecular modeling studies
and synthesis of novel quinoxaline derivatives with potential anticancer activity as
inhibitors of c-Met kinase, Bioorg. Med. Chem. 23 (20) (2015) 6560–6572, https://
doi.org/10.1016/j.bmc. 2015.09.023.
[23] P. Sanna, A. Carta, M. Loriga, S. Zanetti, L. Sechi, Synthesis of substituted 2-
ethoxycarbonyl-and 2-carboxyquinoxalin-3-ones for evaluation of antimicrobial
and anticancer activity, IL Farmaco 53 (7) (1998) 455–461, https://doi.org/10.
1016/s0014-827x(98)00044-5.
[47] A. Al-Marhabi, H.-A. Abbas, Y.A. Ammar, Synthesis, Characterization and Biological
Evaluation of Some Quinoxaline Derivatives: A Promising and Potent New Class of
Antitumor and Antimicrobial Agents, Mol. 20 (11) (2015) 19805–19822, https://
doi.org/10.3390/molecules 201119655.
[24] P. Sanna, A. Carta, M. Loriga, S. Zanetti, L. Sechi, Synthesis of 3, 6, 7-substituted-
quinoxalin-2-ones for evaluation of antimicrobial and anticancer activity. Part 2, IL
Farmaco 54 (3) (1999) 161–168, https://doi.org/10.1016/s0014-827x(99)
00010-5.
[48] H. A. S. Abbas, A. R. Al-Marhabi, Y. A. Ammar, Design, synthesis and biological
evaluation of 2,3-disubstituted and fused quinoxalines as potential anticancer and
antimicrobial agents, Acta Pol. Pharm. Drug Res. 74 (2017) 445-458, https://
ptfarm.pl/pub/File/Acta_Poloniae/2017/2/445.pdf.
[25] P. Sanna, A. Carta, M. Loriga, S. Zanetti, L. Sechi, Preparation and biological eva-
luation of 6/7-trifluoromethyl (nitro)-, 6, 7-difluoro-3-alkyl (aryl)-substituted-qui-
noxalin-2-ones. Part 3, IL Farmaco 54 (3) (1999), 169-177. doi: 10.1016/s0014-
827x(99)00011-7.
[49] M.F. El Shehry, S.Y. Abbas, A.M. Farrag, S.I. Eissa, S.A. Fouad, Y.A. Ammar, Design,
synthesis and biological evaluation of quinoxaline N-propionic and O-propionic
hydrazide derivatives as antibacterial and antifungal agents, Medicinal Chem. Res.
27 (2018) 2287–2296, https://doi.org/10.1007/s00044-018-2235-4.
[50] G. Olayiwola, C.A. Obafemi, F.O. Taiwo, Synthesis and neuropharmacological ac-
tivity of some quinoxalinone derivatives, Afr. J. Biotechnol. 6 (6) (2007) 777–786
https://www.ajol.info/index.php/ajb/article/view/56902/45311.
[51] A.M.S. El-Sharief, M.M. Ali, Y.A. Ammar, A.A. Abd El-Salam, Synthesis of some
quinoxaline-6-morpholyl sulphonamide derivatives, Pak. J. Sci. Ind. Res. 39 (1996)
5–10.
[26] A. Carta, P. Sanna, M. Loriga, M.G. Setzu, P. La Colla, R. Loddo, Synthesis and
evaluation for biological activity of 3-alkyl and 3-halogenoalkyl-quinoxalin-2-ones
variously substituted. Part 4, IL Farmaco 57 (1) (2002) 19–25, https://doi.org/10.
1016/s0014-827x(01)01153-3.
[27] B.G. Katzung, A.J. Trevor (Eds.), Basic & clinical pharmacology, McGraw-Hill
Education, New York, 2015, pp. 619–620.
[28] S. Joshi, N. Khosla, P. Tiwari, In vitro study of some medicinally important Mannich
bases derived from antitubercular agent, Bioorg. Med. Chem. 12 (3) (2004)
571–576, https://doi.org/10.1016/j. bmc.2003.11.001.
[29] N. Anand, Sulfonamides and Sulfones. In Burger’s Medicinal Chemistry and Drug
Discovery, Wolff, M. E. Ed., John Wiley & Sons: New York, (1996) 527–573.
[30] C.T. Supuran, A. Scozzafava, A. Casini, Carbonic anhydrase inhibitors, Med. Res.
Rev. 23 (2) (2003) 146–189, https://doi.org/10.1002/med.10025.
[31] Y.A. Ammar, S.Y. Abbas, M.A.M.Sh. El-Sharief, M.A. Salem, A.R. Mohamed,
Synthesis and characterization of new imidazolidineiminothione and bis-imidazo-
lidine-iminothione derivatives as potential antimicrobial agents, Eur. J. Chem. 8 (1)
(2017) 76–81, https://doi.org/10.5155/eurjchem.8.1.76-81.1542.
[32] A.M.S. El-Sharief, Y.A. Ammar, A. Belal, M.A.M.S. El-Sharief, Y.A. Mohamed,
A.B.M. Mehany, G.A.M. Elhag Ali, A. Ragab, Design, synthesis, molecular docking
and biological activity evaluation of some novel indole derivatives as potent an-
ticancer active agents and apoptosis inducers, Bioorg. Chem. 85 (2019) 399–412,
https://doi.org/10.1016/j.bioorg.2019.01.016.
[52] F. Rahim, K. Zaman, H. Ullah, M. Taha, A. Wadood, M.T. Javed, W. Rehman,
M. Ashraf, R. Uddin, I. Uddin, H. Asghar, A.A. Khan, K.M. Khan, Synthesis of 4-
Thiazolidinone Analogs as Potent in Vitro Anti-urease Agents, Bioorg. Chem. 63
(2015) 123–131, https://doi.org/10.1016/j.bioorg.2015.10.005.
[53] R.I. Lehrer, T. Ganz, M.E. Selsted, B.M. Babior, J.T. Curnutte, Neutrophils and host
defense, Ann. Intern. Med. 109 (2) (1988) 127–142, https://doi.org/10.7326/0003-
4819-109-2-127.
[54] H. Wagner, K. Jurcic, Immunologic studies of plant combination preparations. In-
vitro and in-vivo studies on the stimulation of phagocytosis, Arzneimittel-Forschung
41 (1991) 1072–1076 https://europepmc.org/article/med/1799388.
[55] J. G. Cappuccino, N. Sherman, Microbiology, Laboratory Manual. India: Pearson
Education, Inc. New Delhi, (2004) 282–283. ISBN-13: 9780321840226.
[56] K. Cooper, The theory of antibiotic inhibition zones, Analy. Micro. (1963) 1–86,
https://doi.org/10.1016/b978-1-4832-3129-7.50037-3.
[33] Y.A. Ammar, A.M.Sh. El-Sharief, A. Belal, S.Y. Abbas, Y.A. Mohamed,
A.B.M. Mehany, A. Ragab, Design, synthesis, antiproliferative activity, molecular
docking and cell cycle analysis of some novel (morpholinosulfonyl)isatins with
potential EGFR inhibitory activity, Eur. J. Med. Chem. 156 (2018) 918–932,
https://doi.org/10.1016/j.ejmech.2018.06.061.
[57] P. Akbay, A.A. Basaran, U. Undeger, N. Basaran, In vitro immunomodulatory ac-
tivity of flavonoid glycosides from Urtica dioica L, Phyto. Res. 17 (1) (2003) 34–37,
https://doi.org/10.1002/ptr.1068.
[58] A. Başaran, I. Ceritoğlu, Ü. Ündeğer, N. Basaran, Immunomodulatory activities of
some Turkish medicinal plants, Phytotherapy Res.: Inter. J. Dev. Med. Sci. Res.
Plants Plant Products 11 (8) (1997) 609–611, https://doi.org/10.1002/(sici)1099-
1573(199712)11:8<609::aid-ptr165>3.0.co;2-0.
[34] Y.A. Ammar, A.M.Sh. El-Sharief, Y.A. Mohamed, A.B.M. Mehany, A. Ragab,
Synthesis, spectral characterization and pharmacological evaluation of novel thia-
zole-oxoindole hybrid compounds as potent anticancer agent, Al-Azhar Bull. Sci. 29
(2A) (2018) 25–37, https://doi.org/10.21608/ABSB.2018.33767.
[59] J.P. Coleman, C.J. Smith, Microbial Nucleic Acid and Protein Synthesis, Reference
Module Biomed. Sci., Elsevier (2014), https://doi.org/10.1016/B978-0-12-801238-
3.05147-3.
[35] A. Weber, A. Casini, A. Heine, D. Kuhn, C.T. Supuran, A. Scozzafava, G. Klebe,
Unexpected Nanomolar Inhibition of Carbonic Anhydrase by COX-2-Selective
Celecoxib: New Pharmacological Opportunities Due to Related Binding Site
Recognition, J. Med. Chem. 47 (3) (2004) 550–557, https://doi.org/10.1021/
jm030912m.
[60] A.I. El-Batal, M.H. El-Sayed, B.M. Refaat, A.A. Askar, Marine Streptomyces cyaneus
Strain Alex-SK121 Mediated Eco-friendly Marine Streptomyces cyaneus Strain Alex-
SK121 Mediated Eco-friendly Synthesis of Silver Nanoparticles Using Gamma
Radiation, Br. J. Pharm. Res. 4 (21) (2014) 2525–2547, https://doi.org/10.9734/
BJPR/2014/12224.
[36] S.K. Marvadi, V.S. Krishna, D. Sriram, S. Kantevari, Synthesis of novel morpholine,
thiomorpholine and N-substituted piperazine coupled 2-(thiophen-2-yl)
17

Y.A. Ammar, et al.
BioorganicChemistry104(2020)104164
[61] M.M. Ghorab, A.M. Soliman, M.S. Alsaid, A.A. Askar, Synthesis, antimicrobial ac-
tivity and docking study of some novel 4-(4,4-dimethyl-2,6dioxocyclohexylidene)
methylamino derivatives carrying biologically active sulfonamide moiety, Arab. J.
Chem. (2017), https://doi.org/10.1016/j.arabjc.2017.05.022.
Chem. Res. 26 (11) (2016) 2909–2919, https://doi.org/10.1007/s00044-017-
1990-y.
[65] M.A. Salem, A. Ragab, A.A. Askar, A. El-Khalafawy, A.H. Makhlouf, One-pot
synthesis and molecular docking of some new spiropyranindol-2-one derivatives as
immunomodulatory agents and in vitro antimicrobial potential with DNA gyrase
inhibitor, Eur. J. Med. Chem. 188 (2020) 111977, , https://doi.org/10.1016/j.
ejmech.2019.111977.
[62] A.S. Hassan, A.A. Askar, E.S. Nossier, A.M. Naglah, G.O. Moustafa, M.A. Al-Omar,
Antibacterial Evaluation, In Silico Characters and Molecular Docking of Schiff Bases
Derived from 5-aminopyrazoles, Mol. 24 (17) (2019) 3130, https://doi.org/10.
3390/molecules24173130.
[66] M.A. Salem, A. Ragab, A. El-Khalafawy, A.H. Makhlouf, A.A. Askar, Y.A. Ammar,
Design, synthesis, in vitro antimicrobial evaluation and molecular docking studies
of indol-2-one tagged with morpholinosulfonyl moiety as DNA gyrase inhibitors,
Bioorg. Chem. 96 (2020) 103619, , https://doi.org/10.1016/j.bioorg.2020.103619.
[67] R.L. Baehner, Quantitative Nitroblue Tetrazolium Test in Chronic Granulomatous
Disease, N. Engl. J. Med. 278 (18) (1968) 971–976, https://doi.org/10.1056/
nejm196805022781801.
[63] M. P. Wikler, M.A., Hindler, J. F., Cockerill, F. R., Patel, J. B., Bush, K., Powell, M.,
Dudley, M. N., Turnidge, J. D., Elopoulos, G. M. and Weinstein, Clinical and
Laboratory Standards Institute, in: Methods Dilution Antimicrob. Susceptibility
Tests Bact. That Grow Aerob. Approv. Stand, CLSI Doc. M07-A8, ISBN ISBN 1-
56238-689-1, Wayne, PA, USA., 2008.
[64] A.S. Hassan, D.M. Masoud, F.M. Sroor, A.A. Askar, Synthesis and biological eva-
luation of pyrazolo[1,5-a]pyrimidine-3-carboxamide as antimicrobial agents, Med.
[68] https://www.rcsb.org/structure/4DUH last access 20 Jan 2020.
18