1,2,3-Triazoles based 3-substituted 2-thioquinoxalines: Synthesis, anti-bacterial activities, and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2019.127262

New 1,2,3-triazoles based on 3-Substituted 2-thioquinoxalines were synthesized via a copper-catalyzed click reaction. The new 1,2,3-triazoles were screened for their in vitro antibacterial activities and were subjected to molecular docking studies. The starting materials included, 3-substituted-2-propargylthioquinoxalines were prepared from 2,3-dichloroquinoxaline, aliphatic amines, sodium sulfide, and propargyl bromide. The click of 2-propargythioquinoxalines with aryl azides catalyzed by copper(II) salen complex afforded novel 1,2,3-triazole-linked thioquinoxalines derivatives. The use of salen copper(II) complex as a catalyst, increased the reaction rate and reduced the loading of the toxic copper species. Results obtained from the in vitro anti-bacterial activities of the synthesized compounds revealed that compounds 7e, 7g, and 7k are active against Bacillus subtilis and Micrococcus luteus bacteria. Furthermore, in silico molecular docking results stipulated a sign of good correlation between experimental activity and calculated binding affinity. Docking studies proved 7e as the most potent compound. In addition, the binding maps exhibited activities that may attribute to the existence of electron donating and lipophilic group at para position of phenyl ring and hydrophobic interactions and hydrogen bond with the protein active sites.

Full text of DOI:10.1016/j.molstruc.2019.127262

Journal Pre-proof
1,2,3-Triazoles based 3-substituted 2-thioquinoxalines: Synthesis, anti-bacterial
activities, and molecular docking studies
Ali Keivanloo, Mahsa Fakharian, Saghi Sepehri
PII:
S0022-2860(19)31371-7
DOI:
https://doi.org/10.1016/j.molstruc.2019.127262
MOLSTR 127262
Reference:
To appear in: Journal of Molecular Structure
Received Date: 26 July 2019
Revised Date: 18 October 2019
Accepted Date: 19 October 2019
Please cite this article as: A. Keivanloo, M. Fakharian, S. Sepehri, 1,2,3-Triazoles based 3-substituted 2-
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Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.127262.
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1,2,3-Triazoles Based 3-Substituted 2-Thioquinoxalines: Synthesis, Anti-
bacterial Activities, and Molecular Docking Studies
Ali Keivanloo*,a Mahsa Fakharian,a Saghi Sepehri b
aFaculty of Chemistry, Shahrood University of Technology, Shahrood 36199-95161, Iran.
b
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical
Sciences.
E-mail addresses: akeivanloo@yahoo.com; keivanloo@shahroodut.ac.ir
Abstract: New 1,2,3-triazoles based on 3-Substituted 2-thioquinoxalines were synthesized
via a copper-catalyzed click reaction. The new 1,2,3-triazoles were screened for their in vitro
antibacterial activities and were subjected to molecular docking studies. The starting
materials included, 3-substituted-2-propargylthioquinoxalines were prepared from 2,3-
dichloroquinoxaline, aliphatic amines, sodium sulfide, and propargyl bromide. The click of
2-propargythioquinoxalines with aryl azides catalyzed by copper(II) salen complex afforded
novel 1,2,3-triazole-linked thioquinoxalines derivatives. The use of salen copper(II) complex
as a catalyst, increased the reaction rate and reduced the loading of the toxic copper species.
Results obtained from the in vitro anti-bacterial activities of the synthesized compounds
revealed that compounds 7e, 7g, and 7k are active against Bacillus subtilis and Micrococcus
luteus bacteria. Furthermore, in silico molecular docking results stipulated a sign of good
correlation between experimental activity and calculated binding affinity. Docking studies
proved 7e as the most potent compound. In addition, the binding maps exhibited activities
that may attribute to the existence of electron donating and lipophilic group at para position
of phenyl ring and hydrophobic interactions and hydrogen bond with the protein active sites.
1

Keywords: Click chemistry, 1,2,3-Triazole, Quinoxaline, Schiff base complex, Molecular
docking.
1. Introduction
In medicinal chemistry, quinoxaline derivatives are a class of compounds of major interest,
due to the fact that they have a broad scope of biological activities [1]. These include the
antiviral [2], anti-inflammatory [3-4], antimycobacterial [5-7], antiprotozoal [8] and
anticancer [9-11] activities. The molecular structures of several antibiotics including
actinoleutin, echinomycin, and levomycin, constitute of quinoxaline ring moiety. They
exhibit anti-tumors activities and also inhibition of the growth of bacteria [12]. According to
clinical test studies, various anti-tumor drugs containing a quinoxaline ring have resulted
activity toward solid tumors [13-14]. The 1,2,3-triazoles are important heterocyclic
compounds that are easily synthesized by click chemistry. This compounds can be used for
diverse applications and shows multifarious applications in organic chemistry, medicinal
chemistry, drug discovery, agrochemicals, and dyes [15-18].
The 1,3-dipolar cycloaddition reaction of organic azides and terminal alkynes in thermal
conditions usually affords a mixture of 1,4- and 1,5-regioisomers of the 1,2,3-triazoles.
Synthesis of 1,4-regioisomer by great regioselectivity was reported by Sharpless and Meldal
in their independent works from the reaction of an azide and a terminal alkyne in the presence
of a copper(I) catalyst under mild conditions [19-20]. Recently, several groups have focused
on the rate acceleration of click reaction by the usage of ligands [21-24].
Schiff base ligands, such as salen and salophen were prepared by the reaction of an aldehyde
and an amine in a variety of reaction conditions [25]. These ligands with four coordinating
positions can stabilize many transition metals in different oxidation numbers and have an
important role in a large variety of valuable catalytic reactions [26]. Transition metal complex
2

from Schiff bases have important different catalytic activities, including the asymmetric
epoxidation of olefines [27], oxidation of alcohols [28, 29] and other important catalytic
transformations [30, 31]. In continuation of our recent studies for the preparation of novel
heterocyclic compounds [32-38], we have described here a successful procedure for the
synthesis of 1,2,3-triazoles based 3-substituted 2-thioquinoxalines catalyzed by copper(II)
salen complex. The new 1,2,3-triazoles were screened for their in vitro antibacterial activities
and were subjected to molecular docking studies.
2. Materials and methods
2.1. Reagents and instruments
All the compounds and reagents used in this research work purchased from Merck, Across,
and Fluka. Melting points were Measured by Bamstead Electrothermal. NMR spectra were
1
obtained as DMSO-d6 solutions using a Bruker 300 MHz NMR spectrometer. H NMR and
13C NMR signals were reported relative to Me4Si (δ 0.0). Multiplicities were described using
the following abbreviations: s = singlet, d = doublet, t = triplet, and m = multiplet. Mass
spectra were recorded using a 5975C spectrometer supplied from Agilent Technologies
Company. IR spectra were recorded on a Shimadzu IR-470 (KBr discs).
2.2.General procedure for the synthesis of 3-chloroquinoxalin-2-amines (3a, b)
Aliphatic amines (0.2 mmol) were added to a mixture of 2,3-dichloroquinoxaline (0.1 mmol)
in EtOH (5 mL) and stirred under reflux for 4 h. The solvent was evaporated and the residue
was washed with water (2×10 mL). This was followed by drying to produce a pure desired
product. Further purification was not necessary [33].
2.3.General procedure for the synthesis of 3-substituted quinoxaline-2-thiols (4a-c)
3

The mixture of 2,3-dichloroquinoxaline (1) or 3-chloroquinoxalin-2-amines (3a, b) (1 mmol)
and Na2S.9H2O (1 mmol) was stirred in DMF (5 mL) at room temperature for 3 h. After
completion of the reaction, H2O (10 mL) was added and then acidified to achieve a reaction
mixture to a pH level of 3. The precipitate was filtered off, and washed with water (2×10 mL)
and then dried to produce a pure desire product. Further purification was not necessary [45].
2.4. General procedure for the synthesis of 2- substituted -3-(prop-2-yn-1-
ylthio)quinoxaline (5a-c)
Sodium (1.2 mmol) was dissolved in MeOH (10 mL). Compound 4 (1 mmol) and propargyl
bromide (1.2 mmol) was added to the reaction vessel respectively. The reaction mixture was
stirred at room temperature for 5 h. After completion of the reaction, the solvent was
evaporated, and the residue was washed with water (2×10 mL) and dried to produce a pure
desired product. Further purification was not necessary [46].
2.5. General experimental procedure for the synthesis of 1,2,3-triazole-linked quinoxaline
(7a-k)
To a mixture of 2-chloro-3-propargyl thioquinoxaline or its 2-amine substituted derivatives
(5a-c), (0.5 mmol) and azide (6a-i) (0.5 mmol) in ethanol (10 mL) were added, Salen-Cu(II)
complex (3 mol%) and sodium ascorbate (6 mol%). The resulting mixture was stirred at 50
oC for 3 h. Upon completion of the reaction (TLC), the solvent was evaporated under the
reduced pressure, and the residue was washed with an ammonia solution (1/1) and water
respectively. The pure product (7a–k) was obtained by crystallization from ethanol.
2-chloro-3-(((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7a)
Orange powder solid (90% yield): mp 218-220 ºC; 1H NMR (300 MHz, DMSO-d6) δ 4.74 (s,
2H, CH2), 7.76-8.01 (m, 4H, CH of quinoxaline), 8.16-8.37 (m, 3H, ArH), 8.67 (s, 1H, ArH),
13
9.04 (s, 1H, CH of triazole); C NMR (75 MHz, DMSO-d6) δ 25.3, 115.2, 123.1, 123.5,
4

126.5, 127.9, 128.3, 130.1, 131.5, 131.9, 137.5, 139.2, 141.0, 144.5, 144.7, 148.9, 154.1; IR:
3158, 2928, 2872, 1610, 1530, 1337, 1230, 1156 cm-1. m/z [M]+ 398. Anal. calcd. for
C17H11ClN6O2S: C, 51.20; H, 2.78; N, 21.07; S, 8.04; found: C, 51.24; H, 2.80; N, 21.05; S,
8.02.
2-chloro-3-(((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7b)
Orange powder solid (90% yield): mp 220-223 ºC; 1H NMR (300 MHz, DMSO-d6) δ 4.75 (s,
2H, CH2), 7.77-8.06 (m, 4H, CH of quinoxaline), 8.19 (d, J = 8.7 Hz, 2H, ArH), 8.42 (d, J =
8.7 Hz, 2H, ArH), 9.01 (s, 1H, CH of triazole); 13C NMR (75 MHz, DMSO-d6) δ 54.9, 120.9,
121.0, 122.8, 125.9, 126.9, 127.5, 127.6, 129.0, 137.9, 138.9, 141.2, 145.6, 147.0, 148.3,
154.0; IR: 3138, 2924, 2852, 1599, 1520, 1331, 1244, 1182 cm-1. m/z [M]+ 398. Anal. calcd.
for C17H11ClN6O2S: C, 51.20; H, 2.78; N, 21.07; S, 8.04; found: C, 51.27; H, 2.75; N, 21.02;
S, 8.08.
2-chloro-3-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7c)
1
Light Brown powder solid (80% yield): mp 215-218 ºC; H NMR (300 MHz, DMSO-d6) δ
4.73 (s, 2H, CH2), 7.48-7.83 (m, 5H, ArH), 7.85-8.20 (m, 4H, CH of quinoxaline), 8.83 (s,
1H, CH of triazole); 13C NMR (75 MHz, DMSO-d6) δ 67.9, 120.4, 120.5, 122.6, 127.9,
128.4, 129.1, 130.1, 130.3, 131.5, 137.0, 139.1, 141.1, 144.5, 154.2; IR: 3096, 3124, 2934,
2867, 1620, 1445, 1234, 1167 cm-1. m/z [M]+ 398. Anal. calcd. for C17H12ClN5S: C, 57.71; H,
3.42; N, 19.79; S, 9.06; found: C, 57.74; H, 3.45; N, 19.75; S, 9.05.
2-chloro-3-(((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7d)
1
Light Brown powder solid (75% yield): mp 225-228 ºC; H NMR (300 MHz, DMSO-d6) δ
4.72 (s, 2H, CH2), 7.53-7.95 (m, 4H, CH of quinoxaline), 7.82-8.18 (m, 4H, ArH), 8.89 (s,
1H, CH of triazole); 13C NMR (75 MHz, DMSO-d6) δ 25.3, 119.1, 120.3, 122.8, 127.9,
128.4, 128.9, 130.1, 131.5, 132.0, 134.6, 138.0, 139.1, 141.0, 144.5, 154.1; IR: 3156, 3124,
5

2934, 2867, 1590, 1435, 1244, 1177 cm-1. m/z [M]+ 387. Anal. calcd. for C17H11Cl2N5S: C,
52.59; H, 18.26; N, 18.04; S, 8.26; found: 52.65; H, 18.23; N, 18.11; S, 8.25.
2-chloro-3-(((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7e)
1
Light Brown powder solid (80% yield): mp 225-228 ºC; H NMR (300 MHz, DMSO-d6) δ
2.28 (s, 3H, CH3), 4.75 (s, 2H, CH2), 7.73 (d, J = 7.8 Hz, 2H, ArH), 7.74 (d, J = 7.8 Hz, 2H,
ArH), 8.01 (d, J = 8.4 Hz, 2H, ArH), 8.18 (d, J = 8.4 Hz, 2H, ArH), 8.77 (s, 1H, CH of
13
triazole); C NMR (75 MHz, DMSO-d6) δ 21.0, 25.4, 120.3, 122.5, 127.8, 128.3, 130.0,
130.6, 131.4, 134.7, 138.7, 139.1, 141.0, 143.9, 144.5, 154.2; IR: 3166, 3124, 2924, 2845,
1599, 1425, 1193, 1157 cm-1. m/z [M]+ 367. Anal. calcd. for C18H14ClN5S: C, 58.77; H,
3.84; N, 19.04; S, 8.72; found: C, 58.71; H, 3.90; N, 19.00; S, 8.75.
2-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)-3-chloroquinoxaline (7f)
Cream powder solid (75% yield): mp 200-202 ºC; 1H NMR (300 MHz, DMSO-d6) δ 4.61 (s,
2H, CH2-S), 5.54 (s, 2H, CH2-N), 7.25-7.28 (m, 5H, ArH), 7.78-8.17 (m, 4H, CH of
13
Quinoxaline), 7.82-8.18 (m, 4H, ArH), 8.22 (s, 1H, CH of triazole); C NMR (75 MHz,
DMSO-d6) δ 25.5, 53.2, 124.7, 127.6, 128.2, 128.4, 128.5, 129.1, 130.0, 131.5, 136.5, 139.1,
140.9, 143.0, 144.5, 154.3; IR: 3134, 3114, 2928, 2887, 1621, 1445, 1256, 1277, 1145 cm-1.
m/z [M]+ 367. Anal. calcd. for C18H14ClN5S: C, 58.77; H, 3.84; N, 19.04; S, 8.72; found: C,
58.80; H, 3.81; N, 19.10; S, 8.70.
2-chloro-3-(((1-(4-chloro-3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7g)
Orange powder solid (73% yield): mp 230-232 ºC; 1H NMR (300 MHz, DMSO-d6) δ 4.74 (s,
2H, CH2), 7.76-7.89 (m, 4H, CH of quinoxaline), 7.97- 8.23 (m, 3H, ArH), 8.62 (s, 1H, ArH),
13
8.94 (s, 1H, CH of triazole); C NMR (75 MHz, DMSO-d6) δ 25.2, 117.3, 122.9, 124.9,
125.0, 127.9, 128.3, 130.1, 131.4, 133.5, 136.1, 139.1, 141.0, 144.4, 144.9, 148.4, 154.0; IR:
3144, 3034, 2921, 2845, 1599, 1478, 1296, 1277, 1132 cm-1. m/z [M]+ 432. Anal. calcd. for
6

C17H10Cl2N6O2S: C, 47.13; H, 2.33; N, 19.40; S, 7.40; found: C, 47.22; H, 2.31; N, 19.30; S,
7.49.
2-chloro-3-(((1-(2-chloro-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (6h)
1
Light brown powder solid (70% yield): mp 225-228 ºC; H NMR (300 MHz, DMSO-d6) δ
4.76 (s, 2H, CH2), 7.57-7.79 (m, 4H, CH of quinoxaline), 7.91- 8.32 (m, 3H, ArH), 8.45 (s,
1H, CH of triazole); 13C NMR (75 MHz, DMSO-d6) δ 25.2, 124.0, 126.3, 126.8, 127.9,
128.3, 129.5, 129.5, 130.1, 131.5, 139.1, 139.5, 141.0, 143.6, 144.5, 148.6, 154.1; IR: 3145,
2925, 2872, 1599, 1530, 1351, 1246, 1134 cm-1. m/z [M]+ 432. Anal. calcd. for
C17H10Cl2N6O2S: C, 47.13; H, 2.33; N, 19.40; S, 7.40; found: C, 47.26; H, 2.37; N, 19.34; S,
7.35.
2-chloro-3-(((1-(4-chloro-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxaline (7i)
1
Light brown powder solid (70% yield): mp 228-231 ºC; H NMR (300 MHz, DMSO-d6) δ
4.77 (s, 2H, CH2), 7.64-8.00 (m, 4H, CH of quinoxaline), 8.02-8.24 (m, 2H, ArH), 8.66 (s,
1H, ArH), 8.97 (s, 1H, CH of triazole); IR: 3123, 2912, 2845, 1612, 1467, 1251, 1246, 1164
cm-1. m/z [M]+ 432. Anal. calcd. for C17H10Cl2N6O2S: C, 47.13; H, 2.33; N, 19.40; S, 7.40;
found: C, 47.02; H, 2.35; N, 19.34; S, 7.43.
4-(3-(((1-(2-chloro-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxalin-2-
yl)morpholine (7j)
Light yellow powder solid (80% yield): mp 215-218 ºC; 1H NMR (300 MHz, CDCl3) δ 3.46
(s, 2H, CH2-N), 3.93 (s, 2H, CH2-O), 4.74 (s, 2H, CH2-S), 7.39-7.91 (m, 4H, CH of
13
Quinoxaline), 7.94- 8.31 (m, 3H, ArH), 8.43 (s, 1H, CH of triazole); C NMR (75 MHz,
CDCl3) δ 24.5, 49.9, 66.4, 124.0, 126.3, 126.5, 127.5, 129.0, 129.3, 129.4, 138.5, 138.9,
139.5, 144.1, 148.5, 150.7, 153.6; IR: 3123, 3034, 2967, 2923, 1602, 1535, 1350, 1267, 1145
7

cm-1. m/z [M]+ 483. Anal. calcd. for C21H18ClN7O3S: C, 52.12; H, 3.75; N, 20.26; S, 6.62;
found: C, 52.20; H, 3.71; N, 20.19; S, 6.59.
N-ethyl-3-(((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)quinoxalin-2-amine (7k)
Light yellow powder solid (80% yield): mp 230-233 ºC; 1H NMR (300 MHz, CDCl3) δ 1.34
(t, j = 7.2 Hz, 3H, -NHCH2CH3), 3.63 (q, j = 6.7 Hz, 2H, -NHCH2CH3), 3.77-4.82 (m, 3H,
CH2-S and -NHCH2CH3), 7.39-7.90 (m, 6H, CH of quinoxaline and ArH), 8.14 (s, 1H, CH
13
of triazole), 7.36-8.39 (m, 2H, ArH); C NMR (75 MHz, CDCl3) δ 14.7, 24.1, 36.1, 120.9,
122.9, 124.2, 125.9, 126.0, 127.3, 128.6, 136.6, 139.9, 141.2, 145.4, 146.1, 147.1, 148.9; IR:
3406, 3080, 2972, 2927, 1595, 1518, 1338, 1227, 1045 cm-1. . m/z [M]+ 407. Anal. calcd. for
C19H17N7O2S: C, 56.01; H, 4.21; N, 24.06; S, 7.87; found: C, 56.10; H, 4.22; N, 24.05; S,
7.90.
2.6. Anti-bacterial Assay
The anti-bacterial activities of the new compounds were evaluated biologically using a well-
diffusion method against M. luteus (ATCC 4698), P. aeruginosa (ATCC 27853), and B.
subtilis (DSM 6887). First, the nutrient agar and the nutrient broth cultures were prepared
according to the manufactures’ instructions, which were then incubated at 37 ˚C. After
incubation for an appropriate time period, a suspension of 40 µL of each bacterium was
added to the nutrient agar plates. Cups (5 mm in diameter) were cut in the agar, using a
sterilized glass tube. Each agar palate well-received 30 µL of the test compounds at a
concentration of 1000 µg mL-1 in DMSO. Following this, the plates were incubated at 37 ˚C
for 24 h, after which time, the inhibition zone was measured in mm. The results obtained
were reported as the inhibition zone in mm. The anti-bacterial activity of each compound was
compared with that for tetracycline as the standard drug. DMSO was used as the negative
control.
8

2.7. Molecular Docking Studies
Molecular docking calculation was accomplished using AutoDock version 4.2. The three-
dimensional (3D) protein structures of M. luteus (PDB ID: 4EWP) and B. subtilis (PDB ID:
1SCJ), determined by X-ray crystallography, was retrieved from the RCSB Protein Databank
(http://www.rcsb.org/pdb/home/home.do.). Then, removed the extra crystallized water
molecules by Accelrys discovery studio visualizer 4.0 (DSVisualizer, Accelrys Software Inc.,
San Diego, CA, USA, 2014.) and added polar hydrogens and charges to the molecule by
AutoDockTools version 1.5.6rc3 for correction of protein structures. Correction of the
ionization and tautomeric states of residues and modification of the total Kollman charges on
the protein structure are carried out in protonation step. All two-dimensional (2D) structures
of the ligands were drawn by the ChemDraw program (ChemDraw Ultra 10.0,
CambridgeSoft.). Each structure was energy minimized by the MM+ force field and PM3
semi-empirical techniques in HyperChem8 software. Then, partial charges of atoms were
calculated using the Gasteiger-Marsili procedure. The resulting protein structure was used as
an input for the Auto-Grid program. All maps were calculated with 0.375 Å spacing between
grid points. Each docking was performed by 150 runs of the AutoDock search by the
Lamarckian genetic algorithm (LGA). The factors for LGA were defined as follows: a
maximum number of 2,500,000 energy evaluations; a maximum number of generations of
27,000; mutation and crossover rates of 0.02 and 0.8, respectively. Lastly, the conformation
of the lowest predicted. Graphic manipulations and visualizations were done by Accelrys
discovery studio visualizer 4.0 (DSVisualizer, Accelrys Software Inc., San Diego, CA, USA,
2014.), Autodock Tools [47] and LigPluse 2012 (https://www.ebi.ac.uk/thornton-
srv/software/LIGPLOT/, 2012) [48] software.
9

3. Results and Discussion
3.1 Chemistry
In this research work, we report the synthesis of 1,2,3-triazoles based 3-substituted 2-thio
quinoxalines by the reaction of 2-chloro-3-propargyl thioquinoxaline, and its 2-amine
drivatives with aromatic azides catalyzed by copper(II) salen complex in the click reaction
condition (Scheme 1).
Scheme 1: Synthesis of 1,2,3-triazoles based quinoxaline scsffold by the click reaction in the
presence of copper(II) salen complex.a
aReaction conditions: 5 (0.5 mmol), 6 (0.5 mmol), Salen- Cu(II) (3 mol%), NaAsc (6 mol%),
ETOH (4 mL), 50 oC.
The starting materials include, 3-substituted-2-propargylthioquinoxalines (5a-c) were
prepared from 2,3-dichloroquinoxaline, aliphatic amines, sodium sulfide and propargyl
bromide that as shown in Figure 1
10

Figure 1: Synthesis of 3-substituted-2-propargyl thioquinoxalines from 2,3-
dichloroquinoxaline, aliphatic amines, sodium sulfide and propargyl bromide.
The reaction of 2,3-dichloroquinoxaline with ethyl amine or morpholine afforded 3-
chloroquinoxaline-2-amines (3a-b), by substituting one of the chlorine atoms. The reaction of
2,3-dichloroquinoxaline or 3-chloroquinoxaline-2-amines, with Na2S.9H2O in DMF at room
temperature, produced substituted thioquinoxalines (4a-c). All thioquinoxaline compounds
were reacted with propargyl bromide in the presence of NaOMe in MeOH at room
temperature and propargyl thioquinoxalines were formed (5a-c) in quantitative yield.
Structures and properties of the synthesized substituted quinoxalines are shown in figure 2.
11

Figure 2: Structures and properties of the synthesized substituted quinoxalines.
In continuation of our study, we decided to synthesis of 1,2,3-triazoles based 3-substituted 2-
thioquinoxalines by the click reaction of 2-chloro-3-propargyl thioquinoxaline and its 2-
amine substituted derivatives with aromatic azides. The reaction of 2-chloro-3-propargyl
thioquinoxaline (5a) with 1-azido-3-nitrobenzene (6a) was examined as a model of the
reaction to find the optimized reaction condition (Table 1). The reaction was executed with
various polar protic and aprotic solvents including H2O, DMF, EtOH, and CH3CN. All the
examined solvents were effective, with the exception of water, due to the low solubility of the
starting materials. The greatest yield of the reaction was obtained by the use of EtOH as a
solvent. This produced the final product with a 90% yield (Table 1, entry 3). We found,
Cu(OAc)2 was the most effective catalyst after the screening of a variety of copper salts
(Table 1, entries 3, 4, 9, 13, 15).
12

Table1: Effect of catalyst, solvent, and temperature for the synthesis of 1,2,3-triazole-linked
quinoxalines via click reaction.a
Entry
Catalyst (mol%)
Additive
Solvent
T/
Time/h
Yield (%)
(mol%)
1
2
CuSO4 (10)
CuCl2 (10)
-
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH/H2Ob
H2O
78
78
78
50
78
r.t
5
5
70
60
90
70
65
50
60
30
80
70
50
65
70
60
85
65
90
90
75
80
-
3
Cu(OAc)2 (10)
Cu(OAc)2 (10)
Cu(OAc)2 (5)
Cu(OAc)2 (10)
Cu(OAc)2 (10)
Cu(OAc)2 (10)
Cu(OAc)2 (10)
Cu(OAc)2 (5)
Cu(OAc)2 (10)
CuI (10)
-
5
4
-
5
5
-
5
6
-
10
10
10
5
7
-
80
100
70
70
70
70
78
78
78
78
50
50
50
50
8
-
9
-
DMF
10
11
12
13
14
15
16
17
18
19
20
-
DMF
5
-
CH3CN
DMF
5
-
5
Cu(OAc)2 (5)
Cu(OAc)2 (5)
Cu(OAc)2 (5)
Cu(OAc)2 (5)
Salen-Cu(II) (5)
Salen-Cu(II) (3)
Salen-Cu(II) (2)
Salophen-Cu(II) (5)
L3 (5)
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
5
L1 (5)
3
L2 (5)
3
L4 (5)
4
-
-
-
-
3
3
3
3
aReaction conditions: 5a (0.5 mmol), 6a (0.5 mmol), catalyst, NaAsc (twice of the copper salt), solvent (4 mL),
and temperature.
bRatio of EtOH to H2O is 1:1.
13

In classical click reactions, large quantities of copper salt are commonly used often in
stoichiometric amounts [39]. In order to reduce the loading of the toxic copper salt in the
click reactions, a number of researchers used a variety of nitrogen-containing ligands [40-43].
In this work, to reduce loading of the catalyst, we applied the Schiff bases and other nitrogen-
containing ligands (Fig. 3).
Fig. 3: Ligands and complexes used for click reactions
These included salophene (L1), salen (L2), metformine (L3) and phenylenediamine (L4) as
an additive (Table 1, entries 13-16). Also, we examined copper Schiff base complexs
including, salophen-Cu(II) and Salen-Cu(II) complexs (Table 1, entries 17-20). We have
shown that under catalytic conditions the Salen-Cu(II) complex serves as an effective catalyst
for the click reaction when compared to other ligands used. By decreasing the amount of the
Salen-Cu(II) complex to 3 mol%, the reaction performed efficiently and produced the final
product in a 90% yield in EtOH at 50 oC (Table 1, entry 18).
14

To study the generality and extent of this reaction procedure, different aromatic azides
including both electron-donating or electron-withdrawing functional groups were utilized as
the substrates for the synthesis of 1,2,3-triazoles based 3-Substituted 2-thioquinoxalines
(7a-k) under the optimized reaction conditions. The obtained results are shown in Table 2.
The results show that the products were formed with high yields.
Table 2: Synthesis of 1,2,3-triazoles-linked quinoxaline scaffold by the click reaction in the
presence of copper(II) salen complex.
Entry
X
R
Time (h)
Product
7a
Yield
(%)
90
1
2
3
4
5
6
7
8
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3-NO2-C6H4
4-NO2-C6H4
C6H5
3
3
90
80
75
80
75
73
70
7b
3.5
4
7c
3-Cl-C6H4
CH3-C6H4
C6H5CH2
7d
3
7e
4
7f
4-Cl-3-NO2-C6H3
2-Cl-C6H4
3
7g
3.5
7h
15

9
Cl
4-Cl-2-NO2-C6H3
2-Cl-4-NO2-C6H3
4-Cl-C6H4
3.5
3.5
3.5
70
80
80
7i
7j
10
11
O(CH2CH2)2N
C2H5N
7k
aReaction conditions: 5 (0.5 mmol), 6 (0.5 mmol), Salen-Cu(II) (3 mol%), NaAsc (6 mol%),
ETOH (4 mL), 50 oC.
Spectroscopic data and mass analysis were successfully used to assignment the structure of
1
the new compounds. In the H NMR spectrum for compound 7e, a singlet signal at δ 2.28
with three protons area is characteristic of a methyl group. Another singlet at δ 4.75 with two
protons area is attributed to the CH2 group. Two doublets at δ 7.38 and δ 7.74, with an equal
coupling constant (J = 7.8) and two protons area for each of them are characteristic of four
aromatic protons of the phenyl ring. The protons of quinoxaline ring appeared as two
multiplets at δ 7.98-8.03 and δ 8.15-8.20 with two protons area for each of them. A singlet
13
peak at δ 9.04 is typical of a triazole proton. In the C NMR spectrum of this compound, a
total of 16 peaks were observed for all carbon atoms.
3.2. Anti-bacterial evaluation
The in vitro anti-bacterial properties of newly synthesized 1,2,3-triazoles based 3-substituted
2-thioquinoxalines were screened against Micrococcus luteus, Pseudomonas aeruginos, and
Bacillus subtilis as the Gram-positive and Gram negative bacteria by the well-diffusion
method at a concentration of 1000 mg mL-1. DMSO and Tetracycline were applied as the
negative and positive control, against the bacterial strains. The results were shown in Table 3.
Results obtained showed, only 7e, 7g, and 7k are active against Bacillus subtilis and
16

Micrococcus luteus. According to these results, 7g showed the highest anti-bacterial activity
(Table 3).
Table 3. Anti-bacterial activities of synthesized compounds (1000 ꢀg mL-1) as inhibition
zone in mm.
Entry
Compound
B. subtilis
M. luteus
Ps. aeruginos
1
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7c
7d
3
10
-
14
-
7e
4
7f
5
15
-
18
-
7g
6
7h
7i
7
-
-
8
12
-
16
-
7k
9
DMSO
Tetracycline
10
20
55
3.3. Molecular docking study
Computational techniques have developed crucial in comprehension the structural and
physicochemical features of the drug compounds. Therefore, these techniques help to the
researchers in the discovery and development of active novel molecules [44]. In this work,
the docking was performed using the Autodock 4.2 software in order to forecast the binding
modes of screened compounds in active sites. Thus, investigated score docking (∆Gbind),
hydrogen bonds, hydrophobic interactions, and cation-π stacking of compounds in active
17

sites. The best score docking and strong interactions with active sites residues was checked
for all protein-compound complexes (the top-ranked solution). Molecular docking analysis of
synthesized triazole based on quinoxaline derivatives exposed that they docked well into the
active sites and revealed appropriate interactions with the essential amino acids (Tables 4 and
5).
Table 4. Binding free energy (ꢁGbind), intermolecular hydrogen bonds, hydrophobic contacts and
cation-π interactions in ligands 7c-k at Micrococcus luteus
Compound
∆Gbind
Hydrogen bond
Hydrophobic contacts
Cation-π
(kcal/mol)
contacts
-7.87
Cys123 Asn306, Leu167, Gln238, Ala277,
Val243, Gly337, Ile200, Phe336, Ile164,
Ala338, Ser308, Leu236, Val92, Ala122
Cys123 Leu167, Ile164, Gln238, Phe336,
Asn306, Ala277, Gly337, Val243,
Ile200, Ser308, Leu236, Val92, Ala338,
Ser201
-
-
7c
-8.22
7d
7e
7f
-8.52
-7.80
-7.78
Cys123 Val243, Phe336, Gly337, Ala277,
Leu167, Gln238, Ile164, Asn306,
Ile200, Leu236, Ala338, Val92, Ala122,
Ser308
-
-
-
Ala338, Cys123 Leu167, Gln238, Asn306, Ala277,
Val243, Phe336, Ile164, Gly337, Ile200,
Ser308, Ala122, Val156, Leu236,
Val92, Met202
Asn306, Gln238 Ile281, Leu167, Ala277, Leu168,
7g
18

Ile164, Val243, Ala122, Cys123, Ile200,
Val92, Ala338, Leu236, Met202,
Ser201
-8.15
-7.90
Gln238, Asn306 Ile281, Asn278, Leu167, Val243,
Ala277, Leu168, Ile164, Ala122,
Cys123, Ile200, Ser308, Ser201, Val92,
Ala338, Leu236, Ser201, Met202
-
-
7h
Gln238, Asn306 Asn278, Leu167, Leu168, Ile164,
Ala277, Ile281, Val243, His275,
Cys123, Ala338, Ser201, Ala122,
Leu236, Val92, Ile200, Met202
7i
-7.57
-6.25
His275, Asn306 Met202, Trp197, Val92, Ser201,
Leu236, Leu153, Ile200, Gly339,
Ala338, Ala121, Ala122, Cys123,
-
-
7k
Cys123 Ala277, Leu167, Leu153, Val92,
Ala338, Ile200, Met202, Val243,
Ser201
Tetracycline
Table 5. Binding free energy (ꢁGbind), intramolecular hydrogen bonds, hydrophobic contacts and
cation-π interactions in ligands 7c-k at Bacillus subtilis
Compound
∆Gbind
Hydrogen bond
Hydrophobic contacts
Cation-π
(kcal/mol)
contacts
-4.36
Ser173 Asp140, Pro172, Val143, Asn243,
Ile175, Arg247, Val148, Val149
-
-
7c
-5.07
-
Pro172, Ser173, Val143, Thr174,
7d
19

Ile175, Val149, Ala244, Arg247,
Asn243, Val148
-5.47
-5.25
-4.71
-4.04
-
-
Asp197, Glu195, Pro172, Val143,
Arg247, Ala244, Asn243, Val148,
Ser173
-
-
7e
7f
Asp140, Pro172, Ser173, Ala244,
Val143, Ile147, Ile175, Val148, Arg247,
Asn243, Val149
Val149 Ile147, Val149, Ser173, Val143,
Val148, Ala244, Thr174, Asn243,
Arg247, Pro172, Asp197
Arg247
Lys136
7g
7h
Asn243, Val149 Val148, Ala244, Ile147, Val143,
Arg247, Ser173, Pro172, Lys136,
Asp140
-3.12
-4.86
-3.10
Lys136 Glu195, Asp197, Pro172, Arg247,
Ser173, Asp140
-
-
-
7i
7k
Ser173 Asp140, Pro172, Arg247, Glu195,
Asp197
-
Glu195, Asp197, Val148, Arg247,
Pro172
Tetracycline
Molecular docking experimental data of 1,2,3-triazoles based on quinoxaline scaffold
exposed that compounds docked well into the active site and showed favorable interactions
with the crucial residues. The most biologically active compound, 7e, having methyl group at
para phenyl ring substituted to triazole ring showed a hydrogen bond with Cys123 (NH…S,
2.42 Å) at M. luteus whereas, this compound did not show any hydrogen bond in B. subtilis
20

active site (Figure 5). Also, molecular docking results showed that 7e had the highest ∆Gbind
among compounds against M. luteus and B. subtilis proteins (Tables 4 & 5). It seems that,
existence electron donating group at para position increased antibacterial activity and ∆Gbind
(e.g. Compound 7e vs 7i). Moreover, the phenyl ring bearing a substituent showed better
antibacterial activity as well as free binding energy than two substituents.
Figure 5. Compound 7e from docking results in (A) M. luteus and (B) B. subtilis binding sites
Compound 7g bearing p-chloro-m-nitrophenyl ring moiety showed poor antibacterial activity
and low free binding energy. Molecular docking results displayed that the nitro group of the
phenyl ring is able to form an additional hydrogen bond (Figure 6). Presence an extra
hydrogen bond might have negative effect. In addition, existence two electron withdrawing
groups at para and meta positions of the phenyl ring are inappropriate compared with an
electron donating group at the same position.
21

Figure 6. Compounds 7e (A and C) and 7g (B and D) in M. luteus and B. subtilis binding sites,
respectively
Replacing of the p-methylphenyl moiety at triazol ring of 7e with p-nitrophenyl moiety at the
same position of 7k reduced the ∆Gbind and antibacterial activity. According to the docking
and antibacterial activity data, presence of an electron withdrawing group such as NO2 group
at the para position of phenyl ring had a negative effect on both antibacterial activity and free
binding energy value.
22

Docking results exposed that compounds 7g and 7h formed a cation-π stacking interaction
with Arg247 and Lys136 amino acids of B. subtilis active site, respectively. While these
compounds did not participate in this contact with M. luteus amino acids active site (Figure
7). Also, other compounds in both protein did not participate in this contact.
Figure 7. Compounds 7g and 7h at B. subtilis binding site. Orientations of 7g and 7h phenyl rings and
Arg247 and Lys136 amine groups possible cation-π stacking interactions.
Three compounds 7e, 7g and 7k showed higher antibacterial activity than tetracycline as the
drug standard. Tetracycline was docked into M. luteus and B. subtilis active sites. This
compound formed a hydrogen bond with Cys123 in M. luteus active site whereas, it did not
form any hydrogen bond in B. subtilis active site. New 1,2,3-triazole based on quinoxaline
compounds showed more ∆Gbind than tetracycline at two proteins. All results have tabulated
in tables 4 and 5.
4. Conclusion
23

The click reaction of 2-chloro-3-propargylthio quinoxaline and its 2-amine substituted
derivatives with aromatic azides afforded new 1,2,3-triazoles based 3-Substituted 2-
thioquinoxalines. We investigated the effects of Schiff base and other nitrogen-containing
ligands including, salophene, salen, metformine, and phenylenediamine on the reaction.
Also, we examined copper Schiff complexs including, Salophen-Cu(II) and Salen-Cu(II)
complexs involving the reaction in order to reduce the loading of the toxic copper catalyst.
We found that Salen-Cu(II) complex is a more effective catalyst. This due to the speed up
the transformation and the reduced the amount of toxic copper catalyst to 3 mol%. The In
vitro antibacterial activities of the novel 1,2,3-triazoles were screened and the molecular
docking was performed through the Autodock 4.2 software. The results revealed that
compounds 7e, 7g, and 7k are active against Bacillus subtilis and Micrococcus luteus
bacteria. Moreover, based on the results of antibacterial activity and molecular docking
calculations of the synthesized compounds, the structure activity relationship (SAR) can be
summarized, as the change of substituents at positions of the quinoxaline and triazole rings.
This change has an effect on antibacterial activity. Docking results showed that higher
lipophilicity character and the presence of electron donating group at para position of
phenyl ring, has constructive impacts on both antibacterial activity and free binding energy
of these compounds.
Acknowledgment
The authors wish to express their thanks to the Research Council of the Shahrood University
of Technology for the financial support of this research work
.
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