New quinoxalin-1,3,4-oxadiazole derivatives: Synthesis, characterization, in vitro biological evaluations, and molecular modeling studies

Article abstract of DOI:10.1002/ardp.202000471

A new series of quinoxalin-1,3,4-oxadiazole (10a–l) derivatives was synthesized and evaluated against some metabolic enzymes including human carbonic anhydrase (hCA) isoenzymes I and II (carbonic anhydrases I and II), cholinesterase (acetylcholinesterase

Full text of DOI:10.1002/ardp.202000471

Received: 18 December 2020
DOI: 10.1002/ardp.202000471
Revised: 17 April 2021
Accepted: 23 April 2021
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F U L L P A P E R
New quinoxalin‐1,3,4‐oxadiazole derivatives: Synthesis,
characterization, in vitro biological evaluations, and
molecular modeling studies
Roghieh Mirzazadeh1 | Mohammad S. Asgari2 | Ebrahim Barzegari3
Keyvan Pedrood4 | Maryam Mohammadi‐Khanaposhtani5 | Maedeh Sherafati4
Bagher Larijani4 | Hossein Rastegar6 | Hojjat Rahmani7
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Mohammad Mahdavi4 | Parham Taslimi8
| Eda M. Üç9 | İlhami Gulçin9
1Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
2Department of Chemistry, Iran University of Science and Technology, Tehran, Iran
3Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
4Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
5Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
6Cosmetic Products Research Center, Iranian Food and Drug Administration, MOHE, Tehran, Iran
7Department of Health Management and Economics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
8Department of Biotechnology, Faculty of Science, Bartin University, Bartin, Turkey
9Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey
Correspondence
Mohammad Mahdavi, Endocrinology and
Metabolism Research Center, Endocrinology
and Metabolism Clinical Sciences Institute,
Tehran University of Medical Sciences,
1411713137 Tehran, Iran.
Abstract
A new series of quinoxalin‐1,3,4‐oxadiazole (10a–l) derivatives was synthesized and
evaluated against some metabolic enzymes including human carbonic anhydrase
(hCA) isoenzymes I and II (carbonic anhydrases I and II), cholinesterase (acet-
ylcholinesterase and butyrylcholinesterase), and α‐glucosidase. Obtained data
revealed that all the synthesized compounds were more potent as compared with
the used standard inhibitors against studied target enzymes. Among the synthesized
compounds, 4‐fluoro derivative (10f) against hCA I, 4‐chloro derivative (10i)
against hCA II, 3‐fluoro derivative (10e) against acetylcholinesterase and butyr-
ylcholinesterase, and 3‐bromo derivative (10k) against α‐glucosidase were the most
potent compounds with inhibitory activity around 1.8‐ to 7.37‐fold better than
standard inhibitors. Furthermore, docking studies of these compounds were
performed at the active site of their target enzymes.
Email: momahdavi@tums.ac.ir
Parham Taslimi, Department of Biotechnology,
Faculty of Science, Bartin University, 74100
Bartin, Turkey.
Email: parham_taslimi_un@yahoo.com
K E Y W O R D S
1,3,4‐oxadiazole, carbonic anhydrase, cholinesterase, quinoxalin, α‐glucosidase
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch Pharm. 2021;e2000471.
wileyonlinelibrary.com/journal/ardp
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1
INTRODUCTION
expression of these CA isoforms is found in several human cells and
tissues, which is indicative of their crucial physiological roles. The
CA‐catalyzed reaction is involved in numerous biological and pa-
thological mechanisms, including transport and respiration of carbon
dioxide and bicarbonate in metabolizing lungs and tissues; electro-
lyte secretion; CO2 and pH homeostasis, in diverse organs and
tissues; bone resorption; calcification; biological reactions (like lipo-
genesis, gluconeogenesis, and ureagenesis); tumorigenicity; and
virulence of diverse pathogens.[24–28]
Heterocycle aromatic scaffolds are privileged blocks in drug design
and discovery. The most widely used heterocycles in the design of
drug‐like candidates are mono‐, bi‐, and tricyclic aromatic structures
containing nitrogen, oxygen, and both atoms. One of the most im-
portant monocyclic heterocycles in the design of biologically active
compounds is oxadiazole. This heterocycle in two forms, 1,2,4‐ and
1,3,4‐oxadiazole, has been found in compounds possessing anti-
depressant, analgesic, antiviral, anti‐inflammatory, anticonvulsant,
antiallergic, antipsychotic, antimicrobial, antimycobacterial, anti-
tumor, and antitubercular activities.[1] Furthermore, several deriva-
tives of oxadiazole derivatives with high inhibitory activity against
metabolic enzymes including human carbonic anhydrase I (hCA I),
human carbonic anhydrase II (hCA II), acetylcholinesterase (AChE),
butyrylcholinesterase (BChE), and α‐glucosidase have been
reported.[2–5] Another important heterocycle in the design of new
bioactive compounds is quinoxalin, which has a bicyclic aromatic
structure with two nitrogen atoms. Several derivatives of this
scaffold with biological activities such as antiproliferative,
anti‐α‐glucosidase, anti‐acetylcholinesterase, and anti‐nitric oxide
synthase have been reported.[6–8]
Hence, in continuation of our efforts for developing new and
potent inhibitors against metabolic enzymes, herein, we synthe-
sized a novel series of quinoxalin‐1,3,4‐oxadiazole derivatives and
evaluated their inhibitory effects against hCA I, hCA II, AChE,
BChE, and α‐glucosidase. In addition to in vitro enzymatic inhibi-
tion assays, new quinoxalin‐1,3,4‐oxadiazole derivatives were
evaluated by molecular modeling method for clarifying interaction
modes of these compounds in the active sites of the mentioned
enzymes.
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2
RESULTS AND DISCUSSION
Chemistry
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α‐Glucosidase (EC. 3.2.1.20) is a key enzyme of the glycoside
hydrolase class that cleavages α‐1,4‐linked terminal nonreducing in
the carbohydrate molecules and converted them into glucose.
Inhibition of this enzyme is an important therapeutic method for
decreasing the glucose level in type 2 diabetes.[9–12]
2.1
The synthetic procedure for quinoxalin‐1,3,4‐oxadiazole derivatives
(10a–l) has been depicted in Scheme 1. This procedure was started
with the reaction between 3,4‐diaminobenzoic acid (1) and benzil (2)
in acetic acid to produce 2,3‐diphenylquinoxaline‐6‐carboxylic acid
(3). This compound in the presence of H2SO4 in ethanol converted to
an ester (4). Then, the obtained ester reacted with hydrazine (5) to
give 2,3‐diphenylquinoxaline‐6‐carbohydrazide (6). This compound
easily participated in the cyclization reaction in the presence of CS2
(7) and KOH at reflux condition and afforded 1,3,4‐oxadiazole deri-
vative (8). Finally, compound 8 was reacted with benzyl bromide
derivatives (9a–l) in the presence of K2CO3 in dimethylformamide
(DMF) at room temperature to give the corresponding products
(10a–l) in good yields (89–95%).
Alzheimer's disease (AD) is related to the reduction of acet-
ylcholine (ACh) effects in the brain.[13] Indeed, ACh effects are
regulated by the hydrolytic action of the AChE enzyme. In addition,
central palliative therapy of AD is the administration of AChE en-
zyme inhibitors; however, extra mechanisms are explained and also
tested for producing favorable medicinal strategies.[14] Two im-
portant cholinesterase enzymes are present in the brain, one pri-
marily neuronal and AChE enzyme‐addicted, and the second
principally glial and BChE‐dependent, which can modify AChE in
hydrolyzing brain ACh. In the status of blood, AChE enzyme is re-
corded mostly in red blood cell membranes, whereas BChE enzyme is
recorded primarily in the blood plasma.[15–17] However, recently,
several studies have been reported showing that inhibition of
carbonic anhydrases, given the role that these enzymes play in
neurodegeneration, can be a useful method for AD treatment.[18]
CA enzymes are extensively recorded metalloenzymes in both
eukaryotic and prokaryotic cells. In addition, these isoforms effi-
ciently perform the reversible hydration of carbon dioxide to H+ ions
and bicarbonate and play a very important role in regulating plenty
of physiological mechanisms.[19,20] These isoenzymes are well‐
studied drug targets for diverse disturbances like sleep apnea, epi-
lepsy, glaucoma, and high‐altitude sickness. Indeed, CAs also catalyze
plenty of other reactions, like conversion of cyanamide to urea,
sulfonyl chlorides to sulfonic acids, cyanate to carbamic acid, and
aldehydes to alcohols. They are also esterases with esters of sulfonic,
carboxylic, and phosphoric acid compounds.[21–23] The prevalent
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2.2
Bioactivity results
The newly synthesized quinoxalin‐1,3,4‐oxadiazole derivatives
(10a–l) were evaluated against metabolic enzymes hCA I, hCA II,
AChE, BChE, and α‐glucosidase. As can be seen in Scheme 1, various
derivatives of the quinoxalin‐1,3,4‐oxadiazole scaffold were pre-
pared by using substituted benzyl bromides with electron‐donating
and electron‐withdrawing substituents.
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2.2.1
Inhibition results of hCA I and II isoenzymes
The α‐class CA enzymes are 16 zinc‐containing metalloisoenzymes
that differ in tissue expression function, kinetics, and patterns.

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SCHEME 1 Synthesis of quinoxalin‐1,3,4‐oxadiazole derivatives (10a–l): (a) acetic acid, reflux, 1 h; (b) ethanol, H2SO4, reflux, 24 h;
(c) ethanol, reflux, 3 h; (d) KOH, reflux, 8 h; (e) K2CO3, dimethylformamide, room temperature, 6 h
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AChE and BChE inhibition results
The sluggishness and overexpression of diverse CA isoforms are
responsible for plenty of diseases in human beings. Owing to the role
of CA I and CA II in numerous, significant, normal biological me-
chanisms like the arrangement of the acid–base homeostasis, they
are considered as worthy drug targets in cerebral edema, glaucoma,
and epilepsy.[29–37] Our enzymatic evaluation results indicated that
novel quinoxalin‐1,3,4‐oxadiazole derivatives (10a–l) had an effec-
tive inhibition profile against hCA I isoform. These compounds in-
hibited hCA I with Ki values in the range of 103.28 9.38 to
683.27 103.26, and therefore are more potent than positive con-
trol acetazolamide with a Ki value of 760.52 34.13. Among these
compounds, 4‐fluoro derivative (10f) with 7.3‐fold higher inhibitory
activity than acetazolamide was the most potent compound. The
movement of fluoro substituent at 4 to 3‐position, as in compound
10e, slightly decreased inhibitory activity, whereas changing the
position of fluoro at 4 to 2, as in compound 10d, created a significant
decrease in anti‐hCA I activity. As can be seen in Table 1, 4‐chloro
derivative (10i) had approximately similar inhibition effect as
4‐fluoro derivative (10f). Other chloro derivatives, 2‐chloro deriva-
tive (10g) and 3‐chloro derivative (10h), were weaker than 4‐fluoro,
3‐fluoro, and 4‐chloro derivatives but stronger than remaining syn-
thesized compounds against hCA I. The absence of a substituent on
the pendant phenyl ring and/or introduction of 2‐methyl, 3‐methyl,
2‐bromo, 3‐bromo, and 4‐bromo substituent on the pendant
phenyl ring did not improve inhibitory activity, and it dramatically
decreased.
2.2.2
The differences in the amino acid residues of the active sites of AChE
and BChE determine different specificities for substrates and in-
hibitors for these enzymes. These are key components of cholinergic
brain synapses and neuromuscular connections.[38–40] The main
function of AChE and BChE is to catalyze the hydrolysis of the
neurotransmitter ACh and nerve impulse termination at cholinergic
synapses. Thus, compounds acting as cholinesterase inhibitors can be
considered potential drugs in the treatment of AD, other neurolo-
gical disorders, and as therapeutic agents. Due to the composition of
their active sites, the two enzymes may differ in selectivity and
specificity for inhibitor binding. It is also well documented that BChE
reacts with a wider range of ligands than AChE and is less selective.
Recently, there is a major interest in the extension of selective BChE
inhibitors.[41–47] As can be seen in Table 1, all the novel quinoxalin‐
1,3,4‐oxadiazole derivatives (10a–l) were more potent than positive
control tacrine against AChE and BChE. Among the synthesized
compounds, respectively, 3‐fluoro derivative (10e), 2‐chloro deriva-
tive (10g), and 3‐chloro derivative (10h) were the most potent
compounds against AChE, and 3‐fluoro, 3‐chloro, and 2‐chloro deri-
vatives were the most potent compounds against BChE. These
compounds were almost 1.4‐ to 2‐fold more potent than tacrine. The
use of other substitutions, whether electron‐donating or electron‐
withdrawing, did not increase the inhibitory activity of the remaining
synthesized compounds against AChE and BChE.
In terms of CA II inhibitory activity, all the synthesized
compounds were more potent than acetazolamide. As can be seen
in Table 1, the most potent compounds were 4‐chloro, 4‐fluoro,
3‐chloro, 2‐chloro, and 3‐fluoro derivatives: 10i, 10f, 10h,
10g, and 10e, respectively, and inhibitory activities of the
remaining compounds were not comparable to the mentioned
compounds.
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2.2.3
α‐Glucosidase inhibition results
α‐Glucosidase inhibition helps monosaccharides to be released
slowly into the bloodstream after eating. The type 2 hyperglycemia
state in diabetes mellitus occurs after meals, under the influence of

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insulin resistance. To control the hyperglycemic state after a meal,
α‐glucosidase inhibitors are used as therapeutic agents.[48,49] Our
newly synthesized compounds (10a–l) were also evaluated against
α‐glucosidase, and our obtained results revealed that all these
compounds with IC50 values 28.47 5.30 to 100.23 15.11 nM
showed greater inhibitory activities than positive control acarbose
(IC50 value = 1060.3 nM[50,51]). Among the synthesized compounds,
3‐bromo derivative (10k) and 3‐chloro derivative (10h) were the
most potent compounds and 2‐fluoro derivative (10d) and 4‐bromo
derivative (10l) were the less potent compounds against
α‐glucosidase. As can be seen in Table 1, 2‐chloro derivative (10g)
and 2‐bromo derivative (10j) had similar inhibitory activities.
Inhibitory activities of the latter compounds were greater than
4‐chloro, 3‐fluoro, and 4‐fluoro derivatives (10i, 10e, and 10f,
respectively). However, unsubstituted compound 10a was more
potent than 2‐methyl and 3‐methyl derivatives (10b,c).
10e in the active site of BChE is also shown in Figure 1b. Compound
10e formed interactions with AS residues Trp82 and Phe329 and CT
residues Ser198 and His438. This compound also interacted with the
active site residues Gly117 and Ser287 via 3‐fluoro substituent.
Compound 10e also formed interactions with Leu286, Val288,
Trp231, and Leu125 in the BChE active site.
As can be seen in Table 1, compounds 10f and 10i were the
most potent compounds against hCA I and hCA II, respectively.
The interaction mode of compound 10f in the hCA I active site
demonstrated that this compound formed two hydrogen bonds
with important active site residues Thr199 and His200 via
1,3,4‐oxadiazole ring. The latter ring also created a π–cation inter-
action with His200 (Figure 2a).[53] Furthermore, compound 10f
formed several hydrophobic interactions with Ala132, Leu131,
Pro202, Ala135, Leu198, Val207, Ala121, and Val143. Compound
10i as the most active compound against hCA II can interact with
Zn265 through pendant phenyl rings attached to the quinoxalin ring
(Figure 2b). These phenyl rings also formed π–π interactions with
His94. Furthermore, in Figure 2b, several weak hydrophobic
interactions were observed between compound 10i and active site
residues Ala65, Leu198, Leu204, Phe20, and Trp5.
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2.3
Docking results
To evaluate the interaction modes of the most active compounds in
the active site of target enzymes AChE, BChE, hCA I, hCA II, and
α‐glucosidase, docking studies were carried out by using Auto-
DockTools (1.5.6). Compound 10e as the most potent compound
against cholinesterase enzymes AChE and BChE was docked in the
active site of these enzymes. Important components in the active site
of cholinesterase enzymes are the anionic site (AS), catalytic triad
(CT), and peripheral anionic site (PAS).[52] Molecular modeling of
compound 10e in the active site of AChE predicted that sulfur atom
and 3‐fluorophenyl ring of this compound interacted with AS residue
Trp84 (Figure 1a). Compound 10e also formed two other interac-
tions with AS residues Phe300 and Phe301 via 1,3,4‐oxadiazole ring
and phenyl ring of quinoxalin ring and an interaction with PAS re-
sidue Trp279 via 2,3‐diphenylquinoxalin moiety. Furthermore,
3‐fluoro substituent of this compound formed interactions with ac-
tive site residues Ser124 and Gly117. The binding pose of compound
The most potent, new α‐glucosidase inhibitors 10k and 10h were
docked in the active site of the target enzyme. As can be seen in
Figure 3a, 2,3‐diphenylquinoxalin moiety of the most potent com-
pound 10k interacted with active site residues Glu304, Pro309,
Val316, and Ala326. 1,3,4‐Oxadiazole ring of this compound inter-
acted with Arg312 via hydrophobic interaction. 3‐Bromophenyl ring
formed following interactions with α‐glucosidase active site:
a
π–anion interaction with Asp408 through phenyl ring and two hy-
drophobic interactions with Tyr313 and Arg439 through 3‐bromo
substituent. The interaction mode of compound 10h as the second
potent compound against α‐glucosidase is shown in Figure 3b. As can
be seen in this figure, 2,3‐diphenylquinoxalin moiety of compound
10h interacted with Glu304 and Pro309; however, this moiety es-
tablished two additional hydrophobic interactions with Val316 and
Ala326. 1,3,4‐Oxadiazole ring in compound 10k, similar to compound
FIGURE 1 Interaction modes of compound 10e in the active sites of (a) acetylcholinesterase and (b) butyrylcholinesterase

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FIGURE 2 Interaction modes of compound 10f in the active site of human carbonic anhydrase (hCA) I (a) and compound 10i in the active
site of hCA II (b)
FIGURE 3 Interaction mode of (a) compound 10k and (b) compound 10h in the active site of α‐glucosidase
10h, interacted with Arg312. Arg312 formed an additional hydro-
phobic interaction with 3‐chlorophenyl ring of compound 10h in
comparison to compound 10k.
square fluctuation (RMSF) is a measure of dynamics along the se-
quence. For the AChE–10e complex, small motions were observed
throughout the sequence, except a few local peaks (Figure 4c). The
dynamic range was not more than 4 nm for any of the ligand atoms
(Figure 4c, inset).
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2.4
Molecular dynamics (MD) simulation of
Analyses of deviations for the BChE–10e protein–ligand
complex indicated that the structure of BChE becomes relatively
stable after ~15 ns (Figure 4e). RMSD of the ligand indicated an
almost stable ligand conformation during 30 ns of MD simulation
(Figure 4e, inset). Structural fluctuations for the protein residues
indicated slightly higher dynamics in the C‐terminal half of the
sequence and more stability at the N‐terminal side (Figure 4f). The
pattern of dynamics for the ligand 10e demonstrated similarity to
that in the AChE–10e complex (Figure 4f, inset), which may be due
to the similar structure of ligand binding sites between AChE
and BChE.
protein–ligand complexes
Simulations for the four protein–ligand systems were performed to
evaluate their structural stability under dynamic conditions. Final
coordinates of all complexes (Figures 4a,d and 5a,d) indicated that
the ligand remains bound to the protein for all the systems after
30 ns of MD simulation. The root mean square deviation (RMSD)
analysis for the AChE–10e complex revealed that the protein finds a
sustained dynamics after ~15 ns (Figure 4b). The ligand also shows a
limited range of deviations after 10 ns (Figure 4b, inset). Root mean

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FIGURE 4 (a) Final coordinates, (b) Cα‐RMSD, and (c) residue RMSF for AChE in complex with 10e. (d) Final coordinates, (e) Cα‐RMSD, and
(f) residue RMSF for BChE in complex with 10e. All insets show the corresponding plot for the ligand atoms. AChE, acetylcholinesterase; BChE,
butyrylcholinesterase; RMSD, root mean square deviation; RMSF, root mean square fluctuation
FIGURE 5 (a) Final coordinates, (b) Cα‐RMSD, and (c) residue RMSF for hCA I in complex with 10f. (d) Final coordinates, (e) Cα‐RMSD, and
(f) residue RMSF for hCA II in complex with 10i. All insets show the corresponding plot for the ligand atoms. hCA, human carbonic anhydrase;
RMSD, root mean square deviation; RMSF, root mean square fluctuation

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The complex of hCA I with 10f indicated a remarkably stable
dynamics achieved after ~5 ns and preserved until the end of the MD
simulation (Figure 5b). The ligand also had a stable structure all
through the simulation time (Figure 5b, inset). Fluctuations along the
amino acid sequence of hCA I were generally limited, but they were
higher in both N‐ and C‐terminal regions (Figure 5c). The ligand did
not have fluctuations exceeding 0.3 nm for any of its atoms
(Figure 5c, inset). The pattern of dynamics along the time for hCA II
in complex with 10i was similar to that of hCA I, and the structure
had a stable trend from ~5 ns until the end of the 30 ns time of the
simulation (Figure 5e). The ligand structure was also stable, with
small deviations during the simulation time (Figure 5e, inset). Ana-
lysis of regional fluctuations for hCA II demonstrated small devia-
tions in the N‐terminal region of the protein, but the fluctuations
were significantly limited in the remaining regions of the protein
structure (Figure 5f). The ligand had RMSF values smaller than
0.3 nm for most of its atoms (Figure 5f, inset).
obtained white precipitates were filtered off to give pure
2,3‐diphenylquinoxaline‐6‐carboxylic acid (3).
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4.2
Synthesis of ethyl
2,3‐diphenylquinoxaline‐6‐carboxylate (4)
A mixture of 2,3‐diphenylquinoxaline‐6‐carboxylic acid (3) (5 mmol)
in ethanol (20 ml) and H2SO4 was refluxed for 24 h. Then, it was
poured into water and the obtained white precipitates were filtered
off to produce pure ethyl 2,3‐diphenylquinoxaline‐6‐carboxylate (4).
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4.3
Synthesis of
2,3‐diphenylquinoxaline‐6‐carbohydrazide (6)
A mixture of 2,3‐diphenylquinoxaline‐6‐carboxylate (4) (5 mmol) and
hydrazine (5) (15 mmol) in ethanol was refluxed for 24 h. Then, the
reaction mixture was poured into cold water. Formed precipitates
were filtered, and pure obtained compound 2,3‐diphenylquinoxaline‐
6‐carbohydrazide 6 entered the next step of the procedure.
In general, the MD simulations showed that all the
protein–ligand complexes preserved their binding in a dynamic so-
lution environment. This is indicative of a potent and favorable in-
teraction between each of the proteins with its corresponding ligand.
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3
CONCLUSION
4.4
Synthesis of 5‐(2,3‐diphenylquinoxalin‐6‐yl)‐
1,3,4‐oxadiazole‐2‐thiol (8)
Novel quinoxalin‐1,3,4‐oxadiazole derivatives (10a–l) were synthe-
sized and evaluated for their inhibitory activities against metabolic
enzymes AChE, BChE, hCA I, II, and α‐glucosidase. All the title
compounds showed better enzymatic inhibitory activities than ta-
crine as standard ChEI, acetazolamide as standard CA inhibitor,
and acarbose as standard α‐glucosidase inhibitor. Among the
tested derivatives, compound 10e with around twofold higher
inhibitory activities than tacrine was the most potent compound
against AChE and BChE, and compound 10k with 4.45‐fold
higher inhibitory activities than acarbose was the most potent
compound against α‐glucosidase. Moreover, compounds 10f
and 10i, respectively, with 7.37‐ and 6.8‐fold higher inhibitory
activities than acetazolamide, were the most potent compounds
against hCA I and II isoenzymes.
A mixture of 2,3‐diphenylquinoxaline‐6‐carbohydrazide (6) (5 mmol)
and CS2 (7) (10 mmol) in presence of potassium hydroxaide (5 mmol)
in ethanol was refluxed for 8 h. Then, the reaction mixture was
poured into water and filtered to give pure compound 8.
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4.5
General procedure for the synthesis of
2‐(arylthio)‐5‐(2,3‐diphenylquinoxalin‐6‐yl)‐1,3,4‐
oxadiazole derivatives (10a–l)
A mixture of 5‐(2,3‐diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole‐2‐thiol
(8) (1 mmol) and appropriate benzyl bromides (9a–l) (1 mmol) in the
presence of K2CO3 (1.5 mmol) in DMF was stirred at room tem-
perature for 6 h. Then, the reaction mixture was added to cold water
and precipitates were collected by filtration. These precipitates were
recrystallized in ethanol to obtain pure products (10a–l).
Molecular docking studies of these compounds at the active site
of their related target enzymes were performed, and obtained re-
sults revealed these compounds interacted with important residues
in the target enzymes.
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4.5.1
2‐(Benzylthio)‐5‐(2,3‐diphenylquinoxalin‐
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4
EXPERIMENTAL
6‐yl)‐1,3,4‐oxadiazole (10a)
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4.1
Synthesis of 2,3‐diphenylquinoxaline‐6‐
White solid; isolated yield: 94%; mp 187–189°C; IR (KBr, υ): 3057,
;
2922, 1738 cm−1 1H nuclear magnetic resonance (NMR) (400 MHz,
carboxylic acid (3)
dimethyl sulfoxide [DMSO]‐d6) δ 8.67 (d, J = 1.9 Hz, 1H, H‐22), 8.39
(dd, J = 8.8, 1.9 Hz, 1H, H‐20), 8.34 (d, J = 8.8 Hz, 1H, H‐19),
7.57–7.48 (m, 6H, Ar), 7.47–7.33 (m, 8H, Ar), 7.33–7.26 (m, 1H,
Ar), 4.68 (s, 2H, CH2); 13C NMR (101 MHz, DMSO‐d6) δ 165.10
A solution of 3,4‐diaminobenzoic acid (1) (5 mmol) and benzil (2)
(5 mmol) in acetic acid (20 ml) was heated at reflux condition for 1 h.
Then, the reaction mixture was poured into water and the

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(C‐26), 164.77 (C‐23), 155.15, 155.01, 142.22, 140.62, 138.79,
137.10, 130.91, 130.24, 130.21, 130.20, 129.68, 129.63, 129.59,
129.10, 128.62, 128.61, 128.30, 127.63, 127.62, 127.57, 127.56,
124.68, 36.33 (C‐29); anal. calcd. for C29H20N4OS: C, 73.71; H, 4.27;
N, 11.86; found: C, 73.77; H, 4.23; N, 11.89.
4.5.5
2‐(2,3‐Diphenylquinoxalin‐6‐yl)‐5‐[(3‐
fluorobenzyl)thio]‐1,3,4‐oxadiazole (10e)
White solid; isolated yield: 90%; mp 156–158°C; IR (KBr, υ): 3057,
2923, 1734 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.66 (d, J = 1.8 Hz,
1H, H‐22), 8.38 (dd, J = 8.8, 1.8 Hz, 1H, H‐20), 8.32 (d, J = 8.8 Hz, 1H,
H‐19), 7.55–7.48 (m, 4H, Ar), 7.47–7.26 (m, 9H, Ar), 7.22–7.10 (m,
1H, Ar), 4.68 (s, 2H, CH2); 13C NMR (101 MHz, DMSO‐d6) δ 165.17
(C‐26), 164.58 (C‐23), 163.68, 161.26, 155.12, 154.98, 142.21,
140.60, 140.15, 140.07, 138.79, 131.09, 131.07, 131.00, 130.87,
130.24, 130.23, 130.22, 129.68, 129.67, 129.63, 128.60, 127.59,
125.74, 125.71, 125.70, 124.65, 116.49, 116.27, 115.25, 115.04,
35.62 (C‐29); anal. calcd. for C29H19FN4OS: C, 71.00; H, 3.90; N,
11.42; found: C, 71.06; H, 3.921; N, 11.44.
|
4.5.2
2‐(2,3‐Diphenylquinoxalin‐6‐yl)‐5‐[(2‐
methylbenzyl)thio]‐1,3,4‐oxadiazole (10b)
White solid; isolated yield: 95%; mp 163–165°C; IR (KBr, υ): 3057,
2922, 1735 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.68 (d, J = 1.9 Hz,
1H, H‐22), 8.40 (dd, J = 8.7, 1.9 Hz, 1H, H‐20), 8.33 (d, J = 8.7 Hz, 1H,
H‐19), 7.56–7.47 (m, 5H, Ar), 7.45–7.37 (m, 5H, Ar), 7.27–7.22 (m,
2H, Ar), 7.20–7.14 (m, 2H, Ar), 4.74–4.65 (m, 2H, CH2), 2.44 (s, 3H,
CH3); 13C NMR (101 MHz, DMSO‐d6) δ 165.14 (C‐26), 164.56 (C‐23),
155.00, 142.23, 140.62, 138.80, 137.41, 135.32, 134.22, 130.99,
130.92, 130.79, 130.61, 130.25, 130.22, 129.68, 129.63, 129.32,
128.79, 128.61, 128.17, 127.63, 127.60, 126.63, 126.29, 124.69,
34.98 (C‐29), 19.28 (C‐36); anal. calcd. for C30H22N4OS: C, 74.05; H,
4.56; N, 11.51; found: C, 74.06; H, 4.59; N, 11.44.
|
4.5.6
2‐(2,3‐Diphenylquinoxalin‐6‐yl)‐5‐[(4‐
fluorobenzyl)thio]‐1,3,4‐oxadiazole (10f)
White solid; isolated yield: 91%; mp 215–217°C; IR (KBr, υ): 3058,
2924, 1735 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.63 (d, J = 1.8 Hz,
1H, H‐22), 8.42–8.20 (m, 2H, H‐20, H‐19), 7.58 (dd, J = 8.4, 5.5 Hz,
2H, Ar), 7.52–7.47 (m, 3H, Ar), 7.45–7.34 (m, 7H, Ar), 7.28–7.12 (m,
2H, Ar), 4.66 (s, 2H, CH2); 13C NMR (101 MHz, DMSO‐d6) δ 165.11
(C‐26), 164.66 (C‐23), 163.33, 160.90, 155.08, 154.95, 142.19,
140.59, 133.46, 133.44, 133.43, 131.88, 131.80, 131.74, 131.66,
131.56, 131.48, 130.86, 130.24, 130.21, 129.68, 129.63, 128.60,
127.56, 124.64, 116.02, 115.80, 35.49 (C‐29); anal. calcd. for
|
4.5.3
2‐(2,3‐Diphenylquinoxalin‐6‐yl)‐5‐[(3‐
methylbenzyl)thio]‐1,3,4‐oxadiazole (10c)
White solid; isolated yield: 89%; mp 150–152°C; IR (KBr, υ): 3050,
2924, 1739 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.66 (d, J = 1.9 Hz,
1H, H‐22), 8.39 (dd, J = 8.8, 1.9 Hz, 1H, H‐20), 8.33 (d, J = 8.8 Hz, 1H,
H‐19), 7.56–7.48 (m, 4H, Ar), 7.47–7.34 (m, 8H, Ar), 7.25 (t,
J = 7.5 Hz, 1H, Ar), 7.12 (d, J = 7.6 Hz, 1H, Ar), 4.63 (s, 2H, CH2), 2.31
(s, 3H, CH3); 13C NMR (101 MHz, DMSO‐d6) δ 165.06 (C‐26), 164.80
(C‐23), 155.14 (C‐7), 155.00 (C‐8), 142.21, 140.62, 138.80, 138.27,
136.94, 130.89, 130.25, 130.22, 130.18, 129.68, 129.67, 129.64,
129.62, 129.03, 128.96, 128.61, 127.59, 127.55, 126.65, 124.69,
36.34 (C‐29), 21.43 (C‐36); anal. calcd. for C30H22N4OS: C, 74.05; H,
4.56; N, 11.51; found: C, 74.02; H, 4.59; N, 11.50.
C
29H19FN4OS: C, 71.00; H, 3.90; N, 11.42; found: C, 70.98; H, 3.93;
N, 11.49.
|
4.5.7
2‐[(2‐Chlorobenzyl)thio]‐5‐(2,3‐
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10g)
White solid; isolated yield: 93%; mp 159–161°C; IR (KBr, υ): 3052,
2923, 1731 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.66 (d, J = 1.8 Hz,
1H, H‐22), 8.40–8.29 (m, 2H, H‐20, H‐19), 7.71–7.67 (m, 1H, Ar),
7.55–7.49 (m, 5H, Ar), 7.44–7.35 (m, 8H, Ar), 4.74 (s, 2H, CH2); 13C
NMR (101 MHz, DMSO‐d6) δ 165.30 (C‐26), 164.21 (C‐23), 155.12
(C‐7), 154.97 (C‐9), 142.22, 140.58, 138.77, 134.33, 133.84, 132.42,
132.15, 132.06, 130.87, 130.53, 130.23, 130.20, 130.14, 130.00,
129.92, 129.62, 129.13, 128.59, 127.99, 127.63, 127.59, 124.63,
34.71 (C‐29); anal. calcd. for C29H19ClN4OS: C, 68.70; H, 3.78; N,
11.05; found: C, 68.76; H, 3.72; N, 11.09.
|
4.5.4
2‐(2,3‐Diphenylquinoxalin‐6‐yl)‐5‐[(2‐
fluorobenzyl)thio]‐1,3,4‐oxadiazole (10d)
White solid; isolated yield: 91%; mp 215–217°C; IR (KBr, υ): 3053,
2924, 1730 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.63 (d, J = 1.8 Hz,
1H, H‐22), 8.36 (dd, J = 8.8, 1.8 Hz, 1H, H‐20), 8.30 (d, J = 8.8 Hz, 1H,
H‐19), 7.62 (td, J = 7.7, 1.8 Hz, 1H, Ar), 7.52–7.47 (m, 4H, Ar),
7.46–7.35 (m, 7H, Ar), 7.28–7.16 (m, 2H, Ar), 4.69 (s, 2H, CH2); 13C
NMR (101 MHz, DMSO‐d6) δ 165.25 (C‐26), 164.24 (C‐23), 162.14,
159.69, 155.09, 154.95, 142.20, 140.57, 138.77, 132.04, 132.01,
130.86, 130.84, 130.76, 130.24, 130.21, 129.68, 129.63, 128.60,
127.58, 127.55, 125.14, 125.11, 124.60, 124.11, 123.97, 116.15,
115.94, 30.27 (C‐29); anal. calcd. for C29H19FN4OS: C, 71.00; H,
3.90; N, 11.42; found: C, 71.08; H, 3.91; N, 11.45.
|
4.5.8
2‐[(3‐Chlorobenzyl)thio]‐5‐(2,3‐
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10h)
White solid; isolated yield: 95%; mp 186–188°C; IR (KBr, υ): 3051,
2921, 1732 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.66 (d, J = 1.9 Hz,
1H, H‐22), 8.42–8.29 (m, 2H, H‐20, H‐19), 7.65 (t, J = 1.8 Hz, 1H, Ar),

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MIRZAZADEH ET AL.
|
7.55–7.48 (m, 6H, Ar), 7.47–7.31 (m, 7H, Ar), 4.67 (s, 2H, CH2); 13C
NMR (101 MHz, DMSO‐d6) δ 165.19 (C‐26), 164.56 (C‐23), 155.14
(C‐7), 154.98 (C‐8), 142.21, 140.61, 139.95, 138.79, 133.48, 130.95,
130.93, 130.87, 130.25, 130.22, 130.21, 129.68, 129.63, 129.62,
129.47, 128.60, 128.31, 128.22, 128.21, 127.60, 127.59, 124.65,
35.49 (C‐29); anal. calcd. for C29H19ClN4OS: C, 68.70; H, 3.78; N,
11.05; found: C, 68.69; H, 3.81; N, 11.07.
35.42 (C‐29); anal. calcd. for C29H19BrN4OS: C, 63.16; H, 3.47; N,
10.16; found: C, 63.18; H, 3.39; N, 10.19.
|
4.5.12
2‐[(4‐Bromobenzyl)thio]‐5‐(2,3‐
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10l)
White solid; isolated yield: 92%; mp 207–209°C; IR (KBr, υ): 3057,
;
2923, 1731 cm−1 1H NMR (400 MHz, CDCl3) δ 8.77 (d, J = 1.9 Hz,
|
4.5.9
2‐[(4‐Chlorobenzyl)thio]‐5‐(2,3‐
1H, H‐22), 8.43 (dd, J = 8.8, 1.9 Hz, 1H, H‐20), 8.29 (d, J = 8.8 Hz, 1H,
H‐19), 7.61–7.54 (m, 4H, Ar), 7.54–7.46 (m, 2H, Ar), 7.48–7.34 (m,
8H, Ar), 4.54 (s, 2H, CH2); 13C NMR (101 MHz, CDCl3) δ 165.33 (C‐
26), 164.49 (C‐23), 154.94 (C‐7), 154.76 (C‐8), 142.51, 140.87,
138.56, 138.54, 134.69, 132.00, 130.91, 130.47, 129.88, 129.86,
129.46, 129.34, 129.28, 128.54, 128.41, 128.39, 127.86, 127.21,
124.55, 122.27, 36.07 (C‐29); anal. calcd. for C29H19BrN4OS: C,
63.16; H, 3.47; N, 10.16; found: C, 63.11; H, 3.41; N, 10.17.
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10i)
White solid; isolated yield: 96%; mp 162–164°C; IR (KBr, υ): 3054,
2921, 1738 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.64 (d, J = 1.8 Hz,
1H, H‐22), 8.37 (dd, J = 8.8, 1.8 Hz, 1H, H‐20), 8.32 (d, J = 8.8 Hz, 1H,
H‐19), 7.57 (d, J = 8.4 Hz, 2H, Ar), 7.53–7.50 (m, 4H, Ar), 7.44–7.38
(m, 7H, Ar), 7.30 (d, J = 8.4 Hz, 1H, Ar), 4.66 (s, 1H, CH2); 13C NMR
(101 MHz, DMSO‐d6) δ 165.16 (C‐26), 164.59 (C‐23), 155.11 (C‐7),
154.96 (C‐8), 142.20, 140.60, 138.79, 138.77, 136.95, 136.40,
132.90, 132.44, 131.67, 131.48, 131.15, 130.87, 130.54, 130.24,
130.22, 129.69, 129.64, 129.05, 128.86, 128.60, 127.59, 124.64,
35.49 (C‐29); anal. calcd. for C29H19ClN4OS: C, 68.70; H, 3.78; N,
11.05; found: C, 68.63; H, 3.84; N, 11.03.
|
4.6
Bioactivity assays
The inhibitory effects of novel 2‐(arylthio)‐5‐(2,3‐diphenylquinoxalin‐
6‐yl)‐1,3,4‐oxadiazole derivatives (10a–l) on both hCA I and II iso-
enzymes have been described in the study of Verpoorte et al.[54] and
according to an esterase assay using p‐nitrophenyl acetate substrate.
The measurements were spectrophotometrically recorded at
348 nm.[55–57] However, the inhibitory effects of AChE from electric
eel (Electrophorus electricus) and BChE from equine serum were de-
termined according to the method of Ellman et al.[58] They were
recorded spectrophotometrically at 412 nm using acetylthiocholine
iodide and butyrylcholine iodide, and recorded as enzymatic reaction
substrates according to previous studies. 5,5ʹ‐Dithio‐bis(2‐nitro‐
|
4.5.10
2‐[(2‐Bromobenzyl)thio]‐5‐(2,3‐
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10j)
White solid; isolated yield: 94%; mp 211–213°C; IR (KBr, υ): 3051,
2920, 1734 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.69 (d, J = 1.9 Hz,
1H, H‐22), 8.41 (dd, J = 8.8, 1.9 Hz, 1H, H‐20), 8.34 (d, J = 8.8 Hz, 1H,
H‐19), 7.76–7.68 (m, 2H, Ar), 7.55–7.48 (m, 4H, Ar), 7.45–7.37 (m,
7H, Ar), 7.34–7.26 (m, 1H, Ar), 4.74 (s, 2H, CH2); 13C NMR (101 MHz,
DMSO‐d6) δ 165.35 (C‐26), 164.19 (C‐23), 155.18 (C‐7), 155.03 (C‐
8), 142.26, 140.62, 138.80, 135.95, 133.45, 133.44, 132.21, 132.20,
130.91, 130.74, 130.73, 130.25, 130.22, 129.64, 129.62, 128.62,
128.58, 127.67, 127.67, 124.68, 124.67, 124.57, 37.34 (C‐29); anal.
calcd. for C29H19BrN4OS: C, 63.16; H, 3.47; N, 10.16; found: C,
63.14; H, 3.45; N, 10.16.
benzoic acid) was used as
a substrate to measure AChE
activity.[59–61] In addition, the inhibitory effect of these compounds
on the activity of α‐glucosidase from Saccharomyces cerevisiae was
assessed using the p‐nitrophenyl‐D‐glycopyranoside substrate
(p‐NPG) according to the analysis of Tao et al.[62] First, 200 μl of
phosphate buffer was mixed with 40 μl of enzyme in phosphate
buffer (0.15 U/ml, pH 4.7). In addition, after pre‐incubation, 50 μl of
p‐NPG was added to phosphate buffer (5 mM, pH 7.4) and incubated
again at 30°C. According to the previous studies, adsorption was
measured at 405 nm by spectroscopy.[63,64] IC50 values were ob-
tained from activity (%) versus compounds plots, whereas Ki values
were obtained from the Lineweaver–Burk graph. All the experiments
were performed at least three times (n = 3). We calculated all the
results in an Excel file and drew the graphs, and then calculated Ki
values according to the competition or noncompetition.
|
4.5.11
2‐[(3‐Bromobenzyl)thio]‐5‐(2,3‐
diphenylquinoxalin‐6‐yl)‐1,3,4‐oxadiazole (10k)
White solid; isolated yield: 92%; mp 173–175°C; IR (KBr, υ): 3052,
2921, 1737 cm−1; 1H NMR (400 MHz, DMSO‐d6) δ 8.63 (d, J = 1.9 Hz,
1H, H‐22), 8.35 (dd, J = 8.7, 1.9 Hz, 1H, H‐20), 8.29 (d, J = 8.7 Hz, 1H,
H‐19), 7.79 (t, J = 1.8 Hz, 1H, Ar), 7.55 (d, J = 7.7 Hz, 1H, Ar),
7.52–7.47 (m, 5H, Ar), 7.44–7.30 (m, 7H, Ar), 4.65 (s, 2H, CH2); 13C
NMR (101 MHz, DMSO‐d6) δ 165.15 (C‐26), 164.54 (C‐23), 155.06
(C‐7), 154.92 (C‐8), 142.17, 140.57, 140.20, 138.76, 132.34, 131.21,
131.10, 131.08, 130.83, 130.24, 130.21, 130.20, 129.68, 129.67,
129.63, 129.62, 128.68, 128.59, 128.58, 127.55, 124.60, 122.06,
|
4.7
Docking study
Docking studies of compound 10e as the most potent compound
against AChE (PDB Code: 1EVE) and BChE (PDB Code: 1P0I),

MIRZAZADEH ET AL.
11 of 12
|
compound 10f as the most potent compound against hCA I (PDB
Code: 4WR7), compound 10i as the most potent compound against
hCA II (PDB code: 5AML), and compounds 10k and 10h as most
potent compounds against α‐glucosidase (modeled enzyme) were
performed using AutoDockTools 1.5.6.[65] The 3D structures of the
selected entries were generated by MarvineSketch 5.10.4, and then
the pdbqt formats of target enzymes were provided by Auto-
DockTools. AutoDockTools parameters for each of the target en-
zymes were set as follows:
ORCID
Parham Taslimi
http://orcid.org/0000-0002-3171-0633
REFERENCES
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(1) AChE: box size: 40 × 40 × 40 Å, the center of box: x = 2.023,
y = 63.295, z = 67.062
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(5) α‐glucosidase: box size: 40 × 40 × 40 Å, the center of box:
x = 12.5825, y = −7.8955, z = 12.519. Each docked system was
performed by 50 runs of the AUTODOCK search (by the
Lamarckian genetic algorithm). Finally, the best pose of each
studied compound was analyzed by Discovery Studio 2019
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2019, 352, e1800359.
|
4.7.1
MD simulations
[15] F. Erdemir, D. Barut Celepci, A. Aktaş, Y. Gök, R. Kaya, P. Taslimi,
Y. Demir, İ. Gülçin, Bioorg. Chem. 2019, 91, 103134.
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2019, 125, 12.
To study the stability of the protein structures bound to their cor-
responding ligands, MD simulations were implemented for the four
protein–ligand systems using the docked complexes as initial co-
ordinates. Simulations were performed by Gromacs software version
2020, applying Gromos‐54a7 force field. Periodic boundary conditions
were applied to the systems in all directions.[66] The ligand topologies
defined by PRODRG were corrected by using other residues as
template.[67,68] Adequate number of counterions were added to neu-
tralize the total charge of each system. To eliminate local clashes,
energy minimization was performed using the steepest descent algo-
rithm, which was continued until the maximum force was
<1000 kJ·mol−1·nm−1. Solvent equilibration in the system was achieved
by applying position restraints. In this step, isothermal–isochoric con-
ditions (NVT ensemble) were applied for 200 ps using Nose–Hoover
temperature bath to establish a temperature of 300 K. Then, pressure
equilibrium was implemented under isothermal–isobaric conditions
(NPT ensemble) using Parrinello–Rahman barostat for 200 ps to reach
the pressure of 1 bar. During the simulation, bond constraints were
applied by the LINCS algorithm. The range for van der Waals interac-
tions was set as 1 nm. Long‐range electrostatics calculated by the
particle‐mesh Ewald method was also measured up to a radius of 1 nm.
All the systems were simulated for 30 ns with 2‐fs time steps, saving
data every 10 ps. Output coordinates and trajectories were analyzed by
visual inspection, RMSD, and RMSF. Structures were visualized with
PyMol (www.pymol.org).
[17] E. Bursal, A. Aras, Ö. Kılıç, P. Taslimi, A. C. Gören, İ. Gulçin, J. Food
Biochem. 2019, 43, e12776.
[18] G. Provensi, F. Carta, A. Nocentini, C. T. Supuran, F. Casamenti,
M. B. Passani, S. Fossati, Int. J. Mol. Sci. 2019, 20, 4724.
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2019, 33, e22261.
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[21] C. Çağlayan, Y. Demir, S. Küçükler, P. Taslimi, F. M. Kandemir,
İ. Gulçin, J. Food Biochem. 2019, 43, e12720.
[22] K. Küçükoğlu, H. İ. Gül, P. Taslimi, İ. Gülçin, C. T. Supuran, Bioorg.
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[23] A. Maharramov, R. Kaya, P. Taslimi, M. Kurbanova, A. Sadigova,
V. Farzaliyev, A. Sujayev, İ. Gulçin, Arch. Pharm. 2019, 352,
e1800317.
[24] E. Güzel, U. M. Koçyiğit, B. S. Arslan, M. Ataş, P. Taslimi,
F. Gökalp, M. Nebioğlu, İ. Şişman, İ. Gulçin, Arch. Pharm. 2019,
352, e1800292.
[25] İ. Timur, U. M. Koçyiğit, T. Dastan, S. Sandal, A. O. Ceribasi,
P. Taslimi, İ. Gülçin, M. Koparir, M. Karatepe, M. Ciftci, J. Biochem.
Mol. Toxicol. 2019, 33, e22239.
[26] Ç. Bayrak, P. Taslimi, H. S. Kahraman, İ. Gülçin, A. Menzek, Bioorg.
Chem. 2019, 85, 128.
[27] B. Yiğit, R. Kaya, P. Taslimi, Y. Işık, M. Karaman, M. Yiğit, İ. Özdemir,
İ. Gulçin, J. Mol. Struct. 2019, 1179, 709.
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