New 1,2,3-triazole–(thio)barbituric acid hybrids as urease inhibitors: Design, synthesis, in vitro urease inhibition, docking study, and molecular dynamic simulation

Article abstract of DOI:10.1002/ardp.202000023

A new series of 1,2,3-triazole–(thio)barbituric acid hybrids 8a–n was designed and synthesized on the basis of potent pharmacophores with urease inhibitory activity. Therefore, these compounds were evaluated against Helicobacter pylori urease. The obtained result demonstrated that all the synthesized compounds, 8a–n, were more potent than the standard urease inhibitor, hydroxyurea. Moreover, among them, compounds 8a, 8c–e, 8g,h, and 8k,l exhibited higher urease inhibitory activities than the other standard inhibitor used: thiourea. Docking studies were performed with the synthesized compounds. Furthermore, molecular dynamic simulation of the most potent compounds, 8e and 8l, showed that these compounds interacted with the conserved residues Cys592 and His593, which belong to the active site flap and are essential for enzymatic activity. These interactions have two consequences: (a) blocking the movement of a flap at the entrance of the active site channel and (b) stabilizing the closed active site flap conformation, which significantly reduces the catalytic activity of urease. Calculation of the physicochemical and topological properties of the synthesized compounds 8a–n predicted that all these compounds can be orally active. The ADME prediction of compounds 8a–n was also performed.

Full text of DOI:10.1002/ardp.202000023

Received: 5 February 2020
Revised: 26 May 2020
Accepted: 30 May 2020
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DOI: 10.1002/ardp.202000023
F U L L P A P E R
New 1,2,3‐triazole–(thio)barbituric acid hybrids as urease
inhibitors: Design, synthesis, in vitro urease inhibition,
docking study, and molecular dynamic simulation
Mohammad S. Asgari1 | Homa Azizian2 | Mohammad Nazari Montazer3
Maryam Mohammadi‐Khanaposhtani4 | Mehdi Asadi3 | Saghi Sepehri5
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Parviz R. Ranjbar1 | Rahmatollah Rahimi6 | Mahmood Biglar7 | Bagher Larijani7
Massoud Amanlou3 | Mohammad Mahdavi7
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1School of Chemistry, College of Science, University of Tehran, Tehran, Iran
2Department of Medicinal Chemistry, School of Pharmacy‐International Campus, Iran University of Medical Sciences, Tehran, Iran
3Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran
4Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
5Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran
6Department of Chemistry, Iran University of Science and Technology, Tehran, Iran
7Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
Correspondence
Massoud Amanlou, Department of Medicinal
Abstract
Chemistry, Faculty of Pharmacy and
A new series of 1,2,3‐triazole–(thio)barbituric acid hybrids 8a–n was designed and
Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences,
synthesized on the basis of potent pharmacophores with urease inhibitory activity.
Tehran, Iran.
Therefore, these compounds were evaluated against Helicobacter pylori urease. The
obtained result demonstrated that all the synthesized compounds, 8a–n, were more
potent than the standard urease inhibitor, hydroxyurea. Moreover, among them,
compounds 8a, 8c–e, 8g,h, and 8k,l exhibited higher urease inhibitory activities than
the other standard inhibitor used: thiourea. Docking studies were performed with
the synthesized compounds. Furthermore, molecular dynamic simulation of the most
potent compounds, 8e and 8l, showed that these compounds interacted with the
conserved residues Cys592 and His593, which belong to the active site flap and are
essential for enzymatic activity. These interactions have two consequences: (a)
blocking the movement of a flap at the entrance of the active site channel and (b)
stabilizing the closed active site flap conformation, which significantly reduces the
catalytic activity of urease. Calculation of the physicochemical and topological
properties of the synthesized compounds 8a–n predicted that all these compounds
can be orally active. The ADME prediction of compounds 8a–n was also performed.
Email: Amanlou@tums.ac.ir
Mohammad Mahdavi, Endocrinology and
Metabolism Research Center, Endocrinology
and Metabolism Clinical Sciences Institute,
Tehran University of Medical Sciences, Tehran
1417653761, Iran.
Email: momahdavi@tums.ac.ir
K E Y W O R D S
1,2,3‐triazole, barbituric acid, docking study, molecular dynamic simulation, thiobarbituric acid,
urease inhibition
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Arch Pharm. 2020;e2000023.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
https://doi.org/10.1002/ardp.202000023

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ASGARI ET AL.
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1
INTRODUCTION
urease. Furthermore, docking and molecular dynamic studies were
performed to further investigate the interaction, orientation, and
conformation of these compounds at the active site of urease.
Gastric disorders such as ulceration, gastritis, gastric carcinomas, and
primary gastric lymphomas are etiologically related with Helicobacter
pylori infections.[a] H. pylori is a Gram‐negative microaerophilic bac-
terium that infects up to 50% of the world's human population.[2] The
use of a triple therapy containing two antibiotic and a proton pump
inhibitor is the most common treatment for H. pylori infection.
However, drug resistance of H. pylori against antibiotics is rising
sharply, and the design of new agents with various therapeutic me-
chanisms in this area is an attractive target for pharmacists.[3] Urease
(EC 3.5.1.5) is the main enzyme for the hydrolysis of urea into am-
monia, and given that this process is an essential pathway for the
energy supply of bacteria, inhibition of urease can be useful for the
elimination of bacteria. In this regard, several studies have demon-
strated that urease inhibitors can be useful for treatment of H. pylori
infection.[4]
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The synthetic route for the preparation of 1,2,3‐triazole–(thio)‐
barbituric acid hybrids 8a–n has been depicted in Scheme 1. Initially,
a mixture of 4‐hydroxy‐3‐methoxybenzaldehyde 1 and propargyl
bromide 2 in the presence of K2CO3 in dimethylformamide was
stirred for 3 hr at room temperature to afford 3‐methoxy‐4‐(prop‐2‐
ynyloxy)benzaldehyde 3. In contrast, benzyl azides 5a–i were pro-
duced in situ from reaction between benzyl bromides 4a–i and NaN3
in the presence of NEt3. Then, 1,2,3‐triazole derivatives 6a–i were
synthesized via a click reaction between benzyl azides 5a–i, com-
pound 3, sodium ascorbate, and CuSO4·5H2O at room temperature
for 16–24 h. Finally, compounds 6 reacted with barbituric acid 7a or
thiobarbituric acid 7b in ethanol and piperidine to give the desired
compounds 8a–n.
Barbituric acid derivatives have various biological properties
such as sedative–hypnotic, antimicrobial, antifungal, anticancer, an-
ticonvulsant, and anti‐urease properties.[5–10] However, another
important scaffold in designing new bioactive compounds is the
1,2,3‐triazole ring.[11] There are several reports in the literature
concerning urease inhibitory activity of barbituric acid derivatives
and 1,2,3‐triazole derivatives.[12–17] For example, our research team
has already reported 5‐methylene (thio)barbituric acid derivative A
and 1,2,3‐triazole derivative B with a high inhibitory activity against
H. pylori urease (Figure 1).[15,16] Therefore, in continuation of our
efforts to derive new potent urease inhibitors, 1,2,3‐triazole–(thio)
barbituric acid hybrids 8a–n were designed and synthesized via a
click reaction. These compounds were evaluated against H. pylori
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2.2
Urease inhibition
1,2,3‐Triazole–(thio)barbituric acid hybrids 8a–n were evaluated for
their in vitro inhibitory activities against H. pylori urease in compar-
ison with standard inhibitors hydroxyurea and thiourea (Table 1). The
IC50 values of compounds 8a–n (IC50 values = 8.10–76.53 µM)
FIGURE 1 The design strategy for
1,2,3‐triazole–(thio)barbituric acid
hybrids 8a–n

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SCHEME 1 The synthesis procedure for compounds 8a–n. Reagents and conditions: (a) K2CO3, dimethylformamide, room temperature, 3hr;
(b) NaN3, NEt3, H2O/t‐ (1:1), room temperature, 1hr; (c) sodium ascorbate, CuSO4·5H2O, room temperature, 16–24hr; (d) ethanol, piperidine,
room temperature, 24hr
demonstrated that they were all superior to that of hydroxyurea
(IC50 value = 100 0.2 µM). Also, 8 compounds 8a, 8c–e, 8g,h, and
8k,l of 14 synthesized compounds were more potent than thiourea
against urease (IC50 value = 22.0 0.03 µM). Among the synthesized
compounds, the most potent compounds were compounds 8l and 8e
with IC50 values = 8.102 and 8.30 µM, respectively.
altered to optimize the anti‐urease activity. Among the barbituric
acid derivatives, the most potent compound was compound 8e with
4‐chloro substituent on pendant phenyl ring. A change in the position
of chlorine atom on the phenyl ring from C4 to C2, producing com-
pound 8d (IC50 value = 10.22 µM), slightly diminished the inhibitory
activity, whereas the introduction of the second chloro substituent
on C2 of pendant phenyl ring of compound 8e, as in 2,4‐dichloro
derivative 8f, led to a significant decrease in the activity inhibitory
activity. The second most potent compound in this series was
Structurally, the title compounds are divided into two series:
barbituric acid derivatives 8a–i and thiobarbituric acid derivatives
8j–n. In each series, the substituent on the pendant phenyl ring was
TABLE 1 The urease inhibitory activity of 1,2,3‐triazole–(thio)barbituric acid hybrids 8a–n
Compound
X
R
IC50 (µM)a
Compound
X
O
S
S
S
S
S
–
R
IC50 (µM)a
8a
8b
8c
8d
8e
8f
O
O
O
O
O
O
O
O
2‐CH3
2‐F
9.32 0.16
45.03 0.41
16.76 0.25
10.22 0.13
8.30 0.09
31.58 0.32
14.81 0.15
13.63 0.24
8i
4‐NO2
2‐CH3
2‐F
2‐Cl
4‐Cl
3‐Br
–
24.38 0.22
47.96 0.53
12.01 0.21
8.10 0.17
25.70 0.32
76.53 0.46
100 0.20
8j
4‐F
8k
2‐Cl
8l
4‐Cl
8m
2,4‐Dichloro
3‐Br
8n
8g
8h
Hydroxyurea
Thiourea
4‐Br
–
–
22.0 0.03
aValues are the mean standard error of the mean. All experiments were performed at least three times.

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2‐methyl derivative 8a. The replacement of 2‐methyl substituent
with 2‐fluoro substituent dramatically decreased activity against
urease, as observed in compound 8b. The movement of fluoro sub-
stituent from 2‐position to 4‐position of pendant phenyl group
caused a significant increase in the inhibitory activity. Bromosub-
stituted compounds 8g and 8h showed an approximately same in-
hibitory activity against urease. Inhibitory activities of these
compounds were more than thiourea, and the inhibitory activity of
4‐nitro derivative 8i was slightly lower than thiourea.
manner, as with increasing concentrations of this compound, Vmax
was not affected, but there was an increase in Km. Furthermore, the
value of the inhibition constant (Ki) for compound 8l was calculated
from secondary replotting of Lineweaver–Burk plots, and Ki was
found to be 9.7 0.02 μM (Figure 2b).
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2.4
Docking study
Among the thiobarbituric acid derivatives 8j–n, the most potent
compound was 2‐chloro derivative 8l. This compound was also the
most potent compound among the synthesized compounds 8a–n. The
replacement of chloro substituent with fluoro or methyl substituent,
respectively, led to a slight decrease and a significant decrease in
inhibition, whereas movement of chloro substituent from C2 to C4 of
pendant phenyl ring, as in compound 8m, led to a significant decrease
in the inhibitory activity. Among the synthesized compounds, the less
potent compound was 3‐bromo derivative 8n of thiobarbituric series.
The comparison of IC50 values of barbituric acid derivatives with
their corresponding thiobarbituric acid analogs against H. pylori ur-
ease revealed that barbituric acid analogs 8a, 8e, and 8g, respec-
tively, with substituents 2‐CH3, 4‐Cl, and 3‐Br were more active than
their thiobarbituric acid analogs 8j, 8m, and 8n. In contrast, H. pylori
urease inhibitory activity of 2‐fluoro and 2‐chloro derivatives 8b and
8d of barbituric acid series was less than their thiobarbituric acid
analogs 8k and 8l.
A docking study was carried out to understand the interaction modes
of the synthesized compounds in the urease active site, and these
interactions were compared with standard drug interaction in the
active site. For this purpose, a crystal structure of urease from jack
bean (JBU) with PDB ID: 4H9M was retrieved from RCSB Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do).[18,19] Acet-
ohydroxamic acid (AHA) as a urease inhibitor is bound to the active
site of JBU. Therefore, the reliability of the applied docking protocol
was assessed by redocking of AHA into the active site of JBU. The
key characteristic of a good docking program is its ability to re-
produce the experimental or crystallographic binding modes of li-
gands. To test this, a ligand is taken out of the X‐ray structure of its
protein–ligand complex and redocked into its binding site. The
docked binding mode is then compared with the experimental
binding mode, and the root mean square deviation (RMSD) is calcu-
lated; a prediction of a binding mode is considered successful if the
RMSD is below a certain value (usually <2.0 Å). The superimposed
structures between the docked and the crystallographic AHA over
JBU with an acceptable RMSD value within the cut‐off limit (1.02 Å)
are shown in Figure 3. This protocol was then similarly applied to all
synthesized compounds, 8a–n.
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2.3
Kinetic study of urease inhibition
To determine urease inhibition type, a kinetic study was performed
on the most potent compound 8l. Moreover, urea was selected as a
substrate. As can be seen in Figure 2a, Lineweaver–Burk plots de-
monstrated that compound 8l inhibited urease in a competitive
The performed docking procedure was then applied to eval-
uate the interaction between newly synthetized compounds,
8a–n, at the JBU active site in comparison to thiourea as a re-
ference urease inhibitor. The top scoring pose of all compounds
FIGURE 2 (a) Lineweaver–Burk plots for the inhibition of urease by compound 8l; (b) secondary replotting of Lineweaver–Burk plots for
determination of Ki

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FIGURE 3 The location of jack bean urease active site over C‐terminal (αβ)8 TIM barrel domain. The inset shows a close‐up representation
of the active site; the acetohydroxamic acid co‐crystallized and the corresponding redocked form are represented in green and cyan color,
respectively
was analyzed inside the binding site of JBU. In the binding model,
all the compounds are successfully occupied in the bi‐nickel ac-
tive site cavity.
Additionally, Figure 4b demonstrates that the thiobarbituric acid
ring appears to make some distance deviation from the Ni–Ni center,
which is due to the steric character of the sulfur atom.
Figure 4 shows that the fitting‐in mechanism of barbituric acid
and thio‐barbiturate moiety in the active site of the enzyme is quite
similar. The 1,2,3‐triazol–benzylidene moiety adapts by a flexible
conformation in the large hydrophobic opening of the active site
pocket (Figure 4a), whereas the (thio)barbituric acid ring tends to
orient toward the two nickel atoms through the ionized Ni (Ni2+) and
the carbonyl group at C4 position of the (thio)barbiturate ring
(Figure 4b), which is similar to the behavior of the carbonyl oxygens
in the AHA.
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2.5
Molecular dynamic simulation
To understand the criteria for rational designing of urease inhibitors,
it is necessary to uncover the structural perturbations incurred by
the most potent compounds (compounds 8e and 8l) over urease and
the effect of these compounds on the active site environment in
comparison to thiourea as a urease inhibitor standard.
FIGURE 4 (a) A representation of the
docking poses of compounds relative to
bi‐nickel center and active site flap over jack
bean urease active site. (b) A close‐up
illustration of the (thio)barbiturate ring. The
active site flap is shown in green color

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complexed with thiourea maintained an overall stability after 10 ns of
MD simulation time with higher fluctuation stabilizing at an average
of 3.75 Å (Figure 5, green line), whereas the bound state of com-
pounds 8e and 8l displayed a longer equilibration time (after 7 ns of
MD simulation) with obviously lower fluctuations (3 and 2.6 Å for the
complex of 8e–urease and 8l–urease, respectively; Figure 5, yellow
and red lines).
Root mean square fluctuation (RMSF) refers to the fluctuation of
the Cα atom coordinates from its average position throughout the si-
mulation. This indicates the flexibility of the protein backbone structure.
Loops with loosely organized structure have high RMSF value while
helixes and sheets represent lower RMSF value. Comparing RMSF va-
lues of urease–compound complexes shows that the residues of the flap
region covering the active site, 590–606, would have significantly lower
RMSF value in urease–8e and urease–8l rather than urease–thiourea
complex (Figure 6a). Based on crystal structures of the ureases, besides
the conserved residues in the active site, most of ureases share con-
served residues that make up the mobile flap, which covers the active
site.[20] In JBU, the residues comprising the mobile flap are 590–606 on
α subunit part of the enzyme.
FIGURE 5 Root mean square deviation (RMSD) of the urease Cα
complexed with thiourea (in green) and compounds 8e (in yellow) and
8l (in red) for over 30‐ns MD simulation time
RMSD of the protein's backbone from its initial to final con-
formation is applied over 30 ns molecular dynamic (MD) simulation
to study the stability of the protein–ligand complex. The RMSD value
of each urease–ligand complex indicates that the employed simula-
tion time has been enough to obtain an equilibrium structure over
the simulation time (Figure 5). Thus, the structures at the MD equi-
librium state are used to investigate the structural specificity of the
ligand–protein complexes. The RMSD simulation showed that urease
The other regions that have lower RMSF values in urease–8e and
urease–8l complex were residues 435–440, 474, and 636–637
(Figure 6b).
Furthermore, Figure 6b shows that compounds 8e and 8l well
occupied the active pockets of urease and tightly anchored the
FIGURE 6 (a) RMSF of the urease Cα complexed with thiourea (in green), compounds 8e (in yellow) and 8l (in red); (b) ligand binding location
for over 30 ns MD simulation time. The α‐helical and β‐strand regions are highlighted in red and blue backgrounds, respectively

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FIGURE 7 (a) MD simulated flap distance between Ala440 and Ile599 in thiourea (blue), compound 8e (yellow), and compound 8l (red) in
urease‐bound state. (b) Representative snapshots from MD simulations where the flap covering the active site can adopt open conformations of
the flap (blue) and closed conformation (yellow and red). The zoomed image of the active site residues shows catalytic residues in green and nickel
atoms in purple
helix‐turn‐helix motif over the active site cavity (vertical green line),
which reduces the flexibility of flap residues (590–609) by interacting
with key amino acid residues and results in the inhibition of urease
activity.
Figure 8c shows barbituric acid ring of compound 8e tightly
coordinated along the metal bi‐nickel center for the whole simulation
time. For the first 20 ns of MD simulation time, the 5‐benzylidine
moiety and C4 carbonyl group of barbituric acid ring formed a π–π
stack and an H‐bond with His593 and Cys592, respectively.
Figure 8d shows that the mentioned contacts disappeared and were
substituted by interactions with Phe605 and Arg609 from one part
alongside with Arg349 and Ala440 at the other part of the active site
cavity for the rest of the MD simulation time.
To study the behavior of the active site covering flap over the
course of the MD simulation, the distances between Ile599 at the tip
of the flap and Ala440 at the entrance of the active site channel in
the urease–compound complexes are recorded and analyzed.
Figure 7a displays representative Ile599–Ala440 separations for the
urease–compound complex. In the thiourea‐bound state, the se-
paration distance of these residues is about 32 Å, corresponding as
open flap conformation, whereas in the case of compounds 8e and 8l,
this distance varies only slightly, oscillating within 25 and 21 Å,
respectively, which is related to the closed flap conformation
(Figure 7b). Based on this closed flap conformation, it is proposed
that compounds 8e and 8l inhibit urease activity by stabilizing the
reaction intermediate during the ureolytic reaction.[21]
Compound 8l depicts a similar orientation and interaction with
8e (metal coordination forming through thiobarbituric acid ring,
H‐bond, and π–π stacking with Cys592 and His593, respectively),
except that the two former interactions persisted for higher MD
simulation time (48% and 56%, respectively; Figure 8e,f).
It is noteworthy that Cys592 is one of the key residues in the
active site. Ligand interacting with Cys592 and His593 seems to be
most important for urease inhibition due to a decrease in the flex-
ibility of mobile flap covering the active site entrance, followed by
inhibition of the ureolytic activity.
The molecular interactions of thiourea and the compounds 8e
and 8l over the binding site of JBU are represented in Figure 8.
Thiourea was found to be deeply bound in the active site due to
its smaller size, and it interacted with an ionic coordinated complex
(two Ni ions complexed with all of their coordinated residues in-
cluding His407, His409, His519, and His545; Figure 8a) for the first
quarter part of MD simulation time, and for the rest of the simulation
time, it formed an H‐bond with Thr442, Thr467, and Cys405 through
both of its NH2 groups (Figure 8b).
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2.6
Screening of pharmacokinetic properties
To predict the pharmacokinetic properties of target compounds
8a–n, some of physicochemical properties of these compounds are
calculated and listed in Table 2. Physicochemical properties

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FIGURE 8 A two‐dimensional (left columns) and timeline (right columns) representation of ligand–residue interactions that occur during the
simulation time, which include urease‐bound state of (a,b) thiourea, (c,d) compound 8e, and (e,f) compound 8l
included molecular weight, the number of H‐bond acceptors
(HBA), the number of H‐bond donors (HBD), and octanol/water
partition coefficients (logP). Moreover, the numbers of rotatable
bonds (RBC) as a topological factor were collected (Table 2). One
of the rules used to predict whether a drug is orally active is
Lipinski's rule of 5 (MW ≤ 500, HBA ≤ 10, HBD ≤ 5, and logP ≤ 5).[22]
A drug candidate should not violate more than one of Lipinski's rules
to be active orally. As can be seen in Table 2, all the synthesized
compounds passed Lipinski's rule. The RBC is a very good descriptor
for oral bioavailability of drug candidates, and previous data revealed
that compounds with RBC < 10 have a high oral bioavailability in
rats.[23] As can be seen in Table 2, all the title compounds 8a–n have
RBC = 7, so it is expected that these compounds will have a good oral
bioavailability.

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TABLE 2 Physicochemical properties of the synthesized
compounds 8a–n
TABLE 4 Toxicity prediction of the synthesized compounds 8a–n
Entry Ames test Carcino mouse Carcino rat hERG inhibition
Compound
MW
HBA
7
HBD
2
LogP
2.27
1.95
1.95
2.35
2.35
2.9
RBC
7
8a
8b
8c
8d
8e
8f
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Negative
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Positive
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
8a
8b
8c
8d
8e
8f
447.45
451.41
451.41
467.87
467.87
502.31
512.32
512.32
478.42
463.51
467.48
483.93
483.93
528.38
7
2
7
Positive
7
2
7
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
7
2
7
7
2
7
7
2
7
8g
8h
8i
8g
8h
8i
7
2
2.62
2.62
1.82
2.82
2.49
2.89
2.89
3.16
7
7
2
7
9
2
7
8j
8j
6
2
7
8k
8l
8k
8l
6
2
7
6
2
7
8m
8n
8m
8n
6
2
7
6
2
7
Abbreviations: HBA, number of H‐bond acceptors; HBD, number of
H‐bond donors; logP, the octanol–water partition coefficient;
MW, molecular weight; RBC, number of rotatable bonds.
8a–n have poor permeability to Caco‐2 cells and skin. Moreover,
these compounds have high human oral absorption and protein‐
binding plasma. An in silico ADME study also predicted that these
compounds did not have an inhibition effect on CYP3A4, CYP2C19,
and CYP2D6; however, they probably inhibited CYP2C9. Further-
more, compounds 8a–n can be substrates for CYP3A4. In contrast,
these compounds are not a substrate for CYP2D6.
Absorption, distribution, metabolism, excretion, and toxicity
(ADMET) prediction of the synthesized compounds 8a–n was per-
formed using online software PreADMET, and the obtained results
are presented in Table 3. As can be seen in this table, compounds
TABLE 3 ADME prediction of the synthesized compounds 8a–n
Skin
permeabilitya
CYP3A4
CYP3A4
CYP2C9
CYP2C19
inhibition
CYP2D6
CYP2D6
Entry Caco‐2a HIA%a
PBP%a
substrate
inhibition
inhibition
substrate
inhibition
8a
8b
8c
8d
8e
8f
20.665 94.9864 −3.94844
20.907 94.3737 −4.21172
87.9195 Substrate
89.6537 Substrate
87.4573 Substrate
88.7605 Substrate
88.0221 Substrate
88.4792 Substrate
87.4305 Substrate
87.4278 Substrate
89.8316 Substrate
97.7060 Substrate
87.2295 Substrate
88.8011 Substrate
88.8991 Substrate
89.9210 Substrate
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
20.014 94.3737 −4.24112
20.883 96.7318 −4.004
20.305 96.7318 −4.03017
20.586 97.4518 −3.97189
20.648 97.3802 −3.89729
20.593 97.3802 −3.89845
17.469 71.9521 −3.78887
21.045 97.7060 −3.94447
21.014 97.5517 −4.20359
21.505 97.8670 −3.99523
20.987 97.8673 −4.02237
21.539 97.5362 −3.88953
8g
8h
8i
8j
8k
8l
8m
8n
Abbreviations: ADME, absorption, distribution, metabolism, excretion; PBP, protein‐binding plasma; HIA, human oral absorption.
aThe recommended ranges for Caco‐2: <25 is poor and >500 is great; HIA: >80% is high and <25% is poor; skin permeability: −8.0 to −1.0; and PBP: >80%
is high and <25% is poor.

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General procedure for the synthesis of
1,2,3‐triazole derivatives (6a–i)
In terms of toxicity, PreADMET software predicted that all the
synthesized compounds are mutagens and have medium risk for
cardiotoxicity (hERG inhibition; Table 4). As can be seen in Table 4,
compounds 8d–f, 8j, and 8l,m do not have carcinogenic effects on
mouse and rat, whereas compounds 8b,c have carcinogenic effects on
both of them. Furthermore, compounds 8a, 8g–i, 8k, and 8n do not
have carcinogenic effects on mouse.
4.1.2
A mixture of 3‐methoxy‐4‐(prop‐2‐ynyloxy)benzaldehyde 3 (1 mmol),
sodium ascorbate, and CuSO4·5H2O (7 mol%) was added to the
freshly prepared benzyl azide derivatives 5a–i, and the obtained
mixture was stirred at room temperature for 16–24 hr. Then, the
reaction mixture was poured into crushed ice and precipitated pro-
ducts 6a–i were filtered off, washed with cold water, and purified by
recrystallization (ethyl acetate).
|
2.7
Cytotoxicity
Cytotoxicity of the most active compounds, 8a, 8e, and 8l, was
evaluated against the human normal cell line HDF using 3‐(4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay.
This study revealed that at 200 µM, test compounds were non‐
cytotoxic against the HDF cell line.
|
4.1.3
General procedure for the synthesis of
1,2,3‐triazole–(thio)barbituric acid hybrids 8a–n
The mixture of 1,2,3‐triazole derivatives 6a–i (0.5 mmol), (thio)bar-
bituric acid derivatives 7a,b (0.5 mmol), ethanol (2 ml), and piperidine
(2 drops) was stirred at room temperature for 24 hr. Then, the re-
action mixture was poured into cold ware and obtained precipitates
were dried after filtration to obtain pure products 8a–n.
|
3
CONCLUSION
In general, the 1,2,3‐triazole ring and (thio)barbituric acid as effective
urease inhibitor pharmacophores were attached together. Fourteen
derivatives of the 1,2,3‐triazole–(thio)barbituric scaffold were syn-
thesized and their inhibitory activity against H. pylori urease was
determined. The obtained results demonstrated that all the synthe-
sized compounds were more potent than standard inhibitor hydro-
xyurea and among them compounds 8a, 8c–e, 8g,h, and 8k,l were also
more potent than standard inhibitor thiourea. Docking study of
compounds 8a–n showed that all these compounds successfully oc-
cupied the active site cavity of urease. Molecular dynamic simulation
of the most potent compounds 8e and 8l proposed that these com-
pounds inhibited urease activity by stabilizing the reaction inter-
mediate during the ureolytic reaction. Furthermore, the title
compounds 8a–n can be orally active.
5‐(3‐Methoxy‐4‐{[1‐(2‐methylbenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐
benzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8a)
White solid; yield: 66%, mp: 185–190°C. IR (KBr): 1,702 (C═O), 1,627
.
(C═C alkene), 2,940 (C–H aromatic) cm−1 1H NMR (500 MHz, di-
methyl sulfoxide [DMSO]‐d6) δ 2.21 (s, 3H, CH3), 3.60 (s, 3H, OCH3),
5.03 (S, 2H, N–CH2), 5.67 (s, 2H, O–CH2), 5.91 (s, 1H, alkene), 6.55 (d,
J = 6.3 Hz, 1H, Ar), 6.63 (s, 1H, Ar), 6.87 (d, J = 7.5 Hz, 1H, Ar), 7.07 (d,
J = 6.4 Hz, 1H, Ar), 7.32–7.12 (m, 3H, Ar), 8.13 (s, 1H, triazole), 9.99 (s,
2H, NH) ppm. 13C NMR (126 MHz, DMSO) δ 18.09, 50.39, 54.8, 61.4,
111.1, 113.1, 115.0, 115.1, 122.4, 122.7, 124.4, 130.2, 137.8, 142.8,
144.3, 147.9, 150.2, 158.5, 160.5, 163.7, 165.3 ppm. Anal. calcd. for
C23H21N5O5: C, 61.74; H, 4.73; N, 15.65. Found: C, 61.58; H, 4.83;
N, 15.49.
5‐(4‐{[1‐(2‐Fluorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8b)
White solid; yield: 59%; mp: 208–213°C. IR (KBr): 1,705 (C═O),
|
4
EXPERIMENTAL
1,636 (C═C alkene), 2,924 (C–H aromatic) cm−1 1H NMR (500 MHz,
.
|
4.1
Chemistry
DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.03 (s, 2H, N–CH2), 5.67 (s, 2H,
O–CH2), 5.92 (s, 1H, alkene), 6.56 (d, J = 8.1 Hz, 1H, Ar), 6.64 (s, 1H,
Ar), 6.89 (d, J = 8.2 Hz, 1H, Ar), 7.24 (dt, J = 15.3, 8.4 Hz, 2H, Ar), 7.33
(t, J = 7.3 Hz, 1H, Ar), 7.45–7.38 (m, 1H, Ar), 8.23 (s, 1H, triazole),
10.08 (s, 2H, NH) ppm. 13C NMR (126 MHz, DMSO) δ 46.3, 54.8,
61.4, 111.1, 113.1, 115.0, 115.1, 122.4, 122.7, 124.4, 130.2, 137.8,
142.8, 144.3, 147.9, 150.2, 158.5, 160.5, 163.7, 165.3 ppm. Anal.
calcd. for C22H18FN5O5: C, 58.54; H, 4.02; N, 15.51. Found: C, 58.41;
H, 4.16; N, 15.39.
|
4.1.1
General
Melting points were taken on a Kofler hot‐stage apparatus. 1H and 13C
nuclear magnetic resonance (NMR) spectra (provided as Supporting In-
formation Data) were recorded on Bruker FT‐500, using tetra-
methylsilane as an internal standard. The infrared (IR) spectra were
obtained on a Nicolet Magna FT‐IR 550 spectrophotometer (KBr disks).
Elemental analyses were carried out with an Elementar Analysensysteme
GmbH VarioEL CHN mode. Compounds 3 and 5a–i were obtained ac-
cording to described pathway in our newly published article.[24,25]
The InChI keys of the investigated compounds, together with
some biological activity data, are provided as Supporting Informa-
tion Data.
5‐(4‐{[1‐(4‐Fluorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8c)
White solid; yield: 79%, mp: 116–118°C. IR (KBr): 1,716 (C═O), 1,649
(C═C alkene), 2,921 (C–H aromatic) cm−1 1H NMR (500 MHz,
.

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DMSO‐d6) δ 3.59 (s, 3H, OCH3), 5.01 (s, 2H, N–CH2), 5.81 (s, 2H,
O–CH2), 5.92 (s, 1H, alkene), 6.53 (d, J = 8.3 Hz, 1H, Ar), 6.59 (s, 1H,
Ar), 6.89 (d, J = 8.4 Hz, 1H, Ar), 7.06 (s, 1H, Ar), 7.39 (d, J = 8.7 Hz, 1H,
Ar), 7.48 (t, J = 8.0 Hz, 2H, Ar), 8.18 (s, 1H, triazole), 9.86 (s, 2H, NH).
13C NMR (126 MHz, DMSO) δ 44.07, 48.02, 54.95, 112.16, 113.17,
118.30, 120.30, 120.32, 124.28, 124.86, 125.22, 128.42, 129.83,
131.07, 131.15, 135.39, 136.38, 142.26, 144.55, 148.00, 172.27,
173.62 ppm. Anal. calcd. for C22H18FN5O5: C, 58.54; H, 4.02; N,
15.51. Found: C, 58.39; H, 4.15; N, 15.68.
DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.04 (s, 2H, N–CH2), 5.62 (s, 2H,
O–CH2), 5.92 (s, 1H, alkene), 6.56 (s, 1H, Ar), 6.64 (s, 1H, Ar), 6.87 (s,
1H, Ar), 7.30 (s, 1H, Ar), 7.32 (d, J = 7.7 Hz, 1H, Ar), 7.55 (s, 2H, Ar),
8.28 (s, 1H, triazole), 10.13 (s, 2H, NH). 13C NMR (126 MHz, DMSO) δ
51.4, 54.9, 61.5, 111.1, 113.2, 121.3, 124.3, 126.5, 130.2, 130.4,
137.7, 138.1, 142.9, 144.3, 147.9, 150.2, 163.0, 165.9 ppm. Anal.
calcd. for C22H18BrN5O5: C, 51.58; H, 3.54; N, 13.67. Found: C,
51.41; H, 3.67; N, 13.84.
5‐(4‐{[1‐(4‐Bromobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8h)
White solid; yield: 78%, mp: 211–213°C. IR (KBr): 1,688 (C═O), 1,639
5‐(4‐{[1‐(2‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8d)
White solid; yield: 76%, mp: 196–200°C. IR (KBr): 1,680 (C═O), 1,645
(C═C alkene), 2,922 (C–H aromatic) cm−1 1H NMR (500 MHz,
.
(C═C alkene), 2,926 (C–H aromatic) cm−1
.
1H NMR (500 MHz,
DMSO‐d6) δ 3.59 (s, 3H, OCH3), 5.02 (s, 2H, N–CH2), 5.59 (s, 2H,
O–CH2), 5.92 (s, 1H, alkene), 6.56 (s, 1H, Ar), 6.64 (s, 1H, Ar), 6.89 (d,
J = 8.2 Hz, 1H, Ar), 7.28 (d, J = 8.1 Hz, 2H, Ar), 7.57 (d, J = 8.1 Hz, 2H,
Ar), 8.25 (s, 1H, triazole), 10.10 (s, 2H, NH). 13C NMR (126 MHz,
DMSO) δ 51.4, 55.4, 61.7, 111.8, 113.4, 118.8, 121.4, 124.0, 129.7,
131.1, 134.9, 137.7, 142.9, 144.3, 147.9, 150.2, 163.3, 165.5 ppm.
Anal. calcd. for C22H18BrN5O5: C, 51.58; H, 3.54; N, 13.67. Found: C,
51.33; H, 3.39; N, 13.51.
DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.05 (s, 2H, N–CH2), 5.72 (s, 2H,
O–CH2), 5.92 (s, 1H, alkene), 6.57 (d, J = 8.3 Hz, 1H, Ar), 6.64 (s, 1H,
Ar), 6.89 (d, J = 8.4 Hz, 1H, Ar), 7.19 (d, J = 8.6 Hz, 1H, Ar), 7.38 (dt,
J = 19.7, 7.1 Hz, 2H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 8.22 (s, 1H,
triazole), 10.06 (s, 2H, NH). 13C NMR (126 MHz, DMSO) δ 43.3, 50.0,
54.9, 61.4, 90.5, 111.0, 111.1, 113.1, 113.2, 118.3, 124.5, 127.2,
129.0, 132.0, 133.0, 137.8, 142.9, 144.5, 147.9, 150.2, 163.2, 163.7,
165.3 ppm. Anal. calcd. for C22H18ClN5O5: C, 56.48; H, 3.88; N,
14.97. Found: C, 56.57; H, 3.74; N, 14.86.
5‐(3‐Methoxy‐4‐{[1‐(4‐nitrobenzyl)‐1H‐1,2,3‐triazol‐5‐yl)methoxy}‐
benzylidene}pyrimidine‐2,4,6(1H,3H,5H)‐trione (8i)
5‐(4‐{[1‐(4‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8e)
White solid; yield: 70%, mp: 189–194°C. IR (KBr): 1,705 (C═O), 1,597
White solid; yield: 70%, mp: 201–207°C. IR (KBr): 1,336–1,352
(NO2), 1,678 (C═O), 1,605 (C═C alkene), 3,035 (C–H aromatic) cm−1. 1H
NMR (500 MHz, DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.13 (s, 2H, N–CH2),
5.99–5.72 (m, 3H), 7.09 (s, 1H, Ar), 7.45–7.29 (m, 1H, Ar), 7.56 (d,
J = 8.2 Hz, 2H, Ar), 8.48–8.17 (m, 4H), 10.02–9.52 (m, 2H, NH). 13C NMR
(126 MHz, DMSO) δ 47.6, 51.1, 55.0, 61.6, 112.5, 113.0, 118.7, 123.4,
124.0, 125.4, 128.5, 130.8, 137.8, 138.5, 139.2, 143.4, 149.8, 150.3,
163.9, 165.3, 167.4 ppm. Anal. calcd. for C22H18N6O7: C, 55.23; H, 3.79;
N, 17.57. Found; C, 55.37; H, 3.84; N, 17.65.
(C═C alkene), 2,966 (C–H aromatic) cm−1 1H NMR (500 MHz,
.
DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.04 (s, 2H, N–CH2), 5.70 (s, 2H,
O–CH2), 5.91 (s, 1H, alkene), 6.88 (s, 1H, Ar), 7.29 (s, 2H, Ar), 7.47 (s,
2H, Ar), 7.69 (s, 1H, Ar), 8.24 (s, 1H, triazole), 9.86 (s, 1H, NH), 10.06
(s, 1H, NH). 13C NMR (126 MHz, DMSO) δ 49.5, 54.9, 60.9, 109.2,
111.1, 112.2, 113.2, 118.3, 124.5, 125.8, 127.3, 128.5, 130.9, 133.0,
137.6, 142.5, 144.9, 147.4, 150.3, 162.1, 165.3 ppm. Anal. calcd. for
C22H18ClN5O5: C, 56.48; H, 3.88; N, 14.97. Found: C, 56.31; H, 3.71;
N, 15.08.
5‐(3‐Methoxy‐4‐{[1‐(2‐methylbenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐
benzylidene)‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐dione (8j)
White solid; yield: 86%, mp: 96–98°C. IR (KBr): 1,715
5‐(4‐{[1‐(2,4‐Dichlorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8f)
White solid; yield: 74%, mp: 200–203°C. IR (KBr): 1,700 (C═O), 1,626
(C═C alkene), 2,926 (C–H aromatic) cm−1. 1H NMR (500 MHz, DMSO‐d6)
δ 3.81 (s, 3H, OCH3), 5.27 (s, 2H, N–CH2), 5.73 (s, 2H, O–CH2), 5.99 (s,
1H, alkene), 7.32 (s, 1H, Ar), 7.40 (s, 2H, Ar), 7.48 (s, 1H, Ar), 7.56 (s, 1H,
Ar), 7.72 (s, 1H, Ar), 8.32 (s, 1H, triazole), 9.65–10.00 (m, 2H, NH). 13C
NMR (126 MHz, DMSO) δ 49.6, 54.9, 61.1, 109.7, 112.2, 125.0, 125.2,
127.4, 128.6, 129.4, 131.5, 133.5, 141.6, 146.2, 152.2, 166.8, 168.7 ppm.
Anal. calcd. for C22H17Cl2N5O5: C, 52.61; H, 3.41; N, 13.94. Found: C,
52.75; H, 3.62; N, 14.05.
(C═O), 1,646 (C═C alkene), 2,928 (C–H aromatic) cm−1 1H NMR
.
(500 MHz, DMSO‐d6) δ 2.30 (CH3, s, 3H), 3.59 (s, 3H, OCH3),
5.03 (s, 2H, N–CH2), 5.61 (s, 2H, O–CH2), 5.93 (s, 1H, alkene), 6.89 (d,
J = 8.4 Hz, 1H, Ar), 7.06 (s, 2H, Ar), 7.18 (s, 1H, Ar), 7.23 (s, 2H, Ar), 7.32 (s,
1H, Ar), 8.12 (s, 1H, triazole), 10.04 (s, 2H, NH) ppm. 13C NMR (126 MHz,
DMSO) δ 18.07, 43.92, 50.36, 54.95, 113.04, 118.28, 120.33, 122.44,
124.12, 125.70, 127.74, 128.07, 128.17, 129.85, 129.91, 129.95, 133.60,
133.71, 135.71, 142.62, 154.63, 161.75 ppm. Anal. calcd. for
C23H21N5O4S: C, 59.60; H, 4.57; N, 15.11. Found: C, 59.74; H, 4.68;
N, 15.01.
5‐(4‐{[1‐(2‐Fluorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐
dione (8k)
5‐(4‐{[1‐(3‐Bromobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)pyrimidine‐2,4,6(1H,3H,5H)‐trione (8g)
White solid; yield: 75%, mp: 212–214°C. IR (KBr): 1,692 (C═O), 1,646
White solid; yield: 87%; mp: 108–110°C. IR (KBr): 1,713 (C═O),
(C═C alkene), 1,156, 2,925 (C–H aromatic) cm−1
.
1H NMR (500 MHz,
1,653 (C═C alkene), 2,919 (C–H aromatic) cm−1 1H NMR (500 MHz,
.

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|
Biological evaluations
DMSO‐d6) δ 3.71 (s, 3H, OCH3), 5.11 (s, 2H, N–CH2), 5.74–5.61 (m,
3H), 7.07 (s, 2H, Ar), 7.22 (t, J = 7.6 Hz, 1H, Ar), 7.27 (d, J = 9.5 Hz, 1H,
Ar), 7.34–7.30 (m, 2H, Ar), 7.44–7.40 (m, 1H, Ar), 8.25 (s, 1H, tria-
zole), 10.11 (m, 2H, NH) ppm. 13C NMR (126 MHz, DMSO) δ 42.09,
46.03, 52.96, 109.02, 110.11, 111.14, 111.79, 116.31, 118.36,
122.29, 122.88, 126.46, 127.84, 129.08, 133.41, 136.23, 140.27,
142.54, 145.98, 149.26, 161.21, 170.28 ppm. Anal. calcd. for
C22H18FN5O4S: C, 56.53; H, 3.88; N, 14.98. Found: C, 56.69; H, 3.74;
N, 15.07.
4.2
|
4.2.1
Urease inhibitory assay
All chemicals and urease (JBU; EC 3.5.1.5) were purchased from
Sigma‐Aldrich. Urease inhibition assay of compounds 8a–n was
screened at the concentration of 0–10 mg/ml by the modified spec-
trophotometric method developed by Berthelot using alkaline
phenol–hypochlorite reaction.[26] This method is based on the reac-
tion of ammonia (NH3) with hypochlorite (OCl−) to form a mono-
chloramine and a subsequent reaction of monochloramine with
phenol to form blue‐colored indophenols whose absorbance is mea-
sured at 625 nm.[16] The assay mixture solution consisted of urea
(850 μl, 30 mM, in phosphate buffer 100 mM, pH 7.4) and test com-
pounds (100 μl [0–10 mg/ml], phosphate buffer 100 mM, pH 7.4) with
a total volume of 950 μl. The reactions were initiated by the addition
of 15 μl of urease enzyme solution in phosphate buffer (100 mM, pH
7.4, 3 mg/ml). Urease activity was determined by measuring ammonia
concentration after 30 min of enzymatic reaction. The ammonia
concentration was determined by adding 100 μl incubated solution to
a mixture of 500 μl of solution A (containing 5.0 g phenol and 25 mg
of sodium nitroprusside in 500 ml distilled water) and 500 μl of so-
lution B (contained 2.5 g sodium hydroxide and 4.2 ml of sodium
hypochlorite [5% chlorine] in 500 ml distilled water) and again in-
cubated at 37°C for 30 min. The absorbance of developed blue‐
colored indophenols was read at 625 nm. The activity of uninhibited
urease was designated as the control activity of 100%. Hydroxyurea
and thiourea were used as positive controls. The percentage of in-
hibition was determined according to this formula: [1 − (T/C)] × 100,
where T is the absorbance of the test compound in the presence of
enzyme and C is the absorbance of the solvent as negative control in
the presence of enzyme. The mean and standard error of the mean
was calculated from data of three independent experiments and
calculated by SPSS v16. IC50 values for all compounds 8a–n were
calculated using GraphPad Prism 5 software (GraphPad Software
Inc., San Diego, CA).
5‐(4‐{[1‐(2‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐
dione (8l)
White solid; yield: 68%, mp > 250°C. IR (KBr): 1,703 (C═O), 1,643
(C═C alkene), 2,928 (C–H aromatic) cm−1
.
1H NMR (500
MHz, DMSO‐d6) δ 3.61 (s, 3H, OCH3), 5.05 (s, 2H, N–CH2), 5.72 (s,
2H, O–CH2), 5.92 (s, 1H, alkene), 6.57 (d, J = 8.3 Hz, 1H, Ar), 6.64
(s, 1H, Ar), 6.89 (d, J = 8.4 Hz, 1H, Ar), 7.18 (s, 1H, Ar), 7.43–7.30
(m, 2H, Ar), 7.52 (d, J = 7.8 Hz, 1H, Ar), 8.22 (s, 1H, triazole),
10.09 (s, 2H, NH). 13C NMR (126 MHz, DMSO) δ 50.0, 54.9, 61.5,
111.1, 113.4, 118.8, 124.5, 127.2, 129.8, 132.0, 137.8, 142.7,
144.3, 148.0, 150.2, 163.3, 165.3 ppm. Anal. calcd. for
C22H18FN5O4S: C, 54.60; H, 3.75; N, 14.47. Found: C, 54.72; H,
3.63; N, 14.58.
5‐(4‐{[1‐(4‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐
dione (8m)
White solid; yield: 76%, mp: 111–113°C. IR (KBr): 1,706 (C═O),
1,648 (C═C alkene), 2,926 (C–H aromatic) cm−1
.
1H NMR
(500 MHz, DMSO‐d6) δ 3.60 (s, 3H, OCH3), 5.02 (s, 2H, N–CH2),
5.61 (s, 2H, O–CH2), 5.92 (s, 1H, alkene), 6.53 (d, J = 9.3 Hz, 1H,
Ar), 6.91 (d, J = 9.1 Hz, 1H, Ar), 7.07 (d, J = 4.5 Hz, 1H, Ar), 7.35 (d,
J = 8.0 Hz, 4H, Ar), 8.24 (s, 1H, triazole), 10.02 (s, 2H, NH) ppm.
13C NMR (126 MHz, DMSO) δ 51.01, 55.89, 62.51, 112.13,
114.24, 119.29, 125.47, 128.19, 130.66, 130.83, 133.00, 133.74,
138.83, 143.71, 145.27, 148.97, 165.04, 167.20, 170.06 ppm.
Anal. calcd. for C22H18FN5O4S: C, 54.60; H, 3.75; N, 14.47. Found
C, 54.47; H, 3.76; N, 14.61.
|
4.2.2
Kinetic study
5‐(4‐{[1‐(3‐Bromobenzyl)‐1H‐1,2,3‐triazol‐5‐yl]methoxy}‐3‐
methoxybenzylidene)‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐
dione (8n)
To determine the type of urease inhibition of the synthesized com-
pounds, Lineweaver–Burk plots were used according to literature.[27]
Urease inhibition was measured by varying the concentrations of
urea (1–4 mM) in the presence of different concentrations of the
most potent compound 8l (0, 5, 10, 15 µM). The inhibitory constant
(Ki) was determined from secondary replotting of Lineweaver–Burk
plots. All experiments were conducted in triplicate.
White solid; yield: 73%, mp: 215–217°C. IR (KBr): 1,701 (C═O),
1,630 (C═C alkene), 2,924 (C–H aromatic) cm−1
.
1H NMR
(500 MHz, DMSO‐d6) δ 3.73 (s, 3H, OCH3), 5.19 (s, 2H, N–CH2),
5.65 (s, 2H, O–CH2), 5.92 (s, 1H, alkene), 7.24 (s, 1H, Ar), 7.33 (s,
3H, Ar), 7.41 (s, 1H, Ar), 7.48 (s, 1H, Ar), 7.64 (s, 1H, Ar), 8.24 (s,
1H, triazole), 9.78 (s, 2H, NH). 13C NMR (126 MHz, DMSO) δ 49.6,
54.9, 61.1, 109.2, 112.2, 125.0, 125.2, 127.4, 128.6, 129.4, 131.5,
133.5, 141.6, 146.2, 148.8, 152.2, 166.4, 168.7, 172.7 ppm. Anal.
calcd. for C22H18BrN5O4S: C, 50.01; H, 3.43; N, 13.25. Found: C,
54.15; H, 3.55; N, 13.37.
|
4.2.3
Molecular modeling procedure
To find out the interaction mode of designed molecules over urease
enzyme, Maestro Molecular Modeling platform (version10.5) by

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Schrödinger LLC was employed.[28] The X‐ray crystallographic structure
of JBU (in complex with AHA) was downloaded from the Protein Data
Bank (PDB ID: 4H9M; www.rcsb.org). As urease is reported to be func-
tionally active in the monomeric state, all the docking studies were per-
formed on a single monomer. In addition, prosthetic group and cofactors
were not directly involved in urease inhibition, so they were totally re-
moved before docking investigation. Water molecules and co‐crystallized
ligands were removed from the enzymes' crystallographic structures. The
2D structures of all synthesized compounds were drawn in Marvin
15.10.12.0 program (http://www.chemaxon.com) and converted into a
pdb file. The Protein Preparation Wizard and the LigPrep module were
used to prepare protein and ligand structure properly.[12] The missing
side chains of the proteins were filled using the Prime tool and missing
residues were updated.
During the simulation, every 1,000 ps of the actual frame was stored.
The dynamic behavior and structural changes of the systems were
analyzed by the calculation of the RMSD and RMSF. Subsequently,
the energy‐minimized structure calculated from the equilibrated
trajectory system was evaluated for investigation of each
ligand–protein complex interaction.
|
4.2.5
Prediction of pharmacokinetic properties
The MW and logP values of the synthesized compounds 8a–n were
calculated using ChemDraw 18.2. HBD and HBA were calculated by
the means of MarvineSketch 5.10.4. RBC was calculated using
AutoDock Tools (ver. 1.5.6). Furthermore, ADMET prediction of the
synthesized compounds was performed by preADMET online server
(http://preadmet.bmdrc.org/).[29]
The accurate side chain, backbone conformational changes, or
both during ligand binding at the active site of urease enzyme
were predicted by induced fit docking (IFD) method using Glide
software (Schrödinger LLC, 2018). The AHA binding site was used to
generate the grid for IFD calculation. The maximum 20 poses with
receptor and ligand van der Waals radii of 0.7 and 0.5, respectively,
were considered. Residues within 5 Å of the AHA at the active site
were refined, followed by side chain optimization. Structures whose
Prime energy was more than 30 kcal/mol were eliminated on the
basis of extra precious Glide docking.
|
4.2.6
Cytotoxicity
In vitro cytotoxicity was determined by the MTT assay in triplicate
according to the literature.[30]
ORCID
Saghi Sepehri
http://orcid.org/0000-0001-7340-0468
Mohammad Mahdavi
http://orcid.org/0000-0002-4171-7310
|
4.2.4
Molecular dynamic simulation
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How to cite this article: Asgari MS, Azizian H, Nazari
Montazer M, et al. New 1,2,3‐triazole–(thio)barbituric acid
hybrids as urease inhibitors: Design, synthesis, in vitro urease
inhibition, docking study, and molecular dynamic simulation.
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