Dihydropyrimidones: A ligands urease recognition study and mechanistic insight through in vitro and in silico approach

Article abstract of DOI:10.1007/s00044-020-02643-z

Scaffold varied dihydropyrimidone derivatives 1–20 were evaluated for their selective urease inhibitory kinetics potential. Compounds 1, 2, 3, 4, 5, 6, and 12 were found to be the most promising urease inhibitors and showed the inhibition (K_i v

Full text of DOI:10.1007/s00044-020-02643-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02643-z
ORIGINAL RESEARCH
Dihydropyrimidones: A ligands urease recognition study and
mechanistic insight through in vitro and in silico approach
1 ●
2 ●
3 ●
1 ●
2,4 ●
Farman Ali Khan Shahbaz Shamim Nisar Ullah Muhammad Arif Lodhi Khalid Mohammed Khan
Kanwal2 Farman Ali Sahib Gul Afridi Shahnaz Perveen Ajmal Khan
2 ●
1 ●
5 ●
6
●
Received: 5 July 2020 / Accepted: 23 September 2020
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Scaffold varied dihydropyrimidone derivatives 1–20 were evaluated for their selective urease inhibitory kinetics potential.
Compounds 1, 2, 3, 4, 5, 6, and 12 were found to be the most promising urease inhibitors and showed the inhibition (Ki
values) within the range of 9.9 ± 0.5 to 18.3 ± 0.4 µM. Lineweaver–Burk plot, Dixon plot and their secondary replots confirm
that all these molecules have followed competitive mode of inhibition. Docking arrangements (MOE) revealed that all the
ligands bind in the active site and therefore compete with substrate urea. Molecular docking studies of all compounds have
confirmed the binding interactions of various ligands with the amino acid residues as well as Ni atoms of active site.
Furthermore, these compounds 1–20 were also tested for their cytotoxicity against human neutrophils and plants and were
found to be non-toxic.
●
●
●
●
Keywords Dihydropyrimidones Enzyme kinetics Michaelis–Menten kinetics Neutrophil based cytotoxicity
Phytotoxicity
Introduction
atoms in its core structure. It catalyzes the hydrolysis of
urease into NH3 and carbamate which spontaneously
hydrolyzed further and generates another molecule of
ammonia along with carbon dioxide [1–4]. The speed of
this reaction is approximately 10 times to that of un-
catalyzed reaction. In soluble form, these two molecules of
ammonia and a molecule of carbonic acid are in equilibrium
with their protonated and deprotonated state which results in
a net increase of pH to some extent [5].
Urease (urea amidohydrolase, EC 3.5.1.5) belongs to the
family of metalloenzyme that comprises of two nickel
1
4
*
*
Muhammad Arif Lodhi
arifbiochem@hotmail.com
Ajmal Khan
Ureases are widely distributed in nature and can be found
in various plants, fungi, algae, yeasts, bacteria, some
invertebrates, and in soil [6–8]. All these ureases have same
common catalytic functions but different protein structures.
Plants need nitrogen-containing nutrients for their growth
and germination, urea is the most common fertilizer that
provides essential nutrients to the plants for growth and can
be easily metabolized with the action of urease enzyme that
is present in plants and the microbes as well in the soil [9].
Thus, it is clear that urease plays a vital role in nitrogen
metabolism of many plants as well as microorganisms [10].
Moreover, the importance of urease enzyme in plant
growth and germination there are various side effects that
are associated with the hyperactivity of this enzyme in
agriculture. The hyperactivity of urease produces an excess
amount of ammonia thus leading to environmental
ajmalchemist@yahoo.com
1
2
Department of Biochemistry, Abdul Wali Khan University,
Mardan, Khyber Pakhtunkhwa 23200, Pakistan
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
3
4
Chemistry Department, King Fahd University of Petroleum &
Minerals, Dhahran 31261, Saudi Arabia
Department of Clinical Pharmacy, Institute for Research and
Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal
University, P.O. Box 1982, Dammam 31441, Saudi Arabia
5
6
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr.
Salimuzzaman Siddiqui, Karachi 75280, Pakistan
Natural and Medical Sciences Research Center, University of
Nizwa, Birkat-ul-Mouz 616, Nizwa, Sultanate of Oman

Medicinal Chemistry Research
problems and economical loss [8]. The high concentration
of ammonia in soil at first diminishes the availability of urea
as nutrient for growth of plants, secondly toxicity of
ammonia affect the germination of seeds, and seedling
growth thus resulting in the depriving and death of plants
nitrate trihydrate (Cu(NO ) .3H O) at 80–90 °C. The tar-
3
2
2
geted compounds were characterized by spectroscopic
1
13
techniques like IR, EI-MS, H-, and C-NMR (Table 1).
The detail spectroscopic data were published in our pre-
vious article [16].
[
11]. In order to recover the efficacy of urea and to control
the economic loss, various compounds like acetohy-
droxamic acids, phosphoramides, and phenylpho-
sphorodiamidate etc were used to inhibit the unusual
hydrolysis as well as to increase the time for the absorbance
of surface-applied urea into the soil [12–14].
Biology
Urease enzyme exists in plants, bacteria, fungi, and soil.
Urease catalyzes the hydrolysis of urea to produce ammonia
and carbamate ions. These carbamate ions are then
Similarly, in the field of medicinal and veterinary sci-
ences, urease acts as a virulent factor to produce various
human pathogeneses like urolithiasis, gastric, and peptic
ulcers which leads to carcinoma if left untreated [15–18].
The other pathogenic conditions associated with urease are
pyelonephritis, urinary catheter encrustation, hepatic ence-
phalopathy, and hepatic coma [8, 18–20].
decomposed to produce NH3 and CO . X-ray crystal-
2
lographic studies of urease enzyme have indicated the
presence of two nickel (ІІ) atoms in their active site which
are bridged through oxygen. The Ni ions also showed
coordination with N-atoms of imidazole, carboxylate group,
and H O molecule [25]. The coordination mechanism of
2
inhibitors with the active site of enzyme must be clearly
understood in order to recognize the inhibition potential of
these inhibitors. Since 1920, the scientists are trying to
discover an accurate mechanism of action of urease but till
to date it remain a matter of scientific debate [26]. During
the period of 1950–1970, when the structure, mechanism
and biocatalysis of enzymes were studied, jack bean urease
was considered to be the most proficient, stable, and highly
specific biocatalyst [26–28]. The initial mechanism of
urease was proposed by Zerner and his coworkers, who
Nevertheless, the most efficient approach to control the
complications of urease in agriculture as well as in human
beings is to discover a variety of potent and safe urease
inhibitors. To date many natural products as well as syn-
thetic compounds have been reported as possible inhibitors
of urease. Many synthetic compounds like hydroxyurea,
flurofamide, and hydroxamic acid have shown potential
inhibition against this enzyme. However, after in vivo trials,
some of these compounds have been banned due to their
toxic or unstable nature; for example, acetohydroxamic acid
was confirmed as teratogenic in rats [21–24]. In the same
way, most of the identified inhibitors were harmful,
unstable, expensive, and toxic at high concentration or may
have any other side effects. Therefore, there is a direct need
to design, establish, synthesize, and explore new classes of
compounds against urease enzyme to identify promising
candidates for drug development. Our research group had
explored the synthetic dihydropyrimidone derivatives as
potential urease inhibitors. All these compounds were
synthesized and their in vitro urease inhibition is already
reported [16]. The basic purpose of our current investigation
is to study kinetics, molecular docking, and toxicities of
suggested that one nickel atom activates the H O molecules
2
while the other Ni activates the substrate urea [5]. The three
dimensional study of urease active site indicates that car-
boxylate moiety of cysteine residue keep the urea in a stable
resonance form. It has already been known that active site
of urease isolated from different sources have closely rela-
ted sequence of amino acids.
The synthetic molecules 1–20 have basic skeleton of
dihydropyrimidone with variable substituents at aryl part.
All these compounds have inhibited the enzyme very
strongly. Kinetically, it was also identified that the ligands
have shown the strength of inhibition in a concentration
dependent approach. The type of inhibition was determined
by Lineweaver–Burk plots, the reciprocal of the rate of the
reaction was plotted against the reciprocal of substrate
concentrations to monitor the effect of inhibitor on both Km
and Vmax. Type of inhibition determines the inhibition
pathway of enzyme as well as the binding moieties of
inhibitors. In all these cases, Vmax remained fixed, while Km
values were increased, which indicates that enzyme was
inhibited all the time at the active site. Three different
synthetic dihydropyrimidone derivatives to gain
a
mechanistic insights and considerably high efficacy without
any side effects in order to get rid of several health, agri-
culture, environmental, and economical problems.
Results and discussion
Chemistry
methods were applied to calculate the K values. First, the
slope of each line in the Lineweaver–Burk plots was plotted
i
Dihydropyrimidone derivatives (1–20) were synthesized in
one pot by reacting urea, acetylacetone and aryl aldehydes
in solvent-free condition in presence of catalyst “copper
against various concentrations of inhibitors. In a second
method the K values were calculated when different con-
i
centrations of inhibitors were plotted against 1/Kmapp, which

Medicinal Chemistry Research
Table 1 Chemical structures of different dihydropyrimidone derivatives 1–20
Compounds
1
Molecular
formula
Compoun
ds
Molecular
formula
R
R
C
13
H
13
N
2
O
3
11
C
16
H
19
N
2
O
4
2
3
4
5
C
13
H
13
N
2
O
3
12
C
15
H
H
H
H
16ClN
14ClN
12ClN
2
2
2
O
4
3
2
C
13
H
13
N
2
O
4
13
14
15
C
14
C
13
C
15
O
O
C
13
H13BrNO
3
C
C
C
14
H
H
H
15
N
N
N
2
O
O
O
4
16BrN
2
2
O
4
3
6
7
14
15
2
4
16
17
C
14
H14BrN
O
15
17
2
5
C
14
H
14FN
2
O
3
8
9
C
14
C
15
C
15
H
15
H
17
H
17
N
2
N
2
N
2
O
3
O
3
O
4
18
19
20
C
14
H
14FN
2
O
3
C
14
H
12
F
3
N
2
O
2
1
0
C
16
H
18
N
2
O
2

Medicinal Chemistry Research
Table 2 Inhibitory potential of
dihydropyrimidones (1–20)
against jack bean urease
Enzyme Compounds Ki (µM)
SEM
Km (mM) Km (mM)
Vmax (µmol/
min)
Vmaxapp Type of
−1
±
app
inhibition
Urease
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
12.3 ± 0.2
12.7 ± 0.7
13.4 ± 1.1
9.9 ± 0.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
7.6
9.0
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
106
103
108
107
102
107
103
107
104
102
102
103
107
107
105
107
103
107
106
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
9.5
6.9
11.1 ± 0.3
18.3 ± 0.4
22.2 ± 0.6
30.3 ± 0.5
24.5 ± 1.1
26.5 ± 0.1
24.2 ± 0.1
13.0 ± 0.9
11.9 ± 1.1
16.7 ± 0.4
11.2 ± 0.3
19.1 ± 0.4
14.0 ± 0.6
10.2 ± 0.5
14.0 ± 1.1
40.0 ± 0.1
8.0
10.7
6.6
8.8
10.1
8.4
0
1
2
3
4
5
6
7
8
9
0
7.3
9.1
9.4
5.9
8.0
10.7
6.5
8.9
10.1
9.4
was intercept on x-axis. Thirdly, direct measurement of Ki
values was made from Dixon plots as an intercept on x-axis.
From the Lineweaver–Burk plots, Dixon plots along with
their secondary replots, it was also confirmed that the entire
series have inhibited the enzyme competitively with low Ki
values (9.09 ± 0.05–40.0 ± 0.1 µM) which alternatively
confirm their strength of inhibition. Furthermore, Km, Ki,
Vmax, Vmaxapp and Kmapp values of each compound along
with their type of inhibition are listed in Table 2. These four
types of graphs for compound 20 are presented in Fig. 1.
For structure-based drug designing, prediction of accu-
rate protein-ligand interaction geometries were required.
Thus, for this purpose molecular docking studies of all
compounds (1–20) were performed, which helps to generate
suitable configurations and conformations of ligands that
fits well in the active site of urease and to measure their
bond lengths to confirm their strength as well as to support
the type of inhibition. Initially, redocking of native inhibitor
K (dissociation constant or inhibition constant) was
i
determined from nonlinear regression analysis by Dixon
plot and secondary Lineweaver–Burk plot at various con-
centrations of compounds (1–20), Km (Michaelis–Menten
constant) is equal to the reciprocal of x-axis intersection,
Vmax (maximal velocity) is equal to the reciprocal of y-axis
intersection of each line for each concentration of com-
pounds (1–20) in the Lineweaver–Burk plot. The Vmaxapp is
equal to the reciprocal of y-axis intersection of each line for
each concentration of compounds (1–20) in Dixon plot
(Each point in Lineweaver–Burk and represents the mean of
three determinations).
Molecular docking plays a key role in drug designing by
providing the binding mechanism of ligands with their
target proteins. In the present study, molecular docking of
synthetic analogs 1–20 was performed by using MOE
2009–2010 software. It has been proved that some of the
compounds adjusted well in the active pocket of enzyme by
making strong interactions with specific amino acid residues
and Ni ions. Docking results of most active compounds
have been discussed (Table 3).
(
acetohydroxamic acid) was proceeded several times so as
to validate the docking protocol. Then all the compounds
were subjected to molecular docking to understand about
the in-depth mechanism of inhibition. It was also repeated
several times and lastly the best images for each compound
with more interactions and less docking scores were
selected for further analysis. From the docking poses, it was
identified that almost all compounds showed coordination
with the nickel metallocentre and some have proved as
potent competitive inhibitors of jack bean urease (Table 3).
Compound 1 has inhibited the urease very strongly by
adjusting itself in the active pocket through four strong
interactions with different amino acid residues. His323 has
made a very strong acidic interaction with the carbonyl
oxygen of pyrimidone moiety showing bond length of
1.74 Å. Kcx220 and His222 have given a couple of polar
interactions with the hydrogen of hydroxy group in the

Medicinal Chemistry Research
0
.015
0.026
Fig. 1 Steady-state inhibition of
urease by compound 20, A is the
Lineweaver–Burk plot of
reciprocal of initial velocities
versus reciprocal of four fixed
Jack bean urease concentrations
in absence (■) and presence of
0
0
.0095
.0085
0
.013
0
0
.0075
1
2.5 µM (□), 25.0 µM (●),
0
.0065
0
5
0 µM (○) of compound 20. B
is the Dixon plot of reciprocal of
the initial velocities versus
various concentrations of
compound 20 at fixed urease
concentrations, (■) 50 µM, (□)
-0.2 -0.1
A)
0
0.1
0.2
-70 -60 -50 -40 0 40 50
1
/S
Inhibitor
(B)
(
2
6
5.0 µM, (●)12.5 µM and (○)
.2 µM. C is the, 1/ Kmapp
16
versus various concentrations of
compound 20. D, is the, 1/Slope
versus various concentrations of
compound 20
8
6
4
2
0
12
8
4
-
40 -20 0 20 40 60 80
Inhibitor
-40
0
40 60
80
Inhibitor
(
C)
(D)
range of 1.79 and 3.57 Å, respectively. The last strong back
bone donor interaction was shown by Ala170 with the same
hydroxy hydrogen at a bond distance of 2.80 Å as shown in
Fig. 2 (2D and 3D). It is also clear from the figure that
Kcx220 on the other side is attached with Ni ion of active
site. The second Ni ion is also present in the nearby vicinity,
which greatly support the competitive type of inhibition.
Figure 3 (2D and 3D) showed that compound 2 have four
different interactions with urease enzyme in three different
ways. Kcx220 and Asp363 interacts through their carboxyl
oxygen giving two strong polar interactions with the
hydrogen of hydroxy group showing bond distances of 1.75
and 2.95 Å, respectively. On the other side, Kcx220 and
His249 are in bidentate interaction with Ni ion of the active
site through carboxyl oxygen of Kcx220 and imidazole ring
of His249. Third interaction was observed between the
hydroxyl oxygen of R group and another Ni ion at a dis-
tance of 2.84 Å which on the other side is attached with the
imidazole ring of His139. The fourth very strong back bone
donor interaction was given by the first NH group of pyr-
imidone moiety and carboxyl oxygen of Ala366 in the
range of 1.88 Å.
The carboxyl oxygen of the Kcx220 also showed interaction
with the Ni ion of the active site. Subsequently, acidic
interaction was shown by hydroxyl oxygen of the ligand
and terminal hydrogen of Arg339 in the bond range of
2.02 Å. The remaining three strong interactions were asso-
ciated with the back bone pyrimidone ring; the NH
hydrogen of the skeletal pyrimidone moiety and both the
hydroxy hydrogen of the R group interact with the carboxyl
oxygen of the Ala366, Gly280 and Ala170, respectively.
The estimated bond lengths in this case were 1.87, 2.18 and
2.50 Å, respectively, as shown in Fig. 4 (2D and 3D).
Compound 5 has inhibited the enzyme via four inter-
actions of compound with different amino acid residues
of the active pocket. Ala366 and Gly280 through their
carboxyl oxygen atoms interact with first NH group of
pyrimidone moiety and hydroxy of the ligand, respec-
tively. The bond lengths identified in this case were 1.90
and 3.14 Å, respectively. The acidic interaction was
observed between Arg339 and hydroxy oxygen of the
ligand with a bond distance of 2.07 Å. The last interaction
was established between the methoxy oxygen and Ni ion
of the active site at a distance of 2.90 Å. The Ni ion on the
other side was also involved in the interaction between
the carboxyl and NH group of the imidazole ring of
Kcx220 and His249, respectively, as it is clear from the
Fig. 5 (2D and 3D).
Compound 3 interacts with the active site of enzyme
through five strong interactions. A strong polar interaction
was observed between the carbonyl oxygen of Kcx220 and
hydroxyl hydrogen of the ligand at a distance of 1.67 Å.

Medicinal Chemistry Research
Table 3 Docking score and
molecular interactions of
compound 1–20
Compounds
1
Docking score
Ligand atom
Receptor atom
Bond type
Distance
−11.7450
N3
OD2-ASP 224
O-KCX220
NE2-HIS323
O-ALA 366
O-KCX220
O-ALA366
SG-CYS322
O-KCX220
NE2-HIS323
O-ALA366
SG-CYS322
O-KCX220
NE2-HIS323
O-ALA 366
SD-MET367
NE2-HIS323
O-ALA366
O-ALA366
NH2-ARG339
O-LYS169
H-donor
3.19
2.46
2.73
2.87
2.60
2.85
3.88
2.54
3.29
2.89
3.92
2.36
3.30
2.78
3.14
3.46
3.35
2.91
2.79
3.05
2.88
3.05
2.60
2.60
3.09
2.89
2.90
2.95
2.49
3.72
2.34
3.01
2.80
2.68
2.80
3.11
2.96
O18
O7
H-donor
H-acceptor
H-donor
2
3
−10.7937
−12.2799
N5
O18
N5
H-donor
H-donor
O7
H-donor
O19
O10
N5
H-donor
H-acceptor
H-donor
4
−12.0683
−9.8294
O7
H-donor
O18
O10
N5
H-donor
H-acceptor
H-donor
5
O18
O10
N5
H-donor
6
7
8
−11.6182
−10.3886
−9.7147
H-acceptor
H-donor
N5
H-donor
O10
N5
H-acceptor
H-donor
9
−10.3906
−10.4989
−10.5493
O10
N3
NE2-HIS323
OD2-ASP224
NE2-HIS323
O-ALA366
NE2-HIS222
O-ALA366
O-ALA366
O-ALA366
NE2-HIS222
OD2-ASP363
O-ALA170
NE2-HIS323
O-ALA366
O-ALA170
NE2-HIS324
O-LYS169
H-acceptor
H-donor
1
0
1
O7
H-acceptor
H-donor
1
N5
O10
N5
H-acceptor
H-donor
12
13
14
15
16
−10.9343
−10.3310
−9.4726
N5
H-donor
CL18
O7
Halogen bond
H-acceptor
H-donor
−10.0607
−10.3970
N3
N5
H-donor
O10
N5
H-acceptor
H-donor
17
18
19
20
−9.8301
−9.4302
−7.6193
−10.0191
N5
H-donor
O10
N5
H-acceptor
H-donor
O10
NE2-HIS323
H-acceptor
Similarly, compound 6 was found to be potent inhibitor
and five interactions were observed in three different ways.
The hydrogen bonding interactions of ligand were observed
with the NH group of imidazole ring of His139 and two
carboxyl groups of Asp363 and Kcx220, respectively. The
estimated bond lengths were 1.90, 2.65 and 3.08 Å,
respectively. Ni also showed strong bonding interactions
with the hydroxyl oxygen of compound 6 with the bond
length of 2.71 Å. The second Ni of the active site showed
weak interaction with the alkoxy ring of the ligand in the
bond range of 4.27 Å. These interactions of both the Ni ions
and amino acid residues of active site are shown in Fig. 6
(2D and 3D) which strongly supports the competitive type
of inhibition.
Compound 12 also showed three different interactions.
Ala366 through its carboxyl oxygen interacts with the NH
hydrogen of pyrimidone moiety with bond length of 1.91 Å.
A hydrogen bond interaction was observed between the
Arg339 and methoxy oxygen at a distance of 2.07 Å, while
the Ni ion interacts with the methoxy oxygen in the bond
range of 3.00 Å. On the other hand, Ni ion was also inter-
acting with His249 and Kcx220 which represents its

Medicinal Chemistry Research
Fig. 2 Binding modes of compound 1 against jack bean urease in (A) 2D and (B) 3D
Fig. 3 Binding modes of compound 2 against jack bean urease in (A) 2D and (B) 3D
stability and active site inhibition as indicated in Fig. 7 (2D
and 3D).
Most of them have low efficiency, various side effects on
human health [6–8, 29, 30] and are also involved in causing
environmental pollution [25]. This potent class of dihy-
dropyrimidones has no phytotoxic effects on plants with the
evidence of results as indicated in Table 5. They have
proved to be potential therapeutic agents to treat numerous
urease associated problems.
The toxicity effect of synthetic molecules 1–20 was
tested against human neutrophils by using a standard
operational protocol and acetohydroxamic acid was used as
standard. Acetohydroxamic acid is a well-known urease
inhibitor, which is most widely used to treat several urease
associated diseases. The viability results of human neu-
7
trophils (1 × 10 cells per mL) against synthetic derivatives
(
200 µg/mL) are listed in Table 4. The results indicated that
Conclusion
all synthetic molecules possess non-cytotoxic profile against
human neutrophil cells with the exception of compound 1
which showed some toxicity against these cells.
Several classes of urease inhibitors have been discovered
till now in the field of medicine as well as in agriculture.
In the present work, we have performed the kinetics stu-
dies on 5-acetyl-6-methyl-4-aryl-3,4-dihydropyrimidin-2
(1H)-ones skeleton and have confirmed their competitive
type of inhibition. Molecular docking and kinetics results

Medicinal Chemistry Research
Fig. 4 Binding modes of compound 3 against jack bean urease in (A) 2D and (B) 3D
Fig. 5 Binding modes of compound 5 against jack bean urease in (A) 2D and (B) 3D
both are in line with each other. Both experiments confirm
that dihydropyrimidones 1–20 interfere with the substrate
entry into active site. Molecular docking revealed that
different R groups attached with basic dihydropyrimidone
is the major active pharmacophore. These compounds
were also found to have nontoxic effects against human
neutrophil cells and plants. From the above results it can
be suggested that it is viable lead molecules for the dis-
covery of active therapeutic agents against ureases. For
future prospect, in vivo studies on animal models will
further validate these results and might be helpful in
developing new drug candidate.
Materials and methods
General
All the reagents and chemicals used in this study were of
analytical grade. Deionized H O was used for the
2

Medicinal Chemistry Research
Fig. 6 Binding modes of compound 6 against jack bean urease in (A) 2D and (B) 3D
Fig. 7 Binding modes of compound 12 against jack bean urease in (A) 2D and (B) 3D
preparation of various solutions at room temperature. Type
X urease (Cat. No. U4002-50 KU), urea (Cat. No. U5378),
phenol (Cat. No. 16017), dipotassium hydrogen phosphate
Germany. Fetal bovine serum (Cat. No. A11-104) was
purchased from PAA, Austria. MTT (Cat. No. 1945921)
was obtained from MP Biomedicals, France.
(
Cat. No. 16788-57-1), DMEM-high glucose (Cat. No.
D5648), and penicillin/streptomycin (Cat. No. PO781) were
purchased from Sigma-Aldrich, USA. NaOCl (Cat. No.
General procedure for the synthesis of dihydropyrimidone
derivatives (1–20)
2
30394M) and methanol (Cat. No. 20864.320/0823601)
from BDH Lab. Supplies, UAE. Sodium hydroxide (Cat.
No. S41298-4J) was purchased from Unichem, India.
Thiourea (Cat. No. 1079791000) was provided by Merck,
Equimolar quantities (1 mmol) of urea, acetylacetone,
and aryl aldehyde derivatives were mixed and stirred
at 80–90 °C. After 5 min, copper nitrate trihydrate

Medicinal Chemistry Research
Table 5 Results of LemnaWelv. Phytotoxicity assay
Table 4 Viability of human neutrophils (1 × 107 cells/mL) in the
presence of compound 1–20
Compounds
Concentration of compounds (μg/mL)
Compounds
Conc. µg/mL
Viability [%]
1
000
100
10
1
2
3
4
5
6
7
8
9
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
41.16 ± 2.5
67.77 ± 3.1
80.41 ± 4.0
70.35 ± 4.2
61.28 ± 3.5
68.94 ± 4.0
66.91 ± 4.3
59.84 ± 5.2
72.0 ± 4.5
1
2
3
4
5
6
7
8
9
54.0
21.5
5.4
85.54
89.02
46.41
80.65
23.5
28.9
30.23
4.00
9.60
17.50
13.00
45.06
25.03
6.4
100
31.4
61.6
100
60.0
100
100
13.00
37.50
36.20
13.99
52.00
75.08
25.5
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
69.05 ± 4.9
56.16 ± 2.5
64.77 ± 3.6
59.01 ± 4.1
64.34 ± 4.3
64.21 ± 3.7
69.04 ± 2.0
64.01 ± 5.3
55.84 ± 3.2
52.0 ± 4.0
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
88.2
63.0
61.54
67.02
66.5
27.4
65.2
69.0
50.0
65
37.41
70.35
69.0
20.3
20.5
20.27
6.04
8.40
7.20
9.00
35.01
9.02
16.0
10.03
34.10
27.20
10.23
41.07
31.08
41.3
60.06 ± 4.2
88.04 ± 5.0
63.2
68.9
Acetohydroxamic acid
Phosphoroamide
Mean ± SD of three experiments
(Molecular Devices., CA, USA) and the reactions were
{
Cu(NO ) .3H O} (10 mol %) was added to the reaction
performed in triplicate. Thiourea was used as standard.
Analysis of the results was carried out by using the Soft
Max Pro6.3 software (Molecular Device, USA). %age
inhibition was measured by using the formula:
3
2
2
mixture, in a solvent free condition. Reaction progress
was monitored by TLC After completion of the reaction,
the solid products were extensively washed with distilled
water, hexane and recrystallized from ethanol to afford
target compounds (1–20) (Scheme 1).
100 À ðO:Dtest well=O:DcontrolÞ Â 100
Urease inhibition assay (Indophenol’s method)
Determination of kinetics parameters
Initially, 25 μL of jack bean urease solution and 5.0 μL of a
test compound (0.5 mM in solvent methanol) one unit per
well were taken and the mixture was incubated at 30 °C for
IC50 values represent inhibitory potential of compounds.
Concentration of the test compounds that inhibited the
hydrolysis of urea (substrate) up to 50% urea was measured
by monitoring the effects of their different concentrations
(from high to low) in the assay. These calculations were
carried out by using the EZ-Fit Enzyme Kinetics Program
(Perrella Scientific Inc., Amherst, USA). ES represent the
complex of Jack bean urease and urea, while P stand for the
1
5 min in 96-well plate. Then 55 μL of potassium phosphate
buffer (4 mM, pH 6.8) which contained urea (100 mM) was
added to each well and it was again incubated at 30 °C for
the next 15 min. Activity of urease was determined by the
measurement of NH formed through the above method as
3
mentioned earlier by Weatherburn [31]. After that, each
product obtained as
a
result of reaction. The
well of the plate was inoculated with 45 μL of phenol
Lineweaver–Burk plot was then applied to identify the type
reagent (1% w/v phenol + 0.005% w/v Na -nitroprusside)
of inhibition, while dissociation and inhibition constants
2
and 70 μL of alkali reagents (0.5% w/v sodium hydroxide +
(K ) values were calculated by applying the Dixon plots and
i
0
.1% sodium hypochlorite). Finally, the reaction mixture
secondary replots [32, 33]. Non-linear regression equation
was incubated third time for 50 min at 30 °C. The absor-
bance was measured on microplate readers at 630 nm
was applied to find out the K , K , and V
Lineweaver–Burk plot was used for the determination of Ki
values.
m
i
max

Medicinal Chemistry Research
Scheme 1 General structure of
synthetic compounds 1–20
values in such a way that first at each intersection point of
lines of every inhibitor concentration on y-axis, the values
of 1/Vmaxapp was calculated. In the subsequent proceeding,
the slope of each line on the Lineweaver–Burk plot obtained
as a result of inhibitor concentration was plotted against
various concentrations of inhibitor.
Protein Data Bank (PDB) [37]. Receptor protein having
H O molecules were extracted. Like ligands, Protonate 3D
2
Option was applied for 3D protonation. Similarly, the
energy of urease protein was also minimized by applying
the default parameters of energy minimization algorithm
with gradients of 0.05 and force field Amber99, and then
saved in pdb file format. Later on, docking of all ligands
was carried out in the binding pocket of urease by following
the default parameters of MOE-Dock Program. In order to
increase the accuracy of protocol, re-docking was repeated
[38]. After complete docking of all the ligands, the most
excellent 2D as well as 3D interaction images were chosen
for their specific types of interactions and to measure their
bond lengths, respectively.
Statistical analysis
Each experiment was performed in triplicate and their
results were reported as a mean of three. SoftMax Pro 6.3
Software (Molecular Devices, CA, USA) was used for the
analysis of these results. The graphs were plotted through
GraFit program [34]. By using the same program, we have
obtained the values of correlation coefficients, intercepts,
slopes, and their standard errors from its linear regression
analysis. The relationship for each and every line in all the
graphs was more than 0.99. Each point in the graphs
represents the mean of three experiments.
Cytotoxicity evaluation
The cytotoxicity of urease inhibitors 1–20 were tested
against neutrophil cell lines. We used the following steps
for the evaluation of cytotoxicity.
Molecular docking
(
і) Isolation of human neutrophils Heparinized fresh
Molecular docking was performed to determine an accurate
prediction about the binding orientation of active site resi-
dues and potential inhibitors which is important for
structure-based drug designing. These results were then
correlated with the experimental values. Molecular Oper-
ating Environment (MOE version 2013.08) [35] docking
software was used to perform such studies. Ligands as well
as receptor protein (urease) preparation are the two basic
steps prior to perform docking.
venous blood was taken from hale and hearty young
volunteers in a City Clinical Laboratory, Dabgari Garden
Peshawar, Khyber Pakhtunkhwa, Pakistan. Isolation of
neutrophils was carried out through Siddiqui et al. method
[39]. Accordingly, mixing of whole blood was made with
Ficoll-Paque (Pharmacia Biotech Amersham, Uppsala).
When sedimentation occurred, the unnecessary red blood
cells (RBCs) were layered in a buffy coat way on a 3.0 mL
cushion of Ficoll. Centrifugation was then carried out for
30 min at a rate of 1500 rpm. Supernatant was discarded,
(
a) Ligands preparation Before docking, structures of all
while pellets were collected. Furthermore, it was mixed
with 0.83% of hypotonic ammonium chloride solution in
order to lyse the RBCs. Again, the solution was centrifuged
and Modified Hank’s Solution (MHS) was used for the
washing of collected neutrophils. Later on, resuspension of
neutrophil cells was performed in the same solution at the
the identified ligands were constructed through ChemDraw
Ultra 12.0 (Cambridge Soft-2006, Cambridge, USA) [36],
saved in the format of mol file which were then opened in
MOE. Protonate 3D Option was applied for 3D protonation.
The energies of identified ligands were minimized by
applying the default parameters of energy minimization
algorithm already adjusted in MOE (gradients: 0.05, force
field: MMFF94x). Database of this series of compounds
was created in mdb file format, in which all the ligands with
their 3D structures were saved.
7
rate of 1 × 10 cells per mL.
(іі) Assay procedure for neutrophil based cytotoxicity I-
7
solated human neutrophils (1 × 10 cells per mL) were
treated first with compounds under trial for 30 min and then
with 0.25 mM WST-1(Dojindo Laboratories Kumamoto,
Japan) on shaking water bath at 37 °C [40]. Change in
absorbance was calculated at 450 nm after incubation for
(
(
b) Preparation of receptor protein 3D structure of urease
4UBP) having resolution of 1.55 Å, was retrieved from

Medicinal Chemistry Research
3
h by using 96-well plate reader (Spectra-MAX-340,
mononuclear Cu (II) complexes based on hydrazone ligands.
Bioorg Med Chem Lett. 2016;26:4925–4929.
Molecular Devices, CA, USA). Here the OD represents the
mean of 5 investigational replicates. Percent (%) viability of
cells was measured as:
2
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Overexpression of Helicobacter pylori–associated urease mRNAs
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%
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fðOD test compound  100=OD controlÞ À 100g À 100:
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Phytotoxicity evaluation
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Some of the urease inhibitors are used in agriculture to
reduce the pH of soil and/or to control the loss of urea.
Therefore, all of the identified inhibitors were checked for
their phytotoxic effects according to the modified assay of
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8
. Mobley H, Island MD, Hausinger RP. Molecular biology of
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CH OH) were dissolved in sterilized E-medium. Later on,
3
9
inhibitors having fixed concentrations (made by the dilution
of stock solution) were taken in sterilized and clean conical
flasks and allowed their evaporation for overnight. In each
of the flask, 20 mL of sterilized E-medium and 10 mL
Lemna aequinocitalis Welv plants were inoculated, already
having a rosette of three fronds. For negative control only
1
0. Arfan M, Ali M, Ahmad H, Anis I, Khan A, Choudhary MI, et al.
Urease inhibitors from Hypericum oblongifolium WALL. J
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CH OH was added in few flasks and for positive control
3
phosporoamide (commercially available urease inhibitors)
was used. The flasks were kept in Fisons Fi-Totron 600H
growth cabinet at 30 °C for a whole week having the
environmental conditions (light intensity: 9000 lux, relative
humidity: 56 ± 10 rh) at a short-day span of 12 h. All the
experiments were performed in triplicates. Growth rate of
Lemna aequinocitalis Welv was measured by counting the
number of fronds per dose present in flasks containing
inhibitors, while the growth inhibition was calculated with
the reference to negative control.
(
Citrullus vulgaris). J Enzym Inhib Med Chem. 2004;19:381–387.
1
1
3. Rao D, Ghai S. Effect of phenylphosphorodiamidate on urea
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et al. Synthesis and characterization of new thiosemicarbazones,
as potent urease inhibitors: in-vitro and in-silico studies. Bioorg
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1
5. Macegoniuk K, Grela E, Palus J, Rudzinska-Szostak E, Gra-
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derivatives as a new class of bacterial urease inhibitors. J Med
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1
6. Shamim S, Khan KM, Salar U, Ali F, Lodhi MA, Taha M, et al. 5-
Acetyl-6-methyl-4-aryl-3, 4-dihydropyrimidin-2 (1H)-ones: As
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Acknowledgements The authors are thankful to Higher Education
Commission (HEC) Pakistan for financial support under “National
Research Support Program for Universities” (Project No. 5079) and
King Fahad University of Petroleum and Minerals (KFUPM) Saudi
Arabia (Project No. SB191017).
17. Khan I, Khan A, Halim SA, Saeed A, Mehsud S, Csuk R, et al.
Exploring biological efficacy of coumarin clubbed thiazolo [3, 2–b]
[1, 2, 4] triazoles as efficient inhibitors of urease: a biochemical and
in silico approach. Int J Biol Macromol. 2020;142:345–354.
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Developing new hybrid scaffold for urease inhibition based on
carbazole-chalcone conjugates: synthesis, assessment of ther-
apeutic potential and computational docking analysis. Biorg. Med
Chem. 2019;27:115123.
Compliance with ethical standards
1
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
19. Khan I, Ali S, Hameed S, Rama NH, Hussain MT, Wadood A,
et al. Synthesis, antioxidant activities and urease inhibition of
some new 1, 2, 4-triazole and 1, 3, 4-thiadiazole derivatives. Eur J
Med Chem. 2010;45:5200–5207.
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Synthesis of β-ketosulfone derivatives as new non-cytotoxic
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