Structure based virtual screening-driven identification of monastrol as a potent urease inhibitor

Article abstract of DOI:10.1016/j.jmgm.2013.04.006

Virtual screening uses computer based methods to discover new ligands on the basis of biological structures. Among all virtual screening methods structure based docking has received considerable attention. In an attempt to identify new ligands as urease i

Full text of DOI:10.1016/j.jmgm.2013.04.006

Journal of Molecular Graphics and Modelling 43 (2013) 47–57
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Graphics and Modelling
journal homepage: www.elsevier.com/locate/JMGM
Structure based virtual screening-driven identification of monastrol
as a potent urease inhibitor
Umer Rashida,b, Iram Batoola, Abdul Wadoodc, Ajmal Khand, Zaheer ul-Haqd,
Muhammad Iqbal Chaudharyd, Farzana Latif Ansaria,∗
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
c Department of Biochemistry, Abdul Wali Khan University, Mardan 23200, Pakistan
d Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Virtual screening uses computer based methods to discover new ligands on the basis of biological struc-
tures. Among all virtual screening methods structure based docking has received considerable attention.
In an attempt to identify new ligands as urease inhibitors, structure-based virtual screening (SBVS) of an
in-house database of 10,000 organic compounds was carried out. The X-ray crystallographic structure
of Bacillus pasteurii (BP) in complex with acetohydroxamic acid (PDB Code 4UBP) was used as a protein
structure. As a starting point, ∼10,000 compounds of our in-house database were analyzed to check
redundancy and the compounds found repeated were removed from the database. Finally 6993 com-
pounds were docked into the active site of BP urease using GOLD and MOE-Dock software. A remarkable
feature of this study was the identification of monastrol, a well-known KSP inhibitor already in clinical
trials, as a novel urease inhibitor. The hits identified were further evaluated by molecular docking and on
examination of the affinity predictions, twenty-seven analogs of monastrol were synthesized by a mul-
ticomponent Biginelli reaction followed by their in vitro screening as urease inhibitors. Finally twelve
compounds were identified as new urease inhibitors. The excellent in vitro activity suggested that these
compounds may serve as viable lead compounds for the treatment of urease related problems.
© 2013 Elsevier Inc. All rights reserved.
Received 15 December 2012
Received in revised form 26 February 2013
Accepted 23 April 2013
Available online 30 April 2013
Keywords:
Urease inhibitors
Structure-based virtual screening
Monastrol
Dihydropyrimidines
Docking
1. Introduction
dataset by means of high-throughput docking to an X-ray structure
or model of the target receptor [5–9].
Computational approaches have become a key component of
many drug discovery programs both in academia and industry.
Over the last few decades, modern computer aided drug design
tools established a novel platform for researchers. Time and cost
required for chemical synthesis and in vitro testing has been dra-
matically reduced due to these computational technologies [1–4].
Virtual screening (VS) or in silico screening, is an increasingly used
method for the identification of novel leads from large chemical
libraries. Both ligand-based virtual screening (LBVS) and structure-
based virtual screening (SBVS) protocols have emerged as popular
tools in early drug discovery and development process and among
all VS methods, protein-structure-based docking has received sig-
nificant attention. Structure-based virtual screening (also known as
molecular docking) is most commonly employed for the prediction
of binding modes and binding affinities of each compound in the
The hydrolytic enzyme urease (amidohydrolase, EC 3.5.1.5), a
nickel containing metalloenzyme, is responsible for the catalytic
hydrolysis of urea to volatile ammonia and carbon dioxide. The
enzyme releases ammonia and carbamate, which in turn sponta-
neously generates the products [10–12]. Interestingly, both these
molecules (urea and urease) symbolize milestone molecules in
initial biological research. The very first organic compound syn-
thesized was urea and the first enzyme which was crystallized in
history was urease [13,14]. Furthermore, this enzyme was identi-
fied as metalloenzyme as it contains nickel atom [15].
For its enzymatic hydrolysis, urease utilizes an active site con-
taining a binuclear nickel center bridged by a carbamylated lysine
and a hydroxide ion as shown by the crystallographic structures
resolved for bacterial ureases from Klebsiella aerogenes, Bacillus pas-
teurii and Helicobacter pylori [16–18]. These ureases have a nearly
superimposable active site, which implies that it is common to all
ureases, including jack bean urease whose crystallographic struc-
ture has not been resolved to date. A number of proposals including
computational studies have been made regarding its possible reac-
tion mechanism and consensus has been reached regarding the
∗
Corresponding author. Tel.: +92 51 90642109; fax: +92 51 90642241.
E-mail address: fla qau@yahoo.com (F.L. Ansari).
1093-3263/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jmgm.2013.04.006

48
U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
initial coordination of urea in its active site through the urea
oxygen attacking the vacant coordination site on nickel atom, how-
ever, there are divergent proposals regarding the subsequent steps
[19–25].
thousand compounds were modeled and energy minimized using
energy minimization algorithm implemented on Molecular Oper-
ating Environment (MOE) and were saved in a separate database.
Both these sets of compounds were then added to MOE database.
SBVS experiment was then performed using 10,000 compounds
present in our in-house database against urease. Before SBVS
experiment, the compounds of the database were checked for
redundancy and repeated compounds were removed from the
database. Then, Lipinski’s rule-of-five (Ro5) was applied on the
database for drug-likeness. As a result of this filtering protocol
6993 compounds were found to be drug-like compounds. Finally
6993 compounds were docked by MOE-Dock and GOLD docking
programs separately.
Urease is involved in human and animal pathogenicity of
hepatic encephalopathy, hepatic coma urolithiasis, pyelonephri-
tis, gastric and peptic ulcers, and urinary catheter encrustation
are caused by ammonia produced by ureases [10,16]. Urease
inhibitors can be considered as a tool to control the dam-
aging effect of ureolytic bacterial infections in humans which
occur commonly in the developed countries [26]. There are a
number of classes of compounds that are known to show signif-
icant inhibitory activity against urease enzyme. Urease inhibitors
can be broadly classified into two categories: (1) organic com-
pounds and (2) organometallic compounds. The former category
includes hydroxamic acid and its derivatives [27,28], triazoles,
coumarines [29–31], isatins, semicarbazones [32], Schiff bases
[33,34], urea derivatives [35,36], oxadiazoles [37], and piperidines
[38]. The latter category includes organophosphorous/phosphinic
inhibitors [26,39], phosphoramides [40] and metal complexes of
organotin(IV), vanadium(IV), bismuth(III), copper(II), cadmium(II)
copper(II) and zinc(II) [41–46]. Besides, some natural products such
as gallocatechin, a naturally occurring flavonoid has been reported
as inhibitors of H. pylori urease [47].
All available structurally diverse classes of urease inhibitors
are not satisfactorily significant to the binding cavity as they can-
not interact with the key amino acid residues and the metal ions
present in the urease active site. Therefore, the search of new and
more effective urease inhibitors to diversify the current urease
inhibitors is essential. We have extensively studied the binding
modes and QSAR of different new classes (both from natural and
synthetic origin) of urease inhibitors in our ongoing drug dis-
covery program. The identified inhibitors were studied for their
binding affinities and structure-activity relationships using state-
of-the-art computational techniques [48,49]. In continuation to
discovering new and diverse classes of compounds as drug candi-
dates, we carried out a SBVS experiment on an in-house database of
compounds and identified monastrol, a 3,4-dihydropyrimidine-2-
thione derivative, as a potent candidate for urease inhibition. Mayer
et al. reported the screening of a large library of compounds where
racemic monastrol was identified as KSP inhibitor (IC50 = 30 nM)
and hence may be considered as a lead for the development of
new anticancer drugs [50]. During the drug discovery paradigm
it is quite often recommended to start the designing of a drug
from a compound that already shows some activity against a given
receptor or for a given disease. Since monastrol is a dihydropyrimi-
dine (DHPM) derivative that may also be classed as cyclic thiourea,
therefore, it was logical that DHPM scaffold may be a promising
candidate for urease inhibition.
2.2. Molecular docking
As a first step, the ability of the docking algorithm was vali-
dated to reproduce the co-crystallized pose of three known urease
inhibitors (e.g. bis-coumarin, imidazol and hydroxamic acid) in the
urease pocket. This yielded a good agreement between the docked
and crystal structures as these studies of the known inhibitors fur-
ther confirmed the interaction region of the ligands and a favorable
interaction with urease. As the next step, we evaluated whether this
approach was agreeable to distinguish between known inhibitors
and randomly chosen 3000 drug-like decoys from our in-house
database. To this end we docked the ‘validation set or decoy set’ into
the binding site of urease, as a positive control. Two separate dock-
ing runs were carried out, one with MOE-Dock and the other with
GOLD docking program [51,52]. There were three possible scoring
functions in MOE-Dock i.e. London dG, affinity dG and ASE, while
two scoring functions of Gold were applied. In addition to scoring
functions there were several other parameters needed to be set
for attaining appropriate binding pose and ranking. For example,
it was observed that out of few available placement methods in
MOE dock (Triangle Matcher, Alpha Triangle, Alpha PMI, Proxy Tri-
angle, FlexX and pharmacophore) only Triangle Matcher was found
satisfactory in order to reproduce the binding mode of one of the
known inhibitors i.e. acetohydroxamic acid, which was retrieved
from Protein Data Bank (PDB ID 4UBP) [53]. Similarly, every sco-
ring function implemented in docking programs was evaluated to
analyze the ability to place the correct docking pose in the top rank-
ings. London dG scoring function implemented in MOE dock was
able to place all known inhibitors within the top 1% of the whole
decoy set tested. Gold fitness function in GOLD docking program
also exhibited similar enrichment like London dG in MOE dock.
The next phase of the screening process involved the docking of
each molecule in the database into the binding site of the target.
This docking process employed the prediction of favorable confor-
mation of the ligand in the target binding site.
The in silico successful compounds were docked at the puta-
tive urease active site to examine if they fulfilled the requirements
presented in the search queries in their docked conformations.
Two independent docking experiments were carried out by using
default parameters (unless explicitly mentioned) with MOE-Dock
and GOLD docking program separately.
2. Results and discussion
2.1. Virtual screening
The three dimensional coordinates of the target protein (PDB ID
4UBP) was retrieved from protein data bank. All water molecules
were removed from the protein and the 3D protonation of the pro-
tein molecule was carried out using MOE. The protein molecule
was subjected to energy minimization using energy minimization
algorithm implemented in MOE with default parameters. The min-
imized protein structure was then used for further study.
With the aim to identify novel and potent urease inhibitors VS of
in-house database against urease enzyme was carried out. In a pre-
vious study a 3D-structure database of six thousand compounds
was developed. In the present report 3D structures of new four
Applying the post-docking filtering strategy of both Gold and
MOE, 200 compounds were identified as the top ranked hits which
led to the identification of few structurally similar compounds
ranked in the top list.
All the selected compounds were visually inspected based on
our previous dry and wet lab experience for improbable docking
orientations in the active site of urease. The resulting compounds
were further analyzed for their interactions with the key amino acid
residues such as Ala170. His137, His139, Lys220, His249, His275,
Gly280, Cys322, His323, His324, Arg339, Ala363, Asp363, thereby,
short listing those compounds which showed the known key

U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
49
Table 1
Table 2
Scoring functions and IC50 of initial hits [29].
Inhibition of urease activity by monastrol derivatives of Sets 1–3.
S. no.
Compounds
Fitness score
IC50 (␮M)
No.
R1
R2
R3
R4
X
IC50 (␮M)
Set 1
1
2
3
4
5
6
7
8
GOLD
MOE
H
H
H
H
H
H
H
H
H
H
H
H
C6H5
C6H5
OEt
Me
OEt
OEt
OEt
Me
OEt
OEt
OEt
OEt
OEt
OEt
Me
Me
Me
Me
Me
Me
Me
OEt
CF3
Me
Me
Me
S
S
S
S
S
S
S
S
S
S
S
S
61.56
5.6
5.36
5.4
11.76
29.93
31.26
NI
1
2
3
4
5
6
7
8
9
AAB429a
AAB539a
AAD355a
8da
56.28
41.38
55.93
60.59
47.20
54.00
66.00
48
−13.4425
−11.0070
−15.6482
−10.8738
−09.0359
−13.2499
−09.0144
−08.3252
NDb
110.0 3.06
238.5 2.04
31.2 0.47
34.10 1.24
150.23 2.10
11.76 1.66
17.67 1.09
45.0 2.43
21.0 0.01
3-Br·C6H4
3-OMe·C6H4
3-OH·C6H4
3-OH·C6H4
3-OMe,4-OH·C6H3
3-OH·C6H4
3-OH·C6H4
4-OMe·C6H4
4-NMe2·C6H4
3-Thienyl
14ia
UR A2 (monastrol)
FBTZ1
FBTZ2
9
NI
Thiourea
NDb
10
11
12
15.16
133.03
NI
a
Previously reported.
ND: not determined.
b
Set 2
13
14
15
16
17
18
19
20
21
22
23
24
25
H
H
H
H
H
H
H
H
H
H
H
Me
C6H5
C6H5
C6H5
OEt
Me
Ph
OEt
OEt
Ph
OEt
OEt
OEt
OEt
OEt
OEt
OEt
Me
Me
Ph
Me
Me
Ph
Me
OEt
OEt
Me
Me
Me
Me
O
O
O
O
O
O
O
O
O
O
O
O
O
22.76
NI
NI
NI
NI
41.3
NI
58.26
425.06
NI
interactions with active site residues and Ni798 and Ni799 present
in the binding site. As a result, thereof, several compounds were
eliminated during docking and post docking filtering strategies
[54].
3-OH·C6H4
3-OMe·4-OH·C6H3
3-OMe·4-OH·C6H4
4-NMe2·C6H4
4-NMe2·C6H4
4-OH·C6H4
4-Cl·C6H4
3-Pyridyl
C6H5
2.3. In vitro urease inhibition studies
The in vitro urease inhibitory activity of the rigorously screened
twenty candidates was carried out according to literature proce-
dure [55] using thiourea as standard. Seven compounds showed
urease inhibitory activity with IC50 values ranging from 31.2 to
238.16 ␮M. The scoring functions and IC50 of initial hits are given
in Table 1.
NI
NI
NI
CH2·C6H5
C6H5
Set 3
26
27
H
H
4-OMe·C6H4
OEt
OEt
OEt
Me
–
–
NI
NI
3-OMe, 4-OH·C6H3
The observed activities of compounds AAB429, AAB539,
AAD355, 8d and 14i [29,56–58] were 110.0 3.06 ␮M,
NI: no inhibition observed.
238.16 2.04 ␮M,
31.2 0.47 ␮M,
34.10 1.24 ␮M,
and
derivatives. The use of ultrasound waves has gradually been
introduced in organic synthesis as a green alternative over the
last three decades [60]. A large number of organic reactions can
be carried out under milder conditions, in shorter reaction times
and providing higher yield by ultrasound irradiation. This one pot
cyclocondensation of three building blocks of Biginelli reaction led
to different elements of diversity at C-2, C-4, C-5 and C-6 as shown
in Scheme 1.
150.23 2.10 ␮M respectively. The structures of initial hits
are provided as supporting information. Among the eight hits
identified AAB429, AAB539, AAD355 were commercially available
while the details of other two synthetic compounds (FBTZ1 and
FBTZ2) will be reported in a separate publication. This screening
resulted in a new class of compounds called dihydropyrimidines
and monastrol (URA2) was a member of this important class of
heterocycles. We chose UR A2 as the scaffold for further investiga-
tion based on following rationale: (1) reasonably active compound
(IC50 = 11.76 ␮M) and (2) easy availability of starting materials
and (3) one step synthetic derivitization through one pot Biginelli
reaction.
2.5. In silico studies
After careful inspection of the initial results, supported by vir-
tual docking experiments, we initially focused on the synthesis of
monastrol analogs with different substituents at meta-position of
the C-4 phenyl ring and measured their capacity to inhibit urease.
The results of urease inhibition of these compounds are shown in
Table 2.
As mentioned earlier, monastrol (5) was found as one of
the initial hits with IC50 11.76 ␮M in SBVS experiment, how-
ever, its synthetic analogs 3 and 4 with 3-bromophenyl and
3-methoxyphenyl respectively, were found to be stronger urease
inhibitors with IC50 5.36 ␮M and 5.4 ␮M respectively. Moreover, of
the two regioisomeric DHPMs 4 and 9, the m-isomer (IC50 5.4 ␮M)
2.4. Chemistry
Having identified monastrol as the initial hit in SBVS exper-
iment, a variety of DHPM derivatives were synthesized through
one pot Biginelli reaction [59] for exploration of SAR around
all the six diversity points of DHPM scaffold. These nonplanar
heterocyclic compounds have received considerable attention of
the pharmaceutical industry because of their interesting multi-
faceted pharmacological profiles. Following the Biginelli strategy,
we carried out a sonocatalyzed synthesis of three sets of DHPM
R2
NH2
NH2
NH
R2
O
O
O
HN
R1
X
HN
R1
R3
N
R3
NH
R2
R3
+
SnCl2, ACN
R4
N
R1
NH2
R4
N
R1
X
DMF/NaHCO3
)))) 70oC
CHO
)))) 70oC
R4
O
Set 3 (26-27)
Set 1 (1-11) X=S
Set 2 (12-25) X=O
Scheme 1. Synthesis of compounds 1–27.

50
U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
Fig. 1. Modeled mode of binding of acetohydroxamic acid (HAE800) in 4UBP active site.
was found to be more active than the p-isomer (IC50 = 15.16 ␮M).
It may, therefore, be generalized that 3-substituted DHPMs with
a thiocarbonyl group at C2 position exhibited stronger urease
inhibitory activity with the exception of unsubstituted DHPM 2
(IC50 = 5.6 ␮M). These compounds are more potent as compared
to the standard i.e. thiourea (IC50 of 21 ␮M). However, DHPM-2-
ones were found to be far less active than their thione counterparts
(Table 1), while 2-amino-4-aryl-1,4-dihydropyrimidines (Set 3)
were not found to have any urease inhibitory activity.
All DHPMs that were evaluated for their potential as urease
inhibitors were docked in BP urease. In vitro urease inhibition stud-
ies were carried out using thiourea as standard, however, as its
X-ray crystallographic structure in complex with Jack bean urease
is not present in Protein Data Bank, therefore, the structure of the
complex of acetohydroxamic acid (HAE) and BP urease was used
to perform the docking simulation study. The active site of urease
is located within the cavity or the crevice in its internal territory
in which HAE molecule chelates with two nickel ions via hydroxyl
oxygen as shown in Fig. 1.
studies were carried out on both enantiomers of the synthesized
DHPMS using GOLD. It was interesting to note that with only few
exceptions, the (S) isomer was found to have a higher GOLD fitness
score compared to its (R) isomer. For example, Gold score for S-
monastrol was found to be 58.43 compared to its (R) enantiomer
i.e. 53.95 which reflects a stronger binding of the (S) isomer in the
active site of 4UBP due to the formation of a more stable complex
with both Ni798 and Ni799 atoms with 3-OH substituent.
Moreover, the orientation of the aryl ring in the active site also
seems to play an important role in stabilizing ligand–enzyme com-
plex. A careful glance at the superimposed docking poses of both
(R) and (S) monastrol reveals that the phenyl group anchors itself in
such a way that enables a stronger interaction of 3-OH group with
both Ni798 and Ni799 while similar interactions are not possible
in the (R) isomer (Fig. 3).
Parallel studies when conducted on inactive compounds, for
example compound 14, revealed that there was no Ni metal liga-
tion in the ligand–enzyme complex which appeared to be essential
for stabilizing a ligand–enzyme complex (Fig. 4).
A comparison of ligand–enzyme complex of monastrol (5) with
crystallographic complex of HAE revealed a resemblance in the
fitting-in mechanism of the two compounds. The coordination pat-
tern of compound 5 via its 3-hydroxyphenyl group at C4 mimicked
the coordination of HAE, and similar active site residues, as those in
HAE, were observed to stabilize the enzyme ligand complex (Fig. 2).
It is well documented in literature that the pharmacological
activity of DHPMs is dependent on the absolute configuration at
stereogenic center at C-4 and the control of the stereochemistry at
C-4 has essential importance as it acts like a molecular gear of chiral
regulation during drug-receptor recognition [61,62]. For example,
it is exclusively the (S)-monastrol that shows KSP inhibition [63].
The access to enantiomerically pure DHPMs is, therefore, a prereq-
uisite for the development of useful drugs of this structural type.
Since the present study encompasses the synthesis and in vitro
evaluation of racemic DHPMs as urease inhibitors, exploiting the
predictive power of in silico tool, we were interested in exploring
the probable binding modes of enantiomerically pure (R) and (S)
DHPMs into the active site of urease. Therefore, molecular docking
3. Conclusion
This paper presents the first report on the identification of
monastrol (a KSP inhibitor) as a strong candidate for urease inhibi-
tion in a SBVS experiment. A variety of monastrol derivatives were
synthesized which were found to be even stronger urease inhibitors
than thiourea. It is, therefore, anticipated that this scaffold may
serve as a lead compound and its further modification could give
rise to improved inhibitory activity and for developing antiulcer
drugs. Having these in vitro results, in vivo studies on animal models
will also be carried out.
4. Materials and methods
4.1. Virtual screening
Recently we have developed our in-house database of ∼10,000
compounds. The compounds included in our dataset were either

U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
51
Fig. 2. Molecular level interactions of DHPM 5 (monastrol) in the active site of 4UBP.
of synthetic or natural sources. All these compounds are struc-
turally well elucidated and most of them fulfill the drug-like criteria
according to Lipinski rule of five with the exceptions of naturally
isolated compounds. In a previous report, a 3D-structure database
of six thousands compounds of an in-house database was devel-
oped. During present study, two-dimensional structures of over
four thousand additional compounds were received from our com-
pound bank initially in the CDX format that was later converted to
MOL2 format using Molecular Operating Environment (MOE) soft-
ware and was saved in a separate database. By using the “washing”
module of MOE physiologically relevant protonation state of all the
compounds was produced.
A number of X-ray crystallographic structures of urease are
available in the Protein Data Bank (PDB). All the structures of ure-
ase enzyme were retrieved from the PDB and extensively studied
for the selection of suitable structure for the structure-based virtual
screening. The criteria used for the selection of best X-ray structure
were: (1) highest resolution, (2) complex with potent inhibitors,
and (3) explaining the geometry of nickel ions and their interac-
tions with the inhibitor. On the basis of these criteria the X-ray
Fig. 3. Superimposition of monastrol (5), S (green) and R (brown) at the putative active site of urease. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)

52
U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
Fig. 4. Binding interactions of DHPM 14 at urease active site.
crystallographic structure of B. pasteurii (BP) urease in complex
The software GOLD uses a genetic algorithm (GA) to explore
the full range of ligand conformational flexibility with the par-
tial flexibility of protein. The GOLD scoring function includes the
terms for hydrogen-bonding, vdW, and intramolecular energies.
The vdW interactions for the protein-ligand complex are described
by 8–4 potential. A 12–6 potential was used for the ligand steric
energies that also includes the torsional energies and the active
with acetohydroxamic acid was selected for structure-based vir-
˚
tual screening. The resolution of this structure (1.55 A) was found
to be the highest among all the X-ray structure of BPU. This complex
structure was prepared for molecular docking by removing solvent
molecules followed by 3D protonation and energy minimization
using MOE.
˚
site with a 10 A radius sphere was defined. For each independent
GA run, a maximum number of 100,000 GA operations were per-
formed on a single population of 100 individuals. Operator weights
for crossover, mutation, and migration were set to 95, 95, and 10,
respectively. To allow for poor nonbonded contacts at the start of
each GA run, the maximum distance between hydrogen donors and
4.2. Molecular docking
MOE-Dock tool was employed to explore suitable binding
conformations between small compounds and a macromolecular
protein. It uses a Monte Carlo Simulated Annealing method for
docking a ligand in the binding site of a receptor molecule. The
program forms a 3D grid encompassing the center of the docking
site. The target compounds were built using the builder inter-
face of the MOE program and subjected to energy minimization.
The crystal structure of protein complex urease with HAE was
obtained from PDB (PDB ID 4UBP). The edited crystal structure
after removing water molecules was imported into MOE and all
hydrogen atoms were added to the structure with their standard
geometry followed by their energy minimization using MOPAC 7.0.
The resulting model was subjected to systematic conformational
search at default parameters with RMS gradient of 0.01 kcal/mol
using Site Finder. The enzyme was searched for its active site and
dummy atoms were created from the resulting alpha spheres. The
backbone and residues were kept fixed and the energy minimi-
zation was performed. Root mean square deviation (RMSD) values
were used to compare the ligand between the predicted and its
corresponding crystal structure. The resulting docked poses with
˚
fitting points was set to 4.0 A, and non-bonded vdW energies were
˚
cut-off at 2.5 A. Finally twenty top ranked poses were saved.
4.3. Chemistry
All the reagents and anhydrous solvents were purchased from
standard commercial vendors and were used without any further
purification.
Sonication was performed in Elma E 30 H (Germany) ultrasonic
cleaner with a frequency of 37 KHz and a nominal power of 250 W.
1H and 13C NMR spectra were recorded in DMSO-d6 on a Bruker
spectrometer at 300 and 75 MHz respectively using tetramethyl-
silane (TMS) as internal reference. Chemical shifts are given in ı
scale (ppm). Mass spectra were recorded on Agilent technologies
6890N Gas chromatography (GC) and an inert mass selective detec-
tor 5973 GC–mass spectrometer. Melting points were determined
in open capillaries using Gallenkamp melting point apparatus (MP-
D). The progress of all the reactions was monitored by TLC on
2.0 cm × 5.0 cm aluminum sheets precoated with silica gel 60F254
with a layer thickness of 0.25 mm (Merck).
˚
RMSD less than 1.5 A were clustered together and the lowest energy
minimized pose was used for further analysis. All DHPMs were
docked following the same protocol. Ten different conformations
were selected for each ligand. All other parameters were main-
tained at their default settings. The best conformation of each of the
ligand–enzyme complex was selected based on energetic grounds.
The resulting ligand–enzyme complex model was then used for cal-
culating the energy parameters using MMFF94x force field energy
calculation and predicting the ligand–enzyme interactions at the
active site.
4.3.1. General method for the synthesis of
3,4-dihydropyrimidine-2-ones and
3,4-dihydropyrimidine-2-thiones
A mixture of an aldehyde (10 mmol), a diamino compound
(12 mmol), a dicarbonyl compound (10 mmol, mL), SnCl2·2H2O
(10 mol%) and acetonitrile (10 mL) was mixed in a pyrex tube. The

U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
53
mixture was then irradiated in ultrasonic bath at 70–75 ◦C. The
reaction was monitored by TLC. After the completion of the reac-
tion, the resulting precipitate was filtered and crude product was
recrystallized from an appropriate solvent or purified through col-
umn chromatography.
4.3.1.6. 5-Aceto-4-(3-hydroxyphenyl)-6-methyl-3,4-
dihydropyrimidin-2(1H)-thione (6). 6 was prepared according
to the general procedure by using 3-hydroxybenzaldehyde,
thiourea, acetylacetone. Yield 69%. m.p. 191 ◦C. 1H NMR (300 MHz,
DMSO-d6): ı 2.20 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.34 (d, 1H,
J = 3.3 Hz, CH), 6.67 (s, 1H, Ar-H), 6.86 (d, 2H Ar-H), 7.11 (t, 1H,
Ar-H), 9.68 (br s, 1H, NH), 9.96 (br s, 1H, OH), 10.42 (br s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6): ı 18.1 (CH3), 29.4 (CH3CO),
54.1 (CH-Ar), 101.0 (C-COMe), 123.0–143.5 (C-aromatic), 147.9
(CH3C C), 157.4 (C-OH), 181.7 (C S), 194.4 (COMe); EIMS calcu-
lated for C13H14N2O2S (M+•) 262. CHNS Analysis calculated for
C13H14N2O2S, C = 59.52; H = 5.38; N = 10.68; O = 12.20; S = 12.22
Found: C = 59.59%; H = 5.41%; N = 10.54%; O = 12.21; S = 12.25.
4.3.1.1. 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-
dihydropyrimidin-2(1H)-thione (1). 1 was prepared according
to the general procedure by using benzaldehyde, thiourea, ethyl
acetoacetate. Yield: 74%. m.p. 217 ◦C [64,65]. 1H NMR (300 MHz,
DMSO-d6): ı 1.10 (t, 3H, J = 7.2 Hz, OCH2CH3), 2.21 (s, 3H, CH3),
4.01 (q, 2H, J = 7.2 Hz, OCH2), 5.18 (d, 1H, J = 3.3 Hz, CH), 7.20–7.37
(m, 5H, Ar-H), 9.63 (br s, 1H, NH), 10.32 (br s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6): ı 14.3 (OCH2CH3), 18.5 (CH3), 54.8 (CH-
Ar), 59.9 (OCH2), 100.4 (C-COOEt), 126.0–129.0 (C-aromatic),
147.4 (CH3C C), 164.2 (COOEt), 181.6 (C S); EIMS calculated for
4.3.1.7. 5-(Ethoxycarbonyl)-4-(4-hydroxy-3-methoxyphenyl)-6-
methyl-3,4-dihydropyrimidin-2(1H)-thione (7). 7 was prepared
according to the general procedure by using vanillin, thiourea,
ethyl acetoacetate. Yield 68%. m.p. 210 ◦C. 1H NMR (300 MHz,
DMSO-d6): ı 1.03 (t, 3H, J = 7.2 Hz, CH3), 2.28 (s, 3H, CH3), 3.70 (s,
3H, OCH3), 4.18 (q, 2H, J = 7.5 Hz, OCH2CH3), 5.30 (d, 1H, J = 3.6 Hz,
CH), 6.40 (s, 1H, Ar-H), 6.45 (d, 1H, H-aromatic), 6.50 (d, 1H, Ar-H),
9.71 (br s, 1H, NH), 10.45 (br s, 1H, NH), 10.60 (s, 1H, OH). 13C
NMR (75 MHz, DMSO-d6): ı 14.9 (OCH2CH3), 17.9 (CH3), 53.1
(CH-Ar), 56.7 (OCH3), 60.4 (OCH2), 100.1 (C-COOEt), 119.7–139.0
(C-aromatic), 144.3 (C-aromatic-OH), 148.2, (CH3C C), 151.2
(C-aromatic-OCH3), 165.3 (COOEt), 180.6 (C S); EIMS calculated
for C14H18N2O4S (M+•) 322.
C
14H16N2O2S (M+•) 276.
4.3.1.2. 5-Aceto-6-methyl-4-phenyl-3,4-dihydropyrimidin-
2(1H)-thione (2). 2 was prepared according to the general
procedure by using benzaldehyde, thiourea, acetylacetone. Yield
69%. m.p. 183 ◦C [66]. 1H NMR (300 MHz, DMSO-d6): ı 2.20 (s, 3H,
CH3), 2.29 (s, 3H, CH3CO), 5.10 (d, 1H, J = 3.3 Hz, CH), 7.0–7.6 (m,
5H, Ar-H), 8.87 (br s, 1H, NH), 10.00 (br s, 1H, NH).). 13C NMR (75
MHz, DMSO-d6): ı 18.4 (CH3), 28.7 (CH3CO), 54.6 (CH-Ar), 101.4
(C-COMe), 125.0–129.0 (C-aromatic), 147.8 (CH3C C), 180.2 (C S),
194.4 (COMe); EIMS calculated for C13H14N2OS (M+•) 246.
4.3.1.3. 4-(3-Bromophenyl)-5-(ethoxycarbonyl)-6-methyl-3,4-
dihydropyrimidin-2(1H)-thione (3). 3 was prepared according to
the general procedure by using 3-bromobenzaldehyde, thiourea,
ethyl acetoacetate. Yield 60%. m.p. 182 ◦C. 1H NMR (300 MHz,
DMSO-d6): ı 0.93 (t, 3H, J = 6.9 Hz, OCH2CH3), 2.20 (s, 3H, CH3),
3.98 (q, 2H, J = 7.3 Hz, OCH2), 5.18 (d, 1H, J = 3.3 Hz, CH), 6.61 (s,
1H, Ar-H), 6.67 (d, 2H, Ar-H), 7.12 (t, 1H, Ar-H), 9.60 (br s, 1H,
NH), 10.52 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): 14.9
(OCH2CH3), 18.1 (CH3), 53.4 (CH-Ar), 59.7 (OCH2), 101.4 (C-COOEt),
119.7–144 (C-aromatic), 124.2 (C-aromatic-Br), 147.1 (CH3C C),
164.1 (COOEt), 179.8 (C S); EIMS calculated for C14H15N2O2SBr
(M+•) 355.
4.3.1.8. 5-(Ethoxycarbonyl)-4-(3-hydroxyphenyl)-6-ethoxy-3,4-
dihydropyrimidin-2(1H) thione (8). 8 was prepared according to
the general procedure by using 3-hydroxybenzaldehyde, thioures,
diethylmalonate. Yield 71%. m.p. 213 ◦C. 1H NMR (300 MHz, DMSO-
d6): ı 1.08 (t, 6H, J = 7.1 Hz, OCH2CH3), 4.18 (q, 4H, J = 7.1 Hz, OCH2),
5.25 (d, 1H, J = 3.3 Hz, CH), 6.62 (d, 2H, J = 8.5 Hz, Ar-H), 7.05 (d, 2H,
J = 8.5 Hz, Ar-H), 9.64 (br s, 1H, NH), 9.75 (s, 1H, OH) 10.40 (br s,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 183.1 (C S), 166.2 (C O),
158.0 (C-OH), 156.7 (C N), 129.6 (C-aromatic): EIMS calculated
for C15H18N2O4S (M+•) 322.
4.3.1.9. 5-(Ethoxycarbonyl)-4-(3-nitrophenyl)-6-
(trifluoromethyl)-3,4-dihydropyrimidin-2(1H)-thione
(9). 9
4.3.1.4. 5-(Ethoxycarbonyl)-4-(3-methoxyphenyl)-6-methyl-3,4-
dihydropyrimidin-2(1H)-thione (4). 4 was prepared according to
the general procedure by using 3-methoxybenzaldehyde, thiourea,
ethyl acetoacetate. Yield 64%. m.p. 153 ◦C. 1H NMR (300 MHz,
DMSO-d6): ı 1.09 (t, 3H, J = 7.2 Hz, CH3), 2.27 (s, 3H, CH3), 3.80 (s,
3H, OCH3), 4.10 (q, 2H, J = 7.1 Hz, OCH2CH3), 5.18 (d, 1H, J = 3.3 Hz,
CH), 6.40 (s, 1H, Ar-H), 6.45 (d, 1H, H-aromatic), 6.50 (d, 1H, Ar-H),
9.37 (br s, 1H, NH), 10.25 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): ı 14.9 (OCH2CH3), 18.1 (CH3), 53.7 (CH-Ar), 56.8 (OCH3),
59.8 (OCH2), 99.3 (C-COOEt), 119.6–140.0 (C-aromatic), 148.2,
(CH3C C), 150.9 (C-aromatic-OCH3), 165.1 (COOEt), 182.3 (C S);
EIMS calculated for C15H18N2O3S (M+•) 306.
was prepared according to the general procedure by using 3-
nitrobenzaldehyde, thiourea, ethyl trifluoroacetoacetate. Yield
71%. m.p. 221 ◦C. 1H NMR (300 MHz, DMSO-d6): ı 1.12 (t, 3H,
J = 7.2 Hz, CH3), 4.03 (q, 2H, J = 7.2 Hz, OCH2CH3), 5.24 (d, 1H,
J = 3.3 Hz, CH), 7.61 (m, 2H, Ar-H), 7.76 (br s, 1H, NH), 9.05 (br s,
1H, NH), 8.09 (m, 2H, Ar-H). 13C NMR (75 MHz, DMSO-d6): 14.7
(OCH2CH3), 52.6 (CH-Ar), 60.4 (OCH2), 98.1 (C-COOEt), 119.7–144.1
(C-aromatic), 139.0 (C-CF3), 146.0 (C-NO2), 148.9, (CH3C C), 164.1
(COOEt), 180.1 (C S); EIMS calculated for C14H12 F3N3O4 S (M+•
375.
)
4.3.1.10. 5-(Ethoxycarbonyl)-4-(4-methoxyphenyl)-6-methyl-
3,4-dihydropyrimidin-2(1H)-thione (10). 10 was prepared
according to the general procedure by using anisaldehyde,
thiourea, ethyl acetoacetate. Yield 77%. m.p. 153 ◦C [68]. 1H NMR
(300 MHz, DMSO-d6): ı 1.14 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.29 (s,
3H, CH3), 3.72 (s, 3H, OCH3), 4.04 (q, 2H, J = 7.1 Hz, OCH2), 5.17
(d, 1H, J = 3.3 Hz, CH), 7.22 (d, 2H, J = 8.6 Hz, Ar-H), 7.35 (d, 2H,
J = 8.6 Hz, Ar-H), 9.61 (br s, 1H, NH), 10.39 (br s, 1H, NH). 13C
NMR (75 MHz, DMSO-d6): ı 14.8 (OCH2CH3), 18.4 (CH3), 54.2
(CH-Ar), 56.4 (OCH3), 60.1 (OCH2), 99.8 (C-COOEt), 119.0–141.0
(C-aromatic), 149.0, (CH3C C), 150.7 (C-aromatic-OCH3), 165.4
4.3.1.5. 5-(Ethoxycarbonyl)-4-(3-hydroxyphenyl)-6-methyl-3,4-
dihydropyrimidin-2(1H) thione (5). 5 was prepared according to
the general procedure by using 3-hydroxybenzaldehyde, thiourea,
ethyl acetoacetate. Yield 79%. m.p. 185 ◦C [67]. 1H NMR (300 MHz,
DMSO-d6): ı 1.12 (t, 3H, J = 7.3 Hz, OCH2CH3), 2.27 (s, 3H, CH3), 4.05
(q, 2H, J = 7.3 Hz, OCH2), 5.09 (d, 1H, J = 3.3 Hz, CH), 6.67 (s, 1H, Ar-H),
6.86 (d, 2H Ar-H), 7.11 (t, 1H Ar-H), 9.61 (br s, 1H, NH), 9.96 (br s,
1H, OH) 10.29 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): ı 14.1
(OCH2CH3), 18.4 (CH3), 54.1 (CH-Ar), 59.4 (OCH2), 99.4 (C-COOEt),
122.7–141.0 (C-aromatic), 145.4 (CH3C C), 157.4 (C-OH), 164.5
(COOEt), 182.6 (C S); EIMS calculated for C14H16N2O3S (M+•) 292.
(COOEt), 182.1 (C S); EIMS calculated for C15H18N2O3S (M+•
)
306.

54
U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
4.3.1.11. 5-(Ethoxycarbonyl)-6-methyl-4-(4-N,N-
dimethylphenyl)-3,4-dihydropyrimidin-2(1H)-thione
152.1 (C O), 157.3 (C-OH), 164.5 (COOEt); EIMS calculated for
14H16N2O4 (M+•) 276.
(11). 11
C
was prepared according to the general procedure by using 4-(N,N-
dimethyl)benzaldehyde, thiourea, ethyl acetoacetate. Yield 78%.
m.p. 226 ◦C. 1H NMR (300 MHz, DMSO-d6): ı 1.11 (t, 3H, J = 7.1 Hz,
OCH2CH3), 2.25 (s, 3H, CH3), 3.67 (s, 6H, N(CH3)2), 4.03 (q, 2H,
J = 7.1 Hz, OCH2), 5.34 (d, 1H, J = 3.3 Hz, CH), 7.12 (d, 2H, Ar-H),
7.60 (d, 2H, Ar-H), 9.63 (br s, 1H, NH), 10.36 (br s, 1H, NH). 13C
NMR (75 MHz, DMSO-d6): 180.6 (C S), 165.2 (C O), 119–136
4.3.1.17. 5-(Ethoxycarbonyl)-4-(4-hydroxy-3-methoxyphenyl)-
6-methyl-3,4-dihydropyrimidin-2(1H)-one (17). 17 was prepared
according to the general procedure by using vanillin, urea, ethyl
acetoacetate. Yield 87%. m.p. 232–233 ◦C [68]. 1H NMR (300 MHz,
DMSO-d6): ı 1.14 (t, 3H, J = 7.1 Hz, CH3), 2.24 (s, 3H, CH3), 3.73 (s,
3H, OCH3), 4.10 (q, 2H, J = 7.1 Hz, OCH2CH3), 5.16 (d, 1H, J = 3.3 Hz,
CH), 6.40 (s, 1H, Ar-H), 6.45 (d, 1H, H-aromatic), 6.50 (d, 1H,
Ar-H), 7.79 (br s, 1H, NH), 8.97 (br s,1H, NH), 10.67 (s, 1H OH).
13C NMR (75 MHz, DMSO-d6): ı 14.3 (OCH2CH3), 18.2 (CH3), 53.6
(CH-Ar), 56.4 (OCH3), 60.1 (OCH2), 101.4 (C-COOEt), 119.7–144.
(C-aromatic), 144.1 (C-aromatic-OH), 147.9, (CH3C C), 151.3
(C O), 151.2 (C-aromatic-OCH3), 164.1 (COOEt); EIMS calculated
for C15H18N2O5 (M+•) 306.
(C-aromatic), 44.2 (NCH3): EIMS calculated for C16H21N3O2S (M+•
)
319.
4.3.1.12. 5-(Ethoxycarbonyl)-6-methyl-4-(3-thienyl)-3,4-
dihydropyrimidin-2(1H)-thione (12). 12 was prepared according
to the general procedure by using thiophene-3-carboxaldehyde,
thiourea, ethyl acetoacetate. Yield 56%. m.p. 256 ◦C. 1H NMR
(300 MHz, DMSO-d6): ı 1.08 (t, J = 7.5 Hz, OCH2CH3), 2.25 (s, 3H,
CH3), 4.18 (q, 2H, J = 7.0, OCH2), 5.18 (d, 1H, J = 3.3 Hz, CH), 6.66 (d,
1H, Ar-H), 7.00 (d, 1H, Ar-H), 9.58 (br s, 1H, NH), 10.42 (br s, 1H, NH).
13C NMR (75 MHz, DMSO-d6): ı 14.5 (OCH2CH3), 18.3 (CH3), 54.6
(CH-Ar), 59.8 (OCH2), 99.7 (C-COOEt), 121.0–138.0 (C-aromatic),
148.6, (CH3C C), 164.7 (COOEt), 180.6 (C S); EIMS calculated for
4.3.1.18. 5-Benzoyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenyl-
3,4-dihydropyrimidin-2(1H)-one (18). 18 was prepared according
to the general procedure by using vanillin, urea, dibenzoylmethane.
Yield 66% 74%. m.p. 222 ◦C. 1H NMR (300 MHz, DMSO-d6): ı 3.73
(s, 3H, OCH3), ı 5.10 (d, 1H, J = 3.3 Hz, CH), 6.40 (s, 1H, Ar-H), 6.45
(d, 1H, H-aromatic), 6.50 (d, 1H, Ar-H), 7.22–7.35 (m, 10H, Ar-H),
7.63 (br s, 1H, NH), 9.10 (br s, 1H, NH), 10.12 (s, 1H, OH). 13C
NMR (75 MHz, DMSO-d6): ı 54.1 (CH-Ar), 101.2 (C-COPh), 144.1
(C-aromatic-OH), 146.5 (CH3C C), 151.2 (C-aromatic-OCH3), 152.4
(C O), 193.1 (COPh); EIMS calculated for C24H20N2O4 (M+•) 400.
C
12H14N2O2S2 (M+•) 282.
4.3.1.13. 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-
dihydropyrimidin-2(1H)-one (13). 13 was prepared according to
the general procedure by using benzaldehyde, urea, ethyl acetoac-
etate. Yield 97%. m.p. 202 ◦C [64]. 1H NMR (300 MHz, DMSO-d6):
ı 1.09 (t, 3H, J = 7.2 Hz, OCH2CH3), 2.21 (s, 3H, CH3), 4.01 (q, 2H,
J = 7.2 Hz, OCH2), 5.14 (d, 1H, J = 3.3 Hz, CH), 7.22–7.33 (m, 5H, Ar-H),
7.76 (br s, 1H, NH), 9.22 (br s, 1H, NH) 13C NMR (75 MHz, DMSO-
d6): ı 14.5 (OCH2CH3), 18.2 (CH3), 54.4 (CH-Ar), 59.6 (OCH2),
98.1 (C-COOEt), 126.7–129.0 (C-aromatic), 148.8 (CH3C C), 152.6
(C O), 165.6 (COOEt): EIMS calculated for C14H16N2O3 (M+•) 260.
4.3.1.19. 5-(Ethoxycarbonyl)-6-methyl-4-(4-N,N-
dimethylphenyl)-3,4-dihydropyrimidin-2(1H)-one
(19). 19
was prepared according to the general procedure by using 4-
(N,N-dimethyl)benzaldehyde, urea, ethyl acetoacetate. Yield 89%.
m.p. 259–260 ◦C [73]. 1H NMR (300 MHz, DMSO-d6): ı 1.12 (t,
3H, J = 6.9 Hz, OCH2CH3), 2.30 (s, 3H, CH3), 3.37 (s, 6H, N(CH3)2),
4.09 (q, 2H, J = 7.2 Hz, OCH2), 5.04 (d, 1H, J = 3.3 Hz, CH), 6.62 (d,
2H, J = 8.5 Hz, Ar-H), 7.05 (d, 2H, J = 8.5 Hz, Ar-H), 7.81 (br s, 1H,
NH), 9.11 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): ı 14.1
(OCH2CH3), 17.9 (CH3), 44.3 (N(CH3)2), 54.4 (CH-Ar), 59.2 (OCH2),
100.6 (C-COOEt), 126.7–129.0 (C-aromatic), 147.6 (CH3C C), 149.6
(C-N(CH3)2), 151.8 (C O), 164.9 (COOEt); EIMS calculated for
C16H21N3O3 (M+•) 303.
4.3.1.14. 5-Aceto-6-methyl-4-phenyl-3,4-dihydropyrimidin-
2(1H)-one (14). 14 was prepared according to the general
procedure by using benzaldehyde, urea, acetylacetone. Yield 91%.
m.p. 241–242 ◦C [69,70]. 1H NMR (300 MHz, DMSO-d6): ı 2.07
(s, 3H, CH3), 2.32 (s, 3H, CH3CO), 5.10 (d, 1H, J = 3.3 Hz, CH), 7.69
(br s, 1H, NH), 9.20 (br s, 1H, NH), 7.0–7.6 (m, 5H, Ar-H). 13C NMR
(75 MHz, DMSO-d6): ı 17.9 (CH3), 29.4 (CH3CO), 53.3 (CH-Ar),
101.1 (C-COMe), 120.2–127.0 (C-aromatic), 145.9 (CH3C C), 152.9
(C O), 194.7 (COMe); EIMS calculated for C13H14N2O2 (M+•) 230.
4.3.1.20. 5-(Ethoxycarbonyl)-6-methyl-4-(4-N,N-
dimethylphenyl)-3,4-dihydropyrimidin-2(1H)-one
(20). 20
was prepared according to the general procedure by using 4-(N,N-
dimethyl)benzaldehyde, urea, diethylmalonate. Yield 67%. m.p.
202–205 ◦C. 1H NMR (300 MHz, DMSO-d6): ı 1.14 (t, 6H, J = 7.1 Hz,
OCH2CH3), 3.37 (s, 6H, N(CH3)2), 4.10 (q, 4H, J = 7.1 Hz, OCH2),
5.10 (d, 1H, J = 3.3 Hz, CH), 6.62 (d, 2H, J = 8.5 Hz, Ar-H), 7.05 (d,
2H, J = 8.5 Hz, Ar-H), 7.61 (br s, 1H, NH), 9.11 (br s, 1H, NH). 13C
NMR (75 MHz, DMSO-d6): ı 12.4, 14.7 (OCH2CH3), 44.4 (N(CH3)2),
54.4 (CH-Ar), 59.4, 60,8 (OCH2), 101.2 (C-COOEt), 125.0–141.0
(C-aromatic), 145.9 (CH3C C), 149.3 (C-N(CH3)2), 152.5 (C O),
164.2 (COOEt); EIMS calculated for C17H23N3O4 (M+•) 333.
4.3.1.15. 5-Benzoyl-4,6-diphenyl-3,4-dihydropyrimidin-2(1H)-
one (15). 15 was prepared according to the general procedure
by using benzaldehyde, urea, dibenzoylmethane. Yield 67%. m.p.
213 ◦C [71]. 1H NMR (300MHz, DMSO-d6): ı 5.10 (d, 1H, J = 3.3 Hz,
CH), 7.22–7.35 (m, 15H, Ar-H), 7.78 (br s, 1H, NH), 9.20 (br s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6): ı 53.6 (CH-Ar), 101.4 (C-COPh),
120.2–142.0 (C-aromatic), 145.9 (CH3C C), 152.9 (C O), 192.4
(COPh); EIMS calculated for C23H18N2O2 (M+•) 354.
4.3.1.16. 5-(Ethoxycarbonyl)-4-(3-hydroxyphenyl)-6-methyl-
3,4-dihydropyrimidin-2(1H)-one (16). 16 was prepared according
to the general procedure by using 3-hydroxybenzaldehyde, urea,
ethyl acetoacetate. Yield 89%. m.p. 160–162 ◦C [72]. 1H NMR
(300 MHz, DMSO-d6): ı 1.14 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.18 (s,
3H, CH3), 4.03 (q, 2H, J = 7.3 Hz, OCH2), 5.07 (d, 1H, J = 3.3 Hz, CH),
6.61– 6.67 (m, 3H, Ar-H), 7.12 (t, 1H, 8.1 Hz, Ar-H), 7.72 (br s, 1H,
NH), 9.16 (br s, 1H, NH), 9.3 (s, 1H, OH). 13C NMR (75 MHz, DMSO-
d6): ı 14.1 (OCH2CH3), 18.0 (CH3), 54.9 (CH-Ar), 59.2 (OCH2),
98.7 (C-COOEt), 126.7–129.0 (C-aromatic), 147.6 (CH3C C),
4.3.1.21. 5-(Ethoxycarbonyl)-6-ethoxy-4-(4-hydroxyphenyl)-
3,4-dihydropyrimidin-2(1H)-one (21). 21 was prepared according
to the general procedure by using 4-hydroxybenzaldehyde, urea,
diethylmalonate. Yield 71%. m.p. 188 ◦C. 1H NMR (300 MHz, DMSO-
d6): ı 1.18 (t, 6H, J = 7.1 Hz, OCH2CH3), 3.90 (q, 4H, J = 7.2 Hz, OCH2),
5.10 (d, 1H, J = 3.3 Hz, CH), 6.62 (d, 2H, J = 8.5 Hz, Ar-H), 7.05 (d, 2H,
J = 8.5 Hz, Ar-H), 7.77 (br s, 1H, NH), 9.11 (br s, 1H, NH), 9.96 (br s, 1H,
OH). 13C NMR (75 MHz, DMSO-d6): ı 12.7, 14.1 (OCH2CH3), 17.8
(CH3), 54.2 (CH-Ar), 59.5, 60.6 (OCH2), 98.8 (C-COOEt), 126.7–138.0

U. Rashid et al. / Journal of Molecular Graphics and Modelling 43 (2013) 47–57
55
(C-aromatic), 147.6 (CH3C C), 152.1 (C O), 157.9 (C-OH), 165.1
(COOEt); EIMS calculated for C15H18N2O5 (M+•) 306.
(300 MHz, DMSO-d6): ı 1.18 (t, 6H, J = 7.1 Hz, OCH2CH3), 3.90 (q,
4H, J = 7.1 Hz, OCH2), 3.72 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 4.01 (q,
4H, OCH2), 4.98 (s, 1H, CH), 5.87 (s, 2H, NH2), 6.66 (d, 1H, Ar-H),
7.00 (d, 1H, Ar-H), 7.1 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
ı 12.4, 14.7 (OCH2CH3), 17.9 (CH3), 54.8 (CH-Ar), 56.7 (OCH3),
59.4, 60.1 (OCH2), 100.2 (C-COOEt), 126.7–139.0 (C-aromatic),
147.9 (CH3C C), 150.3 (C-aromatic-OCH3), 160.5 (C-NH2), 165.1
(COOEt); EIMS calculated for C16H21N3O4 (M+•) 319.
4.3.1.22. 4-(4-Chlorophenyl)-5-(ethoxycarbonyl)-6-methyl-3,4-
dihydropyrimidin-2(1H)-one (22). 22 was prepared according to
the general procedure by using 4-chlorobenzaldehyde, urea, ethyl
acetoacetate. Yield 70%. m.p. 218–219 ◦C [73]. 1H NMR (300 MHz,
DMSO-d6): ı 0.97 (t, 3H, J = 7.1 Hz, OCH2CH3), 4.10 (q, 4H, J = 7.2 Hz,
OCH2), 5.04 (d, 1H, J = 3.3 Hz, CH), 7.14–7.24 (m, 4H, Ar-H). 7.63 (br
s, 1H, NH), 9.15 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): ı 14.9
(OCH2CH3), 17.9 (CH3), 54.9 (CH-Ar), 60.3 (OCH2), 101.4 (C-COOEt),
126.7–137.0 (C-aromatic), 148.9 (CH3C C), 151.7 (C O), 164.1
(COOEt); EIMS calculated for C14H15N2O3Cl (M+•) 294.
4.3.2.2. Ethyl
methyl-1,4-dihydropyrimidine-5-carboxylate
2-amino-4-(4-hydroxy-3-methoxyphenyl)-6-
(27). 27 was
prepared according to the general procedure by using vanillin,
guanidine HCl, ethyl acetoacetate. Yield 65%. m.p. 196 ◦C. 1H NMR
(300 MHz, DMSO-d6): ı 1.11 (t, 3H, J = 7.1 Hz, CH3), 2.25 (s, 3H,
CH3), 3.71 (s, 3H, OCH3), 4.01 (q, 2H, J = 7.1 Hz, OCH2CH3), 5.24 (s,
1H, J = 2.24 Hz, CH), 5.78 (s, 2H, NH2), 6.40 (s, 1H, Ar-H), 6.45 (d,
1H, H- aromatic), 6.50 (d, 1H, Ar-H), 7.79 (br s, 1H, NH), 8.97 (br
s,1H, NH), 10.51 (s, 1H OH). 13C NMR (75 MHz, DMSO-d6): ı 14.7
(OCH2CH3), 18.5 (CH3), 54.6 (CH-Ar), 56.2 (OCH3), 60.4 (OCH2),
100.8 (C-COOEt), 119.7–144 (C-aromatic), 144.3 (C-aromatic-OH),
147.2 (CH3C C), 151.2 (C-aromatic-OCH3), 161.5 (C-NH2), 164.1
(COOEt); EIMS calculated for C15H19N3O4 (M+•) 305.
4.3.1.23. 5-(Ethoxycarbonyl)-6-methyl-4-(3-pyridyl)-3,4-
dihydropyrimidin-2(1H)-one (23). 23 was prepared according
to the general procedure by using pyridine-3-carboxaldehyde,
urea, ethyl acetoacetate. Yield 79%. m.p. 213–214 ◦C [74]. 1H NMR
(300 MHz, DMSO-d6): ı 1.08 (t, J = 7.5Hz), 2.28 (s, 3H, CH3), 4.01 (q,
2H, J = 7.1 Hz, OCH2CH3), 5.14 (d, 1H, J = 3.3 Hz, CH), 7.35–7.62 (m,
2H, Ar-H), 7.76 (br s,1H, NH), 9.22 (br s,1H, NH). 8.5 (s, 2H, Ar-H),
13C NMR (75 MHz, DMSO-d6): ı 14.1 (OCH2CH3), 17.9 (CH3), 54.1
(CH-Ar), 59.4 (OCH2), 98.1 (C-COOEt), 126.7–129.0 (C-aromatic),
145.9 (CH3C C), 151.2 (C O), 165.1 (COOEt); EIMS calculated for
C13H15N3O3 (M+•) 261.
4.4. In vitro urease inhibition studies
The shortlisted compounds in initial hits and other synthesized
compounds were screened according to literature procedure [55].
The reaction mixtures comprising 25 ␮L of enzyme (jack bean ure-
ase) solution and 55 ␮L of buffer containing 100 mM urea were
incubated with 5 ␮L of test compounds (0.5 mM concentration) at
30 ◦C for 15 min in 96-well plates. Urease activity was determined
by measuring ammonia production using the indophenol method
in which 45 ␮L each phenol reagent (1% (w/v) phenol and 0.005%
(w/v) sodium nitroprusside) and 70 ␮L of alkali reagent (0.5% (w/v)
NaOH and 0.1% active chloride NaOCl) were added to each well.
The increasing absorbance at 630 nm was measured after 50 min,
using a microplate reader (Molecular Device, USA). All reactions
were performed in triplicate in a final volume of 200 ␮L. The results
(change in absorbance per min) were processed by using soft Max
Pro software (molecular Device, USA). The entire assay was per-
formed at pH 6.8. Percentage inhibition was calculated using the
following formula:
4.3.1.24. 5-Aceto-1,6-dimethyl-4-phenyl-3,4-dihydropyrimidin-
2(1H)-one (24). 24 was prepared according to the general
procedure by using benzaldehyde, N-methylurea, acetylacetone.
Yield 73%. m.p. 154–155 ◦C [66]. 1H NMR (300 MHz, DMSO-d6):
ı 2.10 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.93 (s, 3H, N-CH3), 5.14
(d, 1H, J = 3.3 Hz, CH), 7.69 (br s, 1H, NH), 7.0–7.6 (m, 5H, Ar-H).
13C NMR (75 MHz, DMSO-d6): ı 18.1 (CH3), 28.7 (CH3CO), 30.3
(N-CH3), 54.1 (CH-Ar), 99.7 (C-COMe), 127.0–129.4 (C-aromatic),
148.2 (CH3C C), 152.2 (C O), 195.7(COMe); EIMS calculated for
C13H14N2O2 (M+•) 244.
4.3.1.25. 1-Benzyl-5-(ethoxycarbonyl)-6-methyl-4-phenyl-3,4-
dihydropyrimidin-2(1H)-one (25). 25 was prepared according to
the general procedure by using benzaldehyde, N-benzylurea, ethyl
acetoacetate. Yield 71%. m.p. 88–89 ◦C [75]. 1H NMR (300 MHz,
DMSO-d6): ı 0.97 (t, 3H, CH3), 2.35 (s, 3H, CH3), 4.03 (q, 2H,
J = 7.1 Hz, OCH2CH3), 4.59 (d, 1H, J = 16.8 Hz, CH), 4.93 (d, 1H,
J = 16.8 Hz, CH), 5.24 (d, 1H, J = 3.3 Hz, CH), 7.93 (br s, 1H, NH),
7.06–7.35 (m, 10H, Ar-H). 13C NMR (75 MHz, DMSO-d6): ı 14.7
(OCH2CH3), 17.9 (CH3), 47.2 (N-CH2), 54.4 (CH-Ar), 60.4 (OCH2),
100.0 (C-COOEt), 127.3–139.8 (C-aromatic), 147.6 (CH3C C), 152.1
(C O), 165.6 (COOEt); EIMS calculated for C21H22N2O3 (M+•) 350.
ꢀ
ꢁ
OD test well
OD control
100 −
× 100
Thiourea was used as the standard urease inhibitor.
Appendix A. Supplementary data
4.3.2. General procedure for the synthesis of
2-amino-1,4-dihydropyrimidines
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.jmgm.
2013.04.006.
A mixture of an aldehyde (10 mmol), guanidine hydrochloride
(10 mmol), 1,3-dicarbonyl compound (10 mmol) and sodium bicar-
bonate (3.35 g, 40 mmol), in DMF (20 mL) was taken in a Pyrex
tube and irradiated in an ultrasonic bath at 70–75 ◦C. The reaction
was monitored by TLC. After the completion of the reaction, the
resulting precipitate was filtered and crude product was recrystal-
lized from an appropriate solvent or purified with silica gel column
chromatography. The purity of compounds was checked by TLC in
different solvent systems. All the products were confirmed by MS
1H NMR and 13C NMR data.
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