Design, synthesis, molecular docking studies and in vitro screening of ethyl 4-(3-benzoylthioureido) benzoates as urease inhibitors

Article abstract of DOI:10.1016/j.bioorg.2013.10.001

Thioureas are exceptionally versatile building blocks towards the synthesis of wide variety of heterocyclic systems, which also possess extensive range of pharmacological activities. The substituted benzoic acids were converted into corresponding acid chlorides, these acid chlorides were then treated with potassium thiocyanate in acetone and then the reaction mixture was refluxed for 1-2 h afford ethyl 4-(3-benzoylthioureido)benzoates thioureas in good yields. All the newly synthesized compounds were evaluated for their urease inhibitory activities and were found to be potent inhibitors of urease enzyme. Compounds 1f and 1g were identified as the most potent urease inhibitors (IC₅₀ 0.21 and 0.13 μM, respectively), and was 100-fold more potent than the standard inhibitors. Further molecular docking studies were carried out using the crystal structure of urease to find out the binding mode of the inhibitors with the enzyme.

Full text of DOI:10.1016/j.bioorg.2013.10.001

Bioorganic Chemistry 52 (2014) 1–7
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, molecular docking studies and in vitro screening
of ethyl 4-(3-benzoylthioureido) benzoates as urease inhibitors
Aamer Saeed a, Muhammad Siraj Khan b, Hummera Rafique c, Mohammad Shahid d, Jamshed Iqbal b,
⇑
a Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan
b Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
d Fraunhofer Institute SCAI, Department of Bioinformatics, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2013
Available online 30 October 2013
Thioureas are exceptionally versatile building blocks towards the synthesis of wide variety of heterocy-
clic systems, which also possess extensive range of pharmacological activities. The substituted benzoic
acids were converted into corresponding acid chlorides, these acid chlorides were then treated with
potassium thiocyanate in acetone and then the reaction mixture was refluxed for 1–2 h afford ethyl 4-
(3-benzoylthioureido)benzoates thioureas in good yields. All the newly synthesized compounds were
evaluated for their urease inhibitory activities and were found to be potent inhibitors of urease enzyme.
Keywords:
Antioxidant activity
Ethyl 4-(3-benzoylthioureido)benzoates
Molecular docking
Compounds 1f and 1g were identified as the most potent urease inhibitors (IC50 0.21 and 0.13 lM,
respectively), and was 100-fold more potent than the standard inhibitors. Further molecular docking
studies were carried out using the crystal structure of urease to find out the binding mode of the inhib-
itors with the enzyme.
Urease inhibition
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
agrochemicals [10–12]. 1-Benzoyl-3-(4,6-disubstituted)pyrimi-
dine-2-yl-thioureas have excellent herbicidal activities. Some
Urease (EC 3.5.1.5; urea amidohydrolase) is a metal containing
enzyme that catalyzes the hydrolysis of urea into ammonia and
carbamates [1,2]. Ureases are widespread in nature among plants,
bacteria, fungi, algae and invertebrates. Urease producing bacteria
have a harmful effect on human health. In humans the urease of
Helicobacter pylori causes infections of gastrointestinal and urinary
tract [3], such as stomach cancer and the peptic ulcer [4]. Other
urease associated diseases include hepatic encephalopathy,
urolithiasis, urinary catheter encrustation, pyelonephritis and he-
patic coma [5]. H. pylori survive at the low pH of stomach during
colonization, which ultimately leads to gastric and peptic ulcers
and, in some cases it may lead to cancer [6]. Strategies based on
urease inhibition are the major treatment of diseases caused by
H. pylori and associated urease producing bacteria. Ureases are
inhibited by different classes of compounds [7]. Thioureas and
their derivatives [8,9] were found to have a significant inhibitory
activity against the urease enzyme. Thioureas attracted worldwide
attention due to their number of applications and diverse biologi-
cal significance, possess a wide range of biological applications
including antiviral, antibacterial, antifungal, antitubercular,
antithyroidal, herbicidal, insecticidal activities and they act as
acylthioureas have been found to possess pesticidal effects and it
also promotes plant growth [10]. Halo-thiophene and pyridyl
substituted heterocyclic thioureas are inhibitors of non-nucleoside
HIV-1 reverse transcriptase [13,14]. Various thiourea derivatives
act as potential neuraminidase inhibitors of influenza virus [15].
Some thioureas have been shown to have notable positive effect
on the germination of maize seeds as well as on the chlorophyll
contents in seedling leaves. Thioureas serve as well-known
chelating agents for transition metals complexes [16]. N,N-Dial-
kyl-N0-benzoylthioureas act as selective complexing agents for
the enrichment of platinum metals even from strongly interfering
matrixes [17].
The complexes of thiourea derivatives also show broad spec-
trum of biological activities. High stability and simple synthesis
made it possible to use the N-benzoylthiourea group as modifier
of silica for complexation gas chromatography. By taking the
advantage of the chelating properties of N-benzoylthioureas Cu(II)
ions were bonded with silica to give a packing
p-type interaction
with absorbates having electron donor properties [18]. The high
selectivity of this packing permitted the separation of complex mix-
tures including isomers of different classes of organic compounds.
Silica was chemically modified with N-acyl-N0-benzoylthiourea
group. The resulting material was observed to behave as a selective
mean of preconcentration of Cd(II), Zn(II) and Cu(II) from ethanol
fuel by means of column technique. Ethanolic solutions having 6
⇑
Corresponding author. Fax: +92 992 383441.
E-mail address: drjamshed@ciit.net.pk (J. Iqbal).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.10.001

2
A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
micro mol of the metal ion were percolated through the column and
retention of 100% were achieved for all metals [19]. N-(substituted-
phenyl)-N-phenylthioureas have been developed as anion-binding
site in a hydrogen-bonding receptors, calyx, arenes containing thio-
2.2. Urease inhibition assay
All of the newly synthesized ethyl 4-(3-benzoylthioureido) ben-
zoate derivatives were found to be potent inhibitors of urease of
Canavalia ensiformis (Table 1). Compounds 1f and 1g were the most
ureas as neutral receptors towards
a,a-dicarboxylate anions [20].
Benzoylthiourea of general formula (RANH(C@S)ANH)nAZ where
R = alkyl, cycloalkyl, aryl, alkenyl, etc. and Z is aromatic ring are
used in the optical recording medium which is heat resistance, plas-
ticizer resistant, moisture resistance and water resistance. Thiourea
derivatives serves as precursors and are important intermediate in-
volved in the synthesis of a variety of heterocyclic systems with
wide range of biological and synthetic applications i.e. iminothiazo-
lidinones, thiohydantoins, imidazole-2-thiones, thiazolines, thia-
zolamines, etc. [21,22].
potent compounds among the series with IC50 = 0.21 and 0.13 lM,
respectively. Compound 1f having mono methoxy was slightly less
potent than 1g having di methoxy groups as a substituent. All of
the investigated compounds were 100-fold more than active than
the positive controls, thiourea (IC50 = 20.3
lM) and acetohydroxa-
mic acid (IC50 = 43.2 M). Halogenated substituted thiourea deriv-
l
atives were slightly less active than methoxy substituted
compounds. In brief, the compounds having electron donating
(AOCH3) substituents were slightly more potent inhibitors than
electron withdrawing substituents (ACl or AF).
In the present study, ethyl 4-(3-benzoylthioureido) benzoate
derivatives were synthesized and evaluated for urease inhibitory
activity. The obtained results showed high activity of the synthe-
2.3. Antioxidant activity assay
sized thiourea derivatives with IC50 of 0.13 lM for the most potent
compound. The binding mode of the compounds was predicted by
molecular docking studies. Moreover, the compounds were tested
against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
activities.
Thiourea derivatives and their metal complexes had shown sig-
nificant antioxidant activity [23–25]. Most of the investigated ethyl
4-(3-benzoylthioureido) benzoate derivatives showed good anti-
oxidant activities (Table 1). Compounds 1a, 1b, 1c, 1d, 1f and 1j
with IC50 values 4.13, 4.98, 4.10, 1.93, 2.17 and 7.16 lM, respec-
tively. The ethyl 4-(3-benzoylthioureido) benzoate derivatives
have the ability to donate hydrogen when reacted with DPPH rad-
ical to form radical hybrid, like thiosemicarbazones [26]. Propyl
gallate and 2-tert-butyl-4-hydroxyanisole were used as reference
standards in the antioxidant assay. The tested compounds were
more potent antioxidant as compared to reference standards, ex-
cept compounds 1e and 1h. Compound 1d (IC50 = 1.93 lM) was
the most potent compound with methyl group as substituent
showing good antioxidant activity even at very low concentrations.
2. Results and discussion
2.1. Chemistry
The substituted benzoic acids were converted into correspond-
ing acid chlorides by their reaction with thionyl chloride. These
acid chlorides were then treated with potassium thiocyanate in
acetone and then reaction mixture was refluxed with 1–2 h affor-
ded aminoethylbenzoate to afford 1-aroyl-3-(p-ethylbenzo-
ato)thioureas, which were further purified by recrystallization
from ethanol. The synthetic pathway is illustrated in Scheme 1.
In the IR spectra, the characteristic absorptions for the NAH
protons of ethyl 4-(3-benzoylthioureido) benzoates (1a–j) ap-
peared in the range of 3298–3332 cmꢀ1. The ꢀes1ter carbonyl carbon
absorption peaks observed at 1722–1729 cm and the amidic car-
2.4. Docking and pose ranking
The results of re-docking experiment showed that the enzyme
structure and its binding site were well prepared and the docking
program reproduced the natively bound conformations of the co-
crystallized bound ligand. The docking results for the compounds
1a–1j were achieved, which showed that there were different con-
formations (poses) predicted for each compound. Table 2 shows
the docking scores obtained with FlexX and the rank number of
best pose obtained with NNScore [27] algorithm for the com-
pounds screened against the enzyme. Docking scores of the com-
pounds range between ꢀ12 and ꢀ24 kcal/mol, however, filtering
the poses for the compounds by NNScore showed there could be
other conformations that might interact favorably with the en-
zymes. With the exception of two compounds (1b and 1d), the dif-
ference in the docking scores for the first docking pose and the
predicted favorable pose for all compounds was approximately
bonyl absorption appeared at 1671–1688 cmꢀ1
.
The synthesis of ethyl 4-(3-benzoylthioureido) benzoates (1a–j)
was also confirmed by 1H NMR due to the presence of a two
characteristic broad band singlet for ANH protons at
d
11.31–11.42 ppm and another ANH peak observed at d 10.23–
10.38 ppm. In 13C NMR spectra the characteristics carbonyl carbon
peaks of ester moeity observed in the range of d 165.4–166.2 ppm
and amide carbonyl carbon appeared at d 167.2–168.2 ppm. The
(C@S) carbon signal appeared in the range of d 180.4–181.2 ppm.
Mass spectroscopy and elemental analysis of ester thioureas also
confirm their synthesis successfully. General structure for the ser-
ies of compounds synthesized is as below.
O
C
COCl
COOH
N C S
SOCl2
Reflux
KSCN
Acetone
R
R
O
R
H2N
COC2H5
reflux 1-2 hr
O
H
S
C
O
C
H
N
C
N
OC2H5
R
R = H, 3-Cl, 2,4-Dichloro, 4-CH3 3-CH 4-OCH3 3,4-Dimethoxy, 3-OCH3 2-Br, 2-F.
,
,
,
,
3
Scheme 1. Ethyl 4-(3-benzoylthioureido) benzoates (1a–j).

A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
3
Table 1
Urease inhibition and antioxidant activity of the compounds (1a–1j).
O
C
S
C
O
C
NH
NH
OCH2CH3
R
Compounds
R
Urease inhibition IC50
(
lM) SEM
Antioxidant IC50 (lM) SEM or %
1a
1b
1c
1d
1e
1f
1g
1h
H
3-Cl
0.51 0.071
0.26 0.032
0.28 0.23
0.67 0.19
1.07 0.12
0.21 0.48
0.13 0.061
0.35 0.015
0.47 0.053
0.73 0.059
4.13 0.06
4.98 0.27
4.10 0.49
1.93 0.18
27.9 2.15%
2.17 0.01
12.8 1.06
12.8 1.3%
14.5 0.36
7.16 0.41
–
2,4-diCl
4-CH3
3-CH3
4-OCH3
3,4-diOCH3
2-Br
1i
1j
2-OCH3
2-F
Thiourea
AHA
PG
20.3
5
43.2 2.6
–
–
–
24
3
TBH
27.6 2.1
– Not determined, AHA, acetohydroxamic acid, PG, n-propyl gallate, TBH, 2-tert-butyl-4-hydroxyanisole.
observer a good correlation of the docking scores with the activity
values because of the limitations in the scoring functions of the
docking programs. For this purpose, more rigorous methods such
as molecular dynamics followed by molecular mechanics based
free energy of binding calculation would be required. However, a
bottleneck in using such sophisticated methods is the time con-
suming and compute expensive charge derivation methods.
Table 2
Docking scores and best pose ranks for the compounds docked against the urease
enzyme.
Compound Docking scores
(kcal/mol)
Pose ranks in
top 10
Docking score for
selected pose
1a
1b
1c
1d
1e
1f
1g
1h
1i
ꢀ16.60
ꢀ17.13
ꢀ12.51
ꢀ13.59
ꢀ17.48
ꢀ24.56
ꢀ20.52
ꢀ13.11
ꢀ14.64
ꢀ15.40
3
9
2
8
7
2
2
6
5
9
ꢀ14.62
ꢀ10.82
ꢀ10.41
ꢀ9.38
2.5. Binding mode analysis
ꢀ14.68
ꢀ22.99
ꢀ20.18
ꢀ12.07
ꢀ13.20
ꢀ12.17
A common binding mode was observed for the compounds
docked to the urease enzyme. The hydrophobic opening of urease
pocket is composed of several hydrophobic residues that include
Ala440, Asp494, Aala636, and Met637. As shown in the interaction
diagrams in Figs. 2 and 3, the aromatic ring of the benzoic acid
moiety of all compounds form hydrophobic interactions with these
residues. Aromatic interactions are also formed between the phe-
nyl tail of the compounds and the above mentioned hydrophobic
residues as well as two histidine residues (His492 and His519) that
are located in the entrance of the enzyme active site. His492 is also
involved in making hydrogen bonds with the carboxyl oxygen. The
preferred binding modes selected by the external scoring function
are resulting in a binding mode that is favored by most of the com-
pounds except the compounds 1f and 1g. 1f and 1g adapt confor-
mations that are flipped opposite with respect to the
conformations of all other compounds. Both of these compounds
form hydrogen bonds with Arg439. Although, in all cases, all the
compounds interacts with only one metal atom and adapt confor-
mations where the oxygen of the compound occupies the middle of
both nickel atoms.
1j
2 kcal/mol. For the two compounds, a larger difference of approx-
imately 4–6 kcal/mol was observed.
The docked poses of all compounds are shown in Fig. 1. It has
been observed that the top ranked selected poses with high pre-
dicted activity of all compounds interact within the active site of
the enzyme in a similar way. The strict pharmacophoric criterion
is not fulfilled during the docking calculations, because no docking
predictions were made when the strict metal pharmacomphore
constraints were specified. However, when the two metal pharma-
cophore criteria was relieved and set to optional, interaction with
one of the two nickel atoms was observed. The oxygen atom in
benzoic acid moiety of all compounds interacts with the second
nickel atom. The same oxygen atom is involved in hydrogen bond-
ing interaction with His492 in the active site. Another hydrogen
bond interaction is observed between the amine nitrogen and
Asp494. These three interactions are common in all the predicted
poses of the compounds. Aromatic interactions of the benzene ring
with two alanine residues (Ala440 and Ala636) and Met637 are
formed which helps in tightly holding the compounds towards
the nickel center in the active site. However, the predicted confor-
mation of 1f is compound is opposite as compared to other com-
pounds, in which the oxygen atom in the phenyl moiety make
interactions with the nickel atom and His494.
3. Experimental
3.1. General
1H NMR were recorder on a Brucker AM-300 spectrophotome-
ter and chemical shifts of 1H NMR are reported in parts per million
(ppm). The melting point was determined on Staurt SMP3 melting
point apparatus and is uncorrected. FTIR spectra were recorded
using Shimadzu IR 460 spectrophotometer by Attenuated Total
Reflectance (ATR) method. The elemental analysis was performed
on LECO-932 CHNS analyzer.
The calculated docking scores of the first predicted poses of
each compound show a small correlation of 0.28 with the activity
values, however, the correlation is slightly increased to 0.33 if the
docking scores of the selected poses are used. It is unlikely to

4
A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
Fig. 1. Predicted docked poses of the compounds inside the active site of urease enzyme. The docked conformations of the compounds are shown in stick representation and
the two nickel atoms are represented by blue spheres. Hydrogen bonding and aromatic interactions with the surrounding residues are shown with dotted lines. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Interaction diagrams for compounds 1a and 1e. Hydrogen bond and metal interactions are shown with dotted lines. Green lines represent the hydrophobic residues
located in the active site. (Left): interaction diagram of compound 1a showing interactions with active site residues. (Right): interaction diagram of 1e showing interactions
with active site residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.2. Synthesis of ethyl 4-(3-benzoylthioureido) benzoates (1a–j)
stirring followed by reflux for 1–2 h. The mixture was poured into
crushed ice when the ethyl 4-(3-benzoylthioureido) benzoates
(1a–j) precipitated as solid. The solid product was filtered, then
dried and recrystallized from suitable solvent.
Variously substituted benzoic acids (0.02 mol) were treated
with 2 mL (1.3 eq) of thionylchloride through a dropping funnel
and the reaction mixture was heated under reflux for 2–3 h yields
corresponding acid chlorides. To a stirred solution of potassium
thiocyanate (1.2 g, 0.02 mol) in 10 mL dry acetone, placed in round
bottom flask fitted with a reflux condenser. (1.5 mL, 0.02 mol) of
freshly prepared acid chlorides was added drop wise. After the ini-
tial reaction had subsided a solution of ethyl 4-animobenzoate
(0.02 mol) in acetone (20 mL) was added slowly with constant
3.2.1. Ethyl 4-(3-benzoylthioureido) benzoate (1a)
Yield = 74%; Rfa = 0.8; M.P. = 140–142 °C; IR (KBr)
m
max: 3298
(NAH), 1724 (ester C@O), 1675 (amide C@O), 1583 (C@C), 1272
(C@S), 1152 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.31 (1H, s,
;
ANH), 10.24 (1H, s, ANH), 7.92 (1H, d, J = 7.8 Hz, H-2,H-6), 7.56–
7.87 (5H, m, Ar), 7.47 (1H, d, J = 7.8 Hz, H-3,H-5), 4.21 (2H, q,

A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
5
Fig. 3. Interaction diagrams for compounds 1f and 1g. Hydrogen bond and metal interactions are shown with dotted lines. Green lines represent the hydrophobic residues
located at the opening of the active site. (Left): interaction diagram of compound 1f showing interactions with active site residues. (Right): Interaction diagram of 1g showing
interactions with active site residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
J = 7.2 Hz, ACH2), 2.21 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d
ppm): 180.4 (C@S), 167.3 (amide C@O), 165.4 (ester C@O), 138.6
(C-4), 135.4 (C-1’), 132.6 (C-4’), 130.5 (C-2,C-6), 129.4 (C-3’,C-5’),
128.7 (C-2’,C-6’), 127.6 (C-3,C-5), 126.7 (C-1), 60.2 (AOCH2), 14.3
(ACH3); EIMS (70 eV): m/z (%); [M+] 328 (42%); Anal. Calcd for C17-
H16N2O3S; C, 62.19; H, 4.88; N, 8.54; S, 9.76. Found: C, 62.11; H,
4.64; N, 8.33; S, 9.54.
(C@S), 1154 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.34 (1H, s,
;
ANH), 10.27 (1H, s, ANH), 7.88 (1H, d, J = 7.8 Hz, H-2,H-6), 7.77
(1H, d, J = 7.4 Hz, H-2’,H-6’), 7.68 (1H, d, J = 7.8 Hz, H-3,H-5), 7.57
(1H, d, J = 7.4 Hz, H-3’,H-5’), 4.24 (2H, q, J = 7.2 Hz, ACH2), 2.62
(3H, s, ArACH3), 2.25 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d
ppm): 180.6 (C@S), 167.5 (amide C@O), 165.4 (ester C@O), 138.7
(C-4), 137.8 (C-4’), 132.6 (C-1’), 131.4 (C-2,C-6), 130.3 (C-3’,C-5’),
128.2 (C-2’,C-6’), 127.6 (C-3,C-5), 126.7 (C-1), 60.4 (2H, s, AOCH2),
24.3 (ArACH3), 14.4 (ACH3); EIMS (70 eV): m/z (%); [M+] 342 (61%);
Anal. Calcd for C18H18N2O3S; C, 63.16; H, 5.26; N, 7.02; S, 9.36.
Found: C, 63.07; H, 5.14; N, 6.95; S, 9.21.
3.2.2. Ethyl 4-[3-(3-chlorobenzoyl)thioureido]benzoate (1b)
Yield = 78%; Rfa = 0.7; M.P. = 156–158 °C; IR (KBr)
m
max: 3302
(NAH), 1727 (ester C@O), 1673 (amide C@O), 1578 (C@C), 1280
(C@S), 1153 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.37 (1H, s,
;
ANH), 10.32 (1H, s, ANH), 7.95 (1H, d, J = 7.8 Hz, H-2,H-6), 7.84
(1H, d, J = 2.4 Hz, H-2’), 7.76 (1H, d, J = 7.4 Hz, H-6’), 7.66 (1H, d,
J = 7.8 Hz, H-3,H-5), 7.58 (1H, dd, J = 7.2,2.4 Hz, H-4’), 7.49 (1H,
dd, J = 7.2,7.4 Hz, H-5’), 4.26 (2H, q, J = 7.2 Hz, ACH2), 2.26 (3H, t,
J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d ppm): 180.8 (C@S), 167.5
(amide C@O), 165.6 (ester C@O), 138.7 (C-4), 136.4 (C-1’), 135.5
(C-3’), 133.7 (C-4’), 130.7 (C-2,C-6), 129.4 (C-5’), 128.5 (C-2’),
127.4 (C-3,C-5), 126.5 (C-1), 125.8 (C-6’), 60.3 (AOCH2), 14.2
(ACH3); EIMS (70 eV): m/z (%); [M+] 362.5 (53%); Anal. Calcd for
3.2.5. Ethyl 4-[3-(3-methylbenzoyl)thioureido]benzoate (1e)
Yield = 78%; Rfa = 0.7; M.P. = 196–198 °C; IR (KBr)
m
max: 3322
(NAH), 1722 (ester C@O), 1671 (amide C@O), 1588 (C@C), 1284
(C@S), 1156 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.35 (1H, s,
;
ANH), 10.31 (1H, s, ANH), 7.86 (1H, d, J = 7.8 Hz H-2,H-6), 7.75
(1H, d, J = 7.2 Hz, H-6’), 7.66 (1H, d, J = 2.4 Hz, H-2’), 7.58 (1H, dd,
J = 7.4,7.2 Hz, H-5’), 7.53 (1H, dd, J = 7.4,2.4 Hz, H-4’), 7.46 (1H, d,
J = 7.8 Hz, H-3,H-5), 4.23 (2H, q, J = 7.2 Hz, ACH2), 2.61 (3H, s,
ArACH3), 2.24 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d ppm):
180.5 (C@S), 167.6 (amide C@O), 165.7 (ester C@O), 138.9 (C-4),
137.8 (C-3’), 134.5 (C-1’), 133.2 (C-4’), 130.5 (C-2,C-6), 129.3
(C-5’), 128.2 (C-2’), 127.1 (C-3,C-5), 126.4 (C-1), 125.4 (C-6’), 60.5
(-OCH2), 24.1 (ArACH3), 14.3 (ACH3); EIMS (70 eV): m/z (%); [M+]
342 (53%); Anal. Calcd for C18H18N2O3S; C, 63.15; H, 5.25; N,
7.01; S, 9.35. Found: C, 63.08; H, 5.17; N, 6.94; S, 9.24.
C17H15N2O3SCl; C, 56.27; H, 4.14; N, 7.72; S, 8.83. Found: C,
56.14; H, 4.06; N, 7.53; S, 8.64.
3.2.3. Ethyl 4-[3-(2,4-dichlorobenzoyl)thioureido]benzoate (1c)
Yield = 80%; Rfa = 0.75; M.P. = 117–118 °C; IR (KBr)
m
max: 3309
(NAH), 1723 (ester C@O), 1678 (amide C@O), 1586 (C@C), 1283
(C@S), 1157 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.42 (1H, s,
;
ANH), 10.38 (1H, s, ANH), 7.97 (1H, d, J = 7.8 Hz, H-2,H-6), 7.86
(1H, d, J = 2.3 Hz, H-3’), 7.78 (1H, d, J = 7.2 Hz, H-6’), 7.67 (1H, dd,
J = 7.2,2.3 Hz, H-5’), 7.54 (1H, d, J = 7.8 Hz, H-3,H-5), 4.27 (2H, q,
J = 7.2 Hz, ACH2), 2.28 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d
ppm): 181.2 (C@S), 167.8 (amide C@O), 166.2 (ester C@O), 139.5
(C-4), 138.4 (C-4’), 135.2 (C-2’), 132.3 (C-3’), 131.7 (C-1’), 130.8
(C-6’), 130.3 (C-2,C-6), 128.1 (C-5’), 127.2 (C-3,C-5), 126.8 (C-1),
60.8 (AOCH2), 14.5 (ACH3); EIMS (70 eV): m/z (%); [M+] 397
(37%); Anal. Calcd for C17H14N2O3SCl2; C, 51.38; H, 3.53; N, 7.05;
S, 8.06. Found: C, 51.27; H, 3.35; N, 6.93; S, 7.93.
3.2.6. Ethyl 4-[3-(4-methoxybenzoyl)thioureido]benzoate (1f)
Yield = 76%; Rfa = 0.65; M.P. = 207–208 °C; IR (KBr)
m
max: 3315
(NAH), 1726 (ester C@O), 1674 (amide C@O), 1587 (C@C), 1282
(C@S), 1165 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.38 (1H, s,
;
ANH), 10.29 (1H, s, ANH), 7.86 (1H, d, J = 7.8 Hz, H-2,H-6), 7.73
(1H, d, J = 7.5 Hz, H-2’,H-6’), 7.64 (1H, d, J = 7.5 Hz, H-3’,H-5’),
7.54 (1H, d, J = 7.8 Hz, H-3,H-5), 4.27 (2H, q, J = 7.2 Hz, ACH2),
3.72 (3H, s, AOCH3), 2.25 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3,
d ppm): 180.6 (C@S), 167.4 (amide C@O), 165.5 (ester C@O), 139.6
(C-4’), 138.5 (C-4), 134.5 (C-1’), 130.7 (C-2,C-6), 129.5 (C-2’,C-6’),
127.2 (C-3,C-5), 126.7 (C-1), 117.4 (C-3’,C-5’), 60.6 (AOCH2), 56.5
(Ar-OCH3), 14.2 (ACH3); EIMS (70 eV): m/z (%); [M+] 358 (46%);
Anal. Calcd for C18H18N2O4S; C, 60.33; H, 5.03; N, 6.70; S, 8.94.
Found: C, 60.27; H, 4.92; N, 6.58; S, 8.86.
3.2.4. Ethyl 4-[3-(4-methylbenzoyl)thioureido]benzoate (1d)
Yield = 71%; Rfa = 0.7; M.P. = 223–224 °C; IR (KBr)
mmax: 3329
(NAH), 1726 (ester C@O), 1681 (amide C@O), 1584 (C@C), 1286

6
A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
3.2.7. Ethyl 4-[3-(3,4-dimethoxybenzoyl)thioureido]benzoate (1g)
Yield = 72%; Rfa = 0.6; M.P. = 232–234 °C; IR (KBr)
max: 3321
(NAH), 1724 (ester C@O), 1678 (amide C@O), 1585 (C@C), 1267
(C@S), 1147 (CAN) cmꢀ1 1H NMR, (CDCl3, d ppm): 11.40 (1H, s,
contained 40
K2HPO4, pH 8.2) containing 100 mM urea, 10
valia ensiformis, 5 U/mL) and 10 L of the test compound. The assay
mixture was incubated at 37 °C for 10 min in a 96-wellplate. A
40 L of alkali reagent (0.5%, w/v NaOH, 0.1% active chloride NaO-
Cl) and 40 L of the phenol reagents (1%, w/v phenol, 0.005%, w/v
sodium nitroprusside) were then added to the each well. The
absorbance was measured at 625 nm by using microplate reader
(Bio-TekELx 800™, Instruments, Inc. USA). Percentage inhibition
was calculated using the formula 100 ꢀ (OD test well/OD con-
trol) ꢁ 100. The IC50 values were determined by using PRISM 4.0
(GraphPad, San Diego, California, USA).
l
L of buffer (0.01 M LiCl2, 1 mM EDTA, 0.01 M
m
l
L of enzyme (Cana-
l
;
ANH), 10.32 (1H, s, ANH), 7.90 (1H, d, J = 7.8 Hz, H-2,H-6), 7.84
(1H, d, J = 7.4 Hz, H-6’), 7.73 (1H, d, J = 7.8 Hz, H-3,H-5), 7.65 (1H,
s, Ar-H-2’), 7.56 (1H, d, J = 7.5 Hz, H-5’), 4.27 (2H, q, J = 7.2 Hz,
ACH2), 3.78 (3H, s, 3-OCH3), 3.75 (3H, s, 4-OCH3), 2.25 (3H, t,
J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d ppm): 180.7 (C@S), 167.7
(amide C@O), 165.8 (ester C@O), 139.8 (C-4’), 138.7 (C-3’), 137.6
(C-4), 130.8 (C-2,C-6), 128.3 (C-1’), 127.1 (C-3,C-5), 126.5 (C-1),
121.6 (C-6’), 117.4 (C-5’), 115.5 (C-2’), 60.6 (AOCH2), 56.5
(ArAOCH3), 14.2 (ACH3); EIMS (70 eV): m/z (%); [M+] 388 (33%);
Anal. Calcd for C19H20N2O5S; C, 58.76; H, 5.15; N, 6.18; S, 8.25.
Found: C, 58.57; H, 5.09; N, 6.11; S, 8.17.
l
l
3.4. Antioxidant assay
3.2.8. Ethyl 4-[3-(2-bromobenzoyl)thioureido]benzoate (1h)
Yield = 74%; Rfa = 0.67; M.P. = 213–215 °C; IR (KBr)
m
max: 1728
(ester C@O), 3317 (NAH), 1683 (amide C@O), 1576 (C@C), 1262
(C@S), 1165 (CAN) cmꢀ1 1H NMR (CDCl3,
ppm): 11.34
The free radical scavenging capacity of the compounds was
measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods de-
scribed by Choudhary et al. with some modification [29]. The assay
involved the reaction of the test compounds with 1,1-diphenyl-2-
picrylhyrazyl free radical. The reaction was carried out for 30 min
at 37 °C with 100 mM DPPH. Decrease in absorbance was mea-
sured at 515 nm using micro plate reader (Bio-TekELx 800™,
Instruments, Inc. USA). Percent RSA (radical scavenging activity)
was determined for the samples using a DMSO treated control
group in comparison. The following equation was used for calcula-
tion of RSA (%) = 100 ꢀ {(OD test well/OD control) ꢁ 100}.
;
d
(1H, s, ANH), 10.23 (1H, s, ANH), 7.85 (1H, d, J = 7.8 Hz, H-2,H-6),
7.63–7.81 (4H, m, Ar), 7.56 (1H, d, J = 7.8 Hz, H-3,H-5), 4.23 (2H,
q, J = 7.2 Hz, ACH2), 2.26 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3,
d ppm): 180.4 (C@S), 167.2 (amide C@O), 165.6 (ester C@O), 138.6
(C-4), 137.5 (C-1’), 135.4 (C-4’), 132.7 (C-3’), 131.2 (C-2,C-6), 130.5
(C-6’), 128.4 (C-5’), 127.3 (C-3,C-5), 126.8 (C-1), 121.4 (C-2’), 60.4
(AOCH2), 14.3 (ACH3); EIMS (70 eV): m/z (%); [M+] 406 (46%); Anal.
Calcd for C17H15N2O3SBr; C, 50.25; H, 3.69; N, 6.90; S, 7.88. Found:
C, 50.17; H, 3.56; N, 6.76; S, 7.74.
3.2.9. Ethyl 4-[3-(3-methoxybenzoyl)thioureido]benzoate (1i)
Yield = 73%; Rfa = 0.6; M.P. = 173–174 °C; IR (KBr)
m
max: 1725
(ester C@O), 3325 (NAH), 1685 (amide C@O), 1581 (C@C), 1264
(C@S), 1148 (CAN) cmꢀ1 1H NMR (CDCl3,
ppm): 11.36
3.5. Structure selection and preparation for docking studies
;
d
Molecular docking studies were conducted to observe the inter-
actions and selectivity of the compounds against urease enzyme.
The three dimensional, high-resolution structure of urease enzyme
(PDB Code: 3LA4) was selected from the Protein Data Bank and
prepared for molecular modeling. The X-ray structure has well de-
fined binding site, as the crystal structure of the enzyme is co-crys-
tallized with a bound ligand.
(1H, s, ANH), 10.27 (1H, s, ANH), 7.87 (1H, d, J = 7.8 Hz, H-2,H-6),
7.57–7.77 (4H, m, Ar), 7.53 (1H, d, J = 7.8 Hz, H-3,H-5), 4.25 (2H,
q, J = 7.2 Hz, ACH2), 3.73 (3H, s, AOCH3), 2.26 (3H, t, J = 5.6 Hz,
ACH3); 13C NMR (CDCl3, d ppm): 180.5 (C@S), 167.6 (amide
C@O), 165.7 (ester C@O), 138.7 (C-3’), 137.6 (C-4), 134.2 (C-4’),
131.3 (C-2,C-6), 129.1 (C-6’), 127.4 (C-3,C-5), 126.6 (C-1), 122.5
(C-5’), 118.4 (C-1’), 116.5 (C-2’), 60.7 (AOCH2), 56.4 (Ar-OCH3),
14.5 (ACH3); EIMS (70 eV): m/z (%); [M+.] 358 (54%); Anal. Calcd
for C18H18N2O4S; C, 60.34; H, 3.02; N, 6.70; S, 8.93. Found: C,
60.23; H, 4.93; N, 6.53; S, 8.81.
Before studying the enzyme in molecular modeling experiment,
the enzyme structure was prepared. The structure was protonated
with Protonate3D [30] algorithm implemented in the molecular
modeling tool MOE [31]. Missing atoms of some residues were
completed with utility programs built-in in AmberTools-1.5 pack-
age [32]. However, the missing residues were not added because
they are far away from the binding site. Accurate charge derivation
for such a metal complex is much time consuming and requires
expensive computations if quantum mechanical optimization
methods are used. Therefore, before minimization process, +2
charges were assigned to the both nickel atoms present in the ac-
tive site of the urease. It was made sure that the modified residues
are not excluded during structure preparation. The carbamylated
lysine and methylated cysteine residues were retained as such.
The structure was energy minimized using Amber 99 force field
with all the heteroatoms and solvent molecules present in the en-
zyme structure in MOE environment. The backbone atoms were re-
strained with a small force constant of 100 in order to avoid
collapse of the binding pockets during energy minimization calcu-
lations. The force field based energy minimization algorithms were
used which includes steepest decent followed by conjugate gradi-
ent optimization methods. An RMS gradient of 0.05 was specified
that would terminate the energy minimization process if the root
mean square gradient falls below 0.05. After minimization, the
co-crystallized ligands and other solvent molecules were removed
before performing docking calculations.
3.2.10. Ethyl 4-[3-(2-fluorobenzoyl)thioureido]benzoate (1j)
Yield = 68%; Rfa = 0.7; M.P. = 123–124 °C; IR (KBr)
m
max: 1729
(ester C@O), 3332 (NAH), 1688 (amide C@O), 1591 (C@C), 1271
(C@S), 1156 (CAN) cmꢀ1 1H NMR (CDCl3, d ppm): 11.42 (1H, s,
;
ANH), 10.34 (1H, s, ANH), 7.93 (1H, d, J = 7.8 Hz, H-2,H-6), 7.64–
7.89 (4H, m, Ar), 7.58 (1H, d, J = 7.8, Hz H-3,H-5), 4.26 (2H, q,
J = 7.2 Hz, ACH2), 2.28 (3H, t, J = 5.6 Hz, ACH3); 13C NMR (CDCl3, d
ppm): 181.4 (C@S), 168.2 (amide C@O), 166.5 (ester C@O), 140.6
(C-2’), 138.7 (C-4), 134.7 (C-4’), 131.2 (C-2,C-6), 129.3 (C-6’),
127.3 (C-3,C-5), 126.7 (C-1), 126.3 (C-1’), 125.6 (C-5’), 117.6 (C-
3’), 61.2 (AOCH2), 14.8 (ACH3); EIMS (70 eV): m/z (%); [M+] 346
(24%); Anal. Calcd for C17H15N2O3SF; C, 58.96; H, 4.34; N, 6.94; S,
9.25. Found: C, 58.87; H, 4.21; N, 6.73; S, 9.12.
3.3. Assay for urease inhibition
The in vitro studies were carried out for the synthesized com-
pounds by testing them against the jack bean urease. The enzyme
activity was determined by measuring the absorbance and quanti-
fication of ammonia was done by using the indophenols method as
described by Weatherburn [28]. In brief, the assay mixture

A. Saeed et al. / Bioorganic Chemistry 52 (2014) 1–7
7
3.6. Preparation of compounds
Two possible binding modes can be observed in the predicted
docked conformations, however, the interactions and their orienta-
tion in the active site pocket of the enzyme remains the same. The
identified thiourea derivatives can be utilized in further optimiza-
tion of biological activity using structural variations in the main
skeleton. The antioxidant activity of most of the compounds was
more than the standard drugs. Therefore, the investigated radical
scavengers can be further evaluated in vivo for their efficacy to pre-
vent or control the oxidative stress in diseased models.
The 3D structural coordinates of the compounds were gener-
ated for all the compounds using MOE followed by assignment of
protonation and ionization states in physiological pH range by
using the ‘‘wash’’ module. Afterwards, the compounds structures
were energy minimized with MMFF94x force field for screening
them against the urease enzyme by docking.
3.7. Docking studies
Acknowledgment
The docking calculations were performed by FlexX [33] docking
engine inside the molecular modeling tool LeadIT [34]. For the
docking studies, the binding site of the enzyme was defined by
including the surrounding amino acid residues around 7.5 Å radius
of the co-crystallized bound ligand. The metal atoms were also in-
cluded in the binding pocket definition. The modified carboxylated
LYS residue (KCX490) and methylated CYS residue (CME207) were
retained in the structure during docking calculations. Pharmaco-
phoric constraints were defined for the two metal atoms. The con-
straints were selected to be first essential and then optional to
check if the interactions with the metals will be predicted during
the docking calculations. The default docking and scoring parame-
ters were chosen for the docking calculations and the top 10
docked conformations were retained for analyzing the interactions
and binding modes of individual compounds. In order to make sure
that the protein structure is prepared well for docking, the co-crys-
tallized bound ligand was re-docked to reproduce the crystal struc-
ture’s binding mode.
This work was financially supported by COMSTECH–TWAS and
German-Pakistani Research Collaboration Programme.
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3.8. Pose ranking and selection
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An efficient synthesis of some ethyl 4-(3-benzoylthioureido)
benzoate thioureas has been carried out, which possess significant
antiurease and antioxidant activities. Compounds 1f and 1g
showed significant activity against the urease. The docking studies
conducted on the urease, it can be concluded that the predicted
binding poses that are filtered by the external scoring function re-
sults in interactions that are observed in most of the compounds.
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