Synthesis, biological assay in vitro and molecular docking studies of new Schiff base derivatives as potential urease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.09.009

A series of new and novel Schiff base derivatives were synthesized and investigated as potential new inhibitors of Jack bean urease. The most potent compounds were 3f with (K_i = 0.09 μM) and 3k (K_i = 0.122 μM). A pure competitive mec

Full text of DOI:10.1016/j.ejmech.2011.09.009

European Journal of Medicinal Chemistry 46 (2011) 5473e5479
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological assay in vitro and molecular docking studies of new Schiff
base derivatives as potential urease inhibitors
,
Muhammad Adil S. Aslama, Shams-ul Mahmoodb, Mohammad Shahidc, Aamer Saeedb, Jamshed Iqbala
*
a Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Postal Code 22060, Pakistan
b Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan
c Fraunhofer Institute SCAI, Department of Bioinformatics, Sankt Augustin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new and novel Schiff base derivatives were synthesized and investigated as potential new
inhibitors of Jack bean urease. The most potent compounds were 3f with (Ki ¼ 0.09 M) and 3k
M). A pure competitive mechanism of inhibition was observed. Molecular docking studies
were also performed to illustrate the binding mode of the compounds. Docking studies were performed
on both enzymes from Jack bean urease and H. pylori urease. It was observed that both share the same
binding mode. The binding sites of the two urease structures also aligned very well indicating the
similarity in binding sites of the enzymes.
Received 1 March 2011
Received in revised form
6 September 2011
Accepted 7 September 2011
Available online 16 September 2011
m
(Ki ¼ 0.122
m
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Competitive inhibition
Docking studies
Electrostatic potential
Schiff base
Thiourea derivatives
Urease inhibition
1. Introduction
be the main cause of increase in pH and major contributing factor in
pathologies caused by Helicobacter pylori (H. pylori) and permits
Urease (urea amidohydrolase EC3.5.1.5) is
a
large hetero-
bacteria to endure acidic pH of the stomach during colonization. The
enzymatic activity of urease plays an effective role in pathogenesis of
urolithiasis, pyelonephrities, ammonia and hepatic encephalopathy,
hepatic coma and urinary catheter encrustation [2,7e9]. Urease
inhibitors are known to lessen the environmental issues and
augment the uptake of urea nitrogen by plants [10e14]. Urease
inhibitors are known to be potent antiulcer drugs [15]. Strategies
based on urease inhibition are the main treatment of diseases caused
by urease producing bacteria.
Several classes of compounds are known to show significant
inhibitory activities against urease enzyme, among them hydroxa-
mic acids are the best recognized urease inhibitors [2,16,17]. Whilst,
phosphoramidates being the most potent compounds [18,19].
However, because of teratogenicity of hydroxamic acid in rats [20]
and degradation of phosphoramidates at low pH [21] prevented
their use as a drug in vivo. Polyphenols, another class of compounds
that showed enzyme inhibitory activities [22]. Gallocatechin [23],
a polyphenol which is extracted from green tea and quercetin [24],
a naturally occurring flavonoids have been reported as inhibitors of
H. pylori urease. A few triazoles have also been reported as inhibitors
of urease [25]. Recently, some complexes of Schiff bases with metal
ions showed significant inhibitory activity against urease [26,27]
polymeric enzyme which belongs to the super family of amidohy-
drolases and phosphotriestreases [1]. It catalyzes the hydrolysis of
urea to ammonia and carbamate. The active site of urease possesses
metal center i.e. two nickel (II) atoms [2]. It is widely distributed in
a variety of bacteria, fungi, algae, and plants [3,4]. The order of amino
acids and their enzymatic mechanism in all the ureases is preserved,
Ciurli and his co-workers also proposed an efficient and workable
enzymatic reaction mechanism [5]. The active center of Urease traps
three water molecules and a hydroxide ion links between two nickel
ions. Meanwhile, urea displaces the water molecules and links itself
with the two nickel ions [2,6]. Urea being bi-dentate is held firmly by
the network of hydrogen bonding. This state activates urea molecule
and the result is attack of Ni bridging hydroxide ion developing
a tetrahedral like transition state. Consequently, ammonia is dis-
charged from the active site accompanied by negatively charged
carbamate. The latter is further broken down instantly, generating
another molecule of ammonia. This reaction mechanism is known to
* Corresponding author. Tel.: þ92 992 383591 96; fax: þ92 992 383441.
E-mail addresses: drjamshed@ciit.net.pk, jamshediqb@gmail.com (J. Iqbal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.009

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M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
were ascertained on the basis of their consistent IR, 1H NMR, 13C
NMR, elemental analysis and mass spectroscopic studies. In
a typical case (3j) the presence of C]S functional group was
marked by the appearance of stretching bands at 1110, at 1602 for
CN, at 1590 cmꢀ1 for AreCC and a stretching band around 3385 and
3229 cmꢀ1, indicated the presence of NH2 and NH linkage of the
hydrazide moiety. The aromatic nitro stretching around 1343 cmꢀ1
(symmetric NO2 stretching) and 1538 cmꢀ1 (asymmetric NO2
stretching) depicts the presence of nitro functional group in
compound (3feg). The characteristic azomethine protons were
observed in the range of 7.5e8.1 ppm. In 1H NMR of (3j), the
appearance of a singlet at
display of singlets at 10.4 and 7.50 were noticed for NH and NH2
respectively. In 13C NMR signals at
179.0 (CS) and 153 (CN)
d 8.11 for azomethine proton (CHN) and
d
d
Fig. 1. Synthesis of 1-(substituted benzylidene) thiosemicarbazides derivatives (3aer).
confirmed formation of the semicarabazone.
along with other metal complexes [28]. However, as these metal
complexes have heavy metal atoms, therefore, they can be toxic to
human body [29], hence such molecules cannot be used as drugs.
Schiff base hydrazones are well known class of compounds, possess
various activities like antimicrobial [30,31], antimycobacterial [32],
antitumor [33], anti-inflammatory [34], trypanocidal [35], anti-HIV
[36], antimalarial [37] and antidiabetic activities [38]. However, the
present study was aimed at design, synthesis, and evaluation of
novel Schiff base analogs as inhibitors of urease. The inhibitory
constants of the novel Schiff based compounds were in micromolar
range.
2.2. Urease inhibition assays (in vitro)
Novel Schiff base compounds were evaluated against Jack bean
urease in vitro. Initially, the compounds were screened at
a concentration of 1 mM. The compounds which exhibited more
than 50% inhibition were further selected for complete character-
ization. All of the Schiff base compounds were potent inhibitors of
Jack bean urease as they were structurally similar with the
substrate of urease i.e. urea. Thiourea was used as the reference
compound for the assay, its affinity is also included in Table 1. The
synthesized compounds bear various functional groups at phenyl
ring. The obtained results were shown in Table 1. The screened
compounds exhibited excellent inhibitory activity in micromolar
range. Among the tested compounds 3f was the most potent with Ki
2. Results and discussion
2.1. Chemistry
being 0.09
0.122 and 0.127
moderate activity was exhibited by 3a, 3n, 3m, 3p and 3q ranging
from 0.125 to 0.200 M. Among the synthesized analogs, 3b, 3c, 3d,
3e, 3g, 3i, 3j, 3l, 3o and 3r were least potent with Ki values greater
than 0.200 M.
m
M. Other potent compounds were 3k and 3h having Ki,
The synthesis of target semicarbazide hydrazones was carried
out as outlined in Fig. 1. The reaction of semicarbazide in dry
ethanol with suitably substituted aromatic aldehydes in the pres-
ence of catalytic amount of sulphuric acid afforded the hydrazones
(3aep) in case of (3r) the aliphatic hydrazone respectively.
The target compounds were obtained in appreciable yields and
were purified by recrystallization from ethanol. The structures
mM respectively. Upon further modification,
m
m
The activities of compounds were greatly varied by changing the
substitution at meta and para position. In case of highly potent 3f
Table 1
Inhibition of Jack bean urease by Studied Compounds.
IC50
(
mM)
Ki (mM)
Entry
2
3
4
5
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
Cl
0.215 ꢁ 0.09
0.670 ꢁ 0.04
0.410 ꢁ 0.07
0.767 ꢁ 0.08
0.707 ꢁ 0.09
0.102 ꢁ 0.50
0.547 ꢁ 0.06
0.177 ꢁ 0.03
0.887 ꢁ 0.01
0.989 ꢁ 0.09
0.127 ꢁ 0.01
0.457 ꢁ 0.01
0.263 ꢁ 0.006
0.330 ꢁ 0.15
0.682 ꢁ 0.04
0.220 ꢁ 0.02
0.233 ꢁ 0.06
0.746 ꢁ 0.01
21.0 ꢁ 11
0.170 ꢁ 0.08
0.625 ꢁ 0.07
0.398 ꢁ 0.04
0.670 ꢁ 0.06
0.770 ꢁ 0.14
0.090 ꢁ 0.06
0.497 ꢁ 0.09
0.127 ꢁ 0.03
0.807 ꢁ 0.05
0.910 ꢁ 0.11
0.122 ꢁ 0.11
0.407 ꢁ 0.11
0.152 ꢁ 0.004
0.191 ꢁ 0.08
0.391 ꢁ 0.02
0.127 ꢁ 0.01
0.134 ꢁ 0.03
0.430 ꢁ 0.06
19.6 ꢁ 0.45
Cl
Cl
Br
Br
NO2
OeCH3
OeCH3
NO2
OeCH3
OeCH3
Ne(CH3)2
OeCH3
OeCH3
OeCH2-ph
eOH
OeCH2-ph
OeCH3
eOH
eOH
OeCH3
eOH
3q
3r
Thiourea (Standard)

M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
5475
which bears a NO2 substituent at meta position that is most effec-
tive as compared to 3g when it is present at para position. However,
when the substituent was changed to activating group N-(CH3)2 in
3k, the activity was reduced. Upon further modification in the
substituent on aryl ring in 3h, the activity further declined. Among
the compounds containing halogens inductive effect plays its role,
chloro at ortho (3a) exhibited good activity, Ki being 0.170
mM,
compared to 3b and 3c where it is present on meta and para
positions respectively. The electron donating group show moderate
activity.
2.3. Molecular docking studies
In the docking study, the X-ray crystal structure of Jack bean
urease (entry 3LA4 in the Protein Data Bank) was used. In order to
give an explanation and understanding of potent inhibitory activity
observed, molecular docking of the most potent compound 3f into
binding site of Jack bean urease was carried out. The binding model
of compound 3f and Jack bean urease was shown in Fig. 2. In the
docked conformation of inhibitor 3f (Fig. 2) Ni atoms coordinate
with the sulphur atom, similarly hydrogen bonding can be
observed by Arg439 and Cme592 residues with oxygen of nitro
group, whereas Ala440 with Hydrogen of inhibitor. In Fig. 3,
another interaction diagram can be seen for inhibitor 3l. The
docking results for the compounds are given in Table 2, which are
ranked on the basis of their FlexX docking scores.
Basically the scoring functions are physics base ‘molecular
mechanics force fields’ that approximate the energy of the
masquerade (pose) a low energy i.e. a negative value indicates
a firm system hence a possible binding mode. The FlexX Docking
score is tabulated in Table 2, along with the rank of each molecule.
As 3f gives the lowest score i.e. ꢀ29.52, it suggests that this is the
strongest binding inhibitor. The binding score ranges from ꢀ10
to ꢀ30 proposing that the binding differs as the functional group is
varied. Other molecules show a greater value than ꢀ29.52, giving
an indication that no other molecule is as strong as 3f.
Fig. 3. Docked conformation of 3l in Jack bean Urease.
preparation, FlexX [39] offers an automated pharmacophore query
generation through FlexX-Pharm [40] module to filter the docking
solutions on the basis of pharmacophoric constraints. The two
Nickel atoms were set to be used as essential part of the pharma-
cophore query that was desired to be present in the predicted
docking solutions. When more pharmacophoric constraints were
added together with the metal pharmacophores, it did not produce
the docking solutions for all of the compounds. Therefore, inter-
actions constraints with the other active site residues were set as
optional. However, these interactions constraints can be very useful
in filtering the good binders in the predicted conformational space.
An interaction diagram for compound 3f is shown in Fig. 2.
Fig. 4 shows the binding modes of all compounds in the top 10
docked solutions. Out of the top 10 predictions, all compounds have
a similar binding mode in the 1st ranked docked solution, except 3f
and 3q whose ranks were 7 and 9 respectively. This also indicates
that all the compounds fulfilled the metal pharmacophoric
constraints and the thiourea moiety interacts with the two nickel
atoms. The aromatic rings make similar stack of interactions with
HIS593, PHE605 and LEU523 on one side and HIS593, ARG439,
ALA440 and ALA636 on the other side in the opening of the active
site pocket. The compounds also exhibit flexibility at the wide
opening part of the urease binding pocket and adopt different
The molecular docking studies showed that all the compounds
interact with the bi-nickel center of the urease enzyme. The top 10
docking poses were retained for each compound to analyze the
interactions of the docked conformations within the active site.
Single atoms were included in the active site during receptor
Table 2
FlexX Docking Score.
S.No
ID
Rank
Docking Score
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
1
1
1
1
1
7
1
1
1
1
1
1
1
1
1
ND
9
1
ꢀ22.04
ꢀ14.41
ꢀ22.62
ꢀ14.78
ꢀ22.64
ꢀ29.52
ꢀ26.62
ꢀ24.68
ꢀ11.28
ꢀ23.28
ꢀ17.17
ꢀ28.54
ꢀ14.63
ꢀ18.76
ꢀ23.80
e
9
10
11
12
13
14
15
16
17
18
ꢀ19.32
ꢀ21.23
Fig. 2. Docked conformation of 3f in Jack bean Urease.

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M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
except HIS519 which is protonated at epsilon position. The nega-
tively charged S atoms of the thiourea derivatives are interacting
with the positively charged metal atoms in almost all of the pre-
dicted docked poses. The negatively charged S atoms and the O
atoms of the two negatively charged KCX490 and ASP633 residues
creates an overall negative charge distribution around the metal
center in the urease enzyme which is shown from the electrostatic
charge distribution around the compounds. Therefore, the elec-
trostatic potential surface representation shows the favorable
binding modes of the thiourea compound derivatives.
2.4. Kinetics of inhibition
The most potent compound, 3f was further investigated to
determine the kinetics of inhibition. Enzyme kinetics was deter-
mined in the absence and in the presence of various concentrations
of inhibitor. It was found that the Vmax value did not significantly
change in the presence of the inhibitor. However, Km value
increased with increasing inhibitor concentrations indicating that
3f exhibit a competitive mechanism of inhibition. Fig. 6, showed
LineweavereBurk plots for compound 3f. The both plots visualize
the competitive mechanism of inhibition.
Fig. 4. Predicted docked conformations of all compounds inside the binding pocket of
Jack bean urease. The blue spheres indicate the metal pharmacophores around the two
nickels (Niþ2). The dotted lines indicate various types of interactions of the compounds
with the active site residues including hydrogen bonding and aromatic interactions.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
2.5. Conclusions
conformations, but the aromatic interactions with histidine ring of
HIS593 on one side and ARG439 and ALA440 on the other side is
common to majority of the compounds. Similarly, most of the
compounds make hydrogen bonding interactions with the side
chains of CME592, ARG439 and ALA440 residues at the entrance of
the binding pocket.
Fig. 5 shows the electrostatic charge distribution of the pre-
dicted docked compound poses inside the bi-nickel center of the
urease enzyme. The bi-nickel center of the enzyme forms a metal
complex of the modified Lysine residue KCX490 (LYSINE NZ-
Corboxylic acid), ASP633 and four histidine residues (HIS519,
HIS545, HIS407 and HIS409) around the two positively charged
Niþ2 atoms. All the histidines are protonated at delta positions
New series of Schiff base (3aer) derivatives were synthesized
and evaluated for Jack bean urease inhibition activity. All of the
compounds were potent inhibitors of Jack bean urease due to the
similarity of their basic skeleton with urease substrate. The
compound (3f) showed excellent inhibitor activity with competi-
tive mechanism of inhibition. Furthermore, molecular dockings of
different inhibitors into Jack bean urease active site was performed.
The binding pattern of this potential inhibitor could help to
understand the inhibitory mechanism. Scaffold of Schiff base
urease inhibitors can be utilized in further optimization to improve
potency and selectivity by variations in the basic skeleton. Elec-
trostatic potential was also determined.
Fig. 5. Electrostatic potential surface of predicted docked poses of all 3-series compounds inside the Jack bean urease. The electrostatic charge distribution of functional group is
shown clearly towards the bi-nickel center of the enzyme. The two nickels are represented by small spheres in light blue color. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
5477
3.2.1.3. (E)-1-(4-chlorobenzylidene)thiosemicarbazide (3c). (64%):
m.p 210 ꢂC; Rf: 0.46; IR (Pure)
: 3375 (NH2), 3230 (NeH), 1603 (C]
N), 1587 (AreC]C), 1090 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.6 (s, 1H, NH), 7.93 (s,1H, CH]N), 7.6e7.3 (m, 4H, Ar-
H), 7.1 (s, 2H, eNH2); 13C NMR (75 MHz)
: 181.0 (1C, C]S), 143 (1C,
C]INS–>N), 129 (2C, Ar), 130 (2C, Ar), 131 (Ar), 136 (Ar),; Anal.
Calcd. For C8H8N3SCl: C, 44.95; H, 3.74; N, 19.66; S, 14.98; Found: C,
44.94; H, 3.73; N, 19.63; S, 14.97; GCeMS m/z: 213 (M$þ, 100).
30
20
10
y
0.001
0.01
0
;
d
d
3.2.1.4. (E)-1-(3-bromobenzylidene)thiosemicarbazide (3d). (86%):
m.p 212 ꢂC; Rf: 0.45; IR (Pure)
y
: 3370 (NH2), 3235 (NeH), 1608 (C]
N), 1585 (AreC]C), 1100 (C]INS–>S), cmꢀ1; 1H NMR (Acetone-d6,
300 MHz) : 10.6 (s, 1H, NH), 7.96 (s, 1H, CH]N), 7.8e7.2(m, 4H,
AreH), 7.2 (s, 2H, eNH2); 13C NMR (75 MHz)
: 182.0 (1C, C]S), 144
d
d
(1C, C]N), 123.1 (Ar), 128.4 (Ar), 131 (Ar), 133.0 (Ar), 134.2 (Ar),
136.0 (Ar); Anal. Calcd. For C8H8BrN3S: C, 37.22; H, 3.12; N, 16.28; S,
12.42; Found: C, 37.21; H, 3.11; N, 19.26; S, 12.41; GCeMS m/z: 258
(M$þ, 100).
-0.00075
-0.00025
0.00025
0.00075
0.00125
1/[Urea](µM)
Fig. 6. LineweavereBurk plots of inhibition of Jack bean urease by compound 3f;
3.2.1.5. (E)-1-(4-bromobenzylidene)thiosemicarbazide (3e). (75%):
m.p 197 ꢂC; Rf: 0.18; IR (Pure)
y
: 3380 (NH2), 3240 (NeH), 1605 (C]
N), 1585 (AreC]C),1105 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.4 (s, 1H, NH), 7.93 (s, 1H, CH]N), 7.8e7.6 (m, 4H,
AreH), 7.3 (s, 2H, eNH2); 13C NMR (75 MHz)
: 181.0 (1C, C]S), 143
concentration of inhibitor 3f:
0
mM,
0.01
mM,
0.001 mM.
;
d
3. Experimental work
d
(1C, C]N), 131.4 (2C, Ar), 131.9 (2C, Ar), 125 (Ar), 132.7 (Ar); Anal.
Calcd. For C8H8BrN3S: C, 37.22; H, 3.12; N, 16.28; S, 12.42; Found: C,
37.20; H, 3.10; N, 19.27; S, 12.40; GCeMS m/z: 258 (M$þ, 100).
3.1. Materials
Melting points were recorded using a digital Gallenkamp
(SANYO) model MPD BM 3.5 apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were determined in CDCl3 or acetone-d6
solutions at 300 MHz and 75.4 MHz respectively using Bruker AM-
300 spectrophotometer. FT IR spectra were recorded using an FTS
3000 MX spectrophotometer; Mass spectra (EI, 70 eV) on a GCeMS
3.2.1.6. (E)-1-(3-nitrobenzylidene)thiosemicarbazide
m.p 218 ꢂC; Rf: 0.26; IR (Pure)
: 3375 (NH2), 3245 (NeH), 1606 (C]
N), 1590 (AreC]C), 1090 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.3 (s, 1H, NH), 7.99 (s, 1H, CH]N), 8.5e7.6 (m, 4H,
AreH), 7.1 (s, 2H, eNH2); 13C NMR (75 MHz)
: 180.0 (1C, C]S), 143
(3f). (64%):
y
;
d
d
instrument and elemental analyses with
analyzer.
a LECO-183 CHNS
(1C, C]N), 123.1 (Ar), 124.4 (Ar), 129.1 (Ar), 134.3 (Ar), 135.2 (Ar),
148.2 (Ar); Anal. Calcd. For C8H8N4O2S: C, 42.85; H, 3.60; N, 24.99; S,
14.30; Found: C, 42.84; H, 3.58; N, 24.97; S, 14.28; GCeMS m/z: 224
(M$þ, 100).
3.2. Chemistry
3.2.1. General procedure for synthesis of 1-(substituted benzylidene)
thiosemicarbazides (Schiff bases)
3.2.1.7. (E)-1-(4-nitrobenzylidene)thiosemicarbazide
m.p 225 ꢂC; Rf: 0.22; IR (Pure)
: 3379 (NH2), 3243 (NeH), 1608 (C]
N), 1580 (AreC]C), 1110 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.5 (s, 1H, NH), 8.00 (s, 1H, CH]N), 8.6e7.6 (m, 4H,
AreH), 7.2 (s, 2H, eNH2); 13C NMR (75 MHz)
: 181.0 (1C, C]S), 143
(1C, C]N), 121.2 (2C, Ar), 130.1 (2C, Ar), 139.5 (Ar), 150.5 (Ar); Anal.
Calcd. For C8H8N4O2S: C, 42.85; H, 3.60; N, 24.99; S, 14.30; Found: C,
42.86; H, 3.57; N, 24.98; S, 14.27; GCeMS m/z: 224 (M$þ, 100).
(3g). (87%):
y
The thiosemicarbazide (1.0 mmol) was added in a solution of
suitably substituted benzaldehyde (1.0 mmol) in absolute ethanol
(10 ml) using conc. sulphuric acid in catalytic amount. The reaction
mixture was refluxed for 3e6 h and completion was monitored by
TLC. The reaction mixture was concentrated and resulted solid
product was separated and recrystallized from ethanol.
;
d
d
3.2.1.1. (E)-1-(2-chlorobenzylidene)thiosemicarbazide (3a). (65%):
3.2.1.8. (E)-1-(3-methoxybenzylidene)thiosemicarbazide
m.p 195 ꢂC; Rf: 0.6; IR (Pure)
y
: 3365 (NH2), 3220 (NeH),1600 (CN),
1585 (AreC]C), 1080 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.4 (s, 1H, NH), 7.91 (s, 1H, CH]N), 7.7e7.62 (m, 4H,
AreH), 7.1 (s, 2H, eNH2); 13C NMR (75 MHz)
: 180.0 (C]S), 141
(3h). (73%): m.p 187 ꢂC; Rf: 0.53; IR (Pure)
y
: 3365 (NH2), 3225
(NeH), 1600 (C]N), 1575 (AreC]C), 1095 (C]S), cmꢀ1 1H NMR
(Acetone-d6, 300 MHz) : 10.1 (s,1H, NH), 8.1 (s,1H, CH]N), 7.9e7.1
(m, 4H, AreH), 7.1 (s, 2H, eNH2); 13C NMR (75 MHz)
: 179.0 (1C,
;
;
d
d
d
d
(C]N), 127 (Ar), 129 (Ar), 130 (Ar), 132 (Ar), 134 (Ar); Anal. Calcd.
For C8H8N3SCl: C, 44.95; H, 3.74; N, 19.66; S, 14.98; Found: C, 44.97;
H, 3.72; N, 19.64; S, 14.94; GCeMS m/z: 213 (M$þ, 100).
C]S), 150 (1C, C]N), 113.1 (Ar), 116.1 (Ar), 121.1 (Ar), 129.3 (Ar),
135.2 (Ar), 140.2 (Ar); Anal. Calcd. For C9H11N3OS: C, 51.65; H, 5.30;
N, 20.08; S, 15.32; Found: C, 51.66; H, 5.29; N, 20.07; S, 15.30;
GCeMS m/z: 209 (M$þ, 100).
3.2.1.2. (E)-1(-(3-chlorobenzylidene)thiosemicarbazide (3b). (62%):
m.p 199 ꢂC; Rf: 0.5; IR (Pure)
y
: 3385 (NH2), 3229 (NeH), 1602 (C]
N), 1590 (AreC]C),1073 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.7 (s, 1H, NH), 7.92 (s, 1H, CH]N), 7.9e7.66 (m, 4H,
AreH), 7.4 (s, 2H, eNH2); 13C NMR (75 MHz)
: 179.0 (1C, C]S),
3.2.1.9. (E)-1-(4-methoxybenzylidene)thiosemicarbazide (3i). (61%):
;
m.p 180 ꢂC; Rf: 0.37; IR (Pure)
y
: 3380 (NH2), 3230 (NeH), 1606 (C]
N), 1580 (AreC]C), 1105 (C]S), cmꢀ1 1H NMR (Acetone-d6,
300 MHz) : 10.2 (s, 1H, NH), 8.0 (s, 1H, CH]N), 7.9e7.1 (m, 4H,
AreH), 7.1 (s, 2H, eNH2); 13C NMR (75 MHz)
: 179.0 (1C, C]S), 149
d
;
d
d
140.99 (1C, C]N), 127 (Ar), 129 (Ar), 131 (Ar), 134 (Ar), 135 (Ar);
Anal. Calcd. For C8H8N3SCl: C, 44.95; H, 3.74; N, 19.66; S, 14.98;
Found: C, 44.94; H, 3.73; N, 19.65; S, 14.96; GCeMS m/z: 213
(M$þ, 100).
d
(1C, C]N), 114.2 (2C, Ar), 129.4 (2C, Ar), 125 (Ar), 140.1 (Ar); Anal.
Calcd. For C9H11N3OS: C, 51.65; H, 5.30; N, 20.08; S, 15.32; Found: C,
51.64; H, 5.28; N, 20.06; S, 15.31; GCeMS m/z: 209 (M$þ, 100).

5478
M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
3.2.1.10. (E)-1-(3,4,5-trimethoxybenzylidene)thiosemicarbazide
(3j). (64%): m.p 198 ꢂC; Rf: 0.25; IR (Pure)
: 3370 (NH2), 3235
(NeH), 1610 (C]N), 1590 (AreC]C), 1110 (C]S), cmꢀ1 1H NMR
(Acetone-d6, 300 MHz) : 10.4 (s, 1H, NH), 8.11 (s, 1H, CH]N), 7.5 (s,
2H, eNH2),7.1 (s, 2H, AreH), 3.86(s,6H,OeCH),3.6(s,3H,OeCH); 13C
NMR (75 MHz) : 179.0 (1C, C]S), 153 (1C, C]N), 110.2 (2C, Ar),
140.4 (2C, Ar),141 (Ar),130 (Ar), 59.3 (OeMe), 55.65 (2C, OMe); Anal.
Calcd. For C11H15N3O3S: C, 49.06; H, 5.61; N,15.60; S,11.91; Found: C,
49.07; H, 5.60; N, 15.59; S, 11.90; GCeMS m/z: 269 (M$þ, 100).
(DMSO-d6, 300 MHz)
2H, eNH2), 7.2e6.8 (m, 3H, AreH), 5.30 (s, 2H, OeH); 13C NMR
(75 MHz) : 179.0 (C]S),150 (Ar),145 (C]N),144 (Ar),124 (Ar),122
(Ar), 119 (Ar); Anal. Calcd. For C8H9N3O2S: C, 45.49; H, 4.29; N,
19.89; O, 15.15; S, 15.18; Found: C, 45.48; H, 4.28; N, 19.88; O, 15.14;
S, 15.16; GCeMS m/z: 211.04 (M þ, 100).
d: 11.2 (s, 1H, NH), 8.31 (s, 1H, CH]N), 8.10 (s,
y
;
d
d
d
3.2.1.17. (E)-2-(4-hydroxy-3,5-dimethoxybenzylidene)hydrazine-
carbothioamide (3q). (71%): m.p 104 ꢂC; Rf: 0.17; IR (Pure)
y: 3449
(NH2), 3311 (NeH), 1619 (C]N), 1585 (AreC]C), 1114 (C]S),
3.2.1.11. (E)-1-(4e(N,N-dimethylamino)benzylidene)thio-
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz)
d: 11.0 (s, 1H, NH), 8.21 (s, 1H,
CH]N), 8.0 (s, 2H, eNH2), 7.1 (s, 2H, AreH), 5.25 (s, 1H, OeH), 3.80
semicarbazide (3k). (79%): m.p 205 ꢂC; Rf: 0.38; IR (Pure)
y
: 3413
(NH2), 3265 (NeH), 1588 (C]N), 1575 (AreC]C), 1168 (C]S),
cmꢀ1 1H NMR (Acetone-d6, 300 MHz)
: 10.2 (s, 1H, NH), 8.10 (s,
1H, CH]N), 7.4e7.1 (m, 4H, AreH), 7.0 (s, 2H, eNH2) 3.1 (s, 6H, N-
CH3); 13C NMR (75 MHz)
: 180.0 (1C, C]S), 152 (1C, C]N), 120.2
(s, 6H, CH3); 13C NMR (75 MHz)
d: 180.0 (C]S), 148 (Ar), 146 (C]
;
d
N), 139 (Ar), 128 (Ar), 104 (Ar), 55.0 (CeO); Anal. Calcd. For
C10H13N3O3S: C, 47.05; H, 5.13; N, 16.46; O, 18.80; S, 12.56; Found:
C, 47.02; H, 5.11; N, 16.44; O, 18.79; S, 12.55; GCeMS m/z: 255.07
(M þ, 100).
d
(2C, Ar), 129.4 (2C, Ar), 124 (Ar), 140.1 (Ar), 40.1 (2C, NeC); Anal.
Calcd. For C10H14N4S: C, 54.03; H, 6.35; N, 25.20; S, 14.42; Found: C,
54.00; H, 6.34; N, 25.18; S, 14.41; GCeMS m/z: 222 (M$þ, 100).
3.2.1.18. (E)-2-((E)-but-2-enylidene) hydrazinecarbothioamide (3r).
(48%): m.p 164 ꢂC; Rf: 0.44; IR (Pure)
y: 3378 (NH2), 3160 (NeH),
3.2.1.12. (E)-1-((E)-3-phenylallylidene)thiosemicarbazide
1600 (C]N), 1584 (C]C), 1105 (C]S), cmꢀ1; 1H NMR (DMSO-d6,
(3l). (46%): m.p 190 ꢂC; Rf: 0.57; IR (Pure)
y
: 3413 (NH2), 3265
(NeH), 1588 (C]N), 1575 (AreC]C), 1168 (C]S), cmꢀ1 1H NMR
(Acetone-d6, 300 MHz) : 10.2 (s, 1H, NH), 8.10 (s, 1H, CH]N), 7.64
300 MHz) d: 11.14 (s, 1H, NH), 8.04 (s, 1H, CH]N), 7.6 (s, 2H, eNH2),
;
6.13 (m, 2H, CH]CH), 1.82 (d, 3H, CH3); 13C NMR (75 MHz)
d: 178.0
d
(C]S), 145 (C]N), 138 (C]C), 128 (C]C), 18.89 (CeH); Anal. Calcd.
For C5H9N3S: C, 41.93; H, 6.33; N, 29.34; S, 22.39; Found: C, 41.92; H,
6.32; N, 29.33; S, 22.38; GCeMS m/z: 143.05 (M þ, 100).
Solvent system for Rf (petroleum ether: ethyl acetate 1:1)
(d, 1H, -J ¼ 16.0 Hz, CH]CH) 7.30e7.14 (br m, 5H, Ar), 7.0 (s, 2H,
eNH2), 6.39 (d, 1H, J ¼ 16.0 Hz, (CHeC]N); 13C NMR (75 MHz)
d:
182.0 (C]S), 152 (C]N), 126.2 (2C, Ar), 128.4 (2C, Ar), 127 (Ar),
134.9 (Ar), 142.8 (CH]C), 117.6 (CeC]N), 40.1 (2C, CeN); Anal.
Calcd. For C10H14N4S: C, 54.03; H, 6.35; N, 25.20; S, 14.42; Found: C,
54.00; H, 6.34; N, 25.18; S, 14.41; GCeMS m/z: 222 (M$þ, 100).
3.3. Assay for urease inhibition
The urease activity was determined by measuring amount of
ammonia being produced using indophenol method described by
3.2.1.13. (E)-2-(2-(benzyloxy)benzylidene)hydrazinecarbothioamide
(3m). (49%): m.p 163ꢂC; Rf: 0.62; IR (Pure)
y
: 3425(NH2), 3248(NeH),
Weatherburn [41]. The assay mixture, containing 10
(5 U/mL) and 10 L of test compound in 40 L buffer (100 mM urea,
0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl2, pH 8.2), were incu-
bated for 30 min at 37 ꢂC in 96-well plates. Briefly, 40
L each of
phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitro-
prusside) and 40 L of alkali reagent (0.5%, w/v NaOH and 0.1%
mL of enzyme
1593 (C]N), 1525 (AreC]C), 1089 (C]S), cmꢀ1; 1H NMR (DMSO-d6,
m
m
300 MHz) d: 11.50 (s, 1H, NH), 8.56 (s, 1H, CH]N), 8.13 (s, 2H, eNH2),
7.9e6.9 (m, 9H, AreH), 5.18 (s, 2H, CH2); 13C NMR (75 MHz)
d: 180.0
m
(C]S), 159 (Ar), 145 (C]N), 135 (Ar), 132 (Ar), 128 (Ar), 127 (Ar), 121
(Ar),120 (Ar),112 (Ar), 70.3 (C-O); Anal. Calcd. For C15H15N3OS:C,63.13;
H, 5.30; N, 14.73; O, 5.61; S, 11.24; Found: C, 63.10; H, 5.29; N, 14.72; O,
5.60; S, 11.22; GCeMS m/z: 285.09 (M þ, 100).
m
active chloride NaOCl) were added to each well. The absorbance at
625 nm was measured after 30 min, using a microplate reader (Bio-
Tek ELx 800Ô, Instruments, Inc. USA). All reactions were performed
in triplicate. Percentage inhibition was calculated by using the
formula 100 ꢀ (ODtestwell/ODcontrol) ꢃ 100. Thiourea was used as the
standard inhibitor of urease. The ChengePrusoff equation was used
to calculate the Ki values from the IC50 values, determined by the
non-linear curve fitting program PRISM 4.0 (GraphPad, San Diego,
3.2.1.14. (E)-2-(3-(benzyloxy)-4-methoxybenzylidene)hydrazine-
carbothioamide (3n). Oil; (45%); Rf: 0.5; IR (Pure) y: 3420 (NH2),
3244 (NeH), 1590 (C]N), 1522 (AreC]C), 1084 (C]S), cmꢀ1; 1H
NMR (DMSO-d6, 300 MHz) d: 11.40 (s, 1H, NH), 8.46 (s, 1H, CH]N),
8.03 (s, 2H, eNH2), 7.9e7.0 (m, 8H, AreH), 5.16 (s, 2H, CH2), 3.80 (s,
3H, CH3); 13C NMR (75 MHz)
d: 179.0 (C]S), 150 (Ar), 148 (Ar), 146
California, USA). At 37 ꢂC, one
mmol of ammonia being produced per
(C]N), 136 (Ar), 130 (Ar), 128 (Ar), 126 (Ar), 120 (Ar), 119(Ar), 112
(Ar), 109 (Ar), 70.3 (CeO), 55 (CeH); Anal. Calcd. For C16H17N3O2S:
C, 60.93; H, 5.43; N, 13.32; O, 10.15; S, 10.17; Found: C, 60.92; H,
5.42; N, 13.31; O, 10.13; S, 10.16; GCeMS m/z: 315.10 (M þ, 100).
minute by enzyme is known as one unit of enzyme at pH 8.2.
3.4. Protocol of docking study
In the docking study, the X-ray crystal structure of Jackbean
urease (entry 3LA4 in the Protein Data Bank) was used. The protein
structure was prepared prior to be used in docking study. The
structure preparation includes the addition of hydrogens and force-
field based parameterization of atoms followed by energy mini-
mization. Protonate3D [42] algorithm implemented in MOE [43]
was applied to protonate the protein structures and AMBER99
force-field was selected for energy minimization. It was made sure
to set up the correct metal states for the two Ni atoms in the
binding site of the enzyme. By using Protonate3D, the four
surrounding histidine residues in the active site pocket were
protonated according to the bound state of the two Nickel ions.
After protonation, the crystal structures were energy minimized.
Protein heavy atoms were restrained during the minimization to
3.2.1.15. (E)-2-(4-hydroxybenzylidene)hydrazinecarbothioamide
(3o). (40%): m.p 230 ꢂC; Rf: 0.36; IR (Pure)
y: 3466 (NH2), 3357
(NeH), 1600 (C]N), 1580 (AreC]C), 1163 (C]S), cmꢀ1; 1H NMR
(DMSO-d6, 300 MHz) d: 10.7 (s, 1H, NH), 8.30 (s, 1H, CH]N), 8.05 (s,
2H, eNH2), 7.8e6.8 (m, 4H, Ar-H), 5.25 (s, 1H, OeH); 13C NMR
(75 MHz) : 180.0 (C]S), 159 (Ar),146 (C]N),130 (Ar),126 (Ar), 116
d
(Ar); Anal. Calcd. For C8H9N3OS: C, 49.21; H, 4.65; N, 21.52; O, 8.19;
S, 16.42; Found: C, 49.20; H, 4.64; N, 21.51; O, 8.20; S, 16.41;
GCeMS m/z: 195.05 (M þ, 100).
3.2.1.16. (E)-2-(2,3-dihydroxybenzylidene)hydrazinecarbothioamide
(3p). (45%): m.p 220 ꢂC; Rf: 0.07; IR (Pure)
y: 3378 (NH2), 3300
(NeH), 1613 (C]N), 1599 (AreC]C), 1200 (C]S), cmꢀ1; 1H NMR

M.A.S. Aslam et al. / European Journal of Medicinal Chemistry 46 (2011) 5473e5479
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RMSD value was 0.9 indicating the goodness of alignment. The
binding sites of the two ureases also aligned very well indicating
the similarity in binding sites. Re-docking of the bound ligands was
performed with both structures to test the docking parameters and
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Jack bean urease was defined by 10 Å spacing of the residues
surrounding the bound PO4 and similarly, HAE (the co-crystallized
ligand in bacterial urease structure) was used to define the binding
site in the H. Pylori urease. The geometry parameters of the metal
atoms were set to auto. The default docking parameters were
chosen for scoring function and other chemical parameters rele-
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Acknowledgements
This work was financially supported by COMSTECHeTWAS and
the Higher Education Commission (HEC) of Pakistan through
project No. 20-1264/R & D/08/3302 under the National Research
Support Program for Universities.
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