Synthesis, molecular docking studies, and in vitro screening of sulfanilamide-thiourea hybrids as antimicrobial and urease inhibitors

Article abstract of DOI:10.1007/s00044-012-0376-4

A series of novel hybrid molecules containing sulfanilamide and thiourea templates was designed and synthesized. These compounds were screened for antimicrobial and urease inhibitory activities. Some of the compounds revealed promising antibacterial and a

Full text of DOI:10.1007/s00044-012-0376-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3653–3662
DOI 10.1007/s00044-012-0376-4
ORIGINAL RESEARCH
Synthesis, molecular docking studies, and in vitro screening
of sulfanilamide-thiourea hybrids as antimicrobial
and urease inhibitors
•
Aamer Saeed Sumera Zaib
•
•
Arshid Pervez Amara Mumtaz
•
•
Mohammad Shahid Jamshed Iqbal
Received: 13 September 2012 / Accepted: 15 November 2012 / Published online: 29 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of novel hybrid molecules containing
sulfanilamide and thiourea templates was designed and syn-
thesized. These compounds were screened for antimicrobial
and urease inhibitory activities. Some of the compounds
revealed promising antibacterial and antifungal activities.
Fascinatingly, the majority of the compounds exhibited
potential urease inhibitory activities with IC50 ranging from
0.20 to 7.50 lM. Compound 2b was identified as the most
potent urease inhibitor (IC50 0.20 lM), and was 100-fold
more potent than thiourea, the standard inhibitor. Molecular
docking studies of compounds were performed on 3D crystal
structures of Jack bean and Helicobacter pylori ureases.
crucial point around the world (Gould, 2008). The
increasing prevalence of multidrug resistant strains of
bacteria and the recent emergence of new strains with
reduced susceptibility to antibiotics raises the specter of
untreatable bacterial infections (Sieradzki et al., 1999).
Therefore, there is an urgent need to develop anti-infective
agents for the treatment of bacterial diseases. Currently, the
research has been focused toward development of new
antibacterial agents which may result in the discovery of
novel effective compounds (Nitta et al., 2002).
The limitations of current antifungal drugs, increased
incidence of systemic fungal infections, and rapid devel-
opment of drug resistance have highlighted the need for the
discovery of new antifungal agents, preferably with novel
mechanisms of action (Kontoyiannis et al., 2003). While
synthetic efforts remain the mainstream in antifungal drug
discovery, as evidenced by the fact that 18 out of 23
antifungal drugs approved from 1980 to 2002 are synthetic
(83 % belong to single azole class) (Newman et al., 2003).
Urease (EC 3.5.1.5; urea amidohydrolase) is a metal
containing enzyme that catalyzes the hydrolysis of urea into
ammonia and carbon dioxide (Dixon et al., 1975). The
activity of bacterial ureases has been shown to be important
virulence factor in the development of many harmful clin-
ical conditions for human and animal health as well as
agriculture (Mobley et al., 1995). Bacterial ureases have
been reported to be involved in the formation of infectious
stones and development of peptic ulcers and stomach cancer
(Montecucco and Rappuoli, 2001). Ureases also contribute
to the development of urolithiasis, pyelonephritis, hepatic
encephalopathy, hepatic coma, and urinary catheter encrus-
tation (Mobley and Hausinger, 1989). Therefore, it is very
much desired to find the potential urease inhibitors. We
have reported different types of compounds as urease
inhibitors (Aslam et al., 2011; Hanif et al., 2012a, b).
Keywords
1-Aroyl-3-(4-aminosulfonylphenyl)thioureas ꢀ
Sulfanilamide-thiourea hybrids ꢀ Antibacterial activity ꢀ
Molecular docking ꢀ Urease inhibition
Introduction
Over the last decade, the number of antibiotic-resistant
Gram-negative and Gram-positive bacteria is reaching its
A. Saeed ꢀ A. Mumtaz
Department of Chemistry, Quaid-I-Azam University,
Islamabad, Pakistan
S. Zaib ꢀ A. Pervez ꢀ J. Iqbal (&)
Department of Pharmaceutical Sciences, COMSATS Institute
of Information Technology, Abbottabad 22060, Pakistan
e-mail: drjamshed@ciit.net.pk
M. Shahid
Department of Bioinformatics, Fraunhofer Institute SCAI,
Sankt Augustin, Germany
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Med Chem Res (2013) 22:3653–3662
Sulfanilamide and its derivatives have extensively been
of sulfanilamide has been incorporated into the thiourea
nucleus leaving sulfonamide function intact, in contrast to
most of the known sulfonyl urea/thiourea derivatives in
which the sulfonamide group is masked by being a part of
the urea/thiourea moiety (Saczewski et al., 2010; Faidallah
et al., 2002). In the present investigation, novel sulfonyl
thioureas derivatives were synthesized and evaluated for
in vitro antibacterial properties against four pathogenic
bacterial strains and their urease inhibitory activity.
used in chemotherapy due to their efficacy against broad
range of microorganisms. Figure 1 shows some bioactive
molecules reported in literature.
Sulfonyl ureas are very potent hypoglycemic agents due to
their ability to stimulate the release of insulin from the pan-
creatic islets (Gribble and Reimann, 2003; Drews, 2000).
Besides antibacterial and hypoglycemic activities, sulfona-
mides exhibit diuretic (Maren, 1976), anticarbonic anhydrase
(Al-Rashida et al., 2011), antithyroid (Thornber, 1979), and
antitumor activities (Abbate et al., 2004). Sulfonamides are also
found to exhibit remarkable antimalarial activity (Ryckebusch
et al., 2002; Krungkrai et al., 2005; Klingenstein et al., 2006).
Several antiplasmodial 7-chloro-4-aminoquinolyl-derived sul-
fonamides, ureas, thioureas, and amides, have been synthesized
and tested against chloroquine resistant and chloroquine sen-
sitive Plasmodium falciparum (Koch, 2001).
Results and discussion
Chemistry
The development of solvent-free organic synthesis is of
current interest on account of many advantages, for
example reduced pollution, low cost, simplicity in process
and handling, potential applications in combinatorial
chemistry and chemical industry. Thus, a variety of freshly
prepared aroyl/acyl isothiocyanates (1a–1t) were treated
with sulfanilamide in 1:1 molar ratio under neat conditions
without using any catalyst to furnish the corresponding
1-aroyl/alkanoyl-3-(4-aminosulfonylphenyl)thioureas (2a–2t)
in high yields (Scheme 1).
Thiourea derivatives are potent antitrypanosoma (Aly
et al., 2010), influenza virus neuraminidase inhibitor (Nair
and Sobhia, 2008), antifungal against plant pathogenic
fungi (Ramadas et al., 1998), herbicidal (Yonova and
Stoilkova, 2004), and anticancer activities (Bestmann et al.,
1992). In the title compounds, the sulfanilamide moiety is
appended to the thiourea nucleus to combine their useful
effects in a single structural unit. The aim of the synthesis is
to reduce the toxicity of the parent compound and improve
the therapeutic effect. The title compounds constitute a
unique class of sulfonyl thioureas in which the anilino group
Stretching vibrations attributable to free and associated
NH at 3,212–3,350, C=O at 1,655–1,675, C=S at
1,244–1,263 in addition to those for the aromatic ring at
Fig. 1 Some structurally
related bioactive compounds
Cl
SO2NH2
N
N
O
S
O
S
SO2NH2
S
N
H
N
H
N
H
Cl
N
H
N
H
N
H
R
SO2NH2
S
N
H2N
O
N
N
H
R'
N
Scheme 1 Synthetic pathway
to 1-aroyl-3-(4-
aminosulfonylphenyl)thioureas
SO2NH2
S
O
H2N
SO2NH2
C
N
N
R
S
R
H
H
RT to 60-70°C, 1-2 h.
(1a-t)
(2a-t)
#
2a
R=
H
2b 4-ClC6H5
2c 4-CH3C6H5
2e 3-ClC6H5
2g benzyl
2d 4-NO2C6H5
2f 3-NO2C6H5
2h 2-CH3C6H5
2j 3-CH3OC6H5
2i 3-CH3C6H5
2k 2,3-(CH3O)2C6H54 2l 2-Cl,5-NO2C6H4
2m 3-FC6H5
2o 4-Cl, 2-NO2C6H4
2q 3,5-(CH3O)2C6H4
2s 2-phenylbutanoyl
2n 2,3,4-(CH3O)3C6H3
2p C11H23
2r 2-furanyl
2t 2,4-(Cl)2C6H4
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Med Chem Res (2013) 22:3653–3662
3655
1,581–1,590 cm-1 could be identified in the IR spectra of
the substituted aroyl thioureas, however, the presence of
sulfonamido group leads to further complexity. In com-
pounds containing the thioamide group (HNCS), a set of
fundamentals involving the N–C and C=S bonds are
identified known as ‘‘thioamide’’ bands: I, II, III, and IV.
These bands have a large contribution from C–N stretching
and (N–H) deformation (I), (C–N) and (C–S) stretchings (II
and III) and (C–S) (IV) stretching and usually appear
around 1470, 1250, 1080, and 750 cm-1, respectively
Table 1 The MIC (lg/mL) values of 1-aroyl-3-(4-aminosulfonyl-
phenyl)thiourea derivatives (2a–2t) against different bacterial strains
and standard ciprofloxacin
Compounds
Bacterial strains and their MIC (lg/mL)
E. coli
B. subtilis
S. aureus
S. flexneri
2a
0.313
0.313
0.313
0.313
0.156
0.313
0.313
0.313
1.250
0.313
0.313
0.313
0.313
0.313
0.156
0.313
0.313
0.313
1.250
0.313
0.156
0.313
0.156
0.313
0.156
0.156
0.313
0.313
0.625
0.313
0.313
0.313
0.156
0.313
0.156
0.156
0.313
0.313
0.625
0.313
0.313
0.625
0.313
0.156
0.313
0.156
0.156
0.313
0.313
0.625
0.625
0.313
0.313
0.156
0.313
0.156
0.156
0.313
0.313
0.625
0.625
0.313
0.156
0.156
0.313
0.156
0.313
0.156
0.313
0.313
0.313
0.313
0.313
0.156
0.313
0.156
0.313
0.156
0.313
0.313
0.313
0.313
0.313
0.313
2b
2c
2d
1
2e
(Saeed et al., 2010). In H NMR the characteristic broad
2f
singlets for protons of N1 and N3 are observed at d 12.76
and 9.19, respectively besides the signals for aromatic
protons. 13C NMR showed the peaks around d 170 and 179
for C=O and C=S, respectively (Saeed et al., 2011) except
for the alkanoyl derivative 2p, where these signals appear
at d 174 and 182, respectively.
2g
2h
2i
2j
2k
2l
Antibacterial activity
2m
2n
Antibacterial activity of the synthesized 1-aroyl-3-(4-amin-
osulfonylphenyl) thiourea derivatives was tested against
different strains of both Gram-positive bacteria viz. Staph-
ylococcus aureus, Bacillus subtilis, and two Gram-negative
bacteria namely, Escherichia coli, and Shigella flexneri.
Ciprofloxacin was used as a standard drug for the assay. The
activities are reported in Table 1, the minimum inhibitory
concentrations (MICs) were determined using the broth
micro-dilution method, as recommended by the National
Committee for Clinical Laboratory Standards.
2o
2p
2q
2r
2s
2t
Ciprofloxacin
MIC minimum inhibitory concentration
All of the tested compounds 2a–2t showed potent anti-
bacterial activity against the tested strains. In particular,
compounds 2e and 2o, which possess a 3-chloro and 4-choro-
2-nitro groups respectively, on the aroyl ring, were found to
have the most potent activity (MIC: 0.156 lg/mL) against
E. coli and were equipotent in vitro as ciprofloxacin (MIC:
0.156 lg/mL). In addition, these compounds were two times
more potent than reference drug against S. flexneri, as cip-
rofloxacin showed MIC: 0.313 lg/mL, whereas 2e and 2o
showed MIC: 0.156 lg/mL. The antibacterial activity of 2i
and 2s having comparatively non polar 3-methyl and
2-phenylbutanyl groups, respectively, was disappointing as
these compounds were less potent (MIC: 1.250 lg/mL) than
reference drug against E. coli (0.156 lg/mL). In case of
Gram-positive organisms, most of the compounds exhibited
activity better than or comparable to that of the reference
(0.625 and 0.156 lg/mL for B. subtilis and S. aureus,
respectively). Some of the compounds such as 2b, 2d, 2e, 2l,
2n, and 2o having polar or electron withdrawing substituents
showed excellent inhibitory potency (MIC: 0.156 lg/mL)
against B. subtilis, fourfold more potent than reference drug.
However, all these compounds were equipotent as reference
drug against S. aureus. In contrast, a few compounds such as
2h, 2i, 2r, and 2s having non polar 2- or 3-methyl, 2-furanyl
or a 2-phenylbutanyl substituent, were far less active than
ciprofloxacin in inhibiting the growth of S. aureus. In gen-
eral, the analogs bearing chloro and nitro, both groups
together, or a trimethoxy group on aroyl ring exhibited more
activity than the reference drug against the tested Gram-
positive and Gram-negative organisms.
Antifungal activity
Antifungal activity of the synthesized 1-aroyl-3-(4-amin-
osulfonylphenyl)thiourea derivatives was tested against
different strains viz. Aspergillus niger, Aspergillus flavus,
Aspergillus pterus, and White rot. The activities are rep-
resented in Table 2, the zone of inhibition was determined
using the hyphal extension inhibition assay.
Among all the tested compounds 2a–2j, 2n, 2o, 2r, and
2s showed no antifungal activity against the tested strains.
Compounds 2k and 2t showed activity against only one
strain. 2l, 2m, and 2q showed significant antifungal activity
against the tested strains and showed moderate zone of
inhibition. Whereas 2p among all thiourea derivatives
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Med Chem Res (2013) 22:3653–3662
Table 2 The zone of inhibition (mm) values of 1-aroyl-3-(4-amin-
osulfonylphenyl)thiourea derivatives (2a–2t) against different fungal
strains
Table 3 Urease inhibition activities of the compounds (2a–2t) and
standard inhibitor (thiourea)
Compounds
Ki SEM (lM)
(or % inhibition)
Compounds
Fungal strains and zone
of inhibition
2a
1.31 0.23
0.20 0.01
4.32 0.19
7.50 1.17
1.50 0.06
0.45 0.03
1.60 0.33
53.00 7 %
0.89 0.07
0.65 0.02
0.82 0.01
0.44 0.01
1.49 0.21
1.34 0.14
5.59 0.21
1.30 0.20
1.21 0.05
1.14 0.11
1.23 0.16
1.91 0.18
23.0 3.3
A. niger
A. flavus
A. pterus
W. rot
2b
2c
2k
2l
–
–
0.9 0.44
–
2d
2e
1.6 0.73 2.1 0.09
2.4 0.78 2.8 0.15
–
–
–
2m
2p
2q
2t
0.7 0.32
2f
4.6 0.34 5.1 0.62 4.9 0.71 2.3 1.02
1.5 0.06 1.9 0.69 2.7 0.81 0.9 0.12
2g
2h
2i
0.8 0.39
–
–
–
Terbinafine 5.3 0.48 5.7 0.72 5.2 0.38 5.9 0.19
– No activity
2j
2k
2l
exhibited excellent antifungal activity against tested strains
and showed maximum zone of inhibition comparable to
that of standard Terbinafine.
2m
2n
2o
2p
2q
2r
In vitro urease inhibitory activity
The inhibitory potential of the synthesized compounds was
evaluated against Jack bean urease and is shown in
Table 3. The substitution of different groups at phenyl ring
resulted in variable inhibition patterns depending upon type
and position of substituent. Some of the substituents at
phenyl ring, increased inhibition potency of compounds
and some resulted in lowering in inhibition capacity.
Compounds have their activities in the range of
0.20 0.01–7.50 1.17 lM (Table 3). Compound 2a
having phenyl ring with no substitution was found to have
Ki value 1.31 0.23 lM. Compounds 2b and 2l bearing a
4-chloro, and 2-chloro-5-nitro groups, respectively, on
aroyl ring were found to be the most active inhibitors, with
Ki values of 0.20 0.01 and 0.44 0.01 lM, respec-
tively. They were also found to be 100- and 50-fold more
active than the standard inhibitor (thiourea, Ki 23
3.3 lM), respectively. It is interesting to note that com-
pound 2b having chloro group at para position was most
active while 2l having chloro at ortho position and electron
withdrawing nitro group at meta position was relatively
less active. While compound 2f having nitro group at meta
position and 2i, 2j, and 2k having electron donating
3-methyl, 3-methoxy, and 2,3-dimethoxy groups, respec-
tively are slightly less active than 2b and 2l.
2s
2t
Thiourea
exception was compound 2h having a 2-methyl group,
which exhibited weak inhibition of urease. The potential
inhibitory activities of the 1-aroyl-3-(4-aminosulfonylphe-
nyl)thiourea derivatives may be due to their structural
similarity with the thiourea, which is a natural substrate of
urease.
Thus, the biological behavior of a molecule was not
determined by the influence of a single parameter or vari-
able. Furthermore, in most cases, the presence of various
groups in a compound does not allow to accurately explain
the kind and intensity of its biological activity. However,
taking the necessary precautions, allows us to make some
general remarks on the structure and activity.
Docking
The docking results for the compounds are ranked on the
basis of their docking scores obtained with structures,
3LA4 and 4UBP, docking scores are presented in Table 4.
Both structures were aligned using Chimera program with
an alignment RMSD of 0.6, which indicates a very good
alignment of the conserved residues in the active site
pockets of both structures as shown in Fig. 2. Acetohy-
droxamic acid (PDB code HAE) was used as a reference
ligand to define the active site pocket for docking in the
Comparing 2c and 2i having electron releasing methyl
group on para-, ortho-, and meta-positions, respectively,
showed Ki values 4.32 0.19 and 0.89 0.07 lM,
respectively indicating that methyl group is most effective
when present at meta position. All the target compounds
were very potent inhibitors of urease, in most cases
even more than the standard inhibitor, thiourea. The only
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Med Chem Res (2013) 22:3653–3662
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Table 4 FlexX Docking Score of compounds 2a–2t
Compound Ki
Rank Docking score
(kcal/mol) 3LA4
Rank Docking score
(kcal/mol) 4UBP
2a
2b
2c
2d
2e
2f
1.31 1
0.20 1
-36.14
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-37.39
-37.51
-37.30
-37.40
-38.28
-38.50
-35.62
-37.77
-37.99
-37.17
-35.33
-38.55
-38.10
-34.87
-37.27
-35.20
-35.88
-37.75
-37.18
-35.29
-36.52
-36.28
-37.72
-38.42
-37.41
-38.44
-38.40
-38.29
-38.51
-35.51
-35.69
-38.91
-35.08
-37.72
-26.28
-37.82
-34.56
-35.84
-36.73
4.32 1
7.50 2
1.50 1
0.45 1
1.60 1
2g
2h
2i
53.0
1
0.89 1
0.65 1
0.82 1
0.44 1
1.49 1
1.34 1
5.59 2
1.30 1
1.21 1
1.14 1
1.23 1
1.91 1
2j
2k
2l
Fig. 3 Predicted docked conformations of all compounds (2a–2t)
inside the binding pocket of Jack bean urease. The blue spheres
indicate the metal pharmacophores around the two nickels (Ni2?).
The dotted lines indicate various types of interactions of the
compounds with the active site residues including hydrogen bonding
and aromatic interactions. The docked poses are shown in sticks
representation
2m
2n
2o
2p
2q
2r
2s
docking predictions were produced in the presence of metal
pharmacophoric constraints. When the said constraints
were relieved, the docking predictions were obtained with
the similar binding modes in both urease structures.
Thiourea is having hydrogen bonding interaction with ASP
residue (ASP633 in Jack bean urease, ASP363 in bacterial
urease), GLY residue (GLY550 in Jacbean urease,
GLY280 in bacterial urease), and ALA residue (ALA636
in Jack bean urease, ALA170 in bacterial urease) as shown
in Figs. 3 and 4.
2t
aligned structures of both ureases. The same reference
ligand was re-docked to both structures and the RMSD of
0.80 was obtained for the PDB structure 3LA4 while
RMSD of 0.35 was obtained for the structure 4UBP.
Docking of thiourea
Thiourea was docked to both aligned structures of Jack
bean as well as bacterial urease. It was observed that no
The top scoring poses of all compounds were analyzed
inside the binding site of the two enzymes for binding
Fig. 2 Aligned pocket residues
of both urease structures. Two
nickel atoms overlapping each
other with Acetohydroxamic
acid (PDB code HAE) bound
between the two nickel atoms.
The surrounding HIS, ASP, and
ALA residues of both urease
structures are also shown in the
alignment
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Med Chem Res (2013) 22:3653–3662
Fig. 4 Left binding mode of the
docked Thiourea inside the
active pocket of Jack bean
urease. Right the binding mode
of Thiourea inside the active
pocket of bacterial urease
Fig. 5 Interaction diagrams
showing similar interactions of
Thiourea to same residues in
both urease structures. Left
interaction diagram of Thiourea
inside the pocket of Jack bean
urease. Right interaction
diagram of Thiourea inside the
bacterial urease structure
modes. Similar binding modes have been obtained for all
the compounds in both urease structures as shown in Fig. 5.
The similarity in binding modes in both cases might
explain the negligible differences in the docking scores that
were ranging from -26 to -38 in Jack bean urease and -
34 to -38 in Helicobacter pylori urease.
Figure 5 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 2d and 2o whose ranks
were 2 by 3LA4 and all compounds have a similar binding
mode in the 1st ranked docked solution by 4UBP. This also
indicates that all the compounds fulfilled the metal phar-
macophoric constraints and the thiourea moiety interacts
with the two nickel atoms.
Fig. 6 Interaction diagram can be seen for inhibitor 2b showing
hydrogen bonding by Oxygen of Asp494 and Ala440 residues and
nitro group of His593
Figures 6, 7, 8, and 9 show the interaction diagrams of
four compounds, in which 2e and 2o are most potent
against bacterial strains and 2b and 2l shows excellent
activity against urease.
It has been observed in all docking results from both
structures that the sulfonamide moiety of the 1-aroyl-3-(4-
aminosulfonylphenyl)thiourea derivatives was interacting
with the bi-nickel center of the enzyme as shown in
Fig. 10. The sulfur atom of the sulfonamide moiety was
Fig. 7 Interaction diagram can be seen for inhibitor 2e showing
hydrogen bonding by Oxygen of Asp494 and Ala440 residues
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Med Chem Res (2013) 22:3653–3662
3659
Fig. 8 Interaction diagram can
be seen for inhibitor 2l (2Cl,
5-NO2) showing hydrogen
bonding by Oxygen of Ala 636,
Arg439, and Cme592 residues
and nitro group of His593
placed nearly at the same position where the phosphorus of
PO4 was located in the crystal structure. All of the com-
pounds were having interactions with both nickel atoms.
The rest of the R-groups of the compounds possess free
conformations toward the opening of the binding site.
Conclusions
In conclusion, several sulfanilamide-thiourea hybrids (2a–
2t) were synthesized which differ from most of the well-
known sulfonyl thiourea derivatives in having the free
anilino nitrogen of sulfanilamide being incorporated into
the thiourea nucleus leaving the sulfonamide amino group
intact. The novel structural series exhibited excellent
antibacterial activity against the entire Gram-positive and
Gram-negative bacteria. Compounds 2e and 2o were found
to be much more potent antibacterial agents than the
standard drug ciprofloxacin. Antifungal activity results
showed that compounds 2l, 2m, 2p, and 2q were potent
antifungal agents. Compounds 2b and 2l were found to be
the most active urease inhibitors, 100- and 50-fold more
active than the standard inhibitor, respectively. Further-
more, proposed scaffold of urease inhibitor offers the
possibility of convenient further modifications and exten-
sions that could give rise to the structures with improved
inhibitory activity against urease enzyme.
Fig. 9 Interaction diagram can be seen for inhibitor 2o showing
hydrogen bonding by Oxygen of Arg439, Cme 592, and Ala440
residues and nitro group of His593
Experimental
Materials
Melting points were recorded using a digital Gallenkamp
(SANYO) model MPD.BM 3.5 apparatus and are uncor-
rected. 1H NMR and 13C NMR spectra were determined at
300 and 75.4 MHz, respectively as CDCl3 using a Bruker
NMR spectrometer. FTIR spectra were recorded on an FTS
3000 MX spectrophotometer. Mass Spectra (EI, 70 eV) on
Fig. 10 Electrostatic potential surface of predicted docked poses of
all 1-aroyl-3-(4-aminosulfonylphenyl)thiourea compounds (2a–2t)
inside the Jack bean urease. The electrostatic charge distribution of
functional group is shown clearly toward the bi-nickel center of the
enzyme. The two nickels are represented by small spheres in light
blue color
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Med Chem Res (2013) 22:3653–3662
a MAT 312 instrument, and elemental analyses were
conducted using a LECO-183 CHNS analyzer.
for testing in the same medium and were grown at 37 °C.
Then the cells were suspended, in saline solution, to pro-
duce a suspension of about 10-5 CFU/mL (colony-forming
units per mL). Serial dilutions of the test compounds,
previously dissolved in N,N-dimethylformamide (DMF),
were prepared in test tubes to final concentrations of 2.5,
1.25, 0.625, 0.313, and 0.156 lg/mL. 100 lL of a 24 h old
inoculum was added to each tube. The MIC, defined as the
lowest concentration of the test compound, which inhibited
the visible growth after 18 h, was determined visually after
incubation for 24 h at 37 °C. Tests using DMF as negative
control were carried out in parallel. Ciprofloxacin was used
as control drug. Because the MIC values were not specta-
cular, no statistical calculations were made.
General procedure for synthesis of 1-aroyl-3-(4-
aminosulfonylphenyl)thioureas
A stirred mixture of freshly prepared suitably substituted
benzoyl/acyl isothiocyanates (1a–1t) (10 mmol) were
warmed with sulfanilamide (10 mmol) at 60–70 °C for
1–2 h. The progress of reaction was followed by TLC
examination using hexane–EtOAc (4:1). On completion,
the reaction mixture was poured into cold water and the
precipitated sulfanilamido-thioureas (2a–2t) were filtered.
Recrystallization from EtOH or MeOH furnished the
desired products.
Antifungal assay
1-(Phenylacetyl)-3-(4-aminosulfonylphenyl) thiourea
(2g) (52 %): m.p 145–146 °C; IR (Pure) t: 3212 (NH),
1661 (C=O), 1591 (C=C), 1482 (thioamide I), 1334 (C–S),
1263 (thioamide II), 1158, 1082 (thioamide III), 749 (thi-
Synthetic 1-aroyl-3-(4-aminosulfonylphenyl)thiourea deriva-
tives were screened for antifungal activity by agar well-
diffusion method (Rojas et al., 2006) with little modifications,
with sterilecorkborer ofsize6.0 mm. Theculturesof48 hold
grown on potato dextrose agar (PDA) were used for inocula-
tion of fungal strains viz, A. niger, A. flavus, A. pterus, and
W. rot on PDA plates. An aliquot (20 lL) of inoculum was
introduced to molten PDA and poured into a petri dishby pour
plate technique. After solidification, the appropriate wells
were made on agar plates using cork borer. In agar well-
diffusion method, 50 lL of sample was introduced serially
after successful completion of one compound analysis.
Incubation period of 24–48 h at 28 °C was maintained for
observation of antifungal activity. The antifungal activity
was evaluated by measuring zones of inhibition of fungal
growth surrounding the sample compound. The complete
antifungal analysis was carried out under strict aseptic
conditions. The zones of inhibition were measured with
antibiotic zone scale in mm and the experiment was carried
out in triplicates.
1
oamide IV) cm-1; H NMR (d ppm, 300 MHz) d: 12.9 (s,
1H, NH), 11.7 (s, 1H, NH), 7.1–7.6 (m, 5H, Ar), 7.62 (d,
2H, Ar), 6.5 (d, 2H, Ar), 4.3 (s, 2H, CH2); 13C NMR (d
ppm) (75.4 MHz) d: 180 (C=S), 173 (C=O), 139 (C–S),
136.2 (C–CO), 135.4 (C–C) 133 (Ar), 132 (Ar), 131 (Ar),
129 (Ar), 128 (Ar), 127 (Ar), 126 (Ar), 59 (CH2); CHNS
analysis Calcd/Obs.: C, 51.56; H, 4.33; N, 12.03; S, 18.35;
Found: C, 52.1; H, 4.99; N, 12.78; S, 18.8; EIMS m/
z (%):349 [M?], 214 (8), 171 (52), 91 (100).
1-(2-Furanyl)-3-(4-aminosulfonylphenyl)
thiourea
(2r) (68 %): m.p 169–170 °C; IR (Pure) t: 3245 (NH),
1660 (C=O), 1590 (C=S), 1531 (thioamide I), 1330 (C–S),
1234 (thioamide II), 1151, 1097 (thioamide III), 747 (thi-
oamide IV) cm-1; 1H NMR (d ppm, 300 MHz) d: 12.78 (s,
1H, NH), 11.6 (s, 1H, NH), 7.51 (d, 1H, furanyl), 6.7 (d,
1H, furanyl), 6.67 (d, 1H, furanyl),7.62 (d, 2H, Ar), 6.5 (d,
2H, Ar);13C NMR (d ppm) (75.4 MHz) d: 180 (C=S), 173
(C=O), 144.1 (furanyl), 139 (C–N), 136 (C–CO), 133 (Ar),
131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar), 126 (Ar),124 (Ar),
109.4 (furanyl), 108.7 (furanyl); CHNS analysis Calcd/
Obs.: C, 44.43; H, 3.73; N, 14.13; S, 21.57; Found: C,
44.39; H, 3.81; N, 14.07; S, 21.64; GC–MS m/z: 297.02
(Mꢀ?, 100).
Urease inhibition assay
Urease inhibition activities were determined by measuring
the release of ammonia by the indophenol method,
(Weatherburn, 1967). The amount of ammonia liberated
was assessed by measuring indophenol blue formed in
Berthelot reaction over 30 min incubation at 30 °C fol-
lowing the changes in absorbance at 625 nm. In brief, the
assay mixture containing 10 lL of enzyme (5 U/mL) and
10 lL of test compound in 40 lL buffer (100 mM urea,
0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl2, pH 8.2),
were incubated for 30 min at 30 °C in 96-well plates. A
40 lL each of phenol reagents (1 %, w/v phenol and
0.005 %, w/v sodium nitroprusside) and 40 lL of alkali
Antibacterial activity
The antibacterial activity was evaluated in vitro by MIC
using the serial tube dilution method (Zampini et al.,
2009). For the assay, two Gram-positive bacteria namely,
S. aureus, B. subtilis, and two Gram-negative bacteria
namely, E. coli and S. flexneri were used. Bacterial strains
stored in Muller-Hinton broth (Merck), were subcultured
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Med Chem Res (2013) 22:3653–3662
3661
reagent (0.5 %, w/v NaOH and 0.1 % active chloride
NaOCl) were added to each well. The absorbance was
measured after 30 min, using a microplate reader (Bio-Tek
ELx 800TM, Instruments, Inc. USA). All reactions were
performed in triplicate. Percentage inhibition was calcu-
lated using the formula 100 - (OD test well/OD con-
trol) 9 100. Thiourea was used as the standard inhibitor of
urease. The Cheng-Prusoff equation was used to calculate
the Ki values from IC50 values, determined by the nonlinear
curve fitting program PRISM 5.0 (GraphPad, San Diego,
California, USA). Following equation was used:
small molecule includes generation of 3D confirmations of
the molecules, ionization, and protonation states as well as
generation of possible tautomeric forms at variable pH
values. A molecule with different ionization state will
interact differently inside the binding pocket of the protein.
The ligand molecules were also prepared with MOE, by
implementing MMFF94x force-field and using the ‘‘wash’’
module by setting up the parameters for ionization and
protonation states. The resulting molecule structures were
then energy minimized before screening against the pre-
pared target structures.
Ki ¼ ðIC50Þ=ð1 þ ð½Sꢁ=½KmꢁÞÞ
Docking
where Ki = inhibition constant, IC50 = the inhibitory
strength of the compound, [S] = concentration of sub-
strate, [Km] = substrate concentration, where enzyme
activity is at half maximal.
FlexX program was used in the docking study. Before
carrying out the dockings of small molecules into the
protein structures, the non-standard protein residues (KCX
and CME) and the metal ions were included in the binding
site specification. The binding site for Jack bean urease was
defined by 10 Angstrom spacing of the residues sur-
rounding the bound PO4 and similarly, HAE (the co-crys-
tallized ligand in bacterial urease structure) was used to
define the binding site in the H. pylori urease. The geom-
etry parameters of the two metal atoms were set to be
derived automatically by FlexX and the metal pharmaco-
phores were defined in spherical form to interact in all
directions instead of statistically derived coordinates of the
metal atoms. The default docking parameters were used
and the top 10 solutions were retained for later analysis.
Protocol of docking study
In docking study, the crystal structures of Jack bean Urease
(PDB Code: 3LA4) and H. pylori urease (PDB Code:
4UBP) were used. The protein structures were prepared
prior to be used in docking study. The structure preparation
included addition of hydrogens and force-field based
parameterization of atoms followed by energy minimiza-
tion. Protonate3D (Labute, 2007) algorithm implemented
in MOE (MOE, 2010) 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
these enzymes. By 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 minimi-
zation to allow the flexibility of protein side-chains and
hydrogen atoms only. After minimization, the water mol-
ecules and co-crystallized bound compounds were
removed. The two structures were also aligned using UCSF
Chimera. The RMSD value was 0.6 indicating the good-
ness of alignment. The binding sites of the two ureases also
aligned very well indicating the similarity in binding sites.
Redocking of the bound ligands was performed with both
structures to test the docking parameters and check for the
correct preparation of crystal structures for use in screening
by molecular docking.
Acknowledgments This study was financially supported by
COMSTECH–TWAS and German-Pakistani Research Collaboration
Programme to JI.
References
Abbate F, Casini A, Owa T, Scozzafava A, Supuran CT (2004)
Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer
agent, potently inhibits cytosolic isozymes I and II, and
transmembrane, tumor-associated isozyme IX. Bioorg Med
Chem Lett 14(1):217–223
Al-Rashida M, Ashraf M, Hussain B, Nagra SA, Abbas G (2011)
Discovery of new chromone containing sulfonamides as potent
inhibitors of bovine cytosolic carbonic anhydrase. Bioorg Med
Chem 19(11):3367–3371
Aly AA, Brown AB, Ramadan M, Abdel-Aziz M, Abuo-Rahma GE-
DAA, Radwan MF, Gamal-Eldeen AM (2010) Selectivity of N-
aroyl-N0-arylthioureas towards 2-(1,3-dioxo-1H-inden-2(3H)-
ylidene)malononitrile. New synthesis of (Z)-N-((E)-4-amino-1-
aryl-5-cyano-6-oxo-1H-indeno[1,2-d][1,3]- thiazepin-2(6H)-yli-
dene)-4-arylamides of antitumor and antioxidant activities.
J Heterocycl Chem 47(3):503–508. doi:10.1002/jhet.344
Aslam MA, Mahmood SU, Shahid M, Saeed A, Iqbal J (2011)
Synthesis, biological assay in vitro and molecular docking
studies of new Schiff base derivatives as potential urease
inhibitors. Eur J Med Chem 46(11):5473–5479
Compounds library preparation
Before proceeding to perform docking compounds inside
the protein structures, it is also of much importance to
prepare the small molecules for docking. Preparation of
123

3662
Med Chem Res (2013) 22:3653–3662
¨
Bestmann HJ, Kern F, Schafer D, Witschel MC (1992) 3,4-
Dihydroisocoumarins, a new class of ant trail pheromones.
Angew Chem Int Ed Engl 31(6):795–796. doi:10.1002/anie.
199207951
Montecucco C, Rappuoli R (2001) Living dangerously: how Helico-
bacter pylori survives in the human stomach. Nat Rev Mol Cell
Biol 2(6):457–466. doi:10.1038/35073084
Nair PC, Sobhia ME (2008) Quantitative structure activity relation-
ship studies on thiourea analogues as influenza virus neuramin-
idase inhibitors. Eur J Med Chem 43(2):293–299
Newman DJ, Cragg GM, Snader KM (2003) Natural products as
sources of new drugs over the period 1981–2002. J Nat Prod
66(7):1022–1037. doi:10.1021/np030096l
Dixon NE, Gazzola TC, Blakeley RL, Zermer B (1975) Letter: Jack
bean urease (EC 3.5.1.5). A metalloenzyme. A simple biological
role for nickel? J Am Chem Soc 97(14):4131–4133
Drews J (2000) Drug discovery: a historical perspective. Science
287(5460):1960–1964 8361 [pii]
Faidallah HM, Albar HA, Makki MSI, Sharshira EM (2002) Synthesis
of novel cyclic benzenesulfonylurea and thiourea derivatives.
Phosphorus Sulfur Silicon Relat Elem 177(3):685–693. doi:
10.1080/10426500210275
Gould IM (2008) The epidemiology of antibiotic resistance. Int J
Antimicrob Agents 32(Suppl 1):S2–S9
Nitta T, Arai T, Takamatsu H, Inatomi Y, Murata H, Iinuma M,
Tanaka T, Ito T, Asai F, Ibrahim I, Nakanishi T, Watabe K
(2002) Antibacterial activity of extracts prepared from tropical
and subtropical plants on methicillin-resistant Staphylococcus
aureus. J Health Sci 48(3):273–276. doi:10.1248/jhs.48.273
Ramadas K, Suresh G, Janarthanan N, Masilamani
S (1998)
Gribble FM, Reimann F (2003) Differential selectivity of insulin
secretagogues: mechanisms, clinical implications, and drug
interactions. J Diabetes Complications 17(2 Suppl):11–15
Hanif M, Saleem M, Hussain MT, Rama NH, Zaib S, Aslam MAM,
Jones PG, Iqbal J (2012a) Synthesis, urease inhibition, antiox-
idant and antibacterial studies of some 4-amino-5-aryl-3H-1,2,4-
triazole-3-thiones and their 3,6-disubstituted 1,2,4-triazolo[3,4-
b]1,3,4-thiadiazole derivatives. J Braz Chem Soc 23:854–860
Hanif M, Shoaib K, Saleem M, Hasan Rama N, Zaib S, Iqbal J
(2012b) Synthesis, urease inhibition, antioxidant, antibacterial,
and molecular docking studies of 1,3,4-oxadiazole derivatives.
ISRN Pharmacol 2012:928901. doi:10.5402/2012/928901
Klingenstein R, Melnyk P, Leliveld SR, Ryckebusch A, Korth C
(2006) Similar structure-activity relationships of quinoline
derivatives for antiprion and antimalarial effects. J Med Chem
49(17):5300–5308. doi:10.1021/jm0602763
Antifungal activity of 1,3-disubstituted symmetrical and unsym-
metrical thioureas. Pestic Sci 52(2):145–151. doi:10.1002/
(sici)1096-9063(199802)52:2\145:aid-ps677[3.0.co;2-j
Rojas JJ, Ochoa VJ, Ocampo SA, Munoz JF (2006) Screening for
antimicrobial activity of ten medicinal plants used in Colombian
folkloric medicine: a possible alternative in the treatment of non-
nosocomial infections. BMC Complement Altern Med 6:2
Ryckebusch A, Deprez-Poulain R, Debreu-Fontaine MA, Vandaele R,
Mouray E, Grellier P, Sergheraert C (2002) Parallel synthesis
and anti-malarial activity of a sulfonamide library. Bioorg Med
Chem Lett 12(18):2595–2598
Saczewski F, Kuchnio A, Samsel M, Lobocka M, Kiedrowska A,
Lisewska K, Saczewski J, Gdaniec M, Bednarski PJ (2010)
Synthesis of novel aryl(heteroaryl)sulfonyl ureas of possible
biological interest. Molecules 15(3):1113–1126
¨
Saeed A, Erben MF, Florke U (2010) Effect of fluorine substitution on
Koch KR (2001) New chemistry with old ligands: N-alkyl- and N,N-
dialkyl-N0-acyl(aroyl)thioureas in co-ordination, analytical and
process chemistry of the platinum group metals. Coord Chem
Rev 216–217:473–488. doi:10.1016/s0010-8545(01)00337-x
Kontoyiannis DP, Mantadakis E, Samonis G (2003) Systemic
mycoses in the immunocompromised host: an update in
antifungal therapy. J Hosp Infect 53(4):243–258
Krungkrai J, Scozzafava A, Reungprapavut S, Krungkrai SR,
Rattanajak R, Kamchonwongpaisan S, Supuran CT (2005)
Carbonic anhydrase inhibitors. Inhibition of Plasmodium falci-
parum carbonic anhydrase with aromatic sulfonamides: towards
antimalarials with a novel mechanism of action? Bioorg Med
Chem 13(2):483–489
Labute P (2007) Protonate 3D: assignment of macromolecular
protonation state and geometry. Chemical Computing Group
Inc., Montreal
Maren TH (1976) Relatons between structure and biological activity
of sulfonamides. Annu Rev Pharmacol Toxicol 16:309–327. doi:
10.1146/annurev.pa.16.040176.001521
Mobley HL, Hausinger RP (1989) Microbial ureases: significance,
regulation, and molecular characterization. Microbiol Rev 53(1):
85–108
Mobley HL, Island MD, Hausinger RP (1995) Molecular biology of
microbial ureases. Microbiol Rev 59(3):451–480
MOE (2010) The molecular operating environment version 201010.
Chemical Computing Group Inc., Montreal. http://wwwchem
compcom
the crystal structures and vibrational properties of phenylthiourea
isomers. J Mol Struct 982(1–3):91–99. doi:10.1016/j.molstruc.
2010.08.012
Saeed A, Mumtaz A, Ishida H (2011) Synthesis, characterization of
some new 1-aroyl-3-(4-aminosulfonylphenyl)thioureas and crys-
tal structure of 1-(3,4,5-trimethoxybenzoyl)- 3-(4-aminosulfo-
nylphenyl)thiourea. J Sulfur Chem 32(1):45–54. doi:10.1080/
17415993.2010.541461
Sieradzki K, Roberts RB, Haber SW, Tomasz A (1999) The
development of vancomycin resistance in a patient with meth-
icillin-resistant Staphylococcus aureus infection. N Engl J Med
340(7):517–523. doi:10.1056/NEJM199902183400704
Thornber CW (1979) Isosterism and molecular modification in drug
design. Chem Soc Rev 8(4):563–580
Weatherburn MW (1967) Phenol-hypochlorite reaction for determi-
nation of ammonia. Anal Chem 39(8):971–974. doi:10.1021/
ac60252a045
Yonova PA, Stoilkova GM (2004) Synthesis and biological activity of
urea and thiourea derivatives from 2-aminoheterocyclic com-
pounds. J Plant Growth Regul 23(4):280–291. doi:10.1007/
s00344-003-0054-3
Zampini IC, Cuello S, Alberto MR, Ordonez RM, D’Almeida R,
Solorzano E, Isla MI (2009) Antimicrobial activity of selected
plant species from ‘‘the Argentine Puna’’ against sensitive and
multi-resistant bacteria. J Ethnopharmacol 124(3):499–505
123