New aminobenzenesulfonamide-thiourea conjugates: Synthesis and carbonic anhydrase inhibition and docking studies

Article abstract of DOI:10.1016/j.ejmech.2014.03.023

A variety of 1-substituted-3-(3-aminosulfonylphenyl)thioureas (3a-k) and two new 1-aroyl-3-(4-aminosulfonylphenyl)thiourea derivatives (5a and 5b) were synthesized by reaction of 3-aminobenzenesulfonamide and 4- aminobenzenesulfonamide respectively with f

Full text of DOI:10.1016/j.ejmech.2014.03.023

European Journal of Medicinal Chemistry 78 (2014) 140e150
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New aminobenzenesulfonamideethiourea conjugates: Synthesis and
carbonic anhydrase inhibition and docking studies
,
c
c
d
Sumera Zaib a, Aamer Saeed b , Karin Stolte , Ulrich Flörke , Mohammad Shahid ,
*
,e,
Jamshed Iqbal a
*
a Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
b Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
c Department Chemie, Fakultät für Naturwissenschaften, Universität Paderborn, Warburgerstrasse 100, D-33098 Paderborn, Germany
d Department of Bioinformatics, Fraunhofer Institute SCAI, Sankt Augustin, Germany
e Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
A
variety of 1-substituted-3-(3-aminosulfonylphenyl)thioureas (3aek) and two new 1-aroyl-3-
Received 25 December 2013
Received in revised form
16 February 2014
Accepted 8 March 2014
Available online 12 March 2014
(4-aminosulfonylphenyl)thiourea derivatives (5a and 5b) were synthesized by reaction of 3-amino-
benzenesulfonamide and 4-aminobenzenesulfonamide respectively with freshly prepared aroyl/hetero-
aryl isothiocyanates in dry acetonitrile. FTIR, 1H NMR, 13C NMR, GCeMS and elemental analyses data
confirmed the assigned structures to the synthesized compounds. Further structure of compound (3g)
was also confirmed by single crystal XRD analysis. The compounds were investigated as inhibitors of the
bovine erythrocyte carbonic anhydrase isoform II (bCA II). The inhibition constants of these compounds
against bCA II were in the range 0.011e17.1
(3-aminosulfonylphenyl)thiourea derivatives 3h and 5a were the most potent inhibitors with IC50 of 0.052
and 0.011 M, respectively. In silico docking and molecular dynamics simulation studies were performed
Keywords:
mM. Among the evaluated compounds, 1-substituted -3-
1-Aroyl/heteroaryl-3-(3-
aminosulfonylphenyl)thioureas
3-Aminobenzenesulfonamide
Carbonic anhydrase
m
against bCA II and human CA II enzymes to rationalize the inhibitory properties of these compounds.
Molecular dynamics simulation
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
and fluid transport. The result is a reduction in intraocular pressure
(IOP). Acetazolamide is a well-known example of clinically estab-
The zinc metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1)
catalyzes the rapid reversible conversion of carbon dioxide and
water into a proton and the bicarbonate ion [1e3]. Carbonic
anhydrases are involved in crucial physiological processes con-
nected with transport of CO2/bicarbonate, homeostasis, electrolyte
secretion in a variety of tissues/organs, biosynthetic reactions (such
as gluconeogenesis, ureagenesis and lipogenesis), respiration,
calcification, tumorigenicity, and bone resorption [1e7]. Deficiency
of carbonic anhydrase II is the leading defect in the syndrome of
osteoporosis, cerebral calcification and renal tubular acidosis [8].
Inhibition of carbonic anhydrase in the ciliary processes of the eye
decreases aqueous humor secretion, most likely by slowing the
formation of bicarbonate ions with consequent decrease in sodium
lished carbonic anhydrase inhibitor [9]. Sulfonylureas are amongst
the most familiar sulfonamide derivatives possessing potent hy-
poglycemic activity due to their ability to stimulate the release of
insulin from the pancreatic islets. Carbutamide, tolbutamide,
chlorpropamide and tolazamide belong to the first generation of
sulfonylureas while second generation include glyburide and gli-
pizide [10e12]. Sulfonamides are well known for their diuretic [13],
anti-carbonic anhydrase [14] and antimalarial activity [15e17].
Among the broad spectrum of activities exhibited by sulfon-
amides, their role as inhibitors of the zinc containing metal-
loenzyme carbonic anhydrase (CA) is presumably most widely
studied. Many sulfonamide CA inhibitors have been used as ther-
apeutic agents against various diseases including glaucoma, gastro-
duodenal ulcers, acidebase disequilibria, and various neurological
disorders [18e21].
Thus 4-(3,4-dichloro-phenylureido)thioureido-benzene sulfon-
amide possessing a thiourea scaffold is an effective in vitro inhibitor
of carbonic anhydrase; inhibited the ex vivo growth of Plasmodium
falciparum [22]. Several antiplasmodial 7-chloro-4-aminoquinolyl-
* Corresponding author. Centre for Advanced Drug Research, COMSATS Institute
of Information Technology, Abbottabad 22060, Pakistan.
E-mail addresses: aamersaeed@yahoo.com (A. Saeed), drjamshed@ciit.net.pk,
jamshediqb@gmail.com (J. Iqbal).
http://dx.doi.org/10.1016/j.ejmech.2014.03.023
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
141
derived sulfonamides, ureas, thioureas and amides have been
synthesized and tested against chloroquine-resistant (CQR) and
chloroquine susceptible (CQS) P. falciparum [23].
d 8.1e9.1 and 10.8e12.1 respectively. The typical differences in the
aromatic region of 3-amino and 4-aminobenzenesulfonamides
were also noted. Thus, in case of 4-amino compounds (5a,b) two
doublets are observed around 7.81 and 7.90 for aromatic protons,
while 3-aminobenzenesulfonamide derivatives (3aek), display
three to four different signals due to nearly all unequivalent aro-
matic protons.
Previously we have shown sulfanilamideethiourea hybrids to
be effective antimicrobial agents and urease inhibitors [24]. Herein
we report synthesis of isomeric 3-aminobenzenesulfonamide
appended to thiourea nucleus and their comparison with 4-
aminobenzenesulfonamide derivatives. In the present work, we
have expanded this new class of inhibitors by synthesizing novel 1-
substituted-3-(3-aminosulfonylphenyl)thioureas (3aek) and two
new 1-aroyl-3-(4-aminosulfonylphenyl)thiourea derivatives (5a
and 5b) and determined their inhibition activity against bCA II.
Some of the structurally related sulfonamides reported in literature
are presented in Fig. 1.
Fig. 2 shows the molecular structure of 1-(2-furanyl)-3-(3-
aminosulfonylphenyl) thiourea (3g) and Table 1 summarizes the
main geometric parameters. The furanyl-thiourea moiety is almost
planar with a torsion angle O2eC5eC4eC3 of 5.5(3)ꢀ. Thiourea
torsion angles are O2eC5eN1eC6 1.1(3)ꢀand N2eC6eN1eC5
ꢁ1.3(3)ꢀ. An intramolecular N2eH.O2 hydrogen bond is con-
nected with that conformation. The furanyl and phenyl rings make
a dihedral angle of 22.3(1)ꢀ. Other geometric parameters lie in
expected ranges. Somewhat related molecular structures are
MUWCUD [28] or FAQPOE [29], both with the aminosulfonyl group
in para-position and p-tolyl or trimethoxyphenyl instead of the
furanyl group, respectively. The crystal packing of (3g) (Fig. 3)
shows intermolecular N1eH.S1 (ꢁx þ 1, ꢁy, ꢁz) and N3e
H31.O4 (x, ꢁy þ 0.5, zꢁ0.5) interactions that link molecules into
pairs of centrosymmetric dimers that are stacked along [001].
2. Results and discussion
2.1. Chemistry
A variety of aroyl/heteroaryl isothiocyanates (1aek) were pre-
pared in situ by reaction of corresponding acid chlorides with an
equimolar quantity of potassium thiocyanate in dry acetonitrile.
Treatment of isothiocyanates with 3-aminobenzenesulfonamide (2)
in dry acetonitrile in 1:1 M ratio furnished corresponding 1-aroyl/
heteroaryl 3-(3-aminosulfonylphenyl)thioureas (3aek) in 80e91%
yield (Scheme 1). Two new 1-aroyl-3-(4-aminosulfonylphenyl)
thioureas 5a and 5b were similarly synthesized by reaction of
thiophene-2-carbonyl isothiocyanate and coumarin-3-carbonyl
isothiocyanate respectively with 4-aminobenzenesulfonamide (4)
(Scheme 2).
2.2. Carbonic anhydrase inhibition
All synthesized compounds were tested for their ability to act as
carbonic anhydrase (CA) inhibitors against bovine CA II (bCA-II)
isozyme. CA inhibition data for these compounds is given in Table 2.
CA inhibitory activities of these compounds were investigated
against the standard clinically used inhibitor acetazolamide. The
compounds 3ae3c differ only in the relative position of the chloro
group on the 3-(3-aminosulfonylphenyl)thioureas ring, have
FTIR spectra of the aroyl/heteroaryl substituted thioureas 3aek
and 4aeb were in accordance with those reported for similar
compounds [25e27]. In 1H NMR spectra the characteristic singlets
for N1 and N3 protons were found in a relatively wide range of
shown IC50 values 13.5 ꢂ 1.40, 0.22 ꢂ 0.02 and 5.64 ꢂ 0.09
mM,
respectively. Among these chloro group substitution at position 3
Fig. 1. Structures of some pharmacologically important related compounds reported in literature.

142
S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
Scheme 1. Synthetic route to 3-aminobenzenesulfonamideethiourea conjugates (3ae3k).
Scheme 2. 4-aminobenzenesulfonamideethiourea conjugates (5a, 5b).
shown maximum IC50 value. For compound 3e, chloro group was
M was
shown. In case of 3d and 3f where di-choloro and di-nitro sub-
stituents were present, a significant decrease in inhibition was
noticed. As 3d having di-chloro group attached to the 3-(3-
aminosulfonyl-phenyl)thioureas was the least active with IC50
replaced by nitro group and IC50 value of 0.49 ꢂ 0.01
m
value of 17.1 ꢂ1.81
mM. Compound 3f has di-nitro group attached to
3-(3-aminosulfonylphenyl)thioureas possessed IC50 value of
Fig. 2. Molecular structure of 1-(2-furanyl)-3-(3-aminosulfonylphenyl)thiourea (3g). Displacement ellipsoids are drawn at the 50% probability level.

S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
143
Table 1
Selected bond distances (A) and bond angles ( ) for (3g).
Table 2
ꢀ
ꢀ
Bovine carbonic anhydrase II inhibition by compounds (3ae3k, 5ae5b).
S1eC6
1.652(2)
N1eC6eN2
113.8(2)
Compounds
% Inhibition
IC50 ꢂ SEM (
mM)
C6eN1
C6eN2
C5eN1
C5eO2
S2eC11
S2eO3
S2eO4
S2eN3
1.407(2)
1.343(2)
1.375(2)
1.231(2)
1.614(2)
1.436(2)
1.432(2)
1.614(2)
O2eC5eN1
C6eN2eC7
N3eS2eC11
123.8(2)
131.9(2)
108.95(9)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
5a
32.8 ꢂ 0.02a
78.1 ꢂ 0.01a
81.2 ꢂ 0.01b
29.9 ꢂ 0.01b
70.4 ꢂ 0.02a
36.4 ꢂ 0.05a
74.6 ꢂ 0.02b
73.6 ꢂ 0.01b
79.6 ꢂ 0.01b
86.3 ꢂ 0.01b
39.8 ꢂ 0.02b
75.9 ꢂ 0.03a
43.6 ꢂ 0.01a
81.9 ꢂ 0.03b
13.5 ꢂ 1.40
0.22 ꢂ 0.02
5.64 ꢂ 0.09
17.1 ꢂ 1.81
0.49 ꢂ 0.01
14.2 ꢂ 1.22
9.76 ꢂ 0.75
0.052 ꢂ 0.01
5.58 ꢂ 0.69
1.28 ꢂ 0.09
13.4 ꢂ 1.02
0.011 ꢂ 0.001
11.3 ꢂ 1.32
0.96 ꢂ 0.18
N2eH.S1
2.79
164.3
2.19(3)
166(2)
N3eH31.O4
14.2 ꢂ 1.22
mM. In general, compounds containing substituents
5b
either chloro or nitro group at meta position were more active.
Acetazolamide
Compounds 3h and 5a were the most potent inhibitors having IC50
a
0.1 mM.
1 mM.
values of 0.052 ꢂ 0.01 and 0.011 ꢂ 0.001
mM, respectively. Com-
b
pound 3h having substituent attached to ortho position at 3-(3-
aminosulfonylphenyl)thioureas ring possessed IC50 value of
0.052 ꢂ 0.01
position of 3-(4-aminosulfonylphenyl)thioureas ring had IC50 value
of 0.011 ꢂ 0.001 M. In general most of compounds in the series
have comparable IC50 values to that of standard inhibitor
(0.96 ꢂ 0.18 M) used for comparison. However, presence of
additional coumarin ring in 3k and 5b decreased the activity to
13.4 ꢂ 1.02 and 11.3 ꢂ 1.32 M, respectively. Substitution of pivaloyl
group at 3-(3-aminosulfonylphenyl)thioureas (3j) has enhanced
M) com-
M).
mM. whereas 5a having substituent attached to meta
docking and the rank number of best pose obtained with NNScore
algorithm for the compounds screened against both bCA II and
hCA II enzymes. Docking scores of the compounds were not used
to filter the poses of compounds docked into both enzymes;
however, filtering the poses for the compounds by NNScore
showed there could be other conformations that might interact
favorably with the enzymes. The favorable binding modes for most
of the compounds revealed the interaction of the deprotonated
nitrogen of the sulfonamide moiety of the compounds. It was,
therefore, observed that selected pose was not always the first
ranking pose based on docking scores.
m
m
m
the biological activity showing IC50 value (1.28 ꢂ 0.09
m
parable to the standard inhibitor acetazolamide (0.96 ꢂ 0.18
m
2.3. Docking and pose ranking
2.3.1. Binding mode analysis
The results of re-docking experiments showed that the struc-
tures were well prepared and the docking program reproduced the
natively bound conformations of the co-crystallized bound ligand
D7A in both enzyme structures. The NH atom of the sulfa-
moylbenzamide moiety was in coordination with the Zn2þ atom in
the re-docking of both enzymes, which was exactly the same in
the crystal structure of human carbonic anhydrase II (hCA II).
Similarly, the docking results for the compounds 3ae3k, 5ae5b
were obtained for both enzymes, which showed that there were
different conformations (poses) predicted for individual com-
pounds. Table 3 shows the docking scores obtained with FlexX
A common binding mode was observed for the compounds
docked to both enzymes. The selected docked conformations
form the same interactions with the metal atom and the impor-
tant residues in the active catalytic site of bCA II. The deproto-
nated N of sulfonamide moiety coordinates well with the Zn2þ
and the aromatic ring is involved in hydrophobic and aromatic
interactions with His63, His93, His95 and Thr198. The oxygen
atom of the sulfonamide part is also involved in hydrogen bond
interactions with Thr197 and Thr198. This is similar to the
interaction of co-crystalized ligand D7A with Thr199 in hCA II
enzyme. Another frequent interaction observed in most of the
Fig. 3. Crystal packing of 1-(2-furanyl)-3-(3-aminosulfonylphenyl)thiourea (3g) viewed along c-axis showing hydrogen bonding pattern as dotted lines. H-atoms not involved are
omitted.

144
S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
Table 3
equilibration results showed that the complexes were equilibrated
as shown in Fig. 7, and the enzymeeligand complexes can be
further simulated to obtain enough samples for computing free
energy of binding values as well as determine the stability of the
binding modes. Equilibration plots for all enzymeeligand com-
plexes of bCA II and hCA II are shown in Fig. 8.
Docking scores and best pose ranks for the compounds (3ae3k, 5ae5b) screened
against bCA II and hCA II.
Compounds
Docking scores (kcal/mol)
Pose ranks in top 10
bCA II
hCA II
bCA II
hCA II
3a
3b
3c
3d
3e
3f
3h
3i
3j
ꢁ24.82
ꢁ24.25
ꢁ21.12
ꢁ24.18
ꢁ23.40
ꢁ20.53
ꢁ24.45
ꢁ18.73
ꢁ18.67
ꢁ25.30
ꢁ24.21
ꢁ22.82
ꢁ18.57
ꢁ21.03
ꢁ23.57
ꢁ9.75
8
8
7
9
The distance plots shown in Fig. 9 represent the interatomic
distances between the N atom of the sulfonamide part of the
molecules and the Zn2þ atom during the time period of MD
simulation. The distances show that the initial bound conformation
of the compounds in which the N atom coordinates with the Zn2þ
atom remains the same during the simulation, however, for two
compounds 3c and 3f, their distances increases from the Zn2þ atom
in the beginning of MD simulation in the case of bCA II complex.
However, the conformation changes back again after 2 ns simula-
tion. Similarly the distance between these atoms for compound 3f
inside hCA II enzyme also changes after 4 ns of MD simulation.
These findings suggests that for some compounds, the conforma-
tional changes should be observed for a longer simulation time to
find the comparatively favorable conformation that persists longer
during the simulation time.
4
1
2
6
ꢁ30.56
ꢁ22.02
ꢁ18.01
ꢁ17.31
ꢁ21.55
ꢁ24.09
ꢁ27.74
ꢁ20.18
7
1
3
7
10
8
9
3
1
1
3k
5a
5b
9
8
10
5
8
4
docked compounds is the hydrogen bonding interaction of the
two nitrogen atoms in the thiourea moiety of the compounds
which form hydrogen bonds with Gln91 residue as shown in
Fig. 4. The preferred binding modes selected by external scoring
function reveal that most of the compounds interact in a very
similar way in the catalytic site of the enzyme. The correct co-
ordination with the Zn2þ, aromatic interaction with His93 and
hydrogen bond interaction with Thr198 is also similar to the in-
teractions made by the respective native bound ligand of the hCA
II enzyme as shown in Fig. 5. Hydrophobic interactions are also
observed with Val90 and Phe129 situated at the entrance of the
catalytic site of the enzyme. It can be concluded that the pre-
dicted binding poses that are filtered by the external scoring
function are interacting in a similar way and these binding modes
are favored according to the nature of the active site pockets of
both enzymes as indicated in Fig. 6.
2.3.3. Free energy of binding
The free energy of binding values estimated with MM-PBSA
method for all compounds against both bCA II and hCA II en-
zymes are given in Table 4. The estimated
DG values showed that
the most of the compounds interact with a good binding energy
range inside the active catalytic sites of both enzymes. Further-
more, the free energy of binding values also correlate positively
with the experimentally derived
D
G values from IC50
(mM) values.
The observed correlations of estimated
DG and experimental
DG is
0.52 for bCA II and 0.66 for hCA II, respectively. These results show
that MM-PBSA based method to systematically evaluate free energy
of binding for a series of compounds can provide a good estimate of
the relative binding free energies.
2.3.2. Molecular dynamics simulations
Individual molecular dynamics simulations were conducted for
all the selected poses of the compounds against both bCA II and hCA
II enzymes. Each proteineligand complex was parameterized for
the high-ranking pose that was filtered and selected by NNScore.
The pre-equilibration steps (temperature and density) and final
3. Conclusion
Several novel 1-aroyl-3-(3-aminosulfonylphenyl)thiourea de-
rivatives (3aek) and two new 1-aroyl-3-(4-aminosulfonylphenyl)
thioureas (5a and 5b) were synthesized which differ from most of
Fig. 4. Binding modes and interactions of the compounds inside the active site of bCA II. Compounds are shown in CPK model and the imported co-crystal ligand D7A is shown in
ball & sphere model in green color. Blue circles represent the coordination geometry around the Zn2þ atom. Left: Preferred binding modes for the compounds 3ae3k docked into the
active site of bCA II. Right: Predicted conformation of compound 3a inside the active site of bCA II. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
145
Fig. 5. Interaction diagrams of the selected docked conformations for compounds 3a and 3e inside the active site of bCA II enzyme. Hydrogen bond interactions are shown with
dotted lines in red and hydrophobic interactions are drawn with green lines. (Left): Interaction diagram of compound 3a. (Right): Interaction diagram of compound 3e. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Interaction diagrams of the selected docked conformations for compounds 3b and 3d inside the active site of bCA II enzyme. Hydrogen bond interactions are shown with
dotted lines in red and hydrophobic interactions are drawn with green lines. (Left): Interaction diagram of compound 3b. (Right): Interaction diagram of compound 3d. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. Density plots showing density equilibrations for all individual enzymeeligand complexes. (Left) Density plots for all compounds during pre-equilibration step for bCA II
complexes. (Right): Density plots for all compounds during pre-equilibration step for hCA II complexes.

146
S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
Fig. 8. Equilibration plots for all enzymeeligand complexes of bCA II and hCA II. RMSD deviations in backbone atoms show that the complexes start to equilibrate after 2 ns of MD
simulation. (Left): Backbone RMSD plots of compounds simulated with bCA II. (Right): Backbone RMSD plots of compounds simulated with hCA II.
Fig. 9. Plots showing distance measurements between ligand’s N atom of sulfonamide group and Zn2þ atom for all enzymeeligand complexes of bCA II and hCA II. (Left): Distance
plots for compounds simulated with bCA II. (Right): Distance plots of compounds simulated with hCA II.
the known sulfonyl thiourea derivatives in having the anilino ni-
trogen of sulfanilamide being incorporated into the thiourea nu-
cleus leaving the sulfonamide amino group intact. Some of the
compounds investigated exhibited excellent CA inhibitory activity
in the lower micromolar range. Compounds 3h and 5a were potent
CA II inhibitors with IC50 values 0.052 ꢂ 0.01 and 0.011 ꢂ 0.001
mM,
respectively. Further, these finding suggest that novel thioureas
derivatives may be used for further investigation on a wide range of
CA isozymes which are important drug targets in drug
development.
Table 4
Experimental and estimated free energy (DG values) calculated for all compounds simulated with bCA II and hCA II enzymes.
Compounds
Experimental
G (kcal/mol)
Estimated
MM-PBSA
D
G (kcal/mol)
Estimated
MM-GBSA
DG (kcal/mol)
Estimated
MM-PBSA
D
G (kcal/mol)
Estimated
MM-GBSA
DG (kcal/mol)
D
bCA II
bCA II
bCA II
hCA II
hCA II
3a
3b
3c
3d
3e
3f
3h
3i
3j
3k
5a
5b
ꢁ6.58
ꢁ8.99
ꢁ7.09
ꢁ6.44
ꢁ8.52
ꢁ6.55
ꢁ9.86
ꢁ7.09
ꢁ7.09
ꢁ7.96
ꢁ6.68
ꢁ10.80
e
ꢁ8.64
ꢁ15.5
ꢁ50.72
ꢁ56.11
ꢁ56.99
ꢁ52.79
ꢁ51.82
ꢁ58.06
ꢁ54.71
ꢁ55.7
ꢁ9.52
ꢁ17.25
ꢁ13
ꢁ55.09
ꢁ71.32
ꢁ62.28
ꢁ11.45
ꢁ62.61
ꢁ47.21
ꢁ49.42
ꢁ64.97
ꢁ60.64
ꢁ55.41
ꢁ47.29
ꢁ65.94
0.44
ꢁ14.34
ꢁ9.93
ꢁ2.38
ꢁ10.33
ꢁ10.17
ꢁ13.85
ꢁ13.23
ꢁ15.27
ꢁ8.51
ꢁ14.77
ꢁ4.23
ꢁ10.48
ꢁ10.98
ꢁ12.51
ꢁ11.63
ꢁ12.94
ꢁ18.26
0.66
ꢁ48.81
ꢁ52.63
ꢁ62.95
ꢁ62.25
0.26
ꢁ15.81
ꢁ18.96
0.52
Correlation

S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
147
4. Experimental
Found C, 45.51; H, 3.32; Cl, 9.53; N, 11.30; S, 17.29%; EIMS m/z
(%):369 [Mþ], 371, 171 (56), 119 (100), 91 (65).
4.1. Instrumentation
4.2.4. 1-(3,4-Dichlorobenzoyl)-3-(3-aminosulfonylphenyl)
Melting points were recorded using a digital Gallenkamp
(SANYO) model MPD.BM 3.5 apparatus and are uncorrected. 1H
NMR spectra were determined as CDCl3 solutions at 300 MHz using
a Bruker AM-300 spectrophotometer using TMS as an internal
reference and 13C NMR spectra were determined at 75 MHz in
DMSO-d6. FTIR spectra were recorded on an FTS 3000 MX spec-
trophotometer. Mass Spectra (EI, 70 eV) on a MAT 312 instrument,
and elemental analyses were conducted using a LECO-183 CHNS
analyzer.
thiourea (3d)
Yield: (83%); m.p 180 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]O),
1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
;
d
ppm, J Hz): 12.91 (s, 1H, NH), 11.93 (s, 1H, NH), 7.85 (m, 1H, Ar), 7.51
(m, 1H, Ar), 7.41 (m, 3H, Ar, SO2NH2), 7.62 (d, 2H, Ar, J ¼ 6.3), 6.5 (d,
2H, Ar, J ¼ 5.9);13C NMR (
d ppm): 180 (C]S), 173 (C]O), 144.1, 139
(CeN), 136 (CeCO), 133 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar),
126 (Ar), 124 (Ar), 109.4, 108.7. Anal. Calcd. For C14H11Cl2N3O3S2: C,
41.59; H, 2.74; N, 10.39; S, 15.86%; Found: C, 41.62; H, 2.71; N, 10.43,
S, 15.81%; GCeMS m/z: 402.96, 404.96 (M$þ).
4.2. Synthesis of 1-aroyl/heteroaryl-3-(3-aminosulfonylphenyl)
thioureas (3ae3m) and 1-heteroaryl-3-(4-aminosulfonylphenyl)
thioureas (5a, 5b)
4.2.5. 1-(3-Nitrobenzoyl)-3-(3-aminosulfonylphenyl)thiourea (3e)
Yield: (88%); m.p 260 ꢀC; IR (Pure)
y
: 3350 (NH), 1670 (C]O),
1594 (C]C), 1462 (thioamide I), 1326 (CeS), 1257 (thioamide II),
1155, 1072 (thioamide III), 750 (thioamide IV) cmꢁ1 1H NMR (
A solution of suitably substituted aroyl//heteroaryl/acyl chloride
(10 mmol) in dry acetonitrile (50 mL) was added dropwise to a
suspension of potassium thiocyanate (10 mmol) in acetonitrile
(30 mL) and the reaction mixture was refluxed for 30 min to afford
isothiocyanates (1aek). After cooling to room temperature, a so-
lution of 3-aminobenzenesulfonamide (2) (10 mmol) or 4-
aminobenzenesulfonamide (4) (10 mmol) (in case of 5a and 5b)
in dry acetonitrile (10 mL) was added and the resulting mixture
refluxed for 1e3 h. The reaction mixture was then poured into cold
water and the precipitated thioureas (3ae3m and 5a, 5b) were
recrystallized using aqueous ethanol.
;
d
ppm, J Hz): 12.2 (s, 1H, NH), 11 (s, 1H, NH), 7.69 (d, 2H, Ar, J ¼ 6.2),
7.42 (m, 3H, Ar, SO2NH2), 6.54 (d, 2H, Ar, J ¼ 5.2), 6.42 (d, 2H, Ar,
J ¼ 4.9), 6.2 (d, 2H, Ar, J ¼ 5.9); 13C NMR (
d ppm) (75 MHz) d: 179
(C]S), 171 (C]O), 150.8 (CeNO2), 139 (CeS), 136 (CeCO), 133 (Ar),
132 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar), 126 (Ar), 124 (Ar), 122
(Ar); Anal. Calcd. For C14H12N4O5S2: C, 44.20; H, 3.18; N, 14.73; S,
16.86%; Found C, 44.20; H, 3.18; N, 14.73; S, 16.86%; EIMS m/z (%):
380 [Mþ], 214 (1), 171 (56), 119 (100), 91 (65).
4.2.6. 1-(3,5-Dinitrobenzoyl)-3-(3-aminosulfonylphenyl)thiourea
(3f)
4.2.1. 1-(2-Chorobenzoyl)-3-(3-aminosulfonylphenyl)thiourea (3a)
Yield: (88%); m.p 194e196 ꢀC; IR (Pure)
y
: 3350 (NH), 1670 (C]
O), 1594 (C]C), 1462 (thioamide I), 1326 (CeS), 1257 (thioamide II),
1155, 1072 (thioamide III), 750 (thioamide IV) cmꢁ1 1H NMR (
Yield: (87%); m.p. 341 ꢀC; IR (Pure) cmꢁ1: 3293 (NH), 1665 (C]
y
O), 1591 (C]C), 1542 (thioamide I), 1332 (CeS), 1245 (thioamide II),
;
d
1158,1091 (thioamide III), 750 (thioamide IV) 1H NMR (
d
ppm, J Hz):
ppm, J Hz): 12.2 (s, 1H, NH), 11 (s, 1H, NH), 7.69 (d, 2H, Ar, J ¼ 6.2),
12.81 (s, 1H, NH), 11.7 (s, 1H, NH), 7.42 (SO2NH2), 7.9 (d, 2H, Ar,
7.44 (SO2NH2), 6.54 (d, 2H, Ar, J ¼ 5.2), 6.42 (d, 2H, Ar, J ¼ 4.9), 6.2 (d,
J ¼ 6.1), 7.15 (d, 2H, Ar, J ¼ 6.5), 7.62 (d, 2H, Ar, J ¼ 6.3), 6.5 (d, 2H, Ar,
2H, Ar, J ¼ 5.9); 13C NMR (
d ppm) (75 MHz) d: 179 (C]S),171 (C]O),
J ¼ 5.9) 13C NMR (
d ppm) 180 (C]S), 173 (C]O), 157 (CeCl), 139 (Ce
150.8 (CeNO2), 139 (CeS), 136 (CeCO), 133 (Ar), 132 (Ar), 131 (Ar),
N), 136 (CeCO), 133 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar), 126
(Ar), 124 (Ar), 122 (Ar), 121 (Ar). Anal. Calcd. For C14H12ClN3O3S2: C,
45.46; H, 3.27; N, 11.36; S, 17.34%; Found C, 45.39; H, 3.23; N, 11.27;
S, 17.36%; EIMS m/z (%): 369 [Mþ], 171 (56), 119 (100).
129 (Ar), 128 (Ar), 126 (Ar), 124 (Ar), 122 (Ar); Anal. Calcd. For
C
14H12N4O5S2: C, 44.20; H, 3.18; N, 14.73; S, 16.86%; Found C, 44.20;
H, 3.18; N,14.73; S,16.86%; EIMS m/z (%): 380 [Mþ], 214 (1),171 (56),
119 (100), 91 (65).
4.2.2. 1-(3-Chorobenzoyl)-3-(3-aminosulfonylphenyl)thiourea (3b)
4.2.7. 1-(2-Furanylcarbonyl)-3-(3-aminosulfonylphenyl)thiourea
Yield: (87%); m.p. 221e222 ꢀC IR (Pure) cmꢁ1: 3293 (NH), 1665
y
(3g)
Yield: (78%); m.p 200e203 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]
O), 1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
(C]O), 1591 (C]C), 1542 (thioamide I), 1332 (CeS), 1245 (thio-
amide II), 1158, 1091 (thioamide III), 750 (thioamide IV) 1H NMR (
d
ppm, J Hz): 12.81 (s, 1H, NH), 11.7 (s, 1H, NH), 7.9 (d, 2H, Ar, J ¼ 6.1),
7.15 (d, 2H, Ar, J ¼ 6.5), 7.62 (d, 2H, Ar, J ¼ 6.3), 6.5 (d, 2H, Ar, J ¼ 5.9)
;
d
ppm, J Hz): 12.78 (s, 1H, NH), 11.6 (s, 1H, NH), 7.51 (d, 1H, furanyl,
J ¼ 5.1), 7.42 (SO2NH2), 6.7 (d, 1H, furanyl, J ¼ 6.5), 6.67 (d, 1H,
13C NMR (
d ppm) 180 (C]S), 173 (C]O), 157 (CeF), 139 (CeN), 136
furanyl, J ¼ 6.5), 7.62 (d, 2H, Ar, J ¼ 6.3), 6.5 (d, 2H, Ar, J ¼ 5.9); 13
C
(CeCO), 133 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar), 126 (Ar), 124
(Ar), 122 (Ar), 121 (Ar). Anal. Calcd. For C14H12ClN3O3S2: C, 45.46; H,
3.27; N, 11.36; S, 17.34%; Found C, 45.42; H, 3.36; N, 11.29; S, 17.38%;
EIMS m/z (%): 369 [Mþ], 371, 171 (56), 119 (100).
NMR (d ppm): 180 (C]S), 173 (C]O), 144.1 (furanyl), 139 (CeN),
136 (CeCO), 133 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127 (Ar), 126 (Ar),
124 (Ar), 109.4 (furanyl), 108.7 (furanyl); Anal. Calcd. For
C
11H11N3O3S2: C, 44.43; H, 3.73; N, 14.13; S, 21.57%; Found: C, 44.39;
4.2.3. 1-(4-Chorobenzoyl)-3-(3-aminosulfonylphenyl)thiourea (3c)
H, 3.81; N, 14.07; S, 21.64%; GCeMS m/z: 297.02 (M$þ, 100).
Yield: (71%); m.p 221 ꢀC; IR (Pure)
y
: 3287 (NH), 1665 (C]O),
1590 (C]C), 1525 (thioamide I), 1325 (CeS), 1254 (thioamide II),
1158, 1011 (thioamide III), 754 (thioamide IV) cmꢁ1 1H NMR (
4.2.8. 1-(2-Thienylcarbonyl)-3-(3-aminosulfonylphenyl)thiourea
;
d
(3h)
ppm, J Hz): 12.8 (s, 1H, NH), 11.3 (s, 1H, NH), 7.9 (d, 2H, Ar), 7.11 (d,
2H, Ar, J ¼ 5.5), 7.42 (SO2NH2), 7.62 (d, 2H, Ar, J ¼ 5.9), 6.45 (d, 2H,
Yield: (78%); m.p 102 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]O),
1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
d
Ar, J ¼ 4.5); 13C NMR (
d
ppm) (75 MHz)
d
: 179 (C]S),171 (C]O),139
;
(CeS),138.1 (CeCl),136 (CeCO),133 (Ar),131 (Ar),129 (Ar),128 (Ar),
ppm, J Hz): 11.8 (s, 1H, NH), 9.6 (s, 1H, NH), 7.51 (d, 1H, thiophenyl,
J ¼ 5.3), 7.42 (SO2NH2), 6.73 (d, 1H, thiophenyl, J ¼ 6.51), 6.67 (d, 1H,
thiophenyl, J ¼ 6.5), 7.62 (d, 2H, Ar, J ¼ 6.3), 6.51 (d, 2H, Ar, J ¼ 5.9);
127 (Ar), 126 (Ar), 124 (Ar), 122 (Ar), 121 (Ar); Anal. Calcd. For
C
14H12ClN3O3S2: C, 45.46; H, 3.27; Cl, 9.59; N, 11.36; S, 17.34%;

148
S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
13C NMR (
d
ppm): 180 (C]S), 173 (C]O), 144.1 (thiophenyl),
J ¼ 6.3), 6.51 (d, 2H, Ar, J ¼ 5.9);13C NMR (
d ppm): 180.7 (C]S), 171.4
139 (CeN), 136 (CeCO), 133 (Ar), 131 (Ar), 129 (Ar), 128 (Ar), 127
(Ar), 126 (Ar), 124 (Ar), 107.9 (thiophenyl), 108.2 (thiophenyl); Anal.
Calcd. For
(C]O), 144.1 (thiophenyl), 139 (CeN), 137.3, 136.4, 133 (Ar), 131
(Ar), 129 (Ar), 128 (Ar). Anal. Calcd. For C12H11N3O3S3: C, 42.22; H,
3.25; N, 12.31; S, 28.17; Found: C, 42.19; H, 3.21%; N, 12.36; S,
28.21%; GCeMS m/z: 342.4 (M$þ).
C12H11N3O3S3: C, 42.22; H, 3.25; N, 12.31; S,
28.17%; Found: C, 42.22; H, 3.25; N, 12.31; S, 28.17%; GCeMS m/z:
342 (M$þ, 13).
4.3. Crystallographic study of 1-(2-furanyl)-3-(3-
4.2.9. 1-(1-Adamantylcarbonyl)-3-(3-aminosulfonylphenyl)
aminosulfonylphenyl)thiourea (3g)
thiourea (3i)
Yield: (78%); m.p 197e198 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]
O), 1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
Colorless crystal, size 0.40 ꢃ 0.08 ꢃ 0.06 mm3, monoclinic space
group P21/c with Z ¼ 4, a ¼ 12.8063(10), b ¼ 14.3698(11),
3
ꢀ
103.271(29ꢀ),
V
¼
1351.90(18) A ;
ꢀ
;
d
c
¼
7.5479(6) A,
b
¼
ppm, J Hz): 12.71 (br s, 1H, NH, D2O exchangeable); 7.63 (br s, 1H,
NH, D2O exchangeable); 7.23e7.33 (m, 2H, Ar); 7.38e7.43 (m, 2H,
Ar), 7.39 (SO2NH2), 8.40e8.48 (m, 1H, Ar); 2.08 (s, 3H, adamantane-
CH), 1.69 (s, 6H, adamantane-CH2), 1.58 (q, 6H, adamantane-CH2,
J ¼ 8.6 Hz); 13C NMR (75 MHz, CDCl3): 179.6 (C]S); 170.12 (C]O);
143.05 (C-9); 41.51, 39.25, 38.69, 38.49, 36.44, 36.14, 28.05, 27.86,
27.78, (adamantane-C); Anal. Calcd for C18H23N3O3S2: C, 54.94; H,
5.89; N, 10.68; S, 16.29%; Found: C, 54.94; H, 5.89; N, 10.68; S,
16.29%. GCeMS m/z: 393.2 (M$þ, 54).
Dc ¼ 1.599 Mg/m3,
m
¼ 0.414 mmꢁ1, F(000) ¼ 672. The intensity
data were recorded using a Bruker SMART CCD area-detector
diffractometer with graphite monochromated MoKa radiation
ꢀ
(
l
¼ 0.71073 A) at T ¼ 130(2) K. 12,615 reflections collected
1.6 >
Q s(I). Structure
> 27.9ꢀ; 3218 independent reflections I > 2
solution by direct method [29], full-matrix least squares refinement
[29]based on F2 and 198 parameters. All but H-atoms were refined
anisotropical, hydrogen atoms were clearly located from difference
Fourier maps, refined at idealized positions riding on the carbon or
nitrogen atoms with isotropic displacement parameters
ꢀ
ꢀ
4.2.10. N-(3-Sulfamoylphenylcarbamothioyl)pivalamide (3j)
Uiso(H) ¼ 1.2Ueq(C/N) and CeH 0.95 A, NeH 0.88 A; H(N3) posi-
tions were refined freely. Refinement converged at R1 ¼ 0.039
Yield: (83%): m.p 109 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]O),
1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
[I > 2
s
(I)], wR2 ¼ 0.099 [all data] and S ¼ 1.032; min./max.
DF
3
ꢀ
;
d
ꢁ0.37/0.45 e/A .
ppm, J Hz): 12.91 (s, 1H, NH), 11.93 (s, 1H, NH), 8.13 (1H, brs), 7.59
Crystallographic data (excluding structure factors) for the
structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publi-
cation no. CCDC-954814. Copies of available material can be ob-
tained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by
e-mailing data_request@ccdc.cam.ac.uk, or contacting the Cam-
bridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: þ44 1223 336033.
(1H, t J ¼ 7.8 Hz), 7.68 (1H, d, J ¼ 7.5 Hz), 7.81 (1H, d, J ¼ 7.5 Hz), 8.13
(1H, brs) 7.42 (SO2NH2), 1.26 (s, 3H, Bu); 13C NMR (
d
ppm): 185.5
t
(C]S), 184.9 (C]O), 126.79, 126.81, 126.84, 133.0, 134.6, 143.6,
149.6, 44.67 (merged with DMSO-d6 signal). 31.5; Anal. Calcd. For
C
12H17N3O3S2: C, 45.70; H, 5.43; N, 13.32; S, 20.33%; Found: C,
45.67; H, 5.45; N, 13.29; S, 20.28%; GCeMS m/z: 315.0 (M$þ).
4.2.11. 1-(-Oxo-2H-chromene-3-carbonyl)-3-(3-
aminosulfonylphenyl)thiourea (3k)
4.4. In vitro carbonic anhydrase inhibition
Yield: (68%); m.p. 192e196 ꢀC; IR (pure, cmꢁ1): 3250 (NH), 1725,
1685 (2C]O), 1558 (C]C), 1253 (C]S); 1H NMR (300 MHz):
d
12.20
Carbonic anhydrase inhibition was measured by the method
[30] after standardization of reaction conditions like enzyme conc.,
substrate conc., buffer pH, duration of reaction etc. The method is
based on the principle that p-nitrophenyl acetate is hydrolyzed by
CA to form p-nitrophenol which was determined spectrophoto-
(s, 1H, NH), 11.84 (s, 1H, NH), 10.75 (s, 1H, CeH, H-4), 7.70 (d, 1H,
J ¼ 7.5 Hz, H-8), 7.43e7.37 (m, 4H, H-7, H-6, SO2NH2), 6.89 (d, 1H,
J ¼ 7.8 Hz, H-5); 13C NMR (75.5 MHz, DMSO):
d 187.0, 168.3, 159.5,
150.2, 138.6, 128.4, 126.6, Anal. Calcd. For C17H13N3O5S2: C, 50.61; H,
3.25; N, 10.42; S, 15.89%; Found: C, 50.57; H, 3.21; N, 10.40; S,
15.92%; GCeMS m/z: 403.1 (M$þ).
metrically. Reaction mixture contained 60
buffer (pH 7.6 containing 0.1 mM ZnCl2), 10
compound in 1% DMSO and 10
m
L of 50 mM Trisesulfate
L (0.5 mM) test
L (50 U) bovine enzyme per well.
m
m
4.2.12. 1-(-Oxo-2H-chromene-3-carbonyl)-3-(4-
aminosulfonylphenyl)thiourea (5a)
Contents were mixed and preincubated at 25 ꢀC for 10 min. Plates
were pre-read at 348 nm using a 96 well plate reader. Substrate, p-
nitrophenyl acetate was prepared (6 mM stock using <5% aceto-
Yield: (68%); m.p. 206 ꢀC; IR (pure, cmꢁ1): 3250 (NH), 1725, 1685
(2C]O), 1558 (C]C), 1253 (C]S); 1H NMR (300 MHz):
d
12.20 (s,
nitrile in buffer and used fresh) and 20
achieve 0.6 mM concentration per well. Total reaction volume was
made to 100
L. After30 min incubation at 25 ꢀC, contents were
mL was added per well to
1H, NH), 11.84 (s, 1H, NH), 10.75 (s, 1H, CeH, H-4), 7.70 (d, 1H,
J ¼ 7.5 Hz, H-8), 7.81 (d, 2H, Ar, J ¼ 6.5), 7.89 (d, 2H, Ar, J ¼ 6.1), 6.89
m
(d, 1H, J ¼ 7.8 Hz, H-5); 13C NMR (75.5 MHz, DMSO):
d
187.0, 168.3,
mixed and read at 348 nm. Suitable controls with DMSO and
known inhibitor acetazolamide were included in the assay. Results
reported are mean of three independent experiments (ꢂSEM) and
expressed as percent inhibitions calculated by the formula, Inhi-
bition (%) ¼ [100ꢁ(Abs of test comp/Abs of control) ꢃ 100]. IC50
values of selected compounds exhibiting >50% activity at 0.5 mM
were calculated after suitable dilutions.
159.5, 150.2, 142.6, 129.4, 126.6, 122.0, 126.6, 119.2, 118.20, 115.20
Anal. Calcd. For C17H13N3O5S2: C, 50.61; H, 3.25; N, 10.42; S, 15.89%;
Found: C, 50.54; H, 3.19; N, 10.39; S, 15.92%; GCeMS m/z: 403.6
(M$þ).
4.2.13. 1-(2-Thienylcarbonyl)-3-(4-aminosulfonylphenyl)thiourea
(5b)
Yield: (78%); m.p 225 ꢀC; IR (Pure)
y
: 3245 (NH), 1660 (C]O),
1590 (C]S), 1531 (thioamide I), 1330 (CeS), 1234 (thioamide II),
1151, 1097 (thioamide III), 747 (thioamide IV) cmꢁ1 1H NMR (
4.5. Molecular modeling studies of carbonic anhydrase
;
d
4.5.1. Structure selection and preparation
ppm, J Hz): 11.8 (s, 1H, NH), 9.81 (s, 1H, NH), 7.51 (d, 1H, thiophenyl,
J ¼ 5.3), 7.97 (d, 2H, Ar, J ¼ 6.1), 7.15 (d, 2H, Ar, J ¼ 6.5),6.73 (d, 1H,
thiophenyl, J ¼ 6.51), 6.67 (d, 1H, thiophenyl, J ¼ 6.5), 7.62 (d, 2H, Ar,
Molecular modeling and docking studies were carried out on
bovine CA-II (bCA II) and human CA-II (hCA II) enzymes. In order to
carry out efficient and accurate modeling studies, high-resolution

S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
149
3D structures of both enzymes were selected from the Protein
DataBank [31]. A high-resolution crystal structure of 1.95 A (PDB
calculated by the Antechamber module of Amber and the param-
eters for the ligands were adapted from the general amber force
field (GAFF) library. The docked enzymeeligand complexes were
solvated using the LeaP module of Amber by using an octahedron
ꢀ
Code: 1V9E) was selected for bCA II and a high-resolution crystal
ꢀ
structure of 1.03 A (PDB Code: 3V7X) was selected for hCA II. The
ꢀ
hCA II x-ray structure contains a co-crystalized ligand N-[2-(3,4-
dimethoxyphenyl)ethyl]-4-sulfamoylbenzamide (PDB Code: D7A)
in its active site. As the structure of bCA II enzyme does not include
a co-crystallized ligand, the co-crystalized ligand of hCA II is used
for binding site definition and interaction studies.
box of the TIP3P water model with a distance of 8 A between the
edge of the box and the protein. Using counter sodium ions to
neutralize the negatively charged enzymeeligand complexes
neutralized the solvated complexes but most of the enzymeeligand
complexes were already in neutral charge form. This is because
the þ1 charge of the enzyme was neutralized by the addition of
negatively charge ligand molecule. Each individual enzymeeligand
solvated complex system was minimized with 2500 steps of con-
jugate gradient minimization followed by 2500 steps of steepest
decent minimization.
The enzyme structure was protonated with Protonate3D [32]
algorithm implemented in MOE [33]. Protonate3D correctly iden-
tified and assigned the protonation states for important residues in
the active site and the ligand atoms. Correct charges for the zinc
metal (Zn2þ), the correct protonation states were assigned for the
surrounding histidine residues (His94, His96 and His119) and the
nitrogen atom in the sulfamoylbenzamide moiety of D7A ligand.
After adding hydrogen atoms and determination of protonation
states, the enzyme structures were energy minimized. Gradient-
based energy minimization with Amber99 force field was per-
formed with all the heteroatoms and solvent molecules present in
the 3D structures and the minimization was allowed to terminate if
the root mean square gradient falls below 0.1. The backbone atoms
were restrained with a small force constant of 100 in order to avoid
collapse of the binding pockets during energy minimization
calculations.
The equilibration steps included 50 ps of heating (from 0 to
300 K temperature), 50 ps of density equilibration with weak re-
straints on the complex atoms followed by 3 ns of constant pressure
equilibration at 300 K. The equilibration process was carried out
using Sander program of Amber12 package. Molecular dynamics
simulations were performed by using Particle Mesh Ewald (PME)
MD program of Amber12 package on the equilibrated complexes.
With a step size of 2 fs, the simulations were per-formed for 2 ns
time scale and atomic coordinates after each 500 step were saved.
The SHAKE algorithm was applied to constrain bonds involving
ꢀ
hydrogen atoms with 8 A van der Waals cut off using the PME
method.
4.5.2. Compounds preparation
The 3D structural coordinates of the compounds were generated
for all the compounds using MOE followed by assignment of pro-
tonation and ionization states in physiological pH range by using
the ‘‘wash” module. Then compounds’ structures were energy
minimized with the MMFF94x force field for the docking studies
and structures were prepared by using utility programs present in
AmberTools [34] to derive AM1-BCC charges and force-field related
parameters for the individual compounds.
4.5.6. Free energy of binding
The binding free energies were estimated by using Molecular
Mechanics/PoissoneBoltzmann Surface Area (MM/PBSA) and Mo-
lecular Mechanics/Generalized Born Surface Area (MM/GBSA) cal-
culations. MM/PBSA and MM/GBSA based energies were calculated
for the snapshots extracted from the 2 ns trajectories that included
2000 snapshots recorded at 1 ps time interval. The interaction
energies and salvation free energies were computed for the com-
plex, enzyme and ligand at each 5th frame (snapshot) in the tra-
jectory to obtain statistically meaningful results. Experimental IC50
4.5.3. Docking studies
The binding sites of both enzymes were defined by including
values were transformed to
DG by the formula:
D
G ¼ ꢁRT ln Ki
ꢀ
residues surrounding around 7.5A radius of the co-crystallized
Where R is gas constant, T is temperature in Kelvin (300 K) and
Ki (in nM) is the experimental affinity value. A comparison of
estimated binding energy values with that of experimentally
bound ligands for docking studies. The same co-crystallized
ligand (D7A) was used in both enzyme structures for determining
the active site. Docking was performed by LeadIT [35] program that
utilizes FlexX for the docking calculations. The default docking and
scoring parameters were chosen for the docking calculations and
the top 10 docked conformations were retained for analyzing the
interactions and binding modes.
determined
DG values for the compounds was carried out to
observe correlation between the predicted and experimentally
determined binding energy values.
Acknowledgment
4.5.4. Pose ranking
This work was financially supported by COMSTECHeTWAS and
German-Pakistani Research Collaboration Programme.
The predicted docked poses of each compound were evaluated
for interactions with the enzyme to select the most favorable
binding mode of a compound. The evaluation was performed by
using re-scoring of the docked poses by an external scoring func-
tion, NNScore. Sets of top ranking poses were retained for all
compounds against both enzymes, which were further evaluated
for stability and binding energy estimation with molecular dy-
namics simulations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.023.
References
4.5.5. Molecular dynamics simulations
[1] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for in-
hibitors and activators, Nature Reviews Drug Discovery 7 (2008) 168e181.
[2] C. Temperini, A. Innocenti, A. Scozzafava, S. Parkkila, C.T. Supuran, The
coumarin-binding site in carbonic anhydrase accommodates structurally
diverse inhibitors: the antiepileptic lacosamide as an example and lead
molecule for novel classes of carbonic anhydrase inhibitors, Journal of Me-
dicinal Chemistry 53 (2009) 850e854.
Molecular dynamics (MD) simulations were performed on the
ligand-docked structures of bCA II and hCA II complexes. The force
field charges for the protein structures were calculated by using
Amber [36] force field ff99SB. The force field charges and param-
eters for the zinc metal complex were derived from ZAFF (Zinc
Amber
Force
Field)
[http://pubs.acs.org/doi/abs/10.1021/
[3] F. Mincione, A. Scozzafava, C.T. Supuran, Antiglaucoma Carbonic Anhydrase
Inhibitors as Ophthalomologic Drugs, Wiley, Hoboken (NJ), 2009.
ct1002626]. AM1-BCC charges were used for the ligands

150
S. Zaib et al. / European Journal of Medicinal Chemistry 78 (2014) 140e150
[4] A. Thiry, J.M. Dogne, B. Masereel, C.T. Supuran, Targeting tumor-associated
[19] I. Nishimori, T. Minakuchi, T. Kohsaki, S. Onishi, H. Takeuchi, D. Vullo,
A. Scozzafava, C.T. Supuran, Carbonic anhydrase inhibitors: the beta-carbonic
anhydrase from Helicobacter pylori is a new target for sulfonamide and sul-
famate inhibitors, Bioorganic & Medicinal Chemistry Letters 17 (2007) 3585e
3594.
[20] M.A. Ilies, C.T. Supuran, A. Scozzafava, Carbonic anhydrase inhibitors. Part 91.
Metal complexes of heterocyclic sulfonamides as potential pharmacological
agents in the treatment of gastric acid secretion imbalances, Metal-Based
Drugs 7 (2000) 57e62.
[21] M. Zareef, R. Iqbal, N.G. De Dominguez, J. Rodrigues, J.H. Zaidi, M. Arfan,
C.T. Supuran, Synthesis and antimalarial activity of novel chiral and achiral
benzenesulfonamides bearing 1, 3, 4-oxadiazole moieties, Journal of Enzyme
Inhibition and Medicinal Chemistry 22 (2007) 301e308.
[22] J. Krungkrai, S.R. Krungkrai, C.T. Supuran, Carbonic anhydrase inhibitors: in-
hibition of plasmodium falciparum carbonic anhydrase with aromatic/het-
erocyclic sulfonamides-in vitro and in vivo studies, Bioorganic & Medicinal
Chemistry Letters 18 (2008) 5466e5471.
[23] J. Botet, L. Mateos, J.L. Revuelta, M.A. Santos, A chemogenomic screening of
sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers
ABZ2, the gene encoding a fungal aminodeoxychorismate lyase, Eukaryotic
Cell 6 (2007) 2102e2111.
[24] A. Saeed, S. Zaib, A. Pervez, A. Mumtaz, M. Shahid, J. Iqbal, Synthesis, molec-
ular docking studies, and in vitro screening of sulfanilamide-thiourea hybrids
as antimicrobial and urease inhibitors, Medicinal Chemistry Research 22
(2013) 3653e3662.
[25] A. Saeed, M.F. Erben, U. Flörke, Effect of fluorine substitution on the crystal
structures and vibrational properties of phenylthiourea isomers, Journal of
Molecular Structure 982 (2010) 91e99.
[26] H.M. Faidallah, H.A. Albar, M.S. Makki, E. Sharshira, Synthesis of novel cyclic
benzenesulfonylurea and thiourea derivatives, Phosphorus, Sulfur, and Silicon
and the Related Elements 177 (2002) 685e693.
carbonic anhydrase IX in cancer therapy, Trends in Pharmacological Sci-
ences 27 (2006) 566e573.
[5] P. Ebbesen, E.O. Pettersen, T.A. Gorr, G. Jobst, K. Williams, J. Kieninger,
R.H. Wenger, S. Pastorekova, L. Dubois, P. Lambin, B.G. Wouters, T. Van Den
Beucken, C.T. Supuran, L. Poellinger, P. Ratcliffe, A. Kanopka, A. Gorlach,
M. Gasmann, A.L. Harris, P. Maxwell, A. Scozzafava, Taking advantage of tumor
cell adaptations to hypoxia for developing new tumor markers and treatment
strategies, Journal of Enzyme Inhibition and Medicinal Chemistry 24 (Suppl. 1)
(2009) 1e39.
[6] M. Hilvo, A.M. Salzano, A. Innocenti, M.S. Kulomaa, A. Scozzafava, A. Scaloni,
S. Parkkila, C.T. Supuran, Cloning, expression, post-translational modifications
and inhibition studies on the latest mammalian carbonic anhydrase isoform,
CA XV, Journal of Medicinal Chemistry 52 (2009) 646e654.
[7] V. Alterio, R.M. Vitale, S.M. Monti, C. Pedone, A. Scozzafava, A. Cecchi, G. De
Simone, C.T. Supuran, Carbonic anhydrase inhibitors: X-ray and molecular
modeling study for the interaction of a fluorescent antitumor sulfonamide
with isozyme II and IX, Journal of the American Chemical Society 128 (2006)
8329e8335.
[8] A.A. Sh Ali, S.A. Al-Mashta, Cerebral calcification, osteopetrosis and renal
tubular acidosis: is it carbonic anhydrase-II deficiency? Saudi Journal of Kid-
ney Diseases and Transplantation: An Official Publication of the Saudi Center
for Organ Transplantation, Saudi Arabia 24 (2013) 561e565.
[9] B.S. Avvaru, S.A. Busby, M.J. Chalmers, P.R. Griffin, B. Venkatakrishnan,
M. Agbandje-McKenna, D.N. Silverman, R. McKenna, Apo-human carbonic
anhydrase II revisited: implications of the loss of a metal in protein structure,
stability, and solvent network, Biochemistry 48 (2009) 7365e7372.
[10] F.M. Gribble, F. Reimann, Differential selectivity of insulin secretagogues:
mechanisms, clinical implications, and drug interactions, Journal of Diabetes
and its Complications 17 (2003) 11e15.
[11] J. Drews, Drug discovery: a historical perspective, The New York Academy of
Sciences 287 (2000) 1960e1964.
[27] A. Saeed, A. Mumtaz, U. Florke, Synthesis, characterization and crystal struc-
ture of 1-(4-methylbenzoyl)-3-(4 aminosulfonylphenyl) thiourea, European
Journal of Chemistry 1 (2010) 73e75.
[28] A. Saeed, A. Mumtaz, H. Ishida, Synthesis, characterization of some new 1-
aroyl-3-(4-aminosulfonylphenyl) thioureas and crystal structure of 1-(3, 4,
5-trimethoxybenzoyl)-3-(4-aminosulfonylphenyl) thiourea, Journal of Sulfur
Chemistry 32 (2011) 45e54.
[12] A. Mastrolorenzo, A. Scozzafava, C.T. Supuran, Antifungal activity of silver and
zinc complexes of sulfadrug derivatives incorporating arylsulfonylureido
moieties, European Federation for Pharmaceutical Sciences: Official Journal of
the European Federation for Pharmaceutical Sciences 11 (2000) 99e107.
[13] T.H. Maren, Relatons between structure and biological activity of sulfon-
amides, Annual Review of Pharmacology and Toxicology 16 (1976) 309e327.
[14] A. Ryckebusch, R. Deprez-Poulain, M.A. Debreu-Fontaine, R. Vandaele,
E. Mouray, P. Grellier, C. Sergheraert, Parallel synthesis and anti-malarial ac-
tivity of a sulfonamide library, Bioorganic & Medicinal Chemistry Letters 12
(2002) 2595e2598.
[15] J. Krungkrai, A. Scozzafava, S. Reungprapavut, S.R. Krungkrai, R. Rattanajak,
S. Kamchonwongpaisan, C.T. Supuran, Carbonic anhydrase inhibitors. Inhibi-
tion of plasmodium falciparum carbonic anhydrase with aromatic sulfon-
amides: towards antimalarials with a novel mechanism of action? Bioorganic
& Medicinal Chemistry 13 (2005) 483e489.
[16] R. Klingenstein, P. Melnyk, S.R. Leliveld, A. Ryckebusch, C. Korth, Similar
structure-activity relationships of quinoline derivatives for antiprion and
antimalarial effects, Journal of Medicinal Chemistry 49 (2006) 5300e5308.
[17] K. Ekoue-Kovi, K. Yearick, D.P. Iwaniuk, J.K. Natarajan, J. Alumasa, A.C. de Dios,
P.D. Roepe, C. Wolf, Synthesis and antimalarial activity of new 4-amino-7-
chloroquinolyl amides, sulfonamides, ureas and thioureas, Bioorganic & Me-
dicinal Chemistry 17 (2009) 270e283.
[18] M.A. Santos, S. Marques, D. Vullo, A. Innocenti, A. Scozzafava, C.T. Supuran,
Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-associated iso-
forms I, II, and IX with iminodiacetic carboxylates/hydroxamates also incor-
porating benzenesulfonamide moieties, Bioorganic & Medicinal Chemistry
Letters 17 (2007) 1538e1543.
[29] G.M. Sheldrick, A short history of SHELX, Acta Crystallogr. Sect A, Foundations
of Crystallography 64 (2008) 112e122.
[30] M. al-Rashida, M. Ashraf, B. Hussain, S.A. Nagra, G. Abbas, Discovery of new
chromone containing sulfonamides as potent inhibitors of bovine cytosolic
carbonic anhydrase, Bioorganic & Medicinal Chemistry 19 (2011) 3367e3371.
[31] D.S. Goodsell, Computational docking of biomolecular complexes with Auto-
Dock, Cold Spring Harbor Protocols 2009 (2009) pdb.prot5200.
[32] P. Labute, Protonate 3D, 2007. http://www.chemcomp.com/journal/proton.htm.
[33] MOE (The Molecular Operating Environment) Version 2010.10, Chemical
Computing Group Inc. http://www.chemcomp.com.
[34] D.A. Case, T.E. Cheatham 3rd, T. Darden, H. Gohlke, R. Luo, K.M. Merz Jr.,
A. Onufriev, C. Simmerling, B. Wang, R.J. Woods, The Amber biomolecular
simulation programs, Journal of Computational Chemistry 26 (2005) 1668e
1688.
[35] http://www.biosolveit.de/LeadIT/.
[36] D.A. Case, T. Darden, T.E. Cheatham III, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, M. Crowley, R.C. Walker, W. Zhang, K.M. Merz, B. Wang, S. Hayik,
A. Roitberg, G. Seabra, I. Kolossvary, K.F. Wong, F. Paesani, J. Vanicek, X. Wu,
S.R. Brozell, T. Steinbrecher, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak,
G. Cui, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, P.A. Kollman, AMBER11,
University of California, San Francisco, 2010.