New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis, in vitro biological evaluation, and molecular docking studies

Article abstract of DOI:10.1016/j.bmc.2020.115329

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed K_i of 0.04 μM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn²⁺ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.

Full text of DOI:10.1016/j.bmc.2020.115329

Journal Pre-proofs
New Thiopyrimidine-Benzenesulfonamide Conjugates as Selective Carbonic
Anhydrase II Inhibitors: Synthesis, in vitro Biological Evaluation, and Molec-
ular Docking Studies
Heba T. Abdel-Mohsen, Ahmed M. El Kerdawy, Mohamed A. Omar,
Emanuela Berrino, Ahmed S. Abdelsamie, Hoda I. El Diwani, Claudiu T.
Supuran
PII:
S0968-0896(19)31716-X
https://doi.org/10.1016/j.bmc.2020.115329
BMC 115329
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 October 2019
4 December 2019
14 January 2020
Please cite this article as: H.T. Abdel-Mohsen, A.M. El Kerdawy, M.A. Omar, E. Berrino, A.S. Abdelsamie, H.I.
El Diwani, C.T. Supuran, New Thiopyrimidine-Benzenesulfonamide Conjugates as Selective Carbonic
Anhydrase II Inhibitors: Synthesis, in vitro Biological Evaluation, and Molecular Docking Studies, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115329
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New Thiopyrimidine-Benzenesulfonamide
Conjugates as Selective Carbonic Anhydrase
II Inhibitors: Synthesis, in vitro Biological
Evaluation, and Molecular Docking Studies
Leave this area blank for abstract info.
Heba T. Abdel-Mohsen*a, Ahmed M. El Kerdawy b,c, Mohamed A. Omara, Emanuela Berrinod, Ahmed S.
Abdelsamiea,e, Hoda I. El Diwania, Claudiu T. Supuran*d
aDepartment of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries
Research, National Research Centre, El‐Buhouth St., Dokki, P.O. Box 12622, Cairo, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo,
P.O. Box 11562, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, New Giza, km 22 Cairo–
Alexandria Desert Road, Cairo, Egypt
dUniversità degli Studi di Firenze, Department NEUROFARBA – Pharmaceutical and Nutraceutical section,
University of Firenze, via Ugo Schiff 6, I-50019, Sesto Fiorentino, Firenze, Italy.
eHelmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research
(HZI), Department of Drug Design and Optimization, Campus Building E8.1, 66123, Saarbrücken, Germany.
2

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
New Thiopyrimidine-Benzenesulfonamide Conjugates as Selective Carbonic
Anhydrase II Inhibitors: Synthesis, in vitro Biological Evaluation, and Molecular
Docking Studies
Heba T. Abdel-Mohsen*a, Ahmed M. El Kerdawy b,c, Mohamed A. Omara, Emanuela Berrinod, Ahmed S.
Abdelsamiea,e, Hoda I. El Diwania and Claudiu T. Supuran*d
aDepartment of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries Research, National Research Centre, El‐Buhouth
St., Dokki, P.O. Box 12622, Cairo, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, New Giza, km 22 Cairo–Alexandria Desert Road, Cairo, Egypt
dUniversità degli Studi di Firenze, Department NEUROFARBA – Pharmaceutical and Nutraceutical section, University of Firenze, via Ugo Schiff 6, I-50019, Sesto
Fiorentino, Firenze, Italy.
eHelmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI), Department of Drug Design and Optimization,
Campus Building E8.1, 66123, Saarbrücken, Germany.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed,
synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was
based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group
(ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with
various electronic and hydrophobic environments as a tail. The designed and synthesized
compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA
XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and
hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed
Ki of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I,
hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising
inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site
Zn2+ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through
hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with
the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively.
These results indicated that the designed and synthesized series is an interesting scaffold that can
be further optimized for the development of selective antiglaucoma drugs.
Available online
Keywords:
Synthesis
Thiopyrimidine-Benzenesulfonamide Conjugates
Carbonic anhydrase
Molecular Docking
2009 Elsevier Ltd. All rights reserved.
1. Introduction
I, CA II, CA III and CA XIII isoforms are present in the cytosol;
while CA IV, CA IX, CA XII, CA XIV and CA XV are
Carbonic anhydrases (CAs, EC 4.2.1.1) are a class of
metalloenzymes that use zinc as a metal cofactor to catalyze the
reversible inter-conversion of carbon dioxide and bicarbonate ion
[1,2]. To date more than sixteen human carbonic anhydrase (hCA)
isoforms have been discovered [3,4]. They have different
molecular features, organs and tissues distribution, expression
levels as well as different kinetic properties [5]. For instance, CA

Corresponding author. ht.abdel-mohsen@nrc.sci.eg and
hebabdelmohsen@gmail.com (Heba T. Abdel‐Mohsen);
claudiu.supuran@unifi.it (Claudiu T. Supuran)
membrane bound. Besides, CA VA and CA VB are present in the
mitochondria [3-5]. hCAs are involved in different physiological
as well as pathological processes [6-8]. Thus, they are considered
a well-established therapeutic target for treatment of a wide range
of pathological disorders [9-11]. For instance, CA II contributes in
the regulation of the bicarbonate concentration in the eyes; hence,
its inhibition is regarded as a successful strategy for the intraocular
3

pressure reduction in glaucoma [12-14]. On the other hand, the
extracellular isoforms CA IX and CA XII are strongly upregulated
in different cancers such as breast, renal, colorectal, and non-small
cell lung cancer in response to hypoxia. They play an outstanding
role in controlling the cellular pH system that protects the tumor
cells from the hypoxia-induced acidosis; hence, maintain cell
viability and proliferation. For that reason, targeting CA IX and
CA XII is proposed to be a promising approach for the
development of novel anticancer agents for the treatment of
hypoxic tumors [15-19].
therapeutic applications [33]. Pyrimidine ring is one of the most
common N-based heterocyclic systems that play an important role
in the different metabolic as well as cellular processes [34].
Therefore, pyrimidines and fused pyrimidines are considered
privileged scaffolds for the development of various small
molecules with promising therapeutic applications [35-38].
Against this background, some studies reported the structural
hybridization of classical CA inhibitors that exhibit
a
benzenesulfonamide scaffold as ZBG, with the pyrimidine or
fused pyrimidine moieties as a suitable tail for the synthesis of
different carbonic anhydrase inhibitors of different therapeutic
targets [39-41]. Nocentini et al. [39] reported the discovery of a
new series of antitumor hCA inhibitors by incorporating the uracil
or purine moieties to the classical benzenesulfonamide scaffold,
for example, compounds VII and VIII showed Ki = 4.8 and 25.7
nM, respectively, against hCA IX and Ki = 0.85 and 17.7 nM,
respectively, against hCA II. In addition, compound IX displayed
Ki = 8.2 nM against CA II using the stopped flow CO2 hydration
assay [39] (Figure 2). Also, El-Azab et al. [40] reported the
synthesis and the promising CA inhibitory activity of a series of
benzene sulfonamide-quinazoline conjugates. For example,
compound X revealed Ki = 0.73 nM against CA II (Figure 2).
Moreover, Casini et al. [41] reported the potent CA inhibitory
Generally, the catalytic active site of mammalian α-CAs is a
cone-shaped cavity that is partitioned into two conserved
environments, hydrophobic and hydrophilic walls, with a Zn2+ ion
at the bottom. The Zn2+ ion is tetrahedrally coordinated with three
histidine residues (His94, His96, and His119) and a solvent
molecule; it can bind to hydroxide ion followed by reaction with
carbon dioxide to yield bicarbonate [20-22]. In this way, CA
inhibitors should contain certain group, zinc binding group (ZBG),
that can successfully coordinate with the binding site Zn2+ ion.
ZBG is coupled with variable chemical moieties which are known
as the compound “tail” that facilitate the interactions with specific
residues within the active site. Although the carbonic anhydrase
binding site is highly conserved among the different isoforms,
there is variability in the polarity and hydrophobicity of its
peripheral part [20-24].
activity of
a
series of 4-(2-aminopyrimidin-4-yl-amino)-
benzenesulfonamides. For instance, compounds XI and XII
showed Ki = 1 and 4 nM, respectively against CA II and
demonstrated a strong topical antiglaucoma properties (Figure 3).
Over the years, sulfonamides and their bioisosteres, viz.,
sulfamates and sulfamides, are considered as the main investigated
and clinically applied class of hCA inhibitors [11]. Acetazolamide
(AAZ) (I), methazolamide (II), ethoxzolamide (III),
dichlorphenamide (IV), dorzolamide (V) and sulthiame (VI), are
examples of CA inhibitors that are clinically used for the treatment
of glaucoma, epilepsy, and obesity [25-30]. Despite this achieved
progress, most of the classical sulfonamide-like CA inhibitors are
unselective toward the different hCA isoforms. Hence, their long-
term application is associated with off-target side effects [25].
Consequently, the development of isozyme-selective inhibitors is
regarded as a major challenge and has been under extensive study
in the last few years [31].
O
N
N
N
O
O
H2NO2S
N
NH
N
NH
O
NH
O
H2NO2S
VII
VIII
NH2
O
N
H2NO2S
NH
N
O
N
HN
H2NO2S
NH
N
O
N
N
S
H
IX
X
O
O
N
N
N
N
N
S
Figure 2. Examples for reported pyrimidine- and fused
pyrimidine–sulfonamide conjugates VII-X as CA inhibitors
SO2NH2
N
SO2NH2
N
SO2NH2
S
S
H
EtO
Acetazolamide (AAZ, I
Methazolamide II
( )
Ethoxzolamide III)
(
)
Recently, it was reported that some 2-thioxo-1,2,3,4-
tetrahydropyrimidines displayed carbonic anhydrase inhibitory
properties [42]. For instance, XIII and XIV showed Ki of 30.63
and 49.78 nM, respectively, against CA II [42]. Guided by the
structures of XI and XII [41] as well as XII and XIV [42], isosteric
replacement of the 2-pyrimidine benzenesulfonamide moiety of
XI and XII with an alkylated thiopyrimidine moiety was carried
out in order to design a hybridized series of pyrimidine-
sulfonamide conjugates incorporating primary and secondary
benzenesulfonamide scaffold as ZBG, the alkylated
thiopyrimidine moiety as a spacer, and 4-(un)substituted phenyl
moiety with various electronic and hydrophobic environments as
a tail (Figure 3). It was considered for a long time that primary
sulfonamides act as the most effective CA inhibitors because of
their ability to coordinate the Zn2+ ion in the deprotonated form
making highly stable enzyme-inhibitor adducts. [26,43]. Recent
data confirmed on the other hand the ability of secondary
benzenesulfonamides to act as potent and selective hCA inhibitors,
in some cases showing comparable activity to that of primary
sulfonamides [43]. In CA II, the substitution on the secondary
sulfonamide amino group is accommodated in the small
hydrophobic pocket defined by Trp209, Val143 and Val121,
which represents the CO2 binding pocket [44-46]. Hence, this
NHEt
SO2NH2
SO2NH2
N
SO2NH2
S
S
Me
S
Cl
SO2NH2
O
O
O
O
Cl
Dichlorphenamide IV
Dorzolamide
V
Sulthiame VI)
(
(
)
(
)
Figure 1. Structures of clinically used CA inhibitors
The knowledge of the CA active site topology presents a
prospective strategy for the development of selective inhibitors.
The amino acid residues that exist in and around the upper part of
the CA active site form several selective binding sub-pockets
which can be utilized for designing selective inhibitors. The tail
approach, which is based on introducing different moieties to the
aromatic sulfonamide ring that selectivity forces the interaction
with isoform unique residues in the peripheral part of the active
site, is regarded as a successful approach for targeting the CA
selectivity [20-24, 32].
Structural hybridization, which covalently links two or more
pharmacophores into a single structural framework, is considered
a successful approach for the discovery of new scaffolds with
4

sulfonamide moiety on the CA inhibition profile in comparison to
the corresponding primary sulfonamide. On the other hand, the
incorporation of the selected alkylated thiopyrimidine moiety will
have a twofold impact on the designed structures a) It affords an
additional hydrophobicity to the parent compounds XI, XII and so
stronger binding to the hydrophobic hCA II sub-pockets, and b) It
has the required flexibility needed to accommodate the distal 4-
(un)substituted phenyl moiety properly in hCA II secondary
recognition site. The designed and synthesized conjugates were
subsequently evaluated in vitro for their inhibitory activity on the
cytosolic hCA I and hCA II and on the transmembrane hCA IX
and hCA XII isoforms. Moreover, docking simulation studies were
carried out to study their binding mode in the hCA II active site
and to investigate their structure activity relationship (SAR).
H
N
H
H
N
N
N
O
S
S
CF3
N
O
O
NH
H2NO2S
O
XI
XIII
H
H
H
N
N
N
N
O
S
S
N
N
O
O
NH
H2NO2S
O
XII
XIV
Replacement of pyrimidine
sulfonamide of XI and XII
with alkylated thiopyrimidine
2. Results and discussion
2.1. Chemistry
Thr199
O
N
H
H
secondary
recognition
site
N
N
S
O
H
The synthesis of the target pyrimidine-benzenesulfonamide
conjugates 6-12 was carried out through initial reaction of
thiouracil (1) with different 2-bromoacetophenone derivatives 2a-
g under basic conditions to afford the corresponding intermediates
3 [47,48]. The intermediates 3 were subsequently chlorinated by
the reaction with phosphorus oxychloride to give 4 [47]. Reaction
of 4 with different benzenesulfonamides in acetic acid under reflux
gave the target products 6-12 in good yields.
O
N
R
S
R
N
His94
His96
O
Zn (II)
His119
Zn binding group
spacer
tail
2.2. Biological evaluation
Figure 3. The design strategy for the novel pyrimidine-
sulfonamide conjugates as hCA inhibitors
The synthesized pyrimidine-sulfonamide conjugates 6-12 as
well as acetazolamide; AAZ (I), the positive control, were
evaluated for their potency to inhibit cytosolic hCA I (associated
with edema), cytosolic hCA II (associated with glaucoma), and
transmembrane hCA IX and hCA XII (associated with tumors)
neglected class turned to attract the researchers aiming at the
discovery of novel CA inhibitors [43]. Based on these recent facts,
we were curious in this study to examine the effect of introducing
five different substitutions, namely acetyl, thiazolyl, 5-
methylisoxazol-3-yl, pyridyl and pyrimidinyl groups to the
O
H
O
H
Br
O
N
S
anhydrous K2CO3
EtOH, reflux, 4h
O
N
S
NH
R1
N
R1
1
2a-g
3a-g
R2HNO2S
NH2
O
Cl
N
S
5a-g
POCl3
80 °C, 30 min
N
acetic acid, reflux, 4h
R1
R
2 = H
5e
R
2 = 2-pyridyl
4a-g
5a
5b
5c
5d
2 = COCH3
2 = 2-thiazolyl
2 = 5-methylisoxazol-3-yl
R
2 = 2-pyrimidinyl
5f
R
5g
R
2 = 5-methyl-2-pyrimidinyl
R
R
O
H
N
N
S
N
R2HNO2S
R1
6-12
1 = H, R2 = H
1 = F, R2 = H
1 = Cl, R2 = H
1 = Br, R2 = H
1 = CF3, R2 = H
1 = OCH3, R2 = H
1 = F, R2 = 5-methylisoxazol-3-yl
1 = Cl, R2 = 5-methylisoxazol-3-yl
1 = OCH3, R2 = 5-methylisoxazol-3-yl
1 = H, R2 = 2-pyridyl
1 = Cl, R2 = 2-pyridyl
1 = OCH3, R2 = 2-pyridyl
1 = OCH3, R2 = 2-pyrimidinyl
1 = H, R2 = 4-methyl-2-pyrimidinyl
1 = F, R2 = 4-methyl-2-pyrimidinyl
1 = Cl, R2 = 4-methyl-2-pyrimidinyl
1 = CF3, R2 = 4-methyl-2-pyrimidinyl
R
1 = OCH3, R2 = 4-methyl-2-pyrimidinyl
R
1 = H
1 = F
1 = Cl
1 = Br
1 = CF3
1 = OCH3
6a
6b
6c
6d
6e
6f
9a
R
R
2-4a
2-4b
2-4c
2-4d
2-4e
2-4f
R
9b
9c
R
R
R
R
R
R
10a
10b
10c
11
R
R
R
R
R
R
R
R
1 = Ph, R2 = H
1 = Cl, R2 = COCH3
R
R
1 = Ph
6g
7
R
2-4g
R
12a
12b
12c
12d
12e
R
1 = H, R2 = 2-thiazolyl
1 = F, R2 = 2-thiazolyl
1 = Cl, R2 = 2-thiazolyl
1 = CF3, R2 = 2-thiazolyl
1 = OCH3, R2 = 2-thiazolyl
R
8a
8b
8c
8d
8e
R
R
R
R
R
R
R
5
Scheme 1. Synthesis of pyrimidine-sulfonamide conjugates 6-12

isoforms by the application of stopped flow carbon dioxide
hydration assay [49]. The inhibition results are depicted in table 1.
derivatives are slightly less potent than AAZ (Ki = 0.25 μM). An
apparent decrease in the inhibitory potency was observed in case
of using p-chlorophenyl group 6c (Ki = 1.94 μM) as a tail, whereas,
loss of inhibitory activity was found in case of incorporating a
biphenyl group 6g (Ki > 100 μM) as a tail. In the case of
pyrimidine-secondary benzenesulfonamide conjugates 7-12 a
marked decrease or loss of the activity was observed in
comparison to the equivalent primary sulfonamide derivatives 6.
The only exception was 8c (Ki = 1.75 μM) which showed a slight
increase in the potency in comparison to 6c (Ki = 1.94 μM).
Structure -Activity Relationship
The
obtained
results
show
that
the
primary
benzenesulfonamide-pyrimidine conjugates 6 strongly inhibited
the cytosolic isoform hCA I with Ki ranged from 0.10 μM to 0.87
μM. The only exceptions are 6c and 6g which showed moderate
(Ki = 1.94 μM) to complete loss (Ki > 100 μM) of inhibitory
activity, respectively.
Nearly, all the pyrimidine-primary benzenesulfonamide
conjugates 6 strongly inhibited the cytosolic isoform hCA II with
inhibition potential ranging between Ki = 0.04 μM to 0.09 μM in
comparison to the reference drug AAZ (Ki = 0.012 μM).
Compound 6g was the only exception which showed a moderate
inhibition activity with Ki of 0.73 μM. Compounds 6e and 6f with
4-(trifluoromethyl)phenyl and p-methoxyphenyl substituents,
respectively, are the most potent derivatives with Ki of 0.04 μM.
Comparison of the effect of the phenyl group substitution at 4-
position on the inhibitory activity showed that the 4-fluorophenyl
6b and unsubstituted phenyl 6a derivatives possessed inhibition
potential of Ki = 0.10 μM and Ki = 0.27 μM, respectively,
compared to AAZ (Ki = 0.25 μM) against the cytosolic isoform h
CA I. The p-bromophenyl 6d (Ki = 0.41 μM), p-methoxyphenyl 6f
(Ki = 0.63 μM) and p-(trifluoromethyl)phenyl 6e (Ki = 0.87 μM)
Table 1: Dissociation constants Ki (μM) for CA isoforms in response to 6-12 and AAZ using a stopped flow CO2
hydrase assay [49] and selectivity ratio for hCA II.
Dissociation constants Ki (μM) for CA
Selectivity ratio for hCA II
isoforms
Entry Compound
hCA I
0.27
hCA II
0.05
hCA IX
0.16
hCA XII
0.53
0.82
0.40
0.94
0.90
0.71
0.70
1.35
0.41
>100
3.18
0.36
>100
>100
48.44
0.54
9.31
4.0
I/II
IX/II
3.2
XII/II
1
6a
5.4
1.1
10.6
9.1
2
6b
0.10
0.09
0.05
0.07
0.04
0.04
0.73
0.34
0.54
0.57
0.85
0.83
0.88
80.52
70.15
6.76
45.41
55.9
>100
>100
74.6
3.15
21.79
59.06
7.07
0.012
0.82
9.1
3
6c
1.94
>100
>100
39.20
5.17
38.8
>2000
>1428
980
8
4
6d
0.41
5.9
13.42
22.5
17.8
0.96
3.97
0.75
>175.4
3.74
0.43
>113.6
>1.24
0.69
0.08
0.21
0.072
nd
5
6e
0.87
21.8
6
6f
0.63
15.8
129.3
0.32
7
6g
>100
74.6
0.23
>137
219
8
7
>100
>100
>100
0.55
294.1
185.2
>175.4
0.65
9
8a
73.6
136.3
>175.4
2.05
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
8b
>100
1.75
8c
8d
84.5
>100
>100
92.12
8.94
101.8
>113.6
>1.24
>1.42
7.27
>120.5
>113.6
1.14
8e
>100
>100
>100
49.16
>100
>100
>100
>100
>100
64.4
9a
9b
0.13
9c
>100
>100
>100
>100
2.51
>14.79
>2.20
>1.79
nd
10a
10b
10c
11
>2.20
>1.79
nd
0.80
7.09
3.15
0.31
6.29
6.44
8.67
0.0057
nd
nd
nd
12a
12b
12c
12d
12e
AAZ
4.48
>1.34
20.44
>4.59
>1.69
>14.14
20.83
0.06
0.04
0.10
0.29
0.11
1.23
0.48
4.87
1.54
>100
>100
>100
0.25
83.81
0.49
3.85
0.008
0.07
0.53
0.025
2.08
6

This is followed by compounds 6a and 6c with unsubstituted
phenyl and p-chlorophenyl groups, respectively, showing Ki of
0.05 μM. Whereas compounds 6d and 6b exhibiting p-
bromophenyl group and p-fluorophenyl moiety as a tail displayed
Ki = 0.07 and 0.09 μM, respectively. A decrease in the inhibitory
activity to a sub-micromolar range (Ki = 0.34-0.88 μM) was
observed by the incorporation of a secondary sulfonamide moiety
having acetyl 7 or thiazolyl 8 groups. However, a very weak to
complete disappearance of inhibitory activity was observed in
using secondary sulfonamides having 5-methylisoxazol-3-yl 9 (Ki
= 6.76-80.52 μM), pyridyl 10 (Ki = 45.41 - > 100 μM), 2-
pyrimidinyl 11 (Ki > 100 μM) or 4-methyl-2-pyrimidinyl 12 (Ki =
3.15 - 74.6 μM) moieties.
II selectivity index. Compounds 6a and 6b showed more than 3-
and 9-fold higher selectivity toward hCA II over the membrane
associated isoform hCA IX. However, an apparent increase in the
selectivity was observed by introducing chloro, bromo,
trifluoromethyl or methoxy groups at the 4 position of the phenyl
moiety in 6c, 6d, 6e, 6f, respectively, these derivatives
demonstrated >2000, >1428, 980, and 129 hCA IX/hCA II
selectivity index, respectively. For series 7-12, most of the
compounds showed higher selectivity toward the hCA II over hCA
IX, for instance, the derivative 7 showed 294-fold higher
selectivity toward hCA II over hCA IX. The secondary
sulfonamide derivatives 8c, 9b, 12a, 12d, and 12e are the only
exceptions with higher selectivity toward hCA IX over hCA II.
Studying the selectivity of the synthesized series toward hCA II
over hCA XII showed that derivatives having primary sulfonamide
groups 6a-f have 8- to 22-fold higher selectivity toward hCA II
over hCA XII. Compound 6g is the only exception; it displayed
higher selectivity toward hCA IX and hCA XII over hCA II.
Compound 8b showed more than 175 higher selectivity toward
hCA II over hCA XII. Secondary sulfonamides exhibiting
thiazolyl 8a,d, 5-methylisoxazol-3-yl 9b,c, pyridyl 10a-c,
pyrimidinyl 11 and 4-methyl-pyrimidinyl substituent 12a-d
showed higher selectivity toward hCA XII over hCA II.
The membrane bound tumor associated isoform hCA IX is
highly inhibited by the primary sulfonamides 6a and 6g with Ki =
0.16 and 0.23 μM, respectively and they were the most potent
compounds among the series. This is followed by compound 6b
which exhibit Ki = 0.82 μM. An increase in the potency of 6c (Ki
> 100 μM) was observed by incorporating a 4-methyl-2-
pyrimidinyl in 12c (Ki = 83.81 μM), 5-methylisoxazol-3-yl in 9b
(Ki = 8.94 μM) and reaches its maximum potency by incorporating
2-thiazolyl group in 8c (Ki = 0.55 μM). Also, huge increase in the
potency of the primary sulfonamides 6e (Ki = 39.20 μM) and 6f
(Ki = 5.17 μM) was observed by the introduction of 4-methyl-2-
pyrimidinyl substituent in 12d (Ki = 0.49 μM) and 12e (Ki = 0.53
μM), respectively. These results confirm our assumptions that the
incorporation of secondary sulfonamides can be advantageous in
some cases.
These results indicated that the synthesized 2-
thioxopyrimidine-benzensulfonamide conjugates 6 are interesting
scaffold with higher selectivity toward hCA II. Hence, it can be
further optimized for the development of new drugs that can be
applied for the treatment of glaucoma. In addition, the
incorporation of secondary sulfonamides results in an increase in
the inhibitory activity in some cases against CA IX and CA XII
while no apparent increase in the potency was observed against
CA I and CA II.
Weak hCA XII inhibitory potency was observed by all the
synthesized compounds. Primary sulfonamides 6 showed activity
in the range of Ki = 0.40 to 0.94 μM. A slight increase in the
potency was observed by introduction of 2-thiazolyl moiety in 8a
(Ki = 0.41 μM) in comparison to the primary sulfonamide
derivative 6a (Ki = 0.53 μM). However, a decrease in the potency
was observed by incorporating a pyridyl group in 10a (Ki = 9.31
μM) or 4-methyl-2-pyrimidinyl group in 12a (Ki = 3.15 μM). An
increase in the potency was observed by introduction of 4-methyl-
2-pyrimidinyl group in 12b (Ki = 0.31 μM) in comparison to 6b
(Ki = 0.82 μM) while total loss in the potency was observed by
including 2-thiazolyl moiety in 8b (Ki > 100 μM) or 5-
methylisoxazol-3-yl group in 9a (Ki > 100 μM). A decrease in
potency was observed by introducing an acetyl group in 7 (Ki =
1.35 μM), 2-thiazolyl group in 8c (Ki = 3.18 μM), 5-
methylisoxazol-3-yl group in 9b (Ki = 48.44 μM), pyridyl group
in 10b (Ki = 4.0 μM), 4-methyl-2-pyrimidinyl group in 12c (Ki =
6.29 μM) in comparison to 6c (Ki = 0.40 μM). Incorporation of
thiazolyl moiety in 8d (Ki = 0.36 μM) resulted in more than two-
fold increase in potency in comparison to 6e (Ki = 0.90 μM),
whereas, huge decrease in the potency appeared with compound
exhibiting 4-methyl-2-pyrimidinyl moiety 12d (Ki = 6.44 μM).).
A slight increase in the potency appeared in the secondary
sulfonamide exhibiting 5-methylisoxazol-3-yl group 9c (Ki = 0.54
μM) when compared to the primary sulfonamide congener 6f (Ki
= 0.71 μM), whereas, a slight decrease to complete loss of the
activity appeared by the introduction of pyridyl group in 10c (Ki =
0.80 μM), 2-pyrimidinyl moiety in 11 (Ki = 7.09 μM), 4-methyl-
2-pyrimidinyl group in 12e (Ki = 8.67 μM) and thiazolyl group in
8e (Ki > 100 μM) (Table 1).
2.4. Molecular docking study
Docking simulations were carried out to study the binding
pattern of the newly synthesized sulfonamide conjugates in the
active site of the target anhydrase CA II to elucidate their structure
activity relationship (SAR). The molecular docking protocol was
first validated by running self-docking of the co-crystalized ligand
(AAZ) in the active site of CA II (PDB ID: 3HS4) [50]. The self-
docking validation reproduced the co-crystallized ligand’s
experimental binding mode indicating that the docking protocol
used is adequate for the intended docking analysis. This is shown
by the small RMSD between the experimental co-crystallized
ligand pose and the docked pose of 0.867Å; and by the capability
of the docking pose to reproduce all the key interactions achieved
by the co-crystallized ligands in the active site with Zn2+, Thr199
and Thr200 (Figure 4).
Table 2 shows the predicted docking score of the most potent
compounds 6a-g. Generally, as can be seen in figure 5, the
sulfonamide moiety acted as the ZBG interacting agent with the
active site Zn2+ ion. Moreover, it interacts through hydrogen
bonding with the key amino acids Thr199 and Thr200. In addition,
the linker NH between the benzenesulfonamide and the pyrimidine
moieties establishes a hydrogen bonding interaction with Gln92
backbone carbonyl group. This additional interaction could be
responsible for the potent effect of series 6 on CA II and their
selectivity toward its inhibition. Through hydrophobic interaction,
the benzenesulfonamide and the thiopyrimidine moieties interact
with the hydrophobic side chains of the amino acids
Val121/Leu198 and Ile91/Phe131, respectively (For further details
See SI).
Most of the newly synthesized derivatives exhibited a
preferential selectivity for the glaucoma associated isoform hCA
II over hCA I and hCA IX. In series 6, the derivatives 6a and 6d
displayed 5-fold higher selectivity toward hCA II over hCA I.
Introduction of p-chloro, p-CF3, p-methoxy, p-Ph in 6c, 6e, 6f and
6g, respectively, resulted in about 15 to more than 137 hCA I/hCA
7

(A)
(B)
Figure 5. 2-Dimensional diagram (A) and 3-dimensional
representation (B) of compound 6e showing its interaction with the
CA II active site.
Although the secondary sulfonamides 7, 8, 9, 10, 11 and 12 could
accomplish the planned hydrophobic interaction by their
substituent on the sulfonamide amino group with the hydrophobic
side chains of the CO2 pocket Val121, Leu141, Val143, Leu198,
Val207 and Trp209, they could not achieve a high inhibitory
activity as appeared in their experimental results. This could be
attributed to their inability to achieve the proper coordination
Figure 4. 2-Dimensional interaction diagram showing the AAZ
docking pose interactions with the key amino acids in the CA II
active site (PDB ID: 3HS4).
Table 2. Docking energy scores (S) in kcal/mol for the newly
synthesized 6a-g in CA II active site
Compound Energy score (S) kcal/mol
6a
6b
−9.44
−9.31
−9.33
−9.93
−10.1
−10.23
−9.95
−7.68
6c
6d
6e
6f
6g
AAZ
Compounds 6e and 6f, which showed the most potent inhibitory
activity of CA II with Ki of 0.04 μM, are showing the most
negative energy score of −10.10 and −10.23 kcal/mol,
respectively. Compound 6e shows this high activity due to the
additional polar interaction taking place between its polar fluorine
atoms of its CF3 with the positively charged guanidinium group of
Arg58 (Figure 5A). Whereas, the high activity of compound 6f
could be attributed to the additional H-bond interaction performed
by the p-methoxy group on the distal phenyl group with Asn67
(For further details see SI).
geometry with the Zn2+ cation and the twisting of their whole
structure within the binding site relative to the common binding
pattern of CA inhibitors (For further details see SI)
3. Conclusion
In summary, a molecular hybridization approach of a
benzensulfonamide moiety (ZBG), an alkylated thiopyrimidine
moiety (spacer) and an (un)substituted phenyl moiety (tail) was
implemented for the design of a new series of thiopyrimidine-
benzenesulfonamides 6-12. The designed and synthesized
compounds 6-12 were examined for their inhibitory activity on
four carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA
II, hCA IX and hCA XII using stopped-flow CO2 hydration assay.
Series 6 exerted a promising activity and selectivity toward the
cytosolic isoforms hCA I and hCA II versus the membrane bound
isoforms hCA IX and hCA XII. The thiopyrimidine-
benzenesulfonamides 6e and 6f showed Ki of 0.04 uM against hCA
II and 15.8- to 980-fold selectivity towards hCA II over the other
tested isoforms. Molecular docking simulations of the newly
8

synthesized compounds in the active site of hCA II attributed the
promising hCA II inhibitory activity of series 6 to the ability of
their to interact with the key elements in the active site viz. the Zn2+
ion and the key amino acids Thr199 and Thr200. Through
hydrophobic interaction, the benzene sulfonamide and the
thiopyrimidine moieties interact with the hydrophobic side chains
of the amino acids Val121/Leu198 and Ile91/Phe131, respectively.
Moreover, a hydrogen bond interaction is formed between the NH
group at the 4-position of the pyrimidine moiety and Gln92
backbone carbonyl group. This additional interaction could be
responsible for the potent effect of series 6 on CA II and their
selectivity toward its inhibition. Molecular docking studies
rationalized the potent inhibitory activity of compound 6e to the
additional polar interaction between the polar fluorine atoms of its
CF3 with the positively charged guanidinium group of Arg58.
Whereas, the high activity of compound 6f can be attributed to the
additional H-bond interaction performed by the p-methoxy group
on the distal phenyl group with Asn67.
brown powder (159.43 mg, 80%); mp 243-245 °C; ύ max (atr)/cm-
1 3391, 2973, 2924, 1640, 1573 and 1456; δH (500 MHz; DMSO-
d6) 4.98 (2H, s, CH2), 6.78 (1H, d, 3J = 5.0 Hz), 7.23 (2H, br.), 7.51
(2H, d, 3J = 8.0 Hz), 7.57 (2H, t like, 3J = 7.0 Hz), 7.68-7.72 (3H,
m), 8.03 (2H, d, J = 7.0 Hz ), 8.19 (1H, d, J = 5.0 Hz ), 11.01
ppm (1H, br.); δC (125 MHz; DMSO-d6) 39.00, 104.06, 119.97,
126.56, 128.28, 128.94, 133.96, 135.36, 138.58, 141.13, 150.87,
159.39, 167.48, 192.82 ppm; Anal. Calcd for C18H16N4O3S2: C,
53.99; H, 4.03; N, 13.99. Found: C, 53.58; H, 4.33; N, 14.15.
3
3
4-((2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)benzenesulfonamide (6b)
Following the general procedure III, a mixture of 4b (141.36
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6b as
a pale brown powder (175.11 mg, 84%); mp 216-218 °C; ύ max
(atr)/cm-1 3345, 3263, 3066, 3013, 2959, 1686, 1636, 1597, 1574,
1562, 1505; δH (500 MHz; DMSO-d6) 4.82 (2H, s, CH2), 6.55 (1H,
d, 3J = 5.5 Hz), 7.19 (2H, br.), 7.38 (2H, t, 3J = 7.5 Hz), 7.54 (2H,
d, 3J = 7.5 Hz), 7.69 (2H, d, 3J = 7.5 Hz), 8.12-8.13 (3H, m), 10.00
ppm (1H, br.); δC (125 MHz; DMSO-d6) 38.40, 103.93, 115.94 (d,
J = 21.8 Hz), 118.96, 126.60, 131.29 (d, J = 9.5 Hz), 132.48 (d, J
= 2.6 Hz), 137.32, 142.26, 155.76, 159.29, 165.27 (d, J = 251.4
Hz), 169.18, 192.20 ppm; Anal. Calcd for C18H15FN4O3S2: C,
51.66; H, 3.61; N, 13.39. Found: C, 51.36; H, 3.85; N, 13.62.
4. Experimental
4.1. Chemistry
4.1.1. General remarks
Chemicals used for synthesis and biological experiments were
purchased from commercial suppliers. Precoated silica gel 60
F245aluminium plates (Merck) were used for analytical thin layer
chromatography (TLC). Melting points were measured using open
capillary tubes on a Stuart SMP30 melting point apparatus and
they were uncorrected. Spectral data and elemental analysis of the
synthesized compounds were measured in the Micro analytical
labs, National Research Centre, Cairo, Egypt. IR spectra (4000–
400 cm-1) were recorded applying KBr pellets in a Jasco FT/IR
4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)benzenesulfonamide (6c)
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6c as a
pale brown powder (136 mg, 63%), mp 229-231 °C; ύ max
(atr)/cm-1 3378, 3290, 2923, 1689, 1631, 1568 and 1498; δH (400
MHz; DMSO-d6) 4.81 (2H, s, CH2), 6.55 (1H, d, 3J = 5.6 Hz), 7.19
(2H, s), 7.59-7.63 (4H, m), 7.71 (2H, d, 3J = 8.8 Hz), 8.05 (2H, d,
3J = 8.8 Hz), 8.11 (1H, d, 3J = 5.6 Hz), 9.99 ppm (s, 1H); Anal.
Calcd for C18H15ClN4O3S2: C, 49.71; H, 3.48; N, 12.88. Found: C,
49.34; H, 3.79; N, 12.61.
1
6100 Fourier transform infrared spectrophotometer. HNMR and
13CNMR spectra were measured at 500 (125) MHz and 400 (100)
MHz on Bruker instruments, using DMSO-d6 as a solvent.
General procedure I for the synthesis of 3a-g
As previously reported [47], a mixture of 2-thiouracil (1), 2-
bromoacetophenone derivatives 2a-g, anhydrous K2CO3 and
ethanol was heated under reflux for 4h. The reaction mixture was
cooled, poured on ice / water and neutralized with 2N HCl. The
formed precipitate was filtered and dried to yield 3a-g.
Analytically pure products of 3a-g were obtained by
crystallization form methanol.
4-((2-((2-(4-Bromophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)benzenesulfonamide (6d)
Following the general procedure III, a mixture of 4d (171.81
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6d as
a pale brown powder (170 mg, 71%); mp 232-234 °C; ύ max
(atr)/cm-1 3383, 3290, 3153, 3077, 2958, 1688, 1632, 1568, 1499
and 1464; δH (400 MHz; DMSO-d6) 4.80 (2H, s, CH2), 6.55 (1H,
d, 3J = 6.0 Hz), 7.19 (2H, br.), 7.62 (2H, d, 3J = 8.8 Hz), 7.72 (2H,
d, 3J = 8.4 Hz), 7.77 (2H, d, 3J = 8.4 Hz), 7.97 (2H, d, 3J = 8.4 Hz),
General procedure II for the synthesis of 4a-g
As previously reported [47], a round bottom flask was charged
with 3a-g and POCl3. The mixture was stirred at 80 °C for 30 min.
Subsequently, the reaction was cooled down and poured portion
wise on ice. The formed precipitate was collected by filtration
followed by drying to obtain 4a-g.
3
8.11 (1H, d, J = 6.0 Hz ), 10.00 ppm (1H, s); δC (100 MHz;
DMSO-d6) 38.37, 103.98, 119.07, 126.68, 127.85, 130.27, 131.96,
134.79, 137.41, 142.33, 155.81, 159.32, 169.09, 193.11 ppm;
Anal. Calcd for C18H15BrN4O3S2: C, 45.10; H, 3.15; N, 11.69.
Found: C, 45.35; H, 3.44; N, 11.45.
General procedure III for the synthesis 6-12
A
mixture of 4a-g (0.50 mmol), benzenesulfonamide
derivatives 5a-g (0.50 mmol) and acetic acid (15 mL) was refluxed
for 4h. Then, the reaction was left to cool followed by pouring on
ice-water. The formed precipitate was collected by filtration and
left to dry. Analytically pure compounds 6-12 were afforded by
crystallization from methanol.
4-((2-((2-Oxo-2-(4-(trifluoromethyl)phenyl)ethyl)thio)pyri-
midin-4-yl)amino)benzene sulfonamide (6e)
Following the general procedure III, a mixture of 4e (166.36
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6e as a
pale brown powder (177 mg, 76 %), mp 105-107 °C; ύ max
(atr)/cm-1 3448, 3317, 3082, 2909, 1701, 1647 and 1504; δH (500
MHz; DMSO-d6) 4.86 (2H, s, CH2), 6.55 (1H, d, 3J = 5.5 Hz), 7.19
(2H, br.), 7.60 (2H, d, 3J = 8.0 Hz), 7.72 (2H, d, 3J = 7.5 Hz), 7.93
(2H, d, 3J = 7.5 Hz), 8.11 (1H, d, 3J = 5.5 Hz), 8.22 (2H, d, 3J = 8.0
Hz), 10.01 ppm (1H, br.); δC (125 MHz; DMSO-d6) 38.62, 103.99,
4-((2-((2-Oxo-2-phenylethyl)thio)pyrimidin-4-yl)amino)benz-
enesulfonamide (6a)
Following the general procedure III, a mixture of 4a (132.36 mg,
0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and acetic
acid (15 mL) was refluxed for 4h. Work up afforded 6a as a pale
9

119.04, 123.73 (q, J = 271.3 Hz), 125.84 (q, 3J = 3.6 Hz), 126.64,
129.06, 132.88 (q, 3J = 31.9 Hz), 137.41, 139.02, 142.27, 155.78,
159.31, 168.96, 193.39 ppm; Anal. Calcd for C19H15F3N4O3S2: C,
48.71; H, 3.23; N, 11.96. Found: C, 48.42; H, 3.52; N, 11.74.
4-((2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)-N-(thiazol-2-yl)benzenesulfonamide (8b)
Following the general procedure III, a mixture of 4b (141.36
mg, 0.50 mmol), sulfathiazole (5c) (127.66 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 8b as
a pale brown powder (140.29 mg, 56%); mp 187-189 °C; ύ max
(atr)/cm-1 3433, 3089, 2925, 1632 and 1460; δH (400 MHz; DMSO-
d6) 4.82 (2H, s, CH2), 6.53 (1H, d, 3J = 5.0 Hz), 6.79 (1H, d, 3J =
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)benzenesulfonamide (6f)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6f as a
pale brown powder (100 mg, 47%); mp 220-222 °C; ύ max
(atr)/cm-1 3379, 3271, 3093, 3043, 2916, 1674, 1605, 1574 and
1508; δH (400 MHz; DMSO-d6) 3.87 (3H, s, OCH3), 4.81 (2H, s,
3
5.5 Hz), 7.21-7.22 (1H, m), 7.35-7.39 (2H, m), 7.45 (2H, d, J =
7.6 Hz), 7.63 (2H, d, 3J = 7.2 Hz), 8.12-8.14 (3H, m), 9.97 (1H, s)
and 11.77 ppm (1H, br.); Anal. Calcd for C21H16FN5O3S3: C,
50.29; H, 3.22; N, 13.96. Found: C, 50.11; H, 3.41; N, 13.65.
3
3
CH2), 6.57 (1H, d, J = 5.6 Hz), 7.08 (2H, d, J = 8.4 Hz), 7.18
(2H, br), 7.53 (2H, d, 3J = 8.4 Hz), 7.69 (2H, d, 3J = 8.4 Hz), 8.02
(2H, d, 3J = 8.8 Hz), 8.14 (1H, d, 3J = 5.6 Hz), 10.14 ppm (1H, s);
δC (100 MHz; DMSO-d6) 38.43, 55.58, 103.94, 114.17, 119.32,
126.63, 128.45, 130.67, 137.70, 141.94, 154.24, 159.35, 163.61,
168.84, 191.57 ppm; Anal. Calcd for C19H18N4O4S2: C, 53.01; H,
4.21; N, 13.01. Found: C, 53.28; H, 4.51; N, 13.33.
4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(thiazol-2-yl)benzene-sulfonamide (8c)
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfathiazole (5c) (127.66 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 8c as a
white powder (180.00 mg, 70%), mp 143-145 °C; ύ max (atr)/cm-
1 3436, 2924, 1679, 1634, 1570 and 1535; δH (500 MHz; DMSO-
d6) 4.81 (2H, s, CH2), 6.53 (1H, d, 3J = 5.5 Hz), 6.79 (1H, d, 3J =
3.5 Hz), 7.21 (1H, d, 3J = 4.0 Hz), 7.52 (2H, d, 3J = 8.0 Hz), 7.63
(2H, d like, 3J = 8.0 Hz), 7.66 (2H, d like, 3J = 8.0 Hz), 8.06 (2H,
d, 3J = 8.0 Hz), 8.10 (1H, d, 3J = 5.5 Hz) and 9.99 ppm (1H, s); δC
(125 MHz; DMSO-d6) 38.49, 103.97, 107.93, 118.82, 125.15,
126.86, 129.00, 130.18, 134.41, 135.60, 138.72, 142.47, 155.77,
159.28, 168.63, 169.16 and 192.67 ppm; Anal. Calcd for
C21H16ClN5O3S3: C, 48.69; H, 3.11; N, 13.52. Found: C, 48.41; H,
3.42; N, 13.32.
4-((2-((2-([1,1'-Biphenyl]-4-yl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)benzenesulfonamide (6g)
Following the general procedure III, a mixture of 4g (170.41
mg, 0.50 mmol), sulfanilamide (5a) (86.10 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 6g as a
pale brown powder (125 mg, 53%); mp 263-265 °C; ύ max
(atr)/cm-1 3413, 3290, 3189, 3051, 2924, 1635, 1594, 1550 and
1495; δH (400 MHz; DMSO-d6) 5.02 (2H, s, CH2), 6.82 (1H, d, 3J
= 6.0 Hz), 7.26 (2H, br.), 7.44 (2H, d, 3J = 7.0 Hz), 7.51 (2H, like,
3
3J = 7.2 Hz), 7.63 (2H, d, J = 8.0 Hz), 7.77-7.79 (3H, m), 7.87
4-((2-((2-Oxo-2-(4-(trifluoromethyl)phenyl)ethyl)thio)pyrimi-
din-4-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide (8d)
(2H, d, 3J = 7.6 Hz), 8.11 (2H, d, 3J = 7.6 Hz), 8.21 (1H, d, 3J = 6.0
Hz) and 11.12 ppm (1H, s); δC (100 MHz; DMSO-d6) 38.87,
104.07, 120.36, 126.67, 126.84, 127.09, 127.23, 128.51, 129.09,
134.12, 138.83, 138.96, 141.12, 145.42, 150.49, 159.46, 167.35,
192.44 ppm; Anal. Calcd for C24H20N4O3S2: C, 60.49; H, 4.23; N,
11.76. Found: C, 60.25; H, 4.54; N, 11.61.
Following the general procedure III, a mixture of 4e (166.36
mg, 0.50 mmol), sulfathiazole (5c) (127.66 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 8d as
a pale brown powder (187.34 mg, 68%); mp 222-224 °C; ύ max
(atr)/cm-1 3483, 3421, 3294, 3089, 3043, 1686, 1620 and 1562; δH
(500 MHz; DMSO-d6) 4.86 (2H, s, CH2), 6.54 (1H, d, 3J = 5.0 Hz),
6.80 (1H, br.), 7.22 (1H, br.), 7.54 (2H, d, 3J = 8.0 Hz), 7.68 (2H,
d, 3J = 8.0 Hz), 7.94 (2H, d, 3J = 7.5 Hz), 8.10 (1H, d, 3J = 5.0 Hz),
8.24 (2H, d, 3J = 7.5 Hz), 10.00 (1H, s) and 12.61 ppm (1H, br.);
δC (125 MHz; DMSO-d6) 38.65, 104.02, 107.94, 118.90, 123.73
(q, J = 271.3 Hz), 124.33, 125.84 (q, J = 3.6 Hz), 126.85, 129.08,
132.92 (q, J = 31.9 Hz), 135.38, 138.98, 142.55, 155.78, 159.27,
168.57, 169.00 and 193.29 ppm; Anal. Calcd for C22H16F3N5O3S3:
C, 47.91; H, 2.92; N, 12.70. Found: C, 47.75; H, 2.77; N, 12.93.
N-((4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)phenyl)sulfonyl) acetamide (7)
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfacetamide (5b) (107.12 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 7 as a
white powder (120 mg, 50%); mp 247-249 °C; ύ max (atr)/cm-1
3439, 2926, 1684, 1635 and 1564; δH (400 MHz; DMSO-d6) 1.89
(3H, s, CH3), 4.83 (2H, s, CH2), 6.56-6.58 (1H, m), 7.74-7.75 (2H,
m), 7.61-7.69 (4H, m), 8.04-8.06 (2H, m), 8.13-8.14 (1H, m),
10.12 (1H, s), 11.68 ppm (1H, br.); Anal. Calcd for
C20H17ClN4O4S2: C, 50.37; H, 3.59; N, 11.75. Found: C, 50.65; H,
3.28; N, 11.91.
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)-N-(thiazol-2-yl)benzenesulfonamide (8e)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfathiazole (5c) (127.66 mg, 0.50 mmol), acetic
acid (15 mL) was refluxed for 4h. Work up afforded 8e as a white
powder (160.00 mg, 62%), mp 139-141 °C; ύ max (atr)/cm-1 3483,
3421, 3090, 2900, 1670, 1636 and 1597; δH (400 MHz; DMSO-d6)
3.89 (3H, s, OCH3), 4.83 (2H, s, CH2), 6.60 (1H, br.), 6.81 (1H, d,
3J = 4.8 Hz), 7.10 (2H, d, 3J = 8.8 Hz), 7.22 (1H, d, 3J = 4.4 Hz),
7.44 (2H, d, 3J = 8.8 Hz), 7.65 (2H, d, 3J = 8.8 Hz), 8.04 (2H, d, 3J
= 8.8 Hz), 8.14 (1H, d, 3J = 5.6 Hz), 10.31 (1H, br.) and 12.62 ppm
(1H, br.); Anal. Calcd for C22H19N5O4S3: C, 51.45; H, 3.73; N,
13.64. Found: C, 51.21; H, 3.45; N, 13.91.
4-((2-((2-Oxo-2-phenylethyl)thio)pyrimidin-4-yl)amino)-N-
(thiazol-2-yl)benzenesulfonamide (8a)
Following the general procedure III, a mixture of 4a (132.36
mg, 0.50 mmol), sulfathiazole (5c) (127.66 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 8a as a
pale brown powder (185.21 mg, 77%); mp 181-183 °C; ύ max
(atr)/cm-1 3444, 3348, 3101, 2812, 1678, 1566 and 1539; δH (400
MHz; DMSO-d6) 4.84 (2H, s, CH2), 6.53 (1H, d, 3J = 5.6 Hz), 6.82
3
3
3
(1H, d, J = 4.8 Hz), 7.24 (1H, d, J = 4.4 Hz), 7.45 (2H, d, J =
8.8 Hz), 7.54 (2H, t like, 3J = 7.6 Hz), 7.64-7.68 (3H, m), 8.05 (2H,
d, 3J = 8.0 Hz), 8.12 (1H, d, 3J = 6.0 Hz), 9.97 (1H, s) and 12.60
ppm (1H, br.); Anal. Calcd for C21H17N5O3S3: C, 52.16; H, 3.54;
N, 14.48. Found: C, 52.31; H, 3.37; N, 14.20.
4-((2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (9a)
Following the general procedure III, a mixture of 4b (141.36
mg, 0.50 mmol), sulfamethoxazole (5d) (126.64 mg, 0.50 mmol)
10

and acetic acid (15 mL) was refluxed for 4h. Work up afforded 9a
as a pale brown powder (111.00 mg, 44%); mp 217-219 °C; ύ max
(atr)/cm-1 3437, 2924, 1635, 1570 and 1500; δH (400 MHz; DMSO-
d6) 2.29 (3H, s, CH3), 4.82 (2H, s, CH2), 6.06 (1H, br.), 6.55 (1H,
d, 3J = 4.8 Hz), 7.27 (2H, t, 3J = 8.0 Hz), 7.37 (2H, t, 3J = 8.0 Hz),
7.53 (2H, d, 3J = 8.0 Hz), 7.72 (2H, d, 3J = 8.0 Hz), 8.13 (1H, br.),
10.08 (1H, s) and 11.26 ppm (1H, s); Anal. Calcd for
C22H18FN5O4S2: C, 52.90; H, 3.63; N, 14.02. Found: C, 52.65; H,
3.89; N, 14.42.
for C23H18ClN5O3S2: C, 53.96; H, 3.54; N, 13.68. Found: C, 53.71;
H, 3.81; N, 13.95.
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)-N-(pyridin-2-yl)benzenesulfonamide (10c)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfapyridine (5e) (124.65 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 10c as
a white powder (145.23 mg, 57%), mp 141-143 °C; ύ max (atr)/cm-
1
3431, 2973, 2930, 1670, 1635, 1600, 1501 and 1464; δH (400
4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (9b)
MHz; DMSO-d6) 3.87 (3H, s, OCH3), 4.79 (2H, s, CH2), 6.53 (1H,
d, 3J = 5.6 Hz), 6.85 (1H, t, 3J = 6.4 Hz), 7.05-7.10 (3H, m), 7.53
(2H, d, 3J = 8.8 Hz), 7.66 (2H, d like, 3J = 8.8 Hz), 7.69-7.75 (1H,
m), 7.99-8.00 (1H, m), 8.05 (2H, d, 3J = 8.4 Hz), 8.12 (1H, d, 3J =
5.6 Hz), 10.01 (1H, s) and 11.63 ppm (1H, br.); δC (100 MHz;
DMSO-d6) 38.29, 55.54, 103.94, 113.69, 114.09, 118.78, 127.70,
127.82, 128.21, 128.53, 130.65, 142,71, 142.75, 155.50, 155.52,
159.25, 161.13, 163.55, 169.32 and 191.60 ppm; Anal. Calcd for
C24H21N5O4S2: C, 56.79; H, 4.17; N, 13.80. Found: C, 56.41; H,
4.35; N, 13.72
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfamethoxazole (5d) (126.64 mg, 0.50 mmol)
and acetic acid (15 mL) was refluxed for 4h. Work up afforded 9b
as a white powder (189 mg, 73%), mp 135-137 °C; ύ max (atr)/cm-
1
3309, 3163, 3086, 1678, 1620, 1566 and 1500; δH (400 MHz;
DMSO-d6) 2.29 (3H, s, CH3), 4.86 (2H, s, CH2), 6.08 (1H, s), 6.65
(1H, d, 3J = 6.0 Hz), 7.57 (2H, d, 3J = 8.4 Hz), 7.62 (2H, d, 3J = 8.0
Hz), 7.75 (2H, d, 3J = 8.4 Hz), 8.05 (2H, d, 3J = 8.0 Hz), 8.15 (1H,
d, 3J = 5.6 Hz), 10.46 (1H, s) and 11.29 ppm (1H, s); Anal. Calcd
for C22H18ClN5O4S2: C, 51.21; H, 3.52; N, 13.57. Found: C, 51.54;
H, 3.28; N, 13.32.
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(pyrimidin-2-yl)benzenesulfonamide (11)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfadiazine (5f) (125.14 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 11 as a
pale brown powder (180.34 mg, 71%), mp 165-167 °C; ύ max
(atr)/cm-1 3437, 3092, 2930, 1635, 1577 and 1498; δH (500 MHz;
DMSO-d6) 3.86 (3H, s, OCH3), 4.86 (2H, s, CH2), 6.65 (1H, d, 3J
= 5.0 Hz), 7.02 (1H, br.), 7.08 (2H, d, 3J = 8.0 Hz), 7.69-7.74 (4H,
m), 8.03 (2H, d, 3J = 8.0 Hz), 8.16 (1H, d, 3J = 5.0 Hz), 8.46-8.47
(2H, m) and 10.52 ppm (1H, s); δC (125 MHz; DMSO-d6) 38.61,
55.57, 104.12, 114.14, 115.75, 119.06, 128.39, 128.76, 130.66,
133.52, 142.85, 153.68, 156.93, 158.32, 159.31, 163.63, 168.63,
172.04 and 191.44 ppm; Anal. Calcd for C23H20N6O4S2: C, 54.32;
H, 3.96; N, 16.53. Found: C, 54.11; H, 3.69; N, 16.86
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (9c)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfamethoxazole (5d) (126.64 mg, 0.50 mmol)
and acetic acid (15 mL) was refluxed for 4h. Work up afforded 9c
as a white powder (135.60 mg, 53%), mp 128-130 °C; ύ max
(atr)/cm-1 3422, 3086, 2972, 1673, 1603, 1567 and 1501; δH (500
MHz; DMSO-d6) 2.30 (3H, s, CH3), 3.88 (3H, s, OCH3), 4.79 (2H,
s, CH2), 6.05 (1H, s), 6.55 (1H, d, 3J = 5.0 Hz), 7.08 (2H, d, 3J =
8.0 Hz), 7.49 (2H, d, 3J = 8.0 Hz), 7.72 (2H, d, 3J = 8.5 Hz), 8.04
(2H, d, 3J = 8.0 Hz), 8.15 (1H, d, 3J = 5.0 Hz), 10.08 (1H, s) and
11.26 ppm (1H, s); δC (125 MHz; DMSO-d6) 12.08, 38.29, 55.55,
95.25, 104.12, 114.08, 118.87, 127.90, 128.56, 130.66, 131.83,
143.76, 155.96, 157.59, 159.23, 163.53, 169.50, 170.21 and
191.62 ppm; Anal. Calcd for C23H21N5O5S2: C, 54.00; H, 4.14; N,
13.69. Found: C, 54.35; H, 4.36; N, 13.25.
N-(4-Methylpyrimidin-2-yl)-4-((2-((2-oxo-2-phenylethyl)thio)
pyrimidin-4-yl)amino)benzenesulfonamide (12a)
Following the general procedure III, a mixture of 4a (132.36
mg, 0.50 mmol), sulfamerazine (5g) (132.15 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 12a as
a pale brown powder (162.22 mg, 66%); mp 238-240 °C; ύ max
(atr)/cm-1, 3438, 3351, 3099, 3044, 2924, 1679, 1611 and 1566; δH
(500 MHz; DMSO-d6) 2.29 (3H, s, CH3), 4.85 (2H, s, CH2), 6.55
(1H, d, 3J = 5.5 Hz), 6.88 (1H, d, 3J = 3.5 Hz), 7.53 (2H, t, 3J = 7.5
Hz), 7.66-7.69 (5H, m), 8.03 (2H, d, 3J = 7.5 Hz), 8.13 (1H, d, 3J =
5.5 Hz), 8.28 (1H, d, 3J = 3.5 Hz), 10.06 (1H, br.) and 11.60 ppm
(1H, br.); δC (125 MHz; DMSO-d6) 23.27, 38.56, 104.08, 114.82,
118.39, 126.32, 128.25, 128.46, 128.81, 128.99, 133.75, 135.68,
143.15, 155.66, 156.58, 159.25, 169.21, 172.03 and 193.49 ppm;
Anal. Calcd for C23H20N6O3S2: C, 56.08; H, 4.09; N, 17.06. Found:
C, 56.35; H, 4.27; N, 17.35.
4-((2-((2-Oxo-2-phenylethyl)thio)pyrimidin-4-yl)amino)-N-
(pyridin-2-yl)benzene sulfonamide (10a)
Following the general procedure III, a mixture of 4a (132.36
mg, 0.50 mmol), sulfapyridine (5e) (124.65 mg, 0.5 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 10a as
a pale brown powder (128.17 mg, 54%); mp 198-200 °C; ύ max
(atr)/cm-1 3437, 2927, 1673, 1633, 1569 and 1499; δH (400 MHz;
DMSO-d6) 4.84 (2H, s, CH2), 6.53 (1H, d, 3J = 5.2 Hz), 6.84-6.86
(1H, m), 7.07 (1H, d, 3J = 8.4 Hz), 7.55 (4H, d like, 3J = 6.8 Hz),
7.67 (4H, d, 3J = 7.2 Hz), 7.99-8.01 (1H, m), 8.05 (2H, d, 3J = 6.8
Hz), 8.11 (1H, d, 3J = 5.2 Hz), 10.00 (1H, s) and 11.71 ppm (1H,
br.); Anal. Calcd for C23H19N5O3S2: C, 57.85; H, 4.01; N, 14.67.
Found: C, 57.62; H, 4.31; N, 14.43.
4-((2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (12b)
4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(pyridin-2-yl)benzenesulfonamide (10b)
Following the general procedure III, a mixture of 4b (141.36
mg, 0.50 mmol), sulfamerazine (5g) (132.15 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 12b as
a pale brown powder (150.36 mg, 59%); mp 187-189 °C; ύ max
(atr)/cm-1 3436, 3099, 2925, 1676, 1600 and 1503; δH (500 MHz;
DMSO-d6) 2.29 (3H, s, CH3), 4.82 (2H, s, CH2), 6.54 (1H, d, 3J =
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfapyridine (5e) (124.65 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 10b as
a white powder (130.25 mg, 51%), mp 213-215 °C; ύ max (atr)/cm-
1 3432, 2925, 1674, 1636 and 1569; δH (400 MHz; DMSO-d6) 4.81
(2H, s, CH2), 6.53-6.54 (1H, m), 6.85-6.86 (1H, m), 7.09-7.11 (1H,
m), 7.50-7.52 (2H, m), 7.63-7.68 (3H, m), 7.75-7.76 (2H, m), 8.00-
8.10 (4H, m), 10.00 (1H, s) and 11.74 ppm (1H, br.); Anal. Calcd
3
5.0 Hz), 6.88 (1H, br.), 7.36 (2H, t like, J = 8.5 Hz), 7.69-7.74
(4H, m), 8.11-8.12 (3H, m), 8.29 (1H, d, 3J = 4.0 Hz), 10.04 (1H,
s) and 11.54 ppm (1H, br.); δC (125 MHz; DMSO-d6) 23.25, 38.43,
104.07, 114.88, 115.90 (d, J = 21.88 Hz), 118.47, 128.98, 131.28
11

(d, J = 9.6 Hz), 132.47 (d, J = 2.6 Hz), 143.19, 155.87, 156.59,
157.59, 159.23, 165.27 (d, J = 251.3 Hz), 168.27, 169.19, 172.03
and 192.24 ppm; Anal. Calcd for C23H19FN6O3S2: C, 54.11; H,
3.75; N, 16.46. Found: C, 54.42; H, 3.47; N, 16.19.
for each inhibitor. Concurrently, the un-catalyzed rates were
similarly determined and subtracted from the total observed rates.
A Stock solution of (0.1 mM) of the synthesized compounds or
AAZ in distilled-deionized water was prepared. Further dilutions
up to 0.01 nM were carried out using distilled-deionized water.
Before starting the assay, the tested inhibitors and enzyme
solutions were incubated together for 15 min at room temperature,
in order to allow the generation of the E-I complex. The inhibition
constants were calculated by the application of non-linear least-
squares methods using the Cheng-Prusoff equation. The results
were presented as the mean from at least three different
experiments.
4-((2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4-yl)-
amino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (12c)
Following the general procedure III, a mixture of 4c (149.59
mg, 0.50 mmol), sulfamerazine (5g) (132.15 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 12c as
a pale brown powder (180.46 mg, 69%), mp 211-213 °C; ύ max
(atr)/cm-1 3428, 3054, 2970, 2926, 1626, 1566 and 1499; δH (500
MHz; DMSO-d6) 2.29 (3H, s, CH3), 4.81 (2H, s, CH2), 6.55 (1H,
d, 3J = 5.5 Hz), 6.87 (1H, d, 3J = 4.0 Hz), 7.61 (2H, d, 3J = 8.0 Hz),
7.73 (2H, d, 3J = 8.0 Hz), 7.80 (2H, d, 3J = 8.0 Hz), 8.04 (2H, d, 3J
= 8.0 Hz), 8.11 (1H, d,3J = 5.5 Hz), 8.28 (1H, d,3J = 4.0 Hz), 10.06
(1H, s) and 11.70 ppm (1H, br.); δC (125 MHz; DMSO-d6) 23.27,
38.43, 104.12, 114.81, 118.55, 128.19, 128.54, 128.95, 129.03,
130.15, 134.42, 138.64, 142.34, 155.00, 155.79, 156.61, 159.23,
169.06 and 192.84 ppm; Anal. Calcd for C23H19ClN6O3S2: C,
52.42; H, 3.63; N, 15.95. Found: C, 52.74; H, 3.81; N, 15.68.
5.3. Molecular Docking
All studies of molecular modeling were carried out using the
software Molecular Operating Environment (MOE, 2010.10). All
minimizations were performed with MOE until an RMSD gradient
of 0.1 kcal∙mol−1Å−1 with MMFF94x force field and the partial
charges were automatically calculated. The X-ray crystallographic
structure of CAII co-crystallized with acetazolamide AAZ as
inhibitor (PDB ID: 3HS4) [50] were downloaded from the protein
data bank [https://www.rcsb.org/]. First, removal of water
molecules and ligands not engaged in binding in the active site
were carried out. The enzyme structure was then prepared with
default alternatives for the simulation of molecular docking using
Protonate 3D protocol in MOE. The enzyme structure was then
prepared for the molecular docking simulation using Protonate 3D
protocol in MOE with default options. The co-crystalized AAZ
was used to locate the binding site for molecular docking. Triangle
Matcher placement method and London dG scoring function were
used for docking using Zn2+ metal ion chelation as a constrain for
molecular docking. Docking protocol was first validated by self-
docking of the co-crystallized ligand AAZ in the enzyme active
site giving a docking pose with an energy score (S) = −7.68
kcal/mol and an RMSD of 0.867 Å from the co-crystalized ligand
pose (Figure 4). The validated molecular docking protocol was
then used to study the ligand-enzyme interactions of the newly
synthesized sulphonamide conjugates in the enzyme active site to
predict their binding pattern and to elucidate their SAR (Figure 5)
(For further details see SI) .
N-(4-Methylpyrimidin-2-yl)-4-((2-((2-oxo-2-(4-(trifluorometh-
yl)phenyl)ethyl)thio)pyrimidin-4-yl)amino)benzenesulfonamide
(12d)
Following the general procedure III, a mixture of 4e (166.36
mg, 0.50 mmol), sulfamerazine (5g) (132.15 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 12d as
a pale brown powder (184.65 mg, 66%); mp 210-212 °C; ύ max
(atr)/cm-1 3440, 2926, 2857, 1675, 1629 and 1572; δH (400 MHz;
DMSO-d6) 2.28 (3H, s, CH3), 4.86 (2H, s, CH2), 6.55 (1H, br.),
6.87 (1H, br.), 7.69-7.92 (8H, m), 8.11-8.28 (2H, m), 10.04 (1H,
s) and 11.82 ppm (1H, br.); Anal. Calcd for C24H19F3N6O3S2: C,
51.42; H, 3.42; N, 14.99. Found: C, 51.78; H, 3.15; N, 14.65.
4-((2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4-
yl)amino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (12e)
Following the general procedure III, a mixture of 4f (147.38
mg, 0.50 mmol), sulfamerazine (5g) (132.15 mg, 0.50 mmol) and
acetic acid (15 mL) was refluxed for 4h. Work up afforded 12e as
a white powder (190.44 mg, 73%), mp 230-232 °C; ύ max (atr)/cm-
Conflict of interest
1
3434, 3087, 2926, 2855, 1678, 1605 and 1565; δH (400 MHz;
The authors have no conflict of interest to declare.
Acknowledgments
DMSO-d6) 2.29 (3H, s, CH3), 3.86 (3H, s, OCH3), 4.79 (2H, s,
CH2), 6.54 (1H, d, 3J = 5.6 Hz), 6.87 (1H, d,3J = 5.0 Hz), 7.07 (2H,
d, 3J = 8.8 Hz), 7.69-7.74 (4H, m), 8.02 (2H, d, 3J = 8.8 Hz), 8.13
(1H, d, 3J = 5.6 Hz), 8.28 (1H, d, 3J = 5.2 Hz), 10.04 (1H, s) and
11.52 ppm (1H, br.); δC (125 MHz; DMSO-d6) 23.27, 38.23, 55.59,
104.03, 106.88, 114.11, 118.52, 121.77, 128.55, 129.02, 130.65,
143.21, 155.80, 159.26, 160.43, 163.57, 169.37, 171.16 and
191.88 ppm; Anal. Calcd for C24H22N6O4S2: C, 55.16; H, 4.24; N,
16.08. Found: C, 55.44; H, 4.51; N, 16.38.
Special thanks to Science and Technology Development Fund
(STDF) for supporting this work through the project ID 15063.
Three-dimensional molecular graphics were generated using the
UCSF Chimera package [51]. Chimera is developed by the
Resource for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco, funded by grants from the
National Institutes of Health National Center for Research
Resources (2P41RR001081) and National Institute of General
Medical Sciences (9P41GM103311).
5.2. Biological evaluation
5.2.1. Carbonic anhydrase inhibition
Photophysics stopped-flow instrument was utilized for
evaluating the CA catalyzed CO2 hydration activity [49]. Phenol
red (0.2 mM) was employed as an indicator. The assay was carried
out at absorbance maximum of 557 nm, using 20 mM Hepes (pH
7.5) as a buffer, and 20 mM Na2SO4 for keeping the ionic strength
of the used buffer constant. Initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10-100s were applied.
Subsequently, CO2 concentrations ranging from 1.7 to 17 mM
were applied for the calculating of the kinetic parameters and
inhibition constants. Minimum six traces of the initial 5 to 10% of
the reaction have been utilized for evaluating the initial velocity
References and notes
1. Supuran CT, De Simone G. Carbonic anhydrases: an overview, in: G. De
Simone, Claudiu T. Supuran (Eds.), Carbonic Anhydrases as Biocatalysts
–
from Theory to Medical and Industrial Applications, Elsevier,
Amsterdam, 2015.
2. Guler OO, Capasso C, Supuran CT. A magnificent enzyme superfamily:
carbonic anhydrases, their purification and characterization, J. Enzym.
Inhib. Med. Chem. 2016; 31: 689–694.
3. Lindskog S. Structure and mechanism of carbonic anhydrase, Pharmacol.
Ther. 1997; 74: 1–20.
12

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Supplementary Material
13

New
Thiopyrimidine-
Benzenesulfonamide Conjugates as
Selective Carbonic Anhydrase II
Inhibitors: Synthesis, in vitro Biological
Evaluation, and Molecular Docking
Studies
Heba T. Abdel-Mohsen*a, Ahmed M. El Kerdawy
b,c, Mohamed A. Omara, Emanuela Berrinod,
Ahmed S. Abdelsamiea,e, Hoda I. El Diwania and
Claudiu T. Supuran*d
aDepartment of Chemistry of Natural and Microbial Products, Division of
Pharmaceutical and Drug Industries Research, National Research Centre,
El‐Buhouth St., Dokki, P.O. Box 12622, Cairo, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo
University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza
University, New Giza, km 22 Cairo–Alexandria Desert Road, Cairo, Egypt
dUniversità degli Studi di Firenze, Department NEUROFARBA
–
Pharmaceutical and Nutraceutical section, University of Firenze, via Ugo
Schiff 6, I-50019, Sesto Fiorentino, Firenze, Italy.
eHelmholtz Institute for Pharmaceutical Research Saarland (HIPS)
-
Helmholtz Centre for Infection Research (HZI), Department of Drug Design
and Optimization, Campus Building E8.1, 66123, Saarbrücken, Germany.
Conflict of interest
The authors have no conflict of interest to declare.
14

 Compounds 6e and 6f showed Ki of 0.04 µM
against hCA II.
Highlights
New Thiopyrimidine-
 Compounds 6e and 6f demonstrated
selectivity of 15.8- to 980-fold towards hCA
II over hCA I, hCA IX, hCA XII isoforms.
 Molecular docking simulations of 6a-g with
hCA II revealed key H-bonding interactions
with the sulfonamide group and hydrophobic
interactions with the benzenesulfonamide
and thiopyrimidine moieties.
Benzenesulfonamide Conjugates as
Selective Carbonic Anhydrase II
Inhibitors: Synthesis, in vitro Biological
Evaluation, and Molecular Docking
Studies
Heba T. Abdel-Mohsen*a, Ahmed M. El
Kerdawy b,c, Mohamed A. Omara, Emanuela
Berrinod, Ahmed S. Abdelsamiea,e, Hoda I. El
Diwania and Claudiu T. Supuran*d
aDepartment of Chemistry of Natural and Microbial
Products, Division of Pharmaceutical and Drug
Industries Research, National Research Centre,
El‐Buhouth St., Dokki, P.O. Box 12622, Cairo,
Egypt
bDepartment of Pharmaceutical Chemistry, Faculty
of Pharmacy, Cairo University, Kasr El-Aini Street,
Cairo, P.O. Box 11562, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty
of Pharmacy, New Giza University, New Giza, km 22
Cairo–Alexandria Desert Road, Cairo, Egypt
dUniversità degli Studi di Firenze, Department
NEUROFARBA
–
Pharmaceutical
and
Nutraceutical section, University of Firenze, via
Ugo Schiff 6, I-50019, Sesto Fiorentino, Firenze,
Italy.
eHelmholtz Institute for Pharmaceutical Research
Saarland (HIPS) - Helmholtz Centre for Infection
Research (HZI), Department of Drug Design and
Optimization, Campus Building E8.1, 66123,
Saarbrücken, Germany.
 A
new
series
of
thiopyrimidine-
conjugates was
benzenesulfonamide
synthesized and evaluated for their CA
inhibitory activity.
 Compounds 6a-g displayed promising
inhibitory activity against cytosolic isoforms
hCA I and hCA II.
15