Synthesis and human carbonic anhydrase I, II, IX, and XII inhibition studies of sulphonamides incorporating mono-, Bi- and tricyclic imide moieties

Article abstract of DOI:10.3390/ph14070693

New derivatives were synthesised by reaction of amino-containing aromatic sulphonamides with mono-, bi-, and tricyclic anhydrides. These sulphonamides were investigated as human carbonic anhydrases (hCAs, EC 4.2.1.1) I, II, IX, and XII inhibitors. hCA I w

Full text of DOI:10.3390/ph14070693

pharmaceuticals
Article
Synthesis and Human Carbonic Anhydrase I, II, IX, and XII
Inhibition Studies of Sulphonamides Incorporating Mono-,
Bi- and Tricyclic Imide Moieties
Kalyan K. Sethi 1,
*
, KM Abha Mishra 1, Saurabh M. Verma 2, Daniela Vullo 3, Fabrizio Carta 3
and Claudiu T. Supuran 3,
*
1
2
3
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research Guwahati,
Assam 781101, India; mishraabha590@gmail.com
Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra,
Ranchi 835215, India; smverma@bitmesra.ac.in
Neurofarba Department, Università degli Studi di Firenze, Sezione di Farmaceutica e Nutraceutica, Via Ugo
Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; daniela.vullo@unifi.it (D.V.); farizio.carta@unifi.it (F.C.)
Correspondence: kalyansethi@gmail.com (K.K.S.); claudiu.supuran@unifi.it (C.T.S.)
*
Abstract: New derivatives were synthesised by reaction of amino-containing aromatic sulphonamides
with mono-, bi-, and tricyclic anhydrides. These sulphonamides were investigated as human carbonic
anhydrases (hCAs, EC 4.2.1.1) I, II, IX, and XII inhibitors. hCA I was inhibited with inhibition con-
stants (Kis) ranging from 49 to >10,000 nM. The physiologically dominant hCA II was significantly
inhibited by most of the sulphonamide with the Kis ranging between 2.4 and 4515 nM. hCA IX and
hCA XII were inhibited by these sulphonamides in the range of 9.7 to 7766 nM and 14 to 316 nM,
respectively. The structure–activity relationships (SAR) are rationalised with the help of molecular
docking studies.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Sethi, K.K.; Mishra, K.A.;
Verma, S.M.; Vullo, D.; Carta, F.;
Supuran, C.T. Synthesis and Human
Carbonic Anhydrase I, II, IX, and XII
Inhibition Studies of Sulphonamides
Incorporating Mono-, Bi- and Tricyclic
Imide Moieties. Pharmaceuticals 2021,
14, 693. https://doi.org/10.3390/
ph14070693
Keywords: human carbonic anhydrase inhibitors; benzenesulphonamide; anhydride; docking; SAR
1. Introduction
The zinc metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitously
found in most of the living creatures. CAs are encoded by eight genetically distinct CA
Academic Editor:
Jean Jacques Vanden Eynde
families [
differ by the oligomeric arrangement, three dimensional structures, location in cell, tissues
distribution, and catalytic properties [ ]. There are five cytosolic, five membrane bound,
two mitochondrial, and a secreted hCA isozyme [ ]. Three acatalytic hCA isoforms
are present whose functions are less well understood [ ]. hCAs catalyses the interconver-
1–3]. Human CAs (hCAs) belong to the α-CA. There are 15 such isoforms, which
Received: 30 June 2021
Accepted: 14 July 2021
Published: 19 July 2021
2
1–3
3
sion between CO2 and HCO3, being involved in many physiological processes, including
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
−
breathing, transport of CO2/HCO3 , pH homeostasis, biosynthetic reactions, electrolyte se-
cretion, and calcification. hCA inhibitors usually bind to the Zn2+ ion, although compounds
possessing different inhibition mechanisms were also described [1–3].
Several studies demonstrated that abnormal expression levels of hCA enzymes are
associated with different human diseases, making them a prominent target for the design
of modulators of their activity with biomedical applications [2,4–7]. hCA inhibitors were
clinically used as diuretics, for the management of glaucoma, epilepsy, and altitude sickness,
whereas their use in obesity, in the management of tumours and other pathologies were
validated recently (Table 1) [8–19]. The larger number of hCA isoforms and the new
applications of their inhibitors lead to the search for compounds with improved specificity
and selectivity profile for the targeted isoform, to avoid severe adverse effects due to
inhibition of other isoenzymes which are not relevant in the considered pathology [1–3].
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
The various hCA isoenzymes and their organ and tissue distribution, kinetics, and affinity
towards primary sulphonamides are presented in Table 1. The 3D structure elucidation
Pharmaceuticals 2021, 14, 693. https://doi.org/10.3390/ph14070693
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 693
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of many isoforms among which hCA I, II, IX, and XII has been reported [20] and showed
a rather close similarity in structure, i.e., a central twisted β-sheet bounded by helical
connections and added β-strands (Figure 1) [21].
The binding pocket is present in a large, conical cavity, which is ~12 Å wide and ~13
Å deep. The catalytic Zn2+ is present at the bottom of the cavity, being in a tetrahedral
geometry, coordinated by three histidine residues and a water molecule/hydroxide ion as
ligands (Figure 1) [21,22].
Figure 1. 3D multiple superpositions of four α-hCAs crystal structures (high sequence alignment homology) [23]. The
multiple superpositions involved the following crystal structures: 1AZM (hCA I); 1ZFQ (CA II); 3IAI (CA IX); and 1JD0 (CA
XII) with the Zn ion shown as red sphere, and its coordinating residues His 94, His 96, and His 119 shown in pink. The
protein backbone is shown in green [1,20].
Table 1. Main CA isoforms considered here and their distribution, location, off-targets, kinetic parameter [Kcat/Km
−1
s
−1)], and affinity towards sulphonamides [1,8–10,16–19].
(M
Affinity for
Sulphonamides
hCA
Distribution
Localization
Disease Involved
Offtargets
Kcat/Km
erythrocytes,
gastrointestinal (GI) tract,
eyes
retinal/cerebral
oedema
All other
isoforms
5.0 × 107
hCA I
cytosol
medium
glaucoma
oedema
hCA I
All other
isoforms
unknown
unknown
unknown
eyes, erythrocytes, bone
osteoclasts, GI tract,
kidneys, testis, lung,
brain
1.5 × 108
cytosol
very high
hCA II
epilepsy
altitude sickness
cancer
5.5 × 107
3.5 × 107
hCA IX
GI mucosa, tumours
transmembrane
transmembrane
cancer
hCA I and II
high
hCA I and II All
other isoforms
except hCA II
cancer
glaucoma
retinopathies
renal, germinal epithelia,
intestinal mucosa, eyes,
tumours
very high
hCA XII
hCAs I and II are involved in glaucoma, cerebral and macular oedema, epilepsy, and
mountain sickness [ 10 17 18]. hCA IX and XII are validated targets for hypoxia-induced
8–
,
,
cancers, being strongly overexpressed in many types of such tumours [2,16].
CA inhibitors (CAIs) coordinating to the zinc ion have been developed by incor-
porating various zinc bindings groups [1]. The classical sulphonamide one, is still a

Pharmaceuticals 2021, 14, 693
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key player in this field for the development of many classes of therapeutic agents [1,2].
Sulphonamides bind the Zn2+ ion of the enzyme by replacing the OH− ion coordinated to
the zinc (Figure 1) [20].
We report here the design of novel sulphonamide CAIs based on the “tail” approach [21
]
which consists of inserting moieties/fragments at the terminal part of the CAI molecule,
to induce the desired selectivity or physico-chemical properties (Figure 2). Various drug
design methods, i.e., ligand-based drug design (CoMFA/CoMSIA), pharmacophore-based
drug design, and structure-based drug design (docking study) have been employed to
design CAIs. It is rational drug design which helps to find new druggable targets, i.e.,
organic small molecule sulphonamides for enzyme inhibition. A validated CAI showing
acquiescence to the overall pharmacophore feature of is shown in Figure 2 [21–23]. These
approaches may produce more potent, more specific, acceptable water/lipid-soluble, and
good penetrable hCA inhibitors. Thus, the tails selected to be incorporated for the synthesis
of sulphonamides considered here are various aliphatic/aromatic anhydrides, which may
lead to new pharmacological properties [22–28].
Figure 2. Resulting designed structural element (HIT) as hCA inhibitor from the outcome of ligand and structure-based
drug design (CoMFA, CoMSIA, and pharmacophore modelling) [21].

Pharmaceuticals 2021, 14, 693
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2. Results and Discussion
2.1. Chemistry
Several sulphonamide derivatives incorporating phthalimido moieties were reported
earlier by our groups [21 23]. The compounds obtained from 3 and 4-nitrophthalic an-
hydride, 4,5,6,7-tetrabromophthalic anhydride, and 4,5,6,7-tetrachlorophthalic anhydride
showed good activity, acting as nanomolar inhibitors against hCA I, II, IX, and XII [22 23].
–
,
Sulphonamides containing phthalimide moieties were reported to possess antiglaucoma
activity in a rabbit model of the disease [22].
The synthesis of a series of 13 novel sulphonamides 1–13 is shown in Scheme 1. The
anhydrides reacted with the free NH2 group of aromatic sulphonamides and formed the
corresponding imides (Figure 3). The reaction between amino group and anhydrides
2
involves SN type reaction mechanism which resulted in rearrangement via a transition
state (Figure 3).
Scheme 1. Synthesis of sulphonamide derivatives 1–13 incorporating phthalic anhydride moiety to different benzene
sulphonamides [22 24 29]. n = 0 ( 10 11 12, and 13); n = 1 ( ); n = 2 ( ). -(CH2)n = para for 11 12
and 13; -(CH2)n = meta for ; -(CH2)n = ortho for 10 in the benzene ring. X = F ( 11); X = Cl ( 12); X = Br ( 13); Z = F ( ) in
–
,
1
,
6,
9,
,
,
7
2,
8
1,
2,
3,
4,
5
,
6,
7
,
8,
,
,
9
5,
3,
4,
5
the benzene ring of anhydride. i = glacial acetic acid; ii = stirring with heating.
2.2. Carbonic Anhydrases Inhibition Studies
The hCA I, II, IX, and XII inhibition results of sulphonamides 1–13, are showed in
Table 2. The inhibition studies of standard AAZ and other clinically used sulphonamides/
sulfamate are also considered for comparison and development of SAR. AAZ is a clinically
used drug for the adjunctive treatment of many diseases (Table 1) [8–10,17,18].

Pharmaceuticals 2021, 14, 693
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Figure 3. The proposed steps of the reaction mechanism.
Table 2. Human CAs inhibition data [30], selectivity ratios and docking scores of sulphonamides 1–13 and other clinically
used CAIs.
Ki (nM) *
Selectivity Ratio #
Comp. Code
1
Structures/Name
hCA I
hCA II
hCA IX
hCA XII
A
B
49 ± 3.2
13 ± 0.9
23 ± 1.8
742 ± 33
7766 ± 145
66 ± 5
0.002
0.196
2
3
309 ± 23
258 ± 12
7417 ± 348
933 ± 56
69 ± 4.5
80 ± 7
0.003
0.795
0.333
9.275
4
5
6
463 ± 29
400 ± 31
332 ± 17
44 ± 1.9
4.6 ± 0.3
7.1 ± 0.4
4034 ± 237
4898 ± 300
9.7 ± 0.8
87 ± 6
75 ± 4
33 ± 2
0.011
0.0
0.506
0.061
0.215
0.732
7
427 ± 31
5.2 ± 0.4
103 ± 5
300 ± 18
0.05
0.017

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Table 2. Cont.
Ki (nM) *
Selectivity Ratio #
Comp. Code
8
Structures/Name
hCA I
hCA II
hCA IX
hCA XII
A
B
326 ± 26
2.9 ± 0.2
53 ± 3
226 ± 13
0.055
0.013
0.088
9
444 ± 35
27.7 ± 1.5
498 ± 34
559 ± 26
316 ± 28
309 ± 24
0.056
8077
10
>10,000
4515 ± 325
14.616
11
12
368 ± 15
159 ± 12
3.4 ± 0.2
4.9 ± 0.3
88 ± 7
14 ± 1
0.039
0.1
0.243
0.223
49 ± 0.4
22 ± 1.7
13
281±15
2.4±0.1
12 ± 0.8
40±1.5
99±3.5
0.06
0.5
0.024
2.10
AAZ
250 ± 14
25 ± 1.1
5.7 ± 0.2
45,000 ±
BRZ
3 ± 0.1
9 ± 0.4
37 ± 1.4
52 ± 1.1
3 ± 0.2
0.1
0.2
1
3000
50,000 ±
DZA
3.5 ± 0.2
2.57
4500
EZA
IND
25 ± 1.8
31 ± 1.6
8 ± 0.4
34 ± 1.9
24 ± 1.4
22 ± 0.9
3.4 ± 0.2
0.2
0.6
0.36
4.41
15 ± 0.7
MZA
SLP
50 ± 3
14 ± 0.9
40 ± 2.1
27 ± 1.5
46 ± 3.2
3.4 ± 0.1
3.9 ± 0.2
0.5
0.9
4.11
1200 ± 69
10.25
TPM
ZNS
250 ± 16
10 ± 0.6
35 ± 1.2
58 ± 3.5
5.1 ± 0.3
3.8 ± 0.2
0.2
6.9
2.63
11,000 ±
56 ± 1.5
0.003
680
#
* Mean
±
standard error, from three different assays. Isozyme selectivity ratio A = hCA II/IX and B = hCA II/XII. AAZ = Acetazo-
lamide; BRZ = Brinzolamide; DZA = Dorzolamide; EZA = Ethoxzolamide; IND = Indisulam; MZA = Methazolamide; SLP = Sulpiride;
TPM = Topiramate; and ZNS = Zonisamide.

Pharmaceuticals 2021, 14, 693
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The following SAR was observed for this series of synthesised sulphonamides, after
comparison with the clinically used drugs shown in Table 2:
i.
The synthesised sulphonamides inhibited the cytosolic hCA I, with inhibition con-
stants (Ki) ranging from 49 nM to >10,000 nM (Table 2). The 4-(4-sulfo-1,8-napthalic-
1,3-dioxopyridine) potassium benzene sulphonamide 1 (Ki of 49 nM) was the most
potent inhibitor of the series, whereas the compound 3-chloro-4-(4,5,6,7-tetrachloro-
1,3-dioxoisoindolin-2-yl) benzene sulphonamide 12 with an inhibition constant of
159 nM better than AAZ and TPM (Table 2). In comparison to BRZ, CLX, DZR,
SLP, and VLX, all the synthesised compounds showed significant inhibition constant
(Table 2). The compound 10 is the weakest inhibitor that can be contemplated as a
positive feature of hCA I because it is abundantly found in RBCs and is undoubtedly
an off-target for other CAIs [
benzene and bulky aromatic group (-CH2CH2- or -CH2-) leads to a decreased activity
of compounds and with 427 nM to 332 nM, respectively compared to compound
. The position of -SO2NH2 plays an important role in increase or decrease of the
activities. The decreased activity of compounds 10 according to position of
2,28]. Increasing the chain length of carbon between the
7
8
6
6
> 9 >
-SO2NH2 in the benzene ring are 4th > 3rd > 2nd (Kis 332 nM to >10,000 nM). It is very
clear that –SO2NH2 present in the 2nd position comparatively very less active than
3rd than 4th due to steric hindrance. The electronegativity of halogen (F > Cl > Br) to
the benzene ring was found to influence the hCA I inhibition activity of compounds
11 (368 nM), 12 (159 nM), and 13 (281 nM).
ii. hCA II was significantly inhibited by many of the new sulphonamides, which exhib-
ited Kis ranging from 2.4 to 4515 nM (Table 2). Most of the compounds showed signif-
icant inhibition constants than clinically used standard AAZ and other sulphonamide
drugs. Compound 13 significantly inhibits hCA II with a Ki of 2.4 nM than the
other sulphonamides of the series. Although the compounds showed excellent to
moderate inhibition activity for hCA II. The SAR is straightforward. The increased
chain length of carbon between the benzene and bulky aromatic group (-CH2CH2- or
-CH2-) results in increased activities of the compounds 1 < 2 < 3 with Kis of 7.1 nM,
5.2 nM, and 2.9 nM, respectively. Due to steric hindrance, the hCA II inhibition activ-
ity was lost, i.e., the Ki of ortho, meta, and para position of –SO2NH2 in compound
10 < 9 < 6 was 4515, 27.7, and 7.1 nM, respectively). The electronegativity of halogen
(F > Cl > Br) attached to the benzene ring was found to influence the hCA II inhibi-
tion of compounds 11 (3.4 nM), 12 (4.9 nM), and 13 (2.4 nM). Similarly, benzthiazole
sulphonamide analogues 3 (742 nM) and 4 (44 nM). More electronegativity of halogen
lesser is the potency/efficacy. Overall, most of these sulphonamides showed a potent
action of inhibition against hCA II. hCA II is the main off-target isoform among
several hCAs [28].
iii. hCA IX was moderately to poorly inhibited by the sulphonamide (1–13) with Kis
ranging from 9.7 to 7766 nM (Table 2). Compound
6 potentially inhibited hCA
IX with a Ki of 9.7 nM than the standard AAZ. Although the sulphonamides are
moderate to poorly effective against hCA IX, but the SARs are generated for the
future development of more potent hCA IX inhibitors. The increase in the chain
length of the carbon between the two bulky groups (-CH2CH2- or -CH2-) results in
decreased activities of the compounds, i.e., compound
than compounds (Ki = 103 nM) and (Ki = 53 nM). The steric hindrance effects
cause a significant loss of the hCA IX inhibition properties of sulphonamides, i.e.,
–SO2NH2 at ortho, meta, and para in compound (Ki 498 nM) < 10 (Ki = 559 nM)
6 (Ki = 9.7 nM). The electronegativity of halogen (F > Cl > Br) in the benzene ring
6 (Ki = 9.7 nM) is more potent
7
8
9
≥
<
was found to be influencing the hCA IX inhibition of compounds 11 (88 nM), 12
(49 nM), and 13 (40 nM). More electronegative halogen atom attached to the benzene
ring, reduce the hCA IX inhibition properties.
iv. hCA XII was moderately inhibited by these sulphonamides (Table 2). The inhibition
constant showed Kis ranging from 14 to 316 nM. Compound 11 was the most potent

Pharmaceuticals 2021, 14, 693
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hCA XII inhibitor (Ki = 14 nM) of the series. The SARs of these sulphonamides were
developed for hCA XII inhibition. The increased carbon chain length (-CH2CH2- or
-CH2-) between benzene and anhydride decreased the activities of compounds
with Kis of 33 nM, 226 nM, and 300 nM, respectively. Steric hindrance responsible
for the loss of hCA XII inhibition properties, i.e., –SO2NH2 present in the ortho, para,
and meta substituted sulphonamides 10 (Kis in the range of 33, 309, and
6 > 8 >
7
9
<
< 6
316 nM), respectively. The electronegativity of halogen (F > Cl > Br) in the benzene
ring was found to be influencing the hCA XII inhibition of compounds 11 (14 nM),
12 (22 nM), and 13 (99 nM). More the electronegativity of halogen attached to the
benzene ring, increase the hCA XII inhibition properties.
v.
The close structural similarities in hCAs were the major challenge in finding novel
selective hCA isoenzyme inhibitors. Thus, calculation of the selectivity ratios has
been done (Table 2). The selectivity ratios for the tumour-associated isoforms hCA IX
and XII over hCA II, ranged from 1 to 8.077 nM and 0.013 to 14.616 nM, respectively
for the synthesised sulphonamide reported here (Table 2). Some compounds were
observed to have high selectivity ratios for hCA IX and XII over hCA II. Compound
10 was highly selective for hCA IX inhibition over hCA II. Similarly, compounds
3
and 10 were highly selective for hCA XII inhibition over hCA II. However, most of
the compounds had a low selectivity ratio, indicating that these are more selective for
inhibition of hCA I and II than hCA IX and CA XII.
2.3. Docking Studies
All the synthesised sulphonamides and clinically used sulfamate/sulphonamides
were docked to different hCA isoforms of interest, i.e., 1AZM (hCA I), 1ZFQ (hCA II),
3IAI (hCA IX), and 1JD0 (hCA XII) [20] to rationalize the observed SARs and to find
out the binding mode and other interactions. The Extra Precision docking results of the
co-crystallised ligand AAZ were between
obtained ranging from 1.8–2.3 which is better for docking studies.
−
3.8 to
−
4.8 (Table 3) and the RMSD values
Table 3. Human CAs docking scores of sulphonamides 1–13 and other clinically used CAIs.
Docking Score (Glide XP)
1AZM 1ZFQ 3IAI 1JD0
Docking Score (Glide XP)
1AZM 1ZFQ 3IAI 1JD0
Comp.
Code
Comp.
Code
1
2
3
4
5
6
7
8
9
−
−
−
−
−
−
−
−
−
−
−
8.124
5.822
4.331
4.156
5.760
5.827
5.314
6.118
5.922
4.138
5.999
−
9.218
5.079
4.148
−
7.749
5.264
3.922
4.067
5.293
4.771
5.118
4.900
4.951
4.842
5.392
−
4.209
4.948
4.013
4.257
4.159
4.533
3.241
4.870
4.467
1.699
3.343
12
13
−
−
5.886
6.072
−
4.679
4.654
−
5.134
4.595
−
3.626
3.647
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
AAZ
BRZ
DZA
EZA
IND
MZA
SLP
TPM
ZNS
−4.8
−5.5
−5.0
−5.7
−6.5
−4.9
−6.5
−6.1
−5.7
−3.8
−5.3
−4.5
−4.1
−5.0
−3.8
−6.0
−4.7
−3.7
−4.4
−4.9
−4.8
−5.1
−6.8
−4.9
−5.7
−5.0
−4.7
−4.1
−5.1
−4.4
−4.2
−5.2
−3.5
−4.9
−4.4
−4.5
−3.94
−
4.542
4.597
4.599
4.661
5.149
3.853
4.713
−
−
−
−
−
−
10
11
AAZ = Acetazolamide; BRZ = Brinzolamide; DZA = Dorzolamide; EZA = Ethoxzolamide; IND = Indisulam;
MZA = Methazolamide; SLP = Sulpiride; TPM = Topiramate; and ZNS = Zonisamide.
The docking results of the synthesised sulphonamides are listed in Table 3. The
synthesised compounds showed good binding interactions in the catalytic site of all hCAs.
For example, AAZ, the co-crystallised ligand showed a docking score of
−
4.4 with the hCA
IX protein (pdb code: 3IAI) binding interactions with various amino acids. It formed a
hydrogen bond with THR 199, THR 200, and two water molecules in the catalytic domain
(Figure 4a). The NH− of SO2NH2 interacted with Zn2+ (Figure 4a). Similarly, compound
1
showed the docking score of
H of -SO2NH2 bind to SER 6 (2.130 Å), O of C=O bind to HIS 64 (1.87 Å), and O of -C=O
bind to THR 199 (2.156 Å). In most of the cases, it was observed that the SARs tally the
−
7.749 with the hCA IX protein (pdb code: 3IAI) in which the

Pharmaceuticals 2021, 14, 693
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docking results obtained. For example, compounds
8,
6, and
7
showed the hCA inhibition
of 326 nM, 332 nM, and 427 nM, respectively. Comparing them to the docking scores,
they are in the same order, i.e., compounds , and have the docking scores of 6.118,
5.827, and 5.314, respectively (Table 3). The selected compounds showed good binding
interaction with the hCA I, II, IX, and XII.
8,
6
7
−
−
−
Figure 4. Protein ligand interactions in the 3IAI active site. (
THR 199, THR 200, and two water molecules; ( ) Compound
forms H-bonds with HIS 64 (1.87 Å), and THR 199 (2.156 Å).
a
) AAZ coordinates to the zinc ion and formed H-bond with
b
1, the deprotonate sulphonamide binds to the zinc ion and
Most abundant cytosolic hCA I, which is mainly present in the erythrocytes, GI tract,
and the eye is strongly inhibited by compounds , and 12 compared to the standard
1, 3
sulphonamide AAZ (Table 2). Thus, these compounds can be considered as candidates
for the treatment of retinal/cerebral oedema, ischemic diabetic cardiomyopathy, coronary
revascularization, diabetic retinopathy, etc. [
isozyme abundantly present in the erythrocytes, GI tract, eye, bone-osteoclasts, kidney,
lung, testis, and brain [ ] was significantly inhibited by compounds 11 12
and 13 compared to the standard AAZ (Table 2). These compounds can be much more
useful for the treatment of oedema [ ], glaucoma [ ], epilepsy [10], cancer [16], altitude
sickness [17], and acute altitude sickness (AAS) [32].
Compound also showed significant hCA IX inhibition activity. It can be further
8,9,18,31]. Similarly, hCA II, the cytosolic
2
1
,
5,
6,
7
,
8,
,
,
8
9
6
developed to a clinically meaningful agent for the treatment of hypoxia-induced cancer,
i.e., gliomas/ependymomas, mesotheliomas, carcinomas of the bladder, uterine cervix,
papillary/follicular carcinomas, nasopharyngeal carcinoma, head and neck, oesophagus,
lungs, breast, brain, vulva, squamous/basal cell carcinomas, and kidney tumours [2,16,22].
The compounds are moderately potent for the inhibition of transmembrane hCA XII. The
information would be very much useful for the design and development of more potent
druggable molecules.
BRZ and DZA are potent hCA II inhibitors used clinically for the treatment of glau-
coma or ocular hypertension. Compared to BRZ compounds
terms of hCA II inhibition. Similarly, in comparison to DZA compounds
8
and 13 are more potent in
, and 11
5, 6, 7, 8
are significantly inhibited hCA II (Table 2). Thus, these compounds can be developed as
drug molecules for the treatment of glaucoma or ocular hypertension. TPM and ZNS are
clinically used for the treatment of epilepsy, migraines, Parkinson’s disease, etc. Some of

Pharmaceuticals 2021, 14, 693
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the sulphonamides are better than these clinically used hCA inhibitors (Table 2). Further
pre-clinical and clinical trials are required to test these potent hCA inhibitors for their
clinical importance [2,33–36]. Patients affected by COVID-19 could show a dysregulated
acid–base status probably influenced by the CA activity, which is highly increased in
patients affected by COVID-19 infection. Thus, these compounds could be useful as an
adjunctive pharmacological treatment for COVID-19 [37].
3. Materials and Methods
3.1. Reagents and Instruments
All the reagents and solvents employed in this study were purchased in India and
Italy. Para aminobenzene sulphanilamide (Sigma-Aldrich, Bangalore, India), 4-sulfo-
1,8-napthalic anhydride potassium salt (Sigma-Aldrich, Bangalore, India), tetrachloroph-
thalic anhydride (Sigma-Aldrich, Bangalore, India), tetrabromophthalic anhydride (Sigma-
Aldrich, Bangalore, India), maleic anhydride (Sigma-Aldrich, Bangalore, India),
4-nitrophthalic anhydride (Sigma-Aldrich, Bangalore, India), glacial acetic acid (Merck,
Bangalore, India), chloroform, methanol (Merck, Bangalore, India), DMSO (CDH), and
silica gel 60 F254 plates (Merck Art.1.05554, Bangalore, India) were procured in India.
2-amino benzene sulphanilamide, 3-amino benzene sulphanilamide, para methyl amino
benzene sulphanilamide, para ethyl amino benzene sulphanilamide, 3-fluro para-amino
benzene sulphanilamide, 3-chloro para-amino benzene sulphanilamide, and 3-bromo para-
amino benzene sulphanilamide was arranged from Florence laboratory, Italy. TLC spots
were observed under the wavelength of 254 nm and 365 nm and/or by using ninhydrin
stain solution if required. FT-IR spectra were taken from Bruker, Alpha II and 8400S,
1
Shimadzu. H and 13C NMR spectra were characterised with the help of Bruker DRX-400
spectrometer, where DMSO-d6 was used as solvent and tetramethylsilane for the internal
standard. Chemical shifts of 1H NMR spectra are expressed in
δ (ppm) downfield from
tetramethylsilane and coupling constants (J) are expressed in Hertz. High-resolution elec-
tron ionization mass spectra were performed with the help of Water MicroMass ZQ, Waters
Alliance, Newtown, PA, USA. CHNSO elemental analysis was performed with the help of
Elementar, Vario EL III (Thermo Scientific™, Santa Clara, CA, USA).
3.2. Chemistry
Benzothiazole sulphonamide (6-sulfamido-2-amino-1,3-benzothiazole) was synthe-
sised using benzene sulphonamide and potassium thiocyanate in brominated glacial acetic
acid. The mixture was stirred for 20 h to get yellow coloured benzothiazole sulphonamide
product. The reaction mixture was then added into cold water, made alkaline with 50%
aqueous ammonia, and precipitated solids were filtered out and dried. The dried solids
obtained were re-crystallised with dry ethanol [38]. After that, desired sulphonamide and
respective anhydride in glacial acetic acid were refluxed and stirred under the nitrogen
environment for a suitable time. The reactions were monitored from time to time with the
help of TLC till the completion of the reaction. A total of compounds 1–13 were synthesised
(Scheme 1) [22–26].
3.2.1. Synthesis of 4-(4-Sulfo-1,8-napthalic-1,3-dioxopyridine) Potassium
Benzenesulphonamide (1)
A reaction containing 0.002 moles (0.344 g) of 4-aminobenzenesulphonamide and 0.002
moles (0.631 g) of 4-sulfo-1,8-napthalic anhydride potassium salt in glacial acetic acid (as sol-
◦
vent) was stirred at 130 C for 12 h under the nitrogen environment to produce 0.002 moles
of 4-(4-sulfo-1,8-napthalic-1,3-dioxopyridine) potassium benzenesulphonamide (1) (0.939
g). The reaction was checked every 30 min by using TLC (solvent system—chloroform:
methanol; 1:1). After 12 h and the confirmation of the completion of the reaction by TLC,
the reaction mixture was then cooled, and cold water (30 mL) was added. Filtration of
the product followed by washing with cold water frequently 4 to 5 times and further
recrystallization in ethanol provided compound [22–26].

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White crystalline and solid; yield% = 80%; m.p. = 277 ◦C; solubility; insoluble: acetic
acid glacial; partially soluble: ethanol; fully soluble: water, DMSO and methanol, chloro-
form. IRvmax (cm−1; KBr pellets); 1783.25, 1770.71 (C=O imide); 1304.89, 1159.26 (S=O) and
3525.99 (NH2), 9.40. Mass (ESI+); m/z: 431.0812 [M-K]+. 1H NMR (400 MHz, DMSO
−
d6):
δ
7.441 (s, 2H, SO2NH2), 7.848
7.580 7.601, 8.192–8.211, 9.264–9.286 (d, 3H, Ar
2H, Ar
−
7.888, 7.91
−
7.931 (d, 2H, Ar
−
H from benzenesulphonamide),
−
−
H from napthalic ring), 8.416
−
8.462 (t,
−
H from napthalic ring).13C NMR (100 MHz, DMSO
d6): 123.36, 124.03, 125.95,
127.30, 127.71, 128.66, 129.53, 130.88, 131.17, 131.38, 135.38, 140.03, 144.80, 151.11, 164.24,
164.68. Elem. Anal. calculated for C18H11KN2O7S2: C, 45.95; H, 2.36; K, 8.31; N, 5.95; O,
23.80; S, 13.63; found C, 45.91; H, 2.35; N, 5.91; O, 23.74; S, 13.62.
−
3.2.2. Synthesis of 4-(2-(2,5-Dioxo-2H-pyrrol-1(5H)-yl) Ethyl) Benzenesulphonamide (2)
A reaction containing 0.002 moles (0.401 g) of 4-(2-aminoethyl) benzenesulphonamide
and 0.002 moles (0.196 g) of furan-2,5-dione (maleic anhydride) in glacial acetic acid (as sol-
◦
vent) was stirred at 130 C for 12 h under the nitrogen environment to produce 0.002 moles
of 4-(2-(2,5-dioxo-2H-pyrrol-1(5H)-yl) ethyl)benzenesulphonamide (2) (0.560 g). The re-
action was checked every 30 min by using TLC (solvent system—chloroform: methanol;
1:1). After 12 h and the confirmation of the completion of the reaction by TLC, the reaction
mixture was then cooled, and cold water (30 mL) was added. Filtration of the product
followed by washing with cold water frequently 4 to 5 times and further recrystallization
in ethanol provided compound [22–26].
White crystalline and solid; yield% = 95%; m.p. = 210 ◦C; solubility; insoluble: water,
acetic acid glacial; partially soluble: ethanol and methanol; fully soluble: DMSO, chloro-
form. DMSO and methanol, chloroform. IRvmax (cm−1; KBr pellets); 1770.71, 1701.27 (C=O
imide); 1336.71, 1151.54 (S=O) and 3363.97 (NH2), 2987.84 (aliphatic CH2). Mass (ESI-)
m/z: 278.6341 [M 7.025 (s, 2H, SO2NH2), 7.335
H]. 1H NMR (400 MHz, DMSO-d6):
(d, 2H, Ar H from pyrrol), 7.386 7.407, 7.745 7.765 (d, 4H, Ar H from nitrobenzene),
2.922 2.958, 3.695 d6): 34.33,
3.731 (t, 2H, CH2 aliphatic). 13C NMR (100 MHz, DMSO
;
−
δ
−
−
−
−
−
−
−
38.99, 126.63, 130.06, 135.38, 143.26, 143.28, 171.66. Elem. anal. calculated for C12H12N2O4S:
C, 51.42; H, 4.32; N, 9.99; O, 22.83; S, 11.44; found C, 51.41; H, 4.317; N, 9.99; O, 22.83;
S, 11.34.
3.2.3. Synthesis of 2-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl)
Benzo[d]Thiazole-5-Sulphonamide (3)
A reaction containing 0.002 moles (0.558 g) of 2-aminobenzo[d]thiazole-5-sulphonamide
and 0.002 moles (0.572 g) of 4,5,6,7-tetrachlorophthalic anhydride in glacial acetic acid (as sol-
vent) was stirred at 130◦ C for 12 h under nitrogen environment to produce 0.002 moles of 2-
(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) benzo[d]thiazole-5-sulphonamide (3) (0.989 g).
The reaction was checked every 30 min by using TLC (solvent system—chloroform:
methanol; 1:1). After 12 h and the confirmation of the completion of the reaction by
TLC, the reaction mixture was then cooled, and cold water (30 mL) was added. Filtration
of the product followed by washing with cold water frequently 4 to 5 times and further
recrystallization in ethanol provided compound [22–26].
White amorphous and solid; yield% = 87.96%; m.p. = 302.2 ◦C; solubility; insoluble:
acetic acid glacial; partially soluble: ethanol, methanol; fully soluble: water, DMSO and
chloroform. IRvmax (cm−1; KBr pellets); 1730.21, 1776.50 (C=O imide); 1159.26, 1340.57
(S=O) and 3265.59 (NH2). Mass (ESI+); m/z: 498.3121 [M+1]. 1H NMR (400 MHz,
DMSO
zothiazole sulphonamide), 8.754
13C NMR (100 MHz, DMSO
d6): 121.51, 123.81, 125.21, 128.8, 129.73, 129.91, 133.46, 134.11,
134.71, 140.2, 141.88, 151.62, 155.45.
−
d6):
δ
7.537 (s, 2H, SO2NH2), 8.022
−
8.048, 8.239
−
8.261 (d, 2H, Ar
−
H from ben-
−
8.758 (s, H, Ar
−
H from benzothiazole sulphonamide).
−

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3.2.4. Synthesis of 2-(4,5,6,7-Tetrabromo-1,3-dioxoisoindolin-2-yl)
Benzo[d]Thiazole-5-Sulphonamide (4)
A reaction containing 0.002 moles (0.558 g) of 2-aminobenzo[d]thiazole-5-sulphonamide
and 0.002 moles (0.919 g) of 4,5,6,7-tetrabromophthalic anhydride in glacial acetic acid (as
◦
solvent) was stirred at 130 C for 12 h under nitrogen environment to produce 0.002 moles of
2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) benzo[d]thiazole-5-sulphonamide
(4)
(1.341 g). The reaction was checked every 30 min by using TLC (solvent system—chloroform:
methanol; 1:1). After 12 h and the confirmation of the completion of the reaction by TLC, the
reaction mixture was then cooled, and cold water (30 mL) was added. Filtration of product
followed by washing with cold water frequently 4 to 5 times and further recrystallization
in ethanol provided compound [22–26]
◦
Reddish white amorphous and solid; yield% = 51%; m.p. = 291.6 C; solubility;
insoluble: water, acetic acid glacial; partially soluble: ethanol and methanol; fully sol-
uble: DMSO, chloroform. IRvmax (cm−1; KBr pellets); 1739.85, 1786.14 (C=O imide);
1338.64, 1163.11 (S=O) and 3306.10 (NH2). Mass (ESI+); m/z: 674.6411 [M+1]. 1H
NMR (400 MHz, DMSO
Ar H from benzothiazole sulphonamide), 8.745
sulphonamide).13C NMR (100 MHz, DMSO
d6): 121.48, 122.41, 123.74, 125.17, 131.54,
−
d6):
δ
7.531 (s, 2H, SO2NH2), 8.018
−
8.044, 8.225
−
8.246 (d, 2H,
−
−
8.749 (s, H, Ar
−
H from benzothiazole
−
133.49, 136.98, 138.55, 141.8, 151.67, 155.66, 161.29, 166.76.
3.2.5. Synthesis of 3-Fluoro-4-(5-Nitro-1,3-dioxoisoindolin-2-yl) Benzenesulphonamide (5)
A reaction containing 0.002 moles (0.380 g) of 4-amino-2-fluorobenzenesulphonamide
and 0.002 moles (0.386 g) of 5-nitroisobenzofuran-1,3-dione in glacial acetic acid (as solvent)
was stirred at 130 ◦C for 12 h under nitrogen environment to produce 0.002 moles of 3-
fluoro-4-(5-nitro-1,3-dioxoisoindolin-2-yl) benzenesulphonamide (5) (0.720 g). The reaction
was checked every 30 min by using TLC (solvent system—chloroform: methanol; 1:1).
After 12 h and the confirmation of the completion of the reaction by TLC, the reaction
mixture was then cooled, and cold water (30 mL) was added. Filtration of the product
followed by washing with cold water frequently 4 to 5 times and further recrystallization
in ethanol provided compound [22–26].
◦
White crystalline and solid; yield% = 85%; m.p. = 232 C; solubility; insoluble:
water, acetic acid glacial; partially soluble: ethanol; fully soluble: DMSO, chloroform and
methanol. IRvmax (cm−1; KBr pellets); 1788.70, 1733.10 (C=O imide); 1349.25, 1162.15 (S=O)
and 3369.75 (NH2), 1541.18, 1386.86 (NO2). Mass (ESI-); m/z: 364.1251 [M
NMR (400 MHz, DMSO d6): 7.689 (s, 2H, SO2NH2), 7.834 7.872, 7.895 7.916 (d, 2H,
Ar H from benzenesulphonamide), 7.936–7.94 (s, H, Ar H from benzenesulphonamide),
8.314 8.335, 8.699 8.773 (d, 2H, Ar H from nitrobenzene), 8.694 8.695 (s, 2H, Ar
from nitrobenzene). 19F NMR (300 MHz, DMSO -116.373.13C NMR (100 MHz,
d6):
DMSO d6): 114.95, 115.17, 119.61, 122.82, 122.95, 123.26, 123.30, 126.31, 131.08, 132.15,
−
H]−. 1H
−
δ
−
−
−
−
−
−
−
−
−
H
−
δ
−
133.81, 137.01, 147.40, 147.46, 152.69, 156.30, 158.84, 164.96, 165.21.
3.2.6. Synthesis of 4-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl) Benzenesulphonamide (6)
An amount of 0.344 g (0.002 moles) 4-amino benzenesulphonamide stirred under
nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhydride to
produce 0.880 g (0.002 moles) of 4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) benzene-
sulphonamide (6
) in the presence of glacial acetic acid as solvent for 2 h at 130 ◦C. The
reaction was monitored each 30 min with the help of TLC (chloroform: methanol; 3:1). After
that the mixture was cooled and 30 mL of cold water was added. The product was filtered
and washed with cold water repeatedly for 4/5 times. The product was recrystallised in
ethanol to purify the synthesised compound 6.
White crystalline and solid; yield% = 98%; mp = 315–320 ◦C (decompose); solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol; fully soluble: DMSO
and methanol, chloroform. IRvmax (cm−1; KBr pellets); 1778.43, 1716.70 (C=O imide);
1
1321.28, 1163.11 (S=O) and 3414.12 (NH2). Mass (ESI+); m/z: 438.8015 [M+H]+. H NMR

Pharmaceuticals 2021, 14, 693
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(400 MHz, DMSO-d6): δH 7.533 (s, 2H, SO2NH2), 7.699–7.678 (d, 2H, Ar-H from ben-
zenesulphonamide), 8.050–8.029 (d, 2H, Ar-H from benzenesulphonamide). 13C NMR
(100 MHz, DMSO-d6): δC 167.29, 144.38, 138.98, 134.49, 128.9, 128.8, 128.23, 127.04.
3.2.7. Synthesis of 4-((4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl) Methyl)
Benzenesulphonamide (7)
An amount of 0.445 g (0.002 moles) of 4-(aminomethyl)benzenesulphonamide hy-
drochloride stirred under nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-
tetrachlorophthalic anhydride to produce 0.980 g (0.002 moles) of 4-(2-(4,5,6,7-tetrachloro-
1,3-dioxoisoindolin-2-yl) ethyl)benzenesulphonamide hydrochloride (7) in the presence of
glacial acetic acid as solvent for 1.5 h at 130 ◦C. The reaction was monitored each 30 min
with the help of TLC (chloroform: methanol; 1:1). After that the mixture was cooled and
30 mL of cold water was added. The product was filtered and washed with cold water re-
peatedly for 4/5 times. The product was recrystallised in ethanol to purify the synthesised
compound 7.
White crystalline and solid; yield% = 90%; mp = 305 ◦C; Solubility; insoluble: water,
acetic acid glacial; partially soluble: ethanol and methanol; fully soluble: DMSO, chloro-
form. IRvmax (cm−1; KBr pellets); 1774.57, 1714.77 (C=O imide); 1338.64, 1161.19 (S=O) and
1
3350.46 (NH2). Mass (ESI+); m/z: 451.8231 [M], 452.8101 [M+H]+. H NMR (400 MHz,
DMSO-d6): δH 4.891 (s, 2H, CH2), 7.38 (s, 2H, SO2NH2), 7.565, 7.586 (d, 2H, Ar-H from
benzenesulphonamide), 7.81, 7.831 (d, 2H, Ar-H from benzenesulphonamide). 13C NMR
(100 MHz, DMSO-d6): δC 164.28, 144.26, 140.63, 139.1, 129.49, 129.1, 128.89, 126.81, 42.1.
3.2.8. Synthesis of 4-(2-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl) Ethyl)
Benzenesulphonamide (8)
An amount of 0.400 g (0.002 moles) of 4-(2-aminoethyl) benzenesulphonamide stirred
under nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic an-
hydride to produce 0.936 g (0.002 moles) of 4-(2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)ethyl)benzenesulphonamide (
8
) in the presence of glacial acetic acid as solvent for
◦
1.5 h at 130 C. The reaction was monitored each 30 min with the help of TLC (chloroform:
methanol; 1:1). After that the mixture was cooled and 30 mL of cold water was added. The
product was filtered and washed with cold water repeatedly for 4/5 times. The product
was recrystallised in ethanol to purify the synthesised compound 8.
White crystalline and solid; yield% = 98%; mp = 286 ◦C; solubility; insoluble: water,
acetic acid glacial; partially soluble: ethanol, methanol; fully soluble: DMSO and chloro-
form. IRvmax (cm−1; KBr pellets); 1776.50, 1712.92 (C=O imide); 1303.92, 1159.29 (S=O)
and 3387.11 (NH2), 2955.04 (aliphatic CH2). Mass (ESI+); m/z: 465.2237 [M]+. H NMR
1
(400 MHz, DMSO-d6): δH 3.011–3.048 (t, 2H, CH2), 3.849–3.885 (t, 2H, CH2), 7.347 (s,
2H, SO2NH2), 7.473–7.494 (d, 2H, Ar-H from benzenesulphonamide), 7.763–7.785 (d, 2H,
Ar-H from benzenesulphonamide). 13C NMR (100 MHz, DMSO-d6): δC 164.19, 143.41,
138.143.21, 139.17, 130.16, 129.23, 129.03, 126.81, 34.13.
3.2.9. Synthesis of 3-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl) Benzenesulphonamide (9)
An amount of 0.344 g (0.002 moles) of 3-amino benzenesulphonamide stirred under
nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhydride to
produce 0.880 g (0.002 moles) of 3-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) benzene-
sulphonamide (9
) in the presence of glacial acetic acid as solvent for 2 h at 130 ◦C. The
reaction was monitored each 30 min with the help of TLC (chloroform: methanol; 3:1). After
that the mixture was cooled and 30 mL of cold water was added. The product was filtered
and washed with cold water repeatedly for 4/5 times. The product was recrystallised in
ethanol to purify the synthesised compound 9.
White crystalline and solid; yield% = 92%; mp = 308 ◦C; Solubility; insoluble: water,
acetic acid glacial; partially soluble: ethanol and methanol; fully soluble: DMSO, chloro-
form. IRvmax (cm−1; KBr pellets); 1782.29, 1718.68 (C=O imide); 1301.99, 1132.25 (S=O) and
1
3356.25 (NH2). Mass (ESI+); m/z: 437.7912 [M], 438.7103 [M+H]+. H NMR (400 MHz,

Pharmaceuticals 2021, 14, 693
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DMSO-d6): δH 7.61 (s, 2H, SO2NH2), 7.7–7.72 (s, H, Ar-H from benzenesulphonamide),
7.799–7.84 (t, H, Ar-H from benzenesulphonamide), 7.969–7.988 (d, 2H, Ar-H from benzene-
sulphonamide). 13C NMR (100 MHz, DMSO-d6): δC 163.42, 145.99, 139.4, 132.56, 131.68,
130.92, 129.39, 126.86, 125.6.
3.2.10. Synthesis of 2-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl) Benzenesulphonamide (10)
An amount of 0.344 g (0.002 moles) of 2-amino benzenesulphonamide stirred under
nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhydride to
produce 0.880 g (0.002 moles) of 2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) benzene-
sulphonamide (10) in the presence of glacial acetic acid as solvent for 2 h at 130 ◦C. The
reaction was monitored each 30 min with the help of TLC (chloroform: methanol; 3:1). After
that the mixture was cooled and 30 mL of cold water was added. The product was filtered
and washed with cold water repeatedly for 4/5 times. The product was recrystallised in
ethanol to purify the synthesised compound 10.
White crystalline and solid; yield% = 90%; mp = 313 ◦C; Solubility; insoluble: water,
acetic acid glacial; partially soluble: ethanol and methanol; fully soluble: DMSO and
chloroform. IRvmax (cm−1; KBr pellets); 1782.29, 1712.85 (C=O imide); 1305.85, 1139.97
1
(S=O) and 3387.11 (NH2). Mass (ESI+); m/z: 437.7154 [M]+, 438.7152 [M+H]+. H NMR
(400 MHz, DMSO-d6): δH 7.661 (s, 2H, SO2NH2), 7.802–7.844 (t, 2H, Ar-H from benzene-
sulphonamide), 8.102–8.107, 8.1–22-8.126 (d, 2H, Ar-H from benzenesulphonamide). 13C
NMR (100 MHz, DMSO-d6): δC 165.71, 163.11, 143.27, 139.53, 134.05, 132.74, 131.63, 129.62,
129.48, 129.26.
3.2.11. Synthesis of 3-Fluoro-4-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl)
Benzenesulphonamide (11)
An amount of 0.380 g (0.002 moles) of 4-amino-2-fluoro benzenesulphonamide stirred
under nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhy-
dride to produce 0.916 g (0.002 moles) of 3-fluoro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl) benzenesulphonamide (11) in the presence of glacial acetic acid as solvent for 12 h
◦
at 130 C. The reaction was monitored each 30 min with the help of TLC (chloroform:
methanol; 3:1). After that the mixture was cooled and 30 mL of cold water was added. The
product was filtered and washed with cold water repeatedly for 4/5 times. The product
was recrystallised in ethanol to purify the synthesised compound 11.
◦
White crystalline and solid; yield% = 95%; mp = 278 C; Solubility; insoluble: wa-
ter, acetic acid glacial; partially soluble: ethanol; fully soluble: DMSO, chloroform and
methanol. IRvmax (cm−1; KBr pellets); 1790.00, 1720.56 (C=O imide); 1301.99, 1168.90
1
(S=O) and 3385.18 (NH2). Mass (ESI+); m/z: 455.7312 [M]+, 456.7317 [M+H]+. H NMR
(400 MHz, DMSO-d6): δH 7.704 (s, 2H, SO2NH2), 7.796–7.835 (m, H, Ar-H from ben-
zenesulphonamide), 7.893–7.941 (d, 2H, Ar-H from benzenesulphonamide). 13C NMR
(100 MHz, DMSO-d6): δC 162.74, 159.07, 156.53, 147.76, 139.89, 132.35, 129.78, 123.52,
122.74, 115.39.
3.2.12. Synthesis of 3-Chloro-4-(4,5,6,7-Tetrachloro-1,3-Dioxoisoindolin-2-yl)
Benzenesulphonamide (12)
An amount of 0.412 g (0.002 moles) of 4-amino-2-chloro benzenesulphonamide stirred
under nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhy-
dride to produce 0.949 g (0.002 moles) of 3-chloro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)be◦nzenesulphonamide (12) in the presence of glacial acetic acid as solvent for 12 h
at 130 C. The reaction was monitored each 30 min with the help of TLC (chloroform:
methanol; 3:1). After that the mixture was cooled and 30 mL of cold water was added. The
product was filtered and washed with cold water repeatedly for 4/5 times. The product
was recrystallised in ethanol to purify the synthesised comp◦ound 12.
White crystalline and solid; yield% = 94%; mp = 303 C; solubility; insoluble: wa-
ter, acetic acid glacial; partially soluble: ethanol; fully soluble: DMSO, chloroform and

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methanol. IRvmax (cm−1; KBr pellets); 1788.07, 1722.49 (C=O imide); 1305.87, 1172.76 (S=O)
and 3387.11 (NH2). Mass (ESI+); m/z: 472.6115 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): δH 7.724 (s, 2H, SO2NH2), 7.861–7.882 (d, H, Ar-H from benzenesulphonamide),
8.017–8.042 (m, H, Ar-H from benzenesulphonamide), 8.155–8.16 (d, H, Ar-H from ben-
zenesulphonamide). 13C NMR (100 MHz, DMSO-d6): δC 162.64, 147.69, 140.16, 133.91,
133.12, 132.82, 129.91, 129.11, 129.31, 126.65.
3.2.13. Synthesis of 3-Bromo-4-(4,5,6,7-Tetrachloro-1,3-Dioxoisoindolin-2-yl)
Benzenesulphonamide (13)
An amount of 0.502 g (0.002 moles) of 4-amino-2-bromo benzenesulphonamide stirred
under nitrogen environment with 0.572 g (0.002 moles) of 4,5,6,7-tetrachlorophthalic anhy-
dride to produce 1.038g (0.002 moles) of 3-bromo-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl) benzenesulphonamide (13) in the presence of glacial acetic acid as solvent for 12 h
◦
at 130 C. The reaction was monitored each 30 min with the help of TLC (chloroform:
methanol; 3:1). After that the mixture was cooled and 30 mL of cold water was added. The
product was filtered and washed with cold water repeatedly for 4/5 times. The product
was recrystallised in ethanol to purify the synthesised compound 13.
White crystalline and solid; yield% = 90%; mp = 265 ◦C; Solubility; insoluble: Wa-
ter, acetic acid glacial; partially soluble: ethanol; fully soluble: DMSO, chloroform and
methanol. IRvmax (cm−1; KBr pellets); 1772.64, 1720.56 (C=O imide); 1300.00, 1170.83 (S=O)
and 3400.0 (NH2). Mass (ESI+); m/z: 516.5214 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): δH 7.715 (s, 2H, SO2NH2), 7.842–7.863 (d, H, Ar-H from benzenesulphonamide),
8.051–8.077 (d, H, Ar-H from benzenesulphonamide), 8.292–8.297 (d, H, Ar-H from ben-
zenesulphonamide). 13C NMR (100 MHz, DMSO-d6): δC 162.64, 147.69, 140.16, 133.91,
133.12, 132.82, 129.91, 129.11, 129.31, 126.65.
3.3. CA Inhibition Assay
To check the CA-catalyzed CO2 hydration activity an applied photo physics stopped-
flow instrument has been used. A phenol red indicator (0.2 mM) has been used at the
λ
max
of 557 nm, with 20 mM Hepes buffer (pH 7.5) and Na SO (20 mM), following the
2 4
initial rates of the CA-catalysed CO2 hydration reaction for 10 to100 s. To determine the
kinetic parameters and inhibition constants, 1.7 to 17 mM CO2 concentrations were used.
To determine each inhibitor’s initial velocity minimum of six traces have been used from
the initial 5–10% of the reaction. A similar process has been applied to determine the
uncatalyzed rates. Further, the uncatalyzed rates were deducted from the total observed
rates found. 0.1 mM solution of stock inhibitors were prepared, and dilutions of up to
0.01 nM were done. A preincubation of inhibitor and CA solutions was done for 15 min at
room temperature for allowing the formation of the enzyme-inhibitor adduct. The PRISM
3 software was used to obtain inhibition constants by nonlinear least-squares methods [30].
3.4. Molecular Docking Study
Molecular docking experiment was executed in Maestro Glide of Schrödinger [39,40].
GLIDE XP was used for the study of protein-ligand interactions of the hCA isozymes. The
best pose of each ligand was ranked, and the docking score is based on Chem Score. The
co-crystallised ligand with RMSD value of 1.5 Å, representing that GLIDE is an impeccable
platform for docking study of hCAIs.
4. Conclusions
The synthesis and characterization of a series of sulphonamides 1–13 are described.
2
The reaction between amino group and anhydrides involves SN type reaction mechanism,
in which amino-containing sulphonamide derivatives react with aliphatic/aromatic an-
hydrides. These sulphonamides were tested against four physiologically similar hCA I,
II, IX, and XII isoforms. The relationships between the chemical structure of a molecule
and its hCA inhibition activity (SAR) were established and rationalised with the help of
molecular docking studies. These sulphonamides were potent inhibitors of hCA I and II

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in nM inhibition constant. The compounds inhibited the transmembrane isoforms hCA
IX and XII with moderate to high nM concentration. The sulphonamides reported here
had a moderate to poor selectivity ratio. Therefore, these are better hCA I and II selective
inhibitors than hCA IX and XII. Such compounds may be useful as an interesting candi-
date for the drug development of highly selective and efficacious novel sulphonamides
for hypoxia-induced cancer drug therapy and retinopathies. The sulphonamides which
potentially inhibited hCA I and II can be further developed as a novel interesting clinical
candidate for the treatment of retinal/cerebral oedema, ischemic diabetic cardiomyopa-
thy, coronary revascularization, diabetic retinopathy, epilepsy, cancer, altitude sickness,
COVID-19, etc.
Author Contributions: K.K.S. Conceptualization, methodology, data analysis, writing-original draft
preparation, review, and editing; K.A.M. software and editing; S.M.V. conceptualization, guidance;
D.V. enzyme investigation; F.C. spectral investigation, review, and editing; C.T.S. funding acquisi-
tion, writing—review and editing. All authors have read and agreed to the published version of
the manuscript.
Funding: Florence laboratory work was financed by the Italian Ministry for University and Research
(MIUR), grants PRIN prot. 2017XYBP2R and FISR2019_04819 BacCAD.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data available within the article.
Acknowledgments: This work is acknowledged to U. S. N. Murty, Director, NIPER-Guwahati for
his constant support for the research and development. We are also very much thankful to the
Vice-Chancellor and Head, Department of Pharmaceutical Sciences & Technology, BIT Mesra, Ranchi
for creating opportunities for some part of research work over there. The work from Florence
laboratory was financed by the Italian Ministry for University and Research (MIUR), grants PRIN
prot. 2017XYBP2R and FISR2019_04819 BacCAD.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Abbreviations
AAZ
ASN
BRZ
BZA
CA
Acetazolamide
asparagines
brinzolamide
benzolamide
carbonic anhydrase
C:H:N:S:O carbon:hydrogen:nitrogen:sulphur:oxygen
CAI
carbonic anhydrase inhibitor
central nervous system
dimethyl sulfoxide
Dorzolamide
CNS
DMSO
DZA
E-I
enzyme-inhibitor
EZA
FT-IR
GLN
hCA
HIS
ethoxzolamide
Fourier transform infrared
glutamine
human carbonic anhydrase
Histidine
HRMS
IND
high-resolution mass spectrometry
indisulam
mM
milimolar
MZA
NMR
methazolamide
nuclear magnetic resonance

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nM
nanomolar
PDB
protein data bank
RMSD Root mean square deviation
SAR
SLP
THR
TRP
TPM
UV
structure–activity relationship
Sulpiride
threonine
tryptophan
topiramate
ultraviolet
XP
Zn
Extra precision
Zinc
ZNS
zonisamide
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