Carbonic anhydrase inhibitors: Synthesis and inhibition of the cytosolic mammalian carbonic anhydrase isoforms I, II and VII with benzene sulfonamides incorporating 4,5,6,7-tetrachlorophthalimide moiety

Article abstract of DOI:10.1016/j.bmc.2013.06.035

A series of 4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzene sulfonamides incorporating primary amino moieties with 4,5,6,7-tetrachlorophthalic anhydride. These sulfonamides were assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some of these compounds showed very good in vitro human carbonic anhydrase (hCA) isoforms I, II and VII inhibitory properties, with affinities in the low nanomolar range. Inhibition activities against hCA I were in the range of 159-444 nM; against hCA II in the range of 2.4-4515 nM, and against hCA VII in the range of 1.3-469 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoform being established.

Full text of DOI:10.1016/j.bmc.2013.06.035

Bioorganic & Medicinal Chemistry 21 (2013) 5168–5174
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors: Synthesis and inhibition
of the cytosolic mammalian carbonic anhydrase
isoforms I, II and VII with benzene sulfonamides
incorporating 4,5,6,7-tetrachlorophthalimide moiety
a
Kalyan K. Sethi a, , Saurabh M. Verma , Muhammet Tanç b,c, Fabrizio Carta b, Claudiu T. Supuran b,c,
⇑
⇑
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, India
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, Sesto Fiorentino, Firenze 50019, Italy
c Università degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical Chemistry, Via Ugo Schiff 6, Sesto Fiorentino, Florence 50019, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds
1–8) was synthesized by reaction of benzene sulfonamides incorporating primary amino moieties with
4,5,6,7-tetrachlorophthalic anhydride. These sulfonamides were assayed as inhibitors of the metalloen-
zyme carbonic anhydrase (CA, EC 4.2.1.1). Some of these compounds showed very good in vitro human
carbonic anhydrase (hCA) isoforms I, II and VII inhibitory properties, with affinities in the low nanomolar
range. Inhibition activities against hCA I were in the range of 159–444 nM; against hCA II in the range of
2.4–4515 nM, and against hCA VII in the range of 1.3–469 nM. The structure–activity relationship (SAR)
with this series of sulfonamides is straightforward, with the main features leading to good activity for
each isoform being established.
Received 27 May 2013
Revised 12 June 2013
Accepted 14 June 2013
Available online 26 June 2013
Keywords:
Human carbonic anhydrase inhibitors
Benzenesulfonamide
4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl
benzenesulfonamide
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
by using a metal hydroxide nucleophilic mechanism and other
and are involved in physiological processes connected with respi-
Carbonic anhydrases (CAs) are metalloenzymes present in al-
most all living organism.1,2 There are five genetically distinct CA
ration and transport of CO2 or bicarbonate ion, CO2 homeostasis,
electrolyte secretion in many tissues, biosynthetic reactions, calci-
fication, etc. Inhibition and activation of these enzymes are well
understood processes, with most classes of inhibitors binding to
the metal centre, and activators binding at the entrance of the ac-
tive site cavity.1–3
families;
green plants); the b-CAs (bacteria, algae and chloroplasts of
monocotyledons and dicotyledons); the -CAs (archaea and some
a-CAs (vertebrates, bacteria, algae and cytoplasm of
c
bacteria); the d-CAs (marine diatoms) and f-CAs (bacteria, chemo-
lithotrophs and marine cyanobacteria).1,3 Human CAs (hCAs) all
Several studies demonstrated important roles of CAs in a variety
of physiological processes, and showed that abnormal levels or
activities of these enzymes have been often associated with differ-
ent human diseases.2 In the last few years, several CA isozymes
have become interesting targets for the design of inhibitors or
activators with biomedical applications.4–7 Indeed originally CA
inhibitors (CAIs) were clinically used (Fig. 1) as diuretic,8 antiglau-
coma,9 and anticonvulsant,10 whereas their employment as
antiobesity drugs11 or in the management of hypoxic tumors were
only recently validated.12–16 Examples of clinically used CAIs:
acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA,
dichlorophenamide DCP, dorzolamide DZA, brinzolamide BRZ, ben-
zolamide BZA, topiramate TPM, zonisamide ZNS, sulpiride SLP,
indisulam IND, celecoxib CLX and valdecoxib VLX. However, be-
cause of the large number of hCA isoforms,1–3 there is a constant
need to improve the inhibition and selectivity profile of the so
belong to the
a-family and are present in fifteen isoforms, which
differ by molecular features, oligomeric arrangement, cellular
localization, distribution in organs and tissues, expression levels,
and kinetic properties (Table 1).2 There are five cytosolic forms
(CA I, CA II, CA III, CA VII and CA XIII), five membrane associated
isozymes (CA IV, CA IX, CA XII, CA XIV and CA XV), two mitochon-
drial forms (CA VA and CA VB), and a secreted CA isozyme (CA
VI).1–3 There are three additional ‘acatalytic’ CA isoforms (CA VIII,
CA X, and CA XI) whose functions remain unclear.3 CA which cata-
lyze the interconversion between carbon dioxide and bicarbonate
⇑
Corresponding authors. Tel.: +91 898 751 1080; fax: +91 651 227 5401 (K.K.S.);
tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: kalyansethi@gmail.com (K.K. Sethi), claudiu.supuran@unifi.it
(C.T. Supuran).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.035

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
5169
Table 1
Organ/tissue distribution, subcellular localization, CO2 hydrase activity, and affinity for sulfonamides of the human CA isozymes (hCA I, II, VII)1
CA
Organ/tissue distribution
Subcellular localization
Catalytic activity (CO2 hydration)
Affinity for sulfonamides
CA I
CA II
Erythrocytes gastrointestinal tract, eye
Erythrocytes, eye, gastrointestinal tract,
bone osteoclasts, kidney, lung, testis, brain
Central nervous system, liver
Cytosol
Cytosol
Low
High
Medium
Very high
CA VII
Cytosol
High
Very high
H
O
CH3
N
O
S
N
O
S
S
N
O
O
CH3
S
S
H2N
H2N
H2N
S
CH3
O
N
N
N
O
N
CH3
O
O
AAZ
MZA
EZA
O
O
CH3
O
S
S
CH3
S
O
O
NH
NH2
H2N
S
O
S
O
O
O
O
O
NH2
O
N
O
O
HN
DZA
S
S
O
O
CH3
TPM
BRZ
O
Cl
O
NH2
O
S
N
N
O
S
O
N
H
O
O
N
H
H2N
H2N
S
NH
S
H3CO
O
O
O
SLP
IND
NH2
ZNS
O
S
O
F
F
N
N
F
H3
C
O
CLX
S
O
N
H2N
O
VLX
Figure 1. Clinically used sulfonamides and sulfamates.
far developed CAIs, to avoid side effects due to inhibition of iso-
forms not involved in a certain pathology.2
add to the metal co-ordination sphere, leading to pentaco-ordi-
nated Zn (II) complexes.3,4
A brief presentation of the hCA isoforms (hCA I, hCA II, hCA VII)
as drug targets/off-targets is shown in Table 2. Specifically, CA I is
found in many tissues, but a study from Feener’s group19 demon-
strated that this enzyme is involved in retinal and cerebral edema,
and its inhibition may be a valuable tool for fighting these condi-
tions. CA II is involved in several diseases, such as glaucoma, ede-
ma, epilepsy, and altitude sickness.8–10,17,18 CA VII has been noted
for its contributions to epileptic activity together with CA II and
XIV.10
The potent CAIs incorporate various zinc-binding groups (ZBGs)
and thus belong to various chemotypes, with the classical sulfon-
amide one, still constituting the main player in the field. The sul-
fonamides led to the development of several classes of
pharmacological agents. Sulfonamide inhibitors directly bind to
the metal ion within the enzyme active site, by substituting the
zinc-bound hydroxide ion (Fig. 2). Other CAIs, such as some anions,
These three isoforms are highly related to the central nervous
system and responsible for many disease explained earlier and
the similar compounds reported earlier were tested as CA I, II
Table 2
hCA Isoforms as drug targets/offtargets in various diseases1
Isoform Disease in which is
involved
Possible offtargets among other
hCAs
CA I
CA II
Retinal/cerebral edema8,9
Glaucoma9
Unknown
hCA I
Unknown
Unknown
Unknown
Unknown
Edema8
Epilepsy10
Altitude sickness17,18
Epilepsy10
CA VII

5170
K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
O
R
O
similar compounds reported earlier by reaction of aromatic/het-
Tetrahedal adduct
(Sulfonamide)
erocyclic sulfonamides with phthalic anhydride, were tested as
CA I, II and IV inhibitors, and showed excellent in vitro activity as
well as antiglaucoma effects in rabbits.21
S
-HN
Zn2+ His 119
His 94
His 96
3. CA inhibition studies
E-Zn+2-OH2 + I
Substitution Reaction
(I = CA Inhibitors)
The CA inhibition on hCA I, II, VII data of compounds 1–8, the
standard AZM and other clinically used sulfonamides/sulfamate
are shown in Table 3. Acetazolamide is clinically used for the
adjunctive treatment of drug-induced edema, edema caused by
congestive heart failure, petit mal and other types of epilepsies.8–10
It has also been used to lower the intraocular pressure prior to sur-
gery in acute conditions of angle-closure glaucoma, besides open-
angle and secondary glaucoma and altitude sickness (Table 3).17,18
The following structure–activity relationship (SAR) was ob-
served for this series of compounds:
E-Zn+2-I
+ H2O
Figure 2. Sulfonamide binds to the Zn2+ ion of the enzyme by substituting the
nonprotein zinc ligand to generate a tetrahedral adduct.1
and IV inhibitors, and showed excellent in vitro activity as well as
antiglaucoma effects in rabbits which made an interest.8–10,17–19,21
We report here the design of novel sulfonamide CAIs. The drug de-
sign has been based on the ‘tail’ strategy reported previously20
which consists in attaching moieties that induce the desired phys-
ico-chemical properties to the molecules of aromatic sulfonamides
possessing free amino groups. These moieties should produce, in-
ter alia, high affinity to the CA active site, acceptable water/lipid
solubility, and good penetrability through the biological
membranes, to the molecules of the newly obtained CAIs. The tails
chosen to be incorporated in the compounds reported here are of
the 4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfon-
amide (tetrachlorophthalimide) type, since they may lead to inter-
esting pharmacological properties for the new CAIs containing
them. The compounds reported here were obtained by reaction
of various benzene sulfonamides with 4,5,6,7-tetrachlorophthalic
anhydride.21–25
i. The cytosolic, widespread isoforms hCA I was moderately
inhibited by the synthesized sulfonamides, with inhibition
constants in the range of 150–>10,000 nM (Table 3). The best
inhibitor in the series was the 3-chloro-4-(4,5,6,7-tetra-
chloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
7 (Ki
of 150 nM), whereas most other substitution patterns led
to compounds with inhibition constants similar to that of
acetazolamide (Table 3). The weak inhibition of this isoform
may be considered a positive feature of this class of CAIs
since hCA I, highly abundant in red blood cells, is undoubt-
edly an offtarget when considering other applications of
the CAIs.26
ii. The physiologically dominant cytosolic isoform hCA II was
generally strongly inhibited by the synthesized sulfonamide,
which showed Kis in the range of 2.4–4515 nM (Table 3). SAR
is rather straightforward. For example, against hCA II, com-
pound 8 was a potent inhibitor, with a Ki of 2.4 nM. The sub-
stitution patterns at the aromatic ring, such as halogens, led
to compounds with similar or slightly improved inhibitory
activity over the sulfanilamide derivative 1 (Kis in the range
of 2.4–4.9 nM). Some steric hindrance effects led to a loss of
the hCA II inhibition, such as for example those present in
the meta- or ortho-substituted sulfonamides 4, 5. These com-
pounds showed Kis in the range of 27.7–4515 nM. The linker
between the two bulky groups (–CH2– or –CH2CH2–) led to
an increase of the activity of compounds 2 and 3 compared
to the sulfanilamide derivative 1. Overall, these sulfona-
mides showed a good inhibitory action towards hCA II,
except for the orthanilamide derivative 5 which was a weak
inhibitor.26
2. Chemistry
The lead molecules for the present drug design study were the
sulfonamides incorporating phthalimido moieties reported earlier
by one of these groups.21 The 4,5,6,7-tetrachlorophthalic anhydride
moiety is more hydrophobic compared to the phthalimide one
contained in the derivatives reported previously, which showed
interesting antiglaucoma properties in an animal model of the
disease.21
A simple chemistry has been used to prepare the novel sulfon-
amides of types 1–8 incorporating tetrachlorophthalic anhydride
moieties to simple aromatic sulfonamides (Scheme 1). Simple
aromatic sulfonamides having free amino groups were converted
to the corresponding phthalimide by reaction with 4,5,6,7-tetra-
chlorophthalic anhydride.21 It should be mentioned that the
Cl
Cl
O
Cl
n(H2C)
NH2
X
O
N
Cl
n(H2C)
O
S
Cl
Cl
Glacial acetic acid
Reflux
Cl
+
O
H2N
O
O
S
O
O
H2N
O
X
Cl
4,5,6,7-tetrachlorophthalic anhydride
Benzenesulfonamide
Sulfonamide derivatives
n= 0 for 1, 4, 5, 6, 7, 8); n = 1 for 3; n = 2 for 2
X= H (1, 2, 3, 4, 5); F (6); Cl (7); Br (8)
-(CH2)n- = in para position for 1, 2, 3, 6, 7, 8; in meta position for 4; in ortho position for 5
Scheme 1. Scheme for the synthesis of sulfonamides 1–8 incorporating 4,5,6,7-tetrachloropthalic anhydride moiety to benzene sulfonamides.

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
5171
Table 3
hCA I, II, VII Inhibition data with sulfonamides 1–8, standard AZM and other clinically used CAIs, by a stopped-flow CO2 hydrase assay method27
Compd. code
Structures/name
Ki (nM)*
hCA I
hCA II
hCA VII
Cl
Cl
O
O
O
Cl
Cl
H2N
S
N
1
332
326
7.1
2.1
3.5
O
Cl
Cl
O
S
O
O
Cl
Cl
H2N
N
2
2.9
O
O
Cl
Cl
N
O
3
427
5.2
3.1
Cl
S
O
H2N
O
Cl
Cl
O
O
Cl
N
4
444
27.7
3.5
Cl
O
S
Cl
O
H2N
Cl
Cl
O
O
Cl
Cl
N
5
>10,000
4515
46.9
O
S
O
NH2
Cl
O
F
O
S
Cl
Cl
H2N
H2N
H2N
N
6
7
8
368
159
281
3.4
4.9
2.4
1.3
4.7
2.2
O
O
O
Cl
Cl
Cl
O
S
Cl
Cl
N
O
O
Cl
Cl
O
Br
O
Cl
Cl
S
N
O
O
Cl
AAZ
MZA
EZA
DZA
BRZ
TPM
SLP
IND
ZNS
CLX
VLX
Acetazolamide {N-(5-sulfamoyl-1, 3, 4-thiadiazol-2-yl) acetamide}
250
50
25
12
14
8
9
3
10
40
15
35
21
43
2.5
2.1
0.8
3.5
2.8
Methazolamide
Ethoxzolamide
Dorzolamide
Brinzolamide
Topiramate
Sulpiride
Indisulam
Zonisamide
Celecoxib
50,000
45,000
250
1200
31
56
50,000
54,000
0.9
3630
122
117
2,170
3900
Valdecoxib
*
Mean from three different assays, errors were in the range of 5–10% of the reported values (data not shown).

5172
K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
iii. The cytosolic isoform hCA VII was also strongly inhibited by
was recrystallized in ethanol to purify the synthesized com-
the synthesized sulfonamide, which showed Kis in the range
of 1.3–46.9 nM (Table 3). Compound 6 of the series was the
most potent inhibitor with a Ki of 1.3 nM, but most substitu-
tion patterns to the aromatic ring of sulfanilamide, such as
the various halogens, led to compounds with similar inhibi-
tory activity (Kis in the range of 1.3–4.7 nM). The steric hin-
drance of the ortho-substituted derivative compound 5 led
to a weaker hCA VII inhibition (Ki = 46.9 nM). A linker
between the two bulky moieties present in these com-
pounds did not dramatically change the hCA VII inhibitory
power of the compounds (compared to compounds 1, 2
and 3).
pounds.21–25
White crystalline and solid; % yield = 98%; mp = 315–320 °C;
solubility; insoluble: water, acetic acid glacial; partially soluble:
ethanol; fully soluble: DMSO and methanol, chloroform. IRvmax
(cmÀ1; KBr pellets); 1778.43, 1716.70 (C@O imide); 1321.28,
1163.11 (S@O) and 3414.12 (NH2). MS (ESI+); m/z: 440.8 [M],
436.8 [MÀH]À, 438.8 [M+1]. 1H NMR (400 MHz, DMSO-d6): d
7.533 (s, 2H, SO2NH2), 7.699–7.678 (d, 2H, ArÀH from benzenesul-
fonamide), 8.050–8.029 (d, 2H, ArÀH from benzenesulfonamide).
13C NMR (100 MHz, DMSO-d6): 167.29, 144.38, 138.98, 134.49,
128.9, 128.8, 128.23, 127.04.
5.2.2. Synthesis of 4-(2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)ethyl)benzenesulfonamide (2)
0.002 mol (0.400 g) of 4-(2-aminoethyl)benzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.572 g) of
4,5,6,7-tetrachlorophthalic anhydride to produce 0.002 mol of
4-(2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)ethyl)benzene-
sulfonamide (2) (0.936 g) in the presence of glacial acetic acid as
solvent for 1.5 h at 130 °C. The reaction was monitored in 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 recrystallized in ethanol to purify the
synthesized compounds.21–25
4. Conclusions
A small series of 4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl
benzenesulfonamide was prepared and investigated for the inhibi-
tion of three physiologically relevant CA isoforms CA I, CA II and CA
VII. These compounds were generally relevantly less potent inhib-
itors of CA I, but many of them were effective, low nanomolar CA II
and CA VII. Such compounds may constitute interesting candidates
for the development of novel antiglaucoma, antiepileptic, edema,
or altitude sickness drugs.
White crystalline and solid; % yield = 98%; mp = 286 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol,
methanol; fully soluble: DMSO and chloroform. IRvmax (cmÀ1; KBr
pellets); 1776.50, 1712.92 (C@O imide); 1303.92, 1159.29 (S@O)
and 3387.11 (NH2), 2955.04 (aliphatic CH2). MS (ESI+); m/z:
465.2 [M], 283.2 [MÀ184.24]À, 450.8 [MÀ16.02]À. 1H NMR (400
MHz, DMSO-d6): d 7.347 (s, 2H, SO2NH2), 7.473–7.494 (d, 2H, ArÀH
from benzenesulfonamide), 7.763–7.785 (d, 2H, ArÀH from ben-
zenesulfonamide), 3.011, 3.029, 3.048 (t, 2H, CH2), 3.849, 3.868,
3.885 (t, 2H, CH2). 13C NMR (100 MHz, DMSO-d6): 164.19,
143.41, 138.143.21, 139.17, 130.16, 129.23, 129.03, 126.81, 34.13.
5. Experimental section
5.1. Reagents and instruments
All reagents and solvents were of commercial quality and used
without further purification, unless otherwise specified. All reac-
tions were carried out under an inert atmosphere of nitrogen.
p-Amino benzene sulphanilamide (Sigma–Aldrich), 4,5,6,7-tetra-
chlorophthalic anhydride (Sigma–Aldrich), acetic acid glacial, chlo-
roform, methanol, DMSO (Central Drug House), silica gel 60 F254
plates (Merck Art.1.05554). Spots were visualized under 254 nm
(short) and 365 nm (long) UV illumination and/or by ninhydrin
solution spraying. FT-IR spectra were recorded on 8400S, Shima-
dzu. 1H and 13C NMR spectra were recorded on Bruker DRX-400
spectrometer using DMSO-d6 as solvent and tetramethylsilane as
internal standard. For 1H NMR spectra, chemical shifts are ex-
pressed in d (ppm) downfield from tetramethylsilane, and coupling
constants (J) are expressed in Hertz. Electron ionization mass spec-
tra were recorded in positive or negative mode on a Water Micro-
Mass ZQ.
5.2.3. Synthesis of 4-((4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)methyl)benzenesulfonamide hydrochloride (3)
0.002 mol (0.445 g) of 4-(aminomethyl)benzenesulfonamide
hydrochloride stirred under nitrogen environment with 0.002
mol (0.572 g) of 4,5,6,7-tetrachlorophthalic anhydride to produce
0.002 mol of 4-(2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-
yl)ethyl)benzenesulfonamide (3) (0.955 g) in the presence of
glacial acetic acid as solvent for 1.5 h at 130 °C. The reaction was
monitored in each 30 min with the help of TLC (chloroform meth-
anol; 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 recrystallized in
ethanol to purify the synthesized compounds.21–25
5.2. Chemistry
A mixture of selected sulfonamide and tetrachlorophthalic
anhydride in glacial acetic acid was refluxed with stirring under
nitrogen environment for desirable time to complete the reaction
lead to compounds 1–8.21–25
White crystalline and solid; % yield = 90%; mp = 305 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol
and methanol; fully soluble: DMSO, chloroform. IRvmax (cmÀ1
;
KBr pellets); 1774.57, 1714.77 (C@O imide); 1338.64, 1161.19
(S@O) and 3350.46 (NH2). MS (ESI+); m/z: 454.7 [M], 450.8
[MÀH]À, 452.8 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.38 (s,
2H, SO2NH2), 7.565, 7.586 (d, 2H, ArÀH from benzenesulfonamide),
7.81–7.831 (d, 2H, ArÀH from benzenesulfonamide), 4.891 (s, 2H,
CH2). 13C NMR (100 MHz, DMSO-d6): 164.28, 144.26, 140.63,
139.1, 129.49, 129.1, 128.89, 126.81, 42.1.
5.2.1. Synthesis of 4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)benzenesulfonamide (1)
0.002 mol (0.344 g) of 4-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.572 g) of 4,5,6,
7-tetrachlorophthalic anhydride to produce 0.002 mol of 4-
(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
(1) (0.880 g) in the presence of glacial acetic acid as solvent for 2 h
at 130 °C. The reaction was monitored in 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
5.2.4. Synthesis of 3-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)benzenesulfonamide (4)
0.002 mol (0.344 g) of 3-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.572 g) of 4,5,6,

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
5173
7-tetrachlorophthalic anhydride to produce 0.002 mol of 3-
(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
(4) (0.880 g) in the presence of glacial acetic acid as solvent for 2 h
at 130 °C. The reaction was monitored in 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 recrystallized in ethanol to purify the synthesized com-
pounds.21–25
DMSO-d6): 162.74, 159.07, 156.53, 147.76, 139.89, 132.35,
129.78, 123.52, 122.74, 115.39.
5.2.7. Synthesis of 3-chloro-4-(4,5,6,7-tetrachloro-1,3-
dioxoisoindolin-2-yl)benzenesulfonamide (7)
0.002 mol (0.412 g) of 4-amino-2-chlorobenzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.572 g) of
4,5,6,7-tetrachlorophthalic anhydride to produce 0.002 mol of
3-chloro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzene-
sulfonamide (7) (0.949 g) in the presence of glacial acetic acid as
solvent for 12 h at 130 °C. The reaction was monitored in 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 recrystallized in ethanol to purify the
synthesized compounds.21–25
White crystalline and solid; % yield = 94%; mp = 303 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRvmax (cmÀ1; KBr
pellets); 1788.07, 1722.49 (C@O imide); 1305.87, 1172.76 (S@O)
and 3387.11 (NH2). MS (ESI+); m/z: 470.6 [MÀH]À, 472.6 [M+1].
1H NMR (400 MHz, DMSO-d6): d 7.724 (s, 2H, SO2NH2), 7.861,
7.882 (d, 2H, ArÀH from benzenesulfonamide), 8.017–8.042 (d,
2H, ArÀH from benzenesulfonamide), 8.155 (s, 2H, ArÀH from ben-
zenesulfonamide). 13C NMR (100 MHz, DMSO-d6): 162.64, 147.69,
140.16, 133.91, 133.12, 132.82, 129.91, 129.11, 129.31, 126.65.
White crystalline and solid; % yield = 92%; mp = 308 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol
À1
and methanol; fully soluble: DMSO, chloroform. IRvmax (cm
;
KBr pellets); 1782.29, 1718.68 (C@O imide); 1301.99, 1132.25
(S@O) and 3356.25 (NH2). MS (ESI+); m/z: 440.7 [M], 436.8
[MÀH]À, 438.7 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.61 (s,
2H, SO2NH2), 7.7, 7.72 (d, 2H, ArÀH from benzenesulfonamide),
7.799–7.84 (d, 2H, ArÀH from benzenesulfonamide), 7.969–7.84
(d, 2H, ArÀH from benzenesulfonamide), 7.98–7.988 (d, 2H, ArÀH
from benzenesulfonamide). 13C NMR (100 MHz, DMSO-d6): 163.42,
145.99, 139.4, 132.56, 131.68, 130.92, 129.39, 126.86, 125.6.
5.2.5. Synthesis of 2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-
2-yl)benzenesulfonamide (5)
0.002mol(0.344g)of2-aminobenzenesulfonamidestirredunder
nitrogen environmentwith0.002 mol(0.572 g) of4,5,6,7-tetrachlor-
ophthalic anhydride to produce 0.002 mol of 2-(4,5,6,7-tetrachloro-
1,3-dioxoisoindolin-2-yl)benzenesulfonamide (5) (0.880 g) in the
presenceofglacialaceticacidassolventfor2hat130 °C. Thereaction
was monitored in 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
waterrepeatedlyfor4/5times.Theproductwasrecrystallizedineth-
anol to purify the synthesized compounds.21–25
White crystalline and solid; % yield = 90%; mp = 313 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol
and methanol; fully soluble: DMSO and chloroform. IRvmax
(cmÀ1; KBr pellets); 1782.29, 1712.85 (C@O imide); 1305.85,
1139.97 (S@O) and 3387.11 (NH2). MS (ESI+); m/z: 440.7 [M],
436.7 [MÀH]À, 438.7 [M+1]. 1H NMR (400 MHz, DMSO-d6): d
7.661 (s, 2H, SO2NH2), 7.802–7.844 (d, 2H, ArÀH from benzenesul-
fonamide), 8.102–8.126 (d, 2H, ArÀH from benzenesulfonamide).
13C NMR (100 MHz, DMSO-d6): 165.71, 163.11, 143.27, 139.53,
134.05, 132.74, 131.63, 129. 62, 129.48, 129.26.
5.2.8. Synthesis of 3-bromo-4-(4,5,6,7-tetrachloro-1,3-
dioxoisoindolin-2-yl)benzenesulfonamide (8)
0.002 mol (0.502 g) of 4-amino-2-bromobenzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.572 g) of
4,5,6,7-tetrachlorophthalic anhydride to produce 0.002 mol of 3-
bromo-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzene-
sulfonamide (8) (1.038 g) in the presence of glacial acetic acid as
solvent for 12 h at 130 °C. The reaction was monitored in 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 recrystallized in ethanol to purify the
synthesized compounds.21–25
White crystalline and solid; % yield = 90%; mp = 265 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRvmax (cmÀ1; KBr
pellets); 1772.64, 1720.56 (C@O imide); 1300.00, 1170.83 (S@O)
and 3400.0 (NH2). MS (ESI+); m/z: 518.5 [M], 514.5 [MÀH]À,
516.5 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.715 (s, 2H, SO2-
NH2), 7.842, 7.863 (d, 2H, ArÀH from benzenesulfonamide),
8.051–8.077 (d, 2H, ArÀH from benzenesulfonamide), 8.292 (s,
2H, ArÀH from benzenesulfonamide). 13C NMR (100 MHz, DMSO-
d6): 162.64, 147.69, 140.16, 133.91, 133.12, 132.82, 129.91,
129.11, 129.31, 126.65.
5.2.6. Synthesis of 3-fluoro-4-(4,5,6,7-tetrachloro-1,3-dioxois-
oindolin-2-yl)benzenesulfonamide (6)
0.002 mol (0.380 g) of 4-amino-2-fluorobenzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.572 g) of
4,5,6,7-tetrachlorophthalic anhydride to produce 0.002 mol of
3-fluoro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzene-
sulfonamide (6) (0.916 g) in the presence of glacial acetic acid as
solvent for 12 h at 130 °C. The reaction was monitored in 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 recrystallized in ethanol to purify the
synthesized compounds.21–25
White crystalline and solid; % yield = 95%; mp = 278 °C; solubil-
ity; insoluble: water, acetic acid glacial; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRvmax (cmÀ1; KBr
pellets); 1790.00, 1720.56 (C@O imide); 1301.99, 1168.90 (S@O)
and 3385.18 (NH2). MS (ESI+); m/z: 458.7 [M], 454.7 [MÀH]À,
456.7 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.704 (s, 2H, SO2-
NH2), 7.796–7.818 (d, 2H, ArÀH from benzenesulfonamide),
7.835 (s, 2H, ArÀH from benzenesulfonamide), 7.893–7.941 (d,
2H, ArÀH from benzenesulfonamide). 13C NMR (100 MHz,
5.3. CA inhibition assay
An applied photophysics stopped-flow instrument has been
used for assaying the CA-catalyzed CO2 hydration activity. Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as a buffer and 20 mM Na2SO4 (for maintaining a
constant ionic strength), following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10À100 s. The CO2
concentrations ranged from 1.7–17 mM for the determination of
the kinetic parameters and inhibition constants. For each inhibitor,
at least six traces of the initial 5À10% of the reaction have been
used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the to-

5174
K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5168–5174
6. Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T. Curr. Pharm. Des.
2008, 14, 615.
7. Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T. Med. Res. Rev. 2006,
26, 767.
8. (a) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 641; (b) Kaur, I. P.; Smitha, R.;
Aggarwal, D.; Kapil, M. Int. J. Pharm. 2002, 248, 1.
9. Mincione, F.; Scozzafava, A.; Supuran, C. T. In Drug Design of Zinc-Enzyme
Inhibitors: Functional, Structural, and Disease Applications; Supuran, C. T.,
Winum, J. Y., Eds.; Wiley: Hoboken, 2009; pp 139–154.
tal observed rates. Stock inhibitor solutions (0.1 mM) were pre-
pared in distilledÀdeionized water, and dilutions of up to 0.01
nM were made thereafter with distilledÀdeionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min to 72
h at room temperature (15 min) or 4 °C (all other incubation times)
prior to the assay, to allow the formation of the EÀI complex or the
eventual active site-mediated hydrolysis of the inhibitor. The inhi-
bition constants were obtained by nonlinear least-squares meth-
ods using PRISM 3.27–29
10. Thiry, A.; Dogne`, J. M.; Masereel, B.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7,
855.
11. De Simone, G.; Di Fiore, A.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 655.
12. Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.; Scozzafava, A.;
Wouters, B. G.; Lambin, P. Radiother. Oncol. 2009, 92, 423.
13. Ahlskog, J. K.; Dumelin, C. E.; Trüssel, S.; Marlind, J.; Neri, D. Bioorg. Med. Chem.
Lett. 2009, 19, 4851.
Abbreviations
CA, carbonic anhydrase; hCA, human carbonic anhydrase;CAI,
carbonic anhydrase inhibitor; SAR, structure-activity relationship;
Zn, Zinc; AZM, Acetazolamide; MZA, methazolamide; EZA, ethox-
zolamide; DCP, dichlorophenamide; DZA, Dorzolamide; BRZ, brin-
zolamide; BZA, benzolamide; TPM, topiramate; ZNS, zonisamide;
SLP, Sulpiride; IND, indisulam; COX2, cyclo-oxygenase enzyme 2;
CLX, celecoxib; VLX; valdecoxib; TLC, thin layer chromatography;
UV, ultraviolet; FT-IR, Fourier transform infrared; DMSO, dimethyl
sulfoxide; NMR, Nuclear Magnetic Resonance.
14. Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.;
Bühlmann, P.; Supuran, C. T.; Neri, D. ACS Chem. Biol. 2011, 6, 336.
15. Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759.
16. Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767.
17. Basnyat, B.; Gertsch, J. H.; Johnson, E. W.; Castro-Marin, F.; Inoue, Y.; Yeh, C.
High Alt. Med. Biol. 2003, 4, 45.
18. Hackett, P. H.; Roach, R. C. N. Eng. J. Med. 2001, 345, 107.
19. Gao, B. B.; Clermont, A.; Rook, S.; Fonda, S. J.; Srinivasan, V. J.; Wojtkowski, M.;
Fujimoto, J. G.; Avery, R. L.; Arrigg, P. G.; Bursell, S. E.; Aiello, L. P.; Feener, E. P.
Nat. Med. 2007, 13, 181.
20. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran,
C. T. J. Med. Chem. 1999, 42, 2641.
21. Scozzafava, A.; Mincione, F.; Menabuoni, L.; Supuran, C. T. Drug Des. Disc. 2001,
17, 337–348.
Acknowledgments
22. (a) Maresca, A.; Vullo, D.; Scozzafava, A.; Manole, G.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2013, 28, 392; (b) Maresca, A.; Scozzafava, A.; Vullo, D.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 384.
23. (a) Liu, F.; Martin-Mingot, A.; Lecornué, F.; Maresca, A.; Thibaudeau, S.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 886; (b) Ekinci, D.;
Kurbanoglu, N. I.; Salamci, E.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2012, 27, 845.
This work is acknowledged to our Vice-Chancellor of BIT Mesra,
Ranchi, Dr. P. K. Barahi, who has given all the infrastructural
opportunity for research. Work from the Florence laboratory was
financed by two FP7 EU projects, Metoxia and Dynano.
24. (a) Vullo, D.; Leewattanapasuk, W.; Mühlschlegel, F. A.; Mastrolorenzo, A.;
Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 2647; (b)
Vullo, D.; Isik, S.; Del Prete, S.; De Luca, V.; Carginale, V.; Scozzafava, A.;
Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2013, 23, 1636; (c)
Monti, S. M.; De Simone, G.; Dathan, N. A.; Ludwig, M.; Vullo, D.;
Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013,
23, 1626.
References and notes
1. Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev.
2012, 112, 4421.
2. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Fut. Med.
Chem. 2011, 3, 1165.
3. (a) Mack, E. T.; Snyder, P. W.; Perez-Castillejos, R.; Bilgicer, B.; Moustakas, D. T.;
Butte, M. J.; Whitesides, G. M. J. Am. Chem. Soc. 2012, 134, 333; (b) Breton, S. J.
Pancreas 2001, 2, 159.
4. Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2003, 46, 2197.
25. Supuran, C. T. Front. Pharmacol. 2011, 2, 34.
26. Supuran, C. T. Carbonic Anhydrases: Off-Targets, Add-on Activities, or
Emerging Novel Targets? In Polypharmacology in Drug Discovery; Peters, J. U.,
Ed.; Wiley: Hoboken, 2012; pp 457–489.
27. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
5. Abbate, F.; Winum, J. Y.; Potter, B. V.; Casini, A.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 231.
28. Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668.
29. Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 6208.