Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I, II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

Article abstract of DOI:10.1016/j.bmc.2014.01.031

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295-10,000 nM), but were more effective hCA II inhibitors (K _is of 1.7-887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90-3746 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.

Full text of DOI:10.1016/j.bmc.2014.01.031

Bioorganic & Medicinal Chemistry 22 (2014) 1586–1595
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors: Synthesis and inhibition of the
human carbonic anhydrase isoforms I, II, IX and XII with benzene
sulfonamides incorporating 4- and 3-nitrophthalimide moieties
a
Kalyan K. Sethi a, , Saurabh M. Verma , Muhammet Tanç b,c, Gaultier Purper c, Gaetan Calafato 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, 50019 Sesto Fiorentino (Firenze), Italy
c Università degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), 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 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was
synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These
new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmem-
brane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak
hCA I inhibitors (Kis in the range of 295–10,000 nM), but were more effective hCA II inhibitors (Kis of
1.7–887 nM). The tumor-associated hCA IX was also inhibited, with Kis in the micromolar range, whereas
against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity rela-
tionship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good
activity for each isoforms being established. The high sequence hCA alignment homology and molecular
docking studies was performed in order to rationalize the activities reported and binding mode to differ-
ent hCA as inhibitors.
Received 31 December 2013
Revised 18 January 2014
Accepted 20 January 2014
Available online 31 January 2014
Keywords:
Human carbonic anhydrase inhibitors
Benzenesulfonamide
Nitro-1,3-dioxoisoindolin-2-yl
benzenesulfonamide
Structure activity relationship
Sequence alignment
Docking
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
b-CAs (bacteria, algae and chloroplasts of monocotyledons and
dicotyledons); the
(diatoms and other marine eukaryotes) and f-CAs (diatoms).1,3 Hu-
man CAs (hCAs) all belong to the -family and are present as
c-CAs (archaea and some bacteria); the d-CAs
Carbonic anhydrases (CAs) also known as carbonate dehydrata-
ses are zinc (Zn+2) metalloenzymes present in almost all living
a
organism.1,2 There are five genetically distinct CA families;
a
-CAs
fifteen isoforms, which differ by molecular features, oligomeric
arrangement, cellular localization, distribution in organs and tis-
sues, 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 (CA IV, CA IX, CA XII, CA XIV and CA XV), two
mitochondrial (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) which are also cytosolic.3
CAs catalyze the interconversion between carbon dioxide and
bicarbonate by using a metal hydroxide nucleophilic mechanism,
and are involved in physiological processes connected with respi-
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
active site cavity.1–3
(vertebrates, bacteria, algae and cytoplasm of green plants); the
Abbreviations: CA, carbonic anhydrase; hCA, human carbonic anhydrase; CAI,
carbonic anhydrase inhibitor; SAR, structure–activity relationship; Zn, zinc; AZM,
acetazolamide; MZA, methazolamide; EZA, ethoxzolamide; DCP, dibromophena-
mide; DZA, dorzolamide; BRZ, brinzolamide; BZA, benzolamide; TPM, topiramate;
ZNS, zonisamide; SLP, sulpiride; IND, indisulam; CLX, celecoxib; VLX, valdecoxib;
CNS, central nervous system; VHL, von Hippel–Lindau protein; HIF, hypoxia
inducible factor; PDB, protein data bank; XP, extra precision; RMSD, root mean
square deviation; HIS, Histidine; ASN, asparagines; GLN, glutamine; THR, threonine;
TRP, tryptophan; TLC, thin layer chromatography; UV, ultraviolet; FT-IR, Fourier
transform infrared; DMSO, dimethyl sulfoxide; NMR, nuclear magnetic resonance;
nM, nanomolar; mM, milimolar; E–I, enzyme–inhibitor; C:H:N:S:O,
carbon:hydrogen:nitrogen:sulfur:oxygen.
⇑
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).
http://dx.doi.org/10.1016/j.bmc.2014.01.031
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
1587
Table 1
Different hCA isoforms (hCA I, II, VII, IX and XII), organ/tissue distribution, drug targets/off targets in various diseases, kinetic parameters for CO2 hydration reaction and affinity
for sulfonamides1
Isoforms Organ/tissue localization
Subcellular
localization
Disease in which is
involved
Off-targets other hCAs Kcat/Km
Affinity for
sulfonamides
(MÀ1 sÀ1
)
hCA I
Erythrocytes, GI tract, Eye
Cytosol
Cytosol
Retinal/cerebral
edema8,9,18
Glaucoma9
Edema8
Unknown
5.0 Â 107
Medium
hCA II
Erythrocytes, GI tract, eye, boneosteoclasts,
kidney, lung, testis, brain
hCA I
1.5 Â 108
Very high
Unknown
Unknown
Unknown
Unknown
hCA I, hCA II
Epilepsy10
Altitude sickness17
Cancer16
hCA IX
hCA XII
Tumors, GI mucosa
Renal, intestinal, reproductive epithelia, eye,
tumors
Transmembrane Cancer16
Transmembrane Cancer16 Glaucoma9
Retinopathies19
1.5 Â 108
Very high
Low
hCA I, hCA II Unknown 3.5 Â 107
Unknown
Kcat/Km results in the rate constant that measures catalytic efficiency.
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 anticonvulsant10 agent, whereas their employment as
antiobesity drugs11 or in the management of hypoxic tumors12–16
were only recently validated.4–19 Examples of clinically used CAIs:
Acetazolamide AAZ, Methazolamide MZA, Ethoxzolamide EZA,
Dichlorophenamide DCP, Dorzolamide DZA, Brinzolamide BRZ,
Benzolamide BZA, Topiramate TPM, Zonisamide ZNS, Sulpiride
SLP, Indisulam IND, Celecoxib CLX and Valdecoxib VLX. However,
due to the large number of hCA isoforms, there is a constant need
to improve the inhibition and selectivity profiles of the so far
developed CAIs, to avoid side effects due to inhibition of isoforms
not involved in a certain pathology.1–3
Derivatives D1 and D2 (Fig. 1) are investigational agents for tar-
geting the tumor-associated isoform CA IX. The most useful CAIs for
understanding the function of this protein in vivo were the fluores-
cent sulfonamide compound of type D1 that binds only to CA IX un-
der hypoxic conditions in vivo.2a Compound D2 belongs to a class of
positively charged trimethylpyridinium derivative, membrane-
impermeable compounds. CA IX selective sulfonamide inhibitors
(D1 and D2) reduced the medium acidity by inhibiting the catalytic
activity of the enzyme, and thus the generation of H+ ions, binding
specifically only to hypoxic cells expressing CA IX.2a Derivatives D1
and D2 also participate in many other favourable interactions
including stacking between the trimethylpyridinium ring of the
O
H
N
CH3
O
S
N
S
N
O
S
O
S
O
CH3
S
S
N
H2N
H2N
H2N
CH3
N
O
N
O
N
CH3
O
O
AAZ
MZA
CH3
EZA
O
O
O
S
S
CH3
S
NH
O
O
O
H2N
O
O
NH2
S
O
O
S
NH2
O
O
O
N
HN
S
DZA
S
O
O
O
O
TPM
CH3
BRZ
Cl
O
O
O
NH2
N
S
N
O
S
O
S
O
N
H
O
H2N
N
H
H2N
NH
S
H3CO
O
O
O
IND
ZNS
SLP
SO2NH2
SO2NH2
S
NH2
S
HN
N
H
O
O
F
F
ClO4-
N
COOH
N
F
N+
H3
C
O
N
CLX
O
N
S
HO
O
O
D1
H2
VLX
O
D2
Figure 1. Carbonic anhydrase inhibitors of the sulfonamide and sulfamates type.

1588
K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
Figure 2. Sequence alignment for human
a
-CAs for which the crystal structures were reported with following PDB entries: 1AZM (hCA I); PDB: 1ZFQ (CA II); PDB: 1IAI (CA IX)
and PDB: 1JD0 (CA XII).20a–d
H
y
e
d
i
d
r
s
o
c
i
p
b
h
o
i
l
h
i
c
p
o
s
r
i
d
d
e
y
H
H
N
O
O
S
O
Thr 199
Tetrahedal adduct
(Sulfonamide & bioisosters)
NH-
O
Zn2+ His 119
H
O
His 94
O-
Glu 106
His 96
E-Zn+2-OH2 + I
E-Zn+2-I
+ H2O
Substitution Reaction
(Ia=.CA Inhibitors)
Figure 3. (a) Sulfonamide binding interactions to the Zn2+ ion and amino acid residues from the CA active site. Sulfonamides substitute the non-protein zinc ligand to
generate a tetrahedral Zn(II) adduct;1 (b) Homology modelling showing the helix and b-strand regions of five hCA (PDB files): 1AZM (hCA I); 1ZFQ (hCA II); 1IAI (hCA IX) and
1JD0 (hCA XII). The zinc ligands (His 94, 96 and 119) and AAZ or EZA bound within the active sites are also shown.20a–d

K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
1589
Hydrophobic
moiety
ATP synthesis, enzyme function and the proliferation, migration,
invasion and metastasis of tumor cells; consequently, a tight regu-
lation of these processes has evolved. Changes in the pHi as low as
0.1–0.2 pH units also trigger mechanisms of alternative splicing of
extracellular matrix components that generate different isoforms of
tenascin and fibronectin, which are typical of tumor cells and not
normal cells. These alternatively spliced proteins are not involved
in pH regulation but they may constitute a novel antitumor mech-
anism.2,16 Possible off-targets among other hCAs for CA IX involved
hCA I, and hCA II. CA IX expression is strongly increased in many
types of tumors, such as gliomas/ependymomas, mesotheliomas,
papillary/follicular carcinomas, carcinomas of the bladder, uterine
cervix, nasopharyngeal carcinoma, head and neck, breast, oesopha-
gus, lungs, brain, vulva, squamous/basal cell carcinomas, and
kidney tumors, among others. In some cancer cells, the von Hip-
pel-Lindau protein (VHL) gene is mutated leading to the strong
upregulation of CA IX (up to 150-fold) as a consequence of constitu-
tive hypoxia inducible factor (HIF) activation.2,16
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
sulfonamides led to the development of several classes of pharma-
cological agents. Sulfonamide CAIs directly bind to the metal ion
within the enzyme active site (1AZM; hCA I, 1FZQ; hCA II, 1IAI;
hCA IX and 1JD0; hCA XII), by substituting the zinc-bound hydrox-
ide ion (Fig. 3).20a–d
Scafflold
94 His
Zn+2
96 His
O
S
O
N
119 His
H
N
O
NO2
O
Hydrophobic linker
Sulfonamide
Figure 4. Representative virtual compound showing compliance to the general
pharmacophoric features of sulfonamide compounds acting as carbonic anhydrase
inhibitors.
inhibitor and the phenyl ring of Phe131 amino acid.2a Thus such
structures can be used for the rational drug design of more selective
and potent isozyme IX inhibitors.
A brief presentation of the hCA isoforms (CA I, CA II, IX and XII)
as drug targets/off-targets is shown in Table 1. To date the three-
dimensional structures of most hCA isoforms have been deter-
mined. The analysis of these structures shows that independently
on their sub-cellular localization and, as expected on the basis of
their high sequence alignment homology (Fig. 2), these enzymes
present a rather similar structure, characterized by a central
twisted b-sheet surrounded by helical connections and additional
b-strands (Fig. 3). The active site is located in a large, conical cavity,
approximately 12 Å wide and 13 Å deep, which spans from the
protein surface to the centre of the molecule. The catalytic zinc
ion is located at the bottom of this cavity, exhibiting a tetrahedral
coordination with three conserved His residues and water mole-
cule/hydroxide ion as the fourth ligand. In complex with sulfon-
amide inhibitors, a co-crystallized AAZ/EZA replacing the water
molecule/hydroxide ion as ligand could also be seen (Fig. 3).
Specifically, hCA I found in many tissues its precise physiological
function is unknown. However, a study from Feeener’s group19
demonstrated that this enzyme is involved in retinal and cerebral
edema, and its inhibition may be a valuable tool for fighting these
conditions. hCA II is ubiquitouos, being involved in several diseases,
such as glaucoma, edema, epilepsy, and altitude sickness.8–10,17,18
hCA IX is a therapeutic/imaging target for hypoxic cancers. hCA IX
expression is increased upon hypoxia and is associated with poor
prognosis, tumor progression and extracellular tumor acidification.
Key pH regulators in tumor cells include:Àisoforms CA II, IX and XII,
isoforms of anion exchangers, Na+/HCO3 co-transporters, Na+/H+
exchangers, monocarboxylate transporters and the vacuolar ATP-
ase.16 In tumor cells, these processes are even more complex owing
to the internal compartment being slightly more alkaline (pH 7.4 or
more) and the external compartment being more acidic than in nor-
mal cells. A variation in the pHi/pHe ratio as low as 0.1 pH units may
disrupt important biochemical and/or biological processes such as
These isoforms are highly related and responsible for many dis-
eases (Table 1) and the similar compounds reported earlier were
tested as CA I and II inhibitors, and showed excellent in vitro activ-
ity as well as antiglaucoma effects in rabbits.8–10,17–19,22
We report here the design of novel sulfonamide CAIs. The drug
design has been based on the ‘tail’ strategy reported previously,21
which consists in attaching moieties that induce the desired phys-
ico-chemical properties to the molecules of aromatic sulfonamides
possessing free amino groups (Fig. 4). These moieties should pro-
duce, inter alia, high affinity to the CA active site, acceptable
water/lipid solubility, and good penetrability through the biologi-
cal membranes, to the molecules of the newly obtained CAIs. The
tails chosen to be incorporated in the compounds reported here
are of the nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide (4-
and 5-nitrophthalimide) type, since they may lead to interesting
pharmacological properties for the new CAIs containing them.
The compounds reported here were obtained by reaction of various
benzene sulfonamides with 3- and 4-nitrophthalic anhydride.22–27
2. Chemistry
The lead molecules for the present drug design study were the
sulfonamides incorporating phthalimido moieties reported earlier
O
n(H2C)
NH2
O
N
n(H2C)
NO2
O
S
Glacial acetic acid
Reflux
+
O
H2N
O
O
S
NO2
O
O
H2N
O
Benzenesulfonamide
3- and 4-nitrophthalic
anhydride
Sulfonamide derivatives
Scheme 1. Scheme for the synthesis of sulfonamides 1–8 incorporating 3- and 4-nitrophthalic anhydride moiety to different benzene sulfonamides.22,29 Reagents and
conditions: n = 0 (1, 4, 5, 7 and 8); n = 1 (2, 6); n = 2 (3). –(CH2)n– = in para position for 1, 2, 3 and 6; in meta position for 4 and 7; in ortho position for 5 and 8 in the benzene
ring. 5-NO2 (1, 2, 3, 4, and 5); 4-NO2 (6, 7, and 8) in the benzene ring of anhydride.

1590
K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
by our groups.22a–c The 3- and 4-nitrophthalic anhydride moieties
are interesting due to the presence of the nitro group, which led to
compounds with interesting antitumor properties for a series of
ureido-substituted sulfonamides.22d
A simple chemistry has been used to prepare the novel sulfon-
amides of types 1–8 incorporating 3 and 4-nitrophthalicanhydride
moieties (Scheme 1). Aromatic sulfonamides having free amino
groups were converted to the corresponding phthalimide by
reaction with 3- and 4-nitrophthalicanhydride.22a–c It should be
mentioned that the similar compounds reported earlier by reaction
of aromatic/heterocyclic sulfonamides with phthalic anhydride
derivatives, were tested as CA I, II,IV, IX and XII inhibitors, and
showed excellent in vitro activity22a–c as well as antiglaucoma
effects in rabbits.22a
ward. The increased carbon chain length between the two
bulky groups (–CH2– or –CH2CH2–) led to decreased activi-
ties of compounds 1 > 2 > 3 with Kis of 4.3, 238 and
589 nM, respectively. Compounds having –SO2NH2 in the
ortho, meta and para substituted sulfonamides influence on
the hCA II inhibition due to steric hindrance effects. That is
compounds 5 < 4 < 1 (Kis of >10,000, 887 and 4.3 nM, respec-
tively) and exactly reverse in case of compounds 6 < 7 < 8
(Kis of 34, 3.9 and 1.7 nM, respectively) may be due to pres-
ence of –NO2 functional group in the 4th position. Position of
–NO2 to the benzene ring slightly change in the activity
(5th < 4th) that is compound 2 (238 nM) <6 (34 nM), 4
(887 nM) >7 (3.9 nM) and 5 (>10,000 nM) >8 (1.7 nM). Over-
all, most of these sulfonamides showed a potent inhibitory
action towards hCA II, which is the main target isoform for
designing antiglaucoma drugs, and the off-target one when
other applications of the CAIs are envisaged.28
3. CA inhibition studies
iii. The transmembrane isoform hCA IX was poorly inhibited by
this new class of sulfonamides (1–8) CAIs, which showed Kis
in the range of 873–>10,000 nM against hCA IX (Table 2).
Compound 6 was the most potent hCA inhibitor, with a Ki
of 873 nM then the other compounds of the series. The com-
pounds are less effective inhibitors for hCA IX and SARs are
straightforward. The increased carbon chain length between
the two bulky groups (–CH2– or –CH2CH2–) led to decreased
activities of compounds 1 > 2 > 3 with Kis of 8654, 8837 and
>10,000 nM, respectively of the derivatives. Some steric hin-
drance effects led to a loss of the hCA II inhibition, such as for
example –SO2NH2 present in the ortho, meta and para substi-
tuted sulfonamides 5 < 4 < 1 (Kis in the range of >10,000,
8837 and 8654 nM) resulted due to steric hindrance and
exactly reverse in 6 < 7 < 8 (Kis in the range of 873–
8352 nM) may be due to 4th position of –NO2 functional
group. Position of –NO2 to the benzene ring slightly improve
the activity 5th < 4th that is compound 2 (8837 nM) <6
(873 nM), 4 (10,000 nM) >7 (8352 nM) and 5 (>10,000 nM)
>8 (7820 nM). Overall, most of these sulfonamides showed
a very poor inhibitory action towards hCA IX.
iv. The transmembrane isoforms hCA XII on the other hand was
moderately inhibited by most of the new class of sulfon-
amide CAIs, which showed Kis in the range of 90–
>10,000 nM against hCA XII (Table 2). Compound 1 was
the most potent hCA inhibitor, with a Ki of 90 nM then the
other compounds of the series. The compounds are average
inhibitors for hCA IX and SARs is straightforward. The
increased carbon chain length between the two bulky
groups (–CH2– or –CH2CH2–) led to decreased activities of
compounds 1 > 2 > 3 with Kis of 90, 119 and 620 nM, respec-
tively of the derivatives. Some steric hindrance effects led to
a loss of the hCA XII inhibition, such as for example –SO2NH2
present in the ortho, meta and para substituted sulfonamides
5 < 4 < 1 (Kis in the range of 3746, 96 and 90 nM) resulted
due to steric hindrance and exactly in 6 > 7 > 8 (Kis in the
range of 491, 838, >10,000 nM). Position of –NO2 to the ben-
zene ring slightly improve the activity 5th > 4th that is com-
pound 2 (90 nM) >6 (491 nM), 4 (96 nM) >7 (838 nM) and 5
(3746 nM) >8 (>10,000 nM). Overall, most of these sulfona-
mides showed a moderate inhibitory action towards hCA XII.
v. The selectivity ratio for inhibiting the tumor-associated iso-
forms hCA IX and XII over the cytosolic off-target hCA II,28
was in the range of 0.00–0.08 and 0.0002–9.24 for the new
sulfonamide reported here (Table 2). For hCA IX the selectiv-
ity ratio is too poor then hCA XII in which some the com-
pounds shown a good result, since most sulfonamide CAIs
(D1, D2, acetazolamide, ethoxzolamide, dichlorophenamide,
The CA inhibition on cytosolic hCA I and II membrane bound IX
and XII (tumor associated human CA) data of compounds 1–8, the
standard AZM and other clinically used sulfonamides/sulfamate
are shown in Table 2. Acetazolamide is clinically used for the
adjunctive treatment of drug-induced edema, edema caused by con-
gestive heart failure, petit mal and other types of epilepsies.8–10 It
has also been used to lower the intraocular pressure prior to surgery
in acute conditions of angle-closure glaucoma, besides open-angle
and secondary glaucoma and altitude sickness (Table 1).17,18
The following structure–activity relationship (SAR) was
observed for this series of compounds:
i. The cytosolic isoform hCA I was inhibited by the synthesized
sulfonamides, with inhibition constants in the range of
295 nM to >10,000 nM (Table 2). The best inhibitor in the
series
was
the
4-(2-(5-nitro-1,3-dioxoisoindolin-2-
yl)ethyl)benzenesulfonamide 3 (Ki of 295 nM), whereas some
of the other substitution patterns led to compounds 1, 2, 6, 4
and 7 with inhibition constants 318, 403, 490, 501 and
522 nM, respectively. The inhibition constants are somewhat
close similar to that of clinically used acetazolamide/topira-
mate and better then sulpiride/dorzolamide/brinzolamide
(Table 2). The weak inhibition of this isoform (compounds 5
and 8) may be considered a positive feature of this class of
CAIs since hCA I, highly abundant in red blood cells, is
undoubtedly an off-target when considering other applica-
tions of the CAIs.2,28 The increased carbon chain length
between the two bulky groups (–CH2– or –CH2CH2–) led to
an increased activities of compounds 1 to 3 with 295 nM to
403 nM, respectively of the derivatives. The position of –SO2-
NH2 matters in the increase and decrease of activities. The
decreased activity of compounds 1, 4, 5, 7, 8 according to posi-
tion of –SO2NH2 in the benzene ring are 4th to 2nd (Kis
295 nM to >10,000 nM). It is very clear from the above inhibi-
tion data that, compounds having –SO2NH2 group in the ortho
position of the benzene ring compatatively very less active
then meta and then para position, respectively due to
increased steric hindrance. Position of –NO2 to the benzene
ring slightly alter in the hCAI activities (5th > 4th) that is com-
pound 2 (403 nM) >6 (490 nM) and 4 (501 nM) >7 (522 nM).
ii. The physiologically dominant cytosolic isoform hCA II was
inhibited with low nanomolar concentration by the synthe-
sized sulfonamide in comparison to standard clinically used
AAZ and other clinically used sulfonamide, Kis in the range of
1.7–>10,000 nM (Table 2). Against hCA II, compound 8 was
the most potent hCA inhibitor, with a Ki of 1.7 nM then the
other compounds of the series. The compounds are excellent
to moderate inhibitors for hCA II and SARs are straightfor-

K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
1591
Table 2
hCA I, II, IX and XII inhibition data with sulfonamides 1–8, standard AZM and other important CAIs, by a stopped-flow CO2 hydrase assay method30
Comp. code Structures/name
Ki (nM)*
Selectivity ratio#
Docking score (Glide XP)
1AZM 1ZFQ 3IAI 1JD0
hCA I
318
hCA II hCA IX hCAXII
A
B
O
O
H2N
S
N
1
4.3
8654
90
0.0005 0.58
À5.647 À5.132 À5.246 À4.244
O
NO2
O
O
N
O
2
3
4
403
295
501
238
589
887
8837
119
0.026
2
À5.931 À4.624 À5.515 À4.746
À6.001 À5.509 À5.086 À5.046
À5.776 À4.781 À5.526 À4.751
S
O
H2N
H2N
O
NO2
O
S
O
N
>10,000 620
0.06
0.95
O
NO2
O
O
O
N
10,000 96
0.088
9.24
NO2
O
S
O
H2N
O
O
N
5
>10,000 >10,000 >10,000 3746
0.00
2.669
À3.796 À3.856 À3.601 À3.618
NO2
O
S
O
NH2
O
N
O
6
490
34
873
491
0.039
0.069
À5.845 À5.112 À5.565 À5.335
S
O
H2N
O
O2N
O
O
N
7
522
3.9
8352
838
0.0005 0.005
À5.909 À5.103 À4.471 À4.801
O
S
NO2
O
H2N
(continued on next page)

1592
K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
Table 2 (continued)
Comp. code Structures/name
Ki (nM)*
Selectivity ratio#
Docking score (Glide XP)
1AZM 1ZFQ 3IAI 1JD0
hCA I
hCA II hCA IX hCAXII
A
B
O
O
N
8
>10,000 1.7
7820
>10,000 0.0002 0.0002 À3.823 À4.794 À4.493 À4.377
O
S
NO2
O
NH2
AAZ
MZA
EZA
DZA
BRZ
TPM
SLP
IND
ZNS
CLX
VLX
D1
Acetazolamide
Methazolamide
Ethoxzolamide
Dorzolamide
Brinzolamide
Topiramate
Sulpiride
Indisulam
Zonisamide
Celecoxib
250
50
25
50,000
45,000
250
1200
31
12
14
8
9
3
10
40
15
35
25
27
34
52
37
58
46
24
5.1
16
27
24
14
5.7
3.4
22
3.5
3
3.8
3.9
3.4
0.5
0.5
0.2
0.2
0.1
0.2
0.9
0.6
2.10
4.11
0.36
2.57
1
2.63
10.25
4.41
0.003
1.16
3.30
9
À4.8
À4.9
À5.7
À5.0
À5.5
À6.1
À6.5
À6.5
À5.7
À6.5
À6.1
À3.9
À4.1
À3.8
À3.8
À4.1
À4.5
À5.3
À4.7
À6.0
À5.0
À3.7
À4.7
À4.1
À4.4
À4.9
À4.4
À4.9
À5.1
À4.8
À4.9
À5.0
À5.7
À6.8
À4.7
À4.7
À4.5
À5.0
À5.1
À4.1
À3.5
À4.2
À4.4
À5.1
À4.4
À4.9
À5.2
À4.5
À3.3
À4.0
À5.3
À5.0
56
11,000 6.9
50,000 21
54,000 43
1300
4000
18
13
5
1.3
1.6
1.9
1.5
Valdecoxib
—
—
45
21
D2
7
3
*
Mean from three different assays, errors were in the range of 5–10% of the reported values (data not shown).
The Ki ratios are indicative of isozyme selectivity for transmembrane CAs IX, XII; A (hCA II/hCA IX) and B (hCA II/hCA XII).
#
Figure 5. Docked conformations in the CA IX (PDB: 3IAI) catalytic site. (a) AZM in the binding pocket formed H-bond to HIS 64, ASN 62, GLN 67; (b) Compound 6 (highest
docking score À5.565) formed hydrogen bond to THR 119, THR 200 and GLN 67, GLN 92.
etc) show better hCA II than hCA IX inhibitory action.2 In the
new series reported here, most of the compounds had a poor
selectivity ratio, being thus relatively better CA I and II selec-
tive rather then hCA IX but being thus somewhat highly CA
XII selectivity (compared to the inhibition of CA I and II).
scores of the synthesized compounds are so good enough then
standard AZM and other clinically used sulfonamides/sulfamate,
D1 and D2 also (Table 2). Figure 5 showed the docked conforma-
tions of AZM and compound 6 (highest docking score) in catalytic
site in the binding pocket of CA IX (PDB: 3IAI).
The main objective of our molecular docking study was to ratio-
nalize the SARs reported over here and Zn+2 binding mode interac-
tions to different hCA as inhibitors. In each and every case we have
observed that the SAR tally to the results obtained by means of
docking study. For examples: compounds 5 and 8 showed the low-
est docking scores found in each and every case. The increased car-
bon chain length between the two bulky groups (–CH2– or –
CH2CH2–) led to improve docking score of the synthesized deriva-
tives. Some steric hindrance effects result loss of the hCAI docking
score, for example –SO2NH2 present in the ortho-, meta- and para-
substituted sulfonamides 5 < 4 < 1. Addition of –NO2 to the 4th and
5th position to the compounds provide the clear indication of var-
iable scores of compounds (Table 2) and interaction to different
amino acid (Fig. 5).
4. Docking studies
Molecular docking of eight compounds and other clinically used
sulfonamides/sulfamate were performed to rationalize the SARs
reported and to study the Zn+2 binding mode to different hCA as
inhibitors. Docking study was performed in the crystal structure
of hCA preferably in 1AZM (hCA I), 1FZQ (hCA II), 3IAI (hCA IX)
and 1JD0 (hCA XII).20a–d All the ‘A’ chain of the hCA crystal struc-
tures catalytic domains was considered for docking study. The
Glide (XP) score of the co-crystallized ligand AZM is À3.8 to À4.8
and the RMSD values range from 1.8 to 2.3 which is considered
as good for docking of the ligands (Table 2). Most of the docking

K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
1593
nol; Fully Soluble: DMSO and methanol, chloroform. IRm ;
max (cmÀ1
5. Conclusions
KBr pellets); 1780.36, 1716.70 (C@O imide); 1338.64, 1161.19
(S@O) and 3363.97 (NH2), 1539.25, 1390.72 (NO2). MS (ESI+); m/
z: 346.08 [MÀH]À, 378.08 [MÀH]À+MeOH. 1H NMR (400 MHz,
DMSO-d6) d: 7.527 (s, 2H, SO2NH2), 7.715–7.749, 8.023–8.056 (d,
2H, Ar–H from benzenesulfonamide), 8.278–8.299, 8.73–8.735 (d,
2H, Ar–H from nitrobenzene), 8.654–8.659 (s, H, Ar–H from nitro-
benzene). 13C NMR (100 MHz, DMSO-d6) d: 119.21, 125.95, 127.40,
128.43, 130.84, 133.92, 135.32, 137.16, 144.57, 152.56, 166.05.
Elem. Anal. calcd for C14H9N3O6S: C, 48.42; H, 2.61; N, 12.10; O,
27.64; S, 9.23; found C, 50.50; H, 2.647; N, 12.80; O, 23.883; S,
10.17.
A small series of 4- and 5-nitro-1,3-dioxoisoindolin-2-yl ben-
zenesulfonamide (compounds 1–8)was prepared and investigated
for the inhibition of four physiologically relevant hCA isoforms
hCA I, hCA II, hCA IX and hCA XII. These compounds were generally
potent inhibitors of CA I, but many of them were relevantly good to
moderate effective with nanomolar hCA II. Such compounds may
constitute interesting candidates for the development of novel anti-
glaucoma, antiepileptic, edema, or altitude sickness drugs. The com-
pounds inhibit the transmembrane isoforms hCA IX with high
nanomolar concentration and hCA XII with moderately nanomolar
concentration. In the new series reported here, most of the com-
pounds had a poor selectivity ratio, being thus relatively better
hCA I and II selective rather then hCA IX but being thus somewhat
high CA XII selectivity (compared to the inhibition of hCA I and II).
Such compounds may promote interesting candidates for the devel-
opment of more selective and potent novel hypoxia induced cancer
drug therapy and retinopathies. The compounds showed better hCA
I and II may be further interesting candidates for the development of
retinal/cerebral edema, glaucoma, altitude sickness.
6.2.2. Synthesis of 4-((5-nitro-1,3-dioxoisoindolin-2-
yl)methyl)benzenesulfonamide (2)
0.002 mol (0.444 g) of 4-(aminomethyl)benzenesulfonamide
hydrochloride stirred under nitrogen environment with
0.002 mol (0.386 g) of 5-nitroisobenzofuran-1,3-dione to produce
0.002 mol of 4-((5-nitro-1,3-dioxoisoindolin-2-yl)methyl)ben-
zenesulfonamide (2) (0.722 g) in the presence of glacial acetic acid
as solvent for 12 h at 130 °C. The reaction monitored in each
30 min with the help of TLC (Chloroform/Methanol; 1:1). After
completion of reaction, the reaction 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 from ethanol leading to compound 2.22,29
White crystalline and solid; % yield = 75%; mp = 214 °C; Solubil-
ity; Insoluble: acetic acid glacial; Partially Soluble: ethanol, meth-
6. Experimental section
6.1. 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 nitrogen atmosphere. These
are the following chemicals obtained from different sources that
is p-amino benzene sulphanilamide (Sigma–Aldrich), 3- and 4-
nitrophthalic anhydride (Sigma–Aldrich), acetic acid glacial, chloro-
form, 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, Shimadzu.
1H and 13C NMR spectra were recorded on Bruker DRX-400 spec-
trometer using DMSO-d6 as solvent and tetramethylsilane as inter-
nal standard. For 1H NMR spectra, chemical shifts are expressed in d
(ppm) downfield from tetramethylsilane, and coupling constants (J)
are expressed in Hertz. Electron ionization mass spectra were re-
corded in positive or negative mode on a Water MicroMass ZQ.
CHNSO elemental analysis recorded by Elementar, Vario EL III.
anol; Fully Soluble: water, DMSO and chloroform. IRmmax (cmÀ1
;
KBr pellets); 1768.78, 1707.06 (C@O imide); 1310.10, 1157.33
(S@O) and 3365.90 (NH2), 1537.32, 1350.30 (NO2), 2990.10 (ali-
phatic CH2). MS (ESI+); m/z: 360.17 [MÀH]À. 1H NMR (400 MHz,
DMSO-d6) d: 7.375 (s, 2H, SO2NH2), 7.569–7.59, 7.806–7.827 (d,
2H, Ar–H from benzenesulfonamide), 8.189–8.21, 8.675–8.7 (d,
2H, Ar–H from nitrobenzene), 8.566–8.57 (s, H, Ar–H from nitro-
benzene), 4.933 (s, 2H, CH2 aliphatic). 13C NMR (100 MHz,
DMSO-d6) d: 41.95, 118.96, 125.68, 126.84, 128.81, 130.61,
134.05, 137.31, 140.91, 144.21, 152.29, 166.8 Elem. Anal. calcd
for C15H11N3O6S: C, 49.86; H, 3.07; N, 11.63; O, 26.57; S, 8.87;
found C, 51.92; H, 3.104; N, 12.21; O, 23.052; S, 9.714.
6.2.3. Synthesis of 4-(2-(5-nitro-1,3-dioxoisoindolin-2-
yl)ethyl)benzenesulfonamide (3)
0.002 mol (0.401 g) of 4-(2-aminoethyl)benzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.386 g) of
5-nitroisobenzofuran-1,3-dione to produce 0.002 mol of 4-(2-
6.2. Chemistry
(5-nitro-1,3-dioxoisoindolin-2-yl)ethyl)benzenesulfonamide
(3)
A mixture of selected sulfonamide and 3- or 4-nitrophthalic
anhydride in glacial acetic acid was refluxed with stirring under
nitrogen environment for desirable time to complete the reaction
leading to compounds 1À8.22,29
(0.750 g) in the presence of glacial acetic acid as solvent for 12 h
at 130 °C. The reaction monitored in each 30 min with the help
of TLC (Chloroform/Methanol; 1:1). After completion of reaction,
the reaction 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 from eth-
anol leading to compound 3.22,29
White crystalline and solid; % yield = 88%; mp = 207 °C; Solubil-
ity; Insoluble: water, acetic acid glacial; Partially Soluble: ethanol
and Methanol; Fully Soluble: DMSO, chloroform. DMSO and meth-
anol, chloroform. IRmmax (cmÀ1; KBr pellets); 1770.71, 1705.13
(C@O imide); 1332.86, 1151.54 (S@O) and 3369.75 (NH2),
1539.25, 1350.30 (NO2), 2949.26 (aliphatic CH2). MS (ESI+); m/z:
376.08 [MÀH]À. 1H NMR (400 MHz, DMSO-d6) d: 7.338 (s, 2H, SO2-
NH2), 7.455–7.475, 7.741–7.762 (d, 2H, Ar–H from benzenesulfon-
amide), 8.14–8.16, 8.641–8.666 (d, 2H, Ar–H from nitrobenzene),
8.516–8.521 (s, H, Ar–H from nitrobenzene), 3.05–3.085, 3.914–
3.95 (d, 2H, CH2 aliphatic). 13C NMR (100 MHz, DMSO-d6) d:
34.18, 39.91, 118.78, 125.47, 126.7, 130.1, 130.56, 133.8, 137.04,
6.2.1. Synthesis of 4-(5-nitro-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (1)
0.002 mol (0.344 g) of 4-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.386 g) of 5-nitro-
isobenzofuran-1,3-dione to produce 0.002 mol of 4-(5-nitro-1,
3-dioxoisoindolin-2-yl)benzenesulfonamide (1) (0.695 g) in the
presence of glacial acetic acid as solvent for 12 h at 130 °C. The reac-
tion monitored in each 30 min with the help of TLC (Chloroform/
Methanol; 3:1). After completion of reaction, the reaction mixture
was cooled and 30 ml of cold water was added. The product was fil-
tered and washed with cold water repeatedly for 4/5 times. The
product was recrystallized from ethanol leading to compound 1.22,29
White crystalline and solid; % yield = 88%; mp = 250.2 °C; Solu-
bility; Insoluble: water, acetic acid glacial; Partially Soluble: etha-

1594
K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
143.22, 152.35, 166.7, 166.97. Elem. Anal. calcd for C16H13N3O6S: C,
51.20; H, 3.49; N, 11.19; O, 25.57; S, 8.54; found C, 53.41; H, 3.661;
N, 11.80; O, 21.691; S, 9.438.
as solvent for 12 h at 130 °C. The reaction monitored in each
30 min with the help of TLC (Chloroform/Methanol; 3:1). After
completion of reaction, the reaction 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 from ethanol leading to compound 6.22,29
White crystalline and solid; % yield = 76%; mp = 221 °C; Solubil-
ity; Insoluble: Water, acetic acid glacial; Partially Soluble: ethanol;
6.2.4. Synthesis of 3-(5-nitro-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (4)
0.002 mol (0.344 g) of 3-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.386 g) of 5-nitro-
isobenzofuran-1,3-dione to produce 0.002 mol of 3-(5-nitro-1,
3-dioxoisoindolin-2-yl)benzenesulfonamide (4) (0.694 g) in the
presence of glacial acetic acid as solvent for 12 h at 130 °C. The reac-
tion monitored in each 30 min with the help of TLC (Chloroform/
Methanol; 3:1). After completion of reaction, the reaction mixture
was cooled and 30 ml of cold water was added. The product was fil-
tered and washed with cold water repeatedly for 4/5 times. The
product was recrystallized from ethanol leading to compound 4.22,29
Reddish white crystalline and solid; % yield = 73%; mp = 265 °C;
Solubility; Insoluble: Water, Acetic acid glacial; Partially Soluble:
ethanol and Methanol; Fully Soluble: DMSO, chloroform. IRmmax
(cmÀ1; KBr pellets); 1791.93, 1716.70 (C@O imide); 1346.36,
1116.82 (S@O) and 3369.75 (NH2), 1533.46, 1386.86 (NO2). MS
(ESI+); m/z: 346.08 [MÀH]À. 1H NMR (400 MHz, DMSO-d6) d:
7.604 (s, 2H, SO2NH2), 7.741–7.769, 7.96–7.987 (d, 2H, Ar–H from
benzenesulfonamide), 7.801–7.84 (t, H, Ar–H from benzenesulfon-
amide), 8.02–8.029 (s, H, Ar–H from benzenesulfonamide), 8.271–
8.279, 8.72–8.755 (d, 2H, Ar–H from nitrobenzene), 8.645–8.646 (s,
H, Ar–H from nitrobenzene). 13C NMR (100 MHz, DMSO-d6)
d:119.13, 125.32, 125.88, 126.57, 130.76, 131.46, 132. 86, 133.99,
137.25, 145.88, 152.51, 165.94, 166.18. Elem. Anal. calcd for C14H9-
N3O6S: C, 48.42; H, 2.61; N, 12.10; O, 27.64; S, 9.23; found C, 50.45;
H, 2.829; N, 12.83; O, 23.791; S, 10.10.
Fully Soluble: DMSO, chloroform and methanol. IRm
max (cmÀ1; KBr
pellets); 1770.71, 1708.99 (C@O imide); 1336.71, 1157.33 (S@O)
and 3358.18 (NH2), 1535.39, 1346.36 (NO2). MS (ESI+); m/z:
360.17 [MÀ1]À. 1H NMR (400 MHz, DMSO-d6) d: 7.375 (s, 2H, SO2-
NH2), 7.567–7.588, 7.803–7.824 (d, 2H, Ar–H from benzenesulfon-
amide), 8.094–8.133, 8.23–8.25 (t, 2H, Ar–H from nitrobenzene),
8.335–8.337 (d, H, Ar–H from nitrobenzene), 4.89 (s, 2H, CH2 ali-
phatic). 13C NMR (100 MHz, DMSO-d6) d:41.92, 124.13, 126.8,
127.94, 128.83, 129.32, 134.54, 137.17, 140.86, 144.18, 145.31,
164.11, 166.75. Elem. Anal. calcd for C15H11N3O6S: C, 49.86; H,
3.07; N, 11.63; O, 26.57; S, 8.87; found C, 50.59; H, 2.646; N,
11.60; O, 26.053; S, 9.111.
6.2.7. Synthesis of 3-(4-nitro-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (7)
0.002 mol (0.344 g) of 3-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.386 g) of 4-nitro-
isobenzofuran-1,3-dione to produce 0.002 mol of 3-(4-nitro-1,
3-dioxoisoindolin-2-yl)benzenesulfonamide (7) (0.694 g) in the
presence of glacial acetic acid as solvent for 12 h at 130 °C. The reac-
tion monitored in each 30 min with the help of TLC (Chloroform/
Methanol; 3:1). After completion of reaction, the reaction mixture
was cooled and 30 ml of cold water was added. The product was fil-
tered and washed with cold water repeatedly for 4/5 times. The
product was recrystallized from ethanol leading to compound 7.22,29
White crystalline and solid; % yield = 65%; mp = 214.2 °C; Solu-
bility; Insoluble: Water, acetic acid glacial; Partially Soluble: etha-
6.2.5. Synthesis of 2-(5-nitro-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (5)
0.002 mol (0.344 g) of 2-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.386 g) of 5-nitro-
isobenzofuran-1,3-dione to produce 0.002 mol of 2-(5-nitro-1,
3-dioxoisoindolin-2-yl)benzenesulfonamide (5) (0.694 g) in the
presence of glacial acetic acid as solvent for 12 h at 130 °C. The reac-
tion monitored in each 30 min with the help of TLC (Chloroform/
Methanol; 3:1). After completion of reaction, the reaction mixture
was cooled and 30 ml of cold water was added. The product was fil-
tered and washed with cold water repeatedly for 4/5 times. The
product was recrystallized from ethanol leading to compound 5.22,29
White crystalline and solid; % yield = 97%; mp = 260 °C; Solubil-
ity; Insoluble: water, acetic acid glacial; Partially Soluble: ethanol
and methanol; Fully Soluble: DMSO and chloroform. IRmmax
(cmÀ1; KBr pellets); 1791.93, 1728.28 (C@O imide); 1342.50,
1170.83 (S@O) and 3410.26 (NH2), 1541.18, 1388.79 (NO2). MS
(ESI+); m/z: 346.8 [MÀH]À. 1H NMR (400 MHz, DMSO-d6) d:
7.632 (s, 2H, SO2NH2), 7.787–7.862 (t, 2H, Ar–H from benzenesul-
fonamide), 7.69–7.719, 8.105–8.129 (d, 2H, Ar–H from benzenesul-
fonamide), 8.269–8.290, 8.7–8.75 (d, 2H, Ar–H from nitrobenzene),
8.64 (s, 2H, Ar–H from nitrobenzene). 13C NMR (100 MHz, DMSO-
d6) d: 119.28, 126.02, 129.46, 130.68, 131.4, 132.75, 133.89,
134.32, 137.5, 143.14, 152.51, 165.80, 166.05. Elem. Anal. calcd
for C14H9N3O6S: C, 48.42; H, 2.61; N, 12.10; O, 27.64; S, 9.23; found
C, 51.38; H, 2.355; N, 12.37; O, 23.998; S, 9.897.
nol; Fully Soluble: DMSO, chloroform and methanol. IRm ;
max (cmÀ1
KBr pellets); 1780.36, 1716.70 (C@O imide); 1338.64, 1159.26
(S@O) and 3362.04 (NH2), 1541.18, 1388.79 (NO2). MS (ESI+); m/
z: 346.17 [MÀ1]À, 364 [MÀ1]À+H2O (adduct). 1H NMR (400 MHz,
DMSO-d6) d: 7.597 (s, 2H, SO2NH2), 7.711–7.739, 7.951–7.978 (d,
2H, Ar–H from benzenesulfonamide), 7.785–7.824 (t, H, Ar–H from
benzenesulfonamide), 7.994–7.998 (s, 2H, Ar–H from benzenesul-
fonamide), 8.29–8.33, 8.38–8.41 (d, 2H, Ar–H from nitrobenzene),
8.25–8.29 (t, H, Ar–H from nitrobenzene). 13C NMR (100 MHz,
DMSO-d6) d: 123.86, 125.59, 126.61, 128.01, 129.35, 130.72,
131.7, 132.76, 134.49, 137.37, 145.44, 145.84, 163.34, 165.89.
Elem. Anal. calcd for C14H9N3O6S: C, 48.42; H, 2.61; N, 12.10; O,
27.64; S, 9.23; found C, 49.15; H, 2.829; N, 12.82; O, 26.091; S, 9.11.
6.2.8. Synthesis of 2-(4-nitro-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (8)
0.002 mol (0.344 g) of 2-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.386 g) of 4-nitro-
isobenzofuran-1,3-dione to produce 0.002 mol of 2-(4-nitro-1,
3-dioxoisoindolin-2-yl)benzenesulfonamide (8) (0.694 g) in the
presence of glacial acetic acid as solvent for 12 h at 130 °C. The reac-
tion monitored in each 30 min with the help of TLC (Chloroform/
Methanol; 3:1). After completion of reaction, the reaction mixture
was cooled and 30 ml of cold water was added. The product was fil-
tered and washed with cold water repeatedly for 4/5 times. The
product was recrystallized from ethanol leading to compound 8.22,29
White crystalline and solid; % yield = 65%; mp = 252.4 °C; Solu-
bility; Insoluble: Water, acetic acid glacial; Partially Soluble: etha-
6.2.6. Synthesis of 4-((4-nitro-1,3-dioxoisoindolin-2-
yl)methyl)benzenesulfonamide (6)
0.002 mol (0.444 g) of 4-(aminomethyl)benzenesulfonamide
hydrochloride stirred under nitrogen environment with
0.002 mol (0.386 g) of 4-nitroisobenzofuran-1,3-dione to produce
0.002 mol of 4-((5-nitro-1,3-dioxoisoindolin-2-yl)methyl)ben-
zenesulfonamide (6) (0.722 g) in the presence of glacial acetic acid
nol; Fully Soluble: DMSO, chloroform and methanol. IRm ;
max (cmÀ1
KBr pellets); 1793.86, 1732.13 (C@O imide); 1301.99, 1114.89
(S@O) and 3385.18 (NH2), 1533.46, 1363.72 (NO2). MS (ESI+); m/

K. K. Sethi et al. / Bioorg. Med. Chem. 22 (2014) 1586–1595
1595
z: 346.17 [MÀH]À. 1H NMR (400 MHz, DMSO-d6) d: 7.633 (s, 2H,
SO2NH2), 7.773–7.849 (t, 2H, Ar–H from benzenesulfonamide),
7.603–7.706, 8.094–8.118 (d, H, Ar–H from benzenesulfonamide),
8.163–8.202 (t, 2H, Ar–H from nitrobenzene), 8.308–8.33,
8.39–8.43 (d, H, Ar–H from nitrobenzene). 13C NMR (100 MHz,
DMSO-d6) d: 124.28, 128, 21, 129.41, 129.51, 131.4, 132.84,
133.88, 134.79, 137.33, 143.22, 145.53, 163.04, 165.73. Elem. Anal.
calcd for C14H9N3O6S: C, 48.42; H, 2.61; N, 12.10; O, 27.64; S, 9.23;
found C, 48.47; H, 2.81; N, 12.83; O, 25.68; S, 10.21.
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; p 139.
10. Thiry, A.; Dognè, 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.
6.3. CA inhibition assay
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. (a) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (b) Guler, O. O.; De
Simone, G.; Supuran, C. T. Curr. Med. Chem. 2010, 17, 1516; (c) De Simone, G.;
Supuran, C. T. Biochim. Biophys. Acta 2010, 1804, 404; (d) Battke, C.; Kremmer,
E.; Mysliwietz, J.; Gondi, G.; Dumitru, C.; Brandau, S.; Lang, S.; Vullo, D.;
Supuran, C. T.; Zeidler, R. Cancer Immunol. Immunother. 2011, 60, 649.
17. (a) Basnyat, B.; Gertsch, J. H.; Johnson, E. W.; Castro-Marin, F.; Inoue, Y.; Yeh, C.;
High Alt Med. Biol. 2003, 4, 45; (b) Hackett, P. H.; Roach, R. C. N. Eng. J. Med.
2001, 345, 107.
18. 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.
19. Ogilvie, J. M.; Ohlemiller, K. K.; Shah, G. N.; Ulmasov, B.; Becker, T. A.; Waheed,
A.; Hennig, A. K.; Lukasiewicz, P. D.; Sly, W. S. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 8514.
20. (a) Chakravarty, S.; Kannan, K. K. J. Mol. Biol. 1994, 243, 298; (b) Honndorf, V.S.;
Heine, A.; Klebe, G.; Supuran, C.T. http://dx.doi.org/10.2210/pdb1zfq/pdb.; (c)
Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De
Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233; (d) Whittington, D. A.;
Waheed, A.; Ulmasov, B.; Shah, G. N.; Grubb, J. H.; Sly, W. S.; Christianson, D. W.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9545.
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 10–100 s. The CO2 con-
centrations ranged from 1.7 to 17 mM for the determination of the
kinetic parameters and inhibition constants. For each inhibitor, at
least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total ob-
served rates. Stock inhibitor solutions (0.1 mM) were prepared in
distilled–deionized water, and dilutions of up to 0.01 nM were
made thereafter with distilled–deionized water. Inhibitor and en-
zyme 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 E–I complex or
the eventual active site-mediated hydrolysis of the inhibitor. The
inhibition constants were obtained by nonlinear least-squares
methods using PRISM 3.30
21. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran,
C. T. J. Med. Chem. 1999, 42, 2641.
22. (a) Scozzafava, A.; Mincione, F.; Menabuoni, L.; Supuran, C. T. Drug Des. Disc.
2001, 17, 337; (b) Sethi, K. K.; Verma, S. M.; Tanç, M.; Carta, F.; Supuran, C. T.
Bioorg. Med. Chem. 2013, 21, 5168; (c) Sethi, K. K.; Vullo, D.; Verma, S. M.; Tanç,
M.; Carta, F.; Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 5973; (d) Pacchiano, F.;
Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.; Dedhar, S.; Supuran,
C. T. J. Med. Chem. 2011, 54, 1896.
Acknowledgments
23. (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.
24. (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.
25. (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.
This work is acknowledged to our Vice-Chancellor and Head
Department of Pharmaceutical Sciences BIT Mesra, Ranchi who
has given all the infrastructural opportunity and finance support
for research. Work from the Florence laboratory was financed by
two FP7 EU projects, Metoxia and Dynano. Docking of some inhib-
itors within the various isoforms active sites were also performed.
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