Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I, II, VII, IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

Article abstract of DOI:10.1016/j.bmc.2013.07.044

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. 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, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47-190 nM) and poor hCA VII inhibitors (Kis in the range of 54-175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1-197 nM with high selectivity ratio. 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 study of compounds was performed to rationalize the SAR reported over here.

Full text of DOI:10.1016/j.bmc.2013.07.044

Bioorganic & Medicinal Chemistry 21 (2013) 5973–5982
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, VII, IX and XII with
benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide
moiety
b
a
Kalyan K. Sethi a, , Daniella Vullo , 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, 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 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds
1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic
anhydride moiety. 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, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds
were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA
II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to
acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and
234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. 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 study of compounds was performed to rationalize the SAR reported
over here.
Received 9 July 2013
Revised 24 July 2013
Accepted 25 July 2013
Available online 2 August 2013
Keywords:
Human carbonic anhydrase inhibitors
Benzenesulfonamide
4,5,6,7-Tetrabromo-1,3-dioxoisoindolin-2-
yl benzenesulfonamide
Structure–activity relationship
Docking
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
of monocotyledons and dicotyledons); the c-CAs (archaea and
some bacteria); the d-CAs (marine diatoms) and f-CAs (bacteria,
Carbonic anhydrases (CAs) are zinc (Zn+2) metalloenzymes
present in almost all living organism.1,2 There are five genetically
chemolithotrophs and marine cyanobacteria).1,3 Human CAs
(hCAs), all belong to the
a-family and are present in fifteen iso-
distinct CA families;
plasm of green plants); the b-CAs (bacteria, algae and chloroplasts
a
-CAs (vertebrates, bacteria, algae and cyto-
forms (out of 16 mammalian isoforms), which are differ by molec-
ular features, oligomeric arrangement, cellular localization,
distribution in organs and tissues, expression levels, and kinetic
properties (Table 1).2 There are five cytosolic forms (hCA I, hCA
II, hCA III, hCA VII and hCA XIII), four membrane associated iso-
zymes (hCA IV, hCA IX, hCA XII and hCA XIV), two mitochondrial
forms (hCA VA and hCA VB), and a secreted hCA isozyme (hCA
VI).1–3 There are three additional ‘acatalytic’ hCA isoforms (hCA
VIII, hCA X, and hCA XI) whose functions are remained unclear.3
hCAs which catalyze the interconversion between carbon dioxide
and bicarbonate by using a metal hydroxide nucleophilic mecha-
nism and are involved in many physiological and pathological pro-
cesses connected with respiration and transport of CO2 or
bicarbonate ion, CO2 homeostasis, electrolyte secretion in many
tissues, biosynthetic reactions, calcification, tumorigenicity, and
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; COX, cyclo-oxygenase enzyme;
CLX, celecoxib; VLX, valdecoxib; CNS, central nervous system; VHL, von Hippel–
Lindas protein; HIF, hypoxia inducible factor; PDB, protein data bank; XP, extra
precision; RMSD, root mean square deviation; HIS, histidine; ASN, asparagine; 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.
⇑
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.2013.07.044

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K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
Table 1
Organ/tissue distribution, subcellular localization, CO2 hydrase activity, and affinity for sulfonamides of the human CA isozymes (hCA I, II, VII, IX and XII)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
Cytosol
Cytosol
Low
High
Medium
Very high
CA VII
CA IX
CA XII
Central nervous system, liver
Tumors, Gastrointestinal mucosa
Kidney, Intestine, Reproductive epithelia, Eye, Tumors
Cytosol
Transmembrane
Transmembrane
High
High
Low
Very high
High
Very high
O
H
N
CH3
O
S
N
S
O
S
O
S
O
CH3
S
S
N
H2N
H2N
H2N
CH3
N
O
N
N
O
N
AAZ
O
CH3
O
O
MZA
EZA
CH3
O
O
O
O
S
S
CH3
O
S
NH
NH2
H2N
S
O
O
O
O
O
S
NH2
O
N
O
HN
S
DZA
S
O
O
O
CH3
BRZ
TPM
Cl
O
O
S
O
NH2
N
N
O
S
O
S
O
N
H
O
H2N
N
H
H2N
NH
S
H3CO
O
O
O
SLP
IND
ZNS
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 sulfonamides and sulfamates.
so forth. 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
because of the large number of hCA isoforms,1–3 there is a constant
need to improve the inhibition and selectivity profile of the so far
developed CAIs, to avoid side effects due to inhibition of isoforms
not involved in a certain pathology.2
Several studies demonstrated the important roles of hCAs in a
variety of physiological processes, and showed that abnormal lev-
els or activities of these enzymes have been often associated with
different human diseases.2 In the last few years, several hCA iso-
zymes have become interesting targets for the design of inhibitors
or activators with biomedical applications.4–7 Indeed originally
hCA inhibitors (hCAIs) were clinically used (Fig. 1) as diuretic,8
antiglaucoma,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
hCAIs: acetazolamide AZM, methazolamide MZA, ethoxzolamide
EZA, dibromophenamide DCP, dorzolamide DZA, brinzolamide
BRZ, benzolamide BZA, topiramate TPM, zonisamide ZNS, sulpiride
SLP, indisulam IND, celecoxib CLX and valdecoxib VLX. However,
Derivatives D1 and D2 (Fig. 1) are investigational agents for
targeting the tumor-associated isoform hCA IX. The most useful
CAIs for understanding the function of this protein in vivo were
the fluorescent sulfonamide compounds of type D1.2a Compound
D2 belongs to a class of positively charged trimethylpyridinium
derivative.2a 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 specif-
ically only to hypoxic cells expressing hCA IX.2a It also partici-
pates in many other favorable interactions including stacking
between the trimethylpyridinium ring of the inhibitor (D2) and
the phenyl ring of Phe131.2a Thus; such structures can be used
for the rational drug design of more selective and potent isozyme
IX inhibitors.

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
5975
A brief presentation of the hCA isoforms (hCA I, hCA II, hCA VII,
hCA IX and hCA XII) as drug targets/off-targets is shown in Table 2.
To date the three-dimensional structures of all these isoforms have
been determined. The analysis of these structures shows that inde-
pendently on their sub-cellular localization and, as expected on
the basis of their high sequence alignment homology (Fig. 2), these
enzymes present similar types of 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, con-
ical 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 tetrahe-
dral coordination with three conserved His residues and a co-crys-
tallized AZM/EZA replacing water molecule/hydroxide ion as
ligand (Fig. 3).
Specifically, hCA I is found in many tissues, but a study from
Feener’s group19 demonstrated that this enzyme is involved in ret-
inal and cerebral edema, and its inhibition may be a valuable tool
for fighting these conditions. Similarly hCA II is involved in several
diseases, such as glaucoma, edema, epilepsy, and altitude sick-
ness.8–10,17,18 hCA VII mainly found in CNS has been noted for its
contributions to epileptic activity together with hCA II and XIV.10
Table 2
hCA isoforms as drug targets/offtargets in various diseases1
Isoform Disease in which is
involved
Possible offtargets among other
hCAs
CA I
Retinal/Cerebral
edema8,9,19
Glaucoma9
Edema8
Unknown
CA II
hCA I
hCA IX belongs to the highly active human
a-CAs, its catalytic
Unknown
Unknown
Unknown
Unknown
hCA I, hCA II
hCA I, hCA II
Unknown
properties for the CO2 hydration reaction being comparable with
those of the highly evolved catalyst hCA II.16 Carbonic anhydrase
IX is considered as a potential target for hypoxia induced cancer
therapy. hCA IX expression is increased upon hypoxia and has been
proposed as a therapeutic target since it has been associated with
poor prognosis, tumor progression and pH regulation. Key pH
Epilepsy10
Altitude sickness17,18
Epilepsy10
CA VII
CA IX
CA XII
Cancer16
Cancer16
Glaucoma9
Figure 2. Structure-based sequence alignment of
a-CAs with known three-dimensional structure. The following PDB entries were used in the alignment: 1AZM (hCA I); PDB:
1ZFQ (CA II); PDB: 3MDZ (hCA VII); PDB: 1IAI (CA IX) and PDB: 1JD0 (CA XII).20a–e

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K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
Br
O
Br
Tetrahedal adduct
(Sulfonamide)
n(H2C)
N
Br
O
O
Br
S
X
-HN
O
Zn2+ His 119
His 94
His 96
E-Zn+2-OH2 + I
E-Zn+2-I
+ H2O
Substitution Reaction
(I = CA Inhibitors)
(a)
(b)
Figure 3. (a) Sulfonamide binds to the Zn2+ ion of the enzyme by substituting the non-protein zinc ligand to generate a tetrahedral adduct.1 (b) Crystal structures alignment
showing helix and b-strand regions of five hCA of PDB: 1AZM (hCA I); PDB: 1ZFQ (CA II); PDB: 3MDZ (hCA VII); PDB: 1IAI (CA IX) and PDB: 1JD0 (CA XII) binds to standard
sulfonamide co-crystalized ligand AAZ and EZA to the Zn and His 94, His 96, and His 119 in the binding pocket.20a–e
regulators in tumor cells include: isoforms II, IX and XII of carbonic
anhydrase, isoforms of anion exchangers, Na+/HCO3À co-transport-
ers, Na+/H+ exchangers, monocarboxylate transporters and the vac-
uolar ATPase.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 normal 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 ATP synthesis, enzyme function and
the proliferation, migration, invasion and metastasis of tumor
cells; consequently, a tight regulation 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 compo-
nents that generate different isoforms of tenascin and fibronectin,
which are typical of tumor cells and not normal cells. These alter-
natively spliced proteins are not involved in pH regulation but they
may constitute a novel antitumor mechanism.2,16 hCA IX isoform is
a dynamic drug target and a key feature of many hypoxia induced
tumors.2,16 Possible offtargets among other hCAs for hCA IX in-
volved hCA I, hCA II (Table 2). hCA IX expression is strongly in-
creased 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, , lungs, brain, vulva, squamous/basal cell carcinomas, and
kidney tumors, among others. In some cancer cells, the von Hip-
pel–Lindas protein (VHL) gene is mutated leading to the strong
upregulation of hCA IX (up to 150-fold) as a consequence of consti-
tutive hypoxia inducible factor (HIF) activation.2,16
The potent hCAIs incorporate various zinc-binding groups
(ZBGs) and thus belong to various chemotypes with the classical
sulfonamide one, still constituting the main player in the field.
The sulfonamides led to the development of several classes of
pharmacological agents. Sulfonamide inhibitors directly bind to
the metal ion within the enzyme active site (i.e. 1AZM; hCA I,
1FZQ; hCA II, 3MDZ; hCA VII, 3IAI; hCA IX and 1JD0; hCA XII), by
substituting the zinc-bound hydroxide ion (Fig. 3).20a–e Other
hCAIs, such as some anions, add to the metal co-ordination sphere,
leading to pentaco-ordinated Zn (II) complexes.3,4
Some of these hCA isoforms are highly related and responsible
for many diseases explained earlier and the similar compounds re-
ported earlier were tested as hCA I, II and IV inhibitors, and showed
excellent in vitro activity as well as antiglaucoma effects in rabbits
which made an interest.8–10,17–19,22 We reported here the structure
based virtual evidence of the design of novel sulfonamide hCAIs.
The drug design has been based on the ‘tail’ strategy reported pre-
viously21 which consists in attaching moieties that induce the de-
sired physico-chemical properties to the molecules of aromatic
sulfonamides possessing free amino groups. These moieties should
Br
Br
O
Br
n(H2C)
NH2
X
O
N
Br
n(H2C)
O
S
Br
Br
Glacial acetic acid
Reflux
Br
+
O
H2N
O
O
S
O
O
H2N
O
X
Br
4,5,6,7-tetrabromophthalic anhydride
Benzenesulfonamide
Sulfonamide derivatives
Scheme 1. Scheme for the synthesis of sulfonamides 1–8 incorporating 4,5,6,7-tetrabromopthalic anhydride moiety to benzene sulfonamides. 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.

Table 3
hCA I, II, VII, IX and XII inhibition data with sulfonamides 1–8, standard AZM and other important CAIs, by a stopped-flow CO2 hydrase assay method28
Compounds
code
Structures/name
Kia (nM)
Selectivity ratio
Docking score (Glide XP)
hCA I
hCA II
68
hCA VII
hCA IX
9.1
hCA XII
hCA II/hCA IX
1AZM
1ZFQ
3MDZ
3IAI
1JD0
Br
Br
O
O
O
Br
Br
H2
N
S
N
1
2
3
432
54
59
77
6.1
7.5
5.8
5.0
À5.6
À4.7
À4.6
À4.7
À3.2
O
O
Br
Br
N
O
251
185
71
80
12.3
15.9
12.3
10.2
À6.0
À6.1
À4.7
À4.8
À5.7
À5.3
À5.4
À5.2
À4.6
À5.0
Br
S
O
H2N
O
Br
Br
O
S
O
O
Br
Br
H2
N
N
O
Br
Br
O
O
Br
N
4
5
340
79
80
24
19
3.3
0.8
À5.8
À3.9
À5.2
À4.8
À5.7
À3.2
À5.0
À4.6
À4.3
À4.1
Br
O
S
Br
O
H2
N
Br
O
O
Br
N
>10,000
190
175
234
197
Br
O
S
Br
O
NH2
Br
O
F
O
Br
Br
H2N
S
N
6
7
770
208
96
55
84
72
9.4
8.5
7.5
5.9
10.2
À5.9
À6.0
À4.7
À4.7
À4.5
À4.9
À4.7
À5.2
À3.2
À3.6
O
O
O
Br
Br
Cl
O
S
Br
Br
H2N
N
6.5
O
O
Br
(continued on next page)

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K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
produce, inter alia, high affinity to the hCA active site, acceptable
water/lipid solubility, and good penetrability through the biologi-
cal membranes, to the molecules of the newly obtained hCAIs.
The tails chosen to be incorporated in the compounds reported
here are of the 4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (tetrabromophthalimide) type, since they
may lead to interesting pharmacological properties for the new
hCAIs containing them. The compounds reported here were ob-
tained by reaction of various benzene sulfonamides with 4,5,6,7-
tetrabromophthalic anhydride.22–26
2. Chemistry
The lead molecules for the present drug design study were the
sulfonamides incorporating phthalimide moiety reported earlier
by one of these groups.22 The 4,5,6,7-tetrabromophthalic anhy-
dride 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.22a
A simple chemistry has been used to prepare the novel sulfon-
amides of types 1–8 incorporating tetrabromophthalimide moiety
to simple aromatic sulfonamides (Scheme 1). Simple aromatic sul-
fonamides having free amino groups were converted to the corre-
sponding phthalimide by reaction with 4,5,6,7-tetrabromophthalic
anhydride.22a,b It should be mentioned that the similar compounds
reported earlier by reaction of aromatic/heterocyclic sulfonamides
with phthalic anhydride derivatives, were tested as hCA I, II IV and
VII inhibitors, and showed excellent in vitro activity as well as anti-
glaucoma effects in rabbits.22
3. hCA inhibition studies
The hCA inhibition on cytosolic hCA I, II, VII and membrane
bound IX and XII (tumor associated hCA) data of compounds 1–8,
the standard AZM and other clinically used sulfonamides/ sulfa-
mate 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 epilep-
sies.8–10 It has also been used to lower the intraocular pressure
prior to surgery in acute conditions of angle-closure glaucoma, be-
sides 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:
i. The cytosolic, widespread isoforms hCA I was inhibited by the
synthesized sulfonamides, with inhibition constants Kis in
the range of 143 to >10,000 nM (Table 3). The best inhibitor
in the series was the 3-bromo-4-(4,5,6,7-tetrabromo-1,3-
dioxoisoindolin-2-yl)benzenesulfonamide 8 (Ki of 143 nM),
whereas most other substitution patterns led to compounds
3, 7 and 2 with inhibition constants 185, 208 and 251 nM,
respectively are good/similar to that of acetazolamide
(Table 3). The relatively weak inhibition of this isoform (com-
pounds 4, 1, 6, 5) may be considered a positive feature of this
class of CAIssincehCAI, highlyabundantin redbloodcellsand
is undoubtedly an offtarget when considering other applica-
tions of the CAIs.2,27 The increased carbon chain length
between the two bulky groups (–CH2– or –CH2CH2–) led to
an increased activities of compounds 1 < 2 < 3 with 432, 251
and 185 nM respectively of the derivatives. The position
of –SO2NH2 in the benzene ring matters in the increase and
decrease of activities. The increased activity of compounds
according to position of –SO2NH2 in the benzene ring are 4th
to 2nd (Kis 432 nM to >10,000 nM). Addition of halogen (F,

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
5979
Figure 4. Docked conformations in the CA IX (PDB: 3IAI) catalytic site. (a) Acetazolamide in the binding pocket formed H-bond to HIS 64, ASN 62, GLN 67; (b) compound 3
(highest docking score) formed hydrogen bond to THR 190 and TRP 5.
Cl and Br) to the 3rd position of the benzene ring scaffold
improved the activities (Kis 770 < 208 < 145 nM) with respect
to increase the electro negativity of the halogen groups that is
the compounds 6, 7 and 8.
iv. The transmembrane isoforms hCA IX (tumor associated) was
effectively inhibited by this new class of sulfonamide, which
showed Kis in the range of 8.5–234 nM (Table 3). SAR is
rather straightforward. For example, against hCA IX, in com-
parisons to AZM the compounds 7 and 8 were potent inhib-
itors, with a Ki of 8.5 and 8.8 nM, respectively. Addition of
halogen (F, Cl and Br) to the 3rd position to the compounds
6, 7 and 8 scaffold ‘benzene’ carrying sulfonamide improved
the activities (Kis in the range of 9.4–8.8 nM) with respect to
increase in the electro negativity of the halogen groups. The
increased carbon chain length between the two bulky
groups (–CH2– or ÀCH2CH2–) led to an decreased activities
of compounds 1 > 2 > 3 with Kis of 9.1, 12.3 and 15.9 nM,
respectively of the derivatives. Some steric hindrance effects
led to a loss of the hCA IX inhibition activities, for example –
SO2NH2 present in the ortho-, meta- and para-substituted
sulfonamides 5 < 4 < 1 (showed Kis of 234, 24 and 9.1 nM).
v. The transmembrane isoforms hCA XII (tumor associated) on
the other hand also effectively inhibited by this new class of
sulfonamide hCAIs, which showed Kis in the range of 5.9–
197 nM in comparisons to AZM (Table 3). Here also the SARs
are straightforward. For example, against hCA XII, com-
pounds 1, 7 and 8 was potent inhibitor, with a Kis of 6.1,
5.9 and 8.8 nM, respectively. The increased carbon chain
length between the two bulky groups (–CH2– or –CH2CH2–)
led to decreased activities of compounds 1 > 2 > 3 with Ki
of 6.1, 12.3 and 10.2 nM respectively of the derivatives.
Some steric hindrance effects led to a loss of the hCA XII
inhibition activity, for example –SO2NH2 present in the
ortho-, meta- and para-substituted sulfonamides 5 < 4 < 1
(showed Kis of 197, 19 and 6.1 nM). Addition of halogen
(F, Cl and Br) to the 3rd position to the compounds 6, 7
and 8 scaffold ‘benzene’ carrying sulfonamide improved
the activity of compounds. The improved activities (Kis in
the range of 7.5–6.5 nM) with respect to increase in the elec-
tro negativity of the halogen groups.
ii. The physiologically dominant cytosolic isoform hCA II was
generally moderately inhibited by the synthesized sulfona-
mides compared to standard AZM, which showed Kis in
the range of 47–190 nM (Table 3). Against hCA II, compound
8 was found to be good inhibitor, with a Ki of 47 nM then the
other compounds of the series. Although the compounds are
moderate inhibitors for hCA II but SARs is rather straightfor-
ward. Addition of halogen (F, Cl and Br) to the 3rd position to
the benzene ring that is compounds 6, 7 and 8 improved the
activities (Kis 96 < 55 < 47 nM) with respect to increase in
the electro negativity of the halogen groups. The increased
carbon chain length between the two bulky groups (–CH2–
or –CH2CH2–) led to an decreased activities of compounds
1 > 2 > 3 with Kis of 68, 71 and 80 nM, respectively of the
derivatives. The steric hindrance effects led to a loss of the
hCA II inhibition, such as for example –SO2NH2 present in
the ortho-, meta- and para-substituted sulfonamides
5 < 4 < 1 (Kis in the range of 190, 79 and 68 nM). Overall,
these sulfonamides show a moderate inhibitory action
towards hCA II, which is the main offtarget isoform among
the various hCAs.27
iii. The cytosolic isoform hCA VII was also poorly inhibited
by the synthesized sulfonamide, Kis in the range of 54–
175 nM (Table 3) then the standard AZM. Compound 1
of the series was the most potent inhibitor with a Ki of
54 nM, but most substitution patterns to the aromatic
ring of sulfonamide, such as the various halogens, led
to compounds with improved inhibitory activity (Kis in
the range of 84 < 72 < 68 nM). The steric hindrance 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 175,
80 and 54 nM). The increased carbon chain length
between the two bulky groups (–CH2– or –CH2CH2–) led
to decreased activities of compounds 1 > 2 > 3 with 54,
59 and 77 nM, respectively of the derivatives. Overall,
these sulfonamides show a poor inhibitory action towards
hCA VII, which is one of a major isoform among the var-
ious hCAs in CNS.1
vi. The selectivity ratio for inhibiting the tumor-associated iso-
forms hCA IX over the cytosolic offtarget one hCA II,27 was in
the range of 0.8–10.2 for the new sulfonamide reported here
(Table 3). This is a good result, since most sulfonamide hCAIs
(D1, D2, acetazolamide, ethoxzolamide, dichlorophenamide,
etc.) show better hCA II than hCA IX inhibitory action.2 In the
new series reported here, only compound 5 had a low selec-

5980
K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
tivity ratio in the of 0.8, being thus relatively little hCA IX-
ma–Aldrich), 4,5,6,7-tetrabromophthalic anhydride (Sigma–
Aldrich), glacial acetic acid, Bromoform, 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 illu-
mination 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 spectrometer using DMSO-d6 as sol-
vent and tetramethylsilane as internal standard. For 1H NMR spec-
tra, chemical shifts are expressed in d (ppm) downfield from
tetramethylsilane, and coupling constants (J) are expressed in
Hertz. Electron ionization mass spectra were recorded in positive
or negative mode on a Water MicroMass ZQ.
selective. The remaining ones had selectivities ratios in the
range of 3.3–10.2, being thus highly selective hCA IX/XII
inhbitors (compared to the inhibition of hCA II and I).
4. Docking studies
Molecular docking of eight synthesized compounds and other
clinically available sulfonamides/sulfamate was performed to ratio-
nalize the SARs reported. Docking study was performed preferably
by interest in crystal structures of hCA preferably in 1AZM (hCA I),
1FZQ (hCA II), 3MDZ (hCA VII), 3IAI (hCA IX) and 1JD0 (hCA
XII).20a–e All the ‘A’ chain of the crystal structures catalytic domains
of the hCA was considered for docking. The Glide (XP) score of the co-
crystallized ligand AZM is À3.8 to À4.8 and the RMSD values ranges
from 1.8 to 2.3 which is considered as good for docking of the ligands
(Table 3). Most of the docking scores of the synthesized compounds
are so good enough then standard AZM and other clinically used sul-
fonamides/sulfamate, D1 and D2 also (Table 3). The docked confor-
mations of AZM (Fig. 4) and compound 2 (highest docking score) in
catalytic site in the binding pocket of hCA IX (PDB: 3IAI).
6.2. Chemistry
A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl ben-
zenesulfonamide derivatives (compounds 1–8) was synthesized
by refluxing mixture of selected sulfonamide and tetrabromoph-
thalic anhydride in glacial acetic acid as solvent with stirring under
nitrogen environment for desired reaction time.22–26
6.2.1. Synthesis of 4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (1)
The main objective of our molecular docking study of com-
pounds was to rationalize the in vitro SARs reported over here. In
each and every case we have observed that the concluded SAR of
the compounds tally to the results obtained by means of docking
study. For examples: in case of compound 5, the docking scores
found to be lowest in each and every case. The increased carbon
chain length between the two bulky groups (–CH2– or –CH2CH2–
) led to improve docking scores in case of hCA I and decrease the
docking scores in hCA II, hCA VII, hCA IX and hCA XII of the synthe-
sized derivatives. Some steric hindrance effects led to a loss of
docking scores, for example –SO2NH2 present in the ortho-, meta-
and para-substituted sulfonamides decrease in scores of com-
pounds 5 < 4 < 1. Addition of halogen (F, Cl and Br) to the 3rd
position to the compounds 6, 7 and 8 scaffold ‘benzene’ carrying
sulfonamide improved the scores of compounds with respect to
increase in the electro negativity of the halogen groups.
0.002 mol (0.344 g) of 4-aminobenzenesulfonamide stirred un-
der nitrogen environment with 0.002 mol (0.927 g) of 4,5,6,7-
tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol of
4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
(1) (1.236 g) in the presence of glacial acetic acid as solvent for 2 h
at 130 °C. The reaction 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 for further purification.22–26
White crystalline and solid; yield = 75%; mp = 284 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially poluble: ethanol;
fully soluble: DMSO and methanol, chloroform. IRmmax (cmÀ1; KBr
pellets); 1774.56, 1718.63 (C@O imide); 1313.57, 1157.33 (S@O)
and 3486.18 (NH2). MS (ESI+); m/z: 612.7 [MÀH]À, 614.7 [M+1].
1H NMR (400 MHz, DMSO-d6): d 7.532 (s, 2H, SO2NH2), 7.666–
7.687 (d, 2H, Ar–H from benzenesulfonamide), 8.018–8.040 (d,
2H, ArÀH from benzenesulfonamide). 13C NMR (100 MHz, DMSO-
d6):121.88, 127.46, 128.81, 131.99, 135.30, 137.72, 144.79, 163.63.
5. Conclusions
A small series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl
benzenesulfonamide was prepared and investigated for the inhi-
bition of the five physiologically relevant hCA isoforms hCA I,
hCA II, hCA VII, hCA IX and hCA XII. These compounds were
generally relevantly good to moderate potent inhibitors of hCA
I, but were moderate to poorly effective with low nanomolar
in hCA II and poorly inhibit hCA VII. Such compounds may con-
stitute interesting candidates for the development of novel anti-
glaucoma, antiepileptic, edema, or altitude sickness drugs. The
compounds are excellent inhibitors of transmembrane isoforms
hCA IX and hCA XII effectively in low nanomolar and with high
selectivity ratio by this new class of sulfonamide hCAIs. Such
potent compounds may promote as novel and interesting candi-
dates for the development of more selective and potent hypoxia
induced cancer drug therapy.
6.2.2. Synthesis of 4-((4,5,6,7-tetrabromo-1,3-dioxoisoindolin-
2-yl)methyl)benzenesulfonamide hydrochloride (2)
0.002 mol (0.445 g) of 4-(aminomethyl)benzenesulfonamide
hydrochloride stirred under nitrogen environment with 0.002 mol
(0.927 g) of 4,5,6,7-tetrabromo isobenzofuran-1,3-dione to produce
0.002 mol of 4-(2-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)ethyl)
benzenesulfonamide (2) (1.264 g) in the presence of glacial acetic
acid as solvent for 1.5 h at 130 °C. The reaction 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 for further
purification.22–26
White crystalline and solid; yield = 94%; mp = 293°C; solubility;
insoluble: water, glacial acetic acid; partially soluble: ethanol and
methanol; fully soluble: DMSO, chloroform. IRm
max (cmÀ1; KBr pel-
6. Experimental section
lets); 1766.85, 1712.86 (C@O imide); 1338.64, 1159.26 (S@O) and
3356.25 (NH2). MS (ESI+); m/z: 626.7 [MÀH]À, 628.7 [M+1]. 1H
NMR (400 MHz, DMSO-d6): d 7.375 (s, 2H, SO2NH2), 7.56, 7.581
(d, 2H, Ar–H from benzenesulfonamide), 7.804–7.825 (d, 2H, Ar–
6.1. Reagents and instruments
All reagents and solvents were of commercial quality and used
without further purification, unless otherwise specified. All
reactions were carried out under an inert atmosphere of nitrogen.
The list of chemicals used are p-amino benzene sulfanilamide (Sig-
H
from benzenesulfonamide), 4.880 (s, 2H, CH2). 13C NMR
(100 MHz, DMSO-d6): 41.79, 121.07, 126.33, 128.38, 131.71,
136.86, 140.24, 143.75, 164.12.

K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
5981
6.2.3. Synthesis of 4-(2-(4,5,6,7-tetrabromo-1,3-
dioxoisoindolin-2-yl)ethyl)benzenesulfonamide (3)
0.002 mol (0.400 g) of 4-(2-aminoethyl)benzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.927 g) of
4,5,6,7-tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol
614.6 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.646 (s, 2H, SO2-
NH2), 7.663–7.668 (d, 2H, Ar–H from benzenesulfonamide),
7.772–7.854 (t, 2H, Ar–H from benzenesulfonamide), 8.097–8.12
(d, 2H, ArÀH from benzenesulfonamide). 13C NMR (100 MHz,
DMSO-d6): 121.93, 129.6, 131.6, 131.53, 132.38, 132.77, 134.0,
137.69, 139.03, 143.23, 163.49.
of
4-(2-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)ethyl)ben-
zenesulfonamide (3) (1.292 g) in the presence of glacial acetic acid
as solvent for 1.5 h at 130 °C. The reaction 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 for further
purification.22–26
6.2.6. Synthesis of 3-fluoro-4-(4,5,6,7-tetrabromo-1,3-
dioxoisoindolin-2-yl)benzenesulfonamide (6)
0.002 mol (0.380 g) of 4-amino-2-fluorobenzenesulfonamide
stirred under nitrogen environment with 0.002 mol (0.927 g) of
4,5,6,7-tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol
of
3-fluoro-4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)ben-
White crystalline and solid; yield = 99%; mp = 278 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially soluble: ethanol,
methanol; fully soluble: DMSO and chloroform. IRmmax (cmÀ1; KBr
pellets); 1772.64, 1716.70 (C@O imide); 1334.78, 1156 (S@O) and
3358.18 (NH2), 2953.12 (aliphatic CH2). MS (ESI+); m/z: 658.5
[MÀH+H2O]. 1H NMR (400 MHz, DMSO-d6): d 7.351 (s, 2H, SO2-
NH2), 7.476À7.497 (d, 2H, Ar–H from benzenesulfonamide),
7.772–7.793 (d, 2H, Ar–H from benzenesulfonamide), 3.004,
3.022, 3.041 (t, 2H, CH2), 3.837, 3.857, 3.874 (t, 2H, CH2). 13C
NMR (100 MHz, DMSO-d6): 33.63, 120.93126.32, 129.65, 131.48,
136.87, 142.80, 142.91, 164.01.
zenesulfonamide (6) (1.272 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; 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 for further
purification.22–26
White crystalline and solid; yield = 90%; mp = 281 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRmmax (cmÀ1; KBr
pellets); 1780.36, 1726.35 (C@O imide); 1339.61, 1158.29 (S@O)
and 3372.65 (NH2). MS (ESI+); m/z: 632.66 [M], 632.66 [M+1]. 1H
NMR (400 MHz, DMSO-d6): d 7.785 (s, 2H, SO2NH2), 7.785–7.824
(d, 2H, Ar–H from benzenesulfonamide), 7.884–7.907 (d, 2H, Ar–
H from benzenesulfonamide), 7.931 (s, 2H, Ar–H from benzenesul-
fonamide). 13C NMR (100 MHz, DMSO-d6): 114.95, 122.10, 123.27,
131.95, 138.00, 147.48, 156.40, 158.94, 162.98.
6.2.4. Synthesis of 3-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (4)
0.002 mol (0.344 g) of 3-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.927 g) of 4,5,6,7-
tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol of 3-
(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
(4) (1.236 g) in the presence of glacial acetic acid as solvent for 2 h
at 130 °C. The reaction 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 for further purification.22–26
Reddish white crystalline and solid; yield = 92%; mp = 306 °C;
solubility; insoluble: water, glacial acetic acid; partially soluble:
ethanol and methanol; fully soluble: DMSO, chloroform. IRmmax
(cmÀ1; KBr pellets); 1770.64, 1716.70 (C@O imide); 1338.64,
1161.19 (S@O) and 3367.82 (NH2). MS (ESI+); m/z: 612.7 [MÀH]À,
614.6 [M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.6 (s, 2H, SO2NH2),
7.689–7.694 (d, 2H, Ar–H from benzenesulfonamide), 7.706–7.714
(d, 2H, Ar–H from benzenesulfonamide), 7.787–7.828 (t, 2H, Ar–H
from benzenesulfonamide), 7.958–7.977 (d, 2H, Ar–H from ben-
zenesulfonamide). 13C NMR (100 MHz, DMSO-d6): 121.81, 125.78,
126.87, 130.94, 131.88, 132.2, 132.98, 137.71, 146.0, 163.89.
6.2.7. Synthesis of 3-chloro-4-(4,5,6,7-tetrabromo-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 (0.927 g) of
4,5,6,7-tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol
of 3-chloro-4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)ben-
zenesulfonamide (7) (1.305 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; 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 for further
purification.22–26
White crystalline and solid; yield = 92%; mp = 265 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRmmax (cmÀ1; KBr
pellets); 1780.36, 1722.49 (C@O imide); 1339.61, 1166.97 (S@O)
and 3366.86 (NH2). MS (ESI+); m/z: 646.83 [MÀ1], 648.83 [M+1].
1H NMR (400 MHz, DMSO-d6): i 7.717 (s, 2H, SO2NH2), 7.851–
7.872 (d, 2H, Ar–H from benzenesulfonamide), 8.008–8.034 (d,
2H, Ar–H from benzenesulfonamide), 8.152 (s, 2H, Ar–H from ben-
zenesulfonamide). 13C NMR (100 MHz, DMSO-d6): 122.24, 126.42,
128.09, 131.69, 133.77, 136.96, 138.27, 147.48, 162.84, 166.77.
6.2.5. Synthesis of 2-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-
yl)benzenesulfonamide (5)
0.002 mol (0.344 g) of 2-aminobenzenesulfonamide stirred
under nitrogen environment with 0.002 mol (0.927 g) of 4,5,6,7-
tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol of 2-
(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)benzenesulfonamide
(5) (1.236 g) in the presence of glacial acetic acid as solvent for 2 h
at 130 °C. The reaction 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 for further purification.22–26
White crystalline and solid; yield = 80%; mp = 272 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially soluble: ethanol
and methanol; fully soluble: DMSO and chloroform. IRmmax
(cmÀ1; KBr pellets); 1770.71, 1708.99 (C@O imide); 1340.57,
1172.76 (S@O) and 3390.97 (NH2). MS (ESI+); m/z: 612.6 [MÀH]À,
6.2.8. Synthesis of 3-bromo-4-(4,5,6,7-tetrabromo-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 (0.927 g) of
4,5,6,7-tetrabromo isobenzofuran-1,3-dione to produce 0.002 mol
of 3-bromo-4-(4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl)ben-
zenesulfonamide (8) (1.394 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; 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

5982
K. K. Sethi et al. / Bioorg. Med. Chem. 21 (2013) 5973–5982
4. Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2003, 46, 2197.
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.
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; p 139.
4/5 times. The product was recrystallized in ethanol for further
purification.22–26
White crystalline and solid; yield = 90%; mp = 255 °C; solubil-
ity; insoluble: water, glacial acetic acid; partially soluble: ethanol;
fully soluble: DMSO, chloroform and methanol. IRmmax (cmÀ1; KBr
pellets); 1767.82, 1723.45 (C@O imide); 1340.57, 1120.86 (S@O)
and 3379.40 (NH2). MS (ESI+); m/z: 690.75 [MÀH]À, 692.75
[M+1]. 1H NMR (400 MHz, DMSO-d6): d 7.708 (s, 2H, SO2NH2),
7.832–7.853 (d, 2H, Ar–H from benzenesulfonamide), 8.043–
8.069 (d, 2H, Ar–H from benzenesulfonamide), 8.288 (s, 2H, Ar–H
from benzenesulfonamide). 13C NMR (100 MHz, DMSO-d6):
122.25, 124.26, 126.98, 131.90, 132.95, 136.99, 138.37, 147.57,
162.77, 166.74.
`
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.
6.3. hCA 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 Discovery 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. Basnyat, B.; Gertsch, J. H.; Johnson, E. W.; Castro-Marin, F.; Inoue, Y.; Yeh, C.
High Alt. Med. Biol. 2003, 4, 45.
An Applied Photophysics stopped-flow instrument has been
used for assaying the hCA-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 hCA-cat-
alyzed CO2 hydration reaction for a period of 10–100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each inhib-
itor, 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 to-
tal observed rates. Stock inhibitor solutions (0.1 mM) were pre-
pared in distilled–deionized water, and dilutions of up to
0.01 nM were made thereafter with distilled–deionized 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 E–I complex or the eventual active site-mediated hydrolysis
of the inhibitor. The inhibition constants were obtained by nonlin-
ear least-squares methods using PRISM 3.28–30
18. Hackett, P. H.; Roach, R. C. N. Engl. 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. (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)
Ugochukwu, E.; Shafqat, N.; Pilka, E.; Chaikuad, A.; Krojer, T.; Muniz, J.; Kim, J.;
Bray, J.; Bountra, C.; Arrowsmith, C. H.; Weigelt, J.; Edwards, A.; von Delft, F.;
Carpenter, E.P.; Yue, W. W.; Oppermann, U. http://dx.doi.org/10.2210/
pdb3mdz/pdb.; (d) Ban, N.; Escobar, C.; Garcia, R.; Hasel, K.; Day, J.;
Greenwood, A.; McPherson, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1604; (e)
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.
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. Discov.
2001, 17, 337; (b) Sethi, K. K.; Verma, S. M.; Tanç, M.; Carta, F.; Supuran, C. T.
Bioorg. Med. Chem. 2013, 21, 5168.
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. 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.
Acknowledgments
This work is acknowledged to our vice-chancellor of BIT Mesra,
Ranchi, Dr. P. K. Barahi, who has given all the infrastructural oppor-
tunity for research. Work from the Florence laboratory was fi-
nanced by two FP7 EU projects, Metoxia and Dynano.
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.
References and notes
26. Supuran, C. T. Front. Pharmacol. 2011, 2, 34.
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. Future Med.
Chem. 2011, 3, 1165; (c) Rummer, J. L.; McKenzie, D. J.; Innocenti, A.; Supuran,
C. T.; Brauner, C. J. Science 2013, 340, 1327.
27. 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; p 457.
28. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
29. Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668.
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.
30. Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 6208.