Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/membrane-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating hydrazino moieties

Article abstract of DOI:10.1021/jm0494826

Targeting proteins overexpressed in hypoxic tumors is as an important means of controlling cancer disease. One such protein is the carbonic anhydrase (CA) isoenzyme IX, which in some types of tumors is overexpressed 150-200-fold. We report here a series of sulfonamide derivatives, prepared from 2-carbohydrazido- and 4-carbohydrazido-benzenesulfonamides, which were further derivatized by reaction with aryl isocyanates or arylsulfonyl isocyanates. Several low nanomolar CA IX inhibitors were detected in this way. SAR is discussed for the diverse types of inhibitors and their affinity for different isozymes, with the aim of obtaining isozyme-specific CA IX inhibitors, with putative applications as antitumor drugs.

Full text of DOI:10.1021/jm0494826

J. Med. Chem. 2005, 48, 2121-2125
2121
Carbonic Anhydrase Inhibitors: Synthesis and Inhibition of Cytosolic/
Membrane-Associated Carbonic Anhydrase Isozymes I, II, and IX with
Sulfonamides Incorporating Hydrazino Moieties
Jean-Yves Winum,†,‡ Jean-Michel Dogn e´ , Angela Casini, Xavier de Leval, Jean-Louis Montero,
†,§
†
†,§
‡
†
†
†
,†
Andrea Scozzafava, Daniela Vullo, Alessio Innocenti, and Claudiu T. Supuran*
Polo Scientifico, Laboratorio di Chimica Bioinorganica, Universit a` degli Studi di Firenze, Room 188, Via della Lastruccia 3,
5
0019 Sesto Fiorentino (Florence), Italy, Laboratoire de Chimie Biomol e´ culaire, UMR 5032, Ecole Nationale Sup e´ rieure de
Chimie de Montpellier, Universit e´ Montpellier II, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France, and
Department of Medicinal Chemistry, Natural and Synthetic Drugs Research Centre, University of Li e` ge, 1, av. de l’H oˆ pital,
B36 Sart-Tilman, B-4000 Li e` ge, Belgium
Received June 30, 2004
Targeting proteins overexpressed in hypoxic tumors is as an important means of controlling
cancer disease. One such protein is the carbonic anhydrase (CA) isoenzyme IX, which in some
types of tumors is overexpressed 150-200-fold. We report here a series of sulfonamide
derivatives, prepared from 2-carbohydrazido- and 4-carbohydrazido-benzenesulfonamides, which
were further derivatized by reaction with aryl isocyanates or arylsulfonyl isocyanates. Several
low nanomolar CA IX inhibitors were detected in this way. SAR is discussed for the diverse
types of inhibitors and their affinity for different isozymes, with the aim of obtaining isozyme-
specific CA IX inhibitors, with putative applications as antitumor drugs.
Introduction
to a hypoxia response element in the CA9 gene pro-
moter.1 CA IX was proposed to serve as a marker of
tumor hypoxia, and its predictive and prognostic poten-
tial has been demonstrated in numerous clinical studies
,2
It has only recently been discovered that invasive
growth and metastatic spread of many tumors types are
closely associated with hypoxia. Tumor hypoxia is the
1
(
reviewed in ref 2) CA XII is present in many normal
result of the abnormal process of neoplastic growth and
crucially depends on oxygen/nutrients supply from the
2
tissues and overexpressed in some tumors. It is also
induced by hypoxia, but the underlying molecular
mechanism remains undetermined. Both CA IX and CA
XII are negatively regulated by von Hippel Lindau
tumor suppressor protein, and their expression in renal
cell carcinomas is related to inactivating mutation of
1
host. Thus, changes in tumor metabolism and microen-
vironment connected with adaptation of cells to hypoxia
are important components of tumor progression.1,2 Hy-
poxic conditions elicit cellular responses designed to
improve cell oxygenation and survival by means of
several mechanisms such as neoangiogenesis, improved
glycolysis, and enhanced energy production, as well
as upregulation of molecules related to cell survival/
2
VHL gene. The high catalytic activity of these two CA
isoforms supports their role in acidification of tumor
microenvironment that facilitates acquisition of meta-
static phenotypes.2 Therefore, modulation of extra-
cellular tumor pH via inhibition of CA activity repre-
sents a promising approach to anticancer therapy.2
Sulfonamide CA inhibitors (CAIs) were shown to com-
promise tumor cell proliferation and invasion in vitro
and improve the effect of conventional chemotherapy
in vivo.2 However, their precise targets are not known
in detail at this moment, but it is presumed that these
two tumor-associated CA isozymes, i.e., CA IX and XII,
may represent important molecules for targeting cancer
cells, by an unconventional therapeutic approach.2
In previous work from this laboratory, we showed that
CA IX is a target for which drugs can be developed.6
In such papers we have explored the design of potent
and preferably selective sulfamate/sulfonamide CA IX
inhibitors belonging to various chemical classes.6 It
was thus observed among others that unlike for other
-4
1
apoptosis. The most important molecule regulating the
mammalian response to hypoxia is the heterodimeric
protein hypoxia-inducible factor 1 (HIF-1), which in turn
up-regulates genes involved in adaptation responses to
-4
1
hypoxic conditions. Two such genes encode for the
transmembrane carbonic anhydrase (CA, EC 4.2.1.1)
isozymes CA IX and CA XII, containing extracellular
enzyme active sites. These CAs appear to participate
in tumorigenetic processes via their ability to catalyze
hydration of CO2 to bicarbonate and protons, regulating
-5
-5
2
in this way the intratumoral pH. In addition, CA IX,
possessing a unique N-terminal domain, has a capacity
to perturb E-cadherin-mediated cell-cell adhesion via
interaction with â-catenin and may potentially contrib-
-9
2
ute to tumor invasion. CA IX shows restricted expres-
-9
sion in normal tissues but is tightly associated with
different types of tumors, mostly due to its strong
induction by tumor hypoxia that involves HIF-1 binding
CA isozymes (such as for example CA I, II, or V among
others),1
0-13
aromatic sulfonamides are generally better
*
Corresponding author. Phone: +39-055-4573005; Fax: +39-055-
CA IX inhibitors, as compared to the heterocyclic
derivatives. Thus, it appeared of interest to explore
other chemical scaffolds incorporating aromatic (ben-
zene) sulfonamide derivatives that led to the best CAIs
4
573385; E-mail: claudiu.supuran@unifi.it.
†
Universit a` degli Studi di Firenze.
‡
Universit e´ Montpellier II.
§
University of Li e` ge.
1
0.1021/jm0494826 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/25/2004

2
122 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Winum et al.
targeting CA IX reported up to now.13 In this work we
consider several simple approaches to obtain such
compounds. A common denominator of all derivatives
reported in this paper is the presence of a hydrazine
moiety in their molecule. This was considered to be
beneficial for CA IX targeting compounds, for several
reasons: (i) the facile synthesis and subsequent deriva-
tization of such compounds, leading to a chemical
diversity necessary when such new targets are consid-
Scheme 1
1
4
ered; (ii) the hydrazine moiety is susceptible to redox
chemistry which may be advantageous in the hypoxic
1
,2
environment present in some tumors; and (iii) the
presence of this moiety in some recently approved drugs,
such as for example the HIV protease inhibitor ataza-
navir, which has been shown to be devoid of major
toxicity and induced excellent bioavailability to this
drug, which is the first protease inhibitor for once-a-
day oral dosing.1
5
Chemistry. Benzenesulfonamide derivatives show
well-known CA inhibitory properties, and a wide range
of such compounds have been used in the design of
inhibitors with various medicinal chemistry applica-
1
6-19
tions.
A drawback of some of these simple deriva-
tives (such as orthanilamide, the halogenated sulfanil-
amides, the 4-amino-1,3-benzene-disulfonamides, etc.)
is constituted sometimes by the rather low reactivity of
amino groups grafted on the aromatic ring, which are
deactivated for nucleophilic substitution reactions by the
presence of the sulfamoyl moiety/moieties. Thus, we
decided to investigate whether the substitution of the
amino moiety of some of these derivatives with the
hydrazine one may lead to an enhanced reactivity, to
further derivatize them for obtaining CAIs with various
applications.
acid. The diazonium salts 11-13 were then reduced to
hydrazines 14-16 and 18 with tin(II) chloride or sodium
sulfite in acidic medium,18 and the last compound has
also been acetylated with acetic anhydride at room
temperature, leading to compound 19 (Scheme 2).
CA Inhibition. Data of Table 1 show CA I, II, and
IX inhibition with the new compounds reported here,
as well as clinically used CAIs, such as acetazolamide
(AAZ), methazolamide (MZA), ethoxzolamide (EZA),
dichorophenamide (DCP), dorzolamide (DZA), and brin-
zolamide (BRZ). Indisulam (E7070) (IND), an antitumor
2
0
Two approaches have been considered here for achiev-
ing this goal: (i) starting from commercially available
simple derivatives, such as 4-carboxybenzenesulfona-
mide 1 or 2-carboxymethylbenzenesulfonamide 5, the
corresponding hydrazides 2 and 6, respectively, have
1
6,17
been obtained,
by reaction of the corresponding
methyl ester with hydrazine hydrate. The two hy-
drazides 2 and 6 were then further derivatized, by
2
1
reaction with aryl isocyanates, leading to ureas 3a-j,
or by reaction with arylsulfonylisocyanates,22 leading
to sulfonyl ureas 4a-e and 7a-e, respectively (Scheme
1
). Indeed, in previous work from this laboratory it has
been demonstrated that both ureido- as well as aryl-
sulfonylureido-substituted aromatic/heterocyclic sul-
fonamides lead to potent CAIs targeting isozymes I, II,
2
1,22
and IV,
and this has thereafter been rationalized
after the report of the X-ray crystal structure of some
of these derivatives (or their structurally related con-
geners) with hCA II. In such adducts it has been
observed that the ureido/carboxamido part of the inhibi-
tor participates in several favorable interactions with
amino acid residues at the entrance of the active site,
ensuring in this way a stabilized E-I complex, and as
a consequence potent CA inhibitory properties to these
2
3
compounds; (ii) the conversion of the amino to the
hydrazine moiety, in simple compounds such as sulf-
anilamide, halogenosulfanilamides, or orthanilamide
sulfonamide in phase II clinical trials for which we
recently demonstrated potent CA inhibitory properties,
has also been included for comparison in this study.5
Furthermore, the X-ray crystal structure of IND in
adduct with isozyme hCA II has recently been reported
by our group.10
d,10,24
(derivatives 8-10 and 17) by means of the diazonium
salts, which have been prepared (but not isolated) by
reaction of the amine with in situ generated nitrous

Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2123
Scheme 2
Table 1. Inhibition Data for Derivatives 1-19 Investigated in
the Present Paper and Standard Sulfonamide CAIs, against
Isozymes I, II, and IX
KIa (nM)
compd
R
hCA Ib
hCA IIb
hCA IXc
AAZ
MZA
EZA
DCP
DZA
BRZ
IND
1
-
-
-
-
-
-
-
-
-
250
50
12
14
25
27
25
8
34
1200
50000
38
50
9
3
52
e
nt
37
31
15
24
3400
2950
1150
1450
1200
760
258
124
13
346
175
7.9
8.3
8.5
3.2
8.6
5.4
7.9
7.0
24
2
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
3,4-Cl2C6H3
4-Ac-C6H4
4-EtOOC-C6H4
4-O2N-C6H4
4-Br-C6H4
4-Ph-C6H4
4-PhO-C6H4
4-PhCH2-C6H4
4-Ad-C6H4
9-fluorenyl
Ph
2-Me-C6H4
4-Me-C6H4
4-F-C6H4
4-Cl-C6H4
-
15
9.0
5.1
7.3
11
865
1160
1450
1300
2400
1360
1250
800
18
15
The following SAR should be noted from data of Table
: (i) the derivatives 1-19 investigated here showed
d
33
1
j
4a
25
13
inhibitory activity against all three investigated CA
isozymes, but affinity was in the micromolar range for
the cytosolic isozyme hCA I, and generally in the (low)
nanomolar range for the other cytosolic isozyme, hCA
II, as well as the transmembrane, tumor-associated
isozyme hCA IX. Thus, against hCA I, these compounds
showed KIs in the range of 0.55-48.3 µM, against hCA
II in the range of 5.1-420 nM, and against hCA IX in
the range of 3.2-680 nM; (ii) the best hCA I inhibitors
were some of the compounds designed starting from
67
35
4
4
4
4
5
6
7
7
7
b
c
d
e
54
32
700
9.8
9.5
9.1
420
338
107
92
5.8
6.2
5.4
680
547
76
600
550
35800
24500
4800
4700
3400
4900
3600
3900
3300
2800
45400
36000
48300
-
Ph
a
b
c
2-Me-C6H4
4-Me-C6H4
4-F-C H
83
85
70
7d
7e
6
4
97
64
4-Cl-C6H4
94
72
1
1
1
1
4
5
6
7
-
-
-
-
-
-
83
265
136
149
33
4
3
-sulfamoylbenzenecarbohydrazide 2, such as the ureas
51
d and 3e, as well as the sulfonylureas 4b-e, which
76
showed KIs in the range of 0.55-0.865 µM, whereas
other substitution patterns, or derivatives designed
starting from 6 as well as the simple hydrazine deriva-
tives 14-16 and 18, 19, were much less effective
inhibitors; (iii) against hCA II, the best inhibitors were
the ureas 3a-h, and the sulfonylureas 4c-e, which
showed KIs in the range of 5.1-15 nM. It may be
observed that again these compounds are derived from
295
78
18
48
175
1
9
264
a Errors in the range of 5-10% of the reported value (from three
b
different assays). Human cloned isozyme, by the CO2 hydration
c
method. Catalytic domain of human, cloned isozyme, by the CO2
d
e
hydration method. Ad ) 1-adamantyl. nt ) not tested.
the enzyme active site. This was also the reason why a
smaller number of such derivatives has been synthe-
sized, and our main interest has been concentrated on
the para-substituted compounds; (iv) against hCA IX,
the best inhibitors in the investigated series were again
the ureas 3a-j and the sulfonylureas 4c-e, which
showed KIs in the range of 3.2-24 nM, being more
effective than the clinically used sulfonamides (aceta-
zolamide-brinzolamide), and better or equally potent
hCA IX inhibitors as indisulam IND, the sulfonamide
4
-sulfamoylbenzenecarbohydrazide 2, and that the na-
ture of the group present in the original isocyanate/
sulfonylisocyanate (R), is the most important factor
influencing CA II inhibitory properties. Thus, such
4-substituted-phenyl-, 3,4-dichlorophenyl-, or biphenyl-
moieties seem to be quite beneficial for the hCA II
inhibitory properties, whereas bulkier moieties (ada-
mantly, fluorenyl, etc.), such as in 3g, 3i, and 3j, lead
to a decreased activity. The simple derivatives 1, 2, and
14-18 were less effective hCA II inhibitors as compared
with the most impressive in vivo antitumor properties
reported up to now.5
d,10,24
Thus, the nitrophenyl- (3d)
to the above-mentioned compounds. Some of the best
hCA II inhibitors among the new derivatives reported
here showed the same level of hCA II inhibition as some
of the clinically used compounds (acetazolamide, met-
hazolamide, dorzolamide, indisulam, etc.), whereas
dichlorophenamide, a clinically used systemic antiglau-
and biphenyl-substituted ureas (3f) as well as the
chlorophenylsuylfonylureido derivative 4e, showed in-
hibition constants in the range of 3.2-5.4 nM, being
among the most potent hCA IX inhibitors detected so
far, and their potency compared to that of indisulam is
5-7-fold increased. As a whole, almost all substitution
patterns of ureas 3 reported here were beneficial for the
hCA IX inhibitory properties, and generally all these
compounds were better hCA IX than hCA II inhibitors
(except 3e), a situation which is reversed as compared
to the clinically used derivatives (including indisulam)
which are all better hCA II than hCA IX inhibitors
(Table 1). The simple derivatives 1 and 2, as well as
4
coma drug, is slightly less effective as hCA II inhibitor,
Table 1. It should also be mentioned that the sulfony-
lureas isomeric to 4a-e, of types 7a-e, obtained from
the o-carbohydrazidobenzenesulfonamide 6, were much
less effective hCA II inhibitors as compared to the para-
substituted corresponding compounds. This may be due
to the sterical impairment of the ortho-substituent for
the binding of such compounds to the Zn(II) ion within

2
124 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Winum et al.
the ortho-substituted derivatives 7a-e, or the hydra-
zines 15-19, were weaker hCA IX inhibitors, generally
with inhibition constants in the range of 33-346 nM.
concentrations of urea, when the yields in active protein were
rather low, and the procedure much longer. The obtained CA
IX was further purified by sulfonamide affinity chromatogra-
20
phy, the amount of enzyme being determined by spectropho-
metric measurements and its activity by stopped-flow experi-
Experimental Section
6
,7
2
ments, with CO as substrate. The specific activity of the
To a stirring 0.1 M solution of methyl 2-(aminosulfonyl)-
benzoate (commercially available, Sigma-Aldrich, Milan, Italy)
or methyl 4-(aminosulfonyl)benzoate (prepared by esterifica-
tion of the corresponding carboxylic acid with 1 equiv of EDCI
in a mixture methylene chloride-methanol 1:1) in methanol
was added 10 equiv of hydrazine hydrate. The solution was
allowed to stir at room-temperature overnight then concen-
trated in vaccuo. The residue was coevaporated several times
with toluene until obtaining the desired compound as a white
powder, which was recrystallized in ethanol. Characterization
of all the compounds is available as Supporting Information
to this article.
obtained enzyme was the same as the one previously re-
ported,6,7 but the yields in active protein were 5-6 times higher
per liter of culture medium). An SX.18MV-R Applied Photo-
physics stopped-flow instrument has been used for assaying
the CA CO2 hydration activity assays.32 Phenol red (at a
concentration of 0.2 mM) has been used as indicator, working
at the absorbance maximum of 557 nm, with 10 mM HEPES
(pH 7.5) as buffer, 0.1 M Na2SO
the ionic strength), following the CA-catalyzed CO
reaction for a period of 10-100 s. Saturated CO solutions in
4
(for maintaining constant
2
hydration
2
32
water at 20 °C were used as substrate. Stock solutions of
inhibitor (1 mM) were prepared in distilled-deionized water
with 10-20% (v/v) DMSO (which is not inhibitory at these
concentrations), and dilutions up to 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and en-
zyme solutions were preincubated together for 15 min at room
temperature prior to assay, to allow for the formation of the
E-I complex. Triplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the paper are the mean of such results.
General Procedure for the Coupling of Sulfamoylben-
zoic Acid Hydrazides 2 and 6 with Arylsulfonyl Iso-
cyanate and Aryl Isocyanate. To a stirred solution of
sulfamoylbenzoic acid hydrazide 2 or 6 (1 equiv) in acetone
was added at room temperature the sulfonyl isocyanate or aryl
isocyanate (1 equiv). The reaction was monitored by TLC until
starting material was consumed. Then the mixture was
concentrated under vacuum, and the residue was triturated
with ethylic ether. After filtration and washing with methylene
chloride, the resulting powder can be purified by column
chromatography to yield the expected product in 70% yield.
Synthesis of Hydrazinobenzenesulfonamides 14-16
and 18. Concentrated (36%) hydrochloric acid (6 mL) and 10
g of ice were added to 1 g of 4-amino-3-chloro/fluorobenzene-
sulfonamide (9 or 10). The suspension was cooled on ice and
stirred. An amount of 1 g of sodium nitrite was dissolved into
a minimal amount of water (2 mL), and this solution was
added dropwise to the benzenesulfonamide solution (temper-
ature < 5 °C). At the end of this step, 3 g of sodium sulfite
was poured in the medium, and the obtained suspension was
stirred for 12 h. The suspension was then dried by solvent
evaporation under depression. The residue was suspended in
acetone, and the insoluble part was harvested by filtration.
This precipitate was dissolved in a minimal amount of water,
and the pH was adjusted to 7 with sodium hydroxide. The
solution was then extracted three times with diethyl ether,
and the organic phases were collected, dried on magnesium
sulfate, and evaporated under depression. The dried residue
was dissolved in acetone, and drops of hydrochloric acid were
added in order to precipitate the hydrazine hydrochlorides 15
and 16 which were filtered and washed with acetone. The
purity of the final compound was verified by TLC (MeOH/
Acknowledgment. This research was financed in
part by the 6th Framework Program of the European
Union (EUROXY project). J.Y.W. is grateful to CSGI,
University of Florence, and University of Montpellier
II for a travel grant to Florence.
Supporting Information Available: Complete charac-
terization of the compounds described in this paper. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) H o¨ pfl, G.; Ogunshola, O.; Gassmann, M. HIFs and tumors -
Causes and consequences. Am. J. Physiol. Regul. Integr. Comput.
Physiol. 2004, 286, R608-R623.
(
2) Pastorekova, S.; Pastorek, J. Cancer-related carbonic anhydrase
isozymes and their inhibition. In Carbonic anhydrase - Its
inhibitors and activators; Supuran, C. T., Scozzafava, A., Con-
way, J.; Eds., CRC Press: Boca Raton, FL, 2004; pp 255-282.
(3) Supuran, C. T.; Scozzafava, A.; Conway, J., Eds. Carbonic
anhydrase - its inhibitors and activators; CRC Press (Taylor
and Francis Group): Boca Raton, FL, 2004; pp 1-363, and
references therein.
CHCl
3
: 3/7).
(
4) (a) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors
and their therapeutic potential. Expert Opin. Ther. Pat. 2000,
10, 575-600; (b) Supuran, C. T.; Scozzafava, A. Applications of
carbonic anhydrase inhibitors and activators in therapy. Expert
Opin. Ther. Pat. 2002, 12, 217-242; (c) Supuran, C. T.; Scoz-
zafava, A.; Casini, A. Carbonic anhydrase inhibitors. Med. Res.
Rev. 2003, 23, 146-189; (d) Scozzafava, A.; Mastrolorenzo, A.;
Supuran C. T. Modulation of carbonic anhydrase activity and
its applications in therapy. Expert Opin. Ther. Pat. 2004, 14,
667-702.
CA Inhibition. Human CA I, CA II, and CA IX cDNAs were
expressed in Escherichia coli strain BL21 (DE3) as previously
2
0,25
6,7
described.
A variant of the previously published CA IX
purification protocol has been used for obtaining high amounts
of hCA IX needed in these experiments. The cDNA of the
catalytic domain of hCA IX (isolated as described by Pastorek
2
5
et al. ) was amplified by using PCR and specific primers for
the glutathione S-transferase (GST)-Gene Fusion Vector pGEX-
(
5) Scozzafava. A.; Owa, T.; Mastrolorenzo, A.; Supuran, C. T.
Anticancer and antiviral sulfonamides. Curr. Med. Chem. 2003,
3
3
X. The obtained fusion construct was inserted in the pGEX-
X vector and then expressed in Escherichia coli BL21 Codon
1
0, 925-953.
Plus bacterial strain (from Stratagene). The bacterial cells
were sonicated and then suspended in the lysis buffer (10 mM
Tris pH 7.5, 1 mM EDTA pH 8, 150 mM NaCl and 0.2% Triton
X-100). After incubation with lysozyme (approximately 0.01
g/L), the protease inhibitors Complete were added to a final
concentration of 0.2 mM. The obtained supernatant was then
applied to a prepacked Glutathione Sepharose 4B column and
extensively washed with buffer, and the fusion (GST-CA IX)
protein was eluted with a buffer consisting of 5 mM reduced
glutathione in 50 mM Tris-HCl pH 8.0. Finally the GST part
of the fusion protein was cleaved with thrombin. The advan-
(
6) Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.; Scozzafava,
A.; Supuran, C. T. Carbonic anhydrase inhibitors: Inhibition of
cytosolic isozymes I and II and the transmembrane, tumor-
associated isozyme IX with sulfamates including EMATE also
acting as steroid sulfatase inhibitors. J. Med. Chem. 2003, 46,
2
197-2204.
(
7) Vullo, D.; Franchi, M.; Gallori,E.; Pastorek, J.; Scozzafava, A.;
Pastorekova, S.; Supuran, C. T. Carbonic anhydrase inhibitors.
Inhibition of cytosolic isozymes I and II and transmembrane,
cancer-associated isozyme IX with anions. J. Enzyme Inhib. Med.
Chem. 2003, 18, 403-406.
(
8) Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.; Scozzafava,
A.; Supuran, C. T. Carbonic anhydrase inhibitors: Inhibition of
transmembrane, tumor-associated isozyme IX and cytosolic
isozymes I and II with aliphatic sulfamates. J. Med. Chem. 2003,
46, 5471-5477.
6
,7
tage of this method over the previous one, is that CA IX is
not precipitated in inclusion bodies from which it has to be
isolated by denaturing-renaturing in the presence of high

Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2125
(
9) (a) Weber, A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran, C. T.;
Scozzafava, A.; Klebe, G. Unexpected nanomolar inhibition of
carbonic anhydrase by COX-2 selective Celecoxib: New phar-
macological opportunities due to related binding site recognition.
J. Med. Chem. 2004, 47, 550-557. (b) Pastorekova, S.; Casini,
A.; Scozzafava, A.; Vullo, D.; Pastorek, J.; Supuran, C. T.
Carbonic anhydrase inhibitors: The first selective, membrane-
impermeant inhibitors targeting the tumor-associated isozyme
IX. Bioorg. Med. Chem. Lett. 2004, 14, 869-873. (c) Casey, J.
R.; Morgan, P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Design of selec-
tive, membrane-impermeant inhibitors targeting the human
tumor-associated isozyme IX. J. Med. Chem. 2004, 47, 2337-
(20) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors.
Perfluoroalkyl/aryl-substituted derivatives of aromatic/hetero-
cyclic sulfonamides as topical intraocular pressure lowering
agents with prolonged duration of action. J. Med. Chem. 2000,
4
3, 4542-4551.
(
21) (a) Supuran, C. T.; Scozzafava, A.; Jurca, B. C.; Ilies, M. A.
Carbonic anhydrase inhibitors. Part 49. Synthesis of substituted
ureido- and thioureido derivatives of aromatic/heterocyclic sul-
fonamides with increased affinities for isozyme I. Eur. J. Med.
Chem. 1998, 33, 83-93. (b) Casini, A.; Scozzafava, A.; Mincione,
F.; Menabuoni, L.; Ilies, M. A.; Supuran, C. T. Carbonic anhy-
drase inhibitors. Water soluble 4-sulfamoylphenyl-thioureas as
topical intraocular pressure lowering agents with long lasting
effects. J. Med. Chem. 2000, 43, 4884-4892.
2347.
(
10) Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer
agent, potently inhibits cytosolic isozymes I and II, and trans-
membrane, tumor-associated isozyme IX. Bioorg. Med. Chem.
Lett. 2004, 14, 217-223.
11) Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Inhibition of
mitochondrial isozyme V with aromatic and heterocyclic sul-
fonamides. J. Med. Chem. 2004, 47, 1272-1279.
(
22) (a) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors.
Arylsulfonylureido and arylureido-substituted aromatic and
heterocyclic sulfonamides: towards selective inhibitors of car-
bonic anhydrase isozyme I. J. Enzyme Inhib. 1999, 14, 343-
(
3
63. (b) Popescu, A.; Simion, A.; Scozzafava, A.; Briganti, F.;
Supuran, C. T. Carbonic anhydrase inhibitors. Schiff bases of
some aromatic sulfonamides and their metal complexes: towards
more selective inhibitors of carbonic anhydrase isozyme IV. J.
Enzyme Inhib. 1999, 14, 407-423.
(
12) de Leval, X.; Ilies, M.; Casini, A.; Dogn e´ , J.-M.; Scozzafava, A.;
Masini, E.; Mincione, F.; Starnotti, M.; Supuran C. T. Carbonic
anhydrase inhibitors: Synthesis and topical intraocular pressure
lowering effects of fluorine-containing inhibitors devoid of
enhanced reactivity. J. Med. Chem. 2004, 47, 2796-2804.
13) Vullo, D.; Scozzafava, A.; Pastorekova, S.; Pastorek, J.; Supuran,
C. T. Carbonic anhydrase inhibitors: Inhibition of the tumor-
associated isozyme IX with fluorine-containing sulfonamides.
The first subnanomolar CA IX inhibitor discovered. Bioorg. Med.
Chem. Lett. 2004, 14, 2351-2356.
(23) (a) Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T. Carbonic
anhydrase inhibitors: X-ray crystallographic structure of the
adduct of human isozyme II with the perfluorobenzoyl analogue
of methazolamide. Implications for the drug design of fluorinated
inhibitors. J. Enzyme Inhib. Med. Chem. 2003, 18, 303-308. (b)
Casini, A.; Abbate, F.; Scozzafava, A.; Supuran, C. T. Carbonic
anhydrase inhibitors: X-ray crystallographic structure of the
adduct of human isozyme II with a bis-sulfonamide - two heads
are better than one? Bioorg. Med. Chem. Lett. 2003, 13, 2759-
2763. (c) Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors: X-ray crystallographic structure
of the adduct of human isozyme II with a topically acting
antiglaucoma sulfonamide. Bioorg. Med. Chem. Lett. 2004, 14,
(
(
14) Melkko, S.; Scheuermann, J.; Dumelin, C. E.; Neri D. Encoded
self-assembling chemical libraries. Nat. Biotechnol. 2004, 22,
568-574.
(
15) Havlir, D. V.; O’Marro, S. D. Atazanavir: New Option for
Treatment of HIV Infection. Clin. Infect. Dis. 2004, 38, 1599-
1604.
(
16) Garg, H. G.; Arora, V. Chemistry and biological activity of N1-
acyl-4-arylazopyrazoles. J. Pharm. Sci. 1972, 61, 130-132.
17) Whitehead, C. W.; Traverso, J. J. The reaction of saccharin with
amines. N-Substituted-3-amino-1,2-benzisothiazole-1,1-dioxides.
J. Org. Chem. 1960, 25, 413-416.
18) Crippa, G. B.; Maffei, S. Derivati solfonamidici del pirazolo. 1-(p-
solfonamidofenil)-3-metil-5-pirazolone. Gazz. Chim. Ital. 1941,
2
357-2361.
(
(
24) Supuran, C. T. Indisulam: an anticancer sulfonamide in clinical
development. Expert Opin. Investig. Drugs 2003, 12, 283-287.
25) Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.; Zelnik,
V.; Opavsky, R.; Zatovicova, M.; Liao, S.; Portetelle, D.; Stan-
bridge, E. J.; Zavada, J.; Burny, A.; Kettmann, R. Cloning and
characterization of MN, a human tumor-associated protein with
a domain homologous to carbonic anhydrase an a putative helix-
loop-helix DNA binding segment. Oncogene 1994, 9, 2877-2888.
(
(
71, 97-99.
(
19) Supuran, C. T.; Casini, A.; Scozzafava, A. Development of
sulfonamide carbonic anhydrase inhibitors (CAIs). In Carbonic
anhydrase - Its inhibitors and activators; Supuran, C. T.,
Scozzafava, A., Conway, J.; Eds., CRC Press: Boca Raton, FL,
2004; pp 67-147.
JM0494826