Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

Article abstract of DOI:10.1016/j.bmcl.2004.09.062

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.

Full text of DOI:10.1016/j.bmcl.2004.09.062

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5775–5780
Carbonic anhydrase inhibitors: synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and
IX with sulfonamides derived from 4-isothiocyanato-benzolamide
Alessandro Cecchi,a Jean-Yves Winum,a,b Alessio Innocenti,a Daniela Vullo,a
Jean-Louis Montero,b Andrea Scozzafavaa and Claudiu T. Supurana,*
a
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
b
´ ´ ´
Universite Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032, Ecole Nationale Superieure de Chimie de Montpellier,
8rue de l’Ecole Normale, 34296 Montpellier Cedex, France
Received 24 June 2004; revised 3 September 2004; accepted 17 September 2004
Abstract—A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide
and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new
derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the
cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds
showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6–62nM against
hCA I, 0.5–1.7nM against hCA II and 3.2–23nM against hCA IX, respectively. These derivatives are interesting candidates for
the development of novel therapies targeting hypoxic tumors.
Ó 2004 Elsevier Ltd. All rights reserved.
1.Introduction
N
N
N
O
N
N
O
O
O
O
N
EtO
S
S NH2
S
Me
N
H
S
O
NH2
S
S
O
NH2
Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs)
are widely used therapeutic agents in the management or
prevention of many diseases.1–4 This is mainly due to the
wide distribution of the 14 presently described human
CA isozymes in many cells, tissues, and organs, where
they play crucial physiological functions.1–4 Still, the
available pharmacological agents are far from being per-
fect, as they possess many undesired side effects, mainly
due to their lack of selectivity for the different isozymes.
Thus, development of isozyme-specific or at least organ-
selective inhibitors would be highly beneficial both for
obtaining novel types of drugs, devoid of major side ef-
fects, as well as for physiological studies in which spe-
cific/selective inhibitors may constitute valuable tools
for understanding the physiology/physiopathology of
these enzymes.1–4
Me
O
AZA
MZA
EZA
NH2
SO2NH2
O
S O
H
O
N
S
Cl
N
H
O
O
Cl
S NH2
O
Cl
IND
DCP
N
N
N
N
O
O
S
O
O
S
N
R
N
H
S
S
S
O
NH2
S
NH2
H
O
O
O
BZA
R = COOH CBA
R = NH2
ABA
Sulfonamide CAIs such as acetazolamide AZA, meth-
azolamide MZA, ethoxzolamide EZA, or dichlorophen-
amide DCP among others, played a crucial role in the
understanding of renal physiology and pharmacology,
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.062

5776
A. Cecchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5775–5780
and led to the development of widely used diuretic
drugs such as the benzothiadiazine and high ceiling diu-
retics,1–5 but more recently, also to other types of phar-
macological agents, as it will be shown here shortly.
Thus, a recent and new field in CAI research has been
opened by the report of the potent antitumor properties
of a rather large number of sulfonamide CAIs, as well as
by the isolation of some CA isozymes predominantly
present in tumor cells, such as CA IX and CA XII.6–10
The mechanisms by which such compounds inhibit
tumor cell growth only now begin to be understood,
and important advances in this direction have recently
been achieved, since several laboratories are involved in
the synthesis, evaluation and in vitro/in vivo antitumor
testing of novel types of CAIs with potential application
as anticancer therapeutic agents.6–10 Indeed, a compound
of this type––indisulam IND––has progressed to Phase II
clinical trials for the treatment of solid tumors.11,12
of this disease (the above-mentioned isozymes are the
main ones involved in aqueous humor secretion within
the eye and are the targets of antiglaucoma sulfon-
amides).1–4 Continuing this work and the same type of
chemistry,13 but using as lead molecule benzolamide
BZA, an orphan drug belonging to the family of CAIs,14
we report here novel thioureido-containing benzol-
amide-like compounds, which were designed mainly as
inhibitors of the transmembrane, tumor-associated iso-
zyme CA IX. These compounds were also tested for
their inhibitory properties against the major cytosolic
isoforms CA I and II.
2.Chemistry
Benzolamide BZA has already been used by our group as
lead compound for developing potent topically acting
antiglaucoma CAIs.15 Indeed, a 4-carboxybenzolamide
(CBA) derivative in which the carboxylic acid moiety
has been converted to the diethylaminoethyl-carbox-
amide was shown not only to act as an excellent antiglau-
coma compound in an animal model of the disease (due
to its low nanomolar affinity for isozyme hCA II––KI of
1.4nM)15 but also to bind within the active site of hCA II
In previous work from this laboratory,13 we have devel-
oped thioureido-containing sulfonamide CAIs derived
from simple aromatic sulfonamides, such as sulfanil-
amide and homosulfanilamide, which showed excellent
CA I, II, and IV inhibitory properties and were effective
topically acting antiglaucoma agents in an animal model
N
N
O
N
N
O
O
O
S
SO2NH2
S
SO2NH2
S
N
S
O
S
N
S
H
H
N
N
N
H
N
H
N
H
N
H
A1
A2
N
N
N
O
N
O
O
O
H
S
SO2NH2
S
SO2NH2
N
S
N
H
S
N
S
N
S
H
N
N
N
N
H
N
N
H
H
H
A3
A5
A4
N
N
O
O
N
N
O
O
S
SO2NH2
S
N
H
S
F
S
SO2NH2
O
S
N
S
N
H
N
H
N
H
N
H
N
H
A6
A8
N
N
N
O
N
O
O
O
O2N
S
SO2NH2
OH
S
SO2NH2
F
S
N
S
S
N
S
H
H
H
F
F
N
N
N
N
H
N
H
H
H
OH
F
F
A7
N
S
N
O
O
S
SO2NH2
S
N
H
N
H
N
H
N
N
H
N
H
N
H
N
S
S
N
SO2NH2
S
O
O
N
A9

A. Cecchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5775–5780
5777
HIS 4
N
4.27
N
3.44
N
2.97
O
H
H
N
4.30
2.92
H
O
H
TRP 5
PHE 20
HN
O
THR 200
PHE 131
3.6 - 4.7
O
2.50
H
O
H
N
N
H
S
O
2.79
N
THR 199
HO
2.91
O
VAL 121
N
2.90
2.69
O
H
S
2.71
H
O
H
GLU 106
N
S
N
O
H
2.48
O
O
2.50
NH2
N
1.76
Zn2+
N
3.05
O
HIS 96
GLN 92
N
N
N
N
HIS 94
HIS 119
Figure 1. Detailed schematic representation for the binding within the hCA II active site of a 4-carboxybenzolamide (CBA)-derived inhibitor––the
˚
2-N,N-diethylaminoethylamide of 5-(4-carboxybenzenesulfonamido-1,3,4-thiadiazole-2-sulfonamide (figures represent distances in A). Reproduced
with permission from Elsevier from Ref. 16.
in a completely different manner as compared to other
such derivatives for which the X-ray crystal structures
in complexes with hCA II have been reported.16 Thus,
it has been observed that the inhibitor bound within
the enzyme active site was in the sulfonylimido-4H-d2-
1,3,4-thiadiazoline tautomeric form (Fig. 1), with its
deprotonated primary sulfonamide moiety being coordi-
nated to the Zn(II) ion of the enzyme, also participating
to the classical hydrogen bond network involving amino
acid residues Thr 199 and Glu 106.16 The 1,3,4-thiadiaz-
oline fragment of the inhibitor was shown to make two
hydrogen bonds with the active site residue Thr 200,
the secondary sulfonamide moiety participated in two
hydrogen bonds involving a water molecule and the resi-
due Gln 92, whereas the phenyl ring of the inhibitor par-
ticipated to an edge-to-face interaction with the phenyl
ring of Phe 131, the two cycles being almost perfectly per-
pendicular to each other. The tertiary amine fragment of
the carboxamido tail and the carboxamido moiety itself
made hydrogen bonds with water molecules present at
the rim of the active site entrance and van der Waals con-
tacts with His 4, Trp 5, and Phe 20 (Fig. 1).16 All these
multiple interactions never evidenced previously in
CA–sulfonamide complexes, explained the very high
affinity of this inhibitor for the hCA II active site and
prompted us to use such benzolamide-like derivatives
for the drug design of the compounds reported here.
key intermediate 4-isothiocyanato-benzolamide A, by
the procedure previously described for the preparation
of the isothiocyanato derivatives of sulfanilamide or
homosulfanilamide.13 This compound was then reacted
with different aliphatic, heterocyclic, or aromatic
amines/hydrazines/diamines 1–9 (commercially available
derivatives, chosen in such a way as to possess hetero-
atoms able to participate in hydrogen bonds or other
interactions with amino acid residues situated at the
rim of the active site entrance of CAs, as those shown
in Fig. 1), affording thioureas A1–A9.13,18
3.Carbonic anhydrase inhibition
Inhibition data against three physiologically relevant
isozymes, that is, the cytosolic isozymes hCA I and II
and the membrane-bound, tumor-associated isozyme
hCA IX (of human origin all of them) with the new com-
pounds A1–A9 as well as the standard, clinically used
CAIs acetazolamide AZA, methazolamide MZA, eth-
oxzolamide EZA, dichlorophenamide DCP, and indisu-
lam IND are shown in Table 1.19
The following SAR should be noted from data of Table
1: (i) sulfonamides A1–A9 behave as effective inhibitors
of all the investigated isozymes, typically showing low
nanomolar affinity for all of them, similarly with the
lead molecules benzolamide BZA and aminobenzol-
amide ABA from which they were obtained. This may
be explained taking into account the X-ray crystal data
of the adduct of a structurally related such sulfonamide
in complex with hCA II, mentioned above (Fig. 1),
which evidenced a large number of favorable interactions
The new compounds reported here, of type A1–A9 have
been prepared from aminobenzolamide ABA,17 as
shown in Scheme 1.
Reaction of aminobenzolamide ABA17 with thiophos-
gene in the presence of hydrochloric acid afforded the

5778
A. Cecchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5775–5780
O
O
N
N
O
CSCl2
HCl
O
N
N
S
S
N
H
SO2NH2
N
H
SO2NH2
S
S
A
ABA
SCN
H2N
R-NH2
1 - 9
O
O
N
N
S
N
H
SO2NH2
S
S
R
N
H
N
H
A1 - A9
Scheme 1.
Table 1. Inhibition data for sulfonamides A1–A9 investigated in the
present paper and standard sulfonamide CAIs, against isozymes hCA
I, II, and IX
very small modification in the molecule of a CAI leads
to dramatic changes in the biological activity. The most
active compounds against this isozyme, that is, A1, A3,
and A6–A8 were generally 10 times more efficient than
ABA, already a potent hCA I inhibitor (KI of 6nM),
whereas the ABA–isothiocyanate A was a less effective
inhibitor (KI of 15nM). It should also be mentioned that
these compounds are much more effective hCA I inhibi-
tors than the clinically used derivatives AZA–IND
(Table 1), being in fact among the best CA I inhibitors
ever reported; (iii) against hCA II, all the new deriva-
tives A1–A9 showed excellent inhibitory properties, with
KI values in the range of 0.5–0.9nM. It is practically
almost impossible to discuss these results, as all these
derivatives showed a very potent and homogenous
behavior as CA II inhibitors, being almost twice as effec-
tive as compared to ABA or ABA–isothiocyanate A (KIs
of 1.7–2nM). So potent hCA II inhibitors were so far re-
ported only in the preceding paper in which we designed
benzolamide-like derivatives with potent antiglaucoma
properties.15,16 The new compounds A1–A9 are 10–20
times more effective CA II inhibitors as compared to
the clinically used drugs (Table 1); (iv) very good inhib-
itory properties were also shown by compounds A1–A9
against the tumor-associated isozyme hCA IX, with KI
in the range of 3.0–20nM. Thus, the lead compound
BZA is not a very effective CA IX inhibitor, similarly
to ABA (KI of 38–42nM). The isothiocyanate A is al-
ready a better inhibitor (KI of 23nM), whereas these
properties are further augmented for the thioureas A1–
A9. Among these derivatives, compounds A5–A8 show
KI values in the range of 18–20nM, being more potent
than the clinically used compounds acetazolamide or
indisulam (Table 1). Two other derivatives, A1 and A2
show a further enhancement of activity (KI of 10–
13nM), whereas the most potent inhibitors were A3,
A4, and A9, with inhibition constants in the range of
3.0–4.6nM. It is thus clear that all the substitution pat-
terns investigated here may lead to potent hCA IX
inhibitors; (v) the compounds investigated here are
much better hCA II than hCA IX inhibitors. Indeed,
the selectivity ratios shown in Table 1 against the two
sulfonamide-avid isozymes, hCA II and IX, are ꢀ1,
Inhibitor
KI* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA IXb
KI (hCA II)/KI
(hCA IX)
AZA
MZA
EZA
DCP
IND
BZA
ABA
A
900
780
25
12
14
25
27
34
50
24
42
38
23
13
10
3.2
3.0
19
18
20
18
4.6
0.48
0.52
0.23
0.76
0.62
0.21
0.05
0.07
0.06
0.09
0.21
0.30
0.04
0.02
0.03
0.03
0.19
8
38
1200
31
15
9
15
6
15
2.0
1.7
0.9
0.9
0.7
0.9
0.8
0.5
0.6
0.6
0.9
A1
A2
0.6
58
A3
A4
0.6
60
A5
A6
24
0.7
0.7
0.6
62
A7
A8
A9
* Errors in the range of 5–10% of the reported value (from three dif-
ferent assays).
a Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme, by the CO2 hydration
method.26
between the inhibitor and key amino acid residues pre-
sent within the active site of the enzyme; (ii) against
hCA I, the sulfonamides A1–A9 reported here showed
inhibition constants in the range of 0.6–62nM. Thus,
four such derivatives, that is, A2, A4, A5, and A9 were
medium potency inhibitors, with KI values in the range
of 24–62nM. These compounds incorporate moieties
rather similar to that of other derivatives described here,
which showed KI values in the range of 0.6–0.7nM. For
example, A2 and A3 differ only by the heteroatom pre-
sent in the six-membered ring (with an N-Me group in
A3 substituting the oxygen atom of A2) and the differ-
ence of activity between these two compounds is almost
10-fold. It is rather difficult to explain these results with-
out X-ray crystal structures, which only prove that a

A. Cecchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5775–5780
5779
Cancer Drug Targets 2002, 2, 55–75; (c) Abbate, F.;
Casini, A.; Owa, T.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
proving that these compounds cannot be considered as
CA IX-selective inhibitors.
8. (a) Teicher, B. A.; Liu, S. D.; Liu, J. T.; Holden, S. A.;
Herman, T. S. Anticancer Res. 1993, 13, 1549–1556; (b)
Owa, T.; Yoshino, H.; Okauchi, T.; Yoshimatsu, K.;
Ozawa, Y.; Sugi, N. H.; Nagasu, T.; Koyanagi, N.; Kitoh,
K. J. Med. Chem. 1999, 42, 3789–3799.
4.Conclusions
A small library of sulfonamides has been obtained using
benzolamide as lead compound. Aminobenzolamide
was treated with thiophosgene followed by reaction of
the thiocyanato intermediate with aliphatic/aromatic
amines, or hydrazines, leading to thioureas incorporat-
ing aliphatic, aromatic, or heterocyclic moieties. The
new derivatives have been investigated as inhibitors of
the cytosolic isozymes hCA I and II, as well as the
tumor-associated isozyme hCA IX. The new compounds
showed excellent inhibitory properties against all three
isozymes with inhibition constants in the range of 0.6–
62nM against hCA I, 0.5–1.7nM against hCA II and
3.2–23nM against hCA IX, respectively. These deriva-
tives are interesting candidates for the development of
novel therapies targeting hypoxic tumors.
9. (a) Weber, A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran,
C. T.; Scozzafava, A.; Klebe, G. J. Med. Chem. 2004, 47,
550–557; (b) Pastorekova, S.; Casini, A.; Scozzafava, A.;
Vullo, D.; Pastorek, J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 869–873; (c) Casey, J. R.; Morgan,
P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. J. Med. Chem. 2004, 47, 2337–2347.
10. (a) Franchi, M.; Vullo, D.; Gallori, E.; Pastorek, J.;
Russo, A.; Scozzafava, A.; Pastorekova, S.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2003, 18, 333–338; (b)
Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozzaf-
ava, A.; Pastorekova, S.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2003, 18, 403–406; (c) Ilies, M. A.; Vullo, D.;
Pastorek, J.; Scozzafava, A.; Ilies, M.; Caproiu, M. T.;
Pastorekova, S.; Supuran, C. T. J. Med. Chem. 2003, 46,
2187–2196; (d) Winum, J.-Y.; Vullo, D.; Casini, A.;
Montero, J.-L.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2003, 46, 2197–2204.
Acknowledgements
11. Supuran, C. T. Expert Opin. Invest. Drugs 2003, 12, 283–
287.
This research was financed in part by a Sixth Frame-
work Programme of the European Union (EUROXY
project). J.Y.W. is grateful to CSGI, University of Flor-
ence and University of Montpellier II for a travel grant
to Florence. Special thanks are addressed to Professor
Raffaelo Giannini and to Dr. Cristina Vettori (Univer-
sity of Florence) for their invaluable help.
12. Ozawa, Y.; Sugi, N. H.; Nagasu, T.; Owa, T.; Watanabe,
T.; Koyanagi, N.; Yoshino, H.; Kitoh, K.; Yoshimatsu, K.
Eur. J. Cancer 2001, 37, 2275–2282.
13. (a) Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni,
L.; Ilies, M. A.; Supuran, C. T. J. Med. Chem. 2000, 43,
4884–4892; (b) Casini, A.; Scozzafava, A.; Mincione, F.;
Menabuoni, L.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2002, 17, 333–343.
14. Maren, T. H. Benzolamide––A Renal Carbonic Anhyd-
rase Inhibitor. In Orphan Drugs; Karch, F. E., Ed.;
Dekker: New York, 1982; pp 89–115.
References and notes
15. Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni, L.;
Starnotti, M.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2003, 13, 2867–2873.
16. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 2357–2361.
1. Carbonic Anhydrase––Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC (Taylor
and Francis Group): Boca Raton, FL, 2004; pp 1–376,
and references cited therein.
2. (a) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2000, 10, 575–600; (b) Supuran, C. T.; Scozzafava, A.
Expert Opin. Ther. Pat. 2002, 12, 217–242; (c) Supuran, C.
T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23,
146–189; (d) Scozzafava, A.; Mastrolorenzo, A.; Supuran,
C. T. Expert Opin. Ther. Pat. 2004, 14, 667–702.
3. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199–229; (b)
Supuran, C. T.; Vullo, D.; Manole, G.; Casini, A.;
Scozzafava, A. Curr. Med. Chem.—Cardiovasc. Hematol.
Agents 2004, 2, 49–68.
4. Supuran, C. T.; Casini, A.; Scozzafava, A. Development
of Sulfonamide Carbonic Anhydrase Inhibitors (CAIs). In
Carbonic Anhydrase––Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC: Boca
Raton, FL, USA, 2004; pp 67–148.
5. Maren, T. H. Physiol. Rev. 1967, 47, 595–781.
6. Pastorekova, S.; Pastorek, J. Cancer-Related Carbonic
Anhydrase Isozymes. In Carbonic Anhydrase––Its Inhibi-
tors and Activators; Supuran, C. T., Scozzafava, A.,
Conway, J., Eds.; CRC: Boca Raton, FL, USA, 2004;
pp 253–280.
7. (a) Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran,
C. T. Curr. Med. Chem. 2003, 10, 925–953; (b) Casini, A.;
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Curr.
17. Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med.
Chem. 1998, 33, 739–752.
18. Reaction of the isothiocyanate A with amines 1–9 has been
done in water or N,N-diethylacetamide as solvents, as
previously described.13
5-(4-[3-(2-Dimethylamino-ethyl)-thioureido]-benzenesul-
fonylamino)-1,3,4-thiadiazole-2-sulfonamide A1: H NMR
1
(DMSO-d6, 400MHz): d 10.1 (s, 1H), 8.1 (s, 1H), 7.8 (s,
2H), 7.7 (d, 2H, J = 8.6Hz), 7.5 (d, 2H, J = 8.6Hz), 3.8 (m,
2H), 3.2 (m, 2H), 2.8 (s, 6H), 2.2 (s, 1H); MS ESI+ m/z 466
(M+H)+. ESIÀ m/z 464 (MÀH)À.
5-[4-(3-Morpholin-4-yl-thioureido)-benzenesulfonylamino]-
1
1,3,4-thiadiazole-2-sulfonamide A2: H NMR (DMSO-d6,
400MHz): d 10 (s, 1H), 9.6 (s, 1H), 8.4 (s, 2H), 7.9 (d, 2H,
J = 8.6Hz), 7.8 (d, 2H, J = 8.6Hz), 3.7 (m, 5H), 2.8 (m,
4H), 2.8 (s, 6H), 2.2 (s, 1H); MS ESI+ m/z 480 (M+H)+,
502 (M+Na)+. ESIÀ m/z 478 (MÀH)À.
5-(4-[3-(4-Methyl-piperazin-1-yl)-thioureido]-benzenesul-
1
fonylamino)-1,3,4-thiadiazole-2-sulfonamide A3: H NMR
(DMSO-d6, 400MHz): d 9.1 (s, 1H), 9.6 (s, 1H), 7.8 (s,
1H), 7.7 (m, 5H), 3.2–3 (m, 8H), 2.8 (s, 3H). MS ESIÀ m/z
491 (MÀH)À.
5-(4-[3-(2-Morpholin-4-yl-ethyl)-thioureido]-benzenesul-
1
fonylamino)-1,3,4-thiadiazole-2-sulfonamide A5: H NMR
(DMSO-d6, 400MHz): d 10.1 (s, 1H), 8.1 (s, 1H), 7.9

5780
A. Cecchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5775–5780
(s, 2H), 7.7 (d, 2H, J = 7.8Hz), 7.5 (d, 2H, J = 7.8Hz), 3.9
enzyme being determined by spectrophotometric meas-
urements and its activity by stopped-flow experiments,
with CO2 as substrate.26 The specific activity of
the obtained enzyme was the same as the one previously
reported,9,10 but the yields in active protein were 5–6 times
higher per liter of culture medium). An SX.18MV-R
Applied Photophysics stopped-flow instrument has been
used for assaying the CA-catalyzed CO2 hydration activ-
ity.26 Phenol red (at a concentration of 0.2mM) has been
used as indicator, working at the absorbance maximum of
557nm, with 10mM Hepes (pH7.5) as buffer, 0.1M
Na2SO4 (for maintaining constant the ionic strength),
following the CA-catalyzed CO2 hydration reaction for a
period of 10–100s. Saturated CO2 solutions in water at
20°C were used as substrate.26 Stock solutions of inhibitor
(1mM) were prepared in distilled–deionized water with
10–20% (v/v) DMSO (which is not inhibitory at these
concentrations) and dilutions up to 0.01nM were done
thereafter with distilled–deionized water. Inhibitor and
enzyme solutions were preincubated together for 15min at
room temperature prior to assay, in order 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. The activity of these enzyme preparations seem to
be rather similar with the in vivo activity of CA IX in
tumors as recently proved by CA inhibitor binding studies
and disturbance of tumor pH following CA IX inhibi-
(m, 4H), 3.8 (m, 2H), 3.2 (m, 6H), 2.2 (s, 1H); MS ESI+
m/z 508 (M+H)+, 530 (M+Na)+. ESIÀ m/z 506 (MÀH)À.
5-(4-[3-(2-Fluorophenyl)-thioureido]-benzenesulfonylami-
no)-1,3,4-thiadiazole-2-sulfonamide
A6:
1H
NMR
(DMSO-d6, 400MHz): d 10.3 (s, 1H), 9.8 (s, 1H), 8.5 (s,
2H), 7.8 (m, 4H), 7.6 (t, 1H, J = 7.7Hz), 7.3–7.2 (m, 3H);
MS ESI+ m/z 489 (M+H)+, 511 (M+Na)+. ESIÀ m/z 487
(MÀH)À.
5-[4-(3-pentafluoroanilyl)-thioureido)-benzenesulfonylami-
no]-1,3,4-thiadiazole-2-sulfonamide
A7:
1H
NMR
(DMSO-d6, 400MHz): d 10.4 (s, 1H), 10 (s, 1H), 8.5 (s,
1H), 8.45 (s, 2H), 7.85 (d, 2H, J = 8.4Hz), 7.8 (d, 2H,
J = 8.4Hz). MS ESI+ m/z 576 (M+H)+, 598 (M+Na)+.
ESIÀ m/z 574(MÀH)À.
19. Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids
pACA/hCA I and pACA/hCA II described by LindskogÕs
group.20 Cell growth conditions were those described in
Ref. 21 and enzymes were purified by affinity chromato-
graphy according to the method of Khalifah et al.22
Enzyme concentrations were determined spectrophoto-
metrically at 280nm, utilizing a molar absorptivity of
49mMÀ1 cmÀ1 for CA I and 54mMÀ1 cmÀ1 for CA II,
respectively, based on Mr = 28.85kDa for CA I, and
29.3kDa for CA II, respectively.23,24 A variant of the
previously published9,10 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 et al.25) was
amplified by using PCR and specific primers for the
glutathione S-transferase (GST)-Gene Fusion Vector
pGEX-3X. The obtained fusion construct was inserted in
the pGEX-3X vector and then expressed in Escherichia
coli BL21 Codon Plus bacterial strain (from Stratagene).
The bacterial cells were sonicated, then suspended in the
lysis buffer (10mM Tris pH7.5, 1mM EDTA pH8,
150mM NaCl, and 0.2% Triton X-100). After incubation
with lysozime (approx. 0.01g/L) the protease inhibitors
CompleteTM were added to a final concentration of 0.2mM.
The obtained supernatant was then applied to a prepacked
Glutathione Sepharose 4B column, extensively washed
with buffer and the fusion (GST-CA IX) protein was
eluted with a buffer consisting of 5mM reduced glutathi-
one in 50mM Tris–HCl, pH8.0. Finally the GST part of
the fusion protein was cleaved with thrombin. The
advantage of this method over the previous one,9,10 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 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 chromatography,22 the amount of
ˇ
´
tion as compared to normal tissue pH (Svastova, E.;
´
´
´
ˇ
´
Hulıkova, A.; Rafajova, M.; ZatÕovicova, M.; Gibaduli-
´
nova, A.; Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran,
C. T.; Pastorek, J.; Pastorekova, S. FEBS Lett., in
´
press).
20. Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.;
Jonsson, B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure–
Function Relations in Human Carbonic Anhydrase II as
Studied by Site-Directed Mutagenesis. In Carbonic Anhyd-
rase––From Biochemistry and Genetics to Physiology and
`
Clinical Medicine; Botre, F., Gros, G., Storey, B. T., Eds.;
VCH: Weinheim, 1991; pp 1–13.
21. Behravan, G.; Jonsson, B. H.; Lindskog, S. Eur. J.
Biochem. 1990, 190, 351–357.
22. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson,
S. M. Biochemistry 1977, 16, 2241–2247.
23. Lindskog, S.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A.
1964, 70, 2505–2508.
24. Steiner, H.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem.
1975, 59, 253–259.
25. Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.;
Zelnik, V.; Opavsky, R.; Zatovicova, M.; Liao, S.;
Portetelle, D.; Stanbridge, E. J.; Zavada, J.; Burny, A.;
Kettmann, R. Oncogene 1994, 9, 2877–2888.
26. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573.