Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Article abstract of DOI:10.1016/j.bmcl.2004.01.095

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6- tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.

Full text of DOI:10.1016/j.bmcl.2004.01.095

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2351–2356
Carbonic anhydrase inhibitors: inhibition of the tumor-associated
isozyme IX with fluorine-containing sulfonamides. The first
subnanomolar CA IX inhibitor discovered
Daniela Vullo,a Andrea Scozzafava,a Silvia Pastorekova,b Jaromir Pastorekb and
Claudiu T. Supurana,*
aUniversitꢀa degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
bInstitute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 842 45 Bratislava, Slovak Republic
Received 24 November 2003; revised 23 January 2004; accepted 26 January 2004
Abstract—Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as
CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme
CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetra-
fluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these
compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other
physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX
inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM
against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were
detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of
novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
excretion sites (lungs, kidneys), facilitated CO2 elimi-
nation in capillaries, and pulmonary microvasculature,
elimination of Hþ ions in the renal tubules, and col-
lecting ducts, as well as reabsorption of bicarbonate in
the brush border, and thick ascending Henle loop in
kidneys.1–4 CA II is also involved in the bone develop-
ment and function, such as the differentiation of osteo-
clasts, or the provision of acid for bone resorption in
osteoclasts.1–4 Different CAs are involved in the secre-
tion of electrolytes in many other tissues/organs, such
as: cerebrospinal fluid formation, by providing bicar-
bonate and regulating the pH in the choroid plexus;
gastric acid production in the stomach parietal cells; bile
production, pancreatic juice secretion, intestinal ion
transport, etc.1–4 Some isozymes, such as CA V are in-
volved in molecular signalling processes, such as insulin
secretion signalling in pancreas b-cells, whereas CA II
and V are involved in important metabolic processes, as
they provide bicarbonate for gluconeogenesis, fatty
acids de novo biosynthesis or pyrimidine base synthe-
sis.5 Finally, some isozymes (such as CA IX, CA XII,
CARP VIII) are highly abundant in tumors, being
involved in oncogenesis and tumor progression, and
Inhibitors of the metallo-enzyme carbonic anhydrase
(CA, EC 4.2.1.1) are clinically used mostly as antiglau-
coma agents, as they are highly effective in reducing
elevated intraocular pressure, after systemic adminis-
tration of drugs such as acetazolamide AAZ, methazol-
amide MZA, ethoxzolamide EZA or dichlorophenamide
DCP, or after topically administered sulfonamides, such
as dorzolamide DZA, or brinzolamide BRZ.1–4 CA
inhibitors (CAIs) are also useful for the treatment or
prevention of other diseases, since different CA isozymes
are widely distributed in higher vertebrates.1–4 In these
organisms, including humans, the physiological functions
of CAs have widely been investigated over the last years.
Thus, isozymes CA I, II, and IV are involved in respi-
ration and regulation of the acid/base homeostasy.
These complex processes involve both the transport
of CO2/bicarbonate between metabolizing tissues and
* Corresponding author. Tel.: +39-055-457-3005; fax: +39-055-457-
3385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.095

2352
D. Vullo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2351–2356
Me
N
N
N
N
N
MeCONH
SO2NH2
MeCON
NHEt
SO2NH2
EtO
S
SO2NH2
S
S
AAZ
SO2NH2
MZA
EZA
NHEt
SO2NH2
SO2NH2
N
S
MeO(CH2)3
S
Cl
SO2NH2
S
Me
S
O
O
Cl
O
O
DCP
DZA
BRZ
H
O
O
N
S
N
Cl
H
SO2NH2
E7070
generally lacking from the corresponding non-cancerous
cells.2–7 Thus, it is not at all surprising that many CAIs
were recently shown to possess potent antitumor prop-
erties in vitro and in vivo8–10 with one sulfonamide of
this type, E7070 in Phase II clinical trials as antitumor
agent.11–13
of the molecule has been analyzed. Thus, several
unprecedented strong hydrogen bonds involving the
imino nitrogen, carbonyl oxygen, a fluorine atom in
meta belonging to the inhibitor, and two water mole-
cules, as well as Gln 92of the enzyme active site were
evidenced. A stacking interaction of the perfluorophenyl
ring of the inhibitor and the aromatic ring of Phe 131
was also observed for the first time in a CA––sulfon-
amide adduct.20 All these findings proved that the
polyfluorophenyl tails of such CAIs are indeed beneficial
for obtaining very potent and bioavailable CAIs.
We have recently reported the first inhibition studies of
the transmembrane, tumor-associated isozyme IX, with
both sulfonamide and sulfamates, and detected several
low nanomolar inhibitors, potentially useful for the
development of novel antitumor therapies.14–18 Here we
continue these investigations, reporting the synthesis
and CA IX inhibitory properties of a series of fluori-
nated sulfonamides. We decided to investigate this type
of compounds due to the fact that in a previous con-
tribution from our laboratory,19 it was shown that by
attaching perfluoroalkyl/aryl-carboxamido/sulfonamido
tails, such as for example perfluorobutylsulfonyl, per-
fluorooctylcarboxamido, perfluorophenylcarboxamido
or perfluorophenylsulfonyl, to the aromatic/heterocyclic
sulfonamides also incorporating derivatizable amino
moieties, of types 1–13, very effective CAIs can be
obtained, which also showed good water solubility and
efficacy as topically acting antiglaucoma agents in an
animal model of the disease. The best CAIs in that series
of derivatives were those incorporating the perfluori-
nated aromatic moieties (such as C6F5CO and
C6F5SO2), a fact that was then explained after the report
of the high resolution X-ray crystal structure of isozyme
CA II with one of these agents, the perfluorobenzo-
ylated analog of methazolamide.20 This compound is
almost 10 times a more effective CA II inhibitor (KI of
1.5 nM) as compared to methazolamide (KI of 14 nM).
Its binding to the enzyme active site was shown to be
similar to that of other sulfonamide inhibitors, consid-
ering the interactions of the sulfonamide zinc anchoring
group and thiadiazoline ring contacts,21 but differs
considerably when the perfluorobenzoylimino fragment
2. Chemistry
A problem related to the pentafluorophenyl-containing
CAIs previously reported by this group,19 was the high
reactivity of the fluorine atom in para to the sulfon-
amido/carboxamido moiety, which was shown to
covalently bind to thiol reagents, such as Cys 239 of
b-tubulin, glutathione, cysteine itself, etc., by means of
nucleophilic aromatic substitution reactions, in which
the most reactive fluorine atom of the sulfonamide
reacted with the thiol group of the Cys residue, leading
to modification of the thiol reagent/protein.22–25 In
order to prepare fluorine-containing CAIs devoid of
enhanced reactivity, we report here derivatives lacking
the para-fluorine reactive group, with two types of
such derivatives being prepared: the 2,3,5,6-tetrafluor-
ophenyl-carboxamides A1–A13, and the 2,3,5,6-tetra-
fluorophenyl-sulfonamides B3, B5, and B9, respectively
(Scheme 1).
The carboxamides A1–A13 were easily prepared from
the commercially available 2,3,5,6-tetrafluorobenzoic
acid 14, which has been converted to the corresponding
acyl chloride 15 by treatment with thionyl chloride,

D. Vullo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2351–2356
2353
SO2NH2
SO2NH2
SO2NH2
NHR
SO2NH2
NHR
CH2NHR
4
NHR
2
1
3
SO2NH2
SO2NH2
SO2NH2
SO2NH2
F
Br
Cl
CH2CH2NHR
NHR
NHR
NHR
5
6
7
8
H3C
N
N
N
N
RNH
RNH
SO2NH2
RN
SO2NH2
S
S
9
10
O
S
H
N
SO2NH2
( )n
O
11: n = 0; 12: n = 1; 13: n = 2
1-13: R = H
F
F
F
F
F
SO2
CO
B4, B5, B9: R =
A1-A13: R =
F
F
F
3. CA inhibition
COCl
COOH
F
F
F
F
F
F
SOCl2
1-13
CA inhibition data against three human isozymes, hCA
I, hCA II, and hCA IX for the prepared compounds and
standard inhibitors are shown in Table 1. All the new
compounds reported here act as better CAIs than the
parent sulfonamides from which they were obtained
(data not shown).15 The following SAR can be drawn
from the data of Table 1: (i) with few exceptions (com-
pounds A6, A9, A10, and B9), the CAIs investigated
here are more potent CA IX inhibitors than CA II (or
CA I) inhibitors, a fact rarely observed up to now for
other investigated sulfonamides/sulfamates.14–18 Indeed,
the selectivity ratio toward CA IX as compared to CA II
is less than 1 for these four compounds, as well as for the
clinically used derivatives, meaning that all of them
possess a higher affinity for CA II, and not for CA IX,
the target enzyme for developing novel antitumor ther-
apies. On the contrary, the remaining 12derivatives
reported here showed selectivity ratios higher than unity,
with several such derivatives (A2, A7, A8, and A11)
possessing selectivity ratios in the range of 4.75–26.25,
whereas the remaining ones present this parameter
around 1.05–2.72. A first conclusion may be that the
heterocyclic derivatives (the clinically used sulfonamides
A1-A13
NaOH
F
F
14
15
SO2Cl
F
F
F
F
F
F
F
ClSO3H
SOCl2
4, 5 or 9
B4, B5, B9
Py
F
17
16
Scheme 1.
followed by reaction with the amino-sulfonamides 1–13,
as previously reported.19;26 For the preparation of the
sulfonamides B3, B5, and B9, 1,2,4,5-tetrafluorobenzene
16 was transformed to the monosulfonyl chloride by
treatment with chlorosulfonic acid/thionyl chloride,
followed by reaction with the amino-sulfonamides 4, 5
or 9, as previously reported for structurally-related
compounds.19;27

2354
D. Vullo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2351–2356
Table 1. CA inhibition data with standard inhibitors and the new sulfonamides reported in the present study, against human isozymes I, II, and IX
c
Selectivity ratiob
Inhibitor
KI (nM)
hCA Ia
hCA IIa
hCA IXa
KI (hCA II)/KI (hCA IX)
Acetazolamide
Methazolamide
9005
780
122 0.48
14
27
34
0.51
0.23
0.76
Ethoxzolamide
Dichlorophenamide
Dorzolamide
25
8
38
1200
50,000
––
50
9
520.17
37
24
Brinzolamide
E7070
A1
3
15
0.08
0.62
4.0
31
1500
1700
975
38
21
9.5
0.8
A2
A3
26.25
16
13
15.21.05
9.5
A4
A5
980
900
1.36
127.3
1.5
76
1.64
24
16
A6
A7
760
0.06
4.75
17.5
0.16
0.13
1080
1870
250
A8
A9
84
4.8
2.1
1.4
19
12.6
10.5
3.25.93
5.5
A10
A11
A12
A13
B4
270
6300
6100
6000
860
15
13
2.72
1.71
7.6
128.5
9
0.7
1.41
7.0
4.3
B5
B9
750
13
1.28
0.16
a Human (cloned) isozymes,28 by the CO2 hydration method for CA IX,29 and esterase method for CA I and II.30
b Selectivity ratio toward hCA IX as compared to hCA II.
c Errors in the range of 5–10% of the reported value (from three different assays).
and E7070) as well as A9, A10, and B9, and the fluorine-
sulfanilamide derivative A6, show higher affinity for the
cytosolic isozyme CA II, whereas the remaining aro-
matic derivatives investigated here show higher affinity
for the transmembrane, tumor-associated isozyme CA
IX; (ii) the affinity of the new sulfonamides reported
here for hCA I was medium-low, except for one deriv-
ative, B9, which behaved as a very potent CA I inhibitor
(KI of 13 nM). Indeed, the other fluorinated sulfon-
amides showed KI values in the range of 250–6100 nM,
being as inhibitory (or slightly less) as the clinically used
compounds (except dorzolamide, which is a very weak
CA I inhibitor); (iii) against hCA II, the new compounds
reported here generally behave as very potent inhibitors,
with KI values in the range of 0.7–16 nM, except for
compounds A1, A2, A7, A8, and A9, which were slightly
less effective inhibitors, with KI values in the range of
19–84 nM; (iv) against hCA IX, some of these com-
pounds are the most effective inhibitors ever reported up
to now,14–18 with the first subnanomolar inhibitor
detected (compound A2, with a KI of 0.8 nM, and an
excellent selectivity ratio toward CA IX as compared to
CA II, of 26.25). Other derivatives, such as A1, A4, A5,
A8, A11–A13, B4, B5, and B9, showed KI values in the
range of 3.2–9.5 nM, being very potent CA IX inhibi-
tors. The less effective CA IX inhibitors were A3, A6,
A7, A9, and A10, which possess KI values in the range of
10.5–24 nM, that is, they appreciably inhibit the tumor-
associated isozyme, being more potent than the
clinically used sulfonamides, and equipotent to the anti-
tumor sulfonamide in clinical trials, E7070 (Table 1). It
may be observed that the best and more selective CA IX
inhibitors incorporate metanilamide, sulfanilyl-sulfanil-
amide, and sulfanilyl-homosulfanilamide moieties
(compounds A2, A11, and A12) and tetrafluorobenzoyl
tails; (v) the compounds incorporating tetra-
fluorosulfonyl moieties were slightly more effective CAIs
as compared to the corresponding derivatives incorpo-
rating tetrafluorobenzoyl moieties, but the differences of
activity were insignificant.
4. Conclusions
In a series of 2,3,5,6-tetrafluorophenyl-carboxamido/
sulfonamido aromatic/heterocyclic sulfonamides, very
potent inhibitors against the transmembrane, tumor-
associated isozyme CA IX were detected, some of them
showing also selectivity ratios favorable to CA IX over
CA II, the other physiologically relevant isozyme with
high affinity for sulfonamide inhibitors. The first sub-
nanomolar and rather selective CA IX inhibitor has
also been discovered, as the 2,3,5,6-tetrafluorobenzoyl
derivative of metanilamide showed an inhibition con-
stant of 0.8 nM against hCA IX, and a selectivity ratio
of 26.25 against CA IX over CA II. Several other low
nanomolar CA IX inhibitors were detected among the
new derivatives reported here. Correlated with the easy
preparation of such compounds, the present derivatives
constitute valuable candidates for the development of
novel antitumor therapies based on the selective inhi-
bition of tumor-associated CA isozymes.

D. Vullo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2351–2356
2355
15. Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozza-
fava, A.; Pastorekova, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 1005–1009.
16. 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.
References and notes
1. (a) Maren, T. H. In Carbonic Anhydrase––from Biochem-
istry and Genetics to Physiology and Clinical Medicine;
ꢀ
Botre, F., Gros, G., Storey, B. T., Eds.; VCH: Weinheim,
1991; pp 186–207; (b) Wistrand, P. J.; Lindqvist, A. In
17. Franchi, M.; Vullo, D.; Gallori, E.; Pastorek, J.; Russo,
A.; Scozzafava, A.; Pastorekova, S.; Supuran, C. T.
J. Enzym. Inhib. Med. Chem. 2003, 18, 333–338.
18. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.; Scozza-
fava, A.; Supuran, C. T. J. Med. Chem. 2003, 46, 5471–5477.
19. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 2000, 43,
4542–4551.
Carbonic Anhydrase––from Biochemistry and Genetics to
ꢀ
Storey, B. T., Eds.; VCH: Weinheim, 1991; pp 352–378.
2. (a) Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther.
Patents 2000, 10, 575–600; (b) Supuran, C. T.; Scozzafava,
A. Exp. Opin. Ther. Patents 2002, 12, 217–242.
3. (a) Supuran, C. T.; Scozzafava, A. Curr. Med. Chem.
Imm., Endoc. Metab. Agents 2001, 1, 61–97; (b) Supuran,
C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23,
146–189.
Physiology and Clinical Medicine; Botre, F., Gros, G.,
20. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
J. Enzy. Inhib. Med. Chem. 2003, 18, 303–308.
21. (a) Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.;
Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3583–
3587; (b) Casini, A.; Abbate, F.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2003, 13, 2759–2763; (c)
Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David,
4. Carbonic Anhydrase––its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC: Boca
Raton, Florida, 2004; pp 1–368, and references cited
therein.
5. Supuran, C. T. Exp. Opin. Ther. Patents 2003, 13, 1545–1550.
6. (a) 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; (b) Opavsky,
R.; Pastorekova, S.; Zelnik, V.; Gibadulinova, A.; Stan-
bridge, E. J.; Zavada, J.; Kettmann, R.; Pastorek, J.
Genomics 1996, 33, 480–487; (c) Pastorekova, S.; Parkkila,
S.; Parkkila, A. K.; Opavsky, R.; Zelnik, V.; Saarnio, J.;
Pastorek, J. Gastroenterology 1997, 112, 398–408.
7. (a) Liao, S. Y.; Brewer, C.; Zavada, J.; Pastorek, J.;
Pastorekova, S.; Manetta, A.; Berman, M. L.; DiSaia, P.
J.; Stanbridge, E. J. Am. J. Pathol. 1994, 145, 598–609; (b)
Liao, S. Y.; Aurelio, O. N.; Jan, K.; Zavada, J.;
Stanbridge, E. J. Cancer Res. 1997, 57, 2827–2831; (c)
Vermylen, P.; Roufosse, C.; Burny, A.; Verhest, A.;
Bossehaerts, T.; Pastorekova, S.; Ninane, V.; Sculier, J.
P. Eur. Respir. J. 1999, 14, 806–811; (d) Turner, J. R.;
Odze, R. D.; Crum, C. P.; Resnick, M. B. Hum. Pathol.
1997, 28, 740–744.
8. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925–953.
9. Casini, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran,
C. T. Curr. Cancer Drug Targets 2002, 2, 55–75.
10. (a) 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; (b) Supuran, C. T.
Exp. Opin. Invest. Drugs 2003, 12, 283–287.
11. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
12. (a) Funahashi, Y.; Sugi, N. H.; Semba, T.; Yamamoto, Y.;
Hamaoka, S.; Tsukahara-Tamai, N.; Ozawa, Y.; Tsu-
ruoka, A.; Nara, K.; Takahashi, K.; Okabe, T.; Kamata,
J.; Owa, T.; Ueda, N.; Haneda, T.; Yonaga, M.; Yoshi-
matsu, K.; Wakabayashi, T. Cancer Res. 2002, 62, 6116–
6123; (b) 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) van Kesteren, C.; Beijnen, J. H.; Schellens, J. H.
Anticancer Drugs 2002, 13, 989–997; (b) Punt, C. J.;
Fumoleau, P.; van de Walle, B.; Faber, M. N.; Ravic, M.;
Campone, M. Ann. Oncol. 2001, 12, 1289–1293; (c)
Raymond, E.; ten Bokkel Huinink, W. W.; Taieb, J.;
Beijnen, J. H.; Faivre, S.; Wanders, J.; Ravic, M.;
Fumoleau, P.; Armand, J. P.; Schellens, J. H. J. Clin.
Oncol. 2002, 20, 3508–3521.
€
S.; Waldeck, H.; Schafer, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 841–845.
22. Medina, J. C.; Shan, B.; Beckmann, H.; Farrell, R. P.;
Clark, D. L.; Learned, R. M.; Roche, D.; Li, A.; Baichwal,
V.; Case, C.; Baeuerle, P. A.; Rosen, T.; Jaen, J. C. Bioorg.
Med. Chem. Lett. 1998, 8, 2653–2656.
23. Medina, J. C.; Roche, D.; Shan, B.; Learned, R. M.;
Frankmoelle, W. P.; Clark, D. L.; Rosen, T.; Jaen, J. C.
Bioorg. Med. Chem. Lett. 1999, 9, 1843–1846.
24. Shan, B.; Medina, J. C.; Santha, E.; Frankmoelle, W. P.;
Chou, T. C.; Learned, R. M.; Narbut, M. R.; Scott, D.;
Wu, P.; Jaen, J. C.; Rosen, T.; Timmermans, P. B. M.;
Beckmann, H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
5686–5691.
25. (a) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T.
Exp. Opin. Ther. Patents 2001, 11, 765–787; (b) Scozza-
fava, A.; Casini, A.; Supuran, C. T. Curr. Med. Chem.
2002, 9, 1167–1185.
26. (a) Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J.
Med. Chem. 1998, 33, 739–751; (b) Supuran, C. T.; Clare,
B. W. Eur. J. Med. Chem. 1999, 34, 41–50; (c) Clare, B.
W.; Supuran, C. T. Eur. J. Med. Chem. 1999, 34, 463–474.
27. Synthesis of 2,3,5,6-tetrafluorobenzenesulfonyl chloride:
Chlorosulfonic acid of 5 equiv (97 g; 55.36 mL) were added
to 25 g of 1,2,4,5-tetrafluorobenzene and the obtained
solution was heated at 150 ꢁC. After 2h, the heating was
halted in order to allow the solution to reach room
temperature and 2equiv of thionyl chloride (39.96 g;
24.30 mL) were added. The obtained mixture was heated
for 3 h at 150 ꢁC. The brown solution obtained was then
added dropwise under stirring to a mixture of 250 g ice and
100 mL water. The obtained suspension was extracted
three times with 50 mL of ethyl acetate. The organic
fractions were collected and dried by solvent evaporation
under depression. The obtained brown oil was collected
and stored at )20 ꢁC. The purity of the final compound
was verified by TLC (MeOH/CHCl3: 3/7).
Acylation/sulfonylation of amino-sulfonamides: Sulfon-
amide 1–13 of 5 mmol were dissolved in 15 mL solution of
2.5 M NaOH and cooled to 2–5 ꢁC in a salt–ice bath.
Sulfonyl/acyl chloride 15, 17 of 5 mmol were added in
small portions, concomitantly with 10 mL of a 2M NaOH
solution, maintaining the temperature under 10 ꢁC. The
reaction mixture was then stirred at room temperature for
5–10 h (TLC control), then the pH was adjusted to 2with
5 N HCl, and the precipitated sulfonamides were filtered,
and recrystallized from aqueous ethanol. Alternative
method: Amino sulfonamide 1–15 of 1 g was dissolved in
14. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
2197–2204.

2356
D. Vullo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2351–2356
10 mL of N,N-dimethylacetamide and 2equiv of 2,3,5,6-
Stratagene). The obtained construct was inserted in the
pCAL-n-FLAG vector and then cloned and expressed in
Escherichia coli strain BL21-GOLD(DE3) (from Strata-
gene). The bacterial cells were lyzed and homogenated in a
buffered solution (pH 8) of 4 M urea and 2% Triton X-100,
as described by Wingo et al.31 The homogenate thus
obtained was extensively centrifuged (11,000 g) in order to
remove soluble and membrane associated proteins as well
as other cellular debris. The resulting pellet was washed by
repeated homogenation and centrifugation in water, in
order to remove the remaining urea and Triton X-100.
Purified CA IX inclusion bodies were denaturated in 6 M
guanidine hydrochloride and refolded into the active form
by snap dilution into a solution of 100 mM MES (pH 6),
tetrafluorobenzenesulfonyl chloride, and 1 equiv of pyri-
dine were added to the solution which was left on ice
under stirring. After 60 min of stirring, 50 mL of water
were added to the solution, which was then extracted three
times with 25 mL of ethyl acetate. The organic fractions
were collected and extracted three times with 15 mL of 1 N
hydrochloric acid aqueous solution. The organic phase
was dried on magnesium sulfate. After charcoal treatment,
the organic phase was dried under depression. The purity
of the final compound was verified by TLC (MeOH/
CHCl3: 3/7).
4-[2,3,5,6-Tetrafluorobenzenesulfonylamidoethyl]-benzene-
sulfonamide B5, colorless crystals, mp 187–188 ꢁC, IR
(KBr) cmÀ1: 1154 and 1163 (SO2sym), 1350 and 1378 (SOa2s),
3365 (NH, NH2);
500 mM
L-arginine, 2mM ZnCl 2, 2mM EDTA, 2mM
reduced glutathione, 1 mM oxidized glutathione. Active
hCA IX was extensively dialyzed into a solution of 10 mM
Hepes (pH 7.5), 10 mM tris HCl, 100 mM Na2SO4, and
1 mM ZnCl2. The protein was further purified by sulfon-
amide affinity chromatography,29 the amount of enzyme
was determined by spectrophometric measurements and
its activity by stopped-flow measurements, with CO2 as
substrate.29 The activity of this preparation was identical
to that reported by Wingo et al., with kcat=KM of
F
F
16
15
14
13
2
7
8
9
10
3
1
4
11
12
SO2NH CH2 CH2
SO2NH2
5
6
F
F
1H NMR (DMSO-d6, d ppm, J Hz): 8.79 (t, H-8, 5.5); 8.13
(tt, H-4, 7.5, 10.3); 7.69 (d, H-13-15, 8.4); 7.38 (d, H-12-16,
8.4); 7.30 (s, NH2); 3.29 (br q, H-9, 5.5, 6.9); 2.83 (t, H-10,
6.9). H-4 is coupled with the 2F from positions 3 and 5,
J ¼ 10:3 Hz, as well as with the two F from positions 2
and 6, J ¼ 7:5 Hz; N–H is coupled with CH2-9
(J ¼ 5:5 Hz), whereas CH2-9 appears as a broad quartet
because it is also coupled with CH2-10, which appears as a
triplet (JðvicÞ ¼ 6:9 Hz); 13C NMR (DMSO-d6, d ppm, J
Hz): 120.87 (t, C-1, J(19F–13C) ¼ 15.3); 110.74 (t, C-4,
J(19F–13C) ¼ 23.5); 145.63 (dddd, C-3, 4.4, 10.8, 15.2,
248.4); 142.85 (ddt, C-2, 3.3, 15.7, 254.5); 129.12 (C-12-
16); 125.49 (C-13-15); 142.59 (C-11 or C-14); 142.21 (C-14
or C-11); 43.36 (C-9); 34.69 (C-10). C-1 ¼ 128.5 +
15.5 )2 · 13.0 + 2 ·1.6 ¼ 121.2; C-2 ¼ 128.5 + 34.8)13.0 +
1.6 )4.4 ¼ 147.5; C-3 ¼ 128.5 + 34.8 )13.0 + 0.3 + 1.6 )4.4 ¼
152.2; C-4 ¼ 128.5 )2·13.0 + 2 ·1.6 + 3.1 ¼ 108.8ppm (the
detailed spectrum is presented as this type of compounds
show a very complicated behavior due to the multiple
couplings between the protons, fluorine and carbon atoms,
and no literature data for similar compounds are available
as far as we know). Anal. (C14H12F4N2O4S2) C, H, N.
55 lMÀ1sÀ1
.
29. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573, An
SX.18MV-R Applied Photophysics stopped-flow instru-
ment has been used. Phenol red (at a concn of 0.2mM) 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 Na2SO4 (for maintaining constant the ionic
strength), following the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. Saturated CO2 solutions
in water at 20 ꢁC were used as substrate. Stock solutions of
inhibitors were prepared at a concn of 1–3 mM (in DMSO/
water 1:1, v/v) and dilutions up to 0.1 nM done with the
assay buffer mentioned above. Inhibitor and enzyme
solutions were preincubated together for 10 min 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.
30. A stopped-flow variant of the Poker and Stone spectro-
photometric method (Pocker, Y.; Stone, J.T. Biochemistry
1967, 6, 668–678) has been employed, using an SX.18MV-
R Applied Photophysics stopped-flow instrument, as
described previously.21
28. The cDNA of the catalytic domain of hCA IX (isolated as
described by Pastorek et al.6a) was amplified by using PCR
and specific primers for the vector pCAL-n-FLAG (from
31. Wingo, T.; Tu, C.; Laipis, P. J.; Silverman, D. N. Biochem.
Biophys. Res. Commun. 2001, 288, 666–669.