Carbonic anhydrase inhibitors: Design of spin-labeled sulfonamides incorporating TEMPO moieties as probes for cytosolic or transmembrane isozymes

Article abstract of DOI:10.1016/j.bmcl.2008.05.051

A series of spin-labeled sulfonamides incorporating TEMPO moieties were synthesized by a procedure involving the formation of a thiourea functionality between the benzenesulfonamide and free radical fragment of the molecules. The new compounds were tested as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and showed efficient inhibition of the physiologically relevant isozymes hCA II and hCA IX (hCA IX being predominantly found in tumors) and moderate to weak inhibitory activity against hCA I. Some derivatives were also selective for inhibiting the tumor-associated isoform over the cytosolic one CA II, and presented significant changes in their ESR signals when complexed to the enzyme active site, being interesting candidates for the investigation of hypoxic tumors overexpressing CA IX by ESR techniques, as well as for imaging/treatment purposes.

Full text of DOI:10.1016/j.bmcl.2008.05.051

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3475–3480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbonic anhydrase inhibitors: Design of spin-labeled sulfonamides
incorporating TEMPO moieties as probes for cytosolic or transmembrane
isozymes
Alessandro Cecchi a, Laura Ciani b, Jean-Yves Winum c, Jean-Louis Montero c, Andrea Scozzafava a,
a,
Sandra Ristori b, , Claudiu T. Supuran
*
*
a Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Room 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
b Università degli Studi di Firenze, Department of Chemistry & CSGI, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
c Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of spin-labeled sulfonamides incorporating TEMPO moieties were synthesized by a procedure
involving the formation of a thiourea functionality between the benzenesulfonamide and free radical
fragment of the molecules. The new compounds were tested as inhibitors of the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1) and showed efficient inhibition of the physiologically relevant isozymes hCA II
and hCA IX (hCA IX being predominantly found in tumors) and moderate to weak inhibitory activity
against hCA I. Some derivatives were also selective for inhibiting the tumor-associated isoform over
the cytosolic one CA II, and presented significant changes in their ESR signals when complexed to the
enzyme active site, being interesting candidates for the investigation of hypoxic tumors overexpressing
CA IX by ESR techniques, as well as for imaging/treatment purposes.
Received 20 March 2008
Revised 12 May 2008
Accepted 14 May 2008
Available online 17 May 2008
Keywords:
Carbonic anhydrase
Sulfonamide
Tumor-associated isozyme
Spin label
Ó 2008 Elsevier Ltd. All rights reserved.
TEMPO
Diagnostic tool
In previous work from this laboratory,1,2 we reported that fluo-
rescein-labeled sulfonamides act as very potent inhibitors of the
zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1).3 These fluores-
cein derivatives are now in early phases of development for appli-
cations as diagnostic tools and therapeutic agents for tumors
overexpressing CA isozymes IX and XII (among the 16 presently
known in mammals).3–5 With the aid of such fluorescent inhibitors
it was possible to prove the involvement of CA IX in tumor acidifi-
cation processes which lead to an excessive production of H+ ions
in the extracellular space where the active site of CA IX and XII are
situated,4,5 and the possibility to reverse this deleterious phenom-
enon by blocking the enzyme activity.1–5 The success of this ap-
proach has encouraged us to now investigate alternative
possibilities for the labeling of CAs present in tumors (i.e., CA IX
and XII) or related isoforms, eventually present in the cytosol (CA
I, II, VII, and XIII) or mitochondria (CA VA and VB) of eukaryotic
cells.3 Introduction of a spin label in the molecules of sulfonamides
acting as well-known inhibitors of these enzymes3 seemed to us a
straightforward continuation of our previous work.1,2 In this letter
we report the synthesis, CA inhibitory activity against some phys-
iologically relevant isoforms (CA I and II, cytosolic isozymes, and
CA IX, transmembrane, tumor-associated isoform)3 as well as the
ESR properties for a series of benzenesulfonamides incorporating
2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) and thioureido
moieties.
Spin-labeled sulfonamides were in fact reported in the 70s,6
when little was known on the binding of sulfonamides within
the CA active site, as no X-ray crystallographic structures of any
isoform alone or in complex with inhibitors were available at that
time. Some of the compounds A–E reported earlier6–8 incorporated
either five-membered (pyrrolidine-N-oxide) or six-membered
(piperidine-N-oxide) radical moieties in their molecule, together
with the classical benzenesulfonamide warheads known to coordi-
nate (in deprotonated form, as sulfonamidate anion) to the Zn(II)
ion present in the enzyme active site and crucial for its catalytic
activity (Chart 1).3 These compounds were basically used for gain-
ing insight on the topology of the CA active site and no detailed
inhibition studies are available with them, also because only iso-
forms I and II were known in the period when they were reported.6
Furthermore, the possibility of exploiting the ESR signals of these
earlier compounds of types A–E for imaging/treatment purposes
has never been taken into account. There is in fact a stringent need
* Corresponding authors. Tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: sandra.ristori@unifi.it (S. Ristori), claudiu.supuran@unifi.it
(C.T. Supuran).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.051

3476
A. Cecchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3475–3480
O
N
N
O
SO2NH2
N
SO2NH2
S
n
H
N
O
N
NH
O
A
NH
C
B
O
SO2NH2
O
O
HN
O
SO2NH2
m
n = 0, 1
O
N
m = 1, 2, 3
N
O
D
HN
N
SO2NH2
O
N
E
Chart 1. Spin-labeled sulfonamides A–E reported earlier.
of CAIs which can be used for the selective labeling of CA isozymes
involved in various pathological processes,3,7,8 as exemplified
among others also by the very recent report of Christianson’s group
of a 129Xe-cryptophane-sulfonamide biosensor complexed to hCA II
which can be used as a diagnostic tool for 129Xe-magnetic reso-
nance imaging (MRI).8
pared in acceptable yields for the subsequent synthesis of com-
pounds 2 and 3, an alternative method was employed, that is,
reaction of TEMPO isothiocyanate (obtained from amino-TEMPO
and CSCl2 similar to what is described above)12,13 with the corre-
sponding amines (Scheme 1).16,17
The inhibition data of the new sulfonamides 1–10 and standard,
clinically used inhibitors such as acetazolamide AAZ, methazola-
mide MZA, ethoxzolamide EZA, dichlorophenamide DCP, and
indisulam IND (Chart 2) against the cytosolic isozymes hCA I,
hCA II, and the transmembrane tumor-associated isozyme hCA IX
(h = human isoform) are presented in Table 1.18 The following
structure–activity relationships (SARs) should be noted: (i) against
the ubiquitous, house-keeping, and physiologically relevant iso-
form hCA II the new sulfonamides 1–10 showed good inhibitory
activity, with inhibition constants (Ki values) in the range of 12–
165 nM. The derivatives with a meta substitution pattern on the
benzenesulfonamide ring showed the least efficient inhibitory
activity (Kis of 165 and 152 nM, respectively, for derivatives 4
and 7). Compounds 1–3 showed a compact behavior of efficient
CAIs, with Kis of 28–42 nM. An increased affinity for hCA II has
been observed for the halogeno-substituted derivatives 5 and 6,
with the chlorine-substituted benzenesulfonamide 6 being a very
efficient CAI, comparable to the clinically used compounds AAZ,
MZA, and EZA (Table 1). The sulfanilyl-sulfonamides 8–10 were
slightly less efficient hCA II inhibitors as compared to 6, but they
appreciably inhibited the enzyme with inhibition constants in
the range of 20–47 nM. Thus, we evidenced various types of substi-
tution patterns of the TEMPO-containing CAIs that lead to efficient,
low nanomolar inhibitors of the physiologically relevant isozyme
hCA II; (ii) the inhibition data of compounds 1–10 against the tu-
mor-associated isozyme hCA IX (our target for imaging or treat-
ment purposes)1 showed them to possess excellent inhibitory
activity, with Kis values in the range of 7–220 nM (Table 1). Similar
to the hCA II inhibition discussed above, the meta substitution pat-
tern present in derivatives 4 and 7 led to the least effective inhib-
itory properties (Kis of 132 and 220 nM, respectively) for the
corresponding sulfonamides. The other compounds reported here
were much better hCA IX inhibitors as compared to 4 and 7, with
inhibition constants in the range of 7–41 nM. SAR was rather sim-
The design of the new CA inhibitors (CAIs) 1–10 reported here
was approached by the classical tail strategy,2,3 that is, maintaining
the benzenesulfonamide head present in the spin-labeled com-
pounds A–E6 reported earlier, and by incorporation of a thiourea
linker between this and the free radical TEMPO tail (Scheme 1),
which was not present in any of the earlier derivatives. The ben-
zenesulfonamide moiety was chosen due to its ability to tightly
bind to the zinc ion within the enzyme active site, as shown by
extensive X-ray crystallographic work from this and other
groups.1–3,8–11 Furthermore, the phenyl ring belonging to the bene-
zenesulfonamide moiety may be substituted in positions 3 and 4
(with respect to the sulfamoyl group) with halogen atoms, as some
of these derivatives were shown earlier to lead to potent CAIs.1–3
The central thiourea linker motif was chosen because it was shown
earlier that compounds incorporating it act as potent hCA I, II, and
IX inhibitors.12,13 We then employed the 2,2,6,6-tetramethyl-
piperidine-1-oxyl (TEMPO) moiety as the tail group. This free rad-
ical moiety is active in the ESR experiments, and possesses a simple
spectrum.14 In addition, it is stable, it induces a relatively good
water solubility to the compounds incorporating it, and its spectral
features (such as line width and intensity) can be regulated by tis-
sue oxygen or redox status in in vivo experiments. This latter prop-
erty also renders newly synthesized molecules containing this
radical scaffold to be incorporated in spin probes that interact with
biomolecules such as enzymes.14,15
The compounds 1–10 were prepared as shown in Scheme 1 by
employing two alternative but similar reaction methods (depend-
ing on the stability of the isothiocyanate intermediates). The first
method consisted in the reaction of aminosulfonamides with thio-
phosgene in acidic medium which leads to the corresponding iso-
thiocyanate-sulfonamides.12,13 Reaction of these intermediates
with amino-TEMPO in acetonitrile led to thioureas 1 and 4–10.
As the precursor sulfonamide isothiocyanates could not be pre-

A. Cecchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3475–3480
3477
S
SO2NH2
C
C
H2N
SO2NH2
CSCl2, HCl
N
N
n
n
n
X
X
or
or
S
H2N
H
N
SO2NH2
CSCl2, HCl
H
N
SO2NH2
S
S
O
n
O
O
O
S
C
NH2
N
CSCl2, NaHCO3
H2O/CHCl3
CH3CN
NEt3
CH3CN
N
O
N
O
O
O
X
S
S
N
S
n
SO2NH2
H
N
H
HN
n
SO2NH2
HN
N
H
N
O
1-6
N
O
7-10
-SO2NH2 position
X
n
1
2
3
4
5
6
7
8
9
10
p
p
p
m
p
p
m
p
p
p
H
H
H
H
F
Cl
-
0
1
2
0
0
0
0
0
1
2
-
-
-
Scheme 1. Preparation of TEMPO-tailed sulfonamides 1–10.
Me
N
N
S
N
N
N
MeCONH
SO2NH2
SO2NH2
EtO
S
SO2NH2
MeCON
SO2NH2
S
AAZ
MZA
EZA
SO2NH2
O
NH
S
HN
Cl
Cl
SO2NH2
O
Cl
DCP
IND
Chart 2. Structures of clinically used CAIs AAZ-DCP and of the antitumor sulfonamide in clinical development indisulam (IND).
ilar to what is discussed above for hCA II, except that the best
inhibitor was in this case 9, which with its elongated molecule
may probably better fill the hCA IX active site which is presumed
to be larger than that of hCA II;20 (iii) against the slow cytosolic iso-

3478
A. Cecchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3475–3480
Table 1
structurally similar, more elongated compounds of types 8–10,
with an internal secondary sulfonamide group, compound 9
showed a better inhibition constant (Ki 89 nM) as compared to
its shorter or longer congener, suggesting the importance of the
overall length of the inhibitor for an optimal interaction with the
enzyme active site (in this case the CH2 linker present in 9 led to
the best such interactions).9,11 Derivatives 1–10 showed an overall
inhibition profile against hCA I similar to that of the clinically used
sulfonamides AAZ, EZA, and IND (Table 1); (iv) among all the free
radical inhibitors synthesized here, compounds 2, 5, 8, and 9
showed good selectivity for the inhibition of the tumor-associated
isoform hCA IX over the cytosolic one hCA II, with the best selectiv-
ity ratio of 2.9 achieved for compound 9 (Table 1 and Fig. 1). All
these compounds were obviously much better hCA IX inhibitors
than hCA I inhibitors (Table 1). These are not at all trivial results,
since few compounds reported in the literature selectively inhibit
the tumor-associated isoform over the cytosolic CAs. As also seen
from data of Figure 1, all the clinically used compounds AAZ-IND
show selectivity ratios for the inhibition of CA IX over CA II less
than 1 (thus being better CA II than CA IX inhibitors), whereas
many of the new nitroxylated CAIs reported here (e.g., 2, 4, 5, 7,
8, and 9) have a selectivity ratio >1. This means that these com-
pounds will preferentially bind CA IX (already overexpressed in
hypoxic tumors)1,2 and much less CA II, which is a house-keeping
enzyme, necessary for many physiological processes of the cell.1–3
The ESR spectra of the nitroxide derivatives reported here were
recorded both in the absence and in the presence of enzyme (Fig.
2A and B). hCA II was used as model enzyme for this study owing
to both the good inhibitory activity of most compounds synthe-
sized in this work, and to its ease of handling and low cost as com-
pared to hCA IX, the actual diagnostic target for which these
compounds were designed. ESR experiments were crucial for
assessing the mobility rates of TEMPO-CAIs in solution and upon
binding to the enzyme. Figure 2A and B shows the ESR spectra of
compounds 1 and 6, both free in solution and complexed to the en-
zyme, respectively. The signals of the spin-labeled CAIs dissolved
in buffer were superposable, and this was in agreement with the
fact that the two compounds 1 and 6 only differ by the presence
of a chlorine atom which is placed far from the free radical piper-
idin-N-oxide moiety. Both radicals underwent rapid motion in buf-
CA inhibition data of compounds 1–10 and standard CAIs (AAZ–IND) against human
isoforms hCA I, II (cytosolic), and IX (transmembrane, tumor-associated)
Compound
Ki* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA IXb
KIhCAII/KIhCA
IX
AAZ
MZA
EZA
DCP
IND
1
2
3
4
5
900
780
25
1200
31
179
204
182
2070
233
128
784
365
89
12
14
8
25
27
34
50
24
41
35
39
132
22
14
220
30
7
0.48
0.52
0.23
0.76
0.62
1.00
1.20
0.72
1.25
1.68
0.86
1.55
1.57
2.86
0.80
38
15
41
42
28
165
37
12
152
47
20
33
6
7
8
9
10
170
41
a
Human, recombinant isozymes.
b
Catalytic domain of human, cloned isoform.13
*
Errors in the range of 5–10% of the data shown, from three different assays, by a
CO2 hydration stopped-flow assay.18
form hCA I, whose physiological function is less well defined as
compared to those of isoforms II and IX discussed above,1–3 sulfon-
amides 1–10 showed inhibition constants in the range of 89–
2070 nM. Derivatives 4, 7, and 8 were the least effective hCA I
inhibitors (Kis of 365–2070 nM), while the remaining compounds
possessed medium efficacy as CAIs against this isoform (Ki 89–
233 nM). The reduced affinity of some of the first such molecules
against hCA I is probably due to the fact that they possess a bulky
moiety meta to the sulfamoyl zinc binding group of the benzene-
sulfonamide warhead, a trend we have observed for other classes
of compounds bearing this substitution pattern.19 The inhibition
constants of compounds 1, 2, and 3 (Kis of 179–204 nM) were also
very similar (as for the inhibition of the other two isozymes dis-
cussed here). This demonstrates that the presence/absence of a
CH2 or CH2CH2 spacer between the benzenesulfonamide scaffold
and thiourea moiety does not lead to important differences in
the inhibitory properties of these derivatives. However, for the
3.5
3
non nitroxylated CAIs
nitroxylated CAIs
2.5
2
1.5
1
0.5
0
AAZ MZA EZA DCP IND
1
2
3
4
5
6
7
8
9
10
Figure 1. Histogram representing the enhanced selectivity ratio for the inhibition of the tumor-associated isozyme hCA IX over the cytosolic, house-keeping isoform hCA II
with some of the TEMPO-labeled, nitroxylated sulfonamides 1–10 over the classical, clinically used CAIs AAZ-IND (non-nitroxylated CAIs).

A. Cecchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3475–3480
3479
can be envisaged,3,21,22 considering also their potential as biore-
ductive drugs.
compound 1 in buffer (2 10-5 M)
compound 1 + hCA II (11:1 mol/mol)
A
In conclusion, we report here a novel series of spin-labeled sul-
fonamides incorporating TEMPO moieties which have been synthe-
sized by a procedure involving the formation of a thiourea
functionality between the benzenesulfonamide and free radical
fragment of the molecules. These new sulfonamides showed mod-
erate–weak inhibitory activity against isozyme hCA I, but were in
general much more efficient inhibitors of hCA II and hCA IX, the
last one being predominantly found in tumors. Some of these com-
pounds showed significant changes in their ESR signals when com-
plexed within the enzyme active site, making them potential
candidates of interest for the investigation of hypoxic tumors over-
expressing CA IX by ESR techniques for imaging and diagnostic
purposes.
Acknowledgments
3360
3380
3400
3420
Special thanks are addressed to Dr. Sally-Ann Poulsen (Griffith
University, Brisbane, Australia) for critically reading and correcting
our manuscript. This research was financed in part by a grant of the
6th Framework Programme of the European Union (EUROXY
project).
Magnetic Field, G
compound 6 in buffer (2 10-5 M)
compound 6 + hCA II (1:1 mol/mol)
B
References and notes
1. Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.; De
Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329.
2. (a) Cecchi, A.; Hulikova, A.; Pastorek, J.; Scozzafava, A.; Winum, J. Y.; Montero, J.
L.; Supuran, C. T. J. Med. Chem. 2005, 48, 4834; (b) Cecchi, A.; Supuran, C. T. Curr.
Pharm. Des. 2008, 14, 699.
3. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T.;
Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336; (c) Thiry, A.; Dogné, J. M.;
Masereel, B.; Supuran, C. T. Trends Pharmacol. Sci. 2006, 27, 566.
4. (a) Svastova, E.; Hulikova, A.; Rafajova, M.; Zatovicova, M.; Gibadulinova, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S.
FEBS Lett. 2004, 577, 439; (b) Supuran, C. T. Expert Opin. Investig. Drugs 2003, 12,
283; (c) Pastorekova, S.; Kopacek, J.; Pastorek, J. Curr. Top. Med. Chem. 2007, 7,
865.
5. (a) Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van Zandvoort, M. A. M. J.;
Pastoreková, S.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol.
2007, 83, 367; (b) Pastoreková, S.; Zatovicova, M.; Pastorek, J. Curr. Pharm. Des.
2008, 14, 685.
3360
3380
3400
3420
Magnetic Field,G
6. (a) Taylor, J. S.; Mushak, P.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A. 1970, 67,
1410; (b) Mushak, P.; Coleman, J. E. J. Biol. Chem. 1972, 247, 373; (c) Chignell, C.
F.; Starkweather, D. K.; Erlich, R. H. J. Med. Chem. 1972, 15, 876; (d) Erlich, R. H.;
Starkweather, D. K.; Chignell, C. F. Mol. Pharmacol. 1972, 9, 61; (e) Hower, J. F.;
Henkens, R. W.; Chesnut, D. B. J. Am. Chem. Soc. 1971, 93, 6665.
Figure 2. ESR spectra of compounds 1 (A), and 6 (B) in solution without enzyme
(dotted line) and in the presence of hCA II at a molar ratio of 1:1 (continuous line).
7. (a) Supuran, C. T. Br. J. Urol. Int. 2008, 101, 39; (b) Parkkila, S. Br. J. Urol. Int. 2008,
101, 16; (c) Oostewijk, E. Br. J. Urol. Int. 2008, 101, 2; (d) Pastoreková, S.;
Ratcliffe, P. J.; Pastorek, J. Br. J. Urol. Int. 2008, 101, 8.
fer solution (in the absence of enzyme). The measured hyperfine
coupling constant was typical of a polar medium, being hANi = 17.1.
When in the presence of hCA II, the ESR spectrum of compounds 1
and 6 significantly changed to a slow motion signal, indicating that
binding within the enzyme active site has efficiently occurred. It
should be noted that a very small fraction of labeled compounds
did not interact with the enzyme. This fraction was found to be
of the order of 0.1–3%, as measured from double integration of
the different ESR peaks. The exact amount of non-interacting TEM-
PO-CAIs (whose corresponding peak is indicated by an arrow in
Figure 2A and B) depended both on the structure of the nitroxide
derivative and the molar ratio with respect to hCA II. However,
as mentioned above, in all cases the ESR line shape of TEMPO-CAIs
in the presence of hCA II was completely dominated by the spec-
trum of the bound molecules, which showed the anisotropic fea-
tures characteristic to restricted motional conditions.6–8 A similar
behavior was found for the vast majority of compounds 1–10 (data
not shown) proving that these free radicals were able to interact
strongly with the amino acid residues lining the cavity of the
hCA II enzyme, and making them promising molecules for the
investigation of various CA isozymes by means of ESR techniques.
Both imaging as well as therapeutic applications of such molecules
8. Aaron, J. A.; Chambers, J. M.; Jude, K. M.; Di Costanzo, L.; Dmochowski, I. J.;
Christianson, D. W. J. Am. Chem. Soc. 2008. doi:10/1021/ja802214x.
9. (a) Boriack, P. A.; Christianson, D. W.; Kingery-Wood, J.; Whitesides, G. M. J.
Med. Chem. 1995, 38, 2286; (b) Kim, C. Y.; Chang, J. S.; Doyon, J. B.; Baird, T. T.;
Fierke, C. A.; Jain, A.; Christianson, D. W. J. Am. Chem. Soc. 2000, 122, 12125; (c)
Antel, J.; Weber, A.; Sotriffer, C. A.; Klebe, G. In Carbonic anhydrase – Its
inhibitors Anhydrase—Its Inhibitors and activatorsActivators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton, 2004; pp 45–65.
10. (a) Di Fiore, A.; De Simone, G.; Menchise, V.; Pedone, C.; Casini, A.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 1937; (b) Weber, A.; Casini,
A.; Heine, A.; Kuhn, D.; Supuran, C. T.; Scozzafava, A.; Klebe, G. J. Med. Chem.
2004, 47, 550; (c) Menchise, V.; De Simone, G.; Alterio, V.; Di Fiore, A.; Pedone,
C.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2005, 48, 5721.
11. (a) Abbate, F.; Coetzee, A.; Casini, A.; Ciattini, S.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 337; (b) Alterio, V.; De Simone, G.; Monti, S.
M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 4201; (c)
Güzel, Ö.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Salman, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2008, 18, 152.
12. (a) Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1998, 13, 103; (b) Supuran, C.
T.; Scozzafava, A.; Jurca, B. C.; Ilies, M. A. Eur. J. Med. Chem. 1998, 33, 83; (c)
Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni, L.; Ilies, M. A.; Supuran, C. T.
J. Med. Chem. 2000, 43, 4884; (d) Innocenti, A.; Casini, A.; Alcaro, M. C.; Papini,
A. M.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2004, 47, 5224.
13. (a) Cecchi, A.; Winum, J. Y.; Innocenti, A.; Vullo, D.; Montero, J. L.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 5775; (b) Puccetti, L.;
Fasolis, G.; Cecchi, A.; Winum, J. Y.; Gamberi, A.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 2759.

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A. Cecchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3475–3480
14. Wee, V. T.; Feldmann, R. J.; Tanis, R. J.; Chignell, C. F. Mol. Pharmacol. 1976, 12,
832.
15. Sotgiu, A.; Mader, K.; Placidi, G.; Colacicchi, S.; Ursini, C. L.; Alecci, M. Phys. Med.
Biol. 1998, 43, 1921.
mixture was stirred at room temperature for 48 h. The reaction mixture was
worked up as described above.16 4-[({[(1-Oxyl-2,2,6,6-tetramethylpiperidin-4-
yl)amino]carbono thioyl} amino)methyl]benzene sulfonamide (2): MS:ESI+ m/z
422 (M+Na)+; ESIꢀ m/z 398 (MꢀH)ꢀ; 4-[({[(1-oxyl-2,2,6,6-tetramethylpiperidin-
4-yl)amino]carbono thioyl} amino)ethyl]benzene sulfonamide (3): MS:ESI+ m/z
414 (M+H)+, 426 (M+Na)+..
16. Synthesis of thioureido-sulfonamides 1, 4–10. The isothiocyanate sulfonamides13
(1 mmol) were dissolved in 25 mL of MeCN and a solution of amino-TEMPO in
MeCN (1 mmol) was added dropwise. The reaction was monitored by TLC and
when amino-TEMPO was no longer present, the solution was filtered and the
precipitated solid washed with Et2O. The product was purified by flash
chromatography; yields were in the range of 51–92%. 4-({[(1-Oxyl-2,2,6,6-
tetramethylpiperidin-4-yl)amino]carbonothioyl} amino)benzene sulfonamide (1):
MS:ESI+ m/z 408 (M+Na)+, 793 (2M+Na)+; 3-({[(1-oxyl-2,2,6,6-tetramethyl-
piperidin-4-yl)amino]carbonothioyl}amino)benzene sulfonamide (4); MS:ESI+ m/z
386 (M+H)+, 408 (M+Na)+; ESIꢀ m/z 384 (MꢀH)ꢀ, 769 (2MꢀH)ꢀ: 3-fluoro-4-
({[(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)amino]carbonothioyl} amino)benzene
sulfonamide (5); MS:ESI+ m/z 404 (M+H)+, 426 (M+Na)+; ESIꢀ m/z 402 (MꢀH)ꢀ,
805 (2MꢀH)ꢀ: 3-chloro-4-({[(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)amino]-
carbonothioyl} amino)benzene sulfonamide (6); MS:ESI+ m/z 420 (M+H)+, 442
(M+Na)+: N-[3-(aminosulfonyl)phenyl]-4-{[(1-oxyl-2,2,6,6-tetramethylpiperidin-
4-yl)amino]carbono thioyl} amino)benzene sulfonamide (7); MS:ESI+ m/z 543
(M+H)+; ESIꢀ m/z 541 (MꢀH)ꢀ;N-[4-(aminosulfonyl)phenyl]-4-{[(1-oxyl-2,2,6,6-
tetramethylpiperidin-4-yl)amino]carbono thioyl}amino)benzene sulfonamide (8);
18. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics (Oxford,
UK) stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes
(pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining the ionic strength constant),
following the CA-catalyzed CO2 hydration reaction.15 The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5–10% of
the reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in
distilled–deionized water with 10–20% (v/v) DMSO (which is not inhibitory at
these concentrations) and dilutions up to 0.1 nM were done thereafter with
distilled–deionized water. Inhibitor and enzyme solutions were preincubated
together for 15 min at room temperature prior to assay, in order to allow for
the formation of the E–I complex. The inhibition constants were obtained by
non-linear least-squares methods using PRISM 3, and represent the mean from
at least three different determinations.13
19. (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.;
Supuran, C. T. J. Med. Chem. 2000, 43, 4542; (b) de Leval, X.; Ilies, M.; Casini, A.;
Dogné, J. M.; Scozzafava, A.; Masini, E.; Mincione, F.; Starnotti, M.; Supuran, C.
T. J. Med. Chem. 2004, 47, 2796.
20. (a) Pastorekova, S.; Casini, A.; Scozzafava, A.; Vullo, D.; Pastorek, J.; Supuran, C.
T. Bioorg. Med. Chem. 2004, 14, 869; (b) Wilkinson, B. L.; Bornaghi, L. F.;
Houston, T. A.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2006,
49, 6539.
MS:ESI+ m/z 543 (M+H)+;
N-[4-(aminosulfonyl)benzyl]-4-{[(1-oxyl-2,2,6,6-
tetramethylpiperidin-4-yl)amino]carbono thioyl}amino)benzene sulfonamide (9);
MS:ESI+ m/z 577 (M+Na)+; N-{2-[4-(aminosulfonyl)phenyl]ethyl} -4-{[(1-oxyl-
2,2,6,6-tetramethylpiperidin-4-yl)amino]carbono
thioyl}
amino)benzene
sulfonamide (10): MS:ESI+ m/z 569 (M+H)+.
17. The TEMPO isothiocyanate was prepared cf. Zakrzewski, J.; Hupko, J.; Kryczka,
K. Monatsh. Chem. 2003, 134, 843. In 50 mL round-bottomed flask,
a
thiophosgene (0.43 mL, 5.50 mmol) was added in a mixture of 3 mL of NaHCO3
4.7 M and 2.5 mL of CH2Cl2. The solution was stirred vigorously at room
temperature and then amino-TEMPO (0.8 g, 4.74 mmol) was added dropwise.
The aqueous layer was extracted with CH2Cl2, the organic layers collected, and
dried over anhydrous Na2SO4 and evaporated. The obtained oil was purified by
flash-chromatography and an orange solid was obtained (0.64 g, 63%). MS:ESI+
m/z 214 (M+H)+; ESIꢀ m/z 212 (MꢀH)ꢀ: Synthesis of thioureido-sulfonamides 2
and 3. One equivalent of amino-sulfonamides in 20 mL of MeCN was treated
with a solution of TEMPO isothiocyanate (213 mg, 1 mmol) in MeCN, and the
21. (a) Johansson, E.; Parkinson, G. N.; Denny, W. A.; Neidle, S. J. Med. Chem. 2003,
46, 4009; (b) Parkinson, G. N.; Skelly, J. V.; Neidle, S. J. Med. Chem. 2000, 43,
3624.
22. D’Ambrosio, K.; Vitale, R. M.; Dogné, J. M.; Masereel, B.; Innocenti, A.;
Scozzafava, A.; De Simone, G.; Supuran, C. T. J. Med. Chem. 2008, 51.
doi:10.1021/jm800121c.