Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides - A new zinc-binding function in the design of inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.02.091

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 μM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K_Is in the range of 5.8-8.2 μM) but more inhibitory against hCA II (K_Is in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K_Is in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.

Full text of DOI:10.1016/j.bmcl.2005.02.091

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2353–2358
Carbonic anhydrase inhibitors: synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II,
IX, and XII with N-hydroxysulfamides—a new
zinc-binding function in the design of inhibitors
a,b
Jean-Yves Winum, Alessio Innocenti, Jihane Nasr, Jean-Louis Montero,
a
b
b
a
Andrea Scozzafava, Daniela Vullo and Claudiu T. Supuran
a
a,
*
a
Universit a` degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
0019 Sesto Fiorentino, Florence, Italy
Universit e´ Montpellier II, Laboratoire de Chimie Biomol e´ culaire, UMR 5032, Ecole Nationale Sup e´ rieure de Chimie de Montpellier,
rue de l’Ecole Normale, 34296 Montpellier Cedex, France
5
b
8
Received 20 December 2004; revised 23 February 2005; accepted 28 February 2005
Abstract—A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-
binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated
(
hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent
inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM–4.05 lM.
Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K s in the range of
.8–8.2 lM) but more inhibitory against hCA II (K s in the range of 50.5–473 nM). The same situation was true for the tumor-asso-
I
5
I
ciated isozymes, with K s in the range of 353–790 nM against hCA IX and 372–874 nM against hCA XII. Some sulfamides/sulfa-
I
mates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in
some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors
targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides,
or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are
engaged, based on recent X-ray crystallographic data.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1.Introduction
aromatic and heterocyclic sulfonamides, sulfamates, sulf-
1
–4
amides, and some of their derivatives.
The catalytic
Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread
enzymes, present in mammals in at least 15 different iso-
forms. Some of these isozymes are cytosolic (CA I, CA
II, CA III, CA VII, CA XIII), others are membrane-
bound (CA IV, CA IX, CA XII, and CA XIV), CA
VA, and CA VB are present in mitochondria throughout
the body, whereas CA VI is secreted in the saliva and
and inhibition mechanisms of these enzymes are under-
stood in great detail, and this was helpful for the design
of potent inhibitors, some of which possess clinical appli-
cations for the treatment or prevention of a multitude of
1
diseases.
Recently, the involvement of some CAs and their sulfon-
amide inhibitors in cancer has been investigated: many
potent CA inhibitors (CAIs) were shown to inhibit the
growth of several tumor cell lines in vitro and in vivo,
constituting thus interesting leads for developing novel
1
–4
milk. Three cytosolic acatalytic forms are also known
CARP VIII, CARP X, and CARP XI). The catalytically
(
active isoforms, which play important physiological and
patho-physiological functions, are strongly inhibited by
5
,6
7
antitumor therapies. Indeed, Svastova et al. showed
that the acidic extracellular pH, which is a typical attri-
bute of the tumor microenvironment, is generated by the
activity of one of the tumor-associated CA isozymes,
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
573385; e-mail: claudiu.supuran@unifi.it
4
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.091

2354
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2353–2358
that is, CA IX, and that this acidification can be per-
turbed by deletion of the enzyme active site and inhib-
ited by CA IX-selective inhibitors of the sulfonamide
type, which bind only to hypoxic cells containing CA
IX (the involvement of the other tumor-associated iso-
zyme, that is, CA XII, in such processes has not been
investigated for the moment). Thus, it appears of critical
importance to pursue the development of CAIs targeting
the tumor-associated CA isozymes CA IX and XII,
eventually belonging to novel classes of compounds, less
investigated up to now.
and hCA II inhibitors belonging to the sulfamide class
can be obtained. Indeed, in a series of aromatic sulf-
amides possessing the general formula X–C H –
6
4
NHSO NH , compounds with inhibition constants in
2
2
the range of 8–39 nM against hCA I and 7–36 nM against
4
hCA II have been detected. Furthermore, some of the
corresponding sulfamates have also been reported subse-
1
6
quently, being shown that in some cases the sulfamides
are more potent hCA I, hCA II, or hCA IX inhibitors as
compared to them (e.g., in the case of the 4-trifluoro-
methyl-phenyl, pentafluorophenyl-, or 2-naphthyl-sulf-
amides, among others). Thus, the statement of
1
5
Among the various classes of CAIs reported by this
group in the last years, there were also some compounds
incorporating modified sulfonamide/sulfamate moieties,
Maryannof et al. that sulfamides cannot lead to the de-
sign of potent CAIs can be definitively rebutted. Contin-
uing our work in finding novel ZBFs for the design of
CAIs, we report here that N-hydroxysulfamides consti-
tute interesting such candidates. A small library of such
derivatives has been synthesized and tested for the inhibi-
tion of four physiologically relevant isozymes, the cyto-
solic, ubiquitous hCA I and II, as well as the
transmembrane, tumor-associated isozymes hCA IX
and XII (h means isozyme of human origin), comparing
the activity of these new CAIs with that of the sulfamates
and sulfamides investigated in more detail in recent years.
8
such as N-hydroxysulfonamides, phosphorylated sul-
9
,10
11
fonamides,
or phosphorylated sulfamates. The clas-
sical, clinically used CAIs belong to the unsubstituted
aromatic/heterocyclic sulfonamide types possessing the
1
–3
very simple general formula RSO NH ,
2
but it has
been shown mostly by this group that many other
zinc-binding functions (ZBFs) except the sulfonamide
2
8
–12
one can lead to potent CAIs.
Some of the most inves-
tigated such moieties are shown in Figure 1, and they in-
clude the sulfamate one, the phosphorylated sulfamate,
the sulfamide, the sulfonylated sulfamide/sulfamate,
the sulfamide SchiffÕs base, the N-hydroxy-, N-amino-,
N-cyano-, or N-phosphorylated sulfonamide, the urea,
2.Chemistry
1
,13,14
0
hydroxyurea, or hydroxamate ones.
The N-hydr-
The N-alkyl-N -hydroxysulfamide derivatives 4a–f were
oxysulfamides have not been investigated up to now
for their interaction with CAs, although this ZBF is
included in Figure 1.
prepared using the reaction sequence summarized in
Scheme 1. The commercially available O-(tert-butyldi-
methylsilyl) hydroxylamine 1 was sulfamoylated with
N-Boc-protected sulfamoyl chloride, prepared ab initio
by reacting chlorosulfonyl isocyanate with tert-buta-
1
5
Recently, it has been claimed that sulfamides are inef-
fective as ZBFs for the design of CAIs, based on two
examples of sugar sulfamates and the corresponding sulf-
amides (topiramate, RWJ-37947 and their corresponding
sulfamide derivatives), and two bicyclic compounds
possessing the general formula RCH –XSO NH where
1
7
nol. The key intermediate 2 was allowed to react with
different aliphatic alcohols R–OH, with R = octyl, decyl,
dodecyl, benzyl, biphenylmethyl, under standard Mits-
unobu conditions to afford compounds 3 in very good
yield (>90% for each compound). Compounds 3 were
then fully deprotected using trifluoroacetic acid (TFA)
2
2
2
X = O or NH, and R = benzofuran and benzodioxolan.
This claim is as a matter-of-fact not true, since it has been
0
with 5% of water to yield the N-alkyl-N -hydroxysulf-
amides 4b–f. N-Hydroxysulfamide 4a, previously
4
demonstrated earlier by us that low nanomolar hCA I
O
S
O
S
O
S
O
S
NH2
O
NH2
O
N
H
PO H2
N
H
NHR
3
O
O
O
O
R = H, OH
O
O
S
O
O
S
O
S
N
S
NH2
N
H
S
NH2
N
H
OH
O
O
O
O
O
Schiff's base
O
O
O
O
S
N X
H
HO
HO
H N
NH2
N
NH2
2
N
H
R
O
H
X = OH, NH , CN, PO H
Urea
Hydroxyurea
2
3
3
Hydroxamate
Substituted sulfonamides
Figure 1. Zinc-binding functions (ZBFs) for the design of potent CAIs: sulfonamides, sulfamates, sulfamides, substituted sulfonamides/sulfamates/
sulfamides, SchiffÕs bases, urea, hydroxyurea, and hydroxamates. All these groups bind in deprotonated form, as anions to the Zn(II) ion within the
enzyme active site, presumably in monodentate fashion.

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2353–2358
2355
O
O
O
O
O
i
S
iii
S
Si O NH2
Si O
N
H
N
H
HO N
NH2
O
H
1
2
4a
ii
O
O
O
O
O
S
R
iii
S
HO N
N
Si O N
H
N
R
O
H
H
R= octyl
decyl
dodecyl
benzyl
4b
3b
3c
3d
3e
3f
4c
4d
4e
biphenylmethyl 4f
0
Scheme 1. Synthesis of N-alkyl-N -hydroxysulfamides. Reagents and conditions: (i) chlorosulfonyl isocyanate, t-BuOH, CH
2
Cl
2
; (ii) R–OH, PPh
3
,
2
DIAD, THF, Mitsunobu conditions; (iii) TFA–H O 5%.
1
2
reported (in the salt form) by MonteroÕs group and pre-
pared with a modified procedure, was obtained effi-
mic acid (as anions) act as very weak, millimolar inhibi-
tors, whereas N-hydroxysulfamide 4a showed an
enhanced affinity, with an inhibition constant of
4.05 lM. The substituted alkyl/aryl–alkyl derivatives
4b–e on the other hand are less efficient hCA I inhibitors
as compared to the parent hydroxysulfamide 4a, show-
1
8
ciently in high yield upon treatment of 2 with a
mixture of TFA–H O 5%. All these compounds were
2
fully characterized by NMR spectroscopy and mass
1
spectrometry.
9
ing K s in the range of 5.8–8.2 lM; (ii) against the physio-
I
logically most relevant isozyme, hCA II, the
unsubstituted parent compound 4a again behaves as a
more potent inhibitor as compared to sulfamide or sulf-
3
.CA inhibition data
Inhibition data against isozymes hCA I, II, IX, and XII
with N-hydroxysulfamides 4 as well as sulfamide and
sulfamic acid or organic sulfamides/sulfamates 5 and 6
amic acid (millimolar inhibitors, with K s in the range
I
of 0.39–1.13 mM), with an inhibition constant of
566 nM. For this isozyme, the substituted derivatives
4b–e lead to enhanced inhibition, with K s in the range
2
0
as standard inhibitors are presented in Table 1.
I
of 50.5–473 nM. Thus, the n-decyl- and the benzyl deriva-
tives 4b and 4d are medium potency inhibitors (K s in
the range of 313–473 nM), whereas the slight increase
The following SAR is evidenced from the data of Table
: (i) against isozyme hCA I both sulfamide and sulfa-
I
1
Table 1. Inhibition of isozymes hCA I, II, IX, and XII with sulfamide, sulfamic acid, N-hydroxysulfamides 4, sulfamides 5, and the corresponding
sulfamates 6
RNHSO NHOH RNHSO NH ROSO NH
2
2
2
2
2
4
5
6
a
I
Inhibitor
R
K
(nM)
b
b
c
c
hCA I
hCA II
hCA IX
hCA XII
6
5
6
3
3
H
H
2
NSO
NSO
2
NH
d
3
2
—
—
0.31 · 10
0.21 · 10
4050
5800
6000
8200
8100
8
1.13 · 10
0.39 · 10
566
473
89.6
313
50.5
7
9.6 · 10
9.2 · 10
865
506
485
790
353
26
13.2 · 10
10.7 · 10
1340
874
539
633
372
48
6
3
3
2
H
4
4
4
4
4
5
6
5
6
5
6
5
6
a
H
b
n-C10
H
H
23
25
c
d
e
a
a
b
b
c
c
d
d
n-C12
PhCH
4-PhC
2
6
H
4 2
CH
4-CF
4-CF
3
C
C
6
6
H
H
4
3
4
369
20
138
16
54
30
103
45
4-CNC
4-CNC
6
H
4
6
H
4
480
34
149
32
41
40
76
19
C
C
6
F
F
5
6
5
415
39
113
36
47
38
34
30
2-Naphthyl
2-Naphthyl
103
63
40
62
Data for compounds 4 against all isozymes, as well as those regarding hCA IX inhibition with sulfamides 5, and hCA XII inhibition with all these (4–
4
21
6) compounds are new. Sulfamide/sulfamic acid hCA I, II, and IX data were previously published, as well as the inhibition data of sulfamides 5,
and sulfamates 6 against two isozymes (hCA I and II).
22
a
Errors in the range of 5–10% of the shown data, from three different assays.
Human recombinant isozymes.
b
c
20
Catalytic domain of the human recombinant isozyme, CO
As sodium salt.
2
hydrase assay method.
d

2356
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2353–2358
4
in size to n-dodecyl- or a supplementary phenyl moiety
in 4d, leading to the biphenyl-methyl derivative 4e, has
as consequence a dramatic increase in inhibitory power,
the later two compounds being efficient hCA II inhibi-
I and II inhibitors (the sulfamides 5) or as hCA I, II,
2
2
and IX inhibitors (the sulfamates 6). Although the
general trend is that sulfamates are slightly more effi-
cient hCA I and II inhibitors as compared to the corre-
sponding sulfamides possessing the same substitution
tors (K s in the range of 50.5–89.6 nM); (iii) sulfamide
I
4
,22
and sulfamic acid are much more potent hCA IX inhibi-
tors as compared to their affinity for the cytosolic
pattern,
this finding cannot be generalized since we
have observed many cases in which the reverse is true,
that is, sulfamides being more inhibitory than the corre-
sponding sulfamates. In Table 1 several such examples
are provided in which pairs of sulfamides/sulfamates 5
and 6, respectively, have been tested as inhibitors of iso-
zymes I, II, IX, and XII. Thus, it may be observed that
for the 4-trifluoromethylphenyl-, 4-cyano-phenyl-, penta-
fluorophenyl-, or 2-naphthyl-substituted derivatives
among others, sulfamides 5a–d are much more effective
CAIs as compared to the corresponding sulfamates 6a–d
against all investigated isozymes, and of particular impor-
tance are the hCA IX and hCA XII such data, since sulf-
amides and sulfamates have not been investigated up to
now for the inhibition of these tumor-associated iso-
zymes. It may be observed that sulfamides 5 behave as
2
1
isozymes hCA I and II, with K s in the range of 9.2–
I
9
.6 lM. N-Hydroxysulfamide 4a is also a more potent
hCA IX inhibitor as compared to sulfamide or sulfamic
acid, with an inhibition constant of 865 nM, whereas its
substituted derivatives 4b–e showed an even increased
affinity for this tumor-associated isozyme, with K s in
I
the range of 353–790 nM (Table 1). Again the best
inhibitors were the n-dodecyl- and biphenyl-methyl
derivatives (4c and 4e) whereas the n-decyl- and benz-
yl-substituted compounds showed a less efficient inhibi-
tion profile, similarly with the situation observed for
hCA II; (iv) similarly to hCA IX, hCA XII shows higher
affinity for sulfamide and sulfamic acid, as compared to
the cytosolic isozymes I and II, with K s in the range of
I
1
0.7–13.2 lM. Sulfamic acid is slightly more inhibitory
effective hCA IX inhibitors, with K s in the range of
I
than sulfamide (which is in fact true against all isozymes
investigated up to now). N-Hydroxysulfamide 4a is
more potent an inhibitor as compared to the above-
26–40 nM, whereas sulfamates 6 are slightly less effective
(K s in the range of 40–54 nM). The same is true against
hCA XII, with sulfamides 5 showing K s in the range of
I
I
mentioned compounds, with a K of 1340 nM, whereas
I
19–48 nM, and sulfamates 6 in the range of 34–103 nM.
The first partial conclusion is that potent hCA I, II, IX,
and XII inhibitors can be designed both with sulfamide
or sulfamate (or sulfonamide) ZBFs. Indeed, consider-
ing detailed X-ray crystallography work from this labo-
ratory, mainly on isozymes I and II in adducts with
many structural types of sulfonamide and sulfamate
its derivatives 4b–e showed better hCA XII inhibitory
properties, with K s in the range of 372–874 nM. In this
I
small library of derivatives the best substitution patterns
for obtaining potent CA II, IX or XII inhibitors
were identical, that is, the n-dodecyl- and biphenyl-
methyl-substituted compounds were the most effective
as CAIs.
2
3–30
inhibitors,
it appears that a multitude of factors
influence the binding of such a compound within the en-
zyme active site (Fig. 2). Thus, the ZBF of the inhibitor
(irrespective which one of those shown in Fig. 1 is con-
sidered) is deprotonated in order to be able to coordi-
nate monodentately to the Zn(II) ion from the active
site, and also participates in a network of hydrogen
bonds with the neighboring amino acid residues
At this point it is important to re-examine various ZBFs
4
for the design of CAIs, since our results that sulfamides
may lead to effective such inhibitors were recently con-
1
5
tested by Maryannof et al. In Table 1 we also show
some examples of sulfamides and the corresponding sulf-
amates, which have been tested up to now only as hCA
Hydrophilic
part of active site
His 64
Asn 62
TAIL
Asn 67
Leu 198
O
Glu 106
O
SCAFFOLD
Gln 92
N
O
Pro 202
Leu 204
H
O
Val 135
Hydrophobic part
ZBF
H
of active site
Phe 131
N
Thr 199
2
+
Zn
His 119
His 96
His 94
Figure 2. The general structure of a CAI complexed to the enzyme (a-CA) active site: ZBF = zinc-binding function; the organic scaffold may be
present or absent; the tail too. These structural elements interact both with the hydrophobic as well as the hydrophylic halves of the active site
(representative amino acid residues involved in binding are shown), whereas ZBF interacts with the Zn(II) ion as well as the neighboring residues Thr
199 and Glu 106.

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2353–2358
2357
1
(
conserved in all a-CAs) Thr 199 and Glu 106. These
References and notes
fundamental interactions have already been observed
in the X-ray crystal structure of the adducts of hCA II
with the simplest compounds incorporating a sulfon-
amide moiety, that is, sulfamide and sulfamic acid, re-
1. Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T.
J. Enzyme Inhib. Med. Chem. 2004, 19, 199–229.
2
3
4
. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
2003, 23, 146–189.
. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925–953.
. Casini, A.; Winum, J. Y.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 837–
1
2
ported by us. However, these compounds are weak
CAIs (in the milli- to micromolar range), since other
interactions with the enzyme active site are precluded
due to their intrinsic simplicity, but these X-ray struc-
tures provided strong evidence that such compounds
may be used as lead molecules for designing CAIs with
enhanced affinity for various isozymes, which we con-
firmed subsequently for derivatives possessing also an
8
40.
5. Supuran, C. T.; Scozzafava, A. Eur. J. Med. Chem. 2000,
35, 867–874.
6. Supuran, C. T.; Scozzafava, A. J. Enzyme Inhib. 2000, 15,
4
,22,25
5
97–610.
organic scaffold and sulfamide/sulfamate ZBFs.
7
. Svastova, E.; Hulikova, A.; Rafajova, M.; ZatÕovicova,
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
Lett. 2004, 577, 439–445.
Indeed, a potent CAI should possess such an organic
scaffold (Fig. 2) possibly substituted with various tails
that should enable its interaction with both the hydro-
phobic as well as hydrophilic halves of the enzyme active
8
. Mincione, F.; Menabuoni, L.; Briganti, F.; Mincione, G.;
Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1998, 13,
2
3–30
site.
It has been observed that the amino acid resi-
dues belonging to the two above-mentioned active site
regions (i.e., His 64, Asn 62, Asn 67, and Gln 92 for
the hydrophilic half, and Phe 131, Val 135, Leu 202,
Leu 204, and Leu 198, respectively, for the hydrophobic
2
67–284.
9. Fenesan, I.; Popescu, R.; Scozzafava, A.; Crucin, V.;
Mateiciuc, E.; Bauer, R.; Ilies, M. A.; Supuran, C. T. J.
Enzyme Inhib. 2000, 15, 297–310.
0. Fenesan, I.; Popescu, R.; Supuran, C. T.; Nicoara, S.;
Culea, M.; Palibroda, N.; Moldovan, Z.; Cozar, O. Rapid
Commun. Mass Spectrom. 2001, 15, 721–729.
1. Bonnac, L.; Innocenti, A.; Winum, J. Y.; Casini, A.;
Montero, J. L.; Scozzafava, A.; Barragan, V.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2004, 19, 275–278.
2. Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.;
Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3583–
1
–3
1
1
1
1
half) are generally conserved in all CA isozymes.
With such a multitude of interactions between the inhib-
itor and the active site, low nanomolar inhibitors may be
obtained belonging to different classes of compounds, in
addition to the classical sulfonamide CAIs.
4.Conclusions
3
3. Briganti, F.; Mangani, S.; Scozzafava, A.; Vernaglione,
587.
A small library of N-hydroxysulfamides was synthesized
by an original approach in order to investigate whether
this ZBF is efficient for the design of inhibitors targeting
the cytosolic (hCA I and II) and transmembrane, tumor-
associated (hCA IX and XII) isozymes. The parent
derivative, N-hydroxysulfamide was a more potent
inhibitor as compared to sulfamide or sulfamic acid
against all isozymes, with inhibition constants in the
range of 473 nM–4.05 lM. Its substituted n-decyl-, n-
dodecyl-, benzyl-, and biphenylmethyl-derivatives were
G.; Supuran, C. T. J. Biol. Inorg. Chem. 1999, 4, 528–536.
14. Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2003,
1, 2241–2246.
1
1
1
1
5. Maryanoff, B. E.; McComsey, D. F.; Costanzo, M. J.;
Hochman, C.; Smith-Swintowsky, V.; Shank, R. P. J.
Med. Chem. 2005, 48, in press.
6. Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
2
7. Winum, J.-Y.; Toupet, L.; Barragan, V.; Dewynter, G.;
197–2204.
Montero, J.-L. Org. Lett. 2001, 3, 2241–2243.
18. Hajri, A.-H. D. G.; Criton, M.; Dilda, P.; Montero, J.-L.
Heteroatom. Chem. 2001, 12, 1–5.
less inhibitory against hCA I (K s in the range of 5.8–
I
8
the range of 50.5–473 nM). The same situation was true
.2 lM) but more inhibitory against hCA II (K s in
I
1
9. Experimental procedure for the synthesis of compound 2:
To a solution of tert-butanol (1.2 equiv) in 8 mL of
methylene chloride was added at 0 ꢁCchlorosulfonyl
isocyanate (1.2 equiv). The resulting solution was then
added to a solution of O-(tert-butyldimethylsilyl)hydrox-
ylamine (1 equiv) and triethylamine (3 equiv) in methylene
chloride (30 mL). The reaction mixture was stirred at
room temperature for 3 h, then poured in ethyl acetate,
and washed several times with water. The organic phase
was dried on anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by silica gel chroma-
for the tumor-associated isozymes, with K s in the range
I
of 353–790 nM against hCA IX and 372–874 nM
against hCA XII. Some sulfamides/sulfamates possess-
ing the same substitution pattern have also been investi-
gated for the inhibition of these isozymes, being shown
that for these particular cases sulfamides are more effi-
cient inhibitors as compared to the corresponding sulfa-
mates. Potent CA inhibitors targeting the cytosolic or
tumor-associated CA isozymes can thus be designed
from various classes of sulfonamides, sulfamides, or sulf-
amates and their derivatives.
tography to afford the expected compound in 61% yield.
1
H NMR (DMSO-d , 250 MHz) d 10.55 (s, 1H), 8.75 (s,
6
+
H), 0.65 (s, 9H), 0.4 (s, 9H), 0.05 (s, 6H). MS ESI m/z
1
3
+
ꢀ
ꢀ
49 (M+Na) . ESI m/z 325 (MꢀH) .
General procedure for the synthesis of compounds 3: To a
solution of compound 2 (1 equiv), alcohol R–OH (1 equiv)
and triphenylphosphine (1.1 equiv) in 5 mL of THF was
added di-isopropylazodicarboxylate DIAD (1.1 equiv).
The mixture was stirred at room temperature for 2 h,
then concentrated in vacuo. The residue was purified by
Acknowledgments
This research was financed in part by a grant of the 6th
Framework Programme of the European Union (EUR-
OXY project).

2358
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2353–2358
1
silica gel chromatography to yield compound 3 as a yellow
Compound 4f: mp:149–151 ꢁC; H NMR (DMSO-d
400 MHz) 9.4 (d, 1H, J = 3.1 Hz), 9.2 (d, 1H,
J = 3.1 Hz), 8.1 (t, 1H, J = 6.3 Hz), 8–7.5 (m, 9H), 4.3
6
,
1
oil. Compound 3b: H NMR (DMSO-d
6
, 250 MHz) d 8.8
d
(
s, 1H), 3.5 (t, 2H, J = 5.1 Hz), 1.3 (s, 9H), 1.1–1.2 (m,
1
3
1
2H), 0.8 (s, 9H), 0.7 (t, 3H, J = 3.3 Hz), 0.1 (s, 6H); MS
(d, 2H, J = 6.3 Hz); CNMR (DMSO- d
140.4, 139.5, 138.3, 129.4, 128.6, 127.8, 127.1, 127.0, 126.9,
45.9; MS ESI m/z 301 (M+Na) .
6
, 400 MHz) d
+
+
ꢀ
ꢀ
ESI m/z 461 (M+Na) . ESI m/z 437 (MꢀH) . Com-
1
pound 3c: H NMR (DMSO-d
+
+
6
, 250 MHz) d 8.7 (s, 1H),
.6 (t, 2H, J = 2.5 Hz), 1.4 (s, 9H), 1.1–1.3 (m, 16H), 0.8 (s,
3
9
20. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573. An
SX.18MV-R Applied Photophysics (Oxford, UK)
stopped-flow instrument has been used for measuring the
initial velocities by following the change in absorbance of
a pH indicator. 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 Na SO (for maintaining constant the ionic
+
H), 0.7 (t, 3H, J = 3.3 Hz), 0.1 (s, 6H); MS ESI m/z 489
+
ꢀ
ꢀ
1
(
M+Na) . ESI m/z 465 (MꢀH) . Compound 3d:
H
NMR (DMSO-d , 250 MHz) d 9.6 (s, 1H), 3.5 (t, 2H,
6
J = 2.6 Hz), 1.4 (s, 9H), 1.2 (m, 20H), 0.8 (s, 9H), 0.7 (t,
3
+
+
H, J = 7.4 Hz), 0.1 (s, 6H); MS ESI m/z 517 (M+Na) .
ꢀ
ꢀ
1
ESI m/z 493 (MꢀH) . Compound 3e: H NMR (DMSO-
d , 250 MHz) d 9.9 (s, 1H), 7.4 (m, 5H), 4.75 (s, 2H), 1.4 (s,
6
2
4
+
+
H), 0.9 (s, 9H), 0.15 (s, 6H); MS ESI m/z 439 (M+Na) .
9
strength), following the CA-catalyzed CO
reaction for a period of 10–100 s. Saturated CO
in water at 20 ꢁCwere used as substrate. The CO
concentrations ranged from 1.7 to 17 mM for the deter-
mination of the kinetic 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 uncata-
lyzed rates were determined in the same manner and
subtracted from the total obseverd rates. The kinetic
2
hydration
ꢀ
ꢀ
1
ESI m/z 415 (MꢀH) . Compound 3f: H NMR (DMSO-
2
solutions
d
6
, 250 MHz) d 9.7 (s, 1H), 7.1–7.8 (m, 9H), 4.9 (s, 2H), 1.6
2
+
s, 9H), 0.9 (s, 9H), 0.5 (s, 6H); MS ESI m/z 515
(
(
+
ꢀ
ꢀ
M+Na) . ESI m/z 491(MꢀH) .
General procedure for the synthesis of compounds 4:
Compound 3 was dissolved and stirred at 0 ꢁCin a
mixture of TFA–H O 5%. The reaction was monitored by
2
TLCuntil the complete disappearance of starting material.
The mixture was then diluted with ethyl acetate, washed
with a saturated aqueous solution of NaHCO , and then
constants kcat and kcat/K
m
were obtained by nonlinear
least-squares methods using SigmaPlot. Stock solutions of
inhibitors were prepared at a concentration of 1–3 mM (in
DMSO–water 1:1, v/v) and dilutions up to 0.01 nM done
with the assay buffer mentioned above. K
inhibitors were determined by using Lineweaver–Burk
3
washed several times with water. The organic layer was
dried over anhydrous sodium sulfate and concentrated in
vacuo. The residue was purified by silica gel chromato-
graphy to give compound 4 in moderate to good yield. For
the synthesis of compound 4a, the same procedure was
I
s of the
4
–7
plots, as reported earlier.
21. Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2005, 15, 567–571.
22. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
2197–2204.
23. Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David,
S.; Waldeck, H.; Sch a¨ fer, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 841–845.
24. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C . T.Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
25. Abbate, F.; Winum, J.-Y.; Potter, B. V. L.; Casini, A.;
Montero, J.-L.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2004, 14, 231–234.
26. Weber, A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran, C.
T.; Scozzafava, A.; Klebe, G. J. Med. Chem. 2004, 47,
550–557.
27. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2003, 18, 303–308.
28. Casini, A.; Abbate, F.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett 2003, 13, 2763–2769.
29. Abbate, F.; Coetzee, A.; Casini, A.; Ciattini, S.; Scozzaf-
ava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14,
337–341.
used starting from compound 2. Compound 4a: mp: 75–
1
8
1
4
0 ꢁC; H NMR (DMSO-d , 400 MHz) d 9 (s, 1H), 8.4 (s,
6
ꢀ
ꢀ
H), 6.7 (s, 2H); MS ESI m/z 110 (MꢀH) . Compound
1
6
b: mp: 75–77 ꢁC; H NMR (DMSO-d , 400 MHz) d 8.8
(
J = 6.6 Hz), 1.4 (s, 2H), 1.2–1.3 (m, 10H), 0.9 (t, 3H,
s, 1H), 7.2 (t, 1H, J = 5.7 Hz), 2.85 (q, 2H J = 13.3 Hz,
1
J = 6.4 Hz); CNMR (DMSO- d
3
6
, 400 MHz) d 43.2, 32.1,
+
0.0, 29.5, 26.4, 27.1, 22.9, 14.8; MS ESI m/z 247
3
(
(
+
1
M+Na) . Compound 4c: mp: 89–92 ꢁC; H NMR
DMSO-d , 400 MHz) d 9.1 (d, 1H, J = 3.3 Hz), 8.75 (d,
6
1
2
3
3
H, J = 3.3 Hz), 7.3 (t, 1H, J = 5.8 Hz), 2.9 (q,
H,J = 13.2 Hz, J = 6.4 Hz), 1.1–1.5 (m, 16H), 0.9 (t,
1
H, J = 6.6 Hz); CNMR (DMSO- d
3
6
, 400 MHz) 42.8+,
1.8, 29.6, 29.5, 29.4, 29.2, 29.1, 26.7, 22.6, 14.4; MS ESI
+
1
m/z 275 (M+Na) . Compound 4d: mp: 95–97 ꢁC;
NMR (DMSO-d , 400 MHz) 9.1 (d, 1H, J = 3.2 Hz), 8.75
d, 1H, J = 3.3 Hz), 7.35 (t, 1H, J = 5.8 Hz), 2.9 (q, 2H,
J = 13.4 Hz, J = 6.9 Hz), 1.1–1.5 (m, 20H), 0.8 (t, 3H,
H
6
(
1
3
J = 6.3 Hz); CNMR (DMSO- d
29.6, 29.5, 29.4, 29.2, 29.1, 26.6, 26.2, 25.9, 22.6, 14.4; MS
6
, 400 MHz) d 42.8, 31.8,
+
+
1
ESI m/z 275 (M+Na) . Compound 4e: mp: 88–93 ꢁC; H
NMR (DMSO-d , 400 MHz) d 9.1 (s, 1H), 8.9 (s, 1H), 7.8
t, 1H, J = 6.3 Hz), 7.1–7.4 (m, 5H), 4 (d, 2H, J = 6.3 Hz);
6
(
1
3
CNMR (DMSO- d
6
, 400 MHz) d 139.1, 129.3, 129.1,
30. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 2357–2361.
+
28.6, 127.9, 127.4, 46.3; MS ESI m/z 225 (M+Na) .
+
1