Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, tumor-associated isozyme IX with sulfamates including EMATE also acting as steroid sulfatase inhibitors

Article abstract of DOI:10.1021/jm021124k

A series of sulfamates or bis-sulfamates incorporating aliphatic, aromatic, polycyclic (steroidal), and sugar moieties in their molecules has been synthesized and assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more precisely of the cytosolic isozymes CA I and II, and the transmembrane, tumor-associated isozymes CA IX. Some of these compounds were previously reported to act as inhibitors of steroid sulfatases, among which estrone sulfatase (ES) and dehydroepiandrosterone sulfatase (DHEAS) are the key therapeutic targets for estrogen-dependent tumors. Very potent (nanomolar) inhibitors were detected against the three investigated CA isozymes. Best CA I inhibitors were phenylsulfamate and some of its 4-halogeno derivatives, as well as the aliphatic compound n-octyl sulfamate. Against CA II, low nanomolar inhibitors (1.1-5 nM) were phenylsulfamate and some of its 4-halogeno/nitro derivatives, n-octyl sulfamate, and estradiol 3,17β-disulfamate among others. All the investigated sulfamates showed efficient CA IX inhibitory properties, with inhibition constants in the range of 18-63 nM. The best CA IX inhibitor detected so far was 4-chlorophenylsulfamate. These data are critical for the design of novel antitumor properties, mainly for hypoxic tumors that overexpress CA IX, which are nonresponsive to radiation or chemotherapy. The antitumor properties of the ES/DHEAS inhibitors in clinical trials may on the other hand also be due to their potent inhibitory properties of CA isozymes involved in tumorigenicity, such as CA II and CA IX.

Full text of DOI:10.1021/jm021124k

J . Med. Chem. 2003, 46, 2197-2204
2197
Ca r bon ic An h yd r a se In h ibitor s. In h ibition of Cytosolic Isozym es I a n d II a n d
Tr a n sm em br a n e, Tu m or -Associa ted Isozym e IX w ith Su lfa m a tes In clu d in g
EMATE Also Actin g a s Ster oid Su lfa ta se In h ibitor s
J ean-Yves Winum,† Daniela Vullo,‡ Angela Casini,‡ J ean-Louis Montero,† Andrea Scozzafava,‡ and
Claudiu T. Supuran*,‡
Universite´ Montpellier II, Laboratoire de Chimie Biomole´culaire, UMR 5032, Ecole Nationale Supe´rieure de Chimie de
Montpellier, 8 rue de l'Ecole Normale, 34296 Montpellier Cedex, France, and Universita` degli Studi di Firenze, Dipartimento
di Chimica, Laboratorio di Chimica Bioinorganica, Room 188, Via della Lastruccia 3, 1-50019 Sesto Fiorentino (Firenze), Italy
Received December 18, 2002
A series of sulfamates or bis-sulfamates incorporating aliphatic, aromatic, polycyclic (steroidal),
and sugar moieties in their molecules has been synthesized and assayed as inhibitors of the
zinc enzyme carbonic anhydrase (CA), and more precisely of the cytosolic isozymes CA I andII,
and the transmembrane, tumor-associated isozymes CA IX. Some of these compounds were
previously reported to act as inhibitors of steroid sulfatases, among which estrone sulfatase
(ES) and dehydroepiandrosterone sulfatase (DHEAS) are the key therapeutic targets for
estrogen-dependent tumors. Very potent (nanomolar) inhibitors were detected against the three
investigated CA isozymes. Best CA I inhibitors were phenylsulfamate and some of its 4-halogeno
derivatives, as well as the aliphatic compound n-octyl sulfamate. Against CA II, low nanomolar
inhibitors (1.1-5 nM) were phenylsulfamate and some of its 4-halogeno/nitro derivatives, n-octyl
sulfamate, and estradiol 3,17â-disulfamate among others. All the investigated sulfamates
showed efficient CA IX inhibitory properties, with inhibition constants in the range of 18-63
nM. The best CA IX inhibitor detected so far was 4-chlorophenylsulfamate. These data are
critical for the design of novel antitumor properties, mainly for hypoxic tumors that overexpress
CA IX, which are nonresponsive to radiation or chemotherapy. The antitumor properties of
the ES/DHEAS inhibitors in clinical trials may on the other hand also be due to their potent
inhibitory properties of CA isozymes involved in tumorigenicity, such as CA II and CA IX.
In tr od u ction
gies, such as epilepsy, genetic hemiplegic migraine and
ataxia, tardive diskinesia, hypokalemic periodic paraly-
sis, essential tremor and Parkinson’s disease, and
mountain sickness. Accordingly, drugs belonging to this
pharmacological class are under constant develop-
ment.2,3
Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a very
simple physiological reaction, the interconversion be-
tween carbon dioxide and the bicarbonate ion, and are
involved in crucial physiological processes connected
with respiration and transport of CO2/bicarbonate be-
tween metabolizing tissues and the lungs, pH and CO2
homeostasis, electrolyte secretion in a variety of tissues/
organs, biosynthetic reactions (such as gluconeogenesis,
lipogenesis, and ureagenesis), bone resorption, calcifica-
tion, tumorigenicity, and many other physiologic or
pathologic processes.1-3 Thus, it is not surprising that
many of their isozymes (14 are presently known in
higher vertebrates) have emerged as interesting targets
for the design of inhibitors with clinical applications.2,3
Examples of potent, clinically used inhibitors of CAs are
represented by acetazolamide (AAZ), methazolamide
(MZA), and ethoxzolamide (EZA), which all contain a
terminal sulfonamide as anchoring group to coordinate
the catalytic zinc ion of the enzyme, and possess the
general formula RSO2NH2 (where R is generally an
aromatic/heterocyclic moiety).1-3 These sulfonamides
are used clinically as antiglaucoma agents, but also for
the therapy of other diseases, e.g., increased intracranial
pressure, various neurological/neuromuscular patholo-
Recently, the X-ray crystallographic structure of
sulfamic acid (H2NSO3H) bound to the physiologically
relevant isozyme hCA II has been reported by this
group.4 It was shown that sulfamic acid binds to the
2-
zinc ion of the enzyme as a dianion, via its (NH)SO3
sulfamate bianionic species (Zn-N distance of 2.07 Å),
whereas the NH moiety of the inhibitor also participates
in a hydrogen bond with Thr1990γ. An additional
hydrogen bond is formed from this NH to an adjacent
water molecule at 2.75 Å distance. The second nearest
contact of the ligand to Zn, an oxygen atom, leading to
extra-coordination, is at a distance of about 3.07 Å from
the metal ion, whereas the remaining two oxygens of
the SO3 moiety are involved in two other hydrogen
bonds, one with the backbone NH group of Thr 199 at
a distance of 2.99 Å and the other to a water molecule
at a distance of 2.87 Å. This extended extra-coordination
results in a distorted tetrahedral arrangement around
the metal ion, the remaining three ligands of zinc being
His 94, His 96, and His 119 (as in the uninhibited
enzyme).4 In summary, this very simple inhibitor shows
a large number of favorable contacts in the binding
pocket of CA and may be used as a lead molecule, since
* Corresponding author. Tel: +39-055-457 3005; fax: +39-055-
457 3385; e-mail: claudiu.supuran@unifi.it.
† Universite´ Montpellier II.
‡ Universita` degli Studi di Firenze.
10.1021/jm021124k CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/19/2003

2198 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Winum et al.
Ch a r t 1
Sch em e 1
F igu r e 1. Binding of topiramate (TPM) to the hCA II active
site: the seven hydrogen bonds with critical amino acid
residues of the active site and the zinc coordination are
emphasized, with the corresponding distances (Å).
sulfamic acid itself is a weak inhibitor (KI of 21 µM
against hCA I, and of 97 µM against hCA II, for the
esterase activity of these enzymes).5 Indeed, we have
recently reported the strong CA II inhibitory properties
of a series of sugar sulfamates, among which topiramate
(TPM), which acts as a low nanomolar inhibitor.6 Its
X-ray structure in the complex with hCA II revealed a
very tight association of the inhibitor to the enzyme,
with a network of seven hydrogen bonds (to four amino
acid residues and one water molecule) fixing topiramate
within the active site, in addition to the Zn(II) coordina-
tion through the ionized sulfamate moiety (Figure 1).6
jugated steroids (Scheme 1).7,12 Inhibition of these
enzyme by sulfamates such as EMATE, COUMATE, as
well as many other such agents all possessing the
general formula R-OSO2NH2 (where R is generally an
aromatic moiety/polycyclic ring system)7-11 is of critical
interest for designing novel therapies against breast
cancer,7-11 androgen-dependent skin diseases,13 in cog-
nitive dysfunctions in which DHEAS inhibition was
shown to enhance learning and spatial memory,14 or for
the design of immune modulators, as it was shown that
ES/DHEAS inhibition has a role in regulating T-helper
cell function.15
Sulfamates on the other hand also act as potent
inhibitors of steroid sulfatases, among which estrone
sulfatase (ES) and dehydroepiandrosterone sulfatase
(DHEAS) are the key therapeutic targets for estrogen
dependent tumors.7-11 ES/DHEAS catalyze the hydroly-
sis of estrone sulfate (E1S) and DHEA-sulfates (DH-
EAS), respectively, releasing the corresponding uncon-
Considering the similarity between the two classes
of inhibitors of the two types of enzymes mentioned
here, i.e., sulfonamides/sulfamates as CA inhibitors, and
sulfamates as ES/DHEAS inhibitors, as well as the fact
that some CA isozymes, such as CA IX and CA XII are
predominantly found in cancer cells, lacking from their
normal counterparts,16 it appeared of interest to inves-

Inhibition of Cytosolic Isozymes I, II, and IX with Sulfamates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2199
Ta ble 1. Inhibition Data for Derivatives 1-26 Investigated in
the Present Paper and Standard Sulfonamide CA Inhibitors,
against Isozymes I, II, and IX
Ch a r t 2
.
inhibitor
KIa (nM)
no.
R
hCA Ib hCA IIa hCA IXc
acetazolamide
methazolamide
ethoxzolamide
sulfamic acid
-
-
-
900
780
25
21 000
12
14
8
25
27
34
nt
97 000
(H2NSO3H)
topiramate
1
2
3
4
5
6
7
-
Ph
250
2.1
3.8
113
4.6
7.3
9.5
33
5
nt
63
59
50
18
19
23
34
51
45
36
41
33
54
47
39
37
40
25
43
30
58
32
44
39
32
26
1.3
1.9
95
1.1
1.5
3.8
1.6
98
4-Me-C6H4
4-Ph-C6H4
4-Cl-C6H4
4-Br-C6H4
4-I-C6H4
4-MeO-C6H4
4-PhO-C6H4
4-AcNH-C6H4 37
4-O2N-C6H4
4-NC-C6H4
4-t-Bu-C6H4
4-CF3-C6H4
C6F5
8
9
115
18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
40
480
43
369
415
432
1.5
149
2.9
138
113
125
138
63
2.7
76
10
5
13
C6Cl5
2,4,6-Cl3C6H2 454
2-naphthyl
n-C8H17
103
3.5
105
37
6
15
A
A (EMATE)
A
A
A
A
A
A
31
27
15
29
23
278
315
28
a
Errors in the range of 5-10% of the reported value (from three
different assays). bHuman (cloned) isozymes, by the esterase
method. Catalytic domain of human, cloned isozyme, by the CO2
c
hydration method. A: See structure in the text. nt ) not tested.
tigate in detail the interaction of sulfamates with some
CA isozymes. Here we report the finding that many
sulfamates known to act as potent ES/DHEAS inhibitors
also show very high (nanomolar) affinity for three CA
isozymes, the cytosolic CA I and CA II, and the trans-
membrane, tumor-associated isozyme CA IX.
Sch em e 2
Resu lts
Ch em istr y. The sulfamates 1-26 investigated here
as CAIs are shown in Table 1. They were prepared as
shown in Scheme 2,17-27 by reaction of the corresponding
alcohols/phenols with sulfamoyl chloride.
Ca r bon ic An h yd r a se In h ibitor y Activity. Inhibi-
tion data against three CA isozymes, the cytosolic hCA
I and hCA II,28-33 as well as the transmembrane, tumor-
associated isozyme hCA IX, are shown in Table 1.34-36
Standard, clinically used sulfonamide CAIs, such as
acetazolamide, methazolamide, and ethoxzolamide, were
also included in these assays, for comparison.
Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)-1,4-di-
hydropyridin-1- ylsulfonyl]azanide) was allowed to react
with the requisite alcohol or phenol, but unfortunately,
no reaction was observed even after heating or base
addition. This reagent is in fact effective only for
sulfamoylation of amines. It appears that alcohol and
phenol functions are not enough nucleophilic to react
efficiently with this new sulfamoylating agent. We then
conducted experiments using the unstable N-(tert-
butoxycarbonyl)sulfamoyl chloride (obtained in situ from
chlorosulfonyl isocyanate and tert-butyl alcohol). Under
these conditions, N-Boc-sulfamate derivatives were
obtained but some of them were not stable during
Discu ssion
Ch em istr y. We attempted to synthesize sulfamates
1-25 by an original procedure using a new sulfamoyl-
ating reagent previously described by Winum et al.17
followed by removal of the protecting group. (N-(tert-

2200 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Winum et al.
purification or Boc removal especially those with electron-
withdrawing groups on the phenyl ring, the Boc-NH-
SO2 acting as a good leaving group. So, we chose to
prepare sulfamates 1-25 by the procedure described by
Okada et al.,18 employing the reaction of phenols/
alcohols with sulfamoyl chloride in N,N-dimethylaceta-
mide as solvent. This procedure proved to be very
efficient, and the different derivatives were obtained in
high yields after purification either by crystallization
or silica gel chromatography (Scheme 2). Sulfamoyl
chloride was prepared from chlorosulfonyl isocyanate
and formic acid as described by Appel et al.19 All
compounds were characterized by 1H and 13C NMR, IR,
and mass spectroscopy. Some of these compounds were
previously reported in the literature,20-27 but they have
not been tested as CA inhibitors (CAIs) in earlier
studies. A rather large number of substitution patterns
(R moieties) have been used in compounds 1-26 (aro-
matic, substituted-aromatic, aliphatic, steroidal back-
bones, etc.) together with some disulfamates which were
also prepared, to detect best substitution pattern(s) for
efficient CA inhibition.
as the two sugar sulfamates 24 and 25 (with an activity
comparable again with that of topiramate).
Isozym e II. This is considered the most important
isozyme as target for obtaining inhibitors with phar-
macological applications. All the sulfamates investigated
here generally showed good CA II inhibitory properties,
considering especially the very weak inhibition produced
by the lead molecule, sulfamic acid (inhibition constant
of 97 µM). Again a rather wide range of activities were
evidenced, as follows: (i) very potent CA II inhibitors,
with inhibition constants in the range of 1.1 - 15 nM
proved to be compounds 1, 2, 4-7, 10, 12, 18, 20-22,
and 24, which are all more effective or at the same levels
of inhibition as the clinically used compounds aceta-
zolamide, methazolamide, ethoxzolamide, or topiramate
(Table 1). The best CA II inhibitor was the simple
4-chlorophenylsulfamate 4, but very similar inhibition
was produced by the structurally related halogenophe-
nyl sulfamates 5 and 6, the unsubstituted derivative 1,
the 4-methyl/methoxy- or 4-nitro-substituted compounds
2, 7, and 10, as well as the tert-butyl derivative 12. The
very strong inhibitory activity of the simple, aliphatic
sulfamate 18 is noteworthy (in fact this is the unique
such derivative investigated up to now), which is among
the best inhibitors detected here and belongs to a class
of compounds which deserves much more attention. The
steroidal sulfamates 20, 21, and 22 also showed excel-
lent CA II inhibitory activity, with the best inhibitor
being the bis-sulfamate 21, followed by EMATE 20 and
the monosulfamate 22. It is interesting to note that the
EMATE corresponding nonaromatic sulfamate 23 was
2.7 times less effective as a CA II inhibitor as compared
to EMATE; (ii) less potent inhibitory activity was shown
by compounds 9, 17, 19, 23, 25, and 26, with inhibition
constants in the range of 18-76 nM. These compounds
belong either to the aromatic type of sulfamates (9, 17,
and 19) but possess bulkier substituents/rings as com-
pared to 1 (or in the case of 19, two sulfamate moieties)
or to the polycyclic/sugar type of derivatives (23 and 25).
Compound 26 also showed this type of behavior; (iii) the
most ineffective CA II inhibitors (compounds 3, 8, 11,
13-16) showed inhibition constants in the range of 95-
149 nM and belong to the phenylsulfamate 1 type of
derivative, substituted with moieties which led to an
enhanced loss of affinity for the active site, such as
4-phenyl-/phenoxy-; 4-trifluoromethyl-; 4-cyano-; per-
fluorophenyl-/perchlorophenyl- or 2,4,6-trichlorophenyl.
SAR is difficult to draw from all these data, but one may
speculate that for substituted-phenyl sulfamates derived
from 1, best activity against CA II is observed for the
halogeno-substituted compounds (together with the
unsubstituted lead) and that generally bulky groups in
position 4 (phenyl/phenoxy - but not tert-butyl!) are
detrimental to activity. One must also mention that all
sulfamates 1-26 showed higher affinity for CA II as
compared to CA I, a behavior also observed for sulfona-
mide CAIs.1-3
Car bon ic An h ydr ase In h ibitor y Activity. Isozym e
I. As seen from data of Table 1, sulfamates 1-26
investigated here are much more effective CA I inhibi-
tors as compared to the lead molecule from which they
were obtained, i.e., sulfamic acid (inhibition constant
of 21 µM). A large range of activities was observed for
the different substitution patterns of the investigated
sulfamates, as follows: (i) several derivatives, such as
1, 2, 4-6, 18, 21, and 22, acted as very potent CA I
inhibitors, with inhibition constants in the range of 2.1-
15 nM. It is interesting to note that this potent inhibi-
tory activity is seen for simple aromatic derivatives
(such as the phenyl, 4-tolyl or 4- halogenophenyl-
substituted sulfamates), the aliphatic derivative 18, or
the condensated polycyclic derivatives 21 and 22. It
should be noted that these compounds behave as much
more potent CA I inhibitors as compared to the clinically
used derivatives acetazolamide, methazolamide, ethox-
zolamide, or topiramate (Table 1); (ii) derivatives 7, 9,
10, 12, 20, 23, and 26 also showed potent CA I inhibitory
activity, with inhibition constants in the range of 28-
43 nM, being slightly less effective than the derivatives
discussed above. Again SAR is difficult to envisage, since
simple aromatic (7, 9, 10, 12), polycyclic (20 and 23), or
imidazolyl-phenyl-aliphatic (26) derivatives showed com-
parable activity. Comparing all the derivatives dis-
cussed up to now, it is clear that phenylsulfamate 1 is
the best CA I inhibitor observed in this series, and that
any substitution at its aromatic ring is detrimental to
activity. Still, moieties that lead only to a slight decrease
of activity were 4-methyl-, 4-halogeno-, and to a higher
degree, 4-methoxy-, 4-acetamido-, 4-nitro-, and 4-tert-
butyl; (iii) the other investigated derivatives showed
inhibition constants in the range of 103-480 nM against
hCA I, being better inhibitors than acetazolamide and
methazolamide and showing a potency comparable to
that of topiramate. Among these derivatives, it is
obvious that other substitution patterns of the phenyl-
sulfamate lead 1 were highly detrimental to inhibitory
activity, such as 4-phenyl-, 4-phenoxy-, 4-cyano-, 4-tri-
fluoromethyl- and perfluorophenyl-/perchlorophenyl-.
The bis-sulfamate 19 was also weakly active, as well
Isozym e IX. The inhibition profile of this isozyme
with the sulfamates 1-26 is very interesting, differing
substantially from the inhibition profiles of the cytosolic
isozymes I and II discussed earlier. Thus, as a first
observation, all the investigated sulfamates as well as
the three clinically used inhibitors (acetazolamide,
methazolamide, and ethoxzolamide) showed inhibition

Inhibition of Cytosolic Isozymes I, II, and IX with Sulfamates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2201
constants in the range of 18-63 nM. The range of
activity is thus much more limited as compared to that
of the other two isozymes CA I and II investigated here
(Table 1). Several compounds, such as acetazolamide,
methazolamide, and the sulfamates 4-6, 18, 20, and
26 showed very strong CA IX inhibitory properties (KIs
in the range of 18-30 nM). Again it is rather difficult
to draw SAR conclusions, since these compounds belong
to different structural subtypes, with both halogenophe-
nyl (4-6), aliphatic (18 and 26), and steroidal sulfa-
mates showing strong inhibitory properties. It is note-
worthy that 1, which was one of the best CA I and CA
II inhibitors among the investigated compounds, is in
fact the worst CA IX inhibitor, although it appreciably
inhibits this isozyme, too. In this case, all substitutions
of 1 led to more effective inhibitors, culminating in the
three halogenophenyl derivatives 4-6 which were the
best compounds in the series. Still, other substitution
patterns were equally effective, with the 4-methoxy-,
4-nitro-, 4-tert-butyl-, and the 2,4,6-trichloro- derivatives
leading to some of the best inhibitors (KIs in the range
of 33-37 nM, comparable with that of ethoxzolamide).
A very interesting behavior against CA IX was shown
by the steroidal sulfamates 20-23. Thus, EMATE 20
and the monosulfamate 22 are quite potent inhibitors
(KIs in the range of 30-32 nM), whereas 21 (which
showed the best CA II and CA I inhibitory properties)
was the worst CA IX inhibitor in this subseries (KI of
58 nM), being less effective than 23 (KI of 44 nM). The
two sugar sulfamates 24 and 25 also showed good CA
IX inhibitory properties, with inhibition constants in the
range of 32-39 nM.
Data presented here are particularly important for
the design of compounds able to interfere with the
development of cancer cells. Thus, it has recently been
demonstrated that the tumor-associated isozyme CA IX
may be of considerable value as markers of tumor
progression.37 This is mostly due to its induction by
hypoxia, a clinically important factor of tumor biology
that significantly affects treatment outcome and disease
progression.37 Strong association between CA IX expres-
sion and intratumoral hypoxia has been demonstrated
in the cervical, breast, head and neck, bladder, and
nonsmall cell lung carcinomas (NSCLC).38-41 Further-
more, such hypoxic tumors overexpressing CA IX do not
respond to radiation or chemotherapy. It would be thus
critically important to inhibit this isozyme in order to
test whether such malignancies become again respon-
sive to the anticancer therapy. It must also be stressed
that some sulfonamide CAIs show themselves antitumor
properties in vitro and in vivo, although the mechanism
by which they exert this property is rather unclear for
the moment.42-45 Another point raised by our work is
whether the antitumor properties of the steroid sulfa-
tase inhibitors are due only to the inhibition of ES/
DHEAS. Our data strongly suggest that this may be in
part due to inhibition of some CA isozymes important
for the tumor growth, such as CA IX and/or CA II (CA
I), and that the mechanims of action of the inhibitors
in clinical trials may be much more complicated as
compared to those originally thought.
synthesized and assayed as inhibitors of the cytosolic
isozymes CA I and II, and the transmembrane, tumor-
associated isozymes CA IX. Some of these compounds
were previously reported to act as inhibitors of steroid
sulfatases, among which ES and DHEAS are the key
therapeutic targets for estrogen-dependent tumors. Very
potent (nanomolar) inhibitors were detected against the
three investigated CA isozymes. The best CA I inhibitors
were phenylsulfamate and some of its 4-halogeno de-
rivatives, as well as the aliphatic compound n-octyl
sulfamate. Against CA II, low nanomolar inhibitors
(1.1-5 nM) were phenylsulfamate and some of its
4-halogeno-/nitro-derivatives, n-octyl sulfamate, and
estradiol 3,17â-disulfamate among others. All the in-
vestigated sulfamates showed efficient CA IX inhibitory
properties, with inhibition constants in the range of 18-
63 nM. The best CA IX inhibitor detected so far was
4-chlorophenylsulfamate. The inhibition profile of the
three investigated isozymes with this type of compounds
was rather different, allowing us to hope that the
preparation of CA IX-selective inhibitors is possible.
These data are critical for the design of novel antitumor
therapies, mainly for hypoxic tumors that overexpress
CA IX, which are nonresponsive to radiation or chemo-
therapy. The antitumor properties of the ES/DHEAS
inhibitors in clinical trials may on the other hand also
be due to their potent inhibitory properties of CA
isozymes involved in tumorigenicity, such as CA II and
CA IX.
Exp er im en ta l Section
Gen er a l. All reagents and solvents were of commercial
quality and used without further purification. All reactions
were carried out under an inert atmosphere of nitrogen. TLC
analyses were performed on silica gel 60 F254 plates (Merck
Art.1.05554). Spots were visualized under 254 nm UV il-
lumination, or by ninhydrin solution spraying. Melting point
were determined on a Bu¨chi Melting Point 510 and are
uncorrected. Infrared spectra were recorded on a Perkin-Elmer
FT-IR spectrometer S1000. 1H and 13C NMR spectra were
recorded on a Bruker AC-200 and/or Bruker DRX-400 spec-
trometer using DMSO-d6 as solvent and tetramethylsilane as
internal standard. For 1H NMR spectra, chemical shifts are
expressed in δ (ppm) downfield from tetramethylsilane, and
coupling constants (J ) are expressed in hertz. Electron ioniza-
tion mass spectra (30 eV) were recorded in positive or negative
mode on a Water MicroMass ZQ.
P r ep a r a tion of Su lfa m a tes. Gen er a l P r oced u r e. Sulfa-
mates were prepared by reacting the corresponding alcohol
or phenol (1 equiv) with sulfamoyl chloride (3 equiv) in N,N-
dimethylacetamide.18 (Sulfamoyl chloride was prepared from
chlorosulfonyl isocyanate and formic acid as described in ref
19). After completion of the reaction (TLC monitoring), the
mixture was diluted with ethyl acetate and washed several
times with water. The organic extract was dried (MgSO4) and
concentrated under vacuum. The residue was purified either
by crystallization from ether/pentane or by chromatography
on silica gel.
Phenylsulfamate 1: mp 77-79 °C (lit. mp 77.6-81.2 °C);9c
1H NMR (DMSO-d6, 250 MHz) δ 8 (s, 2H), 7.55-7.2 (m, 5H);
13C NMR (DMSO-d6, 400 MHz) δ 151, 130.6, 127.4, 123; IR
(KBr) 3418, 3305, 3071, 1550, 1486, 1370, 1182 cm-1; MS m/z
196 (M + Na)+.
p-Methylphenylsulfamate 2: mp 75-76 °C (lit. mp 80 °C);20
1H NMR (DMSO-d6, 250 MHz) δ 7.9 (s, 2H), 7.25 (d, J ) 7.2
Hz, 2H), 7.15 (d, J ) 7.2 Hz, 2H), 3.12 (s, 3H, Me); 13C NMR
(DMSO-d6, 400 MHz). 148.9, 136.7, 130.9, 122.8, 20.7; IR (KBr)
3381, 3275, 3056, 2920, 1598, 1535, 1355, 1178 cm-1; MS m/z
210 (M + Na)+.
Con clu sion s. A series of sulfamates or bis-sulfa-
mates incorporating aliphatic, aromatic, polycyclic (ste-
roidal), and sugar moieties in their molecules has been

2202 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Winum et al.
p-Biphenylsulfamate 3: mp 160-162 °C (lit. mp 165 °C);20
1H NMR (DMSO-d6, 250 MHz) δ 8.1 (s, 2H), 7.7 (m, 4H), 7.4
(m, 5H); 13C NMR (DMSO-d6, 400 MHz) δ 150.5, 140, 139.4,
129.8, 128.9, 128.5, 127.6, 123.5; IR (KBr) 3418, 3290, 3071,
1542, 1482, 1377, 1175 cm-1; MS m/z 272 (M + Na)+.
p-Chlorophenylsulfamate 4: mp 99-100 °C (lit. mp 104
°C);20 1H NMR (DMSO-d6, 250 MHz) δ 8.1 (s, 2H), 7.55 (d, J )
8.5 Hz, 2H), 7.3 (d, J ) 8.7 Hz, 2H); 13C NMR (DMSO-d6, 400
MHz) δ 149.7, 130.5, 124.9, 117.7; IR (KBr) 3396, 3275, 3094,
1486, 1362, 1175, 1085 cm-1; MS m/z 230 (M + Na)+.
2-Naphthylsulfamate 17: mp 111-112 °C (lit. mp 112 °C);23
1H NMR (DMSO-d6, 250 MHz) δ 8.2 (s, 2H), 8 (m, 4H), 7.55
(m, 3H); 13C NMR (DMSO-d6, 400 MHz) δ 148.6, 134.1, 132.1,
130.6, 128.6, 128.5, 127.7, 127, 122.5, 120; IR (KBr): 3411,
3267, 3094, 1550, 1362, 1167 cm-1; MS m/z 246 (M + Na)+.
n-Octylsulfamate 18: mp 53-57 °C; 1H NMR (DMSO-d6,
250 MHz) δ 7.2 (s, 2H), 4 (t, J ) 6.4 Hz, 2H), 1.6 (m, 2H), 1.3
(m, 10H), 0.85 (t, J ) 6.6 Hz, 3H); 13C NMR (DMSO-d6, 400
MHz) δ 69.8, 32, 29.4, 29.3, 29.1, 26.1, 22.9, 14.7; IR (KBr)
3373, 3283, 2920, 1546, 1351, 1182 cm-1; MS m/z 232 (M +
Na)+.
p-Bromophenylsulfamate 5: mp 111-113 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 8.1 (s, 2H), 7.65 (d, J ) 8.5 Hz, 2H),
7.25 (d, J ) 8.5 Hz, 2H); 13C NMR (DMSO-d6, 400 MHz) δ
150.2, 133.5, 125.3, 119.9; IR (KBr) 3381, 3275, 3094, 1535,
1482, 1355, 1171, 1066 cm-1; MS m/z 276 (M + Na)+.
5-Pentylresorcinylsulfamate 19: mp 77-79 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 8.1 (s, 4H), 7.1 (s, 3H), 2.6 (t, J ) 7.8
Hz, 2H), 1.6 (m, 2H), 1.3 (m, 4H), 0.85 (t, J ) 6.4 Hz, 3H); 13
C
NMR (DMSO-d6, 400 MHz) δ 151.1, 146.4, 12, 35.5, 31.6, 31,
22.7, 14.7; IR (KBr) 3403, 3358, 3275, 3086, 2943, 1598, 1531,
1370, 1186, 1103 cm-1; MS m/z 361 (M + Na)+.
p-Iodophenylsulfamate 6: mp 134-136 °C; 1H NMR (DMSO-
d6, 250 MHz) δ 8.1 (s, 2H), 7.8 (d, J ) 8.3 Hz, 2H), 7.15 (d, J
) 8.3 Hz, 2H); 13C NMR (DMSO-d6, 400 MHz) δ 150.8, 139.4,
125.5, 92.5; IR (KBr) 3373, 3275, 3086, 1535, 1475, 1355, 1178;
MS m/z 322 (M + Na)+.
p-Methoxyphenylsulfamate 7: mp 62-64 °C (lit. mp 65
°C);21 1H NMR (DMSO-d6, 250 MHz) δ 7.9 (s, 2H), 7.2 (d, J )
8.9 Hz, 2H), 7.0 (d, J ) 8.9 Hz, 2H), 3.5 (MeO); 13C NMR
(DMSO-d6, 400 MHz) δ 158.4, 144.4, 124.2, 115.4, 37.8; IR
(KBr) 3381, 3267, 3094, 2966, 1598, 1542, 1362, 1246, 1156
cm-1; MS m/z 226 (M + Na)+.
Estrone sulfamate 20: mp 197-200 °C (lit. mp 199-202
°C);24 1H NMR (DMSO-d6, 400 MHz) δ 7.9 (s, 2H), 7.35 (d, J )
8.5 Hz, 1H), 7.1 (d, J ) 8.5 Hz, 1H), 7 (s, 1H), 2.85 (d, J ) 4.6
Hz, 2H), 2.45 (m, 2H), 2.25 (m, 1H), 2 (m, 3H), 1.8 (d, J ) 7.4
Hz, 1H), 1.5 (m, 6H), 0.85 (s, 3H); 13C NMR (DMSO-d6, 400
MHz) δ 148.9, 138.9, 138.8, 127.4, 122.7, 120.1, 50.3, 48.1, 44.4,
38.3, 36.2, 32.1, 29.8, 26.6, 26.2, 21.9, 14.3; IR (KBr) 3411,
3252, 2943, 1711, 1490, 1377, 1178 cm-1; MS m/z 372 (M +
Na)+.
Estradiol 3,17â-disulfamate 21: mp 186-190 °C (lit. mp
189-194 °C);25 1H NMR (DMSO-d6, 400 MHz) δ 7.9 (s, 2H),
7.4 (s, 2H), 7.35 (d, J ) 8.5 Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H),
6.95 (s, 1H), 4.35 (t, J ) 8.1 Hz, 1H), 2.8 (m, 2H), 2.3 (m, 1H),
2.2 (m, 2H), 2 (m, 1H), 1.85 (m, 1H), 1.7 (m, 2H), 1.3 (m, 6H),
0.75 (s, 3H); 13C NMR (DMSO-d6, 400 MHz) δ 148.8, 138.9,
138.8, 127.4, 122.7, 120.1, 88.2, 49.4, 44.2, 44.1, 38.6, 36.7, 29.8,
28.3, 27.2, 26.3, 23.4, 12.4; IR (KBr) 3381, 3320, 3094, 2928,
1546, 1377, 1190 cm-1; MS m/z 453 (M + Na)+.
Estradiol 17â-sulfamate 22: mp 157-159 °C (lit. mp 158-
161 °C);25 1H NMR (DMSO-d6, 400 MHz) δ 8.95 (s, 1H), 7.35
(s, 2H), 7.05 (d, J ) 8.4 Hz, 1H), 6.45 (dd, J ) 2.3 Hz, 8.3 Hz,
1H), 6.4 (d, J ) 2.2 Hz, 1H), 4.3 (t, J ) 8.5 Hz, 1H), 2.7 (m in ,
2H), 2.15 (m, 3H), 1.95 (m, 1H), 1.7 (m, 3H), 1.25 (m, 6H), 0.75
(s, 3H); 13C NMR (DMSO-d6, 400 MHz) δ 160.5, 142.6, 135.6,
131.7, 120.5, 118.3, 93, 54.2, 48.8, 48.2, 43.8, 41.5, 34.6, 33,
32.3, 31.3, 28.2, 17.2; IR (KBr) 3404, 3281, 3094, 2930, 1503,
1348, 1180 cm4; MS m/z 374 (M + Na)+.
p-Phenoxyphenylsulfamate 8: mp 109-111 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 8 (s, 2H), 7.35 (m, 2H), 7.1 (m, 2H);
13C NMR (DMSO-d6, 400 MHz) δ 157.4, 155.7, 130.9, 124.8,
124.5, 120.4, 119.4; IR (KBr) 3381, 3298, 3071, 1587, 1490,
1355, 1250, 1175 cm-1; MS m/z 288 (M + Na)+.
p-Acetamidophenylsulfamate 9: mp 175-177 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 10 (s, 1H), 8 (s, 2H), 7.6 (d, J ) 7.2
Hz, 2H), 7.2 (d, J ) 7.2 Hz, 2H), 2 (s, 3H); 13C NMR (DMSO-
d6, 400 MHz) δ 169.2, 146.1, 138.5, 123.4, 120.9, 24.7; IR (KBr)
3373, 3184, 3090, 2996, 1666, 1606, 1546, 1370, 1156 cm-1
;
MS m/z 253 (M + Na)+.
p-Nitrophenylsulfamate 10: mp 100-102 °C (lit. mp 94-
96 °C);22 1H NMR (DMSO-d6, 250 MHz) δ 8.4 (s, 2H), 8.35 (d,
J ) 8.7 Hz, 2H), 7.55 (d, J ) 8.7 Hz, 2H); 13C NMR (DMSO-
d6, 400 MHz) δ 164.7, 127, 123.7, 116.6; IR (KBr) 3418, 3260,
3109, 1613, 1527, 1385, 1325, 1197 cm-1; MS m/z 241 (M +
Na)+.
p-Cyanophenylsulfamate 11: mp 152-153 °C (lit. mp 155
°C);21 1H NMR (DMSO-d6, 250 MHz) δ 8.3 (s, 2H), 7.95 (d, J )
8.7 Hz, 2H), 7.45 (d, J ) 8.7 Hz, 2H); 13C NMR (DMSO-d6,
400 MHz) δ 162.4, 135.2, 123.9, 117.2, 101.8; IR (KBr): 3373,
3252, 2241, 1602, 1501, 1373, 1197 cm-1; MS m/z 221 (M +
Na)+.
p-tert-Butylphenylsulfamate 12: mp 95-97 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 8 (s, 2H), 7.5 (d, J ) 8.7 Hz, 2H), 7.2
(d, J ) 8.7 Hz, 2H), 1.25 (s, 9H); 13C NMR (DMSO-d6, 400
MHz) δ 149.8, 148.7, 127.3, 122.5, 35.1, 32; IR (KBr) 3381,
3290, 3079, 2958, 1550, 1381, 1178 cm-1; MS m/z 252 (M +
Na)+.
Dehydroepiandrosterone sulfamate 23: mp 160-162 °C; 1H
NMR (DMSO-d6, 400 MHz): δ 7.4 (s, 2H), 5.4 (t, J ) 3.7 Hz,
1H), 4.25 (m, 1H), 2.45 (m, 4H), 2 (m, 5H), 1.7 (m, 4H), 1.45
(m, 2H), 1.3 (m, 2H), 1 (m, 5H), 0.8 (s, 3H); 13C NMR (DMSO-
d6, 400 MHz): δ 140.3, 122.9, 80.2, 51.6, 50.4, 47.6, 39.3, 37.3,
36.9, 36.1, 31.9, 31.7, 31.1, 29, 22.2, 20.7, 19.7, 14; IR (KBr):
3373, 3275, 2943, 1730, 1568, 1381, 1182 cm-1; MS m/z 390
(M + Na)+.
1,2:5,6-Di-O-isopropylidene-R-D-glucofuranose 3-sulfamate
24: mp 136-138 °C (lit. mp 140 °C);26 1H NMR (DMSO-d6,
250 MHz) δ 7.8 (s, 2H), 6 (d, J ) 3.7 Hz, 1H), 4.8 (m, 2H), 4.25
(m, 2H), 4 (m, 1H), 3.85 (m, 1H), 1.45 (s, 3H), 1.35 (s, 3H),
1.25 (s, 6H); 13C NMR (DMSO-d6, 400 MHz) δ 112.2, 108.8,
105.2, 83.2, 81.5, 79.6, 73.2, 65.6, 27.2, 26.8, 25.9; IR (KBr):
3350, 3237, 2988, 1568, 1381, 1216, 1085 cm-1; MS m/z 362
(M + Na)+.
p-Trifluoromethylphenylsulfamate 13: mp 100-102 °C; 1H
NMR (DMSO-d6, 250 MHz) δ 8.2 (s, 2H), 7.85 (d, J ) 8 Hz,
2H), 7.5 (d, J ) 8 Hz, 2H); 13C NMR (DMSO-d6, 400 MHz) δ
161.5, 153.8, 127.8, 123.8, 116.4; IR (KBr) 3381, 3275, 3079,
1613, 1538, 1366, 1163, 1062 cm-1; MS m/z 240 (M - H)-.
1,2:3,4-Di-O-isopropylidene-R-D-galactopyranose 6-sulfa-
mate 25. Oil colorless (lit. syrup);26 1H NMR (DMSO-d6, 250
MHz) δ 7.5 (s, 2H), 5.5 (d, J ) 4.9 Hz, 1H), 4.6 (dd, J ) 2.4
Hz, 1H), 4.4 (dd, J ) 2.4 Hz, 1H), 3.9-4.3 (m, 4H), 1.45 (s,
3H), 1.35 (s, 3H), 1.3 (s, 6H); 13C NMR (DMSO-d6, 400 MHz)
δ 109.5, 108.9, 96.4, 71, 70.8, 70.4, 68.6, 66.6, 26.7, 26.6, 25.6,
Pentafluorophenylsulfamate 14: mp 104-106 °C; 1H NMR
(DMSO-d6, 250 MHz) 9.2 (s, 2H); 13C NMR (DMSO-d6, 400
MHz) δ 138.3, 136, 133.6, 131.6; IR (KBr) 3381, 3305, 3094,
1527, 1370, 1223, 1141 cm-1; MS m/z 262 (M - H)-.
Pentachlorophenylsulfamate 15: mp 210-213 °C (lit. mp
215 °C)21; 1H NMR (DMSO-d6, 250 MHz) δ 8.8 (s, 2H); 13C
NMR (DMSO-d6, 400 MHz) δ 151.1, 131, 122.9, 122; IR (KBr)
3381, 3283, 1535, 1381, 1190 cm-1; MS m/z 344 (M - H)-.
25; IR (CH2Cl2) 3404, 3338, 2970, 1604, 1384, 1186, 1069 cm-1
;
MS m/z 362 (M + Na)+.
Sulfamate 2627 was a gift from Prof. T. H. Maren (University
of Florida at Gainesville).
2,4,6-Trichlorophenylsulfamate 16: mp 143-144 °C; 1H
NMR (DMSO-d6, 250 MHz) δ 8.5 (s, 2H), 7.8 (s, 2H); 13C NMR
(DMSO-d6, 400 MHz) δ 149.3, 129.9, 128.9, 124.1; IR (KBr)
3373, 3275, 3071, 1565, 1445, 1377, 1235, 1193 cm-1; MS m/z
298 (M + Na)+.
CA In h ibition . 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 Lind-
skog’s group.28 Cell growth conditions were those described in

Inhibition of Cytosolic Isozymes I, II, and IX with Sulfamates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2203
ref 29, and enzymes were purified by affinity chromatography
according to the method of Khalifah et al.30 Enzyme concentra-
tions were determined spectrophotometrically at 280 nm,
utilizing a molar absorptivity of 49 mM-1 cm-1 for CA I and
54 mM-1 cm-1 for CA II, respectively, based on Mr ) 28.85
kDa for CA I, and 29.3 kDa for CA II, respectively.31,32
Refer en ces
(1) Supuran, C. T.; Scozzafava, A. Applications of carbonic anhy-
drase inhibitors and activators in therapy. Exp. Opin. Ther. Pat.
2002, 12, 217-242.
(2) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors.
Curr. Med. Chem.-Immunol., Endoc. Metab. Agents 2001, 1, 61-
97.
The cDNA of the catalytic domain of hCA IX (isolated as
described by Pastorek et al.33) was amplified by using PCR
and specific primers for the vector pCAL-n-FLAG (from
Stratagene, Milan, Italy). The obtained construct was inserted
in the pCAL-n-FLAG vector and then cloned and expressed
in Escherichia coli strain BL21-GOLD(DE3) (from Stratagene).
The bacterial cells were lysed and homogenated in a buffered
solution (pH 8) of 4 M urea and 2% Triton X-100, as described
by Wingo et al.34 The homogenate thus obtained was exten-
sively centrifuged (11 000g) 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, to remove the remaining urea and
Triton X-100. Purified CA IX inclusion bodies were denatur-
ated in 6 M guanidine hydrochloride and refolded into the
active form by snap dilution into a solution of 100 mM MES
(pH 6), 500 mM L-arginine, 2 mM ZnCl2, 2 mM EDTA, 2 mM
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 HC1, 100 mM Na2SO4, and l mM ZnCl2.
The amount of protein was determined by spectrophometric
measurements and its activity by stopped-flow measurements,
with CO2 as substrate.
(3) Supuran C. T.; Scozzafava, A. Carbonic anhydrase inhibitors and
their therapeutic potential. Exp. Opin. Ther. Pat. 2000, 10, 575-
600.
(4) Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.; Stubbs,
M.; Klebe, G. Nonaromatic sulfonamide group as an ideal anchor
for potent human carbonic anhvdrase inhibitors: Role of hydro-
gen-bonding networks in ligand binding and drug design. J . Med.
Chem. 2002, 45, 3583-3587.
(5) Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors. Part 37. Novel classes of carbonic
anhydrase inhibitors and their interaction with the native and
cobalt- substituted enzyme: kinetic and spectroscopic investiga-
tions. Eur. J . Med. Chem. 1996, 31, 1001-1010.
(6) Casini, A.; Antel, J .; Abbate, F.; Davids, S.; Waldeck, H.;
Scozzafava, A.; Schafer, S.; Supuran, C. T. Carbonic anhydrase
inhibitors: SAR and X-ray crystallographic study for the inter-
action of sugar sulfamates/sulfamides with isozymes I, II and
IV. Bioorg. Med. Chem. Lett. 2003, 13, 841-845.
(7) (a) Smith, H. J .; Nicholls, P. J .; Simons, C.; Le Lain, R. Inhibitors
of steroidogenesis as agents for the treatment of hormone-
dependent cancers. Exp. Opin. Ther. Pat. 2001, 11, 789-824.
(b) Ahmed, S.; Owen, C. P.; J ames, K.; Sampson, L.; Patel, C.
K. Review of estrone sulfatase and its inhibitors-an important
new target against hormone dependent breast cancer. Curr. Med.
Chem. 2002, 9, 263-273.
(8) (a) Woo, L. W. L.; Purohit, A.; Malini, B.; Reed, M. J .; Potter, B.
V. L. Potent active site-directed inhibition of steroid sulphatase
by tricyclic coumarin-based sulphamates. Chem. Biol. 2000, 7,
773-791. (b) Poirier, D.; Ciobanu, L. C.; Maltais, R. Steroid
sulfatase inhibitors. Exp. Opin. Ther. Pat. 1999, 9, 1083-1099.
(c) Ciobanu, L. C.; Luu-The, V.; Poirier, D. Nonsteroidal com-
pounds designed to mimic potent steroid sulfatase inhibitors.
J . Steroid Biochem. Mol. Biol. 2002, 80, 339-353. (d) Malini,
B.; Purohit, A.; Ganeshapillai, D.; Woo, L. W.; Potter, B. V.; Reed,
M. J . Inhibition of steroid sulphatase activity by tricyclic
coumrnarin sulphamates. J . Steroid Biochem. Mol. Biol. 2000,
75, 253-258.
(9) Ahmed, S.; J ames, K.; Owen, C. P. inhibition of estrone sulfatase
(ES) by derivatives of 4-[(aminosulfonyl)oxy]benzoic acid. Bioorg.
Med. Chem. Lett 2002, 12, 2391-2394. (b) Ahmed, S.; J ames,
K.; Owen, C. P.; Patel, C. K.; Sampson, L. The mechanism of
irreversible inhibition of estrone sulfatase (ES) through the
consideration of a range of methane- and amino-sulfonate-based
compounds. Bioorg. Med. Chem. Lett 2002, 12, 1279-1282. (c)
Ahmed, S.; Owen, C. P.; J ames, K.; Patel, C. K.; Patel, M. Acid
dissociation constant, a potential physicochemical factor in the
inhibition of the enzyme estrone sulfatase (ES). Bioorg. Med.
Chem. Lett. 2001, 11, 899-902. (d) Ahmed, S.; J ames, K.; Owen,
C. P.; Patel, C. K. Design, synthesis and biochemical evaluation
of AC ring mimics s novel inhibitors of the enzyme estrone
sulfatase. Bioorg. Med. Chem. Lett. 2002, 12, 1343-1346. (e)
Ahmed, S.; Owen, C. P.; J ames, K.; Patel, C. K.; Patel, M. Novel
inhibitors of the enzyme estrone sulfatase (ES). Bioorg. Med.
Chem. Lett. 2001, 11, 841-844.
Initial rates of 4-nitrophenylacetate hydrolysis catalyzed by
different CA isozymes were monitored spectrophotometrically,
at 400 nm, with a Cary 3 instrument interfaced with an IBM-
compatible PC.35 Solutions of substrate were prepared in
anhydrous acetonitrile; the substrate concentrations varied
between 2 × 10-2 and 1 × 10-6 M, working at 25 °C. A molar
absorption coefficient c of 18 400 M-1 cm-1 was used for the
4-nitrophenolate formed by hydrolysis, in the conditions of the
experiments (pH 7.40), as reported in the literature.35 Non-
enzymatic hydrolysis rates were always subtracted from the
observed rates. Triplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the paper are the mean of such results. Stock solutions of
inhibitor (1 mM) were prepared in distilled-deionized water
with 10-20% (v/v) DMSO (which is not inhibitory at these
concentrations) and dilutions up to 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and en-
zyme solutions were preincubated together for 10 min at room
temperature prior to assay, to allow for the formation of the
E-I complex. The inhibition constant KI was determined as
described by Pocker and Stone, for isozymes I and II.35 Enzyme
concentrations were 3.2 µM for CA II, 10.1 µM for CA I.
An SX.1 8MV-R Applied Photophysics stopped-flow instru-
ment has been used for assaying the CA IX CO2 hydration
activity.36 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 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.36 Stock solutions of inhibitors were prepared at a
concentration of 1-3 mM (in DMSO-water 1:1, v/v) and
dilutions up to 0.1 nM done with the assay buffer mentioned
above. Enzyme concentration was 0.1 µM, and inhibition
constants were calculated as described in ref 36.
(10) Golob, T.; Liebl, R.; von Angerer, E. Sulfamoyloxy-substituted
2-phenylindoles: Antiestrogen- based inhibitors of the steroid
sulfatase in human breast cancer cells. Bioorg. Med. Chem. 2002,
10, 3941-3953.
(11) (a) Nussbaumer, P.; Bilban, M.; Billich, A. 4,4′-Benzophenone-
O,O′-disulfamate: a potent inhibitor of steroid sulfatase. Bioorg.
Med. Chem. Lett. 2002, 12, 2093-2095. (b) Nussbaumer, P.;
Lehr, P.; Billich, A. 2-Substituted 4-(thio)chromenone 6-O-
sulfamates: potent inhibitors of human steroid sulfatase. J . Med.
Chem. 2002, 45, 4310-4320.
(12) Hernandez-Guzman, F. G.; Higashiyama, T.; Osawa, Y.; Ghosh,
D. Purification, characterization and crystallization of human
placental estrone/dehydroepiandrosterone sulfatase, a membrane-
bound enzyme of the endoplasmic reticulum. J . Steroid Biochem.
Mol. Biol. 2001, 78, 441-450.
(13) Hoffmann, R.; Happle, R. Current understanding of androgenetic
alopecia. Part I: ethiopathogenesis. Eur. J . Dermatol. 2000, 10,
319-327.
(14) Wolf, O. T.; Kirschbaum, C. Actions of dehydroepiandrosterone
and its sulfate in central nervous system: effect on cognition
and emotion in animals and humans. Brain Res. Rev. 1999, 30,
264-288.
(15) Suitters, A. J .; Shaw, S.; Wales, M. R.; Porter, J . P.; Leonard,
J .; Woodger, R.; Brand, H.; Bodmer, M.; Foulkes, R. Immune
enhancing effects of dehydroepiandrosterone and dehydroepi-
androsterone sulphate and the role of steroid sulfatase. Im-
munology 1997, 91, 314-321.
Ack n ow led gm en t. This research was financed by
a grant from the Italian CNRstarget project Biotech-
nology. Thanks are addressed to Drs. Silvia Pastorekova
and J aromir Pastorek (Slovak Institute of Virology,
Bratislava, Slovakia) for the gift of the CA IX cDNA and
helpful discussions.

2204 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Winum et al.
(16) Chegwidden, W. R.; Spencer, I. M.; Supuran, C. T. The roles of
carbonic anhydrase isozymes in cancer. In Gene Families:
Studies of DNA, RNA, Enzymes and Proteins; Xue, G., Xue, Y.,
Xu, Z., Holmes, R., Hammond, G. L., Lim, H. A., Eds.; World
Scientific: Singapore, 2001; pp 157-170.
(17) Winum, J .-Y.; Toupet, L.; Barragan, V.; Dewynter, G.; Montero,
J .-L. N-(tert-Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)-
1,4-dihydropyridin-1-ylsulfonyl]azanide: a new sulfamoylating
agent. Structure and reactivity towards amines. Org. Lett. 2002,
3, 2241-2243.
(18) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method
for sulfamoylation of a hydroxyl group. Tetrahedron Lett. 2000,
41, 7057-7051.
(19) Appel, R.; Berger, G. Hydrazinsulfonsaiire-amide, I. U¨ ber das
hydrazodisulfamid. Chem. Ber. 1958, 91, 1339-1341.
(20) Hedayatullah, M.; Guy, A. Synthese et reduction d’azidosulfates
d’aryle. Tetrahedron Lett. 1975, 29, 2455-2458.
(21) Lohaus, G. Preparation and reactions of aryloxysulfonyl isocy-
antes. Chem. Ber. 1972, 105, 2791-2799.
(22) Spillane, W. J .; Hogan, G.; McGrath, P. Aminolysis and hydroly-
sis of sulfamate esters: substantial NdS bonding in the transi-
tion state leading to N)sulfonylamines. J . Phys. Org. Chem.
1995, 8, 610-616.
(23) Eberhard, O.; Roman, M. Sulfamic acid esters. German Patent
2,418,217, 1975. [Chem. Abstr. 1976, 84, 16980q].
(24) Schwarz, S.; Thieme, I.; Richter, M.; Undeutsch, B.; Henkel, H.;
Elger, W. Synthesis of estrogen sulfamates: compounds with a
novel endocrinological profile. Steroids 1996, 61, 710-717.
(25) Ro¨mer, J .; Steinbach, J .; Kasch, H.; Scheller, D. Preparation and
Characterization of the sulphamates of estra-3,17â-diols. Rapid
conversion of 16R-fluoroestradiol into 16R-fluoroestradiol- 3,17â-
disulphamates J . Prakt. Chem. 1999, 341, 574-583.
(26) Kochetkov, N. K.; Usov, A. I.; Deryabin, V. V. Monosaccharides
XXVIII. Synthesis of monosaccharide sulfamates from salts of
monosaccharide hydrogen sulfates. J . Gen. Chem. USSR (Engl.
Transl.) 1972, 42, 2755-2757.
(36) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. J . Biol. Chem. 1971, 246, 2561-2573.
(37) (a) Wykoff, C. C.; Beasley, N.; Watson, P. H.; Turner, K. J .;
Pastorek, J .; Sibtain, A.; Wilson, G. D.; Turley, H.; Talks, K. L.;
Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P.; Harris, A. L. Hypoxia-
inducible expression of tumor-associated carbonic anhydrases.
Cancer Res. 2000, 60, 7075-7083. (b) Ho¨ckel, M.; Vaupel, P.
Tumor hypoxia: definitions and current clinical, biologic and
molecular aspects. J . Natl. Cancer Inst. 2001, 93, 266-276.
(38) (a) Loncaster, J . A.; Harris, A. L.; Davidson, S. E.; Logue, J . P.;
Hunter, R. D.; Wykoff, C. C.;Pastorek, J .; Ratcliffe, P. J .;
Stratford, I. J .; West, C. M. Carbonic anhydrase (CA IX)
expression, a potential new intrinsic marker of hypoxia: cor-
relations with tumor oxygen measurements and prognosis in
locally advanced carcinoma of the cervix. Cancer Res. 2001, 61,
6394-6399. (b) Chia, S. K.; Wykoff, C. C.; Watson, P. H.; Han,
C.; Leek, R. D.; Pastorek, J .; Gatter, K. C.; Ratcliffe, P.; Harris,
A. L. Prognostic significance of
a novel hypoxia-regulated
marker, carbonic anhydrase IX, in invasive breast carcinoma.
J . Clin. Oncol. 2001, 19, 3660-3668.
(39) (a) Wykoff, C. C.; Beasley, N.; Watson, P. H.; Campo, L.; Chia,
S. K.; English, R.; Pastorek, J .; Sly, W. S.; Ratcliffe, P. J .; Harris,
A. L. Expression of the hypoxia-inducible and tumor-associated
carbonic anhydrases in ductal carcinoma in situ of the breast.
Am. J . Pathol. 2001, 158, 1011-1019. (b) Beasley, N.; Wykoff,
C. C.; Watson, P. H.; Leek, R.; Turley, H.; Gatter, K.; Pastorek,
J .; Cox, G. J .; Ratcliffe, P. J .; Harris, A. L. Carbonic anhydrase
IX, an endogenous hypoxia marker, expression in head and neck
squamous cell carcinoma and its relationship to hypoxia, necro-
sis, and microvessel density. Cancer Res. 2001, 61, 5262-5267.
(40) (a) Koukourakis, M. I.; Giatromanolaki, A.; Sivridis, E.; Simo-
poulos, K.; Pastorek, J .; Wykoff, C. C.; Gatter, K. C.; Harris, A.
L. Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor
vascularization and resistance of squamous cell head and neck
cancer to chemoradiotherapy. Clin Cancer Res. 2001, 7, 3399-
3403. (b) Turner, K. J .; Crew, J . P.; Wykoff, C. C.; Watson, P.
H.; Poulsom, R.; Pastorek, J .; Ratcliffe, P. J .; Cranston, D.;
Harris, A. L. The hypoxia-inducible genes VEGF and CA9 are
differentially regulated in superficial vs invasive bladder cancer.
Br. J . Cancer 2002, 86, 1276-82.
(27) Lo, Y. S.; Nolan, J . C.; Maren, T. H.; Welstead, W. J .; Gripshover,
D. F.; Shamblee, D. A. Synthesis and physicochemical properties
of sulfamate derivatives as topical antglaucoma agents. J . Med.
Chem. 1992, 35, 4790-4794.
(28) Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.; J onsson,
B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure-function relations
in human carbonic anhydrase II as studied by site-directed
mutagenesis. In Carbonic anhydrase - From biochemistry and
genetics to physiology and clinical medicine; Botre`, F., Gros, G.,
Storey, B. T., Eds.; VCH: Weinheim, 1991; pp 1-13.
(29) Behravan, G.; J onsson, B. H.; Lindskog, S. Fine-tuning of the
catalytic properties of carbonic anhydrase. Studies of a Thr200-
His variant of human isoenzyme II. Eur. J . Biochem. 1990, 190,
351-357.
(30) Khalifah, R. G.; Strader, D. J .; Bryant, S. H.; Gibson, S. M.
Carbon-13 nuclear magnetic resonance probe of active site
ionization of human carbonic anhydrase B. Biochemistry 1977,
16, 2241-2247.
(31) Lindskog, S.; Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad. Sci. U.S.A. 1964, 70, 2505-2508.
(32) Steiner, H.; J onsson, B. H.; Lindskog, S. The catalytic mecha-
nism of carbonic anhydrase. Hydrogen-isotope effects on the
kinetic parameters of the human C isoenzyme. Eur. J . Biochem.
1975, 59, 253-259.
(33) Pastorek, J .; Pastorekova, S.; Callebaut, I.; Mornon, J . P.; Zelnik,
V.; Opavsky, R.; Zatovicova, M.; Liao, S.; Portetelle, D.; Stan-
bridge, E. J .; Zavada, J .; Bumy, A.; Kettmann, R. Cloning and
characterization of MN, a human tumor-associated protein with
a domain homologous to carbonic anhydrase and a putative
helix-loop-helix DNA binding segment. Oncogene 1994, 9, 2877-
2888.
(41) (a) Giatromanolaki, A.; Koukourakis, M. I.; Sivridis, E.; Pastorek,
J .; Wykoff, C. C.; Gatter, K. C.; Harris, A. L. Expression of
hypoxia-inducible carbonic anhydrase-9 relates to angiogenic
pathways and independently to poor outcome in nonsmall cell
lung cancer. Cancer Res. 2001, 61, 7992-7998. (b) Bartosova,
M.; Parkkila, S.; Pohlodek, T. J .; Karttunen, T. J .; Galbavy, S.;
Mucha, V.; Harris, A. L.; Pastorek, J .; Pastorekova, S. Expression
of carbonic anhydrase IX in breast is associated with malignant
tissues and is related to overexpression of c-erbB2. J Pathol.
2002, 197, 314-321.
(42) (a) Parkkila, S.; Rajaniemi, H.; Parkkila, A. K.; Kivela, J .;
Waheed, A.; Pastorekova, S.; Pastorek, J .; Sly, W. S. Carbonic
anhydrase inhibitor suppresses invasion of renal cancer cells in
vitro. Proc. Natl. Acad Sci. U.S.A. 2000, 97, 2220-2224. (b)
Xiang, Y.; Ma, B.; Li, T.; Yu, H. M.; Li, X. J . Acetazolamide
suppresses tumor metastasis and related protein expression in
mice bearing Lewis lung carcinoma. Acta Pharmacol. Sin. 2002,
23, 745-751.
(43) (a) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibi-
tors: synthesis of N-morpholylthiocarbonylsulfenylamino aro-
matic/heterocyclic sulfonamides and their interaction with
isozymes I, II and IV. Bioorg. Med. Chem. Lett. 2000, 10, 1117-
1120. (b) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase
inhibitors: aromatic sulfonamides and disulfonamides act as
efficient tumor growth inhibitors. J . Enzyme Inhib. 2000, 15,
597-610.
(44) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors-
Part 94. 1,3,4-Thiadiazole-2- sulfonamide derivatives as anti-
tumor agents? Eur. J . Med. Chem. 2000, 35, 867-874.
(45) Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwidden, W. R.;
Scozzafava, A. Carbonic anhydrase inhibitors: sulfonamides as
antitumor agents? Bioorg. Med. Chem. 2001, 9, 703-714.
(34) Wingo, T.; Tu, C.; Laipis, P. J .; Silverman, D. N. The catalytic
properties of human carbonic anhydrase IX. Biochem. Biophys.
Res. Commun. 2001, 288, 666-669.
(35) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed
hydrolysis of p-nitrophenyl acetate. Biochemistry 1967, 6, 668-
678.
J M021124K