Carbonic Anhydrase Inhibitors: Inhibition of Transmembrane, Tumor-Associated Isozyme IX, and Cytosolic Isozymes I and II with Aliphatic Sulfamates

Article abstract of DOI:10.1021/jm030911u

A series of aliphatic sulfamates and related derivatives incorporating cyclic/polycyclic (steroidal) 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 isozyme CA IX. The most potent CA I inhibitor was n-tetradecyl sulfamate and some (substituted)benzyl/phenethyl sulfamates (inhibition constants in the low micromolar range). Against CA II, low nanomolar inhibitors (0.7-3.4 nM) were n-decyl sulfamate and the (substituted)benzyl/phenethyl derivatives mentioned above. Effective CA II inhibition was also observed for the hydroxy/keto derivatives of dehydroepiandrosterone sulfamate. Efficient CA IX inhibitory properties, with inhibition constants in the range of 9-23 nM, were observed for the aliphatic sulfamates C₁₀-C₁₆ (with the most potent inhibitor being the n-dodecyl derivative) and the (substituted)benzyl/phenethyl sulfamates. The inhibition profile of the three investigated isozymes with this type of compound was rather different, allowing us to hope that the preparation of CA IX-selective inhibitors is possible (selectivity ratios toward hCA IX versus hCA II in the range of 5-63 has been observed for some of these compounds, whereas for the clinically used sulfonamides this parameter is in the range of 0.23-0.51). 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 chemotherapy.

Full text of DOI:10.1021/jm030911u

J . Med. Chem. 2003, 46, 5471-5477
5471
Ca r bon ic An h yd r a se In h ibitor s: In h ibition of Tr a n sm em br a n e,
Tu m or -Associa ted Isozym e IX, a n d Cytosolic Isozym es I a n d II w ith Alip h a tic
Su lfa m a tes
J ean-Yves Winum,† Daniela Vullo,‡ Angela Casini,‡ J ean-Louis Montero,† Andrea Scozzafava,‡ and
Claudiu T. Supuran*,‡
Laboratoire de Chimie Biomole´culaire, UMR 5032, Ecole Nationale Supe´rieure de Chimie de Montpellier,
Universite´ Montpellier II, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France, and Dipartimento di Chimica,
Laboratorio di Chimica Bioinorganica, Rm. 188, Universita` degli Studi di Firenze, Via della Lastruccia 3,
I-50019 Sesto Fiorentino, Firenze, Italy
Received J une 2, 2003
A series of aliphatic sulfamates and related derivatives incorporating cyclic/polycyclic (steroidal)
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 isozyme CA IX. The most potent CA I inhibitor was
n-tetradecyl sulfamate and some (substituted)benzyl/phenethyl sulfamates (inhibition constants
in the low micromolar range). Against CA II, low nanomolar inhibitors (0.7-3.4 nM) were
n-decyl sulfamate and the (substituted)benzyl/phenethyl derivatives mentioned above. Effective
CA II inhibition was also observed for the hydroxy/keto derivatives of dehydroepiandrosterone
sulfamate. Efficient CA IX inhibitory properties, with inhibition constants in the range of 9-23
nM, were observed for the aliphatic sulfamates C10-C16 (with the most potent inhibitor being
the n-dodecyl derivative) and the (substituted)benzyl/phenethyl sulfamates. The inhibition
profile of the three investigated isozymes with this type of compound was rather different,
allowing us to hope that the preparation of CA IX-selective inhibitors is possible (selectivity
ratios toward hCA IX versus hCA II in the range of 5-63 has been observed for some of these
compounds, whereas for the clinically used sulfonamides this parameter is in the range of
0.23-0.51). 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 chemotherapy.
In tr od u ction
XII are negatively regulated by von Hippel Lindau
tumor suppressor protein, and their expression in renal
cell carcinomas is related to inactivating mutation of
the VHL gene.7,8 The high catalytic activity of these two
isozymes supports their role in the acidification of tumor
microenvironment that facilitates acquisition of the
metastatic phenotype.7,8 Therefore, modulation of the
extracellular tumor pH via inhibition of CA activity
represents a promising approach to novel anticancer
therapies.6-8 Indeed, sulfonamide/sulfamate CA inhibi-
tors (CAIs) with nanomolar activity against the tumor-
associated isozyme IX were recently reported by this
group, both among the classical sulfonamide drugs such
as acetazolamide (AZA), methazolamide (MZA), and
In a previous paper1 we showed that sulfamates or
bis-sulfamates incorporating aromatic, aliphatic, poly-
cyclic (steroidal), and sugar moieties previously reported
to act as inhibitors of steroid sulfatases, enzymes
considered to be the key therapeutic targets for estrogen
dependent tumors,2 also act as very potent inhibitors
of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1)
and, more precisely, of the cytosolic isozymes CA I and
II and the transmembrane, tumor-associated isozyme
CA IX.3-5 Indeed, CAs are critical in a multitude of
physiological and physiopathological processes, includ-
ing tumorigenesis.3-8 Recently, at least two tumor-
associated CA isozymes have been identified (CA IX and
CA XII)8 among the 14 isoforms isolated in humans.3-5
Among them, CA IX shows restricted expression in
normal tissues but is tightly associated with different
types of tumors, mostly due to its strong induction by
tumor hypoxia that involves hypoxia inducible factor
type 1 (HIF-1) binding to a hypoxia response element
in the CA9 gene promoter.7,8 CA XII is present in many
normal tissues and overexpressed in some tumors, also
being induced by hypoxia, but the underlying molecular
mechanism remains undetermined. Both CA IX and CA
ethoxzolamide (EZA), as well as for derivatives designed
de novo for this target.1,9 Among the sulfamates recently
investigated as CAIs,1 most were aromatic derivatives.
The unique exception was constituted by n-octyl sulfa-
mate (n-C8H17-OSO2NH2), which was one of the most
* Correspondence 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/jm030911u CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/07/2003

5472 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Winum et al.
Ta ble 1. Inhibition Data for Derivatives 1-29 Investigated in
the Present Paper and Standard Sulfonamide CA Inhibitors,
against Isozymes I, II, and IX
acetazolamide, methazolamide, and ethoxzolamide were
also included in these assays, for comparison.
RO-SO2NH2
1-29
d
KI
selectivity ratioc
KI(hCA II)/
KI(hCA IX)
inhibitor
R group
hCA Ia hCA IIa hCA IXb
(µM) (nM) (nM)
compd
AZA
MZA
EZA
1
2
3
4
5
6
7
0.90 12 25
0.48
0.51
0.23
0.78
0.025
40
14
8
6000
5500
750
>10000 >1000
70
58
0.7
4.7
10
87
97
27
34
>1000
>1000
>1000
Me
Et
n-Pr
i-Pr
38
3.70
690
3.10
0.71
0.53
0.43
0.27
0.15
58
n-Bu
>1000
126
23
17
9
n-C5H11
n-C10H21
n-C11H23
n-C12H25
n-C14H29
n-C16H33
n-C18H37
CF3CH2
0.46
0.03
0.27
1.11
5.80
4.41
1.07
1.84
23.88
63.38
5.66
2.28
9.32
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
15
22
65
7.8
129
845
8000
120
458
335
142
279
386
633
>1000
Discu ssion
n-C6F13CH2CH2 400
n-C8F17CH2CH2 >1000 9000
Ch em istr y. The sulfamates 1-29 investigated here
were prepared by the classical procedure1 involving the
reaction of the appropriate alcohol with sulfamoyl
chloride.11-16 Some of these derivatives have been
previously reported in the literature,11-16 but no data
regarding their interaction with CA isozymes are avail-
able. Compounds 1-29 have been thoroughly character-
ized by physicochemical methods (see Experimental
Section for details) that confirmed the proposed struc-
tures.
(CF3)2CH
3.54
1580
883
5900
570
CH2dCHCH2CH2 800
CHtCCH2CH2
ClCH2CH2CH2
c-C5H9
c-C6H11
PhCH2
PhCH2CH2
p-Me-C6H4CH2
p-Ph-C6H4CH2
990
4.62
>1000 >10000 >1000
59
0.76
0.41
0.10
60
>1000
14
12
13
3.4
1.1
2.7
0.24
0.09
0.20
>1000 >10000 >1000
4500 >1000
>1000 >10000 >1000
fluorenylmethyle 38
cholesteryle
e
e
0.40
0.41
13
23
65
76
0.20
0.30
Car bon ic An h ydr ase In h ibitor y Activity. Isozym e
I. As seen from the data of Table 1, sulfamates 1-29
investigated here show a varied degree of CA I inhibi-
tory activity, as follows: (i) Derivatives 15, 20, 25, and
27 showed almost no inhibitory activity against this
isozyme, possessing KI values in the millimolar range,
whereas compounds 4, 14, 17, and 18 were also very
weak inhibitors, with KI values in the range of 400-
990 µM. It may be seen that all these compounds share
some structural features: they either contain bulky
aliphatic/steroidal moieties (such as isopropyl, cyclo-
pentyl, and cholestryl), polyfluorinated long aliphatic
chains (such as in compounds 14 and 15), or unsatur-
ated aliphatic moieties (such as in 17 and 18). These
data are rather surprising, especially when comparing
the KI values of the isopropyl and n-propyl derivatives
(4 and 3, respectively) or of the cyclopentyl and cyclo-
hexyl derivatives (20 and 21, respectively). In both
cases, the first compound of the pair is much less active
than the second one, although they are isomers (the case
of 4 and 3) or close analogues (the case of 20 and 21).
Such huge differences of activity between structurally
very similar compounds have never been detected up
to now either for sulfonamide or sulfamate CAIs,1,3-5
and they are difficult to explain at this moment, with
no X-ray structures available for adducts of such inhibi-
tors with CA isozymes. The data of the polyfluorinated
derivatives 14 and 15 are also surprising for at least
two reasons: on one hand, the corresponding perhydro
derivatives n-octyl sulfamate1 and compound 7 are very
potent CA I inhibitors, and on the other one, the
presence of polyfluorinated tails in aliphatic,25 aro-
matic,26 and heterocyclic26 sulfonamides was shown to
a
Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme, by the CO2 hydration
method. c Selectivity ratio toward hCA IX as compared to hCA II.
d
Errors in the range of 5-10% of the reported value (from three
different assays). e See structure in the text.
potent inhibitors in the entire series of investigated
compounds, with inhibition constants of 3.5 nM against
isozyme hCA I, 2.7 nM against isozyme hCA II, and 25
nM against the tumor-associated isozyme hCA IX,
respectively.1 Considering this simple compound as lead
molecule, we decided to investigate a larger series of
aliphatic sulfamates and related derivatives, to detect
potent CA I, II, and IX inhibitors belonging to this class.
Such compounds may exert a dual antitumor mecha-
nism of action, by inhibiting at least two enzymes
involved in tumorigenesis, CAs and steroid sulfatas-
es.1,10
Resu lts
Ch em istr y. The sulfamates 1-29 investigated here
as CAIs are shown in Table 1. They were prepared as
described previously,1 by reaction of the corresponding
alcohols with sulfamoyl chloride. Some of these deriva-
tives were previously reported in the literature,11-16 but
they were not investigated as CAIs until the present
study.
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 II17-22 as well as the transmembrane, tumor-
associated isozyme hCA IX, are shown in Table 1.23,24
Standard, clinically used sulfonamide CAIs, such as

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5473
lead to very potent inhibitors, typically in the low
nanomolar range. Thus, definitively, the presence of
long aliphatic polyfluorinated groups is detrimental for
the CA I inhibitory properties of sulfamate CA inhibi-
tors. (ii) Several derivatives, such as 1, 2, 11-13, 21,
and 26, acted as moderate CA I inhibitors, with inhibi-
tion constants in the range of 7.8-65 µM. These
derivatives incorporate either small aliphatic moieties
(methyl, ethyl, or trifluoroethyl), very long aliphatic
chains (C16 and C18), or cyclic moieties (cyclohexyl and
fluorenemethyl). (iii) Derivatives 3, 5, 16, and 19 also
showed good CA I inhibitory activity, with inhibition
constants in the range of 3.10-4.62 µM, being more
effective than the derivatives discussed above. These
compounds incorporate medium aliphatic chains (n-
propyl and n-butyl moieties) or halogenated groups such
as 3-chloropropyl or hexafluoro-isopropyl. (iv) The most
potent CA I inhibitors were compounds 6-10, 22-24,
and 28, 29, with inhibition constants in the range of
0.10-0.76 µM, on the same order of magnitude as the
clinically used compounds acetazolamide and meth-
azolamide. These inhibitors incorporate long aliphatic
chains (from C5 to C14), benzyl, or (substituted)phen-
ethyl moieties. It is interesting to note how the CA I
inhibitory properties augment with the elongation of the
aliphatic chain from C1 to C14 (n-alkyl chains), this last
sulfamate being one of the best CA I inhibitors in the
entire series. A further elongation to the C16 and C18
sulfamates leads to a drastic diminution of the CA I
inhibitory properties, the same situation being also true
for the polyfluorination of the alkyl chains, as mentioned
above. Another optimal substitution pattern for obtain-
ing potent CA I inhibitors of this type consists of the
benzyl/phenethyl type of sulfamates (compounds 22-
24) or the dehydroepiandrosterone derivatives 28 and
29 (Table 1).
21, 28, and 29. It may be observed that an elongation
of alkyl chain in the aliphatic sulfamates C4 and C5 is
further beneficial for the CA II inhibitory effects, these
properties varying quite linearly from C1 to C10, which
is the best (subnanomolar) inhibitor in the series (see
later in the text). The further elongation starts then to
be detrimental, with the C11-C18 derivatives gradually
acting as weaker inhibitors, but some of them (for
example 9) are of the same potency as the clinically used
sulfonamides acetazolamide, methazolamide, or ethox-
zolamide (see Table 1). The cyclohexyl sulfamate 21 and
the dehydroepiandrosterone derivatives 28 and 29 also
show good CA II inhibitory properties, especially the
alcohol 28, which is as good as acetazolamide as a hCA
II inhibitor. (iv) Very potent CA II inhibitors were
sulfamates 7, 8, and 22-24, with inhibition constants
in the range of 0.7-4.7 nM. These compounds incorpo-
rate the medium to long aliphatic chains C10 and C11
as well as the benzyl/phenethyl moieties which were also
optimal for obtaining CA I inhibitors. It is important to
note that for the aliphatic derivatives, the best CA I
inhibitor was the sulfamate C14 (10), whereas the best
CA II inhibitor was the sulfamate C10 (7). Finally, as it
will be discussed shortly, the best CA IX inhibitor was
the sulfamate C12 (9).
Isozym e IX. The inhibition profile of this isozyme
with the sulfamates 1-29 is very interesting, differing
substantially from the inhibition profiles of the cytosolic
isozymes I and II discussed above (Table 1). Thus, (i)
derivatives 1-5, 19-21, and 25-27 were ineffective CA
IX inhibitors, with KI values >1 µM. It may be observed
that these compounds incorporate either the small
aliphatic chains C1-C4, the cyclic moieties (cyclopentyl
and cyclohexyl), or the bulky biphenylbenzyl, fluoren-
emethyl, or cholesteryl moieties, which also led to weak
CA I and CA II inhibitors. (ii) Medium potency inhibi-
tors were derivatives 6 and 12-18, with inhibition
constants in the range of 120-633 nM. These inhibitors
incorporate medium aliphatic chains (C5), long aliphatic
chains (C18), polyfluorinated alkyl, and unsaturated
alkyl moieties. It is very interesting to note that
although the polyfluorinated sulfamates 14 and 15 are
quite inefficient CA I and CA II inhibitors, they show
much higher affinity for CA IX, a finding impossible to
explain at this point. Also the alkenyl/alkynyl deriva-
tives 17 and 18 showed higher affinity for CA IX than
for the cytosolic isozymes I and II, which may be
considered an encouraging result for the design of CA
IX-specific inhibitors, a goal of critical importance for
the potential use of CAIs as antitumor agents.6 (iii)
Strong CA IX inhibitors were compounds 7-11, 22-
24, 28, and 29, with inhibition constants in the range
of 9-76 nM. The SAR is rather clear in this subseries,
as best activity is observed again, as for CA II, for
sulfamates with an elongated aliphatic chain (from C10
to C12), whereas a further elongation of the molecule
(C14-C18) leads to decreased activity. The best CA IX
inhibitor is the C12 sulfamate (9, KI of 9 nM) followed
by the benzyl/phenethyl type of derivatives (22-24),
which showed KI values in the range of 12-14 nM. All
these sulfamates are at least 2 times more effective CA
IX inhibitors as compared to the clinically used sulfona-
mides acetazolamide, methazolamide, and ethoxzola-
mide, some of which were shown to inhibit tumor cell
Isozym e II. This is considered the most important
isozyme as target for obtaining inhibitors with phar-
macological applications. The sulfamates 1-29 inves-
tigated here showed a very large variation of the CA II
inhibitory properties, a feature rarely seen for a ho-
mologous series of CA inhibitors. As for isozyme I, again
a rather wide range of activities were evidenced, as
follows: (i) Weak inhibitors, with inhibition constants
in the range from 1.5 to >10 µM, were compounds 1, 2,
4, 14-16, 18, 20, and 25-27. As mentioned for isozyme
I, these weak inhibitors belong to several subcategor-
ies: small aliphatic derivatives (methyl, ethyl, and
isopropyl sulfamates); polyfluorinated aliphatic sulfa-
mates (14-16); unsaturated derivatives; and the bulky
compounds incorporating cyclopentyl, biphenylylmethyl,
fluorenylmethyl, and cholesteryl moieties, respectively.
The huge difference in activity between the cyclopentyl
and cyclohexyl sulfamates 20 and 21 are again dramatic
and difficult to explain. It may also be seen that the
elongation of the aliphatic chain already for the first
members of the series (methyl and ethyl) is beneficial
for the CA II inhibitory effects. (ii) A group of four
compounds, i.e., 3, 13, 17, and 19, showed moderate CA
II inhibitory properties, with inhibition constants in the
range of 570-883 nM. They incorporate C2-C4 aliphatic
chains, possibly substituted with halogens or unsatur-
ated. (iii) Potent inhibitors, with inhibition constants
in the range of 13-129 nM, were derivatives 5, 6, 9-12,

5474 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Winum et al.
growth in vitro and in vivo.6-8 It should also be stressed
that isozymes CA II and CA IX generally show quite
different inhibition profiles with this type of inhibitors.
For example, in addition to the compounds mentioned
above, showing higher avidity for CA IX, other inhibitors
(such as 22-24 and 28, 29) possess higher affinity for
CA II than for CA IX. Thus, in a rather simple series of
sulfamate derivatives, most of which are aliphatic,
cyclic, or hydroxy/keto derivatives of dehydroepiandro-
sterone sulfamate, quite diverse and unexpected inhibi-
tion profiles were observed for the two major cytosolic
isozymes CA I and CA II and the transmembrane,
tumor-associated isozyme CA IX.
therapies, mainly for hypoxic tumors that overexpress
CA IX, which are nonresponsive to radiation or chemo-
therapy.
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 points
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-250 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.
Selectivity tow a r d h CA IX over h CA II. As seen
from data reported here and also previous studies of our
group,1,9 both isozymes hCA II and hCA IX show high
affinity for sulfonamide inhibitors, and hence, it may
be rather difficult to detect compounds with specificity
toward one of this isozyme. In this specific case, the
target isozyme for designing novel antitumor therapies
is obviously hCA IX, and the problem is the affinity of
the potent hCA IX inhibitors also for hCA II. As seen
from data of Table 1, most inhibitors reported here
(compounds 1-7 and 19-29) showed a selectivity ratio
hCA II over hCA IX of less than 1, which means that
they act as more potent inhibitors toward isozyme II
than toward isozyme IX. This feature is also shared by
the clinically used sulfonamides acetazolamide, meth-
azolamide, and ethoxzolamide, which showed selectivity
ratios in the range of 0.48-0.23. Four of the new
inhibitors reported here, i.e., 9, 12, 13 and 17, showed
selectivity ratios in the range of 1.07-2.28, being thus
slightly better hCA IX inhibitors than hCA II inhibitors.
Finally, six other compounds, 10, 11, 14-16, and 18,
showed selectivity ratios in the range of 5.66-63.38, and
they may be considered as the most “CA IX-selective”
inhibitors reported up to now. Among them, the last four
compounds were medium-potency CA IX inhibitors, and
thus they may be considered as leads for developing new
low nanomolar CA IX-selective inhibitors, but com-
pounds 10 and 11 are low nanomolar inhibitors per se,
showing at least a 4-5 times higher affinity for CA IX
over CA II.
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 1-29 were prepared by reacting the requisite alcohol
(1 equiv) with sulfamoyl chloride (3 equiv) in N,N-dimethyl-
acetamide11 (sulfamoyl chloride was prepared from chlorosul-
fonyl isocyanate and formic acid as described in ref 12) 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 concen-
trated under vacuum. The residue was purified either by
crystallization from ether/pentane or by chromatography on
silica gel.
Meth yl su lfa m a te (1): mp 23-25 °C (lit.13,14 mp 22-24 °C,
1
25-27 °C); H NMR (DMSO-d6, 250 MHz) δ 7.45 (s, 2H), 3.7
(s, 3H); 13C NMR (DMSO-d6, 400 MHz) δ 56.3; IR (CH2Cl2)
3448, 3338, 2970, 1542, 1380, 1183 cm-1; MS ESI- m/z 110
(M - H)-.
Eth yl su lfa m a te (2): mp 37-39 °C (lit.14 mp 40-41°C); 1H
NMR (DMSO-d6, 250 MHz) δ 7.40 (s, 2H), 4.07 (q, 2H, J ) 7.4
Hz, J ) 1.4 Hz), 1.26 (t, 3H, J ) 7.4 Hz); 13C NMR (DMSO-d6,
400 MHz) δ 66.0, 15.3; IR (KBr) 3375, 3286, 1556, 1355, 1172
cm-1; MS ESI+ m/z 148 (M + Na)+, ESI- m/z 124 (M - H)-.
n-P r op yl su lfa m a te (3): mp 21-22 °C (lit.13 mp 22-23 °C);
1H NMR (DMSO-d6, 250 MHz) δ 7.38 (s, 2H), 3.97 (t, 2H, J )
6.4 Hz), 1.64 (m, 2H), 1.25 (t, 3H, J ) 7.3 Hz); 13C NMR
(DMSO-d6, 400 MHz) δ 71.3, 22.6, 10.8; IR (CH2Cl2) 3441,
3338, 2963, 1545, 1373, 1179 cm-1; MS ESI+ m/z 162 (M +
Na)+, ESI- m/z 138 (M - H)-.
Isop r op yl su lfa m a te (4): mp <18 °C; 1H NMR (DMSO-
d6, 250 MHz) δ 7.34 (s, 2H), 4.62 (m, 1H), 1.28 (d, 6H, J ) 6.2
Hz); 13C NMR (DMSO-d6, 400 MHz) δ 75.3, 23.2; IR (CH2Cl2)
3433, 3338, 2963, 1542, 1373, 1241, 1179 cm-1; MS ESI+ m/z
162 (M + Na)+, ESI- m/z 138 (M - H)-.
Con clu sion s. A series of sulfamates incorporating
aliphatic, cyclic, and steroidal moieties in their mol-
ecules has been synthesized and assayed as inhibitors
of the cytosolic isozymes CA I and II and the trans-
membrane, tumor-associated isozymes CA IX. The most
potent CA I inhibitor was n-tetradecyl sulfamate and
some (substituted)benzyl/phenethyl sulfamates. Against
CA II, low-nanomolar inhibitors (0.7-3.4 nM) were
n-decyl sulfamate and the (substituted)benzyl/phenethyl
derivatives mentioned above. Good CA II inhibition was
also observed for the hydroxy/keto derivatives of dehy-
droepiandrosterone sulfamate. Efficient CA IX inhibi-
tory properties, with inhibition constants in the range
of 9-23 nM, were observed for the aliphatic sulfamates
C10-C16 (with the best inhibitor being the n-dodecyl
derivative), and the (substituted)benzyl/phenethyl sulfa-
mates mentioned above. The inhibition profile of the
three investigated isozymes with this type of compound
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
n -Bu tyl su lfa m a te (5): mp 26-27 °C (lit.14 mp 27-28 °C);
1H NMR (DMSO-d6, 250 MHz) δ 7.38 (s, 2H), 4 (t, 2H, J ) 6.4
Hz), 1.6 (m, 2H), 1.36 (m, 2H), 0.9 (t, 3H, J ) 7.2 Hz); 13C
NMR (DMSO-d6, 400 MHz) δ 69.5, 31.1, 19.1, 14.2; IR (CH2-
Cl2) 3441, 3330, 2977, 1545, 1377, 1179 cm-1; MS ESI+ m/z
176 (M + Na)+, ESI- m/z 152 (M - H)-.
1
n -P en tyl su lfa m a te (6): mp 37-39 °C; H NMR (DMSO-
d6, 250 MHz) δ 7.4 (s, 2H), 4 (t, 2H, J ) 6.4 Hz), 1.64 (m, 2H),
1.3(m, 4H), 0.88 (t, 3H, J ) 6.6 Hz); 13C NMR (DMSO-d6, 400
MHz) δ 69.8, 28.8, 28.1, 22.4, 14.6; IR (KBr) 3375, 3279, 2933,
2860, 1545, 1465, 1347, 1175, 1040 cm-1; MS ESI+ m/z 190
(M + Na)+, ESI- m/z 166 (M - H)-.
n -Decyl su lfa m a te (7): mp 69-71 °C (lit.16 mp 81.2-83.6
°C); 1H NMR (DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 4 (t, 2H, J )
6.4 Hz), 1.6 (m, 2H), 1.28 (s, 14H), 0.86 (t, 3H, J ) 6 Hz); 13C
NMR (DMSO-d6, 400 MHz) δ 69.8, 32.1, 29.8, 29.6, 29.4, 29.2,
25.9, 22.9, 14.7; IR (KBr) 3367, 3279, 2919, 2845, 1542, 1468,
1344, 1175 cm-1; MS ESI+ m/z 260 (M + Na)+, ESI- m/z 236
(M - H)-.
n -Un d ecyl su lfa m a te (8): mp 76-77 °C (lit.16 mp 77.8-
1
79.6 °C); H NMR (DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 4.0 (t,

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5475
2H, J ) 6.4 Hz), 1.65 (m, 2H), 1.25 (m, 14H), 0.85 (t, 3H, J )
5.7 Hz); 13C NMR (DMSO-d6, 400 MHz) δ 69.7, 32.2, 29.9, 29.8,
29.6, 29.4, 29.2, 25.9, 22.9, 14.7; IR (KBr) 3375, 3286, 2926,
2845, 1545, 1468, 1351, 1179 cm-1; MS ESI- m/z 250 (M -
H)-.
3367, 3264, 3147, 2963, 2867, 1534, 1443, 1340, 1179, 1153,
1069 cm-1; MS ESI+ m/z 188 (M + Na)+, ESI- m/z 164 (M -
H)-.
1
Cycloh exyl su lfa m a te (21): yellow oil; H NMR (DMSO-
d6, 250 MHz) δ 7.4 (s, 2H), 4.4 (m, 1H), 1.2 (m, 10H); 13C NMR
n -Dod ecyl su lfa m a te (9): mp 79-80 °C (lit.16 mp 82-84
(DMSO-d6, 400 MHz) δ 79.5, 32.6, 25.3, 23.9; IR (CH2Cl2) 3433,
1
3338, 2941, 1380, 1183 cm-1
ESI-
Ben zyl su lfa m a te (22): mp 74-76 °C; H NMR (DMSO-
d6, 250 MHz) δ 7.65 (s, 2H), 7.4 (s, 5H), 5.1 (s, 2H); 13C NMR
(DMSO-d6, 400 MHz) δ 137.5, 129.4, 71.1; IR (KBr) 3360, 3279,
1542, 1457, 1340, 1183 cm-1; MS ESI+ m/z 210 (M + Na)+,
ESI- m/z 186 (M - H)-.
; MS ESI+ m/z 202 (M + Na)+,
°C); H NMR (DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 3.95 (t, 2H,
m/z 178 (M - H)-.
J ) 6.4 Hz), 1.6 (m, 2H), 1.3 (m, 16H), 0.85 (t, 3H, J ) 6 Hz);
13C NMR (DMSO-d6, 400 MHz) δ 69.7, 32.2, 29.95, 29.9, 29.85,
29.8, 29.6, 29.4, 29.1, 25.9, 22.9, 14.7; IR (KBr) 3375, 3286,
2919, 2845, 1542, 1468, 1351, 1179 cm-1; MS ESI+ m/z (M +
Na)+, ESI- m/z 264 (M - H)-.
1
n -Tetr a d ecyl su lfa m a te (10): mp 84-85 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 3.9 (t, 2H, J ) 6.4 Hz), 1.6
(m, 2H), 1.35 (m, 20H), 0.85 (t, 3H, J ) 5.9 Hz); 13C NMR
(DMSO-d6, 400 MHz) δ 69.7, 32.2, 30, 29.95, 29.9, 29.8, 29.6,
29.4, 29.2, 25.9, 23, 14.7; IR (KBr) 3375, 3286, 2911, 2852,
1542, 1468, 1351, 1183 cm-1; MS ESI- m/z 292 (M - H)-.
n -Hexa d ecyl su lfa m a te (11): mp 88-89 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 4 (t, 2H, J ) 6.4 Hz), 1.6
(m, 2H), 1.3 (m, 26H), 0.85 (t, 3H, J ) 6 Hz); 13C NMR (DMSO-
d6, 400 MHz) δ 69.7, 32.2, 30, 29.95, 29.9, 29.8, 29.6, 29.4, 29.2,
25.9, 22.9, 14.7; IR (KBr) 3367, 3286, 2919, 2845, 1538, 1468,
1347, 1183 cm-1; MS ESI- m/z 320 (M - H)-.
2-P h en yleth yl su lfa m a te (23): oil; 1H NMR (DMSO-d6,
250 MHz) δ 7.5 (s, 2H), 7.3 (m, 5H), 4.25 (t, 2H, J ) 6.8 Hz),
3 (t, 2H, J ) 6.6 Hz); 13C NMR (DMSO-d6, 400 MHz) δ 138.2,
129.7, 129.2, 127.3, 70, 35.3; IR (CH2Cl2) 3441, 3338, 2963,
1545, 1377, 1183 cm-1; MS ESI+ m/z 224 (M + Na)+, ESI- m/z
200 (M - H)-.
p-Meth ylben zyl su lfa m a te (24): mp 23-25 °C (lit.13,14 mp
22-24 °C, 25-27 °C); 1H NMR (DMSO-d6, 250 MHz) δ 7.6 (s,
2H), 7.3 (d, 2H, J ) 8.2 Hz), 7.22 (d, 2H, J ) 8.3 Hz), 5.0 (s,
2H), 2.3 (s, 3H); 13C NMR (DMSO-d6, 400 MHz) δ 138.9, 132.4,
129.9, 129.4, 71.1, 21.6; IR (KBr) 3375, 3279, 3139, 1545, 1336,
1179, 1069 cm-1; MS ESI+ m/z 224 (M + Na)+, ESI- m/z 200
(M - H)-.
n -Octa d ecyl su lfa m a te (12): mp 91-92 °C; 1H NMR
(CDCl3-DMSO-d6, 250 MHz) δ 7.15 (s, 2H), 3.95 (t, 2H, J )
6.5 Hz), 1.6 (m, 2H), 1.2 (m, 30H), 0.8 (t, 3H, J ) 6.3 Hz); 13
C
p-Bip h en ylylm eth yl su lfa m a te (25): mp >220 °C; 1H
NMR (DMSO-d6, 250 MHz) δ 7.8-7.4 (m, 11H), 5.12 (s, 2H);
13C NMR (DMSO-d6, 400 MHz) δ 141.3, 140.4, 134.7, 129.9,
129.8, 128.5, 127.6, 127.5; IR (KBr) 3367, 3279, 1545, 1487,
1358, 1175, 1076, 1051 cm-1; MS ESI+ m/z 286 (M + Na)+,
ESI- m/z 262 (M - H)-.
NMR (CDCl3-DMSO-d6, 400 MHz) δ 69.8, 32.2, 30, 29.95, 29.9,
29.8, 29.6, 29.4, 29.2, 25.9, 23, 14.6; IR (KBr) 3375, 3286, 2911,
2852, 1538, 1472, 1351, 1183 cm-1; ESI- m/z 348 (M - H)-.
1H,1H-P er flu or oeth yl su lfa m a te (13): mp 51-53 °C (lit.15
mp 50-51 °C); 1H NMR (DMSO-d6, 250 MHz) δ 8 (s, 2H), 4.64
(q, 2H, J ) 8.2 Hz, 17 Hz); 13C NMR (DMSO-d6, 400 MHz) δ
119.6-127.8, 65.1-64; IR (KBr) 3397, 3294, 1531, 1366, 1311,
1271, 1179, 1047 cm-1; MS ESI+ m/z 202 (M + Na)+, ESI- m/z
178 (M - H)-.
F lu or en ylm eth yl su lfa m a te (26): mp 150-152 °C; 1H
NMR (DMSO-d6, 250 MHz) δ 7.9 (d, 2H, J ) 6.4 Hz), 7.7 (d,
2H, J ) 6.8 Hz), 7.6 (s, 1H), 7.5-7.3 (m, 4H), 4.42 (s, 2H); 13
C
NMR (DMSO-d6, 400 MHz) δ 143.9, 141.6, 128.7, 128.1, 125.9,
121.1, 70.6, 47.1; IR (KBr) 3375, 3264, 1534, 1450, 1344, 1164
cm-1; MS ESI+ m/z 298 (M + Na)+.
1H,1H,2H,2H-P er flu or ooctyl su lfa m a te (14): mp 59-61
°C; 1H NMR (DMSO-d6, 250 MHz) δ 7.66 (s, 2H), 4.3 (t, 2H, J
) 5.6 Hz), 2.75 (m, 2H); 13C NMR (DMSO-d6, 400 MHz) δ 106-
121, 61.6, 30.6; IR (KBr) 3397, 3286, 1560, 1369, 1238, 1186,
1139, 1076 cm-1; MS ESI+ m/z 465 (M + Na)+, ESI- m/z 442
(M - H)-.
Ch olester yl su lfa m a te (27): mp 167-168 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 5.35 (m, 1H), 3.3 (m, 1H),
1(s, 3H), 0.9 (s, 3H), 0.8 (d, 6H, J ) 6.6 Hz), 0.6 (s, 3H), 0.6-
1.8 (m, 28H); 13C NMR (DMSO-d6, 400 MHz) δ 140.1, 123.2,
80.2, 56.9, 56.5, 50.3, 42.6, 39.2, 37.4, 36.7, 36.6, 36.2, 32.2,
29, 28.6, 28.3, 24.2, 23.4, 23.1, 19.7, 19.3, 12.4; IR (KBr) 3375,
3272, 2948, 1465, 1347, 1197, 1161 cm-1; MS ESI+ m/z 488
(M + Na)+, ESI- m/z 464 (M - H)-.
1H,1H,2H,2H-P er flu or od ecyl su lfa m a te (15): mp 119-
1
121 °C; H NMR (DMSO-d6, 250 MHz) δ 7.6 (s, 2H), 4.28 (t,
2H, J ) 5.8 Hz), 2.65 (m, 2H); 13C NMR (DMSO-d6, 400 MHz)
δ 122-107, 61.5, 30.5; IR (KBr) 3404, 3301, 1556, 1369, 1208,
1146 cm-1; MS ESI+ m/z 565 (M + Na)+, ESI- m/z 542 (M -
H)-.
7-r-Hyd r oxyd eh yd r oep ia n d r oster on e su lfa m a te (28):
mp 131-133 °C; 1H NMR (DMSO-d6, 400 MHz) δ 7.2 (s, 2H),
5.3 (d, 1H, J ) 4.3 Hz), 4.1 (m, 1H), 3.55 (t, 1H, J ) 3.9 Hz),
2.2 (m, 3H), 1.8 (m, 5H), 1.6 (m, 1H), 1.45-1.15 (m, 7H), 0.9
(m, 2H), 0.75 (s, 3H), 0.6 (s, 3H); 13C NMR (DMSO-d6, 400
MHz) δ 172.2, 142.6, 126.9, 80.1, 60.5, 47.2, 45.3, 42.5, 39.1,
37.7, 37.6, 36.9, 36.1, 31.8, 28.9, 22.1, 21.6, 20.4, 18.5, 13.8;
IR (KBr) 3492, 3338, 3176, 3066, 2948, 1721, 1575, 1465, 1377,
1267, 1172 cm-1; MS ESI+ m/z 406 (M + Na)+, ESI- m/z 382
(M - H)-.
1H-P er flu or oisop r op yl su lfa m a te (16): mp 50-52 °C; 1H
NMR (DMSO-d6, 250 MHz) δ 8.54 (s, 2H), 6.20 (m, 2H); 13C
NMR (DMSO-d6, 400 MHz) δ 117.1-125.6, 70.8-72.1; IR
(KBr) 3397, 3294, 2985, 1538, 1377, 1355, 1307, 1245, 1194,
1106, 1069 cm-1; MS ESI- m/z 446 (M - H)-.
1
Bu t-3-en yl su lfa m a te (17): yellow oil; H NMR (DMSO-
d6, 250 MHz) δ 7.45 (s, 2H), 5.80 (m, 1H), 5.1 (m, 2H), 4.05
(m, 2H), 2.4 (m, 2H); 13C NMR (DMSO-d6, 400 MHz) δ 134.7,
118.3, 68.7, 33.5; IR (CH2Cl2) 3433, 3338, 3073, 2977, 1549,
1373, 1241, 1186 cm-1; MS ESI+ m/z 174 (M + Na)+, ESI- m/z
150 (M - H)-.
7-Oxodeh ydr oepian dr oster on e su lfam ate (29): mp 180-
1
182 °C; H NMR (DMSO-d6, 400 MHz) δ 7.45 (s, 2H), 5.7 (s,
1H), 4.3 (m, 1H), 2.65 (m, 3H), 2.5 (t, 1H, J ) 11.2 Hz), 2.4
(dd, 1H, J ) 19.2 Hz, J ) 8.26 Hz), 2 (m, 3H), 1.5-1.8 (m,
7H), 1.2 (m, 5H), 0.8 (s, 3H); 13C NMR (DMSO-d6, 400 MHz)
δ 219.8, 201.1, 165.7, 126.6, 78.8, 50.1, 48.1, 45.8, 44.6, 38.8,
38.7, 36.2, 35.8, 31.2, 28.6, 24.6, 20.9, 17.6, 14.3; IR (KBr) 3367,
3213, 3095, 2948, 1725, 1673, 1377, 1300, 1183 cm-1; MS ESI+
m/z 404 (M + Na)+.
Bu t-3-yn yl su lfam ate (18): mp 51-53 °C; 1H NMR (DMSO-
d6, 250 MHz) δ 7.55 (s, 2H), 4.05 (t, 2H, J ) 6.2 Hz), 2.85 (s,
1H), 2.58 (t, 2H, J ) 6 Hz); 13C NMR (DMSO-d6, 400 MHz) δ
81.2, 73.5, 67.5, 19.5; IR (KBr) 3375, 3272, 2970, 1545, 1362,
1175 cm-1; MS ESI+ m/z 172 (M + Na)+, ESI- m/z 148 (M -
H)-.
3-Ch lor op r op yl su lfa m a te (19): yellow oil; 1H NMR
(DMSO-d6, 250 MHz) δ 7.45 (s, 2H), 4.15 (t, 2H, J ) 6 Hz),
3.70 (t, 2H, J ) 6.4 Hz), 2.10 (m, 2H); 13C NMR (DMSO-d6,
400 MHz) δ 66.6, 42.1, 32.2; IR (CH2Cl2) 3433, 3338, 2963,
1542, 1377, 1179 cm-1; MS ESI+ m/z 196 (M + Na)+, ESI- m/z
172 (M - H)-.
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.17 Cell growth conditions were as described,18 and
enzymes were purified by affinity chromatography according
to the method of Khalifah et al.19 Enzyme concentrations 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
Cyclop en tyl su lfa m a te (20): mp 58-60 °C; 1H NMR
(DMSO-d6, 250 MHz) δ 7.4 (s, 2H), 4.9 (m, 1H), 1.9-1.4 (m,
8H); 13C NMR (DMSO-d6, 400 MHz) δ 83.7, 33.4, 23.6; IR (KBr)

5476 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Winum et al.
29.3 kDa for CA II, respectively.20,21 The cDNA of the catalytic
(6) (a) Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C. T.
Anticancer and antiviral sulfonamides. Curr. Med. Chem. 2003,
10, 925-953. (b) Casini, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. Sulfonamides and sulfonylated derivatives as
anticancer agents. Curr. Cancer Drug Targets 2002, 2, 55-75.
(7) (a) 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 (b) 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. (c) Ho¨ckel, M.; Vaupel, P. Tumor hypoxia: Defini-
tions and current clinical, biologic and molecular aspects. J . Natl.
Cancer Inst. 2001, 93, 266-276.
(8) Pastorekova, S.; Pastorek, J . Cancer-related carbonic anhydrase
isozymes and their inhibition. In Carbonic anhydrase, its inhibi-
tors and activators; Supuran, C. T., Scozzafava, A., Conway J .,
Eds.; Taylor & Francis: London & New York, 2003; in press.
(9) (a) Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J .; Scozzafava,
A.; Pastorekova, S.; Supuran, C. T. Carbonic anhydrase inhibi-
tors. Inhibition of the tumor-associated isozyme IX with aromatic
and heterocyclic sulfonamides. Bioorg. Med. Chem. Lett. 2003,
13, 1005-1009. (b) Ilies, M. A.; Vullo, D.; Pastorek, J .; Scoz-
zafava, A.; Ilies, M.; Caproiu, M. T.; Pastorekova, S.; Supuran,
C. T. Carbonic anhydrase inhibitors. Inhibition of tumor-
associated isozyme IX with halogenosulfanilamide and halo-
genophenylaminobenzolamide derivatives. J . Med. Chem. 2003,
46, 2187-2196. (c) Franchi, M.; Vullo, D.; Gallori, E.; Pastorek,
J .; Russo, A.; Scozzafava, A.; Pastorekova, S.; Supuran, C. T.
Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes
I and II and transmembrane, cancer-associated isozyme IX with
lipophilic sulfonamides. J . Enzymol. Inhib. Med. Chem. 2003,
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J .; Leese, M. P.; Ganeshapillai, D.; Woo, L. W. L.; Potter, B. V.
L.; Reed, M. J . Inhibition of carbonic anhydrase II by steroidal
and nonsteroidal sulphamates. Biochem. Biophys. Res. Commun.
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(11) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method
for sulfamoylation of a hydroxyl group. Tetrahedron Lett. 2000,
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(12) Appel, R.; Berger, G. Hydrazinsulfonsau¨re-amide, I. U¨ ber das
hydrazodisulfamid. Chem. Ber. 1958, 91, 1339-1341.
(13) Spillane, W. J .; Taheny, A. P.; Kearns, M. Versatile synthesis
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(14) Appel, R.; Senkpiel, W.; Sulfamates. Z. Anorg. U. Allg. Chem.
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J . Steroid Biochem. Mol. Biol. 2002, 80, 429-440.
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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
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Storey, B. T., Eds.; VCH: Weinheim, 1991; pp 1-13.
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catalytic properties of carbonic anhydrase. Studies of a Thr200-
His variant of human isoenzyme II. Eur. J . Biochem. 1990, 190,
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Carbon-13 nuclear magnetic resonance probe of active site
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anhydrase. Proc. Natl. Acad Sci. U.S.A. 1964, 70, 2505-2508.
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2877-2888.
domain of hCA IX (isolated as described by Pastorek et al.22
)
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 E. coli strain BL21-GOLD(DE3) (from
Stratagene). The bacterial cells were lysed and homogenized
in a buffered solution (pH 8) of 4 M urea and 2% Triton X-100,
as described by Wingo et al.23 The homogenate thus obtained
was extensively 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 denaturated 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, and 1 mM oxidized
glutathione. Active hCA IX was extensively dialyzed into a
solution of 10 mM Hepes (pH 7.5), 10 mM Tris HCl, 100 mM
Na2SO4, and 1 mM ZnCl2. The protein was further purified
by sulfonamide affinity chromatography,23 the amount of
enzyme was determined by spectrophometric measurements,
and its activity was determined by stopped-flow measure-
ments, with CO2 as substrate.24 The activity of this preparation
was identical to that reported by Wingo et al.,23 with kcat/KM
of 55 µM-1 s-1. An SX.18MV-R Applied Photophysics stopped-
flow instrument was used for assaying the CA CO2 hydration
activity assays.24 Phenol red (at a concentration of 0.2 mM)
was used as indicator, working at the absorbance maximum
of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2-
SO4 (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.24 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 enzyme solutions were preincubated together for
10 min at room temperature prior to assay, to allow for the
formation of the E-I complex. Triplicate experiments were
done for each inhibitor concentration, and the values reported
throughout the paper are the mean of such results. Enzyme
concentrations were 1.0 µM for hCA I, 10 nM for hCA II, and
0.1 µM for hCA IX. Inhibition constants were calculated as
described.24
Ack n ow led gm en t. This research was financed in
part by a grant from the Italian CNRstarget project
Biotechnology. 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.
Refer en ces
(1) Winum, J .-Y.; Vullo, D.; Casini, A.; Montero, J .-L.; Scozzafava,
A.; Supuran, C. T. Carbonic anhydrase inhibitors: Inhibition of
cytosolic isozymes I and II and the transmembrane, tumor-
associated isozyme IX with sulfamates including EMATE also
acting as steroid sulfatase inhibitors. J . Med. Chem. 2003, 46,
2197-2204.
(2) (a) Smith, H. J .; Nicholls, P. J .; Simons, C.; Le Lain, R. Inhibitors
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