Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with bis-sulfamates

Article abstract of DOI:10.1016/j.bmcl.2004.11.058

A series of bis-sulfamates incorporating aliphatic, aromatic, or betulinyl moieties in their molecules was obtained by reaction of the corresponding diols/diphenols with sulfamoyl chloride. The library of bis-sulfamates thus obtained was tested for the inhibition of three physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K_I s in the range of 79 nM-16.45 μM, hCA II with K_I s in the range of 6-643 nM, and hCA IX with K_I s in the range of 4-5400 nM. Several low nanomolar hCA IX inhibitors were detected, such as 1,8-octylene-bis-sulfamate or 1,10-decylene-bis-sulfamate (K_I s in the range of 4-7 nM), which showed good selectivity ratios (in the range of 3.50-3.85) for hCA IX over hCA II inhibition. The most selective hCA IX inhibitor was phenyl-1,4-dimethylene-bis-sulfamate (K _I of 61.6 nM), which was a 10.43 times better hCA IX than hCA II inhibitor. These derivatives are interesting candidates for the development of novel antitumor therapies targeting hypoxic tumors, since hCA IX is highly overexpressed in such tissues, and its presence is correlated with bad prognosis and unfavorable clinical outcome.

Full text of DOI:10.1016/j.bmcl.2004.11.058

Bioorganic & Medicinal Chemistry Letters 15 (2005) 579–584
Carbonic anhydrase inhibitors: synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes
I, II, and IX with bis-sulfamates
Jean-Yves Winum,a,b Silvia Pastorekova,c Lydia Jakubickova,c Jean-Louis Montero,b
Andrea Scozzafava,a Jaromir Pastorek,c Daniela Vullo,a
Alessio Innocentia and Claudiu T. Supurana,*
a
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
b
´ ´
Universite Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032, Ecole Nationale Superieure de Chimie de Montpellier,
´
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
cInstitute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 842 45 Bratislava, Slovak Republic
Received 3 September 2004; revised 8 November 2004; accepted 18 November 2004
Available online 16 December 2004
Abstract—A series of bis-sulfamates incorporating aliphatic, aromatic, or betulinyl moieties in their molecules was obtained by reac-
tion of the corresponding diols/diphenols with sulfamoyl chloride. The library of bis-sulfamates thus obtained was tested for the
inhibition of three physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II,
and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with KI s in the range of
79 nM–16.45 lM, hCA II with KI s in the range of 6–643 nM, and hCA IX with KI s in the range of 4–5400 nM. Several low nanomolar
hCA IX inhibitors were detected, such as 1,8-octylene-bis-sulfamate or 1,10-decylene-bis-sulfamate (KI s in the range of 4–7 nM),
which showed good selectivity ratios (in the range of 3.50–3.85) for hCA IX over hCA II inhibition. The most selective hCA IX
inhibitor was phenyl-1,4-dimethylene-bis-sulfamate (KI of 61.6 nM), which was a 10.43 times better hCA IX than hCA II inhibitor.
These derivatives are interesting candidates for the development of novel antitumor therapies targeting hypoxic tumors, since hCA IX
is highly overexpressed in such tissues, and its presence is correlated with bad prognosis and unfavorable clinical outcome.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
hypoxia.3 Tumor hypoxia is the result of the abnormal
process of neoplastic growth and crucially depends on
oxygen/nutrients supply from the host.3 Thus, changes
in tumor metabolism and microenvironment connected
with adaptation of cells to hypoxia are important com-
ponents of tumor progression.3,4 Hypoxic conditions
elicit cellular responses designed to improve cell
oxygenation and survival by means of several mecha-
nisms such as neoangiogenesis, improved glycolysis
and enhanced energy production, as well as upregula-
tion of molecules related to cell survival/apoptosis.3
The most important molecule regulating the mammalian
response to hypoxia is the heterodimeric protein hypoxia-
inducible factor 1 (HIF-1), which in turn up-regulates
genes involved in adaptation responses to hypoxic
conditions.3 Two such genes encode for the transmem-
brane hCA isozymes IX and XII, containing extracellu-
lar enzyme active sites. These hCAs appear to
participate in tumorigenetic processes via their ability
In previous work from these laboratories,1,2 we showed
that the simple sulfamates possessing the general for-
mula R–OSO2NH2 (R = aliphatic C5–C16 saturated or
unsaturated chains; simple aromatic-phenyl or substi-
tuted-phenyls––as well as polycyclic, mainly steroidal
moieties) act as efficient inhibitors of several isozymes
of carbonic anhydrase (CA, EC 4.2.1.1), among which
is also the tumor-associated, transmembrane one CA
IX, which is upregulated by hypoxia and overexpressed
in many tumor types.3–5 Indeed, it has only recently
been discovered that invasive growth and metastatic
spread of many tumors are closely associated with a re-
duced oxygenation of the tissue, and more precisely with
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.058

580
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 579–584
to catalyze hydration of CO2 to bicarbonate and pro-
tons, regulating in this way the intratumoral pH.4 In
addition, hCA IX, possessing a unique N-terminal
domain, has a capacity to perturb E-cadherin mediated
cell–cell adhesion via interaction with b-catenin and may
potentially contribute to tumor invasion.4 hCA 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 in-
volves HIF-1 binding to a hypoxia response element in
the CA9 gene promoter.3,4 hCA IX was proposed to
serve as a marker of tumor hypoxia and its predictive
and prognostic potential has been demonstrated in
numerous clinical studies (reviewed in Ref. 4) hCA XII
is present in many normal tissues and overexpressed in
some tumors.4 It is also induced by hypoxia, but the
underlying molecular mechanism remains undeter-
mined. Both hCA IX and hCA XII are negatively regu-
lated by von Hippel Lindau tumor suppressor protein
and their expression in renal cell carcinomas is related
to inactivating mutation of VHL gene.4 The high cata-
lytic activity of these two CA isoforms supports their
role in acidification of tumor microenvironment that
facilitates acquisition of metastatic phenotypes.3,4
Therefore, modulation of extracellular tumor pH via
inhibition of CA activity represents a promising ap-
proach to anticancer therapy.4–6 Sulfonamide CA inhibi-
tors were shown to compromise tumor cell proliferation
O
Cl
S
O
17
NH2
O
O
S
HO
OH
( )n
O
S
NH2
H2N
O
( )n
O
O
DMA
n = 2 - 16
n = 2 - 16
16a-i
1-9
Scheme 1.
CA inhibitors,1,2 by reacting the appropriate diol/diphe-
nol with sulfamoyl chloride in N,N-dimethylacetamide
as solvent.14–16 Sulfamoyl chloride has been generated
from chlorosulfonyl isocyanate and formic acid.16 Thus,
for the aliphatic compounds 1–9, reaction of the diols
16a–i with sulfamoyl chloride 17 afforded the bis-sulfa-
mates in high yields (Scheme 1).
By using the appropriate diphenol/diol, compounds 10–
15 were prepared in a similar manner as 1–9 discussed
above.17
3. Carbonic anhydrase inhibition
Inhibition data against three physiologically relevant
isozymes, that is, the cytosolic isozymes hCA I and II
and the membrane-bound, tumor-associated isozyme
hCA IX (all of them of human origin) with sulfamates
1–15 as well as the standard, clinically used CA inhibi-
tors acetazolamide AAZ, methazolamide MZA, ethoxzo-
lamide EZA, dichlorophenamide DCP, dorzolamide
DZA, brinzolamide BRZ, and indisulam IND are shown
in Table 1.18
and invasion in vitro and improve the effect of conven-
4–7
tional chemotherapy in vivo.
However, their precise
targets are not known in detail at this moment, but it
is presumed that these two tumor-associated CA iso-
zymes, that is, hCA IX and XII, may represent impor-
tant enzymes for targeting cancer cells, by an
unconventional therapeutic approach.4–7
We recently showed that hCA IX was a druggable tar-
get.8–13 In previous works we have explored the design
of potent and possibly selective sulfamate or sulfon-
amide hCA IX inhibitors belonging to various chemical
classes.1,2,8–13 Among the best hCA IX inhibitors de-
tected so far were some aliphatic and aromatic simple
sulfamates, such as the n-C8–C16 alkylsulfamates, or
the benzyl/phenethyl sulfamates, which showed inhibi-
tion constants against hCA IX in the range of
9–25 nM.1,2 In these studies, only one aromatic bis-sulfa-
mate and a steroidal-bis-sulfamate have been investi-
gated for the inhibition of hCA IX, more precisely
5-n-pentyl-resorcinyl-bis-sulfamate, which showed effec-
tive hCA IX inhibitory properties (KI of 43 nM),1 and
estradiol-3,17b-disulfamate (KI of 58 nM),1 whereas no
such aliphatic compounds have been studied. Thus, we
decided to extend our investigations to this type of com-
pounds, and here we present the synthesis and hCA I, II,
and IX inhibitory properties of a series of bis-sulfamates
incorporating both aliphatic and aromatic as well as
polycyclic moieties.
The following SAR should be noted from data of Table
1: (i) sulfamates 1–15 inhibited the three investigated CA
isozymes with different affinities, most of them behaving
as medium potency hCA I inhibitors (KI s in the range
of 79 nM–16.45 lM), whereas against hCA II the KI s
varied in the range of 6–643 nM, and against hCA IX
KI s were in the range of 4–5400 nM; (ii) for hCA I,
the best inhibitors in this series were resorcinyl-bis-sulfa-
mate 13, the catechol-bis-sulfamate derivative 11, the
betulinyl-bis-sulfamate 14 as well as the other resorcinyl
derivative 15, which showed inhibition constants in the
range of 79–105 nM, being much more effective com-
pared to most of the clinically used sulfonamides (except
ethoxzolamide and indisulam). Among the n-aliphatic
derivatives, good activity was shown by the C6 and C7
bis-sulfamates 2 and 3 (KI s in the range of 136–
145 nM), which was strongly diminished for the shorter
(1), longer (4–7) or branched compounds (8 and 9). Phen-
yl-1,4-dimethylene-bis-sulfamate 10 was the most inef-
fective hCA I inhibitor in this series of derivatives
(KI > 20 lM); (iii) against hCA II, the best inhibitors
were the aliphatic derivatives 1–4 (n-C4–C8 chain), the
aromatic ones 11–13, betulinyl-bis-sulfamate 14 as well
as estradiol-3,17b-bis-sulfamate (previously investi-
gated),1 which showed inhibition constants in the range
of 5–15 nM, of the same order of magnitude as the
2. Chemistry
Bis-sulfamates 1–15 investigated here were prepared as
reported earlier for the monosulfamates investigated as

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 579–584
581
Me
N
N
N
N
N
MeCONH
SO2NH2
S
MeCON
SO2NH2
S
EtO
S
SO2NH2
AAZ
SO2NH2
MZA
EZA
NHEt
NHEt
S
SO2NH2
SO2NH2
N
S
MeO(CH2)3
S
Cl
SO2NH2
S
Me
O
O
Cl
O
O
DCP
DZA
BRZ
SO2NH2
H
N
O
OSO2NH2
S
Cl
N
O
H
H2NO2SO
IND
14
Table 1. Inhibition data for bis-sulfamates 1–15 investigated in the present paper and standard sulfonamide CA inhibitors, against isozymes I, II, and IX24
H2NO2S–O–R–O–SO2NH2
1–15
Inhibitor
R
KI* (nM)
Selectivity ratio
KI(hCA II)/KI(hCA IX)
hCA Ia
hCA IIa
hCA IXb
AAZ
MZA
EZA
DCP
DZA
BRZ
IND
1
900
780
12
14
25
27
0.48
0.52
0.23
0.76
0.17
0.08
0.62
0.0018
0.63
0.79
3.85
3.50
2.63
1.07
0.31
0.18
10.43
0.62
0.21
0.09
0.52
1.76
0.08
25
8
38
34
50
1200
50,000
NT
9
52
37
3
15
31
548
24
(CH2)4
(CH2)6
9.7
8.3
8.0
14.6
24.5
84.2
563
417
380
643
6.0
15.0
9.4
14.7
76.3
5.0
5400
13.2
10.1
4.0
2
136
145
3
(CH2)7
(CH2)8
4
378
890
5
(CH2)10
(CH2)12
(CH2)16
7.0
6
1350
16,450
6490
872
32.1
525
1320
2020
61.6
9.6
7
8
MeCHCHMe
CH2–CMe2–CH2
4-CH2–C6H4–CH2
4-t-Bu-1,2-phenylene
4-Me-1,2-phenylene
1,3-C6H4
9
10
>20,000
93
267
11
12
70.8
100
28.0
43.1
58.0
13
14
79
108
Betulinylc
d
15
5-Pentyl-1,3-C6H3
105
6000
Estradiol-3,17-disulfamated
a Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme, by the CO2 hydration method.
c See structure in the text.
d From Ref. 1.
* Errors in the range of 5–10% of the reported value (from three different assays).

582
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 579–584
clinically used sulfonamides AAZ–IND (Table 1). It is
interesting to note that the C-5 monosulfamate previo-
usly investigated behaved as a medium potency hCA II
inhibitor (KI of 58 nM),2 whereas the C10–C12 mono-
sulfamates were very effective inhibitors (KI s in the range
of 0.7–10 nM).2 Thus, the presence of a second sulfa-
mate moiety is beneficial for increasing hCA II inhibi-
tory properties for compounds with a medium length
chain (of four to eight carbon atoms) as compared to
the corresponding monosulfamates, whereas for longer
chain derivatives, the monosulfamates are better inhibi-
tors than the corresponding bis-sulfamates (the entire
series of C1–C18 monosulfamates has been investigated
in the previous work).2 Another group of bis-sulfamates,
such as 5, 6, and 15 showed medium potency hCA II
inhibitory properties, with KI s in the range of 24.5–
84.2 nM. The remaining compounds, possessing long
alkyl chains (7), branched chains (8 and 9) as well as
phenyl-1,4-dimethylene-bis-sulfamate 10 showed weak
hCA II inhibitory effects (KI s in the range of 380–
643 nM); (iv) against hCA IX, the best inhibitors were
the aliphatic derivatives 2–5 incorporating C6–C10
chains, and the aromatic bis-sulfamate 11 possessing a
bulky tert-butyl tail, which showed KI s in the range
4.0–13.2 nM, being much more effective than the clini-
cally used sulfonamides AAZ–IND (KI s in the range
of 24–50 nM). Another group of bis-sulfamates includ-
ing 6, 10, 12–15, and estradiol-3,17b-bis-sulfamate be-
haved as medium potency hCA IX inhibitors, with KI s
in the range of 32–100 nM. Except 6, all these com-
pounds are either aromatic or polycyclic. The most inef-
fective hCA IX inhibitors were the C16 bis-sulfamate 7,
and the branched derivatives 8 and 9 (KI s in the range
of 525–2020 nM). Thus, SAR becomes rather clear: best
hCA IX inhibition is achieved with aliphatic sulfamates
incorporating C6–C10 n-alkyl chains (with the optimum
of activity for the C8-bis-sulfamate) as well as with 1,2-
phenylene-bis-sulfamates also possessing a bulky ali-
phatic moiety (the presence of the tert-butyl moiety in
11 leads to a 7-fold increase of hCA IX inhibitory prop-
erties as compared to the corresponding methyl-substi-
tuted compound 12); (v) since both isozymes II and IX
show good affinity for most of the sulfamates investi-
gated here, the selectivity of these inhibitors for the
tumor-associated isozyme IX is a critical issue for the
drug design of such derivatives. As seen from data of
Table 1, the clinically used sulfonamides AAZ–IND
show better hCA II than hCA IX inhibitory properties,
with selectivity ratios in the range of 0.08–0.62. Many of
the bis-sulfamates investigated here, such as 1–3, 8, 9,
11–14, and estradiol-3,17b-bis-sulfamate also show
selectivity ratios <1. However, six of the new derivatives
have this parameter >1. Thus, the potent aliphatic hCA
IX inhibitors 4–6 showed selectivity ratios in the range
of 2.63–3.50, whereas compound 10 was a 10.43 times
a better hCA IX than hCA II inhibitor (but it is a med-
ium potency hCA IX inhibitor). Compounds 7 and 15
are only slightly better hCA IX than hCA II inhibitors
(selectivity ratios of 1.07–1.76).
each isozyme is best inhibited by a certain structural
type of such compounds, with hCA I being best inhib-
ited by resorcinyl-1,3-bis-sulfamate (KI of 79 nM),
hCA II by estradiol-3,17b-bis-sulfamate, 4-t-butyl-1,2-
phenylene-bis-sulfamate and the aliphatic C4–C7 bis-
sulfamates (KI s in the range of 5–9.7 nM) and CA IX
by the C7–C10 aliphatic bis-sulfamates (KI s in the range
of 4-10 nM). Some of these derivatives are also more
selective CA IX compared to CA II inhibitors (selectiv-
ity ratios in the range of 2.63–10.43). Since CA IX is
highly overexpressed in many hypoxic tumors, such
compounds represent valuable candidates for the poten-
tial development of novel non-classical anti-tumor
therapies.
Acknowledgements
This research was financed in part by a 6th Framework
Program of the European Union (EUROXY project).
J.Y.W. is grateful to CSGI, University of Florence
and University of Montpellier II for a travel grant to
Florence.
References and notes
1. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
2197–2204.
2. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
5471–5477.
ˇ
´
´
´
´
ˇ
´
3. (a) Svastova, E.; Hulıkova, A.; Rafajova, M.; ZatÕovicova,
´
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
´
Lett. 2004, 577, 439–445; (b) Ho¨pfl, G.; Ogunshola, O.;
Gassmann, M. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 2004, 286, R608–R623.
4. (a) Pastorekova, S.; Pastorek, J. Cancer-Related Carbonic
Anhydrase Isozymes. In Carbonic Anhydrase––Its Inhibi-
tors and Activators; Supuran, C. T., Scozzafava, A.,
Conway, J., Eds.; CRC: Boca Raton, FL, USA, 2004;
pp 253–280; (b) Pastorekova, S.; Parkkila, S.; Pastorek, J.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19,
199–229.
5. (a) Carbonic Anhydrase––Its Inhibitors and Activators;
Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC:
Boca Raton, FL, 2004; pp 1–376, and references cited
therein; (b) Winum, J.-Y.; Scozzafava, A.; Montero, J.-L.;
Supuran, C. T. Expert Opin. Ther. Pat. 2004, 14, 1273–
1308.
6. (a) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2000, 10, 575–600; (b) Supuran, C. T.; Scozzafava, A.
Expert Opin. Ther. Pat. 2002, 12, 217–242; (c) Supuran, C.
T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23,
146–189; (d) Scozzafava, A.; Mastrolorenzo, A.; Supuran,
C. T. Expert Opin. Ther. Pat. 2004, 14, 667–702.
7. (a) Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran,
C. T. Curr. Med. Chem. 2003, 10, 925–953; (b) Teicher, B.
A.; Liu, S. D.; Liu, J. T.; Holden, S. A.; Herman, T. S.
Anticancer Res. 1993, 13, 1549–1556; (c) Casini, A.;
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Curr.
Cancer Drug Targets 2002, 2, 55–75; (d) Owa, T.;
Yoshino, H.; Okauchi, T.; Yoshimatsu, K.; Ozawa, Y.;
Sugi, N. H.; Nagasu, T.; Koyanagi, N.; Kitoh, K. J. Med.
Chem. 1999, 42, 3789–3799.
In conclusion, we report the synthesis and CA I, II, and
IX inhibitory properties for a series of aliphatic, aro-
matic, and polycyclic bis-sulfamates. SAR showed that

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 579–584
583
8. (a) Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.;
Scozzafava, A.; Pastorekova, S.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2003, 13, 1005–1009; (b) Ilies, M. A.;
Vullo, D.; Pastorek, J.; Scozzafava, A.; Ilies, M.; Caproiu,
M. T.; Pastorekova, S.; Supuran, C. T. J. Med. Chem.
2003, 46, 2187–2196.
9. (a) Franchi, M.; Vullo, D.; Gallori, E.; Pastorek, J.;
Russo, A.; Scozzafava, A.; Pastorekova, S.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2003, 18, 333–338; (b)
Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozza-
fava, A.; Pastorekova, S.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2003, 18, 403–406.
10. Weber, A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran, C.
T.; Scozzafava, A.; Klebe, G. J. Med. Chem. 2004, 47,
550–557.
11. (a) Pastorekova, S.; Casini, A.; Scozzafava, A.; Vullo, D.;
Pastorek, J.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2004, 14, 869–873; (b) Casey, J. R.; Morgan, P. E.; Vullo,
D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J.
Med. Chem. 2004, 47, 2337–2347; (c) Vullo, D.; Scozza-
fava, A.; Pastorekova, S.; Pastorek, J.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 2351–2356.
1,10-Decylene-bis-sulfamate 5: mp 111–112 ꢁC (lit. 102–
105 ꢁC)16; 1H NMR (DMSO-d6, 400 MHz): d 7.4 (s, 4H), 4
(t, 4H, J = 6.4 Hz), 1.6 (m, 4H), 1.3 (m, 12H); 13C NMR
(DMSO-d6, 400 MHz): d 69.4, 29.3, 28.9, 28.7, 25.5; MS
ESI+ m/z 355 (M+Na)+, 687 (2M+Na)+. ESIÀ m/z 331
(MÀH)À, 663 (2MÀH)À.
1,12-Dodecylene-bis-sulfamate 6: mp 121–122 ꢁC; 1H
NMR (DMSO-d6, 400 MHz): d 7.4 (s, 4H), 4 (t, 4H,
J = 6.4 Hz), 1.6 (m, 4H), 1.3 (m, 16H); 13C NMR (DMSO-
d6, 400 MHz): d 69.4, 29.45, 29.4, 29, 28.7, 25.5; MS ESI+
m/z 383 (M+Na)+. ESIÀ m/z 359 (MÀH)À, 719 (2MÀH)À.
1,16-Hexadecylene-bis-sulfamate 7: mp 126–127 ꢁC; 1H
NMR (DMSO-d6, 400 MHz): d 7.4 (s, 4H), 4 (t, 4H,
J = 6.4 Hz), 1.6 (m, 4H), 1.3 (m, 24H); 13C NMR (DMSO-
d6, 400 MHz): d 69.4, 29.56, 29.54, 29.48, 29.42, 29, 28.8,
25.5; MS ESI+ m/z 439 (M+Na)+, 855 (2M+Na)+. ESIÀ
m/z 415 (MÀH)À, 831 (2MÀH)À.
2,3-Butylene-bis-sulfamate 8: mp 153–155 ꢁC (lit. 115–
1
117 ꢁC)16; H NMR (DMSO-d6, 400 MHz): d 7.5 (s, 4H),
4.5 (m, 2H), 1.35 (d, 6H, J = 6 Hz); 13C NMR (DMSO-d6,
400 MHz): d 77.7, 16.8; MS ESI+ m/z 271 (M+Na)+, 519
(2M+Na)+. ESIÀ m/z 247 (MÀH)À, 495 (2MÀH)À.
2,2-Dimethyl-1,3-propylene-bis-sulfamate 9: mp <30 ꢁC;
1H NMR (DMSO-d6, 400 MHz): d 7.5 (s, 4H), 3.8 (s,
4H), 1 (s, 6H); 13C NMR (DMSO-d6, 400 MHz): d 73.6,
35.2, 21.2; MS ESI+ m/z 285 (M+Na)+, 547 (2M+Na)+.
ESIÀ m/z 261 (MÀH)À, 523 (2MÀH)À.
12. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
13. Supuran, C. T.; Casini, A.; Scozzafava, A. Development
of Sulfonamide Carbonic Anhydrase Inhibitors (CAIs). In
Carbonic Anhydrase––Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC: Boca
Raton, FL, USA, 2004; pp 67–148.
Phenyl-1,4-dimethylene-bis-sulfamate 10: mp >220 ꢁC
1
decomp.; H NMR (DMSO-d6, 400 MHz): d 7.6 (s, 4H),
14. Okada, M.; Iwashita, S.; Koizumi, N. Tetrahedron Lett.
2000, 41, 7051–7057.
15. Appel, R.; Berger, G. Chem. Ber. 1958, 91, 1339–
1341.
7.45 (s, 4H), 5.1 (s, 4H); 13C NMR (DMSO-d6, 400 MHz):
d 135.5, 128.9, 70.3; MS ESI+ m/z 319 (M+Na)+. 4-tert-
Butyl-1,2-phenylene-bis-sulfamate 11: mp 127–128 ꢁC; 1H
NMR (DMSO-d6, 400 MHz): d 8.1 (s, 4H), 7.5 (m, 1H),
7.4 (m, 2H), 1.4 (s, 9H); 13C NMR (DMSO-d6, 400 MHz):
d 150.4, 142, 140, 124.5, 123.3, 121.1, 34.9, 31.5; MS ESI+
m/z 347 (M+Na)+, 670 (2M+Na)+. ESIÀ m/z 323
(MÀH)À, 647 (2MÀH)À.
16. Hirsch, A. F.; Kasulanis, C.; Kraft, L.; Mallory, R. A.;
Powell, G.; Wong, B. J. Med. Chem. 1981, 24, 901–903.
17. General procedure for the preparation of sulfamates 1–15.
Sulfamates 1–15 were prepared by reacting the commer-
cially available appropriate diol or biphenol (1 equiv) with
sulfamoyl chloride (5 equiv) in N,N-dimethylacet-
amide.1,14 Sulfamoyl chloride was prepared from chloro-
sulfonyl isocyanate and formic acid as described in Ref.
15. 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.
4-Methyl-1,2-phenylene-bis-sulfamate 12: mp 149–151 ꢁC;
1H NMR (DMSO-d6, 400 MHz): d 8.1 (s, 2H), 8.05
(s, 2H), 7.35 (d, 1H, J = 3.8 Hz), 7.3 (d, 1H, J = 1.6 Hz),
7.15 (dd, 1H, J = 3.8 Hz, J = 1.6 Hz); 13C NMR
(DMSO-d6, 400 MHz): d 142.3, 140.3, 137.3, 127.8, 124,
123.6, 20.9; MS ESI+ m/z 305 (M+Na)+, 587 (2M+Na)+.
ESIÀ m/z 280 (MÀH)À, 562 (2MÀH)À.
Resorcinyl-bis-sulfamate 13: mp 117–118 ꢁC; 1H NMR
(DMSO-d6, 400 MHz): d 8.1 (s, 4H), 7.25 (m, 3H), 6.7 (m,
1H); 13C NMR (DMSO-d6, 400 MHz): d 158.9, 130.8, 121,
116.8; MS ESI+ m/z 291 (M+Na)+, 559 (2M+Na)+. ESIÀ
m/z 267 (MÀH)À, 534 (2MÀH)À.
1,4-Butylene-bis-sulfamate 1: mp 134–135 ꢁC (lit. 126–
129 ꢁC);16 1H NMR (DMSO-d6, 400 MHz): d 7.45 (s, 4H),
4 (s, 4H), 1.7 (s, 4H); 13C NMR (DMSO-d6, 400 MHz): d
68.9, 25.3; MS ESI+ m/z 271 (M+Na)+, 519 (2M+Na)+.
ESIÀ m/z 247 (MÀH)À, 495 (2MÀH)À.
Betulinyl-bis-sulfamate 14: mp 116–117 ꢁC; 1H NMR
(DMSO-d6, 400 MHz): d 7.4 (s, 2H), 7.35 (s, 2H), 4.7 (s,
1H), 4.6 (s, 1H), 4.1 (d, 1H, J = 9.3 Hz), 4 (dd, 1H,
J = 14.1 Hz, J = 9.6 Hz), 3.75 (d, 1H, J = 9.2 Hz), 2.95 (s,
1H), 2.8 (s, 1H), 2.45 (m, 1H), 2–0.8 (m, 42H); 13C NMR
(DMSO-d6, 400 MHz): d 170, 150.1, 110.5, 88, 67.5, 55.4,
50, 48.6, 47.6, 46.4, 42.7, 38.6, 37.9, 37.6, 36.9, 34.9, 34,
29.3, 28, 26.8, 25.1, 24.3, 21.8, 21.2, 20.8, 19.7, 19.2, 18.2,
16.6, 16.2, 16.1, 15.1, 14.8; MS ESI+ m/z 624 (M+Na)+.
ESIÀ m/z 599 (MÀH)À.
1,6-Hexylene-bis-sulfamate 2: mp 128–129 ꢁC; 1H NMR
(DMSO-d6, 400 MHz): d 7.4 (s, 4H), 4 (t, 4H, J = 5.8 Hz),
1.65 (m, 4H), 1.35 (m, 4H); 13C NMR (DMSO-d6,
400 MHz): d 69.4, 28.6, 25; MS ESI+ m/z 299 (M+Na)+,
575 (2M+Na)+. ESIÀ m/z 275 (MÀH)À, 551 (2MÀH)À.
1,7-Heptylene-bis-sulfamate 3: mp 94–95 ꢁC; 1H NMR
(DMSO-d6, 400 MHz): d 7.4 (s, 4H), 4 (t, 4H, J = 6.5 Hz),
1.65 (m, 4H), 1.3 (m, 6H); 13C NMR (DMSO-d6,
400 MHz): d 69.4, 28.7, 28.4, 25.4; MS ESI+ m/z 313
(M+Na)+, 603 (2M+Na)+. ESIÀ m/z 289 (MÀH)À, 579
(2MÀH)À.
5-Pentyl-resorcinyl-bis-sulfamate 15 was prepared as
described in Ref. 1.
18. 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 earlier by
Behravan, G.; Jonsson, B. H.; Lindskog, S. Eur. J.
Biochem. 1990, 190, 351–357, and enzymes were purified
by affinity chromatography according to the method of
Khalifah et al.19 Enzyme concentrations were determined
1,8-Octylene-bis-sulfamate 4: mp 97–98 ꢁC; 1H NMR
(DMSO-d6, 400 MHz):
d 7.4 (s, 4H), 4 (t, 4H,
J = 6.4 Hz), 1.6 (m, 4H), 1.3 (m, 8H); 13C NMR
(DMSO-d6, 400 MHz): d 69.4, 28.8, 28.7, 25.4; MS ESI+
m/z 327 (M+Na)+, 631 (2M+Na)+. ESIÀ m/z 303
(MÀH)À, 607 (2MÀH)À.
spectrophotometrically at 280 nm, utilizing
a molar

584
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 579–584
absorptivity of 49 mMÀ1 cmÀ1 for CA
I
and
obtained enzyme was the same as the one previously
reported,1,2 but the yields in active protein were 5–6 times
higher per liter of culture medium.
54 mMÀ1 cmÀ1 for CA II, respectively, based on
Mr = 28.85 kDa for CA I, and 29.3 kDa for CA II,
respectively.20,21 A variant of the previously published1,2,11
CA IX purification protocol has been used for obtaining
high amounts of hCA IX needed in these experiments. The
catalytic domain of hCA IX cloned into pGEX-4T-1
vector (details described previously in Zatovicova et al.22)
was expressed in Escherichia coli BL21 Codon Plus
bacterial strain (Stratagene). The bacterial cells were
resuspended in the lysis buffer (10 mM Tris pH 7.5,
1 mM EDTA pH 8, 150 mM NaCl and 0.2% Triton X-
100) and incubated for 20 min on ice with lysozyme
(Sigma) in final concentration 1 mg/mL. To suspension
was added COMPLETE cocktail of protease inhibitors
(Roche) and bacterial cells were sonicated (5 · 30 s). The
obtained lysate was centrifuged for 30 min at 10,000 rpm,
at +4 ꢁC and the supernatant was then applied to a
prepacked Glutathione Sepharose 4B column (Amer-
sham), extensively washed with the lysis buffer followed
by phosphate buffer-saline, pH 7.4, and the fusion protein
was eluted with a buffer consisting of 5 mM reduced
glutathione in 50 mM Tris–HCl, pH 8.0. Finally the GST
part of the fusion protein was cleaved with thrombin
(Sigma). The advantage of this method over the previous
one,1,2,11 is that CA IX is not precipitated in inclusion
bodies from which it has to be isolated by denaturing–
renaturing in the presence of high concentrations of urea,
when the yields in active protein were rather low, and the
procedure much longer. The obtained CA IX was further
purified by sulfonamide affinity chromatography,19 the
amount of enzyme being determined by spectrophotomet-
ric measurements and its activity by stopped-flow exper-
iments, with CO2 as substrate.23 The specific activity of the
19. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S.
M. Biochemistry 1977, 16, 241–2247.
20. Lindskog, S.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A.
1964, 70, 2505–2508.
21. Steiner, H.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem.
1975, 59, 253–259.
ˇ
ˇ
´
´
´
´
22. ZatÕovicova, M.; Tarabkova, K.; Svastova, E.; Gibaduli-
´ˇ
´
nova, A.; Mucha, V.; Jakubıckova, L.; Biesova, Z.;
Rafajova, M.; Gut, M. O.; Parkkila, S.; Parkkila, A. K.;
´
´
´
Waheed, A.; Sly, W. S.; Horak, I.; Pastorek, J.; Pastore-
´
kova, S. J. Immunol. Methods 2003, 282, 117–134.
23. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573.
24. An SX.18MV-R Applied Photophysics stopped-flow
instrument has been used for assaying the CA catalyzed
CO2 hydration activity.23 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 con-
stant 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.23
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 15 min at room temperature prior to assay, in
order to allow for the formation of the E–I complex.
Triplicate experiments were done for each inhibitor
concentration, and the values reported throughout the
paper are the mean of such results.