Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties

Article abstract of DOI:10.1016/j.bmcl.2004.07.087

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, 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_Is in the range of 75-136 nM, hCA II with K_Is in the range of 13-278 nM, and hCA IX with K_Is in the range of 0.12-549 nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K_Is in the range 0.12-0.34 nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.

Full text of DOI:10.1016/j.bmcl.2004.07.087

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5427–5433
Carbonic anhydrase inhibitors: synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II,
and IX with sulfonamides incorporating 1,2,4-triazine moieties
Vladimir Garaj,a Luca Puccetti,b Giuseppe Fasolis,b Jean-Yves Winum,a,c
Jean-Louis Montero,c Andrea Scozzafava,a 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
bSan Lazzaro Hospital, Division of Urology, Via P. Belli 26, 12051 Alba, Cuneo, Italy
c
´ ´
Universite Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032, Ecole Nationale Superieure de Chimie de Montpellier,
´
8rue de l’Ecole Normale, 34296 Montpellier Cedex, France
Received 2 June 2004; revised 7 July 2004; accepted 28 July 2004
Available online 28 August 2004
Abstract—A series ofbenzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of
cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfon-
amides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic
alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three
physiologically relevant carbonic anhydrase (CA, 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 KIs in the range of75–136nM, hCA II with KIs in the
range of13–278nM, and hCA IX with KIs in the range of0.12–549nM. The first hCA IX-selective inhibitors were thus detected, as
the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity
ratios for CA IX over CA II inhibition in the range of166–706. Furthermore, some ofthese compounds have subnanomolar affinity
for hCA IX, with KIs in the range 0.12–0.34nM. These derivatives are interesting candidates for the development of novel uncon-
ventional anticancer strategies targeting the hypoxic areas oftumors. Clear renal cell carcinoma, which is the most lethal urologic
malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading can-
didate ofsuch novel therapies.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
prove cell oxygenation and survival by means of
several mechanisms such as neoangiogenesis, improved
glycolysis, and enhanced energy production, as well as
upregulation ofmolecules related to cell survival/apop-
tosis.1 Nonterminally differentiated proliferating hyp-
oxic cells are significant for the disease outcome
because oftheir resistance to radiotherapy and possibly
other cytotoxic treatments.1c The most important mole-
cule 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.1 Two such
genes encode for the transmembrane carbonic anhyd-
rase (CA, EC 4.2.1.1) isozymes CA IX and CA XII, con-
taining extracellular enzyme active sites. These CAs
appear to participate in tumorigenetic processes via their
It has only recently been discovered that invasive growth
and metastatic spread ofmany tumors types are closely
associated with hypoxia.1 Tumor hypoxia is the result of
the imbalance between neoplastic cells growth and the
recruitment ofnew blood supply ofr the maintenance
1
ofoxygen and nutrients, termed tumor angiogenesis.
Thus, changes in tumor metabolism and microenviron-
ment connected with adaptation ofcells to hypoxia are
1,2
important components oftumor progression. Hyp-
oxic conditions elicit cellular responses designed to im-
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.087

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V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
ability to catalyze hydration ofCO to bicarbonate and
pounds have been used in the design ofinhibitors with
various medicinal chemistry applications.15 Here we
decided to investigate another approach for obtaining
such derivatives, taking advantage ofthe versatile (and
facile) chemistry involving cyanuric chloride 1 (2,4,6-tri-
chloro-1,3,5-triazine),14 and its reactions with various
nucleophiles. Indeed, DÕAlelio and White14 reported al-
ready in 1959 the reaction of 1 with sulfanilamide, as
well as the subsequent substitution ofthe remaining
chlorine atoms with alcohols and amines (ammonia),
but the small number ofprepared derivatives has never
been investigated for their CA inhibitory properties.
Here we re-explored these derivatives reported in the
previous study,14 and extend the series ofinvestigated
compounds, including some sulfanilamide analogues
in our work (Scheme 1).
2
protons, regulating in this way the intratumoral pH.2 In
addition, CA IX, possessing a unique N-terminal do-
main, has a capacity to perturb E-cadherin mediated
cell–cell adhesion via interaction with b-catenin and
may potentially contribute to tumor invasion.2 CA IX
shows restricted expression in normal tissues but is
tightly associated with different types oftumors, 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.1,2 CA IX was proposed to serve
as a marker oftumor hypoxia and its predictive and
prognostic potential has been demonstrated in clinical
studies (reviewed in Ref. 2) CA XII is expressed in many
normal tissues and overexpressed in some tumors.2 It is
also induced by hypoxia, but the underlying molecular
mechanism remains undetermined. Both CA IX and
CA XII are negatively regulated by von Hippel Lindau
(VHL) tumor suppressor protein and their expression
in clear renal cell carcinomas (RCC) is related to inacti-
vating mutation of VHL gene.2 Necrosis and oxidative
stress also regulate CA IX expression in RCC.2c The
high catalytic activity ofthese two CA isoofrms sup-
ports their role in acidification oftumor microenviron-
ment, which enhances tumor growth and progression
Reaction ofcyanuric chloride 1 with sulfanilamide 2,
homosulfanilamide 3 or 4-aminoethyl-benzenesulfon-
amide 4, in a 1:1 molar ratio, afforded the dichlorotri-
azine-substituted sulfonamides 5–7 (ofwhich only
5
has been reported previously).14 Reaction ofthese key
intermediates 5–7 with water afforded derivatives 8–10,
whereas with methylamine, they were converted to the
bis-dimethylamino-triazino-substituted compounds 11–
13 (Scheme 1). Alternatively, the chlorine atoms of 5–7
may be substituted by alkoxy moieties, by reaction with
alcohols in the presence ofsodium hydroxide, when
derivatives 14–19, incorporating methoxy and ethoxy
moieties have been obtained, ofwhich 14 and 17 have
been reported earlier14 (Scheme 1).16
2–4
with earlier acquisition ofmetastatic phenotypes.
Therefore, modulation of extracellular tumor pH via
inhibition ofCA activity represents a promising novel
approach to anticancer therapy.2–4 Sulfonamide CA
inhibitors were shown to compromise in vitro tumor
cells proliferation and invasiveness, such as renal cell
carcinoma cell lines and improve the effect ofconven-
tional chemotherapy in vivo.2–5 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, CA IX and XII, may represent important
molecules for targeting cancer cells, by an unconven-
tional therapeutic approach.2–5
3. CAinhibition
Data of Table 1 show CA I, II, and IX inhibition with
the compounds reported here oftypes 5–19, the parent
sulfonamides used in the synthesis of types 2–4, as well
as clinically used CAIs, such as acetazolamide AAZ,
methazolamide MZA, ethoxzolamide EZA, dichloro-
phenamide DCP, dorzolamide DZA, and brinzolamide
BRZ.17 Indisulam (E7070) IND, an antitumor sulfon-
amide in phase II clinical trials for which we recently
demonstrated potent CA inhibitory properties has also
been included for comparison in this study.5d,10,25 Fur-
thermore, the X-ray crystal structure of IND in adduct
with isozyme hCA II has recently been reported by
our group.10
In previous work from this laboratory, we showed that
CA IX is a druggable target.6–9 In such papers we have
explored the design ofpotent and preefrably selective
sulfamate/sulfonamide CA IX inhibitors belonging to
various chemical classes.6–9 It was thus observed among
others that unlike for other CA isozymes (such as for
example CA I, II, or V among others)10–13 aromatic
sulfonamides are generally better CA IX inhibitors,
as compared to the heterocyclic derivatives. Thus, it
appeared ofinterest to explore other chemical scaffolds
incorporating aromatic (benzene) sulfonamide deriva-
tives that led to the best CAIs targeting CA IX reported
up to now.13 In this work we consider a new such
approach, taking advantage ofthe afcile and versatile
chemistry ofcyanuric chloride (2,4,6-trichloro-1,3,5-tri-
azine),14 which was used to generate a library ofaro-
matic sulfonamides possessing various substituents at
the triazine moiety.
The following SAR should be noted from data of Table
1: (i) against the cytosolic, slow isozyme hCA I, the three
parent sulfonamides 2–4 act as weak inhibitors, with KIs
in the range of21–28 lM, whereas the new derivatives
incorporating triazine moieties, oftypes 5–19 show deci-
sively better inhibitory properties, with KIs in the range
of75–136nM. Basically, all these derivatives show a
rather similar inhibitory activity against this isozyme,
with rather small variations when different substitution
patterns at the triazine ring or the length ofthe spacer be-
tween this and the benzenesulfonamide moieties are con-
sidered. As a whole, the best hCA I inhibitors were the
amines 11–13, the chloroderivative 7, and the ethoxy-
derivative 19, but as mentioned above, differences of
2. Chemistry
Benzenesulfonamide derivatives show well-known CA
inhibitory properties, and a wide range ofsuch com-

V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
5429
NH2
O
S
O
NH2
O
S
O
( )n
N
NH2
H2O or MeNH2
NH
O
S
O
( )n
N
NH2
Cl
X
N
X
2: n = 0
3: n = 1
4: n = 2
( )n
N
N
N
11: n = 0, X = NHMe
12: n = 1, X = NHMe
13: n = 2, X = NHMe
8: n = 0, X = OH
9: n = 1, X = OH
10: n = 2, X = OH
NH
Cl
N
Cl
N
1
NH2
Cl
N
Cl
O
S
O
5: n = 0
6: n = 1
7: n = 2
ROH/NaOH
( )n
N
NH
N
RO
OR
N
17: n = 0, R = Et
18: n = 1, R = Et
19: n = 2, R = Et
14: n = 0, R = Me
15: n = 1, R = Me
16: n = 2, R = Me
Scheme 1.
Table 1. Inhibition data for derivatives 2–19 investigated in the present paper and standard sulfonamide CAIs, against isozymes I, II, and IX17
Compound
KI* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA IXb
KI (hCA II)/KI (hCA IX)
AAZ
MZA
EZA
DCP
DZA
BRZ
IND
2
250
50
12
14
8
25
27
0.48
0.52
25
34
50
0.23
0.76
1200
50,000
nt
38
9
52
37
0.17
0.08
3
31
15
300
170
160
106
13
21
278
130
98
23
15
27
36
112
21
20
84
23
24
0.62
1.02
28,000
25,000
21,000
120
136
75
294
103
33
3
1.65
4.84
4
5
0.15
124
138
142
360
549
153
1.3
1.1
150
155
162
0.12
0.34
12
706.67
0.10
0.15
6
7
8
103
94
1.95
0.36
9
10
96
83
0.17
0.15
11
12
90
88
11.53
24.54
0.24
13
14
85
15
16
116
105
78
0.72
0.13
17
18
166.67
247.05
1.91
110
82
19
* Errors in the range of5–10% ofthe reported value (from three different assays).
a Human cloned isozyme, by the CO2 hydration method.
b Catalytic domain ofhuman, cloned isozyme, by the CO hydration method; nt = not tested.
2

5430
V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
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
O
N
S
Cl
N
H
O
IND
activity are unexpectedly small. Thus, these compounds
are weaker hCA I inhibitors as compared to clinically
used derivatives methazolamide, ethoxzolamide, and
indisulam, but more effective as compared to acetazol-
amide, dichlorophenamide, or dorzolamide (Table 1);
(ii) against isozyme hCA II, one ofthe physiologically
most relevant CAs, the parent sulfonamides 2–4 show
medium potency activity, with KIs in the range of160–
300nM, whereas the new derivatives 5–19 show a better
inhibitory power, with KIs in the range of13–278nM. It
may be observed that the increase ofinhibitory power
was not very significant in some cases, comparing
the parent sulfonamides and the triazinyl-substituted
derivatives, such as, for example, in the case of the hyd-
roxy-derivatives 8–10, which show only slightly better
inhibitory activity as compared to the corresponding
parent sulfonamides. The corresponding chloroderiva-
tives 5–7 already show a better inhibitory power than
derivatives 8–10, whereas for the methylamino-, meth-
oxy-, and ethoxy-substituted derivatives 11–19, a clear-
cut SAR is somehow more difficult to draw. All these
derivatives are better hCA II inhibitors as compared to
the parent sulfonamides 2–4 or the hydroxy-triazines
8–10 discussed above. Still, for the alkoxy-derivatives,
the homosulfanilamides 15 and 18 were much less active
as compared to the corresponding sulfanilamides (14 and
17) or 4-aminoethylbenzenesulfonamides (16 and 19).
On the contrary, for the amines 11–13, the best inhibi-
tory activity was shown just by the homosulfanilamide
12. It is quite difficult to rationalize these results, without
knowing the X-ray crystal structure ofthese adducts, but
such a situation has not been encountered up to now in
other libraries of aromatic sulfonamides derived from
these three derivatives (2–4), containing the same substi-
tution patterns.15 It may also be observed that the com-
pounds investigated here, ofthe type 5–19 are generally
weaker hCA II inhibitors as compared to the clinically
used sulfonamides, with several exceptions, such as 6
or 12 among others (Table 1); (iii) against the tumor-
associated isozyme hCA IX, the activity ofthe new sul-
fonamides 5–19 reported here was even more intriguing.
Thus, several subnanomolar hCA IX inhibitors were de-
tected, such as the chloro-derivative 5 and the ethoxy-
derivatives 17 and 18, whereas two other compounds,
the amines 12 and 13 were also very effective inhibitors,
with KIs in the range of1.1–1.3nM. Except for the other
ethoxy-derivative, 19, which is a potent inhibitor (KI of
12nM—being more potent than the clinically used sul-
fonamides, including indisulam), the other triazine-sub-
stituted derivatives investigated here showed modest or
weak CA IX inhibitory properties, with KIs in the range
of124–549nM, similarly with two ofthe parent sulfon-
amides, sulfanilamide 2, and homosulfanilamide 3 (com-
pound 4 is a rather good hCA IX inhibitor). On the other
hand, it should be noted the dramatic difference ofhCA
IX inhibitory power ofthe amines 11–13, with one such
derivative (11) being rather inactive, whereas the other
two behaving as very potent inhibitors. The differences
between the methoxy-derivatives 14–16 and the corre-
sponding ethoxy-derivatives 17–19 are again very impor-
tant, and cannot be explained at this moment; (iv) the
three CA isozymes investigated here showed a very dif-
ferent behavior toward these inhibitors, with isozyme
IX being generally the most prone to be inhibited by
some ofthem, ofllowed by isozyme II and isozyme I;
(v) the selectivity ofsome ofthese inhibitors against
hCA IX over hCA II is quite interesting, and favorable
for considering the design of isozyme-specific CA IX
inhibitors. Thus, from data of Table 1 it may be observed
that the clinically used derivatives are all more inhibitory
against hCA II than against hCA IX, having selectivity
ratios <1, in the range of0.08–0.76 (the most hCA II
selective inhibitor is brinzolamide, whereas the most
hCA IX ÔselectiveÕ is dichlorophenamide). On the con-
trary, the three aromatic sulfonamides used for the prep-
aration ofthe new derivatives reported here, oftype 2–4,

V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
5431
are all better hCA IX than hCA II inhibitors, but their
Acknowledgements
selectivity ratios are not very high, being in the range
of1.02–4.84. For the compounds 5–19 investigated here,
the selectivity ratio for the two isozymes discussed in de-
tail vary over a very large range, that is, between 0.10 and
706.67. This is the highest such variation seen up to now
for all the investigated CA IX inhibitors,6–10 and we con-
sider this as a very significant result in the search ofCA
IX-specific CA inhibitors. Thus, three ofthe compounds
investigated here, namely 4, 17, and 18, may really be
considered as CA IX-selective inhibitors, as they inhibit
this isozyme 166–706 times better than hCA II (whereas
their selectivity ratios over hCA I are even higher, but
hCA I is known to be an isozyme with lower affinity
for sulfonamide inhibitors).15 Other two compounds,
the amines 12 and 13, are also 11.5–24.5 times better
hCA IX than hCA II inhibitors, which is also an impor-
tant result, since selectivity ratios ofthis amplitude were
rarely seen up to now for other investigated CA IX inhib-
itors.6–10 The other investigated compounds were on the
other hand better hCA II than hCA IX inhibitors,
showing selectivity ratios <1 (Table 1). We are unable
to explain these very high differences ofselectivity of
our compounds for hCA IX, an isozyme for which the
X-ray crystal structure is not available at this moment.
This research was financed in part by a 6th Framework
Programme ofthe European Union (EUROXY pro-
ject). V.G. is grateful to the Italian Embassy in Slovakia
for a travel and research grant at the University of Flor-
ence. J.Y.W. is grateful to CSGI, University of Florence
and University ofMontpellier II ofr a travel grant to
Florence.
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We report here a series ofaromatic, benzenesulfonamide
derivatives incorporating triazine moieties in their
molecules. They were obtained by reaction ofcyanuric
chloride with sulfanilamide, homosulfanilamide, or 4-
aminoethylbenzenesulfonamide. The dichlorotriazinyl-
benzenesulfonamides obtained in this way were
subsequently derivatized by reacting them with various
nucleo- philes, such as water, methylamine, or aliphatic
alcohols (methanol and ethanol). The library ofsulfon-
amides incorporating triazinyl moieties was tested for
the inhibition ofthree physiologically relevant CA iso-
zymes, the cytosolic hCA I and II, and the transmem-
brane, tumor-associated hCA IX. The new compounds
reported here inhibited hCA I with KIs in the range
of75–136nM, hCA II with KIs in the range of13–
278nM, and hCA IX with KIs in the range of0.12–
549nM. The first hCA IX-selective inhibitors were
detected, as the chlorotriazinyl-sulfanilamide as well as
the bis-ethoxytriazinyl derivatives ofsulafnilamide and
homosulfanilamide showed selectivity ratios for CA IX
over CA II inhibition in the range of166–706, having
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nanomolar affinity for hCA IX, with KIs in the range
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5432
V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
550–557; (b) Pastorekova, S.; Casini, A.; Scozzafava, A.;
pACA/hCA I and pACA/hCA II described by LindskogÕs
group.18 Cell growth conditions were those described in
Ref. 19 and enzymes were purified by affinity chromato-
graphy according to the method ofKhalaifh et al.
Enzyme concentrations were determined spectrophoto-
Vullo, D.; Pastorek, J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 869–873; (c) Casey, J. R.; Morgan,
P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. J. Med. Chem. 2004, 47, 2337–2347.
20
10. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
11. Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2004, 47, 1272–1279.
metrically at 280nm, utilizing a molar absorptivity
of49mM À1 cmÀ1 for CA I and 54mMÀ1 cmÀ1 for CA
II, respectively, based on Mr = 28.85kDa for CA I, and
29.3kDa for CA II, respectively.21,22 A variant ofthe
previously published6,7 CA IX purification protocol has
been used for obtaining high amounts of hCA IX needed
in these experiments. The cDNA ofthe catalytic domain
ofhCA IX (isolated as described by Pastorek et al. 23) was
amplified by using PCR and specific primers for the
glutathione S-transferase (GST)-Gene Fusion Vector
pGEX-3X. The obtained fusion construct was inserted in
the pGEX-3X vector and then expressed in E. coli BL21
Codon Plus bacterial strain (from Stratagene). The bac-
terial cells were sonicated, then suspended in the lysis
buffer (10mM Tris pH7.5, 1mM EDTA pH8, 150mM
NaCl, and 0.2% Triton X-100). After incubation with
lysozime (approx. 0.01g/L) the protease inhibitors
CompleteTM were added to a final concentration of
0.2mM. The obtained supernatant was then applied
´
12. de Leval, X.; Ilies, M.; Casini, A.; Dogne, J.-M.; Scozzaf-
ava, A.; Masini, E.; Mincione, F.; Starnotti, M.; Supuran,
C. T. J. Med. Chem. 2004, 47, 2796–2804.
13. Vullo, D.; Scozzafava, A.; Pastorekova, S.; Pastorek, J.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14,
2351–2356.
14. DÕAlelio, G. F.; White, H. J. J. Org. Chem. 1959, 24,
643–644.
15. Supuran, C. T.; Casini, A.; Scozzafava, A. Development
ofSulfonamide 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 67–147.
16. Synthesis ofthe key intermediate 5–7:14 An acetone
solution containing 0.1mol ofeither sualfnilamide
2,
homosulfanilamide (HCl salt) 3, or 4-(2-aminoethyl)-
benzenesulfonamide 4 (17.2g, 22.3g, and 20.0g, respec-
tively), was dropped into a solution of0.1mol (18.5g) of
to a prepacked Glutathione Sepharose 4B column,
extensively washed with buffer and the fusion (GST-
CA IX) protein was eluted with a buffer consisting of
5mM reduced glutathione in 50mM Tris–HCl, pH8.0.
Finally the GST part ofthe usfion protein was
cleaved with thrombin. The advantage ofthis method
over the previous one,6,7 is that CA IX is not precipi-
tated in inclusion bodies from which it has to be isolated
by denaturing–renaturing in the presence ofhigh con-
centrations ofurea, 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,20 the amount ofenzyme being
determined by spectrophometric measurements and its
activity by stopped-flow experiments, with CO2 as sub-
strate.25 The specific activity ofthe obtained enzyme was
the same as the one previously reported,6,7 but the yields in
active protein were 5–6 times higher per liter ofculture
medium. AnSX.18MV-R Applied Photophysics stopped-
flow instrument has been used for assaying the CA-
catalyzed CO2 hydration activity.25 Phenol red (at a
concentration of0.2mM) has been used as indicator,
working at the absorbance maximum of557nm, with
10mM Hepes (pH7.5) as buffer, 0.1M Na2SO4 (for
maintaining constant the ionic strength), following the
CA-catalyzed CO2 hydration reaction for a period of 10–
100s. Saturated CO2 solutions in water at 20°C were used
as substrate.24 Stock solutions ofinhibitor (1mM) were
prepared in distilled-deionized water with 10–20% (v/v)
DMSO (which is not inhibitory at these concentra-
tions) and dilutions up to 0.01nM were done thereafter
with distilled-deionized water. Inhibitor and enzyme
solutions were preincubated together for 15min 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 ofsuch
results.
14
cyanuric chloride 1 in 100mL ofacetone.
The temper-
ature was maintained at 0–5°C. The mixture was stirred
of r 0.5h and then a solution of0.1mol (4.0g) ofsodium
hydroxide in 60mL ofwater was added dropwise. In the
case ofthe reaction with homosulafnilamide hydrochlo-
ride, 0.2mol (8.0g) ofNaOH were used. Stirring was
continued for an additional 0.5h. Ice-water (200mL) was
added to the reaction mixture and the solid was filtered.
The product was washed with cold water until free of
chloride ions. The product was purified by dissolving in
hot acetone and precipitated with ice-water, as described
by DÕAlelio and White.14
Reaction ofintermediates 5–7 with alcohols or water:
0.005mol ofthe appropriate intermediate 5–7 was added
to a suspension/solution of0.01mol ofNaOH in the
corresponding alcohol (methanol or ethanol) or water.
The mixture was refluxed for 4–6h. The alcoxide deriva-
tives 14–19 were isolated by precipitation with water and if
necessary, purified by dissolving in hot acetone and re-
precipitation with water. Reaction ofintermediates 5–7
with methylamine: 0.005mol ofthe appropriate interme-
diate 5–7 was added to a solution of0.01mol of
methylamine hydrochloride in water. The mixture was
heated to reflux and 0.02mol ofaqueous sodium hydrox-
ide was slowly added to the mixture. Refluxing was
continued for 3h. The insoluble products 11–13 were
easily isolated by filtration and crystallized from ethanol–
water 1:1.
2,4-Dimethylamino-6-(4-sulfamoylanilino)-1,3,5-triazine
1
11: mp 232–234°C; H NMR (DMSO-d6, 250MHz) d 9.3
(s, 1H), 8 (d, 2H, J = 8Hz), 7.7 (d, 2H, J = 8Hz), 7.2 (s,
2H), 6.8 (m, 2H), 2.75 (m, 6H); MS ESI+ m/z 310 (M+H)+,
332 (M+Na)+, 619 (2M+H)+, 641 (2M+Na)+. ESIÀ m/z
308 (MÀH)À, 617 (2MÀH)À.
2,4-Diethoxy-6-(4-sulfamoylanilino)-1,3,5-triazine 17: mp
1
208–210°C (lit.14 mp 210–211°C); H NMR (DMSO-d6,
18. Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.;
Jonsson, B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure-
Function Relations in Human Carbonic Anhydrase II as
Studied by Site-Directed Mutagenesis. In Carbonic Anhyd-
rase—from Biochemistry and Genetics to Physiology and
250MHz) d 10.3 (s, 1H), 7.9 (d, 2H, J = 7.2Hz), 7.8 (d,
2H, J = 7.2Hz), 7.2 (s, 2H), 4.4 (q, 4H, J = 6.8Hz), 1.35 (t,
6H, J = 5Hz); MS ESI+ m/z 362 (M+Na)+, 701 (2M+
Na)+. ESIÀ m/z 338 (MÀH)À, 677 (2MÀH)À.
`
Clinical Medicine; Botre, F., Gros, G., Storey, B. T., Eds.;
VCH: Weinheim, 1991; pp 1–13.
17. Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids

V. Garaj et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5427–5433
5433
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Biochem. 1990, 190, 351–357.
20. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S.
M. Biochemistry 1977, 16, 2241–2247.
21. Lindskog, S.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A.
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23. Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.;
Zelnik, V.; Opavsky, R.; Zatovicova, M.; Liao, S.;
Portetelle, D.; Stanbridge, E. J.; Zavada, J.; Burny, A.;
Kettmann, R. Oncogene 1994, 9, 2877–2888.
24. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–
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22. Steiner, H.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem.
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