Carbonic anhydrase inhibitors: Novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX

Article abstract of DOI:10.1016/j.bmcl.2005.04.056

A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4- aminoethylbenzenesulfonamide. The prep

Full text of DOI:10.1016/j.bmcl.2005.04.056

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3102–3108
Carbonic anhydrase inhibitors: Novel sulfonamides
incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic
and tumour-associated carbonic anhydrase isozymes I, II and IX
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, Alba, Cuneo, Italy
´ ´
Universite Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032, Ecole Nationale Superieure de Chimie de Montpellier,
c
´
8 rue de lÕEcole Normale, 34296 Montpellier Cedex, France
Received 5 January 2005; revised 29 March 2005; accepted 12 April 2005
Available online 17 May 2005
This paper is dedicated to the memory of Mircea D. Banciu (1942–2005)
Abstract—A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This
series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide.
The prepared dichlorotriazinyl-benzenesulfonamides were subsequently derivatized by reacting them with various nucleophiles, such
as ammonia, hydrazine, primary and secondary amines, amino acid derivatives or phenol. 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, tumour-associated hCA IX. The new compounds inhibited hCA I with inhibition
constants in the range of 31–8500 nM, hCA II with inhibition constants in the range of 14–765 nM and hCA IX with inhibition
constants in the range of 1.0–640 nM. Structure–activity relationship was straightforward and rather simple in this class of CA
inhibitors, with the compounds incorporating compact moieties at the triazine ring (such as amino, hydrazino, ethylamino, dimeth-
ylamino or amino acyl) being the most active ones, and the derivatives incorporating such bulky moieties (n-propyl, n-butyl, dieth-
ylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy) being less effective hCA I, II and IX inhibitors. Some of the new
derivatives also showed selectivity for inhibition of hCA IX over hCA II (selectivity ratios of 23.33–32.00), thus constituting excel-
lent leads for the development of novel approaches for the management of hypoxic tumours.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
vertebrates),2–5 that is, the cytosolic CA I and II (of hu-
man origin, hCA I and hCA II) and the tumour-associ-
ated transmembrane isozyme hCA IX.2–5 Indeed, the
levels of hCA IX—the best studied tumour-associated
CA at this moment—dramatically increase in response
to hypoxia, a characteristic of many tumours, via a di-
rect transcriptional activation of the CA9 gene by the
hypoxia inducible factor HIF-1,4 being also proven that
the expression of this protein in tumours is generally a
sign of poor prognosis.4 Recently, we and Pastorekova
and co-workers6 showed that hCA IX is involved in
the tumour acidification processes, providing H+ ions
to the extracellular milieu by means of the CO2 hydra-
tion reaction to bicarbonate and protons. The pH of tu-
mours is in fact more acidic by 0.5–1.0 pH unit than that
In a previous contribution from this laboratory1 it
was reported that triazinyl-containing sulfonamides
obtained from cyanuric chloride (2,4,6-trichloro-1,3,5-
triazine) and amino-benzenesulfonamides, also incorpo-
rating hydroxy, alkoxy or amino moieties, act as highly
effective inhibitors of the zinc enzyme carbonic anhy-
drase (CA, EC 4.2.1.1).2–5 The designed inhibitors1 have
been tested for the inhibition of three physiologically rel-
evant CA isozymes (of the 15 currently known in higher
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.056

V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
3103
of the surrounding normal tissue,4 and this acidic envi-
ronment seems to play a very important role both in
the growth, dissemination and propagation of tumour
cells and in their nonresponsiveness to chemo- and
radiotherapy.4,6,7 We have also proved6 that inhibition
of hCA IX in transfected cells or in cultured tumour
cells by means of potent CA IX inhibitors developed
in our laboratory leads to a diminution of the acidifying
effects in these cells, with restoration of a more physio-
logic pH. This constitutes the proof-of-concept that
inhibition of the tumour-associated CAs (two such iso-
zymes are known at this moment, hCA IX and hCA
XII)4 may lead to novel therapeutic approaches in the
fight against hypoxic tumours, which are generally less
responsive or nonresponsive to all the classical chemo-
therapeutic drugs or to radiotherapy.6
paper we continue the investigation of this class of deriv-
atives, reporting the synthesis and hCA I, II and IX
inhibitory properties of a large series of such novel
derivatives.
2. Chemistry
Considering the versatile chemistry of cyanuric chloride
1 (2,4,6-trichloro-1,3,5-triazine),1,8 and its reactions with
various nucleophiles such as amines, amino-sulfon
amides, alcohols, phenols, etc., we extend here our pre-
vious investigations1 in the design of novel CA inhibi-
tors9 containing triazinyl moieties (Scheme 1).
Reaction of cyanuric chloride 1 with sulfanilamide 2,
homosulfanilamide 3 or 4-aminoethyl-benzenesulfon
amide 4, in a 1:1 molar ratio, afforded the dichlorotri-
azine-substituted key intermediates 5–7 reported previ-
ously (Scheme 1).1 Reaction of 5–7 with primary
Since the sulfonamides incorporating triazinyl moieties
previously reported1 were among the most potent and
selective hCA IX inhibitors obtained up to now, in this
Scheme 1.

3104
V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
amines in molar ratios of 1:2 afforded the bis-amino-tri-
azinyl derivatives 8. A large series of such compounds
have been obtained, since in the previous report only
three amino-derivatives were synthesized—the methyl-
amino ones—which were among the best hCA IX inhib-
itors in the series of investigated triazinyl-sulfonamides.
Diverse alkyl amines possessing both normal- and
branched C2–C4 chains (ethyl, n-propyl, iso-propyl, n-
butyl) have been employed in the reaction, in order to
prepare a large library of such derivatives and detect
the best substitution pattern for the CA inhibitory prop-
erties of these derivatives. Some heterocyclic amines
have also been included in our study, such as pyrid
oxal-amine or N-aminoethyl-piperazine among others
(leading to derivatives 8s–x); but a limited number of
such compounds have been prepared, since due to steric
impairment of these rather bulky derivatives it is proba-
ble that they should not fit easily within the enzyme ac-
tive site (see discussion later in the text). However, it is
interesting to note that although these two compounds
possess different nucleophilic moieties in their molecules
in addition to the NH2 one, unexpectedly only the pri-
mary amine reacted with 5–7, leading to only one reac-
tion product in each case (8s–x) that could be separated
and purified without problems. Similarly, reaction of 5–
7 with secondary amines containing 2–4 carbon atoms in
their molecules led to a smaller series of tertiary bis-
amines of type 9, incorporating derivatives with both
identical substituents (R = R0 in structure 9) as well as
compounds with such different moieties (Scheme 1 and
Table 1). The key intermediates 5–7 have also been re-
acted with sodium phenoxide, leading to the bis-ethers
10–12 (in the previous report only the reaction with alk-
oxides has been investigated).1 Finally, the reaction of
the intermediates 5–7 with amino acids and some of
their derivatives has also been investigated. However,
only four mono-substituted derivatives 13–16 could be
obtained at this moment, when working at 1:1 or 1:2
molar ratios between the halogeno-substituted interme-
diates 5–7 and the amino acid derivative. Thus, the sec-
ond chlorine atom from 5–7 is probably deactivated to
the nucleophilic substitution with amino acid deriva-
tives; however, this reaction deserves further investiga-
tion, as only
a limited number of amino acid
derivatives was obtained in this study (Scheme 1).10
3. CA inhibition
Among the physiologically most relevant CA isozymes
in humans are the cytosolic ubiquitous hCA I and
II,2,3 as well as the tumour-associated transmembrane
isozyme IX, which plays a relevant role in tumourigene-
sis, as mainly investigated by Pastorek and co-work-
ers.4,6 Data of Table 1 show hCA I, II and IX
inhibition with the new compounds reported here of
types 8–16, as well as clinically used CA inhibitors, such
as acetazolamide AAZ, methazolamide MZA, ethoxzol-
amide EZA, dichlorophenamide DCP, dorzolamide
DZA and brinzolamide BRZ.9 Indisulam (E7070)
IND, an antitumour sulfonamide in phase II clinical tri-
als for which we recently demonstrated potent CA
inhibitory properties, has also been included for com-
parison in this study.11,12 Furthermore, the X-ray crystal
structure of IND in adduct with isozyme hCA II has
recently been reported by our group.11
The following SAR should be noted from data of Table
1:13 (i) against the cytosolic, slow isozyme hCA I, the
new derivatives incorporating triazine moieties of types
8–16 showed all types of inhibitory properties, with
KIs in the range of 31–8500 nM. Thus, a first group of
derivatives, such as 8s–x, behaved as quite weak hCA
I inhibitors, with KIs in the range of 1560–8500 nM. It
may be observed that all these compounds incorporate
two bulky piperazinyl-ethyl- or pyridoxal amine moie-
ties in their molecules, which are obviously too bulky

V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
3105
Table 1. Inhibition data for derivatives 8–16 investigated in the present paper and standard sulfonamide CA inhibitors, against isozymes hCA I, II
and IX, and their selectivity ratios for isozyme IX over isozyme II13
Compound
R,R0
n
KI* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA IXb
KI (hCA II)/KI (hCA IX)
AAZ
MZA
EZA
DCP
DZA
BRZ
IND
8a
250
50
12
14
8
25
27
0.48
0.52
0.23
0.76
0.17
0.08
0.62
13.33
21.00
12.00
19.28
23.84
5.60
7.93
2.92
2.87
2.77
0.87
0.80
0.66
0.79
0.83
0.80
1.24
1.13
1.23
25
34
50
1200
50,000
NT
31
38
9
52
37
3
15
16
21
18
27
31
14
23
38
46
50
49
63
83
146
175
163
216
326
338
24
H
1
2
0
1
2
1
2
0
1
2
0
1
2
0
1
2
0
0
1
87
95
1.2
1.0
1.5
1.4
1.3
2.5
2.9
13
8b
8c
H
NH2
93
8d
8e
NH2
NH2
104
113
105
112
235
247
241
368
425
540
561
622
613
558
1560
1635
8f
8g
Et
Et
8h
8i
i-Pr
i-Pr
16
18
8j
8k
i-Pr
n-Pr
56
78
8m
8n
n-Pr
n-Pr
125
185
210
204
174
287
274
8o
8p
n-Bu
n-Bu
8q
8r
n-Bu
Et2NCH2CH2
8s
[HN(CH2CH2)2N]CH2CH2
[HN(CH2CH2)2N]CH2CH2
8t
8u
8v
8x
0
1
2
3500
4200
8500
450
596
765
386
423
640
1.16
1.40
1.19
9a
9b
9c
9d
9e
9f
Me, Me
Me, Me
Me, Me
Et, Et
Et, Et
Et, Et
Me, n-Pr
Me, n-Pr
Me, n-Pr
—
0
63
75
39
33
35
65
62
77
39
33
36
138
124
83
29
32
33
28
1.5
1.3
26.00
25.38
23.33
11.01
8.26
1
2
76
1.5
5.9
0
128
130
156
176
180
198
875
631
549
35
1
7.5
6.3
2
12.22
3.71
2.61
9g
9h
9i
0
10.5
12.6
13.7
254
197
113
1.7
1
2
2.62
0.54
10
11
12
13
14
15
16
—
—
—
—
—
—
—
—
—
0.63
0.73
—
—
17.05
32.00
23.57
23.33
33
39
1.0
1.4
—
—
31
1.2
NT = not tested.
* Errors in the range of 5–10% of the reported value (from three different assays).
a Human cloned isozyme, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme, by the CO2 hydration method.

3106
V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
for their favourable binding within the active site of the
enzyme. Several other compounds, such as the bis-
amines 8k–r and the bis-ethers 10–12, were more effec-
tive hCA I inhibitors, showing KIs in the range of
368–875 nM. Again the bulky moieties (phenoxy in
10–12 or n-propyl/n-butyl in 8k–r) are detrimental to
the good inhibitory activity of these compounds, which
is again probably due to steric hindrance effects. Good
hCA I inhibitory activity, with KIs in the range of
104–247 nM, has been detected for derivatives 8d–j
and 9d–i. These compounds incorporate less bulky moi-
eties than the previously discussed ones, such as hydra-
zino, ethylamino, isopropylamino, N,N-diethylamino-
or N-methyl-N-propylamino. Finally, the best hCA I
inhibitors in this series were 8a–c, 9a–c and 13–16, which
showed KIs in the range of 31–95 nM, in the same range
as the potent, clinically used inhibitors ethoxzolamide,
methazolamide and indisulam (Table 1). It is easy to ob-
serve that the best inhibitors in the series incorporate the
most compact substituents at the triazine ring, such as
amino, hydrazine (in 8a–c), dimethylamino (in 9a–c) as
well as the monosubstituted amino acid derivatives
(13–16), which contain a chlorine atom and an amino
acyl moiety, being thus among the less bulky ones in
the entire series. Generally (although there are many
exceptions) the sulfanilamide derivatives (n = 0 in struc-
tures 8–16) were more effective hCA I inhibitors than the
corresponding homosulfanilamides (n = 2), which in
turn were more active inhibitors than the corresponding
derivatives of 4-aminoethylbenzenesulfonamide (n = 2);
(ii) against isozyme hCA II, one of the physiologically
most relevant CAs, the new derivatives 8–16 also
showed interesting inhibitory power, with KIs in the
range of 14–765 nM. SAR is also in this case similar
to what was discussed for hCA I above, rather straight-
forward and simple, with the derivatives incorporating
the compact moieties at the triazine ring being the most
active ones, and the derivatives incorporating bulky
moieties the most ineffective inhibitors. As for hCA I,
generally activity diminished with increasing n from 0
(sulfanilamide derivatives) to 2 (4-aminoethylbenzene-
sulfonamide derivatives) for all compounds 8–16 investi-
gated here (but more exception as for hCA I can be
easily observed from data of Table 1). Thus, the most
ineffective hCA II inhibitors were the bulky derivatives
8n–x and 10–12 (KIs in the range of 83–765 nM), fol-
lowed by the less bulky derivatives 8h–m, 9 and 13–16,
which are medium potency—effective hCA II inhibitors
(KIs in the range of 28–77 nM). The most effective hCA
II inhibitors (with potencies comparable to those of the
clinically used sulfonamides shown in Table 1) were 8a–
g, with KIs in the range of 14–27 nM; (iii) against the tu-
mour-associated isozyme hCA IX, the derivatives 8–16
investigated here showed inhibition constants in the
range of 1.0–640 nM. Considering the triazine ring sub-
stituents, SAR is similar to what disclosed above for the
inhibition of hCA I and II, with the bulky compounds
being the most ineffective inhibitors, and those incorpo-
rating compact, smaller moieties being the most effective
hCA IX inhibitors. This is clearly due to the fact that the
bulky derivatives difficultly may bind within the enzyme
active site. On the other hand, unlike as with hCA I and
II, generally the sulfanilamide derivatives were less effec-
tive hCA IX inhibitors than the corresponding homo-
sulfanilamides, which in turn were less inhibitory than
the corresponding derivatives with n = 2 (of course, also
in this case several exceptions may be observed from
data of Table 1, but the general trend was different for
the transmembrane isozyme than for the cytosolic ones
discussed above). The less effective hCA IX inhibitors
were again the amines/ethers incorporating the bulky
n-propyl, n-butyl, diethylaminoethyl, piperazinyl-ethyl,
pyridoxalamine or phenoxy moieties (8n–x and 10–12),
which showed KIs in the range of 113–640 nM. Medium
potency hCA IX inhibitors were two n-propyl deriva-
tives (8k and 8m) with inhibition constants of 56–
78 nM, whereas the remaining compounds (incorporat-
ing compact substituents at the triazine ring) showed
very effective hCA IX inhibitory properties, with KIs
in the range of 1.0–18 nM, similar to the previously re-
ported derivatives belonging to this class.1 Thus, the
drug design of triazinyl sulfonamides may indeed lead
to very effective hCA IX inhibitors, both belonging to
the amine, amino acid or alkoxy1 derivatives. The best
hCA IX inhibitors were the amino, hydrazine, ethyl
amino, dimethylamino and amino acid derivatives,
which showed inhibition constants in the range of 1–
3 nM, being among the most potent hCA IX inhibitors
reported up to now;1,16,17 (iv) since hCA II is a ubiqui-
tous enzyme showing high affinity for sulfonamide
inhibitors, a critical issue in the design of inhibitors of
other CA isozymes which show potential as medicinal
chemistry targets regards the selectivity of an inhibitor
for the target isozyme over hCA II. In this case, the
selectivity ratios for hCA IX over hCA II of the new
inhibitors are shown in Table 1. It may be observed that
all the clinically used sulfonamides and a small number
of the new inhibitors reported here (e.g., 8k–q and 10–
12) behave better as hCA II than hCA IX inhibitors, with
selectivity ratios <1. Another group of new derivatives,
such as 8h–j, 8r–x and 9g–i, were better hCA IX than
hCA II inhibitors, but with a modest selectivity, in the
range of 1.13–3.71. Finally, the remaining new derivatives
showed good or excellent selectivity ratios, being 5.60–
32.00 times more effective hCA IX than hCA II inhibitors.
Among the most selective hCA IX inhibitors were some of
the amino acid derivatives (14–16) or the secondary
amines 9a–c (selectivity ratios in the range of 23.33–
32.00). As some of these compounds are also very effective
hCA IX inhibitors (in the low nanomolar range), it is
obvious that this class of derivatives affords potent and
quite selective hCA IX inhibitors, with potential to be
developed for the management of hypoxic tumours.
4. Conclusions
We report here a series of aromatic benzenesulfonamide
derivatives incorporating triazine moieties in their mole-
cules. They were obtained by reaction of cyanuric
chloride with sulfanilamide, homosulfanilamide or 4-
aminoethylbenzenesulfonamide. The dichlorotriazinyl-
benzenesulfonamides obtained in this way were sub
sequently derivatized by reacting them with various
nucleophiles, such as ammonia, hydrazine, primary
and secondary amines, amino acid derivatives or phenol.

V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
3107
Talks, K. L.; Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P. J.;
Harris, A. L. Cancer Res. 2000, 60, 7075; (b) Potter, C. P.
S.; Harris, A. L. Br. J. Cancer 2003, 89, 2; (c) Robertson,
N.; Potter, C.; Harris, A. L. Cancer Res. 2004, 64, 6160.
8. DÕAlelio, G. F.; White, H. J. J. Org. Chem. 1959, 24, 643.
9. 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, p 67.
The library of sulfonamides incorporating triazinyl moi-
eties was tested for the inhibition of three physiologi-
cally relevant CA isozymes, the cytosolic hCA I and
II, and the transmembrane, tumour-associated hCA
IX. The new compounds reported here inhibited hCA
I with KIs in the range of 31–8500 nM, hCA II with
KIs in the range of 14–765 nM and hCA IX with inhibi-
tion constants in the range of 1.0–640 nM. SAR was
straightforward and rather simple in this class of CA
inhibitors, with the derivatives incorporating compact
moieties at the triazine ring (such as amino, hydrazino,
ethylamino, dimethylamino or amino acyl) being the
most active ones, and the derivatives incorporating such
bulky moieties (n-propyl, n-butyl, diethylaminoethyl,
piperazinylethyl, pyridoxal amine or phenoxy) being
the most ineffective hCA I, II and IX inhibitors. Some
of the new derivatives also showed selectivity for inhibi-
tion of hCA IX over hCA II, thus constituting excellent
leads for the development of novel approaches in
the management of hypoxic tumours.
10. Synthesis of the key intermediate 5–7:1 An acetone
solution containing 0.1 mol of either sulfanilamide 2,
homosulfanilamide (HCl salt) 3 or 4-(2-aminoethyl)benz-
enesulfonamide 4 (17.2, 22.3 and 20.0 g, respectively), was
dropped into a solution of 0.1 mol (18.5 g) of cyanuric
chloride 1 in 100 mL of acetone.1,8 The temperature was
maintained at 0–5 °C. The mixture was stirred for 0.5 h
and then a solution of 0.1 mol (4.0 g) of sodium hydroxide
in 60 mL of water was added dropwise. In the case of the
reaction with homosulfanilamide hydrochloride, 0.2 mol
(8.0 g) of NaOH was used. Stirring was continued for an
additional 0.5 h. Ice water (200 mL) 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.8 Reaction of intermediates 5–7 with ammonia,
amines or phenol: 0.005 mol of the appropriate interme-
diate 5–7 was added to a suspension/solution of 0.01 mol
of nucleophile in water. The mixture was refluxed for 4–
6 h and the corresponding amount of a NaOH solution
dissolved in a small volume of water was added dropwise.
The desired derivatives, 8–12 precipitated after cooling the
reaction mixture at 4 °C, were filtered and recrystallized
from MeOH–water (1:1) or EtOH–water (1:1 or 1:2).
Yields were generally in the range of 70–95%. Reaction of
intermediates 5–7 with amino acid derivatives: 0.005 mol
of the halogeno intermediates 5–7 was added to a solution
of 0.005 mol (or 0.01 mol) of the appropriate amino acid
derivative in water. The mixture was heated to reflux and
0.005 or 0.01 mol of aqueous NaOH was slowly added to
the mixture. Refluxing was continued for 4–5 h. If
necessary, the pH of the mixture was changed to neutral
using diluted (5%) HCl water solution, and the insoluble
products were easily isolated by filtration and recrystal-
lized from MeOH or EtOH. Yields were in the range of
50–65%. The purity of all products was checked by TLC
using methanol as mobile phase and confirmed by 1H
NMR and MS. Some representatives of this class could
not be obtained in pure enough state and were not
included in Table 1 (e.g., the reaction products of 5 with
ammonia or ethylamine among others).
Acknowledgments
This research was financed in part by a Sixth Frame-
work Programme of the European Union (EUROXY
project) and by Miroglio S.p.A. (Alba, Cuneo, Italy).
J.Y.W. is grateful to CSGI, University of Florence
and University of Montpellier II, for a travel grant to
Florence. V.G. is grateful to the Italian Embassy in Slo-
vakia for a travel and research grant at the University of
Florence and to EC for his stage in the frame of ÔMol-
magÕ, Marie Curie Training Site programme HPMT-
CT-2000-00179.
References and notes
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J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004,
19, 199.
4-(4,6-Bis-isopropylamino-[1,3,5]triazin-2-yl-amino)-ben-
zenesulfonamide 8h: mp 129–131 °C, 1H NMR (DMSO-d6,
250 MHz): d 9.2 (s, 1H), 8 (d, 2H, J = 8 Hz), 7.7 (d, 2H,
J = 8 Hz), 7.2 (s, 2H), 6.7 (m, 2H), 4.1 (m, 2H), 1.15 (d,
12H, J = 6 Hz); MS ESI+ m/z 366 (M+H)+, 731 (2M+H)+.
ESIꢀ m/z 364 (MꢀH)ꢀ, 729 (2MꢀH)ꢀ.
11. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217.
12. Supuran, C. T. Expert Opin. Invest. Drugs 2003, 12, 283.
13. hCA I and hCA II cDNAs were expressed in Escherichia coli
strain BL21 (DE3) from the plasmids pACA/hCA I and
pACA/hCA II described by LindskogÕs group.14 Cell
growth conditions were those described in Ref.14 and
enzymes were purified by affinity chromatography accord-
ing to the method of Khalifah et al.15 Enzyme concentra-
tions were determined spectrophotometrically at 280 nm,
utilizing a molar absorptivity of 49 mMꢀ1 cmꢀ1 for CA I
5. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925.
ˇ
6. Svastova, E.; Hulikova, A.; Rafajova, M.; ZatÕovicova,
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
Lett. 2004, 577, 439.
7. (a) Wykoff, C. C.; Beasley, N. J.; Watson, P. H.; Turner,
K. J.; Pastorek, J.; Sibtain, A.; Wilson, G. D.; Turley, H.;

3108
V. Garaj et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3102–3108
and 54 mMꢀ1 cmꢀ1 for CA II, respectively, based on
15. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S.
M. Biochemistry 1977, 16, 2241.
Mr = 28.85 for CA I, and 29.3 for CA II, respectively. A
variant of the previously published16,17 CA IX purification
protocol has been used for obtaining high amounts of hCA
IX needed in these experiments. The cDNA of the catalytic
domain of hCA IX (isolated as described by Pastorek
et al.18) 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 bacterial
cells were sonicated, then suspended in the lysis buffer
(10 mM Tris pH 7.5, 1 mM EDTApH 8, 150 mMNaCl and
0.2% Triton X-100). After incubation with lysozime
(approx. 0.01 g/L) the protease inhibitors CompleteTM were
added to a final concentration of 0.2 mM. The obtained
supernatant was then applied 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 5 mM reduced glutathione in 50 mM Tris–
HCl, pH 8.0. Finally the GST part of the fusion protein was
cleaved with thrombin. The advantage of this method over
the previous one16,17 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 concentra-
tions 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 chromato
graphy,15 the amount of enzyme being determined by
spectrophotometric measurements and its activity by
stopped-flow experiments, with CO2 as substrate.19 The
specific activity of the obtained enzyme was the same as the
one previously reported,16,17 but the yields in active protein
were 5–6 times higher per liter of culture medium.
16. Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozzaf-
ava, A.; Pastorekova, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 1005.
17. (a) Pastorekova, S.; Casini, A.; Scozzafava, A.; Vullo, D.;
Pastorek, J.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2004, 14, 869; (b) Casey, J. R.; Morgan, P. E.; Vullo, D.;
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J.
Med. Chem. 2004, 47, 2337.
18. 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.
19. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An
SX.18MV-R Applied Photophysics stopped-flow instru-
ment was used for measuring the initial velocities for
the CO2 hydration reaction catalyzed by different CA
isozymes, by following the change in absorbance of a
pH indicator. Phenol red (at
a concentration of
0.2 mM) was used as indicator, working at the absor-
bance maximum of 557 nm, with 10 mM Hepes
(pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining
constant the ionic strength), following the CA-catalyzed
CO2 hydration reaction for
a period of 10–100 s.
Saturated CO2 solutions in water at 20 °C were used
as substrate. The CO2 concentrations ranged from 1.7
to 17 mM for the determination of the inhibition
constants. For each inhibitor at least six traces of the
initial 5–10% of the reaction were used to determine
the initial velocity. The uncatalyzed rates were deter-
mined in the same manner and subtracted from the
total observed rates. The kinetic constants kcat and kcat
/
Km were obtained by nonlinear least-squares methods
using PRISM 3. Stock solutions of inhibitors were
prepared at a concentration of 1–3 mM (in DMSO–
water 1:1, v/v) and dilutions up to 0.01 nM done with
the assay buffer mentioned above. KIs of the inhibitors
were determined by using Lineweaver–Burk plots, as
reported earlier,16,17 and represent the means from at
least three different determinations.
14. 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 Anhy-
drase—From Biochemistry and Genetics to Physiology and
`
Clinical Medicine; Botre, F., Gros, G., Storey, B. T., Eds.;
VCH: Weinheim, 1991, pp 1–13.