Carbonic anhydrase inhibitors: Inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with benzo[b]thiophene 1,1-dioxide sulfonamides

Article abstract of DOI:10.1016/j.bmcl.2005.04.078

A series of selected benzo[b]thiophene-5- and 6-sulfonamide derivatives previously reported to show cytotoxic activity and some others newly synthesized has been tested for the interactions with several CA isozymes, some of which are known to be involved

Full text of DOI:10.1016/j.bmcl.2005.04.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4872–4876
Carbonic anhydrase inhibitors: Inhibition
of cytosolic/tumor-associated carbonic anhydrase isozymes I, II,
and IX with benzo[b]thiophene 1,1-dioxide sulfonamides
a
Alessio Innocenti, Raquel Villar, Victor Martinez-Merino, Marıa J. Gil,
b
b
b
´
Andrea Scozzafava,a Daniela Vulloa and Claudiu T. Supurana,*
a
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Room 188,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
bUniversidad Publica de Navarra, Departamento de Quimica Aplicada, Campus Arrosadia, E-31006 Pamplona, Spain
Received 13 January 2005; revised 27 April 2005; accepted 28 April 2005
Available online 13 September 2005
Abstract—A series of selected benzo[b]thiophene-5- and 6-sulfonamide derivatives previously reported to show cytotoxic activity
and some others newly synthesized has been tested for the interactions with several CA isozymes, some of which are known to
be involved in tumorigenesis (hCA IX), whereas others are ubiquitously found in many normal tissues (the cytosolic isoforms
hCA I and II). The unsubstituted sulfonamides inhibited hCA I with inhibition constants in the range of 63–138 nM, hCA II with
inhibition constants in the range of 6.3–8.8 nM, and hCA IX with inhibition constants in the range of 2.8–15 nM, being thus more
active than clinically used inhibitors such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide or indisulam (E
7070). Some of these derivatives also showed some selectivity for the inhibition of the tumor-associated (hCA IX) over the cytosolic
isozyme hCA II. Although these derivatives may act on many targets other than the CAs (such as the NADH oxidase) or may
induce apoptosis by accumulation of reactive oxygen species, it is quite important to try to decipher as many as possible of the
potential mechanisms that lead to derivatives with potent antitumor activity in order to develop novel therapeutic strategies for
the management of cancer.
ꢀ 2005 Elsevier Ltd. All rights reserved.
It was known for several years that many sulfonamides
possessing carbonic anhydrase (CA, EC 4.2.1.1)1–3
inhibitory properties also inhibit in various degrees the
growth of tumor cells in vitro and in vivo.4–6 The precise
isozyme(s) involved in such processes, among the 15
presently characterized human CAs, were not known
until recently, but the discovery of CA IX7,8 and then
of CA XII9 as isozymes predominantly present in tu-
mors offered a starting point for more detailed studies
in the field.10 Another issue little understood in the first
years of ÔCA–tumors connectionÕ research was why var-
ious tumor cell lines belonging to the same tumor type
(for example, leukemia, non-small cell lung cancer,
ovarian, melanoma, colon, CNS, renal, prostate or
breast cancer) showed very different sensitivity to inhibi-
tion by sulfonamides, with GI50 (molarity of inhibitor
producing a 50% inhibition of tumor cell growth) values
typically in the range of 30 lM–10 nM.4,5,11 It was dis-
covered only later that CA IX/XII are not present in
all tumor types,1–3 and furthermore, that the levels of
isozyme IX—the best studied one at this moment—dra-
matically increase in response to hypoxia via a direct
transcriptional activation of the CA9 gene by the hypox-
ia inducible factor HIF-1.12–16 It was proven thereafter
that the expression of CA IX in tumors is a sign of poor
prognosis.12–16
Recently, the involvement of sulfonamide CA inhibitors
in cancer has been investigated in more detail: Svastova
et al.17 showed that the acidic extracellular pH, which is
a typical attribute of the tumor microenvironment, is
generated by the activity of one of the tumor-associated
CA isozymes, i.e., CA IX, and that this acidification can
be perturbed by deletion of the enzyme active site and
inhibited by potent CA IX inhibitors of the sulfonamide
type, which bind only to hypoxic cells containing CA IX
(the involvement of the other tumor-associated isozyme,
i.e., CA XII, in such processes has not been investigated
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.078

A. Innocenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4872–4876
4873
at the moment). Thus, it appears of critical importance
to pursue the development of CA inhibitors targeting
the tumor-associated CA isozymes CA IX and XII,
eventually belonging to novel classes of compounds, less
investigated up to now.
Inhibition data against isozymes hCA I, II, and IX with
derivatives 1–3 as well as standard sulfonamide inhibi-
tors are presented in Table 1.
The following SAR is evidenced from the data of Table
1: (i) Against isozyme hCA I the primary sulfonamides
1a, 1b, 2a–c, and 3a showed moderate inhibitory activi-
ty, with inhibition constants in the range of 63–138 nM,
being thus less inhibitory than the very potent hCA I
inhibitors ethoxzolamide and indisulam, but much bet-
ter inhibitors than the clinically used drugs acetazola-
mide, methazolamide, and dichlorophenamide (KIÕs in
the range of 780–1200 nM). N-substitution of the sulfa-
moyl moiety of 1 with ethyl or cyclopropylmethyl moi-
eties (1c and 1d) led to a decrease of the hCA I
inhibitory properties (KIÕs in the range of 493–
530 nM), but these derivatives were anyhow much better
inhibitors than AZA and MZA. Thus, this is one of the
first reports showing that some N-substituted sulfona-
mides may act as moderate hCA I inhibitors. The much
bulkier benzyl (1e) or 3-methoxyphenyl (3b) substituents
in such derivatives led, on the other hand, to a total loss
of the CA inhibitory activities, not only against hCA I
but also against the other two isozymes investigated
here. Obviously, these bulky groups impede the binding
of these sulfonamides to the Zn(II) ion within the en-
zyme active site. (ii) Against the ubiquitous, physiologi-
cally relevant isozyme hCA II, again the unsubstituted
sulfonamides 1a, 1b, 2a–c, and 3a showed very good
inhibition, with KIÕs in the range of 6.3–8.8 nM. Thus,
the nature of the ring (benzo[b]thiophene or the corre-
sponding dihydro ring) or the position of the sulfamoyl
moiety (5- or 6-substituted sulfonamides) attached to
this ring does not show major influence on the excellent
inhibitory properties of these derivatives against hCA II.
The presence of the lipophilic R1 moiety in compounds
2b and 2c was also beneficial for the CA inhibitory prop-
erties probably due to the interaction of these moieties
with the hydrophobic half of the active site, as seen in
many sulfonamide/sulfamate–hCA II adducts for which
the X-ray crystal structure has been reported.24 All these
compounds showed an activity comparable to that of
One of our groups reported18 recently the synthesis and
potent cytotoxic activity of some benzo[b]thiophene sul-
fonamide derivatives, which showed good activity against
a wide range of tumor types, with GI50 values in the low
nanomolar range. Some of these compounds possessed
unsubstituted sulfamoyl or N-substituted such moieties
with rather compact groups (ethyl, cyclopropyl, etc.),
and it appeared thus of interest to investigate their inter-
action with several CA isozymes, some of which, as shown
above, were involved in tumorigenesis. We report here an
inhibition study of the cytosolic ubiquitous isozymes CA I
and II, as well as of the tumor-associated isozyme CA IX
(all of human origin) with a selected small library of
benzo[b]thiophene sulfonamide derivatives reported ear-
lier18 to possess potent in vitro antitumor properties,
and compared with other derivatives without cytotoxic
activity against human tumoral cell lines.
The standard, clinically used CA inhibitors (acetazola-
mide AZA, methazolamide MZA, ethoxzolamide
EZA, and dichlorophenamide DCP) were commercially
available from Sigma–Aldrich, whereas indisulam
(IND), a compound in phase II clinical trials as an
antitumor agent,10,19,20 was prepared as reported
earlier. The benzo[b]thiophene sulfonamide derivatives
1a–e, 2a, and 3a–b (Table 1) were obtained from the
corresponding 6-aminobenzo[b]thiophene 1,1-dioxide
derivative, its 2,3-dihydro derivative, and 5-amino-
benzo[b]thiophene 1,1-dioxide, respectively, following
the method described by us before.18 The 6-amino-2,3-
dihydrobenzo[b]thiophene 1,1-dioxide21 was obtained
by reduction of 6-amino-benzo[b]thiophene-1,1-dioxide
using NaOH and an excess of Zn powder.22 Compounds
2b and 2c from Table 1 were prepared from 1a by
reaction with the corresponding alcohol in diluted
KOH.23
N
N
N
O
N
N
O
O
O
O
N
EtO
S
S NH2
S
Me
N
S
NH2
S
S
NH2
H
Me
O
O
O
AZA
MZA
EZA
NH2
SO2NH2
O
S O
H
O
N
S
N
Cl
O
O
H
Cl
S NH2
O
Cl
IND
DCP

4874
A. Innocenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4872–4876
Table 1. Inhibition data for sulfonamides 1–3 investigated in the present paper and standard sulfonamide CA inhibitors, against isozymes hCA I, II,
and IX30
R1
R1
RHNO2S
S
O
S
O
S
H2NO2S
O
RHNO2S
O
O
O
3
1
2
Inhibitor
R
R1
KI* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA IXb
KI (hCA II)/KI (hCA IX)
AZA
MZA
EZA
DCP
IND
1a
—
—
900
780
25
12
14
25
27
0.48
0.52
0.23
0.76
0.62
0.50
0.63
1.20
6.25
—
—
—
8
38
34
50
1200
31
—
H
15
24
15
H
Me
63
85
7.5
8.3
436
475
>1000
8.8
6.3
7.9
7.4
>1000
1b
1c
H
Et
13
H
530
493
>1000
72
362
76
1d
1e
c-PrCH2
H
PhCH2
—
—
H
>1000
10
2a
2b
H
0.88
2.03
2.82
0.57
—
O-n-C4H9
OCH2Ph
—
104
138
75
3.1
2.8
13
2c
3a
—
H
3-MeO-C6H4
3b
—
>1000
>1000
a Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme, by the CO2 hydration method.29
* Errors in the range of 5–10% of the reported value (from three different assays).
ethoxzolamide, one of the best hCA II inhibitors
known,1–3 being more active than AZA, MZA, DCP
or IND, all inhibitors in clinical use/clinical trials for
the management of diverse CA-related disorders, tu-
mors included.1–3 As for hCA I, the substituted deriva-
tives incorporating not very bulky moieties at the
sulfonamide group (1c and 1d) showed weak hCA II
inhibitory activity (KIÕs in the range of 436–475 nM),
whereas the much bulkier derivatives (1e and 3b) were
devoid of activity (KIÕs >1000 nM). (iii) The tumor asso-
ciated isozyme hCA IX was also inhibited well by the
unsubstituted sulfonamides 1a, 1b, 2a–c, and 3a, which
showed KIÕs in the range of 2.8–15 nM. It is obvious that
SAR is very much similar to what was mentioned above
for isozyme II, but these compounds are better inhibi-
tors than all the clinically used derivatives, which
showed inhibition constants in the range of 24–50 nM.
It is also interesting to note the rather good hCA IX
inhibitory properties of the cyclopropylmethyl-substi-
tuted compound 1d, which with a KI of 76 nM behaves
as a moderate inhibitor. The N-ethyl-derivative 1c, was
on the other hand, a much weaker hCA IX inhibitor,
with a KI of 362 nM. The two bulky compounds 1e
and 3b were devoid of hCA IX inhibitory effects. (iv)
A critical issue in the design of CA inhibitors is repre-
sented by the specificity of the inhibitor for the target
isozyme over the ubiquitous ones (hCA I and II, widely
distributed throughout the body).25,26 In the case of
most of the compounds designed here (1a–1c, 2a, and
3a), as for the clinically used inhibitors, no selectivity
of the active compounds for hCA IX over hCA II has
been observed (Table 1). Indeed, the selectivity ratio of
most of the compounds investigated here was in the
range of 0.50–1.20. The compound with a good selectiv-
ity ratio for the inhibition of hCA IX was 1d (selectivity
ratio of 6.25), but this derivative is only a moderate hCA
IX inhibitor. The highly active hCA IX inhibitors 2b
and 2c showed an interesting selectivity for inhibiting
this isozyme over hCA II, being 2.03–2.82 times better
inhibitors of the tumor associated than the cytosolic
isozyme (Table 1). (v) However, the cytotoxicity of
benzo[b]thiophenesulfonamide 1,1-dioxides against
HT-29, CCRF-CEM, K-562, HTB-54 and MEL-AC hu-
man tumoral cell lines or normal human lung fibroblasts
(reported in the previous contributions)18,21 seems to
follow different patterns of action, probably due to the
interaction of these compounds with other enzymes than
the CAs. Thus, 1a, 1c, 1e, 3a, and 3b were cytotoxic,
whereas compounds 1b and 2a were inactive.18,21 The
substitution on position 3 of the benzo[b]thiophene
nucleus or the reduction of its 2,3-double bond leads
to non-cytotoxic derivatives. Furthermore, it has been
demonstrated that benzo[b]thiophenesulfonamide 1,1-
dioxides produce their antitumor effect through inhibi-
tion of a NADH oxidase activity found in the plasma
membrane and conditioned culture medium of different
kinds of tumour cells,27,28 as well as through a process of
apoptosis by accumulation reactive oxygen species
(ROS).29,30 In conclusion, many times the antitumor
activity of a compound may include the interaction with
more than one target, leading to complex pharmacolog-
ical and biochemical processes that in the end lead to a

A. Innocenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4872–4876
4875
diminished growth of the tumor cell.31 However, it is
quite important to try to decipher as many as possible
of these potential mechanisms that lead to derivatives
with potent antitumor activity.
9. Tureci, O.; Sahin, U.; Vollmar, E.; Siemer, S.; Gottert, E.;
Seitz, G.; Parkkila, A. K.; Shah, G. N.; Grubb, J. H.;
Pfreundschuh, M.; Sly, W. S. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 7608.
10. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925.
A series of benzo[b]thiophene-5- and 6-sulfonamide
derivatives previously reported to show cytotoxic activ-
ity and some others newly synthesized has been tested
for the interactions with several CA isozymes, some of
which are known to be involved in tumorigenesis
(hCA IX), whereas others are ubiquitously found in
many normal tissues (the cytosolic isoforms hCA I
and II). The unsubstituted sulfonamides inhibited hCA
I with inhibition constants in the range of 63–138 nM,
hCA II with inhibition constants in the range of 6.3–
8.8 nM, and hCA IX with inhibition constants in the
range of 2.8–15 nM, being thus more active than clini-
cally used inhibitors such as acetazolamide, methazola-
mide, ethoxzolamide, dichlorophenamide or indisulam
(E 7070). Some of these derivatives also showed some
selectivity for the inhibition of the tumor-associated
(hCA IX) over the cytosolic isozyme hCA II. The 2,3-
dihydrobenzo[b]thiophene derivatives could be consid-
ered as new lead compounds for the design of CAIs
because of their absence of cytotoxicity, whereas substi-
tutions on the sulfonamide group could model a certain
degree of selectivity toward some CA isozymes (e.g., CA
IX vs CA II). Although these derivatives may act on
many targets other than the CAs (such as the NADH
oxidase) or may induce apoptosis by accumulation reac-
tive oxygen species, it is quite important to try to deci-
pher as many as possible of the potential mechanisms
that lead to derivatives with potent antitumor activity
in order to develop novel therapeutic strategies for the
management of cancer.
11. Casini, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran,
L. T. Curr. Cancer Drug Targets 2002, 2, 55.
12. Giatromanolaki, A.; Koukourakis, M. I.; Sivridis, E.;
Pastorek, J.; Wykoff, C. C.; Gatter, K. C.; Harris, A. L.
Cancer Res. 2001, 61, 7992.
13. Loncaster, J. A.; Harris, A. L.; Davidson, S. E.; Logue, J.
P.; Hunter, R. D.; Wycoff, C. C.; Pastorek, J.; Ratcliffe, P.
J.; Stratford, I. J.; West, C. M. Cancer Res. 2001, 61, 6394.
14. Chia, S. K.; Wykoff, C. C.; Watson, P. H.; Han, C.; Leek,
R. D.; Pastorek, J.; Gatter, K. C.; Ratcliffe, P.; Harris, A.
L. J. Clin. Oncol. 2001, 19, 3660.
15. Beasley, N. J.; Wykoff, C. C.; Watson, P. H.; Leek, R.;
Turley, H.; Gatter, K.; Pastorek, J.; Cox, G. J.; Ratcliffe,
P.; Harris, A. L. Cancer Res. 2001, 61, 5262.
16. Ivanov, S.; Liao, S. Y.; Ivanova, A.; Danilkovitch-
Miagkova, A.; Tarasova, N.; Weirich, G.; Merrill, M. J.;
Proescholdt, M. A.; Oldfield, E. H.; Lee, J.; Zavada, J.;
Waheed, A.; Sly, W.; Lerman, M. I.; Stanbridge, E. J. Am.
J. Pathol. 2001, 158, 905.
17. 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.
18. Villar, R.; Encio, I.; Migliaccio, M.; Gil, M. J.; Martinez-
Merino, V. Bioorg. Med. Chem. 2004, 12, 963.
19. Supuran, C. T. Expert Opin. Investig. Drugs 2003, 12, 283.
20. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217.
´
´
21. Martınez-Merino V, Gil MJC, Encıo I, Migliaccio M,
Arteaga C. 2000. PCT Patent WO 00/63202.
22. Physical properties of 6-amino-2,3-dihydrobenzo[b]thio-
phene-1,1-dioxide. IR (KBr, cmꢀ1): 1280 (SO2), 3390,
3480 (NH2); 1H NMR (CDCl3 d): 7.10 (d, J = 8 Hz,1H),
6.92 (d, J = 2.2 Hz, 1H), 6.83 (dd, J = 8 Hz, J0 = 2.2 Hz,
1H), 3.92 (s, 2H), 3.44 (t, J = 7 Hz, 2H), 3.22 (t, J = 7 Hz,
2H).
Acknowledgments
23. Example of synthesis for 2b. To a stirred solution of 0.66 g
of 1a (2.68 mmol) in 5 mL of 1-butanol, a solution of
0.35 g of KOH in 2 mL of 1-butanol was drop wise added,
and the stirring was continued at room temperature for
1.5 h. Subsequently, butanol was removed in vacuum and
the solid residue was dissolved in 10 mL of water. The
aqueous layer was washed with ether (2 · 10 mL) and
acidified with HCl to reach pH 2. The solid material was
collected by filtration and recrystallised from water to give
2b (0.32 g, 37% yield): Mp 159–160 ꢁC; IR (KBr, cmꢀ1):
1147, 1345 (SO2), 3319, 3247 (NH2); 1H NMR
(CDCl3 d): 8.29 (s, 1H, H-7), 8.19 (d, J = 8.06 Hz, 1H),
7.80 (d, 1H), 5.27 (t, J = 6.23 Hz, 1H); 4.97 (s, 2H), 3.99–
3.89 (dd, J = 13.18 Hz, 1H), 3.66 (t, J = 6.23 Hz, 2H),
3.56–3.44 (dd, 1H), 1.73–1.37 (m, 4H), 0.95 (t,
J = 7.33 Hz, 3H).
This research was financed in part by a grant of the 6th
Framework Programme of the European Union (EUR-
OXY project). R.V. is indebted to the Navarra Govern-
ment for a grant.
References and notes
1. Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T.
J. Enzyme Inhib. Med. Chem. 2004, 19, 199.
2. Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C.
T. Med. Res. Rev. 2005, 25, 186.
3. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
2003, 23, 146.
4. Supuran, C. T.; Scozzafava, A. Eur. J. Med. Chem. 2000,
35, 867.
24. (a) Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.;
Stubbs, M.; Klebe, G. J. Med. Chem. 2002, 45, 3583; (b)
Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David,
S.; Waldeck, H.; Scha¨fer, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 841; (c) Weber, A.; Casini, A.;
Heine, A.; Kuhn, D.; Supuran, C. T.; Scozzafava, A.;
Klebe, G. J. Med. Chem. 2004, 7, 550; (d) Abbate, F.;
Casini, A.; Scozzafava, A.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2003, 18, 303; (e) Abbate, F.; Coetzee,
A.; Casini, A.; Ciattini, S.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 337; (f) Abbate, F.;
5. Supuran, C. T.; Scozzafava, A. J. Enzyme Inhib. 2000, 15,
597.
6. Mastrolorenzo, A.; Scozzafava, A.; Supuran, C. T. Eur. J.
Pharm. Sci. 2000, 11, 325.
7. Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.;
Zelnik, V.; Opavsky, R.; ZatÕovicova, M.; Liao, S.;
Portetelle, D.; Stanbridge, E. J., et al. Oncogene 1994, 9,
2877.
8. Opavsky, R.; Pastorekova, S.; Zelnik, V.; Gibadulinova,
A.; Stanbridge, E. J.; Zavada, J.; Kettmann, R.; Pastorek,
J. Genomics 1996, 33, 480.

4876
A. Innocenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4872–4876
Casini, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 2357.
25. Supuran, C. T., Scozzafava, A., Conway, J., Eds. Carbonic
Anhydrase—Its Inhibitors and Activators; CRC Press:
Boca Raton, London, New York, 2004.
26. Supuran, C. T. In Carbonic Anhydrase—Its Inhibitors and
Activators; Supuran, C. T., Scozzafava, A., Conway, J.,
Eds.; CRC Press: Boca Raton, 2004; pp 1–23.
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.
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 temper-
ature 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 means of such results. The
cloned enzymes were obtained as reported earlier by this
group.17,30
´
´
27. Alonso, M. M.; Encıo, I.; Martınez-Merino, V.; Gil, M.;
Migliaccio, M. Brit. J. Cancer 2001, 85, 1400.
´
´
28. Encıo, I.; Morre, D. J.; Villar, R.; Gil, M. J.; Martınez-
Merino, V. Br. J. Cancer 2005, 92, 690.
29. Alonso, M. M.; Asumendi, A.; Villar, J.; Gil, M. J.;
´
Martınez-Merino, V.; Encıo, I.; Migliaccio, M. Oncogene
2003, 22, 3759.
´
30. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573,
An SX.18MV-R Applied Photophysics stopped-flow
instrument has been used for assaying the CA-catalyzed
CO2 hydration activity. Phenol red (at a concentration
of 0.2 mM) has been used as an indicator, working at
the absorbance maximum of 557 nm, with 10 Hepes
(pH 7.5) as buffer, 0.1 Na2SO4 (for maintaining constant
31. (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; (c) Vullo, D.; Scozzafava, A.;
Pastorekova, S.; Pastorek, J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 2351.