Carbonic anhydrase inhibitors: Synthesis and inhibition studies against mammalian isoforms I-XV with a series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides

Article abstract of DOI:10.1016/j.bmc.2008.09.032

A series of 2-(hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy- functionalities, as well as the perfluorophenyl moiety have been synthesized and

Full text of DOI:10.1016/j.bmc.2008.09.032

Bioorganic & Medicinal Chemistry 16 (2008) 9113–9120
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors: Synthesis and inhibition studies against
mammalian isoforms I–XV with a series of 2-(hydrazinocarbonyl)-3-
substituted-phenyl-1H-indole-5-sulfonamides
Özlen Güzel a,b, Alessio Innocenti b, Andrea Scozzafava b, Aydın Salman a, Seppo Parkkila c,
Mika Hilvo c, Claudiu T. Supuran b,
*
a Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Institute of Medical Technology and School of Medicine, University of Tampere and Tampere University Hospital, Biokatu 6, FIN-33520 Tampere, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2008
Revised 8 September 2008
Accepted 10 September 2008
Available online 13 September 2008
A series of 2-(hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-5-sulfonamides possessing various 2-,
3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy- functionalities, as well as the
perfluorophenyl moiety have been synthesized and evaluated as inhibitors of 13 catalytically active,
mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, CA I–CA XV (of human (h) or murine
(m) origin). The new compounds were ineffective inhibitors of isozymes hCA III, hCA IV, hCA VA, hCA
VB, hCA VI and mCA XIII, moderate inhibitors of hCA I, hCA VII, hCA IX and mCA XV, and excellent,
low-nanomolar inhibitors of hCA II and hCA XIV. The substitution pattern of the aromatic group in the
3-position of the indole ring influenced biological activity and isozyme inhibition profiles in this series
of sulfonamides. Some of the best and most selective hCA XIV and mCA XV inhibitors ever reported have
been identified in this study.
Keywords:
Carbonic anhydrase
Sulfonamide
Indole-5-sulfonamide
Isoforms CA I–CA XV
CA XIV-selective inhibitor
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
be exploited for the design of effective inhibitors incorporating
such bicyclic moieties.
In a recent preliminary work from this group,1 we investigated
the interaction between 2-(hydrazinocarbonyl)-3-phenyl-1H-in-
dole-5-sulfonamide (compound L), and 12 carbonic anhydrase
(CA, EC 4.2.1.1) isoforms of mammalian (human or murine) origin.
This sulfonamide behaved as a very potent inhibitor of CA I and II
(KIs of 7.2–7.5 nM), a medium potency inhibitor of CA VII, IX, XII
and XIV and a weak inhibitor against the other isoforms, making
it thus an interesting clinical candidate for situations in which a
strong inhibition of CA I and II is needed. Furthermore, the inhibi-
tion profile of L was quite distinct from that of the clinically used
compounds such as acetazolamide AZA, methazolamide, MZA,
ethoxzolamide EZA or dichlorophenamide DCP which promiscu-
ously inhibit most of these mammalian isoforms in the low-nano-
molar range.2 Indeed, many of these CAs are medicinal chemistry
targets for the development of diuretics, antiglaucoma, antiobesity,
anticonvulsant or anticancer drugs/diagnostic tools.2–4
As shown in Figure 1, where a schematic representation for the
binding of sulfonamide L to the human CA II (hCA II) active site is
presented, the inhibitor coordinates to the Zn(II) ion within the en-
zyme active site in the deprotonated state, the same SO2NHÀ func-
tionality of L also making a hydrogen bond with the OH of Thr199.
The CONHNH2 fragment of the inhibitor then participates in a net-
work of three hydrogen bonds with a water molecule (Wat101)
and two amino acid residues known to interact with various inhib-
itors bound to the hCA II active site, that is, Asn62 and Asn67.5–7 A
host of van der Waals interactions between the inhibitor and ami-
no acid residues lining the active site also favorably influence the
strong binding of L to the enzyme cavity (data not shown).1 But
what is more interesting from the drug design point of view, the
3-phenyl moiety of L is accommodated in a hydrophobic region
of the active site1 where enough additional space is available for
various moieties to be introduced as substituents to the phenyl
ring, allowing thus for interesting structure–activity correlations.
Using this X-ray crystal structure as a starting point and the obser-
vation that probably the 3-phenyl moiety of L can be further
substituted without losing the potent CA inhibitory properties,
we report here the synthesis and inhibitory properties against all
the mammalian catalytically active CA isozymes (CA I–XV) of a ser-
ies of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-
The crystal structure of the hCA II adduct with sulfonamide L
reported by us,1 also revealed many favorable interactions be-
tween the inhibitor and the enzyme which explain its strong
low-nanomolar affinity for isoform II (KI of 7.2 nM) but may also
* Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.032

9114
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
H3C
N
N
N
N
H2NO2S
NHNH2
O
CH3CONH
SO2NH2
S
CH3CON
SO2NH2
S
N
H
AZA
MZA
L
SO2NH2
N
Cl
SO2NH2
O
S
SO2NH2
Cl
EZA
DCP
stitutedphenyl)propionates 3, by literature procedures.9 Diazotiza-
tion of sulfanilamide 4 led to the diazonium salt 5 which has been
coupled with the key intermediates 3 allowing the preparation of
the hydrazones 6, which were cyclized in the presence of concen-
trated acid (HCl) to the indoles 7. The last step consisted in conver-
sion of the ester moieties of 7 to the corresponding hydrazides by
treatment with hydrazine hydrate at reflux, leading thus to the de-
sired series of compounds 8a–8n (Scheme 1).
O
O
NH2
HN
Wat101
Asn67
O
H
H
O
NH2
N
H
O
We have chosen the various substituents of the 3-phenyl group
of indoles 8a–8n by considering both the limited available space
within the hydrophobic pocket of the enzyme active site, as dis-
cussed above (see Fig. 1), as well as general medicinal chemistry
considerations, for example, moieties that may increase lipo- or
hydrosolubility of the new compounds, and eventually also inter-
acting in a positive manner with amino acid residues present in
the active site region where this moiety of the inhibitor lies (such
as among others Trp5, Asn62, His64 and Pro201).1,5–7 Thus, we
have incorporated 2-, 3- or 4-substituted phenyl groups possessing
methyl-, halogeno- and methoxy- functionalities in the 3-position
of the indolesulfonamides 8, as well as the perfluorophenyl moiety,
since such substitution patterns would not lead to excessively
bulky groups, and presumably, these derivatives will bind to the
enzyme similarly to the lead molecule L. On the other hand, their
different chemical nature, ensued by the presence of the additional
functionality in the 3-phenyl ring may lead to diverse interactions
of compounds 8 with amino acid residues within the various iso-
forms active sites cavity, and possibly to an inhibition profile
which will be different for this new series of compounds as com-
pared to the lead L or the classical sulfonamide inhibitors AZA–
DCP (see discussion later in the text). In fact, the main interest in
this class of compounds is that of detecting derivatives with a more
isoform-selective profile as compared to the clinically used sulfon-
amides.2–4
NH2
O
NH
N
H
Thr199
NH
OH
Asn62
O
S
-
O
O
NH
Zn2+
His119
His94
His96
Figure 1. Schematic interactions to which L participates when bound within the
hCA II active site, as determined by X-ray crystallography (PDB code 3B4F).1
Hydrogen bonds to which several moieties of the inhibitor participate with amino
acid residues 62, 67 and 199 from the enzyme active site and a water molecule
(Wat101) are shown as dotted lines.
sulfonamides. In contrast to the lead molecule L, these compounds
incorporate 3-phenyl moieties variously substituted in different
positions with methyl-, halogeno (fluoro-, chloro- and bromo-),
and methoxy groups or the 3-perflorophenyl group.
2. Results and discussion
2.1. Chemistry
2.2. Carbonic anhydrase inhibition
Inhibition data against the 13 catalytically active mammalian
(h = human, m = murine) CA isoforms, that is, CA I–CA XV, with
the new sulfonamides 8a–8n reported here as well as the lead L
are shown in Table 1 and Figure 2.
The following structure–activity relationship (SAR) can be
drawn by considering data of Table 1: (i) Unlike the lead L, which
behaves as a very potent hCA I inhibitor (KI of 7.5 nM), the congen-
ers 8a–8n reported here showed moderate–weak inhibitory activ-
ity against this isozyme, with inhibition constants in the range of
104–659 nM. The most effective hCA I inhibitors in this series of
derivatives incorporated 2-Me-, 4-Me-, 3-F-, 4-F-, 4-Cl, 2-Br-phe-
nyl and pentaflurophenyl groups (all of them with KIs around
Sulfonamide L was previously reported by Salman’s group,8
being easily prepared from sulfanilamide as starting material.
Diazotization of sulfanilamide followed by condensation of the dia-
zonium salt with ethyl 2-benzylacetoacetate led to an intermediate
which was cyclized in acidic medium with formation of the ethyl
ester derivative of L, which was then converted to the lead com-
pound by treatment with hydrazine.8 We used a similar approach
for the preparation of the series of congeners of L bearing different
moieties in position 3 of the indole ring (Scheme 1).
Condensation of ring-substituted benzyl bromides 1 with ethyl
acetoacetate 2 gave the key intermediates, ethyl 2-acetyl-3-(sub-

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
9115
R
OC2H5
CH3
OC2H5
CH3
i
R
+
Br
O
O
O
O
1
3
2
R
H2NO2S
H2NO2S
H2NO2S
OC2H5
CH3
iii
+
ii
-
+
N2Cl
O
O
NH2
5
4
R
R
H2NO2S
OC2H5
O
v
iv
N
N
H
N
COOC2H5
H
6
7
R
H2NO2S
HN NH2
N
O
H
8a-n
Scheme 1. Preparation of 2-(hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-5-sulfonamide derivatives 8a–n. Reagents and conditions: (i) EtOH, Na, (ii) NaNO2, 37% HCl,
0 °C; (iii) KOH, 0 °C; (iv) 37% HCl, reflux, 4 h; (v) H2NNH2ÁH2O, reflux, 6 h.
Table 1
Inhibition of CA isozymes I–XV (of human = h, and murine = m origin) with sulfonamides 8a–n and the lead molecule L as standard13
R
H2NO2S
NHNH2
O
N
H
8
Compound 8
R
KI (nM)#,*
KI (nM)#
hCA I
hCA II
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IX**
hCA XII**
mCA XIII
hCA XIV
mCA XV
L
H
7.5
107
730
104
621
116
108
640
311
112
110
510
659
342
110
7.2
11.6
48.4
60.5
36.0
8.6
15.5
38.8
9.2
11.6
48.5
54.1
40.8
7.4
1.4 Â 106
7.8 Â 106
6.9 Â 106
6.7 Â 106
7.3 Â 106
7.1 Â 106
7.5 Â 106
7.4 Â 106
6.2 Â 106
6.9 Â 106
3.9 Â 106
5.5 Â 106
5.2 Â 106
5.5 Â 106
5.4 Â 106
9000
9096
7760
3035
9500
6260
3650
8680
6140
4030
7325
5525
2912
5740
5215
1100
775
748
727
804
784
705
855
743
658
842
790
619
791
750
1100
906
814
690
941
910
576
937
844
506
947
858
672
924
862
2650
7384
6325
4230
7610
6070
3980
7440
5570
3525
6950
5760
4450
7490
5620
89
80
69
62
85
69
54
82
72
61
83
72
58
69
68
102
79
77
61
75
71
65
77
73
59
74
70
63
71
70
110
76
68
55
83
69
58
76
70
56
77
682
60
78
79
2633
894
820
738
893
801
740
935
816
734
992
867
759
810
734
48
66
64
47
28
59
54
49
58
53
47
70
57
47
49
48
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
2-Me
3-Me
4-Me
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
3-OMe
F5
9.3
8.5
7.6
9.2
8.4
7.8
9.5
8.5
7.4
9.3
8.5
7.0
8.8
8.7
7.0
a
From Ref. 1.
#
Errors in the range of 5% of the reported data from three different assays.
h = human; m = murine isozyme.
Catalytic domain.
*
**
110 nM), whereas the remaining compounds were typically three
ble 1). It is thus clear that any substitution in the 3-phenyl moiety
to six times less effective, with KIs in the range of 310–659 nM (Ta-
of the lead L has as a consequence a drastic loss of hCA I inhibitory

9116
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
9.00
8.50
8.00
8.50-9.00
8.00-8.50
7.50-8.00
7.00-7.50
6.50-7.00
6.00-6.50
5.50-6.00
5.00-5.50
7.50
7.00
6.50
L
8b
8d
6.00
5.50
8f
8h
5.00
8j
8l
8n
Figure 2. 3D-graph showing isozyme type on the x-axis versus compound L and 8a–8n along the abscissa versus ÀlogKI on the vertical axis.
power, the most effective inhibitors among derivatives 8 reported
here (i.e, sulfonamides 8a and 8c) being 14.2 times weaker inhib-
itors against this isozyme as compared to L. This may be due to
the fact that the hCA I active site is rather restricted (as compared
to that of hCA II for example) due to the presence of the bulky res-
idue His200 (which in CA II is Thr) near the Zn(II) ion of the en-
zyme, at the bottom of the active site.10,11
(iii) As most sulfonamides investigated up to now,12 derivatives
8 reported here as well as the lead molecule L, show very weak hCA
III inhibitory activity, with inhibition constants in the range of 1.4–
7.8 mM. The clinically used sulfonamides also inhibit this isoform
in the millimolar range.12
(iv) The membrane-bound isoform hCA IV is also not very sen-
sitive to all sulfonamide inhibitors, as shown earlier by us.13 Thus,
(ii) Against the physiologically dominant isoform hCA II, the
new compounds 8 investigated here showed interesting inhibitory
activity, with inhibition constants in the range of 7.0–60.5 nM.
Thus, similarly to the lead L (KI of 7.2 nM), several of the new deriv-
atives showed excellent inhibitory activity. These compounds
incorporate 2-Me-, 3-F-, 3-Cl-, 4-Cl-, 3-methoxy-phenyl and per-
fluorophenyl moieties in position 3 of the indole ring. Thus, for
hCA II which possesses a wider active site cavity as compared to
hCA I,5–7,10,11 many substitution patterns at the 3-phenyl ring are
tolerated without loss of inhibitory activity, but also without a
net gain. Indeed, all these derivatives mentioned above ( 8a, 8e,
8h, 88i, 8m and 8n) showed comparable activity with the lead L,
making thus SAR rather complicated. It is for example obvious that
the phenyl ring can bear a small and compact group in either
ortho-, meta- or para-positions, while maintaining very potent CA
II inhibitory activity, but many of the various isomers (compare
8a, 8b and 8c; 8d–8f; 8g–i, and 8j–8l, respectively) show very dif-
ferent CA II inhibitory activity. Less effective CA II inhibition was
observed for derivatives 8b, 8c, 8d, 8g and 8j–8l, which showed
KIs in the range of 36–60.5 nM, appreciably inhibiting the enzyme.
The only compound with an intermediate activity between the
very efficient and less efficient CA II inhibitors was the 4-fluoro-
phenyl substituted analogue 8f, which with a KI of 15.5 nM is a po-
tent CA II inhibitor, comparable with the clinically used
compounds AZA–DCP (data not shown).2–4
the lead L behaves as a weak hCA IV inhibitor,1 with a KI of 9.0
l
M.
The same trend has been observed here for its congeners 8, which
possess inhibition constants in the range of 2.9–9.5 M (Table 1). It
l
may be observed that some of the substitution patterns present in
the new derivatives reported here, such as the 4-Me- or 4-Br-phe-
nyl ones of 8c and 8l, led to a threefold improvement of the hCA IV
inhibitory activity of these derivatives over the lead L, with KIs of
2.9–3.0 lM.
(v) Against the two mitochondrial isoforms hCA VA and hCA VB,
the lead molecule L showed weak inhibitory activity (KIs of
1.1 l
M).1 While this activity was improved against both isoforms
and with all the new congeners of L reported here, they still remain
weak–medium potency inhibitors of hCA VA (KIs in the range of
0.619–0.855
lM) and hCA VB (KIs in the range of 0.506–
0.941 M). A second salient feature is that SAR is very flat for these
l
two isoforms, with all the compounds possessing a rather similar
inhibitory activity. This is probably due to the particular active site
environments of these isozymes which are not so well studied at
this moment.14
(vi) The secretory isoform hCA VI15 was also weakly inhibited
by L (KI of 2.65
7.490 M, Table 1). However, in contrast to the results or for hCA
l
M)1 and by its congeners 8a–8n (KIs of 3.525–
l
VA and VB inhibition discussed above, in this case all substitution
patterns present in the new sulfonamides 8 were detrimental to
the hCA VI inhibitory activity as compared to the lead L, with the

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
9117
new derivatives being generally 1.34–2.13 times less inhibitory as
compared to L.
Identifying highly potent and isoform-selective inhibitors as the
ones reported here for the first time, may thus help understanding
the involvement and physiological function of this isoform.
(xii) The last mammalian isoform discovered so far, mCA XV,23
was never investigated up to now for its interaction with sulfon-
amide inhibitors, except acetazolamide (which is a moderate
inhibitor, with a KI of 72 nM).22b Data of Table 1 show that many
of the new compounds investigated here as well as the lead L,
show better mCA XV inhibitory activity as compared to the clini-
cally used drug acetazolamide, with KIs in the range of 28–
70 nM. The substitution pattern leading to the most effective CA
XV inhibitor is that present in 8c, with a 4-tolyl group in the 3-po-
sition of the indole ring, whereas the one leading to the CA XV
‘worst’ inhibitor (which has the same potency as acetazolamide)
is the 2-bromophenyl substitution present in 8j (KI of 70 nM).
The 3D plot of Figure 2 shows the variation of the inhibitory
power of these derivatives (as ÀlogKI on the z-axis) as a function
of the substitution pattern of compounds L and 8a–8n (y-axis)
for the 13 CA isozymes investigated here, CA I–CA XV, shown on
the x-axis. As far as we know, this is the first study reporting the
full inhibition profile against all 13 mammalian CA isozymes for
a series of congeneric sulfonamides bearing various substitution
patterns. The complicated landscape observed in Figure 2 is a clear
proof that achieving some isoform-selective sulfonamide CA inhib-
itors is possible, since the blue, CA XIV peaks of most of these new
derivatives are quite prominent. This type of study, although time
consuming and difficult to be done (and presented) might shed
new light on specificity issues when designing inhibitors of en-
zymes which possess a large number of isoforms, as the mamma-
lian CA family
(vii) The brain-associated cytosolic isoform hCA VII16was mod-
erately inhibited by the lead L (KI of 89 nM) and by its congeners
8a–8n, with inhibition constants in the range of 54–85 nM. It may
be observed again a rather flat SAR with all these compounds
behaving as effective, but not very potent hCA VII inhibitors, irre-
spective of the substitution pattern of the 3-phenyl moiety of the
indole ring, a similar trend to that observed for the inhibition of
hCA VA and hCA VB (but compounds 8a–8n are much better inhib-
itors of this cytosolic isoform as compared to the mitochondrial
ones discussed above).
(viii) The tumor-associated isozyme hCA IX17,18 was modestly
inhibited by the lead L and its congeners 8, with inhibition con-
stants in the range of 59–102 nM. SAR was rather flat, with all
the new derivatives 8 being more effective inhibitors than the lead,
but the increase in the inhibitory properties were rather modest,
the best new CA IX inhibitor being the 4-Cl-phenyl derivative 8i,
with a KI of 59 nM.
(ix) The second transmembrane, tumor-associated isoform, hCA
XII19 was weakly inhibited by the lead L (KI of 110 nM), but all the
new compounds 8 (except the meta-bromophenyl-substituted one,
8k) showed better inhibitory activities against this medicinally rel-
evant isoform, with inhibition constants in the range of 55–83 nM.
It is difficult to explain the weak inhibitory activity of 8k compared
to its isomers 8j and 8l, which are 8.85 and 11.36 times better
inhibitors, respectively. Some of these compounds were roughly
two times better CA XII inhibitors as compared to the lead L, with
KIs around 55–58 nM (Table 1).
(x) The lead L was an ineffective inhibitor of the last cytosolic
isoform, mCA XIII20 (KI of 2.633
lM) whereas its substituted cong-
eners 8 behaved as more effective inhibitors with KIs in the range
of 734–992 nM. Again there are no significant differences of activ-
ity for the various substitution patterns of the 3-phenyl group in
compounds 8, except for this enhancement of the inhibitory power
as compared to the lead L.
3. Conclusions
Using 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide
as the lead molecule, a compound for which the X-ray crystal
structure in adduct with hCA II has been reported, a series of cong-
eners possessing various 3-aryl groups has been synthesized and
evaluated as inhibitors of 13 catalytically active, mammalian CA
isoforms, that is, CA I–CA XV (of human or murine origin). The
new compounds reported here incorporated in the 3-position of
the indole ring 2-, 3- or 4- substituted phenyl groups with
methyl-, halogeno- and methoxy-functionalities, as well as the
perfluorophenyl moiety. The new compounds were ineffective
inhibitors of isozymes hCA III, hCA IV, hCA VA, hCA VB, hCA VI
and mCA XIII, moderate inhibitors of hCA I, hCA VII, hCA IX and
mCA XV, and excellent inhibitors of hCA II and hCA XIV. The substi-
tution pattern of the aromatic group in the 3-position of the indole
ring generally influenced biological activity and isozyme inhibition
profiles in this series of sulfonamides. Some of the best and most
selective hCA XIV and mCA XV inhibitors ever reported have been
identified in this study.
(xi) Although the lead L behaved as a medium potency inhibitor
of the transmembrane isoform hCA XIV21 (KI of 48 nM), all the new
derivatives 8 investigated here showed excellent, low-nanomolar
inhibitory properties against this physiologically relevant isoform,
with KIs in the range of 7.0–9.5 nM. This is a very interesting result,
as very few low-nanomolar hCA XIV inhibitors were reported up to
now2,21 and this isoform is quite abundant in several organs (such
as the brain, kidneys, liver, etc.)21 where its functions are not very
well understood. In addition, some of these derivatives (e.g., 8l) can
be indeed considered as isoform XIV selective CA inhibitors, since
the selectivity ratios for the inhibition of CA XIV over CA VII (the
next most inhibited isozyme by this compound among the 13
CAs investigated here) was of 8.28. Indeed, CA XIV shows a strong
expression in the cortex region of kidneys, where it is restricted to
the proximal tubules of S1 and S2 segments.22 The expression is
high in the apical plasma membrane, while the basolateral mem-
brane shows lower signal. CA XIV is also expressed in the initial
portion of the thin descending limbs of Henle. The high expression
of CA XIV in the proximal tubulus suggested that this isozyme may
have an important role in the bicarbonate reabsorption in the kid-
neys.22 CA XIV has also been found to be expressed on neuronal
membranes and axons in both mouse and human brain. The most
intense expression has been observed on large neuronal bodies and
axons in the anterolateral part of the pons and medulla oblongata.
In addition, CA XIV was found to be expressed in the hippocampus,
corpus callosum, cerebellar white matter and peduncles, pyramidal
tract and choroid plexus.22 CA XIV has been also identified in the
murine liver, where it was found to be expressed at the plasma
membrane of hepatocytes.22 It may be observed that the physio-
logical role of this isoform is not well understood for the moment.
4. Experimental
4.1. Chemistry
Buffers, and chemicals were from Sigma–Aldrich (Milan, Italy)
of highest purity available, and were used without further purifica-
tion. All CA isozymes were recombinant ones produced and puri-
fied in our laboratory as described earlier.14–16
4.1.1. Ethyl 2-acetyl-3-(substitutedphenyl)propionate 39
An ethanolic solution of sodium ethoxide was prepared by the
addition of sodium (0.5 g, 22 mmol) to dry ethanol (20 mL). Ethyl
acetoacetate (10.4 g, 80.0 mmol) was added to the reaction

9118
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
mixture, and the solution was stirred for 10 min. at room temper-
ature. Substitutedbenzyl bromide (5 g, 20 mmol) was added, and
the reaction mixture was heated under reflux for 15 h. The mixture
was concentrated under reduced pressure and the residue was
taken up in ether (100 mL). The ether solution was washed with
water (50 mL) and was dried. The residue after removal of solvent
under reduced pressure was purified by fractional distillation.
CONH). Anal. Calcd for C16H16N4O3S (344.388): C, 55.80, H, 4.68;
N, 16.27; S, 9.31. Found: C, 55.54; H, 4.33; N, 16.24; S, 9.49.
4.1.4.4. 3-(2-Fluorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8d
. Yield 60%; mp 246–8 °C; IR(KBr) (t
, cmÀ1),
1644 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.53 (2H, s,
NHNH2), 7.21 (2H, s, SO2NH2), 7.38 (2H, t, J = 7.81 Hz, Ar-H),
7.50–7.56 (2H, m, Ar-H), 7.67 (1H, d, J = 8.78 Hz, indole C7–H),
7.76 (1H, dd, J = 8.78, 1.46 Hz, indole C6–H), 7.92 (1H, s, indole
C4–H), 9.14 (1H, s, CONH), 12.24 (1H, br s, indole NH). Anal. Calcd
for C15H13FN4O3S (348.352): C, 51.72, H, 3.76; N, 16.08; S, 9.20.
Found: C, 51.69; H, 3.54; N, 15.75; S, 8.84.
4.1.2. 2-Substitutedbenzyl-2-[N-(4-sulfonamidophenyl)hydra-
zono]ethanoates 6
To a solution of 0.01 mol sulfanilamide in 4 ml of 37% HCl, 10 ml
of 7% NaNO2 aqueous solution was added dropwise at 0 °C. This
solution, containing diazonium salt, was poured into an ice-cold
mixture of 2.3 g (a little excess of 0.01 mol) ethyl 2-substitutedb-
enzylacetoacetate, 10 ml of EtOH, 20 ml of H2O and 2.7 g of KOH.
The mixture was kept cold overnight. The hydrazone produced as
an oil was separated, dissolved in Et2O, washed with H2O and dried
over anhydrous Na2SO4. Et2O was distilled, the oily residue was
treated with 5 ml of 37% HCl and set aside for 5 h at room temper-
ature. The resulting solid substance was recrystallized from EtOH.
4.1.4.5. 3-(3-Fluorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8e
. Yield 47%; mp 274–7 °C; IR(KBr) (t
, cmÀ1),
1645 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.52 (2H, s,
NHNH2), 7.15 (2H, s, SO2NH2), 7.20 (1H, td, J = 8.78, 2.44 Hz, Ar-
H), 7.29 (2H, t, J = 6.84 Hz, Ar-H), 7.52 (1H, q, J = 6.88 Hz, Ar-H),
7.58 (1H, d, J = 8.78 Hz, indole C7–H), 7.69 (1H, dd, J = 8.79,
1.46 Hz, indole C6–H), 8.03 (1H, d, J = 1.46 Hz, indole C4–H), 9.19
(1H, s, CONH), 12.13 (1H, br s, indole NH). Anal. Calcd for
C15H13FN4O3S (348.352): C, 51.72, H, 3.76; N, 16.08; S, 9.20. Found:
C, 52.17; H, 4.07; N, 15.88; S, 8.84.
4.1.3. Ethyl 5-(aminosulfonyl)-3-substitutedphenyl-1H-indole-
2-carboxylates 78
A mixture of 0.01 mol ethyl 2-substitutedbenzyl-2-[N-(4-sul-
fonamidophenyl)hydrazono]ethanoate 6 and about 10 ml of 37%
HCl was heated on a water bath for 4 h, cooled and poured into
100 ml of H2O, the crude product was filtered, washed with H2O,
and recrystallized from EtOH.
4.1.4.6. 3-(4-Fluorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8f. Yield 48%; mp 269–71 °C; IR(KBr) (t
, cmÀ1),
1645 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.49 (2H, s,
NHNH2), 7.13 (2H, s, SO2NH2), 7.31 (2H, t, J = 8.78 Hz, Ar-H),
7.50 (2H, t, J = 6.84 Hz, Ar-H), 7.57 (1H, d, J = 8.30 Hz, indole C7–
H), 7.68 (1H, dd, J = 8.78, 1.47 Hz, indole C6–H), 7.98 (1H, d,
J = 0.98 Hz, indole C4–H), 9.02 (1H, s, CONH), 12.10 (1H, br s, in-
dole NH). Anal. Calcd for C15H13FN4O3S (348.352): C, 51.72, H,
3.76; N, 16.08; S, 9.20. Found: C, 51.79; H, 3.53; N, 15.79; S, 9.13.
4.1.4. 2-(Hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-
5-sulfonamides 8a–8n
Ethyl 5-(aminosulfonyl)-3-substitutedphenyl-1H-indole-2-car-
boxylate 7 (0.01 mol, 3.45 g) was dissolved in 20 ml of EtOH,
4 ml of H2NNH2.H2O was added and refluxed for 6 h, cooled and
kept cold overnight. The resulting crystals were filtered off, washed
with Et2O and recrystallized from EtOH/DMF.
4.1.4.7. 3-(2-Chlorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8g
. Yield 35%; mp 259–61 °C; IR(KBr) (t
, cmÀ1),
1640 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.45 (2H, s,
NHNH2), 7.13 (2H, s, SO2NH2), 7.45 (3H, d, J = 2.93 Hz, Ar-H),
7.58–7.62 (2H, m, indole C7-H and Ar-H), 7.69 (2H, d, J = 8.78 Hz in-
dole C4-H and indole C6–H), 8.72 (1H, s, CONH), 12.10 (1H, br s, in-
dole NH). Anal. Calcd for C15H13ClN4O3S (364.806): C, 49.39, H,
3.59; N, 15.36; S, 8.79. Found: C, 48.98; H, 3.76; N, 15.50; S, 8.40.
4.1.4.1. 3-(2-Methylphenyl)-2-(hydrazinocarbonyl)-1H-indole-5-
sulfonamide 8a. Yield 29%; mp 249–50 °C; IR (KBr) (t
, cmÀ1), 1646
(C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 2.04 (3H, s, phenyl 2-
CH3), 4.46 (2H, s, NHNH2), 7.11 (2H, s, SO2NH2), 7.26 (1H, d,
J = 7.81 Hz, Ar-H), 7.32 (1H, d, J = 7.32 Hz, Ar-H), 7.35–7.41 (2H, m,
Ar-H), 7.60 (2H, d, J = 9.28 Hz, indole C4,7–H), 7.67 (1H, dt, J = 8.30,
1.71 Hz, indole C6–H), 8.03 (1H, s, CONH), 12.16 (1H, br s, indole
NH). Anal. Calcd for C16H16N4O3S (344.388): C, 55.80, H, 4.68; N,
16.27; S, 9.31. Found: C, 55.66; H, 4.89; N, 16.59; S, 9.61.
4.1.4.8. 3-(3-Chlorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8h
.
Yield 59%; mp 301–4 °C; IR(KBr) (t
, cmÀ1),
1643 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.52 (2H, s,
NHNH2), 7.16 (2H, s, SO2NH2), 7.42 (2H, t, J = 9.76 Hz, Ar-H),
7.49–7.52 (2H, m, Ar-H), 7.59 (1H, d, J = 8.78 Hz, indole C7–H),
7.70 (1H, dd, J = 8.79, 1.46 Hz, indole C6–H), 8.00 (1H, s, indole
C4–H), 9.23 (1H, s, CONH), 12.60 (1H, br s, indole NH). Anal. Calcd
for C15H13ClN4O3S (364.806): C, 49.39, H, 3.59; N, 15.36; S, 8.79.
Found: C, 49.26; H, 3.70; N, 14.98; S, 8.42.
4.1.4.2. 3-(3-Methylphenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8b
. Yield 58%; mp 313–4 °C; IR (KBr) (t
, cmÀ1),
1643 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 2.38 (3H, s,
phenyl 3-CH3), 4.48 (2H, s, NHNH2), 7.13 (2H, s, SO2NH2), 7.21
(1H, d, J = 7.32 Hz, Ar-H), 7.25 (1H, d, J = 7.81 Hz, Ar-H), 7.29 (1H,
s, Ar-H), 7.38 (1H, t, J = 7.32 Hz, Ar-H), 7.56 (1H, d, J = 8.79 Hz, in-
dole C7–H), 7.67 (1H, dd, J = 8.78, 1.46 Hz, indole C6–H), 7.96 (1H,
d, J = 1.46 Hz, indole C4–H), 8.74 (1H, s, CONH), 12.06 (1H, brs., in-
dole NH). Anal. Calcd for C16H16N4O3S (344.388): C, 55.80, H, 4.68;
N, 16.27; S, 9.31. Found: C, 55.31; H, 4.63; N, 16.65; S, 9.47.
4.1.4.9. 3-(4-Chlorophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8i. Yield 70%; mp 273–5 °C; IR(KBr) (t
, cmÀ1),
1640 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.50 (2H, s,
NHNH2), 7.13 (2H, s, SO2NH2), 7.48 (2H, d, J = 8.79 Hz, Ar-H),
7.53 (2H, d, J = 8.30 Hz, Ar-H), 7.58 (1H, d, J = 8.30 Hz, indole C7–
H), 7.68 (1H, dd, J = 8.78,1.46 Hz, indole C6–H), 8.01 (1H, d,
J = 0.98 Hz, indole C4–H), 9.14 (1H, s, CONH), 12.15 (1H, br s, in-
dole NH). Anal. Calcd for C15H13ClN4O3S (364.806): C, 49.39, H,
3.59; N, 15.36; S, 8.79. Found: C, 49.05; H, 3.77; N, 14.98; S, 8.43.
4.1.4.3. 3-(4-Methylphenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8c
. Yield 19%; mp 262–4 °C; IR(KBr) (t
, cmÀ1),
1643 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 2.38 (3H, s,
phenyl 4-CH3), 4.47 (2H, s, NHNH2), 7.11 (2H, s, SO2NH2), 7.30
(2H, d, J = 7.81 Hz, Ar-H), 7.36 (2H, d, J = 7.81 Hz, Ar-H), 7.56 (1H,
d, J = 8.79 Hz, indole C7–H), 7.67 (1H, dd, J1 = 8.79, J2 = 1.46 Hz, in-
dole C6–H), 7.97 (1H, d, J = 1.47 Hz, indole C4–H), 8.73 (1H, s,
4.1.4.10. 3-(2-Bromophenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8j. Yield 85%; mp 260–2 °C; IR(KBr) (t
, cmÀ1),

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 9113–9120
9119
1639 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.46 (2H, s,
NHNH2), 7.13 (2H, s, SO2NH2), 7.38 (1H, td, J = 7.32, 1.95 Hz, Ar-
H), 7.43 (1H, dd, J = 7.32, 1.47 Hz, Ar-H), 7.50 (1H, t, J = 7.32 Hz,
Ar-H), 7.60 (1H, d, J = 8.79 Hz, Ar-H), 7.64 (1H, s, indole C4–H),
7.69 (1H, dd, J = 8.78, 0.98 Hz, indole C6–H), 7.77 (1H, d,
J = 7.81 Hz, indole C7–H), 8.55 (1H, s, CONH), 12.10 (1H, br s, indole
NH). Anal. Calcd for C15H13BrN4O3S (409.257): C, 44.02, H, 3.20; N,
13.69; S, 7.83. Found: C, 43.65; H, 2.92; N, 13.34; S, 7.45.
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. KIs were obtained from Lineweaver–Burk plots, as reported
earlier.12–16 All CAs used here were recombinant proteins obtained
as reported earlier by our groups.12–22
Acknowledgments
4.1.4.11. 3-(3-Bromophenyl)-2-(hydrazinocarbonyl)-1H-indole-
This research was financed in part by two grants of the 6th
Framework Programme of the European Union (EUROXY and DeZ-
nIT projects). Ö.G. is grateful to TUBITAK (Ankara, Turkey) for the
providing financing under the Contract No. 2219/2008.
5-sulfonamide 8k. Yield 64%; mp 302–3 °C; IR(KBr) (t
, cmÀ1),
1641 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.51 (2H, s,
NHNH2), 7.16 (2H, s, SO2NH2), 7.42–7.46 (2H, m, Ar-H), 7.56–7.58
(1H, m, Ar-H), 7.59 (1H, d, J = 8.30 Hz, indole C7–H), 7.65 (1H, s,
Ar-H), 7.69 (1H, dd, J = 8.30, 1.47 Hz, indole C6–H), 7.99 (1H, d,
J = 1.46 Hz, indole C4–H), 9.23 (1H, s, CONH), 12.60 (1H, br s, indole
NH). Anal. Calcd for C15H13BrN4O3S (409.257): C, 44.02, H, 3.20; N,
13.69; S, 7.83. Found: C, 43.75; H, 3.03; N, 13.47; S, 7.50.
References and notes
1. Güzel, Ö.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Salman, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2008, 18, 152.
2. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T.;
Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.
4.1.4.12. 3-(4-Bromophenyl)-2-(hydrazinocarbonyl)-1H-indole-
3. (a) Supuran, C. T.; Scozzafava, A.; Conway, J. Carbonic Anhydrase—Its Inhibitors
and Activators; CRC Press: Boca Raton, New York, London, 2004; (b) Winum, J.
Y.; Rami, M.; Scozzafava, A.; Montero, J. L.; Supuran, C. Med. Res. Rev. 2008, 28,
445; (c) Köhler, K.; Hillebrecht, A.; Schulze Wischeler, J.; Innocenti, A.; Heine,
A.; Supuran, C. T.; Klebe, G. Angew. Chem. Int. Ed. 2007, 46, 7697.
4. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2004, 19, 199; (b) Supuran, C. T.; Scozzafava, A.; Casini, A. Development
of sulfonamide carbonic anhydrase inhibitors. In Carbonic Anhydrase—Its
Inhibitors and Activators; Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC
Press: Boca Raton, 2004; pp 67–147.
5. (a) De Simone, G.; Vitale, R. M.; Di Fiore, A.; Pedone, C.; Scozzafava, A.; Montero,
J. L.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2006, 49, 5544; (b) De Simone, G.;
Di Fiore, A.; Menchise, V.; Pedone, C.; Antel, J.; Casini, A.; Scozzafava, A.; Wurl,
M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 2315; (c) Winum, J. Y.;
Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.; Ciattini, S.; Montero, J. L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 7024.
6. (a) Boriack, P. A.; Christianson, D. W.; Kingery-Wood, J.; Whitesides, G. M.
J. Med. Chem. 1995, 38, 2286; (b) Kim, C. Y.; Chang, J. S.; Doyon, J. B.; Baird, T. T.;
Fierke, C. A.; Jain, A.; Christianson, D. W. J. Am. Chem. Soc. 2000, 122, 12125; (c)
Grüneberg, S.; Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3588.
7. (a) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.;
De Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329; (b) Casini, A.;
Antel, J.; Abbate, F.; Scozzafava, A.; David, S.; Waldeck, H.; Schafer, 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, 47, 550;
(d) Menchise, V.; De Simone, G.; Alterio, V.; Di Fiore, A.; Pedone, C.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2005, 48, 5721.
5-sulfonamide 8l. Yield 59%; mp 261–3 °C; IR(KBr) (t
, cmÀ1),
1641 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.50 (2H, s,
NHNH2), 7.14 (2H, s, SO2NH2), 7.42 (2H, d, J = 8.78 Hz, Ar-H), 7.58
(1H, d, J = 8.78 Hz, indole C7–H), 7.66–7.70 (3H, m, indole C6-H
and Ar-H), 8.01 (1H, d, J = 1.46 Hz, indole C4–H), 9.15 (1H, s, CONH),
12.16 (1H, brs., indole NH). Anal. Calcd for C15H13BrN4O3S
(409.257): C, 44.02, H, 3.20; N, 13.69; S, 7.83. Found: C, 43.64; H,
3.09; N, 13.32; S, 7.56.
4.1.4.13. 3-(3-Methoxyphenyl)-2-(hydrazinocarbonyl)-1H-indole-
5-sulfonamide 8m. Yield 70%; mp 286–8 °C; IR(KBr) (t
, cmÀ1),
1643 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 3.80 (3H, s,
phenyl 3-OCH3), 4.50 (2H, s, NHNH2), 6.96–6.98 (1H, m, Ar-H),
7.03–7.04 (2H, m, Ar-H), 7.14 (2H, br s, SO2NH2), 7.41 (1H, t,
J = 7.30 Hz, Ar-H), 7.57 (1H, d, J = 8.78 Hz, indole C7–H), 7.67 (1H,
dd, J = 8.30, 1.47 Hz, indole C6–H), 8.01 (1H, d, J = 1.47 Hz, indole
C4–H), 8.86 (1H, s, CONH), 11.43 (1H, br s, indole NH). Anal. Calcd
for C16H16N4O4S (360.387): C, 53.32, H, 4.47; N, 15.55; S, 8.90.
Found: C, 53.33; H, 4.26; N, 15.41; S, 8.83.
8. Ergenç, N.; Salman, A.; Bankaog˘lu, G. Pharmazie 1990, 45, 346.
9. Beckwith, A. L. J.; O’Shea, D. M.; Westwood, S. W. J. Am. Chem. Soc. 1988, 110,
2565.
10. Temperini, C.; Innocenti, A.; Guerri, A.; Scozzafava, A.; Rusconi, S.; Supuran, C.
T. Bioorg. Med. Chem. Lett. 2007, 17, 2210.
11. Temperini, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2006, 16,
5152.
12. (a) Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Cecchi, A.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. 2007, 15, 7229; (b) Innocenti, A.; Firnges, M.
A.; Antel, J.; Wurl, M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2005, 15, 1149.
4.1.4.14. 3-(2,3,4,5,6-Pentafluorophenyl)-2-(hydrazinocarbon-
yl)-1H-indole-5-sulfonamide 8n. Yield 52%; mp 86–7 °C; IR(KBr)
(t
, cmÀ1), 1658 (C@O); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 4.43
(2H, s, NHNH2), 7.10 (2H, s, SO2NH2), 7.52 (1H, d, J = 8.79 Hz, indole
C7–H), 7.59 (1H, dd, J = 8.78, 3.42 Hz, indole C6–H), 7.66–7.69 (1H,
m, indole C4–H), 9.28 (1H, s, CONH), 12.20 (1H, br s, indole NH).
Anal. Calcd for C15H9F5N4O3S (420.313): C, 42.86, H, 2.16; N,
13.33; S, 7.63. Found: C, 42.45; H, 2.55; N, 13.25; S, 7.32.
13. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
14. (a) Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava, A.; Supuran, C. T.
J. Med. Chem. 2004, 47, 1272; (b) Nishimori, I.; Vullo, D.; Innocenti, A.;
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J. Med. Chem. 2005, 48, 7860.
15. Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Scozzafava, A.; Supuran, C. T.
J. Med. Chem. 2007, 50, 381.
4.2. CA catalytic/inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of vari-
ous CA isozymes as reported by Khalifah.13 Phenol red (at a con-
centration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as
buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic
strength; these anions are not inhibitory in the used concentra-
tion),12–15 following the CA-catalyzed CO2 hydration reaction for
a period of 5–10 s. Saturated CO2 solutions in water at 25 °C were
used as substrate. Stock solutions of inhibitors were prepared at a
concentration of 10 mM (in DMSO/water 1:1, v/v) and dilutions up
to 0.01 nM done with the assay buffer mentioned above. At least
seven different inhibitor concentrations have been used for mea-
suring the inhibition constant. Inhibitor and enzyme solutions
were preincubated together for 10 min at room temperature prior
16. Vullo, D.; Voipio, J.; Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.; Kaila, K.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 971.
17. (a) Pastorekova, S.; Zatovicova, M.; Pastorek, J. Curr. Pharm. Des. 2008, 14, 685;
(b) Pastorekova, S.; Pastorek, J. Cancer-related carbonic anhydrase isozymes
and their inhibition. In Carbonic Anhydrase—Its Inhibitors and Activators;
Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton (FL),
2004; pp 255–281.
18. (a) Svastova, E.; Hulikova, A.; Rafajova, M.; Zatovicova, M.; Gibadulinova, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S.
FEBS Lett. 2004, 577, 439; (b) Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.;
Scozzafava, A.; Pastorekova, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13,
1005; (c) Cecchi, A.; Hulikova, A.; Pastorek, J.; Pastorekova, S.; Scozzafava, A.;
Winum, J.-Y.; Montero, J.-L.; Supuran, C. T. J. Med. Chem. 2005, 48, 4834.
19. Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.; Scozzafava, A.; Pastorekova,
S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 963.
20. (a) Lehtonen, J. M.; Parkkila, S.; Vullo, D.; Casini, A.; Scozzafava, A.; Supuran, C.
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9120
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