Carbonic anhydrase inhibitors. Interaction of 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12 mammalian isoforms: Kinetic and X-ray crystallographic studies

Article abstract of DOI:10.1016/j.bmcl.2007.10.110

2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA, EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applications, suc

Full text of DOI:10.1016/j.bmcl.2007.10.110

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 152–158
Carbonic anhydrase inhibitors. Interaction of
2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12
mammalian isoforms: Kinetic and X-ray crystallographic studiesq
a
b
b
b
¨
Ozlen Guzel, Claudia Temperini, Alessio Innocenti, Andrea Scozzafava,
¨
Aydın Salmana and Claudiu T. Supuranb,*
aIstanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
b
Received 30 September 2007; revised 29 October 2007; accepted 30 October 2007
Available online 4 November 2007
Abstract—2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA,
EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applica-
tions, such as CA I, II, VA, VB, VII, IX, and XII among others. This sulfonamide is a 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 ubiquitous isoforms,
making it thus a very interesting clinical candidate for situations in which a strong inhibition of CA I and II is needed. The crystal
structure of the hCA II adduct of this sulfonamide revealed many favorable interactions between the inhibitor and the enzyme which
explain its strong low nanomolar affinity for this isoform but may also be exploited for the design of effective inhibitors incorpo-
rating bicyclic moieties.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Known for decades, the metallo-enzymes carbonic
anhydrases (CAs, EC 4.2.1.1) are present in prokaryotes
and eukaryotes and encoded by at least four evolution-
arily un-related gene families: the a-CAs in vertebrates,
bacteria, algae, and cytoplasm of green plants; the b-
CAs predominantly in bacteria, algae, and chloroplasts;
the c-CAs mainly in archea and some bacteria, and the d-
CAs in diatoms and other simple marine metazoa.1–3 In
humans, 15 isozymes are presently known, 12 of which
are catalytically active (CAs I–IV, CA VA, CAVB,
CAVI, CA VII, CA IX, and CAs XII–XIV), whereas
the CA-related proteins (isoforms CA VIII, X, and XI)
are devoid of catalytic activity.1–3 In recent years, it has
emerged that in addition to their well-known role for
the development of diuretics and topically acting anti-
glaucoma drugs,4,5 inhibitors of these enzymes may lead
to novel applications, mainly as antiobesity, anticonvul-
sant, and/or anticancer agents or diagnostic tools.6–10 In
fact two mitochondrial isoforms (CA VA and CA VB)
are involved in lipogenesis and their inhibition leads to
diminished fatty acid biosynthesis,6 whereas at least
two transmembrane, tumor associated isozymes (CA
IX and CA XII) are highly overexpressed and involved
in signaling/pH regulation processes within many types
of hypoxic tumors.8–10 Little is understood for the mo-
ment regarding the brain CAs involved in epileptogene-
sis, since a large number of isoforms are present within
the CNS, among which are CA I, II, IV, VB, VII, and
XIV.7 Specific inhibitors for many such isoforms have
been developed and some of them are in clinical evalua-
tions.5–12 In addition, many CAs isolated from other
organisms than the vertebrates were shown to be targets
for the drug design recently, such as among others the a-
CAs from Plasmodium falciparum and Helicobacter py-
lori,13,14 the b-CAs from H. pylori, Mycobacterium tuber-
culosis, Candida albicans, Cryptococcus neoformans,
etc.15–18 Work is in progress in several laboratories for
developing stronger and more specific inhibitors target-
ing these enzymes, that would lead to conceptually novel
therapies.15–19 In fact a major drawback of many of the
clinically used sulfonamide CA inhibitors (CAIs), such
Keywords: Carbonic anhydrase; Sulfonamide; X-ray crystallography;
Isozyme selective inhibitor; Diabetic retinopathy; Isoform I.
q
The coordinates of the hCA II–indolesulfonamide adduct have been
deposited in PDB, ID code 3B4F.
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.110

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Ozlen Gu¨zel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 152–158
153
as among others ethoxzolamide 1, dorzolamide 2, brin-
zolamide 3 (used as antiglaucoma drugs)5, and zonisa-
mide (an antiepileptic drug),20,21 is their promiscuous
inhibition of many of the 12 catalytically active mamma-
lian isoforms. Thus, screening of large libraries of deriv-
atives or the rational drug design of inhibitors targeting a
precise isoform proved ultimately to be potent tools for
obtaining derivatives with some degree of selectivity for
inhibiting some CA isoforms.22,23
2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide
5,26 a bicyclic compound structurally related with ethox-
zolamide 1, but also possessing two side chains similarly
to 2 and 3, with a different orientation as compared to
all these clinically used compounds, attracted our atten-
tion as a possible candidate for obtaining CAIs with dif-
ferent inhibition profiles as compared to the presently
available compounds. Sulfonamide 5 was previously re-
ported by Salman’s group,26 being easily prepared from
NHEt
NHEt
N
EtO
S
SO2NH2
N
MeO(CH2)3
S
S
SO2NH2
Me
S
S
SO2NH2
O
O
O
O
1
3
2
SO2NH2
H2NO2S
NHNH2
O
N
O
N
H
4
5
Ethoxzolamide 1, a first generation, very potent CAI
against most isoforms,19 has been used as lead molecule
for the design of second generation such compounds,
among which dorzolamide 2 and brinzolamide 3 are
widely used as topically acting antiglaucoma agents.5 A
more recent such bicyclic sulfonamide CAI, zonisamide
4, has been discovered serendipitously to possess signifi-
cant anticonvulsant activity and is presently used as an
antiepileptic.20,21 No X-ray crystal structures of 1 in com-
plex with CAs are available for the moment, however, the
other three bicyclic derivatives 2–4 were crystallized in ad-
ducts with various isoforms, such as CA II and IV among
others, by our and Christianson’s groups.21,24,25
sulfanilamide as shown in Scheme 1. Diazotization of
sulfanilamide followed by condensation of the diazo-
nium salt with ethyl 2-benzylacetoacetate leads to an
intermediate which was cyclized in acidic medium with
formation of the ethyl ester derivative of 5, which was
converted to 5 by treatment with hydrazine (Scheme
1).26
Sulfonamide 5 has been investigated for the inhibition of
the 12 catalytically active mammalian CA isoforms CA I–
XIV (h, human; m, mouse isozyme) (Table 1). Data for
the structurally related, clinically used derivatives 1–4
are also included for comparison, as they were published
C6H5
OC2H5
H2NO2S
H2NO2S
ii
i
CH
+
3
-
+
N2Cl
NH2
O
O
C6H5
H2NO2S
H2NO2S
OC2H5
O
iii
iv
N
C6H5
COOC2H5
N
H
N
H
C6H5
H2NO2S
HN NH2
N
H
O
5
Scheme 1. Preparation of the indole-5-sulfonamide derivative 5. Reagents and conditions: (i) NaNO2, 37% HCl, 0 ꢁC; (ii) KOH, 0 ꢁC; (iii) 37% HCl,
reflux, 4 h; (iv) H2NNH2ÆH2O, reflux, 6 h.

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Ozlen Gu¨zel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 152–158
154
Table 1. Inhibition data with the clinically used derivatives 1–4 as well as sulfonamide 5 against 12 mammalian a-CA isoforms
Isozymec
KId (nM)
1
2
3
4
5
hCA Ia
hCA IIa
25
8
50,000
9
45,000
3
56
7.5
35
7.2
hCA IIIa
hCA IVa
hCA VAa
hCA VBa
hCA VIa
hCA VIIa
hCA IXb
hCA XIIb
mCA XIIIa
hCA XIVb
1.1 · 106
93
25
7.7 · 105
8500
42
1.1 · 105
3950a
50
2.2 · 106
8590a
20
1.4 · 106
9000
1100
1100
2650
89
19
43
33
10
30
0.9
6033a
89a
0.8
34
3.5
52
2.8
37
117a
5.1
102
110
22
3.5
18
3.0
10
11,000
430a
5250a
50a
25
2633
48
27
24
Data for the inhibition of these CAs with compounds 1–4 are from Ref. 28.
a Recombinant enzyme. Data reported for the first time here.
b Catalytic domain.
c h, human; m, murine isozyme.
d Errors in the range of 5% of the reported data from three different assays, by a stopped flow CO2 hydration method.27
earlier by our group.28 It may be observed that except CA
III, which has a low affinity for all sulfonamide inhibi-
tors,28b the other 11 investigated isoforms are generally
all well inhibited by compounds 1–5. Ethoxzolamide is
a promiscuous, potent inhibitor of all of them, with
KIs < 50 nM for inhibition of all these CAs (Table 1).
However, dorzolamide 2 and brinzolamide 3, which have
been obtained through rational drug design and compar-
ison of the X-ray crystal structures of their various cong-
eners with isoforms CA I and II,19,24,25 already show a
better inhibition profile as compared to dorzolamide,
since they are also weak inhibitors of other two isoforms,
hCA I and IV (in addition to hCA III) (Table 1). How-
ever, both compounds generally act as very potent, non-
selective inhibitors of isozymes CA II, VA, VB, VI–
XIV, with inhibition constants in the range of 0.9–
50 nM. Zonisamide 4 is a rather special case among
sulfonamide CAIs, being one of the very few aliphatic
sulfonamides possessing a heterocyclic tail investigated
in detail as an inhibitor.21 Data of Table 1 show that zon-
isamide is a good hCA I, II, VA, and IX inhibitor (KIs in
the range of 5.1–56 nM), a medium potency CA VI and
VII inhibitor (KI of 89–117 nM), and a quite weak inhib-
itor of isozymes CA III, IV, VB, XII, XIII, and XIV, with
KIs > 430 nM (Table 1). These data clearly show that it is
possible to design inhibitors with a certain degree of selec-
tivity for target isozymes, zonisamide for example being a
rather CA IX-selective compound, the next best inhibited
isozymes being CA VA and CA II, but the selectivity ratio
for the inhibition of the tumor-associated isoform IX over
the mitochondrial one CA VA is around 4, and versus the
cytosolic isozyme CA II is around 7. Thus, this sulfon-
amide is four times a better CA IX than CA VA inhibitor,
and a seven times better CA IX than CA II inhibitor. The
newly investigated bicyclic sulfonamide 5 also shows a
completely different inhibition profile as compared to
the other structurally related compounds 1–4 discussed
here (Table 1). Thus, 5 is a very strong, nanomolar CA
I and II inhibitor (KIs of 7.2–7.5 nM), an ineffective CA
III, IV, VA, VB, VI, and XIII inhibitor (KIs > 1100 nM),
and a medium potency CA VII, CA IX, CA XII, and CA
XIV inhibitor (KIs in the range of 48–110 nM). Since CA
VII is present only in the brain,7 CA IX and XII are gen-
erally restricted to tumors8 and CA XIV to the liver and
kidneys (with a rather low level of expression),10 com-
pounds such as 5, which have lower affinity for ubiquitous
isozymes such as CA IV, VA, VB, VI, and XIII but a very
high one for hCA I and II, may have better profiles when
used for the management of diseases due to the imbalance
of CA activity (e.g., glaucoma and other eye diseases). In-
deed, recently, in a seminal paper, Feener’s group29 re-
ported elevated levels of CA I in vitreous from
individuals with diabetic retinopathy, the enzyme being
involved in retinal hemorrhage and erythrocyte lysis char-
acteristics of this disease. It was proved that CA I induced
alkalinization of vitreous which increased kallikrein
activity and generation of factor XIIa. Such results prove
that inhibition of CA I and/or kallikrein-mediated innate
inflammation could provide new therapeutic opportuni-
Table 2. Crystallographic parameters and refinement statistics for the
hCA II–5 adduct
Parameter
Value
Crystal parameter
Space group
Cell parameters
P21
a = 42.00 A
˚
˚
b = 41.35 A
˚
c = 72.34 A
b = 104.35ꢁ
˚
Data collection statistics (20.0–1.89 A)
No. of total reflections
No. of unique reflections
Completenessa (%)
88,138
19,119
99.14 (99.78)
10.53 (1.88)
11.0 (46.0)
F2/sig(F2)
R-sym (%)
˚
Refinement statistics (20.0–1.89 A)
R-factor (%)
R-freeb (%)
Rmsd of bonds from ideality (A)
19.6
23.9
0.009
1.54
˚
Rmsd of angles from ideality (ꢁ)
a Values in parentheses relate to the highest resolution shell (2.00–
1.89).
b Calculated using 5% of data.

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Ozlen Gu¨zel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 152–158
155
far less understood.1,2,28a In fact, CA I has a rather low
catalytic activity as compared to CA II (also a ubiquitous
isoforms present in the same tissue in which CA I is foun-
d)1,2,28a and is also inhibited to a large extent by physio-
logical levels of chloride and bicarbonate (CA II is
much more resistant to inhibition by these anions).1,2,28a
As a consequence, many of the CAIs developed as anti-
glaucoma drugs in the 90s, among which dorzolamide 2
Table 3. Strong interactions in which compound 5 participates with
amino acid residues/metal ion from the hCA II active site, in the hCA
II–5 adduct
3
O
1
H2N
3
4
S
NHNH2
O
2
4
N
O
H
5
Figure 1. Electron density map of 5 (in yellow) bound within the hCA
II active site. The Zn(II) ion of the enzyme, its three histidine ligands
(His94, 96, and 119), residues involved in the binding of the inhibitor
(Asn62 and Asn67) as well as one water molecule participating in
hydrogen bonds with the CO moiety of the inhibitor are also shown.
˚
Distance (A)
Atom of 5
hCA II residue
N1
O3
N1
O2
O3
N2
N3
O4
N4
N4
N4
Zn
Zn
1.98
3.16
2.99
2.70
3.16
2.75
2.91
3.05
2.41
3.10
2.76
Oc1Thr199
N Thr199
Zn
ties for the treatment of hemorrhage-induced retinal and
cerebral edema.29 It should be stressed that untill this
study was published, CA I was considered a typical anti-
target among the many CA isoforms,1,2,28a since this en-
zyme is ubiquitous in the gastrointestinal tract and
blood, being present at very high concentrations in many
such tissues, but its precise physiological function being
w101
Od1 Asn67
w101
Od1 Asn67
Nd2 Asn67
Nd2 Asn62
Figure 2. Detailed schematic representation for interactions in which sulfonamide 5 (in yellow) participates when bound to the hCA II active site. It
should be noted that the bound inhibitor is oriented toward the hydrophilic half of the enzyme active site cavity. The Zn(II) ion is the central violet
sphere.

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Ozlen Gu¨zel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 152–158
156
˚
and brinzolamide 3, were designed in such a way as to act
as very weak CA I inhibitors (see Table 1 and discussion
above). However, these last data obtained recently29
show that potent CA I inhibitors might be quite impor-
tant for the management of such terrible diseases as dia-
betic retinopathy and cerebral edema.29 Few potent CA
I inhibitors are described in the literature,1,2,28a and com-
pound 5 investigated here is one of the most potent such
CAIs.
ation for Ca atoms of 0.31 A only. Examination of the
initially calculated electron density maps in the active
site region showed the clear evidence for the binding
of one inhibitor molecule within the active site cavity.
The electron density of all moieties of the inhibitor is
in fact very well defined (Fig. 1).
The tetrahedral geometry of the Zn2+ binding site
and the key hydrogen bonds between the SO2NH2
moiety of 5 and enzyme active site are all retained
with respect to other hCA II-sulfonamide/sulfamate/
sulfamide complexes structurally characterized so
far (Figs. 1 and 2).5,19,21,23 In particular, the ionized
nitrogen atom of the sulfonamide group of 5 is coor-
In order to better understand the effective hCA II inhib-
itory activity of 5 and also to learn some lessons for the
drug design of new CAIs based on such bicyclic ring sys-
tems, we report the X-ray crystal structure of the hCA
II–5 adduct (Table 2).30 The three-dimensional structure
of the enzyme was very similar to that of hCA II without
any ligand bound,19,21,23–25 as judged by an r.m.s. devi-
˚
dinated to the zinc ion at a distance of 1.98 A. This
nitrogen atom is also hydrogen bonded to the hydro-
˚
xyl group of Thr199 (N–Thr199OG = 2.70 A), which
Figure 3. Superposition of the: (A) hCA II–dorzolamide 2 (gold), PDB entry 1cil,24 with hCA II–5 (blue) adducts, and (B) hCA II–brinzolamide 3
(yellow), PDB entry 1a42,25 with hCA II–5 (blue) adducts. Compound 5 binds in distinct active site regions of the enzyme cavity as compared to
dorzolamide/brinzolamide.

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Ozlen Gu¨zel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 152–158
157
˚
in turn interacts with the Glu106OE1 atom (2.50 A,
Acknowledgments
data not shown). One oxygen atom of the coordi-
nated sulfamoyl moiety is hydrogen bonded to the
This research was financed in part by two grants of the
6th Framework Programme of the European Union
(EUROXY and DeZnIT projects).
˚
backbone amide of Thr199 (ThrN–O2 = 2.99 A),
whereas the second oxygen atom of this moiety is
2+
3.16 A away from the catalytic Zn ion, being con-
˚
sidered as weakly coordinated to the metal
ion.1,1,19,21,23 All these interactions have also been
observed in the adducts of hCA II with other sulfon-
amide inhibitors, such as dorzolamide 2, brinzola-
mide 3 or zonisamide 4, but the corresponding
distances are of course different.19,21–25 The bicyclic
heterocyclic scaffold of 5 is accommodated perfectly
within the active site channel, being oriented towards
the hydrophilic half of it, and participating to several
favorable interactions with various amino acid resi-
dues and water molecules (Figs. 1 and 2). It should
be noted that only the terminal amino moiety, the
oxygen atom of the CONHNH2, and the endocyclic
NH indole group participate in such strong interac-
tions, more precisely, the oxygen (CO) atom makes
References and notes
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3. Ohradanova, A.; Vullo, D.; Kopacek, J.; Temperini, C.;
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˚
a hydrogen bond with water 101 (of 3.05 A) which
also makes a second hydrogen bond with the endocy-
˚
clic NH moiety of 5 (of 2.75 A). The terminal NH2
moiety on the other hand participates in three hydro-
gen bonds with the oxygen atom of Asn67 (of
˚
2.41 A), with the NH2 moiety of the same amino acid
˚
residue (of 3.10 A), and with the NH2 moiety of the
˚
neighboring Asn62 (of 2.76 A) (Table 3). The 3-phe-
nyl-indole scaffold of the inhibitor makes a host of
6. (a) Supuran, C. T. Expert Opin. Ther. Pat. 2003, 13, 1545;
(b) De Simone, G.; Supuran, C. T. Curr. Top. Med. Chem.
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˚
favorable van der Waals interactions (< 4 A) with
amino acids lining the hCA II cavity (data not
shown) which also contribute to the tight binding of
5 within the active site.
´
7. Thiry, A.; Dogne, J.-M.; Masereel, B.; Supuran, C. T.
Curr. Top. Med. Chem. 2007, 7, 855.
´
8. Thiry, A.; Dogne, J.-M.; Masereel, B.; Supuran, C. T.
Trends Pharmacol. Sci. 2006, 27, 566.
In order to try to understand the different inhibition
profiles of compound 5 and dorzolamide 2 or brinzola-
mide 3, we have also superposed the 3 D structures of
the three sulfonamides complexed within the hCA II ac-
tive site (Fig. 3). These figures show that although all
three compounds have an excellent affinity for hCA II,
in the low nanomolar range, their binding to the enzyme
is rather different, since only the SO2NH2 moieties of the
three inhibitors are superposable. We estimate that the
degree of isozyme selectivity observed with sulfonamide
5 as compared to other CAIs investigated up to now
(e.g., 2–4) is due to the particular orientation which 5
adopts when bound to the CA II active site, which is
characteristic only to this sulfonamide (see also Fig. 3).
ˇ
´
9. Svastova, E.; Hulıkova, A.; Rafajova, M.; Zatovicova,
´
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
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´
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A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
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´
10. Pastorekova, S.; Kopacek, J.; Pastorek, J. Curr. Top. Med.
Chem. 2007, 7, 865.
11. Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van
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Zandvoort, M. A. M. J.; Pastorekova, S.; Scozzafava, A.;
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fonamide 5 with all the catalytically active mammalian
CA isoforms. This sulfonamide is a potent inhibitor of
CA I and II, a medium potency inhibitor of CA VII,
IX, XII, and XIV, and a weak inhibitor against the other
ubiquitous isoforms, making it thus a very interesting
clinical candidate for situations in which a strong inhibi-
tion of CA I and II is needed. The high resolution crystal
structure of the hCA II–5 adduct revealed many favor-
able interactions between the sulfonamide 5 and the en-
zyme which explain its strong low nanomolar affinity
for this isoform but may also be exploited for the design
of effective inhibitors incorporating bicyclic moieties.
16. Mogensen, E. G.; Janbon, G.; Chaloupka, J.; Steegborn,
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of Sulfonamide Carbonic Anhydrase Inhibitors. In Car-
bonic Anhydrase—Its Inhibitors and Activators; Supuran,
C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca
Raton, 2004; pp 67–147.
20. Biton, V. Clin. Neuropharmacol. 2007, 30, 230.
21. 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.
22. (a) 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; (b) Winum, J. Y.;
Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.;
Ciattini, S.; Montero, J. L.; Scozzafava, A.; Supuran, C.
27. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An
Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration
activity. Phenol red (at a concentration of 0.2 mM) has
been used as indicator, working at the absorbance
maximum of 557 nm, with 10 mM Hepes (pH 7.5) as
buffer, 0.1 M Na2SO4 (for maintaining constant the ionic
strength), following the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the
kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the
reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared
in distilled-deionized water and dilutions up to 0.1 nM
were done thereafter with distilled-deionized water. Inhib-
itor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to
allow for the formation of the E-I complex. The inhibition
constants were obtained by non-linear least-squares meth-
ods using PRISM 3, as reported earlier,12–14 and represent
the mean from at least three different determinations.
Enzyme concentrations in the assay system were in the
range of 7.1–13 nM.12–14
T. J. Med. Chem. 2006, 49, 7024; (c) Gruneberg, S.;
¨
Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3588.
23. (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) Antel, J.; Weber, A.; Sotriffer, C.
A.; Klebe, G. Multiple Binding Modes Observed in X-ray
Structures of Carbonic Anhydrase Inhibitor Complexes
and Other Systems: Consequences for Structure-based
Drug Design. In Carbonic Anhydrase—Its Inhibitors and
Activators; Supuran, C. T., Scozzafava, A., Conway, J.,
Eds.; CRC Press: Boca Raton, 2004; pp 45–65.
28. (a) Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem.
2007, 15, 4336; (b) Nishimori, I.; Minakuchi, T.; Onishi,
S.; Vullo, D.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. 2007, 15, 7229.
24. Kim, C. Y.; Whittington, D. A.; Chang, J. S.; Liao, J.;
May, J. A.; Christianson, D. W. J. Med. Chem. 2002, 45,
888.
29. Gao, B. B.; Clermont, A.; Rook, S.; Fonda, S. J.;
Srinivasan, V. J.; Wojtkowski, M.; Fujimoto, J. G.;
Avery, R. L.; Arrigg, P. G.; Bursell, S. E.; Aiello, L. P.;
Feener, E. P. Nat. Med. 2007, 13, 181.
25. Boriack-Sjodin, P. A.; Zeitlin, S.; Chen, H. H.; Crenshaw,
L.; Gross, S.; Dantanarayana, A.; Delgado, P.; May, J. A.;
Dean, T.; Christianson, D. W. Protein Sci. 1998, 7, 2483.
26. Compound 5 has been prepared as reported in: Ergenc¸,
N.; Salman, A.; Bankaog˘lu, G. Pharmazie 1990, 45, 346.
2-Benzyl-2-[N-(4-sulfonamidophenyl)hydrazono]ethanoate.
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. The solution containing the diazonium
salt was poured into an ice-cold mixture of 2.3 g (a little
excess of 0.01 mol) ethyl 2-benzylacetoacetate, 10 ml of
C2H5OH, 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 (C2H5)2O, washed with
H2O, and dried over anhydrous Na2SO4. (C2H5)2O was
distilled, the oily residue was treated with 5 ml of 37%
HCl, and set aside for 5 h at room temperature. The
resulting solid substance was recrystallized from C2H5OH.
Ethyl 5-(aminosulfonyl)-3-phenyl-1H-indole-2-carboxylate.
A mixture of 0.01 mol ethyl 2-benzyl-2-[N-(4-sulfonamid-
ophenyl)hydrazono]ethanoate 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 C2H5OH. 2-
(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide.
3.45 g (0.01 mol) ethyl 5-(aminosulfonyl)-3-phenyl-1H-
indole-2-carboxylate was dissolved in 20 ml of C2H5OH,
4 ml of H2NNH2ÆH2O was added and refluxed for 6 h,
cooled, and kept cold overnight. The resulting crystals
were filtered off, washed with (C2H5)2O, and recrystallized
from C2H5OH/DMF.
30. The hCA II–5 complex was crystallized as previously
described. Diffraction data were collected under cryo-
genic conditions (100 K) on a CCD Detector KM4
˚
CCD/Sapphire using CuKa radiation (1.5418 A). The
˚
unit cell dimensions were determined to be: a = 42.00 A,
˚
˚
b = 41.35 A, c = 72.34 A, and a = c = 90ꢁ, b = 104.35ꢁ in
the space group P21. Data were processed with
CrysAlis RED (Oxford Diffraction 2006).31 The struc-
ture was analyzed by difference Fourier technique,
using the PDB file 1CA2 as starting model. The
refinement was carried out with the program REF-
MAC532; model building and map inspections were
performed using the COOT program.33 The final model
of the complex hCA II–5 had an R-factor of 19.6%
˚
and R-free 23.9% in the resolution range 20.0–1.89 A,
with
a rms deviation from standard geometry of
˚
0.009 A in bond lengths and 1.54ꢁ in angles. The
correctness of stereochemistry was finally checked using
PROCHECK.34 Coordinates and structure factors have
been deposited with the Protein Data Bank (PDB ID
3B4F). Crystallographic parameters and refinement
statistics are summarized in Table 2.
31. Oxford Diffraction. CrysAlis RED. 2006, Version
1.171.32.2. Oxford Diffraction Ltd, UK.
32. Jones, T. A.; Zhou, J. Y.; Cowan, S. W.; Kjeldoard, M.
Acta Cryst. 1991, A47, 110.
33. Emsley, P.; Cowtan, K. Acta Cryst. 2004, D60, 2126.
34. Laskowski, R. A.; MacArthur, M. W.; Moss, D.
S.; Thornton, J. M. J. Appl. Crystallogr. 1993, 26,
283.