Synthesis, structure-activity relationship studies, and X-ray crystallographic analysis of arylsulfonamides as potent carbonic anhydrase inhibitors

Article abstract of DOI:10.1021/jm300112w

A series of arylsulfonamides has been synthesized and investigated for the inhibition of some selected human carbonic anhydrase isoforms. The studied compounds showed significant inhibitory effects in the nanomolar range toward druggable isoforms (hCA VII, hCA IX, and hCA XIV) (K_i values from 4.8 to 61.7 nM), whereas they generally exhibited significant selectivity over hCA I and hCA II, that are ubiquitous and considered off-target isoforms. On the basis of biochemical data, we herein discussed structure-affinity relationships for this series of arylsulfonamides, suggesting a key role for alkoxy substituents in CA inhibition. Furthermore, X-ray crystal structures of complexes of two active inhibitors (I and 2a) with hCA II allowed us to elucidate the main interactions between the inhibitor and specific amino acid residues within the catalytic site.

Full text of DOI:10.1021/jm300112w

Article
pubs.acs.org/jmc
Synthesis, Structure−Activity Relationship Studies, and X-ray
Crystallographic Analysis of Arylsulfonamides as Potent Carbonic
Anhydrase Inhibitors†
Rosaria Gitto,*,‡ Francesca M. Damiano,*,‡ Pavel Mader,§ Laura De Luca,‡ Stefania Ferro,‡
§,#
Claudiu T. Supuran,⊥ Daniela Vullo,⊥ Jirí Brynda,§,# Pavlína Rezacova, and Alba Chimirri‡
̌
̌
́
̌
́
‡Dipartimento Farmaco-Chimico, Universita
̀
di Messina, Viale Annunziata, I-98168 Messina, Italy
§Department of Structural Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech
Republic
⊥Universita
̀ ̀
degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Universita di Firenze, Italy
#Structural Biology Team, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague,
Czech Republic
S
* Supporting Information
ABSTRACT: A series of arylsulfonamides has been synthesized and
investigated for the inhibition of some selected human carbonic
anhydrase isoforms. The studied compounds showed significant
inhibitory effects in the nanomolar range toward druggable isoforms
(hCA VII, hCA IX, and hCA XIV) (Ki values from 4.8 to 61.7 nM),
whereas they generally exhibited significant selectivity over hCA I and
hCA II, that are ubiquitous and considered off-target isoforms. On the
basis of biochemical data, we herein discussed structure−affinity
relationships for this series of arylsulfonamides, suggesting a key role
for alkoxy substituents in CA inhibition. Furthermore, X-ray crystal
structures of complexes of two active inhibitors (I and 2a) with hCA II allowed us to elucidate the main interactions between the
inhibitor and specific amino acid residues within the catalytic site.
brain where it is considered involved in generating neuronal
excitation and seizures.9,10 Recently, the hCA VII involvement
in neuropathic pain control was proposed.11 This could
represent an interesting pharmacological mechanism for the
design of new pain killers useful for therapeutic applications.
hCA IX is expressed in a limited number of normal tissues
whereas it is overexpressed in many solid tumors and
considered involved in critical processes connected with cancer
progression. The expression of hCA IX is strongly up-regulated
by hypoxia via the hypoxia inducible factor-1 (HIF-1)
transcription factor. The overexpression of hCA IX induces
the pH imbalance of tumor tissue contributing significantly to
the extracellular acidification of solid tumors; thus, hCA IX
inhibitors could specifically bind hypoxic tumor cells expressing
this isoform. Consequently, they have been proposed as
antitumor agents.12−19 hCA XIV is a transmembrane isozyme
with the active site oriented extracellularly; it is highly abundant
in neurons and axons in the murine and human brain, where it
seems to play an important role in modulating excitatory
synaptic transmission.20
INTRODUCTION
■
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc
metalloenzymes that catalyze a very simple physiological
reaction, the interconversion between carbon dioxide and the
bicarbonate ion. To date, 16 human CA (hCA) isoforms have
been identified that display significant differences in catalytic
activity, subcellular localization, and tissues expression.1 They
play relevant roles in a large series of physiological processes
such as intracellular and extracellular pH homeostasis, cell
proliferation and differentiation, modulation of neuronal
transmission, and several biochemical pathways where either
CO2 or bicarbonate is required. Furthermore, this family of
enzymes is involved in some pathological pathways and thus are
interesting targets for the treatment of glaucoma, cancer,
obesity, and epilepsy. On this basis, the development of CA
inhibitors (CAIs) possessing high potency and selectivity
against specific isoforms represents an attractive strategy to
obtain new active compounds able to avoid side effects, thus
improving therapeutic safety.1−8 Among the different mamma-
lian isoforms, the hCA VII, hCA IX, and hCA XIV have
recently been shown to be druggable targets. hCA VII is one of
the least investigated cytosolic CA isoforms; it presents a
limited distribution, being mainly expressed in the cortex,
hippocampus, and thalamus regions within the mammalian
Received: January 26, 2012
Published: March 26, 2012
© 2012 American Chemical Society
3891
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
Chart 1. Well-Known CAIs and Previously Reported Arylsulfonamides24
The catalytic domain of CA is generally composed of three
histidine residues (His94, His96, and His119) coordinating the
zinc(II) ion. So the active zinc ion can bind hydroxide ion that
reacts with carbon dioxide to give bicarbonate.21 This implies
that the CAIs are generally molecules containing specific
chemical groups able to coordinate the zinc ion of the catalytic
binding site thus blocking the CA enzymatic activity. The most
well-known CAIs contain a sulfamide/sulfonamide/sulfamate
moiety (Chart 1, acetazolamide, topiramate, and zonisamide)
that in deprotonated form binds the active zinc ion in place of
physiological substrates in the catalytic cycle of CAs.22 We
recently reported that some sulfamide-based heterocyclic
compounds such as N-sulfonamide-1,2,3,4-tetrahydroisoquino-
lines can bind the CA catalytic site, displaying significant
inhibitory effects as well as very high selectivity toward hCA
VII, hCA IX, and hCA XIV.23−26 The CA interaction has been
demonstrated by crystallographic data of hCA II in complexes
with some active N-sulfonamide-1,2,3,4-tetrahydroisoquinolines
(PDB codes 3IGP and 3PO6);25,27 from this study, their
sulfonamide moiety has been demonstrated to be bound within
the catalytic site through zinc ion and specific amino acid
residues. Moreover, by means of docking studies we explored
the involvement of other amino acid residues driving the
isoform selectivity against hCA IX and hCA XIV.25 We also
found that the arylsulfonamide derivatives I−IV (Chart 1)24
were active and selective CAIs, showing significant inhibitory
effects at nanomolar concentration and a good selectivity ratio
over hCA I and hCA II isozymes that are abundant in many
tissues and are considered responsible for some unwanted side
effects of CAIs currently used in therapy. Particularly, it was
highlighted that 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquino-
lin-1-yl)benzenesulfonamide (III, Chart 1) displayed very
interesting inhibitory effects against hCA VII isoform (Ki
value of 4.6 nM) and low hCA II activity (Ki value of 3640
nM).24
Chart 2. Designed Compounds
controlling the activity/selectivity against some CA isoforms
(hCA I, hCA II, hCA VII, hCA IX, and hCA XIV). Therefore,
in the current paper both biochemical screening and X-ray
crystal structures of some active derivatives in complex with
hCA II have been carried out to explore the CA inhibitory
effects and the different protein interactions of this new series
of arylsulfonamides (1−4, Chart 2).
CHEMISTRY
■
As depicted in Scheme 1, a series of arylsulfonamides
derivatives (1−4) was prepared by following a previously
reported procedure24 with slight modifications. By coupling of
4-(aminosulfonyl)benzoic acid (6) with suitable 2-(3′-
alkoxyphenyl)ethylamine derivatives 5a−c, we obtained the
corresponding 4-(aminosulfonyl)-N-[2-(3′-phenylethyl]-
benzamides 1a−c that were cyclized by treatment with
phosphorus oxychloride, giving the 3,4-dihydroisoquinoline
derivatives 2a−c; then by reduction of the intermediates 2a−c
with sodium borohydride we prepared the 1,2,3,4-tetrahydroi-
soquinolines 3a−c as racemic mixture. Successively, the
treatment of compounds 3a−c with an excess of sulfamide
yielded 1-[4-(aminosulfonyl)phenyl]-6-alkoxy-1,2,3,4-tetrahy-
droisoquinolinesulfonamides 4a−c. Finally, by dealkylation of
the suitable alkoxy parent compounds, we obtained in good
yields the corresponding hydroxyl analogues (1d, 2d, 3d, and
4d).
In this context we herein report the synthesis of 16
arylsulfonamides 1a−d, 2a−d, 3a−d, and 4a−d (Chart 2)
strictly related to our previously studied compounds I−IV. Our
idea was first and foremost to understand the role of methoxy
substituents. So we planned the synthesis of monomethoxy
analogues (R= MeO) as well as of other monalkoxy derivatives
(R = EtO or PrO) and of the corresponding hydroxyl ones (R
= OH). In this way we hoped to explore how these
modifications could influence the interaction with hydro-
philic/hydrophobic areas of the catalytic pocket, thus
3892
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
a
arylsulfonamides (1a−d, 2a−d, 3a−d, and 4a−d) have been
determined using a stopped-flow assay28 monitoring CA-
catalyzed hydration of carbon dioxide and are reported in Table
1. These results were compared with data previously obtained
for the corresponding dimethoxy analogues (I−IV) and the
reference compounds acetazolamide (AAZ), topiramate
(TPM), and zonisamide (ZNS). From the data of Table 1
some relevant structure−activity relationships (SARs) can be
highlighted for the four classes of synthesized compounds. In
the benzamide series (i.e., compounds 1a−d) the removal of
one methoxy substituent generally gave an improvement in
selectivity with respect to dimethoxy derivative I that resulted
in a very potent inhibitor (Ki 2.7−8.7 nM) against all studied
isoforms. In particular the 3-methoxy derivative 1a was a more
selective inhibitor than 1b−d toward the druggable isoform
hCA IX; the 3-ethoxy-substituted 1b was optimal to improve
hCA VII activity whereas the 3-propoxy derivative 1c behaved
like the dimethoxy analogue I, thus showing high inhibitory
efficacy against hCA VII and hCA IX and no selectivity over
hCA II. The substitution with a 3-hydroxyl group provided an
increase in hCA II activity (1d). Among dihydroisoquinoline
derivatives 2a−d, the 6-methoxy derivative 2a demonstrated an
impressive improvement of hCA II affinity (250-fold) when
compared with the corresponding 6,7-dimethoxy analogue II,
thus furnishing some suggestions about the mode of interaction
within the catalytic site (vide infra, X-ray studies). The most
interesting inhibitor was the 6-ethoxy-substituted compound 2b
that showed high affinity toward hCA VII and hCA IX (Ki 6.5
and 7.2 nM, respectively) and very low efficacy against off-
target isoforms hCA I and II. Compound 2c displayed low
selectivity toward hCA I unlike 2d (R = OH) for which very
Scheme 1
a
Reagents and conditions: (i) HBTU, DMF, NEt3, rt, overnight; (ii)
POCl3, toluene, Δ, 6 h; (iii) NaBH4, CH3OH, rt, 1 h; (iv)
CH3CH(OCH3)2, NH2SO2NH2, MW, two steps of 20 min at 100
°C, 150 W; (v) BBr3 (1 M in DCM), DCM, rt, overnight.
RESULTS AND DISCUSSION
■
All new synthesized compounds were assayed for their ability to
inhibit some human carbonic anhydrases selected among
ubiquitous and druggable isoforms (hCA I, hCA II, hCA VII,
hCA IX, and hCA XIV). The Ki values for the 16
Table 1. Ki Values (nM) against hCA I, hCA II, hCA VII, hCA IX, and hCA XIV Isoforms Shown by Arylsulfonamide
Derivatives Acetazolamide (AAZ), Topiramate (TPM), and Zonisamide (ZNS)
a
Ki (nM)
hCA I
hCA II
hCA VII
hCA IX
hCA XIV
4.3 0.4
b
I
8.7 0.4
3000 76
920 43
1540 39
474 21
514 28
2.7 0.1
5.8 0.3
73.3 6.2
4.6 0.3
7.0 0.5
3.8 0.2
210 14
130 11
b
II
2150 16
3640 127
3130 168
103
112
87.0
50.2
9
8
b
III
b
IV
1a
1b
1c
76.0
5
7
86.7
33.0
7
2
61.7
4
6.5 0.3
8.1 0.3
8.3 0.2
4
4.8 0.5
5.9 0.4
13.9 1.1
10.2 1.0
6.5 0.4
8.0 0.3
8.6 0.7
50.4
24.6
5
2
5.3 0.3
4.9 0.5
8.6 0.6
87.5 3.7
8.8 0.8
31.1 2.5
8.8 0.2
43.5 3.1
8.9 0.8
84.5 6.4
48.5 4.0
33.2 1.9
6.3 0.3
6.7 0.2
12 0.6
10 1.0
35 1.8
51.0
22.4
5
2
1d
2a
41.7
3
681 51
1050 54
83.1 7.1
5.1 0.3
9.2 0.6
667 25
72.3 5.9
24.3 2.1
1210 35
4165 93
6.7 0.1
6.7 0.4
250 12
250 10
56 1.6
18.3 1.2
7.2 0.1
16.1 1.2
51.5 1.0
15.4 1.0
9.2 0.7
9.2 0.8
68.3 3.9
6.5 0.5
52.1 2.8
7.8 0.5
7.9 0.6
25 1.4
8.9 0.5
17.9 1.5
8.7 0.6
46.9 3.7
8.6 0.6
8.7 0.1
15.3 1.3
55.9 1.8
5.2 0.3
6.7 0.4
9.2 0.3
24.8 1.9
41 1.7
2b
2c
2d
3a
53.6
5
5.7 0.5
5.8 0.4
8.2 0.7
34.7 1.5
23.4 1.4
17.8 0.8
7.9 0.7
50.8 2.5
2.5 0.1
0.9 0.1
117 9.1
3b
3c
3d
4a
̀
4b
4c
4d
AAZ
b
b
TPM
58 3.7
1460 62
5250 174
b
ZNS
5.1 0.3
a
Mean standard error, from three different assays. Recombinant full length hCA I, II VII, and XIV and catalytic domain of hCA IX were used.
Data are taken from ref 24.
b
3893
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
high hCA I activity (Ki value of 5.1 nM) was observed (Table
1).
Table 2. Data Collection Statistics and Refinement Statistics
hCA II in complex with I
P21
hCA II in complex with 2a
P21
Among the 1,2,3,4-tetrahydroisoquinolines (3), the mono-
alkoxy derivatives (3a−c) were potent inhibitors of hCA VII,
hCA IX, and hCA XIV isoforms (Ki 5.7−15.4 nM); in
particular, the 6-ethoxy-substituted compound 3b was the best
inhibitor in terms of activity/selectivity. This compound had
inhibition constants ranging from 5.8 to 9.2 nM against the
selected druggable isoforms and showed a good selectivity ratio
exclusively over hCA I when compared with 6,7-dimethoxy
analogue III (see Table 1). Compound 3d (R = OH) exhibited
good inhibitory effects but no significant selectivity. Finally, the
N-sulfonamide-1,2,3,4-tetrahydroisoquinolines 4a−d were ac-
tive against all tested isoforms but displayed very low selectivity
with to respect compound IV.
Overall, the newly synthesized compounds show Ki values
ranging from 4.8 to 68.3 nM toward hCA VII, hCA IX, and
hCA XIV isozymes, displaying in some cases (2−4) a CA
inhibition profile better than that of their parent (2−4 vs II−
IV) and standard compounds. The most effective and selective
inhibitors are ethoxy-substituted derivatives 1b−4b whereas the
presence of a hydroxyl function (e.g., compounds 1d−4d)
generally gives a reduction in activity. Therefore, this study
shows that these new arylsulfonamide derivatives generally
maintain high affinity whereas they behave as weak selective
inhibitors toward only one CA isoform.
To investigate the inhibitor−protein interactions, we
cocrystallized two of the most active inhibitors (compound I
and compound 2a) with the isoform hCA II, and the crystal
structures were determined at atomic resolution (PDB codes
3V7X and 3VBD). The data collection and refinement statistics
are summarized in Table 2. The crystal structures of hCA II in
complex with compounds I and 2a were determined by the
difference Fourier method and refined using data to 1.03 Å and
1.05 Å, respectively. Both complexes crystallized in the
monoclinic P21 space group with one hCA II molecule in the
asymmetric unit and solvent content of 41.2% and 40.5% for
compounds I and 2a, respectively. All hCA II amino acid
residues could be traced into a well-defined electron density
map with the exception of side chains of some terminal and
surface amino acid residues (Ser2, Lys9, and Lys261 in hCA II
in complex with compound I, and Ser2, Lys9, Lys45, and
Lys261 in hCA II in complex with compound 2a), which were
thus not included in the final model.
Compounds I and 2a could be modeled into a well-defined
electron density present within the enzyme active site (see
Figure 1A,B). The high quality of the experimental data allowed
the interpretation of the electron maps by two alternative
conformations of inhibitor. The sulfonamide aryl group of I and
2a is bound in one unambiguous conformation, while both the
2-(3′,4′-dimethoxyphenyl)ethylamine fragment (I) and 6-
methoxy-3,4-dihydroisoquinoline nucleus (2a), that are distal
from sulfonamide group, acquire two different conformations
with equivalent partial occupancies (Figure S1, Supporting
Information).
space group
unit cell
parameters
a, b, c (Å)
42.14, 41.51, 71.84
90, 104.23, 90
42.01, 41.18, 71.76
90, 104.33, 90
α, β, γ (deg)
wavelength (Å)
0.918
0.915
resolution range
(Å)
15.47−1.03 (1.06−1.03)
16.76−1.05 (1.08−1.05)
no. of unique
reflections
117 997 (8733)
109 951 (7248)
multiplicity
3.7 (3.4)
3.5 (2.6)
completeness
(%)
99.8 (100.0)
99.1 (89.0)
a
Rmerge (%)
5.8 (58.4)
9.8 (2.3)
11.3
5.3 (41.3)
9.6 (2.0)
6.0
average I/σ(I)
Wilson B (Å2)
refinement
statistics
resolution range
10.0−1.03
10.0−1.05
no. of
112 061
104 286
reflections in
working set
no. of
5916
5556
reflections in
test set
b
R
(%)
11.65
14.81
0.020
12.71
16.03
0.015
c
Rfree (%)
rmsd bond
length (Å)
mean ADP
protein−
13.9
15.1
inhibitor (Å2)
PDB code
3V7X
3VBD
a
Rmerge = ∑hkl∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑i Ii(hkl), where the Ii(hkl) is
an individual intensity of the ith observation of reflection hkl, and
⟨I(hkl)⟩ is the average intensity of reflection hkl with summation over
all data. R = ||Fo| − |Fc||/|Fo|, where Fo and Fc are the observed and
calculated structure factors, respectively. Rfree is equivalent to R but is
b
c
calculated for 5% of the reflections chosen at random and omitted
from the refinement process.49
and His119), similar to those observed for other arylsulfona-
mides previously reported in the literature.5,29−31 The ionized
nitrogen atom of the sulfonamide moiety is coordinated to the
zinc ion at a distance of about 2.0 Å. The sulfonamide nitrogen
also forms a hydrogen bond with the hydroxyl group of the
Thr199 side chain, and one oxygen atom from the sulfonamide
moiety forms a hydrogen bond with the backbone amine group
of Thr199. The position of the sulfonamide group as well as the
position of the aryl moiety within the enzyme active site is
identical for I and 2a (see Figure 1C,D).
An additional polar interaction of compounds I and 2a with
the enzyme is added through water-mediated interaction of the
amine group present in both inhibitors with the side chain of
Thr200 and main chain of Pro201. In compound I, the
carbonyl group is involved in an additional water-mediated
interaction with the side chain of Gln92 (Figure 1C). Figure 1
shows that the substituents distal from sulfonamide group
interact with residues at the opening of the enzyme active site.
These groups apparently have some degree of freedom in their
binding to the enzyme active site because they are able to
acquire two alternative conformations (see Figure 1C,D).
Compound I binds in an extended conformation in which the
positions of the two alternative conformations are quite similar.
Interactions of compounds I and 2a with the active site of
hCA II are depicted in panels C and E of Figure 1, and the
complete lists of interactions between the two inhibitors and
hCA II are reported in Table S1, Supporting Information.
Major interactions of the inhibitors with the enzyme active site
are mediated through the deeply buried sulfonamide group,
which makes polar interactions with the zinc ion and residues
located at the bottom of the active site cavity (His94, His96,
3894
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
Figure 1. Panels A and B show electron density maps for the inhibitory compounds I (labeled A) and 2a (labeled B). The 2Fo − Fc electron density
map for compounds I and 2a are contoured at 0.9σ and 0.8σ, respectively. The inhibitors are in stick representation with carbon atoms of compound
I colored green and compound 2a magenta (heteroatoms are colored according to standard color coding: oxygen, red; nitrogen, blue; sulfur, yellow).
Carbon atoms with alternative conformations are shown in lighter shades of original colors. Panels C and D show the compounds and active site
residues involved in either direct or water-mediated polar interactions in the active site (same color coding as was used in left-hand panels is used for
inhibitor atoms and amino acid residues). Panels E and F show inhibitors in the active site cavity represented by solvent-accessible surface with
residues involved in van der Waals interactions color coded as follows: residues involved in van der Waals interactions with arylsulfonamide moiety
are colored light brown, residues forming interactions with both alternative conformations are colored yellow, residues forming interactions with
alternative conformation 1 only are colored green, and residues forming interactions with alternative conformation 2 only are colored red.
The dimethoxyphenyl substituent is mostly solvent-exposed,
having only minor van der Waals interactions with specific
residues such as Phe131, Val135, and Pro202. The oxygen of
the 3-methoxy group of conformation 1 makes a water-
mediated hydrogen bond with residues Gly132 and Gln136. On
the contrary, in compound 2a the binding of two alternative
conformations differ substantially and each conformation
occupies a different surface pocket located on the opposite
side of the enzyme active site entrance (see panel D of Figure
1). We found that conformation 1 (magenta) interacts with
residues Ile91, Gln92, and Phe131. On the contrary,
conformation 2 (pink) interacts with the hydrophobic pocket
formed by residues Phe131, Val135, Leu198, Pro202, and
Leu204. Thus, the hCA II/binding was useful to reveal that two
different atropisomers of compound 2a cocrystallized within
the enzymatic catalytic pocket. On the contrary, the existence
both conformers of compound 2a, and the results are reported
in Figure 2A−D. Although the five studied isoforms are similar
and some crucial residues are conserved (Gln92, Leu198,
Pro202), there are several differences in the amino acid
composition within the catalytic site. The weaker Ki value of
compound 2a toward hCA I compared to hCA II could be
mainly attributed to the different hydrophobicity of the area
where the two crucial residues Leu/Phe131 and Ala/Val135
(see Figure 2A) are located. In particular, these residues are
involved in relevant interactions with the hydrophobic portion
of the methoxy substituent on the benzene fused ring of both
2a conformers. So we can speculate that the reduction of
hydrophobicity of hCA I might reduce the affinity of compound
2a.
The superposition between hCA II in complex with 2a and
hCA VII (Figure 2B) confirms that the hydrophobic contact
with Phe131 (Phe133 in hCA VII numbering) seems to play an
important role in the binding mode of compound 2a via the
methoxy group within the catalytic site of hCA VII.
Of particular note are the results of the superposition of the
hCA IX catalytic site and hCA II in complex with compound
2a. In fact, in this case the three hydrophobic residues Leu91,
Val131, and Leu135 create a hydrophobic region (see Figure
2C) that could compensate for the absence of the previously
mentioned crucial Phe residue.
1
of these two atropisomers remained undetected in the H
NMR spectra of compound 2a. Because each conformer
adopted an orientation which ensured the formation of a good
interaction with the hydrophobic pocket, the observed
atropisomerism might not have significant impact on inhibitory
effects.
The crystal structure of inhibitor 2a in complex with hCA II
was employed to decipher the CA inhibitory effects toward all
studied isoforms (see Table 1). Analyzing the differences in the
amino acid composition of the active site of hCA I, hCA II,
hCA VII, hCA IX, and hCA XIV isoforms, we tried to explain
the CA selectivity and the different degree of inhibitory effects
displayed by the most active compounds among this new series
of CAIs.
In a similar way the Leu131 and Ala135 residues model the
hydrophobic area in hCA XIV, creating the surface for inhibitor
2a interaction, thus justifying its high Ki value against this
isoform. Considering the high similarity between hCA I and
hCA XIV, this result appears to be a contradiction; however, it
is possible to hypothesize that the presence of the bulky phenyl
ring of the Phe91 residue in hCA I could lead to a steric clash,
thus precluding the interaction within the catalytic pocket.
We superimposed the coordinates of hCA I (PDB code
3LXE, orange),32 hCA VII (PDB code 3MDZ, magenta), hCA
IX (PDB code 3IAI, cyan),33 and hCA XIV (PDB code 1RJ6,
green)34 onto the coordinates of hCA II (gray) in complex with
3895
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
Figure 2. Representation of (A) hCA I (orange), (B) hCA VII (magenta), (C) hCA IX (cyan), and (D) hCA XIV (green) active sites superimposed
onto hCA II (gray) complexed with compound 2a (both conformers: cyan and yellow, respectively). The zinc is depicted as a black sphere. hCA II
residues are labeled in parentheses.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
Chemistry. All starting materials and reagents were purchased from
Sigma-Aldrich Milan, Italy, and used without further purification.
Microwave-assisted reactions were carried out in a CEM focused
microwave synthesis system. Melting points were determined on a
Buchi B-545 apparatus and are uncorrected. By combustion analysis
(C, H, N) carried out on a Carlo Erba Model 1106 Elemental
Analyzer, we determined the purity of synthesized compounds; the
results confirmed a ≥95% purity. Merck silica gel 60 F254 plates were
used for analytical TLC. Rf values were determined on TLC plates
using a mixture of CH2Cl2/CH3OH (94:6) as eluent. Flash
chromatography (FC) was performed on a Biotage SP1 EXP. 1H
NMR spectra were measured in DMSO-d6 and CD3OD with a Varian
Gemini 300 spectrometer; chemical shifts are expressed in δ (ppm)
and coupling constants (J) in hertz. All exchangeable protons were
confirmed by addition of deuterium oxide (D2O).
General Procedure for the Synthesis of 4-(Aminosulfonyl)-N-[2-
(3′-alkoxy)phenethyl]benzamides (1a−c). A mixture of 4-
(aminosulfonyl)benzoic acid (6) (1 mmol) and N,N,N′,N′-tetrameth-
yl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (1
mmol) in dimethylformamide (1.5 mL) was stirred for 1 h at room
temperature. Successively, the suitable 2-(3′-alkoxyphenyl)ethylamine
(5a−c) (1 mmol) in triethylamine (1 mmol) was added. The reaction
mixture was left overnight and then quenched with water (10 mL) and
The new synthesized arylsulfonamides 1−4 are structurally
related to compounds I−IV that displayed significant CA
inhibitory effects and generally high selectivity toward
druggable isozymes hCA VII, hCA IX, and hCA XIV. We
kept the arylsulfonamide moiety but modified the alkoxy
substituents with the aim to explore their role in the
interactions within the hydrophobic pocket of the enzyme
catalytic site. Overall, the new synthesized compounds 1−4
generally showed lower selectivity when compared to
previously reported I−IV analogues. We interpreted this
finding as evidence that the dimethoxy substitution plays a
key role in controlling the interaction within the catalytic
pocket. The cocrystal structure of compound 2a with hCA II
confirmed that the benzylsulfonamide portion binds the zinc
ion within the catalytic site, thus generating a blockade of
enzymatic activity. Interestingly, the bottom of the hydrophobic
pocket accepts the two alternative conformations generated by
ring flipping. In these two completely different orientations,
each 2a conformer is involved in a number of interactions with
the crucial residues of hCA II, thus suggesting that compound
2a could be an effective inhibitor in both conformations.
3896
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
extracted with ethyl acetate (3 × 5 mL). The combined extract was
dried with Na2SO4, and solvent was removed in vacuo. The residue
was powdered with diethyl ether and crystallized by treatment with
ethanol to give the desired final compounds 1a−c as white crystals.
4-(Aminosulfonyl)-N-[2-(3′-methoxy)phenyl)ethyl]benzamide
(bs, 2H, NH2), 7.29 (bs, 2H, NH2), 7.36 (d, J = 8.2, 2H, ArH), 7.72
(d, J = 8.2, 2H, ArH). Anal. (C16H19N3O5S2) C, H, N.
1-[4-(Aminosulfonyl)phenyl]-6-ethoxy-1,2,3,4-tetrahydroisoqui-
noline-2-sulfonamide (4b). Yield 61%; mp 119−120 °C. Rf = 0.55.
1-[4-(Aminosulfonyl)phenyl]-6-propoxy-1,2,3,4-tetrahydroisoqui-
noline-2-sulfonamide (4c). Yield 89%; mp 79−84 °C. Rf = 0.55.
General Procedure for the Synthesis of Hydroxyl Derivatives 1d,
2d, 3d, and 4d. The suitable alkoxy derivative (1 mmol) was dissolved
in methylene chloride (DCM) (5 mL) and then treated with BBr3 (1
M in DCM) (6 mmol) and stirred overnight. Successively, methanol
(7 mL) was carefully added at 0 °C and the solvents were removed
under reduced pressure. The residue was dissolved in ethyl acetate (10
mL) and washed with water (10 mL × 3). The organic layer was dried
(Na2SO4) and concentrated in vacuo. The crude product was
crystallized from ethanol to give the desired hydroxyl derivative.
4-(Aminosulfonyl)-N-[2-(3′-hydroxy)phenyl)ethyl]benzamide
1
(1a). Yield 85%; mp 172−174 °C. Rf = 0.58. H NMR (DMSO-d6) δ
2.83 (t, J = 7.25, 2H, CH2), 3.49 (m, 2H, CH2), 3.73 (s, 3H, OCH3),
6.76−6.83 (m, 3H, ArH), 7.21 (m, 1H, ArH), 7.47 (bs, 2H, NH2),
7.90 (d, 2H, J = 8.6, ArH), 7.96 (d, 2H, J = 8.6, ArH), 8.75 (bs, 1H,
NH). Anal. (C16H18N2O4S) C, H, N.
4-(Aminosulfonyl)-N-[2-(3′-ethoxy)phenyl)ethyl]benzamide (1b).
Yield 65%; mp 138−140 °C. Rf = 0.58.
4-(Aminosulfonyl)-N-[2-(3′-propoxy)phenyl)ethyl]benzamide
(1c). Yield 56%; mp 146−148 °C. Rf = 0.56.
General Procedure for the Synthesis of 4-(6-Alkoxy-3,4-
dihydroisoquinolin-1-yl)benzenesulfonamides (2a−c). Phosphorus
oxychloride (POCl3, 10 mmol) was added dropwise to a solution of 4-
(aminosulfonyl)-N-[2-(3′-alkoxyphenyl)ethyl]benzamide (1a−c) (1
mmol) in dry toluene (1 mL), and the mixture was refluxed for 6 h
at 110 °C. After cooling, ammonia aqueous solution (10 mL, 10%)
was added to quench the reaction, and the mixture was extracted with
ethyl acetate (3 × 5 mL) and dried over Na2SO4. The solvent was
removed under reduced pressure. The residue was powdered by
treatment with diethyl ether and crystallized from ethanol to give
derivatives 2a−c as yellow crystals.
1
(1d). Yield 78%; mp 194−196 °C. Rf = 0.27. H NMR (DMSO-d6) δ
2.74 (t, J = 7.5, 2H, CH2), 3.44 (m, 2H, CH2), 6.57−7.07 (m, 4H,
ArH), 7.45 (s, 2H, NH2), 7.87 (d, J = 6.7, 2H, ArH), 7.95 (d, J = 6.7,
2H, ArH), 8.72 (bs, 1H, NH), 9.26 (s, 1H, OH). Anal.
(C15H16N2O4S) C, H, N.
4-(6-Hydroxy-3,4-dihydroisoquinolin-1-yl)benzenesulfonamide
1
(2d). Yield 42%; mp 244−246 °C. Rf = 0.11. H NMR (DMSO-d6) δ
2.67 (t, J = 7.2, 2H, CH2), 3.69 (t, J = 7.2, 2H, CH2), 6.65−6.97 (m,
3H, ArH), 7.45 (s, 2H, NH2), 7.68 (d, J = 8.0, 2H, ArH), 7.88 (d, J =
8.0, 2H, ArH), 10.08 (s, 1H, OH). Anal. (C15H14N2O3S) C, H, N.
4-(6-Hydroxy-1,2,3,4-tetrahydroisoquinolin-1-yl)-
benzenesulfonamide (3d). Yield 70%; mp 137−139 °C. Rf = 0.05. 1H
NMR (DMSO-d6) δ 2.65− 3.06 (m, 4H, CH2CH2), 4.96 (s, 1H, CH),
6.38−6.52 (m, 3H, ArH), 7.31 (s, 2H, NH2), 7.42 (d, J = 8.3, 2H,
ArH), 7.75 (d, J = 8.3, 2H, ArH), 9.15 (s, 1H, OH). Anal.
(C15H16N2O3S) C, H, N.
4-(6-Methoxy-3,4-dihydroisoquinolin-1-yl)benzenesulfonamide
1
(2a). Yield 86%; mp 234−236 °C. Rf = 0.56. H NMR (DMSO-d6) δ
2.73 (t, J = 7.25, 2H, CH2), 3.71 (t, J = 7.25, 2H, CH2), 3.81 (s, 3H,
OCH3), 6.84 (dd, J = 2.8, J = 8.5, 1H, ArH), 6.95 (d, J = 2.8, 1H,
ArH), 7.05 (d, J = 8.5, 1H, ArH), 7.44 (bs, 2H, NH2), 7.69 (d, J = 8.2,
2H, ArH), 7.89 (d, J = 8.2, 2H, ArH); Anal. (C16H16N2O3S) C, H, N.
4-(6-Ethoxy-3,4-dihydroisoquinolin-1-yl)benzenesulfonamide
(2b). Yield 46%; mp 200−202 °C. Rf = 0.55.
1-[4-(Aminosulfonyl)phenyl]-6-hydroxy-1,2,3,4-tetrahydroisoqui-
noline-2-sulfonamide (4d). Yield 37%; mp 183−185 °C. Rf = 0.21.1H
NMR (DMSO-d6) δ 2.62− 3.55 (m, 4H, CH2−CH2), 5.84 (s, 1H,
CH), 6.59−6.88 (m, 3H, ArH), 6.96 (bs, 2H, NH2), 7.28 (bs, 2H,
NH2), 7.37 (d, J = 8.3, 2H, ArH), 7.73 (d, J = 8.3, 2H, ArH), 9.35 (bs,
1H, OH). Anal. (C15H17N3O5S2) C, H, N.
4-(6-Propoxy-3,4-dihydroisoquinolin-1-yl)benzenesulfonamide
(2c). Yield 81%; mp 232−234 °C. Rf = 0.55.
General Procedure for the Synthesis of 4-(6-alkoxy-1,2,3,4-
tetrahydroisoquinolin-1-yl)benzenesulfonamides (3a−c). A solution
of compounds 2a−c (1 mmol) and NaBH4 (10 mmol) in methanol
(18 mL) was stirred for 1 h at room temperature. The reaction mixture
was quenched by adding water (10 mL), extracted with ethyl acetate
(3 × 5 mL), dried over Na2SO4, and then concentrated under reduced
pressure. The crude products were crystallized by treatment with a
small amount of diethyl ether to afford the final products 3a−c as
white powders.
CA Inhibition Assay. An 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 indicator, working at the absorbance maximum of 557
nm, with 10−20 mM HEPES (pH 7.5) or Tris (pH 8.3) as buffers, and
20 mM Na2SO4 or 20 mM NaClO4 (for maintaining constant the ionic
strength), following the initial rates of 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 (10 mM) were prepared in distilled−deionized water, and
dilutions up to 0.01 nM were performed thereafter with distilled−
deionized water. Inhibitor 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 nonlinear least-squares methods using PRISM 3, as
reported earlier, and represent the mean from at least three different
determinations. CA isoforms were recombinant ones obtained as
reported earlier by this group.35−38
Protein Crystallography. Protein Crystallization and X-ray
Data Collection. Complexes of human carbonic anhydrase II
(purchased from Sigma, catalog no. C6165) with compounds I
and 2a were prepared by adding a 4-fold molar excess of
inhibitor (dissolved in pure DMSO) to 10 mg mL−1 protein
solution in water without pH adjustment. The best crystals of
the complexes were prepared by the vapor-diffusion hanging
drop method using crystallization conditions recently published
by Behnke et al.39 A 2 μL amount of complex solution was
mixed with 2 μL of precipitant solution containing 3 M
ammonium sulfate, 50 mM Tris-HCl, and 2 mM 4-
4-(6-Methoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)-
benzenesulfonamide (3a). Yield 60%; mp 182−184 °C. Rf = 0.30. 1H
NMR (DMSO-d6)δ 2.68−3.07 (m, 4H, CH2−CH2), 3.70 (s, 3H,
OCH3), 4.99 (s, 1H, CH), 6.50 (d, J = 8.5, 1H, ArH), 6.61 (dd, J = 2.8,
J = 8.5, 1H, ArH), 6.70 (d, J = 2.8, 1H, ArH), 7.30 (bs, 2H, NH2), 7.42
(d, J = 8.24, 2H, ArH), 7.75 (d, J = 8.24, 2H, ArH); Anal.
(C16H18N2O3S) C, H, N.
4 -(6-Ethoxy-1 , 2, 3, 4-tetrahydr oiso quinoli n-1-yl)-
benzenesulfonamide (3b). Yield 89%; mp 212−214 °C. Rf = 0.36.
4-(6-Propoxy-1,2,3,4-tetrahydroisoquinolin-1-yl) benzenesulfo-
namide (3c). Yield 30%; mp 187−189 °C. Rf = 0.33.
Synthesis of 1-[4-(Aminosulfonyl)phenyl]-6-alkoxy-1,2,3,4-tetra-
hydroisoquinoline-2-sulfonamides (4a−c). A mixture of compounds
3a−c (0.5 mmol) and sulfamide (6 mmol) in dimethoxyethane (2
mL) was placed in a cylindrical quartz tube (Ø 2 cm) and then stirred
and irradiated in a microwave oven at 150 W for two steps of 20 min at
100 °C. The reaction was quenched by addition of water (5 mL) and
extracted with ethyl acetate (3 × 5 mL). The organic layer was washed
with an aqueous saturated solution of NaHCO3 (2 × 5 mL), dried
over Na2SO4, and concentrated until dryness under reduced pressure.
The residue crystallized from ethanol to give final compounds 4a−c as
white powders.
1-[4-(Aminosulfonyl)phenyl]-6-methoxy-1,2,3,4-tetrahydroiso-
quinoline-2-sulfonamide (4a). Yield 74%; mp 102−104 °C. Rf = 0.55.
1H NMR (DMSO-d6) δ 2.68−2.73 (m, 1H, CH2CH2), 3.03−3.29 (m,
2H, CH2CH2), 3.33−3.52 (m, 1H, CH2CH2), 3.73 (s, 3H, OCH3),
5.88 (s, 1H, CH), 6.73−6.79 (m, 2H, ArH), 6.95 (m, 1H, ArH), 6.99
3897
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
(hydroxymercuri)benzoate (Sigma, catalog no. 55540) and
equilibrated over a 1 mL reservoir of precipitant solution at 18
°C. The final DMSO concentration in the drop was 2% (v/v).
Crystals with dimensions of 0.5 mm × 0.3 mm × 0.2 mm grew
within 5 days. Before data collection, the crystals were soaked
for 5−10 s in a reservoir solution supplemented with 25% (v/v)
glycerol and stored in liquid N2. Diffraction data were collected
at 100 K at beamlines BL14.1 and BL14.2 of BESSY, Berlin,
equipped with an MX-225 CCD detector from Rayonics
(Evanston, IN). A crystal of hCA II in complex with compound
I diffracted to 1.03 Å, and the diffraction data were processed
using the HKL-3000 suite of programs.40 A crystal of hCA II in
complex with compound 2a diffracted to 1.05 Å, and the
diffraction data were integrated and reduced using MOSFLM41
and scaled using SCALA42 from the CCP4 suite of programs.43
Crystal parameters and data collection statistics for both
complexes are summarized in Table 2.
Structure Determination, Refinement, and Analysis. The
structures of hCA II complexes with compounds I and 2a were
solved using the difference Fourier technique, using the hCA II
structure (Protein Data Bank entry 3PO6) as the initial model. Initial
rigid-body refinement and subsequent restrained refinement for both
hCA II complexes with I and 2a were performed using the program
REFMAC 543 with isotropic ADPs. The structures were refined with
one inhibitor molecule in the enzyme active site with occupancy 1 and
0.84 for compounds I and 2a, respectively. Atomic coordinates and the
geometric library for the inhibitor were generated using the PRODRG
server.44 The Coot program45 was used for inhibitor fitting, model
rebuilding, and addition of water molecules. Final refinement at
resolution was carried out with SHELXL-9746 in conjugate-gradient
least-squares mode, with 5% of the reflections reserved for cross-
validation. Both structures were first refined with isotropic ADPs. After
addition of solvent atoms and metal ions (zinc and mercury), building
inhibitor molecules in the active site, and several alternate
conformations for a number of residues, anisotropic ADPs were
refined for nearly all atoms (with the exception of spatially overlapping
atoms in segments with alternate conformations; also oxygen atoms of
water molecules with an unrealistic ratio of ellipsoid axes were refined
with isotropic ADPs) including the inhibitor molecules. The quality of
the crystallographic model was assessed with MolProbity.47 The final
refinement statistics are summarized in Table 2. All figures showing
structural representations were prepared using PyMOL.48
and AV0Z40550506), and Grant Agency of the Czech Republic
(Grant GA203/09/0820) is gratefully acknowledged. Research
from CTS lab was financed by a seventh FP grant (METOXIA
project). We thank MX 14.1 and 14.2 beamline staff at the
BESSY synchrotron for expert assistance during data collection.
ABBREVIATIONS USED
■
AAZ, acetazolamide; CA, carbonic anhydrase; HIF-1, hypoxia
inducible factor-1; PDB, protein data bank; TPM, topiramate;
ZNS, zonisamide
REFERENCES
■
(1) Supuran, C. T.; Scozzafava, A. Carbonic anhydrases as targets for
medicinal chemistry. Bioorg. Med. Chem. 2007, 15, 4336−4350.
(2) Supuran, C. T. Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nat. Rev. Drug Discovery
2008, 7, 168−181.
(3) Supuran, C. T. Carbonic anhydrase inhibition/activation: trip of a
scientist around the world in the search of novel chemotypes and drug
targets. Curr. Pharm. Des. 2010, 16, 3233−3245.
(4) Supuran, C. T. Carbonic anhydrase inhibitors. Bioorg. Med. Chem.
Lett. 2010, 20, 3467−3474.
(5) Supuran, C. T.; Scozzafava, A.; Casini, A. Carbonic anhydrase
inhibitors. Med. Res. Rev. 2003, 23, 146−189.
(6) Supuran, C. T.; Di Fiore, A.; De Simone, G. Carbonic anhydrase
inhibitors as emerging drugs for the treatment of obesity. Expert Opin.
Emerging Drugs 2008, 13, 383−392.
(7) Supuran, C. T. Diuretics: from classical carbonic anhydrase
inhibitors to novel applications of the sulfonamides. Curr. Pharm. Des.
2008, 14, 641−648.
(8) De Simone, G.; Supuran, C. T. Antiobesity carbonic anhydrase
inhibitors. Curr. Top. Med. Chem. 2007, 7, 879−884.
(9) Thiry, A.; Dogne, J. M.; Supuran, C. T.; Masereel, B. Carbonic
anhydrase inhibitors as anticonvulsant agents. Curr. Top. Med. Chem.
2007, 7, 855−864.
(10) Thiry, A.; Masereel, B.; Dogne, J. M.; Supuran, C. T.; Wouters,
J.; Michaux, C. Exploration of the binding mode of indanesulfona-
mides as selective inhibitors of human carbonic anhydrase type VII by
targeting Lys 91. ChemMedChem 2007, 2, 1273−1280.
(11) Asiedu, M.; Ossipov, M. H.; Kaila, K.; Price, T. J. Acetazolamide
and midazolam act synergistically to inhibit neuropathic pain. Pain
2010, 148, 302−308.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR assignment for compounds 1b,c, 2b,c, 3b,c, and 4b,c,
additional X-ray data, and a complete list of interactions
between hCA II and the two inhibitors I and 2a. This material
is available free of charge via the Internet at http://pubs.acs.org.
(12) Neri, D.; Supuran, C. T. Interfering with pH regulation in
tumours as a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10,
767−777.
(13) Said, H. M.; Supuran, C. T.; Hageman, C.; Staab, A.; Polat, B.;
Katzer, A.; Scozzafava, A.; Anacker, J.; Flentje, M.; Vordermark, D.
Modulation of carbonic anhydrase 9 (CA9) in human brain cancer.
Curr. Pharm. Des. 2010, 16, 3288−3299.
Accession Codes
†Atomic coordinates for the crystal structure of hCA II A with
compounds I and 2a can be accessed using PDB codes 3V7X
and 3VBD, respectively.
(14) Guler, O. O.; De Simone, G.; Supuran, C. T. Drug design
studies of the novel antitumor targets carbonic anhydrase IX and XII.
Curr. Med. Chem. 2010, 17, 1516−1526.
(15) Akurathi, V.; Dubois, L.; Lieuwes, N. G.; Chitneni, S. K.;
Cleynhens, B. J.; Vullo, D.; Supuran, C. T.; Verbruggen, A. M.;
Lambin, P.; Bormans, G. M. Synthesis and biological evaluation of a
99mTc-labelled sulfonamide conjugate for in vivo visualization of
carbonic anhydrase IX expression in tumor hypoxia. Nucl. Med. Biol.
2010, 37, 557−564.
AUTHOR INFORMATION
Corresponding Author
*(R.G.) E-mail: rgitto@unime.it. Phone: 00390906766413.
Fax: 00390906766402. (F.M.D.) E-mail: fdamiano@unime.it.
Phone: 00390906766465. Fax: 00390906766402.
■
(16) Genis, C.; Sippel, K. H.; Case, N.; Cao, W.; Avvaru, B. S.;
Tartaglia, L. J.; Govindasamy, L.; Tu, C.; Agbandje-McKenna, M.;
Silverman, D. N.; Rosser, C. J.; McKenna, R. Design of a carbonic
anhydrase IX active-site mimic to screen inhibitors for possible
anticancer properties. Biochemistry 2009, 48, 1322−1331.
(17) Thiry, A.; Supuran, C. T.; Masereel, B.; Dogne, J. M. Recent
developments of carbonic anhydrase inhibitors as potential anticancer
drugs. J. Med. Chem. 2008, 51, 3051−3056.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this research awarded by MiUR
(Prin2008, grant number No 20085HR5JK_002), Academy
of Sciences of the Czech Republic (Projects AV0Z50520514
3898
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899

Journal of Medicinal Chemistry
Article
(18) Pastorekova, S.; Kopacek, J.; Pastorek, J. Carbonic anhydrase
inhibitors and the management of cancer. Curr. Top. Med. Chem. 2007,
7, 865−878.
(19) De Simone, G.; Vitale, R. M.; Di Fiore, A.; Pedone, C.;
Scozzafava, A.; Montero, J. L.; Winum, J. Y.; Supuran, C. T. Carbonic
anhydrase inhibitors: Hypoxia-activatable sulfonamides incorporating
disulfide bonds that target the tumor-associated isoform IX. J. Med.
Chem. 2006, 49, 5544−5551.
(20) Parkkila, S.; Parkkila, A. K.; Rajaniemi, H.; Shah, G. N.; Grubb,
J. H.; Waheed, A.; Sly, W. S. Expression of membrane-associated
carbonic anhydrase XIV on neurons and axons in mouse and human
brain. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1918−1923.
(21) Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T.
Design of zinc binding functions for carbonic anhydrase inhibitors.
Curr. Pharm. Des. 2008, 14, 615−621.
(22) Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T.
Therapeutic potential of sulfamides as enzyme inhibitors. Med. Res.
Rev. 2006, 26, 767−792.
(23) Gitto, R.; Ferro, S.; Agnello, S.; De Luca, L.; De Sarro, G.;
Russo, E.; Vullo, D.; Supuran, C. T.; Chimirri, A. Synthesis and
evaluation of pharmacological profile of 1-aryl-6,7-dimethoxy-3,4-
dihydroisoquinoline-2(1H)-sulfonamides. Bioorg. Med. Chem. 2009,
17, 3659−3664.
(24) Gitto, R.; Agnello, S.; Ferro, S.; Vullo, D.; Supuran, C. T.;
Chimirri, A. Identification of potent and selective human carbonic
anhydrase VII (hCA VII) inhibitors. ChemMedChem 2010, 5, 823−
826.
(25) Gitto, R.; Agnello, S.; Ferro, S.; De Luca, L.; Vullo, D.; Brynda,
J.; Mader, P.; Supuran, C. T.; Chimirri, A. Identification of 3,4-
Dihydroisoquinoline-2(1H)-sulfonamides as potent carbonic anhy-
drase inhibitors: synthesis, biological evaluation, and enzyme−ligand
X-ray studies. J. Med. Chem. 2010, 53, 2401−2408.
(26) Gitto, R.; Damiano, F. M.; De Luca, L.; Ferro, S.; Vullo, D.;
Supuran, C. T.; Chimirri, A. Synthesis and biological profile of new
1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhib-
itors. Bioorg. Med. Chem. 2011, 19, 7003−7007.
(35) Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.;
Mastrolorenzo, A.; Supuran, C. T. Carbonic anhydrase inhibitors:
inhibition of the transmembrane isozyme XIV with sulfonamides.
Bioorg. Med. Chem. Lett. 2005, 15, 3828−3833.
(36) Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.;
Mastrolorenzo, A.; Supuran, C. T. Carbonic anhydrase inhibitors.
The mitochondrial isozyme VB as a new target for sulfonamide and
sulfamate inhibitors. J. Med. Chem. 2005, 48, 7860−7866.
(37) Innocenti, A.; Vullo, D.; Pastorek, J.; Scozzafava, A.;
Pastorekova, S.; Nishimori, I.; Supuran, C. T. Carbonic anhydrase
inhibitors. Inhibition of transmembrane isozymes XII (cancer-
associated) and XIV with anions. Bioorg. Med. Chem. Lett. 2007, 17,
1532−1537.
(38) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.;
Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Carbonic
anhydrase inhibitors: DNA cloning and inhibition studies of the alpha-
carbonic anhydrase from Helicobacter pylori, a new target for
developing sulfonamide and sulfamate gastric drugs. J. Med. Chem.
2006, 49, 2117−2126.
(39) Behnke, C. A.; Le Trong, I.; Godden, J. W.; Merritt, E. A.;
Teller, D. C.; Bajorath, J.; Stenkamp, R. E. Atomic resolution studies of
carbonic anhydrase II. Acta Crystallogr. Biol. Crystallogr. 2010, 66,
616−627.
(40) Minor, W.; Cymborowski, M.; Otwinowski, Z.; Chruszcz, M.
HKL-3000: the integration of data reduction and structure solution--
from diffraction images to an initial model in minutes. Acta Crystallogr.
Biol. Crystallogr. 2006, 62, 859−866.
(41) Leslie, A. G. Integration of macromolecular diffraction data. Acta
Crystallogr. Biol. Crystallogr. 1999, 55, 1696−1702.
(42) Evans, P. R. Proceedings of the CCP4 Study Weekend. Data
Collection and Processing, Daresbury Laboratory, Warrington, 1993.
(43) The CCP4 suite: programs for protein crystallography. Acta
Crystallogr. Biol. Crystallogr. 1994, 50, 760−763.
(44) Schuttelkopf, A. W.; van Aalten, D. M. PRODRG: a tool for
high-throughput crystallography of protein-ligand complexes. Acta
Crystallogr. Biol. Crystallogr. 2004, 60, 1355−1363.
(45) Emsley, P.; Cowtan, K. Coot: model-building tools for
molecular graphics. Acta Crystallogr. Biol. Crystallogr. 2004, 60,
2126−2132.
(27) Mader, P.; Brynda, J.; Gitto, R.; Agnello, S.; Pachl, P.; Supuran,
C. T.; Chimirri, A.; Rezacova, P. Structural basis for the interaction
between carbonic anhydrase and 1,2,3,4-tetrahydroisoquinolin-2-
ylsulfonamides. J. Med. Chem. 2011, 54, 2522−2526.
(46) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 2008, 64, 112−122.
(28) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561−2573.
(29) Eriksson, A. E.; Jones, T. A.; Liljas, A. Refined structure of
human carbonic anhydrase II at 2.0 A resolution. Proteins 1988, 4,
274−282.
(30) Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T.
Sulfamates and their therapeutic potential. Med. Res. Rev. 2005, 25,
186−228.
(47) Lovell, S. C.; Davis, I. W.; Arendall, W. B., III; de Bakker, P. I.;
Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C.
Structure validation by Calpha geometry: phi,psi and Cbeta deviation.
Proteins 2003, 50, 437−450.
(48) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano
Scientific: Palo Alto, CA, 2002.
(49) Brunger, A. T. Free R value: a novel statistical quantity for
assessing the accuracy of crystal structures. Nature 1992, 355, 472−
475.
(31) Srivastava, D. K.; Jude, K. M.; Banerjee, A. L.; Haldar, M.;
Manokaran, S.; Kooren, J.; Mallik, S.; Christianson, D. W. Structural
analysis of charge discrimination in the binding of inhibitors to human
carbonic anhydrases I and II. J. .Am. Chem. Soc. 2007, 129, 5528−5537.
(32) Alterio, V.; Monti, S. M.; Truppo, E.; Pedone, C.; Supuran, C.
T.; De Simone, G. The first example of a significant active site
conformational rearrangement in a carbonic anhydrase-inhibitor
adduct: the carbonic anhydrase I-topiramate complex. Org. Biomol.
Chem. 2010, 8, 3528−3533.
(33) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.;
Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.;
Scozzafava, A.; Monti, S. M.; De Simone, G. Crystal structure of the
catalytic domain of the tumor-associated human carbonic anhydrase
IX. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233−16238.
(34) Whittington, D. A.; Grubb, J. H.; Waheed, A.; Shah, G. N.; Sly,
W. S.; Christianson, D. W. Expression, assay, and structure of the
extracellular domain of murine carbonic anhydrase XIV: implications
for selective inhibition of membrane-associated isozymes. J. Biol. Chem.
2004, 279, 7223−7228.
3899
dx.doi.org/10.1021/jm300112w | J. Med. Chem. 2012, 55, 3891−3899