Development of potent carbonic anhydrase inhibitors incorporating both sulfonamide and sulfamide groups

Article abstract of DOI:10.1021/jm300818k

A series of compounds incorporating both sulfonamide and sulfamide as zinc-binding groups (ZBGs) are reported as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Crystallographic studies on the complex of hCA II with the lead compound of this series, namely, 4-sulfamido- benzenesulfonamide, revealed the binding of two molecules in the enzyme active site cavity, the first one canonically coordinated to the zinc ion by means of the sulfonamide group and the second one located at the entrance of the cavity. This observation led to the design of elongated molecules incorporating these two ZBGs, separated by a linker of proper length, to allow the simultaneous binding to these different sites. The long inhibitors indeed showed around 10 times better enzyme inhibitory properties as compared to the shorter molecules against four physiologically relevant human (h) isoforms, hCA I, II, IX, and XII.

Full text of DOI:10.1021/jm300818k

Article
pubs.acs.org/jmc
Development of Potent Carbonic Anhydrase Inhibitors Incorporating
Both Sulfonamide and Sulfamide Groups
Katia D'Ambrosio,† Fatma-Zhora Smaine,‡ Fabrizio Carta,§ Giuseppina De Simone,† Jean-Yves Winum,*,‡
and Claudiu T. Supuran*,§
†Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy
‡Institut des Biomolec
Nationale Super
§Universita
degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino
́
ules Max Mousseron (IBMM), UMR 5247, CNRS-UM1-UM2 Bat
ieure de Chimie de Montpellier, 8 rue de l′Ecole Normale, 34296 Montpellier Cedex, France
̂
iment de Recherche Max Mousseron, Ecole
́
̀
(Firenze), Italy
S
* Supporting Information
ABSTRACT: A series of compounds incorporating both sulfonamide and sulfamide as
zinc-binding groups (ZBGs) are reported as inhibitors of the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1). Crystallographic studies on the complex of hCA II with the
lead compound of this series, namely, 4-sulfamido-benzenesulfonamide, revealed the
binding of two molecules in the enzyme active site cavity, the first one canonically
coordinated to the zinc ion by means of the sulfonamide group and the second one located
at the entrance of the cavity. This observation led to the design of elongated molecules
incorporating these two ZBGs, separated by a linker of proper length, to allow the
simultaneous binding to these different sites. The “long” inhibitors indeed showed around
10 times better enzyme inhibitory properties as compared to the shorter molecules against
four physiologically relevant human (h) isoforms, hCA I, II, IX, and XII.
programs and with the help of X-ray crystallography,17 much
INTRODUCTION
■
progress has been achieved.1−3 Indeed, by considering the three
main structural elements present in the molecule of a classical
potent CAI, that is, a zinc-binding group (ZBG), an organic
scaffold, and one or more side chains substituting the scaffold,
important advances have been made in the understanding of
the factors that govern both potency and selectivity against
various hCA isozymes.
Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes
present in most living organisms and encoded by five
evolutionarily unrelated gene families: the α-, β-, γ-, δ- and ζ-
CAs. Human CAs (hCAs) all belong to the α-family and are
present in 15 isoforms, which differ for tissue distribution,
cellular localization, and kinetic properties. All of these enzymes
catalyze the reversible hydration of carbon dioxide to
bicarbonate and proton, a very simple reaction that is, however,
of fundamental importance to many physiological processes
based on gas exchange, ion transport, and pH balance.
Consequently, hCAs are involved in a variety of physiological
processes, and their abnormal levels and/or activities often have
been associated with different human diseases.1−3
In the last years, CA isozymes have become an interesting
target for the design of inhibitors or activators with biomedical
applications.4−7 Indeed, originally, CA inhibitors (CAIs) were
clinically used as diuretic,8 antiglaucoma,9 anticonvulsant,10 and
antiobesity drugs,11 whereas their employment in the manage-
ment of hypoxic tumors was only recently validated,12−14 with
several monoclonal antibodies (Mabs)15 and small molecule
inhibitors in various stages of clinical development.15,16
However, because of the large number of hCA isoforms,1−3
there is a constant need to improve the inhibition and
selectivity profile of the so far developed CAIs, to avoid side
effects due to inhibition of isoforms not involved in a certain
pathology.2 In the last period, by using extensive drug design
The ZBGs leading to potent CAIs may belong to various
functionalities with the classical sulfonamide one still
constituting the main player. The most obvious bioisosteres
of this group are the sulfamate and sulfamide, in which an
additional electron-withdrawing atom/group (O or NH,
respectively) is directly attached to the sulfamoyl function,
generating compounds with general formula R−O−SO2NH2 or
R−NH−SO2NH2. Such compounds were shown to lead
equipotent, and in some cases even stronger, inhibitors than
the corresponding sulfonamides. The X-ray structures of the
two simplest compounds of such family, namely, sulfamide and
sulfamic acid, in adduct with hCA II have been reported,
showing that such molecules were able to participate to a more
complicated hydrogen bond network with respect to that
formed by the classical sulfonamide inhibitors, due to the
presence of the additional oxygen/nitrogen atom.18 At the
Received: February 13, 2012
Published: July 9, 2012
© 2012 American Chemical Society
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Scheme 1. Preparation of Compounds 1−7
Scheme 2. Synthesis of the “Long” CAIs 11 and 12
present time, there are a large number of aromatic, heterocyclic,
aliphatic, and sugar-based sulfamates and sulfamides, which
were shown to possess highly effective inhibitory properties
against all known human isoforms.2 Thus, also, the sulfamates
and the sulfamides constitute a very important class of CAIs,
with some derivatives clinically used for the treatment of
epilepsy and obesity.10,11,19−21
In this paper, we report a new series of CAIs incorporating in
the same molecule two ZBGs, that is, the sulfonamide and
sulfamide ones. The rationale behind our choice was to
understand on one hand which of the two ZBGs could bind the
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metal ion within the enzyme active site and, on the other hand,
how the presence of two ZBGs in the same scaffold may
influence the selectivity profile of the inhibitor against four of
the major isoforms, the cytosolic CA I and II and the
transmembrane, tumor-associated CA IX and XII.1−3,16
Table 1. Crystal Parameters, Data Collection, and
Refinement Statistics for the hCA II−1 Complex
a
crystal parameters
space group
P21
́
unit cell parameters (Å, °)
a = 42.25
b = 41.35
c = 71.96
β = 104.36
RESULTS AND DISCUSSION
■
A straightforward chemistry has been used to prepare
sulfonamides also incorporating sulfamide moieties of types
1−7 (Scheme 1). Simple aromatic sulfonamides possessing
amino- or aminoalkyl moieties of type A, or their sulfonylated
derivatives of types B and C,22,23 were converted to the
corresponding sulfamides by reaction with N-tert-butoxycar-
bonyl sulfamoylchloride [prepared ab initio by reaction of
chlorosulfonyl isocyanate (CSI) with tert-butanol], followed by
deprotection with trifluoroacetic acid (TFA), as reported earlier
for simple amines.24 Two other compounds, 11 and 12 (which
will be called “long” CAIs), have been obtained as depicted in
Scheme 2. N-Acetyl-sulfanilyl chloride was treated with p-
amino-benzoic acid (PABA), leading to 8, which was
deprotected in the presence of concentrated HCl to the
amino derivative 9.25,26 This latter was then sulfamoylated
using sulfamoylchloride as a reagent to give the sulfamide 10.
This compound, possessing a free COOH moiety, was reacted
with 4-aminoethylbenzenesulfonamide and led to the first long
CAI, compound 11 (the condensation was achieved by the
classical dicyclohexyl carbodiimide chemistry). The key
intermediate 8 was also condensed with 4-aminoethylbenzene-
sulfonamide in the same conditions and led to 12, which differs
from 11 by the presence of an acetamido moiety instead of the
sulfamide one.
data collection statistics (20.00−1.50 Å)
temperature (K)
wavelength (Å)
total reflections
unique reflections
completeness (%)
100
1.0000
123249
38111
98.2 (84.0)
5.7 (20.6)
15.6 (4.9)
b
Rmerge (%)
mean I/σ(I)
refinement statistics (20.00−1.50 Å)
c
Rfactor (%)
18.6
21.2
c
Rfree (%)
rmsd from ideal geometry:
bond lengths (Å)
0.006
1.4
bond angles (°)
no. of protein atoms
no. of water molecules
no. of inhibitor atoms
average B factor
2121
445
45
11.75
a
Values in parentheses refer to the highest resolution shell, 1.55−1.50
b
Å. Rmerge = ΣhklΣi|Ii(hkl) − ⟨I(hkl)⟩|/ΣhklΣiIi(hkl), where Ii(hkl) is the
intensity of an observation and ⟨I(hkl)⟩ is the mean value for its
c
unique reflection; summations are over all reflections. Rfactor = Σh||
Fo(h)| − |Fc(h)||/Σh|Fo(h)|, where Fo and Fc are the observed and
calculated structure-factor amplitudes, respectively. Rfree was calculated
with 5% of the data excluded from the refinement.
Before discussing the CA inhibitory data of compounds 1−
12, we shall present a serendipitous crystallographic finding,
which has triggered the preparation of the elongated
compounds described here. We obtained crystals of the
complex of hCA II with the simple sulfonamide-sulfamide
derivative 1 (KI of 74 nM, see later in the text). These crystals
were isomorphous with those of the native protein,27 which was
then used as a starting model for crystallographic refinement
after deletion of nonprotein atoms. Statistics for data collection
and refinement are shown in Table 1. Inspection of the electron
density maps, at various stages of crystallographic refinement,
clearly showed the binding to the enzyme of three inhibitor
molecules: one positioned on the protein surface far away from
the active site, which will not be discussed in the following, and
the other two located in the active site channel (Figure 1). The
first one, entirely defined in the electron density maps (Figure
2), was located at the bottom of the active site and coordinated
to Zn2+ ion through its sulfonamide group. In particular, the
ionized sulfonamide NH− group coordinated to the catalytic
Zn2+ ion with a tetrahedral geometry and donated a hydrogen
bond to the Thr199OG1 atom (2.84 Å). One sulfonamide
oxygen accepted a hydrogen bond from the backbone NH
group of Thr199 (2.92 Å), while the other one was at a distance
of 2.97 Å from the Zn2+ ion. The binding through the
sulfonamide moiety instead of the sulfamide one was probably
due to the lower pKa value of the first one, allowing a much
higher population of the anionic form, which is required for
binding to Zn2+. The benzene ring of the inhibitor made a large
number of strong (<4.5 Å) hydrophobic contacts with residues
Gln92, His94, Val121, Phe131, Leu198, and Thr200. Finally,
the sulfamide group in para-position to the coordinated
sulfamoyl moiety did not interact with the protein but made
Figure 1. Solvent accessible surface of hCA II in its complex with 1.
The two molecules of 1 bound within the active site cavity are shown
in stick representation.
a large number of hydrogen bond with several water molecules
(Figure 3).
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Figure 4. Simulated annealing omit |2Fo − Fc| electron density map,
relative to the inhibitor molecule 1 located on the rim of the active site
cavity. Residues involved in inhibitor recognition are also shown.
two of which with two main chain atoms (Figures 3 and 4).
Additional hydrophobic interactions of the phenyl ring
contributed to the stabilization of the binding, while the
sulfonamide group did not interact directly with the enzyme.
Altogether, these findings suggested that molecules containing a
sulfonamide as ZBG, a linker of proper length, and a sulfamide
able to interact with the border of the active site could show
high affinity for the CA active site. To explore this hypothesis,
we designed compound 11, an elongated CAI, and compound
12, which has the same length as 11 but possesses an acetamide
instead of the sulfamide moiety present in 11.
Figure 2. Simulated annealing omit |2Fo − Fc| electron density map,
relative to the inhibitor molecule 1 coordinated to the catalytic zinc
ion, which is also shown together with its coordinating residues.
The inhibition data of all designed compounds against four
human isoforms, namely, hCA I, II, IX, and XII, are reported in
Tables 2 and 3.28 Both the CO2 hydrase and the esterase assays
(with 4-nitrophenylacetate as substrate) have been used,28 as
one of the reviewers suggested a different binding assay than
monitoring only the inhibition of the physiologic reaction.
Analysis of these inhibition data shows a very interesting
inhibition profile for this newly designed series of compounds.
For the CO2 hydrase assay, starting from the short inhibitors
Table 2. hCA I, II, IX, and XII Inhibition Data with
Sulfonamides 1−12 and Standard Inhibitors AZA, EZA, and
DCP by a Stopped-Flow, CO2 Hydration Assay Method28a
a
KI (nM)
b
b
c
c
hCA I
hCA II
hCA IX
hCA XII
1
1130
1425
72
74
1170
59
64
65
57
54
2
Figure 3. Ribbon diagram of the hCA II−1 complex. The two
inhibitor molecules (green scaffold) bound within the active site are
represented in stick representation. Residues participating in
recognition of the inhibitor molecule bound to the catalytic zinc ion
are reported in cyan, while those interacting with the external inhibitor
molecule are shown in magenta. The active site Zn2+ coordination is
also reported.
3
611
27
580
62
4
633
148
43
9.1
178
5
35
53
6
63
62
49
65
48
7
473
241
237
379
250
25
52
79
10
11
12
AZA
EZA
DCP
57
43
0.6
5.2
12
0.4
4.5
25
0.5
3.9
5.7
56
The second inhibitor molecule, which had lower interaction
affinity with the enzyme (occupation factor = 0.7) (Figure 4),
was located close to the protein surface, with the sulfonamide
moiety pointing toward the interior of the active site and the
sulfamide one oriented toward the external part of the cavity.
The last group was the main player in stabilizing the binding of
this inhibitor, forming three hydrogen bonds with the enzyme,
8
43
1200
38
50
50
a
Errors in the range of 5−10% of the reported value from three
b
c
different determinations. Full length, cytosolic isoform. Catalytic
domain, recombinant enzyme.
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subnanomolar range (KI of 0.6 nM). This compound was also a
subnanomolar inhibitor of hCA IX and XII, whereas its affinity
for hCA I was 237 nM, not being much different from that of
the medium length derivative 5. We thus consider that the
hypothesis that led us to synthesize such a long molecule CAI
has been verified, since we observe a huge inhibitory power
increase (114−160 times) against the three CA isoforms of
interest as drug targets, that is, hCA II, IX, and XII. It is
interesting to note that compound 12, which has the same
length as 11 but incorporates an acetamido instead of the
sulfamide moiety, is still a potent hCA II, IX, and XII inhibitor
(KI values of 3.9−5.2 nM), even if slightly less efficient as
compared to 11. It is thus probable that the sulfamide moiety
present in 11, as the one from the second molecule of 1 bound
to hCA II, participates in interactions with active site residues,
to which the acetamido moiety cannot participate. It also may
be observed that the long compounds 11 and 12 reported here
are much better CAIs against all isoforms as compared to the
clinically used derivatives acetazolamide (AZA), ethoxzolamide
(EZA), and dichlorophenamide (DCP) (Table 2 and Figure 5).
Table 3. hCA I and II Inhibition Data with Sulfonamides 1−
12 and the Standard Inhibitors AZA by a
Spectrophotometric Esterase Assay Method with 4-
Nitrophenylacetate as the Substrate28b
a
KI (nM)
compd
hCA I
hCA II
1
1650
1745
87
123
1239
68
2
3
4
950
203
75
35
5
182
81
6
7
638
327
320
384
342
80
10
11
12
AZA
62
5.3
12.9
159
a
Means from three different measurements.
1−3, it can be observed that compounds 1 and 2 were weak
hCA I inhibitors (KI values of 1.13−1.42 μM), while the
longest molecule 3 was a medium potency inhibitor (KI of 72
nM). Against hCA II, the profile was more complicated, with
compounds 1 and 3 being medium potency inhibitors (KI
values of 59−74 nM), whereas the homosulfanilamide
derivative 2 was a weak inhibitor (KI of 1.17 μM). On the
contrary, the tumor-associated isoforms hCA IX and XII were
inhibited moderately by 1 and 2 (KI values of 64−65 nM
against CA IX and of 54−57 nM against CA XII, respectively),
whereas the longer compound 3 was a weak inhibitor of both
isoforms (Table 2).
Figure 5. Chemical structures of classical CAIs AZA, EZA, and DCP.
The esterase inhibition data of Table 3 (against hCA I and II, as
the transmembrane isoforms hCA IX and XII have much
weaker esterase activity with 4-nitrophenyl acetate as
substrate)28c very much parallel the CO2 hydrase inhibition
data of Table 2, only that generally the KI values are higher for
the esterase method, a fact already reported by Pocker and
Stone in 1967.28b Indeed, the short inhibitors were weaker than
the long-tailed ones, confirming thus (by a distinct method) the
data presented in Table 2 and discussed above.
The medium length compounds 4−7 generally showed an
increase of their inhibitory capability against all isoforms as
compared to the corresponding short inhibitors 1−3 with
which they were structurally related. Indeed, against hCA I and
II, an increase of the length of these molecules was generally
reflected in a decrease of the KI values. For example, by
comparing the inhibition of hCA I with the pair 2 and 5, the
last compound is 9.6 times a better inhibitor as compared to the
short derivative 2. Against hCA II, the same compounds differ
by a factor of 6.6 in inhibiting this isoform. Less differences
were observed for the inhibition of hCA IX and especially hCA
XII, for which some of the short molecules inhibitors (e.g., 1
and 2) showed comparable activity with the longer ones (4−6).
Interestingly, for derivatives 4−7, compound 4, incorporating
the sulfanilamide scaffold, is the best hCA II and IX inhibitor,
even if it has a slightly shorter molecule as compared to its
congeners 5 and 6. Thus, the structure−activity relationship
(SAR) is rather complicated for derivatives 1−7, with the
length of the molecule being an important factor but not the
only one influencing enzyme inhibition. Furthermore, the four
isoforms showed a rather different inhibition profile with these
derivatives. All of these observations also can be applied to
derivative 10, which incorporates PABA instead of the
sulfanilamide moiety. This derivative has only the sulfamide
as ZBG and shows medium potency inhibitory activity against
all isoforms, with hCA IX, XII, and II being better inhibited
than hCA I. The most interesting inhibition profile was
observed with the long CAIs 11 and 12, designed on the basis
of the observation that two molecules of 1 were bound within
the hCA II active site. Indeed, as compared to 1, compound 11
was 123.3 times a better hCA II inhibitor, with an efficacy in the
CONCLUSIONS
■
Here, we reported an interesting case of drug design of CAIs
based on a serendipitous observation of two molecules of 4-
sulfamido-benzenesulfonamide bound within the active site of
hCA II. Although SAR for this family of inhibitors is not at all
simple, an elongation of the molecules gradually led to
increased inhibitory power, with the longest molecules being
subnanomolar or low nanomolar inhibitors of hCA II, IX, and
XII, whereas the inhibition of the offtarget isoform hCA I
remained in the micromolar range.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were of commercial quality
and used without further purification, unless otherwise specified. All
reactions were carried out under an inert atmosphere of nitrogen. TLC
analyses were performed on silica gel 60 F254 plates (Merck
Art.1.05554). Spots were visualized under 254 nm UV illumination
1
or by ninhydrin solution spraying. H and 13C NMR spectra were
recorded on Bruker DRX-400 spectrometer using DMSO-d6 as solvent
and tetramethylsilane as internal standard. For 1H NMR spectra,
chemical shifts are expressed in δ (ppm) downfield from
tetramethylsilane, and coupling constants (J) are expressed in Hertz.
Electron ionization mass spectra were recorded in positive or negative
mode on a Water MicroMass ZQ. The purity of the obtained
compounds was checked by HPLC and was >98%.
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Compounds 1−7 were prepared according the procedure described
in ref 24 (see the Supporting Information). Starting material was
commercially available or synthesized following procedures reported in
refs 22 and 23.
filtered-off, and concentrated in vacuo to give a solid residue that was
purified by silica gel column chromatography eluting with 20%
MeOH/methylene chloride v/v to afford the title compound as a light-
brown solid. Yield: 45%. Silica gel TLC Rf 0.22 (MeOH/methylene
chloride 20% v/v). 1H NMR (400 MHz, DMSO-d6): δ 10,55 (brs, 1H,
exchange with D2O), 10,22 (s, 1H, exchange with D2O), 8.46 (t, 1H, J
= 6.0 Hz, exchange with D2O, CONH), 7.77 (d, 2H, J = 8.8 Hz, Ar−
H), 7.74 (d, 2H, J = 8.8 Hz, Ar−H), 7.69 (d, 2H, J = 8.8 Hz, Ar−H),
7.44 (d, 2H, J = 8.8 Hz, Ar−H), 7.39 (s, 2H, exchange with D2O,
SO2NH2), 7.31 (s, 2H, exchange with D2O, SO2NH2), 7.23 (d, 2H, J =
8.8 Hz, Ar−H), 7.17 (d, 2H, J = 8.8 Hz, Ar−H), 3.48 (q, 2H, J = 6.0
Hz, CH2CH2NH), 2.91 (t, 2H, J = 6.0 Hz, CH2CH2NH). 13C NMR
(100 MHz, DMSO-d6): 166.7, 144.8, 144.7, 143.1, 141.6, 132.4, 130.6,
130.2, 129.4, 129.2, 126.8, 119.2, 117.6, 35.9, 31.8. The synthesis and
characterization of 12 is provided in the Supporting Information.
CA Inhibition. CO2 Hydrase Assay. An Applied Photophysics
stopped-flow instrument has been used for assaying the CA-catalyzed
CO2 hydration activity.28a Phenol red (at a concentration of 0.2 mM)
has been used as an indicator, working at the absorbance maximum of
557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4
(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 (0.1 mM)
were prepared in distilled−deionized water, and dilutions up to 0.01
nM were done thereafter with distilled−deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, to allow for the formation of the E−I
complex. The inhibition constants were obtained by nonlinear least-
squares methods using PRISM 3 and represent the mean from at least
three different determinations. CA isoforms were recombinant ones
obtained in house as reported earlier.
Esterase Assay. Initial rates of 4-nitrophenyl acetate hydrolysis
catalyzed by different CA isozymes were monitored spectrophoto-
metrically, at the isosbestic point (347 nm), with a Cary 3 instrument
interfaced with an IBM compatible PC.28b Solutions of substrate were
prepared in anhydrous acetonitrile; the substrate concentrations varied
between 2.10−2 and 1.10−6 M, working at 25 °C. A molar absorption
coefficient ε of 18400 M−1 cm−1 was used for the 4-nitrophenolate
formed by hydrolysis, in the conditions of the experiments (pH 7.40),
as reported by Pocker and Stone.28b Nonenzymatic hydrolysis rates
were always subtracted from the observed rates. Duplicate experiments
were done for each inhibitor concentration, and the values reported
throughout the paper are the mean of such results. Stock solutions of
inhibitor (1 mM) were prepared in distilled−deionized water with
10% (v/v) DMSO (which is not inhibitory at these concentrations),
and dilutions up to 0.01 nM were done thereafter with distilled−
deionized water. Inhibitor and enzyme solutions were preincubated
together for 10 min at room temperature prior to assay, to allow for
the formation of the E−I complex. The inhibition constant KI was
determined as described by Pocker and stone.28b Enzyme concen-
trations were 3.2 nM for hCA II and 11 nM for hCA I (isoforms hCA
IX and XII have a much lower esterase activity than hCA I and II and
were not assayed by this method).
N-Sulfamoyl-4-aminobenzene Sulfonamide (1). Yield: 80%.
1H NMR (400 MHz, DMSO): δ 10.1 (s, 1H), 7.75 (d, 2H, J = 7.27
Hz), 7.35 (s, 2H), 7.25 (d, 2H, J = 7.27 Hz), 7.2 (s, 2H). MS ESI+/
ESI−: m/z 252.3 (M + H)+, 275.28 (M + Na)+, 250.3 (M − H)−. Anal.
calcd for C6H9N3O4S2: C, 28.68; H, 3.61; N, 16.72. Found: C, 28.72;
H, 3.64; N, 16.73. Detailed characterization of compounds 2−7 is
provided in the Supporting Information.
Synthesis of 4-(4-Acetamidophenylsulfonamido)benzoic
Acid (8).29 N-Acetylsulfanylylchloride (10.48 g, 1.0 equiv) and 4-
aminobenzoic acid (1.0 equiv) were suspended in dry acetone (42.0
mL) containing dry pyridine (8% v/v). The white mixture was stirred
vigorously at r.t. until the starting material was consumed (TLC
monitoring). Then, the reaction was quenched with H2O (60 mL) and
triturated with 1.0 M aqueous hydrochloric acid to afford the titled
product as a white solid that did not required further purification.
Yield: 67%. Silica gel TLC Rf 0.35 (MeOH/methylene chloride 20% v/
1
v). H NMR (400 MHz, DMSO-d6): δ 10,73 (s, 1H, exchange with
D2O), 10,37 (s, 1H, exchange with D2O), 10.00 (brs, 1H, exchange
with D2O), 7.82 (d, 2H, J = 8.4 Hz, Ar−H), 7.78 (m, 4H, Ar−H), 7.21
(d, 2H, J = 8.4 Hz, Ar−H), 2.09 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6): 170.1, 169.3, 144.0, 142.1, 136.3, 132.0, 130.1, 128.5,
119.1, 117.0, 22.5. Data are in agreement with reported data.29
Synthesis of 4-(4-Aminophenylsulfonamido)benzoic Acid
(9).29 4-(4-Acetamidophenylsulfonamido)benzoic acid 8 (1.2 g) was
suspended in 12 M aqueous hydrochloric acid, and the mixture was
heated at reflux for 9 h and cooled down to r.t., and the pH adjusted to
3 by the addition of aqueous concentrated NH4OH. The precipitate
formed was collected by filtration, dried under vacuo, and used for the
next step as it is. Yield: 32%. Silica gel TLC Rf 0.10 (MeOH/
1
methylene chloride 20% v/v). H NMR (400 MHz, DMSO-d6): δ
10,37 (s, 1H, exchange with D2O), 7.83 (d, 2H, J = 8.4 Hz, Ar−H),
7.79 (d, 2H, J = 8.4 Hz, Ar−H), 7.22 (d, 2H, J = 8.4 Hz, Ar−H), 7.58
(d, 2H, J = 8.4 Hz, Ar−H), 6.06 (brs, 2H, NH2). 13C NMR (100 MHz,
DMSO-d6): 170.0, 149.6, 145.0, 132.1, 130.0, 129.2, 118.4, 117.2,
116.8. Data are in agreement with reported data.29
Synthesis of 4-(4-(Sulfamoylamino)phenylsulfonamido)-
benzoic Acid (10).30 4-(4-Aminophenylsulfonamido)benzoic acid 9
(0.1 g, 1.0eq) was dissolved in dry N,N-dimethylacetamide (5.0 mL)
and treated with freshly prepared sulfamoyl chloride until starting
material was consumed (TLC monitoring). Then, the solution was
quenched with slush and extracted with ethyl acetate (4 × 15 mL).
The combined organic layers were washed with brine (3 × 15 mL) and
several times with H2O, dried over Na2SO4, filtered-off, and
concentrated in vacuo to give a white residue that was triturated
from methylene chloride to afford the titled compound as a white
solid. Sulfamoyl chloride was prepared by modification of the method
reported in literature.31 Chlorosulfonylisocyanate (1.0 equiv) was
dissolved into a minimal amount of dry methylene chloride at −10 °C,
and then, formic acid was added dropwise. The white precipitate
formed was treated under vacuo to eliminate all of the solvents and
used as it is. Yield: 56%. 1H NMR (400 MHz, DMSO-d6): δ 10,67 (s,
1H, exchange with D2O), 10,22 (s, 1H, exchange with D2O), 7.79 (d,
2H, J = 8.8 Hz, Ar−H), 7.72 (d, 2H, J = 8.8 Hz, Ar−H), 7.36 (s, 2H,
exchange with D2O, SO2NH2), 7.21 (d, 2H, J = 8.8 Hz, Ar−H), 7.18
(d, 2H, J = 8.8 Hz, Ar−H). 13C NMR (100 MHz, DMSO-d6): 167.8,
144.8, 143.2, 132.3, 131.8, 129.2, 126.4, 119.0, 117.6.
Synthesis of 4-(4-(Sulfamoylamino)phenylsulfonamido)-N-
(4-sulfamoyl-phenethyl)benzamide (11). 4-(4-(Sulfamoylamino)-
phenylsulfonamido)benzoic acid 10 (0.15 g, 1.0 equiv) and 4-(2-
aminoethyl)benzenesulfonamide (0.078 g, 1.0 equiv) were dissolved in
dry acetonitrile (3.0 mL), and the reaction was treated with
dicyclohexylcarbodiimide (0.088 g, 1.1 equiv) and 4-dimethylamino-
pyridine (0.005 g, 0.1 equiv) and was stirred at r.t. until starting
materials were consumed (TLC monitoring). Then, the mixture was
quenched with H2O (20 mL) and was extracted with ethyl acetate (4
× 15 mL). The combined organic layers were dried over Na2SO4,
Crystallization, X-ray Data Collection, and Structure Reso-
lution. The hCA II−1 complex was obtained by adding a 5 M excess
of inhibitor to a 10 mg/mL protein solution in 100 mM Tris-HCl
buffer, pH 8.5. Crystals of the complex were grown at 20 °C by the
vapor diffusion hanging drop method. Drops were prepared by mixing
equal volumes of complex (10 mg/mL in 0.1 M TRIS-HCl pH 8.5)
and precipitant solution, which contained 2.5 M (NH4)2SO4, 0.3 M
NaCl, 100 mM Tris-HCl (pH 8.2), and 5 mM 4-(hydroxymercur-
ybenzoic) acid. Crystals grew within a few days and were isomorphous
to those of the native enzyme.27 X-ray diffraction data were collected
to 1.50 Å resolution at the Synchrotron source Elettra in Trieste, using
a Mar CCD detector. Prior to cryogenic freezing, the crystals were
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Article
transferred to the precipitant solution with the addition of 15% (v/v)
glycerol. Diffracted data were processed using the HKL crystallo-
graphic data reduction package.32 Diffraction data were indexed in the
P21 space group with one molecule in the asymmetric unit. Unit cell
parameters and data reduction statistics are summarized in Table 1.
The enzyme−inhibitor complex structure was analyzed by difference
Fourier techniques, using the atomic coordinates of the native hCA II
(PDB entry 1CA2)27 as a starting model. The refinement was carried
out with the program CNS,33 whereas the model building and map
inspection were performed using the O program.34 Inspection of the
electron density maps, at various stages of crystallographic refinement,
clearly showed the binding of three inhibitor molecules: one
positioned on the protein surface far away from the active site and
the other two located in the active site channel (Figures 1−4). After
initial refinement limited to the enzyme, inhibitor molecules were
gradually built into the model for further refinement. Restraints on
inhibitor bond angles and distances were taken from similar structures
in the Cambridge Structural Database, and standard restraints were
used on protein bond angles and distances throughout refinement.
The ordered water molecules were added automatically and checked
individually. Each peak contoured at 3σ in the |Fo| − |Fc| maps was
identified as a water molecule, provided that hydrogen bonds would be
allowed between this site and the model. The correctness of
stereochemistry was finally checked using PROCHECK.35 Final
refinement statistics are reported in Table 1. Coordinates and
structure factors have been deposited with the Protein Data Bank
(accession code 3V5G). All figures were drawn using Pymol.36
Avidity on Linker Length for a Bivalent Ligand-Bivalent Receptor
Model System. J. Am. Chem. Soc. 2012, 134, 333−345.
(4) Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Inhibition of cytosolic
isozymes I and II and transmembrane, tumor-associated isozyme IX
with sulfamates including EMATE also acting as steroid sulfatase
inhibitors. J. Med. Chem. 2003, 46, 2197−2204.
(5) Abbate, F.; Winum, J. Y.; Potter, B. V.; Casini, A.; Montero, J. L.;
Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors: X-ray
crystallographic structure of the adduct of human isozyme II with
EMATE, a dual inhibitor of carbonic anhydrases and steroid sulfatase.
Bioorg. Med. Chem. Lett. 2004, 14, 231−234.
(6) 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.
(7) 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.
(8) Supuran, C. T. Diuretics: From classical carbonic anhydrase
inhibitors to novel applications of the sulfonamides. Curr. Pharm. Des.
2008, 14, 641−648.
(9) Mincione, F.; Scozzafava, A.; Supuran, C. T. In Drug Design of
Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications;
Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009; pp 139−
154.
̀
(10) Thiry, A.; Dogne, J. M.; Masereel, B.; Supuran, C. T. Carbonic
Anhydrase Inhibitors as Anticonvulsant Agents. Curr. Top. Med. Chem.
2007, 7, 855−864.
ASSOCIATED CONTENT
* Supporting Information
■
(11) De Simone, G.; Di Fiore, A.; Supuran, C. T. Are carbonic
anhydrase inhibitors suitable for obtaining antiobesity drugs? Curr.
Pharm. Des. 2008, 14, 655−660.
S
Detailed characterization of compounds 2−7 and 12. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(12) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C.
T.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Imaging of CA IX with
fluorescent labelled sulfonamides distinguishes hypoxic and (re)-
oxygenated cells in a xenograft tumour model. Radiother. Oncol. 2009,
92, 423−428.
AUTHOR INFORMATION
Corresponding Author
■
(13) Ahlskog, J. K.; Dumelin, C. E.; Trussel, S.; Marlind, J.; Neri, D.
̈
*Tel: +33 467-14-72-34. Fax: +33 467-14-43-44. E-mail:: jean-
yves.winum@univ-montp2.fr (J.-Y.W.). Tel: +39-055-4573005.
Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it
(C.T.S.).
In vivo targeting of tumor-associated carbonic anhydrases using
acetazolamide derivatives. Bioorg. Med. Chem. Lett. 2009, 19, 4851−
4856.
(14) Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.;
Notes
Kalisch, M.; Buhlmann, P.; Supuran, C. T.; Neri, D. Selection of
̈
The authors declare no competing financial interest.
Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded
Compounds. ACS Chem. Biol. 2011, 6, 336−344.
ACKNOWLEDGMENTS
(15) Zatovicova, M.; Jelenska, L.; Hulikova, A.; Csaderova, L.; Ditte,
Z.; Ditte, P.; Goliasova, T.; Pastorek, J.; Pastorekova, S. Carbonic
anhydrase IX as an anticancer therapy target: Preclinical evaluation of
internalizing monoclonal antibody directed to catalytic domain. Curr.
Pharm. Des. 2010, 16, 3255−3263.
■
The financial support by the CNRS/CNR (CoopIntEER
program, Grant No. 131999 to G.D.S. and J.-Y.W.) and from
the EU FP7 project Metoxia (to C.T.S.) are gratefully
acknowledged.
(16) Neri, D.; Supuran, C. T. Interfering with pH regulation in
tumours as a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10,
767−777.
(17) Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, C. T.; De
Simone, G. In Drug Design of Zinc-Enzyme Inhibitors: Functional,
Structural, and Disease Applications; Supuran, C. T., Winum, J. Y., Eds.;
Wiley: Hoboken, 2009; pp 73−138.
(18) Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.; Stubbs, M.
T.; Klebe, G. Nonaromatic sulfonamide group as an ideal anchor for
potent human carbonic anhydrase inhibitors: role of hydrogen-
bonding networks in ligand binding and drug design. J. Med. Chem.
2002, 45, 3583−3587.
(19) De Simone, G.; Supuran, C. T. Antiobesity carbonic anhydrase
inhibitors. Curr. Top. Med. Chem. 2007, 7, 879−884.
ABBREVIATIONS USED
■
CA, carbonic anhydrase; CAI, CA inhibitor; CSI, chlorosulfonyl
isocyanate; hCA, human CA; PABA, p-amino-benzoic acid;
ZBG, zinc-binding group
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