Carbonic anhydrase inhibition with benzenesulfonamides and tetrafluorobenzenesulfonamides obtained via click chemistry

Article abstract of DOI:10.1021/ml500196t

A series of novel benzene- and 2,3,5,6-tetrafluorobenzenesulfonamide was synthesized by using a click chemistry approach starting from azido-substituted sulfonamides and alkynes, incorporating aryl, alkyl, cycloalkyl, and amino-/hydroxy-/halogenoalkyl moi

Full text of DOI:10.1021/ml500196t

Letter
pubs.acs.org/acsmedchemlett
Carbonic Anhydrase Inhibition with Benzenesulfonamides and
Tetrafluorobenzenesulfonamides Obtained via Click Chemistry
Nicolino Pala,† Laura Micheletto,† Mario Sechi,*,† Mayank Aggarwal,‡ Fabrizio Carta,§ Robert McKenna,‡
and Claudiu T. Supuran*,§,∥
†Dipartimento di Chimica e Farmacia, Universita
̀
di Sassari, Via Vienna 2, 07100 Sassari, Italy
‡Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, Florida
32610, United States
§Laboratorio di Chimica Bioinorganica, Universita
(Firenze), Italy
̀
degli Studi di Firenze, Rm 188, Via della Lastruccia 3, 50019 Sesto Fiorentino
∥Neurofarba Dept., Universita
̀
degli Studi di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy
S
* Supporting Information
ABSTRACT: A series of novel benzene- and 2,3,5,6-tetrafluor-
obenzenesulfonamide was synthesized by using a click chemistry
approach starting from azido-substituted sulfonamides and
alkynes, incorporating aryl, alkyl, cycloalkyl, and amino-/
hydroxy-/halogenoalkyl moieties. The new compounds were
medium potency inhibitors of the cytosolic carbonic anhydrase
(CA, EC 4.2.1.1) isoforms I and II and low nanomolar/
subnanomolar inhibitors of the tumor-associated hCA IX and
XII isoforms. The X-ray crystal structure of two such sulfonamides
in adduct with hCA II allowed us to understand the factors
governing inhibitory power.
KEYWORDS: Carbonic anhydrase, click chemistry, human isoform I, human isoform II, human isoform IX, human isoform XII,
benzenesulfonamide
lick chemistry has extensively been used1−4 to obtain
Cinhibitors of the metallo-enzyme carbonic anhydrase (CA,
Ec 4.2.1.1) belonging to the sulfonamide class.5,6 Recently,
thiol-ene click chemistry has been successfully employed to
obtain CA inhibitors (CAIs) of the sulfonamide type, which
again had excellent inhibitory activity against the tumor-
associated isoforms CA IX and XII.7 Among click techniques,
the copper-catalyzed azide−alkyne cycloaddictions (CuAAC)
have acquired a prominent role due to their modularity, the
short reaction times, and increased yields. By exploiting the
high reactivity of aromatic/heterocyclic sulfonamides incorpo-
rating azide/alkyne moieties that were reacted with alkynes/
Figure 1. Stick representation showing (A) compound 5c (green) and
(B) compound 5h (pink) bound in the active site of hCA II. Residues
azides, a large number of compounds possessing a variety of
chemotypes, difficultly available by other procedures, were
are as labeled. The |2Fo − Fc| electron density is contoured at 1.2σ.
synthesized (Figure 1). The obtained compounds were assayed
Active site Zn is shown as a blue sphere. Figure was made using
PyMOL.
as inhibitors of many mammalian CA isoforms of the 16
presently known.8 For example, sulfonamides incorporating
glycosyl moieties (both protected and deprotected at the OH
groups of the sugar) of type C, E, and F,1−4 as well as a
heterocyclic or aromatic groups (Chart 1),9 have been
obtained, many of which showed excellent inhibitory activity
against physiologically/pathologically relevant isoforms such as
CA I, II, IX, and XII.
Received: May 16, 2014
Accepted: June 7, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
(ZBG)] with alkynes 3a−3j (Scheme 1) in the presence of
nanosized metallic copper as catalyst (Scheme 1). Further
Chart 1. Sulfonamides Obtained via Click Chemistry
a
Scheme 1
a
Reagents and conditions: (i) Cu(0) activated nanopowder, TEA,
H2O/tert-butanol, r.t., 18 h for 4, 40 h for 5; (ii) HCl 4 M in dioxane,
room temp, 1.5 h.
derivatives 4k and 5k were obtained by N-Boc deprotection in
acidic conditions from 4j and 5j, respectively.
The azidosulfonamide 1 was prepared starting from
benzenefulfonamide 6 by routine work. The preparation of
the perfluorinated key intermediate 2 started from penta-
fluorobenzenesulfonyl chloride 8, which was converted to the
corresponding sulfonamide 7 by reaction with concentrated
aqueous ammonia. Since the 4-fluoro atom is the most reactive
one for nucleophilic substitution reactions, it has been replaced
by the azido moiety, as depicted in Scheme 2 (see materials and
In addition to generating potent CAIs per se, such as
compounds A−F mentioned above, the click chemistry has also
been employed for introducing linkers between the sulfona-
mide fragment of the molecule and toxins such as duocarmycin,
as in a recent example of hybrid drug possessing CA inhibitory/
toxin fragments for selective tumor targeting.10 Such CAIs were
highly effective in tumor models leading to a synergistic activity
compared to the parent sulfonamide or duocarmycin single
agents, and produced a sustained and potent inhibition of the
tumor growth (in renal cell carcinoma models in mice).
Thus, click chemistry is a powerful tool for generating both
chemical diversity as well as novel applications for targeting
tumors, based on inhibitors of various CA isoforms with
medicinal chemistry applications. However, fluorine has several
properties that make it extremely attractive in drug discovery:
(a) the small atomic size and the length of the C−F bond make
fluorine an unusual substitute of the hydrogen, without
affecting significantly on the molecular geometry; (b) the
high electronegativity induces substantial changes of the
physicochemical properties of the molecules including change
in the lipophilicity, decrease of the pKa, and ability to act as H-
bond acceptor. Indeed, fluorine atoms are able to alter, often
drastically, the binding mode, the affinity, and the selectivity of
the molecule for the respective target. In particular,
perfluorination of benzenes dramatically increases the acidity
of substituents.
In this letter, we used the click-tailing approach for the
synthesis of two homologous series of 4-(R-1H-1,2,3-triazol-1-
yl)-benzenesulfonamides 4 and 2,3,5,6-tetrafluoro-4-(5-R-1H-
1,2,3-triazol-1-il)benzenesulfonamide 5 incorporating a large
variety of different moieties. The new compounds were
investigated for the inhibition of the physiologically dominant,
cytosolic isoforms CA I and II, as well as the transmembrane,
tumor-associated ones CA IX and XII. Although both
benzenesulfonamides5,6 and perfluoro benzenesulfonamides11
were already investigated as CAIs, and their binding to the
enzyme has been elucidated using several experimental
approaches (for example, many X-ray data for adducts of
such compounds with various isoforms are available,12−17), this
is the first time that two homologous series of compounds
acting as CAIs have been compared. The synthesis of the title
compounds 4a−4j and 5a−5j was achieved by reacting the
azides 1 and 2 [incorporating the sulfamoyl zinc-binding group
a
Scheme 2
a
Reagents and conditions: (i) NaN3, tert-butanol, tert-butilnitrite, r.t.,
56 h; (ii) NaN3, acetone, H2O, reflux, 7 h; (iii) 30% aqueous NH4OH,
r.t., 21 h.
methods in Supporting Informations for details). The R moiety
present in alkynes 3, and also in sulfonamides 4 and 5, was
chosen to deeply explore the chemical space. Indeed, the nature
of the R moiety present in the new compounds has been
planned considering various chemical functionalities that
include aromatic, aliphatic, cycloalkyl, and halogeno-, hydroxy-
or aminoalkyl moieties (Table 1), in order to achieve a major
chemical diversity. It is known from earlier work11−17 that the
nature of the tails present in the sulfonamide CAIs strongly
influences their activity since these groups bind in various parts
of the active site, making crucial contacts with amino acid
residues and water molecules from the cavity, thus influencing
the inhibition profile of the compounds in terms of selectivity
against various isoforms (see the crystallographic section in
Supporting Informations).
Inhibition data with the set of compounds 4a−4k and 5a−5k
reported here, against isoforms hCA I, II, IX, and XII, is shown
in Table 1. All these isoforms are either drug targets or off-
targets for various pharmacologic applications of the CAIs.5,6,8
The following structure−activity relationship (SAR) can be
observed:
(i) Against all four isoforms investigated here, the
tetrafluoro-substituted sulfonamides 5 were more
effective CAIs compared to the corresponding benzene-
sulfonamide 4 possessing the same R group at the
triazole ring. This is probably due to the fact that the
fluorine-substituted sulfonamides are more acidic than
the corresponding benzenesulfonamides, which favor
B
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ACS Medicinal Chemistry Letters
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The crystal structures of human CA II in complex with
sulfonamides 5c and 5h have been determined (Figure 2) to
1.5 Å resolution.
Table 1. Inhibition Data against Isoforms hCA I, II
(Cytosolic), IX, and XII (Transmembrane, Tumor-
Associated Enzymes) of Sulfonamides 4a−4k and 5a−5k and
Acetazolamide AAZ (as Standard) by a Stopped-Flow CO2
Hydrase Assay
a
KI (nM)
compd
X
R
hCA I hCA II hCA IX hCA XII
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
H
F
p-Me−C6H4
C6H5
368
176
43.9
50.4
755
142
74.8
66.1
329
235
32.7
168
165
30.1
41.3
648
98.1
56.8
46.9
450
123
26.9
142
12
22.4
25.9
3.0
3.6
6.0
1.4
12.4
2.5
2.3
2.0
62.6
3.0
1.9
3.2
1.9
5.7
0.8
10.8
0.8
0.9
1.1
58.9
2.6
3.6
2.7
5.7
392
cC6H11
357
p-C5H11−C6H4
BrCH2CH2
Cl(CH2)3
Cl(CH2)4
MeOOC
3200
1250
1185
1350
1500
873
5.0
Figure 2. (A) Surface representation of hCA II in complex with 5c
(green) and 5h (pink) extending out of the active site. Zn is shown as
a blue sphere. (B) Zoomed active site details. Figure was made using
PyMOL.
6.9
6.2
4g
4h
4i
5.4
127
8.1
HO−CH2
BocNH−CH2
H2N−CH2
p-Me-C6H4
C6H5
4j
413
10.5
12.1
21.3
24.8
1.5
The structures were solved using protocols as described by us
previously (see Table S1 and experimental details in Supporting
Information). The hydrophobic nonplanar compounds were
found buried deep into the active site, displacing the catalytic
zinc-bound solvent, such that the nitrogen of the sulfonamide
group binds directly to the zinc atom of CA II (distance ≈ 2.0
Å). Hence, the overall Zn coordination can be described as a
distorted tetrahedron. The O atom of the sulfonamide group
lied within hydrogen bonding distance (2.9 Å) from the
backbone N atom of Thr199. The tetrafluorophenyl moiety of
the inhibitors was stabilized by the surrounding hydrophobic
residues (V121, L141, and L198) and also exhibited van der
Waals interactions with the side chains of N62, H64, Q92, H94,
F131, and P202. These interactions are consistent with those
seen with the classical, clinically used sulfonamide CAIs.11−17
However, as the compounds extend out of the active site
(Figure 2A), their tail groups became less ordered, with weaker
density seen for the tails in the difference map (Fo − Fc) for
both compounds. Hence, different orientations were modeled,
and the one that best satisfied the observed data was selected as
the final refined structure. The nonpolar, puckered cyclohexyl
ring in 5c was found in the hydrophobic pocket lined by
residues F131, V135, P202, and L204 (Figure 2A). However,
the tail of 5h was observed to be orientated toward the bulk
solvent, not being involved in any hydrophobic or polar
interactions with the surface of the protein. Indeed, this
compound (5h) was 10.9 times less effective as a hCA II
inhibitor compared to the cyclohexylmethyl substituted
congener 5c (see Table 1). Compounds 5c and 5h bury a
total surface area of 394.3 Å2 (75.0% of its total area) and 328.5
Å2 (67.1% of its total area) with the protein interface and have
average B-factors of 18.3 and 19.7 Å2, respectively. Both
compounds were refined with occupancy of ∼0.70. Some
disordered density that could not be assigned was observed
around the inhibitor in both the crystal structures. Previous
studies have shown a molecule of glycerol bound close to the
sulfonamide inhibitors in the active site of CA II.12,13 In order
to avoid the binding of any unwanted ligand in these crystal
structures, 20% sucrose instead of the normally used glycerol
was used as cryoprotectant prior to freezing the crystals.
4k
5a
5b
5c
5d
5e
5f
765
42.1
41.5
42.5
1450
416
F
F
cC6H11
F
p-C5H11−C6H4
BrCH2CH2
Cl(CH2)3
Cl(CH2)4
MeOOC
38.9
5.3
F
F
652
4.7
5g
5h
5i
F
835
4.5
F
50.1
258
115
12.8
13.6
10.4
25
F
HO−CH2
BocNH−CH2
H2N−CH2
5j
F
44.9
330
5k
AAZ
F
250
a
Errors in the range of 10% of the reported value (from 3 different
assays).
deprotonation of the sulfamoyl group (i.e., ZBG) in
coordinating the metal ion;
(ii) The cytsolic isoforms hCA I and II were moderately
inhibited by most of these sulfonamides, with inhibition
constants ranging between 41.5 and 1500 nM against
hCA I and ranging between 30.1 and 755 nM against
hCA II.
(iii) The tumor associated isoforms were inhibited in the low
nanomolar−subnanomolar range by sulfonamides 4 and
5, with inhibition constants ranging between 1.5 and 38.9
nM against hCA IX and between 0.8 and 12.4 nM against
hCA XII. Just two compounds (4h and 5h), possessing
the electron withdrawing carboxymethyl group as R
moiety, were less active against the two tumor-associated
isoforms.
(iv) R groups leading to effective hCA IX/XII inhibitors were
all those present in derivatives 4 and 5, except COOMe,
whereas the most effective hCA II inhibitors incorpo-
rated cyclohexylmethyl and phenyl moieties. The best
hCA I inhibitors contained tosyl, phenyl, cyclohex-
ylmethyl, and COOMe moieties (Table 1).
C
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ACS Medicinal Chemistry Letters
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(6) Supuran, C. T. Structure-based drug discovery of carbonic
anhydrase inhibitors. J. Enzyme Inhib. Med. Chem. 2012, 27, 759−772.
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Tars, K.; Supuran, C. T. 5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-
sulfonamides strongly inhibit human carbonic anhydrases I, II, IX and
XII: solution and X-ray crystallographic studies. Bioorg. Med. Chem.
2013, 21, 5130−5138.
In conclusion, a series of novel benzene- and 2,3,5,6-
tetrafluorobenzenesulfonamides, synthesized by using click
chemistry approaches, which incorporated aryl, alkyl, cycloalkyl,
and amino-/hydroxy-/halogenoalkyl moieties, were synthesized
and evaluated as CA inhibitors. The new compounds were
medium potency inhibitors of the cytosolic CA isoforms I and
II and low nanomolar/subnanomolar inhibitors of the tumor-
associated hCA IX and XII isoforms. The X-ray crystal structure
of two such sulfonamides in adduct with hCA II allowed us to
understand the factors governing inhibitory power.
(9) Saada, M. C.; Ombouma, J.; Montero, J. L.; Supuran, C. T.;
Winum, J. Y. Thiol-ene click chemistry for the synthesis of highly
effective glycosyl sulfonamide carbonic anhydrase inhibitors. Chem.
Commun. 2013, 49, 5699−5701.
(10) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.; Supuran,
C. T.; Neri, D. A small-molecule drug conjugate for the treatment of
carbonic anhydrase IX expressing tumors. Angew. Chem., Int. Ed. 2014,
53, 4231−4235.
ASSOCIATED CONTENT
* Supporting Information
Full characterization of compounds 4a−4k and 5a−5k and
crystallographic parameters for the two hCA II adducts (with
5c and 5h) are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
(11) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Perfluoroalkyl/aryl-substituted derivatives of aromatic/heterocyclic
sulfonamides as topical intraocular pressure lowering agents with
prolonged duration of action. J. Med. Chem. 2000, 43, 4542−4551.
(12) Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B. S.;
Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T.
Sulfonamides incorporating 1,3,5-triazine moieties selectively and
potently inhibit carbonic anhydrase transmembrane isoforms IX, XII
and XIV over cytosolic isoforms I and II: solution and X-ray
crystallographic studies. Bioorg. Med. Chem. 2011, 19, 3105−3119.
(13) Biswas, S.; Aggarwal, M.; Guzel, O.; Scozzafava, A.; McKenna,
R.; Supuran, C. T. Conformational variability of different sulfonamide
inhibitors with thienyl-acetamido moieties attributes to differential
binding in the active site of cytosolic human carbonic anhydrase
isoforms. Bioorg. Med. Chem. 2011, 19, 3732−3738.
(14) Avvaru, B. S.; Wagner, J. M.; Maresca, A.; Scozzafava, A.;
Robbins, A. H.; Supuran, C. T.; McKenna, R. Carbonic anhydrase
inhibitors. The X-ray crystal structure of human isoform II in adduct
with an adamantyl analogue of acetazolamide resides in a new
hydrophobic binding pocket. Bioorg. Med. Chem. Lett. 2010, 20, 4376−
4381.
(15) Di Fiore, A.; Maresca, A.; Alterio, V.; Supuran, C. T.; De
Simone, G. Carbonic anhydrase inhibitors: X-ray crystallographic
studies for the binding of N-substituted benzenesulfonamides to
human isoform II. Chem. Commun. 2011, 47, 11636−11638.
(16) Pinard, M. A.; Boone, C. D.; Rife, B. D.; Supuran, C. T.;
McKenna, R. Structural study of interaction between brinzolamide and
dorzolamide inhibition of human carbonic anhydrases. Bioorg. Med.
Chem. 2013, 21, 7210−7215.
Accession Codes
PDB accession code for adducts of CA II with 5c and 5h are
4DZ7 and 4DZ9, respectively.
AUTHOR INFORMATION
Corresponding Authors
*(M.S.) E-mail: mario.sechi@uss.it.
*(C.T.S.) E-mail: claudiu.supuran@unifit.it.
■
Author Contributions
The manuscript was written through contributions of all
authors.
Funding
This work was financed by two EU grants (Metoxia and
Dynano (to C.T.S.)), by the Fondazione Banco di Sardegna (to
M.S.), and by the NIH grant GM25154 (to R.M.).
Notes
The authors declare the following competing financial
interest(s): C.T.S. is a coauthor on many patents claiming
carbonic anhydrase inhibitors. The compounds from this paper
are not patented.
ABBREVIATIONS
■
CA, carbonic anhydrase; AAZ, acetazolamide; NMR, nuclear
magnetic resonance; TLC, thin layer chromatography; SAR,
structure−activity relationship; ZBG, zinc-binding group
(17) Marini, A. M.; Maresca, A.; Aggarwal, M.; Nencetti, S.;
Orlandini, E.; Da Settimo, F.; Salerno, S.; Simorini, F.; La Motta, C.;
Taliani, S.; Nuti, E.; McKenna, R.; Rossello, A.; Supuran, C. T.
Tricyclic sulfonamides incorporating benzothiopyrano[4,3-c]pyrazole
and pyridothiopyrano[4,3-c]pyrazole effectively inhibit α- and β-
carbonic anhydrase: X-ray crystallography and solution investigations
on 15 isoforms. J. Med. Chem. 2012, 55, 9619−9629.
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