New superacid synthesized (fluorinated) tertiary benzenesulfonamides acting as selective hCA IX inhibitors: Toward a new mode of carbonic anhydrase inhibition by sulfonamides

Article abstract of DOI:10.1039/c3cc40858b

Tertiary substituted (fluorinated) benzenesulfonamides were synthesized in superacid HF/SbF₅ and tested as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). Strong selectivity toward tumor-associated hCA IX, without inhibiting the off

Full text of DOI:10.1039/c3cc40858b

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Claudiu. T. Supuran, Sébastien Thibaudeau et al.
New superacid synthesized (fluorinated) tertiary benzenesulfonamides
acting as selective hCA IX inhibitors: toward a new mode of
carbonic anhydrase inhibition by sulfonamides
1359-7345(2013)49:54;1-Y

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New superacid synthesized (fluorinated) tertiary
benzenesulfonamides acting as selective hCA IX
inhibitors: toward a new mode of carbonic anhydrase
inhibition by sulfonamides†
Cite this: Chem. Commun., 2013,
49, 6015
Received 31st January 2013,
Accepted 5th March 2013
a
a
b
b
DOI: 10.1039/c3cc40858b
´
`
Benoˆıt Metayer, Agnes Mingot, Daniella Vullo, Claudiu. T. Supuran* and
´
a
Sebastien Thibaudeau*
www.rsc.org/chemcomm
Tertiary substituted (fluorinated) benzenesulfonamides were synthe-
sized in superacid HF/SbF5 and tested as inhibitors of human carbonic
anhydrases (hCAs, EC 4.2.1.1). Strong selectivity toward tumor-
associated hCA IX, without inhibiting the offtarget hCA II, was
observed, pointing out to a new mechanism of action compared to
classical sulfonamides.
Scheme 1 4-Aminobenzenesulfonamide inhibition data.
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metallo-
enzymes that catalyse the reversible hydratation of carbon of new chemotypes, with lower affinity for the CA zinc binding site,
dioxide to bicarbonate and protons.1 Human CAs (hCAs) exist in order to be more selective toward the different isoforms.4 Such a
in sixteen isoforms, belonging to the so-called a-class and differ strategy led to the discovery of new chemotypes that selectively
widely in their cellular localization (cytosol, mitochondria, cell inhibit some targeted hCA isoforms. Recently, several classes of
membrane).2 Sulfonamides and their bioisosteres (sulfamates, such agents have been detected. For example, coumarins,
sulfamides, etc.) constitute the most investigated inhibitors thiocoumarins10 and more recently sulfocoumarins,11 located
of these enzymes,3 with useful therapeutic applications.4 at the entrance of the enzyme active site, were found to act as
Crystallographic studies of many sulfonamide adducts with selective inhibitors of the hCA IX isozyme. In the sulfonamide
several CA isozymes largely show the anchoring properties series, despite the recently discovered zinc-binding ureido-
of these types of inhibitors.3,5 In their deprotonated forms, substituted benzenesulfonamides,12 few other benzenesulfon-
sulfonamides bind the Zn2+ ion of the active site, disrupting the amides were reported to be hCA IX selective inhibitors. In due
catalytic process.6 Most of the side-effects of the sulfonamide- course of our work on the discovery of new sulfonamide type
based CA inhibitors7 are due to the inhibition of the cytosolic inhibitors,13 a series of 4-aminobenzenesulfonamides were
catalytically highly active isoform CA II, which is abundant in recently reported to be selective nanomolar inhibitors of
many tissues/organs and involved in numerous physiological tumor-associated isoforms IX and XII (Scheme 1).14
functions (pH homeostasis, secretion of electrolytes, transport
Whereas the allylic substituted benzenesulfonamide A showed
of anions, etc.).4,8 Among CAs, being hypoxia overexpressed, the only a micromolar level of inhibition toward hCA IX, its fluorinated
plasma membrane associated isoforms hCA IX and XII are now analogue B inhibited hCA IX at the nanomolar level, but also the
well-established anticancer drug targets.9As a consequence, in offtarget hCA II. Interestingly, a chlorinated analogue showed a
the quest for new anti-cancer agents, the selective inhibition of similar level of inhibition for hCA IX with selectivity toward hCA II.
hCA IX and XII over the offtarget hCA II is essential. Therefore, Even more surprisingly, the secondary sulfonamide C and its tertiary
much effort was dedicated in the last few years to the development substituted analogue D showed similar inhibition properties.14 This
similarity between secondary sulfonamides and tertiary ones (which
a
cannot act on a deprotonated form) and the high level of inhibition
and selectivity for hCA IX could not come from the inhibition of the
enzyme through the ‘‘classical’’ zinc binding mode.
Here we investigate in detail this hypothesis, and report
the evaluation of superacid synthesized (fluorinated) tertiary
substituted benzenesulfonamide inhibitors. For the first time
in the benzenesulfonamide family, hCA IX nanomolar selective
´
Superacid Group in ‘‘Organic Synthesis’’ Team – Universite de Poitiers,
CNRS UMR 7285 IC2MP, 4, avenue Michel Brunet, F-86022 Poitiers Cedex,
France. E-mail: sebastien.thibaudeau@univ-poitiers.fr; Fax: +33-549453501;
Tel: +33-549454588
b
`
Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188,
Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
† Electronic supplementary information (ESI) available: Experimental procedures
and spectral data. See DOI: 10.1039/c3cc40858b
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 6015--6017 6015

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these molecules as hCA IX and XII inhibitors. For example, for
the N-allylic derivatives, whereas the chlorinated analogue 3d
inhibits almost equally hCA I, IX and XII, its brominated
analogue 3c is less effective as an inhibitor of hCA XII
(Table 1, entries 7 and 8). Interestingly the fluorinated analogue
3e possesses a higher affinity for the tumor associated isozymes
over the cytosolic isozyme CA I (Table 1, entry 9). Analogously,
for the b-fluorinated derivatives, the aromatic substitution
with a fluorine atom strongly alters hCA XII inhibition
(Table 1, entry 17). This effect of the halogen on the inhibitor
activity against hCAs is difficult to be explained at this stage,
but can be compared to similar reported effects of halogen on
sulfanilamide derivatives.17 Fluorination of the allylic side
chain also strongly influences the inhibitory profile of the
tested inhibitors. Fluorination of the aliphatic chain increases
hCA IX inhibition selectivity for derivatives 4d, 4f and 4h–i,
whereas a similar modification of the brominated analogue 3c
improved hCA XII inhibition (Table 1, entry 15). The strongest
effect of side chain fluorination can be noticed for substrates 3e
and 4e. Derivative 3e can be considered as an excellent selective
hCA IX inhibitor, whereas the inhibitory profile of 4e places it
in the not yet reported class of selective benzenesulfonamide
hCA I inhibitors. The role of fluorine in medicinal chemistry is
well recognized.18 For the nitrogen containing compounds,
the modification of the basicity of the nitrogen containing
proximal functions, by using fluorine’s strong electron with-
drawing effect,19 has been largely used in medicinal chemistry
SAR studies.20 It is also accepted that through a fluorine gauche
effect and through electrostatic interactions, the fluorine atoms
can strongly modify the preferred conformation of nitrogen
containing biomolecules.21 However, the reported fluorine effect
could not be attributed to a modification of the interaction of the
molecule with the Zn2+ ion, as tertiary benzenesulfonamides
cannot interact with the metal ion due to steric impairment. In
addition, a strong influence of the modifications of the aromatic
ring on the inhibition properties has been noticed. This strong
effect of aromatic substitution is comparable to what was recently
reported on non-zinc-binding inhibitors such as (thio)coumarin
derivatives.10,22 As a consequence, the reported behavior of tertiary
substituted benzenesulfonamides reported here probably correlates
with a new mode of inhibition for these ‘‘classical’’ inhibitors.
A series of new tertiary (fluorinated) benzenesulfonamide inhi-
bitors were synthesized and explored as inhibitors of several
physiologically/pathologically relevant CA isozymes with the aim
of discovering inhibitors that could selectively target the tumor
associated isozymes CA IX and XII. All the reported compounds
showed good nanomolar inhibition of hCA IX and XII. In addition,
all these compounds are ineffective inhibitors of the widespread
hCA II isoform (offtarget), making them excellent candidates in the
quest for hCA IX inhibitors with no side-effects. A strong influence
of both aliphatic and aromatic modifications on CA inhibition
selectivity has also been shown, presumably due to a new mode of
action of tertiary benzenesulfonamide inhibitors. This study led to
the discovery of a new hCA IX highly selective fluorinated inhibitor
which could be considered as a lead compound in the discovery
of benzenesulfonamide non-zinc-binding inhibitors, with an anti-
cancer pharmacological profile.
Scheme 2 Synthesis of tertiary substituted benzenesulfonamides.
CA inhibitors with no measurable affinity for the off target
hCA II have been identified.
Starting from commercially available aniline derivatives 1,
sulfonylation with sulfonylchloride derivatives allowed the
synthesis of benzenesulfonamide derivatives 2 (Scheme 2).
Then, allylic substitution allowed the synthesis of a large
variety of tertiary substituted benzenesulfonamides 3. Using a
superacid catalyzed15 hydrofluorination process,16 a series
of fluorinated analogues 4 could be obtained. The N-allylic
derivatives 3a–i and the fluorinated analogues 4b–i were then
tested as hCA inhibitors against hCA I, hCA II, hCA IX and hCA
XII and compared to the previously reported compounds A–D
(Table 1). All tertiary benzenesulfonamides were found to be
ineffective as hCA II inhibitors (or a very weak inhibitor in the
case of compounds 3b and 3g). Exceptionally, whereas they do
not inhibit the highly catalytically active hCA II, all the tested
compounds inhibited the tumor associated isoform IX at the
nanomolar level (KIs of 8.5–95.3 nM). To the best of our
knowledge, these new tertiary benzenesulfonamide inhibitors
are the first sulfonamide type inhibitors showing such a
selectivity between the widespread offtarget isoform II and
the targeted cancer-related isoform IX, making them new lead
structures in the design of potent new anticancer agents. What
also has to be noticed is the impact of the substitution pattern
of the aromatic ring with halogen atoms on the efficiency of
Table 1 hCA inhibition data of benzenesulfonamides A–D, 3 and 4
Substrate
R1 R2
KIa (nM)
Selectivityb,c
I/
hCA hCA hCA hCA I/
II/ II/
IX XII
Entry
I
II IX XII IX XII
1
2
3
4
5
6
7
8
A
B
C
D
7136 4708 3462 1277 2.1 5.6
174 75 83 39.7 2.1 4.4
5626 5176 90 96
6796 4995 73.2 79.8 92.8 85.2 68.2 62.6
1.4 3.7
0.9 1.9
62 58.6 57.5 54
d
e
e
3a NO2
3b Me
3c Br
3d Cl
3e F
3f Me
3g CF3
3h Ome Me 72.5
3i COMe Me 81.7
4b Me
4c Br
4d Cl
4e
4f Me
4g CF3
4h Ome Me 78.6
4i COMe Me 89.1
Me 9.20
/
95.3 52.0 0.1 0.18
/
/
NO2 83.9 975 73.3 1327 1.1 0.06 13.3 0.7
d
d
d
d
e
e
e
e
e
e
e
e
Me 33.2
Me 81.7
Me 238
Me 75.1
/
/
/
/
34.4 837 0.9 0.04
71.9 195.6 1.1 0.42
8.31 82.5 28 2.8
70.1 17.7 1.1 4.2
/
/
/
/
/
/
/
/
9
10
11
12
13
14
15
16
17
18
19
20
21
Me 58.7 366 86.9 83.6 0.7 0.7
4.2 4.4
d
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
/
/
/
/
/
/
/
/
/
/
49.6 96.4 1.5 0.7
33.3 60.7 2.4 1.3
4.2 75.9 0.9 0.05
8.5 71.6 0.9 0.1
9.1 100.0 8.5 0.8
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
d
d
d
d
d
d
d
d
d
NO2 3.8
Me 7.5
Me 77.3
Me 9.8
Me 73.1
Me 9.3
F
86.1 3511 0.1 0.003 /e
e
9.3 33.6 7.8 2.2
81.2 80.4 0.1 0.1
33.3 60.7 2.4 1.3
9.6 83.8 9.3 1.1
/
/
/
/
e
e
e
a
Errors in the range of Æ5% of the reported data from 3 different assays.
b
c
Selectivity ratio between hCA I (hCA II) and IX. Selectivity ratio between
d
e
hCA I (hCA II) and XII. /: Not active. /: Not determined >1000.
c
6016 Chem. Commun., 2013, 49, 6015--6017
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C. T. Supuran, J. Med. Chem., 2004, 47, 5224; F. Carta, C. T. Supuran
and A. Scozzafava, Expert Opin. Ther. Pat., 2012, 22, 79.
8 C. T. Supuran, Bioorg. Med. Chem. Lett., 2010, 20, 3467.
9 D. Neri and C. T. Supuran, Nat. Rev. Drug Discovery, 2011, 10, 767
and references cited therein.
This work was generously supported by the CNRS, the
University of Poitiers, the French National Research Agency
(VINFLUSUP project: ANR JCJC SIMI 7 no. ANR-11-JS07-003-01)
and by an FP7 EU grant (METOXIA).
10 A. Maresca, C. Temperini, H. Vu, N. B. Pham, S. A. Poulsen,
A. Scozzafava, R. J. Quinn and C. T. Supuran, J. Am. Chem. Soc.,
2009, 131, 3057; A. Maresca, C. Temperini, L. Pochet, B. Masereel,
A. Scozzafava and C. T. Supuran, J. Med. Chem., 2010, 53, 335;
F. Carta, A. Maresca, A. Scozzafava and C. T. Supuran, Bioorg. Med.
Chem., 2012, 20, 2266.
Notes and references
1 V. M. Krishnamurthy, G. K. Kaufman, A. R. Urbach, I. Gitlin,
K. L. Gudiksen, D. B. Weibel and G. M. Whitesides, Chem. Rev.,
2008, 108, 946; C. T. Supuran, in Drug Design of Zinc-Enzyme 11 K. Tars, D. Vullo, A. Kazaks, J. Leitans, A. Lends, A. Grandane,
Inhibitors: Functional, Structural, and Disease Applications,
ed. C. T. Supuran and J. Y. Winum, Wiley, Hoboken (NJ), 2009,
R. Zalubovskis, A. Scozzafava and C. T. Supuran, J. Med. Chem.,
2013, 56, 293.
pp. 15–38; J. F. Domsic, B. S. Avvaru, C. U. Kim, S. M. Gruner, 12 F. Pacchiano, F. Carta, P. C. McDonald, Y. Lou, D. Vullo,
M. Agbandje-McKenna, D. N. Silverman and R. J. McKenna, J. Biol.
Chem., 2008, 283, 30766.
2 C. T. Supuran, Curr. Pharm. Des., 2008, 14, 641; C. T. Supuran, A. Di 13 F. Liu, A. Martin-Mingot, F. Lecornue, M.-P. Jouannetaud, A. Maresca,
A. Scozzafava, S. Dehdar and C. T. Supuran, J. Med. Chem., 2011,
54, 1896.
´
Fiore and G. De Simone, Expert Opin. Emerging Drugs, 2008, 13, 383;
G. De Simone, A. DiFiore and C. T. Supuran, Curr. Pharm. Des., 2008,
14, 655; F. Mincione, A. Scozzafava and C. T. Supuran, Antiglaucoma
Carbonic Anhydrase Inhibitors as Ophthalomologic Drugs, in Drug
S. Thibaudeau and C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2012,
27, 886; C. Alp, A. Maresca, N. A. Alp, M. S. Gu¨ltekin, D. Ekinci,
A. Scozzafava and C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2013,
28, 294.
Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease 14 G. Compain, A. Martin-Mingot, A. Maresca, S. Thibaudeau and
Applications, ed. C. T. Supuran and J. Y. Winum, Wiley, Hoboken C. T. Supuran, Bioorg. Med. Chem., 2012, 21, 1555.
(NJ), 2009, pp. 139–154; J. Krungkrai and C. T. Supuran, Curr. Pharm. 15 in Superacids, ed. G. A. Olah, G. K. S. Prakash, A. Molnar and
Des., 2008, 14, 631; J. Borras, A. Scozzafava, L. Menabuoni, J. Sommer, Wiley Interscience, New York, 2nd edn, 2009.
F. Mincione, F. Briganti, G. Mincione and C. T. Supuran, Bioorg. 16 S. Thibaudeau, A. Martin-Mingot, M.-P. Jouannetaud, O. Karam and
Med. Chem., 1999, 7, 2397; A.-K. Hariu, E. Bootorabi, M. Kuushahti,
C. T. Supuran and S. Parkkila, J. Enzyme Inhib. Med. Chem., 2013,
28, 231.
3 V. Alterio, A. Di Fiore, K. D’Ambrosio, C. T. Supuran and
G. De Simone, in Drug Design of Zinc-Enzyme Inhibitors: Functional,
Structural, and Disease Applications, ed. C. T. Supuran and
F. Zunino, Chem. Commun., 2007, 3198; G. Compain, K. Jouvin,
A. Martin-Mingot, G. Evano, J. Marrot and S. Thibaudeau, Chem.
Commun, 2012, 48, 5196; F. Liu, A. Martin-Mingot, M.-P. Jouannetaud,
F. Zunino and S. Thibaudeau, Org. Lett., 2010, 12, 868; F. Liu, A. Martin-
Mingot, M.-P. Jouannetaud, C. Bachmann, G. Frapper, F. Zunino and
S. Thibaudeau, J. Org. Chem., 2011, 76, 1460.
J. Y. Winum, Wiley, Hoboken (NJ), 2009, pp. 73–138; C. Temperini, 17 M. A. Ilies, D. Vullo, J. Pastorek, A. Scozzafava, M. Ilies, M. T. Caproiu,
A. Cecchi, A. Scozzafava and C. T. Supuran, Org. Biomol. Chem., 2008, S. Pastorekova and C. T. Supuran, J. Med. Chem., 2003, 46, 2187.
6, 2499–2506; A. Di Fiore, A. Maresca, V. Alterio, C. T. Supuran and 18 K. L. Kirk, J. Fluorine Chem., 2006, 127, 1013; J.-P. Begue and D. Bonnet-
´
´
G. De Simone, Chem. Commun., 2011, 47, 11636–11638.
4 V. Alterio, A. Di Fiore, K. D’Ambrosio, C. T. Supuran and G. De Simone,
Chem. Rev., 2012, 112, 4421.
Delpon, J. Fluorine Chem., 2006, 127, 992; S. Purser, P. R. Moore,
S. Swallow and V. Gouverneur, Chem. Soc. Rev., 2008, 37, 320.
19 D. O’Hagan, Chem. Soc. Rev., 2008, 37, 308.
5 V. Alterio, M. Hilvo, A. Di Fiore, C. T. Supuran, P. Pan, S. Parkkila, 20 M. Morgenthaler, E. Schweiser, F. Hoffman-Roder, F. Benini,
A. Scaloni, J. Pastorek, S. Pastorekova, C. Pedone, A. Scozzafava,
S. M. Monti and G. De Simone, Proc. Natl. Acad. Sci. U. S. A., 2009,
106, 16233; A. Di Fiore, E. Truppo, C. T. Supuran, V. Alterio,
N. Dathan, F. Bootorabi, S. Parkkila, S. M. Monti and G. De Simone,
Bioorg. Med. Chem. Lett., 2010, 20, 5023.
6 C. T. Supuran, Nat. Rev. Drug Discovery, 2008, 7, 168–181.
7 A. Di Fiore, S. M. Monti, M. Hilvo, S. Parkkila, V. Romano,
A. Scaloni, C. Pedone, A. Scozzafava, C. T. Supuran and
G. De Simone, Proteins, 2009, 74, 164; J. Y. Winum, C. Temperini,
R. E. Martin, G. Jaeschke, B. Wagner, H. Fisher, S. Bendels,
D. Zimmerili, J. Schneider, F. Hiedrich, M. Kansy and K. Muller,
ChemMedChem, 2007, 2, 1100; W. H. Hagmann, J. Med. Chem., 2008,
51, 4359.
21 For recent examples please see: I. Yamamoto, G. P. Deniau,
N. Gavande, M. Chebib, G. A. R. Johnston and D. O’Hagan, Chem.
Commun., 2011, 47, 7956; I. Yamamoto, M. J. Jordan, N. Gavande,
M. R. Doddareddy, M. Chebib and L. Hunter, Chem. Commun., 2012,
48, 829.
K. El Cheikh, A. Innocenti, D. Vullo, S. Ciattini, J. L. Montero, 22 A. Maresca, A. Scozzafava and C. T. Supuran, Bioorg. Med. Chem.
A. Scozzafava and C. T. Supuran, J. Med. Chem., 2006, 49, 7024;
A. Innocenti, A. Casini, M. C. Alcaro, A. M. Papini, A. Scozzafava and
Lett., 2010, 20, 7255; A. Maresca and C. T. Supuran, Bioorg. Med.
Chem. Lett., 2010, 20, 4511.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 6015--6017 6017