Thiol-ene click chemistry for the synthesis of highly effective glycosyl sulfonamide carbonic anhydrase inhibitors

Article abstract of DOI:10.1039/c3cc42541j

Thiol-ene click chemistry has been applied for obtaining sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors incorporating sugar moieties. Most of these new compounds were moderate CA I inhibitors, effective CA II inhibitors, and low nanomolar/subn

Full text of DOI:10.1039/c3cc42541j

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Thiol–ene click chemistry for the synthesis of highly
effective glycosyl sulfonamide carbonic anhydrase
inhibitors†
Mohamed-Chiheb Saada,a Joanna Ombouma,a Jean-Louis Montero,a
Claudiu T. Supuran*b and Jean-Yves Winum*a
Cite this: Chem. Commun., 2013,
49, 5699
Received 8th April 2013,
Accepted 30th April 2013
DOI: 10.1039/c3cc42541j
www.rsc.org/chemcomm
Thiol–ene click chemistry has been applied for obtaining sulfon-
amide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors incorporating
sugar moieties. Most of these new compounds were moderate CA I
inhibitors, effective CA II inhibitors, and low nanomolar/subnanomolar
inhibitors of the tumor-associated isoforms CA IX and XII.
The classical carbonic anhydrase (CA, EC 4.2.1.1) inhibitors
(CAIs) are the sulfonamides and their bioisosteres (sulfamates,
sulfamides, etc.).1–4 However, most of these compounds indis-
criminately inhibit many of the 16 CA isoforms known to date
in mammals.1–3 Thus, efforts have been made to find different
classes of sulfonamides with selectivity for isoforms of interest
for medicinal chemistry, such as the tumor-associated ones CA
IX and XII, which are overexpressed in hypoxic tumors.4–6 Click
chemistry, in its classical form of reacting azides with primary
alkynes, has been much used to design novel sulfonamides
incorporating 1,2,3-triazole moieties, many of which showed
interesting inhibition profiles against isoforms such as CA VA,
VB, IX and XII.7–10 However, variants of this click reaction were
not reported so far for the preparation of sulfonamide CA
inhibitors (CAIs).
Scheme 1 Synthesis of the thiol–sulfonamide intermediate 2 and click chemistry
reactions leading to compounds 5–7.
Here we report the use of thiol–ene click chemistry11 for
designing novel sulfonamide CAIs (Scheme 1). The first key
synthon was the b-mercaptopropionamide of 4-(2-aminoethyl)- As the CAIs incorporating sugar moieties were shown to lead to
benzenesulfonamide 2, obtained by routine procedures as water soluble, isoform-selective compounds (and also to a large
shown in Scheme 1, by reaction of 4-(2-aminoethyl)benzene- chemical diversity, inherent to sugar chemistry)12,13 we used
sulfonamide with the disulfide of b-mercaptopropionic acid sugar scaffolds to which double bonds were appended,
in the presence of thionyl chloride, leading to 1 which was as the ene component for the click chemistry (Scheme 1).
reduced with dithiothreitol to generate the mercapto-derivative 2. Peracetylated sugars were converted to O-allyl ether glycosides 3,
which in the presence of AIBN and UV light (or heating), reacted
with thiol 2, to generate the thioethers 5a–5e. In order to obtain
a
´
Institut des Biomolecules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2,
more chemical diversity, the C-glycosides 4a–4e were obtained by
reacting peracetylated sugars with allyltrimethylsilane in the
presence of boron trifluoride as catalyst. These alkenes were then
converted to thioethers 6a–6e by reaction with 2 under free-radical
ˆ
´
Batiment de Recherche Max Mousseron, Ecole Nationale Superieure de Chimie de
Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France.
E-mail: jean-yves.winum@univ-montp2.fr; Fax: +33 (0)467144344;
Tel: +33 (0)467147234
b
`
Universita degli Studi di Firenze, Neurofarba Department, Section of
´
conditions (AIBN and UV irradiation). Zemplen deacetylation of
Pharmaceutical Sciences, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
glycosidic derivatives 5 and 6 led to the free sugar compounds
7a–7e and 8a–8e. The rationale for this drug design was rather
straightforward. We used the 4-aminoethyl-benzenesulfonamide
E-mail: claudiu.supuran@unifi.it; Fax: +39-055-4573385; Tel: +39-055-4573005
† Electronic supplementary information (ESI) available: Experimental data for
compounds 5a–8e. See DOI: 10.1039/c3cc42541j
c
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Chem. Commun., 2013, 49, 5699--5701 5699

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scaffold due to the fact that many compounds generated Thus, a group of compounds, among which are 5a, 5b, 7b, 7c,
from this derivative led to highly effective CAIs against physio- 5e, 8b, 6c, and 8c, were medium potency hCA I inhibitors with
logically relevant isoforms.13,14 The b-mercaptopropionic acid inhibition constants in the range of 79–98 nM. It may be
moiety has been attached due to the fact that the SH group is observed that these are glucose, galactose and mannose deri-
necessary for the ene–thiol click chemistry, and we showed vatives. Some of them incorporate peracetylated sugar moieties
earlier that this moiety is also not detrimental to CA inhibitory (5a, 5b, 5e, 6c) whereas others contain the free OH moieties of
properties of compounds incorporating it.15 In order to the deprotected sugars. Both O-glycosides and C-glycosides
generate chemical diversity, five different peracetylated mono- showed this activity, which makes the structure–activity rela-
or disaccharides (in pyranosyl or furanosyl form) have been tionship (SAR) rather complicated.
used in the syntheses, i.e., glucopyranose, galactopyranose,
Indeed, the remaining derivatives were much weaker as hCA I
mannopyranose, ribofuranose and cellobiose. We have investi- inhibitors with KIs in the range of 170–870 nM (thus being less
gated earlier the CA inhibitory properties of sulfanilamides efficient CAIs (except for compounds 7e and 8c) compared to the
derivatized with such sugar moieties,12c which not only showed clinically used sulfonamide AAZ, Table 1). In several cases, the
excellent inhibitory properties against physiologically relevant peracetylated compound was a more effective hCA I inhibitor
isoforms, but also possessed very good water solubility and compared to the deacetylated one (e.g., compare the pairs 5a–7a;
in vivo activity as antiglaucoma agents in an animal model of 5e–7e; 6c–8c) but most of the time, for the remaining pairs, the
this disease.12c
deprotected, free sugar derivative was the best inhibitor com-
The new compounds reported here were characterized pared to the peracetylated one. There were no substantial
extensively by spectral and physico-chemical methods which differences in activity between the O- and C-glycosides. Clearly
confirmed their structures (see ESI† for details).
the nature of the sugar incorporated into the scaffold of
Sulfonamides of types 5a–8e reported here were assayed as these sulfonamides was the main determinant of the inhibitory
inhibitors of four physiologically relevant CA isoforms, the activity, as for similar derivatives reported earlier by this and
cytosolic hCA I and II (h = human isoform), and the trans- other groups.7–10,12,13
membrane, tumor-associated hCA IX and XII (Table 1). The
(ii) The physiologically dominant cytosolic isoform hCA II
clinically employed sulfonamide acetazolamide (AAZ, 5-acetamido- was highly inhibited by several of the new sulfonamides
1,3,4-thiadiazole-2-sulfonamide) has been used as standard in reported here (5a, 5b, 7c, 5e, 7e, 6b, 8b, 6c, 8c, and 8d) which
these measurements, for comparison reasons.
The following should be noted for the CA inhibitory proper- of 12 nM). These derivatives incorporate all the five sugar
ties of the compounds 5–8 reported here: moieties considered here for obtaining CAIs, as well as both
had KIs in the range of 7.4–10.5 nM (comparable to that of AAZ,
(i) Against the abundant, cytosolic isoform hCA I (present O-glycoside and C-glycoside derivatives. The remaining deriva-
mainly in red blood cells and the gastrointestinal tract),1–3 tives were medium efficient hCA II inhibitors, with KIs in the
sulfonamides 5a–8e showed medium-weak potency as CAIs. range of 37.3–66.1 nM (Table 1). As for the SAR discussed above
for hCA I inhibition, there are no regularities regarding the
peracetylated/deacetylated compounds in their interaction with
Table 1 CA inhibition of isoforms hCA I, II, IX and XII with sulfonamides 5–8
the enzyme. In some cases the peracetylated compound was
more effective as an hCA II inhibitor compared to the deacetyl-
ated one (5a, 5b, 5e, 6c, 6e) whereas in other cases the opposite
was true. Both O-glycosides and C-glycosides were present in
the group of highly effective and medium potency hCA II
inhibitors. One can conclude, as for hCA I, that the main factor
influencing hCA II inhibitory activity is the nature of the sugar
moiety present in these sulfonamides.
(iii) The tumor-associated transmembrane isoforms hCA IX
and hCA XII were both potently inhibited by all sulfonamides
reported here, which showed inhibition constants >10 nM,
more precisely, in the range of 0.69–8.2 nM against hCA IX,
and of 0.54–16.5 nM against hCA XII, respectively (Table 1). As
these are validated antitumor/antimetastatic targets,5–7 we esti-
mate these results to be highly significant. SAR is again not very
simple for the inhibition of these isoforms. For example, the
five subnanomolar hCA IX inhibitors, 7a, 7b, 7c, 8b and 8c, all
incorporate free sugar moieties, and not the peracetylated ones.
In fact the corresponding peracetylated compounds are almost
one order of magnitude weaker CAIs compared to the depro-
tected ones. These compounds are glucose, galactose and
mannose derivatives, but one may see that the ribose and
cellobiose derivatives also show significant hCA IX inhibition
reported in this communication, by a CO2 hydrase stopped-flow assay (ESI)
KIa (nM)
Compound
hCA I
hCA II
hCA IX
hCA XII
5a
7a
5b
7b
5c
7c
5d
7d
5e
7e
6a
8a
6b
8b
6c
8c
6d
8d
6e
8e
AAZ
96
870
94
88
778
98
703
399
97
188
660
418
217
79
87
96
544
170
435
713
250
9.5
66.1
9.5
63.2
53.5
9.7
64.6
38.5
9.3
10.2
61.0
37.3
9.8
7.4
8.5
9.4
45.0
10.5
39.5
60.8
12
6.9
0.82
7.1
0.92
7.9
0.79
5.8
6.8
7.6
7.5
7.4
8.3
6.6
0.69
8.2
0.75
7.7
8.2
5.8
7.5
25
5.5
0.66
6.7
0.8
16.5
0.64
7.6
7.5
6.4
5.2
6.9
8.5
7.5
0.54
6.9
0.63
7.0
8.3
6.5
6.4
5.7
a
Mean from 3 different assays, errors in the range of Æ10% of the
reported values (data not shown). Acetazolamide (5-acetamido-1,3,4-
thiadiazole-2-sulfonamide, AAZ) was used as control.
c
5700 Chem. Commun., 2013, 49, 5699--5701
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Table 2 Selectivity ratios of sulfonamides 5–8 for the inhibition of hCA II vs. hCA
with excellent hCA IX and XII inhibitory properties, and
selectivity for inhibiting the tumor-associated over cytosolic
isoforms.
IX and hCA XII, respectively
Selectivity ratio
Compound
KI(hCA II)/KI(hCA IX)
KI(hCA II)/KI(hCA XII)
Notes and references
5a
7a
5b
7b
5c
7c
5d
7d
5e
7e
6a
8a
6b
8b
6c
8c
6d
8d
6e
8e
AAZ
1.37
80.6
1.33
68.69
6.77
12.27
11.13
5.66
1.22
1.36
8.24
4.49
1.48
10.72
1.03
12.53
5.84
1.28
6.81
8.10
0.48
1.72
100.15
1.41
79
3.24
15.15
8.5
5.13
1.44
1.96
8.84
4.38
1.30
13.70
1.23
14.92
6.42
1.26
6.07
9.5
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2.1
(although, not in the subnanomolar but the low nanomolar
range). As for the other isoforms discussed above, O-glycosides
and C-glycosides were present in the group of highly effective
and slightly less effective hCA IX inhibitors.
For hCA XII, the subnanomolar inhibitors (7a, 7b, 7c, 8b
and 8c) were exactly the same as the hCA IX subnanomolar
inhibitors, which is not so much unexpected considering
that the two isoforms have a rather high degree of homology
(at least in the active site amino acid residues).16 In fact the SAR
for inhibition of the two transmembrane isoforms is rather
similar (Table 1).
As hCA II is a very active and ubiquitous CA isoform, with
important physiological functions in many tissues/organs, one
of the main problems when designing new CAIs is finding
compounds which are selective for the target isoform over hCA II
(selectivity towards hCA I is a less important issue due to the fact
that hCA I is catalytically less efficient compared to hCA II, and
also notably inhibited by the chloride and bicarbonate present in
plasma).1,2 Table 2 shows thus the selectivity ratios for inhibiting
the transmembrane isoforms hCA IX and XII over the cytosolic
one hCA II. It may be seen for example that the clinically used
compound AAZ is not hCA IX selective (over hCA II) and has only
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´
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factor of 2.1). However, many of the compounds reported in this
communication do show indeed excellent selectivity ratios for
inhibiting hCA IX over hCA II and hCA XII over hCA II. For
example, compounds 7a, 7b, 7c, 5d, 8b and 8c showed selecti-
vity ratios for inhibiting hCA IX over hCA II in the range of
10.72–80.6. As these compounds were also not highly effective as
hCA I inhibitors, they can be indeed considered as hCA IX-selective
compounds. For the inhibition of hCA XII over hCA IX, selectivity
ratios in the range of 13.70–100.15 were registered for compounds
7a, 7b, 7c, 8b and 8c (Table 2).
M. Starnotti, J.-L. Montero, A. Scozzafava and C. T. Supuran, Bioorg. Med.
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15 M. Stiti, A. Cecchi, M. Rami, M. Abdaoui, A. Scozzafava, Y. Guari,
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16 V. Alterio, M. Hilvo, A. Di Fiore, C. T. Supuran, P. Pan, S. Parkkila,
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In conclusion, we have reported a series of sulfonamides
incorporating sugar moieties, obtained by thiol–ene click chemistry,
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 5699--5701 5701