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

Article abstract of DOI:10.1016/j.bmc.2013.06.041

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium-weak hCA I inhibitors (K_Is in the range of 224-7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2-7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4-239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.

Full text of DOI:10.1016/j.bmc.2013.06.041

Bioorganic & Medicinal Chemistry 21 (2013) 5130–5138
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
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
Janis Leitans a, Agnese Sprudza b, Muhammet Tanc c,d, Igor Vozny b, Raivis Zalubovskis b,
,
⇑
Kaspars Tars a, Claudiu T. Supuran c,d,
⇑
a Biomedical Research and Study Center, Ratsupites 1, LV 1067 Riga, Latvia
b Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia
c Università degli Studi di Firenze, CSGI, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, Sesto Fiorentino, 50019 Florence, Italy
d Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Sesto Fiorentino, 50019 Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl
moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl
azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC
4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmem-
brane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors
(KIs in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors
(KIs of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with KIs ranging between 5.4 and 811 nM,
whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the
adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyano-
phenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly
hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible
for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the
two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriaz-
olyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one point-
ing towards the hydrophilic half. These data may be used for the structure-based drug design of even
more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 18 May 2013
Accepted 16 June 2013
Available online 27 June 2013
Keywords:
Carbonic anhydrase
Enzyme inhibitor
Thiophene-2-sulfonamide
Click chemistry
X-ray crystallography
Sulfonamide
1. Introduction
The inhibition and activation of CAs are well understood pro-
cesses, with most classes of inhibitors binding to the metal cen-
Carbonic anhydrase (CAs, EC 4.2.1.1) catalyze the interconver-
sion between CO2 and bicarbonate by using a metal hydroxide
ter,1–3 whereas activators bind at the entrance of the active site
cavity and participate in proton shuttling processes between the
metal ion – bound water molecule and the environment.7 This
leads to the enhanced formation of the metal hydroxide, catalyti-
cally active species of the enzyme.7 Inhibitors generally bind to
the metal ion from the enzyme active site in deprotonated state
(as anions),1–3 although alternative inhibition mechanisms in
which the inhibitor interacts with the zinc-coordinated water mol-
ecule/hydroxide ion,8–10 or does not interact at all with it,11–13
have been recently described. The zinc-binders are the most inves-
tigated classes of CA inhibitors (CAIs), and they include the sulfon-
amides and their isosteres,1–3,14,15 the dithiocarbamates,16 the
xanthates,17 and the hydroxamates/carboxylates18,19 (although
some carboxylates may bind to the enzyme through the alternative
inhibition mechanisms, which do not involve directly the metal
ion).8,9,11–13
nucleophilic mechanism.1–3 They constitute
a
superfamily of
metalloenzymes with five distinct genetic families known to date,
the -, b-, -, d- and f-CAs, in organisms all over the tree of life.
a
c
These enzymes differ in their preference for metal ions used within
the active site for performing the catalysis: Zn(II) ions may be used
by all five classes mentioned above, but the
c-CAs are probably
Fe(II) enzymes (being active also with bound Zn(II) or Co(II)
ions),4,5 whereas the f-class uses Cd(II) or Zn(II) to perform the
physiologic reaction catalysis.6
⇑
Corresponding authors. Tel.: +371 67014826; fax: +371 67550338 (R.Z.); tel.:
+39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: raivis@osi.lv (R. Zalubovskis), claudiu.supuran@unifi.it
(C.T. Supuran).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.041

J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
5131
Sulfonamide/sulfamate CAIs are in clinical use for decades as
antiglaucoma,20 diuretic,21 antiobesity,22 and antitumor agents,23
targeting diverse of the various 16 CA isoforms known to date in
humans.1–3 Recently, targeting CAs from parasites (bacteria, fungi
and/or protozoa among others) with specific inhibitors was pro-
posed as an alternative for designing antiinfective agents with a
new mechanism of action.24
Due to the high number of CA isoforms present in most organ-
isms, and because the clinically used sulfonamides such as aceta-
zolamide AAZ, methazolamide MZA; ethoxzolamide EZA, or
dichlorophenamide DCP do not show selectivity for inhibiting
any of the isoforms with pharmacologic applications,1–3 there is a
constant search for new compounds belonging to the CAI class,
and the sulfonamides remain one of the most investigated such
chemotype, due to their strong affinity for CAs, ease of synthesis,
stability and lack of toxicity.1–3
first step involving the Sonogashira reaction of bromide 1 with tri-
methylsilylacetylene, whereas the second one, the trimethylsilyl
(TMS) protecting group cleavage. Unfortunately several attempts
to perform Sonogashira reaction with 1, under various conditions
did not provide the cross-coupling product, and only unreacted
bromo derivative 1 was detected. The most probable cause of this
lack of reactivity might be the unprotected sulfonamide group,
therefore protection of sulfonamide NH2 employing N,N-dimethyl-
formamide dimethyl acetal was done as reported by Shepard
et al.25 and the protected intermediate 2 was isolated in 93% yield
(Scheme 1). Reaction of compound 2 with trimethylsilylacetylene
under Sonogashira condition afforded the cross-coupling product
3 in a 82% yield. The desired acetylene intermediate 4 was obtained
by treatment of compound 3 with TBAF, when TMS cleavage and
the simultaneous deprotection of the sulfonamide group took
place, affording the key intermediate 4 in 50% yield.
Continuing our interest in sulfonamides as CAIs, we report here
the synthesis of a series of 5-substituted-(1,2,3-triazol-4-yl)thio-
phene-2-sulfonamides, prepared by click chemistry from thioph-
enesulfonamides incorporating an alkyne functionality in the 5th
position of the heterocyclic ring and aryl azides.
Cu(I) catalyzed azide–alkyne cycloaddition or ‘click chemistry’
is a powerful tool for the regioselective synthesis of 1,4-substituted
1,2,3-triazoles.27 After the first attempts to synthesize compound 5
under click chemistry conditions from alkyne 4 and phenylazide it
was observed that the reaction takes place in 99% conversion yield,
t
in 1:1 mixture BuOH/H2O and in the presence of sodium ascor-
bate, at 40 °C in seven days. However, when these reaction condi-
tions were modified by the recently published discovery of using
acetic acid as an additive in the Cu(I) catalyzed azide–alkyne cyclo-
addition,28 the reaction took place with full conversion in 30 min at
room temperature and compound 5 was isolated with a 96% yield.
With these optimal conditions in hand, the entire series of triazoles
5–19 was prepared in 54–96% yields (Scheme 1).
Apart the phenyl group present in compound 5, we incorpo-
rated various aryl such moieties in the new derivatives reported
here, of types 5–19, as it has been observed earlier29,30 that the nat-
ure of the tails present in sulfonamide CAIs strongly influence the
inhibitory properties and isoform selectivity of such derivatives.
Thus we have chosen variously mono/-di-substituted phenyl
2. Results and discussion
2.1. Chemistry
Thiophene-2-sulfonamides were already investigated in the
early 90s by Shepard et al.25 in the search of topically-acting anti-
glaucoma CAIs.26 One of the intermediates reported in this early
paper was the dimethylacetamide protected sulfonamide group
of 5-bromo-thiophene-2-sulfonamide 1. This compounds has been
used for the preparation of 4-acetylene-thiophene-2-sulfonamide
(4), the alkyne component used for click chemistry purposes in this
work.27,28 Compound 4 may be prepared in a two step process: the
TMS
CuI, Pd(PPh3)2Cl2
Et3N
O
O
O
DMF DMA
Br
S
Br
S
S
N
NH2
N
S
S
S
MeCN, RT
93%
DMF, RT
82%
TMS
O
O
O
N
N
2
3
1
Ar-N3
O
TBAF
O
N
N
S
S
NH2
S
NH2
S
N
THF, RT
50%
O
CuSO4* 5H2O
Na ascorbate, AcOH
t-BuOH/H2O, RT
O
Ar
5-19
4
54-96%
Me
;
Br
Me
Me
Me
CF3
;
OMe
Ar =
;
;
;
;
;
Me
5 (96%)
6 (80%)
7 (80%)
8 (70%)
9 (69%)
10 (89%)
11 (70%)
12 (54%)
CN
;
Cl
Cl
Cl
CF3
CN
F
O
S
Cl
;
Me
;
F
OMe
I
;
;
;
.
O
13 (92%)
14 (85%)
15 (89%)
16 (90%)
17 (93%)
18 (72%)
19 (73%)
Scheme 1. Preparation of sulfonamides 5–19 from 1-bromo-thiophene-5-sulfonamide 1 by using click chemistry.

5132
J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
moieties (in diverse positions of the ring with respect to the azide
moiety) as well as the naphthyl moiety (Scheme 1). In this way we
introduced chemical diversity in the small library of thiophene-2-
sulfonamide derivatives 5–19 reported here.
incorporate phenyl, 4-methoxyphenyl-, 3-bromophenyl and 4-
iodophenyl moieties. Another group of derivatives, among which
7, 11, 13, 14 and 16, showed inhibition constants in the range of
29.4–89.1 nM. They incorporate 4-tolyl, 4-trifluoromethylphenyl,
3-cyanophenyl, 3,4-dichlorophenyl and 3-chloro-4-methoxy-
phenyl moieties. It is obvius from these data that even small mod-
ifications of the Ar group from compounds 5–19, lead to important
changes in their affinity for CA IX (but not CA II, as mentioned
above). For example, introduction of a 3-Cl atom in 9, leading thus
to 16, had as a consequence a 12.8-times loss of hCA IX inhibitory
activity of 16 compared 9 (Table 1). Weaker hCA IX inhibition was
observed for compounds 6, 8, 10, 15, 18 and 19, which had KIs in
the range of 101–811 nM. Apparently, bulkier Ar moieties (as the
1-naphthyl one present in 10, the tert-butyl-phenyl present in 8,
etc.) lead to a loss of the hCA IX inhibitory activity for this series
of sulfonamide CAIs.
(iv) hCA XII was also effectively inhibited by all sulfonamides
investigated here, with KIs in the range of 3.4–239 nM (Table 1).
Most of the new sulfonamides were highly effective, low nanomo-
lar hCA XII inhibitors, for example, compounds 5–7, 9, 10, 13, 15,
17 and 18 (KIs range of 3.4–9.6 nM). Several other derivatives, such
as 8, 12, 14, and 16 showed inhibition constants of 24.7–58.8 nM.
Only two derivatives (11 and 19, both possessing CF3 moieties in
their molecules) were less effective as hCA XII inhibitors, with KIs
of 107.5–239 nM (Table 1). Thus, as for hCAII; most of the substi-
tution patterns present in these new sulfonamides as Ar moieties
(except those from 11 and 19) lead to quite effective hCA XII
inhibitors.
2.2. Carbonic anhydrase inhibition
Sulfonamides 5–19 reported here and acetazolamide AAZ as
standard drug were assayed as inhibitors of four physiologically
relevant CA isoforms, the cytosolic hCA I and II, as well as the trans-
membrane, tumo-associated ones CA IX and XII.31 It should be
mentioned that CA II and XII are targets for developing antiglauco-
ma drugs,20 whereas CA IX (and XII) are antitumor drug targets.23
hCA I is rather widespread in many tissues in humans, and it may
be considered as an offtarget isoform.1–3
The following structure–activity relationship (SAR) can be evi-
denced from data of Table 1 regarding the CA inhibitory properties
of sulfonamides 5–19:
(i) hCA I was moderately inhibited by sulfonamides 5–19, which
showed inhibition constants in the range of 224–7544 nM, being
thus similar (or less effective as hCA I inhibitors) to acetazolamide
AAZ (KI of 250 nM). The best inhibitors were 13 and 18 (incorporat-
ing 3-cyanophenyl and 4-methylsulfonyl-2-fluorophenyl moieties
as Ar group) which showed (KIs in the range of 224–289 nM,
whereas the remaining compounds in the series were less effec-
tive, with KIs in the range of 499–7544 nM (Table 1). It may be thus
considered that the relatively weak inhibitory properties against
the widespread isoform hCA I constitute a favorable feature of this
class of CA is reported here.
(ii) The physiologically dominant isoform hCA II was highly
inhibited by the new class of sulfonamides investigated here, with
a flat (but excellent) inhibition profile. Indeed, the KIs only ranged
between 2.2 and 7.7 nM, making all these compounds highly effec-
tive hCA II inhibitors. Unexpectedly (however, see the X-ray crys-
tallographic part for an explanation of this finding) all the
substitution patterns present in compounds 5–19 lead to highly
effective CAIs. All these compounds were better hCA II inhibitors
compared to the clinically used drug AAZ (Table 1).
(v) Except for the offtarget isforms hCA I, which is generally
poorly inhibited by sulfonamides 5–19, the remaining three iso-
forms (hCA II, IX and XII) are well inhibited by the compounds re-
ported here, with hCA II inhibition being in the low nanomolar
ramnge for all compounds, followed by hCA XII inhibition (most
derivatives low nanomolar inhibitors), whereas for hCA IX both
highly effective as well as less effective inhibitors have been de-
tected in this small series of derivatives.
2.3. Protein X-ray crystallography
(iii) A more complicate SAR has been evidenced for the inhibi-
tion of the tumor-associated isoform hCA IX (Table 1). Several com-
pounds, such as 5, 9, 12 and 17, showed highly effective hCA IX
inhibitory properties, with KIs in the range of 5.4–10.9 nM. They
In order to rationalize some of the very interesting inhibition
data reported above, we have resolved the high resolution X-ray
crystal structures for the adducts of two of these inhibitors (10
and 13) bound to hCA II (Tables 2 and 3 and Figs. 1–3).
The crystal structure of hCA II in complex with compound the
1-naphthyl derivative 10 was solved at 1.35 Å resolution (Table 2).
The obtained electron density clearly revealed the expected fea-
tures of inhibitor (Fig. 1). Apart from interaction between the
deprotonated sulfonamide moiety and the metal ion, and the
hydrogen bond between the NH moiety of the sulfonamide and
the OH of Thr199 (present in all other sulfonamide–hCA II com-
plexes)1a,2a,3a,30,32 10 was not involved in other polar contacts with
protein. This is one of the few cases in which positioning of the li-
gand within the hCA II active site was achieved solely by hydro-
phobic and van der Waals interactions (Fig. 1). Interestingly, the
binding mode of 10 is quite different from that, observed in an-
other similar compound 13, (Fig. 2, see latter in the text). The
naphthyl moiety of 10 fits perfectly well in a hydrophobic pocket
of the protein evidenced earlier for the binding of other sulfon-
amide CAIs,1a,2a,3a,30,32 with residues Phe131, Val135, Leu204 and
Pro202 being in van der Waals contact with the naphthyl tail of
the inhibitor (Fig. 2).
Table 1
Inhibition data of isoforms hCA I, II, IX and XII with sulfonamides 5–19 and
a
standard, clinically used sulphonamide (acetazolamide AAZ), by a stopped-flow CO2
hydrase assay31
Compound
KI (nM)a
hCA I
hCA II
hCA IX
hCA XII
5
6
7
8
499
1434
526
2540
746
6.1
3.8
3.5
2.9
6.3
2.4
2.9
7.7
4.5
3.2
3.0
2.7
2.9
2.7
2.2
12
7.2
560
43.6
713
6.4
101
89.1
10.9
29.4
63.5
395
81.9
5.4
7.3
6.2
8.8
45.4
8.4
7.4
107.5
30.3
6.0
24.7
6.6
58.8
9.6
3.4
9
10
11
12
13
14
15
16
17
18
19
AAZ
750
3055
2420
289
2132
2464
4273
709
224
7544
250
140
811
25
The crystal structure of CA II in complex with compound 13 was
solved at 1.82 Å resolution (Table 3). The obtained electron density
was good for most of the molecule except for the phenylcyano
group, where it was weaker but still interpretable (Fig. 2). Along
the interactions with the metal ion and Thr199 (as mentioned
239
5.6
a
Errors were in the range of 5–10% of the reported values, from three different
assays.

J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
5133
Table 2
Data processing, refinement and validation statistics for the hCA II–10 complex
Space group
P21
Cell dimensions
a (Å)
42.3
b (Å)
41.5
c (Å)
72.2
b (°)
104.3
Resolution (Å)
20–1.34
1.34–1.41
50,792
2615
Highest resolution shell (Å)
Number of reflections
Number of reflections in test set
Completeness (%)
Rmerge
<I/rI>
Average multiplicity
R-factor
Rfree
93 (80*)
0.05 (0.17)
12.7 (4.3)
2.0 (1.2)
0.13 (0.19)
0.18 (0.24)
11.7
Average B factor (Å2)
Average B factor for inhibitor (Å2)
<B> from Wilson plot (Å2)
Number of protein atoms
Number of inhibitor atoms
Number of solvent molecules
R.m.s. deviations from ideal values
Bond lengths (Å)
Figure 1. Binding of 10 within the hCA II active site. The zinc ion is shown as a gray
sphere and its coordinating histidines (His94, 96 and 119) are shown in green.
Residues 131, 135, 204 and 205 participating in hydrophobic and van der Waals
contacts with inhibitor are indicated. For the sake of clarity, 2FoÀFc electron density
is shown only for ligand, contoured at 1 and calculated in the absence of ligand. The
figure prepared by using Pymol (DeLano The PyMOL Molecular Graphics System San
Carlos, CA, USA, DeLano Scientific).
11.0
8.5
2094
24
305
0.022
2.41
0.41
Bond angles (°)
Outliers in Ramachandran plot (%)
a
Values in parenthesis are for the high resolution bin.
Table 3
Data processing, refinement and validation statistics for the hCA II–13 complex
Space group
P21
Cell dimensions
a (Å)
42.4
b (Å)
41.5
c (Å)
72.2
104.2
b (o)
Resolution (Å)
20–1.82
1.82–1.86
20,916
1122
Highest resolution shell (Å)
Number of reflections
Number of reflections in test set
Completeness (%)
Rmerge
<I/rI>
Average multiplicity
R-factor
Rfree
99.3 (86.8*)
0.17 (0.36)
21.9 (3.4)
7.5 (2.8)
0.14 (0.21)
0.20 (0.30)
12.2
Average B factor (Å2)
Average B factor for inhibitor (Å2)
<B> from Wilson plot (Å2)
Number of protein atoms
Number of inhibitor atoms
Number of solvent molecules
R.m.s. deviations from ideal values
Bond lengths (Å)
15.6
8.1
2080
22
Figure 2. Binding of 13 within the hCA II active site. The zinc ion is shown as a gray
sphere and its coordinating histidines (His94, 96 and 119) are shown in green.
Residues 92, 121, 131, 141 and 198 participating in hydrogen bonding, hydrophobic
296
and van der Waals contacts with the inhibitor are also indicated.
A glycerol
molecule bound to the enzyme is shown as a thin stick model with sky blue carbon
atoms. For the sake of clarity, 2FoÀFc electron density is shown only for ligand,
contoured at 1 and calculated in the absence of ligand. The figure was prepared by
using Pymol (DeLano, The PyMOL Molecular Graphics System San Carlos, CA, USA,
DeLano Scientific).
0.019
2.14
0.40
Bond angles (°)
Outliers in Ramachandranplot (%)
a
Values in parenthesis are for the high resolution bin.
above for 10), compound 13 was involved in another polar contact
with the protein, that is, a hydrogen bond with Gln92, an amino
acid residues known to be involved in the binding of sulfonamide
CAIs.3a,30,32 A number of hydrophobic and van der Waals interac-
tions are present as well in this adduct, and involved the following
amino acid residues: Val121, Leu141, Phe131, and Leu198 (Fig. 2).
In the electron density map of active site, we also located a glycerol
molecule, in a near identical location as reported earlier in a num-
ber of CA II structures by McKenna’s group.33 As shown by these
researchers, the glycerol is bound within the enzyme active site
during the cryo-protection of the protein crystal, before freezing,
and that does not affect the binding of the sulfonamide inhibitor.33
It should be also noted the very different orientation of the two
inhibitors 10 and 13, when bound to the hCA II active site (Fig. 3).
Only the sulfamoyl moieties of the two compounds are superim-
posable, whereas the rest of the molecule is not (even at the level
of the thiophene ring, rather close to the metal ion). Indeed, the
aryl–triazolyl moieties of the two compounds adopt highly diverse
orientation within the hCA II active site cavity, being completely
non-superimposable. The 3-cyanophenyl group of 13 is oriented
more towards the hydrophilic half of the active site and the triazole
ring participates in the hydrogen bonding with the oxygen of
the CONH2 moiety of Gln92 (Figs. 2 and 3). On the contrary, the
naphthyl (and triazole ring) of 10 are orientated towards the

5134
J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
Figure 3. Comparison of the binding modes of 10 and 13 when bound to hCA II. Sulfonamide 10 is shown with black carbons and 13 with gray carbons. The protein is shown
as a surface model, coloured according to surface atoms (carbon-green, nitrogen-blue, oxygen-red) and the zinc ion is sown as a gray sphere. Interactions of the sulfonamide
group with the metal ion and a hydrogen bond of the triazole ring of 13 to the oxygen of the Gln92 residue are also shown.
hydrophobic half of the hCA II active site, occupying completely
this binding pocket. The perfect fit of the aryl–triazolyl moiety of
these compounds within the hCA II active site, may explain the
excellent inhibition profile of all these compounds against the
dominant CA isoforms, as shown above in Section 2.2.
dried by standard procedures prior to use; petroleum ether of boil-
ing range 40–60 °C was used. Flash chromatography was carried
out using Merck silica gel (230–400 mesh) or reversed phase
PR18 (25–40 lm). Thin-layer chromatography was performed on
silica gel, spots were visualized with UV light (254 and 365 nm).
Melting points were determined on an OptiMelt automated melt-
ing point system. IR spectra were measured on a Shimadzu FTIR
IR Prestige-21 spectrometer. NMR spectra were recorded on Varian
Mercury (400 MHz) spectrometer with chemical shifts values (d) in
ppm relative to TMS using the residual DMSO-d6 signal as an inter-
nal standard. HRMS data were obtained with a Q-TOF micro high-
resolution mass spectrometer with ESI (ESI+/ESIÀ).
3. Conclusions
We report here a series of 2-thiophene-sulfonamides incorporat-
ing 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click
chemistry from 5-ethynylthiophene-2-sulfonamide and substi-
tuted aryl azides. The new sulfonamides were investigated as inhib-
itors of the zinc metalloenzyme CA, and more specifically against
the cytosolic isoforms hCA I and II and the transmembrane, tu-
mor-associated ones hCA IX and XII: The new compounds were
medium–weak hCA I inhibitors (inhibition constants in the range
of 224–7544 nM), but were compactly, highly effective, low nano-
molar hCA II inhibitors (KIs in the range of 2.2–7.7 nM). The tu-
mor-associated hCA IX was inhibited with KIs ranging between 5.4
and 811 nM, whereas hCA XII with inhibition constants in the range
of 3.4–239 nM. The X-ray crystal structure of the adducts of two
such compounds complexed to hCA II (one incorporating 1-naphthyl,
the other one 3-cyanophenyl moieties) evidenced the reasons of
the high affinity for this enzyme. Highly favorable, predominantly
hydrophobic interactions between the sulfonamide scaffold and
the hCA II active site were responsible for the binding, in addition
to the coordination of the sulfamoyl moiety to the zinc ion. The tails
of the two inhibitors adopted very diverse orientations when bound
to the active site, with the naphthyltriazolyl moiety orientated towards
the hydrophobic half of the active site, and the 3-cyanophenyl
pointing towards the hydrophilic half. These data may be used for
the structure-based drug design of even more effective hCA II inhib-
itors, with potential use as antiglaucoma agents or as diuretics.
5-Bromo-N-[(E)-(dimethylamino)methylidene]thiophene-2-sul-
fonamide (2)25
:
To a solution of 5-bromothiophene-2-sulfonamide (1) (7.00 g,
28.91 mmol) in acetonitrile (50 mL) N,N-dimethylformamide di-
methyl acetal (5.50 mL, 41.34 mmol) was added. The mixture
was stirred at room temperature for 24 h. Solvent evaporation in
vacuum afforded 2 (7.98 g, 93%) as light yellow solid. Mp 104–
105 °C. IR (KBr, cmÀ1
) mmax: 1631 (N@C), 1331 (S@O), 1142
(S@O), 1127 (S@O); 1H NMR (400 MHz, DMSO-d6) d: 2.94 (s, 3H),
3.16 (s, 3H), 7.27 (d, J = 3.9 Hz, 1H), 7.36 (d, J = 3.9 Hz, 1H), 8.21
(s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 35.3, 41.1, 117.1, 130.6,
131.0, 145.7, 160.0.
N-[(dimethylamino)methylidene]-5-[2-(trimethylsilyl)ethy-
nyl)]thiophene-2-sulfonamide (3):
To a solution of compound 2 (2.00 g, 6.73 mmol) in dry DMF CuI
(0.13 g, 0.68 mmol), Pd(PPh3)2Cl2 (0.24 g, 0.34 mmol), trimethylsi-
lylacetylene (1.35 mL, 9.46 mmol) and Et3N (4.70 mL, 33.78 mmol)
were added. The mixture was stirred at room temperature under
an argon atmosphere for 48 h, diluted with EtOAc (30 mL), filtered
though a celite plug. Saturated aqueous NH4Cl (50 mL) was added
and the product was extracted with EtOAc (3 Â 50 mL). The organic
extract was washed with H2O (2 Â 50 mL) and brine (50 mL), dried
over Na2SO4, solvent driven off in vacuum. Purification of the crude
product by silica gel chromatography (EtOAc/PE 2:1) afforded 3
(1.75 g, 82%) as light brown solid. Rf = 0.7 (EtOAc/PE 2:1). Mp
4. Experimental protocols
4.1. Chemistry
120.5–121.5 °C. IR (KBr, cmÀ1) IR (KBr, cmÀ1
) mmax: 2147 (C„C),
1635 (N@C), 1345 (S@O), 1148 (S@O); 1H NMR (400 MHz,
DMSO-d6) d: 0.24 (s, 9H), 2.95 (s, 3H), 3.17 (s, 3H), 7.32 (d, J =
Reagents and starting materials were obtained from commer-
cial sources and used as received. The solvents were purified and

J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
5135
3.9 Hz, 1H), 7.43 (d, J = 3.9 Hz, 1H), 8.22 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) d: À0.45, 35.3, 41.1, 96.0, 101.9, 126.0, 130.0, 133.2,
145.7, 160.0; HRMS (ESI) [M+H]+: m/z Calcd for (C12H19N2O2S2Si)
315.0657. Found 315.0652.
J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.78 (s, 2H), 7.79–7.83 (m,
2H), 9.29 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 20.7, 120.1,
120.2, 124.2, 130.4, 130.9, 134.1, 136.9, 138.9, 141.4, 144.4; HRMS
(ESI) [M+H]+: m/z Calcd for (C13H13N4O2S2) 321.0480. Found
321.0527.
5-Ethynylthiophene-2-sulfonamide (4)34
:
To a solution of compound 3 (1.00 g, 3.18 mmol) in dry THF
(30 mL) 1 M TBAF in THF (6.36 mL, 6.36 mmol) was added. The
mixture was stirred at room temperature for 3 h, diluted with
H2O (30 mL), extracted with EtOAc (3 Â 25 mL). The organic phase
was washed with brine (25 mL), dried over Na2SO4, solvent was
driven off in vacuum. Purification of the crude product by silica
gel chromatography (EtOAc/PE 1:1) afforded 4 (0.30 g, 50%) as
brown solid. Rf = 0.8 (EtOAc/PE 1:1). Mp 125.5–126.5 °C. IR (KBr,
5-[1-(4-tert-Butylphenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sul-
fonamide (8):
Compound 8 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 1-azido-4-tert-butylbenzene38 (0.19 g,
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a light
brown solid (0.27 g, 70%). Mp 213–214 °C. IR (KBr, cmÀ1
) mmax:
3303 (NH), 1341 (S@O), 1152 (S@O); 1H NMR (400 MHz, DMSO-
d6) d: 1.34 (s, 9H), 7.50 (d, J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H),
7.62–7.67 (m, 2H), 7.78 (s, 2H), 7.81–7.86 (m, 2H), 9.31 (s, 1H);
13C NMR (100 MHz, DMSO-d6) d: 31.0, 34.6, 120.0, 120.2, 124.2,
126.7, 130.8, 134.0, 136.9, 141.3, 144.3, 151.8; HRMS (ESI)
[M+H]+: m/z Calcd for (C16H19N4O2S2) 363.0949. Found 363.0952.
5-[1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sul-
fonamide (9):
cmÀ1
) mmax: 3330 (NH), 3233 (NH), 2100 (C„C), 1341 (S@O),
1150 (S@O); 1H NMR (400 MHz, DMSO-d6) d: 4.81 (s, 1H), 7.38
(d, J = 3.9 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.83 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d: 75.4, 87.6, 125.4, 130.0, 133.3, 146.5.
General procedure for the synthesis of 1,2,3-triazolylderivatives of
sulfamoylthiophene
To
a
solution of 5-ethynylthiophene-2-sulfonamide (4)
(1 equiv) in 1:1 tBuOH/H2O (10 mL per 1.07 mmol of 4) corre-
sponding arylazide (1 equiv) and solution of CuSO4Á5H2O
(1.3 equiv) in H2O (1.5 mL per 1.36 mmol) were added. The mix-
ture was stirred at room temperature for 5 min before solution of
sodium ascorbate (4 equiv) in H2O (2 mL per 4.27 mmol) was
added. The mixture was stirred at room temperature for another
15 min before AcOH (20 equiv) was added and stirring was contin-
ued for 30 min. Solvent was driven off in vacuum and the crude
product was purified by reversed phase chromatography (C-18,
H2O–MeCN gradient MeCN 10–90%) with followed re-crystalliza-
tion from MeCN/H2O.
5-(1-Phenyl-1H-1,2,3-triazol-4-yl)thiophene-2-sulfonamide (5)
Compound 5 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), azidobenzene35,36 (0.13 g, 1.07 mmol),
CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate (0.85 g,
4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a yellowish solid
Compound 9 was prepared according to the general procedure
from 4 (0.30 g, 1.60 mmol), 1-azido-4-methoxybenzene36 (0.24 g,
1.60 mmol), CuSO4Á5H2O (0.51 g, 2.04 mmol), sodium ascorbate
(1.27 g, 6.41 mmol) and AcOH (1.83 mL, 32.04 mmol) as a light
brown solid (0.37 g, 69%). Mp 258-259 °C. IR (KBr, cmÀ1
) mmax:
3337 (NH), 3233 (NH), 1340 (S@O), 1151 (S@O); 1H NMR
(400 MHz, DMSO-d6) d: 3.85 (s, 3H), 7.15–7.20 (m, 2H), 7.49 (d,
J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.77 (br s, 2H), 7.81–7.86
(m, 2H), 9.25 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 55.6,
115.0, 120.3, 122.0, 124.1, 129.7, 130.8, 137.0, 141.2, 144.3,
159.6; HRMS (ESI) [M+H]+: m/z Calcd for (C13H13N4O3S2)
337.0429. Found 337.0435.
5-[1-(Naphthalen-1-yl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (10):
Compound 10 was prepared according to the general procedure
from
4
(0.20 g, 1.07 mmol), 1-azidonaphthalene39 (0.18 g,
(0.31 g, 96%). Mp 287–288 °C. IR (KBr, cmÀ1
)
m
max: 3324 (NH),
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
3238 (NH), 1335 (S@O), 1151 (S@O); 1H NMR (400 MHz, DMSO-
d6) d: 7.46 (d, J = 3.7 Hz, 1H), 7.50 (d, J = 3.7 Hz, 1H), 7.51–7.56
(m, 1H), 7.61–7.67 (m, 4H), 7.92–7.96 (m, 2H), 9.32 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d: 120.0, 120.2, 124.1, 129.1, 129.6,
130.0, 135.6, 136.4, 141.7, 147.0; HRMS (ESI) [M+H]+: m/z Calcd
for (C12H11N4O2S2) 307.0323. Found 307.0325.
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a brown
solid (0.34 g, 89%). Mp 228–229 °C. IR (KBr, cmÀ1
) mmax: 3307
(NH), 1346 (S@O), 1153 (S@O); 1H NMR (400 MHz, DMSO-d6) d:
7.55 (d, J = 3.9 Hz, 1H), 7.57–7.60 (m, 1H), 7.61 (d, J = 3.9 Hz, 1H),
7.63–7.76 (m, 3H), 7.80 (s, 2H), 7.84 (dd, J = 7.3, 1.2 Hz, 1H),
8.14–8.17 (m, 1H), 8.22–8.26 (m, 1H), 9.22 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 122.0, 124.1, 124.3, 124.7, 125.5, 127.3,
127.8, 128.3, 128.4, 130.7, 130.9, 132.9, 133.7, 137.0, 140.9,
144.4; HRMS (ESI) [M+H]+: m/z Calcd for (C16H13N4O2S2)
357.0480. Found 357.0488.
5-[1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (6):
Compound 6 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 1-azido-3-methylbenzene37 (0.14 g,
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a yellow-
5-{1-[4-(Trifluoromethyl)phenyl]-1H-1,2,3-triazol-4-yl}thiophene-
2-sulfonamide (11):
ish solid (0.27 g, 80%). Mp 222–223 °C. IR (KBr, cmÀ1
)
m
max: 3309
Compound 11 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 1-azido-4-(trifluoromethyl)benzene40
(0.20 g, 1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium
ascorbate (0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol)
as a light brown solid (0.28 g, 70%). Mp 252–253 °C. IR (KBr,
(NH), 1340 (S@O), 1151 (S@O); 1H NMR (400 MHz, DMSO-d6) d:
2.43 (s, 3H), 7.33–7.37 (m, 1H), 7.50 (d, J = 3.9 Hz, 1H), 7.51–7.54
(m, 1H), 7.60 (d, J = 3.9 Hz, 1H), 7.70–7.74 (m, 1H), 7.76–7.79 (m,
3H), 9.33 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 20.9, 117.3,
120.2, 120.6, 124.2, 129.7, 129.8, 130.8, 136.3, 136.8, 139.8,
141.4, 144.4; HRMS (ESI) [M+H]+: m/z Calcd for (C13H13N4O2S2)
321.0480. Found 321.0505.
cmÀ1 3320 (NH), 1340 (S@O), 1152 (S@O); 1H NMR
) mmax:
(400 MHz, DMSO-d6, d): 7.53 (d, J = 3.8 Hz, 1H), 7.61 (d, J =
3.8 Hz, 1H), 7.80 (s, 2H), 8.02–8.07 (m, 2H), 8.17–8.23 (m, 2H),
9.51 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 120.5, 120.7, 123.8
(q, J = 273 Hz), 124.5, 127.4 (q, J = 3.8 Hz), 129.0 (q, J = 33.0 Hz),
130.9, 136.4, 139.1, 141.7, 144.7; HRMS (ESI) [M+H]+: m/z Calcd
for (C13H10F3N4O2S2) 375.0197. Found 375.0250.
5-[1-(3-Bromophenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (12):
Compound 12 was prepared according to the general procedure
from 4 (0.30 g, 1.60 mmol), 1-azido-3-bromobenzene41 (0.32 g,
1.60 mmol), CuSO4Á5H2O (0.51 g, 2.04 mmol), sodium ascorbate
5-[1-(4-Methylphenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (7):
Compound 7 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 1-azido-4-methylbenzene35 (0.14 g,
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a light
brown solid (0.27 g, 80%). Mp 275–276 °C. IR (KBr, cmÀ1
)
mmax
:
3324 (NH), 3233 (NH), 1340 (S@O), 1153 (S@O); 1H NMR
(400 MHz, DMSO-d6) d: 2.39 (s, 3H), 7.41–7.46 (m, 2H), 7.50 (d,

5136
J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
(1.27 g, 6.41 mmol) and AcOH (1.83 mL, 32.04 mmol) as a yellow-
ish solid (0.33 g, 54%). Mp 246–247 °C. IR (KBr, cmÀ1
max: 3304
124.2, 129.8, 130.9, 136.8, 141.3, 144.4, 154.9; HRMS (ESI)
[M+H]+: m/z Calcd for (C13H12ClN4O3S2) 371.0039. Found 371.0047.
5-[1-(4-Iodophenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (17):
)
m
(NH), 1342 (S@O), 1152 (S@O); 1H NMR (400 MHz, DMSO-d6) d:
7.49 (d, J = 3.9 Hz, 1H), 7.57–7.63 (m, 2H), 7.72–7.76 (m, 1H),
7.79 (br s, 2H), 7.97–8.01 (m, 1H), 8.18–18.21 (m, 1H), 9.43 (s,
1H); 13C NMR (100 MHz, DMSO-d6) d: 119.2, 120.5, 122.5, 122.8,
124.3, 130.9, 131.8, 131.9, 136.5, 137.4, 141.5, 144.6; HRMS (ESI)
[M+H]+: m/z Calcd for (C12H10BrN4O2S2) 384.9429. Found
384.9454.
Compound 17 was prepared according to the general procedure
from 4 (0.10 g, 0.53 mmol), 1-azido-4-iodobenzene35,36 (0.13 g,
0.53 mmol), CuSO4Á5H2O (0.17 g, 0.68 mmol), sodium ascorbate
(0.42 g, 2.14 mmol) and AcOH (0.61 mL, 10.68 mmol) as a light
brown solid (0.20 g, 90%). Mp 274–275 °C. IR (KBr, cmÀ1
) mmax:
5-[1-(3-Cyanophenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sulfon-
amide (13):
3349 (NH), 3246 (NH), 1342 (S@O), 1150 (S@O); 1H NMR
(400 MHz, DMSO-d6) d: 7.50 (d, J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz,
1H), 7.74–7.77 (m, 2H), 7.78 (s, 2H), 7.99–8.03 (m, 2H), 9.37 (s,
1H); 13C NMR (100 MHz, DMSO-d6) d: 94.9, 120.2, 122.1, 124.4,
130.9, 136.0, 136.6, 138.8, 141.6, 144.5; HRMS (ESI) [M+H]+: m/z
Calcd for (C12H10IN4O2S2) 432.9290. Found 432.9306.
Compound 13 was prepared according to the general procedure
from
4
(0.20 g, 1.07 mmol), 3-azidobenzonitrile37 (0.15 g,
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a yellow
solid (0.32 g, 92%). Mp 243–244 °C. IR (KBr, cmÀ1
)
m
max: 3331
5-{1-[2-Fluoro-4-(methylsulfonyl)phenyl]-1H-1,2,3-triazol-4-
yl}thiophene-2-sulfonamide (18):
(NH), 3232 (NH), 2235 (C„N), 1336 (S@O), 1157 (S@O); 1H NMR
(400 MHz, DMSO-d6) d: 7.50 (d, J = 3.9 Hz, 1H), 6.61 (d, J = 3.9 Hz,
1H), 7.80 (s, 2H), 7.86 (app t, J = 8.2 Hz, 1H), 8.00–8.04 (m, 1H),
8.32 (ddd, J = 8.2, 2.4, 1.2 Hz, 1H), 8.46–8.48 (m, 1H), 9.45 (s,
1H); 13C NMR (100 MHz, DMSO-d6) d: 112.9, 117.8, 120.6, 123.7,
124.5, 124.9, 130.9, 131.4, 132.7, 136.4, 136.8, 141.6, 144.7; HRMS
(ESI) [M+H]+: m/z Calcd for (C13H10N5O2S2) 332.0276. Found
332.0335.
Compound 18 was prepared according to the general procedure
from
4
(0.10 g, 0.53 mmol), 1-azido-2-fluoro-4-(methylsulfo-
solution of
nyl)benzene (0.12 g, 0.53 mmol) prepared: To
a
2-fluoro-4-(methylsulfonyl)aniline (1.00 g, 5.28 mmol) in mixture
of H2O (6.7 mL) and conc. HCl (3.3 mL) aqueous 4 M NaNO2
(1.72 mL, 6.87 mmol) dropwise was added at 0 °C. The mixture
was stirred at 0 °C for 10 min before aqueous 4 M NaN3 (1.59 mL,
6.36 mmol) dropwise was added at 0 °C and stirring was continued
at the same temperature for 1 h. H2O (15 mL) was added and the
product was extracted with Et2O (2 Â 25 mL). The organic phase
was washed with H2O (25 mL) and dried over Na2SO4. Solvent
evaporation afforded 1-azido-2-fluoro-4-(methylsulfonyl)benzene
(0.89 g, 78%) as orange solid.
5-[1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl]thiophene-2-sul-
fonamide (14):
Compound 14 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 4-azido-1,2-dichlorobenzene37 (0.20 g,
1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a white
solid (0.34 g, 85%). Mp 260–261 °C. IR (KBr, cmÀ1
)
m
max: 3325
IR (KBr, cmÀ1
) mmax: 2142 (N3), 2101 (N3), 1318 (S@O), 1148
(NH), 3218 (NH), 1343 (S@O), 1155 (S@O); 1H NMR (400 MHz,
DMSO-d6) d: 7.49 (d, J = 3.9 Hz, 1H), 7.60 (d, J = 3.9 Hz, 1H), 7.79
(s, 2H), 7.93 (d, J = 8.7 Hz, 1H), 7.99 (dd, J = 8.7, 2.4 Hz, 1H), 8.29
(d, J = 2.4 Hz, 1H), 9.44 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
120.3, 120.6, 122.0, 124.4, 130.9, 131.4, 131.9, 132.4, 135.8,
136.4, 141.6, 144.7; HRMS (ESI) [M+H]+: m/z Calcd for (C12H9Cl2N4-
O2S2) 374.9544. Found 374.9597.
(S@O); 1H NMR (400 MHz, DMSO-d6) d: 3.26 (s, 3H), 7.58 (app t,
J = 8.3 Hz, 1H), 7.77 (ddd, J = 8.3, 2.0, 0.8 Hz, 1H), 7.88 (dd, J =
10.6, 2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d: 43.3, 115.7 (d,
J = 21.9 Hz), 122.3 (d, J = 1.6 Hz), 124.3 (d, J = 3.6 Hz), 133.1 (d, J =
0.9 Hz), 137.7 (d, J = 3.9 Hz), 153.0 (d, J = 252 Hz).), CuSO4Á5H2O
(0.17 g, 0.68 mmol), sodium ascorbate (0.42 g, 2.14 mmol) and
AcOH (0.61 mL, 10.68 mmol) as a light brown solid (0.15 g, 72%).
5-[1-(3-Chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]thiophene-
2-sulfonamide (15):
Mp 279–280 °C. IR (KBr, cmÀ1
) mmax: 3343 (NH), 3261 (NH), 1349
(S@O), 1298 (S@O), 1164 (S@O), 1133 (S@O); 1H NMR (400 MHz,
DMSO-d6) d: 7.59–7.62 (m, 2H), 7.80 (s, 2H), 8.01–8.05 (m, 1H),
8.20–8.28 (m, 2H), 9.27 (d, J = 2.3 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) d: 43.1, 116.7 (d, J = 22.9 Hz), 123.3 (d, J = 5.5 Hz),
124.4 (d, J = 3.5 Hz), 125.0, 126.6, 128.3 (d, J = 10.8 Hz), 130.9,
136.1, 141.5, 142.9 (d, J = 6.2 Hz), 144.8, 153.1 (d, J = 257 Hz);
HRMS (ESI) [M+H]+: m/z Calcd for (C13H12FN4O4S3) 403.0005.
Found 402.9996.
Compound 15 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 4-azido-2-chloro-1-fluorobenzene42
(0.18 g, 1.07 mmol), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium
ascorbate (0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol)
as a light yellow solid (0.34 g, 89%). Mp 240–241 °C. IR (KBr,
cmÀ1 3314 (NH), 1340 (S@O), 1152 (S@O); 1H NMR
) mmax:
(400 MHz, DMSO-d6) d: 7.48 (d, J = 3.9 Hz, 1H), 7.60 (d, J = 3.9 Hz,
1H), 7.72 (app t, J = 9.0 Hz, 1H), 7.79 (s, 2H), 7.99 (ddd, J = 9.0,
4.3, 2.7 Hz, 1H), 8.24 (dd, J = 6.3, 2.7 Hz, 1H), 9.38 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d: 118.3 (d, J = 22.8 Hz), 120.7, 120.9
(d, J = 19.3 Hz), 121.2 (d, J = 7.9 Hz), 122.7, 124.4, 130.9, 133.3 (d,
J = 3.1 Hz), 136.5, 141.5, 144.6, 157.1 (d, J = 249 Hz); HRMS (ESI)
[M+H]+: m/z Calcd for (C12H9ClFN4O2S2) 358.9839. Found
358.9866.
5-{1-[4-Cyano-3-(trifluoromethyl)phenyl]-1H-1,2,3-triazol-4-
yl}thiophene-2-sulfonamide (19):
Compound 19 was prepared according to the general procedure
from 4 (0.20 g, 1.07 mmol), 4-azido-2-(trifluoromethyl)benzoni-
trile (0.23 g, 1.07 mmol) prepared: To a solution of 4-amino-2-(trif-
luormetyl)benzonitrile (1.00 g, 5.37 mmol) in mixture of H2O
(6.7 mL) and conc. HCl (3.3 mL) aq 4 M NaNO2 (1.75 mL, 6.98 mmol)
dropwise was added at 0 °C. The mixture was stirred at 0 °C for
10 min before aq 4 M NaN3 (1.61 mL, 6.44 mmol) dropwise was
added at 0 °C and stirring was continued at the same temperature
for 1 h. H2O (15 mL) was added and the product was extracted with
Et2O (2 Â 25 mL). The organic phase was washed with H2O (25 mL),
dried over Na2SO4. Solvent evaporation afforded 4-azido-2-(trifluo-
romethyl)benzonitrile (0.71 g, 62%) as light brown solid. IR (KBr,
5-[1-(3-Chloro-4-methoxyphenyl)-1H-1,2,3-triazol-4-yl]thio-
phene-2-sulfonamide (16):
Compound 16 was prepared according to the general procedure
from 4 (0.10 g, 0.53 mmol), 4-azido-2-chloro-1-methoxybenzene43
(0.10 g, 0.53 mmol), CuSO4Á5H2O (0.17 g, 0.68 mmol), sodium
ascorbate (0.42 g, 2.14 mmol) and AcOH (0.61 mL, 10.68 mmol)
as a light brown solid (0.20 g, 90%). Mp 262–263 °C. IR (KBr,
cmÀ1
)
mmax
:
3332 (NH), 1336 (S@O), 1156 (S@O); 1H NMR
cmÀ1 max: 2229 (C„N), 2133 (N3); 1H NMR (400 MHz, DMSO-
) m
(400 MHz, DMSO-d6) d: 3.95 (s, 3H), 7.39 (d, J = 9.0 Hz, 1H), 7.47
(d, J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.78 (s, 2H), 7.90 (dd,
J = 9.0, 2.7 Hz, 1H), 8.05 (d, J = 2.7 Hz, 1H), 9.31 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 56.7, 113.6, 120.4, 120.5, 121.9, 122.0,
d6) d: 7.61–7.66 (m, 2H), 8.17 (d, J = 8.2 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 103.7 (q, J = 2.2 Hz), 115.4, 118.3 (q, J =
4.8 Hz), 122.1 (q, J = 274 Hz), 123.7, 132.5 (q, Jv32.7 Hz), 137.0,
145.8.), CuSO4Á5H2O (0.34 g, 1.36 mmol), sodium ascorbate

J. Leitans et al. / Bioorg. Med. Chem. 21 (2013) 5130–5138
5137
(0.85 g, 4.27 mmol) and AcOH (1.22 mL, 21.36 mmol) as a yellow
solid (0.31 g, 73%). Mp 254–255 °C. IR (KBr, cmÀ1
max: 3368
References and notes
)
m
(NH), 3276 (NH), 2230 (CN), 1340 (S@O), 1138 (S@O); 1H NMR
(400 MHz, DMSO-d6) d: 7.52 (d, J = 3.9 Hz, 1H), 7.62 (d, J = 3.9 Hz,
1H), 7.81 (s, 2H), 8.45–8.56 (m, 3H), 9.65 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 108.2, 115.0, 118.5 (q, J = 4.9 Hz), 120.9,
122.0 (q, J = 274 Hz), 124.1, 124.7, 131.0, 132.7 (q, J = 32.7 Hz),
136.0, 137.6, 139.4, 142.0, 145.0; HRMS (ESI) [M+H]+: m/z Calcd
for (C14H9F3N5O2S2) 400.0150. Found 400.0161.
1. a Aggarwal, M.; Kondeti, B.; McKenna, R. Bioorg. Med. Chem. 2013, 21, 1526; b
Aggarwal, M.; McKenna, R. Expert Opin. Ther. Pat. 2012, 22, 903; c Supuran, C. T.;
Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (d) Pastorekova, S.;
Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19,
199; e Jain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D. W. J. Med.
Chem. 1994, 37, 2100; f Baranauskiene, L.; Hilvo, M.; Matuliene, J.; Golovenko,
D.; Manakova, E.; Dudutiene, V.; Michailoviene, V.; Torresan, J.; Jachno, J.;
Parkkila, S.; Maresca, A.; Supuran, C. T.; Grazulis, S.; Matulis, D. J. Enzyme Inhib.
Med. Chem. 2010, 25, 863.
2. a Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; b Supuran, C. T. Front.
Pharmacol. 2011, 2, 34; c Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27,
759.
4.2. Protein production and purification
3. a Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem.
Rev. 2012, 112, 4421; b Capasso, C.; De Luca, V.; Carginale, V.; Cannio, R.; Rossi,
M. J. Enzyme Inhib. Med. Chem. 2012, 27, 892.
4. a Alber, B. E.; Ferry, J. G. Proc. Natl. Acad. Sci. USA 1994, 91, 6909; b Kisker, C.;
Schindelin, H.; Alber, B. E.; Ferry, J. G.; Rees, D. C. EMBO J. 1996, 15, 2323; c
Zimmerman, S. A.; Tomb, J. F.; Ferry, J. G. J. Bacteriology 2010, 192, 1353.
5. a Ferry, J. G. Bioorg. Med. Chem. 2013, 21, 1392; b Tripp, B. C.; Bell, C. B., 3rd;
Cruz, F.; Krebs, C.; Ferry, J. G. J. Biol. Chem. 2004, 279, 6683.
hCA I, hCA II, hCA IX and hCA XII were produced and purified as
described earlier by our groups.8,9,11
4.3. CA inhibition assay
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.31 Phenol
red (at a concentration of 0.2 mM) has been used as indicator, work-
ing at the absorbance maximum of 557 nm, with 20 mM Hepes (pH
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the io-
nic 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 deter-
mined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in dis-
tilled-deionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. 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 non-linear least-squares
methods using PRISM 3, as reported earlier,8 and represent the
mean from at least three different determinations. All CA isofoms
were recombinant ones obtained in-house as reported earlier.8,9,11
6. a Viparelli, F.; Monti, S. M.; De Simone, G.; Innocenti, A.; Scozzafava, A.; Xu, Y.;
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V.; Langella, E.; Viparelli, F.; Vullo, D.; Ascione, G.; Dathan, N. A.; Morel, F. M.
M.; Supuran, C. T.; De Simone, G.; Monti, S. M. Biochimie 2012, 94, 1232.
7. Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C.
T. Biochemistry 1997, 36, 10384.
8. a Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med. Chem. 2010, 53, 5511; b Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.;
Mühlschlegel, F. A.; Vullo, D.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med.
Chem. 2011, 54, 1682; c Kazancıog˘lu, E. A.; Güney, M.; Sßentürk, M.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2012, 27, 880.
9. Tars, K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis,
R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2013, 56, 293.
10. a Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 1583; b Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg.
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C. T. Bioorg. Med. Chem. 2010, 18, 14.
11. a Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.;
Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; b Maresca, A.;
Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2010, 53, 335.
12. a Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
7255; b Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.;
Dedhar, S.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2011, 54, 8271; c Bonneau,
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28, 397.
13. a Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2012,
20, 2266; b Davis, R. A.; Vullo, D.; Maresca, A.; Supuran, C. T.; Poulsen, S. A.
Bioorg. Med. Chem. 2013, 21, 1539.
14. a Vullo, D.; Luca, V. D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.;
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15. a Di Fiore, A.; Maresca, A.; Alterio, V.; Supuran, C. T.; De Simone, G. Chem.
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Expert Opin. Ther. Pat. 2012, 22, 747.
4.4. Crystallization and data collection
Protein was concentrated to 10.8 mg/ml in 20 mM tris–HCl pH
8.0 using 10 kDa cutoff Amicon concentrator. Crystallization was
done by sitting drop technique in 96-well MRC plates (Molecular
Dimensions). 1
l
l of protein was mixed with 1
l
l of bottom solu-
l of
tion, (1.5 M Na citrate, 80 mM tris–HCl pH 9.0) and 0.2
l
16. a Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C.
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2012, 55, 1721.
100 mM inhibitor in 100% DMSO. The obtained crystals were
flash-frozen in liquid nitrogen. Data were collected at beamline
I911-3, MAX-lab synchrotron, Lund, Sweden.
4.5. Structure determination
17. Carta, F.; Akdemir, A.; Scozzafava, A.; Masini, E.; Supuran, C. T. J. Med. Chem
2013, 56, 4691.
Images were processed by MOSFLM44 and scaled by SCALA.45
The structure was refined by REFMAC46 using unliganded CA II mu-
tant (PDB code 3DC3)9 as an initial model. The parameter files for
sulfonamides 10 and 13 were generated by LIBCHECK.47 The ligand
was fitted in electron density in COOT (Emsley and Cowtan
2004),48 followed by further REFMAC runs. Data scaling, refine-
ment, and validation statistics are listed in Table 1. Atomic coordi-
nates and structure factors were deposited in PDB with accession
code 4bf1 and 4bf6.
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2012, 48, 8838.
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Acknowledgment
This work was financed by two FP7 EU grant (METOXIA and
DYNANO).

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