Exploring structural properties of potent human carbonic anhydrase inhibitors bearing a 4-(cycloalkylamino-1-carbonyl)benzenesulfonamide moiety

Article abstract of DOI:10.1016/j.ejmech.2018.11.073

Guided by the crystal structure of 4-(3,4-dihydroquinolin-1(2H)-ylcarbonyl)benzenesulfonamide 3 in complex with hCA II (PDB code 4Z0Q), a novel series of cycloalkylamino-1-carbonylbenzenesulfonamides was designed and synthesized. Thus, we replaced the qui

Full text of DOI:10.1016/j.ejmech.2018.11.073

European Journal of Medicinal Chemistry 163 (2019) 443e452
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Exploring structural properties of potent human carbonic anhydrase
inhibitors bearing a 4-(cycloalkylamino-1-carbonyl)
benzenesulfonamide moiety
,
,
Maria Rosa Buemi a 1, Anna Di Fiore b 1, Laura De Luca a, Andrea Angeli c,
Francesca Mancuso a, Stefania Ferro a, Simona Maria Monti b, Martina Buonanno b,
Emilio Russo d, Giovanbattista De Sarro d, Giuseppina De Simone b, Claudiu T. Supuran c,
,
*
Rosaria Gitto a
a
ꢀ
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Universita degli Studi di Messina, Viale Palatucci, Polo
didattico SS, Annunziata, 98168, Messina, Italy
b Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, 80134, Napoli, Italy
c
ꢀ
Dipartimento NEUROFARBA, Universita di Firenze, Via Ugo Schiff 6, 50019, Sesto Fiorentino, Italy
d Pharmacology Chair, Dept. of Science of Health School of Medicine, University of Catanzaro, Campus Universitario "Salvatore Venuta", Viale Europa - Loc.
Germaneto, 88100, Catanzaro, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Guided by the crystal structure of 4-(3,4-dihydroquinolin-1(2H)-ylcarbonyl)benzenesulfonamide 3 in
complex with hCA II (PDB code 4Z0Q), a novel series of cycloalkylamino-1-carbonylbenzenesulfonamides
was designed and synthesized. Thus, we replaced the quinoline ring with an azepine/piperidine/piper-
azine nucleus and introduced further modifications on cycloalkylamine nucleus by means the installa-
tion of hydrophobic/hydrophilic functionalities able to establish additional contacts in the middle area of
the enzyme cavity. Among the synthesized compounds, the derivatives 7a, 7b, 8b exhibited a remarkable
inhibition for hCA II and the brain-expressed hCA VII in subnanomolar range. The binding of these
molecules to the target enzymes was characterized by means of a crystallographic analysis, providing a
clear snapshot of the most important interactions established by this class of inhibitors into the hCA II
and hCA VII catalytic site. Notably, our results showed that the benzylpiperazine tail of compound 8b is
oriented both in hCA II and in hCA VII toward a poorly explored region of the active site. These features
should be further investigated for the design of new isoform selective CA inhibitors.
Received 7 September 2018
Received in revised form
13 November 2018
Accepted 29 November 2018
Available online 1 December 2018
Keywords:
Carbonic anhydrases
CA inhibitors
Benzenesulfonamides
X-ray crystallography
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
four different classes: cytosolic (CA I, II, III, VII and XIII), mito-
chondrial (CA VA and VB), secretory (CA VI), and membrane asso-
Human Carbonic Anhydrases (hCAs, EC 4.2.1.1) are zinc-
containing enzymes, which catalyze a very simple reaction: the
reversible hydration of carbon dioxide in bicarbonate and proton
[1]. Twelve catalytically active isoforms have been so far identified
that based on the different cellular distribution can be grouped in
ciated (CA IV, IX, XII and XIV) CAs [2e5]. These isoforms differ also
for catalytic efficiency and tissue distribution [6]. Several CA-
isoforms play important roles in different pathological processes
related to cancer, epilepsy, obesity, glaucoma, etc [7e14]. Therefore,
these enzymes have become relevant targets for the design of in-
hibitors with biomedical applications. In details, hCA II and hCA VII
are involved in GABA-mediated neuronal excitation [15], hCA IX
and hCA XII have been characterized as tumor markers [8,10],
whereas hCA XIV seems to play an important role in modulating
excitatory synaptic transmission [16]. Four different classes of CA
inhibitors (CAIs) [1] have been characterized so far: molecules that
bind to the catalytic zinc ion, molecules anchoring to the zinc-
coordinated water molecule/hydroxide ion, compounds occluding
Abbreviations: AAZ, acetazolamide; CA, carbonic anhydrase; CAI, carbonic
anhydrase inhibitors; PTZ, pentylenetetrazole; TPM, topiramate.
* Corresponding author.
E-mail address: rgitto@unime.it (R. Gitto).
These authors contributed equally to the work. The manuscript was written
1
through contributions of all authors. All authors have given approval to the final
version of the manuscript.
https://doi.org/10.1016/j.ejmech.2018.11.073
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

444
M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
2. Results and discussion
2.1. Chemistry
By coupling the commercially available 4-sulfamoylbenzoic acid
(4) and suitable cycloalkylamines, the title 4-(piperidine-1-
carbonyl)benzenesulfonamides (5 a-e), 4-(azepane-1-carbonyl)
benzenesulfonamide (6a), 4-(4-phenylpiperidine-1-carbonyl)ben-
zenesulfonamides (7 a-f), 4-(4-benzylpiperazine-1-carbonyl)ben-
zenesulfonamides (8 a-c), and 4-(4-phenylpiperazine-1-carbonyl)
benzenesulfonamide (8 d) have been readily obtained (Scheme 1).
This reaction was generally carried out at room temperature, in
alkaline medium and in presence of N,N,N,N-tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (pathway
ii) except for compounds 5a and 5d for which carbonylimidazole
has been used (pathway i) to optimize the yield. The chemical
characterization of arylsulfonamides 5 a-e, 6a, 7 a-f, and 8 a-d was
supported by elemental analyses and spectroscopic measurements.
1H NMR spectra data of representative compounds are shown in
Supporting Material. In the 1H NMR spectra of all studied com-
pounds the aromatic and aliphatic protons were observed in the
expected regions. A distinguished feature of these spectra was the
presence of the typical broad single peaks which were observed for
cycloakylamine protons. NMR signals for four protons of benze-
nesulfonamide motif appeared as doublets at 7.5 and 7.8 ppm while
the two protons of eSONH2 group was observed as broad single
peak at 7.4 ppm.
Chart 1. Chemical structures of well-known hCAIs (1e2) and 4-(3,4-dihydroquinolin-
1(2H)-ylcarbonyl)benzenesulfonamide (3).
the active site entrance, and compounds binding out of the active
site. Among them, the most investigated CAIs are those possessing
a sulfonamide/sulfamate zinc binder group (ZBG), of which acet-
azolamide (AAZ, 1) and topiramate (TPM, 2) (Chart 1) are two
clinically used representatives.
It is well known that the presence of benzenesulfonamide as
ZBG provides potent CAIs, whereas the affinity towards the
different isoforms can be tuned by the nature of additional chem-
ical fragment linked to benzenesulfonamide moiety [17]. In the
course of our efforts to identify novel selective CAIs we have syn-
2.2. CA inhibition assay
The CA inhibitory effects of all synthesized compounds 5 a-e, 6
thesized and tested
a
series of quinolone/isoquinoline-
arylsulfonamides showing high affinity toward hCAs [11,18e26].
Among them, we have recently reported the discovery of a set of
heterocyclic-N-carbonylbenzenesulfonamides as a class of potent
inhibitors of druggable CA isoforms (hCA II, hCAVII, hCA IX and hCA
XIV) as found for our prototype 4-(3,4-dihydroquinolin-1(2H)-
ylcarbonyl)benzenesulfonamide 3 (Chart 1) [18,23,26,27]. To deci-
pher the mode of action of these molecules we analyzed the crystal
structures of several derivatives in complex with hCA II, thus
identifying the network of interactions in the CA cavity [17].
Interestingly, the high resolution crystal structure of the hCA II/3
adduct (PDB code 4Z0Q) revealed that the benzenesulfonamide
portion of the inhibitor is well anchored to the active site cavity
establishing many stabilizing interactions with enzyme residues,
whereas the heterocyclic fragment shows a high level of confor-
mational flexibility. With the aim to improve our knowledge about
the structure-affinity relationships (SARs) of CAIs bearing a ben-
zenesulfonamide moiety, we designed a new class of compounds
strictly related to the prototype 3, containing azepine/piperidine/
piperazine nucleus in place of the quinoline ring. Our design was
based on the visual inspection of the binding pose in the hCA II
active site for compound 3 for which the heterocyclic portion was
positioned into the hydrophobic region formed by residues Phe131
and Pro202. Therefore, we have decorated the cycloalkylamine core
with hydrophobic substituents able to establish additional contacts
in the middle area of CA cavity thus controlling affinity and isoform
selectivity toward several druggable CA isoforms, for which we
addressed our biological screening by a stopped flow CO2 hydration
assay. Then, X-ray crystallography has been employed to get in-
formation on action mechanism of the newly designed molecules.
Finally, we evaluated the ability of the most promising and selective
hCA VII inhibitors to exert effects in pentylenetetrazole (PTZ)
seizure tests as an in vivo model of epilepsy.
Scheme 1. Reagents and conditions: i) carbonylimidazole (DCI), THF, r.t., 3h, then
RR’NH, DMF r.t., 2h; ii) RR’NH, HBTU, DMF, TEA, r.t., overnight.

M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
445
Table 1
structural modifications was observed for inhibitors 7 a-f. The
screening of piperazinyl derivatives 8 a-d revealed that all com-
pounds generally displayed inhibitory effects towards studied iso-
forms at nanomolar concentration. It appears that the installation
of a 4-fluorobenzyl moiety optimizes the affinity toward hCA I and
hCA II isoforms for compound 8b.
Ki values (nM) against hCA I, hCA II, hCA VII, hCA IX, hCA XII and hCA XIV isoforms
showed by derivatives 3, 5 a-e, 6a, 7a-f, 8a-d, AAZ 1 and TPM 2.
Ki (nM)a
hCA I
hCA II
hCA VII
hCA IX
hCA XII
hCA XIV
5a
5b
5c
5d
5e
6a
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
1
9.2
4.4
5.7
0.63
6.1
0.79
1.5
3.8
0.5
0.6
0.6
0.7
0.6
0.6
3.0
0.5
5.7
2.0
12.1
10
8.3
4.6
3.1
2.6
2.4
3.4
7.0
16.8
3.3
22.6
24.9
4.7
18.4
3.1
33.1
45.1
28.2
4.8
25.8
58
7.9
7.8
4.5
3.1
9.4
2.7
6.6
7.8
8.5
8.1
6.4
59.4
3.8
5.6
8.5
7.3
5.7
ND
ND
23.8
31.4
41.7
13.7
53.7
37.9
46.9
52.5
7.8
2.3. In vivo testing
187
7.7
75.5
85.0
1.7
9.3
6.6
6.3
6.5
16.5
6.9
0.63
13.3
0.45
2.6
To evaluate the in vivo effects of the most efficacious inhibitors 7a,
7d, 8b, and 8c against the brain expressed hCAVII isoform, we carried
out additional pharmacological studies. Specifically, we studied the
ability of these compounds to prevent seizures in mice using pen-
tylenetetrazole (PTZ) as chemical convulsant agent. The four selected
compounds were tested at the doses of 20 and 30 mg/kg that were
chosen according to the knowledge that the ED50 of TPM (2) and AAZ
(1) in this model are comprised between 22 and 27 mg/kg [28]. Un-
fortunately, none of the tested compounds was able to prevent the
occurrence of clonus, whereas 2 protects against PTZ-induced shocks
and convulsions [28]. At the dose of 20 mg/kg of tested compounds,
some signs of sedation were observed, and this effect was confirmed
at the higher dose of 30 mg/kg by which animals were clearly
sedated. We thought that this event could be related to a toxic effect,
therefore no higher doses were used. Considering latency to clonus
(time to reach a stage 3 seizure after PTZ administration), all com-
pounds were able to increase significantly (p < 0.05) this parameter,
although no significant differences were observed at the doses and
between the tested molecules. All tested compounds seem to have
some effects on neuronal activity considering the signs of sedation
and the increase in latency; however, the unexpected in vivo effects
limitfurtherdetailedstudiesinthis model. The lack ofanticonvulsant
effect suggests that the hCA VII inhibitory properties do not produce
antiepileptic activity for this class of compounds at tested doses.
However, wecannotexcludethatapharmacologicaleffectcouldhave
onset at higher concentrations.
1.0
0.55
0.68
4.4
10.4
7.8
7.8
9.5
2.5
0.9
7.7
11.0
29.8
34.6
15.2
42.8
41.5
41.0
1460
39.0
7.7
6.8
0.69
50.1
91.1
250
250
78.6
2
3
6.8
8.5
9.4
a
Errors in the range of 10% of the reported value, from 3 different assays. Re-
combinant full-length hCA I, hCA II and hCA VII and catalytic domains of hCA IX, hCA
XII and hCA XIV were used.
a, 7 a-f, and 8 a-d were measured for human CA I, CA II, CAVII, CA IX,
CA XII and CA XIV by a stopped flow CO2 hydration assay. The ob-
tained results are summarized in Table 1 and compared with Ki
values of prototype 3 and two well-known inhibitors AAZ 1 and
TPM 2 as reference compounds. As shown in Table 1, the four 4-
(piperidine-1-carbonyl)benzenesulfonamides (5a-e) and the su-
perior cyclohomologous 4-(azepane-1-carbonyl)benzenesulfona-
mide (6a) displayed significant inhibitory effects towards all
studied CA isoforms when compared to well-known inhibitors 1
and 2.
They generally demonstrated high inhibitory activity at low
nanomolar concentration, except for outlier compounds 5c, 5e and
6a against hCA I. Moreover, these compounds reported in Table 1
were poorer inhibitors of hCA XIV in respect to other studied iso-
forms. By deleting the benzene fused ring of prototype 3 we
increased the affinity toward hCA II by an order of magnitude for 4-
(2-methylpiperidine-1-carbonyl)benzenesulfonamide (5b) and 4-
(4-methylpiperidine-1-carbonyl)benzenesulfonamide (5d), sug-
gesting an optimized orientation within hCA II active site cavity.
Notably, for the 4-(4-benzylpiperidine-1-carbonyl)benzenesulfo-
namide (5e) an improvement of affinity toward druggable isoform
hCA VII has been found (Ki value of 0.63 nM), thus suggesting a
crucial role of additional 4-benzylpiperidine group to benzene-
sulfonamide moiety to address hCA VII selectivity.
As shown in Table 1, by incorporating a phenyl moiety at C-4
position of piperidine nucleus, a further increased affinity against
hCA II was observed for compound 7a in comparison with analo-
gous 5a (Ki values of 0.5 vs 5.7 nM) and hCA VII (Ki values of 0.45 nM
vs 8.3 nM). By introducing additional hydroxyl group, nitrile or
acetyl functionalities at C-4 position, no significant improvement of
hCA II inhibition was observed; as result the SAR analysis was very
flat for compounds 7 a-f as hCA II inhibitors. In contrast, the affinity
against hCA VII was optimized for compounds 7d (R1 ¼ OH and
R2 ¼ 4-BrC6H4) and 7e (R1 ¼ CN and R2 ¼ Ph) showing Ki values of
0.55 and 0.68 nM, respectively.
2.4. Structural analysis
To better characterize the inhibition mechanism of the newly
designed and synthesized compounds, we solved the crystal
structures of adducts that some representative molecules of the
series, namely 7a, 7b and 8b, form with isozymes hCA II and hCA
VII. The data collection and refinement statistics for these six crystal
structures are summarized in Table 2 and deposited in to PDB under
codes 6H2Z, 6H33, 6H34, 6H36, 6H37 and 6H38.
Since compounds 7a-7f displayed very high inhibitory activity
against hCA II and hCA VII with respect to the prototype 3, we
initially solved the crystallographic structure of the lead compound
7a in complex with both enzymes. Inspection of (Fo-Fc) and (2Fo-
Fc) electron density maps at various stages of the crystallographic
refinement showed features compatible with the presence of
compound 7a within the active site of both hCA II and hCA VII
(Fig. 1A and B).
These maps were very well defined in both cases, thus indi-
cating absence of inhibitor flexibility within enzyme active sites,
differently from what observed for the prototype 3 within the hCA
II active site [17]. As commonly observed for sulfonamide-based
hCA inhibitors, in both adducts compound 7a was anchored to
the catalytic zinc ion via the primary sulfonamide group, displacing
the zinc-bound solvent molecule and forming hydrogen bond in-
teractions with Thr199 (Fig. 2A and B) [1]. The benzene-
sulfonamide core established strong hydrophobic interactions
(distance <4.0 Å) with residues His94, Leu198 and Val121 in the
case of hCA II and with His94, Val121 Leu198 and Thr200 in the case
of hCA VII (Fig. 2A and B).
Notably, compounds 7a, 7d and 7e demonstrated high inhibi-
tory effects toward brain-distributed hCA VII with Ki values com-
parable to those of the anticonvulsant agent TPM 2. For other
studied isoforms, no clear correlation between Ki values and

446
M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
Table 2
Data collection statistics and refinement statistics.
hCA II in complex with
hCA VII in complex with
7a
7b
8b
7a
7b
8b
Cell parameters
Space group
P21
P21
P21
P21212
P21212
P21212
Cell dimensions (Å,ꢀ)
a ¼ 42.4
b ¼ 41.1
c ¼ 71.7
a ¼ 42.3
b ¼ 41.4
c ¼ 72.0
a ¼ 42.3
b ¼ 41.3
c ¼ 72.1
a ¼ 66.0
b ¼ 88.1
c ¼ 44.0
a ¼ 66.0
b ¼ 88.6
c ¼ 44.1
a ¼ 66.1
b ¼ 89.2
c ¼ 44.2
b
1
¼ 104.2
b
1
¼ 104.2
b
1
¼ 104.3
Number of independent molecules
Data collection statistics
Resolution limits (Å)
Total reflections
Unique reflections
Redundancy
1
1
1
34.7e1.94
70015
17564
29.1e1.58
136858
30160
29.1e1.55
121412
33156
31.1e1.85
91414
21202
30.9e1.90
69324
19303
34.0e1.70
138766
28527
4.0
4.5
3.7
4.3
3.6
4.9
Completeness (%)
98.6 (90.2)
0.114 (0.418)
0.131 (0.521)
0.063 (0.306)
11.5 (2.3)
90.7 (77.2)
0.045 (0.147)
0.050 (0.173)
0.021 (0.089)
29.9 (9.0)
93.8 (82.5)
0.055 (0.228)
0.062 (0.280)
0.028 (0.161)
21.8 (4.4)
94.1 (88.9)
0.067 (0.459)
0.074 (0.573)
0.032 (0.338)
18.0 (2.2)
92.1 (69.4)
0.046 (0.181)
0.052 (0.213)
0.024 (0.108)
22.3 (7.4)
96.6 (90.2)
0.046 (0.503)
0.051 (0.590)
0.021 (0.301)
27.3 (2.6)
R-merge*
Rmeasx
Rpim¶
<I>/<s(I)>
Refinement statistics
Resolution limits (Å)
R-work** (%)
34.7e1.94
17.9
21.4
29.1e1.58
16.6
19.4
29.1e1.55
17.1
20.6
31.1e1.85
19.5
23.3
30.9e1.90
19.4
23.6
34.0e1.70
19.3
24.5
R-free** (%)
r.m.s.d. from ideal geometry:
Bond lengths (Å)
Bond angles (ꢀ)
0.006
1.4
0.008
1.5
0.009
1.6
0.008
1.5
0.007
1.5
0.006
1.5
Number of protein atoms
Number of inhibitor atoms
Number of water molecules
Average B factor (Å2)
All atoms
Protein atoms
Inhibitor atoms
Waters
PDB accession code
2039
24
158
2093
25
225
2063
26
229
2066
24
116
2057
25
115
2055
26
144
16.04
15.42
16.97
23.30
6H2Z
12.51
11.52
15.50
21.33
6H33
11.88
10.78
20.29
20.57
6H34
22.91
22.61
21.45
28.68
6H36
19.45
19.22
19.60
23.68
6H37
21.27
20.75
19.92
28.32
6H38
*R-merge ¼ ShklSijIi(hkl)-<I(hkl)>j/ShklSiIi(hkl), where Ii(hkl) is the intensity of an observation and <I(hkl)> is the mean value for its unique reflection; summations are over all
reflections;
xRmeas ¼ Shkl{N(hkl)/[N(hkl)-1]}1/2xSijIi(hkl)-<I(hkl)>j/ShklSiIi(hkl);
¶Rpim ¼ Shkl{1/[N(hkl)-1]}1/2xSijIi(hkl)-<I(hkl)>j/ShklSiIi(hkl);
**R-
work ¼ ShkljjFo(hkl)j ꢁ jFc(hkl)jj/ShkljFo(hkl)j calculated for the working set of reflections. R-free is calculated as for R-work, but from data of the test set that was not used for
refinement (Test Set Size (%) ¼ 5.7% for hCA II/7a, 3.5% for hCA II/7b, 3.0% for hCA II/8b, 5.0% for hCA VII/7a and hCA VII/7b, 4.0% for hCA VII/8b). Values in parentheses are
referred to the highest resolution shell (2.01e1.94 Å for hCA II/7a, 1.64e1.58 Å for hCA II/7b and 1.61e1.55 Å for hCA II/8b, 1.92e1.85 Å for hCA VII/7a, 1.97e1.90 Å for hCA VII/
7b, 1.76e1.70 Å for hCA VII/8a).
Moreover, the carbonyl group was hydrogen bonded to Gln92 in
the hCA VII active site, but not in the case of hCA II. Finally, 4-
phenylpiperidine substituent was oriented both in hCA II and in
hCA VII toward the hydrophobic region of the active site estab-
lishing many hydrophobic interactions with enzyme residues.
Overall, the interactions established by 7a with the two isoforms
were very similar, with the exception of the hydrogen bond, which
the inhibitor formed with Gln92 in hCA VII. However, it appears
that this hydrogen bond does not contribute significantly to the
binding affinity since the Ki values of the inhibitor against the two
enzymes are comparable. We also studied compound 7b bearing an
additional hydroxyl group at C-4 position to respect parent com-
pound 7a; it has been found that the installation of a polar moiety
did not cause significant changes in the interaction mode of the
inhibitor with hCA II and hCA VII active sites (Fig. 1C, D, 2C and 2D)
when compared with compound 7a. This evidence results in good
agreement with the very similar Ki values against hCA II (0.5 nM
and 0.6 nM for 7a and 7b, respectively); whereas the slight diver-
gence found for Ki values against hCAVII (0.45 nM and 2.6 nM for 7a
and 7b, respectively) are not consistent with their very similar
binding pose within the hCA II and hCAVII active sites. Thus, we can
speculate that the higher affinity of 7a with respect to 7b for the
brain enzyme has to probably be ascribed to other molecular fac-
tors that cannot be detected by crystallographic studies.
molecules of the series, namely compound 8b, in complex with hCA
II and hCA VII. Surprisingly, despite kinetic experiments indicated
for this molecule a higher binding affinity for hCA II with respect to
hCA VII, the electron density maps of the inhibitor were better
defined in the case of the adduct with the brain enzyme with
respect to the adduct with the ubiquitous hCA II (Fig. 1E and F). The
analysis of the structures of the two adducts provided an expla-
nation to this apparent inconsistency. Indeed, in both cases the
inhibitor is bound to the enzyme active site establishing the ca-
nonical interactions of the benzenesulfonamide moiety with CA
active site. Moreover, polar and hydrophobic interactions between
the phenyl-carbonyl-piperazine moieties and residues which
delimit the cavity (see Fig. 3A and B) are detectable. However, in the
case of hCA VII/8b adduct, additional interactions are established
between the inhibitor and residue R159 of a symmetry-related
molecule. Thus, it is evident that the lower conformational flexi-
bility of 8b in hCA VII active site is related to the crystal packing and
thus it is not present in solution.
Finally, it is worth noting that both in hCA II/8b and in hCA VII/
8b the benzylpiperazine tail is oriented toward a rather unexplored
region of the active site located at the border of its hydrophobic
portion (Fig. 4) [29]. Indeed, it has been reported that the majority
of hCAIs orient their tail toward either the hydrophobic or the
hydrophilic region of the catalytic cavity [1,33]. Due to this unusual
feature, these piperazine-derivatives should be further investigated
to address selectivity issues.
Finally, to investigate on the binding mode of the piperazine
derivatives we solved the structure of one of the most active

M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
447
Fig. 1. Electron density of inhibitors. Sigma-A weighted j2Fo-Fcj simulated annealing omit map of inhibitors bound in hCA II and hCA VII active sites. A) hCA II/7a, B) hCA VII/7a, C)
hCA II/7b, D) hCA VII/7b, E) hCA II/8b and F) hCA VII/8b. Zinc ion is represented as a gray sphere. The figure is contoured to the 1.0
s level and was generated by using PyMOL (https://
pymol.org).

448
M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
Fig. 2. Piperidine-1-carbonyl-benzenesulfonamides bound to the active site of hCA II (green) and hCA VII (cyan). A and B: compound 7a (magenta). C and D: compound 7b (salmon).
Residues involved in hydrophobic interactions and hydrogen bonds (black dashed lines) are shown. The figure was made using PyMol. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)
3. Conclusions
from common commercial suppliers. Microwave-assisted reactions
were carried out in a Focused Microwawe TM Synthesis System,
Model Discover (CEM Technology Ltd Buckingham, UK). Melting
points were determined on a Buchi B-545 apparatus (BUCHI
Labortechnik AG Flawil, Switzerland) and are uncorrected. By
combustion analysis (C, H, N) carried out on a Carlo Erba Model
1106-Elemental Analyzer we determined the purity of synthesized
compounds; the results confirmed a ꢂ95% purity. Merck Silica Gel
60 F254 plates were used for analytical TLC (Merck KGaA, Darm-
stadt, Germany). For detection, iodine vapor and UV light (254 nm)
were used. Flash Chromatography (FC) was carried out on a Biotage
SP1 EXP (Biotage AB Uppsala, Sweden). 1H NMR and 13C NMR
spectra were measured in dimethylsulfoxide-d6 (DMSO‑d6) and
CDCl3 with a Varian Gemini 300 spectrometer (Varian Inc. Palo Alto,
In conclusion, a series of new 4-(cycloalkylamino-1-carbonyl)
benzenesulfonamide derivatives was synthesized and tested by a
stopped flow CO2 hydration assay. The most potent compounds
demonstratedexcellentinhibitoryeffectsagainstseveralCAisozymes
at subnanomolar concentration. The findings were corroborated by
crystallographic structures of the hit compounds in complex with
hCAII and hCA VII isoforms. Our structural investigation confirmed
that the combination of the crucial benzenesulfonamide moiety with
a suitable aromatic tail can offer the optimization of affinity toward
both isoforms. Notably, we found that the rim of catalytic cavity of
these isoforms might be exploited to reinforce the network of in-
teractions. Overall, we envision that these structural information for
hCA VII adducts will furnish suggestions for design new potent and
selective inhibitors against this brain-expressed CA isoform.
California USA); chemical shifts are expressed in
d (ppm) and
coupling constants (J) in hertz. All exchangeable protons were
confirmed by addition of D2O. Rf values were determined on TLC
plates using a mixture of DCM/MeOH (96/4) as eluent.
4. Experimental section
4.1. Chemistry
4.1.1. Synthetic procedures for compounds 5 a-e, 6a, 7 a-f, 8 a-d
Pathway i: To a solution of 4-(aminosulfonyl)benzoic acid (4)
(6 mmol) in THF (15 mL) the carbonylimidazole (6 mmol) (CDI) at
All reagents were used without further purification and bought

M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
449
Fig. 3. . (4-Fluorophenyl-piperazine-1-carbonyl)benzenesulfonamide 8b (orange) bound to the active site of hCA II (green) (A) and hCA VII (cyan) (B). Residues involved in hy-
drophobic interactions and hydrogen bonds (black dashed lines) are shown. In (B) residue R159 belonging to a symmetry-related molecule is colored in violet. The figure was made
by using PyMol. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Pathway ii: A mixture of 4-(aminosulfonyl)benzoic acid (4)
(2 mmol) and N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)ura-
nium hexafluorophosphate (HBTU) (2 mmol) in DMF(2 mL) was
stirred at room temperature for 1 h. Then, a solution of the
appropriate amine derivative (2 mmol) in TEA (2 mmol) was added
dropwise. The reaction mixture was left overnight and then
quenched with water (10 mL) and extracted with EtOAc (3 ꢃ 5 mL).
The organic phase was dried with Na2SO4 and the solvent was
removed in vacuo. The residue was purified by flash chromatog-
raphy (DCM/MeOH 96:4), crystallized by treatment with a mixture
of Et2O and EtOH (1:1) to give the desired final compounds 5b, 5c,
6a, 7a-f and 8a-d as white crystals. For compounds 5 a-d, 6a, 8a and
8d registered CAS numbers have been already assigned. However,
their synthetic procedures, chemical properties and structural
characterization are not available in literature.
4.1.1.1. 4-(Piperidine-1-carbonyl)benzenesulfonamide
(5a);CAS:4302-78-7. Yield: 40%; M.p.: 206-207ꢀC; Rf ¼ 0.45.1H NMR
(DMSO‑d6): (
d
) 1.43e3.57 (m, 10H), 7.45 (bs, 2H, NH2), 7.53 (d, J¼8.2,
2H, ArH). 7.83 (d, J¼8.2, 2H, ArH). Anal. Calcd for C12H16N2O3S: C:
53.71; H:6.01; N:10.44. Found: C:53.81; H: 6.11; N 10.54.
4.1.1.2. 4-(2-Methylpiperidine-1-carbonyl)benzenesulfonamide
(5b);CAS 1097096-08-6. Yield: 60%; M.p.: 186e187 ꢀC; Rf ¼ 0.47.1H
NMR (DMSO‑d6): (
d
) 1.15 (d, J ¼ 7.1, 3H, CH3),1.43e3.40 (m, 9H), 7.42
Fig. 4. Structural superposition of compound 8b when bound to hCA II (green) with
the same compound when bound to hCA VII (magenta). The surface representation of
hCA II is also shown with hydrophobic region of the active site in red and the hy-
drophilic one in blue. The figure was made using PyMol. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of
this article.)
(bs, 2H, NH2), 7.50 (d, J¼8.2, 2H, ArH). 7.83 (d, J¼8.2, 2H, ArH). Anal.
Calc for C13H18N2O3S: C:55.30; H: 6.43; N:9.92. Found: C:55.35;
H:6.40; N:9.97.
4.1.1.3. 4-(3-Methylpiperidine-1-carbonyl)benzenesulfonamide
(5c);CAS 4367-75-3. Yield: 35%; M.p.: 190e191 ꢀC; Rf ¼ 0.49.1H
NMR (DMSO‑d6): (
d
) 1.06 (d, J ¼ 7.1, 3H, CH3), 1.10e2.94 (m, 9H),
0 ꢀC was added. The obtained mixture was stirred at room tem-
perature for 3h and then the appropriate amine derivative
(15 mmol) in DMF (5 mL) was added dropwise. The reaction
mixture was stirred at room temperature for 2h. The solvent was
removed in vacuo; by adding of aqueous solution of NaHCO3 (5 mL)
we obtained compounds 5a and 5d as crude products, which were
purified through crystallization by a mixture of Et2O and EtOH
(1:1).
7.43 (bs, 2H, NH2), 7.52 (d, J¼8.2, 2H, ArH), 7.84 (d, J¼8.2, 2H, ArH).
Anal. Calcd for C13H18N2O3S: C:55.30; H: 6.43; N:9.92. Found:
C:55.40; H:6.53; N:10.02.
4.1.1.4. 4-(4-Methylpiperidine-1-carbonyl)benzenesulfonamide
(5d);CAS
1096951e92e6. Yield: 40%; M.p.: 212e213 ꢀC;
Rf ¼ 0.51.1H NMR (DMSO‑d6): (
) 0.90 (d, J ¼ 7.0, 3H, CH3), 1.06e4.44
d
(m, 9H), 7.44 (bs, 2H, NH2), 7.53 (d, J¼8.2, 2H, ArH). 7.84 (d, J¼8.2,

450
M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
2H, ArH). Anal. Calcd for C13H18N2O3S: C:55.30; H:6.43; N:9.92.
Found: C:55.40; H:6.40; N:9.80.
C:60.15; H:5.89; N:11.69. Found: C:60.25; H:5.99; N:11.99.
4.1.1.14. 4-[4-[(4-Fluorophenyl)methyl]piperazine-1-carbonyl]benze-
4.1.1.5. 4-(4-Benzylpiperidine-1-carbonyl)benzenesulfonamide (5e).
nesulfonamide (8b). Yield 30%; M.p. 206e207 ꢀC; Rf ¼ 0.45.1H NMR
Yield 44%; M.p.: 156e157 ꢀC; Rf ¼ 0.59.1H NMR (DMSO‑d6): (
d)
(CDCl3): (d) 2.35e3.62 (m, 8H), 2.49 (s, 2H, CH2Ph), 7.11e7.36 (m,
1.12e1.66 (m, 4H), 2.52 (m, 2H, CH2Ph), 2.62e2.44 (m, 5H),
7.15e7.16 (m, 3H, ArH), 7.24e7.27 (m, 2H, ArH), 7.42 (bs, 2H, NH2),
7.52 (d, J¼8.2, 2H, ArH), 7.84 (d, J¼8.2, 2H, ArH). Anal. Calcd for
4H, ArH), 7.46 (bs, 2H, NH2), 7.57 (d, J¼8.2, 2H, ArH), 7.85 (d, J¼8.2,
2H, ArH). Anal. Calcd for C18H20FN3O3S: C:57.28; H:5.34; N:11.13.
Found: C:57.55; H:5.43; N:11.34.
C
19H22N2O3S: C:63.66; H:6.19; N:7.81. Found: C:63.56; H:6.09;
N:7.71.
4.1.1.15. 4-(4-Benzhydrylpiperazine-1-carbonyl)benzenesulfonamide
(8c). Yield 60%; M.p.: 228e230 ꢀC; Rf ¼ 0.58.1H NMR (DMSO‑d6):
4.1.1.6. 4-(Azepane-1-carbonyl)benzenesulfonamide
(6a)CAS
(d) 2.26e4.32 (m, 9H), 7.16e7.39 (m, 10H, ArH), 7.41(bs, 2H, NH2),
1015627e49e2. Yield: 30%; M.p.: 200e201 ꢀC; Rf ¼ 0.53.1H NMR
7.52 (d, J¼8.2, 2H, ArH), 7.81 (d, J¼8.2, 2H, ArH). 13C NMR
(DMSO‑d6): (
d
) 1.50e3.53 (m, 12H), 7.43 (bs, 2H, NH2), 7.51 (d, J¼7.6,
(DMSO‑d6): (d) 172.8, 149.9, 147.6, 144.14, 133.8, 132.8, 132.19,
2H, ArH). 7.83 (d, J ¼ 7.6, 2H, ArH). Anal. Calcd for C13H18N2O3S:
131.08, 130.8, 79.9, 79.8. Anal. Calcd for C24H25N3O3S: C:66.18;
H:5.79; N:9.65. Found: C:66.28; H:5.69; N:9.55.
C:55.30; H:6.43; N:9.92. Found: C:55.40; H: 6.53, N:9.82.
4.1.1.7. 4-(4-Phenylpiperidine-1-carbonyl)benzenesulfonamide (7a).
4.1.1.16. 4-(4-Phenylpiperazine-1-carbonyl)benzenesulfonamide
Yield: 73%; M.p.: 222e223 ꢀC; Rf ¼ 0.55.1H NMR (DMSO‑d6): (
d)
(8d);
CAS:1015405e80e7. Yield 84%; M.p.: 231e232 ꢀC;
1.86e4.60 (m, 9H), 7.18e7.28 (m, 5H, ArH), 7.45 (s, 2H, NH2), 7.61 (d,
J¼8.00, 2H, ArH), 7.86 (d, J¼8.00, 2H, ArH). Anal. Calcd for
Rf ¼ 0.59.1H NMR (DMSO‑d6): (
d) 3.10e3.89 (m, 8H), 6.80e7.24 (m,
5H, ArH), 7.42 (bs, 2H, NH2), 7.58 (d, J¼8.2, 2H, ArH), 7.84 (d, J¼8.2,
2H, ArH). Anal. Calcd for C17H19N3O3S: C:59.11; H:5.54; N:12.16.
Found: C:58.81; H:5.24; N:12.06.
C
18H20N2O3S: C:62.77; H:5.85; N:8.13. Found: C:63.10; H:5.65;
N:8.47.
4.1.1.8. 4-(4-Hydroxy-4-phenylpiperidine-1-carbonyl)benzenesulfo-
4.2. CA inhibition assay
namide (7b). Yield: 34%; M.p.: 135e137 ꢀC; Rf ¼ 0.37.1H NMR
(DMSO‑d6): (
d
) 1.51e4.45 (m, 8H), 5.20 (bs, 1H, OH), 7.18e7.32 (m,
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity. Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 10e20 mM
Hepes (pH 7.5) or Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or
20 mM NaClO4 (for maintaining constant the ionic strength),
following the initial rates of the CA-catalysed CO2 hydration reac-
tion for a period of 10e100 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 5e10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same
manner and subtracted from the total observed rates. Stock solu-
tions of inhibitor (10 mM) were prepared in distilled-deionized
water and dilutions up to 0.01 nM were done thereafter with
distilled-deionized water. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to
assay, 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, and represent the mean
from at least three different determinations. CA isoforms were re-
combinant ones obtained as reported earlier by this group [30e32].
3H, ArH), 7.45 (bs, 2H, NH2), 7.51 (m, 2H, ArH), 7.63 (d, J¼8.2, 2H,
ArH), 7.86 (d, J¼8.2, 2H, ArH). Anal. Calcd for C18H20N2O4S: C:59.98;
H:5.59; N:7.77. Found: C:60.18; H:5.79; N:7.87.
4.1.1.9. 4-[4-(4-Chlorophenyl)-4-hydroxypiperidine-1-carbonyl]ben-
zenesulfonamide (7c). Yield: 31%; M.p.: 215e217 ꢀC; Rf ¼ 0.38.1H
NMR (DMSO‑d6): (d) 1.52e4.44 (m, 8H), 5.31 (bs, 1H, OH), 7.36 (d,
J¼8.5, 2H, ArH), 7.44 (bs, 2H, NH2), 7.54 (d, J¼8.5, 2H, ArH), 7.63 (d,
J¼7.6, 2H, ArH), 7.86 (d, J¼7.6, 2H, ArH). Anal. Calcd For
C
18H19ClN2O4S: C:54.75; H:4.85; N:7.09. Found: C:54.93; H:5,13; N:
7.37.
4.1.1.10. 4-[4-(4-Bromophenyl)-4-hydroxy-piperidine-1-carbonyl]
benzenesulfonamide
(7d). Yield:
35%;
M.p.:
219e220 ꢀC;
Rf ¼ 0.40.1H NMR (DMSO‑d6): (
d) 1.48e4.45 (m, 8H), 5.31 (s, 1H,
OH), 7.44 (bs, 2H, NH2), 7.49e7.52 (m, 4H, ArH), 7.65 (d, J¼8.8, 2H,
ArH), 7.87 (d, J¼8.8, 2H, ArH). Anal. Calcd for C18H19BrN2O4S:
C:49.21; H:4.36; N:6.38. Found: C:49.58; H:4.67; N:6.44.
4.1.1.11. 4-(4-Cyano-4-phenylpiperidine-1-carbonyl)benzenesulfona-
mide (7e). Yield: 22%; M.p.: 285e286 ꢀC; Rf ¼ 0.59.1H NMR
(DMSO‑d6): (d) 2.07e4.67 (m, 8H), 7.35e7.42 (m, 3H, ArH), 7.42 (bs,
2H, NH2), 7.45e7.47 (m, 4H, ArH), 7.66 (d, J¼8.2, 2H, ArH), 7.87 (d,
J¼8.2, 2H, ArH). Anal. Calcd for C19H19N3O3S: C: 61.77; H:5.18;
N:11.37. Found: C:6.83; H:5.47; N:11.62.
4.3. Anticonvulsant test
Male ICR CD-1 mice (n ¼ 80) were purchased from Charles River
Laboratories s.r.l. (Calco, Lecco, Italy). Animals were housed four/
five per cage and kept under controlled environmental conditions
(60 5% humidity; 22 2 ꢀC; 12/12 h reversed light/dark cycle;
lights on at 20.00). Animals were allowed free access to standard
laboratory chow and tap water. The study was approved by the local
ethics committee and all procedures involving animals and their
care were in compliance with international and national regula-
tions (EU Directive 2010/63/EU for animal experiments, ARRIVE
guidelines and the Basel declaration including the 3R concept).
Male ICR CD-1 mice (30e35 g, 35e40 days old) were pretreated
with vehicle or drugs (groups of 10 mice per dose) 30 min before
the i.p. administration of pentylenetetrazole (PTZ) 60 mg/kg
(inducing clonus in 80% of mice) in order to test the efficacy against
4.1.1.12. 4-(4-Acetyl-4-phenyl-piperidine-1-carbonyl)benzenesulfo-
namide (7f). Yield: 20%; M.p.: 233e234 ꢀC; Rf ¼ 0.52.1H NMR
(DMSO‑d6): (d) 1.89 (s, 3H, CH3), 1.96e3.92 (m, 8H), 7.28e7.38 (m,
5H, ArH), 7.44 (bs, 2H, NH2), 7.55 (d, J¼8.2, 2H, ArH), 7.83 (d, J¼8.2,
2H, ArH). Anal. Calcd for C20H22N2O4S: C:62.16; H:5.74; N:7.25.
Found: C:62.10; H:5.84; N:7.15.
4.1.1.13. 4-(4-Benzylpiperazine-1-carbonyl)benzenesulfonamide
(8a); CAS 1032228e25e3. Yield 40%; M.p.: 204e205 ꢀC;
Rf ¼ 0.43.1H NMR (DMSO‑d6): (
d) 2.32e3.60 (m, 8H), 3.47 (s, 2H,
CH2Ph), 7.22e7.26 (m, 5H, ArH), 7.44 (bs, 2H, NH2), 7.56 (d, J¼8.2,
2H, ArH), 7.83 (d, J¼8.2, 2H, ArH). Anal. Calcd for C18H21N3O3S:

M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
451
clonus, as previously described [28].
Appendix A. Supplementary data
4.4. X-ray crystallography
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.11.073.
Crystals of the hCA II-7b and hCA II-8b adducts were obtained
by co-crystallization. In particular, complexes were prepared by
adding a 5 M excess of the inhibitor to a 10 mg/mL protein solution.
This mixture, equilibrated for 1 h at room temperature, was used
for the crystallization experiments. Drops were prepared by mixing
References
[1] V. Alterio, A. Di Fiore, K. D'Ambrosio, C.T. Supuran, G. De Simone, Multiple
binding modes of inhibitors to carbonic anhydrases: how to design specific
drugs targeting 15 different isoforms? Chem. Rev. 112 (2012) 4421e4468.
[2] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for in-
hibitors and activators, Nat. Rev. Drug Discov. 7 (2008) 168e181.
[3] C. Lomelino, R. McKenna, Carbonic anhydrase inhibitors: a review on the
progress of patent literature (2011-2016), Expert Opin. Ther. Pat. 26 (2016)
947e956.
[4] C.T. Supuran, Advances in structure-based drug discovery of carbonic anhy-
drase inhibitors, Expet Opin. Drug Discov. 12 (2017) 61e88.
[5] C.T. Supuran, G. De Simone, Carbonic Anhydrases as Biocatalysts: from Theory
to Medical and Industrial Applications, Elsevier, 2015.
1 ml of enzyme/inhibitor solution with 1 ml of precipitant solution
containing 1.3 M sodium citrate, 0.1 M TriseHCl, pH 8.5 and further
equilibrated over a well containing 1 mL of precipitant buffer.
Crystals appeared in the drops within 2e3 days and grew to a
maximum dimension of 0.2 ꢃ 0.3 ꢃ 0.2 mm3 in about one week.
Crystals of the hCA II-7a adduct were instead obtained by using the
soaking technique. In particular, native hCA II crystals were grown
at room temperature by the hanging drop vapor diffusion tech-
nique using a protein concentration of 10 mg/mL and 1.3 M sodium
citrate, 0.1 M TriseHCl, pH 8.5 as precipitant solution. A few hCA II
[6] C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist?
J. Enzym. Inhib. Med. Chem. 31 (2016) 345e360.
[7] A. Waheed, W.S. Sly, Carbonic anhydrase XII functions in health and disease,
Gene 623 (2017) 33e40.
crystals were then transferred in a 2 ml drop of freshly prepared
[8] C.T. Supuran, V. Alterio, A. Di Fiore, D.A. K, F. Carta, S.M. Monti, G. De Simone,
Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and
cancer stem cells: three for the price of one, Med. Res. Rev. 38 (2018)
1799e1836. https://doi.org/10.1002/med.21497.
[9] V. Alterio, D. Esposito, S.M. Monti, C.T. Supuran, G. De Simone, Crystal struc-
ture of the human carbonic anhydrase II adduct with 1-(4-sulfamoylphenyl-
ethyl)-2,4,6-triphenylpyridinium perchlorate, a membrane-impermeant, iso-
form selective inhibitor, J. Enzym. Inhib. Med. Chem. 33 (2018) 151e157.
[10] S.M. Monti, C.T. Supuran, G. De Simone, Anticancer carbonic anhydrase in-
hibitors: a patent review (2008 - 2013), Expert Opin. Ther. Pat. 23 (2013)
737e749.
[11] E. Bruno, M.R. Buemi, L. De Luca, S. Ferro, A.M. Monforte, C.T. Supuran,
D. Vullo, G. De Sarro, E. Russo, R. Gitto, Vivo evaluation of selective carbonic
anhydrase inhibitors as potential anticonvulsant agents, ChemMedChem 11
(2016) 1812e1818.
[12] C.T. Supuran, Carbonic anhydrase inhibitors as emerging drugs for the treat-
ment of obesity, Expet Opin. Emerg. Drugs 17 (2012) 11e15.
[13] C.T. Supuran, A. Di Fiore, G. De Simone, Carbonic anhydrase inhibitors as
emerging drugs for the treatment of obesity, Expet Opin. Emerg. Drugs 13
(2008) 383e392.
precipitant solution containing also the inhibitor at the concen-
tration of 2.5 mM and glycerol (15% v/v) as cryoprotectant. These
crystals were kept in the soaking solution for one night and finally
flash-frozen in liquid nitrogen. A very similar experimental strategy
was used to obtain crystals of the hCA VIIeinhibitor complexes. For
crystallographic studies a mutated form of hCA VII, where the
cysteine residues in position 183 and 217 were mutated to serines,
was used, since we previously reported that this mutant was more
suitable for crystallization experiments [33,34] hCA VII crystals
were grown at room temperature by the vapor diffusion hanging
drop method. Equal volumes of protein (5 mg/mL in 0.02 M Tris-HCl
pH 8.0 and 0.1 M NaCl) and precipitant solutions (25% v/v Peg3350,
0.2 M Ammonium acetate and 0.1 M Tris pH 8.5) were mixed and
equilibrated against 1 mL reservoir containing the same precipitant
solution. A few crystals of hCA VII were then transferred into a 2 ml
[14] G. De Simone, A. Scozzafava, C.T. Supuran, Which carbonic anhydrases are
targeted by the antiepileptic sulfonamides and sulfamates? Chem. Biol. Drug
Des. 74 (2009) 317e321.
[15] E. Ruusuvuori, K. Kaila, Carbonic anhydrases and brain pH in the control of
neuronal excitability, Subcell. Biochem. 75 (2014) 271e290.
[16] J.R. Casey, W.S. Sly, G.N. Shah, B.V. Alvarez, Bicarbonate homeostasis in
excitable tissues: role of AE3 Cl-/HCO3- exchanger and carbonic anhydrase
XIV interaction, Am. J. Physiol. Cell Physiol. 297 (2009) C1091eC1102.
drop of the freshly prepared precipitant solution containing the
inhibitor at a concentration of 40 mM for inhibitors 7a and 7b and
10 mM for 8b. Moreover, 25% (v/v) glycerol was used as cryopro-
tectant agent. These crystals were kept in the soaking solution
overnight and then flash-frozen in a gaseous nitrogen stream prior
to the diffraction experiment. All X-ray data sets were collected at
100 K by a copper rotating anode generator developed by Rigaku
and equipped with a Rigaku Saturn CCD detector. Diffraction data
were indexed, integrated and scaled using the HKL2000 software
package [35]. Data collection statistics are reported in Table 2. The
initial phases of all structures were calculated using the atomic
coordinates of hCA VII and hCA II (PDB accession code 6G4T and
1CA2 for hCAVII and II, respectively) with waters removed [34e36].
The structures were refined using the CNS program [37,38] as
previously described [39e41], whereas model building and map
inspections were performed using the program O [42]. Topology
files for inhibitors 7a, 7b e 8b generated using the PRODRG server
[43].
[17] M.R. Buemi, L. De Luca, S. Ferro, E. Bruno, M. Ceruso, C.T. Supuran,
ꢁ
ꢁ
ꢂ
ꢂꢁ
ꢂ
K. Pospísilova, J. Brynda, P. Rezacova, R. Gitto, Carbonic anhydrase inhibitors:
design, synthesis and structural characterization of new heteroaryl-N-
carbonylbenzenesulfonamides targeting druggable human carbonic anhy-
drase isoforms, Eur. J. Med. Chem. 102 (2015) 223e232.
[18] L. De Luca, F. Mancuso, S. Ferro, M.R. Buemi, A. Angeli, S. Del Prete, C. Capasso,
C.T. Supuran, R. Gitto, Inhibitory effects and structural insights for a novel
series of coumarin-based compounds that selectively target human CA IX and
CA XII carbonic anhydrases, Eur. J. Med. Chem. 143 (2018) 276e282.
[19] E. Bruno, M.R. Buemi, A. Di Fiore, L. De Luca, S. Ferro, A. Angeli, R. Cirilli,
D. Sadutto, V. Alterio, S.M. Monti, C.T. Supuran, G. De Simone, R. Gitto, Probing
molecular interactions between human carbonic anhydrases (hCAs) and a
novel class of benzenesulfonamides, J. Med. Chem. 60 (2017) 4316e4326.
[20] L. De Luca, S. Ferro, F.M. Damiano, C.T. Supuran, D. Vullo, A. Chimirri, R. Gitto,
Structure-based screening for the discovery of new carbonic anhydrase VII
inhibitors, Eur. J. Med. Chem. 71 (2014) 105e111.
[21] R. Gitto, F.M. Damiano, P. Mader, L. De Luca, S. Ferro, C.T. Supuran, D. Vullo,
J. Brynda, P. Rezacova, A. Chimirri, Synthesis, structure-activity relationship
studies, and X-ray crystallographic analysis of arylsulfonamides as potent
carbonic anhydrase inhibitors, J. Med. Chem. 55 (2012) 3891e3899.
[22] R. Gitto, F.M. Damiano, L. De Luca, S. Ferro, D. Vullo, C.T. Supuran, A. Chimirri,
Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as
selective carbonic anhydrase inhibitors, Bioorg. Med. Chem. 19 (2011)
7003e7007.
Notes
The authors declare no competing financial interest.
Acknowledgments
[23] P. Mader, J. Brynda, R. Gitto, S. Agnello, P. Pachl, C.T. Supuran, A. Chimirri,
P. Rezacova, Structural basis for the interaction between carbonic anhydrase
and 1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamides, J. Med. Chem. 54 (2011)
2522e2526.
[24] R. Gitto, S. Agnello, S. Ferro, D. Vullo, C.T. Supuran, A. Chimirri, Identification of
potent and selective human carbonic anhydrase VII (hCA VII) inhibitors,
ChemMedChem 5 (2010) 823e826.
Financial support for this research by by Fondo di Ateneo per la
ꢀ
Ricerca (PRA grant number ORME09SPNC - Universita degli Studi di
ꢀ
Messina. We thank Mr. Maurizio Amendola and Mr. Giosue Sor-
rentino for their skillful technical assistance with X-ray
measurements.
[25] R. Gitto, S. Agnello, S. Ferro, L. De Luca, D. Vullo, J. Brynda, P. Mader,

452
M.R. Buemi et al. / European Journal of Medicinal Chemistry 163 (2019) 443e452
C.T. Supuran, A. Chimirri, Identification of 3,4-Dihydroisoquinoline-2(1H)-
[34] M. Buonanno, A. Di Fiore, E. Langella, K. D'Ambrosio, C.T. Supuran, S.M. Monti,
G. De Simone, The crystal structure of a hCA VII variant provides insights into
the molecular determinants responsible for its catalytic behavior, Int. J. Mol.
Sci. 19 (2018).
sulfonamides as potent carbonic anhydrase inhibitors: synthesis, biological
evaluation, and enzyme–ligand X-ray studies, J. Med. Chem. 53 (2010)
2401e2408.
[26] R. Gitto, S. Ferro, S. Agnello, L. De Luca, G. De Sarro, E. Russo, D. Vullo,
C.T. Supuran, A. Chimirri, Synthesis and evaluation of pharmacological profile
of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamides, Bio-
org. Med. Chem. 17 (2009) 3659e3664.
[27] R. Gitto, F.M. Damiano, L. De Luca, S. Ferro, D. Vullo, C.T. Supuran, A. Chimirri,
Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as
selective carbonic anhydrase inhibitors, Bioorg. Med. Chem. 19 (2011)
7003e7007.
[35] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in
oscillation mode, Methods Enzymol. 276 (1997) 307e326.
[36] A.E. Eriksson, T.A. Jones, A. Liljas, Refined structure of human carbonic
anhydrase II at 2.0 A resolution, Proteins 4 (1988) 274e282.
[37] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-
Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice,
T. Simonson, G.L. Warren, Crystallography & NMR system: a new software
suite for macromolecular structure determination, Acta Crystallogr. Sect. D
Biol. Crystallogr. 54 (1998) 905e921.
[28] E. Russo, A. Constanti, G. Ferreri, R. Citraro, G. De Sarro, Nifedipine affects the
anticonvulsant activity of topiramate in various animal models of epilepsy,
Neuropharmacology 46 (2004) 865e878.
[38] A.T. Brunger, Version 1.2 of the crystallography and NMR system, Nat. Protoc.
2 (2007) 2728e2733.
[29] G. De Simone, V. Alterio, C.T. Supuran, Exploiting the hydrophobic and hy-
drophilic binding sites for designing carbonic anhydrase inhibitors, Expet
Opin. Drug Discov. 8 (2013) 793e810.
[30] A. Innocenti, D. Vullo, J. Pastorek, A. Scozzafava, S. Pastorekova, I. Nishimori,
C.T. Supuran, Carbonic anhydrase inhibitors. Inhibition of transmembrane
isozymes XII (cancer-associated) and XIV with anions, Bioorg. Med. Chem. Lett
17 (2007) 1532e1537.
[39] A. Di Fiore, A. Maresca, V. Alterio, C.T. Supuran, G. De Simone, Carbonic
anhydrase inhibitors: X-ray crystallographic studies for the binding of N-
substituted benzenesulfonamides to human isoform II, Chem. Commun. 47
(2011) 11636e11638.
[40] A. Di Fiore, A. Maresca, C.T. Supuran, G. De Simone, Hydroxamate represents a
versatile zinc binding group for the development of new carbonic anhydrase
inhibitors, Chem. Commun. 48 (2012) 8838e8840.
[31] I. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mastrolorenzo,
C.T. Supuran, Carbonic anhydrase inhibitors: inhibition of the transmembrane
isozyme XIV with sulfonamides, Bioorg. Med. Chem. Lett 15 (2005)
3828e3833.
[32] I. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mastrolorenzo,
C.T. Supuran, Carbonic anhydrase inhibitors. The mitochondrial isozyme VB as
a new target for sulfonamide and sulfamate inhibitors, J. Med. Chem. 48
(2005) 7860e7866.
[41] A. Di Fiore, A. Vergara, M. Caterino, V. Alterio, S.M. Monti, J. Ombouma,
P. Dumy, D. Vullo, C.T. Supuran, J.Y. Winum, G. De Simone, Hydroxylamine-O-
sulfonamide is a versatile lead compound for the development of carbonic
anhydrase inhibitors, Chem. Commun. 51 (2015) 11519e11522.
[42] T.A. Jones, J.Y. Zou, S.W. Cowan, M. Kjeldgaard, Improved methods for building
protein models in electron density maps and the location of errors in these
models, Acta Crystallogr. Section A, Found. Crystallogr. 47 (Pt 2) (1991)
110e119.
[33] A. Di Fiore, E. Truppo, C.T. Supuran, V. Alterio, N. Dathan, F. Bootorabi,
S. Parkkila, S.M. Monti, G. De Simone, Crystal structure of the C183S/C217S
mutant of human CA VII in complex with acetazolamide, Bioorg. Med. Chem.
Lett 20 (2010) 5023e5026.
[43] A.W. Schuttelkopf, D.M. van Aalten, PRODRG: a tool for high-throughput
crystallography of protein-ligand complexes, Acta Crystallogr. Sect. D Biol.
Crystallogr. 60 (2004) 1355e1363.