In Vivo Evaluation of Selective Carbonic Anhydrase Inhibitors as Potential Anticonvulsant Agents

Article abstract of DOI:10.1002/cmdc.201500596

Epilepsy is a common neurological disorder caused by an imbalance between inhibitory and excitatory neurotransmission. It is well known that neuronal excitability is related to γ-aminobutyric acid (GABA)ergic depolarization. HCO₃^?-dependent depolarization can be suppressed by membrane-permeable inhibitors of carbonic anhydrase. We previously identified some isoquinoline sulfonamides as potent and selective inhibitors of the human carbonic anhydrase II and VII (hCA II and hCA VII) isoforms. Given that hCA II and hCA VII are specific isoforms involved in GABA-mediated neuronal excitation, we hypothesized that they could represent the biological target for the development of new anticonvulsant agents. Therefore, for selected isoquinoline sulfonamides, we preliminarily tested their ability to prevent audiogenic seizures in DBA/2 mice. All compounds were evaluated after intraperitoneal administration, and some of them proved to protect the mice against convulsions. Among this series of compounds, several derivatives showed combined in vivo efficacy with inhibitory effects toward the targeted carbonic anhydrases (i.e., hCA II and hCA VII). Specifically, the most interesting molecule was 1-(4-aminophenyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamide (6), which proved to be a more active and selective hCA VII inhibitor than the reference compound topiramate. Further studies to explore the in vivo pharmacokinetic profile of the most active compounds may help to provide insight into the future design of selective hCA VII inhibitors.

Full text of DOI:10.1002/cmdc.201500596

DOI: 10.1002/cmdc.201500596
Full Papers
In Vivo Evaluation of Selective Carbonic Anhydrase
Inhibitors as Potential Anticonvulsant Agents
Elvira Bruno,*[a] Maria R. Buemi,[a] Laura De Luca,[a] Stefania Ferro,[a] Anna-Maria Monforte,[a]
Claudiu T. Supuran,[b] Daniela Vullo,[b] Giovambattista De Sarro,[c] Emilio Russo,[c] and
Rosaria Gitto*[a]
Dedicated to Prof. Alba Chimirri on the occasion of her retirement
Epilepsy is a common neurological disorder caused by an im-
balance between inhibitory and excitatory neurotransmission.
It is well known that neuronal excitability is related to g-amino-
ity to prevent audiogenic seizures in DBA/2 mice. All com-
pounds were evaluated after intraperitoneal administration,
and some of them proved to protect the mice against convul-
sions. Among this series of compounds, several derivatives
showed combined in vivo efficacy with inhibitory effects
toward the targeted carbonic anhydrases (i.e., hCA II and
hCA VII). Specifically, the most interesting molecule was 1-(4-
aminophenyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sul-
fonamide (6), which proved to be a more active and selective
hCA VII inhibitor than the reference compound topiramate.
Further studies to explore the in vivo pharmacokinetic profile
of the most active compounds may help to provide insight
into the future design of selective hCA VII inhibitors.
À
butyric acid (GABA)ergic depolarization. HCO3 -dependent de-
polarization can be suppressed by membrane-permeable in-
hibitors of carbonic anhydrase. We previously identified some
isoquinoline sulfonamides as potent and selective inhibitors of
the human carbonic anhydrase II and VII (hCA II and hCA VII)
isoforms. Given that hCA II and hCA VII are specific isoforms in-
volved in GABA-mediated neuronal excitation, we hypothe-
sized that they could represent the biological target for the de-
velopment of new anticonvulsant agents. Therefore, for select-
ed isoquinoline sulfonamides, we preliminarily tested their abil-
Introduction
Epilepsy is one the most common neurological disorders that
can be related to an imbalance between inhibitory and excita-
tory neurotransmission. Marketed antiepileptic drugs (AEDs)
predominantly target voltage-gated cation channels, inhibit
glutamatergic neurotransmission, or improve g-aminobutyric
acid (GABA)-mediated inhibition.[1] These AEDs generally offer
appreciable advantages in terms of their pharmacokinetic pro-
files, tolerability, and lower potential for drug–drug interac-
tions. However, approximately 30% of patients with epilepsy
still suffer from seizures. Therefore, there is a substantial need
for the development of more efficacious AEDs for patients
with refractory seizures. Recent progress in the identification of
new potential molecular targets has allowed the development
of new and more efficacious AEDs.[2,3]
It is well known that GABAergic transmission can exert both
seizure-suppressing and seizure-promoting actions.[4,5] Activa-
tion of the GABAA receptor opens channels that are permeable
to ClÀ and to a lesser extent to HCO3À. Owing to the activity of
a specific chloride-ion transporter, the intracellular concentra-
tion of ClÀ is generally low, so the GABAA-receptor-mediated
current results in hyperpolarization. However, depolarizing
À
GABA effects contribute to HCO3 efflux. Specifically, pro-
[a] Dr. E. Bruno, Dr. M. R. Buemi, Prof. L. De Luca, Dr. S. Ferro,
Prof. A.-M. Monforte, Prof. R. Gitto
longed activation of GABAA receptors can lead to an excess
amount of ClÀ ions in postsynaptic neurons, and this decreases
the driving force of the ClÀ ions. Under these conditions, the
Department of Chemical, Biological, Pharmaceutical, and Environmental
Sciences (CHIBIOFARAM)
University of Messina, Viale Annunziata, 98168 Messina (Italy)
E-mail: brunoe@unime.it
À
HCO3 current dominates; this depolarizes the postsynaptic
À
neurons, as intracellular HCO3 is consistently replenished by
rgitto@unime.it
the activity of carbonic anhydrase.[6] Thus, HCO3À-dependent
depolarization can be suppressed by membrane-permeable in-
hibitors of carbonic anhydrase (CA). By this mechanism, brain
CAs are known to modulate neuronal signaling. The CAs are
metalloenzymes that catalyze the reversible hydration of
carbon dioxide to bicarbonate and a proton, so they are also
known as carbonate dehydratases. The family of human car-
bonic anhydrases (hCAs, EC 4.2.1.1) comprises 16 different a-
CA isoforms, of which there are several cytosolic isoforms (i.e.,
[b] Prof. C. T. Supuran, Dr. D. Vullo
Neurofarba: Department of Neuroscience, Psychology, Pharmaceuticals and
Child Health, Section of Pharmaceutical and Nutraceutical Sciences
University of Florence, 50019 Sesto Fiorentino, Florence (Italy)
[c] Prof. G. De Sarro, Prof. E. Russo
Science of Health Department, School of Medicine, University of Catanzaro
Viale Europa Localitꢀ Germaneto, 88100 Catanzaro (Italy)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201500596.
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CA I–III, CA VII, and CA XIII), five membrane-bound isoforms
(i.e., CA IV, CA IX, CA XII, CA XIV, and CA XV), two mitochondrial
isoforms (i.e., CA VA and CA VB), and one secreted into saliva
(i.e., CA VI). The hCAs are involved in various physiological pro-
cesses, such as gluconeogenesis, lipogenesis, and ureagenesis.
However, abnormal levels or activities of this family of enzymes
have often been associated with different diseases such as
cancer, epilepsy, obesity, glaucoma, and so on.[7–12] Particularly,
hCA II and hCA VII enhance bicarbonate-driven GABAergic exci-
tation during intense GABAA-receptor activation. Several CA in-
hibitors (CAIs, Figure 1) such as topiramate (TPM, 1), acetazol-
amide (AAZ, 2), and zonisamide (3) have a long history as anti-
convulsants, but their molecular targets and mechanisms of
action at the neuronal network level are still poorly under-
stood.
The catalytic domain of all human CAs contains a common
motif including a zinc(II) ion that is essential for catalysis and
located at the bottom of a deep cleft.[13] The active zinc ion is
coordinated by three histidine residues (i.e., His94, His96, and
His119) and water/hydroxide ions react with carbon dioxide to
give bicarbonate. The classical CAIs such as compounds 1–3
(Figure 1) contain a sulfamide, sulfonamide, or sulfamate
moiety able to coordinate the zinc ion within the catalytic
binding site, which thus blocks the enzymatic activity of the
CA.[14–27] X-ray crystallography has confirmed that the selectivity
of the hCA isoform is generally controlled by the remaining
molecular frame.[26,28–35] Therefore, the high selectivity toward
several isoforms is conditioned by favorable interactions be-
tween the hydrophobic/hydrophilic residues on the CA cleft
and suitable functional groups of the most selective
hCAIs.[36–40]
A large amount of heterocyclic compounds containing a sul-
fonamide moiety have been extensively studied as CAIs.[41,42]
Structure–affinity relationships have demonstrated that both
the inhibitory effect and the isoform selectivity are dependent
on the nature of the heterocyclic moiety targeting the hydro-
philic and hydrophobic walls of the catalytic site.[42] We previ-
Figure 1. Carbonic anhydrase inhibitors as anticonvulsants.
Table 1. Inhibition of hCA II and VII isoforms and anticonvulsant effects in DBA/2 mice of test compounds 4–22 and reference compound TPM (1).
[b]
Compd
Structure
R3
Ki [nm][a]
ED50 [mmol(kgi.p.)À1
]
R1
R2
R4
hCA II
hCA VII
Clonic
>100[43]
Tonic
4
5
6
7
8
9
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
OMe
OMe
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
Cl
NH2
H
Cl
NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
H
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
H
15700[43]
5300[43]
11300[43]
422[46]
7.1
8.2
6.0
302
37.5
7.82 (6.66–9.19)[43]
7.25 (4.54–11.6)[43]
3.60 (1.45–8.97)[43]
82.6 (74.3–91.9)
23.5 (14.4–38.5)[43]
17.7 (9.80–30.1)[43]
93.1 (82.5–105)
104[46]
>100
>100
222[46]
14.8
>100
>100
>100
75.2 (46.9–121)
48.2 (30.3–76.6)
61.3 (45.0–83.4)
39.4 (23.0–67.6)
79.0 (53.9–116)
65.6 (43.6–98.7)
88.2 (55.9–139)
10
11
12
13
14
15
16
17
18
19
20
21
22
TPM (1)
3640[27]
3130[27]
4.6[27]
7.0[27]
5.7[44]
23.4[44]
5.5[27]
5.4[27]
6.4[27]
6.7[27]
16.3[27]
10.2[27]
13.6[27]
71.5[27]
698[27]
0.9[43]
SO2NH2
H
8.8[44]
92.9 (61.2–141)
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
48.5[44]
32.8[27]
94.5[27]
87.3[27]
>100
>100
>100
>100
>100
>100
H
Me
Et
nPr
>100
>100
56.9 (40.9–79.2)
1975[27]
235000[27]
820[27]
cPr
58.0 (39.7–84.7)
63.8 (45.1–90.2)
>100
>100
47.7
37.5 (27.0–52.0)
51.4 (38.5–68.6)
nBu
cPent
cHex
350000[27]
650[27]
>100
18890[27]
10[43]
51.5 (34.1–77.7)
29.2
[a] Errors in the range of Æ10% of the reported value, from three different assays; recombinant full-length hCA II and hCA VII were used. [b] All data were
calculated following the Litchfield and Wilcoxon method;[61] at least 32 animals were used to calculate each ED50 value. 95% confidence limits are given in
parentheses. i.p.: intraperitoneal.
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ously demonstrated that a sulfonamide functional
group linked to an isoquinoline scaffold could consti-
tute a promising template for the development of
CAIs that exert anticonvulsant effects.[40,43] Specifically,
1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-
sulfonamide derivatives 4–6 proved to protect
against audiogenic seizures in DBA/2 mice;[43] as
shown in Table 1, the most active compound, 1-(4-
aminophenyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-
2(1H)-sulfonamide (6), was more efficacious than the
reference compound topiramate (1).[43] The aim of
this work was to assess the anticonvulsants effects of
other isoquinoline sulfonamides already reported as
potent CAIs toward selected druggable CA isoforms.
In this study, we especially focused our attention on
determining the inhibitory effects toward the hCA II
and hCA VII isozymes.
Results and Discussion
Considering that sulfonamide-containing compounds
are potential binders to the catalytic site of CAs,[41,42]
the simplest approach in the design of CA inhibitors
is the introduction of a sulfonamide “warhead” as
a metal-binding group (MBG) targeting zinc ions.
Moreover, by searching for CA isoform selectivity we
designed and synthesized a series of isoquinoline sul-
fonamides. The synthetic procedures employed to
obtain designed isoquinoline–sulfonamides 4–22 as
racemic mixtures are described in Scheme 1.[25,27,44–46]
The experimental details as well as careful structural
Scheme 1. Reagents and conditions: a) microwave: 5 min, 908C, 1.38 MPa, 150 W; b) TFA,
MW: 5 min, 908C, 1.38 MPa, 150 W; c) CH3CH(OCH3)2, NH2SO2NH2, heat, 24–48 h or two
steps under the same conditions: 20 min, 1008C, 1.38 MPa, 150 W; d) HBTU, DMF, NEt3,
RT, 2 h; e) POCl3, heat, toluene, 2 h; f) NaBH4, CH3OH, 2 h. HBTU: 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate.
characterization of compounds 4–22 can be found in the Sup-
porting Information.
peritoneally 30 min before auditory stimulation, and the results
are expressed as ED50 values for the inhibition of clonic and
tonic seizures; the results were compared with the data ob-
tained in the same assay for anticonvulsant topiramate (1),
which is currently employed for the treatment of epileptic seiz-
ures.[43] We found that dimethoxy analogues 4–6 were more ef-
ficacious anticonvulsants than reference compound 1 and
monomethoxy analogues 7–9, which thus suggests that the
in vivo effects might be correlated with the significant effects
of hCA VII inhibition that have been observed for compounds
4–6. Interestingly, most active anticonvulsants 4–6 proved to
be inefficacious to inhibit hCA II (Ki >5000 nm). This unexpect-
ed isoform selectivity might be considered an advantage for
the development of CA inhibitors lacking off-target effects re-
lated to activity toward ubiquitous isoforms such as hCA II. Op-
timal compound 6, which bears a 4-aminophenylgroup at the
C1 position of the isoquinoline scaffold, displayed the highest
selectivity for the hCA VII isoform (~2000-fold) over the hCA II
isoform and showed relevant anticonvulsant effects in compar-
ison with that shown by reference compound 1.
The co-crystal structure of CA inhibitor 16 in complex with
hCA II confirmed that the sulfonamide moiety coordinates to
the zinc ion and interacts with the Thr199 residue located in
the catalytic binding pocket (PDB ID: 3PO6, Figure 2).[26] As
shown in Figure 2, the oxygen atom of the methoxy substitu-
ent at the C6 position of the isoquinoline nucleus forms a H-
bond with the crucial Asn67 residue, whereas the C7 methoxy
group is located in the hydrophobic area lined by the Gln92,
Val121, and Phe131 residues.
Therefore, 1-aryl-6-methoxy-3,4-dihydroisoquinoline-2(1H)-
sulfonamides 7–13 were rationally designed to bind the zinc
ion through the sulfonamide moiety as a MBG. Specifically, we
probed the impact of removing the 7-methoxy substituent
during the recognition process of the enzymatic binding site.
Additionally, we introduced another sulfonamide group as
a MBG in the para position of the C1 phenyl substituent (e.g.,
11 and 13). As shown in Table 1, monomethoxy compounds
7–9 are less efficacious inhibitors against the hCA VII isoform
than corresponding dimethoxy analogues 4–6, which were
proven to be potent hCA VII inhibitors at nanomolar concen-
trations (Ki <8.2 nm).
We also investigated the CA inhibitory effects for isoquino-
lines 10–13 bearing a 4-benzenesulfonamide fragment as the
MBG.[27,44] Consistent with previous results, dimethoxy isoqui-
nolines derivatives 10 and 11 were more selective inhibitors of
hCA VII than monomethoxy analogues 11–13.[44] Despite the
fact that compounds 11–13 were found to be very active
Compounds 4–9 were also tested in vivo, and Table 1 sum-
marizes the anticonvulsant effects against audiogenic seizures
in DBA/2 mice. All tested compounds were administered intra-
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To gain knowledge of the relationships between hCA VII in-
hibition and anticonvulsant properties, we extended the
in vivo screening to nine other isoquinoline sulfonamides that
had exhibited potent inhibitory effects toward hCA VII as well
as high selectivity over hCA II.[25] Specifically, Table 1 displays
the experimental data of in vivo screening for 1-(cyclo)alkyl-
3,4-dihydroisoquinoline-2(1H)-sulfonamides 14–21. In addition,
we evaluated the in vivo effectiveness of weaker hCA VII inhibi-
tor 22. Despite a relevant inhibition of the neuronal hCA VII
isoform (IC50 ꢀ16.3 nm), a low in vivo efficacy was found for
tested isoquinolines 14–20. The most active compounds were
isoquinolines 19 and 20, which proved to be efficacious
against both the clonic and tonic phases of audiogenic seiz-
ures at doses similar to those of topiramate (1). Notably, com-
pound 20 also exhibited excellent selectivity (>2500-fold) for
the hCA VII isoform over the ubiquitous hCA II isoform.
Considering that the presence of a sulfonamide moiety as
a MBG might cause a significant improvement in the hydro-
philic properties of the tested compounds, which would thus
impair their ability to cross the blood–brain barrier (BBB),[47] we
performed an in silico prediction of the physicochemical prop-
erties. Thus, we calculated the logD value (pH 7.4)[48] for each
compound, and the data are displayed in Table 2. As expected
these sulfonamide derivatives generally showed low lipophilici-
ty (<2.5), and the ranking score of their anticonvulsant effects
does not fit with the lipophilic properties. Especially, there is
no correlation between anticonvulsant efficacy and lipophilic
properties of most active anticonvulsants 5, 6, and 19
(logD7.4 =1.81, À0.05, and 0.65, respectively), which thus sug-
gests the role of other biopharmaceutical parameters. It is well
known that other calculated physicochemical properties such
Table 2. Calculated physicochemical properties and lipophilic indices.
[a]
Compd clogD7.4
clogP[a]
PSA [ꢁ2][b] logBB[c] LE[d] LLE[e]
4
5
6
7
8
1.22
1.81
À0.05
1.41
2.00
0.15
1.645Æ0.49
2.410Æ0.50
0.440Æ0.50
2.153Æ0.47
2.918Æ0.48
0.948Æ0.48
1.042Æ0.56
À0.188Æ0.51
1.653Æ0.54
0.320Æ0.49
0.560Æ0.42
0.055Æ0.47
0.653Æ0.48
1.162Æ0.48
1.672Æ0.48
1.044Æ0.48
2.181Æ0.48
2.098Æ0.48
2.625Æ0.48
2.157Æ0.67
90.2
90.2
116
81.0
81.0
À0.95
À0.83
À1.51
À0.73
0.62
0.47 6.92
0.45 6.27
0.46 8.17
0.41 5.11
0.45 5.42
0.48 7.67
0.49 8.32
0.30 8.84
0.52 8.91
0.41 8.13
0.82 8.33
0.64 8.47
0.60 8.48
0.47 7.35
0.45 6.43
0.47 7.34
0.50 5.98
0.43 5.36
0.36 3.81
0.57 6.07
9
107
99.0
159
89.8
150
71.8
90.2
90.2
90.2
90.2
90.2
90.2
90.2
90.2
À1.30
À1.17
À2.24
À1.29
À2.16
À0.84
À1.19
À1.10
À1.02
À0.94
À1.04
À0.86
À0.88
À0.80
À1.36
10
11
12
13
14
15
16
17
18
19
20
21
22
TPM (1)
0.01
À0.69
À0.67
À0.50
0.08
À0.21
0.29
0.82
1.35
0.65
1.88
Figure 2. a) Binding of compound 16 (green) to the hCA II catalytic site. Key
residues of the pocket are presented with PyMOL (http://www.pymol.org).
Hydrogen bonds are illustrated by cyan lines, whereas the zinc metal ion is
shown as a gray sphere. b) Schematic drawing of types of interactions of
compound 16 generated by using LigPlot (see Supporting Information).
Dashed lines are hydrogen bonds, and eyelashes show residues involved in
hydrophobic interactions.
1.78
2.34
2.97
124
[a] LogP and logD7.4 data were predicted from a commercially available
software package; see Ref. [48]. [b] PSA values were predicted by using
suitable software; see Ref. [49]. [c] LogBB=À0.0148PSA+0.152logP+
0.139. (Clark equation); see Ref. [51]. [d] LE=À1.4logKi/(number of heavy
atoms); see Ref. [55]. LLE=ÀlogKiÀlogD7.4; see Refs. [54],[55].
hCA VII inhibitors, they generally failed to prevent seizures in
DBA/2 mice.
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as the polar surface area (PSA),[49] lipophilicity (clogP), and de-
rived values such as logBB[50,51] could be useful parameters to
understand the ability of a small molecule to penetrate the
brain.
lines 5, 6, and 19. We hypothesize that the lack of in vivo effec-
tiveness might be related to poor brain penetration. Further
in-depth studies are in progress to explore the in vivo pharma-
cokinetic profile of the most active compounds.
The in silico determination of these parameters is also sum-
marized in Table 2 for all tested compounds, and the values
are compared to the predicted parameters for reference com-
pound 1. In general, central nervous system (CNS) drugs pos-
sess clogP values ranging from 2 to 4[52] and PSA values
<76 ꢁ2.[53] Moreover, the logBB threshold values were set to
0.3 and À1, for which compounds with logBB>0.3 cross the
BBB easily and compounds with logBB<À1 are poorly brain
distributed. As shown in Table 2, compounds 4, 5, 7, 8, 14, 18,
and 20–22 were estimated to have the ability to cross the
BBB; thus, they possess logBB values within the recommended
threshold (>À1). Among them, we found that sulfonamides 4
and 5 protected DBA/2 mice in tonic seizures. On the contrary,
compounds 6, 9–13, 15–17, and 19 displayed logBB parame-
ters below À1.
Experimental Section
Chemistry
Sulfonamide derivatives 4–6,[43] 7–9,[46] 10 and 11,[27] 12 and 13,[44]
and 14–22[25] were synthesized (Scheme 1) and purified by follow-
ing previously reported procedures, and the spectral data are in ac-
cordance with the literature (see the Supporting Information). All
starting materials and reagents were purchased from Sigma–Al-
drich, Milan, Italy, and were used without further purification. Mi-
crowave-assisted reactions were performed in a CEM focused mi-
crowave synthesis system. Melting points were determined with
a Bꢂchi B-545 apparatus. Combustion analysis (C, H, N) was per-
formed with a Carlo Erba Model 1106 Elemental Analyzer from
which we determined the purities of the synthesized compounds;
the results were within Æ0.4% of the theoretical values. Merck
silica gel 60F254 plates were used for analytical TLC. Rf values were
determined on TLC plates by using a mixture of CH2Cl2/MeOH
(94:6) as the eluent. Flash chromatography (FC) was performed
Therefore, for most interesting anticonvulsant 6 the predic-
tion of brain penetration is not coherent with the in vivo ef-
fects. Considering the failure of the in silico prediction of brain
penetration of active anticonvulsant 6, we can hypothesize
that there are other ADME properties that influence the bio-
availability of these potent hCA VII inhibitors.
with a Biotage SP1 EXP system. H NMR and 13C NMR spectra were
1
measured in CDCl3 (SiMe4 as internal standard) or [D6]DMSO with
a Varian Gemini 300 spectrometer; chemical shifts are expressed in
d (ppm) and coupling constants (J) are expressed in Hz. All ex-
changeable protons were confirmed by the addition of deuterium
oxide (D2O).
With the aim to design new hCA VII inhibitors, for which the
balance of lipophilic and hydrophilic features should be opti-
mized, we calculated the values of the two lipophilic indices,
ligand efficiency (LE) and lipophilic efficiency (LLE), for all
tested compounds.[54,55] Specifically, LE and LLE are parameters
that are useful to estimate the role of the molecular size with
respect to biological effects. In fact, the LE index correlates the
number of heavy atoms with the inhibitory effects and the LLE
index combines potency and lipophilicity. It is well known that
good CNS drug candidates generally possess LE values ranging
from 0.3 to 0.5 and LLE values above 5. As shown in Table 2,
the LE and LLE values are optimized for all compounds with
the exception of inhibitor 22, which has low activity.
Thus, for compounds 4–21 the very low Ki values against
the target enzyme seem to overcome the high hydrophilicity
conferred by the MBG portion and, thus, good LLE values are
predicted. The calculated values of the LE and LLE indices sug-
gest the design of new isoquinoline sulfonamides bearing ad-
ditional hydrophobic molecular fragments.
Biological methods
CA inhibition assay: An Applied Photophysics stopped-flow instru-
ment was used for assaying the CA-catalyzed CO2 hydration activi-
ty. Phenol red (at a concentration of 0.2 mm) was used as the indi-
cator, working at the absorbance maximum of l=557 nm with
10–20 mm
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
(HEPES, pH 7.5) or Tris (pH 8.3) as buffer, and 20 mm Na2SO4 or
20 mm NaClO4 (to maintain a constant ionic 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 inhibi-
tion constants. For each inhibitor, at least six traces of the initial 5–
10% of the reaction were used to determine the initial velocity.
The uncatalyzed rates were determined in the same manner and
were subtracted from the total observed rates. Stock solutions of
each inhibitor (10 mm) as racemic mixture were prepared in dis-
tilled deionized water, and dilutions up to 0.01 nm were done
thereafter with distilled deionized water. Inhibitor and enzyme sol-
utions were pre-incubated together for 15 min at room tempera-
ture prior to the assay to allow formation of the enzyme–inhibitor
complex. The inhibition constants were obtained by nonlinear
least-squares methods by using PRISM 3, as reported earlier, and
represent the mean from at least three different determinations.
CA isoforms were recombinant, obtained as reported earlier by this
group.[56–59]
Conclusions
Encouraged by significant selectivity and relevant inhibitory ef-
fects toward the human carbonic anhydrase VII (hCA VII) iso-
form, we herein explored the anticonvulsant effects of a series
of isoquinoline sulfonamides (e.g., compounds 4–22). As
a result, we found that for this class of compounds there is not
a clear correlation between hCA VII inhibition and anticonvul-
sant effects toward audiogenic seizures in DBA/mice after in-
traperitoneal administration. Despite the fact that studied sul-
fonamides 4–22 were generally potent inhibitors of hCA VII,
they failed to prevent seizures with the exception of isoquino-
Anticonvulsant activity in DBA/2 mice: All experiments were per-
formed with DBA/2 mice, which are genetically susceptible to
sound-induced seizures.[60] DBA/2 mice (8–12 g; 22–25 days old)
were purchased from Harlan Italy (Corezzano, Italy). Groups of 10
mice of either sex were exposed to auditory stimulation for 30 min
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pounds 4–22. The compound was administered intraperitoneally
(0.1 mL per 10 g mouse body weight) as a freshly prepared solu-
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and procedures described in this paper were approved by the
local ethical committee of the University of Catanzaro. All efforts
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Acknowledgements
Financial support for this research was awarded by the Italian
Ministry of Education, University and Research (MiUR).
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Keywords: anticonvulsants · carbonic anhydrases · inhibitors ·
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Received: December 23, 2015
Revised: February 5, 2016
Published online on && &&, 0000
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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*
Something on the brain: We evaluated
the anticonvulsant effects of sulfona-
mide derivatives showing high selectivi-
ty toward the human carbonic anhy-
drase VII isoform. The most active com-
pound is a potentially useful lead struc-
ture for further development of anticon-
vulsants.
&& – &&
In Vivo Evaluation of Selective
Carbonic Anhydrase Inhibitors as
Potential Anticonvulsant Agents
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