Carbonic anhydrase inhibitors: Aromatic and heterocyclic sulfonamides incorporating adamantyl moieties with strong anticonvulsant activity

Article abstract of DOI:10.1016/j.bmc.2004.03.008

A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid. Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl isothiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental logk ^′ IAM and theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (logk^′ IAM>1.35). The anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intraperitoneal injection (30mgkg^-1), compounds A8 and A9 exhibited a high protection against electrically induced convulsions (>90%). Their ED₅₀ was 3.5 and 2.6mgkg^-1, respectively.

Full text of DOI:10.1016/j.bmc.2004.03.008

Bioorganic & Medicinal Chemistry 12 (2004) 2717–2726
Carbonic anhydrase inhibitors: aromatic and heterocyclic
sulfonamides incorporating adamantyl moieties with strong
anticonvulsant activity
a,
b,
b
c
*
Marc A. Ilies, Bernard Masereel, St ꢀe phanie Rolin, Andrea Scozzafava,
a
d
c,
*
Gheorghe C ^a mpeanu, Valentin C ^ı mpeanu and Claudiu T. Supuran
a
c
University of Agricultural Sciences and Veterinary Medicine, Faculty of Biotechnologies, Department of Chemistry, B-dul Marasti nr.
9, 71331 Bucharest, Romania
University of Namur, Department of Pharmacy, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium
Universit ꢁa degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino
Firenze), Italy
5
b
(
d
‘C.D. Nenitzescu’ Institute of Organic Chemistry, Splaiul Independentei 202B, Bucharest, Romania
Received 23 January 2004; accepted 5 March 2004
Available online 1 April 2004
Abstract—A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/
heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid.
Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl iso-
thiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in
important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and
0
methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental log k IAM and
0
theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (log k IAM > 1.35). The
anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intra-
peritoneal injection (30 mg kg ), compounds A8 and A9 exhibited a high protection against electrically induced convulsions
(
ꢀ
1
ꢀ1
>90%). Their ED50 was 3.5 and 2.6 mg kg , respectively.
2004 Elsevier Ltd. All rights reserved.
ꢀ
1
. Introduction
acid (valproate) among others. Their use was also
proved useful in the treatment of other diseases such as
neuropathic pain and bipolar disorders, conditions
Epilepsy is a chronic neurological disorder characterized
by seizures generated by the sudden, massive, synchro-
nous excitation of neurons in the brain. In general, the
amplitude of seizure can range from brief cessation of
responsiveness (absence seizure) to severe tonic-clonic
muscle spasms with a loss of consciousness (general
4
–8
associated with significant morbidity and mortality.
The newest generation of anticonvulsants, including
vigabatrin, lamotrigine, gabapentin, tiagabine, topira-
mate, felbamate, zonisamide, or oxcarbazepine, repre-
sents a step forward toward better anticonvulsant
9–12
1–3
seizure).
Several generations of anticonvulsants are
drugs.
Nowadays the marketed anticonvulsants are
available, such as phenytoin, phenobarbital, benzo-
diazepines, etosuximide, carbamazepine, and valproic
estimated to be effective in approximatively 90% of the
epileptic patients. However, their use is associated with
dose-related toxicity and idiosyncratic side effects, and
the presently available drugs should thus be opti-
9–12
mized.
*
Corresponding authors. Tel./fax: +32-81-724338 (B.M.); tel.: +39-55-
57-3005; fax: +39-55-457-3385 (C.T.S.); e-mail addresses:
4
þ
Beside its ability to block the voltage Na -channel, to
potentiate GABAergic transmission and to block the
kainate/AMPA receptor, topiramate TPM occupies a
bernard.masereel@fundp.ac.be; claudiu.supuran@unifi.it
Present address: Texas A&M University at Galveston, Department
of Marine Sciences, Galveston, TX, USA.
0
968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.008

2718
M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
3
2
particular place among new anticonvulsants due to its
ability to inhibit carbonic anhydrase.
ylcarboxamido-1,3,4-thiadiazole-2-sulfonamide Ada-AZA
also showed promising in vivo anticonvulsant proper-
13–20
The enzyme,
ꢀ
30
catalyzing the interconversion of CO2 and HCO3 is
quite abundant in the brain, being present in the glia and
neurons, mainly as the cytosolic isozymes CA II, CA VII
ties. Considering the last derivative as lead compound,
we decided to further investigate this class of com-
pounds. We report here a new series of aromatic/het-
erocyclic sulfonamides incorporating the adamantyl
moiety in their molecule, as well as their CA inhibi-
tory and anticonvulsant properties in the MES test in
rats.
16–18;21
and the membrane-bound isoform CA XIV.
Although their function in the brain was not well
established, it is known that these proteins are involved
in the secretion of cerebrospinal fluid (CSF), being
present in the choroid plexus of vertebrates in high
1
6–18;21;22
amount.
of the brain CAs increases the cerebral blood flow with
It also has been proven that inhibition
2. Results
16;22
the concomitant raising of the CO partial pressure.
2
Kaila and co-workers,
et al. revealed that CA inhibition might serve as an
anticonvulsant mechanism (at least in some forms of
epilepsy), taking into account that the contribution of
23–26
27
Rivera et al., and Grover
2.1. Chemistry
28
The chemical structures of sulfonamides 1–9 on which
the adamantyl-containing moieties A–D have been
attached are shown below. These new sulfonamides
obtained by attaching moieties A–D of to the amino/
imino group of sulfonamides 1–9 will be designated
as (A–D) 1–9 (Chart 1), as for other such derivatives
ꢀ
HCO3 current to the ionic current through c-amino-
butirate A (GABA ) receptors on dendrites is increased
during periods of high-frequency receptor activation.
A
ꢀ
4;25;28
The excitatory effect of HCO is blocked by membrane-
1
permeant CA inhibitors.
3
18;33;34
synthesized by means of the tail approach.
Two synthetic approaches have been used for the
preparation of the new sulfonamides reported here: (i)
reaction of 1-adamantyl carboxylic acid or 1-adaman-
tylacetic acid chlorides with sulfonamides 1–9 in the
O
S
NH2
O
O
O
N
N
N N
O
O
O
O
O
N
S
presence of a base, as reported previously by this
33;34
Me
N
H
S
S NH2
S
O
NH2
O
Me
O
O
group, leading to amides A1–A9 and B1–B9, and (ii)
reaction of adamantyl isocyanate or isothiocyanate with
O
AZA
MZA
TPM
sulfonamides 1–9, leading to derivatives C1–C9 and D6,
35;36
O
N
N
O
S
D7
(Scheme 1).
O
NH2
R
N
H
S
S
NH2
O
O
N
O
SO NH
SO NH
2
2
2
2
SO NH
SO NH
2 2
2
2
Val-AZA: R = (n-C H ) CH
3
7 2
Piv-AZA: R = (CH ) C
3
3
ZNS
Ada-AZA: R = 1-adamantyl
t-Boc-AZA: R = (CH ) CO
F
Cl
Br
3
3
NH2
NH2
NH2
3
NH2
4
1
2
SO NH
2
2
SO NH
SO NH
Classical CA inhibitors (CAIs) such as acetazolamide
AZA and methazolamide MZA have been used for
2
2
2
2
18;29a
decades as anticonvulsants
marginal role in therapy at present.
although they occupy a
Generally, the
29b
I
NH2
lipophilicity improve brain penetration of the drug, so
that methazolamide MZA or the lipophilic tert-butoxy-
carbonyl derivative of acetazolamide tBOC-AZA are
more effective as anticonvulsants than acetazolamide,
CH NH
CH CH NH
2 2 2
2
2
6
5
7
H C
3
N
N
N
N
29
which is very hydrophilic. A similar effect is observed
in the case of zonisamide ZNS, a derivative for which the
CA inhibitory properties have not been investigate in
H N
SO NH
2
S
2
2
HN
SO NH
S
2
2
8
9
18
detail.
Considering the interesting anticonvulsant
activity of several CAIs, a series of aromatic/heterocyclic
sulfonamides incorporating valproyl moieties were pre-
CO
CO
30
pared recently. The valproyl derivative of acetazol-
amide (5-valproylamido-1,3,4-thiadiazole-2-sulfonamide)
Val-AZA was one of the best hCA I and hCA II inhib-
itors in this series and exhibited very strong anticonvul-
sant properties in a maximal electroshock seizure (MES)
A
B
NHCO
NHCS
30
C
test in mice. It was also observed that some structur-
ally related derivatives such as 5-pivaloylamido-1,3,
D
31
4
-thiadiazole-2-sulfonamide Piv-AZA and 5-adamant-
Chart 1.

M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
2719
O
O
SOCl
O
2
(
)n
N
H
A
2 2
SO NH
(
)n
( )n
Cl
H
2
N
A
SO
2
NH
2
n = 0 : A1-A9
n = 1 : B1-B9
X
NCX
N
H
N
A
2 2
SO NH
H
X = O : C1-C9
X = S : D6, D7
Scheme 1.
2
.2. Carbonic anhydrase inhibitory activity
2.3. Lipophilicity
The new sulfonamides reported here were assayed for
inhibition of two CA isozymes (CA I, and II) known to
play a critical role in electrolyte secretion/CO transport
In each series of tail (A–D), the lipophilicity of the most
active compounds A6–A9, B6–B9, C6–C9, and D6–D7
was expressed as their capacity factor (log k ) measured by
liquid chromatography at pH 7.40 by using an immobi-
0
2
18
processes in a variety of tissues, using the esterase activ-
ity of these enzymes (with 4-nitrophenylacetate as chro-
mogenic substrate). The inhibition data are shown in Table
lized artificial cellular membrane (IAM) stationary phase
(Table 2).
the software
3
7;38
Their ClogP has been also calculated with
39
37
1.
C
hem
D
raw
U
ltra 6.0.1. (Table 2).
Table 1. Inhibition data of CA isozymes I and II for derivatives reported in the present paper
Inhibitor
I
K (nM)
a
a
hCA I
hCA II
TPM Topiramate
AZA Acetazolamide
250 ꢁ 13
900 ꢁ 40
780 ꢁ 33
5 ꢁ 0.2
12 ꢁ 1
MZA Methazolamide
ZNS Zonisamide
14 ꢁ 0.8
b
c
Nt
35 ꢁ 2
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
B6
B7
B8
B9
C1
C2
C3
C4
C5
C6
C7
C8
C9
D6
D7
18,600 ꢁ 235 (28,000)
3400 ꢁ 123 (8300)
3500 ꢁ 95 (9800)
1600 ꢁ 42 (6500)
1550 ꢁ 74 (6000)
5500 ꢁ 240 (25,000)
4300 ꢁ 200 (21,000)
850 ꢁ 41 (8600)
265 ꢁ 14 (300)
49 ꢁ 3 (60)
62 ꢁ 4 (110)
35 ꢁ 3 (40)
41 ꢁ 2 (70)
87 ꢁ 6 (170)
69 ꢁ 6 (160)
10 ꢁ 0.8 (60)
12 ꢁ 1 (19)
233 ꢁ 21 (300)
36 ꢁ 3 (60)
39 ꢁ 2 (110)
32 ꢁ 3 (40)
28 ꢁ 3 (70)
47 ꢁ 1 (170)
35 ꢁ 3 (160)
8 ꢁ 1 (60)
770 ꢁ 38 (9300)
15,100 ꢁ 460 (28,000)
1800 ꢁ 62 (8300)
2000 ꢁ 95 (9800)
1300 ꢁ 47 (6500)
1050 ꢁ 58 (6000)
2300 ꢁ 76 (25,000)
1000 ꢁ 55 (21,000)
340 ꢁ 13 (8600)
265 ꢁ 14 (9300)
7 ꢁ 0.6 (19)
276 ꢁ 18 (300)
53 ꢁ 4 (60)
68 ꢁ 7 (110)
39 ꢁ 2 (40)
48 ꢁ 4 (70)
93 ꢁ 7 (170)
76 ꢁ 5 (160)
13 ꢁ 1 (60)
14 ꢁ 0.8 (19)
134 ꢁ 11 (170)
125 ꢁ 14 (160)
22,400 ꢁ 755 (28,000)
3650 ꢁ 120 (8300)
3700 ꢁ 83 (9800)
1870 ꢁ 94 (6500)
1700 ꢁ 50 (6000)
5840 ꢁ 230 (25,000)
4800 ꢁ 200 (21,000)
810 ꢁ 33 (8600)
800 ꢁ 27 (9300)
7800 ꢁ 400 (25,000)
6560 ꢁ 330 (21,000)
The data in parenthesis represent inhibition by the parent sulfonamide 1–9. Mean ꢁ standard deviation (from three different measurements).
a
Human (cloned) isozymes.
Not tested.
Unpublished data from this laboratory.
b
c

2720
M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
Table 2. Extent of protected mice (in %) against convulsions in the MES test at 3 h following intraperitoneal injection of 30 mg/kg, phospholipo-
0
philicity (log k IAM) and lipophilicity (ClogP) of selected CA inhibitors
Inhibitor
MES test % of protected mice
Lipophilicity
0
log k IAM
ClogP
a
TPM Topiramate
AZA Acetazolamide
MZA Methazolamide
83
þ0.04
ꢀ1.13
þ0.09
þ1.77
þ1.84
þ1.53
þ2.99
þ2.39
þ2.71
þ2.15
þ3.37
þ2.04
þ2.37
þ1.69
þ2.01
þ2.16
þ2.65
––
100
a
A6
A7
A8
A9
B6
B7
B8
B9
C6
C7
C8
C9
D6
D7
48
50
92
96
50
67
75
42
58
75
47
73
58
44
þ1.35
þ1.39
þ1.55
þ2.20
þ1.75
þ1.59
þ2.22
þ2.32
þ1.66
þ1.76
þ2.16
þ1.73
þ2.21
þ2.22
a
Values from Ref. 30.
2
.4. Maximal electroshock seizures test
adamantyl isocyanate and adamantyl isothiocyanate,
respectively, as reported for other CAIs incorporating
these functionalities.
35;36
The anticonvulsant activity of the most active CA
inhibitors reported here was examined at 30 mg/kg (ip)
in mice by using the MES test, which detects drugs that
40;41
prevent spread of tonic-clonic seizures.
Three hours
3.2. Carbonic anhydrase inhibitory activity
following intraperitoneal administration of 30 mg/kg,
the electrical stimulus was delivered and the extent of
protected mice was noted (Table 2).
The data of Table 1 show that the new inhibitors pre-
pared by attaching diverse adamantyl-containing moie-
ties to aromatic/heterocyclic sulfonamides 1–9, are
systematically more effective as compared to their parent
sulfonamide from which they were prepared, toward the
two investigated isozymes, hCA I and hCA II. The
enhanced inhibitory power of these compounds is pre-
sumably due to the interaction of the relatively bulky
adamantyl moiety with the enzyme active site. The fol-
lowing structure–activity relationships can be evidenced
on hCA: (i) the adamantylacetamides B1–B9 were more
effective CAIs than the corresponding adamantylcarb-
oxamides A1–A9, which were in turn more effective
inhibitors than the corresponding ureas C1–C9. These
were more effective CAIs as compared to the corre-
sponding thioureas D6, D7. Thus, the best tail for
inducing good CA inhibitory properties was the ada-
mantylacetamide one of type B; (ii) heterocyclic sul-
fonamides incorporating heads 8 and 9 were more
effective CAIs as compared to the aromatic derivatives
incorporating heads 1–7. Among these last derivatives,
the scaffolds of halogenated sulfanilamides 2–5 and
3. Discussion
3
.1. Chemistry
30
In a previous work it was showed that some sulfon-
amide CAIs incorporating lipophilic moieties, such as
the valproyl, pivaloyl, or adamantyl residues connected
to the structure of acetazolamide, exhibited good anti-
convulsant activity in experimental animals. These data
prompted us to extend our investigations in this field,
reporting a new series of sulfonamides incorporating
adamantyl moieties attached to the scaffolds of aro-
matic/heterocyclic sulfonamides of type 1–9, known to
18;30
act as good CAIs.
The amides A1–A9 and B1–B9 incorporating the ada-
mantyl/adamantylmethyl moieties were obtained from
the corresponding acids, which were converted to the
acyl chlorides (with SOCl
sulfonamides 1–9 in the presence of base (triethylamine,
2
), by reaction with amino-
4-aminoethyl-benzenesulfonamide
7
generally lead
to better inhibitors than the simple sulfanilamide scaf-
fold 1; (iii) among the halogenated sulfonamides, the
bromo 4 and the iodo 5 compounds are more active; (iv)
CA II was much more sensitive to inhibition by these
sulfonamides as compared to isozyme CA I, a fact well
33;34
pyridine, or sodium bicarbonate).
The amides were
obtained in good yields by this simple method. It has
been demonstrated that in such conditions only the
acylation reaction of the amino moiety takes place,
without the concomitant N-acylation of the sulfonamide
18
documented for many types of sulfonamide inhibitors.
33;34
group.
It should also be mentioned that some of the new sul-
fonamides reported here, such as A8, A9, B8, B9, C8,
and C9 possess CA I and CA II inhibitory properties of
similar or better potency than the clinically used
Ureas C1–C9 and thioureas D6, D7 were analogously
obtained by reaction of aminosulfonamides 1–9 with

M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
2721
inhibitors acetazolamide AZA and methazolamide
MZA, being slightly less effective than topiramate TPM.
Due to the low expression of CA I and to the abundance
of CA II in the brain, the selectivity toward CA II iso-
zyme should be favorable to the anticonvulsant activity.
enough such molecule would not have difficulties in
crossing the blood-brain barrier and reach the brain
CAs, which represent the target of these CAIs. Thus, the
lipophilicity measurements/calculations are really criti-
cal for detecting good anticonvulsant candidates among
the compounds synthesized in this work.
The ratio of the K values (hCA I/hCA II) of A8
I
(
ratio¼85) is higher than that of acetazolamide
(
ratio¼75), the most specific hCA I inhibitor, whereas
3.4. Maximal electroshock seizures test
A9 (ratio¼64) and C9 (ratio¼57) were more selective
for hCA II than topiramate (ratio¼50).
Compounds characterized by a high CA II inhibitory
potency and by a lipophilicity propitious to brain pen-
etration were evaluated for their anticonvulsant prop-
erties in the MES-test. Intraperitoneally administered in
mice at 30 mg kg , their anticonvulsant activity was
evaluated 3 h later and defined as a protective index
against convulsions induced by an electrical stimulus.
With a protective index ranging from 42% to 58%,
compounds A7, B9, C8, and D7 can be regarded as poor
anticonvulsant drugs as well as the aminomethylbenz-
enesulfonamides (A6, B6, C6, D6). The molecules B7,
B8, C7, and C9 can be considered as moderately active
3
.3. Lipophilicity
ꢀ
1
Generally, the crossing of the blood-brain barrier is
related to lipophilicity, this parameter has been deter-
mined for the best CA II inhibitors (A6–A9, B6–B9, C6–
C9, and D6, D7), as well as for the reference CA
inhibitors (TPM, AZA, MZA). On one hand, their
lipophilicity was theoretically calculated and expressed
as ClogP; on the other hand, their lipophilicity was
experimentally determined by using IAM columns and
expressed as their capacity factor (log k IAM) (Table
2
of phosphatidylcholine immobilized on a silica support
and mimic very closely the lipid environment of a fluid
cell membrane. It has been demonstrated that the log k
IAM value represents the so called ‘phospholipophilic-
ity’. This value is better than the octanol–water partition
coefficient (log P) or than D log P to predict the blood-
brain barrier crossing. Indeed, IAM chromatography
takes into account hydrophobic, ion pairing, and
hydrogen bonding interactions. Considering the ada-
mantyl side chain, the thioureas compounds (D6, D7)
were more lipophilic than their ureas counterparts (C6,
0
(protective index: 67–75%). Methazolamide MZA and
38
both adamantylcarboxamido derivatives A8 and A9
exhibited a high protection against induced convul-
sions (>90%). Dose–response curves (30, 20, 10, 5,
). These IAM columns are coated with a monolayer
ꢀ
1
0
2.5 mg kg ) were performed with these two last com-
pounds. The ED50 of 4-methylthiadiazolidine A9 and
ꢀ
1
thiadiazole A8 were 2.6 and 3.5 mg kg , respectively.
Both molecules were the most selective inhibitors of the
series for hCA II mainly located in the brain.
In conclusion this work led to the design, the synthesis
and the discovery of two lipophilic anticonvulsant
compounds characterized by a high inhibitory potency
of hCA II and a selectivity for this isozyme when com-
pared to their activity on hCA I. Further in vivo
experiment (time-course activity, other epileptic models)
are warranted to confirm their interest in the treatment
of central nervous disease such as epilepsy, or depression
among others.
0
C7) whatever the method used. As expected, the log k
IAM and the ClogP of adamantylacetamides B6–B9
were higher than that of adamantylcarboxamides A6–
0
A9 (D log k IAM ¼ þ0:1 to þ0.7; DClogP ¼ þ0:4 to
0
.9). By using the chromatographic method, the phos-
pholipophilicity of both types of benzenesulfonamide
moieties is quite similar (compare A6–A7, B6–B7, C6–
C7, and D6–D7). On the contrary, the ClogP of amino-
ethyl-benzenesulfonamides 7 are higher than that of the
aminomethylbenzenesulfonamides 6 (DClogP ¼ þ0:1 to
þ0.5). Similarly, the ClogP value of the 4-methylthia-
diazoline derivatives (A9, B9, C9) is higher that that of
their thiadiazole counterparts (A8, B8, C8; DClogP ¼
þ0:1 to þ0.5). Taking all these results, a poor correla-
4. Experimental
4.1. General
0
tion (p < 0:05) is observed between log k IAM and
Melting points: heating plate microscope (not cor-
ꢀ
1
ClogP values. The ClogP value of all the ada-
mantyl derivatives investigated are much higher
rected); IR spectra: KBr pellets, 400–4000 cm Karl
Zeiss Jena UR-20 spectrometer; NMR spectra: Varian
1
(
(
ClogP > þ1:53) than that of methazolamide MZA
ClogP ¼ þ0:09) or topiramate (ClogP ¼ þ0:04) mar-
Gemini 300BB apparatus, operating at 300 MHz for H
NMR and at 75 MHz for C NMR (chemical shifts are
13
keted as anticonvulsant. Thus both sets of values are
features of compounds able to cross the blood-brain
expressed as d values relative to Me Si as internal
4
standard for proton spectra and to the solvent resonance
for carbon spectra). Assignments were made by means
of chemical shifts, peak integrals, selective deuteration,
37
barrier well, as was the case of the reference CA
inhibitors TPM, AZA, or MZA. It should be mentioned
that at the physiological pH, due to the acidity of the
sulfonamide/sulfamate moieties, all these drugs are
found as sulfonamidate/sulfamate anions. Still, as the
anion is in equilibrium with the free acid (sulfonamide/
sulfamate) that is able to cross membranes, a lipophilic
1
1
model spectra, APT, COSY ( H– H), and HETCOR
1
13
( H– C) experiments. Elemental analysis (ꢁ0.4% of the
theoretical values, calculated for the proposed struc-
tures): Carlo Erba Instruments CHNS Elemental Ana-
lyzer, Model 1106.

2722
M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
All reactions were monitored by thin-layer chromato-
graphy (TLC), using 0.25 mm-thick precoated silica gel
plates (E. Merck) eluted with MeOH/CHCl3 1/4 v/v
unless specified otherwise. 1-Adamantane carboxylic
acid, 1-adamantyl-acetic acid, adamantyl isocyanate,
adamantyl isothiocyanate, homosulfanilamide (hy-
drochloride), and 4-(2-aminoethyl)benzenesulfonamide
were from Sigma-Aldrich Chemical Co. Thionyl chlo-
ride, triethylamine, pyridine, were from Aldrich, Fluka,
or Merck. Acetonitrile, tetrahydrofurane, methanol,
ethanol, chloroform, hexane (E. Merck, Darmstadt,
Germany), or other solvents used in the synthesis were
double distilled and kept on molecular sieves in order to
maintain them in anhydrous conditions. 5-Amino-1,3,4-
thiadiazole-2-sulfonamide was obtained from acetazol-
amide (Sigma) by deprotection with concentrated
hydrochloric acid followed by neutralization, using a
from the same solvent. Yields were in the range of 30–
90%, depending on the reactivity of the corresponding
aminosulfonamide. Moreover, due to the low reactivity
of adamantyl isothiocyanate and its limited thermal
stability, the corresponding thioureas could be obtained
just in the case of very reactive aminosulfonamides.
4
.2.2.1. 4-(Adamantan-1-yl-carboxamido)-benzenesulf-
1
onamide A1. White crystals, mp 281–284 ꢁC (EtOH); H
NMR (DMSO-d ), d, ppm: 9.43 (s, 1H, NH), 7.84 (d,
J ¼ 8:9 Hz, 2H, H3,5-Ph), 7.74 (d, J ¼ 8:9 Hz, 2H,
H2,6-Ph), 7.23 (s, 2H, SO NH ), 2.01 (br s, 3H, 3CHb-
6
2
2
Ad), 1.91 (d, J ¼ 3:4 Hz, 6H, 3CH
2
a-Ad), 1.69 (t,
c-Ad); C NMR (DMSO-d ), d,
ppm: 176.4 (CO), 142.4 (C4-Ph), 138.2 (C1-Ph), 126.3
2C, C3,C5-Ph), 119.6 (2C, C2,C6-Ph), 41.1 (Cq-Ad),
8.1 (3Ca-Ad), 36.0 (3Cc-Ad), 27.6 (3Cb-Ad); IR (KBr),
13
J ¼ 3:3 Hz, 6H, 3CH
2
6
42;43
previously reported procedure.
5-Imino-4-methyl-
hydrochloride,
was similarly prepared from methazolamide (Sigma).
2
(
D -1,3,4-thiadiazoline-2-sulfonamide
3
ꢀ
1
cm : 1160 (mSO sym), 1325 (mSO as), 1530 (dNH), 1660
Halogenosulfonamides were prepared by literature
procedures,
2
2
44–46
(mC@O),
(C H N O S) C, H, N.
2850
(mCH2sym),
2910
(mCH2as);
Anal.
optimized in our laboratory. Pre-
parative column chromatography was performed on
silica gel 60 (0.063–0.200 mm)––from Merck––eluted
17 22 2 3
4.2.2.2. 4-(Adamantan-1-yl-carboxamido)-3-fluorobenz-
3
with MeOH/CHCl 2/8 v/v.
enesulfonamide A2. White crystals, mp 163–166 ꢁC (dec)
1
(
6
EtOH); H NMR (DMSO-d ), d, ppm: 9.22 (s, 1H,
NH), 7.73 (dd, 1H, J ¼ 8:5, 7.4 Hz, H5-Ph), 7.66 (dd,
4
4
.2. Chemistry
.2.1. General procedure for the synthesis of adamantyl-
1
2
3
3
H, J ¼ 8:4, 2.2 Hz, H6-Ph), 7.61 (dd, 1H, J ¼ 9:2,
.2 Hz, H2-Ph), 7.43 (br s, 2H, SO NH ), 2.02 (br s, 3H,
2
2
CHb-Ad), 1.92 (br s, 6H, 3CH
2
a-Ad), 1.70 (br s, 6H,
6
carboxamides A1–A9 and B1–B9. An amount of 5 mmol
aminosulfonamide was suspended/dissolved in 20 mL
anhydrous MeCN and 0.78 mL (0.56 g, 5.5 mmol) tri-
ethylamine was added under stirring. The mixture was
cooled to 0–5 ꢁC, then a solution of 5 mmol adamant-
anecarboxyl chloride/adamantaneacetyl chloride (gen-
erated from the corresponding acids and thionyl
chloride), dissolved in 3 mL MeCN, was added dropwise
during 15 min. The reaction was stirred at room tem-
perature for 3 h or until a reasonable conversion was
reached (TLC control). The solvent was evaporated in
vacuum and the resulted product was treated with
13
CH
2
c-Ad); C NMR (DMSO-d ), d, ppm: 176.06
(
J
CO), 154.20 (d, JCipso-F ¼ 248:6 Hz, C3-Ph), 140.9 (d,
Cmeta-F ¼ 3:0 Hz, C1-Ph), 129.45 (d, JCortho-F ¼ 11:8 Hz,
C4-Ph), 124.5 (C6-Ph), 121.63 (d, JCmeta-F ¼ 3:4 Hz, C5-
Ph), 113.15 (d, JCortho-F ¼ 23:02 Hz, C2-Ph), 41.03 (d,
J ¼ 6:6 Hz, Cq-Ad), 38.52 (d, J ¼ 3:4 Hz, 3Ca-Ad),
ꢀ
1
3
6.16 (3Cc-Ad), 27.75 (3Cb-Ad); IR (KBr), cm : 1165
(
(
mSO2sym), 1330 (mSO2as), 1540 (dNH), 1665 (mC@O), 2850
mCH2sym), 2910 (mCH2as); Anal. (C H FN O S) C, H, N.
17 21
2
3
4.2.2.3. 4-(Adamantan-1-yl-carboxamido)-3-clorobenz-
enesulfonamide A3. White crystals, mp 222–224 ꢁC
2
0 mL water. The crude product was filtered off, washed
with water, and dried. Yields were in the range of 50–
0%, depending on the reactivity of the corresponding
1
(EtOH); H NMR (DMSO-d ), d, ppm: 9.06 (s, 1H,
6
9
NH), 7.89 (d, J ¼ 1:8 Hz, 1H, H2-Ph), 7.78 (d,
aminosulfonamide. The resulted compounds were fur-
ther purified by recrystallization (from ethanol) or by
column chromatography, when the fractions containing
the desired compound were collected, evaporated to
dryness, and the obtained product was recrystallized
from the same solvent.
J ¼ 8:6 Hz, 1H, H5-Ph), 7.74 (dd, J ¼ 8:6, 1.8 Hz, 1H,
H6-Ph), 7.49 (s, 2H, SO NH ), 2.03 (br s, 3H, 3CHb-
2 2
Ad), 1.94 (br s, 6H, 3CH
Ad); C NMR (DMSO-d ), d, ppm: 176.01 (CO),
2
a-Ad), 1.71 (br s, 6H, 3CH
2
c-
13
6
141.66 (C4-Ph), 138.32 (C1-Ph), 128.10 (C3-Ph), 127.16
(C2-Ph), 126.66 (C5-Ph), 124.82 (C6-Ph), 40.97 (Cq-
Ad), 38.39 (3C, 3Ca-Ad), 36.00 (3C, 3Cc-Ad), 27.63
ꢀ
1
(
3C, 3Cb-Ad); IR (KBr), cm : 1160 (mSO sym), 1335
2
4
.2.2. General procedure for the synthesis of adamantyl
ureas C1–C9 and thioureas D6, D7. Aminosulfonamide
5 Mmol) dissolved in 15 mL anhydrous tetrahydrofu-
(mSO as), 1540 (dNH), 1665 (mC@O), 2850 (mCH sym), 2910
2
2
(mCH as); Anal. (C17
2 3
H21ClN O S) C, H, N.
2
(
rane, were treated under stirring with 5 mmol adamantyl
isocyanate/isothiocyanate dissolved in 3 mL THF. The
mixture was refluxed overnight or until a reasonable
conversion was reached (TLC control). Then the solvent
was evaporated in vacuum and the resulted crude
product purified by recrystallization from ethanol or by
column chromatography followed by recrystallization
4.2.2.4.
4-(Adamantan-1-yl-carboxamido)-3-bromo-
benzenesulfonamide A4. White crystals, mp 218–221 ꢁC
(dec) (EtOH); H NMR (DMSO-d ), d, ppm: 9.01 (s,
1
6
1H, NH), 8.04 (d, J ¼ 1:8 Hz, 1H, H2-Ph), 7.79 (dd,
J ¼ 8:4, 1.8 Hz, 1H, H6-Ph), 7.75 (d, J ¼ 8:4 Hz, 1H,
H5-Ph), 7.49 (s, 2H, SO NH ), 2.03 (br s, 3H, 3CHb-
2 2
Ad), 1.94 (br s, 6H, 3CH
2
a-Ad), 1.71 (br s, 6H, 3CH c-
2

M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
2723
1
3
Ad); C NMR (DMSO-d
41.78 (C4-Ph), 139.55 (C1-Ph), 129.65 (C2-Ph), 126.77
C5-Ph), 125.34 (C6-Ph), 118.35 (C3-Ph), 40.95 (Cq-
Ad), 38.39 (3C, 3Ca-Ad), 35.93 (3C, 3Cc-Ad), 27.56
6
), d, ppm: 175.79 (CO),
4.2.2.9. 5-(Adamantan-1-yl-carboximido)-4-methyl-
D -1,3,4-thidiazoline-2-sulfonamide A9. White crystals,
2
1
(
1
mp 230–231 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm:
6
8.43 (s, 2H, SO NH ), 3.94 (s, 3H, N–CH
3H, 3CHb-Ad), 1.88 (br s, 6H, 3CH
2
2
3
), 2.00 (br s,
a-Ad), 1.69 (br s,
ꢀ
1
(
(
(
3C, 3Cb-Ad); IR (KBr), cm : 1165 (mSO sym), 1330
mSO2as), 1540 (dNH), 1665 (mC@O), 2850 (mCH2sym), 2910
CH as); Anal. (C17
2
2
13
6H, 3CH c-Ad); C NMR (DMSO-d ), d, ppm: 186.6
2
6
m
H
21BrN
2
O
3
S) C, H, N.
(CO), 164.8 (C@N), 157.5 (C–SO
2
NH
2
), 42.0 (Cq-Ad),
2
3
8.9 (3Ca-Ad), 38.0 (N–CH ), 36.2 (3Cc-Ad), 27.6
3
ꢀ
1
(
3Cb-Ad); IR (KBr), cm : 1175 (mSO2sym), 1360 (mSO2as),
4
.2.2.5. 4-(Adamantan-1-yl-carboxamido)-3-iodobenz-
enesulfonamide A5. White crystals, mp 250–253 ꢁC (dec)
6
EtOH); H NMR (DMSO-d ), d, ppm: 8.93 (s, 1H,
1
600 (mC@O), 2850 (mCH sym), 2900 (mCH as); Anal.
2
2
14 20 4 3 2
(C H N O S ) C, H, N.
1
(
NH), 8.24 (d, 1H, J ¼ 2:0 Hz, H2-Ph), 7.78 (dd, 1H,
J ¼ 8:4, 2.0 Hz, H6-Ph), 7.63 (d, 1H, J ¼ 8:4 Hz, H5-
Ph), 7.44 (s, 2H, SO NH ), 2.04 (br s, 3H, 3CHb-Ad),
4
.2.2.10. 4-(Adamantan-1-yl-methylcarboxamido)-benz-
enesulfonamide B1. White crystals, mp 260–262 ꢁC
EtOH); H NMR (DMSO-d ), d, ppm: 10.10 (s, 1H,
NH), 7.74 (s, 4H, AA BB , Ph), 7.23 (s, 2H, SO NH ),
2.08 (s, 2H, CH
m, 6H, 3CH a-Ad), 1.61 (m, 6H, 3CH c-Ad);
2
2
1
(
1
.95 (br s, 6H, 3CH
ꢀ
2
a-Ad), 1.71 (br s, 6H, 3CH
2
c-Ad);
6
0 0
1
IR (KBr), cm : 1165 (mSO sym), 1330 (mSO as), 1540 (dNH),
2 2
2
2
2
-Ad), 1.93 (br s, 3H, 3CHb-Ad), 1.63
13
1
665 (mC@O), 2860 (mCH2sym), 2910 (mCH2as); Anal.
S) C, H, N.
(
C
(
C
17
H
21IN
2
O
3
2
2
NMR (DMSO-d ), d, ppm: 169.73 (CO), 142.05 (C4-
6
Ph), 138.07 (C1-Ph), 126.60 (2C, C3,C5-Ph), 118.55 (2C,
C2,C6-Ph), 50.80 (CH -Ad), 42.01 (3Ca-Ad), 36.37
4
.2.2.6. 4-(Adamantan-1-yl-carboxamido-methyl)-benz-
2
enesulfonamide A6. White crystals, mp 250–252 ꢁC
EtOH); H NMR (DMSO-d
(
3Cc-Ad), 32.79 (Cq-Ad), 28.01 (3Cb-Ad); IR (KBr),
1
(
6
), d, ppm: 8.08 (t, 1H,
ꢀ1
cm : 1160 (mSO sym), 1335 (mSO as), 1530 (dNH), 1640
2
2
J ¼ 5:8 Hz, NH), 7.74 (d, J ¼ 8:3 Hz, 2H, H3,5-Ph),
(
(
mC@O),
2850
C H N O S) C, H, N.
(mCH2sym),
2900
(mCH2as);
Anal.
7
.36 (d, J ¼ 8:3 Hz, 2H, H2,6-Ph), 7.30 (s, 2H,
18
24
2
3
SO NH ), 4.29 (d, 2H, J ¼ 3:1 Hz, CH -Ph), 1.97 (br s,
2
2
2
3
6
(
H, 3CHb-Ad), 1.81 (br s, 6H, 3CH
2
a-Ad), 1.67 (br s,
4.2.2.11. 4-(Adamantan-1-yl-methylcarboxamido)-3-
clorobenzenesulfonamide B3. White crystals, mp 196–
13
H, 3CH
2
c-Ad); C NMR (DMSO-d ), d, ppm: 177.0
6
CO), 144.4 (C4-Ph), 142.3 (C1-Ph), 127.1 (2C, C2,C6-
Ph), 125.6 (2C, C3,C5-Ph), 41.6 (Cq-Ad), 39.8 (CH
Ph), 38.7 (3Ca-Ad), 36.1 (3Cc-Ad), 27.6 (3Cb-Ad); IR
KBr), cm : 1160 (mSO2sym), 1330 (mSO2as), 1540 (dNH),
615 (mC@O), 2850 (mCH sym), 2920 (mCH as); Anal.
1
1
1
99 ꢁC (EtOH); H NMR (DMSO-d
6
), d, ppm: 9.61 (s,
2
-
H, NH), 7.95 (d, J ¼ 8:5 Hz, 1H, H5-Ph), 7.88 (d,
J ¼ 2:1 Hz, 1H, H2-Ph), 7.74 (dd, J ¼ 8:5, 2.1 Hz, 1H,
H6-Ph), 7.47 (s, 2H, SO NH ), 2.20 (s, 2H, CH -Ad),
.93 (br s, 3H, 3CHb-Ad), 1.64 (m, 12H, 3CH a-Ad-
ꢀ
1
(
1
2
2
2
2
2
1
2
18 24 2 3
(C H N O S) C, H, N.
1
3
þ3CH
2
c-Ad); C NMR (DMSO-d ), d, ppm: 169.75
6
(CO), 140.87 (C4-Ph), 138.07 (C1-Ph), 126.84 (C2-Ph),
125.74 (C3-Ph), 125.67 (C5-Ph), 124.82 (C6-Ph), 50.00
4
.2.2.7.
4-[(Adamantan-1-yl-carboxamido)-2-ethyl]-
benzenesulfonamide A7. White crystals, mp 268–271 ꢁC
(CH
2
-Ad), 41.84 (3C, 3Ca-Ad), 36.43 (3C, 3Cc-Ad),
1
ꢀ1
(
EtOH); H NMR (DMSO-d
J ¼ 8:3 Hz, 2H, H3,5-Ph), 7.47 (t, 1H, J ¼ 5:6 Hz, NH),
.35 (d, J ¼ 8:3 Hz, 2H, H2,6-Ph), 7.30 (s, 2H,
SO NH
), 3.27 (q, 2H, J ¼ 5:6 Hz, 2-Et), 2.77 (t, 2H,
6
), d, ppm: 7.73 (d,
32.80 (Cq-Ad), 28.10 (3C, 3Cb-Ad); IR (KBr), cm :
1160 (mSO sym), 1330 (mSO as), 1540 (dNH), 1665 (mC@O),
2850 (mCH2sym), 2910 (mCH2as); Anal. (C H ClN O S) C,
H, N.
2
2
7
18
23
2
3
2
2
J ¼ 7:1 Hz, 1-Et), 1.94 (br s, 3H, 3CHb-Ad), 1.72 (d,
6
H, J ¼ 2:5 Hz, 3CH a-Ad), 1.64 (br s, 6H, 3CH c-Ad);
2
6
2
4.2.2.12. 4-(Adamantan-1-yl-methylcarboxamido)-3-
iodobenzenesulfonamide B5. White crystals, mp 222–
1
3
C NMR (DMSO-d
), d, ppm: 176.9 (CO), 143.9 (C4-
Ph), 141.9 (C1-Ph), 129.1 (2C, C2,C6-Ph), 125.6 (2C,
C3,C5-Ph), 39.9 (2-Et), 39.8 (1-Et), 38.7 (3Ca-Ad), 36.1
1
2
1
7
25 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm: 9.56 (s,
6
H, NH), 8.24 (br s, 1H, H6-Ph), 7.67 (m, 1H, H5-Ph),
(
3Cc-Ad), 34.8 (Cq-Ad), 27.6 (3Cb-Ad); IR (KBr),
2 2 2
.42 (s, 2H, SO NH ), 2.15 (s, 2H, CH -Ad), 1.95 (br s,
ꢀ
1
cm : 1160 (mSO sym), 1335 (mSO as), 1540 (dNH), 1635
2
2
3H, 3CHb-Ad), 1.64 (m, 12H, 3CH a-Adþ3CH c-Ad);
2
2
(
N O S) C, H, N.
mC@O), 2850 (mCH2sym), 2900 (mCH2as); Anal. (C H -
13
19
26
C NMR (DMSO-d ), d, ppm: 169.89 (CO), 143.29
6
2
3
(C4-Ph), 142.16 (C1-Ph), 136.68 (C2-Ph), 126.85 (C5-
Ph), 126.31 (C6-Ph), 50.53 (CH -Ad), 42.70 (3Ca-Ad),
6.90 (3Cc-Ad), 33.32 (Cq-Ad), 28.58 (3Cb-Ad); IR
2
3
4
diazole-2-sulfonamide A8. White crystals, mp 246–
.2.2.8.
5-(Adamantan-1-yl-carboxamido)-1,3,4-thi-
ꢀ
1
(KBr), cm : 1160 (mSO sym), 1330 (mSO as), 1535 (dNH),
2
2
1
1660 (mC@O), 2850 (mCH2sym), 2910 (mCH2as); Anal.
2
1
3
3
1
48 ꢁC (dec) (EtOH); H NMR (DMSO-d ), d, ppm:
6
(C H IN O S) C, H, N.
18 23 2 3
2.66 (s, 1H, NH), 8.29 (s, 2H, SO
CHb-Ad), 1.94 (br s, 6H, 3CH a-Ad), 1.68 (br s, 6H,
CH c-Ad); C NMR (DMSO-d ), d, ppm: 176.7 (CO),
2 2
64.5 (C–NH), 161.9 (C–SO NH ), 40.8 (Cq-Ad), 37.4
2
NH
2
), 2.00 (br s, 3H,
2
13
4.2.2.13. 4-(Adamantan-1-yl-methylcarboxamido-
methyl)-benzenesulfonamide B6. White crystals, mp
2
6
1
(
3Ca-Ad), 35.7 (3Cc-Ad), 27.4 (3Cb-Ad); IR (KBr),
204–208 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm: 8.31
6
ꢀ
1
cm : 1170 (mSO2sym), 1360 (mSO2as), 1515 (dNH), 1670
(t, 1H, J ¼ 5:9 Hz, NH), 7.76 (d, J ¼ 8:2 Hz, 2H, H3,5-
(
mC@O), 2865 (mCH2sym), 2915 (mCH2as); Anal. (C H N -
Ph), 7.42 (d, J ¼ 8:2 Hz, 2H, H2,6-Ph), 7.31 (s, 2H,
13
18
4
O
3
S
2
) C, H, N.
SO
2
NH
2
), 4.30 (d, 2H, J ¼ 5:9 Hz, NH–CH
2
), 1.91 (br s,

2724
M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
6
2
H, 3CH
2
a-Ad), 1.68 (br s, 1H, 1CHb-Ad), 1.64 (br s,
135.8 (C1-Ph), 126.8 (2C, C2,C6-Ph), 116.5 (2C, C3,C5-
Ph), 50.1 (Cq-Ad), 41.5 (3Ca-Ad), 36.0 (3Cc-Ad), 28.9
13
H, 2CHb-Ad), 1.57 (br s, 6H, 3CH
2
c-Ad); C NMR
ꢀ
1
(
DMSO-d ), d, ppm: 170.14 (CO), 143.95 (C4-Ph),
(3Cb-Ad); IR (KBr), cm : 1160 (mSO2sym), 1325 (mSO2as),
1540 (dNH), 1660 (mC@O), 2850 (mCH sym), 2915 (mCH as);
6
1
42.55 (C1-Ph), 127.53 (2C, C3,C5-Ph), 125.61 (2C,
C2,C6-Ph), 49.90 (CH -Ad), 42.16 (3Ca-Ad), 41.67 (Cq-
Ad), 38.96 (2Cc-Ad), 32.33 (Cc-Ad), 28.05 (3Cb-Ad);
2
2
2
23 3 3
Anal. (C17H N O S) C, H, N.
ꢀ
1
IR (KBr), cm : 1175 (mSO sym), 1365 (mSO as), 1530 (dNH),
1
2
2
4.2.2.18. 4-(3-Adamantan-1-yl-ureido)-3-clorobenzene-
665 (mC@O), 2850 (mCH sym), 2900 (mCH as); Anal.
2
2
sulfonamide C3. White crystals, mp 156–159 ꢁC (dec)
(
C H N O S) C, H, N.
19
26
2
3
1
(
6
EtOH); H NMR (DMSO-d ), d, ppm: 8.37 (d,
J ¼ 8:8 Hz, 1H, H5-Ph), 8.20 (s, 1H, NH-Ph), 7.78 (d,
4.2.2.14. 4-[(Adamantan-1-yl-methylcarboxamido)-2-
ethyl]-benzenesulfonamide B7. White crystals, mp 219–
J ¼ 2:0 Hz, 1H, H2-Ph), 7.64 (dd, J ¼ 8:8, 2.0 Hz, 1H,
2 2
H6-Ph), 7.30 (s, 2H, SO NH ), 7.04 (s, 1H, Ad-NH),
1
2
1
7
22 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm: 7.78 (t,
6
2
1
.03 (br s, 3H, 3CHb-Ad), 1.95 (br s, 6H, 3CH
2
a-Ad),
6
H, J ¼ 5:8 Hz, NH), 7.73 (d, J ¼ 8:2 Hz, 2H, H3,5-Ph),
13
.63 (br s, 6H, 3CH c-Ad); C NMR (DMSO-d ), d,
2
.39 (d, J ¼ 8:2 Hz, 2H, H2,6-Ph), 7.79 (s, 2H,
ppm: 152.88 (CO), 140.01 (C4-Ph), 136.71 (C1-Ph),
26.62 (C2-Ph), 125.14 (C6-Ph), 119.93 (C5-Ph), 119.18
C3-Ph), 50.31 (Cq-Ad), 41.39 (3C, 3Cc-Ad), 36.02 (3C,
SO NH
t, 2H, J ¼ 7:1 Hz, CH
2
2
), 3.24 (dd, 2H, J ¼ 7:1, 5.8 Hz, NH–CH
2
), 2.78
1
(
2
-Ph), 1.88 (br s, 3H, 3CHb-Ad),
(
1
s, 2H, CH
.79 (s, 2H, CH -Ad), 1.65 (br s, 2H, CH a-Ad), 1.61 (br
ꢀ1
2
2
3Ca-Ad), 28.91 (3C, 3Cb-Ad); IR (KBr), cm : 1160
2
a-Ad), 1.56 (br s, 2H, CH
2
a-Ad), 1.49 (m,
(
mSO sym), 1320 (mSO as), 1535 (dNH), 1660 (mC@O), 2850
2
2
13
6
(
H, 3CH
CO), 143.76 (C4-Ph), 142.01 (C1-Ph), 129.08 (2C,
C3,C5-Ph), 125.59 (2C, C2,C6-Ph), 50.04 (CH -Ad),
), 36.45 (3Cc-Ad),
4.92 (CH -Ph), 32.11 (Cq-Ad), 28.01 (3Cb-Ad); IR
2
c-Ad); C NMR (DMSO-d
6
), d, ppm: 169.92
(
mCH2sym), 2910 (mCH2as); Anal. (C H ClN O S) C, H, N.
17
22
3
3
2
4.2.2.19. 4-[(3-Adamantan-1-yl-ureido)-methyl]-benz-
4
3
2.09 (3Ca-Ad), 39.50 (NH-CH
2
enesulfonamide C6. White crystals, mp 226–228 ꢁC
2
ꢀ
1
1
(dec) (EtOH); H NMR (DMSO-d ), d, ppm: 7.75 (d,
(
KBr), cm : 1160 (mSO2sym), 1320 (mSO2as), 1535 (dNH),
6
J ¼ 8:3 Hz, 2H, H3,5-Ph), 7.38 (d, J ¼ 8:3 Hz, 2H,
1
625 (mC@O), 2850 (mCH sym), 2900 (mCH as); Anal.
2
2
H2,6-Ph), 7.29 (s, 2H, SO
NH–CH
2
NH
), 5.68 (s, 1H, NH-Ad), 4.21 (d, 2H, J ¼ 6 Hz,
2
), 6.20 (t, 1H, J ¼ 6 Hz,
(
20 28 2 3
C H N O S) C, H, N.
2
NH–CH ), 1.99 (br s, 3H, 3CHb-Ad), 1.87 (br s, 6H,
2
4
.2.2.15.
5-(Adamantan-1-yl-methylcarboxamido)-
,3,4-thidiazole-2-sulfonamide B8. White crystals, mp
13
3
CH
2
a-Ad), 1.60 (br s, 6H, 3CH
), d, ppm: 156.9 (NHCO), 145.4 (C4-Ph),
42.3 (C1-Ph), 127.2 (2C, C2,C6-Ph), 125.6 (2C, C3,C5-
2
c-Ad); C NMR
1
2
1
(DMSO-d
6
1
32–234 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm:
2.94 (s, 1H, NH), 8.30 (s, 2H, SO NH ), 2.28 (s, 2H,
6
1
2
2
Ph), 49.6 (CH
2
-Ph), 42.2 (Cq-Ad), 42.0 (3Ca-Ad), 36.1
CH -Ad), 1.92 (br s, 3H, 3CHb-Ad), 1.67 (s, 2H, CH a-
2
2
ꢀ1
(
(
(
3Cc-Ad), 28.9 (3Cb-Ad); IR (KBr), cm : 1160
mSO2sym), 1335 (mSO2as), 1550 (dNH), 1640 (mC@O), 2850
Ad), 1.62 (br s, 2H, CH
Ad), 1.55 (br s, 2H, CH
2
a-Ad), 1.58 (br s, 6H, 3CH
a-Ad); C NMR (DMSO-d
2
c-
13
2
6
),
m
25 3 3
CH sym), 2910 (mCH as); Anal. (C18H N O S) C, H, N.
2
2
d, ppm: 170.00 (CO), 164.25 (C–NH), 160.78 (C–
SO NH ), 48.83 (CH -Ad), 41.82 (3Ca-Ad), 36.21 (3Cc-
Ad), 33.04 (Cq-Ad), 27.95 (3Cb-Ad); IR (KBr), cm :
2
2
2
ꢀ
1
4.2.2.20. 4-[2-(3-Adamantan-1-yl-ureido)-ethyl]-benz-
enesulfonamide C7. White crystals, mp 218–220 ꢁC
1
160 (mSO sym), 1320 (mSO as), 1530 (dNH), 1640 (mC@O),
2
2
1
2
H, N.
850 (mCH2sym), 2910 (mCH2as); Anal. (C H N O S ) C,
(dec) (EtOH); H NMR (DMSO-d ), d, ppm: 7.74 (d,
14
20
4
3
2
6
J ¼ 8:3 Hz, 2H, H3,5-Ph), 7.37 (d, J ¼ 8:3 Hz, 2H,
H2,6-Ph), 7.29 (s, 2H, SO
NH–CH ), 5.55 (s, 1H, NH-Ad), 3.19 (q, 2H,
2
NH
2
), 5.65 (t, 1H, J ¼ 5:8 Hz,
4
.2.2.16.
methyl-D -1,3,4-thidiazoline-2-sulfonamide B9. White
5-(Adamantan-1-yl-methylcarboximido)-4-
2
2
J ¼ 6:6 Hz, NH–CH
2
), 2.72 (t, 2H, J ¼ 7 Hz, CH
2
-Ph),
1
1.98 (br s, 3H, 3CHb-Ad), 1.84 (br s, 6H, 3CH a-Ad),
crystals, mp 216–217 ꢁC (EtOH); H NMR (DMSO-d ),
d, ppm: 8.43 (s, 2H, SO
2
6
13
1.59 (br s, 6H, 3CH c-Ad); C NMR (DMSO-d ), d,
ppm: 157.0 (NHCO), 144.1 (C4-Ph), 141.9 (C1-Ph),
NH
2
), 3.92 (s, 3H, N–CH
3
), 2.31
2
6
2
(
1
s, 2H, CH
2
-Ad), 1.91 (br s, 3H, 3CHb-Ad), 1.61 (br s,
13
129.1 (2C, C2,C6-Ph), 125.7 (2C, C3,C5-Ph), 49.4 (CH2-
2H, 3CH a-Adþ3CH c-Ad); C NMR (DMSO-d ), d,
2
2
6
NH), 42.0 (3Ca-Ad), 40.1 (Cq-Ad), 36.1 (3Cc-Ad), 35.9
ppm: 180.5 (CO), 163.5 (C@N), 157.5 (C–SO NH ),
5
2
2
ꢀ
1
(
CH -Ph), 28.9 (3Cb-Ad); IR (KBr), cm : 1160
3.5 (CH
2
–CO), 42.1 (3Ca-Ad), 38.2 (N–CH
3
), 36.3
2
(
m
SO sym), 1340 (mSO as), 1530 (dNH), 1640 (mC@O), 2850
(
3Cc-Ad), 33.0 (Cq-Ad), 28.1 (3Cb-Ad); IR (KBr),
2
2
ꢀ
1
(mCH2sym), 2910 (mCH2as); Anal. (C H N O S) C, H, N.
cm : 1180 (mSO2sym), 1325 (mSO2as), 1590 (mC@O), 2850
) C, H, N.
19 27
3
3
22 4 3 2
(mCH sym), 2900 (mCH as); Anal. (C15H N O S
2
2
4.2.2.21. 5-(Adamantan-1-yl-ureido)-1,3,4-thidiazole-
4
.2.2.17. 4-(3-Adamantan-1-yl-ureido)-benzenesulfon-
2-sulfonamide C8. White crystals, mp 278–280 ꢁC (dec)
1
amide C1. White crystals, mp 277–279 ꢁC (dec)
EtOH); H NMR (DMSO-d ), d, ppm: 8.64 (s, 1H,
(EtOH); H NMR (DMSO-d
NH-thiadiazole), 8.23 (s, 2H, SO NH ), 6.50 (s, 1H,
6
), d, ppm: 10.86 (s, 1H,
1
(
6
2
2
NH-Ph), 7.64 (d, J ¼ 8:8 Hz, 2H, H3,5-Ph), 7.47 (d,
NH-Ad), 2.04 (br s, 3H, 3CHb-Ad), 1.93 (br s, 6H,
13
J ¼ 8:8 Hz, 2H, H2,6-Ph), 7.14 (s, 2H, SO
2
NH ), 6.02 (s,
H, NH-Ad), 2.03 (br s, 3H, 3CHb-Ad), 1.93 (br s, 6H,
2
3CH
2
a-Ad), 1.63 (br s, 6H, 3CH
2
c-Ad); C NMR
1
3
(DMSO-d ), d, ppm: 163.3 (C–NH), 162.7 (C–SO NH ),
6
2
2
13
CH
2
a-Ad), 1.63 (br s, 6H, 3CH
2
c-Ad); C NMR
151.5 (NHCO), 50.9 (Cq-Ad), 41.2 (3Ca-Ad), 35.8 (3Cc-
ꢀ
1
(
DMSO-d
6
), d, ppm: 153.5 (NHCO), 143.7 (C4-Ph),
Ad), 28.9 (3Cb-Ad); IR (KBr), cm : 1180 (mSO sym),
2

M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
2725
1
2
340 (mSO as), 1540 (dNH), 1680 (mC@O), 2850 (mCH sym),
Initial rates of 4-nitrophenyl acetate hydrolysis cata-
lyzed by different CA isozymes were monitored spec-
trophotometrically, at 400 nm, with a Cary 3 instrument
interfaced with an IBM compatible PC. Solutions of
substrate were prepared in anhydrous acetonitrile; the
2
2
910 (mCH as); Anal. (C13
19 5 3 2
H N O S ) C, H, N.
2
52
2
5-(Adamantan-1-yl-ureido)-4-methyl-D -
4.2.2.22.
,3,4-thidiazoline-2-sulfonamide C9. White crystals, mp
1
2
ꢀ2
substrate concentrations varied between 2 · 10 and
1
09–211 ꢁC (EtOH); H NMR (DMSO-d ), d, ppm: 8.29
ꢀ6
6
1
· 10 M, working at 25 ꢁC. A molar absorption
coefficient e of 18,400 M cm was used for the 4-ni-
trophenolate formed by hydrolysis, in the conditions of
52
the experiments (pH 7.40), as reported in the literature.
(s, 2H, SO
CH
2
NH
2
), 7.18 (s, 1H, NH-Ad), 3.77 (s, 3H, N–
ꢀ1
ꢀ1
3
), 2.01 (br s, 3H, 3CHb-Ad), 1.94 (br s, 6H, 3CH a-
2
13
Ad), 1.62 (br s, 6H, 3CH c- Ad); C NMR (DMSO-d ),
2
6
d, ppm: 161.8 (C@N), 159.9 (C–SO
2
NH
2
), 156.0
Nonenzymatic hydrolysis rates were always subtracted
from the observed rates. Triplicate experiments were
done for each inhibitor concentration, and the values
reported throughout the paper are the mean of such
results. Stock solutions of inhibitor (1 mM) were pre-
pared in distilled–deionized water with 10–20% (v/v)
DMSO (which is not inhibitory at these concentrations)
and dilutions up to 0.01 nM were done thereafter with
distilled–deionized water. Inhibitor and enzyme solu-
tions 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 constant
(
NHCO), 50.3 (Cq-Ad), 41.1 (3Ca-Ad), 36.1 (3Cc-Ad),
ꢀ
1
2
8.9 (3Cb-Ad); IR (KBr), cm : 1175 (mSO2sym), 1300
(
(
mSO2as), 1595 (mC@O), 2850 (mCH2sym), 2910 (mCH2as); Anal.
) C, H, N.
14 21 5 3 2
C H N O S
4
.2.2.23.
4-[(3-Adamantan-1-yl-thioureido)-methyl]-
benzenesulfonamide D6. White crystals, mp 203–205 ꢁC
1
(
dec) (EtOH); H NMR (DMSO-d
J ¼ 8:3 Hz, 2H, H3,5-Ph), 7.69 (t, J ¼ 5:6 Hz, 1H, NH–
CH
), 7.41 (d, J ¼ 8:3 Hz, 2H, H2,6-Ph), 7.31 (s, 2H,
SO NH
), 7.11 (s, 1H, NH-Ad), 4.70 (d, 2H, J ¼ 5:6 Hz,
CH -Ph), 2.14 (br s, 6H, 3CH a-Ad), 2.03 (br s, 3H,
6
), d, ppm: 7.76 (d,
2
2
2
52
K
Enzyme concentrations were 3.3 nM for hCA II, and
I
was determined as described by Pocker and Stone.
2
2
13
3
(
1
CHb-Ad), 1.62 (br s, 6H, 3CH
DMSO-d ), d, ppm: 181.01 (C@S), 143.82 (C4Ph),
42.53 (C1-Ph), 127.51 (2C, C3,C5-Ph), 125.67 (2C,
2
c-Ad); C NMR
9
nM for hCA I.
6
C2,C6-Ph), 52.90 (CH -Ph), 45.88 (Cq-Ad), 43.01 (Ca-
2
4
.4. Lipophilicity
Ad), 41.15 (2C, 2Ca-Ad), 35.95 (2C, 2Cc-Ad), 34.91
ꢀ
1
(
(
(
Cc-Ad), 29.04 (3Cb-Ad); IR (KBr), cm : 1160
The retention factor was determined with a immobilized
artificial membrane (IAM) column (1 cm, £ ¼ 3 mm,
Regis Technologies, Morton Grove, IL, USA) con-
nected to a HPLC system (HP 1100 series, Agilent
Technologies) equipped with a diode-array detector.
The mobile phase was 0.01 M PBS (pH 7.4) at a flow of
0.7 mL/min. The column was maintained at 37 ꢁC and
mSO2sym), 1300 (mC@S), 1325 (mSO2as), 1540 (dNH), 2850
m
CH sym), 2910 (mCH as); Anal. (C18
H
25
N
3
O
2
S
2
) C, H, N.
2
2
4
.2.2.24.
4-[2-(3-Adamantan-1-yl-thioureido)-ethyl]-
benzenesulfonamide D7. White crystals, mp 186–188 ꢁC
1
(
dec) (EtOH); H NMR (DMSO-d
J ¼ 8:2 Hz, 2H, H3,5-Ph), 7.40 (d, J ¼ 8:2 Hz, 2H,
H2,6-Ph), 7.30 (s, 2H, SO NH
), 7.20 (t, J ¼ 5:0 Hz, 1H,
NH–CH
), 6.96 (br s, 1H, NH-Ad), 3.59 (dt, J ¼ 7:0,
.0 Hz, 2H, NH–CH
), 2.85 (t, J ¼ 7:1 Hz, 2H, CH
Ph), 2.11 (br s, 6H, 3CH a-Ad), 2.01 (br s, 3H, 3CHb-
6
), d, ppm: 7.75 (d,
0
the drugs detected at 254 nm. The retention factor (log k
2
2
IAM) is expressed as (t ꢀ t Þ=t were t is the retention
R
0
0
R
7;38
3
ꢀ4
Drugs (10 M)
2
time of the drug and t the void time.
0
5
2
2
-
were dissolved in the mobile phase containing 1%
DMSO as void marker. Values are means of three
injections, the standard deviation is less than 1%. For
each drug, the ClogP value was calculated by using the
2
13
Ad), 1.60 (br s, 6H, 3CH
d, ppm: 180.66 (C@S), 143.77 (C4-Ph), 142.07 (C1-Ph),
29.14 (2C, C3,C5-Ph), 125.77 (2C, C2,C6-Ph), 52.72
CH -NH), 43.96 (CH -Ph), 41.19 (3C, 3Ca-Ad), 35.98
3C, 3Cc-Ad), 35.44 (Cq-Ad), 29.04 (3C, 3Cb-Ad); IR
KBr), cm : 1160 (mSO2sym), 1310 (mC@S), 1320 (mSO2as),
550 (dNH), 2850 (mCH sym), 2910 (mCH as); Anal.
2
6
c-Ad); C NMR (DMSO-d ),
39
1
program ChemDraw Ultra 6.0.1.
(
(
(
2
2
ꢀ
1
4.5. Maximal electroshock seizures test
1
2
2
(
C
19
H
27
N
3
O
2
S
2
) C, H, N.
Each compound was intraperitoneally injected to OF1
male mice (26–29 g; Iffa-Credo, Brusseles, Belgium) at a
dose volume of 3 mL/kg. Three hours later, an electrical
stimulus (50 mA; 60 Hz) was delivered for 0.2 s through
corneal electrodes. Protection against seizures was
defined as the abolition of the hind limb tonic extension
4
.3. Enzyme preparations
Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids
pACA/hCA I and pACA/hCA II described by Lindskog
et al. Cell growth conditions were those described by
this group, and enzymes were purified by affinity
chromatography according to the method of Khalifah
and results expressed as the percentage of animals who
40;41
47
did not convulse.
The preliminary screening was
4
8
conducted with groups of 8–12 mice at an intraperito-
neal dose of 30 mg/kg. For compounds A8 and A9,
dose–response were performed at a dosage of: 20, 10, 5
49
et al. Enzyme concentrations were determined spectro-
photometrically at 280 nm, utilizing a molar absorptivity
and 2.5 mg/kg. Nonlinear regression (GraphPad prism
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
of 49 mM cm for CA I and 54 mM cm for CA II,
respectively, based on M
¼ 28:85 kDa for CA I, and
9.30 kDa for CA II, respectively.
software 3.0) was used to determine the intraperitoneal
dose (ED50) protecting 50% of mice against electrically
induced convulsions.
r
50;51
2

2726
M. A. Ilies et al. / Bioorg. Med. Chem. 12 (2004) 2717–2726
28. Grover, L. M.; Lambert, N. A.; Schwartzkroin, P. A.;
Acknowledgements
Teyler, T. J. J. Neurophysiol. 1993, 69, 1541–1555.
9. (a) Tanimukai, H.; Inui, M.; Hariguchi, S.; Kaneko, Z.
Biochem. Pharmacol. 1965, 14, 961–970; (b) Chufan, E. E.;
Pedregosa, J. C.; Baldini, O. N.; Bruno-Blanch, L.
Farmaco 1999, 54, 838–841.
0. Masereel, B.; Rolin, S.; Abbate, F.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2002, 45, 312–320.
1. Almajan, L. G.; Supuran, C. T. Rev. Roumaine Chim.
2
This work was financed in part by the Romanian Grants
ANSTI-6138 and CNFIS-141 and by the Italian CNR
(
Target projects Biotechnologies). Thanks are addressed
to Mrs. Maria Maganu, Mr. Adrian Popa, and Mr.
Nicolae Foldesi for expert technical assistance.
3
3
1
997, 42, 593–597.
References and notes
32. Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.;
Mincione, G.; Ilies, M. A. Eur. J. Med. Chem. 1998, 33,
247–254.
33. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42,
2641–2650.
1
2
3
. Levy, R. H.; Mattson, R. H.; Meldrum, B. S. Antiepileptic
Drugs; 4th ed.; Raven: New York, 1995.
. Unverferth, K.; Rundfeldt, C. Pharmaceuticals 2000, 2,
469–488.
. Barrios, I. A.; Bruno-Blanch, L. E.; Estiu, G. L. J. Mol.
Struct. (Theochem) 2002, 580, 243–250.
. Soderpalm, B. Eur. J. Pain 2002, 6, 3–9.
34. Scozzafava, A.; Briganti, F.; Mincione, G.; Menabuoni,
L.; Mincione, F.; Supuran, C. T. J. Med. Chem. 1999, 42,
3690–3700.
35. Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni, L.;
Ilies, M. A.; Supuran, C. T. J. Med. Chem. 2000, 43, 4884–
4892.
36. Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1999, 14,
343–363.
37. Ducarme, A.; Neuwels, M.; Goldstein, S.; Massingham,
R. Eur. J. Med. Chem. 1998, 33, 215–223.
38. Taillardat-Bertschinger, A.; Galland, A.; Carrupt, P. A.;
Testa, B. J. Chromatogr. A 2002, 953, 39–53.
4
5
. Berk, M.; Segal, J.; Janet, L.; Vorster, M. Drugs 2001, 61,
1
407–1414.
. Nicholson, B. Int. Congress Symp. Ser.––Royal Soc. Med.
001, 248, 33–41.
. MacDonald, K. J.; Young, L. T. CNS Drugs 2002, 16,
49–562.
6
7
2
5
8
9
. Taylor, C. P. Mech. Med. Neuropathic Pain 2002, 211–232.
. Bialer, M.; Johannessen, S. I.; Kupferberg, H. J.; Levy,
R. H.; Loiseau, P., et al. Epilepsy Res. 1999, 34, 1–41.
1
1
1
1
1
0. Duncan, J. S. Br. J. Clin. Pharmacol. 2002, 53, 123–131.
1. Perucca, E. Fund. Clin. Pharmacol. 2001, 15, 405–417.
2. Tomson, T. Curr. Sci. 2002, 82, 698–706.
3. Perucca, E. Pharmacol. Res. 1997, 35, 241–256.
4. Dodgson, S. J.; Shank, R. P.; Maryanoff, B. E. Epilepsia
39.
C
hemDraw Ultra 6.0.1, available from CambridgeSoft
Com, Cambridge, MA.
40. Masereel, B.; Lambert, D.; Dogn ꢀe , J. M.; Poupart, J. H.;
Delarge, J. Epilepsia 1997, 38, 334–337.
41. Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hesie, B.
J.; Kupferberg, H. J.; Scoville, B.; White, B. G. Cleve.
Clin. Q 1984, 51, 293–305.
42. Jitianu, A.; Ilies, M. A.; Briganti, F.; Scozzafava, A.;
Supuran, C. T. Metal-Based Drugs 1997, 4, 1–7.
43. Jitianu, A.; Ilies, M. A.; Scozzafava, A.; Supuran, C. T.
Main Group Metal Chem. 1997, 20, 151–156.
44. Cingolani, E. Gazz. Chim. Ital. 1948, 78, 275–282.
45. Scudi, J. V. J. Am. Chem. Soc. 1937, 59, 1480–1483.
46. Ilies, M. A.; Vullo, D.; Pastorek, J.; Scozzafava, A.; Ilies,
M.; Caproiu, M. T.; Pastorekova, S.; Supuran, C. T.
J. Med. Chem. 2003, 46, 2187–2196.
47. Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.;
Jonsson, B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure–
Function Relations in Human Carbonic Anhydrase II as
Studied by Site-Directed Mutagenesis. In Carbonic Anhy-
drase––from Biochemistry and Genetics to Physiology and
Clinical Medicine; Botr ꢁe , F., Gros, G., Storey, B. T., Eds.;
VCH: Weinheim, 1991; pp 1–13.
2000, 41, S35–S39.
1
1
1
1
1
5. Shank, R. P.; Gardocki, J. F.; Streeter, A. J.; Maryanoff,
B. E. Epilepsia 2000, 41, S3–S9.
6. Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther. Pat.
000, 10, 575–600.
7. Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther. Pat.
002, 12, 217–242.
8. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
003, 23, 146–189.
2
2
2
9. Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David,
S.; Waldeck, H.; Sch €a fer, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 841–845.
2
0. Herrero, A. I.; Del Olmo, N.; Gonzalez-Escalada, J. R.;
Solis, J. M. Neuropharmacology 2002, 42, 210–220.
1. Carbonic Anhydrase––Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC (Taylor
2
&
2. Maren, T. H. Physiol. Rev. 1967, 47, 595–781.
Francis Group): Boca Raton, FL, 2004; pp 1–368.
2
2
3. Pasternack, M.; Voipio, J.; Kaila, K. Acta Physiol. Scand.
48. Behravan, G.; Jonsson, B. H.; Lindskog, S. Eur. J. Bio-
chem. 1990, 190, 351–357.
1993, 148, 229–231.
2
4. Lee, J.; Taira, T.; Pihlaja, P.; Ransom, B. R.; Kaila, K.
Brain Res. 1996, 706, 210–216.
49. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson,
S. M. Biochemistry 1977, 16, 2241–2247.
2
2
5. Voipio, J.; Kaila, K. Prog. Brain Res. 2000, 125, 329–338.
6. Uusisaari, M.; Smirnov, S.; Voipio, J.; Kaila, K. Neuro-
pharmacology 2002, 43, 563–572.
50. Lindskog, S.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A.
1964, 70, 2505–2508.
51. Steiner, H.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem.
1975, 59, 253–259.
52. Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668–
678.
2
7. Rivera, C.; Voipio, J.; Payne, J. A.; Ruusuvuori, E.;
Lahtinen, H.; Lamsa, K.; Pirvola, U.; Saarma, M.; Kaila,
K. Nature 1999, 397, 251–255.