Carbonic anhydrase inhibitors: Anticonvulsant sulfonamides incorporating valproyl and other lipophilic moieties

Article abstract of DOI:10.1021/jm0109199

A series of aromatic/heterocyclic sulfonamides incorporating valproyl moieties were prepared to design antiepileptic compounds possessing in their structure two moieties known to induce such a pharmacological activity: Valproic acid, one of the most widely used antiepileptic drugs, and the sulfonamide residue included in acetazolamide and topiramate, two carbonic anhydrase inhibitors with antiepileptic properties. Some of these derivatives showed very high inhibitory potency against three carbonic anhydrase (CA) isozymes, such as CA I, CA II, and CA IV, involved in important physiological processes. Topiramate, a recently developed antiepileptic drug possessing a sulfamate moiety, also shares this property, although earlier literature data reported this compound to be a weak-moderate CA I, II, and IV inhibitor. The valproyl derivative of acetazolamide (5-valproylamido-1,3,4-thiadiazole-2-sulfonamide, 6M) was one of the best hCA I and hCA II inhibitor in the series and exhibited very strong anticonvulsant properties in an MES test in mice. In consequence, other 1,3,4-thiadiazolesulfonamide derivatives possessing potent CA inhibitory properties and substituted with different alkyl/arylcarboxamido/sulfonamido/ureido moieties in the 5 position have been investigated for their anticonvulsant effects in the same animal model. It was observed that some lipophilic derivatives, such as 5-benzoylamido-, 5-toluenesulfonylamido-, 5-adamantylcarboxamido-, and 5-pivaloylamido-1,3,4-thiadiazole-2-sulfonamide, show promising in vivo anticonvulsant properties and that these compounds may be considered as interesting leads for developing anticonvulsant or selective cerebrovasodilator drugs.

Full text of DOI:10.1021/jm0109199

312
J . Med. Chem. 2002, 45, 312-320
Ca r bon ic An h yd r a se In h ibitor s: An ticon vu lsa n t Su lfon a m id es In cor p or a tin g
Va lp r oyl a n d Oth er Lip op h ilic Moieties
Bernard Masereel,† Ste´phanie Rolin,† Francesco Abbate,‡ Andrea Scozzafava,‡ and Claudiu T. Supuran*,‡,§
Department of Pharmacy, University of Namur, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium, Laboratorio di
Chimica Inorganica e Bioinorganica, Universita` degli Studi di Firenze, Via Gino Capponi 7, I-50121, Florence, Italy, and
CSGI, c/ o Universita` degli Studi di Firenze, Via Gino Capponi 7, I-50121 Florence, Italy
Received May 7, 2001
A series of aromatic/heterocyclic sulfonamides incorporating valproyl moieties were prepared
to design antiepileptic compounds possessing in their structure two moieties known to induce
such a pharmacological activity: valproic acid, one of the most widely used antiepileptic drugs,
and the sulfonamide residue included in acetazolamide and topiramate, two carbonic anhydrase
inhibitors with antiepileptic properties. Some of these derivatives showed very high inhibitory
potency against three carbonic anhydrase (CA) isozymes, such as CA I, CA II, and CA IV,
involved in important physiological processes. Topiramate, a recently developed antiepileptic
drug possessing a sulfamate moiety, also shares this property, although earlier literature data
reported this compound to be a weak-moderate CA I, II, and IV inhibitor. The valproyl
derivative of acetazolamide (5-valproylamido-1,3,4-thiadiazole-2-sulfonamide, 6M) was one of
the best hCA I and hCA II inhibitor in the series and exhibited very strong anticonvulsant
properties in an MES test in mice. In consequence, other 1,3,4-thiadiazolesulfonamide
derivatives possessing potent CA inhibitory properties and substituted with different alkyl/
arylcarboxamido/sulfonamido/ureido moieties in the 5 position have been investigated for their
anticonvulsant effects in the same animal model. It was observed that some lipophilic
derivatives, such as 5-benzoylamido-, 5-toluenesulfonylamido-, 5-adamantylcarboxamido-, and
5-pivaloylamido-1,3,4-thiadiazole-2-sulfonamide, show promising in vivo anticonvulsant proper-
ties and that these compounds may be considered as interesting leads for developing
anticonvulsant or selective cerebrovasodilator drugs.
In tr od u ction
well as different neurological/neuromuscular pathologies
such as epilepsy,1,4,6 genetic hemiplegic migraine and
ataxia,7 tardive dyskinesia,8 hypokalemic periodic pa-
ralysis,9,10 essential tremor and Parkinson’s disease,11
and mountain sickness,12,13 among others.
Carbonic anhydrase (CA, EC 4.2.1.1), a widely occur-
ring enzyme in higher vertebrates, is also quite abun-
dant in the brain, being present in the glia but not
neurons, mainly as the cytosolic isozymes CA II, CA VII,
and the mebrane-bound isoform CA XIV.1-3 The func-
tion of this enzyme in the brain is not well defined, but
since the choroid plexus of all vertebrates has a 10 times
higher concentration of CA than the eye (a tissue very
rich in this enzyme) and since the cerebrospinal fluid
(CSF) contains a high amount of bicarbonate, it is
obvious that CAs (catalyzing with high efficiency the
reversible interconversion between carbon dioxide and
bicarbonate) are involved in the secretion of this fluid
(as they are analogously involved in the secretion of the
ocular fluid).1-3 It has been proved that inhibition of
the brain CAs causes a selective increase of the cerebral
blood flow, with the concomitant raising of the carbon
dioxide partial pressure.1-4 As a consequence, CA
inhibitors are useful in the treatment of conditions
associated with increased intracranial pressure,1-5 as
Several sulfonamide CA inhibitors such as acetazol-
amide 1,1,4 methazolamide 2,1,4 topiramate 3,14 and
zonisamide 415 were and are still used as antiepileptic
drugs. The anticonvulsant effects of these or related
sulfonamides are probably due to CO2 retention second-
ary to inhibition of the red cell and brain enzymes,1,4
but other mechanisms of action, such as blockade of
sodium channels and kainate/AMPA receptors, as well
as enhancement of GABA-ergic transmission, were also
hypothesized/proved for some of these drugs.14 Lipo-
philic derivatives, such as methazolamide 21,4 or the tert-
butoxycarbonyl derivative of acetazolamide 516 are more
effective anticonvulsants than acetazolamide itself,
proving in this way that the penetrability of the drug
to brain is an important factor influencing biological
activity. Acetazolamide and methazolamide are still
clinically used nowadays in some forms of epilepsy, but
they are considered to belong to a minor class of anti-
epileptic agents.16 The recently developed drug, topira-
mate 3, is a very effective antiepileptic,14 and it also acts
as a strong CA inhibitor with a potency similar to that
of acetazolamide against the physiologically important
* To whom correspondence should be addressed. Phone: +39-
055-2757551. Fax: +39-055-2757555. E-mail: claudiu.supuran@
unifi.it.
† University of Namur.
‡ Universita` degli Studi di Firenze.
§ CSGI.
10.1021/jm0109199 CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/19/2001

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 313
isozyme CA II (unpublished results from this laboratory;
see later in the text).
A widely used antiepileptic drug, on the other hand,
is valproic acid (or its sodium salt), VPA 6.17 A large
variety of VPA derivatives or related compounds with
potent antiepileptic properties have been reported,18-21
among which are many VPA amides, esters, and phos-
phonate analogues, but no sulfonamides incorporating
this moiety have been prepared and assayed as anti-
convulsants up to now.
Considering our interest in developing sulfonamide
CA inhibitors with different biological activity,22-26 we
investigated the possibility of designing anticonvulsant
sulfonamides by using acetazolamide 1, topiramate 3,
and valproic acid 6 as lead molecules. Thus, aromatic/
heterocyclic sulfonamides incorporating VPA moieties
in their molecule are reported here, together with their
inhibitory properties against several physiologically
relevant CA isozymes. Some of the best in vitro CA
inhibitors were then tested in vivo for their anticonvul-
sant activity by a maximal electroshock seizure (MES)
test in rats. Structure-activity relationship data were
then used for the evaluation of other anticonvulsant
sulfonamides incorporating lipophilic moieties other
than the valproyl one. Among the new derivatives
investigated here, some compounds showed a more
potent anticonvulsant activity compared to the clinically
used drug topiramate.
inhibition of three CA isozymes (CA I, II, and IV)29-34
known to play a critical role in electrolyte secretion/CO2
transport processes in a variety of tissues,1 using the
Resu lts
Ch em istr y. The chemical structures of sulfonamides
A-T on which the valproyl moiety has been attached
are shown. The sulfonamides obtained by attaching the
valproyl moiety of 6 to the amino/hydroxy group of
sulfonamides A-T will be designated as 6A-6T.
Two synthetic approaches have been used for the
preparation of the new sulfonamides reported here: (i)
reaction of valproic acid 6 with sulfonamides A-T in
the presence of carbodiimides, as reported previously
by this group,23 leading to amides (6A-6P ) or esters
(6Q-6T) (Woltersdorf et al.28b showed that in the condi-
tions used here, only the O-acylation reaction occurs,
without the concomitant acylation of the sulfonamide
moiety) and (ii) reaction of valproyl chloride with
sulfonamides A-T in the presence of base such as
tertiary amines, sodium bicarbonate, etc.25-28 By similar
procedures, some other sulfonamides of types 7-15
investigated here for their anticonvulsant properties
were also obtained.
esterase activity of these isozymes with 4-nitrophenyl-
acetate as substrate, and the inhibition data are shown
in Table 1.35,36
Ca r bon ic An h yd r a se In h ibitor y Activity. The
new sulfonamides reported here were assayed for

314 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Masereel et al.
Ta ble 1. Inhibition Data of Carbonic Anhydrase for
Ta ble 2. Extent of Protected Mice (in %) against Convulsions
in the MES Test at 0.5 and 3 h following Intraperitoneal
Injection of 30 or 10 mg/kg of CA Inhibitora
Derivatives Reported in the Present Papera
KI (nM)
mp
(°C)
30 mg/kg
0.5 h
10 mg/kg
no.
inhibitor
hCA Ib
hCA IIb
bCA IVc
220
240
54
ntd
130
compound
3 h
0.5 h
3 h
1
2
3
4
acetazolamide 900
methazolamide 780
topiramate
zonisamide
5
12
14
5
4300d
9
methazolamide 2
83 (12)
100 (6)
75 (4)
96 (14)
25 (6)
83 (6)
92 (6)
33 (6)
8 (6)
83 (6)
75 (6)
67 (6)
25 (6)
100 (12)
83 (6)
60 (5)
93 (14)
67 (6)
25 (6)
75 (6)
25 (6)
33 (6)
96 (12)
100 (12)
38 (12)
33 (6)
75 (8)
62 (8)
88 (8)
50 (8)
250
ntd
460
topiramate 3
5b
6M
7
25 (8)
12 (8)
100 (14)
62 (8)
6A
6B
6C
6D
6E
6F
23000 (45400) 255 (295) 950 (1310) 204-6
19000 (25000) 221 (240) 740 (2200) 233-5
22500 (28000) 240 (300) 630 (3000) 278-9
35600 (78500) 300 (320) 950 (3200) 288-9
3700 (25000) 96 (170) 210 (2800) 255-6
3500 (21000) 84 (160) 200 (2500) 234-6
8
9
10
11
12
13
14
15
25 (8)
44 (8)
93 (8)
87 (8)
6G
6H
6I
6J
6K
6L
6M
6N
6O
1000 (8300)
950 (9800)
600 (6500)
550 (6000)
970 (6100)
800 (8400)
50 (8600)
70 (9300)
360 (455)
62 (70)
45 (60)
50 (110) 125 (320)
110 (180)
240-1
246-7
244-5
252-4
298-9
281-3
274-6
218-20
36 (40)
43 (70)
26 (28)
70 (75)
6 (60)
10 (19)
9 (3)
49 (66)
85 (125)
155 (175)
133 (160)
25 (540)
42 (355)
76 (125)
16 (19)
a
Rate of mice (n ) 6-14 for each group) protected against
b
seizures induced by an electroshock (50 mA; 0.2 s). Data from
ref 16 (obtained at a dose of 315 µmol/kg of compound 5).
Ta ble 3. Time-Course Activity of 6M (30 mg/kg ip) in the ME
Test with Six to Eight OF1 Male Mice for Each Time
>300
6P
7 (9)
189-91
176-8
164-5
213-4
201-3
time (h)
MES test
% of protected mice
6Q
50 (55)
7 (8)
15 (17)
6R
50 (50)
7 (7)
13 (15)
0.5
1
7.5/8 + 6/6
6.5/8
96
81
6S
1500 (24000) 115 (125) 333 (560)
1300 (18000) 844 (110) 313 (450)
6T
2
3
4
8/8
7/8 + 6/6
4/8
100
93
50
728
900
38
5
10
2
4
51
21
3
841
926a
1042
1143
1225b
1344
1426a
1545
6
5/8
63
260
3
850
110
7
1.2
4
10
9
120
12
65
46
6
Ta ble 4. Lipophilicity (CLogP) Data for Compounds 1-15
Investigated in This Work
5
compound
ClogP a
compound
ClogP a
5.5
1.5
4
1
2
3
4
5
6M
7
-1.1308
0.0880
0.0415
-0.3630
0.3178
1.8231
-0.3342
0.3581
9
10
11
12
13
14
15
0.4788
1.3691
0.9413
1.5281
0.1061
0.0388
-0.2233
a
The data in parentheses represent inhibition by the parent
sulfonamide A-T). b Human (cloned) isozymes. c From bovine lung
d
microsomes, by the esterase method. nt ) not tested. Data from
ref 37.
Ma xim a l Electr osh ock Seizu r es Test. The anti-
convulsant activity of the most active CA inhibitors was
examined at 10 and 30 mg/kg (ip) in mice by using the
MES test, which detects drugs that prevent spread of
tonic-clonic seizures.37 At the desired time following ip
administration (0.5 or 3 h), the electrical stimulus was
delivered and the extent of protected mice was noted
(Table 2). The time course activity of the most active
compound (6M) was then evaluated up to 6 h after
injection (Table 3).
8
a
Calculated with the program ChemDraw Ultra 6.0.1.
prompted us to prepare the even more lipophilic valproic
acid derivatives of the aromatic/heterocyclic sulfona-
mides A-T, previously used to design CA inhibitors
with topical antiglaucoma activity.22-26
The valproyl moiety was introduced into the mol-
ecules of these new derivatives either by reacting the
free acid 6 with amines (A-P ) or alcohols (Q-T) in the
presence of carbodiimides23,28 or by reacting valproyl
chloride (obtained from 6 and SOCl2)28 with amines/
alcohols A-T in the presence of bases (triethylamine,
pyridine, or sodium bicarbonate).23,28 The amides/esters
6A-6T were obtained in good yields by both these
very simple methods. It has been demonstrated by
Woltersdorf et al.28b that under such conditions only
the O-acylation reaction takes place without the con-
comitant N-acylation of the sulfonamide moiety.
Ca r bon ic An h yd r a se In h ibitor y Activity. The
data of Table 1 show that the new inhibitors prepared
by attaching valproyl moieties to aromatic/heterocyclic
sulfonamides A-T are generally more effective, com-
pared to their parent sulfonamide from which they were
prepared, toward the three investigated isozymes hCA
I, hCA II, and bCA IV (h ) human; b ) bovine isozyme).
The enhanced inhibitory power of these compounds is
ClogP Ca lcu la tion s. The lipophilicity of some of the
target compounds used for the in vivo measurements
reported above has been calculated (Table 4) with the
program ChemDraw Ultra 6.0.1.
Discu ssion
Ch em istr y. Few sulfonamides with anticonvulsant
properties have been reported. Thus, Ganz et al.27
showed that some substituted benzenesulfonamides
such as 3-chloro-4-phenacetamidobenzenesulfonamide
exhibited good anticonvulsant activity in experimental
animals, activity that was decreased by substituting the
SO2NH2 moiety and increased by introducing lipophilic
substituents to the aromatic ring. More recently, Chufan
et al.16 demonstrated that the more liphophilic aceta-
zolamide analogue 5 shows better anticonvulsant prop-
erties than the lead molecule 1 itself. All these data

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 315
F igu r e 1. hCA II/6M adduct. The inhibitor has been docked within the enzyme active site by using the acetazolamide/hCA II
adduct (PDB file 1AZM).38a The Zn(II) ion (central violet sphere), its three histidine ligands (in green, His 94, His 96, and His
119), and the proton shuttle residues His 64 and His 4 (situated at the entrance of the active site) are evidenced. The valproyl
moiety of the inhibitor points toward the hydrophobic half of the active site.
presumably due to the interaction of the relatively long
valproyl moiety incorporated in their molecules with
hydrophobic patches within the enzyme active site, as
observed for inhibitors previously reported by this
group,1,2,22-25 a fact explained thereafter by detailed
QSAR models.26 This is particularly well exemplified for
the inhibitor 6M, one of the most potent in the new
series reported here, which has been docked within the
hCA II active site, using the X-ray coordinates of the
acetazolamide/hCA II adduct (Figure 1).38a It may be
seen that the deprotonated sulfonamide moiety of the
inhibitor is coordinated to the Zn(II) ion of the enzyme
and its NH moiety donates a hydrogen bond to the
Oγ of Thr 199, which in turn donates a hydrogen bond
to the carboxylate group of Glu 106, as for other hCA
II/sulfonamide adducts previously reported.1,2,38 One of
the oxygen atoms of the SO2NH moiety also participates
in a hydrogen bond with the backbone NH moiety of
Thr 199.1,2,38 This binding is in fact expected because
the new inhibitor 6M has a carboxamidothiadiazole
sulfonamide moiety similar to acetazolamide (1) and its
derivative (5). In contrast to acetazolamide, whose
acetamido moiety does not participate in any particular
interaction with active site residues,38a the long hydro-
phobic valproyl moiety of the inhibitor 6M is oriented
toward the hydrophobic half of the active site, being in
van der Waals contact with the following amino acid
residues: Val 121, Phe 130, Val 135, Leu 141, Val 143,
Val 207, and Pro 201, all of which line the hydrophobic
half of the CA active cavity.38,39a The two hydrophilic,
catalytically relevant residues His 64 and His 4 (the CA
II proton shuttle residues)39b are also shown in Figure
1, since they are two of the most important residues
situated on the hydrophilic half of the active site. Thus,
it is obvious that the extensive hydrophobic interactions
between the valproyl moiety of 6M and amino acid
residues situated in the hydrophobic half of the active
site cavity have as an effect that 6M (KI ) 6 nM) is twice
more potent a hCA II inhibitor compared to acetazola-
mide 1 (KI ) 12 nM) (see Table 1).
The nature of the sulfonamide attached to the val-
proyl moiety in the new derivatives 6A-6T reported
here greatly influenced the CA inhibitory power of these
compounds. Among the synthesized derivatives, the
heterocyclic sulfonamides (6M-6R) were more active
than the aromatic ones (6A-6L, 6S, 6T). The efficiency
of the obtained inhibitor generally varied in the follow-
ing way based on the parent sulfonamide from which it
was prepared: the derivatives of p-hydrazinobenzene-
sulfonamide < the orthanilamides < the metanilamides
< the sulfanilamides < the homosulfanilamides < the
p-aminoethylbenzenesulfonamides = the halogeno-
substituted sulfanilamides = the 1,3-benzene-disulfona-
mides < 4-methyl-δ2-1,3,4-thiadiazoline-2-sulfonamide
< the benzothiazole-2-sulfonamides = the 1,3,4-thia-
diazole-2-sulfonamides. All three CA isozymes investi-

316 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Masereel et al.
gated here were susceptible to inhibition with this type
of sulfonamide. hCA II and bCA IV were the most
sensitive, whereas hCA I was generally less susceptible
to inhibition compared to the first two isozymes.
class of derivatives and thus to better understand SAR
for obtaining anticonvulsant sulfonamides/sulfamates
(Table 4).
Ma xim a l Electr osh ock Seizu r es Test. The MES
test has been performed with compounds 6M and 7-15
as well as methazolamide 2 and topiramate 3 as
standards. Thus, compounds with powerful CA inhibi-
tory properties (KI values against hCA II ranging from
1 to 14 nM) and characterized by a wide range of
physicochemical properties (ClogP in the range 1.8 to
-0.36) were included in these experiments (Tables 2 and
3). Each CA inhibitor (30 or 10 mg/kg) was injected
intraperitoneally to mice at a dose volume of 3 mL/kg.
At the desired time following injection (0.5 or 3 h), an
electrical stimulus was delivered and the protection
extent against convulsions was registered.
It must also be stressed that topiramate 3, a lipophilic
sugar derivative possessing a sulfamate moiety, tested
under the same conditions as the other sulfonamides
investigated here, behaved as a very strong CA inhibi-
tor, being together with the valproyl derivative 6M one
of the most effective inhibitor in the investigated series
(Table 1). Thus, both 3 and 6M are more effective
inhibitors than acetazolamide 1 or methazolamide 2
against all three CA isozymes investigated in the
present study. Against isozyme II the two compounds
practically possess the same efficacy (KI values of 5 nM
for 3 and 6 nM for 6M), whereas the latter compound
is a much more potent CA I and CA IV inhibitor
compared to topiramate (Table 1). Our results are thus
in contradiction with the early report of Shank’s group
that topiramate is a weak CA inhibitor but consistent
with the presence of CA II in the brain.14a It is also
true that the same group recently reported different
results, showing that 3 is a much stronger CA inhibitor,
but with an efficacy 10 times lower than that of
acetazolamide, against a large number of CA isozymes.40
Obviously, the assay methods of this and the above-
mentioned study40 are different, as different as the
source of enzymes. Our data clearly show that topira-
mate is a potent CA II inhibitor, and this statement is
also very much supported by the side effects seen in
many patients treated with this antiepileptic drug,14d
which are typical for the strong sulfonamide CA inhibi-
tors used as systemic antiglaucoma agents (such as
acetazolamide, methazolamide) and include paresthe-
sias, nephrolithiasis, and weight loss, among others.1
We must also mention that zonisamide 4 has also been
reported to act as a weak CA inhibitor,37 but since this
compound is not available yet in Europe, we did not test
its effect on the three CA isozymes of our interest and
the inhibition data of Table 1 are taken from the
literature.37 Being an aliphatic sulfonamide derivative,
4 is expected to act as a weaker CA inhibitor compared
to the aromatic/heterocyclic sulfonamides or the sulfa-
mate investigated here.1b
The data of Table 2 show that several of the investi-
gated compounds, such as 6M, 8, 9, 12, and 13, similar
to methazolamide 2 and topiramate 3, efficiently protect
mice against seizures induced by electroshock at a
dosage of 30 mg/kg. The protection rate was of 75-100%
at 0.5 h and 25-100% at 3 h after drug administration.
All these compounds were quite lipophilic, possessing
ClogP in the range 0.35-1.82. Other investigated sul-
fonamides, such as 7, 10, 14, and 15, showed less
effective protection against electroshock-induced sei-
zures, with a protection rate ranging from 25% to 67%
at 0.5 h and from 25% to 67% at 3 h drug injection
(Table 2). These derivatives were generally less lipo-
philic than the first subseries mentioned above (CLogP
of -0.33 to 0.038) except for 10, which has a lipophilicity
comparable to that of the compounds discussed earlier
(CLogP of 1.36). The least effective sulfonamide was 11,
which produced a protection of 8% at 0.5 h and 33% at
3 h following administration, although this is a rather
lipophilic derivative, possessing a ClogP of 0.94. The
compounds of the first group (2, 3, 6M, 9, 12, 13) were
also investigated at a decreased dosage of 10 mg/kg
(Table 2). In these experiments, methazolamide pro-
duced a protection of 75% at 0.5 h and 88% at 3 h
postadministration, being slightly more effective than
topiramate (protection of 62% at 0.5 h and 50% at 3 h
postadministration). The most effective compounds in
the investigated series were 6M, 12, and 13, which
showed a rate of protection in the range 25-44% at 0.5
h and 87-100% at 3 h after administration. Less
effective was 9, showing a protection of 12% after 0.5 h
and 62% after 3h. The correlation of the lipophilicity
(ClogP), in vitro CA inhibition, and in vivo anticonvul-
sant activity of these compounds is thus not really
straightforward. It seems that a strong CA inhibitor
(with an inhibition constant against hCA II in the 1-15
nM range) also possessing a good lipophilicity should
intrinsically lead to powerful anticonvulsant activities
(such as in the case of 6M, the best anticonvulsant in
this series, possessing a ClogP of 1.82, or 12, with a
ClogP of 1.52). On the other hand, our data also show
that parameters other than CA inhibition and lipophi-
licity may strongly influence the in vivo anticonvulsant
properties, since some less lipophilic compounds (such
as 13, ClogP of 0.10) showed in vivo activity similar to
that of 6M and 12, whereas many quite lipophilic
compounds (for example, 10, ClogP of 1.36) possessed a
rather diminished anticonvulsant activity. Plasma pro-
Since the most effective CA inhibitors 3 and 6M
showed very good anticonvulsant properties in the MES
test (see later in the text and Table 2), several other
thiadiazolesulfonamides (7-15) structurally related to
6M were also included in the study. The CA inhibitory
properties of these derivatives (Table 1) show all of
them to be low nanomolar inhibitors of hCA II (KI
values ranging from 1.2 to 10 nM), similar to deriva-
tive 5 reported by Chufan et al.,16 and to appreciably
inhibit the other two isozymes hCA I and bCA IV.
These compounds (7-15), reported previously by our
group,25b,26a,28,39,41-45 were generally designed in order
to obtain novel antiglaucoma sulfonamides, and they
possess different alkyl/arylcarboxamido, arylsulfonamido,
or arylsulfonylureido moieties in the 5 position of
the thiadiazole ring. These moieties were chosen in
such a way as to ensure a wide range of lipophilicities
for these thiadiazolesulfonamides (ClogP data in the
range 1.8 to -0.36), since this property seems to be
crucial1 for the biological activity/pharmacology of this

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 317
tein binding of these sulfonamides is probably another
important parameter influencing their bioavailability,
since it is known, for example, that acetazolamide or
methazolamide indeed binds to plasma proteins to
different degrees.4 It can be envisaged, on the other
hand, that all these compounds possess pKa values in
the range 7.4-7.6 (for the deprotonation of the SO2NH2
moiety),2,4 and thus, we think that this parameter may
not vary considerably in the series of derivatives inves-
tigated in vivo.
The excellent anticonvulsant activity of the derivative
6M, which incorporates in its structure moieties present
in two antiepileptic drugs (acetazolamide and valproic
acid), prompted us to investigate the time course activity
up to 6 h after injection in mice (Table 3). The obtained
data clearly show this sulfonamide to be highly effective
in protecting mice from electroshock-induced seizures
over a long period of time, with a protection rate of
93-100% in the first 3 h and 50-63% in the next 3 h.
Although other mechanisms of action should be not
excluded, we suggest that the interesting anticonvulsant
properties of some of these sulfonamides investigated
here are due to inhibition of brain CAs, particulary CA
II. In this regard, it is also important to observe that
some of the most lipophilic inhibitors, such as 6M, 8, 9,
12, and 13, showed the best anticonvulsant properties,
which constitutes clear-cut proof that penetration through
the blood-brain barrier is an important factor influenc-
ing bioavailability of the drug, although other factors
(such as drug binding to plasma proteins) may also
considerably influence activity in vivo.
At this point, we also want to stress that inhibition
of brain CAs causes a selective increase of cerebral blood
flow, with a concomitant increase of the carbon dioxide
partial pressure.46 For instance, administration of 12
mg/kg of acetazolamide to rabbits induced an 80%
increase in the brain blood flow.47 In consequence, these
vasodilatory properties of acetazolamide are increas-
ingly being used as a diagnostic tool for testing the
cerebrovascular reserve capacity in patients with chronic
cerebrovascular disease by using both magnetic reso-
nance imaging (MRI) and positron emission tomo-
graphic (PET) methods, devised in order to develop
diagnostic tools that exploit these valuable properties
of the sulfonamide CA inhibitors.47-51 The use of more
potent CA inhibitors and more lipophilic derivatives
than acetazolamide may be of interest for developing
novel diagnostic tools for monitoring cerebrovascular
diseases too.
data reported this compound to be a weak or moderate
CA inhibitor. The valproyl derivative of acetazolamide
(5-valproylamido-1,3,4-thiadiazole-2-sulfonamide, 6M)
was the best inhibitor in the series and exhibited very
strong anticonvulsant properties in the MES test in
mice. In consequence, several other 1,3,4-thiadiazole-
sulfonamide derivatives possessing potent CA inhibitory
properties and substituted with different alkyl/arylcar-
boxamido/sulfonamido/ureido moieties in the 5 position
have been investigated for their anticonvulsant effects
in the same animal model. It was observed that some
derivatives, such as 5-benzoylamido-, 5-toluenesulfonyl-
amido-, 5-adamantylcarboxamido-, and 5-pivaloylamido-
1,3,4-thiadiazole-2-sulfonamide, show promising in vivo
anticonvulsant properties and that these compounds
may be considered as interesting leads for developing
anticonvulsant or selective cerebrovasodilator drugs. A
straightforward correlation among anticonvulsant activ-
ity, CA inhibition, and lipophilicity could not be ration-
alized for this series of compounds, proving thus that
many of the enigmas surrounding sulfonamide antiepi-
leptic drugs still remained unresolved for the moment.
Exp er im en ta l P r otocols
Ch em istr y. Melting points were recorded with a heating
plate microscope and are not corrected. IR spectra were
recorded in KBr pellets with a Carl Zeiss IR-80 instrument.
1H NMR spectra were recorded in DMSO-d6 or TFA as
solvents, with a Bruker CPX200 or Varian 300 instrument.
Chemical shifts are reported as δ values relative to Me4Si as
internal standard. Elemental analyses were done by combus-
tion for C, H, N with an automated Carlo Erba analyzer and
were (0.4% of the theoretical values. All reactions were
monitored by thin-layer chromatography (TLC) using 0.25 mm
precoated silica gel plates (E. Merck). Sulfonamides A-T used
in the syntheses were either commercially available com-
pounds (from Sigma-Aldrich, Milan, Italy, or Acros, Milan,
Italy) or were prepared as described previously.22-26 Topira-
mate was from J anssen Pharmaceutica (Topamax), whereas
acetazolamide, methazolamide, and valproic acid were from
Sigma-Aldrich (Milan, Italy). Other organic/inorganic reagents
were from Sigma-Aldrich, Fluka, or E. Merck and were used
without additional purification. Acetone, acetonitrile, DMF,
and other solvents (E. Merck) used in the synthesis/chroma-
tography were doubly distilled and kept on molecular sieves
in order to maintain them under anhydrous conditions.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6A-6T. Meth od A. An amount of 2 mmol of sulfonamide A-T
was dissolved/suspended in 50 mL of anhydrous acetonitrile
and then treated with a stoichiometric amount (290 mg )
322 µL, 2 mmol) of valproic acid 6. An amount of 380 mg
(2 mmol) of EDCI‚HCl (or the equivalent amount of diisopropyl-
carbodiimide) was then added, and the reaction mixture was
magnetically stirred at room temperature for 15 min. Then a
total of 30 µL (2 mmol) of triethylamine was added and stirring
was continued for 8-16 h at 4 °C (TLC control). The solvent
was evaporated in vacuo and the residue taken up in 50 mL
of water when the lipophilic valproic acid derivatives precipi-
tated and were filtered. The obtained crude sulfonamides were
crystallized from ethanol or mixtures of EtOH/water. Yields
were in the range 85-95%.
Con clu sion s
We prepared a series of aromatic/heterocyclic sulfona-
mides incorporating valproyl moieties to design anti-
convulsant compounds possessing in their structure
moieties known to induce such a biological action (it
is well-known that valproic acid is one of the most
widely used antiepileptic drug, whereas some sulfona-
mides, such as acetazolamide and topiramate, also
possess this pharmacological action). Some of these
derivatives showed very good inhibitory properties
against three CA isozymes involved in important physi-
ological processes, such as CA I, CA II, and CA IV. A
particularly interesting discovery was that topiramate
also shares this property, although earlier literature
Meth od B. An amount of 2 mmol of sulfonamide A-T was
dissolved/suspended in 50 mL of anhydrous acetonitrile and
then treated with a stoichiometric amount (360 mg, 2 mmol)
of valproyl chloride28 dissolved in a small volume of the same
solvent. A stoichiometric amount of base (Et3N, pyridine, or
sodium bicarbonate) was then added, and the reaction mixture
was stirred at room temperature for 3-6 h (TLC control). The
solvent was then evaporated in vacuo, the residue was taken
up in 50-100 mL of cold water, and the crude precipitate

318 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Masereel et al.
obtained was filtered and crystallized from the same solvents
mentioned above. Yields were in the range 90-95%.
(s, 1H, H-7 of benzothiazole), 8.15 (s, 2H, SO2NH2). Anal.
(C15H20N2O4S2) C, H, N.
4-(Va lp r oyla m id o)b en zen esu lfon a m id e, 6C (Met h od
A). White crystals, mp 278-9 °C (EtOH). IR (KBr), cm-1: 1160
(SO2sym), 1335 (SO2as), 1585 (amide II), 1640 (amide I). 1H
NMR (DMSO-d6), δ, ppm; J , Hz: 0.89 (t, 6H, 2Me), 1.10-1.70
(m, 8H, 2 CH2CH2), 2.55 (m, 1H, CH), 7.23 (s, 2H, SO2NH2),
7.78 (d, 2H, AA′BB′, 8.9), 7.91 (d, 2H, AA′BB′, 8.9), 10.03 (s,
1H, CONH). 13C NMR (DMSO-d6), δ, ppm: 6.35 (CH3), 20.36
(CH2), 20.59 (CH2), 30.66 (CH), 119.20 (C2/C3 -Ph), 126.5 (C3/
C2 -Ph), 137.92 (C1/C4 -Ph), 142.39 (C4/C1 -Ph), 159.27
(CONH). Anal. (C14H22N2O3S) C, H, N.
En zym e P r ep a r a tion s. 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.29 (the two plasmids were a gift from Prof. Sven Lindskog,
Umea University, Sweden). Cell growth conditions were those
described by this group,30 and enzymes were purified by
affinity chromatography according to the method of Khalifah
et al.31 Enzyme concentrations were determined spectro-
photometrically at 280 nm, utilizing a molar absorptivity of
49 mM-1 cm-1 for CA I and 54 mM-1 cm-1 for CA II on the
basis of Mr ) 28.85 kDa for CA I and Mr ) 29.30 kDa for CA
II.32,33 CA IV was isolated from bovine lung microsomes as
described by Maren et al., and its concentration has been
determined by titration with ethoxzolamide.34
4-(Va lp r oyla m id om et h yl)b en zen esu lfon a m id e,
6E
(Meth od B). White crystals, mp 255-6 °C (EtOH). IR (KBr),
cm-1: 1160 (SO2sym), 1330 (SO2as), 1560 (amide II), 1630
(amide I). 1H NMR (DMSO-d6), δ, ppm; J , Hz: 0.89 (t, 6H,
2Me), 1.10-1.70 (m, 8H, 2 CH2CH2), 2.55 (m, 1H, CH), 4.49
(d, 2H, 6.0, CH2-C6H4), 7.31 (s, 2H, SO2NH2), 7.46 (d, 2H,
AA′BB′, 8.3), 7.78 (d, 2H, AA′BB′, 8.3), 8.62 (t, 1H, NHCO,
6.1). 13C NMR (DMSO-d6), δ, ppm: 6.38 (CH3), 20.33 (CH2),
20.65 (CH2), 30.62 (CH), 41.58 (CH2), 125.68 (C2/C3 -Ph),
121.56 (C5-pyrrole), 127.41 (C3/C2 -Ph), 142.53(C1/C4 -Ph),
144.19 (C4/C1 -Ph), 160.75 (CONH). Anal. (C15H24N2O3S)
C, H, N.
Initial rates of 4-nitrophenyl acetate hydrolysis catalyzed
by different CA isozymes were monitored spectrophotometri-
cally at 400 nm with a Cary 3 instrument interfaced with an
IBM compatible PC.35 Solutions of substrate were prepared
in anhydrous acetonitrile; the substrate concentrations varied
between 2 × 10-2 and 1 × 10-6 M, working at 25 °C. A molar
absorption coefficient ꢀ of 18 400 M-1 cm-1 was used for the
4-nitrophenolate formed by hydrolysis under the conditions
of the experiments (pH 7.40) as reported in the literature.35
Nonenzymatic hydrolysis rates were always subtracted from
the observed rates. Duplicate 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 prepared 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 there-
after with distilled-deionized water. Inhibitor and enzyme
solutions were preincubated together for 10 min at room
temperature prior to assay to allow for the formation of the
E/I complex. The inhibition constant KI was determined as
described by Pocker and Stone.35 Enzyme concentrations were
3.5 nM for hCA II, 12 nM for hCA I, and 36 nM for bCA IV
(this isozyme has a decreased esterase activity,36 and higher
concentrations had to be used for the measurements).
4-(Valpr oylam idoeth yl)ben zen esu lfon am ide, 6F (Meth -
od A). White crystals, mp 234-6 °C (EtOH/water 1:1). IR
(KBr), cm-1: 1160 (SO2sym), 1330 (SO2as), 1560 (amide II),
1610 (amide I). 1H NMR (DMSO-d6), δ, ppm; J , Hz: 0.89 (t,
6H, 2Me), 1.10-1.70 (m, 8H, 2 CH2CH2 of valproic acid), 2.55
(m, 1H, CH), 2.90 (t, 2H, 7.2), 3.47 (q, 2H, 6.5), 7.29 (s, 2H,
SO2NH2), 7.42 (d, 2H, AA′BB′, 8.2), 7.75 (d, 2H, AA′BB′, 8.2),
8.08 (t, 1H, NHCO, 5.7). 13C NMR (DMSO-d6), δ, ppm: 6.30
(CH3), 20.35 (CH2), 20.58 (CH2), 30.72 (CH), 35.14 (CH2-Ph),
39.70, (N-CH2), 125.68 (C2/C3 -Ph), 129.14 (C3/C2 -Ph),
141.99 (C1/C4 -Ph), 143.89 (C4/C1 -Ph), 160.66 (CONH).
Anal. (C16H26N2O3S) C, H, N.
4-(Va lp r oyla m id o)-3-flu or ob en zen esu lfon a m id e, 6G
(Meth od B). White crystals, mp 240-1 °C (EtOH/water, 1:2).
IR (KBr), cm-1: 1160 (SO2sym), 1320 (SO2as), 1600 (amide
1
II), 1650 (amide I). H NMR (DMSO-d6), δ, ppm; J , Hz: 0.89
(t, 6H, 2Me), 1.10-1.70 (m, 8H, 2 CH2CH2), 2.55 (m, 1H, CH),
7.46 (s, 2H, SO2NH2), 7.65 (ddd, 1H, H2-Ph, 6.1, 2.0), 7.68
(dtr, 1H, H6-Ph, 8.4, 2.0), 7.92 (ddtr, 1H, H5-Ph, 8.3, 4.1,
Ma xim a l Electr osh ock Seizu r es Test. Each compound
was intraperitoneally injected to OF1 male mice (25-28 g; Iffa-
Credo, Les Oncins, France) at a dose volume of 3 mL/kg. At
the desired time, 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. The preliminary screening was conducted with
groups of 6-14 mice at an intraperitoneal dose of 30 mg/kg.
For the most active compounds, the groups contained eight
mice each and the dosage was 10 mg/kg. The electroshock was
applied at 0.5 or 3 h after injection. Then the time-course
activity of 6M was examined up to 6 h after injection of 30
mg/kg.52-54
1.6), 9.84 (s, 1H, NHCO). 13C NMR (DMSO-d6), δ, ppm; J C,F
,
Hz: 6.30 (CH3), 20.28 (CH2), 20.55 (CH2), 30.68 (CH), 113.40
(d, C2-Ph, 22.9), 121.13 (C5-Ph), 123.28 (C6-Ph), 129.39 (d,
C4-Ph, 11.8), 140.81 (d, C1-Ph, 6.2), 153.89 (d, C3-Ph,
249.9), 159.01 (CONH). Anal. (C14H21FN2O3S) C, H, N.
4-(Va lp r oyla m id o)-3-ch lor ob en zen esu lfon a m id e, 6H
(Meth od A). White crystals, mp 246-7 °C (EtOH). IR (KBr),
cm-1: 1160 (SO2sym), 1320 (SO2as), 1580 (amide II), 1660
(amide I). 1H NMR (DMSO-d6), δ, ppm; J , Hz: 0.89 (t, 6H,
2Me), 1.10-1.70 (m, 8H, 2 CH2CH2), 2.55 (m, 1H, CH), 7.49
(s, 2H, SO2NH2), 7.78 (dd, 1H, H6-Ph, 8.4, 2.1), 7.89 (d, 1H,
H5-Ph, 8.5), 7.92 (d, 1H, H2-Ph, 2.1), 9.64 (s, 1H, NHCO).
13C NMR (DMSO-d6), δ, ppm: 6.37 (CH3), 20.29 (CH2), 20.61
(CH2), 30.75 (CH), 123.33 (C6-Ph), 125.08 (C4-Ph), 126.82
(C5-Ph), 127.09 (C2-Ph), 138.14 (C1-Ph), 141.39 (C3-Ph),
158.95 (CONH). Anal. (C14H21ClN2O3S) C, H, N.
Ca lcu la tion s a n d Dock in g. All the molecular mechanics
and dynamics calculations were done with the program MOE
(Molecular Operating Environment).55 The starting point
for modeling the binding of inhibitor 6M within the CA active
site was constituted by the X-ray structure of the adduct of
the structurally related acetazolamide 1 with hCAII reported
by Liljas’s group.38 A total of 30 minimization cycles have
5-Va lp r oyla m id o-1,3,4-th ia d ia zole-2-su lfon a m id e, 6M
(Meth od B). White crystals, mp 274-6 °C (EtOH). IR (KBr),
cm-1: 1170 (SO2sym), 1350 (SO2as), 1580 (amide II), 1630
(amide I). 1H NMR (DMSO-d6), δ, ppm; J , Hz: 0.89 (t, 6H,
2Me), 1.10-1.70 (m, 8H, 2 CH2CH2), 2.55 (m, 1H, CH), 8.31
(s, 2H, SO2NH2), 13.07 (s, 1H, CONH). 13C NMR (DMSO-d6),
δ, ppm: 6.35 (CH3), 20.36 (CH2), 20.59 (CH2), 30.66 (CH),
158.07 (CONH), 161.52 (C2 thiadiazole), 163.38 (C5 thia-
diazole). Anal. (C10H18N4O3S2) C, H, N.
been performed with
a tether constraint on the heavy
atoms gradually lowered till complete relaxation.39a ClogP
qjdata were calculated by using the program ChemDraw Ultra
6.0.1.56
Ack n ow led gm en t. This research was financed by
a grant from the Italian CNRsTarget Project Biotech-
nology and by CSGI (Florence, Italy). Thanks are
addressed to Drs. M.A. Ilies, A. Casini, and M. Barboiu
for expert technical assistance.
2-Su fa m oyl-6-b en zot h ia zolyl Va lp r oa t e 6Q (Met h od
B). White crystals, mp 176-8 °C (EtOH). IR (KBr), cm-1: 1180
1
(SO2sym), 1373 (SO2as), 1748 (COO). H NMR (DMSO-d6), δ,
ppm: 0.88 (t, 6H, 2Me), 1.10-1.73 (m, 8H, 2 CH2CH2), 2.54
(m, 1H, CH), 6.69 (m, 2H, H5, and H6 of benzothiazole), 7.24

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 319
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