Carbonic Anhydrase Inhibitors. Inhibition of Mitochondrial Isozyme V with Aromatic and Heterocyclic Sulfonamides

Article abstract of DOI:10.1021/jm031057+

The first inhibition study of the mitochondrial isozyme carbonic anhydrase (CA) V (of murine origin) with a series of aromatic and heterocyclic sulfonamides is reported. Inhibition data of the cytosolic isozymes CA I and CA II and the membrane-bound isozyme CA IV with these inhibitors are also provided for comparison. Several low nanomolar CA V inhibitors were detected (K _I values in the range of 4-15 nM), most of them belonging to the acylated sulfanilamide, ureido-benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and aminobenzolamide type of compounds. The clinically used inhibitors acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and topiramate on the other hand were less effective CA V inhibitors, showing inhibition constants in the range of 47-63 nM. Some of the investigated sulfonamides, such as the ureido-benzenesulfonamides and the acylated sulfanilamides showed higher affinity for CA V than for the other isozymes, CA II included, which is a remarkable result, since most compounds investigated up to now inhibited the cytosolic isozyme CA II better. These results prompt us to hypothesize that the selective inhibition of CA V, or the dual inhibition of CA II and CA V, may lead to the development of novel pharmacological applications for such sulfonamides, for example in the treatment or prevention of obesity, by inhibiting CA-mediated lipogenetic processes.

Full text of DOI:10.1021/jm031057+

1
272
J . Med. Chem. 2004, 47, 1272-1279
Ca r bon ic An h yd r a se In h ibitor s. In h ibition of Mitoch on d r ia l Isozym e V w ith
Ar om a tic a n d Heter ocyclic Su lfon a m id es
†
‡
‡
§
†
,†
Daniela Vullo, Marco Franchi, Enzo Gallori, J ochen Antel, Andrea Scozzafava, and Claudiu T. Supuran*
Laboratorio di Chimica Bioinorganica, Universit a` degli Studi di Firenze, Rm. 188, via della Lastruccia 3, I-50019 Sesto
Fiorentino (Firenze), Italy, Dipartimento di Biologia Animale e Genetica, Universit a` degli Studi di Firenze, via Romana 17-19,
0122 Firenze, Italy, and Solvay Pharmaceuticals GmbH, Hans B o¨ ckler-Allee 20, D-30173 Hannover, Germany
5
Received October 6, 2003
The first inhibition study of the mitochondrial isozyme carbonic anhydrase (CA) V (of murine
origin) with a series of aromatic and heterocyclic sulfonamides is reported. Inhibition data of
the cytosolic isozymes CA I and CA II and the membrane-bound isozyme CA IV with these
inhibitors are also provided for comparison. Several low nanomolar CA V inhibitors were
detected (K values in the range of 4-15 nM), most of them belonging to the acylated
I
sulfanilamide, ureido-benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and aminoben-
zolamide type of compounds. The clinically used inhibitors acetazolamide, methazolamide,
ethoxzolamide, dorzolamide, brinzolamide, and topiramate on the other hand were less effective
CA V inhibitors, showing inhibition constants in the range of 47-63 nM. Some of the
investigated sulfonamides, such as the ureido-benzenesulfonamides and the acylated sulfa-
nilamides showed higher affinity for CA V than for the other isozymes, CA II included, which
is a remarkable result, since most compounds investigated up to now inhibited the cytosolic
isozyme CA II better. These results prompt us to hypothesize that the selective inhibition of
CA V, or the dual inhibition of CA II and CA V, may lead to the development of novel
pharmacological applications for such sulfonamides, for example in the treatment or prevention
of obesity, by inhibiting CA-mediated lipogenetic processes.
In tr od u ction
histidine residues and a water molecule/hydroxide ion.
The latter is the active species, acting as a potent
At least 14 different R-carbonic anhydrase (CA, EC
.2.1.1) isoforms were isolated in higher vertebrates,
1
,2
nucleophile. For â- and γ-CAs, the zinc hydroxide
mechanism is valid too, although at least some â-class
enzymes do not have water directly coordinated to the
4
where these zinc enzymes play crucial physiological
1
-3
roles.
Some of these isozymes are cytosolic (CA I, CA
4
metal ion. CAs are inhibited primarily by two main
II, CA III, CA VII), others are membrane-associated (CA
IV, CA IX, CA XII and CA XIV), CA V is mitochondrial,
and CA VI is secreted in saliva.
classes of inhibitors: the inorganic anions (such as
cyanide, cyanate, thiocyanate, azide, hydrogen sulfide,
etc.) and the unsubstituted sulfonamides possessing the
general formula RSO2NH2 (R ) aryl, hetaryl, perha-
1
-3
Three acatalytic
forms are also known, which are denominated CA-
related proteins (CARP), CARP VIII, CARP X, and
1
,2
1
-3
loalkyl). Several important physiological and physio-
pathological functions are played by the CA isozymes
present in organisms all over the phylogenetic tree,
related to respiration and transport of CO2/bicarbonate
between metabolizing tissues and the lungs, pH and
CO2 homeostasis, electrolyte secretion in a variety of
tissues/organs, biosynthetic reactions, such as the lipo-
genesis, gluconeogenesis, and ureagenesis among others
(in animals), CO2 fixation (in plants and algae), etc.1
The presence of these ubiquitous enzymes in so many
tissues and in so many different isoforms represents an
attractive goal for the design of inhibitors or activators
CARP XI.
Representatives of the â- and γ-CA family
4
are highly abundant in plants, bacteria, and archaea.
These enzymes are very efficient catalysts for the
reversible hydration of carbon dioxide to bicarbonate,
but at least the R-CAs possess a high versatility, being
able to catalyze different hydrolytic processes, such as
the hydration of cyanate to carbamic acid, or of cyana-
mide to urea, the aldehyde hydration to gem-diols, and
the hydrolysis of carboxylic or sulfonic acids esters, as
well as other less investigated hydrolytic processes, such
as hydrolysis of halogeno derivatives, arylsulfonyl ha-
lides, etc.1 It is not known whether reactions catalyzed
by CAs other than the hydration of CO2/dehydration of
,2,4
,2
1
,2
with biomedical applications.
-
HCO3 may have physiological relevance in systems
Only CA V is present in mitochondria, among the
many R-CA isoforms found in animals. This isozyme was
shown to be involved in several biosynthetic processes,
1
where these enzymes are present. The catalytic mech-
anism of the R-CAs is understood in great detail: the
active site consists of a Zn(II) ion coordinated by three
5
6
such as ureagenesis, gluconeogenesis, and lipogenesis,
both in vertebrates (rodents) and in invertebrates
*
Correspondence author. Tel. +39-055-4573005: Fax: +39-055-
7-10
(locust).
Indeed, in several important biosynthetic
4
573385; E-mail: claudiu.supuran@unifi.it.
†
processes involving pyruvate carboxylase, acetyl CoA
carboxylase, and carbamoyl phosphate synthetases I
and II, bicarbonate, not carbon dioxide, is the real
substrate of these carboxylating enzymes, and the
Laboratorio di Chimica Bioinorganica, Universit a` degli Studi di
Firenze.
‡
Dipartimento di Biologia Animale e Genetica, Universit a` degli
Studi di Firenze.
§
Solvay Pharmaceuticals Gmb.
1
0.1021/jm031057+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/29/2004

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1273
provision of enough bicarbonate is assured mainly by
the catalysis involving the mitochondrial isozyme CA
V (probably assisted by the high activity cytosolic
chlorides in the presence of bases, as reported earlier
for structurally related compounds.
1
6-25
8
,11-14
isozyme CA II).
Considering that inhibition of the mitochondrial en-
zyme with sulfonamides was not investigated up to now,
and that this information may be important for obtain-
1
5
ing inhibitors with new pharmacological applications,
we conducted a detailed such study, using the murine
full length isozymes mCA V. Inhibition data for clasical
sulfonamide CA inhibitors (CAIs) used clinically, such
as acetazolamide 1, methazolamide 2, ethoxzolamide 3,
and dichlorophenamide 4, were obtained, together with
inhibition data for aromatic/heterocyclic sulfonamides
possessing varied structures, designed in order to
investigate the best inhibition/selectivity profiles against
the mitochondrial isoform with respect to the cytosolic
isozymes CA I and II and the membrane-associated
isozyme CA IV, considered up to now to be responsible
CA In h ibition . Inhibition data of four CA isozymes,
i.e., the cytosolic hCA I and II, the membrane-bound
bCA IV, and the mitochondrial mCA V with compounds
1
-46 is shown in Table 1 (h denotes enzyme of human,
for most of the physiological functions of CAs in higher
vertebrates.1
development of novel antiobesity therapies.
-3,14
b of bovine, and m of murine origin).
Such compounds may be useful for the
1
5
Discu ssion
Ch em istr y. Sulfonamides1,2 and sulfamates26,27 are
the most potent CAIs reported up to now against the
physiologically relevant isozymes CA I, II, and IV or the
tumor-associated isozyme CA IX. Thus, we decided to
explore a large series of sulfonamides of types 1-45 as
well as the most potent sulfamate CA II inhibitor
detected up to now, the clinically used antiepileptic drug
Resu lts
Ch em istr y. The structure of sulfonamides 1-45 and
sulfamate 46 investigated for their interaction with
mCA V (as well as with the cytosolic, physiologically
relevant isozymes hCA I and II, and the membrane-
bound isozyme bCA IV) is shown below.
2
6
topiramate 46, against the murine, full length mito-
chondrial isozyme, mCA V. Both aromatic and hetero-
cyclic sulfonamides were included in this study, since
the inhibition profile against the other investigated
isozymes (i.e., CA I, II, IV, and IX) of these compounds
1
,2
is very different. A major concern when designing
CAIs is constituted by the general lack of selectivity of
such compounds for the target isozyme, and as a
consequence, the probable side effects of drugs based
1
,2
on them. With very few exceptions (reviewed in ref
) most sulfonamides undiscriminately inhibit all CA
1
isozymes, although the affinity of particular isoforms
for these inhibitors vary in a well defined manner, with
CA II showing the highest affinity, generally followed
1
,2,26,27
by CA IX and IV,
whereas CA I is usually less
inhibited by sulfonamides but has a higher affinity for
2
8
metal-complexing anions. Up to now, only the mCA V
inhibition data of one sulfonamide inhibitor, acetazola-
2
9
Most of these sulfonamides were previously reported
except for 8-15, 17, 29-31, and 37,
which are new and were prepared by acylation of
mide 1 has been published, and thus it is necessary
to explore as many as possible aromatic/heterocyclic
sulfonamides in order to detect both potent inhibitors,
as well as leads that may show a favorable selectivity
profile toward the mitochondrial isozyme with respect
by our group,1
6-24
sulfanilamide 5, its homologues 6 and 7, or 5-imino-4-
2
25
methyl-δ -1,3,4-thiadiazoline-2-sulfonamide with acyl

1
274 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Vullo et al.
Ta ble 1. Inhibition Data of Compounds 1-46 against Isozymes CA I, II, IV, and V, at 20 °C
selectivity ratio
KI (hCA II)/
KI (mCA V)
selectivity ratio
KI (hCA II)/
KI (mCA V)
KIa [nM]
KIa [nM]
inhibitor hCA Ib hCA IIb bCA IVc mCA Vd
inhibitor hCA Ib hCA IIb bCA IVc mCA Vd
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
900
780
25
12
14
8
220
240
13
58
63
47
0.206
0.222
0.170
0.059
0.220
0.184
0.175
1.160
1.797
1.104
1.101
1.107
2.517
2.395
3.500
5.076
2.176
1.700
11.428
5.250
5.000
0.361
2.130
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
81
69
40
31
187
74
40
28
9
64
39
15
24
26
20
18
103
20
20
18
245
54
76
13
87
83
14
9
5
5
4
6
48
51
63
1.666
1.076
0.450
0.555
0.728
0.650
0.600
0.555
0.489
0.185
0.197
0.153
0.068
0.204
0.107
0.333
0.200
0.200
0.500
0.333
0.187
0.058
0.079
1200
28000
25000
21000
21400
14600
19700
19300
23500
17650
20600
14200
1400
3300
950
38
380
640
1360
920
913
212
74
210
206
233
85
96
18
13
17
10
21
20
15
10
75
13
12
10
120
10
15
2
22
300
170
160
246
133
232
227
258
214
230
63
66
37
17
240
105
75
13
49
3000
2800
2500
2850
2100
2630
2540
2840
2370
2550
980
850
176
600
460
140
150
20
85
119
e
nt
69
66
nt
nt
1000
645
900
250
290
850
270
10
130
185
163
10
30
174
8
8
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
6
17
1.5
3
1
1
2
2
9
3
5
6
5
5
6
1200
800
430
12
103
5
50000
nt
250
43
45
54
36
23
a
Errors in the range of 5-10% of the reported value (from three different assays). b Human (cloned) isozymes, by the 4-nitrophenyl
c
d
acetate esterase method. Bovine isozyme, isolated from lung microsomes, by the esterase method. Full length murine, cloned isozyme,
by the CO2 hydration method. nt ) not tested.
e
to the cytosolic or membrane-bound isoforms mentioned
above. The series of compounds included in our study
comprises sulfonamides 1-45 and sulfamate 46. Most
of these sulfonamides were previously reported by this
rivatives investigated here is associated with long
aliphatic acyl (15), long perfluoroacyl (16), or aromatic
acyl (17, 18, 29) moieties, whereas shorter aliphatic
acyls lead to less effective CA V inhibitors (compounds
8-14). For ureas 19-22, CA V inhibitory activity
increased from the sulfanilamide derivative 19 to the
longer chain analogue 21, with the phenyl moiety of the
second urea nitrogen atom (for example in 21) being
more effective than the corresponding 3,4-dichlorophen-
yl-substituted analogue 22. For the disulfonamides
23-27, good CA V inhibitory activity was correlated
with the presence of 4-phenylsufonylamido- or 4-sub-
stituted-phenylsulfonylamido moieties both for sul-
fanilamide (23, 26, and 27) and its homologues (24 and
25). Less effective was only the 4-acetylamidobenzene-
sulfonylamido-substituted compound 28. Thus, a first
structure-activity relationship (SAR) conclusion is that
sulfanilamides, homosulfanilamides, or 4-aminoethyl-
benzenesulfonamides incorporating long acyl/perfluo-
roacyl, aromatic acyl, aryl ureas, or arylsulfonamido
moieties substituting the free amino moiety of the leads
5-7 show low nanomolar affinity for mCA V. For the
second subseries, of heterocyclic sulfonamides showing
low nanomolar affinity for mCA V, i.e., compounds 35
and 38-43, one may observe that the presence of
perfluorobenzoyl moieties (in 35 and 38) is beneficial
for inducing strong inhibitory properties, whereas the
corresponding benzoyl derivatives 34 and 37, or the two
compounds incorporating other acyls (33 and 36), show
decreased CA V inhibitory properties. The best CA V
inhibitors were, on the other hand, the aminobenzol-
amide derivatives 39-43, which showed KI values in
the range of 4-9 nM. The lead itself, aminobenzolamide
43, is already a very potent CA V inhibitor (KI of 6 nM),
whereas its acetylated derivative 39 is slightly less
effective (KI of 9 nM). The halogenated acetylated
aminobenzolamides 40-42 are, on the other hand, more
effective CA V inhibitors as compared to the parent,
group, mainly in the search of topically acting anti-
glaucoma drugs based on CAIs,1
6-24
but 13 compounds,
such as 8-15, 17, 29-31, and 37, are new and were
obtained from sulfanilamide 5, its close analogues 6 and
2
7
, or 5-imino-4-methyl-δ -1,3,4-thiadiazoline-2-sulfon-
2
5
amide by acylation reactions with aliphatic or aromatic
acyl chlorides in the presence of bases (such as trieth-
ylamine or pyridine), as previously reported by this
group for structurally related CAIs.1
6-25
The new com-
pounds reported here were characterized by standard
physicochemical procedures that confirmed their struc-
tures (see Experimental Section for details).
CA In h ib it ion . Inhibition data of the four CA
isozymes of interest here, i.e., CA I, II, IV, and V with
compounds 1-46 are shown in Table 1. Since most CA
I, II, and IV inhibition data were previously reported,
only the CA V data will be discussed in detail, stressing
on the other hand the selectivity issue of these sulfon-
amides with regards to the mitochondrial isozyme
versus the cytosolic/membrane-associated ones. The
following observations can be made: (i) several highly
potent CA V inhibitors have been detected among the
investigated sulfonamides, such as 15-27, 29-31, 35,
and 38-43, these compounds showing inhibition con-
stants in the range of 6-36 nM. These inhibitors both
belong to the aromatic sulfonamide and heterocyclic
compounds subseries. Thus, the acylated sulfanilamides
1
5-18, the ureas derived from sulfanilamide, homosul-
fanilamide and 4-aminoethylbenzenesulfonamide 19-22,
the disulfonamides 23-27, and the coumarinyl-substi-
tuted benzenesulfonamides 29-31 belong to the first
subseries of potent, low nanomolar CA V inhibitors. It
is obvious from the data of Table 1 that the best CA V
inhibitory activity for the 4-acylated-sulfanilamide de-

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1275
unsubstituted compound 39. One should stress that all
inhibitors discussed at this paragraph show much
stronger CA V inhibitory properties as compared to the
clinically used CAIs, compounds 1-4 (see later in the
text); (ii) a second group of compounds, such as 1-3, 9,
fonamide CAIs, followed by mCA V and bCA IV,
whereas isozyme hCA I has the lowest affinity. Still,
many exceptions from this general behavior are ob-
served (see discussion later in the text). First of all, by
defining a selectivity ratio between isozymes II and V
as the ratio of the corresponding inhibition constants,
one may see (Table 1) that most compounds have this
ratio less than 1, obviously meaning that they better
inhibit the cytosolic isozyme II and not the mitochon-
drial one, CA V. But 17 compounds in the series
investigated here, i.e., 8-21 and 23-25, show a selec-
tivity ratio in the range of 1.1-11.4, possessing thus a
higher affinity for the mitochondrial CA V, over the
rapid cytosolic isozyme CA II. The most CA V selective
inhibitor was urea 19, followed by its close congeners
20 and 21 and the acylated sulfanilamides 15 and 16.
Since some of these compounds are also relatively
efficient CA V inhibitors, with inhibition constants in
the range of 13-20 nM, one may affirm that the
acylated sulfanilamides and the ureido-benzenesulfona-
mides tend to be more CA V selective than the other
derivatives investigated here. The most efficient CA V
inhibitors detected on the other hand, such as 39-43,
all possess selectivity ratios in the range of 0.2-0.5,
being much more efficient CA II inhibitors than CA V
inhibitors, similarly with the clinically used derivatives
1-3 and 44. The most CA II selective inhibitors (over
CA V) were dichlorophenamide 4, the furyl analogue of
methazolamide 36, brinzolamide 45, and topiramate 46,
all of them showing selectivity ratios of 0.058-0.079.
One of the investigated compounds, 22, showed very
efficient CA I inhibitory properties, possessing KI values
of the same order of magnitude for both cytosolic
isozymes CA I and II, a behavior previously observed
for several other ureido-sulfonamide derivatives.1
Finally, most of these sulfonamides showed higher
affinity for CA V over CA IV (such as 1-21, 23-28, 33,
34, and 37), but some derivatives, such as 22, 32, 35,
36, and 39-46, were more effective CA IV inhibitors
and not CA V inhibitors (Table 1).
1
3, 14, 33, 34, 36, 37, and 44-46, are effective mCA V
inhibitors, with KI values in the range of 47-96 nM,
but they are less potent inhibitors as compared to the
derivatives discussed above. In this group we find the
clinically used CAIs acetazolamide 1, methazolamide 2,
ethoxzolamide 3, dorzolamide 44, brinzolamide 45, and
topiramate 46, as well as several close analogues of
acetazolamide and methazolamide (33, 34, 36, and 37).
The remaining compounds are acylated sulfanilamides
incorporating trifluoromethyl (9) n-butyl and tert-butyl
moieties (13 and 14). The main SAR conclusion here is
that surprisingly, it seems that fused bicyclic sulfon-
amides (such as 3, 44 and 45) or sugar-sulfamates
(although only one such compound has been tested, and
maybe the conclusion for this type of derivatives should
be taken with caution) do not lead to the most effective
CA V inhibitors, although for CA II, usually this type
of inhibitors was the most effective (ethoxzolamide,
dorzolamide, brinzolamide, and topiramate are among
the most effective CA II inhibitors detected). Another
conclusion is that the acylating moieties in position 5
for 1,3,4-thiadiazole-2-sulfonamide and the correspond-
ing thiadiazoline-2-sulfonamide derivatives is of criti-
cal importance for the CA V inhibitory properties.
These data show that both acylamido (such as in 35 and
3
8) and especially arylsulfonamido (in 39-43) moieties
are highly beneficial for the CA V inhibitory properties,
with aromatic moieties being preferred over the ali-
phatic ones; (iii) a third group of compounds, including
,17
8
, 10-12, 28, and 32, showed moderate CA V inhibitory
properties, with KI values in the range of 100-245 nM.
These compounds include the acylated sulfanilamides
(
(
derivatives 8, 10-12) incorporating short acyl moieties
C1-C3), one of the disulfonamides (compound 28) and
the only Schiff’s base investigated here, and another
sulfanilamide derivative, compound 32; (iv) the last
group of sulfonamides (compounds 4-7) include the
weak CA V inhibitors demonstrated here, which showed
KI values in the range of 640-1360 nM. All these
compounds are simple aromatic sulfonamides or bis-
sulfonamides, such as dichlorophenamide 4, sulfanil-
amide 5, and its two close homologues 6 and 7. The most
ineffective CA V inhibitor in the entire series of 46
derivatives investigated here was sulfanilamide 5,
whereas the elongation of the molecule in 6 and 7 led
to an enhanced inhibitory activity. The presence of
two sulfonamide moieties, such as in dichlorophen-
amide 4, further increased the CA V inhibitory proper-
ties. It should also be noted that derivatization of the
simple aminosulfonamides 5-7 by a variety of acyl,
arylsulfonyl, or ureido moieties, led to highly potent CA
V inhibitors as compared to the parent sulfonamides,
as illustrated well for compounds 8-32 discussed
earlier.
The compounds investigated here which possess
potent inhibitory activity against CA II and CA V, may
be useful for the prevention and treatment of obesity.15
Indeed, in a recent patent it has been shown that
topiramate, a potent CA II inhibitor and also an efficient
CA V inhibitor, may be useful for this purpose.15 It was
in fact noted that many obese epilepsy patients treated
with this antiepileptic drug lost 10-15% of their body
3
9
weight, and the same effect has also been demon-
4
0
strated in an animal model of obesity, but this “side
effect” of the drug was never explained before the above-
1
5,41
15
mentioned patent was published.
Antel et al.
demonstrated that the antiobesity activity of topiramate
may be due among others to the CA II/CA V inhibition
which has as a consequence a diminished de novo
lipogenesis in adipocytes. Since many of the compounds
investigated here show more potent CA II/V inhibitory
properties in vitro, we expect that they may show better
antiobesity activity, and thus a potential for clinical
4
1
development.
A last important aspect regards the inhibition profiles
of the different CA isozymes investigated here with
these derivatives and the CA V selectivity issues related
to it. Thus, at a first glance, the general trend observed
is that hCA II has the highest affinity for these sul-
The supposed mechanism by which CA V inhibitors
may reduce fat biosynthesis and thus be useful as
antiobesity agents is schematically shown in Figure 1.
Thus, in several important biosynthetic processes in-

1
276 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Vullo et al.
biosynthesis inhibition may be enhanced even more, and
may thus explain the rationale of using CAIs in the
1
5,41
treatment and prophylaxis of obesity.
Con clu sion s
We investigated a large series of aromatic/heterocyclic
sulfonamide and a sulfamate for their inhibitory proper-
ties against the mitochondrial isozyme CA V, comparing
these inhibition data with those for the cytosolic isozymes
CA I and II and the membrane-associated isoform CA
IV. Several low nanomolar CA V inhibitors were de-
tected (KI values in the range of 4-15 nM), most of them
belonging to the acylated sulfanilamide, ureido-ben-
zenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and
aminobenzolamide type of compounds. The clinically
used CAIs acetazolamide, methazolamide, ethoxzola-
mide, dorzolamide, brinzolamide, and topiramate on the
other hand were less effective CA V inhibitors, showing
inhibition constants in the range of 47-63 nM. Some
of the investigated sulfonamides, such as the ureido-
benzenesulfonamides and the acylated sulfanilamides
showed higher affinity for CA V than for the other
isozymes, CA II included, which is a remarkable result,
since most investigated up to now CAIs inhibited better
the cytosolic isozyme CA II. These results prompt us to
hope that the selective inhibition of CA V, or the dual
inhibition of CA II and CA V, may lead to the develop-
ment of novel pharmacological applications of CAIs, for
example for the treatment or prevention of obesity, by
inhibiting CA-mediated lipogenetic processes.
F igu r e 1. The transfer of acetyl groups from the mitochon-
drion to the cytosol (as citrate) for the provision of substrate
for de novo lipogenesis.41 All steps involving bicarbonate also
need the presence of at least two CA isozymes: CA V in the
mitochondrion and CA II in the cytosol (see discussion in the
text).
volving pyruvate carboxylase (PC), acetyl CoA carboxy-
lase (ACC), and carbamoyl phosphate synthetases I and
II, bicarbonate, not carbon dioxide, is the real substrate
of these carboxylating enzymes, and the provision of
enough bicarbonate is assured mainly by the catalysis
involving the mitochondrial isozyme CA V (probably
assisted by the high activity cytosolic isozyme CA II).
Mitochondrial PC is needed for the efflux of acetyl
groups from the mitochondria to the cytosol where the
fatty acid biosynthesis takes place.8,41 Practically, pyru-
vate is carboxylated to oxaloacetate in the presence of
bicarbonate and PC. The bicarbonate needed for this
process is generated under the catalytic influence of the
mitochondrial CA V. The mitochondrial membrane is
impermeant to acetyl-CoA which condenses with oxalo-
acetate to form citrate, which is thereafter translocated
to the cytoplasm by means of the tricarboxylic acid
transporter. In the cytosol, the citrate is cleaved and
regenerates acetyl-CoA and oxaloacetate. As oxaloace-
tate also cannot cross the mitochondrial membrane, its
decarboxylation regenerates pyruvate which can be then
transported into the mitochondria by means of the
pyruvate transporter (Figure 1).8 The acetyl-Co A thus
generated in the cytosol is in fact used for the de novo
lipogenesis, by carboxylation in the presence of ACC and
bicarbonate, with formation of malonyl-Co A. The
bicarbonate needed in this process is furnished by the
CA II catalyzed conversion of CO2 to bicarbonate.
Subsequent steps involving the sequential transfer of
Exp er im en ta l Section
Gen er a l. Melting points: heating plate microscope (not
-1
corrected); IR spectra: KBr pellets, 400-4000 cm Karl Zeiss
J ena UR-20 spectrometer; NMR spectra: Varian Gemini
1
3
7
00BB apparatus, operating at 300 MHz for H NMR and at
13
5 MHz for C NMR (chemical shifts are expressed as δ values
relative to Me Si as internal standard for proton spectra and
4
to the solvent resonance for carbon spectra). Attributions are
1
1
1
13
given based on APT, COSY ( H- H), and HETCOR ( H- C)
experiments. Elemental analysis ((0.4% of the theoretical
values, calculated for the proposed structures): Carlo Erba
Instruments CHNS Elemental Analyzer, Model 1106. All
reactions were monitored by thin-layer chromatography (TLC),
using 0.25 mm-thick precoated silica gel plates (E. Merck)
,41
eluted with MeOH:CHCl 1:4 v/v unless specified otherwise.
3
Sulfonamides/sulfamate 1-7 and 44-46 are commercially
available and were from Sigma-Aldrich, Merck, Alcon, and
J ohnson & J ohnson. Acyl halides used in the syntheses of
compounds 8-15, 17, and 37 were commercially available
(from Sigma-Aldrich or Fluka) and were used without ad-
ditional purification. Coumarin-3-carboxylic acid was a gift
from prof. G. Renzi (University of Florence). Acetonitrile,
triethylamine, pyridine, methanol, ethanol, chloroform, ethyl
acetate (E. Merck, Darmstadt, Germany), or other solvents/
reagents used in the synthesis were double distilled and kept
on molecular sieves in order to maintain them in anhydrous
conditions. Sulfonamides 16, 18, 19-28, 32-36, and 38-43
8
,41
acetyl groups lead to longer chain fatty acids.
As a
whole, two CA isozymes are critical to the entire process
of fatty acid biosynthesis: CA V within the mitochondria
(to provide enough substrate to PC), and CA II within
the cytosol (for providing sufficient substrate to ACC).
It was in fact demonstrated that inhibition of CAs by
sulfonamides (such as for example trifluoromethane-
sulfonamide (TFM), a very potent but unstable CA
16-24
were previously reported by this group.
Ch em ist r y. Gen er a l P r oced u r e for t h e Syn t h esis of
Su lfon a m id es 8-15, 17, 29-31, a n d 37. Method A (Schot-
ten-Baumann synthesis): Amino/imino-sulfonamide (5 mmol)
2
-
25
1
(such as 5-imino-4-methyl-2-sulfonamido-δ 1,3,4-thiadiazoline,
inhibitor ) can decrease lipogenesis in adipocytes in cell
culture.8
-10
sulfanilamide 5, homo-sulfanilamide 6, or 4-aminoethylben-
zenesulfonamide 7) was dissolved in 15 mL of 2.5 M NaOH
and cooled to 2-5 °C in a salt-ice bath. The corresponding acyl
chloride (5 mmol) was added in small portions, concomitantly
with 10 mL of a 2 M NaOH solution, maintaining the
temperature under 10 °C. The reaction mixture was then
In such experiments, an undiscriminate
inhibition of all CA isozymes present in these tissues is
achieved by the used inhibitor, such as for example TFM
8
-10
or acetazolamide.
With more specific CA V inhibi-
tors, as those described here, the process of fatty acid

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1277
4-Pivaloylamido-benzenesulfonamide 14, white crystals, mp
stirred at room temperature for 5-10 h (TLC control) and then
the pH was adjusted to 2 with 5 N HCl, and the precipitated
sulfonamides were filtered and recrystallized from aqueous
ethanol.
Method B: As above, but pyridine was used as solvent and
no other base was necessary. After the reaction was performed,
the excess pyridine was evaporated in vacuo and the reaction
mixture poured in 50 mL of ice-water, and the precipitated
sulfonamides were recrystallized as described above.
Method C: The sulfonamide to be derivatized was suspended/
dissolved in a 1:1 mixture of acetone-water, and the stoichio-
metric amount of acyl chloride and base were added concomi-
-
1
sym
as
162-4 °C; IR (KBr), cm : 1158 (SO
2
), 1374 (SO
); H NMR (DMSO-d ), δ, ppm: 0.95
(s, 9H, t-Bu); 7.02 (s, 2H, SO NH ), 7.80 (d, 2H, AA′BB′, 8.9
Hz), 7.91 (d, 2H, AA′BB′, 8.9 Hz), 8.01 (br s, CONH);
NMR (DMSO-d ), δ, ppm: 9.4 (Me); 40.3 (C tertiary); 126.24
(C2/C3-Ph), 128.33 (C3/C2-Ph), 139.32 (C1/C4-Ph), 141.51
(C4/C1-Ph); 162.39 (CO); Anal. (C11 S) C, H, N.
4-n-Hexanamido-benzenesulfonamide 15, white crystals, mp
2
), 1672
1
(CONH); 3360 (NH, NH
2
6
2
2
1
3
C
6
H
16
N
2
O
3
-
1
sym
as
132-5 °C; IR (KBr), cm : 1159 (SO
2
), 1368 (SO
); H NMR (DMSO-d ), δ, ppm: 0.89
(t, 3H, J ) 6.8 Hz); 1.30 (m, 4H), 1.62 (m, 2H), 3.94 (t, 2H, J
) 6.4 Hz), 7.07 (s, 2H, SO NH ), 7.79 (d, 2H, AA′BB′, 8.9 Hz),
7.90 (d, 2H, AA′BB′, 8.9 Hz), 8.04 (br s, CONH); C NMR
(DMSO-d ), δ, ppm: 14.2 (Me); 19.9 (CH ); 28.7 (CH ); 28.9
(CH ); 42.1 (CH ); 126.24 (C2/C3-Ph), 128.35 (C3/C2-Ph),
139.33 (C1/C4-Ph), 141.70 (C4/C1-Ph); 162.54 (CO); Anal.
S) C, H, N.
4-Benzoylamino-benzenesulfonamide 17, white crystals, mp
2
), 1675
1
(CONH); 3360 (NH, NH
2
6
tantly. The base used may be NaOH, NaHCO
3
, Et
3
N, pyridine,
2
2
1
3
etc. Good results were generally obtained when working with
sodium bicarbonate as base. The reaction mixture was mag-
netically stirred at room temperature for several hours, the
solvent was evaporated, and the reaction products were
recrystallized as described above.
Coumarin-3-carboxylic acid was converted to the corre-
sponding acyl chloride by reaction with thionyl chloride in
benzene and was used for the preparation of derivatives
6
2
2
2
2
(C12
18 2 3
H N O
-
1
sym
as
209-10 °C; IR (KBr), cm : 1154 (SO
2
), 1373 (SO
); H NMR (DMSO-d ), δ, ppm: 7.12
), 7.20-7.55 (m, 5H, Ph); 7.78 (d, 2H, AA′BB′,
2
), 1673
1
(CONH); 3360 (NH, NH
(s, 2H, SO NH
8.9 Hz), 7.92 (d, 2H, AA′BB′, 8.9 Hz), 8.06 (br s, CONH);
NMR (DMSO-d ), δ, ppm: 123.05 (Ph); 126.24 (C2/C3-Ph),
127.41(Ph); 128.35 (C3/C2-Ph), 130.62 (Ph); 139.33 (C1/C4-Ph),
141.70 (C4/C1-Ph); 151.13 (Ph); 166.59 (CO); Anal. (C H12-
2
6
2
9-31 as described above (best yields were obtained by
applying Method C).
-Acetamido-benzenesulfonamide 8, white crystals, mp
2
2
1
3
C
4
6
-
1
sym
as
2
03-4 °C; IR (KBr), cm : 1163 (SO
2
), 1375 (SO
); H NMR (DMSO-d ), δ, ppm: 2.54
s, 3H, Me); 7.09 (s, 2H, SO NH ), 7.81 (d, 2H, AA′BB′, 8.9
2
), 1672
1
(CONH); 3360 (NH, NH
2
6
13
(
2
2
2 3
N O
S) C, H, N.
1
3
Hz), 7.94 (d, 2H, AA′BB′, 8.9 Hz), 8.07 (br s, CONH);
NMR (DMSO-d ), δ, ppm: 21.4 (Me); 126.56 (C2/C3-Ph),
28.45 (C3/C2-Ph), 139.60 (C1/C4-Ph), 141.37 (C4/C1-Ph);
62.39 (CO); Anal. (C S) C, H, N.
-Trifluoroacetamido-benzenesulfonamide 9, white crystals,
C
4-(Coumarin-3-yl-carboxamido)benzenesulfonamide 29, white
-
1
sym
as
6
crystals, 166-8 °C; IR (KBr), cm : 1159 (SO
2
), 1362 (SO
); H NMR (DMSO-d ), δ, ppm:
6.84 (s, 1H, 4H of coumarin); 7.12 (s, 2H, SO NH ), 7.16-7.58
(m, 4H, coumarin); 7.79 (d, 2H, AA′BB′, 8.9 Hz), 7.90 (d, 2H,
2
),
1
1
1
1676 (CONH); 3360 (NH, NH
2
6
8
H
10
N
2
O
3
2
2
4
-
1
sym
as
13
mp 185-6 °C; IR (KBr), cm : 1159 (SO
CONH); 3360 (NH, NH
s, 2H, SO NH
2
), 1374 (SO
); H NMR (DMSO-d ), δ, ppm: 7.33
2
), 7.80 (d, 2H, AA′BB′, 8.9 Hz), 7.95 (d, 2H,
2
), 1673
6
AA′BB′, 8.9 Hz), 8.04 (br s, CONH); C NMR (DMSO-d ), δ,
1
(
(
2
6
ppm: 123.45; 126.24 (C2/C3-Ph), 127.40; 128.35 (C3/C2-Ph),
130.13; 139.33 (C1/C4-Ph), 141.70 (C4/C1-Ph); 151.13; 153.34;
158.50, 166.59 (CONH); 176.33 (CO-O); Anal. (C16H N O S)
12 2 5
C, H, N.
2
1
3
AA′BB′, 8.9 Hz), 8.04 (br s, CONH); C NMR (DMSO-d
ppm: 44.2 (CF ); 126.67 (C2/C3-Ph), 128.26 (C3/C2-Ph), 139.49
C1/C4-Ph), 141.80 (C4/C1-Ph); 163.62 (CO); Anal. (C
S) C, H, N.
-Propionamido-benzenesulfonamide 10, white crystals, mp
6
), δ,
3
(
8
H
7
F
3
-
4-(Cou m a r in -3-yl-ca r boxa m idom et h yl)ben zen esu lfon -
-
1
N
2
O
3
amide 30, white crystals, 169-70 °C; IR (KBr), cm : 1159
sym as 1
4
(SO
(DMSO-d
coumarin); 7.10 (s, 2H, SO
7.79 (d, 2H, AA′BB′, 8.9 Hz), 7.92 (d, 2H, AA′BB′, 8.9 Hz),
8.04 (br s, CONH); 13C NMR (DMSO-d
), δ, ppm: 41.54
(CH ); 123.40; 126.24 (C2/C3-Ph), 127.46; 128.43 (C3/C2-Ph),
130.25; 139.14 (C1/C4-Ph), 141.62 (C4/C1-Ph); 151.76; 153.67;
2
), 1364 (SO
), δ, ppm: 4.49 (d, 2H, 6.0 Hz), 6.84 (s, 1H, 4H of
NH ), 7.16-7.57 (m, 4H, coumarin);
2 2
), 1675 (CONH); 3360 (NH, NH ); H NMR
-
1
sym
as
1
(
(
2
90-1 °C; IR (KBr), cm : 1162 (SO
2
), 1375 (SO
); H NMR (DMSO-d ), δ, ppm: 1.33
t, 3H, Me); 3.27 (q, 2H, CH ); 7.09 (s, 2H, SO NH ), 7.82 (d,
H, AA′BB′, 8.9 Hz), 7.94 (d, 2H, AA′BB′, 8.9 Hz), 8.05 (br s,
2
), 1675
6
1
CONH); 3360 (NH, NH
2
6
2
2
2
2
2
6
1
3
CONH); C NMR (DMSO-d
6
), δ, ppm: 16.1 (Me); 42.5 (CH
26.60 (C2/C3-Ph), 128.13 (C3/C2-Ph), 139.07 (C1/C4-Ph),
41.45 (C4/C1-Ph); 162.13 (CO); Anal. (C S) C, H, N.
-n-Butanamido-benzenesulfonamide 11, white crystals, mp
2
);
2
1
1
14 2 5
158.48, 166.52 (CONH); 176.40 (CO-O); Anal. (C17H N O S)
C, H, N.
4-(Coumarin-3-yl-carboxamidoethyl)benzenesulfonamide
9
H
12
N
2
O
3
4
-
1
sym
as
1
87-8 °C; IR (KBr), cm : 1162 (SO
2
), 1376 (SO
); H NMR (DMSO-d ), δ, ppm: 1.26
t, 3H, J ) 7.3 Hz); 1.62 (m, 2H), 3.43 (t, 2H, J ) 6.2 Hz), 7.11
s, 2H, SO NH ), 7.80 (d, 2H, AA′BB′, 8.9 Hz), 7.92 (d, 2H,
2
), 1675
1
-1
sym
(
(
(
CONH); 3360 (NH, NH
2
6
31, white crystals, 160-1 °C; IR (KBr), cm : 1158 (SO
2
),
); H NMR (DMSO-
), δ, ppm: 2.91 (t, 2H, 7.2 Hz), 3.45 (q, 2H, 6.5 Hz), 6.83 (s,
1H, 4H of coumarin); 7.11 (s, 2H, SO NH ), 7.18-7.58 (m, 4H,
coumarin); 7.79 (d, 2H, AA′BB′, 8.9 Hz), 7.92 (d, 2H, AA′BB′,
8.9 Hz), 8.05 (br s, CONH); 13C NMR (DMSO-d
), δ, ppm:
35.14 (CH ), 39.70, (N-CH ), 123.40; 126.21 (C2/C3-Ph),
27.45; 128.58 (C3/C2-Ph), 130.36; 139.09 (C1/C4-Ph), 141.55
(C4/C1-Ph); 151.41; 153.38; 158.44, 166.19 (CONH); 176.32
(CO-O); Anal. (C H N O S) C, H, N.
as
1
1363 (SO
2 2
), 1675 (CONH); 3360 (NH, NH
d
6
2
2
1
3
AA′BB′, 8.9 Hz), 8.05 (br s, CONH); C NMR (DMSO-d
ppm: 12.1 (Me); 22.7 (CH ); 42.5 (CH ); 126.19 (C2/C3-Ph),
28.17 (C3/C2-Ph), 139.34 (C1/C4-Ph), 141.59 (C4/C1-Ph);
62.76 (CO); Anal. (C10 S) C, H, N.
-Isobutanamido-benzenesulfonamide 12, white crystals, mp
6
), δ,
2
2
2
2
1
1
6
H
14
N
2
O
3
2
2
1
4
-
1
sym
as
1
64-6 °C; IR (KBr), cm : 1158 (SO
2
), 1374 (SO
); H NMR (DMSO-d ), δ, ppm: 1.28
d, 6H, J ) 6.2 Hz); 3.87 (m, 1H), 7.13 (s, 2H, SO NH ), 7.76
d, 2H, AA′BB′, 8.9 Hz), 7.95 (d, 2H, AA′BB′, 8.9 Hz), 8.02 (br
2
), 1673
1
(
(
(
CONH); 3360 (NH, NH
2
6
18 16
2
5
2
2
2
5-Benzoylimino-4-methyl-∆ -1,3,4-thiadiazoline-2-sulfona-
mide, 37, white crystals, mp 280-1 °C (MeOH). IR (KBr),
1
3
-1
sym
as
1
s, CONH); C NMR (DMSO-d
6
), δ, ppm: 22.4 (Me); 67.8 (CH);
26.24 (C2/C3-Ph), 128.36 (C3/C2-Ph), 139.19 (C1/C4-Ph),
41.46 (C4/C1-Ph); 162.50 (CO); Anal. (C10 S) C, H, N.
-n-Pentanamido-benzenesulfonamide 13, white crystals,
cm : 1175 (SO
(d
2 2 6
5H, Ph); 8.51 (s, 2H, SO NH ); C NMR (DMSO-d ), δ, ppm:
2
2
), 1325 (SO ), 1600 (CONH); H NMR
1
1
6
-DMSO), δ, ppm; J , Hz: 4.90 (s, 3H, Me); 7.20-7.55 (m,
13
H
14
N
2
O
3
4
42.13 (Me); 123.13 (Ph); 127.30 (Ph); 130.48 (Ph); 151.04 (Ph);
157.91 (CONH), 164.91 (C-thiadiazole), 164.94 (C-thiadiazole);
Anal. (C H N O S ) C, H, N.
10 10 4 3 2
-1
sym
as
mp 186-7 °C; IR (KBr), cm : 1163 (SO
2
), 1370 (SO
); H NMR (DMSO-d ), δ, ppm: 0.92
t, 3H, J ) 7.1 Hz); 1.35 (m, 2H), 1.58 (m, 2H), 3.87 (t, 2H, J
6.4 Hz), 7.08 (s, 2H, SO NH ), 7.80 (d, 2H, AA′BB′, 8.9 Hz),
.94 (d, 2H, AA′BB′, 8.9 Hz), 8.05 (br s, CONH); C NMR
DMSO-d ), δ, ppm: 14.0 (Me); 19.1 (CH ); 31.1, (CH ); 42.3
CH ); 126.18 (C2/C3-Ph), 128.21 (C3/C2-Ph), 139.30 (C1/
2
), 1675
1
(
(
)
7
(
(
CONH); 3360 (NH, NH
2
6
CA In h ibition . 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 Lind-
2
2
1
3
3
0
6
2
2
skog’s group. Cell growth conditions were those described in
the literature,31 and enzymes were purified by affinity chro-
2
3
2
C4-Ph), 141.64 (C4/C1-Ph); 162.60 (CO); Anal. (C11
C, H, N.
H
16
N
2
O
3
S)
matography according to the method of Khalifah et al.
Enzyme concentrations were determined spectrophotometri-

1
278 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Vullo et al.
cally at 280 nm, utilizing a molar absorptivity of 49 mM cm-1
-1
of the mCA V cDNA and to Prof. G. Renzi (University
of Florence) for the gift of coumarin-3-carboxylic acid.
for CA I and 54 mmol-1 cm for CA II, respectively, based on
-1
M ) 28.85 kDa for CA I, and 29.3 kDa for CA II, respec-
r
33,34
tively.
bCA IV was isolated from bovine lung microsomes
Refer en ces
as described by Maren et al., and its concentration has been
determined by titration with ethoxzolamide.35
(1) (a) Supuran, C. T.; Scozzafava, A.; Conway, J . (Eds.) Carbonic
Anhydrase - Its Inhibitors and Activators; CRC Press: Boca
Raton, FL, 2004; pp 1-369. (b) Supuran, C. T.; Scozzafava, A.;
Casini, A. Carbonic anhydrase inhibitors. Med. Res. Rev. 2003,
The mCAV cDNAs (a gift of Prof. P. Laipis, University of
Florida) was amplified by using PCR and specific primers for
the vector pCAL-n-FLAG (from Stratagene, Milan, Italy). The
obtained construct was inserted in the pCAL-n-FLAG vector
and then cloned and expressed in E. coli strain BL21-GOLD-
2
3, 146-189.
(
2) (a) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors
and their therapeutic potential. Exp. Opin. Ther. Pat. 2000, 10,
575-600. (b) Supuran, C. T.; Scozzafava, A. Applications of
carbonic anhydrase inhibitors and activators in therapy. Exp.
Opin. Ther. Pat. 2002, 12, 217-242.
3) Hewett-Emmett, D. Evolution and distribution of the carbonic
anhydrase gene families. In The Carbonic Anhydrases - New
Horizons, Chegwidden, W. R.; Carter, N.; Edwards, Y., Eds.;
Birkhauser Verlag: Basel, Switzerland, 2000; pp 29-78.
4) Smith, K. S.; Ferry, J . G. Prokaryotic carbonic anhydrases.
FEMS Microbiol. Rev. 2000, 24, 335-366.
2
9
(
DE3) (from Stratagene) as described by Heck et al. In
2
9
contrast to the report of Heck et al., no CA V activity was
found in the cell lysate, but all enzyme was present in inclusion
bodies from which it was purified as described below. The
bacterial cells were lysed by sonification and homogenated in
a buffered solution (pH 8) of 4 M urea and 2% Triton X-100.
The homogenate thus obtained was extensively centrifuged
(
(
(
11 000g) in order to remove soluble and membrane-associated
proteins as well as other cellular debris. The resulting pellet
was washed by repeated homogenation and centrifugation in
water, to remove the remaining urea and Triton X-100.
Purified CA V inclusion bodies were denaturated in 6 M
guanidine hydrochloride and refolded into the active form by
snap dilution into a solution of 100 mM MES (pH 6), 500 mM
L-arginine, 2 mM ZnCl , 2 mM EDTA, 2 mM reduced glu-
2
tathione, and 1 mM oxidized glutathione. Active mCA V were
extensively dialyzed into a solution of 10 mM Hepes (pH 7.5),
(5) Dodgson, S. J . Inhibition of mitochondrial carbonic anhydrase:
a discrepancy examined. J . Appl. Physiol. 1987, 63, 2134-2141.
(6) Dodgson, S. J .; Cherian, K. Mitochondrial carbonic anhydrase
is involved in rat renal glucose synthesis. Am. J . Physiol. 1989,
2
57, E791-E796.
(
7) (a) Spencer, I. M.; Hargreaves, I.; Chegwidden, W. R. Effect of
the carbonic anhydrase inhibitor acetazolamide on lipid synthe-
sis in the locust. Biochem. Soc. Trans. 1988, 16, 973-974. (b)
Chegwidden, W. R.; Spencer, I. M. Carbonic anhydrase provides
bicarbonate for de novo lipogenesis in the locus. Compd. Bio-
chem. Physiol. 1996, 115B, 247-254.
1
2 4 2
0 mM Tris HCl, 100 mM Na SO , and 1 mM ZnCl and
3
2
(8) Chegwidden, W. R.; Dodgson, S. J .; Spencer, I. M. The roles of
carbonic anhydrase in metabolism, cell growth and cancer in
animals. In The Carbonic Anhydrases - New Horizons; Cheg-
widden, W. R., Edwards, Y., Carter, N., Eds.; Birkh a¨ user
Verlag: Basel, 2000; pp 343-363.
further purified by sulfonamide affinity chromatography. The
amount of protein was determined by spectrophometric mea-
surements and its activity by stopped-flow enzymatic assays,
2
with CO as substrate.
Initial rates of 4-nitrophenylacetate hydrolysis catalyzed by
different CA isozymes were monitored spectrophotometrically,
at 400 nm, with a Cary 3 instrument interfaced with an IBM-
(9) Lynch, C. J .; Fox, H.; Hazen, S. A.; Stanley, B. A.; Dodgson, S.
J .; Lanoue, K. F. Role of hepatic carbonic anhydrase in de novo
lipogenesis. Biochem. J . 1995, 310, 197-202.
(10) Hazen, S. A.; Waheed, A.; Sly, W. S.; Lanoue, K. F.; Lynch, C.
J . Differentiation-dependent expression of CA V and the role of
carbonic anhydrase isozymes in pyruvate carboxylation in
adipocytes. FASEB J . 1996, 10, 481-490.
11) Atwood, P. V. The structure and mechanism of action of pyruvate
carboxylase. Int. J . Biochem. Cell Biol. 1995, 27, 231-249.
(12) Alldred, J . B.; Reilly, K. E. Short-term regulation of acetyl CoA
3
6
compatible PC. Solutions of substrate were prepared in
anhydrous acetonitrile; the substrate concentrations varied
-
2
-6
between 2 × 10 and 1 × 10 M, working at 25 °C. A molar
-
1
-1
absorption coefficient ꢀ of 18 400 M cm was used for the
-nitrophenolate formed by hydrolysis, in the conditions of the
(
4
3
6
experiments (pH 7.40), as reported in the literature. Non-
enzymatic hydrolysis rates (which were around 0.5-0.9% of
the catalytic 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 prepared in distilled-deionized water with 10-20%
carboxylase in tissues of higher animals. Prog. Lipid Res. 1997,
3
5, 371-385.
(
13) Forster, R. E.; Dodgson, S. J . Membrane transport and provision
of substrates for carbonic anhydrase in vertebrates. In The
Carbonic Anhydrases - New Horizons; Chegwidden, W. R.,
Edwards, Y., Carter, N., Eds.; Birkh a¨ user Verlag: Basel, 2000;
pp 263-280.
(v/v) DMSO (which is not inhibitory at these concentrations)
(14) (a) Parkkila, S. An overview of the distribution and function of
carbonic anhydrase isozymes in mammals. In The Carbonic
Anhydrases - New Horizons; Chegwidden, W. R., Edwards, Y.,
Carter, N., Eds.; Birkh a¨ user Verlag: Basel, 2000; pp 79-94. (b)
Parkkila, S.; Parkkila, A. K.; Kivel a¨ , J . Role of carbonic anhy-
drase and its inhibitors in biological science related to gastro-
enterology, neurology and nephrology. In Carbonic anhydrase
- Its inhibitors and activators; Supuran, C. T.; Scozzafava, A.;
Conway, J ., Eds., CRC Press: Boca Raton, FL, 2004; in press.
(15) Hebebrand, J .; Antel, J .; Preuschoff, U.; David, S.; Sann, H.;
Weske, M. Method for locating compounds which are suitable
for the treatment and/or prophylaxis of obesity. WO Patent
and dilutions up to 0.01 nM were done thereafter 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
I
inhibition constant K was determined as described by Pocker
3
6
and Stone, for isozymes I, II, and IV. Enzyme concentrations
were 3.0 µM for CA II, 10.5 µM for CA I and 25 µM for CA IV
3
7
(
this isozyme has a decreased esterase activity and higher
concentrations had to be used for the measurements).
An SX.18MV-R Applied Photophysics stopped-flow instru-
0
7821, 2002.
2
ment has been used for assaying the CA IX CO hydration
(
16) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Perfluoroalkyl/aryl-substituted derivatives of aromatic/hetero-
cyclic sulfonamides as topical intraocular pressure lowering
agents with prolonged duration of action. J . Med. Chem. 2000,
3
8
activity. Phenol red (at a concentration of 0.2 mM) has been
used as indicator, working at the absorbance maximum of 557
nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na
2 4
SO (for
maintaining constant the ionic strength), following the CA-
4
3, 4542-4551.
catalyzed CO
Saturated CO
2
hydration reaction for a period of 10-100 s.
solutions in water at 20 °C were used as
(
17) Supuran, C. T.; Scozzafava, A.; J urca, B. C.; Ilies, M. A. Carbonic
anhydrase inhibitors. Part 49. Synthesis of substituted ureido-
and thioureido derivatives of aromatic/heterocyclic sulfonamides
with increased affinities for isozyme I. Eur. J . Med. Chem. 1998,
33, 83-93.
2
substrate. Stock solutions of inhibitors were prepared at a
concentration of 1-3 mM (in DMSO-water 1:1, v/v) and
dilutions up to 0.1 nM done with the assay buffer mentioned
above. Enzyme concentration was 0.1 µM, and inhibition
(18) Clare, B. W.; Supuran, C. T. Carbonic anhydrase inhibitors. Part
61. Quantum chemical QSAR of a group of benzenedisulfona-
mides. Eur. J . Med. Chem. 1999, 34, 463-474.
19) Supuran, C. T.; Briganti, F.; Scozzafava, A. Sulfenamido-
sulfonamides as inhibitors of carbonic anhydrase isozymes I, II
and IV. J . Enzyme Inhib. 1997, 12, 175-190.
constants were calculated as described in the literature.38
(
Ack n ow led gm en t. Thanks are addressed to Dr. P.
Laipis (University of Florida, Gainesville) for the gift

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1279
(
20) Supuran, C. T.; Nicolae, A.; Popescu, A. Carbonic anhydrase
inhibitors. Part 35. Synthesis of Schiff bases derived from
sulfanilamide and aromatic aldehydes: the first inhibitors with
equally high affinity towards cytosolic and membrane-bound
isozymes. Eur. J . Med. Chem. 1996, 31, 431-438.
Inhibition of human and murine mitochondrial isozymes V with
anions. Bioorg. Med. Chem. Lett. 2003, 13, 2857-2861.
(29) Heck, R. W.; Tanhauser, S. M.; Manda, R.; Tu, C.; Laipis, P. J .;
Silverman, D. N. Catalytic properties of mouse carbonic anhy-
drase V. J . Biol. Chem. 1994, 269, 24742-24746.
(
21) Almajan, L. G.; Supuran, C. T. Carbonic anhydrase inhibitors.
Part 30. Complexes of 5-pivaloylamido-1,3,4-thiadiazole-2-sul-
fonamide with trivalent metal ions. Rev. Roumaine Chim. 1997,
(
30) Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.; J onsson,
B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure-function relations
in human carbonic anhydrase II as studied by site-directed
mutagenesis. In Carbonic anhydrase - From biochemistry and
genetics to physiology and clinical medicine; Botr e` , F., Gros, G.,
Storey, B. T., Eds., VCH: Weinheim, 1991; pp 1-13.
31) Behravan, G.; J onsson, B. H.; Lindskog, S. Fine-tuning of the
catalytic properties of carbonic anhydrase. Studies of a Thr200-
His variant of human isoenzyme II. Eur. J . Biochem. 1990, 190,
4
2, 593-597.
(
22) J itianu, A.; Ilies, M. A.; Briganti, F.; Scozzafava, A.; Supuran,
C. T. Complexes with biologically active ligands. Part 9. Metal
complexes containing of 5-benzoylamino- and 5-(3-nitrobenzoyl-
amino)-1,3,4-thiadiazole-2-sulfonamide as carbonic anhydrase
inhibitors. Metal Based Drugs 1997, 4, 1-8.
(
(
23) Ilies, M.; Supuran, C. T.; Scozzafava, A.; Casini, A.; Mincione,
F.; Menabuoni, L.; Caproiu, M. T.; Maganu, M.; Banciu, M. D.
Carbonic anhydrase inhibitors. Sulfonamides incorporating fu-
ran-, thiophene- and pyrrole-carboxamido groups possess strong
topical intraocular pressure lowering properties as aqueous
suspensions. Bioorg. Med. Chem. 2000, 8, 2145-2155.
3
51-357.
(
32) Khalifah, R. G.; Strader, D. J .; Bryant, S. H.; Gibson, S. M.
Carbon-13 nuclear magnetic resonance probe of active site
ionization of human carbonic anhydrase B. Biochemistry, 1977,
16, 2241-2247.
(
24) Ilies, M. A.; Vullo, D.; Pastorek, J .; Scozzafava, A.; Ilies, M.;
Caproiu, M. T.; Pastorekova, S.; Supuran, C. T. Carbonic
anhydrase inhibitors. Inhibition of tumor-associated isozyme IX
with halogenosulfanilamide and halogeno-aminophenylbenzol-
amide derivatives. J . Med. Chem. 2003, 46, 2187-2196.
25) (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Synthesis of water-soluble, topically effective intraocular pres-
sure lowering aromatic/heterocyclic sulfonamides containing
cationic or anionic moieties: is the tail more important than the
ring? J . Med. Chem. 1999, 42, 2641-2650. (b) Scozzafava, A.;
Briganti, F.; Mincione, G.; Menabuoni, L.; Mincione, F.; Supuran,
C. T. Carbonic anhydrase inhibitors: Synthesis of water-soluble,
amino acyl/dipeptidyl sulfonamides possessing long-lasting in-
traocular pressure-lowering properties via the topical route. J .
Med. Chem. 1999, 42, 3690-3700.
26) Casini, A.; Antel, J .; Abbate, F.; Scozzafava, A.; David, S.;
Waldeck, H.; Sch a¨ fer, S.; Supuran, C. T. Carbonic anhydrase
inhibitors: SAR and X-ray crystallographic study for the inter-
action of sugar sulfamates/sulfamides with isozymes I, II and
IV. Bioorg. Med. Chem. Lett. 2003, 13, 841-845.
27) Winum, J .-Y.; Vullo, D.; Casini, A.; Montero, J .-L.; Scozzafava,
A.; Supuran, C. T. Carbonic anhydrase inhibitors: Inhibition of
cytosolic isozymes I and II and the transmembrane, tumor-
associated isozyme IX with sulfamates including EMATE also
acting as steroid sulfatase inhibitors. J . Med. Chem. 2003, 46,
(33) Lindskog, S.; Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad Sci. U.S.A. 1964, 70, 2505-2508.
(34) Steiner, H.; J onsson, B. H.; Lindskog, S. The catalytic mecha-
nism of carbonic anhydrase. Hydrogen-isotope effects on the
kinetic parameters of the human C isoenzyme. Eur. J . Biochem.
1975, 59, 253-259.
(
(
35) Maren, T. H.; Wynns, G. C.; Wistrand, P. J . Chemical properties
of carbonic anhydrase IV, the membrane-bound enzyme. Mol.
Pharmacol. 1993, 44, 901-906.
(
36) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed
hydrolysis of p-nitrophenyl acetate. Biochemistry 1967, 6, 668-
6
78.
(
37) Baird, T. T.; Waheed, A.; Okuyama, T.; Sly, W. S.; Fierke, C. A.
Catalysis and inhibition of human carbonic anhydrase IV.
Biochemistry 1997, 36, 2669-2678.
(
(38) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. J . Biol. Chem. 1971, 246, 2561-2573.
(
39) Gordon, A.; Price, L. H. Mood stabilization and weight loss with
topiramate. Am. J . Psychiatry 1999, 156, 968-969.
(
(40) Picard, F.; Deshaies, Y.; Lalonde, J .; Samson, P.; Richard, D.
Topiramate reduces energy and fat gains in lean (Fa/?) and obese
(fa/ fa) Zucker rats. Obesity Res. 2000, 8, 656-663.
(41) Supuran, C. T. Carbonic anhydrase inhibitors in the treatment
and prophylaxis of obesity. Exp. Opin. Ther. Pat. 2003, 13,
1545-1550.
2
197-2204.
(
28) Franchi, M.; Vullo, D.; Gallori, E.; Antel, J .; Wurl, M.; Scoz-
zafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors.
J M031057+